Cyanoborohydride Research Articles

Development of Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry Methods for Analysis of DNA Adducts of Formaldehyde and Their Application to Rats Treated with N-Nitrosodimethylamine or 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

Reaction of formaldehyde with DNA in vitro produces a variety of adducts among which are observed the cross-link di-(N(6)-deoxyadenosyl)methane (dAdo-CH 2-dAdo, 1) and the hydroxymethyl adduct N(6)-hydroxymethyl-dAdo (N(6)-HOCH 2-dAdo, 2). While the structures of these adducts have been known for decades, there have been no reports of their formation in vivo. Formaldehyde is released during intracellular metabolism of carcinogenic N-nitrosomethyl compounds such as N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), but DNA adducts formed by this pathway have not been previously characterized. In this study, we addressed these questions by developing highly sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring methods for quantitation of adducts 1 and 2, the latter as N(6)-methyl-dAdo ( 3). Considerable effort was devoted to the problem of artifactual formation of 1, which can occur during storage of DNA samples by reaction of dAdo with 2. This problem was solved by the addition of adenosine deaminase during the DNA hydrolysis step, thus removing dAdo as a reactant. The instability of adduct 2 was another potential block to analysis, and this was solved by converting it to 3 with NaBH 3CN. Separate aliquots of DNA were analyzed for adducts 1 and 2, using the [(15)N]-labeled adducts as internal standards. The application of these methods to rat hepatic DNA to which adducts 1 and 3 were added demonstrated accuracy and precision. The detection limits for adducts 1 and 3 were 1-4 adducts per 10 (9) nucleotides using 100-150 microg of DNA. The method was applied to analyze hepatic and pulmonary DNA from rats treated with NDMA and NNK. The results clearly demonstrated the dose-dependent presence of N(6)-HOCH 2-dAdo ( 2) in all DNA samples. The cross-link adduct dAdo-CH 2-dAdo ( 1) was observed in hepatic DNA of NNK-treated rats, with lower amounts in pulmonary DNA. Levels of these adducts were generally less than those of DNA adducts formed by the classical diazohydroxide pathway of nitrosamine metabolism. The results of this study demonstrate for the first time the presence of formaldehyde DNA adducts in tissues of rats treated with carcinogenic nitrosamines and suggest that these adducts may play a role in cancer induction by these compounds.

Synthesis of an anionically substituted nitroindoline-caged GABA reagent that has reduced affinity for GABA receptors

A 7-nitroindolin-1-yl amide of the neuroactive amino acid γ-aminobutryate (GABA) has been synthesised with two phosphate groups attached to the indoline nucleus at 4-alkoxy substituent. The compound is designed to release GABA on a sub-microsecond time scale in response to flash photolysis with near-UV light. The presence of the high negatively charged substituent shows evidence that interaction of the GABA conjugate with ionotropic GABA receptors is much reduced in comparison with an earlier nitroindoline-GABA compound that had no charged groups on the indoline nucleus. In experiments to develop the eventual synthetic route, an unusual reductive cleavage of a TBDMS ether was observed as a significant side reaction during reduction of an indole to an indoline with NaBH 3CN in glacial acetic acid. In the eventual synthetic route, the primary amine of GABA was masked as an azide until the final stage of the synthesis, which overcame significant problems with other forms of amine protection.

Synthesis and antimicrobial activity of 7-fluoro-3-aminosteroids

A series of 7-fluoro-3-aminosteroids were synthesized and their in vitro antimicrobial activities were evaluated against Gram-positive and Gram-negative bacteria. The nucleophilic fluorination of several 7β-hydroxysteroids by diethylaminosulfur trifluoride in n-pentane, followed by reductive amination of the resulting 7-fluoro-3-ketosteroids with spermidine in the presence of NaBH 3CN, afforded 7-fluoro-3-aminosteroids in high yield. Compound 25 showed the highest antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, and Escherichia coli.

