Published on Web 05/10/2003
Efficient Macrocyclization of U-Turn Preorganized Peptidomimetics: The Role of Intramolecular H-Bond and Solvophobic Effects Jorge Becerril,† Michael Bolte,‡ M. Isabel Burguete,† Francisco Galindo,† Enrique Garcı´a-Espan˜a,§ Santiago V. Luis,*,† and Juan F. Miravet*,† Contribution from the Department of Inorganic and Organic Chemistry, UniVersity Jaume, I. E-12080 Castello´ n, Spain, Department of Inorganic Chemistry, UniVersity of Valencia, c/Dr. Moliner 50, 46100, Burjassot, Valencia, and Institut fu¨r Organische Chemie, J. W. Goethe-UniVersita¨t Frankfurt, Marie-Curie-Str. 11, 60439 Frankfurt am Main, Germany Received September 9, 2002; E-mail:
[email protected].
Abstract: Simple peptidomimetic molecules derived from amino acids were reacted with meta- and parabis(bromomethyl)benzene in acetonitrile to very efficiently yield macrocyclic structures. The cyclization reaction does not require high dilution techniques and seems to be insensitive to the size of the formed macrocycle. The analysis of data obtained by 1H NMR, single-crystal X-ray diffraction, fluorescence measurements, and molecular mechanics indicate that folded conformations can preorganize the system for an efficient cyclization. The role played by intramolecular hydrogen-bonding and solvophobic effects in the presence of folded conformations is analyzed.
Introduction
Much work has been devoted in recent years to the preparation and study of peptidomimetics. The interest on this class of compounds has evolved from different areas of research. The analysis of the molecular basis of structural features in proteins, in particular structural motifs associated with folding, has been one of the main driving forces for this interest.1 A second factor has been the development of novel receptors in the fields of Molecular Recognition and Supramolecular Chemistry. In this area, molecules containing peptides or peptide-related fragments have found applications in the self-assembly of nanotubes and rosettes,2a-c modulation of protein-protein interactions,2d-e catalysis 2f-k cation, and recognition.2l-n A third point has been † ‡ §
Department of Inorganic and Organic Chemistry, University Jaume. Institut fu¨r Organische Chemie, J. W. Goethe-Universita¨t Frankfurt. Department of Inorganic Chemistry, University of Valencia.
(1) (a) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173. (b) Gardner, R. R.; Liang, G.-G. B.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117, 3280. (c) Nowick, J. S.; Insaf, S. J. Am. Chem. Soc. 1997, 119, 10 903. (2) (a) Bong, D. T.; Ghadiri, M. R. Angew. Chem., Int. Ed. Engl. 2001, 40, 2163. (b) Ranganthan, D. Acc. Chem. Res. 2001, 34, 919. (c) Kerchoffs, J. M. C.; Crego-Calama, M.; Luyten, I.; Timmerman, P.; Reinhoudt, D. N. Org. Lett. 2000, 2, 4123. (d) Peczuh, M. W.; Hamilton, A. D. Chem. ReV. 2000, 100, 2479. (e) Park, H. S.; Hamilton, A. D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5105. (f) Scarso, A.; Scheffer, U.; Go¨bel M.; Broxterman, Q.; Kaptein, B.; Formaggio, F.; Toniolo, C.; Scrimin, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5144. (g) Gilbertson, S. R.; Collibee, S. E.; Agrakov, A. J. Am. Chem. Soc. 2000, 122, 6522. (h) Dangel, B.; Clarke, M.; Haley, Sames, D.; Polt, R. J. Am. Chem. Soc. 