Synthesis and enzymic hydrolysis of acylated adenosine derivatives

Article abstract of DOI:10.1080/07328300601059284

Various derivatives of adenosine were prepared by acylation of adenosine (6-amino-9-(β-D-ribofuranosyl)purine (1) with different molar equivalents of acetic anhydride and/or pivaloyl chloride in pyridine. Compounds 6-acetylamino-9-[(2,3,5-tri-O-acetyl)-β-D-ribofuranosyl]purine (3), 6-amino-9-[(2,3,5-tri-O-acetyl)-β-D-ribofuranosyl]purine (4), and 6-pivaloylamino-9-[(2,3,5-tri-O-pivaloyl)-β-D-ribofuranosyl]purine (5) were subsequently submitted to hydrolysis catalyzed by a number of hydrolytic enzymes. Regioselective enzymic deacetylation at the primary hydroxyl group of 3 and 4 with butyrylcholinesterase (BChE) produced 6-acetylamino-9-[(2,3-di-O- acetyl)-β-D-ribofuranosyl]purine (9) and 6-amino-9-[(2,3-di-O-acetyl- β-D-ribofuranosyl]purine (10), respectively. All structures were established by ¹H and ¹³C NMR spectroscopies.

Full text of DOI:10.1080/07328300601059284

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Synthesis and Enzymic Hydrolysis of Acylated
Adenosine Derivatives
a
a
Ž. Car a , V. Petrović & S. Tomić
a Faculty of Science , University of Zagreb , Zagreb, Croatia
Published online: 02 Feb 2007.
To cite this article: Ž. Car , V. Petrović & S. Tomić (2006) Synthesis and Enzymic Hydrolysis
of Acylated Adenosine Derivatives, Journal of Carbohydrate Chemistry, 25:8-9, 713-723, DOI:
10.1080/07328300601059284
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Journal of Carbohydrate Chemistry, 25:713–723, 2006
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300601059284
Synthesis and Enzymic
Hydrolysis of Acylated
Adenosine Derivatives
ˇ
´
´
Z. Car, V. Petrovic, and S. Tomic
Faculty of Science, University of Zagreb, Zagreb, Croatia
Various derivatives of adenosine were prepared by acylation of adenosine (6-amino-9-(b-
D-ribofuranosyl)purine (1) with different molar equivalents of acetic anhydride and/or
pivaloyl chloride in pyridine. Compounds 6-acetylamino-9-[(2,3,5-tri-O-acetyl)-b-D-
ribofuranosyl]purine (3), 6-amino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine (4),
and 6-pivaloylamino-9-[(2,3,5-tri-O-pivaloyl)-b-D-ribofuranosyl]purine (5) were sub-
sequently submitted to hydrolysis catalyzed by a number of hydrolytic enzymes. Regio-
selective enzymic deacetylation at the primary hydroxyl group of 3 and 4 with
butyrylcholinesterase (BChE) produced 6-acetylamino-9-[(2,3-di-O-acetyl)-b-D-ribofura-
nosyl]purine (9) and 6-amino-9-[(2,3-di-O-acetyl-b-D-ribofuranosyl]purine (10), respect-
ively. All structures were established by 1H and 13C NMR spectroscopies.
Keywords Acylated adenosine, Hydrolases, Enzymic hydrolysis
INTRODUCTION
High selectivity under mild conditions is characteristic for reactions catalyzed
by enzymes. Thus, enzymes were found to be suitable biocatalysts in the devel-
opment of new synthetic methods in organic synthesis.[1,2] They are especially
interesting in transformations of multifunctional compounds such as nucleo-
sides since time-consuming protection and deprotection steps, possible side
reactions, tedious separation processes, low yields, and other associated
problems can often be avoided. Furthermore, the regioselectivity and/or che-
moselectivity observed in reactions catalyzed by enzymes add to their rising
attractiveness in synthetic organic chemistry.
Nucleosides exhibit a broad spectrum of biologic activities including anti-
viral, anticancer, antibacterial, and antiparasitic activities.[3] These generally
Received December 29, 2005; accepted September 15, 2006.
