Conversion of a primary amine to a labeled secondary amine by the addition of phenolic group and radioiodination

To preserve the nucleophilicity of amino compounds during conjugative radioiodination, a new method for converting primary amines to phenolic secondary amines was developed. Amino acids were used as model compounds for establishing optimal conditions for the reductive amination. In the first step of the reaction, the aldehyde group of 4-hydroxybenzaldehyde (formylphenol) was reacted reversibly with an amino group to form an imine. The irreversible attachment of formylphenol to the amino group was accomplished by reduction of the imine with sodium cyanoborohydride. The pH optimum for the reaction was 5.0. Higher temperature has favorable effects on the rate and extent of the conjugation. Phenolic derivatives of amino compounds suitable for radioiodination are produced by the reactions described.     

Asymmetric reduction of alkyl aryl ketones in the binding domain of bovine serum albumin

The asymmetric reduction of alkyl aryl ketones with sodium borohydride (NaBH₄) or sodium cyanoborohydride (NaBH₃CN) in the presence of bovine serum albumin (BSA) was studied. The asymmetric induction in the product alcohols was largely dependent on the BSA concentration and the pH value of the buffer solution. The maximum optical yields obtained ranged over 14–78%, varying with the alkyl and aryl substituents, using ca. $\text{1}\text{3}$ molar eq of BSA to the ketone and at pH > 9. With two exceptions, the predominant enantiomer had the R configuration in 13 ketones investigated.     

Chemical methods of DNA and RNA fluorescent labeling.

Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3'-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde groups and the hydrazone bonds are stabilized by reduction with sodium cyanoborohydride. Alternatively, DNA can be quantitatively split at the depurinated sites with ethylenediamine. The aldimine bond between the aldehyde group in depurinated DNA or oxidized RNA and ethylenediamine is stabilized by reduction with sodium cyanoborohydride and the primary amine group introduced at these sites is used for attachment of isothiocyanate or succinimide derivatives of fluorescent dyes. The fluorescent DNA labeling can be carried out either in solution or on a reverse phase column. These procedures provide simple, inexpensive methods of multiple DNA labeling and of introducing one fluorescent dye molecule per RNA, as well as quantitative DNA fragmentation and incorporation of one label per fragment. These methods of fluorophore attachment were shown to be efficient for use in the hybridization of labeled RNA, DNA and DNA fragments with oligonucleotide microchips.     

Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride

A procedure is described for radiolabeling proteins in vitro by reductive alkylation. The proteins are treated with [¹⁴C]formaldehyde in the presence of sodium cyanoborohydride, a reducing agent that is stable in aqueous solution at pH 7. The advantage of this procedure is that the reaction can be carried out at neutral pH for extended periods of time and over a wide range of temperatures (0–37°C). Moreover, owing to the flexibility in the reaction conditions afferded by the use of sodium cyanoborohydride, higher incorporation of radiolabel into protein can be attalned.     

Modification of the carbohydrate in ricin with metaperiodate-cyanoborohydride mixtures. Effects on toxicity and in vivo distribution

Attempts to target antibody-ricin conjugates (immunotoxins) to designated cell types in vivo may be thwarted by their rapid clearance by hepatic reticuloendothelial cells which have receptors that recognise oligosaccharide side chains on the toxin. The B-chain of ricin contains high mannose type oligosaccharides and the A-chain contains a complex unit (GlcNAc)2-Fuc-Xyl-(Man)4-6, all of which potentially could be recognised by the reticuloendothelial system. Treatment of ricin with a mixture of sodium metaperiodate and sodium cyanoborohydride at pH 3.5 resulted in oxidative cleavage of the carbohydrates and reduction of the aldehyde groups thus formed to primary alcohols. By conducting the modification procedure at acidic pH, both the possibility of Schiff's base formation between the aldehyde groups and amino groups in the protein and the possibility of non-specific oxidation of amino acids were minimised. The extent of the carbohydrate modification depended on the duration of treatment, resulting maximally in the destruction of 13 of the 18 mannose residues and of all xylose and fucose. The toxicity of the modified toxin to cells in culture declined by up to 90% as the carbohydrate was destroyed. This was not due to a reduced ability of the B-chain to bind to cells or of the A-chain to inactivate ribosomes. In contrast to the in vitro results, the toxicity of the modified toxin to mice and rats was elevated by up to fourfold. The modification greatly reduced the clearance of the toxin by non-parenchymal cells in the liver and prevented the damage to hepatic Kupffer and sinusoidal cells and to the red pulp of the spleen that is inflicted by the native toxin. The elevated toxicity to animals appears to be because the modified toxin evades the reticuloendothelial system and persists in the bloodstream for longer periods, thus resulting in lethal damage to vital tissues in the animal at lower dosage. The results suggest that immunotoxins prepared from modified ricin would not be readily cleared by the reticuloendothelial system and so be more effective at killing their target cells.     

