Cyclic and Acyclic Oxorhenium(V)−Peptide Conjugates as New

Bioconjugate Chem. 2006, 17, 807−814

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Cyclic and Acyclic Oxorhenium(V)-Peptide Conjugates as New Ligands of the Human Cyclophilin hCyp-18 Ce´cile Clavaud, Marion Heckenroth, Charlotte Stricane, Andre´ Me´nez, and Christophe Dugave* CEA/Saclay, De´partement d’Inge´nierie et d’Etudes des Prote´ines, Baˆtiment 152, 91191 Gif-sur-Yvette, France. Received November 17, 2005; Revised Manuscript Received April 7, 2006

Peptide metalloconstructs display interesting conformations, activities, and resistance to proteolysis. However, introduction of a metal core close to the residues that interact with the protein might strongly affect the binding. We investigated the effects of a coordinated oxorhenium core on the binding of model peptides to cyclophilin hCyp-18, a protein implicated in important biological processes and several diseases. For this purpose, we synthesized a series of linear metalloconstructs bearing an oxorhenium(V) core (ReO3+), as well as a peptide cyclized through oxorhenium(V) coordination. All these peptides contain an Ala-Pro-Xaa-pNA moiety (Xaa ) Cys derivative) and are anticipated to bind simultaneously to the S1-S1′ and S2′-S3′ subsites of hCyp-18. Therefore, the metal core is coordinated to both the cysteine residue and exogenous or endogenous NS2 tridentate systems. Cyclization of the peptide through metal coordination did not affect the affinity whereas bimolecular oxorhenium metalloconstructs bind hCyp-18 with a slightly better affinity than the corresponding nonmetalated peptide. Peptide labeling with a 99mTcO3+ core was also carried out successfully.

INTRODUCTION Engineered peptide metalloconstructs bearing a technetium core or a rhenium core have important applications in nuclear medicine due to their radiochemical properties (1, 2), stabilized conformations, and resulting enhanced resistance to proteolysis (3). Technetium and rhenium both belong to the group VIIB transition metals and share common similar coordination geometries, atom size, and bond lengths. They form stable complexes with a variety of organic groups, in particular with thiolates, thioethers, amines, amides, and carboxylates, all of which may be found in peptides or in synthetic bifunctional chelating agents (BFCAs) grafted onto peptides. In a first strategy called the integrated approach, the peptide backbone plus the side chains coordinate the metal core without any exogenous motif, but metalation often results in dramatic changes in geometry and steric hindrance relative to the nonmetalated peptide and thus greatly alters its biological activity (4). Conversely, the peptide can bind the metal core via coordination to a BFCA grafted either onto the N-terminus or onto an amino acid side chain, provided these moieties do not interact with the biological target (5). The latter approach has yielded a large number of peptide and protein radiopharmaceuticals (1, 2). Several reports have described the design, synthesis, and biological characterization of peptide hormones cyclized through rhenium and technetium metal coordination (6). In particular, Quinn and co-workers have designed R-melanotropin (R-MSH) peptide analogues that display an enhanced affinity for the R-MSH receptor relative to the linear peptide, due to additive constraints upon metalation (4, 7-9). A similar strategy was utilized by Gilon’s research team to cyclize a GnRH analogue (10). Sharma et al. also reported the metal-mediated cyclization of melanocortin and tuftsin peptides (11). Recently, Gilon and co-workers have described the synthesis of a library of metalcyclized somatostatin analogues (12). * Corresponding author. Tel: (33) 16908 5225. Fax: (33) 16908 9071. E-mail: [email protected].

We investigated the biochemical activity of short peptides bearing an oxorhenium(V) core coordinated via either an intermolecular (acyclic complexes) or an intramolecular (cyclic complex) NS2/S chelating agent. These metalloconstructs all contain a minimal tripeptide sequence Ala-Pro-Xaa-pNA which specifically binds to cyclophilin hCyp-18, an important peptidyl prolyl isomerase which plays a critical role in protein folding and cell division and communication, and is implicated in a large number of diseases including AIDS, cancer, and neurodegenerative disorders (13). Introduction of a ReO3+/99mTcO3+ metal core inside short ligands of cyclophilin might yield interesting substrate analogues with stabilized, biologically active conformations. Moreover, incorporation of technetium and rhenium radioisotopes might lead to the development of a new generation of radiopharmaceuticals that target hCyp-18. However, no information regarding the effect of the introduction of a rhenium complex close to the active site of hCyp-18 was available. In this study, we investigated the effect of the oxorhenium core on the binding properties of peptides derived from Suc-Ala-Ala-Pro-Phe-pNA 1, a known model substrate of hCyp-18 which displays a moderate affinity for cyclophilin.

