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A method for the selective introduction of the N2-(dimethylamino)methylene group into 8-thio-9-(2-hydroxyethoxymethyl)guanine (1) has been developed. The effect of the N2-amidine protection on the S-alkylation of 1 was studied.
Thienamycin, a natural product produced by Streptomyces cattleya is the first representative of a unique class of beta-lactam antibiotics, the carbapenems. Despite its outstanding potency and antibacterial spectrum, thienamycin was itself unsuited for further development because of its chemical instability in concentrated solution and in the solid state. Synthesis of the amidine derivative, N-formimidoyl thienamycin (imipenem, MK0787) resulted in a crystalline product with much improved stability and with antibacterial properties significantly superior to thienamycin. Imipenem has an unusually broad antimicrobial spectrum. A high order of bactericidal activity is found against Pseudomonas aeruginosa, Serratia, Bacteroides fragilis, enterococci and numerous other species intrinsically resistant to other antibiotics. Imipenem is refractory to hydrolysis by all important classes of bacterial beta-lactamases and thus exhibits no cross-resistance with penicillins or cephalosporins. Imipenem is distinguished from the new generation of extended-spectrum cephems by its unusually high potency against Gram-positive as well as Gram-negative organisms. Offsetting these excellent antimicrobial properties was an unusual susceptibility exhibited by imipenem to renal metabolism in animal species and in man. Very low urinary recoveries resulted without, however, any significant reduction in the serum half-life of imipenem. A brush-border dipeptidase, dehydropeptidase-I, was shown to be responsible for renal metabolism. Metabolism has been countered with the development of cilastatin (MK0791), a substituted amino-propenoate inhibitor of dehydropeptidase which is specific, potent and well matched in its pharmacokinetic properties for co-administration with imipenem. With the imipenem/cilastatin combination, uniformly high urinary concentrations and recovery are obtained regardless of the varying but often extensive metabolism suffered by imipenem in human populations. An additional benefit conferred by cilastatin results from its ability to exclude imipenem competitively from entry into and subsequent metabolism within the proximal tubular epithelium of the kidney. The tubular necrosis induced by imipenem alone when it is administered at very high doses to susceptible mammalian species is thereby eliminated. Thus the imipenem/cilastatin combination affords reliability and enhanced safety in the application of the antibiotic's unusual antibacterial potential in the treatment of difficult infections regardless of the site of disease.
A plasminogen-binding site of human alpha 2-plasmin inhibitor was studied. The chromatogram of digest from the amidinated alpha 2-plasmin inhibitor (67K-daltons, plasminogen-binding form) with trypsin was almost identical with that obtained from the 65K-daltons derivative (non-plasminogen-binding form) treated with the same procedure, except for the three tryptic peptides. One of the three peptides, the deamidinated peptide T-11, was found to have a strong ability to inhibit the interaction of alpha 2-plasmin inhibitor with human plasmin. Moreover, the dissociation constant Kd for interaction between the peptide T-11 and plasmin was estimated to be 5.5 microM, indicating that Kd is about 10-fold lower than that of epsilon-aminocaproic acid. The sequence of the peptide T-11 was determined by the Edman method as follows: NH2-G-D-K-L-F-G-P-D-L-K-L-V-P-P-M-E-E-D-Y-P-Q-F-G-S-P-K-COOH. alpha 2-Plasmin inhibitor and its 65K-daltons derivative were found to have the same NH2-terminal sequence of Asn(Asp)-Gln-Glu-Gln-. These results indicated that the plasminogen-binding site(s) of alpha 2-plasmin inhibitor could be located in the COOH-terminal region of its molecule and that some of epsilon-NH2-groups in the deamidinated peptide T-11 may be involved in the lysine-binding site(s) of plasmin(ogen).
The title compound, C(8)H(8)N(3)S(+).Cl(-).2H(2)O, has been synthesized and characterized both spectroscopically and structurally. The structure consists of 1,3-benzothiazole-6-carboxamidinium cations, chloride anions and water molecules, all interconnected by hydrogen bonds into a three-dimensional network. The 1,3-benzothiazole moiety is inclined to the 6-amidine group by 36.71 (9) degrees.
