Regioselective synthesis of cytarabine monopropionate by using a fungal whole-cell biocatalyst in nonaqueous medium

Article abstract of DOI:10.1016/j.bmcl.2014.05.092

The utilization of a dehydrated fungal biocatalyst of Aspergillus oryzae cells was successfully performed to achieve efficient acylation modification of a polar nucleoside cytarabine (ara-C). Organic solvents showed evident influence on the reaction catalyzed by the A. oryzae whole-cells. Except for hexane-pyridine, the catalytic activity and regioselectivity of the whole-cells clearly increased with increasing the polarity of the hydrophobic organic solvents used. The effects of some crucial factors on the reaction were further examined. The best reaction medium, hydrophobic solvent concentration, vinyl propionate/ara-C ratio, reaction temperature and shaking speed were confirmed as isopropyl ether (IPE)-pyridine, 30% (v/v), 90, 30 °C and 140-180 rpm, respectively. The cell biocatalyst also showed good thermal stabilities in both IPE-pyridine and hexane-pyridine systems. In addition, the desired 3′-O-propional derivative of ara-C was synthesized with the yields of 88.3% and regioselectivity (>70%). The resulting biocatalytic system appears to be an effective alternative, and can thus be employed for application in highly regioselective modification of nucleoside analogues.

Full text of DOI:10.1016/j.bmcl.2014.05.092

Accepted Manuscript
Regioselective synthesis of cytarabine monopropionate by usinga fungal whole-
cell biocatalyst in nonaqueous medium
Mei-yan Yang, Hui Wu, Zhi-hong Lu, Xiao-feng Li, Fu-rao Lai, Guang-lei Zhao
PII:
S0960-894X(14)00601-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.092
BMCL 21706
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 March 2014
22 May 2014
24 May 2014
Please cite this article as: Yang, M-y., Wu, H., Lu, Z-h., Li, X-f., Lai, F-r., Zhao, G-l., Regioselective synthesis of
cytarabine monopropionate by using a fungal whole-cell biocatalyst in nonaqueous medium, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.092
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Regioselective synthesis of cytarabine monopropionate by using
a fungal whole-cell biocatalyst in nonaqueous medium
Mei-yan Yanga, Hui Wub, Zhi-hong Lub, Xiao-feng Lia,b*, Fu-rao Lai, and Guang-lei
Zhaoa**
a
State Key Laboratory of Pulp and Paper Engineering, South China University of
Technology, Guangzhou, 510641, China.
b College of Light Industry and Food Sciences, South China University of Technology,
Guangzhou, 510641, China
Correspondence:
* Xiao-feng Li, Ph.D.
College of Light Industry and Food Sciences, South China University of Technology,
Wushan Road 381, Guangzhou, 510641, China.
Tel: +86-20-2223-6819. Fax: +86-20-8711-2853.
E-mail: xflibio@gmail.com，zhaoguanglei@gmail.com
1

Abstract
The utilization of a dehydrated fungal biocatalyst of Aspergillus oryzae cells was
successfully performed to achieve efficient aylation modification of a polar
nucleoside cytarabine (ara-C). Organic solvents showed evident influence on the
reaction catalyzed by the A. oryzae whole-cells. Except for hexane-pyridine, the
catalytic activity and regioslectivity of the whole-cells clearly increased with
increasing the polarity of the hydrophobic organic solvents used. The effects of some
crucial factors on the reaction were further examined. The best reaction medium,
hydrophobic solvent concentration, vinyl propionate/ara-C ratio, reaction temperature
and shaking speed were confirmed as isopropyl ether (IPE)-pyridine, 30% (v/v), 90,
30°C and 140-180 rpm, respectively. The cell biocatalyst also showed good thermal
stabilities in both IPE-pyridine and hexane-pyridine systems. In addition, the desired
3′-O-propional derivative of ara-C was synthesized with the yields of 88.3% and
regioselectivity (>70%). The resulting biocatalytic system appears to be an effective
alternative, and can thus be employed for application in highly regioselective
modification of nucleoside analogues.
Keywords: Fungal whole-cell; nucleoside; acylation; solvent engineering;
regioselectivity
2

