Synthesis of adenophostin A and congeners modified at glucose

Article abstract of DOI:10.1039/b001386m

A convergent route is described to the super-potent lo-wyo-inositol 1,4,5-trisphosphate receptor agonist adenophostin A (2) and analogues 5 and 7, in which the glucose bisphosphate unit is replaced by corresponding xylose bisphosphate and mannose bisphosphate units respectively. Adenosine was converted into its 2′,3′-O-p-methoxybenzylidene derivative 8ab, which was selectively N⁶-dimethoxytritylated by a transient protection method. 5′-O-Benzylation followed by reductive acetal cleavage gave, after separation from its 3′-O-p-methoxybenzyl isomer, the versatile glycosyl acceptor 5′-O-benzyl-N ⁶-dimethoxytrityl-2′-O-p-methoxybenzyladenosine 13. Coupling of 13 with selectively protected glucopyranosyl, xylopyranosyl or mannopyranosyl dimethyl phosphites gave the required 3′-O-α-pyranosyl adenosine derivatives. Acidic hydrolysis gave corresponding N⁶-unprotected triols which were phosphitylated using bis(benzyloxy)(diisopropylamino)phosphine and imidazolium Inflate without further N⁶-protection. Deprotection gave the target trisphosphates 2,5 and 7. Synthetic adenophostin A (2) was identical with a sample of natural material in all respects. Analogues 5 and 7 will be useful for structure-activity studies on the adenophostins. The Royal Society of Chemistry 2000.

Full text of DOI:10.1039/b001386m

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Synthesis of adenophostin A and congeners modified at glucose
Rachel D. Marwood, Andrew M. Riley, David J. Jenkins and Barry V. L. Potter*
Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology,
University of Bath, Claverton Down, Bath, UK BA2 7AY
1
Received (in Cambridge, UK) 21st February 2000, Accepted 19th April 2000
Published on the Web 25th May 2000
A convergent route is described to the super-potent 1-myo-inositol 1,4,5-trisphosphate receptor agonist
adenophostin A (2) and analogues 5 and 7, in which the glucose bisphosphate unit is replaced by corresponding
xylose bisphosphate and mannose bisphosphate units respectively. Adenosine was converted into its 2Ј,3Ј-O-p-
methoxybenzylidene derivative 8ab, which was selectively N6-dimethoxytritylated by a transient protection method.
5Ј-O-Benzylation followed by reductive acetal cleavage gave, after separation from its 3Ј-O-p-methoxybenzyl isomer,
the versatile glycosyl acceptor 5Ј-O-benzyl-N6-dimethoxytrityl-2Ј-O-p-methoxybenzyladenosine 13. Coupling of 13
with selectively protected glucopyranosyl, xylopyranosyl or mannopyranosyl dimethyl phosphites gave the required
3Ј-O-α-pyranosyl adenosine derivatives. Acidic hydrolysis gave corresponding N6-unprotected triols which were
phosphitylated using bis(benzyloxy)(diisopropylamino)phosphine and imidazolium triflate without further N6-
protection. Deprotection gave the target trisphosphates 2, 5 and 7. Synthetic adenophostin A (2) was identical
with a sample of natural material in all respects. Analogues 5 and 7 will be useful for structure–activity studies on
the adenophostins.
The isolation3 and initial characterisation4,5 of the natural
Introduction
glyconucleotides adenophostins A (2) and B (3) in 1993 was an
extremely important development; 2 and 3 are by far the most
potent Ins(1,4,5)P3 receptor agonists yet identified. They were
reported to elicit Ca2ϩ-release from cerebellar microsomes with
potencies 100-fold higher than Ins(1,4,5)P3,5 and subsequent
studies have demonstrated potencies 10–100 fold greater than
Ins(1,4,5)P3 in other cell types.6,7 The adenophostins are
now finding widespread use as pharmacological tools for the
investigation of cell signalling mechanisms,8 and a potential
application in parthenogenetic oocyte activation in the bio-
technology of animal reproduction has been proposed.9
In 1983 1-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3, 1]
(Fig. 1) was identified as a second messenger responsible for
increasing intracellular Ca2ϩ concentration in stimulated cells
and its role has now been well characterised.1 Ins(1,4,5)P3 inter-
acts with a tetrameric receptor situated in the lipid bilayer of
the endoplasmic reticulum, and the synthesis and biological
evaluation of numerous Ins(1,4,5)P3 analogues have led to a
good understanding of structure–activity relationships at this
receptor.2
The extraordinary activity of the adenophostins was
unexpected, as their structures bear little obvious resemblance
to Ins(1,4,5)P3. Nevertheless, it is clear that the 3Љ,4Љ-bis-
phosphate of 2 and 3 corresponds stereochemically to the crit-
ical 4,5-bisphosphate of Ins(1,4,5)P3, and all three molecules
contain a third phosphate, which enhances affinity for the
receptor.2,5 Several Ins(1,4,5)P3 mimics based on the adeno-
phostins have been prepared,7,10–14 and some structure–activity
relationships have been elucidated. Importantly, it appears that,
for potency to exceed that of Ins(1,4,5)P3, a base (or base surro-
gate) is necessary. We have recently synthesised base-modified
adenophostin analogues14 to explore this requirement.
As part of our continuing research programme investigating
structure–activity relationships of 1 and 2, a route was required
to 2 which would also allow the synthesis of analogues 5 and 7
in which the glucopyranosyl bisphosphate moiety is replaced
with xylopyranosyl and mannopyranosyl bisphosphate units
respectively. Little attention has so far been directed at modifi-
cations to the glucopyranosyl component of the adenophostins,
and 5 and 7 were considered suitable target molecules with
which to begin studies in this area. Assuming that, at the
Ins(1,4,5)P3 receptor binding site, the 5Љ-hydroxymethyl and
2Љ-hydroxy groups of adenophostin A mimic the 3- and 6-OH
groups of Ins(1,4,5)P3 respectively, then 5 can be seen as struc-
turally analogous to 3-deoxy-Ins(1,4,5)P3 (4),15 while 7 is related
to Ins(1,3,6)P3 (6),16 in which the equivalent of the equatorial
6-OH group in Ins(1,4,5)P3 is changed to axial. Both 4 and 6
may interact in novel ways with Ins(1,4,5)P3 receptors as a
Fig. 1 Relationship of 1-myo-inositol 1,4,5-trisphosphate (1) to
adenophostins A (2) and B (3), and of inositol trisphosphates 4 and 6 to
target compounds 5 and 7.
DOI: 10.1039/b001386m
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947
This journal is © The Royal Society of Chemistry 2000
1935

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Scheme 1 Reagents and conditions: i) p-methoxybenzaldehyde, (EtO)3CH, PTSA, 35–40 ЊC, 4 h (90%); ii) (a) Me3SiCl, pyridine, room temp., 2 h;
(b) DMTrCl, room temp., 16 h; (c) conc. aq. NH3, room temp., 30 min (97%); iii) KOH, BnCl, benzene–dioxane (2:1), reflux, 20 min (95%); iv)
DIBAL-H, CH2Cl2, Ϫ78 to Ϫ25 ЊC, 2 h; v) Ac2O, pyridine, room temp., 16 h (93% from 10ab); vi) conc. aq. NH3, MeOH, CHCl3, room temp., 48 h
(100%); vii) (MeO)2PNEt2, 1H-tetrazole, CH2Cl2, room temp., 20 min; viii) (a) 4 Å sieves, dioxane–toluene (3:1), room temp., 2 h; (b) ZnCl2, AgClO4,
dark, 7 h (53%); ix) TFA, CH2Cl2, room temp., 1.75 h (81%); x) (a) (BnO)2PNPri2, imidazolium triflate, CH2Cl2, room temp., 1 h; (b) MCPBA,
Ϫ78 ЊC, 10 min (70%); xi) wet Pd(OH)2/C, MeOH–cyclohexene–H2O (14:7:1), reflux, 2.5 h (92%). Bn = benzyl, DMTr = dimethoxytrityl,
PMB = p-methoxybenzyl.
result of their slight structural differences to Ins(1,4,5)P3; 4 has
been shown to behave as a partial agonist at the Ins(1,4,5)P3
receptors of cerebellar microsomes17 while there is evidence
that 6 may show selectivity for the type 1 Ins(1,4,5)P3 receptor
subtype.18 Both analogues, however, bind with lower affinities
than Ins(1,4,5)P3 itself, and further investigation of 6 in particu-
lar has been limited by its low potency. The glyconucleotides
5 and 7, on the other hand, might be expected to have higher
affinities for Ins(1,4,5)P3 receptors than the corresponding
inositol phosphates, due to the enhancing effect of their adeno-
sine 2Ј-monophosphate (2Ј-AMP) component.
Two previous syntheses of adenophostin A have been
reported. In the first, Hotoda et al.19 glycosylated N6,N6-
dibenzoyl-5Ј-O-monomethoxytrityl-2Ј-O-p-methoxybenzyl-
adenosine with 3,4,6-tri-O-acetyl-2-O-benzyl-α--glucopyr-
anosyl bromide. A rather different approach was reported
by van Straten et al.,20 who first prepared a 3-O-α--gluco-
pyranosyl--ribofuranose derivative, followed by a Vorbrüggen
condensation with an activated adenine derivative. A disadvan-
tage of both of these previous routes was the large number of
protection/deprotection steps between construction of the
pyranosyladenosine unit and the final compound. In the
present work we describe an efficient route to a suitably pro-
tected glycosyl acceptor, which allows a convergent approach
to the synthesis of compounds 2, 5 and 7, with minimal
manipulation between phosphite-mediated coupling and final
trisphosphates. A preliminary report of our synthesis of 2 has
appeared.21
hydride at reduced temperature,24 or with benzyl bromide–silver
oxide,25 or with benzyl trichloroacetimidate,26 or via stannyl
ethers27 were unsuccessful and so an alternative strategy was
sought.
Treatment of adenosine with zinc chloride–p-methoxy-
benzaldehyde gave the 2Ј,3Ј-O-p-methoxybenzylidene deriv-
ative28 8ab (Scheme 1). Precipitation of the partially purified
product from p-methoxybenzaldehyde gave the kinetic product
(endo diastereoisomer) as judged by NMR spectroscopy; sub-
sequent addition of diisopropyl ether to the mother liquor gave
a second crop containing a 2:1 endo:exo diastereoisomeric
mixture. The identities of diastereoisomers were ascertained by
analogy to literature 1H NMR data for the corresponding
benzylidene derivatives which have been unambiguously
assigned by NOE experiments.29 NMR data for 8ab and the
pure endo diastereoisomer 8a are reported for the first time here.
Repeating the reaction at 40–45 ЊC gave an inseparable 3:2
exo:endo diastereomeric mixture, a ratio which, in our hands,
was also obtained when benzaldehyde was substituted for
p-methoxybenzaldehyde.30 This latter result is at variance
with that of Baggett et al.,31 who reported exclusively the exo
diastereoisomer under these conditions. Exposing the pure endo
diastereoisomer 8a to the higher temperature conditions also
gave a 3:2 exo:endo product mixture, suggesting this to be the
equilibrium ratio. An improved yield of 8ab (as a 3:2 exo:endo
mixture) was obtained using p-methoxybenzaldehyde, triethyl
orthoformate and PTSA;28b direct acetal exchange with
p-methoxybenzaldehyde dimethyl acetal in DMF in the pres-
ence of PTSA, successful in the preparation of methyl 2,3-O-p-
methoxybenzylidene-β--ribofuranoside,13 gave no product by
TLC.
Results and discussion
For consistency with selectively protected glucopyranose11
and xylopyranose22 units previously prepared in these labor-
atories, we required an adenosine derivative protected with a
p-methoxybenzyl ether at position 2Ј and a benzyl ether at
position 5Ј together with appropriate N6-protection. Attempts
to 5Ј-O-benzylate the known23 N6-benzoyl-2Ј-O-p-methoxy-
benzyladenosine selectively with benzyl bromide and sodium
Selective protection of the N6-position with a dimeth-
oxytrityl group in the presence of the free primary hydroxy
group was achieved by the sequential treatment of 8ab with
chlorotrimethylsilane, dimethoxytrityl chloride and concen-
trated aqueous ammonia, to give the required product 9ab in
1
97% yield after column chromatography. The H NMR spec-
trum of 9ab revealed a D2O-exchangeable triplet at 5.2 ppm,
1936
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947

