Synthesis of simple adenosine diphosphate ribose analogues

Article abstract of DOI:10.1080/15257770802341350

(Chemical Equation Presented) The synthesis of analogues of adenosine diphosphate ribose and acetylated adenosine diphosphate ribose, modified at the northern pentose, is reported. The stereochemistry at the acetylated centers was chosen to minimize acety

Full text of DOI:10.1080/15257770802341350

Nucleosides, Nucleotides and Nucleic Acids, 27:1127–1143, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802341350
SYNTHESIS OF SIMPLE ADENOSINE DIPHOSPHATE RIBOSE
ANALOGUES
Olivier P. Chevallier and Marie E. Migaud
School of Chemistry and Chemical Engineering, Queen’s University, Belfast,
Northern Ireland, United Kingdom
2
The synthesis of analogues of adenosine diphosphate ribose and acetylated adenosine diphos-
phate ribose, modified at the northern pentose, is reported. The stereochemistry at the acetylated
centers was chosen to minimize acetyl migration and dictated the overall synthetic strategy.
Keywords Acetylated adenosine diphosphate ribose; acetyl migration
Adenosine diphosphate ribose (ADPR), a ubiquitous metabolite of
nicotinamide adenine dinucleotide (NAD), recently has been described as
a calcium channel activator[1,2] directly involved in cell death initiation.[3]
Similarly, another NAD metabolite, O-acetylated ADPR (existing in solution
as a mixture of 2ꢁꢁ -OAc and 3ꢁꢁ-OAc ADPR), which has been shown to directly
regulate the same calcium channel,[4] has also been proposed to mediate
cell survival. Additionally, O-acetylated ADPR has been shown to act as
reporter of NAD-dependent protein deacetylase activities at remote sites[5]
Received 18 February 2008; accepted 19 June 2008.
We are thankful to the European Social Funds for funding O. Chevallier’s studentship.
Address correspondence to Marie E. Migaud, School of Chemistry and Chemical Engineering,
David Keir Building, Stranmillis Road, Queen’s University, Belfast, BT9 5AG, Northern Ireland, U.K..
E-mail: m.migaud@qub.ac.uk
1127

1128
O. P. Chevallier and M. E. Migaud
FIGURE 1 ADPR, acetylated ADPR and synthetic targets.
and has been implicated in chromatin silencing[6] and histone macroH2A
binding.[7]
In all cases, more studies are needed to get a better understanding of
the role of ADPR and of O-acetyl ADPR as potential second messengers
as well as the mechanisms controlling their metabolisms. In order to
investigate the biological targets of ADPR and acetylated ADPR as well as
their metabolism, the chemical synthesis of ADPR-like molecules deemed
the most appropriate approach, as illustrated by a recent report on the
synthesis of non-hydrolysable analogues of O-acetyl ADPR.[8] We recently
reported a study on acetate migration in partially protected furanoside
systems and the products distribution observed under basic, neutral and
acidic conditions depending on the stereochemistry at the C1, C2, and C3
carbon centers of the respective furanosides.[9] The modifications on ADPR
reported here were structurally simple as they involved the reduction of the
furanose ring to a ribitol moiety (3) and the inversions of configuration,
either at the C2ꢁꢁ or at the C3ꢁꢁ position of the northern ribose, as shown by
analogues (4–7) in Figure 1.
Adenosine diphosphate ribitol 3 was prepared by simple treatment at
0◦C of ADPR in water with sodium borohydride. Attempts were made at
synthesizing adenosine diphosphate xylose 6 from the partially protected
3-O-benzyl 5-O-(dibenzylphospho)-xylose 8 (Scheme 1). Unfortunately, the
removal of the benzyl protecting groups under mild hydrogenolysis condi-
tions resulted in phosphate ester migration between the C5 position and
the C3 position via the formation of a cyclic phosphate triester (Scheme
1). Similarly when attempts were made at synthesizing a dibenzylphospho-
triester from 3-O-benzoyl 1,2-O,O-isopropylidene ribose under basic con-
ditions using tetrabenzylpyrophosphate, rapid migration of the benzoate

Simple Adenosine Diphosphate Ribose Analogues
1129
SCHEME 1 Phospho-ester migration occurring in the partially protected xylose intermediate. a) H2;
Pd/C, THF.
moiety and phosphorylation at the C-3 position took place. It therefore was
concluded that adenosine diphosphate xylose 6 should be synthesized from
the acetylated adenosine diphosphate xylose 4 by simple deacetylation as
final synthetic step. A similarly divergent route was used for the preparation
of ADP-arabinose 7 from 5.
SYNTHESIS OF THE PHOSPHOSUGAR PRECURSORS TO 4–7
As had been often observed, the C-3 stereochemistry of a xylofuranose
derivative facilitates acetyl migration from the C-3 hydroxyl to the C-5
primary hydroxyl either in acidic or basic conditions.[9] Consequently, in
order to synthesize a 3-O-acetylated 5-phosphorylated xylose derivative, the
acetyl moiety had to be introduced after the phosphorylation step. This
requirement involved the temporary masking of the C-3 hydroxyl group
prior to phosphorylation. The C-5 trityl furanose 9, prepared from 1,2-O,O-
isopropylidene 5-O-trityl xylose, was deprotected under acidic conditions,
without benzoyl migration, to give the precursor 10 (Scheme 2). Under
SCHEME 2 Synthesis of 3-O-acetyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene α-D-xylofuranoside.
a) HCOOH, 1H-Dowex, CH2Cl2, 62%; b) i. (BnO)2PN(iPr)2 (12); 2,4-DNP, CH2Cl2, ii. tBuOOH, 60%;
c) (BnO)4P2O3, THF, tBuLi, 37%; d) CH3OH, NaOCH3, 96%; e) Ac2O; Pyr, 100%.

1130
O. P. Chevallier and M. E. Migaud
basic phosphorylation conditions, a complex mixture of products was
obtained where only one main compound was identified unambiguously
as the phosphate triester derivative 11 (Scheme 2) and for which the
benzoate ester migrated from the C-3 position to the C-5 primary alco-
hol. Phosphorylation under acidic conditions was then carried out on
the carbohydrate derivative 10. The dibenzylphosphoramidite reagent 12,
prepared in 56% yield from phosphorus trichloride, diisopropylamine and
benzyl alcohol, was used with 2,4-DNP as activator.[10] The phosphite triester
intermediate was then oxidized in situ with tert-butyl hydroperoxide to yield
the dibenzylphosphate triester sugar 13.
Protecting group inter-conversion at the C-3 hydroxyl was conducted
under Zemplen conditions (catalytic sodium methoxide in methanol) to
afford the crude phosphate triester 14. Acetylation of the remaining alcohol
using classic acetylation procedures afforded the phosphorylated xylose 15,
quantitatively.
The strategy developed for the synthesis of the 3-O-acetyl-5-O-
dibenzylphosphate-1,2-O,O-isopropylidene-β-D-arabinofuranose 16 was sim-
ilar to the one developed for the xylose intermediate 15. However, with the
arabinose series, it was possible to functionalize the C3 hydroxyl position
with the required acetyl group before the phosphorylation step as the anti
configuration of the C3 and C5 hydroxyls does not favor an intramolec-
ular acyl migration.9 1,2-O-Isopropylidene-5-O-tert-butyldiphenylsilyl-β-D-
arabinofuranose 17 (Scheme 3) was synthesized in 53% overall yield from
D-arabinose.[11] Acetylation of the C3-hydroxyl was achieved using acetic
anhydride in pyridine to give quantitatively the fully protected sugar 18.
TBAF-promoted cleavage of the silyl ether 18 afforded the desired arabinose
19 in 73% yield while the subsequent phosphorylation yielded the arabino-
furanose 16 in 64% yield.
SCHEME 3 Synthesis of 3-O-acetyl-5-O-dibenzylphosphate-1,2-O,O-isopropyledene β-D-arabino-
furanoside. a) Ac2O, pyr, quant; b) TBAF, THF, 73%; 12, 2,4-DNP, CH2Cl2, 64%.
The acetonide deprotection of the two acetylated compounds 15 and
16 was carried out with aqueous TFA with a subsequent workup requiring
ethyl acetate and an excess of pyridine. This sequence, necessary to prevent
acetate migration and hydrolysis, provided the compounds 20 and 21 in
61% and 55% yield, respectively (Scheme 4).
Removal of the phosphate triester benzyl protecting groups was carried
out on the xylofuranose derivative 20 using Pd/C as catalyst in THF under