Regioselective acylation of only one out of several hydroxyl groups of
polyhydroxyl compounds is a useful reaction for modification of the drug candidates
bearing sugar residues to improve their clinical efficiency, which is also valuable for
new drug/prodrug discovery and development.1, 2 For an example, some nucleoside
analogues with high hydrophilicity, such as aracytosine (ara-C), have major clinical
shortfalls in the treatment of solid tumors, since they can not easily transfer across the
cell membrane by passive diffusion and might undergo a rapid enzymatic deactivation
in plasma. Regioselective acylation of the nucleoside analogues could significantly
improve their antitumor, antiviral and immunosuppressive effects. And even just a
single acylation of those compounds many led to promising candidate compounds,
4
such as the 3’-0-acyl-ara-cytidinesor the antibiotic puromycin.3,
In addition,
regioselective acylation of nucleosides represents a way of introducing protecting
groups commonly used in drug discrovery.3 However, it still remains as a significant
challenge in classic organic chemistry.1, 2, 5
During the past decade enormous efforts have been made in the development of
synthetic methodologies for selective preparation of lipophilic derivatives of
nucleoside analogues.6-8 Among these synthetic tools available to chemists, the use of
enzymes has become one of the most attractive alternatives to the conventional
chemical methods for its high regioselectivity, and environmental friendliness.9
However, in terms of efficiency and cost-effectiveness, the need for isolated enzymes
is disadvantageous.
Biocatalytic reactions can also be performed by use of whole-cell biocatalysts.
Compared to the isolated enzymes, whole-cell biocatalysts offer several benefits to
organic synthesis.9, 10 This type of biocatalysts provide a natural environment for
enzyme location, which protects the cell-bound enzymes from a rapid deactivation in
3

non-aqueous solvents.11 In addition, the use of microbial biocatalysts can eliminate
the need for enzyme purification and immobilization which account for a large part in
the enzyme cost. Until recently the majority of whole-cell mediated bio-processes
were carried out in aqueous media for hydrophobic compounds.12 Only a few
researches have been involved in non-aqueous biocatalysis with whole-cells,
including asymmetric reduction of ketones in neat substrates,13 transesterification of
oils for biodiesel production, etc.7
The finding of microbial strains that are not only tolerant to organic solvents but
possesses good catalytic performance plays an important role in whole-cell
biocatalysis. A wide range of microbial cells have been explored for their capability in
catalysis, including both bacterial and fungal strains. Among them, filamentous fungi
have been attracted much attention. They have been used for fermentative food
processing since ancient times, and thus are considered to be quite safe for use in
pharmaceutical industry. Recently, several strains of filamentous fungi have been
exploited as whole-cell biocatalysts in asymmetric reduction,8 kinetic hydrolysis of
(R,S)-benzyl glycidyl ether,14 etc, in which the cells exhibited moderate to good
catalytic activity and high selectivities. In continuation of our research on the “green”
synthesis of nucleoside derivatives with potential antitumor and antiviral activities,3, 4,
15 we reported here the application of a whole-cell biocatalyst from Aspergillus oryzae
in acylation of the hydrophilic nucleoside ara-C, a traditional clinical agent for
therapy of acute leukemias. The influence of some influential factors, such as the
types of organic solvents, substrate ratio and shaking speed, were also investigated in
4

