American Chemical Society - Home - Division of ... CARB Program/ACS Boston meeting... · American...

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American Chemical Society Division of Carbohydrate Chemistry 240th ACS National Meeting, Boston, MA, August 22-26, 2010 T. Lowary, Program Chair SUNDAY MORNING Wolfrom-Isbell-New Investigator Award Symposium G. Eggleston, Organizer; T. Lowary, Organizer; T. Lowary, Presiding Papers 1-3 SUNDAY AFTERNOON Wolfrom-Isbell-New Investigator Award Symposium G. Eggleston, Organizer; T. Lowary, Organizer; T. Lowary, Presiding Papers 4-9 MONDAY MORNING Synthetic Oligosaccharides and Glycoconjugates for Preventing and Combating Disease L. Wang, Organizer; G. Boons, Organizer; G. Boons, Presiding Papers 10-16 MONDAY AFTERNOON Synthetic Oligosaccharides and Glycoconjugates for Preventing and Combating Disease G. Boons, Organizer; L. Wang, Organizer; L. Wang, Presiding Papers 17-22 MONDAY EVENING Sci-Mix T. Lowary, Organizer Papers 85, 89, 86, 92, 87, 94, 93, 110, 108, 115, 96, 113, 83, 98, 82, 97, 100, 81, 102, 88, 103, 101, 104, 111, 106, 105, 107 TUESDAY MORNING Petite and Sweet: Glyconanotechnology as a Bridge to New Medicines J. Barchi, Organizer; X. Huang, Organizer; J. Barchi, Presiding Papers 23-27 Oligonucleotide Therapeutics

Transcript of American Chemical Society - Home - Division of ... CARB Program/ACS Boston meeting... · American...

Page 1: American Chemical Society - Home - Division of ... CARB Program/ACS Boston meeting... · American Chemical Society Division of Carbohydrate Chemistry 240th ACS National Meeting, Boston,

American Chemical Society Division of Carbohydrate Chemistry 240th ACS National Meeting, Boston, MA, August 22-26, 2010 T. Lowary, Program Chair SUNDAY MORNING Wolfrom-Isbell-New Investigator Award Symposium G. Eggleston, Organizer; T. Lowary, Organizer; T. Lowary, Presiding Papers 1-3 SUNDAY AFTERNOON Wolfrom-Isbell-New Investigator Award Symposium G. Eggleston, Organizer; T. Lowary, Organizer; T. Lowary, Presiding Papers 4-9 MONDAY MORNING Synthetic Oligosaccharides and Glycoconjugates for Preventing and Combating Disease L. Wang, Organizer; G. Boons, Organizer; G. Boons, Presiding Papers 10-16 MONDAY AFTERNOON Synthetic Oligosaccharides and Glycoconjugates for Preventing and Combating Disease G. Boons, Organizer; L. Wang, Organizer; L. Wang, Presiding Papers 17-22 MONDAY EVENING Sci-Mix T. Lowary, Organizer Papers 85, 89, 86, 92, 87, 94, 93, 110, 108, 115, 96, 113, 83, 98, 82, 97, 100, 81, 102, 88, 103, 101, 104, 111, 106, 105, 107 TUESDAY MORNING Petite and Sweet: Glyconanotechnology as a Bridge to New Medicines J. Barchi, Organizer; X. Huang, Organizer; J. Barchi, Presiding Papers 23-27 Oligonucleotide Therapeutics

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M. Manoharan, Organizer; M. Manoharan, Presiding; R. Iyer, Presiding Papers 28-38 TUESDAY AFTERNOON Petite and Sweet: Glyconanotechnology as a Bridge to New Medicines J. Barchi, Organizer; X. Huang, Organizer; J. Barchi, Presiding Papers 39-44 Oligonucleotide Therapeutics M. Manoharan, Organizer; M. Manoharan, Presiding; S. Srivastava, Presiding Papers 45-54 WEDNESDAY MORNING Recognition of DNA: Recent Advances D. Arya, Organizer; D. Arya, Presiding Papers 55-59 General Papers T. Lowary, Organizer; E. Cioffi, Presiding Papers 60-66 WEDNESDAY AFTERNOON General Papers T. Lowary, Organizer; S. Graham, Presiding Papers 67-74 Recognition of DNA: Recent Advances D. Arya, Organizer; D. Arya, Presiding Papers 75-79 WEDNESDAY EVENING General Posters T. Lowary, Organizer Papers 80-115 THURSDAY MORNING General Papers T. Lowary, Organizer; C. Marzabadi, Presiding Papers 116-124 THURSDAY AFTERNOON

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General Papers T. Lowary, Organizer; R. Narain, Presiding Papers 125-135

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CARB: Division of Carbohydrate Chemistry S M T W T General Papers D D

General Posters E

Oligonucleotide Therapeutics** D

Petite and Sweet: Glyconanotechnology as a Bridge to New Medicines D

Recognition of DNA: Recent Advances P

Recognition of DNA: Recent Advances** A

Sci-Mix E

Synthetic Oligosaccharides and Glycoconjugates for Preventing and Combating Disease

D

Wolfrom-Isbell-New Investigator Award Symposium D Legend

A = AM; P = PM; D = AM/PM; E = EVE; AE = AM/EVE; DE = AM/PM/EVE; PE = PM/EVE; *Cosponsored symposium with primary organizer shown in parenthesis; located with primary organizer. **Primary organizer of cosponsored symposium.

   

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CARB Todd Lowary Sunday, August 22, 2010 Oral

Wolfrom-Isbell-New Investigator Award Symposium - AM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Gillian Eggleston, Todd Lowary

Presiders: Todd Lowary

Duration: 9:15 am - 11:20 am

Pres Time

Pub #

Presentation Title

9:15 am Introductory Remarks

9:20 am 1 Developing an enzymatic approach to synthesize heparan sulfates Jian Liu

10:00 am 2 Creating and implementing breakthrough medical technology Dr Robert S Langer

10:40 am 3 Advances in heparin analysis and synthesis Prof. Robert J. Linhardt PhD

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CARB Todd Lowary Sunday, August 22, 2010 Wolfrom-Isbell-New Investigator Award Symposium - PM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Gillian Eggleston, Todd Lowary

Presiders: Todd Lowary

Duration: 1:00 pm - 4:45 pm

Pres Time

Pub #

Presentation Title

1:00 pm 4 Development of a fully synthetic three-component carbohydrate-based cancer vaccine prof. Geert-Jan Boons PhD

1:35 pm 5 Chemoenzymatic synthesis and antibody recognition of HIV-1 V3 glycopeptides Prof. Lai-Xi Wang

2:10 pm 6 Entirely carbohydrate vaccine constructs and their application in probing glycoimmunology Prof. Peter R Andreana PhD

2:45 pm Intermission

3:00 pm 7 Carbohydrate polymer assembly: How do mycobacteria do it? Laura L. Kiessling

3:35 pm 8 Lipopolysaccharide transport and assembly in Escherichia coli Prof. Daniel Kahne

4:10 pm 9 Arrays and automated oligosaccharide synthesis Prof. Nicola L. B. Pohl

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CARB Todd Lowary Monday, August 23, 2010 Oral

Synthetic Oligosaccharides and Glycoconjugates for Preventing and Combating Disease - AM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Lai-Xi Wang, GJ Boons

Presiders: GJ Boons

Duration: 8:00 am - 12:00 pm

Pres Time

Pub #

Presentation Title

8:00 am Introductory Remarks

8:05 am 10 Bioactive N-linked glycopeptides and glycoprotein conjugates Thomas J. Tolbert PhD

8:25 am 11 Efficient chemoenzymatic synthesis of sialyl Lewis antigens using sialyltransferase mutants Professor/Ph.D. Xi Chen PhD, Go Sugiarto, Kam Lau, Lei Zhang, Dr. Shengshu Huang Ph.D.

9:00 am 12 Synthesis of saponin vaccine adjuvants for immunotherapy Prof. David Y Gin PhD

9:35 am 13 Glycosylation engineering of human IgG-Fc for functional studies Prof. Lai-Xi Wang

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10:10 am Intermission

10:30 am 14 Chemoenzymatic synthesis of GPI-anchored proteins and glycoproteins Dr. Xueqing Guo, Dr. Zhimeng Wu, Prof. Zhongwu Guo

10:50 am 15 Cancer relevant epitopes uncovered by synthetic mucin glycopeptides Prof. Shin-Ichiro Nishimura PhD

11:25 am 16 Chemical biology of O-GlcNAc processing enzymes Prof. David J Vocadlo, Dr. Tracey M Gloster, Wesley F Zandberg, Julia E Heinonen, David Shen, Thomas Clark, Dr. Carlos Martinez-Fleites, Yuan He, Prof. Gideon J Davies

CARB Todd Lowary Monday, August 23, 2010 Oral

Synthetic Oligosaccharides and Glycoconjugates for Preventing and Combating Disease - PM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: GJ Boons, Lai-Xi Wang

Presiders: Lai-Xi Wang

Duration: 1:30 pm - 5:05 pm

Pres Time

Pub #

Presentation Title

1:30 pm 17 Synthesis of a single molecule L-rhamnose-containing three component vaccine and evaluation of antigenicity in the presence of anti L-rhamnose antibodies

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M.S. Sourav Sarkar, M.S. Rommel S Talan, Mr. Steven A Lombardo, Prof. Katherine A Wall PhD, Prof. Steven J Sucheck PhD

1:50 pm 18 Design of glycoprotein vaccines against influenza and other viral diseases Prof. Chi-Huey Wong PhD

2:25 pm 19 Modular synthesis of heparan sulfate oligosaccharides for array development Prof. Geert-Jan Boons

3:00 pm Intermission

3:15 pm 20 New targets for antibiotics Professor Suzanne Walker

3:50 pm 21 Challenges and opportunities in natural product glycosylation Jon S Thorson

4:25 pm 22 Glycomimetic and non-carbohydrate inhibitors as probes of the lectin DC-SIGN Professor Laura L Kiessling

5:00 pm Concluding Remarks    

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CARB Todd Lowary Monday, August 23, 2010 Sci-Mix

Sci-Mix - EVE Session

Location: Boston Convention & Exposition Center

Room: Hall C

Organizers: Todd Lowary

Duration: 8:00 pm - 10:00 pm

Pub #

Presentation Title

81 Expression and characterization of enzymes for bioenzymatic synthesis of heparin Priscilla Paul, Wenjing Zhao, Prof. Robert Linhardt, Prof. Jonathan Dordick, Prof. Jian Liu

82 Catalytic conversion of biomass-derived carbohydrates to useful chemicals in one step Dr. Weiran Yang, Prof. Ayusman Sen

83 Behaviour of polysaccharide aggregates in asymmetricalfield-flow fractionation and size-exclusion chromatography Leena Pitkänen, Maija Tenkanen, Päivi Tuomainen

85 Synthesis and structural optimization of antifungal kanamycin B analogs Marina Y FOSSO, Yukie KAWASAKI, Sanjib SHRESTHA, Jon TAKEMOTO, Tom C.-W. CHANG

86 E. coli K5 heparosan production for bioengineered heparin Zhenyu Wang, Mellisa Ly, Fuming Zhang, Zhenqing Zhang, Jonathan S. Dordick, Robert J. Linhardt

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87 Stochastic simulation of lectin microarrays with nanosensor transducers: Potential platforms for optimal, high-throughput screening and profiling of glycoproteins Mr. Nigel F Reuel, Dr. Jin-Ho Ahn PhD, Dr. Michael S. Strano PhD

88 Evaluation of different thioesters for glycocluster synthesis applying native chemical ligation Johannes W. Wehner, Prof. Dr. Thisbe K. Lindhorst

89 NMR for structure elucidation of commercially available heparin polysaccharides Kemal Solakyildirim, Scott A. McCallum, Robert J. Linhardt

92 Study on therelative reactivity of glycosyl acceptors in the glycosylations of2-Azido-2-deoxy-galactosides Jane Kalikanda, Dr. Zhitao Li

93 Synthesis of tailored glycoconjugates for the precise detection of pathogens Ashish A. Kulkarni, Dr. Suri S. Iyer

94 Structure-activity studies of synthetic glycophosphatidylinositol anchored proteins Dr. Carl V Christianson, Prof. Peter H Seeberger

96 Synthesis of a fluorous-tagged disaccharide for the enzymatic preparation of heparin oligosaccharides Sayaka Masuko, Smritilekha Bera, Robert J. Linhardt

97 Preventing the transmission of Plasmodium falciparum through the inhibition of malaria protein binding to placental chondroitin sulfate A Julie M Beaudet, Leandra J Mansur, Bo Yang, Fuming Zhang, Robert J Linhardt

98 Real-time assessment of the morphological change of cellulose in response to enzymatic treatment Chi Nguyen

100 Immobilization of enzymes relevant to bioengineered heparin synthesis Eric R. Sterner, Dr. Robert J. Linhardt, Dr. Jonathan S. Dordick, Dr. Jian Liu, Dr. Fuming Zhang, Wenjing Zhao, Priscilla Paul, Jeff Martin

101 Analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three

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consecutive Tn-antigen clusters by surface plasmon resonance (SPR) and NMR Ayano Takasaki-Matsumoto Ph.D., Shinya Hanashima Ph.D., Ami Aoki, Yoshiki Yamaguchi Ph.D., Reiko Sato, Hiroko Kawakami Ph.D., Mamoru Mizuno Ph.D., Prof. Yoko Fujita-Yamaguchi Ph.D.

102 Modular glycoconjugate tool set for assembly and presentation of multivalent carbohydrate ligands on surfaces Irene Abia, Brian Sanders, Michael D. Best, David C. Baker

103 Analysisof the absorption of low molecular weight heparin in human umbilical cord tissue as a model for the prevention of cancer metastasis Amanda M. Weyers, Thangriala Sudha, Bo Yang, Boyangzi Li, Majde Takieddin, Fuming Zhang, Shaker A. Mousa, Robert J. Linhardt

104 Synthesis and biological evaluation of a Gal(α1-2)GalCer analog Yanke Liang, Prof. Amy R Howell PhD

105 Thiol-click chemistry approach to glycomimetics: Novel stereoselective synthesis of (1-3)-S-thiodisaccharides Prof. Zbigniew J. Witczak, Irena Bak-Sypien

106 High-throughput glycoarray for monitoring immune responses to a cancer vaccine Dr. Christopher Campbell M.D., Ph.D., Dr. Yalong Zhang Ph.D., Dr. Olaf Ludek Ph.D., Mr. David Farnsworth, Dr. Jeffrey Gildersleeve Ph.D.

107 NMR spectroscopic studies of APF: A small glycopeptide possessing potent antiproliferative activity Dr Kristie M Adams PhD, Dr Piotr Kaczmarek PhD, Dr Susan K Keay MD, PhD, Dr Joseph J Barchi, Jr PhD

108 Structural and quantitative analysis of disaccharides using CE with LIF detection Yuqing Chang, Tatiana Laremore, Fuming Zhang, Robert J Linhardt

110 Carbon nanotubes and chitosan as possible scaffolds for bone tissue regeneration Julia Stone, Yetunde Olusanya, Whitney Jones, Pasakorn Traisawatwong, Melisa Stewart, Cordella Kelly-Brown, Laura Carson Ph.D., Aderemi Oki Ph.D., E. Gloria C. Regisford Ph.D.

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111 Biosynthesis of heparin by metabolic engineering of Chinese hamster ovary cells Leyla Gasimli, Jongyoun Baik PhD, Dr. Susan Sharfstein PhD, Prof. Robert J. Linhardt PhD

113 Design, synthesis, and characterization of sulfated N-aryl aminoglycosides Amanda M. Fenner, Robert J. Kerns PhD

115 On-line microflow high-performance liquid chromatography with nano-electrospray ionization mass spectrometry for heparan sulfate disaccharide analysis Dr. Bo Yang, Kemal Solakyildirim, Jeffrey G. Martin, Tatiana Laremore, Prof. Robert J. Linhardt

   

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CARB Todd Lowary Tuesday, August 24, 2010 Oral

Petite and Sweet: Glyconanotechnology as a Bridge to New Medicines - AM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Joseph Barchi, Xuefei Huang

Presiders: Joseph Barchi

Duration: 8:30 am - 11:35 am

Pres Time

Pub #

Presentation Title

8:30 am Introductory Remarks

8:35 am 23 Toxicity and functional assessment using polysaccharide-based magnetic iron oxide nanoparticles for cell labeling in vivo and in vitro Yoshitaka Miyamoto PhD, Yumie Koshidaka, Hiroaki Saito, Yukimasa Kagami, Katsutoshi Murase, Noritada Kaji, Hiroshi Yukawa, Hirofumi Noguchi MD, PhD, Hisashi Iwata MD, Yoshinobu Baba PhD, Shuji Hayashi MD, PhD

9:05 am 24 Glycodendrimers for the mediation of cancer cellular aggregation processes Dr. Mark L. Wolfenden, Julie J Sprenger, Michael P Capp, Dr. Pratima Nangia-Makker, Dr. Avraham Raz, Dr. Mary J Cloninger

9:35 am 25 Glyconanoparticles: Multibiofunctional nanomaterials for biomedical applicationsProf. Soledad Penades

10:05 am Intermission

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10:35 am 26 New glycopeptide-based nanoparticle constructions for anticancer therapy Dr. Joseph John Barchi Jr, Dr. Ray Brinas, Dr. Andreas Sundgren, Dr. Padmini Sahoo, Ms. Amy Houghten, Ms Susan Morey, Mr Michael Sanford, Dr. Howard Young

11:05 am 27 Functionalized catanionic surfactant vesicles: A new approach to carbohydrate vaccines Juhee Park, Lenea Rader, Glen Thomas, Douglas English, Lindsey Zimmerman, Daniel C. Stein, Philip DeShong

CARB Todd Lowary Tuesday, August 24, 2010 Oral

Petite and Sweet: Glyconanotechnology as a Bridge to New Medicines - PM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Joseph Barchi, Xuefei Huang

Presiders: Joseph Barchi

Duration: 1:30 pm - 5:05 pm

Pres Time

Pub #

Presentation Title

1:30 pm 39 Sugar and proteins: Applications of bioconjugates

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Professor Benjamin G. Davis

2:00 pm 40 Sialic acid recognition: Particles, chips, and cells Prof. Robert A. Field

2:30 pm 41 Functionalized nanoparticle for protein detection Prof. Chun-Cheng Lin

3:00 pm Intermission

3:30 pm 42 Nanotechnology-based tools for the glycomics revolution: Silicon nanophotonic carbohydrate biosensors Prof. Daniel M Ratner PhD

4:00 pm 43 Targeted glyco-magnetic nanoprobes for detection and molecular imaging of atherosclerosis Kheireddine El-Boubbou PhD, Dr. Medha Kamat PhD, Prof. David Zhu PhD, Ruiping Huang, Dr. George Abela MD, Prof. Xuefei Huang PhD

4:30 pm 44 Carbohydrates on nanoparticles, surfaces and polymers: From basics to diagnostics applications Prof. Dr. Peter H. Seeberger

5:00 pm Concluding Remarks    

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CARB Todd Lowary Tuesday, August 24, 2010 Oral

Oligonucleotide Therapeutics - AM Session

Location: Boston Convention & Exhibition Center

Room: Room 252A

Cosponsored by:

ANYL, BIOL, MEDI, ORGN

Organizers: Muthiah Manoharan

Presiders: Muthiah Manoharan, Radhakrishnan Iyer

Duration: 8:40 am - 11:45 am

Pres Time

Pub #

Presentation Title

8:40 am Introductory Remarks

8:45 am 28 Novel synthesis of 2'-deoxynucleoside 5'-triphosphates without nucleoside protection and DNA polymerase recognition Julianne M. Caton-Williams PhD, Matthew R. Smith, Zhen Huang PhD

9:00 am 29 Activation of retinoic acidinducible gene (RIG-I) by short oligonucleotides Radhakrishnan Iyer, John Coughlin, Seetharamaiyer Padmanabhan, Brent Korba, Sua Myong

9:15 am 30 Solid-phase synthesis of 5'-di- and tri-phosphates andtheir modified analogs of DNA, RNA and chemically modified oligonucleotides Ivan Zlatev, Thomas Lavergne, Sudhakar Takkellapati, Rajenda Pandey,

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Yupeng Fan, Marija Prhavc, Kathy Mills, G. Rajeev Kallanthottathil, Françoise Debart, Jean-Jacques Vasseur, François Morvana, Muthiah Manoharan

9:30 am 31 Drug discovery harnessing RNA interference Muthiah Manoharan

9:45 am 32 Parallel high throughput synthesis of chemically modified 21-27mersiRNA sequences Jason Costigan, Satya Kuchimanchi, Sarfraz Shaikh, Keri Dufault, Jack de Groot, Rachel Meyers

10:00 am 33 New developments in the synthesis of oligoribonucleotides: Use of dimer/trimer blocks in combination with an “ionic tag” soluble support Matthew R Hassler, Dr. Nandyala Mallikarjuna Reddy, Dr. Tak-Hang Chan, Dr. Masad J Damha

10:15 am Break

10:30 am 34 Advanced process of RNA synthesis Dr. Anuj Mohan

10:45 am 35 Novel method for the confirmation of siRNA sequence byLC-MS/MS Gary Lavine, Matthias Kretschmer, James McArdle, Satya Kuchimanchi, Veeragu Murugaiah, Muthiah Manoharan

11:00 am 36 Evaluation of Canonical vs.Dicer-substrate siRNAs in vitro and in vivo Don Foster, Sayda Elbashir, Satya Kuchimanchi, Greg Hinkle, William Cantley, Rick Duncan, Geff Cole, Chris Sherill, Kathy Mills, Mara Broberg, Jeff Rollins, Klaus Charisse, Muthiah Manoharan

11:15 am 37 Modulation of thermal stability can enhance the potency of siRNA Haripriya Addepalli, Meena Meena, Chang G. Peng, Gang Wang, Yupeng Fan, Klaus Charisse, K. Narayanannair Jayaprakash, G. Rajeev Kallanthottathil, Rajendra Pandey, Gary Lavine, Ligang Zhang, Kerstin Jahn-Hofmann, Philipp Hadwiger, Muthiah Manoharan, Martni Maier

11:30 am 38 Carbohydrate conjugation tosiRNA for tissue-specific delivery

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G. Rajeev Kallanthottathil, K. Narayanannair Jayaprakash, Maria Frank-Kamenetsky, Gang Wang, Tianlei Lei, Mariano Severgnini, William Querbes, Jim Butler, Alfica Sehgal, Tomoko Nakayama, Klaus Charrise, Martin Maier, Kevin Fitzgerald, Muthiah Manoharan

CARB Todd Lowary Tuesday, August 24, 2010 Oral

Oligonucleotide Therapeutics - PM Session

Location: Boston Convention & Exhibition Center

Room: Room 252A

Cosponsored by:

ANYL, BIOL, MEDI, ORGN

Organizers: Muthiah Manoharan

Presiders: Muthiah Manoharan, Suresh Srivastava

Duration: 2:00 pm - 4:50 pm

Pres Time

Pub #

Presentation Title

2:00 pm 45 Efficient synthesis of siRNA-folic acid conjugates Rajendra Pandey, Muthusamy Jayaraman, Anna Borodovsky, David Butler, Shigeo Matsuda, Gang Wang, Martin Maier, Bo Peng, Marjorie Solomon, Sergey Shulga-Morskoy, Klaus Charrise, David Bumcrot, Kristina Yucius, Victor Kotelianski, G. Rajeev Kallanthottathil, Muthiah Manoharan

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2:15 pm 46 Combining 2'-TBDMS and 2'-ALE chemistry for on-column site specific modifications of RNA Dr. Jeremy G Lackey PhD, Dr. Richard Johnsson PhD, Prof. Masad J Damha PhD, FCIC

2:30 pm 47 Synthesis and evaluation of bicyclic ketal-based cationiclipids for the delivery of siRNA via lipid nanoparticle delivery systems Muthusamy Jayaraman, David Butler, Laxman Eltepu, Martin Maier, Tom Madden, Michael Hope, Ying Tam, Barbera Mui, Andrew Sprague, Akin Akinc, Soma De, G. Rajeev Kallanthottathil, Muthiah Manoharan

2:45 pm 48 Development of a stability-indicating, ion-pair RP-HPLCmethod for separation and quantitative determination of two siRNA duplexes in aliposome Veeravagu Murugaiah, William Zedalis, Gary Lavine, Klaus Charrise, Muthiah Manoharan

3:00 pm 49 Novel applications of aerosol-based detectors for theanalysis of non-chromophore, multi-lipid, drug delivery vehicles William Zedalis, Matthias Kretschmer, Muthiah Manoharan

3:15 pm Intermission

3:30 pm 50 Optimizing the LALassay for detection of bacterial endotoxin in conjugated and formulated siRNAs Mara Broberg, Kathy Mills, Klaus Charisse, William Zedalis, Muthiah Manoharan

3:45 pm 51 Conjugationstrategies for RNAs using copper-catalyzed click chemistry Chang Geng Peng, Takeshi Yamada, Shigeo Matsuda, Haripriya Addepalli, Rowshon Alam, Narayanannair Jayaprakash, Muthusamy Jayaraman, David Butler, Rajendra Pandey, Kathy Mills, Martin Maier, Klaus Charrise, G. Rajeev Kallanthottathil, Muthiah Manoharan

4:00 pm 52 Non-nucleoside building blocks for copper-assistedand copper-free click chemistry for synthesis of oligonucleotide conjugates K.N. Jayaprakash, Chang G. Peng, Takeshi Yamada, David Butler, G Rajeev Kallanthottathil, Martin Maier, Muthiah Manoharan

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4:15 pm 53 Solid-support immobilized, reusable Cu (I)catalyst for "click reactions" of oligonucleotides with ligands Laxman Eltepu, Chang G. Peng, K. Narayanannair Jayaprakash, Takeshi Yamada, Muthusamy Jayaraman, G. Rajeev Kallanthottathil, Muthiah Manoharan

4:30 pm 54 Synthesis of oligo spermine-containingoligonucleotides for siRNA delivery Shigeo Matsuda, Gang Wang, Ligang Zhang, Tianlei Lei, Rowshon Alam, Chang G Pang, K. N. Jayaprakash, Takeshi Yamada, David Butler, Maria Frank-Kamenetsky, Martin Maier, Klaus Charrise, Kevin Fitzgerald, G. Rajeev Kallanthottathil, Muthiah Manoharan

4:45 pm Concluding Remarks  

   

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CARB Todd Lowary Wednesday, August 25, 2010 Oral

Recognition of DNA: Recent Advances - AM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Cosponsored by:

MEDI

Organizers: Dev Arya

Presiders: Dev Arya

Duration: 8:00 am - 11:35 am

Pres Time

Pub #

Presentation Title

8:00 am Introductory Remarks

8:05 am 55 Allosteric modulation of DNA by minor groove binding polyamides David M Chenoweth PhD, Prof. Peter B Dervan PhD

8:45 am 56 Strong and selective molecular recognition of the DNA minor groove: Compound and DNA chemistry and unusual conformational matching W. David Wilson, Rupesh Nanjunda, Arvind Kumar, Manoj Munde, Yang Liu, Rebecca Hunt, David W. Boykin

9:25 am Intermission

9:35 am 57 Toward DNA recognition by a Janus Wedge approach Prof. Larry W McLaughlin PhD, Ayan Pal, Dr. Han Chen PhD, Dr. Meena Meena PhD

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10:15 am 58 Recognition of DNA major groove Prof. Dev P Arya, Dr. Liang Xue, Mr. Sunil Kumar

10:55 am 59 Identification and cleavage site analysis of DNA sequences bound strongly by bleomycin Prof. Sidney M Hecht PhD

 

CARB Todd Lowary Wednesday, August 25, 2010 Oral

Recognition of DNA: Recent Advances - PM Session

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Dev Arya

Presiders: Dev Arya

Duration: 1:30 pm - 5:05 pm

Pres Time

Pub #

Presentation Title

1:30 pm 75 Role of DNA topography in recognition by proteins and small molecules Prof. Thomas D. Tullius

2:10 pm 76 Selective modulation of DNA polymerase activity by fixed-conformation nucleoside analogs Prof. Martin Egli, Dr. Robert L. Eoff, Dr. Victor E. Marquez, Prof. F. Peter Guengerich

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2:50 pm 77 Structural mechanisms underlying DNA replication and human DNA mismatch repair Prof. Lorena S. Beese PhD, Elizabeth McSweeney, Prof Paul L Modrich PhD, Jillian Orans PhD, Quincy Tseng, Weina Wang

3:30 pm Intermission

3:45 pm 78 Manipulating the electrostatic potential in the DNA grooves: Effect on thermodynamic stability, ion binding, hydration and structure Professor Barry Gold PhD

4:25 pm 79 Sequence-dependent recognition of minor groove width Dr. Barry Honig

   

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CARB Todd Lowary Wednesday, August 25, 2010 Oral

General Papers - AM Session

Glycobiology

Location: Boston Convention & Exhibition Center

Room: Room 252A

Organizers: Todd Lowary

Presiders: Eugene Cioffi

Duration: 9:00 am - 11:35 am

Pres Time

Pub #

Presentation Title

9:00 am 60 Synthesis and characterization of chain-end functionalizable glycopolymer and its oriented glyco-macroligand formation Satya Nandana Narla, Xue-Long Sun PhD

9:20 am 61 Separation and characterization of the glycosaminoglycan components of proteoglycans Mellisa Ly, Tatiana N Laremore PhD, Kemal Solakyildirim MS, Professor Robert J. Linhardt PhD

9:40 am 62 Synthesis of nucleotide activated L-sugars from polyols via bioconversion Dr. Ryan D Woodyer PhD

10:00 am Intermission

10:15 am 63 Glycomorphology of the pulmonary vasculature: Endothelial cell glycocalyx and

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endothelial barrier function Professor Eugene Cioffi

10:35 am 64 Multidimensional glycan arrays for enhanced lectin and antibody profiling Dr. Yalong Zhang, Dr. Christopher Campbell MD, PhD, Dr. Qian Li, Dr. Jeffrey Gildersleeve

10:55 am 65 Peptide nucleic acids bind strongly and sequence selectively to double helical RNA Mr Thomas T Zengeya, Dr Ming Li PhD, Dr Eriks Rozners PhD

11:15 am 66 Post surface function method for preparation of liposomal glyco-conjugates MS Hailong Zhang, Dr. Yong Ma, Prof. Xue-Long Sun

