Synthesis of 8-vinyladenosine 5′-di- and 5′-triphosphate: Evaluation of the diphosphate compound on ribonucleotide reductase

Article abstract of DOI:10.1016/S0040-4020(03)01151-7

The synthesis of 5′-di- and 5′-triphosphate of 8-vinyladenosine to be tested on ribonucleotide reductases requires the modification of known methods. The phosphate group was introduced by treatment with an in situ generated chlorophosphite. Protection of the 2′,3′ diol with acetyl groups suppressed depurination during acid removal of the phosphotriester protecting groups. The di- and triphosphate compounds were obtained by treatment of the activated adenylic acid with phosphate or pyrophosphate anions followed by removal of the acetate protecting groups. Preliminary studies were conducted on Escherichia coli ribonucleotide reductase and have shown that the diphosphate compound is efficiently reduced.

Full text of DOI:10.1016/S0040-4020(03)01151-7

Tetrahedron 59 (2003) 7315–7322
Synthesis of 8-vinyladenosine 50-di- and 50-triphosphate:
evaluation of the diphosphate compound on
ribonucleotide reductase
Pascal Lang,a Catherine Gerez,b Denis Tritsch,a Marc Fontecave,b Jean-Franc¸ois Biellmanna
and Alain Burgera
,
*
a
´ ´ ´
Laboratoire de Chimie Organique Biologique associe au CNRS (UMR 7509), Faculte de Chimie, Universite Louis Pasteur,
1 rue Blaise Pascal, 67008 Strasbourg, France
Laboratoire de Chimie et de Biochimie des Centres Redox Biologiques, DRDC-CB, CEA/CNRS/Universite Joseph Fourier, (UMR 5047),
17 rue des Martyrs, 38054 Grenoble Cedex 09, France
b
´
Received 16 April 2003; revised 13 June 2003; accepted 23 July 2003
Abstract—The synthesis of 50-di- and 50-triphosphate of 8-vinyladenosine to be tested on ribonucleotide reductases requires the modi0fic0ation
of known methods. The phosphate group was introduced by treatment with an in situ generated chlorophosphite. Protection of the 2 ,3 diol
with acetyl groups suppressed depurination during acid removal of the phosphotriester protecting groups. The di- and triphosphate
compounds were obtained by treatment of the activated adenylic acid with phosphate or pyrophosphate anions followed by removal of the
acetate protecting groups. Preliminary studies were conducted on Escherichia coli ribonucleotide reductase and have shown that the
diphosphate compound is efficiently reduced.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
molecules and thus their analogues could have important
therapeutic and diagnostic applications. Nevertheless, the
preparation of di- and triphosphate of nucleoside analogues
remains a difficult task and none of the methods described is
universally satisfactory.5 Basically, their synthesis can be
classified in two approaches. One involves the displacement
of a 50-O-leaving group by a nucleophilic phosphorylating
reagent, while the other consists of reacting the 50 hydroxyl
function with an electrophilic phosphorylating reagent.
Among the large number of C-8-substituted purine nucleo-
side analogues prepared and so far screened against different
tumor cell lines and viruses, 8-vinyladenosine has shown
significant anti-tumoral and anti-viral activity.1,2 In connec-
tion with 8-vinyladenosine, we and others2,3 have proposed
that 8-vinyladenosine, after activation by the kinase to its di-
or triphosphate may act as a radical trap of the proposed C-20
radical intermediate mediated by ribonucleotide reductase.4
Trapping of the incipient radical may lead to inhibition of
this crucial enzyme in DNA replication and thus may
account for the biological activity. Evaluation of 8-vinyl-
adenosine on ribonucleotide reductases requires the
preparation of its di- and triphosphate analogues.
Initially, we applied known and conventional methods that
we expected compatible with the chemical reactivity of
8-vinyladenosine. The double bond of 8-vinyladenosine and
8-vinyl purine is electron deficient and is known to add
nucleophiles.1,2,6,7 In a first attempt, the Poulter method was
chosen.8,9 This method, based on the displacement of a
50-O-tosyl group by a di- or triphosphate reagent, has been
efficiently used for the preparation of natural di- and
triphosphate nucleosides.
2. Results and discussion
Nucleoside di- and triphosphate are ubiquitous biological
The tosyl intermediate 4 required for condensation was
prepared from the known protected 8-vinyladenosine 11 as
shown in Scheme 1. The 50-silyl protecting group of
compound 1 was selectively cleaved in 78% yield by
treatment with CF3COOH/H2O (95:5) at 08C. Introduction
of the tosyl group as described by Poulter using tosyl
chloride and 4-dimethylaminopyridine as base failed. Thus,
Keywords: di- and tri-phosphate synthesis; ribonucleotide reductase;
sensitive base.