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confirming that the 5Ј-hydroxy group remained unprotected.
Although transient protection is well established for N6-
benzoylation,32 to the best of our knowledge this is the first
example of its application to N6-dimethoxytritylation. Benzyl-
ation of the 5Ј-hydroxy group was achieved in 95% yield using
benzyl chloride–potassium hydroxide,33 a superior method in
this case to sodium hydride–benzyl bromide. Substitution at
position 5Ј rather than N6 was confirmed by the 13C NMR
spectrum of 10ab, which indicated a methylene carbon at
73.5 ppm, typical of benzyl ether methylenes and by typical34
deshielding of the 5Ј-methylene carbon to 70.1 ppm.
Treatment of 10ab with DIBAL-H in dichloromethane at low
temperature gave an inseparable mixture of the 2Ј- and 3Ј-O-p-
methoxybenzyl ethers in a 3:2 ratio. The temperature of this
reaction was crucial; repeating it at 0 ЊC gave only traces of
these products. Similarly, using the established reductive cleav-
age reagents LiAlH4–AlCl3, BH3ؒN(CH3)3–AlCl3, NaBH3CN–
(CH3)3SiCl, NaBH3CN–HCl or NaBH3CN–TFA also gave very
poor results. Acetylation of the product mixture gave acetates
11 and 12 which could be separated by column chromato-
graphy. Introduction of an acetyl ester also facilitated identifi-
cation of the regioisomers; the 1H NMR spectra of both 11 and
12 exhibited a deshielded doublet of doublets corresponding to
methines geminal to acetates, the positions of which were iden-
tified by 2D COSY experiments. The 3Ј-O-acetyl derivative 11
was treated with methanolic ammonia to give the required
glycosyl acceptor 13.
being isolated first as the free acid before being converted into
1
the sodium salt. Both the H and 31P NMR spectra and the
negative ion FAB mass spectrum of 2 were in full agreement
with those reported previously for both natural3,4 and syn-
thetic19,20 adenophostin A. Synthetic 2 had the same retention
time as a sample of natural adenophostin A in analytical
reverse phase HPLC and eluted as a single peak (using an ODS
column and eluting with a gradient of 10–30% acetonitrile
and 0.05 mol dmϪ3 phosphate buffer, containing 0.1% w/v of
tetrabutylammonium hydrogensulfate).
The successful glycosylation, phosphorylation and deprotec-
tion strategies developed in the synthesis of 2 were now adapted
to prepare the other targets 5 and 7. 2-O-Benzyl-3,4-O-di-O-p-
methoxybenzyl--xylopyranose22 (19, Scheme 2) was phos-
With 13 in hand, attention turned to coupling with an
appropriate activated derivative of 2,6-di-O-benzyl-3,4-di-O-p-
methoxybenzyl--glucopyranose11 (14). Phosphite glycosyl-
ation methodology was selected for three reasons. First, use
of the trichloroacetimidate derivative of 14 had proved dis-
appointing with model adenosine acceptors;30 second, phos-
phites tend to give good selectivity for α-anomeric products35,36
and third, the 3Ј-hydroxy of adenosine is relatively unreactive
and a phosphite donor had been employed successfully to
glycosylate an acid-sensitive, unreactive alcohol in a previous
example.37 Reaction of 14 with dimethoxy(diethylamino)-
phosphine36 and 1H-tetrazole smoothly gave dimethyl phos-
phites 15, confirmed by 1H and 31P NMR spectroscopy to be a
1:1 anomeric mixture. Using a carefully controlled ratio and
quantity of silver perchlorate–zinc chloride as promoter, alco-
hol 13 was glycosylated with 15 to give 16 in 53% yield. The 1H
NMR spectrum of 16 displayed a doublet at 5.2 ppm (J 3.4 Hz)
corresponding to H-1Љ and thereby confirming preparation
of the α-glucopyranosyl anomer; none of the corresponding
β-anomer was detected.
Deprotection of the three p-methoxybenzyl ethers and the
N-dimethoxytrityl group of 16 was achieved in one step with
10% TFA in dichloromethane to give triol 17 in 81% yield. Note
the short reaction time (1.75 h) compared to the 41 h required
by a DDQ-mediated protection of a 2Ј-O-p-methoxybenzyl
ether in the synthesis of 2 by Hotoda et al.19 Triol 17 was phos-
phitylated using bis(benzyloxy)(diisopropylamino)phosphine
and imidazolium triflate,38 a method which obviates the need
for base protection. The intermediate trisphosphite triester was
oxidised with MCPBA, then quenched at Ϫ78 ЊC to avoid pos-
sible oxidation of the adenine base. The 31P NMR spectrum of
the product 18 confirmed the presence of three phosphate tri-
esters, while the presence of the free unphosphorylated amino
group was substantiated by the presence of a broad singlet in
the 1H NMR spectrum at 6.1 ppm.
Scheme 2 Reagents and conditions: i) (MeO)2PNEt2, 1H-tetrazole,
CH2Cl2, room temp., 20 min; ii) (a) 13, 4 Å sieves, dioxane–toluene
(3:1), room temp., 2 h; (b) ZnCl2, AgClO4, dark, 9 h (46%); iii) TFA,
CH2Cl2, room temp., 1.75 h (81%); iv) (a) (BnO)2PNPri2, imidazolium
triflate, CH2Cl2, room temp., 1.5 h; (b) MCPBA, Ϫ78 ЊC, 10 min (68%);
v) wet Pd(OH)2/C, MeOH–cyclohexene–H2O (11:5:1), reflux, 2.5 h
(85%). Bn = benzyl, DMTr = dimethoxytrityl, PMB = p-methoxy-
benzyl.
phitylated in a similar fashion to 14, giving glycosyl donor 20 as
1
a 2:3 α:β anomeric mixture as indicated by H and 31P NMR
spectroscopy. Glycosylation of 13 with 20 gave an inseparable
1:1 anomeric mixture (21ab) as judged by integral ratios of the
H-1Љ anomeric protons in the 1H NMR spectrum. Treatment of
this mixture with 10% TFA in dichloromethane gave the
required α-coupled triol 22 and its β-anomer 23, which were
readily separated by column chromatography. The anomeric
1
configurations of 22 and 23 were easily distinguished using H
NMR spectroscopy by comparing the coupling constants of the
H-1Љ protons: that of 22 resonated at 5.17 ppm with a relatively
small axial–equatorial coupling constant of 3.8 Hz; that of 23
resonated at 4.46 ppm with a larger axial–axial coupling con-
stant of 7.9 Hz. Compound 22 was phosphitylated and oxidised
to give 24 without prior protection of position N6, as described
for 17, and was smoothly deprotected similarly to 18 to give
5. The purity of 5 (xylo-adenophostin) was confirmed by
analytical HPLC under similar conditions to those described
for 2.
Attempts to deprotect 18 with sodium in liquid ammonia, or
catalytic hydrogenation over palladium black or palladium on
carbon were unsuccessful. Catalytic hydrogenation over 20%
palladium hydroxide allowed isolation of the target compound
2, but in poor yield, and after the rather long reaction time of 5
days. However, complete deprotection of 18 in high yield was
readily achieved by catalytic transfer hydrogenation.39 The
crude product was purified by ion exchange chromatography,
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947
1937

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pyranosides with consequent reduction in the yield of 26.
The use of BF3ؒEt2O as catalyst and lower temperatures44 in
place of CSA and reflux was not successful, at least on this
scale, with equilibration being incomplete after 3 days at
room temperature. Benzylation of 26 with benzyl bromide
and sodium hydride in DMF gave the fully protected com-
pound 27.
At this stage, one strategy would be to remove the allyl group
from 27 and convert the product into a glycosyl donor with the
BDA protection still in place. However, it has been shown
that BDA protection of positions 3 and 4 in mannose has a
deactivating effect on thioglycoside glycosyl donors,45 and it
was felt that, in the present case, similar effects might lead to
difficulties at the critical coupling reaction with 13. Accord-
ingly, the BDA group was removed from 27 using TFA, giving
the 3,4-diol 28, which was then alkylated with p-methoxybenzyl
chloride and sodium hydride to give 29. The allyl protection at
the anomeric position of 29 was selectively cleaved using a
catalytic amount of PdCl2 in methanol at room temperature.46
The reaction mixture became increasingly acidic as the reaction
progressed, but it was found that this method was compatible
with moderately acid-labile protecting groups (BDA or
p-methoxybenzyl), providing that the acid was neutralised
before work-up. Mannopyranose 30 was obtained as a 3:1 mix-
ture of α- and β-anomers as judged by 1H NMR spectroscopy.
Phosphitylation of 30 with 1H-tetrazole and dimethoxy-
(diethylamino)phosphine in dichloromethane as for 14 and 19
Scheme 3 Reagents and conditions: i) AllOH, HCl, 50–60 ЊC, 5 h (α-
anomer by crystallisation, 61%); ii) butanedione, (MeO)3CH, CSA,
MeOH, reflux, 10 h (78%); iii) NaH, BnBr, DMF, 0 ЊC to room temp.,
14 h (88%); iv) TFA, H2O, CH2Cl2, 15 min (85%); v) NaH, PMBCl,
DMF, room temp., 17 h (66%); vi) PdCl2, MeOH, room temp., 3 h
(79%); vii) (MeO)2PNEt2, 1H-tetrazole, CH2Cl2, room temp., 20 min;
viii) (a) 13, 4 Å sieves, dioxane–toluene (3:1), room temp., 2 h;
(b) ZnCl2, AgClO4, dark, 8 h (61%); ix) TFA, CH2Cl2, room temp., 5 h
(82%); x) (a) (BnO)2PNPri2, imidazolium triflate, CH2Cl2, room temp.,
1 h; (b) MCPBA, Ϫ78 ЊC, 10 min (56%); xi) wet Pd(OH)2/C, MeOH–
cyclohexene–H2O (10:5:1), reflux, 2.5 h (71%). All = allyl, Bn = benzyl,
DMTr = dimethoxytrityl, PMB = p-methoxybenzyl.
1
then gave glycosyl donor 31. The 31P and H NMR spectra of
31 indicated that the α- and β-anomers were present in a ratio
of approximately 10:1.
Glycosylation of 13 with 31 proceeded smoothly, as
expected, to give exclusively the α-coupled product 32. Cleavage
of the acid-labile protecting groups in 32 was achieved with
10% TFA in dichloromethane. The reaction was significantly
slower than the equivalent deprotection of 16 and of 21ab, but
was complete after 5 hours. Monitoring of the reaction by TLC
indicated that cleavage of the dimethoxytrityl group took place
rapidly, suggesting that one of the p-methoxybenzyl groups in
32 was refractory to the reaction conditions. The triol 33 was
isolated in good yield, indicating that extended exposure to the
acidic reaction conditions had not been detrimental to the
glycosidic or nucleosidic linkages. Phosphitylation of 33 and
oxidation of phosphites as described for 17 and 22 furnished
the fully protected trisphosphate 34. However, deprotection of
34 by catalytic transfer hydrogenation for 10 hours as described
for 18 and 24 gave a product contaminated by a minor impurity,
clearly apparent in the 31P and 1H NMR spectra. It was
thought that this impurity may have arisen from intramolecular
phosphate migration, which would be favoured by the cis-
relationship of O-2 and O-3 on the mannopyranosyl ring. The
reaction was therefore repeated for a shorter time (2.5 hours)
and pure 7 was isolated after ion exchange chromatography
followed by conversion into the sodium salt. The purity of
7 (manno-adenophostin) was confirmed by analytical HPLC
as described for 2 and 5.
Synthetic adenophostin A (2) was shown to be equipotent
with naturally occurring adenophostin A in evoking Ca2ϩ
release from the intracellular stores of permeabilised cells.21
Full biological evaluation of 2, 5 and 7 will be reported else-
where. The synthesis of these three related glyconucleotides by
a convergent approach employing the versatile glycosyl
acceptor 13 demonstrates the value of this strategy, not only as
an efficient route to adenophostin A but also to the first adeno-
phostin analogues with modified pyranosyl rings. Because the
glucopyranosyl ring of the adenophostins is analogous to
the inositol ring of Ins(1,4,5)P3, this approach may allow the
design of novel glyconucleotide counterparts to conventional
inositol-based polyphosphates, potentially with enhanced
affinities for Ins(1,4,5)P3 receptors, for intervention in the poly-
phosphoinositide pathway of cellular signalling.
The selectively protected intermediate (30, Scheme 3),
required for the synthesis of 7 was not known, and a route to it
was developed from allyl α--mannopyranoside (25). Although
25 was first described as a crystalline solid (mp 98–99 ЊC, [α]D
ϩ99, no spectroscopic data given),40 a later report gave signifi-
cantly different physical properties (mp 138–139 ЊC, [α]D
ϩ51.6)41. In other cases, crystalline 25 was not isolated.42 In our
hands, the successful preparation of 25 was found to require a
careful choice of conditions. Various combinations of temper-
ature, amount and type of acid catalyst and work-up were
tried before procedures were established that gave crystalline
25 in good yield and uncontaminated by unreacted hexose,
β-anomer or other by-products. In all cases the material
resisted crystallisation until it had been further purified by
flash chromatography on a short column of silica, and a
final recrystallisation was still required to remove traces of
β-anomer. In the present case, this was necessary because it was
found that the presence of any impurities made purification
after the next step difficult.
Selective protection of the two equatorial hydroxy groups at
positions 3 and 4 in 25 was readily accomplished using the
butane diacetal (BDA) protecting group.43 Initially this was
achieved by acid-catalysed reaction of 25 with 2,2,3,3-tetra-
methoxybutane (TMB) in methanol according to the original
procedure reported by Montchamp et al.43 for methyl α-manno-
pyranoside. However, it was later found that identical results
could be achieved more conveniently by using the simplified
method of Hense et al.,44 which employs commercially available
butanedione in place of TMB. After 1 hour, two major prod-
ucts were present in the reaction mixture, but after 10 hours,
only the 3,4-diacetal 26 was detected by TLC. Longer reaction
times led to the gradual accumulation of methyl manno-
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J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947

