NH COOH

RH

Cα

PG1 NH2COO

RH

Cα

PG2

NH CO

RH

Cα

PG1 NH COO

RH

Cα

PG2

COUPLINGREAGENTS

Coupling Reagents

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COUPLING REAGENTS AND ADDITIVES OFFERED BY BACHEM

The coupling reaction i.e. the formation of an amide bond between amino acids and/or peptides is the crucial step in peptide synthesis. The reaction consists of two con-secutive steps:1. Activation of the carboxy moiety2. Acylation of the amino groupDuring the first step the protected amino acid (or pep-tide) reacts with a so-called coupling reagent yielding a reactive intermediate. The chemistry behind and the most important coupling reagents will be presented in this brochure.

PEPTIDE SYNTHESISOur comprehensive offer of coupling reagents, amino acids derivatives and further building blocks, and resins at www.bachem.com will fulfill the needs of solid-phase and solution peptide synthesis. If the product you need is lacking, please let us know.

Activation during Peptide Synthesis

The formation of an amide bond between a carboxylic acid moiety and an amino func-tion of two amino acids is the core reaction in peptide synthesis:

could be isolated if desired. During the second step (II. in Fig. 1), A is attacked by a nucleophile such as the α-amino group of a carboxy-protected amino acid. Steps I and II can be performed either consecutively or, with certain types of coupling reagents, as a one-pot reaction.

Over the years, numerous activation methods have been developed, such as the generation of carboxylic halides (chlorides, fluorides), carboxylic azides, symmetrical or mixed anhydrides or the use of carbodi-imides (DCC, DIC, EDC · HCl) with or without additives.

The first step of this condensation reaction, the activation of the carboxyl moiety (I. in Fig. 1) is often the critical one. Depend-ing on the type of activating reagent, the intermediate A is a stable compound which

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If the amino compound contains other func-tional groups able to take part in the coupl-ing reaction, use of a stable preactivated carboxylic compound A is recommended. Active esters such as the pentafluorophe-nyl (OPfp) and hydroxysuccinimido (OSu or NHS) esters are reactive amino acid deriva-tives finding broad application in peptide synthesis. For our comprehensive offer of active esters please see our online shop.

Coupling ReagentsCarbodiimides have been used as activa-tors for decades in solid-phase and solution peptide synthesis. They still hold their place, though in recent years two classes of cou-pling reagents became popular, the phos-phonium- and the aminium-(imonium-)type reagents such as BOP, PyBOP, PyBrOP,

TBTU, HBTU, HATU, COMU, and TFFH. These compounds achieve high coupling rates ac-companied by few undesired side reactions.In contrast to activation by carbodiimides, peptide couplings using the latter com-pounds require the presence of a base. Di-isopropylethylamine (DIPEA) and N-methyl-morpholine (NMM) are the most frequently used ones in Fmoc/tBu-based solid-phase synthesis. In cases of a markedly increased risk of racemization, the weaker base sym.-collidine has been recommended for substituting DIPEA or NMM. Racemization is one of the main side reac-tions, when activating carboxyl groups of amino acids (except for glycine or proline). Electron-withdrawing groups bound to the α-amino moiety (e.g. acyl, peptidyl (i.e.peptide fragments)) increase the

NONE OF THE COUPLING REAGENTS IS APPLICABLE TO ALL TYPES OF COUPLING REACTIONS

Fig. 1.Activation and cou-pling of a protected amino acid.PG, PG’: protecting groupsAct: activating group

Coupling Reagents

4

tendency to racemize considerably. Ure-thane derivatives, which include standard α-amino protecting groups as Fmoc, Boc, and Z, of amino acids usually retain their optical purity upon activation. The mecha-nism of racemization (Fig. 2) involves the abstraction of the α-hydrogen from the α-carbon atom of the activated amino acid, either by direct formation of an enolic in-termediate (direct α-abstraction, path A) or by formation of a 5-membered oxazolinone ring (path B), which isomeric aromatic con-figuration is readily formed in the presence of bases.Many other side reactions have been described so far, depending on the type of side chain functionalities, the combination of protecting groups, the reactivity of the carboxyl group, or the basicity of the amino function. They will be mentioned later as special information for the respective cou-pling reagent.Every coupling reagent has its individual advantages and drawbacks. None of them will render good results for all types of amino acid derivatives!This brochure gives information on a large number of currently available coupling reagents, their scope and limitations. We hope that it can help the chemist to make the appropriate choice.