Simple Adenosine Diphosphate Ribose Analogues
1131
SCHEME 4 Acetonide removal with no acetyl migration. a) TFA/H2O; EtOAc/Pyr; 20, 61%; 21, 55%.
balloon pressure of hydrogen at room temperature. The products of this
hydrogenolysis were identified as the 3-O-acetyl derivative 22(α and β) and
the 2-O-acetyl 23 (α and β) (Scheme 5). Similar conditions were used on
the arabinofuranose 21 to give a similar mixture of acetylated materials 24(α
and β) and 25(α and β). This acetyl migration was thought to be generated
by the acidity of the reaction condition due to the intrinsic acidity of the
catalyst and the formation of the acidic phosphate monoester. Yet, the same
mixture of acetylated compounds was obtained when Pd(OH)2 was used as
catalyst.
SCHEME 5 Hydrogenolysis and mixture of acetyl compounds. a) H2, Pd(OH)2; THF.
The reaction was then carefully monitored by TLC and stopped after
1
total disappearance of the starting material, about 30 minutes. The H-
NMR and 13C-NMR of the reaction mixtures were taken immediately
after filtration of the catalyst and removal of the solvent. These analyses
showed the unique presence of the expected phosphorylated and acetylated
carbohydrate 22 as a mixture of α/β anomers. However, these compounds
rearranged rapidly in aqueous solution to give the same distribution of
acetylated products as observed previously indicating that the acetyl migra-
tion occurred after the removal of the benzyl protecting groups. Under the
same conditions, arabinose 21 presented similar reactivity. In hindsight, the
mixture of acetylated products can be explained by the fact that unlike the
partially protected furanosides,[9] mutarotation equilibrium exists between
the cyclic form and the open form of C1-deprotected-pentoses. Acetyl
migrations occurred in arabinose and xylose when the hydroxyls at C2 and
C3 were no longer sterically restricted in a trans-configuration imposed by
the furanoside structure.

1132
O. P. Chevallier and M. E. Migaud
The monoacetylated furanosyl precursors 22/23 and 24/25 were used to
access the acetylated ADP-xylose 4 and ADP-arabinose 5. The condensation
between the free acid phosphate monoesters 22/23 and 24/25 and adeno-
sine monophosphomorpholidate (AMP-morpholidate) was carried out in
anhydrous pyridine. Once the reaction reached equilibrium and pyridine
evaporated, the unreacted monophosphates were removed using DSC-SAX
pre-packed anion exchange column eluted first with water and then with a
formic acid buffer (50 mM, pH 4). The fractions containing pyrophosphate
products were identified by HPLC using an anion exchange SAX column
(KH2PO4, 50 mM, 5% MeOH, pH 3.5) and were pooled together before
being purified further by semi-preparative RP-C18-HPLC chromatography
column eluted with an ammonium formate buffer (HCOOH, 50 mM, 5%
MeOH, pH 6). The pure acetylated ADP-xylose 4 and acetylated ADP-
arabinose 5 were isolated in 4% and 9% yields, respectively (Table 1).
Such moderate yields for the formation of the target compounds are
not unusual for a pyrophosphate linkage[8] and can be explained by the
poor solubility in anhydrous pyridine of the ester phosphate precursors
leading to heterogeneous reaction conditions and formation of an impor-
tant amount of side-products. However, prior modification of the phosphor-
sugar counter ion to improve solubility proved difficult since exchange for
a more lipophilic cation such as a triethylammonium or an octylammonium
salt, under nucleophilic, protic conditions (methanol, triethylamine or
octylamine) would have resulted in the rapid hydrolysis of the acetate
moiety, already quite labile under neutral conditions.
In conclusion, we have synthesized five analogues of ADPR, two of
which are also analogues of acetylated ADPR. As for acetylated ADPR, the
acetylated xylose and arabinose parents are present in solution as mixture
of regioisomers due to the facile acyl migration of the acetyl group that can
occur when the sugars are ring-opened.
EXPERIMENTAL SECTION
General Experimental Protocol
All reactions requiring anhydrous or inert conditions were carried out
1
in oven dried glassware under a positive atmosphere of argon. H, 13C,
and 2D (H-COSY, HMQC) NMR spectra were all recorded on Bru¨ker
1
avance DPX 300 and Bru¨ker avance DPX 500. TMS (0 ppm, H NMR),
CDCl3 (77 ppm, 13C NMR), and HMPA (26.7 ppm, 31P NMR) were used
as internal references. The chemical shifts (δ) are reported in ppm (parts
per millions). HPLC runs were performed on a Varian Workstation system
with UV detector operating at 254 nm. UV quantification was carried out by
using the absorbance of a solution of final compounds in water at their λmax
and found to be in all cases at 254 nm. The extinction coefficient used to

Simple Adenosine Diphosphate Ribose Analogues
1133
TABLE 1 Characterisation of compounds 5-7
Retention time
(minutes)
Compound
31P NMR, ppm
Isolated yield (%)
4
6.17
−10.1
6.52
2.71
2.62
−9.9
−10.0
−10.0
9
2
7
HPLC conditions: RP-C18 analytical column Supelco, 4.6 × 150 mm, 5 mm, formic acid 50
mM, pH 6, 5%; MeOH, 1 ml/min, UV detector: 254 nm.
calculate the solution concentration was that of ADPR, that is, 14.3 103 M−1
cm−1. By comparison to concentration obtained by phosphorus content
assay, this extinction coefficient was consistent for compound 3 and was
used for the subsequent evaluations. Reversed-phase chromatography was
run on RP-C18 column (analytical column, 4.6 × 150 mm, 5 µm, Supelco;
semi-preparative column, 10 × 250 mm, 10 µm, Phenomex luna) using
ammonium formate buffer (50 mM, pH = 6.0, 5% MeOH) at a flow rate
of 1 mL/min. Mass spectra were recorded on a VG Autospec spectrometer,
Varian Workstation 1200 (ES). Optical rotations were measured on a Perkin-
Elmer 341 polarimeter.

