Cyanoborohydride Research Articles

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.

New and efficient routes for the synthesis of murrayaquinone A and murrayanine

Three new routes were established for the synthesis of biologically active murrayaquinone A and a new method was developed for synthesis of murrayanine from the same starting material as 4-hydroxy carbazole. During the synthetic course a few novel observation were recorded, which include two one pot reaction sequences and C–N bond cleavage by sodium cyanoborohydride.

SURFACE MODIFICATION OF ALUMINUM OXIDE FOR BIOSENSING APPLICATION

The surface modification for immobilization of antibodies on the aluminum oxide surface was developed for biosensing application. The aluminum oxide has been received much attention since it has various advantages including low cost, hardness, resistance to organic solvents, and high capacitance. In addition, the aluminum oxide can prevent the leakage current in the electrical detection. For development of the protein anchorable surface, the aluminum oxide chip was treated with oxygen plasma and directly immersed in ethanol and toluene solution of 3-aminopropyltriethoxysilane (APTES). Next, this amine modified chip was immersed in the glutaraldehyde solution that contained sodium cyanoborohydride. Finally, the fluorescent materials, the Alexa 488, and the FITC conjugated anti-IgG were reacted to the aldehyde-modified surface. To verify the modified surface, the intensity of the fluorescence was measured and compared. The higher intensity of the fluorescence was observed when the APTES was dissolved in toluene rather than in ethanol. From the highest intensity of the fluorescence observed, the FITC conjugated anti-IgG was well immobilized on the modified surface of the aluminum oxide with APTES and glutaraldehyde. Thus, the proposed surface modification method can immobilize the proteins including antibodies, and can be applied for the various biological researches including biosensors and biochips.

ChemInform Abstract: A New and Convenient Method for Reduction of Oximes to Amines with NaBH3CN in the Presence of MoCl5/NaHSO4·H2O System.

During the past decades, sodium borohydride has played an important role for the reduction of functional groups in modern organic synthesis. This reagent is a relatively mild reducing agent and mostly used for the reduction of aldehydes and ketones in protic solvents. In order to control the reducing power of NaBH4, hundreds of substituted boron hydrides have been made and introduced in chemical literature and many of them are now commercially available. In fact, advances in such field have been realized by replacement of one or more hydride with other substituents, change of sodium cation to other metal, quaternary ammonium and phosphonium cations, a concurrent cation and hydride exchange, ligand metal borohydrides and finally combination of the hydride transferring agents with metals, metal salts, Lewis acids and solid supports. In this context, NaBH3CN carrying an electron withdrawing cyanide group is a remarkable stable and selective reducing agent and has been found many applications in organic synthesis. It is also well known that the reducing capability of NaBH3CN in reduction reactions is greatly depended to use low pH values (3-4). In spite of the great convenience of NaBH3CN in synthetic organic chemistry, however, this reagent suffers from harsh reaction conditions (strongly acidic media), limitation to use acid-sensitive functional groups and formation of some side products. In line of the outlined strategies and our research interest to develop mild and efficient protocols for sodium cyanoborohydride reduction of functional groups in the absence of Bronsted acidic media, herein, we wish to introduce a new and convenient method for reduction of various aldoximes and ketoximes to their corresponding amines with NaBH3CN/ MoCl5/NaHSO4·H2O system in refluxing EtOH or DMF (Scheme 1). Results and Discussion

Study on Galactose–Poly(Ethylene Glycol)–Poly(L-Lysine) as Novel Gene Vector for Targeting Hepatocytes In Vitro

