Role of the thiosugar ring in the inhibitory activity of salacinol, a potent natural α-glucosidase inhibitor.

Herein, ring-cleaved (24) and truncated (25) analogues of an azasugar, 1-deoxynojirimycin (23), exhibited inhibitory activity (Ki = 4-10 µM) equal to that of the parent compound (1, Ki = 14 µM). Based on this structure-activity relationship (SAR), four ring-cleaved (26a-26c and 27c) and three truncated (28a-28c) analogues of salacinol (1), a potent thiosugar-ring-containing α-glucosidase inhibitor, were synthesised. Bioassay results revealed that all the synthetics were inactive, indicating that the 5-membered thiosugar ring of 1 played an essential role in the potent activities of sulfonium-type inhibitors. The present findings are interesting and important in understanding the function of salacinol, considering that the observed inhibitory activity trend was contrary to the SAR observed in aza-compounds (23, 24, and 25) in a previous study, which suggested that the cyclic structure did not contribute to their strong inhibitory activity.

Radical Nitrososulfonation of Propargyl Alcohols: Thiazolidine-2,4-dione-Assisted Synthesis of 5-Alykyl-4-sulfonylisoxazoles.

In this study, we discover a good NO/HNO precursor, N-hydroxypyridinesulfonamide, and the regioselective radical nitrososulfonylation reaction of propargyl alcohols. Direct and unique isoxazole synthesis afforded a good-to-high yield of 5-alkyl-3-aryl-4-pyridinesulfonylisoxazoles. Copper-catalyzed aerobic oxidation could efficiently proceed in the presence of thiazolidine-2,4-dione. This work provides a powerful method for the synthesis and functionalization of alkyl-substituted isoxazoles and explores a new investigation route for drug-drug discovery.

Total Synthesis of Calanthoside, a Potential Hair Growth Stimulant: A Facile Synthetic Approach via One-Pot S- and O-Glucosidic Bond Formation.

The first total synthesis of calanthoside (1), which exhibits potent proliferative activity against human hair follicle dermal papilla cells, has been achieved in seven steps with an overall yield of 43% on a gram scale starting from anthranilic acid (11). The synthetic strategy features a one-pot process involving thioglucoside bond formation via nucleophilic substitution reaction and enol-glucosylation for building the S-,O-bisdesmoside structure of 1. Moreover, the one-pot reaction showed broad substrate adaptability to several sugar donors other than d-glucose, thus affording S,O-bisglycoside intermediates in ∼84% yield.

Biosynthetic diversification of non-ribosomal peptides through activity-based protein profiling of adenylation domains.

We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid (O-allyl-L-serine) was successfully incorporated into gramicidin S.

Structure-activity relationship study of 4,5-didehydroguadiscine, an aporphine alkaloid showing potent melanogenesis-inhibitory activity in B16 melanoma cells.

Although 4,5-didehydroguadiscine (12a), an alkaloid with potent melanogenesis-inhibitory activity isolated from Hornschuchia obliqua (Annonaceae), consists of an aporphine nucleus with an aromatized B-ring, to date, it has not been utilized as a template for structure-activity relationship (SAR) studies of pharmacological activities because of its exceptional structure. Accordingly, herein, five analogs (12b-12f) of 12a and five benzylisoquinoline analogs (13b-13f) lacking the C11a-C11b bond of 12b-12f were prepared. The inhibitory effects of 12b-12f and 13b-13f on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were examined and compared with those of 12a. Melanogenesis-inhibitory activities of 12b-12f were the same as that of 12a, whereas the melanogenesis-inhibitory activities of 13b-13f were significantly inferior to those of 12a and 12b-12f. These results suggest that the C11a-C11b bond plays an essential role in the melanogenesis-inhibitory activities of 12a-12e.

Chemoproteomics profiling of surfactin-producing nonribosomal peptide synthetases in living bacterial cells.

Much of our current knowledge on nonribosomal peptide synthetases (NRPSs) is based on studies in which the full NRPS system or each protein domain is expressed in heterologous hosts. Consequently, methods to detect the endogenous activity of NRPSs, under natural cellular conditions, are needed for the study of NRPS cell biology. Here, we describe the in vivo activity-based protein profiling (ABPP) for endogenous NRPSs and its applications to the study of their activities in bacteria. Remarkably, in vitro and in vivo ABPP in the context of the surfactin producer Bacillus subtilis enabled the visualization, tracking, and imaging of an endogenous SrfAB-NRPS with remarkable selectivity and sensitivity. Furthermore, in vivo, ABPP allowed the discovery of the degradation processes of the endogenous SrfAB-NRPS in the context of its native producer bacteria. Overall, this study deepens our understanding of the properties of NRPSs that cannot be addressed by conventional methods.

Total Synthesis of Decahydroquinoline Poison Frog Alkaloids ent-cis-195A and cis-211A.

