Coupling Methods for Peptide Synthesis

N. Leo Benoiton,
University of Ottawa, Ottawa, Ontario, Canada
doi: 10.1002/9780470048672.wecb101

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Article Contents
Peptide-Bond Formation Activated Forms Auxiliary Nucleophiles Coupling Methods Tactics for Avoiding Aggregation

Peptide-bond formation requires activation of a carboxyl group followed by aminolysis of the activated carbonyl. The reagents and methods employed for activation, the intermediates and activated forms generated and their aminolysis are described. Rationalization of the strategy employed to avoid isomerization during coupling is presented. Approaches for minimizing aggregation that interferes with coupling are also given.

N-Alkylation of the Peptide Bond Other Peptide-Bond Forming Approaches

Peptides are employed for therapeutic purposes and for research in the development of new drugs, vaccines, biomarkers, diagnostic agents, and so on. The demand for these peptides has experienced double-digit annual growth over recent years (1). Most peptides are obtained by chemical synthesis. Chain assembly is effected by successive addition of single residues always starting from the carboxy-terminus, which eliminates the danger that isomerization might occur during coupling. If the peptide is long enough, then two or more segments may be combined to produce the nal product. Moreover, the chain may be assembled in solution or on a solid phase. Many methods are available for linking two amino acids together. Each has its own peculiarities as well as favorable and unattractive features. Some methods of coupling are of general use, some are not applicable to solid-phase synthesis, and some are employed only for specic purposes. The choice of method depends on circumstances that vary considerably; every method seems to nd its place among the armaments employed for synthesis. An intriguing observation is that ve different coupling methods are involved in the synthesis in kilogram amounts of the nine-residue drug Atisoban (2).

Peptide-Bond Formation
A peptide bond is formed by reaction of the carboxyl group of one amino acid with the amino group of a second amino acid (Fig. 1A). A combination of the two occurs when the carboxyl group is activated, which means it has been rendered decient in electrons or electrophilic, and the amino group is electron rich or nucleophilic. The amino group is nucleophilic when it is not protonated; the carboxyl group is activated by afxing an electron-withdrawing moiety to it. The latter is achieved by use of compounds called coupling reagents or by one or more chemical reactions. Peptide-bond formation is slower when the activated residue is valine, isoleucine, or threonine protected on the side chain; these are known as hindered

residues. During the process of aminolysis, a competing reaction may occur (Fig. 1B) because the carbonyl on the amino group of the activated residue is also nucleophilic. The result is the formation of a ve-membered cyclic structure, which is referred to as a 5(4H )-oxazolone that contains nitrogen and oxygen atoms and a carbonyl group, with substituents on carbons 2 and 4. The substituent at C-4 is the side chain of the amino acid; the substituent at C-2 originates from the acyl group of the activated residue. When the acyl group is another amino-acid residue, that is, if the activated component is a peptide, then the structure is a 2-alkyl-5(4H )-oxazolone that is chirally unstable. It readily tautomerizes (Fig. 1F) to give a mixture of the two enantiomers of the oxazolone. When the acyl group is that of the protector of an amino acid, the oxazolone that is formed is a 2-alkoxy-5(4H )-oxazolone, which is chirally stable and does not isomerize (3). These alkoxycarbonyl protectors (ROCO, often referred to as urethane protectors, which is incorrect; urethane = ROCONH-), namely benzyloxycarbonyl (Cbz or Z; benzyl = C6 H5 CH2 ) cleaved by hydrogenolysis, tert -butoxycarbonyl (Boc) cleaved by acidolysis, and 9-uorenylmethoxycarbonyl (Fmoc) cleaved by beta -elimination have been designed, in that order, so that the activated residue does not enantiomerize during couplings. The effect of the tautomerization of the 2-alkyl-5(4H )-oxazolone is the production (Fig. 1E ) of some other isomer of the peptide product because the oxazolone also undergoes aminolysis. Hence, coupling of an N -alkoxycarbonylamino acid occurs without enantiomerization of the residue because even if the 2-alkoxy-5(4H )-oxazolone is formed, it does not isomerize. However, there is a danger to produce epimeric peptides during the coupling of an N -protected peptide because of the severe tendency of the activated peptide to form the 2-alkyl-5(4H )-oxazolone that isomerizes readily and is also a precursor of the product. In all cases, the product of isomerization is a mixture of epimeric peptides. When the activated residue is isoleucine or threonine that have two stereogenic centers and when the activated component is a peptide, which does 1

