Cyanoborohydride Research Articles

Evidence for an imidazoline by-product from glycans using tandem mass spectrometry

Herein is reported the separation and identification of a previously unknown imidazoline by-product originating from the fluorescent labeling procedure when applied to enzymatically released N-linked glycans of a human IgG1. The imidazoline by-product was generated via the reductive amination procedure with either sodium cyanoborohydride or 2-picoline borane. Using ultra performance liquid chromatography (UPLC) in conjunction with hydrophilic interaction-based chromatography (HILIC), the 2-aminobenzoic acid (2-AA)-labeled glycans were well-resolved from imidazoline by-products to facilitate direct identification utilizing electrospray ionization mass spectrometry (ESI–MS) with fragmentation. It was found that this minor species (∼2%) was 18.0105u less than the neighboring peak GlcNAc2Man3GlcNAc2Fuc peak, abbreviated as A2G0F at 1582.5899u. While this mass loss corresponds to the mass of a water molecule, the molecular location of loss of water was not straightforward in consideration of the biantennary A2G0F structure.Model studies were carried out using A2G0F standard and N-acetyllactosamine to identify the impurity as an imidazoline ring structure located at the reducing end of the glycan as confirmed by high resolution mass fragment ions. Imidazoline content decreased when the reductant concentration was increased. To conclude, evidence for the imidazoline structure was accomplished through high resolution, high accuracy mass spectrometry (HRAM), and experiments showing chemical susceptibility and isotopically labeled tracers. This study is the first to identify these minor species which likely impact all N-acetylglucosamine-type N-linked glycans from biologics.

Reductive amination derivatization for the quantification of garlic components by isotope dilution analysis.

In this work, we synthesized internal standards for four garlic organosulfur compounds (OSCs) by reductive amination with 13C, D2-formaldehyde, and developed an isotope dilution analysis method to quantitate these organosulfur components in garlic samples. Internal standards were synthesized for internal absolute quantification of S-allylcysteine (SAC), S-allylcysteine sulfoxide (alliin), S-methylcysteine (SMC), and S-ethylcysteine (SEC). We used a multiple reaction monitoring (MRM) to detect 13C, D2-formaldehyde-modified OSCs by ultrahigh-performance liquid phase chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and obtained MS spectra showing different ratios of 13C, D2-formaldehyde-modified and H2-formaldehyde-modified compounds. The resulting labeled and unlabeled OSCs were exhibited correlation coefficient (R2) ranged from 0.9989 to 0.9994, respectively. The average recoveries for four OSCs at three concentration levels ranged from 89% to 105%. By 13C, D2-formaldehyde and sodium cyanoborohydride, the reductive amination-based method can be utilized to generate novel internal standard for isotope dilution and to extend the quantitative application.

Evaluation of Structure, Chaperone-Like Activity and Allergenicity of Reduced Glycated Adduct of Bovine β-casein.

Milk has a potent reducing environment with an important quantity of sugar levels. In the current study β-casein was glycated in the presence of D-glucose and sodium cyanoborohydride as a reducing agent. Then, the reduced glucitol adduct of β-casein was used for the structural and functional analyses using different spectroscopic techniques. The results of fluorescence and far ultraviolet circular dichroism assessments suggest important structural alteration upon non-enzymatic glycation of β-casein. In addition, the chaperone activity, micellization properties and antioxidant activity of this protein were altered upon glucose modification. Also, as a result of reduced glycation, the allergenicity profile of this protein remained largely unchanged. Additional to its energetic and nutritional values, β-casein has important functional properties. The native structure of this protein is important to perform accurately its biological functions. Non-enzymatic glycation under reducing state was capable to alter both structural and functional aspects of β-casein. Due to effective reducing environment and significant quantity of reducing sugar of human milk, similar structural and functional alterations are most likely to occur upon reducing glycation of β-casein in vivo. Also, these changes might be even intensified during chronic hyperglycemia in diabetic mothers.

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization.

