Efficiently regioselective glucosylation of estrogen analogues mediated by fungus Rhizopus oryzae AS 3.2380

Article abstract of DOI:10.1016/j.catcom.2017.04.027

Compared with estrogens, estrogen 3β-glucosides display more superiority in uptake and metabolism, which are thought to be potential replacement therapy agents for female. In the present research, various estrogen 3β-glucosides (1a–6a) were obtained using the biotransformation experiment of estrogen substrates mediated by Rhizopus oryzae AS 3.2380. The characteristics of estrogens glucosylation with high specificity and regio-selectivity were observed. An optimal incubation medium was determined as the pH 0.25 7.0 (sodium phosphate buffer), 20 0.25 g/L (D-glucose) and 24 0.25 h incubation time with the yields 0.25 > 0.25 70%. A environment friendly preparative method for these glucosides was also established using MCI (Middle chromatogram isolated gel) column chromatography eluted with ethanol aqueous, which could give estrogen 3β-glucosides with the purity 0.25 > 0.25 95%. This study suggested that R. oryzae AS 3.2380 could be an efficient engineering bacteria for the glucosylation of chemical substance. Glucosyltransferase also could be prepared from R. oryzae AS 3.2380.

Full text of DOI:10.1016/j.catcom.2017.04.027

Accepted Manuscript
Efficiently regioselective glucosylation of estrogen analogues
mediated by fungus Rhizopus oryzae AS 3.2380
Ping Li, Chengpeng Sun, Yan Wang, Shumei Wang, Chunyan
Yan, Sa Deng, Xiaokui Huo, Lei Feng, Chao Wang, Yan Tian,
Baojing Zhang, Xiaochi Ma
PII:
DOI:
Reference:
S1566-7367(17)30162-0
doi: 10.1016/j.catcom.2017.04.027
CATCOM 5012
To appear in:
Catalysis Communications
Received date:
Revised date:
Accepted date:
16 February 2017
26 March 2017
14 April 2017
Please cite this article as: Ping Li, Chengpeng Sun, Yan Wang, Shumei Wang, Chunyan
Yan, Sa Deng, Xiaokui Huo, Lei Feng, Chao Wang, Yan Tian, Baojing Zhang, Xiaochi
Ma , Efficiently regioselective glucosylation of estrogen analogues mediated by fungus
Rhizopus oryzae AS 3.2380. The address for the corresponding author was captured
as affiliation for all authors. Please check if appropriate. Catcom(2017), doi: 10.1016/
j.catcom.2017.04.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Efficiently regioseletive glucosylation of estrogen analogues mediated by fungus
Rhizopus oryzae AS 3.2380
Ping Lia, Chengpeng Suna, Yan Wanga, Shumei Wangb, Chunyan Yanb, Sa Denga,
Xiaokui Huoa, Lei Fenga, Chao Wanga,* wach_edu@sina.com, Yan Tiana, Baojing
Zhanga, Xiaochi Maa,b maxc1978@163.com
aCollege of Pharmacy,
Academy of Integrative Medicine,
Dalian Medical University,
Dalian 116044, P.R. China
bKey Laboratory of Digital Quality Evaluation of Chinese Materia Medica of State
Administration of TCM,
Guangdong Pharmaceutical University,
Guangdong 510006, P.R. China
*Corresponding authors.

ACCEPTED MANUSCRIPT
Abstract
Compared with estrogens, estrogen 3β-glucosides display more superiority in uptake
and metabolism, which are thought to be potential replacement therapy agents for
female. In the present research, various estrogen 3β-glucosides (1a–6a) were obtained
using the biotransformation experiment of estrogen substrates mediated by Rhizopus
oryzae AS 3.2380. The characteristics of estrogens glucosylation with high specifity
and regio-selectivity were observed. An optimal incubation medium was determined
as the pH 7.0 (sodium phosphate buffer), 20 g/L(D-glucose) and 24 h incubation time
with the yields > 70%. A environment friendly preparative method for these
glucosides was also established using MCI (Middle chromatogram isolated gel)
column chromatography eluted with ethanol aqueous, which could give estrogen
3β-glucosides with the purity > 95%. This study suggested that R. oryzae AS 3.2380
could be an efficient engineering bacteria for the glucosylation of chemical substance.
Glucosyltransferase also could be prepared from R. oryzae AS 3.2380.
Keywords: Estrogen, estrogen 3β-glucoside, glucosylation, Rhizopus oryzae AS
3.238

