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

Abstract

We report a simple, one‐step enzymatic synthesis of the blue fluorescent noncanonical amino acid β‐(1‐azulenyl)‐l‐alanine (AzAla). By using an engineered tryptophan synthase β‐subunit (TrpB), stereochemically pure AzAla can be synthesized at scale starting from commercially available azulene and l‐serine. Mutation of a universally conserved catalytic glutamate in the active site to glycine has only a modest effect on native activity with indole but abolishes activity on azulene, suggesting that this glutamate activates azulene for nucleophilic attack by stabilization of the aromatic ion.

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

“…Trp analogs are valuable chiral precursors to pharmaceuticals as well as versatile molecular probes, but their chemical synthesis is challenged by stereoselectivity requirements and functional group incompatibility. This has spurred enzyme engineers to evolve TrpB variants capable of producing Trp analogs 20 , 21 , 25 , 26 , but the capabilities of available TrpBs are still limited. Compared to existing engineered TrpBs, our panel of variants has substantially higher activity for the synthesis of Trp and Trp analogs at moderate temperatures from almost all indole analogs tested and also accepts indole analogs, such as 5-CF 3 (Fig.…”

“…All samples except those containing azulene were analyzed at 277 nm, representing the isosbestic point between indole and Trp and allowing estimation of yield by comparing the substrate and product peak areas for indole analogs 21 . Azulene yield was estimated as described previously 25 . Nucleophile retention times were determined though injection of authentic standards and product retention times were identified by extracting their expected mass from the mass spectrum.…”

Tryptophan Synthase: Biocatalyst Extraordinaire

“…We hypothesized that the electron accumulation of the Cpstabilized by the tropylium system could promote nucleophilic attack by azulene on the amino-acrylate to form the ncAA β-(1-azulenyl)-Lalanine (Scheme 7, AzAla, 30). [80] AzAla is a blue Trp isostere whose fluorescent properties have been leveraged for spectroscopic studies, [81][82][83] but whose use was limited due to its difficult, time-sensitive, multi-step synthesis. [84] Figure 1.…”

“…TrpB-catalyzed synthesis of β-(1-azulenyl)-L-alanine (AzAla) (30) from azulene (29) and serine. [80] Azulene was tested against a diverse panel of engineered TrpB variants, and nearly every enzyme demonstrated significant activity for the reaction, the exception being variants bearing a glycine mutation at the highly conserved catalytic glutamate (E104G, PfTrpB numbering). We reasoned that the catalytic glutamate may be important for stabilizing the tropylium cation to facilitate the nucleophilic attack from Cp- (Figure 2).…”

“…Although azulene has no heteroatoms, it experiences a permanent dipole readily apparent in its resonance structure, which is a cycloheptatrienyl cation (tropylium) fused to a cyclopentadienyl anion (Cp − ). We hypothesized that the electron accumulation of the Cp − stabilized by the tropylium system could promote nucleophilic attack by azulene on the amino‐acrylate to form the ncAA β‐(1‐azulenyl)‐ l ‐alanine (Scheme 7, AzAla, 30 ) [80] . AzAla is a blue Trp isostere whose fluorescent properties have been leveraged for spectroscopic studies, [81–83] but whose use was limited due to its difficult, time‐sensitive, multistep synthesis [84] …”