rethinking amide bond synthesis

8/3/2019 Rethinking Amide Bond Synthesis

1/9

REVIEWdoi:10.1038/nature10702

Rethinking amide bond synthesisVijaya R. Pattabiraman

1

& Jeffrey W. Bode1

One of the most important reactions in organic chemistryamide bond formationis often overlooked as acontemporary challenge because of the widespread occurrence of amides in modern pharmaceuticals and biologicallyactive compounds. But existing methods are reaching their inherent limits, and concerns about their waste and expenseare becoming sharper. Novel chemical approaches to amide formation are therefore being developed. Here we reviewand summarize a new generation of amide-forming reactions that may contribute to solving these problems. We alsoconsider their potential application to current synthetic challenges, including the development of catalytic amideformation, the synthesis of therapeutic peptides and the preparation of modified peptides and proteins.

Amide linkages1 are not only the key

chemical connections of proteins butthey are also the basis for some of the

mostversatileand widely usedsynthetic polymers.Chemical reactions for their formation are amongthe most executed transformations in organic chemistry (Fig. 1). Theprevalence of amide functionality, particularly in peptides and proteins2,sometimes gives the incorrect impression that there are no remainingsynthetic challenges. This is surprising, as it is often the case that evensimple amides resist formation, forcing practitioners to resort to evermore exotic and expensive reagents for their synthesis. Furthermore,the favourable properties of amides, such as high polarity, stability andconformational diversity, make it one of the most popular and reliablefunctional groups in all branches of organic chemistry.Improvedmethodsfor the synthesis of amide functionality, whether catalytic and waste-free

or chemoselective and suitable for fragment coupling, are in greatdemand.

In living systems, most amide bonds are

formed by the complex factories that areribosomes. Long, complex proteins are assembledamino acid by amino acid, using a templatedamidation of amines and the active esters of

amino acid monomers and RNA (Fig. 2a)3. Synthetic chemists, by con-trast, do not have the luxury of workingon this single-moleculescale, andinstead deal with trillions of molecules that must be coaxed into precisereaction trajectories. This strategy necessitates that nearly every func-tional group be protected by a bulky hydrophobic appendage, leadingto a reliable, but rather wasteful approach to peptide synthesis, in whichdozens of molecules are sacrificed to form just one amide bond4.

Thecurrent methods for amide formationare remarkably general butat the same time widely regarded as expensive and inelegant. Notsurprisingly, in 2007 the American Chemical Society Green Chemistry

Institute (comprising members from major pharmaceutical industriesworldwide) voted amide formation avoiding poor atom economyreagents as the top challenge for organic chemistry5. Furthermore, eventhe best stoichiometric reagents often fail for the synthesis of stericallyhindered amides. The issues of waste and expense associated with amideformation are compounded when applied to peptide synthesis, and areresponsible for the great cost of commercial therapeutic peptides. Thechemical synthesis of proteins is largely prohibited by limitationsinherent to traditional amide formation, although advances in thechemoselective ligation of unprotected peptide fragments demonstratehow advances in amide-forming methodologies can have far-reachingimpacts across scientific disciplines.

The state of the art in amide bond formation

Before considering emerging methodologies for amide bond construc-tion, we should take stock of the successes of existing amide-formingreactions. Acylation of amines with activated carboxylic acids is themost common reaction performed in the synthesis of modern phar-maceuticals, accounting for 16% of all reactions, according to a recentlyanalysed data set of medicinal chemistry campaigns6. In the peptidefield, the development of solid-phase peptide synthesis7 and subsequentimprovements in coupling reagents, protecting groups, resins and chro-matographic methods have made the synthesis of small amounts (51,000mg) of moderately sized (3050 residues) peptides routine andcommonplace (Fig. 2b). Hydrophobic peptide sequences and challengeswith non-canonical amino acids still create problems, but there arefewpeptides of this size that cannotbe produced by a skilled practitioneron a laboratory scale. For the synthesis or semi-synthesis of proteins,

1Laboratorium fur Organische Chemie, ETH Zurich, 8093 Zurich, Switzerland.

R2

H2NR1

O

OHR2N

H

R1

O

Carboxylic acid Free amine New amide bond

Coupling

reagents

Base,solvent

O

NH

O

NH

R1

O

OA*

R2

H2N

+A* OA*

a

b

Figure 1 | Chemical structure of amides and the conventional chemicalmethod for amide bond synthesis. a, Resonancestructuresof an amidegroup.b, The conventional method for formation of an amide bond. It involvesactivation of a carboxylic acid by an activating group (A*), followed bynucleophilic displacement by a free amine to generate a new amide bond in thepresence of coupling reagent, base and solvent. R1 and R2, small molecules,peptides or proteins.

2011: YEAR OF CHEMISTRYCelebrating the central sciencenature.com/chemistry2011

2 2 / 2 9 D E C E M B E R 2 0 1 1 | V O L 4 8 0 | N A T U R E | 4 7 1

Macmillan Publishers Limited. All rights reserved2011
http://www.nature.com/doifinder/10.1038/nature10702http://www.nature.com/doifinder/10.1038/nature10702


2/9

native chemical ligation8 (NCL) has revolutionized the field and madepossible access to materials once thought impossible (Fig. 2c). Examplesinclude the preparation of enantiomeric proteins such as snow fleaantifreeze protein9, plectasin10 and the 203 amino acid HIV-1 proteasecovalent dimer11. An often overlooked achievement in synthetic amidechemistry is the access to high-molecular-weight amide-based polymers,including nylons and aramids, made possible by advances in polymer-ization techniques, among others12.

Emerging chemical methods for amide bond formation

The limits of traditional amide-formingreactions and NCL will continueto be pushed and tested, but the next breakthrough may come from new,

unexpected and selective methods for amide bond formation. Althoughmany of the emerging methods described below are still in their infancy,they reflect a growing creativity and urgency among synthetic organicchemists in addressing what is increasingly recognized as an unmet syn-thetic need. This emerging generation of new amide formations mayprovide the basis for important catalytic methods or novel fragmentcoupling strategies.

The conventional approach to amide formation is the condensation ofan amine with a carboxylic acid via an active ester. New methods foramide formation fall intotwo broad categories classified by their reactionpartners. In the first, aminesserve as one of the reaction partners andareacylated by either a catalytic or oxidative fashion. In the second, novelcombination of reaction partners lead to amide products by mechanisti-cally unique pathways.

