Improved synthesis and isolation of 2'-O-methyladenosine: Effective and scalable enzymatic separation of 2'/3'-O-methyladenosine regioisomers

Article abstract of DOI:10.1002/ejoc.200900348

An efficient separation of a mixture of 2'/3'-O-methyladenosine regioisomers (1 + 2: 1:1) has been developed by selective enzymatic acylation using immobilized Pseudomonas cepacia lipase (PSL-C) in combination with acetonoxime levulinate as acyl donor. Th

Full text of DOI:10.1002/ejoc.200900348

FULL PAPER
DOI: 10.1002/ejoc.200900348
Improved Synthesis and Isolation of 2Ј-O-Methyladenosine: Effective and
Scalable Enzymatic Separation of 2Ј/3Ј-O-Methyladenosine Regioisomers
Saúl Martínez-Montero,[a] Susana Fernández,[a] Tatiana Rodríguez-Pérez,[a]
Yogesh S. Sanghvi,[b] Ke Wen,[c] Vicente Gotor,*[a] and Miguel Ferrero*[a]
Keywords: Nucleosides / Enzymes / Isomers / Separation of isomers / Acylation / Green chemistry
An efficient separation of a mixture of 2Ј/3Ј-O-methyladen-
3Ј-O-methyladenosine was isolated in 81% yield and 97%
osine regioisomers (1 + 2; 1:1) has been developed by selective purity from the aqueous layer. Hydrolysis of acylated prod-
enzymatic acylation using immobilized Pseudomonas cepa-
cia lipase (PSL-C) in combination with acetonoxime levulin-
ate as acyl donor. The 3Ј-hydroxy group of 2Ј-O-methyladen-
osine (1) was acylated with high selectivity (ca. 70%),
whereas an equal amount of 3Ј-O-methyladenosine (2) in the
same solution resulted in minor acylation of 5Ј-hydroxy
group (ca. 8%). The differential behavior of both regioiso-
mers towards enzymatic acylation allowed to develop a sepa-
ration protocol. Upon extraction of the acylated products, the
ucts in organic layer furnished 2Ј-O-methyladenosine in 67%
yield and 99% purity. The separation process was success-
fully applied to the crude reaction mixture of methylated
products (ca. 3:1 of 1/2) on 5-g scale. We also report on the
use of methyl p-toluenesulfonate as a safe reagent for 2Ј-O-
methylation of adenosine.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
O-methyl nucleosides, these protocols are much less efficient
for purine analogues.[5] In the latter case, the problems de-
rive from the inherent reactivity of the purine bases towards
alkylation and the need for selective protection of the 3Ј-
and/or 5Ј-hydroxy groups.
Introduction
In recent years, several oligonucleotide-based drugs have
entered human clinical trials for the treatment of a variety
of viral, infectious or cancer-related diseases.[1] Among
these, 2Ј-O-methylribonucleotides have been extensively
used as a second-generation chemistry to elicit high nucle-
ase resistance, cellular uptake and improved binding affinity
for the RNA target.[2] Additionally, methylribonucleotides
have found applications in studying pre-mRNA splicing, ex-
amining the structures of spliceosomes and preparing nucle-
ase-resistant hammerhead ribozymes.[3] Also, 2Ј-O-alkylri-
bonucleosides are present in RNAs as minor components.[4]
The importance of methylribonucleotides, which consti-
tutes a significant portion of the raw material cost in the
preparation of oligonucleotides, has stimulated large effort
towards the synthesis of 2Ј-O-methylated nucleosides as a
key building blocks using several synthetic approaches. Al-
though some of these strategies have been successfully im-
plemented for the production of pyrimidine-containing 2Ј-
Since the late 1950s, several protocols for the synthesis
2Ј-O-methylribonucleosides have been reported. In 1965,
Broom and Robins reported the first direct synthesis of 2Ј-
O-methyladenosine (1) by methylation with diazomethane
in DME (Figure 1).[6] Alternatively, the use of methyl iodide
in combination with sodium hydride[7] or silver oxide[8] has
also been explored on nucleobase-protected ribonucleo-
sides. However, these approaches suffer from the toxicity of
the methylating reagents and their incompatibility with
large-scale applications. Moreover, the low yields of the
product obtained under these conditions and the concomi-
tant formation of the 3Ј-O-methyl isomer (2) further com-
plicates purification.
[a] Departamento de Química Orgánica e Inorgánica and Instituto
Universitario de Biotecnología de Asturias, Universidad de
Oviedo,
33006 Oviedo (Asturias), Spain
Fax: +34-98-510-3448
E-mail: vgs@uniovi.es
Figure 1. Structures of 2Ј-O-methyladenosine and 3Ј-O-methyl-
adenosine.
MFerrero@uniovi.es
[b] Rasayan Inc.,
2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA
[c] Chemistry Department, East China Normal University,
Shanghai, China
Other procedures utilized metal catalysts (stannous chlo-
ride,[9] copper,[10] iron,[11] silver, and strontium acetylacet-
onates[5d]) to activate the cis-diol system in order to sup-
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900348.
Eur. J. Org. Chem. 2009, 3265–3271
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3265

V. Gotor, M. Ferrero et al.
FULL PAPER
press methylation of the purine base. The acetylacetonates 5Ј, and 8% towards 3Ј,5Ј) and Candida antarctica lipase B
used herein are very expensive, thus hindering scale-up (CAL-B) was found to be totally regioselective towards the
attempts. Alternative approaches toward the synthesis of 2Ј- acylation of 5Ј-hydroxy group.
