Chemical and Enzymatic Synthesis of 1-Deoxy-D-xylulose-5-phosphate

Full text of DOI:10.1021/jo971933p

J . Org. Chem. 1998, 63, 2375-2377
2375
Sch em e 1
Ch em ica l a n d En zym a tic Syn th esis of
1-Deoxy-D-xylu lose-5-p h osp h a te
Sean V. Taylor, Long D. Vu, and Tadhg P. Begley*
Department of Chemistry, Cornell University,
Baker Laboratory, Ithaca, New York 14853
Ulrich Scho¨rken, Sigrid Grolle, Georg A. Sprenger,*
Stephanie Bringer-Meyer, and Hermann Sahm
Institut fu¨r Biotechnologie, 1 des Forschungszentrums
J u¨lich GmbH, Postfach 1913, D-52425 J u¨lich, Germany
Received October 20, 1997
1-Deoxy-D-xylulose-5-phosphate 1 or the corresponding
nonphosphorylated sugar, 2, has been identified as a
precursor in three major biosynthetic pathways (Scheme
1). In higher plants, algae, and bacteria, a nonmeva-
lonate pathway to isopentenyl diphosphate 3 which
utilizes 1 or 2 has been demonstrated.1 In bacteria, 1 or
2 is involved in the biosynthesis of thiamin pyrophos-
phate 4.2 In Escherichia coli, it has been demonstrated
that one of the enzymes required for the biosynthesis of
the thiazole moiety of thiamin (ThiSG) catalyzes the
epimerization of 1 but not 2. This suggests that 1 is the
precursor to the thiazole.3 Last, labeling studies have
shown that 1 or 2 is the precursor to pyridoxal 5.4 While
numerous routes to 2 have been published,5 a synthesis
of 1 has not yet been described. In this paper we report
a chemical and an enzymatic syntheses of 1. The
availability of 1 should facilitate studies on the mecha-
nistic enzymology of all three biosynthetic pathways.
The chemical synthesis of 1 is outlined in Scheme 2.
Benzylation of diethyl-D-tartrate 6 followed by LAH
reduction gave 8.6 Phosphorylation of 8 using dibenzyl
phosphorochloridate resulted in a mixture of 9, the cor-
responding diphosphate, and starting material 8, which
Sch em e 2
(1) (a) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H.
Biochem. J . 1993, 295, 517. (b) Rohmer, M.; Seemann, M.; Horbach,
S.; Bringer-Meyer, S.; Sahm, H. J . Am. Chem. Soc. 1996, 118, 2564.
(c) Eisenreich, W.; Menhard, B.; Hylands, P. J .; Zenk, M. H.; Bacher,
A. Proc. Natl. Acad. Soc. U.S.A. 1996, 93, 6431. (d) Schwender, J .;
Seemann, M.; Lichtenthaler, H. K.; Rohmer, M. Biochem. J . 1996, 316,
73. (e) Lichtenthaler, H. K.; Schwender, J .; Disch, A.; Rohmer, M. FEBS
Lett. 1997, 400, 271. (f) Duvold, T.; Cali, P.; Bravo, J .-M.; Rohmer, M.
Tetrahedron Lett. 1997, 38, 6181. (g) Eisenriech, W.; Sagner, S.; Zenk,
M.; Bacher, A. Tetrahedron Lett. 1997, 38, 3889. (h) Schwender, J .;
Zeidler, J .; Gro¨ner, R.; Mu¨ller, C.; Focke, M.; Braun, S.; Lichtenthaler,
F. W.; Lichtenthaler, H. K. FEBS Lett. 1997, 414, 129. (i) Arigoni, D.;
Sagner, S.; Latzel, C.; Eisenreich, W.; Bacher, A.; Zenk, M. H. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 10600. (j) Putra, S. R.; Lois, L. M.;
Campos, N.; Boronat, A.; Rohmer, M. Tetrahedron Lett. 1998, 39, 23.
(k) Knoss, W.; Reuter, B.; Zapp, J . Biochem. J . 1997, 326, 449.