A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao

Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 +/- 2 torr and a Hill coefficient of 2.3 +/- 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen-ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5-phosphate (PLP) shows three major bands for the cyanoborohydride-reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed-bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 +/- 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse-phase chromatography indicates that the addition of PLP to HbAo is directed solely to the beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.     

Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.

Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum characteristic of pyridoxal-containing enzymes, and it has now been found to contain bound pyruvate, the carbonyl function of which is essential for catalytic activity. The decarboxylase is inactivated by treatment with a number of reagents that attack carbonyl groups, including sodium borohydride. Reduction with tritiated borohydride leads to the introduction of stably bound radioactivity, which, after acid hydrolysis, has been identified as tritiated lactate by several chromatographic procedures and by treatment with lactate dehydrogenase. The enzyme resists inactivation by cyanoborohydride in the absence of substrate, but is readily inactivated by this reagent in the presence of phosphatidylserine. Under the conditions of treatment of neutral pH, cyanoborohydride does not react with carbonyl residues at an appreciable rate, but reduces imino groups much more rapidly. This finding, together with demonstrated dependence of the enzyme upon the carbonyl residue of pyruvate for activity, strongly suggests that a Schiff base is formed by addition of the amino group of phosphatidylserine to the pyruvate residue of the enzyme as an essential step in the action of the decarboxylase.     

Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity.

Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P. F., Chlumsky, L. J., Daubner, S. C., and O'Malley, K. L. (1990) J. Biol. Chem. 265, 2042-2047). The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of [32P]phosphate. Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7. Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate. Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7. Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold. Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold. The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells. In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions. These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines.     

Chelating derivatives of chitosan obtained by reaction with ascorbic acid

Ascorbic acid immediately dissolves Euphausia superba chitosan upon mixing and forms chitosan ascorbate; during the 6-h period after dissolution in water at pH 5–7, ascorbate is oxidized to dehydroascorbate which undergoes Schiff reaction with the amino groups of chitosan, thus yielding a viscous solution of a polymeric ketimine. The latter is characterized by infrared spectrometry, circular dichroism spectropolarimetry, viscometry and alkalimetry. When brought into contact with transition metal ions, the chitosan ascorbate ketimine yields insoluble metal chelates. Upon reduction with sodium cyanoborohydride, the water-insoluble N-[2-(1,2-dihydroxyethyl)tetrahydrofuryl] chitosan (NDTC) is obtained, which shows enhanced capacity for uranium, up to 800 mg U/g from solutions at pH 4·5.     

Neurospora tryptophan synthase. Characterization of the pyridoxal phosphate binding site

Tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities. Tryptophan synthase from Neurospora crassa, a homodimer of two 75-kDa subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microM. The spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were characterized and compared to those previously established for the heterotetrameric (alpha 2 beta 2) enzyme from Escherichia coli. The Schiff base formed between pyridoxal phosphate and the enzyme was readily reduced by sodium borohydride, but not sodium cyanoborohydride. The active site residue that binds pyridoxal phosphate, labeled by reduction of the Schiff base with tritium-labeled sodium borohydride, was determined to be lysine by high performance liquid chromatography analysis of the protein hydrolysate. A 5400-dalton peptide containing the reduced pyridoxal phosphate moiety was generated by cyanogen bromide treatment, purified and sequenced. The sequence is 85% homologous with the corresponding sequence obtained for yeast tryptophan synthase (Zalkin, H., and Yanofsky, C. (1982) J. Biol. Chem. 257, 1491-1500); the lysine derivatized by pyridoxal phosphate is located at the same relative position as that in the yeast and E. coli enzymes.     

Physicochemical characterization of poly(ethylene glycol)-modified anti-GAD antibodies.

