Synthesis of alpha-P-modified nucleoside diphosphates with ethylenediamine

This report describes a one-pot synthesis of alpha-P-borano-, alpha-P-thio-, and alpha-P-seleno-modified nucleoside diphosphate analogues that are otherwise difficult to obtain. The key step involves the intramolecular nucleophilic attack by an amino group in 5 to remove the gamma-phosphate. The absolute configurations of P-diastereomers were confirmed by analysis of their 1H NMR. Affinity studies revealed that the nucleoside boranodiphosphates are potentially useful in antiviral research.     

A combination chemical and enzymatic approach for the preparation of azole carboxamide nucleoside triphosphate

Alternative substrates for DNA and RNA polymerases offer an important set of biochemical tools. Many of the standard methods for nucleoside triphosphate synthesis fail in the cases of nonpurine and nonpyrimidine nucleosides. An efficient preparation of the 5'-O-tosylates for both the deoxy- and ribonucleosides enabled preparation of the diphosphate esters by displacement with tris(tetra-n-butylammonium) pyrophosphate. Enzymatic synthesis of the azole carboxamide deoxyribonucleoside triphosphate was based on ATP as the phosphate donor, nucleoside diphosphate kinase as the catalyst, coupled with phosphoenol pyruvate (PEP) and pyruvate kinase as an ATP regeneration system. Ribonucleoside triphosphate synthesis required PEP as the phosphate donor and pyruvate kinase as the catalyst. An optimized purification procedure based upon boronate affinity gel was developed to yield highly purified nucleoside triphosphates. The strategy outlined here provides a new and efficient method for preparation of nucleoside 5'-triphosphate and is likely applicable to a broad variety of base and sugar modified nucleoside analogues.     

An improved P(V)-N activation strategy for the synthesis of nucleoside diphosphate 6-deoxy-l-sugars

Four nucleoside diphosphate 6-deoxy-l-sugars have been efficiently synthesized by coupling sugar-1-phosphates prepared from the H-phosphonate precursors with nucleoside 5′-phosphoropiperidates in the presence of 4,5-dicyanoimidazole (DCI) as the activator. Compared to the conventional 1H-tetrazole-promoted phosphoromorpholidate method, the new phosphoropiperidate/DCI system significantly shortened the reaction time and afforded nucleoside diphosphate sugars in excellent isolated yields.     

2-Trimethylsilylethyl sulfides in the von Braun cyanogen bromide reaction: selective preparation of thiocyanates and application to nucleoside chemistry

Mixed 2-(trimethylsilyl)ethyl sulfides were synthesized and used in the von Braun cyanogen bromide reaction for preparing selectively thiocyanates in high yield. We show here that this cleavage reaction is highly selective in methanol in comparison with the reaction of the corresponding non-silyl sulfide analogues. This reaction was applied to the synthesis of nucleosidic thiocyanates such as the new nucleosides 14 and 18 in the search for mechanism-based inhibitors of ribonucleoside diphosphate reductase and bioactive molecules. The selective cleavage is possible for sulfides bearing hydroxyl functions and aromatic rings. The reactions of cyanogen bromide as cyanating and brominating agent were observed for the first time under the same conditions with the naphthoxyhexyl 2-trimethylsilylethyl sulfide 7, which, treated with cyanogen bromide in dichloromethane, led selectively to the p-bromonaphthoxyhexyl thiocyanate 10 in 89% yield. Another reaction induced by cyanogen bromide was observed in dichloromethane with the 2-(trimethylsilylethyl)thio nucleoside 13, which gives the corresponding symmetrical disulfide 21 in good yield.     

A highly selective and sensitive fluorescence sensing system for distinction between diphosphate and nucleoside triphosphates

Among the numerous chemosensors available for diphosphate (P(2)O(7)(4-), PPi) and nucleoside triphosphates (NTPs), only a few can distinguish between PPi and NTPs. Hence, very few bioanalytical applications based on such selective chemosensors have been realized. We have developed a new fluorescence sensing system for distinction between PPi and NTPs based on the combination of two sensors, a binuclear Zn(II) complex (1·2Zn) and boronic acid (BA), in which one chemosensor (1·2Zn) shows signal changes depending on the PPi (or NTP) concentration, and the other (BA) blocks the signal change caused by NTPs; this system enables the distinction of PPi from NTPs and is sensitive to nanomolar concentrations of PPi. The new sensing system has been successfully used for the direct quantification of RNA polymerase activity.     

