Strategies for Peptide Synthesis: An Overview

R H2N O OH H2N R'

O OH

Peptide Coupling Reagent

H2N

H N O R'

O OH

-H2O

Han, S., Kim, Y. Tetrahedron, 2004, 60, 2447-2467 Albericio, F. Current Opinion in Chemical Biology, 2004, 8, 211-221 Humphrey, J., Chamberlin, R. Chem. Rev., 1997, 97, 2243-2266

Outline
1) Introduction - General Strategy 2) Coupling reagents - Carbodiimides - Uronium Reagents - Phosphonium Reagents - Organophosphorous Reagents - Acid Halogenating reagents - Racemization Pathways - Racemization Suppressants 3) Difficult Couplings - ,-disubstituted Amino Acids - Peptide Macrocylizations - N-methyl Amino Acids - Segment Condensations 4) Applications - Process Scale Solid Phase Peptide Synthesis

Introduction

- Amide Bonds are Ubiquitous in Nature - A large number of Natural products are based upon a peptide framework and exhibit a spectrum of biological activity - Currently there are many peptide therapeutics in development - The current pursuit of non-natural amino acid mimics makes coupling chemistry paramount for drug discovery and scientific advancement -There is no single strategy for amide bond formation that is a magic bullet -Advances in coupling chemistry have made formation of the most difficult amide bonds possible

General Strategy For Peptide Bond Formation

R''' H2N R R'HN O OH OR'' O X O R'HN O R O OR'' R'''

Peptide Bond Forming Reagent

R R'HN

H N

X = Activating Group

Carbodiimides: Representative Examples/ Comparisons

H3C N H3C N

$2.25/gram
N C N CH3 C

$0.25/gram
N

H3C H3C N

$0.90/gram
C N CH3 CH3

EDC

DCC

DIC

Water Soluble by-product is easily removed in aqueous work-up

Urea formed is partially soluble in many solvents and hard to purify via column chromatography

Urea formed is soluble in most organics. This is advantageous in solid phase synthesis.

CH3 H3C N C N CH3 H3C N CH3 C N CH3

CIC
CH3 H3C N CH3 C N CH3

BMC
H3C CH3 O O N C N H3C O O CH3

BEC

BDDC

N,N-dicyclopentylcarbodiimide

Carbodiimides: Basic Structure and Mechanism

R R +HN OO BocHN BocHN O RHN O N N H N RHN C N R O O X HN N R R N H O N H R O BocHN X H2N R BocHN O N H R

X= Activator
O N O N H

Product

N-acyl Urea Formation

Byproduct

Common Activators: Accelerate Reaction and Suppress Byproduct Formation

O N O OH N N H3C N CH3 OH N N

HOSu
OH N N N N

DMAP
O F N O OH F

HOBt
F F

OH F

HOAt

N, hydroxyphthalimide

PfpOH

Lou Carpino: Peptide Giant UMass, Amherst

- Developed benzotriazole based aminium reagent, HATU, and elucidated the active form of the coupling agent - Introduced HOAt as an efficient additive for coupling reactions -Introduced the widely used Fmoc protecting group -Pioneered the use of amino acid fluorides as coupling agents

Uronium reagents: Basic Structure and Reactive Species

H3C O N N N N CH3 CH3 N CH3 H3C N CH3 O N N N N CH3

HBTU

CH3

Uronium

Guanidinium

- Originally the uronium isomer was thought to be the active species -Upon solving of the x-ray crystal structure, it was found that the guanidinium species was predominate -However, the uronium could be prepared and was found to be more reactive than the guanidinium salt - Original attempts to classify the reactive species were misguided based on known thermodynamic stabilities -The two forms are readily distinguished by a shift in the IR absorption from ~1710 cm-1 (Uronium) to ~1670 cm-1 (guanidinium)

