Chemical Synthesis of Bacterial Siderophores and ...
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Open Access Theses Theses and Dissertations
January 2015
Chemical Synthesis of Bacterial Siderophores andApplications in Pathogen DetectionGregory Gene JarvisPurdue University
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Recommended CitationJarvis, Gregory Gene, "Chemical Synthesis of Bacterial Siderophores and Applications in Pathogen Detection" (2015). Open AccessTheses. 1110.https://docs.lib.purdue.edu/open_access_theses/1110
PURDUE UNIVERSITY GRADUATE SCHOOL
Thesis/Dissertation Acceptance
To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.
Gregory G. Jarvis
CHEMICAL SYNTHESIS OF BACTERIAL SIDEROPHORES AND APPLICATIONSIN PATHOGEN DETECTION
Master of Science
Philip S. Low
Christine A. Hrycyna
David Thompson
Philip S. Low
R. E. Wild 03/09/2015
i
CHEMICAL SYNTHESIS OF BACTERIAL SIDEROPHORES AND APPLICATIONS
IN PATHOGEN DETECTION
A Thesis
Submitted to the Faculty
of
Purdue University
by
Gregory G. Jarvis
In Partial Fulfillment of the
Requirements for the Degree
of
Master of Science
May 2015
Purdue University
West Lafayette, Indiana
ii
ACKNOWLEDGEMENTS
I would like to take this opportunity to thank all of my family, friends, and
colleagues who have supported me throughout my time studying chemistry at Purdue. I
sincerely appreciate the time that post doctoral researchers Rajesh Pandey, Madduri
Srinivasarao, and Srinivasa Tenneti have willingly dedicated to mentoring me during my
time at Purdue, allowing me thrive as an independent researcher. I especially want to
thank my parents, Gary and Terri Jarvis, who have always been there to provide sound
wisdom and guidance through the arduous times that accompany graduate school. They
have always encouraged me to follow my own path, allowing me to become the
individual that I am today.
iii
TABLE OF CONTENTS
Page
LIST OF FIGURES ............................................................................................................ v
LIST OF SCHEMES.......................................................................................................... vi
LIST OF ABBREVIATIONS ........................................................................................... vii
ABSTRACT ....................................................................................................................... ix
CHAPTER 1. AN IMPROVED SYNTHESIS OF PETROBACTIN, A BACILLUS ANTHRACIS SIDEROPHORE……..…………………………………………........................1
1.1 Abstract ........................................................................................................ 1 1.2 Introduction .................................................................................................. 1 1.3 Experimental Section ................................................................................... 3 1.3.1 Chemicals and Materials .......................................................................... 3 1.3.2 Equipment ................................................................................................. 3 1.3.3 Synthesis of 3-(dibenzylamino)propyl methanesulfonate (3) .................. 3 1.3.4 Synthesis of tert-Butyl (4-((3- (dibenzylamino)propyl)amino)butyl)carbamate (5) .................................. 4 1.3.5 Synthesis of tert-butyl (4-((3-aminopropyl)amino)butyl)carbamate (6).. 4 1.3.6 Synthesis of methyl 3,4-bis(benzyloxy)benzoate (9) .............................. 4 1.3.7 Synthesis of tert-Butyl (4-((3-(3,4-
bis(benzyloxy)benzamido)propyl)amino)butyl)carbamate (10) .............. 5 1.3.8 Synthesis of N-(3-((4-aminobutyl)amino)propyl)-3, 4-bis(benzyloxy)benzamide (11) ................................................................ 5 1.3.9 Synthesis of 2-tert-Butyl-1,3-di-N-(hydroxyl)-Succinimidyl Citrate (13) ................................................................................................ 6 1.3.10 Synthesis of tert-butyl 4-((4-((3-(3,4- bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-(2-((4-((3-(3,4- bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-oxoethyl)-2- hydroxy-4-oxobutanoate (14) .................................................................. 6 1.3.11 Synthesis of Petrobactin (1) ...................................................................... 6 1.4 Results and Discussion ................................................................................. 7 1.5 Conclusion .................................................................................................... 8
iv
Page CHAPTER 2. CHEMICAL SYNTHESIS OF STAPHYLOFERRIN A AND ITS EVALUATION FOR USE IN A PATHOGEN-DETECTION DEVICE…………….…….10
2.1 Abstract ...................................................................................................... 10 2.2 Introduction ................................................................................................ 10 2.3 Experimental Section ................................................................................. 12 2.3.1 Chemicals and Materials ........................................................................ 12 2.3.2 Equipment ............................................................................................... 13 2.3.3 Synthesis of Staphyloferrin A ................................................................ 13 2.3.4 Preparation of Staphyloferrin A-BSA Conjugate ................................... 15 2.3.5 Preparation of Staphyloferrin A-BSA-patterned Chips .......................... 15 2.3.6 Growth of S. aureus ................................................................................ 16 2.3.7 Capture and Imaging of Chips ............................................................... 17 2.4 Results and Discussion ............................................................................... 17 2.5 Conclusion .................................................................................................. 18
CHAPTER 3. SELECTIVE CAPTURE OF S. AUREUS USING IMMOBILIZED SIDEROPHORE STAPHYLOFERRIN A…………………………………………………...21
3.1 Abstract ...................................................................................................... 21 3.2 Introduction ................................................................................................ 21 3.3 Experimental Section ................................................................................. 23 3.3.1 Chemicals and Materials ........................................................................ 23 3.3.2 Equipment ............................................................................................... 23 3.3.3 Synthesis of Staphyloferrin A ................................................................ 24 3.3.4 Preparation of Staphyloferrin A-BSA conjugate .................................... 25 3.3.5 Preparation of Staphyloferrin A-BSA-patterned chips ........................... 25 3.3.6 Growth of S. aureus ................................................................................ 25 3.3.7 Capture and Imaging of Chips ............................................................... 26 3.3.7.1 Evaluation of Limit of Detection ......................................................... 26 3.3.7.2 Time-course Study ............................................................................... 26 3.3.7.3 Evaluation of Chip Specificity ............................................................ 27 3.4 Results and Discussion ............................................................................... 27 3.5 Conclusion .................................................................................................. 29
