Inl. J . Peptide Protein Res. 40, 1992, 380-382
Conformation of cyclosporin A in polar solvents SO0 YOUNG KO and CLAUD10 DALVIT
Preclitiical Research. Sarido: Phart?ia A G , Basel, Switzerland
Received 27 February. accepted for publication 22 March 1992
A major conformation of cyclosporin A in methanol and in aqueous methanol was revealed by some simple NMR experiments. Thus, a stepwise transition of cyclosporin A conformation from 100% CDC13 to 100% CD30D was followed by 'H NMR, which showed that the chloroform conformation of cyclosporin A was still the major one in methanol. Employing the same technique, it was also shown that the chloroform conformation of cyclosporin A was one of the major conformations in 50"; aqueous methanol. This may be the first experimental determination of a major conformation of cyclosporin A in polar solvents. Key words: conformation; cyclosporin A; NMR spectroscopj ; polar solvents
Cyclosporin A, cyclo-(MeBmt-Abu-Sar-MeLcu-Val- NMR studies, which is different from the crystalline MeLeu-Ala-[ D 1-Ala-MeLeu-MeLeu-MeVal) unde- conformation, and is perhaps more compatible with the capeptide, is an immunosuppressant known by thc receptor-bound conformation (5). trade name Sandimune@. Its conformation was first Prompted by this report, we now disclose our earlier determined by X-ray crystallography. NMR spectros- findings on the major conforniation of cyclosporin A in copy analysis soon confirmed that this unusual mole- methanol and in aqueous methanol, and some very cule assumes the same conformation in chloroform (1). simple NMR experiments which yielded these findings. Conformations in other non-polar organic solvents (e.g., benzene, tetrahydrofuran) were shown to be simRESULTS AND DISCUSSION ilar to the one observed in chloroform (2). On the other hand, cyclosporin A exhibits many dif- Proton NMR spectra of cyclosporin A in CDC13 and ferent conformations in polar solvents such as metha- in CD3OD are shown in Figs. 1 and 2, respectively. nol or DMSO, which renders conformational studies in Cyciosporin A exhibits two conformations in chlorothese biologically more relevant solvents very difficult, form in ca. 95:5 ratio. The major conformer assumes if at all possible. It is only sparingly soluble in water. the crystalline conformation, while the minor one has Experimental determination of cyclosporin A conformations in water is, therefore, not possible. Molecular dynamics calculations seem to suggest that the crystalline (hence chloroform) conformation would be still favored in watcr (3). Recently two groups independently completed conformational studies of cyclosporin A when it is bound to its putative rcceptor protein cyclophilin (4). These studies were performed in water, cyclophilin now solubilizing the non-polar undecapeptide. From these state-of-the-art NMR experiments, a hitherto unknown conformation has emerged. These results have rekindled wide interest in the c j B 3 closporin A conformations in polar solvents. One recent report describes a conformation of cyclosporin A FIGURE 1 in aqueous methanol, obtained via two-dimensional ' H NMR spectrum of cyclosporin A in CDC13. 8
380
7
PPM
2
1
0
Cyclosporin A
*
I
I
..__ld.
100% CD30D
8
I
6
5
PPM
4
3
2
1
0
FIGURE 2 'H NMR spectrum of cyclosporin A in CD,OD.