Further studies with adenosine 5-dithiophosphoromorpholidate. A convenient preparation of nucleoside phosphorodithioates by a ‘triester’ approach

Adenosine 5^′-dithiophosphoromorpholidate 1c reacts with orthophosphate to give adenosine 5^′-(α,α-dithio)diphosphate 3c but is not converted into adenosine 5^′-phosphorodithioate 2c by acidic hydrolysis. A new approach to the synthesis of nucleoside phosphorodithioates is described.     

Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.     

Bifunctional abietadiene synthase: free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.     

Enzyme-catalyzed synthesis of nucleoside triphosphates from nucleoside monophosphates

Syntheses of adenosine 5′-triphosphate (ATP) from adenosine 5′-monophosphate (AMP) and ribavirin 5′-triphosphate (RTP) from ribavirin 5′-monophosphate (RMP) (1) were performed using enzymes as catalysts. Synthesis of ATP is based on acetyl phosphate as the phosphate donor, and acetate kinase (Bacillus stearothermophilus, EC 2.7.2.1), adenylate kinase (porcine muscle, EC 2.7.4.3), and inorganic pyrophosphatase (yeast, EC 2.6.1.1) as the catalysts. Three reactions on a 150-mmol scale provided ATP as its barium salt in 82% yield and 67% purity. Synthesis of RTP used phosphoenol pyruvate (PEP) as the phosphate donor, and pyruvate kinase (rabbit muscle, EC 2.7.1.40) and adenylate kinase (rabbit muscle) as the catalysts. A gram-scale reaction provided RTP as its barium salt in 93% yield and 97% purity. This work demonstrates the utility of the autoxidationresistant acetate kinase fromB. stearothermophilus, the value of pyrophosphatase in controlling the level of pyrophosphate in the reactions and the ability of adenylate kinase to accept at least one substrate other than a derivative of adenosine.     

Dual catalytic apparatus of the thiamin diphosphate coenzyme: acid-base via the 1',4'-iminopyrimidine tautomer along with its electrophilic role

It was recently reported (Jordan, F.; Zhang, Z.; Sergienko, E. A. Bioorg. Chem. 2002, 30, 188-198) that addition to the E477Q active-center variant of yeast pyruvate decarboxylase of (a) pyruvate on a rapid-scan UV stopped-flow, or (b) acetaldehyde or benzoylformate on a circular dichroism (CD) instrument, generates a new band with lambda(max) near 300-310 nm. A chemical model demonstrated that the wavelength is appropriate to the 1',4'-iminopyrimidine tautomer of the 4'-aminopyrimidine ring in thiamin diphosphate. Herein, we report the formation of a new positive CD band centered at 305 nm when the Escherichia colipyruvate dehydrogenase complex first E1 subunit and its variants are exposed to phosphonolactylthiamin diphosphate, a stable analogue of the covalent adduct formed between the substrate pyruvate and the C2 atom of thiamin diphosphate. The behavior of this CD band, whether it suggests saturation of the enzyme by phosphonolactylthiamin diphosphate, or its very existence (the band is not seen with the E571A E1 variant, where E571 is hydrogen bonded to the N1' atom of the 4'-aminopyrimidine ring), as well as its position are consistent with its assignment to the 1',4'-imino thiamin diphosphate tautomer on the enzyme, chiral by virtue of its fixed V conformation. The mechanism of binding of phosphonolactylthiamin diphosphate closely resembles that of thiamin diphosphate itself.     

Type II isopentenyl diphosphate isomerase: irreversible inactivation by covalent modification of flavin

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction; whereas, the type II enzyme (IDI-2) requires reduced flavin. Epoxy, diene, and fluorinated substrate analogues, irreversible inhibitors of IDI-1, were analyzed as mechanistic probes for IDI-2. 3,4-Oxido-3-methyl-1-butyl diphosphate (eIPP), 3-methylene-4-penten-1-yl diphosphate (vIPP), and 3-(fluoromethyl)-3-buten-1-yl diphosphate (fmIPP) inactivate IDI-2 through formation of covalent adducts with the reduced flavin. UV-visible spectra of the inactivated complexes are consistent with modification of the isoalloxazine ring at position N5. vIPP and fmIPP are also alternate substrates with isomerization competing with alkylation of the flavin cofactor. (Z)-3-(Fluoromethyl)-2-buten-1-yl diphosphate ((Z)-fmDMAPP) and (Z)-3-(difluoromethyl)-2-buten-1-yl diphosphate ((Z)-dfmDMAPP) are alternate substrates, which are isomerized to the corresponding IPP derivatives. The rates of isomerization of fmIPP and (Z)-fmDMAPP are approximately 50-fold less than IPP and DMAPP, respectively. dfmIPP is not an irreversible inhibitor. These studies indicate that the irreversible inhibitors inactivate the reduced flavin required for catalysis by electrophilic alkylation and are consistent with a protonation-deprotonation mechanism for the isomerization catalyzed by IDI-2.     