LIST OF REFERENCES .................................................................................................. 35
PUBLICATIONS .............................................................................................................. 41
v
LIST OF FIGURES
Figure ............................................................................................................................. Page 1.1 Structure of petrobactin, 1 ............................................................................................ 8
2.1 Capture of S. aureus using a lab-on-a-chip device consisting of SA-BSA stamped on a gold substrate ............................................................................................................ 20
3.1 Chemical structure of ferric-staphyloferrin A with a short peptide linker for use in capture studies (15) ........................................................................................................... 30
3.2 The microcontact printing process used on gold chips to produce our lab-on-a-chip device. ............................................................................................................................... 32
3.3 Capture efficiency based on concentration of S. aureus ............................................. 33
3.4 Time-course study to examine the effect of bacterial exposure time to the chip ....... 33
3.5 Specificity of capture device when tested against a variety of human pathogens ...... 34
vi
LIST OF SCHEMES
Scheme Page
1.1 Chemical synthesis of the first building block of petrobactin ...................................... 8
1.2 Chemical synthesis of catechol subunit of petrobactin ................................................. 9
1.3 Chemical synthesis of functional petrobactin ............................................................... 9
2.1 Chemical synthesis of SA using SPPS techniques ..................................................... 19
3.1 Solid-phase synthesis of 15 ......................................................................................... 31
vii
LIST OF ABBREVIATIONS
PCR: Polymerase chain reaction
Sb(OEt)3: Antimony triethoxide
TEA: Triethylamine
DMSO: Dimethyl sulfoxide
EtOAc: Ethyl acetate
i-PrNH2: Isopropylamine
AcOH: Acetic acid
MRSA: Methicillin-resistant Staphylococcus aureus
VRSA: Vancomycin-resistant Staphylococcus aureus
SA: Staphyloferrin A
ABC transporter: ATP-binding cassette transporter
SPPS: Solid-phase peptide synthesis
3-t-Bu-citrate: 3-(tert-butoxycarbonyl)-3-hydroxypentanedioic acid
BSA: Bovine serum albumin
EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
PDMS: Polydimethylsiloxane
PBS: Phosphate-buffered saline
DCM: Dichloromethane
viii
THF: Tetrahydrofuran
DMF: N,N-Dimethylformamide
PyBOP: Benzotriazole-1yl-oxytripyrrolidinophosphonium hexafluorophosphate
DIPEA: N,N-Diisopropylethylamine
MeOH: Methanol
(Ph3P)4Pd0: Tetrakis(triphenylphosphine)palladium(0)
PhSiH3: Phenylsilane
TFA: Trifluoroacetic acid
TIPS: Triisopropylsilane
LoD: Limit of detection
ix
ABSTRACT
Jarvis, Gregory G. M.S., Purdue University, May 2015. Chemical Synthesis of Bacterial Siderophores and Applications in Pathogen Detection. Major Professor: Philip S. Low. Bacterial siderophores are a class of small organic compounds that are produced
endogenously by bacteria to chelate essential ferric iron from their surroundings. Because
the bioavailability of ferric iron is extremely low is physiological pH, the host of a
parasitic infection can be a poor source of soluble Fe3+. Consequently, bacteria rely upon
siderophore transport systems to efficiently scavenge the nutrient. Nearly every bacterial
species ever isolated has evolved a set of unique enzymatic pathways for de novo
synthesis of these compounds, thus exists an inimitable relationship between a genus or
species of bacteria and the siderophores it produces.
Our goal was to exploit the highly-refined interaction that takes place between a
siderophore and its corresponding membrane-bound transporter by utilizing it as means
to capture and identify a pathogen more rapidly and economically than standardized PCR
methodology. To achieve this, we developed a robust lab-on-a-chip device that consists
of a surface-mounted siderophore that is capable of capturing its cognate pathogen with a
high degree of accuracy. With this device, bacterial capture occurred in a predetermined
pattern, and was verified using a benchtop light microscope. This simple and relatively
inexpensive method of pathogen detection may allow for widespread use of the device,
while simultaneously reducing the time it takes to diagnose bacterial infections.
x
Until recently, researchers have obtained siderophores for research using
complicated, time-consuming techniques that involve isolating siderophore from large
bacterial supernatants followed by extensive purification. Our goal was to avoid
disadvantages associated with siderophore isolation methodology by chemically
synthesizing siderophores. This would allow us to circumvent the use of potentially
hazardous biologics while simultaneously improving product yield and drastically
simplifying the purification process. The following work describes an improved approach
for chemical synthesis of petrobactin, a Bacillus anthracis siderophore, as well as the first
chemical synthesis of staphyloferrin A (SA), a siderophore of Staphylococcus aureus. We
also outline the implementation of SA in an S. aureus detection device and report on its
efficacy.
1
CHAPTER 1. AN IMPROVED SYNTHESIS OF PETROBACTIN, A BACILLUS ANTHRACIS SIDEROPHORE
1.1 Abstract
A B. anthracis siderophore, petrobactin, was synthesized chemically with 65%
total yield. The synthesis includes application of a novel amide-ester exchange reaction
using Sb(OEt)3 under neat conditions, which considerably improved the chemical yield of
functional petrobactin when compared to other reported syntheses.
1.2 Introduction
Petrobactin, 1, is an iron-chelating siderophore produced by pathogenic bacterium
Bacillus anthracis (see figure 1.1).1 It has been shown to be a high-affinity scavenger of
sparingly-soluble ferric iron from the extracellular environment of a B. anthracis
infection.2-4 Since human pathogens require iron as an essential nutrient, the
reinternalization of the ferrisiderophore complex is crucial to the survival of the bacterial
cell.5, 6
In order to study siderophore-based iron acquisition systems of B. anthracis in the
most efficient manner possible, the availability of research quantities of petrobactin
becomes a legitimate concern. Typically, siderophores are isolated from supernatants of
bacterial cultures and purified several times to obtain a product worth of research.7
Because of the complexity of this process, overall yield can be low, so researchers must
2
extract from large cultures of bacteria to obtain appreciable amounts of the material.
