Chemoenzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-d-erythritol: a substrate for IspE

Article abstract of DOI:10.1016/j.tetlet.2008.05.074

Enantiomerically pure 2-C-methyl-d-erythritol 4-phosphate 1 (MEP) is synthesized from 1,2-O-isopropylidene-α-d-xylofuranose via facile benzylation in good yield. Subsequently, 1 is used for enzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-d-erythritol

Full text of DOI:10.1016/j.tetlet.2008.05.074

Tetrahedron Letters 49 (2008) 4461–4463
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Tetrahedron Letters
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Chemoenzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol: a
substrate 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Enantiomerically pure 2-C-methyl-D-erythritol 4-phosphate 1 (MEP) is synthesized from 1,2-O-isopro-
Received 23 April 2008
Revised 14 May 2008
Accepted 14 May 2008
Available online 20 May 2008
pylidene-
synthesis of 4-diphosphocytidyl-2-C-methyl-
methyl- -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.
a
-
D
-xylofuranose via facile benzylation in good yield. Subsequently, 1 is used for enzymatic
D-erythritol 2 (CDP-ME) using 4-diphosphocytidyl-2-C-
D
Ó 2008 Elsevier Ltd. All rights reserved.
About 450,000 people a year are infected with multi-drug resis-
tant tuberculosis (MDR-TB), which is resistant to the main first-
line drugs isoniazid and rifampin. In addition extensively drug
resistant tuberculosis (XDR-TB), which is resistant to isoniazid
and rifampin and resistant to any fluoroquinolone and at
least one of three injectable second-line drugs (i.e., amikacin, kana-
mycin, or capreomycin) has been reported in 37 countries in all
regions of the world since 2006. Moreover human immunodefi-
ciency virus—tuberculosis (HIV-TB) co-infection is also a big chal-
lenge besides the XDR-TB.1 Yet no new anti-TB drug has been
introduced since the 1960s. In this context, designing and develop-
ing a new anti-TB drug is very important.
4-diphosphate 8 (HMBPP) by IspG. IspH (LytB) catalyzes the syn-
thesis of isopentenyl diphosphate 9 (IPP) and its isomer dimethyl-
allyl diphosphate 10 (DMAPP).
Since the MEP pathway is not found in mammalian cells, it is
considered an attractive target for the development of antimicrobi-
als, antimalarials, and herbicidal agents,3 a hypothesis that is being
explored by an increasing number of researchers. A major difficulty
hindering this research is the shortage of pure substrates. In this
regard, access to MEP pathway intermediates and their analogues
is essential to ongoing biochemical investigations and develop-
ment of high throughput screens to attempt to identify leads for
synthesis of new therapeutics. Recently, we have described assays
for mycobacterial Dxs, IspC, and IspD.4 In order to study mycobac-
terial IspE, we were in need of compound 2.
To date two different biosynthetic pathways have been reported
leading to isopentenyl diphosphate, the universal precursor of iso-
prenoids. The mevalonate pathway2 is found in animals, whereas
the non-mevalonate or methylerythritol phosphate (MEP) path-
way is found in many bacteria, some protozoa, and plants (Scheme
However, the reported chemical synthesis of 2 is only 50% enan-
tiomerically pure.5 Whereas when synthesized enzymatically
starting with the formation of 5 by condensation of 3 and 4 cata-
lyzed by Dxs, the ultimate yield of 1 is very low.6 In addition this
enzymatic method is time consuming and expensive. Herein, we
report a chemoenzymatic method to synthesize 2 in good yield.
To initiate the synthesis of 2 (Scheme 2), we synthesized enan-
tiopure 1. Many procedures are available for the synthesis of 1.
However, only one procedure is reported for synthesis of enantio-
merically pure 1 from commercially available 1,2-O-isopropyl-
1).3 In the MEP pathway, 1-deoxy-
D-xylulose 5-phosphate 5 (DXP)
is made by condensing pyruvate 3 and glyceraldehyde 3-phos-
phate 4 catalyzed by DXP synthase (Dxs). Subsequently, 1 is syn-
thesized by intramolecular rearrangement and reduction of 5
catalyzed by IspC. Then 1 is coupled with cytidine triphosphate
(CTP) using IspD to produce 2. 2 is subsequently phosphorylated
by
synthase (IspE) to form 4-diphosphocytidyl-2-C-methyl-
tol-2-phosphate 6 (CDP-ME2P) and cyclized by IspF to form 2-C-
methyl- -erythritol 2,4-cyclodiphosphate 7 (ME-CPP). The cyclic
4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate
D-erythri-
idene-a-D
-xylofuranose.7
Dibenzyl phosphochloridate8 was synthesized by a modified
procedure of chlorinating dibenzyl phosphite 11, in toluene and
benzene using N-chlorosuccinamide (NCS). Dibenzyl phosphochlo-
ridate 12 in pyridine was used to selectively protect the primary
D
diphosphate is transformed into 1-hydroxy-2-methyl-2-E-butenyl
alcohol of 1,2-O-isopropylidene-
5-dibenzylphosphate-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.).
E-mail addresses: Praba@colostate.edu (P. Narayanasamy), Dean.Crick@
colostate.edu (D. C. Crick).
a-D-xylofuranose 14.
Then the free secondary alcohol 14 was oxidized to ketone 15
quantitatively with pyridinium dichromate (PDC).7
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.074

