Synthesis and biological activity of some new leucine-enkephalin analogues

The opioid pentapeptide leucine-enkephalinamide and eleven of its analogues have been synthesised by the solid phase technique employing mostly 9-fluoroenylmethyloxycarbonyl amino acid active esters in the presence of 1-hydroxybenzotriazole. Both the conventional chloromethylated copolystyrene-2% divinylbenzene resin as well asp-alkoxybenzyl alcohol resin were employed and it was observed that yields were uniformly better with the latter resin. The analogues were made by affecting single or multiple replacements of amino acids involving positions 1,2 and 5. Some of the analogues were found to be more potent than morphine in the guinea pig ileum assay.     

Purification of opioid-binding materials from rat brain

Opiate receptors were solubilized from rat brain with digitonin and treated with an affinity resin, AH-Sepharose coupled with [D-Ala2, Leu5]enkephalin. The eluted materials from the resin with the opioid peptide had opioid-binding activity. Based on the protein content of the purified materials, 450-fold purification over the solubilized receptors was achieved. Analyses by gel electrophoresis revealed that the purified materials were rich in one polypeptide with a molecular weight of 62,000.     

Synthesis of novel basic head-to-side-chain cyclic dynorphin A analogs: strategies and side reactions

Novel N-terminus-to-side-chain cyclic analogs of the opioid peptide dynorphin (Dyn) A-(1-11)NH(2) were prepared that retain the basicity of the N-terminal amine and restrict the backbone conformation around the important Tyr(1) residue. Cyclic peptides were synthesized in which the N-terminal amine and the N(epsilon)-amine of a Lys at position 3 or 5 were attached to the alpha-carbon and carbonyl of an acetyl group, respectively. Several synthetic strategies were explored with detailed analysis of the side reactions in order to obtain the desired cyclic peptides. One of the side reactions observed involved premature loss of the N-terminal 9-fluorenylmethoxycarbonyl (Fmoc) group during the neutralization step following deprotection of the Mtt (4-methyltrityl) protecting group from the side chain of Lys. The successful strategy involved the synthesis of the linear peptide up through Gly(2) and functionalization through the N(epsilon)-amine of Lys. A linear N-terminal alkylated analog was prepared by alkylation of the peptide on the resin with an equimolar amount of bromoacetamide, followed by treatment of the peptide with Fmoc-OSu prior to cleavage from the resin to facilitate separation by reversed phase high performance liquid chromatography of unreacted peptide from the desired alkylated product. The novel N-terminal cyclic Dyn A analogs and the linear analog were evaluated for their opioid receptor affinities. These peptides exhibited large losses in affinity for opioid receptors; the low affinity of the linear N-terminal alkylated peptide suggested that the alpha-acetamide group on the N-terminal amine resulted in unfavorable interactions with opioid receptors.     

Novel opioid peptides derived from casein (beta-casomorphins). I. Isolation from bovine casein peptone

A material which displayed opioid activity in the guinea pig ileum longitudinal muscle-myenteric plexus preparation was extracted from an enzymatic casein digest into chloroform/methanol. The extract was roughly purified by adsorption/desorption procedures using charcoal and Amberlite XAD-2 resin as adsorbents. A high degree of purity was achieved by high-pressure liquid chromatography of the material on muBondapak C18 and mu-Porasil columns and finally by gel filtration chromatography on a Bio-Gel P-2 column. Several pronase-resistant compounds with opioid activity were obtained.     

Fmoc-amino acid chlorides in solid phase synthesis of opioid peptides.

Fmoc-amino acid chlorides were employed in the solid phase synthesis of the opioid peptides [Leu]enkephalin, [Leu]enkephalin amide, and dermorphin. The conventional polystyrene-based Merrifield resin or Wang's resin served as solid support. A binary salt of either triethylamine or diisopropylethylamine in the presence of 1-hydroxybenzotriazole or pivalic acid was used for acylation. The coupling rates were quite fast, being comparatively faster when 1-hydroxybenzotriazole was used along with triethylamine or diisopropylethylamine. The peptides obtained in good yields showed, after purification, biological and spectral properties identical with those of the natural peptides.     