1134
O. P. Chevallier and M. E. Migaud
Synthetic Procedures
Adenosine-diphospho-ribitol 3
To a solution of commercially available ADPR (63 mg, 112.7 µmol)
dissolved in mQ water (10 mL) was added NaBH4 (5 mg, 132.1 mmol) at 0
C. The resulting solution was allowed to warm up at room temperature and
kept under stirring for 14 hours. The reaction mixture was then neutralized
with Dowex (H+ form) and filtered. The resin was rinsed with mQ water
(5 mL×3) and freeze-dried to afford 3 as a fluffy powder (79.6 µmol, 71%
quantified by UV).
1H NMR (D2O) 8.49 (s, 1H, H2); 8.21 (s, 1H, H8); 6.13 (d, J = 6.0
ꢁ
Hz, 1H, H1 ); 4.68 (dd, J = 3.4 Hz, J = 6.3 Hz, 1H); 4.55–4.52 (m, 1H);
4.42–4.40 (m, 1H); 4.24–4.21 (m, 2H); 4.13–4.09 (m, 1H); 4.07–3.99 (m,
2H); 3.89–3.82 (m, 2H); 3.79–3.72 (m, 2H). 13C NMR (D2O) δ 156.0 (CAde);
ꢁ
153.2 (×2) (CAde); 149.4 (CAde); 140.7 (CAde); 119.0 (C1 ); 90.5, 85.9, 81.1,
ꢁ
ꢁꢁ
72.5, 72.0, 71.4 (Cribo); 67.4 (d, J = 4.8 Hz, C5 or C5 ); 66.6 (d, J = 5.3 Hz,
C5 or C5 ); 62.8 (CCH2). 31P NMR (D2O) δ -9.3(d, JP-O-P = 21.1 Hz); -9.9 (d,
ꢁ
ꢁꢁ
JP-O-P = 20.8 Hz).
HRMS m/z 562.0942 (calcd for C15H26O14N5P2([M+H]+) 562.0952).
3-O-Benzyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-
xylofuranose.
A
solution of 3-O-benzyl-1,2-O,O-isopropylidene-α-D-
xylofuranose[12] (180 mg, 0.64 mmol) in THF (20 mL) was treated
at -78◦C with ter-BuLi (415 µL of a 1.7 M solution in pentane, 0.71
mmol). This resulting mixture was stirred for 30 minutes and a solution
of TBPP (380 mg, 0.71 mmol) in THF (10 mL) was added to the
reaction still at −78◦C. The reaction mixture was stirred for 1 hour at
−78◦C, before being allowed to warm up to room temperature. After
14 hours under stirring at room temperature, the reaction mixture
was then diluted with Et2O (20 mL) and NH4Claq. (30 mL) was then
slowly added. The aqueous layer was extracted with Et2O. The combined
organic layers were then washed with NH4Cl(aq), water and brine;
dried, filtered, and concentrated under reduced pressure to afford
the crude phosphorylated sugar. Purification by flash chromatography
(gradient from 75/25 to 45/55 v/v petroleum ether/ethyl acetate) gave
3-O-benzyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-xylofuranose
(286 mg, 83%) as a colorless oil.
1H NMR (CDCl3) δ 7.36–7.28 (m, 15H, HAr); 5.93 (d, J1,2 = 3.7 Hz, 1H,
H1); 5.05–5.03 (m, 4H, HCH2Bn); 4.61 (AB, JA = 11.8 Hz, 1H, HCH2Bn); 4.59
(d, J1,2 = 3.8 Hz, 1H, H2); 4.45 (AB, JA = 11.8 Hz, 1H, HCH2Bn); 4.37 (ddd,
ꢁ
J3,4 = 3.3 Hz, J4,5 = 6.0 Hz, J4,5 = 6.1 Hz, 1H, H4); 4.27 (ABX, J4,5 = 6.0 Hz,
ꢁ
ꢁ
ꢁ
ꢁ
J5,5 = 10.3 Hz, 1H, H5); 4.23 (ABX, J4,5 = 6.1 Hz, J5,5 = 10.3 Hz, 1H, H5 );
3.92 (d, J3,4 = 3.3 Hz, 1H, H3); 1.46 (s, 3H, CH3Iso); 1.31 (s, 3H, CH3Iso). 13
C
NMR (CDCl3) δ 137.0, 135.7, 135.6, 128.5, 128.4, 128.3, 127.9, 127.8, 127.5
(CAr); 111.8 (CqIso); 105.1 (C1); 82.0 (C2); 81.2 (C3); 78.6 (d, JP-O-C-C = 8.5

Simple Adenosine Diphosphate Ribose Analogues
1135
Hz, C4); 71.8 (CCH2Bn); 69.3 (d, JP-O-C = 5.1 Hz, CCH2Bn); 69.2 (d, JP-O-C = 5.3
Hz, CCH2Bn); 65.0 (d, JP-O-C = 5.3 Hz, C5); 26.7 (CMeIso); 26.2 (CMeIso). 31P
NMR (CDCl3) δ 0.2. HRMS m/z 541.5670 (calcd for C29H34O8P ([M+H]+)
541.5633).
3-O-Benzyl-5-O-dibenzylphosphate-D-xylofuranose 8. 3-O-Benzyl-5-O-
dibenzylphosphate-1,2-O,O-isopropylidene-α-D-xylofuranose (189 mg, 0.35
mmol) was treated with a mixture of TFA/H2O (8 mL/2 mL) at 0◦C.
The resulting mixture was stirred for 90 minutes and was then diluted
with EtOAc (25 mL). The resulting solution was carefully poured in a
saturated solution of Na2CO3 (30 mL) at 0◦C. The aqueous layer was
extracted with EtOAc (20 mL × 4) and the combined organic fractions
were dried, filtered, and concentrated under reduced pressure to yield
the crude xylofuranose derivative. Purification by flash chromatography
(gradient from 1/0 to 97/ v/v CHCl3/ethanol) afforded the pure
3-O-benzyl-5-O-dibenzylphosphate-D-xylofuranose 8 (122 mg, 70%) as a
mixture of anomers (α/β:7/3).
1H NMR (CDCl3) 7.31–7.27 (m, 15H, HAr); 5.46 (t, J = 4.8 Hz, 0.7H,
H1α); 5.18 (d, J = 9.6 Hz, 0.3H, H1β); 5.04–4.99 (m, 4H, HCH2Bn); 4.66 (AB,
JA = 12.0 Hz, 0.7H, HαCH2Bn); 4.62 (AB, JA = 11.6 Hz, 0.3H, HβCH2Bn); 4.50
(AB, JA = 11.6 Hz, 0.3H, HβCH2Bn); 4.48 (AB, JA = 12.0 Hz, 0.7H, HαCH2Bn);
4.44–4.40 (m, 1H, H4); 4.35–4.29 (m, 0.3H, H5β); 4.26–4.25 (m, 0.2H, H2β);
ꢁ
4.24–4.20 (m, 0.3H, H5 β); 4.18–4.15 (m, 2.1H, H2α, H3α, H5α,); 4.00–3.99
(m, 1 H, H5 α, H3β). 13C NMR (CDCl3) δ 137.6, 137.0, 135.5, 135.6, 128.5,
ꢁ
128.4, 128.0, 127.9, 127.8, 127.7, 127.5 (CAr), 103.4 (C1β); 96.2 (C1α); 83.1
(C3α); 82.5 (C2β); 79.2 (d, JP-O-C-C = 7.1 Hz, C4β); 78.1 (C3β); 76.6 (d, JP-O-C-C
= 7.7 Hz, C4α); 75.2 (C2α); 72.5 (CCH2βBn); 71.8 (CCH2αBn); 69.6 (CCH2βBn);
69.5 (CCH2βBn); 69.5 (CCH2αBn); 69.4 (CCH2αBn); 67.4 (d, JP-O-C = 5.6 Hz, C5β);
66.8 (d, JP-O-C = 5.6 Hz, C5α). 31P NMR (CDCl3) δ 0.2 (β), 0.0 (α).HRMS m/z
501.1701 (calcd for C26H30O8P ([M+H]+) 501.1678).
3-O-Benzoyl-1,2-O,O-isopropylidene-5-O-trityl-α-D-xylofuranose 9.[13] To
a solution of 1,2-O,O-isopropylidene-5-O-trityl-α-D-xylofuranose[12] (17.9
g, 41.4 mmol) in dry DCM/dry pyridine (50 mL/30 mL) was added
dropwise at 0◦C a solution of benzoyl chloride (5.8 mL, 49.9 mmol) in
dry DCM (40 mL). The resulting solution was stirred overnight at room
temperature. The reaction mixture was then concentrated under reduced
pressure to afford a yellowish oil. This residue was partitioned between
water (100 mL) and CHCl3 (100 mL). The organic layer was then washed
with NaHCO3 (100 mL), water, brine, dried filtered, and concentrated
under vacuum to afford the crude fully protected furanoside as a yellow
oil. Purification by flash chromatography (gradient 1/0 to 8/2 v/v hex-
ane/ethyl acetate) afforded 3-O-benzoyl-1,2-O,O-isopropylidene-5-O-trityl-α-
D-xylofuranose 9 (21.3 g, 95%).
1H NMR (CDCl3) δ 7.83–7.81 (m, 2H, HBz); 7.57–7.54 (m, 1H, HBz);
7.40–7.37 (m, 8H, HAr); 7.19–7.14 (m, 9H, HAr); 5.95 (d, J1,2 = 3.7 Hz, 1H,

