Lara Malins Baran Group Meeting Highlights in …...Lara Malins Baran Group Meeting Highlights in...

Lara Malins Baran Group Meeting04/23/16Highlights in Peptide and Protein Synthesis

"My entire yearning is directed toward the synthesis of the first enzyme. If its preparation falls into my lap with the synthesis of a natural protein material, I consider my mission fulfilled."

Emil Fischer

Peptides: The Greatest Hits

Peptides: Greatest Hits Collection(1882-present)

Scope of this meeting: (aka Lara's peptide mixtape):

www.thepeptides.com

The PepTides - "Love Question Mark" (2013)

...Rugby league player suspended after admitting to the "use and trafficking of peptides"

(August 2013)

• Brief historical perspective on peptide synthesis, outlining challenges and discoveries in the early years (1882-1960s)• Development of solid-phase peptide synthesis (SPPS)• Advances in coupling reagents and protecting groups• Chemoselective ligation chemistry for the synthesis of large peptides and proteins• Perspectives and unmet challenges• A collection of some personal favorites!

Peptides in the news:

• Non-amide bond forming ligation methods (e.g. bioconjugation, see Sella GM, May 2014 - ADCs)• Biological expression or semi-synthetic approaches (e.g. expressed protein ligation, unnatural amino acid incorporation - cf. P. Schultz)• Unnatural amino acid/peptide derivatives (!-peptides, peptoids, peptidomimetics)• Post-translational modifications (glycoslyation, sulfation, phosphorylation) and the synthesis of post- translationally-modified peptides or proteins

The Golden Oldies:Peptides as Therapeutic Leads:• ~100 therapeutic peptides currently on market• Octreotide, Goselerin, Leuprolide - top sellers• Approval rate for peptides (20%) vs. for small molecules (10%)• Anticipated global market of US$23.7 billion by 2020

Octreotide(Novartis)

"peptides can assist you during your journey to health and well-being" (https://peptidesdirect.com.au)

-EuPA Open Proteomics 2014, 4, 58 HN N

HO

HN

O

O

HN O

HN

ONH

OHN

S

SO

NH2

NH

NH2

OHHO

OH

O

NH O

OH

HN

O

O

NH O

NHN

O

NH2HN

NH

NH

OH

O

NH

OHN O

NH

O

HNNH

N

HNO

NH

O

NH2

Goselerin(AstraZeneca)

Topics not covered:

1882

1901

1932

1953

1963 present

1970

1992

1994dipeptideGly-Gly

Cbz

oxytocin

SPPS

Fmoc NCL

ligationTimeline:

Theodor Curtius

H2N OAg

O+ Ph Cl

O

Ph NH

O HN

OOH

O

Ph OH

O

+

+ 2AgCl

22

Ph NH

ON3

OH2N

HN

OOEt

O

NH

HN

OOEt

OOHN

O

Ph

NH

HN

ON3

OOHN

O

Ph

1) H2NNH22) HNO2

A

ANH

HN

ONH

OOHN

O

Ph

Acyl azide coupling

HN

OOEt

O

pentaglycine peptide

J. Prakt. Chemie 1882, 26, 145

J. Prakt. Chemie 1904, 70, 57

Theodor Curtius

- Emil Fischer, 1905(letter to Adolph von Baeyer)

HN NH

O

Oconc. HCl

"H3N

HN

OOH

O

Cl

Ber. Dtsch. Chem. Ges. 1901, 34, 2868


NH O

ClO

Br

H2NO

OEt

NH O

OEtOH

N

OBr N

H O

OHOH

N

OH2N

base/NH3

Emil Fischer

Acid chlorides in peptide synthesis and the #-bromoacyl method

glycylglycine

leucylglycylglycine"masked" Leu amine

H2NHN

O

NHO

N

O

NHO

HN

O

NH2

O O

HN

NH2

O

O

HN

ONH

O

OH

H2N

SS

Total Synthesis of Oxytocin

Vincent du Vigneaud• Head of Biochemistry - Cornell University Medical College• Studied the biochemical importance of sulfur-containing compounds (insulin, penicillin, oxytocin, vasopressin)• First total chemical synthesis of a bioactive peptide hormone• Nobel Prize in Chemistry (1955)

J. Am. Chem. Soc. 1953, 75, 4879 (Vigneaud)• Prepared using a 3-fragment condensation approach• Coupling methods: acid chlorides, anhydrides, pyrophosphite method• Purified by counter-current distribution (CCD) • No HPLC, mass spec, high resolution NMR!• Overall yield <<1%

CCD apparatus(invented by Lyman Craig)

Practical Limitations: • lack of readily cleavable N-terminal protecting groups • limited access to enantiopure amino acids

Octadecapeptide - Fischer

Leu-(Gly)3-Leu-(Gly)3-Leu-(Gly)8-Gly


NH O

HN

OHN

OBr

O

Cl


NH

R

O

PGHN

R'

OOR* N

R

O

OR*

OPGHN

R'

N

O OPGHN

R'

R

N

O OHPGHN

R'R

H N

O OPGHN

R'

R

Racemization through oxazolone formation (major)

O

O

NH

R O

O

NH

RO

O

NH

R

Cbz (Z) BocFmoc

CarpinoJACS 1957, 79, 4427McKay and AlbertsonJACS 1957, 79, 4686

Bergmann and ZervasBer. Dtsch. Chem. Ges.

