Stereoselective synthesis of 2-deoxy-2-fluoroarabinofuranosyl-α-1- phosphate and its application to the synthesis of 2′-Deoxy-2′- fluoroarabinofuranosyl purine nucleosides by a chemo-enzymatic method

Article abstract of DOI:10.1080/15257770903396741

Stereoselective introduction of a phosphate moiety into 2-deoxy-2-fluoroarabinofuranose derivatives at the anomeric position was investigated by two methods. One involved a stereoselective hydrolysis of 1-bromo-derivative, and the consecutive phosphorylation of 2-deoxy-2-fluoro- α-D-arabinofuranose via a phosphoramidite derivative. The other method involved stereoselective α-phosphorylation of the 1-bromo-derivative at the 1-position. The resulting α-1-phosphate was utilized to prepare 2′-deoxy-2′-fluoroarabinofuranosyl purine nucleosides by an enzymatic glycosylation reaction. This chemo-enzymatic method will be applicable to the synthesis of some 2′F-araNs, and three important 2′F-araNs were actually obtained in 30-40% yields from 1,3,5-tri-O-benzoyl-2-deoxy-2- fluoro-α-D-arabinose with high purity.

Full text of DOI:10.1080/15257770903396741

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Stereoselective Synthesis of 2-Deoxy-2-
Fluoroarabinofuranosyl-α-1-Phosphate
and Its Application to the Synthesis of
2′-Deoxy-2′-Fluoroarabinofuranosyl
Purine Nucleosides by a Chemo-
Enzymatic Method
Kohei Yamada a , Noritake Matsumoto a & Hiroyuki Hayakawa a
a Biochemicals Division, Yamasa Corporation, Choshi, Chiba, Japan
Version of record first published: 24 Nov 2009
To cite this article: Kohei Yamada, Noritake Matsumoto & Hiroyuki Hayakawa (2009): Stereoselective
Synthesis of 2-Deoxy-2-Fluoroarabinofuranosyl-α-1-Phosphate and Its Application to the Synthesis of
2′-Deoxy-2′-Fluoroarabinofuranosyl Purine Nucleosides by a Chemo-Enzymatic Method, Nucleosides,
Nucleotides and Nucleic Acids, 28:11-12, 1117-1130
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Nucleosides, Nucleotides and Nucleic Acids, 28:1117–1130, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903396741
STEREOSELECTIVE SYNTHESIS OF
2-DEOXY-2-FLUOROARABINOFURANOSYL-α-1-PHOSPHATE
AND ITS APPLICATION TO THE SYNTHESIS OF
2ꢀ-DEOXY-2ꢀ-FLUOROARABINOFURANOSYL PURINE
NUCLEOSIDES BY A CHEMO-ENZYMATIC METHOD
Kohei Yamada, Noritake Matsumoto, and Hiroyuki Hayakawa
Biochemicals Division, Yamasa Corporation, Choshi, Chiba, Japan
Stereoselective introduction of a phosphate moiety into 2-deoxy-2-fluoroarabinofuranose deriva-
2
tives at the anomeric position was investigated by two methods. One involved a stereoselective
hydrolysis of 1-bromo-derivative, and the consecutive phosphorylation of 2-deoxy-2-fluoro-α-D-
arabinofuranose via a phosphoramidite derivative. The other method involved stereoselective α-
phosphorylation of the 1-bromo-derivative at the 1-position. The resulting α-1-phosphate was utilized
to prepare 2ꢁ-deoxy-2ꢁ-fluoroarabinofuranosyl purine nucleosides by an enzymatic glycosylation
reaction. This chemo-enzymatic method will be applicable to the synthesis of some 2ꢁF-araNs, and
three important 2ꢁF-araNs were actually obtained in 30–40% yields from 1,3,5-tri-O-benzoyl-2-
deoxy-2-fluoro-α-D-arabinose with high purity.
Keywords Chemo-enzymatic synthesis; α-phosphorylation; purine nucleoside phos-
phorylase; 2ꢁ-deoxy-2ꢁ-fluoroarabinofuranosyl nucleosides
INTRODUCTION
Arabinose-derived antisense oligonucleotides (2ꢁ-deoxy-2ꢁ-fluoro-β-D-
arabinofuranose nucleic acids, 2ꢁF-ANA) have efficient gene silencing abili-
ties and potential for therapeutic use.[1] 2ꢁ-Deoxy-2ꢁ-fluoroarabinofuranosyl
nucleosides (2ꢁF-araNs) are key materials for 2ꢁF-ANA. However, there
are no efficient methods to make 2ꢁF-araNs except that 2ꢁ-deoxy-2ꢁ-
Received 24 August 2009; accepted 8 October 2009.
The authors are grateful to Prof. Dr. M.J. Damha of McGill University for helpful discussions
and providing us authentic samples. They are also grateful to Drs. T. Hamamoto, K. Okuyama, and
T. Noguchi of Yamasa Corporation for helpful discussion and providing us with NPases; and to Dr. K.
Kodama of Yamasa Corporation for helpful discussions and for supporting this work.
Address correspondence to Kohei Yamada, Biochemicals Division, Yamasa Corporation, 2-10-1
Araoicho, Choshi, Chiba 288-0056, Japan. E-mail: k yamada@yamasa.com
1117