Coupling Reagents and Additives

1. Carbodiimides

DCC(Dicyclohexylcarbodiimide)

This popular condensation reagent has been applied for coupling since 1955 and is still much in use today. DCC is routinely applied in solution as well as in solid-phase peptide synthesis, mostly in combination with additives such as HOBt or HOSu in order to reduce epimerization in the case of peptides or racemization in the case of amino acids. Couplings in solution with amino acid derivatives provided as salts (e.g. hydrochlorides) require one equivalent of a tertiary base (as NMM). Else, the reac-tion does not require additional base, so that racemization can be kept minimal.Application of DCC in solid-phase synthesis is limited due to the by-product dicyclohex-ylurea (DCU) with is formed simultaneously with the coupling reaction (see Fig. 3). This by-product is sparingly soluble in most solvents, and therefore DCC should better be replaced by other carbodiimides e.g. DIC (diisopropylcarbodiimide).

Fig. 2.Mechanism of base-catalyzed racemization during activation.

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However, in the manufacture of active esters, DCC is still one of the reagents of choice. In solution, the low solubility of DCU turns into an advantage of this activation method.Application of preformed OPfp esters re-duces the risk of concomitant racemization during couplings. Fmoc-AA-OPfp-esters can be isolated and purified by crystal-lization. They are stable but highly reactive building blocks routinely used for couplings in fully automated SPPS.

Literature:J. C. Sheehan and G. P. Hess, J. Am. Chem. Soc. 77, 1067 (1955)G. W. Anderson and F. M. Callahan, J. Am. Chem. Soc. 80, 2902 (1958)I.Schön and L.Kisfaludy, Synthesis 303 (1986)

DIC(Diisopropylcarbodiimide)

DIC (or DIPCDI) is a useful reagent for au-tomated SPPS, because the corresponding urea is soluble in standard solvents such as isopropanol and can be washed out more readily than the one obtained from DCC. If base-free conditions are required as to minimize racemization, the combination of DIC and HOBt (or HOAt, Oxyma Pure) is still one of the best methods e.g. for coupling Fmoc-Cys(Trt)-OH.

Literature:A. Williams and I. T. Ibrahim, Chem. Rev. 81, 589 (1981)L. A. Carpino, A. El-Faham, Tetrahedron 55, 6813 (1999)

EDAC · HCl, EDC · HCl, WSC· HCl (Q-1955)(N-(3-Dimethylaminopropyl)-N’-ethylcar-bodiimide · HCl)

This water-soluble carbodiimide was es-pecially designed for couplings in aqueous solution, even though the stability of the

Fig. 3.Carbodiimide-mediated amide bond formation.

Coupling Reagents

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reagent under these conditions is limited. Normally, EDAC-mediated couplings are performed in polar solvents such as DMF or NMP or even in methylene chloride. EDAC has also been employed in SPPS on highly polar resins compatible with water-con-taining solvents. Contrary to DCU, the urea formed from EDAC is readily soluble. EDAC is the reagent of choice for the con-jugation of peptides, labels, small organic molecules etc. to proteins.

Literature:J. C. Sheehan, P. A. Cruickshank, G. L. Boshart, J. Org. Chem. 26, 2525 (1961)K. D. Kopple and D.E.Nitecki, J. Am. Chem. Soc. 84, 4457 (1962)J. C. Sheehan, J. Preston, P. A. Cruickshank, J. Am. Chem. Soc. 87, 2492 (1965)K. Hojo, H. Ichikawa, Y. Fukumori, K. Kawa-saki, Int. J. Pept. Res. Ther. 14, 373 (2008)Y. J. Pu, R. K. Vaid, S. K. Boini, R. W. Towsley, C. W. Doecke, D. Mitchell, Org. Process Res. Dev. 13, 310 (2009)

In addition to racemization another side reaction with carbodiimides is an O-N-migration of the activated carboxyl func-tion forming an N-acyl urea (see Fig. 3). This stable compound is unable to take part in further couplings reaction. As this side reaction depends on the temperature and coupling behavior of the correspond-ing amino function, low temperatures are always recommended in carbodiimide-mediated couplings.Due to the fact, that all carbodiimides are condensing reagents, side reactions of un-protected amino acid-side chains (Asn, Gln), such as conversion of amides to nitriles have been observed. Appropriate side-chain protecting groups will prevent these side reactions.Other side reactions depending on the na-ture of additives are not a special problem of carbodiimides and will be described later.

Fig. 4.Mechanism of the DCC/HOBt-mediated peptide coupling.

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2. Additives

Additives such as HOBt, HOAt or Oxyma pure® are strongly recommended in all cases of amide bond formations with car-bodiimides, in order to enhance the reactiv-ity and also to reduce formation of epimers as well as N-acylureas.

HOBt(1-Hydroxybenzotriazole)

N-Hydroxybenzotriazole, developed by W. König and R. Geiger in 1970 was the most popular additive during the last decades. Today, HOBt is still one of the most effective suppressors of racemization in carbodi-imide-mediated reactions.The most important drawback of the addi-tive lies in its explosive character, especially in water-free form. Therefore, its availability is more restricted today and it should be substituted by more stable additives in the future.The mechanism of activation by HOBt used in combination with DCC is shown in Fig. 4.

