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Biomedical subjects

G Barany

Publications and source records attributed to G Barany.

72 records · Page 4Linked to original sources
PubMed1987

A convenient general method for synthesis of N alpha- or N omega-dithiasuccinoyl (Dts) amino acids and dipeptides: application of polyethylene glycol as a carrier for functional purification.

N alpha-Dithiasuccinoyl (Dts) amino acids needed for solid-phase peptide synthesis have been prepared in good yields and excellent purities by a new method that exploits the solubility properties of polyethylene glycol (PEG; bifunctional with average molecular weight 2000 was found to be optimal). Suitably side-chain protected amino acid derivatives are first reacted with a polymeric xanthate, following which the free alpha-carboxyl is blocked by silylation and the Dts heterocycle is elaborated in the same pot by reaction with chlorocarbonylsulfenyl chloride. Upon aqueous work-up, the polymeric carrier removes any urethane blocked amino acids which arise during the process. Exaggerated conditions were explored to prove the power of this functional purification approach, and mechanisms of formation of polymer-bound urethanes are proposed and supported by solution model studies. The preparation and characterization of the companion N-(iso-propyldithio)carbonyl derivative of proline is also presented.

Amino Acids↗
PubMed1986

Synthesis in vitro of a seven amino acid peptide encoded in the leader RNA of Rous sarcoma virus.

Sequences of avian retroviral RNAs suggest that short open reading frames in the putatively untranslated leader sequences might direct the synthesis of small peptides. Previous analyses indicate that translation of Rous sarcoma virus (RSV) RNA in vitro faithfully reflects translation of the viral RNA in the chick cell. Accordingly, we sought to determine if the heptapeptide LP1, encoded in the open reading frame closest to the 5' end of RSV RNA, could be synthesized in vitro since this would strongly suggest that it might also be synthesized in vivo. Here we confirm that RSV RNA directs the synthesis of LP1 in rabbit reticulocyte lysates. LP1 is rapidly degraded in the lysate by an aminopeptidase activity. On the basis of the following observations, we propose that the open reading frame encoding LP1 plays a role in the life cycle of avian retroviruses. The LP1 open reading frame is ubiquitous with respect to position and length in 12 strains of avian retrovirus. In the amino acid sequences of the 12 strains, only three of the seven residues are invariant. On the basis of the conservation of the -3 and +4 nucleotides flanking the AUG codon, the strengths of initiation for translation of LP1 are approximately the same in the different viruses. The LP1 open reading frame is positioned in front of sites on retrovirus RNA that are required for initiation of cDNA synthesis and for packaging of the RNA into mature virus.

Amino Acid Sequence↗
PubMed1986

Effects of allyl methyl trisulfide on glutathione S-transferase activity and BP-induced neoplasia in the mouse.

Allyl methyl trisulfide (AMT), a constituent of garlic oil, was studied for its effects on glutathione S-transferase (GST) activity and on benzo[a]pyrene (BP)-induced neoplasia of the forestomach and lungs of female A/J mice. AMT induced increased GST activity in the forestomach, small bowel mucosa, liver, and lung. The forestomach and small bowel mucosa responded to a single low dose of AMT (3.0 mumol) given by oral intubation, whereas liver and lung were less reactive. A dose schedule of two administrations of 15 mumol AMT given 48 hours apart gave close-to-maximum induction in all four tissues and was chosen for investigation of its inhibitory effects. With this dose schedule, AMT produced an inhibition of BP-induced neoplasia of the forestomach as shown by a greater than 70% reduction in the number of tumors found at the completion of the experiment. Inhibition of pulmonary neoplasia did not occur. AMT is a member of a new class of naturally occurring chemicals that have the capacity to inhibit chemical carcinogenesis.

Allyl Compounds↗
PubMed1985

Improved approach for anchoring N alpha-9-fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solid-phase peptide synthesis.

