Application of the fast protein liquid chromatographic system and MonoQ HR 5/5 anion exchanger to the separation of nucleotides

The applicability of a new type of anion exchanger, MonoQ HR 5/5, and the Pharmacia-LKB fast protein liquid chromatographic (FPLC) system to the separation of nucleotides is described. The elution characteristics of adenosine-5'-, cytidine-5'-, uridine-5'-, guanosine-5'-mono-, -di- and -triphosphates and inositol-5'-monophosphate reference compounds, and of nucleotides originating from various biological samples, are optimized by varying the concentration gradient programme with ammonium phosphate buffer. Some practical examples of biological interest for monitoring the metabolic changes of nucleotides are presented.     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.

     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.     

Vanadyl ion complexes with nucleotides

Summary Complexes of adenosine-5-triphosphate, adenosine-5-monophosphate, guanosine-5-monophosphate, inosine-5-monophosphate, cytidine-5-monophosphate and uridine-5-monophosphate with vanadyl ion, have been studied in the solid state by i.r. spectroscopy and magnetochemically. All complexes have normal magnetic moments, very close to the spin-only values. From the i.r. spectra it is suggested that the vanadyl ion is interacting with adenosine-5-triphosphate, through the N-1 of the purine ring, with adenosine-5-monophosphate, guanosine-5-monophosphate, inosine-5-monophosphate, through the N-7 of the purine ring, with cytidine-5-monophosphate through the N-3 of the pyrimidine ring, and most probably through the phosphate group with uridine-5-monophosphate. The complexes of vanadyl ion with the nucleotides are probably polymeric.     

Resolution of crystalline ribonuclease by paper electrophoresis

1. Crystalline ribonuclease samples obtained from different commercial sources in addition to one prepared in the laboratory were resolved into their components, RNases I, II, III and IV, by a new two-dimensional electrophoretic technique 2. RNase I and RNase II liberated more uridyhc acid and cytidylic acid from yeast nbonucleic acid, and demonstrated a greater enzymic activity on undine-2', 3'-phosphate and cytidine-2',3'-phosphate, than either RNase III or RNase IV RNase III and RNaso IV liberated more adenylic acid and guanylic acid from yeast ribonucleic acid, and showed a greater enzymic activity on adenosine-2',3'-phosphate and guanosine-2', 3 '-phosphate than cither RNase I and RNase II 3. The degree of heterogeneity of the RNase samples studied revealed the age of the preparation 4. It is thus demonstrated, that certain of the activities of “crystalline nbonucleasc” reside in four different protein entities, and some activity toward punne nucleotidc esters existed in two of the four protein entities     

Total synthesis of 2',3',4',5',5' '-(2)h(5)-ribonucleosides: the key building blocks for NMR structure elucidation of large RNA

The diastereospecific chemical syntheses of uridine-2',3',4',5',5' '-(2)H(5) (21a), adenosine-2',3',4',5',5' '-(2)H(5) (21b), cytidine-2',3',4',5',5' '-(2)H(5)(2)H(5) (21c), and guanosine-2',3',4',5',5' '-(2)H(5) (21d) (>97 atom % (2)H at C2', C3', C4', and C5'/C5' ') have been achieved for their use in the solution NMR structure determination of oligo-RNA by the Uppsala "NMR-window" concept (refs 4a-c, 5a, 6), in which a small (1)H segment is NMR-visible, while the rest is made NMR-invisible by incorporation of the deuterated blocks 21a-d. The deuterated ribonucleosides 21a-d have been prepared by the condensation of appropriately protected aglycone with 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-alpha/beta-D-ribofuranose-2,3,4,5,5'-(2)H(5) (19), which has been obtained via diastereospecific deuterium incorporation at the C2 center of appropriate D-ribose-(2)H(4) derivatives either through an oxidation-reduction-inversion sequence or a one-step deuterium-proton exchange in high overall yield (44% and 24%, respectively).     