CARB Todd Lowary Wednesday, August 25, 2010 Oral

General Papers - PM Session

Glycobiology and Computation

Location: Boston Convention & Exhibition Center

Room: Room 252A

Organizers: Todd Lowary

Presiders: Steven Graham

Duration: 1:00 pm - 3:55 pm

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Pres Time

Pub #

Presentation Title

1:00 pm 67 Approach to study carbohydrate-carbohydrate interactions involved in myelin using glycolipids assay in microtiter plate Jingsha Zhao, Prof. Amit Basu

1:20 pm 68 WITHDRAWN

1:40 pm 69 Synthesis and characterization of protein glycopolymer conjugate Valentinas Gruzdys, Xue-Long Sun PhD

2:00 pm 70 Robust analytical development for oligonucleotide manufacture Dr. Ipsita Roymoulik

2:20 pm Intermission

2:35 pm 71 Glycomics studies on central nervous system Dr. Fuming Zhang, Dr. Zhenling Liu, Kemal Solakyildirim, Dennis Pu, Prof. Robert J. Linhardt

2:55 pm 72 Computational studies on the interactions of mannose with DOPC and POPC phospholipids Dr Parthasarathi R, Dr Gnanakaran S

3:15 pm 73 Optimizatin of analysis of glycosaminoglycans in biological samples Boyangzi Li, Prof. Robert J Linhardt PhD

3:35 pm 74 Conformational analysis of nucleosides and nucleotides: PSEUROT 2010 Steven M. Graham

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CARB Todd Lowary Wednesday, August 25, 2010 Poster

General Posters - EVE Session

Location: Boston Convention & Exposition Center

Room: Hall C

Organizers: Todd Lowary

Duration: 8:00 pm - 10:00 pm

Pub #

Presentation Title

80 Glycosylation of substituted 4H-1,2,4-triazole-3-thiol Professor El Sayed H. El Ashry PhD,DSc

81 Expression and characterization of enzymes for bioenzymatic synthesis of heparin Priscilla Paul, Wenjing Zhao, Prof. Robert Linhardt, Prof. Jonathan Dordick, Prof. Jian Liu

82 Catalytic conversion of biomass-derived carbohydrates to useful chemicals in one step Dr. Weiran Yang, Prof. Ayusman Sen

83 Behaviour of polysaccharide aggregates in asymmetricalfield-flow fractionation and size-exclusion chromatography Leena Pitkänen, Maija Tenkanen, Päivi Tuomainen

84 Key new observations in the synthesis of thiosialosides Ms Ines F Oliveira, Dr Goreti R Morais PhD, Mr Bradley R Springett, Dr Robert A Falconer PhD

85 Synthesis and structural optimization of antifungal kanamycin B analogs Marina Y FOSSO, Yukie KAWASAKI, Sanjib SHRESTHA, Jon TAKEMOTO, Tom C.-W. CHANG

86 E. coli K5 heparosan production for bioengineered heparin

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Zhenyu Wang, Mellisa Ly, Fuming Zhang, Zhenqing Zhang, Jonathan S. Dordick, Robert J. Linhardt

87 Stochastic simulation of lectin microarrays with nanosensor transducers: Potential platforms for optimal, high-throughput screening and profiling of glycoproteins Mr. Nigel F Reuel, Dr. Jin-Ho Ahn PhD, Dr. Michael S. Strano PhD

88 Evaluation of different thioesters for glycocluster synthesis applying native chemical ligation Johannes W. Wehner, Prof. Dr. Thisbe K. Lindhorst

89 NMR for structure elucidation of commercially available heparin polysaccharides Kemal Solakyildirim, Scott A. McCallum, Robert J. Linhardt

90 Syntheses of C-5-spirocyclic C-glycoside SGLT2 inhibitors Benjamin A. Thuma, Dr. Vincent Mascitti PhD, Dr. Ralph P. Robinson PhD, Cathy Préville, Matthew R. Reese, Dr. Robert J. Maguire PhD, Christopher L. Carr, Michael T. Leininger, André Lowe, Claire M. Steppan

91 Co-axial cellulose nanofibers for electrical applications Minoru Miyauchi, Dr. Jianjun Miao PhD, Dr. Trevor J. Simmons PhD, Prof. Jonathan S. Dordick PhD, Prof. Robert J. Linhardt PhD

92 Study on therelative reactivity of glycosyl acceptors in the glycosylations of2-Azido-2-deoxy-galactosides Jane Kalikanda, Dr. Zhitao Li

93 Synthesis of tailored glycoconjugates for the precise detection of pathogens Ashish A. Kulkarni, Dr. Suri S. Iyer

94 Structure-activity studies of synthetic glycophosphatidylinositol anchored proteins Dr. Carl V Christianson, Prof. Peter H Seeberger

95 Chemoenzymatic synthesis of heparan sulfate Dr Renpeng Liu, Prof Jian Liu PhD, Dr Yongmei Xu

96 Synthesis of a fluorous-tagged disaccharide for the enzymatic preparation of heparin oligosaccharides

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Sayaka Masuko, Smritilekha Bera, Robert J. Linhardt

97 Preventing the transmission of Plasmodium falciparum through the inhibition of malaria protein binding to placental chondroitin sulfate A Julie M Beaudet, Leandra J Mansur, Bo Yang, Fuming Zhang, Robert J Linhardt

98 Real-time assessment of the morphological change of cellulose in response to enzymatic treatment Chi Nguyen

99 Chemoselective glycosylation of hemoglobin as a potential oxygen therapeutic Thomas J Styslinger, Ning Zhang, Andre F. Palmer PhD, Peng G. Wang PhD

100 Immobilization of enzymes relevant to bioengineered heparin synthesis Eric R. Sterner, Dr. Robert J. Linhardt, Dr. Jonathan S. Dordick, Dr. Jian Liu, Dr. Fuming Zhang, Wenjing Zhao, Priscilla Paul, Jeff Martin

101 Analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters by surface plasmon resonance (SPR) and NMR Ayano Takasaki-Matsumoto Ph.D., Shinya Hanashima Ph.D., Ami Aoki, Yoshiki Yamaguchi Ph.D., Reiko Sato, Hiroko Kawakami Ph.D., Mamoru Mizuno Ph.D., Prof. Yoko Fujita-Yamaguchi Ph.D.

102 Modular glycoconjugate tool set for assembly and presentation of multivalent carbohydrate ligands on surfaces Irene Abia, Brian Sanders, Michael D. Best, David C. Baker

103 Analysisof the absorption of low molecular weight heparin in human umbilical cord tissue as a model for the prevention of cancer metastasis Amanda M. Weyers, Thangriala Sudha, Bo Yang, Boyangzi Li, Majde Takieddin, Fuming Zhang, Shaker A. Mousa, Robert J. Linhardt

104 Synthesis and biological evaluation of a Gal(α1-2)GalCer analog Yanke Liang, Prof. Amy R Howell PhD

105 Thiol-click chemistry approach to glycomimetics: Novel stereoselective synthesis of (1-3)-S-thiodisaccharides Prof. Zbigniew J. Witczak, Irena Bak-Sypien

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106 High-throughput glycoarray for monitoring immune responses to a cancer vaccine Dr. Christopher Campbell M.D., Ph.D., Dr. Yalong Zhang Ph.D., Dr. Olaf Ludek Ph.D., Mr. David Farnsworth, Dr. Jeffrey Gildersleeve Ph.D.

107 NMR spectroscopic studies of APF: A small glycopeptide possessing potent antiproliferative activity Dr Kristie M Adams PhD, Dr Piotr Kaczmarek PhD, Dr Susan K Keay MD, PhD, Dr Joseph J Barchi, Jr PhD

108 Structural and quantitative analysis of disaccharides using CE with LIF detection Yuqing Chang, Tatiana Laremore, Fuming Zhang, Robert J Linhardt

109 Development of a novel cancer vaccine based on multivalent presentation of tumor-associated carbohydrate antigens on gold nanoparticle scaffolds Raymond P Brinas Ph.D., Andreas Sundgren Ph.D., Micah Maetani, Omar Abbudayyeh, Howard A Young Ph.D., Michael Sanford, Joseph J Barchi Ph.D.

110 Carbon nanotubes and chitosan as possible scaffolds for bone tissue regeneration Julia Stone, Yetunde Olusanya, Whitney Jones, Pasakorn Traisawatwong, Melisa Stewart, Cordella Kelly-Brown, Laura Carson Ph.D., Aderemi Oki Ph.D., E. Gloria C. Regisford Ph.D.

111 Biosynthesis of heparin by metabolic engineering of Chinese hamster ovary cells Leyla Gasimli, Jongyoun Baik PhD, Dr. Susan Sharfstein PhD, Prof. Robert J. Linhardt PhD

112 Purification strategies for separation of capsular polysaccharide from fermentation broth Ujjwal Bhaskar, Zhenyu Wang, Dr. Fuming Zhang, Prof. Jonathan S. Dordick PhD, Prof. Robert J. Linhardt PhD

113 Design, synthesis, and characterization of sulfated N-aryl aminoglycosides Amanda M. Fenner, Robert J. Kerns PhD

114 Photo-click immobilization of carbohydrates on polymeric surfaces: An effortless method to functionalize surfaces for biomolecular recognition studies Oscar Norberg, Lingquan Deng, Mingdi Yan, Olof Ramström

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115 On-line microflow high-performance liquid chromatography with nano-electrospray ionization mass spectrometry for heparan sulfate disaccharide analysis Dr. Bo Yang, Kemal Solakyildirim, Jeffrey G. Martin, Tatiana Laremore, Prof. Robert J. Linhardt

   

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CARB Todd Lowary Thursday, August 26, 2010 Oral

General Papers - AM Session

Synthetic Chemistry

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Todd Lowary

Presiders: Cecilia Marzabadi

Duration: 8:45 am - 12:00 pm

Pres Time

Pub #

Presentation Title

8:45 am 116 De novo synthesis of a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) building block for the preparation of the zwitterionic polysaccharide A1 (PS A1) repeating subunit of Bacteroides fragilis Dr. Rajan Pragani PhD, Prof. Dr. Peter H Seeberger PhD

9:05 am 117 Automated solution-phase synthesis of alpha-galactosides Rajarshi Roychoudhury, Prof. Nicola L. B. Pohl

9:25 am 118 Study of the protecting group participation and stereoselectivity of 2-azido-2-deoxygalactopyranosyl donors Prof. Zhitao Li

9:45 am 119 Reactions of glycals with ortho-substituted benzanilines Prof. Cecilia H Marzabadi, Katherine Kochalski

10:05 am 120 Novel glycolipids in CD1d-mediated immunity: Synthesis of new agonists for CD1d

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Justyna Wojno, John-Paul Jukes, Paolo Polzella, Vincenzo Cerundolo, Liam R Cox, Gurdyal S Besra

10:25 am Intermission

10:40 am 121 Automated synthesis of systematic di- and tri-saccharide libraries Xin Liu, Dr. Beatrice Y. M. Collet, Shu-Lun Tang, Sahana K. Nagappayya, Rajarshi Roychoudhury, Dr. Xueshu Li, Heather Edwards, Prof. Nicola L. B. Pohl

11:00 am 122 Probe of activated glycoside building block stability for automated solution-phase synthesis of carbohydrate libraries Dr. Beatrice Y. M. Collet, Xin Liu, Shu-Lun Tang, Heather Edwards, Sahana Nagappayya, Lin Liu, Prof. Nicola L. B. Pohl

11:20 am 123 Synthesis of unnatural glycosaminoglycans and evaluation of their interaction with proteins Dr. Smritilekha Bera PhD

11:40 am 124 Grafting of cellulose esters by single electron transfer living radical polymerization Set-LrP Dr Petr Vlcek, Vladimir Raus, Dr Miroslav Janata, Dr Jaroslav Kriz, Petra Latalova, Eva Cadova

CARB Todd Lowary Thursday, August 26, 2010 Oral

General Papers - PM Session

Polysaccharides

Location: Boston Convention & Exhibition Center

Room: Room 251

Organizers: Todd Lowary

Presiders: Ravin Narain

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Duration: 1:00 pm - 4:55 pm

Pres Time

Pub #

Presentation Title

1:00 pm 125 Solvation and hydrolysis of cellulose with transition metal salts Dr. Veronika Viner, Dr. Benjamin G. Harvey, Dr. Roxanne L Quintana

1:20 pm 126 Chemical derivatization of glucan microparticles for targeted delivery Dr. Ernesto R Soto, Dr. Gary R Ostroff

1:40 pm 127 Degradation mechanism and crystal structure change of cellulose during TEMPO-NaOCl-NaBr selective oxidation and its biomedical application as hemostatics Dr.,Associate Prof. Bin Sun PhD, Wenxiu Han, Wei Wang, Chunju Gu, Jinhong Ma, Meifang Zhu, Borun Liang

2:00 pm 128 Temperature-dependent chain-collapse behavior of cellulose ethers in dilute solution Hongwei Shen, Robert Sammler, David Redwine, David Meunier, Meinolf Brackhagen

2:20 pm 129 Improving dyeability of nylon wiith nano-chitosan Ms Ho Yan Wong, Ms Hoi Ying Lui, Ms Li Wei Liu, Dr Yau Shan Szeto PhD

2:40 pm 130 Modeling of Congo red adsorption on a surface of crystalline cellulose using molecular dynamics Dr. Miroslaw Wyszomirski, Dr. Karim Mazeau

3:00 pm Intermission

3:15 pm 131 Evaluation of glycopolymers and glyconanoparticles for biosensing and gene delivery applications Prof. Ravin Narain PhD, Miss Marya Ahmed

3:35 pm 132 Application of data mining techniques to differentiate glucose-containing disaccharide ions fragmented via infrared-multiple photon dissociation using tunable lasers and Fourier transform ion cyclotron resonance mass spectrometry Dr. Sarah Stefan PhD, Mohammad U. Ehsan, Dr. Alexander Aksenov PhD, Dr.

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Vladimir Boginksi PhD, Dr. Brad Bendiak PhD, Dr. John Eyler PhD

3:55 pm 133 Influence of the structure of phosphoramidates on flame retardant properties of cellulose Victoria Salimova, Dr. Sabyasachi Gaan, Dr. Joëlle Grützmacher

4:15 pm 134 High-flux thin-film nanofibrous composite ultrafiltration membranes containing cellulose barrier layer Dr. Hongyang Ma, Prof. Benjamin S. Hsiao, Prof. Benjamin Chu

4:35 pm 135 Discovery of 6-deoxy-D-altrose in nature Professor Masakuni Tako PhD, Takuya Yogi, Professor Ken Izumori PhD, Professor Hideharu Ishida PhD, Professor Makoto Kiso PhD

 

 

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CARB 1

Developing an enzymatic approach to synthesize heparan sulfates

Jian Liu(1), [email protected], Eshelman School of Pharmacy, Chapel Hill North Carolina 27599, United States . (1) Med Chem and Nat Prod, University of North Carolina, Chapel Hill North Carolina 27599, United States

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Synthesis of heparan sulfate using enzymes provides a promising approach because of the high regioselectivity of heparan sulfate biosynthetic enzymes. The synthesis of heparan sulfate involves up to 15 specialized enzymes, including sulfotransferases, an epimerase and glycosyltransferases. All of these enzymes have been expressed effectively in bacteria and in yeast in our lab. Using these enzymes, we are able to synthesize oligosaccharides with different sulfation patterns and sizes. Furthermore, we demonstrated the feasibility of engineering the substrate specificity of heparan sulfate sulfotransferases using a structurally guided mutagenesis approach. The engineered enzymes have provided us the opportunity to prepare the sulfated polysaccharides that can not be achieved by the wild type enzymes. Overall, our method offers a generic for developing carbohydrate-based therapeutics.

CARB 2

Creating and implementing breakthrough medical technology

Robert S Langer(1), [email protected], 45 Carleton Street, Cambridge MA 02142, United States . (1) Massachusetts Institute of Technology, United States

Several different case studies in the areas of drug delivery, medical devices and biotherapeutics will be discussed. Each study will be examined in terms of the process and excitement of discovery, initial resistance by the scientific community to the discovery in some cases, the way very broad patents were received, how the technologies were transferred to companies, and the way they have been or are trying to be commercialized. Among those that will be discussed are studies involving heparinase and heparin in which Bob Linhardt played a seminal role.

CARB 3

Advances in heparin analysis and synthesis

Robert J. Linhardt(1), [email protected], Center for Biotechnology and Interdisciplinary Studies, 110 8th Street, Troy NY 12180, United States . (1)

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Departements of Chemistry, Biology and Chemical Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Heparin, a member of the heparan sulfate (HS) family of proteoglycans (PGs), is an important therapeutic agent. Disaccharide analysis on HS requires 104 cells using LC-MS and oligosaccharide mapping affords critical structural information. Sequencing of oligo/polysaccharides is now possible using FTICR-MS with EDD leading to structure-activity relationships (SAR). Improved SAR facilitates new drug development, safer heparins, and has led to an early understanding of the PG-ome and its importance in ESC differentiation. Novel approaches are currently underway in our laboratory to explore HS biosynthesis, including the metabolic engineering of CHO cells and the construction of an artificial Golgi on a digital microfluidic platform. Finally, research is now being directed to develop large-scale enzyme-assisted synthesis of HS/heparin aimed at replacing animal sourced pharmaceutical heparin with a bioengineered product.

CARB 4

Development of a fully synthetic three-component carbohydrate-based cancer vaccine

Geert-Jan Boons(1), [email protected], 315 Riverbend Rd, Athens GA 30606, United States . (1) complex Carbohydrate Research Center, University of Georgia, Athens Georgia 30606, United States

The mucin MUC1 is over-expressed by as much as 50-fold and aberrantly glycosylated by the majority of carcinomas. Recently, a National Cancer Institute translational research working group determined that MUC1 is one of the most promising targets for cancer vaccine development. However, a MUC1-based cancer vaccine that can elicit robust humoral and cellular immune responses has not been reported. We show here that an appropriately glycosylated MUC1 peptide covalently linked to promiscuous helper T-epitope and a Toll-like receptor (TLR) agonist can elicit robust humoral and cellular immune responses and was efficacious in reversing tolerance and generating a therapeutic response in a mouse model of mammary cancer. The superior properties of the vaccine candidate are attributed to the local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells, and avoidance of epitope suppression.

CARB 5

Chemoenzymatic synthesis and antibody recognition of HIV-1 V3 glycopeptides

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Lai-Xi Wang(1), [email protected], 725 W. Lombard Street, Baltimore MD 21201, United States . (1) Institute of Human Virology and Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore MD 21201, United States

A typical HIV-1 gp120 envelope glycoprotein carries more than 20 N-glycans. These oligosaccharides are essential for the virus to evade immune response and meanwhile can serve as ligands for dendritic cell-mediated HIV-1 transmission. The third variable domain (V3) of gp120 is a predominant neutralization determinant that carries three conserved N-glycans within or around the loop. To elucidate the roles of these N-glycans in antibody recognition, we have synthesized a series of homogeneous, full-size V3 glycopeptides by a chemoenzymatic approach and evaluated their binding to two broadly neutralizing antibodies: the V3-speicific antibody 447-52D and the carbohydrate-specific antibody 2G12. Our studies indicate that the complex type N-glycan at N301 has moderate effects on the recognition of V3 to 447-52D, while the V3 domain facilitates the binding of antibody 2G12 to the two high-mannose type N-glycans at the V3 base. This study provides new insights in HIV-1 vaccine design.

CARB 6

Entirely carbohydrate vaccine constructs and their application in probing glycoimmunology

Peter R Andreana(1), [email protected], 5101 Cass Ave, Detroit MI 48202, United States . (1) Chemistry, Wayne State University, Detroit MI 48202, United States

For effective immunity, a strong and long-term response MUST be generated through both cellular and humoral arms of the immune system, involving class II major histocompatibility complex (MHCII) proteins, CD4+ T-cells and B-cells in a T-cell-dependent cascade. A new class of bacterial polysaccharides, characterized by an alternating zwitterionic charge motif on adjacent monosaccharides, has been shown to stimulate T- and B-cell immune responses effectively. We hypothesize that chemically conjugating tumor-associated carbohydrate antigens (TACAs) to zwitterionic polysaccharides (ZPSs) will lead to carbohydrate vaccines that can stimulate strong immunity. A process for evaluating immunogenicity, specificity, and function of the novel construct will include isolation, purification, chemical modification of PS A1 and subsequent in vivo mouse studies. ELISA, FACS and cytotoxicity studies will reveal that an immune response is specific for the conjugated Tn antigen and that cell killing is achieved using a complement dependent approach.

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CARB 7

Carbohydrate polymer assembly: How do mycobacteria do it?

Laura L. Kiessling(1), [email protected], 1101 University Ave., Madison WI 53706, United States . (1) Departments of Chemistry and Biochemistry, University of Wisconsin-Madison, Madison WI 53706, United States

Carbohydrate polymers are the most abundant organic compounds on this planet. They mediate fundamental processes in humans but also can be essential for pathogen survival. A key component of the mycobacterial cell wall is a polysaccharide that contains galactofuranose (Galf) residues. Because Galf residues are not constituents of human glycoproteins or glycolipids, the enzymes mediating their incorporation should serve as antimicrobial targets. Blocking Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), would be valuable because TB results in approximately 2 million deaths/year. To this end, we are exploring the synthesis of the galactan portion of the cell wall. One key enzyme in this pathway is the bifunctional glycosyltransferase, GlfT2, that assembles a polysaccharide composed of alternating 1,5- and 1,6-linked Galf residues. Our investigations were driven by several questions: How does a single glycosyltransferase generate both types of Galf linkages? Does the enzyme have multiple active sites? Does elongation occur by a process that is distributive or processive? How is polysaccharide length controlled in the absence of a template? Can inhibitors that block this enzyme be found? We have explored these questions using a range of approaches that span biology and chemistry, and our results will be discussed. We anticipate that the strategies developed to investigate the molecular mechanism of GlfT2 can be readily applied to probe a wide variety of enzymatic polymerization reactions, including those that result in polysaccharide production.

CARB 8

Lipopolysaccharide transport and assembly in Escherichia coli

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Daniel Kahne(1), [email protected], 12 Oxford St, Cambridge MA 02138, United States . (1) Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA 02138, United States

The outer membrane of Gram-negative bacteria contains an outer leaflet composed of lipopolysaccharide (LPS) that is transported to this location by a pathway that is essential for viability. It has been suggested that inhibitors of this pathway could be useful antibiotics. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane, transport across the periplasm, and insertion into the outer leaflet of the outer membrane. I will talk about the mechanism of LPS transport and assembly and the development of tools that could lead to the discovery of inhibitors of this process.

CARB 9

Arrays and automated oligosaccharide synthesis

Nicola L. B. Pohl(1), [email protected], 2756 Gilman, Ames IA 50011, United States . (1) Department of Chemistry and The Plant Sciences Institute, Iowa State University, Ames IA 50011, United States

Practical methods to enable the automated solid-phase synthesis of peptides and nucleic acids have been crucial to driving genomics and proteomics. Unfortunately, solid-phase methods usually require large excesses of building blocks to achieve the high yields needed when intermediates cannot be purified and therefore are essentially limited to building blocks that are made in a few steps. This talk will discuss the development of an alternative solution-phase automation strategy to iterative synthesis that is based on soluble fluorocarbon tags and the interface of this fluorous synthesis strategy with the formation of small molecule microarrays. The scope and current limitations of this method will also be presented.

CARB 10

Bioactive N-linked glycopeptides and glycoprotein conjugates

Thomas J. Tolbert(1), [email protected], 800 E. Kirkwood Ave., Chemistry Building, Bloomington IN 47405, United States . (1) Department of Chemistry, Indiana University, Bloomington IN 47405, United States

N-linked glycosylation is a highly prevalent posttranslational modification that is also present in a large fraction of FDA approved protein therapeutics. We have recently developed methods for the synthesis of homogeneous N-linked glycopeptides (1) and glycoproteins (2) and are applying these techniques to the

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study of bioactive glycopeptides and semisynthetic glycoprotein conjugates. These studies will be presented with a focus on the effects of N-linked glycosylation on diabetes related peptides and glycosylated antibody fragments. N-linked glycosylation has proven to have some unexpected effects on the physical properties and bioactivity of glycoconjugates in these systems that may be useful in the development of glycopeptide and glycoprotein therapeutics. (1) Journal of the American Chemical Society 2010, 132, 3211-3216. (2) Journal of the American Chemical Society 2009, 131, 13616-13618.

CARB 11

Efficient chemoenzymatic synthesis of sialyl Lewis antigens using sialyltransferase mutants

Xi Chen(1), [email protected], One Shields Avenue, Davis CA 95616, United States ; Go Sugiarto(1); Kam Lau(1); Lei Zhang(1); Shengshu Huang(1). (1) Department of Chemistry, University of Califorina-Davis, Davis California 95616, United States

Sialic acids are a family of nine-carbon monosaccharides mainly found as the terminal residues of glycans on cell surfaces (1). More than 50 structurally distinct sialic acids have been found in nature (2, 3). Sialyl Lewisx is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation (4) and cancer metastasis (5). Sialyl Lewisa is also involved in cancer progression (6). One of our research goals is to develop efficient chemoenzymatic method for the synthesis of sialyl Lewisx and sialyl Lewisa oligosaccharides containing structurally diverse sialic acid residues. The general biosynthetic pathway of sLex and sLea involves sialylation followed by fucosylation. In order to obtain sLex and sLea containing structurally diverse sialic acids, a more efficient approach would be to carry out the fucosylation before the sialylation so that different sialic acid forms can be introduced in the last glycosylation step. However, most sialyltransferases reported can not tolerate fucosylated substrates except for a viral alpha2,3-sialyltransferase (v-ST3Gal I) from myxoma virus (7). We have successfully cloned and expressed the v-ST3Gal I in E. coli. The enzyme has been used in the sialylation of Lewisx antigen. In addition, mutants of Pasteurella multocida sialyltransferase 1 (PmST1), a multifunctional alpha2,3-sialyltransferase that has excellent expression level, solubility, and activity towards non-fucosylated substrates, have been designed and generated for direct sialylation of fucosylated oligosaccharides. 1. Varki, A. (1997) Sialic acids as ligands in recognition phenomena, FASEB J. 11, 248–255. 2. Angata, T., and Varki, A. (2002) Chemical diversity in the sialic acids and

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related alpha-keto acids: an evolutionary perspective, Chem. Rev. 102, 439–469. 3. Schauer, R. (2000) Achievements and challenges of sialic acid research, Glycoconjugate J. 17, 485–499. 4. Rosen, S. D. (2004) Ligands for L-selectin: homing, inflammation, and beyond, Annu. Rev. Immunol. 22, 129–156. 5. Fukuda, M. (1996) Possible roles of tumor-associated carbohydrate antigens, Cancer Res. 56, 2237-2244. 6. Ugorski, M., and Laskowska, A. (2002) Sialyl Lewis(a): a tumor-associated carbohydrate antigen involved in adhesion and metastatic potential of cancer cells, Acta Biochim. Pol. 49, 303–311. 7. Sujino, K., Jackson, R. J., Chan, N. W., Tsuji, S., and Palcic, M. M. (2000) A novel viral alpha2,3-sialyltransferase (v-ST3Gal I): transfer of sialic acid to fucosylated acceptors, Glycobiology 10, 313–320.

CARB 12

Synthesis of saponin vaccine adjuvants for immunotherapy

David Y Gin(1), [email protected], 1275 York Ave., Box 379, New York NY 10065, United States . (1) Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering, New York NY 10065, United States

Saponin natural products constitute some of the most promising adjuvants for immune response potentiation and dose-sparing in several experimental vaccine therapies. For example, melanoma, breast cancer, small cell lung cancer, prostate cancer, HIV-1, and malaria are among the numerous maladies targeted in vaccine clinical trials using Quillaja saponins as critical adjuvants for immune response augmentation. Recent synthetic efforts directed at the chemical synthesis and evaluation of adjuvant-active natural and non-natural saponins will be described.

CARB 13

Glycosylation engineering of human IgG-Fc for functional studies

Lai-Xi Wang(1), [email protected], 725 W. Lombard St, Baltimore MD 21201, United States . (1) Institute of Human Virology and Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, Baltimore MD 21201, United States

Monoclonal antibodies (MAbs) of the immunoglobulin G (IgG) type are an important class of therapeutic glycoproteins. Compelling evidence has indicated that the fine structures of the conserved N-glycan attached to the Fc domain are responsible for the effector functions of MAbs, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and activation of apoptosis. However, the structural heterogeneity of Fc glycosylation

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poses a big challenge in understanding the functional roles of IgG-Fc glycosylation. In this paper, we describe a chemoenzymatic method for IgG-Fc glycosylation engineering that involves expression of human IgG-Fc in yeast and subsequent glycan remodelling through endoglycosidase-catalyzed transglycosylation. This method permits the synthesis of various homogeneous glycoforms of human IgG-Fc. The affinity of different glycoforms to key Fc receptors is being evaluated by SPR analysis and will be discussed.

CARB 14

Chemoenzymatic synthesis of GPI-anchored proteins and glycoproteins

Zhongwu Guo(1), [email protected], 5101 Cass Avenue, Detroit Michigan 48202, United States ; Xueqing Guo(1); Zhimeng Wu(1). (1) Department of Chemistry, Wayne State University, Detroit Michigan 48202, United States

Many surface proteins/glycoproteins are anchored onto the cell membrane via glycosylphosphatidylinositols (GPIs) – a class of complex glycolipids. GPI-anchored proteins/glycoproteins play an important role in many biological processes. To study these processes, it is necessary to have access to homogeneous and structurally defined GPI-linked proteins, glycoproteins, and derivatives, which is currently difficult to achieve both biologically and chemically. To address the issue, we developed a practical method for GPI-linked protein/glycoprotein synthesis based on sortase-mediated site-specific ligation of proteins/glycoproteins and chemically synthetic GPIs, as outlined in

. In specific, sortase A (SrtA) of Staphylococcus aureus was used to couple GPIs and proteins/glycoproteins. SrtA recognizes a pentapeptide LPXTG near the substrate protein C-terminus, breaks the peptide bond between T and G, and then links the protein to a GPI analogue. The method has been employed to prepare a number of GPI-linked peptides, glycopeptides, and proteins and is expected to be generally applicable.