EXPERIMENTAL PROCEDURES Materials and Methods. All reagents and solvents were purchased from Sigma-Aldrich, Fisher, or Novabiochem and were of the highest purity available. THF was distilled from sodium/benzophenone immediately prior to use. 99mTc was used as a commercial 99Mo/99mTc eluate (Schering). Reaction progress and flash chromatography elution (Merck silica gel 40-63 µm) were monitored by analytical thin-layer chromatography (Merck 60F254). Spots on TLC were visualized using UV light (254 nm), and spots were revealed by ninhydrin or phosphomolybdic acid. RP-HPLC analysis and purification were conducted on a Waters 600 Millennium chromatography system coupled to a Waters diode array detector. Separations were achieved on a Vydac C18 column eluted with an isocratic system (100% A) from 0 to 5 min, a linear gradient system (100% A to 100% B) from 5 to 25 min, and then an isocratic system (100% B) at a flow rate of 1 mL‚min-1 (analytical column 4.5 × 250 mm, 5

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µm) or 4 mL‚min-1 (semipreparative column 10 × 250 mm, 10 µm) (A, 0.1% aqueous solution of trifluoroacetic acid; B, acetonitrile). 1H and 13C NMR spectra were recorded on Bruker AVANCE 250 and 400 NMR spectrometers; δ and J are reported in ppm relative to TMS and Hz, respectively. Electrospray mass spectrometry (ES/MS) was performed on a Quattro II (Micromass, Altricham, U.K.). (βAla-Ala-Pro-Cys-pNA)2 2. (Boc-Cys-pNA)2, Boc-Pro-OH, Boc-Ala-OH and Boc-βAla-OH were coupled successively by the DCC, HOBt method to give the disulfide peptide (Boc-βAlaAla-Pro-Cys-pNA)2. This peptide was deprotected in 4 N HCl/ dioxane and purified by HPLC, to give peptide 2. HPLC tr ) 18.6 min. 1H NMR (D2O): δ 8.12 + 7.56 (2d, J ) 9.1 Hz, 8H), 4.72 (m, 2H), 4.55 (m, 2H), 4.43 (m, 2H), 3.84-3.63 (2m, 4H), 3.35-3.04 (2m, 8H), 2.66 (m, 4H), 2.27 (m, 2H), 2.041.87 (m, 6H), 1.31 (d, 6H). 13C NMR (D2O): δ 174.9, 174.0, 172.5, 170.9, 144.3, 143.7, 125.7, 121.2, 65.0, 61.2, 54.3, 50.0, 48.4, 39.4, 36.2, 32.3, 18.2, 24.1, 16.0. ES/MS (positive ionization): m/z 960.4 (M + H+, 100%). N-tert-Butyloxycarbonyl-bis(thioacetoethyl)amine 6. Diethanolamine (21.1 g, 200 mmol) was treated with di-tertbutyldicarbonate (44.1 g, 200 mmol) and DIPEA (71.1 mL, 400 mmol) in methanol (250 mL) overnight at room temperature. After removal of the solvent, the residue was dissolved in ethyl acetate and the organic layer was washed successively with 10% citric acid, saturated sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, and the solvent was evaporated in vacuo. The crude product was purified by silica gel flash chromatography (eluent: ethyl acetate:hexane 10:90) to give 6 (40.6 g, 99%). 1H NMR (CDCl3): δ 4.27 (bs, 2H), 3.78 (bs, 4H), 3.42 (bs, 2 × 2H), 1.46 (s, 9H). 13C NMR (CDCl3): δ 156.59, 80.50, 61.04, 52.49, 28.58. Diisopropylazodicarboxylate (24.87 mL, 120 mmol) was added dropwise to a solution of triphenylphosphine (31.79 g, 120 mmol) in dry THF (250 mL) at 0 °C. After formation of the white ylide precipitate, the solution was stirred for 30 min. A solution of N-tert-butyloxycarbonyl-diethanolamine (8.21 g, 40 mmol) in 40 mL of dry THF and thioacetic acid (9 mL, 120 mmol) were added successively. The reaction was stirred for 1 h at 0 °C and then overnight at room temperature. After reduction of the volume, the residue was washed and dried as described above. The crude product was purified twice by silica gel flash chromatography (eluent: ethyl acetate:hexane; first purification 10:90, second purification 5:95) to provide the bisthioacetate 5 (11.1 g, 87%). 1H NMR (CDCl3): δ 3.40-3.35 (t, J ) 6.6 Hz, 4H, 3.06-3.00 (bt, J ) 7.3 Hz, 4H), 2.34 (s, 3H), 1.47 (s, 9H). 13C NMR (CDCl3): δ 155.2, 80.3, 47.6, 47.4, 30.7, 28.4, 27.9, 27.4. ES/MS (positive ionization): m/z 322.1 (M + H+). Elemental anal. (%): calcd C (48.57), H (7.21), N (4.36), S (19.95); found C (48.49), H (7.04), N (4.42), S (20.22). N-tert-Butyloxycarbonyl-bis(thioethyl)amine Disulfide 7. Sodium methanolate (1.39 g, 25.7 mmol) was added to a solution of 6 (4.12 g, 12.8 mmol) in methanol (100 mL). The mixture was stirred over 5 days at room temperature in an open flask. The solvent was removed under reduced pressure, and the crude product was purified by silica gel flash chromatography (eluent: ethyl acetate:hexane 20:80) to provide disulfide 7 (1.96 g, 71%). 1H NMR (CDCl3): δ 3.75-3.68 (bq, J ) 5.7 Hz, 4H), 3.02-2.93 (dt, J ) 5.7 Hz, J′ ) 10.1 Hz, 4H), 1.47 (s, 9H). 13C NMR (CDCl3): δ 154.98, 79.94, 51.21, 50.77, 38.10, 28.49. ES/MS (positive ionization): m/z 236.7 (M + H+). Elemental anal. (%): calcd C (45.93), H (7.28), N (5.95), S (27.25); found C (46.25), H (7.21), N (6.13), S (26.81). N-tert-Butyl-bis(thioethyl)glycinate Disulfide 3a. Compound 7 (1.96 g, 8.4 mmol) was treated with a 50:50 trifluoroacetic acid:DCM solution for 30 min. Then the solvent was evaporated in vacuo and a solution of tert-butyl-bromoacetate

Clavaud et al.