When dealing with highly pathogenic B. anthracis, siderophore isolation methodology
can pose significant health risks to researchers.8
In order to address these problems, members of the scientific community have
since devised methods to produce petrobactin utilizing both enzymatic and chemical
synthesis techniques.9-13 These techniques, however, involve difficult, inefficient
reactions that require refining in order to improve overall petrobactin yield. Consequently,
we chose to undertake the task of optimizing petrobactin chemical synthesis so
quantifiable amounts of the siderophore can be produced in a reasonably straightforward,
convergent synthesis.14
3
1.3 Experimental Section
1.3.1 Chemicals and Materials
Spectrograde solvents were used without further purification. Anhydrous DCM,
DMSO, DMF, EtOAc, MeOH, H2SO4, methanesulfonyl chloride, 3-(dibenzylamino)-1-
propanol, N-Boc-1,4-butanediamine, 3,4-dihydroxybenzoic acid, PhSiH3, DIPEA, TEA,
triphenylphosphine, and (Ph3P)4Pdo were purchased from Sigma Aldrich (St. Louis, MO).
Antimony (III) ethoxide was purchased from ProChem, Inc. (Rockford, IL). PyBOP was
purchased from GenScript (Piscataway, NJ).
1.3.2 Equipment
All NMR spectra were recorded on either 400 MHz Varian or 500 MHz Bruker
spectrometers.
1.3.3 Synthesis of 3-(dibenzylamino)propyl methanesulfonate (3)
Methanesulfonyl chloride was added dropwise to the cooled solution of 3-
(dibenzylamino)-1-propanol (2) and TEA in DCM. The mixture was stirred at 0oC. After
completion of the reaction (2 h) water was added and the crude mixture was extracted
with DCM, washed with brine, dried over anhydrous Na2SO4 and concentrated. The
residue was purified by column chromatography (NH- silica gel, hexanes/EtOAc 9.5:0.5)
to afford 3 as a colorless gum.
4
1.3.4 Synthesis of tert-Butyl (4-((3-(dibenzylamino)propyl)amino)butyl)carbamate (5)
A mixture of 3, N-Boc-1,4-butanediamine (4) and DIPEA in dry DMSO was
stirred at 80oC under nitrogen environment for 24 h. The reaction was quenched with
water. The layers were separated and the aqueous layer was extracted several times with
EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, and
concentrated. The concentrated residue was purified by column chromatography (NH-
silica gel, hexanes/EtOAc 9.5:0.5 to 7:3) to afford 5 as a colorless gum.
1.3.5 Synthesis of tert-butyl (4-((3-aminopropyl)amino)butyl)carbamate (6)
A mixture of 5 and Pd/C (10%) in ethanol was refluxed under H2(g) for 4 h. The
suspension was filtered over celite and the filtrate was concentrated to give the crude
product, 6, which was used in the next step.
1.3.6 Synthesis of methyl 3,4-bis(benzyloxy)benzoate (9)
To a solution of 3,4-dihydroxybenzoic acid (7) in MeOH was added a catalytic
amount of concentrated H2SO4 and the mixture was refluxed for 8h. The reaction mixture
was cooled to room temperature and solvent was removed by rotary evaporation. The
remaining residue was dissolved in EtOAc and washed with saturated NaHCO3 and brine.
The combined extract were dried over Na2SO4, and concentrated to give the crude methyl
ester 8. To the residue was added a solution of benzyl bromide and K2CO3 in DMF. The
mixture was heated at 120oC. After 36 h, the reaction mixture was cooled to ambient
temperature and poured into ice-cold water. The mixture was then passed through cotton
5
to remove K2CO3. The mixture was extracted with EtOAc 3 times. The organic layers
were washed with ice-cold 1 M HCl, water, brine, dried over Na2SO4, and concentrated.
The residue was purified by column chromatography (silica gel, hexanes/EtOAc 9.5:0.5)
to afford 9 as a colorless gum.
1.3.7 Synthesis of tert-Butyl (4-((3-(3,4-bis(benzyloxy)benzamido)propyl)amino)butyl)carbamate (10)
To a mixture of 6, 9 was added with Sb(OEt)3 in neat conditions at room
temperature and the flask was purged with N2(g). The mixture was heated at 80oC for 24
h. After cooling to room temperature, the mixture was quenched with MeOH and filtered
through pad of celite using CHCl3, MeOH, then i-PrNH2. The resulting filtrate was
concentrated with a rotary evaporator and the resulting residue was purified by column
chromatography on Cromatorex®-NH-DM1020 silica gel using a mixture of
EtOAc:MeOH (100:0 to 100:1) as a mobile phase to give 10.
1.3.8 Synthesis of N-(3-((4-aminobutyl)amino)propyl)-3, 4-bis(benzyloxy)benzamide (11)
TFA was added dropwise to the cooled solution of 10 in DCM. The mixture was
stirred at 0oC. After completion of reaction (2 h), reaction mixture was concentrated and
it was used as the TFA salt without any purification.
6
1.3.9 Synthesis of 2-tert-Butyl-1,3-di-N-(hydroxyl)-Succinimidyl Citrate (13)
13 was synthesized in 4 steps from citric acid (12) according to previously
described procedures.15
1.3.10 Synthesis of tert-butyl 4-((4-((3-(3,4-bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-(2-((4-((3-(3,4-
bis(benzyloxy)benzamido)propyl)amino)butyl)amino)-2-oxoethyl)-2-hydroxy-4-oxobutanoate (14)
11 was dissolved in dioxane and DCM and TEA was added to the stirring flask to
facilitate deprotection of the TFA salt. 13 was then added to the flask and the reaction
was allowed to proceed for 1.5 h after which the solvent was removed by rotary
evaporation. The residue was dissolved in EtOAc and extracted 3 times with H2O. The
organic layer was isolated and dried over MgSO4. The mixture was filtered through
cotton and the filtrate was concentrated using rotary evaporation to yield 14.