(2) Himmeldirk, K.; Kennedy, I. A.; Hill, R. E.; Sayer, B. G.; Spenser,
I. D. Chem. Commun. 1996, 1187.
was recycled. Swern oxidation of 9 gave 10. Methyl
addition using the in situ generated methyl cerium
reagent gave 11, which was oxidized to 12 using the
Dess-Martin reagent. Ketone 12 is stable when stored
at -20 °C and was quantitatively deprotected using
catalytic hydrogenation to 1 in an overall yield of 5%.
E. coli 1-deoxy-D-xylulose-5-phosphate synthase cata-
lyzes the condensation of pyruvate 16 and glyceraldehyde
3-phosphate 14 to give 1 (Scheme 3). This thiamin-
dependent enzyme has been overexpressed at a high level
(∼10% of total soluble protein) and purified7 and can be
used for the enzymatic synthesis of 1.
(3) (a) Taylor, S. V.; Chiu, H.-J .; Backstrom, A. D.; Begley, T. P.
Unpublished Results. (b) Begley, T. P. Nat. Prod. Rep. 1996, 177.
(4) Hill, R. E.; Himmeldirk, K.; Kennedy, I. A.; Pauloski, R. M.;
Sayer, B. G.; Wolf, E.; Spenser, I. D. J . Biol. Chem. 1996, 271, 30426.
(5) (a) David, S.; Estramareix, B.; Fischer, J . C.; The´risod, M. J .
Chem. Soc., Perkin Trans.
1 1982, 2131. (b) Backstrom, A. D.;
McMordie, R. A. S.; Begley, T. P. J . Carbohydr. Chem. 1995, 14, 171.
(c) Kennedy, I. A.; Hemscheidt, T.; Britten, J . F.; Spenser, I. D. Can.
J . Chem. 1995, 73, 1329. (d) Piel, J .; Boland, W. Tetrahedron Lett. 1997,
38, 6387.
(6) (a) Kalinowski, H.-O.; Crass, G.; Seebach, D. Chem. Ber. 1981,
114, 477. (b) Cunningham, A. F., J r.; Ku¨ndig, E. P. J . Org. Chem. 1988,
53, 1823.
(7) Sprenger, G. A.; Scho¨rken, U.; Wiegert, T.; Grolle, S.; De Graaf,
A. A.; Taylor, S. V.; Begley, T. P.; Bringer-Meyer, S.; Sahm, H. Proc.
Natl. Acad. Sci. U.S.A.. 1997, 94, 12857.
S0022-3263(97)01933-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/18/1998

2376 J . Org. Chem., Vol. 63, No. 7, 1998
Notes
exchange column was from Pharmacia Biotech, (Uppsala, Swe-
den). The Aminex 87 H HPLC column was from Bio-Rad
Laboratories GmbH (Munich, Germany). The Centriprep-30
ultrafiltration membrane was from Amicon (Witten, Germany).
Rabbit muscle aldolase, triosephosphate isomerase, and alkaline
phosphatase were from Boehringer Mannheim (Mannheim,
Germany). The fructose-1,6-diphosphate was purchased from
Biomol (Hamburg, Germany).
Sch em e 3
(2S,3S)-2,3-Bis(ben zyloxy)su ccin ic Acid Dieth yl Ester 7.
Thallous ethoxide (3.87 g, 0.0155 mol, 1.1 mL) and benzyl
bromide (3.99 g, 0.0233 mol, 2.77 mL) were added to a stirred
solution of diethyl-D-tartrate (1.6 g, 0.00776 mol) in dry aceto-
nitrile (20 mL) at 45 °C. Stirring was continued for 48 h at 55
°C and then for 12 h at room temperature. The reaction mixture
was filtered through Florisil to remove thallium bromide, and
the acetonitrile was removed in vacuo. Flash chromatography
(15:85 ethyl acetate:hexane) of the resulting oil gave 2.00 g of a
white solid in 88% yield (Rf ) 0.32, 15:85 ethyl acetate:hexane).
