Synthesis and properties of human β-endorphin-(1–17) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–17) (I), [D-Ala²]-β_h-EP-(1–17) (II), [Gln⁸]-β_h-EP-(1–17) (III) and [D-Ala², Gln⁸]-β_h-EP-(1–17) (IV). Measurement in a radio-receptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 33; II, 47; III, 889; IV, 123; β_h-endorphin, 2253.     

Human β-endorphin

Three analogs of human β-endorphin (β_h-EP) were synthesized by the solidphase method: [Gln⁸, Trp²⁷]-β_h-EP (I), [Gln⁸,Arg⁹,Trp²⁷]-β_h-EP (II), and [Gln⁹,Arg¹¹,Trp²⁷]-β-EP (III). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 778; II, 467; III, 449. Relative potencies in an analgesic assay were: β-EP, 100; I, 114; II, 165; III, 83. The 8–11 segment of β_h-EP can tolerate a net increase in charge of + 2 without diminishing analgesic potency. The substitution of Glu⁸ may be one of the more dependable means of designing β-endorphin antagonists.     

Synthesis and properties of human β-endorphin-(1–9) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–9) (I), [D-Ala²]-β_h-EP-(1–9) (II), [Gln⁸]-β_h-EP-(1–9) (III), and [D-Ala², Gln⁸]-β_h-EP-(1–9) (IV). Measurement in a radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 76; II, 100; III, 200; IV, 200. Two new amino acid derivatives were prepared and used for synthesis of the analogs: N^α-t-butyloxycarbonyl-O-(cyclopentyl) -tyrosine and N^α-t-butyloxycarbonyl-γ-(cyclopentyl)-glutamic acid.     

β-Endorphin: synthesis and properties of analogs modified in positions 8 and 31

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: [Gln^8.31]-β_h-EP(I), [Arg⁸, Gln³¹] -β_h-EP(II), [Ala⁸, Gln³¹] -β_h-EP (III), and [Val⁸, Gln³¹]-β_h-EP(IV). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 200; II, 150; III, 150; IV, 120. Relative potencies in an analgesic assay were: β_h-EP, 100; 1,236; II, 254;III, 116; IV, 121. The side-chain of Glu-8 in β_h-EP can be replaced by a variety of structures without diminishing biological activity.     

β-Endorphin: synthesis and properties of human β-endorphin-(1–28) and its analogs

Three analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–28) (II), [D-Ala², Gln⁸] - β_h-EP-(1–28) (III). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 85; II, 380; III, 146. Relative potencies in an analgesic assay were: β_h-EP; 100; I, 18; II, 36; III, 13.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

β-Endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human β-endorphin (β_h-EP) (I, [Gln⁸, Gly³¹]-β_h-EP-Gly-Gly-NH₂; II, [Arg^{9,12,24,28,29}]-β_h-EP and III, [Cys^11,26, Phe²⁷, Gly³¹]-β_h-EP), which have been shown to possess potent inhibiting activity to β_h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were β_h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: β_h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.     

Chemical synthesis of human β-endorphin(1–27) analogs by peptide segment coupling

[Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂, two analogs of human β-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides, Msc-[Gly⁸]β_hEP(1–8)SH and Msc-[l -Leu⁸]β_hEP(1–8)SH, were synthesized by standard solid phase method on 4-[α-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[α-(Boc-l -Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)^{9, 19, 24}]-β_hEP(9–27)NH₂ [Gly⁸]β_hEP(1–27)NH, and [l -Leu⁸]β_hEP(1–27)NH₂ were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly⁸]β_hEP(1–27)NH₂ and [l -Leu⁸]β_hEP(1–27)NH₂ in the segment coupling reaction were approximately 18%. Less than 1 % of racemization of Leu-8 occurred during coupling of Msc-[l -Leu⁸]β_hEP(1–8)SH to H-[Lys(Cit)^{9, 19, 24}]-β_h EP(9–27)NH₂. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of β-endorphin(1–27) and its analogs influenced the receptor binding activity.     

β-Endorphin

A double-headed analog of human β-endorphin (β_h-EP), N, N'-bis (β-endorphinyl)-cystine (II), has been synthesized by the solid-phase method, along with β_h-EP-Cys(CH₂CONH₂)-OH (I) and (Tyr³¹]-β_h-EP (III). Their relative potencies in a radioreceptor-binding assay were: B_h-EP, 100; II, 235; I, 170; and III, 204. In the tail-flick test for analgesic activity their relative potencies were: β_hEP, 100; II, 86; I, 93; and III, 116.     