Synthesis of methylene- and difluoromethylenephosphonate analogues of uridine-4-phosphate and 3-deazauridine-4-phosphate

Cytidine triphosphate synthetase (CTPS) catalyzes the formation of cytidine triphosphate from glutamine, uridine-5'-triphosphate (UTP), and adenosine-5'-triphosphate. Inhibitors of CTPS are of interest because of their potential as therapeutic agents. One approach to potent enzyme inhibitors is to use analogues of high energy intermediates formed during the reaction. The CTPS reaction proceeds via the high energy intermediate UTP-4-phosphate (UTP-4-P). Four novel analogues of uridine-4-phosphate (U-4-P) and 3-deazauridine-4-phosphate (3-deazaU-4-P) were synthesized in which the labile phosphate ester oxygen was replaced with a methylene and difluoromethylene group. The methylene analogue of U-4-P, compound 1, was prepared by a reaction of the sodium salt of tert-butyl diethylphosphonoacetate with protected, 4-O-activated uridine followed by acetate deprotection and decarboxylation. It was found that this compound undergoes relatively facile dephosphonylation presumably via a metaphosphate intermediate. The difluoromethylene derivative, compound 2, was prepared by electrophilic fluorination of protected 1. This compound was stable and did not undergo dephosphonylation. Synthesis of the methylene analogue of 3-deazaU-4-P, compound 3, was achieved by ribosylation of protected 4-(phosphonomethyl)-2-hydroxypyridine. Electrophilic fluorination was also employed in the preparation of protected 4-(phosphonodifluoromethyl)-2-hydroxypyridine which was used as the key building block in the synthesis of difluoro derivative 4. These compounds represent the first examples of a nucleoside in which the base has been chemically modified with a methylene or difluormethylenephosphonate group.     

Combinatorial multinuclear NMR and X-ray diffraction studies of uranium(VI)-nucleotide complexes

The complex formation of uranium(VI) with four nucleotides, adenosine- (AMP), guanosine- (GMP), uridine- (UMP), and cytidine-monophosphate (CMP), has been studied in the alkaline pH range (8.5-12) by (1)H, (31)P, (13)C, and (17)O NMR spectroscopy, providing spectral integral, chemical shift, homo- and heteronuclear coupling, and diffusion coefficient data. We find that two and only two complexes are formed with all ligands in the investigated pH region independently of the total uranium(VI) and ligand concentrations. Although the coordination of the 5'-phosphate group and the 2'- and 3'-hydroxyl groups of the sugar unit to the uranyl ions is similar to that proposed earlier ("Feldman complex"), the number and the structures of the complexes are different. The uranium-to-nucleotide ratio is 6:4 in one of the complexes and 3:3 in the other one, as unambiguously determined by a combinatorial approach using a systematic variation of the ratio of two ligands in ternary uranium(VI)-nucleotide systems. The structure of the 3:3 complex has been determined by single-crystal diffraction as well, and the results confirm the structure proposed by NMR in aqueous solution. The results have important implications on the synthesis of oligonucleotides.     

新型离子交换硅胶键合相的制备及评价

二甲基氯硅烷与硅胶表面反应,形成牢固的ＳｉＨ键之后,连接上活泼的中间体——烯丙基缩甘油醚作为柔软的分子臂,最后接上二乙基氨基,由此制得了新型的离子交换硅胶键合相。经漫反射红外光谱、元素分析和高效液相色谱法对键合相进行了鉴定和评价。结果表明：键合反应按预定路线进行,键合相具有较好的色谱性能。此种方法可有效地运用于无孔硅胶填料的制备。     

Separation of nucleoside mono-, di- and triphosphates by capillary electrophoresis via cadmium complexation

Summary The CE separation of twelve nucleotides (5′-mono-, di-, triphosphates of adenosine, guanosine, cytidine and uridine) was improved by adding cadmium ion to the ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM). Cadmium ion acts as a complexing agent for some nucleotides (ATP, CTP, GTP, UTP, GDP). In order to accelerate the separation, the electroosmotic flow was reversed by flushing the fused-silica capillary with 0.2 % aqueous solution of the polycationic surfactant hexadimethrine bromide. A good separation of the twelve nucleotides studied was then achieved on a dynamically coated capillary in less than 5 min by using an ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM) to which 2 mM cadmium ion has been added. High peak efficiencies were obtained (210 000 theoretical plates) and the resolution between two adjacent peaks was always greater than 1.5.     

THE VACUUM ULTRAVIOLET PHOTOCHEMISTRY OF NUCLEOTIDES

Abstract— Photodestruction of nucleotides by vacuum UV (λ < 200 nm) and near UV radiation in thin films has been studied; the products were analyzed by thin-layer chromatography. Vacuum UV photolysis leads to the rupture of the N-glycosidic bond with liberation of bases, the quantum yield of the process being about 2 × 10^-4 for guanosine-5'-monophosphate. No bases have been found among the products of near UV photolysis.
Vacuum UV irradiation of aqueous solutions of adenosine-5'-monophosphate and guanosine-5'-monophosphate produces, along with bases, some other fluorescent substances. The identity of photoproducts of vacuum UV photolysis with those of γ-radiolysis is established. The mechanism of photochemical conversion is discussed.     

