Journal of Saudi Chemical Society (2012) 16,97–116 King Saud University Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com REVIEW ARTICLE Recent development in peptide coupling reagents Tarfah I. Al-Warhi a,*, Hassan M.A. Al-Hazimi b, Ayman El-Faham b,c a King Saud University, Women Students-Medical Studies and Sciences Sections, Chemistry Department, College of Science, P.O. Box 22452, Riyadh 11495, Saudi Arabia b King Saud University, College of Science, Chemistry Department, P.O. Box 2455, Riyadh 11451, Saudi Arabia c Alexandria University, Faculty of Science, Chemistry Department, P.O. Box 426, Ibrahimia, Alexandria 21321, Egypt Received 30 October 2010; accepted 27 December 2010 Available online 5 January 2011 KEYWORDS Abstract Two decades of domination of benzotriazole-based chemistry stimulated the progress in Peptide coupling reagents; peptide synthesis to a high level of effectiveness. However, the growing need for new and more com- Peptide bond; plex peptide structures, particularly for biomedical studies and, very recently, for the large-scale Carbodiimides; production of peptides as drugs, required manufacturing peptide products by efficient synthetic Phosphonium salts; strategies, at reasonably low prices. Therefore, the search for new, more versatile and low-cost Aminium salts; reagents becomes a great challenge. Several comprehensive review articles summarized the great Fluoroformamidinium cou- effort undertaken, but up to now, no versatile coupling reagent useful for both amide and ester pling reagents; bond formation, as well as for solution and solid-phase peptide synthesis has been yet developed. Organophosphorus reagents; The most-widely used coupling reagents are carbodiimides on one hand and phosphonium and Triazine coupling reagents aminium salts on the other. Herein in this review article, we summarized the recent development in peptide coupling reagents during the last two decades. ª 2011 King Saud University. Production and hosting by Elsevier B.V.Open access under CC BY-NC-ND license. * Corresponding author. E-mail address: [email protected] (T.I. Al-Warhi). 1319-6103 ª 2011 King Saud University. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license. Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2010.12.006 Production and hosting by Elsevier 98 T.I. Al-Warhi et al. Contents 1. Introduction . 98 2. Chemistry . 98 2.1. Peptide bond formation . 98 2.2. Coupling reagents and activation . 100 2.2.1. Carbodiimides (Rich and Singh, 1979) ............................................. 100 2.2.2. Phosphonium and aminium salts . 103 2.2.3. Fluoroformamidinium coupling reagents. 106 2.2.4. Organophosphorus reagents. 108 2.2.5. Triazine coupling reagents . 109 References . 113 1. Introduction length of the peptide chain increases, the protocols routinely used on an automatic multiple peptide solid-phase synthe- The synthesis of proteins has challenged chemists for over a sizer take advantage of potent coupling reagents and a large century. Among many methodologies used or currently being excess of the acylating mixture. On the other hand, it is used, chemical synthesis offers attractive advantages over the well-known that over activation may lead to undesired side molecular biology and genetic engineering approaches. reactions. Moreover, the high cost of some protected amino Chemical synthesis confirms the covalent structure and pro- acids (particularly the un-natural ones) and coupling re- vides material both for three-dimensional structure determi- agents suggests maintaining their consumption to a reason- nation by NMR or X-ray crystallography and for ably low level, compatibly with the success of the biological evaluation. The synthesis of analogues allows the synthesis. In automatic multiple solid-phase synthesis, used relation between molecular structure and pharmacological to obtain simultaneously peptides not only of different se- activity to be determined, and in favorable cases, compounds quences but also of different length, the coupling reagents with activity, selectivity, or biostability superior to the origi- should be efficient in terms of yield and preservation of opti- nal natural product could be produced on an industrial scale cal purity of the final products. In particular, the coupling if desired (Lloyd-Williams et al., 1997). Since the difference reagents should be applied repetitively under standard condi- between peptides and proteins is essentially one of size or tions to a wide range of substrates including less reactive of the length of the amino acid backbone, the problems in- and sterically hindered amino acids. In addition, using an volved in the chemical synthesis of proteins are basically automatic multiple peptide synthesizer, coupling reagents, those of the synthesis of peptides. preferably commercially available and easy-to-use, should Since the first simple peptides were synthesized by The- have the following characteristics: fast reactions at room odor Curtius and Emil Fisher a century ago, research in temperature, good solubility in the common solvents, and the