N-terminal tetrapeptide of dermorphin and D-Arg-substituted tetrapeptides: inactivation process of the antinociceptive activity by peptidase

Degradation products of the N-terminal tetrapeptide of dermorphin, H-Tyr-D-Ala-Phe-Gly-OH (ALPG) and D-Arg2-substituted tetrapeptide analogs of dermorphin, H-Tyr-D-Arg-Phe-Gly-OH (ARPG), H-Tyr-D-Arg-Phe-Gly-NH2 (TDAPG-NH2) and H-Tyr-D-Arg-Phe-beta-Ala-OH (TDAPA) by enkephalin degrading enzymes were studied by using reversed-phase high-performance liquid chromatography. After 5 and 25 hr incubations of the peptides with solubilized enzymes of mouse brain or spinal cord, liberation of the appreciable Tyr1 residue was observed in ALPG but not in ARPG, TDAPG-NH2 and TDAPA. When ARPG and TDAPG-NH2 were incubated with enzymes for 25 hr, a main degradation product was the N-terminal tripeptide produced from the hydrolysis of Phe3-Gly4 bond. Conversely, TDAPA did not produce the N-terminal tripeptide after 25 hr incubation with enzymes. In the enzyme assay, Tyr1-D-Arg2 bond of ARPG, TDAPG-NH2 and TDAPA was more stable than that of ALPG to the cleavage by aminopeptidase M (AP-M). Phe3-Gly4 bond of ALPG, ARPG and TDAPG-NH2 were easily hydrolyzed by carboxypeptidase Y (CP-Y) within 3 hr incubation, whereas the hydrolysis of Phe3-beta-Ala4 bond of TDAPA by CP-Y was not observed after 3 hr incubation. The present results and previous behavioural data suggest that a potent and prolonged antinociceptive activity of the D-Arg-substituted tetrapeptides is mainly attributed to the stability of Tyr1-D-Arg2 bond against aminopeptidase of peptidases.     

Dehydro-dermorphins. II. Synthesis and biological activity of unsaturated dermorphin hexa and heptapeptides

The Phe3 and/or Tyr5 residues in dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and its N-terminal hexapeptide-amide were replaced by delta-Phe or by Phe5 in order to examine the effect on opioid activity. On GPI preparation, the substitution of Phe5 for Tyr5 was well tolerated, whereas the hexa and heptapeptides containing delta Phe in position 3 and/or 5 displayed low potency. The unsaturation at position 3 alone or at positions 3 and 5 was particularly detrimental to mu activity. In the tail flick test, the influence of unsaturation or substitution at positions 3 and 5 generally matched the results of the in vitro assay. Dehydropeptides showed comparatively low antinociceptive effects and [Phe5] analogues displayed about 50% of the analgesic potency of the original peptides.     

Dermorphin tetra- and heptapeptide analogues containing a [3,4] amide bond replacement by a carbon-carbon double and single bond.

Seven dermorphin hepta- and tetrapeptide analogues containing [3,4] amide bond replacement by a carbon-carbon double and single bond were prepared. 1H NMR studies of the pseudoheptapeptide in DMSO indicate the presence of extended conformations with stacking of the side chains in the N-terminal part and an inverse gamma-turn around Ser7 in the conformational equilibrium. The binding data show that the affinity of the analogues for the mu-receptor is only slightly diminished in the D-Ala2 series and is more affected in the D-Arg2 series. Since the Gly4NH is not present in these compounds we conclude that this NH is not required to stabilize the bioactive conformation nor is it directly involved in binding to the receptor.     

The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis

The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.     

