Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry

A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with 2H2O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using [2H]diethylene glycol and [2H]hydrazine hydrate afforded [11,11,12,12,23,23(-2)H]lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-[11,11,12,12(-2)H]pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-[11,11,12,12(-2)H] progesterone (XIV), consisting of 0.3% 2H0-, 1.1% 2H1-, 8.6% 2H2-, 37.1% 2H3-, 52.1% 2H4-, and 0.8% 2H5-species.     

Chain dynamics of selectively deuterated fatty acids in high-density lipoproteins studied by deuterium NMR

Deuterium order parameters have been determined for approximately 5 mol% selectively deuterated palmitic acid incorporated into the outer monolayer of high-density lipoproteins (HDL3). The values are SCD = 0.38 for [2,2-2H2]palmitic acid, 0.38 for [4,4-2H2]palmitic acid, 0.37 for [5,5,6,6-2H4]palmitic acid, 0.23 for [11,11,12,12-2H4]palmitic acid, and 0.05 for [16,16,16-2H3]palmitic acid. Comparison of the acyl chain order parameters in HDL3 with acyl chain order parameters determined recently [Parmar, Y.I., Wassall, S.R., & Cushley, R.J. (1984) J. Am. Chem. Soc. 106, 2434-2435] for approximately 5 mol% deuterated palmitic acid in sonicated unilamellar vesicles, composed of the same ratio of phosphatidylcholine/sphingomyelin (85/15 w/w) found in HDL3, shows that acyl chain order in the HDL3 monolayer is approximately 3-5 times higher than in the vesicle bilayer. The acyl chain order in the lipoprotein monolayer is approximately 1.5-2 times higher than in the bilayer of phosphatidylcholine multilamellar dispersions. Deuterium longitudinal relaxation times have been measured for deuterated palmitic acid in HDL3, and the values T1 approximately 16 ms for C2H2 and 170 ms for C2H3 groups are a factor of more than 2 times smaller than found in phospholipid bilayers.     

Incorporation of bile acid of low concentration into model and biological membranes studied by 2H and 31P NMR

We have analyzed the manner of incorporation of bile acid into lipid bilayers and resultant perturbation of the bilayer structure with lower bile acid/lipid ratios relevant to the physiological conditions (approximately 1 mM) by 2H and 31P NMR methods, as an aid to understanding the possible role as an endogenous tumor promoter in colon cancer besides the primary physiological function of solubilizing lipids. On the basis of the 2H quadrupole splittings of [6,6,7,7,8-2H5]deoxycholate and [11,11,12,12-2H4]chenodeoxycholate in the presence of lamellar multibilayers of egg yolk lecithin, these bile acids were found to be incorporated in such a manner that the B-D rings lie parallel with the normal of the bilayers when the ratio of the bile acid to lipid is low (less than 0.11). When the ratio is increased, these bile acid molecules are not dispersed entirely in the bilayer but aggregate to form micelles with lipids. Further, we studied the resultant perturbation of the multibilayers of egg yolk lecithin analyzed by using the 2H quadrupole splitting of [18,18,18-2H3]stearic acid as a probe and by 31P chemical shift anisotropy. We found that the bilayer structure is retained even at the bile acid-to-lipid ratio of 0.25, although a small amount of an isotropic phase appeared such as small vesicles and micelles. The molecular ordering of fatty acyl chains was rather enhanced by the presence of 1 mM deoxycholate in erythrocyte ghosts as seen from the 2H quadrupole splitting of [16,16,16-2H3]palmitic acid, although deoxycholate caused hemolysis in this condition. The former observation can be explained by the way the lipid-protein interaction is modified by deoxycholate located in the interface between the lipids and proteins.     

The isolation and characterization of 60 nm vesicles (''nanovesicles'') produced during ionophore A23187-induced budding of human erythrocytes.

1. Penicillin N was synthesized by coupling alpha-amino-alpha-p-nitrobenzyl-N-p-nitro-benzyloxycarbonyl-D-adipate with 6-aminopenicillanic acid benzyl ester, followed by removal of the protecting groups through hydrogenolysis. 2. alpha-Amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate was prepared by treating alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-glutamic acid with [14C]diazomethane followed by rearrangement with silver trifluoromethanesulphonate. 3. Coupling of alpha-amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate with 6-aminopenicillanic acid benzyl ester gave triprotected [10-14C]penicillin N. 4. 3H was introduced at C-6 of the Schiff's base derivative (10) by oxidation followed by reduction with NaB3H4. 5. The so-derived (6 alpha-3H)-labelled Schiff's base was hydrolysed to give 6-amino [6 alpha-3H]penicillanic acid benzyl ester p-toluenesulphonic acid salt, which after coupling as the free amine with alpha-amino-alpha-p-nitrobenzyl-N-pnitrobenzyloxycarbonyl-D-adipate and then hydrogenolysis, yielded [6alpha-3H]penicillin N. 6. Triprotected [10-14C]penicillin N and triprotected [6alpha-3H]penicillin N in admixture were hydrogenolysed to give [10-14C,6alpha-3H]penicillin N.     

