Di-tert-butyl diethylphosphoramidite as the phosphitylating reagent in the preparation of 3-deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate

Article abstract of DOI:10.1016/S0008-6215(01)00089-1

3-Deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate were prepared from a common intermediate 3-deoxy-3-C-methylene-1,2-O-isopropylidene-α-D-ribo-hexofuranose. The preparation of the phosphorylated unsaturated sugars employed di-tert-butyl diethylphosphoramidite as the phosphitylating reagent. The removal of all the protecting groups was done under acidic conditions in the ultimate step. The unsaturated sugar phosphates were competitive inhibitors but neither substrates nor inactivators of glucose-6-phosphate and ribose-5-phosphate isomerases.

Full text of DOI:10.1016/S0008-6215(01)00089-1

www.elsevier.nl/locate/carres
Carbohydrate Research 332 (2001) 141–149
Di-tert-butyl diethylphosphoramidite as the
phosphitylating reagent in the preparation of
3-deoxy-3-C-methylene-
3-deoxy-3-C-methylene-
D
-ribo-hexose-6-phosphate and
-erythro-pentose-5-phosphate
D
Alain Burger,* Denis Tritsch, Jean-Franc¸ois Biellmann
Laboratoire de Chimie Organique Biologique associe´ au CNRS UMR 7509, Faculte´ de Chimie,
Uni6ersite´ Louis Pasteur, 1 rue Blaise Pascal, F-67008 Strasbourg, France
Received 28 February 2001; accepted 21 March 2001
Abstract
3-Deoxy-3-C-methylene-
were prepared from a common intermediate 3-deoxy-3-C-methylene-1,2-O-isopropylidene-a-
D
-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-
D
-erythro-pentose-5-phosphate
-ribo-hexofuranose.
D
The preparation of the phosphorylated unsaturated sugars employed di-tert-butyl diethylphosphoramidite as the
phosphitylating reagent. The removal of all the protecting groups was done under acidic conditions in the ultimate
step. The unsaturated sugar phosphates were competitive inhibitors but neither substrates nor inactivators of
glucose-6-phosphate and ribose-5-phosphate isomerases. © 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Carbohydrates; Enzyme inhibitors; Phosphoric acids and derivatives; Phosphorylation
proposed (Scheme 2, A).1 Later, other groups
have proposed that the interconversion of al-
dose and ketose sugars follows rather through
a hydride transfer mechanism (Scheme 2, B).2
To get further information about the enzyme
mechanism, we have extended our study to
1. Introduction
The glucose analogues, 3-deoxy-3-C-methy-
lene-
oxy-3-C-fluoromethylene-
substrates of -xylose isomerase (or
D
-ribo-hexose (1) (Scheme 1) and 3-de-
D
-ribo-hexose, are
-glucose
D
D
isomerase) (EC 5.3.1.5) isolated from Strepto-
myces rubiginosus.1 The produced ketose
derivatives inactivate the enzyme by an out/in
mechanism and by a kcat mechanism, respec-
tively, with the remarkable partition ratio of
1.1 The inactivation of the enzyme was due to
the reaction with His-54. Based on the crystal-
lographic structure of the native and the alkyl-
ated enzyme, a proton-transfer mechanism in-
volving a histidine as the general base, was
Scheme 1. Structures of a kcat inhibitor of
and of the putative kcat inhibitors of phosphoglucose and
D-xylose isomerase
* Corresponding author. Tel.: +33-3-90241692.
E-mail address: aburger@chimie.u-strasbg.fr (A. Burger).
phosphoribose isomerases.
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00089-1

142
A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
Scheme 2. Proposed enzymatic mechanisms for the isomerisation of an aldose to a ketose. The ene-diol and the hydride shift
mechanisms are shown in A and B, respectively.
phosphoglucose isomerase (EC 5.3.1.9) and
phosphoribose isomerase (EC 5.3.1.6). These
enzymes perform the interconversion of gluc-
ose-6-phosphate and fructose-6-phosphate
and of ribose-5-phosphate and ribulose-5-
phosphate, respectively, via a proton-transfer
mechanism.3 For such a study, we required
the phosphates of unsaturated carbohydrate
derivatives 2 and 3. We report here the synthe-
sis of sodium salts of sugar phosphates 2 and
3 and the evaluation of their biological activ-
ity on phosphoglucose and phosphoribose
isomerases.
unsaturation6 and the O-6-phosphate func-
tionalities.7
We initially expected to prepare selectively
the di-tert-butyl phosphate at O-6 by treat-
ment of diol 4 with di-tert-butyl diethyl-
phosphoramidite8 and 1H-tetrazole followed
by oxidation with tert-butyl hydroperoxide.
