Pramod B. Shinde

Anti-inflammatory constituents of the red alga Gracilaria verrucosa and their synthetic analogues.

A chemical study on the anti-inflammatory components of the red alga Gracilaria verrucosa led to the isolation of new 11-deoxyprostaglandins ( 1- 4), a ceramide ( 5), and a C 16 keto fatty acid ( 6), along with known oxygenated fatty acids ( 7- 14). Their structures were elucidated on the basis of NMR and MS data. The absolute configurations of compounds 1- 5 were determined by Moshers method. The anti-inflammatory activity of the isolated compounds ( 1- 14) was evaluated by determining their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Compounds 9 and 10 exhibited the most potent activity. In the evaluation of these two compounds and derivatized analogues ( 15- 40), the anti-inflammatory activity was enhanced in some synthetic analogues. These enone fatty acids were investigated as potential anti-inflammatory leads for the first time.

Cytotoxic bromotyrosine derivatives from a two-sponge association of Jaspis sp. and Poecillastra sp.

Bioassay-guided chemical investigation of the lipophilic extract of a two-sponge association (Jaspis sp. and Poecillastra sp.) led to the isolation of two new bromotyrosine derivatives (1 and 2), along with known derivatives (3-12). Cyclobispsammaplin A (1) is a cyclic derivative of the previously reported bispsammaplin A (13), while psammaplin M (2) is composed of beta-alanine (or aspartic acid) unit. Compounds 3, 4, 6, 10, and 12 are isolated for the first time from a sponge belonging to the subclass Tetractinomorpha. Structure elucidation was performed by a combination of high resolution mass and 2D NMR (principally COSY, HMBC, HSQC, and NOESY) spectroscopy. Compounds 1-4, 6, 10, and 12 were evaluated for cytotoxicity against a small panel of five human solid tumor cell lines and their activity was compared in relevance to their structure.

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5′-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure–activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.

Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17.

Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogs possessing unnatural sugars replacing native D-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as D-quinovosyl-10-deoxymethynolide, L-rhamnosyl-10-deoxymethynolide, L-olivosyl-10-deoxymethynolide, and D-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar D-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.

Bile acid derivatives from a sponge-associated bacterium Psychrobacter sp.

In our search for bioactive metabolites from a marine sponge-associated bacterium Psychrobacter sp., a new bile acid derivative (1), which was assumed to be an artifact, were isolated along with six known (2–7) compounds by bioactivity-guided fractionation. Elucidation of the structure of the new compound was done using a combination of NMR (1H, 13C, HMBC, HSQC, and COSY) and MS spectroscopy. Compound 1 exhibited moderate suppressive effects on both NO and IL-6 production at a concentration of 200 μM (87.3 μg/mL) without significant cytotoxicity against cells. Compounds 2–5 and 7 showed selective inhibitory activity against several human pathogenic bacterial strains at the low concentration of 30 μg/well. In a cytotoxicity evaluation, only compound 7 showed mild cytotoxicity against five human solid tumor cell lines (A-549, SK-OV-3, SK-MEL-2, XF-498, and HCT-15) with ED50 values in the range of 11–14 μg/mL.

Characterization of FK506 Biosynthetic Intermediates Involved in Post-PKS Elaboration

The post-PKS modification steps of FK506 biosynthesis include C9-oxidation and 31-O-methylation, but the sequence of these reactions and the exact route have remained unclear. This study details the post-PKS modification pathways in FK506 biosynthesis through the identification of all intermediates and in vitro enzymatic reactions of the cytochrome P450 hydroxylase FkbD and the methyltransferase FkbM. These results complete our understanding of post-PKS modification steps to FK506 showing the substrate flexibility of two enzymes involved and the existence of two parallel biosynthetic routes to FK506.

A non-immunosuppressive FK506 analogue with neuroregenerative activity produced from a genetically engineered Streptomyces strain†

FK506 exhibits neuroprotective and neuroregenerative activities in addition to its clinically important immunosuppressant properties. The macrolide ring of FK506 is biosynthesized by a hybrid polyketide synthase/nonribosomal peptide synthetase system and is further modified via C-9 oxidation by FkbD and 31-O-methylation by FkbM. A new FK506 analogue, 9-deoxo-prolyl-FK506 (1), was isolated from the fkbD deletion mutant of Streptomyces sp. KCTC11604BP, and its biological activities were evaluated. The in vitro immunosuppressive activity was significantly reduced, but in vitro neurite outgrowth activity similar to FK506 was maintained. These results demonstrate the potential of pathway engineering for the modification of structurally complex natural products, such as FK506, to create improved biological agents.

New Glycerides from a Two-Sponge Association of Jaspis sp. and Poecillastra sp.

A chemical investigation of the MeOH extract of a two-sponge association (Jaspis sp. and Poecillastra sp.), collected from Jeju Island, Korea, led to the isolation of two new lysophosphatidylglycerols (1 and 2) along with known lysophosphatidylglycerol (3) and glycolipids (4 and 5). Their structures were elucidated on the basis of NMR and MS spectroscopic data.