Aleksandra Olma

Position Three in Vasopressin Antagonist Tolerates Conformationally Restricted and Aromatic Amino Acid Substitutions: A Striking Contrast with Vasopressin Agonists

We report the solid‐phase synthesis and some pharmacological properties of 12 position three modified analogues (peptides 1–12) of the potent non‐selective antagonist of the antidiuretic (V2‐receptor), vasopressor (V1a‐receptor) responses to arginine vasopressin (AVP) and of the uterine contracting (OT‐receptor) responses to oxytocin (OT), [1(‐β mercapto‐β,β‐pentamethy lenepropionic acid)‐2‐O‐ethyl‐d‐tyrosine 4‐valine] arginine vasopressin [d(CH2)5D‐Tyr(Et) 2VAVP] (A) and two analogues of (B) (peptides 13,14), the 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid3 (Tic3) analogue of (A). Peptides 1–12 have the following substituents at position three in (A): (1) Pro; (2) Oic; (3) Atc; (4) D‐Atc; (5) Aic; (6) D‐Phe; (7) Ile; (8) Leu; (9) Tyr; (10) Trp; (11) Hphe; (12) [HO]Tic; Peptide (13) is the Tyr‐NH2 9 analogue of (B): Peptide (14) is the D‐Cys 6 analogue of (B). All 14 new peptides were evaluated for agonistic and antagonistic activities in in vivo V2 and V1a assays and in in vitro (no Mg2+) n oxytocic assays. With the exception of the D‐Phe3 peptide (No. 6), which exhibits very weak V2 agonism (…0.0017 u/mg), none of the remaining 13 peptides exhibit any agonistic activities in these assays. In striking contrast to their deleterious effects on agonistic activities in AVP, the Pro3, Oic3, Tyr3, Trp3 and Hphe3 substitutions in (A) are very well tolerated, leading to excellent retention of V2, V1a and OT antagonistic potencies. All are more potent as V2 antagonists than the Ile3 and Leu3 analogues of (A). The Tyr‐NH29 and D‐Cys6 substitutions in (B) are also well tolerated. The anti‐V2 pA2 values of peptides 1–5 and 7–14 are as follows (1) 7.77±0.03; (2) 7.41± 0.05; (3) 6.86±0.02; (4) 5.66±0.09; (5) …5.2; (7) 7.25± 0.08; (8) 6.82±0.06; (9) 7.58±0.05; (10) 7.61±0.08; (11) 7.59±0.07; (12) 7.20±0.05; (13) 7.57±0.1; (14) 7.52± 0.06. All analogues antagonize the vasopressor responses to AVP, with anti‐V 1a pA2 values ranging from 5.62 to 7.64, and the in vitro responses to OT, with anti‐OT pA2 values ranging from 5.79 to 7.94. With an anti‐V2 potency of 7.77±0.03, the Pro3 analogue of (A) is surprisingly equipotent with (A), (anti‐V2 pA2=7.81±0.07). These findings clearly indicate that position three in AVP V2/V1a antagonists, in contrast to position three in AVP agonists, is much more amenable to structural modification than had heretofore been anticipated. Furthermore, the surprising retention of V2 antagonism exhibited by the Pro3, Oic3, Tyr3, Trp3 and Hphe3 analogues of (A), together with the excellent retention of V2 antagonism by the Tyr‐NH29 and D‐Cys6 analogues of (B) are promising new leads to the design of potent and possibly orally active V2 antagonists for use as pharmacological tools and/or as radioiodinatable ligands and for development as potential therapeutic agents for the treatment of the hyponatremia caused by the syndrome of the inappropriate secretion of the antidiuretic hormone (SIADH).

An investigation of position 3 in arginine vasopressin with aliphatic, aromatic, conformationally-restricted, polar and charged amino acids.