Literature:W. König and R. Geiger, Chem. Ber. 103, 788 (1970)W. König and R. Geiger, Chem. Ber. 103, 2024 (1970)

HOBt-6-sulfonamidomethyl resin · HCl (200-400 mesh) (D-2435)(1-Hydroxybenzotriazole-6-sulfonamido-methyl resin · HCl)

“Polymeric HOBt”, a highly efficient poly-meric auxiliary for the synthesis of amides. Simple filtration allows the separation of the product from the polymer.

Literature:I.E. Pop, B.P. Déprez, A.L. Tartar, J. Org. Chem. 62, 2594 (1997)

HOOBt (HODhbt)(Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzo-triazine)

ODhbt esters, generated during couplings with a carbodiimide and HOOBt or DEPBT-mediated couplings, are more reactive than HOBt esters. The release of the benzotri-azine can be monitored spectrophotometri-cally.

Literature:E. Atherton, S. Cameron, M. Meldal, R.C. Sheppard, J. Chem. Soc. Chem. Commun. 1763 (1986)

HOSu (Q-1800)(N-Hydroxysuccinimide)

In contrast to HOBt and HODhbt, HOSu is completely shelf-stable. It is also a well-known additive in carbodiimide-mediated reactions since many years. On the other hand HOSu forms stable and isolable active esters, which are applicable in organic as well as in aqueous solution.A drawback of HOSu is due to its hydroxam-ic acid structure susceptible to the Lossen-rearrangement. This side reaction can be observed under condensation conditions and leads to introduction of an additional β-alanine.

Literature:G. W. Anderson, J. E. Zimmerman, F. M. Cal-lahan, J. Am. Chem. Soc. 86, 1839 (1964)F. Weygand, D. Hoffmann, E. Wünsch, Z. Naturforsch. 21b, 426 (1966)S. J. Davies and A. K. Mohammed, J.Chem. Soc. Perkin Trans. I, 2982 (1981)

Coupling Reagents

8

HOAt(1-Hydroxy-7-aza-1H-benzotriazole)

More than HOBt, this additive accelerates coupling reactions and suppresses racemi-zation. However, the explosive properties of this compound restrict its applications and availability.

Literature:L. A. Carpino, A. El-Faham, Tetrahedron 55, 6813 (1999)J. Klose, A. El-Faham, P. Henklein, L. A. Car-pino, M. Bienert, Tetrahedron Lett. 40, 2045 (1999)L. A. Carpino, H. Imazumi, B. M. Foxman, M. J. Vela, P. Henklein, A. El-Faham, J. Klose, J. Bienert, Org. Lett. 2, 2253 (2000)

Oxyma Pure® (Q-2750)(Ethyl 2-cyano-2-(hydroximino)acetate)

A trademark of Luxembourg Bio Technolo-gies Ltd, Rechovot, IsraelThis more recently developed additive is a non-explosive alternative to HOBt or HOAt, and allows high coupling rates at low race-mization when applied in combination with carbodiimides.In practice, Oxyma Pure can be used in an identical manner as HOBt in DMF on auto-mated synthesizers.

Literature:R. Subirós-Funosas, R. Prohens, R. Barbas, A. El-Faham, F. Albericio, Chem. Eur. J. 15, 9394 (2009)S. N. Khattab, Bull. Chem. Soc. Jpn. 83, 1374 (2010)R. Subirós-Funosas, A. El-Faham, F. Alberi-cio, Peptide Science 98, 89 (2012)

DMAP(4-(N,N-Dimethylamino)pyridine)

Esterifications of carboxylic acids with primary or secondary, aliphatic alcohols employing carbodiimides proceed smoothly, when performed in non-polar solvents (DCM, toluene,…). Nevertheless, a remark-able acceleration is observed, if catalysts as DMAP are present, forming highly reactive intermediates. In the case of amino acid compounds, racemization is often a main disadvantage of this coupling method, which could be reduced sometimes by running the reaction at low temperatures or applying the alcoholic partner in high concentrations.Literature:S. S. Wang, J. P. Tam, B. S. Wang, R. B. Merri-field, Int. J. Pept. Protein Res. 18, 459 (1981)J.-P. Gamet, R. Jacquier, J. Verducci, Tetra-hedron, 40, 1995 (1984)K. Takeda, A. Ayabe, M. Suzuki, Y. Konda, Y. Harigaya, Synthesis 689 (1991)C. Grondal, Synlett 1568 (2003)

3. Phosphonium Reagents

BOP (Q-1980)(Benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate)

BOP was the first of a broad range of phos-phonium type coupling reagents. It was introduced by Castro et al. already in 1975. BOP provides excellent coupling behavior and good solubility in most of the common solvents, in solid-phase as well as in solu-tion. It converts carboxyl groups into -OBt esters and has no guanylation-activity to amino functions as aminium-compounds

9

like TBTU. It is a useful reagent for lactoni-zation, selective esterification or amidation of α-amino acids without racemization. However, its severe drawback is the toxicity problem due to the carcinogenic HMPA (hexamethylphosphoramide) formed as by-product during the reaction.