The title compound, [Li(4)(C(21)H(46)N(8)Si(2))(2)], has been prepared and its structure determined. The whole molecule can be regarded as a molecular cage made up of the central eight-membered ring and six contiguous rings of three types. As the first example of a bridged amidinate-dicyanamide framework, this noteworthy structure and its electronic features are presented.
In the title compound, C(16)H(18)N(6)O, an N-carbonylimidazole derivative of pyrazoline-1-carboximidamide, the pi-electron density of the N atom in the 1-position on the pyrazoline ring is delocalized through the amidine moiety and the adjacent carbonyl group. The imidazole ring, though coplanar with the rest of the molecule, is deconjugated. The pyrazoline ring adopts a flat-envelope conformation, having the substituted phenyl ring oriented perpendicular to the mean plane of the heterocycle. Both of the two potential hydrogen-bond donors are involved in intramolecular hydrogen-bonding interactions.
In the asymmetric unit of the title compound, C10H15N4O2+.H2PO4-, there are two protonated aminoguanidinium cations and two dihydrogenphosphate anions. The positive charge on the protonated amidine group is delocalized over the three C-N bonds in a manner similar to that found in guanidinium salts. The aminoguanidinium cations are found to be the E-isomer structures. Intramolecular interactions of the N-H...N type are observed, leading to the formation of five-membered rings. Extensive networks of O-H...O, N-H...O and C-H...O hydrogen bonds stabilize the three-dimensional network. In the crystal structure, pi-pi interactions between the benzene rings, with a distance of 3.778 (2) angstroms between the ring centroids, also affect the packing of the molecules.
A fluorescent in-situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single-cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3-D morphology perfectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single-stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly-(A)+ RNA was performed with a poly-biotin-d(U)-tailed oligo-d(T) probe. Bound probe was detected using streptavidin-fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di-amidine-2-phenyl indol. Multi-parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly-(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly-(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.
Two new ACTH-analogues, an octadecapeptide amide and a tetracosapeptide containing the lower homologue of arginine (norarginine) in position 8, have been synthesized by the generally accepted method. Special attention was paid to the synthesis of the required tetrapeptide representing the 7-10 sequence, which was obtained either by direct introduction of L-nitronorarginine or by amidination of the gamma-amino function in a protected peptide containing alpha, gamma-diaminobutyric acid. Biological activity determination showed that the shortening e arginine side chain in position 8 results in the formation of active ACTH-analogues.
The specific inhibition of trypsin-like serine proteases has become an important therapeutic target. These proteases have been implicated in several physiological and pathophysiological processes, including blood coagulation, digestion, and inflammation. Proteases of this class cleave polypeptide chains C-terminal to a basic residue (arginine or lysine). It has been shown that selectivity for a particular serine protease can be conferred based upon the structural moiety incorporated in the P1 position. In this regard, the three isomers (ortho, meta, and para) of amidinophenylalanine represent modified arginine residues and are important synthetic targets. Herein, a convenient asymmetric synthesis of (S)-Nalpha-(tert-butyloxycarbonyl)-2-, (S)-Nalpha-(tert-butyloxycarbonyl)-3-, and (S)-Nalpha-(tert-butyloxycarbonyl)-4-amidinophenyl-alanine N,O-dimethylamides (Weinreb amides) will be described. These derivatives represent key synthetic intermediates for the synthesis of enzyme inhibitors because the amidine can be readily orthogonally protected, while the Weinreb amide is easily converted to a variety of electrophilic carbonyls via reduction to the corresponding aldehyde or by reaction with various lithiated heterocycles. Likewise, the Weinreb amide can be reduced to the aldehyde and subsequently oxidized to the corresponding carboxylate, which is suitable for solid- or solution-phase peptide synthetic strategies.