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Coupling Methods for Peptide Synthesis

Figure 1 Coupling in peptide synthesis. Aminolysis (A) of the activated residue gives the L-L peptide. A competing intramolecular reaction (B) may occur giving the 5(4H)-oxazolone. When R = alkoxy (BzlO, tBuO, or FmO), the oxazolone is aminolyzed (E) giving the L-L peptide. When R = peptidyl (-Xaa-NHCHR3 ) the oxazolone tautomerizes (F) and is then aminolyzed (E, E ) giving also the D-L peptide.

not have glycine at the terminus, the process is an epimerization. When the activated component bears an N -alkoxycarbonyl group and the amino acid is other than glycine, isoleucine, or threonine, the process is an enantiomerization (4), but it is also referred to as racemization. The process of isomerization of an activated peptide is also often referred to as racemization, but it is careless usage of the term and an unwarranted extension of the meaning of the word.

Activated Forms
A multitude of activated forms of amino acids and peptides is available (Fig. 2), which have varying stabilities. Some are shelf-stable reagents; some can be prepared and manipulated, but they are not stable enough to be stored; and others are intermediates that have been postulated to explain the results. In fact, the most popular coupling reagents generate activated forms that have never been detected; hence, they are hypothesized structures. Noteworthy among these structures is the O -acylisourea that comes from carbodiimides (5), the acyloxyphosphonium cation that comes from the phosphonium-salt reagents, and the O -acyluronium cation that comes from the uronium-salt reagents (Fig. 2). The mixed (MxAn) and symmetrical (SyAn) anhydrides are compounds that can be puried and characterized, but they are usually prepared and used immediately. The SyAn is the anhydride formed from the combination of two molecules of N -alkoxycarbonylamino acid. The MxAn is formed by reaction of the latter with an alkyl chloroformate (chlorocarbonate). Both structures occasionally surface as intermediates in reagent-mediated reactions, namely the SyAn in carbodiimide-mediated reactions and the MxAn in EEDQ-mediated (see Fig. 4) reactions. Acyl azides and halides are prepared in solution and used immediately. Some activated esters are shelf-stable reagents. They are of two kinds, which originate from substituted phenols and substituted hydroxamic acids (Fig. 3).

Auxiliary Nucleophiles
Peptide-bond formation involves reaction of the nucleophilic amino group of one residue with the activated carboxyl group 2

of a second residue (Fig. 1). In many cases, the activated residue reacts rst with another molecule to produce a compound that is activated by another moiety; the latter then undergoes aminolysis to give the peptide. These molecules are referred to as auxiliary nucleophiles. The original ones were incorporated into coupling reactions when it was realized that their presence had benecial effects. The common auxiliary nucleophiles (Fig. 3) (69), which are also known as additives, appear in Fig. 4. Each possesses a hydroxyl group; all except the HOSu are slightly acidic with p Ks in the 3.44.6 range. They participate in coupling reactions in one of two forms. Either they are added in their original form HOW, or they are present by virtue of the fact that the coupling reagent employed is composed of the deprotonated moiety -OW attached to a positively charged atom that is a strong electrophile (Fig. 5). HOSu serves primarily as a nucleophile for the preparation of activated esters (10); that is, it is added to an activated amino acid without the simultaneous presence or subsequent addition of an amino-bearing residue. The other auxiliary nucleophiles are added to carbodiimide-mediated reactions to suppress the side reactions of isomerization (7) (Fig. 1) and N -acylurea formation (Fig. 4). They protonate the intermediates that are formed, namely the O -acylisourea (Fig. 4) and the oxazolone (Fig. 1), which prevents their rearrangement and tautomerization, respectfully, that lead to undesired consequences. The protonated intermediates are strong nucleophiles that readily yield the peptide product. Hence, the additives trap activated intermediates that prevent the side reactions. When the additives are incorporated into coupling reagents, they are released as the anion OW as soon as the reaction is initiated. The anion is a strong nucleophile that can react with or trap any activated intermediates that are formed. The compounds generated are activated esters that undergo aminolysis to give the peptide. The traditional additive has been HOBt (7); HOAt (HOaBt) (9) is of more recent development. HOObt (8) was not employed in the past because of the known side reaction of aminolysis at the carbonyl of the additive. However, this reaction occurs in negligible amount, and the additive has received renewed interest. Although it was dormant for many years, HOcBt (7) has surfaced with a vengeance as it is being employed in multikilogram amounts in the synthesis of cephalosporin and the fusion inhibitor Fuzeon. It and HOAt