methodology of peptide synthesis has undergone dra- stability of their solutions for several days. matic development. However, it is only over the last few Synthesis in solution is only rarely a practical alternative decades, especially after Bruce Merrifield’s invention of so- for large peptides, since it is slow, labor-intensive, and dogged lid-phase peptide synthesis (SPPS) (Merrifield, 1963) that by the problems of racemization and poor solubility of the syn- reliable methods for the preparation of the peptide have thetic intermediates. been achieved. Modern synthetic methods, whether manual or machine-assisted, using solid supports or in solution, al- 2. Chemistry low many peptides to be synthesized without undue diffi- culty. However, long peptides and proteins, or those 2.1. Peptide bond formation peptides having a high incidence of the more sensitive amino acids, still present difficulties. Side reactions can always oc- Success in the chemical synthesis of peptides, as well as peptide cur even in quite simple sequences, but for larger molecules libraries, relies on an efficient combination of protecting much more serious complications can manifest themselves. groups and coupling reagents (Scheme 1). In solid-phase synthesis, incomplete deprotections and cou- The formation of a peptide bond between two amino acids pling reactions tend to become more pronounced as the involves two steps. The first step is the activation of the R R R O 1 Coupling Reagent R O HO R1O NHR2 2 NH2 NHR N O O H O R Scheme 1 Peptide bond formation. Recent development in peptide coupling reagents 99 O - O H O H activation Path A H R' N R' N R' N OH X X H O H R O R Base O R Activated Intermediate (X = activating moiety) BH+ Path B Base H O O N R R' N H X R' N R' X O O O H R H O R Oxazolone Base H2N-R'' R N R N O H R' R' R' N O O O O NHR'' H O R BH+ H H2N-R'' N R D,L-Peptide R' O O Scheme 2 Mechanism of racemization. carboxyl group of one residue; this step accounts for a key step which has been most extensively developed, in recent years in the synthesis of a large number of bioorganic molecules, in (Albericio and Carpino, 1997; Humphrey and Chamberlin, particular during peptide synthesis (Sheehan and Hess, 1955). 1997; Lloyd-Williams et al., 1997; Albericio and Kates, If the activation of carboxylic acid is slow, the coupling re- 2000; Albericio et al., 2001; Han and Kim, 2004; Benoiton, agents will be degraded and will no longer be able to activate 2006). the carboxyl function. This is crucial for cyclization steps or in An essential feature of all coupling methods is that in addi- convergent strategies during the fragment coupling steps be- tion to giving peptide bonds in good yield, the configurational cause yields tend to be lower than those of other couplings integrity of the carboxylic component must be maintained. (El-Faham and Albericio, 2008). This duality, good yield and absence of racemization is often The second step is the nucleophilic attack of the amino difficult to achieve, since usually the best methods involve con- group of the other amino acid derivative at the active carbox- version of the acid to a derivative bearing a good leaving ylic group (Albericio and Carpino, 1997; Albericio et al., 1998, group. Such leaving groups tend to increase the acidity of 2001; Albericio and Kates, 2000; Han and Kim, 2004; Benoi- the a-proton and favor formation of an oxazolone, as shown ton, 2006; El-Faham et al., 2006). This process is an energy- in Scheme 2. Loss of configuration is especially prominent if requiring reaction (Bodanszky, 1993), therefore, one of the oxazolone formation occurs, but also can occur at stages of carboxylic groups must be activated before the reaction can the activated carboxyl derivatives. occur. Unfortunately, this activation step, along with the next In stepwise solid-phase peptide synthesis (Merrifield, 1985) coupling reaction, brings up a loss of configuration at the car- the problem of racemization is less dramatic than that for boxyl residue undergoing the activation process. other strategies. Several parameters have been used to deal Peptides are, therefore, assembled by amide bond forma- with such side reactions during peptide-coupling reactions. A tion between optically active monomers. This being so, the key issue is Na-protecting group of the amino acid to be cou- possibility of loss of chiral integrity must be considered, and pled (the one with the carboxylic function activated) is nor- understanding the mechanisms of racemization is surely neces- mally a urethane function such as, t-butoxycarbonyl (Boc, 1) sary for its prevention. Two major pathways for the loss of (McKay and Albertson, 1957), 9-fluorenylmethyloxycarbonyl configuration, both base-catalyzed, have been recognized: (a) (Fmoc, 2)(Carpino, 1987) or the recent type of base-sensitive direct enolization, and (b) 5(4H)-oxazolone formation amino protecting groups 1,1-dioxobenzo[b]thiophene-2-ylm- (Scheme 2)(Antonovics and Young, 1967; Carpino, 1988; ethyloxy-carbonyl (Bsmoc, 3)(Carpino et al., 1997, 1999), 2- Bodanszky, 1993; Lloyd-Williams et al., 1997).