Cyanoborohydride Research Articles

(±)‐1,2‐Dialkyl‐5‐nitro‐2,3‐dihydro‐1H‐indoles by a tandem reductive amination‐SNAr reaction

Abstract magnified imageA tandem reductive amination‐SNAr reaction has been applied to the synthesis of (±)‐1,2‐dialkyl‐5‐nitro‐2,3‐dihydro‐1H‐indoles. Treatment of a series of 2‐fluoro‐5‐nitrobenzyl ketones with primary amines and sodium cyanoborohydride in methanol at room temperature provided good yields of the target heterocycles. The reaction is sensitive to steric hindrance and proceeds best with less hindered ketone substrates using primary amines that are unbranched at the α carbon. J. Heterocyclic Chem., (2009).

Stereoselective Synthesis of Unnatural β-Amino Acids from Nitroethane via 5,6-Dihydro-4H-1,2-oxazin-3-ylacetates

The reduction of easily available methyl 5,6-dihydro-4H-1,2-oxazin-3-ylacetates provides an efficient route to different diastereomerically pure unnatural β-amino acids. A two-step protocol including reduction of the C=N bond of the initial oxazine with sodium cyanoborohydride during the first step and hydrogenation of the N-O bond during the second step produced the target β-amino acid esters with higher stereoselectivity than obtained with conventional catalytic hydrogenation.

Evaluation of the DNA Damaging Potential of Cannabis Cigarette Smoke by the Determination of Acetaldehyde Derived N2-Ethyl-2′-deoxyguanosine Adducts

Acetaldehyde is an ubiquitous genotoxic compound that has been classified as a possible carcinogen to humans. It can react with DNA to form primarily a Schiff base N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) adduct. An online column-switching valve liquid chromatography tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) method was developed for the determination of N(2)-ethylidene-dG adducts in DNA following reduction with sodium cyanoborohydride (NaBH(3)CN) to the chemically stable N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) adduct. Accurate quantitation of the adduct was obtained by the addition of the [(15)N(5)]N(2)-ethyl-dG stable isotope-labeled internal standard prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides with the incorporation of NaBH(3)CN in the DNA hydrolysis buffer. The method required 50 microg of hydrolyzed DNA on column for the analysis, and the limit of detection for N(2)-ethyl-dG was 2.0 fmol. The analysis of calf thymus DNA treated in vitro with acetaldehyde (ranging from 0.5 to 100 mM) or with the smoke generated from 1, 5, and 10 cannabis cigarettes showed linear dose-dependent increases in the level of N(2)-ethyl-dG adducts (r = 0.954 and r = 0.999, respectively). Similar levels (332.8 +/- 21.9 vs 348.4 +/- 19.1 adducts per 10(8) 2'-deoxynucleosides) of N(2)-ethyl-dG adducts were detected following the exposure of calf thymus DNA to 10 tobacco or 10 cannabis cigarettes. No significant difference was found in the levels of N(2)-ethyl-dG adducts in human lung DNA obtained from nonsmokers (n = 4) and smokers (n = 4) with the average level observed as 13.3 +/- 0.7 adducts per 10(8) 2'-deoxynucleosides. No N(2)-ethyl-dG adducts were detected in any of the DNA samples following analysis with the omission of NaBH(3)CN from the DNA hydrolysis buffer. In conclusion, these results provide evidence for the DNA damaging potential of cannabis smoke, implying that the consumption of cannabis cigarettes may be detrimental to human health with the possibility to initiate cancer development.

Improved method to measure aldehyde adducts to N-terminal valine in hemoglobin using 5-hydroxymethylfurfural and 2,5-furandialdehyde as model compounds

Hemoglobin (Hb) adducts are used to measure reactive compounds/metabolites in vivo. Schiff base adducts from aldehydes to N-termini in Hb have been measured by GC–MS/MS after stabilisation through reduction, and detachment by a modified Edman procedure. This paper describes a further development using 5-hydroxymethylfurfural (HMF) and its probable metabolite, 2,5-furandialdehyde (FDA), as model compounds. Reference compounds were synthesized and characterized. The conditions for the reduction of the Schiff bases were optimized using NaBH 3CN as a mild reducing agent, and steps used in the earlier method could be deleted. The adduct from FDA could not be specifically analysed, as selective reduction of the imine could not be achieved. In a few samples of human blood, background levels of 10–35 pmol/g globin of the HMF adduct were observed. Half-lifes of the reversible Schiff base adduct from HMF were determined to 3.4 h at 37 °C and 10.9 h at 25 °C. The developed method showed good sensitivity and reproducibility for the analysis of the Schiff base from HMF, with improvements regarding simplicity of work-up procedures due to mild conditions. The developed method could be explored for application to adducts from other aldehydes bound as Schiff bases to N-termini in Hb.