1932, 65, 1192Carpino

JACS 1970 , 92, 5748

H2/PdNa/NH3 (l)HBr/AcOH

TFAHCl

base (amines)NH3 (l)

DeprotectionConditions:

Essential protecting groups (urethanes)

Ph NH

OCl

O

A convenient finding...

R

Ph NH

ONHR'

O

RH2NR'

racemizationbenzoyl

O NH

OCl

O

R

configurationallystable

PhCbz

O NH

ONHR'

O

R

Ph

H2NR'

NH

R'

O

OHO

R''O

Boc, Fmoc, Cbz (racemization-suppressing

urethane group)

NH

R

O

HN

HN

R'

O

O

R''O

CN

3

2 1

chain growth

OMe

OH2N

R

O

HN

1 OMe

O

23

NH

R

O

HN

R'

O

O

R''O2

3 OH

N-acyl groupracemization-prone

H2N1

OMe

O

NH

R

O

HN

HN

R'

O

O

R''O2 1

OMe

O3

CNchain growth

X

C-to-N:

N-to-C:

epimerization at C-2

Unidirectional chain growth:

Oxytocin

J. Am. Chem. Soc. 1959, 81, 5688 (Vigneaud)


Improved oxytocin synthesis published in 1959: stepwise, p-nitrophenyl ester couplings, C-to-N elongation, 38% overall yield

I

II

III

du Vigneaud

Racemization through enolization (minor)

NH

R

O

PGHN

R'

OOR*

NH

R

O

PGHN

R'

OOR*

2


Bruce Merrifield and Solid-Phase Peptide Synthesis (SPPS)

• Biochemist by training, moved to Rockefeller Institute for Medical Research (1949)• Work on the structure of peptide growth factors required the synthesis of a pentapeptide:

29 May 1959 - Merrifield records concept of SPPS in his lab book

"my overall yield of pentapeptide was 7%, and it took me 11 months. Certainly, an experienced peptide chemist would have done better, but not without considerable effort."

O

OLinker N

HO

O

LinkerNH

OH

OLoad resin

DeprotectN-terminus

H2NLinker

NH

OH

O

NH

O

O

LinkerOH

NLinkerPeptide 1Repeat: deprotect

and couple

i) Deprotect (global)ii) Cleave from resin

Peptide 1H2NO

OH

= solid support (resin)

= N-terminal amino protecting group

= side-chain protecting groups

Synthetic peptide product

coupleamino acid

Solid-phase peptide synthesis (SPPS)

iii) HPLC purify

Common linkers:

RCl

Trityl linker (R = H)2-Cl trityl linker (R = Cl)

ClMerrifield

(chloromethyl)

Linker =O

NH2OMe

OMe

Rink amide linker

HN

PAM linker(4-hydroxymethyl-phenylacetamidomethyl)

OOH

Common Resins:

Cle.g.

Merrifield linker

Cl

DVBcross-link

Merrifield resin

• Resin loading: 0.2 - 1.5 mmol/gram (number of functionalizable sites)• "Swellability": determines appropriate solvents

Properties to consider:

CbzHN O

ONHEt3

1)

O

O

NHEt3

2) O

OCbzHN

1) Cbz removal: 30% HBr in AcOH

(2-4 h)Cl

H2NO

NH O

HN

OOHN

H O

J. Am. Chem. Soc. 1963, 85, 2149 (Merrifield)

1984 - Awarded the Nobel Prize for SPPS

A reviewer's thoughts: the approach is "a travesty...not chemistry at all, a concept which should be surpressed by the community."

