orthogonal ligation strategies for peptide and protein orthogonal ligatio… · unprotected peptide...

Orthogonal LigationStrategies for Peptideand Protein

James P. TamQitao Yu

Zhenwei MiaoDepartment of Microbiology

and Immunology,Vanderbilt University,

A5119 MCN,Nashville,

TN 37232-2363

Abstract: This review focuses on the concept, criteria, and methods of an orthogonal amideligating strategy suitable for syntheses of peptides, peptide mimetics, and proteins. Utilizingunprotected peptides or proteins derived from chemical or biosynthetic sources, this ligationstrategy has been shown to be general and exceptionally mild. Its orthogonality in ligating twounprotected segments with free N-terminal (NT)-amines at a specific NT-amine is achieved througha chemoselective capture step and then an intramolecular acyl transfer reaction. Both couplingreagents for enthalpic activation and protection schemes therefore become unnecessary. More thana dozen orthogonal ligation methods based on either imine or thioester captures have beendeveloped to afford native and unusual amino acids at ligation sites of linear, branched, or cyclicpeptides. Because unprotected peptides and proteins of different sizes and forms can be obtainedfrom either chemical or recombinant sources, orthogonal ligation removes the size limitationimposed on the chemical synthesis of a protein with a native or non-native structure. Furthermore,by using building blocks from biosynthetic sources, orthogonal ligation provides a unifyingoperational concept for both total and semisynthesis of peptides and proteins.© 2000 John Wiley& Sons, Inc. Biopoly 51: 311–332, 1999

INTRODUCTION

Chemical syntheses of peptides and proteins havebeen useful for structure–function studies that havemade possible the discovery of new therapeuticagents. They fall broadly into two approaches. Thestepwise approach, often by solid-phase peptide syn-thesis,1–3 employs repetitive steps to assemble pro-tected amino acids. In very lengthy syntheses (e.g.,proteins) multiple purification steps to remove heter-ogeneous byproducts may be required.4–6 In the seg-ment approach that ligates blocks of premade andpurified peptides, usually in their protected forms,purification is simpler.7–19Both approaches have beensuccessful in preparing proteins of 70 to;150 amino

acids and contain strategies that are technologicallyfeasible for preparing proteins extending beyond thisrange. However, they have not been developed to thepoint of becoming routine and practical for synthesesof proteins with.150 amino acid residues. Recently,new segment ligation methods that efficiently coupleunprotected peptide segments to form these largerpeptides and proteins have been realized. These liga-tion methods yielding either amide or nonamidebonds at the ligation sites. Of these two ligationmethods forming an amide bond regiospecific to thedesired N-terminal (NT)-amine between unprotectedpeptide segments containing two free NT-amines rep-resent a significant methodological advance. We will

Correspondence to: James P. Tam; e-mail: [email protected] (Peptide Science), Vol. 51, 311–332 (1999)© 2000 John Wiley & Sons, Inc.

311

refer to this group of methods as the orthogonal liga-tion strategy in accordance with other orthogonal con-cepts in chemistry, including orthogonal protectionschemes20 and orthogonal activation21 and coupling22

in organic chemistry that distinguish two functionalsites based on chemoselectivity.

This paper reviews the considerable progress thathas been achieved in the past six years in developingnew orthogonal ligation methods in peptide synthesis.A major part of this review deals with our own worksince methods developed by others have already beenreviewed in great depth.23 For the sake of complete-ness, we have also included those results that areeither submitted for publication or are in press.

CONCEPTUAL FRAMEWORK OFORTHOGONAL LIGATION

The conceptual framework of orthogonal ligationstrategy is distinguished mechanistically from othersegment strategies by a cascade reaction consisting oftwo reaction sequence of a nonamide capture and anintramolecular acylation (Figure 1). It contains thefollowing elements.

1. Free peptides and their esters as buildingblocks derived from chemical or biosyntheticsources. Ligation is performed without a pro-tection scheme or coupling reagent, and inaqueous conditions.

2. Complementary amine- or acyl-nucleophile andelectrophile pair. Four functional groups(amine, ester, nucleophile, and electrophile) areclustered in the reaction center in the two-stepligation sequence. An nucleophile (or electro-phile) is placed proximally near the reactingNT-amine of one segment, (e.g.,2 as an amine-nucleophile or -electrophile pair) and the com-plementary reactive electrophile (or nucleo-phile) at the acylating C-terminal (CT) ester(e.g.,1 as an acyl-nucleophile or -electrophilepair) of another segment.

3. Intermolecular chemoselective capture. The re-action is initiated by a chemoselective captureof the nucleophile and electrophile pair thatforms a nonamide covalent intermediate3.

4. Intramolecular acyl transfer to form amide. Thecapture reaction brings the NT-amine and CT-ester into close proximity, which results in an

FIGURE 1 General concept of orthogonal ligation. X and Y represent a nucleophile and anelectrophile (see Tables II and III). Z is the bond of the capture intermediate. R1–R4 represent sidechains.

312 Tam, Yu, and Miao

intramolecular acyl transfer to form the amide4and5.

5. Products. The products contain an amide bondand a peptide backbone structure. However,some ligation methods yield imidic bond withunusual side chains5. All products usually donot need a deprotecting step after the ligationreaction and, in some cases, can be used directlyfor biological assays.

In the literature, orthogonal ligation has also beencalled chemoselective, capture-activation,24 native,25

intramolecular,26 or biomimetic ligation27 because itcontains attributes of all or part of these descriptions.To gain a better understanding of the conceptual ap-proach of orthogonal ligation, we will provide a briefdescription of its background and the advantages ofeach of these elements.

Ligation Strategies without a ProtectingGroup Scheme

A fundamental difference between orthogonal ligationand conventional segment methods of peptide synthe-sis is the absence of a protection scheme. This in turnleads to a tactical difference of activation in the cou-pling reaction. Enthalpic activation by a couplingreagent in conventional segment strategies requiresthe use of a protection scheme to block other com-peting functional groups. However, these schemesoften result in poor solubility of protected segments,low coupling efficiency, and possible racemizationdue to overactivation of the acylating carboxylgroup.5–13 Significant efforts by many laboratorieshave been made to overcome these limitations. Theuse of partial or minimal protecting-group strategies,

powerful solubilizing solvents, and novel protectinggroups to disrupt aggregations due to ordered struc-tures have resulted in the successful syntheses of largepeptides.14–19

Partly because of these limitations and partly be-cause of new developments in biochemistry, alterna-tive ligation strategies have emerged that do not relyon multitiered protecting groups and enthalpic activa-tion. These include ligation strategies based on en-zymes, entropic activation, chemoselectivity, priorthiol capture, and orthogonality (Table I).

Enzymatic syntheses often use minimally pro-tected peptide segments and the specificity of an en-zyme to accomplish the coupling reaction under mildconditions.28–33Recent innovations such as syntheticligases based on mutated subtilisins and amide-bondforming catalytic antibodies hold significant prom-ise.34–36Chemoselective ligation, often based on thioland imine chemistries, employs a pair of mutuallyreactive nucleophile and electrophiles to couple twounprotected peptide segments, usually as a nonamidelinkage as surrogate bonds at ligation sites.37 Exam-ples include hydrazone,38–40 oxime,41 thioether,42,43

and thioester.44–48 An advantage of these peptide-bond replacement methods is the greater efficiency ofligation reaction deriving from the high and specificreactivity between the two functional groups that isoften not seen in amino acids. Although this strategyhas found extensive applications and successes inprotein chemistry for conjugation reactions, semisyn-theses, and synthesis of peptide dendrimers,37 a lim-itation is that some of these linkages are not as stableas an amide bond at acidic38 or basic pH.44

Ligation strategies based on entropic activationhave been developed in semi- and total syntheses.Semisynthetic methods rely on the complementarity