Reconstitution of partially purified opioid receptors with a GTP-binding protein

Partially purified opioid receptors, obtained from rat brains using an affinity resin, AF-Amino Toyopearl with [D-Ala2, Leu5]enkephalin, were reconstituted with an inhibitory GTP-binding protein (Gi). In the reconstituted system, the displacement curve for the binding of a delta-agonist, [D-Ala2, D-Leu5]enkephalin, showed two states, high and low affinity binding ones, with different affinities for the agonist. The high affinity binding was eliminated by the addition of a guanine nucleotide analog to the system. These results directly showed that opioid receptors, at least the delta-type, could interact with Gi.     

Peptides Derived from α-hydroxymethylserine: Aspects of solid-phase synthesis

Summary The solid-phase synthesis of peptides derived from the sterically hindered α-hydroxymethylserine (HmS) was investigated. The acid-sensitive,O,O-isopropylidene (Ipr) protection of HmS is compatible with the Fmoc chemistry, represented here by the Fmoc-HmS(Ipr)-OH and Fmoc-HmS(Ipr)-F derivatives. Three analogs of the opioid pentapeptide DADLE with a single or two consecutive HmS residue(s) were synthesized using Wang resin as the solid support. The HATU method has been shown to effectively accomplish ‘difficult’ couplings with the HmS(Ipr) residue. Wang resin is not suitable, for the synthesis of sequences with a C-terminal HmS because of the easy formation of the diketopiperazine resulting from the cyclization of the susceptible dipeptide sequence AA-HmS(Ipr) bound to the resin. A further drawback of the Wang resin methodology is the increased danger of the undersired N→O-acyl shift, when long-lasting acidic cleavage is applied. These side reactions are totally suppressed when the 2-chlorotrityl polystyrene is used as a solid support. The mild conditions (AcOH/TFE/DCM) applied for the peptide detachment from this resin do not affect the Ipr protection, affording highly pure fragments with HmS(Ipr) residues suitable for post-cleavage condensation, cyclization or controlled side-chain deprotection. This approach is documented by the efficient synthesis of linear and cyclic analogs of the opioid hexapeptide DTLET containing two residues of HmS or HmS(Ipr) in positions 2 and 6.     

Early Onset and Accumulation of Rem Sleep in Depression: A Study of the Phase-Advance Hypothesis

–Twenty-two depressed subjects who met criteria for major depressive disorder were grouped according to their initial REM latency. Subjects with short (≥ 60 min) initial REM latency were separated from those with normal (< 60 min) initial REM latency. Subjects with short initial REM latency were found to have earlier onsets to at least two subsequent REM periods. The number of minutes of REM sleep accumulated were also plotted against elapsed time after sleep onset. The short-latency group accumulated REM sleep earlier than, but at about the same rate as, the normal latency group. These data support the phase-advance hypothesis of REM sleep in depression.     

Synthesis of peptidomimetics: An evaluation of the p-nitrophenyl carbamate of ethylenediamine

The application of p-nitrophenyl carbamate of Boc-ethylenediamine forthe solid-phase synthesis of peptidomimetics was examined. The per step yield of coupling was estimated using mass spectrometry, based on the repeated coupling of the same monomer in the synthesis of alkylurea oligomers. Introduction of the urea moiety at the terminus of the chain adjacent to the resin was accomplished by the use of the BHA resin in the assembly of the chain and liquid HF in the cleavage step. In addition, an alkylurea oligomer was treated with bis(p-nitrophenyl) carbonate followed by ammonolysis in order to obtain a urea moiety at the terminus distant from the resin. Aside from the expected oligomer, a product was obtained in which the terminal alkylurea had undergone cyclization. Finally, four peptidomimetics, analogues of 1–4 enkephalin fragment, containing up to four alkylurea units instead of glycine residues, were synthesized. Two of these peptidomimetics were examined for opioid activity and turned out to be active in the guinea pig ileum assay.     