Several Fmoc-amino acids have been esterified by use of N,N-dimethylformamide dineopentyl acetal to 2,4,5-trichlorophenyl 3'-(4''-hydroxymethyl-phenoxy)propionate, and the resultant handle derivatives were purified and then quantitatively coupled onto aminomethyl supports. Compared to literature methodology, the present procedure is preferred because: (i) extra steps to selectively protect and liberate the carboxyl of the handle are circumvented; and (ii) the additional methylene group spacer reflecting substitution of a propionyl group for an acetyl group in the handle changes the electronic parameters of the resultant p-alkoxybenzyl ester sufficiently so that the rates of acidolytic cleavage of the anchoring linkage are 2- to 3-fold increased and useful improvements in yields can be achieved.

Amino Acids↗
PubMed1985

Inhibition of carcinogenesis by some minor dietary constituents.

Previous work has shown that food contains a large number of minor dietary constituents that can inhibit the occurrence of cancer. Additional inhibitors from four different natural sources will be the subject of this presentation. 1. Citrus fruit oils. Orange, tangerine, lemon, and grapefruit oils given p.o. induce increased glutathione (GSH) S-transferase activity in tissues of the mouse. When fed in the diet prior to and during the course of administration of benzo(a)pyrene (BP), the four citrus fruit oils inhibit formation of tumors of both the forestomach and lungs of mice. When fed either before or after the administration of 7,12-dimethylbenz(a)anthracene (DMBA) orange oil inhibits mammary tumor formation. 2. Garlic oil. Allyl methyl trisulfide (AMT), a constituent of garlic oil, has been synthesized recently. When given p.o. 96 and 48 hr prior to BP, AMT inhibits the occurrence of forestomach tumors in mice. 3. Green coffee beans. Two diterpene esters, kahweol palmitate and cafestol palmitate, which are potent inducers of GSH S-transferase activity have been isolated from coffee beans. When administered p.o. prior to DMBA the two diterpene esters inhibit mammary tumor formation. 4. Cruciferous vegetables. Several glucosinolates occur in cruciferous vegetables. Efficient procedures for the isolation of these compounds have been developed recently. The inhibitory effects of three of these i.e. glucobrassicin, glucotropaeolin, and glucosinalbin were studied in several animal models. Glucobrassicin caused substantial inhibition of BP-induced neoplasia of the lung and forestomach of the mouse and DMBA-induced neoplasia of the breast in rats. Glucosinalbin and glucotropaeolin are less active in these systems. In addition to protective effects, indoles derived from the hydrolysis of glucobrassicin have potential harmful properties. The implications of multiple properties and factors which may determine their consequence will be discussed.

Animals↗
PubMed1984

Application of N,N-dimethylformamide dineopentyl acetal for efficient anchoring of N alpha-9-fluorenylmethyloxycarbonylamino acids as p-alkoxybenzyl esters in solid-phase peptide synthesis.

The attachment of Fmoc-amino acids onto p- alkoxybenzyl alcohol resins via DCC-DMAP coupling suffers from two different problems: formation of dimers and racemization. The use of N,N-dimethylformamide dineopentyl acetal for the preparation of Fmoc- aminoacyloxybenzyl handles is the basis of a safe and efficient anchoring method that avoids both problems.

4-Aminopyridine↗
PubMed1982

Chemistry of carbamoyl disulfide protected derivatives of proline.

High-yield methods have been devised for the synthesis of a series of alkyl and aryldithiocarbonyl derivatives of the alpha-imino acid proline. The carbamoyl disulfide protecting groups were rapidly and quantitatively removed under a variety of reductive conditions, suggesting their potential value for orthogonal schemes of peptide synthesis.

Carbamates↗
PubMedDate not supplied

Toward new designed proteins derived from bovine pancreatic trypsin inhibitor (BPTI): covalent cross-linking of two 'core modules' by oxime-forming ligation.