(1.9 mL, 12.6 mmol) and DIPEA (2.2 mL, 12.6 mmol) in DCM (60 mL) was added. The mixture was stirred overnight at room temperature. After removal of the solvent in vacuo the crude product was purified by silica gel flash chromatography (eluent: ethyl acetate:hexane 30:70) to provide the disulfide 3a (1.72 g, 83%). 1H NMR (CDCl3): δ 3.44 (s, 2H), 3.343.29 (bt, J ) 2.9 Hz, 4H), 2.91-2.87 (bt, J ) 4.5 Hz, 4H), 1.47 (s, 9H). 13C NMR (CDCl3): δ170.98, 81.15, 57.24, 55.85, 39.86, 28.24. ES/MS (positive ionization): m/z 250.0 (M + H+). Elemental anal. (%): calcd C (48.16), H (7.68), N (5.62), S (25.71); found C (48.22), H (7.56), N (5.55), S (25.51). General Procedure for the Preparation of Compounds 3b and 3c. Bromoacetyl bromide (90 µL, 1 mmol) and DIPEA (190 µL, 1 mmol) were successively added to a solution of tertbutylamine or piperidine in DCM (10 mL) to provide the bromoacetamides 8b and 8c. The mixture was stirred for 30 min at 0 °C. The solvent was removed under reduced pressure. Compound 7 was treated with a 50:50 trifluoroacetic acid:DCM solution (20 mL) for 30 min. After removal of the solvent in vacuo and chase of toluene on the crude product, the residue was dissolved in methanol (5 mL). Solutions of bromoacetamide and amine resulting from the acidolysis of 7 were mixed together and treated overnight with sodium methanolate (118 mg, 2.1 mmol). After removal of the solvent in vacuo the crude product was purified by silica gel flash chromatography. N-tert-Butyl-bis(thioethyl)glycinamide Disulfide 3b. tertButylamine (106 µL, 1.03 mmol) was treated as describe above (eluent: ethyl acetate:hexane 50:50) to provide the disulfide 3b (59 mg, 54%). 1H NMR (CDCl3): δ 7.35 (s, 1H), 3.36 (s, 2H), 3.31-3.23 (bt, J ) 5.4 Hz) + 2.91-2.87 (bt, J ) 5.9 Hz) (4H), 1.38 (s, 9H). 13C NMR (CDCl3): δ 169.81, 62.50, 57.48, 51.22, 39.67, 28.96. ES/MS (positive ionization): m/z 248.6 (M + H+). Elemental anal. (%): calcd C (48.35), H (8.12), N (11.28), S (25.82); found C (48.41), H (8.69), N (10.97), S (25.44). N-Piperidyl-bis(thioethyl)glycinamide Disulfide 3c. Piperidine (105 µL, 1.03 mmol) was treated as describe above (eluent: ethyl acetate:hexane 50:50) to give disulfide 3c (78 mg, 29%). 1H NMR (CDCl3): δ 3.57 (s, 2H), 3.57-3.48 + 3.43-3.39 (2bt, 4H), 3.39-3.29 (bt, J ) 5.8 Hz) + 2.98-2.90 (bt, J ) 5.8 Hz) (4H), 1.61 + 1.23 (2m, 6H). 13C NMR (CDCl3): δ 168.8, 57.2, 55.87, 46.5, 42.9, 39.9, 26.5, 25.8, 24.6. ES/MS (positive ionization): m/z 260.8 (M + H+). Elemental anal. (%): calcd C (50.73), H (7.74), N (10.76), S (24.63); found C (50.43), H (8.09), N (10.43), S (24.22). N-Bis-(thioethyl)glycine Disulfide Hydrochloride 9. Compound 3a (1.72 g, 6.9 mmol) was treated with 50/50 1 N HCl in acetonitrile for 1 h. After evaporation of the solvent in vacuo, toluene was chased twice over the residue. The hydrochloride salt 9‚HCl was quantitatively isolated (1.57 g, 100%). 1H NMR (D2O): δ 4.30 (s, 2H), 3.97-3.94 (bt, J ) 5.6 Hz, 4H), 3.313.26 (bt, J ) 5.9 Hz, 4H). 13C NMR (D2O): δ 169.0, 58.9, 34.6. ES/MS (positive ionization): m/z 192.9 (M +H+). Elemental anal. (%): calcd C (31.37), H (5.26), N (6.10), S (27.91); found C (31.13), H (5.53), N (5.76), S (27.63). Methyl N-E-Bis-(thioethyl)glycyl(N-r-tert-butyloxycarbonyl)lysinate Disulfide 10. Compound 9 (101.5 mg, 0.4 mmol), HATU (205 mg, 0.6 mmol), and DIPEA (313 µL, 1.6 mmol) were added to a solution of Boc-Lys-OMe (104.1 mg, 0.4 mmol) in anhydrous DMF (14 mL), and the mixture was stirred overnight at room temperature. After removal of the solvent under reduced pressure, the crude product was purified twice by silica gel flash chromatography (eluent: dichloromethane: methanol; first purification: 95:5, second purification: 98:2) to give 10 (149.7 mg, 86%). 1H NMR (CDCl3): δ 7.50 (s, 1H), 5.08 (bd, 1H), 4.28 (m, 1H), 3.74 (s, 3H), 3.38 (s, 2H), 3.323.24 (m, 6H), 2.92-2.88 (m, 4H), 1.82-1.50 (3m, 3 × 2H),