´
Address correspondence to S. Tomic, Faculty of Science, University of Zagreb, Horvato-
vac 102A, HR-10000 Zagreb, Croatia. E-mail: stomic@chem.pmf.hr
713

ˇ
Z. Car et al.
714
result from their ability to inhibit specific enzymes.[3] The rising focus on
nucleoside chemistry has also been provoked by the discovery that some
nucleoside analogs are potential anti-HIV agents[4–6] and that antisense and
antigene oligonucleotides are potential and selective inhibitors of gene
expression.[7–9] Although several chemical methods were used for the regiose-
lective modifications[10,11] of nucleoside’s sugar moiety, enzymic methods offer
significant advantages.[1,2,12–14]
Partially deprotected 50-adenosines are well-known common intermediates
in oligonucleotide synthesis.[15,16] Their conjugates with carbohydrates were
also studied. Thus, 6-acetylamino-9-[(2,3-di-O-acetyl)-b-D-ribofuranosyl]-
purine has been used in the synthesis of 50-O-glycosylnucleosides.[17] Many
natural antibiotics possessing significant antitumor and antiviral activities
have the structure of a nucleoside connected to oligosaccharides.[17] 6-Amino-
9-[(2,3-di-O-acetyl)-b-D-ribofuranosyl]purine is a starting material in the
synthesis of adenosine a-P-boranophosphoglucose, which can be useful in
investigations of the stereochemistry and mechanism of action of enzymes
such as glycogen and starch synthetases, glycosidyl, and nucleosidyl trans-
ferases.[18] It has also been shown that the free 50-hydroxyl group of partially
acetylated adenosines plays a crucial role in the catalytic action at the
adenosine deaminase (ADA) active site.[19]
In continuation of our interest in enzymic transformations of carbo-
hydrates,[20,21] in this work we turn to acylated adenosine derivatives in an
attempt to perform regioselective enzymically catalyzed deprotection of the 50-OH.
RESULTS AND DISCUSSION
Preparation of Acylated Adenosine Derivatives
Conventional acetylation of adenosine 1 with 10 molar equivalents of Ac2O
in pyridine for 24 h followed by column chromatography on silica gel produced
the pentaacetate 2 (29%) and the tetraacetate 3 (67%) as the major product
(Table 1).
Treatment of 1 with six molar equivalents of the same acylating agent for 24 h
resulted in the formation of the triacetate 4 (70%) as the sole reaction product.
Pivaloylation of 1 was achieved by using 10 equivalents of pivaloyl chloride
in pyridine for 48 h at 508C. It was noticed, by TLC monitoring, that during the
first 24 h several products were formed. After 48 h only tetrapivalate 5 was
isolated in 70% yield.
Gradual addition of seven molar equivalents of pivaloyl chloride to a
solution of 4 in pyridine for 48 h produced 6-pivaloylamino-9-[(2,3,5-tri-O-
acetyl)-b-D-ribofuranosyl]purine 6 (66%). Some unreacted 4 (33%) was recov-
ered from the reaction mixture as well.

Acylated Adenosine Derivatives
Table 1: Acylated adenosine derivaties.
715
R1
R2
R3
R4
R5
1
2
3
4
5
H
H
H
H
H
Ac
H
H
H
H
H
H
H
Ac
Ac
Ac
Piv
Ac
H
Ac
Ac
Ac
Ac
Ac
Ac
Piv
Ac
Ac
H
Ac
Ac
Ac
Piv
Ac
Ac
Ac
H
Ac
Ac
H
Piv
Piv
Ac
Ac
Ac
H
6
7a
8a
9a
10a
Ac
Ac
H
H
aCompounds prepared by enzymic hydrolysis.
Enzymic Deacylations
Tetraacetate 3, triacetate 4, and tetrapivalate 5 were submitted to
hydrolysis catalyzed by various commercially available enzymes. To improve
solubility of acylated derivatives 3 and 5, organic solvent (Me2SO or DMF)
was added to the phosphate-buffered (pH 7) incubation mixtures (Table 2).
