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Tetrahedron Letters 49 (2008) 4461–4463
Chemoenzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol: asubstrate for IspE
Prabagaran Narayanasamy *, Hyungjin Eoh, Dean C. Crick *
Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences. Colorado State University, 1682 Campus Delivery,Fort Collins, CO 80523-1682, USA
Enantiomerically pure 2-C-methyl-D-erythritol 4-phosphate 1 (MEP) is synthesized from 1,2-O-isopro-
pylidene-a-D-xylofuranose via facile benzylation in good yield. Subsequently, 1 is used for enzymatic
synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2 (CDP-ME) using 4-diphosphocytidyl-2-C-
methyl-D-erythritol synthase (IspD). The chemoenzymatically synthesized 2 can be used as substrate
for assay of IspE and for high throughput screening to identify IspE inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
About 450,000 people a year are infected with multi-drug resis-
4-diphosphate 8 (HMBPP) by IspG. IspH (LytB) catalyzes the syn-
tant tuberculosis (MDR-TB), which is resistant to the main ﬁrst-
thesis of isopentenyl diphosphate 9 (IPP) and its isomer dimethyl-
line drugs isoniazid and rifampin. In addition extensively drug
resistant tuberculosis (XDR-TB), which is resistant to isoniazid
Since the MEP pathway is not found in mammalian cells, it is
and rifampin and resistant to any ﬂuoroquinolone and at
considered an attractive target for the development of antimicrobi-
least one of three injectable second-line drugs (i.e., amikacin, kana-
als, antimalarials, and herbicidal a hypothesis that is being
mycin, or capreomycin) has been reported in 37 countries in all
explored by an increasing number of researchers. A major difﬁculty
regions of the world since 2006. Moreover human immunodeﬁ-
hindering this research is the shortage of pure substrates. In this
ciency virus—tuberculosis (HIV-TB) co-infection is also a big chal-
regard, access to MEP pathway intermediates and their analogues
lenge besides the Yet no new anti-TB drug has been
is essential to ongoing biochemical investigations and develop-
introduced since the 1960s. In this context, designing and develop-
ment of high throughput screens to attempt to identify leads for
ing a new anti-TB drug is very important.
synthesis of new therapeutics. Recently, we have described assays
To date two different biosynthetic pathways have been reported
for mycobacterial Dxs, IspC, and IspD.In order to study mycobac-
leading to isopentenyl diphosphate, the universal precursor of iso-
terial IspE, we were in need of compound 2.
prenoids. The mevalonate is found in animals, whereas
However, the reported chemical synthesis of 2 is only 50% enan-
the non-mevalonate or methylerythritol phosphate (MEP) path-
tiomerically pure.Whereas when synthesized enzymatically
way is found in many bacteria, some protozoa, and plants (
starting with the formation of 5 by condensation of 3 and 4 cata-
In the MEP pathway, 1-deoxy-D-xylulose 5-phosphate 5 (DXP)
lyzed by Dxs, the ultimate yield of 1 is very low.In addition this
is made by condensing pyruvate 3 and glyceraldehyde 3-phos-
enzymatic method is time consuming and expensive. Herein, we
phate 4 catalyzed by DXP synthase (Dxs). Subsequently, 1 is syn-
report a chemoenzymatic method to synthesize 2 in good yield.
thesized by intramolecular rearrangement and reduction of 5
To initiate the synthesis of 2 (), we synthesized enan-
catalyzed by IspC. Then 1 is coupled with cytidine triphosphate
tiopure 1. Many procedures are available for the synthesis of 1.
(CTP) using IspD to produce 2. 2 is subsequently phosphorylated
However, only one procedure is reported for synthesis of enantio-
merically pure 1 from commercially available 1,2-O-isopropyl-
synthase (IspE) to form 4-diphosphocytidyl-2-C-methyl-D-erythri-
tol-2-phosphate 6 (CDP-ME2P) and cyclized by IspF to form 2-C-
Dibenzyl phosphochloridatewas synthesized by a modiﬁed
methyl-D-erythritol 2,4-cyclodiphosphate 7 (ME-CPP). The cyclic
procedure of chlorinating dibenzyl phosphite 11, in toluene and
diphosphate is transformed into 1-hydroxy-2-methyl-2-E-butenyl
benzene using N-chlorosuccinamide (NCS). Dibenzyl phosphochlo-ridate 12 in pyridine was used to selectively protect the primaryalcohol of 1,2-O-isopropylidene-a-D-xylofuranose 13 yielding
* Corresponding authors. Tel.: +1 970 491 6789; fax: +1 970 491 1815 (P.N.); tel.:
+1 970 491 3308; fax: +1 970 491 1815 (D.C.C.).