Literature:B. Castro, J. R. Dormoy, G. Evin, C. Selve, Tetrahedron Lett. 14, 1219 (1975)B. Castro, G. Evin, C. Selve, R. Seyer,Synthesis, 413 (1977)J.-A. Fehrentz and B. Castro, Synthesis, 676 (1983)R. P. McGeary, Tetrahedron Lett. 39, 3319 (1998)A. Wahhab and J. Leban, Tetrahedron Lett. 40, 235 (1999)

PyBOP® (Q-2715)(Benzotriazol-1-yloxy-tripyrrolidino-phos-phonium hexafluorophosphate)

A trademark of Merck KGaA, Darmstadt, GermanyIntroduced as a non-toxic version of BOP, PyBOP has the same effective coupling properties in solid phase as BOP. PyBOP has been used as well for obtaining peptide thioesters.

Literature:J. Martinez, J. Laur, B. Castro, J. Med. Chem. 28, 1874 (1985)J. Coste, D. Le-Nguyen, B. Castro, Tetrahedron Lett. 31, 205 (1990)T. Høeg-Jensen M. H. Jakobsen, C. E. Olsen, A. Holm, Tetrahedron Lett. 32, 7617 (1991)R. von Eggelkraut-Gottanka, A. Klose, A. G. Beck-Sickinger, M. Beyermann, Tetrahedron Lett. 44, 3551 (2002)

PyBrOP®

(Bromo-tripyrrolidino-phosphonium hexa-fluorophosphate)

A trademark of Merck KGaA, Darmstadt, Germany This reagent was developed by J. Coste, to overcome the lack of PyBOP®, as well as other HOBt-containing coupling reagents, in incomplete couplings to N-methyl-amino-acids. Further good results were reported in coupling of Aib-derivatives.Additionally, due to its high reactivity, PyBrOP® is not a standard coupling reagent for all amino acids. The formation of oxazo-lones during prolonged couplings accompa-nied by higher racemization limits the use of the coupling reagent.

Literature:J. Coste, M.-N. l. Dufour, A. Pantaloni, B. Castro, Tetrahedron Lett. 31, 669 (1990)J. Coste, E. Frérot, P. Jouin, B. Castro, Tetrahedron Lett. 32, 1967 (1991)S. Gazal, G. Gellerman, E. Glukhov, C. Gilon, J. Pept. Res. 58, 527 (2001)

PyAOP(7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate)

The HOAt-analog to PyBOP®, developed by L. Carpino gave also remarkable faster cou-pling rates as PyBOP®, due to the enhanced electron withdrawing effect of the corre-sponding formed -OAt active esters during coupling reaction.

Coupling Reagents

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Contrary to HATU PyAOP cannot react with amino groups yielding guanidines.

Literature:F. Albericio, M. Cases, J. Alsina, S. A. Triolo, L. A. Carpino, S. A. Kates, Tetrahedron Lett. 38, 4853 (1997)F. Albericio, J. M. Bofill, A. El-Faham, S. A. Kates, J. Org. Chem. 63, 9678 (1998)

PyOxim (Q-2760)(Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluo-rophosphate)

This recently developed coupling reagent contains Oxyma pure as part of the mol-ecule instead of the explosive compounds HOBt or HOAt. This improvement of safety in handling is accompanied with an ac-celerated reactivity in couplings as well as a minimized allergenic potential, making PyOxim to one of the best coupling reagents in solid-phase reactions. The only drawback is the formation of tris-pyrrolidinophos-phamide as side product, which can cause problems in separation when the reagent is applied in solution.

Literature:R. Subiros-Funosas, A. El-Faham, F. Albericio, Org. Biomol. Chem. 8, 3665 (2010)

DEPBT (Q-2565)(3-(Diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazin-4(3H)-one)

Contrary to the other described phosphoni-um reagents, DEPBT is a mixed anhydride of HOOBt and dietylphosphoric acid. It medi-ates amide bond formations with a remark-able resistance to racemization. Therefore

it is the reagent of choice for coupling the racemization prone Fmoc-His(Trt)-OH.

