The art of stitching proteins: D‐Cys‐ε‐Lys and its diastereomer L‐Cys‐ε‐Lys read through the UAG codon (see scheme). As the resulting proteins can participate in native chemical ligation (NCL), this process provides a means to prepare proteins chemoselectively modified (e.g. ubiquitinated) through a peptidic side chain located at the ε position of a rationally selected lysine residue.

A short synthetic approach toward a novel class of chiral nucleophilic catalysts, the dissymmetry of which stems from restricted rotation about an Ar-Ar bond, has been developed. The key steps of the synthesis include preparation of a nucleophilic 1-methyl-2-pyrrolino[3,2-c]pyridine core 16 by ortho-lithiation and creation of the biaryl axes via Suzuki cross-coupling reactions. Comparative HPLC studies of racemization for configurationally labile biaryls 31, 38, and 43 containing 1-methyl-2-pyrrolino[3,2-c]pyridine, 4-(dimethylamino)pyridine, and 4-(1-pyrrolidino)pyridine cores, respectively, have demonstrated that a pyrrolidino substituent ortho to the biaryl axis is optimal for slowing Ar-Ar rotation. Biaryls containing all three cores have been shown to retain DMAP-like catalytic activity in the acylation of a hindered alcohol. Biaryls 55 and 56, which are configurationally stable at ambient temperature, have also been prepared via modification of configurationally labile derivatives. Compounds 55 and 56 in optically pure form should provide a useful starting point for studies on catalytic asymmetric acyl transfer using atropisomeric analogues of DMAP.

A concise seven-step synthesis of atropisomeric 3-aryl analogues of DMAP from 4-pyridone 8 has been developed. A representative compound of this class, biaryl (+/-)-15, has been resolved using CSP HPLC and shown to be an efficient nucleophilic catalyst for kinetic resolution of a series of secondary alcohols on both an analytical and preparative scale (stereoselectivity factors, s = 8.9-29).

The discovery of pyrrolysine (1, Figure 1), the 22nd genetically encoded amino acid, [1,2] and its subsequent incorporation into recombinant proteins in E. coli [3] have laid the foundation for the future development of novel biotechnologies and tactics in protein research. In principle, incorporation of the genes encoding the pyrrolysine tRNA synthase (PylS) and its cognate tRNA (PylT) could enable both bacteria [4,5] and eukaryotes [6] to use the UAG codon for the production of proteins containing pyrrolysine (1) and its analogues. Herein, we describe the synthesis of a new pyrrolysine congener that can be incorporated into recombinant protein and selectively modified with azide-containing dyes by copper(I) click chemistry. We demonstrate the usefulness of this technology for monitoring conformational changes of calmodulin (CaM) by Förster resonance energy transfer (FRET) measurements.To harness the unique UAG-codon-pyrrolysine system for biochemical applications, we decided to search for functionalized pyrrolysine surrogates that would be both more stable and amenable for further post-translational modification (i.e., tagging). Our primary focus centered on derivatives of the THF-containing amino acid 2 because of the steric and electronic similarity between the THF ring and the 3,4-dihydro-2H-pyrrole ring present in 1. Recently, in collaboration with the Krzycki group, we demonstrated that Methanosarcina barkeri PylS could charge PylT with the parent pyrrolysine analogue 2.[7] We subsequently postulated that this framework could be used as a starting point for the design of other functionalizable pyrrolysine analogues.Herein, we report on the synthesis of the pyrrolysine analogue 3, bearing a terminal alkyne functionality, that not only reads through the UAG codon in E. coli overexpressing the Methanosarcina mazei PylS and PylT, but also allows for site-specific modification of the resulting protein through the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.[8] The taggable pyrrolysine analogue 3 was prepared from the known tetrahydrofuran 4[9] by a seven-step sequence (Scheme 1). Thus, desilylation of 4 with TBAF/AcOH in THF [10] or AcCl in MeOH [11] gave alcohol 5 in 76 % or 89 % yield, respectively. Its subsequent oxidation with H 5 IO 6 in the presence of a catalytic amount of CrO 3[12] provided acid 6 that, in turn, was coupled (BOP/NMM) with Boc-Lys-OtBu to give amide 7 in 68 % yield over the two steps. NMO/OsO 4 -mediated dihydroxylation of 7 furnished an approximately 1:1 mixture of the corresponding diastereomeric diols 8 (93 %) that were oxidatively cleaved with NaIO 4 to give aldehyde 9 in 94 % yield. Subsequent Ohira-Bestmann alkynylation [13,14] with diazoketophosphonate 10 [15] in the presence of K 2 CO 3 led to the protected target 11 (70 %). Its final treatment with neat TFA cleanly freed the desired pyrrolysine analogue 3.To test if 3 could be recognized by a pyrrolysyl-tRNA synthetase and incorporated into recombinant protein in vivo,

Expedient five-step syntheses of a cyclic bis(benzimidazole)-based amide 5 and two sterically more hindered analogues 23-24 have been developed. These amides are chiral due to the inherent ruffling of the macrocyclic plane. Racemization of the optical antipodes of these compounds has been studied using dynamic chiral stationary phase HPLC. These studies reveal that, while the parent amide 5 racemizes rapidly, for the sterically more hindered amides 23-24, the rate of racemization is significantly reduced. Bis(benzimidazole)-based amides 5 and 23-24 form stable Ni(II) complexes 25-27, respectively. Like their parent ligands, complexes 25-27 are chiral due to their highly ruffled geometry. Studies of these complexes by chiral stationary phase HPLC reveal that metalation leads to a much lower rate of racemization. Incorporation of a strap can slow racemization even further. A series of strapped cyclic amides 54-57, along with their corresponding dimers 58-61, have been prepared. The rate of racemization for amides 54-57 is strongly dependent on the length of the strap. X-ray single-crystal structure analysis of the Ni(II) complex of strapped amide 54 reveals that the bis(benzimidazole) core retains its highly ruffled shape, with the two phenyl rings of the macrocycle located anti to the strap. Chiral separation of strapped ligands 54-57 and their corresponding Ni(II) complexes is shown to be facile by chiral stationary phase HPLC.