Covalent inactivation of Mycobacterium thermoresistibile inosine-5'-monophosphate dehydrogenase (IMPDH).

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in nucleotide biosynthesis. Because of its critical role in purine biosynthesis, IMPDH is a drug design target for immunosuppressive, anticancer, antiviral and antimicrobial chemotherapy. In this study, we use mass spectrometry and X-ray crystallography to show that the inhibitor 6-Cl-purine ribotide forms a covalent adduct with the Cys-341 residue of Mycobacterium thermoresistibile IMPDH.

Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides.

Theories about the origin of life require chemical pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Separate pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.

Prebiotic stereoselective synthesis of purine and noncanonical pyrimidine nucleotide from nucleobases and phosphorylated carbohydrates.

According to a current "RNA first" model for the origin of life, RNA emerged in some form on early Earth to become the first biopolymer to support Darwinism here. Threose nucleic acid (TNA) and other polyelectrolytes are also considered as the possible first Darwinian biopolymer(s). This model is being developed by research pursuing a "Discontinuous Synthesis Model" (DSM) for the formation of RNA and/or TNA from precursor molecules that might have been available on early Earth from prebiotic reactions, with the goal of making the model less discontinuous. In general, this is done by examining the reactivity of isolated products from proposed steps that generate those products, with increasing complexity of the reaction mixtures in the proposed mineralogical environments. Here, we report that adenine, diaminopurine, and hypoxanthine nucleoside phosphates and a noncanonical pyrimidine nucleoside (zebularine) phosphate can be formed from the direct coupling reaction of cyclic carbohydrate phosphates with the free nucleobases. The reaction is stereoselective, giving only the ß-anomer of the nucleotides within detectable limits. For purines, the coupling is also regioselective, giving the N-9 nucleotide for adenine as a major product. In the DSM, phosphorylated carbohydrates are presumed to have been available via reactions explored previously [Krishnamurthy R, Guntha S, Eschenmoser A (2000) Angew Chem Int Ed 39:2281-2285], while nucleobases are presumed to have been available from hydrogen cyanide and other nitrogenous species formed in Earth's primitive atmosphere.

6-Substituted 2-Aminopurine-2'-deoxyribonucleoside 5'-Triphosphates that Trace Cytosine Methylation.

Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2'-deoxy 5-methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O(6) -alkylated 2'-deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6-substituted 2-aminopurine-2'-deoxyribonucleoside 5'-triphosphates modified at position 6 with various functionalities. We found that sensing of 5-methylation by this class of nucleotides is more general, not being restricted to O(6) -alkyl modification of dGTP but also applying to other functionalities.

Synthesis and biological profiling of 6- or 7-(het)aryl-7-deazapurine 4'-C-methylribonucleosides.

The synthesis and biological activity profiling of a large series of diverse pyrrolo[2,3-d]pyrimidine 4'-C-methylribonucleosides bearing an (het)aryl group at position 4 or 5 is reported as well as the synthesis of several phosphoramidate prodrugs. These compounds are 4'-C-methyl derivatives of previously reported cytostatic hetaryl-7-deazapurine ribonucleosides. The synthesis is based on glycosylation of halogenated 7-deazapurine bases with 1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-methyl-ß-d-ribofuranose followed by cross-coupling and nucleophilic substitution reactions. The final compounds showed low cytotoxicity and several derivatives exerted antiviral activity against HCV or Dengue viruses at micromolar concentrations.

Synthesis and antiretroviral activity of novel 4'-fluoro-5'-deoxyphosphonic acid carbocyclic nucleoside analogs.

Novel 5'-deoxycarbocyclic purine phosphonic acid analogs with the 4'-electropositive moiety, fluorine were designed, and synthesized from glyceraldehyde. The cyclopentenol intermediate, 9, was successfully synthesized by the ring-closing metathesis of divinyl 8. The condensation reaction of cyclopentanol 15 with purine bases under Mitsunobu conditions successfully afforded the desired phosphonate analogs. The synthesized nucleoside phosphonic acid analogs, 19, 22, 26, and 29, were subjected to antiviral screening against human immunodeficiency virus (HIV)-1. Guanine phosphonic acid analog 29 showed significant anti-HIV activity (EC50 = 10.3 µM).