1118
K. Yamada et al.
fluoroarabinofuranosyl pyrimidine nucleosides can be prepared chemically.
In general, it is difficult to prepare the purine 2ꢁF-araNs chemically from
natural ribo-nucleosides, because purine-base migration to the 2ꢁ-position
occurs, accompanied with an activation of the 2ꢁ-hydroxyl group during
fluorination.[2] (A facile method to introduce fluorine at the up-side of
the 2ꢁ-carbon of the 6-chloropurine riboside derivative was reported. See
ref.[2b]) Additionally, an attempt to condense 2-deoxy-2-fluoroarabinose
derivatives with purine bases failed to prevent formation of some isomers
(stereo- and positional-isomer),[3,4] in contrast to pyrimidine bases,[5] and
the separation of these isomers was difficult. Recently, two groups modified
this coupling method and its stereoselectivity was moderately improved.[6,7]
William and co-workers studied the effects of solvents and/or additives,
and the high stereoselectivity at the anomeric position was achieved in
the synthesis of the antineoplastic agent clofarabine.[8] These chemical
methods, however, cannot suppress the production of undesired isomers,
and they must be removed from the target nucleoside by various purification
steps.
Therefore, a stereoselective and regioselective construction of a β-
glycosidic linkage in the purine 2ꢁF-araNs is a challenging assignment.
We decided to investigate the application of the enzymatic glycosylation
reaction to synthesize the purine 2ꢁF-araNs. There are a few reports for
the preparation of the 2ꢁ-fluoro-nucleosides by the use of enzymes. The
nucleoside phosphorylase-catalyzed trans-glycosylation,[9] which includes
phosphorolysis and glycosylation steps, is one of the effective methods
to synthesize stereoselectively β-nucleoside analogues. Tuttle[10] had suc-
ceeded in the enzymatic trans-glycosylation to obtain an adenine derivative
from 2ꢁ-deoxy-2ꢁ-fluororibosyl thymine or an arabinosyl analog (Scheme 1).
However, this approach, consisting of two enzymatic reactions catalyzed
by thymidine phosphorylase (TPase) and purine nucleoside phosphorylase
(PNPase), would not meet our goal for a practical synthesis. Large amounts
of enzyme and a longer reaction time were required, compared with the
same reaction of natural nucleosides, to obtain acceptable yields. (Tuttle[10]
and coworkers solved this problem by using a large excess of immobilized
SCHEME 1 The nucleoside phosphorylase-catalyzed trans-glycosylation of pyrimidine and purine
nucleosides, containing a fluoro-substituent at the 2ꢁ-position.

2ꢁ-Deoxy-2ꢁ-fluoroarabinofuranosyl Purine Nucleosides
1119
enzymes.) This is because the enhanced stability of the glycosidic linkage
by fluoro-substitution at the 2-position of the sugar[3,11] would hold up the
reaction.
The rate-determining step in the two-step enzymatic reactions would
be phosphorolysis, to give α-1-phosphate. If we can make α-1-phosphate by
chemical modification in a stereoselective fashion, we are able to develop
a new chemo-enzymatic approach including the chemical phosphorylation
and the enzymatic glycosylation to synthesize 2ꢁF-araNs in an efficient
manner. This method features one enzymatic process, which is essential
to obtain the desired β-purine-nucleoside, and the α-configuration of the
1-phosphate group in the sugar moiety is critical, because a nucleoside
phosphorylase (NPase) discriminates only α-1-phosphate as a substrate.
To achieve our goal, we had to explore the stereoselective synthesis of
α-1-phosphorylated 2-fluoroarabinofuranose (1) from the corresponding
1-bromide (Scheme 2). In this article, we describe a stereoselective α-
phosphorylation at the anomeric position of the 2-fluoro-sugar and its
application to the synthesis of the purine 2ꢁF-araNs.
SCHEME 2 Stereoselective α-phosphorylation at an anomeric position.
RESULTS AND DISCUSSION
Howell[5] reported that bromination of an anomeric mixture of 1-O-
acetyl-2-deoxy-2-fluoroarabinofuranose derivative gave only the α-anomer.
This indicates that the configuration of an electronegative substituent at
the anomeric position of the 2-fluoroarabinofuranose derivative is greatly
affected by the stereo-electronic effect and the steric effect of the β-fluoro
group, in addition to an anomeric effect. Therefore, we tried to synthesize α-
1-phosphate using this property. (More recently, β-selective phosphorylation
of 3 was reported; see ref.[12]) Similar to the coupling reaction with a purine
base,[8] direct phosphorylation of 1-bromide (3)[5] with phosphoric acid
in polar solvents like CH3CN or DMF did not proceed stereoselectively
(α/β = <1) due to the coexistence of SN1 and SN2 mechanisms. On the
contrary, hydrolysis of 3 under mild basic conditions gave α-1-hydroxyl
sugar (4) exclusively (α-anomer: J 1,2 = ∼0 Hz, β-anomer: J 1,2 = 3.4 Hz)
at an 86% yield (Scheme 3). The stereochemistry was indeed confirmed by
converting 4 into the known α-1-O-benzoate (2)[5] by benzoylation of the
hydroxy group at the 1-position. Unfortunately, the direct phosphorylation

