Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens

Article abstract of DOI:10.1021/jo501529k

The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts d-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it

Full text of DOI:10.1021/jo501529k

Article
pubs.acs.org/joc
Synthesis of Methylerythritol Phosphate Analogues and Their
Evaluation as Alternate Substrates for IspDF and IspE from
Agrobacterium tumefaciens
Sergiy G. Krasutsky,‡,§ Marek Urbansky,†,∥ Chad E. Davis,†,⊥ Christian Lherbet,‡,# Robert M. Coates,*,†
and C. Dale Poulter*,‡
‡Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States
†Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: The methylerythritol phosphate biosynthetic
pathway, found in most Bacteria, some parasitic protists, and
plant chloroplasts, converts D-glyceraldehyde phosphate and
pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP), where it intersects with the
mevalonate pathway found in some Bacteria, Archaea, and
Eukarya, including the cytosol of plants. D-3-Methylerythritol-
4-phosphate (MEP), the first pathway-specific intermediate in
the pathway, is converted to IPP and DMAPP by the
consecutive action of the IspD-H proteins. We synthesized five
D-MEP analoguesD-erythritol-4-phosphate (EP), D-3-meth-
ylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate
(EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)and studied
their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from
Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues,
except MTP, and their products were substrates for the three consecutive enzymes.
cyclomethylerythrityl diphosphate (cMEDP) synthase (IspF).
Subsequently, the cyclic diphosphate is opened reductively by
hydroxydimethylallyl diphosphate (HDMAPP) synthase
(IspG), and the hydroxyl group is removed by a second
reduction by IspH to give a mixture of IPP and DMAPP.3 The
steps between MEP and IPP/DMAPP and the genes encoding
the biosynthetic enzymes are shown in Scheme 1.
The ispD, ispE, and ispF genes encode monofunctional
enzymes in most MEP-dependent organisms. However, there
are several reports of fused ispD and ispF that encode
bifunctional IspDF proteins in bacteria, including several
genera of α-proteobacteria belonging to the order Rhizobiales.4
The IspDF protein catalyzes the first and last steps in the
conversion of MEP to cMEDP, and the missing ispE activity
can be provided in vivo with an enzyme from the same or
different organisms. Time course studies with IspDF and IspE
from Agrobacterium tumefacians indicate that the enzymes in
combination efficiently convert MEP to cMEDP without any
evidence for substrate channeling between the individual active
sites in the protein complex.5
INTRODUCTION
■
The isoprenoid biosynthetic pathway produces over 60 000
small-molecule metabolites that perform numerous essential
functions in all forms of life.1 A few examples include electron
transfer (ubiquinones), cellular membranes (sterols and
hopanes), hormones (sterols, sesquiterpenes, diterpenes),
pheromones (monoterpenes), photosynthesis (carotenoids
and chlorophylls), and signal transduction (isoprenylated
proteins). Isoprenoid molecules are synthesized from (R)-
mevalonate (MVA)2 or D-methylerythritol phosphate (MEP)3
by two fundamentally different pathways. The MVA pathway
provides isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP) for isoprenoid biosynthesis in most
Eukarya, including all mammals and the cytosol and
mitochondria in plants, Archaea, and a few Bacteria. IPP and
DMAPP are synthesized from MEP in most Bacteria and
Apicomplexa, a group of parasitic protists.
MEP is synthesized in two steps from pyruvate and D-
glyceraldehyde phosphate via D-deoxyxylulose phosphate
catalyzed by deoxyxylulose phosphate synthase (DXS) and
deoxyxylulose keto-reductase (DXR or IspC), respectively.3
MEP is converted to cyclic methylerythritol diphosphate
(cMEDP) in three steps catalyzed by diphosphocytidylmethy-
lerythritol (CDP-ME) synthase (IspD), diphosphocytidylme-
thylerythrityl phosphate (CDP-MEP) synthase (IspE), and
Since the MEP pathway is orthogonal to the MVA pathway,
it is an attractive target for development of small-molecule
Received: July 10, 2014
Published: September 3, 2014
© 2014 American Chemical Society
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Scheme 1. Biosynthesis of IPP and DMAPP from MEP
Figure 1. MEP analogues.