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dd, J 2.2, 6.6, 3Ј-H), 5.28 (1 H, t, J 5.4, D2O exch., 5Ј-OH), 5.47
Experimental
(1 H, dd, J 2.9, 6.3, 2Ј-H), 5.96 (1 H, s, p-methoxybenzylidene
CH), 6.30 (1 H, d, J 2.9, 1Ј-H), 6.99 (2 H, m, meta-H of
p-methoxybenzylidene ring), 7.38 (2 H, s, D2O exch., NH2),
7.50 (2 H, m, ortho-H of p-methoxybenzylidene ring) and 8.17
and 8.38 (2 H, 2 s, 2-H and 8-H); δC (d6-DMSO; 100 MHz) 55.83
(OCH3), 62.05 (C-5Ј), 83.12 (C-3Ј), 84.32 (C-2Ј), 86.79 (C-4Ј),
90.30 (C-1Ј), 107.29 (p-methoxybenzylidene CH), 114.44 (meta-
C of p-methoxybenzylidene ring), 119.38 (C-5), 128.44 (ipso-C
of p-methoxybenzylidene ring), 129.24 (ortho-C of p-methoxy-
benzylidene ring), 140.67 (C-8), 149.34 (C-4), 153.36 (C-2),
156.36 (C-6) and 161.02 (para-C of p-methoxybenzylidene
ring); m/z (FABϩ) 386 [(M ϩ 1)ϩ, 100%].
Materials and methods
Chemicals were purchased from Aldrich, Fluka, Lancaster and
Sigma chemical companies. Dichloromethane was dried over
calcium hydride, distilled and stored over 4 Å molecular sieves.
N,N-Dimethylformamide (DMF) was dried over barium oxide,
distilled under reduced pressure and stored over 4 Å molecular
sieves. Pyridine was dried over potassium hydroxide pellets, dis-
tilled and stored over potassium hydroxide pellets. Toluene,
dioxane and triethylamine were purchased in anhydrous form.
TLC was performed on precoated plates (Merck aluminium
sheets silica 60 F254, Art. No. 5554). Products were visualised
by UV light (254 nm) or by dipping in a solution of phos-
phomolybdic acid in methanol followed by heating. Flash
chromatography was carried out using Merck-Kieselgel 60
(0.040–0.063 mm) under pressure.
Method B. A mixture of adenosine (20.0 g, 0.07 mol),
p-methoxybenzaldehyde (400 cm3), triethyl orthoformate (100
cm3) and dry PTSA (40.3 g, 0.26 mol) was stirred at 35–40 ЊC
for 4 h under N2, whereupon the resulting purple solution was
poured into 0.3 mol dmϪ3 aqueous NaHCO3 solution (1000
cm3) and stirred vigorously for 15 min. Ethyl acetate (300 cm3)
was added and the resulting organic layer was set aside. The
aqueous layer was extracted with more ethyl acetate (300 cm3)
and the combined organic layers were washed with saturated
aq. NaCl (200 cm3), dried (MgSO4), filtered and concentrated.
Diisopropyl ether (400 cm3) was added and the suspension was
refrigerated at 4 ЊC for 48 h. The product was isolated as a white
powder (26.0 g as a mixture of endo and exo isomers, 2:3
endo:exo, by 1H NMR, 90%).
Data for mixture of diastereoisomers 8ab: δH (d6-DMSO; 400
MHz) 3.57–3.62 (2 H, m, 5Ј-HA, 5Ј-HB), 3.77 (1.8 H, s,
OCH3exo), 3.79 (1.2 H, s, OCH3endo), 4.26–4.31 (0.6 H, m,
4Ј-Hexo), 4.37–4.40 (0.4 H, m, 4Ј-Hendo), 5.05–5.10 (1 H, m,
3Ј-H), 5.16 (0.6 H, t, J 5.6, D2O exch., 5Ј-OHexo), 5.29 (0.4 H, t,
J 5.6, D2O exch., 5Ј-OHendo), 5.45–5.49 (1 H, m, 2Ј-H), 5.97 (0.4
H, s, p-methoxybenzylidene CHendo), 6.19 (0.6 H, s, p-methoxy-
benzylidene CHexo), 6.28 (0.6 H, d, J 2.9, 1Ј-Hexo), 6.30 (0.4 H,
d, J 2.9, 1Ј-Hendo), 6.95–7.02 (2 H, m, meta-H of p-methoxy-
benzylidene ring), 7.39–7.52 (4 H, m, simplifies to 2 H, m on
D2O exch., ortho-H of p-methoxybenzylidene ring, NH2) and
8.18 and 8.39 (2 H, 2 s, 2-H, 8-H); δC (d6-DMSO; 100 MHz)
55.21 (OCH3), 61.57 (C-5Ј), 80.47, 82.81, 84.45 (C-4Ј, C-3Ј,
C-2Ј all exo), 82.59, 83.67, 86.30 (C-4Ј, C-3Ј, C-2Ј all endo),
87.98 (C-1Јexo), 89.55 (C-1Јendo), 102.87 (p-methoxybenzylidene
CHexo), 106.56 (p-methoxybenzylidene CHendo), 113.66 (ortho-
C of p-methoxybenzylidene ringexo), 113.79 (ortho-C of
p-methoxybenzylidene ringendo), 119.14 (C-5), 128.07 (ipso-C
of p-methoxybenzylidene ring), 128.51, (meta-C of p-methoxy-
benzylidene ring), 139.72 (C-8exo), 139.88 (C-8endo), 148.81
(C-4), 152.68 (C-2), 156.18 (C-6), 160.26 (para-C of p-methoxy-
benzylidene ringexo) and 160.38 (para-C of p-methoxybenzyl-
idene ringendo).
1H and 13C NMR spectra were recorded on either JEOL
GX270 or EX 400 or Varian Mercury 400 spectrometers.
Chemical shifts were measured in ppm relative to internal
tetramethylsilane or HDO. 31P NMR spectra were recorded on
JEOL GX270 or EX400 spectrometers and 31P NMR chemical
shifts were measured in ppm and denoted positive downfield
from external 85% H3PO4. J values are given in Hz. Proton
assignments were established with 2D COSY experiments, and
the number of protons attached to carbon atoms was estab-
lished by DEPT experiments. Mps (uncorrected) were deter-
mined using a Reichert-Jung Thermo Galen Kofler block.
Microanalysis was carried out at the University of Bath Micro-
analysis Service. Low resolution mass spectra were recorded at
the University of Bath Mass Spectrometry Service using ϩve
and Ϫve fast atom bombardment (FAB) with m-nitrobenzyl
alcohol (NBA) as the matrix. High resolution accurate mass
spectra were recorded at the University of Bath Mass Spec-
trometry Service. Optical rotations were measured at ambient
temperature using an Optical Activity Ltd. AA-10 polarimeter
in a cell volume of 1 cm3 or 5 cm3, and [α]D values are given in
10Ϫ1 deg cm2 gϪ1. Ion exchange chromatography was performed
on an LKB-Pharmacia Medium Pressure Ion-Exchange
Chromatograph using either Q Sepharose Fast Flow resin and
gradients of triethylammonium bicarbonate as eluent, or MP1
AG ion exchange resin and a gradient of 150 mM aqueous TFA
as eluent. Synthetic phosphates were quantified by total phos-
phate assay.47
2Ј,3Ј-O-p-Methoxybenzylideneadenosine (8ab)
Method A. A suspension of zinc chloride (70 g, 0.50 mol) in
p-methoxybenzaldehyde (350 cm3) was stirred for 30 min under
N2, whereupon adenosine (25.0 g, 0.09 mol) was added and the
mixture was stirred for 18 h. The resulting viscous cream-
coloured suspension was divided into two. Each half was
poured into 400 cm3 of chilled water and this mixture was
extracted with chloroform (400 cm3, 300 cm3, 200 cm3). The
combined organic extracts were washed with water (300 cm3),
dried (MgSO4), filtered and concentrated. The suspensions
from each half, containing product and p-methoxybenzalde-
hyde, were recombined and refrigerated at 4 ЊC overnight.
Precipitated product was recovered by filtration (13.8 g, endo
isomer by 1H NMR), and remaining product left in the filtrate
was precipitated by addition of diisopropyl ether (300 cm3) and
refrigeration (10.0 g mixture of endo and exo isomers, 2:1
endo:exo, as indicated by 1H NMR integral ratio of p-methoxy-
benzylidene CH). Combined yield (23.80 g, 66%); Rf 0.26 (ethyl
acetate–ethanol, 9:1).
N6-Dimethoxytrityl-2Ј,3Ј-O-p-methoxybenzylideneadenosine
(9ab)
To a suspension of 8ab (3.83 g, 8.78 mmol, concentrated from
3 × 20 cm3 dry pyridine) in dry pyridine (50 cm3) under N2 was
added chlorotrimethylsilane (2.79 cm3, 22.0 mmol), whereupon
the starting material dissolved. The solution was stirred for 2 h,
dimethoxytrityl chloride (7.83 g, 22.0 mmol) was added and the
resultant orange mixture was stirred overnight. The mixture
was cooled to 0 ЊC and the reaction was quenched by addition
of water (5 cm3). The cooling bath was removed and after 10
min concentrated aq. NH3 (20 cm3) was added. The solution
was stirred for a further 30 min, then was partitioned between
5% (w/v) aq. NaHCO3 (150 cm3) and CH2Cl2 (150 cm3). The
aqueous layer was back-extracted with CH2Cl2 (2 × 50 cm3) and
the combined organic layers were dried (MgSO4), filtered and
concentrated. The dark orange oil thus obtained was subjected
to flash chromatography (eluent ethyl acetate–hexane, 3:2) to
Data for endo diastereoisomer 8a: mp 215–217 ЊC (from
ethanol) (Found: C, 55.9; H, 4.9; N, 18.1. Calcd for
C18H19N5O5: C, 56.1; H, 5.0; N, 18.2%); δH (d6-DMSO; 400
MHz) 3.63–3.65 (2 H, m, simplifies on D2O exch., 5Ј-HA,
5Ј-HB), 3.77 (3 H, s, OCH3), 4.35–4.39 (1 H, m, 4Ј-H), 5.06 (1 H,
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947
1939

View Article Online
give the title compound as a pale cream foam (6.60 g, 97%); Rf
0.29 (ethyl acetate–hexane, 3:2).
p-methoxybenzylidene CH), 6.27 (1 H, d, J 2.5, 1Ј-H), 6.78–
6.94 (7 H, m, ArCH), 7.20–7.48 (15 H, m, ArCH) and 7.96 and
8.05 (2 H, 2 s, 2-H, 8-H); δC (CDCl3; 100.4 MHz) 55.21
(2 × OCH3), 55.34 (OCH3), 70.10 (C-5Ј), 70.64 (DMTr Cq),
73.45 (OCH2Ar), 82.79, 84.86, 85.57 (C-2Ј, C-3Ј, C-4Ј), 90.89
(C-1Ј), 107.66, (p-methoxybenzylidene CH), 113.13, 113.89
(meta-C of p-methoxyphenyl rings), 121.32 (C-5), 126.81,
127.72, 127.78, 127.89, 128.14, 128.31, 128.47, 128.78, 130.10
(ArCH, ipso-C of p-methoxybenzylidene ring), 137.31 (ipso-C
of Bn ring), 137.45 (ipso-C of DMTr p-methoxyphenyl rings),
138.81 (C-8), 145.43 (ipso-C of DMTr phenyl ring), 148.39
(C-4), 152.47 (C-2), 154.15 (C-6), 158.26 (para-C of DMTr
p-methoxyphenyl rings) and 160.88 (para-C of p-methoxy-
benzylidene ring); m/z (FABϩ) 688 [(M ϩ H)ϩ, 26%] and 303
(100).
Data for endo diastereoisomer 9a: (Found: Mϩ, 687.267.
Calcd for C39H37N5O7 Mϩ: 687.269); δH (d6-DMSO; 400 MHz)
3.49–3.61 (2 H, m, 5Ј-HA, 5Ј-HB), 3.71 (6 H, s, 2 × OCH3 of
DMTr), 3.78 (3 H, s, OCH3 of p-methoxybenzylidene), 4.35–
4.39 (1 H, m, 4Ј-H), 5.03 (1 H, dd, J 2.3, 6.5, 3Ј-H), 5.22 (1 H, t,
J 6.6, D2O exch., 5Ј-OH), 5.48 (1 H, dd, J 2.8, 6.5, 2Ј-H), 5.95
(1 H, s, p-methoxybenzylidene CH), 6.30 (1 H, d, J 2.8, 1Ј-H),
6.82–6.86 (4 H, m, ArCH), 6.98–7.01 (2 H, m, ArCH), 7.19–
7.29 (9 H, m, ArCH), 7.48 (1 H, br s, D2O exch., NH), 7.51–
7.96 (2 H, m, ArCH) and 8.32 and 8.48 (2 H, 2 s, 2-H, 8-H); δC
(d6-DMSO; 100 MHz) 55.36 (2 × OCH3), 55.58 (OCH3), 61.76
(C-5Ј), 69.93 (DMTr Cq), 82.84, 84.01, 86.70 (C-4Ј, C-3Ј, C-2Ј),
90.01 (C-1Ј), 106.95 (p-methoxybenzylidene CH), 113.35,
114.15 (meta-C of p-methoxyphenyl rings), 120.97 (C-5),
126.86, 128.07, 128.34, 128.71, 128.91, 130.12 (ArCH, ipso-C
of p-methoxybenzylidene ring), 137.52 (ipso-C of DMTr
p-methoxyphenyl rings), 140.85 (C-8), 145.57 (ipso-C of DMTr
phenyl ring), 148.31 (C-4), 151.79 (C-2), 153.97 (C-6), 158.04
(para-C of DMTr p-methoxyphenyl rings) and 160.75 (para-C
of p-methoxybenzylidene ring); m/z (FABϩ) 688 [(M ϩ H)ϩ,
26%] and 303 (100).
Data for mixture of diastereoisomers 10ab, endo:exo 2:3:
δH (CDCl3; 400 MHz) 3.67 (0.4 H, 0.5 ABX, 2JAB 13.9, 3JAX 4.2,
5Ј-HAendo), 3.68–3.72 (1.6 H, m, 5Ј-HAexo, 5Ј-HB), 3.77, 3.81 and
3.82 (6 H, s, 2 × OCH3 of DMTr), 3.81 and 3.82 (3 H, 2 s,
OCH3 of p-methoxybenzylidene), 4.46 (0.4 H, AB, JAB 12.2,
OCHHArendo), 4.48–4.54 (2.2 H, m, OCH2Arexo, OCHHArendo
,
4Ј-Hexo), 4.65 (0.4 H, q, J 2.4, 4Ј-Hendo), 5.04 (0.4 H, dd, J 2.4,
6.4, 3Ј-Hendo), 5.13 (0.6 H, dd, J 3.7, 6.1, 3Ј-Hexo), 5.45 (0.6 H,
dd, J 2.9, 6.4, 2Ј-Hexo), 5.51 (0.4 H, dd, J 2.2, 6.6, 2Ј-Hendo), 5.95
(0.4 H, s, p-methoxybenzylidene CHendo), 6.13 (0.6 H, s,
p-methoxybenzylidene CHexo), 6.21 (0.6 H, d, J 2.4, 1Ј-Hexo),
6.27 (0.4 H, d, J 2.5, 1Ј-Hendo), 6.78–6.94 (7 H, m, ArCH), 7.20–
7.48 (15 H, m, ArCH), 7.03 and 7.95 (1 H, 2 s, 2-H or 8-H) and
7.96 and 8.05 (1 H, 2 s, 2-H or 8-H); δC (CDCl3; 100.4 MHz)
55.21 (2 × OCH3), 55.34 (OCH3), 70.10 (C-5Ј), 70.64 (DMTr
Cq), 73.45 (OCH2Ar), 81.33, 84.00 and 84.27 (C-2Ј, C-3Ј,
C-4Ј all exo), 82.79, 84.86, 85.57 (C-2Ј, C-3Ј, C-4Ј all endo),
89.98 (C-1Јexo), 90.89 (C-1Јendo), 104.26 (p-methoxybenzylidene
CHexo), 107.66, (p-methoxybenzylidene CHendo), 113.13, 113.89
(meta-C of p-methoxyphenyl rings), 121.32 (C-5), 126.81,
127.72, 127.78, 127.89, 128.14, 128.31, 128.47, 128.78 and
130.10 (ArCH, and ipso-C of p-methoxybenzylidene ring),
137.31 (ipso-C of Bn ring), 137.45 (ipso-C of DMTr
p-methoxyphenyl rings), 138.81 (C-8), 145.43 (ipso-C of DMTr
phenyl ring), 148.39 (C-4), 152.47 (C-2), 154.15 (C-6), 158.26
(ipso-C of DMTr p-methoxyphenyl rings) and 160.88 (para-C
of p-methoxybenzylidene ring).
Data for mixture of diastereoisomers 9ab, endo:exo 2:3: δH
(CDCl3; 270 MHz) 3.77–3.84 (1 H, m, 5Ј-HAexo, 5Ј-HAendo), 3.77
(6 H, s, 2 × OCH3 of DMTr), 3.80 (3 H, s, OCH3 of p-
methoxybenzylidene), 3.94–4.00 (1 H, m, 5Ј-HB), 4.53 (0.4 H,
m, 4Ј-Hexo), 4.68 (0.6 H, m, 4Ј-Hendo), 5.16–5.18 (1 H, m, 3Ј-H),
5.26–5.35 (1 H, m, 2Ј-H), 5.98 (0.4 H, d, J 4.95, 1Ј-Hendo), 6.00–
6.02 (1 H, m, p-methoxybenzylidene CHendo, 1Ј-Hexo), 6.25 (0.6
H, s, p-methoxybenzylidene CHexo), 6.77–7.02 (7 H, m, ArCH),
7.21–7.50 (10 H, m, ArCH), 7.76, 7.83 (1 H, 2 s, 2-H or 8-H)
and 8.01 and 8.02 (1 H, 2 s, 2-H or 8-H); δC (CDCl3; 100 MHz)
55.59 (OCH3), 63.41 (C-5Јexo), 63.72 (C-5endo), 71.12 (DMTr
Cq), 80.52, 84.21, 86.43 (C-2Ј, C-3Ј, C-4Ј all endo), 83.07, 84.01,
85.91 (C-2Ј, C-3Ј, C-4Ј all exo), 92.26 (C-1Јexo), 94.47 (C-1Јendo),
104.93 (p-methoxybenzylidene CHexo), 107.85 (p-methoxy-
benzylidene CHendo), 113.41, 114.15 (meta-C of p-methoxy-
phenyl rings), 122.43 (C-5exo), 122.56 (C-5endo), 127.12, 127.97,
128.14, 128.28, 128.83, 128.93, 130.27 (ArCH, and ipso-C
of p-methoxybenzylidene ring), 137.30 (ipso-C of DMTr
p-methoxyphenyl rings), 139.49, (C-8exo), 139.81 (C-8endo),
145.28 (ipso-C of DMTr phenyl ring), 147.31 (C-4endo), 147.45
(C-4exo), 152.02 (C-2endo), 152.20 (C-2exo), 154.73 (C-6exo), 154.76
(C-6endo), 158.46 (para-C of DMTr p-methoxyphenyl rings),
160.79 (para-Cendo of p-methoxybenzylidene ring) and 161.00
(para-Cexo of p-methoxybenzylidene ring).
3Ј-O-Acetyl-5Ј-O-benzyl-N6-dimethoxytrityl-2Ј-O-p-methoxy-
benzyladenosine (11) and 2Ј-O-acetyl-5Ј-O-benzyl-N6-
dimethoxytrityl-3Ј-O-p-methoxybenzyladenosine (12)
To a solution of 10ab (1.00 g, 1.29 mmol) in CH2Cl2 at Ϫ78 ЊC
under N2 was added a solution of DIBAL-H in CH2Cl2 (1 mol
dmϪ3; 6.5 cm3, 6.50 mmol) dropwise. The reaction mixture was
allowed to warm slowly to Ϫ25 ЊC over 2 h, after which time
TLC (ethyl acetate–pentane, 1:1) indicated conversion into two
products (Rf 0.51 and 0.39). The reaction mixture was cooled
to Ϫ78 ЊC and quenched by addition of ethyl acetate (5 cm3).
Diethyl ether (100 cm3) was added and the cooling bath was
removed. Ice-cold NaOH solution (1 mol dmϪ3) was added
until there were two clear layers; the resulting aqueous layer was
discarded and the organic layer was washed with water (75
cm3), dried (MgSO4), filtered and concentrated to a yellow oil
which was dissolved in pyridine (10 cm3) and acetic anhydride
(5 cm3) and stirred overnight. The reaction mixture was concen-
trated, and then concentrated repeatedly from toluene. The
residue was subjected to flash chromatography (eluent ethyl
acetate–hexane, 2:3, then 1:1, then 3:2). Initial fractions con-
tained pure 11, while further fractions contained a mixture of
the two regioisomers. The material in these fractions was
repeatedly re-chromatographed to completely separate the two
regioisomers. Yield of 11 (0.58 g, 55% from 10ab); Rf 0.35
(CHCl3–acetone, 19:1) (Found: C, 70.0; H, 5.8; N, 8.5. Calcd
for C48H47N5O8: C, 70.1; H, 5.8; N, 8.5%); δH (CDCl3; 400
5Ј-O-Benzyl-N6-dimethoxytrityl-2Ј,3Ј-O-p-methoxybenzylidene-
adenosine (10ab)
A solution of 9ab (6.06 g, 8.83 mmol) in benzene (85 cm3) and
dioxane (41 cm3) under N2 was sequentially treated with KOH
powder (85%, 14.6 g, 221 mmol) and benzyl chloride (3.05 cm3,
26.5 mmol) and the resultant mixture was heated under reflux
for 20 min, then was cooled and partitioned between diethyl
ether (200 cm3) and iced water (150 cm3). The ethereal layer was
washed with water (2 × 75 cm3), and the combined aqueous
layers were extracted with ether (100 cm3). The combined ether-
eal layers were dried (MgSO4), filtered and concentrated to
give a yellow oil which was subjected to flash chromatography
(eluent ethyl acetate–hexane, 1:1) to yield the title compound as
a pale cream foam (6.53 g, 95%).
Data for endo diastereoisomer 10a: (Found: C, 70.8; H,
5.5; N, 8.85. Calcd for C46H43N5O7: C, 71.0; H, 5.6; N,
9.0%); δH (CDCl3; 400 MHz) 3.68–3.72 (2 H, m, 5Ј-HA, 5Ј-HB),
3.77 (6 H, s, 2 × OCH3 of DMTr), 3.82 (3 H, s, OCH3 of
p-methoxybenzylidene), 4.46, 4.49 (2 H, AB, JAB 12.2,
OCH2Ar), 4.65 (1 H, q, J 2.4, 4Ј-H), 5.04 (1 H, dd, J 2.4, 6.4,
3Ј-H), 5.51 (1 H, dd, J 2.2, 6.6, 2Ј-H), 5.95 (1 H, s,
1940
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947