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538μmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0mmol/g of dry polymer compared to 1.5mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K m value of 2.61mM compared to the soluble enzyme. After 7cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

Synthesis and intramolecular cyclization of some derivatives of 3-[(2,3-dihydro-1Н-1,5-benzodiazepin-4-yl)methylidene]indolin-2-one

Isatin and its 5-bromo derivative react with о-phenylenediamine and acetone in formic acid; products of this three-component condensation were isolated as perchlorates, from which for the first time bases were obtained, derivatives of 3-[(2,3-dihydro-1Н-1,5-benzodiazepin-4-yl)methylidene]indolin-2-one. The reduction of these derivatives with sodium cyanoborohydride is followed by intramolecular cyclization affording a bridged 2,3,4,5-tetrahydro-1,4-methano-1Н-1,5-benzodiazepine system.

ChemInform Abstract: Asymmetric Diels—Alder Cycloadditions of Trifluoromethylated Dienophiles under Trienamine Catalysis.

β-Trifluoromethyl (CF3 ) enones were proved to act as good dienophiles in asymmetric normal-electron-demand Diels-Alder cycloadditions with 2,4-dienals under trienamine catalysis with a chiral secondary amine. The sequential reductive amination transformations with benzylamine produced cis- and trans-fused chiral trifluoromethylated octahydroisoquinolines in a diastereodivergent manner by using NaBH(OAc)3 and NaBH3 CN as the reductants, respectively. Moreover, other types of activated alkenes that bear a CF3 group have also been successfully utilized to construct a diverse range of chiral cyclic frameworks in high stereoselectivity.

Asymmetric hydrogenation of N-heterocycles with sodium cyanoborohydride and S-(–)-binol

Asymmetric hydrogenation of N-heterocycles with sodium cyanoborohydride and S-(–)-binol

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.

Reactions between arylhydrazinium chlorides and 2-chloroquinoline-3-carbaldehydes: molecular and supramolecular structures of a hydrazone, a 4,9-dihydro-1H-pyrazolo[3,4-b]quinoline and two 1H-pyrazolo[3,4-b]quinolines

Hydrazone derivatives exhibit a wide range of biological activities, while pyrazolo[3,4-b]quinoline derivatives, on the other hand, exhibit both antimicrobial and antiviral activity, so that all new derivatives in these chemical classes are potentially of value. Dry grinding of a mixture of 2-chloroquinoline-3-carbaldehyde and 4-methylphenylhydrazinium chloride gives (E)-1-[(2-chloroquinolin-3-yl)methylidene]-2-(4-methylphenyl)hydrazine, C17H14ClN3, (I), while the same regents in methanol in the presence of sodium cyanoborohydride give 1-(4-methylphenyl)-4,9-dihydro-1H-pyrazolo[3,4-b]quinoline, C17H15N3, (II). The reactions between phenylhydrazinium chloride and either 2-chloroquinoline-3-carbaldehyde or 2-chloro-6-methylquinoline-3-carbaldehyde give, respectively, 1-phenyl-1H-pyrazolo[3,4-b]quinoline, C16H11N3, (III), which crystallizes in the space group Pbcn as a nonmerohedral twin having Z' = 3, or 6-methyl-1-phenyl-1H-pyrazolo[3,4-b]quinoline, C17H13N3, (IV), which crystallizes in the space group R\overline{3}. The molecules of compound (I) are linked into sheets by a combination of N-H...N and C-H...π(arene) hydrogen bonds, and the molecules of compound (II) are linked by a combination of N-H...N and C-H...π(arene) hydrogen bonds to form a chain of rings. In the structure of compound (III), one of the three independent molecules forms chains generated by C-H...π(arene) hydrogen bonds, with a second type of molecule linked to the chains by a second C-H...π(arene) hydrogen bond and the third type of molecule linked to the chain by multiple π-π stacking interactions. A single C-H...π(arene) hydrogen bond links the molecules of compound (IV) into cyclic centrosymmetric hexamers having \overline{3} (S6) symmetry, which are themselves linked into a three-dimensional array by π-π stacking interactions.

Synthesis and Anticancer Activities of Novel Guanylhydrazone and Aminoguanidine Tetrahydropyran Derivatives.