A non-viral gene-delivery system has been used to deliver plasmid DNA into specific cell types because of its safety and ease of manufacture. Receptor-mediated gene transfer is currently a promising gene-delivery technique. To specifically target genes to asialoglycoprotein receptor of hepatocytes, a galactose moiety was combined into the poly(ethylene glycol) (PEG)-terminal end by reductive coupling using lactose and sodium cyanoborohydride. A synthesis method of conjugating poly(L-lysine) (PLL) derivatives with terminally galactose-graft-PEG was developed using ring-opening polymerization of N ε -benzyloxycarbonyl-L-lysine-N α -carboxyan-hydride (Z-Lys-NCA) initiated onto galactose graft amine-terminated PEG (galactose–PEG–NH2) as a macro-initiator. The synthesis was characterized with 1H-, 13C-NMR, IR and UV spectroscopy, and all of them successfully verified the formation of the co-polymers. The gel-retardation assay of the complexes between galactose–PEG–PLL and plasmid DNA indicated that these polymeric gene carriers demonstrated the potent ability to condense plasmid DNA electrostatically as well as PLL. The particle size and zeta potential of polymer/DNA complexes were measured, and their cytotoxicity and transfection efficiency in different cells were evaluated. The results indicate that galactose–PEG–PLL can form a complex with plasmid DNA and serve as an effective gene-delivery carrier with lower cytotoxicity compared to that of PLL. Transfection experiments clearly showed that galactose–PEG–PLL effectively delivered DNA into hepatoma cells in vitro. Such data demonstrates that galactose and its complex with plasmid DNA may serve as a safe and effective gene-transfer system targeting hepatocytes.

Replication of biosynthetic reactions enables efficient synthesis of A-factor, a γ-butyrolactone autoinducer from Streptomyces griseus

We report a concise synthesis of A-factor, the prototypical γ-butyrolactone signalling compound of Streptomyces bacteria. In analogy to enzymatic reactions in A-factor biosynthesis, our synthesis features a tandem esterification-Knoevenagel condensation yielding a 2-acyl butenolide and a surprising, chemoselective conjugate reduction of this α,β-unsaturated carbonyl compound using sodium cyanoborohydride.

Immobilization of 2-mercaptoethylamine on oxidized chitosan: a substantially mucoadhesive and permeation enhancing polymer

Thiolated chitosans are mucoadhesive polymers that are able to increase the paracellular permeability and improve the oral bioavailability of hydrophilic and macromolecular compounds. Due to the low hydratability of most thiolated chitosans at pH > 6.5, it was the aim of this study to develop a thiomer as a permeation enhancer for mucosal membranes where a comparatively high pH is prevalent. For this purpose chitosan was oxidized by means of sodium periodate to cleave the polysaccharide structure. The intermediate polymer was coupled with 2-mercaptoethylamine and reduced with sodium cyanoborohydride to obtain the secondary amine moiety. The permeation enhancing effect of 2-mercaptoethylamine-functionalized chitosan conjugate (chitosan-2-MEA) was assessed in Ussing-type chambers using FD4 as a model compound. Further in vitro characterization was conducted regarding water absorbing capacity, cohesive properties, mucoadhesion and cytotoxic effects on Caco-2 cells. By means of fundamental alteration of chitosan's backbone and following thiol immobilization, the paracellular transport of FD4 across rat intestinal mucosa was 5.1-fold improved compared with control (buffer) only. Mucoadhesion could be 20-fold improved likely based on increased chain flexibility and disulfide formation between chitosan-2-MEA and mucus glycoproteins. The novel thiomer showed excellent hydratability in aqueous media even up to pH 11 and was proven to be non-toxic. According to the above-mentioned properties, chitosan-2-MEA seems to be a promising mucoadhesive and permeation enhancing excipient for the development of drug delivery systems.

Reactive Aldehyde Metabolites from the Anti-HIV Drug Abacavir: Amino Acid Adducts as Possible Factors in Abacavir Toxicity