Characterization of opioid peptides in the rat stomach

A combination of several chromatographic and assay systems was used to characterize the opioid peptides in rat stomach extracts. Partial purification of opioid material in acetic acid extracts of the corpus plus antrum regions of the rat stomach was carried out by gel filtration chromatography on Sephadex G-50, followed by adsorption onto Amberlite XAD-2 resin. A single peak in opioid activity was determined by both radioreceptor assay (RRA) and bioassay. By high performance liquid chromatography, this peak was resolved into five distinct components, characterized by RRA and (or) radioimmunoassay, with retention times corresponding to methionine enkephalin (met-enk), leucine enkephalin, met-enk-arg6-gly7-leu8, met-enk-arg6-phe7, and dynorphin 1-13. Closer examination of the dynorphin component revealed the presence of dynorphins 1-17, 1-13, and 1-8. Trypsin digestion of the partially purified (Sephadex G-50 and Amberlite XAD-2 chromatographed) extract resulted in an overall increase in opioid activity, suggesting the presence of larger, possibly precursor forms.     

Synthesis of peptidomimetics: An evaluation of the p-nitrophenyl carbamate of ethylenediamine

Summary The application of p-nitrophenyl carbamate of Boc-ethylenediamine for the solid-phase synthesis of peptidomimetrics was examined. The per step yield of coupling was estimated using mass spectrometry, based on the repeated coupling of the same monomer in the synthesis of alkylurea oligomers. Introduction of the urea moiety at the terminus of the chain adjacent to the resin was accomplished by the use of the BHA resin in the assembly of the chain and liquid HF in the cleavage step. In addition, an alkylurea oligomer was treated with bis(p-nitrophenyl) carbonate followed by ammonolysis in order to obtain a urea moiety at the terminus distant from the resin. Aside from the expected oligomer, a product was obtained in which the terminal alkylurea had undergone cyclization. Finally, four peptidomimetics, analogues of 1–4 enkephalin fragment, containing up to four alkylurea units instead of glycine residues, were synthesized. Two of these peptidomimetics were examined for opioid activity and turned out to be active in the guinea pig ileum assay.     

Effects of bromocriptine and alpha-flupenthixol on sleep in REM sleep deprived rats.

Administration of bromocriptine mesylate (5 mg/kg, i.p.), a dopamine receptor stimulant, to rats which were deprived of REM sleep for 24 hours resulted in a significant increase in wakefulness as well as significant reduction of REM sleep during the first 5 hours of EEG recording. These effects were completely abolished by pretreatment with α-flupenthixol (0.2 mg/kg, i.p.), a dopamine receptor blocker. The loss of REM sleep has not been regained during the next 25 hours of EEG recording suggesting that the stimulation of dopamine receptors reduced REM sleep without causing subsequent REM rebound. These data raise questions on the negative dopamine control of REM sleep and on the potential use of dopamine stimulants in clinical situations characterized by excessive REM or by REM sleep dysfunction (narcolepsy).     

Ureido group containing cyclic dermorphin(1-7) analogues: synthesis, biology and conformation.

Six cyclic peptides related to dermorphin(1-7) have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid agonist activities were observed, depending on the size of the ring. The results were compared with those obtained earlier for 1-4 dermorphin analogues. The conformations of all six dermorphin analogues were studied. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. On the basis of NMR data, an ensemble of conformations was obtained for each peptide. The opioid activity profiles of the compounds are discussed in the light of the structural data.     

An ether stressor increases REM sleep in rats: possible role of prolactin

Sleep alterations after a 1-min exposure to ether vapor were studied in rats to determine if this stressor increases rapid eye-movement (REM) sleep as does an immobilization stressor. Ether exposure before light onset or dark onset was followed by significant increases in REM sleep starting approximately 3-4 h later and lasting for several hours. Non-REM (NREM) sleep and electroencephalographic slow-wave activity during NREM sleep were not altered. Exposure to ether vapor elicited prolactin (Prl) secretion. REM sleep was not promoted after ether exposure in hypophysectomized rats. If the hypophysectomy was partial and the rats secreted Prl after ether exposure, then increases in REM sleep were observed. Intracerebroventricular administration of an antiserum to Prl decreased spontaneous REM sleep and inhibited ether exposure-induced REM sleep. The results indicate that a brief exposure to ether vapor is followed by increases in REM sleep if the Prl response associated with stress is unimpaired. This suggests that Prl, which is a previously documented REM sleep-promoting hormone, may contribute to the stimulation of REM sleep after ether exposure.     

