Diastereoselective enzymatic preparation of acetylated pentofuranosides carrying free 5-hydroxyl groups

Article abstract of DOI:10.1016/j.tetasy.2009.07.001

Methyl 3-O-acetyl-2-deoxy-α-d-ribofuranoside, 1,3-di-O-acetyl-2-deoxy-α-d-ribofuranose and 1,2,3-tri-O-acetyl-α-d-arabinofuranose were diastereoselectively prepared (de = 100%) from anomeric mixtures of the corresponding 5-acetylated compounds through Can

Full text of DOI:10.1016/j.tetasy.2009.07.001

Tetrahedron: Asymmetry 20 (2009) 1813–1816
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Diastereoselective enzymatic preparation of acetylated pentofuranosides
carrying free 5-hydroxyl groups
Esteban D. Gudiño a, Adolfo M. Iribarren a,b, Luis E. Iglesias a,
*
a Universidad Nacional de Quilmes, Roque Sáenz Peña 352—(1876) Bernal, Provincia de Buenos Aires, Argentina
b INGEBI (CONICET)—Vuelta de Obligado 2490—(1428) Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Methyl 3-O-acetyl-2-deoxy-a-D-ribofuranoside, 1,3-di-O-acetyl-2-deoxy-a-D-ribofuranose and 1,2,3-tri-
O-acetyl-a-D-arabinofuranose were diastereoselectively prepared (de = 100%) from anomeric mixtures
of the corresponding 5-acetylated compounds through Candida antarctica B lipase (CAL B)-catalysed
alcoholysis.
Received 9 June 2009
Accepted 2 July 2009
Available online 28 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
lation of 1,3,5-tri-O-acetyl-2-deoxy-a, b-D-ribofuranose 4a,b and
methyl 2,3,5-tri-O-acetyl- , b- -arabinofuranoside 5a,b afforded
a
D
It is well known that stereoselective reactions are powerful
transformations in organic synthesis, since they avoid undesired
products and difficult separation procedures; consequently, they
are valuable tools in the chemistry of polyhydroxylated com-
pounds such as carbohydrates. Among these compounds, partially
acetylated derivatives of anomerically pure sugars are important as
synthetic precursors of oligosaccharides, glycopeptides and
nucleosides.1
Nowadays, the efficient synthesis of partially acylated carbohy-
drates can be achieved by the use of hydrolytic enzymes such as
lipases, which catalyse regioselective acylation and deacylation
reactions.2–4 Most of the acylated carbohydrates obtained through
this methodology are derived from hexopyranose moieties;3–6 in
contrast, pentofuranoses3,4,7–13 and in particular, the stereoselec-
tive enzymatic acylation and deacylation of their anomeric mix-
tures,3,7,11 have been reported to a lesser extent.
the corresponding 5-O-deacetylated furanosides 10 and 11.
These results prompted us to study in more detail the stereose-
lectivity of these enzymatic deacetylations; herein we report the
results obtained (Scheme 2).
2. Results and discussion
Experiments of CAL-B-catalysed alcoholysis of 2-deoxy 3a,b and
4a,b and arabino substrates 5a,b and 6a,b were studied at alcohol/
substrate ratios (A/S) = 1200, 120 and 3; a high excess was assessed
since it provided regioselectivity in the CAL B-catalysed alcoholysis
of acylated nucleosides14–17 and methyl 2,3,5-tri-O-acetyl-
a,b-D-
ribofuranoside.18 Under these conditions, Table 1 shows the A/S ra-
tios and times allowing maximum production of monodeacetylat-
ed diastereomerically pure products 9a, 10a and 12a from the
corresponding anomeric substrates 3a,b, 4a,b and 6a,b. No signifi-
cant further conversion of the 5-O-monodeacetylated product was
observed after the times indicated in Table 1.