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2
3
MHz) 2.14 (3 H, s, CH3CO), 3.67 (1 H, JAB 10.7, JAX 3.1,
5Ј-HA), 3.74–3.79 (1 H, m, 5Ј-HB, obscured by OCH3), 3.75
(3 H, s, OCH3), 3.76 (6 H, s, 2 × OCH3), 4.34–4.39 (2 H, AB,
JAB 12.0, OCHHAr, overlapping with 4Ј-H), 4.50 (1 H, AB,
JAB 12.0, OCHHAr), 4.54 and 4.58 (2 H, AB, JAB 11.7,
OCH2Ar), 4.68 (1 H, dd, J 5.3, 6.4, 2Ј-H), 5.43 (1 H, dd,
J 2.8, 5.1, 3Ј-H), 6.12 (1 H, d, J 6.7, 1Ј-H), 6.66–6.69 (2 H, m,
ArCH), 6.79–6.83 (4 H, m, ArCH), 6.88 (1 H, br s, D2O
exch., NH), 6.95–6.99 (2 H, m, ArCH), 7.22–7.38 (14 H, m,
ArCH) and 7.77 and 8.06 (2 H, s, 2-H, 8-H); δC (CDCl3; 100
MHz) 21.36 (CH3CO), 55.59 (3 × OCH3), 69.96 (C-5Ј), 70.96
(DMTr Cq), 72.14, 79.01 and 82.44 (C-2Ј, C-3Ј, C-4Ј), 72.86
and 74.06 (2 × OCH2Ar), 86.38 (C-1Ј), 113.36 and 113.95
(meta-C of p-methoxyphenyl rings), 121.20 (C-5), 127.00,
128.01, 128.08, 128.23, 128.82, 129.02, 129.73 and 130.32
(ArCH, ipso-C of PMB ring), 137.49 (ipso-C of Bn ring),
137.68 (ipso-C of DMTr p-methoxyphenyl rings), 138.23
(C-8), 145.65 (ipso-C of DMTr phenyl ring), 149.05 (C-4),
152.60 (C-2), 154.19 (C-6), 158.38 (para-C of DMTr
p-methoxyphenyl rings), 159.60 (para-C of PMB ring) and
170.39 (CH3CO); m/z (FABϩ) 822 [(M ϩ H)ϩ, 30%], 303 (100)
and 91 (17).
p-methoxyphenyl rings) and 159.82 (para-C of PMB ring); m/z
(FABϩ) 780 [(M ϩ H)ϩ, 32%], 303 (100) and 91 (21).
2,6-Di-O-benzyl-3,4-di-O-p-methoxybenzyl-D-glucopyranosyl
dimethyl phosphite (15)
To a mixture of glucopyranose 1411 (2.00 g, 3.33 mmol) and
1H-tetrazole (0.35 g, 5.00 mmol) in CH2Cl2 (20 cm3) under N2
was added dimethoxy(diethylamino)phosphine36 (0.72 cm3,
4.33 mmol). The mixture was stirred at room temperature for 20
min, when TLC (ethyl acetate–hexane, 1:3) indicated complete
conversion into product (Rf 0.66). The reaction mixture was
partitioned between diethyl ether (150 cm3) and water (100
cm3). The resulting ethereal layer was washed with saturated aq.
NaCl (100 cm3), dried (MgSO4), filtered and concentrated to
1
give a colourless, mobile oil which was shown by H NMR
spectroscopy to be a 1:1 anomeric mixture, and which was used
for the next step without further purification; δH (CDCl3; 400
MHz) 3.48–3.74 (11 H, m, 2 × POCH3α, 2 × POCH3β, 2-H,
3-H, 5-H, 6-HA, 6-HB), 3.77, 3.78, 3.78 and 3.79 (6 H, 4 s,
4 × 1.5 OCH3), 3.94–3.99 (1 H, m, 4-H), 3.94–3.99 (3 H, m,
3 × OCHHAr), 4.70–4.91 (5 H, m, 5 × OCHHAr), 4.94 (0.5 H,
t, J 8.07, 1-Hβ), 5.54 (0.5H, dd, J 3.18, JH-P 8.55, 1-Hα), 6.80–
6.85 (4 H, m, 2 × meta-H of PMB rings), 7.05–7.12 (2 H, m,
ortho-H of PMB rings) and 7.20–7.38 (12 H, m, ArCH); α and
β subscripts denote signals arising from α- and β-anomers
Yield of 12 (0.39 g, 38% from 10ab); Rf 0.30 (CHCl3–acetone,
2
19:1); δH (CDCl3; 400 MHz) 3.54 (1 H, 0.5 ABX, JAB 10.7,
3JAX 3.9, 5Ј-HA), 3.77–3.79 (1 H, m, 5Ј-HB, obscured by OCH3),
3.77 (3 H, s, OCH3), 3.78 (6 H, s, 2 × OCH3), 4.22–4.24 (1 H, m,
4Ј-H), 4.34 (1 H, AB, JAB 10.7, OCHHAr), 4.46–4.58 (4 H, m,
3 × OCHHAr, 3Ј-H), 5.72 (1 H, dd, J 2.9, 4.9, 2Ј-H), 6.17 (1 H,
d, J 2.9, 1Ј-H), 6.78–6.87 (9 H, m, simplifies to 8 H, m on D2O
exch., ArCH, NH), 7.17–7.35 (14 H, m, ArCH) and 8.05 and
8.07 (2 H, 2 s, 2-H, 8-H); δC (CDCl3; 100 MHz) 20.81 (CH3CO),
55.23 (3 × OCH3), 68.40 (C-5Ј), 70.61 (DMTr Cq), 72.89 and
73.39 (2 × OCH2Ar), 74.41, 75.33 and 81.55 (C-2Ј, C-3Ј, C-4Ј),
87.09 (C-1Ј), 113.15 and 113.84 (meta-C of p-methoxyphenyl
rings), 126.81 (C-5), 127.72, 127.87, 128.51, 128.78, 129.30,
129.86 and 130.10 (ArCH, ipso-C of PMB ring), 137.43 (ipso-C
of Bn ring), 137.53 (ipso-C of DMTr p-methoxyphenyl rings),
138.57 (C-8), 145.11 (ipso-C of DMTr phenyl ring), 148.00
(C-4), 152.49 (C-2), 153.66 (C-6), 158.26 (para-C of DMTr
p-methoxyphenyl rings), 159.66 (para-C of PMB ring) and
169.91 (CH3CO).
1
respectively; δP (CDCl3; 36 MHz; H decoupled) 141.14 (OPβ-
(OCH3)2) and 142.31 (OPα(OCH3)2).
2Љ,5Ј,6Љ-Tri-O-benzyl-3Ј-O-ꢀ-D-glucopyranosyl-2Ј,3Љ,4Љ-tri-O-p-
methoxybenzyl-N6-dimethoxytrityladenosine (16)
A mixture of dimethyl phosphite 15 (2.42 g, 3.50 mmol) and
acceptor 13 (1.36 g, 1.75 mmol) in dioxane (18 cm3) and toluene
(6 cm3) under N2 was stirred with 4 Å molecular sieves (approx.
1.8 g) for 2 h, when dry zinc chloride (0.57 g, 4.20 mmol) and
silver perchlorate (1.74 g, 8.40 mmol) were added. The flask was
wrapped in foil to exclude light, and stirring was continued for
7 h. Solid NaHCO3 (1.50 g) and water (60 cm3) were added
and the reaction mixture was diluted with ethyl acetate (80 cm3)
and was stirred for a further 30 min, then was filtered through a
Celite pad, and the residue was well washed with ethyl acetate.
Water (50 cm3) was added to the filtrate and the resulting aque-
ous layer was discarded. The organic layer was washed with
saturated aq. NaCl (70 cm3), dried (MgSO4), filtered and con-
centrated and the residue was subjected to flash chromato-
graphy (eluent ethyl acetate–hexane, 3:7, then 1:1) to yield the
title compound as a colourless oil (1.26 g, 53%); Rf 0.33 (ethyl
acetate–hexane, 2:3); [α]D21 ϩ10.0 (c 1.5, CHCl3) (Found: C, 71.9;
H, 6.2; N, 5.0. Calcd for C82H83N5O14: C, 72.3; H, 6.2; N, 5.1%);
δH (CDCl3; 400 MHz) 3.43–3.79 (7 H, m, 2Љ-H, 4Љ-H, 5Ј-HA,
5Ј-HB, 5Љ-H, 6Љ-HA, 6Љ-HB), 3.69, 3.77, 3.78 and 3.79 (15 H, 4 s,
5 × OCH3), 3.96 (1 H, t, J 9.3, 3Љ-H), 4.34–4.76 (14 H, m,
9 × OCHHAr, 2Ј-H, 3Ј-H, 4Ј-H), 4.87 (1 H, AB, JAB 10.3,
OCHHAr), 5.22 (1 H, d, J 3.4, 1Љ-H), 6.21 (1 H, d, J 4.9, 1Ј-H),
6.65–6.67 (2 H, m, ArCH), 6.79–6.86 (10 H, m, ArCH), 7.01–
7.05 (4 H, m, ArCH), 7.21–7.36 (24 H, m, ArCH), and 7.89 and
8.04 (2 H, 2 s, 2-H, 8-H); δC (CDCl3; 100 MHz) 55.44, 55.49
(2 × OCH3), 68.48 (C-6Љ), 69.52 (C-5Ј), 70.87 (DMTr Cq),
71.09 (C-5Љ), 72.36 and 72.50 (2 × OCH2Ar), 73.01 (C-3Ј),
73.67, 73.75, 74.97 and 75.59 (4 × OCH2Ar), 77.31 (C-4Љ),
79.65 (C-2Ј), 79.83 (C-2Љ), 81.66 (C-3Љ), 82.32 (C-4Ј), 87.10
(C-1Ј), 96.35 (C-1Љ), 113.38, 113.94 and 114.00 (meta-C of
p-methoxyphenyl rings), 121.60 (C-5), 127.04, 127.90, 127.99,
128.04, 128.12, 128.19, 128.28, 128.52, 128.61, 128.74, 129.07,
129.80 and 130.36 (ArCH), 129.34, 130.66 and 131.30
(3 × ipso-C of PMB ring), 137.75, 137.88, 138.08 and 138.39
(ipso-C of DMTr p-methoxyphenyl rings, 3 × ipso-C of ben-
zyl rings), 139.07 (C-8), 145.76 (ipso-C of DMTr phenyl
ring), 148.83 (C-4), 152.51 (C-2), 158.49 (para-C of DMTr
p-methoxyphenyl rings) and 159.37, 159.46 and 159.61
5Ј-O-Benzyl-N6-dimethoxytrityl-2Ј-O-p-methoxybenzyladeno-
sine (13)
A solution of 11 (3.85 g, 4.69 mmol), in methanol (60 cm3)
chloroform (30 cm3) and concentrated aq. NH3 (15 cm3) was
stirred in a sealed flask for 48 h. The solvents were evaporated
and the residue was repeatedly concentrated from chloroform
and then subjected to flash chromatography (eluent ethyl
acetate–hexane, 3:2) to yield the title compound as a white foam
in quantitative yield; Rf 0.39 (ethyl acetate–hexane, 1:1)
(Found: C, 70.6; H, 5.9; N, 8.9. Calcd for C46H45N5O7: C, 70.8;
H, 5.8; N, 9.0%); δH (CDCl3; 400 MHz) 3.66 (1 H, 0.5 ABX,
2JAB 10.8, 3JAX 3.5, 5Ј-HA), 3.77 (3 H, s, OCH3), 3.78–3.82 (1 H,
3
0.5 ABX obscured by OCH3, JBX 2.6, 5Ј-HB), 3.78 (6 H, s,
2 × OCH3), 4.18–4.21 (1 H, m, 4Ј-H), 4.34 (1 H, t, J 5.0, 3Ј-H),
4.47 (1 H, t, J 4.7, 2Ј-H), 4.56 and 4.60 (2 H, AB, JAB 12.0,
OCH2Ar), 4.57 and 4.66 (2 H, AB, JAB 11.7, OCH2Ar), 6.16
(1 H, d, J 4.1, 1Ј-H), 6.71–6.84 (7 H, m, ArCH), 6.93 (1 H, br s,
D2O exch., NH), 7.11–7.38 (15 H, m, ArCH) and 7.97 and 8.07
(2 H, 2 s, 2-H, 8-H); δC (CDCl3; 100 MHz) 55.59 (3 × OCH3),
69.69 (C-5Ј), 70.33, 81.24 and 84.20 (C-2Ј, C-3Ј, C-4Ј), 70.96
(DMTr Cq), 72.97 and 73.90 (2 × OCH2Ar), 87.03 (C-1Ј),
113.35 and 114.18 (meta-C of p-methoxyphenyl rings), 121.30
(C-5), 127.00, 127.94, 128.06, 128.13, 128.77, 128.82, 129.02
and 130.33 (ArCH and ipso-C of PMB ring), 137.67 (ipso-C of
DMTr p-methoxyphenyl rings), 137.72 (ipso-C of Bn ring),
138.53 (C-8), 145.63 (ipso-C of DMTr phenyl ring), 148.62
(C-4), 152.49 (C-2), 154.19 (C-6), 158.38 (C-4 of DMTr
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947
1941

