Design of Benzophenone-Containing Photoactivatable Linear

J. Med. Chem. 2005, 48, 3379-3388

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Design of Benzophenone-Containing Photoactivatable Linear Vasopressin Antagonists: Pharmacological and Photoreactive Properties Sylvie Ponthieux,†,§ Joe¨lle Cabot,‡,§ Bernard Mouillac,‡,* Rene´ Seyer,‡ Claude Barberis,‡ and Eric Carnazzi† CNRS UPR9023 and INSERM U469, IGF, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France Received August 3, 2004

We designed and synthesized new photoactivatable linear vasopressin analogues containing benzophenone photophores. All compounds were monitored and purified using RP-HPLC and characterized by mass spectrometry. Affinity and selectivity were determined in CHO cells expressing either human V1a, V1b or V2 receptor subtypes. Within the series, compounds 6 (PhCH2CO-LBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)-NH2) and 9 (PhCH2CO-DBpa-Phe-Gln-AsnArg-Pro-Arg-Tyr(3I)-NH2), containing a benzoylphenylalanine residue (Bpa), were selected and their antagonistic properties determined (Kinact ) 1.87 and 0.35 nM, respectively). The dissociation constant of the most potent candidate (compound 9) was further calculated from saturation experiments using the 125I derivative (Kd ) 0.07 ( 0.01 nM). Photolabeling experiments using radioactive compound 9 as a probe were specific and UV-dependent and allowed the identification of two bands at molecular masses around 85-90 kDa and 46 kDa, respectively, as previously described using two photoreactive linear azidopeptide antagonists (Phalipou et al. J. Biol. Chem. 1997, 272, 26536-26544 and Phalipou et al. J. Biol. Chem. 1999, 274, 23316-23327). The results suggest therefore that compound 9 is a potent new tool for the accurate mapping of the human V1a receptor antagonist binding site. Introduction Vasopressin and oxytocin are two neuropeptides involved in several physiological functions and act through the binding to different subtypes of the seven-transmembrane G-protein-coupled receptor family.1 Oxytocin acts via a single receptor functionally coupled to phospholipase C and stimulates the contraction of uterine and mammary myocytes during parturition and lactation, respectively. Vasopressin exerts its biological functions upon binding to three receptor subtypes, identified as V1a, V1b and V2. Both V1a and V1b subtypes are coupled to phospholipase C producing inositol phosphates as second messengers. Binding to the V2 receptor subtype results in the production of cyclic adenosine monophosphate upon activation of adenylyl cyclase. Among its biological functions, vasopressin exerts an antidiuretic effect on the kidney, following binding to the V2 receptor subtype, and regulates blood pressure and vasoconstriction by interacting with the V1a receptor subtype. The V1b receptor subtype is responsible for stimulating corticotropin release from the pituitary. To date, several members of this receptor family have been cloned from various mammalian species.2-5 For years our laboratory has been interested in the structure-activity relationships of the oxytocin and vasopressin peptide family, to allow a better understanding of the molecular mechanisms involved in the ligandreceptor interactions. In addition, the precise mapping of a receptor-binding site is of great importance for the rational design of potential therapeutic agents, as well as for the development of selective analogues to study the receptor subtypes involved in specific functions. * To whom correspondence should be addressed. Phone: 33 (0)4 67 14 29 22. Fax: 33 (0)4 67 54 24 32. E-mail: [email protected]. † CNRS UPR9023. ‡ INSERM U469. § These two authors contributed equally to this work.

In the past few years we have identified several contact regions of the ligand-receptor binding site for the human V1a receptor subtype using site-directed mutagenesis, molecular modeling and receptor photolabeling approaches.6-10 For this purpose we previously developed a series of linear antagonists bearing a photoreactive arylazido group at the N-terminal position of the peptide ligands,9 although it has been described previously that this position is quite sensitive to chemical modifications.11 Upon photoactivation, one of these antagonists was shown to be covalently attached to the upper part of the seventh transmembrane domain of the human V1a receptor stably expressed in Chinese hamster ovary cells.7 We further mapped the receptorbinding site by positioning the azido group near the C-terminal part of the antagonist, on the side-chain of a lysine residue.8 Photolabeling experiments indicated that the new ligand was covalently attached in a region spanning the first extra-cellular loop of the human V1a receptor. A third peptide antagonist with a cyclic structure and having an azido group on the C-terminal residue allowed us to determine another contact point in the extracellular region of the third transmembrane domain. Combined with the data obtained from the mutagenesis and modeling experiments,6 these experimental results are in perfect agreement with the 3D model of the peptide binding pocket of the V1a receptor and clearly prove that photolabeling is an essential approach for the direct identification of specific ligandreceptor contact regions. To further investigate the mapping of the V1a receptor-binding site, we designed and synthesized a new series of photoactivatable vasopressin analogues containing the benzophenone group, which has, to date, never been described for the AVP/OT family. The affinity of each compound was determined from com-

10.1021/jm040871+ CCC: $30.25 © 2005 American Chemical Society Published on Web 04/07/2005

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Scheme 1. Peptide Sequences of Vasopressin Analogues Bearing the Photoreactive Benzophenone Groupa

a Compounds were numbered (left part of the scheme) and aligned to allow a comparison of the relative position of the photoreactive benzophenone groups with the azido function of the selected parent probe, the azidophenylbutyryl-linear antagonist.9 Inhibition constants (Ki) for the human receptor, except for the parent probe (rat liver V1a receptor), are listed on the right of each analogue structure. The asterisk (*) indicates the photoreactive moeity of each compound. Bzbz: benzoylbenzoyl group, L and DBpa: p-benzoylphenylalanine residue of either L- or D-configuration. Y ) N-terminal modification of compounds 4-9.