*
´
´
Corresponding author. Present address: Departement Recepteurs et
Proteines Membranaires (UPR 9050 du CNRS), Ecole Superieure de
´
´
´
´
Biotechnologie de Strasbourg, Universite Louis Pasteur, Rue Sebastien
Brant, 67400 Illkirch, France. Tel.: þ33-3-90-24-47-19; fax: þ33-3-90-
24-47-26; e-mail: aburger@esbs.u-strasbg.fr
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)01151-7

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P. Lang et al. / Tetrahedron 59 (2003) 7315–7322
Scheme 1. First attempt to synthesize 8-vinyladenosine diphosphate.
the tosylate 3 was prepared by an alternative procedure.
Treatment of the alcohol 2 with methyllithium in THF at
low temperature and reaction with para-toluenesulfonyl
chloride afforded compound 3 in 60% yield. Removal of the
silyl protecting groups was realised in a mixture of
hydrofluoric acid (2%) in water and acetonitrile at room
temperature. The modest yield is explained by the
difficulties encountered during purification due to the low
solubility of product 4 in organic solvents. Compound 4 was
treated by tris(tetra-butylammonium) hydrogen-pyro-
phosphate in acetonitrile but no product was detected even
after two day stirring. From this first attempt, it turned out
that nucleophilic attack on C-50 of 8-vinyladenosine is
dramatically retarded, since this compound remained
unaffected while, in a control experiment, tosyladenosine
was consumed after two days. We then focused our attention
on another approach.
POCl3 in the presence of Proton Spongew was slow and
afforded four major products in addition to other minor
products; whereas, with adenosine, the reaction proceeded
to completion. Again this procedure was unsuitable for
substrate 6.
We then explored the phosphoramidite chemistry by
procedures previously developed for the phosphate of
AZT11 and modified our synthetic scheme in introducing
first the phosphate and later the vinyl group (Schemes 2
and 3).
Selective deprotection of the 50 protecting group of known
compound 81 with TFA in CH2Cl2 and water afforded the
alcohol 9 in 72% yield. When compound 9 was reacted with
Perich and Johns phosphitylating reagent,12 the reduced
product 11 was obtained in a low yield (30%) rather than the
expected iodo compound 10. Dehalogenation at C-8 was
supported by 1H NMR analysis in CDCl3, a new signal was
observed at d 8.09 ppm compatible to the chemical shift
assigned for the C-8 proton of adenosine derivatives. We
have already observed in our laboratory the reduction of an
8-iodoadenine derivative when treated with triethyl phos-
phite in acidic medium.13 Other authors have also reported
We first tried the procedure developed by Kovacs and
10
¨
¨
Otvos for the preparation of acid-sensitive nucleotides in
order to prepare the triethyl ammonium salt of the
monophosphate intermediate 7 as shown in Figure 1.
Reaction in trimethyl phosphate of 8-vinyladenosine 6 with
Figure 1. Second attempt.

P. Lang et al. / Tetrahedron 59 (2003) 7315–7322
7317
Still conditions using tributylvinyltin and a catalytic amount
of Pd(PPh3)4 at 908C.3,16 The silyl protecting groups were
cleaved with Bu4NF quantitatively. The next step was to
remove the tert-butyl phosphate protecting groups of
compound 12 but attempts with CF3COOH in water or
CH2Cl2 failed. Likely depurination reaction took place
during isolation of the adenylic acid as evidenced by
TLC and 1H NMR. Depurination in acid medium of
adenosine is well known and glycosidic bond cleavage is
even more pronounced for some 8-substituted adenosine
derivatives.17–21
We anticipated that protection of the 20,30 diol 10 with acid
resistant and electron attracting protecting groups should
retard glycosidic bond cleavage during acid removal of the
phosphate triester protecting groups.22 The silyl groups of
compound 10 were then removed quantitatively with
fluoride anion. Protection of the hydroxyl functions by
acetyl groups under standard conditions gave the diacetate
13 in 83% yield. The vinyl moiety was then introduced as
described above. Removal of the tert-butyl groups was
realised quantitatively in a mixture of TFA/H2O (95:5). The
desired compound was obtained by simple evaporation of
the volatiles and was sufficiently pure to be used for the next
step as evidenced by TLC and 1H NMR. This result showed
that protection of the 20,30-hydroxyl functions by acetyl
groups suppressed depurination during acidic treatment of
the phosphotriester.
Scheme 2. Synthesis of 8-iodoadenosine phosphotriester intermediate.
the reduction of 8-iodoadenosine derivatives with different
reagents as for example with thiourea.14 Furthermore,
oxidation of phosphite with iodine is frequently used in
oligonucleotide solid phase synthesis and its mechanism has
been described. We overcame this problem by introducing
the phosphate group on alcohol 9 in pyridine with an in situ
generated chlorophosphite.15 Under these conditions, the
phosphate triester 10 was isolated in 55% yield.
Starting from nucleoside monophosphate, many chemical
methods are available for the preparation of the correspond-
ing nucleoside di- and triphosphates.5 For this purpose,
The vinyl moiety was then introduced in 80% yield under
Scheme 3. Synthesis of 8-vinyladenosine di- and triphosphate.