Only in hydrolysis of 3 catalyzed by BChE the addition of organic solvents
was not needed. Parallel controls containing the substrate in reaction
medium but without the enzyme were also prepared to exclude any nonenzy-
mic processes.
Table 2: Enzyme-catalyzed hydrolysis of tetraacetate 3.
Organic
solvent/mL
Reaction
time/h
Product
(Yield)
Enzyme/mg
PPL/1.7
PLE/1.1
BChE/2.6
Subtilisin/3.5
a-Chymotrypsin/1
Me2SO/200
Me2SO/200
—
DMF/500
DMF/200
4
2.5
2
48
48
Unselective hydrolysis
7 1 8 (54%; 7 : 8 ¼ 2.7 : 1)
9 (89%)
—
—

ˇ
Z. Car et al.
716
It was shown that BChE catalyzes the selective hydrolysis of the 50-OAc
in the tetraacetate 3 yielding 89% of 6-acetylamino-9-[(2,3-di-O-acetyl)-b-D-
ribofuranosyl]purine 9. The reaction was carried out in phosphate buffer
without the addition of organic solvents (Sch. 1).
Hydrolysis of tetraacetate 3 catalyzed by porcine liver esterase (PLE)
(Sch. 1) produced the regioisomeric mixture of 6-acetylamino-9-[(3,5-di-O-
acetyl)-b-D-ribofuranosyl]purine 7 and 6-acetylamino-9-[(2,5-di-O-acetyl)-b-D-
1
ribofuranosyl]purine 8 in a ratio of 2.7 : 1 as determined by H NMR. Total
yield of 54% was obtained in 2.5 h. It is possible, however, that in this
reaction selective hydrolysis of the 20-OAc occurs in the first step liberating
the 20-OH group, followed by the 30 ! 20 acetyl migration. This type of
migrations are known and were previously reported.[19,22]
Hydrolysis of 3 with porcine pancreas lipase (PPL) as a catalyst in 4 h
resulted in a mixture of several partially acetylated products. Some unreacted
starting material was detected as well. No reaction occurred when
Scheme 1

Acylated Adenosine Derivatives
717
a-chymotrypsin or subtilisin were used as possible catalysts under the same
reaction conditions.
Regioselective hydrolyses of 20,30,50-tri-O-acetylribonucleosides have
already been described using subtilisin and lipase from Candida antarctica
(CAL) as catalysts,[14,19] and the corresponding 20,30-di-O-acetylribonucleosides
were obtained in relatively good yields in slow reactions (16–24 h). We found
that BChE catalyzes the selective hydrolysis of the 50-OAc in the triacetate 4
as well. In 4.5 h 39% of diacetate 10 was obtained (Table 3). Prolongation of
reaction times resulted in loss of selectivity. This is often the case when
using enzymes as catalysts in transformations of compounds that are not
their natural substrates. While stereospecific reactions occur with enzymes’
natural substrates, only stereoselectivity can be expected in transformations
of nonnatural substrates, and enzymes with time start recognizing, in
smaller degrees, other similar structures leading to the decrease of selectivity.
Triacetate 4 underwent unselective deacetylation in reactions catalyzed by
PPL and PLE and no reaction was observed using a-chymotrypsin.
Tetrapivalate 5 was also subjected to hydrolyses under similar reaction
conditions and with the same enzymes as 3 and 4, but no reactions were
observed. The most probable cause for this unreactivity is the very low solubi-
lity of 5 even in incubation mixtures in which up to 30% (v/v) of organic solvent
was added.
EXPERIMENTAL
General Methods for Synthesis and Characterization
of Adenosine Derivatives
All solvents were reagent grade and distilled before use. Column chromato-
graphy was performed on silica gel (Merck) and TLC on Merck silica gel (60 F
254) plates (0.25 mm) with solvent A, EtOAc-C6H6-EtOH (10 : 2 : 1.5); solvent
B, EtOAc-MeOH (5 : 1); solvent C, EtOAc-C6H6 (10 : 1); and solvent D, EtOAc-
C6H6 (5 : 1). Visualisation was effected by use of UV light and by charring
with H2SO4. Melting points were determined with a Bu¨chi B-40 apparatus
Table 3: Enzyme-catalyzed hydrolysis of triacetate 4.
