Direct formation and isolation of unprotected α-and ß-d-ribopyranosyl urea, α-and ß-d-ribofuranosyl urea, and a ribosyl-1,2-cyclic carbamate in carbohydrate melts.

Solvent-free melts of unprotected d-ribose and urea generated mainly C1- substituted ribosyl products. The remarkable resolving power of a graphitised-carbon HPLC column allowed products of the reaction formed over a range of heating times and temperatures to be monitored. Heating an uncatalysed mixture of d-ribose and urea at temperatures between 75 °C and 90 °C resulted in complex mixtures of compounds; after 19 h heating at 90 °C, up to ten components could be resolved. At shorter heating times and lower temperatures, the composition and distribution of products varied. By manipulation of the reaction time and temperature, and with the addition of an acid catalyst, it was possible to optimise the yields of selected products. Thus, the acid-catalysed reaction after 1-2 h at 80 °C gave optimal yields of α- and ß-d-ribopyranosyl urea, whereas the uncatalysed reaction after 22 h at 75-78 °C in addition produced significant amounts of α-d-ribofuranosyl-1,2- cyclic carbamate [glyco-1,2-oxazolidin-2-one] plus the α- and ß-ribofuranosyl ureas. The five compounds were isolated and characterised, demonstrating the significant advantages of this approach; its simplicity, and the ability to produce multiple compounds of biological interest in a single step. LC/MS was used to identify tentatively several other components of the reaction mixture. The unprotected title compounds were prepared, isolated and characterised with water as the only solvent.

Metal oxide-based dispersive solid-phase extraction coupled with mass spectrometry analysis for determination of ribose conjugates in human follicular fluid.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. The pathogenesis of PCOS remains unclear and early diagnosis of PCOS is challenging. Follicular fluid provides a unique window in the critical processes during oocyte and follicular maturation, and the metabolic level of follicular fluid has important impact on the developmental potential of oocytes and subsequent embryos. Previous studies demonstrated some modified ribonucleosides in biological fluids were diseases related metabolites. In this respect, analysis of endogenous modified ribonucleosides in follicular fluids will facilitate the investigation of follicular development. Here, we developed a strategy for determination of ribose conjugates from follicular fluid using metal oxide-based dispersive solid-phase extraction (DSPE) coupled with liquid chromatography-multiple reaction monitoring-mass spectrometry analysis (DSPE-LC-MRM-MS/MS). Cerium dioxide (CeO2) was used to selectively recognize and capture cis-diol containing ribose conjugates from complex biological samples under basic environment. The trapped ribose conjugates were then easily released under acidic environment. The results showed that 50 potential ribose conjugates were detected in follicular fluid by the developed DSPE-LC-MRM-MS/MS method. We then further investigated the contents change of the detected ribose conjugates in follicular fluid from PCOS patients. The results indicated that the follicular fluid from healthy controls and PCOS patients can be clearly differentiated with the partial least squares-discriminate analysis (PLS-DA) based on the detected ribose conjugates. In addition, the contents of 8 ribose conjugates were significantly different between PCOS patients and healthy controls, which could potentially serve as the indicator of PCOS.

Enantioresolution and quantification of monosaccharides by comprehensive two-dimensional gas chromatography.

This work presents the development of a simple and efficient analytical protocol for the direct enantioselective resolution of sugars. A racemic mixture of the C3 sugar d,l-glyceraldehyde and the C5 monosaccharides d,l-arabinose, d,l-ribose, d,l-xylose, and d,l-lyxose was subjected to derivatization with trifluoroacetic anhydride, and corresponding derivatives were separated on a ß-cyclodextrin column with excellent resolution factors. Even though each aldopentose shows beside the linear form four predominant cyclic hemiacetals being the α- and ß-furanose along with the α- and ß-pyranose, we show that the overall enantiomeric excess of each compound can be precisely determined. Moreover, the measured detection limit for derivatized aldopentoses ranges from 0.015 to 0.019pmol on the column, while the quantification limit varies from 0.5 to 0.64pmol on the column.

Simultaneous Enantioseparation of Aldohexoses and Aldopentoses Derivatized With L-Tryptophanamide by Reversed Phase HPLC Using Butylboronic Acid as a Complexation Reagent of Monosaccharides.