Monoclonal antibodies against glutamic acid decarboxylase (anti-GAD) were modified with poly(ethylene glycol) (PEG), and the resulting conjugates were characterized. Monoclonal anti-GAD antibodies were purified from ATCC HB184 hybridoma cells by either cell culture supernatant or ascites fluid from BALB/c mice. Polyclonal rabbit IgG antibodies were also used as a model protein. Polyclonal rabbit IgG or purified anti-GAD was modified by PEG (MW = 5000 or 20000 Da) through either the lysine residues or through the carbohydrate moiety. Lysine modification was performed in PBS (pH 7.4) or 0.1 M borate (pH 9.2) by adding a molar excess (5-80) of a succinimidyl activated propionic acid terminated mPEG (SPA-PEG) while stirring at room temperature. Carbohydrate modifications were performed in PBS (pH 6.2) by first oxidizing the antibody with sodium periodate followed by incubation with hydrazide-terminated PEG followed by reduction with sodium cyanoborohydride. The degree of modification was assessed by 1H NMR or TNBS (trinitrobenzenesulfonic acid). Circular dichroism (CD) spectra were obtained for lysine-modified rabbit IgG at various degrees of modification ranging from 5 to 60 PEG per antibody. Binding was assessed using an ELISA method with GAD or rabbit anti-mouse-IgG (H+L) coated plates. The TNBS and 1H NMR analysis of the modified antibody showed reasonably similar results from 5 to 60 PEG per antibody. The 1H NMR method showed greater sensitivity at low modifications (below 20:1) and was fairly linear up to about 60 PEG per antibody. The CD spectra of the polyclonal rabbit IgG showed only small differences at variously modified antibody. The binding affinity of anti-GAD is lower for all PEG modifications with respect to unmodified anti-GAD. Modifications at pH 7.4 show lower binding to GAD than modifications at pH 9.2. Binding to GAD or anti-mouse-IgG is decreased as the degree of modification is increased. Lysine modifications showed lower binding to GAD or anti-mouse-IgG than carbohydrate modifications. Binding to GAD or anti-mouse-IgG is lower for PEG20000-modified anti-GAD with respect to PEG5000-modified anti-GAD.     

Amine boranes as alternative reducing agents for reductive alkylation of proteins

The use of several commercially available amine-borane complexes was investigated for the reductive methylation of amino groups of several proteins. An earlier study in our laboratory, using turkey ovomucoid as the model protein, showed that dimethylamine borane is a slightly weaker reducing agent, but a good, less toxic substitute for sodium cyanoborohydride (K. F. Geoghegan, J. C. Cabacungan, H. B. F. Dixon, and R. E. Feeney, 1981, Int. J. Peptide Protein Res.17, 345–352). N-α-Acetyl-l-lysine, poly-l-lysine, turkey ovomucoid, bovine serum albumin, chicken ovalbumin, β-lactoglobulin, casein, and soybean protein were reductively methylated with dimethylamine borane and trimethylamine borane. The latter produced a consistently lower degree of modification even in the presence of sodium dodecyl sulfate. In a comparison that included the boranes triethylamine, t-butylamine, morpholine, and pyridine, pyridine borane was found to be slightly stronger than sodium cyanoborohydride. In a pH 7 solution containing 2 mmN-α-acetyl-l-lysine and 20 mm formaldehyde, complete dimethylation was achieved with about 10 mm pyridine borane after 2 h incubation at 22°C, while more than 15 mm was necessary with sodium cyanoborohydride. Like dimethylamine borane, both pyridine borane and triethylamine borane showed a reducing capacity at pH 7 which was as high as that at pH 9. Reductive alkylation under neutral to mild acid conditions allows modification of alkaline labile proteins and also limits the side reactions between proteins and formaldehyde.     

Fluorometric assay of pyridoxal and pyridoxal 5'-phosphate.

A new and very sensitive fluorometric method for the determination of pyridoxal and pyridoxal 5′-phosphate is reported. The specificity is based on the reductive amination of pyridoxal and its 5′-phosphate with methyl anthranilate and sodium cyanoborohydride at pH 4,5 to 5,0. Separation of the highly fluorescent methyl-N-pyridoxyl anthranilate was achieved by a combination of column and thin-layer chromatography on silica gel. This method has been applied to the assay of pyridoxal and pyridoxal 5′-phosphate in seruum.     