Reaction
Enzyme/mg
time/h
Product (Yield)
PPL/1.9
PLE/1.2
BChE/14.5
a-Chymotrypsin/1
4
Unselective hydrolysis
Unselective hydrolysis
10 (39%)
3.5
4.5
48
—

ˇ
Z. Car et al.
718
and are uncorrected. Optical rotations were measured at ꢀ208C using the
Optical Activity AA-10 Automatic Polarimeter. 1H and 13C NMR spectra
(300 MHz or 600 MHz, Me2SO or CDCl3, internal Me4Si) were recorded with
a Bruker AV300 spectrometer.
6,6-Diacetylamino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine (2)
and 6-acetylamino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine (3).
To a solution of 1 (100 mg, 0.37 mmol) in dry pyridine (1 mL) acetic anhydride
(354 mL, 3.74 mmol) was added. The mixture was stirred at ambient tempera-
ture for 24 h and the reaction stopped by addition of 96% EtOH. Water was
added and the mixture of solvents evaporated under reduced pressure. The
remaining traces of water were removed by codistillation with toluene.
Column chromatography (solvent A) of the residue gave, firstly, 6,6-diacetyla-
mino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine 2 (51 mg, 29%) as a white
syrup;[23] [a]D 218.58 (c 0.87, CHCl3); Rf ꢀ 0.46 (solvent A). 1H NMR (300 MHz,
CDCl3): 2.12 (s, 3 H, OAc) 2.14 (s, 3 H, OAc), 2.17 (s, 3 H, OAc), 2.38 (s, 6 H,
NAc2), 4.37–4.51 (m, 3 H, H-40, H-50a, H-50b), 5.69 (app t, 1 H, J ¼ 5.22 Hz,
J ¼ 4.94 Hz, H-30), 5.98 (app t, 1 H, J ¼ 5.50 Hz, J ¼ 5.22 Hz, H-20), 6.27 (d, 1
H, J ¼ 4.95 Hz, H-10), 8.30 (s, 1 H, H-8), 8.99 (s, 1 H, H-2); 13C NMR
(600 MHz, CDCl3): 20.27, 20.38, 20.61 (CH3CO, 20-OAc, 30-OAc, 50-OAc),
26.20 (2 CH3CO, NAc2), 62.86 (C-50), 70.43 (C-30), 72.98 (C-20), 80.54 (C-40),
86.70 (C-10), 130.77 (C-5), 141.19 (C-8), 151.07 (C-4), 152.84 (C-2), 153.15
(C-6), 169.20, 169.39, 170.10 (C55O, 20-OAc, 30-OAc, 50-OAc), 171.67 (2 C55O,
NAc2).
Eluted next was 6-acetylamino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]-
purine 3 as a white syrup[24] (109 mg, 67%); [a]D 217.38 (c 1.33, CHCl3);
Rf ꢀ 0.26 (solvent A). 1H NMR (300 MHz, CDCl3): 2.09 (s, 3 H, OAc), 2.12
(s, 3 H, OAc), 2.17 (s, 3 H, OAc), 2.64 (s, 3 H, NAc), 4.35–4.48 (m, 3 H, H-40,
H-50a, H-50b), 5.68 (app t, 1 H, J ¼ 5.22 Hz, J ¼ 4.95 Hz, H-30), 5.98 (app t,
1 H, J ¼ 5.50 Hz, J ¼ 5.49 Hz, H-20), 6.24 (d, 1 H, J ¼ 5.22 Hz, H-10), 8.26
(s, 1 H, H-8), 8.71 (s, 1 H, H-2), 9.28 (br s, 1 H, NH); 13C NMR (300 MHz,
CDCl3): 20.23, 20.36, 20.57 (CH3CO, 20-OAc, 30-OAc, 50-OAc), 25.60 (CH3CO,
NAc), 62.86 (C-50), 70.41 (C-30), 72.87 (C-20), 80.18 (C-40), 86.25 (C-10), 122.05
(C-5), 141.49 (C-8), 149.38 (C-4), 150.91 (C-6), 152.43 (C-2), 169.24, 169.46,
170.23, 170.92 (C55O, 20-OAc, 30-OAc, 50-OAc, NAc).
