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.     

The synthesis of 2′-homouridine,its incorporation into a dinucleoside monophosphate and hydrolytic behaviour of the dimer

An efficient route to 2′-homouridine (1), a new nucleoside analogue, is reported that is based on an ene reaction. This nucleoside has been incorporated into a dinucleoside monophosphate and hydrolytic studies on the dimer show that it does not behave like a ribonucleotide.     

General one-step synthesis of free hexofuranosyl 1-phosphates using unprotected 1-thioimidoyl hexofuranosides

A general one-step strategy is developed for the synthesis of hexofuranosyl 1-phosphates starting from new unprotected glycofuranosyl donors. It required first the preparation of new 1-thiohexofuranosides bearing a thioimidoyl heterocycle as a leaving group. The presence of sulfur and/or nitrogen atom(s) on the aglycon allowed remote activation of these thioglycofuranosides by anhydrous phosphoric acid and led to the target phosphates 9, 27, 29, and 30 in good to excellent selectivities and, more importantly, with very limited or no ring expansion. Moreover, this one-step phosphorylation reaction could be significantly improved by avoiding any tedious protecting group manipulations on negatively charged compounds and by focusing on a simple but general procedure of purification. This approach was applied to the diastereocontrolled synthesis of d-galacto- and d-glucofuranosyl 1-phosphates and also to the preparation of rare epimer and/or deoxy counterparts, that is, d-manno- and d-fucofuranosyl derivatives.     

Kinetic and thermodynamic analysis of the hydrolytic ring-opening of the malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)- pyrimido[1,2-alpha]purin-10(3H)-one

3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is the major reaction product of deoxyguanosine with malondialdehyde or base propenals. M1dG undergoes hydrolytic ring-opening to N2-oxopropenyl-deoxyguanosine (N2OPdG) under basic conditions. We report that ring-opening of M1dG as a nucleoside or in oligonucleotides is a reversible second-order reaction with hydroxide ion. NMR and UV analysis revealed N2OPdG(-) to be the only product of M1dG ring-opening in basic solution. The rate constant for reaction of M1dG with hydroxide is 3.8 M(-1) s(-1), and the equilibrium constant is calculated to be 2.1 +/- 0.3 x 10(4) M(-1) at 25 degrees C. Equilibrium constants determined by spectroscopic analysis of the reaction end-point or by thermodynamic analysis of rate constants determined over a range of temperatures yielded a value 2.5 +/- 0.2 x 10(4) M(-1). Kinetic analysis of ring-opening of M1dG in oligonucleotides indicated the rate constant for ring-opening is decreased 10-fold compared to that in the nucleoside. Flanking purines or pyrimidines did not significantly alter the rate constants for ring-opening, but purines flanking M1dG enhanced the rate constant for the reverse reaction. A mechanism is proposed for ring-opening of M1dG under basic conditions and a role is proposed for duplex DNA in accelerating the rate of ring-opening of M1dG at neutral pH.     

Enzyme-catalyzed synthesis of furanosyl nucleotides

A bacterial alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase was found to couple four hexofuranosyl-1-phosphates, as well as a pentofuranosyl-1-phosphate, with deoxythymidine 5'-triphosphate, providing access to furanosyl nucleotides. The enzymatic reaction mixtures were analyzed by electrospray ionization mass spectrometry and NMR spectroscopy to determine the anomeric stereochemistry of furanosyl nucleotide products. This is the first demonstration of a nucleotidylyltransferase discriminating between diastereomeric mixtures of sugar-1-phosphates to produce stereopure, biologically relevant furanosyl nucleotides.     

Acetyl derivatives of nucleoside 5′-triphosphates,I

Acetyl derivatives of nucleoside 5′-phosphates and nucleoside 5′-triphosphates (both ribo and deoxyribo series) were prepared by accomplishing partial (O-) and full (N, O-) acetylation of the basic compounds with acetic anhydride in pyridine medium.     

Biased Borate Esterification during Nucleoside Phosphorylase-Catalyzed Reactions: Apparent Equilibrium Shifts and Kinetic Implications**

Biocatalytic nucleoside (trans-)glycosylations catalyzed by nucleoside phosphorylases have evolved into a practical and convenient approach to the preparation of modified nucleosides, which are important pharmaceuticals for the treatment of various cancers and viral infections. However, the obtained yields in these reactions are generally determined exclusively by the innate thermodynamic properties of the nucleosides involved, hampering the biocatalytic access to many sought-after target nucleosides. We herein report an additional means for reaction engineering of these systems. We show how apparent equilibrium shifts in phosphorolysis and glycosylation reactions can be effected through entropically driven, biased esterification of nucleosides and ribosyl phosphates with inorganic borate. Our multifaceted analysis further describes the kinetic implications of this in situ reactant esterification for a model phosphorylase.     

