An improved preparation of D-glyceraldehyde 3-phosphate and its use in the synthesis of 1-deoxy-D-xylulose 5-phosphate

Article abstract of DOI:10.1002/hlca.200900441

D-Glyceraldehyde 3-phosphate (=D-GAP; 2) was prepared by an improved chemical method (Scheme 2), and it was then employed to synthesize 1-deoxy-d-xylulose 5-phosphate (=DXP; 3) which is enzymatically one of the key intermediates in the MEP (4) terpenoid b

Full text of DOI:10.1002/hlca.200900441

Helvetica Chimica Acta – Vol. 93 (2010)
1745
An Improved Preparation of d-Glyceraldehyde 3-Phosphate and Its Use in
the Synthesis of 1-Deoxy-d-xylulose 5-Phosphate
a
a
a
b
a
a
by Heng Li ), Jie Tian ), Hui Wang ) ), Shao-Qing Yang ), and Wen-Yun Gao* )
a) College of Life Sciences/National Engineering Research Center for Miniaturized Detection Systems,
Northwest University, 229 North Taibai Road, Xiꢀan 710069, P. R. China
(
phone: þ 86-29-88303446 ext. 852; fax: þ 86-29-88303551; e-mail: gaowenyun@nwu.edu.cn.)
b
)
Department of Chemistry and Chemical Engineering, Baoji College of Arts and Sciences,
Gaoxin Street, Baoji 721013, P. R. China
1
d-Glyceraldehyde 3-phosphate (¼d-GAP; 2) was prepared by an improved chemical method
Scheme 2), and it was then employed to synthesize 1-deoxy-d-xylulose 5-phosphate (¼ DXP; 3) which is
(
enzymatically one of the key intermediates in the MEP (4) terpenoid biosynthetic pathway (Scheme 1).
The recombinant DXP synthase of Rhodobacter capsulatus was used to catalyze the condensation of d-
glyceraldehyde 3-phosphate (2) and pyruvate (¼2-oxopropanoate; 1) to produce the sugar phosphate 3
(
Scheme 2). The simple two-step chemoenzymatic route described affords DXP (3) with more than 70%
overall yield and higher than 95% purity. The procedure may also be used for the synthesis of isotope-
labeled DXP (3) by using isotope-labeled pyruvate.
Introduction. – Terpenoid natural products are found essentially in all forms of life.
The more than tens of thousands of naturally occurring terpenes comprise physiolog-
ically important compounds such as vitamines A and D, cholesterol, steroid hormones,
chlorophylls, carotenoids, and pharmaceutically active compounds like taxol, artemi-
sinin, and ginkgolides, just to mention a few. The pioneering studies of Bloch and co-
workers by using yeast and animal cells have led to the elucidation of the mevalonate
(
MVA) pathway of terpenoid biosynthesis [1]. Recently, Arigoni, Rohmer, and co-
workers independently made the discovery by studies on certain bacteria that there is a
second biochemical pathway of terpenoid biosynthesis, namely the 2-methyl-d-
erythritol 4-phosphate (¼ MEP; 4) pathway (Scheme 1) [2][3]. In this nonclassic
biosynthetic pathway, the key intermediate is 1-deoxy-d-xylulose 5-phosphate
(
¼ DXP; 3) which is converted into MEP (4). The first committed intermediate 3 of
this route to 4 by a two-step process catalyzed by the DXP reductoisomerase (DXR) in
the present of NADPH, is biosynthesized from pyruvate (¼2-oxopropanoate; 1) and d-
glyceraldehyde 3-phosphate (¼d-GAP; 2) by the catalytic action of DXP synthase
(
¼ DXS), a thiamine diphosphate dependent enzyme. Subsequently, 4 is transformed
by four consecutive enzyme reactions into 3-methylbut-3-en-1-yl diphosphate
(
¼ isopentenyl diphosphate ¼ IPP; 5) and 3-methylbut-2-en-1-yl diphosphate
(¼dimethylallyl diphosphate; 6), the two universal building blocks for the terpenoids
[
4]. Recent research has established that all enzymes of this new pathway are
unexplored targets for the design of novel types of antibacterial and antiparasitic drugs
5][6]. The concept has been validated by the discovery that fosmidomycin, a long-
[
ꢁ
2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

