| dc.description.abstract | The thesis titled "Synthesis and Evaluation of Catalytic Supramolecular Aggregates" describes the efficiency of various organized assemblies consisting of several newly developed amphiphiles that are covalently attached with nucleophilic moieties. These systems were employed as putative mimics of hydrolase enzymes. In this thesis, the synthesis and detailed kinetic characterization of different supramolecular aggregates such as micelles, microemulsions, vesicular and metalloaggregate systems have been described.
The thesis has been divided into five chapters. Chapter 1 gives an introduction to the general area of aggregate chemistry in an enzyme (hydrolase) modeling perspective. It gives a comprehensive account of the research done by various other groups working in the area of supramolecular and bioorganic chemistry toward the development of enzyme mimics.
Early part of the work carried out in our laboratory toward the above-mentioned objectives is presented in Chapter 2. This part of the work deals with the utilization of cationic aggregate-bound monoperoxyphthalate (MPP) as an excellent catalyst for the hydrolysis of both alkanoate and phosphotriester substrates. We considered the use of MPP for this purpose on the basis of its pronounced ?-nucleophilic character (Fig. 1). Moreover, the presence of a preformed COO? anion in MPP makes it bind cationic aggregate surface effectively in a mildly alkaline pH condition.
Figure 1. Cationic Aggregate Bound Monoperoxyphthalate.
Our studies revealed that cetyltrimethylammonium chloride (CTACI) micelles bound MPP in aqueous buffered media at pH 8.5 are very effective for the cleavage of p-nitrophenyl diphenyl phosphate (PNPDPP) and also carboxylate ester p-nitrophenyl acetate (PNPA). The reactions of MPP in other organized assemblies such as microemulsions and vesicles have also been studied, which showed impressive rate acceleration over background (MPP alone in buffer, in the absence of any cationic surfactant).
The crucial turnover experiments with excess substrate showed ‘burst’ kinetics which indicated a two-step mechanism, where an intermediate is first generated. These phosphorylated or acylated intermediates accumulate under the reaction conditions. The presence of the phosphorylated MPP (Scheme 1) was detected by analyzing the products by ³¹P-NMR spectroscopy. The acetylated MPP was similarly isolated by reacting MPP with PNPA and analyzing the products by ¹H-NMR spectroscopy.
Chapter 3 describes the synthesis and catalytic properties of four new dialkylaminopyridine (DAAP) functionalized surfactants 1–4 with different headgroup charges. Comicellar systems of these functional surfactants with host cationic surfactant CTABr in aqueous buffer (pH = 8.5–9.0) were used to cleave p-nitrophenyl alkanoates of varying hydrophobicities and also phosphotriester PNPDPP. The catalytic systems especially 1/CTABr and 3/CTABr conferred significantly greater reactivity toward the esters derived from alkanoic acids of moderate chain lengths (n = 6–8) during the hydrolytic cleavage relative to their shorter or longer chain counterparts. These catalytic systems comprising coaggregates of either 1/CTABr or 3/CTABr conformed to Michaelis–Menten kinetic scheme and clearly demonstrated turnover behavior in the presence of excess substrates. Various Michaelis–Menten parameters such as Km, Vmax, kcat and kcat/Km were determined from the respective Lineweaver–Burk plots.
Chapter 4 deals with more complex multiphase assemblies, the microemulsion (ME) systems which are optically stable dispersions and are formed spontaneously when water, hydrocarbon (oil), surfactant and a cosurfactant (usually a short-chain alcohol) are mixed in definite proportions. The MEs used were oil-in-water (O/W), water-in-oil (W/O) and a bicontinuous (BC) ME where oil and water are in nearly comparable amounts. All the MEs were stabilized by cationic surfactant CTABr, n-BuOH (cosurfactant), cyclohexane (oil) and pH 8.5 buffer (aqueous phase). Since these have both aqueous and oil phases, the sites of solubilization are quite different for the catalyst and the substrate depending on their intrinsic hydrophobicities.
For convenience, Chapter 4 has been divided into Sections I and II. Section I deals with the reactions of p-nitrophenyl alkanoate esters with non-amphiphilic dimethylaminopyridine (DMAP), 5 and its related mono- and dianionic water-soluble derivatives 6 and 7 in the above-mentioned three different ME media. The second-order rate constants in ME media were determined over a phase volume (?) of approximately 0.13–0.46. Our results indicated that while the non-amphiphilic DAAP catalysts were partitioned between the micellar and aqueous pseudophases in MEs, the hydrophobic substrates were found to be mainly confined to oil-rich phases. The main effects of ME media on the hydrolysis reaction are both due to electrostatic and substrate partitioning.
Section II describes the reaction of the amphiphilic DAAPs (1–4) towards cleavage of p-nitrophenyl alkanoates of varying hydrophobicities in different MEs as described above. In order to examine their esterolytic activities, individual DAAP surfactants were separately doped into each ME and the resulting kinetic data for esterolysis were compared with that of non-amphiphilic DMAP, 5. Results obtained in this study are markedly different from that of the same DAAP amphiphiles solubilized in CTABr micelles. Thus the least hydrophobic substrate, i.e., PNPA was found to be more susceptible to hydrolysis than the alkanoate esters having long alkyl chains under these conditions. The turnover behavior of the DAAPs was however maintained in these MEs.
Chapter 5 invokes metallomicelles as reaction-specific catalysts. In this section an attempt has been made to mimic metalloprotease activities. First, the synthesis of the lipophilic ligands 8–12 was described. The Cu²? complexes of these ligands were utilized as catalysts for cleavages of p-nitrophenyl hexanoate and PNPDPP under near-neutral (physiological) conditions of pH 7.6. The stoichiometries of the complexes with Cu²? with these ligands (8–12) were determined by the kinetic version of the Job plots. The experiments indicated a 2:1 ratio of ligand:Cu²? for the ortho-substituted dialkylaminopyridine-based ligands 8–10, and 1:1 complexes for the bispicolylamine-based ligands 11 and 12. The complexes with other transition metal ions such as Co²?, Ni²? and Zn²? did not give very effective formulations for esterolysis. EPR studies were also carried out to characterize the nature of the coordination geometries of the complexes. The systemic pKa values of the effective nucleophile, i.e., the hydroxymethyl group of all these coordination compounds were determined by pH–rate constant profile. Evidences for the turnover behavior in the esterolysis reactions of the excess substrates by Cu²? complexes of ligands 8 and 9 were obtained from the observations of burst-type kinetic behavior. However, a fast turnover without any ‘burst’ was seen with aggregates composed of Cu²? complexes of ligands 10, 11 and 12. | |
