Enzymes are increasingly applied to organic synthesis because of their high enantioselectivity, chemoselectivity, and environmental friendliness. Hydrolytic enzymes are the largest class used for biotransformations, and subtilisin is one of the commonly used serine proteases for organic synthesis. We hypothesized that we could expand the usefulness of subtilisin to hydrolyze unnatural substrates. The X-ray crystal structure of subtilisin shows that it has a large open active site. Previous workers focused on hydrolysis of natural substrates and close analogs, but the open active site suggests that subtilisin should accept bulky substrates that do not resemble peptides. / The Diels-Alder reaction is important in organic synthesis because it has the potential to create several stereocenters in a single step. We resolved a bulky secondary alcohol ester of a spiro chiral auxiliary for the Diels-Alder reaction on a gram scale using subtilisin Carlsberg. The reaction proceeded with high enantioselectivity and yield. Cholesterol esterase showed high enantioselectivity but with the opposite enantiopreference, consistent with mirror image arrangement for the active sites of subtilisins and lipases/esterases. We also used molecular modeling to identify the molecular basis of enantioselectivity of subtilisin Carlsberg toward this secondary alcohol. / Resolution of tertiary alcohol esters is difficult because they are also very bulky, but important because there are only a few synthetic and biocatalytic methods to prepare enantiopure tertiary alcohols. We discovered several proteases that hydrolyze esters of tertiary alcohols, one of which was subtilisin Carlsberg. This is the first examination of protease hydrolysis of tertiary alcohol esters. Substrate studies and molecular modeling explained their reactivity and enantioselectivity. / Sulfinamidies are also bulky substrates, but surprisingly subtilisin catalyzed a catalytic promiscuous reaction of N-acyl sulfinamides. Subtilisin Carlsberg-catalyzed hydrolysis of N-acyl sulfinamides favored cleavage of the sulfinamide (S(O)-N) bond with a minor amount of the expected carboxamide (C(O)-N) bond. The sulfinamide hydrolysis was enantioselective and confirmed by product isolation from the S-N sulfinamide cleavage. In contrast, the related subtilisins BPN' and E favored the C-N carboxamide hydrolysis. / Further examination using electrospray-mass spectrometry revealed a sulfinyl-enzyme intermediate located at the active site, analogous to an acyl-enzyme. This suggested an analogous mechanism to the amide/ester hydrolysis. Substrate variation indicated a substrate reversal was responsible for the change in reactivity by binding in the S1 acyl pocket. Three mutations of subtilisin BPN' towards subtilisin Carlsberg increased the S-N hydrolysis by 14-fold, indicating that only a few mutations were responsible for the catalytic promiscuity. The substrate specificity and mutagenesis were consistent with a reversed orientation for the sulfinyl hydrolysis reactions. / The active site of subtilisin is indeed versatile for unnatural substrates and unnatural reactions. We expanded the applications of subtilisin to resolve bulky substrates, such as chiral auxiliaries and tertiary alcohol esters, and also to perform a new catalytic promiscuous sulfinamide S-N bond hydrolysis. Based on these results, subtilisin should prove useful for future resolutions of bulky substrates, and also in catalytic promiscuous reactions where unusual reactive centers are hydrolyzed.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.102684 |
| Date | January 2006 |
| Creators | Mugford, Paul F. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry.) |
| Rights | © Paul F. Mugford, 2006 |
| Relation | alephsysno: 002572051, proquestno: AAINR27821, Theses scanned by UMI/ProQuest. |
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