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Helvetica Chimica Acta – Vol. 93 (2010)
Scheme 1. 1-Deoxy-d-xylulose 5-Phosphate (3) as Precursor for Terpenoids via the MEP (4) Pathway
known antibiotic and its acetyl congener FR900098 are indeed inhibitors of DXR, the
second enzyme of the MEP pathway [3], and are active against bacteria as well as the
malaria parasite [7 – 10]. To find new DXR inhibitors and determine their bioactivity,
there is now a demand for larger quantities of DXP (3), the natural substrate of DXR.
Over the last two decades, some chemical and enzymatic methods including
labeling with stable or radioactive isotopes, have been established by several groups
due to the biological importance of DXP [11]. Generally, two strategies were exploited
in the chemical synthesis of this sugar phosphate. Efficient approaches to the target
compound employed commercially available starting materials which already possess
the required configuration at certain C-atoms corresponding to the two asymmetric
centers of DXP [12 – 16]. These 6 – 8 steps reaction sequences afforded optically pure
final products with low to medium overall yields. In addition, the iterative protection
and deprotection steps were often critical in these schemes, decreasing the overall yield
and requiring purification of the final product. A second method involved the
preparation of the required DXP C-atom chain from achiral precursors. In this case, the
two chiral centers of the target molecule were produced by asymmetric dihydroxylation
of an achiral a,b-unsaturated pentan-2-one derivatives with a chiral osmium tetraoxide
complex (5 – 7 steps, 84 – 87% ee values, and 6.5 – 23% overall yields) [17 – 19].
In comparison to chemical syntheses, the enzymatic procedures always have the
combined advantages of i) short reaction time, ii) easy access to isotope-labeled
products from commercially available precursors, iii) virtually perfect stereoselectivity,
iv) the simplicity of the one-pot reaction conditions in aqueous solution. Therefore,
enzymatic synthesis of DXP (3), especially multiple-isotope-labeled DXP with the
recombinant DXS represents an attractive alternative to chemical synthesis. However,
the enzyme-mediated DXP synthetic methodology has not been fully explored up to
now. Taylor and co-workers [12] reported an enzymatic route in which 3 was produced
from d-fructose 1,6-diphosphate and pyruvate in 47% yield by the co-action of
aldolase, triosephosphate isomerase (TIM), and partially purified DXS of Escherichia
coli. The medium yield may result from the BaCl precipitation/redissolution steps for
2
the purification of the final product from the reaction buffer. Another way was set up
by Kis, Eisenreich, and co-workers [20] who used recombinant DXS of Bacillus subtilis

Helvetica Chimica Acta – Vol. 93 (2010)
1747
to prepare isotope-labeled DXP from isotope-labeled glucose and/or isotope-labeled
pyruvate as starting materials with an overall yield of ca. 50%. Totally 7 enzymes and an
ATP-recycling system were involved in this cascade reaction, and the ion-exchange
process for purification of the final product from the reaction buffer was really time-
consuming.
There should be a most straightforward way to prepare DXP (3) enzymatically
from pyruvate (1) and d-GAP (2; Scheme 1). But this strategy is always hampered by
the lack of good-quality d-GAP since commercially available 2 is either racemic or
enantiomerically impure and expensive. Although d-GAP plays a central role in
fermentation and the glycolysis of carbohydrates, the methods for the preparation of
this triose phosphate are rare. Ballou and Fischer published the first chemical synthesis
of this bioactive compound in which d-mannitol was used as starting material [21]:
Starting from 1 kg of d-mannitol only few grams of the target compound 2 were
obtained after a 9-step conversion. A practical preparation of d-GAP (2) was reported
by Barker and co-workers [22]. In this method, d-fructose 6-phosphate was oxidized by
Pb(OAc) to produce 2-O-glycoloyl-d-glyceraldehyde 3-phosphate and after subse-
4
quent mild acid hydrolysis of this intermediate and anion exchange with DEAE
Sephadex, d-GAP was obtained in 81% yield and at least 95% purity. The main
problem of this process is that the purification step took a too long time, which can lead
to partial decomposition of the unstable phosphate 2 even when operating at 48.
In this article, an improved procedure for the purification of d-GAP (2) is disclosed.
Meanwhile, an enzymatic preparation of DXP (3) by using 2, recombinant DXS of R.
capsulatus, and commercially available pyruvate (1) was established (Scheme 2). DXP
could thus be obtained in more than 70% overall yield and high purity (> 95%). This
route can also be used for the preparation of isotope-labeled DXP if isotope-labeled
pyruvate is used.
Scheme 2. Chemical Synthesis of d-Glyceraldehyde 3-Phosphate (2) and Enzymatic Preparation of 1-
Deoxy-d-xylulose 5-Phosphate (3)
Results and Discussion. – Basically, the synthesis of d-GAP (2) followed the
published procedure with minor improvements [22], and the crude product obtained as
yellowish oil could be used for the preparation of DXP (3) without further workup.
First, the DEAE Sephadex purification of the product 2 was done in accordance with
the described method, but either the yield or the purity was lower than the published
ones. To solve the problem, we tried elution buffers with different pH values and
different total volumes. The data (see Table) showed that the best results were obtained
when an elution buffer of pH 6.5 and 500 ml of total volume were selected. The yield of
d-GAP (2) at pH 7.0 and 500 ml total volume was a little better, but the product was