an extra cis peptide bond between residues 3 and 4 (6). On the other hand, the spectrum in methanol looks much more complicated. More than 40 N-methyl peaks are detected (2.5-3.5 ppm), indicating the presence of at least six different conformations. However, there exists one dominant set of signals (marked *), accounting for ca. 30% of the whole population. This set of signals could result either from a single major conformer or from a number of conformers under fast equilibria. Direct comparison of the two spectra in Figs. 1 and 2 is not possible due to solvent effects on chemical shift. We added to a CDC13 solution of cyclosporin A (20 mM) a CD30D solution of the same concentration in small portions. The transition from 100% CDC13 to 100% CD30D took place over 17 steps. A proton NMR spectrum was recorded at each step. Methods of sample preparation (inverse addition or dissolution of cyclosporin A in a pre-mixed CDC13-CD30D solvent), aging of the sample, or lower concentration (down to 1 mM) were found to make little difference. Under these conditions, it was possible to follow the conformational changes from chloroform to methanol solution. Selected spectra are shown in Fig. 3. It is clear from Fig. 3 that the chloroform conformation survives the solvent change, and ends up to be the most favored conformation in methanol, and that the signals seen to be dominant in Fig. 2 (marked *) are indeed attributable to this single conformer. The conformation of cyclosporin A in aq. methanol was studied in a similar way. Thus, D20 was added in small portions to a CD3OD solution of cyclosporin A. Due to the insolubility of cyclosporin A in water, we did not attempt to maintain a constant concentration in this study. The transition from 100% CD30D (10 mM) to 50% D20/CD3OD (5 mM) took place over 5 steps, beyond which point, cyclosporin A precipitated out. A proton NMR spectrum was recorded at each step (Fig. 4). The chloroform conformation, which was the major one (ca. 30%) in 100% methanol, is again retained in 50 % water/methanol. Although its population diminishes a little, and a new conformation emerges
70% CD30D/CDC13
3.50
a25
PPM
3.00
2.75
FIGURE 3 IH NMR spectra of cyclosporin A from CDCl3 to CD,OD.
(marked +), the chloroform conformation is still one of the major conformations present in up to 50% water/ methanol, accounting for ca. 20% of the whole population. In conclusion, our simple NMR studies have revealed that the crystalline conformation of cyclosporin A is still a favored conformation in polar solvents such as methanol and aqueous methanol. This may be the first experimental confirmation of the results of the molecular dynamics calculation carried out by Lautz etal. (3). While there certainly exist at least five other conformations (and probably more) in methanol and in aqueous methanol, it is remarkable that the crystalline conformation is preserved in these polar solvents to the extent that it is. EXPERIMENTAL PROCEDURES Instruments The 1D proton NMR spectra were recorded at 300°K on a Brucker AM500 spectrometer equipped with an Aspect 3000 computer. 381
S.Y. KO and C, Dalvit CD30D; 81..5~,CD3OD; 87.2% CD30D; 90.7% CD3OD; 100% CD3OD. Proton NMR spectra were also recorded, at selected solvent compositions, with samples prepared by inverse additions (i.e. additions of CsA/CDC13solution to CsA/ CD3OD solution), or by dissolving CsA in a premixed CDC13-CD30D solvent. Effects of sample concentration were checked at selected solvent compositions with samples in 20 mM, 10 mM, 6.7 mM and 1.0 mM. Effects of sample ageing were checked at selected solvent compositions after leaving the sample for 1 h, 2 h, and overnight at room temperature. Samples of CsA in CD3OD/D20 were prepared by adding portions of D2O to CD30D solution of CsA (10 mM). Proton NMR spectra were recorded at the following solvent compositions and sample concentrations: 100% CD3OD, 10 mM; 10% D20,9.0 mM; 20% D20,g.O mM; 30% D20,7.0 mM; 40% Dz0,6.0 mM; 509, D20, 5 . 0 m ~ . REFERENCES Petcher, T.J., Weber, H.P. & Riiegger, A. (1976)Helv. Chim. Acta 59. 1480: Kcssler, H., Loosli, H.R. & Oschkinat, H. (1985) Helv. Chim. A m 68, 661; Loosli, H.R., Kessler, H., Oschkinat, H., Webcr, H.P., Petcher, T.J. & Widmer, A. (1985)Helv. Chim. Acta 68, 682 Kessler, H., Gehrke, M., Lautz, J., KBck, M., Seebach, D. &
T i i*' 3.5a
315
ppy
3.w
**
1/
2.75
FIGURE 4 'H NMR spectra of cyclosporin A from CD,OD to 50°, CD,ODDzO.
