View Online PAPER www.rsc.org/obc | Organic & Biomolecular Chemistry PyOxP and PyOxB: the Oxyma-based novel family of phosphonium salts† Ramon Subiros-Funosas,´ a,b Ayman El-Faham*a,c,d and Fernando Albericio*a,b,e Received 12th March 2010, Accepted 19th May 2010 First published as an Advance Article on the web 17th June 2010 DOI: 10.1039/c003719b Recent studies described the great impact of a non-benzotriazolic family of coupling reagents based on ethyl 2-cyano-2-(hydroxyimino)acetate, Oxyma, as a powerful coupling methodology for peptide synthesis. Here we present the synthesis and evaluation of the derived phosphonium salts O-[(1-cyano-2-ethoxy-2-oxoethylidene)amino]-oxytri(pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyOxP) and tetrafluoroborate (PyOxB). Both coupling reagents exhibited higher capacity to suppress racemization in various peptide models and enhanced solubility in DMF and DCM than benzotriazole-based reagents. In addition, the hexafluorophosphate analog PyOxP, combined excellent stability with outstanding efficiency in the assembly of demanding penta and decapeptides that include consecutive Aib residues. Cyclization models revealed the advantages of PyOxP, which rendered a higher percentage of cyclic material than other known potent phosphonium salts. Introduction side reactions, usually during slow couplings, such as cyclizations or in the introduction of hindered residues. In these situations, an Success in peptide synthesis is highly dependent on the coupling excess of such a coupling reagent usually causes the attachment of 1,2 strategy used to form the peptide bond. Traditionally, carbodi- a guanidine moiety to the free N-terminus of the growing peptide imides were considered an efficient approach to assemble low chain, thereby blocking any further reaction.11 Moreover, these to moderate sequences, in consumption with additives (HOBt 1, reagents are costly for peptide synthesis purposes. Phosphonium HOAt 2 and 6-Cl–HOBt 3; Fig. 1 left), which lower the likelihood salts differ from the former in the nature of the electrophilic 3–5 of racemization and other side reactions. However, the need for core as they have a positively charged phosphorus center and stronger activating reagents led to the introduction of stand-alone they do not undergo the above mentioned termination of the onium salts (mainly aminium/uronium and phosphonium ones), peptide chain when added in excess. Thus phosphonium salts which have become the preferred coupling choice in solution- and show similar potency to their aminium/uronium counterparts.12 Downloaded by Universitat de Barcelona on 30 June 2011 6 solid-phase peptide synthesis. These types of onium salts were first introduced by Kenner Published on 17 June 2010 http://pubs.rsc.org | doi:10.1039/C003719B The most powerful onium salts are the aminium/uronium and coworkers in 1969, although they did not gain general ones. Their structure comprises a carbocation skeleton and an acceptance until the 1990s, when many became commercially additive, usually benzotriazole-based (HATU 4, HBTU 5/TBTU available.13 The most predominant phosphonium salts display a 7–9 6,HCTU7/TCTU 8; Fig. 1 center). The tetramethylamino tris(pyrrolidino)phosphonium-type electrophilic core (PyAOP 12, group included in the carbocation skeleton of these classical PyBOP 13, PyClock, 14;Fig.2)14–16 while their tris(dimethylamino) coupling reagents has recently evolved to a dimethylmorpholino analogs, of which BOP is the most representative, are slightly less 10 moiety (HDMA 9, HDMB 10, 6-Cl–HDMB 11;Fig.1right), reactive and also originate HMPA as a byproduct, which is a thereby resulting in improved stability and solubility in common carcinogenic and toxic phosphoramide.17–19 solvents used in peptide synthesis, as well as an increase in In both classes of onium salts, aminium/uronium and phos- optical purity and reactivity. The high reactivity of both types phonium, the same trend is observed: HOAt (2)analogsshowed of aminium/uronium salts may also lead to the appearance of superiority to HOBt (1) and 6-Cl–HOBt (3) ones in activation and coupling steps and also in reducing the loss of optical a Institute for Research in Biomedicine, Barcelona Science Park, purity.12 In addition, derivatives of 6-Cl–HOBt (3) are generally Baldiri Reixac 10, 08028, Barcelona, Spain. E-mail: aymanel_faham@ 9 hotmail.com, [email protected]; Fax: +34 93-403-71-26; Tel: +34 less hazardous and more reactive than HOBt (1) counterparts. 