Improved straightforward chemical synthesis of dihydroxyacetone phosphate through enzymatic desymmetrization of 2,2-dimethoxypropane-1,3-diol

Article abstract of DOI:10.1021/jo048697k

Dihydroxyacetone phosphate (DHAP) was synthesized in high purity and yield in four steps starting from dihydroxyacetone dimer (DHA) (47% overall yield). DHA was converted into 2,2-dimethoxypropane-1,3-diol, which was desymmetrized by acetylation with lipase AK. The alcohol function was phosphorylated to give dibenzyl phosphate ester 4. From 4, two routes were investigated for large-scale synthesis of DHAP. First, acetate hydrolysis was performed prior to hydrogenolysis of the phosphate protective groups. The acetal hydrolysis was finally catalyzed by the phosphate group itself. Second, acetate and acetal hydrolysis were performed in one single step after hydrogenolysis.

Full text of DOI:10.1021/jo048697k

is still a matter of research, as attested by the many
enzymatic3 or synthesis procedures4 recently described.
The main drawbacks to enzymatic reactions are often
related to the high cost of enzymes, specific equipment,3f
and difficult isolation of the final desired compound from
excess DHAP precursors such as glycerol.3e Moreover,
substrate inhibition phenomena often limit the final
DHAP concentration to 100 mM.3f In contrast, chemical
syntheses yield stable precursors (Figure 1) that can be
stored in large quantities and converted into DHAP just
before use in enzymatic aldol reactions.
Improved Straightforward Chemical
Synthesis of Dihydroxyacetone Phosphate
through Enzymatic Desymmetrization of
2,2-Dimethoxypropane-1,3-diol
Franck Charmantray, Lahssen El Blidi,
Thierry Gefflaut, Laurence Hecquet, Jean Bolte,* and
Marielle Lemaire*
Laboratoire SEESIB, UMR 6504 CNRS,
Universite´ Blaise Pascal, 24 avenue des Landais,
63177 Aubie`re Cedex, France
Compound 1 is the most widely used and is readily
prepared from dihydroxyacetone dimer (DHA).4e Com-
pound 2 has been obtained from commercially available
1,3-dibromoacetone in a few steps.4c A three-step prepa-
ration of a cyclic phosphate 3 as an alternative DHAP
precursor has recently been described.4b However, from
1, 2, and 3, DHAP is formed in moderate yield with
several impurities, mainly inorganic phosphate. More-
over, these syntheses are not highly reproducible. The
dimethyl acetal precursor 4 used by Ballou et al.5 and
Valentin et al.4d appears the most suited for achieving
the last steps in high yield. However, its preparation from
dibromoacetone4d or 3-chloropropane-1,2-diol5 involves too
many steps for a large-scale application.
The present study describes a fundamental improve-
ment in DHAP chemical synthesis via 4 in terms of time,
yield, purity and reproducibility. The route for preparing
DHAP is depicted in Scheme 1.
We report a three-step synthesis of compound 4 from
DHA with only one short purification step and its one-
pot conversion into DHAP on large scale.
Results and Discussion. Dihydroxyacetone dimethyl
acetal 6 was prepared according to a slight modification
of the previously described method.4b,6 To use the crude
diol 6 in the next step, neutralization and removal of
TsOH was achieved with the basic Amberlyst A26 resin
(OH- form) instead of Na2CO3. In the second step,
monofunctionalization of the diol was performed via a
lipase-catalyzed transesterification with use of vinyl
acetate as the acyl donor.7 Of the lipases tested (Amano
AK: Pseudomonas fluorescens lipase; Amano PS: Burk-
holderia cepacia lipase; CAL: Candida antartica lipase;
CRL: Candida rugosa lipase; CCL: Candida cylindracea
lipase; PPL: porcine pancreas lipase; and WGL: wheat
germ lipase), Amano AK gave the best results: most of
the diol 6 was promptly converted into monoacetate 7
before the slow appearance of the undesired diacetate.