1120
K. Yamada et al.
SCHEME 3 Reagents and conditions: a) 30% HBr-AcOH, CH2Cl2; b) Et3N, H2O, DMF (86% from 2);
c) (i-Pr2N)2PCl, Et3N, CH3CN; d) 2-cyanoethanol, 1H-tetrazole; e) t-BuOOH (51% from 4); f) NH4OH,
MeOH; g) adenine, PNPase, Tris-HCl (pH = 7.0), 50◦C (50% from 5); h) 2,6-diaminopurine, PNPase,
Tris-HCl (pH = 7.0), 50◦C (58% from 5).
of 4 using POCl3 was not effective because of the immediate formation of
1-chloride, even under a low temperature. Next, we tried to introduce a
phosphate group at the 1-position using phosphoramidite-chemistry. The
reaction of 2-cyanoethyl N,N,Nꢁ,Nꢁ-tetraisopropylphosphorodiamidite with
4 catalyzed by 1H-tetrazole, followed by the substitution reaction with 2-
cyanoethanol, gave the corresponding bis(2-cyanoethyl) 1-phosphite, but
the configuration at the 1-position was shown by 31P NMR analysis to be
an approx. 1:1 mixture of anomers (138.61 and 137.91 ppm). On the
other hand, treatment of 4 with bis(diisopropylamino)chlorophosphine
under basic conditions, followed by phosphitylation and oxidation gave
protected α-1-phosphate (5) stereoselectively in 51% yield (α/β = 21).
After deprotection by NH4OH, we could obtain the desired α-1-phosphate
(1) in quantitative yield (Scheme 3). Thus, we could prepare a pure α-
1-phosphate (1). We also examined the stability of the α-1-phosphate (1)
because the enzymatic reaction of the fluorinated sugar would require a
longer reaction time compared to natural substrates. The aqueous solution
of α-1-phosphate (1) under neutral pH was allowed to stand for 1 week at
50◦C, but no decomposed products were observed by 1H NMR analysis. This
result indicated that the 1-phosphate was sufficiently stable for subsequent
enzymatic glycosylation.
Three 2ꢁF-araNs containing adenine, 2,6-diaminopurine and guanine
are key intermediates for the preparation of 2ꢁF-ANA, as described in ref.[1]
We examined the PNPase-catalyzed glycosylation reaction of α-1-phosphate
(1) with adenine and 2,6-diaminopurine except guanine, because guanine
was not soluble in the reaction media. 2,6-Diaminopurine is equivalent
to guanine after it is deaminated. Therefore, we optimized the enzymatic

2ꢁ-Deoxy-2ꢁ-fluoroarabinofuranosyl Purine Nucleosides
1121
FIGURE 1 Time-course of the enzymatic glycosylation of α-1-phosphate (1) with adenine (◦) or 2,6-
diaminopurine (•). These reactions were performed in a mixture of α-1-phosphate (1) (2.4 mM), purine
base (5.0 mM) and PNPase (15 units/ml) in Tris-HCl buffer (5.0 mM, pH 7.0) at 50◦C.
reaction using adenine and 2,6-diaminopurine. The solubility of purine
bases in aqueous media influences the yield and reaction time. Therefore,
we chose the PNPase prepared from Bacillus stearothermophilus, having a
higher optimal temperature[13] so that we could carry out the enzymatic
reaction at 50◦C. α-1-Phosphate (1) was treated with two purines at 50◦C in
the presence of the PNPase (the crude extract from the cell culture was
used for the reaction without purification of the enzyme); the reactions
were monitored by HPLC analysis for 120 hours. The conversions of two
nucleobases to the corresponding 2ꢁF-araNs were monitored by HPLC
analyses.
As shown in Figure 1, the reaction with adenine or 2,6-diaminopurine
reached to plateau after 60 hours, and the corresponding nucleosides
were formed at yields of 50% to 60% from 5. This result demonstrates
the potency of the chemo-enzymatic synthesis of 2ꢁF-araNs. (In addi-
tion to adenine and 2,6-diaminopurine, we tested the reactions with
2-aminopurine, 2-fluoroadenine, 2-amino-6-chloropurine, 6-chloropurine,
and 2,6-dichloropurine. We made sure these reactions proceeded, except
for 6-cholropurine and 2,6-dichloropurine.)
A short and simple introduction of the phosphate group into the 2-
deoxy-2-fluoroarabinofuranose derivative at an anomeric position would be
required for the practical preparation of 2ꢁF-araNs. Therefore, we examined
the direct α-phosphorylation of 1-bromide (3). As shown in Scheme 4, a
typical phosphorylation condition in CH3CN gave α,β-1-phosphate (6) at a
ratio of α/β = 0.6 at a 47% yield, and the dimer (7) was formed in 45%
yield as a byproduct. The yield of α,β-1-phosphate (6) was improved to