New acylation reactions of amines

Amidation by catalytic acylation of amines with carboxylic acids.Traditional amide synthesis relies on a coupling reagent to convert anunreactive carboxylic acid into an activated carboxylate for reaction withamines to give amides. In the absence of a coupling reagent, the carboxylicacidand the amine simply form a carboxylate-ammonium salt,rather thanan amide product, owing to the unfavourable thermodynamics of theamide-formingreaction.Despite this, several promisingcatalysts for directamide synthesis from carboxylic acid and amine in the absence of a coup-ling reagent have begun to appear13,14. Most prominent arethe boronic acidcatalysts, first reported in ref. 15, and with recent improvements1618

(Fig. 3a). In all these cases, the boronic acid takes the role of a couplingreagent in generating an active ester suitable for amidation in a waste-free

catalytic manner.

Amidation by catalytic generation of activated carboxylates. Agrowing and important concept in organic chemistry is the concept ofredox economy, in which internal exchange of oxidation states betweenadjacent functional groups within or between reactants provides accessto a reactive intermediate without the need for stoichiometric reagents.In the context of amide bond formation, this has been achieved by thecatalytic generation of activated carboxylates from functionalizedaldehydes, such as formylcyclopropanes, a,b-unsaturated aldehydes19

and their more conveniently prepared a-hydroxyenone surrogates20,a-haloaldehydes and epoxyaldehydes21. These processes work particu-larly well with aN-heterocyclic carbene (NHC) catalyst and a co-catalystto generate an activated carboxylate, which is then converted to an

amide with a variety of amines (Fig. 3b). Such reactions make possibleamide formation using only catalytic amounts of reagents, and offer apromising solution to the recognized problem of side-product genera-tion in traditional amide formations. The mechanistic basis for thesecatalytic reactions requires more elaborate starting materials than clas-sical acylating agents generated from carboxylic acids. When used toaccess targets forwhich theclassical monomers arenot readily available,such asb-aminoacids, the initial synthetic burdenfor the preparation ofthese functionalized monomers is acceptable, particularly if the sub-sequent amide formations do not require stoichiometric couplingreagents. Enantioselective variants of new NHC-catalysed reactionsfor amidation with functionalized aldehyde monomers are an area ofintense interest, suggesting that these reactions may one day lead tocatalytic, enantioselective peptide synthesis.

Catalytic, oxidative amidation of amines. An alternative redoxapproach to amide formation is acylation of amines from alcohols oraldehydes along with a stoichiometric oxidant. NHC catalysts havebeen used for amide formation from simple aldehydes, stoichiometricoxidants and a suitable co-catalyst (Fig. 3b)22. Several metalcatalysts andoxidants23,24 have also been identified for this process, with the mostpromising being palladium25 and copper/silver26 (Fig. 4a).

An intriguing example of oxidativeamidation from the alcohol oxida-tion state is catalytic ruthenium promoted coupling of amines and alco-hols27. In general, this reaction proceeds in a clean atom-economicalmanner without any acid, base or additives, and generates molecularhydrogen as theonly by-product (Fig. 3c). Thus, although it is formally anoxidativeamidation,no stoichiometric oxidant is needed. Mechanistically,the ruthenium pincer-type catalyst promotes dehydrogenation of the

alcohol to aldehyde, which then participates in amidation with amine

mRNA

5

3

O

OA*AA1 LinkerH2N

Active ester(5 to 10 equivalents) Base, solvent

Solidsupport

O

LinkerNH

O

OA*

Active ester(4 to 9 equivalents)

+ Base

+ Solvent

By-products

New amide bond

Solid-phase peptide synthesis

Fragment 1 Fragment 2

b Solid-phase peptide synthesisa Ribosomal protein synthesis c Chemoselective amide forming ligation

Solvent,

with or withoutadditives

Solidsupport

Psite

Asite

AA4AA5

AA6AA

7AA8

AA3

AA2

AA1

Largerribosomalsubunit

Smallerribosomalsubunit

AA2AA3

AA4

AA5

AA1

AA2AA

3

AA4 AA5

AA1

AA1AA2 AA

3

AA4 AAn AA1 AA2

AA3 AA4 AAn

AA1AA

2

AA3

AA4

AAnAA

1

AA2 AA3

AA4 AAn

Polypeptide

Figure 2 | Representation of protein and peptide synthesis by biochemicaland chemical methods. a, Simplifiedcartoon depiction of protein synthesisbyribosomes. Proteins are assembled by templated amidation with unprotectedamino acids (blue spheres, AAx). The A site (grey, right) serves as the entrypoint for aminoacyl transfer RNA and elongation of the polypeptide occursalongthe P site (grey, left) by formation of a new amide linkage. This process ishighly efficient and readily produces very high-molecular-weight proteins.mRNA,messenger RNA. b, Schematic illustration of routinelyused solid-phasepeptide synthesis. Peptides are assembled on a solid support with active esters

generated from fully protected amino acid monomers, coupling reagent andbase. Peptides between 30 and 50 residues are routinely produced with naturalor unnatural amino acids. c, Scheme depicting the concept of chemoselectiveamide forming ligation for the synthesis of complex peptides, proteins andglycopeptides. Two uniquely reactive functional groups (represented by filledred circle and matching grey shape) on unprotected fragments reactchemoselectively to readily produce proteins up to 200 amino acid residueswith fewer by-products and waste.

RESEARCH REVIEW

4 7 2 | N A T U R E | V O L 4 8 0 | 2 2 / 2 9 D E C E M B E R 2 0 1 1



3/9


4/9

for the synthesis of difficultN-methylated peptides, phosphonium-basedcoupling reagents and bis(trichloromethyl)carbonate have been themethods of choice and now isonitrile chemistry presents interestingpossibilities34.

Several new chemoselective approaches to amides from thioacidshave recently appeared (Fig. 5b), based on pioneering, but long-overlooked chemistry3538. These include the coupling of thioacids withazides39 to give amides without the use of activating or coupling agents.

Mechanistically, the reaction between thioacid and azide is believed togenerate a thiatriazoline intermediate that breaks down to an amide,liberating nitrogen and elemental sulphur. Thioacids, in contrast tocarboxylic acids, havebeen shown40,41 to react with isonitriles at ambienttemperature to produce a thioformimidate carboxylate mixed anhydrideintermediate, which could then be reactedwith a peptide amine to give anamide42. In related reactions, thioacids have been reacted with electron-deficient sulphonamides43,44, isocyanates and isothiocyanates to produceamide linkages45. These reactions can even be performed in the presenceof unprotected alcohols, partially protected peptides and highly hinderedamino acids, andmay hold promise for thechallenging area of glycopep-tide synthesis.

