Presence Of Sodium Cyanoborohydride Research Articles

A high-specificity aniline-based mass tag for aldehyde detection.

We studied an aldehyde-labeling reagent, N-{2-[(4-aminophenoxy)methyl]benzyl}-N,N-diethylethanaminium bromide (CAX-A), containing an aniline functional group for the detection of aldehydes with high specificity. Six standard aldehydes were labeled by CAX-A and analyzed using LC-ESI-Orbitrap-MS. The aldehydes (each 40 nmol) were derivatized with CAX-A in the presence of sodium cyanoborohydride at room temperature overnight. The labeling reaction was applied to two urine samples for the detection of putative aldehydes. All six standard CAX-aldehyde derivatives were detected as precursor ions by dilution to 830 fmol/injection (signal-to-noise [S/N] ratio 587-1573). A total of 2184 MS1 features were detected overall in urine and blanks, of which 14 were putative aldehydes found only in urine. CAX-A can provide three levels of specificity for aldehyde detection. First is the known labeling specificity of the aniline functional group for aldehydes, which we confirmed here by observing a significant peak only from the aldehyde (S/N = 3388) when a mixture of an aldehyde, a ketone (no peak), and a quinone (S/N = 2.3) was tested. Second is the ease of formation of an analyte-characteristic first product ion (via anchimeric-assisted loss of triethylamine as a neutral) in MS2 from a CAX-labeled analyte. Third is the formation of a characteristic second product ion via loss of CO in MS3. CAX-A enables the specific, convenient detection of putative aldehydes in urine.

Dextran-Coated Latex Nanoparticles via Photo-RAFT Mediated Polymerization Induced Self-Assembly.

Polysaccharide coated nanoparticles represent a promising class of environmentally friendly latex to replace those stabilized by small toxic molecular surfactants. We report here an in situ formulation of free-surfactant core/shell nanoparticles latex consisting of dextran-based diblock amphiphilic copolymers. The synthesis of copolymers and the immediate latex formulation were performed directly in water using a photo-initiated reversible addition fragmentation chain transfer-mediated polymerization induced self-assembly strategy. A hydrophilic macromolecular chain transfer-bearing photosensitive thiocarbonylthio group (eDexCTA) was first prepared by a modification of the reducing chain end of dextran in two steps: (i) reductive amination by ethylenediamine in the presence of sodium cyanoborohydride, (ii) then introduction of CTA by amidation reaction. Latex nanoparticles were then formulated in situ by chain-extending eDexCTA using 2-hydroxypropyl methacrylate (HPMA) under 365 nm irradiation, leading to amphiphilic dextran-b-poly(2-hydroxypropyl methacrylate) diblock copolymers (DHX). Solid concentration (SC) and the average degree of polymerization -- of PHPMA block (X) were varied to investigate their impact on the size and the morphology of latex nanoparticles termed here SCDHX. Light scattering and transmission electron microscopy analysis revealed that SCDHX form exclusively spherical nano-objects. However, the size of nano-objects, ranging from 20 nm to 240 nm, increases according to PHPMA block length.

High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.

We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution.

Amine Chemistry Method for Selective Enrichment of N-Linked Glycopeptides for Glycoproteomics Analysis.

An amine chemistry method was developed for the extraction of N-glycopeptides using amine-functionalized beads for glycoproteomics analysis. Two reductive amination reactions between primary amine and aldehyde were employed in this approach. The first one was to block the primary amines in the peptides by addition of formaldehyde and sodium cyanoborohydride into the peptide sample, and the second one was to couple the glycopeptides onto solid phase beads by incubating the glycopeptides containing aldehyde groups (oxidized by periodate) with the amine-functionalized beads in the presence of sodium cyanoborohydride. It was demonstrated that the blocking of primary amines in the peptides by the first reductive amination reaction prior to the periodate oxidation made the amine chemistry method very efficient and sensitive. This new method was validated by analysis of glycoprotein standards as well as proteome samples. It was found that this new method led to significant increase in the identification of N-glycosites compared with the conventional hydrazide chemistry method.

