Bifunctional pyridoxal derivatives as efficient bioorthogonal reagents for biomacromolecule modifications.

Two types of pyridoxal analogs, azido pyridoxal (PL-N3) and carboxyl pyridoxal (PL-COOH), were developed as novel bifunctional bioorthogonal molecules. These molecules showed fast imine formation with hydrazinyl groups and stable covalent linkages via azido/carboxyl groups, and thus were of great use for site-specific peptide and protein modifications.

Chiral Pyridoxal-Catalyzed Asymmetric Biomimetic Transamination of α-Keto Acids.

A series of chiral pyridoxals 8 and 9 have been developed from commercially available pyridoxine and (S)-α,α-diarylprolinols. The pyridoxals exhibited good catalytic activity in an asymmetric transamination of α-keto acids with 2,2-diphenylglycine (7f) as the amine source to give various α-amino acids in 29-85% yields with 53-80% ee's. The current asymmetric transamination has successfully mimicked a complete biological transamination process characterized by two half-transaminations, a small chiral pyridoxal molecule acting as the catalyst, and enantioselective control.

Iron(III) complexes of a pyridoxal Schiff base for enhanced cellular uptake with selectivity and remarkable photocytotoxicity.

Iron(III) complexes of pyridoxal (vitamin B6, VB6) or salicylaldehyde Schiff bases and modified dipicolylamines, namely, [Fe(B)(L)](NO3) (1-5), where B is phenyl-N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1), (anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (pybpa in 3, 5) (H2L(1) is 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylpyridine (1-3) and H2L(2) is 2-[(2-hydroxyphenyl-imino)methyl]phenol), were prepared and their uptake in cancer cells and photocytotoxicity were studied. Complexes 4 and 5, having a non-pyridoxal Schiff base, were prepared to probe the role of the pyridoxal group in tumor targeting and cellular uptake. The PF6 salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate FeN4O2 core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible Fe(III)/Fe(II) response near -0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in various cancer cells with IC50 values ranging from 0.4 to 5 µM with 10-fold lower dark toxicity. The cell death proceeded by the apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display 3-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5 are distributed throughout the cells without any specific localization pattern.

Stereoselective preparation of pyridoxal 1,2,3,4-tetrahydro-ß-carboline derivatives and the influence of their absolute and relative configuration on the proliferation of the malaria parasite Plasmodium falciparum.

We have selectively synthesized by Pictet-Spengler condensation of tryptophan and pyridoxal the four stereoisomers of a pyridoxal ß-carboline derivative that was designed to inhibit the proliferation of Plasmodium falciparum. Biological investigation of the four compounds revealed that they all inhibit the growth of P. falciparum. With an IC50 value of 8 ± 1 µM, the highest inhibitory effect on the proliferation of the parasite was found for the 1,3-trans-substituted tetrahydro-ß-carboline that was obtained from d-tryptophan. Lower activity was found for its enantiomer, while the two diastereomeric cis-products were markedly less effective. Apparently a distinct spacial orientation of the carboxyl group of the substituted tetrahydropyridine unit of the compounds is needed for high activity, while the absolute configuration of the molecules is of lesser importance.

Synthesis and structure-activity relationships of carboxylic acid derivatives of pyridoxal as P2X receptor antagonists.

Carboxylic acid derivatives of pyridoxal were developed as potent P2X(1) and P2X(3) receptor antagonists with modifications of a lead compound, pyridoxal-5'-phosphate-6-azophenyl-2',5'-disulfonate (5b, iso-PPADS). The designing strategies included the modifications of aldehyde, phosphate or sulfonate groups of 5b, which may be interacted with lysine residues of the receptor binding pocket, to weak anionic carboxylic acid groups. The corresponding carboxylic acid analogs of pyridoxal-5'-phosphate (1), 13 and 14, showed parallel antagonistic potencies. Also, most of 6-azophenyl derivatives (24-28) of compound 13 or 14 showed potent antagonistic activities similar to that of 5b at human P2X(3) receptors with 100 nM range of IC(50) values in two-electrode voltage clamp (TEVC) assay system on the Xenopus oocyte. The results indicated that aldehyde and phosphoric or sulfonic acids in 5b could be changed to a carboxylic acid without affecting antagonistic potency at mouse P2X(1) and human P2X(3) receptors.