1.3.11 Synthesis of Petrobactin (1)
14 was dissolved in DCM/H2O (100:1) and the mixture was cooled to 0°C. TFA
was added to the solution and the reaction was allowed to warm to room temperature.
After 2 h, the solvent was removed by rotary evaporation, and placed under high vacuum
to ensure complete removal of TFA. Benzylated petrobactin was recovered and
redissolved in a mixture of AcOH/H2O (20:1) in a flask purged with H2. 10% Pd/C was
added to the flask and the reaction was stirred for 2h under H2. The mixture was filtered
through cotton and the filtrate was extracted 3 times with EtOAc. The pH of the aqueous
7
layer was neutralized with 1M K2CO3 and the resulting solution was lyophilized to yield
petrobactin (1).
1.4 Results and Discussion
To synthesize 1 (see figure 1.1), we designed a convergent synthesis using 3
building blocks that were individually modified and protected for later assembly. In order
to prepare the first building block, outlined in Scheme 1.1, we started with commercially
available 2, which was O-mesylated, providing the terminal hydroxyl with an adequate
leaving group for subsequent nucleophilic substitution. The mesylated product, 3, was
reacted with the mono-protected diamine, 4, to yield a protected version of the first
building block, compound 5. In order to deprotect the terminal amine group, a
debenzylation reaction was performed using catalytic hydrogenation to yield 6 as a free
primary amine.
Scheme 1.2 demonstrates preparation of the protected dihydroxybenzoic acid
moeity required for petrobactin synthesis. Commercially available 7 was refluxed in
H2SO4/MeOH to esterify the aryl carboxylic acid, yielding 8. The catechol component of
compound 8 was then protected using benzyl bromide to produce compound 9.
Assembly of these two building blocks was performed in the first step of Scheme
1.3, where 6 and 9 were coupled using Sb(OEt)3 by heating the reaction in neat
conditions . This important step in petrobactin synthesis notoriously produces low-yields
of 10 using standard peptide-coupling reagents, so we modified the reaction to include a
high-yielding Sb(OEt)3-mediated ester amide transformation reaction based on
procedures outlined in the literature.12, 16 Boc was removed from the terminal amine with
T
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. This compo
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1
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1.5 Conclu
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9
10
CHAPTER 2. CHEMICAL SYNTHESIS OF STAPHYLOFERRIN A AND ITS EVALUATION FOR USE IN A PATHOGEN-DETECTION DEVICE
2.1 Abstract
Siderophores are unique molecules that can be used to capture bacteria that
express a specific receptor for them. In order to test this hypothesis, we synthesized a
siderophore unique to S. aureus called staphyloferrin A, and stamped it in a distinctive
pattern on a gold chip using microcontact printing. Upon introduction of the chip to a
solution containing S. aureus, capture of the pathogen was witnessed within the confines
of pattern where SA was stamped. These experiments provide evidence of an interaction
that occurrs between S aureus and SA. These findings are significant because they imply
that siderophores are capable of capturing bacteria on a small device that can be imaged
using optical microscopy.
2.2 Introduction
S. aureus is a gram-positive bacterium that commonly resides on human epithelial
tissues and nasal mucosa. It is a community microbe that thrives in environments with
unsanitary conditions, and is believed to be a primary causative factor of an estimated
500,000 surgical site infections of hospital patients in the United States alone.17 Among
the most pathogenic of these infections are caused by MRSA and VRSA – antibiotic-
11
resistant mutants of S. aureus that require rigorous and potentially toxic treatment
regimens.18-20
In order to effectively treat nosocomial pathogens like S. aureus, we must focus our
priorities on early detection. The ability to diagnose communicable diseases in the early
stages of progression is a powerful tool we have against prohibiting rapid transmission of
the disease throughout exposed populations. Diagnosis involves isolation of the bacteria,
pre-enrichment, and characterization using RT-PCR, a process that consumes a
significant amount of time that could be spent treating the patient and preventing disease
communication.21
To expedite the process of pathogen detection and identification, we have
developed a device with the sole purpose of specifically capturing S. aureus. It is a lab-
on-a-chip device that can be imaged with a benchtop optical microscope at low
magnification.22, 23 The device was designed to allow for clear distinction between a
blank chip and a chip containing captured bacteria. With dark field microscopy, the
bacteria can be easily detected as they scatter light and appear as bright yellow spots
against a black background.
It is exceedingly important that this device is able to capture S. aureus with a high
degree of specificity, since the capturing of other strains of bacteria can give misleading
data to clinicians, which can effectively lead to disease misdiagnosis and subsequent
mistreatment. We attempted to overcome this problem by taking advantage of a particular
interaction that occurs between the ABC transporter HtsA, located on the outer
membrane of S. aureus, and its cognate siderophore SA.24-26
12
Siderophores are small organic chelators that bacteria biosynthesize and release
into their extracellular environment for sequestration of ferric iron.27 The iron-
siderophore chelate is reinternalized by bacterial cells using a specialized receptor
complex. Once inside the cell, Fe(III) is reduced to Fe(II) and released from the
siderophore to be incorporated into essential metabolic processes.28 Herein, we wish to
report on the methods developed to chemically synthesize SA using SPPS, and the
implementation of the siderophore in a pathogen detection device.22
2.3 Experimental Section
2.3.1 Chemicals and Materials
Fmoc-D-Ornithine(Alloc)-OH was purchased from Iris Biotech (Marktredwitz,
Germany). Dichloromethane (DCM), methanol (MeOH), and dimethylformamide (DMF)
were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Triphenylphosphine (PPh3),
tetrahydrofuran (THF), diisopropyl azodicarboxylate, Stratospheres™ PL-Wang resin,
piperidine, diisopropylethylamine (DIPEA), phenylsilane (PhSiH3),
tetrakis(triphenylphosphine)palladium ((Ph3P)4Pd0), citric acid, trifluoroacetic acid (TFA),
iron(III) chloride, bovine serum albumin (BSA), phosphate-buffered saline (PBS), and
2,2’-bipyridine were purchased from Sigma-Aldrich (Saint Louis, MO). Benzotriazol-1-
yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was purchased from
GenScript (Piscataway, NJ). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was
purchased from Bio-Rad (Hercules, CA).