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P. Lang et al. / Tetrahedron 59 (2003) 7315–7322
Hoard and Ott’s procedure was applied on phosphate 15.23
The acetyl groups were retained for monophosphate
activation with carbonyl diimidazole in order to avoid
formation of cyclocarbonate at 20 and 30.24 The stability of
the vinyl group was checked under the conditions used for
removal of the acetate protecting groups. When 8-vinyl-
adenosine was treated with ammoniac TLC and UV showed
that the vinyl group remained unaffected under these
conditions. After condensation with phosphate or pyro-
phosphate anion on the phosphorimidazolate intermediate,
the ester protecting groups were removed with ammonia as
previously described for solid phase synthesis of nucleoside
triphosphates.25,26 Finally, the sodium salts of the di- and
triphosphate of 8-vinyladenosine 5 and 16 were obtained by
ion exchange chromatography in 9 and 4% overall yield,
respectively (calculated from compound 14). The structures
of products 5 and 16 were ascertained from UV, MS, 1H and
31P NMR analyses.
not shown). Moreover, the reduction of 8-vinylADP only
occurred when dGTP was present (data not shown). These
preliminary results (Fig. 2) demonstrate that 8-vinylADP is
recognized by protein R1 substrate sites and efficiently
reduced in the presence of dGTP, which is the specific
allosteric effector that drives the reduction of the natural
substrate ADP. The presence of the vinyl group at the
8-position of adenosine thus does not strongly affect the
interaction of the corresponding nucleotide with ribonucleo-
tide reductase. Similar results were previously obtained with
the 8-azido analogue.28
3. Conclusion
In conclusion, we have described the synthesis of the
diphosphate and the triphosphate derivative of the 8-vinyl-
adenosine by modifications of known methods. This new
approach may give access to a wide range of new C-8
substituted purine phosphate derivatives because of the
versatility of the palladium catalysed reaction used for the
introduction of the functionality. Preliminary studies were
conducted with the synthesized diphosphate compound on
purified E. coli ribonucleotide reductase and have shown
that the compound is efficiently reduced. These experimen-
tal data indicate that the anti-tumoral and anti-viral activity
of 8-vinyladenosine can unlikely result from inhibition of
ribonucleotide diphosphate reductase. Therefore, the mech-
anism(s) by which 8-vinyladenosine exerts its cytotoxic
effects remains to be solved. Another possible mechanism
of action might be the inhibition of DNA synthesis at the
polymerase level by 20-deoxy-8-vinyladenosine 50-tri-
phosphate after enzymatic phosphorylation and reduction
of the adenosine analogue.
Ribonucleotide reductase catalyses the conversion of
ribonucleotides to deoxyribonucleotides and thus plays a
central role in DNA biosynthesis. In mammals, viruses and
some bacteria, such as Escherichia coli, the enzyme consists
of two subunits named R1 and R2, both required for the
activity.27 The protein R1 contains the binding sites for the
substrates which are the purine and pyrimidine ribonucleo-
side diphosphates and also binding sites for nucleoside
triphosphates which function as allosteric effectors.
Since 8-vinyladenosine has shown anti-tumoral and anti-
viral activity, we started to investigate the reaction of its
diphosphate analogue with ribonucleotide reductase. The
model for this study is the E. coli enzyme. Figure 2 shows
that in the presence of the allosteric effectors ATP and
dGTP and an electron transfer chain consisting of
thioredoxin and thioredoxin reductase, pure preparations
of R1 and R2 are able to catalyse the reduction of
8-vinylADP 5 by NADPH.
4. Experimental
4.1. General
The reaction can be monitored spectrophotometrically,
since oxidation of NADPH results in the loss of its strong
absorption at 340 nm. No oxidation of NADPH could be
detected when protein R1 or protein R2 was omitted (data
Melting points were measured on a Reichert microscope
and were uncorrected. UV spectra were determined on a
Hewlett–Packard 8451A. IR spectra were recorded on
a Brucker FT IFS25. NMR spectra were recorded on a
Brucker SY (200 or 300 MHz) apparatus. For 1H NMR the
residual proton signal of the deuteriated solvent was used as
an internal reference; for CDCl3 (d¼7.26 ppm), 2H6-DMSO
(d¼2.50 ppm). For 13C NMR, the central 13C signal of the
deuteriated solvent was used as an internal reference; for
2H6-DMSO (d¼39.46 ppm) and for CD3OD (d¼49 ppm).
For 31P NMR, the signal of H3PO4 (85%) was used as an
external reference (d¼0 ppm). The chemical shifts are
reported in ppm downfield from TMS or external reference.
MS were measured on a ZAB apparatus (Fison) by fast atom
bombardment (FAB, matrix nitrobenzylalcohol) or on a
Bio-Q quadrupole mass spectrometer (Fison) by electron
spray mass analysis. Microanalyses were performed by the
Strasbourg service of the CNRS analytical department.