marielle.lemaire@chimie.univ-bpclermont.fr
Received July 28, 2004
Abstract: Dihydroxyacetone phosphate (DHAP) was syn-
thesized in high purity and yield in four steps starting from
dihydroxyacetone dimer (DHA) (47% overall yield). DHA was
converted into 2,2-dimethoxypropane-1,3-diol, which was
desymmetrized by acetylation with lipase AK. The alcohol
function was phosphorylated to give dibenzyl phosphate
ester 4. From 4, two routes were investigated for large-scale
synthesis of DHAP. First, acetate hydrolysis was performed
prior to hydrogenolysis of the phosphate protective groups.
The acetal hydrolysis was finally catalyzed by the phosphate
group itself. Second, acetate and acetal hydrolysis were
performed in one single step after hydrogenolysis.
Four dihydroxyacetone phosphate-dependent aldolases
are known to catalyze the condensation of a variety of
aldehydes with dihydroxyacetone phosphate (DHAP) to
give monosaccharides and other chiral compounds of
related structures.1 These DHAP aldolases have shown
complementary diastereoselectivity for the two stereo-
centers connected by the newly formed C-C bond, and
three of them are useful catalysts for applications in
organic synthesis. Of these, the glycolytic enzyme fructose-
1,6-bisphosphate aldolase has been the most widely used
over the last 20 years.2 DHAP is the essential donor
substrate, and although commercially available it is too
expensive for large-scale synthesis. Large-scale DHAP
production requires efficient and reliable syntheses. This
* Address correspondence to this author. Phone: 33-4-73-40-75-84.
Fax: 33-4-73-40-77-17.
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ed.; Drauz, K., Waldmann, H., Eds.; Wiley-VCH: Weinheim, Germany,
2002; Vol. 2, pp 931-974. (b) Phung, A. N.; Zannetti, M. T.; Whited,
G.; Fessner, W. D. Angew Chem., Int. Ed. 2003, 42, 4821-4824. (c)
Fessner, W.-D.; Helaine, V. Curr. Opin. Biotechnol. 2001, 12, 574-
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Silvestri, M. G.; Desantis, G.; Mitchell, M.; Wong, C.-H. Top. Stere-
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C.; Wong, C.-H. J. Org. Chem. 2003, 68, 6828-6831. (g) Espelt, L.;
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10.1021/jo048697k CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/12/2004
9310
J. Org. Chem. 2004, 69, 9310-9312

FIGURE 1. Structures of dihydroxyacetone phosphate (DHAP) precursors.
SCHEME 1. Dihydroxyacetone Phosphate Synthesisa
a Reagents and conditions: (a) HC(OMe)3, MeOH, cat. TsOH, rt (ref 2b); (b) lipase AK (Amano), vinyl acetate, isopropyl ether, 30 °C
(61% from 5); (c) P(OBn)3, I2, Pyr, CH2Cl2, -30 °C (93%); (d) NaOH, MeOH, rt; (e) H2, Pd/C, MeOH, rt; (f) H2O, 45 °C (84% from 4); (g)
H2, Pd/C, MeOH, rt; (h) H2O, 65 °C (62% from 4).
The monoester 7 was distilled under reduced pressure
and isolated in 61% yield from 5. Phosphorylation of
compound 7 was performed following the procedure
described by Gefflaut et al.4c The choice of benzyl
preferred to phenyl for phosphate protecting groups was
based on the facile hydrogenolysis of the former with no
need for pressure and expensive PtO2 catalyst. Two
equivalents of the phosphorylating agent, dibenzyl phos-
phoroiodidate (DBPI) generated from P(OBn)3 and I2,
were necessary to give the precursor 4 in 93% yield after
short silica gel column purification.
From 4, two routes to prepare DHAP were explored.