In our laboratory it was found that the Candida antarctica B lipase
(CAL B)-catalysed alcoholysis of peracetylated nucleosides regiose-
lectively affords the corresponding 50-O-deacylated products;14–17
we extended the scope of this biotransformation to pentofurano-
sides. In a previous paper,18 we reported the different behaviours
Since alcoholysis of 4a,b at the high A/S ratio employed for 3a,b
(entry 1) afforded low substrate conversion, other experimental
conditions were tested; at a lesser alcohol excess in dichlorometh-
ane (entry 2) a better yield of 10a could be obtained; replacement
of dichloromethane by acetonitrile, petroleum ether or dioxane
gave no appreciable reaction. With the tetraacetylated arabinof-
uranose 6a,b the best conditions for the formation of the free 5-hy-
droxyl derivative 12a involved ethanol at A/S = 120 and
dimethylformamide as a cosolvent (entry 3).
In every case, products 9, 10 and 12 consisted only of the 5-O-
deacetylated regioisomer, without removal of the more reactive
anomeric acetate of 4 and 6. Their 1H NMR analysis showed H-5 sig-
nals at ca. 3.7–3.9 ppm (Section 4.3.2); this as well as a shift towards
higher fields of ca. 0.5–0.6 ppm in comparison with H-5 of substrates
of methyl 2,3,5-tri-O-acetyl-
methyl 2,3,5-tri-O-acetyl-b-
alcoholysis. While the enzymatic deprotection of the former pro-
ceeded regioselectively to afford methyl 2,3-di-O-acetyl- -ribo-
furanoside 7a in high yield, the alcoholysis of the b-diastereomer
was less selective, leading to fully deacetylated methyl b- -ribofur-
a
-D
-ribofuranoside 1a (Scheme 1) and
D
-ribofuranoside 1b in CAL B-catalysed
a-D
D
anoside. Recently, in work aimed at the synthesis of nucleosides
from furanosides,19 we have reported that CAL B-catalysed deacety-
* Corresponding author. Fax: +54 11 4365 7132.
E-mail address: leiglesias@unq.edu.ar (L.E. Iglesias).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.07.001

1814
E. D. Gudiño et al. / Tetrahedron: Asymmetry 20 (2009) 1813–1816
R5
X
R3
R4
R2
O
Y
R1
1a X = H, Y = OCH3 ; R1 = R4 = R5 = OAc ; R2 = R3 = H
7a X = H, Y = OCH3 ; R1 = R4 = OAc ; R2 = R3 = H; R5 = OH
7b X = OCH3, Y = H ; R1 = R4 = OAc ; R2 = R3 = H; R5 = OH
8a X = H, Y = OAc; R1 = R4 = OAc ; R2 = R3 = H; R5 = OH
8b X = OAc, Y = H; R1 = R4 = OAc ; R2 = R3 = H; R5 = OH
9a X = H , Y = OCH3; R4 = OAc; R1 = R2 = R3 = H; R5 = OH
10a X = H , Y = OAc; R4 = OAc; R1 = R2 = R3 = H; R5 = OH
11a X = H , Y = OCH3; R2 = R4 = OAc; R1 = R3 = H; R5 = OH
11b X = OCH3, Y = H; R2 = R4 = OAc; R1 = R3 = H; R5 = OH
12a X = H, Y = OAc; R2 = R4 = OAc; R1 = R3 = H; R5 = OH
1b X = OCH3, Y = H ; R1 = R4 = R5 = OAc ; R2 = R3 = H
2a X = H, Y = OAc; R1 = R4 = R5 = OAc ; R2 = R3 = H
2b X = OAc, Y = H; R1 = R4 = R5 = OAc ; R2 = R3 = H
3a X = H , Y = OCH3; R4 = R5 = OAc; R1 = R2 = R3 = H
3b X = OCH3 , Y = H; R4 = R5 = OAc; R1 = R2 = R3 = H
4a X = H , Y = OAc; R4 = R5 = OAc; R1 = R2 = R3 = H
4b X = OAc , Y = H; R4 = R5 = OAc; R1 = R2 = R3 = H
5a X = H , Y = OCH3; R2 = R4 = R5 = OAc; R1 = R3 = H
5b X = OCH3, Y = H; R2 = R4 = R5 = OAc; R1 = R3 = H
6a X = H, Y = OAc; R2 = R4 = R5 = OAc; R1 = R3 = H
6b X = OAc, Y = H; R2 = R4 = R5 = OAc; R1 = R3 = H
Scheme 1.