View Article Online
(3 × para-C of PMB ring); m/z (FABϩ) 1362 [(M ϩ H)ϩ, 8%],
129.10 (ArCH), 135.16–135.32 (2 × ipso-C of benzylphospho
303 (100) and 121 (75).
ring with C-P coupling), 135.88, 136.03 and 136.38 (4 × ipso-C
of benzylphospho ring with C-P coupling), 137.55, 137.81 and
138.21 (3 × ipso-C of Bn rings), 139.60 (C-8), 150.26 (C-4),
153.06 (C-2) and 155.57 (C-6); δP (CDCl3; 162 MHz, 1H
decoupled) Ϫ1.31, Ϫ2.02 and Ϫ2.19 (3 s); m/z (FABϩ) 1478
[(M ϩ H)ϩ, 6%] and 91 (100).
2Љ,5Ј,6Љ-Tri-O-benzyl-3Ј-O-ꢀ-D-glucopyranosyladenosine (17)
TFA (7 cm3) was added to a solution of 16 (1.28 g, 0.94 mmol)
in CH2Cl2 (63 cm3) and the resulting bright orange solution was
stirred at room temperature under N2 for 1.75 h before being
poured into saturated aq. NaHCO3 (500 cm3). CH2Cl2 (150
cm3) was added and the colourless mixture was stirred vigor-
ously for 15 min. The organic layer was collected and the aque-
ous layer was back-extracted with CH2Cl2 (2 × 150 cm3). The
combined organic layers were dried (MgSO4), filtered and con-
centrated. The resulting crude product was purified by flash
chromatography (eluent ethyl acetate–ethanol, 14:1) to yield
the title compound as a white solid (530 mg, 81%); Rf 0.28 (ethyl
acetate–ethanol, 14:1); mp 95–115 ЊC (from ethanol); [α]D25 Ϫ2.3
(c 2.6, CHCl3) (Found C, 63.3; H, 5.9; N, 9.8. Calcd for
C37H41N5O9: C, 63.5; H, 5.9; N, 10.0%); δH ((CD3)2CO; 400
MHz) 3.45–3.51 (2 H, m, 2Љ-H, 4Љ-H), 3.66–3.81 (4 H, m, 5Ј-HA,
5Ј-HB, 6Љ-HA, 6Љ-HB), 3.93–3.96 (1 H, m, 5Љ-H), 4.04 (1 H, t,
J 9.0, 3Љ-H), 4.38–4.39 (1 H, m, 4Ј-H), 4.50–4.56 (4 H, m,
2 × OCH2Ar), 4.61–4.64 (2 H, m, 3Ј-H, OH), 4.81–4.87 (5 H,
m, OCH2Ar, 2Ј-H, 2 × OH), 5.22 (1 H, d, J 3.9, 1Љ-H), 6.12
(1 H, d, J 5.4, 1Ј-H), 6.91 (2 H, br s, NH2), 7.20–7.42 (15 H, m,
ArCH), 8.21 and 8.25 (2 H, 2 s, 2-H, 8-H); δC ((CD3)2CO; 100
MHz) 70.68 (C-6Љ), 70.74 (C-5Ј), 71.51 (C-4Љ), 73.21 (C-5Љ),
73.75, 73.88 and 73.96 (3 × OCH2Ar), 74.30 (C-3Љ), 75.11
(C-2Ј), 78.49 (C-3Ј), 80.23 (C-2Љ), 83.05 (C-4Ј), 89.14 (C-1Ј),
99.10 (C-1Љ), 120.35 (C-5), 128.11, 128.25, 128.35, 128.46,
129.02, 129.08 and 129.15 (ArCH), 139.19 and 139.78 (3 × ipso-
C of Bn rings), 140.03 (C-8), 150.73 (C-4), 153.77 (C-2) and
157.04 (C-6); m/z (FABϩ) 700 [(M ϩ H)ϩ, 100%] and 91 (99).
3-O-ꢀ-D-Glucopyranosyladenosine 2Ј,3Љ,4Љ-trisphosphate
(adenophostin A) (2)
A mixture of 18 (59 mg, 0.04 mmol) and wet 20% palladium
hydroxide on carbon (177 mg), in methanol (7 cm3), cyclo-
hexene (3.5 cm3) and water (0.5 cm3) was heated under reflux for
2.5 h. After cooling the reaction mixture was filtered through a
membrane filter and the catalyst was washed copiously with
methanol and water. Concentration of the filtrate afforded a
residue which was applied to an MP1 AG ion exchange resin
column and eluted with a gradient of 0–100% 150 mmol dmϪ3
aq. TFA. Concentration of the appropriate fractions (being
careful to keep the temperature below 20 ЊC) gave the title com-
pound as the free acid (24 mg, 92%), which was dissolved in
water and eluted through a short column of Naϩ Diaion WK-
40 ion exchange resin to give, after concentration, the sodium
salt (Found: MϪ, 668.039. Calcd for C16H25N5O18P3 (M Ϫ H)Ϫ:
668.040); δH (D2O; 400 MHz) 3.60–3.73 (6 H, m, 2Љ-H, 5Ј-HA,
5Ј-HB, 5Љ-H, 6Љ-HA, 6Љ-HB), 3.97 (1 H, q, J 8.9, 4Љ-H), 4.28 (1 H,
m, 4Ј-H), 4.37 (1 H, q, J 9.3, 3Љ-H), 4.48 (1 H, m, 3Ј-H), 5.10–
5.14 (2 H, m, 1Љ-H, 2Ј-H), 6.18 (1 H, d, J 6.4, 1Ј-H) and 8.24 and
1
8.34 (2 H, 2 s, 2-H, 8-H); δP (D2O; 162 MHz; H decoupled)
0.32, 0.87 and 1.22 (3 s); λmax (H2O) 259 nm, ε 15 400, pH 7.5;
m/z (FABϪ) 668 [(M-H)Ϫ, 100%], 266 (34) and 113 (44).
2-O-Benzyl-3,4-di-O-p-methoxybenzyl-D-xylopyranosyl
dimethyl phosphite (20)
2Љ,5Ј,6Љ-Tris-O-[bis(benzyloxy)phosphoryl]-3Ј-O-ꢀ-D-gluco-
pyranosyl adenosine (18)
To a mixture of xylopyranose 1922(1.23 g, 2.57 mmol) and 1H-
tetrazole (0.27 g, 3.85 mmol) in CH2Cl2 (12 cm3) under N2 was
added dimethoxy(diethylamino)phosphine36 (0.55 cm3, 3.34
mmol). The mixture was stirred for 20 min, when TLC (ethyl
acetate–toluene, 1:4) indicated complete conversion into prod-
uct (Rf 0.63). The reaction mixture was partitioned between
diethyl ether (100 cm3) and water (75 cm3). The resulting ether-
eal layer was washed with saturated aq. NaCl (75 cm3), dried
(MgSO4), filtered and concentrated to give a clear runny oil,
which was used without further purification; δH (CDCl3; 400
MHz) 3.23 (0.6 H, q, J 9.8, 4-Hβ), 3.40–3.95 (11.4 H, m, 2-H,
3-H, 4-Hα, 5-HA, 5-HB, P(OCH3)2 with C-P coupling), 3.77 (3.6
H, s, OCH3α), 3.77 (2.4 H, s, OCH3β), 4.54–4.90 (6.6 H, 1-Hβ,
3 × OCH2Ar), 5.42 (0.4 H, dd, J 3.4, JH-P 8.6, 1-Hα), 6.81–6.89
(4 H, m, ArCH) and 7.22–7.37 (9 H, m, ArCH); δP (CDCl3;
161.7 MHz; 1H decoupled) 140.88 OPβ(OMe)2 and 142.05
OPα(OMe)2; α and β subscripts denote signals arising from
α- and β-anomers respectively.
A mixture of 17 (100 mg, 0.14 mmol), bis(benzyloxy)(diiso-
propylamino)phosphine (0.16 cm3, 0.47 mmol) and imidazo-
lium triflate 38 (100 mg, 0.46 mmol) in CH2Cl2 (3 cm3) under N2
was stirred for 30 min, after which time TLC (ethyl acetate–
hexane, 7:3) indicated some starting material remaining; there-
fore a further 1.0 equiv. each of bis(benzyloxy)(diisopropyl-
amino)phosphine and imidazolium triflate was added. TLC
after a further 30 min indicated complete conversion to the
trisphosphite (Rf 0.68). Water (1 drop) was added and the solu-
tion was cooled to Ϫ78 ЊC. MCPBA (139 mg, 0.49 mmol) was
added and stirring was continued for 10 min. 10% (w/v) Aq.
Na2SO3 (15 cm3) and ethyl acetate (20 cm3) were added and the
mixture was allowed to warm to room temperature. The result-
ing organic layer was washed with 15 cm3 each of saturated aq.
NaHCO3 and saturated aq. NaCl and then dried (MgSO4), fil-
tered and concentrated to give an oil which was subjected to
flash chromatography (eluent chloroform–acetone, 4:1, then
7:3) to yield the title compound as a colourless oil (148 mg,
70%); Rf 0.11 (chloroform–acetone 9:1); [α]D20 ϩ11.7 (c 3.0, in
CHCl3) (Found C, 63.9; H, 5.7; N, 4.6. Calcd for C79H80-
N5O18P3: C, 64.1; H, 5.45; N, 4.7%); δH (CDCl3; 400 MHz)
3.54–3.69 (4 H, m, 2Љ-H, 5Ј-HA, 5Ј-HB, 6Љ-HA, 6Љ-HB), 3.83–3.86
(1 H, m, 5Љ-H), 4.30 (1 H, AB, JAB 11.7, OCHHAr), 4.36–4.81
(14 H, m, 4Ј-H, 3Ј-H, 4Љ-H, 11 × OCHHAr), 4.88–5.07 (8 H, m,
3Љ-H, 7 × OCHHAr), 5.33 (1 H, d, J 3.4, 1Љ-H), 5.62–5.65 (1 H,
m, 2Љ-H), 6.09 (2 H, br s, NH2), 6.35 (1 H, d, J 6.3, 1Ј-H), 6.95–
7.40 (45 H, m, ArCH), 7.92 and 8.24 (2 H, 2 s, 2-H, 8-H);
δC (CDCl3; 100 MHz) 68.51 (C-6Љ), 68.28–70.07 (C-5Ј,
6 × POCH2Ar with C-P coupling), 70.27 (C-5Ј), 71.77, 73.53
and 73.76 (3 × OCH2Ar), 73.87 and 74.54 (C-3Ј, C-4Ј with C-P
coupling), 77.00 (C-2Љ), 77.40 (C-2Ј with C-P coupling), 78.28
(C-3Љ with C-P coupling), 82.60 (C-4Ј), 85.78 (C-1Ј), 95.60
(C-1Љ), 119.98 (C-5), 127.80, 127.93, 128.02, 128.17, 128.26,
128.48, 128.61, 128.65, 128.68, 128.74, 128.79, 129.03 and
2Љ,5Ј-Di-O-benzyl-2Ј,3Љ,4Љ-tri-O-p-methoxybenzyl-N6-
dimethoxytrityl-3Ј-O-D-xylopyranosyladenosine (21ab)
A mixture of dimethyl phosphite 20 (1.47 g, 2.57 mmol) and 13
(1.00 g, 1.28 mmol) in dioxane (15 cm3) and toluene (5 cm3)
under N2 was stirred with 4 Å molecular sieves (approx. 1.50 g)
for 2 h, and then dry zinc chloride (0.42 g, 3.08 mmol) and silver
perchlorate (1.26 g, 6.16 mmol) were added. The flask was
wrapped in foil to exclude light and stirring was continued for
9 h. Solid NaHCO3 (1.00 g) and water (30 cm3) were added
and the reaction mixture was diluted with ethyl acetate (40 cm3).
After stirring for a further 30 min the mixture was filtered
through a Celite pad, and the residue was well washed with
ethyl acetate. Water (30 cm3) was added to the filtrate and the
resulting aqueous layer was discarded. The organic layer was
washed with saturated aq. NaCl (75 cm3), dried (MgSO4),
1942
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947