Literature:C.-X. Fan, X.-L. Hao, Y.-H. Ye, Synth. Com-mun. 26, 1455 (1996)H. Li, X. Jiang, Y. H. Ye, C. Fan, T. Romoff, M. Goodman, Org. Lett. 1, 91 (1999)M. Mergler, F. Dick, T. Vorherr, Innovation and Perspectives in Solid Phase Synthesis and Combinatorial Libraries, 7th International Symposium, Southampton, p. 235, R. Epton, ed. Mayflower Worldwide (2002)F. Dettner, A. Hänchen, D. Schols, L. Toti, A. Nusser, R. D. Süssmuth, Angew. Chem. Int. Ed. Engl. 48, 1856 (2009)

Phosphonium-based coupling reagents do not affect amino groups and are well suited to be used as cyclization reagents, when applied in excess.Two classes of building blocks should be disclaimed from coupling procedures with phosphonium reagents:a. Phosphorylated amino acids (Fmoc-Ser(PO,OH,OBzl)-OH,…) can undergo unde-sired couplings between reagent and the unprotected phosphoryl-side chain.b. Compounds containing nucleotides (e.g. PNA-building blocks) with oxo-functions like guanine, etc. are prone to be attackedunder structural rearrangement.

Literature:S. Pritz, Y. Wolf, C. Klemm, M. Bienert, Pep-tides 2006, Proceedings of the 29th Euro-pean Peptide Symposium, Gdansk, Poland, p. 472, edited by K. Rolka, P. Rekowski and J. Silbering (2006)J. W. Perich, N. J. Ede, S. Eagle, A. M. Bray, Lett. Pept. Sci. 6, 91 (1999)

4. Aminium/Uronium-Imonium Reagents

TBTU (BF4-) (Q-1665)/ HBTU (PF6

-)(2-(1H-Benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium tetrafluoroborate/hexafluorophosphate)

11

These two compounds, only differing in their counterions (BF4-, PF6-) have nearly identical chemical properties and belong to the most popular coupling reagents. Their application is widespread in solid-phase reactions as well as in solution, because all resulting by-products are soluble in water as well as in standard organic solvents (see Fig. 5). For couplings of phosphory-lated amino acids TBTU and HBTU are the reagents of choice.

Literature:P. Henklein, C. Mügge, B. Costisella, V. Wray, T. Domke, A. El-Faham, Y. Lee, S A. Kates, L. A. Carpino, Innovation and Perspectives in Solid-Phase Synthesis and Combinatorial Libraries, 5th International Symposium, London, p. 309, R. Epton, ed. Mayflower Scientific (1998)R. Knorr, A. Trzeciak, W. Bannwarth, D. Gillessen, Peptides 1988, Proceedings of the 20th European Peptide Symposium, Tübingen, p. 37, G. Jung and E. Bayer, eds. W. de Gruyter, Berlin (1989)R. Knorr, A. Trzeciak, W. Bannwarth, D. Gil-lessen, Tetrahedron Lett. 30, 1927 (1989)I. Abdelmoty, F. Albericio, L. A. Carpino, B. M. Foxman, S. A. Kates, Lett. Pept. Sci. 1, 57 (1994)J. M. Matsoukas, D. Panagiotopoulos, M. Keramida, T. Mavromoustakos, R. Yam-dagni, Q. Wu, G. J. Moore, M. Saifeddine, M. D. Hollenberg, J. Med. Chem. 39, 3585 (1996)R. F. Poulain, A. L. Tartar, B. P. Déprez, Tetra-hedron Lett. 42, 1495 (2001)

A well-known limitation of aminium/uronium-derivatives is a possible reaction with free amino groups yielding guanidines, when the coupling reagent is applied in excess (Fig. 6). This side reaction is normally inhibited by application of a slight excess of carboxyl compound in relation to coupling reagent and by a short period of preactiva-tion before adding to the amino compound.

HCTU(2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium hexafluoro-phosphate)

The replacement of HOBt by 6-Chloro-HOBt as part of the molecule, leads to higher reaction rates and improved results in the synthesis of difficult peptides.

Literature:O. Marder, Y. Shvo, F. Albericio, Chimica Oggi 20, 37 (2002)O. Marder and F. Albericio, Chimica Oggi 21, 6 (2003)

Fig. 5.Coupling with TBTU in the presence of DIPEA.

Coupling Reagents

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HDMC (Q-2765)(N-[(5-Chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uronium hexafluorophosphate N-oxide)

When modifying the uronium part of HCTU by introduction of a morpholine moiety, a further increase of reactivity can be gained. Even coupling rates of HATU could be ex-ceeded in some cases by HDMC.

Literature:A. El-Faham and F. Albericio, J. Org. Chem. 73, 2731 (2008)

TATU (BF4-) (Q-2150)/ HATU (PF6

-) (Q-2780)(2-(7-Aza-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethylaminium tetrafluoroborate/hexafluorophosphate)

The HOAt analogs to HBTU/TBTU are highly efficient coupling reagents for solid- and

solution-phase reactions. Contrary to HOBt-based reagents they have been used successfully in couplings of N-methyl-amino acids.