1H NMR (400 MHz, CDCl3) δ: 7.29 (m, 10H), 4.87 (d, J ) 12.1
Hz, 2H), 4.45 (d, J ) 12.1 Hz, 2H), 4.39 (s, 2H), 4.12 (m, 4H),
1.18 (t, J ) 7.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ: 169.22,
137.10, 128.47, 128.39, 128.05, 78.47, 73.3, 61.43, 14.20. MS
(CI + CH4): 295 (M+ - 91).
(2R,3R)-2,3-Bis(ben zyloxy)bu ta n e-1,4-diol 8. Lithium alu-
minum hydride (452 mg, 0.0119 mol) was added over 30 min to
a stirred solution of diester 7 (2.00 g, 0.00518 mol) in dry THF
(35 mL) at 0 °C. The mixture was warmed to room temperature
and stirring was continued for 3 h. The reaction mixture was
cooled to 0 °C, quenched by slow addition of THF (35 mL), water
(0.5 mL), 2M sodium hydroxide (0.5 mL), and water (0.5 mL),
and filtered. The filtrate was dried over anhydrous magnesium
sulfate, and THF was removed in vacuo to give 1.30 g of a white
solid in 83.3% yield. 1H NMR (400 MHz, CDCl3) δ: 7.34 (m,
10H), 4.66 (s, 4H), 3.75 (m, 6H), 2.57 (br s, 2H). 13C NMR (100
MHz, CDCl3) δ: 138.10, 128.73, 128.17, 128.14, 79.07, 72.75,
60.94. MS (CI + CH4): 303.
(2R,3R)-P h osp h or ic Acid Diben zyl Ester 2,3-Bis(ben zyl-
oxy)-4-h yd r oxybu tyl Ester 9. Dibenzyl phosphorochloridate
(0.0028 mol) dissolved in CCl4 (6 mL) was added to a stirred
solution of diol 8 (650 mg, 0.00215 mol) in pyridine (25 mL) at
0 °C. The reaction was stirred at room temperature for 12 h
and quenched by addition of water (2 mL), and the solvents were
removed in vacuo. Flash chromatography (20:80 ethyl acetate:
methylene chloride) gave 479 mg of a yellow oil in 40% yield (Rf
) 0.22, 25:75 ethyl acetate:methylene chloride). 1H NMR (400
MHz, CDCl3) δ: 7.32 (m, 20H), 5.04 (m, 4H), 4.61 (m, 4H), 4.29
(m, 1H), 4.16 (m, 1H), 3.77 (m, 2H), 3.61 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ: 138.08, 137.94, 135.89 (d), 128.73, 128.7,
128.62, 128.57, 128.24, 128.08, 128.03, 78.38, 77.97, 77.89, 73.27,
73.00, 69.49 (d), 67.00 (d), 61.37. 31P NMR (160 MHz, CDCl3)
δ: -0.151.
Treatment of 13 and 16 with rabbit muscle aldolase,
triosephosphate isomerase, and partially purified 1-deoxy-
D-xylulose-5-phosphate synthase gave 1 in 47% yield,
which was easily purified by precipitation as its barium
salt using a variation of the method of Cardini and
Leloir.8 The in situ generation of 14 from fructose-1,6-
diphosphate 13 is preferable to directly adding it to the
reaction mixture, since commercially available prepara-
tions of 14 are enantiomerically impure and expensive.
The synthetic and biological methods reported in this
study are simple and should be easily reproduced by
organic chemists and biochemists for use in studies on
the biosynthesis of isoprenoids, thiamin, and pyridoxol.
In addition, isotopically labeled 1 should be readily
attainable using these routes and commercially available
labeled precursors.
Exp er im en ta l Section
Gen er a l. Mass spectra were obtained in the Mass Spectrom-
etry Laboratory, School of Chemical Sciences, University of
Illinois. 1H and 13C NMR were collected on a Varian XL-400
spectrometer. Chemical shifts are reported relative to δ(CHCl3)
) 7.24 ppm and δ(H20) ) 4.65 ppm. 31P spectra were calibrated
against an external H3PO4 standard set at δ ) 0.00 ppm and
were not decoupled. The 13C spectrum of compound 1 was
calibrated against an internal standard of ethanol at 56.80 and
16.15 ppm. Unless stated otherwise, all reagents and chemicals
were purchased from commercial sources and used as supplied.