Scheme 2. Synthesis of EP and EEP
inhibitors as antibacterial, antiparasitic, and herbicidal agents.6
Most of the reports of inhibitors and alternate substrates are for
the DXS,7 DXR,8 IspD,9,10 IspG,11 and IspH9 proteins found in
the early and later parts of the pathway. In a recent report, high
throughput screening identified a triazolopyrimidine inhibitor
of IspD, and subsequent synthetic work provided structurally
related submicromolar inhibitors with herbicidal activity.12
Studies with DXR13 indicate that the enzyme accepts a
number of analogues as alternate substrates and synthesizes
products that are potentially inhibitors of downstream enzymes.
We now report the synthesis of five analogues of MEP and
showed that four of these molecules and their products are
alternate substrates for the three consecutive reactions catalyzed
by Agrobacterium tumefaciens IspDF and IspE.
RESULTS AND DISCUSSION
■
Synthesis of MEP Analogues. Five MEP analogues, D-
erythritol phosphate (EP), D-methylthreitol phosphate (MTP),
D-ethylerythritol phosphate (EEP), D-aminodeoxymethylery-
thritol phosphate (NMEP), and D-methylerythritol thiophos-
phate (MESP), were synthesized for this study (Figure 1). EP
and EEP were synthesized from 1,3-O-benzylidene-D-eryth-
rulose and 4-(t-butyldimethylsilyl) ether (1) by the dioxanone
approach outlined in Scheme 2.14 The five-step route to MTP
was described previously.14
was seen for the related addition of methylmagnesium
bromide.14 Desilylation with Bu4NF gave diol 6. The diol was
lithiated with n-butyllithium and phosphorylated regioselec-
tively at the primary hydroxyl group with freshly prepared
dibenzyl chlorophosphate. Catalytic hydrogenolysis of the
dibenzyl phosphate and benzylidene ring gave EEP.
NMEP was synthesized from (Z)-4-(tert-butyldimethylsila-
nyloxy)-3-methyl-but-2-en-1-ol (8)16 (Scheme 3). Our original
strategy involved phosphorylation of 8 using the benzyl
phosphite-iodine procedure,17 removal of the TBDMS group,
and introduction of the amino group by a Mitsunobu
displacement18 with phthalimide, followed by hydrolysis of
the imide. However, our attempts to hydrolyze the phthalimide
group prior to a Sharpless dihydroxylation19 failed, presumably
because of the reactivity of the phosphate triester. Alcohol 8
was then protected as a THP ether; the TBDMS protecting
group was removed; and nitrogen was introduced at C1 by a
Mitsunobu displacement.17 The phthalimide moiety was
The route to EP from dioxanone 1 is shown in Scheme 2.
Reduction of 1 with NaBH4 in methanol, followed by
desilylation with tetrabutylammonium fluoride, gave diol 3
with NMR spectral data in accord with the literature.15
Phosphorylation of the primary hydroxyl group with dibenzyl
chlorophosphate in pyridine and hydrogenolysis of the benzyl
and benzylidene protecting groups gave EP, whose NMR
parameters matched the reported values.10
EEP was prepared by a similar set of reactions. Treatment of
ketone 1 with ethylmagnesium bromide proceeded with highly
stereoselective axial-face addition to the carbonyl group.
Previously, an “axial”/“equatorial” stereoselectivity of 20:1
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the crude product, the orthoester was unstable on a silica
column and was isolated in 51% yield. Reduction of the methyl
ester with LiBH4 proceeded in excellent yield (NMR), but
again the resulting alcohol was unstable on silica and was
obtained in 61% yield after purification. To improve yields, we
omitted purification steps for reactions 2 and 3, and pure
tosylated orthoester (steps 2−4) was obtained in an overall
89% yield. The orthoester protecting group was removed in
two steps by a mild treatment with aq. HCl to give a formate
ester, followed by hydrolysis with ammonia in CH3OH to give
diol tosylate 22. Treatment of 22 with tBuOK and removal of
the silyl blocking group gave epoxide 24, which was opened
with inorganic thiophosphate to give MESP. An NMR analysis
of the C1 Mosher’s ester of diol 24 indicated a 17:1 ratio of
2S,3R and 2R,3S enantiomers.20
Scheme 3. Synthesis of NMEP
Evaluation of EP, MTP, and EEP as Alternate
Substrates for IspDF and IspE. EP, MTP, and EEP, along
with MEP as a control, were incubated with different
combinations of [α-32P]CTP, [γ-32P]ATP, IspDF, and IspE.