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Coupling Methods for Peptide Synthesis

Figure 2 Activated forms of N-alkoxycarbonylamino acids (RCO = R1 OCONHCHR2 CO) and N -protected peptides. Some are aminolyzed (A) to give the peptide, and some may generate (B), a second activated form that is also a source of the product. Experience indicates that acyl halides and symmetrical anhydrides of peptides do not exist.

Figure 3 Auxiliary nucleophiles (additives) that suppress side reactions by reacting with activated intermediates, and the two acylated forms of benzotriazole that are produced.

are often superior to HOBt as additives; their hydroxyl groups are slightly more acidic, which provides better leaving groups in the esters. HOcBt is less expensive. The addition of HOBt to a carbodiimide-mediated reaction minimizes the dehydration of side-chain amides that occurs in its absence.

form is employed as soon as it is prepared. Examples include MxAn as well as SyAn and azides.

Dialkylcarbodiimides
The compounds implicated in carbodiimide-mediated reactions (11, 12) appear in Fig. 4A. The reaction is postulated to be initiated by protonation of one of the slightly basic nitrogen atoms by the acid followed by attack at the central carbon atom by the acid anion that generates the O -acylisourea (OAIU). Aminolysis the OAIU gives the peptide with liberation of N,N -dialkylurea. A side reaction of N -acyl-N,N -dialkylurea (NAU) formation by O -to-N acyl shift of the OAIU can occur. The latter is avoided by adding an auxiliary nucleophile that protonates intermediates. The acyl shift is promoted by heat and tertiary amine that assures a deprotonated nitrogen atom, and delay in the aminolysis caused by bulkiness in the reacting residues. The three common carbodiimides are dicyclohexylcarbodimide (R4 = R5 = C6 H11 , DCC) (11) giving urea DCU, diisopropylcarbodimide (R4 = R5 = C(CH3 )2 , DIC) giving urea 3

Coupling Methods
Peptide-bond formation is achieved in two ways. In the rst approach, a reagent is added to a mixture that contains a component with a free carboxyl group and one with a free amino group. Such coupling reagents include carbodiimides, EEDQ (Fig. 4), and onium-salt based reagents (Fig. 5). The other approach involves preparing the activated component after which the amino-bearing component is added. Here, one can distinguish two separate cases. In one case, the activated form is a shelf-stable compound such as an activated ester or an N -carboxyanhydride (NCA). In the other case, the activated

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Coupling Methods for Peptide Synthesis

DIU, and ethyl(dimethylaminopropyl)carbodiimide hydrochloride (R4 = C2 H5 , R5 = (HCl . (CH3 )2 NCH2 CH2 CH2 , EDC) giving urea EDU (12). All carbodiimides are allergenic, which cause skin irritation, so they should be handled with care. DCC is an inexpensive, brittle solid that produces DCU that is bulky and insoluble in most solvents. DIC is a moderately expensive liquid employed in solid-phase synthesis because DIU is soluble in organic solvents. Contact of eyes with fumes from spills of DIC can cause temporary blindness. The soluble EDC is excellent for synthesis in solution because EDU and the NAU are soluble in water. It is available as crystals that are expensive. Reactions are usually effected by adding the reagent to a mixture of the reacting components and the auxiliary nucleophile if desired. In a variant, the acid is allowed to react with itself and possibly an additive after which the amino-containing component is added. In this case, the N -alkoxycarbonylamino acid is converted into the SyAn and the activated ester, respectively, prior to the aminolysis. The efciency of a carbodiimide-mediated coupling of a peptide that is effected in the presence of an auxiliary nucleophile is enhanced by the presence of cupric ion (13). Cupric ion prevents a 2,4-dialkyl-5(4H )-oxazolone from isomerizing (13).