The unstable five membered cyclic phosphate
59 was isolated instead of the desired com-
pound. The proposed cyclic structure for 5
1
was supported by H NMR and FABMS
data. One plausible reason for the formation
2. Results and discussion
Synthesis.—The synthesis of the two target
compounds could be realised from the com-
mon starting material 4.4 Compound 4 was
prepared by known procedures.4,5 We first
envisioned to prepare the 6-phosphate glucose
derivative using the dibenzylphosphate strat-
egy of Mu¨ller and Schmidt for the preparation
of a sugar diphosphate nucleoside.5 In their
synthesis, the hydrogenolysis over palladium
of a 1-dibenzylphosphate-3-C-methylene-hex-
opyranosyl intermediate afforded selectively
the corresponding 1-phosphate-3-C-methyl-
ene-hexopyranosyl compound. We prepared
the dibenzylphosphate derived from 4. How-
ever, in our hands and under various condi-
tions, reduction of the double bond occurred
with hydrogenolysis of the benzyl protecting
groups. To overcome this limitation, a new
approach was elaborated and applied to 4 as
shown in Scheme 3. We hoped that the acidic
removal of the phosphate tert-butyl protecting
groups could be compatible with the
Scheme 3. (a) (i) (t-BuO)2Et2NP, 1H-tetrazole, THF, −25 °C
to rt, 3 h; (ii) t-BuOOH, 4 equiv Et3N. (b) (i) t-BuPh2SiCl,
NEt(i-Pr)2, DMF, 3 h; (ii) 4 equiv ClCH2OCH3, 5 equiv
NEt(i-Pr)2, 4 °C to rt, 12 h. (c) Bu4NF, THF, 1 h. (d) (i)
(t-BuO)2Et2NP, 1H-tetrazole, THF, −25 °C to rt, 3 h; (ii) 3
equiv H2O2, 9 equiv Et3N. (e) (i) 0.3 equiv CF3CO2H, water,
7 days; (ii) 2 equiv NaOH, water.

A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
143
gration of the silyl group to the vicinal
secondary alcohol was observed.11 For this
reason we preferred the more bulky tert-
butyldiphenylsilyl group because of its lower
tendency to migrate.12 Selective protections of
the diol 4 were accomplished in one pot in
90% overall yield. Successive treatment with
tert-butylchlorodiphenylsilane and Hu¨nig’s
base,13 and chloromethyl methylether14 af-
forded the silyl ether at O-6 and the MOM
acetal at O-5, respectively. Removal of the
silyl moiety of ether 6 under standard condi-
tions with the fluoride anion12 furnished the
alcohol 7. Preparation of the 6-phosphotri-
ester 8 was first tried using the original condi-
tions.8 Successive treatment of alcohol 7 with
di-tert-butyl diethylphosphoramidite in the
presence of 1H-tetrazole and m-chloroperoxy-
benzoic acid (mCPBA) afforded the desired
compound along with a complex mixture of
polar products as evidenced by TLC. De-tert-
butylation of acid sensitive phosphate triesters
was already observed. Surprisingly, it was
mentioned for silica-gel chromatography but
not for phosphite oxidation with mCPBA.15
Decomposition of the phosphate triester 8 was
avoided when triethylamine was added to neu-
tralise 1H-tetrazole in excess and hydrogen
peroxide was used instead of mCPBA. Under
these conditions, the triester 8 was isolated in
high yield. Removal of all the protecting
groups of compound 8 was performed in the
last step under acidic conditions with 0.3
equiv of trifluoroacetic acid in water. Depro-
tection of the phosphate triester and acetonide
groups was faster than removal of the MOM
Scheme 4. Proposed mechanism for the intramolecular trans-
esterification.
Scheme 5. (a) (i) NaIO4, 1:1 water–EtOH, 1 h; (ii) NaBH4,
4 °C, 2 h. (b) (i) (t-BuO)2Et2NP, 1H-tetrazole, THF, −25 °C
to rt, 3 h; (ii) 3 equiv H2O2, 9 equiv Et3N. (e) (i) 0.3 equiv
CF3CO2H, water, 32 h; (ii) NaOH, water.
of by-product 5 is related to Watanabe’s
observation10 that 1H-tetrazole catalysed
transesterification of different trialkyl phos-
phite with an alcohol. However, decomposi-
tion of an alkyl di-tert-butyl phosphite to the
corresponding alkyl tert-butyl hydrogenphos-
phonate was observed rather than transester-
ification. Nevertheless, the kinetically more
favourable intramolecular reaction with the
vicinal alcohol likely explains why transester-
ification occurred with the di-tert-butyl phos-
phite derived from 4 (Scheme 4). Acidic
hydrolysis of the cyclophosphate triester gave
a complex mixture of products that we were
unable to separate. Thus, use of Perich and
Johns phosphitylating reagent8 required prior
to protect the secondary vicinal alcohol. This
was accomplished by the sequence of reactions
depicted in Scheme 3.
1
group as evidenced by H NMR. After com-
pletion, the volatiles were removed under vac-
uum to give the corresponding phosphate
monoester that was pure enough to continue
without chromatography. The disodium salt 2
was obtained in 96% yield after addition of
two equivalents of sodium hydroxide.
The same methodology was used for the
preparation of the second target compound 3
(Scheme 5).