J. Org. Chem. 2001, 66, 5245-5247 Angew. Chem. Int. Ed. 2002, 41, 442

Uronium reagents: Overview/Cost Analysis

O N N N N H3C N CH3 N CH3 H3C N CH3 N N CH3 H3C N CH3

~$25/gram App. Bio. PF6

O N N

~$10/gram Aldrich
Cl

O N N N N

~$2/gram Pep. Intl. PF6

CH3

PF6

CH3

CH3

CH3

HATU

HBTU

HCTU

-HATU is the most reactive uronium reagent, however it can be cost prohibitive on large scales and is often used only as a last resort -HBTU is the more cost effective alternative and is acceptable for most coupling applications, however lower yielding couplings can become problematic on industrial scales and with long peptides - HCTU has been developed as an effective alternative to HATU on industrial scales, the higher reactivity of this species is attributed to the more reactive Cl-HOBt intermediate BF4
NMe2 Me2N O O N O O

BF4 - TSTU and TNTU are useful alternatives under aqueous reaction conditions
Me2N

NMe2 O N

TSTU

TNTU

Uronium reagents: Mechanism and Side Reactions

O N N N N H3C N CH3 N CH3 O R R' N H O O R

HOAt

CH3 R' NH H N N O R Me2N R NMe2 O O N N O N N O R O N N N

O N N N O Me2N NMe2

Intramolecular general base catalysis enhances reactivity

Guanidine Formation
H3C O R'O R NH2 O N N N N N CH3 R'O R

H3C N N N CH3 O

HAMDU

Guanidine Side product

This side reaction highlights the importance of stoichiometry and pre-activation of the acid component. This can generally be avoided with the proper precautions.

Uronium reagents: Efficiency Comparison

CH3 O O N H

CH3 OH H2N

O O

Fmoc-Deg-Phe-OFm

O PF6 N N N N N N H3C N N

Coupling Agent

HPLC Yield

PF6

N N

HATU HBTU HAPyU HAMDU HDTU

94 85 92 57 64

CH3

HAPyU
O N O

HAMDU
CH3 N CH3 PF6

J. Org. Chem. 1998, 63, 9678-9683

N

N N H3C CH3

HDTU

Phosphonium Reagents: The Basics

Common Reagents
N O PF6 X P(NMe2)3 N X N N P(NMe2)3 PF6 N X P N N PF6 PF6 X O N N N P N N

X=Cl, CloP X=Br, BroP

X=CH, BOP X=N, AOP

X=Cl, PyCloP X=Br, PyBroP

X=CH, PyBOP X= N, PyAOP

- Reagents bearing the dimethyl amine moiety produce HMPA as a toxic byproduct, and thus their pyrrolidine based analogues are preferred - Halogenophosphonium reagents have been shown to be more efficient coupling reagents in the coupling of N-methylated amino acids

H3C O H N Boc OH N CH3

CH3 OCH3 O

PyBroP= 79% PyCloP= 85% PyBOP= 26%

Boc-Pro-MeVal-OMe

"Difficult" because of steric hindrance

J. Org. Chem. 1994, 59, 2437-2446

Phosphonium Reagents: Thioamide Formation

O R SH H2N R' S O N H R' R N H R'

PyBOP
R

2.5 : 1 (68% overall yield of both products) - Thioamides are useful probes in peptide structure and function, particularly for the elucidation of the contribution of backbone hydrogen bonding - These peptide analogues are not readily accessed through thioacylation in an analogous fashion to standard peptides - This example uses the oxophillicity of the phosphonium coupling reagent to direct thioamide formation preferentially over amide formation -Interestingly, this selectivity is reversed (1:24) with the use of PyBrOP

Note: Thioamides are also readily prepared using Lawesson's reagent

OCH3 O R N H H3CO R S S P P S S R S N H R

J. Org. Chem. 1994, 59, 1257-1263

Organophosphorous Reagents
Acylating Reagent IBCF Organophosphorous Reagent
OH

O i-BuO O

O R

O R

EtO EtO

O P O

O R

DEPC

Amine Attack - Originally developed as an alternative to the mixed anhydride method

Amine Attack

-The use of organophosphorous reagents gives enhanced regioselectivity toward the carbonyl of the phosphoric-carboxylic mixed anhydride