1136
O. P. Chevallier and M. E. Migaud
H1); 5.66 (d, J3,4 = 3.0 Hz, 1H, H3); 4.68–4.66 (m, 1H, 1H, H4); 4.64 (d,
ꢁ
J1,2 = 3.7 Hz, 1H, H2); 3.58 (ABX, J4,5 = 7.9 Hz, J5,5 = 9.1 Hz, 1H, H5); 3.41
ꢁ
ꢁ
ꢁ
(ABX, J4,5 = 5.6 Hz, J5,5 = 9.1 Hz, 1H, H5 ); 1.61 (s, 3H, CH3Iso); 1.34 (s, 3H,
CH3Iso). 13C NMR (CDCl3) δ 164.9 (COBz); 143.3 (CTr ); 133.0, 129.6 (CBz);
128.4 (CTr ); 128.2 (CBz); 127.6, 126.8 (CTr ); 111.9 (CqIso); 104.7 (C1); 86.7
(CqTr); 83.2 (C2); 77.9 (C4); 76.3 (C3); 60.2 (C5); 26.5 (CMeIso); 26.0 (CMeIso).
20
HRMS m/z 559.2114 (calcd for C34H32O6Na([M+Na]+) 559.2097). [α]D
= -42.9 (c = 0.3, MeOH)
3-O-Benzoyl-1,2-O,O-isopropylidene-α-D-xylofuranose
10.[13]
3-O-
Benzoyl-1,2-O,O-isopropylidene-5-O-trityl-α-D-xylofuranose 9 (17.2 g, 32.1
mmol) was dissolved in DCM (100 mL). To this resulting solution was added
Dowex (H+ form) and formic acid (5 mL) and the suspension was then kept
under stirring overnight at room temperature. The solvent was removed
under vacuum to afforded the crude 3-O-benzoyl-1,2-O-isopropylidene-
α-D-xylofuranoside as a yellow oil. Purification by flash chromatography
(gradient 9/1 to 1/1 v/v hexane/ethyl acetate) afforded 2 fractions, the
3-O-benzoyl-1,2-O,O-isopropylidene-α-D-xylofuranose 10 (5.9 g, 62%) and
the starting material 9 (3.9 g, 23%).
1H NMR (CDCl3) δ 8.03–7.99 (m, 2H, HBz); 7.62–7.57 (m, 1H, HBz);
7.48–7.43 (m, 2H, HBz); 6.02 (d, J1,2 = 3.7 Hz, 1H, H1); 5.45 (d, J3,4 = 2.8
Hz, 1H, H3); 4.72 (d, J1,2 = 3.7 Hz, 1H, H2); 4.51 (ddd, 1H, J3,4 = 2.8 Hz,
ꢁ
ꢁ
J4,5 = J4,5 = 6.2 Hz, 1H, H4); 3.89 (ABX, J4,5 = 6.3 Hz, J5,5 = 11.9 Hz, 1H,
ꢁ
ꢁ
ꢁ
H5); 3.71 (ABX, J4,5 = 6.1 Hz, J5,5 = 11.9 Hz, 1H, H5 ); 1.55 (s, 3H, CH3Iso);
1.34 (s, 3H, CH3Iso). 13C NMR (CDCl3) δ 166.2 (COBz); 133.8, 129.9, 128.7
(CBz); 112.5 (CqIso); 104.8 (C1); 83.7 (C2); 79.9 (C4); 77.4 (C3); 59.9 (C5);
20
26.7 (CMeIso); 26.4 (CMeIso). [α]D = -10.9 (c = 0.4, MeOH). HRMS m/z
295.3113 (calcd for C15H19O6 ([M+H]+) 295.3151).
5-O-Benzoyl-3-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-
xylofuranose 11. A solution of 3-O-benzoyl-1,2-O-isopropylidene-α-D-
xylofuranose 10 (137 mg, 0.46 mmol) in THF (20 mL) was treated at -78◦C
with t-BuLi (328 µL of a 1.7 M solution in pentane, 0.56 mmol). This
resulting mixture was stirred for 30 minute and a solution of TBPP (301
mg, 0.56 mmol) in THF (10 mL) was added to the reaction still at -78◦C.
The reaction mixture was stirred for 1 hour at -78◦C, before being warmed
up to room temperature. After 8 hours under stirring at room temperature,
the reaction mixture was diluted with Et2O (20 mL) and NH4Claq. (30
mL) was then slowly added. The aqueous layer was extracted with Et2O.
The combined organic layers were then washed with NH4Claq, water and
brine; dried, filtered, and concentrated under reduced pressure to afford
the crude phosphorylated sugar. Purification by flash chromatography
(gradient from 75/25 to 45/55 v/v petroleum ether/ethyl acetate) gave 5-
O-benzoyl-3-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-xylofuranose
11 (94 mg, 37%) as a colorless oil.

Simple Adenosine Diphosphate Ribose Analogues
1137
1H NMR (CDCl3) δ 8.06–8.03 (m, 2H, HBz); 7.58–7.53 (m, 1H, HBz);
7.43–7.38 (m, 2H, HBz); 7.35–7.28 (m, 10H, HAr); 5.83 (d, J1,2 = 3.7 Hz, 1H,
H1); 5.04–4.99 (m, 4H, HCH2Bn); 4.87 (dd, J3,4 = 2.3 Hz, J3,4 = 7.8 Hz, 1H,
ꢁ
H3); 4.57 (d, J1,2 = 3.7 Hz, 1H, H2); 4.54–4.47 (m, 3H, H4, H5 , H5); 1.48 (s,
3H, CH3Iso); 1.26 (s, 3H, CH3Iso). 13C NMR (CDCl3) δ 166.0 (COBz); 135.4
(d, JP-O-C-C = 6.5 Hz, CAr); 135.3 (d, JP-O-C-C = 6.6 Hz, CAr); 133.1, 129.8, 128.7,
128.6, 128.4, 128.0 (CAr); 112.3 (CqIso); 104.8 (C1); 83.2 (d, JP-O-C = 1.6 Hz,
C2); 79.8 (d, JP-O-C = 5.5 Hz, C3); 77.2 (d, JP-O-C-C = 7.3 Hz, C4); 69.9 (d, JP-O-C
= 5.6 Hz, CCH2Bn); 69.8 (d, JP-O-C = 5.9 Hz, CCH2Bn); 61.6 (C5); 26.6 (CMeIso);
26.2 (CMeIso). 31P NMR (CDCl3) δ -0.2.
3-O-Benzoyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-
xylofuranose 13. To a solution of 3-O-benzoyl-1,2-O-isopropylidene-α-
D-xylofuranose 10 (2.9 g, 9.8 mmol) and dibenzylphosphoramidate (3.7 g,
10.8 mmol) in dry DCM (30 mL) was added dropwise a solution of 2,4-DNP
(2.0 g, 10.8 mmol) in dry DCM (20 mL). This resulting mixture was stirred
overnight at room temperature, then a solution of ter-BuOOH in toluene
(6 ml of a 3 M solution) was added. The resulting solution was stirred for
20 minutes. After dilution with DCM (30 mL), the reaction mixture was
washed with a sodium thiosulfate solution (50 mL), water (30 mL x 3),
dried, filtered, and concentrated under vacuum to afford the crude fully
protected phosphorylated precursor. Purification by flash chromatography
(gradient 85/15 to 6/4 v/v hexane/ethyl acetate) yielded 3-O-benzoyl-5-
O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-xylofuranose 13 (3.4 g,
63%) as a yellow oil.
1H NMR (CDCl3) δ 7.99–7.97 (m, 2H, HBz); 7.60–7.56 (m, 1H, HBz);
7.47–7.41 (m, 2H, HBz); 7.32–7.27 (m, 10H, HAr); 5.94 (d, J1,2 = 3.7 Hz, 1H,
H1); 5.40 (d, J3,4 = 3.1 Hz, 1H, H3); 4.59 (d, J1,2 = 3.7 Hz, 1H, H2); 4.53
ꢁ
(ddd, 1H, J3,4 = 3.1 Hz, J4,5 = 6.1 Hz, J4,5 = 6.3 Hz, 1H, H4); 4.24 (ABX,
ꢁ
ꢁ
ꢁ
J4,5 = 6.3 Hz, J5,5 = 10.8 Hz, 1H, H5); 4.19 (ABX, J4,5 = 6.1 Hz, J5,5 = 10.8
Hz, 1H, H5 ); 1.49 (s, 3H, CH3Iso); 1.28 (s, 3H, CH3Iso). 13C NMR (CDCl3)
ꢁ
δ 165.0 (COBz); 135.7 (d, JP-O-C-C = 6.1 Hz, CAr); 135.6 (d, JP-O-C-C = 6.2 Hz,
CAr); 133.5, 129.7, 129.0, 128.6, 128.5,128.4, 127.9 (CAr); 112.4 (CqIso); 105.0
(C1); 83.3 (C2); 77.6 (d, JP-O-C-C = 8.5 Hz, C4); 76.4 (C3); 69.4 (d, JP-O-C = 6.0
Hz, CCH2Bn); 69.3 (d, JP-O-C = 5.8 Hz, CCH2Bn); 64.5 (d, JP-O-C = 5.1 Hz, C5);
20
26.7 (CMeIso); 26.2 (CMeIso). 31P NMR (CDCl3) δ -0.4. [α]D = -7.6 (c = 1.1,
MeOH). HRMS m/z 555.1788 (calcd for C29H32O9P([M+H]+) 555.1784).
5-O-Dibenzylphosphate-1,2-O,O-isopropylidene-α-D-xylofuranose
14.[14]
3-O-Benzoyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-
D-xylofuranose 13 (3.2 g, 5.8 mmol) was dissolved in MeOH (50
ml). To the resulting solution was added MeONa (130 µL of a 25%
wt solution, 0.62 mmol) and was left under stirring for 4 hours.
The reaction mixture was then neutralized with Dowex (H+ form),
filtered. The resin was rinsed with MeOH (50 mL×3). The combined

