Methyl Ester Moiety Research Articles

Synthesis, in vitro anti-breast cancer activity, and intracellular decomposition of amino acid methyl ester and alkyl amide phosphoramidate monoesters of 3'-azido-3'-deoxythymidine (AZT).

We report the synthesis and anticancer activity of a series of AZT phosphoramidate monoesters containing amino acid methyl ester (3a-11a) and N-alkyl amide (3b-11b, 9c-9f) moieties. The aromatic amino acid methyl esters were found to be more cytotoxic than the aliphatic analogues toward MCF-7 cells (human pleural effusion breast adenocarcinoma cell line). A marked stereochemical preference for the L-amino acid stereochemistry was also observed in MCF-7 cells. There was no consistent enhancement of cytotoxicity of the methyl amides over the corresponding methyl esters. AZT and the two AZT aromatic amino acid methyl ester phosphoramidates 8a and 9a were found to be more cytotoxic toward MCF-7 cells than to CEM cells (human T-cell lymphoblastic leukemia). The selective cytotoxicity toward MCF-7 cells may be associated with greater intracellular levels of phosphoramidate monoester and/or phosphorylated AZT.

A conserved threonine in the second extracellular loop of the human EP 2 and EP 4 receptors is required for ligand binding

G protein coupled receptors for prostaglandins are activated when agonists are bound to a binding pocket formed in part by the seven transmembrane domains. Recent studies have determined that substitution of a conserved threonine in the second extracellular loop of the prostaglandin EP 3 receptor resulted in increased affinity for ligands with a C1 methyl ester moiety. The homologous threonine in the second extracellular loop of the human prostaglandin EP 2 and EP 4 receptors was mutated to alanine. When expressed in COS1 cells, detectable radioligand binding at both of these receptors bearing the threonine to alanine substitution (EP 2T185A; EP 4T168A) was abolished, as well as the receptors' ability to stimulate intracellular [cAMP]. In contrast, EP 2 and EP 4 receptors bearing conservative threonine to serine mutations (EP 2T185S; EP 4T168S) displayed K d values for [ 3 H ]prostaglandin E 2 similar to wild type receptors: 8.8±0.7 nM for EP 2T185S compared to 12.9±1.2 nM for EP 2 wild type; 2.0±0.8 nM for EP 4T168S compared to 0.9±0.3 nM for the EP 4 wild type receptor. The EC 50 values for cAMP stimulation were 1.3±0.6 nM for EP 2 wild type; 2.7±1.3 nM for EP 2T185S; 1.1±0.3 nM for EP 4 wild type; and 1.4±0.33 nM for EP 4T168S. These studies suggest a critical role for the hydroxyl moiety on these conserved threonine residues at position 168/185 of the second extracellular loop in prostaglandin receptor–ligand interactions.

Synthesis of new phosphonamidate and phosphinamide desmuramyldipeptide analogs

New desmuramyldipeptide analogs with modified peptide backbones were synthesized. The peptide bond between Ala and Glu was replaced by a phosphonamidate methyl ester moiety. The amide bond at the end of the acyclic side chain was replaced by a phosphinamide moiety.

Reaction of „Jordan′s Cation”︁ [Cp2ZrCH3(thf)]+ with Amino Acid‐Derived Isocyanates—A First Synthetic Route to Oligopeptide‐Modified Group 4 Bent Metallocene Cation Systems

AbstractThe organometallic cation [Cp2ZrCH3(thf)]+, employed as the tetraphenylborate salt (1), reacts cleanly in 1:1 stoichiometry with the isocyanates 2 derived from valine methyl ester or valylvaline methyl ester, respectively. In each case addition of the Zr–CH3 group to the isocyanate sp‐carbon center is observed with formation of a functionalized zirconocene cation derivative containing a chelating N‐metallated N‐acetylvaline methyl ester (3a) or N‐acetylvalylvaline methyl ester (3b) moiety, respectively, coordinated in the bent metallocene s̀‐ligand plane. The spectroscopic data of 3, supported by an X‐ray crystal structure analysis of the zirconated dipeptide derivative 3b, have revealed the presence of chelating (η1‐O:η1‐N)‐coordination of the terminal N‐acetyl groups in addition to a Zr–O=C interaction with the adjacent valyl amido group.