Design and synthesis of a library of tertiary amides: Evaluation as mimetics of the melanocortins’ active core

Two hundred and ten tertiary amides were prepared on solid phase. Diamines were coupled to activated carboxylated Wang polymer, and the polymeric substituted benzyloxycarbonyl protected diamines obtained were reacted with aldehydes or ketones in trimethyl orthoformate giving resin attached Schiff bases. Coupled resins were then reduced to secondary amines by sodium cyanoborohydride in 4% acetic acid/trimethyl orthoformate, followed by acylation with the carboxylic acid in the presence of PyBroP and diisopropylethylamine. Cleavage of tertiary amides from the resin was made by trifluoroacetic acid in the presence of scavengers (mainly 1,2-ethanedithiol). When indole derivatives were prepared, parallel alkylation with the linker fragment occurred, giving derivatives of 2-(4-hydroxybenzyl)-indole as side products. Solution synthesis or mixed liquid/solid phase preparation of title substances proved to be advantageous in cases when the above method did not give acceptable results. According to this approach an efficient formation of Schiff bases was achieved in the presence of TiCl 4. Substances were isolated by reversed phase chromatography; in some cases isomers were additionally separated by chiral chromatography on Chirobiotic T. When tested on human recombinant melanocortin receptors all the tertiary amides showed some binding affinities; for the highest affinity compounds the K is reached 400 nM on MC 1, 2 μM on MC 3 and 1 μM on MC 4 and MC 5 receptors. cAMP assays of some of the title compounds showed that the tertiary amides are melanocortin receptor antagonists on the four MC receptor subtypes.

The synthesis of facial amphiphile 3α,7α-diaminocholestane

The facial amphiphile 3α,7α-diaminocholestane 3 was synthesized from 3β-acetoxy-7-ketocholestane 1 through a stepwise reductive amination. The reductive amination of 1 with NH 4OAc in the presence of NaBH 3CN, and protection with Boc 2O yielded 7α-( tert-butyloxycarbonyl)-aminocholestane 4 in 86% yield. The reductive amination of 6, which was obtained from 4 after hydrolysis and subsequent oxidation, with NH 4OT f in the presence of NaBH(OEh) 3 provided 3 in 75% yield after protection with Boc 2O.

A Stereoselective Entry into Functionalized 1,2-Diamines by Zinc-Mediated Homologation of α-Aminoacids

A general, stereoselective synthesis of 4,5-disubstituted imidazolidines-2-ones from alpha-aminoacids has been developed: the key steps are a Blaise reaction of bromoacetate on alpha-aminonitriles and further reduction. Although reduction with sodium cyanoborohydride afforded a mixture of cis and trans isomers 6a-e with moderate to good stereoselectivity, reduction with sodium in liquid ammonia gave the trans isomers 8a-e with complete stereoselectivity. Acidic hydrolysis of the urea gave 4-amino-pyrrolidinones, which can be precursors to beta,gamma-diaminoacids or 3-aminopyrrolidines.

Synthesis of substituted 3-arylpiperidines and 3-arylpyrrolidines by radical 1,4 and 1,2-aryl migrations

A route to 3-arylpiperidines and 3-arylpyrrolidines involving radical 1,4- and 1,2-aryl migrations has been explored. For the piperidines, the first route requires a xanthate addition to an N-allylarylsulfonamide, followed by acetylation and treatment with lauroyl peroxide to give the corresponding 1,4-aryl transfer product. This compound can be converted into the desired piperidine derivative following acidic hydrolysis. For the second approach to piperidines, addition of an α-keto xanthate to olefins of type 14 causes 1,2-aryl migration leading to an α,β-unsaturated ester, which can be converted into a piperidine by the action of ammonia or a primary amine and sodium cyanoborohydride. Substituted 3-arylpyrrolidines can be obtained by simply starting with an α-amido substituted xanthate.

C‐C Bond Formation by Radical Cyclization: Regioselective Synthesis of [6,6] Pyrano pyran System

: A number of 4‐aryloxymethyl pyrano[3,2‐c][1]benzopyran‐5‐(2H)‐ones 3(a–f) were prepared by refluxing 4‐chloromethyl pyrano[3,2‐c][1]benzopyran‐5‐(2H)‐ones 1(a,b) with o‐bromophenols 2(a–c) in acetone in the presence of anhydrous potassium carbonate and sodium iodide in 70–80% yield. Compounds 3(a–f) were then subjected to radical cyclization by refluxing with tri‐n‐butyltin chloride, sodium cyanoborohydride, and azobisisobutyronitrile (AIBN) in dry benzene for 4–5 h to give a cis‐diastereomeric mixture of [6,6]pyranopyran derivatives in 80–85% yield.