Transamidation reactions

An interesting reaction in the context of amide chemistry is an amide-metathesis or transamidation process that would exchange the consti-tuents of two different amide groups. Efforts towards this goal havebeen reported46. Although secondary and tertiary amides are extremelyinert under normal conditions, they can be activated in the presenceof aluminium, zirconium or hafnium-amido catalysts to undergotransamidation and amide metathesis reactions. These reactions could

allow secondary and tertiary amides to be used as substrates in dynamiccovalent chemistry and oligoamide synthesis. Although the reportedconditions are not yet suitable for reactions on proteins or unprotectedpeptides, one could imagine applications such as chemoselectivechemical protein splicing to excise a protein segment of interest froma full-length protein for further studies and manipulations. As moremild and chemoselective metal-catalysed or enzymatic transamidationreactions become available, this method could become a valuable tool in

protein engineering or in the preparation of bio-inspired materials.

New methods for chemoselective ligationOne of the greatest needs in new methods for amide formation is thediscovery of chemoselective amide-forming ligations, ideally those thatcan lead to amide products in the presence of unprotected amines,carboxylic acids and alcohols. NCL8, in which a carboxy (C)-terminalpeptide or protein thioester reacts with an N-terminal cysteine peptideor protein to give an amide-linkage, represents a nearly ideal chemo-selective amide formation, in that it operates under mild aqueousconditions without complications from unprotected side chains(Fig. 6a). Its limitation is the need for an N-terminal cysteine residueand occasional difficulties in preparing the C-terminal thioesters, par-ticularly by Fmoc solid-phase peptide synthesis. To circumvent these

problems, approaches have been developed for NCL with cysteine-likeamino acid residues47 and for accessing C-terminal thioesters48,49. Thewide utility and reliability of NCL has elevated it to a privileged placeamong chemical methods for peptide, protein and glycopeptide syn-thesis. Inspired by these advances, chemists have sought to identifynew amide-ligation strategies that mirror the functional group toleranceandrapidreaction rates of NCL. Given therare occurrence of cysteine in

b

R2

NC

R1

O

SH

R1

O

NH

R2

Isonitrile

Azide

Thioacid

25 C

R2

NCO

Isocyanate

R1

O

NH

R3

NH

S

O O

R2 Ar N N

N R2

Sulphonamide

R1

O

NH

R2 R

1

O

NH

R2

Base,

25 C

SO2,

R1

= Amino acid, peptide

R2

= Amino acid, Ar, aliphatic,

saccharide

R1

= Ar, peptide

R2

= Ar, SO2Ar, SO

2-peptide

COOAr, alkene

BaseN

2

R1 = Amino acid, peptide

R2

= cyclohexyl, t-butyl

R3

= Saccharide, amino acid

R1

= Amino acid, saccharide

R2

= Amino acid, saccharide,

Ar, aliphatic

25 C

COS

H2N-R3

a

R2

NC

R1

O

NH

R2

Isonitrile

1. Microwave

150 C

2. NaOMe,MeOH

R1

= Amino acid

R2

= Saccharide

R1

O

OH

Carboxylicacid

R2

NC

Isonitrile

1. Microwave

150 C

2. Reduction

R1

O

NR

2

CH3

R1, R2 = Amino acid,

peptide, N-methylated

peptide

Figure 5 | Emerging reactions for chemoselective amide bond formationwith carboxylic acid, thioacid and amine surrogates. a, Direct condensationof carboxylicacid (red,middle)with isonitriles under microwaveconditions for

synthesisof aminoacids,N-methylated peptidesand glycopeptides. b, Reaction

of a thioacid (red, middle) with various amine surrogates, such as an azide, anelectron-deficient sulphonamide, isocyanate and isonitrile, to give, in manycases, a native amide linkage containing amino acid, peptide, saccharide,

aromatic and aliphatic compounds. t-, tertiary.

RESEARCH REVIEW




5/9

many proteins, ligation at serine or threonine residues has also gainedattention. In a recent example, C-terminal O-salicyaldehyde ester wasfound to undergo ligation to give an amide with an N-terminal serine orthreonine peptide with excellent reaction rates and selectivity50.

The two most promising reactions to emerge from the efforts toidentify new chemoselective ligations are the Staudinger51 ligation(Fig. 6b) and the a-ketoacidhydroxylamine decarboxylative ligation(Fig. 6c)52. The traceless Staudinger amide-forming ligation, firstreported in refs 53 and 54, involves the reaction of a peptidylC-terminal phosphinothioester with a N-terminal peptide azide. Thisliberates a molecule of nitrogen and leads to an iminophosphoraneintermediate, which breaks down to give the new amide bond. Severalapplications of this reaction have appeared, including the synthesis of

RNase A55,56 in conjunction withNCL and the chemoselective formationof peptide macrocycles57. Restrictions of the Staudinger ligation includeits limited functional group tolerance, which necessitates partial or com-plete protection of peptide side chains.

Thea-ketoacidhydroxylamine ligation takes advantage of a selectivereaction between a C-terminal peptide ketoacid and an N-terminalpeptide hydroxylamine58. The initial reaction between these twouniquely reactive functionalities is believed to produce a metastablehemiaminal intermediate that undergoes subsequent reaction to releasea molecule each of carbon dioxide and water to produce a new amidebond. In a recent utilization of this ligation method, human glucagon-like peptide 1 (GLP1; also known as GCG), a 30-amino-acid therapeuticpeptide, was synthesized in good yields and purity with fully unprotectedpeptide sidechains59. Thehighly chemoselective nature of thea-ketoacid

hydroxylamine ligation suggests an unconventional reaction mechanism,

which when fully identified could help in fine-tuning the reactivefunctionalities for developing new variants of this unusual ligation. Aninteresting off-shootof thea-ketoacidhydroxylamineligation is the selec-tive reaction between a a-ketophosphonic acid and N-(benzoyloxy)amineto generate an amide group in aqueous conditions without couplingreagents60. Application ofa-ketoacidhydroxylamine ligation and its var-iants for the synthesis ofa-peptides, b-peptides61 and glycopeptides62 havebegun to appear.