Synthesis of Thio‐Linked Analogues of Lewis X and Sialyl Lewis X

AbstractThe synthesis of thio‐linked Lewis X and sialyl Lewis X‐derived epitopes 3–5 has been achieved using a small number of building blocks. The key building‐block was 1‐O‐silyl‐protected 4‐S‐acetyl‐2,6‐di‐O‐benzoyl‐3‐S‐(2,3,4‐tri‐O‐acetyl‐α‐L‐fucopyranosyl)‐3,4‐dithio‐β‐D‐glucopyranose (15), which was obtained from the fucosyl donor 6 together with 3‐thiogalactose 7 as the acceptor. Their acid‐catalyzed S‐glycosylation afforded the thio‐linked disaccharide 8 which was subsequently converted to the 4a‐O‐unprotected derivative 12. Conversion to the 4a‐triflate followed by treatment with KSAc in tetrahydrofuran led, under inversion of configuration, to 15 in good overall yield. Selective removal of the S‐acetyl group followed by base‐promoted S‐glycosylation with acetobromogalactose gave the acyl‐protected Lewis X analogue 25. Acetobromogalactose gave the acyl‐protected Lewis X analogue 25. Transformation into trichloroacetimidate 27, followed by acid‐catalyzed S‐glycosylation of heptylthiol and complete deacylation afforded target molecule 3. Similarly, acid‐catalyzed reaction of donor 27 and the 3b,4b‐O‐unprotected lactose derivative 31 as acceptor led to pentasaccharide 32, complete deacylation of which afforded target molecule 4. Transformation of 15 into the donor trichloroacetimidate 34, followed by acid‐catalyzed S‐glycosylation of heptylthiol afforded thioglycoside 35. Selective removal of the S‐acetyl group and subsequent base‐promoted S‐glycosylation with the known donor 37 furnished the thio‐linked tetrasaccharide 38. Cleavage of all the O‐acyl groups and hydrolysis of the methyl ester moiety afforded the sialyl Lewis X analogue 5.

Lipoconjugates: structure-activity studies for pheromone analogues of Ustilago maydis with varied lipophilicity.

The synthesis, biological activities and conformational behaviour of a variety of analogues of the mating pheromones of the basidomycete Ustilago maydis are reported. The pheromone analogues derived from the two allelic forms H-G-R-D-N-G-S-P-I-G-Y-S-S-Xaa-Z (a1) and H-N-R-G-Q-P-G-Y-Y-Xaa-Z (a2), with Xaa-Z being an unidentified lipophilic cysteine derivative, all differ in the C-terminal residue and include -Cys(farnesyl)-OMe, -Cys(farnesyl)-OH, -Cys(prenyl)-OMe, -Cys-OMe, -Cys(n-dodecyl)-OMe and the unnatural residues -Ahds-OMe (Ahds=alpha-aminohexadecanoic acid), -Ahds-OH, -Ads-OMe (Ads=alpha-aminodecanoic acid) and -N-Hdg-OMe (N-Hdg=N-hexadecylglycine). The synthesis of the unnatural methyl ester analogues was carried out by condensation of the fully protected fragments Fmoc-G-R(Pmc)-D(tBu)-N(Trt)-G-S(tBu)-P-I-G-Y(tBu)-S(tBu)-S(tBu)-OH (a1') and Fmoc-N(Trt)-R(Pmc)-G-Q(Trt)-P-G-Y(tBu)-Y(tBu)-OH (a2') respectively, prepared by Fmoc-SPPS, with the appropriate methylester compounds and subsequent deprotection with TFA/scavenger and piperidine. Synthesis and physicochemical properties of the unnatural lipophilic amino acid methylesters are described. The preparation of the cysteine analogues was performed by condensation of a1' or a2' with H-Cys(Trt)-OMe and subsequent deprotection with TFA/scavenger. Alkylation of the thiol function and Fmoc-deprotection was achieved in a novel one-pot reaction by treatment with alkyl bromide and DIPEA, quenching with EDT and Fmoc removal by addition of 20% piperidine (v/v). Hydrolysis of the methyl esters was carried out by treatment with NaOH in MeOH/H2O. The results of the biological assay reveal an increase in activity with increasing chain length of the lipophilic anchor, with alkyl being better than prenyl and sulfur being not essential, while the position of the anchor is optimal at C7 and the methyl ester moiety is important. NMR studies of two chosen analogues in DMSO and SDS/water demonstrate that the lipophilic C-terminal residue has no influence on the structural behaviour of the peptides. Chemical-shift and NOE patterns indicate a main all-trans conformation of the peptide backbone and a weakly populated cis conformation around the Xaa Pro peptide bond in all eight cases without formation of a defined folded structure. No evidence is seen that the membrane-simulating system SDS/water has a structure-inducing effect on the bound peptide. We therefore conclude that the lipomodification in mating pheromones of U. maydis acts to increase the effective concentration of the drug in the target cell membrane without additional structure-inducing or receptor-binding effects.

