Acyl,pseudotetra-, tri- and dipeptide active-core analogs of insect neuropeptides

Pseudopeptides of the achetakinin insect neuropeptide family were synthesized by replacing the amino acid blocks Phe-, Phe-Tyr-, and Phe-Tyr-Pro- of the active-core pentapeptide Phe-Tyr-Pro-Trp-GIy-NH₂ with hydrocinnamic acid, 6-phenylhexanoic acid, and both 9-phenylnonanoic and 6-phenylhexanoic acid, respectively. All four of these analogs retained myotropic activity, demonstrating that the active core could be reduced from a pentapeptide to a modified dipeptide. Most notable of these was the pseudotetrapeptide hydrocinnamyl-Tyr-Pro-Trp-Gly-NH₂, which retained 70% of the potency and over 85% of the maximal activity of the parent pentapeptide. The N-terminal amino group, the phenol ring of the Tyr residue, the sulfate moiety and the Gly residue of the insect sulfakinin active core Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂ were all replaced by dodecanedioic acid. The resulting pseudotetrapeptide, dodecandioyl-His-Nle-Arg-Phe-NH₂, elicited myostimulatory activity. Conversely, the related acyl pseudopentapeptide azelayl-Gly-His-Nle-Arg-Phe-NH₂ proved myoinhibitory. A possible explanation for these disparate biological responses is discussed. These acyl pseudopeptides are important advances towards the eventual development of stable, potent mimetic agonists and antagonists of insect neuropeptides.     

From peptide libraries to optimized nonpeptide ligands in the search for S‐farnesyltransferase inhibitors

Abstract: A complete 331 776‐member library of tetrapeptides made of 24 amino acid building blocks was synthesized robotically on solid phase and subjected to a deconvolution based on the inhibitory potency of the sublibraries in a HPLC assay of the S‐farnesyltransferase activity in vitro. One of the non‐natural peptide and noncysteine‐containing leads Nip‐Trp‐Phe‐His (Nip = p‐nitrophenyl‐l ‐alanine) was optimized chemically to give a proteolytically stable pseudopeptide with a 200‐fold potency compared with the original lead. The final compound was converted to the C‐terminal ethyl ester: p‐F‐C₆H₄‐CO(CH₂)₂‐CO‐Bta‐d ‐Pheψ[CH2NH]His‐OEt (Bta = benzothienyl‐l ‐alanine) and shown to behave as a prodrug which was hydrolyzed back to the C‐terminal acid following cell penetration. The method confirmed that several structurally original leads can be discovered in large libraries when deconvolution relies upon a highly specific assay and that these leads can be optimized by chemical modification to impart the final compound the desired pharmacological and pharmacokinetic properties.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists

Abstract: A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C‐terminal hexapeptide sequence Tyr‐Phe‐Ser‐Pro‐Arg‐Leu‐NH₂ of PBAN1‐33NH₂; whereas library II (d ‐Phe library) was based on the sequence of the PBAN lead linear antagonist Arg‐Tyr‐Phe‐d ‐Phe‐Pro‐Arg‐Leu‐NH₂. In both libraries the Pro residue was replaced by the BBC building unit N^α‐(ω‐aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N‐terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl₃I for selective deprotection of the Boc group from the building unit prior to on‐resin amino‐end to backbone‐nitrogen (AE‐BN) cyclization, during solid‐phase synthesis with Fmoc chemistry.     

A bifunctional heterodimeric insect neuropeptide analog

The synthesis and biological activity of a bifunctional, heterodimeric insect neuropeptide analog are described. The heterodimer is composed of the C-terminal pentapeptide active core regions of the leucokinin/achetakinin and pyrokinin neuropeptide families linked via their N-terminal amino groups with a succinyl diacid moiety. Members of the leucokinin/achetakinin family can induce fluid secretion in malpighian tubules of the house cricket, Acheta domesticus, whereas the pyrokinins demonstrate activity in a cricket oviduct myotropic bioassay. No cross-activity is observed for the two neuropeptide families in these bioassays. However, the heterodimer elicits responses in both Acheta bioassays. Such a bifunctional analog may in future serve as a template for the design of stable, bifunctional pest insect control agents of greater efficiency.     

New N-terminal modified proctolin analogues

Five proctolin analogues modified in position 1 of the peptide chain by Cit (I), Lys (II), His (III), Phe(p-NH₂) (IV) and γ-Abu (V) were synthesized by conventional liquid phase method. Biological activity of the obtained peptides was investigated by the cardioexcitatory test on two insect species, cockroach, Periplaneta americana L. and yellow mealworm, Tenebrio molitor L.     