Reversed-phase HPLC study on the in vitro enzymic degradation of dermorphin

A high-performance liquid chromatographic (HPLC) method for the separation of the opioid heptapeptide dermorphin and related fragments has been developed. The chromatographic system was applied in the study of the kinetics of degradation of dermorphin (Der) in various tissues. Der was found to be extremely resistant to human and rat plasma (T 1/2 greater than 180 min). Upon incubation with homogenates of rat brains and kidneys, Der was cleaved with a half-life of 20.8 +/- 2.2 min and 2.4 +/- 0.3 min respectively. The catabolite formed was identified, in both tissues, as the N-terminal tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH. The stability to rat kidney and brain of the N-terminal hexa- and pentapeptides and of the [4 psi 5, NHCO] Der analogue was also investigated. The nature of the enzyme systems involved in the in vitro degradations is discussed.     

Dermorphin tetrapeptide analogues with 2',6'-dimethylphenylalanine (Dmp) substituted for aromatic amino acids have high mu opioid receptor binding and biological activities

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.     

Dimeric dermorphin analogues as mu-receptor probes on rat brain membranes. Correlation between central mu-receptor potency and suppression of gastric acid secretion

The opioid receptor preference for dermorphin and several dimerized structural analogues was investigated using rat brain synaptosomes and correlated with the potencies of intracerebroventricularly administered dimeric dermorphin peptides to inhibit gastric acid secretion. The carboxyl terminus of dermorphin or amino-terminal dermorphin analogues was bridged by dihydrazide or (poly)ethylenediamine structures. Synaptosomal membranes were prepared for radioligand binding assay in the presence of soybean trypsin inhibitor and preincubated to remove endogenously bound opioid peptides before storage at -70 degrees C. Specific radiolabeled agonists used in the radioligand binding assays were [D-Ala2,N-methyl-Phe4,Gly-ol5] [3H] enkephalin for mu-receptors and [D-Ala2,D-Leu5] [3H]enkephalin for delta-receptors. delta-Receptor binding assays were conducted in the presence of 2.6 microM [N-Me-Phe3,D-Pro4]morphiceptin to suppress peptide binding to mu-receptors. [D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin and dermorphin had affinities of 1.39 and 1.22 nM for mu-receptors and 355.8 and 178.6 nM for delta-receptors, respectively. Affinities of dimeric-dermorphin0 for mu- and delta-receptors, and the mu-selectivity ratio, exceeded values characteristic of dermorphin. The dimerized amino-terminal dermorphin analogues are peptides whose receptor binding differed from the parent molecule; e.g. the affinity of dimeric tetrapeptides toward mu-receptors was reduced but was increased for delta-receptors relative to monomeric dermorphin-(1-4)-amide. Dimeric tetradermorphin linked by a bridge containing 12 methylene units (di-tetra-dermorphin12), exhibited a dramatic loss in the mu-selectivity ratio as a result of diminished mu-affinity. On the other hand, substitution of Gly4 by Sar in di-tetra-dermorphin2 enhanced binding to mu-receptors: substitution of D-Arg2 for D-Ala resulted in an increased binding to mu-receptors while decreasing binding to delta-receptors, yielding a peptide with the highest mu-selectivity ratio. These substitutions of D-Arg2 and Sar4 in dimeric amino-terminal dermorphin pentapeptides enhanced binding to both mu- and delta-receptors relative to dermorphin-(1-5)-amide, but led to a decrease in its mu-selectivity ratio. Several dimeric dermorphin analogues exhibited an enhanced mu-selectivity ratio relative to their monomeric analogues. Dimeric peptides, which had a relatively high affinity for mu-receptors, were effective in the suppression of gastric acid secretion.     

Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors. III. Probable binding conformations of mu-agonists with phenylalanine in position 3.