6-(Hetero)Arylpurine nucleotides as inhibitors of the oncogenic target DNPH1: synthesis, structural studies and cytotoxic activities.

The 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) has been proposed as a new molecular target for cancer treatment. Here, we describe the synthesis of a series of novel 6-aryl- and 6-heteroarylpurine riboside 5'-monophosphates via Suzuki-Miyaura cross-coupling reactions, and their ability to inhibit recombinant rat and human DNPH1. Enzymatic inhibition studies revealed competitive inhibitors in the low micromolar range. Crystal structures of human and rat DNPH1 in complex with one nucleotide from this series, the 6-naphthylpurine derivative, provided detailed structural information, in particular regarding the possible conformations of a long and flexible loop wrapping around the large hydrophobic substituent. Taking advantage of these high-resolution structures, we performed virtual docking studies in order to evaluate enzyme-inhibitor interactions for the whole compound series. Among the synthesized compounds, several molecules exhibited significant in vitro cytotoxicity against human colon cancer (HCT15, HCT116) and human promyelocytic leukemia (HL60) cell lines with IC50 values in the low micromolar range, which correlated with in vitro DNPH1 inhibitory potency.

On the use of metal purine derivatives (M=Ir, Rh) for the selective labeling of nucleosides and nucleotides.

The reactions of neutral or cationic IrIII and RhIII derivatives of phenyl purine nucleobases with unsymmetrical alkynes produce new metallacycles in a predictable manner, which allows for the incorporation of either photoactive (anthracene or pyrene) or electroactive (ferrocene) labels in the nucleotide or nucleoside moiety. The reported methodology (metalation of the purine derivative and subsequent marker insertion) could be used for the postfunctionalization and unambiguous labeling of oligonucleotides.

Advances in chemical synthesis of structurally modified bioactive RNAs.

Methods for the chemical synthesis of RNA have been available for almost half century, and presently, RNA could be chemically synthesized by automated synthesizers, using protected ribonucleosides preactivated as phosphoramidites, which has already been covered by many reviews. In addition to advancement on synthetic methods, a variety of modifications have also been made on the synthesized oligonucleotides, and previous reviews on the general synthesis of RNAs have not covered this area. In this tutorial review, three types of modifications have been summarized standing at the viewpoint of medicinal chemistry: (1) modifications on nucleobase, comprising substituent introduction and replacement with pseudobase; (2) modifications on ribose, consisting of modifications on the 2', 3' or 5'-position, alternation of configuration, and conformational restriction on ribose; (3) modifications on internucleoside linkages, including amide, formacetal, sulfide, sulfone, ether, phosphorothiolate and phosphorothioate linkages. Synthetic methods achieving these modifications along with the functions or values of these modifications have also been discussed and commented on.

The selective synthesis of metallanucleosides and metallanucleotides: a new tool for the functionalization of nucleic acids.

Nucleobases team up: the efficient and selective preparation of purine-derived metallanucleosides, metallanucleotides, and metalladinucleotides having M-C bonds (M=Ir(III), Rh(III)) is reported for the first time. The results presented may be applied to the synthesis of functionalized nucleic acids, or DNA/RNA-modified segments.

Gram-scale chemical synthesis of 2'-deoxynucleoside-5'-o-triphosphates.

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2'-deoxynucleoside-5'-O-triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved "one-pot, three-step" synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2'-deoxyguanosine-5'-O-triphosphate (dGTP) and 2'-deoxyadenosine-5'-O-triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2'-deoxycytidine-5'-O-triphosphate (dCTP), thymidine-5'-O-triphosphate (TTP), and 2'-deoxyuridine-5'-O-triphosphate (dUTP).

An improved protection-free one-pot chemical synthesis of 2'-deoxynucleoside-5'-triphosphates.

A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 '-deoxyguanosine-5 '-triphosphate (dGTP) and 2 '-deoxyadenosine-5 '-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 '-deoxycytidine-5 '-triphosphate (dCTP), thymidine-5 '-triphosphate (TTP), and 2 '-deoxyuridine-5 '-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved "one-pot, three step" Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%-70%).

Synthesis of cyclic di-nucleotidic acids as potential inhibitors targeting diguanylate cyclase.