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CARB 15

Cancer relevant epitopes uncovered by synthetic mucin glycopeptides

Shin-Ichiro Nishimura(1), [email protected], N21 W11, Kita-ku,, Sapporo Hokkaido 0010021, Japan . (1) Hokkaido University, Sapporo Hokkaido 0010021, Japan

Robust compound library of synthetic MUC1 glycopeptides allowed for the first time rapid and precise identification of the specific epitope recognized by anti-KL-6 monoclonal antibody, a probe for detecting biomarker of interstitial pneumonia. We revealed that an essential epitope recognized by anti-KL-6 MAb is Pro-Asp-Thr-Arg-Pro-Ala-Pro in which Thr is modified by Neu5Aca2,3Galb1,3GalNAca. Anti-KL-6 MAb could not differentiate this structure from core 2-based glycopeptides involving this epitope and showed a similar binding affinity toward these compounds, indicating that branching at O-6 position of GalNAc does not influence the interaction of anti-KL-6 MAb with MUC1s involving an essential epitope. This is the reason why anti-KL-6 MAb often reacts with tumor-derived MUC1s as well as a biomarker of interstitial pneumonia, namely KL-6 originally discovered as a circulating pulmonary adenocarcinoma-associated antigen. Novel monoclonal antibodies obtained by this epitope reacted specifically with core 1-based structures and did not recognize MUC1s bearing core 2 type O-glycans.

CARB 16

Chemical biology of O-GlcNAc processing enzymes

David J Vocadlo(1)(2), [email protected], 8888 University Drive, Burnaby British Columbia V5A 1S6, Canada ; Gideon J Davies(3); Tracey M Gloster(1); Julia E Heinonen(1); David Shen(2); Carlos Martinez-Fleites(3); Wesley F Zandberg(2); Yuan He(3); Thomas Clark(1). (1) Department of Chemistry, Simon Fraser University, Burnaby British Columbia V5A 1S6, Canada (2) Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby British Columbia V5A 1S6, Canada (3) Department of Chemistry, University of York, York, United Kingdom

A common form of protein glycosylation in which serine and threonine residues of nuclear and cytoplasmic proteins are post-translationally modified with O-linked 2-acetamido-2-deoxy-β-D-glucopyranose residues (O-GlcNAc) is found in the nucleocytoplasm of multicellular eukaryotes. Unlike better known forms of glycosylation occuring within the secretory pathway, O-GlcNAc is a dynamic modification that is turned over more rapidly than the proteins that it ornaments. Dysregulation of O-GlcNAc has been implicated in the etiology of various diseases. Two enzymes process O-GlcNAc; O-GlcNAc transferase (OGT) mediates installation of O-GlcNAc and O-GlcNAcase (OGA) catalyzes removal of

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O-GlcNAc from proteins. Here we describe research into understanding the chemistry and biochemistry of these enzymes as well as how this knowledge has been used to develop chemical tools for manipulating O-GlcNAc levels in tissues.

CARB 17

Synthesis of a single molecule L-rhamnose-containing three component vaccine and evaluation of antigenicity in the presence of anti L-rhamnose antibodies

Sourav Sarkar(1), [email protected], 2801 W. Bancroft St., Toledo OH 43606, United States ; Rommel S Talan(1); Steven A Lombardo(2); Katherine A Wall(2); Steven J Sucheck(1). (1) Department of Chemistry, The University of Toledo, Toledo OH 43606, United States (2) Department of Medicinal and Biological Chemistry, The University of Toledo, Toledo OH 43606, United States

We hypothesized that conjugation of L-rhamnose (Rha) to a carbohydrate antigen would enhance antigenicity of the antigen in mice possessing anti-Rha antibodies. We synthesized a vaccine containing the GalNAc-O-Thr (Tn) tumor specific antigen, a T-cell epitope (YAF) and a Rha moiety. Mice were immunized with Rha-ovalbumin (Rha-OVA). Anti-Rha antibody titers were >100 fold higher in groups of mice immunized with Rha-OVA than the control groups. Mice producing anti-Rha were challenged with Rha-YAF-Tn or YAF-Tn. Sera collected from the groups immunized with Rha-OVA and later challenged with Rha-YAF-Tn showed a two fold increase in anti-Tn titer at 1/100 serum dilution compared to mice not immunized with Rha-OVA. Proliferation of T-cells using cells primed with either Rha-YAF-Tn or YAF-Tn showed a 4-fold increase in the presence of Rha antibodies. The results suggest that T-cells were presented with higher concentrations of Rha-YAF-Tn as a result of the presence of the anti-Rha antibodies.

CARB 18

Design of glycoprotein vaccines against influenza and other viral diseases

Chi-Huey Wong(1)(2), [email protected], 128 Academia Road, Section 2, Nankang District, Taipei 115, Taiwan Republic of China . (1) Academia Sinica, Taipei 115, Taiwan Republic of China (2) The Scripps Research Institute, Taipei 115, Taiwan Republic of China

Protein glycosylation is the most complex post-translational process; more than 90 percent of human proteins are glycosylated. The significance of glycosylation at the molecular level is however not well understood, and as such the pace for the development of carbohydrate-based drug discovery and diagnosis is relatively slow. It is thus important to develop new tools to study the effect of glycosylation on the structure and function of proteins and other biologically

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active molecules. This lecture will focus on the development of new methods for the synthesis of homogenious glycoproteins with well defined glycan structure, glycoarrays for the high-throughput analysis of protein-glycan interaction and design of click-induced fluorescent probes for use to identify new cancer biomarkers for diagnosis and drug discovery. New glycoprotein vaccines have been designed and developed to tackle the problems of flu and breast cancer.

CARB 19

Modular synthesis of heparan sulfate oligosaccharides for array development

Geert-Jan Boons(1), [email protected], 315 Riverbend Rd, Athens GA 30606, United States . (1) Complex Carbohydrate Research Center, University of Georgia, Athens Georgia 30606, United States

Although hundreds of heparan sulfate binding proteins have been identified, and implicated in a myriad of physiological and pathological processes, very little information is known about ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity-relationships, which in turn is due to the structural complexity of natural HS and difficulties of preparing well-defined HS-oligosaccharides. To address this deficiency, we have developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The modular building blocks have been used for the preparation of a library of oligosaccharides, which has been employed to probe ligand requirements of several HS-binding proteins. To facilitate high through put screening, spacer modified HS-oligosaccharides have been employed for microarray fabrication.

CARB 20

New targets for antibiotics

Suzanne Walker(1), [email protected], 200 Longwood Ave, Boston MA 02115, United States . (1) Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston MA 02115, United States

Wall teichoic acids (WTAs) are anionic carbohydrate polymers that are covalently attached to the peptidoglycan layers of many Gram positive organisms, including S. aureus. They comprise 50% of the cell wall mass and have a major influence on cell envelope integrity. They are suggested targets for antibiotics since deleting certain genes in the pathway results in a loss of virulence while deleting others is lethal. We developed a pathway-specific, cell based screen to discovery inhibitors of WTA biosynthesis and have identified a potent WTA-active antibiotic.

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We will describe studies on the mechanism of action of this compound and on the cellular effects of blocking wall teichoic acid biosynthesis. Prospects for the use of WTA inhibitors as antibiotics will be discussed.

CARB 21

Challenges and opportunities in natural product glycosylation

Jon S Thorson(1), [email protected], 777 Highland Avenue, Madison WI 53705-2222, United States . (1) School of Pharmacy, University of Wisconsin, United States

In nature, the attachment of sugars to small molecules is often employed to mediate targeting, mechanism of action, and/or pharmacology. As an alternative to pathway engineering or classical chemical glycosylation strategies, we report merging three promiscuous enzymes (a sugar kinase - 'E1', a nucleotidylyltransferase - 'E2', and a glycosyltransferase – 'E3') with upstream synthetic chemistry and downstream chemoselective ligation provides a powerful method to diversify, via the attachment of variant sugars ('glycorandomize'), complex natural products. The optimization of E1-E3 using enzyme evolution and structure-based enzyme engineering, the glycorandomization proof of concept, the recent advances based upon the reversibility of glycosyltransferase-catalyzed reactions, the evolution of 'flexible' glycosyltransferases and the potential for in vivo glycosylation hosts will be highlighted. In addition, recent developments pertaining to our development of a complementary chemical method ('neoglycorandomization') will be discussed. The impact of the (neo)glycorandomization upon the activity of selected small molecule-based drugs and/or understanding sugar transport may also be presented.

CARB 22

Glycomimetic and non-carbohydrate inhibitors as probes of the lectin DC-SIGN

Laura L Kiessling(1), [email protected], 1101 University Avenue, Madison WI 53706, United States . (1) Departments of Chemistry and Biochemistry, University of Wisconsin-Madison, Madison WI, United States

Carbohydrates act through carbohydrate-binding proteins (lectins) to govern numerous cellular processes, including cell adhesion, recognition, and signaling. Despite the importance of lectins, inhibitors that can be used to interrogate or

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mitigate their function are rare. In principle, carbohydrates themselves can serve as probes, but they have several liabilities, including low affinity and relaxed specificity. We have used high throughput screening and chemical synthesis to identify potent inhibitors of the lectin DC-SIGN. DC-SIGN is a C-type lectin found on the surface of dendritic cells that mediates antigen uptake and dendritic cell–T cell interactions. Ironically, this immune system lectin can be co-opted to facilitate the dissemination of pathogens, including HIV, dengue virus, and Mycobacterium tuberculosis. The identification of DC-SIGN inhibitors provides the means to dissect the function of this lectin and its involvement in a wide range of infectious diseases.

CARB 23

Toxicity and functional assessment using polysaccharide-based magnetic iron oxide nanoparticles for cell labeling in vivo and in vitro

Yoshitaka Miyamoto(1), [email protected], Higashi-ku, Nagoya 461-0047, Japan ; Yumie Koshidaka(1); Hiroaki Saito(2); Yukimasa Kagami(3); Katsutoshi Murase(2); Noritada Kaji(3); Hiroshi Yukawa(1); Hirofumi Noguchi(4); Hisashi Iwata(5); Yoshinobu Baba(3); Shuji Hayashi(1). (1) Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Higashi-ku Nagoya 461-0047, Japan (2) Nagoya Research Laboratory, MEITO Sangyo Co., Ltd., 25-5, Kaechi, Nishibiwajima Kiyosu 452-0067, Japan (3) Department of Applied Chemistry, Nagoya University Graduate School of Engineering, Furo-cho, Chikusa-ku Nagoya 464-8603, Japan (4) Baylor Institute for Immunology Research, Baylor Research Institute, 3434 Live Oak St., Dallas TX 75204, United States (5) Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, 1200 Matsumoto-cho, Kasugai Aichi 487-8501, Japan

It is desireble to develop less-invasive medical treatment and diagnostics for the patient in breakthrough of medical technology. Especially, contrast agents for magnetic resonance imaging (MRI) have been widely used to diagnose blood vessel disorder, internal structure of organs. Recently, we developed a novel contrast agent for MRI using polysaccharide-based magnetic iron oxide nanoparticles. The agent was transduced into each cell efficiently. Additionally, we reported that the effective imaging of transplanted cells was the efficienty labeled using the contrast agent. The purpose of this study is to evaluate the effect of the contrast agent on cell functions in vivo and in vitro. In this experiment, the contrast agent was added to human hepatocellular liver carcinoma cell line (HepG2) and its effect on cell functions was evaluated. As a result, the dependence of the amount of a contrast agent on liver functions was investigated using different contrast agents.

CARB 24

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Glycodendrimers for the mediation of cancer cellular aggregation processes

Mary J Cloninger(1), [email protected], 103 Chemistry and Biochemistry Building, Bozeman Montana 59717, United States ; Mark L. Wolfenden(1); Julie J Sprenger(1); Michael P Capp(1); Pratima Nangia-Makker(2); Avraham Raz(2). (1) Department of Chemistry and Biochemistry, Montana State University, Bozeman Montana 59717, United States (2) Department of Pathology, School of Medicine, Wayne State University, Detroit MI 48201, United States

Galectin-3 is a galactoside-binding protein that is commonly up or down regulated in different cancers and is implicated in tumor formation and proliferation, apoptosis, angiogenesis, and B cell activation. In particular, galectin-3 is thought to be involved in the aggregation processes that lead to tumor proliferation, making this lectin an interesting target for our multivalent carbohydrate functionalized poly(amidoamine) (PAMAM) dendrimers. Capitalizing on the differences in the monovalent binding interaction between galactose, lactose, N-acetylgalactosamine, and N-acetyllactosamine, and also on the range of generations that are readily available for PAMAM dendrimers, a library of glycodendrimers was prepared. This series of glycodendrimers includes compounds having a large range of binding capabilities with galectin-3 and enables attenuation of galectin-3 mediated biological processes. Here, the binding interactions of these glycodendrimers with galectin-3 and the effect that glycodendrimers have on cancer cellular aggregation processes are presented.

CARB 25

Glyconanoparticles: Multibiofunctional nanomaterials for biomedical applications

Soledad Penades(1), [email protected], Paseo Miramon, 182, San Sebastian Guipuzcoa 20009, Spain . (1) Biofunctional Nanomaterials Unit, CIC biomaGUNE, San Sebastian Guipuzcoa 20009, Spain

Our laboratory pioneered the development of a new technology (Glyconanotechnology) for tailoring - in a simple and versatile way – bio-functional gold nanoclusters (glyconanoparticles, GPNs). GNPs present some advantages over other previously prepared colloids as: 1) easy preparation and purification; 2) exceptional small core size and narrow distribution sizes; 3) control over ligands number and nanoparticles size; 4) water solubility; 5) high storage stability without flocculation; and 6) singular physical properties. GNPs with biologically significant carbohydrates and with differing carbohydrate density were prepared to study and intervene in carbohydrate-mediate cell adhesion processes. The methodology includes also the preparation of hybrid

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GNPs incorporating carbohydrates and other molecules such as fluorescent probes, biotin as well as biological molecules such as peptides, DNA and RNA providing with the possibility to create artificial “nanocells”. The manipulation of the metallic cluster to obtain luminescent glyco-quantum dots (semiconductors) and magnetic glyco-nanoparticles for application in cellular labeling and imaging by magnetic resonance (MRI), is comprised within the potential of this technology. Examples of gold GNPs as anti-adhesion agents and as magnetic probes in cellular labeling and tracking by magnetic resonance imaging (MRI) will be highlighted.

CARB 26

New glycopeptide-based nanoparticle constructions for anticancer therapy

Joseph John Barchi(1), [email protected], 376 Boylse Street, PO Box B, Frederick MD 21702, United States ; Ray Brinas(2); Andreas Sundgren(1); Padmini Sahoo(3); Amy Houghten(3); Susan Morey(3); Michael Sanford(2); Howard Young(2). (1) Chemical Biology, National Cancer Institute at Frederick, Frederick MD 21702, United States (2) Experimental Immunology, National Cancer Institute at Frederick, Frederick MD 21702, United States (3) Biotechnical and Clinical Laboratory Sciences, University of Buffalo, Buffalo NY 14214, United States

The surface of mammalian cells is replete with carbohydrates that are presented in a wide range of structural subtypes and are involved in a wealth of biologically important functions. Transformation of normal cells to a neoplastic phenotype involves aberrant gene expression resulting in the presence of very different glycans displayed on the tumor cell surface. Many of these are recognized and targeted by the immune system and are thus referred to as tumor-associated carbohydrate antigens (TACA). Various TACA's have been employed in anti tumor vaccines with mostly disappointing results due to immunological tolerance and lack of a cell mediated response. We propose here a novel strategy for the preparation of fully synthetic immunogens based on a nanoscale inorganic platform that displays tumor glycopeptide antigens in a multivalent fashion. We have linked these to multicomponent gold nanoparticles as “stand alone” immunogens where an appropriate nanoconstruction is used in place of immunostimulating proteins. The talk will concentrate on the development of these and other particles as vaccine constructs and novel entities that elicit tailored cytokine responses when they interact with antigen presenting cells.

CARB 27

Functionalized catanionic surfactant vesicles: A new approach to carbohydrate vaccines

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Philip DeShong(1), [email protected], University of Maryland, Department of Chemistry and Biochemistry, College Park MD 20742, United States ; Juhee Park(1)(2); Lenea Rader(1); Glen Thomas(1); Douglas English(2)(3); Lindsey Zimmerman(4); Daniel C. Stein(4). (1) Department of Chemistry & Biochemistry, University of Maryland, College Park MD 20742, United States (2) SD Nanosciences, Inc., College Park MD 20742, United States (3) Department of Chemistry, Wichita State University, College Park MD 20742, United States (4) Department of Cell Biology & Molecular Genetics, University of Maryland, College Park MD 20742, United States

Vaccines against Gram-negative pathogens (e.g. E. coli, F. tularensis, N. meningitidis, P. aeruginosa) are difficult to produce because their glycosylated cell surfaces (i.e. lipolysaccharides) are poorly immunogenic and fail to elicit a helper T-cell mediated immune response. Catanionic surfactant vesicles are unilamellar vesicles that form spontaneously from a mixture of cationic and anionic surfactants in water. The size and surface charge of the vesicle can be controlled by choice of surfactants, and the external leaflet of the vesicle can be decorated with non-ionic surfactants in concentrations as high as 20 mole percent without affecting the stability of the vesicle. We incorporated a mixture of peptides designed to interact with T-cells and LOS-derived glycoconjugates into catanionic vesicles. The resulting functionalized vesicle was capable of inducing a strong IgG response in mice. The synthesis and characterization of the vesicles will be discussed and biological evaluation of this approach to carbohydrate vaccines will be presented.

CARB 28

Novel synthesis of 2'-deoxynucleoside 5'-triphosphates without nucleoside protection and DNA polymerase recognition

Julianne M. Caton-Williams(1), [email protected], Room 540 General Classroom Building, 38 Peachtree Center Ave., Atlanta Georgia 30303-3083, United States ; Matthew R. Smith(1); Zhen Huang(1). (1) Department of Chemistry and Department of Biology, Georgia State University, Atlanta Georgia 30303, United States

Because nucleosides comprise amino and multi-hydroxyl groups, the synthesis of the nucleoside 5'-triphosphates requires protection and deprotection of these groups, which are achieved in multiple synthetic steps. In order to simplify the triphosphate synthesis and avoid the protection and deprotection reactions, we have developed a convenient strategy to synthesize 2'-deoxynucleoside 5'-triphosphates (dNTPs) without nucleoside protection. The facile synthesis is achieved by generating an in situ, selective phosphorylating reagent that reacts selectively with the 5'-hydroxyl group and finally generates the triphosphates in one-pot. The synthesized triphosphates are of high quality and can be effectively incorporated into DNAs by DNA polymerase. The developed synthetic conditions

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are mild, and this strategy is also useful in one-pot synthesis of modified triphosphates.

CARB 29

Activation of retinoic acid inducible gene (RIG-I) by short oligonucleotides

Radhakrishnan Iyer(1), [email protected], 113 Cedar Street, Milford MA, United States ; John Coughlin(1); Seetharamaiyer Padmanabhan(1); Brent Korba(2); Sua Myong(3). (1) Spring Bank Pharmaceuticals, United States (2) Georgetown University, United States (3) University of Illinois- Urbana-Champaign Department, United States

Retinoic acid inducible gene (RIG-I), a host cellular protein, acts as a “sensor” of viral RNA. Activation of RIG-I results in Interferon (IFN) expression and induction of antiviral effects. Using a novel translocation assay of RIG-I on a dsRNA template, we discovered that short oligonucleotides (SO) cause activation of RIG-I. The RIG-I actives had specific structural and stereochemical attributes. The RIG-I active analog SB 40 had potent antiviral activity against HBV (EC50, 0.5 to 1 micromolar in HepG2.2.15 cell lines) and was synergistic with 3TC and Adefovir. SB 40 showed potent antiviral activity in the HBV transgenic mouse model with an EC50 < 1 mg/kg. A related analog SB 44 had EC50 (of 1~2 micromolar) against HCV (genotypes 1a, 1b) with synergistic antiviral activity with other anti-HCV drugs. The stimulation of RIG-I pathway by SO could be exploited for multiple therapeutic applications including discovery of orally bioavailable broad-spectrum antiviral agents.

CARB 30

Solid-phase synthesis of 5'-di- and tri-phosphates and their modified analogs of DNA, RNA and chemically modified oligonucleotides

Ivan Zlatev(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Thomas Lavergne(2); Sudhakar Takkellapati(1); Rajenda Pandey(1); Yupeng Fan(1); Marija Prhavc(1); Kathy Mills(1); G. Rajeev Kallanthottathil(1); Françoise Debart(2); Jean-Jacques Vasseur(2); François Morvana(2); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States (2) Institut des Biomolécules Max Mousseron, France

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A robust, reproducible, scalable, and automated method for the solid-phase synthesis of 5'-di- and tri-phosphates of DNA, RNA, and their chemically modified analogs that employs 5'-H-phosphonate intermediates is described. 5'-Triphosphates of oligonucleotides of varying lengths and sequences containing different 5'-terminal nucleotides with and without internal sugar and/or backbone modifications (2'-O-Me, 2'-F, 2'-O-MOE, LNA, phosphorothioate) were efficiently prepared as crude products or further purified by HPLC. The synthetic scheme involved the introduction of a 5'-H-phosphonate monoester onto a solid-supported oligonucleotide; the 5'-H-phosphonate monoester was further transformed into either a di- or tri-phosphate. Oligonucleotide 5'-polyphosphates were typically obtained in high yields and acceptable purity. The preparation method was efficiently adapted to a fully automated synthesis cycle using a standard oligonucleotide synthesizer.

CARB 31

Drug discovery harnessing RNA interference

Muthiah Manoharan(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, Qatar . (1) Alnylam Pharmaceuticals, United States

RNA interference (RNAi) is a powerful biological process for specific silencing of mRNAs in diverse eukaryotic cells. By using synthetic short interfering RNAs (siRNAs) this process can be utilized to develop drugs targeting any given gene, including the “undruggable targets”. Desirable "drug-like" properties can be imparted to siRNAs by introducing chemically modified nucleosides and other synthetic building blocks. siRNAs containing chemical modifications show enhanced resistance towards nuclease degradation, less immune stimulation, and reduced "off-target" effects compared to unmodified siRNAs. To achieve in vivo delivery, certain chemical conjugates and novel liposomal formulations are being investigated. A summary of this drug discovery paradigm and progress in the development of several new drug compounds will be presented

CARB 32

Parallel high throughput synthesis of chemically modified 21-27mer siRNA sequences

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Jason Costigan(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Sarfraz Shaikh(1); Keri Dufault(1); Jack de Groot(1); Satya Kuchimanchi(1); Rachel Meyers(1). (1) Alnylam Pharamaceuticals, United States

Methods have been developed for high throughput synthesis of 21 to 27mer siRNA sequences containing different chemical modifications. We have developed procedures that enable rapid generation of modified RNA single strands and duplexes using a MerMade192 synthesis platform. The purpose of this work is to generate high quality duplexes for in vitro screening experiments and bio-analytical assays. Sequences that contain different chemical modifications, including 2'OMe, 2'F, 5' phosphate and 3' cholesterol have been successfully synthesized and fully characterized by LC-MS and analytical ion exchange methods. Cleavage and deprotection methods have been optimized to minimize formation of impurities. Development of high throughput purification processes and annealing methods that complement high throughput synthesis productivity will be discussed.

CARB 33

New developments in the synthesis of oligoribonucleotides: Use of dimer/trimer blocks in combination with an “ionic tag” soluble support

Matthew R Hassler(1), [email protected], 801 Sherbrooke St. W, Montreal Quebec H3A 2K6, Canada ; Nandyala Mallikarjuna Reddy(1); Tak-Hang Chan(1); Masad J Damha(1). (1) Department of Chemistry, McGill University, Montreal Quebec H3A 2K6, Canada

The reality of mainstream RNA based therapeutics is quickly approaching and the demand for these compounds may soon exceed the capabilities of the manufacturers. Large-scale synthesis of short-interfering RNA (siRNA) is generally carried out via iterative step-wise solid phase synthesis. A major drawback of the solution-phase alternative is the rigorous purification required between each step, which can be costly and time consuming. We have demonstrated that by using an “ionic tag” soluble support we are able to overcome some of the limitations of solution-phase synthesis. This allows for selective precipitation of the growing RNA over all other reagents used in the oligonucleotide synthesis cycle. Herein, we will present our recent developments in this area. Additionally, we will describe the synthesis of di- and tri- nucleotide amidite building blocks for large scale RNA solution phase synthesis. siRNAs synthesized by our newly developed method will be compared to traditional solid-phase synthesized siRNAs for purity and biological activity.

CARB 34

Advanced process of RNA synthesis

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Anuj Mohan(1), [email protected], 33 Industrial Way, Wilmington MA 01887, United States . (1) Chemgenes, United States

We have developed a process of RNA synthesis that utilizes 3'- DMT -5'- CED phosphoramidites leading to step-wise coupling efficiency > 99% in the reverse RNA synthesis (5 ->3') resulting in high purity short and long RNA sequences. These new monomers have been found to be distinctly superior to the standard 3'- CED -2'-O-TBDMS phosphoramidites used in conventional RNA synthesis (3' -> 5'). The monomers provide method for synthesis of clean RNA with wide variety of modifications or labels at 3'- end of oligonucleotide. The mechanism of the novel process will be discussed.

CARB 35

Novel method for the confirmation of siRNA sequence by LC-MS/MS

Gary Lavine(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Matthias Kretschmer(1); James McArdle(2); Satya Kuchimanchi(1); Veeragu Murugaiah(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States (2) McArdle & Associates, LLC, United States

We have developed a new procedure for confirmation of the sequence of chemically modified synthetic oligonucleotides by tandem mass spectrometry fragmentation. The method is highly reliable for the interpretation of high-resolution Q-TOF MS/MS data. Deconvolution of the MS/MS data provided simplified peak assignments were used to match expected fragment ions. Data from the MS/MS experiments of three different charge states were combined; evaluation of these complementary data sets provided more complete coverage. To verify the assigned structure, the sequence confirmation was challenged by comparison of the quality of fit to computer-generated sequence perturbations. Through this novel perturbation method we have shown that the sequence of synthetic oligonucleotides can be confirmed using MS/MS data from a Q-TOF instrument.

CARB 36

Evaluation of Canonical vs. Dicer-substrate siRNAs in vitro and in vivo

Don Foster(1); Satya Kuchimanchi(1); Greg Hinkle(1); William Cantley(1); Rick Duncan(1); Geff Cole(1); Chris Sherill(1); Kathy Mills(1); Mara Broberg(1); Jeff Rollins(1); Klaus Charisse(1); Muthiah Manoharan(1); Sayda Elbashir(1). (1) Alnylam Pharmaceuticals, United States

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Canonical siRNAs are duplexes formed by two 21-mers; 19 bases are paired and two nucleotides overhang on each strand (Elbashir SM et al., 2001, RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15:.188-200; Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K and Tuschl T., 2001, Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498). Dicer-substrate siRNAs (DsiRNA) are 25/27-mer duplexes from which 21-mer siRNAs are generated by the action of Dicer in situ (Kim DH, Behlke MA, Rose SD, Chang MS, Choi S and Rossi JJ. 2005, Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23: 222–226).We conducted comprehensive studies in vitro and in vivo to compare canonical siRNA and DsiRNA. We chose two well-characterized genes as targets and designed over 300 compounds, representing both types of siRNAs, to hybridize to these mRNAs. We will present data from this comparative study regarding silencing efficacy, duration of silencing, and other key parameters such as nuclease stability and cytokine induction.

CARB 37

Modulation of thermal stability can enhance the potency of siRNA

Haripriya Addepalli(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Meena Meena(1); Chang G. Peng(1); Gang Wang(1); Yupeng Fan(1); Klaus Charisse(1); K. Narayanannair Jayaprakash(1); G. Rajeev Kallanthottathil(1); Rajendra Pandey(1); Gary Lavine(1); Ligang Zhang(1); Kerstin Jahn-Hofmann(2); Philipp Hadwiger(2); Muthiah Manoharan(1); Martni Maier(1). (1) Alnylam Pharmaceuticals, United States (2) Alnylam Europe, Germany

During RISC assembly, the guide (or antisense) strand must separate from its complementary passenger (or sense) strand to generate the active RISC complex. Although this process is facilitated through sense strand cleavage, there is evidence for an alternate mechanism, in which the strands are dissociated without cleavage of the sense strand. Here we show that the potency of siRNA can be improved by modulating the thermodynamic stability of the duplex formed between sense and antisense strands. We found that placement of thermally destabilizing modifications, such as a non-canonical bases like 2,4-difluorotoluene, or single base pair mismatches in the central region of the sense strand (nucleotides 9 to 12) significantly improved the potency of a well-validated siRNA targeting firefly luciferase. For this particular siRNA, the strongest correlation between the decrease in thermal

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stability and the increase in potency was found at position 10. Controls with a stabilized sugar-phosphate backbone indicated that enzymatic cleavage of the sense strand prior to strand dissociation was not required for the observed silencing activity.

CARB 38

Carbohydrate conjugation to siRNA for tissue-specific delivery

G. Rajeev Kallanthottathil(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; K. Narayanannair Jayaprakash(1); Maria Frank-Kamenetsky(1); Gang Wang(1); Tianlei Lei(1); Mariano Severgnini(1); William Querbes(1); Jim Butler(1); Alfica Sehgal(1); Tomoko Nakayama(1); Klaus Charrise(1); Martin Maier(1); Kevin Fitzgerald(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

Systemic administration of lipophile-conjugated siRNA mediates uptake into cells and causes gene silencing in vivo (Nature Biotech., 2007, 25, 1149; Nature, 2004, 432, 173). To further improve tissue-specific delivery of siRNAs, we conjugated carbohydrate ligands to siRNA with and without lipophilic moieties. These ligands bind specifically to the asialoglycoprotein receptor (ASGPR). ASGPR is a transmembrane glycoprotein of 42 kDa that mediates binding, internalization, and degradation of extracellular glycoproteins with exposed terminal galactose residues. The receptor is highly expressed on the surface of liver hepatocytes; it is present on the sinusoidal and lateral plasma membranes. Among various galactose analogues tested for binding affinity, a triantennary N-acetylgalactosamine (GalNAc) cluster had nanomolar binding affinity to the receptor. We will describe synthesis of multivalent GalNAc-siRNA conjugates for hepatocyte-specific delivery and in vitro receptor binding, uptake, and gene silencing.

