A chimeric opioid peptide with mixed μ agonist/δ antagonist properties

Abstract: There is evidence to indicate that opioid compounds with mixed μ agonist/δ antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective μ agonist H‐Dmt‐D‐Arg‐Phe‐Lys‐NH₂ ([Dmt¹]DALDA) (Dmt = 2′,6′‐dimethyltyrosine) and the potent and selective δ antagonist H‐Tyr‐TicΨ[CH₂‐NH]Cha‐Phe‐OH (TICP[Ψ]) (Cha = cyclohexylalanine), connected ‘tail‐to‐tail’ via a short linker, was synthesized using a combination of solid‐phase and solution techniques. The resulting peptide, H‐Dmt→D‐Arg→Phe→Lys‐NH‐CH₂‐CH₂‐NH‐Phe←Cha[NH‐CH₂]ΨTic←Tyr‐H, showed the expected μ agonist/δ antagonist profile in the guinea‐pig ileum and mouse vas deferens assays. Its μ and δ receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a Ψ (E,CH=CH) or Ψ (CH2CH2) isosteric replacement

The peptide CO–NH function was replaced by a trans carbon-carbon double bond or by a CH₂–CH₂ isostere in enkephalin analogues of DADLE, DCDCE–NH₂ or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly³-Phe⁴ bond was replaced by the isosteres GlyΨ (E,CH=CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or GlyΨ(CH₂CH₂)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH₂–CH₂ replacement. The Gly³Ψ (E,CH=CH)Phe⁴ DCDCE–NH₂ analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Potent in vivo inhibitors of rat renin: Analogues of human and rat angiotensinogen sequences containing different classes of pseudodipeptides at the scissile site*

Using solid-phase methodology we have synthesised peptides based on the 8–14 or 6–14 human and rat angiotensinogen sequences, containing the following different isosteric units at the P₁-P₁’cleavage site: Leu-Ψ[CH₂NH]Leu; Leu-[CH(OH)CH₂]Val; Leu-Ψ[CH(OH)CH₂]Leu and Leu-Ψ[CH(NH₂)CH₂]Val. In vitro, peptide Piv-His-Pro-Phe-His-Leu-Ψ[CH(OH)CH₂]Leu-Tyr-Tyr-Ser-NH₂( XXI ) is the most potent inhibitor of rat plasma renin reported having an IC₅₀ of 0.21 nM; it is a much weaker inhibitor of human renin (IC₅₀ 45 nM). Peptide Boc-His-Pro-Phe-His-Leu-Ψ[CH(OH)CH₂] Leu-Val-Ile-His-NH₂ ( XX ) was a highly effective inhibitor of rat renin in vivo. When infused (1 mg/kg/h) into two-kidney, one-clip chronic renal hypertensive rats, it lowered blood pressure and suppressed both plasma renin and angiotensin II. When given as a bolus (1 mg/kg) there was a divergence between the rapid rebound of renin levels and blood pressure, which remained suppressed. These results indicate that potent in vivo inhibitors of rat renin could be useful not only in examining the role of circulating renin but also in elucidating the equally important involvement of extracirculatory renin pools.     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and ¹H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II ß-turn in the solid state with Met and Aib as the corner residues (øMet =- 51.8^o, øMet = 139.5^o, øAib = 58.1^o, øAib = 37.0^o). A single, weak 4 -> 1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N—O 3.25 Å, N-H—O 128.4^o). ¹Hn.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagneti radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCI₃ and (CD₃)₂SO. Nuclear Overhauser effects observed between Met Cα H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II ß-turns in these solvents.     

Synthesis and biological activity of potent,low molecular weight renin inhibitors*

A series of renin inhibitors containing the dipeptide transition state mimics (2R, 4S, 5S)-5-amino-4-hydroxy-2-methyl-6-cyclohexyl hexanoic acid (Cha-Ψ[CH(OH)CH₂]Ala) and (2R, 45, 5S)-5-amino-4-hydroxy-2-isopropyl-6-cyclohexyl hexanoic acid (Cha-Ψ[CH(OH)CH₂]Val) were prepared. A structure-activity study, using pseudopeptide (Boc-Phe-His-Leu-Ψ[CH(OH)CH₂]Val-Ile-His-OH) as our lead structure, led to a new series of inhibitors, which correspond to tripeptides and contain no natural amino acids. For example, R, S-Bpma-Ape-Cha-Ψ[CH(OH)CH₂]Ala-NH₂ (IC₅₀= 1.26 nM against human plasma renin at pH 6.0; molecular weight = 564) has only two thirds of the molecular weight but twice the potency of our original lead. This new class of low molecular weight renin inhibitor displays excellent specificity toward human renin versus the related aspartic proteinase pepsin and angiotensin-1-converting enzyme. Examples are given of selected inhibitors showing encouraging evidence for intestinal absorption after intracolonic and oral administration in male Sprague-Dawley rats.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal d-Trp or d-Tpi