Unless otherwise indicated, all reagents were obtained from
commercial suppliers and were used without purification.
All experiments sensitive to air or/and to moisture were
carried out under an argon atmosphere in an oven dried
Figure 2. Reduction of ADP (X) or 8-vinylADP (B). Assays were
performed at 378C as described in Section 4, in the presence of 1.4 mM
ATP and 0.6 mM dGTP. Units are defined as nmol of NADPH oxidized per
minute.

P. Lang et al. / Tetrahedron 59 (2003) 7315–7322
7319
(1208C) glassware assembled under a stream of argon.
Anhydrous solvents were freshly distilled before use:
tetrahydrofurane from sodium benzophenone ketyl,
pyridine from CaH2 and DMF from P2O5 (distilled under
reduced pressure). Analytical thin-layer chromatography
was performed on silica gel precoated TLC plates (Merck,
60, F254). Flash chromatography was performed on silica
gel (Merck, 60, 40–63 mesh).
4.1.3. 8-Vinyl-9-(5-O-tosyl-b-D-ribo-pentofuranosyl)-
adenine (4). Compound 3 (0.024 g, 0.036 mmol) was
suspended in acetonitrile and an aqueous solution of HF
(2%) was added (1 mL). After 24 h stirring, the reaction
mixture was neutralised by solid ammonium bicarbonate.
After evaporation to dryness, the crude product was
chromatographed over silica gel with CHCl3/MeOH (9:1)
to give product 4 in 48% yield. 1H NMR (200 MHz,
CDCl3þ5%CD3OD) d 2.33 (3H, s, CH3), 4.10–4.28 (3H,
m, H-40 and H-50), 4.48 (1H, dd, J1¼5.9 Hz, J2¼4.5 Hz,
H-30), 4.87 (1H, dd, J1¼5.0 Hz, J2¼5.9 Hz, H-20), 5.67 (1H,
dd, J1¼11.2 Hz, J2¼1.2 Hz, Holefinic), 5.85 (1H, d,
J¼5.0 Hz, H-10), 6.30 (1H, dd, J1¼17.2 Hz, J2¼1.2 Hz,
Holefinic), 6.81 (1H, dd, J1¼17.2 Hz, J2¼11.2 Hz, Holefinic),
7.25 (2H, d, J¼8.3 Hz, HAr), 7.72 (2H, d, J¼8.3 Hz, HAr),
8.16 (1H, s, H-2).
4.1.1. 8-Vinyl-9-[2,3-di-O-(tert-butyldimethylsilyl)-b-D-
ribo-pentofuranosyl]-adenine (2). Compound 1 (1.91 g;
3.0 mmol) was dissolved in a mixture of trifluoroacetic acid
and water at 08C (12 mL, 95:5). The reaction was monitored
by TLC (ether). When hydrolysis was completed, the
reaction solution was poured into a mixture of CH2Cl2 and a
saturated aqueous solution of NaHCO3 (600 mL, 1:3), and
decanted. The aqueous phase was extracted twice with
CH2Cl2 (2£150 mL). The combined organic layers were
dried (Na2SO4) and filtered. After evaporation to dryness,
the crude product was dissolved in a minimum of CHCl3
and chromatographed over silica gel with ether to give
product 2 in 78% yield. Mp¼238–2408C; 1H NMR
(200 MHz, CDCl3) d 20.62 (3H, s, Si–CH3), 20.14 (3H,
s, Si–CH3), 0.12 (3H, s, Si–CH3), 0.14 (3H, s, Si–CH3),
0.73 (9H, s, Si–C(CH3)3), 0.96 (9H, s, Si–C(CH3)3), 3.70
(1H, m, H-50), 3.93 (1H, m, H-50), 4.15 (1H, m, H-40), 4.33
(1H, d, J¼4.6 Hz, H-30), 5.07 (1H, dd, J1¼7.9 Hz,
J2¼4.6 Hz, H-20), 5.73 (2H, bs, NH2), 5.75 (1H, dd,
J1¼11.1 Hz, J2¼1.4 Hz, Holefinic), 5.98 (1H, d, J¼7.9 Hz,
H-10), 6.49 (1H, dd, J1¼17.2 Hz, J2¼1.4 Hz, Holefinic), 6.82
(1H, dd, J1¼17.2 Hz, J2¼11.1 Hz, Holefinic), 6.88 (1H, m,
OH), 8.30 (1H, s, H-2); 13C NMR, (CDCl3) d 25.5 (p),
25.4 (p), 25.3 (p), 24.50 (p), 17.8 (q), 18.1 (q), 25.9 (p),
25.8 (p), 63.1 (s), 73.9 (t), 74.1 (t), 88.7 (t), 89.5 (t), 120.3
(q), 122.9 (t), 124.9 (s), 149.4 (q), 151.4 (q), 151.9 (t), 155.5
(q); MS (FABþ) MHþ 522. Anal. calcd for C24H43IN5O4-
Si2, C 55.24, H 8.31, N 13.42; found C 55.44, H 8.46, N
13.52.
