Formal synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol from d-(+)-arabitol

Article abstract of DOI:10.1016/j.tet.2012.08.020

2-C-Methyl-d-erythritol-4-phosphate (MEP) is a key chemical intermediate of the non-mevalonate pathway for isoprenoid biosynthesis employed by many pathogenic microbes. MEP is also the precursor for the synthesis of 4-diphosphocytidyl-2-C-methyl d-erythri

Full text of DOI:10.1016/j.tet.2012.08.020

Tetrahedron 68 (2012) 8937e8941
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Tetrahedron
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Formal synthesis of 4-diphosphocytidyl-2-C-methyl
-(þ)-arabitol
D-erythritol from
D
*
Sina I. Odejinmi, Rafael G. Rascon, Wyman Chen, Kent Lai
Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2012
Received in revised form 2 August 2012
Accepted 7 August 2012
Available online 14 August 2012
2-C-Methyl-
pathway for isoprenoid biosynthesis employed by many pathogenic microbes. MEP is also the pre-
cursor for the synthesis of 4-diphosphocytidyl-2-C-methyl -erythritol (CDP-ME), another key in-
termediate of the non-mevalonate pathway. As this pathway is non-existent in higher animals, including
humans, it represents great opportunities for novel antimicrobial development. To facilitate the in-depth
studies of this pathway, we reported here a formal synthesis of CDP-ME through a new synthesis of 2-C-
D-erythritol-4-phosphate (MEP) is a key chemical intermediate of the non-mevalonate
D
Keywords:
MEP
CDP-ME
methyl-
D
-erythritol-4-phosphoric acid from
D-(þ)-arabitol.
Ó 2012 Elsevier Ltd. All rights reserved.
Selective phosphorylation
Dioxanone
Monophosphate
1. Introduction
investigations is 4-diphosphocytidyl-2-C-methyl
ME) kinase.21e38 This enzyme catalyzes the conversion of CDP-ME
to diphosphocytidyl-2-C-methyl -erythritol-2-phosphate (CDP-
D-erythritol (CDP-
Isoprenoids represent one of the most diverse classes of natu-
rally occurring molecules. To date, there are over 30,000 different
compounds identified with representatives involved in cellular
processes, such as bacterial cell wall biosynthesis, photosynthesis,
cell signaling, oxidative phosphorylation, and protein bio-
synthesis.1 All isoprenoids are synthesized from the five-carbon
precursorsdisopentenyl diphosphate (IPP) and dimethylallyl di-
phosphate (DMAPP), which are produced by one of the two bio-
chemical pathways: (a) Mevalonate (MVA) Pathway or (b)
Methylerythritol Phosphate (MEP) Pathway. The MVA pathway for
isoprenoid biosynthesis is seemingly unrelated to the MEP
pathway and is utilized by Archaea, Eukaryea, most Gram-positive
bacteria, such as Staphylococcus aureus.2e6 In contrast, the
MEP pathway is utilized primarily by Gram-negative bacteria
(e.g., Pseudomonas aeruginosa, Yersinia pestis), a few Gram-positive
bacteria (e.g., Mycobacterium tuberculosis), Chlamydia, Protozoa,
and some plants.7e18 Genetic disruptions of either biosynthetic
pathway are lethal to microbes.19,20 Since the MEP pathway is ab-
sent in humans, the enzymes involved in this pathway are ideal
targets for drug discovery of novel antimicrobials agents. One of the
enzymes that has recently become the subject of intense
D
MEP).38 Because of this kinase activity, inhibitors of CDP-ME kinase
can be explored as novel antimicrobial agents. In order to demon-
strate this proof of principle, we have conducted high throughput
screening (HTS) for small molecule inhibitors of various microbial
CDP-ME kinases.39 In these HTS, large milligram quantity of the
substrate, CDP-ME, is required. Therefore, we sought for an efficient
and stereoselective synthesis of 2-C-methyl-D-erythritol-4-
phosphoric acid (1), a convenient precursor for the synthesis of
CDP-ME.