solvents have been tetrahydrofuran and DMF; the favored tertiary amine is N -methylmorpholine (NMM). NMM is believed to act as an acceptor of the acyl group that forms the acylmorpholinium ion (RCO-N+ MM), which is the acylating reagent. It was thought for many years that halo-containing solvents were unsuitable; however, DCM is a good solvent provided the tertiary amine is not triethylamine as this combination retards the formation of the MxAn (19). Strictly anhydrous conditions are also not essential because a MxAn can be puried by washing it with aqueous solutions (19). MxAn have not been employed in solid-phase synthesis because a side reaction of aminlyosis occurs at the wrong carbonyl (Fig. 4B) in the range of 110% that is unavoidable. The larger amounts develop when the activated residue is hindered. The urethane produced is stable to all conditions used for deprotection; hence, the impurity cannot be readily eliminated. Isopropyl chloroformate seems to be a superior reagent for MxAn reactions (20). MxAn can be employed for acylation of an amino acid or peptide with an unprotected carboxyl group in polar or partially aqueous solvent

Phosphonium and uronium salt-based reagents

A revolution in coupling occurred with the introduction of BOP (21) (Fig. 5). Numerous related reagents followed (Fig 5) (22, 23). All reagents react according to the same mechanism. An equi-mixture of the reagent, acid, and amino-containing component is prepared, and two equivalents of a tertiary amine are added. No reaction takes place without the tertiary amine, which creates the carboxylate ion RCO2 . The anion displaces the ring structure of the reagent generating the acyloxy-phosphonium (Fig. 1) or -carbenium cation, never detected but postulated, which then undergoes nucleophilic attack by one or both of the available species, the amino-containing component that produces peptide and the benzotriazolyl BtO or equivalent anion AtO that gives the activated ester RCO2 Bt, RCO2 At, and so on, which undergoes aminolysis to produce peptide. The HOBt/HOAt that is liberated is neutralized by the second equivalent of base that had been added. Many assume that the activated ester is the source of the peptide, but indisputable evidence suggests peptide originates also from some other intermediate (24), which means the initial intermediate. The preferred tertiary amine is diisopropylethylamine (DIEA) and not NMM as originally employed because the more basic amine is necessary to drive the reaction to completion. A variant involves the addition of an equivalent of HOBt or HOAt to encourage formation of the activated ester. However, this ester formation is not always benecial as the benzotriazolyl esters of hindered residues undergo aminolysis much more slowly to the point that the benzotriazolyl ester of N -protected-N -methylvaline is resistant to aminolysis (25). The hexamethyl-substituted reagents generate the toxic hexamethylphosphor-trisamide as side product. The tripyrrolidino-containing reagents generate an amide that is environmentally acceptable. The tetramethylurea that is produced from the uronium-based reagents is soluble in water and easy to dispose of. The phosphonium-based reagents are more reactive than the carbenium-based reagents, hence, they are less stable for storage. For this reason, the carbenium (uronium)-based

1-Ethoxycarbonyl2-ethoxy1,2-dihydroquinoline (EEDQ)
This reagent (14) (Fig. 3B) is employed in the same way as carbodiimides and occasionally by operators who have developed a skin irritation after contact with DCC. The acid displaces the ethoxy group after which spontaneous rearrangement to the MxAn occurs with expulsion of quinoline. Aminolysis of the MxAn gives the peptide. Unavoidable aminolysis at the other carbonyl gives a small amount of irreversibly substituted amino component as impurity.