Rhenium Coordinates Binding to Human Cyclophilin

1.44 (s, 9H). 13C NMR (CDCl3): δ 173.3, 156.5, 150.5, 79.9, 57.3, 52.3, 39.6, 38.6, 32.3, 29.3, 28.3, 22.7. HRMS (TOF/ ES): m/z calcd for C18H33N3O5S2 435.1920; found 435.1913. Sodium N-E-Bis-(thioethyl)glycyl(N-r-tert-butyloxycarbonyl)lysinate Disulfide 11. Compound 10 (71.5 mg, 0.16 mmol)was treated with lithium hydroxide (6.58 mg, 0.16 mmol) in acetonitrile:water 3:1 for 45 min. The solvent was removed to give the lithium salt 11, which was used in the further step without purification. ES/MS (positive ionization): m/z 423.05 (M + H+). (Boc-Lys(COCH2N(CH2CH2S)2)-Ala-Pro-Cys-pNA)2 13. Compound 11 was coupled with (Ala-Pro-Cys-pNA)2 disulfide by the DCC, HOBt method to give peptide 13 (70 mg, 78%). tr ) 22.2 min. 1H NMR (CDCl3): δ 10.16 (s, 2H), 8.22 + 7.85 (2d, J ) 9.1 Hz, 8H), 7.56 + 7.15 (2s, 2 × 2H), 5.22 (s, 2H), 4.71 (m, 2H), 4.11 (m, 4H), 3.65 (m, 4H), 3.46 (m, 8H), 3.26 (m, 8H) 3.11 (m, 4H,), 2.92 (m, 4H), 2.23-1.97 (2m, 2 × 4H), 1.91 (bd, 10H), 1.67 (m, 8H), 1.44 (s, 18H). 13C NMR (CDCl3): δ 157.5, 143.9, 143.7, 125.0, 119.4, 80.1, 61.7, 54.3, 49.2, 34.0, 28.3, 25.6, 24.9. ES/MS (positive ionization): m/z 356.92 (M - 2Boc + 4H+, 100%). (Lys(COCH2N(CH2CH2S)2)-Ala-Pro-Cys-pNA)2 14. Compound 13 (70 mg, 0.04 mmol) was treated with 4 N HCldioxane (5 mL) for 4 h at 0 °C. After removal of the solvent in vacuo, the residue was dissolved in methanol and purified by HPLC (eluent: A, 0.1% aqueous solution of trifluoroacetic acid; B, acetonitrile; A:B 100:0 from 0 to 5 min, 10 min linear gradient from 100:0 to 70:30, 8 min isocratic 70:30, 12 min linear gradient from 70:30 to 0:100, then 0:100 for 5 min at a flow rate of 4 mL‚min-1) to give peptide 14 (13.0 mg, 23%), tr ) 18.6 min. 1H NMR (D2O): δ 8.24 + 7.62 (2d, J ) 7.4 Hz, 8H), 7.56 + 7.15 (2s, 2 × 2H), 4.64 (m, 2H), 4.46 (bt, 2H), 4.11 (m, 2H), 3.83 (s, 6H), 3.68 (m, 4H), 3.57 (s, 8H) 3.32 (m, 4H), 3.07 (m, 12H), 2.30-2.04 (2m, 2 × 4H), 1.4 (m, 8H), 1.8 (bd, 6H). 13C NMR (D2O): δ 143.9, 143.7, 125.0, 119.4, 83.5, 61.71, 54.3, 49.2, 35.6, 28.3, 25.6, 24.9. HRMS (TOF/ES): m/z calcd for C58H86N16O14S6 1422.4988; found 1422.4978. βAla-Ala-Pro-Cys(Acm)-pNA 15. Peptide 2 (96 mg, 0.1 mmol) was treated successively with tributylphosphine (38 µL, 2.2 equiv) in wet THF (6 mL) for 40 min. After removal of the solvent in vacuo, the crude product was treated with iodoacetamide (44 mg, 2.2 equiv) and cesium carbonate (72 mg, 0.22 equiv) in DMF (6 mL) overnight at room temperature. Purification by RP-HPLC using the standard gradient program described above gave compound 15 (40.7 mg, 38%); tr ) 15.8 min. 1H NMR (CD3CN): δ 8.19 + 7.98 (2d, 4H, J ) 9.3 Hz), 6.82 (bs, 1H), 6.49 (bs, 1H), 5.94 (bs, 1H), 4.80-4.72 (m, 2H), 4.55-4.50 (m, 1H), 3.76-3.68 (m, 2H), 3.49-3.37 (m, 4H), 2.96 (s, 2H), 2.13-1.74 (2m, 4H), 1.47 (m, 2H), 1.22 (t, 3H). 13C NMR (CD CN): δ 172.8, 171.9, 169.0, 144.0, 143.5, 124.8, 3 119.6, 61.2, 58.5, 53.7, 47.7, 47.6, 36.5, 36.2, 35.2, 33.9, 25.3, 24.9, 17.5. ES/MS (positive ionization): m/z 538.35 (M + H+, 100%). General Procedure for Preparation of Complexes 4a-c. Solutions of 2 and 3a-c in methanol were treated with 10% tributylphosphine in methanol for 30 min under argon before addition of [nBu4N][ReOCl4] and 10% triethylamine in methanol. A brown precipitate immediately formed. After stirring for 2 h, the crude mixture was centrifuged. The precipitate was washed twice with methanol, dissolved in dimethylsulfoxide, and purified by HPLC. Complex 4a. HPLC tr ) 21.9 min. 1H NMR (D2O): δ 8.17 + 7.68 (2d, J ) 8.0 Hz, 4H), 4.96 (m, 1H), 4.56 (m, H), 4.48 (m, H), 4.22 (bs, 2H), 3.80-3.59 (m, 6H), 3.18 (m, 4H), 2.94 (m, 4H), 2.70 (m, 2H), 2.28-2.02 (m, 4H), 1.45 (s, 9H), 1.33 (m, 3H). ES/MS (positive ionization): m/z 930.2 (185Re, M + H+, 53%) + 932.40 (187Re, M + H+, 100%).