In this paper we present the convenient syntheses of six new guanylhydrazone and aminoguanidine tetrahydropyran derivatives 2–7. The guanylhydrazone 2, 3 and 4 were prepared in 100% yield, starting from corresponding aromatic ketones 8a–c and aminoguanidine hydrochloride accessed by microwave irradiation. The aminoguanidine 5, 6 and 7 were prepared by reduction of guanylhydrazone 2–4 with sodium cyanoborohydride (94% yield of 5, and 100% yield of 6 and 7). The aromatic ketones 8a–c were prepared from the Barbier reaction followed by the Prins cyclization reaction (two steps, 63%–65% and 95%–98%). Cytotoxicity studies have demonstrated the effects of compounds 2–7 in various cancer and normal cell lines. That way, we showed that these compounds decreased cell viabilities in a micromolar range, and from all the compounds tested we can state that, at least, compound 3 can be considered a promising molecule for target-directed drug design.

An unexpected effect of acetal stereochemistry on the course of its reductive cleavage

During the course of studies toward the synthesis of the ‘upenamide BC spirocycle a surprising effect of acetal stereochemistry on the course of its reductive cleavage was observed. Reduction of alpha acetal 6a with diisobutylaluminum hydride led to PMB ether 10, while the corresponding beta acetal 6b was resistant to reduction with diisobutylaluminum hydride, but on treatment with sodium cyanoborohydride–trimethylsilyl chloride afforded PMB ether 8.

Asymmetric Diels-Alder Cycloadditions of Trifluoromethylated Dienophiles Under Trienamine Catalysis.

β-Trifluoromethyl (CF3 ) enones were proved to act as good dienophiles in asymmetric normal-electron-demand Diels-Alder cycloadditions with 2,4-dienals under trienamine catalysis with a chiral secondary amine. The sequential reductive amination transformations with benzylamine produced cis- and trans-fused chiral trifluoromethylated octahydroisoquinolines in a diastereodivergent manner by using NaBH(OAc)3 and NaBH3 CN as the reductants, respectively. Moreover, other types of activated alkenes that bear a CF3 group have also been successfully utilized to construct a diverse range of chiral cyclic frameworks in high stereoselectivity.

C(16)-C(22) oxygen-bridged analogues of ceDAF-12 and LXR ligands

The DAF-12 receptor in nematodes and the Liver X Receptor (LXR) in mammals are structurally related transcription factors that play key roles in determining the life span of the organism. Both types of receptors are activated by oxysterols, cholesterol metabolites with oxidized side chains. Restricting the movement of the oxysterol side chain to certain orientations may have profound effects in the activity profile, however this has not been explored so far. In a first attempt to obtain analogues of natural ligands of DAF-12 and LXR with restricted side chain mobility we introduced a 16,22-oxygen bridge in 26-hydroxycholesterol, a cholestenoic acid and a dafachronic acid (5–7). Diosgenin was used as starting material, the key step to obtain the 16,22 epoxy functionality was the one pot formation and reduction of a cyclic hemiketal via the oxocarbenium ion using sodium cyanoborohydride. All new compounds were characterized by NMR and mass spectrometry and assayed as ceDAF-12 or LXR ligands in transactivation cell-based assays. The dafachronic acid analogue 7 behaved as a ceDAF-12 agonist.

Синтез, противогрибковая и противовирусная активность N-бензильных производных тетраенового макролидного антибиотика люцензомицина

Реакции тетраенового макролидного антибиотика люцензомицина с пара-замещенными бензальдегидами и цианоборогидридом в условиях реакции восстановительного аминирования приводят к образованию его N-бензильных производных. Изучены физико-химические и медико-биологические свойства полученных производных пимарицина. Биологическими исследованиями показано, что N-бензильные производные люцензомицина обладали высокой противогрибковой и противовирусной активностью. Фармакологические испытания показали, что острая токсичность полученных производных люцензомицина была в 6 раз меньше, чем у исходного антибиотика.

Fragmentation and ion mobility properties of negative ions from N-linked carbohydrates: Part 7. Reduced glycans.