petition experiments allowing the preselection of compounds that we then tested for their antagonistic property, receptor subtype selectivity and photolabeling reactivity for the human vasopressin V1a receptor. Results Probe Design and Synthesis. Since ligand photoincorporation is highly dependent on the nature of the reactive intermediate generated upon UV irradia-

tion,12,13 we substituted the N-terminal azidophenylbutyryl group of the previously reported analogue 4-N3Ph(CH2)3CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)NH2 (Parent probe, Scheme 1)9 for a benzophenone group14 according to the following criteria: (i) Benzophenones react preferentially with C-H bonds, thus resulting in high yields of covalent binding as described in the literature12,15 and permitting the identification of local interactions at the amino acid level,15-17 (ii) UV

Photoactivatable Linear Vasopressin Antagonists

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Scheme 2. Synthetic Route for the Probes

irradiations are performed at 365 nm, a wavelength higher than the one needed to activate arylazido compounds (254 nm), thus reducing protein degradation and allowing studies on cell cultures or other living systems, (iii) Benzophenones are chemically stable in reduced ambient light and are compatible with solid-phase peptide synthesis conditions such as HF acidolysis, (iv) Bpa18 are available under radioiodinated or tritiated forms19,20 with the radioisotope incorporated close to the photoreactive carbonyl therefore reducing possible loss of detection due to ligand degradation. In addition, the benzophenone group should fit perfectly in the putative ,hydrophobic pocket. of the vasopressin V1a receptor binding site,8 without altering the affinity of the corresponding ligand.9,11

Peptides were synthesized on a p-methylbenzhydrylamine (MBHA) resin using a Boc/BOP/HF strategy and purified on RP-HPLC. The synthesis of noniodinated precursors of compounds 3, 6 and 9 was completed in solution (Scheme 2). To determine a potential effect of the N-terminal ammonium charge, acetylated and phenylacetylated compounds were also designed. Since we were interested by the pharmacological properties of the radioiodinated compounds only, all analogues were, at first, monoiodinated using ICl (Scheme 2).9 They were then purified on RP-HPLC and characterized by either FAB or MALDI-TOF mass spectrometry (Table 1). Contrary to previous observations made with arylazido analogues,21 the 337-nm laser irradiation in the MALDI-TOF mode did not alter the Bpa-ligands,

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Table 1. Chemical Characteristics of Monoiodinated Compounds 1-9a compd

% MeCN elution (HPLC system II)

formula

exact mass

M + 1, found (FAB)

1 2 3 4 5 6 7 8 9

44 45 43 38 40 45 38 40 45

C70H87I1N16O15 C68H84I1N17O14 C70H89I1N18O14 C60H78I1N17O12 C62H80I1N17O13 C68H84I1N17O13 C60H78I1N17O12 C62H80I1N17O13 C68H84I1N17O13

1518.56 1489.54 1532.59 1355.51 1397.52 1473.55 1355.51 1397.52 1473.55

1519 1490 1533 1356 1398 1475 1356 1398 1475

a Each compound was purified using semipreparative RP-HPLC, and each of the collected fractions was further checked by analytical HPLC. The fractions were collected with respect to purity rather than yield, such that the purity of each compound was determined above 99%.

though this wavelength was very close to the benzophenone maximal absorption wavelength. This would confirm the reversibility of the diradicaloid formation as well as the possibility to increase covalent incorporation through repeated irradiations.12 However, a slight lightinduced deiodination occurred under 337-nm irradiation (not shown). Binding Affinities of Compounds 1-9. To place the benzophenone reactive carbonyl in a similar position to the azido group of our selected photoactivatable parent probe (Scheme 1),9 we first coupled benzoylbenzoic acid directly on a glycine residue used as the hydrophilic spacer and substituted the original Arg6 residue for a lysine residue to allow further biochemical derivatization (Schemes 1 and 2, compound 1). However, the affinity of the corresponding monoiodinated compound 1 (Bzbz-Gly-DTyr(Me)-Phe-Gln-Asn-Lys-Pro-ArgTyr(3I)-NH2), determined in competition experiments using [125I]-HOPhCH2CO-DTyr(Me)-Phe-Gln-Asn-ArgPro-Arg-NH2,22 was nearly 650-fold lower than that of the parent arylazido derivative (Ki ) 115 nM in human, versus Ki ) 0.18 nM, previously measured in rat liver membranes).9 To recover an affinity constant in the nanomolar range, we therefore removed the glycine residue and coupled either benzoylbenzoic acid or benzoylphenylalanine (LBpa)18 directly to the C-terminal H-DTyr(Me)Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 octapeptide segment of the parent probe (Schemes 1 and 2). The corresponding monoiodinated compounds 2 and 3 bound to the V1a receptor with affinity constants of 18 and 462 nM, respectively. Despite a gain of approximately 10-fold for compound 2 (Ki ) 18 nM), the affinity of compound 3, however, was even lower than that of compound 1 (Ki ) 115 nM). Having demonstrated that the receptor binding site tolerated various hydrophobic groups23,24 and various acyl chain lengths at the N-terminal position of previously described vasopressin analogues,9 various shortened ligands were generated by coupling either L or DBpa-residues on the C-terminal H-Phe-Gln-Asn-ArgPro-Arg-Tyr-MBHA resin corresponding to the heptapeptide segment of the parent probe (Schemes 1 and 2). We then compared the affinities of the corresponding monoiodinated derivatives 4 and 7 to the affinities of their respective N-acetylated (compounds 5 and 8) and N-phenylacetylated (compounds 6 and 9) analogues. Interestingly, all compounds of the D-configuration had a better affinity than their enantiomers of the L-configuration, the most striking difference being ob-