Table I Comparison of Different Ligation Strategies

StrategyProtecting

GroupAmideBond

EntropicActivation Comments

1. Enzymatic Yes/no Yes Yes Ligases include native and mutantproteolytic enzymes as well ascatalytic antibody

2. Chemoselective No No No Based on protein conjugationmethods

3. Entropic No Yes Yes Structural complementary required4. Prior thiol

captureNo Yes Yes Template directed

5. Orthogonalligation

No Yes Yes A cascade reaction ofchemoselective capture and acyltransfer, useful for both inter- andintramolecular amide formation

Orthogonal Ligation Strategies 313

of two segments that form a stable noncovalent inter-mediate from which a proximity-driven acylation re-action mediated chemically or enzymatically ensues.An example is the CNBr-nicked recombination ofcytochromec using a homoserine lactone segment.48

At present, this method is limited by the lack ofsufficient knowledge of the protein–protein interac-tions to design segments with complementarity.Bioorganic strategies based on entropic activationhave employed organic templates to facilitate in-tramolecular acylation reactions.49 An ingenious ex-ample was introduced by Kemp and his co-workers intheir “prior thiol capture” strategy.50–52 The capturestep brings together the respective C- and N-terminiof the two peptide segments through a tricyclic tem-plate that enables the subsequent amide bond forma-tion by an intramolecular acyl migration (Figure 2).Such entropic activation, in the words of Kemp, isattributed to the high effective molarity akin to manyintramolecular reactions found in small cyclic organiccompounds.

Orthogonal ligation is the latest strategy developedfor segment synthesis. It employs a two-step reactionsequence of capture and activation. The capture steputilizes the principle of chemoselective ligation toform a covalent intermediate between two peptidesegments. Then, an amide bond via an intramolecularacyl transfer is formed through entropic activations.The intramolecular acylation rate, which is first orderand often spontaneous, minimizes side reactions as-sociated with enthalpic activation methods.

The conceptual framework of orthogonal ligationis similar to the prior thiol capture with three differ-ences: (1) It does not require a template to bridge thetwo peptide segments and is not restricted by a thiolcapture mechanism to form a covalent tricyclicbridged intermediate. (2) Instead of a.12-memberring, the acyl migration is mediated through a morefavorable five- or six-member ring intermediate. (3)Finally, it fully exploits unprotected peptide segmentsas building blocks including those from biosynthetic

sources. These improvements provide a greater ver-satility and practicality to segment ligations.

Complementary Amine- and Acyl-Nucleophile andElectrophile Pairs. For the synthesis of peptides witha native backbone structure, orthogonal ligation be-tween two segments is performed in an NT-amine toCT-carboxyl end fashion. Since such an amide bondformation involves two reactions, capture and acyltransfer, it requires four functional groups, usuallyarranged as a specific and complementary pair oneach segment. Typically, an nucleophile (or electro-phile) is paired with an NT-amine in an amine seg-ment and an electrophile (or nucleophile) is pairedwith a CT-ester in an acyl segment. For simplicity, wedefine the pair bearing the reactivea-amine as theamine-nucleophile or -electrophile and the pair bear-ing the acylating carbonyl as the acyl-nucleophile or-electrophile.

The spatial pairing of these functional pairs oneach peptide segment is of critical importance forimplementing the ligation as a cascade reaction. Akey tactic is to place a nucleophile/electrophile prox-imally to the reacting NT-amine or CT-carbonyl, usu-ally linked by a spacer of 3 atoms or less. The effectof clustering in a reaction center with these fourfunctional groups can afford, after the capture step, acovalent intermediate with the NT-amine positionedat close proximity to the acylating CT-carbonyl toproduce a high effective molarity that enables a spon-taneous intramolecular acylation.

Four paired combinations of an amine- or an acyl-nuclophile/electrophile on an amine or acyl segmenthave been explored (Tables II and III). For the aminesegment, a commonly paired arrangement is an NT-amine with a side-chain nucleophile. This type ofspatial arrangement is commonly found in many nat-urally occurring amino acids11–17. Essentially, alla-amino acids containing heteroatomic side chainsoccupying NT positions can be considered as amine-nucleophiles. Since not all amino acids contain a

FIGURE 2 Prior thiol capture ligation developed by Kemp et al.50–52


side-chain nucleophile and since amine-nucleophileshave different reactivities toward specific acyl-electrophiles, they provide the orthogonality to distin-guish one NT-amine from another. Thus far, aminesegments of NT-Cys11, -Ser 12, -Thr 13, -Trp 15,and -His14, containing weak-base nucleophiles suchas a thiol or hydroxyl on their side chains spatiallyseparated by two atoms from their NT-amines, havebeen found to be most useful. Other amine segmentsof NT-Asp, -Glu, -Lys, and -Arg have not been ex-plored. These amino acids cannot function as thedesired amine-nucleophiles when they are placed ininternal positions of a peptide sequence because theira-amines would be acylated as amides.

Unnatural or modified amino acids also providepotentially rich source of amine-nucleophiles, partic-ularly for syntheses of peptide mimetics and organicmolecules. An example of an unnatural amine-nucleo-phile is found in N-oxyethylthiol glycine18.53 Plac-ing an auxillary nucleophile at the N-terminus such asa thiol nucleophile on thea-amine mimics an NT-Cysand is an attractive option when the side chain doesnot contain a functional group. However, it is neces-sary to remove the N-oxyethylthiol after the ligationreaction to regenerate Gly.

The amine-electrophile contains an NT-amine witha side-chain electrophile (Table II) is the second com-bination of a paired functional arrangement for theamine segment. None of the naturally occurringamino acids contains a reactive amine-electrophilesuitable for the orthogonal ligation scheme. Thus,amine-electrophilic segments either are modified fromexisting naturally occurring amine-nucleophiles or areprepared directly from total syntheses. Examples ofamine-electrophiles includeb-arylsulfenyl 20 oralkoxycarbonyl sufenyl-thiol, b-bromo 19 andg-formyl amino acids21 (Table II). In some cases, theamine-electrophiles can be readily transformedthrough an NT-Cys by a sulfenylating agent.

Amine-electrophiles are prone to undergo a smallring formation between thea-amine and the side-chain electrophile to form a reactive NT-imine, suchas the ready conversion ofb-bromoalanine19 toaziridine 22. However, aziridine turns out to be aninteresting example of an NT-electrophile that hasbeen successfully exploited for orthogonal ligation.54

Similarly, the a-amine g-formyl segment rapidlyisomerizes to the cyclic five-member NT-imine pyr-rolidine, which is effective for ligating with an acyl-nucleophile to form a bicyclic lactam.

Table II Spatial Arrangements of Amine-Nucleophile and Amine-Electrophile Used in Orthogonal Ligation

Entry

Nucleophile/ElectrophileNT-Amino

AcidX 5 R 5 Spacera

Amine-nucleophile 11 SH H 2 Cys12 OH H 2 Ser13 OH Me 2 Thr14 Imidazole H 2 His15 Indole H 2 Trp16 Amide H 2 Asn17 SH 3 Hcy18 SH 3 Thiol-Gly

Amine-electrophile 19 Br H 2 BrAlab

20 SSR H 2 Sulfenyl-Cys21 CHO 3 g-Formyl Abuc

22 Aziridine 1 Aziridine acid

a Spacer, atoms betweena-amine and X.b BrAla, b-bromo alanine.c Abu, a-aminobutyric acid.


For the acyl segment, there are again two combi-nations, acyl-electrophile and acyl-nuclephile. Anacyl-electrophile contains an electrophile paired withan acylating carbonyl, usually as an ester, at theC-terminus (Table III). Similar to the spatial require-ment defined for the amine segments, a spacer#3atoms is favorable for linking these two functionalgroups. Again, similar to the amine-nucleophiles,there are several spatial paired arrangements of theseacyl-nucleophiles. Two useful arrangements are O-glycoaldehyde ester23 and thioester27 in which theelectrophiles are part of the ester moiety. Their cap-ture through either an imine or thioester (Figure 1) byan NT-nucleophile segment such as NT-Cys 11 (Ta-ble II) would result in an O- or S-ester that leads to anacyl migration through a five-membered ring interme-diate. Analogs of glycoaldehyde with different spac-ers linked to an ester have also been explored inligation chemistry24,25(Table III).