The alcohol 916 was obtained in two steps in
83% yield by oxidative cleavage of the diol 4
with sodium periodate and by reduction of the
generated aldehyde with sodium borohy-
dride.17 Phosphorylation and removal of the
protecting groups were realised as described
The tert-butyldimethylsilyl group was re-
ported as a selective protecting group of the
primary alcohol of the 5,6-diol of a gly-
coside.11 However, under basic conditions, mi-

144
A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
urated ketoses. So the absence of inactivation
of phosphoglucose isomerase could be due to
the fact that both compounds 2 and 3 were
not isomerised by the enzyme. Contrarily to
xylose isomerase, phosphoglucose isomerase
has a rather strict substrate specificity. The
enzyme catalyses only the reversible isomerisa-
tion of glucose-6-phosphate and fructose-6-
before. Phosphate 3 was isolated in high yield
as the sodium salt.
Phosphate salts 2 and 3 were characterised
by their spectroscopic and physical data. For
instance in 31P NMR, four resonances of
phosphate 2 were detected on the proton-de-
coupled spectrum at 4.71 (50%), 4.67 (37%),
4.45 (2%) and 4.3 (11%) ppm. On the proton-
coupled spectrum, only the three most abun-
dant isomers were detected each as a triplet
signal with a 3JPH coupling constant of 6.5, 5.6
and 6.9 Hz, respectively, in agreement with
phosphate, but 3-deoxy-3-fluoro- -glucose
D
was reported to be transformed by the enzyme
at a relatively slow rate.22 The two analogues
2 and 3 inhibited reversibly phosphoglucose
isomerase. Inhibition constants of 4–6 mM
were found for both compounds.
The pentose analogue 3 was also tested on
phosphoribose isomerase. It was neither sub-
strate nor inactivator. The enzyme was re-
versibly inhibited by compound 3. The
inhibition constant (about 27 mM) was much
higher than the inhibition constants of known
3
the JPH determined on other phosphoric
mono-esters of primary alcohols.18 In deuter-
ated water, phosphate 2 exists likely as a
mixture of both a and b pyranose and fura-
nose anomers, the a and b pyranose anomers
being probably the most abundant by analogy
to glucose-6-phosphate.19 On the other hand,
two triplet signals were observed in the NMR
spectrum for phosphate 3 at 1.32 (43%) and
competitive inhibitors such as 4-phospho-
erythronic acid (Ki=4.4 mM) or 5-phospho-
ribonic acid (Ki=119 mM).23
D
-
-
3
D
1.18 (57%) ppm with JPH of 6.1 and 5.8 Hz,
respectively. In deuterated water, phosphate 3
exits likely as a mixture of a and b furanose
anomers as observed for ribose-5-phosphate.20
Enzymatic assays.—Phosphoglucose iso-
merase is assayed by a coupled enzymatic test
with glucose-6-phosphate dehydrogenase.21 So
we have first studied the effect of 3-deoxy-3-
In conclusion, the synthetic strategy pre-
sented here employed di-tert-butyl di-
ethylphosphoramidite as the phosphitylating
reagent for the preparation of unsaturated
carbohydrate phosphates. It represents a valu-
able alternative to the dibenzylphosphate one,
which failed in our hands. The phosphate and
sugar protecting groups were removed at the
last step under acidic conditions compatible
with the unsaturation. The unsaturated sugar
phosphates were competitive inhibitors but
neither substrates nor inactivators of glucose-
6-phosphate and ribose-5-phosphate iso-
merases.
C-methylene-
D
-ribo-hexose-6-phosphate
(2)
and 3-deoxy-3-C-methylene-erythro-pentose-
5-phosphate (3) on the glucose-6-phosphate
dehydrogenase activity. Both compounds 2
and 3 were neither substrate nor inactivator of
this enzyme. On the other hand, they were
reversible competitive inhibitors. Inhibition
constants of 14 and 10 mM were found for
compounds 2 and 3, respectively. These inhi-
bitions were too low to affect the activity of
glucose-6-phosphate dehydrogenase during
the determination of the activity of phospho-
glucose isomerase in our experimental
conditions.
3. Experimental
General methods.—Unless otherwise indi-
cated, all reagents were obtained from com-
mercial suppliers and were used without
purification. All experiments sensitive to air
and/or to moisture were carried out under an
Ar atmosphere in an oven dried (120 °C)
glassware assembled under a stream of Ar.
Anhydrous solvents were freshly distilled be-
fore use: THF from sodium benzophenone
ketyl radical, diisopropylethylamine from
CaH2 and DMF from P2O5. Analytical thin-
The incubation of phosphoglucose iso-
merase in the presence of compounds 2 or 3
did not inactivate the enzyme. As mentioned
above, the inactivation of xylose isomerase
with 3-deoxy-3-C-methylene-ribo-hexose (1)
and 3-deoxy-3-C-fluoromethylene-ribo-hexose
requires the transformation of the compounds
by the enzyme to the corresponding a,b-unsat-

A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
145
a 70% solution of t-BuOOH in water (0.32
mL, 2.35 mL) were added to the cooled solu-
tion (−30 °C). The cooling bath was removed
and stirring was continued until conversion
was completed (TLC). The reaction was di-
luted with AcOEt (20 mL) and water. The
phases were separated, the organic phase was
washed with brine (10 mL), dried over
Na2SO4, filtered and evaporated to dryness to
give the crude extract of the unstable cy-
clophosphotriester 5 (720 mg) that decom-
posed quickly if subjected to SiO2
layer chromatography was performed on silica
gel pre-coated TLC plates (E. Merck, 60,
F254). Products were isolated by flash chro-
matography on silica gel (E. Merck, 60, 230–
400 mesh). Melting points were measured on a
Reichert microscope and were uncorrected.