served for the N-acetylated compounds 5 (Ki ) 1492 nM) and 8 (Ki ) 24 nM) with an approximately 60-fold better affinity for compound 8 (D-configuration). In addition, although N-acetylation of compounds 4 (Ki ) 148 nM) and 7 (Ki ) 20 nM) did not enhance the affinity of the resulting compounds 5 (Ki ) 1492 nM) and 8 (Ki ) 24 nM), the incorporation of a phenylacetyl group at the same position strongly increased the affinity of the resulting compounds 6 (Ki ) 2.5 nM) and 9 (Ki ) 0.8 nM) (Scheme 1), suggesting the importance of a lipophilic group for local interactions within the binding pocket. Due to the importance of the stereochemistry of the Bpa-residues in all compounds, we hydrolyzed two of them (compounds 6 and 9), subsequently derivatized the H-Bpa-OH using the Marfey’s reagent,25 and finally controlled unambiguously their optical purity using RP-HPLC (system I, data not shown). Biological Activity of Compounds 6 and 9. We selected phenylacetylated compounds 6 and 9 for their high affinity and determined their activity in CHO cells expressing the human V1a receptor. Both of the compounds were unable to induce inositol phosphate accumulation and were able to inhibit AVP-induced accumulation of inositol phosphates, confirming their antagonistic properties (Figure 1). The results of the inhibition experiments indicated that compound 9 (D-configuration, Kinact ) 0.35 ( 0.14 nM, n ) 3) was more efficient than its L isomer (compound 6, Kinact ) 1.87 ( 0.22 nM, n ) 3) with a nearly 5-fold better Kinact value. Association Kinetics. Since important structural modifications were performed on the parent probe to generate compound 9 (addition of Bpa and of PhCH2CO-), association kinetics of radioactive compound 9 ([125I]9) to the receptor were performed (Figure 2). After a 60 min incubation, 80% of the compound was found to be bound to the human V1a receptor expressed in CHO cells and as such this incubation period was deemed acceptable for the binding assays and for photolabeling experiments. Selectivity and Affinity of Compound 9. Owing to both its high affinity and strong antagonist potency, we determined the selectivity of compound 9 using membrane preparations of CHO cells overexpressing either V1a, V1b or V2 receptor subtypes. As shown in Figure 3, compound 9 displayed a strong selectivity for the vasopressin V1a receptor subtype (Ki ) 0.47 ( 0.04 nM) over either V1b or V2 receptors subtypes (Ki ) 306 ( 8 nM and 2200 ( 1000 nM, respectively). Saturation binding experiments resulted in linear Scatchard representations, suggesting that [125I]9 bound



Figure 2. Association kinetics of radioactive compound 9 ([125I]9) for the V1a receptor. Membrane preparations of CHO cells expressing human V1a receptor were incubated in the presence of [125I]9 and harvested after different incubation times. The specific binding (9) was expressed for each incubation time as the difference between total (4) and nonspecific binding (O) determined in the presence of an excess of the corresponding non radioactive iodinated compound 9. The experiment is representative of three independent experiments each performed in triplicate.

Figure 1. Determination of the antagonist properties of compounds 6 and 9. myo-[2-3H]Inositol prelabeled CHO cells expressing the human V1a receptor were incubated with (all gray-colored bars) or without (basal activity, open bars) vasopressin in the presence of various concentrations of either compound 6 (upper graph) or 9 (lower graph). Results are expressed as disintegrations per min/well (250 000 cells/well) with no correction for the basal activity. Inhibition constants (Kinact) were calculated according to the formula Kinact ) IC50/ (1 + [AVP]/Kact), where [AVP] ) 1 nM, IC50 corresponds to the concentration of analogue yielding 50% inhibition of total specific inositol phosphate accumulation and Kact is the concentration of AVP inducing half-maximal accumulation of inositol phosphates (Kact ) 0.35 nM for AVP-stimulated CHO cells expressing the human V1a receptor).41 These graphs are representatives of three independent experiments each performed in triplicate.

to a single class of binding sites (Figure 4). The calculated Kd value was 0.07 ( 0.01 nM (n ) 8), close to the value determined for the parent azidopeptide (Ki ) 0.18 in rat liver membranes, Scheme 1),9 thus indicating that the initial DTyr2(Me) residue could be substituted for a DBpa derivative (compound 9) without altering the pharmacological properties. Photolabeling Experiments. Photolabeling experiments were performed using membrane preparations of V1a expressing CHO cells. The solubilized proteins were separated using SDS-PAGE and analyzed by autoradiography. As shown in Figure 5 (panel A), the photolabeling was specific since it was completely suppressed in the presence of an excess of either nonradioactive monoiodinated compound 9 (lane 5) or AVP (lane 6). In addition, the labeling was clearly UV-dependent, since its intensity increased according to the exposure time (comparing lanes 2, 3 and 4 corresponding to different irradiation times) and was not observed for nonirradiated samples (lane 1). Two major protein

Figure 3. Binding selectivity of compound 9 by competition experiments. Membrane preparations of CHO cells expressing either human V1a (b), V1b (2) or V2 (0) receptors were incubated in the presence of increasing amounts of compound 9 and a fixed amount of [3H3]AVP. The specific binding measured in the presence of compound 9 was expressed as the fraction of the total binding determined in its absence. The nonspecific binding was determined at the highest concentration of compound 9. The apparent dissociation constants (Ki) were calculated according to the relation Ki ) IC50/(1 + [*L]/ Kd), where IC50 is the concentration of compound 9 yielding 50% inhibition of specific total binding and [*L] and Kd are the respective concentration and affinity constants of [3H3]AVP for its different receptor subtypes (Kd ) 0.7 nM for the V1a receptor, 0.37 nM for the V1b receptor and 1.36 nM for the V2 receptor). Concentration-displacement curves are representative of three independent experiments each performed in triplicate.