6-Amino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine (4). To
a
solution of 1 (110 mg, 0.41 mmol) in dry pyridine (1 mL) acetic anhydride
(235 mL, 2.47 mmol) was added. The mixture was stirred at ambient tempera-
ture for 24 h and the reaction stopped by addition of 96% EtOH. After usual
workup of the reaction mixture, as described in preparation of 2 and 3,
column chromatography (solvent B) of the residue gave white crystalline[24,25]
6-amino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine 4; (137 mg, 85%); mp
174–1758C (from abs. EtOH), lit.[24] 1748C; [a]D 220.98 (c 1.00, CHCl3),

Acylated Adenosine Derivatives
719
lit.[24] 227.98 (CHCl3); Rf ꢀ 0.44 (solvent B). 1H NMR (600 MHz, CDCl3): 2.09
(s, 3 H, OAc), 2.13 (s, 3 H, OAc), 2.15 (s, 3 H, OAc), 4.36–4.40 (m, 1 H, H-40),
4.44–4.48 (m, 2 H, H-50a, H-50b), 5.68 (dd, 1 H, J ¼ 5.45 Hz, J ¼ 4.54 Hz,
H-30), 5.74 (br s, 2 H, NH2), 5.94 (app t, 1 H, J ¼ 5.43 Hz, J ¼ 5.36 Hz, H-20),
6.19 (d, 1 H, J ¼ 5.30 Hz, H-10), 7.97 (s, 1 H, H-8), 8.37 (s, 1 H, H-2);
13C NMR (600 MHz, CDCl3): 20.37, 20.51, 20.74 (CH3CO, 20-OAc, 30-OAc,
50-OAc), 63.05 (C-50), 70.63 (C-30), 73.28 (C-20), 80.44 (C-40), 86.41 (C-10),
120.08 (C-5), 139.42 (C-8), 149.64 (C-4), 151.38 (C-2), 154.60 (C-6), 169.32,
169.52, 170.25 (C55O, 20-OAc, 30-OAc, 50-OAc).
6-Pivaloylamino-9-[(2,3,5-tri-O-pivaloyl)-b-D-ribofuranosyl]purine (5).
To a solution of 1 (200 mg, 0.75 mmol) in dry pyridine (2 mL) pivaloyl
chloride (920 mL, 7.5 mmol) was added. The mixture was stirred at 508C
under reflux for 48 h and the reaction stopped by addition of 96% EtOH.
After evaporation, as described in preparation of 2 and 3, column chromato-
graphy (solvent C) of the residue gave as a main product 6-pivaloylamino-9-
[(2,3,5-tri-O-pivaloyl)-b-D-ribofuranosyl]purine 5; yellow oil (317 mg, 70%);
1
[a]D 238 (c 1.00, CHCl3); Rf ꢀ 0.55 (solvent C). H NMR (300 MHz, CDCl3):
1.13 (s, 9 H, OPiv), 1.23 (s, 9 H, OPiv), 1.27 (s, 9 H, OPiv), 1.40 (s, 9 H,
NPiv), 4.35–4.46 (m, 3 H, H-40, H-50a, H-50b), 5.58 (dd, 1 H, J ¼ 4.95 Hz,
J ¼ 2.75 Hz, H-30), 5.83 (app t, 1 H, J ¼ 6.04 Hz, J ¼ 5.77 Hz, H-20), 6.23 (d, 1
H, J ¼ 6.04 Hz, H-10), 8.15 (s, 1 H, H-8), 8.51 (br s, 1 H, NH), 8.76 (s, 1 H,
13
H-2); C NMR (300 MHz, CDCl3): 26.86, 27.00, 27.09, 27.20 ((CH3)3CCO, 20-
OPiv, 30-OPiv, 50-OPiv, NPiv), 38.67 ((CH3)3CCO, Piv), 38.73 (2 (CH3)3CCO,
Piv), 40.43 ((CH3)3CCO, Piv), 63.12 (C-50), 70.60 (C-30), 73.25 (C-20), 81.18 (C-
40), 85.92 (C-10), 122.71 (C-5), 140.83 (C-8), 149.43 (C-4), 151.48 (C-6), 152.18
(C-2), 175.68, 176.80, 177.01, 177.89 (C55O, 20-OPiv, 30-OPiv, 50-OPiv, NPiv).