Another spatial arrangement of an acyl-electro-phile is the placement of the electrophile on the sidechain of a CT-ester. An example is the formyl groupof -formyl ester26. Such a CT-electrophile can reactwith an NT-Cys to form a 6,5-bicyclic lactam37.

The second combination for an acyl segment is anacyl-nucleophiles. Two forms of this combinationhave been explored. CT-thioacids29 with a thiolatedirectly linked to the acylating carbonyl represent thesimplest form of an acyl-nucleophile. This CT-nu-

cleophile is versatile and has been useful for manytransformations in orthogonal ligation scheme. CT-Cys ester30 or its analogues such as CT-Hcy (homo-cysteine) with the thiolate located at the side chainalso is useful for the synthesis of various bicyclicanalogues38 in the orthogonal ligation schemes.

Convergence of Concepts and Practicein Semi- and Total Synthesis Strategies

The centerpiece of orthogonal ligation is the acylmigration to form an amide bond. Such a mechanismis frequently found in enzymes that make or break anamide bond. Proteases utilize dyad or triad catalyticresidues for intramolecular N- to O- or S-acyl trans-fers and hydrolysis. Indeed, the O- or S-acyl interme-diates have been exploited successfully for enzymaticsynthesis with new engineered enzymes containinghigh ligase activity.34–36,55Recently, intramolecularacyl transfer reactions also have been found in proteinsplicing and autoproteolysis. In protein splicing, aseries of acyl transfers lead to a final covalent O- orS-acyl intermediate resulting in a spontaneous uncata-lyzed O- or S- to N-acyl transfer reaction that formsthe corresponding amide bond.56–58The splicing sitesinvariably involve Xaa-Cys, -Ser, and -Thr, which arealso found as ligation sites in orthogonal ligationmethods. Interestingly, NT-nucleophiles such as NT-

Table III Spatial and Functional Arrangements of Acyl-Nucleophilies and Acyl-Electrophiles Used in OrthogonalLigation

Entry C-Terminal Structure

Nucleophile/Electrophile

Y Spacera

CT-electrophile 23 Glycoaldehyde ester OOCH2CHO 224 b-Formyl ethyl ester OOCH2CH2CHO 325 b-Formyl-iso-butyl ester OOCH2C(CH3)2CHO 326 g-Formyl Abub OCH2CHO 327 Thioester OSR3 028 Perthioester OSSR4 0 or 1

CT-nucleophile 29 Thioacid OSH 030 Cys OSH 2

a Spacer, atoms betweena-carbonyl and the electrophile or nucleophile.b Abu, a-aminobutyric acid; R1 5 side chain of amino acids; R2 5 alkyl; R3, see Figure 9; R4 5 aryl.


Ser and NT-Thr have been found to be the active sitesof hydrolase enzymes. Thus, there is a convergence inthe amide bond formation in both the orthogonalligation and biological processes that utilize the acylmigration as the principle to achieve selectivity.

Nearly all amine segments containing an NT-nu-cleophile or NT-electrophile are prepared by stepwisesolid-phase synthesis on the benzyl or benzhy-drylamine type of functionalized resins that arewidely available commercially. Since these aminesegments are free peptides and their preparations andpurification are similar to conventional methods, botht-butyloyxcarbonyl (t-Boc) and 9-flourenylmethyoxy-carbonyl (Fmoc) chemistries have been used withexcellent results. Syntheses of acyl segments contain-ing a CT-electrophile or CT-nucleophile are also pre-pared by solid-phase methods but may require spe-cially prepared resins, such as thioester resins. Forlarge peptides and protein segments that do not con-tain unusual amino acids in their sequences, bothamine and the acyl peptide thioester can be preparedby recombinant methods.59 Some of these methodsfor preparing CT-electrophile or CT-nucleophile seg-ments will be discussed in the appropriate sections.

Because orthogonal ligation can utilize unpro-tected peptides and their esters of different sizes, andforms containing genetically coded and unusualamino acids from both chemical and recombinantsources, it has the significant advantage of removing

the size limitations in chemical protein syntheses. Bydefinition, semisynthesis employs one of the twobuilding blocks from a biosynthetic or natural sourceand, operationally, orthogonal ligation is identical tosemisynthesis under the framework defined in thisreview. Thus, in terms of chemistry, orthogonal liga-tion unifies the total and semisynthetic methods into asingle operational strategy.

Strategies and Methods of OrthogonalLigation

All orthogonal ligation methods have an intramolec-ular acyl transfer reaction, but their capture methodsvary. They fall into two general strategies (Table IV).In the imine capture, the initial capture product is animine intermediate formed between the amine andacyl segments containing an aldehyde electrophile. Inthe thioester capture ligation, the intermediate is acovalent thioester or perthioester formed by a thio-ester or its analogues and a thiol nucleophile. Morethan a dozen methods utilizing either capture strategyhave been developed to form amide bonds with nativeand non-native side chains. For convenience, we havenamed each method according to its ligation productor the NT-nucleophile when several methods yield acommon product.

Reaction conditions for different orthogonal liga-tion methods are fairly straightforward and reproduc-

Table IV Imine and Thioester Capture Strategies for Orthogonal Ligation Methods

CaptureStrategy Method Segmentsa

Conditions

Ligation ProductpH Solvent

Imine Thiazolidine 11 1 23 Acidic H2O SPro 31Oxazolidine 12 1 23 Acidic pyr/H1 OPro 32Oxazolidine 13 1 23 Acidic pyr/H1 OProMe 33His 14 1 23 Acidic pyr/H1 Triazabicycles 34a,bb

Trp 15 1 23 Acidic pyr/H1 Tryptoline 35b

Asn 16 1 23 Acidic pyr/H1 Tetrohydropyrimidone 36b

Thiazabicycle 11 1 26 Basic H2O b-Turn dipeptide 37b

Pyrroline 21 1 30 Basic H2O Bicyclic lactam 38b

Thioester Cys 11 1 27 Basic H2O Cys 39Perthioester 20 1 29 Acidic H2O Cys 39Hcy 17 1 27 Basic H2O Metc 40Thiol-Gly 18 1 27 Basic H2O Glyd 41BrAla 19 1 29 Acidic H2O Cys 39Aziridine 22 1 29 Acidic H2O Cysd 39His 14 1 28 Acidic H2O His 42

a See Tables II and III.b Structures, see Figures 4 and 5.c After S-methylation, for product see Figure 11.d Sequence dependent.


ible. These reactions are bimolecular, often in a nearlyequal stoichiometric ratio of two segments without anextraneous coupling reagent. They are generally per-formed in an aqueous solution buffered at an appro-priate pH. However, in ligating large peptide seg-ments, strongly denaturing conditions are sometimesemployed to remove the conformational factors thatadversely influence the reaction rates. Specific condi-tions, side reactions, and postligation workups will bediscussed under different ligation methods.

IMINE LIGATION

Imine ligation involves an imine capture step thatresults from an acyl-aldehyde with an NT-amine onan amine-nucleophile segment (Figure 3). This iminethen undergoes a rapid ring-chain tautomerization due

to the addition of nucleophile paired to the NT-amine.The resulting heterocycle facilitates an acyl transfer ofthe ester intermediate to form a proline-like imidicbond. The imine capture is well represented by liga-tion methods involving amine-nucleophile of NT-Cys, -Ser, -Thr, -Trp, -His, and -Asn, yielding avariety of mono-, bi-, and tricyclic side chains at theligation site (Figures 4 and 5).

There is a reverse-imine capture that involves apreformed imine deriving from the intramolecularcyclization of an amine-electrophile pair bearing analdehyde at its side chain (Figure 5). An example ofreverse-imine capture is pyrrolidine ligation involvingan CT-Cys30 and an NT-g-formyl 21 segment thataffords a bicyclic lactam38 at the ligation site. Inreverse-imine capture, the intramolecular ring closureof an NT-aldehyde to an NT-cyclic imine is rapid, sothat it serves as an active intermediate to be captured

FIGURE 3 Imine ligation of N-terminal Cys-, Ser-, and Thr-containing peptides to formpseudoprolines through thiazolidine (Thz) and oxazolidine ring formation.