The total synthesis of two decahydroquinoline poison frog alkaloids ent-cis-195A and cis-211A were achieved in 16 steps (38% overall yield) and 19 steps (31% overall yield), respectively, starting from known compound 1. Both alkaloids were synthesized from the common key intermediate 11 in a divergent fashion, and the absolute stereochemistry of natural cis-211A was determined to be 2R, 4aR, 5R, 6S, and 8aS. Interestingly, the absolute configuration of the parent decahydroquinoline nuclei of cis-211A was the mirror image of that of cis-195A, although both alkaloids were isolated from the same poison frog species, Oophaga (Dendrobates) pumilio, from Panama.

Inhibition of efflux pumps aids small-molecule probe-based fluorescence labeling and imaging in the Gram-negative bacterium Escherichia coli.

A major challenge in fluorescence imaging experiments, which are essential to determine protein activity, expression, and localization, is the penetration of small-molecule probes through the outer membrane permeability barrier of bacteria. Here, we describe a novel strategy for small-molecule probe-based fluorescence protein labeling and imaging in the Gram-negative bacterium Escherichia coli. We targeted a siderophore enterobactin biosynthetic enzyme EntE in E. coli. When coupled with an efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone, small-molecule probes were able to efficiently enter the cells, leading to the fluorescence labeling and imaging of overproduced EntE in E. coli. This study demonstrates that the combination of small-molecule probes with appropriate efflux pump inhibitors may substantially enhance their interaction with the target proteins in live bacteria.

Activity, Binding, and Modeling Studies of a Reprogrammed Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket.

The gatekeeping adenylation (A) domain of the non-ribosomal peptide synthetase (NRPS) selectively incorporates specific proteinogenic/non-proteinogenic amino acid into a growing peptide chain. The EntE of the enterobactin NRPS is a discrete aryl acid A-domain with 2,3-dihydroxybenzoic acid (DHB) substrate specificity. Reprogrammed EntE N235G variant possesses an enlarged substrate recognition site, and is capable of accepting non-native aryl acids. Biochemical characterization of this unique substrate recognition site should provide a better understanding of activi-site microenvironments. Here, we synthesized a non-hydrolysable adenylate analogue with 2-aminobenzoic acid (2-ABA), 3-aminobenzoic acid (3-ABA), and 4-aminobenzoic acid (4-ABA) and used them to calculate the apparent inhibition constants (Kiapp.). Dose-response experiments using 3-ABA-sulfamoyladenosine (AMS) provided Kiapp. values of 596 nM for wild-type EntE and 2.4 nM for the N235G variants. These results suggest that 3-amino group of benzoic acid plays an important role in substrate recognition by the N235G variant. These findings would help designing aryl acid substrates with substituents at the 2- and 3-positions.

Elongation of the side chain by linear alkyl groups increases the potency of salacinol, a potent α-glucosidase inhibitor from the Ayurvedic traditional medicine "Salacia," against human intestinal maltase.

Four chain-extended analogs (12a-12d) and two related de-O-sulfonated analogs (13a and 13c) by introducing alkyl groups (a: R = C3H7, b R = C6H13, c: R = C8H17, d: R = C10H21) to the side chains of salacinol (1), a natural α-glucosidase inhibitor from Ayurvedic traditional medicine "Salacia", were synthesized. The α-glucosidase inhibitory activities of all the synthesized analogs were evaluated in vitro. Against human intestinal maltase, the inhibitory activities of 12a and 13a with seven-carbon side chain were equal to that of 1. In contrast, analogs (12b-12d, and 13c) exhibited higher level of inhibitory activity against the same enzyme than 1 and had equal or higher potency than those of the clinically used anti-diabetics, voglibose, acarbose, and miglitol. Thus, elongation of the side chains of 1 was effective for specifically increasing the inhibitory activity against human intestinal maltase.

Ligand compatibility of salacinol-type α-glucosidase inhibitors toward the GH31 family.

We show that salacinol-type α-glucosidase inhibitors are ligand-compatible with the GH 31 family. Salacinol and its 3'-O-benzylated analogs inhibit human lysosomal α-glucosidase at submicromolar levels. Simple structure-activity relationship studies reveal that the salacinol side-chain stereochemistry significantly influences binding to GH31 α-glucosidases.

Probing the Compatibility of an Enzyme-Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains.

An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

Formal Syntheses of (-)-Lepadiformines A, C, and (-)-Fasicularin.

Marine tricyclic alkaloids lepadiformine and fasicularin, with a unique perhydropyrrolo[2,1- j]quinoline or perhydropyrido[2,1- j]quinoline framework, were synthesized starting from the B ring of the tricyclic system. This approach includes a highly stereocontrolled diallylation of a cyclic enaminoester and subsequent ring-closing metathesis to construct the A/B ring system, which was transformed into key lactams 32 and 33, and amino alcohol 37. Thus, we achieved formal syntheses of (-)-lepadiformines A, C, and (-)-fasicularin in a divergent manner.