1138
O. P. Chevallier and M. E. Migaud
filtrates were then concentrated under reduced pressure to afford
the crude deprotected carbohydrate derivative. Purification by flash
chromatography (gradient from 1/0 to 97/3 v/v CHCl3/ethanol) gave
5-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-xylofuranose 14 (2.5 g,
96%) as a colorless oil.
1H NMR (CDCl3) 7.37–7.32 (m, 10H, HAr); 5.89 (d, J1,2 = 3.5 Hz, 1H,
H1); 5.08–4.98 (m, 4H, HCH2Bn); 4.56 (d, J1,2 = 3.5 Hz, 1H, H2); 4.29–4.23
ꢁ
ꢁ
ꢁ
(m, 1H, H3, H4, H5);4.03 (ABX, J4,5 = 6.3 Hz, J5,5 = 12.4 Hz, 1H, H5 ); 1.49
(s, 3H, CH3Iso); 1.31 (s, 3H, CH3Iso). 13C NMR (CDCl3) δ 135.3 (d, JP-O-C-C
= 6.3 Hz, CAr), 135.2 (d, JP-O-C-C = 6.8 Hz, CAr), 128.8, 128.8, 128.7, 128.6,
128.1, 128.0 (CAr); 111.8 (CqIso); 105.0 (C1); 85.0 (C2); 78.8 (d, JP-O-C-C
=
4.8 Hz,C4); 73.6 (C3); 70.0 (d, JP-O-C = 5.9 Hz, CCH2Bn); 68.9(d, JP-O-C = 5.7
Hz, CCH2Bn); 63.0 (d, JP-O-C = 4.9 Hz, C5); 26.9 (CMeIso); 26.2 (CMeIso). 31P
NMR (CDCl3) δ -0.6. HRMS m/z 451.1511 (calcd for C22H28O8P([M+H]+)
451.1522).
3-O-Acetyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene-α-D-
xylofuranose 15. To
a solution of 5-O-dibenzylphosphate-1,2-O,O-
isopropylidene-α-D-xylofuranose (2.3 g, 5.1 mmol) in pyridine (35
ml) was added acetic anhydride (0.6 ml, 5.6 mmol). The resulting solution
was stirred for 3 hours at room temperature. Then the reaction mixture was
concentrated under vacuum and azeotrope 3 times with toluene to afford
3-O-acetyl-5-O-dibenzylphosphate-1,2-O-isopropylidene-α-D-xylofuranose 15
(2.5 g, 100%) as a colorless oil.
1H NMR (CDCl3) 7.34–7.32 (m, 10H, HAr); 5.90 (d, J1,2 = 3.7 Hz, 1H,
H1); 5.19 (d, J3,4 = 3.1 Hz, 1H, H3); 5.05–5.00 (m, 4H, HCH2Bn); 4.49 (d, J1,2
ꢁ
= 3.7 Hz, 1H, H2); 4.44 (ddd, J3,4 = 3.1 Hz, J4,5 = J4,5 = 6.1 Hz, 1H, H4);
ꢁ
4.16–4.13 (m, 2H, H5, H5 ); 2.02 (s, 3H, CH3Ac); 1.49 (s, 3H, CH3Iso); 1.30
(s, 3H, CH3Iso). 13C NMR (CDCl3) δ 169.6 (COAc); 135.8 (d, JP-O-C-C = 3.8 Hz,
CAr), 135.7 (d, JP-O-C-C = 3.9 Hz, CAr), 128.5, 128.0 (CAr); 112.4 (CqIso); 104.9
(C1); 83.3 (C2); 77.6 (d, JP-O-C-C = 4.8 Hz,C4); 75.8 (C3); 69.5(d, JP-O-C = 5.3
Hz, CCH2Bn); 69.4(d, JP-O-C = 5.2 Hz, CCH2Bn); 64.2 (d, JP-O-C = 5.0 Hz, C5);
20
26.7 (CMeIso); 26.2 (CMeIso); 20.6 (CMeAc). 31P NMR (CDCl3) δ -0.4. [α]D
=
-8.5 (c = 0.8, MeOH). HRMS m/z 493.1662 (calcd for C24H30O9P([M+H]+)
493.1627).
3-O-Acetyl-5-O-dibenzylphosphate-D-xylofuranose 20. 3-O-Acetyl-5-
O-dibenzylphosphate-1,2-O-isopropylidene-α-D-xylofuranose (385 mg,
0.58 mmol) was treated with a mixture TFA/H2O (8 ml/2 ml) at
0◦C. The resulting solution was stirred for 25 minutes and was then
diluted with EtOAc. Pyridine (15 mL) was then slowly added at 0◦C.
The resulting mixture was concentrated under vacuum to give a white
residue which was dissolved with EtOAc(25 mL). This solution was
washed with water (20 mL x 3) dried, filtered, and concentrated under
vacuum to afford the deprotected furanose as an oil. Purification by flash