1122
K. Yamada et al.
SCHEME 4 Substitution reaction of 1-bromide (3) with phosphoric acid.
70% as the amount of phosphoric acid increased, but the α/β-selectivity
of phosphorylation was not changed.
Komatsu[14] has reported the isomerization reaction of the α,β-1-
phosphorylated 2-deoxyribose derivative to give α-1-phosphate. In contrast,
the isomerization reaction of α,β-1-phosphate (6) did not occur at all
because the stability of the glycosidic bond was enhanced due to fluoro-
substitution at the 2-position. (In fact, the anomeric ratio of 6 could not
be changed by the treatment with an excess of phosphoric acid.) We,
therefore, had to control the stereoselectivity in the substitution reaction
of 1-bromide (3). In general, this substitution reaction proceeds through
two mechanisms. One was a nucleophilic displacement of the 1-halogenated
sugar at the anomeric position in an SN2 mechanism;[12] the other was
the generation of an oxonium ion that was trapped subsequently by the
nucleophile.[15] In the latter reaction, it could be expected that the reaction
from the α-face was favored,[5] due to the stereo-electronic effect and
the steric effect of the 2-β-fluoro substituent. Therefore, we searched for
reaction conditions to retain the configuration at the 1-position of α-1-
bromide (3).
We first examined the effect of solvents on the reaction of 1-bromide
(3). The reaction did not proceed stereoselectively in polar or nonpolar
solvents (Table 1, entries 1–3). It is well known that the halide ion-catalyzed
Lemieuxꢁs glycosidation method proceeds in an α-selective manner. How-
ever, it is limited to pyranosides.[15] To suppress the direct SN2 reaction of
α-1-bromide, we added various tetra-n-butylammonium salts to the mixture.
The addition of tetra-n-butylammonium iodide (TBAI)[16] at room temper-
ature gave good results. As shown in Table 1, the α/β-selectivity was raised
from 0.6 to 3.2, when 5.0 equivalents of TBAI was added (entry 1 vs. 5). In
this reaction condition, SN2-type double inversion might be considered in
addition to SN1-type substitution,[5] and the phosphate anion might attack
stereoselectively the C1-carbon from the α-face.
Next, the addition of nitrate and bromide showed effects comparable to
the iodo anion, but triflate and sulfate were not effective at all. On the other
hand, α,β-1-phosphate (6) was not obtained when chloride or fluoride were
added because of the lower reactivity of plausible intermediates (1-halides)
at room temperature.

2ꢁ-Deoxy-2ꢁ-fluoroarabinofuranosyl Purine Nucleosides
1123
TABLE 1 The effect of additive for steroselective phosphorylation
Entry
Solvent
Additive (eq.) n-Bu4N·X
1-Phosphate 6 (%) α/β-ratioa
Dimer 7 (%)
1
2
3
4
5
6
7
8
9
CH3CN
CH2Cl2
DMF
-
-
-
67
77
49
67
0.6
0.7
0.8
1.3
3.2
-
1.9
2.2
2.6
0.9
0.4
9
15
8
N.D.
23
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
X = I (1.0)
X = I (5.0)
X = F (5.0)
X = Cl (5.0)
X = Br (5.0)
X = NO3 (5.0)
X = OTf (5.0)
X = HSO4 (5.0)
63
Not detectedc, d
71c
69
65
68
9
10
11
aThe ratio were estimated by HPLC analysis.
bNot determined.
cThe reaction was carried out at 70◦C.
dA β-1-fluorinated sugar was obtained (85%).
The addition of chloride worked well at an elevated temperature, but
the selectivity was low. (The reaction of 1-chloride proceeded at 70◦C to
afford it at a selectivity of α/β = 1.9.). Consequently, the iodide was the
most effective additive in terms of stereoselectivity, handling, and cost. The
resulting anomeric mixtures of 1-phosphate (1), which were obtained after
the deprotection of the benzoyl groups by NH4OH-MeOH, could be used
for the enzymatic glycosylation reaction without isolating the α-anomer.
SCHEME 5 Reagents and conditions: a) H3PO4, n-Bu3N, TBAI, CH3CN, MS4A; b) H3PO4, 2-butanone,
MS4A, 80◦C; c) NH4OH, MeOH; d) adenine, PNPase, phosphate buffer (pH = 7.0), 50◦C (X = H, 29%
from 3); e) 2,6-diaminopurine, PNPase, phosphate buffer (pH = 7.0), 50◦C (X = NH2, 39% from 3); f)
ADase, 40◦C (X = NH2, 87%).