Homonucleoside phosphonic acid-I: Ageneral synthesis of isosteric phosphonate analogs of nucleoside 3'-phosphates

A general synthetic route to 3'-deoxy-3'-dihydroxyphosphinylmethyl ribonucleosides 3 the isosteric phosphonate analogs of nucleoside 3'-phosphates, is described. This involved alkylation of 1,2;5,6-di-O-isopropylidene-α-D-ribo-hexofuranose-3-ulose 7) with tetraethyl methylenediphosphonate 6, followed by stereoselective catalytic reduction and cleavage of C₆ to generate 3-deoxy-3-diethoxyphosphinylmethyl-1,2-O-isopropylidene-α-D 12a. Benzoylation followed by acetolysis then generated the key crystalline intermediate 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-diethoxyphosphinylmethyl-β-D-ribofuranose 13, This compound, or the related glycosyl chloride, was condensed with several purine and pyrimidine bases and all protecting groups were removed by mild alkaline treatment via a series of intramolecular cyclizations and hydrolysis. In this manner the phosphonate analogs of nucleoside 3'-phosphates derived from adenine, 6-dimethylaminopurine, uracil, thymine, and cytosine were prepared.     

Binding causes the remote [5'-3H]thymidine kinetic isotope effect in human thymidine phosphorylase

The remote 5'-3H V/K kinetic isotope effect (KIE) observed in human thymidine phosphorylase (6.1%) is significantly larger than can be explained by the reaction chemistry. One hypothesis connects the 5'-3H KIE in purine nucleoside phosphorylase to that enzyme's SN1 transition state. The transition state of thymidine phosphorylase, however, is an SN2 nucleophilic displacement. Here we report equilibrium binding isotope effects sufficiently large to explain the presence of this substantial KIE in thymidine phosphorylase.     

Synthesis of glycosyl-1-phosphates via dehydrative glycosylation

[reaction: see text] Direct synthetic access to glycosyl-1-phosphates is accomplished with the dehydrative coupling of carbohydrate hemiacetals and dialkyl phosphates, employing dibenzothiophene-5-oxide and triflic anhydride. The procedure offers a new and versatile method for efficient preparation of a host of glycosyl-1-phosphates of variable structure with good control over anomeric selectivity.     

Target‐Based Whole‐Cell Screening by 1H NMR Spectroscopy

An NMR‐based approach marries the two traditional screening technologies (phenotypic and target‐based screening) to find compounds inhibiting a specific enzymatic reaction in bacterial cells. Building on a previous study in which it was demonstrated that hydrolytic decomposition of meropenem in living Escherichia coli cells carrying New Delhi metallo‐β‐lactamase subclass 1 (NDM‐1) can be monitored in real time by NMR spectroscopy, we designed a cell‐based NMR screening platform. A strong NDM‐1 inhibitor was identified with cellular IC₅₀ of 0.51 μM , which is over 300‐fold more potent than captopril, a known NDM‐1 inhibitor. This new screening approach has great potential to be applied to targets in other cell types, such as mammalian cells, and to targets that are only stable or functionally competent in the cellular environment.     

Bisphosphonate derivatives of nucleoside antimetabolites: hydrolytic stability and hydroxyapatite adsorption of 5'-beta,gamma-methylene and 5'-beta,gamma-(1-hydroxyethylidene) triphosphates of 5-fluorouridine and ara-cytidine

Kinetics of the hydrolytic reactions of four bisphosphonate derivatives of nucleoside antimetabolites, viz., 5-fluorouridine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 4), 5-fluorouridine 5'-beta,gamma-methylene triphosphate ( 5), ara-cytidine 5'-beta,gamma-(1-hydroxyethylidene) triphosphate ( 6), and ara-cytidine 5'-beta,gamma-methylene triphosphate ( 7), have been studied over a wide pH range (pH 1.0-8.5) at 90 degrees C. With each compound, the disappearance of the starting material was accompanied by formation of the corresponding nucleoside 5'-monophosphate, the reaction being up to 2 orders of magnitude faster with the beta,gamma-(1-hydroxyethylidene) derivatives ( 4, 6) than with their beta,gamma-methylene counterparts ( 5, 7). With compound 7, deamination of the cytosine base competed with the phosphate hydrolysis at pH 3-6. The measurements at 37 degrees C (pH 7.4) in the absence and presence of divalent alkaline earth metal ions (Mg (2+) and Ca (2+)) showed no sign of metal ion catalysis. Under these conditions, the initial product, nucleoside 5'-monophosphate, underwent rapid dephosphorylation to the corresponding nucleoside. Hydrolysis of the beta,gamma-methylene derivatives ( 5, 7) to the corresponding nucleoside 5'-monophosphates was markedly faster in mouse serum than in aqueous buffer (pH 7.4), the rate-acceleration being 5600- and 3150-fold with 5 and 7, respectively. In human serum, the accelerations were 800- and 450-fold compared to buffer. In striking contrast, the beta,gamma-(1-hydroxyethylidene) derivatives did not experience a similar decrease in hydrolytic stability. The stability in human serum was comparable to that in aqueous buffer (tau 1/2 = 17 and 33 h with 4 and 6, respectively), and on going to mouse serum, a 2- to 4-fold acceleration was observed. To elucidate the mineral-binding properties of 4- 7, their retention on a hydroxyapatite column was studied and compared to that of zoledronate ( 1a) and nucleoside mono-, di-, and triphosphates.     

