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

Abstract

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 ( T = 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.

Facile in Vitro Biocatalytic Production of Diverse Tryptamines

“…Many substituted indoles and even other amino acids can participate in this versatile reaction, which affords access to complex starting materials for downstream modification . Recently, these efforts have been made even easier by evolving the β‐subunit of tryptophan synthase (TrpB; EC 4.2.1.20) for efficient stand‐alone function . These engineered TrpBs can operate at high temperatures, which is helpful in solubilizing hydrophobic indoles, and provide access to highly complex Trp analogues in good yield.…”

“…To test the promiscuity of Rgn TDC, we sought to measure its kinetic parameters with a complete set of 4‐, 5‐, 6‐, and 7‐chlorotryptophans; however, the requisite enantiopure chlorotryptophans were not readily available. We therefore prepared each Trp analogue from its indole precursor through a simple biocatalytic reaction by using engineered TrpBs from Pyrococcus furiosus with high stand‐alone activity . Surprisingly, the kinetic parameters of Rgn TDC with each chlorotryptophan showed only modest decreases in catalytic efficiency when compared to the native Trp substrate (Table , Figure S3).…”

“…We first tested the enzymes in a cascade reaction at 37 °C, which resulted in full conversion of indole to tryptamine (5000 turnovers, Figure S6), thus demonstrating that the two enzymes can operate efficiently under the same conditions. Although TrpB enzymes have recently been engineered for higher activity at lower temperature, the solubility of substituted indoles drops precipitously at these temperatures, thereby hindering preparative‐scale reactions. Therefore, we adopted a sequential one‐pot, two‐enzyme reaction setup.…”

Direct Enzymatic Synthesis of a Deep‐Blue Fluorescent Noncanonical Amino Acid from Azulene and Serine

“…The significant effect of the E104(105)G mutation suggested that this conserved catalytic residue might be playing an important role in the non‐native azulene reaction. We explored this possibility by examining two engineered variants with and without the E104(105)G mutation: Pf 5G8, which exhibits optimal activity at 75 °C, and Tm 9D8*, which exhibits optimal activity at lower temperatures such as 37 °C . Challenging the enzymes with indole demonstrated that this mutation only modestly decreases the rate of Trp formation (Figure ), with an additional slight decrease in the chemoselectivity of the reaction that leads to the formation of trace amounts of isoTrp (a product of the N‐alkylation of indole, shown in Figure S2 and described previously).…”

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

“…2 We evolved this enzyme to accept a variety of indole analogues and nitroalkanes with activated sp³-carbon atoms as nucleophiles ( Figure 1b). [3][4][5][6][7][8] However, these studies revealed limitations including chemo-and stereoselective control we would need to address in order to realize TrpB's broad potential as a biocatalyst for C-C bond formation. Previous biocatalytic reactions catalyzed by variants of TrpB [5][6]8 and proposed stereo-and chemo-selective reaction using engineered TrpB.…”