1124
K. Yamada et al.
The chemo-enzymatic method was applied to prepare 2ꢁF-araNs con-
taining adenine and guanine moieties[17] (Scheme 5). 1-Bromide (3)
was directly phosphorylated by the method mentioned above. After the
treatment of 1-phosphate mixtures with phosphoric acid in butanone at
80◦C, (α-6 was recovered by the degradation of dimer (7) under this
acidic condition, although isomerization of β to α-6 was not observed.)
α-1-phosphate (6) was produced at a 63% yield (α/β = 3.1), which
was estimated by HPLC analysis. After the benzoyl groups were removed
by NH4OH-MeOH treatment, the crude α-1-phosphate (1) was used for
the enzymatic reaction without separation of the β-isomer. For the large
scale syntheses, the reaction conditions were slightly modified to increase
the concentration of substrates (approximately 6-fold), and decrease the
amount of PNPase (approximately 1/4∼1/9). About 15 mM of α-1 was
treated with 1.3∼2.0 eqivalents of adenine or 2,6-diaminopurine in 5 mM
KH2PO4 at 50◦C in the presence of the PNPase (10∼20 units/ml). In
both reactions, the precipitation of the purine base was observed in each
reaction mixture after 48 hours. Although these enzymatic reactions were
relatively slow to primary reaction conditions (see Figure 1), the desired
2ꢁF-araNs were obtained in similar yields after about 1 week. It should
be noted that any undesired isomers were not produced in contrast to
chemical methods,[6–8] and highly pure 2ꢁF-araNs were obtained at yields
of 30–40% from 3 by a simple purification procedure. Additionally, the
2,6-diaminopurine derivative was easily converted to 2ꢁF-araG in 87% yield
(more than 99% of purity by HPLC analysis) by the use of adenosine
deaminase-catalyzed hydrolysis at the 6-position.18
CONCLUSION
The stereoselective phosphorylation at the anomeric position of the
1-bromo-2-fluoroarabinofuranose derivative (3) was attained chemically in
two ways. Both the 1-bromo-derivative (3) and the 1-hydroxy derivative
(4) were key starting materials for the preparation of α-1-phosphate (1),
which was a good substrate for enzymatic glycosylation. We proved that
α-1-phosphate (1) was recognized as a substrate for the PNPase we se-
lected. Moreover, the addition of TBAI enabled us to substitute the 1-
bromo-group in compound (3) with phosphoric acid directly and stere-
oselectively. The chemo-enzymatic method we developed was applied to
the synthesis of the purine 2ꢁF-araNs. The quality of the desired 2ꢁF-
araNs was excellent after simple purification when it was compared
with the chemical condensation method. We believe that this chemo-
enzymatic method is most useful for the synthesis of the purine 2ꢁF-
araNs and will facilitate the development of 2ꢁF-ANA-oligonucleotides as
drugs.

