Succinyl Derivatives Research Articles

Anti-AIDS agents 38. Anti-HIV activity of 3-O-acyl ursolic acid derivatives.

Based on our previous finding that 3-O-acyl-betulinic and -oleanolic acids, especially the 3-O-(3',3'-dimethyl)-succinyl derivatives (2 and 4), demonstrated potent anti-HIV activity [EC(50) < 0.00035 and 0.00086 microM; therapeutic index (TI) > 20 000 and 22 326, respectively], several 3-O-acyl-ursolic acids were prepared and evaluated for anti-HIV activity. Ursolic acid (6) was equipotent (EC(50) 4.4 microM) with oleanolic acid (EC(50) 3.7 microM), although it was slightly toxic (IC(50) 14.3 microM, TI 3.3). 3-O-Diglycoryl-ursolic acid (10) demonstrated relatively potent anti-HIV activity with an EC(50) of 0. 31 microM and a TI of 155.5. In contrast, 3-O-(3', 3'-dimethylsuccinyl)-ursolic acid (8), which is analogous to the extremely potent anti-HIV betulinic acid and oleanolic acid derivatives 2 and 4, displayed only weak anti-HIV activity (EC(50) 2.1 microM, TI 23.6).

Pine needle abortion in cattle: analysis of isocupressic acid in North American gymnosperms

Isocupressic acid is a labdane resin acid found in Pinus ponderosa needles and causes abortion in late term pregnant cattle. A chemical survey was conducted to determine the phytochemical distribution of isocupressic acid in selected North American shrub and tree species with possible association with cattle abortions. A method of analysis for the evaluation of total available isocupressic acid (including hydrolysed acetyl and succinyl derivatives) was developed based on a base/acid extraction of dry, ground plant material, followed by detection and quantitative determination using capillary gas chromatography of the di-(trimethylsilyl)-derivative. Samples from 23 different species were collected throughout the western and southern regions of the United States and analysed. Isocupressic acid was detected in 15 species, and significant concentrations (>0.5% dry weight) were found in Pinus ponderosa, P. jeffreyi, P. contorta, Juniperus scopulorum, and J. communis. Copyright © 1999 John Wiley & Sons, Ltd.

Pine needle abortion in cattle: analysis of isocupressic acid in North American gymnosperms

Isocupressic acid is a labdane resin acid found in Pinus ponderosa needles and causes abortion in late term pregnant cattle. A chemical survey was conducted to determine the phytochemical distribution of isocupressic acid in selected North American shrub and tree species with possible association with cattle abortions. A method of analysis for the evaluation of total available isocupressic acid (including hydrolysed acetyl and succinyl derivatives) was developed based on a base/acid extraction of dry, ground plant material, followed by detection and quantitative determination using capillary gas chromatography of the di-(trimethylsilyl)-derivative. Samples from 23 different species were collected throughout the western and southern regions of the United States and analysed. Isocupressic acid was detected in 15 species, and significant concentrations (>0.5% dry weight) were found in Pinus ponderosa, P. jeffreyi, P. contorta, Juniperus scopulorum, and J. communis. Copyright © 1999 John Wiley & Sons, Ltd.

Coupling of the antiviral agent zidovudine to polyaspartamide and in vitro drug release studies

A macromolecular prodrug of the known antiretroviral agent zidovudine and α,β-poly( N-2-hydroxyethyl)- dl-aspartamide (PHEA) was synthesized. A succinic spacer was present between the polymer and the drug, and 1,1′-carbonyldiimidazole was used as the coupling agent. In vitro drug release studies at pH 1.1, 5.5 and 7.4 indicated that limited amounts of intact drug were released from the conjugate. At pH 1.1 and 7.4 succinylzidovudine was released, and this was hydrolysed to give free zidovudine. In the presence of α-chymotrypsin, zidovudine was released preferentially in comparison with the succinyl derivative. The amounts of released zidovudine and succinylzidovudine were greater in plasma than in aqueous buffer solutions. These results show that after i.v. administration this drug-polymer conjugate can release zidovudine into the blood circulation for prolonged periods.

Two Isomeric α and β Aspartyl Dodecapeptides and Their Cyclic Amino Succinyl Analogue From the Australian Green Tree Frog Litoria gilleni

Three related peptides, caeridins 1.1-1.3, have been isolated from the green tree frog Litoria gilleni. Caeridins 1.1 and 1.2 are dodecapeptides differing only in having α and β Asp at residue 4 [viz. Gly Leu Leu Asp Gly Leu Leu Gly Thr Leu Gly Leu (NH2)]. Caeridin 1.3 is the corresponding cyclic amino succinyl derivative derived formally by cyclization of Asp(4) and Gly (5). Hydrolysis of caeridin 1.3 yields caeridin 1.1 and 1.2 in the ratio 3:1. This constitutes a rare case of the isolation of three such related peptides from a natural system.