O

OH2N

2) Et3N/DMF

CbzHN OH

ODIC, DMF, rt, 18 h

1)

2) Ac2O/Et3Nrt, 2 h

O

OHN

OCbzHN

SPPSthen

NaOH/EtOH

Seminal Paper

Biopolymers 2008, 90, 175 (Mitchell)Leu-Ala-Gly-Val (8% yield of purified compound)

Merrifield

H2N O O O NH2

H2N O OO

H2N O OO

NH2

NH2

n

n

n

- polystyrene +1-2% divinylbenzene (DVB) cross-link:

- ChemMatrix polyethylene glycol (PEG):

more polar resindecreases hydrophobic aggregation

(PCT Int Appl 2005, WO 2005012277 A1)

J. Am. Chem. Soc. 1966, 88, 5319

For development of solid-phase oligonucleotide synthesis, see:

3

NN

Lara Malins Baran Group Meeting04/23/16

NH

O

O

O Oside-chain

benzyl PGs

N-terminal Boc

O

O

Boc SPPS (Boc/benzyl) Fmoc SPPS (Fmoc/tBu)

NH

O

O

O Oside-chain tBu

or Trt

N-terminal Fmoc

O

O

piperidine labile

TFA labile

TFA labile

HF labile

Pros: • Optimizable to 50-60 residues (less aggregation prone)• Stable thioester linkages

Cons:• HF safety hazard• Limited FG compatibility

Pros: • mild and safe (no HF required) • spectrophotometric quantification of couplings

Cons:• More aggregation prone• Incompatible with base sensitive substrates (e.g. thioesters)

"Assembly of the 124 amino acid residues into the protected, resin-bound straight-chain precursor of RNase required 369 chemical reactions and 11,931 steps of the automated peptide synthesis machine."

J. Am. Chem. Soc. 1969, 91, 501 (Merrifield)

Merrifield and his peptidesynthesis machine

Automation

NH

O

OR H2N

RNH

HN

-CO2

Fmoc Spectrophotometric Quantification

piperidine-fulvene adduct!= 7800 cm-1 (" = 301 nm)

fast

ResinHF labile

ResinTFA labile

"Automated Synthesis of Peptides"Science 1965, 150 , 178

Synthesis of an Enzyme with Ribonuclease A (RNase) Activity

• N-terminal Boc protecting groups• DCC as the coupling reagent - JACS 1955, 77, 1067 (Sheehan)

NN

N

OH

NHHOAt

HOBt

J. Am. Chem. Soc. 1993, 115 , 4397 (Carpino) - 1367 citations

NO

O

HO

HOXt/EDC

HOAt: complete at 22 hHOBt: trace product

OMeH2NO

OHNH O

O+

HNN

H O

O

OMe

Oconditions

DCC, 24 hHOBt, DCC, 24 hHOAt, DCC, 24 hHOAt, DCC, DCM, 24 h

DL-isomer (%)coupling conditions61.5%41.9%14.4%< 1-2%

HOAt as a peptide coupling additive

• superior leaving group• neighboring group effect

O

ONNN

X

via:

NH

R R

N NOH

N NN

N

OO

R

Improvements in Coupling Reagents

HOBt in peptide synthesisChem. Ber. 1970, 103 , 788 (König) - 965 citations

NN

N

OHHOBt

Racemizationsuppressor

Reviews: Chem. Rev. 2011, 111 , 6557 (Albericio); Tetrahedron 2004, 60, 2447 (Han)

Tetrahedron 1991, 47, 259 (Coste)

HOAt:

Oxyma: A Safer Alternative

COOEtNC

NOH

• Slightly more reactive than HOAt• Non-explosive (unlike HOBt, HOAt)

Chem. Eur. J. 2009, 15 , 9394 (Albericio)

COOEtNC

NO K

K-Oxyma: for highly acid-labile resinsEur. J. Org. Chem. 2013, 6372 (Albericio)

• non-acidic alternative to Oxyma

**Final product had enzymatic activity (though less than native RNase)

NN

N

O PNMe2

Me2NNMe2

PF6

BOP

NN

N

O PN

NN

PyBOP

PF6

Br PN

NN

PyBrOP

PF6

Tet. Lett. 1975, 16 , 1219(Castro)

Highlights in Peptide and Protein Synthesis

4

NH NH

N

NH2

OHO

NHNH

NH2Cl

H3N

H3N

NHNH

NH2

ClH3N

N

NH

HN NH2

O

HN

EDC/HOBt

HATU, HBTU, TCTU, DPPA, T3P, EDC, DIC, DCC, BOP, BOP-Cl, PyAOP, PyBOP, PyBrOP, cyanuric chloride

Other attempts:

"Alphabet Soup": Why are there so many coupling reagents?