In addition to the yield of products reported in Table 1, calcu-
lated by taking into account the mass of each substrate as anomer-
ic mixtures, a yield based on the mass of the -anomer content in
the substrate and in the product was determined (% -anomer
recovery, Table 1). This value shows that CAL B-catalysed alcohol-
ysis allows the recovery of the -anomer from anomeric mixtures
in moderate yields. In this way, this enzymatic procedure provides
not only an access to regioselectively 5-O-monodeacetylated prod-
ucts, but also a simple method to resolve pentofuranoside anomer-
ic mixtures, which are usually difficult to separate through
traditional chromatographic procedures.
X
Y
HOCH2
R3
X
Y
AcOCH2
R3
R2
R1
R2
R1
O
O
a
EtOH
a
Candida antarctica B
R4
3,4,6 a,b
3a, 9a X = H , Y = OCH3; R4 = OAc; R1 = R2 = R3 = H
3b
X = OCH3 , Y = H; R4 = OAc; R1 = R2 = R3 = H
4a, 10a X = H , Y = OAc; R4 = OAc; R1 = R2 = R3 = H
4b
X = OAc , Y = H; R4 = OAc; R1 = R2 = R3 = H
6a, 12a X = H, Y = OAc; R2 = R4 = OAc; R1 = R3 = H
6b
X = OAc, Y = H; R2 = R4 = OAc; R1 = R3 = H
R4
lipase
a
9,10,12 a
Concerning the stereoselectivity of CAL B in the catalysis of the
carbohydrate anomers, some papers have reported differences in
the behaviour of pyranose anomers.3,5,20,21 In CAL B-catalysed acy-
lations, the alkyl pyranoside anomers displayed different regiose-
lectivities;5,21 in alcoholyses, differential recognition of
pentaacetylated glucosamine epimers by CAL B has been de-
scribed.20 However, with regard to furanoses, apart from work
reporting the regioselective alcoholysis of the hexose 1,2,3,4,6-
Scheme 2. CAL B-catalysed diastereoselective deacetylation of pentofuranosides 3,
4 and 6.
(datanot shown)isconsistentwithprimaryalcoholmethylene hydro-
gens, indicating that the deacetylation occurred at the 5-acetate.
1H NMR analysis allowed us to evaluate the stereoselectivity of
CAL B-catalysed deacetylation of the anomeric mixtures studied.
penta-O-acetyl-a,b-D
-fructofuranose,11 in which a different regi-
oselectivity in the monodeacetylation of each anomer was ob-
served, to the best of our knowledge no other work on the
stereoselective CAL B-catalysed reaction of furanoses has been re-
ported. In the field of nucleosides, CAL B-catalysed hydrolysis of an
Products 9a, 10a and 12a were obtained exclusively as the a-ano-
mer, indicating a high diastereoselectivity of CAL B in the deacety-
lation of these substrates. The results suggest that pentose
substitution at carbon 2 affects the observed stereoselectivity,
since 2a,b and 5a,b, which share the same 3-substitution pattern
as 3-, 4- and 6a,b, reacted with lower diastereoselectivity. How-
ever, compounds 8 and 11, and the corresponding 5-O-deacety-
a
/b mixture of p-chlorobenzoyl-protected thymidine afforded the
50-deacylated anomer.22
a
3. Conclusion
lated products from 2 and 5, were enriched in the
a-anomer (for
2, b/
a
ratio = 2.6/1.0, for 8, b/
ratio = 1.1/1.0, for 11, b/
a
ratio = 1.0/3.0, at a A/S = 1200; for
In conclusion, this work describes a simple procedure for a
highly stereoselective preparation of three free 5-hydroxyl-acety-
5, b/a
a ratio = 1.0/2.5 at a A/S = 120, using
acetone as a cosolvent).
Table 1
CAL B-catalysed diastereoselective preparation of 9a, 10a and 12a (Scheme 2)
Entry
Substrate
Substrate b/
a
ratioa
T (°C)
t (h)
Alcohol
A/Sb
Product (yield, %)c
ded (%)
a
-Anomer recoverye (%)
1
2
3
3a,b
4a,b
6a,b
1.1/1.0
1.2/1.0
1.3/1.0
30
30
30
24
1.5
24
Ethanol
Ethanolf
Ethanolg
1200
3
120
9a (32)
10a (24)
12a (30)
100
100
100
67
58
69
a
Anomeric ratio, determined by 1H NMR.