FIGURE 4 Imine ligation to form heterocycles with N-terminal Cys, Ser, Thr, His, Trp, and Asn.


by a CT-nucleophile. Bicyclic lactams chemistry iswell established for preparing constrained organicmolecules and peptide mimetics.60 We will not elab-orate on the reverse imine capture in this review.Another ligation involving an imine is formed inCys-aziridine ligation which will be discussed later.

The specificity of the imine ligation is due to thering-chain tautomerization in the heterocyclic ringformation. In normal imine capture, however, ring-chain tautomerization is a part of intermolecular cap-ture reaction. Formation of a heterocyclic ring by anNT-nucleophile containing an NT-weak base such asa thiol or hydroxyl moiety drives the ligation reactionand is a central feature of the imine strategy. Thus, theNT-weak base serves as a specific recognition motiffor the amine segment. The reaction of a acyl-alde-hyde with other amines would form Schiff bases,which are unstable and reversible under the aqueousconditions without an additional reaction by an NT-nucleophile. Since imine ligation is performed underan acidic aqueous condition, amine-nucleophiles witha strongly basic side-chain amine such as Lys or Arg,which would be protonated and are excluded in theligation reactions.

At the ligation site, the l,2-substituted moiety ofmercapto amine of NT-Cys11 or hydroxyl amine ofNT-Ser/Thr12, 13 forms a five-membered-ring thia-zolidine or oxazolidine with the CT-glycoalde-hyde.61,62Reactions with other NT-nucleophiles suchas Trp13, His 14, and Asn16 involving two or morenucleophilic sites yield more complex products. Thestereochemistry of these heterocyclic products34 and36 has not been fully characterized. However, theprinciples of these ligations are very similar and wewill focus our discussion mainly to thiaproline andoxaproline ligations.

In our laboratory, NT-nucleophile segments havebeen prepared by the conventional solid-phase meth-

ods by both Boc and Fmoc chemistry while the prep-aration of the corresponding to a CT-electrophile seg-ment bearing an aldehyde requires special attention.

Thiaproline Ligation

Thiaproline ligation is the first example that demon-strates the principle of orthogonal ligation by couplingan NT-Cys segment11 and a CT-ester glycoaldehyde23 to form a thiaproline at the ligation site (Figure3).63–65 The concept using initial capture and thenproximity-driven acyl transfer and its validation werereported in the Thirteenth American Peptide Sympo-sium held in the summer of 1993.66

The capture between an NT-Cys11 segment and aCT-glycoaldehyde ester23 to form a thiazolidineester is facile and regiospecific. The specificity ofthree aldehyde esters reacting with all 20 NT-aminoacids has been examined by a library containing 400dipeptides under aqueous conditions.69 The fastestcapture reaction was between the glycoaldehyde esterand cysteine to form a stable thiazolidine ester that iscomplete in,l5 min at pH 5 and,5 min underneutral or basic pH. The thiazolidine ligation of NT-Cys 11 is .1000 fold faster then NT-Thr13 andNT–Trp15. NT-Ser12and NT-Asn16are essentiallyunreactive in 72 h. This high reactivity between theCT-aldehyde23 and the NT-Cys11 under aqueousconditions provides the exclusive orthogonality of theligation. Furthermore, performance in acidic condi-tions avoids side reactions of aldehyde with othernucleophiles present in peptides. These features makethe reaction very useful for the purpose of chemose-lective ligation of two molecules, even without theacyl transfer reaction, and can be applied to regiospe-cific conjugation reactions in protein chemistry.68

After heterocyclic ring formation, the secondaryamine in the formed thiazolidine ring acts as a weak

FIGURE 5 Two pathways of imine ligation to form bicyclic lactams.


base and can undergo an O- to N-acyl migrationthrough a favorable five-membered-ring transitionstate at pH 5 to form a proline mimic, 2-hydroxy-methyl thiaproline, at the ligation site.63,64 While thering formation is fast, the rearrangement is generallyslow and constitutes the rate-determining step of theligation. The rate of rearrangement increases with theincreased pH, and depends on the steric and electronicenvironment of the amino and carboxyl components.Small amino acids such as Gly and Ala are the pre-ferred choices. However, other amino acids, such asLeu and Val, have been used successfully in thesynthesis of HIV-1 protease analogues. Reaction ofNT-Cys with the glycoaldehyde23 is favorable andcan be completed within 2 h. Reaction of the otherester aldehydes such as24, which requires a six-membered transition state, is difficult at pH, 9. AtpH 9.5, the reaction takes 72 h to complete with aconsiderable amount of hydrolysis under aqueousconditions. Similarly, reaction with other aldehyde-esters, such as25, is sterically hindered.

The effectiveness of this strategy has been con-firmed in the synthesis of analogues of 50 residueTGFa

63,64 and 99 residue peptide HIV-1 protease.65

The 2-hydroxymethyl thiaproline formed at the liga-tion site can be considered a pseudoproline (SPro)structure. Enzyme activity test shows that the substi-tution of proline with SPro in the sequence of HIV-1protease gives identical activity (Table V).65

Oxaproline and Other HeterocyclicLigations69

Oxaproline ligation involves an NT-Ser12 or NT-Thr13 and a CT-ester glycoaldehyde23 to form an oxa-proline at the ligation site (Figure 3). It proceedsthrough an imine capture, imine-oxazolidine ring-chain tautomerization, and O,N-acyl transfer. Sinceoxazolidine has been found to be 104 times less stablethan the corresponding thiazolidine analogues70,71

and ring closure of the imine to oxazolidine is not a

favored process, the oxazolidine ring-chain tautomer-ization generally favors an open imine form in aque-ous conditions. Thus, in contrast to thiaproline liga-tion, oxaproline ligation does not readily proceed inaqueous conditions. However, it can be achieved un-der nearly nonaqueous conditions. This difference inreactivity provides useful orthogonality in ligatingsegments containing NT-Cys and NT-Thr segments intandem ligation schemes of multiple peptide seg-ments.

Several conditions for oxaproline have been tested.In nearly anhydrous conditions such as 90% DMSOor dimethylformamide in 10% acetate buffer at pH5.5, the selectivity of NT-Thr is at least 100 foldgreater than NT-Ser.67 However, oxaproline ligationsin our laboratory are typically carried out in pyridine–acetic acid mixtures (1:1, mol/mol) that have appro-priate pH and solubilities for unprotected segments toachieve a reasonable reaction rate and minimizes sidereactions.67 The amine segment is used in slight ex-cess. The ligation reaction at room temperature isrelatively slow and required more than 35 h for com-pletion. However, at an elevated temperature of 40°C,the reactions are completed in about 10 h. High per-formance liquid chromatography monitoring (HPLC)showed that the reaction proceeded cleanly and gavepredominantly a single product together with the un-reacted starting materials and hydrolyzed acyl seg-ment as observable byproduct.

Under the condition of a pyridine–acetic acid mix-ture, imine ligations of other NT-nucleophile peptideswith the glycoaldehyde ester were also examined (Ta-ble VI). Rate analysis for 36 h showed that ligationoccurred rapidly with those NT-amino acids such asNT-Cys11, -Ser12, -Thr 13, -Trp 15, and -His14, butslowly with NT-Asn 16. However, NT-Lys and NT-Arg did not participate in the ligation reaction, prob-ably because of the low pKa of the pyridine–aceticacid condition and the protonated state of the stronglybasic side chains of lysine and arginine. Similarly, noligation products were observed with other amine

Table V Kinetic Parameters of the Synthetic HIV-1 PR Analogues on the Hydrolysis of Substrate IV inComparison with Recombinant HIV-1 PR

Protease 38–39 LinkageKm

(mM)Vmax

(mmol/min z mg)Vmax/Km

(mmol/min z mg)

HIV-1 PR –Leu38–Pro39– 10.1 3.43 3.403 105

[SPro39, Abu67,95]– HIV-1 PR –Leu38–SPro39– 11.9 3.96 3.333 105

[Ala38–SPro39, Abu67,95]–HIV-1 PR –Ala38–SPro39– 8.2 2.26 2.763 105

[Leu38–(NHCH2–Thz)39,Abu67,95]–HIV-1 PR –Leu38–(NHCH2–Thz)39– 11.4 1.14 1.003 105

a [SPro] and Thz, see Figure 3.


segments of nonamine-nucleophile such as NT-Ala,Met, and Tyr as well as for those NT-amino acidswith acidic side chains such as Asp. The NT-Ser12,-Thr 13, and -Asn16 segments provided a predomi-nant single ligation product, but those segments withNT-His 14 and Trp 15 at the N-termini gave threemajor isomeric products. The stereochemistries ofthese isomers have not been characterized at this time.