J. Am. Chem. Soc. 2011, 133 , 14710


• Coupling of bulky (and/or non-proteinogenic) amino acids• Considerations for removal of coupling reagent byproducts• Enhancing rate of amide bond formation• Minimizing racemization of activated acid

CbzHN OH

O

R N

NR2

CbzHN O

O

RHN

NR2

CbzHNHN

O

R

NR2

O

CbzHNHN

O

R

NR2

O

Aib couplings with the Aziridine method

Helv. Chim. Acta 1990, 73, 13Helv. Chim. Acta 1987, 70, 102 (Heimgartner)

Et2O, rt, 0.5-6 h

J. Org. Chem. 1992, 57, 5566 (Heathcock)Synthesis of (-)-Mirabazole C - Application of PyBroP

CbzHN OH

O

MeBnS

H3NOMe

O

MeBnS

Cl

HN OMe

O

MeBnS

CbzHNO

MeBnSPyBroP, DIEA

DMAP, DCM, 4 h

90%

1) HBr, HOAc

HN OMe

O

MeBnS

NH O

MeBnS

OCbzHN

MeBnS

A

2) A, PyBroPDIEA, DMAPDCM, 24 h

HN N

H

O

MeBnS

NH O

MeBnS

OHN

MeBnS

O

SBn

S

NNS

S

NMe

Me

S

N

Me S

NNS

S

NMe

Me

S

N

Me

1) Na, NH32) NH4Cl3) TiCl4, DCM NiO2, benzene

reflux, 6 h60%

63%

+ 60%

(-)-Mirabazole C

Case Studies:

Acid chloride, BOP, BOP-Cl, DPPA, DCC were unsuccessful in coupling A

!-aminoisubutyric acid(Aib)

H2NO

OH

HN

O

OHMe

N-Me-valine

Synthesis of Palau'amine

N

N

O

NN

OO N

NBoc H Boc

OH

OH

NBocN

H

NH

OH

MeO2CMe

Boc

NN

Boc H BocF

OH

Synthesis of 5-N-acetylardeemin

DCC/DMAPDCC/HOBt

BOP-Cl

C8

C15b

partial racemization(C15b or C8)

cyanuric fluoridepyridine

DCM, -15 oC

H2N CO2Me

Me+

H-Ala-CO2Me

NaHCO3, H2ODCM

71% (2 steps)

J. Am. Chem. Soc. 1999, 121 , 11953 (Danishefsky)

See also: TFFH as a reagent for acid-fluoride couplings:

JACS 1995, 117 , 5401 (Carpino)

N N

FPF6

TFFH

Choosing a coupling reagent: where to begin?

solid-phase solution-phase

aniline?

acidchloride

base sensitive?

DIC/HOBtDIC/oxyma

bulky AA,difficult

coupling

PyAOPHATU

HOAt/DICPyBrOP

PyBOPHBTU

HOBt/DIC

generalconds bulky AA,

difficultcoupling

HOBt/DIC or DCC

generalconds

HOAt/DICacid fluorideor EDC

(water-soluble)


Start

5


A Few Notable Protecting GroupsReview: Chem. Rev. 2009, 109, 2455 (Albericio)

SO2

OO

BsmocJACS 1997, 119, 9915JOC 1999, 64, 4324

SO2

OO

!-Nsmoc

SO2

OO

"-Nsmoc

> >

JOC 2007, 72, 1729

H

JOC 2007, 72, 1729

Relative sensitivity to piperidine:

5 g Fmoc-Phe-OH: $16.655 g Bsmoc-Phe-OH: $107.26

Price comparison (Alfa Aesar)

SO2

OO

HN Cl

NH

SO2

CH2

H2N Cl

-CO2

N

SO2

N

J. Am. Chem. Soc. 1997, 119, 9915 (Carpino)

Leu-Leu-Leu O

O

O NH

O

SO2

NH2N NH2

NH2

HN

Bsmoc

Me

Bsmoc removalonly

Fm removalonly10% in DCM

2% in DCM

Alternatives to Fmoc

Michaeladdition

Selective removal of Bsmoc or Fm/Fmoc

JOC 2003, 68, 4894 (Enzon Pharmaceuticals, Inc.)

O

O

O

NHO

O

NHO

O

SO2

N NH

O

O

O

NHO

O

H2N

N

N

O2S

O

OHPEG

EDC, DMAPDCM, rt, 12 h

O

O

O

NHO

O

NH

82% (2 steps)

(1 equiv.)

O

PEG

PEG 2'-paclitaxelglycinate

DCM, rt, 3 h

JOC 2003, 68, 4894 (Enzon Pharmaceuticals, Inc.)

O

O

O

NHO

O

NHO

O

SO2

H3C

O

O

OH

O

CH3 OHO

O

HO

CH3CH3

O

OH

NHO

OO

O

BsmocHN OH

O

EDC, DMAPDCM, 10 oC to rt, 1.5 h

Taxol

Application of Bsmoc protection

!"