Symmetrical anhydrides (Fig. 2)

In a variant (15) of the carbodiimide method, two moles of an N -alkoxycarbonylamino acid are reacted with one mole of DCC in dichloromethane (DCM). After 30 minutes, the DCU is removed by ltration and the solution is mixed with the aminolyzing component. The DCM is sometimes replaced with dimethylformamide (DMF) for the aminolysis step. SyAn formation is very slow in DMF. The method is wasteful of substrate but gives clean reactions. Some SyAn have been isolated and stored (16, 17), but the practice is no longer in vogue. SyAn are less reactive and hence more selective than OAIU; they do not acylate hydroxy groups that have not been deprotonated. They are especially effective for acylating secondary amines such as N -methylamino-acid residues.

Mixed anhydrides (Fig. 2)

The MxAn method is popular for synthesis in solution; it is inexpensive and simple to effect. Chloroformate R6 OCOCl [R6 = ethyl, C2 H5 or isobutyl, CH(CH3 )2 ] is added to a small excess of the acid at 5 C in the presence of tertiary amine and after 3060 seconds, the aminolyzing component is added (18). The reaction is complete within 3060 minutes. Traditional 4

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Coupling Methods for Peptide Synthesis

Figure 4 (A) Compounds implicated in carbodiimide-mediated reactions. The acid is postulated to add to the carbodiimide generating the O-acylisourea. Aminolysis occurs at the point indicated by the dashed arrow. (B) Compounds implicated in EEDQ-mediated reactions. The acid is postulated to displace the ethoxy of the reagent generating 6 which rearranges to the MxAn. Aminolysis (a) gives the desired peptide and (b) the ethoxycarbonylated nucleophile as impurity.

Figure 5 Structures and examples of nomenclature for phosphonium and carbonium salt-based reagents. Rules of nomenclature dictate that the latter be named as modied ureas; see text for revised structures; ino indicates a ring linked at the nitrogen atom.

reagents are more attractive for solid-phase synthesis. The chemistry of the uronium-based reagents is complex. In fact, the BTU of HBTU and TBTU does not have the structure given in Fig. 5, but it is 1-(bis(dimethylamino)methylene-1-H -benzotri azolylinium 3-oxide, that is, the tetramethycarbenium moiety is linked to the other nitrogen atom of the ring, analogous to structure 5 (Fig 3) (26). Acylated HOBt had originally been shown to exist in solution as two forms in equilibrium, the ester (RCO2 -Bt) and the N -acyl N -oxide (RCO-BtO) (6). In model experiments, the uronium forms of HBTU and HATU reacted more quickly and led to less epimerization than the guanidinium forms (27). Other reagents are continuously being developed, typical examples being BtO-CH=N+ Me2 . PF6 (28) and PyCOP (Fig. 5) (29). Reagents based on HODhbt Fig 3 have also been described. All have their unique properties. The azabenzotriazolyl-containing reagents seem to be particularly effective for performing difcult couplings (30).

Acyl halides (Fig. 2)

A straightforward method of coupling involves conversion of an Fmoc-amino acid (Fmoc-Xaa-OH) to the chloride FmocXaa-Cl, using thionyl chloride (SO2 Cl) in DCM (31, 32) or triphosgene (O=C(OCCl3 )2 , which is a source of phosgene (COCl2 ) (33). Only the latter allows preparation of derivatives with acid-sensitive side-chain protectors tBu but not trityl. Also available are Cbz, Boc and Fmoc-amino-acid uorides obtained from the acids using cyanuric uoride (cyclic C3 N3 F3 ),