CARB 39

Sugar and proteins: Applications of bioconjugates

Benjamin G. Davis(1), [email protected], Chemistry Research Laboratory, Oxford Oxfordshire OX1 3TA, United Kingdom . (1) Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom

Sugars and Post-Translational Modifications are critical biological markers that modulate the properties of proteins. Our work studies the interplay of proteins,

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sugars and modifications and their utility. (i) Synthetic Biology's development at the start of this century may be compared with Synthetic Organic Chemistry's expansion at the start of the last; after decades of isolation, identification, analysis and functional confirmation the future logical and free-ranging redesign of biomacromolecules offers tantalizing opportunities. New methods are required: despite 80-years-worth of non-specific, chemical modification of proteins, precise methods in protein chemistry remain rare. The development of efficient, complete, chemo- and regio-selective methods, applied in benign aqueous systems to redesign the structure and function of bioconjugates (proteins, polymers, nanoparticles, viruses, nanotubes) will be presented. (ii) Bioconjugate Applications: These conjugates find application in • drug delivery • nanomolar inhibitors of bacterial interactions • gene delivery vehicles • probes of in vivo function • non-invasive presymptopmatic disease diagnosis • localized delivery of unprecedented radio-dose.

[1] B.G.Davis, Chem. Rev. 2002, 102, 579-601. [2] B.G. Davis, Science 2004, 303, 480-482 [3] S.I. van Kasteren, H.B. Kramer, H.H. Jensen, S.J. Campbell, J. Kirkpatrick, N.J. Oldham, D.C. Anthony, B.G. Davis, Nature 2007, 446, 1105-1109. [4] C. Fleming, A. Maldjian, D. Da Costa, P. Penny, R.C. Noble, N.R. Cameron, B.G. Davis, Nat. Chem. Biol. 2005, 1, 270-274 [5] S.I. van Kasteren, H.B. Kramer, D.P. Gamblin, B.G. Davis, Nat. Protoc. 2007, 2, 3185-3194 [6] S.Y. Hong, G. Tobias, B. Ballesteros, F. El Oualid, J.C. Errey, K.J. Doores, A.I. Kirkland, P.D. Nellist, M.L.H. Green , B.G. Davis, J. Am. Chem. Soc. 2007, 129, 10966-10967 [7] G.J.L. Bernardes, J.M. Chalker, J.C. Errey, B.G. Davis, J. Am. Chem. Soc. 2008, 130, 5052-5053

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[8] S.I. van Kasteren, S.J. Campbell, S. Serres, D.C. Anthony, N.R. Sibson, B.G. Davis Proc. Natl Acad. Sci. U.S.A. 2009, 106, 18-23

CARB 40

Sialic acid recognition: Particles, chips, and cells

Robert A. Field(1), [email protected], Norwich Research Park, Colney Lane, Norwich Norfolk NR4 7UH, United Kingdom . (1) Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, United Kingdom

The sialic acids are a family of carbohydrates that are intimately associated with cellular recognition events. Using a combination of glycoarray and glyconanoparticle approaches, we have been investigating sialic acid recognition in the context of the human immune system and infection by the parasitic protozoan <i>Trypanosoma cruzi</i>. On one hand, glycoarray approaches with soluble, recombinant proteins have allowed us to explore similarities and differences in the carbohydrate-binding specificity of human and murine siglecs. On another, glyconanoparticle studies have enabled us ask questions about the structure and sialylation of parasite mucins. The combination of this information is now enabling us to begin to explore host recognition of parasite glycans through cell-based assays.

CARB 41

Functionalized nanoparticle for protein detection

Chun-Cheng Lin(1), [email protected], 101, Sec. 2, Kuang Fu Rd., Hsinchu Taiwan 30013, Taiwan Republic of China . (1) Department of Chemistry, National Tsing Hua University, Hsinchu Taiwan 30013, Taiwan Republic of China

Biomolecule-conjugated nanoparticles (NPs) have been demonstrated to have promising applications on bioanalysis. Due to their large surface area to volume ratio and homogeneity in aqueous solution, functionalized nanoparticles were demonstrated as good carriers for affinity probes in target molecule separation. Our results showed that the ligand affinities with target proteins were significantly enhanced when ligands were assembled on the nanoparticles. The MALDI-TOF MS analysis was applied to identify the target molecules captured on the functionalized nanoparticles. In an attempt to fabricate highly active immunoprobes for serum biomarker detection, we developed a simple and effective method for site-specific and self-oriented immobilization of antibodies on magnetic nanoparticles (MNPs). The functionalized antibody conjugated nanoparticles were further extended as a multiplex probe to identify the biomarkers in human serum. The immobilization strategy was also applied to fabricate Fc-fused lectin microarray.

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CARB 42

Nanotechnology-based tools for the glycomics revolution: Silicon nanophotonic carbohydrate biosensors

Daniel M Ratner(1), [email protected], Box 355061, Dept. of Bioengineering, Seattle WA 98195, United States . (1) Department of Bioengineering, University of Washington, Seattle WA 98195, United States

Spurred by the post-genomic revolution in biological research, glycomics – the comprehensive study of carbohydrates (glycans) in biology – has undergone a rapid expansion, as researchers seek to unravel the many roles played by glycans in nature. This talk describes that application of nanotechnology and biointerfacial engineering to design new tools for glycomics research. Techniques including surface modification via molecular self-assembly, scanning probe microscopy characterization of glycosylated materials and an ultra-sensitive biosensing platform based on silicon nanophotonics will be discussed in the context of enabling glycomics research through nanoscale engineering.

CARB 43

Targeted glyco-magnetic nanoprobes for detection and molecular imaging of atherosclerosis

Kheireddine El-Boubbou(1), [email protected], Michigan State University, Department of Chemistry, East Lansing MI 48824, United States ; Medha Kamat(1); David Zhu(2); Ruiping Huang(2); George Abela(2); Xuefei Huang(1). (1) Department of Chemistry, Michigan State University, East Lansing MI 48823, United States (2) Department of Radiology, Michigan State University, East Lansing MI 48824, United States

Cardiovascular diseases, often associated with atherosclerosis, are the leading cause of morbidity and mortality in the world. Despite the significant progress in cardiology, there remain large unmet needs to early detect atherosclerotic plaques, especially those which are prone to ruptures causing heart attacks and strokes. One of the major causes of such dramatic event is “inflammation” which occurs during early onset of the disease leading to over-expression of cell-adhesion glycoproteins. Our proposed work is based on the surveillance that hyaluronic acid (HA) is upregulated in atherosclerotic lesions and its principal cell-adhesion receptor, CD44 is involved in several atherogenic processes. Thus, we engineered novel hyaluronic superparamagnetic iron oxide nanoparticles (HA-SPIONPs) to be used not only as novel contrast cargo but also as proficient probes to non-invasively target atherosclerotic plaques using magnetic resonance imaging (MRI). The targeting agents on the external surface of the NPs will allow the selective labeling of the plaques. Due to the superparamagnetic nature of the

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NPs, imaging of atherosclerotic plaques in rabbits was successfully examined. We anticipate that such novel nanoprobes will not only deepen our fundamental understanding of the molecular and cellular events characterizing unstable atherosclerotic plaques, but also be potentially developed into a highly innovative therapy for atherosclerosis.

CARB 44

Carbohydrates on nanoparticles, surfaces and polymers: From basics to diagnostics applications

Peter H. Seeberger(1), [email protected], Arnimallee 22, Berlin 14195, Germany . (1) Dept. of Biomolecular Systems, Max-Planck Institute for Colloids and Interfaces, Berlin 14195, Germany

Cell surface oligosaccharides and glycosaminoglycans are important in signal transduction processes of biomedical significance. Described is an integrated platform relying on defined oligosacharides derived by automated solid phase syntehsis that are placed on many different nanoparticles, surfaces and polymers. By tuning the material as well as the carbohydrate, applications in the areas of diagnostics, vaccines and therapeutics. Particular focus will be placed on previously unpublished materials. For Previous work in our laboratory please see: 1. Disney, M.D.; Zheng, J.; Swager, T.; Seeberger, P.H.; Visual Detection of Bacteria with Carbohydrate Containing Fluorescent Polymers, J. Am. Chem. Soc. 2004, 126, 13343-13346. 2. de Paz, J.L.; Noti, C.; Boehm-Knoche, F.; Werner, S.; Seeberger, P.H.; Glycodendrimers Containing Synthetic Heparin Oligosaccharides to Regulate Heparin-Mediated Biological Processes; Chem. Biol. 2007, 14, 879-887. 3. Kikkeri, R.; Lepenies, B.; Adibekian, A.;Laurino, P.; Seeberger, P.H.; In Vitro Imaging and In Vivo Liver Targeting with Carbohydrate Capped Quantum Dots; J. Am.

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Chem. Soc. 2009, 131, 2110-2112. 4. Kikkeri, R.; Gupta, T.; Hossain, L.H.; Kamena, F.; Gorodyska, G.; Beurer, E.; Textor, M.; Seeberger, P.H.; Ru(II)-Glycodendrimers as Probes to Study Lectin-Carbohydrate Interactions and Electrochemically Measure Mono- and Oligosaccharide Concentrations; Langmuir 2010, 26, 1520–1523. 5. Kikkeri, R.; Laurino, P.; Odedra, A.; Seeberger, P.H.; Microreactor Synthesis of Carbohydrate Functionalized Quantum Dots; Angew. Chem. Int. Ed. 2010, 49, in press.

CARB 45

Efficient synthesis of siRNA-folic acid conjugates

Rajendra Pandey(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Muthusamy Jayaraman(1); Anna Borodovsky(1); David Butler(1); Shigeo Matsuda(1); Gang Wang(1); Martin Maier(1); Bo Peng(1); Marjorie Solomon(1); Sergey Shulga-Morskoy(1); Klaus Charrise(1); David Bumcrot(1); Kristina Yucius(1); Victor Kotelianski(1); G. Rajeev Kallanthottathil(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

<tt>Cells and tissues have specific transport systems for nutrients like vitamins. Covalent conjugation of vitamin ligands will facilitate the delivery of siRNAs into cells having corresponding receptors and will enhance silencing of endogenous genes in vivo. Folate receptor/folic acid is a well-studied system with an affinity of 10-10 M. The folate receptor is over-expressed in many types of tumor cells and in activated macrophages—cell types of relevance for disease treatment. Folic acid is an attractive choice for conjugation due to lack of immunogenicity and unlimited availability. Although folic acid has been employed as a targeting moiety for small molecule drugs and diagnostic agents, efficient synthetic methods to conjugate folic acid analogs to siRNAs were lacking. Part of the challenge arose due to the multiple functional groups in folic acid and incompatibility of deprotection conditions after automated RNA synthesis. We have prepared suitably protected folic acid phosphoramidites and solid supports with a variety of linkers. Using these building blocks, significant quantities of folic acid conjugated siRNAs were synthesized. Synthesis, characterization, in vivo stability and uptake properties of these conjugates will be presented. </tt>

CARB 46

Combining 2'-TBDMS and 2'-ALE chemistry for on-column site specific modifications of RNA

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Jeremy G Lackey(1), [email protected], 801 Sherbrooke St. West, Montreal Quebec H3A 2K6, Canada ; Masad J Damha(1); Richard Johnsson(1). (1) Chemistry, McGill University, Montreal Quebec H3A 2K6, Canada

The celllular delivery and stability of siRNA must be overcome before it's therapeutic potential can be realized. Modifications at the 2'-position have been used extensively in this regard. Traditionally, 2'-modified nucleoside phosphoramidites are synthesized via a multi-step synthetic route and then incorporated in the oligonucleotide chain. This process can be tedious and expensive. Here in, we will describe a method for on-column site specific 2'-modification. Various 2'-moieties will be introduced through the combination of 2'-ALE and 2'-TBDMS protecting groups. The characterization and biological activity of these molecules will also be discussed.

CARB 47

Synthesis and evaluation of bicyclic ketal-based cationic lipids for the delivery of siRNA via lipid nanoparticle delivery systems

Muthusamy Jayaraman(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; David Butler(1); Laxman Eltepu(1); Martin Maier(1); Tom Madden(2); Michael Hope(2); Ying Tam(2); Barbera Mui(2); Akin Akinc(1); Soma De(1); G. Rajeev Kallanthottathil(1); Muthiah Manoharan(1); Andrew Sprague(1). (1) Alnylam Pharmaceuticals, United States (2) AlCana Technologies, Canada

The discovery of RNA interference (RNAi) as a means to silence expression of specific genes is potentially a great boon to modern medicine. Synthetic small interfering RNAs (siRNAs) are powerful agents for specifically eliciting the cleavage of mRNAs. A critical hurdle in realizing therapeutic application of siRNA is it's cytosolic delivery across cellular membranes in vivo. Additionally, when administered systemically in unprotected form, nucleic acids suffer from poor pharmacokinetics due to rapid degradation by serum nucleases. Of methods tested so far, liposomal formulation has proven most successful in vivo.1,

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2,3 When administered intravenously in the form of an encapsulated lipid nanoparticle (LNP), siRNAs are delivered to the liver resulting in specific knockdown of hepatocytic targets. To identify the optimal LNPs for the delivery of siRNAs, cationic lipids derived from endo- and exo- cyclic aminodiols were evaluated. In this presentation we discuss design, synthesis and evaluation of the structure activity relationship of these cationic lipids in systemic delivery of siRNA.

CARB 48

Development of a stability-indicating, ion-pair RP-HPLC method for separation and quantitative determination of two siRNA duplexes in a liposome

Veeravagu Murugaiah(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; William Zedalis(1); Gary Lavine(1); Klaus Charrise(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

We have developed a stability-indicating, ion-pair RP-HPLC method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The first phase of the mobile phase system consisted of 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 5% methanol; the second consisted of 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 90% methanol. The column used was an XBridge C18 column (50 mm x 2.1 mm i.d., 2.5 µm particle size) and separation was performed at 60°C. Quantitation was achieved with UV detection at 260 nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110 µg/mL. Accuracy of the method was determined by replicate analysis (n=5) at four concentrations (R2 > 0.996 and RSDs of 1 – 4%). Use of an ion pairing reagent compatible with mass spectrometric detection makes this method amenable to LC-MS impurity profiling.

CARB 49

Novel applications of aerosol-based detectors for the analysis of non-chromophore, multi-lipid, drug delivery vehicles

William Zedalis(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Matthias Kretschmer(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

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Analytical quantification of Lipid Nanoparticles (LNPs), a drug delivery vehicle consisting of multiple lipids, is challenging due to the lack of UV active chromophores on the constituents. A comparison employing three forms of aerosol-based detection for liquid chromatography was performed using evaporative light-scattering detection (ELSD), charged aerosol detection (CAD), or condensation nucleation light-scattering detection (CNLSD). A summary of our findings will be presented.

CARB 50

Optimizing the LAL assay for detection of bacterial endotoxin in conjugated and formulated siRNAs

Mara Broberg(1), [email protected], 300 3rd St., 3rd Floor, Cambridge MA, United States ; Kathy Mills(1); Klaus Charisse(1); William Zedalis(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

Endotoxins, part of the outer cell wall of Gram negative bacteria, potently stimulate the innate immune response. The limulus amoebocyte lysate (LAL) test is the most widely used assay for detection of endotoxin and is an FDA requirement for testing of parental drug product safety. We used a commercially available LAL assay to detect and quantitatively determine the concentration of endotoxin in various conjugated siRNAs and liposome-formulated siRNAs. However, the lipids present in these various conjugates and formulations presented some challenges for the accurate determination of endotoxin levels. We developed some solutions to overcome these problems and summary of our analytical processes will be presented.

CARB 51

Conjugation strategies for RNAs using copper-catalyzed click chemistry

Chang Geng Peng(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Takeshi Yamada(1); Shigeo Matsuda(1); Haripriya Addepalli(1); Rowshon Alam(1); Narayanannair Jayaprakash(1); Muthusamy Jayaraman(1); David Butler(1); Rajendra Pandey(1); Kathy Mills(1); Martin Maier(1); Klaus Charrise(1); G. Rajeev Kallanthottathil(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

To improve the cellular uptake of siRNAs, we conjugated lipophilic molecules,

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carbohydrates, and polyamines to appropriate sites of RNA molecules. A simple approach to achieve conjugation is via “click” chemistry involving the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). We have achieved efficient synthesis of various conjugates by incorporating alkynes at different sites of the sugar residue of the ribonucleoside and reacting them with the desired ligands carrying an azido group. A summary of these synthetic processes are presented.

CARB 52

Non-nucleoside building blocks for copper-assisted and copper-free click chemistry for synthesis of oligonucleotide conjugates

K.N. Jayaprakash(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Chang G. Peng(1); Takeshi Yamada(1); David Butler(1); G Rajeev Kallanthottathil(1); Martin Maier(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

Cu(I)-mediated Huisgen 1,3-dipolar [3 + 2] cycloadditions between azides and alkynes, popularly known as “click reactions,” have been used for the conjugation of number of biomolecules. We developed several non-nucleoside alkyne building blocks suitable for click chemistry based on hydroxyprolinol (see below) One of the main issues in using copper mediated click chemistry for oligonucleotides and other bio-molecular conjugation is that trace amounts of potentially cytotoxic copper present in the product must be removed if these molecules are intended for therapeutic applications. Here, we report the use of copper-free click chemistry for the synthesis of oligonucleotide conjugates. Incorporation of various ligands into oligonucleotides in solution and solid phase will be presented.

CARB 53

Solid-support immobilized, reusable Cu (I) catalyst for "click reactions" of oligonucleotides with ligands

Laxman Eltepu(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Chang G. Peng(1); K. Narayanannair Jayaprakash(1); Takeshi Yamada(1); Muthusamy Jayaraman(1); G. Rajeev Kallanthottathil(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

Cu(I)-catalyzed Huisgen's 1,3-dipolar cycloaddition reaction, often referred as the “Click-reaction,” is often used in the bioconjugation of macromolecules due to its selectivity and

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ease. One of the main limitations of this approach is that trace amounts of cytotoxic Cu ions contaminate the products. Here, we report the synthesis of a Cu-catalyst on solid support and its application in “Click reactions” between small molecules and oligonucleotides. Use of this novel catalyst resulted in an experimentally simplified work-up protocol and prevented the leaching of the Cu metal. The catalyst could be re-cycled providing an environmentally clean process. The application of this novel Cu complex in “Click reactions” of alkyne- and azide-bearing oligonucleotides or functionalized small molecules will be presented.

CARB 54

Synthesis of oligo spermine-containing oligonucleotides for siRNA delivery

Shigeo Matsuda(1), [email protected], 300 3rd St, 3rd Floor, Cambridge MA, United States ; Gang Wang(1); Ligang Zhang(1); Tianlei Lei(1); Rowshon Alam(1); Chang G Pang(1); K. N. Jayaprakash(1); Takeshi Yamada(1); David Butler(1); Maria Frank-Kamenetsky(1); Martin Maier(1); Klaus Charrise(1); Kevin Fitzgerald(1); G. Rajeev Kallanthottathil(1); Muthiah Manoharan(1). (1) Alnylam Pharmaceuticals, United States

To improve the cellular delivery of siRNAs, we synthesized building blocks of multiunits of spermine derivatives and incorporated them into RNA oligonucleotides using phosphoramidite chemistry. Spermine-containing oligonucleotides were also synthesized by post-synthetic 'click' conjugation. A summary of these synthetic efforts for polyamine-conjugated siRNA synthesis are presented.

CARB 55

Allosteric modulation of DNA by minor groove binding polyamides

David M Chenoweth(1), [email protected], 77 Massachusetts Avenue, Bldg. 18-053, Cambridge MA 02139, United States ; Peter B Dervan(1). (1) Chemistry and Chemical Engineering, California Institute of Technology, Pasadena CA 91125, United States

Many human diseases are caused by dysregulated gene expression. The oversupply of transcription factors may be required for the growth and metastatic behavior of human cancers. Cell permeable small molecules that can be programmed to disrupt transcription factor-DNA interfaces could silence aberrant gene expression pathways. Pyrrole-imidazole polyamides are DNA minor groove binding molecules that are programmable for a large repertoire of DNA motifs. Atomic resolution X-ray crystal structures of Py/Im polyamides bound to DNA reveal a large widening of the minor groove and compression of the major groove

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along with bending of the helix axis toward the major groove. These allosteric perturbations of the DNA helix provide a molecular basis for disruption of transcription factor-DNA interfaces by small molecules, a minimum step in chemical control of gene networks.

CARB 56

Strong and selective molecular recognition of the DNA minor groove: Compound and DNA chemistry and unusual conformational matching

W. David Wilson(1), [email protected], 50 Decatur St, NSC, Atlanta GA 30303, United States ; Rupesh Nanjunda(1); Arvind Kumar(1); Manoj Munde(1); Yang Liu(1); Rebecca Hunt(1); David W. Boykin(1). (1) Department of Chemistry, Georgia State University, Atlanta GA, United States

A number of organic cations recognize different DNA base sequences through minor groove complexes and have biological activity against diverse cells from cancer to infectious disease organisms. Although most classical minor groove binding agents specifically interact with AT base pairs, a combination of directed synthesis with a thorough set of biophysical methods has allowed us to discover new types of cooperative binding modes that provide for GC base pair recognition. The GC recognition modes involve a surprising stacking of dications that create an unprecedented tetracationic minor groove complex. Although design of minor groove binding agents to this time has been based on a restricted set of curvature and functional group position rules, we now realize that there are a number of other ways to target the groove. An overview of these new types of DNA complexes will be presented and contrasted with the classical AT specific complexes. Supported by NIH

CARB 57

Toward DNA recognition by a Janus Wedge approach

Larry W McLaughlin(1), [email protected], 2609 Beacon St, Chestnut Hill MA 02467, United States ; Ayan Pal(1); Han Chen(1); Meena Meena(1). (1) Department of Chemistry, Boston College, Chestnut Hill MA 02467, United States

Janus was the Roman god of beginnings and endings who is typically pictured with two faces. DNA recognition by a Janus-Wedge format involves the "wedge residue" inserting itself between the nucleobases of the Watson-Crick target and forming a DNA triplex. The J-W residues in the Janus Wedge triplex are composed of two hydrogen-bonding faces such that after insertion into the DNA duplex, typically through the major groove, the J-W residues are hydrogen-bonded to both W-C faces of the target base pair. The product three-stranded complex contains significantly more interstrand hydrogen bonds than does the target W-C DNA duplex, and should be favored thermodynamically. A key

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component of the recognition motif is to design residues that can discriminate all four base pairs, particularly A-T from T-A and G-C from C-G. Toward this end asymmetric residues will be described, each designed for recognition of one base pair.

CARB 58

Recognition of DNA major groove

Dev P Arya(1), [email protected], 461 Hunter, Clemson SC, United States ; Liang Xue(1); Sunil Kumar(1). (1) Chemistry, Clemson University, Clemson SC 29634, United States

Sequence-specific recognition of duplex DNA can by achieved by small molecules using polyamides that target the DNA minor groove. The major groove of DNA is much richer in information content and a number of DNA-protein interactions take place in the major groove. In this seminar, I will present our results from efforts in targeting the DNA major groove. Our efforts towards the recognition of A and B form DNA will be presented.

CARB 59

Identification and cleavage site analysis of DNA sequences bound strongly by bleomycin

Sidney M Hecht(1), [email protected], Biodesign Institute, Tempe AZ 85287, United States . (1) Center for BioEnergetics, Biodesign Institute, Arizona State University, Tempe AZ 85287, United States

A hairpin DNA library containing a randomized region was used to identify DNAs that bound strongly to bleomycin. Several strongly bound hairpin DNAs were identified and sequenced, and then used to characterize bleomycin-DNA interaction. Sequence analysis indicated that the randomized regions did not contain disproportionate numbers of 5'-GT-3' or 5'-GC-3' sequences, i.e. the DNA sequences cleaved predominantly when excess bleomycin is employed with arbitrarily closen DNAs. Further, the cleavage of these DNAs by Fe-bleomycin also involved atypical sequences, and involved a much larger proportion of alkali labile lesions than is observed when using excess bleomycin and arbitrarily chosen DNAs. The implications of these observations will be discussed.

CARB 60

Synthesis and characterization of chain-end functionalizable glycopolymer and its oriented glyco-macroligand formation

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Satya Nandana Narla(1), [email protected], 2121 Euclid Ave, Cleveland Ohio 44115, United States ; Xue-Long Sun(1). (1) Department of Chemistry, Cleveland State University, Cleveland Ohio 44115, United States

Cell surface carbohydrates act as receptors for a variety of protein ligands and thereby play a significant role in a wide range of biological processes. Therefore, carbohydrate binding interaction by protein has provided a starting point for the development of new therapies and a new protein and cell isolation and targeting concept in biological research and applications. We report a facile synthesis of α,ω-orthogonally functionalizable glycopolymer and its oriented glyco-macrolignand formation for rapid purification and identification of carbohydrate-binding protein applications. Specifically, α-biotin, ω-O-cyanate chain end functionalized glycopolymer was synthesized via cyanoxyl-mediated free-radical polymerization in one-pot fashion. Oriented glyco-macroligand formation and its glyco-affinity capturing of proteins were demonstrated by combining α-biotin end-terminated glycopolymer with streptavidin modified magnetic beads and ω-O-caynate end-terminatedglycipolymer with amine modified silica gel, respectively. The one-pot synthetic approach is expected to open a versatile pathway towards heterotelechelic (bio) functionalized polymers with defined chain end groups. The α,ω-orthogonally functionalizable glycopolymer can be used for both oriented polymer-protein conjugation and polymer surface immobilization for protein drug development and biosensor applications.

CARB 61

Separation and characterization of the glycosaminoglycan components of proteoglycans

Mellisa Ly(1), [email protected], 100 Nyroy Dr. Apt. 5, Troy NY 12180, United States ; Tatiana N Laremore(2); Kemal Solakyildirim(1); Robert J. Linhardt(1)(3)(4)(2). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY 12180, United States (3) Department of Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (4) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Proteoglycans (PGs) are a diverse group of glycoconjugates constituted by various core proteins post-translationally modified with linear, anionic glycosaminoglycans (GAGs) that consist of repeating disaccharide building blocks. Characterization of the GAG component of PGs is important because GAGs often dominate the physical, chemical, and biological properties of PGs. The structure of the chondroitin sulfate GAG chains of bikunin and decorin is investigated. Intact bikunin and decorin GAGs in a narrow molecular weight range were separated by continuous-elution preparative PAGE are analyzed using Fourier transform mass spectrometry. A method to elucidate the varying

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sulfated domains for GAG chains of bikunin and decorin are developed using a combination of continuous-elution preparative PAGE, mass spectrometry, and GAG degrading enzymes, such as hyaluronidase (Streptococcus dysgalactiae) and exolytic chondroitin lyase II (Arthrobacter aurescens). Initial results indicate that there are regions of highly sulfated domains and nonsulfated domains.

CARB 62

Synthesis of nucleotide activated L-sugars from polyols via bioconversion

Ryan D Woodyer(1), [email protected], 801 W Main St, Peoria IL 61606, United States . (1) zuChem, Inc., Chicago IL 60612, United States

Many biologically active natural products owe their bioactivity at least in part to glycosylation. However, no commercially viable biological system exists for site-specific attachment of L-sugar or modified-sugar monomers. We are developing methods for the production of activated l-sugars (in addition to other modified sugars) from inexpensive polyols using a combination of whole cell bioconversion and purified biocatalysts. L-Sugars, namely L-ribose, L-xylose, L-fucose, L-galactose, L-gulose, were prepared from their inexpensive polyol counterparts using a recombinant whole cell E. coli biocatalyst containing and engineered polyol-1-dehydrogenase. Converting these L-sugars into nucleotide activated sugars is achieved using thermostable engineered biocatalysts, sugar-1-kinase and nucleotidyltransferase. Greater than 75% conversion to the corresponding sugar-1-P was achieved for L-glucose, L-arabinose, L-xylose, L-rhamnose, L-mannose, and 6-Azido-D-galactose using an engineered sugar-1-kinase. Gram scale synthesis of L-sugar-1-phosphates has been demonstrated with a space-time yield of 200 g/L*d. Some activated sugars have been prepared using a nucleotidyltransferase and engineering to improve this step is currently underway.

CARB 63

Glycomorphology of the pulmonary vasculature: Endothelial cell glycocalyx and endothelial barrier function

Eugene Cioffi(1), [email protected], 307 University Blvd. N., College of Medicine; MSB 3370, Mobile AL 36688-0002, United States . (1) Department of Pharmacology, University of South Alabama, Mobile AL 36688-0002, United States

The endothelium forms a semi-permeable barrier between constituents of the blood and underlying tissue, consisting of glycoproteins, glycolipids and proteoglycans which coat the cell surface. The carbohydrate network that contributes to the glycocalyx is very complex, and its role in endothelial barrier function is poorly understood.

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We have probed the molecular identities of endothelial cell surface carbohydrates using fluorescently-tagged lectins in a systematic way, revealing the presence of α-2,3-Gal and α-2,6-Gal/GalNAc terminal sialic acid residues that differed dependent upon the cell type. Overall a dramatic loss of cell-cell and cell-matrix adhesion was noted by the formation of large inter-endothelial cell gaps subsequent to a dose-dependent treatment with neuraminidases. In addition, endothelial barrier integrity was also evaluated using ECIS™ experiments, which correlate intracellular resistance with barrier function. Overall these observations suggest that the glycocalyx of endothelial cells contain terminal sialic acid residues that play a significant role in endothelial barrier function.

CARB 64

Multidimensional glycan arrays for enhanced lectin and antibody profiling

Yalong Zhang(1), [email protected], 376 Boyles Street, Frederick Md 21702, United States ; Qian Li(1); Christopher Campbell(1); Jeffrey Gildersleeve(1). (1) National Cancer Institute, Frederick MD 21702, United States

Carbohydrate-protein interactions are important in many biological processes. Multivalency is critical in the formation of a high avidity multivalent complex between carbohydrate ligands and the protein. Since many combinations of the structure, density, spacing and orientation of carbohydrate ligands are to be considered, identifying multivalent inhibitors and probes has been proven challenging. Here, we develop a new format array via spacing neoglycoproteins apart with unconjugated BSA. The array is composed of 591 combinations of glycan structure and presentation. One application of the array was identifying inhibitors for five lectins. A key advantage is that multivalent inhibitors can be identified independent of the structure information. The other application was focus on the effects of neoglycoprotein density on antibody binding. We tested monoclonal antibodies, serum antibodies and serum antibodies induced by a prostate cancer vaccine. It was found that subpopulation antibodies could be distinguished, which could not have been detected otherwise.