Structure-Function Relationships in Human Interleukin-11: IDENTIFICATION OF REGIONS INVOLVED IN ACTIVITY BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

Chemical modification approaches combined with site-directed and deletion mutagenesis have been used to identify functionally critical regions/residues of recombinant human IL-11 (rhIL-11). Incubation of rhIL-11 with iodoacetic acid results in specific alkylation of a single methionine residue, Met58, and a 25-fold reduction of in vitro biological activity on mouse plasmacytoma cells. A similar decrease in activity is observed when Met58 is substituted with Ala, Leu, Gln, Glu, or Lys by site-directed mutagenesis. Treatment of rhIL-11 with succinic anhydride leads to modification of the amino-terminal amino group and partial labeling of 2 lysines, Lys41 and Lys98, and to a 3-fold decrease in activity. The activity losses can be attributed to modification of the lysine residues, since the succinyl derivative of the amino terminus is fully active. In addition, carboxyl-terminal deletion mutagenesis studies have demonstrated that removal of the last 4 residues reduces rhIL-11 activity 25-fold, whereas removal of 8 or more amino acids results in an inactive molecule. Based on secondary structure predictions and the location of exon/intron boundaries in the IL-11 genomic structure, we propose a four-helix bundle topology as a structural model for rhIL-11. This model has been tested by limited proteolysis using three side chain-specific endoproteinases. A limited number of protease-sensitive cleavage sites are present in rhIL-11, and all but two are located in the postulated helix interconnecting loops or at helix termini. alpha-Helices, which in the proposed structure form a compact core of the molecule, are inaccessible to digestion under limiting conditions. According to the model, Met58, Lys41 and Lys98 are located on the surface of the molecule, in agreement with their preferential accessibility to chemical modifications. By analogy with human growth hormone, we postulate that Met58 and the carboxyl terminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys41 and Lys98 may be a part of binding site II.

Differences in the binding affinities of dimeric concanavalin A (including acetyl and succinyl derivatives) and tetrameric concanavalin A with large oligomannose-type glycopeptides

Dimeric derivatives of concanavalin A (Con A) such as acetyl- and succinyl-Con A have been used for years as probes of cellular membranes. The altered binding and biological activities of these derivatives relative to native tetrameric Con A have generally been attributed to their reduced valence. However, the present study shows that acetyl- and succinyl-Con A possess lower affinities than tetrameric Con A toward certain oligomannose-type glycopeptides which are found on the surface of cells. It has previously been shown that native tetrameric Con A possesses 5-30-fold enhanced affinities toward Man7-Man9 oligomannose-type glycopeptides, respectively, relative to Man5 and Man6 oligomannose-type glycopeptides [Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726]. Using titration microcalorimetry and hemagglutination inhibition measurements, methyl alpha-D-mannopyranoside, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (which binds with about 60-fold higher affinity than methyl alpha-D-mannopyranoside and is the major Con A binding epitope on oligomannose-type carbohydrates), and a Man5 oligomannose-type oligosaccharide are shown to bind to underivatized dimeric Con A at pH 5.2 and acetyl- and succinyl-Con A at pH 7.2 with affinities equal to those of native tetrameric Con A. However, a mixture of Man7 and Man8 glycopeptides and a Man9 oligomannose-type glycopeptide were shown to bind to underivatized dimeric Con A and acetyl- and succinyl-Con A with affinities only about 2-fold higher than the Man5 oligosaccharide, in contrast to the higher affinities of native tetrameric Con A for these carbohydrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational studies of anionic melittin analogues: effect of peptide concentration, pH, ionic strength, and temperature--models for protein folding and halophilic proteins.

