Enzymatic approaches to obtain macrolactams with immunomodulatory activity

Dottorato di Ricerca in Biochimica (XXIII ciclo) Enzymatic approaches to obtain macrolactams with immunomodulatory activity Introduction and aim of work In the last years, the use of enzymes, purified from animals, vegetables or microorganisms, to obtain biologically active molecules have been improved due to the many advantages offered by biocatalysis. Enzymes catalyze most biological reactions “in vivo” and they also transform natural and unnatural substrates “in vitro”. They operate under mild conditions, with chemo-, regio-, and stereoselectivity. The aim of our work is to use enzymes to selectively transform bioactive polifunctional molecules; in particular we focalize our attention on lipases (triacylglycerol acylhydrolases, EC 3.1.3.3), ubiquitous enzymes that “in vivo” catalyze the hydrolysis of triacylglycerols. Lipases are probably the most frequently used enzymes, mainly because several preparations from different sources are commercially available, at low cost, with a satisfactory degree of purity and because they retain their activity even in drastic conditions and don’t need cofactors. Since their action occurs at the lipid-water interface, they are stable also in organic solvents. This peculiar property makes them well suitable to application on lipophilic substrate. The first compound that we have studied belongs to the family of macrocycles: we started from ascomycin, a macrolactam isolated from the fermentation broth of a soil fungus, the Streptomyces hygroscopicus, to obtain pimecrolimus, the 32-epi chloro derivative of ascomycin; this is a topical immunomodulator which provides a valid alternative to glucocorticosteroids and cyclosporine A for the topical treatment of atopic dermatitis and other inflammatory dermatoses. In fact pimecrolimus (like many ascomycin analogues) possesses two functional molecular domains: through the “binding domain” binds an immunophilin; the formed complex, through the ”effector domain” interacts with the protein phosphatase calcineurin, the target enzyme, by preventing calcineurin from dephosphorylating the nuclear factor of activated T cells, a transcription factor. This result in the blockage of signal transduction pathways in T cells and the inhibition of the synthesis of inflammatory cytokines, specifically Th1 and Th2-type cytokines. Experimental approach In order to obtain pimecrolimus from ascomycin, the 32-hydroxy group has to be substituted by a chlorine atom, the 24-hydroxy group being not affected. A selective protection of this function is then required. We chosen, as first approach, to submit ascomycin to a lipase catalyzed acylation in transesterification condition, using vinyl acetate as acylating agent, the best method to perform an irreversible reaction. As reported in the Table 1, lipase from Candida cylindracea (CCL), from Pseudomonas fluorescens (PFL) and from porcine pancreas (PPL) afforded negative results while lipase from Candida antarctica B (CAL B) was able to introduce an acyl group only on 32-OH, leaving unreacted the 24 one. These results suggest that the active site of CAL B, containing as for all lipases the triad serine, histidine and aspartate, can accommodate our substrate in a way that leaves only position 32 accessible to the biotransformation: so we planned to take advantage from this regioselectivity with a different approach. Lipase Solvent Acyl Donor Time (h) Conversion(%) CCL Toluene Vinyl Acetate 94 0 PPL Toluene Vinyl Acetate 94 0 PFL Chloroform Vinyl Acetate 100 0 CAL B Acetonitrile Vinyl Acetate 53 30 (32-OAc) CAL B Toluene Vinyl Acetate 53 90 (32-OAc) Table 1 We prepared the 24, 32-diacetate, that by a CAL-B catalyzed removal of 32-acyl group could afford the macrolactam protected only in positon 24. The enzyme was screened with several solvents, either in hydrolysis than in an alcoholysis conditions, and the best result was observed when the 24, 32-diacylated macrolactam was treated with n-octanol in tert-butylmethylether; in fact in this condition a conversion of 80% was observed, after 54 h . Lipase Solvent Alcohol Time (h) Conversion(%) CAL B Toluene H2O 80 0 CAL B Toluene MeOH 48 0 CAL B Toluene n-Butanol 100 0 CAL B Toluene n-Octanol 120 30 CAL B T-butylmethylether n-Octanol 54 80 (24-OAc) Table 2 Finally, after the substitution of 32-OH with an atom of chlorine, the protective group in 24 was removed by an acid hydrolysis giving the desired pimecrolimus. In the future we prospect to use the enzymatically obtained 24-monoacetate to prepare other 32-halogens derivates, such as 32-fluoro and 32-iodo and then value the effect of these atoms on the activity immunomodulatory by the biological assays. References - E. Santaniello, P. Ferraboschi, P. Grisenti, Enzyme Microb. Technol.,1993, 15, 367-381. -A. Horvart, M. A. Grassberger, G. Schulz, E. Haidl, H. Sperner, A. Steck, Tetrahedron, 2000, 56, 7469-7476. -E. Bornhövd, W. Burgdorf, A. Wollenberg, Curr. Op. Invest. Drugs, 2002, 708-712.