ACCEPTED MANUSCRIPT
1. Introduction
Estrogen replacement therapy (estrogens: 17β-estradiol, E2; 17α-estradiol, E3;
estrone, E) for female menopause is clinically carried out, which displayed effects in
reducing vasomotor, postmenopausal bone loss and the incidence of osteoporotic
fractures [1]. However, these oral estrogens for estrogen replacement therapy undergo
extensive first-pass hepatic metabolism, which was associated with cholelithiasis,
hypercoagulability, and hypertension. The addition of the glucose in the 3-position
facilitated the transport of the estrogens into the liver cells, the absorption of estrogens
into the portal system as a conjugated estrogen and thereby avoid most first-pass
hepatic metabolism [2-4]. For example, E2-3-glucoside effectively reduces serum
gonadotropin levels to the premenopausal range, which is effective at reducing
postmenopausal symptoms [4,5].
Generally, estrogen 3β-glucosides are prepared by metabolism or chemical
synthesis. The chimpanzee liver, rabbit liver microsomes and silkworm pupal ovaries
displayed the capability to produce estradiol-3β-D-glucosides by the metabolism
pathway [6-11]. The chemical synthetic method for the synthesis of estrogen
3β-glucoside was established with the synthetic strategies of carbohydrate chemistry
with the yields of 46%-71% [12].
Compared with chemical glycosylation, biotransformed glycosylation is thought to
be an efficient and environment friendly method [13-17]. Microalgae strain
Selenastrum capricornutum has been used to transform environmental pollutant
estrogens, and ethinylestradiol glucoside analogues were obtained [13, 18]. However,

ACCEPTED MANUSCRIPT
regio-selective and specify glucosylation of estrogens with high yields needs to be
investigated systematically. In the present paper, filamentous fungus Rhizopus oryzae
AS 3.2380 has been used to catalyze the glucosylation of estrogens. The high yield,
region selectivity and the optimal incubation conditions for the glucosylation have
been investigated. The convenient and efficient purify method for transformed
products have been established with little organic solvent.
2. Experimental section
2.1. Materials
Chemicals, microorganisms and instruments are described in the supplementary
information.
2.2. Glucosylation experiment
All of the filamentous fungi were pre-incubated in potato medium to get enough
fungi cells. The glucosylation medium was sodium phosphate buffer: 13.62 g
NaH2PO4, 2.36 g NaOH, and 20 g D-glucose in 1000 mL water. 1.5 g R. oryzae AS
3.2380 fungus cells were pre-incubated in the glucosylation medium (200 mL), 30 ºC,
130 rpm for 12 h. Then, estrogen substrates (10 mg) dissolved in acetone were
injected into the medium with the continued incubation for 24 h [19]. When the
fungus cells were filtered, the incubation culture was subjected to a MCI (Middle
chromatogram isolated gel) column, which was eluted by 30% ethanol aqueous, 50%
ethanol aqueous, and 95% ethanol, successively. The estrogen 3β-glucosides could be
obtained in the 50% ethanol aqueous elution with the purity > 95%.
3. Results and discussion