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the d forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin(6-14) and Leu¹³Ψ(CH₂NH)Phe¹⁴ -bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of ¹²⁵I-Tyr⁴ -bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [³H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist d -Tpi⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3095) was slightly more potent in these assays than d -Trp⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3125). Nevertheless, d -Trp⁶ Leu¹³Ψ (CH₂NH)Phe¹⁴-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC₅₀ value less than 1 nm . Our work indicated that the substitution of d -Trp and d -Tpi at position 6 of the pseudononapeptide bombesin analogs (Ψ^13-14), in which the Met¹⁴ residue is replaced by Leu or Phe, results in potent bombesin/GRP antagonists with improved in vivo activity.     

Dehydro-enkephalins

Two kinds of dehydropeptide analogs of enkephalin containing a ΔTyr unit at the N-terminus have been synthesized by coupling Boc-ΔTyr-(Cl₂Bzl)-OH with amino acid amides and tetrapeptide esters using the water soluble carbodiimide-HOBt method. Pentapeptides consisting of ΔTyr¹, and ΔPhe⁴ or ΔLeu⁵ were also prepared. Ultraviolet difference spectroscopy was important in the characterization of the dehydro moieties, ΔTyr, ΔPhe and ΔLeu. Attempts to liberate ΔTyr¹-enkephalins have been unsuccessful because of the instability of an N-terminal ΔTyr residue having p-phenolic group in the side chain.     

Folded conformations of the δ-selective opioid dermenkephalin with head-to-tail interactions. A simulated annealing study through NMR restraints

Despite similar tripeptide N-termini, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH₂), naturally occuring opioid peptides from frog skin, exhibit high affinity but contrasting selectivity for the μ- and δ-opioid receptors, respectively. Structure-activity relationship studies have shown that the N-terminal tripeptide, Tyr-D-Xaa-Phe (where Xaa is either Ala or Met), is necessary for binding with both the μ- and δ-receptors while the nature and/or the conformation of the C-terminus His-Leu-Met-Asp-NH₂ of dermenkephalin are responsible for addressing the peptide to the δ-receptor. In order to examine the conformational characteristics that are related to the selectivity of dermenkephalin towards the δ-receptor, 50 NOE restraints (10 between nonadjacent residues), and 7 dihedral angles, derived from a two-dimensional ¹H-NMR study of dermenkephalin in dimethyl sulfoxide, were used in simulated annealing and energy minimization procedures. Twenty-four resulting conformers (60% of the generated structures) with no severe distance restraint violation were pooled into seven groups and three related families. These 24 conformers show close proximity between the two methionine residues, S-shaped structures, mean planes of N-terminal and C-terminal moieties almost at right angles to each other, a C-terminus region above the plane of the N-terminal region and g^? as preferential orientation in the side chain of the. Aside these similarities, families of conformers differ by the preferential orientation in the side chain of Tyr (t or g^?) and proximity between Tyr and Asp, or Tyr and the C-terminus. In contrast to previous models, practically no β-turn structures exist for dermenkephalin, most of the NH hydrogen bonds participating to γ-turns. The possible relationship between the conformational characteristics of dermenkephalin and the δ-opioid receptor selectivity is discussed. © Munksgaard 1996.     

Effect of sulfate position on rnyotropic activity of the gastrin/CCK-like insect leucosulfakinins

Leucosulfakinin and leucosulfakinin-II are sulfated insect neuropeptides with homology to human gastrin II and cholecystokinin. Biological evaluation of the analog [Ser(SO₃H)², Tyr⁵]leucosulfakinin-II, two cholecystokinin analogs containing key leucosulfakinin amino acid replacements, and a Tyr(SO₃H) position-analog series has demonstrated that while the presence of a sulfate group is critical to leucosulfakinin myotropic activity, its position is not. Most strikingly, the analog [Tyr (SO₃H)⁵,Phe⁶,Nle⁹]leucosulfakinin, in which the sulfate moiety is shifted one position towards the N-terminus relative to the parent peptide, retains a very significant 38% of the parent's threshold activity. A shift in the location of the sulfate group by two to five positions toward the N-terminus led to reduction of potency to a significant but low plateau of activity, whereas a shift by more than one position towards the C-terminus led to a complete loss of activity. The introduction of a single residue (Arg) in the 7 position of CCK-8 transforms this inactive mammalian hormone into a myotropically-active leucosulfakinin analog on the isolated cockroach hindgut.     