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Helvetica Chimica Acta – Vol. 93 (2010)
Table. Optimization of d-GAP (2) Purification on DEAE-Sephadex A-25a)
Buffer and volume
pH Value
5.5
Yield [%]
Purity [%]b)
0
.05m AcONa þ 0.6m AcONa, 250 ml each
.05m AcONa þ 0.6m AcONa, 500 ml each
63
72
83
85
55
62
73
77
90
93
96
86
86
91
91
82
6
6
7
.0
.5
.0
0
5.5
6
6
7
.0
.5
.0
a) Column was operated at 48 with a flow rate of 2.5 ml/min. b) Determined by H-NMR.
1
slightly contaminated by glycolic acid (¼2-hydroxyacetic acid). After the anion-
exchange process, the fractions containing 2 were pooled and treated with Dowex
þ
5
0w ꢀ 8 resin to remove Na , and the acidic eluate was concentrated in vacuo to give
pure 2.
DXP (3) was then prepared conveniently from pyruvat (1), DXS of R. capsulatus,
and crude or purified d-GAP (2). To get a maximum production of 3, the ratio 1/2 was
determined as 2 :1, the concentration of the DXS was determined as 0.83 mg/ml, the pH
of the reaction buffer was adjusted to 7.5, and the incubation was done at 378 overnight.
The final product was also purified by a combination of anion-exchange on DEAE
Sephadex and then cation-exchange on Dowex 50w ꢀ 8.
In conclusion, we have improved the preparation of the biologically important
triose phosphate d-GAP (2) and set up a straightforward two-step chemoenzymatic
synthesis of DXP (3), the key intermediate of the MEP (4) biosynthetic pathway for
terpenoids, by achieving a higher yield and purity of 2. Moreover, isotopically labeled
DXP should be readily accessible by this route from isotope-labeled pyruvate.
We gratefully acknowledge Prof. C. Dale Poulter, Chemistry Department of University of Utah, for
providing the plasmid pet11a-pFMH30. This work was supported by the National Science Foundation of
China (NSFC) Grant 20772095 and the Department of Education, Shaanxi Province Grant 08JK439.
Experimental Part
General. Plasmid pet11a-pFMH30 for the synthesis of recombinant DXS of R. capsulatus was a kind
gift from Prof. C. Dale Poulter, Chemistry Department of the University of Utah. The bacterial strain E.
coli BL21(DE3) was from the stock of this institute, Ni-NTA agarose resin was from Qiagen, sodium
ꢃ
þ
pyruvate was from Fluka, and Dowex 50w ꢀ 8 – 200 resin (H form, 100 – 200 mesh) and DEAE
ꢁ
1
Sephadex A-25 (Cl form) were from Sigma. All other chemicals used were of anal. reagent grade. H-
and P-NMR: Varian-Inova-400 NMR spectrometer in D O; d in ppm, J in Hz. MS: Thermo Fisher LTQ
31
2
XL mass spectrometer; m/z (rel. %).
Preparation of DXS. Recombinant R. capsulatus DXS was prepared according to the published
procedures with plasmid pet11a-pFMH30 transformed E. coli BL21-DE3 competent cells [23][24]. The
yield of the protein was 21 mg per liter medium.
d-Glyceraldehyde 3-Phosphate (¼(2R)-2,3-Dihydroxypropanal 3-Phosphate ¼ (2R)-2-Hydroxy-3-
oxopropyl Dihydrogen Phosphate ¼ d-GAP; 2). At r.t., disodium d-fructose 6-phosphate (1.0 g,
3
.3 mmol) was moistened with H O (1.6 ml) and then dissolved in glacial AcOH (132 ml) with efficient
2