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Cbz-

SPPS

!Protein Functional Domains

Smallest Proteins

Chemical Ligation

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Perspectives on Protein Synthesis


Annu. Rev. Biochem. 2000 69, 923

• SPPS (and subsequent coupling reagent and PG advances) greatly improved the effiency of peptide preparation but did not substantially expand the size of accessible targets

Limitations of Solid- and Solution-phase couplings:

• On-resin aggregation generally limits routine SPPS to ~50 amino acids• Solution-phase fragment condensations (using protected peptides) are plaqued by the poor solubility of protected peptides

6


OOC

SH

NH3

OH

SHCOO

NH3Chemoselective

ligationOOC

SHHO

NH3

OH

SHCOO

NH3

+

HO

Chemical Ligation

• unprotected peptides• aqueous solution• physiological pH

mutually reactivefunctional groups

Amide bonds through acyl transfer

H3NSPh

OClH3N

O

O

HS+

Na2CO3 H2O 0 oC

H2NO

O

S

OH2N

- PhSH

S to Nacyl shift

NH

O

O

HSO

H2N

R

Template

O S

OPG

Thiolcapture

Template

O SH

O

Template

O S

O

H2N

SH

H2NS

Template activation

O to N acyl

transfer

Template

OH S

NH S

O

O

NH

HSRelease template

Template

Prior thiol capture

A A A

A

A

B

B

B

BHO S

PG

=

Tetrahedron Lett. 1981, 22, 185 (Kemp)Tetrahedron Lett. 1981, 22, 181 (Kemp)

O

O

O

O

OAcMe

OMeS S

CO2EtH2N

O

O

OH

OAcMe

OMeS S

CO2Et

HN

O

Tetrahedron Lett. 1981, 22, 181 (Kemp)

t1/2 = 2.7 h (DMSO)28 h (DMF)

• concentration independent O to N acyl transfer

(intramolecular)N to O acyl

transfer• effective local conc. of

intramolecular amine = 0.6 M

J. Org. Chem. 1986, 51, 1829 (Kemp)

OS S

X

Z

O

X = H, Cl, Br, NO2Z = H, Cl

RO

CO2MeH2N

OS S

X

Z

HO

CO2MeHN

OR

DMSO, 25 oC

ring substituents

9.7 x 10-5

4.8 x 10-4

1.1 x 10-3

7.6 x 10-5 7.8 x 10-4

1.2 x 10-3

2.4 x 10-1

3.5 x 10-3

7.6 x 10-3

R

HBrClHBrClNO2HCl2

HHHHHHH3ClCl2

X Zacyloxy

CH3CH3CH3Z-AlaZ-AlaZ-AlaZ-AlaZ-AlaZ-Ala2

k1(s-1) t1/2k1 /kH

(rel rate)

2 h24 min10 min2.5 h15 min10 min2.8 s 33.3 min1.5 min2

1 (ref)4.9111 (ref)10 153200 345 1002

Effectivemolarity (EM)*

4.6

4.55.1

5.78.6 34.9 2.72

*EM determined from the ratio of k1 to k2 (k2 = rate constant for intermolecular aminolysis of each 1- and/or 3-functionalized 4-(acyloxy)dibenzofuran template with H-Cys(Bn)-OEt in DMSO at 25 oC

(deprotection)

(reduction)

HO S

n = 0, 1

R'

PG

Optimized templates - 4,6-dibenzofurans

acyl shift

MRTGGA

O

OO

SHBocHN

S

O

HN

O

H2N

S

O

S

O

O

+

1) HFIP/H2O (thiol capture)2) DMSO (acyl transfer)3) PEt3, DCM/HFIP (template cleavage)

GGANH

HS

OMRT

O

BocHN

S

O

HN

O

PG

PG

4) Dnp-F, DIEA (Cys protection)5) TFA/thioanisole (remove non-Cys PGs)6) Scm-Cl (convert Acm to Scm)

GGANH

DnpS

OMRT

O

H2N

S

O

S

O

O Repeat 1-6 for2 more ligations

Cys(Acm) Cys(Scm)

BPTI 30-5829 residue peptide

Total Synthesis of BPTI - Basic Pancreatic Trypsin Inhibitor (30-58)

Justus Liebigs Ann. Chem. 1953, 583, 129 (Wieland)

J. Org. Chem. 1989, 54, 2803 (Kemp)


Annu. Rev. Biochem. 2000 69, 923Chem. Soc. Rev. 2009, 38, 338

7


6 M Gn•HCl/0.1 M Na2HPO4

pH = 4.3O

SHHN

OBr+A B

O

SHN

OA B

Thioester ligation - "Backbone-engineered" proteinsScience 1992, 256, 221 (Kent)

thioester linkage

H2NO

S

OO

NH

NH O

S

OO

NH

HIV-1 PR(1-50)

