David J. Robins

Detection, characterisation, and quantification of carnosine and other histidyl derivatives in cardiac and skeletal muscle

Isocratic reverse phase analytical high performance liquid chromatography (HPLC) has been used to examine naturally occurring imidazoles of cardiac and skeletal muscles. Elution of muscle extracts with a phosphate buffer mobile phase from columns packed with hypersil ODS (5 micron) resulted in good separation of the skeletal muscle imidazole-containing dipeptides carnosine and anserine. Measured concentrations corresponded to published values. N-Acetyl forms that were not commercially available were prepared from their parent compounds and their identities verified by NMR-spectroscopy. Examination of frog cardiac muscle confirmed the presence of N-acetylhistidine and also indicated the presence of its 1-methyl derivative. Extracts of mammalian cardiac muscle were examined by HPLC which indicated the presence of low concentrations of carnosine but substantial amounts of N-acetyl forms of histidine, 1-methylhistidine, carnosine and anserine. Fractions corresponding to the numerous peaks were examined using staining systems specific for certain chemical features and compared to results obtained for commercial or synthetic standards. Results of these tests supported the chromatographic data. The total concentrations in cardiac muscle of these imidazole-containing substances (approx. 10 mM) is sufficient to alter significantly the sensitivity of their contractile apparatus to calcium ions.

Analysis of Carnosine, Homocarnosine, and Other Histidyl Derivatives in Rat Brain

Isocratic reverse‐phase analytical HPLC has been used to examine naturally occurring imidazoles of rat brain. Elution of brain extracts with a phosphate buffer mobile phase from columns packed with Hypersil ODS (5 μm) resulted in good separation of the well‐documented brain imidazole‐containing dipeptides carnosine and homocarnosine. Measured concentrations corresponded to published values. Several further peaks observed had properties consistent with those of N‐acetyl derivatives of compounds related to carnosine and homocarnosine. N‐Acetyl forms not commercially available were prepared and their identities verified by nuclear magnetic resonance spectroscopy. A number of these had chromatographic properties identical to those of compounds in brain extracts. Fractions corresponding to some of the peaks were examined using staining systems specific for certain chemical features and compared with results obtained for commercial or synthetic standards. The results of these tests supported the chromatographic data. Thus, chromatographic and microchemical evidence is presented for the existence of N‐acetyl forms of histidine, 1‐methylhistidine, carnosine, anserine, and homocarnosine in rat brain.

Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave Km values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and Ki values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a Ki of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.

SYNTHESIS OF A KEY INTERMEDIATE IN THE DIAMINOPIMELATE PATHWAY TO L-LYSINE: 2,3,4,5-TETRAHYDRODIPICOLINIC ACID

Abstract 2,3,4,5-Tetrahydrodipicolinic acid ( 3 ) is a key intermediate in the diaminopimelate (DAP) pathway to l -lysine ( 7 ). It was synthesized as the stable racemic potassium salt ( 25 ) from dipicolinic acid ( 14 ) by esterification, hydrogenation to the cis -diester ( 17 ), followed by elimination of p -toluenesulfinic acid from the N -toluenesulfonyl derivative ( 24 ) of dimethyl cis -piperidine-2,6-dicarboxylate with concomitant cleavage of the ester groups. (±)-2,3,4,5-Tetrahydrodipicolinic acid is unstable in neutral or acidic solution, and in basic solution it exists in equilibrium with the corresponding enamine ( 27 ) and 2-oxo-6-aminoadipate ( 26 ).

Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.

In vivo pharmacology and anti-tumour evaluation of the tyrphostin tyrosine kinase inhibitor RG13022

Amplification and increased expression of many growth factor receptors, including the epidermal growth factor receptor (EGFR), has been observed in human tumours. One therapeutic strategy for overcoming EGF autocrine control of tumour growth is inhibition of EGFR protein tyrosine kinase (PTK). A series of low molecular weight molecules have been identified which inhibit the EGFR PTK in vitro and demonstrate antiproliferative activity against human cancer cell lines with high expression of EGFR. A significant growth delay in squamous cancer xenografts has been reported for one of these compounds, the tyrphostin RG13022. Based on these encouraging results, we sought to confirm the activity of RG13022 in vivo and relate the effects to the in vivo plasma disposition. RG13022 and three additional peaks were detected by HPLC following intraperitoneal administration of 20 mg kg-1 RG13022 in MF1 nu/nu mice. RG13022 demonstrated rapid biexponential elimination from plasma with a terminal half-life of 50.4 min. RG13022 plasma concentrations were less than 1 microM by 20 min post injection. A primary product was identified as the geometrical isomer (E)-RG13022. Both RG13022 and its geometrical isomer inhibited DNA synthesis in HN5 cells after a 24 h in vitro incubation (IC50 = 11 microM and 38 microM respectively). Neither RG13022 nor its geometrical isomer displayed significant cytotoxicity. RG13022 had no influence on the growth of HN5 tumours when administered chronically, starting either on the day of tumour inoculation or after establishment of tumour xenografts. The rapid in vivo elimination of RG13022 has potential significance to the development of this and other related tyrphostin tyrosine kinase inhibitors, as plasma concentrations fell below that required for in vitro activity by 20 min post injection. The lack of in vivo tumour growth delay suggests that a more optimal administration schedule for RG13022 would include more frequent injections or continuous administration. An improved formulation for RG13022 is therefore required before further development of this or other similar protein tyrosine kinase inhibitors can be made. Alternative strategies should also be sought which display longer lasting in vivo exposures.