Synthesis and aqueous solution properties of multinuclear oxo-bridged vanadium(IV/V) complexes

Reaction of the multifunctional phenolic ligands 2,5-bis[N,N-bis(carboxymethyl)aminomethyl]hydroquinone (H6cahq), 2,2'-bis[N,N-bis(carboxymethyl)aminomethyl]-4,4'-isopropylidenediphen ol(H6capd),2,2',2'-tris[N,N-bis(carboxymethyl)aminomethyl]-1,1 ,1-tris(4-hydroxyphenyl)ethane (H9catp) and the monofunctional 2-[N,N-bis(carboxymethyl)aminomethyl]-4-carboxyphenol (H3cacp), with VOSO4 and NaVO3 affords the oxo-bridged mixed-valence vanadium(IV/V) Na6[(VO)4(mu-O)2(mu-cahq)2] x Na2SO4 x 20H2O (1), HnNa(3-n)[(VO)2(mu-O)(mu-cacp)2] (2), HnNa(3-n)[(VO)4(mu-O)2(mu-capd)2] (3), HnNa(9-n)[(VO)6(mu-O)3(mu3-catp)2] (4). In addition to the synthesis, we report the infrared, magnetic, optical and electrochemical properties of these complexes. The hydrolytic stability at different pH values was also investigated using visible spectroscopy.     

N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.     

Stereospecificity of hydrogen transfer in the saccharopine dehydrogenase reaction.

The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety.     

Bis(sulfo-N-succinimidyl) [15N,2H16]doxyl-2-spiro-4'-pimelate, a stable isotope-substituted, membrane-impermeant bifunctional spin label for studies of the dynamics of membrane proteins: application to the anion-exchange channel in intact human erythrocytes

We have synthesized and characterized an isotopically substituted homologue of the membrane-impermeant bifunctional spin label bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP) [Beth et al. (1986) Biochemistry 25, 3824-3832] in which the nitroxide N is substituted with 15N and all of the protons in the doxylpimelate moiety are replaced by deuterons ([15N,2H16]BSSDP). Like its normal isotope homologue, [15N,2H16]BSSDP reacts with the anion-exchange channel in intact human erythrocytes at a site that spans the single extracytoplasmic chymotryptic cleavage site and that overlaps the stilbenedisulfonate site. The narrower line widths in the EPR spectrum of [15N,2H16]BSDP-labeled anion channels allow calculation of a minimum separation of 16 A between spin labels bound at the functionally important stilbenedisulfonate sites on adjacent subunits of an anion channel dimer. The 15N and 2H isotopic substitutions also provide substantial improvement in signal to noise of motionally sensitive regions of the ST-EPR spectrum of [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes. [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes were cross-linked to covalent dimers in the extracytoplasmic domain with the membrane-impermeant cross-linking reagent bis(sulfo-N-succinimidyl) suberate [Staros (1982) Biochemistry 21, 3950-3955], and the saturation-transfer EPR spectrum of these cells was compared with that of cells treated with [15N,2H16]BSSDP but not subsequently cross-linked. The spectra were essentially identical, supporting the hypothesis that anion channel subunits form stable dimers in the membranes of intact erythrocytes.     

The biosynthesis of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Alignment of the butylamine segment and source of the secondary amino nitrogen

The unusual amino acid hypusine is produced in a single protein of mammalian cells by a novel posttranslational event in which a lysine residue is conjugated with the four-carbon moiety from the polyamine spermidine to form an intermediate deoxyhypusine, and in which this intermediate is subsequently hydroxylated. Specifically isotopically labeled precursors of hypusine were used to identify the biosynthetic origin of some of the atoms of hypusine and thus to provide further insight into the mechanism of this in vivo chemical modification reaction. Radiolabel from [1,4-3H] putrescine, [1,8-3H]spermidine, and [5-3H]spermidine entered hypusine during growth of Chinese hamster ovary cells. The occurrence of this label at positions 1 and 4, at position 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine revealed an alignment of atoms identical to that in the butylamine segment of spermidine. Growth of cells with [epsilon-15N]lysine as the source of lysine yielded hypusine enriched in 15N, whereas only isotope-free hypusine during growth by [4-15N]spermidine. These was found in cells whose spermidine was replaced during growth by [4-15N]spermidine. These findings are in accordance with a proposal that the first phase of hypusine biosynthesis, the production of intermediate deoxyhypusine, occurs through transfer of the butylamine moiety from spermidine to the epsilon-amino nitrogen of protein-bound lysine. The technique of thermospray high-performance liquid chromatography/mass spectrometry provided positive identification of 15N in hypusine through final separation and on-column direct analysis of this amino acid. Methods of preparation are given for spermidine of high specific radioactivity, labeled specifically at position 5 with 3H, and for spermidine with 15N at the 4-position.     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible inactivation of beta-adrenergic receptors of C6 glioma cells. Synthesis and study of a thiol derivative of propranolol

