Affinity reagents for cross-linking hemoglobin: bis(phenoxycarbonylethyl)phosphinic acid (BPCEP) and bis(3-nitrophenoxycarbonylethyl)phosphinic acid (BNCEP).

Synthesis and properties of two new macrobiomolecular cross-linking reagents, bis(phenoxycarbonylethyl) phosphinic acid (BPCEP) and bis(3-nitrophenoxycarbonylethyl)phosphinic acid (BNCEP), have been reported. The reagents were successfully employed to cross-link human hemoglobin under oxygenated conditions. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and fast protein liquid chromatography (FPLC) analyses of the reaction products indicated that the cross-link was intramolecular in nature, and that it was between the two beta subunits of hemoglobin in each case. The products were purified by DEAE-cellulose chromatography, and the purified material was employed for oxygen-binding assessments. The oxygen equilibrium curve of the cross-linked material, in each case, was right-shifted toward lower oxygen affinity as desired. The sigmoidal shapes of oxygen curves, in each case, suggested retainment of oxygen-binding cooperativity, although considerably lower than that of the native hemoglobin

Bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP): a novel affinity reagent for the beta-cleft modification of human hemoglobin.

The design, synthesis, and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP, 1) have been reported. The reagent was designed with the aid of molecular modeling, employing crystal coordinates of human hemoglobin A0. It was synthesized in three steps commencing from 4-t-butoxycarbonylphenol. The tri-sodium salt of 1 was employed to cross-link human oxyHb. While SDS-PAGE analyses of the modified hemoglobin product pointed to the molecular mass range of 32 kDa, the HPLC analyse suggested that the cross-link had formed between the beta 1-beta 2 subunits. The oxygen equilibrium measurements of the modified hemoglobin at 37 degrees C showed significantly reduced oxygen affinity (P50 = 31.3 Torr) as compared with that of cell-free hemoglobin (P50 = 6.6 Torr). The sigmoidal shape of O2 curves of the modified Hb pointed to reasonable retainment of oxygen-binding cooperativity after the cross-link formation. Molecular dynamics simulation studies on the reagent-HbA0 complex suggested that the most likely amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the-chains, and Lys-144 on the other. These predictions were consistent with the results of MALDI-MS analyses of the peptide fragments obtained from tryptic digestion of the cross-linked product.

Bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP): a new reagent for hemoglobin modification.

The synthesis and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP; 2) has been reported. The reagent was synthesized in four steps from hydroxybenzoic acid. The tri-sodium salt of BCCEP was employed to cross-link oxyHb, and the product was purified by DEAE-cellulose chromatography. The purified material was analyzed by SDS-PAGE, IEF, and HPLC analyses, which clearly showed the formation of covalent, intramolecular cross-links. While SDS-PAGE analyses of individual bands pointed to the molecular weight range of 32 kDa, the HPLC analyses suggested that the cross-links had formed between beta 1-beta 2 subunits. The oxygen equilibrium measurements and the Hill plots were performed on the purified bands to assess oxygen affinity as well as cooperativity of oxygen binding of the modified hemoglobins. All bands corresponding to modified hemoglobins showed significantly reduced oxygen affinity as compared with that of cell-free hemoglobin, as desired. The modified hemoglobins, however, exhibited somewhat reduced oxygen-binding cooperativity as contrasted with human stroma-free hemoglobin. Molecular dynamics simulation studies (Insight II/Discover/Biosym) on the Reagent-HbA0 complex suggested that the most likely amino acid residues involved in the cross-linking are Lys82 or N-terminal Val1 on one of the beta chains, and Lys144 on the other.

The use of fluorophore-containing spin traps as potential probes to localize free radicals in cells with fluorescence imaging methods.

Central to the study of free radical processes is the ability to identify and localize their cellular site of formation. Under the best of experimental conditions, spin trapping/ESR spectroscopy can only characterize intracellular production of specific free radicals and confocal microscopy can only localize the site of their formation. In this article, we report on the development of a fluorophore-containing nitrone, alpha-[4-[5-((2-carboxy)phenyl)-5-hydroxy-4-oxo-3-phenyl)-2-pyrrolin+ -1-yl]phenyl]-N-(tert-butyl)nitrone sodium salt (4). This nitrone (4) reacts with alpha-hydroxyethyl radical with a second order rate constant of 1.7 x 10(5) M-1 s-1 to give a characteristic ESR spectrum. However, we were unable to decrease the fluorescence emission, due in part to the small concentration of nitroxide generated from the reaction of alpha-hydroxyethyl radical with nitrone (4). Using the fluorophore-containing nitroxide (7) as a model, we found that only 12% of the nitroxide needs to be reduced to give an almost 400% increase in the fluorescent emission of (7). Our findings suggest new approaches to the development of various fluorophore-containing nitrones that can both characterize specific free radicals and localize their site of intracellular formation.

A fluorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils.

Toward the development of a fluorescence assay in combination with confocal microscopy to image free radicals generated by cells, we synthesized a fluorophore-nitroxide, 5-((2-carboxy)phenyl)-5-hydroxy-1-((2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)methyl)-3-phenyl-2-pyrrolin-4-one sodium salt, and tested the applicability of this probe to detect oxygen-centered free radicals. The reaction of the fluorophore-nitroxide with superoxide (10 microM/min) generated either by the reaction of xanthine oxidase on xanthine or by PMA-activated neutrophils in the presence of cysteine (200 microM) resulted in a loss of electron spin resonance (ESR) signal intensity concurrent with an increase in fluorescence emission. The decrease in ESR signal and the augmentation in fluorescence emission were inhibited by the addition of superoxide dismutase. This fluorophore-nitroxide also reacted with methyl radical generated by the reaction of hydroxyl radical with DMSO (0.14 M). In this case a loss in ESR signal intensity concomitant with an increase in fluorescence emission which were inhibited by catalase (300 U/ml), was recorded. These results clearly demonstrated the feasibility of using fluorescence methodology in conjunction with a fluorophore-nitroxide to detect oxygen-centered free radicals in biological systems.

Brain targeting of anti-HIV nucleosides: synthesis and in vitro and in vivo studies of dihydropyridine derivatives of 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine.

A significant number of patients with AIDS and AIDS-related complex develop neurological complications. Therefore, it is critical that anti-HIV agents penetrate the blood-brain barrier and suppress viral replication in the brain. In an effort to increase the brain delivery of anti-HIV nucleosides, in vitro and in vivo pharmacokinetics of dihydropyridine derivatives of 3'-azido-2',3'-dideoxyuridine (AzddU, AZDU, or CS-87) and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) have been studied. In vitro studies of the prodrugs (AzddU-DHP and AZT-DHP) in human serum, mouse serum, and mouse brain homogenate indicated that the rates of serum conversion from prodrugs to parent drugs are species dependent: mouse brain homogenate greater than mouse serum greater than human serum. Half-lives in human serum, mouse serum, and mouse brain homogenate are 4.33, 0.56, 0.17 h, respectively, for AzddU and 7.70, 1.40, and 0.18 h, respectively, for AZT. In vivo studies of AzddU-DHP and AZT-DHP showed that the prodrugs have areas under the serum concentration-time curves (AUC) similar to those of the parent drugs. The AUC in serum for AzddU following prodrug administration is 25.79 micrograms h/mL, which is similar to the value of 25.83 micrograms h/mL when AzddU was administered. Analogously, the serum AUCs for AZT when AZT-DHP and AZT were administered are 25.38 and 26.64 micrograms h/mL, respectively. However, the brain AUCs for both AzddU and AZT derived from prodrugs, being 11.43 and 11.28 micrograms h/mL, respectively, are greater than the brain AUCs for AzddU (2.09 micrograms h/mL) and AZT (1.21 micrograms h/mL) when the parent drugs were administered. Thus, the relative brain exposure (re) for AzddU (5.47) and AZT (9.32) indicate a significant increase in exposure to the anti-HIV nucleosides following prodrug administrations. The results of extended half-lives of the synthesized prodrugs in human serum along with the higher re values in vivo warrant studies in larger animals to determine the potential usefulness of the prodrugs in humans.

Synthesis and pharmacological study of antihyperlipaemic activity of 2-substituted thieno(2,3-d)pyrimidin-4(3H)-ones.

A series of 2-substituted thieno(2,3-d)pyrimidin-4-(3H) ones (1-15) was prepared by the reaction of thiophene ortho-aminoester (IV) with a variety of nitriles (V) under acidic conditions, and screened for antihyperlipaemic activity in various animal models. While most of these compounds were found active, 2-chloromethyl-5,6,7,8-tetrahydrobenzo(b)thieno(2,3-d) pyrimidin-4(3H)-one (5) was found to be the most active of all. The serum triglyceride lowering activity exhibited by 5 was found comparable to that of clofibrate and riboflavin tetrabutyrate. Compound 5 was also found to be safe as indicated by acute and chronic toxicity studies in mice and rats.