Selectivity in Enrichment of cAMP-dependent Protein Kinase Regulatory Subunits Type I and Type II and Their Interactors Using Modified cAMP Affinity Resins

cAMP regulates cellular functions primarily by activating PKA. The involvement of PKAs in various signaling pathways occurring simultaneously in different cellular compartments necessitates stringent spatial and temporal regulation. This specificity is largely achieved by binding of PKA to protein scaffolds, whereby a distinct group of proteins called A kinase anchoring proteins (AKAPs) play a dominant role. AKAPs are a diverse family of proteins that all bind via a small PKA binding domain to the regulatory subunits of PKA. The binding affinities between PKA and several AKAPs can be different for different isoforms of the regulatory subunits of PKA. Here we employ a combination of affinity chromatography and mass spectrometry-based quantitative proteomics to investigate specificity in PKA-AKAP interactions. Three different immobilized cAMP analogs were used to enrich for PKA and its interacting proteins from several systems; HEK293 and RCC10 cells and rat lung and testis tissues. Stable isotope labeling was used to confidently identify and differentially quantify target proteins and their preferential binding affinity for the three different cAMP analogs. We were able to enrich all four isoforms of the regulatory subunits of PKA and concomitantly identify more than 10 AKAPs. A selective enrichment of the PKA RI isoforms could be achieved; which allowed us to unravel which AKAPs bind preferentially to the RI or RII regulatory domains of PKA. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual specificity with a potential preference for RI. For some of these AKAPs our data provide the first insights into their specificity.

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.

Reduction of 2-amino-1-(4-nitrophenyl)-1 H-pyrido[3,2-b]indole-3-carbonitrile by sodium borohydride and sodium cyanoborohydride

The reduction of pyridoindole derivative 1 by sodium borohydride in methanol gives 4,5-dihydropyridoindole 2. On treatment of 5H-pyrido[3,2-b]indolium chloride 3 with sodium cyanoborohydride in methanol in the presence of hydrogen chloride, reduction of the pyridine ring is accompanied by reduction of the CN group, resulting in the formation of tetrahydropyridoindole 4. Compound 4 reacts with DMF dimethyl acetal to yield amidine 6, and refluxing of 4 with acetic anhydride results in tetrahydropyridine ring cleavage yielding indolylacrylonitrile 9. The hydrochloride and chloride of compounds 4 and 6 were obtained

Diastereoselective Synthesis of γ-Amino Acids and Their Derivatives from Nitroethane via Intermediacy of 5,6-Dihydro-4H-1,2-oxazines Bearing the CH2CH(CO2Me)2 Substituent at C3

Stereoselective two-step reduction of available 2-[(5,6-dihydro-4H-1,2-oxazin-3-yl)methyl]malonates provides an efficient route to derivatives of different γ-amino acids. The mechanism and stereochemistry of the first step, reduction of the C=N bond with sodium cyanoborohydride, is discussed.

Facilitated Monocyte-Macrophage Uptake and Tissue Distribution of Superparmagnetic Iron-Oxide Nanoparticles