Partially protected ligation strategies

Chemoselective amide-formation with partially protected fragmentsare especially useful for the assembly of complex homogeneousglycoproteins. In general, the syntheses of long glycopeptides with

highly glycosylated side chains are difficult to prepare by conventionalsolid-phase chemistries whencompared to peptides of similar length. Tocircumvent this problem, chemoselective glycoprotein ligation usingrelatively small and readily accessible glycopeptide fragments hasemerged as the preferred strategy. In addition to many establishedand emerging methods63, the silver-ion-mediated condensation ofpartially protected peptide amines with peptide thioacids or thioester,commonly known as the thioester ligation, has proven to be one of themost dependable methods for the synthesis of very large glycoproteins.Following preliminary reports on reactions of thioacids in ref. 35, it wasshown64 that thioesters could also be used for the ligation in thepresenceof an activating agent such as 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). In these methods, silver(I) reacts with the sul-phur atom of a peptide thioacid or thioester to generate a reactive

intermediate, which is then displaced by a nucleophilic amine leading

(3:1) DMA/DMSOCO

2, H

2O

HN

HO

c Ketoacid-hydroxylamine ligation d Thioester ligation

O

S H2N

CONH2

Sac SacSac Sac

Sac Sac

Sac

Sac Sac

Sac SacSac SacSac

1. HOOBt, DIEA,AgCl, DMSO2. Five iterations

FmocHN

Sac SacSac Sac

Sac Sac

Sac

Sac Sac

Sac SacSac SacSac

FmocHN

O

HN

5

23 kDa MUC2 glycoprotein

O

O

OH

HN

O

Human glucagon-like peptide 1, residues 736

b Traceless Staudinger ligation

6 M guanidine HCl,pH 7.6 buffer,phenyl mercaptan

Human interleukin 8, residues 172

O

SBn H2N

HS

O

O

HN

HS

O

a Native chemical ligation (NCL)

N3

O

S PPh2

PG PG PG PG

PG PG

1. (10:1) DMF/H2O

2. Cleavage/deprotection

O

HN

(solidsupport)

Ribonuclease A, residues 110124

Interleukin-8 (1-33)Interleukin-8 (35-72)

GLP-1 (22-36)

Interleukin-8(1-33)Interleukin 8 (133)Interleukin 8 (3572)

Interleukin-8 (35-72)Interleukin-8(1-33) Interleukin 8 (3572)

Interleukin-8(35-72)Interleukin-8 (1-33)Interleukin 8 (133)

Interleukin-8 (35-72)Interleukin-8(1-33) GLP1 (2236)

GLP-1 (22-36)Interleukin-8 (35-72)Interleukin-8(1-33) GLP1 (2236)

GLP-1(22-36) Interleukin-8(35-72) Interleukin-8 (1-33)GLP1 (721)

GLP-1(22-36) Interleukin-8(35-72) Interleukin-8 (1-33)GLP1 (721)

GLP-1(22-36) Interleukin-8(35-72) Interleukin-8 (1-33)RNase A (110111) GLP-1 (22-36)Interleukin-8 (35-72)

Interleukin-8(1-33)RNase A (112124)

GLP-1 (22-36)Interleukin-8 (35-72)Interleukin-8(1-33)RNase A (112124)

GLP-1(22-36) Interleukin-8(35-72) Interleukin-8 (1-33)RNase A (110111)

Interleukin-8 (35-72)Interleukin-8(1-33) MUC2 repeat unit

Interleukin-8 (35-72)Interleukin-8(1-33) MUC2 repeat unit

Interleukin-8(35-72)Interleukin-8 (1-33)

MUC2 repeat unit

Interleukin-8(35-72)Interleukin-8 (1-33)MUC2 repeat unit

Figure 6 | Methods for chemoselective amide forming ligation for peptides,proteins and glycopeptides. a, Synthesis of human interleukin 8 by nativechemical ligation (NCL) of a C-terminal peptide thioester and an N-terminalcysteine residue to give a native amide linkage. b, Traceless Staudinger ligationbetween a C-terminal phosphinothioester and an azide for the synthesis of afragment of RNase A. c, Decarboxylative amide ligation between a C-terminalpeptide a-ketoacid and an N-terminal hydroxylamine to form a native amidebond. In ac, the numbers in parentheses represent the amino acid residues in

the N to C direction. d, Chemoselective silver-promoted thioester ligation of apartially protected C-terminal peptide thioester and an N-terminal amine toobtain a fully synthetic 23 kDa MUC2 repeat glycoprotein. PG, protectinggroup; DMF, N,N-dimethylformamide; Fmoc, 9-fluorenylmethyloxycarbonyl;Sac, monosaccharide; HOOBt, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine; DIEA, N,N-diisopropylethylamine; DMSO, dimethylsulphoxide; MUC2, mucin 2 protein.

REVIEW RESEARCH




6/9


7/9

deprotection step. For these reasons, a fragment-coupling strategy is notalways applicable for therapeutic peptide synthesis. A second challengeconcerns the expense and chemical waste generated by iterative peptidecouplings, which often require large excesses of protected amino acidmonomers, coupling agents and additives. As an example, the synthesisof 1,000 kg of Fuzeon (containing 36 amino acids) requires about45,000 kg of raw materials, not including the solvents used in the syn-thesis or purification74.

In this regard, the evolving reactions for chemoselective amide-ligation offer great potential to improve the synthesis of therapeuticpeptides in high purity and on a commercial scale. Ligations of fullyunprotected peptide fragments offer considerable advantages over thecoupling of fully or partially protected fragments, including obviatingthe need for post-coupling deprotections, the ability to work underaqueous conditions, and facile purification of the ligated product fromany unreacted fragments. Ligation reactions are often suited forcouplings at near equimolar ratios of reactant, whereas traditional,coupling-reagent-based approaches often require an excess of thecarboxylic acid fragment. NCL comes closest to these ideals, but therequirement for a N-terminal cysteine or a cysteine surrogate invitesthe identification of new variants of this reaction and the continueddevelopment of novel ligation chemistries described above.

Synthesis of modified peptides. An importantand syntheticallychallengingarea is the identification and incorporation of modifica-tions to a-peptide-based structures that increase metabolic stability,potency, bioavailability and permeability. Although most small ormedium-sized peptides composed entirely of natural amino acids havelimited stability, relatively modest modifications (including cyclizationor the incorporation of backbone methyl groups) can dramaticallyimprove their therapeutic index. For example, there is currently greatexcitement surrounding cyclotides, which are short peptides cyclizedhead to tail by an amide linkage and further cross-linked by disulphides(Fig. 8a)75,76. The synthesis of such architectures, and cyclic peptides ingeneral, by traditional methods is very challenging, as it often involves

fully protected peptides. This could prevent the formation of its nativeconformation for correct head to tail cyclization or disulphide forma-tion. Advances in NCL have provided ways to synthesize diverse cyclicpeptides and cyclotides.Conversely, new chemoselectiveligation methodsdistinct fromNCL could provide accessto circularpeptidesand cyclotide-type peptide architectures that do not have a disulphide or a cysteine.

The most established class of modified peptides currently used astherapeutic agents are natural and unnatural N-methylated peptides,

exemplified by cyclosporineA. N-methylation of single or multipleback-bone amides has evolved as one of the favourite strategies to enhancepotency, stability and structural rigidity. Synthetically, the increasedsteric demands ofN-alkyl amino acids often cause great difficulties foramide bond formation (Fig. 8d)77. They often require expensive couplingreagents and longer reaction times, risking epimerization and prematurecleavage of the peptide from the resin by diketopiperazine formation.In this regard, new chemical processes that deliver N-methyl peptides,such as the reaction of thioacids with isonitriles followed by reduction,promise improved synthetic access to this class of compounds.

