Engineered enzymes and method for the synthesis of diverse tyrosine analogs

A method for preparing a tyrosine or a tyrosine analog or a salt thereof, the method comprising:

• combining in a reaction mixture:
• (i) a first substrate;
• (ii) a second substrate;
• and (iii) an engineered tryptophan synthase β-subunit (TrpB) comprising an amino acid substitution at a position corresponding to amino acid residue E105 of SEQ ID NO:
• 1;
• and
• maintaining the reaction mixture under conditions sufficient to form the tyrosine or a tyrosine analog or a salt thereof.

A method of preparing a compound of Formula I, II, or III:

• or a salt thereof,
• wherein R is (A), (B), or (C):
• the method comprising:
• combining in a reaction mixture:
• (i) a 3-hydroxy amino acid, (ii) phenol or a phenol analog, and (iii) an engineered tryptophan synthase α-subunit (TrpB) comprising an amino acid subtitution at a position corresponding to amino acids residue E105 of SEQ ID NO:
• 1;
• and
• maintaining the reaction mixture under conditions sufficient to form the compound according to Formula I, II, or III;
• wherein:
• R1 is hydrogen or C1-8 alkyl, which is optionally substituted with one or more R1a;
• each R1a is independently selected from the group consisting of hydrogen, 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 hydrogen and C1-6 alkyl;
• each R1c is independently selected from the group consisting of hydrogen, —OH, halogen, C1-6 alkyl, C1-6 alkoxy;
• R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, halogen, C1-8 alkyl, which is optionally substituted with one or more R2a;
• each R2a is independently selected from the group consisting of hydrogen, halogen, —OH, —CN, —N3, —NO2, C1-12 alkyl, C2-12 alkenyl, C1-12 alkynyl, C1-12 alkoxy, C1-12 thioalkoxy, —N(R2b)2, —C(O)R2c, —C(O)N(R2b)2, —NR2bC(O)R2c, and —OC(O)R2c;
• each R2b is independently selected from the group consisting of hydrogen and C1-6 alkyl;
• each R2c is independently selected from the group consisting of hydrogen, —OH, halogen, C1-6 alkyl, C1-6 alkoxy;
• R6 is hydrogen or C1-8 alkyl, which is optionally substituted with one or more R3a;
• each R3a is independently selected from the group consisting of hydrogen, halogen, —OH, —CN, —N3, —NO2, C1-12 alkyl, C2-12 alkenyl, C1-12 alkynyl, C1-12 alkoxy, C1-12 thioalkoxy,
• —N(R3b)2, —C(O)R3c, —C(O)N(R3b)2, —NR3bC(O)R3c, and —OC(O)R3c;
• each R3b is independently selected from the group consisting of hydrogen and C1-6 alkyl;
• each R3c is independently selected from the group consisting of hydrogen, —OH, halogen, C1-6 alkyl, C1-6 alkoxy;
• n is selected from 0, 1, 2, 3, 4, or 5;
• m is selected from 0, 1, 2, 3, 4, 5, or 6;
• X is —C(R7) or —N;
• wherein R7 is hydrogen or C1-8 alkyl, which is optionally substituted with one or more R4a;
• each R4a is independently selected from the group consisting of hydrogen, halogen, —OH, —CN, —N3, —NO2, C1-12 alkyl, C2-12 alkenyl, C1-12 alkynyl, C1-12 alkoxy, C1-12 thioalkoxy, —N(R4b)2, —C(O)R4c, —C(O)N(R4b)2, —NR4bC(O)R4c, and —OC(O)R4c;
• each R4b is independently selected from the group consisting of hydrogen and C1-6 alkyl;
• and
• each R4c is independently selected from the group consisting of hydrogen, —OH, halogen, C1-6 alkyl, C1-6 alkoxy.