Use of Onium Salt-Based Coupling Reagents in Peptide Synthesis 1 Fernando Albericio,* ,2a Josep M. Bofill, 2a Ayman El-Faham, 2b and Steven A. Kates* ,2c Department of Organic Chemistry, University of Barcelona, E-08028 Barcelona, Spain; Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003; and PerSeptive Biosystems, Inc., 500 Old Connecticut Path, Framingham, Massachusetts 01701 Received April 29, 1998 Peptide coupling methods derived from onium salts based on 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) are becoming incorporated in synthetic strategies more frequently than the classical carbodiimide methods. We have correlated the reactivity of various onium salts derived from HOXt (X ) A, B), with the structure of the reagents in question. Thus, we confirmed that the aza derivatives are more reactive than the parent benzotriazole derivatives in both activation and coupling. In addition, the activation step is determined by the structure of the carbon skeleton. Thus, pyrrolidino derivatives appear to be reagents of choice relative to the piperidino analogues or those derived from trialkylamines. Furthermore although phosphonium salts are slightly less reactive than the corresponding aminium/uronium salts, the former should be used for the activation of hindered species, since the latter may lead to the formation of guanidino derivatives. Introduction The success of peptide synthesis is based upon the proper management of temporary and permanent pro- tecting groups and the efficiency of the coupling reagents chosen to elongate the peptide chain. 3 During the past few years, there has been an evolution in the develop- ment of new activation methods and their application to both solution and solid-phase methodology. 4 The formerly predominant carbodiimide and active ester techniques have been replaced with onium salts based upon 1-hy- droxybenzotriazole (HOBt, 1) 5 and 7-aza-1-hydroxyben- zotriazole (HOAt, 2). 6 HOAt-based reagents have been shown to be more efficient than the corresponding HOBt analogues in terms of coupling yield and reduced loss of configuration at the C-terminal carboxy acid residue. 6,7 To correlate the reactivity of different onium salts (3, 4) based upon HOXt where X ) A, B, model studies were designed to investigate the stability of these reagents in solution and determine the ease of carboxylic acid activa- tion as well as the nature and extent of any undesired side reactions. Furthermore, since practical use of these reagents may require storage in solution for prolonged times, the present study provides guidelines for optimum handling of this class of compounds. The nature of the different reagents, phosphonium (4) vs aminium/uronium (3), 8 N-alkyl substituents (R), and OAt vs OBt influences are studied (Figure 1). (1) Abbreviations used for amino acids and the designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in: J. Biol. Chem. 1972, 247, 977-983. The following additional abbreviations are used: ACP, acyl carrier protein; AOP, (7- azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro- phosphate; Boc, tert-butyloxycarbonyl; BOP, benzotriazol-1-yl-N-oxy- tris(dimethylamino)phosphonium hexafluorophosphate; DCC, N,N′- dicyclohexylcarbodiimide; Deg, diethylglycine; DIEA, N,N-diiso- propylethylamine; DMAP, N,N-dimethyl-4-aminopyridine; DMF, N,N- dimethylformamide; Fm, 9-fluorenylmethyl; Fmoc, 9-fluorenylmethyl- oxycarbonyl; HAMDU, O-(7-azabenzotriazol-1-yl)-1,3-dimethyl-1,3- dimethyleneuronium hexafluorophosphate; HAMTU, O-(7-azabenzo- triazol-1-yl)-1,3-dimethyl-1,3-trimethyleneuronium