BackgroundWe posit that the same mononuclear phagocytes (MP) that serve as target cells and vehicles for a host of microbial infections can be used to improve diagnostics and drug delivery. We also theorize that physical and biological processes such as particle shape, size, coating and opsonization that affect MP clearance of debris and microbes can be harnessed to facilitate uptake of nanoparticles (NP) and tissue delivery.MethodsMonocytes and monocyte-derived macrophages (MDM) were used as vehicles of superparamagnetic iron oxide (SPIO) NP and immunoglobulin (IgG) or albumin coated SPIO for studies of uptake and distribution. IgG coated SPIO was synthesized by covalent linkage and uptake into monocytes and MDM investigated related to size, time, temperature, concentration, and coatings. SPIO and IgG SPIO were infused intravenously into naïve mice. T2 measures using magnetic resonance imaging (MRI) were used to monitor tissue distribution in animals.ResultsOxidation of dextran on the SPIO surface generated reactive aldehyde groups and permitted covalent linkage to amino groups of murine and human IgG and F(ab')2 fragments and for Alexa Fluor® 488 hydroxylamine to form a Schiff base. This labile intermediate was immediately reduced with sodium cyanoborohydride in order to stabilize the NP conjugate. Optical density measurements of the oxidized IgG, F(ab')2, and/or Alexa Fluor® 488 SPIO demonstrated ∼50% coupling yield. IgG-SPIO was found stable at 4°C for a period of 1 month during which size and polydispersity index varied little from 175 nm and 200 nm, respectively. In vitro, NP accumulated readily within monocyte and MDM cytoplasm after IgG-SPIO exposure; whereas, the uptake of native SPIO in monocytes and MDM was 10-fold less. No changes in cell viability were noted for the SPIO-containing monocytes and MDM. Cell morphology was not changed as observed by transmission electron microscopy. Compared to unconjugated SPIO, intravenous injection of IgG-SPIO afforded enhanced and sustained lymphoid tissue distribution over 24 hours as demonstrated by MRI.ConclusionsFacilitated uptake of coated SPIO in monocytes and MDM was achieved. Uptake was linked to particle size and was time and concentration dependent. The ability of SPIO to be rapidly taken up and distributed into lymphoid tissues also demonstrates feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy.

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.

Unusual Enantiopure Heterocyclic Skeletons by Lewis Acid Promoted Rearrangements of 1,3‐Dioxolanyl‐Substituted 1,2‐Oxazines

AbstractLewis acid promoted rearrangements of different 4‐alkoxy‐substituted 1,2‐oxazines syn‐1 are reported. Depending on the nature of this alkoxy group different reaction pathways are possible either providing bicyclic 1,2‐oxazinones 2 or the novel tricyclic products 3–5. A mechanistic rational describing the role of the 4‐alkoxy group is presented. The key step for formation of tricyclic skeletons 3–5 is a 1,2‐alkyl shift. Hydrogenation reactions of these tricyclic compounds gave unsaturated 1,2‐oxazines 12 and 13 or tetrahydrofurans 15a–c/16a–c depending on the time of hydrogenolysis. Tetrahydrofuryl‐annulated 1,2‐oxazine 12 was used for further transformations into complex substituted tetrahydrofurans. Reduction with sodium cyanoborohydride and subsequent cleavage of the N,O‐bond by hydrogenation furnished aminofuran derivative 19. Alternatively treatment with a strong base such as n‐butyllithium afforded imidoester 21 via a Beckmann‐type fragmentation.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Fluorescent detection of α-aminoadipic and γ-glutamic semialdehydes in oxidized proteins

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. α-Aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.

Involvement of Highly Sulfated Chondroitin Sulfate in the Metastasis of the Lewis Lung Carcinoma Cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.

Rearrangement of fused tetracyclic heterocycles induced by alkyl halides and formation of a new type of ‘proton sponge’

Intramolecular criss-cross cycloaddition of 3-substituted symmetrical homoallenyl azines 2 by heating in xylene lead to interesting fused heterocyclic systems consisting of four five-membered rings with two nitrogen atoms in the skeleton 3. These compounds 3 were found to be sensitive to attack by alkyl halides. Their presence, depending on the reaction conditions, resulted in a new type of rearrangement leading to compounds 4 and 5, respectively. With an excess of alkyl halide and in the presence of NaBH 3CN a new structure 6 with signs of the molecule corresponding a ‘proton sponge’ moiety was created. The scope of the rearrangement and reaction products structure was investigated.

Intermolecular Cope-Type Hydroamination of Alkenes and Alkynes using Hydroxylamines

The development of the Cope-type hydroamination as a method for the metal- and acid-free intermolecular hydroamination of hydroxylamines with alkenes and alkynes is described. Aqueous hydroxylamine reacts efficiently with alkynes in a Markovnikov fashion to give oximes and with strained alkenes to give N-alkylhydroxylamines, while unstrained alkenes are more challenging. N-Alkylhydroxylamines also display similar reactivity with strained alkenes and give modest to good yields with vinylarenes. Electron-rich vinylarenes lead to branched products while electron-deficient vinylarenes give linear products. A beneficial additive effect is observed with sodium cyanoborohydride, the extent of which is dependent on the structure of the hydroxylamine. The reaction conditions are found to be compatible with common protecting groups, free OH and NH bonds, as well as bromoarenes. Both experimental and theoretical results suggest the proton transfer step of the N-oxide intermediate is of vital importance in the intermolecular reactions of alkenes. Details are disclosed concerning optimization, reaction scope, limitations, and theoretical analysis by DFT, which includes a detailed molecular orbital description for the concerted hydroamination process and an exhaustive set of calculated potential energy surfaces for the reactions of various alkenes, alkynes, and hydroxylamines.

