An efficient synthesis of 3'-O-triazole modified guanosine-5'-O-monophosphate using click chemistry.

First chemical synthesis of 3'-O-1,2,3-triazolyl-guanosine-5'-O-monophosphate by copper catalyzed click chemistry is described. The present cycloaddition reaction involves, in situ generation of azide from the corresponding bromide followed by copper catalyst cycloaddition with 3'-O-propargyl guanosine monophosphate in water, in the presence of catalytic amount of ß-cyclodextrin. The CuAAC reaction is highly regioselective forming 1,4-cycloadduct with good yield and high purity. The final compound, 3'-O -triazole substituted guanosine monophosphate has the potential to use in various biomolecules such as labeled nucleic acids, mRNA dinucleotide cap analogs for molecular biology and their applications in the therapeutic field.

Design, synthesis and antiviral evaluation of 2'-C-methyl branched guanosine pronucleotides: the discovery of IDX184, a potent liver-targeted HCV polymerase inhibitor.

BACKGROUND: Ribonucleoside analogs possessing a ß-methyl substituent at the 2'-position of the d-ribose moiety have been previously discovered to be potent and selective inhibitors of hepatitis C virus (HCV) replication, their triphosphates acting as alternative substrate inhibitors of the HCV RdRp NS5B. Results/methodology: In this article, the authors detail the synthesis, anti-HCV evaluation in cell-based replicon assays and structure-activity relationships of several phosphoramidate diester derivatives of 2'-C-methylguanosine (2'-MeG). CONCLUSION: The most promising compound, namely the O-[S-(hydroxyl)pivaloyl-2-thioethyl]{abbreviated as O-[(HO)tBuSATE)]} N-benzylamine phosphoramidate diester derivative (IDX184), was selected for further in vivo studies, and was the first clinical pronucleotide evaluated for the treatment of chronic hepatitis C up to Phase II trials.

Synthesis of a biotinylated photocleavable nucleotide monophosphate for the preparation of natively folded RNAs.

RNAs are involved in many functional roles in the cell, and this functional diversity is predicated on RNAs adopting requisite three-dimensional architectures. Preparing such "natively folded" RNAs with a homogeneous population is sometimes problematic for structural or enzymatic studies. Yet, standard methods for RNA preparations denature the RNA and create a heterogeneous population of conformers. Therefore, preparation of "natively folded" RNAs without going through the process of denaturing and refolding is important to obtain maximal biological function. Here, we present a simple strategy using "click" chemistry to couple biotin to a "caged" photocleavable (PC) guanosine monophosphate (GMP) in high yield. This biotin-PC-GMP is readily accepted by T7 RNA polymerase to transcribe "natively folded" RNAs ranging in size from 27 to 493 nucleotides. This facile strategy allows efficient biotinylation of RNA and provides a traceless means to remove the biotin after the purification. Such preparation of natively folded RNAs should benefit biophysical and therapeutic applications.

5'-Guanosine monophosphate mediated biocompatible porous hydrogel of ß-FeOOH-viscoelastic behavior, loading, and release capabilities of freeze-dried gel.

The present manuscript reports the characterization, optimization of rheological properties, and loading and release capabilities of 5'-GMP mediated ß-FeOOH hydrogel. Circular dichroism (CD) analysis indicates it to contain mainly the left-handed helix similar to that of Z-DNA. The highest viscosity (>300 cP) corresponds to the sample containing 2.5 × 10(-3) mol dm(-3) of 5'-GMP (SP2H). Field emission scanning electron microscope (FESEM) and transmission electron microscope (TEM) studies indicate the freeze-dried (FD) SP2H to be porous in nature, which is also supported by its high Brunauer-Emmett-Teller (BET) surface area of 226 m(2)/g as compared to that of SP3H (75 m(2)/g). Selected area electron diffraction (SAED) analysis and Raman spectroscopy show it to contain ß-FeOOH phase. The FD SP2H exhibits the high swelling ratio (326%) and loading capacity for methylene blue (MB) dye. It displays a controlled and efficient release (>90%) for optimized [MB] (2.5 × 10(-4) mol dm(-3)) in 48 h. The low toxicity of as synthesized FD SP2H nanostructures against MDA-MB-231 (breast cancer cells) up to 100 µg/mL suggests its biocompatible nature. The high porosity, surface area, % swelling, and loading and release performance of the hydrogel indicate its potential for drug delivery and other biological applications.