Sample preparation Samples of cyclosporin A(CsA) in CDCl3/CD3OD were prepared by adding, to a CDCl3 solution of CsA (20mM), portions of a CD3OD solution of CsA (20mM). Proton NMR spectra were recorded at the following solvent compositions: loo%, CDCh; 4.8% CD30D; 9.1% CD3OD; 13.0% CD3OD; 16.7% CD3OD; 23.1% CD30D; 28.6% CD3OD; 37.59, CD30D; 44.4% CD30D; 50.0% CD30D; 56.5% CD30D; 63.0% CD30D; 70.0% CD3OD; 76.2";
382
Thaler, A. (1990) Biochem. Pharmacol. 40, 169 Lautz, J., Kessler, H., Kaptein, P. & van Gusteren, W.F. (1987) J . Comptrrer-Aided Mol. Design 1, 2 19 Weber, C., Wider, G., Freyberg, B.v., Traber, R., Braun, W., Widmer, H. & Wiithrich, K. (1991) Biochemistry 30, 6563; Fesik, S.W., Gampe, Jr., R.T., Eaton, H.L., Gemmecker, G., Olcjniczak, E.T., Neri, P., Holzman, T.F., Egan, D.A., Edalji, R., Simmer. R., Helfrich, R., Hochlowski, J. & Jackson, M. (1991) Biuchemi.vto.30.6574: Fesik, S.W., Gampe, Jr., R.T., HolLman, T.F., Egan, D.A., Edalji, R., Luly, J.R., Simmer, R., Helfrich, R., Kishore, V. & Rich, D.H. (1990) Science 250, 1406 Hsu, V.L., Heald, S.L., Harding, M.W., Handschumacher, R.E. & Armitage, I.M. (1990) Biochem. Phurmacol. 40, 131 Seebach, D., KO, S.Y., Kessler, H., KBck, M., Reggelin, M., Schmieder, P., Walkinshaw, M.D., BBlsterli, J.J. & Bevec, D. (1991) Heh. Chim.Acru 74, 1953
.4ddrcss. Son Yowig K o
Sandoz Institute for Medical Research
5 Gowcr Place London WClE 6BN UK
Conformation of cyclosporin A in polar solvents SO0 YOUNG KO and CLAUD10 DALVIT
Preclitiical Research. Sarido: Phart?ia A G , Basel, Switzerland
Received 27 February. accepted for publication 22 March 1992
A major conformation of cyclosporin A in methanol and in aqueous methanol was revealed by some simple NMR experiments. Thus, a stepwise transition of cyclosporin A conformation from 100% CDC13 to 100% CD30D was followed by 'H NMR, which showed that the chloroform conformation of cyclosporin A was still the major one in methanol. Employing the same technique, it was also shown that the chloroform conformation of cyclosporin A was one of the major conformations in 50"; aqueous methanol. This may be the first experimental determination of a major conformation of cyclosporin A in polar solvents. Key words: conformation; cyclosporin A; NMR spectroscopj ; polar solvents
Cyclosporin A, cyclo-(MeBmt-Abu-Sar-MeLcu-Val- NMR studies, which is different from the crystalline MeLeu-Ala-[ D 1-Ala-MeLeu-MeLeu-MeVal) unde- conformation, and is perhaps more compatible with the capeptide, is an immunosuppressant known by thc receptor-bound conformation (5). trade name Sandimune@. Its conformation was first Prompted by this report, we now disclose our earlier determined by X-ray crystallography. NMR spectros- findings on the major conforniation of cyclosporin A in copy analysis soon confirmed that this unusual mole- methanol and in aqueous methanol, and some very cule assumes the same conformation in chloroform (1). simple NMR experiments which yielded these findings. Conformations in other non-polar organic solvents (e.g., benzene, tetrahydrofuran) were shown to be simRESULTS AND DISCUSSION ilar to the one observed in chloroform (2). On the other hand, cyclosporin A exhibits many dif- Proton NMR spectra of cyclosporin A in CDC13 and ferent conformations in polar solvents such as metha- in CD3OD are shown in Figs. 1 and 2, respectively. nol or DMSO, which renders conformational studies in Cyciosporin A exhibits two conformations in chlorothese biologically more relevant solvents very difficult, form in ca. 95:5 ratio. The major conformer assumes if at all possible. It is only sparingly soluble in water. the crystalline conformation, while the minor one has Experimental determination of cyclosporin A conformations in water is, therefore, not possible. Molecular dynamics calculations seem to suggest that the crystalline (hence chloroform) conformation would be still favored in watcr (3). Recently two groups independently completed conformational studies of cyclosporin A when it is bound to its putative rcceptor protein cyclophilin (4). These studies were performed in water, cyclophilin now solubilizing the non-polar undecapeptide. From these state-of-the-art NMR experiments, a hitherto unknown conformation has emerged. These results have rekindled wide interest in the c j B 3 closporin A conformations in polar solvents. One recent report describes a conformation of cyclosporin A FIGURE 1 in aqueous methanol, obtained via two-dimensional ' H NMR spectrum of cyclosporin A in CDC13. 8
380
7
PPM
2
1
0
Cyclosporin A
*
I
I
..__ld.