A Highly Stereoselective Synthesis of (−)-(ent)-Julifloridine from the Cyclization of an Alanine-Derived Chloroamine with an Acetylenic Sulfone

The cyclization of gamma-chloroamine 11, derived from l-alanine, and acetylenic sulfone 12 afforded the dehydropiperidine 19 via conjugate addition followed by intramolecular alkylation of the corresponding sulfone-stabilized anion. An unexpected acid-catalyzed desulfonylation of 19 occurred in one step via desilylation and tautomerization of the enamine moiety to the corresponding aldehyde, followed by elimination of p-toluenesulfinic acid. The highly stereoselective reduction of the resulting unsaturated aldehyde 25 with sodium cyanoborohydride produced piperidine 23 with a diastereomeric ratio of >98:2. (-)-(ent)-Julifloridine (8) was obtained by Swern oxidation of 23, followed by Wittig olefination and hydrogenation/debenzylation.

Regioselective ring openings in the 3,5-dioxa-8-azabicyclo[5.2.0]nonane ring system

In order to distinguish primary and secondary hydroxyl groups in the presence of a β-lactam moiety benzylidene acetal protection and reductive cleavage were used. When the reductive ring opening was carried out using diisobutylaluminium hydride, the benzylidene acetal group remained intact, while the β-lactam was reduced to the corresponding β-amino alcohol. The acetal cleavage with sodium cyanoborohydride was successful, resulting mainly in a β-lactam compound bearing the benzyloxy group on the secondary carbon atom.

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Stereoselective reductions of N-Boc-hexahydro-1 H-indolin-5(6 H)-ones

We report the divergent effects of a 3a-methyl and 3a-phenyl substituent on the chemoselectivity and stereoselectivity of reduction of the enamide moiety of N-Boc-hexahydro-1 H-indolin-5(6 H)-ones. Under ionic reduction conditions (triethylsilane/trifluoroacetic acid) the enamide group of 3a-methyl- N-Boc-hexahydro-1 H-indolin-5(6 H)-one was reduced to afford exclusively a cis ring-fused product. For the 3a-phenyl substituted analogue more forcing conditions (sodium cyanoborohydride at pH 2–2.5) were required and resulted in the selective reduction of the enamide group to give a trans ring-fused product as well as reduction of the ketone group.

A new procedure for the synthesis of peptide-derived Amadori products on a solid support

A new and straightforward solid-phase synthesis of a series of site-specific Amadori-modified peptides is described. The method involves reductive alkylation of the ε-amino groups of lysine with 2,3:4,5-di- O-isopropylidene-β- d-arabino-hexos-2-ulo-2,6-pyranose in the presence of sodium cyanoborohydride on a solid support.

Identification of the Non-lysosomal Glucosylceramidase as β-Glucosidase 2

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid beta-glucosidase, being beta-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985-2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.

Chemo- and regioselective reductive opening of azetidinium ions

Enantiomerically pure azetidinium trifluoromethanesulfonates were opened by various hydride reagents. LiAlH 4 and NaBH 3CN reacted with a complete regioselectivity and the latter reagent also reacted in a chemoselective way, leaving unaffected an ester or a cyano moiety present in the substrate. This reaction provides 1,2-diamines, 1,2- and 1,4-amino alcohols or α-amino esters by combining proper choice of substrate and hydride reagent.

Synthesis of 4-amino-3-oxo-tetrahydroazepino[3,4- b]indoles: new conformationally constrained Trp analogs

The synthesis of tryptophan analogs is reported in which the conformation has been constrained by formation of a seven-membered lactam. Boc-protected 2′-formyl tryptophan was obtained by SeO 2 oxidation of Boc-tetrahydro-β-carboline-3-carboxylic acid. Reductive amination was performed with a variety of amines and amino acid esters using sodium cyanoborohydride, followed by ring closure to the target compounds. The constrained Trp derivative has been incorporated into the endomorphin-1 opioid peptide sequence to probe the bioactive conformation.