2ꢁ-Deoxy-2ꢁ-fluoroarabinofuranosyl Purine Nucleosides
EXPERIMENTS
1125
Physical data were measured as follows: Melting points were determined
on a Yanagimoto MP-500D micromelting point apparatus (Yanagimoto
1
Analytical Instruments Corp., Kyoto, Japan), and were uncorrected. H
NMR spectra were recorded at 500 MHz on a Bruker AV-500 instrument
in CDCl3, DMSO-d6 or D2O as the solvent, with tetramethylsilane or 3-
(trimethylsilyl)propionic acid-d4 sodium salts as the internal standards. 31P
NMR spectra were recorded at 203 MHz on a Bruker AV-500 instrument
in CDCl3 or D2O as the solvent, with 85% H3PO4 as the external standard
(Bruker, Yokohama, Japan). Ultraviolet (UV) spectra were recorded with
a Shimadzu UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto,
Japan). Mass spectra were obtained in the fast atom bombardment (FAB)
mode on a JEOL JMS-AX500 instrument. High-resolution ESI mass spectra
were recorded on a JEOL JMS-T100LP instrument (JEOL, Tokyo, Japan).
Thin laser chromatography (TLC) was carried out on Merck precoated
plates (Kieselgel 60F254). Purine nucleoside phosphorylase was prepared in
the laboratory of Yamasa Corp. (Chosi, Japan). The activity of the enzyme
was measured by the method of the literature.[13] One unit of the enzyme
was defined as the amount of enzyme forming a µmol of adenine per minute
under the reaction condition. Adenosine deaminase (from calf intestine)
was purchased from Roche Diagnostics Corp. (Manheim, Germany), and
used without further purification.
2-Deoxy-2-fluoro-3,5-di-O-benzoyl-α-D-arabinofuranose (4)
To a solution of 1-bromide (3) (387 mg, 0.91 mmol) in DMF (15
mL), Et3N (0.76 mL, 0.71 mmol) and H2O (0.5 mL) were added, and
the mixture was stirred at room temperature for 30 minutes. Solvents were
removed under reduced pressure, and the residue was purified by column
chromatography over silica gel (2.3 × 14 cm, CHCl3) to give 4 (282 mg,
1
86%) as a colorless oil: H NMR (CDCl3) δ 8.08–8.01 (4H, m), 7.63–7.41
(6H, m), 5.70 (1H, dd, J = 3.6 and 10.2 Hz), 5.50 (1H, dd, J = 4.3 and 22.0
Hz), 5.18 (1H, d, J = 49.1 Hz), 4.77–4.60 (3H, m), 2.89 (1H, t, J = 3.4 Hz);
FAB MS (m/z) 361 (M++H). Anal. Calcd for C19H17FO6: C, 63.33; H, 4.76.
Found: C, 63.42; H, 4.72.
Bis(2-cyanoethyl)-2-deoxy-2-fluoro-3,5-di-O-benzoyl-α-
D-arabinofuranosyl-1-phosphate (5)
To a solution of 1-hydroxysugar (4) (1.01 g, 2.8 mmol) in acetonitrile
(20 mL), Et3N (1.2 mL, 8.4 mmol) and (i-Pr2N)2PCl (1.69 g, 5.6 mmol)
were added. After 1 hour of stirring at room temperature, 2-cyanoethanol
(1.9 mL, 28.0 mmol) and 1H-tetrazole (981 mg, 14.0 mmol) were added

1126
K. Yamada et al.
to the mixture. After 1.5 hours of stirring at room temperature, 70%
t-BuOOH (2.5 ml) was added to the mixture and stirred for 30 minutes. The
mixture was extracted with AcOEt, and the organic phase was washed with
water (twice), saturated NaHCO3 and brine, and then dried (Na2SO4). The
solvent was removed under reduced pressure, and the residue was purified
by column chromatography over silica gel (3.6 × 16 cm, 50–100% AcOEt
1
in hexane) to give 5 (784 mg, 51%) as a colorless oil: H NMR (CDCl3) δ
8.07–8.05 (4H, m), 7.65–7.43 (6H, m), 6.14 (1H, dd, J = 4.2 and 8.3 Hz),
5.56 (1H, dd, J = 3.9 and 20.9 Hz), 5.34 (1H, d, J = 48.4 Hz), 4.82 (1H,
q, J = 3.9 Hz), 4.78 (1H, dd, J = 3.5 and 12.2 Hz), 4.68 (1H, dd, J = 4.6
and 12.2 Hz), 4.32–4.27 (4H, m), 2.78–2.68 (4H, m); 31P NMR (CDCl3) δ
-3.73; FAB MS (m/z) 343 (M-OPO(OCE)2). Anal. Calcd for C25H24FN2O9P:
C, 54.95; H, 4.43; N, 5.13. Found: C, 54.97; H, 4.59; N, 4.98.
Disodium (2-deoxy-2-fluoro-α-D-arabinofuranosyl)-1-phosphate
(1)
To a solution of 1-phosphate ester (5) (590 mg, 1.08 mmol) in MeOH-
THF (1:1, 6 mL), 28% NH4OH (6 mL) was added. After 1 h of stirring
at room temperature, the solvent was removed under reduced pressure.
Then 28% NH4OH (10 mL) was added to the residue and the mixture was
stirred at room temperature overnight. After concentration, the residue was
dissolved in water (20 mL) and washed with AcOEt (20 mL). The solvent
was removed under reduced pressure and the residue was dissolved in 100
mL of water. The solution was passed through an ion-exchange resin (30
mL, PK 216 Na form, Mitsubishi Chemical Corp., Tokyo, Japan) to give a
1
sodium salt of 1 (295 mg, 99%) as an amorphous foam: H NMR (D2O)
δ 5.70 (1H, dd, J = 6.9 and 9.8 Hz), 5.02 (1H, d, J = 50.5 Hz), 4.26–4.17
(2H, m), 3.87 (1H, dd, J = 3.2 and 12.4 Hz), 3.74 (1H, dd, J = 5.3 and 12.4
Hz); 13C NMR (D2O) δ 103.65 (dd, J = 7.1 and 180.1 Hz), 103.17 (dd, J =
3.7 and 36.7 Hz), 87.51 (d, J = 2.5 Hz), 77.73 (d, J = 27.1 Hz), 63.94; 31P
NMR (D2O) δ 2.19; FAB MS (m/z) 277 (M++H); HR-ESIMS (m/z). Calcd
for [C5H9FO7P]−:231.0070; found: 231.0062.
2-Deoxy-3,5-di-O-benzoyl-2-fluoro-α(and
β)-D-arabinofuranosyl-1-phosphate (6)
To a solution of 1.0 M tri-n-butylammonium phosphate in acetonitrile
(0.75 mL, 0.75 mmol), MS4A(50 mg) and various tetra-n-butylammonium
salts (shown in Table 1) were added. After 40 minutes of stirring at room
temperature, a solution of 1-bromide 3 (50 mg, 0.12 mmol) in acetonitrile
(0.5 mL) was added to the mixture at 0◦C or room temperature. The α,β-
ratio of reactions shown in Table 1 were determined by HPLC analysis under
the following conditions: detector, UV 230 nm/column, Shiseido CAPCELL