N2-tert-Butoxycarbonyl-N5-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-(S)-2,5-diaminopentanoic Acid

N2-tert-Butoxycarbonyl-N5-[N-(9-fluorenylmethyloxycarbonyl)-2-aminoethyl]-(S)-2,5-diaminopentanoic acid (5) has been synthesized by the reaction of N2-tert-butoxycarbonyl-L-2,5-diaminopentanoic acid (Boc-L-ornithine, 3) and N-Fmoc-2-aminoacetaldehyde (N-Fmoc-glycinal, 4) in the presence of sodium cyanoborohydride in methanol containing 1% acetic acid at room temperature.

Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate.

D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI-TOF analyses, an increase in apparent molecular weight (SDS-PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P-BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P-BSA antiserum subjected to caprylic acid precipitation followed by hapten-affinity chromatography; its affinity constant is 7.1 × 10(8) M(-1). Rbt-5-P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to D-ribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P-specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P-protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P-specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide.

Mouvements en miroir chez un adulte

A mild procedure for the direct reaction of aromatic and aliphatic aldehyde bisulfite adducts with primary and secondary amine hydrochlorides in the presence of sodium cyanoborohydride in methanol is reported.

Direct reductive alkylation of amine hydrochlorides with aldehyde bisulfite adducts

A mild procedure for the direct reaction of aromatic and aliphatic aldehyde bisulfite adducts with primary and secondary amine hydrochlorides in the presence of sodium cyanoborohydride in methanol is reported.

A microscopic perspective of a microreactor

The use of microreactors in (bio)chemical processes has been gaining relevance in the last decade. The low consumption of reagents, the possibility of continuous operation and the faster translation from lab- to production scale are some of the several advantages of these devices. The whole results in cost reductions in process development. Enzyme catalyzed reactions have proved to be an excellent alternative to the use of chemicals due to its ability to catalyze the most complex chemical processes under benign experimental and environmental conditions. In this way, enzymes may be crucial to the implementation of a much more sustainable chemical industry.The present work is within such scope, using as model system the immobilization of invertase in glass (silica) microchannels, for the production of fructose syrups through sucrose hydrolysis. The immobilization of the enzyme was achieved through treatment of the substrate with a sequence of coatings (Figure 1). Activation of the inner surface of the microchannels with 3-aminopropyltriethoxysilane (APTES) was followed by the introduction of a spacer, glutaraldehyde. Lastly the enzyme solution was introduced in the presence of sodium cyanoborohydride, in order to enhance the stability of the support-enzyme binding.The characterization of the coatings at each stage of the immobilization protocol was carried out to confirm the change of the microchannel surface. Such studies were performed using Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) analysis (Figure 1).The results obtained, namely the shifts on the surface roughness corroborate that the several coatings were successfully applied and the enzyme immobilized. Moreover, the immobilization approach used proved to be highly effective, resulting on successful continuous use of the microreactor for a period of 30 days with roughly constant full conversion of a sucrose solution of 50g/l, at a flow rate of 7µl/minute (Figure 2).Future work will involve a more extensive characterization of the several coatings by Quartz Crystal Microbalance which will be decisive to achieve a better comprehension of the coating phenomena and hence optimize the immobilization process.The authors would like to thank Fundação para a Ciência e a Tecnologia, Portugal, for financial support through contracts under the program Ciência 2007 awarded to P. Fernandes, for the doctoral grant SFRH/BD/74818/2010 awarded to F. Carvalho and for the doctoral grant SFRH/BD/71990 /2010 awarded to Patrizia Paradiso.