Theoretical conformational analysis was carried out for several tetrapeptide analogues of beta-casomorphin and dermorphin containing a Phe residue in position 3. Sets of low-energy backbone structures of the mu-selective peptides [N-Me-Phe3, D-Pro4]-morphiceptin and Tyr-D-Orn-Phe-Asp-NH2 were obtained. These sets of structures were compared for geometrical similarity between themselves and with the low-energy conformations found for the delta-selective peptide Tyr-D-Cys-Phe-D-Pen-OH and nonactive peptide Tyr-Orn-Phe-Asp-NH2. Two pairs of geometrically similar conformations of mu-selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the mu-receptor, were selected as two alternative models of probable mu-receptor-bound backbone conformations. Both models share geometrical similarity with the low-energy structures of the linear mu-selective peptide Tyr-D-Ala-Phe-Phe-NH2. Putative binding conformations of Tyr1 and Phe3 side chains are also discussed.     

Proton magnetic resonance studies on dermorphin and its peptide fragments

Dermorphin (Tyr? D-Ala? Phe? Gly? Tyr? Pro? Ser? NH₂), a potent natural peptide opioid, its synthetic L-Ala² analog, and all the N fragments from the tripeptide (Tyr? D -Ala? Phe? NH₂) to the parent hexapeptide amide were characterized for the first time by means of proton nmr spectroscopy at 11.74 T. Assignments of most protons of dermorphin were facilitated by the study of the N-terminal fragments. Comparison of spectroscopic parameters with relative pharmacological activity is proposed as a possible means of studying flexible agonists in solution.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

Enkephalin degradation in mouse brain studied by a new H.P.L.C. method: further evidence for the involvement of carboxydipeptidase

The degradation of the Met-enkephalin by peptidases present in striatum extracts was followed by a new H.P.L.C. method, based on ligand exchange chromatography. An aminopeptidase activity is present in both the soluble and the particulate fractions of mouse striatum. In addition we confirmed the presence, mainly in the particulate fraction, of a carboxydipeptidase releasing TyrGlyGly and PheMet from Met-enkephalin. The tetrapeptide GlyGlyPheMet also appears to be cleaved by the same enzyme activity.     

Separate metabolic pathways for Leu-enkephalin and Met-enkephalin-Arg6-Phe7 degradation by rat striatal synaptosomal membranes

Metabolism of Leu-enkephalin and Met-enkephalin-Arg⁶-Phe⁷ was studied using synaptosomal plasma membranes prepared from rat corpus striatum and whole brain. Cleavage of the pentapeptide was mediated largely by an aminopeptidase leading to the release of Tyr and Gly-Gly-Phe-Leu. Bestatin, an aminopeptidase inhibitor, prevented the release of Tyr and the tetrapeptide, but not secondary cleavage at the Gly Phe site leading to the release of Tyr-Gly-Gly and Phe-Leu. Cleavage at the latter site was inhibited by low concentrations of Thiorphan, an inhibitor of a non-aminopeptidase enkephalinase. MK-421, an inhibitor of the angiotensin converting enzyme, acted only at high substrate concentrations of Leu-enkephalin, indicating that the converting enzyme has a relatively low affinity for the pentapeptide. In contrast to the pentapeptide the major products found upon incubation of heptapeptide with synaptosomal plasma membrane were Arg-Phe and Met-enkephalin. Product release was inhibited by low concentrations of MK-421 but not by Thiorphan, indicating that the cleavage of the heptapeptide was mediated by the angiotensin converting enzyme. This pathway may represent a mechanism for the formation of Met-enkephalin from larger precursors present in striatum and other regions of the central nervous system.     

Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme

Highly purified human brain cathepsin H (EC 3.4.22.16) was used to study its involvement in degradation of different brain peptides. Its action was determined to be selective. On Leu-enkephalin, dynorphin (1-6), dynorphin (1-13), alpha-neoendorphin, and Lys-bradykinin, it showed a preferential aminopeptidase activity by cleaving off hydrophobic or basic amino acids. It showed no aminopeptidase activity on bradykinin, which has Pro adjacent to its N-terminal amino acid, on neurotensin with blocked N-terminal amino acid, or on dermorphin with second amino acid D-alanine. After prolonged incubation, cathepsin H acted as an endopeptidase. Dermorphin and dynorphin (1-13) were cleaved at bonds with Phe in the P2 position, while dynorphin (1-6), alpha-neoendorphin, bradykinin and Lys-bradykinin were cleaved at bonds with Gly in the P2 position. Further on, it was shown that human brain cathepsin H activity could be controlled in vivo by cystatin C in its full-length form or its [delta1-10] variant, already known to be co-localized in astrocytes, since the Ki values for the inhibition are in the 10(-10) M range.     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Raman and surface-enhanced Raman spectroscopy investigation of vasopressin analogues containing 1-aminocyclohexane-1-carboxylic acid residue

