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Separation of silybin diastereoisomers

Silybin diastereoisomers

Silybin diastereoisomers are preparatively unseparable, only analytically (HPLC).

Chemistry of silybin is currently performed with the natural silybin, e.g., mixture of A & B; so far no substantial difference in chemical reactivity (in isotropic milieu) was found.

Both diastereomers differ in the interactions with bio-receptors (3D structures).

Preparatory separation of silybin

Possible solution
Preparatory HPLC (low capacity, time consuming, rather expensive – unacceptable for pharmacological production)
Derivatization with a „chiral handle“ (better capacity than HPLC, however, the separation is rather critical)
Biocatalytic discrimination

Biocatalytic discrimination

Table 1. Evaluation of diastereoselec­tivity of selected hydrolases for silybin (1) transesterifi­cationa and 23-O-acetylsilybin (2) alcoholysis (a)

Enzyme Reaction Type Time Conversion E (b)
Lipase PS Transesterification 48 39.7 1.6 (B)
Lipase CV 48 14.4 2.4 (A)
Lipase A 48 8.4
Candida rugosa Lipase 3 16.8
Novozym 435 3 32.9
Lipase CAL-A 48 33.0 2.0 (A)
Alcaligenes sp. Lipase 18 47.9 1.0 (-)
Subtilisin 48 37.2 1.7 (B)
Acylase Amano 3000 48 2.9 1.7 (B)
Lipase A Alcoholysis 48 5.3 1.8 (B)
Candida rugosa Lipase 5 29.0
Novozym 435 3 33.7
Alcaligenes sp. Lipase 48 3.4 1.5 (B)
Subtilisin 48 2.6 1.1 (B)
Protease N 48 4.0 1.1 (B)
Protease P 48 14.7 1.1 (B)
Acid protease II 48 4.3 1.2 (B)
Acylase Amano 3000 48 8.3 1.4 (B)

Scheme 1. Exploitation of Novozym 435 for the preparation of 1a and 1b

Exploitation of Novozym 435 for the preparation of 1a and 1b

Conclusions

Collaboration

Financial support

This work was supported by the grant P207/10/0288 (R.G.) from the Czech Science Foundation, ESF COST Chemistry actions CM0701 and CM0602 (grants MSMT OC08049 and LC06010), by institutional research concept AV0Z50200510, and by the bilateral Czech-Italian Inter-Academic Project between CNR and AVČR (D.M. + V.K.).

References

  1. Monti D., Gažák R., Marhol P., Biedermann D., Purchartová K., Fedrigo M., Riva S., Křen V. Enzymatic Kinetic Resolution of Silybin Diastereoisomers. J. Nat. Prod. 2010; 73: 613–619
  2. Gažák R., Marhol P., Purchartová K., Monti D., Biedermann D., Riva S., Cvak L., Křen V. Large-scale separation of silybin diastereoisomers using lipases. Process Biochem. 2010, in press

Institute of Microbiology - Czech Academy of Sciences