Comparative Phosphoproteomics of Zebrafish Fyn/Yes Morpholino Knockdown Embryos

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

Dimethylthexylsilyl 2-acetamido-3- O-allyl-2-deoxy-6- O-(4-methoxybenzyl)-β- d-glucopyranoside, dimethylthexylsilyl 3,4,6-tri- O-benzyl-β- d-mannopyranosyl-(1→4)-2-acetamido-3- O-allyl-2-deoxy-6- O-(4-methoxybenzyl)-β- d-glucopyranoside, and dimethylthexylsilyl 2- O-(benzylsulfonyl)-3,4,6-tri- O-benzyl-β- d-mannopyranosyl-(1→4)-2-acetamido-3- O-allyl-2-deoxy-6- O-(4-methoxybenzyl)-β- d-glucopyranoside: synthesis of authentic samples

Dimethylthexylsilyl 2-acetamido-3- O-allyl-2-deoxy-6- O-(4-methoxybenzyl)-β- d-glucopyranoside was prepared by reduction of the corresponding 4,6- O-(4-methoxybenzylidene) acetal with sodium cyanoborohydride and trifluoroacetic acid. This alcohol was coupled to 2- O-benzoyl-3,4,6-tri- O-benzyl-α- d-glucopyranosyl trichloroacetimidate to give a β-glucoside that was converted to dimethylthexylsilyl 3,4,6-tri- O-benzyl-β- d-mannopyranosyl-(1→4)-2-acetamido-3- O-allyl-2-deoxy-6- O-(4-methoxybenzyl)-β- d-glucopyranoside by saponification, Dess–Martin oxidation, and sodium borohydride reduction. Sulfonylation then gave dimethylthexylsilyl 2- O-(benzylsulfonyl)-3,4,6-tri- O-benzyl-β- d-mannopyranosyl-(1→4)-2-acetamido-3- O-allyl-2-deoxy-6- O-(4-methoxybenzyl)-β- d-glucopyranoside.

Synthesis and Reactivity of Novel α,α,β- and α,α,δ-Trichlorinated Imines

A variety of different N-(2,2,3-trichloropropylidene)amines, N-(2,2,3-trichlorobutylidene)amines, and N-(2,2,5-trichloropentylidene)amines were synthesized for the first time, and their reactivity with regard to hydride reagents was investigated. In this way, N-(2,2,5-trichloropentylidene)amines were evaluated as substrates for the synthesis of piperidines, and N-(2,2,3-trichloropropylidene)amines and N-(2,2,3-trichlorobutylidene)amines were reduced efficiently into the corresponding novel beta,beta,gamma-trichloroamines by means of sodium cyanoborohydride in methanol in the presence of acetic acid. Furthermore, N-(2,2,3-trichloropropylidene)amines were transformed into 2-(chloromethyl)aziridines by lithium aluminium hydride in Et2O, and N-(2,2,5-trichloropentylidene)acetamide was used for the First time as a suitable Substrate for the addition of oxygen, nitrogen, and Sulfur nucleophiles in good yields.

Selective Reduction of 7-Demethylsinomenine

Abstract Several borohydride reagents: sodium borohydride, sodium cyanoborohydride, soudium acetoxyborohydride and soudium triacetoxyborohydride were screened, respectively, for the reduction of 7-demethylsinomenine, which was an α,β-dicarbonyl compound derived from sinomenine. Highly regio- and stereo-selectivity was acquired when sodium cyanoborohydride or NaBH(OAc) 3 was used. The product was structurally confirmed as 7R configuration by NMR, X-ray crystal diffraction analysis. Some preliminary discussion was also made on the mechanism of the selective reduction.

Automated Online Sequential Isotope Labeling for Protein Quantitation Applied to Proteasome Tissue-specific Diversity

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure, which allows protein quantitation using differential isotopic dimethyl labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.