New Alloferon Analogues: Synthesis and Antiviral Properties

We have extended our study on structure/activity relationship studies of insect peptide alloferon (H‐His‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH) by evaluating the antiviral effects of new alloferon analogues. We synthesized 18 alloferon analogues: 12 peptides with sequences shortened from N‐ or C‐terminus and 6 N‐terminally modified analogues H‐X¹‐Gly‐Val‐Ser‐Gly‐His‐Gly‐Gln‐His‐Gly‐Val‐His‐Gly‐OH, where X¹ = Phe ( 13 ), Tyr ( 14 ), Trp ( 15 ), Phg ( 16 ), Phe(p‐Cl) ( 17 ), and Phe(p‐OMe) ( 18 ). We found that most of the evaluated peptides inhibit the replication of Human Herpesviruses or Coxsackievirus B2 in Vero, HEp‐2 and LLC‐MK₂ cells. Our results indicate that the compound [3‐13]‐alloferon ( 1 ) exhibits the strongest antiviral activity (IC₅₀ = 38 μm ) among the analyzed compound. Moreover, no cytotoxic activity against the investigated cell lines was observed for all studied peptides at concentration 165 μm or higher.     

Cyclic enkephalin analogs containing various para‐substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists*

Abstract: The cyclic enkephalin analog H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ is a highly potent opioid agonist with IC₅₀s of 35 pm and 19 pm in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe¹‐analog of this peptide showed 370‐fold and 6790‐fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr¹ hydroxyl‐group in the interaction with μ and δ opioid receptors. In the present study, the effect of various substituents (‐NH₂, ‐NO₂, ‐CN, ‐CH₃, ‐COOH, ‐COCH₃, ‐CONH₂) introduced in the para‐position of the Phe¹‐residue of H‐Phe‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ on the in vitro opioid activity profile was examined. Most analogs showed enhanced μ and δ agonist potencies in the two bioassays, except for the Phe(pCOOH)¹‐analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH₃)¹‐ and the Phe(pCONH₂)¹‐analogs. The latter compound showed subnanomolar μ and δ agonist potencies and represents the most potent enkephalin analog lacking the Tyr¹ hydroxyl‐group reported to date. Taken together, these results indicate that various substituents introduced in the para‐position of Phe¹ enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure‐activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Transport characteristics of peptides and peptidomimetics: I. N‐methylated peptides as substrates for the oligopeptide transporter and P‐glycoprotein in the intestinal mucosa

Abstract: Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N‐Methylation has been used successfully in peptide‐based drug design in an attempt to improve the metabolic stability of a peptide‐based lead compound. However, the effect of N‐methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P‐glycoprotein (P‐gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N‐acetylated and C‐amidated) analogs of H‐Ala‐Phe‐Ala‐OH with N‐methyl groups on either the Ala‐Phe or Phe‐Ala peptide bond were synthesized. Transport studies using Caco‐2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N‐methylation of the Ala‐Phe peptide bond of H‐Ala‐Phe‐Ala‐OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N‐methylation of the Phe‐Ala peptide bond of H‐Ala‐Phe‐Ala‐OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N‐methylation of the Phe‐Ala peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ decreased the substrate activity of the molecule for the efflux transporter P‐gp. In contrast, N‐methylation of the Ala‐Phe peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ increased the substrate activity of the molecule for P‐gp.     

Further studies on proctolin analogues modified in position 2 of the peptide chain and their influence on heart-beat frequency of insects

Seven proctolin analogues (I-VII) modified in position 2 of the peptide chain by Phe (p-guanidino) (I), Phe (p-OEt) (II), Tyr (3′-NH₂) (III), Tyr (3′-NO₂) (IV), Afb (p-OH) (V) (Afb = 3-amino-4-phenyl-l -butyric acid), Afb (p-NH₂) (VI), Afb (p-NO₂) (VII), and the tetrapeptide Tyr (3′-NH₂)-Leu-Pro-Thr (VIII) were synthesized by the classic liquid-phase method. The biological effects of the peptides were investigated in cardioexcitatory tests on two insect species, the cockroach Periplaneta americana L., and the yellow mealworm, Tenebrio molitor L. Within physiological concentrations (10⁻⁹ - 10⁻⁷ M) peptides II, III, and IV stimulated the heart action of P. americana like proctolin itself. Under identical conditions, in the case of T. molitor, only peptide III showed cardiostimulatory properties, whereas other compounds (including II and IV) were inactive at concentrations up to 10⁻⁷m . Results reported here reflect, with reference to the analogues I-VII, selective recognition of receptors on myocardium of both insect species. The tetrapeptide VIII revealed a weak deacceleratory effect on P. americana and T. molitor heart action.     