Melittin (MLT), a 26-residue cationic (net charge +5 at pH 7.2) peptide from bee venom, is well known to be a monomeric, approximately random coil; but when its charges are reduced by titration, by acetylation (net charge +2) or succinylation (net charge -2), or by screening by salt, it goes over to tetrameric alpha-helix. The conversion is promoted by raising the peptide concentration. The tetramer is held together by hydrophobic forces. We have changed the net charge to -6 by acylation with acetylcitric anhydride (a new acylating agent); this anionic derivative forms tetrameric helix at neutral pH, without salt, and at relatively low concentration, conditions under which the cationic MLT does not become helical. Thus, a high net charge is not sufficient to prevent association and helix formation. We have synthesized an anionic melittin analogue of MLT (E-MLT; net charge -4) in which all five lysine and arginine residues are replaced with glutamate, and acetyl and succinyl derivatives of E-MLT (net charges -5 and -6). All three of these are resistant to helix formation. They require much higher NaCl or NaClO4 concentration for helix formation than does MLT. Even CaCl2, MgCl2, and spermine.4HCl are less effective in helicizing E-MLT than MLT. MLT, at pH 7.2, shows increasing helix as the peptide concentration increases (8-120 microM), but E-MLT and its acyl derivatives do not. MLT and acylated MLTs in the helical tetramer show both cold- and heat-induced unfolding, with maximum stability near room temperature. At high temperature, a significant amount of residual structure remains. Heating (to 100 degrees C) monomeric MLT (i.e., MLT at low concentration) or E-MLT results in a monotonic increase in negative ellipticity. In 1.0 M NaCl, E-MLT (at sufficiently high concentration) also shows cold and hot unfolding. The results are discussed in respect to charge-charge and charge-dipole interactions, and hydrophobic effects. E-MLT is also discussed in relation to proteins of halophilic bacteria, which have higher proportions of anionic residues than do corresponding proteins of nonhalophiles.

Antiidiotypic antibodies against anti-cGMP polyclonal antibodies

Affinity-purified polyclonal anti-cGMP antibodies were obtained from rabbit serum after immunization by succinyl derivative of cGMP coupled to bovine serum albumin. These antibodies were used to raise antiidiotypic antibodies in rats. Putative antiidiotypic serum inhibited the binding of [ 3H]cGMP to affinity-purified anti-cGMP antibodies. The influence of immunoglobulins isolated from antidiotypic serum on the ion conductance of rod outer segment plasma membrane fragments from frog retina was studied in patch-clamp experiments. These immunoglobulins increased the conductance of ion channels acting like a natural agonist (cGMP). Preimmune immunoglobulins did not act. The data obtained suggest that antiidiotypic antibodies interact with regulatory cGMP-binding sites of the plasma membrane channels.

Capillary electrophoresis analysis of concanavalin A and its succinyl derivative

A high-performance capillary electrophoresis (CE) system has been developed for concanavalin A (Con A) and its succinyl derivative (SCA). Under the CE system, the tetramer, trimer, dimer, monomer and protein fragments of CA were separated in less than 50 min. SCA was resolved into more than 10 components which were believed to be isomeric succinyl derivatives of Con A. The CE system is a simple and sensitive analytical technique to profile the composition of Con A and SCA preparations. The minimum concentration of Con A in water detectable by the CE system is 1 microgram ml-1.

Resin acids of Pinus ponderosa needles

Resin acids of ponderosa pine needle oleoresins are characterized by labdane diterpenes. Imbricataloic and/or acetyl- and succinyl-isocupressic acids predominate. Succinyl derivatives of neutral diterpenes were also found.

Modification of sturgeon protamines with succinic anhydride

The modification of the α- and e-NH2 groups of sturins A and B — protamines fromAcipenser guldenstadti — with succinic anhydride has been studied. Succinylation was carried out at 20°C, pH from 7.5 to 10.5, using a 50 to 180-fold excess of succinic anhydride. The modification products were separated by reversed-phase HPLC on a Zorbax ODS column. A linear relationship has been established between the retention times of the succinyl derivatives and the number of succinyl groups introduced. The influence of the pH and of the excess of succinic anhydride on the modification of the sturins has been studied by the method of experimental planning and mathematical modeling. The optimum conditions have been found for the succinylation of the proteins: a 150- to 180-fold excess of succinic anhydride and pH values of 9.8–10.2 for sturin A and of 8.4–9.4 for sturin B.

Synthesis of 5′ -0-Succinyl-2′, 3′-Cyclic Adenosine Monophosphate

Abstract 5′-0-Succinyl-2′, 3′-cyclic adenosine monophosphate was synthesized in preparation for 2′,3′-cyclic nucleotide antibody production. Succinylation was performed with [14C]labeled and unlabeled succinic anhydride and morpholinodicyclohexylcarbodiimide. The products, resolved on QAE Sephadex, were purified and characterized by comparison with authentic 3′,5′-cyclic AMP succinyl derivatives. Yield: 73% monosuccinyl-(5′ -0)-2′,3′ -cyclic AMP; 10% disuccinyl-(N, 5′-0)-2′,3′-cyclic AMP. The phosphodiester ring in the monosuccinyl derivative was shown to be intact by its susceptibility to alkaline phosphatase only after exposure to 2′,3′- cyclic nucleotide 3′-phosphodiesterase.