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Complex 4b. HPLC tr ) 19.5 and 21.0 min. 1H NMR (D2O): δ 8.23 + 7.68 (2d, J ) 7.5 Hz, 4H), 4.80 (m, 1H), 4.64 (m, 1H), 4.48 (m, 1H), 4.22 (s, 2H), 3.68-3.59 (m, 6H), 3.16 (m, 4H), 2.70 (m, 4H), 2.29 (m, 2H), 2.20-2.05 (m, 4H), 1.41 (m, 12H). ES/MS (positive ionization): (first isomer) m/z ) 929.1 (185Re, M + H+, 53%) + 931.00 (M + H+, 100%); (second isomer) m/z 929.1 (185Re, M + H+, 50%) + 931.00 (M + H+, 100%). Complex 4c. HPLC tr ) 18.9 and 20.5 min. 1H NMR (D2O): δ 8.26 + 7.70 (2d, J ) 8.9 Hz, 4H), 4.97 (m, 1H), 4.56 (m, H), 4.47 (m, H), 4.39 (s, 2H), 4.11 (m, 2H), 3.76-3.58 (m, 8H), 3.24 (m, 6H), 3.15 (m, 2H), 2.83 + 2.70 (2m, 2 × 1H), 2.021.92 (m, 4H), 1.86-1.54 (bs, 6H), 1.38 (m, 3H). ES/MS (positive ionization): (first isomer) m/z ) 941.2 (185Re, M + H+, 52%) + 943.2 (187Re, M + H+, 100%); (second isomer) m/z 941.2 (185Re, M + H+, 59%) + 943.2 (187Re, M + H+, 100%). Complex 5. A solution of 14 (4.1 mg, 2.9 µmol) in methanol (115 µL) was treated with a 10% methanolic solution of tributylphosphine (15 µL) for 30 min under argon. Then methanolic solutions of [nBu4N]ReOCl4 (43 µL, 5.7 µmol) and 10% Et3N (35 µL, 22.8 mol) were added successively. A brown precipitate immediately formed. After stirring for 2 h, the crude mixture was centrifuged. The precipitate was washed twice with methanol, then dissolved in DMSO, and purified by HPLC. tr ) 19.9 min. 1H NMR (d6-DMSO): δ 10.5 (bs, 1H), 8.23 + 7.87 (2d, J ) 7.2 Hz, 4H), 7.82 (s, 1H), 4.63 (m, 1H), 4.55 (bt, 1H), 4.35-4.31 (m, 2H), 3.62 (s, 3H), 3.19 (m, 2H), 3.102.99 (m, 4H), 2.89 (m, 2H), 2.10-1.80 (2m, 2 × 2H), 1.561.53 (m, 4H), 1.15 (bd, 3H). ESMS (positive ionization): m/z 913.1 (185Re, M + H+, 52%) + 915.2 (187Re, M + H+, 100%). Determination of Dissociation Constants (Kd). Fluorimetric determination of Kd values was done using a JASCO FP-750 spectrofluorimeter equipped with a 200 µL thermostated cell. Increasing concentrations of rhenium complexes (dissolved in DMSO) were added to solutions of hCyp-18 (1.1 µM, 180 µL) in HEPES buffer (35 mM, pH 7.8). Fluorescence at 345 nm (λexcitation ) 285 nm, λemission ) 324 nm) was recorded at 20.0 ( 0.1 °C. Kd values were obtained as the fitted points of inflection from the sigmoid log F/C profiles. A concentrationdependent fluorescence enhancement was observed with CsA whereas a concentration-dependent fluorescence quenching was observed with pNA derivatives. Reaction with Glutathione. A solution of complexes 4a-c or 5 (500 µL, 50 µM) in a 35 mM HEPES buffer pH 7.4 was treated by an aqueous solution of glutathione (100 µL, 60 mM) at room temperature. For blank, distilled water was used instead of glutathione. All the solutions were purged with argon prior to use. Aliquots of the mixture (30 µL) were analyzed by RPHPLC after 5, 15, 30, 60, and 120 min. 99mTc Radiolabeling Experiments. The [99mTcVO](NS /S) 2 mixed ligand complexes 16a-c and 17 were obtained by ligandexchange reaction using the [99mTcVO]gluconate precursor which was produced by mixing solutions of stannous chloride (4 µL, 2 mg‚mL-1) in HCl 0.1 N, 50 mM gluconate (20 µL), and a [99mTc]pertechnetate solution (200 µL, 11-30 MBq) eluted from a 99Mo/99mTc Elumatic III generator, Schering. The resulting [99mTcVO]gluconate solution was treated for 1 h at 50 °C with disulfides 2, 3a-c, or 14 (100 µL of a 20 mM solution) previously reduced as reported above. Aliquots of these solutions were analyzed by RP-HPLC.