Synthesis of 4-pyridoxolactone from pyridoxine using a combination of transformed Escherichia coli cells.

We developed a simple and efficient synthesis for 4-pyridoxolactone starting with pyridoxine and using a whole-cell biotransformation by two transformed Escherichia coli cell types. One set of transformed cells expressed pyridoxine 4-oxidase, catalase, and chaperonin, while the second set expressed pyridoxal 4-dehydrogenase. With this combination of cells, pyridoxine was first oxidized to pyridoxal, which was then dehydrogenated to 4-pyridoxolactone by pyridoxine 4-oxidase and pyridoxal 4-dehydrogenase, respectively. In a reaction mixture containing the two transformed cell types, 10 mM of pyridoxine was completely converted into 4-pyridoxolactone at 30 degrees C in 24 h. When starting with 80 mM of pyridoxine, it was necessary to add 0.5 mM or more of NAD(+) to complete the reaction.

A molecularly imprinted polymer-based synthetic transaminase.

The design, synthesis, and evaluation of a molecularly imprinted polymer transaminase mimic is described. Methacrylic acid-ethylene glycol dimethacrylate copolymers were synthesized using, as a template, a transition state analogue (TSA) for the reaction of phenylpyruvic acid and pyridoxamine to yield phenylalanine and pyridoxal. Polymer suitability was established on the basis of (1)H NMR studies of template-functional monomer interactions. Polymer recognition characteristics were examined in a series of HPLC studies using the polymers as chromatographic stationary phases. Selectivity for the TSA, relative to substrates and products, was observed in both aqueous and nonpolar media. In the latter case (chloroform/AcOH, 96:4), an enantioseparation factor (alpha) of 2.1 was obtained, and frontal chromatographic studies revealed the presence of 11.9 +/- 0.2 micromol g(-1) (dry weight) of enantioselective sites. Polymers imprinted with the l-form of the oxazine-based TSA induced a 15-fold enhancement of the apparent reaction rate (app. V(max) 2.5 x 10(-7) mol s(-1); app. K(m) 8.2 x 10(-3) M) and enantioselective production of phenylalanine (32 +/- 4% ee) for reactions conducted in an aqueous buffer system. Substrate selectivity was evident, and a turnover number (k(cat)) of 0.1 s(-)(1) was determined. This is the first example of the catalysis of sigmatropic shifts in aqueous media by molecularly imprinted polymers.

Polymeric and dendrimeric pyridoxal enzyme mimics.

Pyridoxal was covalently attached to polyethylenimine polymers, but the resulting materials were found to degrade rapidly. In comparison, the dendrimeric pyridoxals, which possess only one pyridoxal unit at the core of every dendrimer molecule were found to be relatively stable compounds. A total of 12 poly(amidoamine) type dendrimers were synthesized. They range from G1 to G6 with either NMe(2) or NHAc termini. The NMe(2)-terminated pyridoxal dendrimers racemize alpha-amino acids 50-100 times faster than does simple pyridoxal, while the NHAc-terminated pyridoxal dendrimers racemize alpha-amino acids only 3-5 times faster than does simple pyridoxal. Both the NMe(2)- and NHAc-terminated pyridoxal dendrimers decarboxylate 2-amino-2-phenyl-propionic acid 1-3 times faster than simple pyridoxal. The interior polarity in the pyridoxal dendrimers is similar to that of 85:15 water-DMF solution. Furthermore, we successfully incorporated eight lauryl groups to the G5 pyridoxal dendrimer at known positions. The laurylated dendrimer exhibits lower racemization and decarboxylation rates than do the unlaurylated ones, in contrast to the positive rate effects of laurylation in polyethylenimine-pyridoxamines in our previous transamination studies.

