Cysteine proteases are a class of proteolytic enzymes, which are involved in a series of metabolic and catabolic processes, such as protein turnover, digestion, blood coagulation, apoptosis, fertilization and cell differentiation, and the immune response system. The development of novel potent and selective inhibitors for cysteine proteases has therefore gained increasing attention among medicinal chemists. In this thesis we have reported the design, synthesis, and evaluation of several peptidyl inhibitors for clan CA and clan CD cysteine proteases. We have continued the investigation of dipeptidyl vinyl sulfones as potent and selective inhibitors for dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, which is involved in the processing of intracellular proteases, such as granzymes. We have found that DPPI tolerates negatively charged amino acid residues in the P2 position with inhibition rates of 7,600 M-1s-1. Dipeptidyl vinyl sulfones with positively charged amino acid residues at the P1 position, however, do not inhibit DPPI at all. A second project focused on the epoxidation of the double bond of the vinyl sulfone moiety of the dipeptidyl vinyl sulfones. Instead of epoxidizing the double bond, we found that an isomerization had occurred. The newly formed compounds were determined to be allyl sulfones. We tested this new class of inhibitors with clan CA proteases and obtained inhibition rates of 560 M-1s-1 for Cbz-Leu-Phe-AS-Ph with calpain I. Two new classes of compounds for the clan CD protease S. mansoni legumain were designed, synthesized, and evaluated. Aza-peptidyl epoxides were found to be potent and selective inhibitors of S. mansoni legumain with IC50’s as low as 45 nM. Aza-peptide Michael acceptors were derived from the aza-peptide epoxide design and synthesized in an analogous fashion. The aza-peptide Michael acceptors inhibited S. mansoni legumain with even lower IC50’s, as low as 10 nM. However, the aza-peptide Michael acceptors react with thioalkylating agents contained in the buffer, such as DTT. The rates of degradation were determined spectroscopically, and half-lives of 3 to 20 minutes were measured. This observation gave us insights into the enzymatic mechanism and allowed us to determine the point of attack for the legumain active site cysteine thiol.

The C-5 modified pyrimidine analogues are well-known anticancer and antiviral drugs which underscore further development of novel probes to study their physical, chemical, and biological properties. In my dissertation the syntheses and properties of (β-halo)vinyl sulfone and/or (β-keto)sulfone analogues of C-5 modified pyrimidine have been discussed. In the first part of the dissertion, the synthesis of 5-(β-halo)vinyl sulfones either by transition metal-catalyzed or iodine-mediated halosulfonylation reaction of 5-acetylene pyrimidine nucleosides have been explored. The novel (β-chloro/bromo/iodo)vinyl sulfones efficiently undergo addition-elimination reaction with different nucleophiles such as thiols, amines, amino acid, peptides to provide (β-substituted)vinyl sulfone analogues. The rate of these substitution reactions depends on the nature of halogen atom presents at the β-position and increases with the order of I ≥ Br > Cl. (β-chloro/bromo/iodo)vinyl sulfones possess exclusively E stereochemistry while their β-substitued analogues possess either E (for β–thio analogues) or Z (for β–amino analogue) stereochemistry. It has been observed that the vinylic proton of (β-chloro) or (β-amino)sulfone analogue undergoes exchanges with deuterium in polar protic deutorated solvents. The antiproliferative activities of those analogues have been explored and was found that protected 5-(E)-(1-chloro-2-tosylvinyl)-2'-deoxyuridine inhibited the growth of L1210, CEM and HeLa cells in lower micromolar range. In the second part of the dissertation the syntheses and reactivities of 5-(β-keto) sulfone of pyrimidine nucleosides were investigated. Thus, 5-(β-halovinyl)sulfone of uracil and cytosine nucleosides have been efficiently converted into corresponding 5-(β-keto) sulfone analogues by displacement of halogen with ammonia followed by acid-catalyzed hydrolysis of the resulting (β-amino)sulfone analogues. A number of electrophiles were trapped at the acidic α-carbon of the 5-(β-keto)sulfones by treatment with electrophiles such as methyl, benzyl, or allyl halide in the presence of base. The 5-(α-iodo-β-keto)sulfone analogues of uracil nucleosides have been tested as an alternative substrates to probe the incorporation of nucleophiles at α-carbon. In the third part of the dissertation, the synthesis of 5'-phosphates of 5-(β-chloro) and 5-(β-keto) sulfones of 2'-deoxyuridine and their polymerase-catalyzed incorporation into DNA were evaluated. Thus, 5'-O-phosphorylated analogues have been efficiently incorporated into the DNA by human DNA repair polymerase (pol β) or bacterial polymerase (pol I).

The research presented in this thesis focuses on the molecular design of an oxidation-sensitive nanocarrier and its enzyme conjugate with a view of their application in the field of biomaterials. I have polarised our attention on a specific class of polymers, the polysulfides, for their environmental responsiveness (towards oxidising substances, a condition often associated with inflammatory reactions), interesting physico-chemical properties, ease of the preparation and multiple possibilities for further modifications and bioconjugations, which are perfectly suitable for the development as systems for drug delivery applications. In this work we firstly have focused on the synthesis of amphiphilic poly(propylene sulfide)-poly(ethylene glycol) (PPS-PEG) block copolymers by employing vinyl sulfone as the functional group to link the blocks and modify the end of the PEG. This study was followed by an investigation of the macromolecular interchange and payload exchange of the formed polymeric micelles to understand the 'co-formulation' events, employing fluorophores (dansyl groups) and quenchers (dabsyl groups) either as terminal groups in macroamphiphiles or as encapsulated hydrophobic payloads. In another part of the work, I have developed a micellar system with which simultaneously to two of the most important ROS: superoxide and hydrogen peroxide, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity towards superoxide with that of hydrophobic thioethers towards hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then 'mopped-up' by the polysulfides. This mode of operation is functionally analogous to the SOD/catalase combination, with the advantage of being based on a single and more stable system.