13
2.3.2 Equipment
All solid-phase synthesis reactions were performed in a solid-phase peptide
synthesis vessel from Chemglass Life Sciences (Vineland, NJ). Hilic Atlantis Silica
HILIC OBD Preparative HPLC column was purchased from Waters Corporation
(Milford, MA). Gold surface plasmon resonance (SPR) chips were purchased from
Reichert Technologies (Depew, NY). All images were taken with an Olympus BX43
LED Upright Microscope.
2.3.3 Synthesis of Staphyloferrin A22
Fmoc-D-Orn(Alloc)-OH and triphenylphosphine were added to DCM:THF (1:1),
and the solution was cooled to 0°C in an ice bath. DIAD was added dropwise to the
solution to prevent generation of excess heat. The solution was then added to a SPPS
vessel containing StratoSpheresTM PL-Wang resin, 100–200 mesh that was pre-swollen
in DCM for 10 min prior to addition. Ar(g) was bubbled through the solution for 36 h,
allowing maximum loading of the residue onto the resin. The solution was removed from
the SPPS vessel and the resin was washed with 3 x 5 mL DCM, 3 x 5 mL THF, 3 x 5 mL
DMF, and 3 x 5 mL DCM. The resin was treated with a solution of 20% piperidine in
DMF to remove the N-terminal Fmoc (3 x 5 mL) over the course of 30 minutes. The
Fmoc-cleavage solution was drained from the vessel and the resin was washed with 3 x 5
mL DMF. After verifying the presence of a primary amine on the residue with a Kaiser
test, a solution of 3-t-Bu-citrate,15 PyBOP, and DIPEA in 4 mL DMF was introduced to
the vessel. The reaction was bubbled with Ar(g) for 12 h. After removing the coupling
solution and washing the resin with 3 x 5 mL DMF, 3 x 5 mL MeOH, and 3 x 5 mL
14
DCM , a Kaiser test was used to determine completion of the reaction by signifying the
lack of a free primary amine on the resin. Next, a solution of (Ph3P)4Pd0 and PhSiH3 in
DCM was added to the resin to remove Alloc from the ornithine amine. The reaction was
allowed to bubble with Ar(g) for 2 h, the solution was drained, and the resin was washed
with 3 x 5 mL DCM, 3 x 5 mL DCM:DIPEA (19:1), 3 x 5 mL DMF, and 3 x 5 mL DCM.
A positive Kaiser test affirmed the presence of a primary amine on the resin. A solution
of 3-t-Bu-citrate, PyBOP, and DIPEA in DMF was introduced to the vessel. The reaction
was bubbled with Ar(g) for 12 h to ensure reaction completion. After removing the
coupling solution from the vessel, the resin was washed with 3 x 5 mL DMF, 3 x 5 mL
MeOH, and 3 x 5 mL DCM. A negative Kaiser test indicated that the reaction had
reached completion. In order to cleave SA from the resin and simultaneously deprotect
tert-butyl groups present on citrate moieties, a solution of 92.5% TFA, 2.5% DCM, 2.5%
H2O and 2.5% TIPS was added to the vessel and allowed to bubble with Ar(g) for 1 h.
The cleavage solution was drained into a round bottom flask, and combined with two
additional portions of the cleavage solution used to wash the resin remaining in the vessel.
The cleavage solution was concentrated under reduced pressure to approximately 2 mL,
making certain to avoid heating the solution beyond 40° C while evaporating.10 To the
concentrate was added diethyl ether and the precipitate was collected by centrifugation.
After decanting the supernatant, the precipitate was washed with diethyl ether and
centrifuged three additional times to remove any remaining ether-soluble components.
The precipitate was dried under high vacuum to yield the crude mixture of diastereomers
as white powder. The material was purified by preparative HPLC using an Atlantis Silica
HILIC OBD column (19 x 150 mm, 5 μm) with a 10 mM ammonium acetate buffer (pH
15
7) and acetonitrile mobile phase yielding two chromatographically distinguishable
diastereomers of SA.
2.3.4 Preparation of Staphyloferrin A-BSA Conjugate
14 mmol of SA was dissolved in deionized water and gently stirred as a 0.2 M
aqueous FeCl3 solution was added dropwise. The chelation reaction was allowed to stir
for 5 min and lyophilized. In order to prepare “ink” used for PDMS stamping, 96 μmol of
EDC was combined with 28 μmol of staphyloferrin A-Fe in 375 μL of pH 7.4 PBS. This
mixture was stirred at room temperature for 30 minutes to activate the ornithine
carboxylate. A 1 mL solution of 0.9 mmol (60 mg) BSA in PBS was prepared and added
to the reaction and stirred for an additional 2 h. After completion of the reaction, the
mixture was filtered using 0.45 μm membrane filter. The filtrate was collected and either
used for immediate PDMS stamping, or lyophilized for later use.
2.3.5 Preparation of Staphyloferrin A-BSA-patterned Chips
To produce a PDMS stamp that will create a pattern visible by low magnification
optical microscopy, a silicon wafer was etched with 20 μm wide parallel lines with 20 μm
gaps using UV photolithography. The stamp was fabricated by pouring viscoelastic liquid
PDMS over the silicon master and allowing it to cure. The hardened PDMS was
henceforth able to be used for microcontact printing applications. To apply ink to the
stamp, a cotton swab was dipped in 50 uL of SA-BSA ink, rolled onto the textured side
of the PDMS, and allowed to dry in the inverted position after removing excess liquid
with a dry cotton swab. The inked stamp was then placed on a gold-coated glass chip,
16
pressed lightly for 10 seconds, and allowed to sit on top of the chip for an additional 5
minutes. The stamp was carefully removed and the chip was sealed in a sterile Petri dish
for 5 hours at 4°C. To block portions of the chip unstamped by the PDMS, a 5% (w/w)
BSA solution was prepared in pH 7.4 in PBS and applied to the chip for 5 minutes. The
BSA solution was removed with a sterile pipette and the chip was rinsed thoroughly with
3 x 5 mL portions of pH 7.4 PBS.