Simple Adenosine Diphosphate Ribose Analogues
1139
chromatography (gradient from 1/0 to 97/3 v/v CHCl3/ethanol) gave
3-O-acetyl-5-O-dibenzylphosphate-D-xylofuranose 20 (159 mg, 61%) as a
α/β mixture (4/1).
1H NMR (CDCl3) 7.35–7.30 (m, 10H, HAr); 5.43 (t, J = 4.2 Hz, 0.8H,
H1α); 5.29 (s, 0.2H, H1β); 5.17 (dd, J3,4 = 3.5 Hz, J2,3 = 5.1 Hz, 0.8H, H3α);
5.12 (dd, J3,4 = 2.5 Hz, J2,3 = 6.1 Hz, 0.2H, H3β); 5.07–4.98 (m, 4H, HCH2Bn);
4.54–4.49 (m, 1H, H4); 4.29–4.24 (m, 0.2H, H5β); 4.15–4.16 (m, 0.2H, H2β);
ꢁ
ꢁ
4.13–4.11 (m, 1H, H2α, H5 β); 4.06–4.00 (m, 1.6H, H5α, H5 α); 2.03 (s, 0.6H,
CH3Acβ); 2.01 (s, 2.4H, CH3Acα). 13C NMR (CDCl3) δ 170.6 (COAc); 135.7,
135.6, 135.5 (CAr), 128.5, 128.0 (CAr); 102.9 (C1β); 95.9 (C1α); 80.3 (C2β);
78.3 (C3α); 78.2 (C3β); 77.7 (d, JP-O-C-C = 6.3 Hz,C4β); 75.3 (C2α); 75.2 (d,
JP-O-C-C = 6.3 Hz,C4α); 69.8(d, JP-O-C = 5.6 Hz, CCH2Bnβ); 69.6(d, JP-O-C = 5.3
Hz, CCH2Bnα); 69.5(d, JP-O-C = 5.1 Hz, CCH2Bnα); 67.0 (d, JP-O-C = 5.4 Hz, C5β);
65.5 (d, JP-O-C = 5.5 Hz, C5α); 20.6 (CMeAc). 31P NMR (CDCl3) δ 0.3 (β), -0.1
(α). HRMS m/z 453.1328 (calcd for C21H26O9P([M+H]+) 453.1314).
3-O-Acetyl-5-O-phosphate-D-xylofuranose 22. 3-O-Acetyl-acetyl-5-O-
dibenzylphosphate-D-ribofuranose (42 mg, 0.81 mmol) was diluted in
THF (10 ml) at room temperature and added under argon to commercial
Pd(OH)2/C. The mixture was stirred at room temperature under H2
pressure (balloon pressure) for 30 minutes. The catalyst was then filtered
off (AUTOTOP syringe filters, Whatman) and the solvent removed
under reduced pressure to yield quantitatively 3-O-acetyl-5-O-phosphate-
D-ribofuranose as a mixture of α/β anomers (1/1) which rearranged in
solution to yield a mixture of 3-O-acetyl-5-O-phosphate-D-ribofuranose 22
and 2-O-acetyl-5-O-phosphate-D-ribofuranose 23.
1H NMR (D2O) 5.32 (d, J = 4.4 Hz, 0.5H, H1α); 5.17 (s, 0.5H, H1β); 5.15
(dd, J3,4 = 3.6 Hz, J2,3 = 5.6 Hz, 0.5H, H3α); 5.08 (dd, J3,4 = 1.9 Hz, J2,3 = 5.2
Hz, 0.5H, H3β); 4.48–4.42 (m, 1H, H4); 4.03–4.01 (m, 0.5H, H2α); 4.00–3.95
ꢁ
(m, 1H, H2β, H5β); 3.91 (ABX, J4,5 = 4.2 Hz, J5,5 = 11.2 Hz, 0.5H, H5α); 3.88
ꢁꢁ
ꢁ
ꢁ
ꢁ
(ABX, J4,5 = 6.0 Hz, J5,5 = 10.5 Hz, 0.5H, H5 β); 3.81 (ABX, J4,5 = 5.6 Hz,
ꢁ
ꢁ
J5,5 = 11.3 Hz, 0.5H, H5 α); 2.03 (s, 1.5H, CH3Acβ); 2.02 (s, 1.5H, CH3Acα).
13C NMR (D2O) δ 173.9, 173.5 (COAc); 102.2 (C1β); 95.7 (C1α); 79.1 (C2β);
78.8 (d, JP-O-C-C = 8.9 Hz,C4β); 77.6 (C3A); 77.2 (C3β); 75.3 (d, JP-O-C-C = 8.7
Hz,C4α); 74.2 (C2α); 65.0 (d, JP-O-C = 4.9 Hz, C5β); 64.4 (d, JP-O-C = 4.9 Hz,
C5α); 20.5 (CMeAc). 31P NMR (CDCl3) δ 1.6 (br). HRMS m/z 271.0215 (calcd
for C7H12O9P([M-H]) 271.0219).
O-Acetyl-adenosine-diphospho-xylose 4. The product of the hydrogenol-
ysis of 3-O-acetyl-acetyl-5-O-dibenzylphosphate-D-ribofuranose (37 mg, 136.0
µmol) was made anhydrous by repeated dissolution in dry pyridine
and evaporation of the solvent. After each evaporation step, argon was
flushed into the rotary evaporator. AMP-morpholidate (4’-morpholine-N -
N’-dicyclohexylcarboxamidinium salt; 106 mg, 149.6 µmol) was dissolved
in dry pyridine, evaporated to dryness and the process was repeated three

1140
O. P. Chevallier and M. E. Migaud
times with exclusion of moisture. Both components were finally dissolved in
dry pyridine and combined. The reaction mixture was repeatedly sonicated
and flushed with argon. A final amount of pyridine was added and the
reaction mixture (5 mL total volume) was stirred for 2 days. The solvent was
then removed under vacuum and the yellowish residue was dissolved in mQ
water (0.5 mL) to be purified using pre-packed DSC-SAX column (6 mL/1
g) with water (10 mL) and then buffer (formic acid 5 mM, pH = 4.0, 10
mL). The fractions containing pyrophosphate products, identified by HPLC
using anion exchange chromatography (SAX column, 200 × 4.5 mm, 5 µm;
phosphate buffer, KH2PO4 50 mM, pH = 3.5, 5% MeOH) were freeze-dried
to afford a white residue which was purified further using reversed-phase
chromatography. The residue was dissolved in mQ water (0.5 mL) and
purified by HPLC using a semi-preparative column with ammonium formate
buffer (formic acid 50 mM, pH = 6.0, 5% MeOH) eluated at a flow rate of
2.5 mL/minute to yield adenosine-diphospho-xylose 6 (2.7 µmol quantified
by UV, 2%), O-acetyl-adenosine-diphospho-xylose 4 (9.5 µmol quantified by
UV, 7%).
1H NMR (D2O) 8.40 (s, 1H, H2); 8.13 (s, 1H, H8); 6.02 (d, J = 5.3
ꢁ
ꢁꢁ
Hz, 1H, H1 ); 5.62 (m, 1H, H1 ); 4.41–4.39 (m, 2H); 4.27–4.26 (m, 2H);
4.17–4.00 (m, 6H); 1.92, 1.91, 1.90 (s, 3H, CH3Ac). 31P NMR (D2O) δ
-10.1 (br, P-O-P). HRMS m/z 600.0779 (calcd for C17H24O14N5P2([M-H])
600.0744).
Adenosine-Diphospho-Xylose 6
1H NMR (D2O) 8.41 (s, 1H, H2); 8.17 (s, 1H, H8); 6.05 (d, J = 6.0 Hz,
ꢁ
ꢁꢁ
1H, H1 ); 5.10–5.06(m, 1H, H1 ); 4.58–4.55 (m, 2H); 4.47–4.42 (m, 2H);
4.31–4.29 (m, 2H); 4.14–4.01 (m, 4H). 31P NMR (D2O) δ -10.0 (br, P-O-P).
HRMS m/z 558.0635 (calcd for C15H22O14N5P2([M-H]) 558.0639).
3-O-Acetyl-5-O-tert-butyldiphenylsilyl-1,2-O,O-isopropylidene-β-D-
arabinofuranose 18. A solution of 5-O-tert-butyldiphenylsilyl-1,2-O,O-
isopropylidene-β-D-arabinofuranose[11] 17 (7.0 g, 16.3 mmol) in pyridine
(50 ml) with acetic anhydride (1.8 ml, 19.6 mmol) was stirred overnight
at room temperature. The reaction mixture was concentrated under
vacuum and azeotroped 3 times with toluene to afford 3-O-acetyl-5-O-tert-
butyldiphenylsilyl-1,2-O,O-isopropylidene-β-D-arabinofuranose 18 (7.5 g,
98%) as a colorless oil.
1H NMR (CDCl3) δ 7.70–7.67 (m, 4H, HAr); 7.44–7.37 (m, 6H, HAr);
5.89 (d, J1,2 = 4.0 Hz, 1H, H1); 5.34 (d, J3,4 = 1.9 Hz, 1H, H3); 4.56 (d, J1,2
ꢁ
= 4.1 Hz, 1H, H2); 4.22 (ddd, J3,4 = 2.0 Hz, J4,5 = 5.8 Hz, J4,5 = 7.9 Hz, 1H,
ꢁ
ꢁ
H4); 3.87 (ABX, J4,5 = 8.1 Hz, J5,5 = 10.3 Hz, 1H, H5); 3.82 (ABX, J4,5 = 5.8
ꢁ
ꢁ
Hz, J5,5 = 10.3 Hz, 1H, H5 ); 2.10 (CMeAc); 1.32 (s, 3H, CH3Iso); 1.28 (s, 3H,
CH3Iso); 1.07 (s, 9H, tBu). 13C NMR (CDCl3) δ 169.6(COAc); 135.6, 133.2,
129.6, 127.6 (CAr); 112.6 (CqIso); 105.7 (C1); 85.5 (C4); 84.8 (C2); 77.3 (C3);