All reactions were carried out under an inert (nitrogen or argon)
atmosphere with dry, freshly distilled solvents unless stated
otherwise. Dibenzyl phosphorochloridate was prepared accord-
ing to the procedure of Smith.9 Anhydrous cerium chloride was
prepared according to the method of Imamoto et al.10 The Dess-
Martin reagent was prepared according to the original procedure
of Dess and Martin,11 and thoroughly dried in vacuo before use.
EM Science Silica gel 60 F-254 was purchased from Krackeler
Scientific (Albany, New York). For thin-layer chromatography
analysis, Merck precoated glass backed TLC plates (silica gel
60, F-254, 250 µm) were used. The Q-Sepharose HP anion-
(2R,3S)-P h osp h or ic Acid Diben zyl Ester 2,3-Bis(ben zyl-
oxy)-4-oxobu tyl Ester 10. Dimethyl sulfoxide (330 mg, 0.00423
mol, 327 µL) was added with stirring to a solution of oxalyl
chloride (0.00184 mol) in methylene chloride (4.9 mL) at -78
°C. The mixture was stirred for 2.5 min and warmed to -10
°C, and alcohol 9 (450 mg, 0.000799 mol), dissolved in methylene
chloride (5 mL), was added. After 10 min, triethylamine (471
mg, 0.00465 mol, 648 µL) was added, the reaction mixture stirred
for 5 min and then warmed to room temperature. Water (5 mL)
was added, and the resulting mixture was extracted with
chloroform (4 × 5 mL). The organic extracts were combined,
washed with water (2 × 5 mL) and brine (2 × 5 mL), and dried
with anhydrous magnesium sulfate, and the solvent was re-
moved in vacuo. Flash chromatography (15:85 ethyl acetate:
methylene chloride) gave 323 mg of a white solid in 73% yield
(Rf ) 0.40, 15:85 ethyl acetate:methylene chloride). 1H NMR
(400 MHz, CDCl3) δ: 9.60 (d, J ) 1 Hz, 1H), 7.31 (m, 20H), 5.01
(m, 4H), 4.55 (m, 4H), 4.15 (m, 2H), 3.92 (m, 1H), 3.84 (dd, J )
1 Hz, J ) 3.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 202.44,
137.25, 136.94, 135.82 (d), 128.79, 128.74, 128.61, 128.47, 128.44,
128.29, 128.24, 128.18, 128.16, 82.11, 77.35, 77.27, 73.79, 73.32,
69.64 (d), 65.01 (d). 31P NMR (160 MHz, CDCl3) δ: -1.094. MS
(CI + CH4): 561.
(8) Cardini, C. E.; Leloir, L. F. Methods Enzymol. 1957, III, 835.
(9) Smith, M. Biochem. Prepr. 1961, 8, 130.
(10) Imamoto, T.; Takiyama, N.; Nakamura, K.; Hatajima, T.;
Kamiya, Y. J . Am. Chem. Soc. 1989, 111, 4392.
(2R,3R)-P h osp h or ic Acid Diben zyl Ester 2,3-bis(ben zyl-
oxy)-4-h yd r oxyp en tyl Ester 11. Ceric chloride was dried at
140 °C in vacuo for 2 h, cooled to room temperature, dissolved
(11) Dess, D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155.

Notes
J . Org. Chem., Vol. 63, No. 7, 1998 2377
in THF (5 mL), and stirred at room temperature for 2 h. Methyl
iodide (1 g, 7 mmol, 439 µL) was added with stirring to fresh
lithium shavings in diethyl ether (3.5 mL). This mixture was
stirred for 30 min at room temperature under a reflux condenser.