bands were specifically labeled with molecular masses of 85-90 kDa and 46 kDa. The 85-90-kDa value is in agreement with the expected molecular mass for the native glycosylated human V1a receptor. Incubation of the membranes in the presence of protease inhibitors and ZnCl2 resulted in a decrease of the labeling intensity

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Figure 4. Saturation and Scatchard representation of a binding experiment performed with radioactive compound 9 ([125I]9). Membrane were incubated in the presence of increasing amounts of [125I]9. The specific binding (9) was expressed as the difference between total (4) and nonspecific binding (O) determined in the presence of an excess of the corresponding non radioactive iodinated compound 9. The experiment is representative of eight independent experiments each performed in triplicate.

This pattern is in accordance with previous results in which we reported that the 46-kDa protein was a proteolytic fragment generated during the incubation step.7,8,10,26 We determined a covalent incorporation of 2-3% by cutting the gels in 2-mm slices and by measuring their radioactivity content. Discussion

Figure 5. V1a receptor photolabeling using radioactive compound 9. Panel A: Time-dependent photolabeling of V1aexpressing CHO cell membranes. Membranes were incubated with [125I]9 in the absence (lanes 1, 2, 3 and 4) or in the presence of an excess of either nonradioactive monoiodinated compound 9 (lane 5) or AVP (lane 6). Membranes were irradiated for either 30 (lane 2), 50 (lane 3), 80 (lane 4) or 60 min (lanes 5 and 6) or not at all (lane 1). Following irradiation, the membranes were washed, solubilized in Laemmli buffer and subjected to 12% SDS-polyacrylamide gels for radioautography. Panel B: Effect of protease inhibitors on the photolabeling pattern. Membranes were incubated in the presence of ZnCl2 and protease inhibitors and then irradiated for 1 h.

of the 46-kDa protein and a concomitant increase of the labeling intensity of the 85-90-kDa protein (panel B).

Though a high affinity is not a prerequisite for efficient photoaffinity labeling, we have always considered this criteria important since much better results are usually obtained due to low nonspecific interactions.21,27-29 Accordingly, we have reported a series of photoactivatable azidopeptides among which the high affinity analogues allowed us to map three distinct regions of the human vasopressin V1a receptor.7,8,26 Although the covalent binding yields were particularly high for these azidoligands, 15-20% for the two linear antagonists7,8 and 2-3% for the cyclic one, covalent incorporation of azido compounds are significantly lower than those described for benzophenone-containing compounds, mostly because the latter ones incorporate preferentially into CH bonds within a hydrophobic environment.12 To more precisely map the V1a receptor binding-site, we designed a new series of photoactivatable analogues in which benzophenone-supporting residues were introduced in place of the various azidophenylacyl groups of the above-mentioned vasopressin ligands.9 We placed the benzophenone reactive carbonyl in a similar position occupied by the azido group, and for this purpose, added a Gly residue at the N-terminal position of the octapep-



tide segment 2-9 of the selected parent analogue (Scheme 1). This residue served as an anchor for the benzophenone group and was chosen to reduce the overall hydrophobic character of the butyryl chain of the parent analogue. However, this first modification gave compound 1, which was characterized by a very low affinity, suggesting that these modifications generated a steric hindrance altering the binding properties. We therefore removed the Gly spacer as well as the DTyr2(Me) residue of compound 1, hypothesizing that the two aromatic rings of the benzophenone group would be able to ,substitute. the aromatic rings of the two Nterminal residues {4-N3Ph1(CH2)3CO- and DTyr2(Me)}. Although an increase in affinity was observed for compounds 4 and 7 when compared to compound 3, we further acylated their N-terminal free amine to increase their affinity, using hydrophobic groups.11 Thus, the incorporation of a phenylacetyl group on compounds 4 and 7 generated compounds 6 and 9, having the highest binding affinities of the series, with respectively 60-fold and 25-fold increase compared to their free N-terminal amine analogues (compounds 4 and 7, Scheme 1). The high sensitivity related to the stereochemistry of the Bpa-residue introduced in position 2 (compounds 4-9) suggests that close ligand-receptor interactions occur at this position. Therefore, position 2 deserves further mapping reasoning that the closer the photoreactive group would be to the receptor contour, the higher the covalent binding yield would be. Such a sensitivity in regards to the stereochemistry of residues was reported by Escher et al. with angiotensin analogues.30,27,31 In the case of vasopressin, position 2 was described as crucial regarding the biological activity.23,24,32 In particular, antagonistic properties were obtained upon O-alkylation of the Tyr2 residue {Tyr2(Me)}.23,34,35 In agreement with this observation if one considers that the p-benzoyl group of a Bpa-residue is a particular O-alkylation of a Tyr-residue, both compounds 6 and 9 remained antagonists although the stereochemistry of the Bpa-residue in position 2 induced a strong difference in their affinities. To date, no photoreactive benzophenone analogues of either vasopressin or oxytocin have been reported in the literature. Compound 9 allowed an efficient photolabeling of the human V1a receptor, identifying molecular masses similar to those previously labeled with 3-N3Ph(CH2)2CO-DTyr(Me)-Phe-Gln-AsnArg-Pro-Arg-Tyr(3125I)-NH2, a probe which permitted the identification of the 7th transmembrane domain as the covalent binding site.7,8 Although the covalent incorporation yield is low (3%), compound 9 is a new tool to more precisely map the V1a receptor binding site on the basis of the particular photoreactivity of benzophenone groups. Such groups are indeed interesting since they can be activated above 300 nm, causing minimal damages to the protein content. Moreover, the photoreactive intermediates generated as diradicaloids in a triplet state, react preferentially with weak CH bonds, and are able to return to their ground state, permitting repeated irradiation steps. Such reactivity is therefore complementary to that of nitrene intermediates, irreversibly generated from azido groups upon release of a nitrogen molecule, and known to react nearly only with nucleophiles. These observations thus

