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The syntheses of Ac-Asp-Gly-Ser and Ac-Asp-Pro-Leu-Gly-NH2 are described. The two peptides were prepared in solution by stepwise elongation using the DCC method with additives (HOSu or HOBt). The push-pull method was also used in the synthesis of Ac-Asp-Gly-Ser. A racemization test of the amino acids in this peptide has been performed.

Synthesis of the cis - and trans -isomers of the proline-leucine (3- i -Pr-Pro) and proline-phenylalanine (3-Ph-Pro) chimeric amino acids was accomplished by intramolecular reductive-cycloalkylation of the appropriate azido-olefin. These chimeric amino acids were incorporated into the triprolyl analogues of Pro-Leu-Gly-NH 2 : Pro- cis -3- i -Pr-Pro-Pro-NH 2 ( 3 ), Pro- trans -3- i -Pr-Pro-Pro-NH 2 ( 4 ), Pro- cis -3-Ph-Pro-Pro-NH 2 ( 5 ), Pro- trans -3-Ph-Pro-Pro-NH 2 ( 6 ).

A peptidomimetic analogue of Pro-Leu-Gly-NH 2 (PLG), compound 1-[2-pyrrolidinylcarbonyl]-tetrahydro-5'-oxospiro[pyrrolidine-2,6'-pyrrolo[2,1-b]thiazolidine]-3'-carboxamide (3), has been synthesized that contains a highly constrained spiro bicyclic type-II β-turn mimic. Peptidomimetic (3) enhanced the binding of the dopamine receptor agonist ADTN to dopamine receptors by 40% at 10 -6 M. At this same concentration PLG enhanced the binding of ADTN by 26%. Like PLG, (3) exhibited a bell-shaped dose-response curve with the maximum effect occurring at a concentration of 10 -6 M. Because of the highly rigid nature of the spiro bicyclic type-II β-turn constraint found in (3), these results lend strong support for the hypothesis that the biologically active conformation of PLG is a type-II β-turn

In order to investigate the relation between the conformation and the ability to modulate dopamine receptors, conformational free-energy calculations using an empirical potential (ECEPP/3) and hydration shell model were carried out on the tripeptide +H2-Pro-Leu-Gly-NH2 (PLG) and its analogues in which the Leu residue is replaced with Phe, Ahx (L-2-aminohexanoic acid), Ile, Abu and Ala residues in the unhydrated and hydrated states. PLG and two tripeptides possessing Phe and Ahx residues show dopamine receptor modulating activity, while the other tripeptides do not. Irrespective of activities, PLG and its analogues are found to have the high probabilities to form β-bends of types II and I in the unhydrated state. However, in the hydrated state, the β-bend probabilities of the PLG analogues decrease significantly compared with those in the unhydrated state. These results indicate that the β-bend structure is a necessary factor for the PLG analogues to be active, but not a sufficient factor, and that the interactions of water molecules with the backbone may force the tripeptides to be more distorted or extended. The size and the orientation of the hydrophobic moiety of the second residue seem to be of consequence in eliciting the activity of the tripeptides. © Munksgaard 1995.

Two series of conformationally constrained analogues of Pro-Leu-Gly-NH2 (PLG) have been synthesized. In one series of analogues, the Leu-Gly-NH2 dipeptide segment of PLG was replaced with the gamma-lactam residues 3(S)- and 3(R)-amino-2-oxopyrrolidineacetamide and the delta-lactam residue 3(S)-amino-2-oxopiperidineacetamide. The corresponding gamma-lactam analogues of less than Glu-Leu-Gly-NH2 were also synthesized. In a second series of analogues, the glycinamide residue of PLG was replaced with the 2-ketopiperazine, 3(S)-amino-2-pyrrolidone, and 3(S)-amino-2-piperidone residues. The above analogues were tested for their ability to enhance the binding of the dopamine receptor agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) to striatal dopamine receptors. Of the conformationally constrained analogues of PLG synthesized in this study, only the gamma-lactam analogue 3(R)-(N-L-prolylamino)-2-oxo-1-pyrrolidineacetamide (3) was found to possess significant activity. This analogue was 10,000 times more active than PLG, under preincubation conditions. It significantly enhanced the binding of ADTN at concentrations of 10(-9) and 10(-10) M.