The beta-adrenergic receptor of C6 glioma cells contains a disulfide bridge which can be reduced by dithiothreitol (DTT). On intact cells, N-ethylmaleimide (NEM) (5 mM) does not change the affinity of [3H] H2-alprenolol ([3H] DHA) but reduces the total number of beta-adrenergic cell receptors by 21 +/- 3 per cent ; (N = 3). After receptor reduction by DTT, NEM irreversibly blocks the accessibility of the beta-adrenergic receptors to [3H]DHA. On isolated membranes, incubation in the presence of either NEM (5 mM) or isoproterenol (5.10(-7) M) does not significantly modify the total number of beta-adrenergic receptors accessible to [3H]DHA. Incubation of membranes with both NEM and isoproterenol reduces the number of binding sites by 33 +/- 2 per cent ; (N = 3). A thiol derivative of propranolol was synthetized. Its affinity is 10 times lower than that of propranolol. This sulfur derivative reduces the total number of beta-adrenergic receptors by 22 +/- 3 per cent (N = 3) when incubated with the native receptor and by 55 +/- 4 per cent (N = 4) when incubated with the reduced receptor. DTT does not significantly reverse the blockade induced by propranolol-SH. A model is proposed for explaining these results.     

近年来华东地区家鸭中禽流感病毒的亚型分布

[目的]为了研究近年来华东地区家鸭中禽流感病毒的亚型分布情况.[方法]对2002-2006年分离自华东地区家鸭的180株禽流感病毒的HA亚型和其中88株禽流感病毒的NA亚型分别进行了测定.[结果]近年来华东地区家鸭中至少存在9种HA亚型和6种NA亚型组成的H1N1,H3N1,H3N2,H3N8,H4N6,H5N1,H5N2,H6N2,H6N8,H8N4,H9N2,H10N3,H11N2共13种亚型的禽流感病毒.[结论]华东地区家鸭中有多种亚型的禽流感病毒分布,应加强家鸭禽流感的监测和防制工作.     

Structure of a substrate radical intermediate in the reaction of lysine 2,3-aminomutase.

Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize an organic radical that appears in the steady state of the reaction catalyzed by lysine 2,3-aminomutase from Clostridium SB4. Results of a previous electron paramagnetic resonance (EPR) study [Ballinger, M. D., Reed, G. H., & Frey, P. A. (1992) Biochemistry 31, 949-953] demonstrated the presence of EPR signals from an organic radical in reaction mixtures of the enzyme. The materialization of these signals depended upon the presence of the enzyme, all of its cofactors, and the substrate, lysine. Changes in the EPR spectrum in response to deuteration in the substrate implicated the carbon skeleton of lysine as host for the radical center. This radical has been further characterized by EPR measurements on samples with isotopically substituted forms of lysine and by analysis of the hyperfine splittings in resolution-enhanced spectra by computer simulations. Changes in the hyperfine splitting patterns in EPR spectra from samples with [2-2H]lysine and [2-13C]-lysine show that the paramagnetic species is a pi-radical with the unpaired spin localized primarily in a p orbital on C2 of beta-lysine. In the EPR spectrum of this radical, the alpha-proton, the beta-nitrogen, and the beta-proton are responsible for the hyperfine structure. Analysis of spectra for reactions initiated with L-lysine, [3,3,4,4,5,5,6,6-2H8]lysine, [2-2H]lysine, perdeuteriolysine, [alpha-15N]lysine, and [alpha-15N,2-2H]lysine permit a self-consistent assignment of hyperfine splittings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relatively stable N-ligated [2Fe2S](2+) clusters with dipyrromethane capping ligands

[2Fe2S] clusters with terminal N-ligation (His, Arg) and unique functions are increasingly recognized in biological systems. In this work three new [2Fe2S] clusters 1-3 with different 1,1'-dipyrrolmethane derivatives as chelating terminal ligands have been prepared and fully characterized, including by X-ray crystallography: (NEt(4))(2)[L(2)Fe(2)(mu-S)(2)] with L=Me(2)C(C(4)H(3)N)(2) (1), Ph(2)C(C(4)H(3)N)(2) (2), (CH(2))(5)C(C(4)H(3)N)(2) (3). These systems represent rare examples of synthetic [2Fe2S] complexes with N-donor capping ligands. While geometric parameters as well as spectroscopic and electrochemical characteristics of the new complexes are as anticipated, the chelating nature of the terminal ligands in 1-3 imparts a relatively high stability that will be advantageous for reactivity studies of the [2Fe2S] core.     

