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Both isomers of (E)-2,3-methanophenylalanine (delta EPhe), a sterically restricted amino acid, were incorporated into peptides in order to examine their possible enzyme inhibitory activity. Both (2R,3S)- and (2S,3R)- delta EPhe-Phe(or Leu)-OMe were found to inhibit effectively the hydrolysis of Ac-Tyr-OEt by chymotrypsin in a competitive manner. The ester groups of these dipeptides were quite resistant to chymotrypsin hydrolysis, and the delta EPhe-Phe peptide bond was also entirely stable. The inhibition constant (Ki) of the most potent dipeptide of H-(2R,3S)-delta EPhe-Phe-OMe was 0.16 mM at 25 degrees C. The inhibitory action of delta Phe-containing peptides was found to depend on the configuration of the delta Phe residue. The electrophilic nature of the cyclopropane ring which is conjugated with both the phenyl ring and the ester carbonyl group appears to be relevant to the inhibitory activity. Fully irreversible inactivation of chymotrypsin was achieved by its incubation with H-(2R,3S)- delta EPhe-Leu-OMe. An enzyme carboxylate group is thought to be responsible for nucleophilic attack on the cyclopropane ring leading to irreversible inactivation.

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog [D-Ala2, cysteamine 5]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.

An AM-toxin analog, cyclotetradepsipeptide, containing an O-methyl-L-tyrosine residue in place of an L-2-amino-5-arylpentanoic acid residue in AM-toxins has been prepared by a conventional method. The synthesis was attempted through six different routes and the desired analog was obtained by one such procedure. This compound showed extremely weak activity in causing necrosis on apple leaves.

The Gly3 residue in Gly2- and D-Ala2-enkephalins has been replaced by delta Ala3 and Ser3 in order to examine the effect on binding to the delta and mu opiate receptors. [D-Ala2, delta Ala3, Leu5]-enkephalin maintains most of its receptor binding affinities for both sites. It is suggested that the proper conjunctions of positions 2 and 3 of the enkephalin sequence are important to receptor preference and that position 3 may have a very specific interaction with the delta receptor. The in vivo analgesic and CNS activities of the peptides are also discussed.

The fluorescent enkephalins in which an essential Tyr1 residue is replaced by L-1-pyrenylalanine (Pya) were synthesized and examined in the receptor binding assays. [Pya1, Leu5]Enkephalin and its methyl ester showed binding characteristics specific for the opiate receptors, exhibiting a potent inhibition of Tyr1-containing enkephalins. Surprisingly, the methyl ester displayed almost the same potencies to those of DAGO-enkephalin. This analog bound 24-fold more strongly to mu than to delta-receptors. C-terminal free analog Pya1-Enk-OH was delta-preferential with a fairly good affinity. These results indicate that Tyr1 in enkephalin is not necessary to recognition of the opiate receptors.

[delta Ala2, Leu5]-enkephalin has been prepared and shown to be more active than the parent saturated enkephalin in a binding assay using rat brain membranes and [3H]dihydromorphine as a tracer. In a comparison of potencies against [3H]dihydromorphine and [3H]-[D-Ala2, D-Leu5]-enkephalin as tracers, [delta Ala2, Leu5]-enkephalin showed preference for micro opiate receptors, possibly due to the hydrophobicity of the delta Ala2 residue. A synthetic tetrapeptide enkephalin [delta Ala2]-desLeu5-enkephalin had weak activity and high selectivity for the micro receptors. O-Acylation of a serine residue in the peptide was achieved by coupling between the peptide and a carboxylic acid using DCC and a catalytic amount of 4-dimethylaminopyridine.

The receptor binding properties and biological actions of chemically deglycosylated asialo human CG (AHF-hCG) were studied in ovarian granulosa cells from diethylstilbestrol (DES)-treated immature rats. In ovarian homogenates from DES- and FSH-treated rats, the relative binding affinity of AHF-hCG was 2-fold higher than that of native hCG and 14-fold higher than that of ovine LH. When assayed for LH-like activity in granulosa cells from DES plus FSH-treated animals, the deglycosylated hormone behaved as a partial agonist in terms of cAMP formation, but fully stimulated progesterone production to the same extent as that elicited by LH. When added with LH to FSH-treated cells, AHF-hCG inhibited LH-stimulated cAMP formation by 70% but did not alter the elevated level of progesterone production. These findings are consistent with the presence of excess or spare LH receptors in the maturing granulosa cell. When added to freshly prepared granulosa cells which have minimal LH receptors, AHF-hCG decreased FSH-stimulated cAMP production by 20% and reduced progesterone production by 50% and increased cGMP formation by 100% during 48 h of culture. The ability of AHF-hCG to decrease the progesterone response to FSH suggests that no spare FSH receptors are present during granulosa cell differentiation. In contrast, native hCG did not alter FSH-induced cAMP or progesterone production but reduced the cGMP responses to FSH and choleragen. Whereas native hCG displayed negligible binding potency when compared with that of ovine FSH in competition with [125I]iodo-human FSH for ovarian receptors, AHF-hCG bound to FSH receptors with about 5% of the binding affinity of ovine FSH. In choleragen-treated granulosa cells, the increases in cAMP and progesterone synthesis were enhanced by addition of both hCG and AHF-hCG, and cGMP production was increased by AHF-hCG but slightly decreased by hCG. These results indicate that the enhanced LH receptor affinity caused by removal of the sugar moieties from hCG is accompanied by a relatively greater increase in FSH receptor affinity, and that deglycosylated hCG acts as a partial agonist with the ability to modify granulosa cell responses to both LH and FSH.

