Analogs of natural lipids. IV. Synthesis and properties of cyclopentanoid analogs of phosphatidic acid

A new series of phosphatidic acid analogs has been synthesized in which the glycerol moiety of diacylglycerophosphoric acid is replaced by each of the three isomeric cyclopentane-1,2,3-triols (1,3/2, DL-1,2/3, and 1,2,3/0). Of the seven possible configurational and positional phosphatidic acid analogs of this series, five isomers have been obtained and characterized by spectroscopic methods and microanalysis. Four of the five isomers are 1-(or 3-)phosphoryl derivatives, while the fifth is a 2-phosphate. The analogs were prepared in configurationally pure form by unequivocal synthetic procedures involving selectively blocked intermediates: acyl migration was avoided by the use of mild deblocking procedures. The anhydrous lipid products, all of which are dipalmitoyl esters, are solids indefinitely stable at room temperature in the free acid or potassium salt form; they have chromographic mobility and melting points similar to dipalmitoyl glycerophosphoric acid the dipotassium salts bind water of hydration tenaciously, remaining hydrated after drying in vacuo at 100 degrees C. NMR spectra of dimethyl esters of some of the analogs show nonequivalence of the two methyl groups, consistent with the diastereotopic nature of those groups. In addition to their intrinsic interest as conformationally restricted acidic lipids, the analogs are now available as starting materials for the synthesis of the more complex acidic and amphoteric lipids required for our exploitation of these cyclopentanoid analogs as unique probes for the study of lipid-lipid and protein-lipid interactions.     

Analysis of the degradation mechanisms of MHC class I-presented tumor antigenic peptides by high performance liquid chromatography/electrospray ionization mass spectrometry: application to the design of peptidase-resistant analogs

Peptide vaccines based on the use of MHC class I restricted epitopes are currently assayed for anti-tumor and anti-viral immunotherapy. With the aim of designing minimally modified, peptidase-resistant analogs, we developed a rational approach based on a detailed understanding of the degradation mechanism of peptides in serum. Degradation of murine tumor antigen P198 and human tumor antigen MAGE-3.A1 was followed by on line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). This method provided high precision and sensitivity for rapid and direct analysis of degradation fragments in a complex mixture and, very importantly, precise identification of transient degradation fragments present at low concentrations. The design of structurally modified analogs, and the analysis of their degradation by on-line HPLC/ESI-MS, allowed us to to demonstrate the efficiency of local modifications in the protection of a given peptide bond towards a specific peptidase activity.     

8-[18F]fluorooctanoic acid and its beta-substituted derivatives as potential agents for cerebral fatty acid studies: synthesis and biodistribution

Fluorine-18 labeled analogs of 8-fluorooctanoic acid and its structurally modified derivatives with methyl or gem-dimethyl branching or with oxygen substitution at the C3 position were prepared using nucleophilic substitution of the tosylate precursors by [18F]fluoride ion, for evaluation as tracers for cerebral fatty acid metabolism. Tissue distribution studies in rats showed low brain uptakes of these 18F-labeled fatty acid analogs with poor brain-to-blood ratios of activity. The oxygen-substituted analog did not show any significant accumulation of radioactivity in most tissues. The initial brain uptake of activity after injection of ethyl 8-[18F]fluorooctanoate and its free acid remained virtually unchanged over an extended time period, beta-Monomethyl and beta-gem-dimethyl branched analogs had similar brain uptake at the early time period, but they showed rapid clearance of activity from the brain. TLC analysis showed no incorporation of 8-[18F]fluorooctanoic acid and its beta-dimethyl analogs into brain lipids. It was also shown in the metabolite analysis that the labeled metabolites produced from 8-[18F]fluorooctanoic acid are found in blood, and that they could enter the brain to a significant degree. On the contrary, such radioactive metabolites could not be found in the brain in the experiment with the beta-gem-dimethyl branched analog. Thus, the present studies showed that retention of radioactivity in the brain with 8-[18F]fluorooctanoic acid derivatives is mainly attributable to their radioactive metabolites, and that the rapid clearance of beta-branched analogs from the brain is due to the lack of availability as substrates in the cerebral fatty acid metabolism.     