Simple Adenosine Diphosphate Ribose Analogues
1141
63.6 (C5); 26.7 (CtBu); 26.6 (CMeIso); 25.9 (CMeIso); 20.8 (CMeAc); 19.1 (CSi).
HRMS m/z 493.2009 (calcd for C26H34O6NaSi([M+Na]+) 493. 2022).
3-O-Acetyl-1,2-O,O-isopropylidene-β-D-arabinofuranose 19. 3-O-Acetyl-
5-O-tert-butyldiphenylsilyl-1,2-O,O-isopropylidene-β-D-arabinofuranose 18
(5.6 g, 11.9 mmol) was dissolved in THF (100 mL). To this resulting
solution was added TBAF (14.3 ml of 1M solution in THF) and was
left under stirring at room temperature overnight. A saturated solution
of NH4Cl (50 mL) was then added. The aqueous layer was extracted
with chloroform (3 × 50 mL) and the combined organic layers were
washed with water, brine, dried, filtered, and concentrated under vacuum
to afford an oil. Purification by flash chromatography (gradient from
8/2 to 1/1 v/v petroleum ether/ethyl acetate) afforded two fractions
3-O-acetyl-1,2-O,O-isopropylidene-β-D-arabinofuranose 19 (2.0 mg, 73%)
and 3,5-bis-O-acetyl-1,2-O,O-isopropylidene-β-D-arabinofuranose (610 mg,
7%).
1H NMR (CDCl3) δ 5.93 (d, J1,2 = 4.0 Hz, 1H, H1); 5.03 (d, J3,4 = 2.2
ꢁ
Hz, 1H, H3); 4.63 (d, J1,2 = 4.1 Hz, 1H, H2); 4.17 (ddd, J3,4 = 2.2 Hz, J4,5
ꢁ
= 5.2 Hz, J4,5 = 7.3 Hz, 1H, H4); 3.83 (ABX, J4,5 = 7.4 Hz, J5,5 = 11.4
ꢁ
ꢁ
ꢁ
Hz, 1H, H5); 3.82 (ABX, J4,5 = 4.7 Hz, J5,5 = 11.4 Hz, 1H, H5 ); 2.09 (s,
3H, CMeAc); 1.55 (s, 3H, CH3Iso); 1.32 (s, 3H, CH3Iso). 13C NMR (CDCl3) δ
170.1(COAc); 112.9 (CqIso); 105.6 (C1); 86.5 (C4); 84.8 (C2); 77.7 (C3); 62.7
(C5); 26.7 (CMeIso); 26.0 (CMeIso); 20.8 (CMeOAc). HRMS m/z 233.1034 (calcd
for C10H17O6 ([M+H]+) 233.1025).
3,5-Bis-O-acetyl-1,2-O,O-isopropylidene-β-D-arabinofuranose. 1H NMR
(CDCl3) δ 5.94 (d, J1,2 = 3.8 Hz, 1H, H1); 5.10 (d, J3,4 = 1.9 Hz, 1H, H3);
4.60 (d, J1,2 = 3.8 Hz, 1H, H2); 4.32–4.30 (m, 2H, H4, H5); 4.27–4.24 (m, 1H,
H5 ); 2.10 (s, 6H, CMeAc); 1.57 (s, 3H, CH3Iso); 1.32 (s, 3H, CH3Iso). 13C NMR
ꢁ
(CDCl3) δ 170.6, 169.7(COAc); 113.1 (CqIso); 105.9 (C1); 84.4 (C2); 83.0 (C4);
77.4 (C3); 63.8 (C5); 26.7 (CMeIso); 26.0 (CMeIso); 20.8 (CMeAc). HRMS m/z
275.2768 (calcd for C12H19O7 ([M+H]+) 275.2810).
3-O-Acetyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene-β-D-
arabinofuranose 16. To a solution of 3-O-acetyl-1,2-O-isopropylidene-β-
D-arabinofuranose 19 (0.59 g, 2.54 mmol) and dibenzylphosphoramidate
12 (1.2 g, 3.31 mmol) in dry DCM (20 mL) was added dropwise a solution
of 2,4-DNP (0.61 g, 3.31 mmol) in dry DCM (20 mL). This resulting
mixture was stirred for 3 hours at room temperature, and then a solution
of ter-BuOOH in toluene (1.6 ml of a 3 M solution) was added and kept
under stirring for 20 minutes. After dilution with DCM (20 mL), the
reaction mixture was washed with a sodium thiosulfate solution (30 mL),
water (30 mL × 3), dried, filtered, and concentrated under vacuum to
afford the crude fully protected phosphorylated precursor. Purification
by flash chromatography (gradient from 1/0 to 95/5 v/ CHCl3/ethanol)
yielded
3-O-acetyl-5-O-dibenzylphosphate-1,2-O,O-isopropylidene-β-D-
arabinofuranose 16 (800 mg, 64%) as a yellow oil.