51

51

NH O

SHHIV-1 PR(1-50) 51

HO

HIV-1 PR(53-99) O

OBr

HIV-1 PR(53-99) OH

OBr

52

52

HIV-1 PR(1-50) NH O

SO

51 HIV-1 PR(53-99) OH52

HFHF

automatedBoc-SPPS

automatedBoc-SPPS

Synthesis of HIV-1 protease:

6 M Gn•HCl/0.1 M Na2HPO4pH = 4.3, 48 h

[peptides] = 4 mM

assembled HIV-1 PR dimer exhibits enzymatic activity

O

SRHN

OH2N+

HS Transthio-esterification

HN

OH2N

S

OS!N

acyl shift

HN

ONH

HSO

A B

A

A

B

B

thioesterpeptide N-terminal Cys

peptide

Native Chemical Ligation (NCL)

slow fast

O

IL-8 (1-32) S HN

OH2N

+HS

IL-8 (35-72)SH

SHSH

O

NH

HN

O

HS

Folded IL-8

HN 33

34

HN 33

34IL-8 (1-32)

SH

SH

IL-8 (35-72)

SH

HS

6 M Gn•HCl/phosphate buffer

pH = 7.623 oC, 48-72 h

Science 1994, 266, 776 (Kent)

thiol additive*Science 1994, 266, 776 (Kent)

Total Synthesis of Interleukin-8:

*Thiol additive serves as a reductant to prevent disulfide formation and to reverse unproductive thioesterification of internal Cys residues

Rate enhancement with thiol additives

O

STransthio-

esterification+

O

S +HS

Benzyl thioester Thiophenyl thioesterHS

A A

barnase (50-110)*HN

OH2N

HS

49

O

barnase (1-47) SHN 48

+

HN

ONH

HS

49

OHN 48

6 M Gn•HCl/0.1 M Na2HPO4 pH = 7.5

barnase (50-110)*barnase (1-47)

For benzylthiol:

easier to handle(HPLC stable)

more reactivein ligations

HS

complete at t = 7 h< 25% complete at t = 7 h

HS4 vol% or

For PhSH:

J. Am. Chem. Soc. 1997, 119 , 4325 (Dawson, Ghadiri, Kent)

• Construction of native amide bonds in aqueous media• Completely chemoselective (alcohols, amines, acids, internal thiols, etc. are all tolerated) - allows use of unprotected peptides

Comparative synthesis of Barnase (1-110):J. Am. Chem. Soc. 1997, 119 , 4325 (Dawson, Ghadiri, Kent)


8


J. Am. Chem. Soc. 2006, 128 , 6640 (Kent)

O

SR

HS R'S

RHO SR'

SR'

O

pKa < 4 - inefficient at transthioesterification

H2N

HS

O

pKa ! 9inefficient LG

in ligation

Ideal thiol additive: 6 < pKa < 9

Fine-tuning with thiol additives

HS NH2

HS

HSO

HO

NHSHS

HSO

HO

4-mercaptophenylacetic acid (MPAA)

• stable solid• water-soluble• rapid ligation• no epimerization• minimal stench!

A A A

B

Kinetically-controlled ligations

OSA

O

SBH2N

HS

O

NH2

O

latent alkyl thioester

CH2N

HS

O

ligation #1 ligation #2(after activation)

Angew. Chem. Int. Ed. 2006, 45, 3985 (Kent)

- Exploiting thioester reactivity allows for Iterative ligations

PNAS 2007, 104 , 4846 (Kent)

KVFERCELAR TLKRLGMDGY RGISLANWMCLAKWESGYNT RATNYNAGDR STDYGIFQINSRYWCNDGKT PGAVNACHLS CSALLQDNIADAVACAKRVV RDPQGIRAWV AWRNRCQNRDVRQYVQGCGV

Convergent Synthesis of Human Lysozyme

1-29O

S

130 amino acids - 4 fragments, 3 ligations

O

OH

31-64O

SH2N

HS

O

Acm

R

1-29OAcm

31-64O

SNH

HS

O

R

66-94O

SNH

O

RS

96-130H2N

HS

O

66-94NH

O

S O

NH

HS

O

96-130

AcmAcm

Acm Acm

MeONH2, pH = 4Thzremoval

66-94O

NH

HS

O

96-130

Acm Acm

H2N

HS

O

kinetically-controlledligation (KCL)

