Studies on enzyme models.

Abstract

The thesis entitled "Studies on Enzyme Models" is divided into three chapters. The first chapter deals with a general introduction on the mechanism of enzyme action and study of enzyme models mimicking some enzymes. The second chapter deals with a formal introduction to the present work, the design and building of the model systems for mimicking carboxylase and its co-enzyme, thiamine pyrophosphate, for the synthesis of an "unnatural" acyloin for chloramphenicol synthesis. This chapter also deals with the kinetics studies of the mixed acyloin formation. The third chapter deals with the synthesis of "model enzymes" for the synthesis of ephedrine.

Chapter I In the General Introduction, certain known features for the specificity as well as rate acceleration in enzyme catalysis are discussed. Concepts such as proximity effect and intramolecular catalysis, propinquity effect, orbital steering, stereopopulation control, covalent catalysis, etc., are considered. The importance of binding energy in enzyme-substrate complexation and the relevance of hydrophobic association in these processes are also discussed with suitable examples of model systems.

Chapter II The chapter on Mixed Acyloin Condensation describes the objectives and approach to the present studies on mixed acyloin formation involving p-nitrobenzaldehyde and acylglycolaldehyde and the conversion of this acyloin into chloramphenicol esters. The chapter starts with the various methods available for chloramphenicol synthesis, followed by the design and synthesis of model systems for achieving specificity and enhanced rates in the formation of the mixed acyloin 1-p-nitrophenyl-1-hydroxy-3-acyloxy-propane-2-one (1), a key intermediate in the synthesis of chloramphenicol esters. Hydrophobic association was invoked as a major criterion for achieving specificity and rate acceleration. The conversion of (1) to chloramphenicol esters and the successful control of stereochemistry during one of the stages in the conversion to the final compound are also described. NMR studies have shown that the pNB molecule is sandwiched by the rings of the catalyst (2) in water: Catalyst (2): HO—C—OH The hydrophobic association of pNB with the terminal aromatic rings on the catalyst determines two main features of the reaction, viz., the pNB molecule has a greater chance of being associated with the aromatic rings, and the acylglycolaldehyde could be specifically attached to the 2-position of thiazole due to its steric lability. The equilibrium constant and the free energy of association of pNB with 5-(4-methoxybenzhydryl)-4-methyl-5-(hydroxyethyl)-thiazolium chloride (2) have been estimated through the linear enhancement of pNB absorption at 254 nm with graded amounts of the catalyst (2), and the association energy was found to be of the order of –6.2 kcal/mole. Since chloramphenicol is marketed as the palmitate ester, a variation of the above catalyst (2) was used for condensing pNB and palmitoylglycolaldehyde. The presence of the hydrophobic laurylamide chain in the catalyst (3) was expected to form micellar associations with the palmitoyl group on glycolaldehyde, therefore imparting greater specificity to the catalyst. Catalyst (3): Lauryl catalyst structure The lauryl catalyst (3) actually synthesized the palmitoylated carbinol (4) in high yields, with a first-order rate constant. The advantage of the "enzyme" model approach over other methods of chloramphenicol synthesis has been discussed. The basic strategy of conversion of the acyloins (1) and (4) to the corresponding threo dihydroxy amino propane derivatives was to convert them into oximes, interlocking the oxime nitrogen and ?-hydroxy by chelation with zinc so that the addition of a hydride through borohydride leads predominantly to the threo configuration. The intermediate amines were converted to chloramphenicol derivatives by established methods (Chart 1). The chapter concludes with the kinetic measurements of the acyloin formation, bioassay to determine the potency of the antibiotics synthesized in the ultimate stage, and a general discussion on the effect of hydrophobic association and other factors on the specificity and rates of mixed acyloin formation. The rate order of acyloin formation with excess of acetylglycolaldehyde (i.e., pseudo-first order with respect to acetylglycolaldehyde) was found to be first order with respect to pNB, at low catalyst concentrations (0.1 mole/mole of pNB). The rate constant was of the order of 6 × 10?? sec?¹. The rate constant with excess of palmitoylglycolaldehyde and 0.1 mole catalyst was also determined.

Chapter III This chapter begins with an introduction on some of the important commercial processes of the synthesis of the drug ephedrine. This is followed by the design of the catalyst (5) to achieve an acyloin intermediate (6) for the synthesis of ephedrine. Studies on the condensation of a mixed acyloin, involving benzaldehyde and methylglycolaldehyde, have been carried out. Again, the substrate specificity of the catalyst (5) to give the required acyloin (6) is explained by invoking hydrophobic associations of the phenyl ring of benzaldehyde with the aromatic rings on the quaternary nitrogen of (5) and the tolyl group of tosylglycolaldehyde with the phenyl group at the 5-position of the thiazole in the catalyst (5). The association and hence the alignment of the phenyl group of benzaldehyde below the phenyl groups of the catalyst have been proved by NMR studies. The association energy has been evaluated as in the previous case by spectral methods and it has been found to be –7.2 kcals/mole. The chapter also deals with the conversion of the acyloin (6) to ephedrine, speculating the methods available for such a conversion. Here the acyloin was converted into the Schiff’s base with methylamine. It is expected that without interlocking, dipolar repulsion would orient the ?-hydroxy and the ?-anil group in opposite directions. A hydride addition in this system should result in the erythro disposition of the hydroxyl and the amino group in the product. In fact, sodium borohydride neatly hydrogenolysed the tosyluxy residue and gave the erythro product in high yields (7, ii). Structures: (6) CH—C—CH?OTs | | OH CH?

(7) CH—CH—CH? | | OH CH?