2.3.6 Growth of S. aureus
A frozen stock of S. aureus was used to seed a growth culture in standard LB
broth at 37°C on an orbital shaker overnight. 10 μL of the seed culture was transferred to
low-iron LB media, which was prepared by adding 2,2′-bipyridine to a concentration of
0.2 mM. The bacteria were grown to a concentration of 1 x 108 CFU/mL by measuring
optical density at 600 nm with a spectrophotometer and diluted with pH 7.4 PBS to the
appropriate concentration for experiments.
2.3.7 Capture and Imaging of Chips
50 uL of 1 x 106 CFU/mL S. aureus was dispensed onto siderophore-stamped
gold chips and incubated in a closed, sterile Petri dish for 1 h. The solution was removed
with a sterile pipette and the chip was rinsed thoroughly with 3 x 5 mL pH 7.4 PBS and
deionized water respectively. The chips were dried with a gentle stream of Ar(g) to
remove any excess water. Dry chips were imaged using dark field optical microscopy at
10x magnification.
17
2.4 Results and Discussion
In order to exploit the receptor-ligand interaction that exists between SA and
HtsA, we chose to design a synthesis scheme which allowed us to chemically produce SA.
Prior to this study, the siderophore was obtained through isolation from live S. aureus, a
laborious, time-consuming process requiring growth of a large amount of bacteria,
extraction from the bacterial supernatant, and multiple purifications.29 By synthesizing
SA, we are able to considerably simplify this process by using dependable and high-
yielding peptide-coupling strategies on solid-phase resin. Synthesizing the ligand on solid
phase eliminates the need for purification after each reaction, a compulsory practice
associated with conventional solution-phase syntheses. This approach also allows us to
circumvent purification concerns associated with contamination from other chemically-
similar siderophores or small molecules produced by S. aureus that are normally present
in a crude culture supernatant.
Scheme 2.1 demonstrates how SA can be methodically synthesized using SPPS
techniques. After cleavage from the resin and HPLC purification, SA was chelated to
Fe3+ and simultaneously reacted with BSA using traditional EDC-coupling techniques in
an aqueous medium.
A dilute solution of SA-BSA conjugate was stamped onto gold chips using a
patterned PDMS stamp. Portions of the chip where the pattern was not present were
blocked with BSA to ensure there was no reactive gold exposed on the chip. The chip
was exposed to a solution of 106 CFU/mL S. aureus in order to examine its ability to
capture the pathogen for the purpose of identification. The chip was later imaged using
dark field microscopy at 10x magnification. Capture was witnessed and is exemplified in
18
figure 2.1. When compared to images taken of the same chip prior to exposure to S.
aureus, captured bacteria are readily observable as bright yellow clusters that contrast
against the dark background of the chip. Since the SA-BSA conjugate was stamped in a
specific pattern and the capture reflects this pattern, it is discernible that there is no
observable interaction between S. aureus and BSA alone, but exclusively between the
siderophore and its cognate receptor on the cell surface of the pathogen.
2.5 Conclusion
Though the siderophore-patterned chip shows promise as an effective means of
pathogen capture and detection in these and other preliminary studies,23, 30 additional
experiments are required to investigate the limit of detection, the specificity towards S.
aureus versus other human pathogens, and the minimum amount of time required for
discernable capture.
We have learned that the chemical synthesis of SA and the production of the
pathogen capture device are relatively inexpensive and straightforward processes. It can
also be suggested that this method of pathogen detection may significantly reduce the
amount of time required to assay for a specific strain of bacteria when compared to
commonly accepted methods in the literature.31, 32 This is an exceedingly important
characteristic for a pathogen detection system, since rapidly-communicable diseases can
disseminate through a population in a very short period of time. In addition to these
benefits, now that a simple, high-yielding chemical synthesis of SA has been developed,
its other potential applications can be investigated without the need for time-consuming
culture isolation methodology.26, 33, 34 Because of this, we anticipate the use of SA in a
19
variety of other applications, ranging from its use as a constituent in targeted antibiotic
delivery constructs, to its potential function as a metal chelator for use in patients with
iron-overload disorders.35-41
Scheme 2.1. Chemical synthesis of SA using SPPS techniques.22
HO
WangResin-OH
AllocHN O
O
NH
Fmoc-D-Orn(alloc)-OH
DIAD, PPh330 h
20% piperidine in DMF
30 min
AllocHN O
O
NHFmoc
92.5% TFA, 2.5% H2O,
2.5% DCM, 2.5% TIPS1 h
HN O
O
HNO
tBuOOC
OOH
OHO
OHO
tBuOOC
(PPh3)4Pdo, PhSiH3,
DCM
2 h
HN OH
O
HNO
HOOC
OOH
OHO
OHO
HOOC
O
tBuOOC
OOH
OH
OH OH
AllocHN O
O
NH2
PyBOP, DIPEA, DMF12 h
H2N O
O
NH
O
tBuOOC
OOH
OH
HOO
COOtBu
OHO
HO
PyBOP, DIPEA, DMF12 h
HOO
COOtBu
OHO
HO
Figure 2.1stamped omagnifica
. Capture of on a gold subation. (Left)
f S. aureus usbstrate. Imag
Prior to S. a
sing a lab-onges were takeaureus captu
n-a-chip deven using dar
ure. (Right) A
vice consistinrk field micrAfter S. aure
ng of SA-BSoscopy at 10eus capture.2
20
SA 0x 22
21
CHAPTER 3. SELECTIVE CAPTURE OF S. AUREUS USING IMMOBILIZED SIDEROPHORE STAPHYLOFERRIN A
3.1 Abstract
The necessity for a quick and inexpensive diagnostic tool for bacterial disease has
become increasingly essential as dangerous mutants arise through antibiotic resistance
mechanisms and total eradication of some pathogenic strains remains difficult.17-19, 42, 43
In order to address this problem, we developed a pathogen detection device that is
capable of rapidly and specifically capturing S. aureus at concentrations as low as 1 x 102
CFU/mL. This technology takes advantage of an innate high-affinity interaction that is
present between HtsA, a membrane-bound transporter on S. aureus, and staphyloferrin A,
a unique S. aureus siderophore called that has been immobilized on a gold chip.22, 26, 33
3.2 Introduction
Population mobility is an increasingly important concern when it comes to the
rapid spread of communicable diseases throughout the world. Advances in transportation
technology have allowed us to quickly and easily translocate, dramatically increasing the
probability of major disease outbreaks and pandemics.44 Consequently, researchers and
clinicians must lead the movement to develop pathogen detection devices that are not
only capable of specific pathogen identification, but are also inexpensive, simple to use,
and easy to obtain anywhere in the world.