Pyridine and piperidine derivatives as inhibitors of dihydrodipicolinic acid synthase, a key enzyme in the diaminopimelate pathway to l-lysine

Abstract A key step in the diaminopimelate (DAP) pathway to l -lysine ( 7 ) involves condensation of pyruvate with aspartic acid β-semialdehyde ( 1 ) to yield l -2,3-dihydrodipicolinic acid ( 2 ) (DHDPA) catalyzed by DHDPA synthase. The best inhibitors of DHDPA synthase of the thirty pyridine and piperidine derivatives prepared were the N -oxide ( 15 ) of dipicolinic acid and the di-imidate ( 13 ) of dimethyl pyridine-2,6-dicarboxylate each with an IC 50 value of 0.2 mM. The N -oxide ( 15 ) and dinitrile ( 12 ) are non-competitive inhibitors with K i values of 0.29 and 1.25 mM against aspartate semialdehyde and 0.06 and 0.34 mM against pyruvate, respectively.

Biosynthesis of bisdethiobis(methylthio)gliotoxin, a new metabolite of Gliocladium deliquescens

Bisdethiobis(methylthio)gliotoxin (2) has been identified as an amorphous, minor metabolite of Gliocladium deliquescens and characterised via the crystalline bis-4-bromobenzoate of the corresponding didehydro-derivative. The new metabolite has been synthesised from gliotoxin (1) by reduction and methylation. Feeding experiments with [14C]-(1) and -(2), prepared biosynthetically from L-[U-14C]phenylalanine, have shown that bisdethiobis(methylthio)gliotoxin is formed (8.6% incorporation), apparently irreversibly, from gliotoxin in G. deliquescens.

Incorporation of chirally deuterated putrescines into pyrrolizidine alkaloids: A reinvestigation

Abstract Based on previous tracer work and recent enzymatic studies it can be predicted that incorporation of (S)-1-2H]putrescine via the symmetrical intermediate homospermidine into the necine base moiety of pyrrolizidine alkaloids (PAs) should proceed with 50% retention of deuterium. However, values of only 34 to 34.5% retention had been found independently in two laboratories in the past. These results were confirmed in this study. Deuterium isotope effects during homospermidine formation as a reason for the low retention could be excluded by GC mass spectral studies. Doubly-labelled [2H-14C]putrescine was fed to Senecio vulgaris root cultures and by means of quantitative GC mass spectrometry the specific 2H-retention was established for various intermediates of PA-biosynthesis such as putrescine, spermidine and homospermidine. The results clearly indicate that 2H is stereoselectively lost from (S)-[1-2H]-labelled putrescine during its reversible inter-conversion with spermidine. This loss corresponds precisely to the above mentioned difference between measured and predicted 2H-retention. Since (S)-[1-2H]-labelled putrescine is incorporated into spermidine with deuterium retention, it is most likely the 2H is lost during the conversion of spermidine into putrescine. The mechanism of this unusual reaction which is insensitive to β-hydroxyethylhydrazine (a potent diamine oxidase inhibitor) needs to be elucidated.

New co-metabolites of gliotoxin in Gliocladium virens

Chromatographic separation of extracts of Gliocladium virens, grown on a medium containing [35S]sulphate, has led to the identification of 5 co-metabolites of gliotoxin (1a), new to this fungus, viz. the epitrisulphide, glioto din E (1b), the 3-hydroxymethylbut-2-enyl ether (3c), didehydrogliotoxin (5), bas-N-norgliovictin (6), and the 3-methylbut-2-enyl ether of cyclo-(glycyl-L-tyrosyl)(7); the metabolites (3c), (6), and (7) are new natural products. The synthesis of the epitri- and epitetrasulphides, gliotoxin E (1b) and gliotoxin G (1c), from gliotoxin (1a) is described, as is that of the 3-methylbut-2-enyl ether (7) from cyclo-(glycyl-L-tyrosyl). The biosynthetic significance of the dioxopiperazines (6) and (7) is briefly discussed.