Biomimetic Total Synthesis of (+)-Nocardioazine B and Analogs.

Nocardioazines A and B are prenylated, bioactive pyrroloindoline natural products, isolated from Nocardiopsis, with a desymmetrized cyclo-d-Trp-d-Trp DKP core. Based on our deeper biosynthetic understanding, a biomimetic total synthesis of (+)-nocardioazine B is accomplished in merely seven steps and 23.2% overall yield. This pathway accesses regio- and stereoselectively C3-isoprenylated analogs of (+)-nocardioazine B, using the same number of steps and in similar efficiency. The successful strategy mandated that the biomimetic C3-prenylation step be executed early. The use of an unprotected carboxylic acid of Trp led to high diastereoselectivity toward formation of key intermediates exo-12a, exo-12b, and exo-12c (>19:1). Evidence shows that N1-methylation causes the prenylation reaction to bifurcate away to result in a C2-normal-prenylated isomer. Nocardioazine A, possessing an isoprenoidal-epoxide bridge, inhibits P-glycoprotein (P-gp)-mediated membrane efflux, in multidrug-resistant mammalian colon cancer cells. As several P-gp inhibitors have failed due to their toxicity effects, endogenous amino-acid-derived noncytotoxic inhibitors (from the nocardioazine core) are worthy leads toward a rejuvenated strategy against resistant carcinomas. This total synthesis provides direct access to Trp-derived isoprenylated DKP natural products and their derivatives.

Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule.

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.

Phylogenetic sequence analysis and functional studies reveal compensatory amino acid substitutions in loop 2 of human ribonucleotide reductase.

Eukaryotic class I ribonucleotide reductases (RRs) generate deoxyribonucleotides for DNA synthesis. Binding of dNTP effectors is coupled to the formation of active dimers and induces conformational changes in a short loop (loop 2) to regulate RR specificity among its nucleoside diphosphate substrates. Moreover, ATP and dATP bind at an additional allosteric site 40 Å away from loop 2 and thereby drive formation of activated or inactive hexamers, respectively. To better understand how dNTP binding influences specificity, activity, and oligomerization of human RR, we aligned >300 eukaryotic RR sequences to examine natural sequence variation in loop 2. We found that most amino acids in eukaryotic loop 2 were nearly invariant in this sample; however, two positions co-varied as nonconservative substitutions (N291G and P294K; human numbering). We also found that the individual N291G and P294K substitutions in human RR additively affect substrate specificity. The P294K substitution significantly impaired effector-induced oligomerization required for enzyme activity, and oligomerization was rescued in the N291G/P294K enzyme. None of the other mutants exhibited altered ATP-mediated hexamerization; however, certain combinations of loop 2 mutations and dNTP effectors perturbed ATP's role as an allosteric activator. Our results demonstrate that the observed compensatory covariation of amino acids in eukaryotic loop 2 is essential for its role in dNTP-induced dimerization. In contrast, defects in substrate specificity are not rescued in the double mutant, implying that functional sequence variation elsewhere in the protein is necessary. These findings yield insight into loop 2's roles in regulating RR specificity, allostery, and oligomerization.

Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity.

Class I ribonucleotide reductase (RR) maintains balanced pools of deoxyribonucleotide substrates for DNA replication by converting ribonucleoside diphosphates (NDPs) to 2'-deoxyribonucleoside diphosphates (dNDPs). Binding of deoxynucleoside triphosphate (dNTP) effectors (ATP/dATP, dGTP, and dTTP) modulates the specificity of class I RR for CDP, UDP, ADP, and GDP substrates. Crystal structures of bacterial and eukaryotic RRs show that dNTP effectors and NDP substrates bind on either side of a flexible nine-amino acid loop (loop 2). Interactions with the effector nucleobase alter loop 2 geometry, resulting in changes in specificity among the four NDP substrates of RR. However, the functional groups proposed to drive specificity remain untested. Here, we use deoxynucleoside analogue triphosphates to determine the nucleobase functional groups that drive human RR (hRR) specificity. The results demonstrate that the 5-methyl, O4, and N3 groups of dTTP contribute to specificity for GDP. The O6 and protonated N1 of dGTP direct specificity for ADP. In contrast, the unprotonated N1 of adenosine is the primary determinant of ATP/dATP-directed specificity for CDP. Structural models from X-ray crystallography of eukaryotic RR suggest that the side chain of D287 in loop 2 is involved in binding of dGTP and dTTP, but not dATP/ATP. This feature is consistent with experimental results showing that a D287A mutant of hRR is deficient in allosteric regulation by dGTP and dTTP, but not ATP/dATP. Together, these data define the effector functional groups that are the drivers of human RR specificity and provide constraints for evaluating models of allosteric regulation.