Then the free secondary alcohol 14 was oxidized to ketone 15
quantitatively with pyridinium dichromate
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
P. Narayanasamy et al. / Tetrahedron Letters 49 (2008) 4461–4463
Scheme 1. Isoprenoid biosynthesis via the MEP pathway.
Scheme 2. Chemoenzymatic synthesis of CDPME.
P. Narayanasamy et al. / Tetrahedron Letters 49 (2008) 4461–4463
Previously, 3-C-alkyl ribofuranoses were obtained by a stereo-
Thus, we successfully synthesized enantiomerically pure 1,
selective addition reaction with the alkyl group on the b-face of
which could be utilized by mycobacterial IspD to synthesize 2 in
the carbohydrate ring.Accordingly, in the ketone 15, addition of
satisfactory yields. Radiolabel could be introduced during the
the methyl group occurs from the less hindered b-face, leading to
methylation or reduction steps if required. Pure 2 can be used to
the tertiary alcohol 16 with the desired stereochemistry. The alco-
study the kinetic properties of IspE and for high throughput
hol 16 was protected by using benzyl bromide, after activating the
screening to identify IspE inhibitors. Experiments for M. tuberculo-
hydroxyl group to yield 90% of benzylated 17.
sis IspE inhibitors are in progress.
Acetonide deprotection was carried out using 90% aq triﬂuoro-
acetic acid giving rise to two anomers 18, which underwent a so-
dium metaperiodate-mediated glycol oxidative cleavage to givethe aldehyde 19. The aldehyde was reduced with sodium borohy-
This research was supported by NIH/NIAID Grant No. AI65357-
dride forming the MEP precursor,alcohol 20, followed by
hydrogenolysis in water/methanol medium, without acid workupleading to enantiomerically pure 1 ). The chemically syn-
thesized 1 was characterized by NMR, MS, and optical rotation, andthe data were found to be identical with those previously reported
1. (a) Brennan, P. J.; Crick, D. C. Curr. Top. Med. Chem. 2007, 7, 475–488; (b)
Editorial Lancet 2006, 368, 964.; (c) Wright, A. Morbidity and Mortality Weekly
in the Subsequently, chemically synthesized 1 was
Report (MMWR) 2006, 55, 1176; (d) World Health Organization. WHO Report,
used as a substrate for the enzymatic synthesis of 2.
Recombinant Rv3582c, M. tuberculosis IspD, was prepared as
2. Bochar, D. A.; Friesen, J. A.; Stauffacher, C. V.; Rodwell, V. W. Compr. Nat. Prod.
previously described.Brieﬂy, Rv3582c was ampliﬁed using PCR
3. Rohmer, M. Nat. Prod. Rep. 1999, 16, 565–574.
primers, and Expand High Fidelity PCR system (Roche Molecular
4. (a) Eoh, H.; Brown, A. C.; Buetow, L.; Hunter, W. N.; Parish, T.; Kaur, D.;
Biochemicals, Indianapolis, Indiana, USA) (Rv3582c–F: CAT ATG
Brennan, P. J.; Crick, D. C. J. Bacteriol. 2007, 189, 8922–8927; (b) Dhiman, R. K.;
AGG GAA GCG GGC GAA GTA G and Rv3582c–R: CTC GAG TCA
Schaeffer, M. L.; Bailey, A. M.; Testa, C. A.; Scherman, H.; Crick, D. C. J. Bacteriol.
2005, 187, 8395–8402; (c) Bailey, A. M.; Mahapatra, S.; Brennan, P. J.; Crick, D.
CCC GCG GAG TAT AGC TTG), containing NdeI and XhoI restriction
C. Glycobiology 2002, 12, 813–820.
enzyme sites (underlined), respectively. The PCR products were di-
5. Koppisch, A. T.; Poulter, C. D. J. Org. Chem. 2002, 67, 5416–5418.
gested and ligated into the pET28a(+) vector (EMD Biosciences,
6. Kuzuyama, T.; Takagi, M.; Kaneda, K.; Dairi, T.; Seto, H. Tetrahedron Lett. 2000,
Inc., San Diego, CA) and the ligation mixtures were used to trans-
7. Hoefﬂer, J.-F.; Grosdemange, C.-P.; Rohmer, M. Tetrahedron 2000, 56, 1485–
form E. coli DH5a cells (Life Technologies, Rockville, MD) creating
DH5a[pET28a(+)::Rv3582c] for ampliﬁcation. The recombinant
8. Gao, F.; Yan, X.; Shakya, T.; Baettig, O. M.; Brunet, S. A. M.; Berghuis, A. M.;
Wright, G. D.; Auclair, K. J. Med. Chem. 2006, 49, 5273–5281.