ACCEPTED MANUSCRIPT
3.1. Screening experiment for the glucosylation of estrogens
The screening experiment for the glucosylation of estrogens was performed by the
incubation using 20 different filamentous fungi (Supplementary data). The incubation
cultures were analyzed by a HPLC-DAD, which suggested the multi transformation
capability of most fungi. And, major products were observed from the cultures of R.
oryzae AS 3.2380, which were suggested to be estrogen glucosides by LC-MS.
Therefore, the biotransformed glucosylation of estrogens could be conducted using R.
oryzae AS 3.2380. The HPLC chromatograms of the glucosylation experiment of
substrates 1–4 were shown in Fig. 1, which indicated the high yield and selective
glucosylation capability of R. oryzae AS 3.2380.
3.2. Preparation and identification of estrogen glucosides
When the natural estrogens ethinylestradiol (1), 17α-estradiol (2), 17β-estradiol (3),
and estrone (4) as the substrates were transformed by R. oryzae AS 3.2380, the
corresponding
3β-D-glucopyranosyl-ethinylestradiol (1a) [18], 3β-D-glucopyranosyl-17α-estradiol
(2a) [20], 3β-D-glucopyranosyl-17β-estradiol (3a) [20], and
products
were
obtained
and
identified
to
be
1
3β-D-glucopyranosyl-estrone (4a) [6] (Fig. 2), on the basis of HRESIMS, H NMR
(Table 1), and 13C NMR data (Table 2).
Product 5a was obtained from the incubation medium of 5, which had the
1
1
molecular C24H32O8 established by HR-ESIMS, H NMR and 13C NMR. The H
NMR spectrum of 5a displayed the proton signals of 16α-hyroxyestrone, such as the
ABX system [δH 7.17 (d, J = 8.4 Hz), 6.79 (dd, J = 8.4, 2.4 Hz), 6.72 (d, J = 2.4 Hz)],

ACCEPTED MANUSCRIPT
18-methyl group (δH 0.88, 3H, s), and H-16 (δH 4.24 m) (Table 1). The 13C NMR
spectrum also confirmed the presence 16α-hydroxyestrone, which showed 18 carbon
signals, such as the ketone group of C-17 (δC 219.1), oxygenated methine (δC 70.3)
1
(Table 2). Additionally, the H and 13C NMR also suggested the presence of
glucopyranosyl group (δC 100.6, 77.1, 76.7, 73.3, 69.8, 60.7). The proton signal of
terminal hydrogen (H-1') was doublet with the coupling constant as J = 7.8 Hz, which
indicated the β configuration of the glycoside bond. 2D NMR experiments have been
performed to establish the exact structure of 5a, especial the location of
glucopyranosyl group. In the HMBC spectrum of 5a, the oxygenated C-3 was
established by long range correlations from H-1 (δH 7.17), H-2 (δH 6.79), H-4 (δH 6.72)
to C-3 (δC 155.4) (Supplementary data, Fig. S1). The 16-OH and 17-ketone groups
were established by HMBC correlations of H-15/C-16, H-16/C-17, H-18/C-17
(Supplementary data, Fig. S1). For the location of glucopyranosyl group, the long
range correlation from H-1' to C-3 was observed from the HMBC experiment of 5a
(Fig. 2). Therefore, the transformed product 5a was elucidated to be
3β-D-glucopyranosyl-16α-hydroxyestrone as shown in Fig. 2.
Product 6a gave the same molecular formula as that of 5a C24H32O8, which was
established by HR-ESIMS. Similarly, product 6a was elucidated to be an estrogen
3β-D-glucoside analogue by HR-ESIMS and NMR data (Tables 1 and 2) as shown in
Fig. 2.
More estradiol carboxylic esters have been used as the substrates for the
glucosylation transformation of R. oryzae AS 3.2380, including 17β-estradiol

