Incorporation of Amino Acids with Long-Chain Terminal Olefins into Proteins

Abstract

Abstract:The increasing need for site-specific protein decorations that mimic natural posttranslational modifications requires access to a variety of noncanonical amino acids with moieties enabling bioorthogonal conjugation chemistry. Here we present the incorporation of long-chain olefinic amino acids into model proteins with rational variants of pyrrolysyl-tRNA synthetase (PylRS). N ε -heptenoyl lysine was incorporated for the first time using the known promiscuous variant PylRS(Y306A/Y384F), and N ε -pentenoyl lysine was incorporated in significant yields with the novel variant PylRS(C348A/Y384F). This is the only example of rational modification at position C348 to enlarge the enzyme's binding pocket. Furthermore, we demonstrate the feasibility of our chosen amino acids in the thiol-ene conjugation reaction with a thiolated polysaccharide.

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

“…Hydrogels polymerized by the thiol‐ene reaction are of special interest for the encapsulation and delivery of proteins for therapeutic purposes . We chose cysteine‐free superfolder GFP (cfsfGFP) with Sac in place of Arg at position 2 as model protein for the thiol‐ene reaction with the thiol‐containing polymer . In this system, robust fluorescence serves as read‐out for successful bioconjugation.…”

“…ChemBioChem 2017, 18,85-90 www.chembiochem.org tein for the thiol-ene reaction with the thiol-containing polymer. [38,40] In this system, robust fluorescences erves as read-out for successful bioconjugation. After UV-illumination to start the thiol-ener eaction and extensive washing, green fluorescence was retained in the hydrogel (Figures 4A and S10), thus indicating as uccessful reaction (when using non-Sac-containing cfsfGFP,nof luorescencewas observed).…”

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

“…When compared with currently available bio-conjugation methods [47,48] (i.e., copper-catalysed azide-alkyne cycloaddition, copper-free reaction, photo-click reaction) [32,49,50,51], the thiol-ene conjugation reaction (also known as “thiol–ene click chemistry”) [52] might be a reasonable alternative. For example, Exner et al [53] incorporated long chain olefinic amino acids in target GFP and lipase proteins via stop codon suppression and subsequently performed thiol-ene protein conjugation with the anomeric tetrasaccharide. Recently, Met analogue l -homoallylglycine has been incorporated into silk Fibroin, which brought further possible chemistry such as thiol-ene or olefin metathesis to target proteins [54].…”

One‐Pot Synthesis of Unprotected Anomeric Glycosyl Thiols in Water for Glycan Ligation Reactions with Highly Functionalized Sugars

“…Forthe direct attachment of the 1-thio-hexa-hyaluronane 30 (HA-6-SH) to ap rotein, the engineered thermostable lipase TTL from Thermoanaerobacter thermohydrosulfuricus, which contains an N-pentenoyl lysine residue in position 221, was used. [21] Then on-canonical amino acid N-pentenoyl lysine was incorporated into the protein using stop-codon suppression. Upon applying thiol-ene conditions,q uantitative incorporation of the hyaluronan hexasaccharide was accomplished, as indicated by HPLC-MS of the protein (Scheme 3).…”