Then, 500 µL of the resulting methyllithium solution was added
to the cerium chloride-THF mixture at -78 °C and stirred for
1 h. Aldehyde 10, dissolved in THF (5 mL), was added dropwise
to the methyllithium-cerium chloride mixture, and stirring was
continued at -78 °C for 3 h. The reaction mixture was quenched
by addition of saturated ammonium chloride (0.5 mL) and
warmed to room temperature. Water was added (10 mL) and
the aqueous phase was extracted with chloroform (3 × 5 mL).
The organics were combined, extracted with brine (3 × 5 mL),
and dried over anhydrous magnesium sulfate, and the solvents
were removed in vacuo. Flash chromatography (20:80 ethyl
acetate:methylene chloride) gave 105 mg of the two possible
diastereomers as a yellow oil in 33% yield (Rf ) 0.35, 20:80 ethyl
acetate:methylene chloride). 1H NMR (400 MHz, CDCl3) δ: 7.27
(m, 20H), 5.05 (m, 4H), 4.58 (m, 4H), 4.21 (m, 2H), 3.90 (m, 2H),
3.33 (m, 1H), 1.19 and 1.12 (d, J ) 6.3 and 6.4 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ: 138.05, 137.97, 137.92, 137.58,
135.87 (d), 128.72, 128.70, 128.67, 128.65, 128.60, 128.57, 128.53,
128.50, 128.36, 128.27, 128.13, 128.07, 128.06, 128.03, 127.96,
127.91, 81.84, 80.83, 78.31 (d), 77.91 (d), 74.82, 73.66, 73.31,
73.17, 69.50 (d), 69.41, 67.35, 67.27 (d), 66.99 (d), 66.88, 20.35,
19.91. 31P NMR (160 MHz, CDCl3) δ: -0.717, -0.845.
(2R,3S)-P h osp h or ic Acid Diben zyl Ester 2,3-Bis(ben zyl-
oxy)-4-oxop en tyl Ester 12. Dess-Martin reagent (258 mg,
0.000608 mol) and 11 (100 mg, 0.00017 mol) were stirred in
methylene chloride (15 mL) for 12 h at room temperature.
Diethyl ether (10 mL) was added, and the reaction was quenched
by the addition of saturated sodium thiosulfate (5 mL) and
saturated sodium bicarbonate (5 mL). The mixture was stirred
until clear and the aqueous phase was extracted with diethyl
ether (2 × 10 mL). The organics were combined and washed
with saturated sodium bicarbonate (2 × 10 mL), water (10 mL),
and brine (10 mL). The organics were dried over anhydrous
magnesium sulfate, and the solvent was removed in vacuo to
give 73 mg of a yellow oil in 74% yield (Rf ) 0.4, 20:80 ethyl
acetate:methylene chloride). 1H NMR (400 MHz, CDCl3) δ: 7.31
(m, 20H), 4.99 (m, 4H), 4.48 (m, 4H), 4.09 (m, 2H), 3.92 (m, 1H),
3.85 (d, J ) 3.4 Hz, 1H), 2.09 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ: 210.30, 137.43, 137.02, 135.85 (d), 128.84, 128.73,
128.59, 128.49, 128.39, 128.20, 83.74, 78.40 (d), 74.09, 73.73,
69.63, 65.50 (d), 27.96. 31P NMR (160 MHz, CDCl3) δ: -0.385.
MS (CI + CH4): 575.
induced at an OD600 of 0.8 with IPTG (0.4 mm), and harvested
by centrifugation after an additional 4 h of growth. The cell
pellet was resuspended in buffer A (50 mm Tris-HCl, 1 mm DTT,
0.5 mm thiamin pyrophosphate, 5 mm MgCl2, pH 7.5), sonicated
in a Branson sonicator (10 30-s pulses at 40 W output, duty cycle
50%), and centrifuged (1 h at 38000g). Ammonium sulfate (225
g/L, 40% saturation) was added to the resulting cell-free extract
at 4 °C. The mixture was centrifuged (1 h at 38000g), and the
precipitate was dissolved in buffer A, desalted by repeated
ultrafiltration through a YM30 membrane, and applied to a 100
mL bed volume Q-Sepharose HP anion-exchange column. The
enzyme eluted in a linear NaCl gradient at 0.1-0.2 m NaCl.