In this work, Raman spectroscopy (RS) was employed to characterize molecular structures of [Arg8]vasopressin (AVP) and its [Acc2,D-Arg8]AVP, [Acc3]AVP, and [Cpa1, Acc3]AVP analogues. The RS band assignments have been proposed. To determine the mechanism of adsorption of the above-mentioned compounds adsorbed on a colloidal silver surface, surface-enhanced Raman spectra (SERS) were measured. The SERS spectra were used to determine relative proximity of the adsorbed functional groups of [corrected] investigated peptides and their orientation on the silver surface. The AVP and [Acc3]AVP SERS spectra (Acc: 1-aminocyclohexane-1-carboxylic acid) show that the L-tyrosine (Tyr) lies far from the metal surface, whereas the [Cpa1,Acc3]AVP spectrum (Cpa: 1-mercaptocyclohexaneacetic acid) provides evidence that Tyr interacts with the silver surface. These results suggest that [corrected] the binding of the Tyr-ionized phenolic group might be responsible for the selectivity of the analogues. We show that the aromatic ring of L-phenylalanine (Phe) of AVP and [Acc2,D-Arg8]AVP interacts with the silver surface. The strength of this interaction is considerably weaker for [Acc2,D-Arg8]AVP than for AVP. This might be due either to a longer distance between the Phe ring and the silver surface, or to the almost perpendicular orientation of the Phe ring towards the surface. The carbonyl group of the L-glutamine [corrected] (Gln) or L-asparagine [corrected](Asn) of AVP, [Acc2,D-Arg8]AVP, and [Acc3]AVP is strongly bound to the silver surface. We have also found that all peptides adsorb on the silver surface via sulfur atoms of the disulfide bridge, adopting a "GGG" conformation, except [Cpa1,Acc3]AVP, which accepts a "TGG" geometry.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H-Tyr-D-Ala-Phe-Gly-NH2 using the 4-amino-2-benzazepin-3-one scaffold.

The synthesis of conformationally restricted dipeptidic moieties 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba)-Gly ([(4S)-amino-3-oxo-1,2,4,5-tetrahydro-1H-2-benzazepin-2-yl]-acetic acid) and 8-hydroxy-4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Hba)-D-Ala ([(4S)-amino-8-hydroxy-3-oxo-1,2,4,5-tetrahydro-benzo[c]azepin-2-yl]-propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N-acyliminium precursor. Introducing these Aba scaffolds into the N-terminal tetrapeptide of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2)-induced remarkable shifts in affinity and selectivity towards the opioid mu- and delta-receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N-terminal tetrapeptide H-Tyr-D-Ala-Phe-Gly-NH2, which is the minimal subunit of dermorphin needed for dermorphin-like opiate activity.     

Dermorphin-based potential affinity labels for mu-opioid receptors.

Dermorphin and [Lys7]dermorphin, selective micro -opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the 'message' or 'address' sequences as potential peptide-based affinity labels for micro -receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe3 and Phe5 was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine-containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing micro - and delta-opioid receptors. In dermorphin, introduction of the electrophilic groups in the 'message' domain lowered the binding affinity by > 1000-fold; only [Phe(p-NH2)3]dermorphin retained nanomolar affinity for micro -receptors. Modifications in the 'address' region of both dermorphin and [Lys7]dermorphin were relatively well tolerated. In particular, [Phe(p-NH2)5,Lys7]dermorphin showed similar affinity to dermorphin, with almost 2-fold higher selectivity for micro -receptors. [Phe(p-NHCOCH2Br)5]- and [Phe(p-NHCOCH2Br)5,Lys7]dermorphin exhibited relatively high affinity (IC50 = 27.7 and 15.1 nm, respectively) for micro -receptors. However, neither of these peptides inhibited [3H]DAMGO binding in a wash-resistant manner.     