Five analogs of cyclic di-nucleotidic acid including c-di-GMP were synthesized and evaluated for their biological activities on Slr1143, a diguanylate cyclase of Synechocystis sp. Slr1143 was overexpressed from the recombinant plasmid which contained the gene of interest and subsequently purified by affinity chromatography. A new HPLC method capable of separating the compound and product peaks with good resolution was optimized and applied to the analysis of the compounds. Results obtained show that cyclic di-inosinylic acid 1b demonstrates a stronger inhibition on Slr1143 than c-di-GMP and is a potential inhibitor for biofilm formation.

CycloSal-phosphate pronucleotides of cytostatic 6-(Het)aryl-7-deazapurine ribonucleosides: Synthesis, cytostatic activity, and inhibition of adenosine kinases.

A series of cycloSal-phosphate prodrugs of a recently described new class of nucleoside cytostatics (6-hetaryl-7-deazapurine ribonucleosides) was prepared. The corresponding 2',3'-isopropylidene 6-chloro-7-deazapurine nucleosides were converted into 5-O'-cycloSal-phosphates. These underwent a series of Stille or Suzuki cross-couplings with diverse (het)arylstannanes or -boronic acids to yield the protected 6-(het)aryl-7-deazapurine pronucleotides that were subsequently deprotected to give 12 derivatives of free pronucleotides. The in vitro cytostatic effect of the pronucleotides was compared with parent nucleoside analogues. In most cases, the activity of the pronucleotide was similar to or somewhat lower than that of the corresponding parent nucleosides, with the exception of 7-fluoro pronucleotides 13 a, 13 b, and 13 d, which had exhibited GIC(50) values that were improved by one order of magnitude (to the low nanomolar range). The presence of a cycloSal-phosphate group also influenced selectivity toward various cell lines. Several pronucleotides were found which strongly inhibit human adenosine kinase but only weakly inhibit the MTB adenosine kinase.

[Astrobiological investigations on board Russian space crafts].

The possibility of prebiotic synthesis of nucleic acids components (nucleotides) has been demonstrated under condition of the space orbital stations and satellites under effect of all space radiation spectra. Since a lot of different nucleic acids components are known to be present within small space bodies, we have to investigate their chemical complication in respect with such components as nucleotides. The goal of this work is to review our results in the field of prebiotic synthesis of purine and pyrimidine nucleotides on the board of Russian space crafts. The increase in the solid reaction mixtures exposure time leads to degradation of both initial components (nucleosides) and the reaction products (nucleotides). The dominating role of heat energy in the abiogenic reactions has been revealed in laboratory (ground) experiments. Similar set of natural nucleotides has been synthesized under effect of different open space energy sources in both flight and ground experiments. The formation of 5'-nucleotides is a dominating process. All the data are discussed in the context of exobiological investigations on the Earth's orbit.

Incorporation of an inducible nucleotide analog into DNA by DNA polymerases.

Non-natural nucleotides with diverse functionalities are highly useful in many areas of basic research and practical applications. We have previously developed an efficient method for post-synthetic modifications of 2-amino-6-vinylpurine (AVP)-containing oligonucleotides, which permits conjugations of a variety of useful functional appendages to the AVP moiety in DNA. Here we report an investigation on the ability of various DNA polymerases to use 5'-triphosphate of 2'-deoxyribosyl-2-amino-6-(2-methylthioethyl)purine (a stable precursor of AVP) as the substrate for templated DNA synthesis.

The purine machine scores a base hit.

A new synthetic method allows incorporation of 13C or 15N into selected positions within purine nucleotide bases, starting from simple labeled precursors. The procedure harnesses diverse enzymes to support biosynthesis by the pentose phosphate and de novo purine pathways. Selective isotope incorporation should expand the range of RNAs that are amenable to NMR analysis.

Pathway engineered enzymatic de novo purine nucleotide synthesis.

A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+, glucose, and CO2 as stoichiometric precursors in labeled form. Using this approach, U-13C- GTP, U-13C, 15N- GTP, 13C 2,8- ATP, and U-15N- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing 13C 2,8-adenosine and 15N 1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.

Synthesis of modified DNA by PCR with alkyne-bearing purines followed by a click reaction.

Alkyne-bearing deazapurine triphosphates were prepared and successfully incorporated into DNA using the polymerase chain reaction (PCR). The obtained alkyne-labeled DNA was successfully used in a click reaction with galactose azide.