Common Organophosphorous Reagents

O O P N3 H3C H3C

O S P N3 O EtO P EtO CN O O N P Cl O N O

2 DPPA Oil, difficult to handle

MPTA Crystalline, stable

DECP Useful for nucleophillic amines

BOP-Cl Especially good for N-alkyl amino acids

Organophosphorous Reagents: Practical Considerations

Boger's Total Synthesis of Sandramycin
OH HH O N O O CH3 N CH3 N O CH3 CH3 O N O H H3C CH3 O O CH3 N O N O N H N O N CH3 O CH3 CH3 N CH3 O O O N H H O O HO N

N O H2N CO2H N

SESHN

N CH3

N H

Sandramycin

CH3 N O N H

NHSES

H3C

H CH3 CH3 O S O

DPPA, NaHCO3 90%

SESHN H O N O H N O N CH3 O CH3 N CH3 CH3 O N O H

SES=

SiMe3 O

- Macrocyclization was accomplished under mild conditions using NaHCO3 as a base -Use of stronger bases led to decomposition
H3C

CH3 N O N H

NHSES

H CH3 CH3

J. Am. Chem. Soc. 1996, 118, 1629-1644 J. Org. Chem. 1987, 52, 764-769

Acid Halogenation
R''' H2N R R'HN O OH OR'' O X O R'HN O R O OR'' R'''

Acid Halogenating Reagent

R R'HN

H N

X = Cl or F - Acid halides are typically used when steric congestion prohibits the use of standard coupling reagents - Currently there are a number of commercially available Fmoc amino acid fluorides, however Boc and Cbz groups present problems in the coupling of acid halides - For difficult couplings the more reactive and less expensive acid chloride would be preferred, however until recently an appropriate protecting group was not available

Carboxyanhydride Formation: An Unwanted Side reaction

O O H N R H X O O HN O R O O HN O R O

X=Activator

Oxazolonium Ion

N-carboxyanhydride

This side product can be significantly reduced with careful selection of protecting groups for the amine functionality. Boc protected amines form the carboxyanhydride byproduct much more readily than the corresponding Fmoc or Cbz amino acids.

Acc. Chem. Res. 1996, 29, 268-274 J. Am. Chem. Soc. 1996, 118, 9796-9797

Acid Chlorides
H2N R R'HN O OH

R''' OR'' O R R'HN O O OR'' R'''

Acid Halogenating Reagent

R R'HN O Cl

H N

- Acid Chlorides were first introduced for peptide coupling in 1903 by Emil Fischer -However, when the amine functionality is protected as a carbamate, the oxazolinone is readily accessed, leading to racemization and undesirable side products -These problems can be avoided by the use of a sulfonyl protecting group, but deprotection conditions may be too harsh for many peptides -This problem was solved by Vedejs and coworkers

Common reagents to Make Acid Chlorides

Cl N Cl N N Cl O Cl O Cl Cl H3CO N N OCH3 N Cl Cl Cl Cl O O O Cl Cl Cl Cl O S Cl

Cyanuric Chloride

CDMT

BTC
O P Cl Cl

Thionyl Chloride

Oxalyl Chloride

Phosphorous oxychloride (POCl3)

Acid Chlorides: New Sulfonyl Protecting Groups

N H3C N S O S O Cl S N O S O Cl

ThsCl "thisyl chloride"

BtsCl "betsyl Chloride"

- Both sulfonyl chlorides are readily accessed from the commercially available mercapto derivatives -The corresponding sulfonamides are synthesized in high yield for a number of amino acid derivatives, inculding zwitterionic amino acids - Both groups can be selectively removed in the presence of other sulfonamides under reducing conditions including; Zn/HOAc, Al-Hg/ether, and H3PO2

N S

O S O

H N H OH O

1) SOCl2 CH2Cl2 2) Na2CO3 CH2Cl2

H +H3N O CH3 O CH3

H Bts N H

Ph

H N H

O O CH3 CH3

95% Yield 99.75 de (crude)

J. Am. Chem. Soc. 1996, 118, 9796-9797

Acid Fluorides
Common Acid Fluorinating Reagents
F N F N N F Me2N F N NMe2 N F H3C N F N CH3

TFFH

BTFFH

DFIH

Cyanuric Fluoride Useful for in situ generation of acid fluorides in coupling reactions
H3C N H3C S F F F

DAST can be used in the absence of base to promote acid fluoride formation. Particularly useful in preparing Fmoc amino acid fluorides.