2ꢁ-Deoxy-2ꢁ-fluoroarabinofuranosyl Purine Nucleosides
1127
PAK NH2 (4.6 mm × 250 mm; temperature, 30◦C; mobile phase, a mixture
of 120 mM KH2PO4 (50%) and acetonitrile (50%); pH 4; flow rate, 1.0
mL/min). An analytical sample of αβ-6 (α/β = 1.5) was obtained as a 2/3
tri-n-butylammonium salt via purification by ODS column chromatography:
1H NMR (CDCl3) δ 8.09–7.98 (4H, m), 7.56–7.30 (6H, m), 6.03–6.00 (1H,
m), 5.79 (0.4H, dt, J = 4.8 and 16.3 Hz), 5.47 (0.6H, dd, J = 4.2 and 22.1
Hz), 5.40 (0.6H, d, J = 49.1 Hz), 5.15 (0.4H, br d, J = 52.1 Hz), 4.75–4.59
(2.6H, m), 4.34 (0.4H, q, J = 5.5 Hz), 2.91–2.87 (6H, m), 1.68–1.62 (4H, m),
1.36–1.29 (4H, m), 0.90 (6H, t, J = 7.3 Hz); 31P NMR (CDCl3) δ 0.33, -0.35;
HR-ESIMS (m/z). Calcd for [C19H17FO9P]−: 439.0594; found 439.0583.
9-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (2ꢁF-araA)
To the suspension of o-phosphoric acid (2.8 g, 28.3 mmol) and MS4A
(2.0 g) in acetonitrile (10 mL), tri-n-butylamine (13.4 mL, 56.4 mmol)
was added at 0◦C and the mixture was stirred at room temperature for 1
hour. After the addition of TBAI (8.7 g, 23.5 mmol), a solution of 3 (2.0
g, 4.7 mmol) in acetonitrile (20 mL) was added dropwise to the mixture
at room temperature. After 2 hours of stirring at room temperature, the
filtrate was concentrated under reduced pressure. The residue was extracted
with AcOEt (150 mL), and the organic phase was washed with 0.4% HCl
(three times) and dried (Na2SO4). After the solvent was removed under
reduced pressure, the residue was dissolved in 2-butanone (50 mL), and
o-phosphoric acid (7.4 g) and MS4A (2.0 g) were added to the solution.
After 2 hours of stirring at 80◦C, the filtrate was concentrated under reduced
pressure. The residue was extracted with AcOEt (150 mL), and the organic
phase was washed with water and dried using Na2SO4. The solvent was
removed under reduced pressure, the residue was dissolved in methanol
(40 mL) and 28% NH4OH (40 mL) was added to the mixture and then
stirred at room temperature overnight. After concentration, the residue was
dissolved in water (50 mL) and washed with AcOEt (200 mL × 2), and the
water phase was concentrated to give crude 1.
To the solution of 1 in 20 mM KH2PO4 (200 mL, pH 7.6), adenine
(540 mg, 4.0 mmol) and PNPase (2000 units) were added. The mixture was
kept at 50◦C for 144 hours. After dilution, the solution was passed through
a membrane filter (0.45 µm) and applied to reverse-phase ODS column
chromatography (400 mL). After it was washed with 2000 mL of water,
the eluate of 3% aqueous CH3CN was collected and concentrated under
reduced pressure to leave crystalline 2ꢁF-araA (362 mg, 29% from 3): m.p.
232–233◦C (lit.[17a] 232–234◦C); UV (H2O) λmax 258.5 nm (ε = 15 300);1H
NMR (DMSO-d6): δ 8.26 (1H, d, J = 1.7 Hz), 8.17 (1H, s), 7.36 (2H, br
s), 6.41 (1H, dd, J = 4.6 and 14.1 Hz), 6.15 (1H, br), 5.25 (1H, br), 5.22
(1H, dt, J = 4.3 and 52.7 Hz), 4.47 (1H, dt, J = 4.5 and 19.4 Hz), 3.86–3.84
(1H, m), 3.68–3.63 (2H, m); FAB MS (m/z) 270 (M++H). Anal. Calcd for