diethylaminosulfur triuoride (DAST, Et2 NSF3 ), or tetramethyluoroformamidium hexauorophosphate (TFFH, Me4 N2 C+ F . HF6 which is activated in DMF by tertiary amine. The amino-containing component is added after removal of excess reagent and side products. TFFH is employed for generating the uoride in the presence of the incoming nucleophile. The Fmoc-Xaa-Cl are highly activated reagents that generate the strong acid HCl on aminolysis. Neutralization by tertiary amines causes formation of the 5(4H )-oxazolone that is aminolyzed more slowly, so it is best to avoid this structure by use of KOBt for neutralization or by destruction (reduction) of the acid with powdered zinc (34). For this and other reasons, Fmoc-Xaa-Cl are not used routinely, but they are particularly suited for couplings between hindered residues, and for esterifying a hydroxymethyl-linker-resin. Acyl uorides are less reactive, hence, are more stable to oxygen nucleophiles such as water or methanol; they have a lower tendency to cyclize to the 5(4H )-oxazolone and can be employed in the absence of base. Their properties resemble those of activated esters. They are also suited for coupling hindered residues, but the possible deleterious effects of prolonged exposure to base because of their lesser reactivity must not be disregarded.

Acyl azides (Fig. 2)

The preparaton of acyl azides involves conversion of acid RCO2 H to ester RCO2 Me and subsequent hydrazinolysis with 5

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Coupling Methods for Peptide Synthesis

excess N2 H4 to give acyl hydrazide RCONHNH2 (35). The latter is puried by crystallization and treated at +5 C with a solution of aqueous AcOH that contains NaNO2 or tert -butylnitrite. The acyl azide (RCON3 ) that is formed is extracted into an organic solvent, which is dried and added to the aminolyzing component. An acyl azide can be produced directly from the anion of an acid in the presence of the aminolyzing component by use of diphenyl phosphorazidate [(PhO)2 PON3 ] (36), which is a reagent that is especially good for converting a linear peptide to a cyclic peptide. The acyl azide reaction is generally applicable to Boc- or Z-Xaa-OH, which includes instances when Xaa = Ser, Thr, or His with unprotected side chains. For coupling peptides, activation of the hydrazide is performed with tert -butyl nitrite (tBuNO2 ) and nitrosyl chloride (NO2 Cl) (37). Because hydrazine destroys protectors such as triuoroacetyl (CF3 C=O), o -nitrophenysulfanyl (O2 NPhS), and nitro of nitroarginine, a variant that circumvents the problem assembles the chain starting with a protected hydrazide (H-Xaa-NHNH-O2 CR2 ) obtained in solution or on a solid support (H-Xaa-NHNH-linker-resin) (38). Aminolysis of an acyl azide is very slow. tert -Butyl nitrite with HOAt converts the hydrazide to an activated ester that allows aminolysis to occur in 4 hours instead of 23 days (39). Acyl azides are sensitive to tertiary amines; however, they do not form oxazolones, hence, proper manipulation of the coupling species just about guarantees products that are enantiomerically pure. Caution: Azides tend to explode under certain conditions.

an impurity that develops from the condensation of three moles of HONSu, SuNO2 C-NHCH2 CH2 C(=O)-ONSu (42), which is an activated ester that causes the incorporation of beta-alanine into a chain. p -Nitrophenyl esters are employed instead of DCC for activating asparagine and glutamine derivatives during chain assembly to avoid the dehydration of the carboxamide groups by the carbodiimide. Pentauorophenyl esters are employed for temporary protection during the formation of a bond between the anomeric carbon of a hexose and the nitrogen on the side chain of asparagine. Succinimido esters are useful for acylating amino-acid or peptide anions in polar or partially aqueous solvents. 2-Chloro-4,6-dimethoxy-1,3,5-triazine is a reagent that produces superactive triazine esters from acids (43).

Amino-acid N -carboxyanhydrides (Fig. 2)

A unique form of activation of an amino acid but not of a peptide is the N -carboxyanhydride (NCA) (Fig. 2), which is the cyclic anhydride formed between the carboxyl group and a carboxyl group bound to the amino group. These anhydro N -carboxyamino acids are obtained by reacting the amino acid with phosgene in tetrahydrofuran (44). Addition of Xbb-NCA to H-Xaa gives peptide H-Xbb-Xaa after acidiction to release CO2 from the amino-terminus (45). It is possible that the NCA oligomerizes, so the reaction must be effected under very controlled conditions of pH and temperature. For this reason, NCAs are not employed routinely. An option that eliminates the dimerization is use of the N -alkoxycarbonyl- or urethane- protected amino-acid NCA, Boc, Cbz or Fmoc serving as useful substituents (46). These structures also are not employed routinely but have proved advantageous in certain cases. They decompose in the presence of tertiary amine.

