Tautomeric phenomenon of a novel potent immunosuppressant(FK506) in solution. I. Isolation and structure determination of tautomeric compounds.

Abstract

Tautomeric phenomenonwas observed in the HPLCchromatogramobtained from a novel potent immunosuppressant, FK506, in ethanol solution. Two tautomeric compounds derived from FK506 were isolated and purified by HPLC.Their structures were elucidated by spectral analyses. The mechanismsfor this tautomerization were also established based on the results of structure analysis.Cis (major component1*) and trans conformational isomers of FK506 resulting from the restricted rotation of the amide bond in the pipecolic acid moiety ( Fig. 1) have been known to exist in chloroform as the equilibrium mixture1'2) which could be detected by HPLCunder low temperature column condition3*. However, this equilibrium is not detectable on the HPLCchromatogram obtained by increasing the column temperature in order to improve a peak shape, while FK506 in ethanol solution provided three peaks which suggest the existence of another kind of equilibrium phenomenon (Fig. 2). This means that the origin of the equilibrium in ethanol is different from the equilibrium associated with the rotation around the amide bond. Wereport the conclusion that a new equilibrium is explained by tautomerism of FK506; the resulting mixture consists of three species, FK506, Tautomeric compoundsI and II in ethanol solution4*. Herein wereport structures of two tautomeric compounds of FK506in comparisonwith spectral data of FK5064) and postulate a mechanism of interconversion between these three compounds. Materials and Methods Isolation of Tautomeric Compounds

Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis

“…First is a cis-trans conformational isomerization involving restricted rotation across the amide bond in a pipecolic moiety. The second is reported for water containing solutions where epimerization to an intermediate tautomer I (cis) occurs which is then converted into tautomer II (cis-trans) [23]. This gives rise to a problem of a different water content in a sample and in a standard solution during quantitative HPLC assessments.…”

Evaluation of a cyano stationary phase for the determination of tacrolimus, sirolimus and cyclosporin A in whole blood by high-performance liquid chromatography–tandem mass spectrometry

“…Firstly, column temperatures of at least 60 • C have to be employed with C 18 or C 8 stationary phases in order to avoid excessive band broadening of chromatographic peaks. This band broadening stems from the partial separation of the conformers of individual immunosuppressants on HPLC stationary phases [1,[25][26][27]. Such high column temperatures lead to a significant reduction in the lifetime of silica-based columns due to the instability of silica at elevated temperatures [28].…”

Identification of an 18-Methyl Derivative of Tacrolimus API in Streptomyces clavuligerus CKD-1119

“…To establish the analytical conditions of the unknown derivative powder (purity: 90%, 15 mg), the previous studies on structural analysis were used as reference. 6,7 Results and Discussion Fig. 3 to be 818 (Fig.…”

“…Considering all these factors the control of impurities in pharmaceuticals is further open for isolating and identifying process-related impurities using mass spectrometry, nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC) techniques to gain insight in the related field of the pharmaceutical industry. 5 Hence, we present the structural analysis of new derivative as an unknown impurity and it is very difficult to isolate due to their similar chemical structures [5][6][7] from the tacrolimus bulk API recovered through the purification of crude tacrolimus during the fermentation of tacrolimus, the producing strain, Streptomyces clavuligerus CKD-1119, also produces ascomycin and dihydrotacrolimus. Chemical structure of tacrolimus and its related compounds is presented in Fig.…”

“…The NMR data of tacrolimus and ascomycin have been published in previous papers. 6 Sample analysis was performed with CHCl 3 -d solvent conditions.Ascomycin, an active tacrolimus analog in which the C21 allyl group is replaced by an ethyl group, is produced mainly by the fermentation of the streptomycetes strain. To confirm the differences between the 3 compounds, NMR analysis was performed so that values could be assigned to tacrolimus and ascomycin in the NMR spectra.…”