Alcohol/substrate molar ratio.
b
c
d
e
f
Yield determined after isolation by silica gel column chromatography.
Diastereomeric excess, determined by 1H NMR.
Yield calculated on the basis of a-anomer content in isolated product and in the substrate.
Dichloromethane was employed as solvent.
Dimethylformamide was employed as cosolvent.
g

E. D. Gudiño et al. / Tetrahedron: Asymmetry 20 (2009) 1813–1816
1815
lated pentofuranosides, through an enzymatic alcoholysis. By using
this methodology, these products can be prepared in two steps
of the enzyme, monitored by TLC using dichloromethane–metha-
nol 95:5 v/v as the mobile phase.
from the corresponding free
D
-pentofuranose, avoiding an addi-
Control experiments carried out in the absence of the enzyme
showed no appreciable reaction.
tional step involved in traditional synthetic procedures when
applying protecting groups. Moreover, it provides a simple proce-
dure for the separation of anomeric mixtures of these pentofurano-
sides. In this way, the results herein described widen the
applications of CAL B in the field of pentofuranoses.
4.3.2. Preparative procedure
4.3.2.1. Methyl 3-O-acetyl-2-deoxy-a-D-ribofuranoside 9a. Acc-
ording to the analytical procedure, substrate 3a,b (0.31 mmol) was
dissolved in ethanol at an A/S = 1200 (21.6 mL) and CAL B (93 mg)
was added. The reaction mixture was shaken at 200 rpm and 30 °C
for 24 h. Then the lipase was filtered off, washed with dichlorometh-
ane, the resulting filtrates evaporated and the crude product
subsequently purified by silicagel column chromatography using
4. Experimental
4.1. General
dichloromethane–methanol 98:2, affording 9a (32%),
½
a 2D0
ꢁ
¼
NMR spectra were recorded on a Bruker AC-500 spectrometer in
CDCl3, at 500 MHz for 1H and 125 MHz for 13C using TMS and CDCl3
as internal standards, respectively; d are indicated in ppm and J, in
hertz. Optical rotations were measured on a Perkin Elmer Polarim-
eter 343.
All employed reagents and solvents were of analytical grade
and obtained from commercial sources. For the enzymatic alcohol-
yses, absolute ethanol was employed and the solvents tested were
dried and distilled prior to use.
TLC was performed on Silica Gel 60 F254 plates (Merck) and the
resulting plates were developed using ethanol–sulfuric acid 80:20
v/v with heating. Column chromatography was carried out using
silica Gel Merck 60 (0.040–0.063 mm).
Lipase B from C. antarctica (CAL B, Novozym 435, 10,000 PLU/mg
solid; PLU:Propyl Laurate Units), a generous gift from Novozymes
(Brazil), was used without any further treatment or purification.
Enzymatic reactions were carried out in a temperature-con-
trolled incubator shaker (Sontec OS 11, Argentina) at 200 rpm
and 30 °C.
þ129:4 (c 0.06, CH3CH2OH), 1H NMR (500 MHz, CDCl3): 2.03 (ddd,
1H, J = 14.6, 8.7, 5.3 Hz, H-2), 2.09 (s, 3H, CH3), 3,40 (s, 3H, OCH3),
3.76 (dd, 1H, J = 11.8, 3.8 Hz, H-5), 3.82 (dd, 1H, J = 11.8, 3.8 Hz, H-
50), 4.11 (pq, 1H, J = 3.8, 3.8 Hz, H-4), 5.07-5.09 (m, 1H, H-3), 5.10
(d, 1H, J = 5.3 Hz, H-1). 13C NMR (125 MHz, CDCl3): 21.15 (CH3),
39.26 (C-2), 55.08 (OCH3), 62.59 (C-5), 74.27 (C-3), 83.54 (C-4),
104.96 (C-1), 171.40 (CO).