RESULTS AND DISCUSSION Cyclophilin hCyp-18 binds a large variety of proteins and peptides that display a canonical Xaa-Pro sequence, with a marked preference when Xaa is Ala, Gly, and Glu (Figure 1) (13-15). It also accommodates short pseudopeptide analogues of compound 1 (16, 17) as well as peptides derived from the

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Clavaud et al. Scheme 2

Scheme 3

Figure 1. Structure of peptide 1 (R ) CH3) and schematic drawing of hCyp-18 subsites S1-S1′ and S2-S2′. Scheme 1

HIV-1 capsid (18) and cyclopeptides such as cyclosporine (1315). hCyp-18 also binds some non-peptide compounds, in particular the macrolide sanglifehrin (19, 20) and cymbimicins (21), as well as small molecules such as dimedone amino acid derivatives (22), constrained Gly-Pro mimetics (23), and aniline derivatives (24-26). All these compounds interact simultaneously with the two subsites S1-S1′ and S2′-S3′ and usually enhance the fluorescence of Trp121 which is located at the edge of the S2′-S3′ subsite, though peptide 1 causes a fluorescence quenching due to the presence of the p-nitroanilide moiety (13, 17, 18). We investigated the potential of acyclic and cyclic metalloconstructs derived from the model tetrapeptide 1 as hCyp-18 ligands. All these peptides contain a Cys-pNA moiety which is anticipated to interact with the S2′-S3′ subsite via two H-bonds with Trp121 and Arg 148 (27, 28) and provides the S motif for metal coordination. In cyclic metalloconstructs, the complementary tridentate chelating motif N(CH2-CH2-S)2 (noted NS2) is grafted onto the N-terminal sequence of the peptide (P2 position), which does not play a significant part in the binding (Scheme 1) (29, 30). We first checked that coordination of a metal core at P2′ in a linear peptide containing the Ala-Pro sequence did not interfere with the binding of the peptide to hCyp-18. For this purpose, the disulfide tetrapeptide (βAla-Ala-Pro-Cys-pNA)2 2 was readily prepared as a common precursor that could be connected to compounds 3a-c via coordination of an oxorhenium(V) core after reduction of disulfides, in order to provide complexes 4ac. Compounds 3a-c possess the NS2 motif which is connected to 3 different substituents in order to check that variation in this position affects neither the coordination of the metal core nor the biochemical properties of the peptides (Scheme 1). We also investigated the ability of the intramolecular complex 5 to bind to hCyp-18. Compound 5, a cyclic analogue of complexes 4a-c, restricts the positioning of the rhenium core relative to the acyclic rhenium complexes. Several syntheses of functionalized NS2 motifs have been reported in the literature, but they necessitated the use of dangerous reagents such as ethylene sulfide (toxic) (31, 32) or liquid ammonia (toxic, hazardous) (33). Therefore, these

methods were not easily transposable to batch preparation. In particular, the double nucleophilic opening of ethylene sulfide by a variety of amines requires prolonged heating under pressure. Moreover, in our hands, the synthesis of disulfide 3a starting from ethylene sulfide and glycine tert-butyl ester gave very low yields (2-5%). Results seem to depend strongly on the temperature of addition of all reactants and on the presence of polymers. Previous reports have underlined that use of excess ethylene sulfide essentially gives byproducts along with polymers resulting from the opening of ethylene sulfide by the intermediary bis-mercaptan moiety (34). Moreover, we found that the reaction product was not usable in further steps due to the existence of nonseparable side products (Scheme 2). Another approach developed by Chiotellis and co-workers was based on the attractive reductive amination of 2,2′-dithiobis(2-methylpropanal) using sodium cyanoborohydride. However, this synthesis was not compatible with nonsubstituted disulfides (35). All these reasons prompted us to develop a simple and reproducible route to prepare large quantities of disulfides 3a. Selective N-protection of diethanolamine with di-tert-butyl dicarbonate followed by bis-thioacetylation with thioacetic acid, triphenylphosphine, and diisopropylazodicarboxylate (36) gave compound 6 in high yield (Scheme 3). In contrast, derivatization of the diol with 2 equiv of methanesulfonyl chloride or triphenylphosphine/carbon tetrabromide, and substitution with a salt (potassium or diisopropylethylammonium) of thioacetic acid, gave compound 6 in poor to moderate yields. Methanolysis of the bis-thioacetate was carried out for 5 days in an open flask under high dilution conditions (Scheme 3). In fact, removal of the acetyl groups was completed in a few hours but it gave the desired disulfide mixed with the corresponding bis-thiol, which was anticipated to react irreversibly with electrophiles employed in further steps. Under these conditions, precursor 7 was isolated by silica gel flash chromatography in 71% yield. After removal of the Boc group of compound 7 with TFA, N-alkylation with tert-butyl bromoacetate in the presence of diisopropylamine (DIPEA) gave compound 3a in 83% yield. The reaction was carried out reproducibly on the 25-120 mmol scale. Compounds 3b and 3c were obtained as follows: after acidolysis of the Boc group of precursor 6, treatment with bromoacyl-tert-butylamine 8b and bromoacetyl-piperidine 8c (obtained respectively by acylation of tert-butylamine or piperidine with bromoacetyl bromide) and sodium methanolate gave the corresponding disulfides 3b and 3c. Compound 3a was also obtained by this procedure, but the yield was significantly lower than that obtained with DIPEA (Scheme 4).