100% CD30D
8
I
6
5
PPM
4
3
2
1
0
FIGURE 2 'H NMR spectrum of cyclosporin A in CD,OD.
an extra cis peptide bond between residues 3 and 4 (6). On the other hand, the spectrum in methanol looks much more complicated. More than 40 N-methyl peaks are detected (2.5-3.5 ppm), indicating the presence of at least six different conformations. However, there exists one dominant set of signals (marked *), accounting for ca. 30% of the whole population. This set of signals could result either from a single major conformer or from a number of conformers under fast equilibria. Direct comparison of the two spectra in Figs. 1 and 2 is not possible due to solvent effects on chemical shift. We added to a CDC13 solution of cyclosporin A (20 mM) a CD30D solution of the same concentration in small portions. The transition from 100% CDC13 to 100% CD30D took place over 17 steps. A proton NMR spectrum was recorded at each step. Methods of sample preparation (inverse addition or dissolution of cyclosporin A in a pre-mixed CDC13-CD30D solvent), aging of the sample, or lower concentration (down to 1 mM) were found to make little difference. Under these conditions, it was possible to follow the conformational changes from chloroform to methanol solution. Selected spectra are shown in Fig. 3. It is clear from Fig. 3 that the chloroform conformation survives the solvent change, and ends up to be the most favored conformation in methanol, and that the signals seen to be dominant in Fig. 2 (marked *) are indeed attributable to this single conformer. The conformation of cyclosporin A in aq. methanol was studied in a similar way. Thus, D20 was added in small portions to a CD3OD solution of cyclosporin A. Due to the insolubility of cyclosporin A in water, we did not attempt to maintain a constant concentration in this study. The transition from 100% CD30D (10 mM) to 50% D20/CD3OD (5 mM) took place over 5 steps, beyond which point, cyclosporin A precipitated out. A proton NMR spectrum was recorded at each step (Fig. 4). The chloroform conformation, which was the major one (ca. 30%) in 100% methanol, is again retained in 50 % water/methanol. Although its population diminishes a little, and a new conformation emerges
70% CD30D/CDC13
3.50
a25
PPM
3.00
2.75
FIGURE 3 IH NMR spectra of cyclosporin A from CDCl3 to CD,OD.
