Rajesh Viswanathan

Free Radical-Mediated Aryl Amination : Convergent Two-and Three-Component Couplings to Chiral 2,3-Disubstituted Indolines

5-exo-trig Cyclization of an aryl radical to the nitrogen of an azomethine is used as the key annulating step in a modular preparation of 2,3-cis- and trans-disubstituted indolines. The precursors are readily prepared by phase-transfer-catalyzed Michael addition of a glycine Schiff base to a variety of acceptors. When the more reactive alkylidene malonate Michael acceptors are implemented, a one-pot three-component coupling is possible. The net result is a convergent [3 + 2] coupling strategy for the construction of highly functionalized indolines, a substructure occurring in numerous biologically active natural products.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection

Bacteria can utilize a mammalian host siderophore to usurp host iron; however, the host can respond by down-regulating siderophore expression and up-regulating expression of an inhibitory siderophore-binding protein.

Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria

BackgroundThe hapalindole-type family of natural products is a group of hybrid isoprenoid-indole alkaloids, produced solely by members of the Subsection V cyanobacterial strains. This family broadly includes the hapalindoles, welwitindolinones, fisherindoles and ambiguines amongst others, all of which have an isonitrile- or isothiocyanate-containing indole alkaloid skeleton, with a cyclized isoprene unit. The hapalindoles are diversified into the welwitindolinones, fischerindoles and ambiguines through the employment of tailoring oxygenase, methyltransferase and prenyltransferase enzymes. We compare the genetic basis for the biosynthesis of this diverse group of natural products and identify key early biosynthetic intermediates.ResultsWhole genome sequencing of freshwater and terrestrial cyanobacteria Westiella intricata UH strain HT-29-1, Hapalosiphon welwitschii UH strain IC-52-3, Fischerella ambigua UTEX 1903 and Fischerella sp. ATCC 43239 led to the identification of a candidate hapalindole-type gene cluster in each strain. These were compared with the recently published ambiguine and welwitindolinone gene clusters and four unpublished clusters identified within publicly available genomes. We present detailed comparative bioinformatic analysis of the gene clusters and the biosynthesis of a pivotal indole-isonitrile intermediate resulting in both cis and trans geometrical isomers. Enzyme analyses and metabolite extractions from two hapalindole-producing Fischerella strains indicate the presence of cis and trans indole-isonitriles as biosynthetic intermediates in the early steps of the pathway.ConclusionsInterestingly, the organization of the welwitindolinone gene cluster is conserved in all producing strains but distinct from the hapalindole and ambiguine clusters. Enzymatic assays using WelI1 and WelI3 from Westiella intricata UH strain HT-29-1 demonstrated the ability to catalyze the formation of both cis and trans geometrical isomers when using a cell lysate. The enzymatic and metabolic characterization of both cis and trans indole-isonitrile intermediates implies conservation of their stereochemical integrity towards members of the ambiguine and welwitindolinone products. In summary, we present data that supports a unified biosynthetic pathway towards hapalindoles in nine individual species of cyanobacteria. Diversification of the pathway occurs later through the employment of specialized enzymatic steps towards fischerindoles, ambiguines and welwitindolinones.

Branch-Selective Synthesis of Oxindole and Indene Scaffolds: Transition Metal-Controlled Intramolecular Aryl Amidation Leading to C3 Reverse-Prenylated Oxindoles

In an effort to access biologically important scaffolds, a concise branch-selective synthesis of C3 tertiary oxindoles by Cu(I)-catalyzed aryl amidation and 2,2-dimethyl indene by Pd(0)-catalyzed Heck cyclization has been accomplished from acyclic reverse-prenylated intermediates. Oxindole C3-enolate generation using NaH followed by alkylation in the presence of appropriate electrophiles provides a novel route to quaternary C3 reverse-prenylated oxindoles.

Regioselective covalent immobilization of catalytically active glutathione s-transferase on glass slides

The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His(6)-tagged glutathione S-transferase (His(6)-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His(6)-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω-alkyne and azide moieties and covalently attached to the surface by a Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in A(340) upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His(6)-GSTase-CVIA-PE) or azide (His(6)-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His(6)-GSTase-CVIA-F).

Synthetic, potentiometric and spectroscopic studies of chelation between Fe(III) and 2,5-DHBA supports salicylate-mode of siderophore binding interactions

Catecholate type enterobactin, a prototype siderophore, comprises 2,3-dihydroxybenzoic acid (2,3-DHBA) cyclically linked to serine in E. coli. The existence of iron-chelating ligands in humans is a recent discovery, however, the basic chemical interactions between 2,5-dihydroxybenzoic acid and Fe(III) ion remain poorly understood. Achieving an accurate description of the fundamental Fe(III) binding properties of 2,5-DHBA is essential for understanding its role in iron transport mechanisms. Here, we show that 2,5-DHBA binds iron in a salicylate mode via a two-step kinetic mechanism by UV spectroscopy. Complexation between Fe(III) salt and 2,5-DHBA initially occurs at 1:1 ratio (of ligand to metal) and binding resulting in higher-order complexes continues at higher concentrations. Through potentiometric measurements we quantify the distribution of Fe(III)-2,5-DHBA complexes with 1:1, 1:2 and 1:3 stoichiometry. The formation of 1:3 complexes is further supported through high-resolution mass spectrometry. Further, using kinetic and equilibrium UV spectroscopy, we report Fe(III)-2,5-DHBA complex formation at a pH range of 2.5-9.0 at 298.15K in water. Maximum complexation occurred at a pH range of 4.5-6.5 consistent with deprotonation of the carboxylic acid proton. Equilibrium measurements and stopped-flow kinetics show that complexation rate constants were independent of concentrations of 2,5-DHBA. Together the data supports a model in which the rate-determining step involves rearrangement of ligands on an initial complex formed by reversible binding between the carboxylate group of 2,5-DHBA and Fe(III).