The covalent immobilization of microsomal uridine diphospho-glucuronosyltransferase (UDPGT): initial synthesis and characterization of an UDPGT immobilized enzyme reactor for the on-line study of glucuronidation

The microsomal fraction of rat liver containing uridine diphospho-glucuronosyltransferase (UDPGT; EC 2.4.1.17) has been covalently immobilized on a high performance chromatographic support. In this study Nucleosil Si-500 silica was converted into diol-bonded silica and subsequently converted into an aldehyde form through oxidation with sodium periodate. The microsomal fraction was immobilized via Schiff base formation followed by reduction with sodium cyanoborohydride. The resulting immobilized enzyme reactor (IMER) was placed in a multi-dimensional chromatographic system which utilized a mixed mode (C18 and anion exchange) column to trap the parent compound and glucuronide and a C18 column to separate the substrate and product. The IMER system was used for the online glucuronidation of 4-methylumbelliferone (4Me7OHC) and acetaminophen (APAP). The Michaelis-Menten kinetic parameters (Km and Vmax) associated with the formation of 4Me7OHC and APAP glucuronides demonstrated that the immobilization had not significantly affected the enzymatic activity of the UDPGT relative to the non-immobilized enzyme. The IMER retained enzymatic activity for more than 6 weeks. The results of this study demonstrate an easy and convenient way to identify compounds which may be glucuronidated and to synthesize and characterize the resulting products.     

Facile new synthesis and purification of 5,10-methenyltetrahydrofolate from folic acid

A facile new synthesis of tetrahydrofolate is reported starting with folic acid and reducing with sodium cyanoborohydride in glacial acetic acid. Conversion to 5,10-methenyltetrahydrofolate is effected by heating with formic acid in acetic acid, followed by purification on phosphocellulose at acid pH.     

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.     

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.     

6-Acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin is an activator of nitric oxide synthases

6-Acetyl-7,7-dimethyl-7,8-dihydropterin 3 has been shown to be able to substitute for the natural cofactor of nitric oxide synthases, tetrahydrobiopterin 1, in cells and tissues that contain active nitric oxide synthases (NOSs). In both macrophages, which produce iNOS, and endothelial cells, which produce eNOS, in which tetrahydrobiopterin biosynthesis has been blocked by inhibition of GTP cyclohydrolase 1, dihydropterin 3 restored production of nitric oxide by these cells. In tissues, 3 caused relaxation in preconstricted rat aortic rings, again in which tetrahydrobiopterin biosynthesis had been inhibited, an effect that was blocked by the NOS inhibitor, L-NAME. However, dihydropterin 3 was not itself an active cofactor in purified NOS (nNOS) preparations free of tetrahydrobiopterin suggesting that intracellular reduction to 6-acetyl-7,7-dimethyl-5,6,7,8-tetrahydropterin 4 is required for activity. Compound 4 was prepared by reduction of the corresponding 7,8-dihydropterin with sodium cyanoborohydride and has been shown to be a competent cofactor for nitric oxide production by nNOS. Together, the results show that the 7,7-dimethyl-7,8-dihydropterin is a novel structural framework for effective tetrahydrobiopterin analogues.     

Analysis of neutral sugars as glycamines using an amino acid analyzer.

A new method of the analysis of neutral sugars was developed based on the separation of their corresponding glycamines. Sugars and ammonia were combined by means of the reduction with sodlum cyanoborohydride. The glycamines thus obtained were quantitatively analyzed by an automatic amino acid analyzer. Satisfactery results were obtained in the analyses of the constituent sugars of several polysaccharides and glycoconjugates.     

C-Branched pyrrolidines from 2-C-acetylmethyl-glycosylazides. Reduction of imines formed by monosaccharide ring opening

Reduction of the protected 2-C-acetylmethyl-beta-glucopyranosyl azide derivative 1 produced the corresponding beta-glucosylamine 3. Rather than forming a conformationally strained 1,2-trans-fused bicyclic imine, we propose that the beta-glycosylamine underwent anomerization to an acyclic imine (4) followed by an intramolecular ring closure by the 5-hydroxy group. The resultant 2-C-acetylmethyl-alpha-glucopyranosylamine 5, which possesses the 1,2-cis-configuration was immediately converted to a bicyclic imine (2) in excellent yield. Attempts to selectively reduce the C=N double bond of 2 using sodium borohydride and cyanoborohydride failed to produce bicyclic amine 6. Instead, compound 6 underwent another ring-opening elimination and further reduction to produce a C-branched pyrrolidine (8) in good yield. Catalytic hydrogenation of 1 and 2 also provided the C-branched pyrrolidine (10).