O-alkylribonucleosides are based on a glycosylation reac-
In order to develop a biocatalytic process for the separa-
tion[12] that requires multistep synthesis of the carbohydrate tion of a mixture of 2Ј/3Ј-O-methyladenosine, it is essential
precursors, where a mixture of α- and β-anomers was ob- to know the selectivity of the pure 3Ј-isomer 2 under enzy-
tained. Advances in protecting group methodology using matic protocols. Thus, treatment of 3Ј-O-Me-A (2) with le-
3Ј,5Ј-di-O-(tetraisopropyldisiloxane-1,3-diyl) led to the syn- vulinate oxime ester[19] in the presence of CAL-B at 45 °C
thesis of 2Ј-O-methyladenosine building block in seven in THF consumed all of the starting material in 12 h (TLC).
steps from the 6-chloropurine nucleoside.[8b] Once again, After work-up, 1H NMR spectroscopic data of the product
the foregoing methodology required expensive reagents and confirmed the formation of 5Ј-O-levulinyl-3Ј-O-methyl-
complex scale-up processes.
adenosine (3, Scheme 1) as the sole product confirming re-
Although 3Ј-O-methyladenosine (2), the side product ob- gioselective acylation of the 5Ј-hydroxy group. Nucleoside
tained in the direct alkylation protocols, has yet to find ap- 3 was isolated in 86% yield as amorphous solid. Similar
plications in oligonucleotide-based therapeutics, the 5Ј-tri- treatment using PSL-C as biocatalyst indicated that the re-
phosphates of 3Ј-O-methyladenosine and 3Ј-O-methylgua- action was much slower furnishing about 14% of 3 after
nosine are chain terminators and are thus valuable tools for 144 h.
nucleic acid sequencing.[13] In addition, Sharma[14] reported
that 3Ј-O-methyladenosine and to a lesser extent 3Ј-O-
methylguanosine are potent inhibitors of vaccinia virus
growth in -cells and Vero cells. Early viral RNA synthesis
was totally inhibited in 2.5 h post-infection adding 100 µ
of 3Ј-O-methyladenosine to infected cells.
The increasing demand for 1 as a building block for oli-
gonucleotide-based therapeutics and the interesting profile
for 2 motivated us to develop an approach where both
products could be harvested from a single process that is
environmentally safe and scalable. Herein, we describe for
the first time an economical methodology enabling the syn-
thesis and efficient separation of the isomeric mixture of 1
and 2 formed during the direct methylation of inexpensive
adenosine.
Scheme 1. Enzymatic synthesis of 5Ј-O-Lev-3Ј-O-Me-A.
Next, we embarked on the synthesis of 2Ј-O-levulinyl-3Ј-
Results and Discussion
O-methyladenosine (5) for two reasons. First, we wanted to
For the separation of isomeric and racemic mixtures, bio- prepare an authentic reference sample of 5 for comparison
catalysts have become an attractive and powerful tool in with its regio-isomer 5Ј-O-levulinyl-3Ј-O-methyladenosine
organic synthesis due to their high selectivity, mild reaction (3). This exercise will enable us to conclusively demonstrate
conditions and low environmental impact. In our research that the acylation of 3Ј-O-methyladenosine using PSL-C
group, we have developed methodologies for the separation produced only 3 and none of 5. Second, 5 would be a useful
of anomeric and racemic mixtures of nucleosides using li- building block for the preparation of oligonucleotides.
pases in organic solvents. For example, a convenient separa- Therefore, we proposed a two-step protocol for the un-
tion of α/β-mixtures of thymidine nucleosides from an in- equivocal synthesis of 5 shown in Scheme 2.
dustrial waste stream, an anomeric mixture of 2Ј-deoxy-2Ј-
Treatment of 3Ј-O-methyladenosine (2) with 5.2 equiv. of
fluoro-α/β-arabinonucleosides[15] and a parallel kinetic reso- levulinic acid (LevOH) and DCC in the presence of DMAP
lution of /-nucleosides[16] have been accomplished. One furnished 2Ј,5Ј-di-O-levulinyl-3Ј-O-methyladenosine (4) in
common theme in these examples is the selection of levuli- 78% isolated yield. The hydrolysis of 5Ј-acyl group in 4 was
nyl chemistry[17] due to: a) applications for orthogonal pro- carried out with CAL-B as catalyst due to its well-demon-
tection during the solution-phase synthesis of oligonucleo- strated selectivity with a variety of modified nucleosides.[19]
tides; b) prior experience with the lipase-mediated levulinyl- When diester 4 was treated with CAL-B at 45 °C in 0.15 
ation of nucleosides; c) inherently small, atom-efficient and phosphate buffer (pH 7) containing 18% of 1,4-dioxane, to-
inexpensive protecting group. Recently, we reported a gene- tal regioselectivity toward the hydrolysis of the 5Ј-O-levul-
ral chemoenzymatic synthesis of monolevulinyl-protected inyl group was observed, furnishing 2Ј-O-levulinyl-3Ј-O-
nucleosides using commercial lipases.[18] In this study, we methyladenosine (5) in 84% yield.
discovered that immobilized Pseudomonas cepacia lipase
With a clear understanding of the lipase-mediated acyl-
(PSL-C) was moderately regioselective towards the acyl- ation of 2 and reference samples of related products 3–5 on
ation of the 3Ј-hydroxy group of 1 (ca. 80%, 12% towards hand, the stage was set for the separation of 2Ј/3Ј-O-
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Improved Synthesis and Isolation of 2Ј-O-Methyladenosine
Scheme 2. Chemoenzymatic synthesis of 3Ј,5Ј-di-O-Lev-3Ј-O-Me-
A and 2Ј-O-Lev-3Ј-O-Me-A.
methyladenosine mixture (Scheme 3). Treatment of an equi-
molar mixture[20] of 2Ј/3Ј-O-methyladenosine with aceto-
noxime levulinate and PSL-C (type II) afforded after 6 d 3Ј-
O-Lev-2Ј-O-Me-A (6) as the major product, 5Ј-O-Lev-2Ј-
O-Me-A (7), 3Ј,5Ј-di-O-Lev-2Ј-O-Me-A (8), and 5Ј-O-Lev-
3Ј-O-Me-A (3) as minor products with unreacted 3Ј-O-Me-
A (2) (entry 1, Table 1). As we observed earlier (Scheme 1),
the 3Ј-O-Me isomer 2 reacted at a significantly slower rate
furnishing only 5Ј-acylated product 3 under these condi-
tions. Next, we altered several reaction conditions: tempera-
ture, solvent, acylation agent ratio, and different biocata-
lysts in order to increase the formation of 6 and reducing
the amount of compound 3.