Stable Carbon Isotope Measurement of Individual Fatty Acids Using Gas Chromatography/Isotope Ratio Monitoring Mass Spectrometry.

A gas chromatography/isotope ratio monitoring mass spectrometry (GC/IRMS) method was established for the determination of stable carbon isotopic compositions of individual saturated n-fatty acids ranging from C16 to C30. The fatty acids were derivatized to their methyl esters using BF3/methanol, and three n-alkanes were used as internal isotopic standard. Isotopic mass balance calculation indicated that the esterification caused no isotopic fractionation between the methyl ester moiety of fatty acid and methanol used. Therefore, isotopic correction was made by one additional carbon atom with the δ13C value of methanol. Analytical accuracy (deviation of the corrected δ13C values determined by GC/IRMS from those determined by a conventional method) and precision (standard deviation) were satisfactory for 90 to 360 ng of individual standard fatty acids examined. The accuracy was ±0.3‰ for average of several analyses, and the precision within 0.3‰. This method was applied to the δ13C determination of n-fatty acids ranging from C20 to C30 in a marine sediment.

Enantioselective hydrolysis of cyclopentaneacetic acid esters related to methyl jasmonate

The hydrolysis of methyl jasmonate with Aspergillus niger and A. carbonarius is shown to be very dependent upon the particular structure of the substrate. Some variation of the side chain at C-2 is tolerated, whereas the 3-keto group and the methyl ester moieties are particularly important. Porcine pancreatic lipase, Candida lipase, and Rhizopus lipase are unreactive towards butyl jasmonate.

Steric and electronic requirements for muscarinic receptor-stimulated phosphoinositide turnover in the CNS in a series of arecoline bioisosteres

A series of arecoline derivatives was utilized to assess steric and electronic effects important for activating muscarinic receptors in the CNS. Arecoline derivatives in which the methyl ester moiety was replaced by hexyloxy-1,2,5-oxadiazole ( 2b), hexyloxythiophene ( 3b) or hexyloxypyrazine ( 4b) were compared with the hexyloxy-1,2,5-thiadiazole compound ( 1b) (Hexyloxy-TZTP), known from previous work to be active as an M 1 M 3 partial agonist. MNDO calculations showed that the N-S bonds of the alkoxythiadiazole ring were highly polarized with the ability to form H-bonds to the N's. On the other hand, the smaller oxadiazole had lower polarities in the N-O bonds and reduced ability to form H-bonds, the thiophene was of comparable size to the thiadiazole and had large C-S bond polarities without the H-bond capability and the pyrazine had limited ability to form H-bonds. The compounds were compared with respect to their abilities to stimulate phosphoinositide (PI) turnover in the hippocampus of the rat brain. 1b was more active than 2b–4b for stimulating the PI turnover response. The data suggest that the ability to form H-bonds is an important factor for the ability of 1 to stimulate M 1 muscarinic receptors in the CNS.

Modified polysulphone membranes: 1. Pervaporation of water/alcohol mixtures through modified polysulphone membranes having methyl ester moiety

Membranes were prepared from modified Udel P-3500 polysulphone having methyl carboxylate groups, of which the degree of substitution ranged from 0.05 to 1.90 ester groups per repeat unit. The membranes showed permselectivity towards water in the pervaporation of aqueous alcohol solutions.

Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione.

S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)

Sources of propionate for the biogenesis of ethyl-branched insect juvenile hormones: Role of isoleucine and valine

Corpora allata from adult female Manduca sexta biosynthesize the sesquiterpenoid juvenile hormone (JH) III and the unusual ethyl-branched homologue JH II in vitro. We maintained corpora allata in medium 199 using [methyl-(3)H]methionine as the source of the JH methyl ester moiety and as a mass marker. This allowed measurement of the relative contributions of (14)C-labeled precursors to the biogenesis of JH II and III carbon skeletons. We showed efficient incorporation of a propionate equivalent, from isoleucine or valine catabolism, into the ethyl-branched portion of JH II, using double-label liquid scintillation counting of isolated JHs and gas chromatography/mass spectrometry with selected ion monitoring of JH deuteromethoxyhydrin derivatives. Methionine was a poor source of propionate for JH II biosynthesis, while glucose, succinate, threonine, and beta-alanine did not contribute propionate at all. Leucine, isoleucine, and glucose incorporated into JH III and the acetate-derived portion of JH II.

Crystal and molecular structure of aspartame X HCl X 2H2O.

