Cisplatin mediates selective downstream hydrolytic cleavage of Met-(Gly)(n)-His segments (n=1,2) in methionine- and histidine-containing peptides: the role of ammine loss trans to the initial Pt-S(Met) anchor in facilitating amide hydrolysis

The pH- and time-dependent reactions of the antitumor drug cisplatin, cis-[PtCl(2)(NH(3))(2)], with the methionine- and histidine-containing pentapeptides Ac-Met-Gly-His-Gly-Gly-OH, Ac-Met-Gly-Gly-His-Gly-OH and Ac-Gly-Met-Gly-His-Gly-OH (Gly=glycyl, Met=L-methionyl, His=L-histidyl) at 313K have been investigated by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. Cisplatin mediates a rapid "downstream" hydrolytic cleavage of the Met-Gly amide bond in weakly acid solution (pH < or =5) for all three peptides, leading to release of H-Gly-His-Gly-Gly-OH, H-Gly-Gly-His-Gly-OH and H-Gly-His-Gly-OH, respectively, and formation of kappa(2)S,N(M) chelate complexes of the methionine-containing residuals Ac-Met-OH or Ac-Gly-Met-OH. An alternative reaction pathway affords tridentate kappa(3)S,N(M),N(imidazole) macrochelates of the original pentapeptide following ammine loss. The downstream cleavage pathway is competitive with the likewise cisplatin-mediated upstream cleavage of the Ac-Gly linkage in the pentapeptide Ac-Gly-Met-Gly-His-Gly-OH. This leads to formation of both the kappa(3)S,N(M),N(G1) complex of H-Gly-Met-Gly-His-Gly-OH due to upstream cleavage and the analogous tridentate complex for H-Gly-Met-OH due to initial downstream loss of H-Gly-His-Gly-OH followed by upstream loss of acetic acid. As downstream cleavage is not observed for Ac-(Gly)(2)-Met-(Gly)(2)-OH under similar conditions, it may be concluded that rapid histidine imidazole substitution of the ammine ligand in trans-position to an anchoring methionine S atom must assist hydrolytic cleavage of the Met-Gly amide bond.     

Amide and ester derivatives of N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid: the selective inhibitor of glucosamine-6-phosphate synthase

Several amide and ester derivatives of a glutamine analogue, N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (FMDP) (1-8), were synthesized and evaluated for the inhibitory activity in regard to glucosamine-6-phosphate synthase from Candida albicans. The syntheses were accomplished by the reaction of N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (BocFMDP) with the corresponding amines to give the FMDP amides (1-4) or with alkyl halides to give corresponding esters of FMDP (5-8). Among the synthesized compounds, the acetoxymethyl ester of FMDP was the most active inhibitor of the enzyme. Its IC50 value compared to that of FMDP (4 microM) was equal to 11.5 microM. The methyl and allyl esters and the N-hexyl-N-methyl-amide of FMDP exhibited a moderate enzyme inhibitory activity.     

Model-free analysis of a thermophilic Fe(7)S(8) protein compared with a mesophilic Fe(4)S(4) protein

15N T(1), T(2) and (1)H-(15)N NOE were measured for the thermophilic Fe(7)S(8) protein from Bacillus schlegelii and for the Fe(4)S(4) HiPIP protein from Chromatium vinosum, which is a mesophilic protein. The investigation was performed at 276, 300, and 330 K at 11.7 T for the former, whereas only the 298 K data at 14.1 T for the latter were acquired. The data were analyzed with the model-free protocol after correcting the measured parameters for the effect of paramagnetism, because both proteins are paramagnetic. Both thermophilic and mesophilic proteins are quite rigid, with an average value of the generalized order parameter S2at room temperature of 0.92 and 0.94 for Fe(7)S(8) and Fe(4)S(4) proteins, respectively. The analyzed nitrogens for the Fe(7)S(8) protein showed a significant decrease in S2with increasing temperature, and at the highest temperature >70% of the residues had an internal correlation time. This research shows that subnanosecond rigidity is not related to thermostability and provides an estimate of the effect of increasing temperature on this time scale.     

Diepoxybutane forms a monoadduct with B-form (dG-dC)n.(dG-dC)n and a crosslinked diadduct with the left-handed Z-form.