2. Results and discussion
Recently, other research groups have focused on the synthesis of
2-C-methyl-D-erythritol-4-phosphoric acid (9% overall yield after
14 steps)42 or its salts43,46,49 because the availability of MEP path-
way intermediates and their analogs would substantially assist the
ongoing biochemical investigations and the development of novel
antibiotics, therapeutic agents and herbicides.40e49 We preferred to
synthesize 2-C-methyl- -erythritol-4-phosphoric acid (1) for CDP-
D
ME synthesis instead of the its salts because as a free diacid, it could
be used directly for CDP-ME synthesis22 without the need for the
extra steps for its salt preparation (sodium and trialkylammonium)
and of the concerns for the limited solubility of the salts, particu-
larly the sodium salt.45 In our efforts to further simplify the syn-
* Corresponding author. Department of Pediatrics, University of Utah SOM, Rm
2C412, SOM Building, 50 N. Medical Drive, Salt Lake City, UT 84132, USA. Tel.: þ1
801 581 4510; e-mail address: kent.lai@hsc.utah.edu (K. Lai).
thesis of 2-C-methyl- -erythritol-4-phosphoric acid (1), we chose
D
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.08.020

8938
S.I. Odejinmi et al. / Tetrahedron 68 (2012) 8937e8941
to explore its synthesis in 7 steps from
the previously reported 14 steps.42
The synthesis of diol 6 (Scheme 1) has been described beginning
from the condensation of the benzaldehyde (3) and
(2) in the presence of HCl gas to give a stable benzylidene de-
rivative.46,51 We have modified this procedure by the addition of
catalytic.
D
-(þ)-arabitol (2) instead of
Because diol 6 has both primary and secondary hydroxyl func-
tional groups, we anticipated selective phosphorylation43 of its
primary alcohol functionality as the primary hydroxyl group of diol
6 may have greater access to the phosphorylating reagent than its
secondary hydroxyl group. In our experiments, the treatment of the
equal mixture of diol 6 and 7 with dibenzylphosphorochloridate in
the presence of excess pyridine with CH2Cl2 or CHCl3 as a co-
D-(þ)-arabitol
Scheme 1. Benzylidene formation by catalytic HCl.
HCl (12 N) to the mixture of
D
-(þ)-arabitol (2) and benzaldehyde
solvent, and stirring overnight, caused the significant formation
of double phosphorylation product 9 derived from the reaction of
diol 6 with dibenzylphosphorochloridate (Scheme 2). However, the
primary hydroxyl group in diol 7 was unreactive under these
conditions and we were able to isolate the new phosphorylated
products from the previous inseparable diol mixture by flash
chromatography on silica gel column due to relative polarity dif-
ferences in the unreactive polar diol 7 and the less polar phos-
phorylated products 8 and 9 (Scheme 2). The 1H NMR analysis of
the crude mixture obtained after the phosphorylation reactions
revealed that the acetal 1H NMR peak of diol 6 at 5.60 ppm was
transformed to a 1H NMR peak at 5.52 ppm and 5.86 ppm in dif-
ferential proportions based on the reaction conditions, evident of
the formation of two new dioxanes. After purification and further
spectral analysis, the acetal 1H NMR peak at 5.52 ppm was assigned
to monophosphate 8 and the acetal 1H NMR peak at 5.86 ppm was
assigned to bisphosphate 9. Contrary to our expectations, the se-
lectivity of the primary hydroxyl group over the secondary hy-
droxyl group did not improve even at shorter reaction times (1.5 h)
in excess pyridine with either co-solvent.
However, when the co-solvent was changed to toluene, the
monophosphorylated product 8 was formed in 53% yield. Because
of the moderate yield and the longer reaction time needed for this
reaction, we explored new reaction conditions with CHCl3 since the
reaction was usually completed much faster in this solvent.