Three aldohexoses, glucose, galactose, and mannose, and three aldopentoses, arabinose, xylose, and ribose, were derivatized with L-tryptophanamide (L-TrpNH2 ) under alkaline conditions. Using a basic mobile phase (pH 9.2), the three aldohexoses or the three aldopentoses were simultaneously enantioseparated, respectively, but all the six monosaccharides could not be simultaneously enantioseparated. A large amount of nonreacted L-TrpNH2 was detected after the derivatized monosaccharides. In order to widen the separation window, a large portion of nonreacted L-TrpNH2 could be eliminated by liquid-liquid extraction with ethylacetate, and elution order of the derivatized monosaccharides and nonreacted L-TrpNH2 was found to be reversed using a neutral mobile phase. All of the six monosaccharides were simultaneously enantioseparated by reversed phase high-performance liquid chromatography (HPLC) using InertSustainSwift C18 column (4.6 mm i.d. × 150 mm) and a mobile phase containing 180 mM phosphate buffer (pH 7.6), 1.5 mM butylboronic acid, and 5% acetonitrile at 40 °C. Nomenclature of D and L for monosaccharides is based on the configurations of the asymmetric C4 center for aldopentoses and C5 center for aldohexoses. It was found that the enantiomer elution order of these six monosaccharides and fucose in the proposed method conformed to be the absolute configuration of the C2 center.

Compounds from marine-derived Verrucosispora sp. FIM06054 and their potential antitumour activities.

Strain FIM06054 was isolated from a marine sponge sample collected from the East China Sea and was characterised as a strain of Verrucosispora genus on the basis of its 16S rRNA gene sequence. One new compound, FW054-1 (1), together with a known aminofuran compound proximicin A (2), was isolated from the culture broth of Verrucosispora sp. FIM06054. Their structures were elucidated on the basis of spectral analysis. 1 and 2 showed antiproliferative activity against several human tumour cell lines.

Analytical method development for directed enzyme evolution research: a high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers.

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality L-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a ß-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C(18) guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM ß-cyclodextrin was used as electrolyte. 0.35%of D-ribose in L-ribose can be detected which can be translated into 99.3% ee of L-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.

Bi-level optimizing control of a simulated moving bed process with nonlinear adsorption isotherms.

A bi-level optimizing control scheme originally proposed for a simulated moving bed (SMB) with linear isotherms has been extended to an SMB with nonlinear isotherms. Cyclic steady state optimization is performed in the upper level to determine the optimum switching period and time-varying feed/desorbent flow rates, and repetitive model predictive control is run in the lower level for purity regulation, taking the decision variables from the upper level as feed-forward information. Experimental as well as numerical study for an SMB process separating a high-concentration mixture of aqueous L-ribose and L-arabinose solutions showed that the proposed scheme performs satisfactorily against various disturbances. In contrast, an alternative scheme based on an SMB model with linear isotherms showed a limitation in the control performance; this scheme was apt to fail in purity regulation.

HPLC separation of all aldopentoses and aldohexoses on an anion-exchange stationary phase prepared from polystyrene-based copolymer and diamine: the effect of NaOH eluent concentration.

To investigate the separations of all aldopentoses (ribose, arabinose, xylose and lyxose) and aldohexoses (glucose, galactose, allose, altrose, mannose, gulose, idose and talose) on the D6 stationary phase prepared by the reaction of chloromethylated styrene-divinylbenzene copolymer and N,N,N',N'-tetramethyl-1,6-diaminohexane, we examined the effect of varying the concentration of the NaOH eluent on the elution orders. Separations of these aldoses were achieved using a 20 mM NaOH eluent. The elution behaviors of the aldoses were probably due to not only the individual pK(a) values, but also the chemical structures of the cyclic aldoses.

A new isoprenyl phenyl ether riboside from the culture of basidiomycete Laccaria amethystea.

A new isoprenyl phenyl ether riboside, 3-(3-methylbut-2-enyloxy)-4-O-alpha-D-ribofuranose benzoic acid methyl ester (1), was isolated from the culture of basidiomycete Laccaria amethystea. The structure of 1 was elucidated on the basis of extensive spectroscopic analysis.

L-Ribose from L-arabinose by epimerization and its purification by 3-zone simulated moving bed chromatography.

L-Ribose has recently received attention as the starting material for nucleoside drugs. As it is not found in nature, it is being produced by enzymatic or epimerization reaction. We investigated an epimerization reaction by molybdenium oxide and examined the effects of temperature, solvent, and molybdenum oxide amount on epimerization. L-Ribose has a yield of 22% under the conditions of 100 kg/m3 L-arabinose, 20% methanol, 5 kg/m3 MoO3, and 90 degrees C. In addition, simulated moving bed (SMB) that was equipped with three NH2-HPLC columns was used to separate L-arabinose and L-ribose resulting from L-arabinose epimerization. A 3-zone SMB process was developed to eliminate the high pressure problem in the conventional 4-zone SMB. Aspen simulation was performed to determine the operating variables such as switching time, raffinate, and extract flow rates. Experimental purities of extract and raffinate were compared with the theoretical ones and they are found to be fairly well correlated.