ligation

66-94O

NH

HS

O

96-1301-29

O31-64N

H

HS

O

O

NH

HS

O

MPAA

1) ligation: MPAA2) Acm removal: AgOAc, DTT

PNAS 2007, 104 , 4846 (Kent)Convergent Synthesis of Human Lysozyme

66-94O

NH

HS

O

96-1301-29

O31-64N

H

HS

O

O

NH

HS

O

130 amino acids - 4 fragments, 3 ligations

kinetically-controlledligation

native chemicalligation

native chemicalligation

One-pot KCL to homogeneous EPO

1-29O

S

O

OH

Acm

31-78O

SEtH2N

HS

O

80-166H2N

HS

O

Acm

Acm

O

OH

HOHO

NHAc

O O

NHAcHO

OH

O

OH

ONHAc

O

AcHNO

OHOHO

O

HOAcHN

HOHO OH

OHO

AcHN

HOHO

OH

HO2C

CO2H

=

=

1-29

Acm O

NH

HS

O

31-78

Acm O

NH

HS

O

80-166

Acm

KCL, 5 h; then: MPAA,

One-potkinetically-controlled

ligation

Angew. Chem. Int. Ed. 2012, 51, 11576 (Danishefsky)

EPO (1-166) precursor

See also: Science 2013, 342, 1357"At last..."


9

HS R


S S

Thia-zip reaction

Method for the synthesis of cysteine-rich cyclic peptides

cyclopsychotrideJ. Nat. Prod. 1994, 57, 1619

- one of the largest known cyclic peptides (31 AAs)- 6 cysteine residues and 3 disulfide bonds

CSCKSKVCYKNSIPCGESCVFIPCTVTALLG

S SS S

J. Am. Chem. Soc. 1999, 121, 4316 (Tam)

H2N

HS SH SH SH SH SH SR

O H2N

HS SH SH SH SH S O

H2N

HS SH SH SH SH S O

thiolactoneexchange

pH = 7.5(8 M urea)

S

O

NH2

SHSH

acyl shiftsSHSHSH

NH

OHS

SHSH

SHSHSH

S to Nacyl shift irreversible

[O]DMSO

disulfide formation

cyclopsychotride

(20 h - 24 h)

Thia-zip cyclization is 200x faster than unassisted cyclization

blocking terminal thiol prevents

lactam formationblocking internalthiol still leads

to lactam formation

What about Cys-free peptides and proteins?

HS OHN

HS NH

HNHS

HNHS

OMe

HNHS

O2N

HNHSHNHS

MeOOMe

HNHS

MeOOMe

OMe

R R

R

R = H, OMe R = H, OMe

HNHS

NO2

OHN

R

HSA

H2N A

HSN-terminal

Auxiliary-basedCys surrogates

HNHS

JACS 1996, 118 , 5891 Bioorg. Med. Chem. 2001, 9, 2323Tet. Lett. 2001, 42, 1831PNAS 2001, 98, 6554

Tet. Lett. 2003, 44, 6059Bioorg. Med. Chem.

2004, 12 , 2714

Org. Lett. 2000, 2, 23Org. Lett. 2001, 3, 1403

Bioorg. Med. Chem. Lett. 2002, 12 , 1963J. Am. Chem. Soc. 2002, 124 , 4642

=

H2N A

HSSide-chain

Auxiliary-basedCys surrogates

RHN

HS

A

HSO

OHO

NH

OHHO

OHS

OO

ONHAc

OHHO

OHS

ONH

OHSO NH

OHS

O

O

OHO

HN

OH

OHSO

HN

OHO

NH

OHHO

OHSO

HO

JACS 2006, 128 , 5626Chemistry 2007, 13 , 5670

JACS 2006, 128 , 15026JACS 2007, 129 , 7690

Angew. Chem. Int. Ed.2007, 46, 5975 Chem. Commun. 2008, 10 , 1229

=

HSHN

Angew. Chem. Int. Ed.2015, 54, 15055

Relative abundance (Cys) = 1.7%

ReductiveHN

ONH

HSO

A BHN

ONH

O

A B

Cys Ala

Ligation-Desulfurization

Radical

Pd/Al2O3, H2

VA-044, TCEP, tBuSH

Reductive: JACS 2001, 123 , 526 (Dawson) - 373 citationsRadical: ACIE 2007, 46, 9248 (Danishefsky) - 311 citations

NNH

N

NNH

N

VA-044HO2C P CO2H

HO2C

TCEP

BocHNO

OH

RS

O

OHN

TrtS

BocHNO

OH

RSO

OtBu

R

Thiol amino acids:

e.g.Reviews: Curr. Opin. Chem. Biol. 2014, 22, 70Aust. J. Chem. 2015, 68, 521


10

Science 2000, 287, 2007 (Bertozzi)

OMeO

PPh2

O OH

OMeO

PPh2

O O

OH

Staudinger ligationReview: Angew. Chem. Int. Ed. 2004, 43, 3106

sampleN3

OMeO

PPh2

O O

sampleN

MeO

PPh2

O O

sampleNO

O

PPh2

O O

sampleHNO

aza ylide-N2

"Cell-surface engineering"