We report the solid‐phase synthesis and some pharmacological properties of 23 new analogs of arginine vasopressin (AVP) which have the Phe3 residue replaced by a broad variety of amino acids. Peptides 1–9 have at position 3: (1) the mixed aromatic/aliphatic amino acid thienylalanine (Thi) and the aliphatic amino acids; (2) cyclohexylalanine (Cha); (3) norleucine (Nle); (4) Leu; (5) norvaline (Nva); (6) Val; (7) alpha‐aminobutyric acid (Abu); (8) Ala; (9) Gly. Peptides 10–23 have at position 3: the aromatic amino acids, (10) homophenylalanine (Hphe); (11) Tyr; (12) Trp; (13) 2‐naphthylalanine (2‐Nal); the conformationally‐restricted amino acids (14) Pro; (15) 2‐aminotetraline‐2‐carboxylic acid (Atc); the polar amino acids (16) Ser; (17) Thr; (18) Gln; and the charged amino acids (19) Asp; (20) Glu; (21) Arg; (22) Lys; (23) Orn. All 23 new peptides were evaluated for agonistic and, where appropriate, antagonistic activities in in vivo antidiuretic (V2‐receptor) and vasopressor (V1a‐receptor) assays and in in vitro (no Mg2+) oxytocic assays. The corresponding potencies (units/mg) in these assays for AVP are: 323±16; 369±6 and 13.9±0.5. Peptides 1–9 exhibit the following potencies (units/mg) in these three assays: (1) 379±14; 360±9; 36.2±1.9; (2) 294±21; 73.4±2.7; 0.33±0.02; (3) 249±28; 84.6±4.3; 4.72±0.16; (4) 229±19; 21.4±0.6; 2.1±0.2; (5) 134±5; 31.2±0.9; 28.4±0.2; (6) 114±9; 45.3±2.3; 11.3±1.6; (7) 86.7±2.5; 4.29±0.13; 0.45±0.03; (8) 15.5±1.5; 0.16±0.01; ∼0.02; (9) 3.76±0.03; <0.02; in vitro oxytocic agonism was not detected. These data show that the aliphatic amino acids Cha, Nle, Leu, Nva and Val are well‐tolerated at position 3 in AVP with retention of surprisingly high levels of antidiuretic activity. Peptides 2–9 exhibit significant gains in both antidiuretic/vasopressor (A/P) and antidiuretic/oxytocic (A/O) selectivities relative to AVP. [Thi3]AVP appears to be a more potent antidiuretic and oxytocic agonist than AVP and is equipotent with AVP as a vasopressor agonist. The antidiuretic potencies of peptides 10–23 exhibit drastic losses relative to AVP. They range from a low of 0.018±0.001 units/mg for the Lys3 analog (peptide 22) to a high of 24.6±4.6 units/mg for the Hphe3 analog (peptide 10). Their vasopressor potencies are also drastically reduced. These range from a low of <0.002 units/mg for peptide 22 to a high of 8.99±0.44 units/mg for the Atc3 analog (peptide 15). Peptides 10–23 exhibit negligible or undetectable in vitro oxytocic agonism. The findings on peptides 10–23 show that position 3 in AVP is highly intolerant of changes with aromatic, conformationally‐restricted, polar and charged amino acids. Furthermore, these findings are in striking contrast to our recent discovery that position 3 in the potent V2/V1a/OT antagonist d(CH2)5d‐Tyr(Et)2VAVP tolerates a broad latitude of structural change at position 3 with many of the same amino acids, to give excellent retention of antagonistic potencies. The data on peptides 1–4 offer promising clues to the design of more potent and selective AVP V2 agonists. Copyright

Synthesis and receptor binding of opioid peptide analogues containing β3-homo-amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β3‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β3h‐D‐Ala in position 2 or β3hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright

Synthesis and binding properties of endomorphin-2 analogs containing α-hydroxymethyl amino acids

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)-α-hydroxymethyltyrosine in position 1 and (R)- and (S)-α-hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that α-hydroxymethyl substitution of amino acid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d‐Ala‐Phe‐Phe‐NH2 (TAPP). Each amino acid was replaced with an l‐ or d‐β3‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β3h‐ d‐Ala‐Phe‐PheNH2 was found to be as active as the native tetrapeptide. Copyright

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity.

New analogues of deltorphin I (DT I, Tyr‐d‐Ala‐Phe‐Asp‐Val‐Val‐Gly‐NH2), with the d‐Ala residue in position 2 replaced by α‐methyl‐β‐azido(amino, 1‐pyrrolidinyl, 1‐piperidinyl or 4‐morpholinyl)alanine, were synthesized by a combination of solid‐phase and solution methods. All ten new analogues were tested for receptor affinity and selectivity to μ‐ and δ‐opioid receptors. The affinity of analogues containing (R) or (S)‐α‐methyl‐β‐azidoalanine in position 2 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. Peptide II, containing (S)‐α‐methyl‐β‐azidoalanine in position 2, displayed excellent δ‐receptor selectivity with its δ‐receptor affinity being only three times lower than that of DT I. Copyright

The Biological Consequences of Replacing d-Ala in Biphalin with Amphiphilic α-Alkylserines

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, μ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α‐alkylserines in position 2 and 2′. The incorporation of bulky α,α‐disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N‐migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor‐binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α‐alkylserine, as analogues containing (R)‐α‐alkylserines displayed higher μ receptor affinity and selectivity than those incorporating the (S)‐isomers.

Chemistry of α‐hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize “difficult” sequences containing a C‐terminal HmS residue, MeA–HmS or consecutive HmS. Preparative methods for orthogonal N‐ and/or C‐protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid‐phase peptide chemistry protocols allows synthetic flexibility toward HmS‐containing peptides. In the synthesis of the sterically hindered dipeptides with the C‐terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid‐phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C‐terminal HmS(Ipr) or HmS residues, thus precluding their C → N elongation. The successful protocols involve: (i) the 2 + 1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2‐chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N → O‐acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin. Copyright

Biological Consequences of the Incorporation of Amphiphilic Amino Acids into Opioid Peptide Sequences

Hydrophobic and aromatic interactions are most critical for membrane peptide receptor-ligand complex stability. We have hypothesized that proper location of hydrophilic counterparts to lipophilic and/or aromatic residues may stabilize complexation with the receptor pocket. In this work, we are presenting the biological consequences of introduction of a hydroxymethyl group into the α-position of phenylalanine or tyrosine residues of enkephalin or deltorphin analogues. The consequences of such a modification are strongly dependent on the position of the primary amino acid in the peptide chain.

Synthesis, binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3-homo-amino acids.

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β3‐homo‐amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β3‐homo‐Tyr in place of Tyr (Tyr‐d‐Ala‐Phe‐Gly‐β3‐homo‐Tyr‐NH‐)2 showed good μ receptor affinity and selectivity (IC50 = 0.302, IC50 ratio μ/δ = 68) and enzymatic stability in human plasma. Copyright