hexafluorophos- phate; HAPipU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(pentameth- ylene)uronium hexafluorophosphate; HAPyU, 1-(1-pyrrolidinyl-1H- 1,2,3-triazolo[4, 5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluoro- phosphate N-oxide; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5- b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophos- phate N-oxide; HBMDU, O-(benzotriazol-1-yl)-1,3-dimethyl-1,3-dimethyl- eneuronium hexafluorophosphate; HBTU, N-[(1H-benzotriazol-1-yl)- (dimethylamino)methylene]-N-methylmethanaminium hexafluorophos- phate N-oxide; HBPipU, O-(benzotriazol-1-yl)-1,1,3,3-bis(pentameth- ylene)uronium hexafluorophosphate; HDTU, O-(3,4-dihydro-4-oxo- 1,2,3-benzotriazin-3-yl)1,1,3,3-tetramethyluronium hexafluorophosphate; HMPA, hexamethylphosphoric triamide; HOAt, 7-aza-1-hydroxyben- zotriazole (3-hydroxy-3H-1,2,3-triazolo-[4,5-b]pyridine]; HOBt, 1-hy- droxybenzotriazole; HODhbt, 1-oxo-2-hydroxydihydrobenzotriazine; HPLC, high performance liquid chromatography; MeOH, methanol; PAL, 5-[[(4-amino)methyl]-3,5-dimethoxyphenoxy]valeric acid; PEG- PS, poly(ethylene glycol)-polystyrene (graft resin support); PPTS, pyridinium p-toluensulfonate; PS, copoly(styrene-1%-divinylbenzene) support; PyAOP, 7-aza-benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phos- phonium hexafluorophosphate; PyBOP, benzotriazol-1-yl-N-oxy-tris- (pyrrolidino)phosphonium hexafluorophosphate; SPPS, solid-phase peptide synthesis; Su, succinimidyl; TFA, trifluoroacetic acid; TMP, 2,4,6-trimethylpyridine (collidine). Amino acid symbols denote L- configuration unless indicated otherwise. (2) (a) University of Barcelona. (b) University of Massachusetts. (c) PerSeptive Biosystems. (3) (a) Stewart, J. M.; Young, J. D. Solid-Phase Peptide Synthesis, 2nd ed.; Pierce Chemical: Rockford, Illinois, 1984. (b) Merrifield, R. B. Science 1986, 232, 341-347. (c) Barany, G.; Kneib-Cordonier, N.; Mullen, D. G. Int. J. Pept. Protein Res. 1987, 30, 705-739. (d) Atherton, E.; Sheppard, R. C. Solid-Phase Peptide Synthesis: A Practical Approach; IRL: Oxford, 1989. (e) Fields, G. B.; Tian, Z.; Barany, G. In Synthetic Peptides: A User’s Guide; Grant, G. A., Ed.; W. H. Free- man: New York, 1992; pp 259-345. (f) Lloyd-Williams, P.; Albericio, F.; Giralt, E. In Chemical Approaches to the Synthesis of Peptides and Proteins; CRC: Boca Raton, FL, 1997. (4) (a) Albericio, F.; Carpino, L. A. In Methods in Enzymology, Solid- Phase Peptide Synthesis; Fields, G. B., Ed.; Academic Press: Orlando, FL, 1997; Vol. 289, pp 104-126. (b) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243-2266. (5) Ko ¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788-798. (6) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398. (7) (a) Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Carpino, L. A.; Bienert, M. Tetrahedron Lett. 1993, 34, 4781-4784. (b) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J. Chem. Soc., Chem. Commun. 1994, 201-203. (c) Carpino, L.; El-Faham, A.; Albericio, F. Tetrahedron Lett. 1994, 35, 2279-2282. (d) Angell, Y. M.; Garcı ´a-Echeverrı ´a, C.; Rich, D. H. Tetrahedron Lett. 1994, 35, 5981- 5984. (e) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695- 698. (f) Dalcol, I.; Rabanal, F.; Ludevid, M.-D.; Albericio, F.; Giralt, E. J. Org. Chem. 1996, 60, 7575-7581. (g) Angell, Y. M.; Thomas, T. L.; Flentke, G. R.; Rich, D. H. J. Am. Chem. Soc. 1995, 117, 7279-7280. (h) Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.; Kates, S. A. Tetrahedron Lett. 1997, 38, 4853-4856. 9678 J. Org. Chem. 1998, 63, 9678-9683 10.1021/jo980807y CCC: $15.00 © 1998 American Chemical Society Published on Web 12/04/1998