The characteristics of the reactions of DL-diepoxybutane (DEB) with (dG-dC)n.(dG-dC)n in the right-handed B-form or the left-handed Z-form were investigated. DEB does react with right-handed B-DNA since less salt is required to convert the modified B-form to Z-form than for the unmodified DNA. However, the product appears to be a monoadduct rather than the crosslinked diadduct formed with the Z-form. The modified B-form can be isolated, converted to a Z-form with l mM MnCl2, and then this activated complex further reacts intramolecularly to give the crosslinked Z-product. This modified Z-form cannot be reverted to the B-form unless the crosslink is cleaved with periodate. Only MnCl2, and to a lesser extent ZnCl2, was effective in facilitating the intramolecular conversion of the B-DNA monoadduct to the Z-DNA diadduct; lmM MgCl2 and 4M NaCl were ineffective suggesting that somewhat different types of modified left-handed conformations were generated by the different salts. DEB also cleaves DNA under our reaction conditions thus precluding studies with supercoiled recombinant plasmids harboring segments that adopt Z-structures.     

Incorporation of (2S,3S) and (2S,3R) beta-methyl aspartic acid into RGD-containing peptides

We report the synthesis and biological activity of a series of side-chain-constrained RGD peptides containing the (2S,3R) or (2S,3S) beta-methyl aspartic acid within the RGD sequence. These compounds have been assayed for binding to the integrin receptors alpha(IIb)beta3 and alpha(v)beta3 and the results demonstrate the importance of the side-chain orientation of this particular residue within the RGD sequence. Based on our findings, the (2S,3S) beta-methylated analogues of our RGD sequences maintain their binding potency to the integrin receptors while the (2S,3R) beta-methylated analogues exhibit a drastically reduced binding affinity. Our studies demonstrate that the three-dimensional orientation of the aspartyl side chain is a very important parameter for integrin binding and that small changes that affect the side-chain orientations give rise to drastic changes in binding affinity. These results provide important information for the design of more potent RGD mimetics.     

Rabbit reticulocyte lipoxygenase catalyzes specific 12(S) and 15(S) oxygenation of arachidonoyl-phosphatidylcholine

The rabbit reticulocyte lipoxygenase is known to display an unusual facility for oxygenation of esterified polyunsaturated fatty acids, yet the precise structures of the products are not known. With free arachidonate as substrate the enzyme is known to catalyze 15S and 12S oxygenations, and demonstration of a facility for catalysis of these reactions on phospholipids would extend the potential scope of lipoxygenase reactions in cells. We elected to study in detail the reaction of the enzyme with a natural phospholipid, palmitoyl/arachidonoyl-phosphatidylcholine. We determined the nature of the products by initial isolation by RP-HPLC, followed by transesterification and identification of the oxygenated products by HPLC, uv, GC-MS, and steric analysis of hydroxyl configuration by HPLC. The major product was identified as a phosphatidylcholine in which the arachidonate component was converted to the 15(S)-hydroperoxy-eicosatetraenoate. A second oxygenated phospholipid was produced in smaller quantities (2-5% of the latter product) and identified as the 12(S)-oxygenated analog. These products were also identified after reaction of the reticulocyte lipoxygenase with human red cell membranes which were radiolabeled by preincubation with [3H]arachidonic acid. The finding of 12S oxygenation represents the first evidence that a lipoxygenase can control a reaction centered on the 10-carbon of an arachidonoyl phospholipid. This is an important precedent, because hydrogen abstraction from carbon-10 is a critical step in the lipoxygenase-catalyzed synthesis of 8- and 12-hydroperoxy-eicosatetraenoates (HPETEs) and for the conversion of 5- and 15-HPETEs to leukotrienes.     

rRNA-protein neighbourhood in Escherichia coli 70 S ribosomes and 70 S initiation complex. Probing by bifunctional Pt(II)-containing reagent

The cleavable homobifunctional reagent dichloro[N,N,N',N'-tetrakis(2-aminoethyl)-1,6-hexamethylenediamminedi platinum (II)] dichloride was used for studying rRNA-protein cross-links in free 35S-labelled 70 S ribosomes and within initiation complex ribosome.AUGU6.fMet-tRNA(fMet). It was shown that the sets of proteins cross-linked to 16 S and 23 S rRNA in free 70 S ribosomes and in 70 S initiation complex do not differ significantly. The authors are the first to demonstrate most of the 23 S rRNA-protein cross-links and some 16 S rRNA-protein cross-links, in particular those with L7/L12 protein.     