Preparation of the Ester AldehydeSegments

Several methods have been developed to prepare theCT-ester aldehyde segments. In then 1 1 strategy, amasked aldehyde function of an amino acid ester isintroduced either enzymatically or chemically ontothe CT-ester or thioester. The enzymatic addition iseffected by a kinetically controlled aminolysis.64,72

This reaction can be smoothly and efficiently accom-plished in an hour in the presence of a water-miscibleorganic solvent if a high concentration of amino com-ponent is used.72 The CT-peptide ester was synthe-sized by the solid-phase method using a resin newlydeveloped in author’s laboratory. After the cleavageand removal of all protecting groups, this resin pro-vided a peptide containing a Ca ester for an enzyme-catalyzed coupling between the peptide and the smallsubstrate, the dimethoxy ethyl ester of Ala present inlarge excess at high molar concentrations. Under thiscondition, no hydrolysis of other bonds susceptible totrypsin was observed. Since this strategy depends ona specific enzyme such as trypsin (Lys, Arg), chymo-

trypsin (Phe, Tyr, Trp), or V8 (Glu, Asp), a moregeneral chemicaln 1 1 strategy of adding a singleamino acid glycol aldehyde dimethyl acetal derivativeonto a peptide thioester will be described later.65 Thethioester was synthesized by stepwise solid-phasesynthesis using Boc chemistry on a Boc-amino acidthioester resin developed by Hojo and Aimoto.74 Ac-tivation of thioester by Ag1 in the presence of a largeexcess of the masked amino acid aldehyde derivativeefficiently yielded the peptide aldehyde dimethyl ac-etal. Treatment of the acetal by triflouroacetic acid(TFA) at low temperature unmasks the aldehyde func-tion for the next ligation step.

Several direct methods to afford the glycoaldehydeester have also been developed. The most directmethod is through a functionalized resin support suchas a new glyceric ester resin that provides a diolprecursor of peptide C-terminal oxyethyl aldehyde.75

The glyceric ester resin can be prepared by startingfrom benzaldehyde resin with two variations.76 Thefirst method directly attached Fmoc amino acid glyc-eric ester to the resin and the second formed hydroxylacetal on the resin followed by coupling Fmoc aminoacid to the glyceric acetal resin (Figure 6). Fmocchemistry then can be used to assemble the peptidechain. After cleavage by TFA, the diol of the glycericester is converted to glycoaldehyde by oxidation withNaIO4. It should be noted that when the oxidation wasperformed below pH 5, Met was oxidized to Met(O)77

even in the presence of large excess of Met as scav-enger. This side reaction can be minimized by per-

FIGURE 6 On-resin synthesis of peptide glycoaldehydeester.

Table VI Relative Ligation Rates of DifferentN-Terminal Amino Acids in Pyridine–AceticAcid (1:1, mol/mol)

H–Leu–Ile–Asn–Gly–OCH2CHO 1 X–Phe–Lys–Ile–NH23 H–Leu–Ile–Lle–Asn–Gly–[Xaa]–Phe–Lys–Ile–NH2

[Xaa] in ProductaX in

Segment

Yield(%)

at 36 hRelative

Rate

SPro Ser 79.6 1OPro Thr 74.2 0.95OProMe Cys 95.3 .1000b

Triazabicycles His 77.9 1.2Tryptoline Trp 73.3 1.1Tetrahydropyrimidone Asn 44.4 0.3

a Structures, see Figures 3 and 4.b Rate for thiazolidine ester formation (tR 5 31.9min), but the

rate for amide product (tR 5 29.9 min) throughO,N-acyl transferis 0.9 relative to OPro.


forming the reaction at neutral pH. The aldehyde isthen purified by HPLC to remove the formaldehydebyproduct generated during the reaction. The secondmethod uses thioester resin and a transesterificationwith a large excess of thiol glycoacetal. A similarstrategy based on Pd° reduction of a thioester resin,and finally, a direct method to produce the maskedglyceric ester from the free peptide in the presence ofglycerol also have been developed.63

Stereochemistry at C2 Carbon of cProand cis–trans Conformation of PeptideBackbone

In imine captures, the heterocyclic ring formationcreates a new asymmetric carbon at position 2 of thecPro ring (Figure 3). These R and S diastereoisomersare stable and separable by HPLC in thiaproline liga-tion. However, after acyl migration, only one ligationproduct was observed in the HPLC profile. In oxapro-line ligation, no stable oxazolidine ester intermediatesare detected by HPLC.

The C2 stereochemistry has been determined bymeasuring the nuclear Overhauser effect (NOE) crosspeak between the C2-H and C4-H of the oxaprolinering in the two-dimensional (2D)1H-nmr spec-tra.78–83 Two sets of independent nmr data are ob-tained based on the assignment of the 2D doublequantum filtered correlated spectroscopy. These re-sults indicate that the C2 stereocenter is anR rather

than anSepimer (Table VII). Other observable NOEcross peaks also support that C2 centers areRepimers. These include the NOE cross peaks betweenthe C2-hydroxymethyl protons and theaxial C5-H butno NOE peaks between the C5-H and C2-H. Thus, theoxaproline and thiaproline ligations are regiospecificas well as stereospecific and do not produce diaste-reomers during synthesis.

The proposed mechanism of the stereospecificity isthat in oxaproline ligation the O,N-acyl migration isstereospecific but not the oxazolidine formation be-cause the C2-R epimer proceeds at a faster rate thanthe C2-S epimer. The unreacted C2-S epimer under-goes an equilibration to a mixture ofR, S epimersthrough oxazolidine ring-chain tautomerization. Asimilar mechanism for stereospecific control can beinvoked for the thiaproline ligations, i.e., the first stepof ester formation produces a mixture of epimers atC2 and the O,N-acyl migration of theR epimer is fastcompared with theS epimer.

The cis–trans isomer ratios of the Xaa—Pro bondhave been determined by conventional NOE experi-ments.81,82 A typical pattern83 of the Hi-C2-Hi11 (i5 Xaa,i11 5 Pro) NOE cross peak is observed in allpseudoproline-containing peptides. This pattern, i.e.,the Hi-Hi11 in a proline-containing peptide, is char-acteristic of thetransXaa—Pro bond.78–80,83Thecisisomer in each compound is characterized by the NOEcross peak between the Hi and C4-Hi11 (Figure4).79,83 In 27b, 27d,and 27e, NOE cross peaks be-

Table VII Stereochemistry of Model Dipeptides Z–Xaa–cPro–OMe

Structure X R1 R2 C2 cis/trans

Z-A-[OP]-OMe O Me H R 68:32Z-V-[OP]-OMe O iPro H R 56:44Z-A-[OPMe]-OMe O Me Me R 54:46Z-V-[OPMe]-OMe O iPro Me R 43:57Z-V-[SP]-OMe S iPro H R 40:60

a [OP], [OPMe], and [SP], see Figure 3.


tween C2-Hi11 and H-Val or CH3-Val are also ob-served to confirm the assignment ofcis isomers. Inaddition, the NOE cross peaks between the Hi andC2i11-hydroxymethyl protons are observed incis iso-mers. However, thecis–trans ratios vary dependingon the side chains around the pseudoprolinebonds.78–80For example, Val at thei position has anunfavorable effect oncis conformation because of thesteric hindrance between its large side chain and C4-Hi11 (Table V).