Preparation and standardization of U.S. Standard Pertussis Vaccine, Lot No. 11

Novel glutathione (GSH) analogs, previously shown to inhibit glutathione S-transferase (GST) activity at about 1 microM in vitro, were tested for their ability to potentiate the killing of cultured tumor cells by chemotherapeutic drugs. When tested at doses up to 200 microM, the analogs were neither toxic nor capable of potentiating drug toxicity unless the diethyl ester (DEE) form was used for treatment of the cells. HPLC analysis revealed rapid internalization of the DEE and intracellular conversion to a monoethyl ester form that accumulated in the cell, followed by a more gradual loss of the second ester to generate the active parent form. For the four GSH analogs tested, the ability of the DEE forms to potentiate chlorambucil (CMB) toxicity in HT-29 human colon adenocarcinoma cells strongly correlated with the in vitro ability of the parent form to inhibit recombinant human P1-1. This isozyme is the dominant form of GST present in HT-29 cells. Of the four analog DEEs tested, gamma-glutamyl-S-(benzyl)cysteinyl-R(-)-phenyl glycine (TER 117) DEE was the most effective in potentiating CMB toxicity in several cell lines: HT-29, HT4-1 (HT-29 subclone), SKOV-3 ovarian carcinoma, and SK VLB (vinblastine-resistant variant of SKOV-3) cells. gamma-Glutamyl-S-(octyl)cysteinyl-glycine (TER 143) DEE potentiated mitomycin C (MTC) toxicity in HT4-1 and SK VLB cells while TER 117 DEE did not. TER 117 DEE enhanced melphalan effects on xenografts of HT4-1 in mice to a similar extent as that achieved with the previously described nonspecific GST inhibitor, ethacrynic acid. Taken together, our results indicate that cell-permeable analogs of GSH can potentiate cytotoxicity of common chemotherapeutic drugs and this effect has a strong positive correlation with the ability of the analogs to inhibit specific GST isozymes.     

High-affinity alpha-aminobutyric acid A/benzodiazepine ligands: synthesis and structure-activity relationship studies of a new series of tetracyclic imidazoquinoxalines

A series of tetracyclic imidazoquinoxaline analogs was developed which constrain the carbonyl group of the partial agonist 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-[(dimethylamino)carbonyl] - 4,5-dihydroimidazo[1,5-alpha]quinoxaline (2, U-91571) away from the benzene ring. These analogs orient the carbonyl group in the opposite direction of the previously reported full agonist 1-(5- cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihydroimidazo[1,5- alpha]pyrrolo [2,1-c]quinoxalin-10(11H)-one (3, U-89267). A number of approaches were utilized to form the "bottom" ring of this tetracyclic ring system including intramolecular cyclizations promoted by Lewis acids or base, as well as metal-carbenoid conditions. The size and substitution pattern of the additional ring was widely varied. Analogs within this series had high affinity for the benzodiazepine receptor on the alpha-aminobutyric acid A chloride ion channel complex. From TBPS shift and Cl- current assays, the in vitro efficacy of compounds within this class ranged from antagonists to partial agonists with only 18a identified as a full agonist. Additionally, several analogs were quite potent at antagonizing metrazole-induced seizures indicating possible anticonvulsant or anxiolytic activity. Unlike 3, analogs in this series did not have high affinity for the diazepam insensitive alpha 6 beta 2 delta 2 subtype. These results suggest that either constraining the carbonyl group away from the benzene ring or the greater planarity that results from the additional cyclic structure provides analogs with partial agonist properties and prevents effective interaction with the alpha 6 beta 2 delta 2 subtype.     

Reproduction of antiviral effect in an in vivo model of human cytomegalovirus retinal infection

Fibroblast growth factors (FGFs) play important roles in a variety of developmental processes in mammals. The dependence of their activity on heparin binding has been a puzzle that, in recent years, has been the subject of active investigation. Recent structural analyses on complexes of FGFs with heparin fragments or heparin analogs have unveiled the extreme complexity of these systems.     

Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10,000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desNH2Tyr1, D-Ala2, Ala15]-hGRF (1-29)-Gly-Gly-Cys(NH2)-S-Nle-PEG5000 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity.     