The labeling patterns expected from these experiments are
shown in Scheme 5. The reaction mixtures were analyzed by
TLC,4,5 and the results are shown in Figure 2.
Scheme 5. Predicted Pattern for Incorporation of 32P into
the Products from Incubations with IspDF and IspE
removed with hydrazine, and the resulting amine was protected
as a Boc amide. The THP blocking group was removed, and
alcohol 14 was phosphorylated using benzyl phosphite-
iodine.17 A Sharpless dihydroxylation19 gave 16 as a 12:1
mixture of 2R,3S and 2S,3R enantiomers, as judged by chiral
HPLC. NMEP was obtained after removing the benzyl and Boc
protecting groups.
The synthesis of MESP is outlined in Scheme 4. Ester 1716
was asymmetrically dihydroxylated as described for 15.19 Diol
18 was first protected as an orthoester. Although the reaction
proceeded in high yield as judged from an NMR spectrum of
Scheme 4. Synthesis of MESP
Incubation of IspDF with MEP and [α-32P]CTP gave CDP-
ME as the only radioactive product, as expected for catalysis by
the IspD active site in the IspDF fusion protein4 (see lane 3). A
similar incubation with IspDF and IspE with MEP, [α-32P]
CTP, and [γ-32P]ATP gave two 32P-labeled products, cMEDP
(radiolabel from [γ-32P]ATP) and CMP (radiolabel from
[α-32P]CTP) (see lane 4).4 A spot with a smaller Rf in the
region expected for CDP-MEP was not seen.
Related incubations with EP indicated that IspDF gave CDP-
E (lane 5) and IspDF/IspE gave cEDP and CMP (lane 6),
although the reactions appeared to be slower and a spot was
seen for CDP-E. The TLC profiles for EEP suggest that CDP-
EE and CMP have similar Rf values. Incubation of IspDF with
EEP and [α-32P]CTP (lane 9) gives a single spot as expected
for formation of labeled CDP-EE. While a similar incubation
with IspDF/IspE with EEP, [α-32P]CTP, and [γ-32P]ATP gives
two spots, as expected, however the spot assigned to cEEDP
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and cMEDP (lane 8). Similar experiments with EEP, visualized
in lanes 3 and 1, respectively, show similar patterns, except that
the putative spot for CDP-EE has the same Rf value as CMP.
Overlapping Rf’s for CDP-EE and CMP were established by
the experiments visualized in lanes 2, 4, and 5. In lane 2, EEP
was incubated with IspDF, [α-32P]CTP, and [γ-32P]ATP to
give [32P]CDP-EE, with the same Rf for [32P]CMP from
incubation of EEP with IspDF, IspE, [α-32P]CTP, and ATP
seen in lane 4. The spot for [32P]CMP is absent when EEP is
incubated with IspDF, IspE, unlabeled CTP, and [γ-32P]ATP
(lane 5), establishing that CMP and CDP-EE comigrate. Thus,
our TLC studies indicate that the alternate substrates EP and
EEP behave similar to MEP5 for the three consecutive
reactions catalyzed by IspDF and IspE. No clear evidence
was seen for the mandatory CDP-EP and CDP-EEP, although
spots indicative of the intermediates were seen at the leading
edge for the spot for ATP in time course experiments (also see
Figures S1 and S2, Supporting Information). Difficulty in
detecting CDP-EP and CDP-EEP is not surprising. CDP-MEP
migrates at a poorly defined spot at the leading edge of that for
ATP and only constitutes a maximum of ∼10% of the total
radioactivity during time course measurements.
Negative-ion LC−MS analyses of incubation mixtures similar
to those described in Figures 2 and 3 were performed using
single ion monitoring, as previously described for MEP.4,5 They
support the assignments for the products from incubations with
EP or EEP. The reaction mixture from incubation of EP with
CTP and IspDF gave a peak at m/z 506 and expected for CDP-
E (Figure 4A). A similar analysis of the incubation of EP with
CTP, ATP, IspDF, and IspE gave peaks m/z 263 for cEDP
(Figure 4B) and m/z 322 for CMP (data not shown). Related
incubations with EEP gave peaks at m/z 534 for CDP-EE
(Figure 4C), m/z 291 for cEEDP (part D), and m/z 322 for
CMP (data not shown).