DAST

Advantages/Disadvantages of the Approach

- Promotes the formation of amide bonds at sterically hindered sites - Most carbamate protected amino acids are stable compounds that do not form the N-carboxyanhydride byproduct -More water stable than the corresponding acid chlorides -Arg and His are not stable and need to be generated in situ

- The amino acid fluorides show similar reactivity to activated esters and may not be appropriate for difficult couplings at unreactive sites J. Am. Chem. Soc. 1995, 117, 5401-5402 Lett. Pept. Sci. 1996, 2, 285

Acid Fluorides Applied

H3C H3C

H O OH NBoc N H Boc

DCC/DMAP or DCC/HOBt or BOP-Cl Racemization

H3C H3C

O NH

NBoc N H Boc H3C CO2Me

CH3 H2N CO2Me

Cyanuric Fluoride/ CH2Cl2/ Pyr

NaHCO3, H2O CH2Cl2 71%

No Racemization

H3C H3C

O F

NBoc N H Boc

- Danishefsky and co-workers applied an acid fluoride mediated coupling in route to 5-N-acetylardeemin when standard coupling reagents failed to produce a diastereomerically pure compund

J. Am. Chem. Soc. 1999, 121, 11953-11963

Microwave Assisted Synthesis

Solid Phase Synthesis of the Acyl Carrier Peptide
1) Piperidine, DMF, w 1min 2) Asn-Fmoc, PyBOP HOBt, DIEA, DMF, w 2 min

GLY-NHFmoc

GLY-ASN-NHFmoc

VQAAIDYING >95% - Researchers at CEM Corporation were able to rapidly and efficiently assemble a decameric peptide in >95% overall yield with microwave assisted synthesis - The reactions were enhanced through microwave radiation allowing for higher resin substitution, less reagent excess and higher coupling yields due to decreased intramolecular aggregation

Biopolymers, 2003, 71, 361

Reagent Reaction Time(min) Temperature(oC) Solvent PyBOP 20 110 DMF HATU 1.5 110 DMF

Synthesis, 2002, 11, 1592-1596

Racemization Pathways
Oxazolone Formation

H N O X R H O O O N R H N O O H R H2N O R N H H R NHR O

Oxazolone Base

N O O

R O

R O

Oxazole

Epimerization can be controlled with the appropriate rate enhancing, racemization suppressants

Racemization Suppressants
OH N N N N OH N N N N O N N OH

HOBt Most commonly used racemization suppressant. Often used in combination with carbodiimide chemistry

HOAt HOAt is more effective than HOBt due to intramolecular general base catalysis. Caution should be used on large scales, as HOAt is slightly explosive
Ph Ph P Ph

HODhbt HODhbt has yet to find widespread use due to a ring opening side reaction

OH H3C N N EtO2C N

H2F3 Ph

PTF When PTF is used in combination with HBTU, the coupling efficiency is equivalent to that of HATU. Unsuitable for phosphorous reagents because of strong P-F bond

HOCt When used with DIC, HOCt suppressed all racemization except with histidine.