A 25-residue disulfide-cross-linked peptide, termed 'oxidized core module' (OxCM), that includes essentially all of the secondary structural elements of bovine pancreatic trypsin inhibitor (BPTI) most refractory to hydrogen exchange, was shown previously to favor nativelike beta-sheet structure [Carulla, N., Woodward, C., and Barany, G. (2000) Synthesis and Characterization of a beta-Hairpin Peptide That Represents a 'Core Module' of Bovine Pancreatic Trypsin Inhibitor (BPTI). Biochemistry 39, 7927-7937]. The present work prepares to explore the hypothesis that the energies of nativelike conformations, relative to other possible conformations, could be decreased further by covalent linkage of two OxCMs. Optimized syntheses of six approximately 50-residue OxCM dimers are reported herein, featuring appropriate monomer modifications followed by oxime-forming ligation chemistry to create covalent cross-links at various positions and with differing lengths. Several side reactions were recognized through this work, and modified procedures to lessen or mitigate their occurrence were developed. Particularly noteworthy, guanidine or urea denaturants that were included as peptide-solubilizing components for some reaction mixtures were proven to form adducts with glyoxylyl moieties, thus affecting rates and outcomes. All six synthetic OxCM dimers were characterized by 1D (1)H NMR; three of them showed considerable chemical shift dispersion suggestive of self-association and mutual stabilization between the monomer units.

Amino Acid Sequence↗
PubMedDate not supplied

Solid-phase syntheses of heterocycles containing the 2-aminothiophenol moiety.

Efficient and general procedures have been developed for the solid-phase preparation of substituted benzothiazoles (1), 3, 4-dihydro-1,4-benzothiazines (2), 3,4-dihydro-1,4-benzothiazine-1, 1-dioxides (3), 3,4-dihydro-3-oxo-1,4-benzothiazines (4), and 3, 4-dihydro-3-oxo-1,4-benzothiazine-1,1-dioxides (5). All five classes of compounds were prepared from a common intermediate, resin-bound 2-amino-4-carboxythiophenol, in a minimal number of steps. This intermediate was generated by (i) coupling 4-fluoro-3-nitrobenzoic acid onto Wang resin, or onto an amino acid bound to the resin, (ii) substitution of the aryl fluoride with a protected thiol, (iii) reduction of the nitro group, and (iv) removal of sulfur protection. Reaction with the appropriate substrates and reagents to effect cyclization gave the substituted core structures, which were modified further to introduce additional point(s) of diversity. Following cleavages from the solid support, the compounds were obtained in high initial purities and good isolated yields after purification.

Aniline Compounds↗
PubMedDate not supplied

Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. A chemical approach for evaluating the role of disulfide bridges in protein folding and stability.

The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges.

Amino Acid Sequence↗
PubMedDate not supplied

Solid-phase synthesis and biological activity of the parallel dimer of deamino-oxytocin.

The parallel dimer of deamino-oxytocin has been synthesized by a novel solid-phase route. Successive orthogonal deprotection and oxidation reactions, carried out while the peptide remained anchored to a polymeric support, resulted in the formation of two disulfide bridges and conversion, with minimal side reactions, of the linear monomeric precursor to the dimer. The purified dimer showed approximately 0.1% to 2% of the biological activities of the monomer, as well as prolonged action. The time course of response to the deamino-oxytocin dimer differed from that of the monomer (and of oxytocin), and is probably due to slow formation of monomer under the conditions of biological testing.

Amino Acid Sequence↗
PubMedDate not supplied

Optimized preparation of deca(L-alanyl)-L-valinamide by 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis on polyethylene glycol-polystyrene (PEG-PS) graft supports, with 1,8-diazobicyclo [5.4.0]-undec-7-ene (DBU) deprotection.