Conformation-activity relationships of cyclo-constrained micro/delta opioid agonists derived from the N-terminal tetrapeptide segment of dermorphin/deltorphin.

The N-terminal tetrapeptide segments of dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) and deltorphin (Tyr-D-Ala-Phe-Asp/Glu-Val-Val-Gly-NH(2)) are agonists at the opioid receptors micro and delta, respectively. [D-Arg(2), Lys(4)]-dermorphin-(1-4) amide (Tyr-D-Arg-Phe-Lys-NH(2), DALDA) and [Dmt(1)]DALDA (where Dmt is 2',6'-dimethyltyrosine) are among the most potent and selective micro-agonists reported to date, both in vitro (having picomolar micro receptor affinity) and in vivo. In this communication, conformation-activity studies of the following four cyclic analogs of DALDA are presented and discussed: the lead peptide S(2),S(4)-cyclo (Tyr-D-Cys-Phe-Cys-NH(2)), constrained by means of an S(4.2)--S(4.4) disulfide between Cys(2) and Cys(4); its two cis and trans C(4.2)--C(4.4)-olefinic dicarba analogs, and the product of saturation of them both. They are potent nonselective or moderately micro-selective opioid agonists in vitro.They have been synthesized and tested earlier [Berezowska I, Chung NN, Lemieux C, Wilkes BC, and Schiller PW, Acta Biochim Polon 53, 2006, 73-76]. We have studied their conformations using NMR and molecular dynamics. With major conformational constraints imposed by the 11-membered ring spanning residues 2-4, they show well defined conformations of this ring, while the exocylic Tyr(1) and Phe(3) side chains still have significant conformational freedom. The more active and selective micro versus delta disulfide and saturated dicarba agonists seem to have in common: (i) their ring structures more flexilble than those of the other two and (ii) their ring structures similar to each other and more diverse than those in the other two. Given this and the small size of the peptides having confirmed bioactivity profiles, there is a chance that their conformations determined in solution approach receptor-bound conformations. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Dehydro-enkephalins. VIII. Attempted synthesis of dehydrotyrosine-enkephalins

Two kinds of dehydropeptide analogs of enkephalin containing a delta Tyr unit at the N-terminus have been synthesized by coupling Boc-delta Tyr-(Cl2 Bzl)-OH with amino acid amides and tetrapeptide esters using the water soluble carbodiimide-HOBt method. Pentapeptides consisting of delta Tyr1, and delta Phe4 or delta Leu5 were also prepared. Ultraviolet difference spectroscopy was important in the characterization of the dehydro moieties, delta Tyr, delta Phe and delta Leu. Attempts to liberate delta Tyr1-enkephalins have been unsuccessful because of the instability of an N-terminal delta Tyr residue having p-phenolic group in the side chain.     

Calcium-Dependent Release of Tyrosine in Brain Elicited by Stimulation of Neuropeptide Receptors

We sought to establish whether the endogenous opiate-receptor agonist Met-enkephalin (m-ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m-ENK (1-10 microM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylalanine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), taurine (Tau), and glycine (Gly) were similarly increased by 50-150% with m-ENK in slices of CS, but not Cx. The enhanced release of AAs by m-ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5-55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as gamma-aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m-ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8-51%) compared to that of GABA, Gly, Glu, and Tau (49-123%). Nonetheless, m-ENK (10 microM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction.(ABSTRACT TRUNCATED AT 250 WORDS)