FK-506, an immunosuppressive agent used for the prevention of graft rejection fol-lowing organ transplantation, is a mac-rolide antibiotic containing an endocyclic a-ketoamide moiety whose cis and trans forms (Figures 1A and 1B) give rise to two interconverting conformers-1,2,3,4. At room temperature the two conformers have a ra-tio of approximately 3:1 respectively as indicated by 13C NMR5. X-Ray crystal analysis found that crystalline FK-506 ex-ists in the cis conformations.

FK-506 crystals were obtained from Dr. Robert Borris, Merck Sharp & Dohme Re-search Laboratories (Rahway, NJ). HPLC was performed using a Rainin Dynamax®- 60A 8mm C18 column (4.6mm ID x 25cm L) with a guard column (4.6mm ID x 1.5cm L). Column temperature was con-trolled with a CJB - 16 column jacket (Aura Industries, Staten Island, NY) and a refrig-erated circulating bath (RM6, Lauda, Westbury, NY). A high pressure pump (Model SP8700XR, Spectra Physics, San Jose, CA) and an autoinjector (SP8780XR) set for lOmL injections were used. The col-umn was eluted with 85:15 (v:v) acetoni-trile-water at 1mL/min. The eluate was monitored with a Gilson Model 116 vari-able wavelength detector (Gilson Medical Electronics, Middleton, WI) at 210nm.

Reversed-phase HPLC of FK-506 showed that at low temperature (-9°C) the trans and cis conformers are widely separated with retention times of ca. 5.5 and 9 min-utes, respectively. As the column tempera-ture is increased to 5, 20, 35, and 50°C, the retention times decrease and the peaks coalesce till a sharp symmetrical peak is observed at 50°C (Figure 2). The slower eluting peak was ascribed to the cis con-figuration since the ratio of peaks was ca. 3:1 in favor of the later peak which corre-sponds to the 3:1 ratio in favor of the cis conformer found by 13C NMR5.

Furthermore, the chromatogram at -5°C of a fresh solution obtained by dissolving FK-506 in cold methanol (figure 3B) showed essentially only the later peak. The small amount of the trans conformer at 5.5 minutes is due to equilibration during the short dwell time in the non-thermostated injection valve. Figure 3A is the chromato-gram at -5°C of a solution of FK-506 equilibrated at room temperature. Similar chromatographic peak splitting at lower than ambient temperatures has been ob-served for proline-containing peptides 6,7.

The use of preparative low temperature chromatography of FK-506 can make available pure conformers for further ex-

perimentation if their solutions are kept at  -10°C or lower or used rapidly at higher temperatures. In particular the use of the pure conformers can shed light on the mechanism of the FK-506 binding protein (FKBP), which has been characterized as a peptidyl-prolyl -cis -trans isomerase8,9,10,11. Furthermore, if either the cis or the trans conformer is found to have a higher bind-ing constant with FKBP, the conforma-tional stability of that conformer could be enhanced through synthetic chemical ma-nipulations thus possibly providing com-pounds with increased immunosuppressive potencies12,13,14.

Physical constants and spectroscopic data such as (a), ord, cd, and NMR are more meaningful and easier to interpret when determined with the isolated pure con-formers than data obtained with the equi-librium mixture. The increased lipophilicity of the cis conformer, as indicated by its longer retention time (ca. 9 minutes) when compared to the trans conformer (see Figures 2 and 3), has definite implications for the molecular modeling of these structures and must be taken into account in the energy minimization calculation of the spatial disposition of FK-506 components including those not directly attached to the a-ketoamide moiety.

Figure 1. Schematic presentation of FK-506 showing (A) the cis conformation of the a­ketoamide moiety relative to the pipecolic acid moiety and (B) the trans conformation.

Figure 2. HPLC of equilibrated FK-506 at the indicated temperatures on a Dynamax-60A 8mn C18 column (4.6mm ID x 25cm L) and guard module (4.6mm ID x 1.5cm L). Mobile phase: 85:15 (v:v) acetonitrile:water; flow rate: 1m/min; detection: UV @ 210nm, 0.1 AUFS.

Figure 3. HPLC of FK-506 at -5°C. Panel A: equilibrated FK-506 solution; Panel B: FK-506 crystals freshly dissolved in cold methanol. Con­ditions are identical to those described in Fig. 2 except that a manual injector (Rheodyne 7125) was used instead of the autoinjector.

REFERENCES

1. Kino, T., et al., J. Antibiotics, 40, (1987) 1249.

2. Transplantation Proc., 19, Suppplement 6, (1987).

3. Transplantation Proc., 22, (1990) 5.

4. Starlz, T., et al., JAMA, 264, (1990) 63.

5. Tanaka, H., et al., J. Am. Chem. Soc., 109, (1987) 5031.

6. Gesquire, J., et al., J. Chromatogr., 478, (1989) 121.

7. Henderson, D., and Mello, J., J. Chromatogr., 499, (1990) 79.

8. Harrison, R., and Stein, R., Biochemistry, 29, (1990) 1684.

9. Rosen, M., et al., Science, 248, (1990) 863.

10. Justice, R., Jr., et al., Biochem. Bio­phys. Res. Commun., 171, (1990) 445.

11. Schreiber, S.L., Science, 251, (1991) 283.

12. Jones, A., et al., J. Org. Chem., 55, (1990) 2786.

13.     Corey, E.J., and Huang, H., Tetrahe­dron Lett., 30, (1989) 5235.

14.     Askin, D., et al., Tetrahedron Lett., 30, (1989) 6121.     

 * Presented at the 3rd International Symposium on Analytical Methods in Biotechnology, October 22-24, 1990, San Francisco/Burlingame, CA.