[h]D were measured on a Perkin–Elmer
241MC polarimeter. IR spectra were recorded
on a Perkin–Elmer 881 or a Bruker FT
IFS25. NMR spectra were recorded on a
Brucker SY (200 or 300 MHz) apparatus. For
1H NMR the residual proton signal of the
deuterated solvent was used as an internal
reference: for CDCl3 (l 7.26 ppm), C6D6 (l
7.16), and D2O (l 4.90). For 13C NMR, the
13C central signal of the deuterated solvent
was used as an internal reference; for CDCl3
(l 76.9 ppm), C6D6 (l 128.0) and 2-methyl-2-
propanol-d10 (l 40.02). The chemical shifts are
reported in ppm downfield from TMS. For 31P
NMR, H3PO4 (85%) was used as an external
reference (l 0 ppm). MS were measured on a
LKB 9000S apparatus by electron impact (EI,
70 eV), on a Trio 2000 (FISONS, UK) ap-
paratus by chemical ionisation (CI) or on a
ZAB (FISONS, UK) apparatus by fast-atom
bombardment (FAB). Microanalyses were
performed by the Service de Microanalyses de
Strasbourg.
1
chromatography. H NMR analysis indicated
essentially a mixture of two glucose deriva-
tives where phosphate 5 was the major com-
1
pound (70%). H NMR (CD2Cl2): l 5.80 (d, 1
H, J1,2 3.9 Hz, H-1), 5.49 (bs, 1 H, H-unsat),
5.41 (bs, 1 H, H-unsat), 4.91 (m, 1 H, H-2),
4.84 (m, 1 H, H-4), 4.60–4.15 (m, 3 H, H-5,
H-6), 1.50 (s, 9 H, t-Bu), 1.47 (s, 3 H, Me),
1.35 (s, 3 H, Me); (FABMS, NBA): m/z 333
[M−1], 277, 209, 153, 97.
6-O-tert-Butyldiphenylsilyl-3-deoxy-5-O-
methoxymethyl-3-C-methylene-1,2-O-isopropy-
lidene-h-
D
-ribo-hexofuranose
(6).—To
a
stirred solution under Ar of compound 4
(0.649 g, 3 mmol) (dried by azeotropic distilla-
tion with 2×2 mL dry pyridine) in dry DMF
(3 mL) were added t-BuPh2SiCl (0.831 mL,
3.15 mmol) and dry NEt(i-Pr)2 (0.810 mL,
4.75 mmol). The reaction evolution was moni-
tored by TLC with 7:3 hexane–ether as elu-
ents. After 3 h, the reaction was cooled in an
ice bath, NEt(i-Pr)2 (2.76 mL, 15 mmol) was
added followed by a dropwise addition of
ClCH2OCH3 (0.975 mL, 13 mmol) (CAU-
TION, carcinogen and highly toxic24). Stirring
was continued 1 h at 4 °C and overnight at rt.
The reaction was diluted with water (2 mL)
and Et2O (30 mL), the phases were separated,
the aqueous phase was extracted twice with
Et2O (2×15 mL). The combined organic
phases were washed with brine (20 mL), dried
over MgSO4, filtered and evaporated to dry-
ness to give an orange oil. Flash chromatogra-
phy over SiO2 with 17:5 then 4:1 hexane–ether
as eluents furnished compound 6 as a colour-
less oil (1.28 g, 90%). [h]2D1 +61° (c 4.3,
CHCl3); IR (neat): w 3070 and 3049 (w),
1959,1895 and 1830 (w), 1589 (w), 1468 (m),
3-Deoxy-3-C-methylene-1,2-O-isopropyli-
dene-h- -ribo-hexofuranose (4).—Diol 4 was
D
obtained as described:5 [h]D21 +134° (c 4,
CHCl3); lit. [h]D20 +119° (c 1, CHCl3);4 Anal.
Calcd for C10H16O5: C, 55.55; H, 7.46. Found:
C, 55.50; H, 7.50.