The crystal and molecular structure of the hydrochloride salt of the peptide sweetener aspartame (alpha-L-Asp-L-Phe methyl ester) has been determined at 120 K using 3877 reflections with I greater than 2.5 sigma I. Space group P2(1)2(1)2(1), cell dimensions a = 6.768(1), b = 9.796(1) and c = 26.520(3) A; final R factor 0.033. While the N-terminal L-Asp group in the structure of aspartame itself forms a six-membered ring with an intramolecular hydrogen bond between the carboxylate and the protonated amino terminus, the corresponding group in the hydrochloride adopts a completely different conformation with a weak intramolecular hydrogen bond between the carboxyl group and the N atom of the L-Phe residue. The L-Phe methyl ester moiety is rather similar in the two structures. Of the many possible conformations of aspartame, only one may be expected to function as a substrate at the receptor site for sweet taste, and a proposal is made for this active conformation.

Labelling of chlorophylls and precursors by [2-14C]glycine and 2-[1-14C]oxoglutarate in Rhodopseudomonas spheroides and Zea mays. Resolution of the C5 and Shemin pathways of 5-aminolaevulinate biosynthesis by thin-layer radiochromatography.

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway. Both [2-14C]glycine and, to a lesser extent 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, [2-14C]glycine formed 5-[5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochlorophyll a labelling by 2-[1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by [2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-[1-14C]oxoglutarate or [2-14C]glycine as substrates. Only the C5 pathway was detected: 2-[1-14C]oxoglutarate was converted to 5-[5-14C]aminolaevulinate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies [Porra, R.J., Klein, O. and Wright, P. E. (1983) Eur. J. Biochem. 130, 509-516] showing that the C5 pathway formed all the 5-aminolaevulinate for chlorophyll biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Juvenile hormone III biosynthesis : Stoichiometric incorporation of [2- 14C]acetate and effects of exogenous farnesol and farnesoic acid

De novo biosynthesis of the sesquiterpenoid juvenile hormone III (JH- III, C 16JH, methyl-10,11-epoxy-3,7,11-trimethyl-2E,6E- dodecadienoate ) by isolated corpora allata of the adult female cockroach Diploptera punctata has been studied in vitro using a radiochemical assay. Labelling from l-[ methyl- 3H]methionine in the methyl ester moiety was used to quantify biosynthesized JH- III. When corpora allata were incubated in a medium lacking acetate but containing [1- 14C]glucose, carbon from glucose labelled up to 30% of the sesquiterpenoid carbon chain. When the glands were incubated in a medium lacking glucose but containing [2- 14C]acetate, dose-dependent utilization of labelled acetate was observed for the biosynthesis of juvenile hormone and its immediate precursor methyl farnesoate, reaching a plateau at stoichiometry, i.e. when 9 mol of acetate were incorporated for each mole of JH- III synthesized. This is a unique case of incorporation of exogenous, labelled acetate into an isoprenoid without significant dilution. The stoichiometric incorporation of exogenous [2- 14C]acetate was utilized to measure the influence of farnesol and farnesoic acid (two sesquiterpenoid precursors of JH- III) on the carbon flow through this biosynthetic pathway. Both compounds stimulated JH- III biosynthesis and inhibited [2- 14C]acetate incorporation into juvenile hormone and methyl farnesoate in a dose-dependent fashion, resulting in a nearly total suppression of de novo JH- III biosynthesis.

Farnesoic acid stimulation of C 16 juvenile hormone biosynthesis by corpora allata of adult female Diploptera punctata

Biosynthesis of C 16 juvenile hormone (C 16JH) by isolated corpora allata (CA) of the viviparous cockroach Diploptera punctata has been studied by a radiochemical assay. This assay uses the incorporation from methyl labelled l-methionine in the methyl ester moiety of C 16JH. The optimal concentration of l-methionine in the medium has been determined. When varying proportions of (methyl- 3H)- and (methyl- 14C)-labelled l-methionine are used in the assay, no isotope effect was observed indicating that labelled l-methionine can be used as a mass marker to quantify C 16JH synthesis in D. punctata. C 16JH synthesis was stimulated by addition of farnesoic acid (FA) to the medium (maximum at 30–40 μM). When stimulated with 30 μM FA, the C 16JH release rate was directly dependent on C 16JH synthesis rate, and no intraglandular accumulation of either C 16JH or its immediate precursor methyl farnesoate were observed. Spontaneous and FA-stimulated rates of C 16JH release were studied during the first reproductive cycle of the adult female. The two final steps of C 16JH biosynthesis are not physiologically rate-limiting, as the ratio of spontaneous to FA-stimulated C 16JH release (fractional endocrine activity ratio) is always lower than unity. There is a precise relationship between the length of the basal oöcytes and the rate of C 16JH release.

Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester.

Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.