The urgency in devising new synthetic reactions for amide formationis enhanced by the isolation and structural identification of ever morecomplicated natural peptides with highly unusual modifications.Despite their unique biological activities, impressive chemical and

metabolic stability, and complex architectures, none of these structurescan currently be chemically synthesized, limiting opportunities forexploring the structureactivity relationship or developing designedamide structures with these enviable properties. Of particular interestis microcin J25, in which part of the peptide is threaded through a smallmacrocycle formed by an amide bond between a side chain and the Nterminus (Fig. 8b)78. These form compact, globular structures, ideallysuited for mimicking proteinprotein interactions79,80. Subtilosin A81

and thuricin CD82 form another distinct class of naturally producedcyclic peptides, with multiple rings stitched together by a quaternaryamino acid in whicha cysteinesulphur is directly linked to thea-carbonof the peptide backbone (Fig. 8c). As traditional amide-formingreactions have largely failed for the synthesis of such structures83, newamide-forming reactions must now move to the front line in thesesynthetic efforts.Covalently modified synthetic proteins. Recent progress in thechemical synthesis of oligomeric proteinsconsisting of severalcovalently linked protein subunits with biological relevanceillustratesthe power of new amide-forming reactionsto provide access to materialsnot readily available by either synthetic or biotechnological means. Ofparticular interest is ubiquitination, in which proteins are tagged fordestruction by covalent post-translational modification of a lysine sidechain by theattachment of multiple ubiquitin proteins. Theapplication ofa variant of NCLmakespossible syntheticaccess to ubiquitinated proteinsfor investigations of fundamental biological questions8486.

A vital need in this area is a range of methods that would allow thechemical synthesis of much larger peptide structures, as current work islimited to perhaps at most 200residues. Proteinsin this size range canbeobtained by NCL of two large fragments or sequences with orthogonalprotecting groups or kinetically controlled ligations87,88. One way ofpushing thisboundaryfurther wouldbe the identificationof two distinctchemically orthogonal ligation methods that can operate together in thesame flask, thereby allowing more than two fragments to be joinedtogether in a controlled fashion.Such a strategy would reducethe numberof purification, deprotection or manipulation steps necessary for theassembly of larger structures.Functionalized amide-based polymers. Simple polyamides, such asnylon and related structures, have transformed the modern world byproviding tough, durableand inexpensivematerials for widespreadappli-cations.Despite these successes, chemists arefar frommatchingnaturallyoccurring highly versatile polyamides that have both structural as well asfunctional properties. For example,no chemical methods for theproduc-tion of materialswith thestrengthand propertiesof silksor collagenson a

meaningful scale are currently available. The potential applications of

Cyclic peptide(kalata B1)

Knotted peptide

(microcin J25)

Highly N-methylatedcyclic peptide

(cyclosporine A)

Cyclic peptide(subtilosin A)

a c

d

b

Figure 8 | Three-dimensional structures of representative examples forcyclic, knotted and N-methylated peptides. a, Kalata B1 is a cyclic peptide ofthe cyclotide family with six different loops formed by three disulphide bondsand a head to tail amide cyclization. b, Microcin J25 is a knotted peptide withunusually high stability imparted by threading the C terminus through a smallmacrocycle formed by the N terminus and a side-chain carboxylate.c, Subtilosin A represents a distinct class of bacterially derived cyclic peptidewith three unusual side-chain sulphur to backbone a-carbon linkages and ahead to tail cyclization. d, Cyclosporine A, a clinically important cyclic peptide

with a very high proportion ofN-methylated amides.

REVIEW RESEARCH




8/9

chemical methodsfor thecontrolledsynthesis of richly functionalized co-block polyamidesfrom simple starting materials arevast. Examplesof theapplicationof newamide-bondformingreactions to polyamide synthesisare already emerging. Milsteins ruthenium catalyst has been used89 fordirect synthesis of polyamides up to 30 kDa starting from simple diolsand diamines, a process that has promise for low cost production offunctionalized polyamides for biomaterials applications. In anotherimpressive example, peptide self-assembly has been combined with

chemical ligation to form90

polypeptide fibrils and other architectureswith molecular weights $3,000 kDa. This is especially useful, as it pro-vides access to synthetic polypeptides with a defined peptide sequencelarger than any natural protein for applications in biophysics, basicprotein science and cell biology.

Outlook

Over the past 100 years, synthetic chemists have advanced from thepreparation of a dipeptide to the almost routine preparation of syntheticpeptides of 50100 or more amino acids. Structures of 20 to 40 aminoacid residues can be prepared on a multi-kilogram scale. With a newgeneration of reagents and reactions, synthetic chemists will pushfurther towards once unimaginable syntheses.

The adoption of catalytic methods for amide formation will have the

most immediate impact on the contemporary preparation of amide-containing compounds. Such reactions will be both environmentallysustainable and costeffective. Thefirst applications of these new reactionswill be in the synthesis of small molecules, but the principles and under-lying mechanisms can be extended to peptidic structures. This is particu-larly true of peptides containing unnatural amino acid residues, in whichenantioselective variants of the catalytic reactions may provide directaccess to not only the chiral starting material but the key amide linkageas well. The advantages of efficiency and selectivity inherent to catalyticmethods for amide formation will offer new opportunities for enantio-selective and chemoselective reactions, including amidations in the pres-ence of reactive functional groups.

Improved amide-forming reactions that tolerate unprotected func-tional groups will continue the revolution in the preparation of synthetic

proteins that began with NCL. Such reactions are needed to improve theproduction and discovery of the rapidly expanding class of peptidethera-peutic agents currently under development. Such techniques will also aidthe facile preparation of glycoproteins and other post-translationallymodified structures. Among the long-term targets are methods forsynthesis, deconstruction and reassembly of naturally expressedproteins.Thiswould allowa portion of a protein of interestto bereplacedby a synthetic sequence containing isotopically labelled residues forstructural analysis, incorporation of unnatural amino acids for stabilityor enhanced activity, or specific probes for interrogating binding andbiological function.

As we learn better ways to synthesize complex, highly functionalizedamide-based structures without the need for aggressive reactants andcumbersome protecting groups, synthetic amides will provide a new

generation of functional materials with controllable higher-orderproperties, such as adhesion, cell growth regulation and biomineraliza-tion. Until now, synthetic methods have not kept up with the promiseof such materials. Chemists should take note of the challenges andopportunities existingin amide formation, and devise synthetic methodsthat are chemoselective, catalytic and sustainable.

1. Arthur, G. The Amide Linkage: Selected Structural Aspects in Chemistry,Biochemistry, and Materials Science (Wiley-Interscience, 2000).