3-Deoxy-3-C-methylene-1,2-O-isopropyli-
dene-h- -ribo-hexofuranose-5,6-tert-butyl-
D
phosphate (5).—Compound 4 (486 mg, 2.25
mmol) (dried under high vacuum, p=100 Pa,
at 50 °C for 5 h) and anhyd 1H-tetrazole
(dried by sublimation at p=100 Pa in an oil
bath at 85 °C. CAUTION, explosive, the oil
bath temperature was kept below 100 °C24),
were dissolved in dry THF (6 mL). The solu-
tion was cooled at −25 °C. A solution of
di-tert-butyl diethylphosphoramidite7 (586
mg, 2.35 mmol) in dry THF (2 mL) was added
dropwise to the mixture during a 5 min pe-
riod. After addition, the reaction temperature
was allowed to raise slowly, in 3 h to rt. After
30 min, Et3N (1.26 mL, 9 mmol) followed by
1
1427 (m), 1213 (m), 1019 (bs) cm−1; H NMR

146
A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
(CDCl3): l 7.69–7.63 (m, 4 H, Ph), 7.44–7.34
(m, 6 H, Ph), 5.77 (d, 1 H, J1,2 4.3 Hz, H-1),
5.40 (dd, 1 H, J1=J2 1.7 Hz, H-olef), 5.18
(dd, 1 H, J1=J2 1.7 Hz, H-olef), 5.02 (bs, 1
H, H-4), 4.85 (bdd, 1 H, H-2), 4.73 (d, 1 H, J
6.6 Hz, CH2O), 4.69 (d, 1 H, CH2O), 3.90–
3.62 (m, 3 H, H-5, H-6), 3.3 (s, 3 H, OMe),
1.47 (s, 3 H, Me), 1.38 (s, 3 H, Me), 1.04 (s, 9
H, t-Bu); 13C NMR (CDCl3): l 146.4 (q),
135.6 (t), 133.4 (q), 133.2 (q), 129.8 (t), 127.8
(t), 112.6 (q), 112.5 (s), 105.0 (t), 96.9 (s), 81.9
(t), 80.8 (t), 79.7 (t), 63.0 (s), 55.8 (p), 27.7 (p),
26.9 (p), 19.2 (q); MS (IE, 70 eV) 441 (M+−
33, 8), 383 (4), 353 (8), 351 (4), 323 (8), 321
(7), 176; Anal. Calcd for C28H38O6Si: C, 65.78;
H, 8.07. Found: C, 65.92; H, 8.05.
at 50 °C for 3 h) and dry 1H-tetrazole (0.603
g, 8.6 mmol) in dry THF (20 mL) was cooled
at −25 °C. To this solution was added drop-
wise di-tert-butyl diethylphosphoramidite
(0.723 g, 2.9 mmol). After addition, the reac-
tion temperature was allowed to rise slowly to
rt. After 3 h stirring, the reaction was cooled
in an ice bath and Et3N (2.75 mL, 20 mmol)
and 30% H2O2 in water (0.6 mL, 6 mmol)
were added. Stirring was continued at rt until
complete conversion (TLC). The reaction was
diluted with water (5 mL) and AcOEt (20
mL), and the phases were separated. The
aqueous phase was extracted with AcOEt (10
mL). The combined organic phases were
washed with brine (30 mL), dried over
Na2SO4, filtered and evaporated to dryness.
Flash chromatography over SiO2 with 1:4 hex-
ane–ether and ether gave compound 8 (0.912
g, 88%), which crystallised upon standing in
the refrigerator. Mp 48–49 °C; [h]2D3 +100° (c
1.3, AcOEt); IR (KBr): w 3083 (w), 1638 (w),
3-Deoxy-5-O-methoxymethyl-3-C-methyl-
ene-1,2-O-isopropylidene-h- -ribo-hexofur-
D
anose (7).—To a solution under Ar of com-
pound 6 (1.19 g, 2.5 mmol) in dry THF (5
mL) was added Bu4NF·3 H2O (0.867 mg, 2.75
mmol). After 1 h stirring, water (5 mL) was
added and the reaction medium was extracted
twice with AcOEt (2×20 mL). The combined
organic phases were washed with brine (20
mL), dried over MgSO4, filtered and evapo-
rated to dryness. Flash chromatography over
SiO2 with 1:1 hexane–ether and ether gave
compound 7 (0.60 g, 92%) which crystallised
upon standing in the refrigerator. Mp 47–
48 °C; [h]2D1 +170° (c 4.1, CHCl3); IR (neat): w
3472 (bs),3086 (w), 1644 (w), 1457 (m), 1414
1
1393 (m), 1268 (s), 1002 (bs) cm−1; H NMR
(C6D6): l 5.74 (d, 1 H, J1,2 4.2 Hz, H-1), 5.35
(m, 1 H, H-unsat), 5.32 (m, 1 H, H-unsat),
5.14 (m, 1 H, H-4), 4.68 (m, 1 H, H-2), 4.57
(d, 1 H, J 6.7 Hz, CH2O), 4.54 (d, 1 H,
CH2O), 4.38–4.20 (m, 2 H, H-6), 3.98 (bdd, 1
H, J1 5.6, J2 3.6 Hz, H-5), 3.14 (s, 3 H, OMe),
1.46 (s, 3 H, Me), 1.41 (s, 9 H, t-Bu), 1.40 (s,
9 H, t-Bu), 1.31 (s, 3 H, Me); 13C NMR
(C6D6): l 147.7 (q), 112.5 (s), 105.5 (t), 97.0
(s), 82.2 (t), 81.9 (q), 81.8 (q), 80.3 (t), 78.7 (d,
JC-5P 8 Hz, t), 65.4 (d, JC-6P 5 Hz, s), 55.6 (p),
29.9 (p), 29.8 (p), 27.8 (p); (FABMS, NBA):
395 (M−57, 100), 339 (12), 305 (11), 209 (50),
166 (53); Anal. Calcd for C20H37O9P: C, 53.09;
H, 8.24. Found: C, 53.12; H, 8.37.