4462
P. Narayanasamy et al. / Tetrahedron Letters 49 (2008) 4461–4463
GAP
pyruvate
O
DXP
O
P
OH
O
P
-O
O
O
HO
-O
-O
-O
Dxp synthase
or Dxs
reductoisomerase
+
-O
P
O
O
COOH -O
O
H
or IspC
OH
O
OH OH
NH
OH
3
5
4
1
NH
2
2
O
N
N
MEP cytidyl
transferase
P
-
-O
O
O
P
O
O
P
O
P
OH
N
O
O
N
O
P
O
O
O
O
O
O
O
O
O
-
or IspD
-
-
-
O
O
O
O
CDP-ME kinase
OH OH
OH OH
HO
OH
HO
OH
2
or IspE
6
O
P
O
-O
P
P
-
O
O
O
O
-
-
MECDP
synthase
O
O
O
HMBPP
synthase
O
O
P
-
O
9
O
P
-
P
O
IspH
O
O
-
-
O
O
O
O
or IspF
O
P
O
P
or IspG
OH
OH OH
-
8
O
O
7
-
-
O
O
10
Scheme 1. Isoprenoid biosynthesis via the MEP pathway.
O
O PH
O
O
O P Cl
O
NCS
Toluene/Benzene
95 % yield
11
12
O
O
O
HO
O
H
H
H
P
P
O
O
O
PDC
12
OH
OH
H
O
H
O
BnO OBn
H
BnO OBn
H
H
H
O
dcm, AcOH
91 % yield
O
H
pyridine
90 % yield
O
H
O
O
15
14
13
O
O
O
O
H
O
P
H
O
CH MgCl
benzylation
90 % yield
P
3
O
TFA
H
O
BnO OBn
H
BnO OBn
THF
H
HO
o
H
BnO
O
-10 C
O
85 % yield
17
16
80 % yield
O
O
O
BnO CH
O
P
BnO
3
P
NaIO
NaBH
4
O
BnO CH
4
3
BnO
BnO
P
H
OH
BnO OBn
BnO
O
O
MeOH/H O
H
BnO
2
MeOH
86 % yield
OH
O
OH OH
OH
91 % yield
19
20
18
NH
2
N
cytidine
O
P
-O
HO CH
3
Pd/C, H
O
P
O
triphosphate
2
OH
N
O
-O
O
P
O
MeOH/H O
O
O
O
-
2
-
IspD
OH OH
O
O
OH OH
94 % yield
1
HO
OH
2
Scheme 2. Chemoenzymatic synthesis of CDPME.