We varied the concentration of pyridine in the reaction mixture
to 3, 1 and 0.8 equiv in CHCl3 and kept the concentration of
dibenzylphosphorochloridate to 1 equiv. While it remained difficult
to control the monophosphorylation with 3 equiv of pyridine,
(3). This would allow for the simple laboratory condensation of
these reagents and additionally, avoiding the risk of the handling of
hazardous HCl gas. Initially, the addition of excess concentrated HCl
or liquid H2SO4 caused the formation of undesired side reactions.
We soon realized that catalytic amount of HCl (10 mol %), when
added to benzaldehyde before the addition of
D
-(þ)-arabitol,
afforded a benzylidene derivative as a white solid in good yield
(Scheme 1). The purification of the resulting solid mixture was
achieved by simply quenching the reaction mixture after 3 days
under argon atmosphere with dilute KOH solution (10%) followed
by filtration and rinsing of the residue with H2O to afford the
resulting triols 4 and 5 in 73% total yield (Scheme 1). Slightly lower
yield was obtained after a work-up procedure when the reaction
mixture was left to stand for less than a day. Triols 4 and 5 were
identified by comparison of NMR data determined from the mix-
ture with literature data.46 Compared to the NMR spectra data re-
ported for the pure product obtained by using HCl gas,46 we have
observed the triols 4 and 5 obtained as an equal mixture of in-
separable diastereomers when concentrated HCl (12 N) was added
(Scheme 1). In the 1H NMR of the diastereomeric mixture, the 1H
NMR peaks of the benzylidene acetal hydrogens of the two triols
were in equal isomeric ratio at 5.94 ppm and 5.60 ppm and the
chemical shift of the desired dioxane 1H NMR peak ((CD3)2CO) at
5.60 ppm was identical to the reported spectral data of triol 4.46 The
diastereomeric mixture upon treatment with NaIO4 afforded in-
termediate aldehyde products, which were immediately reduced
with NaBH4 to afford an inseparable diastereomeric mixture of
diols 6 and 7 in equal isomeric ratio, in a total of 65% yield (Scheme
1). After the NaBH4 reduction, the chemical shift of the dioxane 1H
NMR peak at 5.60 ppm of the benzylidene acetal hydrogen for diol 6
did not change from the chemical shift value observed for triol 4,
which was in total agreement with the literature data.46
greater selectivity for monophosphate
8 over the undesired
bisphosphate 9 was achieved with 1 and 0.8 equiv of pyridine, re-
spectively. The yield of the desired monophosphate 8 improved to
79% with 1 equiv of pyridine in 2.5 h but TLC analysis showed

S.I. Odejinmi et al. / Tetrahedron 68 (2012) 8937e8941
8939
O
R
OH
R
O
O
O
O
(BnO)2POCl
Dess-Martin Periodinane
51%
6 + 7
Pyr., CHCl3
79%
10
8
+
R
R
R = OPO(OBn)2
O
O
9
Scheme 2. Preparation of dioxanone through a selective monophosphorylation and DesseMartin periodinane oxidation.
a slight formation of bisphosphate 9. Lower yield of 59% of the
monophosphorylated product was obtained with 0.8 equiv of
pyridine.
In the methylation step, the addition of CH3MgBr to dioxanone 10
afforded diol 11 stereoselectively. When the reaction mixture was
set at 0.1 M, the occurrence of side reactions caused significant
reduction in yields. These side reactions were significantly avoided
when the reaction mixture was further diluted to 0.05 M. The 1H
and 13C NMR spectral data are identical to those previously re-
ported through different routes.46 Although dioxanones bearing
Through the continued variation of experimental conditions
with regards to the choice of solvents, temperature and time, we
successfully achieved a selective phosphorylation of diol 6. Al-
though longer reaction times increased the formation of the double
phosphorylated product 9, a major difficulty still lay in the con-
trolling of the amounts of the by-product 9 with bisphosphate
moiety even at shorter reaction times. However, the modification of
the reaction conditions using chloroform as solvent at 0 ꢀC for 2.5 h,
allowed the selective formation of the monophosphorylated
product 8 in 79% yield as a pure isomer based on unrecovered diol 7.
The application of DesseMartin Periodinane (DMP)50,52 reagent
afforded dioxanone 10 in moderate yield of 51% (Scheme 3).