details. In addition, the catalytic properties and stability of the fungi whole-cell
biocatalyst in organic solvents were investigated (Scheme 1). 16, 17
(Insert Scheme 1 here)
Table 1 showed the effects of a series of solvents both pure and binary on the
catalytic behaviors of A.oryzae cells in acylation of ara-C.18 No product was detected
when reactions were carried out in DMSO and DMF which have high polarities [in
terms of ET(30)]. In pyridine, the microbial cells exhibited certain catalytic activity,
which may be due to its lower polarity than DMSO or DMF. Thus a “co-solvent”
strategy was proposed by using the mixture of the polar solvent pyridine and another
hydrophobic solvent as reaction medium. Table 1, entries 3-8 showed that the organic
solvent mixtures had an evident effect on the whole-cell catalytic performances.
Except for hexane-pyridine, the catalytic activity and regioselectivity of the cells
clearly increased with increasing the polarity of the hydrophobic organic solvents
used, suggesting that the polarity of the solvents was a crucial factor affecting the
whole-cell catalyzed reaction and might be used to approximately predict the solvent
effects. Furthermore, two kind organic solvents with a quite similar polarity
(hexane-pyridine and IPE-pyridine) gave significant differences in product yield and
regioselectivity, indicating that the catalytic behaviors of the A.oryzae cells in organic
solvents were not only polarity-dependent. Hence, IPE-pyridine mixture was chosen
as the most suitable reaction solvent due to the best reaction results achieved.
(Insert Table 1 here)
To get a better insight into the fungus whole-cell mediated acylation of ara-C in
5

IPE-pyridine system, the effects of several key variables were further optimized.
Considering that varying the volume ratio of IPE to pyridine enabled the reaction
media to controllable ET(30) values, the influence of IPE content on the reaction was
investigated. As Fig.1a showed, when changing the IPE content from 10% (v/v) up to
30% (v/v), the initial rate, yield and 3′-regioselectivity of the reaction went up
markedly. Further increased in the content of hydrophobic IPE, however, caused
visible insolubility of ara-C in the reaction media. Thus, when the IPE content was
increased from 30% (v/v) to 40% (v/v), both the yield and 3′-regioselectivity of the
reaction decreased.
Fig.1b confirmed that the initial rate and yield of the whole-cell catalyzed reaction
can be greatly boosted by increasing the VP/ara-C ratio from 10 to a high level of 50
(mol/mol), beyond which the initial rate and yield were improved slowly, and then
hardly changed when the ratio was above 90 (mol/mol). Excessive supply of vinyl
acyl donor in the acylation reactions was considered to be necessary for vinyl
ester-involved biotransformation due to the rapid enzymatic hydrolysis of vinyl esters,
a side reaction along with the enzymatic acylation of the nucleoside. As illustrated in
Fig.1c, the cells exhibited catalytic activity in a wide range of temperature (from 20°C
to 50°C). The highest initial rate and yield of the reaction were achieved at 30℃.
Temperature may be not only closely related to the thermodynamic equilibrium of the
reactions but also involved in the deactivation of biocatalyst. It can be found that the
increase of the temperature above 30℃ resulted in significant decrease of the
3'-regioselectivity from 71.3% to 16.1%. This phenomenon suggested that higher
6

temperature influenced the confirmation of the enzymes active center, and thus the
recognition site of the A.oryzae whole-cells on sugar moity of ara-C changed from
3'-OH to 5'-OH.
In biocatalysis, substrates have to diffuse to the active center of enzymes from
reaction media by solutions of the salvation, and the products also need diffuse to the
reaction media from the active center of enzymes. These processes were related to the
mass transfer resistance, viscosity of the media and the solution of substrates and
products in the reaction media and so on. Previous researches showed that the
catalytic efficiency of whole-cell biocatalysts was 1-2 classes lower than free
enzymes19 due to the low permeability of the whole-cell catalysts. Hence, the shaking
speed of the reaction system was particularly important to facilitate the mass transfer
in the reaction. Fig.1d showed that the initial rate of the reaction increased as raising
the shaking speed at the shaking speed of <180 rpm, confirming the existence of mass
transfer resistance in the reaction system. However, the reaction rate decreased when
the shaking speed was above 180 rpm, which was mainly due to that the drastic
shaking made the whole-cell catalysts adhere to the wall of reaction flasks and thus
reduced the efficient cell concentration. Changes in shaking speeds showed little
influence on 3'-regioselectivity of the reaction. For analysis of the statistically
significant difference between the effect of these key variables and biocatalytic
reaction, the Duncan’s post-test at 0.05 level was done. Results showed that the
change of IPE concentration had no significant effect on the reaction, while changes
of VP/ara-C and shaking speed resulted in significant differences of both initial
7

