Room temperature ionic liquids (RTIL’s) are convenient solvents for peptide synthesis !

The chemical peptide coupling with modern coupling agents is efficient in ionic liquids. The reaction rate is fast enough, and the method offers some interests in the case of hindered amino acids, which are not easy to couple under standard conditions. Highly pure crude peptides are obtained in most cases compared to the corresponding coupling in classical solvents. Di-, tetra-, octa-and cyclo-peptides are conveniently synthesized in good yields, and various cheap coupling agents may be used


Introduction
Some years ago, we embarked on the study of alternative methods for peptide coupling, considering that the "classical" methods still remain unsatisfactory in terms of atom economy, convergence and cost. 1 One of these methods relies on the oxaziridine-amide rearrangement, as we were inspired by this interesting and powerful reaction previously studied by Lattes and coworkers. 2,3Indeed, we supposed that this rearrangement would also occur starting from αamino imines, thus leading to peptides after reaction (Scheme 1).
5][6] Later, a variant led us to propose a mild method for the formylation of amino acids. 7Although not general, this method avoids the necessity to use any coupling agent and takes advantage of the reactivity of amino aldehydes to give easy access to the coupling of hindered amino acids. 8,9Inspired by this first set of results, we then examined other alternative methods, such as the in situ ring contraction of heterocyclic enamines yielding peptides of quaternary prolines, 10 and copper (I) mediated formation of peptidic guanidines starting from amidines. 11ore recently, our interest for organic chemistry and asymmetric synthesis in ionic liquids [12][13][14][15][16][17] prompted us to examine these new media in the context of peptide synthesis. 18s well established today, the unique properties of room temperature ionic liquids (RTIL's) present many advantages in the context of green chemistry. 19At the beginning of our study, and rather curiously with regard to the importance of this topic, no report described chemical peptide synthesis in ionic liquids; only a study from Erbeldinger was devoted to enzymatic access to (Z)-aspartame. 202][23] We decided to embark on this approach by considering "modern" coupling agents such as HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and BOP ((Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate) as reagents of choice for this chemistry in ionic liquids, since they present structural similarities with ionic solvents, such as [bmim][PF 6 ] (Scheme 2).Thus we expected these coupling agents to be easily dissolved in ionic liquids, sufficiently stabilized by the solvent, to give slow and selective reactions (for the mechanism, vide infra).The studied amino acids are depicted in Scheme 3: all are commercially available except for MPG and MPBrG which were prepared according to a literature procedure. 25The usual Z and Boc protecting groups were employed for the protection of the amine functions.The carboxylic function of the second amino acid was protected as methyl ester (Scheme 4).The main results for different coupling reactions are given in Table 1, along with a comparison with analogous reactions carried out in classical solvents (dichloromethane or THF).The simplified mechanism for such coupling reactions is presented in Scheme 5. [26][27][28] Scheme 5. Simplified mechanism for peptide coupling using HATU as coupling reagent.We first verified that experiments without any coupling agent, or using DCC, led to no conversion, and selected HATU as coupling reagent (anyway, BOP gave very similar results 18 ).
After the optimization of the reaction conditions and the extraction procedure 18 we were delighted to obtain very good yields in most cases, which compared favorably with that of peptide synthesis in « classical » solvents such as dichloromethane and THF.The main advantage was the higher purity of the crude product observed when the reaction was performed in an ionic liquid with respect to dichloromethane or THF.A more selective chemical pathway, due to the stabilization effect of the ionic liquid on both the coupling reagent and the charged intermediates, could explain this behavior (see Scheme 5).Also, we think that by-products resulting from the reaction of HATU, like tetramethylurea and HOAt, are not extracted and stay in the ionic phase (vide infra).
Moderate yields were observed for the coupling of two quaternary amino acids, but the efficiency was rather similar to molecular solvents.Interested by further studies in chiral recognition by means of hindered peptides, 29,30 we succeeded in obtaining crystals of ZGly-(R)-MPGOMe which were suitable for X-ray analysis (slow crystallization from acetonitrile/water solution).The corresponding ORTEP is presented in Figure 1.In the crystaline state, this compound presents an intramolecular hydrogen bond that stabilizes the structure via a five-membered pseudo cycle.This behavior is rather common in small peptides including quaternary aminoacids. 31