Activated esters (Fig. 2)

Two types of activated esters exist, those in which the activating moiety is a substituted phenol and those in which the activating moiety is a substituted hydroxylamine (Fig. 2). Common examples of the former are p -nitrophenyl, pentachlorophenyl and pentauorophenyl; common examples of the latter are succinimido, benzotriazolyl, 7-azabenzotriazolyl, and 4-oxo-3,4-dihydrobenzotriazinyl (Fig. 3). All (RCO2 W) react with an amino group (NH2 R ) to produce tetrahedral intermediate R( O)C(OW)-N+ H2 R that collapses to the product RC(=O)NHR at a speed that is the limiting rate of the reaction (40). The reaction is catalyzed by mild acid, and the rate is much greater in polar solvents; a difference of a hundred times for reactions in DMF versus DCM is typical. Penta-substituted phenyl esters are more reactive than p -nitrophenyl esters. With the exception of succinimido esters, the aza-containing esters are encountered as intermediates in reactions mediated by carbodiimides or onium-salt reagents (see above). Occasionally, the intermediate is generated intentionally, by adding the amino-containing component after a delay. These esters are highly reactive species because of the phenomenon of anchimeric assistance, which is a favorable juxtaposition of the potentially reactive atoms (41). Succinimido esters (10) are unique in that the moiety liberated, HONSu, is soluble in water; hence, they are easier to purify. They are less reactive and are more stable than other aza-containing esters. Activated esters are usually prepared by reaction of the acid with the phenol or HONSu and a carbodiimide, or by ester interchange. Caution must be taken in the preparation of succinimido esters. Use of an excess of HONSu to drive the reaction can result in the formation of 6

Tactics for Avoiding Aggregation

The coupling of a protected residue to a peptide chain is inefcient or occasionally fails once the chain has been extended to ve or more residues (47). Based on the observation that replacement of the pertinent residue by a secondary amino acid eliminates the difculty (48), it has been postulated that the problem occurs because of the chain folding on itself or the binding of two chains because of the tendency of functional groups to form hydrogen bonds (>C=O - - - NH<). The resulting aggregation interferes with the merger of the reacting groups. Tactics to prevent the formation of hydrogen bonds and hence aggregation have been developed. These tactics involve elimination of the hydrogen atom on a peptide bond at the fth or sixth residue of a chain and the use of microwave energy. Peptides that contain serine and are insoluble in aqueous medium can be administered to an organism in the soluble O -acyl form H-Ser(peptide1 )-peptide2 -OH that immediately undergoes O -to-N acyl shift to H-peptide1 -Ser-peptide2 -OH at above neutral pH (49).

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Coupling Methods for Peptide Synthesis

R3 O Fmoc NCHCOPhF5 CH2 Fmoc O Fmoc O Fmoc

R3 O NCHC CH2 Xaa

OMe 8 R3 O H NCHC CH2 Fmoc Xcc O Xaa Fmoc

OMe 9 R3 O Xcc NCHC CH2 HO Xaa

OMe 10

OMe 11

Figure 6 Synthesis of a peptide chain with a bond protected by the 2-hydroxy-4-methoxybenzyl (Hmb) group.

Fmoc Ser: X = O, Y = H Thr: X = O, Y = CH3

Xbb H3C C H3C

CO2H N X CH C H Y

i H-Xaa ii iii H+

Y HCXH Xbb NHCHC O Xaa

Figure 7 The use of pseudo-prolines. (i) Fmoc-dipeptide containing the pseudo-proline at the COOH-terminus is coupled to a chain, (ii) the chain is extended, and (iii) the original amino-acid residue is regenerated.