Deca(L-alanyl)-L-valinamide is known to be a challenging model target for solidphase peptide synthesis, due to its hydrophobicity and its tendency to form secondary structures which inhibit acylation and deprotection. Here we report systematic studies on the synthesis of this peptide on an automated continuous-flow instrument, using the 9-fluorenylmethyloxycarbonyl (Fmoc) group for N alpha-amino protection and a polyethylene-glycol polystyrene (PEGPS) graft support. The optimal deprotection reagent proved to be DBU-piperidine-DMF (1:1:48, vol/vol/vol). The synthetic peptides were analyzed and characterized by high-performance liquid chromatography and several mass spectrometric techniques.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMedDate not supplied

Syntheses and biological activities of parallel and antiparallel homo and hetero bis-cystine dimers of oxytocin and deamino-oxytocin.

New methods have been developed for the synthesis of disulfide-bridged homo and hetero peptide dimers (both parallel and antiparallel), as exemplified in the oxytocin and deamino-oxytocin families. The linear sequences were assembled by 9-fluorenyl-methyloxycarbonyl (Fmoc) solid-phase synthesis techniques, with orthogonal protection for the two beta-thiols by appropriate combinations of S-[(N'-methyl-N'-phenylcarbamoyl)sulfernyl] (Snm), S-acetamido-methyl (Acm) and S-2, 4, 6-trimethoxybenzyl (Tmob) groups. Two octapeptide-resins gave rise to four different nonapeptide amides, which were brought together in defined ways to provide access to four of the six possible dimeric products. For each dimer, the first disulfide bond was formed in solution by a directed method, and then the second disulfide bond was formed, without purification of the intermediates, by iodine oxidation. Final isolated yields of the desired pure dimers were in the 20% to 40% range. Biological activities ranged from 0.2% to 6% that of oxytocin, depending on the assay, and were in some cases considerably protracted. These data are consistent with the hypothesis that dimers revert slowly to monomers under the testing conditions.

Amino Acid Sequence↗
PubMedDate not supplied

Novel N omega-xanthenyl-protecting groups for asparagine and glutamine, and applications to N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis.

The N alpha-9-fluorenylmethyloxycarbonyl (Fmoc), N omega-9H-xanthen-9-yl (Xan), N omega-2-methoxy-9H-xanthen-9-yl (2-Moxan) or N omega-3-methoxy-9H-xanthen-9-yl (3-Moxan) derivatives of asparagine and glutamine were prepared conveniently by acid-catalyzed reactions of appropriate xanthydrols with Fmoc-Asn-OH and Fmoc-Gln-OH. The Xan and 2-Moxan protected derivatives have been used in Fmoc solid-phase syntheses of several challenging peptides: a modified Riniker's peptide to probe tryptophanalkylation side reactions, Briand's peptide to assess deblocking, at the N-terminus and Marshall's ACP (65-74) to test difficult couplings. Removal of the Asn and Gln side-chain protection occurred concomitantly with release of peptide from the support, under the conditions for acidolytic cleavage of the tris(alkoxy)benzylamide (PAL) anchoring linkage by use of trifluoroacetic acid/scavenger mixtures. For each of the model peptides, the products obtained by the new protection schemes were purer than those obtained with N omega-2,4,6-trimethoxybenzyl (Tmob) or N omega-triphenylmethyl (Trt) protection for Asn and Gln.

Amino Acids↗
PubMedDate not supplied

Intramolecular pyrophosphate formation during N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis of peptides containing adjacent phosphotyrosine residues.

The derivative N alpha-9-fluorenylmethyloxycarbonyl-O-phospho-L-tyrosine [Fmoc-Tyr(PO3H2)-OH] has been used successfully for the solid-phase synthesis of a wide variety of phosphorylated peptides. However, when it is used to incorporate consecutive phosphotyrosine residues, a pyrophosphate linkage can form between the two adjacent tyrosines. Incorporation of unprotected phosphotyrosine during the synthesis of peptides with multiple phosphotyrosine residues has been studied as a function of coupling conditions and the absence or presence of intervening amino acid residues. The pyrophosphate-forming side reaction is more severe with increased coupling times and/or repetitions of coupling and occurs only when the phosphotyrosine residues are directly adjacent to one another.

Amino Acid Sequence↗