suggest that covalent incorporation of Bpa radicals would occur on different but neighboring amino acid residues of the human V1a receptor, resulting in its concise mapping and offering new information on the receptor binding site topology. Experimental Section General. Protected amino acids are: Boc-DTyr(Dcb)-OH, Boc-Lys(Z2Cl)-OH, Boc-Lys(Fmoc)-OH, Boc-Arg(Tos)-OH, BocAsn(Xan)-OH, Boc-Gln(Xan)-OH. Bpa derivatives were obtained from Bachem, BOP, PyBOP and p-methylbenzhydrylamine resin were from either Bachem or Novabiochem. Solidphase peptide synthesis was performed using a manual device as previously described with analytical grade solvents.21 The pH of the organic solutions during solid-phase and solution synthesis was determined using a moistened pH indicator paper. Peptides were deprotected using HF (from Matheson, provided by Interchim) on a Kel-F apparatus (from the Protein Research Foundation). Couplings of the Bpa derivatives to the C-terminal peptides were performed in UV spectroscopy-grade quality Me2SO. All Bpa derivatives were handled in dim light. HPLC. Analytical HPLC monitorings were performed using three systems. Systems I and II. A Merck Lichrospher endcapped C18 column (5-µm particle size, 4 × 250 mm, 100 Å porosity) was fitted to a Shimadzu LC 9A pump monitored by a two-channel analogic recorder (Kipp and Zonen), equipped with two Waters 440 and 441 photometers, operating at 254 and 214 nm, respectively. Elution was usually performed in a high-pressure mode using a static 2 mL mixing chamber using linear gradients of mobile phase B (CF3CO2H:MeCN, 0.05/100, v/v) in mobile phase A (CF3CO2H:H2O, 0.1/100, v/v) at a flow rate of 2 mL/min (System I) or 1 mL/min (System II) with gradients of 1% B/min. The values reported as percent elution correspond to the percent of MeCN of the eluent passing through the detector cell at the time of UV detection and were corrected for the void volume (v0 ≈ 1.7 mL). System III. The radioiodinated compounds were purified on a specific installation including a Lichrocart Purospher end-capped C18 column (250 × 4 mm; 5-µm particle size) connected to a high-pressure Beckman gradient system including two 114 M pumps and a 421 A Controller, a 440 Waters detector set at 254 nm and a LKB fraction collector. Elution was performed with linear gradients of mobile phase B (CF3CO2H:MeCN:H2O, 0.1/60/40, v/v/v) in mobile phase A (CF3CO2H:H2O, 0.1/100, v/v) at a flow rate of 1 mL/min and gradients of 1% B/min. Highly radioactive 0.5-mL fractions were measured on a Mini-Assay type 6-20 γ counter (Numelec) equipped with an attenuator. Semipreparative HPLC was performed at each step of the synthesis utilizing high-pressure gradient mode employing a dual pump system (model 400, Applied Biosystem), driven by a 738A Controller (Applied Biosystem) and fitted with either a Whatman Partisil ODS 3 Magnum 20 column (10-µm particle size, 22 × 500 mm, v0 ) 110 mL) or a Vydac 218TP1022 column (10-µm particle size, 100 Å porosity, 22 × 250 mm, v0 ) 50 mL), each protected with a precolumn. UV detection was performed using a Waters 440 absorbance detector (254 nm) and an ABI 738A spectrophotometric detector (set on 214 nm). Peptide purifications were done using linear gradients of 0.2%, 0.5% or 1%/min of mobile phase B (CF3CO2H:MeCN, 0.05/100, v/v) in mobile phase A (CF3CO2H:H2O, 0.1/100, v/v) at a flow rate of 10 mL/min as previously reported.21 Briefly, the peak corresponding to the desired peptide was collected in several fractions using a Gilson model 202 fraction collector, coupled to the 214-nm detector and operating in the peak detection mode. The fractions were then checked (analytical HPLC), collected with respect to their purity rather than yield, flashfrozen and lyophilized. Mass Spectrometry. FAB-MS was performed at the Universite´ de Montpellier II, using thioglycerol as matrix. For MALDI-TOF the samples were prepared by adding to a 10-5 M peptide solution, an equal volume of a saturated solution of R-cyano-4-hydroxycinnamic acid in CF3CO2H:MeCN:H2O (0.1/50/50, v/v/v). The mix (0.5 µL) was then applied on the target of a Bruker Biflex III mass spectrometer and dried at