A MODEL FOR BIOLOGICAL QUANTUM CONVERSION INVOLVING THE PHOTOOXIDATIVE DEPHOSPHORYLATION OF MENADIOL DIPHOSPHATE

Abstract— The mechanism of the photooxidative dephosphorylation of menadiol diphosphate appears to be invariant with respect to three sensitizers used; namely, riboflavin, biacetyl, and menadiol diphosphate itself. The mechanism involves triplet energy transfer from the sensitizers to oxygen to yield singlet oxygen which oxidizes menadiol diphosphate. The photolysis of menadiol diphosphate in acetic acid has resulted in formation of acetyl phosphate, as determined by paper chromatography. Therefore, we have demonstrated the feasibility of our model for converting light energy into chemical potential ('high-energy' phosphate bond energy or group transfer potential) in the form of acetyl phosphate.     

Structure and Function of a “Head‐to‐Middle” Prenyltransferase: Lavandulyl Diphosphate Synthase

We report the first X‐ray structure of the unique “head‐to‐middle” monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis‐prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S‐thiolo‐dimethylallyl diphosphate and S‐thiolo‐isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure‐based mechanism of this unusual prenyl synthase.     

Mechanism and stereochemistry of diphosphate formation from dioxaphosphorinanes: a critical reassessment.

The mechanism of diphosphate formation from (R)-2-chloro-2-oxo-5,5-dimethyl-4-(R)-phenyl-1,3,2-dioxaphosphorinane (5a) and 2-hydroxy-2-oxo-5,5-dimethyl-4-(R)-phenyl-1,3,2-dioxaphosphorinane (6) has been investigated. The products formed are the ax-ax diphosphate 7a and the ax-eq diphosphate 7b, with no evidence in the 31P NMR spectrum for pentacoordinate chlorooxyanionic phosphoranes 9. The structure of 7bhas been established unambiguously by NMR spectroscopy, mass spectrometry, and elemental analysis, and the structures of 5a and 7a have been confirmed by X-ray crystallography. The mechanism of the crucial diphosphate-forming reaction has been probed using 18O-labeling studies. The 18O-labeling patterns are consistent with the unsymmetric ax-eq diphosphate 7b arising from selective nucleophilic attack of the axial oxygen of 6 on the chloride 5a with inversion of configuration at phosphorus. The symmetric ax-ax diphosphate 7a can be formed directly, as a result of selective nucleophilic attack of the axial oxygen of 6 on the chloride 5a with retention of configuration, but the majority arises indirectly by isomerization of the ax-eq diphosphate 7b. The isomerization apparently involves intermolecular exchange, with nucleophilic attack of the phosphate anion 6 on the equatorially substituted phosphorus atom of 7b with inversion of configuration at phosphorus.     

Biochemistry and molecular genetics of the biosynthesis of the earthy odorant methylisoborneol in Streptomyces coelicolor

Methylisoborneol (2) is a volatile organic compound produced by a wide variety of Actinomycete soil organisms, myxobacteria, and cyanobacteria. It has an unusually low odor threshold and, together with geosmin, is responsible for the characteristic smell of moist soil as well as unpleasant taste and odor episodes associated with public water supplies and contamination of various foodstuffs, including fish, wine, and beer. Despite considerable interest in detection and remediation of methylisoborneol, the biosynthesis of this methylated monoterpene has been obscure. In Streptomyces coelicolor, the sco7700 and sco7701 genes are shown to correspond to a two-gene operon responsible for methylisoborneol biosynthesis. Both genes have been amplified by PCR and the resulting DNA has been cloned and expressed in Escherichia coli. Incubation of recombinant SCO7701 protein, annotated as a possible C-methyltransferase, with geranyl diphosphate (1) and S-adenosylmethionine gave the previously unknown compound, (E)-2-methylgeranyl diphosphate (3). Incubation of 3 in the presence of Mg2+ with recombinant SCO7700, previously annotated only as a possible metal-binding protein or terpenoid synthase, resulted in the formation of 2-methylisoborneol (2). The steady-state kinetic parameters for both biochemical reactions have been determined. Incubation of geranyl diphosphate and S-adenosylmethionine with a mixture of both SCO7700 and SCO7701 resulted in formation of methylisoborneol (2). Cyclization of 2-methylgeranyl diphosphate (3) to methylisoborneol (2) likely involves the intermediacy of 2-methyllinalyl diphosphate.     

