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Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima

Boville, Christina E. and Romney, David K. and Almhjell, Patrick J. and Sieben, Michaela and Arnold, Frances H. (2018) Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima. Journal of Organic Chemistry, 83 (14). pp. 7447-7452. ISSN 0022-3263. PMCID PMC6054569.

[img] PDF (Results of site-saturation mutagenesis libraries; LCMS calibration curves for Chart 1; HPLC data for Figure 2, Chart 1, and indole; NMR spectrum of 4-cyanotryptophan from Scheme 2) - Supplemental Material
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The use of enzymes has become increasingly widespread in synthesis as chemists strive to reduce their reliance on organic solvents in favor of more environmentally benign aqueous media. With this in mind, we previously endeavored to engineer the tryptophan synthase β-subunit (TrpB) for production of noncanonical amino acids that had previously been synthesized through multistep routes involving water-sensitive reagents. This enzymatic platform proved effective for the synthesis of analogues of the amino acid tryptophan (Trp), which are frequently used in pharmaceutical synthesis as well as chemical biology. However, certain valuable compounds, such as the blue fluorescent amino acid 4-cyanotryptophan (4-CN-Trp), could only be made in low yield, even at elevated temperature (75 °C). Here, we describe the engineering of TrpB from Thermotoga maritima that improved synthesis of 4-CN-Trp from 24% to 78% yield. Remarkably, although the final enzyme maintains high thermostability (T50 = 93 °C), its temperature profile is shifted such that high reactivity is observed at ∼37 °C (76% yield), creating the possibility for in vivo 4-CN-Trp production. The improvements are not specific to 4-CN-Trp; a boost in activity at lower temperature is also demonstrated for other Trp analogues.

Item Type:Article
Related URLs:
URLURL TypeDescription Information CentralArticle
Boville, Christina E.0000-0002-2577-9343
Romney, David K.0000-0003-0498-7597
Almhjell, Patrick J.0000-0003-0977-841X
Sieben, Michaela0000-0002-4412-7148
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2018 American Chemical Society. Received: February 25, 2018; Published: April 13, 2018; Publication Date (Web): April 13, 2018. This article is part of the Organic and Biocompatible Transformations in Aqueous Media special issue. Author Contributions: C.E.B. and D.K.R. contributed equally to this work. The authors declare the following competing financial interest(s): The contents of this paper are the subject of a patent application submitted by Caltech, and some authors are entitled to a royalty on revenues arising from that patent. This work was funded by the Jacobs Institute for Molecular Engineering for Medicine (JIMEM) and the Rothenberg Innovation Initiative (RI2) at Caltech. C.E.B. was supported by a postdoctoral fellowship from the Resnick Sustainability Institute, D.K.R. was supported by a Ruth Kirschstein NIH Postdoctoral Fellowship (F32GM117635), and M.S. was supported by a postdoctoral fellowship from the German Academic Exchange Service (DAAD).
Group:Resnick Sustainability Institute
Funding AgencyGrant Number
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Rothenberg Innovation Initiative (RI2)UNSPECIFIED
Resnick Sustainability InstituteUNSPECIFIED
NIH Postdoctoral FellowshipF32GM117635
Deutscher Akademischer Austauschdienst (DAAD)UNSPECIFIED
PubMed Central ID:PMC6054569
Record Number:CaltechAUTHORS:20180808-152215835
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Official Citation:Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima. Christina E. Boville, David K. Romney, Patrick J. Almhjell, Michaela Sieben, and Frances H. Arnold. The Journal of Organic Chemistry 2018 83 (14), 7447-7452 DOI: 10.1021/acs.joc.8b00517
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:88673
Deposited By: Tony Diaz
Deposited On:09 Aug 2018 14:50
Last Modified:20 Aug 2018 17:14

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