1997, 119, 10 865. (i) Jarvo, E. R., Miller, S. J. Tetrahedron 2002, 52, 2481. (j) Adria´n, F.; Burguete, M. I.; Garcı´a, J. I.; Garcı´a, J.; Garcı´a-Espan˜a, E.; Luis, S. V.; Mayoral, J. A.; Royo, A. J.; Sa´nchez, M. C. Eur. J. Inorg. Chem. 1999, 2347. (k) Sansone, F.; Baldini, L.; Casnati, A.; Lazzarotto, M.; Ugozzoli, F.; Ungaro, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4842. (l) Kubik, S.; Goddard, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5127. (m) Kubik, S. J. Am. Chem. Soc. 1999, 121, 5846. (n) Pryor, K. E.; Shipps, G. W.; Skyler, D. A.; Rebek, J. Tetrahedron 1998, 54, 4107. 10.1021/ja0284759 CCC: $25.00 © 2003 American Chemical Society
the observation of important biomedical activities of some natural and synthetic peptidomimetics, as has been the case for the synthesis of novel compounds with anti-HIV activities3a,b or antibacterial properties.3c To stabilize “active” conformations, many of the former examples are based on the preparation of cyclic peptidomimetic structures. In particular, to achieve additional conformational constraints, special attention is focused on the incorporation of aromatic rings into the peptidomimetic backbone. Therefore, different strategies have been described associated to the preparation of cyclic peptides and analogues. For these compounds, the cyclization step is usually the main challenge and their preparation often involves complex synthetic methodologies which require the use of specific reactions, selective protection/deprotection steps and high dilution techniques.4 Under some circumstances, the use of the appropriate templates, in particular metal cations, can greatly improve cyclization steps.5 In connection with those approaches, a simple analysis of the structures of some peptidomimetic molecules being able to form U-turns, suggests that this preorganization (3) (a) Ettmayer, P.; Billich, A.; Hecht, P.; Rosenwirth, B.; Gstach, H. J. Med. Chem. 1996, 39, 3291. (b) Glenn, M. P.; Pattenden, L. K.; Reid, R. C.; Tyssen, D. P.; Tyndall, J. D. A.; Birch, C. J.; Fairlie, D. P. J. Med. Chem. 2002, 45, 371. (c) Fernandez-Lopez, S.; Kim, H. S.; Choi, E. C.; Delgado, M.; Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D. A.; Wilcoxen, K. M.; Ghadiri, M. R. Nature 2001, 412, 452. (4) (a) Scharn, D.; Germeroth, L.; Scheneider-Mergener, J.; Wenschuh, H. J. Org. Chem. 2001, 66, 507. (b) Feng, Y.; Wang, Z.; Burgess, K. J. Am. Chem. Soc. 1998, 120, 10 768. (c) Feng, Y. B.; Burgess, K. Chem. Eur. J. 1999, 5, 3261. (d) Ranganathan, D., Haridas, T. V.; Kurur, S.; Madhusudanan, K. P.; Roy, R.; Kunwar, A. C.; Sarma, A. V. S.; Vairamani, M.; Sarma, K. D. J. Org. Chem. 1999, 64, 3620 (e) Reid, R. C., Kelso, M. J.; Scanlon, M. J., Fairlie, D. P. J. Am. Chem. Soc. 2002, 124, 5673. (5) Hass, K.; Ponikwar, W.; No¨th, H.; Beck, W. Angew. Chem., Int. Ed. Engl. 1998, 37, 1086. J. AM. CHEM. SOC. 2003, 125, 6677-6686
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Scheme 1 a
Table 1. Yields Obtained for the Cyclization Reaction after Chromatographic Purification
a (i) DCC, N-hydroxysuccinimide, THF; (ii) H NCH (CH ) CH NH , 2 2 2 n 2 2 DME; (iii) HBr/AcOH; (iv) aq. NaOH