Negative ion collision-induced dissociation (CID) spectra of released N-glycans provide very informative structural information relating to branching patterns and location of residues such as fucose. For some structural studies, particularly those involving chromatography, glycans are often reduced to avoid production of multiple peaks from α- and β-anomers. We examined the effect of reduction on the production of diagnostic fragment ions and on the ion mobility properties of N-glycans. Released N-glycans from the glycoproteins bovine fetuin, ribonuclease B, chicken ovalbumin, and porcine thyroglobulin were reduced with sodium cyanoborohydride and both negative ion CID spectra and ion mobility properties of their phosphate adducts were examined with a Waters Synapt G2Si travelling-wave ion mobility mass spectrometer with electrospray sample introduction. Estimated collisional cross sections were measured with dextran as the calibrant, Fragment ions were similar to those from the unreduced glycans with the exception that the prominent (2,4) A cleavage ion from the reducing terminus was replaced by a prominent [M-H3 PO4](-) ion. Other ions arising from the chitobiose core were of lower relative abundance than those from the unreduced glycans. Estimated collisional cross sections were similar to those of the unreduced compounds but with symmetrical arrival time distribution (ATD) profiles, unlike those of the unreduced glycans whose peaks often contained prominent asymmetry. This observation showed that this asymmetry was due to anomer separation. Reduction of the reducing terminal GlcNAc residue resulted in fewer diagnostic ions from the chitobiose core but fragmentation of the remainder of the molecules generally paralleled that of the unreduced glycans. Thus, most structural information, with the exception of the linkage position of fucose on the core GlcNAc, was available. ATD peaks were symmetrical with the result that cross sections were more appropriate for data-base searching than those from the non-reduced compounds where asymmetry produced lower precision in the measurement.

Dual hydrophobe and phenylboronic acid-grafted poly(vinylpyrrolidone) polymers as shear-thinning and mucoadhesive eyedrop formulations

Event Abstract Back to Event Dual hydrophobe and phenylboronic acid-grafted poly(vinylpyrrolidone) polymers as shear-thinning and mucoadhesive eyedrop formulations Sahar Mokhtari1, Paniz Sheikholeslami1, Jacqueline Chau1, Ben Muirhead2, Heather Sheardown1, 2 and Todd Hoare1, 2 1 McMaster University, Department of Chemical Engineering, Canada 2 McMaster University, School of Biomedical Engineering, Canada Introduction: Effective drug delivery to the eye is challenging due to the multiple biological barriers promoting rapid clearance, with as little as ~5% of drugs administered via eyedrops reaching the cornea[1]. Shear-associative hydrogels cross-linked via physical interactions that can be broken under stress offer a potential solution given that (a) they are easily to administer (b) they can self-lubricate blinking and (c) they can be engineered with different viscosities to control release kinetics[2],[3]. Adding a mucoadhesive functionality offers further potential to enhance the residence time in the tear film and thus reduce the required frequency of eyedrops. Herein, we describe a physically-gelling hydrogel formulation based on poly(vinylpyrrolidone) (PVP) chemically modified with both hydrophobes (C8 – C18) and phenylboronic acid (PBA) mucoadhesive groups[4] with potential to interact with each layer of the tear film (mucoadhesion to cornea, self-associations between hydrophobes and free mucin-PBA interactions in the aqueous, and hydrophobe assembly in the lipid layer, Fig. 1). Materials and Methods: Functional PVP was prepared via copolymerization of N-vinylpyrrolidone (VP) with N-vinylformamide (VF) followed by hydrolysis of VF residues to primary amines. Alkylchloride (C8 – C18) hydrophobes were then grafted to 15-20 mol% of the free amine groups via condensation, after which 4-formylphenylboronic acid was grafted to the remaining amine groups via reductive amination using sodium cyanoborohydride. Shear thinning and mucoadhesion were measured using rheology, while transmittance was measured using UV/vis spectrophotometry. Drug release kinetics of doxorubicin hydrochloride were measured using UV/vis spectrophotometry. In vitro cytotoxicity to human corneal epithelial cells (HCECs) was assessed using an MTT assay. Anterior segment biocompatibility was tested by administering 0.1 mL of 15 wt% polymer solution to the eyes of New Zealand White rabbits and scoring ocular responses using Draize scoring. Results and Discussion: Polymers containing up to 2.5 mol% hydrophobes and 19 mol% PBA showed transmissions (>90%), refractive indices (~1.348), and cytocompatibilities (>90% cell viability to HCECs, Fig. 2a) suitable for ophthalmic use, even at high concentrations (15 wt%). The polymers are highly shear-thinning, with 3-4 decades of viscosity between zero and infinite shear relatively independent of the PBA content (Fig. 2b). The excess storage modulus associated with polymer-mucin interactions is as high as >1 kPa, suggesting strong mucoadhesion (Fig. 2c). Formulations effectively prolonged release of doxorubicin hydrochloride over ~1 week (Fig. 2d). Draize scoring indicated no significant conjunctival edema or redness, secretion, corneal opacity, or iris involvement; indeed, the treated eyes showed less inflammation than the control eyes, suggesting potential benefits for dry eye treatment. Conclusion: Hydrophobically and PBA-modified PVP can form a highly shear-sensitive self-associative gel that remains transparent, demonstrates both shear thinning and mucoadhesive potential, facilitates controlled drug release, and is biologically well-tolerated in vitro and in vivo. We anticipate this polymer may offer particular utility in eyedrops for either prolonged drug release to the anterior segment and/or alone as a dry eye therapeutic. 20/20: NSERC Ophthalmic Materials Research Network (funding)