ARKAT
Having a method allowing easy access to various dipeptides (including examples with one or two hindered amino acids) in hands, we turned to the extension of the method to tetra-, octaand cyclopeptides (Scheme 6).Scheme 6. Synthesis of tetra-, octa-and cyclopeptides in [bmim][PF 6 ] (65° C, 4d, DIEA), HATU as coupling agent, except for cyclization carried out with BOP.
Interestingly, results for such couplings compared quite well with those observed in molecular solvents.For example, tetrapeptides were usually formed with ca 80% yield and octapeptide with ca 70% when synthesized in THF.Cyclooctapeptide was obtained in the 80-90% range depending on the concentration and conditions of the cyclization reaction. 29,30s mentioned before, one interesting aspect of this method is the high purity of the obtained crude peptides.This was assumed to be due, at least in part, to the ability of ionic solvents to retain the residues of the activation agent in solution.In addition to the benefit for the purification procedure, this could open up the possibility to recycle the activation agent.To this purpose, we decided to examine a series of those reagents to compare both efficiency and reaction rate in [bmim][PF 6 ].We choose the synthesis of dipeptide ZGly-MPBrGOMe as model reaction for this study.Typical results are shown in Table 2.
As previously reported, 18 DCC failed to give any useful coupling but yielded mainly a crude mixture in which many impurities could be detected (not identified).On the other hand, HATU and BOP seem to be excellent reagents in these conditions, according to our working hypothesis (vide supra).In the fifth entry of table 2, the use of chloro-N,N,N',N'tetramethylformamidinium hexafluorophosphate as coupling agent is reported, which is the precursor of HATU and much less expensive.Interestingly, a fairly good coupling yield was observed with this agent.

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In the same series of experiments, we examined the reaction rate at the beginning of the coupling reaction.For this purpose, aliquots of the reaction mixture (2 drops) were quenched at various times with a water/acetonitrile/TFA mixture (55/45/0.1)and analyzed by HPLC (for chromatography conditions, see experimental part).This study showed a rapid coupling when using charged reagents, giving a conversion up to 90 % in 1.5h.Indeed, the efficiency (in terms of conversion rate) decreased in the order CMPI>BOP>HATU.It is noteworthy that this kinetic behavior is not common: indeed, one could expect the opposite order of reactivity when constructing the peptide bond in classical solvents.Quite

Experimental Section
General Procedures.The NMR spectra were recorded on a Brüker AVANCE 300 spectrometer ( 1 H at 300 MHz and 13 C at 75 MHz) in deuterochloroform or deuteromethanol, the chemical shifts are quoted in ppm in δ-values and the coupling constants are quoted in Hz.Optical rotations were measured with a Perkin Elmer polarimeter, with a 1 dm length cell.HPLC were performed on a HP series 1100 apparatus (integrator HP 3395, column Capital HPLC LTD, C18-KL5-25091, 25 cm x 4.6 mm, 1 mL.min -1 , 20 µL, λ 215 nm).Preparation of dipeptides.General procedure.The N-protected amino acid (25mg), the amino ester hydrochloride (1.1 eq.) and the coupling reagent (HATU or BOP) (1.1 eq.) were introduced in a 5 mL flask under nitrogen.0.5 mL of [bmim][PF 6 ] was then added at room temperature.The reaction mixture was mixed and cooled at 0° C. Diisopropylethylamine (3.3 eq.) was then added dropwise.After stirring for 3 days at 65° C, the mixture was washed successively with NaHCO 3 (1M), citric acid (5%), and water.The dipeptide was extracted from the ionic phase with diethylether or toluene, using a continuous liquid-liquid extractor (24h).Spectral data for Boc-Phe-Gly-OMe, 32 Boc-Phe-Phe-OMe 33 and Z-Gly-Gly-OMe 34 where found identical to literature values.