22
To achieve this, we have constructed a lab-on-a-chip device consisting of an
immobilized, S. aureus-specific ligand that captures the pathogen via a receptor-ligand
interaction. The ligand, a siderophore called SA, is biosynthesized by S. aureus, and is
released into the extracellular environment to sequester ferric iron, The ferrisiderophore
complex is then reinternalized by the bacteria, where captured iron can be incorporated
into essential intracellular redox processes.24-27, 34
Because there is a unique interaction between SA and HtsA, a membrane-
spanning ABC transporter responsible for uptake of the ferrisiderophore complex, we
designed a lab-on-a-chip device that uses support-bound SA to capture S. aureus. The
siderophore was loaded onto BSA, which was then reacted with the gold surface of the
chip in a predetermined pattern specified by microcontact PDMS stamping. S. aureus can
be captured by SA present on the chip, and the bacterial cells can be imaged using dark
field microscopy.
After the first solid-phase chemical synthesis of SA was published by our
laboratory in 2014,22 we chose to expand upon this procedure by adding a peptidic spacer
(15) that should improve siderophore loading onto BSA and S. aureus capture efficiency.
We also undertook the task of evaluating the siderophore’s ability to selectively capture
the pathogen in a variety of conditions and exposure times.
23
3.3 Experimental Section
3.3.1 Chemicals and Materials
Fmoc-D-Ornithine(Alloc)-OH was purchased from Iris Biotech (Marktredwitz,
Germany). Dichloromethane (DCM), methanol (MeOH), and dimethylformamide (DMF)
were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Triphenylphosphine (PPh3),
tetrahydrofuran (THF), diisopropyl azodicarboxylate, Stratospheres™ PL-Wang resin,
piperidine, diisopropylethylamine (DIPEA), phenylsilane (PhSiH3),
tetrakis(triphenylphosphine)palladium ((Ph3P)4Pd0), citric acid, trifluoroacetic acid (TFA),
iron(III) chloride, bovine serum albumin (BSA), phosphate-buffered saline (PBS), and
2,2’-bipyridine were purchased from Sigma-Aldrich (Saint Louis, MO). Benzotriazol-1-
yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was purchased from
GenScript (Piscataway, NJ). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was
purchased from Bio-Rad (Hercules, CA).
3.3.2 Equipment
Solid-phase peptide synthesis vessels from Chemglass Life Sciences (Vineland,
NJ) were used in all solid-phase synthesis reactions. Atlantis Silica HILIC OBD
Preparative HPLC column was purchased from Waters Corporation (Milford, MA). Gold
surface plasmon resonance (SPR) chips were purchased from Reichert Technologies
(Depew, NY). Images were taken with an Olympus BX43 LED Upright Microscope.
24
3.3.3 Synthesis of 1522
To a SPPS vessel containing 2-chlorotrityl resin pre-loaded with H-Cys(Trt),
DCM was added and bubbled with N2(g) to swell the resin. The solvent was drained, and
a solution of 20% piperdine in DMF was added to the vessel and bubbled for 30 min to
remove the terminal Fmoc. The solution was drained and the resin was washed with 3 x 5
mL DMF, 3 x 5 mL MeOH, and 3 x 5 mL DCM. A solution of Fmoc-L-Asp(OtBu)-OH,
PyBOP, and DIPEA in DMF was added to the vessel and bubbled with N2(g) for 5 h. The
reaction mixture was drained and the resin was washed with 3 x 5 mL DMF, 3 x 5 mL
MeOH, and 3 x 5 mL DCM. This process of Fmoc deprotection, peptide coupling, and
extensive washing was repeated with the following residues: Boc-L-Dap(Fmoc)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-D-Orn(Alloc)-OH and the first 3-tBu-citrate moiety. A
solution of (PPh3)4Pdo and PhSiH3 in DCM was then added to the reaction vessel to
deprotect the Alloc-protected ornithine amine. The resin was washed and reacted with the
second 3-tBu-citrate moiety using the same conditions as prior peptide couplings. To
cleave compound 1 from the resin, a solution of 95 % TFA, 2.5% ethanedithiol, and 2.5%
TIPS was added to the resin and bubbled with N2(g). The cleavage mixture was collected
and concentrated by rotary evaporation at room temperature.10 A white powder was
precipitated from the crude concentrate using diethyl ether and the precipitate was
washed with additional portions of diethyl ether. The white powder was purified by
HPLC using a Waters Atlantis Silica HILIC OBD Preparative HPLC column with 10
mM ammonium acetate buffer (A) and acetonitrile (B) (95%B - 65%B) over 30 min.
25
3.3.4 Synthesis of 15-BSA conjugate
Refer to section 2.3.4 for details.
3.3.5 Preparation of 15-BSA-patterned chips
See figure 3.2 and section 2.3.5 for details.
3.3.6 Growth of S. aureus
Refer to section 2.3.6 for details.
26
3.3.7 Capture and Imaging of Chips
3.3.7.1 Evaluation of Limit of Detection
50 μL of S. aureus in pH 7.4 PBS was dispensed onto siderophore-patterned gold
chips in a sterile Petri dish at 1 x 106, 1 x 105, 1 x 104, 1 x 103, or 1 x 102 CFU/mL and
incubated for 1 h. The chips were then washed with 3 x 5 mL pH 7.4 PBS and 3 x 5 mL
deionized water to remove any unbound bacteria from the chip. The chips were then
imaged using dark field microscopy at 40x magnification.