Literature:L. A. Carpino, J. Am. Chem. Soc. 115, 4397 (1993)F. Albericio, J. M. Bofill, A. El-Faham, S. A. Kates, J. Org. Chem. 63, 9678 (1998)

COMU (Q-2735)(1-[1-(Cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate)

COMU is a novel coupling reagent with coupling efficiencies comparable to HATU. The incorporation of Oxyma Pure as part of the molecule in place of the explosive compounds HOBt or HOAt results in safer handling in combination with better solubil-ity and a reduced allergenic potential than HBTU/TBTU or HATU. COMU is especially suited for microwave-accelerated SPPS.Recently it has been demonstrated that COMU can be used to prepare esters of all types of alcohols at room temperature under mild conditions in the presence of organic bases.

Fig. 6.N-Terminal gua-nidinylation by the coupling reagent.

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Literature:A. El-Faham and F. Albericio, J. Org. Chem. 73, 2731 (2008)A. El-Faham, R. Subiros Funosas, R. Pro-hens, F. Albericio, Chem. Eur. J. 15, 9404 (2009)R. Subiros-Funosas, G. A. Acosta, A. El-Faham, F. Albericio, Tetrahedron Lett. 50, 6200 (2009)A. El-Faham and F. Albericio, J. Pept. Sci. 16, 6 (2010)J.K. Twibanire and T.B. Grindley et al.Org. Lett. 13, 2988 (2011)

TOTT (Q-2600)(2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate)

TOTT is a thiuronium salt of 2-mercaptopyr-idone-1-oxide which showed good results in couplings of sterically hindered or methyl-ated amino acids, comparable with HATU. Racemization levels of those couplings are reported to be lower as with other reagents.

Literature:M. A. Bailén, R. Chinchilla, D. J. Dodsworth, C. Nájera, J. Org. Chem. 64, 8936 (1999)

TFFH (Q-2280)(Tetramethylfluoroformamidinium hexa-fluorophosphate)

A highly efficient coupling reagent which generates amino acid fluorides in situ. Amino acid fluorides are especially suited for the coupling of sterically hindered α,α-disubstituted amino acids such as Aib.

Literature:L.A. Carpino and A. El-Faham, J. Am. Chem. Soc. 117, 5401 (1995)A. El-Faham, OPPI Briefs 30, 477 (1998)A. El-Faham and S.N. Khattab, Synlett 886 (2009)S. N. Khattab, Bull. Chem. Soc. Jpn. 83, 1374 (2010)

5. Miscellaneous Coupling Reagents

EEDQ (Q-1735)(N-Ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline)

EEDQ is an “old” reagent (developed in 1967), which still is of interest due to its re-

Fig. 7.EEDQ-mediated generation of mixed anhydride.

Coupling Reagents

14

markable properties. It converts the amino acid derivative into an anhydride EEDQ does not require the presence of a tertiary base nor does it affect the amino function or other reactive groups such as hydroxyls (Fig. 7). Albeit the formation of the anhy-dride is slow, it is consumed very rapidly by the amino-component which minimizes racemization during product formation. As an example, EEDQ was the only reagent amongst the tested ones which mediated the coupling 3,5-dinitrobenzoyl-Leu-OH to 3-aminopropyl-silica with negligible level of concomitant racemization.

Literature:B. Belleau and G. Malek, J. Am. Chem. Soc. 90, 1651 (1968)B. Belleau, R. R. Martel, G. Lacasse, M. Men-ard, N. L. Weinberg, Y. G. Perron, J. Am. Chem. Soc. 90, 823 (1968)R. Pougeois, FEBS Lett. 154, 47 (1983)H. Kunz, H. Waldmann, C. Unverzagt, Int. J. Pept. Protein Res. 26, 493 (1985)M. H. Hyun, M. H. Kang, S. C. Han, Tetrahedron Lett. 40, 3435 (1999)A. Yang, A. P. Gehring, T. Li, J. Chromatogr. A 878, 165 (2000)Merck Index, 14th ed. No. 3518, (2006)

T3P(2-Propanephosphonic acid anhydride)

A trademark of EuticalsT3P (or PPA) is a cyclic anhydride of pro-pylphosphonic acid, which is normally used in combination with tertiary amines for solution-phase and cyclization reactions. PPA gives superior results especially for sterically hindered peptides.

Literature:J. Klose, M. Bienert, C. Mollenkopf, D. Wehle, C.-W. Zhang, L. A. Carpino, P. Henklein, Chem. Commun. 1847 (1999)P. Koch, C. Vedder, T. Schaffer, Chimica Oggi 26, 6 (2008)

DMTMM and related compounds(4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salts)

Triazines - a group of coupling reagents, developed by Kaminski et al. - are conden-sation products of substituted cyanuric

Fig. 8.Mechanism of DMTMM-mediated amide bond formation.

