cyclic dipeptide

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7218-7221, August 1992 Biochemistry

Conformation for a fi-cyclodextrin monosubstituted with a cyclic dipeptide (x-ray diffration/water channel)

BENEDETTO Di BLASIO*, VINCENZO PAVONE*, FLAVIA NASTRI*, CARLA ISERNIA*, MICHELE SAVIANO*, CARLO PEDONE*, VINCENZO CUCINOTrAt, GIUSEPPE IMPELLIZZERIt, ENRICO RIZZARELLItt, AND GRAZIELLA VECCHIOt *Dipartimento di Chimica, Universiti di Napoli, via Mezzocannone, 4, 80134 Napoli, Italy; tDipartimento Scienze Chimiche, Universit& di Catania, Catania, Italy; and tIstituto per lo Studio delle Sostanze Naturali di Interesse Alimentare e Chimico Farmaceutico, Consiglio Nazionale delia Ricerche, Catania, Italy

Communicated by Isabella L. Karle, February 21, 1992

The structural characterization of a (3-cycloABSTRACT dextrin monosubstituted with the peptide cyclo(L-HiS-L-Leu) is reported. This work provides an x-ray example of a covalently bound group that folds In such a way that the terminal apolar side chain is retained in the hydrophobic interior of the cone-shaped cyclodextrin cavity. 6-Deoxy-6-cycldo(L-histldyl-Lleucyl)-ig-cydodextrin crystallizes in the space group P 1 with cell dimensions a = 14.728(8) A, b = 15.084(7) A, c = 18.182(10) A, a = 94.36(6), 13 = 95.81(5)0, y = 116.0S(9)°; overall isotropic agreement R = 10.6% for 5703 observed reflections (Fo > 3o). The molecular structure of two indepdent molecules wih the formula C54HN0.3W7.25H20. Each molecule ames a "sleeping swan"-like overall shape with the hydrophobic leucine side chain inserted inside the cavity of the macrocycle. The two independent units give rise to a head-to-tail dimer linked by hydrogen bonds occurring between primary and s hydroxyl groups of the two monomers. The packing of the dimers produces cavities cont water molecules. There are infinite hydroplc channels running in the crystal, which is similar to what is found in the structures of cyclic peptides.

FIG. 1. Atom numbering scheme of cHL-3CD.

conditions in which it is necessary to maintain a high local concentration of inhibitor.

Cyclodextrins (CDs) represent an interesting class of cyclic oligosaccharides constituted by 6-12 D-glucopyranosyl units, linked by a(1-4)-glycosidic bonds (1, 2). Among the known CDs, 1-CD (or cycloheptaamylose) is one of the most widely investigated so far. Crystallographic studies (3-5) have established that CDs are truncated cone-shaped cyclic molecules with glucose units in the 4C1 chair conformation. Because of the presence of the cavity and of primary hydroxyl groups, these cycloamyloses have been used for building molecular models with peculiar properties (6, 7). Furthermore, it has been shown that cyclo(L-histidyl-L-prolyl), a dipeptide with hormonal and neurotropic activity (8-13), inhibits the (Fe+ ascorbic acid)-induced peroxidation of skeletal muscle sarcoplasmic reticulum membrane lipids (14). Bavykina et al. (15) recently published the synthesis of diketopiperazines (DKPs) related to cyclo(L-histidyl-L-prolyl) and attributed the biological behavior ofthis molecule to the antioxidant activity of the cyclodipeptide. This suggests that DKP derivatives may be used as antiinflammatory agents (16-19). In this context, we report here an example of structural characterization by x-ray diffraction analysis of a 13-CD monosubstituted with a potentially bioactive cyclic dipeptide-namely, 6-deoxy-6-cyclo(Lhistidyl-L-leucyl)-1-CD (cHL-,1CD) (Fig. 1). This molecule may provide a useful combination of a cyclopeptide as a bioactive moiety and of the CD cavity as a delivery system. Thus, this compound could be useful in the inflammatory

