Formation of Trideuteromethylated Artifacts of Pyrrole-Containing Natural Products.

Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.

A chemical probe based on the PreQ1 metabolite enables transcriptome-wide mapping of binding sites.

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs.

Recifin A, Initial Example of the Tyr-Lock Peptide Structural Family, Is a Selective Allosteric Inhibitor of Tyrosyl-DNA Phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel ß-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box.

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP.

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Experimental and Chemoinformatics Study of Tautomerism in a Database of Commercially Available Screening Samples.

We investigated how many cases of the same chemical sold as different products (at possibly different prices) occurred in a prototypical large aggregated database and simultaneously tested the tautomerism definitions in the chemoinformatics toolkit CACTVS. We applied the standard CACTVS tautomeric transforms plus a set of recently developed ring-chain transforms to the Aldrich Market Select (AMS) database of 6 million screening samples and building blocks. In 30 000 cases, two or more AMS products were found to be just different tautomeric forms of the same compound. We purchased and analyzed 166 such tautomer pairs and triplets by 1H and 13C NMR to determine whether the CACTVS transforms accurately predicted what is the same "stuff in the bottle". Essentially all prototropic transforms with examples in the AMS were confirmed. Some of the ring-chain transforms were found to be too "aggressive", i.e. to equate structures with one another that were different compounds.

Reactive Species Involved in the Regioselective Photooxidation of Heptamethine Cyanines.

Heptamethine cyanines are important near-IR fluorophores used in many fluorescence applications. Despite this utility, these molecules are susceptible to light-promoted reactions (photobleaching) involving photochemically generated reactive oxygen species (ROS). Here, we have sought to define key chemical aspects of this nearly inescapable process. Near-IR photolysis of a model heptamethine cyanine leads to the regioselective oxidative cleavage of the cyanine polyene. We report the first quantitative analysis of the major reaction pathway following either photolysis or exposure to candidate ROS. These studies clearly indicate that only singlet oxygen (1O2), and not other feasible ROS, recapitulates the direct photolysis pathway. Computational studies were employed to investigate the regioselectivity of the oxidative cleavage process, and the theoretical ratio is comparable to observed experimental values. These results provide a more complete picture of heptamethine cyanine photooxidation, and provide insight for design of improved compounds for future applications.

Neighbor-directed histidine N (τ)-alkylation: A route to imidazolium-containing phosphopeptide macrocycles.

Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.

Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.

Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP.

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.

Enhanced stereoselectivity of a Cu(II) complex chiral auxiliary in the synthesis of Fmoc-L-γ-carboxyglutamic acid.

L-γ-Carboxyglutamic acid (Gla) is an uncommon amino acid that binds avidly to mineral surfaces and metal ions. Herein, we report the synthesis of N-α-Fmoc-L-γ-carboxyglutamic acid γ,γ'-tert-butyl ester (Fmoc-Gla(O(t)Bu)(2)-OH), a suitably protected analogue for Fmoc-based solid-phase peptide synthesis. The residue was synthesized using a novel chiral Cu(II) complex, whose structure-based design was inspired by the blue copper protein rusticyanin. The five-coordinate complex is formed by Shiff base formation between glycine and the novel ligand (S)-2-(N-(2-methylthio)benzylprolyl)aminobenzophenone in the presence of copper. Michael addition of di-tert-butyl methylenemalonate to the α-carbon of the glycine portion of the complex occurs in a diastereoselective fashion. The resulting (S,S)-complex diastereomer can be easily purified by chromatography. Metal complex decomposition followed by Fmoc protection affords the enantiomerically pure amino acid. With the use of this novel chiral complex, the asymmetric synthesis of Fmoc-Gla(O(t)Bu)(2)-OH was completed in nine steps from thiosalicylic acid in 14.5% overall yield.

Using conformationally locked nucleosides to calibrate the anomeric effect: Implications for glycosyl bond stability.