Conformation of dehydropeptides

Six model dipeptide methyl amides containing dehydroaminobutyric acid (Δ Abu) of the type Boc-X-δ^ZAbu-NHCH₃ and Box-X-δ^EAbu-NHCH₃, X = Ala, Val, Phe (Boc =tert-butoxycarbonyl), have been synthesized and their solution conformations explored using 300 MHz ¹H NMR and IR spectroscopy. Studies based on delineation of intramolecularly hydrogen bonded NH groups in CDCl₃ and (CD₃)₂SO revealed that none of the NH groups is appreciably solvent shielded. Difference NOE (Nuclear Overhauser Effect) studies have also failed to detect the presence of any discernible turn structure in these peptides. These studies indicate that the conformational preferences of peptides containing, α, β-dehydroaminobutyric acid are different from those of Δ^ZPhe and Δ^ZLeu. It appears that steric interactions due to the β-substituent in the dehydroamino acid moiety play an important role. Unlike Δ^ZPhe and Δ^ZLeu, which have relatively large β-substituents, phenyl and isopropyl, respectively, and stabilize a β-turn, the β-methyl group of Δ^ZAbu or Δ^EAbu is readily accommodated in extended conformation. Clearly, the size of β-substituent in dehydroamino acid crucially influences the conformational preferences. Thus, it may be possible to use different dehydroamino acids to introduce variable but definite constraints in synthetic peptides.     

Crystal-state structural analysis of two γ-lactam-restricted analogs of Pro-Leu-Gly-NH2

The crystal structures of two analogs of Pro-Leu-Gly-NH, (1), containing a γ-lactam conformational constraint in place of the -Leu-Gly- sequences, are described. The highly biologically active (S,R)-diastere-omer 2a is semi-extended at the C-terminus, with the N-terminal Pro residue in the unusual “C₅” conformation [ψ₁=– 0.8(15)°] stabilized by a (peptide)N-H…N(amino) intramolecular H-bond [the N(3)…N(4) separation is 2.687(11)Å]. Conversely, the N,N′-isopropylidene aminal trihydrate of the (S,S)-diastereomer 2b, compound 3, adopts a β-bend conformation at the C-terminus, as already reported for 1. However, the backbone torsion angles [φ= 57.4(4), ψ₂=– 129.9(3)°; ψ₃= - 92.3(4), ψ₃= 6.4(5)°] lie close to the values expected for the corner residues of an ideal type-II β-bend. A weak intramolecular 4 → 1 H-bond is seen between the Gly carboxyamide anti-NH and Pro C=O groups. In the newly formed 2,2,3,4-tetraalkyl-5-oxo-imidazolidin-1-yl moiety the ψ₁ torsion angle is 12.9(4)° and the intramolecular N(3)…N(4) separation is 2.321(4)Å.     

Conformations of ψ[CH2NH] pseudopeptides

The cyclic pseudopentapeptide cyclo[Gly-Proψ[CH₂NH]Gly-D-Phe-Pro] and its TFA salt were synthesized by solution methods, and their conformations were studied by NMR spectroscopy in both DMSO-d₆ and CDC₁₃. While intramolecular hydrogen bonding is observed with some conformers, the nature of the solvent and the presence of TFA affects the relative structural rigidities of the compounds. No evidence was found for the ψ[CH₂NH] or ψ[CH₂NH⁺₂] units acting as H donors in this series.     

Solid phase synthesis of peptides containing the non-hydrolysable analog of (O)phosphotyrosine,P(CH2PO3H2)Phe

Studies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH₂PO₃H₂) Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the β₂ adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH₂PO₃H₂)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc- p (CH₂PO₃Et₂)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (~60% total yield) and further deprotections provided both the p(CH₂PO₃H₂)Phe and p(CH₂PO₃HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenyl-thiocarbamyl form (PTC).     