The results from various experiments are summarized in
Table 1. Lowering the temperature of the reaction to 30 °C
using PSL-C, type I (a lot with 16% higher activity) the
reaction was slower than PSL-C, type II, but selectivity was
similar (entry 2, Table 1). Use of pure biocatalyst, Chromo-
bacterium viscosum lipase (CVL), neither increased the
selectivity nor the conversion (entry 3, Table 1). Increasing
the enzyme and oxime ester ratios up to 1:3 using faster
PSL-C (type I) offered shorter reaction times but lower
selectivities (entry 4, Table 1). When the acylation was per-
formed with PSL-C (type II), the best selectivity was ob-
Scheme 3. Enzymatic reaction of 1:1 mixture of 2Ј/3Ј-O-Me-A. For
experimental details see Table 1.
Table 1. Enzymatic acylation of 1:1 mixtures of 2Ј-O-Me-A (1) and
3Ј-O-Me-A (2).[a]
Entry Enzyme M/E[b] M/OE[c] T [°C] t [d] 3[d] 6[d] 7[d] 8[d]
1
PSL-C
PSL-CЈ
CVL
PSL-CЈ
PSL-C
PSL-C
PSL-C
PSL-C
1:1.5
1:1.5
1:0.25
1:3
1:3
1:3
1:1.5
1:1.5
1:1.5
1:3
1:3
1:3
55
30
55
30
30
40
30
30
6
7
7
4
5
5
6
5
12
8
66
69
71
55
72
62
71
70
11
11
12
10
11
12
5
11
12
7
19
7
9
6
7
2
3[e]
4
10
16
10
17
18
8
5
6
7[f]
8[g]
1:3
1:3
1:3
1:3
15
[a] The reactions were carried out in THF, 0.20  concentration, at
250 rpm with a 1:1 mixture of 1/2. PSL-CЈ = PSL-C type I, PSL-
C = PSL-C type II. [b] Ratio mixture 1+2/enzyme (w/w). [c] Mix-
ture 1+2/oxime ester. [d] Percentages of compounds in the crude
reaction based on HPLC peak integration. [e] After 30 h at room
temp., the temperature was increased up to 55 °C. [f] 1,4-dioxane
as solvent. [g] The starting mixture 1/2 is 3:1.
served for compound 6 (entry 5, Table 1). Increasing the we were pleased to see that the enzymatic reaction took
reaction temperature to 40 °C resulted in reduced selectivity place in a similar way as it was with the equimolar mixture
(entry 6, Table 1). Change of solvent to 1,4-dioxane pro- (entry 8, Table 1).
vided no further improvement in the formation of 6 (entry
We conclude that the reaction conditions shown in entry
7, Table 1). We selected the reaction conditions described in 5 of Table 1 are the best conditions, among the evaluated,
entry 5 as the best case for further work. Because direct to separate the isomeric mixture of 1 and 2. Upon comple-
alkylation of adenosine offers about 3:1 ratio of 2Ј/3Ј-O- tion of the reaction, the reaction mixture is extracted with
methyladenosine (1/2), we repeated this reaction using the H2O/CH2Cl2 providing unreacted 3Ј-O-Me regioisomer 2
same ratio of two products to validate the real utility of the (81% yield, 97% by HPLC)[21] in the aqueous layer. The
system. Despite of the different ratio of the two products, two 5Ј-O-levulinylated compounds 3 and 7 were easily re-
Eur. J. Org. Chem. 2009, 3265–3271
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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V. Gotor, M. Ferrero et al.
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Scheme 4. Synthesis and enzymatic separation of 2Ј-O-Me-A.
moved by flash chromatography column. The remaining 3Ј- the products were extracted with organic solvent and sub-
O-Lev-2Ј-O-Me-A (6) and 3Ј,5Ј-di-O-Lev-2Ј-O-Me-A (8) jected to a flash chromatography to remove 5Ј-O-acylated
were subjected to hydrolysis with NH4OH/MeOH[22] pro- products 3 and 7. Hydrolysis of the remaining mixture 3Ј-
viding the 2Ј-O-Me-A regioisomer (1) in 67% yield (99% O-Lev-2Ј-O-Me-A (6) and 3Ј,5Ј-di-O-Lev-2Ј-O-Me-A (8)
by HPLC) starting from the initial isomeric mixture.
with NH4OH/MeOH followed by flash chromatography
furnished 2Ј-O-Me-A (1) in 26% yield and 99% of purity
(HPLC).