4.3.2.2. 1,3-Di-O-acetyl-2-deoxy-a-D-ribofuranose 10a. Follow-
ing the above reported protocol, 4a,b (0.31 mmol) was dissolved
in ethanol (A/S = 3, 0.053 mL) and dichloromethane (7.5 mL) and
the mixture shaken for 1.5 h. After the described work up, 10a
(24%) was obtained: ½a D20
ꢁ
¼ þ89:4 (c 0.04, CH3CH2OH), 1H NMR
(500 MHz, CDCl3): 2.08, (s, 3H, CH3), 2.11 (s, 3H, CH3), 2.50 (ddd,
1H, J = 15.0, 7.9, 5.3 Hz, H-2), 3.74–3.83 (m, 2H, H-5, H-50), 4.29
(dd, 1H, J = 6.7, 3.6 Hz, H-4), 5.16-5.19 (m, 1H, H-3), 6.39 (d, 1H,
J = 5.3 Hz, H-1). 13C NMR (125 MHz, CDCl3): 21.04, 21.29 (CH3s),
38.57 (C-2), 62.42 (C-5), 74.00 (C-3), 86.09 (C-4), 98.34 (C-1),
170.48, 171.08 (COs).
4.2. Preparation of the substrates
4.3.2.3. 1,2,3-Tri-O-acetyl-a-D-arabinofuranose 12a. At first, 6a,b
(0.31 mmol) was dissolved in ethanol at an A/S = 120 (2.20 mL)
and 0.20 mL of DMF and the mixture shaken for 24 h. Application
of the above protocol and the involved work up gave 12a (30%):
Substrate 3 was prepared via standard protocols.18 2-Deoxy-
D-ri-
bose in an excess of methanol containing sulfuric acid and copper sul-
fate afforded methyl 2-deoxyriboside, which by subsequent
treatment with acetic anhydride and dimethylaminopyridine in pyr-
idine gave thetriacetylated compound. Purification of thecrude prod-
uct by column chromatography afforded methyl 3,5-di-O-acetyl-2-
½
a 2D0
ꢁ
¼ ꢀ13:3 (c 0.12, CH3CH2OH), 1H NMR (500 MHz, CDCl3):
2.12, 2.13, 2.14 (9H, CH3s), 3.78–3.93 (m, 2H, H-5, H-50), 4.21–
4.25 (m, 1H, H-4), 5.12 (dd, 1H, J = 5.1, 1.8 Hz, H-3), 5.25 (d, 1H,
J = 1.8, H-2), 6.18 (s, 1H, H-1). 13C NMR (125 MHz, CDCl3): 20.59,
20.61, 20.62 (CH3s), 61.23 (C-5), 74.10 (C-3), 76.83 (C-2), 82.50
(C-4), 93.78 (C-1), 169.82, 171.10, 170.14 (COs).
deoxy-a, b-D-ribofuranoside 3a,b, which gave satisfactory NMR data.
Since direct peracetylation of the corresponding
D-pentose with
acetic anhydride in pyridine produces mixtures of furanoses and
pyranoses, in order to prepare 4 and 6, the pentose was first trea-
ted with acetic anhydride in dry tetrahydrofurane and CAL B,23
then further acylated with acetic anhydride in pyridine, following
the previously described protocols.23 Application of this procedure
gave, after purification of the crude products by column chroma-
Acknowledgements
We thank UNQ, CONICET and ANPCyT (PICT 06-36472) for par-
tial financial support. AMI and LEI are research members of CONI-
CET. We are grateful to Novozymes (Brazil) for the generous gift of
CAL B.
tography, the substrates 1,3,5-tri-O-acetyl-2-deoxy-a, b-D-ribofu-
ranose 4a,b and 1,2,3,5-tetra-O-acetyl- , b- -arabinofuranose 6a,b.
a
D
References
4.3. CAL B-catalysed deacetylation of substrates 3, 4 and 6
1. Tanmaya, P. Chem. Rev. 2002, 102, 1623.
2. Bornsheuer, U.; Kazlauskas, R. Hydrolases in Organic Chemistry; Wiley:
Weinheim, 2004. Chapter 5, pp 141–152.