Anal. Calcd. for C30H45O8N5: C, 59.68; H, 7.51; N, 11.60. Found: C, 60.03;
H, 7.82; N, 11.21.
6-Pivaloylamino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine (6). To
a solution of 4 (30 mg, 0.076 mmol) in dry pyridine (0.5 mL) pivaloyl chloride
(66 mL, 0.53 mmol) was added gradually while the mixture was stirred at
ambient temperature for 48 h. The reaction was stopped by addition of 96%
EtOH. After evaporation, as described in preparation of 2 and 3, column
chromatography (solvent D) of the residue gave as a main product 6-pivaloyla-
mino-9-[(2,3,5-tri-O-acetyl)-b-D-ribofuranosyl]purine 6; yellow oil (24 mg,
1
66%); [a]D 26.58 (c 1.24, CHCl3); Rf ꢀ 0.55 (solvent D). H NMR (600 MHz,
CDCl3): 1.40 (s, 9 H, NPiv), 2.08 (s, 3 H, OAc), 2.13 (s, 3 H, OAc), 2.16 (s, 3
H, OAc), 4.38–4.48 (m, 3 H, H-40, H-50a, H-50b), 5.66 (dd, 1 H, J ¼ 5.45 Hz,
J ¼ 4.24 Hz, H-30), 5.94 (app t, 1 H, J¼ 5.53 Hz, J ¼ 5.53 Hz, H-20), 6.24 (d, 1
H, J ¼ 5.56 Hz, H-10), 8.18 (s, 1 H, H-8), 8.59 (br s, 1 H, NH), 8.76 (s, 1 H,
13
H-2); C NMR (300 MHz, CDCl3): 20.24, 20.42, 20.65 (CH3CO, 20-OAc,

ˇ
Z. Car et al.
720
30-OAc, 50-OAc), 27.28 ((CH3)3CCO, NPiv), 40.38 ((CH3)3CCO, NPiv), 62.93 (C-
50), 70.59 (C-30), 73.01 (C-20), 80.46 (C-40), 86.22 (C-10), 123.27 (C-5), 141.11 (C-
8), 149.59 (C-4), 151.60 (C-6), 152.50 (C-2), 169.20, 169.44, 170.17 (C55O, 20-
OAc, 30-OAc, 50-OAc), 175.69 (C55O, NPiv).
Anal. Calcd. for C21H27O8N5: C, 52.82; H, 5.70; N, 14.67. Found: C, 52.74;
H, 6.07; N, 14.30.
Eluted next was some unreacted 4 (10 mg, 33%).