ACCEPTED MANUSCRIPT
17-acetate (7), 17β-estradiol valerate (8), depofemin (9), and 17β-estradiol enanthate
(10). On the basis of spectroscopic data, the transformed products were determined to
be estrogen 3β-glucosides analogues. However, the 17-ester groups of these
glucosides were hydrolyzed. As a result, the glucosylation biotransformation of these
estradiol carboxylic esters yielded the same product 3a.
3.3. Regio-selective and substrate specific characteristics of the glucosylation
As shown in Fig. 2, ten estrogen analogues have been used as the substrates for the
evaluation of glucosylation capability of R. oryzae AS 3.2380. And, the specific
3β-glucoside derivatives were obtained with high yields. Although there were two
hydroxyl groups in the substrates 1–3, 5 and 6, no 17-glucoside was obtained.
However, both 3-glucoside and 17-glucoside derivatives of estrogens were observed
in the previous study of estrogen metabolism in vivo. Therefore, these series estrogens
and their corresponding transformed products indicated the regio-selectivity for the
glucosylation by R. oryzae AS 3.2380.
The present biotransformation indicated the hypothesis that R. oryzae AS 3.2380
possessed the glucosylation capability of phenolic hydroxyl group. So, various
substrates with phenolic hydroxyl groups were incubated in the medium with R.
oryzae AS 3.2380, including caffeic acid, ferulaic acid, 7-hydroxycoumarin,
daphnetin, quercetin, baicalein, and stilboestrol (Supplementary data, Fig. S2).
However, no glucoside derivative was obtained from the culture. Therefore, R. oryzae
AS 3.2380 displayed the glucosylation capability with substrate specificity.
3.4. Glucosylation time-courses of estrogens 1–4

ACCEPTED MANUSCRIPT
The time-courses for the glucosylation of estrogens have been studied with
substrates 1–4 (Fig. 3a). The glucosylation of substrates 1–4 displayed similar time
course, as the time for the highest yields 30 h (1a), 24 h (2a), 24 h (3a), and 36 h (4a).
Comparison of the incubation time for the highest yields showed that epimerides
17α-estradiol (2) and 17β-estradiol (3) had the similar optimal incubation time.
Ethinylestradiol (1) got the highest yield than other estrogens, which indicated that the
sub-structure affected the glucosylation reaction in a minor way. Additionally, the
time-courses showed that the yields of glucosides decreased, when the incubation
time prolonged over 36 h. It suggested that further reaction may conduct on these
estrogen glucosides meditated by R. oryzae AS 3.2380, such as hydrolysis,
hydroxylation. Thus, the incubation time for the preparation of estrogen glucosides
needed to be controlled strictly.
3.5. Incubation conditions investigation for the glucosylation of estrogens
The incubation medium for the glucosylation was sodium phosphate buffer (pH 7.0)
containing glucose. In order to get the best medium for the highest glucosylation
yields, the pH values and the amount of glucose were investigated with substrate 1. As
shown in Fig. 3b, the different concentrations of glucose in the medium were studied
as 2, 10, 20, 40, and 80 g/L. The yields of glucosylation products displayed a
para-curve as the increase of glucose concentration. The highest yields was got at the
approximately 30 g/L glucose for the incubation medium. The reason for the low
yields as the glucose concentration >40 g/L was supposed that more glucose may
inhibit the metabolism function of R. oryzae AS 3.2380. The pH values of incubation

ACCEPTED MANUSCRIPT
buffer were changed by HCl or NaOH aqueous solutions to be 3, 5, 7, 9, 11. When the
medium was <3 or >11, no glucosylation product was observed. And the highest yield
was obtained in the pH 7 incubation medium (Fig. 3c).
3.6. Preparation process study of glucosylation products
The present paper not only gave a glucosylation method of estrogens in
bio-catalytic system, but also established a preparation technique for glucosylation
products in environment friendly way. Therefore, the purify method for estrogen
3-glucosides was designed with no usage of those nocuous organic solvents, such as
methanol, acetonitrile, n-butyl alcohol, ethyl acetate.
After incubation of estrogen substrates in the sodium phosphate buffer with R.
oryzae AS 3.2380, the fungi cells were filtered by filter paper. Then, the buffer
containing transformed products was subjected to a MCI column, eluted by
ethanol-water (30:70), ethanol-water (50:50), and 95 % ethanol successively. The
fraction eluted by 50% ethanol aqueous was evaporated in vacuum, which gave the
estrogen 3β-glucosides with the purity > 95% (Supplementary data, Fig. S3). The
more purify products could be obtained by recrystallization in ethanol.
4. Conclusion
Estrogens as the replacement therapy for female get more and more attention.
Estrogen 3β-glucosides are thought to be potential replacement therapy agents, which
displayed superiorities in metabolism, and uptake. The preparation of estrogen
3β-glucosides by biotransformation gave the high efficient and environmental friendly
advantages. Although ethinylestradiol has been transformed by microalgae, the