View Article Online
filtered and concentrated and the residue was subjected to flash
chromatography (eluent ethyl acetate–hexane, 3:7, then 2:3,
then 1:1) to yield the title compound as an inseparable 1:1
anomeric mixture (0.76 g, 46%); Rf 0.38 (ethyl acetate–toluene,
1:4); selected δH (CDCl3; 400 MHz) 6.13 (0.5 H, d, J 1.5, 1Ј-Hα)
and 6.22 (0.5 H, d, J 4.4, 1Ј-Hβ); α and β subscripts denote
signals arising from α- and β-anomers respectively; m/z (FABϩ)
1246 [(M ϩ H)ϩ, 14%], 303 (100) and 121 (72).
acetate (20 cm3) were added and the mixture was allowed to
warm to room temperature. The resulting organic layer was
washed with 15 cm3 each of saturated aq. NaHCO3 and satur-
ated aq. NaCl, dried (MgSO4), filtered and concentrated to give
a clear oil which was subjected to flash chromatography (eluent
chloroform–acetone, 9:1, then 7:1, then 6:1, then 4:1, then
3:2) to give the title compound as a clear oil (161 mg, 68%);
Rf 0.11 (ethyl acetate–hexane, 7:3); [α]D20 ϩ6.5 (c 1.7, CHCl3)
(Found Mϩ, 1360.425. Calcd for C71H73N5O17P3 (M ϩ H)ϩ:
1360.421); δH (CDCl3; 400 MHz) 3.49 (1 H, dd, J 3.5, 9.4, 2Љ-H),
2Љ,5Ј-Di-O-benzyl-3Ј-O-ꢀ-D-xylopyranosyladenosine (22) and
2Љ,5Ј-di-O-benzyl-3Ј-O-ꢁ-D-xylopyranosyladenosine (23)
2
3.52 (1 H, dd, J = 3J 11.0, 5Љ-Hax), 3.62 and 3.76 (2 H, ABX,
3
3
2
2JAB 10.8, JAX 3.2, JBX 3.4, 5Ј-HA, 5Ј-HB), 3.95 (1 H, dd, J
11.1, 3J 5.9, 5Љ-Heq), 4.33–5.02 (14 H, m, 3Ј-H, 3Љ-H, 4Ј-H, 4Љ-H,
6 × POCH2Ar, 2 × OCH2Ar), 5.29 (1 H, d, J 3.2, 1Љ-H), 5.61–
5.66 (1 H, m, 2Љ-H), 5.98 (2 H, br s, NH2), 6.34 (1 H, d, J 5.9, 1Ј-
H), 6.98–7.39 (40 H, m, ArCH) and 7.93 and 8.25 (2 H, 2 s, 2-H,
8-H); δC (CDCl3; 100.4 MHz) 60.25 (C-5Љ), 69.40–70.23 (C-5Ј,
6 × POCH2Ar with C-P coupling), 72.12 (OCH2Ar), 73.59
(C-4Љ with C-P coupling), 73.97 (OCH2Ar and C-3Ј), 77.07
(C-2Љ), 77.71 (C-2Ј, C-3Љ with C-P coupling), 82.55 (C-4Ј), 86.01
(C-1Ј), 95.81 (C-1Љ), 120.05 (C-5), 127.78, 127.91, 127.98,
128.15, 128.19, 128.22, 128.26, 128.34, 128.48, 128.53, 128.68,
128.72 and 128.79 (ArCH), 135.14–136.23 (6 × ipso-C of
benzylphospho rings), 137.14 and 137.64 (2 × ipso-C of Bn
rings), 139.54 (C-8), 150.14 (C-4), 153.14 (C-2) and 155.54
A solution of 21ab (745 mg, 0.60 mmol) in CH2Cl2 (27 cm3) and
TFA (3 cm3) was stirred for 1 h under N2 before being poured
into saturated aq. NaHCO3 (150 cm3). CH2Cl2 (75 cm3) was
added and mixture was stirred vigorously for 30 min. The
resulting aqueous layer was extracted with CH2Cl2 (2 × 75 cm3)
and the combined organic layers were dried (MgSO4), filtered
and concentrated. The resulting crude product was purified by
flash chromatography (eluent ethyl acetate–methanol, 14:1) to
yield the α-anomer 22 (138 mg, 40%); Rf 0.51 (chloroform–
methanol, 9:1); [α]D18 ϩ22.1 (c 2.0, acetone) (Found Mϩ, 580.240.
Calcd for C29H34N5O8 (M ϩ H)ϩ: 580.240); δH ((CD3)2CO; 400
MHz) 3.12 (1 H, br s, OH), 3.44 (1 H, dd, J 3.7, 9.5, 2Љ-H), 3.60–
3.65 (3 H, m, 4Љ-H, 5Љ-Hax, 5Љ-Heq), 3.74 and 3.83 (2 H, ABX,
2JAB 10.9, JAX 3.8, JBX 3.4, 5Ј-HA, 5Ј-HB), 3.95–3.99 (1 H, m,
3Љ-H), 4.36 (1 H, q, J 4.1, 4Ј-H), 4.57–4.63 (4 H, m, OCH2Ar),
4.71 (1 H, br s, OH), 4.84–4.88 (1 H, m, 2Ј-H obscured by HDO
peak), 5.03 (1 H, br s, OH), 5.17 (1 H, d, J 3.8, 1Љ-H), 6.12 (1 H,
d, J 4.7, 1Ј-H), 6.98 (2 H, br s, NH2), 7.19–7.43 (10 H, m,
ArCH) and 8.22 and 8.26 (2 H, 2 s, 2-H, 8-H); δC ((CD3)2CO;
100 MHz) 62.82 (C-5Љ), 69.91 (C-5Ј), 70.59 (C-4Љ), 73.34 and
73.66 (2 × OCH2Ar), 73.96 (C-3Љ), 74.44 (C-2Ј), 77.47 (C-3Ј),
79.70 (C-2Љ), 82.29 (C-4Ј), 88.66 (C-1Ј), 98.58 (C-1Љ), 119.65
(C-5), 127.72, 127.81, 128.33, 128.40 and 128.51 (ArCH),
138.40 and 138.43 (2 × ipso-C of Bn rings), 139.39 (C-8), 149.89
(C-4), 153.04 (C-2) and 156.28 (C-6); m/z (FABϩ) 580
[(M ϩ H)ϩ, 100%] and 91 (79).
3
3
1
(C-6); δP (CDCl3; 162 MHz; H decoupled) Ϫ0.63 (2 P, s) and
Ϫ0.20 (1 P, s); m/z (FABϩ) 1360 [(M ϩ H)ϩ, 5%] and 91 (100).
3Ј-O-ꢀ-D-Xylopyranosyladenosine 2Ј,3Љ,4Љ-trisphosphate (xylo-
adenophostin) (5)
A mixture of 24 (84 mg, 0.06 mmol) and wet 20% palladium
hydroxide on carbon (252 mg), in methanol (11 cm3), cyclo-
hexene (5 cm3) and water (1 cm3) was heated under reflux for 2.5
h. After cooling the reaction mixture was filtered through a
membrane filter and the catalyst was washed copiously with
methanol and water. Concentration of the filtrate afforded a
clear residue which was applied to an MP1 AG ion exchange
resin column and eluted with a gradient of 0–100% 150 mmol
dmϪ3 TFA. Concentration of the appropriate fractions (being
careful to keep the temperature below 20 ЊC) gave the desired
product as the free acid (33 mg, 85%), which was dissolved in
water and eluted through a short column of Naϩ Diaion WK-40
ion exchange resin to give, after concentration, the sodium salt;
(Found: MϪ, 638.030. Calcd for C15H23N5O17P3 (M Ϫ H)Ϫ:
Further elution gave the β-anomer 23 (143 mg, 41%); Rf 0.43
(chloroform–methanol, 9:1) (Found Mϩ, 580.241. Calcd for
C29H34N5O8 (M ϩ H)ϩ: 580.240); δH (CD3OD; 400 MHz) 3.18
2
(1 H, dd, J = 3J 10.8, 5Љ-Hax), 3.29 (1 H, dd, J 7.6, 9.1, 2Љ-H),
3.46 (1 H, t, J 8.9, 3Љ-H), 3.53–3.58 (1 H, m, 4Љ-H), 3.61 and 3.77
2
3
3
(2 H, ABX, JAB 11.0, JAX 3.1, JBX 2.8, 5Ј-HA, 5Ј-HB), 3.85
(1 H, dd, 2J 11.4, 3J 5.3, 5Љ-Heq), 4.34 (1 H, ddd, J 2.9, 2.9, 5.6,
4Ј-H), 4.43 and 4.48 (2 H, AB, JAB 12.0, OCH2Ar), 4.46 (1 H, d,
J 7.9, 1Љ-H), 4.53 (1 H, t, J 5.1, 3Ј-H), 4.66 (1 H, t, J 4.2, 2Ј-H),
4.78 and 4.86 (2 H, AB, JAB 11.1, OCH2Ar), 6.11 (1 H, d, J 3.8,
1Ј-H), 7.19–7.39 (10 H, m, ArCH) and 8.18 and 8.30 (2 H, 2 s,
2-H, 8-H); δC (CD3OD; 100 MHz) 67.17 (C-5Љ), 70.30 (C-5Ј),
71.22 (C-4Љ), 74.63 and 75.97 (OCH2Ar), 76.11 (C-2Ј), 77.62
(C-3Љ), 79.79 (C-3Ј), 82.81 (C-2Љ), 83.12 (C-4Ј), 90.27 (C-1Ј),
105.19 (C-1Љ), 120.31 (C-5), 128.62, 128.97, 128.01, 129.33 and
129.61 (ArCH), 139.14 and 140.13 (2 × ipso-C of Bn rings),
140.63 (C-8), 150.36 (C-4), 153.91 (C-2) and 157.11 (C-6); m/z
(FABϩ) 580 [(M ϩ H)ϩ, 100%] and 91 (67).
2
638.030); δH (D2O; 400 MHz) 3.50 (1 H, dd, J = 3J 10.8, 5Љ-
Hax), 3.61 (1 H, dd, J 3.4, 9.2, 2Љ-H), 3.65–3.71 (2 H, m, 5Ј-HA,
5Ј-HB), 3.76 (1 H, dd, J 5.6, 11.4, 5Љ-Heq), 4.02–4.10 (1 H, m,
4Љ-H), 4.25–4.32 (2 H, m, 3Љ-H, 4Љ-H), 4.34–4.56 (1 H, m, 3Ј-H),
5.07 (1 H, d, J 3.5, 1Љ-H), 5.08–5.14 (1 H, m, 2Ј-H), 6.16 (1 H, d,
J 6.2, 1Ј-H), 8.23 and 8.33 (2 H, 2 s, 2-H,8-H); δP (D2O; 162
1
MHz; H decoupled) 0.23, 0.67 and 0.78 (3 s); λmax (H2O) 259
nm, ε 15 400, pH 7.5; m/z (FABϪ) 638 [(M Ϫ 1)Ϫ, 100%].
Allyl ꢀ-D-mannopyranoside (25)
Allyl alcohol (160 cm3) and acetyl chloride (5 cm3) were stirred
together for 1 hour after which time -mannose (20.0 g, 111
mmol) was added. The mixture was heated at 50 to 60 ЊC under
N2 with vigorous stirring for 5 h. TLC (ethyl acetate–propan-2-
ol–water, 9:4:2) showed a major product at Rf 0.49. The clear
solution was allowed to cool, triethylamine (20 cm3) was added
and the solvents were removed by evaporation in vacuo at 50 ЊC
to leave an orange oil (≈30 g). Purification by flash chromato-
graphy on silica (300 g) eluting with dichloromethane–acetone
1:2 to 1:4 gave a pale yellow oil (18.4 g) which crystallised on
standing. Recrystallisation from hot acetone (200 cm3) gave the
title compound as colourless needles (15.0 g, 61%); Rf 0.49 (ethyl
acetate–propan-2-ol–water, 9:4:2); mp 100–101.5 ЊC (from
acetone) (lit.,40 98–99 ЊC; lit.,41 138–139 ЊC); [α]D20 ϩ84.5 (c 2.0, in
water) [lit.,40 ϩ99 (in water); lit.,41 ϩ51.6 (c 0.23, in water)]
2Љ,5Ј-Di-O-benzyl-2Ј,3Љ,4Љ-tris-O-[bis(benzyloxy)phosphoryl]-3Ј-
O-ꢀ-D-xylopyranosyladenosine (24)
A solution of triol 22 (101 mg, 0.17 mmol), bis(benzyloxy)-
(diisopropylamino)phosphine (0.19 cm3, 0.58 mmol) and imi-
dazolium triflate 38 (125 mg, 0.58 mmol) in CH2Cl2 (3.5 cm3)
under N2 was stirred for 1 h, after which time TLC (ethyl
acetate–hexane, 7:3) indicated some starting material remain-
ing; therefore a further 1.0 equivalent each of bis(benzyloxy)-
(diisopropylamino)phosphine and imidazolium triflate was
added. TLC after a further 30 min indicated conversion into the
trisphosphite (Rf 0.63). Water (1 drop) was added, the solution
was cooled to Ϫ78 ЊC and MCPBA (170 mg, 0.59 mmol) was
added. After 10 min 10% (w/v) aq. Na2SO3 (15 cm3) and ethyl
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947
1943