Gamma-Glutamyl transpeptidase was isolated from sheep kidney cortex as an apparently homogeneous, highly active protein. At optimal pH and in the absence of acceptors, the enzyme catalyzes the release of about 510 mumol of p-nitroaniline per mg protein per min from the model substrate L-gamma-glutamyl-p-nitroanilide. Polyacrylamide gel electrophoresis in a sodium dodecylsulfate buffer system showed the presence of a large (Mr approximately 65000) and a small (Mr approximately 27000) polypeptide chain. Dissociation into two polypeptide chains was also achieved in 8 M urea. Amidination with dimethylsuberimidate produced a crosslinked protein of molecular weight approximately 90000. In the course of this work a convenient procedure was developed for the determination of gamma-glutamyl transpeptidase activity using L[glycine-2-3H]glutathione as the substrate. In this procedure the release of cysteinyl-[2-3H]glycine from glutathione is followed, after separation of the radioactive di-peptide from unreacted glutathione on a small Dowex-1 acetate column. The reactions with gamma-glutamyl-p-nitroanilide and glutathione are both strongly activated by several metal ions (Ca2+, Mg2+, Na+ and K+) and by a number of amino acids and peptide acceptors. The products of the reaction with glutathione were identified as cysteinylglycine, gamma-glutamylglutathione and glutamate. The formation of these products is consistent with the function of gamma-glutamyl transpeptidase in both the gamma-glutamyl transfer reaction and in the hydrolysis of the gamma-glutamyl bond. The activating effect of metal ions in the reaction with glutathione was shown to be dependent on the acceleration of the transfer reaction; the rate of hydrolysis of the gamma-glutamyl bond remaining unchanged.
Normal erythrocyte catalase, the enzyme present in the blood of Swiss acatalasemic heterozygotes, and their hybrid produced in vitro, were studied after crosslinking with bifunctional reagents. On theoretical grounds [cf. Hajdu, J., Bartha, F. & Friedrich, P. (1976) Eur. J. Biochem. 68, 373--383] it is inferred from the dodecylsulphate gel electrophoretic patterns obtained after treating catalase with diimidates of various chain lengths that the enzyme is an isologous tetramer (D2 symmetry). The minimal distances between crosslinkable primary amino groups across the three domains of bonding are different. Reaction with diimidates causes a moderate loss of enzyme activity in all three enzyme types due to amidination rather than crosslink formation. On the other hand, crosslinking stabilizes the enzyme against urea and heat inactivation. This is most prominent with heterozygote acatalasemic catalase. Crosslinking markedly prevents the development of peroxidase activity that can be elicited in catalases by urea treatment. The role of the quaternary structure of the protein in the relationship between catalase and peroxidase activities is discussed.
We studied the effect of glycerides on pancreatic phospholipase A2 hydrolysis of mixed monomolecular films of trioctanoylglycerol/1,2-didodecanoyl-sn-glycero-3-phosphocholine with the technique of Piéroni and Verger [(1979) J. Biol. Chem. 254, 10090-10094]. The quantity of enzyme adsorbed to the interface was concomitantly determined with [3H]amidinated phospholipase. At phospholipid packing above the critical penetration pressure, triacylglycerol stimulates phosphatidylcholine hydrolysis to a great extent. On the other hand, the activity of pancreatic phospholipase A2 on a mixed film is inhibited by the action of pancreatic lipase. Interface binding of phospholipase A2 to the lipid substrate does not imply activity.
The N-terminal alpha-helical region of phospholipase A2 is an important part of the enzyme for catalytic activity and lipid binding. Porcine pancreatic phospholipase A2 has Arg-Ser at positions 6 and 7, whereas the bovine enzyme has Asn-Gly. To pursue further the effects of these variable residues on differences in enzymatic properties, we prepared and studied the following semisynthetic analogs of epsilon-amidinated phospholipase A2 (AMPA): porcine [Ala7]AMPA, [Gly7]AMPA, [Asn6]AMPA, [Asn6-Gly7]AMPA and bovine [Ser7]AMPA and [Arg6-Ser7]AMPA. As we had previously found for the Asn6 leads to Arg bovine substitution, an Asn6-Gly7 leads to Arg5-Ser7 bovine substitution similarly improves the catalytic activity, the affinity for neutral lipid-water interfaces and the capacity to penetrate lecithin monolayers, while just changing Gly7 leads to Ser produces almost no effect on these properties. Ser7 leads to Ala and Ser7 leads to Gly substitutions in porcine AMPA did not affect penetration or lipid binding, although they did diminish catalytic activity (which is true of all substitutions made in the porcine enzyme). Arg6 leads to Asn substitution in porcine AMPA decreases penetration of lecithin monolayers, but not as much as it was improved by the Asn6 leads to Arg substitution in bovine AMPA. In contrast to the dramatic increase in affinity for lipid-water interfaces of Asn6 leads to Arg substitution in bovine AMPA, no decrease in affinity was found for Arg6 leads to Asn substitution in porcine AMPA. This difference is most likely due to the fact that the porcine enzyme has positively charged Lys and His in place of the Lys10, Glu17 pair that lie very close to residue 6 in the bovine structure. It can thus be conclude that (with the exception of Gly7 leads to Ser in bovine AMPA) all the substitutions tried at positions 6 and 7 in bovine and porcine AMPAs have definite effects on the catalytic activity.
Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527-2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse-phase HPLC. Mass spectroscopy, 1H- and 13C-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of the two lysine residues: [formula: see text]
The anti-bacterial activity of 13 commercially available acaricides, in use in the Caribbean, was tested against Dermatophilus congolensis, an actinomycete involved in streptothricosis of cattle, sheep and goats in the tropics. Acaricides used included organochloride, organophosphorus, carbamate, pyrethroid and amidine compounds. Each acaricide was tested at a typical working dilution used by the farmers, at 25 degrees C and 37 degrees C, in the presence or absence of 15 percent newborn calf serum as a fouling agent. Fouling of acaricides was found to have a marked inhibitory effect on its anti-bacterial activity. Vapona and Cattle Washing Detergent were found to be more active against Dermatophilus congolensis than the other acaricides tested.
1 Changes in the osmotic fragility and critical haemolytic volume of human erythrocytes produced by S-n-decylthiouronium (S-10) and related compounds have been studied. 2 S-10 had a biphasic action on osmotic fragility, protecting erythrocytes against lysis in low concentrations but producing lysis in a concentration of 1 mM or higher. 3 Some lower homologues of S-10 also protected erythrocytes against osmotic lysis, the degree of protection depending on the length of the alkyl chain. 4 Critical haemolytic volume was increased by antihaemolytic concentrations of procaine and chlorpromazine but not by antihaemolytic concentrations of S-10 and related amidines. 5 It is concluded that S-10 and its near homologues penetrate and stabilize erythrocyte membranes, potency increasing with the number of methylene groups in the side-chain up to about ten. The stabilization produced by S-10 apparently differs from that produced by many other lipid-soluble depressant drugs. It may be related to a drug-induced change in the ionic permeability of the membrane.
Interactions between alpha 2-agonists and noradrenaline vasoconstrictor responses were studied in the superior mesenteric arterial bed of the rat by use of perfusion both in situ with blood and in vitro with Krebs-Henseleit solution. Xylazine (1.9 X 10(-6) mol) administered into the perfusion circuit reduced the maximum response to noradrenaline in the in situ preparation by 35% and decreased the pD50 for noradrenaline from 8.5 +/- 0.01 to 7.9 +/- 0.13 (n = 7). Yohimbine (1 mg kg-1, i.v.) gave a small parallel shift in the noradrenaline log dose-response curve and prevented the reduction in the maximum response by subsequent administration of xylazine. In vitro, xylazine (1.9 X 10(-6) mol) also gave a long-lasting reduction of 37% in the maximum response but did not affect the mid-point sensitivity to noradrenaline. Yohimbine (10(-6) M) did not change either of these effects. Clonidine (1.9 X 10(-6) mol) did not affect the maximum response to noradrenaline in vitro but did reduce the pD50 from 7.72 +/- 0.17 to 6.9 +/- 0.17 (n = 6). Yohimbine did not change these effects. Guanfacine (1.8 X 10(-6) mol) had no effect on the sensitivity of the in vitro preparation to noradrenaline but did reduce the maximum response by 20%. Yohimbine (10(-6)M) prevented the depression of the maximum response. It is concluded xylazine and clonidine interfere with noradrenaline induced vasoconstriction only to a limited extent through their interaction with alpha 2-adrenoceptors and that some other, as yet uncharacterized mechanism which may be activated by their aryl amidine structure, is responsible for their in vitro effects.