ACCEPTED MANUSCRIPT
substrates specialty and region selectivity did not investigated [13]. The present paper
developed a glucosylation method for estrogens with high yields (> 60%).
Regio-selective characteristic was also observed for the series of estrogen
3β-glucosides. The glucosylated products of the other hydroxyl groups did not
observed in the incubation medium. And the preparation process for glucoside
analogues was established and optimized in green approach. This research program
supported a glucosylation technique for further development of estrogens.
Acknowledgement
This research program is financially supported by the National Natural Science
Foundation of China (No. 81503201, 81622047 and 81473334), Dalian Outstanding
Youth Science and Technology Talent (2015J12JH201), Distinguished professor of
Liaoning Province, Project sponsored by Liaoning BaiQianWan Talents Program and
Innovation Team of Dalian Medical University for financial support.
Appendix data
Experimental sections and the spectroscopic spectra of estrogen 3β-D-glucosides
could be obtained at the internet.
Supplementary data
Supplementary material
References
[1] P. Villa, F. Sagnella, C. Perri, R. Suriano, B. Costantini, F. Macri, L. Ricciardi, A. Lanzone,
Climacteric: J. Int. Menopause Soc. 11 (2008) 498–508.
[2] J.H. Tong, E.I. Seper, D.S. Layne, D.G. Williamson, Can. J. Biochem. Cell boil. 61 (1983)

ACCEPTED MANUSCRIPT
784–789.
[3] D.G. Williamson, D.S. Layne, Biochem. Biophys. Res. Commun. 64 (1975) 256–261.
[4] A.T. Sweeney, V. Tangpricha, J. Weinberg, A.O. Malabanan, F.N. Chimeh, M.F. Holick,
Translational Res. 148 (2006) 164–170.
[5] M.F. Holick, Method for delivering estrogens for treatment of menopausal symptoms, WO Pat.
9603995, 1996.
[6] P. N. Rao, R.H. Purdy, M.C. Williams, P.H.Jr. Moore, J.W. Goldzieher, D.S. Layne, J. Steroid
Biochem. 10 (1979) 179–185.
[7] D. G. Williamson, D.S. Layne, J. Clin. Endocrinol. Metab. 33 (1971) 157–159.
[8] R.S. Labow, D.G. Williamson, D.S. Layne, D.C. Collins, Can. J. Biochem. 53 (1975) 1028–
1030.
[9] O. Manabu, F. Yoshinori, I. Nobuo, O. Eiji, Gen, Comp. Endocrinol. 61 (1986) 393–401.
[10] D.G. Williamson, A. Polakova, D.S. Layne, Biochem. Biophys. Res. Commun. 42 (1971)
1057–1062.
[11] D.C. Collins, D.G. Williamson, D.S. Layne, J. Biol. Chem. 245 (1970) 873–876.
[12] R.B. Conrow, S. Bernstein, J. Org. Chem. 36 (1971) 863–870.
[13] M.D. Greca, G. Pinto, P. Pistillo, A. Pollio, L. Previtera, F. Temussi, Chemosphere 70 (2008)
2047–2053.
[14] W.M. J. Kloosterman, S. Roest, S.R. Priatna, E. Stavila, K. Loos, Green Chem. 16 (2014)
1837–1846.
[15] W.M.J. Kloosterman, D. Jovanovic, S.G.M. Brouwer, K. Loos, Green Chem. 16 (2014) 203–
210.
[16] M. Ye, J.G. Dai, H.Z. Guo, Y.J. Cui, D.A. Guo, Tetrahedron Lett. 43 (2002) 8535–8538.
[17] C. Wang, X.K. Huo, B.J. Zhang, C.P. Sun, X.G. Tian, S. Deng, B. Li, W. Wang, P.P. Dong,
X.C. Ma, Catal. Commun. 89 (2017) 40–43.
[18] Y. Fujimoto, N. Ikekawa, M. Ogiso, E. Ohnishi, Experientia 42 (1986) 567–568.
[19] C.Z. Zang, S.C. Kan, C.W. Yeh, C.C. Lin, C.J. Shieh, Y.C. Liu, Ultrason. Sonochem. 26
(2015) 415–421.
[20] J.T. Wu, K.I.H. Williams, Steroids 54 (1989) 401–419.

