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

1
Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Str. 10, D-10623 Berlin, Germany
2
Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, D-12489 Berlin, Germany
3
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Campus Berlin-Buch, Robert-Roessle-Str. 10, D-13125 Berlin, Germany
4
Chair of Chemical Synthetic Biology, Department of Chemistry, University of Manitoba, 144 Dysart Rd, Winnipeg, MB R3T 2N2, Canada
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(9), 2299; https://doi.org/10.3390/ijms20092299
Received: 18 April 2019 / Revised: 5 May 2019 / Accepted: 7 May 2019 / Published: 9 May 2019
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Abstract

In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins—a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings. View Full-Text
Keywords: biorthogonal conjugations; deallylation/deprotection; direct sulfhydration/transsulfuration pathway; homocysteine; methionine metabolism; non-canonical amino acids; O-acetyl-homoserine; reprogrammed translation; S-allyl-homocysteine; selective labelling biorthogonal conjugations; deallylation/deprotection; direct sulfhydration/transsulfuration pathway; homocysteine; methionine metabolism; non-canonical amino acids; O-acetyl-homoserine; reprogrammed translation; S-allyl-homocysteine; selective labelling
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Nojoumi, S.; Ma, Y.; Schwagerus, S.; Hackenberger, C.P.R.; Budisa, N. In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation. Int. J. Mol. Sci. 2019, 20, 2299.

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