could favor intramolecular processes over the intermolecular ones and provide simple routes for the preparation of macrocyclic peptidomimetic structures. As a matter of fact, the presence of folded conformations found for acyclic peptidomimetics have been associated by us and other groups to the outcome of macrocyclization reactions.6 The predisposition of different peptidomimetic molecules to fold has been widely studied and several structural units such as urea, amino acids, proline residues, etc., have been associated with the formation of β-γ- or related U-turns in peptides or peptidomimetics.1,7 Here, we report how simple peptidomimetics with C2 symmetry such as 8-12, show a strong tendency to form folded structures under some conditions and how this can be used advantageously for the synthesis of cyclophane peptidomimetics. Results and Discussion
Open-chain peptidomimetic molecules 8-12 can be easily prepared starting from the corresponding diamines and the NCbz protected amino acids 1 through the initial formation of their activated N-hydroxysuccinimide esters 2 (see Scheme 1).8 Overall yields for the preparation of compounds 8-12, after the final deprotection step, were in the range 60-80%, and did not depend very much on the nature and length of the aliphatic spacer. Preliminary molecular mechanics calculations on compounds 8-10 suggested that folded conformations such as I (see Scheme 3) could be prevalent as a result of intramolecular H-bonds and contribute to the appropriate preorganization of the peptidomimetic chain for macrocyclization. Consequently, we evaluated the cyclization reaction shown in the Scheme 2 to test for the preorganization present in those molecules and its dependence on different factors, in particular the amino acid side chain and the length of aliphatic spacer. With this purpose, compounds 8-12 were reacted with different bis(bromomethyl)arenes in order to obtain the corresponding cyclic molecules 13-22. The yields (obtained after chromatographic purification) for the macrocyclization reactions carried out in acetonitrile and using potassium carbonate as a base are shown in Table 1. The reaction can be accelerated by addition of tetrabutylammonium (6) (a) Miller, S. C.; Blackwell, H. E.; Grubbs, R. H. J. Am.Chem. Soc. 1996, 118, 9606. (b) Adria´n, F.; Burguete, M. I.; Luis, S. V.; Miravet, J. F.; Querol, M. Tetrahedron Lett. 1999, 40, 1039. (c) Prabhakaran, E. N.; Rajesh, V.; Dubey, S.; Iqbal, J. Tetrahedron Lett. 2001, 42, 339. (d) Pattarawarapan, R. S.; Roy, S.; Burgess, K. Tetrahedron 2000, 56, 9809. (7) (a) Nakanishi, H.; Kahn, M. In Bioorganic Chemistry: Peptides and Proteines; Hecht, S. M., Ed; Oxford University Press: New York, 1998; Chapter 12. (b) Liu, Z.-P.; Rizo, J.; Gierasch, L. M. In Bioorganic Chemistry: Peptides and Proteines; Hecht, S. M., Ed; Oxford University Press: New York, 1998; Chapter 6. (8) Wagler, T. R.; Fang, Y.; Burrows, C. J. J. Org. Chem. 1989, 54, 1584. 6678 J. AM. CHEM. SOC.
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compd
R
n
yield (%)
13a 14a 15a 16a 17a 13b 14b 15b 16b 17b 18a 19a 20a 21a 22a 18b 19b 20b 21b 22b
-CH2Ph -CH2Ph -CH2Ph -CH2Ph -CH2Ph -CH(CH3)2 -CH(CH3)2 -CH(CH3)2 -CH(CH3)2 -CH(CH3)2 -CH2Ph -CH2Ph -CH2Ph -CH2Ph -CH2Ph -CH(CH3)2 -CH(CH3)2 -CH(CH3)2 -CH(CH3)2 -CH(CH3)2
0 1 2 4 6 0 1 2 4 6 0 1 2 4 6 0 1 2 4 6
65 61 66 65 52 49 61 69 52 55 63 52 63 67 55 64 56 65 60 65