ACCEPTED MANUSCRIPT
Table 1 1H NMR spectroscopic data of estrogen 3β-glucosides 1a–6a (δH in ppm)
No.
1a
2ab
3ab
4aa
5ab
6ab
1
7.20 d (9.0)
7.16 d (9.0)
7.16 d (8.5)
7.19 d (8.5) 7.17 d (8.4)
7.19 d (9.0)
6.86 dd
(9.0, 2.5)
6.86 dd
6.79 dd
6.80 dd
2
4
6
6.78 dd (9.0, 2.0) 6.78 dd (8.5, 1.5)
(8.5, 2.5)
(8.4, 2.4)
(9.0, 1.8)
6.80 d (2.5)
6.20 d (2.0)
6.70 d (1.5)
6.82 d (2.5) 6.72 d (2.4) 6.72 d (21.8)
2.82 m
1.76 m
2.75 m
1.25 m
2.75 m
1.25 m
2.24 m
2.03 m
2.80 m
1.23 m
2.78 m
1.23 m
1.90 m
1.74 m
1.31 m
1.79 m
1.19 m
1.33 m
1.79 m
1.19 m
1.33 m
1.65 m
1.42 m
1.46 m
1.87 m
1.33 m
1.49 m
1.78 m
1.23 m
1.47 m
7
8
9
2.26 m
2.10 m
2.10 m
2.39 m
2.20 m
2.33 m
2.35 m
1.43 m
2.27 m
1.26 m
2.27 m
1.26 m
2.01 m
1.05 m
1.76 m
1.40 m
2.36 m
1.41 m
11
12
14
2.16 m
1.75 m
1.85 m
1.30 m
1.85 m
1.30 m
1.89 m
1.56 m
2.35 m
1.38 m
1.88 m
1.53 m
1.96 m
1.43 m
1.82 m
1.38 m
1.81 m
1.38 m
2.00 m
1.65 m
1.70 m
1.39 m
1.67 m
1.41 m
2.25 dd
(18.0, 7.8)
1.83 m
1.96 m
1.42 m
1.58 m
1.14 m
1.58 m
1.14 m
1.56 m
1.46 m
1.99 m
1.74 m
15
16
2.81 m
1.43 m
1.88 m
1.39 m
1.88 m
1.39 m
2.87 m
2.49 m
4.24 m
0.88 s
17
18
21
1'
3.45 dd (11.5, 5.5)
0.67 s
3.52 m
0.67 s
3.73 d (5.4)
0.66 s
0.87 s
0.91 s
2.89 s
4.84 d (7.0)
3.35-3.45 m
3.35-3.45 m
3.35-3.45 m
3.35-3.45 m
4.76 d (7.5)
3.1-3.3 m
3.1-3.3 m
3.1-3.3 m
3.1-3.3 m
4.76 d (7.5)
3.1-3.3 m
3.1-3.3 m
3.1-3.3 m
3.1-3.3 m
4.84 d (7.0) 4.77 d (7.8)
4.78 d (7.8)
3.14 m
2'
3.3-3.6 m
3.3-3.6 m
3.3-3.6 m
3.3-3.6 m
3.14 m
3.21 m
3.19 m
3.26 m
3'
3.25 m
4'
3.19 m
5'
3.25 m
3.68 m
3.45 m
3.88 dd
(12.0, 2.0)
3.69 dd
3.89 dd (12.0, 1.5)
3.69 dd (12.0, 5.0)
3.68 brd (10.5)
3.52 m
3.68 m
3.44 m
3.68 m
3.44 m
6'