Involvement of two alpha-ketoglutarate-dependent dioxygenases in enantioselective degradation of (R)- and (S)-mecoprop by Sphingomonas herbicidovorans MH.

Cell extracts of Sphingomonas herbicidovorans MH grown on (R)-mecoprop contained an enzyme activity that selectively converted (R)-mecoprop to 4-chloro-2-methylphenol, whereas extracts of cells grown on (S)-mecoprop contained an enzyme activity selective for the S enantiomer. Both reactions were dependent on alpha-ketoglutarate and ferrous ions. Besides 4-chloro-2-methylphenol, pyruvate and succinate were detected as products of the reactions. Labeling experiments with (18)O2 revealed that both enzyme activities catalyzed a dioxygenation reaction. One of the oxygen atoms of pyruvate and one of the oxygen atoms of succinate were derived from molecular oxygen. Analysis of cell extracts obtained from cells grown on different substrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that growth on (R)-mecoprop and (S)-mecoprop caused the appearance of prominent protein bands at 34 and 32 kDa, respectively. Both protein bands were present when cells grew on the racemic mixture. The results demonstrate that S. herbicidovorans initiated the degradation of each enantiomer of mecoprop by a specific alpha-ketoglutarate-dependent dioxygenase. By comparing conversion rates of various phenoxy herbicides, we confirmed that the two enzyme activities were distinct from that of TfdA, which catalyzes the first step in the degradation of 2,4-dichlorophenoxyacetic acid in Ralstonia eutropha JMP134.     

利用荧光手性试剂（S）—TBMB甲酸分析氨基糖的D—和L—构型

D、L型氨基糖经荧光手性试剂 (S) ( ) 2 叔丁基 2 甲基 苯并 1 ,3 二氧杂环戊烷 4 甲酸 [(S) TBMB甲酸 ]标记、完全乙酰基化得到非对映的氨基糖完全乙酰基化N (S) TBMB羧酸衍生物。其1 HNMR图谱 ,特别是强的叔丁基及甲基信号峰被用于分析氨基糖的D、L构型。此外 ,还利用反相HPLC及同样的荧光标识方法创立了简便的高灵敏度氨基糖D、L构型分析方法。其全部分析操作时间在 2h内 ,检测极限为 0 .2 pmol     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Syntheses and characterization of neutral complexes of Zn(II) with mono- and dianionic pyrrole-2-imine ligands [(pyrrole-2-CHN)nR]n− (n = 1, 2)

The reactions of the dianionic [(pyrrole-2-CHN)₂R]^2? ligands [(N′₂N₂)^2?] (R = (R)(S)-1,2-cyclohexane or 1,2-ethane) with Zn(II) yield neutral dimeric [Zn₂(N′₂N₂)₂] complexes. The dimeric nature of the complexes was established by field-desorption mass spectrometry. ¹H NMR studies show that these complexes have dimeric structures in solution in which the (N′₂N₂)^2? ligands act as di-bidentates.The metal centres have tetrahedral geometries and bot have Δ or Λ configurations. The complex with the (R)(S)-1,2-cyclohexanediyl bridges has a rigid structure in solution. Neither intermolecular nor intramolecular exchange processes are observed The ¹H NMR spectrum of the complex with the 1,2-ethanediyl bridging groups shows that at 213 K in CDCl₃ a fast conformational movement is already taking place between two identical structures of the complex. It is not possible to determine whether in this complex intermolecular exchange processes are also taking place.The reactions of the anionic [pyrrole-2-CHNR′]^? ligands [(N′N)^?] (R′ = t-Bu, i-Pr, (S)-CHMePh or 2,6-xylyl) with Zn(II) yield the neutral Zn(N′N)₂ complexes. These complexes were synthesized to study the coordination properties of the [pyrrole-2-CHNR′]^? moieties with Zn(II). A ¹H NMR study established that the zinc centres in the complexes containing the prochiral i-Pr or chiral (S)-CHMePh substituents have tetrahedral geometries with Δ or Λ configurations in CDCl₃ at 213 K. These complexes undergo an intramolecular exchange process at higher temperatures (above 260 K when R′ = i-Pr) which involves inversion of the configuration of the zinc centre. A mechanism for this exchange process is proposed.     