Modified taxols, 4. Synthesis and biological activity of taxols modified in the side chain.

A number of taxol derivatives substituted at the 2' position of the side chain have been prepared and their biological activities determined in KB cell culture and/or the P-388 in vivo assay. The 2'-(t-butyldimethylsilyl)taxol 5 is essentially inactive, indicating the need for a free hydroxyl group at the 2' position for activity. Epimerization of the 2' position occurred on treatment of 2'-acetyltaxol derivatives with 1,5-diazabicyclo[5.4.0]undec-7-ene, but treatment of 2'-(2,2,2-trichloroethyloxycarbonyl) taxol derivatives with DBU yielded the novel cyclization products 11 and 12 and, after deprotection at the 7 position, 13. The derivative 13 is also essentially inactive in the KB test system. Two taxols with increased H2O solubility were prepared, the 2'-(beta-alanyl) derivative 15 and the 2'-succinyl derivative 16. Although both these derivatives were active in vivo and in vitro, the former was too unstable and the latter not active enough to make suitable H2O-soluble derivatives of taxol.

Occurrence of succinyl derivatives in the catabolism of arginine in Pseudomonas cepacia.

Pseudomonas cepacia NCTC 10743 utilizes arginine as the sole source of carbon and nitrogen for growth. Arginine is degraded to glutamate via succinyl derivatives. The catabolic sequence in this pathway is L-arginine----N2-succinylarginine----N2-succinylornithine--- -N2-succinylglutamate semialdehyde----N2-succinylglutamate----glutamate + succinate. The formation of the enzymes responsible for arginine degradation is regulated not only by induction but also by both carbon and nitrogen catabolite repression.

Succinyl derivatives of N‐tris (hydroxymethyl) methyl‐2‐aminoethane sulphonic acid: their effects on the frog neuromuscular junction

Succinic anhydride (SA) dissolved in Ringer solution buffered with N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (SA-TES solution) potentiates the depolarizing action of acetylcholine (ACh, 10-40 microM) on frog muscle and the tension induced by bath application of this agonist. Applied from one side of a double-barrelled micropipette, SA-TES increases the amplitude of iontophoretically elicited ACh potentials. The potentiation of the effects of ACh by SA-TES does not involve changes in either the activity of the ACh esterase or the input resistance of the muscle membrane. For depolarizations of frog sartorius muscle, dose-response relationships obtained for ACh concentrations from 0.5 to 20 microM indicate that SA-TES increases the apparent affinity of ACh by a factor of 3. SA-TES exerts an "accelerating' effect on the responses elicited by bath-applied ACh; i.e., it increases the rate of depolarization when ACh is added to the bath and the rate of repolarization upon washing out. These effects are particularly marked in preparations treated with neostigmine (3 microM). SA-TES does not potentiate the depolarizing action of agonists which do not contain an ester group. Moreover, the time course of the responses elicited by these compounds is not influenced by SA-TES. SA-TES fails to influence significantly the effects of the neurally released transmitter. Only a 10% increase in the average amplitude of the endplate potentials was observed. SA hydrolyzes in about 30 min at room temperature; however the SA-TES solution retains its activity for several weeks. Succinate is inactive, and so is SA in Ringer buffered with phosphate. The SA-TES solution contains seven succinyl-TES derivatives, which were separated by ion-exchange chromatography and paper chromatography. At concentrations between 1 to 150 microM, these succinyl-TES derivatives affected the ACh-induced contraction of frog rectus abdominus muscle. The most abundant derivative potentiated the action of high doses of ACh, but was inhibitory at lower ones. The other derivatives were mostly inhibitory. These results are discussed in terms of two hypotheses. One postulates the presence of a diffusion barrier formed by groups that bind ACh and are saturated by SA-TES. The other assumes that SA-TES acts directly on the ACh receptor exerting its potentiating effect through a cooperative mechanism.

Activation of luteinizing hormone release from pituitary cells by polycations.