Structure-guided design, synthesis, and evaluation of guanine-derived inhibitors of the eIF4E mRNA-cap interaction.

The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5' terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the compound has an IC(50) of 2.5 µM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E inhibitors with physicochemical properties suitable for cellular activity assessment.

Convenient synthesis of a propargylated cyclic (3'-5') diguanylic acid and its "click" conjugation to a biotinylated azide.

The ribonucleoside building block, N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine, was prepared from commercial N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3'-5')guanosine phosphotriester was synthesized from the reaction of N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine with N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3'-/5'-deprotection, and purification. The propargylated guanylyl(3'-5')guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2'-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride affording the desired propargylated c-di-GMP diester, the purity of which exceeded 95%. Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.

Design, synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus.

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2'-beta-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2'-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.

Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers.

The full understanding of dynamics of cellular processes hinges on the development of efficient and non-invasive labels for intracellular RNA species. Light-up aptamers binding fluorogenic ligands show promise as specific labels for RNA species containing those aptamers. Herein, we took advantage of existing, non-light-up aptamers against small molecules and demonstrated a new class of light-up probes in vitro. We synthesized two conjugates of thiazole orange dye to small molecules (GMP and AMP) and characterized in vitro their interactions with corresponding RNA aptamers. The conjugates preserved specific binding to aptamers while showing several 100-fold increase in fluorescence of the dye (the 'light-up' property). In the presence of free small molecules, conjugates can be displaced from aptamers serving also as fluorescent sensors. Our in vitro results provide the proof-of-concept that the small-molecule conjugates with light-up properties can serve as a general approach to label RNA sequences containing aptamers.

Synthesis and evaluation of 2-pyrrolepolyamide-2'-deoxyguanosine 5'-phosphate.

2-Pyrrolepolyamide-2'-deoxyguanosine 5'-phosphate (hybrid 4) was synthesized and evaluated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 exhibited the highest activity compared with the other hybrids (1, 2, and 3) and distamycin A.

The first pure LambdaHT rotamer of a complex with a cis-[metal(nucleotide)2] unit: a cis-[Pt(amine)2(nucleotide)2] LambdaHT rotamer with unique molecular structural features.

cis-[PtA2(nucleotide)2] complexes (A2 stands for two amines or a diamine) have been extensively investigated as model compounds for key cisplatin-DNA adducts. All cis-[metal(nucleotide/nucleoside)2] complexes with guanine and related purines characterized in the solid state thus far have the DeltaHT conformation (head-to-tail orientation of the two bases and right-handed chirality). In sharp contrast, the LambdaHT conformation (left-handed chirality) dominates in acidic and neutral aqueous solutions of cis-[PtA2(5'-GMP)2] complexes. Molecular models and solution experiments indicate that the LambdaHT conformer is stabilized by 5'-phosphate/N1H hydrogen-bond interactions between cis nucleotides with the normal anti conformation. However, this evidence, while compelling, is indirect. At last, conditions have been defined to allow crystallization of this elusive conformer. The structure obtained reveals three unique features not present in all other cis-[PtA2(nucleotide)2] solid-state structures: a LambdaHT conformation, very strong hydrogen-bond interactions between the phosphate and N1H of cis nucleotides, and a very small dihedral angle between the planes of the two guanines lying nearly perpendicular to the coordination plane. These new results indicate that, because there are no local base-base repulsions precluding the LambdaHT conformer, global forces rather than local interactions account for the predominance of the DeltaHT conformer over the LambdaHT conformer in the solid state and in both inter- and intrastrand HT crosslinks of oligonucleotides and DNA.

Efficient preparation of organic substrate-RNA conjugates via in vitro transcription.