Influence of fear conditioning on elicited ponto-geniculo-occipital waves and rapid eye movement sleep

The amygdala plays a central role in fear conditioning, a model of anticipatory anxiety. It has massive projections to brainstem regions involved in rapid eye movement sleep (REM) and ponto-geniculo-occipital (PGO) wave generation. PGO waves occur spontaneously in REM or in response to stimuli. Electrical stimulation of the central nucleus of the amygdala enhances spontaneous PGO wave activity during REM and the amplitude of both the acoustic startle response and the elicited PGO wave (PGOE), a neural marker of alerting. This study examined the effects of fear conditioning on REM and on PGOE. On conditioning days, the number of REM episodes, the average REM duration and the REM percentage were decreased while REM latency was increased. The presentation of auditory stimuli in the presence of a light conditioned stimulus produced PGOE of greater amplitudes. The results suggest that fear, most likely involving the amygdala, can influence REM and brainstem alerting mechanisms.     

The influence of prior wakefulness on REM sleep

Data from studies of naps and of shifted sleep were used to determine the relationship between two measures of rapid eye movement (REM) sleep (percentage of REM in the first 2 hr of sleep and REM latency) and prior wakefulness. For each sample, we calculated the difference between the observed value and that predicted by a cosine function that estimated the circadian rhythm of REM sleep propensity. The difference values were found to correlate reliably with hours and log hours of prior wakefulness. We conclude that while REM sleep is regulated in part by an endogenous circadian oscillator, it is also influenced by the duration of prior wakefulness.     

Dream recall after night awakenings from tonic/phasic REM sleep

Eleven healthy subjects, 9 females and 2 males aged 21-23, were submitted to all night polygraphic recording and awaken in REM (Rapid Eye Movements) sleep, randomly upon tonic or phasic REM. Immediately upon awakening subjects were asked about possible dreaming according to the standardized questionnaire. Seventy-seven dreams, i.e. 79% of all 97 REM awakenings, were reported and analyzed. There were no significant differences in reported frequency of dreamings after awakening, mood and dream content due to phasic/tonic REM sleep. Dreams from phasic REM were a bit more colorful. Predictor of morning remembering of dreams was meaninglessness, not meaningfulness of dreams, and, in lesser extent, good mood, colorfulness, dreams with words and phasic REM sleep.     

Fragment replica-exchange method for efficient protein conformation sampling

The native structure of proteins corresponds to the global minimum of the free energy. The replica-exchange method (REM) has been recently used to search for the energy minimum in a wide protein conformation space. For large systems, however, applying REM can be costly because the number of replicas required for conformational sampling increases. We have developed a variant of REM called fragment REM, which is based on the existence of correlations between the local amino acid sequence and the local structure. Equilibrium distributions for two peptides were computed by conventional molecular dynamics, REM and the proposed REM simulations. We found that the modified REM successfully reduces the number of replicas needed for the simulation.     

Effects of selective sleep deprivation on ventilation during recovery sleep in normal humans.

To assess the effects of selective sleep loss on ventilation during recovery sleep, we deprived 10 healthy young adult humans of rapid-eye-movement (REM) sleep for 48 h and compared ventilation measured during the recovery night with that measured during the baseline night. At a later date we repeated the study using awakenings during non-rapid-eye-movement (NREM) sleep at the same frequency as in REM sleep deprivation. Neither intervention produced significant changes in average minute ventilation during presleep wakefulness, NREM sleep, or the first REM sleep period. By contrast, both interventions resulted in an increased frequency of breaths, in which ventilation was reduced below the range for tonic REM sleep, and in an increased number of longer episodes, in which ventilation was reduced during the first REM sleep period on the recovery night. The changes after REM sleep deprivation were largely due to an increase in the duration of the REM sleep period with an increase in the total phasic activity and, to a lesser extent, to changes in the relationship between ventilatory components and phasic eye movements. The changes in ventilation after partial NREM sleep deprivation were associated with more pronounced changes in the relationship between specific ventilatory components and eye movement density, whereas no change was observed in the composition of the first REM sleep period. These findings demonstrate that sleep deprivation leads to changes in ventilation during subsequent REM sleep.