2. Wieland, T. & Bodanszky, M. The World of Peptides: A Brief History of PeptideChemistry(Springer, 1991).

3. Schmeing, T. M. & Ramakrishnan, V. What recent ribosome structures haverevealed about the mechanism of translation. Nature 461, 12341242 (2009).

4. Coin, I.,Beyermann, M. & Bienert,M. Solid-phasepeptidesynthesis: fromstandardprocedures to thesynthesis of difficult sequences.NatureProtocols2, 32473256(2007).

5. Constable,D. J.C. et al. Key green chemistryresearch areas a perspective frompharmaceutical manufacturers. Green Chem. 9, 411420 (2007).

6. Roughley, S. D. & Jordan, A. M. The medicinal chemists toolbox: an analysis ofreactions used in the pursuit of drug candidates. J. Med. Chem. 54, 34513479(2011).

7. Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide.J. Am. Chem. Soc. 85, 21492154 (1963).Seminal paper describing the concept and utility of solid-phase peptidesynthesis.

8. Dawson, P. E., Muir, T. W., Clarklewis, I. & Kent, S. B. H. Synthesis of proteins bynative chemical ligation. Science 266, 776779 (1994).Seminal paper describing the concept of native chemical ligation for proteinsynthesis.

9. Pentelute, B. L.,Gates, Z. P., Dashnau, J. L.,Vanderkooi, J. M. & Kent,S. B. H. Mirrorimageforms of snow fleaantifreeze protein prepared by total chemical synthesishave identical antifreeze activities.J. Am. Chem. Soc. 130, 97029707 (2008).

10. Mandal, K. etal. Racemiccrystallography of synthetic protein enantiomersused todetermine the X-ray structure of plectasin by direct methods. Protein Sci. 18,11461154 (2009).

11. Torbeev,V. Y. & Kent, S. B. H. Convergent chemical synthesis andcrystalstructureof a 203 amino acid covalent dimer HIV-1 protease enzyme molecule. Angew.Chem. Int. Edn 46, 16671670 (2007).The largest fully chemically synthesized protein using native chemical ligation.

12. Marchildon, K. Polyamides still strong after seventy years. Macromol. React. Eng.5, 2254 (2011).

13. Marcelli, T. Mechanistic insights into direct amide bond formation catalyzed byboronic acids: halogens as Lewis bases.Angew. Chem. Int. Edn 49, 68406843(2010).

14. Ishihara, K. Dehydrative condensation catalyses. Tetrahedron 65, 10851109(2009).

15. Ishihara, K., Ohara, S. & Yamamoto, H. 3,4,5-Trifluorobenzeneboronic acid as an

extremely active amidation catalyst. J. Org. Chem. 61, 41964197 (1996).The first highly active boronic acid catalyst for amide synthesis.

16. Tang, P. Boric acidcatalyzed amideformation from carboxylic acidsand amines:n-benzyl-4-phenylbutyramide. Org. Synth. 81, 262268 (2005).

17. Al-Zoubi, R. M., Marion, O. & Hall,D. G. Direct and waste-free amidations andcycloadditions by organocatalytic activation of carboxylic acids at roomtemperature. Angew. Chem. Int. Edn 47, 28762879 (2008).

18. Charville,H., Jackson,D.,Hodges,G. & Whiting,A.The thermaland boron-catalyseddirect amide formation reactions: mechanistically understudied yet importantprocesses. Chem. Commun. 46, 18131823 (2010).

19. Bode, J. W. & Sohn, S. S. N-Heterocyclic carbene-catalyzed redox amidations ofa-functionalized aldehydes with amines. J. Am. Chem. Soc. 129, 1379813799(2007).Details catalytic, redox neutral amide-forming reactions.

20. Chiang, P.-C., Kim, Y. & Bode, J. W. Catalytic amide formation witha-hydroxyenones as acylating reagents. Chem. Commun. 45664568 (2009).

21. Vora, H. U. & Rovis, T. Nucleophilic carbene and HOAt relay catalysis in an amidebond coupling: an orthogonal peptide bond forming reaction.J. Am. Chem. Soc.

129, 1379613797 (2007).Details an organocatalytic oxidative method for amide formation.22. DeSarkar,S. & Studer,A. Oxidativeamidation and azidationof aldehydesby NHC

catalysis. Org. Lett. 12, 19921995 (2010).23. Allen, C. L. & Williams, J. M. J. Metal-catalysed approaches to amide bond

formation. Chem. Soc. Rev. 40, 34053415 (2011).24. Ekoue-Kovi, K. & Wolf, C. One-pot oxidative esterification and amidation of

aldehydes. Chem. Eur. J. 14, 63026315 (2008).25. Tamaru, Y.,Yamada,Y. & Yoshida,Z. Direct oxidativetransformation of aldehydes

to amides by palladium catalysis. Synthesis 474476 (1983).26. Yoo, W.-J. & Li, C.-J. Highly efficient oxidative amidation of aldehydes with amine

hydrochloride salts. J. Am. Chem. Soc. 128, 1306413065 (2006).27. Gunanathan, C., Ben-David, Y. & Milstein, D. Direct synthesis of amides from

alcohols and amines with liberation of H2. Science 317, 790792 (2007).First report of the use of ruthenium catalysts for direct couplingof alcohols andamines for amide synthesis.

28. Nordstrm,L. U.,Vogt, H. & Madsen,R. Amide synthesis fromalcohols andaminesby the extrusion of dihydrogen.J. Am. Chem. Soc. 130, 1767217673 (2008).

29. Muthaiah, S. et al. Direct amide synthesis from either alcohols or aldehydes withamines: activity of Ru(II) hydride and Ru(0) complexes.J. Org. Chem. 75,30023006 (2010).

30. Chan, W.-K., Ho, C.-M., Wong, M.-K. & Che, C.-M. Oxidative amide synthesis andN-terminala-amino groupligation of peptides in aqueous medium. J. Am. Chem.Soc. 128, 1479614797 (2006).

31. Shen,B., Makley,D. M. & Johnston, J. N. Umpolung reactivity in amide andpeptidesynthesis. Nature 465, 10271032 (2010).

32. Li, X.& Danishefsky,S. J. Newchemistrywithold functionalgroups: onthe reactionof isonitriles with carboxylic acids A route to various amide types.J. Am. Chem.Soc. 130, 54465448 (2008).

33. Wu, X. Y., Stockdill, J. L., Wang, P. & Danishefsky, S. J. Total synthesis ofcyclosporine: access to N-methylated peptides via isonitrile coupling reactions.J. Am. Chem. Soc. 132, 40984100 (2010).