4.1.4. 8-Iodo-9-[2,3-di-O-(tert-butyldimethylsilyl)-b-D-
ribo-pentofuranosyl]-adenine (9). Compound 8 (1.5 g;
2.04 mmol) was dissolved in a mixture of trifluoroacetic
acid and water at 08C (25 mL, 95:5). The reaction was
monitored by TLC (ether/hexane, 1:1). When hydrolysis
was completed, the reaction solution was poured into a
mixture of CH2Cl2 and a saturated aqueous solution of
NaHCO3 (600 mL, 1–3) and decanted. The aqueous phase
was extracted twice with CH2Cl2 (2£150 mL). The
combined organic layers were dried (Na2SO4) and filtered.
After evaporation to dryness, the crude product was
dissolved in a minimum of CHCl3 and chromatographed
over silica gel with ether/hexane (9:1) to give product 9 in
25
72% yield. Mp¼242–2438C; [a]D ¼212 (c 0.33,
MeOH); UV (MeOH); lmax¼267 nm (1¼15500); 1H
NMR (200 MHz, CDCl3) d 20.57 (3H, s, Si–CH3),
20.14 (3H, s, Si–CH3), 0.11 (3H, s, Si–CH3), 0.14 (3H,
s, Si–CH3), 0.78 (9H, s, Si–C(CH3)3), 0.96 (9H, s, Si–
C(CH3)3), 3.69 (1H, m, H-50), 3.91 (1H, m, H-50), 4.14 (1H,
m, H-40), 4.31 (1H, bd,0 J¼4.4 Hz, H-30), 5.30 (1H, dd,
J1¼8 Hz, J2¼4.4 Hz, H-2 ), 5.92 (2H, bs, NH2), 5.96 (1H, d,
J¼8 Hz, H-10), 8.25 (1H, s, H-2); 13C NMR, (CDCl3) d
24.72 (p), 23.86 (p), 23.67 (p), 23.50 (p), 18.7 (q), 19.1
(q), 26.8 (p), 64.0 (s), 74.3 (t), 74.8 (t), 90.4 (t), 93.7 (t),
101.7 (q), 124.4 (q), 150.8 (q), 153.1 (t), 155.4 (q); HRMS
(FABþ) calcd for MHþ 622.1742, found 622.1741. Anal.
calcd for C22H40IN5O4Si2, C 42.50, H 6.49, N 11.27; found
C 42.27, H 6.50, N 11.00.
4.1.2. 8-Vinyl-9-[2,3-di-O-(tert-butyldimethylsilyl)-5-O-
tosyl-b-D-ribo-pentofuranosyl]-adenine (3). Compound
2 (0.104 g, 0.2 mmol) was dissolved in THF and cooled
down at 2788C (2.5 mL), and an ether solution of methyl-
litihum was added (0.14 mL, 0.22 mmol, 1.1 equiv.). After
15 min stirring at 2788C and further 15 min at 48C, a
solution of p-toluenesulfonyl chloride in THF (0.04 g,
0.22 mmol, 1.1 equiv.) was added. The reaction was
followed by TLC (EtOAc). The reaction mixture was
hydrolysed by water (0.1 mL), the organic phase was dried
(Na2SO4) and filtered. After evaporation to dryness, the
crude product was dissolved in a minimum of CHCl3 and
chromatographed over silica gel with ether/hexane (9:1) to
give product 3 in 60% yield. 1H NMR (200 MHz, CDCl3) d
20.45 (3H, s, Si–CH3), 20.12 (3H, s, Si–CH3), 0.13 (6H,
s, Si–CH3), 0.74 (9H, s, Si–C(CH3)3), 0.94 (9H, s, Si–
C(CH3)3), 2.41 (3H, s, CH3), 4.07–4.27 (3H, m, H-40 and
H-50), 4.46 (1H, dd, J1¼4.5 Hz, J2¼2.8 Hz, H-30), 5.30 (1H,
dd, J1¼5.9 Hz, J2¼4.5 Hz, H-20), 5.54 (2H, bs, NH2), 5.75
(1H, dd, J1¼11.0 Hz, J2¼1.5 Hz, Holefinic), 5.85 (1H, d,
J¼5.9 Hz, H-10), 6.49 (1H, dd, J1¼17.1 Hz, J2¼1.5 Hz,
Holefinic), 6.80 (1H, dd, J1¼17.1 Hz, J2¼11.0 Hz, Holefinic),
7.25 (2H, d, J¼8.1 Hz, HAr), 7.72 (2H, d, J¼8.1 Hz, HAr),
8.16 (1H, s, H-2), MS (FABþ) MHþ 676.
4.1.5. 8-Iodo-9-[2,3-di-O-(tert-butyldimethylsilyl)-50-O-
di-(tert-butoxyphosphoryl)-b-D-ribo-pentofuranosyl]-
adenine (10). Compound 9 (1 g, 1.6 mmol) was dried by
repeated co-evaporation with dry pyridine (3£5 mL).