The effect of the interaction between Pro-Leu-Gly-NH2 (PLG) and catecholamines on the release of MSH by rat pituitaries glands was studied in vitro. The catecholamines dopamine (DA) and nor

The effect of chronic administration of morphine to Sprague-Dawley rats on the binding of dopamine receptor agonist, 3H-N-n-propylnorapomorphine (3H-NPA) to striatal membranes was determine

Laser Raman spectra of Pro-Leu-Gly-NH2, the factor that inhibits release of pituitary melanotropin, have been obtained in the solid state, in dimethylsulfoxide and in aqueous solution. The amide I frequencies were observed at 1650 and 1687 cm−1 in the solid state and at 1669 cm−1 in dimethylsulfoxide. The conformation of this tripeptide has been proposed by 1H-NMR studies in [2H]-dimethylsulfoxide and revealed by X-ray analysis to be type II β-turn. These observed amide I frequencies thus are characteristic of type II β-turn conformation. The relatively lower amide I frequency, 1645 cm−1, observed in 2H2O indicates that the conformation of the peptide in aqueous solution could be different from those in solid state and in dimethylsulfoxide. pH changes have no significant effect on aqueous solution spectra, except for the shape of the amide III band. The maximum of the amide III band shifted from 1259 cm−1 at pH 2.0 to 1242 cm−1 at pH 11.7. The amide III peaks in the solid state were at 1234 and 1268 cm−1.

3H-Pro-Leu-Gly-NH2, a melanocyte-stimulating hormone release inhibiting factor, was injected into 5 healthy men. A plot of the time of the disappearance of radioactivity in plasm

The regulation of release of MSH and ACTH seems to be closely related. The effect of Pro-Leu-Gly-NH2, the most active MSH-release inhibiting factor (MIF), was investigated on ACTH-hypersecretion in six patients suffering from primary adrenocortical insufficiency. MIF was infused at constant rates with stepwise increase in concentration (0.25 mg, 2.5 mg, 25.0 mg/30 min). Plasma ACTH-levels were measured radioimmunologically at 10 minutes intervals. In four cases there was a remarkable decrease (P less than 0.005) in the ACTH plasma level of more than 50% as compared to the elevated starting levels of 800 to 1400 pg/ml. It is concluded that under certain circumstances MIF could play a role in the regulation of ACTH-secretion.

The effects of MIF-1 (Pro-Leu-Gly-NH 2 ) on activity and aggression of male Siamese Fighting Fish ( Betta splendens ) were considered. Animals were given intraperitoneal injections of 0.0 or 10.0 mg/kg MIF-1. After a 10-minute delay, they were placed in a 10 gallon aquarium and their activity was monitored for 60 minutes. Although aggressive responses in the presence of suitable opponents were not reliably affected, a significant decrease in general activity was produced. This is compatible with differential effects of MIF-1 across species.

With the idea to give further support that Pro-Leu-Gly-NH 2 (PLG) acts as MIF (the inhibitor factor of MSH), this paper reports the effect of PLG on the secretion of MSH release using a recently developed radioimmunoassay for α-MSH. Pro-Leu-Gly-NH 2 was effective in inhibiting the release of MSH induced by the injection of acid extracts of median eminence (MRF). The rise in plasma MSH by these extracts was not due to their intrinsic content of MSH. Pro-Leu-Gly-NH 2 also inhibited the basal secretion of MSH in male rats. Since PLG also blocked the release of MSH induced by the injection of haloperidol, it is suggested that its effect is not mediated by dopamine.

Several analogues of L-prolyl-L-leucylglycinamide (PLG) were synthesized wherein the prolyl residue was replaced with other heterocyclic amino acid residues. Among the analogues synthesized were D-Pro-Leu-Gly-NH2 (2), less than Glu-Leu-Gly-NH2 (3), Thz-Leu-Gly-NH2 (4), Pip-Leu-Gly-NH2 (5), Aze-Leu-Gly-NH2 (6), L-delta 3,4-Pro-Leu-Gly-NH2 (7), and D-delta 3,4-Pro-Leu-Gly-NH2 (8). These analogues were tested for their ability to enhance the binding of the agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene to central dopamine receptors. Analogues 2, 3, and 5-7 showed activity comparable to that of PLG, while the tripeptides 4 and 8 were found to be inactive. The results show that the N-terminal prolyl residue of PLG is not an essential requirement for this tripeptide's ability to modulate dopamine receptors.