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room temperature. Desorption of the samples was performed using a 337-nm laser, and the positive ion mass spectra were obtained in a linear and reflector mode with an acceleration of 19 kV. External mass calibration was performed with lowmass peptide standards (angiotensine I and II). Amino Acid Analyses. Peptides were hydrolyzed as routinely, derivatized using phenyl isothiocyanate and then analyzed using RP-HPLC.36 The obtained H-Bpa-OH were purified by RP-HPLC, derivatized using Marfey’s reagent (1 g/100 mL Me2CO), and analyzed (system II) for detection of racemization. The D isomer, H-DBpa-Dnp-LAla-NH2 eluted at 37% MeCN (214/254 ) 1.6), followed by the L isomer H-LBpaDnp-LAla-NH2 at 49% (214/254 ) 1.4). Underivatized L- and D-Bpa eluted both at 24% (214/254 ) 0.2) and the Marfey’s reagent at 26% (214/254 ) 0.6). General Protocol for Solid-Phase Peptide Synthesis. The C-terminal octapeptide segment of the parent azidopeptide and noniodinated precursors of 1, 2, 4, 5, 7 and 8 were synthesized on a p-methylbenzhydrylamino polystyrene (pMBHA) using a tert-butyloxycarbonyl strategy with either BOP or PyBOP as coupling reagents, (i-Pr)2EtN as base (added stepwise until pH 8-9 according to a moistened indicator paper) and DMF as solvent.21 Bzbz-Gly-DTyr(Me)-Phe-Gln-Asn-Lys-Pro-Arg-Tyr-NH2‚ 2CF3CO2H was synthesized up until the Gly residue on a p-MBHA resin (4.34 g, 2.5 mmol) using Boc-protected amino acids (5 mmol) and BOP (5 mmol) in CH2Cl2 or DMF. Benzoylbenzoı¨c acid (1.42 mmol) was then coupled to an aliquot of the peptidyl-resin (1 g, ≈0.47 mmol) using BOP (1.42 mmol). The peptidyl-resin was treated twice with piperidine (20% in DMF, for 4 and 16 min, respectively) to remove the Fmoc group of the lysyl side-chain. The noniodinated precursor of compound 1 (341 mg) was obtained by HF treatment of the peptidyl-resin and HPLC purification. H-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚3CF3CO2H was synthesized to an aliquot of the heptapeptidyl-resin (1.5 g, ≈0.67 mmol) using Boc-DTyr(Me)-OH (2.02 mmol) and BOP (2.02 mmol) in CH2Cl2. The octapeptide was obtained by HF treatment of the peptidyl-resin and HPLC purification. Bzbz-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚2CF3CO2H was obtained by coupling benzoylbenzoı¨c acid (0.6 mmol) to an aliquot of the above octapeptidyl-resin (400 mg, ≈0.2 mmol) using BOP (0.6 mmol) in CH2Cl2. The noniodinated precursor of compound 2 (187 mg) was obtained by HF treatment of the peptidyl-resin and HPLC purification. H-LBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚3CF3CO2H was synthesized on an aliquot of the heptapeptidyl-resin (1 g, ≈0.45 mmol) using Boc-LBpa-OH (0.6 mmol) and BOP (0.6 mmol) in CH2Cl2. The noniodinated precursor of compound 4 was obtained by HF treatment of an aliquot of the peptidylresin (≈0.5 g) and HPLC purification. Ac-LBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚2CF3CO2H was synthesized by acetylation of an aliquot of the peptidylresin described for the synthesis of the precursor of compound 4 (0.58 g, ≈0.26 mmol) using acetic anhydride (50% in CH2Cl2) in the presence of (i-Pr)2EtN as base. The noniodinated precursor of compound 5 was obtained by HF treatment of the peptidyl-resin and HPLC purification. H-DBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚3CF3CO2H was synthesized on an aliquot of the heptapeptidyl-resin (2 g, ≈0.9 mmol) using Boc-DBpa-OH (1.8 mmol) and BOP (1.8 mmol) in CH2Cl2. The noniodinated precursor of compound 7 was obtained by HF treatment of an aliquot of the peptidylresin (≈1 g) and HPLC purification. Ac-DBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚2CF3CO2H was synthesized by acetylation of an aliquot of the peptidylresin described for the synthesis of the precursor of compound 7 (1 g, ≈0.45 mmol) using acetic anhydride (50% in CH2Cl2) in the presence of (i-Pr)2EtN as base. The noniodinated precursor of compound 8 was obtained by HF treatment of the peptidyl-resin and HPLC purification. General Protocol for Solution Coupling of the NTerminus Group. Noniodinated precursors of 3, 6 and 9 were synthesized up until the second last residue according to the