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

BACKGROUND: Cyanobacteria 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. RESULTS: A 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. CONCLUSIONS: Genome 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 ß-lactam antibiotics.

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.

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 nature's polyketide tailoring arsenal.

Correction: Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis.

Correction for 'Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis' by Norah Alqahtani et al., Org. Biomol. Chem., 2015, 13, 7177-7192.

Synergism between genome sequencing, tandem mass spectrometry and bio-inspired synthesis reveals insights into nocardioazine B biogenesis.

Marine actinomycete-derived natural products continue to inspire chemical and biological investigations. Nocardioazines A and B (3 and 4), from Nocardiopsis sp. CMB-M0232, are structurally unique alkaloids featuring a 2,5-diketopiperazine (DKP) core functionalized with indole C3-prenyl as well as indole C3- and N-methyl groups. The logic of their assembly remains cryptic. Bioinformatics analyses of the Nocardiopsis sp. CMB-M0232 draft genome afforded the noz cluster, split across two regions of the genome, and encoding putative open reading frames with roles in nocardioazine biosynthesis, including cyclodipeptide synthase (CDPS), prenyltransferase, methyltransferase, and cytochrome P450 homologs. Heterologous expression of a twelve gene contig from the noz cluster in Streptomyces coelicolor resulted in accumulation of cyclo-l-Trp-l-Trp DKP (5). This experimentally connected the noz cluster to indole alkaloid natural product biosynthesis. Results from bioinformatics analyses of the noz pathway along with challenges in actinomycete genetics prompted us to use asymmetric synthesis and mass spectrometry to determine biosynthetic intermediates in the noz pathway. The structures of hypothesized biosynthetic intermediates 5 and 12-17 were firmly established through chemical synthesis. LC-MS and MS-MS comparison of these synthetic compounds with metabolites present in chemical extracts from Nocardiopsis sp. CMB-M0232 revealed which of these hypothesized intermediates were relevant in the nocardioazine biosynthetic pathway. This established the early and mid-stages of the biosynthetic pathway, demonstrating that Nocardiopsis performs indole C3-methylation prior to indole C3-normal prenylation and indole N1'-methylation in nocardioazine B assembly. These results highlight the utility of merging bioinformatics analyses, asymmetric synthetic approaches, and mass spectrometric metabolite profiling in probing natural product biosynthesis.

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

BACKGROUND: The 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. RESULTS: Whole 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. CONCLUSIONS: Interestingly, 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.

Regioselective cope rearrangement and prenyl transfers on indole scaffold mimicking fungal and bacterial dimethylallyltryptophan synthases.

Aromatic prenyltransferases are an actively mined enzymatic class whose biosynthetic repertoire is growing. Indole prenyltransferases catalyze the formation of a diverse set of prenylated tryptophan and diketopiperazines, leading to the formation of fungal toxins with prolific biological activities. At a fundamental level, the mechanism of C4-prenylation of l-tryptophan recently has surfaced to engage a debate between a "direct" electrophilic alkylation mechanism (for wt DMATS and FgaPT2) versus an indole C3-C4 "Cope" rearrangement followed by rearomatization (for mutant FgaPT2). Herein we provide the first series of regioselectively tunable conditions for a Cope rearrangement between C3 and C4 positions. Biomimetic conditions are reported that effect a [3,3]-sigmatropic shift whose two-step process is interrogated for intramolecularity and rate-limiting general base-promoted mechanism. Solvent polarity serves a crucial role in changing the regioselectivity, resulting in sole [1,3]-shifts under decalin. An intermolecular variant is also reported that effectively prenylates the C3 position of l-tryptophan, resulting in products that mimic the structures accessed by bacterial indole prenyltransferases. We report an elaborate investigation that includes screening various substituents and measuring steric and electronic effects and stereoselectivity with synthetically useful transformations.

Exploring bat-inspired cyclic tryptophan diketopiperazines as ABCB1 Inhibitors.

Chemotherapy-induced drug resistance remains a major cause of cancer recurrence and patient mortality. ATP binding cassette subfamily B member 1 (ABCB1) transporter overexpression in tumors contributes to resistance, yet current ABCB1 inhibitors have been unsuccessful in clinical trials. To address this challenge, we propose a new strategy using tryptophan as a lead molecule for developing ABCB1 inhibitors. Our idea stems from our studies on bat cells, as bats have low cancer incidences and high ABCB1 expression. We hypothesized that potential ABCB1 substrates in bats could act as competitive inhibitors in humans. By molecular simulations of ABCB1-substrate interactions, we generated a benzylated Cyclo-tryptophan (C3N-Dbn-Trp2) that inhibits ABCB1 activity with efficacy comparable to or better than the classical inhibitor, verapamil. C3N-Dbn-Trp2 restored chemotherapy sensitivity in drug-resistant human cancer cells with no adverse effect on cell proliferation. Our unique approach presents a promising lead toward developing effective ABCB1 inhibitors to treat drug-resistant cancers.