reaction rate and yield of the reaction. Variation of temperature, however, only led to
significant difference in the initial reaction rate of the reaction.
(Insert Fig.1 here)
From both a practical and a theoretical viewpoint, it was of considerable
importance to understand the stability of the whole-cell biocatalyst in the reaction
media.20 As showed in Fig.2, the A. oryzae whole-cells only had a little loss of
catalytic activity after incubated in 30% (v/v) IPE-pyridine solvent under 30 ℃-35 ℃.
Further improving of the incubation temperature led to the decrease of their relative
activities. When the incubation temperature was as high as 60 ℃, the relative activity
was decreased to 37.8%. Fig.2 also illustrated the deactivation profile of the
whole-cells of A.oryzae by incubating the cells in different co-solvent media with
similar ET(30) values at various temperatures. The relative activity after incubation in
30% (v/v) hexane-pyridine was almost the same like in 30% (v/v) IPE-pyridine.
Furthermore, the Duncan’s post-test showed that there was no statistically significant
differences between them at P<0.05. It demonstrated that the whole-cell biocatalyst
had similar thermal stability in IPE-pyridine to that in hexane-pyridine.
(Insert Fig.2 here)
In conclusions, a fungus whole-cell biocatalytic system was developed for
3′-regioselective synthesis of ara-C propionate. The catalytic behaviors of the
whole-cell biocatalyst from A.oryzae showed a clear solvent dependence within the
range of organic solvents tested. Binary mixture was shown to be more suitable for
the fungus whole-cell catalyzed acylation of hydrophilic substrate than the pure polar
8

