Swati P. Kolet

Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

Biocatalysis: fungi mediated novel and selective 12β- or 17β-hydroxylation on the basic limonoid skeleton

Basic limonoids carrying a 4,4,8-trimethyl-17-furanylsteroid skeleton are a class of triterpenoids and well-known for their insecticidal as well as a vast array of pharmacological activities. Rare and synthetically challenging 12β- and 17β-hydroxylation was achieved on the basic limonoid skeleton to produce a novel series of hydroxylated limonoids using fungi-mediated biocatalysis. The fungal system belonging to the genera of Mucor efficiently converted azadiradione, epoxyazadiradione, gedunin and their derivatives into corresponding 12β- and/or 17β-hydroxy derivatives. The position and stereochemistry of hydroxylation was determined by rigorous spectroscopic and crystallographic studies. This fungi-mediated stereo- and regio-selective hydroxylation process was highly efficient and mild enough to sustain chemically sensitive functional groups around the basic limonoid skeleton. Modifications of specific functional groups and variation in biocatalyst were shown to bring selectivity among 12β- or 17β-hydroxylation.

Expedient preparative isolation, quantification and characterization of limonoids from Neem fruits

Major basic limonoids from Neem fruits such as azadirone, epoxyazadiradione and azadiradione were isolated in preparative scale using an automated and rapid medium pressure liquid chromatography (MPLC)-based protocol. They were individually quantified using HPLC. An LC-ESI-MS/MS-based rapid identification technique was developed for the high-throughput screening of these limonoids in phytochemical extracts.

Biocatalyst mediated production of 6β,11α-dihydroxy derivatives of 4-ene-3-one steroids

Biotransformation of steroids with 4-ene-3-one functionality such as progesterone (I), testosterone (II), 17α-methyltestosterone (III), 4-androstene-3,17-dione (IV) and 19-nortestosterone (V) were studied by using a fungal system belonging to the genera of Mucor (M881). The fungal system efficiently and quantitatively converted these steroids in regio- and stereo-selective manner into corresponding 6β,11α-dihydroxy compounds. Time course experiments suggested that the transformation was initiated by hydroxylation at 6β- or 11α-(10β-hydroxy in case of V) to form monohydroxy derivatives which upon prolonged incubation were converted into corresponding 6β,11α-dihydroxy derivatives. The fermentation studies carried out using 5L table-top fermentor with substrates (I and II) clearly indicates that 6β,11α-dihydroxy derivatives of steroids with 4-ene-3-one functionality can be produced in large scale by using M881.

Mucor hiemalis mediated 14α-hydroxylation on steroids: In vivo and in vitro investigations of 14α-hydroxylase activity

Transformation of testosterone and progesterone into synthetically challenging 14α-hydroxy derivatives was achieved by using fungal strain Mucor hiemalis. Prolonged incubation led to the formation of corresponding 6β/7α,14α-dihydroxy metabolites. The position and stereochemistry of newly introduced hydroxyl group was determined by detailed spectroscopic analyses. The time course experiment indicated that fungal strain initiated transformation by hydroxylation at 14α-position followed by at 6β- or 7α-positions. Studies using cell-free extracts suggest that the 14α-hydroxylase activity is NADPH dependent and belongs to the cytochrome P450 family.

One-Pot Fluorescent Labeling Protocol for Complex Hydroxylated Bioactive Natural Products

Tagging of small bioactive molecules with a fluorophore is a highly sensitive method to trace their cellular activities through real-time visual information. Here we disclose a 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-based, high-yielding, one-pot labeling protocol for hydroxylated molecules using Yamaguchi coupling as the key reaction. This methodology was successfully applied on several sensitive and complex hydroxylated bioactive compounds including 7-deacetylazadiradione, simvastatin, camptothecin, andrographolide, cinchonine, β-dihydroartemisinin, and azadirachtin A. Further, utility of this protocol was illustrated on the cytotoxic activity of azadiradione derivatives against several cancer cell lines through cell imaging of two qualified fluorescent probes.

Biocatalyst mediated regio- and stereo-selective hydroxylation and epoxidation of (Z)-α-santalol

Biocatalyst mediated regio- and stereo-selective hydroxylation and epoxidation on (Z)-α-santalol were achieved for the first time, using a fungal strain Mucor piriformis. Four novel metabolites were characterized as 10,11-cis-β-epoxy-α-santalol, 5α-hydroxy-(Z)-α-santalol, 10,11-dihydroxy-α-santalol and 5α-hydroxy-10,11-cis-β-epoxy-α-santalol. Using Amano PS lipase from Burkholderia cepacia, α- and β-isomers of 10,11-cis-epoxy-α-santalol were resolved efficiently.

Biocatalyst mediated functionalization of salannin, an insecticidal limonoid

Transformation of salannin, an insecticidal C-seco limonoid was investigated using a fungal system, Cunninghamella echinulata. Salannin was efficiently converted into two metabolites, where the C-17 furan moiety was transformed into γ-hydroxybutenolide (salanninolide) and N-(2-hydroxyethyl)-α,β-unsaturated-γ-lactam (salanninactam) analogues. Present studies have indicated salanninolide to be a metabolite in the C-seco limonoid biosynthetic pathway.