THIOESTER LIGATION

The CT-electrophile thioester has been known as anenergy-rich bond since the discovery of the enzymeCoA.84 In nature, thioesters are involved in nonribo-somal peptide synthesis85 through a multienzymethiotemplate system that forms antimicrobial peptidesand in many posttranslational protein modificationssuch as receptor palmitoylation and myristoylation.86

However, these reactions are enzyme mediated anddirect ligation of thioester with an amine segment bychemical means is generally unsuccessful except withNT-Cys and NT-His.87 Previously, Schwyzer88 andsubsequently Blake and Yamashiro89 have shown thatthioester is relatively unreactive to aminolysis and canbe prepared as an activated building block throughstepwise synthesis. However, thioester readily reactswith a thiol through transesterification to form a newthioester. With an NT-Cys, this thioester linkageforming an S-acyl covalent intermediate would spon-taneously undergo an S- to N-acyl migration to forma peptide bond through a five-member ring interme-diate (Figure 7). This reaction, first reported by Wie-

land et al. in 1953,90 involves a CT-thioester and acysteine derivative that results in the formation of acysteine peptide bond between two amino acids. Thesignificance of this finding went unnoticed for 40years but recently was exploited by two laboratoriesfor orthogonal segment ligation.25,54 Since then, thethioester method as a method of capture in orthogonalligation has been applied successfully for cys-teine,25,54methionine,27 glycine,53 and histidine liga-tion of unprotected peptides.

Cys-Thioester Ligation

Cysteinyl ligation involves an CT-thioester27 and anNT-nucleophile Cys11 that produces Cys at the liga-tion site. Cys ligation possesses two ideal elements inthe orthogonal ligation strategy. First, Cys bearing athiol supernucleophile as an NT-nucleophile readilyundergoes a thiol-thioester exchange reaction with aCT-electrophile thioester27 that, after the acyl mi-gration, regenerates the thiol moiety on the NT-nu-cleophile amino acid. This preserves the integrity ofthe side-chain functional group of the NT-nucleophileamino acid at the ligation site. Second, the thioester asa CT-electrophile contains perhaps the most favorablespatial arrangement of these two functional groups,with the alkyl thiolate electrophile directly linked tothe acylating carbonyl. Such an arrangement providesnot only an activated ester but also a highly desirablefive-member ring intermediate for an intramolecularacyl migration after the thioester capture. Conse-quently, Cys ligation by thioester capture has beenextensively exploited in chemical and, more recently,semisyntheses of proteins.91–93

FIGURE 7 Normal and reverse thioester ligation to form a Cys bond.


Several conditions for ligating CT-thioester withNT-cysteine peptide have been reported. Most liga-tions are performed under aqueous conditions buff-ered at pH 7–8 by sodium phosphate. However, Cysligation can occur under acidic conditions.54 In gen-eral, Cys ligation can be and has been performedsuccessfully under nondenaturing conditions. Becauseof the uncertainty of the conformational states of largepeptide segments, ligations have also been routinelyperformed under a strongly denaturing condition of6–7M guanidine HCl.25,94 Although Cys ligation isfairly tolerant to hindered amino acids at CT-thio-esters, unhindered CT-thioesters have given the bestresults.

A detailed study on conditions of Cys ligation haveshown that hydrolysis of thioester, disulfide formationand bisacylated product can be major side reactions(Figure 8).54 Hydrolysis of thioesters usually proceedsslowly at both acidic and basic pH. The rates arehighly unpredictable because they are sequence andconformational dependent. This is particularly truewhen the CT-thioester contains heteroatomic sidechains to assist their hydrolysis. In general, the use ofa basic pH accelerates ligation reactions, thus reduc-ing the hydrolyzed side product.

A critical condition necessary for Cys ligation isthe maintenance of a reducing environment to prevent

N,S-bisacylated byproduct and reduce disulfide for-mation. A combination of R3P and a large excess ofan alkyl thiol improved the yield from 75 to.95% ina model peptide54 and was considered an optimalcondition for thiol–thioester exchange. Tri(2-carbon-ylethyl)phosphine (TCEP)95 is convenient to use be-cause it is water soluble and rapidly reduces theblocked disulfide forms of the NT-Cys segments totheir reactive forms. A large excess of a small thiolsuch as 3-mercaptopropionic acid (MPA) is added toregenerate the NT-Cys nucleophile and free cysteinefrom the bisacylated byproduct. Despite their highsolubility in aqueous conditions, bifunctional thiolcatalysts such as MPA and mercaptoethanol are notideal because of their ability to promote thioesterhydrolysis. We have been examining a diverse groupof nonstenching thiol catalysts that do not have theselimitations and have found that tertiary and quaternaryamine thiols are suitable. Other thiol catalysts such asthiophenol23 and the nonstenching 2-mercaptoethane-sulfonate93 have also been useful.

The regiospecificity of Cys ligation is exception-ally high in aqueous buffer between pH 7–8. At thispH range, the selectivity of a thiol over amines issubstantial because the thiol group is a stronger nu-cleophile. As a result, there are no significant sidereactions in the peptides containing side-chain func-

FIGURE 8 Side reactions in Cys-thioester ligation.


tionalities such as the amine of lysine, thiol of cys-teine, guanidine of arginine, and imidazole of His atinternal positions of the peptide sequence. The selec-tivity of the thiol at the N-terminus over the internalcysteinyl thiol, although not readily apparent, couldbe attributed to two factors. First, only the NT-cys-teine contains ana-amine in a 1,2-relationship, whichcan serve as a general base to facilitatetrans-thioes-terification. The general base assistance of an NT-amine in the rate acceleration of disulfide formationon NT-cysteinyl peptides has been shown to be 3–10-fold faster than the corresponding disulfide formationbetween internal cysteinyl thiols.96 This also providesan explanation for the great susceptibility of an NT-cysteinyl peptide to form a disulfide bond homodimeras compared to those peptides with an internal cys-teine residue. Second, thetrans-thioesterification be-tween N-terminal cysteine and thioester will lead to aproximity-driven S- to N-acyl transfer to form a stablepeptide bond whereastrans-thioesterifications be-tween thiols of internal cysteines are reversible. Thereversibility of S-acylation of an internal cysteine hasbeen demonstrated and can be suppressed with theaddition of an excess of thiol compounds.

It is clear from the above discussion that internalthiols present in NT-Cys and CT-thioester segmentsare reactive moieties that can impede the reaction

course in the Cys or other thioester ligation methods.Thus, ligation reactions often become sluggish whenboth segments contain multiple thiols competing withthe NT-Cys for the thiol–thioester exchange reactions.CT-thioesters with internal thiols quickly undergoring-chain tautomerization to form thiolactones thatcan often serve as the effective acylation moieties inthe capture step.97

Preparation of CT-Thioester Segments

A biosynthetic57 and two chemical methods25,54 forpreparing CT-thioesters of unprotected peptides andproteins have been reported. Since CT-thioesters arecentral to many ligation methods, their preparationsare discussed in detail. Because there has recentlybeen some confusion in the literature regarding thetwo chemical methods to yield the CT-thioester seg-ment in Cys-thioester ligation, it is worthwhile topoint out their differences and advantages.