Abacavir is a nucleoside reverse transcriptase inhibitor marketed since 1999 for the treatment of infection with the human immunodeficiency virus type 1 (HIV). Despite its clinical efficacy, abacavir administration has been associated with serious and sometimes fatal toxic events. Abacavir has been reported to undergo bioactivation in vitro, yielding reactive species that bind covalently to human serum albumin, but the haptenation mechanism and its significance to the toxic events induced by this anti-HIV drug have yet to be elucidated. Abacavir is extensively metabolized in the liver, resulting in inactive glucuronide and carboxylate metabolites. The metabolism of abacavir to the carboxylate involves a two-step oxidation via an unconjugated aldehyde, which under dehydrogenase activity isomerizes to a conjugated aldehyde. Concurrently with metabolic oxidation, the two putative aldehyde metabolites may be trapped by nucleophilic side groups in proteins yielding covalent adducts, which can be at the onset of the toxic events associated with abacavir. To gain insight into the role of aldehyde metabolites in abacavir-induced toxicity and with the ultimate goal of preparing reliable and fully characterized prospective biomarkers of exposure to the drug, we synthesized the two putative abacavir aldehyde metabolites and investigated their reaction with the α-amino group of valine. The resulting adducts were subsequently stabilized by reduction with sodium cyanoborohydride and derivatized with phenyl isothiocyanate, leading in both instances to the formation of the same phenylthiohydantoin, which was fully characterized by NMR and MS. These results suggest that the unconjugated aldehyde, initially formed in vivo, rapidly isomerizes to the thermodynamically more stable conjugated aldehyde, which is the electrophilic intermediate mainly involved in reaction with bionucleophiles. Moreover, we demonstrated that the reaction of the conjugated aldehyde with nitrogen bionucleophiles occurs exclusively via Schiff base formation, whereas soft sulfur nucleophiles react by Michael-type 1,4-addition to the α,β-unsaturated system. The synthetic phenylthiohydantoin adduct was subsequently used as standard for LC-ESI-MS monitoring of N-terminal valine adduct formation, upon modification of human hemoglobin in vitro with the conjugated abacavir aldehyde, followed by reduction and Edman degradation. The same postmodification strategy was applied to investigate the products formed by incubation of abacavir with rat liver cytosol, followed by trapping with ethyl valinate. In both instances, the major adduct detected corresponded to the synthetic phenylthiohydantoin standard. These results suggest that abacavir metabolism to the carboxylate(s) via aldehyde intermediate(s) could be a factor in the toxic events elicited by abacavir administration. Furthermore, the availability of a reliable and fully characterized synthetic standard of the abacavir adduct with the N-terminal valine of hemoglobin and its easy detection in the model hemoglobin modifications support the usefulness of this adduct as a prospective biomarker of abacavir toxicity in humans.

Hydrazide Reactive Peptide Tags for Site-Specific Protein Labeling

New site-specific protein labeling (SSPL) reactions for targeting-specific, short peptides could be useful for the real-time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH(3) in order to specifically select reactive carbonyl-containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the hydrazide reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an overexpressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N-terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents.

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 New and Convenient Method for Reduction of Oximes to Amines with NaBH3CN In the Presence of MoCl5/NaHSO4·H2O System

Various aldoximes and ketoximes were efficiently reduced to their corresponding amines with <TEX>$NaBH_3CN$</TEX> in the presence of <TEX>$MoCl_5/NaHSO_4{\cdot}H_2O$</TEX> system. Reduction reactions were carried out in refluxing EtOH or DMF within 0.3-3.8 h to afford the amines in high to excellent yields.

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.

Reductive aminopropylation of ketene silyl (thio)acetals leading to the synthesis of δ-amino esters

In the presence of silica gel or titanium tetrachloride, ketene silyl acetals or ketene silyl thioacetals underwent 1,4-addition with α,β unsaturated aldimines which possess a large triphenylmethyl group at the imino nitrogens followed by reduction with sodium cyanoborohydride to give aminopropylated products, δ-amino esters, in good yields.

Ammonia Plasma Treated Polyethylene Films for Adsorption or Covalent Immobilization of Trypsin: Quantitative Correlation between X-ray Photoelectron Spectroscopy Data and Enzyme Activity

The ammonia plasma process was used for generating reactive groups, particularly primary amine functions on the surface of polyethylene (PE) films, to immobilize the enzyme trypsin. The attachment of the enzyme was achieved by directly applying an aqueous solution of trypsin to the plasma-activated surface or by using glutaraldehyde as a chemical linker. In both cases, the utilization of sodium cyanoborohydride efficiently stabilized the immobilization. The surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) and enzymatic activity measurements. Active trypsin was successfully immobilized on the surface with a mean activity of 0.09 ± 0.02 U/cm(2). The study of the stability of the immobilized enzyme during repetitive assays showed that some activity could be maintained during several months. An original quantitative correlation between the immobilized enzyme activity and the XPS signal intensity of the S 2p electrons present in the sulfur-containing amino acid residues was evidenced.