Helvetica Chimica Acta – Vol. 93 (2010)
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stirring. Upon dissolution of the salt, conc. H SO (0.36 ml) was added, and Pb(OAc) (5.9 g, 13.3 mmol)
2
4
4
was added during 15 min in 10 portions. After 2 h, oxalic acid dihydrate (1.6 g, 12.7 mmol) was added, and
1
stirring was continued for an additional 30 min. The suspension was centrifuged ) at 4000 rpm at 48 for
1
0 min, and the supernatant was concentrated at 308 in vacuo to ca. 5 ml by means of a rotary evaporator.
The residual solid was washed with H O (3 ꢀ 20 ml), the concentrate and washings were combined, and
2
Ba(OAc) (3.3 g, 12.9 mmol) was added with efficient stirring at 48 for 15 min. The white suspension was
2
centrifuged at 4000 rpm at 48 for 10 min, the residual solid was washed with H O (2 ꢀ 5 ml), and the
2
þ
supernatant and washings were combined and treated with ca. 60 ml of Dowex 50w ꢀ 8 resin (H form).
The suspension was filtrated, and the acidic soln. was concentrated to ca. 3 ml and stored at 258 for 18 h.
Then the soln. was evaporated in vacuo to give crude 2 as yellowish oil. For purification, the crude 2 was
dissolved in H O (2 ml) and the pH of the soln. adjusted to pH 6.5 with 2m NaOH. Then the soln. was
2
applied to a 1.0 ꢀ 18 cm column of DEAE Sephadex A-25 (acetate form) at 48, and the product was eluted
with a linear gradient of AcONa (0.05m, pH 6.5, and 0.6m, pH 6.5, 250 ml each, flow rate 2.5 ml/min). The
þ
combined fractions containing 2 were treated with excess Dowex 50w ꢀ 8 resin (H form) at r.t., and
1
concentrated twice in vacuo at 308 to remove AcOH: oily 2 (0.47 g, 84.8%). H-NMR: 4.95 (d, J ¼ 5.6,
3
1
HꢁC(1)); 4.00 (m, CH (3)); 3.67 (m, HꢁC(2)). P-NMR (121.5 MHz): 2.32 (s). ESI-MS (neg.): 169
2
ꢁ
(
100, [M ꢁ H] ).
1
-Deoxy-d-xyl-2-ulose 5-Phosphate ¼ 1-Deoxy-d-threo-pent-2-ulose 5-Phosphate ¼ (2R,3S)-2,3-Di-
hydroxy-4-oxopentyl Dihydrogen Phosphate ¼ DXP; 3). To a soln. containing 120 mm Tris · HCl, pH 7.5,
1
0 mm MgCl , 2 mm thiamine pyrophosphate, and 2 mm dithiothreitol, purified 2 (100 mg, 0.59 mmol) or
2
þ
crude 2 (145 mg, containing 45 mg of glycolic acid) and sodium pyruvate (1 · Na ; 130.3 mg, 1.18 mmol)
were added. After the pH of the mixture was adjusted to 7.5 with 2m NaOH, the reaction was initiated by
addition of recombinant DXS of R. capsulatus (1 mg, total volume 1.2 ml) and incubated at 378 overnight.
Then the mixture was directly applied to a 1.5 ꢀ 18 cm column of DEAE Sephadex A-25 (formate form,
2
5 ml) at 48 which had been equilibrated with 80 ml of 0.06m HCOONH (pH 8.0) beforehand. The
4
4
product was then eluted with a linear gradient of 0.06 – 0.68m HCOONH (0.06m and 0.62m, pH 8.0,
2
50 ml each, flow-rate 2.5 ml/min, fractions of 15 ml). The combined fractions containing 3 were treated
þ
þ
with excess Dowex 50w ꢀ 8 resin (H form) to remove NH . The acidic eluate was collected on ice and
4
1
concentrated repeatedly in vacuo to remove HCOOH: oily yellowish 3 (105 mg, 83.2%). H-NMR: 2.29
31
(
s, Me(1)); 4.00 (m, CH (5)); 4.35 (ddd, J ¼ 2.0, 2.0, 1.0, HꢁC(4)); 4.48 (d, J ¼ 2, HꢁC(3)). P-NMR
2
ꢁ
(
121.5 MHz): 1.14 (s). ESI-MS (neg.): 213 (100, [M ꢁ H] ).
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Received December 8, 2009