The purification was monitored by SDS-PAGE, which indicated
that the partially purified enzyme was 70% pure.
E n zym a t ic Syn t h esis of 1-Deoxy-D-xylu lose-5-p h os-
p h a te, 1. Fructose-1,6-diphosphate 13 (trisodium salt, 406 mg)
and pyruvate 16 (sodium salt, 220 mg) were dissolved in 36 mL
of buffer A to give final concentrations of 25 mM 13 and 50 mM
16. Triosephosphate isomerase (200 units), rabbit muscle al-
dolase (40 units), and partially purified 1-deoxy-D-xylulose-5-
phosphate synthase (4 mg) in a volume of 4 mL of buffer A were
added. To monitor the reaction progress, samples were removed,
dephosphorylated with alkaline phosphatase, and analyzed for
2 by HPLC (Aminex 87 H column, eluted with 6 mM H2SO4,
and monitored at 195 nm, retention time of 2 was 12 min). After
12 h the reaction was judged to be 80% complete. Another 203
mg of 13 and 110 mg of 16 were added to the reaction mix. After
24 h the reaction was over 90% complete.
P u r ifica tion of th e En zym a tica lly Syn th esized 1-Deoxy-
D-xylu lose-5-p h osp h a te, 1. The reaction mixture was depro-
teinized by ultrafiltration using a Centriprep-30 membrane. A
4 mL portion of 1 M BaCl2 solution was added and the cloudy
mixture was centrifuged for 10 min at 3000 rpm. The pellet
was washed with water (2 × 10 mL) and 1 was precipitated from
the washings by addition of ethanol (60 mL) followed by standing
at -20 °C for 30 min. After centrifugation (10 min, 3000 rpm)
the pellet was washed twice with ice cold 70% ethanol (2 × 5
mL) and dried. The pellet was redissolved in water (8 mL),
insoluble material was removed by centrifugation, and the
solvent removed using a Speed Vac to give 1 as a light orange
powder (707 mg, 47% yield). 1H NMR (400 MHz, D20) δ: 4.35
(s, 1H), 4.17 (t, J ) 5.9 Hz, 1H), 3.71 (m, 2H), 2.12 (s, 3H).12
Ack n ow led gm en t. The authors wish to thank Ally-
son Backstrom for helpful discussions. This work was
supported by a grant from the National Institutes of
Health (T. P. B., DK44083), a National Institutes of
Health Training Grant (S. V. T.), and a grant from the
Deutsche Forschungsgemeinschaft (G. S. and U. S., SFB
380/B21).
1-Deoxy-D-xylu lose-5-p h osp h a te 1. Compound 12 (10 mg,
0.0000174 mol) was dissolved in absolute ethanol (8 mL).
Pearlman’s catalyst (20% Pd(OH)2 on carbon) was added (12 mg)
and the reaction was stirred for 6 h at room temperature under
a positive pressure of hydrogen. The catalyst was removed by
filtering through Celite, and the solvent was removed in vacuo
to give 4 mg of a clear film in 99% yield. 1H NMR (400 MHz,
D20) δ: 4.27 (s, 1H), 4.19 (t, J ) 6.1 Hz, 1H), 3.81 (m, 2H), 2.11
(s, 3H). 13C NMR (100 MHz, D20) δ: 212.08, 76.12, 69.33 (d),
65.17 (d), 25.15. 31P NMR (160 MHz, D2O) δ: 0.837.
J O971933P
(12) The C3 epimer of 1 can be clearly differentiated by NMR from
1 and the stereochemistry at C4 is determined by the stereochemistry
of 14. Therefore, the configuration of the enzymatic product is 3S,4R.
In addition, the enzymatic product is a substrate for ThiSG. The
enantiomer of 1 would not be a substrate.
P a r tia l P u r ifica tion of 1-Deoxy-D-xylu lose-5-p h osp h a te
Syn th a se. E. coli J M109 (pUCBM20dxs)7 was grown in LB
medium supplemented with ampicillin (100 mg/L) at 37 °C,