Scale Up of the Process
The utility of this enzymatic separation was then applied
to a real mixture of regioisomers obtained via a direct alky-
lation process where products were used as such without
Conclusions
Herein, we have described an efficient enzymatic separa-
chromatography. Therefore, methylation of 5 g of adenosine tion of a 2Ј/3Ј-O-Me-A regioisomers mixture among other
(9, Scheme 4) was carried out using safer conditions where alkylated and dialkylated products which are usually ob-
methyl p-toluenesulfonate (pTosOMe) was employed as the tained during the alkylation reactions of adenosine. Thus,
alkylation reagent in the presence of KOH as base and using this methodology it is possible to avoid tedious purifi-
DMSO as solvent. These conditions circumvent the use of cation steps that lead to lower yields. This protocol offers
hazardous reagents such as diazomethane or methyl iodide excellent separation of both regioisomers in good yields and
and NaH traditionally used for direct alkylation procedure. high purities. PSL-C catalyzes the acylation of the 3Ј-hy-
1
The H NMR analysis of the crude product confirmed a droxy group in 2Ј-O-Me-A isomer (1) with high selectivity.
mixture of monoalkylated (2Ј- and 3Ј-O-Me-A) as major However, 3Ј-O-Me-A isomer (2) is only acylated at the 5Ј-
products and trace amount of dialkylated products. The hydroxy group with a low conversion rate. As a result, a
HPLC shows a 74:26 ratio of 2Ј/3Ј-O-Me-A, respectively. simple aqueous/organic extraction of the crude acylated
The enzymatic acylation was carried out directly on the products dissolved the polar 3Ј-O-Me-A (2) in the water
crude products under the conditions described earlier [PSL- layer and the non-polar acylated 6 and 8 were pulled in the
C (1:3, w:w), 30 °C, 3 equiv. of oxime ester, and THF as organic phase. Subsequent hydrolysis of 6 and 8 followed
solvent]. In 5 d 1 was totally converted into acylated prod- by chromatography furnished 2Ј-O-Me-A (2) in 26% yield
ucts. It is noteworthy that there was no difference in the with 99% purity (HPLC). The usefulness of this process
rate of reaction between this protocol containing minor is clearly demonstrated when the enzymatic acylation was
amount of other nucleoside related products and earlier applied to a crude mixture after direct methylation of aden-
procedure where we utilized a clean mixture of 2Ј/3Ј-O-Me- osine (9) using “green” reagents. Despite several impurities
A regioisomers. This experiment indicates that efficiency of were present in the crude reaction mixture, the overall effi-
the immobilized enzyme was not compromised despite of ciency of enzyme was not compromised. This study consti-
the presence of impurities. After completion of the reaction, tutes another fine example of how immobilized enzymes
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Eur. J. Org. Chem. 2009, 3265–3271

Improved Synthesis and Isolation of 2Ј-O-Methyladenosine
gen was added levulinic acid (170 mg, 1.45 mmol), DCC (298 mg,
1.45 mmol), and DMAP (2.8 mg, 0.02 mmol). The reaction was
stirred at room temp. for 2 h. The insoluble material was collected
by filtration and the filtrate was evaporated under vacuum. The
crude was purified by flash chromatography column (gradient el-
uents 1–3% MeOH/CH2Cl2) to give 3Ј,5Ј-di-O-levulinyl-3Ј-O-
methyladenosine (4) in 78% yield. Rf (10% MeOH/CH2Cl2) = 0.52.
1H NMR (CDCl3, 300 MHz): δ = 2.14 (s, 6 H, 2Me-Lev), 2.65 (m,
8 H, 4CH2-Lev), 3.38 (s, 3 H, OMe), 4.36 (m, 4 H, 3Ј-H+4Ј-H+5Ј-
H), 5.86 (dd, JHH = 3.1, JHH = 5.0 Hz, 1 H, 2Ј-H), 6.08 (d, JHH
= 2.9 Hz, 1 H, 1Ј-H), 6.23 (br. s, 2 H, NH2), 7.99 (s, 1 H, 2-H
or 8-H), and 8.29 (s, 1 H, 8-H or 2-H) ppm. 13C NMR (CDCl3,
100.6 MHz): δ = 27.9 (CH2-Lev), 29.9 (Me-Lev), 38.0 (CH2-Lev),
59.3 (OMe), 63.3 (C-5Ј), 74.0 (C-3Ј), 78.1, 80.1 (C-2Ј+C-4Ј), 87.4
(C-1Ј), 119.9 (C-5), 139.4 (C-2 or C-8), 149.3 (C-4), 153.0 (C-8 or
C-2), 155.8 (C-6), 171.7 (C=O), 172.4 (C=O), 206.2 (C=O), and
206.6 (C=O) ppm. MS (API+): m/z = 478 (100) [M + H]+, 380 (12),
282 (8).
can be developed as key tools in organic synthesis offering
economical processes under environmentally friendly condi-
tions.
Experimental Section
General: Pseudomonas cepacia lipase immobilized on ceramic [PSL-
C, Type I (1387 U/g) and Type II (1195 U/g) both from Amano]
were purchased from Aldrich. Chromobacterium viscosum lipase
(CVL, 4100 U/mg) was a gift from Genzyme Co. In order to pre-
pare the artificial mixture of 1 and 2, 3Ј-O-methyladenosine (2) was
purchased from R. I. Chemical Inc. (www.richemical.com). HPLC
analysis was performed at 254 nm using the following method: Me-
diterranea column (250ϫ45 mm), 0.8 mL/min flow, 20 °C, and two
mobile phases (A: MeCN; B: water); for gradient conditions see
Table 2.
3
3
3
Table 2. Details for HPLC gradient.