We envisaged that since our dioxanone monophosphate is a 1,3-
dioxan-5-one derivative, its direct methylation would provide ac-
cess to the tertiary diol 11 as one isomer. When the solution of
dioxanone 10 was treated with Grignard reagent at ꢁ78 ꢀC, only
one desired isomer was predominantly isolated, which was similar
to the results obtained from the works of others (Scheme 3).46,49,50
bulky a-CH2OR side chains where R is trityl, TBDPS (tert-butyldi-
phenylsilyl)50 or TBDMS (tert-butyldimethylsilyl)46,49 groups have
been shown to have bias for axial nucleophilic attacks, the results of
the stereoselectivity on the methylation reactions with phosphate
group in the a-CH2OR side chaincorroborates the observation that
all R groups in the a-CH2OR side chain in 1,3-diaxan-5-ones have
the same preference for axial nucleophilic attacks.50
The hydrogenolysis of compound 11 was conducted in MeOH
under hydrogen atmosphere (balloon) with vigorous stirring for
9 h. After the completion of the hydrogenolysis, phosphoric acid 1
was isolated in quantitative yield. The 1H and 13C spectral data are
in agreement with previous reports42 and used for CDP-ME syn-
thesis without further purification. The final CDP-ME synthesis was
carried out by the experimental conditions developed by Eoh, H.
O
OH
OPO(OH)2
CH3MgBr
60%
O
O
H2, Pd(OH)2/C
100%
Ph
Ph
OH
O
O
OPO(OBn)2
OPO(OBn)2
OH
OH
10
11
1
O
P
O
HO
Ref. 50
HO
P
O
NH2
O
O
O
N
O
O
N
OH
NH4
NH4
HO
O
OH
CDP-ME
Scheme 3. Selective alkylation and global deprotection. 1H and 31P NMR spectra of CDP-ME are given in the Supplementary data.

8940
S.I. Odejinmi et al. / Tetrahedron 68 (2012) 8937e8941
et al. The 1H and 31P NMR spectral data of synthesized CDP-ME
were identical to those reported.22,45
diastereomeric mixture of alcohol 6 and 7 as a white solid
(7.0 g, 65%).
3. Conclusion
4.1.3. Dibenzyl (((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)methyl)
phosphate (8). Liquid (BnO)2POH (6.12 mL, 27.08 mmol) was dis-
solved in dry toluene (70 mL) followed by the addition of N-chlor-
osuccinimide (4.02 g, 30.08 mmol).53 The mixture was stirred for 2 h
and filtered. The residue was washed with toluene followed by the
concentration of the filtrate. The resulting concentrated oil was then
added to the solution of alcohol 6 and 7 (5.27 g, 25.07 mmol, isomer
ratio 1:1) in CHCl3 (50 mL) and brought to 0 ꢀC. Pyridine (2.03 mL,
25.07 mmol) was added dropwise and slowly. The mixture was
allowed to stir for 2.5 h and quenched with satd. NH4Cl. After sep-
aration, aqueous layer was extracted with EtOAc. The combined or-
ganic layer was washed with brine, dried and concentrated in vacuo.
The crude oil was purified by flash chromatography (2:1, 3:2 hex-
ane:EtOAc) to afford monophosphate 8 as a white solid (4.7 g, 79%)
based on 50% of unrecovered starting material. 1H NMR (400 MHz,
We have achieved a stereoselective synthesis of MEP in 7 steps
from
D
-(þ)-arabitol in 11.5% overall yield. Our initial synthetic
strategy embodies the application of catalytic HCl for the prepa-
ration of the protected benzylidene derivative needed as the key
starting material for the synthesis of phosphoric acid 1. The desired
diol in a diastereomeric diol mixture was selectively converted to
a monophosphate through controlled reaction conditions. 2-C-
methyl- -erythritol-4-phosphoric acid (1) was obtained after the
D
subsequent oxidation of the monophosphate with DesseMartin
Periodinane, selective alkylation of the resulting dioxanone with
CH3MgBr and hydrogenolysis.