Complexation behavior of mono- and disaccharides by the vinylbenzeneboronic acid-divinylbenzene copolymer resins packed in a high-performance liquid chromatographic column.

Using an HPLC column packed with monodispersed vinylbenzeneboronic acid-divinylbenzene (V-D) copolymer resins, the elution behaviors of the mono- and disaccharides were studied under different pH mobile phases. The monodispersed V-D copolymer resins were prepared by the copolymerization of 4-vinylbenzeneboronic acid and divinylbenzene in the presence of template silica gel particles (particle size: 5 microm; pore size: 10 nm), followed by dissolution of the template silica gel using a NaOH solution. Similarly, styrene-divinylbenzene (S-D) copolymer resins as the control resins were also synthesized. The transmission electron micrographs of these polymer resins revealed a good monodispersity. The complexation behavior of the saccharides was evaluated by comparison of the peak area eluted through the V-D column for that through the S-D column. Four aldopentoses (D-ribose, D-arabinose, D-xylose, and D-lyxose) and four aldohexoses (D-glucose, D-mannose, D-galactose, and D-talose) were retained completely at pH 11.9. Especially, ribose and talose were totally retained even under acidic and neutral conditions. For the disaccharides, unlike sucrose and maltose, palatinose was completely retained in basic mobile phases.

Boric acid as a mobile phase additive for high performance liquid chromatography separation of ribose, arabinose and ribulose.

A new high performance liquid chromatographic (HPLC) method is described for the analysis of ribose, arabinose and ribulose mixtures obtained from (bio)chemical isomerization processes. These processes gain importance since the molecules can be used for the synthesis of antiviral therapeutics. The HPLC method uses boric acid as a mobile phase additive to enhance the separation on an Aminex HPX-87K column. By complexing with boric acid, the carbohydrates become negatively charged, thus elute faster from the column by means of ion exlusion and are separated because the complexation capacity with boric acid differs from one carbohydrate to another. Excellent separation between ribose, ribulose and arabinose was achieved with concentrations between 0.1 and 10 gL(-1) of discrete sugar.

Selective derivatization and sequestration of ribose from a prebiotic mix.

Observations regarding the catalytic potential of RNA and the role of RNA in biology have formed the basis for the "RNA world" hypothesis, which suggests that a genetic system based on self-replicating polyribonucleotides preceded modern biology. However, attempts to devise a realistic prebiotic synthesis of nucleic acids from simple starting materials have been plagued by problems of poor chemical selectivity, lack of stereo- and regiospecificity, and similar rates of formation and degradation of some of the key intermediates. For example, ribose would have been only a small component of a highly complex mix of sugars resulting from the condensation of formaldehyde in a prebiotic world. In addition, ribose is more reactive and degrades more rapidly compared with most other monosaccharides. This study demonstrates an approach for the preferential sequestration of ribose relative to other sugars that takes advantage of its greater reactivity. Cyanamide reacts especially rapidly with ribose to form a stable bicyclic adduct. This product crystallizes spontaneously in aqueous solution, whereas the corresponding products derived from threose, galactose, glucose, mannose, and each of the other pentoses do not. Furthermore, when employing a racemic mixture of d- and l-ribose, enantiomerically twinned crystals are formed that contain discrete homochiral domains.

Simultaneous catalysis and product separation by cross-linked enzyme crystals.

Crystalline cross-linked xylose isomerase (CLXI, EC 5.3.1.5) and xylanase (CLX, EC 3.2.1.8) were studied in a packed-bed reactor for simultaneous catalytic reaction and separation of substrates from reaction products. Streptomyces rubiginosus xylose isomerase catalyzed a slow isomerization of L-arabinose to L-ribulose and an epimerization to L-ribose. In equilibrium the reaction mixture contained 52.5% arabinose, 22.5% ribulose, and 25% ribose. In a packed-bed column filled with CLXI, a simultaneous reaction and separation resulted in fractions where arabinose concentration varied between 100-0%, ribulose between 0-55%, and ribose between 0-100%. Trichoderma reesei xylanase II hydrolyzed and transferred xylotetraose mainly to xylotriose and xylobiose. In a packed-bed column filled with CLX, xylotetraose rapidly reacted to xylobiose and xylose by a mechanism that is not yet fully understood.

Diastereoisomers of an 'arsenomethionine'-based structure from Sargassum lacerifolium: the formation of the arsenic-carbon bond in arsenic-containing natural products.