1
(m), 1377 (s), 1213 (s), 1019 (bs) cm−1; H
NMR (CDCl3): l 5.82 (d, 1 H, J1,2 4.1 Hz,
H-1), 5.48 (dd, 1 H, J1 2, J2 1.3 Hz, H-olef),
5.29 (dd, 1 H, J1=J2 2 Hz, H-olef), 4.93–4.83
(m, 2 H, H-2, H-4), 4.79 (d, 1 H, J 7 Hz,
CH2O), 4.75 (d, 1 H, CH2O), 3.73–3.62 (m, 3
H, H-5, H-6), 3.45 (s, 3 H, OMe), 1.49 (s, 3 H,
3-Deoxy-3-C-methylene- -ribo-hexose-6-
D
13
Me), 1.38 (s, 3 H, Me); C NMR (CDCl3): l
phosphate disodium salt (2).—A stirred mix-
ture of compound 8 (0.95 g, 2.1 mmol) in
deionised water (5 mL) was degassed three
times under the vacuum of a water pump and
flushed under a stream of Ar. To the mixture
was added CF3COOH (0.05 mL, 0.65 mmol).
After 12 h stirring, a solution was obtained.
146.6 (q), 112.8 (s), 104.6 (t), 97.3 (s), 82.9 (t),
81.8 (t), 80.0 (t), 62.1 (s), 55.9 (p), 27.5 (p),
27.4 (p); MS (IE, 70 eV) 245 (M+−15, 9),
213 (8), 173 (11), 171 (37), 155 (55), 142 (59),
97 (100); Anal. Calcd for C12H20O6: C, 55.37;
H, 7.74. Found: C, 55.11; H, 7.93.
1
3-Deoxy-5-O-methoxymethyl-3-C-methyl-
The reaction was monitored by H NMR as
ene-1,2-O-isopropylidene-h- -ribo-hexofur-
D
followed. An aliquot (0.025 mL) of the reac-
tion was taken and diluted with D2O (0.45
mL) for NMR analysis. The water peak was
suppressed by pre-saturation. Hydrolysis of
anose-6-di-tert-butylphosphate (8).—A stirred
solution under Ar of the alcohol 5 (0.6 g, 2.3
mmol) (dried under high vacuum, p=100 Pa,

A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
147
ethylene glycol (0.1 mL) was added to the
mixture. The mixture was filtered and the
solid was washed twice with EtOH (2×5
mL). The filtrate was used directly for reduc-
tion. It was cooled in an ice bath and NaBH4
(0.2 g, 5.3 mmol) was added portionwise. Af-
ter 2 h stirring, excess NaBH4 was decom-
posed by slow addition of glacial acetic acid
(0.914 mL, 16 mmol) to the cold reaction
medium. The volume was reduced under vac-
uum to about 5 mL (pale orange). The com-
pound was extracted with EtOAc (3×20 mL).
The combined organic phases were washed
with aq Na2S2O4 (10%, 5 mL). The thiosulfate
solution was extracted with AcOEt (10 mL).
The combined organic phases were washed
with brine (20 mL), dried over Na2SO4,
filtered and evaporated to dryness to give 9 as
a pale yellow oil (0.814 g, 83%). Compound 9
was homogeneous by TLC (AcOEt as eluent)
with spectroscopic data identical to the lit.16
[h]2D1 +170° (c 4, CHCl3); lit. [h]2D1 +151° (c
the protecting groups was detected by the
disappearance of the signals corresponding to
the methyls of acetonide, the tert-butyl ester
and MOM group, respectively, at 1.34, 1.43,
1.49 and 3.4 ppm (reference spectrum for t 0
was measured in MeOH-d4) and by the ap-
pearance of the new signals corresponding to
the methyls of tert-BuOH, acetone and
MeOH, respectively, at 1.2, 2.18 and 3.4 ppm.