The delta Phe4-enkephalins have been synthesized and examined in an in vitro receptor binding assay and an in vivo tail flick analgesia test. The delta Phe4 residue was derived from Boc-Gly-Phe(beta-OH)-OH by spontaneous dehydration and azlactonization. The dipeptide azlactone was coupled directly with H-Leu-OBzl to yield a tripeptide which was converted into the pentapeptides after stepwise coupling with two amino acids using the water soluble EDC-HOBt method. Dehydroenkephalins were liberated with hydrogen fluoride in the presence of anisole. In the radioligand binding assay which did not contain an enzyme inhibitor [D-Ala2, delta Phe4, Leu5] enkephalin was almost twice as active as saturated [D-Ala2, D-Leu5]-enkephalin. The delta Phe4-enkephalins exhibited a considerably diminished activity as compared with the saturated peptide in the in vivo analgesic assay. These results are discussed with regard to the enzyme stability and receptor preference of dehydroenkephalins.

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.

We have synthesized the first enkephalin analog containing a "cyclopropyl" phenylalanine (Phe) residue. The E-configuration of this residue is apparently responsible for its low activity in the MVD and GPI muscle assays. The enkephalin is very stable to cleavage by carboxypeptidase Y.

The fluorescent amino acid, L-1-pyrenylalanine (Pya) was incorporated into [D-Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya-enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4-enkephalins showed high specificity for the mu receptors, while Pya5-enkephalins showed high specificity and selectivity for the delta receptors. Particularly, [D-Ala2,Pya5]enkephalin was as potent as the most utilized delta-specific ligand of [D-Ala2,D-Leu5]enkephalin (DADLE), and yet its delta-selectivity was about 5-times greater than that of DADLE. Thus, Pya-enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor-binding assays.

Conformationally restricted enkephalin analogs containing E-cyclopropylphenylalanine (delta EPhe), [D-Ala2, (2R,3S)-delta EPhe4,Leu5]enkephalin and its (2S,3R) isomer, were evaluated in receptor-binding assays using rat brain and in assays using muscle preparations. The (2S,3R) isomer was almost completely inactive in all assays. In contrast, the (2R,3S) isomer showed a very high affinity for the delta and a very weak affinity for the mu receptors in rat brain. The extent of delta affinity and the selectivity of this isomer were almost equal to those of [D-Pen2,D-Pen5]enkephalin. However, the (2R,3S) isomer was inactive in both the mouse vas deferens and guinea pig ileum assays, and showed no antagonistic activity in these tissues. These results indicate that the (2R,3S) isomer interacts with the delta receptors in rat brain, but not with those in the mouse vas deferens, and they suggest that the delta receptors in the central and peripheral nervous systems are different from each other.

[D-Ala(2)(2R,3S)-delta(E)Phe(4)Leu(5)]enkephalin (CP-OH) [delta denoting cyclopropyl; superscript E indicating the E-configuration about the cyclopropane ring], a highly selective opioid ligand for delta receptors in rat brain, but not for those in the mouse vas deferens, was examined for in vivo biological activities by intracerebroventricular administration. CP-OH (5-20 micrograms) showed no analgesic activity in the hot plate (51 degrees C) test using rats. However, it suppressed completely the analgesic effects of intraperitoneally administered morphine (3 mg/kg rat) in a dose-dependent manner. CP-OH showed no binding affinity for brain kappa receptors to which dynorphin, an opioid peptide that inhibits morphine analgesia, binds predominantly. These results suggest that, besides the conventional delta receptors which mediate analgesia, the rat brain contains another delta-like receptor which has a modulatory role to attenuate morphine-induced analgesia mediated through the mu receptors, and that this modulatory receptor does not exist in the mouse vas deferens.

The N-terminal tripeptide enkephalin analogue, Tyr-D-Ala-Gly, was dimerized at the C-terminus systematically with a series of alpha,omega-diaminoalkanes, NH2-(CH2)n-NH2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The binding affinities of dimers for delta opiate receptors in rat brain were evaluated and compared with those for delta receptors in NG108-15 cells. Although the monomeric tripeptide amide was almost inactive, dimers showed a dramatic increase in binding affinity (8-900 times). The enhancement of affinity was apparently related to the number of methylene chains in the crosslinking spacer moiety, and it was maximal at n = 14-18 in the rat brain. In NG cells the activity increased progressively from n = 2 to n = 22 without reaching any apparent peak. These results suggest that delta receptors in rat brain and NG cells may have slight structural differences.