Enzymatic Hydrolysis of (4). Synthesis of 2Ј-O-Levulinyl-3Ј-O-meth-
yladenosine (5): To a solution of 3Ј,5Ј-di-O-levulinyl-3Ј-O-methyl-
adenosine (4) (76 mg, 0.16 mmol) in 1,4-dioxane (0.28 mL) was
added 0.15  phosphate buffer pH 7 (1.30 mL) and CAL-B
(76 mg). The stirred mixture (250 rpm) was allowed to react for
18 h at 45 °C. The enzyme was filtered off and washed with
CH2Cl2, the solvents were distilled under vacuum, and the residue
was dissolved in CH2Cl2 and washed with NaHCO3 (aq.). The
combined organic layers were dried with Na2SO4 and evaporated
to give 51 mg (84%) of 2Ј-O-levulinyl-3Ј-O-methyladenosine (5). Rf
Time [min]
A (%)
B (%)
0.0
8
92
90
80
50
30
15.0
20.0
25.0
30.0
10
20
50
70
Melting points were taken on samples in open capillary tubes and
are uncorrected. IR spectra were recorded on an Infrared Fourier
Transform spectrophotometer using KBr pellets. Flash chromatog-
raphy was performed using silica gel 60 (230–400 mesh). For rou-
tine experiments, 1H, 13C NMR, and DEPT were obtained using
(10% MeOH/CH Cl ) = 0.45; m.p. 140–142 °C. IR (KBr): ν =
˜
2
2
3236, 3125, 2928, 1745, 1713, 1691, and 1616 cm–1. 1H NMR
(CDCl3, 300 MHz): δ = 2.13 (s, 3 H, Me-Lev), 2.59 (m, 2 H, CH2-
3
Lev), 2.68 (m, 2 H, CH2-Lev), 3.45 (s, 3 H, OMe), 3.72 (d, JHH
=
1
300.13 or 400.13 MHz for H, and 75.5 or 100.61 MHz for 13C. A
3
12.0 Hz, 1 H, 5Ј-H), 4.00 (d, JHH = 12.0 Hz 1 H, 5Ј-H), 4.33 (m,
2 H, 3Ј-H + 4Ј-H), 5.78 (t, 3JHH = 6.0 Hz, 1 H, 2Ј-H), 6.01 (d, 3JHH
= 7.0 Hz, 1 H, 1Ј-HЈ), 6.39 (br. s, 2 H, NH2), 7.85 (s, 1 H, 2-H
or 8-H), and 8.26 (s, 1 H, 8-H or 2-H) ppm. 13C NMR (CDCl3,
100.6 MHz): δ = 27.6 (CH2-Lev), 29.7 (Me-Lev), 37.7 (CH2-Lev),
58.6 (OMe), 62.9 (C-5Ј), 74.7 (C-3Ј), 79.3, 85.7 (C-2Ј+C-4Ј), 88.7
(C-1Ј), 120.8 (C-5), 140.4 (C-2 or C-8), 148.6 (C-4), 152.2 (C-8 or
C-2), 155.8 (C-6), 171.6 (C=O), and 206.1 (C=O) ppm. MS
(APCI+): m/z = 380 (100) [M + H]+, 282 (10). C16H21N5O6
(379.37): calcd. C 50.66, H 5.58, N 18.46; found C 50.4, H 5.5, N
18.5.
spectrometer operating at 600.15 and 150.93 MHz for H and 13C,
1
respectively, was used for the acquisition of 1H-1H homonuclear
(COSY and NOESY) and 1H-13C heteronuclear (HSQC and
HMBC) correlations. APCI+ and ESI+ were used to record mass
spectra (MS). Microanalyses were performed on a Perkin–Elmer
model 2400 instrument.
Enzymatic Synthesis of 5Ј-O-Levulinyl-3Ј-O-methyladenosine (3): A
suspension of 3Ј-O-methyladenosine (2) (58 mg, 0.21 mmol), the
oxime ester (106 mg, 0.62 mmol), and CAL-B (58 mg) in dry THF
(1.1 mL) was stirred (250 rpm) under nitrogen for 12 h at 45 °C.
The enzyme was filtered off and washed with CH2Cl2. The solvents
were distilled under vacuum and the residue was purified by flash
chromatography column (4% MeOH/CH2Cl2) to give 67 mg (86%
yield) of 5Ј-O-levulinyl-3Ј-O-methyladenosine (3). Rf (10% MeOH/
Procedure for the Enzymatic Separation of a 1:1 Mixture of 2Ј/3Ј-
O-Methyladenosine: In a standard procedure, THF (5.1 mL) filled
into an Erlenmeyer flask that contained the isomeric mixture of 2Ј/
3Ј-O-methyladenosine (1/2) (300 mg, 1.07 mmol), oxime ester
(547 mg, 3.20 mmol) and PSL-C (900 mg). The reaction mixture
was stirred at 30 °C and 250 rpm under nitrogen and monitorized
by HPLC. The enzyme was filtered off, washed with CH2Cl2 and
MeOH, and the solvents evaporated under vacuum. The residue
was poured in H2O and extracted with CH2Cl2. The aqueous layer
was concentrated and the solid was washed with CH2Cl2 to give
121 mg (81% yield, 97% by HPLC) of 3Ј-O-methyladenosine (2).
The combined organic layers were dried with Na2SO4 and evapo-
rated, and the residue was subjected to flash chromatography col-
umn (4% MeOH/CH2Cl2) in order to remove traces of 5Ј-O-levuli-
nyl-3Ј-O-methyladenosine (3) and 5Ј-O-levulinyl-2Ј-O-methyladen-
osine (7).