Following pathway A, three reactions were consecutively
performed without isolation of the intermediates. First,
a basic hydrolysis of the ester group was carried out with
a stoechiometric amount of NaOH, leading to the crude
alcohol 8. Sodium acetate was simply removed by an
aqueous washing of a DCM solution of 8. An analytical
sample of 8 was also prepared by column chromatogra-
phy. Next, the crude 8 was hydrogenated in the presence
of Pd/C (10%) in MeOH to quantitatively remove the
benzyl groups. Finally, we took advantage of the acidity
of the free phosphate monoester to catalyze the hydrolysis
of the dimethoxy acetal. This reaction was performed in
distilled water at 45 °C. Following pathway B, two steps
were consecutively performed. The hydrogenolysis was
followed by the ester and acetal hydrolyses catalyzed by
the acidic phosphate group at 65 °C. In both cases, the
DHAP was assayed enzymatically.8 The acid hydrolysis
of intermediates 9 and 10 led to different results.
Following route A, hydrolysis of 9 gave a 424 mM DHAP
solution in 50 min (84% yield calculated from 4). This
chemical yield is in the same range as the one previously
described from the same precursor by Valentin et al.4d
From route B, a 62% yield from 4 was reached in 90 min.
The higher temperature and reaction time required for
acetate hydrolysis were responsible for DHAP decompo-
sition, leading to a lower yield. These results are in
agreement with data from the literature.3f As determined
by 13C and 31P NMR, the DHAP sample from route A was
free of inorganic phosphate, and methanol was the sole
impurity detected. At pH 3.5, DHAP was present in its
ketone and hydrate forms as already described in the
literature.4d In conclusion, pathway A is clearly the best
method for preparing DHAP from 4.
This short and facile procedure was performed with
only one distillation and one short chromatography on
silica gel with inexpensive reagents. DHAP overall yield
from DHA was 47%. A DHAP solution of high purity and
concentration can thus be obtained on several gram scale
and stored at -18 °C for several months without notice-
able decomposition. In the case of the use of commercially
available diphenylphosphorochloridate to introduce the
phosphate group, pathway B would be more suitable
owing to the instability of this protection under the basic
conditions necessary for acetate hydrolysis in route A.
Experimental Section
2,2-Dimethoxy-1,3-propanediol (6). This compound was
synthesized by a slight modification of the method described by
Ferroni et al.4b Starting with 20 g (111 mmol) of 5, the reaction
was stopped by adding 2.2 g of Amberlyst A 26 (OH- form). After
neutralization (15 min), the resin was removed by filtration and
the filtrate was evaporated to dryness. Compound 6 was
(8) Bergmeyer, H. U. Methods in Enzymatic Analysis, 34th ed.;
Verlag Chemie: Weinheim, Germany, 1984; Vol. 11, pp 146-147.
J. Org. Chem, Vol. 69, No. 26, 2004 9311

obtained as a crude, slightly yellow solid in quantitative yield
(30 g; 220 mmol).
cyclohexane/AcOEt (4/6) as eluant. 8 was isolated as a colorless
oil in 93% yield (186 mg). IR (film) 3411,1497, 1455, 1381, 1258,
1090, 893 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 10H), 5.05
(m, 4H), 4.01 (d, 2H), 3.62 (s, 2H), 3.25 (s, 6H). 13C NMR (100
MHz, CDCl3) δ 135.5, 128.7-128.0, 100.2, 69.7, 62.4, 59.0, 48.4.
ES+ (MeOH), M + Na+ calcd for C19H25O7NaP 419.1236, found
419.1236. Anal. Calcd for C19H25O7P (396.3): C, 57.57; H, 6.36;
P, 7.81. Found: C, 57.52; H, 6.52; P, 7.80.
3-Hydroxy-2,2-dimethoxypropyl Acetate (7). Lipase Amano
AK (600 mg, 12 000 U) was added to a solution of crude diol 6
(30 g, 220 mmol) dissolved in 450 mL of a mixture of vinyl
acetate and isopropyl ether (2/1). The suspension was stirred
for 18 h at 30 °C. The lipase was removed by filtration through
a membrane (0.2 µm). The filtrate was evaporated to dryness.