CARB 65

Peptide nucleic acids bind strongly and sequence selectively to double helical RNA

Thomas T Zengeya(1), [email protected], 4400 Vestal Parkway East, Binghamton New York 13902-6000, United States ; Ming Li(1); Eriks Rozners(1). (1) Chemistry, State University of New York at Binghamton, Binghamton New York 13902, United States

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Non-coding RNAs play a central role in gene expression, making them an attractive target for molecular recognition. However, because of RNA's conformational flexibility and the non-selective electrostatic interactions dominated by negatively charged phosphate backbone, discovery and designing of molecules that bind selectively to biologically and therapeutically relevant RNAs has been slow. Currently, most drugs that target RNA use hydrophobic and electrostatic interactions to achieve shape selective RNA recognition. For instance, ribosomal RNA is a receptor for antibiotics. However, while many molecules exhit high binding affinity, their selectivity is typically very low. In this presentation, we report that Peptide Nucleic Acids (PNA) bind strongly and sequence selectively to RNA duplexes. Our data suggests that the binding mode is formation of a Hoogsteen triple helix. In contrast to DNA, RNA triple helices have not been extensively studied. Moreover, stable triple helix formation between PNA and RNA has not been previously reported. Our results suggest that relatively small PNA analogues may be designed to recognize short homopurine stretches that are common in non-coding RNAs. It is conceivable that optimization of PNA properties using chemical modifications may provide a novel way to interfere with the function of non-coding RNAs.

CARB 66

Post surface function method for preparation of liposomal glyco-conjugates

Hailong Zhang(1), [email protected], 2121 Euclid Avenue, SI 414, Cleveland OH 44115, United States ; Yong Ma(1); Xue-Long Sun(1). (1) Department of Chemistry, Cleveland State University, Cleveland OH 44115, United States

Cell surface glycans have been attractive biomimetic targets for potential biomedical applications since they act as receptors for a variety of protein ligands and are involved in a wide range of biological processes. Liposome, a spherical closed self-assembled lipid, has been extensively studied as model of cell membrane and mostly as carrier for drug/gene delivery application. Liposome surface functionalization with carbohydrate has been a versatile membrane mimetic approach and facilitates enormous potential applications of liposomes such as for glyco-model system, targeted drug and gene delivery as well as multivalent inhibitors. Conventional methods such as using amide or thiol-maleimide coupling as well as by imine or hydrazone linkage are less efficient and harmful for integrity and thus limit the liposome's biological application. Herein, we developed an efficient and chemoselective liposome surface glyco-functionalization method based on Staudinger ligation, in which carbohydrate derivative carrying a spacer with azide is conjugated onto the surface of preformed liposomes carrying a terminal triphosphine in PBS buffer (pH 7.4) and at room temperature. Specifically, by

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using lactose and heparin as the model of carbohydrates, the effect of reaction conditions on integrity and stability of liposome was investigated by dynamic light scattering and the leakage of entrapped 5,6-carboxyfluorescein, respectively. Furthermore, the density and accessibility of grafted carbohydrate residues on the liposome surface were evaluated. The high specificity and high yield as well as biocompatible reaction condition natures of the Staudinger ligation approach make it an attractive alternative to all current protocols for liposome surface functionalization.

CARB 67

Approach to study carbohydrate-carbohydrate interactions involved in myelin using glycolipids assay in microtiter plate

Jingsha Zhao(1), [email protected], 324 Brook St., Providence RI 02912, United States ; Amit Basu(1). (1) Department of Chemistry, Brown University, Providence RI 02912, United States

Glycolipid assay in the microtiter plate has been utilized to mimic the carbohydrate-carbohydrate interaction involved in myelin. As a model study, carbohydrate-lectin interaction was investigated with fluorescent lectins first. To study the carbohydrate-carbohydrate interaction, fluorescent silica nanoparticles, which have been functionalized with carbohydrates using the copper promoted azide-alkyne cycloaddition, was employed. The interaction between glycolipid in the plate and the carbohydrate on the surface of the fluorescent silica nanoparticles was elucidated by fluorescence spectrometer.

CARB 68

WITHDRAWN

CARB 69

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Synthesis and characterization of protein glycopolymer conjugate

Valentinas Gruzdys(1), [email protected], 2121 Euclid Avenue, SR 382, Cleveland Ohio 44115-2214, United States ; Xue-Long Sun(1). (1) Department of Chemistry, Cleveland State University, Cleveland Ohio 44115, United States

Covalent attachment of synthetic macromolecules is an effective way to improve protein stability with reduced immunogenicity and extended plasma half-lives. In addition, secondary property can be introduced, such as for protein targeted delivery. Glycoengineering aimed adding carbohydrates to proteins to alter their pharmacokinetic properties such as increasing in vivo activity and prolonging the duration of action have been a promising approach for protein therapeutics. In this presentation, a facile synthesis of chain end-functionalized glycopolymer for well-defined protein glycopolymer conjugation is reported. Briefly, an O-cyanate chain end-functionalized glycopolymer presenting multiple copies of lactose epitope units was synthesized via cyanoxyl-mediated free-radical polymerization in one-pot fashion. Bovine serum albumin (BSA) was employed as amine-containing model protein for conjugation reaction, and the resulting protein-glycopolymer conjugate was characterized by SDS-PAGE. The versatility of the synthetic strategy presented in this work was the oriented multivalent carbohydrate modification of protein in straightforward approach and in aqueous conditions.

CARB 70

Robust analytical development for oligonucleotide manufacture

Ipsita Roymoulik(1), [email protected], 155 Fortune Boulevard, Milford MA 01757, United States . (1) Analytical Development, Avecia Oligomedicines, Milford MA 01757, United States

A general overview of the role of Analytical Development in Oligonucleotide manufacture will be provided. Analytical techniques utilized to control the quality of oligonucleotides through robust purity and assay methods during in-process and QC release testing will be emphasized through understanding of typical oligonucleotide physico-chemical properties. The different categories of specifications and approaches to setting those specifications for critical quality attributes using risk and science based analytics will also be discussed. To ensure a back-up supply oligonucleotide therapeutic, sponsors require API through Drug Product quality being assessed at multiple laboratories and on multiple instrument platforms. Some of the key challenges due to the resulting analytical variability encountered during method qualifications and subsequent

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validations will also be discussed through traditional versus statistical approaches.

CARB 71

Glycomics studies on central nervous system

Fuming Zhang(1), [email protected], 110 8th St., Troy NY 12180, United States ; Zhenling Liu(1); Kemal Solakyildirim(1); Dennis Pu(1); Robert J. Linhardt(1). (1) Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Glycosaminoglycans (GAG) are a family of highly sulfated, complex, polydisperse linear polysaccharides that display a variety of important biological roles. They are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth. GAGs from swine brains and spinal cords were isolated and purified with a procedure including defat, proteolysis, anion-exchange chromatography; and methanol precipitation. The isolated GAGs were subjected to carbazole assay to quantify the amount of GAG in each tissue and polyacrylamide gel electrophoresis (PAGE) analysis. The disaccharide composition of chondroitin sulfate and heparan sulfate from the GAGs were determined using liquid chromotography and mass spectrometry (LC-MS). 1H NMR spectroscopy was also used to characterize their structure. Finally the GAGs were biotinylated and immobilized on BIAcore SA biochips. The interactions between the GAGs and proteins related to central nervous system (such as midkine, brain-derived neurotrophic factor (BDNF) and Slit) were investigated using surface plasmon resonance (SPR).

CARB 72

Computational studies on the interactions of mannose with DOPC and POPC phospholipids

Parthasarathi R(1), [email protected], [email protected], T6, MS K710, Los Alamos NM 87545, United States ; Gnanakaran S(1). (1) Theoretical Biology & Biophysics Group, Los Alamos National Laboratory, Los Alamos NM 87545, United States

Many pathogen induced potential causative immune responses are determined by the interaction of a virulence factor containing carbohydrates with membranes, including Mycobacterium tuberculosis in which its cell-wall glycolipid lipoarabinomannan (LAM), appear to be the most potent non-peptidic molecule to modulate the host immune response. In this study we seek a basic understanding of the nature of interactions between mannose and membranes since the initial molecular recognition of mannose molecules capping the LAM at

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the host membrane is a critical step in the origin of pathogenesis. Relationships between physicochemical characteristics of mannose and its interactions with DOPC and POPC lipid molecules are investigated using density functional theory with 6-311++(d,p) basis sets. Carbohydrate-lipid interactions are probed with respect to competing hydrogen bonds to water. Based on these studies, relative strengths of mannose-water, lipid-water and mannose-lipid interactions are identified. Finally, we show that non-covalent interactions beyond hydrogen bonding may influence the stabilization of mannose at the membrane-water interface.

CARB 73

Optimizatin of analysis of glycosaminoglycans in biological samples

Boyangzi Li(1), [email protected], 110 8th Street, Troy NY 12180, United States ; Robert J Linhardt(1). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States

As an important component of biological polysaccharides, glycosaminoglycans (GAGs) function as molecular co-receptors in numerous biological activities, such as cell–cell interaction, cell adhesion and migration, cell signaling, cell growth and differentiation. They mainly locate on the external side of cell membranes and contribute to the composition of extracellular matrix. Highly charged heterogeneous structure as they possess the sequence and composition of GAGs in different biological samples and the same biological samples under different conditions are always in the interests of researchers to understand glycobiology. Here we present our work in optimization of the composition of GAGs from biological samples—from improving extraction method to using different techniques for characterization, including reversed-phase ion-pair chromatography (R-IPC) coupled with electrospray ion mass spectroscopy (ESI-MS), strong anion exchange chromatography (SAX), capillary electrophoresis (CE) and introducing isotopically labeled disaccharide standards as internal standards for R-IPC coupled with ESI-MS.

CARB 74

Conformational analysis of nucleosides and nucleotides: PSEUROT 2010

Steven M. Graham(1), [email protected], 8000 Utopia Parkway, Queens NY 11439, United States . (1) Department of Chemistry, St. John[apos]s University, Queens NY 11439, United States

The biological activity of nucleosides and nucleotides is intimately tied to their conformation equilibrium. Most nucleosides and nucleotides exhibit a two-state 'north-south' equilibrium. In this formalism, 'north' describes a nucleos(t)ide where C2' is puckered 'down' and C3' is puckered 'up'; 'south' describes a

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nucleos(t)ide where C2' is puckered 'up' and C3' is puckered 'down'. The PSEUROT program allows a user to input 1H-1H coupling constants, obtained from NMR spectroscopy, and to obtain as output the ratio of north/south conformers that would have produced the observed coupling constants. The original PSEUROT program, to our knowledge, has not been updated in a systematic fashion in the 30 years subsequent to its first description. This talk will focus on the merits of the use of molecular mechanics (AMBER) methods versus ab initio methods to update the PSEUROT program.

CARB 75

Role of DNA topography in recognition by proteins and small molecules

Thomas D. Tullius(1), [email protected], 590 Commonwealth Avenue, Boston MA 02215, United States . (1) Department of Chemistry and Program in Bioinformatics, Boston University, Boston MA 02215, United States

In the age of genomics, DNA is most often depicted as a string of letters. While this is useful for representing the large amount of information encoded in a genome, the underlying molecular nature of DNA is obscured. Readout of genetic information is based on protein binding to specific sites in genomic DNA, but proteins cannot "read" DNA letters – they discriminate between potential DNA binding sites via the principles of molecular recognition. To introduce a structural dimension to genome analysis, we developed a database of DNA structural patterns, ORChID, based on hydroxyl radical cleavage of DNA. We used ORChID to produce a topographical map of the variation in DNA structure throughout the human genome, at single-nucleotide resolution. I will present recent work in which we use ORChID to assess how DNA topography contributes to the binding of proteins and small molecules to DNA.

CARB 76

Selective modulation of DNA polymerase activity by fixed-conformation nucleoside analogs

Martin Egli(1), [email protected], School of Medicine, 607 Light Hall, Nashville Tennessee 37232-0146, United States ; Robert L. Eoff(1); Victor E.

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Marquez(2); F. Peter Guengerich(1). (1) Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville Tennessee 37232, United States (2) Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, NIH, Frederick Maryland 21702, United States

The ability to selectively modulate the activity of individual DNA polymerases (pols) or specific classes of pols is an attractive strategy for achieving therapeutic benefits such as anti-microbial activity. We have investigated the potential by various pols to selectively incorporate conformationally constrained dATP analogues with bicyclo[3.1.0]hexane (methanocarba-) pseudosugar moieties that are locked into either the N- or S-orientation. All pols tested showed a preference for the N- over the S-conformer, but with variable levels of inhibition relative to native dATP. Surprisingly, human pol iota showed increased activity towards the N-oriented conformer compared with dATP. Most notably, human pol eta was capable of inserting both conformers and is quite proficient at extension from the S-oriented conformer, in contrast to other polymerases tested. Our results expand upon the idea that individual pols utilize distinct mechanisms to achieve the same end and further our ability to develop novel nucleoside analogues that target specific enzymes. This work was supported by US National Institutes of Health grants R01 ES010375 (F.P.G.), F32 CA119776 (R.L.E.), P30 ES000267 (F.P.G., M.E.), and P01 ES05355 (M.E.).

CARB 77

Structural mechanisms underlying DNA replication and human DNA mismatch repair

Lorena S. Beese(1), [email protected], Nanaline Duke Bld., Rm 134; Research Dr.; Box 3711, Durham NC 27710, United States ; Jillian Orans(1); Weina Wang(1); Elizabeth McSweeney(1); Paul L Modrich(1); Quincy Tseng(1). (1) Department of BIochemistry, Duke University Medical Center, Durham NC 27705, United States

Accurate DNA replication and repair are fundamental requirements for genomic stability. DNA polymerases achieve remarkably high replication fidelity, often allowing only a single insertion error for every 10,000 incorporated and additionally discriminating nearly a 1000- fold in favor of the 2'-deoxyribonucleotide triphosphate (dNTP) compared to ribonucleotide triphosphate (rNTP). High-resolution crystal structures of DNA polymerase - substrate complexes provide new insights into mechanisms of nucleotide discrimination. Polymerase complexes with mismatched DNA offer insight into a mechanisms for spontaneous mutation. DNA mismatch repair (MMR) further enhances the fidelity of replication, and initiates an apoptotic response to certain classes of DNA damage. Crystal structures of human MMR proteins MSH2-

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MSH6, MSH2-MSH3, and exonuclease I bound to heteroduplex DNA lesions provide further insight into MMR and DNA recognition.

CARB 78

Manipulating the electrostatic potential in the DNA grooves: Effect on thermodynamic stability, ion binding, hydration and structure

Barry Gold(1), [email protected], 512 Salk Hall, Pittsburgh PA 15216, United States . (1) Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh PA 15216, United States

Cations, which sequence-selectively associate with DNA in both the major and minor grooves, play a significant role in determining DNA conformation and stability. In the major groove, cations are associated with the N7/O6 edge of guanine, while in the minor groove they are found at A-T pairs. Modification of these potential cation binding sites should result in the reorganization of salts and water within the grooves, which in turn would affect local conformation and stability. We report the biophysical characterization of DNA duplexes in which we altered the N-7 position of purines (major groove) and the N-3 position of adenine (minor groove). These modifications either specifically eliminate a natural DNA cation binding sites or introduce a stable cationic appendage to mimic a high occupancy cation binding site. Interpretation of how these major and minor modifications affect DNA structure and stability, and potentially protein recognition of DNA, will be discussed.

CARB 79

Sequence-dependent recognition of minor groove width

Barry Honig(1), [email protected], 1130 St. Nicholas Ave, New York New York 10032, United States . (1) Center for Computational Biology and Bioinformatics and Department of Biochemistry and Molecular Biophysics, Columbia University and Howard Hughes Medical Institute, New York New York 10032, United States

By comprehensively analyzing the three dimensional structures of protein-DNA complexes, we show that the binding of arginines to narrow minor grooves is a widely used mode for protein-DNA recognition that is distinct from previously defined readout mechanisms. Minor groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. The biophysical basis of this recognition mechanism involves an enhancement of the negative potential of DNA, which is strongly correlated with minor groove width. Strikingly, arginines are frequently located at local minima in width and potential. These findings suggest that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for

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the formation of base-specific hydrogen bonds, to achieve DNA binding specificity.

CARB 80

Glycosylation of substituted 4H-1,2,4-triazole-3-thiol

El Sayed H. El Ashry(1), [email protected], Faculty of Science, Alexandria Alexandria 31213, Egypt . (1) Chemistry Department, Alexandria University, Alexandria 31213, Egypt

Glycosylsulfanyl-heterocycles have attracted much attention because of their biological activity and in particular inhibition of the activity of enzymes. Reaction of 5-substituted-4H-1,2,4-triazole-3-thiol with glycosyl halides in presence of triethylamine gave the S-glycosides whereas the respective S,N4-bis(glycosyl) derivatives were synthesized in the presence of potassium carbonate. The S,N2-bis(glycosyl) isomer could also be isolated in minor amounts in some cases. MWI led to higher yields in much less time than the conventional methods and no change in regioselectivity has been noticed.

CARB 81

Expression and characterization of enzymes for bioenzymatic synthesis of heparin

Priscilla Paul(1), [email protected], 110 8th Street, Troy NY 12180, United States ; Wenjing Zhao(1); Robert Linhardt(1); Jonathan Dordick(1); Jian Liu(2). (1) Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) University of North Carolina, United States

The goal of this project is to bioenzymatically synthesize 1 kg of non-animal source heparin that is chemically and biologically equivalent to United States Pharmacopoeia standard heparin. In the biosynthesis, N-sulfated heparosan, produced by E. coli fermentation and subsequent N-deacetylation and sulfation, is modified by several O-sulfotransferases (OST) to produce heparin. 2-OST, 6-OST-1, and 3-OST-1 transfer sulfo groups from 3'phosphoadenosine-5'phosphosulfate (PAPS) to the positions 2-O- of uronic acid, 6-O and 3-O of glucosamine respectively. The expression and characterization of these necessary enzymes are critical to this process. OST have been expressed in E. coli and radiometrically assayed for their activities including 2-OST, 3-OST-1, 6-OST-1, and 6-OST-3. Sulfotransferase activity will be detected using continuous spectrophotometric coupled enzyme assays based on the regeneration of PAPS from desulfated 3'phosphoadenosine-5'phosphate (PAP) by recombinant aryl sulfotransferases. P-nitrophenyl sulfate will be the sulfate donor and visible spectrophotometric indicators of enzyme turnover will be utilized.

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CARB 81

Expression and characterization of enzymes for bioenzymatic synthesis of heparin

Priscilla Paul(1), [email protected], 110 8th Street, Troy NY 12180, United States ; Wenjing Zhao(1); Robert Linhardt(1); Jonathan Dordick(1); Jian Liu(2). (1) Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) University of North Carolina, United States

The goal of this project is to bioenzymatically synthesize 1 kg of non-animal source heparin that is chemically and biologically equivalent to United States Pharmacopoeia standard heparin. In the biosynthesis, N-sulfated heparosan, produced by E. coli fermentation and subsequent N-deacetylation and sulfation, is modified by several O-sulfotransferases (OST) to produce heparin. 2-OST, 6-OST-1, and 3-OST-1 transfer sulfo groups from 3'phosphoadenosine-5'phosphosulfate (PAPS) to the positions 2-O- of uronic acid, 6-O and 3-O of glucosamine respectively. The expression and characterization of these necessary enzymes are critical to this process. OST have been expressed in E. coli and radiometrically assayed for their activities including 2-OST, 3-OST-1, 6-OST-1, and 6-OST-3. Sulfotransferase activity will be detected using continuous spectrophotometric coupled enzyme assays based on the regeneration of PAPS from desulfated 3'phosphoadenosine-5'phosphate (PAP) by recombinant aryl sulfotransferases. P-nitrophenyl sulfate will be the sulfate donor and visible spectrophotometric indicators of enzyme turnover will be utilized.

CARB 82

Catalytic conversion of biomass-derived carbohydrates to useful chemicals in one step

Weiran Yang(1), [email protected], Chemistry Building, #439, University Park PA 16802, United States ; Ayusman Sen(1). (1) Department of Chemistry, The Pennsylvania State University, State College PA 16802, United States

With diminishing fossil fuel reserves, the production of liquid fuels and value-added chemicals directly from biomass is of great current interest. Here, we show that biomass derived carbohydrates such as fructose, glucose, and even cellulose can be catalytically converted to 2,5-dimethyltetrahydrofuran, 2-methyltetrahydrofuran, 2-methyltetrahydropyran, 2-ethyltetrahydrofuran, and 2-methylcyclopentanone in one step. The total yield of these chemicals is relatively high. These chemicals are commercially important as solvents or starting materials. Also, since these liquid chemicals have high energy density and low solubility in water, they can be used as liquid fuels or fuel additives. A multifunctional catalyst composed of a transition metal-based component and an acid was employed.

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CARB 82

Catalytic conversion of biomass-derived carbohydrates to useful chemicals in one step

Weiran Yang(1), [email protected], Chemistry Building, #439, University Park PA 16802, United States ; Ayusman Sen(1). (1) Department of Chemistry, The Pennsylvania State University, State College PA 16802, United States

With diminishing fossil fuel reserves, the production of liquid fuels and value-added chemicals directly from biomass is of great current interest. Here, we show that biomass derived carbohydrates such as fructose, glucose, and even cellulose can be catalytically converted to 2,5-dimethyltetrahydrofuran, 2-methyltetrahydrofuran, 2-methyltetrahydropyran, 2-ethyltetrahydrofuran, and 2-methylcyclopentanone in one step. The total yield of these chemicals is relatively high. These chemicals are commercially important as solvents or starting materials. Also, since these liquid chemicals have high energy density and low solubility in water, they can be used as liquid fuels or fuel additives. A multifunctional catalyst composed of a transition metal-based component and an acid was employed.

CARB 83

Behaviour of polysaccharide aggregates in asymmetrical field-flow fractionation and size-exclusion chromatography

Leena Pitkänen(1), [email protected], P.O. Box 27, Helsinki, Finland ; Maija Tenkanen(1); Päivi Tuomainen(1). (1) Department of Food and Environmental Sciences, University of Helsinki, Helsinki 00014, Finland

Asymmetrical flow field-flow fractionation (AsFlFFF) and high-performance size-exclusion chromatography (HPSEC) are techniques for the separation and characterization of macromolecules; the latter more utilized so far for the analysis of hemicelluloses, such as wood- and cereal-derived xylans. In this paper, we compare the behavior of naturally occurring xylan aggregates in AsFlFFF and HPSEC and their effect on the obtained molar mass distribution and molecular characteristics (molar mass averages, size, intrinsic viscosity). We found out that aqueous xylan solutions contain two types of dense aggregates/molecules: assemblies with size above the size determined for individual xylan chains and some material having very low particle size. Although the amount of aggregates present in xylan solutions is extremely low, their role needs to be understood to avoid erroneous interpretation of AsFlFFF and HPSEC data. Using the data from both separation and detection systems

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complementary information on the solution properties of aqueous polysaccharide solutions could be obtained.

CARB 83

Behaviour of polysaccharide aggregates in asymmetrical field-flow fractionation and size-exclusion chromatography

Leena Pitkänen(1), [email protected], P.O. Box 27, Helsinki, Finland ; Maija Tenkanen(1); Päivi Tuomainen(1). (1) Department of Food and Environmental Sciences, University of Helsinki, Helsinki 00014, Finland

Asymmetrical flow field-flow fractionation (AsFlFFF) and high-performance size-exclusion chromatography (HPSEC) are techniques for the separation and characterization of macromolecules; the latter more utilized so far for the analysis of hemicelluloses, such as wood- and cereal-derived xylans. In this paper, we compare the behavior of naturally occurring xylan aggregates in AsFlFFF and HPSEC and their effect on the obtained molar mass distribution and molecular characteristics (molar mass averages, size, intrinsic viscosity). We found out that aqueous xylan solutions contain two types of dense aggregates/molecules: assemblies with size above the size determined for individual xylan chains and some material having very low particle size. Although the amount of aggregates present in xylan solutions is extremely low, their role needs to be understood to avoid erroneous interpretation of AsFlFFF and HPSEC data. Using the data from both separation and detection systems complementary information on the solution properties of aqueous polysaccharide solutions could be obtained.

CARB 84

Key new observations in the synthesis of thiosialosides

Robert A Falconer(1), [email protected], Richmond Road, Bradford West Yorkshire BD7 1DP, United Kingdom ; Ines F Oliveira(1); Goreti R Morais(1); Bradley R Springett(1). (1) Institute of Cancer Therapeutics, School of Life Sciences, University of Bradford, Bradford BD7 1DP, United Kingdom

The disulfide bond plays an important role in both chemistry and biology, being responsible for the formation of higher order structures in peptides and proteins. Glycosyl disulfides are also useful tools to aid understanding of enzyme systems and are of interest as donors in glycosylation reactions. In our quest to build a library of thiosialosides via the simultaneous selective S-deacetylation of 2-sialyl thioacetate and alkylation, disulfides were observed.

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Further experiments demonstrated that such disulfides resulted from the alkylation of a sialyl acetyl disulfide, identified as a randomly constant by-product during the synthesis of thiosialosides. Formation of this disulfide by-product is extremely variable and dependent on the commercial source of KSAc. We describe our efforts to identify and characterise this by-product, and our efforts towards understanding the mechanism of its formation.

CARB 85

Synthesis and structural optimization of antifungal kanamycin B analogs

Marina Y FOSSO(1), [email protected], 0300 Old Main Hill, Box # 72, Logan UT 84322, United States ; Yukie KAWASAKI(2); Sanjib SHRESTHA(2); Jon TAKEMOTO(2); Tom C.-W. CHANG(1). (1) Department of Chemistry and Biochemistry, Utah State University, Logan UT 84322, United States (2) Department of Biology, Utah State University, Logan UT 84322, United States

Besides their traditional role of antibacterial agents, aminoglycosides, both classical and structurally unusual ones, have been reported to exhibit antifungal activity. This was confirmed as several members of our library of kanamycin B analogs were found to possess significant antifungal activity, notably against Rhodotorula piliminae and Fusarium graminearum, the latter being the major causal agent of wheat head blight and maize ear rot in North America. Based on the leads and following the synthesis of two new kanamycin B analogs, we were able to draw a structure – activity relationship which reveals that attaching an 8-carbon alkyl chain at position O-4´´ of kanamycin B converts it into a fungicide with loss of antibacterial activity. Further effort has then directed to the investigation of the mechanism of action and to the optimization of the synthetic protocol.

CARB 85

Synthesis and structural optimization of antifungal kanamycin B analogs

Marina Y FOSSO(1), [email protected], 0300 Old Main Hill, Box # 72, Logan UT 84322, United States ; Yukie KAWASAKI(2); Sanjib SHRESTHA(2); Jon TAKEMOTO(2); Tom C.-W. CHANG(1). (1) Department of Chemistry and Biochemistry, Utah State University, Logan UT 84322, United States (2) Department of Biology, Utah State University, Logan UT 84322, United States

Besides their traditional role of antibacterial agents, aminoglycosides, both classical and structurally unusual ones, have been reported to exhibit antifungal activity. This was confirmed as several members of our library of kanamycin B

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analogs were found to possess significant antifungal activity, notably against Rhodotorula piliminae and Fusarium graminearum, the latter being the major causal agent of wheat head blight and maize ear rot in North America. Based on the leads and following the synthesis of two new kanamycin B analogs, we were able to draw a structure – activity relationship which reveals that attaching an 8-carbon alkyl chain at position O-4´´ of kanamycin B converts it into a fungicide with loss of antibacterial activity. Further effort has then directed to the investigation of the mechanism of action and to the optimization of the synthetic protocol.

CARB 86

E. coli K5 heparosan production for bioengineered heparin

Zhenyu Wang(1)(2), [email protected], Room 4005, Troy NY 12180, United States ; Mellisa Ly(3)(2); Fuming Zhang(4)(2); Zhenqing Zhang(5); Jonathan S. Dordick(1)(4)(2); Robert J. Linhardt(1)(4)(3)(2). (1) Department of Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY 12180, United States (3) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (4) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States (5) Baxter International Inc., United States

Heparosan is an acidic polysaccharide natural product. It is a critical precursor in heparin biosynthesis and in the chemoenzymatic synthesis of bioengineered heparin. The process of heparosan production by E. coli K5 fermentation significantly affects the cost and the pharmacological properties of the final product bioengineered heparin. In this study, heparosan produced by E. coli K5 fermentation was characterized and the fermentation process was optimized and controlled to produce the ideal bioengineered heparin precursor. E. coli strain improvement by metabolic engineering and gene manipulation was also investigated.

CARB 86

E. coli K5 heparosan production for bioengineered heparin

Zhenyu Wang(1)(2), [email protected], Room 4005, Troy NY 12180, United States ; Mellisa Ly(3)(2); Fuming Zhang(4)(2); Zhenqing Zhang(5); Jonathan S. Dordick(1)(4)(2); Robert J. Linhardt(1)(4)(3)(2). (1) Department of Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY 12180, United States (3) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (4) Department of Chemical

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and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States (5) Baxter International Inc., United States

Heparosan is an acidic polysaccharide natural product. It is a critical precursor in heparin biosynthesis and in the chemoenzymatic synthesis of bioengineered heparin. The process of heparosan production by E. coli K5 fermentation significantly affects the cost and the pharmacological properties of the final product bioengineered heparin. In this study, heparosan produced by E. coli K5 fermentation was characterized and the fermentation process was optimized and controlled to produce the ideal bioengineered heparin precursor. E. coli strain improvement by metabolic engineering and gene manipulation was also investigated.

CARB 87

Stochastic simulation of lectin microarrays with nanosensor transducers: Potential platforms for optimal, high-throughput screening and profiling of glycoproteins

Nigel F Reuel(1), [email protected], 77 Massachusetts Avenue, Room 66-566, Cambridge MA 02139, United States ; Jin-Ho Ahn(1); Michael S. Strano(1). (1) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States

As expressed, glycoprotein therapeutics expand in market share, there is a growing need for high-throughput platforms for profiling (to determine their homogeneity) and screening (to detect any deleterious glycan additions). An array of carefully selected lectins could provide such a platform. A Kinetic Monte Carlo method has been employed to model the interactions of glycoproteins with a lectin array to determine which lectins would be of best use for screening and profiling. Interaction parameters between many lectins and glycans were supplied as dissociation constants (KD). Clear contrast for screening purposes is manifested in both frequency response and average site occupancy. Three current applications of the array are modeled: 1) screening protein therapeutics, 2) differentiating arthritic disease, and 3) evaluating cancer biomarkers. The complete profiling of glycoproteins without a priori knowledge of their synthesis is further discussed, as well as the application of stochastic nanosensors to label free detection.