Org. Lett. 2003, 5, 975-977

- Racemzation suppression agents also work to enhance the reactivity of intermediates in the coupling reaction -Also, Cu(II) salts have been found to act as racemization suppressants in the presence of standard coupling agents (CuCl2, Cu(OBt)2, Cu(OAt)2), J. Pept. Sci. 2001, 7, 115-120

Difficult Couplings: , dialkyl

H3C H2N CH3 CO2H H3C H2N CH3 CO2H

2-aminoisobutyric acid (aib)

-ethylalanine

Synthesis, 1995, 1205-1222

H (CH2)nY

R Xn(H2C)

H (CH2)nY

R Xn(H2C)

R (CH2)nY

Xn(H2C)

Calculated Bond Angle:

111.9o

108.4o

106.5o

- Disubstituted amino acids are particularly challenging because of the propensity for racemization of the penultimate residue, and diketopiperazine formation -Steric congestion also makes these residues less reactive to standard peptide coupling techniques -Both of these are a result of the geminal dialkyl effect -Dialkyl substituents force the peptide from it's preferred straight chain, anti-periplanar conformation into a staggered conformation, decreasing the bond angle in the chain and facilitating ring formation

Diketopiperazine Formation
CH3 N O R O R' NH OBn CH3 R' O

CH3 N R

O CH3

Diketopiperazine Diketopiperazine formation is especially problematic in the synthesis of peptides containing N-methylated amino acids. This is in part due to N-alkyl peptide exhibting a greater preference for the Z-amide.

, dialkyl : Solutions
Barton PTOC Ester
O ZHN ZHN O N S PhS N H CO2Et H3C CH3 O N H CO2Et

95%

Proposed Mechanism

N O O

S SPh R2N R' -O

N O

S NR2 R'

SPh R'

O NR2 N OS SPh

- PTOC esters can be generated in situ and coupled, but for higher yields isolation is recommended -Coupling proceeds under base free conditions, eliminating racemization - Free amines can be used in place of sulfeneamides but yields decrease

Tetrahedron, 1996, 52, 9367-9386

, dialkyl : Solutions
Azirine Method for Solution Phase Coupling
H3C R' ZHN CO2H N CH3 NR2 N H R' NHZ NR2 O O ZHN R' H N O NR2

O H3C CH3

H3C R'

CH3 O

HCl
ZHN

H N

R'

DCC
OH R'

N O NHZ

H2NR'' HOBt

ZHN

H N

O NH R''

O H3C CH3

O H3C CH3

-Azirine Method was used in the formation of hindered bonds in the natural product peptide, Alamethicin -This gave high yields and negligible racemization for the following "difficult" peptides Z-Val-Aib 95% Z-Val-Aib-Aib-NMePh 99%

Helv. Chim. Acta. 1990, 73, 13

Carbodiimides: Macrocyclization
OTBS OTBS

HN Ph O O NH HO TsN NH H N O HN

1) Et2NH 2)CF3CO2H
NPht Ph

HN O O NH HO NH H N O O HN

CO2t-Bu N Fmoc

3)DCC/HOBt MeCN, 60h 41%

TsN

NPht N

Cyclotheonamide A (A serine protease inhibitor, isolated from a marine sponge) Reagent BOP-Cl, DMAP BOP, DMAP EDC, HOBt DCC, HOBt DPPA, NaHCO3 BBC, DIEA Yield 25 38 24 41 25 36

Maryanoff, B.E.; Greco, M.N.; Zhang, H.; Andrade-Gordon, P.; Kauffman, J.A.; Nicolau, K.C., Liu, A.; Brungs, P.H. J. Am. Chem. Soc. 1995, 117, 1225-1239

Macrocyclizations
Silverman's Traceless Linker
O O P NHAc OMe OMe MeO O

Br

1) n-BuLi allylchlorodimethyl silane 82% 2) t-BuLi DMF 82%

H3C Si

CH3

MeO

Br

86%
O

NHAc

1) (S,S)-Et-DuPHOS-Rh, H2 2) (Boc)2O, DMAP 3) Hydrazine, MeOH 93%

BocHN O

H3C Si

CH3 I O H N

OEt

9-BBN, Pd(PPh3)4

J.Am. Chem. Soc. 1999, 121, 8407-8408 Org. Lett. 2000, 2, 3743-3746

H3C Si

CH3

H N O

BocHN O

OMe

Orthogonal Proecting groups allow for chain extension in either direction. Perfect for macrocyclizations