Tris(2,4,6-tribromophenoxy)dichlorophosphorane, (BDCP,
2.81 g, 2.57 mmol) was dissolved in dry pyridine (8 mL)
and di-t-butyl phosphonate (0.32 mL, 1.93 mmol) was
added. The mixture was stirred at room temperature for
15 min and then compound 9 was added (1.0 g, 1.61 mmol).
The resulting solution was stirred at room temperature for
1 h, and then a solution of t-butyl hydroperoxide in water
(1 mL, 7.24 mmol) was added. After stirring for an
additional 4 h, the mixture was diluted with CH2Cl2,
washed with NaHCO3 and water, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The crude
product was dissolved in CHCl3 and purified by chroma-
tography over silica gel with ether/acetone (8:2) to give

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P. Lang et al. / Tetrahedron 59 (2003) 7315–7322
25
product 10 in 55% yield. Mp¼67–688C; [a]D ¼þ1 (c
H-40), 4.60 (1H, dd, J1¼J2¼5.5 Hz, H-30), 5.41 (1H, dd,
J1¼5.5 Hz, J2¼4.4 Hz, H-20), 5.94 (1H, d, J¼4.4 Hz, H-10),
8.22 (1H, s, H-2); 13C NMR (CD3OD) d 30.5 (p), 67.6 (s),
71.7 (t), 72.6 (t), 84.0 (q, d, J¼8 Hz), 84.6 (q, d, J¼6.5 Hz),
94.4 (t), 94.8 (t), 104.2 (q), 151.7 (q), 152.0 (q), 152.9 (t),
155.5 (q); HRMS (FABþ) calcd for MHþ 586.0938, found
586.0927. Anal. calcd for C18H29IN5O7P, C 36.94, H 4.99,
N 11.96; found C 36.87, H 4.97, N 11.68.
1
0.37, MeOH); UV (MeOH); lmax¼265 nm (1¼21300); H
NMR (200 MHz, CDCl3) d 20.38 (3H, s, Si–CH3), 20.08
(3H, s, Si–CH3), 0.17 (6H, s, Si–CH3), 0.79 (9H, s, Si–
C(CH3)3), 0.97 (9H, s, Si–C(CH3)3), 4.06 (1H, m, H-50),
4.40 (1H, m, H-50), 4.22 (1H, m, H-40), 4.60 (1H, dd,
J1¼4.3 Hz, J2¼2.5 Hz, H-30), 5.30 (1H, dd, J1¼6 Hz,
J2¼4.3 Hz, H-20), 5.25 (2H, bs, NH2), 5.88 (1H, d,
J¼6 Hz, H-10), 8.22 (1H, s, H-2); 13C NMR (CDCl3) d
24.2 (p), 23.6 (p), 18.8 (q), 19.0 (q), 26.7 (p), 26.8 (p), 30.7
(p, d, J¼3 Hz), 66.0 (s), 72.7 (t), 73.6 (t), 83.4 (q, d,
J¼8 Hz), 84.7 (q, d, J¼8 Hz), 93.5 (t), 102.6 (q), 124.3 (q),
151.7 (q), 153.3 (t), 155.0 (q); HRMS (FABþ) calcd for
MHþ 814.2657, found 814.2651.
To a solution of the alcohol (0.304 g, 0.52 mmol) in dry
pyridine (6 mL) was added Ac2O (0.294 mL, 3.11 mmol)
and the reaction solution was stirred for 3 h. The reaction
mixture was diluted with CH2Cl2 (50 mL) and washed with
a saturated solution of NaHCO3, dried over Na2SO4, filtered
and concentrated over reduced pressure. The crude product
was purified by chromatography over silica gel with
ether/acetone (8:2) to give compound 13 in 83% yield.
4.1.6. 9-[2,3-di-O-(tert-butyldimethylsilyl)-50-O-di-(tert-
butoxyphosphoryl)-b-D-ribo-pentofuranosyl]-adenine
(11). Compound 9 (0.6 g, 9.65 mmol) was dissolved in dry
THF (8 mL) and di-t-butyl phosphoramidate (0.587 mL,
1.93 mmol) was added and tetrazole (0.27 g, 3.86 mmol)
were added at 2208C. The reaction temperature was
allowed to reach room temperature and the resulting
solution was stirred further at this temperature for 1 h
before a solution of t-butyl hydroperoxide in water
(0.66 mL, 4.8 mmol) was added. After stirring for an
additional 2 h, the mixture was diluted with AcOEt,
quenched with Na2SO3, washed with water, dried over
Na2SO4, filtered, and evaporated under reduced pressure.