Rhenium Coordinates Binding to Human Cyclophilin

Bioconjugate Chem., Vol. 17, No. 3, 2006 811

Scheme 4

Scheme 5

Compound 3a was also used to synthesize peptide 14, the precursor of the cyclic metalloconstruct 5 by the synthetic route depicted in Scheme 5. Unexpectedly, deprotection of the tertbutyl group of 3a with 50% trifluoroacetic acid in dichloromethane gave a complex mixture of compounds resulting putatively from the reaction of the disulfide with the intermediary tert-butyl cation. Therefore, the tert-butyl ester was cleaved under very mild conditions (1 N aqueous HCl in acetonitrile) to give compound 9 in quantitative yields (Scheme 5). Coupling to the side chain of Boc-lysine methyl ester with HATU in DMF gave the corresponding adduct 10 in 86% yield. In contrast, coupling with DCC afforded compound 10 in lower yields. Saponification of the methyl ester gave the modified Boc-lysine 11, which can be used directly in peptide synthesis. Standard coupling of compound 11 with 1/2 equiv of disulfide peptide 12 led to peptide 13 in good yields. Removal of the Boc groups with a 4 N solution of hydrogen chloride in dioxane gave peptide 14, the oxidized precursor of complex 5, which was purified by RP-HPLC. Complexes 4a-c were assembled by reducing peptide 2 on the one hand and compounds 3a-c on the other with tributylphosphine in methanol and then mixing the resulting crude products with tetrabutylammonium tetrachlorooxorhenate(V) to give the corresponding rhenium coordinates 4a-c. After reduction of peptide 14 as reported above, metalation with commercial [Bu4N][ReOCl4] (37) gave the peptide-oxorhenium complex 5. All complexes precipitated and were thoroughly washed with

Figure 2. RP-HPLC chromatogram of peptide 2 (A) and complex 4b: Vydac C18 column (5 µm, 4.6 mm, 250 mm) eluted with a binary gradient system (A, 0.1% aqueous solution of trifluoroacetic acid; B, acetonitrile; A:B 100:0 from 0 to 5 min, 25 min linear gradient from 100:0 to 0:100, then 0:100 for 5 min at a flow rate of 1 mL‚min-1).

methanol and dried prior to characterization by NMR and mass spectrometry. Analysis by RP-HPLC on a C18 column showed that compound 4a appeared as a single peak whereas 4b and 4c were eluted as two peaks that had identical mass spectra, suggesting the existence of at least two diastereomers resulting from either the up or the down RedO position relative to the peptide moiety, or from a syn/anti isomerism about the coordinated tertiary amine (Figure 2). This was not observed with the cyclic complex 5, which gave a single but slightly shouldered peak which might correspond either to the coexistence of several diastereomers that do not separate under these conditions, or might result from a slow cis-trans isomerization about the alanyl-proline peptide bond (38) as suggested by the 400 MHz NMR spectrum (data not shown). In all cases, crude complexes were free of residual reduced or oxidized forms of peptides 2 or 14 as assessed by RP-HPLC and mass spectrometry analysis (Figure 3). In particular, all products’ mass spectra show multipeak motifs that reflect the coexistence of 185Re187Re and 32S-34S isotopic mixtures. In all cases, the experimental profile was identical to the calculated motif (Figure 3). The standard fluorescence titration assay (18) was employed for the determination of the apparent dissociation constants (Kd) of the ligands as previously reported. Excitation of the indole moiety of Trp121 at 285 nm and recording of fluorescence at 324 nm for increasing concentrations in peptide showed a classical sigmoid curve drawn from the relation % fluorescence ) f(log[ligand]). We first ensure that Phe to Cys change at P2′ (disulfide 2 and its thioalkylated equivalent 15) does not perturb the binding of the peptide to hCyp-18. Peptide 15 (βAla-AlaPro-Cys(Acm)-pNA) was simply prepared by reduction of peptide 2 with tributylphosphine and quenching of the free thiol with iodoacetamide. The experiments showed that increasing the concentration of peptides 2 and 15 produces a fluorescence quenching which is very similar to that observed with the model peptide 1 (Figure 4). This suggests that these compounds interact at the S2′-S3′ subsite to produce an extinction of the Trp121 fluorescence and hence that the Phe to Cys mutation has no significant effect on the affinity. The same curve profile was observed with all oxorhenium complexes (Table 1). This clearly shows that coordination of a rhenium core at P2′ does not affect the affinity of the 4-mer peptide sequences for the protein. As anticipated, the “R” group of complexes 4a-c (see Scheme 1) does not seem to influence the binding to hCyp-18 since it might protrude outside the active site. Complex 5 exhibits an affinity for hCyp-18 that is equivalent to that of peptides 1 and 14, suggesting that the rhenium core does not perturb the binding, though the cyclic structure of the complex is assumed to maintain the metal core close to the protein structure. A structural examination of several cyclophilin-ligand complexes, including hCyp-18:1, has shown that the side chain of the P2′ residue cannot interact with the protein since it stands out from the protein:ligand complex (27). This allows a relatively free positioning of the P2′ side chain, and, therefore, the highly polarized oxorhenium core is not anticipated to

812 Bioconjugate Chem., Vol. 17, No. 3, 2006

Clavaud et al.