Synthesis, characterization and biological activity of copper complexes with pyridoxal thiosemicarbazone derivatives. X-ray crystal structure of three dimeric complexes.

A dimeric copper complex of the unsubstituted pyridoxal thiosemicarbazone (H(2)L), [[Cu(HL)(OH(2))](2)]Cl(2).2H(2)O, previously tested on Friend murine cell lines has been recently resynthesized to evaluate its behavior on different murine and human leukemic cell lines and has been compared, in vitro and in vivo, with its monomeric counterpart [Cu(H(2)L)(OH(2))Cl]Cl. On TS/A murine adenocarcinoma cell line in vitro, both compounds significantly inhibit cell proliferation at micromolar concentrations, although the dimeric compound is more active. Despite this cytotoxicity they lack in vivo activity on TLX5 lymphoma. The unsubstituted dimeric [[Cu(HL)(OH(2))](2)]Cl(2).2H(2)O induces apoptosis on CEM and U937 human cell lines, with IC(50) concentrations of 1.2 x 10(-5) and 6.7 x 10(-6) M, respectively, but it is inactive on K562. Moreover, it alters significantly the cell cycle of U937 and CEM lines and decreases the telomerase activity of U937. To verify if other dimeric copper complexes show relevant biological activity new complexes with N-substituted pyridoxal thiosemicarbazones have been synthesized and characterized using spectroscopic techniques. Three of them, namely [Cu(Me(2)-HL)Cl](2).6H(2)O (Me(2)-H(2)L=pyridoxal N1,N1-dimethylthiosemicarbazone) (1), [Cu(MeMe-HL)Cl](2)Cl(2).4H(2)O (MeMe-HL=pyridoxal N1,N2-dimethylthiosemicarbazone) (2), [Cu(Et-H(2)L)Cl](2)Cl(2).2H(2)O (Et-H(2)L=pyridoxal N1-ethylthiosemicarbazone) (3), were also characterized by X-ray diffractometry. These complexes are dimeric and all three present a square pyramidal coordinative geometry with the ligand showing an SNO tridentate behavior. Their biological activities have been tested in vitro on U937, CEM and K562 cell lines to ascertain their effectiveness in comparison to the corresponding unsubstituted complex [[Cu(HL)(OH(2))](2)]Cl(2).2H(2)O. Compound 1 shows weak proliferation inhibition on all three cell lines, but it does not induce apoptosis and it does not inhibit telomerase activity, compound 2 is not effective at low concentration and is toxic at higher doses; compound 3 inhibits CEM cell growth better than complex 1 but it does not exert any other biological effect.

Development of potential iron chelators for the treatment of Friedreich's ataxia: ligands that mobilize mitochondrial iron.

Friedreich's ataxia (FA) is a crippling neurodegenerative disease that is due to iron (Fe) overload within the mitochondrion. One therapeutic intervention may be the development of a chelator that could remove mitochondrial Fe. We have implemented the only well characterized model of mammalian mitochondrial Fe overload to examine the Fe chelation efficacy of novel chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. In this model we utilize reticulocytes treated with the haem synthesis inhibitor succinylacetone which results in mitochondrial Fe-loading. Our experiments demonstrate that in contrast to desferrioxamine, several of the PCIH analogues show very high activity at mobilizing (59)Fe from (59)Fe-loaded reticulocytes. Further studies on these ligands in animals are clearly warranted considering their potential to treat FA.

Development of novel aroylhydrazone ligands for iron chelation therapy: 2-pyridylcarboxaldehyde isonicotinoyl hydrazone analogs.