Mono- and bicyclic analogs of parathyroid hormone-related protein. 1. Synthesis and biological studies

The bioactive conformation of parathyroid hormone-related protein (PTHrP), a single-chain linear peptide structurally similar to parathyroid hormone (PTH), is of considerable interest because PTH and PTHrP both recognize and bind to a shared G-protein-coupled receptor. Both hormones are thought to present a bioactive conformation to the receptor which is substantially alpha-helical in nature. To better characterize this putative biologically relevant conformation, we prepared a series of conformationally constrained analogs of PTHrP with enhanced alpha-helical stability. A combination of structural constraint and helix stabilization was achieved through side chain-to-side chain lactam ring formation between Lys(i) and Asp(i+4) residues (13-to-17 and 26-to-30) along the PTHrP sequence. Mono- and bicyclic analogs derived from the agonist PTHrP-(1-34)NH2 and the antagonist PTHrP-(7-34)NH2 were prepared and characterized in terms of receptor binding and stimulation (or antagonism) of PTH-stimulated adenylyl cyclase activity in osteoblast-like cells. The binding affinity of monocyclic [Lys13,Asp17]-(I) and bicyclic [Lys13,Asp17,Lys26,Asp30]PTHrP-(1-34)NH2 (III) agonists was in the low nanomolar range and similar to that of the parent linear peptide. Furthermore, their efficacy was in the sub-nanomolar range and about 10-fold higher than that of the corresponding linear parent peptide. Analogs I and III are the first cyclic PTH/PTHrP receptor agonists and amongst the most potent PTHrP analogs yet designed. The rank-order of potency in the cyclic antagonist series does not correlate with the binding affinities. In light of the positional dependence and the differential effects of lactam bridge formation on the biological activities of agonist vs antagonists, these analogs may provide insight regarding the biologically relevant conformations of PTHrP-derived ligands [Maretto et al. (1997) Biochemistry 36, 3300-3307].     

Structure-activity relationships of the antimalarial agent artemisinin. 1. Synthesis and comparative molecular field analysis of C-9 analogs of artemisinin and 10-deoxoartemisinin

A series of C-9 beta-substituted artemisinin analogs (2-21) were synthesized via dianion alkylation of the total synthetic intermediate 57 followed by subsequent ozonolysis/acidification, or by alkylation of the enolate derived from (+)-9-desmethylartemisinin, 2. Inactive acyclic analogs 22 and 23 were synthesized by nucleophilic epoxide opening and the ring contracted analog 24 was prepared by an alternate route. 10-Deoxo-9-alkyl derivatives 68 and 70 were synthesized convergently from intermediates in the preparation of 9-alkyl derivatives. In vitro bioassay was conducted in W-2 and D-6 clones of drug resistant Plasmodium falciparum. Comparative molecular field analysis (CoMFA) of the 9-alkyl lactone derivatives provided a model with a cross-validated r2 = 0.793. Inclusion of inactive 1-deoxyartemisinin analogs 26-42 provided a model with a value of 0.857. The activities of a number of other analogs of divergent structure (43-56) were predicted with good accuracy using the CoMFA model.     

Stabilized analogs of thymopentin. 1. 4,5-Ketomethylene pseudopeptides

The pentapeptide, thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5) is known for its activity as an immunomodulating drug, but with limited half-life in plasma. In this first paper of a series of three studies, the synthesis of analogs stabilized at the peptide bond between the C-terminal amino acids via insertion of a ketomethylene moiety is described. N-Blocked pseudopeptides containing Val(k)Phe, Ala(k)Phe, and Val(k)Val units were prepared and attached to chloromethyl Merrifield resin via the carboxy terminal. Removal of the N-BOC group by trifluoroacetic acid was followed by sequential coupling with N-BOC dipeptides of aspartic acid to yield resin-bound N-BOC pseudotetrapeptides. Removal of N-BOC and coupling with N-BOC-r-N-tosylarginine followed by total cleavage of blocking groups and resin by HF afforded the target pseudopentapeptides. The analogs were found to compete favorably with thymopentin for binding to CEM cells, but binding was reduced by about 20-30% on average. All analogs showed significant enhancement of half-life versus thymopentin in mouse serum, but most showed only modest improvement in human serum. Insertion of proline or norleucine at position 2 in the chain caused a substantial increase in half-life (3-4-fold), while N-methylnorleucine conferred complete stability in the analogs.     