1128
K. Yamada et al.
C10H12FN5O3·0.3H2O: C, 43.73; H, 4.62; N, 25.50. Found: C, 43.63; H, 4.46;
N, 25.48.
9-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)guanine (2ꢁF-araG)
To the suspension of o-phosphoric acid (12.7 g, 130 mmol) and MS4A
(10 g) in acetonitrile (49 mL), tri-n-butylamine (61.8 mL, 258 mmol) was
added at 0◦C and the mixture was stirred at room temperature for 1 hour.
After the addition of TBAI (39.9 g, 108 mmol), a solution of 3 (10.3 g,
21.5 mmol) in acetonitrile (85 mL) was added dropwise to the mixture
at room temperature. After 2 hours of stirring at room temperature, the
filtrate was concentrated under reduced pressure. The residue was extracted
with AcOEt (400 mL), and the organic phase was washed with water (400
mL) and dried (Na2SO4). The solvent was removed under reduced pressure
and the precipitated material was removed by suction. After the filtrate
was concentrated, the residue was washed with hexane (15 mL × 5) and
dried under reduced pressure, to give crude 1-phosphate (5). The crude
5 was dissolved in 2-butanone (255 mL) and o-phosphoric acid (34.0 g)
and MS4A (10 g) were added to the solution. After 2 hours of stirring at
80◦C, the filtrate was concentrated under reduced pressure. The residue
was extracted with AcOEt (400 mL), and the organic phase was washed
with water (400 mL) and dried (Na2SO4). The solvent was removed under
reduced pressure, the residue was dissolved in methanol (200 mL) and
28% NH4OH (200 mL) was added to the mixture and then stirred at room
temperature overnight. After concentration, the residue was dissolved in
water (200 mL) and washed with AcOEt (250 mL × 2), and the water phase
was concentrated to give crude 1.
To the solution of 1 in 5 mM KH2PO4 (1 L, pH 7.6), 2,6-diaminopurine
(4.05 g, 27.0 mmol) and PNPase (23460 units) were added. The mixture was
kept at 50◦C for 190 hours. After dilution, the solution was passed through
a membrane filter (0.45 µm) and applied to a column of adsorption
resin (350 mL, Sepabeads SP 206, Mitsubishi Chemical Corp.). After it
was washed with 3 L of water, the eluate of 1–15% aqueous EtOH was
collected and concentrated under reduced pressure to give 2ꢁF-araDAP[19]
as a water solution. (The analytical sample was isolated and the structure was
confirmed by its spectroscopic analyses. See ref.[19]).
To the solution of 2ꢁF-araDAP in 10 mM KH2PO4 (1 L, pH 7.0),
adenosine deaminase (Roche, 1500 units) were added. The mixture was
kept at 40◦C for 2 hours. The solution was passed through a membrane filter
(0.45 µm) and applied to a column of adsorption resin (500 mL, Sepabeads
SP 206, Mitsubishi Chemical Corp.). After it was washed with 3 L of water,
the eluate of 1–10% aqueous EtOH was collected and concentrated under
reduced pressure to leave crystalline 2ꢁF-araG (2.09 g, 34% from 3): m.p.
257–259◦C (lit.[17b] 250—251◦C); UV (H2O) λmax 251.9 nm (ε = 13 300);

2ꢁ-Deoxy-2ꢁ-fluoroarabinofuranosyl Purine Nucleosides
1129
1H NMR (DMSO-d6): δ 10.50 (1H, br s), 7.80 (1H, s), 6.54 (2H, br s), 6.13
(1H, dd, J = 4.2 and 16.0 Hz), 5.94 (1H, d, J = 4.5Hz), 5.11 (1H, dt, J =
3.8 and 52.4 Hz), 5.08 (1H, t, J = 5.7 Hz), 4.37 (1H, dd, J = 3.9 and 17.8
Hz), 3.82–3.79 (1H, m), 3.66–3.57 (2H, m); FAB MS (m/z) 286 (M++H).
Anal. Calcd for C10H12FN5O4·0.3H2O: C, 41.33; H, 4.37; N, 24.10. Found:
C, 41.57; H, 4.33; N, 23.69.
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