Regioselective Acylation of Nucleosides
(Table 2). As shown in Table 2, CAL-B displayed the high-
est catalytic activity in the butanoylation of floxuridine (En-
try 2). Moreover, the initial reaction rate dropped markedly
with the elongation of acyl chain from C4 to C8 (Table 2,
Entries 2–4) and then remained constant (Table 2, En-
tries 5–9). Pleiss et al. described the orientation and binding
of the acyl group in the hydrophobic pocket of CAL-B
through molecular modeling.[13] An acyl group of less than
C4 binds into a narrow cleft at the bottom of the active site.
Sharp distortion would occur if the acyl chain would extend
further from C4, resulting in an increase of the activation
energy and a lower reaction rate. Up to C7, the binding site
is still a narrow cleft, but then it becomes spacious. Like-
wise, it was reported that CAL-B was more specific toward
short chain fatty acids or acyl donors in the synthesis of
glucose esters and in the acylation of β-thymidine.[12,14]
Conclusions
A deep insight was gained into the interactions between
the enzyme and the substrates (nucleosides) in the catalytic
pathway by rational substrate engineering. The results im-
proved and extended the model for the CAL-B-catalyzed
acylation of 2Ј-deoxynucleosides proposed by Lavandera et
al. Besides, the findings provide a guide to control and max-
imize the regioselectivity of the synthetically useful enzyme
by chemical modification or protein engineering ap-
proaches.
Supporting Information (see footnote on the first page of this arti-
cle): General procedure for the acylation of nucleosides, HPLC
analysis conditions, retention times, and characterization data.
Acknowledgments
Table 2. Effect of the acyl chain length on the CAL-B-catalyzed
acylation of floxuridine.[a]
We wish to thank Ms. Xiu-Mei Liu for her help with NMR spec-
troscopy. This work was financially supported by the National Nat-
ural Science Foundation of China (Grant No. 20676043 and
20603036), the Science and Technology Project of Guangdong
Province (Grant No. 2006A10602003), and the Science and Tech-
nology Project of Guangzhou City (Grant No. 2007Z3-E4101).
[1] a) J. A. Montgomery, S. Ananthan, W. B. Parker, J. A. Secrist,
C. G. Temple, Cancer Chemotherapy Agents – Series: ACS Pro-
fessional Reference Book (Ed.: W. O. Foye), American Chemical
Society, Washington DC, 1995, pp. 47–110; b) A. Matsuda, T.
Sasaki, Cancer Sci. 2004, 95, 105–111.
Acyl chain
length
Entry
v0
Time Conversion
5Ј-Regioselectivity
[mM/h] [h]
(%)
(%)[b]
[2] C. Heidelberger, F. J. Ansfield, Cancer Res. 1963, 23, 1226–
1243.
[3] W. H. Prusoff, Biochim. Biophys. Acta 1959, 32, 295–296.
[4] B. A. Chabner, D. P. Ryan, L. Paz-Area, R. Garcia-Carbonero,
P. Calabresi, Goodman & Gilman’s The Pharmacological Basis
of Therapeutics, 10th ed. (Ed.: G. Hardman, L. E. Limbird),
Mc-Graw Hill, New York, 2001, pp. 1389–1459.
[5] a) E. De Clercq, H. J. Field, Br. J. Pharmacol. 2006, 147, 1–11;
b) C. P. Landowski, X. Q. Song, P. L. Lorenzi, J. M. Hilfinger,
G. L. Amidon, Pharm. Res. 2005, 22, 1510–1518; c) B. S. Vig,
P. J. Lorenzi, S. Mittal, C. P. Landowski, H. C. Shin, H. I. Mos-
berg, J. M. Hilfinger, G. L. Amidon, Pharm. Res. 2003, 20,
1381–1388.
[6] a) A. Diaz-Rodriguez, S. Fernandez, Y. S. Sanghvi, M. Ferrero,
V. Gotor, Org. Process Res. Dev. 2006, 10, 581–587; b) J. Gar-
cia, S. Fernandez, M. Ferrero, Y. S. Sanghvi, V. Gotor, J. Org.
Chem. 2002, 67, 4513–4519; c) J. Garcia, S. Fernandez, M. Fer-
rero, Y. S. Sanghvi, V. Gotor, Tetrahedron: Asymmetry 2003,
14, 3533–3540.
1
2
3
4
5
6
7
8
9
C2
C4
C6
44.6
48.5
38.2
22.0
19.6
18.9
19.1
18.8
19.0
2
2
3
4
5
5
5
5
5
99
99
99
99
99
99
99
98
98
65
80
77
76
75
76
77
77
77
C8
C10
C12
C14
C16
C18
[a] The reaction was initiated by adding 200 U of CAL-B into 2 mL
of anhydrous acetone containing 0.04 mmol of floxuridine and
0.24 mmol of vinyl esters at 40 °C and 250 rpm. [b] Defined as the
ratio of the concentration of the desired product to that of all the
products, and determined by HPLC analysis using an SB-C18 col-
umn.
As can be seen in Table 2, the highest 5Ј-regioselectivity
was observed in the butanoylation (80%, Entry 2). As men-
tioned above, there exists a hydrophobic interaction be-
tween the hydrophobic side of the base moiety (C5–C6–R1)
and the acyl portion in the conformation of the 5Ј-acylation
transition state.[12] Compared with the butanoyl group, the
acetyl group would display a weaker interaction, leading to
a lower 5Ј-regioselectivity (65%, Entry 1). CAL-B displayed
slightly lower 5Ј-regioselectivities (75–77%, Entries 3–9) for
the acylation of floxuridine with acyl donors of the chain
length of more than C4. The reason for this might be that
the hydrophobic side of the base moiety of floxuridine is
smaller than the acyl group of more than C4, and cannot
interact with the entire chain. Moreover, a larger acyl group
might cause a steric clash.
[7] a) M. Ferrero, V. Gotor, Monatsh. Chem. 2000, 131, 585–616;
b) M. Ferrero, V. Gotor, Chem. Rev. 2000, 100, 4319–4347; c)
N. Li, D. Ma, M. H. Zong, J. Biotechnol. 2008, 133, 103–109;
d) N. Li, M. H. Zong, X. M. Liu, D. Ma, J. Mol. Catal. B:
Enzym. 2007, 47, 6–12.
[8] O. Kirk, M. W. Christensen, Org. Process Res. Dev. 2002, 6,
446–451.
[9] a) J. Uppenberg, M. T. Hansen, S. Patkar, T. A. Jones, Struc-
ture 1994, 2, 293–308; b) J. Uppenberg, N. Ohrner, M. Norin,
K. Hult, G. J. Kleywegt, S. Patkar, V. Waagen, T. Anthonsen,
T. A. Jones, Biochemistry 1995, 34, 16838–16851.
[10] a) E. M. Anderson, M. Karin, O. Kirk, Biocatal. Biotransform.
1998, 16, 181–204; b) V. Gotor-Fernandez, E. Busto, V. Gotor,
Adv. Synth. Catal. 2006, 348, 797–812.
[11] a) A. Diaz-Rodriguez, S. Fernandez, I. Lavandera, M. Ferrero,
V. Gotor, Tetrahedron Lett. 2005, 46, 5835–5838; b) J. Garcia,
Eur. J. Org. Chem. 2008, 5375–5378
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5377