solid-phase synthesis. The peptidyl-resins were then treated with HF and the intermediates purified by RP-HPLC for solution synthesis in Me2SO using (i-Pr)2EtN as base, which was added stepwise until pH 8-9 according to a moistened indicator paper. H-LBpa-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚ 3CF3CO2H was obtained by coupling Boc-LBpa-OH (0.66 mmol) with octapeptide H-DTyr(Me)-Phe-Gln-Asn-Arg-ProArg-Tyr-NH2‚3CF3CO2H (50 mg, ≈0.33 mmol) using an excess of PyBOP in Me2SO. The noniodinated precursor of compound 3 was further purified by RP-HPLC. PhCH2CO-LBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚ 2CF3CO2H was obtained by coupling purified H-LBpa-PheGln-Asn-Arg-Pro-Arg-Tyr-NH2‚3CF3CO2H (6.8 mg, 4 µmol) with an excess of PhCH2CO2H (1.5 mg, 11 µmol, 2.7 equiv) in Me2SO (100 µL). BOP (2.7 mg) was added progressively until the complete disappearance of the peptide. Following 3 h of coupling, the mixture was acidified using CF3CO2H (10 µL), and the noniodinated precursor of compound 6 was isolated by semipreparative RP-HPLC and lyophilization. PhCH2CO-DBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚ 2CF3CO2H (noniodinated compound 9) was obtained similarly as described for noniodinated compound 6, isolated by semipreparative RP-HPLC and lyophilization. General Procedure of Nonradioactive Iodination. Compounds 1-9 were obtained by ICl iodination in ≈30% MeCN by successive additions of ICl amounts (10-2 M in MeOH), under HPLC monitoring, to form predominantly the monoiodinated (iodination might be performed directly on HPLC-fractions). The monoiodinated compounds were purified on semipreparative RP-HPLC, and then they were collected, lyophilized as trifluoroacetate salts and fully characterized by FAB mass spectrometry (Table 1) and amino acid analysis. Radioiodination Procedure. Noniodinated precursor of 9, PhCH2CO-DBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2‚2CF3CO2H (40 µL, 10-4 M) was diluted with a Pi buffer (60 µL, 0.5 M, pH 6.8) in an Iodo-Gen coated 300 µL Eppendorf (15 µg in 100 µL of CHCl3, previously evaporated to dryness) and incubated with 1 mCi of Na125I for 5 min. The reaction mixture was then removed from the Eppendorf and diluted with HPLC eluent A (900 µL) for immediate HPLC purification (HPLC system III). Fractions (0.5 mL) were collected at the corresponding MeCN elution % of nonradioactive compound 9, and the radioactivity content was measured by γ-counting (2000 Ci/mmol). The compound was then concentrated using a SpeedVac centrifuge and stored in liquid nitrogen. CHO Cell Culture. CHO cells expressing either human V1a, V1b or V2 receptors were cultured as previously described,6 in Petri dishes using Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 4 mM LGln, 500 units/mL penicillin and streptomycin, 0.25 µg/mL amphotericin B and 400 µg/mL geneticin, in an atmosphere of 95% air and 5% CO2 at 37 °C. Cells were eventually treated overnight with 5 mM sodium butyrate before confluence to increase receptor expression.37 Membrane Preparations. CHO cells grown in Petri dishes (15 cm diameter) were rinsed twice with a Pi buffer (10 mL, pH 6.8), harvested in an ice-cold buffer containing 15 mM Tris, 2 mM MgCl2 and 0.3 mM EDTA (pH 7.4) and lysed using a Polytron instrument in two successive steps of 12 and 5 s, respectively. Unbroken cells and large fragments were removed by centrifugation (1500g, 5 min, 4 °C) and the supernatants further centrifuged for 20 min (40000g, 4 °C). Pellets were then resuspended in a 50 mM Tris buffer (12 mL) containing 5 mM MgCl2 (pH 7.4) and centrifuged again. Pellets were then suspended in the Tris buffer (0.5 mL) and homogeneized (Potter, five strokes), and the protein content was determined according to the Bradford method.38 Preparations were used either immediately or stored in liquid nitrogen. Binding Experiments. Affinities of compounds 1-9 for the V1a receptor expressed in CHO cells (2 to 3 µg of protein) were determined by coincubating each compound at a concentration range from 10-6 to 10-12 M with a radioiodinated linear antagonist (45-80 pM).22 The selectivity of compound 9 was



determined by incubation at a concentration range from 10-6 to 10-12 M in competition with [3H3]AVP (2 nM) as the radioligand, and using either V1a, V1b or V2 expressing CHO cell membranes (10 to 15 µg of protein). Nonspecific binding was determined in the presence of AVP (10-5 M). As previously described,6 experiments were performed in triplicate in a 50 mM Tris buffer, 5 mM MgCl2 (pH 7.4), containing 1 mg/mL bovine serum albumin for 1 h at 30 °C. Membranes were harvested over Whatman glass fibers (GF/C) previously soaked for more than 5 h with 10 mg/mL BSA. The binding data were analyzed by nonlinear least-squares regression using the computer program Ligand.39 Association kinetics of radioactive compound 9 ([125I]9) to the receptor were performed according to the above-described protocol.40 Membranes (1 µg) were incubated in the presence of [125I]9 (80 pM) with or without an excess of the corresponding non radioactive compound 9 (0.4 µM). The membranes were harvested at different incubation times over Whatman glass fibers (GF/C) previously soaked with 0.5% polyethyleneimine. The Kd of compound [125I]9 was determined using saturation experiments, according to the protocol described for the competition experiments. Briefly, V1a-expressing CHO membranes (1 to 2 µg of protein) were incubated in the presence of compound [125I]9 ranging from 50 pM to 1.2 nM with or without an excess of the corresponding nonradioactive compound 9 (0.4 µM). Incubations were performed for 1 h at 30 °C in the abovedescribed Tris buffer containing 1 mg/mL bovine serum albumin, and filtration was performed over Whatman glass fibers previously soaked in 0.5% polyethyleneimine. Inositol Phosphate Assays. The antagonist potencies of analogues 6 and 9 were determined by measuring inositol phosphate accumulation, as previously reported.41 Transfected CHO cells were labeled with 1 µCi/mL myo-[2-3H]inositol (1020 Ci/mmol, Dupont New England Nuclear) for 24 h in a serum- and inositol-free medium. Cells were then rinsed twice in Pi buffer and incubated for 10 min in the absence (basal activity) or in the presence of various concentrations of compounds 6 or 9 in a Pi buffer containing 10 mM LiCl. Inositol phosphate production was then induced with vasopressin (1 nM) for an additional incubation period of 15 min. Incubation was stopped by addition of perchloric acid and inositol phosphates were extracted on an anion exchange column (Dowex AG-1 × 8, formate form, 200-400 mesh, Bio-Rad) for determination of the radioactivity content by scintillation counting. The Kinact value was calculated according to the formula Kinact ) IC50/(1 + [AVP]/Kact), where [AVP] ) 1 nM and Kact ) 0.35 nM.41 Photolabeling Experiment of Compound 9. CHO cell membranes expressing the human V1a receptor subtype (500 µg of protein) were resuspended in 50 mM Tris-HCl, 5 mM MgCl2 (4 mL, pH 7.4, buffer A) containing 0.5 mg/mL bovine serum albumin, with or without ZnCl2 (10-4 M) and protease inhibitors (leupeptin, 5 µg/mL; benzamidine, 10 µg/mL; soybean trypsin inhibitor, 5 µg/mL). Incubations were performed in borosilicated tubes for 1 h in darkness at 30 °C in the presence of freshly radioiodinated ligand [125I]9 (1 to 2 nM). Nonspecific interactions were determined in the presence of an excess of either nonradioactive monoiodinated compound 9 (0.4 µM) or AVP (10 µM). Membranes were then washed with cold bovine serum albumin-free buffer A (10 mL) and centrifuged (44000g, 20 min) twice. The pellet was resuspended in cold bovine serum albumin-free buffer A (1 mL) and irradiated for 1 h at 365 nm or as stated otherwise (Blackray UV lamp, 100 W) in an ice-cold siliconized Petri dish. Then the membranes were washed twice with buffer A (1 mL) and solubilized in Laemmli buffer for SDS-PAGE separation (12% crosslinked gels). The migration was performed using prestained standards (Kaleidoscope, from Bio-Rad). Following this, the gel was fixed in MeCO2H:MeOH:Me2SO:H2O, 16/40/2/42 (v/ v), dried under vacuum at 80 °C and finally autoradiographed using Kodak XAR-5 films at -70 °C. The molecular masses were deduced from the log (mass) versus migration plot. To evaluate the covalent binding yield, gels were cut in 2-mm