View Article Online
(Found: C, 49.1; H, 7.3. Calcd for C9H16O6: C, 49.1; H, 7.3%);
1H NMR data were identical to those previously reported;41
δC (D2O; 100 MHz) 61.14 (C-6), 66.98 (C-4), 68.34 (OCH2-
and 73.35 (2 × CH2Ph), 98.46 (C-1), 99.54 and 99.82 (C-2Ј and
C-3Ј), 117.34 (CH CH᎐CH ), 127.27, 127.34, 127.41, 127.52,
127.58, 127.91, 128.16 and 128.49 (Ph CH), 133.85 (CH2-
᎐
2
2
CH᎐CH ), 70.24 and 70.78 (C-2 and C-3), 73.00 (C-5), 99.15
CH᎐CH ) and 138.59 and 138.82 (2 × ipso-C of Ph); m/z
᎐
᎐
2
2
(C-1), 118.58 (OCH CH᎐CH ) and 133.33 (OCH CH᎐CH );
(FABϩ) 537 [(M ϩ Na)ϩ, 36%], 513 [(M Ϫ 1)ϩ, 14], 483
[(M Ϫ OMe)ϩ, 90], 101 (28) and 91 [(C7H7)ϩ, 100].
᎐
᎐
2
2
2
2
m/z (FABϩ) 243 [(M ϩ Na)ϩ, 100%], 221 [(M ϩ 1)ϩ, 14] and
163 [(M Ϫ C3H5O)ϩ, 70]; m/z (FABϪ) 373 [(M ϩ NBA)Ϫ, 100%]
and 219 [(M Ϫ 1)Ϫ, 62].
Allyl 2,6-di-O-benzyl-ꢀ-D-mannopyranoside (28)
To a solution of 27 (8.00 g, 15.5 mmol) in CH2Cl2 (40 cm3) was
added 95% (v/v) TFA in water (40 cm3). TLC (ether–hexane,
1:1) showed complete conversion of 27 into a single product
(Rf 0.24) within 15 minutes. The solvents were removed by
evaporation under reduced pressure to leave an oily residue,
which was taken up in ether (100 cm3), washed with saturated
NaHCO3 (100 cm3) and dried (MgSO4). Concentration by
evaporation under reduced pressure gave a yellow oil which was
purified by flash chromatography using acetate–hexane 1:1 as
eluent to give the diol 28 (5.28 g, 85%) as a colourless oil
(Found: C, 68.6; H, 7.1. Calcd for C23H28O6: C, 69.0; H, 7.05%);
[α]D23 ϩ7.2 (c 2.1, in CHCl3); δH (CDCl3; 270 MHz) 2.64 (1 H, d,
J 8.6, D2O exch, OH), 3.07 (1 H, br s, D2O exch., OH), 3.70–3.85
(6 H, m, 2-, 3-, 4- and 5-H and 6-H2), 3.92–4.00 (1 H, m, CHH-
(2ЈS,3ЈS)-Allyl 3,4-O-(2Ј,3Ј-dimethoxybutane-2Ј,3Ј-diyl)-ꢀ-D-
mannopyranoside (26)
To a solution of 25 (11.0 g, 50.0 mmol), trimethyl orthoformate
(20 cm3) and CSA (500 mg) in methanol (200 cm3) was added
butanedione (5.0 cm3, 57 mmol). The mixture was heated at
reflux under N2. TLC (ethyl acetate) after 1 h showed two major
products (Rf 0.36 and 0.40), but after 10 h only the more polar
product remained. The mixture was allowed to cool, triethyl-
amine (1 cm3) was added, and stirring was continued at room
temperature for a further 1 h. The solvents were removed by
evaporation under reduced pressure, leaving an orange oil.
Purification by flash chromatography on silica eluting with
ethyl acetate–hexane 2:1 gave the diacetal 26 as a hygroscopic
foam (13.1 g, 78%) (Found C, 53.5; H, 8.1. Calcd for C15H26O8:
C, 53.9; H, 7.8%); [α]D23 ϩ232 (c 1.0, in CHCl3); δH (CDCl3; 270
MHz) 1.29 (3 H, s, Me), 1.33 (3 H, s, Me), 2.49 (1 H, t, J 5.1,
D2O exch., 6-OH), 3.11 (1 H, d, J 2.4, D2O exch., 2-OH), 3.27 (3
H, s, OMe), 3.28 (3 H, s, OMe), 3.70–3.90 (3 H, m, 5-H and 6-
CH᎐CH ), 4.13–4.22 (1 H, m, CHHCH᎐CH ), 4.52–4.72 (4 H,
᎐
᎐
2
2
m, 2 × CH2Ph), 4.93 (1 H, br s, 1-H), 5.14–5.20 (1 H, m,
CH CH᎐CH CH ), 5.20–5.29 (1 H, m, CH CH᎐CH -
᎐
᎐
2
cis
trans
2
cis
CHtrans), 5.78–5.94 (1 H, m, CH CH᎐CH ), and 7.25–7.34
᎐
2
2
(10 H, m, Ph); δC (CDCl3; 68 MHz) 69.56, 70.79 and 71.45 (C-3,
C-4 and C-5), 67.81 (CH CH᎐CH ), 70.11 (C-6), 72.87 and
H ,), 3.92–4.22 (5 H, m, 2-, 3- and 4-H and CH CH᎐CH), 4.89
᎐
᎐
2
2
2
2
(1 H, d, J 1.3, 1-H), 5.18–5.23 (1 H, m, CH CH᎐CH CHtrans),
73.43 (2 × CH2Ph), 77.76 (C-2), 96.14 (C-1), 117.29
(CH CH᎐CH ), 127.50, 127.76, 127.86, 128.26 (Ph CH), 133.58
᎐
2
cis
5.24–5.34 (1 H, m, CH CH᎐CH CHtrans) and 5.81–5.90 (1 H,
᎐
᎐
2
cis
2
2
m, CH CH᎐CH ); δ (CDCl3; Me4Si; 100 MHz) 17.70 and
17.79 (2 × Me), 47.92 and 48.12 (2 × OMe), 61.17 (C-6), 62.86
(C-4), 68.16, 69.71 and 70.78 (C-2, C-3 and C-5), 68.16
(CH CH᎐CH ) and 137.61 and 138.09 (2 × ipso-C of Ph);
᎐
2 2
᎐
2
2
C
m/z (FABϩ) 423 [(M ϩ Na)ϩ, 77%], 399 [(M Ϫ 1)ϩ, 14], 343
[(M Ϫ C3H5O)ϩ, 15], 181 (16) and 91 [(C7H7)ϩ, 100]; m/z
(FABϪ) 553 [(M ϩ NBA)Ϫ, 100%] and 399 [(M Ϫ 1)Ϫ, 56].
(CH CH᎐CH ), 99.29 (C-1), 99.82 and 100.35 (C-2Ј and C-3Ј),
᎐
2
2
117.70 (CH CH᎐CH ) and 133.69 (CH CH᎐CH ); m/z (FABϩ)
᎐
᎐
2
2
2
2
691 [(2M ϩ Na)ϩ, 84%], 357 [(M ϩ Na)ϩ, 60], 303 [(M Ϫ
OMe)ϩ, 62] and 101 (100); m/z (FABϪ) 333.1 [(M Ϫ 1)Ϫ, 100%].
Allyl 2,6-di-O-benzyl-3,4-di-O-p-methoxybenzyl-ꢀ-D-manno-
pyranoside (29)
To a solution of 28 (5.00 g, 12.5 mmol) in dry DMF (100 cm3)
at 0 ЊC was added NaH (1.50 g of a 60% dispersion in mineral
oil, 37.5 mmol). The mixture was stirred at 0 ЊC for 30 min and
then p-methoxybenzyl chloride (4.0 cm3, 30 mmol) was added
dropwise over 2 min. The mixture was allowed to reach room
temperature and stirred for 3 h. TLC (ether–hexane, 1:1)
showed conversion into a major product (Rf 0.36), but the reac-
tion was not complete, and so more p-methoxybenzyl chloride
(0.6 cm3, 4 mmol) was added and stirring was continued at
room temperature for a further 14 h. Excess NaH was destroyed
by careful addition of water and solvents were removed by
evaporation under reduced pressure. The residue was par-
titioned between ether and water (100 cm3 of each) and the
organic layer was washed sequentially with 0.1 M HCl, satur-
ated NaHCO3 solution and brine (100 cm3 of each), dried
over MgSO4 and concentrated by evaporation under reduced
pressure to give a yellow oil. Purification by flash column
chromatography using ether–hexane 1:2 as eluent gave the title
compound 29 (5.31 g, 66%) as a colourless oil (Found: C, 73.3;
H, 6.9. Calcd for C39H44O8: C, 73.1; H, 6.9%); [α]D20 ϩ30 (c 1.4, in
CHCl3); δH (CDCl3; 270 MHz) 3.68–3.80 (4 H, m, 2-H, 4-H and
6-H2), 3.77 and 3.80 (6 H, 2 s, 2 × OMe), 3.86–3.98 (3 H, m,
(2ЈS,3ЈS)-Allyl 2,6-di-O-benzyl-3,4-O-(2Ј,3Ј-dimethoxybutane-
2Ј,3Ј-diyl)-ꢀ-D-mannopyranoside (27)
To a solution of 26 (7.10 g, 21.2 mmol) in dry DMF (100 cm3)
at 0 ЊC was added NaH (2.55 g of a 60% dispersion in mineral
oil, 63.7 mmol). The mixture was stirred at 0 ЊC for 1 h and then
benzyl bromide (5.6 cm3, 47 mmol) was added gradually over 1
min. After a further 1 h at 0 ЊC the mixture was allowed to reach
room temperature and stirred for 14 h. Excess NaH was des-
troyed by careful addition of water and solvents were removed
by evaporation under reduced pressure. The residue was par-
titioned between ether and water (100 cm3 of each) and the
organic layer was washed sequentially with 0.1 mol dmϪ3 HCl
and saturated NaHCO3 solution (100 cm3 of each), dried over
MgSO4 and concentrated by evaporation under reduced pres-
sure to give a yellow oil. Purification by flash column chrom-
atography using ether–hexane 1:3 as eluent gave the title
compound (9.62 g, 88%) as a colourless oil, which slowly crystal-
lised; mp 56–58 ЊC (from hexane); [α]D23 ϩ155 (c 1.1, in CHCl3)
(Found C, 67.6; H, 7.3. Calcd for C29H38O8: C, 67.7; H, 7.4%);
δH (CDCl3; 400 MHz) 1.27 (3 H, s, Me), 1.33 (3 H, s, Me), 3.19
and 3.27 (6 H, 2 s, 2 × OMe), 3.72 (1 H, dd, J 1.4, 3.0, 2-H),
3.75–3.78 (2 H, m, 6-H2), 3.91–3.96 (2 H, m, 5-H and CHH-
3-H, 5-H and CHHCH᎐CH ), 4.10–4.19 (1 H, m, CHH-
᎐
2
CH᎐CH ), 4.10 (1 H, dd, J 10.3, 3.0, 3-H), 4.13–4.18 (1 H, m,
CH᎐CH ), 4.38–4.82 (8 H, m, 4 × CH Ar), 4.91 (1 H, d, J 1.6,
᎐
2 2
᎐
2
CHHCH᎐CH ), 4.21 (1 H, t, J 10.3, 4-H), 4.57 and 4.63 (2 H,
1-H), 5.11–5.15 (1 H, m, CH CH᎐CH CHtrans), 5.16–5.24 (1 H,
᎐
2 cis
᎐
2
AB, JAB 12.2, CH2Ph), 4.67 and 4.93 (2 H, AB, JAB 12.2,
CH2Ph), 4.86 (1 H, d, J 1.4, 1-H), 5.12–5.16 (1 H, m, CH2-
m, CH CH᎐CH CH ), 5.76–5.92 (1 H, m, CH CH᎐CH ),
᎐ ᎐
2 cis trans 2 2
6.76–6.82 (2 H, m, meta-H of PMB ring), 6.84–6.89 (2 H, m,
meta-H of PMB ring), 7.04–7.10 (2 H, m, ortho-H of PMB
ring) and 7.24–7.41 (12 H, m, ArH); δC (CDCl3; 68 MHz) 55.22
CH᎐CH CH ), 5.18–5.24 (1 H, m, CH CH᎐CH CHtrans),
᎐
᎐
cis
trans
2
cis
5.80–5.90 (1 H, m, CH CH᎐CH ), 7.22–7.33 (8 H, m, Ph), and
᎐
2
2
7.43 (2 H, d, J 7.3, Ph); δC (CDCl3; 100 MHz) 17.83 (2 × Me),
47.86 and 47.97 (2 × OMe), 63.81 (C-4), 69.13 (C-3), 70.94
(OMe), 67.69 (CH CH᎐CH ), 69.23 (C-6), 71.84, 74.57 and
᎐
2 2
74.68 (C-3, C-4 and C-5), 71.83, 72.52, 73.27 and 74.73
(C-5), 75.79 (C-2), 67.83 (CH CH᎐CH ), 68.84 (C-6), 73.04
(4 × CH2Ar), 79.95 (C-2), 97.08 (C-1), 113.68 (2 × meta-C of
᎐
2
2
1944
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947