Self-catalyzed immobilization of GST-fusion proteins for genome-encoded biochips.

With the surge of proteomic information that has become available in recent years from genome sequencing projects, selective and robust technologies for making protein biochips have become increasingly desirable. Herein, we describe the development of small-molecule SNAr electrophiles (smSNAREs), a new class of capture probes that enables a selective, single-step immobilization for protein biochips. This enzymology-driven approach rides on the binding and catalytic mechanism of SjGST. We have designed and synthesized mechanism-based substrate analogs 3, 4, and 5 as electrophilic precursors for conjugation of glutathione S-transferase (GST) or any of its fusion proteins. Upon evaluating the conjugation of these probes to glutathione in the presence of SjGST via UV­visible spectroscopy (UV­vis) and LC-MS techniques, we found that 3, 4, and 5 were transferable to GSH. Through the anchoring of alkyne 5 as a smSNARE probe on glass surface, we demonstrate the single-step, self-catalyzed immobilization of SjGST. Fluorescence imaging quantitatively revealed an 18-fold increase in selective binding of SjGST over random orientations (due to nonspecific binding) of the protein. Binding between GST and smSNARE surface is robust and does not reverse upon adding up to 100 mM GSH. Further, a 6-fold increase in resolution for the smSNARE surface probe was observed over commonly employed commercially available GSH-epoxy surfaces. Detailed control experiments revealed insights into the reversibility of binding and catalysis of GSH to form conjugation products with 5 in the presence of the enzyme. As an application of this protein capture technology, we printed alkaloid biosynthesis enzyme, isonitrile synthase (IsnA), to result in a biochip. Because proteins bearing a GST-fusion purification tag are commonly created through the pGEX expression system, these findings show broad potential applicability to genome-wide studies and proteomic platforms.

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).

Synthesis of a suite of bioorthogonal glutathione S-transferase substrates and their enzymatic incorporation for protein immobilization.

Label-free protein immobilization allows precise detection of biomolecular events. Preserving enzyme function is intrinsically challenging for these strategies. Considering that glutathione S-transferase (GST) is a broadly employed enzymatic fusion tag, we reported a label-free self-catalyzed immobilization for Schistosoma japonicum GST. We now report the synthesis, structure, and enzymology of a set of 20 smSNAREs (small molecule SNAr-electrophiles). These smSNAREs mimic (electronically) the canonical GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), and bear a wide variety of bioorthogonal functionalities such as alkynes, aldehydes, acetals, and azides. Sixteen analogues including the chloro- and nitro-substituted 1, 3, 5, 6, 7, 11, 12, and 13 participated in the GST-catalyzed conjugation, indicating the substrate tolerance of the enzymatic H-site of SjGST. Using UV-vis spectroscopy, we estimate the efficiency of conjugation as a function of substrate diversity. Using LC-MS, we characterized the conjugates formed under each enzymatic transformation. Significant deviations from the canonical CDNB architecture are tolerated. Relative rates between nitro and chloro substituents indicate the nucleophilic addition step is rate determining. Enzyme immobilization on glass slides is affected by additional surface interactions and therefore does not reflect kinetic profiles observed in solution. This new class of heterobifunctional linkers enables a single-step and uniform protein capture on designer surfaces.

Unveiling an indole alkaloid diketopiperazine biosynthetic pathway that features a unique stereoisomerase and multifunctional methyltransferase.

The 2,5-diketopiperazines are a prominent class of bioactive molecules. The nocardioazines are actinomycete natural products that feature a pyrroloindoline diketopiperazine scaffold composed of two D-tryptophan residues functionalized by N- and C-methylation, prenylation, and diannulation. Here we identify and characterize the nocardioazine B biosynthetic pathway from marine Nocardiopsis sp. CMB-M0232 by using heterologous biotransformations, in vitro biochemical assays, and macromolecular modeling. Assembly of the cyclo-L-Trp-L-Trp diketopiperazine precursor is catalyzed by a cyclodipeptide synthase. A separate genomic locus encodes tailoring of this precursor and includes an aspartate/glutamate racemase homolog as an unusual D/L isomerase acting upon diketopiperazine substrates, a phytoene synthase-like prenyltransferase as the catalyst of indole alkaloid diketopiperazine prenylation, and a rare dual function methyltransferase as the catalyst of both N- and C-methylation as the final steps of nocardioazine B biosynthesis. The biosynthetic paradigms revealed herein showcase Nature's molecular ingenuity and lay the foundation for diketopiperazine diversification via biocatalytic approaches.

Biocatalysts from cyanobacterial hapalindole pathway afford antivirulent isonitriles against MRSA.