solvents. The 3'-regioselectivity and thermal stabilities observed in organic solvents
renders the whole-cell biocatalyst a promising candidate for non-aqueous production
of bioactive nucleoside esters. In addition, the fungus cell biocatalysts can be
produced in abundance via cultivation and avoid tedious purification of free enzymes,
thus enabling the whole-cell catalyst being superior to the free enzymes in cost and
handling. It is hoped that this new whole-cell biotechnological strategy may benefit
some related pharmaceutical processes, making them greener and more cost-effective.
Extension of the whole-cell biocatalytic technology to modification of other bioactive
drugs is currently under development and will be reported in due course.
Acknowledgements
The authors are grateful to the National Natural Science Foundation of China
(Nos. 31270636), a Foundation for Natural Science Foundation of Guangdong
Province (No.S2013010012486), the Project of Department of Education of
Guangdong Province (No. 2012KJCX0006), Program for New Century Excellent
Talents in University (NCET-12-0192) and self determined research funds of SCUT
from the college basic research and operation of MOE (Nos. 2013ZM0044,
2013ZZ0068 and 2013ZM0065) for financial supports.
References
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8. L.C. Rocha, H.V. Ferreira, E.F. Pimenta, R.G.S. Berlinck, M.H.R. Seleghim, D.C.D. Javaroti, L.D.
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13. A. Jakoblinnert, R. Mladenov, A. Paul, F. Sibilla, U. Schwaneberg, M.B. Ansorge-Schumacher,
P.D. de María, Chem. Commun. 2011, 47, 12230-12232.
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Mar. Biotechnol. 2011, 13, 314-320.
15. M. Ferrero, V. Gotor, Monatsh. Chem. 2000, 131, 585-616.
16. Analytical methods: The reaction mixture was analyzed by RP-HPLC on a 4.6mm × 250 mm
(5µm)
Zorbax SB-C18 column (Agilent Technologies Co. Ltd. , Massachusetts, USA) using a Waters
600E pump and a Waters 2996UV/photodiode array detector (Waters Corp., Massachusetts,
USA) at 276 nm. A mixture of ammonium acetate buffer (0.01 M, pH 4.27) and methanol
(80/20, v/v) was used as mobile phase with the flow rate of 0.9 ml/min. The retention times for
ara-C, 3′-O-propionyl ara-C and 5′-O-propionyl ara-C were 3.14, 11.89 and 13.81 min,
respectively. Regioselectivity was defined as the ratio of the HPLC peak area corresponding to
the indicated product to that of all the products formed upon a certain reaction time. The yield
of ara-C to its 5′-O-ester was defined as the ratio: [moles of 5′-regioisomer in the acylation
reaction]/[initial moles of ara-C]. The average error for this determination was less than 1.0%.
The biomass of the strain was calculated in term of dry cell weight.
17. 13C NMR of Ara-C: 13C NMR δ 164.9 (C-4) , 143.4 (C-6), 92.5 (C-5) , 86.5 (C-1′) , 85.3 (C-4′),
76.3 (C-3′) ,74.9 (C-2′) , 61.2 (C-5′); FT-IR (KBr, cm-1) v 3214-3342 (OH,NH), 2922 (CH),
1649 (C=C), 1117/1051 (C-O-C)., M.δ.C. 13C NMR of 5′-O-propionyl ara-C: 13C
NMR(DMSO-d6, 166.10 (C4-N), 155.86 (C=O 2), 143.40(C-6), 93.11(C-5), 86.34 (C-1´),
82.20(C-4´), 77. 25 (C-3´), 74.81( C-2´), 64.20 (C-5´), 27.22 (CH3) , 9.39 (CH3) ESI-MS
((C12H17N3O6, 299.30): m/z(%)=298.90(100) ([M-H]+)., M.δ.C. 13C NMR of
3´-O-propionyl ara-C：：13C NMR(DMSO-d6, 166.10 (C4-N), 155.85(C=O 2), 143.18 (C-6),
93.11 (C-5), 86.34 (C-1´), 83.15 (C-4´), 79. 20 (C-3´), 72.75( C-2´), 61.66(C-5´), 27.20(CH2),
9.40 (CH3)；ESI-MS (C12H17N3O6, 299.30): m/z(%)= 298.90(100) ([M-H]+).
18. Aspergillus oryzae 3.5232 (Collection No. 3.5232) was supplied by CGMCC (China General
Microbiological Culture Collection Center, China). 1-β-D-arabinofuranosylcytosine and vinyl
propionate (VP) was purchased from Sigma (USA). All other chemicals were from
commercial sources and were of the highest purity available. General procedure of whole-cell
acylation of ara-C：2 ml organic solvents containing 20 mM ara-C, 600 mM VP, 4% water and
50 mg/ml freeze-dried Aspergillus oryzae 3.5232 (Collection No.3.5232) were incubated by
shaking (140 rpm) at a fixed temperature for 24 hours. Aliquots were withdrawn at specified
time intervals from the reaction mixture, and then diluted 100 times with water-methanol prior
to HPLC analysis.To structurally characterize the product of the whole-cell catalyzed
acylation of ara-C with VP, the reaction was scaled up. Upon completion of the reaction, the
reaction mixture was centrifuged to remove the cell masses, isolated, and purified by
half-preparation HPLC with a semi-preparation column. The acquisition was concentrated to
10

