Protein Prosthesis:  1,5-Disubstituted[1,2,3]triazoles as cis-Peptide Bond Surrogates

Abstract

Proline is unique among the natural amino acids in the similar propensity of its peptide bond to be in the cis or trans conformation. This attribute affects many processes, including the rate at which proteins fold, their structures, and their activities. Other aliphatic amino acids can serve as mimics for proline residues with trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. Here, 1,5-disubstituted-[1,2,3]-triazoles were assessed as cispeptide bond surrogates. Huisgen's 1,3-dipolar cycloaddition reaction of amino alkynes and azido acids and a Ru(II) catalyst were used to synthesize a variety of Xaa-1,5-triazole-Ala modules in moderate-to-high yields. Two of these modules, along with their 1,4-triazole regioisomers, were installed in a turn region of bovine pancreatic ribonuclease by using expressed protein ligation. The resulting semisynthetic enzymes displayed full enzymatic activity, indicating the maintenance of native structure. The 1,5-triazole surrogates instilled conformational stability that was comparable to that of Xaa-cis-Pro segments, whereas the 1,4-triazoles conferred markedly less stability. The stability conferred by both surrogates was independent of the Xaa residue, eliminating an uncertainty in protein design. We conclude that Xaa-1,5-triazole-Ala modules can serve as viable mimics of Xaa-cis-Pro segments. The possibility of synthesizing this surrogate by the ligation of fragments in situ and the emergence of biocompatible catalysts for that process portends its widespread use.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 trans peptide bonds. In contrast, chemical synthesis is needed to create surrogates for cis prolyl peptide bonds. 1,5-7

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

“…Values of k cat /K M for the enzymatic cleavage of the fluorogenic substrate AUAA were determined as described previously [44]. Activity was measured at 20 °C in 100 mM 2-(N-morpholino)ethanesulfonic acid-NaOH buffer (pH 6.0) containing NaCl (100 mM).…”

Stereoelectronic and steric effects in side chains preorganize a protein main chain

“…The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide. Likewise, proteins containing a cis-peptide bond can be stabilized by constrained amides or isosteres (21)(22)(23). The utility of these substitutions is limited, however, by the difficulty of modifying the backbone of proteins as well as concomitant effects on structure and function (24)(25)(26)(27).…”

Protein Engineering with Non-Natural Amino Acids

“…One potential side reaction is oligomerization of the alkyne. RuAAC has been used to ligate pancreatic ribonuclease with a cis-peptide bond surrogate (Tam et al, 2007).…”