ACCEPTED MANUSCRIPT
(12.0, 5.0)
a
b
Compounds 1a and 4a were measured in CD3OD (500 Mz). Compounds 2a, 3a, 5a, and 6a
were measured in DMSO-d6. (2a, 3a in 500 MHz; 5a, and 6a in 600 MHz).
Table 2 13C NMRspectroscopic data of estrogen 3β-glucosides 1a–6a (δC in ppm).
1a
2ab
3ab
4aa
5ab
6ab
No.
1
2
127.2
115.3
157.0
117.9
139.0
34.1
28.5
41.0
45.2
135.5
30.8
27.7
50.8
48.2
23.7
39.9
80.4
13.3
88.8
75.0
102.6
74.4
78.06
71.5
78.12
62.6
126.0
113.7
155.2
116.3
137.3
29.2
26.8
38.5
43.6
133.5
26.0
36.6
42.8
49.5
22.8
29.9
80.0
11.2
126.0
113.7
155.2
116.3
137.3
29.2
26.8
38.5
43.6
133.5
26.0
36.6
42.8
49.5
22.8
30.0
80.0
11.2
127.2
115.3
157.1
117.9
138.9
27.7
126.1
113.8
155.4
116.4
137.3
29.2
125.9
113.8
155.4
116.3
137.3
29.2
3
4
5
6
7
32.8
25.9
27.2
8
39.8
37.7
37.2
9
45.4
43.5
43.4
10
11
12
13
14
15
16
17
18
20
21
1
2'
135.1
27.1
133.0
31.3
133.2
25.7
36.7
25.2
36.0
49.3
47.3
42.1
51.7
47.0
43.2
22.5
31.4
35.5
30.6
70.3
216.6
83.4
223.7
14.3
219.1
14.1
11.6
100.7
69.8
76.7
73.3
77.0
60.8
100.9
69.8
76.7
73.3
77.0
60.8
102.5
71.5
78.0
75.0
78.1
62.6
100.6
69.8
76.7
73.3
77.1
60.7
100.6
69.8
76.7
73.3
77.1
60.8
3'
4'
5'
6'
a
b
Compounds 1a and 4a were measured in CD3OD (125 Mz). Compounds 2a, 3a, 5a, and 6a
were measured in DMSO-d6. (2a, 3a in 125 MHz; 5a, and 6a in 150 MHz).

ACCEPTED MANUSCRIPT
Figure captions
Fig. 1. The HPLC chromatograms of the glucosylation experiment of substrates 1–4
by R.oryzae AS 3.2380.
Fig. 2. The glucosylation of estrogens 1–10 by R. oryzae AS 3.2380.
Fig. 3. (A) Time-courses of the glucosylation of estrogens by R. oryzae AS 3.2380.
Conditions optimized for the glucosylation of 1. (B) Concentration of glucose in the
medium. (C) pH of the incubation culture.

ACCEPTED MANUSCRIPT
Figure 1

ACCEPTED MANUSCRIPT
Figure 2

ACCEPTED MANUSCRIPT
Figure 3

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Highlight
 An efficient glucosylation of estrogens was developed using Rhizopus oryzae
AS 3.238.
 A Series of estrogens 3β-glucosides were prepared.
 Substrate specifity and region-seletive characteristic were observed.