Figure 2. TLC plate showing the different products following 1 h
incubations with MEP, EP, MTP, or EEP (500 μM) with IspDF (4.8
μM) and [α-32P]CTP (150 μM, 40 μCi/μmol) or IspDF, IspE (6.2
μM), [γ-32P]ATP (150 μM, 320 μCi/μmol), and [α-32P]CTP (150
μM, 40 μCi/μmol). The reactions were quenched with methanol.
Lane 1: MEP, CTP (control); lane 2: MEP, ATP (control); lane 3:
MEP, IspDF, CTP; lane 4: MEP, IspDF, IspE, ATP, CTP; lane 5: EP,
IspDF, CTP; lane 6: EP, IspDF, IspE, ATP, CTP; lane 7: MTP,
IspDF, CTP; lane 8: MTP, IspDF, IspE, ATP, CTP; lane 9: EEP,
IspDF, CTP; lane 10: EEP, IspDF, IspE, ATP, CTP.
has the same Rf value as CMP (lane 10). No spots were seen
with expected Rf values for CDP-EP or CDP-EEP.
The formation of CDP-EE was established by a series of
incubations with EEP and different combinations of labeled
and unlabeled ATP and CTP (Figure 3). To provide a basis for
comparison, we incubated MEP with IspDF, IspE, [α-32P]CTP,
and [γ-32P]ATP, which resulted in well-resolved spots for CMP
and cMEDP (lane 7), while incubation with IspDF and [α-32P]
CTP gave CDP-ME with an Rf distinctive from those of CMP
In contrast to EP and EEP, MTP was not a substrate for
IspD (Figure 2). In addition, MTP at concentrations up to 5
mM did not inhibit turnover of MEP. Thus, it appears that the
threitol diastereomer is not recognized by the active site of
IspD.
Evaluation of NMEP and MESP as Alternate Sub-
strates for IspDF and IspE. MEP and NMEP were incubated
with IspDF, IspE, [α-32P]CTP, and [γ-32P]ATP under
conditions similar to those shown in Figures 2 and 3. TLC
analysis of the products from MEP5 showed the expected
pattern of spots (Figure S3, Supporting Information, lanes 1
and 2). TLC analysis of a similar experiment with NMEP
showed the formation of new radioactive materials with smaller
Rf’s than seen for the corresponding MEP derivatives that were
not resolved into individual peaks (Figure S3, Supporting
Information, lane 4). Related experiments with MEP where
IspDF was replaced with H281S, a mutant which does not have
IspF activity, gave spots for CDP-ME and CDP-MEP but not
cMEDP, as expected (Figure S3, Supporting Information, lane
3). A related incubation of NMEP with [H281S]IspDF and
IspE (Figure S3, Supporting Information, lane 6) and with
[H281S]IspDF (Figure S3, Supporting Information, lane 5)
again gave unresolved spots at lower Rf’s. The product mixtures
were then analyzed by negative ion LC−MS. Selective ion
monitoring at m/z 519 (CDP-NME), m/z 599 (CDP-
NMEDP), and m/z 276 (cNMEDP) gave major peaks at
9.02 min (CDP-NME), 37.06 min (CDP-NMEDP), and 9.37
min (cNMEDP) (Figures S4 and S5, Supporting Information).
Figure 3. TLC plate showing the different products following 1 h
incubations of different combinations of MEP and EEP (500 μM),
IspDF (4.8 μM), IspE (6.2 μM), unlabeled ATP, [γ-32P]ATP (150
μM, 320 μCi/μmol), unlabeled CTP, and [α-32P]CTP (150 μM, 40
μCi/μmol). The reactions were quenched with methanol. Lane 1:
EEP, [α-32P]CTP, IspDF; lane 2: EEP, [α-32P]CTP, [γ-32P]ATP,
IspDF; lane 3: EEP, [α-32P]CTP, [γ-32P]ATP, IspDF, IspE; lane 4:
EEP, [α-32P]CTP, ATP, IspDF, IspE; lane 5: EEP, CTP, [γ-32P]ATP,
IspDF, IspE; lane 6: EEP, [γ-32P]ATP, IspDF; lane 7: MEP, [α-32P]
CTP, [γ-32P]ATP, IspDF, IspE; lane 8: MEP, [α-32P]CTP, IspDF;
lane 9: EEP, [γ-32P]ATP; lane 10: EEP, [α-32P]CTP.