bromide as a phase transfer catalyst but this reagent did not affect the yield of isolated macrocyclic product. The results were quite good for the meta and para substituted aromatic subunits and no significant differences were observed when derivatives of phenylalanine or valine were used. In most cases, yields for the expected product of ca. 80% could be estimated from the crude reaction mixture. The formation of the corresponding 2+2 cyclization compounds was detected in some cases as minor side products. In the case of ortho disubstituted aromatic derivatives the cyclization reactions did not work and 1,2-dihydroisoindol derivatives were obtained, a situation that has been described previously.9 The good results obtained when the components of the reaction were 1,3- or 1,4-bis(bromomethyl)benzene and the diamines 8, 9, and 10 (n ) 0, 1, 2) could, in a first analysis, be ascribed to the above-mentioned preorganization induced by intramolecular H-bonding as shown in I (Scheme 3). Surprisingly, although longer aliphatic chains should disfavor this kind of intramolecular hydrogen-bond formation, and therefore lead to a decrease of the efficiency of macrocycle formation, for compounds 11 and 12, with aliphatic spacers containing six and eight methylene units respectively, cyclization yields were also good and comparable to those obtained for compounds with shorter aliphatic spacers. Those results prompted us to study in more detail the importance of hydrogen bonding in the possible preorganization. In first place, 1H NMR experiments were carried out. The chemical shifts observed in CDCl3 and CD3CN for the signals of the amide protons in compounds 8-12 correspond with those expected for strongly hydrogen-bonded systems (see Table 2). The described chemical shift values were almost insensitive to changes in concentration (see Figure 1) indicating that hydrogen bonding is taking place intramolecularly, as indicated also by the changes observed with temperature.10 It is noticeable that, both in chloroform and acetonitrile, the amide proton resonances appear at lower ppm values as the length of the (9) Yaounanc, J. J.; Le Bris, N.; Le Gall, G.; Cle¨ment, J. C.; Handel, H.; Des Abbayes, H. J. Chem. Soc., Chem. Commun. 1991, 206. (10) (a) Gellman, S. H.; Dado, G. P.; Liang, G. B.; Adams, B. R. J. Am. Chem. Soc., 1991, 113, 1164. (b) Gaemman, K.; Bernhard, J.; Seebach, D.; Perozzo, R.; Scapozza, L.; Folkers, G. HelV. Chim. Acta 1999, 82, 1.
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Table 2. (a) 1H NMR Chemical Shifts for the Amide Protons of Acyclic Compounds 8-12 in CDCl3 and CD3CN compd
R
n
δ (ppm, CDCl3)
δ (ppm, CD3CN)
8a 9a 10a 11a 12a 8b 9b 10b 11b 12b
-CH2Ph -CH2Ph -CH2Ph -CH2Ph -CH2Ph -CH(CH3)2 -CH(CH3)2 -CH(CH3)2 -CH(CH3)2 -CH(CH3)2
0 1 2 4 6 0 1 2 4 6
7.55 7.56 7.30 7.24 7.21 7.70 7.67 7.27 7.31 7.29
7.34 7.40 7.21 7.18 7.16 7.52 7.54 7.34 7.30 7.28
a
Scheme 2 a
The concentration was 10-2 M in all cases.
a Reagents: (i) p-bis(bromomethyl)benzene, CH CN, Bu NBr, K CO , 3 4 2 3 reflux 12 h; (ii) m-bis(bromomethyl)benzene, CH3CN, Bu4NBr, K2CO3, reflux 12 h.
Scheme 3
Figure 1. Plot of 1H NMR chemical shift of amide hydrogen atoms of compounds 8b and 12b in CDCl3 vs logarithm of concentration (M).