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chloride with tertiary amines as NMM or DABCO, able to mediate peptide couplings in water or alcoholic solutions. These reagents proved to be particularly efficient allowing high yields and low racemization levels. For the coupling mechanism of DMTMM see Fig. 8.

Literature:K. Jastrzabek, B. Kolesinska, G. Sabatino, F. Rizzolo, A. Papini, Z. Kaminski, Int. J. Pept. Res .Ther. 13, 229 (2007)Z. J. Kaminski, B. Kolesinska, J. Kolesinska, G. Sabatino, M. Chelli, P. Rovero, M. Blaszc-zyk, M. L. Glowka, A. M. Papini, J. Am. Chem. Soc. 127, 16912 (2005)B. Kolesinska, J. Fraczyk, G. Sabatino, A. Papini, Z. Kaminski, Chimica Oggi 25, 26 (2007).

BTC(bis-Trichloromethylcarbonate or “Triphos-gene”)

The stable trimeric form of phosgene is a well-known reagent for generating acid chlorides from carboxylic acids. Its applica-bility for peptide couplings was evaluated by C. Gilon et al. for solution- and solid-phase couplings. They used collidine as base and THF or DCM as solvents. Other solvents such as DMF or NMP have to be strictly avoided, as they can react with BTC. Care must be taken when handling the reagent, because BTC and even more so phosgene which is formed as intermediate are highly toxic compounds. Beyond these restrictions, BTC is one of the most efficient activation reagents and recommended especially for difficult couplings and less reactive amines such as anilines.

Literature:E. Falb, T. Yechezkel, Y. Salitra, C. Gilon, J. Pept. Res. 53, 507 (1999)B. Thern, J. Rudolph, G. Jung, Tetrahedron Lett. 43, 5013 (2002)

CDI(1,1’-Carbonyldiimidazole)

CDI, as the related compounds disuccinimi-dyl carbonate or 4-nitrophenyl-chlorofor-mate, is a phosgene analog, a derivative of carbonic acid. CDI is not routinely used for peptide couplings. Its field of application lies in the synthesis of ureas and urethanes from amines, alcoholic compounds or resins and linkers.Nevertheless CDI has been successfully used in peptide couplings. The protocol for couplings has to include a preactivation step during which the reactive acid imidaz-olide is formed, which is added to the amino component.

Literature:G.W. Anderson and R. Paul, J. Am. Chem. Soc. 80, 4423 (1958)H. Ogura, T. Kobayashi, K. Shimizu, K. Kaw-abe, K. Takeda, Tetrahedron Lett. 20, 4745 (1979)K. Takeda, Y. Akagi, A. Saiki, T. Tsukahara, H. Ogura, Tetrahedron Lett. 24, 4569 (1983)T. Kamijo, H. Harada, K. Iizuka, Chem. Pharm. Bull. 32, 5044 (1984)

Conclusion

TBTU, HBTU and PyBOP are well suited reagents for most standard coupling reactions. They all contain the potentially explosive HOBt as part of the molecule. The same problem is encountered with the more reactive HOAt-based coupling reagents. Due to safety considerations, all of them will have to be replaced in the future by Oxyma Pure-based reagents such as COMU or PyOxim. More specialized reagents as HATU, HDMC, TOTT or DEPBT can be required to succeed in incorporating amino acid derivatives prone to side reactions. However, “old” re-agents as EEDQ still can be useful in cases where other reagents rendered poor results.Bachem offers a large selection of coupling reagents to meet all requirements of the synthetic chemist.

Coupling Reagents

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For more information as well as many valuable hints regarding properties and ap-plication of coupling reagents the following review articles are recommended:

F. Albericio and L.A. CarpinoCoupling reagents and activation. Methods for solid-phase assembly of peptides.Meth. Enzymol. 289, 104-126 (1997)F. Albericio, R. Chinchilla, D. J. Dodsworth, C. NájeraNew trends in peptide coupling reagents.Org. Prep. Proced. Int. 33, 203-303 (2001)S.-Y. Han and Y.-A. KimRecent development of peptide coupling reagents in organic synthesis.Tetrahedron 60, 2447-2467 (2004)A.R. Katritzky, K. Suzuki, K. SinghN-Acylation in combinatorial chemistry.ARKIVOC 12-35 (2004) C.A.G.N. Montalbetti and V. FalqueAmide bond formation and peptide cou-pling.Tetrahedron 61, 10827-10852 (2005)E. Valeur and M. BradleyAmide bond formation: beyond the myth of coupling reagents.Chem. Soc. Rev. 38, 606-631 (2009)

