A Pyrrolysine Analogue for Protein Click Chemistry

Abstract

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,

Site-specific protein modifications through pyrroline-carboxy-lysine residues

“…This history of evolutionary pressure along with the observation that even the unmodified pyrrolysyl-tRNA synthetase PylS is relatively tolerant of nonpyrrolysine substrates [but generally utilizes them with lower efficiency (35)(36)(37)(38)(39)(40)(41)(42)], makes it an attractive system for the incorporation of unnatural amino acids either using wild-type (35,(38)(39)(40)(41)(42) or mutant pyrrolysyltRNA synthetases (35,39,(43)(44)(45)(46). Pcl mutagenesis differs from the unnatural amino acid technology in that a standard metabolite, D-Orn, is processed by the biosynthesis enzymes of a natural amino acid into a close analog of Pyl.…”

Genetically Encoded Cyclopropene Directs Rapid, Photoclick‐Chemistry‐Mediated Protein Labeling in Mammalian Cells

“…[5] Numerous small organic groups such as ketone, [6] azides, [7] terminal alkynes, [8] and terminal alkenes, [9] as well as larger reactive bioorthogonal groups such as cyclooctyne, [10] norbornene, [11] transcyclooctene, [10b, 11b] tetrazole, [12] and tetrazine [13] have been genetically encoded for site-selective protein labeling in vivo. To track fast protein dynamics in vivo, it is imperative that these genetically encoded bioorthogonal reporters direct fast and selective bioorthogonal labeling with the cognate biophysical probes, preferably with a spatiotemporal control.…”

Biochemical analysis with the expanded genetic lexicon

“…2 (11)(12)(13)(14)(15)(16)(17)) [33, 43,44,[73][74][75][76][77][78]. The azide-alkyne Huisgen cycloaddition ("click" chemistry), in particular, has been widely used to label proteins in vitro and in living cells (Fig.…”