Reaction of nitroso chlorides of the adamantane series with nucleophiles

Reactions of nitroso chlorides of 2-alkylydene adamantanes with O- and N-nucleophiles resulted in α-substituted oximes of the adamantane series. Reaction involved 1,4-nucleophilic addition of the nucleophiles to the generated in situ nitroso alkenes acting as the Michael acceptors. Reduction of the precursors of nitroso alkenes with sodium cyanoborohydride gave 2-substituted adamantylated oximes.

Quantification of plant cell wall monosaccharides by reversed-phase liquid chromatography with 2-aminobenzamide pre-column derivatization and a non-toxic reducing reagent 2-picoline borane

In this report, we described a sensitive method for quantifying plant cell wall monosaccharides using chemical derivatization, reversed-phase high performance liquid chromatography separation with ultraviolet detection (HPLC-UV). Monosaccharides were derivatized with 2-aminobenzamide (2-AB) by reductive amination to increase the hydrophobicity and detected by ultraviolet absorption for HPLC-UV analysis. A non-toxic reductant, 2-picoline borane was utilized to replace the traditionally used sodium cyanoborohydride (NaCNBH3) to avoid the formation of toxic by-products. Experimental conditions were optimized using glucose as a model system to achieve a high reaction yield of 99%. Under the optimized conditions, we demonstrated that the derivatization yields of several saccharides with 2-AB using 2-picoline borane were all slightly higher than those observed using NaCNBH3. In plants, cell wall monosaccharides consist of arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, and xylose. Using our method, we successfully quantified these monosaccharides from Arabidopsis cell wall by HPLC-UV, and we obtained a good linearity at a wide dynamic range over five orders (1pmol through 10nmol of injection amount), a detection limit of ∼0.1pmole, and a high precision and accuracy.

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.

Mass spectrometry signal enhancement by reductive amination

Organosulfur compounds (OSCs) subjected to reductive amination in the presence of formaldehyde exhibited increased mass spectrometry signal intensities. In this study, four OSCs including S-allyl cysteine, S-allylcysteinine sulfoxide, S-methylcysteine and S-ethylcysteine were generated using isotopic formaldehyde, and mass spectrometry signal intensities of modified and unmodified OSCs were compared. This comparison involved tandem mass spectrometry infusion and detection techniques, such as selected ion monitoring (SIM) and multiple reaction monitoring (MRM). The signal intensities of modified OSCs increased from 2.6 to 39.2 fold by infusion, from 50.0 to 479.6 fold by SIM, and from 146.4 to 2494.8 fold by MRM. Compounds bearing primary amine groups reacted with formaldehyde in high yield and underwent reductive amination in the presence of sodium cyanoborohydride to form a dimethyl group on these amine groups. The modified OSCs showed enhanced intensities because the electron donating dimethyl groups increase their basicity. This signal enhancement is expected to improve the limit of detection in absolute quantification and structural characterization. Therefore, reductive amination involving primary amine groups may find application in the enhancement of mass spectrometry signal intensities.