Crystal and molecular structure of the dehydropeptide Ac-ΔPhe-Val-ΔPhe-NH-Me

The dehydropeptide Ac-ΔPhe-l -Val-ΔPhe-NH-Me, containing two dehydrophenylalanine (ΔPhe) residues, crystallizes from methanol/water in space group P2₁2₁2₁ with a= 12.622 (1), b= 12.979 (1), and c= 15.733 (1) Å. In the solid state, the molecular structure is characterized by the presence of two intramolecular hydrogen bonds which form two consecutive β-bends. The (φ,Ψ) torsion angles of the three residues are very similar and close to the standard values of type III β-bends, so the molecular conformation corresponds to an incipient right-handed 3₁₀ -helix, only slightly distorted. In the crystal, the molecules are linked by head-to-tail hydrogen bonds, thus forming continuous helical columns packed in antiparallel mode. There are no lateral hydrogen bonds; the only interactions are hydrophobic contacts between the apolar side chains of neighboring helical columns.     

Pseudopeptides containing the 2‐hydrazonoacyl fragment: analogs of the chemotactic agent HCO‐Met‐Leu‐Phe‐OMe

Abstract: In order to further examine the properties of pseudopeptides containing the 2‐hydrazonoacyl fragment, two new series of analogs of the prototypical chemotactic N‐formyl‐tripeptide HCO‐Met‐Leu‐Phe‐OMe were designed and synthesized. The first group contains the new fragment as the N‐terminal residue and is represented by the N‐aryl derivatives p‐Cl‐C₆H₄‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 2 and 3 ) and by the corresponding N‐aroyl analogs p‐CH₃‐C₆H₄‐CO‐NH‐N=C(R)‐CO‐Leu‐Phe‐OMe ( 4 ). The second group contains the new fragment in place of the central Leu residue and is represented by compounds HCO‐Xaa‐NH‐N=C(R)‐CO‐Phe‐OMe ( 7a and 7b ) where Xaa is Nle and Met, respectively. The conformational and biochemical properties of the new products were examined.     

Structure and conformation of peptides containing the sulphonamide junction

N-acetyl-tauryl-l -phenylalanine methyl ester 1 has been synthesized. The crystal structure and molecular conformation of 1 have been determined. Crystals are monoclinic, space group P2₁ with a = 5.088(2), b = 17.112(17), c = 9.581(6) Å, β= 92.34(4)^M-0, Z = 2. The structure has been solved by direct methods and refined to R = 0.043 for 2279 reflections with I < 1.5σ(I). The sulphonamide junction maintains the peptide backbone folded with Tau and Phe C^α atoms in a cisoidal arrangement, the torsion angle around the S-N bond being 65.4^M-0. In this conformation the p-orbital of the sulphonamide nitrogen lies in the region of the plane bisecting the O-S-O angle, thus favouring dα-pα interactions between nitrogen and sulphur atoms. The S-N bond with a length of 1.618 Å has significant α-bond character. The CO-NH is planar and adopts trans conformation. The Tau residue is extended with the Tau-C^α₁-C^β_a bond anti-periplanar to the S-N bond. The Phe side chain conformation corresponds to the statistically most favoured g^- rotamer and exhibits a χ¹ torsion angle of –67.5^M-0. The packing is characterized by intermolecular H-bonds which the Tau and Phe NH groups form with the acetyl carbonyl and one of the two sulphonamide oxygens, respectively.     

The receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related δ-opioid antagonists contains all trans peptide bonds

Two different models for the receptor-bound conformation of δ-opioid peptide antagonists containing the N-terminal dipeptide segment H-Tyr-Tic (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) have been proposed. Both models are based on spatial overlap of the Tyr and Tic² aromatic rings and N-terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide δ-antagonist naltrindole. However, in one model the peptide bond between the Tyr and Tic² residues assumes the trans conformation, whereas in the other it is in the cis conformation. To distinguish between these two models, we prepared the two peptides H-Tyrψ[CH₂NH]. Tic-Phe-Phe-OH and H-Tyrψ[CH₂NH]. MeTic-Phe-Phe-OH (MeTic = 3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in which a cis peptide bond between the Tyr and Tic (or MeTic) residues is sterically forbidden. Both compounds turned out to be moderately potent δ-opioid antagonists in the mouse vas deferens assay. A molecular mechanics study performed with both peptides resulted in low-energy conformations in which the torsional angle (“ω₁”) of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180°(trans conformation) and which were in good agreement with the proposed model with all trans peptide bonds. Furthermore, this study confirmed that neither of these two peptides could assume low-energy conformations in which “ω₁” had a value of 0°(cis conformation). Conformers with that same bond in the gauche- conformation (“ω₁”= -60“) were also identified, but were higher in energy and showed no spatial overlap with naltrindole. On the basis of these results it is concluded that the receptor-bound conformation of δ-peptide antagonists containing an N-terminal H-Tyr-Tic-dipeptide segment must have all trans peptide bonds. © Munksgaard 1998.     

Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine,thiol and aspartyl proteases

We synthesized short chromogenic peptidyl–Arg–p-nitroanilides containing either (Galβ)Ser or (Glcα,β)Tyr at P₂ or P₃ sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl–X–Arg–pNa and Acetyl–X–Phe–Arg–pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe⁸–His–Leu–Val–Ile–His–Asn¹⁴ of human angiotensinogen, in which [GlcNAcβ]Asn was introduced before Phe⁸ and/or after His¹³ and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]–ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P₃ it is unfavorable, in contrast to the P₂ position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac–(Galβ)Ser–Arg–pNa in comparison to Ac–Ser–Arg–pNa, and by the hydrolysis of Ac–(Glcα,β)Tyr–Arg–pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P^′₄ glycosylated peptide [Abz–F–H–L–V–I–H–(GlcNAcβ)N–E–EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K_m and higher k_cat values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P^′₄ and P₄ glycosylated substrates.     

Synthesis of keto-methylene and dehydro-keto-methylene pseudo-dipeptides

A new synthetic pathway is proposed for the preparation of keto-methylene and dehydro-keto-methylene dipeptide isosteres bearing the general formulas (RS)XxxΨ(COCH₂)(RS)Yyy and (RS)XxxΨ(COCH₂)ΔYyy. The method involves the condensation of an oxazolone derived from an N-benzoyl amino acid ( 3a, b ) via a modified Dakin-West reaction with the appropriately α-substituted succinoyl chloride half ester ( 2a, b ). This last compound is obtained from a Stobbe condensation followed by reaction with oxalyl chloride. The fully protected pseudo-dehydro dipeptides ( 41, b ) thus obtained can then be catalytically hydrogenated to the desired pseudo-dipeptides ( 5a, b ). Acid hydrolysis of ( 4 ) and ( 5 ) gives the pure fully deblocked dipeptide surrogates which are then N-protected for coupling into the desired peptide analogs. For preparative applications, the prior isolation of intermediates is not required throughout the procedure up to the final products. In this manner, the following compounds were prepared: Boc-(RS)PheΨ(COCH₂)(E, Z)Δ Phe-OH ( 9 ), Boc-(RS)Pheψ(COCH₂)(RS)Phe-OH (7a), and Boc-Glyψ(COCH₂)(RS)Leu-OH ( 7b ). Characterization of isolated intermediates and final products was carried out.     

Development of a radioimmunoassay for a pseudononapeptide bombesin/GRP antagonist with antitumor activity

Bombesin-like and GRP-like peptides may act as autocrine growth factors in the proliferation of some cancers. A pseudononapeptide bombesin antagonist, [d -Tpi⁶,Leu¹³φ(CH₂NH)-Le¹⁴]bombesin(6–14), and related analogs synthesized in our laboratory significantly inhibit tumor growth in various cancer models. A radioimmunoassay (RIA), suitable for determination of RC-3095 and its congeners in unextracted serum, was developed in order to facilitate further experimental and clinical evaluation of this bombesin/GKP receptor antagonist for the treatment of various tumors. Antibodies were generated against RC-3095 and Des-Tpi¹-RC-3095, conjugated to bovine serum albumin with glutaraldehyde. Antiserum JH-631b was selected for further experiments based on the antibody characterization. At an antiserum dilution of 1:189000, this antibody bound approximately 50% of 7 fmol of added radiolabeled Tyr¹-RC-3095. The antibody cross-reacted with C-terminal fragments of RC-3095. Fragments without the C-terminus and naturally existing peptides of the bombesin family or structurally unrelated peptides did not cross-react. The minimum detectable dose of RC-3095 was 0.4 pg/tube. Intra- and interassay coefficients of variation ranged from 3.2 to 4.4% and from 5.6 to 12.8%, respectively. The RIA is suitable for direct determination of RC-3095 in serum. The RIA should be of value for monitoring levels of this analog in serum during long-term therapy. © Munksgaard 1995.