Backbone modifications in cyclic pep tides Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement

NMR and X-ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D-Xxx-Pro-Gly-Pro-Gly) possess β- and γ-turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D-Phe-Proψ [CH₂S]Gly-Pro-Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K₂CO₃ and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by ¹H and ¹³C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a γ-turn intramolecular hydrogen bond in the all-amide structure. In CDCl₃, the pseudopeptide adopted the same all-trans conformation as its parent, although the remaining β-turn hydrogen bond was weaker according to Δδ/ΔT_NH measurements. In DMSO-d₆, the all-trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly inter converted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. ¹³C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly¹-Pro² position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine‐containing fMLF analogs

Abstract: In order to explore the properties of chemotactic N‐formylpeptides containing isopeptide bonds within their backbones, a group of lysine‐containing analogs of the prototypical chemotactic tripeptide N‐formylmethionyl‐leucyl‐phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO‐Met or Boc‐Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N^α or N^ε bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO‐Met‐Lys(Z)‐Phe‐OMe ( 2a , b ), RCO‐Met‐Lys(Z‐Phe)‐OMe ( 3a , b ), Z‐Lys(RCO‐Met)‐Phe‐OMe ( 4a , b ), Z‐Phe‐Lys(RCO‐Met)‐OMe ( 5a , b ), RCO‐Met‐Phe‐Lys(Z)‐OMe ( 6a , b ) and Z‐Lys(RCO‐Met‐Phe)‐OMe ( 7a , b ), with R=OC(CH₃)₃ and R=H for compounds a and b , respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b ) and antagonists (compounds a ). All N‐formyl derivatives 2b ? 7b are less potent than fMLF‐OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a ? 7a do not show antagonistic activity towards fMLF induced chemotaxis and O₂^? production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

Detailed SAR and PCA of the Tyrocidines and Analogues Towards Leucocin A‐Sensitive and Leucocin A‐Resistant Listeria monocytogenes

The tyrocidines, antimicrobial cyclic decapeptides from Bacillus aneurinolyticus, have potent activity with drug/disinfectant potential, specifically against Listeria monocytogenes. The tyrocidine activity is dependent on an amphipathic balance. Structure–activity relationship (SAR) analysis combined with principal component analysis showed the best activity correlation with hydropathy and solvent accessible volume (hydrophobicity parameters), M_r and molecular volume (steric/size parameters), coupled with rigid sequence and charge prerequisites. For potent activity against L. monocytogenes strains, there is a prerequisite for a Tyr or Phe in the (W/F)(w/f)NQ(Y/F/W) sequence of the variable pentapeptide and ornithine (Orn, O) as cationic residue in the conserved V(K/O)LfP pentapeptide, particularly with Trp in the aromatic dipeptide moiety of the variable pentapeptide. The roles of Trp and Orn in the tyrocidines were confirmed with most active peptide, tyrocidine B (TrcB) containing Orn and a Trp‐d ‐Phe in the aromatic dipeptide moiety. However, a novel analogue with a trimethylated ornithine and Phe‐d ‐Phe showed an activity rivalling that of TrcB. Our results emphasized that activity is dictated by interplay between the character of the aromatic residues in the variable pentapeptide and the cationic residue. Any residue change resulting in tighter membrane/cell wall interaction is likely to trap tyrocidines and impede their mechanism of action.     

Synthesis,In vitro and Docking Studies of New Flavone Ethers as α‐Glucosidase Inhibitors