Macrocyclizations
Silverman's On resin Cyclization With a Traceless Linker

H3C Si H3C FmocHN O H3C CH3 H N O N H CH3 H N O

CH3 H3C O N H

H N CH3 O O O

1) Pd(PPh3)4 2) Pip/DMF 3) HBTU 4) 50% TFA/CH2Cl2, 36hr

CH3 CH3

- Silverman applied his side chain attachment methodology to the synthesis of a peptide based natural product, sansalcamide A - The ten step synthesis was carried out in 67% overall yield and >95% purity -Alowed for on-resin cyclizations with peptides not bearing polar sidechains -Solid phase methodology eliminates need for high dilution, and reduces dimerization and oligomerization -Especially important because many biologically active peptides are exclusively hydrophobic
H3C CH3 O H3C

H3C NH O O NH H N O CH3 O NH

CH3

CH3 CH3

Sansalcamide A

Org. Lett. 2000, 2, 3743-3746

Macrocyclizations : Tentoxin
Novel Incorporation of Difficult Residues

OH CH3 Boc N CH3 O H3C CH3 H N O N H H N O O O

EDC CuCl CH2Cl2/DMF

CH3 Boc N CH3 O H3C

H N

O N H

H N O

O O

CH3 CH3 Boc N CH3 O H3C CH3 O O N CH3 O O O

MeI K2CO3 18-crown-6 DMF

H N

H N

Tentoxin 1) TFA/H2O 2) DIC/HOBt/DIEA

NMe O N H H N O MeN CH
3

- The N-methyl dehydrophenylalanine could not be installed through standard peptide couplings

H3C CH3

- The dehydro amino acid was installed by an elimination reaction of the unprotected precursor - N-methylation was also carried out regioselectively on solid support due to the enhanced acidity of the amide proton of the dehydro-residue

Org. Lett. 2003, 5, 2115-2118

Difficult Couplings: N-methyl Amino Acids

Acid Catalyzed Cleavage of Imino Acid Sequences

O N HO O CH3

HO

CH3 N R O

O O O

HN

CH3 R

H3O+
OH

O NHMe HO O R

O R

Proceeds through Z-amide conformer. Minor side reaction however and is generally too slow to cause significant problems.

- N-alkyl amino acids are more prone to cleavage and diketopiperazine formation because the Z-amide is more populated than in standard amino acids, making cyclizations favorable -Furthermore, racemization is more problematic because the alpha proton is the most acidic proton whereas in natural amino acids the amide proton would be deprotonated first -The steric bulk of N-alkyl AAs reduces the nucleophillicity of the amine, slowing the reaction rate and leading to undesirable byproducts - It is important to note that in the case of N-methyl AAs, HOBt suppresses the reaction rate and benzotriazole based reagents should be avoided in most cases

Tet. Lett. 1991, 32, 1967

N-methyl Amino Acids: Solutions

BOP-Cl as an Effective Coupling Agent of Me-AAs

BOP-Cl
O O O N O P O Cl R R O Me R N H O O O O P N N O R N Me O R O

2
R O-

Intramolecular Base Catalysis

- Intramolecular base catalysis is proposed to allow for efficient coupling with BOP-Cl where other phosphorous based reagents fail - BOP-Cl allows for one-pot couplings of N-MeAAs because of selective reaction with the carboxylate -primary amines react with BOP-Cl yielding undesired side products - It is critical to employ high purity BOP-Cl and is best to prepare it from ethanol, diethylcarbonate and PCl5, Bull. Soc. Chim. Belg. 1986, 95, 203 - SPPS does not proceed efficiently with BOP-Cl. HOAt based reagents are the preferred reagents for SPPS of Nmethyl AAs and do not suffer the low reactivity of HOBt esters

Segment Condensations

- For large peptides, often times solid phase synthesis is not an operative pathway -Peptides/proteins must be made by convergent methods in which smaller pieces are brought together to form the whole oligomer -There are two ways to accomplish this, solution phase couplings using standard peptide bond forming reagents or native chemical ligation -Solution phase couplings are often made chemoselective by a protein's propensity for folding, this however requires additional protection strategies if multiple segments are to be coupled