The crude product was dissolved in CHCl3 and purified by
chromatography over silica gel with ether/acetone (8:2) to
give product 11 in 30% yield. 1H NMR (200 MHz, CDCl3) d
20.24 (3H, s, Si–CH3), 20.04 (3H, s, Si–CH3), 0.11 (3H,
s, Si–CH3), 0.13 (3H, s, Si–CH3), 0.79 (9H, s, Si–
C(CH3)3), 0.93 (9H, s, Si–C(CH3)3), 4.40–4.10 (4H, m,
H-50, H-40, H-30), 5.30 (1H, dd, J1¼6 Hz, J2¼4.3 Hz, H-20),
5.64 (2H, bs, NH2), 5.97 (1H, d, J¼6 Hz, H-10), 8.09 (1H, s,
H-8), 8.33 (1H, s, H-2).
25
Mp¼74–758C, [a]D ¼þ16 (c 0.28, MeOH); UV
1
(MeOH); lmax¼266 nm (1¼23900); H NMR (200 MHz,
CDCl3) d 1.42 (9H, s, C(CH3)3, 1.45 (9H, s, C(CH3)3), 2.09
(3H, s, CH3), 2.15 (3H, s, CH3), 4.3 (3H, m, H-50, H-40), 5.90
(1H, dd, J1¼J2¼5.8 Hz, H-30), 5.95 (2H, bs, NH2), 5.98
(1H, d, J¼4.5 Hz, H-10), 6.45 (1H, dd, J¼4.5 Hz, J¼5.8 Hz,
H-20), 8.26 (1H, s, H-2); 13C NMR (CDCl3) d 21.4 (p), 21.5
(p), 30.2 30.7 (p), 65.5 (s), 70.8 (t), 71.9 (t), 81.1 (q, d,
J¼8.2 Hz), 83.7 (q, d, J¼8.2 Hz), 90.7 (t), 85.6 (t), 100.6
(q), 123.9 (q), 151.7 (q), 153.8 (t), 155.0 (q), 170.2 (q);
HRMS (FABþ) calcd for MHþ586.0938, found 586.0927.
4.1.9. 8-Vinyl-9-[2,3-di-O-(acetyl)-50-O-di-(tert-butoxy-
phosphoryl)-b-D-ribo-pentofuranosyl]-adenine
(14).
Compound 13 (0.255 g, 0.38 mmol) was dissolved in
anhydrous DMF (5 mL) and the solution was degassed.
Pd(PPh3)4 (10%, 50 mg) and vinyltributyltin (447 mL,
1.52 mmol) were added to the degassed solution under Ar.
The reaction was heated to 908C for 30 min and then the
solution was diluted with CHCl3 (10 mL) and washed with
water (3£100 mL), brine (50 mL) and dried over Na2SO4.
The crude product was purified by chromatography on silica
gel with a mixture of ether/acetone (6:4) to give product 14
4.1.7. 8-Vinyl-9-[50-O-di-(tert-butoxyphosphoryl)-b-D-
ribo-pentofuranosyl]-adenine (12). To a solution of the
8-vinyl protected adenosine derivative (0.93 g, 1.30 mmol)
in dry THF (20 mL) was added Bu4NF (2.2 equiv.) and this
mixture was stirred for 1 h. The reaction mixture was
purified by chromatography over silica gel with acetone to
give the deprotected product in 96% yield. 1H NMR
(200 MHz, CD3OD) d 1.40 (9H, s, C(CH3)3), 1.43 (9H, s,
C(CH3)3), 4.34–4.09 (3H, m, H-50, H-40), 4.57 (1H, dd,
J1¼5.6 Hz, J2¼4.2 Hz, H-30), 5.19 (1H, dd, J1¼J2¼5.4 Hz,
H-20), 5.79 (1H, dd, J1¼11.1 Hz, J2¼1.5 Hz, Holefinic), 6.05
(1H, d, J¼5.2 Hz, H-10), 6.43 (1H, dd, J1¼17.2 Hz,
J2¼1.5 Hz, Holefinic), 7.06 (1H, dd, J1¼17.2 Hz,
J2¼11.1 Hz, Holefinic), 8.16 (1H, s, H-2).
4.1.8. 8-Iodo-9-[2,3-di-O-(acetyl)-50-O-di-(tert-butoxy-
phosphoryl)-b-D-ribo-pentofuranosyl]-adenine (13). To
a solution of compound 10 (0.904 g, 1.17 mmol) in dry THF
(5 mL) was added Bu4NF (2.5 equiv.) and this mixture was
stirred for 1 h. The reaction mixture was purified by
chromatography over silica gel with acetone to give the
deprotected product quantitatively. Mp.1708C (Dec.);
25
in 80% yield. Mp.2108C (Dec.); [a]D ¼þ46 (c 0.29,
MeOH); UV (MeOH); lmax¼293 nm (1¼15100); 1H NMR
(200 MHz, CDCl3) d 1.43 (9H, s, C(CH3)3), 1.46 (9H, s,
C(CH3)3), 2.00 (3H, s, CH3), 2.13 (3H, s, CH3), 4.30 (3H, m,
H-50, H-40), 5.75 (2H, m, Holefinic, H-30), 6.11 (1H, dd,
J1¼J2¼6.15 Hz, H-20), 5.91 (2H, bs, NH2), 6.22 (1H, d,
J¼6.15 Hz, H-10), 6.44 (1H, dd, J1¼17.0 Hz, J2¼1.7 Hz,
Holefinic), 6.97 (1H, dd, J1¼17.0 Hz, J2¼12.3 Hz, Holefinic),
8.30 (1H, s, H-2); 13C NMR (CDCl3) d 20.4 (p), 20.6 (p),
29.8 (p), 65.5 (s), 70.6 (t), 72.2 (t), 81.2 (q, d, J¼9.3 Hz),
83.0 (q, d, J¼9.3 Hz), 83.8 (t), 85.6 (t), 119.3 (q), 123.6 (t),
124.9 (s), 148.8 (q), 150.9 (q), 152.8 (t), 155.1 (q), 169.3
169.6 (q); HRMS (FABþ,) calcd for MHþ 570.2328, found
570.2327.