Synthesis of deuterium-labeled derivatives of dimethylallyl diphosphate

Short practical syntheses for five deuterium-labeled derivatives of dimethylallyl diphosphate (DMAPP) useful for enzymological studies are reported. These include the preparation of the C1-labeled derivatives (R)-[1-2H]3-methylbut-2-enyl diphosphate ((R)-[1-2H]1-OPP) and (S)-[1-2H]3-methylbut-2-enyl diphosphate ((S)-[1-2H]1-OPP), the C2-labeled derivative [2-2H]3-methylbut-2-enyl diphosphate ([2-2H]1-OPP), and the methyl-labeled derivatives (E)-[4,4,4-2H3]3-methylbut-2-enyl diphosphate ((E)-[4,4,4-2H3]1-OPP) and (Z)-[4,4,4-2H3]3-methyl-but-2-enyl diphosphate ((Z)-[4,4,4-2H3]1-OPP).     

荧光素二磷酸酯的合成和荧光分光光度法分析碱性、酸性磷酸酯酶

目前荧光光度法测定碱性和酸性磷酸酯酶的底物常用伞形酮磷酸酯和萘酚As-BI(6-溴-3-羟基-2-萘基邻茴香胺)的磷酸二氢酯.它们具有很高的灵敏度,在荧光免疫分析中被广泛应用. 本文用荧光素和三氯氧磷在吡啶介质中合成了荧光素二磷酸酯,再用氢氧化钠处理为四钠盐.该醇易溶于水,其水溶液无荧光.它在碱性或酸性磷酸酯酶的催化下水解,产生具有很强荧光的荧光素,且荧光强度与酶量成正比,因此可进行碱性或酸性磷酸酯酶的测定.     

Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase

[formula: see text] Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes.     

A soluble form of phosphatase in Saccharomyces cerevisiae capable of converting farnesyl diphosphate into E,E-farnesol

After anion-exchange chromatography, the soluble fraction of a cell-free extract of Saccharomyces cerevisiae showed two phosphatase activity peaks when p-nitrophenyl phosphate (pNPP) was used as the substrate. However, only the second pNPP active peak demonstrated the ability to convert farnesyl diphosphate (FPP) into E,E-farnesol. N-terminal sequence analysis of the purified pNPP/FPP phosphatase revealed that it was a truncated form of alkaline phosphatase Pho8 lacking 62 amino acids from the N-terminus and was designated Pho8Delta62. Although other isoprenyl diphosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) could also be hydrolyzed by Pho8Delta62 to the corresponding alcohols, selectivity was observed among these substrates. The optimum pH was 7.0 for all three isoprenyl diphosphate substrates. Although lower hydrolytic activity was observed for FPP and GGPP at pH 6.0 and 8.5, hydrolysis of GPP was observed only at pH 7.0. Mg2+ and Mn2+ inhibited hydrolysis of FPP and GGPP, and GGPP was more sensitive to Mg2+ inhibition than FPP. The rate of FPP hydrolysis increased in the presence of Triton X-100.     

One-pot synthesis of nucleoside 5'-triphosphates from nucleoside 5'-H-phosphonates

Nucleoside 5'-triphosphates (NTPs) play key roles in biology and medicine. However, these compounds are notoriously difficult to synthesize. We describe a one-pot method to prepare NTPs from nucleoside 5'-H-phosphonate monoesters via pyridinium phosphoramidates, and we used this approach to synthesize ATP, UTP, GTP, CTP, ribavirin-TP, and 6-methylpurine ribonucleoside-TP (6MePTP). Poliovirus RNA-dependent RNA polymerase efficiently employed 6MePTP as a substrate, suggesting that the cognate nucleoside, a poorly understood antiviral agent, may damage viral RNA.