CH Cl ) = 0.35; m.p. 123–125 °C. IR (KBr): ν = 3354, 3173, 2917,
˜
2
2
1711, and 1662 cm–1. H NMR (CDCl3, 300 MHz): δ = 2.17 (s, 3
1
H, Me-Lev), 2.56 (m, 2 H, CH2-Lev), 2.74 (m, 2 H, CH2-Lev), 3.53
3
(s, 3 H, OMe), 4.14 (t, JHH = 4.8 Hz, 1 H, 4Ј-H), 4.38 (m, 3 H,
3
3Ј-H+5Ј-H), 4.83 (t, JHH = 4.8 Hz, 1 H, 2Ј-H), 6.00 (m, 3 H, 1Ј-
H +NH2), 8.04 (s, 1 H, 2-H or 8-H), and 8.30 (s, 1 H, 8-H or 2-
H) ppm. 13C NMR (CDCl3, 100.6 MHz): δ = 27.7 (CH2-Lev), 29.8
(Me-Lev), 37.8 (CH2-Lev), 58.6 (OMe), 63.6 (C-5Ј), 73.5 (C-3Ј),
79.7, 80.2 (C-2Ј+C-4Ј), 89.8 (C-1Ј), 120.0 (C-5), 139.3 (C-2 or C-
8), 149.4 (C-4), 152.9 (C-8 or C-2), 155.6 (C-6), 172.5 (C=O), and
206.5 (C=O) ppm. MS (API+): m/z = 380 (100) [M + H], 282 (10).
C16H21N5O6 (379.37): calcd. C 50.66, H 5.58, N 18.46; found C
50.8, H 5.4, N 18.6.
Synthesis of 3Ј,5Ј-Di-O-Levulinyl-3Ј-O-methyladenosine (4): To a
On the other hand, the first fraction containing 3Ј-O-levulinyl-2Ј-
stirred mixture of 3Ј-O-methyladenosine (2) (80 mg, 0.28 mmol) O-methyladenosine (6) and 3Ј,5Ј-di-O-levulinyl-2Ј-O-methyladeno-
and Et3N (240 µL, 1.70 mmol) in 1,4-dioxane (2.8 mL) under nitro- sine (8) was dissolved in MeOH (9 mL) and then ammonia
Eur. J. Org. Chem. 2009, 3265–3271
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3269

V. Gotor, M. Ferrero et al.
FULL PAPER
(8.4 mL, 30% aq.) was added under continuous stirring. The reac-
tion mixture was stirred at room temp. for 2 h. After evaporation
of the solvents, the crude reaction mixture was separated by flash
column chromatography (4% MeOH/CH2Cl2) to give 100 mg (67%
yield from the initial isomeric mixture, 99% by HPLC) of 2Ј-O-
methyladenosine (1).
2Ј-O-methyladenosine (8) was dissolved in MeOH (65 mL), then
ammonia (61 mL, 30% aq.) was added under continuous stirring.
The reaction mixture was stirred at room temp. for 2 h. After evap-
oration of solvents crude reaction was subjected to flash
chromatography column (4% MeOH/CH2Cl2) to obtain 1.36 g
(26% yield, 99% by HPLC) of 2Ј-O-methyladenosine (1).
2Ј-O-Methyladenosine (1): Rf (10% MeOH/CH2Cl2) = 0.21; m.p.
Supporting Information (see also the footnote on the first page of
204–206 °C, ref.[9b] 203–204 °C or 202–203.5 °C.[9d] IR (KBr): ν =
˜
this article): General spectroscopic and experimental data are
3368, 3263, 3106, 2923, 1689, and 1611 cm–1. 1H NMR ([D4]-
1
shown. Furthermore, copies of H, 13C, and DEPT NMR spectra
3
MeOH, 300 MHz): δ = 3.42 (s, 3 H, OMe), 3.75 (dd, JHH = 2.8,
in addition of some 2D NMR experiments are included. Some
HPLC analyses are shown in support of the purity.
3
3
3JHH = 12.6 Hz, 1 H, 5Ј-H), 3.89 (dd, JHH = 2.7, JHH = 12.6 Hz,
3
3
1 H, 5Ј-H), 4.16 (q, JHH = 2.6 Hz, 1 H, 4Ј-H) 4.43 (t, JHH
5.0 Hz, 1 H, 2Ј-H), 4.49 (apparent dd, JHH = 3.1, JHH = 5.0 Hz,
1 H, 3Ј-H), 6.06 (d, JHH = 5.9 Hz, 1 H, 1Ј-H), 8.19 (s, 1 H, 2-H
=
3
3
3
Acknowledgments
or 8-H), and 8.34 (s, 1 H, 8-H or 2-H) ppm. 13C NMR ([D6]DMSO,
75.5 MHz)): δ = 57.4 (OMe), 61.4 (C-5Ј), 68.7 (C-3Ј), 82.4, 85.7,
86.3 (C-1Ј+C-2Ј+C-4Ј), 119.2 (C-5), 139.6 (C-2 or C-8), 148.9 (C-
4), 152.4 (C-8 or C-2), and 156.0 (C-6) ppm. MS (EI): m/z (%) =
281 (5) [M+], 192 (57), 164 (28), 146 (23), 135 (100). HRMS calcd.
for C11H15N5O4: 281.1119; found 281.1111.
Financial support of this work by the Ministerio de Educación y
Ciencia (MEC) (MEC-07-CTQ-2007-61126) is gratefully acknowl-
edged. S. M.-M. thanks MEC for a predoctoral grant.
[1] For selected recent reviews in this area, see: a) M. Sohail, Drug
Discovery Today 2001, 6, 1260–1261; b) N. M. Dean, Curr.
Opin. Biotechnol. 2001, 12, 622–625; c) S. T. Crooke, Oncogene
2000, 19, 6651–6659; d) D. W. Green, H. Roh, J. Pippin, J. A.
Drebin, J. Am. Coll. Surg. 2000, 191, 93–105; e) E. Koller,
W. A. Gaarde, B. P. Monia, Trends Pharmacol. Sci. 2000, 21,
142–148; f) Y. S. Sanghvi, in Comprehensive Natural Products
Chemistry (Eds.: D. H. R. Barton, K. Nakanishi), vol. 7 (Ed.:
E. T. Kool): DNA and Aspects of Molecular Biology, Pergamon
Press, New York, 1999, p. 285.