Segment Condensations
Native Chemical Ligation (Kent Ligation)
O H3C O N H N O N H O S O O O NH COSR Cys COS-Na+

H2O, pH 7

O H3C O N H N O N H

O S

O O

O NH Cys COS-

1) Convert to thio-ester 2) repeat condensation 3) Cleave Peptide

- Solid phase chemcal ligation avoids the need to protect inernal residues within the peptide segments - Also, tedious purification of intermediates is no longer an issue - Assembly of the peptide components can proceed in either the C to N or N to C directions, the C to N direction however requires an additonal transient protecting group on the N-terminal cysteine - Racemization is not a problem because peptides are coupled under neutral conditions - Coupling using a C-terminal thio acid eliminates cyclization products derived from self-condensation

J. Am. Chem. Soc., 1999, 121, 8720-8727

Segment Condensations: Mechanism

Native Chemical Ligation (Kent Ligation)
O O Peptide SR +H3N -S Peptide2

H2O, pH 7 Chemoselective Reaction

O H2N O Peptide1 S Peptide2

Spontaneous Rearrangement
SH Peptide2 O

O Peptide1 N H

J. Am. Chem. Soc., 1999, 121, 8720-8727

Segment Condensations: Applied

Solid Phase Protein Synthesis Solid Phase Chemical Ligation (SPCL) "Accelerated" SPCL

- GV-PLA2, a 118 residue phospholipase was synthesized using SPCl in four segments with three ligation reactions - The molecule contains six disulfide bonds which could potentially compete with the ligation reaction - The major peak was collected, found to correspond to the mass of the full length peptide, and shown to possess full biological activity - An accelerated method was used in which concentrated solutions of each peptide component were used and ligation reactions proceeded for 1 hour, instead of the usual overnight reaction time

J. Am. Chem. Soc., 1999, 121, 8720-8727

Segment Condensations: Improved

Chemical Ligation of Non-cysteine Containing Peptides
O Peptide1 N H SH Peptide2 O

Selective Desulfurization

O Peptide1 N H Peptide2 O

- Native Chemical Ligation sequence can be carried out followed by a desulfurization step to afford a ligated product linked at an alanine residue - Desulfurization readily occurred in the presence of Raney Ni or with Pd/Al2O3 under hydrogen atmosphere -The products of both reactions could be directly lyophilized and for the most part underwent minimal side reactions

J. Am. Chem. Soc. 2001, 123, 526-533

Peptide Synthesis: Process Considerations

- There are more than 40 chemically synthesized peptide therapeutics on the market today (a four-fold increase from 1990) - All examples at left are potent hormones or hormone analogues that are required in relatively small quantities (<50 kg/ year) - These peptides represent research undertaken at least 10 years ago and hence represent the inefficient, expensive methodology of peptide synthesis in the early 90s - It was once thought that peptide therapeutics could be expected to cost $75-100 per gram per amino acid residue to produce - With current advancements in chemistry and economies of scale, peptides could be produced at <$1 per gram on a multi tonne scale -The economic advancements led to the development of enfuvirtide (a.k.a. Fuzeon or T-20)

Nat. Rev. Drug Disc., 2003, 2, 587-593

Peptide Synthesis: Process Considerations

Key Problems in Process Scale Peptide Synthesis
1) Contaminants and Impurities a) Racemization: Difficult to detect in large peptides -Use proper racemization suppresants, low dielectric constant solvents, and short activation times b) Reaction By-Products: - Ureas, phosphonium salts, and scavengers are the most common byproducts c)Oxidized peptides: Peptides rich in cysteine are easily oxidized and form dimers and higher order oligomers in solution d) Peptide Sequence Failures -Deletions can be minimized by using multiple methods for detection of primary amines (Ninhydrin, Trinitrobenzene Sulfonic Acid, and NF-31)
COO-AA1-AA2-NHFmoc COO-AA1-AA2-NHFmoc COO-AA1-AA2-NHFmoc Piperidine DMF COO-AA1-AA2-NH2 COO-AA1-AA2-NHFmoc COO-AA1-AA2-NH2 Fmoc-NH-AA3-COOH DIEA, HBTU, HOBt