Previous studies have demonstrated that aroylhydrazone iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class have high Fe chelation efficacy both in vitro and in vivo. Depending on their design, these drugs may have potential as agents for the treatment of Fe overload disease or cancer. Considering the high potential of this class of ligands, we have synthesized seven novel aroylhydrazones in an attempt to identify Fe chelators more efficient than desferrioxamine (DFO) and more soluble than those of the PIH class. These compounds belong to a new series of tridentate chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazones (PCIH). In this study we have examined the Fe chelation efficacy and antiproliferative activity of these chelators including their effects on the expression of genes (WAF1 and GADD45) known to be important in mediating cell cycle arrest at G1/S. From seven chelators synthesized, three ligands, namely 2-pyridylcarbox-aldehyde benzoyl hydrazone (PCBH), 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone (PCBBH), and 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH), showed greater Fe chelation activity than DFO and comparable or greater efficiency than PIH. These ligands were highly effective at both mobilizing 59Fe from cells and preventing 59Fe uptake from 59Fe-transferrin and caused a marked increase in the RNA-binding activity of the iron-regulatory proteins (IRP). Our studies have also demonstrated that compared with the cytotoxic Fe chelator, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), these ligands have far less effect on cellular growth and 3H-thymidine, 3H-leucine, or 3H-uridine incorporation. In addition, in contrast to 311, which markedly increased WAF1 and GADD45 mRNA expression, PCBH and PCTH did not have any effect, whereas PCBBH increased the expression of GADD45 mRNA. Collectively, these results demonstrate the potential of several of these ligands as agents for the management of Fe overload disease.

Diorganotin(IV) complexes of pyridoxal thiosemicarbazone: synthesis, spectroscopic properties and biological activity.

The complexes [SnR2(L)] (R = Me, Et, Bu, Ph; H2L = pyridoxal thiosemicarbazone) have been prepared and characterized. In the light of the spectral properties of the complexes in the solid state (IR, mass, Mössbauer) the bideprotonated thiosemicarbazonato anion is O(phenolic)-, N(3)-, S-bonded to the tin atom which probably has trigonal bipyramidal coordination with N(3) atom and R groups occupying equatorial positions. NMR ( 1H, 13C and 119Sn) data in CDCl3 or DMSO-d6 suggest that this coordinative picture remains in these solutions. The ethyl, butyl and phenyl derivatives suppress proliferation of Friend erithroleukaemia cells (FLC). Of the pyridoxal thiosemicarbazone complexes so far evaluated. [SnBu2(L)] and [SnPh2(L)] showed the lowest thresholds for inhibition of FLC proliferation. The effects of these compounds on DMSO-induced differentiation of FLC, DNA synthesis and reverse transcriptase were also assayed.

Synthesis of N-(4'-pyridoxyl)sphingosine and its uptake and metabolism by isolated cells.

N-(4'-pyridoxyl)sphingosine was synthesized and characterized as a stable compound for specialized delivery of a bioactive lipid. It was found to be facilely taken up by hepatocytes although by a mechanism more typical for lipids than the one used by natural vitamin B6. Some of the N-(4'-pyridoxyl)sphingosine was metabolically acted upon inside the cell to release pyridoxal 5'-phosphate and sphingosine, but formation of pyridoxal 5'-phosphate from the synthetic compound was poor compared with natural vitamin forms of B6, which may partly be due to entrapment within cell membranes and to constraints at the level of cytosolic pyridoxal kinase which is responsible for phosphorylation of the vitamin. Unlike the parent long-chain base, the B6 conjugate was not particularly cytotoxic. Furthermore, the compound was neither an activator nor inhibitor of the respiratory burst of human neutrophils. These findings identify N-(4'-pyridoxyl)sphingosine as an interesting tool for studies of the cellular transport, metabolism, and functions of both vitamin B6 and sphingosine.

Synthesis of 6-[18F]fluoropyridoxal and radioactivity distribution in rat at 60 min.