General Methods for Enzymic Deacylations
of Adenosine Derivatives
Lipase from porcine pancreas (PPL, 27 U/mg), butyrylcholinesterase from
equine serum (BChE, 17.6 U/mg), proteinase from Bacillus subtilis (subtilisin,
13.1 U/mg) and a-chymotrypsin from bovine pancreas (61 U/mg) were pur-
chased from Fluka/BioChemika. Esterase from porcine liver (PLE, 41 U/mg)
was obtained from Sigma. All solvents were reagent grade and distilled
before use. Column chromatography was performed on silica gel (Merck) and
TLC monitoring on Merck silica gel (60 F 254) plates (0.25 mm) with solvents
B, C, and solvent E, EtOAc-MeOH (5 : 2). Visualization was effected by use of
UV light and by charring with H2SO4. All experiments were carried out in
phosphate-buffered solutions (0.1 M, pH 7) at 378C. To improve solubility of
adenosine derivatives in this media organic solvents (Me2SO or DMF) were
added in some reaction mixtures (Table 2). Control reactions were performed
parallel to every enzymic hydrolysis and contained all reactants except the
enzyme. pH values of reaction mixtures were monitored periodically by pH-
indicator paper Neutralit (pH ¼ 5.5–9.0, Merck) and adjusted with 0.1 M
NaOH. Reactions were stopped by the addition of 96% EtOH and the
solvents evaporated under reduced pressure, if not stated otherwise.
Enzymic Deacetylations of Tetraacetate 3
Tetraacetate 3 (20 mg, 0.046 mmol) was suspended in a mixture of organic
solvent and phosphate buffer (2 mL) in all experiments except the one with
BChE where only aqueous buffered solution was used (Table 2). Enzymes
were added next. The reactions were monitored by TLC (solvent B).
Column chromatography (solvent B) of the residue with PLE gave the
regioisomeric mixture of two triacetates 7 and 8 (9.8 mg, 54%); Rf ꢀ 0.41
(solvent B) as a main product. 1H NMR (600 MHz, Me2SO): 2.04 (s, 3 H,
OAc), 2.13 (s, 3 H, OAc), 2.26 (s, 3 H, NAc), 4.24–4.40 (m, 3 H, H-40, H-50a,
H-50b), 5.05–5.08 (m, 1 H, H-20), 5.34 (dd, 1 H, J ¼ 5.34 Hz, J ¼ 3.80 Hz,
H-30), 6.03 (d, 1 H, J ¼ 6.17 Hz, H-10), 8.68 (s, 1 H, H-8), 8.70 (s, 1 H, H-2),
13
10.76 (br s, 1 H, NH); C NMR (600 MHz, Me2SO): 20.42, 20.59 (CH3CO, 30-
OAc, 50-OAc), 24.16 (CH3CO, NAc), 63.15 (C-50), 70.76 (C-20), 72.23 (C-30),
79.41 (C-40), 87.57 (C-10), 123.71 (C-5), 142.91 (C-8), 149.61 (C-4), 149.64

Acylated Adenosine Derivatives
721
(C-6), 151.67 (C-2), 168.70, 169.50, 169.96 (C55O, 30-OAc, 50-OAc, NAc) for the
6-acetylamino-9-[(3,5-di-O-acetyl)-b-D-ribofuranosyl]purine 7 and 1H NMR
(600 MHz, Me2SO): 1.98 (s, 3 H, OAc), 2.08 (s, 3 H, OAc), 2.26 (s, 3 H, NAc),
4.24–4.40 (m, 3 H, H-40, H-50a, H-50b), 4.62–4.65 (m, 1 H, H-30), 5.78 (app t,
1 H, J ¼ 5.08 Hz, J ¼ 4.98 Hz, H-20), 6.26 (d, 1 H, J ¼ 4.53 Hz, H-10), 8.66
(s, 1 H, H-8), 8.68 (s, 1 H, H-2), 10.76 (br s, 1 H, NH); 13C NMR (600 MHz,
Me2SO): 20.37, 20.46 (CH3CO, 20-OAc, 50-OAc), 23.55 (CH3CO, NAc), 63.15
(C-50), 68.41 (C-30), 74.30 (C-20), 81.73 (C-40), 85.69 (C-10), 123.60 (C-5),
142.91 (C-8), 149.61 (C-4), 149.64 (C-6), 151.67 (C-2), 168.70, 169.37, 169.92
(C55O, 20-OAc, 50-OAc, NAc) for the 6-acetylamino-9-[(2,5-di-O-acetyl)-b-D-
ribofuranosyl]purine 8.