Development and application of a capillary electrophoresis based method for the assessment of monosaccharide in soil using acid hydrolysis

An efficient methodology for the determination of carbohydrate content in soils, employing acid hydrolysis and subsequent capillary electrophoresis analysis (CE), is here described. Polysaccharides present in soil samples were hydrolyzed, at 100 °C during 4 h, to their monosaccharide form, by addition of 2 mol dm −3 trifluoroacetic acid (TFA) directly to soil. The resulting monosaccharides were then quantitatively derivatized with 4-aminobenzoic acid ethyl ester, via reductive amination with sodium cyanoborohydride, and separated by CE, coupled to an UV–vis diode array set at 300 nm. Separation electrolyte consisted of 5.0 × 10 −2 mol dm −3 sodium tetraborate buffer (pH 10.2) and 6 × 10 −3 mol dm −3 sodium dodecylsulphate. A 78 cm long capillary with an internal and external diameter of 75 and 375 μm, respectively, was used and separation performed at 16 °C, with an applied voltage of 30 kV. Quantification was undertaken using ribose as the internal standard. As an application example, carbohydrate composition (w/w) of a farmyard manure fertilized soil was found to vary between 0.0045 ± 0.0003% (glucose) and 0.0267 ± 0.0002% (arabinose) of the total soil content. Xylose, rhamnose, mannose, fucose and galactose content were also studied in the present work.

Protein−RNA Cross-Linking in the Ribosomes of Yeast under Oxidative Stress

Living systems have efficient degradative pathways for dealing with the fact that reactive oxygen species (ROS) derived from cellular metabolism and the environment oxidatively damage proteins and DNA. But aggregation and cross-linking can occur as well, leading to a series of problems including disruption of cellular regulation, mutations, and even cell death. The mechanism(s) by which protein aggregation occurs and the macromolecular species involved are poorly understood. In the study reported here, evidence is provided for a new type of aggregate between proteins and RNA in ribosomes. While studying the effect of oxidative stress induced in the yeast proteome it was noted that ribosomal proteins were widely oxidized. Eighty six percent of the proteins in yeast ribosomes were found to be carbonylated after stressing yeast cell cultures with hydrogen peroxide. Moreover, many of these proteins appeared to be cross-linked based on their coelution patterns during RPC separation. Since they were not in direct contact, it was not clear how this could occur unless it was through the RNA separating them in the ribosome. This was confirmed in a multiple-step process, the first being derivatization of all carbonylated proteins in cell lysates with biotin hydrazide through Schiff base formation. Following reduction of Schiff bases with sodium cyanoborohydride, biotinylated proteins were selected from cell lysates with avidin affinity chromatography. Oxidized proteins thus captured were then selected again using boronate affinity chromatography to capture vicinal diol-containing proteins. This would include proteins cross-linked to an RNA fragment containing a ribose residue with 2',3'-hydroxyl groups. Some glycoproteins would also be selected by this process. LC/MS/MS analyses of tryptic peptides derived from proteins captured by this process along with MASCOT searches resulted in the identification of 37 ribosomal proteins that appear to be cross-linked to RNA. Aggregation of proteins with ribosomal RNA has not been previously reported. The probable impact of this phenomenon cells is to diminish the protein synthesis capacity.

Use of methanesulfonic acid in the reductive ring-opening of O-benzylidene acetals

Methanesulfonic acid was shown to be an efficient and convenient substitute for ethereal HCl in reductive 4,6- O-benzylidene acetal ring-opening reaction with sodium cyanoborohydride in THF. Normal regioselectivity was observed, the 6- O-benzyl ethers with free 4-OH group being the major products of the reaction.

Reduction of substituted 1,10-phenanthrolines as a route to rigid chiral benzimidazolylidenes

Variously substituted 1,10-phenanthrolines are reduced to octahydrophenanthrolines in moderate to good yields with NaBH 3CN in acetic acid/methanol. The exact solvent composition is important to avoid the formation of tetrahydrophenanthrolines and N-alkylated byproducts, and to optimize the formation of octahydrophenanthrolines. Resolution of a racemic reduction product gives an enantiomerically pure C 2-symmetric diamine from which the corresponding rigid benzimidazolylidene is prepared, whereas reduction of chiral phenanthrolines derived from bicyclic ketones affords diastereomerically pure diamines, which may also be converted to benzimidazolylidenes.