Semiempirical studies on the interactions of dialkyldi(aquo)tin cations, [R2Sn(H2O)2]2+, with selected nucleotides

Semiempirical calculations have been carried out on the interactions of [R₂Sn(H₂O)₂]²⁺, [R = H(CH₂)_n: n = 1–8], mainly with five nucleotides, 5′‐adenosine monophosphate (5′‐AMP), but also with guanosine 5′‐monophosphate (5′‐GMP), cytidine 5′‐monophosphate (5′‐CMP), uridine‐5′‐monophosphate (5′‐UMP) and inosine 5′‐monophosphate (5′‐IMP). The preferred sites of interaction were calculated to be the ribose O2 and O3 hydroxyl oxygens and/or the phosphate oxygens, with the nitrogen sites in the bases the least attractive to the tin compounds. This is in general agreement with experimental findings. Structures of the 1:1 coordination complexes vary from distorted tetrahedral, to distorted trigonal pyramidal to distorted octahedral geometries. Copyright © 2007 John Wiley & Sons, Ltd.     

A rapid and simple chemical method for the preparation of Ag colloids for surface-enhanced Raman spectroscopy using the Ag mirror reaction

Colloidal silver (Ag) nanoparticles (AgNP) have been widely used for surface-enhanced Raman spectroscopy (SERS) applications. We report a simple, rapid and effective method to prepare AgNP colloids for SERS using the classic organic chemistry Ag mirror reaction with Tollens’ reagent. The AgNP colloid prepared with this process was characterized using SEM, and the reaction conditions further optimized using SERS measurements. It was found that Ag mirror reaction conditions that included 20 mM AgNO₃, 5 min reaction time, and 0.5 M glucose produced AgNP colloids with an average size of 319.1 nm (s.d ± 128.1). These AgNP colloids exhibited a significant SERS response when adenine was used as the reporter molecule. The usefulness of these new AgNP colloids was demonstrated by detecting the nucleotides adenosine 5′-mono-phosphate (AMP), guanosine 5′-monophosphate (GMP), cytidine 5′-monophosphate (CMP), and uridine 5′-monophosphate (UMP). A detection limit of 500 nM for AMP was achieved with the as-prepared AgNP colloid. The bacterium Mycoplasma pneumoniae was also easily detected in laboratory culture with these SERS substrates. These findings attest to the applicability of this AgNP colloid for the sensitive and specific detection of both small biomolecules and microorganisms.     

Synthesis and nucleic acid-binding properties of water-soluble porphyrins appending platinum(II) complexes.

We synthesized two water-soluble porphyrins appending platinum(II) complexes [alpha,beta-(4a) and alpha,alpha-(4b) 5,15-bis(2-trans-[PtCl(NH3)2]N-2-aminoethylaminocarbonylphenyl) 2,3,7,8,12,13,17,18-octamethylporphyrin] and studied their reactions with a variety of nucleic acids [disodium adenosine-5'-monophosphate (AMP), disodium guanosine-5'-monophosphate (GMP), disodium thymidine-5'-monophosphate (TMP), disodium cytidine-5'-monophosphate (CMP), synthetic polymer poly(dG)-poly(dC), poly(dA)-poly(dT)] by 1H-NMR, UV-vis and FAB-MS spectroscopies. Based on the denaturation experiments of synthetic nucleic acid polymers, we conclude that the presence of the porphyrins (5.6 microM) does not cause significant changes in the melting temperature of poly(dA)-poly(dT) (28 microM) (deltaT=1 degrees C) and shows reannealing. On the other hand, gradual melting of poly(dG)-poly(dC) (28 microM) occurs at a low temperature (deltaT= -27 degrees C) in the presence of the porphyrins (5.6 microM), and the solutions do not show reannealing phenomena. The results of UV-vis and 1H-NMR experiments revealed that the porphyrins bind to guanine bases and that the porphyrins bind to GMP more strongly than to the other nucleotides. The binding modes between the porphyrins and synthetic nucleic acids are affected more by the coordination of the nucleobase [poly(dG)-poly(dC)] to the Pt(II) in the porphyrins than by Coulomb and hydrophobic interactions.     