N-Alkylation of the Peptide Bond

Pseudo-prolines as alkylated dipeptide precursors

A different approach that involves N -alkylated residues to prevent hydrogen bonding is the use of cyclic structures called pseudo-prolines (51). These structures are compounds formed by reaction of serine, threonine, or cysteine residues with formaldehyde, acetaldehyde, or an acetone equivalent. The products are oxazolidines or thiazolidines that bear a carboxyl group and one or more methyl groups on the other carbon atoms of the rings (Fig. 7). The pseudoprolines are employed in synthesis as the Fmoc-dipeptides, with the cyclic structure at the carboxy-terminus. The Fmoc-amino acid is combined with the terminal residue either before or after formation of the ring. The pseudo-proline-containing dipeptide is coupled to the amino group of the peptide that is being assembled. At the end of the synthesis, the pseudo-proline is converted back to its precursor amino acid by acidolysis (Fig. 7). The strength of acid necessary is dictated by the nature of substituents R1 , R2 that originate from the carbonyl compound and the hetero atom O or S of the ring. Methyl groups render the cyclic structures more sensitive to acid; the thiazolidine ring is much more stable to acid than the oxazolidine ring. 7

Elimination of the hydrogen atom at a peptide residue to prevent hydrogen-bond formation involves replacing it with a functional group that can be removed at the end of the synthesis. The protector must be stable to all the conditions employed during assembly of the chain, be easy enough to introduce on to the amino group, and must allow efcient coupling of the residue bearing between the two N -protectors as well as the subsequent aminolysis reaction by the N -alkylamino-acid derivative. Producing such a protector has not been an easy task. The protector of choice for Fmoc/tBu chemistry is 2-hydroxy-4-methoxybenzyl (Hmb) (50) (see Fig. 6). Coupling is achieved by HOBt-assisted aminolysis by H-Xaa- of the pentauorophenyl ester of the N ,O -disubstituted-derivative 8 giving 9. The two protectors are removed, the next residue is introduced as the SyAn [(Fmoc-Xcc)2 O], which acylates the 2-hydroxy group of the Hmb giving 10. The residue then migrates spontaneously to the amino group via the O -to-N acyl shift giving N -substituted peptide 11. The protector of choice for Boc/Bzl chemistry is 2-hydroxybenzyl.

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Coupling Methods for Peptide Synthesis

Microwave-assisted couplings
The new technology of facilitating chemical reactions by use of microwave energy has been applied to the synthesis of peptides (52, 53). The theory is that the energy excites the growing peptide chain, which prevents aggregation during chain extension. Coupling times are substantially reduced, and products of enhanced purity have been obtained.

Other Peptide-Bond Forming Approaches

A new approach to the synthesis of activated esters of peptides is through alkyl thio esters R(O=C)SR obtained by solid-phase synthesis (54). The segment Fmoc-Xcc-Xxxx -Xaa-S-CH2 spacer--linker-resin is assembled using Boc/Bzl chemistry except for the last residue, thio ester Fmoc-Xcc-Xxxn -Xaa-S-CH2 spacer-H is released by acidolysis with HF at the bond indicated by the arrow and converted into activated peptide Fmoc-Xcc-Xxxn -Xaa-ODhbt by reaction with HODhbt in the presence of silver ion. These esters react with amino components, but side-chain amino groups have to be protected. They are used for chemoselective ligation, in which two unprotected peptides, each possessing a uniquely reactive functional group at one of the two termini, are allowed to combine in denaturing aqueous solvent (55, 56). One type involves reaction of the thio ester with a side-chain sulfur atom at the terminus of the other segment followed by an S -to-N acyl shift.

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Further Reading
F. Albericio and L. A. Carpino, Coupling reagents and activation. Methods Enzymol. 1997;289:104126. N. L. Benoiton, Chemistry of Peptide Synthesis. Boca Raton, FL: Taylor & Francis, 2005. E. Gross and J. Meienhofer, eds., The Peptides: Analysis, Synthesis, Biology, vol 1. New York: Academic Press, 1979.

See Also
Amino Acids, Chemical Properties of Click Peptides, Design and Application of Peptide Combinatorial Libraries Solid-Phase Synthesis of Biomolecules

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