A concise synthetic way has been developed for the preparation of guanosine monophosphate derivatives carrying a decaethylene glycol spacer at their 5'-oxygen to which are attached a range of organic substrates. The four different compounds, prepared via a convergent synthetic strategy, carry a tethered benzylallyl ether residue (1a), an anthracene (1b), a benzyl carbamate residue (1c), or a primary amino group (1d), respectively. All four compounds have been successfully incorporated at the 5'-end of a 25-mer long RNA transcript via T7 RNA polymerase, and no inhibition of chain elongation could be observed. Under proper conditions, 1a and 1b can be incorporated up to 90-95% and 1c up to 68%. The amino-terminated initiator 1d is incorporated less efficiently although still up to 49%. These results show that the more hydrophobic the guanosine monophosphate derivative is, the higher is its enzymatic incorporation.

The sulfinyl moiety as an internal nucleophile. 2. Stereoselective synthesis of (-)-galantinic acid via 1,3-asymmetric induction.

A stereoselective synthesis of (-)-galantinic acid is disclosed. The key steps include hydrolytic kinetic resolution of a racemic epoxide and regio- and stereoselective heterofunctionalization of an olefin, using a pendant sulfinyl group as the nucleophile. The participation of the sulfinyl group was unambiguously proven by conducting the reaction in the presence of H(2)(18)O.

Ground-state stability and rotational activation parameters for individual rotamers of (R,S,S,R)-(N,N'-dimethyl-2,3-diaminobutane)PtG(2) complexes (G = 9-EtG, 3'-GMP, and 5'-GMP).

Rate constants for guanine rotation about the Pt-N7 bond in (R,S,S,R)-Me(2)DABPtG(2) complexes (Me(2)DAB = N,N'-dimethyl-2,3-diaminobutane; G = 9-EtG, 3'-GMP, and 5'-GMP) were evaluated from line-shape analysis of H8 resonances. Three diastereomers, two in head-to-tail (DeltaHT and LambdaHT) and one in head-to-head (HH) conformations, exist in equilibrium in solution. The two guanines are equivalent in DeltaHT and LambdaHT conformers and nonequivalent in the HH form; therefore, four rate constants (k(Delta)(HT), k(Lambda)(HT), k(HH)()s, and k(HH)()d; sub-subscripts s and d stand for H8-shielded and -deshielded guanine, respectively) were evaluated. Activation parameters (DeltaH and DeltaS) were evaluated from the rate constant dependence on temperature. High values of DeltaH (78-93 kJ mol(-)(1)) and DeltaS (51-71 J K(-)(1) mol(-)(1)) were found for G rotation in the preferred DeltaHT rotamer having the six-membered ring of each guanine more canted toward the cis-G and a favorable dipole-dipole internucleotide interaction. Lower values of DeltaH() (64-76 kJ mol(-)(1)) and very small values of DeltaS() (-7-11 J K(-)(1) mol(-)(1)) were found for G rotation in the less favorable LambdaHT rotamer, indicating that the ground-state entropy of this rotamer is close to that of the activated complex and the ground-state enthalpy closer to that of the activated complex than for the DeltaHT rotamer. For the two guanines in the HH rotamer there is no large difference in activation parameters. In general DeltaH falls in the range 66-84 kJ mol(-)(1) (rather close to the values for the LambdaHT rotamer) and DeltaS in the range 14-41 J K(-)(1) mol(-)(1). The equilibrium constant between HT and HH rotamers was also evaluated together with the corresponding thermodynamic parameters (DeltaH and DeltaS). It is found that the low enthalpy is the major stabilizing factor for DeltaHT as compared to HH, while the entropy factor would favor the latter rotamer. In contrast the greater entropy is the stabilizing factor for the LambdaHT rotamer (the second most abundant conformer for 9-EtG and 3'-GMP) over the HH rotamer. In the latter case the enthalpy would favor the HH rotamer. In the case of the 5'-GMP derivative the greater entropy of the LambdaHT rotamer is not such to compensate for the lower enthalpy of the HH rotamer, and the latter remains the second most abundant rotamer. This investigation has allowed for the first time the enthalpic and entropic contributions favoring different rotamers to be distinguished.

Modification of second-sphere communication, leading to an unusually high abundance of the head-to-head conformer of cisplatin cross-link retro models.