In the present work we examined the effect of cationic polymers on the pituitary gonadotrope. Such polymers are widely used to anchor gonadotropes and other cell types to culture dishes and other substrata to which they are not normally adherent. Homopolymers of Lys (eight size classes from 4,000-700,000 daltons) stimulate Ca+2-dependent LH release from pituitary cell cultures. In contrast, release does not occur in response to the epsilon-CBZ or succinyl derivatives (which have no internal charge) or in response to polymers of L-Glu, D-Glu, or Gly. The observation that polymers of D-Lys, L-Lys, and L-Arg all stimulate LH release with similar efficacy and potency indicates that simple charge interactions, rather than interaction with specific polymer-binding sites, are the cause of LH release. Since monomeric Lys neither stimulates LH release nor competitively inhibits release in response to Lys polymers, it appears that multiple charge coordination by Lys polymers is responsible for activation of the release mechanism. Putrescine, spermine, and spermidine (which have more closely spaced charges) do not stimulate LH release, suggesting that a certain minimal distance of charge separation must occur to obtain efficacy. The reduced potency of heteropolymers of Lys (spaced with Ala or Tyr) suggests that a maximal effective distance also exists. Consecutive and concomitant incubation studies indicate that LH released in response to poly-L-Lys or GnRH comes from the same pool as that released by GnRH. The time courses of release are similar for the two compounds.

Synthesis of undecagold cluster molecules as biochemical labeling reagents. 1. Monoacyl and mono[N-(succinimidooxy)succinyl] undecagold clusters.

This paper describes the synthesis and characterization of succinyl, phthalyl, and N-(succinimidooxy)-succinyl derivatives of the undecagold cluster complex tricyanoheptakis[4,4',4"-phosphinidynetris(benzenemethana mine)]undecagold, 1, molecular formula Au11(CN)3[P(C6-H4CH2NH2)3]7. These are useful as electron-dense reagents for labeling biological structures in preparation for electron microscopic analysis. Limited reaction of 1 with succinic or phthalic anhydrides produces a mixture of mono-, bis-, etc. (N-succinyl)-1 or (N-phthalyl)-1, which can be separated by anion-exchange chromatography at pH 11.5. Yields of monoacylated derivatives can be maximized by controlling the ratio of succinic or phthalic anhydride to 1. The remaining 20 primary amino groups can be dialkylated or acetylated, blocking their participation in further chemical modifications of the carboxylic functional group introduced in the succinylation or phthalylation of 1. These carboxyl groups can be activated as N-hydroxysuccinimido esters, which are acylating derivatives of 1. An example is mono[N-(succinimidooxy)-succinyl]icosa(N,N-dimethyl)-1 whose synthesis is described. Bis- and tris(N-succinyl) and -(N-phthalyl) derivatives of 1 are also produced and isolated in usable quantities.

Macrophages: a source of luteotropic cybernins.

A macrophage homogenate contained substances which stimulated primary cultures of mouse granulosa cells to secrete more progesterone. The response to the luteotropic substances was similar to that observed when intact macrophages were co-cultured with granulosa cells. The bioactive polypeptides present in cytosolic and particulate fractions of cell homogenates were non-dialyzable, heat labile and trypsin sensitive. When the surface of intact macrophages was treated with trypsin there was a loss of activity from the particulate fraction suggesting that some luteotropic proteins reside on the plasma membrane of mononuclear phagocytes. Treatment of macrophages with Con A but not the succinyl derivative of the lectin caused a release of luteotropic proteins with apparent molecular weights of 26,000 and 41,000. These findings in conjunction with our prior observation that macrophages must contact granulosa cells to stimulate progesterone secretion suggest that aggregation of mononuclear cell surface proteins may occur when the two cells interact thus resulting in the expression of luteotropic activity. Hence, it appears that macrophages which are found within corpus luteum may be a source of ovarian cybernins. This is the first description that a cell of the immune system can communicate at the molecular level with a steroid secreting cell of the ovary.

Retention behaviour of bile acids in ion suppression and ion-pair chromatography on bonded phases

The retention behaviour of some free bile acids and succinyl derivatives in acidic form at low pH (3) and as ion pairs at higher pH (7.5) in the presence of quaternary alkylammonium has been studied in a reversed-phase chromatographic system using C18 or C8 bonded stationary phases and mixtures of acetonitrile or methanol and aqueous phosphate buffer as the mobile phase. The influence of pH, organic solvent content, quaternary alkylammonium concentration, stationary phase and temperature on retention has been investigated. The more efficient separations for bile acids either by ion suppression or by ion-pair chromatography are presented. The use of UV detection and determination of response for the bile acids, operating at a wavelength in the range 200–220 nm, indicate that high responses and detection limits of 0.02–1 nmole are obtained below 205 nm.