CARB 87

Stochastic simulation of lectin microarrays with nanosensor transducers: Potential platforms for optimal, high-throughput screening and profiling of glycoproteins

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Nigel F Reuel(1), [email protected], 77 Massachusetts Avenue, Room 66-566, Cambridge MA 02139, United States ; Jin-Ho Ahn(1); Michael S. Strano(1). (1) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States

As expressed, glycoprotein therapeutics expand in market share, there is a growing need for high-throughput platforms for profiling (to determine their homogeneity) and screening (to detect any deleterious glycan additions). An array of carefully selected lectins could provide such a platform. A Kinetic Monte Carlo method has been employed to model the interactions of glycoproteins with a lectin array to determine which lectins would be of best use for screening and profiling. Interaction parameters between many lectins and glycans were supplied as dissociation constants (KD). Clear contrast for screening purposes is manifested in both frequency response and average site occupancy. Three current applications of the array are modeled: 1) screening protein therapeutics, 2) differentiating arthritic disease, and 3) evaluating cancer biomarkers. The complete profiling of glycoproteins without a priori knowledge of their synthesis is further discussed, as well as the application of stochastic nanosensors to label free detection.

CARB 88

Evaluation of different thioesters for glycocluster synthesis applying native chemical ligation

Johannes W. Wehner(1), [email protected], Otto-Hahn-Platz 4, Kiel Schleswig-Holstein 24098, Germany ; Thisbe K. Lindhorst(1). (1) Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, Kiel 24098, Germany

Multivalent glycomimetics such as glycoclusters and glycodendrimers [1] are valuable tools for structure-function investigations in the glyco sciences.[2] During the last decade, our group has utilized various chemistries to achieve glycoclusters of different architectures and has tested their biology mainly in bacterial adhesion.[3] In this work it has been the aim to evaluate native chemical ligation (NCL) for the synthesis of clustered glycosides. This approach is appealing, because it does not require protecting groups and can be extended to more complex targets, eventually. Native chemical ligation was first reported in 1953 [4] and utilized in 1994 by S. B. Kent et al. to synthesize peptides [5] and has then been developed into a popular chemoselective method for ligation of large peptides and glycopeptide fragments.[6] Glycocluster synthesis via NCL requires a glycocysteine derivative, which can be reacted with a thioester in a subsequent ligation step. Thus, a number of thioesters with varying polarities (type 1 - 4) was synthesized and ligated with a mannosidic cysteine derivative.

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[1] N. Röckendorf; Th. K. Lindhorst, Top. Curr. Chem. 2001, 217, 201-238; [2] M. Lahmann, Top. Curr. Chem. 2009, 288, 17-65; [3] M. Dubber; O. Sperling; Th. K. Lindhorst, Org. Biomol. Chem. 2006, 4, 3913-3922; [4] T. Wieland; E. Bokelmann; L. Bauer; H. U. Lang; H. Lau, Liebigs Ann. 1953, 583, 129-149; [5] P. E. Dawson; T. W. Muir; I. Clark-Lewis; S. B. Kent, Science 1994, 266, 776-779; [6] C. P. R. Hackenberger; D. Schwarzer, Angew. Chem. 2008, 120, 10182-10228; Angew. Chem. Int. Ed. 2008, 47, 10030-10074.

CARB 88

Evaluation of different thioesters for glycocluster synthesis applying native chemical ligation

Johannes W. Wehner(1), [email protected], Otto-Hahn-Platz 4, Kiel Schleswig-Holstein 24098, Germany ; Thisbe K. Lindhorst(1). (1) Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, Kiel 24098, Germany

Multivalent glycomimetics such as glycoclusters and glycodendrimers [1] are valuable tools for structure-function investigations in the glyco sciences.[2] During the last decade, our group has utilized various chemistries to achieve glycoclusters of different architectures and has tested their biology mainly in bacterial adhesion.[3] In this work it has been the aim to evaluate native chemical ligation (NCL) for the synthesis of clustered glycosides. This approach is appealing, because it does not require protecting groups and can be extended to more complex targets, eventually. Native chemical ligation was first reported in 1953 [4] and utilized in 1994 by S. B. Kent et al. to synthesize peptides [5] and has then been developed into a popular chemoselective method for ligation of large peptides and glycopeptide fragments.[6] Glycocluster synthesis via NCL requires a glycocysteine derivative, which can be reacted with a thioester in a subsequent ligation step. Thus, a number of thioesters with varying polarities (type 1 - 4) was synthesized and ligated with a mannosidic cysteine derivative.

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[1] N. Röckendorf; Th. K. Lindhorst, Top. Curr. Chem. 2001, 217, 201-238; [2] M. Lahmann, Top. Curr. Chem. 2009, 288, 17-65; [3] M. Dubber; O. Sperling; Th. K. Lindhorst, Org. Biomol. Chem. 2006, 4, 3913-3922; [4] T. Wieland; E. Bokelmann; L. Bauer; H. U. Lang; H. Lau, Liebigs Ann. 1953, 583, 129-149; [5] P. E. Dawson; T. W. Muir; I. Clark-Lewis; S. B. Kent, Science 1994, 266, 776-779; [6] C. P. R. Hackenberger; D. Schwarzer, Angew. Chem. 2008, 120, 10182-10228; Angew. Chem. Int. Ed. 2008, 47, 10030-10074.

CARB 89

NMR for structure elucidation of commercially available heparin polysaccharides

Kemal Solakyildirim(1), [email protected], 2100 Massachusetts Ave. Apt#2A, Troy New York 12180, United States ; Scott A. McCallum(2); Robert J. Linhardt(1)(2)(3)(4). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy New York 12180, United States (3) Department of Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States (4) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy New York 12180, United States

Heparin is highly negatively charged, heterogeneous linear polysaccharide that is mainly used in pharmaceutical products as an anticoagulant drug for the treatment of thrombosis, thrombophlebitis, and embolism. Commercial USP heparin is primarily obtained from porcine intestinal mucosa. Recently, heparin was contaminated with oversulfated chondroitin sulfate (OSCS), which caused the death of nearly 100 Americans. However, it may now possible to make a bioengineered heparin from non-animal sources. FDA approval would require a bioengineered heparin to have a structure identical to USP heparin. NMR spectroscopy is a powerful and high-resolution technique capable of detailed structural elucidation of the heparin polysaccharide. Commercial heparin samples, obtained from various manufacturers around the world, were analyzed using 1D 1H and 13C NMR and 2D H-H COSY, TOCSY, HMQC, NOESY NMR experiments. The specific aim of this study is to use NMR to establish the structural features present in USP heparin.

CARB 89

NMR for structure elucidation of commercially available heparin polysaccharides

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Kemal Solakyildirim(1), [email protected], 2100 Massachusetts Ave. Apt#2A, Troy New York 12180, United States ; Scott A. McCallum(2); Robert J. Linhardt(1)(2)(3)(4). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy New York 12180, United States (3) Department of Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States (4) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy New York 12180, United States

Heparin is highly negatively charged, heterogeneous linear polysaccharide that is mainly used in pharmaceutical products as an anticoagulant drug for the treatment of thrombosis, thrombophlebitis, and embolism. Commercial USP heparin is primarily obtained from porcine intestinal mucosa. Recently, heparin was contaminated with oversulfated chondroitin sulfate (OSCS), which caused the death of nearly 100 Americans. However, it may now possible to make a bioengineered heparin from non-animal sources. FDA approval would require a bioengineered heparin to have a structure identical to USP heparin. NMR spectroscopy is a powerful and high-resolution technique capable of detailed structural elucidation of the heparin polysaccharide. Commercial heparin samples, obtained from various manufacturers around the world, were analyzed using 1D 1H and 13C NMR and 2D H-H COSY, TOCSY, HMQC, NOESY NMR experiments. The specific aim of this study is to use NMR to establish the structural features present in USP heparin.

CARB 90

Syntheses of C-5-spirocyclic C-glycoside SGLT2 inhibitors

Benjamin A. Thuma(1), [email protected], Eastern Point Rd., Groton CT 06340, United States ; Ralph P. Robinson(1); Cathy Préville(1); Matthew R. Reese(2); Robert J. Maguire(1); Christopher L. Carr(2); Michael T. Leininger(2); André Lowe(2); Claire M. Steppan(2); Vincent Mascitti(1). (1) CVMED Medicinal Chemistry, Pfizer Global Research and Development, Groton CT 06340, United States (2) Pfizer Global Research and Development, Groton CT 06340, United States

SGLT2 inhibitors theoretically should promote weight loss and control hyperglycemia in a glucose-dependent yet insulin-independent manner, thereby potentially making such therapeutic agents very compelling complements to the current arsenal of anti-diabetic agents. We recently reported that some C-5-spirocyclic C-glycoside derivatives are potent and selective SGLT2 inhibitors.1 In this poster, several syntheses of C-5-spirocycle-containing C-glycosides are presented. Particularly, a multigram-scale synthesis capitalizing on a one pot aldol-Cannizzaro sequence is described. Spiro oxetane formation using an unprotected penta-ol C-glycoside as substrate is also exemplified.2

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1. Robinson, R. P. et al. Bioorg. Med. Chem. Lett. 2010, 20, 1569. 2. Mascitti, V. et al. Tetrahedron Letters 2010, 51, 1880.

CARB 91

Co-axial cellulose nanofibers for electrical applications

Minoru Miyauchi(1)(2), [email protected], 110 8th Street, BT4123, Troy NY 12180, United States ; Jianjun Miao(1)(3)(4)(2); Trevor J. Simmons(1)(4)(2); Jonathan S. Dordick(1)(3)(5)(2); Robert J. Linhardt(1)(3)(4)(5)(2). (1) Department of Center for Nanotechnology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) Department of Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY 12180, United States (3) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States (4) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (5) Department of Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Electrospinning has been widely investigated as simple technique to prepare nanoscale fibers of various polymers. New technologies are still being introduced for electrospinning, for example, dry-jet wet electrospinning using room temperature ionic liquids (RTILs) as solvent. Since RTILs are non-volatile, electrospun fibers are collected into a coagulation bath that is filled with appropriate co-solvent to remove RTILs. Because of the strong dissolving power of RTILs, electrospinning can be applied for many kinds of biomaterials, including unmodified cellulose. Some composite nanofibers were fabricated by adding functional nanomaterials such as nanoparticles and carbon nanotubes into electrospinning solutions. Co-electrospinning techniques were also used to fabricate core-sheath type composite nanofibers. Using these techniques, functional materials, which can normally not be electrospun, could be fabricated into fibers. Specialized cellulose nanofiber for electronic components and their potential applications will be discussed.

CARB 92

Study on the relative reactivity of glycosyl acceptors in the glycosylations of 2-Azido-2-deoxy-galactosides

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Jane Kalikanda(1), [email protected], 4400 Vestal Parkway East, Binghamton NY 13902, United States ; Zhitao Li(1). (1) Department of Chemistry, Binghamton University, Binghamton NY 13902, United States

The reactivity of acceptors depends on the nucleophilicity of the hydroxyl groups in partially protected carbohydrate molecules that in turn depends on their nature, their spatial orientation, conformation of the sugar ring and also on the presence of other protecting groups in the molecule. A better understanding of the relative reactivity of glycosyl acceptors is very helpful for developing a more efficient oligosaccharide synthesis. However, it is often difficult to determine the relative reactivity of acceptors with different protecting groups and from different sugars. We recently observed that the stereoselectivity of 2-azido-2-deoxy-galactoside donors is highly dependent on the reactivity of acceptors. The stereochemical outcome of a series of glycosylation reactions was used to characterize a library of glycosyl acceptors and provided a useful way to quantify the reactivity of acceptors

CARB 93

Synthesis of tailored glycoconjugates for the precise detection of pathogens

Ashish A. Kulkarni(1), [email protected], 506, Riddle Road,, Apt. No. 41, Cincinnati Ohio 45220, United States ; Suri S. Iyer(1). (1) Department of Chemistry, University of Cincinnati, Cincinnati Ohio 45220, United States

Carbohydrate-protein interactions play a very important role in a variety of essential biological processes such as adhesion, cell-cell communication and organ differentiation. Several infectious agents use the cell surface carbohydrates to gain entry and infect the cells. We are harnessing this recognition capability and developing molecules that can be used as integral components of biosensors. This work is highly significant because carbohydrates are highly stable under a variety of conditions, do not suffer from lot-to-lot variation, can be synthesized in large quantities and can be adapted to any platform. Additionally, different strains, including newly emerging strains can be identified rapidly. In this regard, we have developed chemically defined glycoconjugates using a versatile modular approach for capturing toxins, viruses and bacteria. Specifically, we have synthesized carbohydrate based recognition elements and attached these molecules to a scaffold via flexible oligoethylene glycol linkers. The multivalent unit has been covalently linked to a fluorescent molecule or a biotin. The design and synthesis of these multivalent ligands, covalent linkage of the scaffold to the ligands and the reporter to the scaffold will be presented.

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CARB 93

Synthesis of tailored glycoconjugates for the precise detection of pathogens

Ashish A. Kulkarni(1), [email protected], 506, Riddle Road,, Apt. No. 41, Cincinnati Ohio 45220, United States ; Suri S. Iyer(1). (1) Department of Chemistry, University of Cincinnati, Cincinnati Ohio 45220, United States

Carbohydrate-protein interactions play a very important role in a variety of essential biological processes such as adhesion, cell-cell communication and organ differentiation. Several infectious agents use the cell surface carbohydrates to gain entry and infect the cells. We are harnessing this recognition capability and developing molecules that can be used as integral components of biosensors. This work is highly significant because carbohydrates are highly stable under a variety of conditions, do not suffer from lot-to-lot variation, can be synthesized in large quantities and can be adapted to any platform. Additionally, different strains, including newly emerging strains can be identified rapidly. In this regard, we have developed chemically defined glycoconjugates using a versatile modular approach for capturing toxins, viruses and bacteria. Specifically, we have synthesized carbohydrate based recognition elements and attached these molecules to a scaffold via flexible oligoethylene glycol linkers. The multivalent unit has been covalently linked to a fluorescent molecule or a biotin. The design and synthesis of these multivalent ligands, covalent linkage of the scaffold to the ligands and the reporter to the scaffold will be presented.

CARB 94

Structure-activity studies of synthetic glycophosphatidylinositol anchored proteins

Carl V Christianson(1), [email protected], Free University Berlin, Arnimallee 22, Berlin Berlin 12101, Germany ; Peter H Seeberger(1). (1) Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Berlin 12101, Germany

The structural complexity of glycophosphatidylinositol (GPI) anchors has hindered studies relating structural elements to specific biological roles. Access to defined GPI structures through total synthesis1 facilitates the modification of proteins via native chemical ligation (NCL) to probe the importance of glycosylation and lipidation patterns of GPI anchors.2 Here we present studies of GFP modified with fully synthetic GPI anchors (Figure 1.) and other probes in order to gain a better understanding of the role that this posttranslational modification plays in biological processes.

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1. Liu, X.; Kwon, Y. U.; Seeberger, P. H., Convergent synthesis of a fully lipidated glycosylphosphatidylinositol anchor of Plasmodium falciparum. J. Am. Chem. Soc. 2005, 127, (14), 5004-5. 2. Becker,C. F.; Liu, X.; Olschewski, D.; Castelli, R.; Seidel, R.; Seeberger, P. H., Semisynthesis of a glycosylphosphatidylinositol-anchored prion protein. Angew. Chem. Int. Ed. Engl. 2008, 47, (43), 8215-9.

CARB 94

Structure-activity studies of synthetic glycophosphatidylinositol anchored proteins

Carl V Christianson(1), [email protected], Free University Berlin, Arnimallee 22, Berlin Berlin 12101, Germany ; Peter H Seeberger(1). (1) Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Berlin 12101, Germany

The structural complexity of glycophosphatidylinositol (GPI) anchors has hindered studies relating structural elements to specific biological roles. Access to defined GPI structures through total synthesis1 facilitates the modification of proteins via native chemical ligation (NCL) to probe the importance of glycosylation and lipidation patterns of GPI anchors.2 Here we present studies of GFP modified with fully synthetic GPI anchors (Figure 1.) and other probes in order to gain a better understanding of the role that this posttranslational modification plays in biological processes.

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1. Liu, X.; Kwon, Y. U.; Seeberger, P. H., Convergent synthesis of a fully lipidated glycosylphosphatidylinositol anchor of Plasmodium falciparum. J. Am. Chem. Soc. 2005, 127, (14), 5004-5. 2. Becker,C. F.; Liu, X.; Olschewski, D.; Castelli, R.; Seidel, R.; Seeberger, P. H., Semisynthesis of a glycosylphosphatidylinositol-anchored prion protein. Angew. Chem. Int. Ed. Engl. 2008, 47, (43), 8215-9.

CARB 95

Chemoenzymatic synthesis of heparan sulfate

Renpeng Liu(1), [email protected], 303 Beard Hall, Chapel Hill 27599, United States ; Jian Liu(1); Yongmei Xu(1). (1) Eshelman school of Pharmacy, UNC-Chapel Hill, Chapel Hill NC 27599, United States

Heparan sulfate (HS) participates in a variety of biological functions. The specific functions of HS are governed by the unique distributions of sulfated saccharide sequences; however, chemical synthesis of structurally defined HS remains extremely challenging. Here, we describe a method for an enzyme-based de novo synthesis of HS oligosaccharides with defined structures. We use bacteria heparosan glycosyltransferases to build up the HS backbones and position the GlcNS residue by providing unnatural monosaccharide donor, UDP-N-trifluoroacetylglucosamine.

By using this approach, we have prepared four HS octasaccharides with defined distribution of GlcNS residues. By controlling the position of GlcNS residue,we are able to regulate the modification site of O-sulfotransferases and C5-epimerase to prepare oligosaccharides with defined structures. Our method

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could be used as a general approach for synthesizing structurally defined HS oligosaccharides that could aid in the discovery of novel HS-based therapeutic agent.

CARB 96

Synthesis of a fluorous-tagged disaccharide for the enzymatic preparation of heparin oligosaccharides

Sayaka Masuko(1), [email protected], 110 8th Street, Troy NY 12180, United States ; Smritilekha Bera(1); Robert J. Linhardt(1). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Heparin is a highly sulfated glycosaminoglycan (GAG) that is extensively used in medical practice as an anticoagulant. The preparation of heparin and its oligosaccharides can be accomplished by synthetic and enzymatic methods. However, the purification of these modified oligosaccharide products from the reaction mixture is laborious and low-yielding. Therefore, a fluorous tag technique will greatly simplify purification procedures and structural characterization of the products. A heparin disaccharide with a fluorous affinity Froc tag can be chemically synthesized in a small number of steps. The Froc-tagged disaccharide will be placed on the reducing end to accept UDP-sugar building blocks in the enzymatic preparation of heparin and heparin oligosaccharides.

CARB 96

Synthesis of a fluorous-tagged disaccharide for the enzymatic preparation of heparin oligosaccharides

Sayaka Masuko(1), [email protected], 110 8th Street, Troy NY 12180, United States ; Smritilekha Bera(1); Robert J. Linhardt(1). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Heparin is a highly sulfated glycosaminoglycan (GAG) that is extensively used in medical practice as an anticoagulant. The preparation of heparin and its oligosaccharides can be accomplished by synthetic and enzymatic methods. However, the purification of these modified oligosaccharide products from the reaction mixture is laborious and low-yielding. Therefore, a fluorous tag technique will greatly simplify purification procedures and structural characterization of the products. A heparin disaccharide with a fluorous affinity Froc tag can be chemically synthesized in a small number of steps. The Froc-tagged disaccharide will be placed on the reducing end to accept UDP-sugar

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building blocks in the enzymatic preparation of heparin and heparin oligosaccharides.

CARB 97

Preventing the transmission of Plasmodium falciparum through the inhibition of malaria protein binding to placental chondroitin sulfate A

Julie M Beaudet(1)(2), [email protected], 110 8th Street, Troy New York, United States ; Leandra J Mansur(1)(2); Bo Yang(1); Fuming Zhang(3); Robert J Linhardt(1)(2)(3). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, United States (2) Department of Biology, Rensselaer Polytechnic Institute, United States (3) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy New York 12180, United States

One way that Plasmodium falciparum can be transferred is during the birthing process. Infected erythrocytes express the VAR2CSA protein which binds to chondroitin sulfate A (CSA) in the placenta. During birth the bound erythrocytes rupture and the P. falciparum parasite can infect the newborn. Laboratory studies have already shown that bovine tracheal CSA can inhibit the binding of infected erythrocytes to CSA in the placenta. We are isolating bovine placental glycosaminoglycans (GAGs), and can inhibit the infected cells from binding to the placenta using the extracted GAGs. The GAG -bound infected cells can be transported to the spleen thus preventing contact between the parasite and the newborn. The GAGs are isolated from the placenta tissue and purified. Structural analysis of the GAGs is preformed using PAGE, NMR, and LCMS disaccharide analysis techniques. Extracted CSA is then immobilized on an SPR chip in order to perform an activity assay with infected erythrocytes or similar protein compounds.

CARB 97

Preventing the transmission of Plasmodium falciparum through the inhibition of malaria protein binding to placental chondroitin sulfate A

Julie M Beaudet(1)(2), [email protected], 110 8th Street, Troy New York, United States ; Leandra J Mansur(1)(2); Bo Yang(1); Fuming Zhang(3); Robert J Linhardt(1)(2)(3). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, United States (2) Department of Biology, Rensselaer Polytechnic Institute, United States (3) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy New York 12180, United States

One way that Plasmodium falciparum can be transferred is during the birthing process. Infected erythrocytes express the VAR2CSA protein which binds to

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chondroitin sulfate A (CSA) in the placenta. During birth the bound erythrocytes rupture and the P. falciparum parasite can infect the newborn. Laboratory studies have already shown that bovine tracheal CSA can inhibit the binding of infected erythrocytes to CSA in the placenta. We are isolating bovine placental glycosaminoglycans (GAGs), and can inhibit the infected cells from binding to the placenta using the extracted GAGs. The GAG -bound infected cells can be transported to the spleen thus preventing contact between the parasite and the newborn. The GAGs are isolated from the placenta tissue and purified. Structural analysis of the GAGs is preformed using PAGE, NMR, and LCMS disaccharide analysis techniques. Extracted CSA is then immobilized on an SPR chip in order to perform an activity assay with infected erythrocytes or similar protein compounds.

CARB 98

Real-time assessment of the morphological change of cellulose in response to enzymatic treatment

Chi Nguyen(1), [email protected], 3141 Chestnut Street, Philadelphia Pa 19104, United States . (1) Department of Chemistry, Drexel University, Philadelphia Pa 19103, United States

Cellulose is considered a very attractive alternative source of fuels because of the abundant and renewable supply. Thus improving conversion of cellulose from biomass into cellulosic ethanol is of great scientific interest. Presently, one of the key steps of the enzymatic hydrolysis of cellulose, where cellulase breaks down the crystal structure on the surface region of cellulose and exposes cellulose chains for subsequent hydrolysis by cellulase, is still not fully understood. It is hypothesized that this non-hydrolytic enzymatic step could be the rate-limiting step for the entire enzymatic hydrolysis of cellulose. We have developed a nanomechanical approach that allows us to assess the minute change in morphology of cellulose in real time. Here we report our recent progress in using this approach to study the effect of cellulolytic enzyme on cellulose. The results of this study will help to provide a thorough insight into the initial steps of cellulose hydrolysis.

CARB 98

Real-time assessment of the morphological change of cellulose in response to enzymatic treatment

Chi Nguyen(1), [email protected], 3141 Chestnut Street, Philadelphia Pa 19104, United States . (1) Department of Chemistry, Drexel University, Philadelphia Pa 19103, United States

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Cellulose is considered a very attractive alternative source of fuels because of the abundant and renewable supply. Thus improving conversion of cellulose from biomass into cellulosic ethanol is of great scientific interest. Presently, one of the key steps of the enzymatic hydrolysis of cellulose, where cellulase breaks down the crystal structure on the surface region of cellulose and exposes cellulose chains for subsequent hydrolysis by cellulase, is still not fully understood. It is hypothesized that this non-hydrolytic enzymatic step could be the rate-limiting step for the entire enzymatic hydrolysis of cellulose. We have developed a nanomechanical approach that allows us to assess the minute change in morphology of cellulose in real time. Here we report our recent progress in using this approach to study the effect of cellulolytic enzyme on cellulose. The results of this study will help to provide a thorough insight into the initial steps of cellulose hydrolysis.

CARB 99

Chemoselective glycosylation of hemoglobin as a potential oxygen therapeutic

Thomas J Styslinger(1), [email protected], Newman & Wolfrom Lab, 100 W. 18th Avenue, Columbus Ohio 43210, United States ; Ning Zhang(2); Andre F. Palmer(2); Peng G. Wang(1). (1) Department of Chemistry, The Ohio State University, Columbus Ohio 43210, United States (2) Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus Ohio 43210, United States

Hemorrhagic shock is the leading cause of death among trauma patients1. This condition results from the body's inability to compensate and provide adequate tissue perfusion and oxygenation as a result of blood loss. Over the past 20 years, hemoglobin-based oxygen carriers (HBOCs) have been explored as a potential treatment for hemorrhagic shock due to hemoglobin's role in the transportation and storage of oxygen in red blood cells. One attractive potential for the development of HBOCs involves taking advantage of nature's strategy of protein glycosylation for the protection and stabilization of proteins. Previous studies conducted in our labs have shown that site-selective glycosylation of hemoglobin is possible with a lactose derivative and has advantageous effects2 . In the present study we have expanded the glycosylation sites to include lysine residues and developed methodology to facilely broaden the scope of carbohydrates conjugated to hemoglobin. Furthermore, the synthetic route employed avoids the tedious protection and deprotection steps which typically accompany the synthesis of such molecules. The range of carbohydrates being investigated for conjugation and corresponding biological studies includes various oligosaccharides and polysaccharides. 1) Cocchi, M. N., Kimlin, E., Walsh, M., Donnino, M. W., Emerg Med Clin North Am. 2007, 25(3), 623-42.

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2) Zhang, Y., Bhatt, V. S., Sun, G., Wang, P. G., Palmer, A. F., Bioconjugat Chem. 2008, 19, 2221-2230.

CARB 100

Immobilization of enzymes relevant to bioengineered heparin synthesis

Eric R. Sterner(1), [email protected], 110 8th Street, Center for Biotechnology and Interdisciplinary Studies, Troy New York 12180, United States ; Robert J. Linhardt(2); Jonathan S. Dordick(1); Jian Liu(3); Fuming Zhang(2); Wenjing Zhao(4); Priscilla Paul(1); Jeff Martin(5). (1) Department of Chemical and Biochemical Engineering, Rensselaer Polytechnic Institute, Troy New York 12180, United States (2) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States (3) Department of Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill North Carolina 27599, United States (4) Department of Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States (5) Department of Biochemistry and Biophysics, Rensselaer Polytechnic Institute, Troy New York 12180, United States

The immobilization of enzymes on solid resin surfaces is an important technique used as a means to improve the stability and performance of enzymes for a variety of applications, as well as a method to eliminate costly downstream separations steps required for free enzymes in solution. This study aims to investigate the expression, purification, and immobilization of the O-sulfotransferase enzymes relevant to the synthesis of bioengineered heparin. Enzyme immobilization will be performed on micro-particle with intended applications in the medical and biochemistry research and process scale-up. Immobilization on nano-particles will also be considered with applications intended for heparin synthesis on a micro-scale. The kinetics of the free and immobilized enzymes will be determined and compared to illustrate the benefit of immobilized enzymes reactions over free enzyme solutions. A thorough study of the effect of solid resin affinity immobilization on enzyme stability will also be considered and reported.

CARB 100

Immobilization of enzymes relevant to bioengineered heparin synthesis

Eric R. Sterner(1), [email protected], 110 8th Street, Center for Biotechnology and Interdisciplinary Studies, Troy New York 12180, United States ; Robert J. Linhardt(2); Jonathan S. Dordick(1); Jian Liu(3); Fuming Zhang(2); Wenjing Zhao(4); Priscilla Paul(1); Jeff Martin(5). (1) Department of Chemical and Biochemical Engineering, Rensselaer Polytechnic Institute, Troy New York 12180, United States (2) Department of Chemistry and Chemical Biology, Rensselaer

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Polytechnic Institute, Troy New York 12180, United States (3) Department of Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill North Carolina 27599, United States (4) Department of Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States (5) Department of Biochemistry and Biophysics, Rensselaer Polytechnic Institute, Troy New York 12180, United States

The immobilization of enzymes on solid resin surfaces is an important technique used as a means to improve the stability and performance of enzymes for a variety of applications, as well as a method to eliminate costly downstream separations steps required for free enzymes in solution. This study aims to investigate the expression, purification, and immobilization of the O-sulfotransferase enzymes relevant to the synthesis of bioengineered heparin. Enzyme immobilization will be performed on micro-particle with intended applications in the medical and biochemistry research and process scale-up. Immobilization on nano-particles will also be considered with applications intended for heparin synthesis on a micro-scale. The kinetics of the free and immobilized enzymes will be determined and compared to illustrate the benefit of immobilized enzymes reactions over free enzyme solutions. A thorough study of the effect of solid resin affinity immobilization on enzyme stability will also be considered and reported.

CARB 101

Analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters by surface plasmon resonance (SPR) and NMR

Ayano Takasaki-Matsumoto(1), [email protected], 4-1-1 Kitakaname, Hiratsuka Kanagawa 259-1292, Japan ; Shinya Hanashima(2); Ami Aoki(1); Yoshiki Yamaguchi(2); Reiko Sato(3); Hiroko Kawakami(3); Mamoru Mizuno(3); Yoko Fujita-Yamaguchi(1). (1) Department of Applied Biochemistry, Tokai University School of Engineering, Hiratsuka Kanagawa 259-1292, Japan (2) Frontier Research System, RIKEN, Wako Saitama 351-0198, Japan (3) Glyco-organic Chemistry, The Noguchi Institute, Itabashi Tokyo 173-0003, Japan

Tn-antigens (GalNAca-Ser/Thr) are <tt>oncogenic antigens and </tt>are generally masked by covalently linked terminal carbohydrate moieties in normal human tissues but are exposed in most primary and metastatic epithelial malignant tumors. MLS128 recongnizes an epitope consisting of three or two consecutive Tn-antigens (Tn3 or Tn2). Thermodynamic and kinetic analyses of the antigen-antibody interaction between MLS128 and Tn3- or Tn2-peptide, synthesized as described (Sakai et al. J. Biochem. 2010), were conducted by SPR and NMR at varying temperatures. The binding epitopes were analyzed by 1H-NMR as well as TR-NOESY experiments. At varying temperatures from 5 to 35 °C, KD values for both ligands increased while affinities for Tn3 were always

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higher than Tn2 as determined by SPR. NMR experiments supported that the ligand binding site of MLS128 accepts three GalNAc residues on Tn3-peptide, which correlated with the SPR results that the affinity for Tn3-peptide exhibited lower KD value than that of Tn3-peptide.