We report herein the synthesis, α‐glucosidase inhibition and docking studies for a series of 3–15 new flavones. A simple nucleophilic substitution reaction takes place between 3′hydroxyflavone ( 2 ) with halides to afford the new flavones. Chalcone ( 1 ), 3′hydroxyflavone ( 2 ) and the newly synthesized flavones ( 3–15 ) were being evaluated for their ability to inhibit activity of α ‐glucosidase. Compounds 2 , 3 , 5 , 7 – 10 and 13 showed good inhibitory activity with IC₅₀ values ranging between 1.26 and 36.44 μ m as compared to acarbose (IC₅₀ = 38.25 ± 0.12 μ m ). Compounds 5 (5.45 ± 0.08 μ m ), 7 (1.26 ± 0.01 μ m ) and 8 (8.66 ± 0.08 μ m ) showed excellent inhibitory activity, and this may be due to trifluoromethyl substitution that is common for these compounds. Compound 7 , a 2,5‐trifluoromethyl‐substituted compound, recorded the highest inhibition activity, and it is thirty times better than the standard drug. Docking studies for compound 7 suggest that both trifluoromethyl substituents are well positioned in a binding pocket surrounded by Phe300, Phe177, Phe157, Ala278, Asp68, Tyr71 and Asp214. The ability of compound 7 to interact with Tyr71 and Phe177 is extremely significant as they are found to be important for substrates recognition by α ‐glucosidase.     

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).     

Synthesis and pharmacological activity of the N-terminal dermorphin tetrapeptide analogs with CH2-NH peptide bond isosteres

The synthesis of pseudotetrapeptides H-Tyr-D-Ala-Phe-NH-(CH₂)₂-NH₂ (1a), H-Tyr-D-Ala-Phe-ψ(CH₂-NH)-Gly-NH₂ (2a), H-Tyr-D-Ala-ψ(CH₂-NH)-Phe-Gly-NH₂ (3a), and H-Tyr-ψ(CH₂-NH)-D-Ala-Phe-Gly-NH₂ (4a), representing the N-terminal tetrapeptide sequence of dermorphin, in which amide bonds are replaced by CH₂-NH bond, is described. N-acetyl-Tyr and desamino-Tyr pseudopeptide analogs (1-4b), (1-3c) are also described. The analogs were assayed in binding studies based on displacement of μ and δ-receptor selective radiolabels from rat brain membrane and in a bioassay using guinea pig ileum (GPI). Pseudopeptides in which the C-terminal (1a) or D-Ala-Phe (3a) amide bond are substituted, exhibit higher μ-affinities and μ-receptor selectivity than the corresponding Phe-Gly or Tyr-D-Ala analogs (2a, 4a). Acetyl-and desamino-Tyr pseudopeptide analogs (1-4b) and (1-3c) did not exhibit μ and δ-opioid receptor affinity at nM concentration. The relevance of the single peptide replacement and of its association to acetylation or amino group elimination of Tyr, is discussed on the basis of a receptor model for μ and δ opioids.     

Racemization free coupling of peptide segments

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂ has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed α-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt NMM always rendered highly optically impure products containing 10-20% of the d -epimer.     

Role of guanidine group at the N-terminal proctolin chain in cardioexcitatory effects in insects

Six proctolin analogues (I-VI) modified in position 1 of the peptide chain by the following amino acids: homo-Arg, Gac, Gav, Gap, Phe (p-guanidino) and Orn, were synthesized by conventional liquid phase method. The myotropic activity of the obtained peptides was investigated in cardioexcitatory test on two insect species, cockroach, Periplaneta americana L., and yellow mealworm, Tenebrio molitor.     

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 in vitro activities of new tryptophan-modified and thiomethylene-containing pseudopeptide antagonists of the neurokinins

A series of pseudopeptide analogs of the substance P-like hexapeptide Ava-Phe-Phe-Gly-Leu-Met-NH₂ was produced by N_α-protection, introduction of the thiomethylene bond, of d - and non-proteinogenic amino acids, and alteration of the side chain of tryptophan. Synthesis of the pseudopeptides on a solid phase was successfully improved by direct formation of the CH₂—S bond on the resin. However, while thiomethylene formation between leucine and norleucine led to the expected SS diastereoisomer, the major product of the similar coupling between two phenylalanines was the SR isomer. An improved resistance of the analogs to proteolysis was observed, which could be related to the structural changes. Interestingly, these modifications led to three water-soluble and potent neurokinin antagonists on classical in vitro bioassays.     

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO₃H group in the sulfated-tyrosine by the non-hydrolyzable -CH₂SO₃H group was the first described modification of the sulfate ester that does not affect CCK₈ activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH₂SO₃Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH₂SO₃Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK₈ by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK₈ analogues containing the l - or the d -Phe(p-CH₂SO₃Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH₂SO₃Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure l isomer was used. Both CCK₈ analogues displayed high affinities for peripheral and central receptors (KI ～ 1 nm ) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The ?stabilized-CCK₈ peptide”, easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH₂SO₃Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.