plasmids harboring Rv3582c were isolated using a Plasmid Mini-
9. (a) Nakatani, K.; Arai, K.; Terashima, S. Tetrahedron 1993, 49, 1901–1912; (b)
prep Kit (Qiagen, Valencia, CA) and the sequences of the plasmids
Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J. J. Chem. Soc., Perkin
were conﬁrmed by Macromolecular Resources (Colorado State Uni-
10. Crimmins, M. T.; Brown, B. H. J. Am. Chem. Soc. 2004, 126, 10264–10266.
versity). Transformation of BL21 (DE3) (Novagen, Madison, WI)
11. Data for 20: 1H NMR (CDCl3, 300 MHz): d 7.27–7.19 (m, 15H), 5.00–4.96 (m,
4H), 4.43–4.41 (m, 2H), 4.26 (t, 1H, J = 9.9 Hz), 3.98–3.94 (m, 2H), 3.62–3.60 (m,
BL21(DE3)[pET28a(+)::Rv3582c]. Protein expression was induced
2H), 1.10 (s, 3H).; 13C NMR (CDCl3, 75 MHz): 138.5, 135.7, 135.6, 128.6, 128.4,128.1, 128.0, 127.6, 127.3, 78.2, 73.0, 69.6, 64.8, 64.2, 15.4.; IR (neat, cmÀ1):
in the presence of 0.5 mM isopropyl–b–D–thiogalactopyranoside
3588, 2966, 2362, 2336, 1652, 1614. HRMS (ESI) C26H32O7P (M+H+) calcd
(IPTG) at 20 °C for 10 h. The recombinant protein carrying a
487.1880 and found 487.1870; [a]D 10.0 (c 0.5, CHCl3).
hexa–histidine tag was puriﬁed by immobilized metal afﬁnity
12. Koppisch, A. T.; Blagg, B. S. J.; Poulter, C. D. Org. Lett. 2000, 2, 215–217.
chromatography on HIS-selectTM Nickel afﬁnity gel from Sigma–
13. (a) Rohdich, F.; Schuhr, C. A.; Hecht, S.; Herz, S.; Wungsintaweekul, J.;
Eisenrich, W.; Zenk, M. H.; Bacher, A. J. Am. Chem. Soc. 2000, 122, 9571–
Aldrich (St. Louis, MO) using a linear gradient of 50–200 mM imid-
9574; (b) Richard, S. B.; Bowman, M. E.; Kwiatkowski, W.; Kang, I.; Chow, C.;
azole in washing buffer [50 mM 4–morpholine propane sulfonic
Lillo, A. M.; Cane, D. E.; Noel, J. P. Nat. Struct. Biol. 2001, 8, 641–648.
14. Reaction mixtures contained 50 mM Tris–HCl (pH 7.4), 200 lM 2-amino-6-
mercapto-7-methylpurine ribonucleoside (MESG), 1 mM DTT, 100 lM 2-C-
methyl-D-erythritol 4-phosphate (MEP), 100 lM CTP, 0.03 U inorganic
Compound 2 was synthesized enzymaticalwith a maximum
pyrophosphatase, 1U purine nucleoside phosphorylase and 11.6 pmols of
yield of 25% after incubation at 37 °C for 1 h. Formation of 2 is fol-
puriﬁed M. tuberculosis IspD per 50 ll of reaction volume. Reaction mixtureswere incubated at room temperature for 30 min after which their endpoint
lowed by monitoring the formation of PPi released during catalysis
absorbance at 360 nm was determined. Chemically produced 1 was compared
by IspD (EnzChekÒ Phosphate Assay Kit, Invitrogen) although this
with authentic 1. Synthesized 2 was found to generate spectra identical to
is not required in production of 2. When examined by MS and 1H
15. Cassera, M. B.; Gozzo, F. C.; Alexandri, F. L.; Merino, E. F.; Portillo, H. A.; Peres, V.
NMR the product was found to generate spectra identical to
J.; Almeida, I. C.; Eberlin, M. N.; Wunderlich, G.; Weisner, J.; Jomaa, H.; Kimura,
E. A.; Katzin, A. M. J. Biol. Chem. 2004, 51749–51759.
clinical update 74 AUGUST 2004 By Mary Birch Obstructive sleep apnoea and breathing retraining About the author Mary Birch , RN, BA, MBioE, Grad Dip Soc, is a registered Buteyko practitioner. Introduction Obstructive sleep apnoea (OSA) is a sleep disorder where repeated upper airway obstruction during sleep leads to a decrease in blood oxygen saturation and disrupted slee
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