Dawson et al.25 have reported Cys-thioester liga-tion in which the CT-thioester is obtained through anindirect method (referred to as Dawson strategy inFigure 9). They use a stepwise solid phase method togenerate synthetic peptides containing a CT-thioacidthat requires a solution transformation to a thioesterafter HF (anhydrons hydrogen fluoride) cleavage from

FIGURE 9 Comparison of two procedures in preparing Cys-thioester.


the resin support.25,23 The CT-thioacid is preparedfrom the mercaptobenzhydryl resin developed byBlake and Yamashiro,10,11but later modified to avoidthe inconvenience and hazards of hydrogen sulfidegas.98 The peptide CT-thioacid is then converted tothe thioester by one of two methods in solution phase.Nucleophilic reaction of the thioacid with an alkylhalide such as benzylbromide or with a symmetricaldisulfide such as 5, 59-dithiobis(2-nitrobenzoic acid)affords the corresponding peptide thioester.25,94

Independently, we have reported a differentmethod for preparing unprotected peptide CT-esterobtained directly from solid-phase synthesis (referredto as Tam’s strategy in Figure 9).54 In our method, wehave adopted the alkyl thioester resin developed byHojo and Aimoto, who utilized this resin for prepar-ing hydroxysuccimide active ester for conventionalsegment synthesis.74 After the completion of the pep-tide synthesis on solid-phase and HF cleavage, anunprotected peptide CT-thioester is directly obtainedfor orthogonal ligations without the need for solutiontransformation as in the Dawson method. We havesubsequently simplified and generalized the method toprepare the alkyl thioester resin as a 3-mercaptopro-pionyl benzyhydrylamine resin that can used for at-tachment to any activated Boc-amino acid.99 We havefound that even hindered,b-branched Boc-amino ac-ids such as Val and Ile have been successfully at-tached to this new thiol resin.54

Besides having the advantage of convenience, pre-paring peptide CT-thioesters directly from solid phasealso avoids the difficulties involved in handling thio-acids, which are relatively unstable to HF and suscep-tible to hydrolysis or formation of side products dur-ing the purification steps. However, large peptidesegments of CT-thioacids have been successfully pre-pared for orthogonal ligation and in situ transforma-tion of a CT-thioacid to its corresponding ester has theadvantage of “tuning” its reactivity using differentelectrophiles.25,94

Thus far, peptide segments of CT-thioesters orCT-thioacids prepared by solid-phase synthesis haverelied on Boc chemistry because of the susceptibilityof the thioester linkage to repetitive base cleavages inFmoc chemistry. Recently, Li et al.100 reported amodified Fmoc procedure for preparing CT-thioesterbased on a more hindered thioester linker and a lessbasic Fmoc-cleavage procedure. However, we couldnot determine in this report the extent of peptidechains loss due to repetitive base cleavages to estab-lish its usefulness for syntheses of large thioesterpeptide segments. An alternative method that avoidsthe use of HF and Boc chemistry is the use of acombination of very acid-labile Bpoc (or methoxyl

triphenyl) for the NT-amine and the TFA-labiletert-butyl for side-chain combination as a protectionscheme. This combination has been successfullyadopted for the synthesis of CT-phenyl ester for theprior thiol capture.101

Recent developments of preparing the CT-thioestersegments from a biosynthetic source through recom-binant methodologies represent an exciting expansionof the scope of orthogonal ligation for semisynthesis.Since CT-thioesters have been found to be interme-diates in the protein splicing process, biologists havedesigned mutants that trap these intermediates, whichare released as CT-thioesters through thiol–thioesterexchange reactions by an extraneous thiol.59 Proteinexpression kits with several vectors are commerciallyavailable that allow recombinant proteins to be ex-pressed as a CT-thioester. This type of semisyntheticmethod combining the advantages of protein splicingand Cys ligation has been developed by Muir etal.91,92as an “expressed protein ligation” method, andby Xu et al. as an “intein ligation” method.93 Bothlaboratories have validated this semisynthetic ap-proach and have been successful in generating pro-teins up to 600 amino acid residues in length.91–93

Reverse Cys Ligations by NT-Electrophiles and CT-Thioacid54

Methods of forming Cys at the ligation sites havebeen developed utilizing a CT-thioacid nucleophileand an NT-electrophile (Figure 7). For example, theCT-thioacid, a CT-nucleophile, forms a thioester in-termediate with an NT-electrophile segment in thereverse direction. Similar to normal thioester ligation,a thioacid with the thiolate directly linked to theacylating carbonyl produces the favorable geometryof a five-membered intermediate for the facile S,N-acyl migration to form the amide bond. Althoughthere are strategic similarities, a difference betweenthe normal and reverse thioester ligation is that thelatter involves a thioacid and is generally performedin acidic conditions. This difference may have advan-tages in devising tandem ligation strategies for syn-thesis of multiple peptide segments and proteins andfor proteins that are not stable under basic conditions.

Cys-BrAla and Cys-Aziridine Ligations

An amine-electrophile such as NT-b-bromoalanine(BrAla) 19 is readily S-alkylated by a thioacid29 toform a covalent thioester that will rearrange rapidly togive the cysteine at the ligation site (Figure 7).54 Theside reaction in this ligation method is ab-amino acidisomer formation at the ligation site because the N-


terminal BrAla peptide can readily undergo 1,3-elim-ination of HBr at pH. 6 to form an NT-cyclic imine,an aziridine ring. Subsequent ring opening at thea orb position by the thioacid forms the desired peptideproduct with theb-isomer as a byproduct. Whenthioesterification was conducted at pH, 5, the aziri-dine formation was slow and this byproduct was min-imized to ,3%. The dominant thioesterification oc-curred by direct displacement of BrAla. Interestingly,the ring opening becomes regiospecific at theb posi-tion with a steric hindered proline at the second po-sition and only the desireda-peptide is obtained.

Cys-Perthioester Ligation102

Another reverse thioester ligation involves an acyldisulfide intermediate in the capture step. An NT-electrophile containing an activated thiol side chain ofmixed disulfides28 readily undergoes a thio–thiolexchange reaction with the Ca-thiocarboxylic acid ofan acyl segment. The mixed acyl disulfide (perthio-ester) then undergoes a rapid intramolecular S,N-acyltransfer through a six-member ring intermediate.Thiolytic reduction of the resulting hydrodisulfide(S-SH) gives a Cys residue at the ligation site (Figure10).

A characteristic of this reaction is that the captureand S,N-acyl migration can occur under very acidicpH (pH . 4). In our laboratory, we have performedthe reaction at pH, 2 with mixed acetonitrile–watersolution, with the segment concentration of 10; 15mM. After reaction release of yellow color (Npys-H),indicating the occurrence of the capture reaction bysulfur–sulfur exchange, the pH is adjusted to 6, whichpermits treatment of the hydrodisulfide with 1,4-di-thiothreitol to give the final product with Cys at theligation site. The high efficiency of the first capturestep is attributed to the Npys-activated sulfide and tothe supernucleophilicity and low pKa of the thiocar-boxylic acid compared to a normal alkyl thiol. Theacyl transfer is spontaneous and 90% complete in 5min even at acidic pH, 4. The efficiency of this acyl

transfer step is attributed to the activated acyl disul-fide and proximity of the CT-acyl and NT-amine in asix-membered ring intermediate. This orthogonal seg-ment ligation and disulfide reduction is a one-potprocess that does not require isolation of the interme-diates.

An alternative activation method for perthioesterligation has been developed. The thioacid is activatedas a mixed acyl disulfide according to the proceduredescribed by Yamashiro and Li.11 Reaction of themixed disulfide with an NT-nucleophile cysteinyl seg-ment forms a covalent acyl disulfide. However, theactivation of thioacid produces an enthalpically acti-vated CT-acyl disulfide, which must be kept undervery acidic conditions to prevent random acylation.This method has been used for the synthesis a 32-residue peptide102 from two purified segments, theacyl segment (15 residues) containing a thioacid, andthe amino segment containing anNT-Cys(Npys), aswell as analogues of the 99-residue HIV protease.

Methionine Ligation27

The principle of Cys-thioester ligation has been suc-cessfully extended to form a Met at the ligation site.In Met ligation, the latent thiol moiety of Met asNT-homocysteine (Hcy) is exploited for capture bytrans-thioesterification with an acyl segment bearing aCT-thioester to give an S-acyl intermediate. After theS,N-acyl migration and subsequent S-methylation ofthe ligated Hcy residue with excessp-nitrobenezen-sulfonate, Met is obtained at the ligation site (Figure11). Thus, this ligation method requires an additionS-alkylation step after completion of the orthogonalligation scheme. However, it would be useful forMet-containing peptides or proteins without any cys-teinyl residues.

