Synthesis and investigation of viral cysteine protease inhibitors and biosynthetic studies on subtilosin A

• Author / Creator

  Miyyapuram, Venugopal

• ABSTRACT

  This thesis discusses the synthesis and evaluation of cysteine protease inhibitors, the asymmetric reduction of pseudoxazolones, and the study of the mechanism of subtilosin A biosynthesis.

  Five classes of compounds, including pyridinylamines and ethers, have been designed with the aim of developing non-covalent inhibitors of SARS–CoV 3CLpro, a chymotrypsin-like cysteine protease vital to the life cycle of the SARS coronavirus. These compounds were synthesized and screened against SARS–CoV 3CLpro. 3-Bromo-5-[5-(4-nitro-phenyl)-furan-2-ylmethoxy]-pyridine (37), 5-Bromo-N-((5-(4-nitrophenyl)furan-2-yl)methyl)pyridin-3-amine (54), N-((5-(4-Aminophenyl)furan-2-yl)methyl)-5-bromopyridin-3-amine (61) and N-{5-[5-(4-Nitro-phenyl)-furan-2-ylmethylene]-4-oxo-2-thioxo-thiazolidin-3-yl}-acetamide (67) show very good inhibition with IC50 values ranging from 12 μM to 31 μM. Mechanism studies suggest that these compounds are reversible inhibitors.

  Inhibitors against Israel acute paralysis virus (IAPV), which is associated with Colony Collapse Disorder (CCD) in honeybees, have been designed with a glutamine residue at the P1 position based on sequence comparisons between IAPV 3Cpro and known 3C proteases. Two fluorogenic peptide substrates, Abz-QTTTQAG-Y(NO2)-E (95) and Abz-EVSMQVD-Y(NO2)-D (98), were synthesized. However, both compounds show no activity. As a different strategy, a peptidyl fluoromethyl ketone, Ac-Val-Thr(OBn)-Leu-6-fluoro-N,N-dimethyl-S-oxohexanamide (77), incorporating the SARS recognition sequence, has also been synthesized, and testing of its activity is in progress.

  Asymmetric reduction of pseudoxazolones containing an imine moiety can serve as a method for preparing enantiopure amino acids. Several conditions were tried but without success. Although the results are not encouraging, attempts to prepare pseudoxazolone derivatives with increased reactivity led to synthesis of highly substituted olefins.

  Subtilosin A, a ribosomally synthesized antimicrobial cyclic peptide from Bacillus subtilis, is posttranslationally modified to contain unusual thioether cross-links between cysteine sulfurs and alpha-carbons of phenylalanines and threonine. The sbo-alb gene locus is required for the production of subtilosin A as well as for immunity, but the exact roles of these genes are unclear. In order to elucidate the roles of albA, albE and albF genes that may be responsible for the posttranslational modifications, three potential substrates were designed and synthesized. In addition to these biosynthetic studies, subtilosin A1, a T6I mutant of subtilosin A, was purified and characterized. It was found to show hemolytic activity and altered bactericidal activity compared to that of the parent.

• Subjects / Keywords

  • Peptide antibiotics--Synthesis
  • Cyclic peptides--Synthesis
  • Bacillus subtilis--Genetics
  • Cysteine proteinases--Inhibitors--Synthesis
  • SARS (Disease)--Chemotherapy
  • Cysteine proteinases--Inhibitors--Evaluation

• Graduation date

  Fall 2009

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R35P56

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.