Separation and 1H NMR spectra of a pair of arsenic-containing diastereoisomers (1a and 1b) isolated from a brown alga has provided support for their structures (proposed on the basis of NMR spectra of the unseparated mixture). The diastereoisomerism and analogies with nitrogen-containing algal lipids indicated that they were derived from an analogue of methionine in which the dimethylarsinoyl- group had replaced amino. Although S-adenosylmethionine is probably the source of methyl and 5'-deoxyribose-5'-yl groups in arsenic-containing natural products, the arsenic-carbon bonds in some compounds might be formed by a process in which arsenic replaces nitrogen in amino-acid synthesis.

Biotransformation of the antiviral agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and characteristics of a mesoionic ribose metabolite.

The biotransformation of the antiinfluenza agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896, I) was studied. In addition to a urea metabolite (II) formed by transformation of the cyanamide functionality, another highly polar metabolite was found in mouse urine and in BSC-1, MDCK, and other cell culture incubations of [14C]LY217896. Using 13C-labeled LY217896 together with NMR and MS techniques, this highly polar metabolite was identified as a ribose derivative (III), which apparently exists in a mesoionic form (i.e. positive and negative charges within the same ring system). It was also found that this ribose is formed from LY217896 and ribose-1-phosphate in a reaction catalyzed by the enzyme purine nucleoside phosphorylase, but that the reverse reaction (cleavage of the ribose) is not observed under the conditions used. When tested in vitro using the same assay as that used to measure the antiviral activity of LY217896, this ribose and the urea metabolite exhibit essentially no activity. The presence of a ribose has been implicated in the activity of antiviral compounds such as ribavirin and anticancer agents like 2-aminothiadiazole and tiazofurin, which are structurally similar to LY217896. These activities have been postulated to involve either mono- or triphosphorylated forms, or NAD-type analogs. Possible implications of the formation of this mesoionic ribose metabolite for the mechanism of antiviral activity of LY217896 are discussed.

Conversion of D-mannitol to D-ribose: a newly discovered pathway in Escherichia coli.

A mutant (mtlD) strain of Escherichia coli unable to oxidize mannitol-1-phosphate to fructose-6-phosphate was used to study the fate of mannitol-1-phosphate. D-[1-14C]mannitol entered the cells via the phosphotransferase system and was phosphorylated equally at carbon 1 or 6. The label disappeared gradually from the mannitol-1-phosphate pool, and some 60% of the 14C was recovered in nucleic acids. Ribose was isolated from the purified RNA. The 14C label distribution in the isolated ribose precluded a simple hexose-to-pentose conversion by elimination of one terminal carbon from mannitol-1-phosphate. The 14C from mannitol-1-phosphate that did not enter macromolecules was found in CO2 and in some organic, non-phosphorylated compounds that were not identified. We suggest that the de novo synthesis of mannitol-1-phosphate in E. coli may be a reaction specifically dedicated to the biosynthesis of ribose.

Quantitation of xylose from plasma and urine by capillary column gas chromatography.

A specific and sensitive assay for quantitation of xylose from plasma and urine has been developed. Following a clean-up procedure, plasma (0.1 ml) or urine (0.2 ml) samples are concentrated and undergo two sequential derivatization steps. A methyloxime derivative is formed initially, followed by trimethylsilylation of all hydroxyl groups. The derivatized samples are quantitated by capillary column gas chromatography using flame ionization detection. Xylose and the internal standard (2-deoxy-D-ribose) have retention times of 6.5 and 5.2 min, respectively. Other monosaccharides (e.g. ribose, arabinose) do not interfere with the assay. Standard curves are linear and reproducible over a concentration range of 10-200 mg/l for plasma and 100-2000 mg/l for urine. The within-day and day-to-day percentage coefficients of variation were less than 5 and 9%, respectively, for plasma and urine.

Ribose. An oxidation product of glucose 6-phosphate in microsomal fraction.

An oxidative metabolism of glucose 6-phosphate was studied in rat liver microsomal fraction. Although radioactive 14CO2 was formed from [1-14C]glucose 6-phosphate in the microsomal fraction (Hino, Y., and Minakami, S. (1982) J. Biochem. (Tokyo) 92, 547-557), the formation was negligible when [2-14C]glucose 6-phosphate was used as a starting substrate. These results indicated an inability of the microsomal fraction to rearrange [2-14C]glucose 6-phosphate to form [1-14C] glucose 6-phosphate, and it was expected that a certain compound derived from glucose 6-phosphate accumulated as an end-product of the reaction. We, therefore, have tried to identify the product by high performance liquid chromatography, and found that ribose accumulated as the end-product. The formation of ribose was inhibited in the same manner as that of 14CO2 by antibodies against rat liver microsomal hexose-6-phosphate dehydrogenase, and the ratios of ribose to 14CO2 formed in the reaction were 0.5-0.8 on a molar basis. The finding of ribose formation further suggested the involvement of ribose phosphate isomerase and phosphatase activities in the reaction.