Christopher J. Logan

Peptide synthesis. Part 2. Procedures for solid-phase synthesis using Nα-fluorenylmethoxycarbonylamino-acids on polyamide supports. Synthesis of substance P and of acyl carrier protein 65–74 decapeptide

Use of base-labile Nα-9-fluorenylmethoxycarbonylamino-acids in combination with acid-labile t-butyl or p-alkoxybenzyl side-chain or carboxy-terminal (resin-linkage) protecting groups enables solid-phase peptide synthesis to be carried out under exceptionally mild reaction conditions. The repetitive and vigorous acidic treatments required in conventional synthesis are avoided. High-yield syntheses of a decapeptide and of an undecapeptide amide (Substance P) are described using this new combination of protecting groups on polyamide supports.

The incorporation of 3-phenoxybenzoic acid and other xenobiotic acids into xenobiotic lipids by enzymes of the monoacylglycerol pathway in microsomes from adult and neonatal tissues

The incorporation of 3-phenoxybenzoic acid (3PBA) into xenobiotic lipids by enzymes of the monoacylglycerol (MG) pathway was measured using microsomes prepared from rat liver as an enzyme source. The mean activities of the three enzymes involved were: acyl-CoA synthetase, 1.1 nmol/min/mg protein; MG acyltransferase, 75 pmol/min/mg protein; and diacylglycerol acyltransferase, 11.4 pmol/min/mg protein. MG and DG acyltransferase also showed activity with benzoyl-CoA or 1-naphthylacetyl-CoA as acyl donor but none with clofibryl-CoA or 2,4-dichlorophenoxyacetyl-CoA. MG acyltransferase activity, using 3PBA-CoA, was higher in microsomes from rat intestinal mucosa and pig liver, and lower in rat adipose tissue, rat liver and mouse liver. This ranking of activities corresponds to published activities using natural substrates. There was a large increase in MG acyltransferase, using either 3PBA-CoA or palmitoyl-CoA as substrate, in microsomes from the livers of rats 16-18 days old. Lysophosphatidic acid (lyso-PA) and lysophosphatidylethanolamine (lyso-PE), but not other phospholipids or detergents, stimulated MG acyltransferase activity more than two-fold. Lyso-PA (5 microM) increased the Vmax but had little effect on the Km for 2-hexadecylglycerol, whereas 100 microM lyso-PE decreased the Km and had a smaller effect on the Vmax. These results illustrate that the incorporation of xenobiotic acids into diacyl- and triacylglycerol by enzymes of the MG pathway may be a more general phenomenon than was previously suspected and that it may be subject to a variety of developmental and physiological controls.

Studies on the fate of flocoumafen in the Japanese quail (Coturnix coturnix japonica)

1. 14C-Flocoumafen, administered to Japanese quail as a single oral or i.p. dose, was rapidly and extensively eliminated in excreta; most was eliminated within 24 h. Extensive metabolism of the rodenticide was seen, with at least 8 metabolites detected; unchanged flocoumafen comprised 9% dose. The elimination kinetics and metabolic profiles were qualitatively similar after oral and i.p. dosing. 2. The major metabolites (60% dose) were labile to beta-glucuronidase, liberating aglycones with identical chromatographic mobilities to those of the unchanged flocoumafen isomers. 3. Radioactivity was retained mostly in the liver; largely as unchanged flocoumafen associated with the mitochondrial and microsomal fractions. Elimination of radioactivity from most tissues was biphasic with an initially rapid depletion (5 days) followed by a slow terminal elimination phase. The elimination half life from liver was greater than 100 days. 4. Livers of quail receiving extended dietary exposure to flocoumafen at 5, 15 and 50 ppm had concentrations of flocoumafen (1.0 nmol/g) that were independent of dose, indicating a capacity-limited binding site. These hepatic concentrations were similar to those after a single oral dose and were also similar to those in rats. The data indicate the presence in quail liver of a saturable high affinity flocoumafin binding site with similar characteristics and capacity to that in the rat. 5. The selective toxicity of flocoumafen to rats (highly toxic) and quail (moderately toxic) appears to arise from differences in metabolism rather than from anticoagulant binding in the liver. When hepatic binding sites of rats are saturated anticoagulant action becomes lethal, whereas quail are able to survive and extensively metabolize the compound.

The formation of a novel mercapturic acid during the metabolism of an N-methyl aromatic amine, 4-cyano-N,N-dimethylanine

Abstract As part of a collaborative study on the genotoxicity of aromatic amines 1 we have recently investigated the metabolism of 4-cyano- N , N -dimethylaniline (CDA) in rats and mice. We now wish to report the formation of a sulphur containing metabolite, which indicates an important role of glutathione in the metabolism of an aromatic amine N -methyl group.

Structure–Activity Studies of Glucose Transfer: Determination of the Spontaneous Rates of Hydrolysis of Uridine 5′-Diphospho-α-d-glucose (UDPG) and Uridine 5′-diphospho-α-d-glucuronic acid (UDPGA)

Abstract The pH-rate profiles for the hydrolysis of uridine 5′-diphospho-α- d -glucose (UDPG) and uridine 5′-diphospho-α- d -glucuronic acid (UDPGA) in aqueous solution have been measured. The results obtained and a comparison with other data suggests that the mechanism of hydrolysis of each activated glycosyl-donor at pH 1–4 probably involves the slow ionisation, via an S N 1 process, of the neutral molecule to a glycosyl ion and UDP. From these data, the catalytic power ( k cat / k uncat ) of the glycosyltransferases has been estimated for the first time to be in the order of 10 11–13 .

Mixed ion-pair high-performance liquid chromatography of uridine 5′-diphospho-α-d-glucuronic acid and its hydrolysis products

Abstract Although conventional ion-exchange HPLC and ion-pair reversed phase HPLC using tetrabutylammonium hydroxide allowed ready determination of uridine 5′-diphospho-α- d -glucose and its hydrolysis products, neither method was suitable for the determination of uridine 5′-diphospho-α- d -glucuronic acid (α-UDPGA). However, mixed ion-pair reversed-phase HPLC, using a combination of tetrabutylammonium hydroxide and tetraethylammonium hydroxide (1:1), has been found to offer a convenient and effective means of simultaneously determining α-UDPGA and its hydrolysis products, UDP, UMP and cUMP.

Mechanistic studies of biological glycosylation. Determination of the glucosylating reactivity of uridine-5′-diphospho-α-D-glucose (UDPG) and assessment of the catalytic power of the glycosyltransferases.

Abstract Enzymic rate enhancements of the glycosyltransferases are estimated to be in the order of 10 10 , as revealed by a determination for the first time of the magnitude of the spontaneous glucosylating reactivity (which prevails only below pH 3) of the prototypical ‘activated’ co-substrate of biological glycosylation, UDPG.

Addition of amine nitrogen to an unactivated double bond. The mechanisms of the reverse Hofmann elimination

The tertiary amino-group of amine (7) adds to the transannular double bond with a half-life of 3.3 s at 25°. Reaction is general acid catalysed, and a mechanism is proposed in which nucleophilic addition of amine nitrogen to one end of the double bond is concerted with the transfer of a proton from the general acid to the other. The reaction is observed only with systems in which substantial ground state strain is relieved on cyclisation. It gives the products of antiperiplanar addition, and represents the microscopic reverse of the Hofmann elimination. The reverse Hofmann reaction as normally carried out under acidic conditions is a much slower reaction, much less sensitive to structure, which goes by a quite different mechanism. The cyclisation of amine (8), for example, is specific acid catalysed and probably involves a carbonium ion intermediate.