EXPERIMENTAL PROCEDURES The cHL-PCD was synthesized as described (20, 21). The product, characterized by NMR, was homogeneous on HPLC (TLC: RfPrOH/H20/AcOEt/NH3, 5:3:1:1); 1H NMR (at 250 MHz in 2H20) (ppm) 7.66 (s, 1H), 6.87 (s, 1H), 5.4-4.9 (m, 7H), 4.6-4.4 (m, 2H), 4.2 (dd, 1H), 4.05-3.40 (m, 38H), 3.33 (t, 1H), 3.21 (dd, 2H), 3.05-2.80 (m, 2H), 1.5 (m, 2H), 1.0-0.8 (m, 7H). Crystals of cHL-,BCD suitable for x-ray diffraction study were obtained by evaporation of an aqueous solution at room temperature. Most of the crystals were twinned, but a single crystal of dimensions 0.5 x 0.3 x 0.4 mm was isolated and sealed in a boron glass capillary under an argon atmosphere and was used for all x-ray studies. Diffraction data were measured on a four-circle automatic diffractometer with CuKa radiation to a maximum scattering angle of 20 = 1300. A total of 13,537 independent reflections were collected; 5703 observed reflections with F0 > 3o! were used in the subsequent calculations after correction for Lorenz and polarization effects. Cell parameters and diffraction data are reported in Table 1. The space group and unit cell dimensions suggested the presence of two cHL-f3CD independent molecules and a number of water molecules. The initial structure solution was

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Abbreviations: CD, cyclodextrin; cHL-8CD, 6-deoxy-6-cyclo(Lhistidyl-L-leucyl)-f-CD; DKP, diketopiperazine.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Table 1. Crystallographic data for cHL-PCD Molecular formula C54HmN4O36 7.25H20 Molecular size, amu 1367.3 + 130.6 P1 Space group Cell parameters a = 14.728(8) A b = 15.084(7) A c = 18.182(10) A a = 94.36(6)0

Volume, A3 Density (calc), g/ml

z Radiation (CuKa), A Temperature Independent reflections Measured reflections with F. > 3cr(Fo) Final R, % amu, Atomic mass units.

8 = 95.81(5)° y = 116.08(9)0

3576(4) 1.391 2 A = 1.54178 Ambient 13,537 5703 R = 10.6

obtained with the Patterson search program PATSEE (22) by using atomic coordinates of a part (four glucosidic units) of a single 1-CD of the dimer of Hamilton and Sabesan (23) as the input fragment for the rotation search. The best solution ofthis procedure was used to assign the phases to 800 E values and to partially expand with SHELX-86 (24). The E map revealed the atomic position ofthe two independent 13-CD molecules. After several cycles of structure factor calculation followed by Fourier synthesis, the electron-density map showed the two remaining cyclodipeptide moieties (cHL) and the primary hydroxyl 0(6) 3 of one monomer of 8-CD in two statistical positions with occupancy factor equal to 0.5. Successive Fourier difference maps revealed 10 water molecules with an occupancy factor equal to 1.0 and 9 water molecules with approximately half the occupancy factor. The refinement was carried out using full-matrix least-squares calculations (25) with isotropic temperature factors for N, C, and 0 atoms, and the structure was refined to an overall R = 0.106. Hydrogen atoms were introduced in structure factor calculations in their stereochemically expected positions with isotropic temperature factor equal to the equivalent U factor of the bonded atom, except those of the water molecules.§ Most of the

§A list of observed and calculated structure factors is available from B.D.B. and C.P.

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geometric parameters of the two independent cHL-,fCD molecules are within expected values (26). However, the comparison of some chemically equivalent bond distances shows differences greater than ESDs (estimated standard deviations) calculated with the standard least-squares programs. This means, in our opinion, that the ESDs are underestimated, probably because of the complexity of the structure, including the data quality.