Purification of the residue (solvent B) obtained from the incubation
mixture with BChE gave 6-acetylamino-9-[(2,3-di-O-acetyl)-b-D-ribofurano-
syl]purine 9 as a main product; white syrup[17] (16 mg, 89%); [a]D 227.68 (c
1
0.73, acetone); Rf ꢀ 0.33 (solvent B). H NMR (300 MHz, Me2SO): 1.99 (s, 3
H, OAc), 2.15 (s, 3 H, OAc), 2.26 (s, 3 H, NAc), 3.67–3.77 (m, 2 H, H-50a,
H-50b), 4.23–4.27 (m, 1 H, H-40), 5.55 (dd, 1 H, J ¼ 5.18 Hz, J ¼ 2.95 Hz,
H-30), 5.96 (app t, 1 H, J ¼ 6.00 Hz, J ¼ 5.94 Hz, H-20), 6.31 (d, 1 H,
J ¼ 6.55 Hz, H-10), 8.68 (s, 1 H, H-8), 8.72 (s, 1 H, H-2), 10.79 (br s, 1 H, NH);
13C NMR (600 MHz, Me2SO): 20.47, 20.66 (CH3CO, 20-OAc, 30-OAc), 24.18
(CH3CO, NAc), 60.80 (C-50), 70.99 (C-30), 72.49 (C-20), 83.62 (C-40), 85.01
(C-10), 123.56 (C-5), 142.51 (C-8), 149.70 (C-4), 151.45 (C-6), 152.54 (C-2),
169.07, 169.42, 169.64 (C55O, 20-OAc, 30-OAc, NAc).
Enzymic Deacetylations of Triacetate 4
Triacetate 4 (20 mg, 0.051 mmol) was suspended in phosphate buffer
(2 mL) in all experiments except the one with BChE where 4 (100 mg,
0.254 mmol) was dissolved in 5 mL of phosphate buffer. Enzymes were added
next (Table 3) and the reactions were monitored by TLC (solvents B and E).
After evaporation the residue with BChE was extracted with the mixture of
solvents EtOAc/MeOH 5 : 2 (3 ꢀ 10 mL) and combined organic extracts were
again evaporated under reduced pressure. Column chromatography of the
resulting residue (solvent B) gave white crystalline 6-amino-9-[(2,3-di-O-
acetyl)-b-D-ribofuranosyl]purine 10 as a main product; (35 mg, 39%); mp
180.3–181.28C (from acetone/pentane), lit.[26] 181–1828C; [a]D 270.88 (c
0.72, acetone), lit.[26] 278.78 (acetone); Rf ꢀ 0.32 (solvent B), 1H NMR
(300 MHz, Me2SO): 1.98 (s, 3 H, OAc), 2.14 (s, 3 H, OAc), 3.62–3.76 (m, 2 H,
H-50a, H-50b), 4.23–4.26 (m, 1 H, H-40), 5.51 (dd, 1 H, J ¼ 5.42 Hz,
J ¼ 2.43 Hz, H-30), 5.93 (dd, 1 H, J ¼ 6.96 Hz, J ¼ 5.50 Hz, H-20), 6.21 (d, 1
H, J ¼ 7.05 Hz, H-10), 7.45 (br s, 2 H, NH2), 8.16 (s, 1 H, H-8), 8.39 (s, 1 H,
13
H-2); C NMR (300 MHz, Me2SO): 20.10, 20.43 (CH3CO, 20-OAc, 30-OAc),
61.17 (C-50), 71.32 (C-30), 72.25 (C-20), 83.82 (C-40), 85.11 (C-10), 119.16 (C-5),

ˇ
Z. Car et al.
722
139.55 (C-8), 148.84 (C-4), 152.60 (C-2), 156.18 (C-6), 169.16, 169.52, (C55O, 20-
OAc, 30-OAc).
Starting unreacted compound 4 was recovered as well (52 mg, 52%).
ACKNOWLEDGMENTS
We wish to thank the Ministry of Science and Technology of the Republic of
Croatia for support of this work (project 0119401).
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