4.1.10. 8-Vinyl-9-[2,3-di-O-(acetyl)-50-O-(phosphoryl)-b-
D-ribo-pentofuranosyl]-adenine (15). Compound 14
(0.054 g, 0.19 mmol) was dissolved at 08C in a mixture of
trifluoroacetic acid and water (5 mL, 95:5). After 10 min
stirring, the reaction was complete (TLC: i-PrOH/NH4OH/
H2O, 11:7:2) and the solvents were co-evaporated with
cyclohexane (3£10 mL). The crude product was used
25
[a]D ¼24 (c 0.33, MeOH); UV (MeOH); lmax¼265 nm
1
(1¼15600); H NMR (200 MHz, CD3OD) d 1.37 (9H, s,
C(CH3)3), 1.41 (9H, s, C(CH3)3), 4.05–4.32 (3H, m, H-50,
without purification in the next step. H NMR (400 MHz,
1

P. Lang et al. / Tetrahedron 59 (2003) 7315–7322
7321
DMSO-d6) d 2.01 (3H, s, CH3), 2.13 (3H, s, CH3), 4.03-
4.16 (2H, m, H-50), 4.38 (1H, m, H-40), 5.60 (1H, dd,
J1¼J2¼5.95 Hz, H-30), 5.75 (1H, dd, J1¼11.2 Hz,
J2¼1.9 Hz, Holefinic), 6.11 (1H, dd, J1¼J2¼5.5 Hz, H-20),
6.32 (1H, d, J¼5.25 Hz, H-10), 6.40 (1H, dd, J1¼17.0 Hz,
J2¼1.9 Hz, Holefinic), 7.17 (1H, dd, J1¼17.0 Hz,
J2¼11.2 Hz, Holefinic), 7.95 (2H, bs, NH2), 8.26 (1H, s,
H-2).
Activity of the E. coli ribonucleotide reductase was assayed
spectrophotometrically monitoring NADPH consumption at
340 nm (1340¼6220 M21 cm21).31 Standard conditions
were thioredoxin 0.16 mg/mL, thioredoxin reductase
0.028 mg/mL, DTT 0.5 mM, MgCl2 10 mM, NADPH
0.32 mM, ATP 1.4 mM, dGTP 0.6 mM, R1 0.18 mg/mL,
R2 0.038 mg/mL in Hepes buffer 35 mM pH 7.5. The
substrates were ADP and 8-vinyl ADP 5 added at
concentrations indicated in the results section. Units are
defined as nmol of NADPH oxidized per minute.
Typical procedure for the di- and triphosphate synthesis.
The crude product 15 was dissolved in pyridine (5 mL) and
tributylamine (22.8 mL, 0.095 mmol) was added, the
mixture was then co-evaporated with dry pyridine
(3£5 mL) and dry DMF (3£5 mL). Dry DMF (1.5 mL)
and carbonyldiimidazole (0.062 g, 0.38 mmol) were added,
and this mixture was stirred for 3 h before adding methanol
(11.6 mL, 0.28 mmol). After 30 min, tributylammonium
hydrogenphosphate or bis-tributylammonium hydrogen-
pyrophosphate (4 equiv.) was added and the mixture was
stirred for 21 h. The solvent was removed under vacuum at
room temperature and the crude product was chromato-
graphed on a DEAE fractogel column (8£3 cm) with a
linear gradient from 0 to 0.5 M of triethylammonium
bicarbonate (pH 7.5) at a flow rate of 30 mL/h over a 5 h
period. The appropriate fractions were collected and
co-evaporated with methanol. The acetyl groups were
removed with 10 mL of ammoniac (34% in water) and
methanol (1:1). The solvents were evaporated and the crude
product was chromatographed on a DEAE column as
described before. Triethylammonium and protons were
exchanged for sodium by passing the solution through a
Dowex AG 50W-X2 column (Naþ form) and eluting with
water. After freeze-drying of appropriate fractions, the
desired compounds were obtained.
Acknowledgements
We thank Dr. Courtney Aldrich for discussions and
corrections.
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