RESULTS The molecular structure of cHL-f3CD consists of two independent molecules related by a pseudo-21-fold axis almost parallel to the c direction (Fig. 2). The compound crystallizes with 14.5 water molecules in the asymmetric unit. The conformations of the two units appear to be similar as regards the 1-CD; the only major conformational differences occur in the cyclic peptide, where different DKP to 13-CD hydrogen bonding interactions are present. The overall shape of each cHL-,1CD corresponds to a truncated cone with the cyclopeptide moiety forming a folded structure. The terminal part, represented by the hydrophobic leucine side chain, is directed inside the cavity of ,8-CD. The whole molecule of cHL-,8CD assumes a "sleeping swan"-like shape (Fig. 3), with an unusual conformation when compared with those assumed by known substituted 83-CD, where the substituted groups stick out from the cone-shaped torus of the CD. Head-to-tail (narrow side of the truncated cone to the wider one) intermolecular hydrogen bonds, occurring between the primary and secondary hydroxyl groups of the two monomers and symmetry-related molecules, give rise to infinite columns of cHL-13CD dimers along the c axis. The packing of these cylinders produces cavities containing hydrogenbonded water molecules, which, together with P-CD hydroxyl groups, form a continuum hydrophilic channel. Conformation of (8-CD Molecules. 13-CD molecules present only slight differences with respect to uncomplexed hydrated or methylated CDs or CDs monosubstituted with bulky groups (26-30). All glucosidic units are in the 4C1 chair conformation (endocyclic torsion angles all show gauche conformation), and they are related by an approximate sevenfold axis. The best fit of the two independent cHL-13CD molecules and the two 13-CD macro-rings gave mean square deviations of 0.67 and 0.60 A, respectively. The seven 0(4) atoms form a near regular heptagon with side length and radius of 4.34 ± 0.12 and 4.98 ± 0.24 A, respectively, for both A and B molecules. The glucose residues are inclined with

B

A

FIG. 2. Stereoview of the dimer along the b axis.

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Biochemistry: Di Blasio et al.

FIG. 3. Molecular model of cHL-I3CD. The cyclic dipeptide moiety cHL is solid.

respect to the perpendicular axis of the plane formed by 0(4) atoms in such a way that the hydrophobic cavity is narrower at the primary hydroxyl side. The conformation of the (3-CD is stabilized by a ring of seven intramolecular hydrogen bonds 0(2) n... .0(3) n + 1 with all distances in the range 2.70-2.95 A. In the cHL-,(CD, the two (3-CD molecules contain three C(6)-0(6) bonds-namely, C(6)2-0(6)2, C(6)5-0(6)5, C(6)6-0(6)6 for molecule A and C(6)2-{(6)2, C(6)3-0(6)3,


C(6)6-0(6)6 for molecule B-rotated "inward" [(+)gauche] to allow the formation of hydrogen bonds with crystallization water molecules, while the 0(6) 3 in molecule A is disordered over (-)-gauche and (+)-gauche. Conformation of Covalently Substituted cHL. The DKP rings have an asymmetrical conformation (31) (shallow boat or twisted) with the two CP atoms in quasi-axial position, similar to that already found in the crystalline structure of the cHL monohydrate (32). The imidazole group and the DKP ring of the two cHLs are intermolecularly hydrogen bonded to the secondary hydroxyl groups 0(2)H and 0(3)H and intramolecularly bonded with primary hydroxyl groups 0(6)H. The imidazole rings of both cHLs are bonded through the NE to the C(6)1 atom of the p-CD and are oriented "toward" the center of the cavity with the torsion angles O(5)1-C(5)1-0(6)1-N6 corresponding to the (+)-gauche conformation (700 and 740 for molecules A and B, respectively). The imidazole groups are planar and folded over the DKP rings, as is commonly found in cyclic dipeptides of amino acid residues with aromatic side chains (33). The main significant conformational difference of the independent cyclic dipeptides is the torsion angle N8-CY--C~-f-C1 corresponding to 880 in molecule A and to 1170 in molecule B. This is related to a different hydrogen bond involving the carbonyl group of the leucine residue. In molecule A, this group is intramolecularly hydrogen bonded to one of the statistical positions of 0(6)3A, while in molecule B a water molecule (OW3) mediates the interaction with 0(6)3B. The observed interactions stabilize the relative orientation of the imidazole ring and the DKP ring, with respect to the plane of the seven 0(4) atoms, giving rise to a folded structure. This structural feature produces a favorable interaction between the hydrophobic leucine side chain and the inner cavity of 3-CD similar to that occurring in inclusion compounds of (3-CD. The C01 and C1 leucine side chain methyl groups are enclosed in the (-CD cavity; in fact, the distance of these atoms from the mean-square plane of the 0(4) atoms is in the range 0.05-0.07 and 0.08-0.4 A for molecules A and B, respectively. Crystal Packing and Water Structure. In Fig. 4, packing along the c axis is shown. The cHL-/3CD dimers pack along the c axis by intermolecular hydrogen bonds, giving rise to infinite cylinders. Water molecules (OW1, OW2, and OW3)