Compound 7 was purified by distillation under reduced pressure
(0.1 mmHg, 72 °C) and was isolated as a colorless oil in 61%
yield from 5 (23.88 g). Its spectral data are in accordance with
the previously described ones.
3-Acetoxy-2,2-dimethoxypropyl Dibenzyl Phosphate (4).
Iodine (11.37 g, 44.8 mmol) was added to a solution of tribenzyl
phosphite4c (16.57 g, 47.04 mmol) in 100 mL of anhydrous EtOH-
free CH2Cl2 at -30 °C. This solution was then added dropwise
to a solution of 7 (4 g, 22.4 mmol) in a mixture of anhydrous
pyridine (7.26 mL, 90 mmol) and anhydrous CH2Cl2 (120 mL)
at -30 °C, under argon. When the addition was completed, the
mixture was allowed to warm to rt for 30 min and then filtered
on Celite, concentrated to dryness, and dissolved in 180 mL of
an Et2O/water (5/1) mixture. The organic layer was washed with
50 mL of KHSO4 (0.3 M), 50 mL of saturated NaHCO3, and 100
mL of brine, dried over MgSO4, and evaporated to dryness.
Compound 4 was purified on a short column (5 cm diameter;
300 mL of silica gel; eluant cyclohexane/AcOEt 7/3 then 4/6) and
obtained as a colorless oil in 93% yield (9.13 g). Its spectral data
are in accordance with the previously described ones.
Dihydroxyacetone Phosphate (DHAP). Route A: 3-Hy-
droxy-2,2-dimethoxypropyl Dibenzyl Phosphate (8). Aque-
ous NaOH (1 M; 6.84 mL) was added to a solution of 4 (3 g, 6.84
mmol) in 10 mL of methanol. The mixture was stirred at rt for
30 min. After dilution with water (15 mL), alcohol 8 was
extracted with CH2Cl2 (2 × 50 mL). The organic layer was dried
over MgSO4, filtered, and evaporated to dryness to quantitatively
give crude 8.
To the crude compound 8 in 30 mL of methanol was added
140 mg of 10% Pd/C. The mixture was stirred for 45 min at rt
under hydrogen atmosphere, using a balloon. Pd/C was removed
by filtration through a membrane (0.2 µm) and the filtrate was
evaporated to dryness. Distilled water (13.2 mL) was added and
the solution (theor. 518 mM of 9) was stirred at 45 °C. The DHAP
formation was followed by enzymatic assay. Once the maximum
yield (84% in 50 min from 4) was reached, the reaction was
cooled in an ice bath and the pH was adjusted to 3.7 with NaOH
(3N). At pH 3.7, the DHAP solution (424 mM) can be stored at
-18 °C for several months without noticeable degradation.
Route B: To 3 g of compound 4 (6.84 mmol) in 30 mL of
methanol was added 140 mg of 10% Pd/C. The mixture was
stirred for 45 min at rt under hydrogen atmosphere, using a
balloon. Pd/C was removed by filtration through a membrane
(0.2 µm) and the filtrate was evaporated to dryness. Distilled
water (13.2 mL) was added and the solution (theor. 518 mM of
10) was stirred at 65 °C. The DHAP formation was followed by
enzymatic assay. Once the maximum yield (62% in 90 min from
4) was reached, the reaction was stopped as described in route
A.
Supporting Information Available: General experimen-
tal information, Excel graphics for DHAP formation following
route A and B and Wong’s method, DHAP 13C NMR and 31P
NMR spectra in H2O + D2O from route A and 13C NMR
spectrum from route B. This material is available free of charge
via the Internet at http://pubs.acs.org.
Purification of Compound 8 for Full Characterization.
8 (200 mg crude) was purified by flash chromatography with
JO048697K
9312 J. Org. Chem., Vol. 69, No. 26, 2004