*3.9. Calcitonin Gene-Related Peptide Receptors (Antimigraine Drugs): Enzymatic Deracemization Process*

The (*R*)-amino acid (*R*)-2-amino-3-(7-methyl-1 H-indazol-5-yl)propanoic acid (*R*)-**108**, (Figure 30) is a key intermediate needed for synthesis of antagonists of calcitonin gene-related peptide receptors **109** [135] Such antagonists are potentially useful for the treatment of migraine and other maladies [135,136].

(*R*)-Amino acid 108 was prepared in 68% isolated yield with >99% e.e. from racemic amino acid **110** using (*S*)-amino acid oxidase from *Proteus mirabilis* expressed in *Escherichia coli* in combination with a commercially available (*R*)-transaminase using (*R*)-alanine as amino donor [137]. The (*R*)-enantiomer was also prepared in 79% isolated yield with >99% e.e. from the corresponding keto acid **111** using the (*R*)-transaminase with racemic alanine as the amino donor. The rate and yield of this reaction could be accelerated by addition of lactate dehydrogenase (with NAD<sup>+</sup> , formate and formate dehydrogenase to regenerate NADH) to remove the inhibitory pyruvate produced during the reaction. A (*R*)-transaminase was identified and purified from a soil organism identified as *Bacillus thuringiensis* and cloned and expressed in *Escherichia coli*. The recombinant (*R*)-transaminase was very effective for the preparation of (*R*)-**108** and gave a nearly complete conversion of **111** to (*R*)-**108** without the need for additional enzymes for pyruvate removal [137].

**Figure 30.** Calcitonin gene-related peptide receptors (antimigraine drugs): Enzymatic preparation of (*R*)-2-amino-3-(7-methyl-1*H*-indazol-5-yl)propanoic acid.

*3.10. Corticotropin Releasing Factor (CRF)-1 Receptor Antagonist: Enzymatic Resolution by Transaminase* 

(*R*)-amines synthesis for Anxiety and depression are psychiatric disorders that constitute a major health concern worldwide. While numerous marketed treatments exits for both disorders, there continue to be need agents which may have increased efficacy and/or reduced side-effect profiles [138–140]. CRF-1 receptor antagonists have been proposed as novel pharmacological treatments for depression, anxiety and stress disorders [138–141]. (*R*)-sec-butylamine **112** and (*R*)-1-cyclopropylethylamine **113** (Figure 31) are key chiral intermediates for the synthesis of CRF-1 receptor antagonists such as **114** [141,142].

**Figure 31.** Corticotropin releasing factor (CRF)-1 receptor antagonist: Enzymatic synthesis of (*R*)-1-cyclopropylethylamine and (*R*)-*sec*-butylamine.

We have developed enzymatic resolution process for the preparation of (*R*)-sec-butylamine and (*R*)-1-cyclopropylethylamine [143]. Screening was carried out to identify strains useful for the preparation of (*R*)-1-cyclopropylethylamine and (*R*)-*sec*-butylamine from the racemic amines with an (*S*)-specific transaminase. Several *Bacillus megaterium* strains as well as several soil isolates were found to have the desired activity for the resolution of the racemic amines to give the (*R*)-enantiomers. Using an extract of the best strain, *Bacillus megaterium* SC6394, the reaction was shown to be a transamination requiring pyruvate as amino acceptor and pyridoxal phosphate as a cofactor. Initial batches of both amines were produced using whole cells of *Bacillus megaterium* SC6394. The transaminase was purified to homogeneity to obtain *N*-terminal as well as internal amino acid sequences. The sequences were used to design polymerase chain reaction (PCR) primers to enable cloning and expression of the transaminase in *Escherichia coli* SC16578. In contrast to using *Bacillus megaterium* process, pH control and aeration were not required for the resolution of *sec*-butylamine and an excess of pyruvate was not consumed by the recombinant cells. The resolution of *sec*-butylamine (0.68 M) using whole cells of *Escherichia coli* SC16578 was scaled up to give (*R*)-*sec*-butylamine 1/2 H2SO4 in 46.6% isolated yield with 99.2% *e.e*. An alternative isolation procedure was also used to isolate (*R*)-*sec*-butylamine as the free base. Using the same recombinant (*S*)-tansaminase, (*R*)-1-cyclopropylethylamine was obtained in 42% isolated yield (theoretical max. 50%) and 99% e.e. [143].

#### **4. Conclusions**

The production of single enantiomers of drug intermediates is increasingly important in the pharmaceutical industry. Biocatalysis provides organic chemists an alternate opportunity to prepare pharmaceutically important chiral compounds. The examples presented in this review are only from a few selected articles for synthresis of chiral alcohols and unnatural amino acids. Different types of biocatalytic reactions are capable of generating a wide variety of chiral compounds useful in the development of drugs. The use of hydrolytic enzymes such as lipases, esterases, proteases, dehalogenases, acylases, amidases, nitrilases, epoxide hydrolases, and decarboxylases for the resolution of variety of racemic compounds and in the asymmetric synthesis of enantiomerically enriched chiral compounds. Dehydrogenases and aminotransferases has been successfully used along with cofactors and cofactor regenerating enzymes for the synthesis of chiral alcohols, aminoalcohols, amino acids and amines. Aldolases and decarboxylases have been effectively used in asymmetric synthesis by aldol condensation and acyloin condensation reactions. Monoxygenases have been used in enantioselective and regioselective hydroxylation, epoxidation, sulfoxidation and Baeyer-Villiger reactions. Dioxygenases have been used in the chemo-enzymatic synthesis of chiral diols. Enzymatic deracemization, dynamic resolution and stereoinversion, to achieve >50% yield and high e.e. by combination of chemo- and/or biocatalysts in sequential reactions or by a single biocatalyst. In the course of the last decade, progress in biochemistry, protein chemistry, molecular cloning, random and site-directed mutagenesis, directed evolution of biocatalysts under desired process conditions has opened up unlimited access to a variety of enzymes and microbial cultures as tools in organic synthesis. Future of bicatalysis for synthesis of chiral compounds looks very promising.

#### **Acknowledgments**

The author would like to acknowledge Ronald Hanson, Animesh Goswami, Amit Banerjee, Venkata Nanduri, Jeffrey Howell, Steven Goldeberg, Robert Johnston, Mary-Jo Donovan, Dana Cazzulino, Thomas Tully, Thomas LaPorte, Lawrence Parker, John Wasylyk, Michael Montana, Ronald Eiring, Rapheal Ko, Linda Chu, Clyde McNamee, Michael Montana for research collaboration.

#### **Conflict of Interest**

The authors declare no conflict of interest.

#### **References**