0

FIG. 4. Pseudohexagonal crystal packing viewed along the c axis. Water molecules

(0) are also indicated.

Biochemistry: Di Blasio et al. are located between the cylinders and link symmetry-related dimers along the c axis. The water molecule (OW5) is hydrogen bonded to the secondary hydroxyl group 0(3)7A. The average planes of the 0(4) atoms of the two 83-CDs are inclined to the cylinder axis, and the angle between these planes is -44°. This twist produces channels running along the a axis that are available to contain water molecules. The solvent water molecules are involved within the crystal in several different hydrogen-bond interactions. Quite similar water molecule packing schemes were described by Karle and Duesler (34) in the structures of cyclic peptides. In the cHL-j3CD structure, the hyo ophilic channel is filled by the remaining 15 water molecules, 9 of these being statistically disordered on half-occupied sites. The water molecules participate in hydrogen bonds also involving primary and secondary hydroxyl groups of }-CD dimers, giving rise to a complex hydrogen bond network. However, the hydrogen bonds are not randomly distributed but follow a characteristic pattern, constituted by circularly closed structures containing six and eight water molecules. The 0(6)7A makes part of the infinite channel.

CONCLUSION This study provides an example of structural characterization of a p-CD moiety with hydroxyl group 0(6)H functionalized with cyclic dipeptide cHL. The crystal structure confirms that the a-CD is rigid and that only slight changes occur in the conformation when a primary hydroxyl group is substituted with a bulky group. Furthermore, the overall shape of the imidazole-DKP moiety remains the same with respect to that assumed in the solid state by the free cyclopeptide. The stabilization of this conformation is due to intramolecular hydrogen bonds involving polar groups coupled to van der Waals interactions of apolar groups with the hydrophobic interior of the cone-shaped CD cavity. The unusual conformation assumed by the terminal leucine side chain can be explained by the tendency to exclude the hydrophobic groups from a hydrophilic surrounding. We wish to acknowledge Mr. M. Muselli for technical assistance. This research has been partially supported by Consiglio Nazionale delle Ricerche (Rome), Progetto Strategico Cosmetica, and by Consiglio Nazionale della Ricerche Grant 89.5354. 1. Saenger, W. (1980) Angew. Chem. Int. Ed. Engl. 19, 344-362. 2. Jones, S. P., Grant, J. W., Hadgraft, J. & Parr, G. D. (1984) Acta Pharm. Technol. 30, 213-223. 3. Chacko, K. K. & Saenger, W. (1981) J. Am. Chem. Soc. 103, 1708-1715. 4. Lindner, K. & Saenger, W. (1982) Carbohydr. Res. 99, 103108. 5. Stezowki, J. J., Jogun, K. H., Eckle, E. & Bartles, K. (1978) Nature (London) 274, 621-623.