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiolate exchange in [TmR]ZnSR' complexes and relevance to the mechanisms of thiolate alkylation reactions involving zinc enzymes and proteins

The zinc and cadmium thiolate complexes [TmBut]MSCH2C(O)N(H)Ph (M = Zn, Cd) may be obtained via treatment of the respective methyl complex [TmBut]MMe with PhN(H)C(O)CH2SH. The molecular structure of [TmBut]ZnSCH2C(O)N(H)Ph has been determined by X-ray diffraction, thereby demonstrating the presence of an intramolecular N-H S hydrogen bond between the amide N-H group and thiolate sulfur atom. [TmBut]ZnSCH2C(O)N(H)Ph mimics the function of the Ada DNA repair protein by undergoing alkylation with MeI to give [TmBut]ZnI and MeSCH2C(O)N(H)Ph. A series of crossover experiments and 1H NMR magnetization transfer studies establish that thiolate exchange between [TmR]ZnSR' derivatives is facile in this system, an observation that supports the previous suggestion that the alkylation of [TmPh]ZnSCH2C(O)N(H)Ph by MeI may proceed via a sequence that involves dissociation of [PhN(H)C(O)CH2S]-.     

A novel type of oxygenolytic ring cleavage: 2,4-oxygenation and decarbonylation of 1H-3-hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline

Abstract The utilization of quinaldine (2-methylquinoline) by Arthrobacter sp. Rü61a proceeds via 1 H -4-oxoquinaldine, 1 H -3-hydroxy-4-oxoquinaldine, and N -acetyl-anthranilic acid. By analogy, 1 H -4-oxoquinoline is degraded by Pseudomonas putida 33/1 via 1 H -3-hydroxy-4-oxoquinoline and N -formylanthranilic acid. Using the purified enzymes from both organisms, the mode of N -heterocyclic ring cleavage was investigated. The conversions of 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinoline to N -acetyl- and N -formylanthranilic acid, respectively, were both accompanied by the release of carbon monoxide. The enzyme-catalysed transformations were performed in an [¹⁸O]O₂ atmosphere and resulted in the incorporation of two oxygen atoms into the respective products, N -acetyl- and N -formylanthranilic acid, indicating an oxygenolytic attack at C-2 and C-4 of both 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinolone.     

Incorporation of 3H from delta-(L-alpha-amino (4,5-3H)adipyl)-L-cysteinyl-D-(4,4-3H)valine into isopenicillin N.

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.     

Synthetic analogue approach to metallobleomycins: syntheses, structure and properties of mononuclear and tetranuclear gallium(III) complexes of a ligand that resembles the metal-binding site of bleomycin

As part of our interest into the bioinorganic chemistry of gallium, gallium(III) complexes of the peptide ligand N-(2-(4-imidazolyl)ethyl)pyridine-2-carboxamide (pypepH2) resembling a fragment of the metal-binding domain of bleomycins (BLMs), have been isolated. Reaction of pypepH2 with (Et4N)[GaCl4] and Ga(acac)3 [acac- is the acetylacetonate(-1) ion] affords the mononuclear complex [Ga(pypepH)2]Cl.2H2O (1) and the tetranuclear complex [Ga4(acac)4(pypep)4].4.4H2O (2), respectively. Both complexes were characterized by single-crystal X-ray crystallography, IR spectroscopy and thermal decomposition data. The pypepH- ion in 1 behaves as a N(pyridyl), N(deprotonated amide), N(pyridine-type imidazole) chelating ligand. The doubly deprotonated pypep2- ion in 2 behaves as a N(pyridyl), N(deprotonated amide), N(imidazolate), N'(imidazolate) mu2 ligand and binds to one Ga(III) atom at its pyridyl, amide and one of the imidazolate nitrogens, and to a second metal ion at the other imidazolate nitrogen; a chelating acac- ligand completes six coordination at each Ga(III) centre. The IR data are discussed in terms of the nature of bonding and known structures. The 1H NMR spectrum of 1 suggests that the cation of the complex maintains its integrity in dimethylsulfoxide (DMSO) solution. Complexes 1 and 2 are the first synthetic analogues of metallobleomycins with gallium(III).