4. Experimental section
4.1. General
CDCl3):
d
¼7.47e7.25 (m, 15H), 5.52 (s, 1H), 5.10 (m, 4H), 4.22e3.99
(m, 6H). (Presence of impurity peaks in the 1H NMR spectrum at
d
¼0.8. 1.2, 2.1and 3.5 ppm),; 13C NMR (100 MHz, CDCl3):
¼137.3,
d
General experimental procedures. Anhydrous tetrahydrofuran
(THF), toluene, and pyridine were obtained from commercial ven-
dors and used without further purification. Dichloromethane was
distilled freshly from CaH2. Methanol, hexane, and EtOAc were used
directly without further purification. All non-aqueous reactions
were done under an argon atmosphere, in oven-dried or flame-
dried glassware and with magnetic stirring. Flash chromatogra-
135.7, 135.6, 129.2e128.0 (aromatic C), 125.9, 101.3, 77.9 (d,
JCP¼6.9 Hz), 77.2, 72.3, 69.5 (d, JCP¼5.4 Hz), 66.2 (d, JCP¼5.4 Hz), 63.1;
31P NMR (D2O):
d¼0.43 (s); HRMS (ESI/FTMS) calcd for MþH of
C25H27O7P, 471.15; found, 471.16.
4.1.4. Bisphosphonate 9. Liquid (BnO)2POH (7.75 mL, 35.07 mmol)
was dissolved in dry toluene (150 mL) followed by the addition of
N-chlorosuccinimide (5.15 g, 38.57 mmol).53 The mixture was
stirred for 2 h and filtered. The residue was washed with toluene
(20 mL) followed by the concentration of the filtrate. The resulting
concentrated oil was then added to the solution of alcohol 6 and 7
(2.95 g, 14.03 mmol, isomer ratio 1:1) in CHCl3 (25 mL) and
brought to 0 ꢀC. Pyridine (3.40 mL, 42.08 mmol) was added
dropwise. The mixture was allowed to stir 24 h and quenched
with satd. NH4Cl. After separation, aqueous layer was extracted
with EtOAc. The combined organic layer was washed with brine,
dried, and concentrated in vacuo. The crude oil was purified
by flash chromatography (1:1 hexane:EtOAc; 2:2:1 hex-
ane:EtOAc:MeOH) to afford diphosphosphate 9 as a clear oil
phy was done on silica gel with an average 63e200 mm particle size.
The 1H, 13C, and 31P NMR spectra were recorded at 400 MHz with
CDCl3, DMSO, or D2O as solvents. High-resolution and electrospray
(ES) mass spectra were obtained at the University of Utah Mass
Spectrometry Facility.
4.1.1. (1R)-1-((4R,5R)-5-Hydroxy-2-phenyl-1,3-dioxan-4-yl)ethane-
1,2-diol (4)þ(1R)-1-((4S,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)
ethane-1,2-diol (5). The benzylidene protection was modified from
literature procedure.46 In a dry round-bottomed flask containing
benzaldehyde (8.5 mL, 84.45 mmol), HCl (0.59 mL (12 N), 7.1 mmol)
was added and stirred. This was followed by the immediate addi-
tion of
D
-(þ)-arabitol (10.71 g, 70.38 mmol) in spatula batches. The
(6.73 g, 66%). 1H NMR (400 MHz, CDCl3):
5.86 (s, 1H), 5.10e4.95 (m, 8H), 4.12e4.02 (m, 6H); 13C NMR
(100 MHz, CDCl3):
d
¼7.47e7.29 (m, 25H),
mixture was stirred briefly and left to stand for 3 days under argon.
The resulting white solid was scraped out of the flask and quenched
with dilute KOH (10%, 20 mL). The remaining solid particles were
further crushed in the KOH mixture, filtered, and washed with
distilled H2O. After drying, the mixture was obtained as a white
solid (12.38 g, 73%).
d
¼136.5, 135.5 (2C), 129.4, 128.5e127.8 (aro-
matic C), 126.5, 104.2, 76.2e66.2 (8C) (76.1, dd, JCP¼20.3 Hz,
8.4 Hz), (69.4, t, JCP¼5.4 Hz), (66.3, dd, JCP¼12.3 Hz, 5.4 Hz); 31P
NMR (D2O):
d
¼0.25 (s), 0.04 (s); HRMS (ESI/FTMS) calcd for MþH
of C39H40O10P2, 731.22; found, 731.22.