[2] Antisense Drug Technology Principles: Strategies and Applica-
tions (Ed.: S. T. Crooke), Marcel Dekker, New York, 2001.
[3] a) L. Beigelman, J. A. McSwiggen, K. G. Draper, C. Gonzalez,
K. Jensen, A. M. Karpeisky, A. S. Modak, J. Matulic-Admic,
A. B. DiRenzo, P. Haeberli, D. Sweedler, D. Tracz, S. Grimm,
F. E. Wincott, V. G. Thackray, N. Usman, J. Biol. Chem. 1995,
270, 25702–25708; b) A. I. Lamond, B. S. Sproat, U. Rider, Cell
1989, 58, 383–390; c) B. J. Blencowe, B. S. Sproat, U. Rider, S.
Barbino, A. I. Lamond, Cell 1989, 59, 531–539.
3Ј-O-Methyladenosine (2): Rf (10% MeOH/CH2Cl2) = 0.21; m.p.
175–177 °C, ref.[9b] 174–175 °C or 177–178 °C.[9d] IR (KBr): ν =
˜
3365, 3266, 3135, 2924, 1689, and 1614 cm–1. 1H NMR ([D4]-
3
MeOH, 300 MHz): δ = 3.52 (s, 3 H, OMe), 3.73 (dd, JHH = 2.7,
3
3
3JHH = 12.6 Hz, 1 H, 5Ј-H), 3.90 (dd, JHH = 2.7, JHH = 12.6 Hz,
3
3
1 H, 5Ј-H), 3.99 (dd, JHH = 2.7, JHH = 5.1 Hz, 1 H, 3Ј-H), 4.25
(q, JHH = 2.7 Hz, 1 H, 4Ј-H), 4.83 (m, 1 H, 2Ј-H), 5.4 (d, JHH
3
3
=
6.3 Hz, 1 H, 1Ј-H), 8.19 (s, 1 H 2-H or 8-H), and 8.32 (s, 1 H, 8-
H or 2-H) ppm. 13C NMR ([D6]DMSO, 75.5 MHz): δ = 57.5
(OMe), 61.5 (C-5Ј), 72.7 (C-3Ј), 79.9, 83.3, (C-2Ј+C-4Ј), 87.9 (C-
1Ј), 119.3 (C-5), 139.7 (C-2 or C-8), 148.9 (C-4), 152.3 (C-8 or C-
2), and 156.0 (C-6) ppm. MS (EI): m/z (%) = 281 (3) [M+], 266 (9),
192 (3), 178 (18), 164 (63), 148 (10), 135 (100). HRMS calcd. for
C11H15N5O4: 281.1119; found 281.1116.
3Ј-O-Levulinyl-2Ј-O-methyladenosine (6): The isolated compound
was identical with that described in ref.[18d]
[4] R. H. Hall, in: The Modified Nucleosides in Nucleic Acids, Co-
lumbia University Press, New York, 1971.
5Ј-O-Levulinyl-2Ј-O-methyladenosine (7): The isolated compound
was identical with that described in ref.[18b]
[5] For recent accounts on 2Ј-O-methylation of guanosine and
adenosine, see: a) A. R. Kore, G. Parmar, S. Reddy, Nucleosides
Nucleotides Nucleic Acids 2006, 25, 307–314; b) S. Chow, K.
Wen, Y. S. Sanghvi, E. A. Theodorakis, Bioorg. Med. Chem.
Lett. 2003, 13, 1631–1634; c) K. Wen, S. Chow, Y. S. Sanghvi,
E. A. Theodorakis, J. Org. Chem. 2002, 67, 7887–7889; d) L.
Beigelman, P. Haeberli, D. Sweedler, A. Karpeisky, Tetrahedron
2000, 56, 1047–1056.
3Ј,5Ј-di-O-Levulinyl-2Ј-O-methyladenosine (8): The isolated com-
pound was identical with that described in ref.[18d]
Methylation of Adenosine and Enzymatic Isolation of 2Ј-O-Methyl-
adenosine (1): Adenosine (9) (5 g, 18.7 mmol) was dissolved in
DMSO (75 mL). Then KOH (1.92 g, 34.2 mmol) was added under
continuous stirring. After the KOH had dissolved, pTosOMe (4.2 g,
22.5 mmol) was added and the reaction mixture was stirred at room
temp. for 12 h. Then the DMSO was evaporated under vacuum and
acetone (75 mL) was added. The solid was filtered off and the fil-
trate evaporated to furnish 5.7 g of crude product. The crude prod-
uct was placed in an Erlenmeyer flask and PSL-C (17.1 g), oxime
ester (10.39 g, 60.8 mmol), and THF (100 mL) were added. The
reaction mixture was stirred (250 rpm) at 30 °C under nitrogen at-
mosphere for 5 d. The enzyme was filtered off, washed with CH2Cl2
and MeOH, and the solvents evaporated under vacuum. The resi-
due was taken up in H2O and extracted with CH2Cl2. The aqueous
layer contained 3Ј-O-methyladenosine (2), traces of 2Ј-O-methyl-
adenosine (1), and pTosOK. The combined organic layers were
dried with Na2SO4, evaporated, and the residue was subjected to
flash chromatography column (4% MeOH/CH2Cl2) in order to re-
move traces of 5Ј-O-levulinyl-3Ј-O-methyladenosine (3) and 5Ј-O-
levulinyl-2Ј-O-methyladenosine (7). The first fraction containing
3Ј-O-levulinyl-2Ј-O-methyladenosine (6) and 3Ј,5Ј-di-O-levulinyl-
[6] A. D. Broom, R. K. Robins, J. Am. Chem. Soc. 1965, 87, 1145–
1146.