After 7 days, activated charcoal (100 mg) was
added, the mixture was stirred for 1 h and
filtered (Millex-GV® filter unit 22 mm). Freeze-
drying gave a colourless foam which was dis-
solved in deionised water (1 mL). To the
cooled degassed solution at 4 °C under Ar was
added dropwise an aqueous solution of NaOH
(0.1 N, 4 mL). After freeze-drying, the com-
pound was dissolved in deionised water (1.5
mL) and abs EtOH (20 mL) was added, and
the mixture was stored in the refrigerator. The
supernatant was removed. Freeze-drying and
vacuum drying (p=100 Pa, P2O5) afforded
the 6-phosphate glucose analogue 2 (575 mg,
96%). Mp (dec.) 112°C; [h]2D1 +43° (30 min),
+41° (17 h) (c 4.2, water); IR (KBr): w 3396
(bs), 1657 (m), 1095 (s), 978 (s) cm−1;1H
NMR (D2O): l 6.14 (d, J 4 Hz, 1%), 5.86 (bs,
1%), 5.76 (bs, 1%), 5.65–5.35 (complex, 24%),
4.67 (d, J 8 Hz, 3%), 4.52–4.00 (complex,
51%), 3.94 and 3.88 (2 bs, 8%), 3.70–3.52
(complex, 11%) due to the water signal, infor-
mation around 4.9 ppm was missed; 13C NMR
(water+5% tert-BuOH-d10 +2.5% 4 mM
EDTA, Na3) showed two isomers: l 149.2 (q),
147.7 (q), 107.4 (s), 107.2 (s), 100.7 (t), 94.9
(t), 84.4 (s), 81.2 (d, JC-5P 7 Hz, t), 76.1 (d,
JC-5P 8 Hz, t), 75.0 (t), 72.5 (t), 69.6 (t), 69.3
(t), 65.8 (d, JC-6P 5 Hz, t); 31P NMR (D2O+
3% 4 mM EDTA, Na3) showed four isomers:
l 4.72 (t, JH-6P 6.5 Hz, 50%), 4.67 (t, JH-6P 6.5
Hz, 37%), 4.45 (2%), 4.30 (t, JH-6P 6.5 Hz,
11%); (FABMS, thioglycerol) 277 (M−23,
100), 255 (58), 237 (52); Anal. Calcd for
C7H11Na2O8P: C, 28.00; H, 3.96. Found: C,
27.90; H, 4.06.
1
0.8, CHCl3);16 IR, H NMR lit.;16 13C NMR
(CDCl3): l 145.6 (q), 112.6 (q), 112.2 (s), 104.4
(t), 82.1 (t), 80.0 (t), 63.5 (s), 27.4 (p), 27.1 (p);
MS (CI, CH4) 187 (M+ +1, 30), 170 (12), 155
(14), 129 (100), 111 (84).
3-Deoxy-3-C-methylene-1,2-O-isopropyli-
dene - h -
D
- erythro - pentofuranose - 5 - di - tert-
butylphosphate (10).—The triester 10 was
prepared as described for 6. Flash chromatog-
raphy over SiO2 (treated with 1% Et3N) with
1:9 hexane–ether as eluents gave compound
10 (90%). Mp 61–62 °C; [h]2D1 +88° (c 4,
AcOEt); IR (neat): w 2985(s), 2936 and 2876
(m), 1277 (s), 990 (bs) cm−1; 1H NMR (C6D6):
l 5.79 (d, 1 H, J1,2 4.1 Hz, H-1), 5.18 (dd, 1 H,
J1 2.4, J2 1.2 Hz, H-unsat), 4.97 (dd, 1 H, J
1.3 Hz, H-unsat), 4.85 (m, 1 H, H-4), 4.55 (dd,
1 H, H-2), 4.20–3.94 (m, 2 H, H-5), 1.45 (s, 3
H, Me), 1.41 (s, 18 H, t-Bu), 1.25 (s, 3 H,
13
Me); C NMR (C6D6): l 147.2 (q), 112.5 (q),
112.0 (s), 105.3 (t), 82.1 (t), 81.8 (q), 81.7 (q),
78.8 (d, JC-4P 9.7 Hz, t), 68.2 (d, JC-5P 5.6 Hz,
s), 29.9 (p), 29.8 (p), 27.8 (p), 27.5 (p);
(FABMS, thioglycerol) 321 (M−57, 100), 265
(15), 232 (7), 209 (65), 166 (63); Anal. Calcd
for C17H31O7P: C, 53.96; H, 8.26. Found: C,
53.96; H, 8.28.
3-Deoxy-3-C-methylene-1,2-O-isopropyli-
dene-h- -erythro-pentofuranose (9).—A solu-
D
tion of diol 4 (1.15 g, 5.3 mmol) in EtOH (10
mL) was added dropwise to a solution of
NaIO4 (1.3 g, 6.05 mmol) in water (10 mL)
under vigorous stirring. After 1 h stirring,
3-Deoxy-3-C-methylene-
D
-erythro-pentose-
5-phosphate disodium salt (3).—Acidic hydrol-

148
A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
ysis of the phosphate triester 10 was realised
and monitored as described for 6 (32 h reac-
tion time). Compound 3 was isolated quanti-
tatively as the sodium salt. Mp (dec.) 90 °C;
[h]2D1 +96° (15 min), +93° (24 h) (c 2.7,
water); IR (KBr): w 3432 (bs), 1626 (m), 1005
were undertaken by determining the enzy-
matic rate with glucose-6-phosphate dehydro-
genase (125 ng/mL), glucose-6-phosphate
(0.12 mM) and NADP+ (4.3 mM) in the
presence of the analogues at different concen-
trations (10–40 mM). The influence of ribose-
5-phosphate on the activity of the
dehydrogenase was studied similarly.