P. Narayanasamy et al. / Tetrahedron Letters 49 (2008) 4461–4463
4463
Previously, 3-C-alkyl ribofuranoses were obtained by a stereo-
selective addition reaction with the alkyl group on the b-face of
the carbohydrate ring.9 Accordingly, in the ketone 15, addition of
the methyl group occurs from the less hindered b-face, leading to
the tertiary alcohol 16 with the desired stereochemistry. The alco-
hol 16 was protected by using benzyl bromide, after activating the
hydroxyl group to yield 90% of benzylated 17.10
Thus, we successfully synthesized enantiomerically pure 1,
which could be utilized by mycobacterial IspD to synthesize 2 in
satisfactory yields. Radiolabel could be introduced during the
methylation or reduction steps if required. Pure 2 can be used to
study the kinetic properties of IspE and for high throughput
screening to identify IspE inhibitors. Experiments for M. tuberculo-
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 give
the aldehyde 19. The aldehyde was reduced with sodium borohy-
dride forming the MEP precursor,11 alcohol 20, followed by
hydrogenolysis in water/methanol medium, without acid workup
leading to enantiomerically pure 1 (Scheme 2). The chemically syn-
thesized 1 was characterized by NMR, MS, and optical rotation, and
the data were found to be identical with those previously reported
in the literature.7,12,13 Subsequently, chemically synthesized 1 was
used as a substrate for the enzymatic synthesis of 2.
Recombinant Rv3582c, M. tuberculosis IspD, was prepared as
previously described.4a Briefly, Rv3582c was amplified using PCR
primers, and Expand High Fidelity PCR system (Roche Molecular
Biochemicals, Indianapolis, Indiana, USA) (Rv3582c–F: CAT ATG
AGG GAA GCG GGC GAA GTA G and Rv3582c–R: CTC GAG TCA
CCC GCG GAG TAT AGC TTG), containing NdeI and XhoI restriction
enzyme sites (underlined), respectively. The PCR products were di-
gested and ligated into the pET28a(+) vector (EMD Biosciences,
Inc., San Diego, CA) and the ligation mixtures were used to trans-
Acknowledgment
This research was supported by NIH/NIAID Grant No. AI65357-
030010.
References and notes
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
Report (MMWR) 2006, 55, 1176; (d) World Health Organization. WHO Report,
WHO: Geneva, Switzerland, 2007.
2. Bochar, D. A.; Friesen, J. A.; Stauffacher, C. V.; Rodwell, V. W. Compr. Nat. Prod.
Chem. 1999, 2, 15–44.
3. Rohmer, M. Nat. Prod. Rep. 1999, 16, 565–574.
4. (a) Eoh, H.; Brown, A. C.; Buetow, L.; Hunter, W. N.; Parish, T.; Kaur, D.;
Brennan, P. J.; Crick, D. C. J. Bacteriol. 2007, 189, 8922–8927; (b) Dhiman, R. K.;
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.
C. Glycobiology 2002, 12, 813–820.
5. Koppisch, A. T.; Poulter, C. D. J. Org. Chem. 2002, 67, 5416–5418.
6. Kuzuyama, T.; Takagi, M.; Kaneda, K.; Dairi, T.; Seto, H. Tetrahedron Lett. 2000,
41, 703–706.
7. Hoeffler, J.-F.; Grosdemange, C.-P.; Rohmer, M. Tetrahedron 2000, 56, 1485–
1489.
form E. coli DH5a cells (Life Technologies, Rockville, MD) creating
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.
9. (a) Nakatani, K.; Arai, K.; Terashima, S. Tetrahedron 1993, 49, 1901–1912; (b)
Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J. J. Chem. Soc., Perkin
Trans.1 1997, 3423–3433.
10. Crimmins, M. T.; Brown, B. H. J. Am. Chem. Soc. 2004, 126, 10264–10266.
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,
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):
3588, 2966, 2362, 2336, 1652, 1614. HRMS (ESI) C26H32O7P (M+H+) calcd
DH5 [pET28a(+)::Rv3582c] for amplification. The recombinant
a
plasmids harboring Rv3582c were isolated using a Plasmid Mini-
prep Kit (Qiagen, Valencia, CA) and the sequences of the plasmids
were confirmed by Macromolecular Resources (Colorado State Uni-
versity). Transformation of BL21 (DE3) (Novagen, Madison, WI)
with pET28a(+)::Rv3582c afforded the recombinant strain
BL21(DE3)[pET28a(+)::Rv3582c]. Protein expression was induced
in the presence of 0.5 mM isopropyl–b–D–thiogalactopyranoside
(IPTG) at 20 °C for 10 h. The recombinant protein carrying a
hexa–histidine tag was purified by immobilized metal affinity
chromatography on HIS-selectTM Nickel affinity gel from Sigma–
Aldrich (St. Louis, MO) using a linear gradient of 50–200 mM imid-
azole in washing buffer [50 mM 4–morpholine propane sulfonic
acid (MOPS) (pH 7.9), 1 mM MgCl2, 10% glycerol, and 1 mM
b-mercaptoethanol].
487.1880 and found 487.1870; [a] 10.0 (c 0.5, CHCl3).
D
12. Koppisch, A. T.; Blagg, B. S. J.; Poulter, C. D. Org. Lett. 2000, 2, 215–217.
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–
9574; (b) Richard, S. B.; Bowman, M. E.; Kwiatkowski, W.; Kang, I.; Chow, C.;
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
mercapto-7-methylpurine ribonucleoside (MESG), 1 mM DTT, 100
methyl- -erythritol 4-phosphate (MEP), 100 CTP, 0.03
pyrophosphatase, 1U purine nucleoside phosphorylase and 11.6 pmols of
purified M. tuberculosis IspD per 50 l of reaction volume. Reaction mixtures
l
M 2-amino-6-
M 2-C-
inorganic
l
D
lM
U
Compound 2 was synthesized enzymatically14 with a maximum
yield of 25% after incubation at 37 °C for 1 h. Formation of 2 is fol-
lowed by monitoring the formation of PPi released during catalysis
by IspD (EnzChekÒ Phosphate Assay Kit, Invitrogen) although this
is not required in production of 2. When examined by MS and 1H
NMR the product was found to generate spectra identical to
reported data.5,13,15
l
were incubated at room temperature for 30 min after which their endpoint
absorbance at 360 nm was determined. Chemically produced 1 was compared
with authentic 1. Synthesized 2 was found to generate spectra identical to
reported data.
15. Cassera, M. B.; Gozzo, F. C.; Alexandri, F. L.; Merino, E. F.; Portillo, H. A.; Peres, V.
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.