Studies of the enzymatic synthesis of N-protected amino acid-estradiol derivatives in an organic solvent

Article abstract of DOI:10.1016/S0040-4039(00)00797-8

Amino acid-estradiol derivatives were synthesized via protease-catalyzed condensation for the first time and the optimum conditions were studied systematically. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00797-8

Tetrahedron Letters 41 (2000) 5379±5381
Studies of the enzymatic synthesis of N-protected amino
acid-estradiol derivatives in an organic solvent
Ai-Xin Yan,a Guo-Wen Xing,a,y Yun-Hua Ye,a,* Gui-Ling Tian,a
Man-Sau Wongb and Kin-Sing Leeb
aKey Laboratory of Bioorganic Chemistry and Molecular Engineering, Department of Chemistry, Peking University,
Beijing 100871, China
bOpen Laboratory of Chirotechnology and Department of Applied Biology and Technology,
The Hong Kong Polytechnic University, Kowloon, Hong Kong, China
Received 14 March 2000; accepted 16 May 2000
Abstract
Amino acid-estradiol derivatives were synthesized via protease-catalyzed condensation for the ®rst time
and the optimum conditions were studied systematically. # 2000 Elsevier Science Ltd. All rights reserved.
Peptidyl steroids include those compounds in which amino acids or peptides are coupled with
steroids to form amides or esters. Cholyl glycine and cholyl taurine are examples of peptidyl
steroids that play important roles in the process of human life.1 In the previous paper, a series of
amino acid and peptide derivatives of estradiol has been synthesized using di€erent coupling
reagents and their binding anities for the estrogen receptor have been studied.2 In this paper, we
present the results of a study of the synthesis of this kind of compound catalyzed by a protease in
an organic solvent. The advantages of enzymatic methods for peptidyl steroid synthesis include
the use of mild reaction conditions, which are generally racemization free, minimal side chain
protection and high regio- and stereoselectivity.3 Acyl donors with di€erent structures, such as
di€erent N-protecting groups and di€erent esters were studied and are discussed.
P-L-Ala-OR was chosen as the acyl donor and the 17-aminoestra-1,3,5(10)-trien-3-ol (I) or 17-
hydrazonoestra-1,3,5(10)-trien-3-ol (II) as the acyl acceptor substrate. The amide bond formation
between P-L-Ala-OR and the 17-b-amino or 17-b-hydrazono group of estradiol (I,II) was catalyzed
by subtilisin Carlsberg in DMF containing a little water (Fig. 1).
* Corresponding author. E-mail: yhye@pku.edu.cn
y
Present address: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00797-8

5380
Figure 1. Synthetic scheme for P-Ala-estradiol derivatives
In order to investigate the e€ects of di€erent carboxyl components on the yield of the enzymatic
reactions, a series of N-protected alanine esters were prepared4 6 and condensed with compound
I or II. The results are displayed in Tables 1 and 2.
Table 1
E€ects of di€erent protecting groups on the yield of the enzymatic reactions
Table 2
E€ects of di€erent esters on the enzymatic reaction

5381
As shown in Table 1, Boc was better as the amino protecting group for the acyl donor than Z
or Fmoc.{ Since Boc has the smallest size among the three blocking groups, this result suggests
that subtilisin Carlsberg has a small hydrophobic pocket in its S2 position and Boc is more suitable
for the binding between substrate and subtilisin. Table 1 also demonstrates that compound II
acted as a better nucleophile than compound I due to favorable electronic e€ects. Table 2 indicates
that the tri¯uoroethyl ester is the best substrate to be used as the carboxyl component for the
enzymatic reaction. Furthermore, it seemed that electron-withdrawing groups such as tri¯uoroethyl,
phenyl, benzyl and carboxamidomethyl in the carboxyl component were preferable to electron-
donating groups such as methyl and ethyl esters. This result suggests that electronic e€ects play
an important role in the acyl-enzyme intermediate formation process. A previous report7 also
recommended the use of tri¯uoroethyl esters of amino acids as the carboxyl components due to their
ability to facilitate the process of acyl-enzyme formation, the key step in enzymatic condensation.
The optimum molar ratio for the enzymatic synthesis was also studied with the coupling
between Boc-Ala-OCH2CF3 and compound I as a model reaction. The best result was gained
when two equivalents of the carboxyl component were used.
In conclusion, we have successfully obtained N-protected amino acid-estradiol derivatives
catalyzed by subtilisin Carlsberg in DMF. The structures of all the products were con®rmed by
FAB-MS, 13C NMR, [ꢀ]D and elemental analysis. These results indicate that the estradiol derivatives
(I and II) are compatible as nucleophilic substrates for subtilisin and can provide useful information
for broadening the use of protease-catalyzed reactions for routine organic synthesis.
Acknowledgements
This work was supported by the Hong Kong Polytechnic University and National Natural
Science Foundation of China.
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{
Abbreviations. Boc: tert-Butyloxycarbonyl; Z: Benzyloxycarbonyl; Fmoc: 9-Fluorenylmethyloxycarbonyl.