A useful set of empirical rules is put forward to predict the conformations of cyclic tetrapeptides and cyclic tetradepsipeptides on the basis of primary structure, briefly presented as follows: A conformation allowing an intramolecular hydrogen bond (IMHB) of gamma-turn is preferred, and an ester bond always adopts a trans form. On a right-handed peptide ring, the carbonyl group acylating a D residue is oriented to the upper side of the main ring. The carbonyl group acylating a D proline or an N-methyl-D-amino acid residue is oriented to the lower side of the ring, forming a cis bond. The LDDL configurational sequence adopts a cis-trans-cis-trans backbone with Ci symmetry. A glycine residue behaves as a D residue in an L-peptide. Conformations of cyclotetrapeptides containing two glycine residues at diametric positions or containing an N-methyl-dehydroamino acid residue are predicted by use of appendices of rule 5. Almost all conformations of cyclic tetrapeptides are predicted by these rules. Energetical rationalization of the rules and prediction of possible new conformations are described. Conformations of cyclo(-L-Pro-L-Leu-D-Tyr(Me)-L-Ile-)(1) and cyclo (-L-Pro-D-Leu-D-Tyr(Me)-L-Ile)(2) are compared. Results of n.m.r. experiments showed that compound 1 adopts a unique cis-trans-trans-trans backbone with a gamma-turn IMHB, and 2 has a cis-trans-cis-trans backbone with Ci symmetry. These observations confirmed the rules described above. Peptides 1 and 2 are the first diastereomeric peptides with trans (LD) and cis (DD) secondary amide bonds.

Dimeric analogues of the inactive enkephalin fragment Tyr-D-Ala-Gly were synthesized by cross-linking with alkanediamine at the C-terminus. Biological evaluation of these dimers (H-Tyr-D-Ala-Gly-NH)2.(-CH2-)n (DTREn), where n = 0-6, revealed that the fragment inactive for mu receptors was activated by its dimerization, with the maximum activation found with DTRE2, and that the dimer was highly mu-selective. So-called "handicapped" dimers, which lack one of the essential groupings required for enkephalin activity, were found to be far less active, indicating that the dimer interacts bivalently with mu receptors. It seems, therefore, that mu opiate receptors contain at least two equivalent binding sites which are extremely close to each other.

Dimeric analogs of neurokinin A and neurokinin B COOH-terminal heptapeptides were synthesized in order to examine the effect of ligand dimerization on the receptor selection. Dimerization was carried out at the NH2-terminus of peptides with succinic acid, yielding succinyl bis[Asp-Ser-Phe-Val-Gly-Leu-Met-NH2] (D-NKA4-10) and succinyl bis[Asp-Phe-Phe-Val-Gly-Leu-Met-NH2] (D-NKB4-10). In the assay using rat vas deferens (RVD), it was found that the deletion of the NH2-terminal tripeptide from native neurokinin A or B enhances the activity 1.5- to 8-fold, resulting in formation of NK-2 receptor specific ligands NKA4-10 and NKB4-10. When dimeric analogs of these shortened peptides, namely D-NKA4-10 and D-NKB4-10, were examined in RVD and guinea pig ileum (GPI), they were fairly potent in GPI, but not in RVD. Under conditions in which the NK-1 receptors in GPI were desensitized with NK-1 specific substance P methyl ester, dimers reduced the activity drastically, while the corresponding monomers exhibited unchanged activity. These results suggest that dimerization of the COOH-terminal heptapeptide of neurokinins changes the receptor selection of peptides from NK-2 to NK-1, and that the NK-1 receptor has a structure favorable to a dimeric peptide ligand.

To confirm the structure of AM-toxin I (a phytotoxic cyclotetradepsipeptide) the proposed peptide was prepared by a conventional method. The synthetic peptide and natural AM-toxin I were identical as regards t.l.c., u.v., mass spectra and biological activity in causing necrosis on apple leaves. A prepared dimer of AM-toxin I showed extremely weak activity; the relationship between the ring size and biological activity of AM-toxin I is discussed.

DAGO-enkephalin ([ D-Ala2, MePhe4, Gly-ol5]enkephalin), a highly selective ligand for mu opiate receptors, was dimerized with a series of alpha,omega-alkanedioic acids (n = 2-12) at the OH-terminus. In the radioligand receptor binding assays with rat brain, most of the DAGO-enkephalin dimers with cross-linking methylene chain n (DEDn) were more potent than DAGO monomer. For delta receptors, affinity of DEDn was maximized with n = 8, which might be related to an optimal distance between two binding sites. For mu receptors, an increase in chain length resulted in a progressive loss of potency. Although all of DEDn are considerably mu-selective, with a mu/delta ratio of 15-50, DEDn exhibited fairly flat binding curves with 15-50% smaller sloped than that of DAGO, suggesting that the dimers interact more strongly with one of the possible two mu binding sites.