3. Kadereit, D.; Waldmann, H. Chem. Rev. 2001, 101, 3367.
4. La Ferla, B. Monatsh. Chem. 2002, 133, 351.
5. Gonçalves, P. L. M.; Roberts, S. M.; Wan, P. W. H. Tetrahedron 2004, 60, 927.
6. Filice, M.; Fernández-Lafuente, R.; Terreni, M.; Guisán, J. M.; Palomo, J. M. J. Mol.
Catal. B: Enzym. 2007, 49, 12.
4.3.1. Analytical procedure
In a typical analytical protocol, the substrate (10 mg) was dis-
solved in the nucleophilic alcohol at an alcohol/substrate molar ra-
tio (A/S) = 1200 or 120 and CAL B (300 mg mmolꢀ1 substrate) was
added.
When a cosolvent was assayed, the substrate was dissolved in a
mixture of alcohol and 10 % (v/v) of the solvent tested, maintaining
the above reported A/S ratios. An A/S = 3 was also assayed; in this
case, a 0.7 % (v/v) mixture of alcohol and solvent was employed.
The resulting reaction mixtures were shaken at 200 rpm and
30 °C. Samples were taken at different times and, after removal
7. Hennen, W. J.; Sweers, H. M.; Wang, Y. F.; Wong, C. H. J. Org. Chem. 1988, 53,
4939.
8. Fernández-Lorente, G.; Palomo, J. M.; Cocca, J.; Mateo, C.; Moro, P.; Terreni, M.;
Fernández-Lafuente, R.; Guisán, J. M. Tetrahedron 2003, 59, 5705.
9. Chien, T. C.; Chern, J. W. Carbohydr. Res. 2004, 339, 1215.
10. Jun, S. J.; Moon, M. S.; Lee, S. H.; Cheong, C. S.; Kim, K. S. Tetrahedron Lett. 2005,
46, 5063.

1816
E. D. Gudiño et al. / Tetrahedron: Asymmetry 20 (2009) 1813–1816
11. Antona, N.; El-Idrissi, M.; Ittobane, N.; Nicolosi, G. Carbohydr. Res. 2005, 340, 319.
12. Prasad, A. K.; Kalra, N.; Yadav, Y.; Kumar, R.; Sharma, S. K.; Patkar, S.; Lange, L.;
Wengel, J.; Parmar, V. S. Chem. Commun. 2007, 2616.
13. Prasad, A. K.; Kalra, N.; Yadav, Y.; Singh, S. K.; Sharma, S. K.; Patkar, S.; Lange, L.;
Olsen, C. E.; Wengel, J.; Parmar, V. S. Org. Biomol. Chem. 2007, 5, 3524.
14. Zinni, M. A.; Gallo, M.; Iglesias, L. E.; Iribarren, A. M. Biotechnol. Lett. 2000, 22,
361.
18. Iñigo, S.; Taverna Porro, M.; Montserrat, J. M.; Iglesias, L. E.; Iribarren, A. M. J.
Mol. Catal. B: Enzym. 2005, 35, 70.
19. Taverna Porro, M.; Montserrat, J. M.; Iribarren, A. M. Tetrahedron Lett. 2008, 49,
4642.
20. Nicolosi, G.; Spatafora, C.; Tringali, C. Tetrahedron: Asymmetry 1999, 10, 2891.
21. Danieli, B.; Luisetti, M.; Sampagnaro, G.; Carrea, G.; Riva, S. J. Mol. Catal. B:
Enzym. 1997, 3, 193.
15. Zinni, M. A.; Iglesias, L. E.; Iribarren, A. M. Biotechnol. Lett. 2002, 24, 979.
16. Zinni, M. A.; Pontiggia, R.; Rodríguez, S. D.; Montserrat, J. M.; Iglesias, L. E.;
Iribarren, A. M. J. Mol. Catal. B: Enzym. 2004, 29, 129.
22. García, J.; Díaz-Rodríguez, A.; Fernández, S.; Sanghvi, Y. S.; Ferrero, M.; Gotor, V.
J. Org. Chem. 2006, 71, 9765–9771.
23. Prasad, A. K.; Sorensen, M. D.; Parmar, V. S.; Wengel, J. Tetrahedron Lett. 1995,
36, 6163.
17. Zinni, M. A.; Iglesias, L. E.; Iribarren, A. M. J. Mol. Catal. B: Enzym. 2007, 47, 86.