about 1 mL by vacuum rotary evaporation. After crystallization under 4℃, two products were
obtained and then determined by 13C NMR (Bruker AVANCE Digital 400MHz Nuclear
Magnetic Resonance Spectrometer, Bruker Co., Germany) at 100 MHz. The ESI-MS spectra
of the product were recorded on a Thermo LCQ DECA XP Plus ESI Mass Spectrometer with
a spray voltage of 4.5 kV (Thermo Finnigan, USA). All the experiment was repeated twice.
19. R.R. Chen, Appl. Microbiol. Biotechnol. 2007, 74, 730-738.
20. In order to assess the thermal stability of the enzyme, 100 mg aliquots of dried cells were added
into separate screw-capped vials containing 2 ml of the selected medium [30% (v/v)
hexane-pyridine or 30% (v/v) IPE-pyridine] and the mixture was incubated for 6 hours at
various temperatures from 30 to 60 °C. Then the biocatalyst was recovered by filtration from
the solvent mixtures and added to a fresh volume of the same reaction medium containing 20
mM ara-C and 600 mM VP. The assay reaction was then incubated at 140 rpm under 30 ºC.
The relative activity was expressed as the ratio of the residual activity after incubation to the
original activity of the cell biocatalyst in the same reaction system.
11

Figure Caption
Scheme 1 Acylation of ara-C with VP catalyzed by whole-cell（1：Ara-C；2：VP；
3：Vinyl alcohol (byproduct of the reaction); 4: Aldehyde; 5: 3’-O-propionyl ara-C; 6:
5’-O-propionyl ara-C）
Fig.1 (a) Effect of the IPE content on acylation of ara-C catalyzed by A.oryzae cells
(reaction conditions: 50 mg/mL biomass, 20 mM ara-C, 600 mM VP, 4% water, 1 mL
solvent medium, 30 ℃,140 rpm and 24 h). (b) Effects of VP/ara-C ratio (reaction
conditions: 50 mg/mL biomass, 20 mmol/L ara-C, 4% water, 1 mL solvent medium,
30 ℃,140 rpm and 24 h). (c) Effect of reaction temperature (reaction conditions: 50
mg/mL biomass, 20 mmol/L ara-C, 1800 mmol/L VP, 4% water, 1 mL
30%IPE-pyridine, 140 rpm and 24 h). (d) Effects of shaking speed (reaction
conditions: 50 mg/mL biomass, 20 mmol/L ara-C, 1800 mmol/L VP, 4% water, 1 mL
30% IPE-pyridine, 30 ℃and 24 h).
Fig.2 The thermal stabilities of A.oryzae whole-cells in 30% IPE-pyridine and 30%
hexane-pyridine (The reaction conditions: 50 mg/mL biomass, 20 mmol/L ara-C,
1800 mmol/L VP, 4% water, 2 mL 30%IPE-pyridine, 30 ℃, 140 rpm and 24 h)
12

Tables
Table 1 Acylation of ara-C with VP in various pure and binary organic solvents catalyzed by
whole-cells of A.oryzae a
Entry Solventb
ET(30)
V0
Y
3’-Regiosel
ectivity (%)
（kcal/mol）
(mmol/L·h)
(%)
1
2
3
4
5
6
7
8
DMSO
45.10
43.50
40.20
42.01
41.88
40.17
40.06
40.05
0
0
0
DMF
0
0
0
Pyridine
0.52 A
0.56 A
0.88 B
1.27 C
3.06 D
3.30 E
16.05 A
21.36 B
26.82 C
40.60 D
57.37 F
44.92 E
64.16 A
63.97 A
64.98 A
68.83 B
69.08 B
63.53 A
t-Butanol-pyridine
t-Pentyl alcohol-pyridine
THF-pyridine
IPE-pyridine
Hexane-pyridine
a The reaction conditions: 50 mg/mL biomass, 20 mM ara-C, 600 mM VP, 4% water, 1 mL solvent
medium, 30 ℃,140 rpm and 24 h; different capital letters denote significant differences at
Duncan’s post-test between different solvent systems.
b 75% pyridine was added in the binary solvents
13

Figure(s)

Figure(s)

Figure(s)