Figure 3. Electrospray mass spectrum of complex 5 (m/z): Experimental spectrum (A and B); calculated spectrum (C); the existence of several distinct signals of incremented masses relative to the theoretical values may be explained by the presence of rhenium isotopes 185Re and 187Re and sulfur isotopes 32S and 34S. Table 1. RP-HPLC Retention Timesa compound 1 4a 4b 4c 2 15 14 5

HPLC tr (min) 21.9 19.5 + 21.0 18.9 + 20.5 18.5 18.9 17.6 19.9

ES/MS (m/z)b

Kd (µM)

932.2/930.2 931.0/929.1 + 931.0/929.1 943.2/941.2 + 943.1/941.2 960.4 538.35 356.9c 915.2/913.1

135 ( 20 81(5 78 (8 75 ( 5 111 ( 20 195 ( 29 127 ( 14 108 ( 2

a Vydac C18 column: 10 µM, 4.6 mm, 250 mm. Eluted with an isocratic system (100% A) from 0 to 5 min, a linear gradient system (100% A to 100% B) from 5 to 25 min, and then an isocratic system (100% B) at a flow rate of 1 mL‚min-1 (A, 0.1% aqueous solution of trifluoroacetic acid; B, acetonitrile). ES/MS m/z values and apparent affinity constants (Kd) of complexes 4a-c and 5 and peptides 1, 2, 14, and 15, calculated from fluorescence titration experiments b Observed as M + H+. c Observed as M + 4H+.

Figure 4. Fluorimetric determination of the apparent dissociation constants (Kd) of peptides 1 (9), 14 (0), and 15 (b) (A) and complexes 4a (9) and 5 (0) (B).

interact with the protein or therefore to disturb the positioning of the peptide backbone inside the active site. In particular, Arg155 and His126 and Asn102 are located within 6-7 Å of

the rhenium atom (27) and are not anticipated to interact directly with the metal, although deleterious effects of the oxorhenium dipole on the protein structure cannot be precluded. These results indicate that introduction of a ReO3+ core inside peptidic ligands of hCyp-18 does not perturb the interaction. We also investigated the possibility that complexes 4a-c and 5 might interact nonspecifically with hCyp-18. For this purpose we preincubated cyclosporin A (CsA) with hCyp-18 prior to addition of compounds 4a-c and 5. In fact, CsA, a cyclic undecapeptide, binds simultaneously to S1-S1′ and S2′-S3′ subsites and produces an enhancement of fluorescence (39). Therefore, CsA was anticipated to compete with the rhenium complexes. Preincubation with CsA effectively prevented binding of the complexes, and resulted in a concentration-dependent recovery of fluorescence, suggesting that compounds 4a-c and 5 interact with hCyp-18 in a specific way and bind to the S1S1′ and S2′-S3′ subsites simultaneously since they possess both a proline residue and a pNA moiety. We checked that these complexes also showed satisfactory stabilities when incubated with hCyp-18 for 24 h since the peptide adducts were recovered without any detectable degradation as assessed by RP-HPLC. We investigated the resistance of complexes to thiol-contain-

Rhenium Coordinates Binding to Human Cyclophilin

ing reducing agents by treating the complexes 4a-c and 5 with 1 mM glutathione (GSH) in pH 7.4 buffer. As anticipated, complexes 4a-c are sensitive to GSH which replaces the βAlaAla-Pro-Cys-pNA peptide to give the corresponding glutathionecontaining complexes (40). Conversely, complex 5 is extremely resistant to glutathione, although up to 20% of the starting material is degraded in the first 5 min whereas 80% of the complex was recovered after 30 min at 20 °C. The presence of bimolecular head-to-tail complexes might account for this result, since complexation was performed with concentrated solutions. However, only one peak was observed by RP-HPLC and no other rhenium-containing species was detected by mass spectrometry. Another explanation might be the existence of multiple non-interconvertible (up/down-syn/anti-cis/trans) isomers which might differ in their resistance to GSH. Finally, labeling of the peptides was carried out with 99mTc using standard procedures. Pertechnetate obtained from a 99Mo/ 99mTc generator was reduced with stannous chloride at room temperature. The [99mTcVO]gluconate precursor was complexed by an equimolar amount of peptide 2 and compounds 3a-c previously reduced with tributyl phosphine to give complexes 16a-c. Complex 17 was prepared from peptide 14 by a similar procedure. The metalated peptides 16a-c and 17 were analyzed by RP-HPLC and gave elution profiles similar to those of the corresponding rhenium complexes. In conclusion, we synthesized a series of bimolecular acyclic complexes 4a-c and a cyclic peptide complex 5 through coordination of the oxorhenium core. The rhenium complexes were assayed as ligands of cyclophilin and display satisfactory stabilities under the conditions employed for the binding assay. Results clearly indicate that all complexes interact with hCyp18 with an affinity which is equivalent to that of the substrate tetrapeptide 1 and suggest that it is possible to produce novel acyclic and cyclic cyclophilin ligands respectively by assembly or cyclization through metal coordination. The low affinity of these compounds certainly limits their use in vivo, but application of this strategy to more complex peptides such as CsA and SfA analogues might yield interesting radiopharmaceuticals to target cyclophilin.

ACKNOWLEDGMENT We gratefully acknowledge Dr. L. Le Clainche (CEA/Saclay, DIEP) for help in radiolabeling experiments and Dr. J. Barbier and F. Beau (CEA/Saclay, DIEP) for assistance in the analysis of 99mTc complexes. We thank Dr. M. Moutiez (CEA/Saclay, DIEP) for purification of the recombinant cyclophilin hCyp18. We are indebted to Dr. E. Zeckri (CEA/Saclay, DBJC) for NMR experiments at 400 MHz and to Dr. J. Le Gal (CEA/ Saclay, DIEP) for critical reading of the manuscript.

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