Steric and electronic parameters such as the anomeric effect (AE) and gauche effect play significant roles in steering the North ⇆ South equilibrium of nucleosides in solution. Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in "fixed" relationships, either antiperiplanar where the AE is permanently "on", or gauche where the AE is impaired. The consequences of these two alternatives were subject to high-level calculations and measured experimentally by x-ray crystallography, hydrolytic stability of the glycosyl bond, and pKa values.

Inhibition of ABCG2-mediated drug efflux by naphthopyrones from marine crinoids.

Five new naphthopyrones (1-5) along with the known compounds TMC-256A1, 5,8-dihydroxy-6-methoxy-2-propyl-4H-naphtho[2,3-b]pyran-4-one, TMC-256C1, comaparvin, 6-methoxycomaparvin, and 6-methoxycomaparvin 5-methyl ether (6-11) were isolated from crinoids of the family Comasteridae. All compounds were tested for their ability to inhibit the multidrug transporter ABCG2, which plays a role in drug resistance. Six of the seven angular naphthopyrones showed moderate activity with <60% inhibition of ABCG2-mediated transport as compared to the positive control fumitremorgin C. None of the linear naphthopyrones inhibited ABCG2-mediated efflux.

North- and south-bicyclo[3.1.0]hexene nucleosides: the effect of ring planarity on anti-HIV activity.

The syntheses of new conformationally locked North- and South-bicyclo[3.1.0]hexene nucleosides is reported. The North analogues were synthesized by a convergent approach from the known (1S,2R,5R)-5-[(tert-butyldiphenylsilyloxy)methyl]bicyclo[3.1.0]hex-3-en-2-ol by Mitsunobu coupling with the nucleobases. The South analogues were synthesized from their bicyclo[3.1.0]hexane nucleoside precursors by the selective protection of the primary hydroxy group, conversion of the secondary alcohol into a good leaving group, and base-catalyzed elimination to generate the olefin. The transformation of a bicyclo[3.1.0]hexane nucleoside into a bicyclo[3.1.0]hexene nucleoside flattens the five-membered ring of the bicyclic system and rescues anti-HIV activity for North-D4T, North-D4A, and South-D4C. The relationship between planarity and the anti/syn disposition of the nucleobase that is favored by a particular pseudosugar platform are proposed as key parameters in controlling biological activity.

A novel side-bridged hybrid phosphonate/acetate pendant cyclam: synthesis, characterization, and 64Cu small animal PET imaging.

Copper-64 (t(1/2)=12.7h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik-Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with (64)Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the (64)Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.

What are the consequences of freezing the anomeric effect in nucleosides?

The consequences of freezing the orientation of the oxygen's lone pair orbitals--which determines whether the anomeric effect is operative or not--were studied theoretically and experimentally in two oxobicyclo-[3.1.0]hexane nucleosides (1 and 2). The results showed significant differences in the properties of these molecules, which correlated with the magnitude of the n2 --> sigma * delocalization.

Conformationally constrained analogues of diacylglycerol. 29. Cells sort diacylglycerol-lactone chemical zip codes to produce diverse and selective biological activities.

Diacylglycerol-lactone (DAG-lactone) libraries generated by a solid-phase approach using IRORI technology produced a variety of unique biological activities. Subtle differences in chemical diversity in two areas of the molecule, the combination of which generates what we have termed "chemical zip codes", are able to transform a relatively small chemical space into a larger universe of biological activities, as membrane-containing organelles within the cell appear to be able to decode these "chemical zip codes". It is postulated that after binding to protein kinase C (PKC) isozymes or other nonkinase target proteins that contain diacylglycerol responsive, membrane interacting domains (C1 domains), the resulting complexes are directed to diverse intracellular sites where different sets of substrates are accessed. Multiple cellular bioassays show that DAG-lactones, which bind in vitro to PKCalpha to varying degrees, expand their biological repertoire into a larger domain, eliciting distinct cellular responses.

Synthesis of a cross-bridged cyclam derivative for peptide conjugation and 64Cu radiolabeling.

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.