Direct access to side chain N,N′-diaminoalkylated derivatives of basic amino acids suitable for solid-phase peptide synthesis

A simple and efficient one-pot procedure that enables rapid access to orthogonally protected N,N'-diaminoalkylated basic amino acid building blocks fully compatible with standard Boc and Fmoc solid-phase peptide synthesis is reported. Described synthetic approach includes double reductive alkylation of N (α)-protected diamino acids with N-protected amino aldehydes in the presence of sodium cyanoborohydride. This approach allows preparation of symmetrical, as well as unsymmetrical, basic amino acid derivatives with branched side-chains that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analogue in which the lysine residue was substituted with the synthetic derivative N (α)-(9H-fluorenyl-9-methoxycarbonyl)-N (β),N (β) '-bis[2-(tert-butoxycarbonylamino)ethyl]-L-2,3-diaminopropionic acid. This substitution resulted in an analogue with more ordered secondary structure in 2,2,2-trifluoroethanol and enhanced antibacterial activity without altering hemolytic activity.

ChemInform Abstract: Synthesis of Cholic Acid Amino Analogues by Oxime Reduction with TiCl3—NaBH3CN.

Amino analogues of cholic acid have been synthesized by reduction of the corresponding oximes with titanium(iii) chloride in the presence of sodium cyanoborohydride.

A validated bioanalytical HPLC method for pharmacokinetic evaluation of 2‐deoxyglucose in human plasma

2-Deoxyglucose (2-DG), an analog of glucose, is widely used to interfere with glycolysis in tumor cells and studied as a therapeutic approach in clinical trials. To evaluate the pharmacokinetics of 2-DG, we describe the development and validation of a sensitive HPLC fluorescent method for the quantitation of 2-DG in plasma. Plasma samples were deproteinized with methanol and the supernatant was dried at 45°C. The residues were dissolved in methanolic sodium acetate-boric acid solution. 2-DG and other monosaccharides were derivatized to 2-aminobenzoic acid derivatives in a single step in the presence of sodium cyanoborohydride at 80°C for 45 min. The analytes were separated on a YMC ODS C₁₈ reversed-phase column using gradient elution. The excitation and emission wavelengths were set at 360 and 425 nm. The 2-DG calibration curves were linear over the range of 0.63-300 µg/mL with a limit of detection of 0.5 µg/mL. The assay provided satisfactory intra-day and inter-day precision with RSD less than 9.8%, and the accuracy ranged from 86.8 to 110.0%. The HPLC method is reproducible and suitable for the quantitation of 2-DG in plasma. The method was successfully applied to characterize the pharmacokinetics profile of 2-DG in patients with advanced solid tumors.

Design, Synthesis, and Antimicrobial Activity, of New 1,4-disubstituted Octahydroquinoxaline-2,3-dione Derivatives

A series of 1,4-disubstituted octahydroquinoxaline-2,3-dione derivatives was prepared through two steps reaction. The latter involves the formation of N,N-disubstituted cyclohexane-1,2-diamine derivatives (la-j) through reductive alkylation of 1,2-cyclohexanediamine with different aldehydes in presence of sodium cyanoborohydride. Fusion of compounds (1a-j) with diethyl oxalate affording the target compounds (2a-j). Elucidation of structures of compounds (2a-j) was based upon different spectral data as well as the elemental methods of analyses. In addition, mass spectrometry and X-ray diffraction analyses were carried out. Moreover, the lipophilicity of the target compounds as expressed from the Clog P. Most of the test compounds (2a-j) showed weak to moderate antibacterial and antifungal activities against most of the used bacterial and fungal strains in comparison to chloramphenicol and clotrimazole as reference drugs respectively.

Synthesis of cholic acid amino analogues by oxime reduction with TiCl 3–NaBH 3CN

Amino analogues of cholic acid have been synthesized by reduction of the corresponding oximes with titanium(iii) chloride in the presence of sodium cyanoborohydride.

ChemInform Abstract: The Reductive Cleavage of Cyclic Aminol Ethers to N,N-Dialkylamino-Derivatives: Modifications to the Eschweiler-Clarke Procedure.