Preparation of N-vanillyl chitosan and 4-hydroxybenzyl chitosan and their physico-mechanical, optical, barrier, and antimicrobial properties

Chitosan derivatives such as N-vanillyl chitosan and 4-hydroxybenzyl chitosan were prepared by reacting chitosan with 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxybenzaldehyde. Amino groups on chitosan reacts with these aldehydes to form a Schiff base intermediate, which is later on converted into N-alkyl chitosans by reduction with sodium cyanoborohydride. The chemical reaction was monitored by 1H NMR spectroscopy and the absence of aldehydic proton at 9.83ppm in NMR spectra was observed for both the modified chitosan derivatives confirming the reaction. Modified chitosan films were later prepared by solution casting method and their physico-mechanical, barrier, optical and thermal properties were studied. The results clearly indicated significant change in tensile strength, water vapour transmission rate, and haze properties of modified chitosans. Modified chitosan films were also studied for their antimicrobial activity against Aspergillus flavus. The results showed a marked reduction of aflatoxins produced by the fungus in the presence of the N-vanillyl chitosan and 4-hydroxybenzyl chitosan film discs to 98.9% and non-detectable levels, respectively.

Optimization and application of microwave-assisted acid hydrolysis for rapid quantification of protein oxidation markers using LC–MS

Simple and efficient microwave-assisted acid hydrolysis (MAAH) of proteins was used for rapid quantification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) as major protein oxidation markers. The precursor amino acid residues corresponding to AAS and GGS in oxidized proteins were derivatized by reductive amination with sodium cyanoborohydride (NaCNBH3) and p-aminobenzoic acid (ABA) followed by MAAH to generate the marker derivatives AAS–ABA and GGS–ABA. The quantification was performed using electrospray ionization liquid chromatography–mass spectrometry (ESI LC–MS). The important parameters for hydrolysis were optimized, which include the temperature, the reaction time, the acid concentration and volume as well as the microwave power. Compared to the conventional acid hydrolysis of 18–24h using 6–12M HCl at 110°C applied commonly in the literature and also in this work, MAAH of proteins can be completed as fast as in only 2–10min and, additionally, with a 3–5 times higher yield of the final derivatization products. Furthermore, a better agreement between the ratio of the detected derivatization products and the theoretical yields from the studied protein has also been achieved, which indicates that MAAH may serve as a more reliable method of acid hydrolysis for this purpose than that with conventional thermal heating. The MAAH method is demonstrated to be a time-saving, reproducible and efficient technique for studying AAS and GGS as protein oxidation markers using LC–MS.

N-(2-formyl-1-methylimidazol-4-yl)-2,2-dimethylpropanamide: a versatile reagent for preparing imidazole-amine ligands with variable second-coordination spheres

Two synthetic pathways to N-(2-formyl-1-methylimidazol-4-yl)-2,2-dimethylpropanamide from 1-methyl-2-carboxaldehyde are described. The reagent serves as a useful synthon for reductive amination reactions with primary and secondary amines in the presence of sodium cyanoborohydride to yield a series of ligands with second coordination sphere functional groups. Protocols for the syntheses of related imidazole synthons functionalized in the 4-position with amino acids, Schiff bases, and other amides are also reported.

Synthesis of α-trifluoromethyl-phenethylamines from α-trifluoromethyl β-aryl enamines and β-chloro-β-(trifluoromethyl)styrenes

A novel synthetic approach towards α-trifluoromethyl-phenethylamines was elaborated by reduction of the electron deficient β-aryl-α-trifluoromethyl enamines and imines with sodium cyanoborohydride in the presence of trifluoroacetic acid. The starting imines were prepared by the reaction of primary amines with β-aryl-α-trifluoromethyl enamines or β-chloro-β-(trifluoromethyl)styrenes.

Synthesis of amino analogues of cholic acid

Amino analogues of cholic acid were synthesized by reduction of oximes with combination of titanium(III) chloride and sodium cyanoborohydride.

Sodium Cyanoborohydride–Mediated Direct Conversion of Some Heterocyclic Aldehydes to Esters

An exceptional oxidizing behavior of sodium cyanoborohydride is observed where electron-withdrawing heterocyclic aldehydes are directly converted to their corresponding esters on treatment of sodium cyanoborohydride in methanol. The spectral analysis and single-crystal structure confirm the formation of esters.