M.M. Joullié and K.M. LassenEvolution of amide bond formation. ARKIVOC 189-250 (2010)C. Roche, M. Pucheault, M. Vaultier, A. CommerçonOnium salt supported peptide synthesis. Tetrahedron 66, 8325-8334 (2010)A. El-Faham and F. AlbericioPeptide coupling reagents: more than a letter soup.Chem. Rev. 111, 6557-6602 (2011)V.R. Pattabiraman and J.W. BodeRethinking amide bond synthesis.Nature 480, 471-479 (2011)T.I. Al-Warhi, H.M.A. Al-Hazimi, A. El-FahamRecent development in peptide coupling reagents.J. Saudi Chem. Soc. 16, 97-116 (2012)R. Subirós-Funosas, S.N. Khattab, L. Nieto-Rodríguez, A. El-Faham, F. AlbericioAdvances in acylation methodologies en-abled by Oxyma-based reagents.Aldrichim. Acta 46, 21-40 (2013)J. R. Dunetz, J. Magano, G. A. WeisenburgerLarge-Scale Applications of Amide Coupling Reagents for the Synthesis of Pharmaceu-ticals.Org. Proc. Res. Devel.20, 140-177 (2016)

REFERENCES

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COUPLING REAGENTS AND ADDITIVES

We also offer a comprehensive choice of pre-formed active esters of Fmoc-, Boc-, and Z-amino acids:Fmoc-Xaa-OPfp and Fmoc-Xaa-OSuBoc-Xaa-ONp and Boc-Xaa-OSuZ-Xaa-ONp and Z-Xaa-OSufor all strategies of peptide synthesis.Appropriately protected active esters of the proteinogenic amino acids as well as of unusual amino acids can be found in our online shopshop.bachem.com

Coupling Reagents

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COUPLINGREAGENTS

BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexa-fluorophosphate)Q-1980

COMU(1-[1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethyl-amino-morpholino]-uronium hexa-fluorophosphate)Q-2735

DEPBT(3-(Diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazin-4(3H)-one)Q-2565

EDAC · HCl, EDC · HCl, WSC · HCl(N-(3-Dimethylaminopropyl)-N’-ethyl-carbodiimide · HCl)Q-1955

EEDQ(N-Ethoxycarbonyl-2-ethoxy-1,2-dihy-droquinoline)Q-1735

HATU(N-[(7-Aza-1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-meth-ylmethanaminium hexafluorophos-phate N-oxide)Q-2780

HDMC(N-[(5-Chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uronium hexafluorophosphate N-oxide)Q-2765

PyBOP(Benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate)Q-2715

PyOxim((Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate)Q-2760

TATU(N-[(7-Aza-1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-meth-ylmethanaminium tetrafluoroborate N-oxide)Q-2150

TBTU(N-[(1H-Benzotriazol-1-yl)(dimethylamino)-methylene]-N-meth-ylmethanaminium tetrafluoroborate N-oxide)Q-1665

TFFH(Tetramethylfluoroformamidinium hexafluorophosphate)Q-2280

TOTT(2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetra-methylisothiouronium tetrafluorobo-rate)Q-2600

ADDITIVES HOSu(N-Hydroxysuccinimide)Q-1800

Oxyma Pure(Cyano-hydroxyimino-acetic ethyl ester)Q-2750

HOBt-6-sulfonamidomethyl resin · HCl (200-400 mesh)(1-Hydroxybenzotriazole-6-sulfonami-domethyl resin · HCl)D-2435

CALCITONINGENE-RELATEDPEPTIDES

ANTIMICROBIALPEPTIDES

AMYLOID PEPTIDES

CASPASESUBSTRATES INHIBITORS

CYSTEINEDERIVATIVES

GHRELIN,LEPTIN ANDOBESTATIN

1

DAP AND DABDERIVATIVES

ENDOTHELINS

1

FRET SUBSTRATES

DIABETES PEPTIDES

PEPTIDES IN COSMETICS

PARACTIVATINGPEPTIDES

NON-IONIC DETERGENTS

ORTHOGONALITYOF PROTECTINGGROUPS

PEPTIDE YY

VETERINARYPEPTIDES

PRIONPEPTIDES

PSEUDOPROLINEDIPEPTIDES

SECRETASESUBSTRATES INHIBITORS

VIP/PACAP

MATRIXMETALLO-PROTEINASES

LHRHAGONISTS AND ANTAGONISTS

MELANOMA PEPTIDES

NEUROPEPTIDE YN-METHYLATEDAMINO ACIDDERIVATIVES

PRODUCT BROCHURES

2011

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All information is compiled to the best of our knowledge. We cannot be made liable for any possible errors or misprints. Some products may be restricted in certain countries.

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