β-ENDORPHIN: SYNTHESIS OF ANALOGS WITH EXTENSION AT THE CARBOXYL TERMINUS WITH HIGH RADIORECEPTOR BINDING ACTIVITY

Four analogs of human β-endorphin (β_h-EP) have been synthesized: [Gly³¹]-β_h-EP-Gly-NH₂, [CH₃(CH₂)₄NH³¹₂]-β_h-EP, [Gly³¹]-β_h-EP-Gly-Gly-NH₂, and [Gln⁸, Gly³¹]-β_h-EP-Gly-Gly-NH₂. All are more active than β_h-EP in an opiate receptor binding assay. Stepwise extension at the COOH-terminus shows a progressive increase in binding activity. The last analog, which combines extension at the COOH-terminus with elimination of the remaining anionic charge in β_h-EP, is nine times more active than the parent molecule.     

HUMAN β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF ANALOGS WITH SUBSTITUTIONS IN POSITIONS 2, 9, 18, 27 AND 31

Four analogs of the opioid peptide human β-endorphin (β_h-EP) have been synthesized: [d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, [d -Phe¹⁸,Phe²⁷,Gly³¹)-β_h-EP, [d -Thr²,d -Lys⁹,Phe²⁷,Gly³¹]-β_h-EP, and [d -Thr²,d -Phe¹⁸,Phe²⁷,Gly³¹]-β_h-EP. All are practically indistinguishable from β_h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.     

β-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human β-endorphin (β-EP) which contain cystine bridges, [Cys¹⁵-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (I) and [Cys¹⁶-Cys²⁶,Phe²⁷,Gly³¹]-β-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2–2.5 times the opiate receptor binding activity of β-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg^{9,19,24,28,29}Cys(Cam)^11,26, Phe²⁷,Gly³¹] and [Arg^{9,19,24,28,29},Cys-(Cam)^12,26, Phe²⁷, Gly³¹], were shown to have approximately the same opiate receptor activity as β-endorphin.     

β-ENDORPHIN: SYNTHESIS AND BIOLOGICAL ACTIVITY OF SHORTENED PEPTIDE CHAINS

Three analogs of β-endorphin have been synthesized by the solid-phase method: β_c-endorphin-(1–5)-(28–31), β_c-endorphin-(6–31) and β_h-endorphin-(1–5)-(16–31). The analgesic activities of these synthetic peptides relative to that of the parent molecule are reported. All three peptides at high doses exhibit either no or much weaker analgesic activity than β-endorphin. These data suggest that the entire β-endorphin molecule is necessary for full in vivo analgesic activity.     

Preparation and properties of tritiated human [Tyr 18, Trp 27]-β -endorphin

Tritiated [Tyr 18, Trp 27]-βh-EP was prepared from the corresponding diiodotyrosine derivative by catalytic reduction in the presence of carrier free tritium gas. A photoaffinity probe for β-endorphin (β-EP) receptors was prepared by selective modification of [Tyr¹⁸, Trp²⁷]-β_h-endorphin with 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) under acidic conditions to yield [Trp¹⁸-2,4-NAPS-Trp²⁷]-β_h-endorphin (NAPS-β-EP). NAPS-β-EP was purified by high performance liquid chromatography and characterized by ultraviolet absorption spectroscopy and peptide mapping. Tritiated NAPS-β-EP was prepared from tritiated [Tyr¹⁸, Trp²⁷]-β_h-endorphin with 2,4-NAPS-Cl. The ability of NAPS-β-EP to form covalent bonds to macromolecules due to photolysis was established using bovine serum albumin. The efficiency of photolytic cross-linking was 15% and the equilibrium dissociation constant was 1.3 times 10^-5 M.     

Spectroscopic analysis of [Trp3]-β-casomorphin analogs Comparative structure conformation-activity studies

A series of [3-tryptophan]-β-casomorphin-5([Trp³]-β-CM-5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure-conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp³]-β-CM-5, H-Tyr-Pro-Trp-Pro-Gly-OH (I) was modified at Pro-2 and Pro-4 by d -Pro substitutions to provide two diastereometric analogs, [Trp³-d -Pro-4]-β-CM-5 (II) and [d -Pro^2,4,Trp³]-β-CM-5 (III). In the GPI and MVD assays, β-CM-5 effected IC₅₀ values of 1.3 μm and 8.9 μm , respectively, which confirmed its known μ/δ-selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and < 0.005 relative to β-CM-5 on the GPI assay. Compounds I and II exhibited pronounced μ/δ-selectivities (> 18.9- and 12.4-fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of β-CM-5 and its [Trp³]-β-CM-5 analogs showed striking differences in their near-UV and far-UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) α-helicity (maximum at 193 nm and minima at 208 and 222 nm) for β-CM-5 was obtained in trifluoroethanol (TFE); however, none of the [Trp³]-β-CM-5 analogs showed such CD bands. Of potential relevance to γ-turn or C₇ secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent-dependent in H₂O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near-UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp³]-β-CM-5 compounds to be II > I > III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I-III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side-chains, by the Förster method, and were as follows: [Trp³]-β-CM-5, r = 10.6Å; [Trp³, d -Pro⁴]-β-CM-5, r = 9.6Å; and [d -Pro^2,4,Trp³]-β-CM-5, r = 11.0Å. A rank order correlation existed between the Tyr-Trp intramolecular distances and biological activity with shorter distance corresponding to higher biological potency. Furthermore, based on the fluorescence lifetime data analysis (Globals software) of the [Trp³]-β-CM-5 analogs, which were best fitted to a double exponential decay model, the relative ranking of long (> 1.5 ns) lifetime fractions of these three compounds was II > I > III. In summary, detailed spectroscopic analysis of three [Trp³]-β-CM-5 diastereomeric analogs by CD and FET have provided intriguing data indicating a possible structure conformation-activity relationship among these μ/δ-selective opioid-mimetic compounds.     