4.1.2. (4R,5R)-4-(Hydroxymethyl)-2-phenyl-1,3-dioxan-5-ol
(6)þ(4S,5R)-4-(hydroxymethyl)-2-phenyl-1,3-dioxan-5-ol (7). The
solid mixture of 4þ5 (12.38 g, 51.5 mmol, 1:1 isomer ratio) was
added to a flask containing CH2Cl2 (150 mL) and satd. NaHCO3
(50 mL) followed by the addition of NaIO4 (22 g, 103 mmol). The
mixture was stirred for 1.5 h and filtered. After thorough rinsing of
the residue with EtOAc, the filtrate was separated and the aqueous
layer was extracted with EtOAc. The combined organic layer was
washed with brine, dried (MgSO4), and concentrated in vacuo. The
crude oil was dissolved in MeOH (200 mL) and brought to 0 ꢀC.
NaBH4 (2.34 g, 61.80 mmol) was added in small portions slowly to
the cold solution of the crude compound in MeOH. The mixture
was then allowed to stir for 2 h and quenched with satd. NH4Cl.
The mixture was evaporated overnight. The resulting white crude
mixture was dissolved in distilled H2O and extracted with EtOAc.
The combined organic layer was washed with brine, dried
(MgSO4) and concentrated in vacuo and purified by flash
Chromatography (1:1 hexane:EtoAc, 3:7 acetone:CHCl3) to afford
4.1.5. Dibenzyl (((4R)-5-oxo-2-phenyl-1,3-dioxan-4-yl)methyl) phos-
phate (10). DesseMartin Periodinane reagent (2.36 g, 5.56 mmol)
was added to the solution of alcohol 8 (2.18 g, 4.64 mmol) in CH2Cl2
(50 mL) and stirred for 2 h. The mixture was diluted with ether
(100 mL) quenched with a mixture of satd. NaHCO3 (30 mL) and
Na2S2O3 (ca. 5 g) and stirred for about 5 min until the white mixture
was clear. After separation, aqueous layer was extracted with ether
and the combined organic layer was washed with brine, dried
(MgSO4) and concentrated in vacuo. The resulting crude mixture
was purified by flash chromatography (2:1, 1:1 hexane:EtoAc) to
afford dioxalone 10 as a clear oil (1.11 g, 51%).
1H NMR (400 MHz, CDCl3):
5.04 (d, J¼9.6 Hz, 2H), 5.00 (d, J¼7.2 Hz, 2H), 4.63 (m, 1H),
4.50e4.38 (m, 4H),; 13C NMR (100 MHz, CDCl3):
d¼7.53e7.25 (m, 15H), 5.94 (s, 1H),
d¼202.4, 136.4,
135.6, 135.4, 129.3, 128.5e127.8 (aromatic C), 126.1, 99.2, 81.1 (d,
JCP¼6.8 Hz), 72.2, 69.3 (d, JCP¼6.1 Hz), 65.5 (d, JCP¼5.4 Hz).; 31P NMR
(D2O):
469.14; found 469.14.
d
¼-0.19; HRMS (ESI/FTMS) calcd for MþH of C25H25O7P,

S.I. Odejinmi et al. / Tetrahedron 68 (2012) 8937e8941
8941
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(180 mg) was added to the solution of alcohol 11 (455 mg,
0.94 mmol) in MeOH (30 mL) under H2 gas (balloon) and stirred
vigorously for 9 h. The mixture was filtered followed by the rinsing
of the filter cake with MeOH (50 mL). The filtrate was concentrated
and dried in vacuo to afford compound 1 (203 mg, 100%). 1H and 31
P
NMR spectral data are the same as published.42
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