COO-AA1-AA2-AA3-NHFmoc COO-AA1-AA2-AA3NHFmoc COO-AA1-AA2-NHFmoc

Single-Site Deletion

Nat. Rev. Drug Disc., 2003, 2, 587-593 Chimica Oggi. June 2003, 6-11 Anal. Biochem. 1976, 71, 260 Eur. J. Org. Chem. 1999, 2787

COO-AA1-AA2-NH2 COO-AA1-AA2-NH2 COO-AA1-AA2-NH2 Fmoc-NH-AA3-COOH DIEA, HBTU, HOBt

COO-AA1-AA2-AA3-AA3-NHFmoc COO-AA1-AA2-AA3NHFmoc COO-AA1-AA2-NHFmoc

Single-Site Double Hit

Peptide Synthesis: Process Considerations

Key Problems in Process Scale Peptide Synthesis

2) Toxic and Hazardous Reagents Used in Synthesis a) Storage and Bulk Use of Reagents -Triazole based reagents such as HBTU can have stability problems - This is especially pronounced with HOAt which is sensitive to friction and spark, leading to burning and explosion - The use of such reagents may require an expensive safety upgrade b) Hazardous Reagents - Carbodiimides and benzotriazole reagents are known to cause skin irritation and contact dermatitis with prolonged use - They also cause sensitization of the respiratory tract over time 3) Yield -Ramifications of low yielding reactions are obvious, however the extent of the problem is magnified in a step wise synthesis with few purification steps

Nat. Rev. Drug Disc., 2003, 2, 587-593 Chimica Oggi. June 2003, 6-11

Peptide Synthesis: Enfuvirtide

- Enfuvirtide is a 36 residue peptide that selectively inhibits HIV-1 membrane fusion -It has been approved for treatment of HIV patients in the US - Projected requirements are 3 tonnes per year, where patients will receive 180 mg per day or 80g per year - The drug was developed at Trimeris and a streamlined synthesis was developed by Roche on tonne scales -This involved a hybrid solid/solution phase synthesis where small segments were first made by SPPS and then coupled in solution

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-LeuGlu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-NH2

Route 1: Linear Solid Phase Peptide Synthesis - Fmoc SPPS conducted for the 36 residue sequence - Greater than 2 equivalents of Fmoc-AA were used per coupling - Furthermore, upon cleavage from the resin, the peptide was only ~30-40% pure - This required difficult, low throughput chromatographic separation - Overall yield was 6-8% - This was an expensive and inefficient initial synthesis, but allowed access to enough material for clinical trials

Nat. Rev. Drug Disc., 2003, 2, 587-593

Peptide Synthesis: Enfuvirtide

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-LeuGlu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-NH2

Route 2: Combination of SPPS and fragment condensations - Three side chain protected fragments are constructed using a super acid sensitive resin, 2-chlorotrityl resin - Resin is not patent protected and can be easily recycled, also attachment is racemization-free - The three fragments were synthesized using HBTU/HOBt and 1.5 eq of Fmoc protected amino acids, no re-couple cycles were necessary - Each fragment is isolated in >85% yield and >90% purity - Each fragment can be synthesized in one week and in 300-500 kg scales - To make the process efficient solvent recycling must occur, while yields are >99% per coupling, the cost is 75L of solvent per kilogram resin - Five solution phase reactions complete the peptide which is then isolated in 30% overall yield - The segment condensations were optimized to show less than 1% racemization

Nat. Rev. Drug Disc., 2003, 2, 587-593

Conclusions

- Amide Bonds are ubiquitous in nature and elsewhere - Effective methods to make these bonds are critical to extending pharmaceutical and bio-organic research - There however is no ONE method that is effective in every situation - Unfortunately the process of selecting the proper tool is somewhat empirical, however the tool box is well stocked and most difficulties can be overcome