Dopamine transporter ligand binding domains. Structural and functional properties revealed by limited proteolysis

Dopamine transporters (DATs) are members of the Na+- and Cl--dependent neurotransmitter and amino acid transporter family predicted by hydrophobicity analysis to have 12 transmembrane-spanning helices. The structure of DAT was studied using the photoaffinity compounds [125I]1-[2-(diphenylmethoxy)-ethyl]-4-[2-(4-azido-3-iodophenyl) ethyl] piperazine ([125I]DEEP), a 1-(2-diphenylmethoxy)-ethyl-4-(3-phenyl propyl)piperazine (GBR analog), and [125I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), a cocaine analog, which had been shown in a previous study to become incorporated into different regions of the DAT primary sequence. The proximity of the photolabeled binding sites to integral membrane structures was investigated by subjecting photolabeled membrane suspensions to limited proteolysis with trypsin and separately analyzing the resulting membranes and supernatants for the presence of photolabeled DAT fragments. Trypsin treatment of [125I] DEEP-labeled membranes generated labeled 45- and 14-kDa DAT fragments that immunoprecipitated with an epitope-specific antiserum generated against amino acids 42-59 near the first putative transmembrane domain, whereas [125I]RTI 82 was found in 32- and 16-kDa tryptic fragments that precipitated with an antiserum directed against a sequence near transmembrane domain 4 (amino acids 225-238). All of the photolabeled fragments were recovered in the protease-treated membranes, indicating that they possess integral membrane structures that prevent their release from the membrane as soluble forms. The size of the two smallest fragments in conjunction with their retention in the membrane suggests that incorporation of the photoaffinity ligands occurs in or near membrane spanning regions and delineates the maximum possible distance between the transmembrane structures, incorporated photolabel, and antibody epitopes. Carbohydrate analysis of the fragments identified sialic acids and N-linked oligosaccharides exclusively on the 45-kDa [125I]DEEP-labeled fragment, which, based on size, would be expected to contain four consensus glycosylation sites between putative transmembrane domains 3 and 4. Photoaffinity labeling after trypsin treatment of membranes showed that the larger but not the smaller fragments retain binding capacity, as the 45- and 32-kDa fragments were capable of becoming photolabeled. Binding of photoaffinity ligands at these fragments was displaced with the same pharmacology as that of intact DATs. These results verify numerous aspects of DAT structure and topology heretofore only predicted from theoretical considerations and extend our knowledge of DAT structure-function properties.     

Methodological problems in Tourangeau and Ellsworth's study of facial expression and experience of emotion.

Argues that flaws in the method of R. Tourangeau and P. C. Ellsworth's study (see record 1981-00499-001) undermined their test of hypotheses derived from theories of emotion. Tourangeau and Ellsworth did not manipulate facial movements into valid analogs of emotional expressions, so failure to confirm the sufficiency hypothesis is uninformative. (12 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Probing immunogenicity of a T cell epitope by L-alanine and D-amino acid scanning

All residues of the I-Ed restricted fragment 24-36 of a snake toxin were individually changed into L-alanine and the corresponding D-enantiomer. Four analogs substituted with L-Ala at positions 25;30, 31 and 33, and nine analogs substituted with a D-residue along the stretch 25-33 lost most (position 28) or all their capacity to stimulate a toxin-specific T hybridoma. None of these analogs stimulated splenocytes from mice immunized with the peptide 24-36. Only the L-A31 and D-W29 modified analogs could prime a T cell response which, however, showed no cross-reactivity with the native peptide, demonstrating that T cell response selectivity can be deeply modified by mutation or configuration inversion of a single residue. Our data suggest that (i) the region 25-33 is the core of the T epitope that binds to I-Ed, and (ii) Y25 R30 and R33 contribute to the peptide binding by anchoring into pockets of I-Ed. In agreement with T cell priming observations, only the L-A31 and D-W29 modified analogs elicited strong antibody responses, just like the peptide 24-36, whereas nearly all other analogs were less immunogenic. All but the L-Ala30 and L-Ala33 modified analogs were recognized by a 24-36 specific antiserum as well as the native peptide. Altogether, our results show that substitution by D-amino acid in a peptide could be particularly well-suited for either minimizing the risk of hypersensitivity or designing peptidic vaccines.     

Auditory coding, cues, and coherence in phonetic perception.

C. T. Best, M. Studdert-Kennedy, S. Manuel, and J. Rubin-Spitz (1989) reported that listeners given speech labels showed categorical-like perception of a series of complex tone analogs to a /la/-/ra/ speech series, whereas nonspeech listeners were unable to classify the stimuli consistently. In 2 experiments, a new training and testing procedure was used with adult listeners given nonspeech instructions. They classified the /la/-/ra/ tone analogs consistently, showed categorical-like perception, and generalized their training to a new, /li/-/ri/ tone analog series. Two sets of auditory attributes were described for coding the /l/-/r/ distinction, and 1 was shown to quantitatively predict listeners' classification of both series. These results are consistent with models of perception in which a rich, abstract auditory code is computed and forms the basis for both speech and nonspeech auditory categories. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.     