aliphatic spacer increases from ethylenic to butylenic spacers (see the values for 8-10) and remains almost invariable for longer ones (10-12). These data indicate that intramolecularly hydrogen bonded conformations must be predominant in solution but, according to previous work in this field, they are very unlikely to correspond to folded structures such as I (Scheme 3) due to an unfavorable entropy factor.10 To better understand this behavior the model compounds 24 and 25 were studied (see Scheme 4). For compound 24 1H NMR experiments discarded intermolecular association and the chemical shift value for the amide protons (6.2 ppm in CDCl3) shows that hydrogen bonding is not so strong as that found for compounds 8-12. On the other hand, in compound 25 the only intramolecular H-bond interaction that can take place connects the lone pair of the amine group and the amide NH. In this case, the chemical shift of the amide protons appears at 7.2 ppm in CDCl3 and dilution experiments indicate the intramolecular nature of the interaction. Therefore, two types of hydrogen bonds would explain the observed spectra of compounds 8-12 in CDCl3 and CD3CN. The first type would involve the two amide groups and result in a folding in the molecule such as in I (Scheme 3). Its importance would decrease as the aliphatic spacer becomes longer. The second one involves the amine lone pair and the amide NH. This one must be present in all of the studied peptidomimetics independently of the spacer size and would explain the chemical shift values for the amide proton resonance in the molecules with long aliphatic spacers. Hence, the values reported in Table 2 can be rationalized if it is assumed that for compounds 8 and 9 both types of intramolecular H-bonding take
Scheme 4
Scheme 5
place, whereas for larger compounds 10-12 only the last type is present. Consequently, intramolecular hydrogen bonding can be argued as a mean to understand the preorganization that would yield preorganization favorable for the macrocyclization with 8-9 but the same case cannot be done for longer molecules 10-12. Because the key step for the efficiency of the cyclization is the intramolecular reaction of the intermediate species II (see Scheme 5) a computational study about the preorganization of such species was performed. It has been shown previously how J. AM. CHEM. SOC.
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Table 3. Average (C-N) Distances between the Primary Amino Group and Bromomethyl Units in Intermediate Compounds (II) (see Scheme 5) Calculated by Molecular Dynamics Simulationsa precursor of the intermediate (II)
d (Å) in CHCl3
d(Å) in H2O
8a 9a 10a 11a 12a 8b 9b 10b 11b 12b
10.7 11.8 12.5 13.4 15.6 10.7 13.4 13.3 14.7 15.5
8.2 9.7 8.7 9.0 8.2 7.2 8.7 7.4 7.9 8.3
a The molecular dynamics simulations were carried out with MACROMODEL 7.0 (5 ns, AMBER* as force field and GB/SA simulation of solvation).
molecular dynamics simulations can reproduce well the folding induced by intramolecular H-bonding.11 The presence of intramolecular H-bonding such as that shown in II was studied by analysis of 200 structures sampled during a 5 ns molecular dynamics simulation. It was found, in accordance with related studies described previously,10a that hydrogen bonding between the two amido groups in chloroform is only important for the shorter aliphatic species. For the compounds derived from phenylalanine containing spacers with 2, 3, 4, 6, and 8 methylene units the percentage of H-bonded structures found was respectively 25, 10, 7, 1 and 1. Similar results were obtained for the compounds derived from valine. As expected, intramolecular hydrogen bonding was found to be almost negligible when a simulation of water as a solvent was used for the calculations. Additionally, to analyze the ease of the cyclization reaction in these species, the distance between the nitrogen atom of the primary amino group and the carbon atom of the bromomethyl subunit (see distance d in structure II, Scheme 5) was monitored during the MD calculation (see Table 3) both in chloroform and water (acetonitrile parametrization was not available in the molecular mechanics program.) It can be seen that the average distances were always shorter when a simulation of water was employed as a solvent. The differences are significant, especially for the larger molecules, and a shorter C-N distance should correlate with an increased probability for the intramolecular cyclization reaction. Moreover, the distances distribution was always much narrower for the simulations performed in water where a range of preferred distances was found (see as an example Figure 2). Therefore, the calculations suggest that solvophobic effects seem to be a key issue for the efficiency of macrocyclization and could explain the invariance of the yield with the length of the aliphatic spacer. It can be recalled here that hydrophobic effects are accepted to be the driving force for the protein folding in water which results in actiVe conformations.7b Solvophobic effects, although not as strong as in water, are expected to be significant in acetonitrile, the solvent used for the studied reactions. As a matter of fact, significant folding of apolar molecules to yield ordered helical structures has been shown (11) (a) McDonald, D. Q.; Still, W. C. J. Am. Chem. Soc. 1994, 116, 11 550. (b) Burguete, M. I.; Escuder, B.; Garcı´a-Espan˜a, E.; Lo´pez, L.; Luis, S. V.; Miravet, J. F.; Querol, M. Tetrahedron Lett. 2002, 43, 1817. (c) For reviews on molecular dynamics simulations of biomolecules see the special issue on this topic: Acc. Chem. Res. 2002, 35, 321-489. 6680 J. AM. CHEM. SOC.