View Article Online
PMB rings), 117.06 (CH CH᎐CH ), 127.39, 127.50, 127.70,
h at room temperature, and then dry zinc chloride (0.35 g, 2.58
᎐
2
2
127.76, 128.23, 128.26, 129.21, 129.61 (Ar CH), 130.71 (2 ×
ipso-C of PMB rings), 133.78 (CH CH᎐CH ), 138.40 and
138.43 (2 × ipso-C of Ph) and 159.09 (2 × para-C of PMB
rings); m/z (FABϩ) 663 [(M ϩ Na)ϩ, 40%], 639 [(M Ϫ 1)ϩ, 42],
519 [(M Ϫ PMB)ϩ, 88], 121 (100) and 91 [(C7H7)ϩ, 24].
mmol) and silver perchlorate (1.07 g, 5.16 mmol) were added.
The flask was wrapped in foil to exclude light, and stirring was
continued for 8 h. Solid NaHCO3 (1.00 g) and water (30 cm3)
were added and the reaction mixture was diluted with ethyl
acetate (40 cm3). After stirring for a further 30 min the mixture
was filtered through a Celite pad, and the residue was well
washed with ethyl acetate. Water (20 cm3) was added to the
filtrate, and the resulting aqueous layer was discarded. The
organic layer was washed with saturated aq. NaCl (50 cm3),
dried (MgSO4), filtered and concentrated and the residue was
subjected to flash chromatography (eluent ethyl acetate–hexane,
3:7, then 1:1) to yield the title compound as a clear oil (0.89 g,
61%); Rf 0.53 (ethyl acetate–toluene, 1:4); [α]D18 Ϫ1.0 (c 1.0, in
CHCl3) (Found C, 72.1; H, 6.2; N, 5.1. Calcd for C82H83N5O14:
C, 72.3; H, 6.1; N, 5.1%); δH (CDCl3; 400 MHz) 3.55 (1 H,
ABX, JAB10.7, JAX 2.9, 5Ј-HA), 3.59–3.73 (4 H, m, 3Љ-H or
4Љ-H, 5Ј-HB, 6Љ-HA, 6Љ-HB), 3.66 (3 H, s, OCH3), 3.77 (9 H, s,
3 × OCH3), 3.79 (3 H, s, OCH3), 3.83–3.91 (3 H, m, 2Љ-H, 3Љ-H
or 4Љ-H, 5Љ-H), 4.28 (1 H, t, J 2.9, 4Ј-H), 4.37–4.60 (13 H, m, 2Ј-
H, 3Ј-H, 11 × OCHHAr), 4.77 (1 H, AB, JAB 10.3, OCHHAr),
5.05 (1 H, s, 1Љ-H), 6.16 (1 H, d, J 5.4, 1Ј-H), 6.63–6.67 (2 H, m,
ArCH), 6.78–6.81 (4 H, m, ArCH), 6.86–6.90 (2 H, m, ArCH),
6.96–6.99 (2 H, m, ArCH), 7.06–7.08 (2 H, m, ArCH), 7.20–
7.35 (28 H, m, ArCH) and 7.90 and 8.07 (2 H, 2 s, 2-H, 8-H);
δC (CDCl3; 100 MHz) 55.07, 55.15 and 55.20 (5 × OCH3), 69.08
(C-6Љ), 69.52 (C-5Ј), 70.58 (DMTr Cq), 71.75, 72,03, 72.52,
73.29, 73.53 and 74.70 (6 × OCH2Ar), 72.37, 74.28 and 74.59
(C-3Љ, C-4Љ, C-5Љ), 73.21 (C-3Ј), 79.62 (C-2Ј, C-2Љ), 82.56 (C-4Ј),
86.18 (C-1Ј), 97.81 (C-1Љ), 113.06, 113.65 and 113.70 (3 × meta-
C of p-methoxyphenyl rings), 121.00 (C-5), 126.73, 127.40,
127.48, 127.55, 127.60, 127.73, 127.17, 128.24, 128.48, 128.76,
129.16, 129.49, 129.63, 130.05 and 130.49 (ArCH), 128.81,
130.51 and 130.57 (3 × ipso-C of PMB rings), 137.42 and
138.17 (ipso-C of DMTr p-methoxyphenyl rings, 3 × ipso-C of
Bn rings), 138.30 (C-8), 145.43 (ipso-C of DMTr phenyl ring),
148.62 (C-4), 152.29 (C-2), 153.99 (C-6), 158.20 (2 × para-C of
DMTr p-methoxyphenyl rings) and 159.06, 159.11 and 159.41
(para-C of PMB ring); m/z (FABϩ) 1362 (Mϩ, 7%), 436 (6), 303
(100) and 121 (88).
᎐
2
2
2,6-Di-O-benzyl-3,4-di-O-p-methoxybenzyl-D-mannopyranose
(30)
A solution of 29 (2.60 g, 4.06 mmol) in dry methanol (20 cm3)
was stirred vigorously with PdCl2 (100 mg) for 3 hours, after
which time TLC (ethyl acetate–hexane, 1:2) showed the reac-
tion to be essentially complete with conversion of 29 (Rf 0.40)
into a major product with Rf 0.14. Triethylamine (0.5 cm3) was
added and the suspension was filtered through Celite. The fil-
trate was concentrated by evaporation under reduced pressure
and the residue was purified by flash chromatography (eluent
ethyl acetate–hexane, 2:3) to give the mannopyranose 30 (ratio
of α- to β-anomers 3:1) as a colourless glass (1.92 g, 79%)
(Found: C, 71.7; H, 6.7. Calcd for C39H44O8: C, 72.0; H, 6.7%);
δH (CDCl3; 400 MHz) 3.18 (0.75 H, br s, D2O exch., 1-OHα),
3.41 (0.25 H, ddd, J 9.3, 4.4, 2.9, 5-Hβ), 3.56 (0.25 H, dd, J 9.3,
2.4, 3-Hβ), 3.60–3.71 (2 H, m, 6-H2α and 6-H2β), 3.75–3.83 (7.75
H, 2-Hα, 2-Hβ, 4-Hα and 2 × OMe), 3.88 (0.25 H, t, J 9.3, 4-Hβ),
3.92 (0.75 H, dd, J 9.8, 3.4, 3-Hα), 3.97–4.01 (0.75 H, m, 5-Hα),
4.40–4.80 (8 H, m, 4 × CH2Ar in α-anomer, 3.5 × CH2Ar in
β-anomer and H-1β), 5.07 (0.25 H, d, J 11.7, CHCHAr in
β-anomer), 5.23 (0.75 H, br s, D2O exch. gives d, J 1.6, 1-Hα),
6.78–6.81 (2 H, m, meta-H of PMB ring), 6.84–6.88 (2 H, m,
meta-H of PMB ring), 7.06–7.09 (2 H, m, ortho-H of PMB
ring) and 7.25–7.37 (12 H, m, ArH); δC (CDCl3; 68 MHz, data
for α-anomer only) 55.25 (OMe), 69.59 (C-6), 71.84, 72.62,
73.22 and 74.65 (4 × CH2Ar), 71.37, 74.81 and 74.89 (C-3, C-4
and C-5), 79.48 (C-2), 92.68 (C-1), 113.70 and 113.75 (meta-C
of PMB rings), 127.57, 127.83, 128.01, 128.31, 129.25 and
129.64 (Ar CH), 130.60 and 130.67 (2 × ipso-C of PMB rings),
137.98 and 138.40 (2 × ipso-C of Ph) and 159.11 (2 × para-C of
PMB rings); m/z (FABϩ) 623 [(M ϩ Na)ϩ, 74%], 599 [(M Ϫ 1)ϩ,
24], 121 (100); m/z (FABϪ) 753 [(M ϩ NBA)Ϫ, 100%] and
311(52).
2
3
2Љ,5Ј,6Љ-Tri-O-benzyl-3Ј-O-ꢀ-D-mannopyranosyl adenosine (33)
2,6-Di-O-benzyl-3,4-di-O-p-methoxybenzyl-D-mannopyranosyl
A solution of 32 (528 mg, 0.39 mmol) in CH2Cl2 (27 cm3) and
TFA (3 cm3) was stirred for 5 h under N2 before being poured
into saturated aq. NaHCO3 (200 cm3). CH2Cl2 (100 cm3) was
added and the mixture was stirred vigorously for 30 min. The
resulting aqueous layer was extracted with CH2Cl2 (2 × 100
cm3) and the combined organic layers were dried (MgSO4), fil-
tered and concentrated. The resulting crude product was puri-
fied by flash chromatography (eluent ethyl acetate–ethanol,
14:1, then 9:1) to yield the title compound as a white solid
(223 mg, 82%); mp 175–178 ЊC (from ethanol); Rf 0.22 (ethyl
acetate–ethanol, 14:1) (Found Mϩ, 700.296. Calcd for
C37H42N5O9 (M ϩ H)ϩ: 700.298); δH (d6-DMF; 400 MHz) 3.68–
3.86 (6 H, m, 4Љ-H, 5Љ-H, 5Ј-HA, 5Ј-HB, 6Љ-HA, 6Љ-HB), 3.93–3.96
(1 H, m, 3Љ-H), 3.99–4.00 (1 H, m, 2Љ-H), 4.39 (1 H, q, J 3.2,
4Ј-H), 4.34–4.62 (5 H, m, 2 × OCH2Ar, 3Ј-H), 4.71 and 4.80
(2 H, AB, JAB 12.2, OCH2Ar), 4.97–5.03 (2 H, m, 2Ј-H, 3Љ-OH),
5.13 (1 H, d, J 4.7, 4Љ-OH), 5.37 (1 H, s, 1Љ-H), 5.95 (1 H, d,
J 6.7, 2Ј-OH), 6.11 (1 H, d, J 6.4, 1Ј-H), 7.24–7.44 (15 H, m,
ArCH) and 8.22 and 8.34 (2 H, 2 s, 2-H, 8-H); δC (d6-DMF;
100.4 MHz) 68.83 (C-4Љ or C-5Љ), 71.12 and 71.24 (C-5Ј, C-6Љ),
72.49 (C-3Љ), 73.47, 73.63 and 73.73 (3 × OCH2Ar), 74.53 (C-4Љ
or C-5Љ), 74.72 (C-2Ј), 76.59 (C-3Ј), 79.62 (C-2Љ), 83.35 (C-4Ј),
88.10 (C-1Ј), 99.20 (C-1Љ), 120.18 (C-5), 127.86, 127.96, 128.00,
128.11, 128.24, 128.77, 128.91 and 129.03 (ArCH), 139.21,
139.21 and 139.86 (3 × ipso-C of Bn rings), 140.05 (C-8), 150.85
(C-4), 153.62 (C-2) and 157.14 (C-6); m/z (FABϩ) 700
[(M ϩ H)ϩ, 70%] and 91 (100).
dimethyl phosphite (31)
To a mixture of mannopyranose 30 (1.29 g, 2.15 mmol) and
1H-tetrazole (0.23 g, 3.23 mmol) in CH2Cl2 (15 cm3) under N2
was added dimethoxy(diethylamino)phosphine36 (0.46 cm3,
2.80 mmol) and the mixture was stirred at room temperature for
20 min, whereupon TLC (ethyl acetate–toluene, 1:4) indicated
complete conversion into product (Rf 0.69). The reaction mix-
ture was partitioned between diethyl ether (80 cm3) and water
(60 cm3) and the resulting ethereal layer was washed with satur-
ated aq. NaCl (60 cm3), dried (MgSO4), filtered and concen-
trated to give a clear runny oil, which was used without further
purification. δH (CDCl3; 400 MHz) (α-anomer) 3.39–3.44 (6 H,
m, OP(OMe)2 with C-P coupling), 3.65–3.71 (2 H, m, 2-H,
6-HA), 3.76–3.80 (1 H, m, 6-HB overlapping with 2 × OCH3),
3.76, 3.78 (6 H, 2 s, 2 × OCH3), 3.90–4.03 (3 H, m, 3-H, 4-H,
5-H), 4.44–4.82 (8 H, m, 4 × OCH2Ar), 5.52 (1 H, dd, J 1.8, JH-P
8.2, 1-H), 6.78–6.86 (4 H, m, meta-H of PMB rings), 7.08–7.11
(2 H, m, ortho-H of PMB rings) and 7.21–7.39 (12 H, m,
ArCH); δP (CDCl3; 161.7 MHz; 1H decoupled) 140.83
(OPβ(OCH3)2) and 141.14 (OPα(OCH3)2).
2Љ,5Ј,6Љ-Tri-O-benzyl-3Ј-O-ꢀ-D-mannopyranosyl-2Ј,3Љ,4Љ-tri-O-
p-methoxybenzyl-N6-dimethoxytrityladenosine (32)
A mixture of dimethyl phosphite 31 (1.48 g, 2.15 mmol), 13
(0.84 g, 1.08 mmol) and 4 Å molecular sieves (approx. 1.2 g) in
dioxane (12 cm3) and toluene (4 cm3) under N2 was stirred for 2
J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947
1945

View Article Online
2Љ,5Ј,6Љ-Tri-O-benzyl-2Ј,3Љ,4Љ-tris-O-[bis(benzyloxy)phosphoryl]-
3Ј-O-ꢀ-D-mannopyranosyladenosine (34)
and Prize Studentship 044430) for financial support and
Dr M. Takahashi (Sankyo, Japan) for an authentic sample of
natural adenophostin A.
A solution of 33 (100 mg, 0.14 mmol), bis(benzyloxy)(diiso-
propylamino)phosphine (0.16 cm3, 0.49 mmol) and imidazo-
lium triflate 38 (103 mg, 0.47 mmol) in CH2Cl2 (3 cm3) under N2
was stirred for 1 h, after which time TLC (ethyl acetate–hexane,
7:3) indicated conversion to the trisphosphite (Rf 0.67). Water
(1 drop) was added and the solution was cooled to Ϫ78 ЊC,
whereupon MCPBA (144 mg, 0.50 mmol) was added. After 10
min 10% (w/v) aq. Na2SO3 (15 cm3) and ethyl acetate (20 cm3)
were added and the mixture was allowed to warm to room tem-
perature. The resulting organic layer was washed with 15 cm3
each of saturated aq. NaHCO3 and saturated aq. NaCl, dried
(MgSO4), filtered and concentrated to give a clear oil which was
subjected to flash chromatography (eluent chloroform–acetone,
9:1, then 4:1, then 3:2) to give the title compound as a colour-
less oil (119 mg, 56%); Rf 0.29 (ethyl acetate–hexane, 7:3); [α]D20
Ϫ1.9 (c 1.1, in CHCl3) (Found Mϩ, 1480.483. Calcd for
C79H81N5O18P3 (M Ϫ H)Ϫ: 1480.478); δH (CDCl3; 400 MHz)
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2
3
3
3.53, 3.66 (2 H, ABX, JAB 10.9, JAX 3.2, JBX 2.5, 5Ј-HA,
2
3
5Ј-HB), 3.70 (1 H, ABX, JAB 10.8, JAX 5.9, 6Љ-HA), 3.75–3.78
(1 H, m, 6Љ-HB), 3.82–3.86 (1 H, m, 5Љ-H), 4.26–4.29 (1 H, m,
4Ј-H), 4.37–4.44 (5 H, m, 2Љ-H, 2 × OCH2Ar), 4.54 and 4.64
(2 H, AB, JAB 11.7, OCH2Ar), 4.68 (1 H, t, J 4.7, 3Ј-H), 4.79–
5.03 (14 H, m, 6 × OCH2Ar, 3Љ-H, 4Љ-H), 5.30 (1 H, d, J 1.5, 1Љ-
H), 5.44 (1 H, ddd, J 3.5, 3.5, JH-P 8.5, 2Ј-H), 5.86 (2 H, br s,
NH2), 6.22 (1 H, d, J 5.0, 1Ј-H), 7.11–7.32 (45 H, m, ArCH) and
8.00 and 8.23 (2 H, 2 s, 2-H, 8-H); δC (CDCl3; 100.4 MHz)
69.24–70.34 (C-5Ј, C-6Љ, 6 × POCH2Ar with C-P coupling),
72.05 (C-5Љ with C-P coupling), 72.87 (C-4Љ with C-P coupling),
72.96, 73.60 and 73.88 (3 × OCH2Ar), 74.49 (C-4Љ), 76.28 (C-3Љ
with C-P coupling), 76.77 (C-2Љ), 77.67 (C-2Ј), 82.27 (C-4Ј),
86.84 (C-1Ј with C-P coupling), 97.75 (C-1Љ), 119.94 (C-5),
127.50, 127.59, 127.72, 127.87, 127.96, 127.97, 128.05, 128.08,
128.13, 128.20, 128.32, 128.39, 128.49, 128.61, 128.64, 128.69
and 128.73 (ArCH), 135.31–135.95 (6 × ipso-C of benzyl-
phospho ring with C-P coupling), 137.42, 138.14 and 138.39
(3 × ipso-C of Bn rings), 139.15 (C-8), 149.92 (C-4), 153.08
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1
(C-2) and 155.38 (C-6); δP (CDCl3; 162 MHz; H decoupled)
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3%] and 91 (100).
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3-O-ꢀ-D-Mannopyranosyladenosine 2Ј,3Љ,4Љ-trisphosphate
(manno-adenophostin) (7)
A mixture of 32 (61 mg, 0.04 mmol) and wet 20% palladium
hydroxide on carbon (180 mg), in methanol (7.2 cm3), cyclohex-
ene (3.6 cm3) and water (0.7 cm3) was heated under reflux for
2.5 h. After cooling the reaction mixture was filtered through
a membrane filter and the catalyst was washed copiously with
methanol and water. Concentration of the filtrate afforded a
clear residue which was applied to an MP1 AG ion exchange
resin column and eluted with a gradient of 0–100% 150 mmol
dmϪ3 aq. TFA. Concentration of the appropriate fractions
(being careful to keep the temperature below 20 ЊC) gave the
desired product as the free acid (19 mg, 71%), which was dis-
solved in water and eluted through a short column of Naϩ
Diaion WK-40 ion exchange resin to give, after concentration,
the sodium salt (Found: MϪ, 668.039. Calcd for C16H25N5O18P3
(M Ϫ H)Ϫ: 668.040); δH (D2O; 400 MHz) 3.58–3.72 (5 H, m,
5Ј-HA, 5Ј-HB, 5Љ-H, 6Љ-HA, 6Љ-HB), 4.15–4.24 (3 H, m, 2Ј-H,
4Ј-H, 4Љ-H), 4.39–4.48 (2 H, m, 3Ј-H, 3Љ-H), 4.98 (1 H, s, 1Љ-H),
5.08–5.14 (1 H, m, 2Ј-H), 6.11 (1 H, d, J 6.4, 1Ј-H) and 8.24 and
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1
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Acknowledgements
We thank the Wellcome Trust (Programme Grant 045491
1946
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J. Chem. Soc., Perkin Trans. 1, 2000, 1935–1947
1947