Repeated intraventricular injections of human β-endorphin to rats every eight hours for three days resulted in the development of super-sensitivity of brain dopamine receptors as evidenced by an enhanced locomotor activity response to apomorphine, a dopamine receptor agonist. Daily subcutaneous injections of Pro-Leu-Gly-NH2 or its cyclic analog, cyclo (Leu-Gly) blocked the enhanced apomorphine-induced stimulation of locomotor activity in β-endorphin treated rats. Thus, the development of brain dopamine receptor supersensitivity induced by chronic β-endorphin administration may be regulated by the hypothalamic peptides.

The synthesis of several analogues of L-prolyl-L-leucylglycinamide (PLG) was carried out in which the glycinamide residue was replaced with the following cyclic amino acid residues: L- and D-prolinamide, (+)- and (-)-thiazolidine-2-carboxamide, L- and D-3,4-dehydroprolinamide, L-azetidine-2-carboxamide, L-piperidine-2-carboxamide, and L-thiazolidine-4-carboxamide to give PLG analogues 2-10, respectively. The ability of these analogues to enhance the binding of the dopamine agonist ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene) to dopamine receptors was determined by using bovine brain tissue. All of the PLG analogues synthesized in this study enhanced the binding of ADTN to central dopamine receptors. The percent enhancement of ADTN binding produced by analogues 2,3, and 7-10 at various concentrations was comparable to the percent enhancement produced by PLG. The PLG analogues Pro-Leu-(+)-thiazolidine-2-carboxamide (4), Pro-Leu-(-)-thiazolidine-2-carboxamide (5), and Pro-Leu-L-3,4-dehydroprolinamide (6), however, produced significantly greater enhancement (2-3-fold) in ADTN binding than did PLG.

Pro-Leu-Gly-NH2 (MIF) inhibits the tremor induced by oxotremorine. Objective measurement of this tremor permits the drawing of a dose-effect curve. The inhibitory effect of the peptide increases linearly with increasing doses until an optimum is reached (between 30 and 40 mg/kg i.p.). At still higher doses the peptide is inactive. The same phenomenon is observed with analogues of MIF. This finding may have important bearings on the interpretation of clinical and experimental data obtained with MIF.

Single doses of MIF-1 (0.03–2.0 mg/kg, SC) and chronic pretreatments with MIF-1 (0.03–2.0 mg/kg, SC, BID, 3 1 2 days) or pareptide (0.25 mg/kg, SC, BID, 3 1 2 days) did not affect the acute cataleptic response to haloperidol in the mouse. Chronic pretreatment with haloperidol (8.0 mg/kg, IP, BID, 3 days) decreased the duration of catalepsy in mice given smaller challenge doses of haloperidol (2.0 or 3.0 mg/kg, IP) 15 hours after the last pretreatment injections. Administration of either MIF-1 or pareptide to mice also chronically pretreated with haloperidol antagonized the development of tolerance.

Pro-Leu-Gly-NH 2 , tocinoic acid ( Cys-Tyr-Ile-Gln-Asn-Cys -OH), and des-Cys-tocinoic acid (Cys-Tyr-Ile-Gln-Asn-OH) were studied for their effects on release of melanocyte stimulating hormone (MSH) from rat pituitaries. These peptides neither blocked nor stimulated secretion in cell culture systems. Pro-Leu-Gly-NH 2 also failed to alter MSH release when added to whole- and hemi-pituitary incubation systems.

A crystal-state structural analysis of Boc-Pro-Leu-Gly-NH2(2a) hemihydrate and two conformationally restricted analogues that incorporate a γ- or δ-lactam modification at the –Leu-Gly– sequence, (3) and (4) respectively, has been performed by X-ray diffraction. In all three compounds the conformation of the tertiary amide group of the Nα-protecting urethane moiety is cis and the prolyl residue is semi-extended. The basic conformational feature of the published structure of H-Pro-Leu-Gly-NH2(1), the ten-membered ring type-II β-bend structure at the C-terminus, is preserved in the Nα-t-butyloxycarbonyl derivative (2a). However, the γ- and δ-lactam modifications induce drastic changes in the backbone torsion angles of the –Leu-Gly– sequence, eventually promoting the onset of extended conformations.