34. Chatterjee,J., Gilon, C.,Hoffman, A. & Kessler,H. N-Methylationof peptides: a newperspective in medicinal chemistry. Acc. Chem. Res. 41, 13311342 (2008).

35. Blake, J. Peptide segment coupling in aqueous-medium silver ion activation ofthe thiolcarboxyl group. Int. J. Pept. Protein Res. 17, 273274 (1981).

36. Rosen,T.,Lico,I. M.& Chu, D.T. W.A convenientand highlychemoselectivemethodfor the reductive acetylation of azides. J. Org. Chem. 53, 15801582 (1988).

RESEARCH REVIEW




9/9

37. Messeri, T., Sternbach, D. D. & Tomkinson, N. C. O. A novel deprotection/functionalisation sequence using 2,4-dinitrobenzenesulfonamide: Part 1.Tetrahedr. Lett. 39, 16691672 (1998).

38. Messeri, T., Sternbach, D. D. & Tomkinson, N. C. O. A novel deprotection/functionalisation sequence using 2,4-dinitrobenzenesulfonamide: Part 2.Tetrahedr. Lett. 39, 16731676 (1998).

39. Shangguan, N.,Katukojvala, S., Greenberg,R. & Williams,L. J. The reactionof thioacidswith azides:a newmechanism andnew synthetic applications.J. Am.Chem.Soc. 125, 77547755 (2003).

40. Yuan, Y., Chen, J., Wan, Q. A., Wilson, R. M. & Danishefsky, S. J. Toward fullysynthetic, homogeneous glycoproteins: advances in chemical ligation.

Biopolymers 94, 373384 (2010).41. Wang,P. & Danishefsky,S. J. Promising general solutionto theproblemof ligating

peptides and glycopeptides. J. Am. Chem. Soc. 132, 1704517051 (2010).42. Rao,Y.,Li, X.C. & Danishefsky,S. J. Thio FCMAintermediatesas strongacyl donors:

a generalsolution tothe formationof complexamidebonds.J.Am. Chem.Soc. 131,1292412926 (2009).

43. Talan, R.S.,Sanki,A. K.& Sucheck,S. J.Facilesynthesisof N-glycosylamides usinga N-glycosyl-2,4-dinitrobenzenesulfonamide and thioacids. Carbohydr. Res. 344,20482050 (2009).

44. Crich, D., Sana, K. & Guo, S. Amino acid and peptide synthesis andfunctionalization by the reaction of thioacids with 2,4-dinitrobenzenesulfonamides. Org. Lett. 9, 44234426 (2007).

45. Crich, D. & Sasaki,K. Reactionof thioacids withisocyanates andisothiocyanates: aconvenient amide ligation process. Org. Lett. 11, 35143517 (2009).

46. Stephenson, N. A., Zhu, J., Gellman, S. H. & Stahl, S. S. Catalytic transamidationreactions compatible with tertiary amide metathesis under ambient conditions.J. Am. Chem. Soc. 131, 1000310008 (2009).

47. Rohde, H. & Seitz, O. Ligation-desulfurization: a powerful combination in thesynthesis of peptides and glycopeptides. Biopolymers 94, 551559 (2010).

48. Blanco-Canosa, J. B. & Dawson, P. E. An efficient Fmoc-SPPS approach for thegeneration of thioester peptide precursors for use in native chemical ligation.Angew. Chem. Int. Edn 47, 68516855 (2008).

49. Fang, G.-M. et al. Protein chemical synthesis by ligation of peptide hydrazides.Angew. Chem. Int. Edn 50, 76457649 (2011).

50. Li, X., Lam, H. Y., Zhang, Y. & Chan, C. K. Salicylaldehyde ester-inducedchemoselective peptideligations: enablinggeneration of naturalpeptidic linkagesat the serine/threonine sites. Org. Lett. 12, 17241727 (2010).

51. Staudinger, H. & Meyer, J. Uber neue organische phosphorverbindungen III.Phosphinmethylenderivate und phosphinimine. Helv. Chim. Acta 2, 635646(1919).

52. Bode, J. W., Fox, R. M. & Baucom, K. D. Chemoselective amide ligations bydecarboxylative condensations of N-alkylhydroxylaminesand a-ketoacids.Angew.Chem. Int. Edn 45, 12481252 (2006).Seminal paper describing the principle and application of the a-ketoacid-hydroxylamine ligation reaction.

53. Saxon, E.,Armstrong, J. I. & Bertozzi, C. R. A traceless Staudinger ligation for thechemoselective synthesis of amide bonds. Org. Lett. 2, 21412143 (2000).Describes the development of the Staudinger reaction for peptide ligation.

54. Nilsson, B. L., Kiessling, L. L. & Raines, R. T. Staudinger ligation: a peptide from athioester and azide. Org. Lett. 2, 19391941 (2000).The development of traceless Staudinger ligation.

55. Nilsson, B. L., Hondal, R. J., Soellner, M. B. & Raines, R. T. Protein assembly byorthogonalchemical ligationmethods.J.Am. Chem.Soc. 125, 52685269(2003).

56. Kohn, M. & Breinbauer, R. The Staudinger ligation a gift to chemical biology.Angew. Chem. Int. Edn 43, 31063116 (2004).

57. Kleineweischede, R. & Hackenberger, C. P. R. Chemoselective peptide cyclizationby traceless Staudinger ligation.Angew. Chem. Int. Ed 47, 59845988 (2008).

58. Medina, S. I., Wu, J. & Bode, J. W. Nitrone protecting groups for enantiopureN-hydroxyamino acids: synthesis of N-terminal peptide hydroxylamines forchemoselective ligations. Org. Biomol. Chem. 8, 34053417 (2010).

59. Wu, J., Ruiz-Rodriguez, J., Comstock, J. M., Dong, J. Z. & Bode, J. W. Synthesis ofhuman GLP-1 (736) by chemoselective a-ketoacid-hydroxylamine peptideligation of unprotected fragments. Chem. Sci. 2, 19761979 (2011).

60. Arora, J. S.,Kaur, N. & Phanstiel, O. Chemoselective N-acylationvia condensationsof N-(benzoyloxy)aminesand a-ketophosphonic acids under aqueousconditions.J. Org. Chem. 73, 61826186 (2008).

61. Carrillo,N., Davalos, E.A., Russak,J. A.& Bode,J. W.Iterative,aqueous synthesis ofb3-oligopeptides without coupling reagents.J. Am. Chem. Soc. 128, 14521453(2006).

62. Sanki, A. K., Talan, R. S. & Sucheck,S. J. Synthesis of small glycopeptides bydecarboxylative condensation and insight into the reaction mechanism. J. Org.Chem. 74, 18861896 (2009).