TX-1877: design, synthesis, and biological activities as a BRM-functional hypoxic cell radiosensitizer

PURPOSE: 2-Nitroimidazole acetamide TX-1877 and its derivatives (TX-1877 analogs) were designed, synthesized, and evaluated by their in vitro and in vivo radiosensitization, tumor growth control, suppression of lung metastasis, and immunopotentiation, as biological response modifier (BRM)-functional hypoxic cell radiosensitizers. MATERIALS AND METHODS: TX-1877 analogs were designed and synthesized in our laboratory. In vitro radiosensitizing ability was estimated using EMT6/KU cells under hypoxic conditions. In vivo radiosensitization, antimetastasis, and immunopotentiation were evaluated using female C3H/He mice bearing the SCCVII tumor. Days (15 or 10) after the inoculation of 10(5) SCCVII tumor cells into the hinder thigh, a drug (0.4 mg/g) was administered i.p. and local irradiation of 30 Gy was given at 30 min after its administration. Tumor growth was observed for 20 days and mice were euthanized to count the number of metastatic nodules on the surface of the lungs. Tumor tissues were extirpated and stained by the ABC method at 1, 2, and 3 weeks after treatment for immunological evaluation. RESULTS: Novel types of bifunctional radiosensitizers, TX-1877 and its analogs possessing BRM-functions (i.e., antimetastatic and immunopotentiation effects) were developed. In vitro radiosensitizing abilities of TX-1877 and its analogs, with their partition coefficient values of more than 0.050, were comparable to misonidazole (MISO) at their doses of 1 mM. Tumor regrowth was suppressed evidently 20 days after the treatment in the irradiated group with TX-1877 (TX-1877 plus R) and with KIN-806 (KIN-806 plus R). The former group reduced markedly the mean number of metastatic lung nodules regardless of radiation therapy. TX-1877 and KIN-806 plus R induced helper T lymphocytes. The TX-1877, TX-1877 plus R, KIN-806, and KIN-806 plus R enhanced macrophage infiltration for 3 weeks after treatment. CONCLUSION: TX-1877 is an excellent BRM-functional hypoxic cell radiosensitizer, expected to be useful for clinical use.     

Bunyamwera virus-induced polypeptide synthesis

Bunyamwera virus-induced polypeptide synthesis in BSC-1 cell has been studied using polyacrylamide gel electrophoresis and autoradiography. Four virus-induced polypeptides were identified. Their molecular weights were 200 X 10(6) (L), 128 X 10(6) (G1), 31 X 10(6) (G2), and 23 X 10(6) (N). Pulse-chase experiments, short labeling experiments, and experiments using amino acid analogs failed to show evidence of polypeptides processing by proteolytic cleavage. Analysis of the kinetics of synthesis of these polypeptides showed that a clear division into early and late categories could be made, the onset of synthesis of polypeptide N and L rapidly reached a peak and then declined. Polypeptides G1 and G2 were made for several hours; their rate of synthesis then declined. All four polypeptides then continued to be made in relatively small amounts for many hours.     

Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor alpha-subunit, p195-212 and p259-271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195-212 and p259-271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195-212- and p259-271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.     

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: synthesis and fluorine effect on the biological activity

Analogs of Met-enkephalin and [D-Pen2, D-Pen5]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [D-DFAB2, Met5-NH2]enkephalin showed mu and delta agonist potencies comparable to those of natural [Leu5]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While L-amino acid substitution in position 3 of DPDPE usually resulted in higher delta agonist potency than D-amino acid substitution. [D-DFAB3]DPDPE turned out to be a more potent delta agonist than [L-DFAB3]DPDPE. Furthermore, [D-DFAB3]DPDPE showed over 100-fold higher delta agonist potency than [D-Abu3]DPDPE (Abu = 2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a delta opioid receptor subsite.     

General synthesis of 3-substituted alkenyl GABA as potential anticonvulsants

Stereospecific synthesis of cis and trans 3-substituted vinyl-gamma-aminobutyric acid analogs were obtained by either a Claisen rearrangement or a Wittig reaction from common diene precursors.