PLP-dependent H(2)S biogenesis

The role of endogenously produced H(2)S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H(2)S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H(2)S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5'-phosphate-dependent (PLP) enzymes, cystathionine β-synthase and cystathionine γ-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H(2)S can be liberated. In this review, we present an overview of the kinetics of the H(2)S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S) → Rh2(S) → PPD(S) and Rg3(R) → Rh2(R) → PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.     

Solution structure of a trans-opened (10S)-dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation

Two-dimensional NMR was used to determine the solution structure of an undecanucleotide duplex, d(CGGTCACGAGG).d(CCTCGTGACCG), in which (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is covalently bonded to the exocyclic N(6)() amino group of the central deoxyadenosine, dA(6), through trans addition at C10 of the epoxide (to give a 10S adduct). The present study represents the first NMR structure of a benzo[a]pyrene (10S)-dA adduct in DNA with a complementary T opposite the modified dA. Exchangeable and nonexchangeable protons of the modified duplex were assigned by the use of TOCSY (in D(2)O) and NOESY spectra (in H(2)O and D(2)O). Sequential NOEs expected for a B-type DNA conformation with typical Watson-Crick base pairing are observed along the duplex, except at the lesion site. We observed a strong intraresidue NOE cross-peak between H1' and H8 of the modified dA(6). The sugar H2' and H2' ' of dC(5) lacked NOE cross-peaks with H8 of dA(6) but showed weak interactions with H2 of dA(6) instead. In addition, the chemical shift of the H8 proton (7.51 ppm) of dA(6) appears at a higher field than that of H2 (8.48 ppm). These NOE and chemical shift data for the dA(6) base protons are typical of a syn glycosidic bond at the modified base. Restrained molecular dynamics/energy minimization calculations show that the hydrocarbon is intercalated from the major groove on the 3'-side of the modified base between base pairs A(6)-T(17) and C(7)-G(16) and confirm the syn glycosidic angle (58 degrees ) of the modified dA(6). In the syn structure, a weak A-T hydrogen bond is possible between the N3-H proton of T(17) and N7 of dA(6) (at a distance of 3.11 A), whereas N1, the usual hydrogen bonding partner for N3-H of T when dA is in the anti conformation, is 6.31 A away from this proton. The 10(S)-dA modified DNA duplex remains in a right-handed helix, which bends in the direction of the aliphatic ring of BaP at about 42 degrees from the helical axis. ROESY experiments provided evidence for interconversion between the major, syn conformer and a minor, possibly anti, conformer.     

Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar)

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.     

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and intercistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), P. damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).     

The Bis-Trityl Route to (S)-HPMPA

Abstract

(S)-HPMPA, (S)-9-(3-hydroxy-2-phosphonyl)methoxypropyl)-adenine 1, a broad spectrum adenine nucleotide antiviral, was prepared from (S)-DHPA 2. Protection of (S)-DHPA 2 as its′ N,O-di-trityl derivative 3 followed by regioselective 2′-O-alkylation with p-toluene-sulfonyloxymethyldiethylphosphonate yielded bis-trityl-protected diethyl-(S)-HPMPA 4. De-protection and ester cleavage gave (S)-HPMPA 1.     

Acceptor proteins for (ADP-ribose)n in the HeLa S3 cell cycle

The acceptor proteins for (ADP-ribose)n were investigated by using nuclei or chromosomes isolated from specific phases of the cell cycle of HeLa S3 cells. Analysis of HMG proteins and histone H1 by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 increased by 12- and 5-fold, respectively, in the metaphase chromosomes as compared with that in the G1 phase cell nuclei. The degree of (ADP-ribosyl)n-ation of these proteins in the S phase cell nuclei was as low as that in G1 phase cell nuclei. In the G2 phase cell nuclei, the degrees of (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 were about 5- and 2-fold greater, respectively, as compared with that in the G1 phase cell nuclei. The (ADP-ribosyl)n-ation of HMG 1 and 2 was constant through the cell cycle except for a slight decrease in the S phase. The data may imply that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 is linked to chromatin structural changes in mitosis.     

beta-cyclodextrin-immobilized (4S)-phenoxy-(S)-proline as a catalyst for direct asymmetric aldol reactions

beta-Cyclodextrin-immobilized (4S)-phenoxy-(S)-proline was prepared conveniently by simply heating the amino acid and beta-cyclodextrin in ethanol-water (1/1, v/v) and removal of the solvent. This proved to be an efficient catalyst for direct asymmetric aldol reactions, and the catalyst could be recycled four times without loss of enantioselectivity.