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6. D'Souza, V. T. & Bender, M. (1987) Acc. Chem. Res. 20, 146-152. 7. Breslow, R. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 1-60. 8. Peterkofsky, A. & Battaini, F. (1980) Neuropeptides 1, 105118. 9. Bauer, K., Graf, K., Faivre-Bauman, A., Beier, S., TixierVidal, A. & Kleinkauf, H. (1978) Nature (London) 274, 174175. 10. Prasad, C., Matsui, T. & Peterkofsky, A. (1977) Nature (London) 268, 142-144. 11. Bhargava, H. (1981) Life Sci. 28, 1261-1275. 12. Prasad, C., Matsui, T., Williams, J. & Peterkofsky, A. (1978) Biochem. Biophys. Res. Commun. 85, 1582-1587. 13. Kukla, M. J., Brescin, H. J. & Bowden, C. R. (1985) J. Med.

Chem. 28, 1745-1747. 14. Shvachkin, Yu. P., Korshunova, G. A., Bavykin, N. I., Gulyaeva, N. V., Dupin, A. M., Boldyrev, A. A. & Severin, S. E.

(1989) Byull. Eksp. Biol. Med. 108, 671-673. 15. Bavykina, N. I., Gabrielyan, A. E., Korshunova, G. A. & Shvachkin, Y. P. (1990) Zh. Obshch. Khim. 60, 170-175. 16. Godfroid, J. J. & Braquet, P. (1986) Trends Pharmacol. Sci. 7, 368-373. 17. McCord, J. M. (1974) Science 185, 529-531. 18. Greenwald, R. A. & Moy, W. W. (1980) Arthritis Rheum. 23, 455-463. 19. Cheeseman, K. H. (1984) Adv. Inflammation Res. 6, 179-188. 20. Cucinotta, V., D'Alessandro, F., Impellizzeri, G., Pappalardo, G., Rizzarelli, E. & Vecchio, G. (1991) J. Chem. Soc. Chem. Commun., 293-294. 21. Bonomo, R. P., Cucinotta, V., D'Alessandro, F., Impellizzeri, G., Maccarrone, G. & Vecchio, G. (1991) Inorg. Chem. 30, 2708-2713. 22. Egert, E. & Sheldrick, G. M. (1985) Acta Crystallogr. A 41, 262-268. 23. Hamilton, J. A. & Sabesan, M. N. (1982) Acta Crystallogr. B 38, 3063-3069. 24. Sheldrick, G. M. (1986) SHELX-86 in Crystallographic Computing (Oxford Univ. Press, Oxford), pp. 175-179. 25. Sheldrick, G. M. (1975) SHELX-76 Programsfor Crystal Structure Determination (Univ. of Cambridge, Cambridge). 26. Lindner, K. & Saenger, W. (1982) Carbohydr. Res. 99, 103115. 27. Czugler, M., Eckle, E. & Stezowski, J. J. (1981) J. Chem. Soc. Chem. Commun., 1291-1302. 28. Harata, H., Uekama, K., Otagiri, M. & Hirayama, F. (1983) Bull. Chem. Soc. Jpn. 56, 1732-1738. 29. Harata, K. (1984) Chem. Lett., 1641-1645. 30. Kamitori, S., Hirotsu, K. & Higuchi, T. (1987) J. Chem. Soc. Perkin Trans. 2, 7-14. 31. Kolodziejczyk, A. & Ciarkowski, J. (1986) Biopolymers 25, 771-786. 32. Tanaka, I., Iwata, T., Takahashi, N. & Ashida, T. (1977) Acta Crystallogr. B 33, 3902-3904. 33. Ramani, R., Venkatesan, K., Marsh, R. E. & Kung, W. J. H. (1976) Acta Crystallogr. B 32, 1051-1056. 34. Karle, I. L. & Duesler, E. (1977) Proc. Natl. Acad. Sci. USA 74, 2602-2606.