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Figure 2. Distribution of distances (see d in structure II, Scheme 5) found in the molecular dynamics simulations of the intermediate compound derived from 12a using simulations of chloroform and water as the solvent. (MACROMODEL 7.0, AMBER* as force field and GB/SA simulation of solvation).
Figure 3. Representative structures found in the MD simulation of intermediate II (see Scheme 5) derived from 12b using simulation of water (up) and chloroform (bottom). (Surface area was calculated with MACROMODEL 7.0 assuming a probe radius of 1.4 Å. The dotted line indicate the atoms whose distance was monitored during the simulation).
to take place in acetonitrile.12 To illustrate the effect of the solvent in the preorganization, representative molecules from the simulations for the intermediate derived from 12b in chloroform and in water are shown in Figure 3. It can be seen that in chloroform the molecule tends to be extended and fully exposed to the solvent. However, in water the molecule tends to fold back and minimize the exposition to the solvent due the presence of hydrophobic subunits. To get more evidences of the proposed solvent-induced folding, additional experiments were perfomed. First, the reaction to obtain compounds 18a and 22a was carried out in dimethylformamide as the solvent. The change of acetonitrile a polar solvent ( ) 37.5), that at the same time allows for the presence of fairly strong intramolecular hydrogen bonding interactions, for dimethylformamide, a strongly hydrogen bonding solvent with almost same polarity ( ) 36.7), resulted in a significative decrease in reaction yields (due to an important formation of side-products, no starting material was left) both (12) Lahiri, S.; Thompson, J. L.; Moore, J. S. J. Am. Chem. Soc. 2000, 122, 11 315.
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for compound 18a containing a ethylenic spacer (65% yield in acetonitrile, 31% in dimethylformamide) and 22a containing an octamethylenic spacer (53% yield in acetonitrile, 29% in dimethylformamide). Second, the reaction was performed in tetrahydrofurane, a quite less polar solvent ( ) 7.6), where intramolecular hydrogen bonding is expected to be important (the amide proton chemical shift values obtained for compounds 8a and 12a in this solvent are comparable to those obtained in chloroform). In this case, the preparation of compounds 18a and 22a resulted in poor cyclization yields ( 2σ(I)]) 0.064, wR2 ) 0.119, data-parameter-ratio ) 17.0, max. peak in final electron density map ) 0.25 e Å3. Compound 18b. C20H32N4O2, monoclinic, space group P21, a ) 10.1580(6), b ) 18.8852(15), c ) 10.8306(6) Å, β ) 92.105(5)°, V ) 2076.3(2) Å3, Z ) 4, µ ) 0.076 mm-1, Fcalc. ) 1.153 gcm-1, STOEIPDS-II-two-circle diffractometer, Mo KR-radiation, 2θ-range ) 3.76-53.96°, 20 452 reflections collected, 8830 independent reflections (Rint) 0.047), structure solution with SHELXS-90, refinement on F2 with SHELXL-97, R1[I > 2σ(I)]) 0.038, wR2 ) 0.066, data-parameter-ratio ) 17.6, max. peak in final electron density map ) 0.16 e Å3.
Acknowledgment. Financial support was provided by Ministerio de Ciencia y Tecnologı´a of Spain (proyect BQU20001424) and BANCAIXA (P1.1A2000-10). J.B. thanks Ministerio de Educacio´n for a PhD fellowship. Supporting Information Available: X-ray crystallographic data (.cif files) for the compounds 13b and 18b. This material is available free of charge via the Internet at http://pubs.acs.org. JA0284759