(m) cm−1
;
1H NMR (D2O+5% 4 mM
EDTA, Na3): l 5.65–5.46 (complex, 50%),
5.34 (d, J 2 Hz, 7%), 4.19–3.93 (complex,
43%) due to the water signal, information
around 4.9 ppm was missed; 13C NMR
(D2O+2.5% 4 mM EDTA, Na3) showed two
isomers: l 146.7 (q), 146.5 (q), 113.7 (s), 110.0
(s), 102.1 (t), 96.3 (t), 79.7 (d, JC-4P 7.9 Hz, t),
78.9 (d, JC-4P 7.9 Hz, t), 78.0 (t), 73.2 (t), 69.2
(d, JC-5P 5.1 Hz, s), 68.3 (d, JC-5P 5.1 Hz, s); 31P
NMR (D2O+2.5% 4 mM EDTA, Na3)
showed two isomers: l 1.32 (t, JH-6P 6.1 Hz,
43%), 1.18 (t, JH-6P 5.8 Hz, 57%); (FABMS,
thioglycerol) 247 (M−23, 65), 225 (100), 197
Determination of the enzymatic acti6ity of
phosphoglucose isomerase.—The activity was
measured using the coupled test with glucose-
6-phosphate dehydrogenase.21 The concentra-
tion of phosphoglucose isomerase from rabbit
muscle (Sigma) was determined at 280 nm
assuming an absorption coefficient A1% 13.2
and a molecular weight of 132,000 Da for the
dimer.25 For 3-deoxy-3-C-methylene-
hexose-6-phosphate (2), 3-deoxy-3-C-methyl-
ene- -erythro-pentose-5-phosphate (3) and
D
-ribo-
D
phosphoglucose isomerase, inactivation tests
were performed by incubating the analogues
at different concentrations (1–100 mM) with
phosphoglucose isomerase in a 0.1 M tri-
ethanolamine–HCl buffer pH 7.7 at 30 °C
and measuring the residual enzymatic activity
in function of time. Reversible inhibition tests
were undertaken with the analogues and ri-
bose-5-phosphate (10–40 mM). The concen-
tration of fructose-6-phosphate was 1.5 mM.
Enzymatic acti6ity of phosphoribose iso-
merase.—The enzymatic activity was deter-
mined by following the absorbance increase at
282 nm due to the formation of ribulose-5-
phosphate from ribose-5-phosphate (m 58.6
(25),
165
(27);
Anal.
Calcd
for
C6H10NaO7P·H2O: C, 27.08; H, 4.55. Found:
C, 26.95; H, 4.35.
Determination of the acti6ity of glucose-6-
phosphate dehydrogenase.—Glucose-6-phos-
phate (1.2 mM), NADP+ (4.3 mM) and
glucose-6-phosphate dehydrogenase from bak-
ers yeast (125 ng/mL) were incubated at 30 °C
in a 0.1 M triethanolamine–HCl buffer pH
7.7 (total vol 1 mL). The activity was mea-
sured by following the formation of NADPH
at 340 nm with an Uvicon 933 spectrophoto-
meter (Kontron). 3-Deoxy-3-C-methylene-
ribo-hexose-6-phosphate (2), 3-deoxy-3-C-
methylene- -erythro-pentose-5-phosphate (3)
D
-
M
−1cm−1).26 Ribose-5-phosphate (50 mM)
D
was incubated with phosphoribose isomerase
from spinach (Sigma) (1 mg/mL) at 30 °C in a
triethanolamine–HCl buffer pH 7.7 (total vol-
and glucose-6-phosphate dehydrogenase were
incubated at a concentration of 4 mM with
the enzyme (2.5 mg/mL) and NADP+ (4.3
mM) to control if the two sugar phosphates
were substrates of glucose-6-phosphate dehy-
drogenase. The formation of NADPH was
followed at 340 nm. To test if they were able
to inactivate glucose-6-phosphate dehydroge-
nase, compounds 2 and 3 (20 mM) were incu-
bated with glucose-6-phosphate dehydro-
genase (2.5 mg/mL) in the absence or in the
presence of NADP+ (4.3 mM) in a 0.1 M
triethanolamine–HCl, 6.5 mM MgCl2 buffer
pH 7.8 at 30 °C. At given time aliquots (0.5
mL) were withdrawn to determine the residual
enzymatic activity. Reversible inhibition tests
ume 500 ml) for 3-deoxy-3-C-methylene- -ery-
D
thro-pentose-5-phosphate (3) and phos-
phoribose isomerase, the ribose-5-phosphate
analogue was added in the medium to test if
the enzyme is able to isomerise it in the ketose
derivative. The reaction was followed by tak-
ing the absorption spectrum (240–320 nm) as
a function of time. To test if compound 3 is
an irreversible inhibitor of phosphoribose iso-
merase, the ribose-5-phosphate analogue (50
mM) was incubated with the enzyme (1 mg/
mL) in a 0.1 M triethanolamine–HCl buffer
pH 7.8 at 37 °C. At given time, aliquots (0.5

A. Burger et al. / Carbohydrate Research 332 (2001) 141–149
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matic activity. The isomerase rate was mea-
sured with phosphoribose isomerase (1
mg/mL) and ribose-5-phosphate (10 mM) in
the presence of the ribose-5-phosphate ana-
logue at different concentrations (10–40 mM)
to study the reversible inhibition.
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