63. Hirano, K.,Macmillan,D., Tezuka,K., Tsuji, T. & Kajihara,Y. Design andsynthesis ofa homogeneous erythropoietin analogue with two human complex-typesialyloligosaccharides: combined use of chemical and bacterial proteinexpression methods.Angew. Chem. Int. Edn 48, 95579560 (2009).

64. Aimoto,S., Mizoguchi, N.,Hojo, H. & Yoshimura,S. Developmentof a facile methodfor polypeptide-synthesis synthesis of bovine pancreatic trypsin-inhibitor(BPTI). Bull. Chem. Soc. Jpn 62, 524531 (1989).The early application of the thioester ligation method for protein synthesis.

65. Hojo, H. et al. Chemical synthesis of 23 kDa glycoprotein by repetitive segmentcondensation: a synthesis of MUC2 basal motif carrying multiple O-GalNacmoieties. J. Am. Chem. Soc. 127, 1372013725 (2005).

66. Deming, T. J. Synthetic polypeptidesfor biomedical applications. Prog. Polym.Sci.32, 858875 (2007).

67. Rutledge, R. D. & Wright, D. W. Biomineralization: Peptide-mediated Synthesis ofMaterials (Wiley & Sons, 2009).

68. Ouchi,M., Terashima, T. & Sawamoto, M. Transition metal-catalyzed living radicalpolymerization: toward perfection in catalysis and precision polymer synthesis.Chem. Rev. 109, 49635050 (2009).

69. Deming, T. J. Facile synthesis of block copolypeptides of defined architecture.

Nature 390, 386389 (1997).The first organometal initiators for controlled polyamide synthesis.

70. Kramer, J. R. & Deming, T. J. Glycopolypeptides via living polymerization ofglycosylated-L-lysine N-carboxyanhydrides. J.Am. Chem.Soc.132, 1506815071(2010).

71. Sun, H., Zhang, J., Liu, Q., Yu, L. & Zhao, J. Metal-catalyzed copolymerization ofimines andCO: a non-amino acidrouteto polypeptides.Angew.Chem.Int. Edn 46,60686072 (2007).

72. Bray, B.L. Large-scalemanufacture of peptidetherapeuticsby chemical synthesis.Nature Rev. Drug Discov. 2, 587593 (2003).

73. Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Synthetic therapeuticpeptides: science and market. Drug Discov. Today15, 4056 (2010).

74. Marx, V. Roches fuzeon challenge. Chem. Eng. News 83, 1617 (2005).75. Ireland, D. C., Clark, R. J., Daly, N. L. & Craik, D. J. Isolation, sequencing, and

structure activity relationships of cyclotides.J. Nat.Prod. 73, 16101622 (2010).76. Clark, R. J. et al. The engineering of an orally active conotoxin for the treatmentof

neuropathic pain. Angew. Chem. Int. Edn 49, 65456548 (2010).77. Rodriguez, H., Suarez, M. & Albericio, F. A convenient microwave-enhanced solid-

phase synthesis of short chainN

-methyl-rich peptides.J. Pept. Sci.

16, 136140(2010).

78. Rebuffat,S. F. etal. Molecular knots as templates for proteinengineering: thestoryof lasso peptides. J. Pept. Sci. 16, 38 (2010).

79. Vincent, P. A. & Morero, R. D. The structure and biological aspects of peptideantibiotic microcin J25. Curr. Med. Chem. 16, 538549 (2009).

80. Knappe, T. A., Linne, U., Robbel, L. & Marahiel, M. A. Insights into thebiosynthesis and stability of the lasso peptide capistruin. Chem. Biol. 16,12901298 (2009).

81. Kawulka, K. E. et al. Structure of subtilosin A, a cyclic antimicrobial peptide fromBacillus subtilis with unusual sulfur to a-carbon cross-links: formation andreductionof a-thio-a-amino acid derivatives.Biochemistry43, 33853395 (2004).

82. Rea, M. C. et al. Thuricin CD, a posttranslationally modified bacteriocin with anarrow spectrum of activity against Clostridium difficile. Proc. Natl Acad. Sci. USA107, 93529357 (2010).

83. Rosengren, K. J. et al. Microcin J25 has a threaded sidechain-to-backbone ringstructure and not a head-to-tail cyclized backbone. J. Am. Chem. Soc. 125,1246412474 (2003).

84. Kumar, K. S. A., Spasser, L., Erlich, L. A., Bavikar, S. N. & Brik, A. Total chemical

synthesis of di-ubiquitin chains. Angew. Chem. Int. Edn 49, 91269131 (2010).85. Virdee, S., Ye, Y., Nguyen,D. P., Komander,D. & Chin,J. W. Engineereddiubiquitin

synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. NatureChem. Biol. 6, 750757 (2010).

86. Yang, R., Pasunooti, K. K., Li, F., Liu, X.-W. & Liu, C.-F. Synthesis of K48-linkeddiubiquitin using dual native chemical ligation at lysine. Chem. Commun. 46,71997201 (2010).

87. Tam, J. P., Yu, Q. & Yang, J.-L. Tandem ligation of unprotected peptides throughthiaprolyl andcysteinyl bonds inwater.J.Am. Chem.Soc. 123, 24872494(2001).

88. Bang, D., Pentelute, B. L. & Kent, S. B. H. Kinetically controlled ligation for theconvergent chemical synthesis of proteins.Angew.Chem.Int. Edn 45, 39853988(2006).

89. Zeng,H. & Guan, Z.Directsynthesisof polyamidesvia catalytic dehydrogenationofdiols and diamines. J. Am. Chem. Soc. 133, 11591161 (2011).

90. Ryadnov, M. G. & Woolfson, D. N. Self-assembled templates for polypeptidesynthesis. J. Am. Chem. Soc. 129, 1407414081 (2007).

Acknowledgements J.W.B. is grateful for the support of diverse agencies for thedevelopment of new methods for amide formation, including the Arnold and Mabel

Beckman Foundation, the David and Lucille Packard Foundation, ResearchCorporation for Science Advancement, the US National Institutes of Health(NIH-NIGMS) and the Swiss National ScienceFoundation (200021-131957). V.R.P. issupported by an ETH Fellowship. We thank all our past and present co-workers whohave contributed to the discovery and development of new amide-forming reactions.

Author Contributions J.W.B. conceived the outline of this Review; J.W.B. and V.R.P.discussed, planned and wrote it.

Author Information Reprints and permissions information is available atwww.nature.com/reprints . The authors declare no competing financial interests.Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence should be addressed to J.W.B.([email protected]).

REVIEW RESEARCH

http://www.nature.com/reprintshttp://www.nature.com/naturemailto:[email protected]:[email protected]://www.nature.com/naturehttp://www.nature.com/reprints

rethinking amide bond synthesis

Documents