Genome mining for natural product biosynthetic gene clusters in the Subsection V cyanobacteria

BackgroundCyanobacteria are well known for the production of a range of secondary metabolites. Whilst recent genome sequencing projects has led to an increase in the number of publically available cyanobacterial genomes, the secondary metabolite potential of many of these organisms remains elusive. Our study focused on the 11 publically available Subsection V cyanobacterial genomes, together with the draft genomes of Westiella intricata UH strain HT-29-1 and Hapalosiphon welwitschii UH strain IC-52-3, for their genetic potential to produce secondary metabolites. The Subsection V cyanobacterial genomes analysed in this study are reported to produce a diverse range of natural products, including the hapalindole-family of compounds, microcystin, hapalosin, mycosporine-like amino acids and hydrocarbons.ResultsA putative gene cluster for the cyclic depsipeptide hapalosin, known to reverse P-glycoprotein multiple drug resistance, was identified within three Subsection V cyanobacterial genomes, including the producing cyanobacterium H. welwitschii UH strain IC-52-3. A number of orphan NRPS/PKS gene clusters and ribosomally-synthesised and post translationally-modified peptide gene clusters (including cyanobactin, microviridin and bacteriocin gene clusters) were identified. Furthermore, gene clusters encoding the biosynthesis of mycosporine-like amino acids, scytonemin, hydrocarbons and terpenes were also identified and compared.ConclusionsGenome mining has revealed the diversity, abundance and complex nature of the secondary metabolite potential of the Subsection V cyanobacteria. This bioinformatic study has identified novel biosynthetic enzymes which have not been associated with gene clusters of known classes of natural products, suggesting that these cyanobacteria potentially produce structurally novel secondary metabolites.

Novel Quorum-Quenching Agents Promote Methicillin-Resistant Staphylococcus aureus (MRSA) Wound Healing and Sensitize MRSA to β-Lactam Antibiotics

ABSTRACT The dwindling repertoire of antibiotics to treat methicillin-resistant Staphylococcus aureus (MRSA) calls for novel treatment options. Quorum-quenching agents offer an alternative or an adjuvant to antibiotic therapy. Three biaryl hydroxyketone compounds discovered previously (F1, F12, and F19; G. Yu, D. Kuo, M. Shoham, and R. Viswanathan, ACS Comb Sci 16:85–91, 2014) were tested for efficacy in MRSA-infected animal models. Topical therapy of compounds F1 and F12 in a MRSA murine wound infection model promotes wound healing compared to the untreated control. Compounds F1, F12, and F19 afford significant survival benefits in a MRSA insect larva model. Combination therapy of these quorum-quenching agents with cephalothin or nafcillin, antibiotics to which MRSA is resistant in monotherapy, revealed additional survival benefits. The quorum-quenching agents sensitize MRSA to the antibiotic by a synergistic mode of action that also is observed in vitro. An adjuvant of 1 μg/ml F1, F12, or F19 reduces the MIC of nafcillin and cephalothin about 50-fold to values comparable to those for vancomycin, the antibiotic often prescribed for MRSA infections. These findings suggest that it is possible to resurrect obsolete antibiotic therapies in combination with these novel quorum-quenching agents.

Two Distinct Cyclodipeptide Synthases from a Marine Actinomycete Catalyze Biosynthesis of the Same Diketopiperazine Natural Product

Diketopiperazine natural products are structurally diverse and offer many biological activities. Cyclodipeptide synthases (CDPSs) were recently unveiled as a novel enzyme family that employs aminoacyl-tRNAs as substrates for 2,5-diketopiperazine assembly. Here, the Nocardiopsis sp. CMB-M0232 genome is predicted to encode two CDPSs, NozA and NcdA. Metabolite profiles from E. coli expressing these genes and assays with purified recombinant enzymes revealed that NozA and NcdA catalyze cyclo(l-Trp-l-Trp) (1) biosynthesis from tryptophanyl-tRNA and do not accept other aromatic aminoacyl-tRNA substrates. Fidelity is uncommon among characterized CDPSs, making NozA and NcdA important CDPS family additions. Further, 1 was previously supported as a biosynthetic precursor of the nocardioazines; the current study suggests that Nocardiopsis sp. may derive this precursor from both NozA and NcdA. This study offers a rare example of a single bacterium encoding multiple phylogenetically distinct enzymes that yield the same secondary metabolite and provides tools for chemoenzymatic syntheses of indole alkaloid diketopiperazines.

Characterization of the Nocardiopsin Biosynthetic Gene Cluster Reveals Similarities to and Differences from the Rapamycin and FK-506 Pathways

Macrolide‐pipecolate natural products, such as rapamycin (1) and FK‐506 (2), are renowned modulators of FK506‐binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB‐M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A–D (4–7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L‐lysine into the L‐pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP‐binding metabolites and for probing new enzymology in natures polyketide tailoring arsenal.