[7] a) E. Wagner, B. Oberhauser, A. Holzner, H. Brunar, G. Issak-
ides, G. Schaffner, M. Cotton, M. Knollmueller, C. R. Noe,
Nucleic Acids Res. 1991, 19, 5965–5971; b) J. Yano, L. S. Kan,
P. O. P. Ts ’o, Biochim. Biophys. Acta 1980, 629, 178–183.
[8] a) R. P. Hodge, N. D. Sinha, Tetrahedron Lett. 1995, 36, 2933–
2936; b) H. Inoue, Y. Hayase, A. Imura, S. Iwai, K. Miura, E.
Ohtsuka, Nucleic Acids Res. 1987, 15, 6131–6148.
[9] a) H. Cramer, W. Pfleiderer, Helv. Chim. Acta 1996, 79, 2114–
2136; b) T. Pathak, J. Chattopadhyaya, Chem. Scr. 1986, 26,
135–139; c) M. J. Robins, A. S. K. Lee, F. A. Norris, Carbohydr.
Res. 1975, 41, 304–307; d) M. J. Robins, S. R. Naik, A. S. K.
Lee, J. Org. Chem. 1974, 39, 1891–1899; e) M. J. Robins, S. R.
Naik, Biochim. Biophys. Acta 1971, 246, 341–343.
[10] K. Yamauchi, K. Hattori, M. Kinoshita, J. Chem. Soc. Perkin
Trans. 1 1985, 1327–1330.
[11] K. Yamauchi, T. Nakagima, M. Kinoshita, J. Org. Chem. 1980,
45, 3865–3868.
3270
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 3265–3271

Improved Synthesis and Isolation of 2Ј-O-Methyladenosine
[12]
[18] a) A. Díaz-Rodríguez, S. Fernández, Y. S. Sanghvi, M. Ferrero,
V. Gotor, Org. Process Res. Dev. 2006, 10, 581–587; b) J.
García, S. Fernández, Y. S. Sanghvi, M. Ferrero, V. Gotor, Tet-
rahedron: Asymmetry 2003, 14, 3533–3540; c) J. García, S.
Fernández, M. Ferrero, Y. S. Sanghvi, V. Gotor, Nucleosides
Nucleotides Nucleic Acids 2003, 22, 1455–1457; d) J. García, S.
Fernández, M. Ferrero, Y. S. Sanghvi, V. Gotor, J. Org. Chem.
2002, 67, 4513–4519.
a) L. Beigelman, A. Karpeisky, J. Matulic-Adamic, N. Usman,
Nucleosides Nucleotides 1995, 14, 421–424; b) B. S. Ross, R. H.
Springer, G. Vasquez, R. S. Andrews, P. D. Cook, O. L. Ace-
vedo, J. Heterocycl. Chem. 1994, 31, 765–769; c) G. Parmentier,
G. Schmitt, F. Dolle, B. Luu, Tetrahedron 1994, 50, 5361–5368;
d) C. Chavis, F. Dumont, R. H. Wightman, J. C. Zeigler, J. L.
Imbach, J. Org. Chem. 1982, 47, 202–206; e) A. H. Haines, Tet-
rahedron 1973, 29, 2807–2810.
[19] I. Lavandera, J. García, S. Fernández, M. Ferrero, V. Gotor,
Y. S. Sanghvi, in Current Protocols in Nucleic Acid Chemistry,
(Eds: S. L. Beaucage, D. E. Bergstrom, G. D. Glick, R. A.
Jones), John Wiley & Sons, New York, 2005, chapter 2, pp.
2.11.1–2.11.36.
[20] The equimolar mixture of the two nucleosides was prepared by
mixing the equal weight of 2Ј-O-methyladenosine (1) and 3Ј-
O-methyladenosine (2) with an intention of giving both nucleo-
sides an equal chance to compete for enzymatic acylation pro-
cess.
[21] Presence of about 3% of starting material 1 was confirmed
along with 97% of compound 3 in the HPLC analysis.
[22] C. B. Reese, H. Yan, Tetrahedron Lett. 2003, 44, 2501–2504.
Received: March 31, 2009
[13]
[14]
V. D. Axelrod, M. R. Vartikyan, V. A. Aivazashvili, R. Beabeal-
ashvili, Nucleic Acids Res. 1978, 15, 3549–3561.
B. B. Goswami, O. K. Sharma, J. Virol. 1983, 45, 1164–1167.
[15] J. García, A. Díaz-Rodríguez, S. Fernández, Y. S. Sanghvi, M.
Ferrero, V. Gotor, J. Org. Chem. 2006, 71, 9765–9771.
[16] J. García, A. Díaz-Rodríguez, S. Fernández, Y. S. Sanghvi, M.
Ferrero, V. Gotor, Org. Lett. 2004, 6, 9765–9771.
[17] a) For comprehensive literature, see: Current Protocols in Nu-
cleic Acid Chemistry (Eds: S. L. Beaucage, D. E. Bergstrom,
G. D. Glick, R. A. Jones), John Wiley & Sons, New York, 2007,
chapter 2; b) Greene’s Protective Groups in Organic Synthesis,
4th ed. (Eds: T. W. Green, P. G. M. Wuts), John Wiley & Sons,
New York, 2006; c) Protecting Groups, 3rd ed. (Ed.: P. J. Koci-
enski), Georg Thieme, Stuttgart, 2005.
Published Online: Mai 21, 2009
Eur. J. Org. Chem. 2009, 3265–3271
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3271