Method for the synthesis of tryptophan analogs in aqueous solvents at reduced temperatures

A method for preparing a compound according to Formula I:

• wherein R3 is (A) or (B):
• the method comprising:
• combining i) an indole substrate or azulene substrate;
• a serine substrate;
• and an engineered tryptophan synthase (3-subunit (TrpB) selected from the group consisting of a T.maritima TrpB, a P. furiosis TrpB, an A. fulgidus TrpB, a T. naphthophila TrpB, a T. petrophila TrpB, a T. neapolitana TrpB, a C. subterraneus TrpB, a D. tunisiensi TrpB, a D. kuznetsovii TrpB, a P. mobilis TrpB, an A. aeolicus TrpB, an S. azorense TrpB, a T. pseudethanolicus TrpB, a T. thermophilus TrpB, a P. abyssi TrpB, an M. jannaschii TrpB, a T. kodakarensis TrpB, and an M. aeolicus TrpB in a reaction mixture;
• and maintaining the reaction mixture under conditions sufficient to form the compound according to Formula I;
• wherein:
• the engineered TrpB comprises a pyridoxal phosphate (PLP) binding loop mutation, a helix 1 mutation, a strand 7-8 mutation, or a combination thereof;
• and
• wherein the PLP binding loop mutation is a mutation of a residue corresponding to any one of residues G227, G228, G229, and S230 in a T.maritima TrpB, the helix 1 mutation is a mutation at a position corresponding to E30 in a T.maritima TrpB, and the strand 7-8 mutation is a mutation at a position corresponding to 1184 in a T. maritima TrpB;
• R1 is H or C1-8 alkyl, which is optionally substituted with one or more R1a;
• each Rla is independently selected from the group consisting of halogen, —OH, —CN, —N3,
• —NO2, C1-12 alkyl, C6-14 aryl, C2-12 alkenyl, C1-12 alkynyl, C1-12 alkoxy,
• C1-12 thioalkoxy, —N(R1b)2, —C(O)R1c, —C(O)N(R1b)2, —NR1bC(O)R1c, and
• —OC(O)R1c;
• each R1b is independently selected from the group consisting of H and C1-6 alkyl;
• each R1c is independently selected from the group consisting of H, —OH, halogen,
• C1-6 alkyl, C1-6 alkoxy;
• Y1-Y3 and Z1-Z4 are independently selected from the group consisting of CH, CR2, and N;
• each R2 is independently selected from the group consisting of halogen, —OH, —CN, N3,
• —NO2, C1-12 alkyl, C6-14 aryl, C2-12 alkenyl, C1-12 alkynyl, C1-12 alkoxy,
• C7-26 arylalkyloxy, C1-12 thioalkoxy, —N(R2a)2, —C(0)R2b, —C(0)N(R2a)2,
• —NR2aC(O)R2b, and —OC(O)R2b;
• each R2a is independently selected from the group consisting of H and C1-6 alkyl;
• each R2b is independently selected from the group consisting of H, —OH, halogen,
• C1-6 alkyl, C1-6 alkoxy;
• and subscript n is 0, 1, 2, or 3.