slices and their radioactivity content measured using a γ-counter. The covalent binding yield was calculated as the percentage of the total amount of receptor pmol loaded per well.

Acknowledgment. This work was supported by INSERM and CNRS. Thanks are due to Dr. P. Jouin for constant interest and advice, Dr. N. Gale´otti for MALDI-TOF experiments, Dr. G. Niel for suggesting and providing Marfey’s reagent, A. Pantaloni for amino acid analysis, M.-N. Balestre for help in biological tests and Joanne Ryan for corrections of the manuscript. Appendix Abbreviations: Abbreviations used are in accordance with the IUPAC-IUB Commission (Eur. J. Biochem. 1984, 138, 9-37, or http://www.chem.qmul.ac.uk/iupac/AminoAcid/). The following abbreviations were also used: AVP, arginine vasopressin; Boc, tertbutyloxycarbonyl; BOP, (1H-1,2,3-benzotriazol-1-yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate; Bpa, benzoylphenylalanine; BSA, bovine serum albumine; Bzbz-, benzoylbenzoyl; CHO, Chinese hamster ovary; Dcb, 2,6-dichlorobenzyl; DMF, N,N-dimethylformamide; dpm, disintegration per min; EDTA, ethylenediamine tetraacetate; equiv, molar equivalent; FAB, fast atom bombardment; Fmoc, fluorenylmethoxycarbonyl; HPLC, high-performance liquid chromatography; IC50, concentration yielding 50% inhibition of the biological effect; (i-Pr)2EtN, diisopropylethylamine; Kact, activation constant; Kinact, inactivation constant; *L, radiolabeled ligand; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MBHA, p-methylbenzhydrylamine; MS, mass spectroscopy; Pi, phosphate buffer; Pi/NaCl, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PyBOP, (benzotriazolyloxy)tripyrrolidinophosphonium hexafluorophosphate; SPPS, solid-phase peptide synthesis; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)aminomethane; Tos, tosyl; v0, void volume of a chromatographic system; V1a, liver vasopressine receptor subtype; V1b, pituitary vasopressin receptor subtype; V2, kidney vasopressin receptor subtype; Xan, xanthyl; Z2Cl, 2-chlorobenzyloxycarbonyl. References (1) Jard, S.; Elands, J.; Schmidt, A.; Barberis, C. Vasopressin and oxytocin receptors: an overview. Progress in Endocrinology; Elsevier: Amsterdam, 1988; pp 1183-1189. (2) Morel, A.; O’Carroll, A. M.; Brownstein, M. J.; Lolait, S. J. Molecular cloning and expression of a rat V1a arginine vasopressin receptor. Nature 1992, 356, 523-526. (3) Hirasawa, A.; Shibata, K.; Kotosai, K.; Tsujimoto, G. Cloning, functional expression and tissue distribution of human cDNA for the vascular-type vasopressin receptor. Biochem. Biophys. Res. Commun. 1994, 203, 72-79. (4) Hutchins, A. M.; Phillips, P. A.; Venter, D. J.; Burrell, L. M.; Johnston, C. I. Molecular cloning and sequencing of the gene encoding a sheep arginine vasopressin type 1a receptor. Biochim. Biophys. Acta 1995, 1263, 266-270. (5) Thibonnier, M.; Auzan, C.; Madhun, Z.; Wilkins, P.; BertiMattera, L.; Clauser, E. Molecular cloning, sequencing, and functional expression of a cDNA encoding the human V1a vasopressin receptor. J. Biol. Chem. 1994, 269, 3304-3310. (6) Mouillac, B.; Chini, B.; Balestre, M. N.; Elands, J.; TrumppKallmeyer, S.; Hoflack, J.; Hibert, M.; Jard, S.; Barberis, C. The binding site of neuropeptide vasopressin V1a receptor. Evidence for a major localization within transmembrane regions. J. Biol. Chem. 1995, 270, 25771-25777. (7) Phalipou, S.; Cotte, N.; Carnazzi, E.; Seyer, R.; Mahe, E.; Jard, S.; Barberis, C.; Mouillac, B. Mapping peptide-binding domains of the human V1a vasopressin receptor with a photoactivatable linear peptide antagonist. J. Biol. Chem. 1997, 272, 2653626544.

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JM040871+

Design of Benzophenone-Containing Photoactivatable Linear

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