Human retinoblastomas have binding sites for the COOH-terminal segment of human β-endorphin

Two human retinoblastoma cell lines (Y79 and McA) were evaluated for the presence of binding sites for human beta-endorphin (β_h-EP). Using tritiated β_h-EP (³H-β_h-EP) and synthetic β-EP analogues, it was possible to demonstrate binding sites for ³H-β_h-EP with an ED₅₀ of 3.5 nM in Y79 cells and 8 nM in McA cells respectively. The non-opioid segment [β_h-EP-(6–31)] retained about 20% relative potency in Y79 and 40% in McA in displacing the tritiated hormone when compared with β_h-EP. Camel β-EP had a relative potency of less than 1% and β_h-EP-(1–27) was inactive in both cells in doses as high as 4μm. Taken together with previous reports on similar binding sites in human neuroblastoma and glioblastoma cell lines, it appears that cell lines of neural origin have binding sites for the COOH-terminal of human β-EP.     

β-Endorphin: Synthesis and properties of analogs with replacement of lysine residues by arginine

Human β-endorphin analogs, [Arg^{9,19,24,28,29}]-β-endorphin (I) and [Arg^24,28,29]-β-endorphin (II), have been synthesized by the solid-phase method. Peptide II had 86% of the analgesic potency and 216% of the receptor binding activity of the parent molecule. Peptide I had only 18% analgesic potency but its binding activity was more than three time greater than that of human β-endorphin.     

Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH₂, I; cyclo[Lys²⁶-Asp³⁰]-hPTH-(20-34)-NH₂, II; cyclo[Glu²²-Lys²⁶]-hPTH-(20-34)-NH₂, III; cyclo[Lys²⁷-Asp³⁰]-hPTH-(20-34)-NH₂, IV; and [Leu²⁷]-hPTH-(20-34)-NH₂, V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in α-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of α-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV, This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV, The data suggest that in the region bounded approximately by residues 24–32, an amphiphilic α-helix is important for correct functional binding to the PTH receptor.     

β-ENDORPHIN: SYNTHESIS AND ANALGESIC ACTIVITY OF SEVERAL ANALOGS MODIFIED IN POSITIONS 2 AND 5

The solid-phase syntheses of [Sar²]-, [Ala²]-, [D-Leu²]-, [D-Lys²]-β- endorphins and [Pro⁵]-, [Leu⁵]-, [D-Leu⁵]-, [D-Ala², D-Leu⁵]-β-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu² analog to be 48% as potent as β_h-endorphin while the Ala², D-Lys², Leu⁵, and [D-Ala², D-Leu⁵] analogs were 8 to 17% as active. The Sar², D-Leu⁵, and Pro⁵ analogs were less than 1% as potent.     

Conformational studies of highly potent 1‐aminocyclohexane‐1‐carboxylic acid substituted V2 vasopressin agonists

Abstract: Conformational studies of three agonists of V₂ receptor modified with 1‐aminocyclohexane‐1‐carboxylic acid (Acc), [Acc²,DArg⁸]VP, [Acc³]AVP and [Cpa¹,Acc³]AVP, using 2D NMR and theoretical calculations are presented in this paper. It is believed that α,α‐disubstituted amino acids, such as Acc, affect the formation of either 3₁₀ or α‐helical conformation. Moreover, a peptide with Acc may adopt either the γ‐ or an inverse γ‐turn over it. Thus, incorporation of Acc into the arginine‐vasopressin sequence induced C₇‐membered ring conformation with Acc at the top of it, and additional formation of β‐bend involving residues in the 2–5 fragment of the peptides. Furthermore, the analogues are also characterized by type I of β‐turn involving residues Acc³‐Cys⁶ in [Acc³]AVP and [Cpa¹,Acc³]AVP, and by type IV or II′ in [Acc²,DArg⁸]VP. Replacement of Tyr at position 2 of [Acc²,DArg⁸]VP with Acc afforded a hydrogen bond between the guanidine moiety of DArg⁸ and the side chain of either Asn⁵ or Gln⁴. In the remaining analogues, the β‐turn comprising the Cys⁶‐Gly⁹ residues allows the positively charged side chain of Arg⁸ to be directed toward Tyr². The substitution of Cys¹ with Cpa¹ enhances hydrophobic properties of N‐terminal part of the molecule strengthening thereby the affinity to the binding pocket of receptors.