Protein prosthesis: 1,5-disubstituted[1,2,3]triazoles as cis-peptide bond surrogates.

Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation.1 This attribute affects many processes, including the rate at which proteins fold,2 their structures,3 and their activities.4 Other aliphatic amino acids can serve as mimics for proline residues with transpeptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis-prolyl peptide bonds.1,5-7 An effective surrogate for a cis-peptide bond must meet certain criteria. The surrogate must be similar in size to proline, so as not to disturb secondary and tertiary interactions within the folded protein. The surrogate must be able to accommodate adjacent residues, which could be important in protein structure and function. Finally, it is desirable if the surrogate is accessible via a facile synthetic route and amenable to solid-phase peptide synthesis. We reasoned that 1,5-disubstituted[1,2,3]triazoles could meet all of these criteria. In particular, we noted that an Xaa-1,5-triazoleAla module is a remarkable isostere of an Xaa-cis-Pro dipeptide, retaining its stereochemistry and number of non-hydrogen atoms and maintaining similar hybridization (Scheme 1). A regioisomer, 1,4-disubstituted triazoles, had been incorporated into small peptides,7-9 and a simple 1,5-disubstituted triazole induced peptoid oligomers to adopt a turn.10 The synthesis and use of 1,5disubstituted triazoles as cis-peptide bond mimics are, however, unknown. Likewise, neither 1,5nor 1,4-disubstituted triazoles has been incorporated into a protein. Herein, we report on the attainment of these goals. To synthesize the triazole, we employed Huisgen’s 1,3-dipolar cycloaddition reaction.11 Catalysis of this reaction by Cu(I) is known to yield exclusively a 1,4-disubstituted triazole,12 but we required the 1,5-disubstituted regioisomer. There are reports of regioselective formation of 1,5-disubstituted triazoles being mediated by metals13 or stereoelectronic effects,14 but only under harsh conditions. Recently, Ru(II) catalysts had been shown to yield exclusively the 1,5-disubstituted triazole in a mild reaction,15 though this methodology had never been applied to substrates containing amino acids. As a model protein, we chose bovine pancreatic ribonuclease (RNase A; 124 residues), which has been the object of much seminal work in protein chemistry.16 In the native protein, residues Gly112-Asn113-Pro114-Tyr115 form a Type VIb reverse turn (Scheme 1), in which the Asn113-Pro114 peptide is in the cis conformation. We targeted that dipeptide segment for replacement with 1,5-triazole-Ala surrogates. The chemoselectivity of the 1,3-dipolar cycloaddition enables a convergent synthetic route to the requisite triazole (Table 1). The synthesis of the azido acids was achieved by Cu(II)-catalyzed diazo transfer of commercially available amino acids,17 followed by protection of the carboxyl functionality with a t-butyl or benzyl group to give 1 or 2, respectively. The route to the amino alkynes was also general, starting from the Weinreb amide of an amino acid, and followed by reduction to the aldehyde and conversion to the alkyne with the Bestmann-Ohira reagent18 to give 3-8.19 The scope of the Ru(II)-mediated cycloaddition with these R-amino acid derivatives is shown in Table 1. The cycloadditions were effected by the catalyst Cp*RuCl(COD), which had been reported to be effective for the cycloaddition of secondary azides.20 For substrates with a diverse set of proteinogenic functionalities, the reaction afforded the desired regioisomers 9-15 under mild conditions and in moderate-to-high yield. No 1,4-disubstituted regioisomers were detected by the comparison of NMR spectra to those of analogous 1,4-disubstituted triazoles synthesized by Cu(I)-mediated cycloaddition.12 Toluene was used as the solvent, unless the substrate was found to be insoluble (entries 2 and 3). Benzyl protection of the carboxyl group of the azido substrate was necessary for orthogonal deprotection relative to the acid-sensitive † Department of Chemistry, University of WisconsinsMadison. ‡ Martin-Luther University. § Current address: Department of Chemistry, University of California, Berkeley. || Department of Biochemistry, University of WisconsinsMadison. Scheme 1