1142
O. P. Chevallier and M. E. Migaud
1H NMR (CDCl3) 7.30–7.27 (m, 10H, HAr); 5.88 (d, J1,2 = 3.8 Hz, 1H,
H1); 5.08 (m, 1H, H3); 5.05–5.01 (m, 4H, HCH2Bn); 4.54 (d, J1,2 = 3.8 Hz,
ꢁ
1H, H2); 4.23–4.16 (m, 3H, H4, H5, H5 ); 2.00 (s, 3H, CH3Ac); 1.44 (s,
3H, CH3Iso); 1.23 (s, 3H, CH3Iso). 13C NMR (CDCl3) δ 169.2 (COAc); 135.5,
135.4 (CAr); 128.2, 128.1 (CAr); 112.6 (CqIso); 105.6 (C1); 83.9 (C2); 83.3
(d, JP-O-C-C = 8.3 Hz,C4); 76.5 (C3); 69.1(d, JP-O-C = 4.9 Hz, CCH2Bn); 69.0(d,
JP-O-C = 4.6 Hz, CCH2Bn);66.3 (d, JP-O-C = 5.4 Hz, C5); 26.3 (CMeIso); 25.5
(CMeIso); 20.3 (CMeAc). 31P NMR (CDCl3) δ -0.1. HRMS m/z 493.1625 (calcd
for C24H30O9P([M+H]+) 493.1627).
3-O-Acetyl-5-O-dibenzylphosphate-D-arabinofuranose 21. The same pro-
cedure as described for 20 was used for the synthesis of 21 (55%).
1H NMR (CDCl3) 7.34–7.30 (m, 10H, HAr); 5.43 (s, 0.4H, H1α); 5.39
(d, J1,2 = 4.3 Hz, 0.6H, H1β); 5.06–4.99 (m, 4H, HCH2Bn); 4.96 (t, J2,3 = J3,4
= 4.4 Hz, 0.6H, H3β); 4.83 (dd, J2,3 = 1.6 Hz, J3,4 = 4.4 Hz, 0.4H, H3α);
ꢁ
ꢁ
4.33–4.20 (m, 2.4H, H4α, H5β, H5 β, H5α, H5 α); 4.18 (d, J2,3 = 1.8 Hz, 0.4H,
H2α); 4.17 (dd, J1,2 = J2,3 = 4.4 Hz, 0.6H, H2β); 4.00 (ddd, J3,4 = 4.2 Hz,
ꢁ
J4,5 = 4.2 Hz, J4,5 = 7.2 Hz, 1H, H4β); 2.07 (s, 1.8H, CH3Acβ); 2.06 (s, 1.2H,
CH3Acα). 13C NMR (CDCl3) δ 171.0 (COAcα); 170.8 (COAcβ); 135.6, 135.5
(x2), 135.4 (CAr); 128.5, 128.0, 127.9 (CAr); 102.7 (C1α); 96.9 (C1β); 81.0 (d,
JP-O-C-C = 6.2 Hz,C4α); 80.3 (C2α); 78.2 (C3α); 79.8 (d, JP-O-C-C = 5.9 Hz,C4β);
79.1 (C3β); 75.8 (C2β); 69.7(d, JP-O-C = 5.5 Hz, CCH2Bn); 69.6(d, JP-O-C = 5.9
Hz, CCH2Bn); 68.6 (d, JP-O-C = 5.7 Hz, C5β); 67.2 (d, JP-O-C = 5.8 Hz, C5α); 20.7
(CMeAc). 31P NMR (CDCl3) δ -0.4 (β), -0.5 (α). HRMS m/z 453.1315 (calcd
for C21H26O9P([M+H]+) 453.1314).
3-O-Acetyl-5-O-phosphate-D-arabinofuranose 24. The same procedure as
described for 22 was used for the synthesis of 24.
1H NMR (D2O) 5.25 (s, 0.7H, H1α); 5.24 (d, J1,2 = 3.8 Hz, 0.3H, H1β);
5.00 (t, J2,3 = J3,4 = 4.2 Hz, 0.3H, H3β); 4.81 (dd, J2,3 = 1.8 Hz, J3,4 = 4.2
Hz, 0.7H, H3α); 4.32–4.29 (m, 0.7H, H4α); 4.19 (dd, J1,2 = 3.8 Hz, J2,3 = 4.2
ꢁ
Hz, 0.3H, H2β); 4.06 (m, 0.7H, H2α); 4.04–3.90 (m, 2.3H, H4β, H5β, H5 β
,
H5α, H5 α);2.01 (s, 0.9H, CH3Acβ); 2.00 (s, 2.1H, CH3Acα). 13C NMR (D2O)
δ 173.3 (COAc); 103.9 (C1α); 98.0 (C1β); 82.5 (d, JP-O-C-C = 8.3 Hz,C4α); 81.5
(C2α); 80.9 (C3α); 80.8 (d, JP-O-C-C = 8.2 Hz,C4β); 79.9 (C3β); 76.6 (C2β); 69.3
(d, JP-O-C = 5.2 Hz, C5β); 67.7 (d, JP-O-C = 5.1 Hz, C5α); 20.8 (CMeAc). 31P
NMR (CDCl3) δ 1.8 (br). HRMS m/z 271.0217 (calcd for C7H12O9P([M-H])
271.0219).
ꢁ
O-Acetyl-adenosine-diphospho-arabinose 5. The same procedure as de-
scribed for 4 and 6 was used for the synthesis of 5 (9.3 µmol; 9%) and 7 (2.3
µmol; 4%).
1H NMR (D2O) 8.53 (s, 1H, H2); 8.08 (s, 1H, H8); 6.10 (d, J = 5.9 Hz,
ꢁ
ꢁꢁ
ꢁꢁ
1H, H1 ); 5.41 (d, J = 4.5 Hz, 0.5H, H1 ); 5.30 (s, 0.2H, H1 ); 5.24 (s, 0.3H,
ꢁꢁ
H1 ); 4.54–4.48 (m, 2H); 4.40–4.33 (m, 2H); 4.19–4.16 (m, 2H); 4.14–3.94
(m, 4H); 2.09, 2.03, 2.02 (s, 3H, CH3Ac). 31P NMR (D2O) δ -9.9 (br, P-O-P).
HRMS m/z 600.0775 (calcd for C17H24O14N5P2([M-H]) 600.0744).

Simple Adenosine Diphosphate Ribose Analogues
1143
Adenosine-diphospho-arabinose 7
1H NMR (D2O) 8.47 (s, 1H, H2); 8.23 (s, 1H, H8); 6.10 (d, J = 5.9 Hz,
ꢁ
ꢁꢁ
ꢁꢁ
1H, H1 ); 5.20 (d, J = 4.6 Hz, 0.5H, H1 ); 5.18 (s, 0.5H, H1 ); 4.58–4.56 (m,
2H); 4.40–4.33 (m, 2H); 4.36–4.33 (m, 2H); 4.16–3.99 (m, 4H). 31P NMR
(D2O) δ -10.0 (br, P-O-P). HRMS m/z 558.0667 (calcd for C15H22O14N5P2
([M-H]) 558.0639).
REFERENCES
1. Perraud, A.L.; Fleig, A.; Dunn, C.A.; Bagley, L.A.; Launay, P.; Schmitz, C.; Stokes, A.J.; Zhu,
Q.; Bessman, M.J.; Penner, R.; Kinet, J.P.; Scharenberg, A.M. ADP-ribose gating of the calcium-
permeable LTRPC2 channel revealed by Nudix motif homology. Nature 2001, 411, 595–599.
2. Kuhn, F.J.P.; Heiner, I.; Luckhoff, A. TRPM2: a calcium influx pathway regulated by oxidative stress
and the novel second messenger ADP-ribose. Eur. J. Physiol. 2005, 451, 212–219.
3. Hara, Y.; Wakamori, M.; Ishii, M.; Maeno, E.; Nishida, M.; Yoshida, T. H. Y.; Shimizu, S.; Mori, E.;
Kudoh, J.; Shimizu, N.; Kurose, H.; Okada, Y.; Imoto, K.; Mori, Y. LTRPC2 Ca2+-permeable channel
activated by changes in redox status confers susceptibility to cell death. Mol. Cell 2002, 9, 163–173.
4. Grubisha, O.; Rafty, L.A.; Takanishi, C.L.; Xu, X.; Tong, L.; Perraud, A.L.; Scharenberg, A.M.;
Denu, J.M. Metabolite of SIR2 reaction modulates TRPM2 ion channel. J. Biol. Chem. 2006, 281,
14057–14065.
5. Denu, J. Linking chromatin function with metabolic networks: Sir2 (silent information regulator 2)
family of NAD+-dependent deacetylases. Trends Biochem. Sci. 2003, 28, 41–48.
6. Liou, G.G.; Tanny, J.C.; Kruger, R.G.; Walz, T.; Moazed, D. Assembly of the SIR complex and its
regulation by O-acetyl-ADP-Ribose, a product of NAD-dependent histone deacetylation. Cell 2005,
121, 515–527.
7. (a). Kustatscher, G.; Hothorn, M.; Pugieux, C.K.S.; Ladurner, A.G. splicing regulates NAD metabo-
lite binding to histone macroH2A. Nature Structural & Molecular Biology 2005, 12, 624–625 (b). Hoff,
K. G.; Wolberger, C. Getting a grip on O-acetyl-ADP-ribose. Nat. Struct. Mol. Biol. 2005, 12, 560–561.
8. Comstock, L.R.; Denu, J.M. Synthesis and biochemical evaluation of O-acetyl-ADP-ribose and N-
acetyl analogs. Org. Biomol. Chem. 2007, 5, 3087–3091.
9. Chevallier, O.P.; Migaud, M.E. Investigation of acetyl migrations in furanosides. Beilstein J. Org. Chem.
2006, 2:14.
10. Amigues, E.J.; Migaud, M.E. Synthesis of cyclophospho-glucoses and glucitols. Tetrahedron Lett. 2004,
45, 1001–1004.
11. Vazquez-tato, M.P.; Seijas, J.A.; Fleet, G.W.J.; Mathews, C.J.; Hemmings, P.R.; Brown, D. Synthesis
of (3S, 5S)-quinuclidine-3,5-diol and of [3S-(3, 3a, 3a)]-octahydro-2-furo[2,3-c]pyridinol from D-
arabinose. Tetrahedron 1995, 51, 959–974.
12. Paquette, L.A.; Barriault, L.; Pissarnitski, D.; Johnston, J.N. Stereocontrolled elaboration of natural
(−)-polycavernoside A, a powerfully toxic metabolite of the red alga Polycavernosa tsudai. J. Am.
Chem. Soc. 2000, 122, 619–631.
13. Yadav, J.S.; Subba Reddy, B.V. A mild and selective cleavage of trityl ethers by CBr4-MeOH. Carbohydr.
Carbohydr. 2000, 329, 885–888.
14. Hoeffler, J.-F.; Pale-Grosdemange, C.; Rohmer, M. Synthesis of tritium labelled 2-C-methyl-D-
erythritol, a useful substrate for the elucidation of the methylerythritol phosphate pathway for
isoprenoid biosynthesis. Tetrahedron 2000, 56, 1485–1489.