93-403-70-88 This relative performance is connected to the acidity of the bCIBER-BBN, Networking Centre on Bioengineering, Biomaterials and additive, which is linked to the reactivity of the corresponding Nanomedicine Barcelona Science Park, Baldiri Reixac 10, 08028, Barcelona, active ester. Therefore, PyAOP (12) is considered the most reactive Spain phosphonium salt (pK of HOAt=3.28), whereas PyClock (14)is cDepartment of Chemistry, College of Science, King Saud University, a P.O. Box 2455, Riyadh, 11451, Kingdom of Saudi Arabia a good alternative to PyBOP (13), because the presence of Cl at dDepartment of Chemistry, Faculty of Science, Alexandria University, position 6 of the benzotriazole ring results in a better leaving group 20 Ibrahimia, 21321, Alexandria, Egypt (pK a of 6-Cl–HOBt=3.35 vs. pK a of HOBt=4.60). However, eDepartment of Organic Chemistry, University of Barcelona, Mart´ıi additional factors contribute to the great difference in reactivity. Franques´ 1-11, 08028, Barcelona, Spain Thus, it has been proposed that the nitrogen at the 7-position † Electronic supplementary information (ESI) available: 1Hand13C-NMR spectra for PyOxP and PyOxB, and HPLC characterization of racemiza- of HOAt creates a neighboring group effect, which results in tion and coupling efficiency experiments. See DOI: 10.1039/c003719b enhanced reactivity and solubility of its analogs. This journal is © The Royal Society of Chemistry 2010 Org. Biomol. Chem., 2010, 8, 3665–3673 | 3665 View Online Fig. 1 Benzotriazole-based additives (left), tetramethyl (center) and dimethylmorpholino (right) aminium salts. was not tested.26 In the present study, we compared the capacity of O-[(cyano-(ethoxycarbonyl)methyliden)-amino]- yloxytripyrrolidinophosphonium hexafluorophosphate and tetrafluoroborate (PyOxP 17 and PyOxB 18,Fig.4)tolower racemization and enhance coupling extension in linear and cyclic peptide models with the abovementioned benzotriazole-based phosphonium salts PyAOP (12), PyBOP (13) and PyClock (14). Fig. 2 Benzotriazole-based phosphonium salts. Recently, we reported ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma 15, Fig. 3) and its derived uronium salt 1-[(1- (cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino- morpholinomethylene)] methanaminium hexafluorophosphate (COMU 16, Fig. 3) as efficient additive and coupling reagent Downloaded by Universitat de Barcelona on 30 June 2011 21,22 for peptide synthesis. The active esters formed by these Fig. 4 Structure of the hexafluorophosphate and tetrafluoroborate Published on 17 June 2010 http://pubs.rsc.org | doi:10.1039/C003719B reagents are extremely reactive due to the low hindrance and tris(pyrrolidino)phosphonium salts derived from Oxyma. acidity of Oxyma (pK a = 4.60), which results in an excellent leaving group.23 Oxyma (15) and COMU (16) show superiority to HOBt (1) derivatives and a similar or even higher potency than Results and discussion HOAt (2) analogs in suppressing racemization and assembling demanding peptide models. In terms of safety, 15 and 16 also have The Oxyma-based phosphonium salts PyOxP (17) and PyOxB a considerably lower risk of explosion than benzotriazole-based (18) were successfully synthesized in one-pot fashion from Oxyma reagents and are compatible with microwave irradiation.24,25 (15) (Scheme 1). The first step consisted of the rapid formation of the intermediate potassium salt of the oxime with potassium carbonate (K2CO3) after 10 min of gently stirring. Subsequent addition of PyBroP or the analogous tetrafluoroborate salt to the reaction mixture afforded, after 4 h, the desired phosphonium salts 17 and 18, in 76% yield after recrystallization from DCM–hexane. Previous formation of the potassium salt is preferred over the addition of a tertiary base to Oxyma (15), like Et3N, to generate + - the anion, since this method yields Et3N Br , which is difficult to separate from the desired phosphonium salt. Fig. 3 Oxyma and its derived uronium salt, COMU. The stability of phosphonium salts is a basic feature for their optimal performance in peptide synthesis, since they are predom- In view of these outstanding results, the next step was to inantly used in slow-rate couplings, specially cyclizations, and include Oxyma (15) in a tris(pyrrolidino)-type phosphonium therefore a rapid hydrolysis of the reagent dramatically decreases salt, in both hexafluorophosphate and tetrafluoroborate their efficiency. In order to check and compare the stability of the forms. The synthesis and structure determination of the proposed oxime-based phosphonium salts with their benzotriazole hexafluorophosphate version was accomplished by Hoffmann counterparts, we conducted hydrolysis experiments in an NMR et al. in 2003, although its suitability as a coupling reagent tube (3 days in acetone) and in an open vial (5 days in DMF) 3666 | Org. Biomol. Chem., 2010, 8, 3665–3673 This journal is © The Royal Society of Chemistry 2010 View Online Scheme 1 Table 1 Hydrolytic stability (%) of phosphonium salts during NMR and Table 2 Solubility of phosphonium salts in DMF and DCM HPLC assays DMF DCM Method of analysis Entry Coupling Reagent Wt/g mL-1 Molarity Wt/g mL-1 Molarity Entry Coupling Reagent NMR, 3 days (%)a HPLC, 5 h (%)b 1 PyAOP (12) 1.04 1.99 0.92 1.79 1 PyAOP (12)1029 2 PyBOP (13) 0.80 1.54 0.64 1.23 2 PyBOP (13)2039 3 PyClock (14) 0.80 1.44 0.40 0.72 3 PyClock (14)50 35 4 PyOxP (17) 1.36 2.58 1.04 1.97 4 PyOxP (17)6040 5 PyOxB (18) 1.52 3.24 1.32 2.82 5 PyOxB (18)3025 a The samples were dissolved in acetone-d6 and placed in an NMR tube (closed vial).