Efficient synthesis of nucleoside 5′-triphosphates and their β,γ-bridging oxygen-modified analogs from nucleoside 5′-phosphates

Thirteen nucleoside 5′-triphosphates (NTPs) and their β,γ-bridging oxygen-modified analogs (β,γ-CX₂-NTPs, X = H, F, Cl, and Br) have been efficiently synthesized from nucleoside 5′-phosphoropiperidates with 4,5-dicyanoimidazole as the activator. A high-yielding and chromatography-free protocol for the preparation of both natural and base-modified nucleoside 5′-phosphoropiperidates from the corresponding nucleoside 5′-phosphates was also developed.     

Enzymatic Synthesis of α-Glucose-1-phosphate: A Study Employing a New α-1,4 Glucan Phosphorylase from Corynebacterium callunae 1

Abstract

In synthetic pathways to complex carbohydrates such as oligosaccharides or nucleotide sugars the activated sugar 1-phosphates serve as important starting molecules. In this study the enzymatic synthesis of α-glucose-1-phosphate (Glc-1-P) has been investigated using a new bacterial α-glucan phosphorylase from Corynebacterium callunae. The major factors governing the rate of reaction and the attainable degree of substrate conversion have been identified and, accordingly, for optimizing the yield and limiting reaction time for the enzymatic process several points must be considered: (i) the pH-dependent equilibrium of reaction, (ii) product inhibition of the phosphorylase and (iii) enzymatic cleavage of α-1,6 glycosidic linkages present in α-1,4-glucans such as starch or maltodextrins by pullulanases to improve their phosphorolytic conversion. Results obtained in continuous experiments with the phosphorylase retained in an ultrafiltration membrane reactor confirmed the complete operational stability of the enzyme for several days at 30 °C. Since no more than approximately 18 % of the inorganic phosphate can be converted into Glc-1-P an efficient procedure for phosphate and product recovery will be particularly important.     

Quantitative analysis of enzymatic assays using indoxyl-based substrates

Hydrolysis of indoxyl-based substrates by hydrolytic enzymes is a commonly used semiquantitative detection system that generates a water-insoluble indigo dye which is difficult to quantify. This work describes the quantitative analysis and enzyme kinetics for alkaline phosphatase (AP) and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) in solution obtained by applying known solubilization methodology from the textiles industry to the enzymatic product. This proposal is based on the reduction of the tetrahalo-indigo blue dye in a basic medium with the aim of generating its aqueous-soluble parent compound termed indigo white, which gives a rich yellow color in solution and is fluorescent. A quantitative ELISA (where a soluble end product is required) is accomplished for first time using BCIP as substrate.     

Electrochemical biosensor for microRNA detection based on hybridization protection against nuclease S1 digestion

Nuclease S1 can catalyze the nonspecific endo- and exonucleolytic cleavage of single-stranded DNA and RNA to yield nucleoside 5′-phosphates and 5′-phosphooligonucleotides. However, it cannot hydrolyze double-stranded DNA, double-stranded RNA, or DNA-RNA hybrid. Inspired by this specific property, a simple electrochemical method was developed for microRNA detection based on hybridization protection against nuclease S1 digestion. In the absence of hybridization process, the assembled probe DNA on the electrode surface can be easily digested by nuclease S1 and a strong electrochemical signal can be generated due to the decreased repulsive force towards the redox probe. However, after hybridization with target microRNA, the digestion activity of nuclease S1 is inhibited, which can lead to a weak electrochemical signal. Based on the change of the electrochemical signal, the detection of target microRNA-319a can be achieved. Under optimal experiment conditions, the electrochemical signal was proportional to microRNA-319a concentration from 1000 to 5 pM and the detection limit was 1.8 pM (S/N = 3). The developed method also showed high detection selectivity and reproducibility. Furthermore, the proposed method was successfully applied to assay the expression level of microRNA-319a in the leaves of rice seedlings after being incubated with different concentrations of 6-benzylaminopurine.     

Protecting-group-free synthesis of glycosyl 1-phosphates

Glycosyl 1-phosphates enriched in the α-anomer are obtained without the use of protecting groups in two steps starting from the free hemiacetal. Condensation of free hemiacetals with toluenesulfonylhydrazide yields a range of glycosylsulfonohydrazide donors which can be oxidized using cupric chloride in the presence of phosphoric acid and the coordinating additive 2-methyl-2-oxazoline to give useful yields of the fully deprotected glycosyl 1-phosphates.     

A transition state analogue for an RNA-editing reaction

Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR.RNA complexes.     

Non-glycosidic analogues of nucleotides: 2′(R),3′(S),5′-trihydroxypentyl derivatives of adenine and cytosine

Chiral 2′,3′,5′-trihydroxypentyl derivatives of adenine and cytosine in which configurations at C-2′ and C-3′ are opposite to those of the natural nucleosides have been synthesized. The nucleoside analogues were converted into 3′-phosphates and dinucleoside phosphate analogues with 3′–5′ phosphodiester linkages. PMR, UV and CD spectra of the compounds are presented.