The condition used for transesterification is similarto those previously described for cysteinyl peptide. Itis performed at pH 7.6 in phosphate buffer in a highlyreductive environment containing a threefold excessof water-soluble R3P (TCEP) to prevent disulfide for-

FIGURE 10 Perthioester legation to form an Xaa–Cys bond under acidic conditions.


mation and to accelerate the desired reaction.trans-Thioesterification occurs rapidly and the covalentthioester intermediate of two free peptide segments isusually not observed. Performing the reaction under abasic condition permits rapid rearrangement through afavorable six-membered ring transition state in theS,N acyl-migration to form the homocysteinyl prod-uct.

Selective methylation on Cys was previously dem-onstrated by Heinrikson in 1971 with methyl p-nitro-benzenesulfonate as methylation reagent.103 This pro-cedure was adopted for S-methylation of homocystei-nyl peptides using large excess of methylatingreagent. The reaction was acid quenched after 1 h toavoid undesirable methylation on side chains such asthe e-NH2 groups of Lys and imidazole rings of His.Although the thioalkylation was carried out underbasic conditions, other methods of selective S-meth-ylation under neutral or acidic conditions are alsoavailable104 for transforming of Hcy to Met.

Both the ligation and S-methylation proceed withhigh efficiency and regiospecificity. There is no ob-servable random acylation on the side-chain function-alities such as the amine of lysine, guanidine of argi-nine, or imidazole of histidine. This is clearly illus-trated in the synthesis of a 34 residue parathyroidhormone (PTH), which contains three His, three Lys,and two Arg residues. There are, however, four ob-servable side reactions. The most noticeable is theS,N-bisacylated product, which can be minimized byusing an excess of the Hcy peptide segment. Theaddition of a small thiol compounds such as MPAafter the reaction is completed will regenerate thedesired Hcy peptide. The second side reaction is theformation of disulfides of either the starting materialor the product. Again, this side reaction is reversiblein a strong reductive environment using R3P duringthe reaction. The third is hydrolysis of thioester tocarboxylic acid. This side reaction is often acceleratedwhen His is present in the peptide sequence but has

been found to be,5 % in the synthesis of PTH. Thefourth is peptide degradation caused by intramolecu-lar proteolysis of the Hcy—X amide bond via a five-member thiolactone ring formation. The latter twoside reactions are severe at high pH and prolongedreaction conditions, but are insignificant when thereaction is performed at pH, 8.4.

Glycine Ligation53

A ligation method that forms Gly or other smallamino acids at the ligation site has been developed byCanne et al.53 In this method, a peptidea-thioester27reacts, via thiol–thioester exchange, with anNa(ethanethiol) peptide18 to produce the captureproduct. This thioester-linked intermediate then rear-ranges to give the amide-linked product, containingthe analogous N-oxyalkyl compound. Zinc dust isthen added directly to the reversed phase HPLC-purified peptide in the acidic eluant to reduce theN—O bond of the O-alkyl hydroxamate and give theligation product31 with a Gly at the ligation site.

The most notable feature of this method is the slowrearrangement of the initial thioester intermediate.The acyl transfer rearrangement via a six-memberedring coupled to a secondary weak O-alkyl hydroxam-ine is considerably less favored than the facile five-membered ring in the Cys ligation. The subsequentrearrangement to an amide bond was not observed forbulky Ca-substituted amino acids on both sides of theligation site.

Histidine Ligation

To extend the principle of thioester ligation to non-cysteinyl NT-amino acids, other nucleophiles havealso been examined as potential capture devices. Ascheme has been developed by our laboratory usingAg1 ion as a capture device together with amine-

FIGURE 11 Homocysteine ligation to generate methionine at the ligation site after S-methylation.


nucleophiles such as NT-Ser, -Asn, and -Asp.105 An-other scheme involves the imidazole group of the Hisside chain, which is known to be a weak base at acidicpH and can catalyze the acyl transfer in the enzymaticprocess.87 However, ligation scheme that uses NT-His14 as nucleophile and a CT-thioester to form His atthe ligation site is inefficient and activation of CT-ester is necessary.105 Thus, an acyl segment containsa CT-thioacid that can be activated by a thiophilicpromoter such as Ellman’s reagent106 is used for thecapture reaction by the imidazole of the NT-His toform an Xaa–His amide bond after subsequent Nim- toNa-acyl transfer (Figure 12).

At acidic pH and in the absence of the thiol nu-cleophiles, the imidazole moiety would be the solenucleophile. Thus, when the ligation reaction is per-formed at acidic pH in phosphate buffer maintained atpH 5.7, it is generally complete in 8 h. This methodhas been used to generate His-containing peptidessuch as calcitonin and parathyroid hormone with yieldof 60 ; 75 %.

Sequential Orthogonal Ligation

At present, the thioester strategy without any protect-ing group is limited to the ligation of two segmentsbecause the peptide bearing the thioester is prone tocyclization if a free N-terminal cysteine is present.For sequential orthogonal ligations, the N-terminalcysteine of the thioester segment must be thereforeprotected. This protecting group should be compatiblewith Boc chemistry, which is the favored method for

preparing thioester peptides. Furthermore, this pro-tecting group should be removable under mild aque-ous conditions in accordance with the nature of theunprotected peptide. Photolabile and reduction-sensi-tive protecting groups for amines and thiols as well asthiazolidine-based phenacyl protecting groups (Figure13) for the NT-Cys were developed to fulfill theserequirements. Sequential orthogonal ligations wereperformed through a three-segment sequential thio-ester ligation of a humanb-defensin (38aa) and NK-lysin (77aa).107 Another method using base labile2-(methylsulfonyl) ethyloxycarbonyl (Msc) as pro-tecting group was also developed by Muir et al. forsequential ligation.23

CONCLUSIONS AND PERSPECTIVE

For many years, chemical synthesis of large peptidesand proteins has been a daunting task. The present andcontinuing efforts to develop different orthogonal li-gation methods will likely make this task easier. Thusfar, studies have convincingly validated the useful-ness of this strategy, although it is still in its infancy.Research on its development is concentrated in only afew laboratories and has focused on protein synthesis,but it is reasonable to expect that this amide strategycould be widely applicable to peptides and mimetics

FIGURE 12 Histidine ligation.

FIGURE 13 Sequential Cys-thioester ligation employinga photolabile Cys-protecting group.


of all sizes and shapes. For example, the principle oforthogonal ligation has been extended for the synthe-sis of cyclic and branched peptides and peptide mi-metics. In particular, orthogonal ligation methodshave been extended successfully for preparing cyclicpeptides.108–113 Applications into peptide mimeticsand organic synthesis can be envisioned with existingor new combinations of amine- and acyl-nucleophileand electrophile pairs that yield amino acids at theligation sites. Since the building blocks can be ob-tained efficiently by either stepwise solid-phase syn-thesis or recombinant methods, whether they are pep-tides, proteins, or their thioesters, the limitations ofsize to produce synthetic or semisynthetic proteins areno longer a concern. More importantly, ligation, totalsynthesis of peptides and proteins, intein splicing andsynthesis, as well as enzyme processing and synthesisare now related chemically by the ester7 amide acylshifts (Figure 14).

An advantage of orthogonal ligation implicit in itsoperational definition is its application to ligate mul-tiple peptides in tandem without a protectionscheme.23,107Such tandem ligating methods will findapplications for synthesis of peptide dendrimers,structure–function studies and combinatorial libraries.Future applications of this enabling methodologyshould make possible syntheses of both novel de-signed peptides and proteins containing unusualamino acids, building blocks or architectures that arenot readily accessible by recombinant methods.

Note added in proof:The References list publications up toFebruary 1999. Since then many more papers have appeared

in literature. We regret the omission of prior papers onligation chemistry in this review and would appreciate theauthors bringing to our attention their work.

We thank Professor Bruce Merrifield for a critical review ofthis manuscript. This work was in part supported by U.S.Public Health Service Grants CA 36544, GM 157145 andAI 46164.

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