Tuning Pig Liver Esterase Enantioselectivity for the Synthesis of Unnatural Serine and Tyrosine Analogues

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Chemistry and Biochemistry

First Advisor

Douglas S. Masterson

Advisor Department

Chemistry and Biochemistry


The synthesis of disubstituted and homologated unnatural amino acids and the development of a mass spectrometry based assay for enantioselectivity are presented here. Disubstituted and homologated unnatural amino acids have proven to be effective for the treatment of diseases and for use as imaging agents. Peptides that contain unnatural amino acids have also proven to be effective treatments for various disease states including cancer. The potential to evaluate such unnatural amino acids for pharmaceutical properties is limited by the available syntheses. No method currently exists that will allow for the synthesis of homochirally similar unnatural amino acids from a common intermediate. Herein is described a method that will allow for the synthesis of both enantiomers of disubstituted and homologated serine and tyrosine analogues from a common malonate half ester intermediates via several well-known functional group transformations and protecting group chemistry. The described synthetic methodology has resulted in the synthesis of both enantiomers of α-methyl-, β 2,2 -, and β3,3 -serine and both enantiomers of α-methyl-, β 2,2 -, β3,3 -, and γ4,4 - tyrosine analogues and one enantiomer of a γ2,2 - tyrosine analogue. Synthesis of the aforementioned unnatural amino acids requires a chiral malonate half ester. To obtain this half ester intermediate, a malonate diester is enantioselectively hydrolyzed by Pig Liver Esterase (PLE) to give an excess of one enantiomer of a malonate half ester over the other. The enantiomeric composition of the unnatural amino acids is dependent on the enantioselectivity of the enzyme. An increase in the enantioselectivity of the enzyme is needed if less than 97% enantiomeric excess ( ee ) is obtained from the enzymatic hydrolysis. This process can consume a tremendous amount of time and materials. This dissertation also describes the development of a high throughput electrospray-mass spectrometry based assay for determining the enantioselectivity of PLE for malonate diesters. The mass spectrometry assay has been utilized to evaluate the hydrolysis of three malonate diesters under a variety of cosolvent, buffer, and pH conditions. The PLE hydrolysis of a diester that was used to synthesize unnatural serine analogues was improved from 70% to greater than 97% ee . The hydrolysis of another diester that was used to synthesize unnatural tyrosine analogues was improved from 63% to 85% ee . This assay has proven to be a powerful tool for the enhancement of the enantiomeric composition of malonate half esters that can be used for the synthesis of disubstituted and homologated unnatural amino acids.