The reductive cleavage of cyclic aminol ethers to give N-alkylamino- derivatives in very high yields can be achieved using chlorotrimethylsilane in the presence of sodium cyanoborohydride: in the case of cyclic aminol ethers derived from formaldehyde the Eschweiler-Clarke reaction can be carried out in formic acid heated under reflux in the absence of formaldehyde.

Reductive N-alkylation of chitosan with acetone and levulinic acid in aqueous media

Reductive N-alkylation with acetone and levulinic acid in the presence of sodium cyanoborohydride was applied to chitosan to prepare N-isopropyl and 5-methyl-pyrrolidinone chitosans, respectively. These chitosan derivatives were obtained quantitatively, and the highest degrees of substitution (DS) were achieved for chitosan solutions at the initial pH 4.5–5.0. When the molar ratio of the primary amino groups of chitosan, NaBH 3CN and either acetone or levulinic acid was 1:10:3, reaction ratios at the primary amino groups reached about 100% and 41% for N-isopropyl and 5-methyl-pyrrolidinone chitosans, respectively, after the reaction at room temperature for 72 h. No depolymerization occurred on chitosan molecules under the reductive N-alkylation conditions used.

Stereochemical Configuration of 4-Hydroxy-2-nonenal-Cysteine Adducts and Their Stereoselective Formation in a Redox-regulated Protein

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys(73) and, to the lesser extent, the active site Cys(32). More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys(73), whereas Cys(32) exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.

Methods of the site-selective solid phase synthesis of peptide-derived Amadori products

Two procedures of glycated peptides' synthesis have been developed. The first method involves reductive alkylation of the epsilon-amino groups of lysine with 2,3:4,5-di-O-isopropylidene-beta-D-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride on solid support. The second one uses a new fully protected lysine derivative, which is a building block designed for direct introduction of the glycated lysine moiety into a peptide, according to the standard solid phase synthesis protocol. The applicability of the proposed methods for the synthesis of peptide-derived Amadori products is discussed. The structure of the synthesized glycated peptides was confirmed by high-resolution mass spectrometry and enzymatic hydrolysis. Circular dichroism studies, performed in water solution, revealed that the formation of the Amadori rearrangement product in the lysine side chain does not influence significantly the conformational preferences of the peptides studied. However, when the solvent was changed to trifluoroethanol, the glycated peptides preferred beta-turn conformation.

Activity, Splice Variants, Conserved Peptide Motifs, and Phylogeny of Two New α1,3-Fucosyltransferase Families (FUT10 and FUT11)

We report the cloning of three splice variants of the FUT10 gene, encoding for active alpha-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related alpha-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other alpha1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH(2) termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link alpha-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic alpha1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an alpha1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude alpha1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta alpha-N-acetylglucosaminidase.

Assignment of the 1H and 13C NMR spectra of 2-aminobenzamide-labeled xylo-oligosaccharides

Xylose, xylo-oligosaccharides with a degree of polymerization (DP) from 2 to 6, xylo-oligosaccharides carrying a single arabinofuranosyl, glucosyluronic acid, or 4- O-methyl glucosyluronic acid residues and 4-β- d-Xyl p-(1 → 4)- d-Xyl p-(1 → 3)-α- l-Rha p-(1 → 2)-α- d-Gal pA-(1 → 4)- d-Xyl p were labeled at their reducing ends with 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride (NaBH 3CN). These derivatives were then analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESI-MS) and by 1H and 13C NMR spectroscopy. Reacting Xyl 3-Xylitol-2AB with UDP-Xyl in the presence of rice microsomes resulted in the formation of small amounts of Xyl 4–6-Xylitol-2AB showing that the 2AB-labeled compound is an acceptor for xylosyltransferase. The 2AB-labeled xylo-oligosaccharides and the 2AB-labeled xylo-oligosaccharides carrying 4- O-methyl glucuronic acid are fragmented by xylanase and α-glucuronidase present in the culture filtrate of Fomitopsis palustris FFPRI 0507. Thus, the 2AB-labeled xylo-oligosaccharides are useful for studying enzymes involved in xylan degradation and biosynthesis.