The emergence of resistance to frontline antibiotics has called for novel strategies to combat serious pathogenic infections. Methicillin-resistant Staphylococcus aureus [MRSA] is one such pathogen. As opposed to traditional antibiotics, bacteriostatic anti-virulent agents disarm MRSA, without exerting pressure, that cause resistance. Herein, we employed a thermophilic Thermotoga maritima tryptophan synthase (TmTrpB1) enzyme followed by an isonitrile synthase and Fe(II)-α-ketoglutarate-dependent oxygenase, in sequence as biocatalysts to produce antivirulent indole vinyl isonitriles. We report on conversion of simple derivatives of indoles to their C3-vinyl isonitriles, as the enzymes employed here demonstrated broader substrate tolerance. In toto, eight distinct L-Tryptophan derived α-amino acids (7) were converted to their bioactive vinyl isonitriles (3) by action of an isonitrile synthase (WelI1) and an Fe(II)-α-ketoglutarate-dependent oxygenase (WelI3) yielding structural variants possessing antivirulence against MRSA. These indole vinyl isonitriles at 10 µg/mL are effective as antivirulent compounds against MRSA, as evidenced through analysis of rabbit blood hemolysis assay. Based on a homology modelling exercise, of enzyme-substrate complexes, we deduced potential three dimensional alignments of active sites and glean mechanistic insights into the substrate tolerance of the Fe(II)-α-ketoglutarate-dependent oxygenase.

Bioinspired Brønsted Acid-Promoted Regioselective Tryptophan Isoprenylations.

Tryptophan-containing isoprenoid indole alkaloid natural products are well known for their intricate structural architectures and significant biological activities. Nature employs dimethylallyl tryptophan synthases (DMATSs) or aromatic indole prenyltransferases (iPTs) to catalyze regio- and stereoselective prenylation of l-Trp. Regioselective synthetic routes that isoprenylate cyclo-Trp-Trp in a 2,5-diketopiperazine (DKP) core, in a desymmetrizing manner, are nonexistent and are highly desirable. Herein, we present an elaborate report on Brønsted acid-promoted regioselective tryptophan isoprenylation strategy, applicable to both the monomeric amino acid and its dimeric l-Trp DKP. This report outlines a method that regio- and stereoselectively increases sp3 centers of a privileged bioactive core. We report on conditions involving screening of Brønsted acids, their conjugate base as salt, solvent, temperature, and various substrates with diverse side chains. Furthermore, we extensively delineate effects on regio- and stereoselection of isoprenylation and their stereochemical confirmation via NMR experiments. Regioselectively, the C3-position undergoes normal-isoprenylation or benzylation and forms exo-ring-fused pyrroloindolines selectively. Through appropriate prenyl group migrations, we report access to the bioactive tryprostatin alkaloids, and by C3-normal-farnesylation, we access anticancer drimentines as direct targets of this method. The optimized strategy affords iso-tryprostatin B-type products and predrimentine C with 58 and 55% yields, respectively. The current work has several similarities to biosynthesis, such as-reactions can be performed on unprotected substrates, conditions that enable Brønsted acid promotion, and they are easy to perform under ambient conditions, without the need for stoichiometric levels of any transition metal or expensive ligands.

Structure-Guided Synthesis and Mechanistic Studies Reveal Sweetspots on Naphthyl Salicyl Hydrazone Scaffold as Non-Nucleosidic Competitive, Reversible Inhibitors of Human Ribonucleotide Reductase.

Ribonucleotide reductase (RR), an established cancer target, is usually inhibited by antimetabolites, which display multiple cross-reactive effects. Recently, we discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH or E-3a) of human RR (hRR) binding at the catalytic site (C-site) and inhibiting hRR reversibly. We herein report the synthesis and biochemical characterization of 25 distinct analogs. We designed each analog through docking to the C-site of hRR based on our 2.7 Å X-ray crystal structure (PDB ID: 5TUS). Broad tolerance to minor structural variations preserving inhibitory potency is observed. E-3f (82% yield) displayed an in vitro IC50 of 5.3 ± 1.8 µM against hRR, making it the most potent in this series. Kinetic assays reveal that E-3a, E-3c, E-3t, and E-3w bind and inhibit hRR through a reversible and competitive mode. Target selectivity toward the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.

Synthesis of the ABC- and D-ring systems of the indole alkaloid ambiguine G.

A glycine Schiff base Michael addition is used in sequence with free radical-mediated aryl amination to ultimately arrive at the ambiguine G ABC-tricycle convergently. Additionally, thermal Diels-Alder cycloaddition of beta-chloromethacrolein with Cohen's diene is used to diastereoselectively construct the D-cyclohexane ring bearing a neopentyl chloride.