Rapid atropisomerization of cisplatin-DNA cross-link models, cis-PtA(2)G(2) (A(2) = two amines or a diamine, G = guanine derivative, bold font indicating a guanine not linked to another guanine), makes their NMR spectra uninformative. The conformers [two head-to-tail (DeltaHT and LambdaHT) conformers, one head-to-head (HH) conformer] are detected in (CCC)PtG(2) retro models (CCC = chirality-controlling chelates designed to reduce rotation around the G N7 to Pt bond by destabilizing the transition state). Clear trends are found with four CCC ligands, 2,2'-bipiperidine (Bip) and N,N'-dimethyl-2,3-diaminobutane (each with S,R,R,S and R,S,S,R configurations at the chelate ring N, C, C, and N atoms, respectively). S,R,R,S ligands favor left-handed G base canting and the LambdaHT form; R,S,S,R ligands favor right-handed canting and the DeltaHT form. The HH conformer is normally negligible unless G = 5'-GMP. However, understanding this 5'-phosphate effect is complicated by possible interligand interactions of the 5'-phosphate with the N1H of the cis-5'-GMP and a CCC NH; these interactions are referred to as second-sphere (SSC) and first-to-second-sphere (FSC) communication, respectively. We now investigate the four (CCC)PtG(2) models with 1-Me-5'-GMP, a G lacking the N1H needed for SSC. The phosphate location makes FSC possible in the major but not the minor HT form. The major form should increase from pH 3 to pH 7 because the phosphate is deprotonated at pH 7. However, the major DeltaHT form for the R,S,S,R pair did not change in abundance, and the major LambdaHT form for the S,R,R,S pair actually decreased. Thus, FSC is weak. At pH approximately 7 the HH conformer of the S,R,R,S pair had an abundance (40-44%) higher than that in any reported cis-PtA(2)G(2) adduct. FSC involving one 1-Me-5'-GMP could play a role. The high HH abundance and use of a pH jump experiment with (S,R,R,S)-BipPt(1-Me-5'-GMP)(2) allowed us to obtain the first deconvoluted CD spectrum for a cis-PtA(2)G(2) HH conformer. The CD features for the HH conformer are much weaker than for the HT conformers. Our findings are best interpreted to indicate that FSC is not important in aqueous solution, especially for the HT form. Weak FSC is consistent with recent models of the cross-link in duplexes. In contrast, crystals of fluxional models often reveal FSC, but not the more important SSC. SSC was unrecognized until our retro model studies, and the new results reinforce the value of studying retro models for identifying interactions in solution.

Kinetics of template-directed pyrophosphate-linked dideoxyguanylate synthesis as a function of 2-MeImpdG and poly(C) concentration: insights into the mechanism.

Aqueous solutions of deoxyguanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpdG, yield primarily deoxyguanosine 5'-monophosphate, 5'dGMP, and pyrophosphate-linked dideoxyguanylate, dG5'ppdG, abbreviated G2p (see Chart 1). The initial rate of G2p formation, d[G2p]/dt in M h-1, determined at 23 degrees C, pH 7.8, 1.0 M NaCl and 0.2 M Mg2+ by timed high-performance liquid chromatography (HPLC) analysis, exhibits a second-order dependence on 2-MeImpdG concentration, [G]o, indicating a bimolecular mechanism of dimerization in the range 0.02 M < or = [G]o < or = 0.09 M. In the presence of polycytidylate, poly(C), G2p synthesis is accelerated and oligodeoxyguanylate products are formed by incorporation of 2-MeImpdG molecules. The kinetics of G2p formation as a function of both monomer and polymer concentration, expressed in C equivalents, were also determined under the above conditions and exhibited a complex behavior. Specifically, at a constant [poly(C)], values of d[G2p]/dt typically increased with [G]o with a parabolic upward curvature. At a constant [G]o, values of d[G2p]/dt increase with [poly(C)], but level off at the higher poly(C) concentrations. As [G]o increases this saturation occurs at a higher poly(C) concentration, a result opposite to expectation for a simple complexation of two reacting monomers with the catalyst prior to reaction. Nevertheless, these results are shown to be quantitatively consistent with a template-directed (TD) mechanism of dimerization where poly(C) acts as the template to bind 2-MeImpdG in a cooperative manner and lead, for the first time, to the formulation of principles that govern template-directed chemistry. Analysis of the kinetic data via a proposed TD cooperative model provides association constants for the affinity between polymer and monomer and the intrinsic reactivity of 2-MeImpdG toward pyrophosphate synthesis. To the best of our knowledge, poly(C)/2-MeImpdG is the first system that could serve as a textbook example of a TD reaction under conditions such that the template is fully saturated by monomers and under conditions that it is not.

Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring.

GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.

Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate and adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate. New nucleotide affinity labels which react with rabbit muscle pyruvate kinase.

Three new reactive nucleotide analogues with bromo-keto substituents adjacent to a thiophosphate have been synthesized. Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (GMPS-BDB), reacts covalently with rabbit muscle pyruvate kinase with complete inactivation and incorporation of 1.8 mol of reagent/mol of enzyme subunit. By contrast, the mono-keto compound, guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPS-BOP), causes no loss of pyruvate kinase activity. When the analogous adenosyl nucleotide derivatives are incubated with pyruvate kinase, the di-keto compound, adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (AMPS-BDB), rapidly effects inactivation, whereas the mono-keto compound, adenosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (AMPS-BOP), causes no loss of activity. Complete protection against inactivation by GMPS-BDB is provided by phosphoenolpyruvate in the presence of K+ and Mn2+ and the amount of reagent incorporated (0.9 mol/reagent/mol subunit) is reduced to half that observed in the absence of protectants. Gas-phase sequencing of the tryptic peptides purified from inactive GMPS-BDB or AMPS-BDB-modified enzyme gave the cysteine-labeled peptides: C151DENILWLDYK161, and N162IC164K165 as the two major peptide products, with a smaller amount of N43TGIIC48TIGPASR55. Reaction in the presence of the protectants PEP, K+, and Mn2+ yielded Cys164 as the only labeled residue, indicating that inactivation is primarily due to modification of Cys151. We propose that GMPS-BDB (or AMPS-BDB), which may exist in enolized form in aqueous solution, functions as a reactive analogue of phosphoenolpyruvate and GDP (ADP) to target Cys151 in the active site of pyruvate kinase.

Guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate: a new reactive purine nucleotide analog labeling Met-169 and Tyr-262 in bovine liver glutamate dehydrogenase.

A new guanosine nucleotide has been synthesized and characterized: guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPSBOP), with a reactive functional group which can be placed at a position equivalent to the pyrophosphate region of GTP. This new analog is negatively charged at neutral pH and is similar in size to GTP. GMPSBOP has been shown to react with bovine liver glutamate dehydrogenase with an incorporation of 2 mol of reagent/mol of subunit. The modification reaction desensitizes the enzyme to inhibition by GTP, activation by ADP, and inhibition by high concentrations of NADH, but does not affect the catalytic activity of the enzyme. The rate constant for reaction of GMPSBOP with the enzyme exhibits a nonlinear dependence on reagent concentration with KD = 75 microM. The addition to the reaction mixture of alpha-ketoglutarate, GTP, ADP, or NADH alone results in little decrease in the rate constant, but the combined addition of 5 mM NADH with 0.4 mM GTP or with 10 mM alpha-ketoglutarate reduces the reaction rate approximately 6-fold. GMPSBOP modifies peptides containing Met-169 and Tyr-262, of which Tyr-262 is not critical for the decreased sensitivity of the enzyme toward allosteric ligands. The presence of 0.4 mM GTP plus 5 mM NADH protects the enzyme against reaction at both Met-169 and Tyr-262, but yields enzyme with 1 mol of reagent incorporated/mol of subunit which is modified at an alternate site, Met-469. In the presence of 0.2 mM GTP + 0.1 mM NADH, protection against modification of Tyr-262, but only partial protection against labeling of Met-169, is observed. In contrast, the presence of 10 mM alpha-ketoglutarate + 5 mM NADH protect only against reaction with Met-169. The results suggest that GMPSBOP reacts at the GTP-dependent NADH regulatory site [Lark, R. H., & Colman, R. F. (1986) J. Biol. Chem. 261, 10659-10666] of bovine liver glutamate dehydrogenase, which markedly affects the sensitivity of the enzyme to GTP inhibition. The reaction of GMPSBOP with Met-169 is primarily responsible for the altered allosteric properties of the enzyme.