3.3.7.2 Time-Course Study
50 μL of 1 x 106 CFU/mL S. aureus in pH 7.4 PBS was dispensed onto
siderophore-patterned gold chips in a sterile Petri dish and incubated for the following
times: 5 min, 30 min, 1 h, 2 h. Afterwards, the chips were washed with 3 x 5 mL pH 7.4
PBS and 3 x 5 mL deionized water to remove any unbound bacteria from the chip. The
chips were imaged using dark field microscopy at 40x magnification.
27
3.3.7.3 Evaluation of Chip Specificity
50 μL of pH 7.4 PBS containing 1 x 106 CFU/mL S. aureus, E. coli, P. aeruginosa,
S. flexneri, V. cholerae, Y. enterocolitica, or S. enterica was dispensed onto siderophore-
patterned gold chips in a sterile Petri dish and incubated for 1 h. Afterwards, the chips
were each washed with 3 x 5 mL pH 7.4 PBS and 3 x 5 mL deionized water to remove
any unbound bacteria from the chip. The chips were imaged using dark field microscopy
at 10x magnification.
3.4 Results and Discussion
Compound 15 was produced using established solid-phase synthesis techniques,
and the synthetic scheme we developed was based on a similar synthesis of SA published
by our laboratory in 2014.22 Our recent findings have provided us with insight on how to
produce high yields of enantiomerically pure 15 (see figure 3.1).
Beginning with preloaded H-Cys(Trt)-2-chlorotrityl resin, we systematically
added Fmoc-L-Asp(OtBu)-OH, Boc-L-Dap(Fmoc)-OH, and two Fmoc-Glu(OtBu)-OH
residues to the resin respectively using SPPS strategies. SA was modularly synthesized at
the N-terminus of the peptidic spacer with addition of differentially deprotectable Fmoc-
Orn(Alloc)-OH and two 3-t-Bu-citrate residues. Compound 15 was cleaved from the
resin, purified by HPLC, and used in pathogen detection studies in order to assess its
ability to capture S. aureus.
To expand upon previously-published pathogen capture studies conducted using a
lab-on-a-chip device,23, 30, 45 15 was covalently coupled to BSA, mediated by EDC in pH
28
7.4 PBS. This solution was used as ink for microcontact printing in order to produce a
specific pattern of 15-BSA on a gold chip according to figure 3.2 below. The pattern,
imprinted on a PDMS stamp from a silicon master, consists of raised 20 μm wide lines in
parallel with 20 μm gaps, producing a recognizable pattern that is observable with low-
magnification optical microscopy.
After stamping, the gold chips were blocked with a solution of unlabeled BSA,
which reacts with gold exposed on the non-stamped components of the chip. This helps
avoid any potential non-specific interactions between bacteria and the oxidized gold
surface of the chip.45-47 The originally-stamped pattern of 15-BSA was indistinguishable
from BSA used for blocking, but was observable in the following experiments by
imaging S. aureus captured in the same pattern.
To determine the limit of detection (LoD) of this device, we exposed stamped
chips to incrementally decreasing concentrations of S. aureus ranging from 1 x 106
CFU/mL to 1 x 102 CFU/mL. Using dark field microscopy, we imaged the chips at 40x
magnification. Figure 3.3 exhibits visible capture of S. aureus down to 1 x 102 CFU/mL.
This data suggests that future applications of this device may enable early detection of S.
aureus-related infections since pathogens can be effectively detected at early stages of
population propagation without the necessity for pre-enrichment cultures.21 In addition to
having a low limit of detection, the device also proved to capture the pathogen in an hour
or less of exposure time, which is a vast improvement over other pathogen-detection
methods like RT-PCR.48-50 Results of the time-course experiment are shown in figure 3.4.
Upon examination of the specificity of the chip when screened against a panel of
other human pathogens at 1 x 106 CFU/mL, there was clear evidence of S. aureus capture
29
(see figure 3.5). Aside from a low degree of cross reactivity exhibited with S. enterica,51
our construct did not capture any of the other bacterial strains tested. The high degree of
specificity that exists between compound 15 and S. aureus is an important characteristic
that contributes to the simplicity and reproducibility of the assay by discriminating
against false-positive results.
3.5 Conclusion
When taken together, the data collected from these experiments indicate that this
device, which can selectively detect low concentrations of a S. aureus in minimal time,
has enormous potential in the field of medicine. We expect that this device will accelerate
the process of detecting S. aureus in our environment, and hope that it will contribute to
the process of eradicating this dangerous pathogen from affected areas.
Figure 3.1. CChemical strructure of feuse in
erric-staphylon capture stu
oferrin A wiudies (15).
ith a short peeptide linker
30
r for
31
Scheme 3.1. Solid-phase synthesis of 15.
F
Figure 3.2. TThe microcontact printinng process usa-chip devi
sed on gold cice.
chips to prod
duce our lab
32
b-on-
FFigure 3.3. C(B) 1 x 105 C
(F) BS
Figure 3the chi
Capture efficCFU/mL, (C
SA only with
3.4. Time-cop. (A) 2 h, (B
iency based C) 1 x 104 CFh 1 x 106 CFU
ourse study toB) 1 h, (C) 3
on concentrFU/mL, (D) U/mL S. aurmagnificati
o examine th30 min, (D)
magnificat
ration of S. a1 x 103 CFU
reus. All imaion.
he effect of b5 min. All imtion.
aureus. (A) 1U/mL, (E) 1 ages were tak
bacterial expmages were
1 x 106 CFUx 102 CFU/mken at 40x
posure time ttaken at 40x
33
U/mL, mL,
to x
FFigure 3.5. S(A) Before
choler
pecificity ofe capture, (Brae, (G) Y. e
f capture dev) S. aureus,
enterocolitica
vice when te(C) E. coli, a, (H) S. ent
magnificat
ested against (D) P. aerug
terica. All imtion.
a variety ofginosa, (E) Smages were t
f human pathS. flexneri, (Ftaken at 10x
34
hogens. F) V.
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35
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