(marked +), the chloroform conformation is still one of the major conformations present in up to 50% water/ methanol, accounting for ca. 20% of the whole population. In conclusion, our simple NMR studies have revealed that the crystalline conformation of cyclosporin A is still a favored conformation in polar solvents such as methanol and aqueous methanol. This may be the first experimental confirmation of the results of the molecular dynamics calculation carried out by Lautz etal. (3). While there certainly exist at least five other conformations (and probably more) in methanol and in aqueous methanol, it is remarkable that the crystalline conformation is preserved in these polar solvents to the extent that it is. EXPERIMENTAL PROCEDURES Instruments The 1D proton NMR spectra were recorded at 300°K on a Brucker AM500 spectrometer equipped with an Aspect 3000 computer. 381
S.Y. KO and C, Dalvit CD30D; 81..5~,CD3OD; 87.2% CD30D; 90.7% CD3OD; 100% CD3OD. Proton NMR spectra were also recorded, at selected solvent compositions, with samples prepared by inverse additions (i.e. additions of CsA/CDC13solution to CsA/ CD3OD solution), or by dissolving CsA in a premixed CDC13-CD30D solvent. Effects of sample concentration were checked at selected solvent compositions with samples in 20 mM, 10 mM, 6.7 mM and 1.0 mM. Effects of sample ageing were checked at selected solvent compositions after leaving the sample for 1 h, 2 h, and overnight at room temperature. Samples of CsA in CD3OD/D20 were prepared by adding portions of D2O to CD30D solution of CsA (10 mM). Proton NMR spectra were recorded at the following solvent compositions and sample concentrations: 100% CD3OD, 10 mM; 10% D20,9.0 mM; 20% D20,g.O mM; 30% D20,7.0 mM; 40% Dz0,6.0 mM; 509, D20, 5 . 0 m ~ . REFERENCES Petcher, T.J., Weber, H.P. & Riiegger, A. (1976)Helv. Chim. Acta 59. 1480: Kcssler, H., Loosli, H.R. & Oschkinat, H. (1985) Helv. Chim. A m 68, 661; Loosli, H.R., Kessler, H., Oschkinat, H., Webcr, H.P., Petcher, T.J. & Widmer, A. (1985)Helv. Chim. Acta 68, 682 Kessler, H., Gehrke, M., Lautz, J., KBck, M., Seebach, D. &
T i i*' 3.5a
315
ppy
3.w
**
1/
2.75
FIGURE 4 'H NMR spectra of cyclosporin A from CD,OD to 50°, CD,ODDzO.
Sample preparation Samples of cyclosporin A(CsA) in CDCl3/CD3OD were prepared by adding, to a CDCl3 solution of CsA (20mM), portions of a CD3OD solution of CsA (20mM). Proton NMR spectra were recorded at the following solvent compositions: loo%, CDCh; 4.8% CD30D; 9.1% CD3OD; 13.0% CD3OD; 16.7% CD3OD; 23.1% CD30D; 28.6% CD3OD; 37.59, CD30D; 44.4% CD30D; 50.0% CD30D; 56.5% CD30D; 63.0% CD30D; 70.0% CD3OD; 76.2";
382
Thaler, A. (1990) Biochem. Pharmacol. 40, 169 Lautz, J., Kessler, H., Kaptein, P. & van Gusteren, W.F. (1987) J . Comptrrer-Aided Mol. Design 1, 2 19 Weber, C., Wider, G., Freyberg, B.v., Traber, R., Braun, W., Widmer, H. & Wiithrich, K. (1991) Biochemistry 30, 6563; Fesik, S.W., Gampe, Jr., R.T., Eaton, H.L., Gemmecker, G., Olcjniczak, E.T., Neri, P., Holzman, T.F., Egan, D.A., Edalji, R., Simmer. R., Helfrich, R., Hochlowski, J. & Jackson, M. (1991) Biuchemi.vto.30.6574: Fesik, S.W., Gampe, Jr., R.T., HolLman, T.F., Egan, D.A., Edalji, R., Luly, J.R., Simmer, R., Helfrich, R., Kishore, V. & Rich, D.H. (1990) Science 250, 1406 Hsu, V.L., Heald, S.L., Harding, M.W., Handschumacher, R.E. & Armitage, I.M. (1990) Biochem. Phurmacol. 40, 131 Seebach, D., KO, S.Y., Kessler, H., KBck, M., Reggelin, M., Schmieder, P., Walkinshaw, M.D., BBlsterli, J.J. & Bevec, D. (1991) Heh. Chim.Acru 74, 1953
.4ddrcss. Son Yowig K o
Sandoz Institute for Medical Research
5 Gowcr Place London WClE 6BN UK