6-[18F]Fluoropyridoxal was synthesized by the flourination of a propylamine derivative of pyridoxal (pyridoxal Schiff base) with 18F-labelled acetylhypofluorite. Two different fluorinating agents, 5% F2 in N2 and acetylhypofluorite, were investigated with nonradioactive material. The evaluation of reactions in CH3CN and chloroform showed CH3CN to be the better solvent and CH3COOF to be the better fluorinating reagent. The synthesis gave a radiochemical yield of about 18% (expressed at the end of synthesis) and required 35-40 min to complete. The specific activity of the final radiopharmaceutical at the end of the synthesis was about 25.9 GBq/mmol (700 mCi/mmol). The tissue distribution of 6-fluoropyridoxal in rat at 60 min is also reported. A large concentration in liver and kidney indicates that this radiopharmaceutical could be of special interest in the imaging of liver functions. The concentration in the brain might also allow in vivo PET imaging of the 6-(fluoropyridoxal) uptake if a high efficiency PET scanner is used.

Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes.

A new vitamin B6 analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear 1H-19F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDPL showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL [Chang, Y. C., & Graves, D. J. (1985) J. Biol. Chem. 260, 2709-2714], except the Km of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower Vmax value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. 19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate [Chang, Y. C., Scott, R. D., & Graves, D. J. (1986) Biochemistry 25, 1932-1939].(ABSTRACT TRUNCATED AT 250 WORDS)

A new synthetic method for 99mTc labeled N-pyridoxyl-5-methyltryptophan as a hepatoma imaging agent.

N-Pyridoxyl-5-methyltryptophan (5-PMT) was synthesized by a simplified method using sodium borohydride for the reduction of a Schiff base of pyridoxal and 5-methyltryptophan. Lyophilized kits containing 5-PMT, stannous chloride and L-(+)-ascorbic acid were prepared and labeled to afford 99mTc-5-PMT with 96% or higher radiochemical purity analysed by two thin-layer chromatographic solvent systems. 99mTc-5-PMT showed a rapid blood clearance, a faster hepatobiliary transit and a lower renal retention in comparison with 99mTc-5-EHIDA in rats. Eleven (61%) of 18 patients with histologically confirmed hepatocellular carcinoma showed positive images at 2 to 5 h after i.v. injection. The smallest tumor that could be identified was 2 cm in diameter with the best tumor/liver ratio of 4. In conclusion, 99mTc-5-PMT synthesized by sodium borohydride reduction shows great promise as a useful hepatoma imaging agent.

A clinical comparison of 99mTc-diethyl-iminodiacetic acid, 99mTc-pyridoxylideneglutamate, and 131I-rose bengal in liver transplant patients.

The relative merits of three radiopharmaceuticals for evaluating liver transplant patients were determined in paired studies. In 86% of studies both 131I-RB and 99mTc-PG gave similar information for differentiation of hepatocellular disease and billary tract obstruction. 99mTc-PG probably demonstrates the biliary tract and small intestine better early after injection (8%); 131I-RB is probably better in showing the colon at 24 hours when intestinal activity is not seen by 1 hour (6%). 99mTc-diethyl-IDA is superior in all respects when compared to 99mTc-PG. The blood retention method (20 min./5 min.) showed that none of the radiopharmaceuticals was a reliable indicator of hepatocyte function when compared to total serum bilirubin.

4-Halovinyl- and 4-ethynyl-4-deformylpyridoxal derivatives and related analogues as potentially irreversible antagonists of vitamin B6.

Analogues of pyridoxal bearing alpha- and beta-chlorovinyl, beta-bromovinyl, butadienyl, acetyl, and 1-butenyl groups in place of the formyl group have been synthesized by subjecting 3,alpha5-di-O-benzylpyridoxal to appropriate Wittig or Grignard reactions. Similar methods yielded one-carbon homologues of pyridoxal and pyridoxol. Synthesis of the 4-ethynyl analogue of pyridoxal was achieved by dehalogenating blocked beta-halovinyl derivatives. The substituted vinyl and the ethynyl analogues were found to be active as inhibitors of mouse mammary adenocarcinoma cells grown in cell culture at an ID50 of 10(-5)-10(-6) M. The inhibitory activity of the 4-ethynyl analogue could be partially reversed by pyridoxal. This analogue was found to inhibit pyridoxal phosphokinase, and its 5'-phosphate was likewise found to be a potent noncompetitive inhibitor of pyridoxine-P oxidase.