• "Bioorthogonal" tagging• aqueous media• on the surface of living cells• inside living animals (see: Nature 2004, 430, 873) fast


O

S +A B"traceless" auxiliary

PPh2 N3

O

SA PPh2N B

O

NA B

PPh2

HS

O

NH

A B

Ph2P SH

O

-N2

H2O

Org. Lett. 2000, 2, 2141 (Bertozzi)Org. Lett. 2000, 2, 1939 (Raines)

Org. Lett. 2001, 3, 9 (Raines)"Traceless" Staudinger Peptide Ligation

HO PPh2

HS PPh2SH

PPh2

OHPPh2

N

HNPh2P

HS

PAr2 NAr =

J. Am. Chem. Soc. 2007 129 , 11421 (Raines)

NH NH

N

NH2

SO

Ph2P

BH3

NHNH

NH2Cl

N3

N3

NHNH

NH2

ClN3

N

NH

HN NH2

O

HN

DABCO60 oC

20%

Angew. Chem. Int. Ed. 2003, 42, 4373 (Maarseveen)Staudinger Ring-Closure (Medium-Sized Lactams)

J. Am. Chem. Soc. 2011, 133 , 14710

H2O soluble

KAHA (Ketoacid-hydroxylamine) ligationsAngew. Chem. Int. Ed. 2006, 45, 1248 (Bode)

HN

ONH

+R

HN

ONH

ROA B A B

OOH

O

HO-CO2

DMF, 40 oC(50 - 100 mM)

-H2O

BocHN OH

O

R

SNC

Br

BocHN

R

O

S

CN BocHN

R

O

O

OH

HBTU, DIEADCM, rt

Oxone1:1 DMF/H2O

J. Am. Chem. Soc. 2008, 130 , 4253 (Bode)

O O

HN

S

O O

HN

S

Rink resin

CN

PF6

OO

HN

SO

CN

FmocHN

1:2 MeCN/PhMe15 h, 60 oC

PF6 H2C-CN

OO

NH2

SO

CN

HNpeptide

O

O

HNpeptide OH

Fmoc-Phe-OHHATU, HOAt DIEA, DMF

Fmoc-SPPSTFA

OxoneDMF/H2O

cyanosulfur ylideSolid-phase approachOrg. Biomol. Chem. 2009, 7, 2259 (Bode)

modification of cellsurface glycans


11

OHN

O

A B

OOH

O

0.1 - 5 mM20-25 oC

O

BONH

HN

ONH

O

A B

HO

oxazetidine

KAHA: Increased reactivity through ring-strain

A

OOH

O

oxaproline

1) 10 - 15 mM40-60 oC H

N

ONH

O

A B

OH

Nature Chem. 2015, 7, 668 (Bode)

2) pH = 10.5(O to N acyl

shift)

MeO

OMe

NH

OO

MeO

OMe

NO

OH

BF3•OEt2, DCM-40 oC to 0 oC

NO

OHFmoc

Ooxazetidinebuilding block


Perspectives and Future Directions


• Routine preparation of small to medium peptides by SPPS is generally rapid and efficient• Small proteins and enzymes are now accessible by chemical ligation methods (NCL, Staudinger ligation, KAHA, etc) employing unprotected peptides from SPPS

• Rapid and efficient on-resin ligation chemistry: e.g. an automated, programmable solid-phase "ligation machine" for protein synthesis

Some Ideals:

• Unnatural amino acids employed in ligation chemistry (oxazetidines, thiol amino acids, ketoacid precursors) should be commercialized and cost-effective (as accessible as coupling reagents)

• Improvements in coupling reagents and protecting groups allows the construction of most amide bonds (particularly between proteinogenic amino acids)

The State-of-the-art:

Unmet Challenges:• "inaccessible" structures: modified peptides (bearing complex post-translational modifications or non-native structural motifs)

HN N

H

O

O

Ph

NH

HN

O

O

H

S

13

22

S to !-Ccross-links

Subtilosin A

• Facile synthesis of peptides bearing non-proteinogenic or heavily modified amino acids

Astexin-1

"Rethinking amide bond synthesis"Review: Nature 2011, 480, 471 (Bode)

O

HR' R NH2

O

NH

R' R

CuI (1 mol%)AgIO3 (1 mol%)

TBHP

Ar

NHBoc

NO2

BrNIS, baseH2O, 0 oCH2N

Me

O

NH O

OMe HN

Me

O

NH O

OMe

O

Ar

BocHN

+

+

J. Am. Chem. Soc. 2010, 132 , 4098

O

SHBocHNHN

O

OBn

Me

TsOH•

O

NBocHN

Me O

OBn+

CN

CHCl3

Nature 2010, 465, 1027

J. Am. Chem. Soc. 2006, 128 , 13064

12

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