CARB 101

Analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters by surface plasmon resonance (SPR) and NMR

Ayano Takasaki-Matsumoto(1), [email protected], 4-1-1 Kitakaname, Hiratsuka Kanagawa 259-1292, Japan ; Shinya Hanashima(2); Ami Aoki(1); Yoshiki Yamaguchi(2); Reiko Sato(3); Hiroko Kawakami(3); Mamoru Mizuno(3); Yoko Fujita-Yamaguchi(1). (1) Department of Applied Biochemistry, Tokai University School of Engineering, Hiratsuka Kanagawa 259-1292, Japan (2) Frontier Research System, RIKEN, Wako Saitama 351-0198, Japan (3) Glyco-organic Chemistry, The Noguchi Institute, Itabashi Tokyo 173-0003, Japan

Tn-antigens (GalNAca-Ser/Thr) are <tt>oncogenic antigens and </tt>are generally masked by covalently linked terminal carbohydrate moieties in normal human tissues but are exposed in most primary and metastatic epithelial malignant tumors. MLS128 recongnizes an epitope consisting of three or two consecutive Tn-antigens (Tn3 or Tn2). Thermodynamic and kinetic analyses of the antigen-antibody interaction between MLS128 and Tn3- or Tn2-peptide, synthesized as described (Sakai et al. J. Biochem. 2010), were conducted by SPR and NMR at varying temperatures. The binding epitopes were analyzed by 1H-NMR as well as TR-NOESY experiments. At varying temperatures from 5 to 35 °C, KD values for both ligands increased while affinities for Tn3 were always higher than Tn2 as determined by SPR. NMR experiments supported that the ligand binding site of MLS128 accepts three GalNAc residues on Tn3-peptide, which correlated with the SPR results that the affinity for Tn3-peptide exhibited lower KD value than that of Tn3-peptide.

CARB 102

Modular glycoconjugate tool set for assembly and presentation of multivalent carbohydrate ligands on surfaces

Irene Abia(1), [email protected], 1420 Circle Drive, Knoxville TN 37996-1600, United States ; Brian Sanders(1); Michael D. Best(1); David C. Baker(1). (1) Department of Chemistry, University of Tennessee, Knoxville TN 37996-1600, United States

D-Mannose mono-, di- and trisaccharides with thiol-terminated aglycons were synthesized for attachment to gold nanoparticles to mimic carbohydrate-involved

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cell-surface interactions. These molecules were constructed by glycosylation of appropriately protected glycosyl donors and acceptors, followed by free-radical addition to introduce the thiol terminals onto the aglycons. Subsequent deprotection afforded the free-OH saccharides. Tethering chemistries will allow these components to be attached to gold-clad silicon chips, creating carbohydrate surfaces of varying valencies and densities. Propargyl glycosides of mono- and di-mannosides have also been synthesized for tethering via click chemistry. (Supported by NSF DMR-0906752.)

CARB 102

Modular glycoconjugate tool set for assembly and presentation of multivalent carbohydrate ligands on surfaces

Irene Abia(1), [email protected], 1420 Circle Drive, Knoxville TN 37996-1600, United States ; Brian Sanders(1); Michael D. Best(1); David C. Baker(1). (1) Department of Chemistry, University of Tennessee, Knoxville TN 37996-1600, United States

D-Mannose mono-, di- and trisaccharides with thiol-terminated aglycons were synthesized for attachment to gold nanoparticles to mimic carbohydrate-involved cell-surface interactions. These molecules were constructed by glycosylation of appropriately protected glycosyl donors and acceptors, followed by free-radical addition to introduce the thiol terminals onto the aglycons. Subsequent deprotection afforded the free-OH saccharides. Tethering chemistries will allow these components to be attached to gold-clad silicon chips, creating carbohydrate surfaces of varying valencies and densities. Propargyl glycosides of mono- and di-mannosides have also been synthesized for tethering via click chemistry. (Supported by NSF DMR-0906752.)

CARB 103

Analysis of the absorption of low molecular weight heparin in human umbilical cord tissue as a model for the prevention of cancer metastasis

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Amanda M. Weyers(1), [email protected], 110 8th St., Cogswell, Troy NY 12180, United States ; Thangriala Sudha(2); Bo Yang(1); Boyangzi Li(1); Majde Takieddin(2); Fuming Zhang(3); Shaker A. Mousa(2); Robert J. Linhardt(1)(4). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany NY 12208, United States (3) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States (4) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Glycosoaminoglycans (GAGs) mediate a wide variety of biological functions. Low molecular weight heparins (LMWHs) re GAG derivatives exhibiting favorable pharmacological properties are being studied as anti-cancer drugs. Human pancreatic cancer cell adhesion has been shown to be inhibited by LMWH, possibly via the binding of GAG to the endothelial lining of the blood vessel. Here a non-anticoagulant LMWH was perfused through the vein in the umbilical cord tissue samples at different concentrations to determine the absorption of GAG by the blood vessel. Samples were then defatted, proteolyzed, purified by anion-exchange chromatography, and subject to methanol precipitation. Total sulfated GAG content of the isolated samples was quantified via carbazole assay and through complexation with 9-dimethymethylene blue. The polydispersity and molecular weights of the isolates was determined via polyacrylamide gel electrophoresis and GAG structures were determined via liquid chromatography-mass spectroscopy.

CARB 104

Synthesis and biological evaluation of a Gal(α1-2)GalCer analog

Yanke Liang(1), [email protected], 55 N. Eagleville Road, Storrs CT 06269-3060, United States ; Amy R Howell(1). (1) Department of Chemistry, University of Connecticut, Storrs CT 06269-3060, United States

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A synthesis of an analog of the immunostimulatory glycolipid, Gal(α1-2)galactosylceramide, is described. Good reactivity and high α selectivity was achieved utilizing 'armed' donors and 'disarmed' acceptors. Biological evaluation is expected to establish the effect of antigen presenting cells on the cytokine release profile.

CARB 104

Synthesis and biological evaluation of a Gal(α1-2)GalCer analog

Yanke Liang(1), [email protected], 55 N. Eagleville Road, Storrs CT 06269-3060, United States ; Amy R Howell(1). (1) Department of Chemistry, University of Connecticut, Storrs CT 06269-3060, United States

A synthesis of an analog of the immunostimulatory glycolipid, Gal(α1-2)galactosylceramide, is described. Good reactivity and high α selectivity was achieved utilizing 'armed' donors and 'disarmed' acceptors. Biological evaluation is expected to establish the effect of antigen presenting cells on the cytokine release profile.

CARB 105

Thiol-click chemistry approach to glycomimetics: Novel stereoselective synthesis of (1-3)-S-thiodisaccharides

Irena Bak-Sypien(1), 84 W. South Street, Wilkes-Barre PA 18766, United States ; Zbigniew J. Witczak(1). (1) Department of Pharmaceutical Sciences, Wilkes University, Wilkes-Barre PA 18766, United States

During the last decade the concept of click chemistry was transplanted to carbohydrate chemistry [1-3]. While, the original alkyne-azide concept of click- chemistry is well known, the alternative thiol-click is relatively unexplored. Recently, we have developed one-pot, thiol-click procedure for the synthesis of new family of (1-5)-C-thiodisaccharides [3]. Starting template for the specifically developed thiol-click approach is cyanoglycal 1 conveniently prepared in our

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laboratory. Glycal 1 undergoes base catalyzed Michael additions with peracetylated 1-thio-D-glucose 2 via one step thiol-click approach. The exclusive regio- and stereochemistry of the Michael addition reaction proceeds with the formation of 1,4-adduct 3 in high yield. The adduct 3 upon the conventional mild hydrolysis, affords amide derivative suitable for further functionalization with protected aminoacids to form carboamino peptides. Further examples of reactive thiols for quick one-pot thiol-click coupling approach with functionalized enones and their application to synthesis of new families of thio carboamino peptides will be also explored. They will be use as new tools for glycobiology and specifically as new inhibitors of galectin-3 [4].

1. M.Fiore, A. Mara, and A. Dondoni, J. Org.. Chem., 2009, 74, 4222. 2. S. G. Gouin, L. Bultel, C. Falentin and J. Kovensky, Eur. J. Org. Chem. 2007, 1160. 3. Z. J. Witczak, D. Lorchak and N. Nguyen, Carbohydr. Res., 2007, 342, 1929. 4. Galectins, John Wiley & Sons, Hoboken, NJ, 2008, Eds. A. Klyosov, Z.J. Witczak, and D. Platt.

CARB 105

Thiol-click chemistry approach to glycomimetics: Novel stereoselective synthesis of (1-3)-S-thiodisaccharides

Irena Bak-Sypien(1), 84 W. South Street, Wilkes-Barre PA 18766, United States ; Zbigniew J. Witczak(1). (1) Department of Pharmaceutical Sciences, Wilkes University, Wilkes-Barre PA 18766, United States

During the last decade the concept of click chemistry was transplanted to carbohydrate chemistry [1-3]. While, the original alkyne-azide concept of click- chemistry is well known, the alternative thiol-click is relatively unexplored. Recently, we have developed one-pot, thiol-click procedure for the synthesis of new family of (1-5)-C-thiodisaccharides [3]. Starting template for the specifically

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developed thiol-click approach is cyanoglycal 1 conveniently prepared in our laboratory. Glycal 1 undergoes base catalyzed Michael additions with peracetylated 1-thio-D-glucose 2 via one step thiol-click approach. The exclusive regio- and stereochemistry of the Michael addition reaction proceeds with the formation of 1,4-adduct 3 in high yield. The adduct 3 upon the conventional mild hydrolysis, affords amide derivative suitable for further functionalization with protected aminoacids to form carboamino peptides. Further examples of reactive thiols for quick one-pot thiol-click coupling approach with functionalized enones and their application to synthesis of new families of thio carboamino peptides will be also explored. They will be use as new tools for glycobiology and specifically as new inhibitors of galectin-3 [4].

1. M.Fiore, A. Mara, and A. Dondoni, J. Org.. Chem., 2009, 74, 4222. 2. S. G. Gouin, L. Bultel, C. Falentin and J. Kovensky, Eur. J. Org. Chem. 2007, 1160. 3. Z. J. Witczak, D. Lorchak and N. Nguyen, Carbohydr. Res., 2007, 342, 1929. 4. Galectins, John Wiley & Sons, Hoboken, NJ, 2008, Eds. A. Klyosov, Z.J. Witczak, and D. Platt.

CARB 106

High-throughput glycoarray for monitoring immune responses to a cancer vaccine

Christopher Campbell(1), [email protected], 376 Boyles Street, Room 108, Frederick MD 21702, United States ; Yalong Zhang(1); Olaf Ludek(1); David Farnsworth(1); Jeffrey Gildersleeve(1). (1) Chemical Biology Laboratory, National Cancer Institute, Frederick MD 21702, United States

Cancer vaccines lengthen survival in some patients, though others experience little or no benefit. Better understanding of tumor characteristics and host factors that influence the response to vaccination should help explain these survival differences between patients and lead to more widely effective vaccines.

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Important insights will likely come from monitoring humoral responses to carbohydrates. Although ubiquitious in cancer, abnormal tumor-associated carbohydrate antigens are overlooked aspects of antitumor immunity due to technical challenges of studying carbohydrate-protein interactions. We used a high-throughput glycoarray to study humoral responses induced in 28 subjects by a prostate cancer vaccine (PSA TRICOM). Post-vaccination changes in anti-carbohydrate antibodies were detected in the majority of patients, including responses to known tumor-associated carbohydrate antigens and the terminal disaccharide of the Forssman antigen (GalNAcα1-3GalNAcβ). These changes might reflect reactions to vaccine components or antigen spreading. Studies aimed at elucidating the origin of these changes are ongoing.

CARB 106

High-throughput glycoarray for monitoring immune responses to a cancer vaccine

Christopher Campbell(1), [email protected], 376 Boyles Street, Room 108, Frederick MD 21702, United States ; Yalong Zhang(1); Olaf Ludek(1); David Farnsworth(1); Jeffrey Gildersleeve(1). (1) Chemical Biology Laboratory, National Cancer Institute, Frederick MD 21702, United States

Cancer vaccines lengthen survival in some patients, though others experience little or no benefit. Better understanding of tumor characteristics and host factors that influence the response to vaccination should help explain these survival differences between patients and lead to more widely effective vaccines. Important insights will likely come from monitoring humoral responses to carbohydrates. Although ubiquitious in cancer, abnormal tumor-associated carbohydrate antigens are overlooked aspects of antitumor immunity due to technical challenges of studying carbohydrate-protein interactions. We used a high-throughput glycoarray to study humoral responses induced in 28 subjects by a prostate cancer vaccine (PSA TRICOM). Post-vaccination changes in anti-carbohydrate antibodies were detected in the majority of patients, including responses to known tumor-associated carbohydrate antigens and the terminal disaccharide of the Forssman antigen (GalNAcα1-3GalNAcβ). These changes might reflect reactions to vaccine components or antigen spreading. Studies aimed at elucidating the origin of these changes are ongoing.

CARB 107

NMR spectroscopic studies of APF: A small glycopeptide possessing potent antiproliferative activity

Kristie M Adams(1), [email protected], 376 Boyles St, Bldg 376, Frederick Maryland 21702, United States ; Piotr Kaczmarek(1); Susan K Keay(2)(3); Joseph J Barchi, Jr(1). (1) Chemical Biology Laboratory, National Cancer Institute-

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Frederick, Frederick Maryland 21702, United States (2) Department of Medicine, Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore Maryland 21201, United States (3) Research Service, Veterans Affairs Maryland Health Care System, Baltimore Maryland 21201, United States

Antiproliferative factor (APF) is a novel glycopeptide isolated from the urine of patients diagnosed with interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic bladder disease. APF is a negative growth factor that inhibits proliferation of normal bladder epithelial cells at subnanomolar concentrations, leading to the hypothesis that APF could possess anticancer properties. APF was found to markedly inhibit growth of both T24 bladder carcinoma and kidney cancer cell lines. Subsequent structural and SAR studies revealed that APF (as isolated from IC/PBS patients) is a nine-residue peptide containing sialyl-TF antigen α-O-linked to the N-terminal threonine residue, Neu5Acα2-3Galβ1-3GalNAcα-O-TVPAAVVVA. Neither the sialyl group nor the C-terminal alanine residue is necessary to maintain antiproliferative activity, yet other seemingly inconsequential structural changes completely abolish the activity of APF. Thus, we have structurally characterized APF and several APF analogues in aqueous solution using 2D NMR spectroscopic techniques in an attempt to further understand the structure of APF.

CARB 107

NMR spectroscopic studies of APF: A small glycopeptide possessing potent antiproliferative activity

Kristie M Adams(1), [email protected], 376 Boyles St, Bldg 376, Frederick Maryland 21702, United States ; Piotr Kaczmarek(1); Susan K Keay(2)(3); Joseph J Barchi, Jr(1). (1) Chemical Biology Laboratory, National Cancer Institute-Frederick, Frederick Maryland 21702, United States (2) Department of Medicine, Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore Maryland 21201, United States (3) Research Service, Veterans Affairs Maryland Health Care System, Baltimore Maryland 21201, United States

Antiproliferative factor (APF) is a novel glycopeptide isolated from the urine of patients diagnosed with interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic bladder disease. APF is a negative growth factor that inhibits proliferation of normal bladder epithelial cells at subnanomolar concentrations, leading to the hypothesis that APF could possess anticancer properties. APF was found to markedly inhibit growth of both T24 bladder carcinoma and kidney cancer cell lines. Subsequent structural and SAR studies revealed that APF (as isolated from IC/PBS patients) is a nine-residue peptide containing sialyl-TF antigen α-O-linked to the N-terminal threonine residue, Neu5Acα2-3Galβ1-3GalNAcα-O-TVPAAVVVA. Neither the sialyl group nor the C-terminal alanine residue is necessary to maintain antiproliferative activity, yet other seemingly inconsequential structural changes completely abolish the activity of APF. Thus,

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we have structurally characterized APF and several APF analogues in aqueous solution using 2D NMR spectroscopic techniques in an attempt to further understand the structure of APF.

CARB 108

Structural and quantitative analysis of disaccharides using CE with LIF detection

Yuqing Chang(1), [email protected], 110 8th ST, Troy NY 12180, United States ; Tatiana Laremore(1); Fuming Zhang(1); Robert J Linhardt(1). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection can be applied to the structural and quantitative analysis of unsaturated disaccharides. The sensitivity of this technique is attributed to the high intensity of the incident light and the ability to accurately focus the light on the capillary. By labeling the disaccharide reducing groups by proper fluorescent tags, such as fluorophore 2-aminoacridone (AMAC), and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY), hydrazide, the fluorotagged products can be separated by CE with appropriate electrophoretic conditions. The labeling process is simple and the conditions are mild. Using LIF as detection method at 488nm, the sensitivity could be largely improved compared to that was detected by UV detector at 255nm. With this strategy, it is possible to realize high sensitivity in the detection of Δ-disaccharides in trace amount.

CARB 108

Structural and quantitative analysis of disaccharides using CE with LIF detection

Yuqing Chang(1), [email protected], 110 8th ST, Troy NY 12180, United States ; Tatiana Laremore(1); Fuming Zhang(1); Robert J Linhardt(1). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection can be applied to the structural and quantitative analysis of unsaturated disaccharides. The sensitivity of this technique is attributed to the high intensity of the incident light and the ability to accurately focus the light on the capillary. By labeling the disaccharide reducing groups by proper fluorescent tags, such as fluorophore 2-aminoacridone (AMAC), and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY), hydrazide, the fluorotagged products can be separated by CE with appropriate electrophoretic conditions. The labeling process is simple and the conditions are mild. Using LIF as

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detection method at 488nm, the sensitivity could be largely improved compared to that was detected by UV detector at 255nm. With this strategy, it is possible to realize high sensitivity in the detection of Δ-disaccharides in trace amount.

CARB 109

Development of a novel cancer vaccine based on multivalent presentation of tumor-associated carbohydrate antigens on gold nanoparticle scaffolds

Raymond P Brinas(1), [email protected], 376 Boyles St., Frederick MD 21702, United States ; Andreas Sundgren(1); Micah Maetani(1); Omar Abbudayyeh(1); Howard A Young(2); Michael Sanford(2); Joseph J Barchi(1). (1) Chemical Biology Lab, National Cancer Institute, NIH, Frederick MD 21702, United States (2) Laboratory of Experimental Biology, National Cancer Institute, NIH, Frederick MD 21702, United States

We have designed and synthesized vaccine constructs based on gold nanoparticles (Au NPs) bearing multiple copies of a tumor-associated carbohydrate, Thomsen-Friedenreich (TF) disaccharide, which was O-linked to either serine or threonine residues of a specific, thiol functionalized 16-mer peptide repeating unit from a specific tumor-associated mucin (MUC4). The Au NPs were furtherfunctionalized with a segment from a form of the complement-derived protein, C3d, and with the linker that was used to attach the peptides to gold. Initial results on the pro-inflammatory response of pre-stimulated mouse bone marrow-derived macrophages when treated with the vaccine constructs indicated particle-specific modulation of particular cytokine release. Guided by these results, we attempted to optimize responses by exploring differences based on particle size and coating ligand. We will present the details of Au NP synthesis, as well as optimization of our vaccine design based on the immunological assays.

CARB 110

Carbon nanotubes and chitosan as possible scaffolds for bone tissue regeneration

Julia Stone(1), [email protected], PO Box 519 MS 2008, Prairie View TX 77446, United States ; Yetunde Olusanya(2); Whitney Jones(2); Pasakorn Traisawatwong(1); Melisa Stewart(1); Cordella Kelly-Brown(1); Laura Carson(1); Aderemi Oki(3); E. Gloria C. Regisford(2). (1) Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View TX 77446, United States (2) Department of Biology, Prairie View A&M University, Prairie View TX 77446, United States (3) Department of Chemistry, Prairie View A&M University, Prairie View TX 77446, United States

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Carbon nanotubes (CNTs) and chitosan exhibit characteristics necessary for successful bone tissue regeneration. An in vitro model for bone cell growth with CNTs and Chitosan added to cell culture medium of human fetal osteoblastic cells at concentrations of 0, 100, 250, 500, and 1000 ng/ml for 24 and 48 hours. SEM images exhibited no marked changes on CNTs or CTS morphology and composition. Cell proliferation was significantly higher in cells grown in the presence of 500 ng/mL of CNT, compared to controls. Western blot analysis revealed that OPN expression did not exceed that of control cells, although cells treated with 500 ng/ml CNTs showed the greatest expression. Cells treated with CTS had a dose dependent increase in OPN expression, with the highest expression at 1000 ng/ml. These data indicate that CNTs and CTS may be of great potential for therapeutic use in bone regeneration.

CARB 110

Carbon nanotubes and chitosan as possible scaffolds for bone tissue regeneration

Julia Stone(1), [email protected], PO Box 519 MS 2008, Prairie View TX 77446, United States ; Yetunde Olusanya(2); Whitney Jones(2); Pasakorn Traisawatwong(1); Melisa Stewart(1); Cordella Kelly-Brown(1); Laura Carson(1); Aderemi Oki(3); E. Gloria C. Regisford(2). (1) Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View TX 77446, United States (2) Department of Biology, Prairie View A&M University, Prairie View TX 77446, United States (3) Department of Chemistry, Prairie View A&M University, Prairie View TX 77446, United States

Carbon nanotubes (CNTs) and chitosan exhibit characteristics necessary for successful bone tissue regeneration. An in vitro model for bone cell growth with CNTs and Chitosan added to cell culture medium of human fetal osteoblastic cells at concentrations of 0, 100, 250, 500, and 1000 ng/ml for 24 and 48 hours. SEM images exhibited no marked changes on CNTs or CTS morphology and composition. Cell proliferation was significantly higher in cells grown in the presence of 500 ng/mL of CNT, compared to controls. Western blot analysis revealed that OPN expression did not exceed that of control cells, although cells treated with 500 ng/ml CNTs showed the greatest expression. Cells treated with CTS had a dose dependent increase in OPN expression, with the highest expression at 1000 ng/ml. These data indicate that CNTs and CTS may be of great potential for therapeutic use in bone regeneration.

CARB 111

Biosynthesis of heparin by metabolic engineering of Chinese hamster ovary cells

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Leyla Gasimli(1), [email protected], 12 Marshall Str, 1FL Rear apt, Troy NY 12180, United States ; Jongyoun Baik(2); Susan Sharfstein(2); Robert J. Linhardt(1)(3)(4). (1) Department of Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) College of Nanoscale Science and Engineering, State University of New York at Albany, Albany NY 12203, United States (3) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (4) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Heparin(HP), a highly sulfated polysaccharide, is a widely used anticoagulant with direct healthcare applications in the pharmaceutical market. The primary sources of heparin on the market are of animal origin. These supplies inherently involve risk of contamination in addition to uncontrollable qualitative differences among batches. Chinese hamster ovary(CHO) cells have been used as producers of functional proteins on an industrial scale. They naturally produce heparan sulfate(HS), which shares the biosynthesis pathway with HP. Therefore CHO cells are good candidates to produce clean and consistent HP on a large scale. Preliminary results show that genes coding for C5Epimerase, 2-O-sulfotransferase, and 6-O- sulfotransferases are expressed in CHO cells, whereas genes coding for N-deacetylase/N-sulfotransferase-2(NDST2), and 3-O-sulfotransferases(3OST) are not. NDST2 has been already transfected into cells. 3OSTs will also be transfected and overexpressed in cells with the goal that HP will be synthesized on the core protein of HS, greatly simplifying large scale purification.

CARB 111

Biosynthesis of heparin by metabolic engineering of Chinese hamster ovary cells

Leyla Gasimli(1), [email protected], 12 Marshall Str, 1FL Rear apt, Troy NY 12180, United States ; Jongyoun Baik(2); Susan Sharfstein(2); Robert J. Linhardt(1)(3)(4). (1) Department of Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (2) College of Nanoscale Science and Engineering, State University of New York at Albany, Albany NY 12203, United States (3) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States (4) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Heparin(HP), a highly sulfated polysaccharide, is a widely used anticoagulant with direct healthcare applications in the pharmaceutical market. The primary sources of heparin on the market are of animal origin. These supplies inherently involve risk of contamination in addition to uncontrollable qualitative differences among batches. Chinese hamster ovary(CHO) cells have been used as

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producers of functional proteins on an industrial scale. They naturally produce heparan sulfate(HS), which shares the biosynthesis pathway with HP. Therefore CHO cells are good candidates to produce clean and consistent HP on a large scale. Preliminary results show that genes coding for C5Epimerase, 2-O-sulfotransferase, and 6-O- sulfotransferases are expressed in CHO cells, whereas genes coding for N-deacetylase/N-sulfotransferase-2(NDST2), and 3-O-sulfotransferases(3OST) are not. NDST2 has been already transfected into cells. 3OSTs will also be transfected and overexpressed in cells with the goal that HP will be synthesized on the core protein of HS, greatly simplifying large scale purification.

CARB 112

Purification strategies for separation of capsular polysaccharide from fermentation broth

Ujjwal Bhaskar(1), [email protected], 4117, CBIS, 110 8th Street, Troy New York 12180, United States ; Zhenyu Wang(2); Fuming Zhang(2); Jonathan S. Dordick(1)(2); Robert J. Linhardt(1)(2). (1) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy New York 12180, United States (2) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy New York 12180, United States

In the recent past, contamination problems have arisen with heparin derived from porcine tissues. One solution to this issue is the synthesis of a bioengineered version of heparin, which could enable large-scale heparin production. Capsular polysaccharide derived from K5 strain of Escherichia coli provides a polysaccharide precursor for the development of bioengineered heparin. This study deals with the various purification strategies for the separation of the capsular polysaccharide, heparosan, from the fermentation broth of E. coli K5 culture. The purification makes use of the surface charge present on the polysaccharides based on the manipulation of solution pH. Separation methods including ion-exchange chromatography and affinity precipitation are explored in heparosan purification. Affinity precipitation-based separation systems, in place of ion exchange columns, can provide an efficient, lower cost separation. Naturally derived polycations are investigated as a possible mediator of affinity-based separation for heparosan.

CARB 113

Design, synthesis, and characterization of sulfated N-aryl aminoglycosides

Amanda M. Fenner(1), [email protected], 115 S. Grand Ave., S311 PHAR, Iowa City IA 52242, United States ; Robert J. Kerns(1). (1) Division of Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City IA 52242, United States

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Hundreds of eukaryotic and prokaryotic proteins bind to cell surface glycosaminoglycans (GAGs) to promote biological activities. Development of compounds to block GAG-protein interactions has primarily focused on optimizing the degree and orientation of anionic substituents on a scaffold, to mimic GAG structure, but their utility is diminished by non-specific interactions with many proteins. To overcome these limitations, our lab demonstrated that replacing N-sulfo groups on heparin with non-anionic N-arylacyl groups increased affinity and selectivity for binding different heparin-binding proteins. Here we report the preparation and characterization of N-aryl substituted tri- and tetrasaccharides followed by sulfonation of the hydroxyl groups. An LC-MS method to analyze components of the resulting mixtures was developed using ion-pair-RP-HPLC to separate oligosaccharides based on degree and position of sulfates, and ESI-MS to characterize individual molecules. Screening for biological activity indicates the aromatic substituent, core saccharide, and degree of sulfation are all important for differentiation of function.

CARB 113

Design, synthesis, and characterization of sulfated N-aryl aminoglycosides

Amanda M. Fenner(1), [email protected], 115 S. Grand Ave., S311 PHAR, Iowa City IA 52242, United States ; Robert J. Kerns(1). (1) Division of Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City IA 52242, United States

Hundreds of eukaryotic and prokaryotic proteins bind to cell surface glycosaminoglycans (GAGs) to promote biological activities. Development of compounds to block GAG-protein interactions has primarily focused on optimizing the degree and orientation of anionic substituents on a scaffold, to mimic GAG structure, but their utility is diminished by non-specific interactions with many proteins. To overcome these limitations, our lab demonstrated that replacing N-sulfo groups on heparin with non-anionic N-arylacyl groups increased affinity and selectivity for binding different heparin-binding proteins. Here we report the preparation and characterization of N-aryl substituted tri- and tetrasaccharides followed by sulfonation of the hydroxyl groups. An LC-MS method to analyze components of the resulting mixtures was developed using ion-pair-RP-HPLC to separate oligosaccharides based on degree and position of sulfates, and ESI-MS to characterize individual molecules. Screening for biological activity indicates the aromatic substituent, core saccharide, and degree of sulfation are all important for differentiation of function.

CARB 114

Photo-click immobilization of carbohydrates on polymeric surfaces: An effortless method to functionalize surfaces for biomolecular recognition studies

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Oscar Norberg(1), [email protected], Teknikringen 30, Stockholm 100 44, Sweden ; Lingquan Deng(1); Mingdi Yan(2); Olof Ramström(1). (1) Department of Chemistry, KTH - Royal Institute of Technology, Stockholm 100 44, Sweden (2) Department of Chemistry, Portland State University, Portland, United States

Carbohydrates play highly diverse roles in biological systems. To efficiently study the interactions between carbohydrates and proteins, efforts have been made in applying microarray technology to carbohydrates. However, there are numerous obstacles to overcome concerning the manufacture of carbohydrate microarrays, where robust and versatile formats are needed. In the present study a method has been developed for efficient carbohydrate attachment to different polymeric surfaces. A specific bifunctional linker was designed for the immobilization method, efficiently connecting the polymeric substrate to the carbohydrates. The method utilizes efficient photocoupling of stabilized perfluorophenyl azides (PFPAs) and highly chemoselective Copper catalyzed Azide- Alkyne Cycloaddition. The method enables a rapid and convenient protocol to the general attachment of azide-functionalized structures and was used to fabricate a range of surfaces presenting β-D-galactosides, α-D-mannosides and N-acetyl-β-D-glucosamines on different polymeric surfaces. The surfaces were evaluated in real-time studies using a QCM flow-through system with a series of different lectins.