Simultaneous determination of 5'-monophosphate nucleotides in infant formulas by HPLC-MS

A method was developed for simultaneous determination of 5'-monophosphate nucleotides, adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, inosine 5'-monophosphate, and uridine 5'-monophosphate in infant formulas by high-performance liquid chromatography-mass spectrometry equipped with electrospray ionization source. The complete chromatographic separation of five nucleotides was achieved through a Symmetry C(18) column, after a binary gradient elution with water containing 0.1% formic acid and acetonitrile as mobile phase. The multi-reaction monitoring mode was applied for tandem mass spectrometry analysis. The established method was further validated by determining the linearity (R(2) > 0.999), recovery (92.0-105.0%), and precision (relative standard deviation ≤6.97%). To verify the applicability of the method, thirty commercially available infant formulas were randomly purchased from the supermarkets in Hangzhou, China, and then analyzed. The results showed that the developed method is validated, sensitive, and reliable for quantitation of nucleotides in infant formulas.     

Investigation of the torsional angle at the glycosidic bond in 5′-amino-5′-deoxythymidine derivatives by carbon-13 n.m.r. spectroscopy

The conformational preference of the thymine base ring with respect to the sugar ring in β,β,β,-trichloroethyl 5′amino-5′-deoxythymidine-5′-phosphate has been studied by ¹³C n.m.r. spectroscopy. The magnitude of the three bond vicinal coupling constant, J(C-2, H-1′), for β,β,β-trichloroethyl 5′-amino-5′-deoxythymidine-5′-phosphate and the similarity between the chemical shifts for the furanose carbons C-1′, C-2′, and C-3′ in β,β,β-trichloroethyl 5-′-amino-5′-deoxythymidine-5′-phosphate and in β,β,β-trichloroethyl thymidine 5′-phosphate indicate that the amino analogue exists in aqueous solution predominantly in the anti conformation, as is the case with natural nucleotides.     

5′-Phosphouridine as a source of terminal phosphate groups in oligonucleotides

A universal key component is proposed for the preparation of oligonucleotides with 3′- and 5′-terminal phosphate groups — 2′,3′-dibenzoyluridin-5′-yl (4-chlorophenylphosphate) (pU(Bz)₂), which is a potential source of the phosphate group. The condensation ofpU(Bz)₂ with the 5′-OH or the 3′-OH group of a protected oligonucleotide leads to the formation of oligodeoxyribonucleotides with 5′- or 3′-terminal uridine, respectively. The oxidation of the 2′,3′-cis-glycol group of the terminal uridine unit followed by β-elimination forms oligodeoxyribonucleotides with terminal phosphate groups.     

Simple Synthesis of P1P2-Diadenosine 5′-Pyrophosphate

Pyrophosphate-linked coenzymes play essential roles in several biochemical systems. Symmetrical diadenosine-5′-pyrophosphate (Ap2A) has been synthesized from adenosine-5′-phosphate in virtually quantitative yield. The simple procedure is carried out in anhydrous pyridine using adenosine phosphoromorpholidate and adenosine monophosphate bis-(tri-n-butylammonium salt) as coupling reagents.     

Counter-current chromatographic separation of nucleic acid constituents with a hydrophilic solvent system

Nucleic acid constituents such as nucleobases, nucleosides and nucleotides were separated by counter-current chromatography using type J coil planet centrifuge. The separation was performed with a hydrophilic solvent system composed of 1-propanol/800 mM potassium phosphate buffer (pH 7.4) (1:1, v/v) by eluting the lower aqueous phase at a flow-rate of 0.5 ml/min. Eight selected nucleic acid constituents (4.0 mg, 0.5 mg of each), uridine monophosphate (UMP), adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), uridine, urasile, deoxy uridine, adenosine and adenine were well resolved within 160 min.     

Selective acylation of ribonucleotides and ribonucleosides with acylimidazoles

Selective acylation of ribonucleotides and ribonucleosides can be achieved by using N-acylimidazole on a preparative scale with good yields (50–80%). For uridine 3′-phosphate (Up): in the presence of MDCAI, the 2′-O-acyl-derivative is the main product, while in the presence of an excess of TEAH, the 5′-O-acyl-derivative is the main product. For ribonucleosides (U_R or A_R or ψ_R): in the presence of MDCAI, the acylations take place preferably at 2′-OH or 3′-OH of ribonucleosides and only 3′-O-acyl-derivatives can be isolated by crystallization; in the presence of an excess of TEAH. 5′-O-acyl-derivative is obtained as the main product. Arabinonucleoside and deoxyribonucleoside are only slowly acylated to form 5′-O-acyl-derivatives as the main products by acylimidazole in the presence of MDCAI. Possible mechanisms of these acylations have been discussed.