Studies on C -seco limonoids (tetranortriterpenes) from Azadirachta indica A. Juss: a) Isolation and characterization of new C -seco limonoids b) Functionalization of salannin and azadirachtin using Nocardia sp. c) Elucidation of pathways for biotransformation and structural specificity of the organism

Abstract

a) Isolation and characterization of new C seco limonoids b) Functionalization of salannin and azadirachtin using Nocardia sp. c) Elucidation of pathways for biotransformation and structural specificity of the organism

The thesis entitled “Studies on C seco Limonoids (tetranortriterpenes) from Azadirachta indica A. Juss...” is divided into five chapters.

Chapter 1 Provides a general introduction to the chemistry of neem (Azadirachta indica) constituents and their biological activities. It also explains the significance of microbial modification of natural products and the role of biocatalysts in organic chemistry. (Ref: W. Kraus in The Neem Tree, 1995)

Chapter 2 Isolation and characterization of two new azadirachtin analogues from neem seed kernels Extraction of neem seed kernels using modified procedures led to the isolation of azadirachtin, the major active principle. Further purification of more polar fractions yielded two new and previously unknown azadirachtin analogues, co-occurring in minor quantities. Isolation and characterization of 11 epi azadirachtin H Purification by repeated silica gel column chromatography (100-200 mesh) using CHCl -CH CN and ethyl acetate-hexane yielded a microcrystalline solid. Spectral analyses (IR, ¹H NMR, ¹H-¹H COSY, ¹³C NMR, SEFT, NOESY, FAB MS) confirmed a new epimeric analogue of azadirachtin. The ¹H NMR showed a signal at 5.41 (H 11) not present in azadirachtin H. It collapsed to a singlet upon D O exchange, proving coupling with 11 OH (not H 9). NOESY confirmed stereochemistry. The compound was identified as 11 epi azadirachtin H. Isolation and characterization of 11 epi azadirachtin D Semi preparative HPLC ( Bondapak C 18, CH CN-H O) yielded another pure compound lacking one carbomethoxy group and containing an additional methyl group, resembling azadirachtin D. Large upfield shifts in H 30 (~ 0.68) and H 6 (~ 0.5) were observed. NOESY showed strong NOE between the carbomethoxy methyl group and the oriented H 30 and H 6, confirming COOMe orientation and 11 OH. Identified as 11 epi azadirachtin D.

Chapter 3 Part A: Biocatalyst mediated functionalization of ring A in salannin Salannin is a major C seco limonoid with mild insect antifeedant activity. Prior structure-activity studies exist, but none produced derivatives with an , unsaturated ketone in ring A, a structural feature important for biological activity (e.g., gedunin, nimbolide). A gram positive bacterium identified as Nocardia sp. was found to quantitatively convert salannin (1) and 3 deacetylsalannin (2) into 1 detigloyloxy 3 deacetylsalannin 1 en 3 one (3), a previously unknown compound. Identified via ¹H NMR, ¹H-¹H COSY, SEFT, and HRMS. The pathway involves:

Deacetylation formation of 3 deacetylsalannin Oxidation of the C 3 secondary alcohol 3 oxo 3 deacetylsalannin (intermediate) Final transformation to compound 3

Part B: Structural flexibility of the biocatalyst To evaluate substrate flexibility, a series of salannin derivatives (including new ones) were synthesized with minor modifications to the core skeleton. Most were efficiently converted to the corresponding , unsaturated ketones. These results demonstrate the unique ability of Nocardia sp. to functionalize ring A and its wide substrate specificity. The organism preferentially oxidizes C 3 over C 1, indicating its preference for a 1 en 3 one over a 2 en 1 one system.

Chapter 4 Biocatalyst mediated ring A functionalization and ring D expansion in azadirachtin Azadirachtin (4), one of the most complex and potent insect antifeedants, was incubated with resting cells of Nocardia sp., forming two sets of metabolites:

Organic solvent soluble metabolites:

3 deacetylazadirachtin (5) 1 detigloyloxy 3 deacetylazadirachtin 1 en 3 one (6) 1 detigloyloxy 3 deacetyl 11,19 deoxa 12,19 oxa 11 oxo azadirachtin 1 en 3 one (7)

Water soluble metabolite:

1 detigloyloxy 3 deacetyl 4 carbomethoxy 11,19 deoxa 11 oxo 19 hydroxyazadirachtin 1 en 3 one 12 carboxylic acid (8)

These metabolites possess essential biological structural units such as:

, unsaturated ketone keto lactone hydroxy dihydrofuran (key for antifeedant and IGR activity)

A time course study showed the metabolite distribution depended on incubation duration. Cells retained high catalytic efficiency over 30-45 hours and could be reused.

Chapter 5 Preliminary cell free enzyme studies Nocardia sp. was grown in modified mineral salts medium. Cell free extracts from 36 hour cultures converted salannin and azadirachtin into their 3 deacetyl derivatives. However, conversion of 3 deacetylsalannin to 3 deacetyl 3 oxosalannin occurred only when the cells had been previously induced by growth in the presence of azadirachtin or salannin. This indicates:

the secondary alcohol dehydrogenase involved is inducible, not constitutive NAD is the required cofactor