CARB 115

On-line microflow high-performance liquid chromatography with nano-electrospray ionization mass spectrometry for heparan sulfate disaccharide analysis

Bo Yang(1), [email protected]; Kemal Solakyildirim(1); Jeffrey G. Martin(1); Tatiana Laremore(1); Robert J. Linhardt(1). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States

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The heparan sulfate and heparin, as an important kind of glycosaminoglycan (GAG), is composed acidic and linear chains of repeating multiple disaccharide units. They have crucial biological functions by binding to different growth factors, enzymes, morphogens, cell adhesion molecules, and cytokines. The characterization of HS/heparin with various sequences and structures is essential for elucidating the functions corresponding to these GAGs. Due to their structural complexity and heterogeneity, the structural analysis of these GAGs is still a long-standing challenge for analysts. In this work, a highly sensitive and fast method by ion pairing reversed-phase microflow high-performance liquid chromatography (IPRP-Mf-HPLC) with nano-electrospray ionization mass spectrometry was applied for separation and identification of HS/ heparin disaccharides. The new approach allows us to detect approximately 0.1 ng of per disaccharide.

CARB 115

On-line microflow high-performance liquid chromatography with nano-electrospray ionization mass spectrometry for heparan sulfate disaccharide analysis

Bo Yang(1), [email protected]; Kemal Solakyildirim(1); Jeffrey G. Martin(1); Tatiana Laremore(1); Robert J. Linhardt(1). (1) Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy New York 12180, United States

The heparan sulfate and heparin, as an important kind of glycosaminoglycan (GAG), is composed acidic and linear chains of repeating multiple disaccharide units. They have crucial biological functions by binding to different growth factors, enzymes, morphogens, cell adhesion molecules, and cytokines. The characterization of HS/heparin with various sequences and structures is essential for elucidating the functions corresponding to these GAGs. Due to their structural complexity and heterogeneity, the structural analysis of these GAGs is still a long-standing challenge for analysts. In this work, a highly sensitive and fast method by ion pairing reversed-phase microflow high-performance liquid chromatography (IPRP-Mf-HPLC) with nano-electrospray ionization mass spectrometry was applied for separation and identification of HS/ heparin disaccharides. The new approach allows us to detect approximately 0.1 ng of per disaccharide.

CARB 116

De novo synthesis of a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) building block for the preparation of the zwitterionic polysaccharide A1 (PS A1) repeating subunit of Bacteroides fragilis

Rajan Pragani(1)(2), [email protected], Am Muehlenberg 1, Potsdam Brandenburg 14476, Germany ; Peter H Seeberger(1)(2). (1) Department of

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Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Potsdam 14476, Germany (2) Department of Chemistry and Biochemistry, Freie Universitaet Berlin, Potsdam 14476, Germany

Zwitterionic polysaccharide A1 (PS A1) found in Bacteroides fragilis is the putative cause of postoperative intra-abdominal abscesses in humans and has been shown to activate a MHCII mediated T-cell response in the absence of proteins. Total synthesis of defined homogeneous PS A1 fragments would enable a more in-depth understanding of this important immunostimulant. Herein, we present our progress toward the synthesis of the PS A1 repeating subunit. Detailed accounts of the de novo synthesis of the 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) monosaccharide, and its use toward the completion of the PS A1 repeating subunit will be reported.

CARB 117

Automated solution-phase synthesis of alpha-galactosides

Rajarshi Roychoudhury(1), [email protected], 2612 Gilman, Ames IA 50011, United States ; Nicola L. B. Pohl(1). (1) Department of Chemistry and The Plant Sciences Institute, Iowa State University, Ames IA 50011, United States

Human cells do not express alpha-galactoside (alpha-Gal) epitopes. However, constant exposure to this epitope leads to humans producing high levels of antibodies against this carbohydrate motif. These antibodies can be used to target immune responses against other compounds that are conjugated to alpha-Gal. Unfortunately, the synthesis of this motif is particularly challenging as anchimeric assistance cannot be used to set the correct anomeric configuration. Here we present a systematic study of several activated galactose building blocks to ascertain the influence of conformational effects in determining the alpha/beta selectivity in glycosylation reactions. We also demonstrate the incorporation of the most selective building block in the development of the first automated solution-phase synthesis protocol for the incorporation of terminal alpha-galactosides.

CARB 118

Study of the protecting group participation and stereoselectivity of 2-azido-2-deoxygalactopyranosyl donors

Zhitao Li(1), [email protected], Binghamton University, Binghamton NY 13902, United States . (1) Department of Chemistry, Binghamton University, Binghamton NY 13902, United States

Protecting groups have profound effects on the stereoselectivity of glycosylation reactions. These effects include neighboring group participation, electronic effect

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and steric effect. Nonneighboring group participation was recently reported to be responsible for stereoselectivity in some glycosylation reactions. Our computational model study indicated that the nonneighboring group participation may also contribute to the stereoselectivity of glycosylation of 2-azido-2-deoxygalactosyl (GalN3) donors. A series of GalN3 donors with different protecting groups were synthesized and tested in glycosylation reactions. The relationship between the protecting pattern and the stereoselectivity of the reaction is consistent with the predictions based on our computational model. This result suggests that our theoretical model is effective and may be applied to develop more stereoselective donors.

CARB 119

Reactions of glycals with ortho-substituted benzanilines

Cecilia H Marzabadi(1), [email protected], 400 South Orange Ave, South Orange NJ 07079, United States ; Katherine Kochalski(1). (1) Chemistry and Biochemistry, Seton Hall University, South Orange NJ 07079, United States

Previously we reported on the successful reactions of glycals with benzanilines to form carbohydrate-based quinolines and tetrahydroquinolines. We were interested to see the outcome of these addition reactions when a nucleophilic group was substituted on the ortho-position of the aldehyde ring. In this presentation we will discuss the results of these studies and our efforts to prepare pyranobenzopyrans and other heterocycles from carbohydrate precursors. [ACS_F09_Marzabadi]

CARB 120

Novel glycolipids in CD1d-mediated immunity: Synthesis of new agonists for CD1d

Justyna Wojno(1)(2), [email protected], Edgbaston, Birmingham West Midlands B15 2TT, United Kingdom ; John-Paul Jukes(3); Paolo Polzella(3); Vincenzo Cerundolo(3); Liam R Cox(1); Gurdyal S Besra(2). (1) School of Chemistry, University of Birmingham, Birmingham B15 2TT, United Kingdom (2) School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom (3) Tumour Immunology Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX1 3QT, United Kingdom

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The glycolipid α-galactosyl ceramide, α-GalCer, has been shown to stimulate the proliferation of murine spleen cells and activate the immune system through binding to the protein CD1d. The induction of both Th1 and Th2 cytokines by α-GalCer is likely to limit its therapeutic utility. Analogues of α-GalCer have been shown to induce iNKT cell-derived cytokines more selectively through a skewed Th1-Th2 response. The crystal structure of human CD1d in complex with α-GalCer revealed a key hydrogen bond between the N-H of the amide group and the CD1d protein but the C=O is not involved in direct binding. A range of α-galactosyl ceramide and threitol ceramide analogues have been synthesised in which the amide group in our lead has been replaced with different carbonyl functional groups. Additional modifications will also be presented along with the synthesis of labelled derivatives, which will find application in trafficking studies.

CARB 121

Automated synthesis of systematic di- and tri-saccharide libraries

Xin Liu(1), [email protected], 2606 Gilman, Ames IA 50011, United States ; Beatrice Y. M. Collet(2); Shu-Lun Tang(1); Sahana K. Nagappayya(1); Rajarshi Roychoudhury(1); Xueshu Li(1); Heather Edwards(1); Nicola L. B. Pohl(1). (1) Department of Chemistry and The Plant Sciences Institute, Iowa State University, Ames IA 50011, United States (2) LuCella Biosciences, Inc., Ames IA 50011, United States

The lack of availability of small carbohydrate libraries with systematic structural variations has limited a rigorous study of the role of stereochemical features on the biological activity of carbohydrates as well as the development of robust methods such as mass-spectrometry-based carbohydrate sequencing, NMR structural databases, and predictive computational tools for carbohydrate structures. To address these issues as well as probe the scope and limitations of automated oligosaccharide synthesis, the synthesis of the first di- and tri-saccharide library is reported with particular focus on the incorporation of the mass identical monomers glucose, galactose, and mannose. The large-scale preparation and process issues involved in the synthesis of monosaccharide building blocks necessary for the synthesis of the library are also discussed.

CARB 122

Probe of activated glycoside building block stability for automated solution-phase synthesis of carbohydrate libraries

Beatrice Y. M. Collet(1), [email protected], 1030 Roy J. Carver Co-Lab, Ames IA 50011, United States ; Xin Liu(2); Shu-Lun Tang(2); Heather Edwards(2); Sahana Nagappayya(2); Lin Liu(2); Nicola L. B. Pohl(2). (1) LuCella Biosciences, Inc., Ames IA 50011, United States (2) Department of Chemistry and The Plant Sciences Institute, Iowa State University, Ames IA 50011, United States

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Trichloroacetimidate-activated glycosides are commonly used as donor building blocks in the synthesis of carbohydrate-based macromolecules. However the stability of these building blocks is often debated and could potentially limit their use in automated synthesis protocols. In the context of the automated solution-phase synthesis of di- and tri-saccharide libraries, the preparation of a variety of Schmidt trichloroacetimidate donors with systematic structural variations will be reported. The stability of these building blocks at four different storage temperatures will be presented and structure-related patterns in the degradation processes will be proposed.

CARB 123

Synthesis of unnatural glycosaminoglycans and evaluation of their interaction with proteins

Smritilekha Bera(1), [email protected], 110, 8th St, BIOTECH, 4005, Troy NY 1280, United States . (1) Department of chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy NY 12180, United States

Glycosaminoglycans (GAGs) play a wide range of physiological and pathological events as a decisive agent for cell-cell communications, cell proliferation, angiogenesis, inflammatory processes, wound repair and healing, viral invasion as well as a growth factor receptor and enzyme inhibitors and so on. These molecules are linear, anionic and having repetitive disaccharide motifs and discriminated in between and within GAG families by structural architecture with slight variations in stereochemistry, length, and patterns of sulfation. This structural diversity creates an enormous number of protein binding motifs and also has featured daunting challenge to define the structural and functional properties of GAGs, which could be implicated in many diseases and targets of therapeutic approaches. Heparin sulfate, one of the most important natural glycosaminoglycans, is used as anticoagulant drug and exploited in treating cancers and suppressing inflammatory responses and chondroitin sulfate (CS), an another essential glycosaminoglycans play vital roles in biological processes, such as neural development, viral invasion, cancer metastasis, arthritis, and spinal cord injury etc. Although, these GAGs are generally isolated from living animals, synthetic chemistry offers a potentially more reliable and practicable route to homogeneous glycosaminoglycan leading to either exact copies of naturally occurring glycosaminoglycan or, alternatively, incorporating unnatural proteolytically stable glycosidic linkages. Thus in a word, these artificially designed glycosaminoglycans might enhance its in vivo potentiality by retaining the geometric and spatial characteristics of the native polysaccharides and exhibiting stability toward N- and O-glycosyl hydrolase activity or inhibiting these enzymes to some extent. In addition, the artificial linkage may be finely tuned as an inert functionality during synthetic transformations within the synthesis of

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target molecule. Our initial target is the synthesis of unnaturally linked glycosaminoglycans of heparin and condroitin sulfate subsequently the investigation of their interaction with protein.

CARB 124

Grafting of cellulose esters by single electron transfer living radical polymerization Set-LrP

Petr Vlcek(1), [email protected], Heyrovsky Sq. 2, Praha 16206, Czech Republic ; Vladimir Raus(2); Miroslav Janata(3); Jaroslav Kriz(4); Petra Latalova(5); Eva Cadova(6). (1) Department of Controlled Polymerization, Institute of Macromolecular Chemistry, Praha 16206, Czech Republic (2) Department of Controlled Polymerization, Institute of Macromolecular Chemistry, Czech Republic (3) Department of Controlled Polymerization, Institute of Macromolecular Chemistry, Czech Republic (4) Department of structure analysis, Institute of Macromolecular Chemistry, Czech Republic (5) Department of Controlled Polymerization, Institute of Macromolecular Chemistry, Czech Republic (6) Department of Controlled Polymerization, Institute of Macromolecular Chemistry, Czech Republic

Cellulose diacetate and acetate butyrate were functionalized with 2-bromoisobutyrylbromide or dichloroacetylchloride, giving polyfunctional macroinitiators for controlled radical polymerization, having various number of functional groups. Recently, they have been used for controlled grafting by ATRP. Here, the macroiniator were grafted with (meth)acrylates and their mixtures and this polymerization has been initiated with the Percec's system, composed of zero-valent copper (fine powder) and amine-type ligands in polar solvent. The grafting proceeds as a controlled proces, semilogarithmic dependence of monomer consumption on reaction time is linear. The grafts are capable of polymerization of another monomer, giving rise to block copolymer-type grafts. The process is relatively fast and the formed products contain virtually negligible amount of residual catalyst components and, therefore, they do not require additional purification by repeated precipitation or column chromatography. Thus, the SET-LRP is a convenient method for controlled synthesis of graft copolymers with cellulose, and perhaps, also other backbone.

CARB 125

Solvation and hydrolysis of cellulose with transition metal salts

Veronika Viner(1), [email protected], 1900 North Knox Rd., Stop #6303, China Lake CA 93555, United States ; Benjamin G. Harvey(1), [email protected], 1900 North Knox Rd., Stop #6303, China Lake CA 93555, United States ; Roxanne L Quintana(1). (1) Resarch Department, Chemistry Division, NAVAIR-NAWCWD, China Lake CA 93555, United States

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The solvation and hydrolysis of cellulose to sugar oligomers and glucose in concentrated aqueous solutions of FeCl3 and ZnCl2 were studied. The effect of the concentrations of the metal salts and cellulose, reaction temperatures, and incorporation of additional acid catalysts was examined. The salts could be conveniently separated from the products by complexation with ether, allowing for almost quantitative recovery. The products were analyzed by UV-Vis spectroscopy, HPLC, and ICP-AES to determine product distributions, conversions, and the concentrations of metal impurities in the products. High salt concentrations protected the glucose from further reaction, allowing for excellent yields of glucose under moderate conditions. To address more realistic feedstocks, heterogeneous sources of cellulose, including wood and paper, were subjected to optimized hydrolysis conditions. Both feedstocks were readily solvated with moderate conversion to glucose. These results are promising for the development of an efficient system for the conversion of waste biomass to glucose.

CARB 126

Chemical derivatization of glucan microparticles for targeted delivery

Ernesto R Soto(1), [email protected], 373 Plantation Street, Biotech 2, Suite 113, Worcester MA 01604, United States ; Gary R Ostroff(1). (1) Program in Molecular Medicine, University of Massachusetts Medical School, Worcester MA 01604, United States

We have previously reported on targeted delivery of siRNA to macrophages using siRNA encapsulated β-glucan particles (GP). GP are 2-4 μm hollow and porous microspheres that facilitate uptake by cells bearing β-glucan receptors (dectin-1 and CR3) thus providing for targeted delivery to innate immune cells (macrophages, dendritic cells, and neutrophils). To extend the application of the GP delivery technology to non-professional phagocytic cells, we have developed methods to chemically graft targeting ligands to the glucan particle surface to specific cell surface receptors. We will detail the methods and results of chemically modifying GPs with galactose to target particles to hepatocytes via the asialoglycoprotein receptor, or with arginine–glycine–aspartic acid (RGD)-containing peptides to target particles to endothelial cells and fibroblasts via the β1 integrin receptor.

CARB 127

Degradation mechanism and crystal structure change of cellulose during TEMPO-NaOCl-NaBr selective oxidation and its biomedical application as hemostatics

Bin Sun(1), [email protected], 2999 North Renmin Road,Songjiang, Room C402,Buld 5, Shanghai Shanghai 201620, China ; Wenxiu Han(2); Wei Wang(2);

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Jinhong Ma(1); Chunju Gu(1); Meifang Zhu(1); Borun Liang(1). (1) State Key Lab for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, Shanghai 201620, China (2) Key Laboratory of Science and Technology of Eco-textile, Ministry of Education, College of Chemical Science and Engineering, Donghua University, Shanghai 201620, China

The functional modification of cellulose and its application in the field of biomedical materials has been considered as one of the most important research directions, which attracted much attention in recent years. In this contribution,the degradation extent and its mechanism of the cellulose during TEMPO-NaOCl-NaBr selective oxidation process have been explored. The crystal structure and absorbable hemostatic properties of the celluronic acid have been characterized in detail. It is suggested that the significant degradation at the initial stage is certainly associated with the fringed-micelle structure that rayon fiber has and the β-elimination induced by the formation of the aldehyde intermediate in the alkaline solution. The fully oxidized celluronic acid sodium salt exhibits amorphous structure in character whereas the celluronic acid obtained after treating with acid exhibits clear crystal structure in character, which is quite similar to the crystal modification of cellulose �.

The change of the hydrogen bond structure owing to the introduction of the carboxyl group at C6 position was discussed.

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The results upon preliminary animal test indicate that the oxidized cellulose products have good hemostatic and absorbable properties which has rapid hemostasis in 2 minutes, and is basically absorbed in the body of rabbits within 6 days.

CARB 128

Temperature-dependent chain-collapse behavior of cellulose ethers in dilute solution

Hongwei Shen(1), [email protected], 2301 N Brazosport Blvd., B-1220/Rm101, Freeport TX 77541, United States ; Robert Sammler(1); David Redwine(1); David Meunier(1); Meinolf Brackhagen(1). (1) The Dow Chemical Company, Freeport TX 77541, United States

Chain behavior of cellulose ethers (polysaccharides) in dilute aqueous solution was studied using light scattering techniques. Radius of gyration (Rg) and hydrodynamic radius (Rh) were determined for various types of cellulose ethers over a range of concentrations and temperatures. A critical association concentration (CAC) well below its chain overlap concentration (C*) was found. This suggests polymer chain is in an associated state even below C*. Polymer chains at a concentration between CAC and C* are random coils at room temperature but form aggregates as temperature increases above a critical value. Below CAC, at a few ppm, Rg and Rh can decrease significantly with increasing temperature above a critical value and the polymer chain changes from random coils to constrained coils to spherical shape. This is the first experimental evidence on chain collapse for cellulose ethers. The degree of chain collapse is discussed in term of chain stiffness, the persistence length.

CARB 129

Improving dyeability of nylon wiith nano-chitosan

Yau Shan Szeto(1), [email protected], Hung Hom, Kowloon, Hong Kong Special Administrative Region of China; Ho Yan Wong(1); Li Wei Liu(1); Hoi Ying Lui(1). (1) Institute of Textiles $ Clothing, The Hong Kong Polytechnic University, Hung Hom, Hong Kong Special Administrative Region of China

The dyeability of nylon treated with chitosan nano-particles was studied. Nano-particles of chitosan were applied onto the surface of nylon fabric using a pad-dry-cure method. The chitosan-treated and the untreated nylon fabrics were dyed using acid dyes with different numbers of sulphonic acid groups at 1%, 2% and 4% depths respectively. It was found that the rate of dyeing and the final exhaustion of the chitosan-treated nylon fabric were increased. The effect of dye structures on exhaustion was also studied. Regarding the physical property of

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the fabric, the coefficient of friction of the chitosan-treated fabric was similar to that of the control. The colour fastness to water (ISO105-E01:1994) of the dyed fabrics was also assessed. It was shown that treatment with chitosan did not lead to deterioration of the colour fastness to water of nylon. In summary, the study indicated that the depth of dyeing of nylon could be increased with the chitosan treatment.

CARB 130

Modeling of Congo red adsorption on a surface of crystalline cellulose using molecular dynamics

Miroslaw Wyszomirski(1), [email protected], Willowa 2, Bielsko-Biala, Poland ; Karim Mazeau(2). (1) Faculty of Environmental and Materials Sciences, University of Bielsko-Biala, Bielsko-Biala 43-309, Poland (2) Centre de Recherches sur les Macromolécules Végétales CNRS, Grenoble 38041, France

Congo red is an azo-dye which interacts with cellulose surface. Understanding these interactions might help understanding of adsorption of cellulose binding modules of cellulases, because they share the same hydrophobic mechanisms, with implications for cellulose hydrolysis. In our efforts to identify key variables we modeled interactions between Congo red and cellulose microfibril in explicit aqueous environment using molecular dynamics (Accelrys Materials Studio ). We found structural requirements for these interactions to understand ruling mechanisms and to establish likely binding sites on cellulose surfaces.

CARB 131

Evaluation of glycopolymers and glyconanoparticles for biosensing and gene delivery applications

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Ravin Narain(1), [email protected], 116 St and 85 Ave, Edmonton AB, Canada ; Marya Ahmed(1). (1) Chemical and Materials Engineering, University of Alberta, Edmonton AB T6G 2G6, Canada

Synthetic carbohydrate based nano-structured materials are receiving an enormous attention in recent years since those materials have been shown to mimic natural polysaccharides, glycopeptides and glycoproteins in biological systems. Peptides and proteins with carbohydrate moieties attached to their backbone have many desirable biological properties, but their chemical synthesis is a technical challenge. Therefore, designing carbohydrate based materials that can perform similar functions as glycoproteins or can recognize glycoproteins is a viable approach for better understanding of the cellular recognition processes such as uptake, cell growth regulation, differentiation, adhesion, cellular trafficking, inflammation by bacteria and viruses and the immune response. Carbohydrate based materials for biomedical applications have other benefits such as improving the solubility of the materials in physiological conditions, imparting biocompatibility in the materials (especially for drug and gene delivery applications), and enhancing specificity (e.g. glycopolymers with galactose residues have higher recognition towards hepatocytes). Recently, we have made significant advances in the design of biologically relevant glycopolymers and glyconanoparticles. For instance, we have studied the specific interactions of carbohydrate residues with specific types of lectins. We have also studied the biocompatibility, the biomolecular recognition processes, cellular uptake and transfection efficiencies of the glyconanoparticles. In this talk, I will summarize on the preparation and biological applications of well-defined glycopolymers and monodisperse glyconanoparticles.

CARB 132

Application of data mining techniques to differentiate glucose-containing disaccharide ions fragmented via infrared-multiple photon dissociation using tunable lasers and Fourier transform ion cyclotron resonance mass spectrometry

Mohammad U. Ehsan(1), [email protected], PO Box 117200, Gainesville FL 32611, United States ; Sarah Stefan(1); Alexander Aksenov(1); Vladimir Boginksi(1); Brad Bendiak(2); John Eyler(1). (1) Department of Chemistry, University of Florida, Gainesville FL 32611, United States (2) Department of Cell and Developmental Biology, Denver Colorado, United States

Data mining techniques were used to analyze mathematically the infrared multiple photon dissociation (IRMPD) fragmentation patterns of gas-phase lithiated disaccharide isomers irradiated with both a tunable CO2 laser and a free electron laser (FEL). The IR fragmentation patterns over the wavelength range of 9.2 to 10.6 µm have been shown in earlier work to give diagnostic fragmentation and showed distinct correlation with geometry of the anomeric carbon.

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Application of data mining approaches for data analysis allowed for unambiguous determination of anomeric carbon configurations for each disaccharide isomer pair using fragmentation data for a single wavelength. This combination of wavelength-selective IRMPD and data mining may offer a powerful and convenient tool for structural differentiation of gas-phase carbohydrate complexes.

CARB 133

Influence of the structure of phosphoramidates on flame retardant properties of cellulose

Victoria Salimova(1), [email protected], Lerchenfeldstrasse 5, Sankt Gallen Sankt Gallen 9014, Switzerland ; Sabyasachi Gaan(1); Joëlle Grützmacher(2). (1) Department of Advanced Fibers, Empa, Materials Science and Technology, Sankt Gallen 9014, Switzerland (2) Department of Inorganic Chemistry, ETH Swiss Federal Institute of Technology, Zurich 8092, Switzerland

The most effective flame retardants for cellulose contain phosphorus and nitrogen, either in the same compounds (Pyrovatex® CP, Ciba) or separate compounds (Proban® CC, Rhodia). Phosphorus in various chemical states interact with cellulose through acidic intermediates to reduce its flammability, and nitrogen is believed to enhance the action of phosphorus through the synergistic effect. Although numerous investigations have been performed on the reaction mechanisms of phosphorus-containing compounds, the exact mechanism of action was not elucidated so far. It has been shown, that different classes of organophosphorus compounds exhibit different flame retardant behaviour, when combined with cellulose. This research focuses on the effect of phosphoramidate structure on thermal decomposition and burning behavior of cellulose. For this study, dimethyl- (DMP), diethyl- (DEP), di-n-butyl- (DBP), di-isopropyl- (DIP) and diphenyl-phosphoramidates (DPP) were taken. The structures of these phosphoramidates differ in the nature of the ester group that, as it was shown, has a significant influence on the flame retardant properties of treated cellulose. Dimethyl-, di-n-butyl-, and di-isopropylphosphoramidates were synthesized according to the Todd-Atherton reaction. Cellulose in form of cotton fabrics and microcrystalline cellulose (Avicel© PH-101) were used in this study. Fabrics as well as microcrystalline cellulosic powder were impregnated with the phosphoramidates from ethanol solution and dried in the oven at 60ºC for 2 hours. The amount of phosphorus retained on the fabrics and powder was controlled using elemental analysis. The burning behavior of phosphoramidates-treated samples was estimated using Limiting Oxygen Index Test. The highest values of LOI and burning rate were found to be for DMP-treated cellulose. Thermal behaviour was evaluated using thermogravimetric analysis (TGA) and microscale combustion calorimetry (MCC).

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The results from these measurements correlated well with LOI values. DMP-treated cellulose exhibited lowest decomposition temperature, highest yield of char at 600ºC and lowest heat release rates. The surface of the chars left after the LOI test was investigated using Scanning Electron Microscopy (SEM) and elemental analysis. The DMP-, DEP-, DIP- and DPP-chars were shown to have similar morphologies, whereas on the surface of DBP-char some swellings were observed. Elemental analysis of fabrics before combustion and their chars after combustion revealed that during the burning process nothing else but cellulose is burned away, leaving the polymeric coating formed from phosphoramidate. Further attempts to get an insight into the mode of actions of phosphoramidates were contrived using evolved gas analysis techniques such as TGA-FTIR-MS. Here, gases evolved during the TGA were fed to the FT-IR gas cell and mass spectrometer over a pre-heated transfer line. Analysis of those gases revealed that different reactions are taking place phosphoramidates with distinctive structure react with cellulose.

CARB 134

High-flux thin-film nanofibrous composite ultrafiltration membranes containing cellulose barrier layer

Hongyang Ma(1), [email protected], Stony Brook University, Stony Brook University New York 11794, United States ; Benjamin S. Hsiao(1), [email protected], Stony Brook University, Stony Brook New York 11794, United States ; Benjamin Chu(1), [email protected], Stony Brook University, Stony Brook New York, United States . (1) Department of Chemistry, Stony Brook University, Stony Brook New York 11794, United States

A novel class of thin-film nanofibrous composite (TFNC) membrane consisting of a cellulose barrier layer, a nanofibrous mid-layer scaffold, and a melt-blown non-woven substrate was successfully fabricated and tested as an ultrafiltration (UF) filter to separate an emulsified oil and water mixture, a model bilge water for on-board ship bilge water purification. Two ionic liquids: 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate, were chosen as the solvent to dissolve cellulose under mild conditions. The permeation flux of the cellulose-based TFNC membrane was significantly higher (e.g. 10X) than comparable commercial UF membranes (PAN10 and PAN400, Sepro) with similar rejection ratios for separation of oil/water emulsions. As ionic liquids can be recycled and reused without obvious decomposition, the chosen method also demonstrates a benign pathway to fabricate the cellulose barrier layer for other types of membranes.

CARB 135

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Discovery of 6-deoxy-D-altrose in nature

Masakuni Tako(1), [email protected], 1 Senbaru, Nishihara Okinawa 903-0213, Japan ; Takuya Yogi(1); Ken Izumori(2); Hideharu Ishida(3); Makoto Kiso(3). (1) Department of Sub-tropical Bioscience and Biotechnology, University of the Ryukyus, Nishihara Okinawa 903-0213, Japan (2) Rare Sugar Research Center, Kagawa University, Miki Kagawa 761-0761, Japan (3) Department of Applied Biochemistry, Gifu University, Gifu Gifu 501-1112, Japan

A novel acetylfucoidan has been isolated [1,2] and covered by patent [3] from commercially cultured Cladosiphon okamuranus. The acetylfucoidan has now been produced on the industrial scale and used commercially as the supplement of health foods and cosmetics additives in Japan [4]. We report here the isolation of a novel polysaccharide which substituted 6-deoxy-D-altrose from fruiting bodies of a edible mushroom (Lactarius akahatsu). The paper is the second report to identify 6-deoxy-D-altrose in nature[5]. Fruiting bodies of L. akahatsu were collected on January and February, 2008 in Okinawa, Japan. Dried powdered samples were suspended in distilled water and stirred at 100� for 3h to extract a polysaccharide. Total carbohydrate and uronic acid of the polysaccharide was 76.5% and 9.8%, respectively. After acid hydrolysis of the polysaccharide, D-Glc, D-Gal, D-GlcUA and unkown sugar was identified by HPLC and The 6-deoxy sugar residue was detected by 1H- and 13C-NMR spectroscopy of the polysaccharide. The 6-deoxy sugar was identified as a 6-deoxy-D- or L-altrose by 1H- and 13C-NMR, COSY and HMQC spectra. The specific rotation of the sugar was estimated to be +18.2 degree. Thus, the unkown sugar was identified as the 6-deoxy-D-altrose. References:[1] M. Tako et al.: J. Appl. Glycosci., 43, 143-148 (1996).[2] M. Tako et al.: Botanica Marina, 43, 393-398 (2000).[3] M. Tako: Japanese Patent No. 3371124 (2002).[4] M. Tako: Botanica Marina, 46, 461-465 (2003).[5]M. Tako et al.,15th European Carbohydrate Symposium, July 19-24, 2009, Wien, Austria.