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established by incubating MESP with [32P]CTP, [32P]ATP,
and [H281S]IspDF, which only catalyzes the conversion of
MESP to CDP-MES (lane 2), while those for cMESDP and
CMP were established by comparing an incubation with
[α-32P]CMP and [γ-32P]ATP (lane 1) with an incubation with
[α-32P]CMP and unlabeled ATP (lane 3). LC−MS analyses
gave negative ion electrospray spectra for CDP-MES (m/z at
536), CDP-MESP (m/z at 616), and cMESDP (m/z 293).
Conclusions. Five MEP analogues were synthesized in this
study. Four of these and their products were substrates for the
three consecutive reactions catalyzed by IspDF and IspE. EP,
EEP, NMEP, and MESP have topologies that are modestly
different from that of MEP. In EP and EEP, a hydrogen atom
and an ethyl group, respectively, replace the methyl group at C3
in MEP, and both analogues are converted to the
corresponding cyclic diphosphates by ispDF and ispE. From
a topological perspective, cEDP and its subsequent metabolites
are likely alternate substrates for IspG and IspH. However, the
resulting nor-analogues of IPP and DMAPP would be
unreactive competitive inhibitors of IPP isomerase and farnesyl
diphosphate (FPP) synthase, the next two enzymes in the
isoprenoid pathway.21,22 In contrast, EEP would give ethyl
analogues of IPP and DMAPP, which in turn are substrates for
IPP isomerase and farnesyl diphosphate (FPP) synthase.22
Related reactions are used to construct the carbon skeletons of
insect juvenile hormones.23 MTP, the threo diastereomer of
MEP, is not a substrate for ispD and does not inhibit the
enzyme. Conversion of cNMEDP to the amino analogue of
HDMAPP by IspG would produce a potent nanomolar
inhibitor of IspH.24 cMESDP contains a highly conservative
replacement of oxygen by sulfur that should not substantially
impede its conversion to the thiolo analogue of DMAPP, at
which point it becomes a low micromolar inhibitor of FPP
synthase.25 Thus, the ability of IspDF and IspE to process
analogues of MEP presents an opportunity for in vivo synthesis
of inhibitors of downstream enzymes and new metabolites.
Figure 4. LC−MS chromatograms of products detected by single-ion
monitoring of products from the following incubations: (A) EP, CTP,
IspDF; (B) EP, CTP, ATP, IspDF, IspE; (C) same as A with EEP as a
substrate; (D) same as B with EEP as a substrate.
EXPERIMENTAL SECTION
Mass analyzers used for HRMS were TOF (pure samples) or Quad/
TOF (HPLC/MS).
■
Similar experiments were performed for MESP. In this case,
TLC analysis of the products from an incubation with IspDF,
IspE, [α-32P]CTP, and [γ-32P]ATP gave spots for cMESDP
and CMP (Figure 5, lane 1). The Rf of CDP-MES was
Synthesis of Alternate Substrates. 1,3-Benzylidene-D-eryth-
ritol, 4-(t-Butyldimethylsilyl) Ether (2). A solution of dioxanone 1
(2.17 g, 6.73 mmol)14 in MeOH (22 mL) was stirred and cooled at 0
°C as NaBH4 (305 mg, 8.08 mmol) was added portionwise over 10
min. The cooling bath was removed, and the reaction mixture was
allowed to stir 1 h at rt. The reaction was neutralized by the addition of
satd. NH4Cl (10 mL) and H2O (10 mL). The aqueous reaction
mixture was extracted with CH2Cl2 (2 × 100 mL). The organic
extracts were combined, dried (MgSO4), and evaporated to give 2.03 g
of crude oil that was an 8:1 mixture (1H NMR) of monosilyl ether 2
and starting material. Column purification (EtOAc:hexane 21:79) gave
two fractions. After solvent was removed, the first contained 0.58 g of
colorless oil that was a mixture of alcohol 2 (∼40%) and dioxanone 1
(∼40%), based on 1H NMR analysis, and the second contained 1.18 g
of 2 (60%, Corr. for Rec. SM) as a colorless oil: TLC Rf = 0.68 (50:50,
EtOAc:hexane); [α]D = 32.5 (c = 1.0 in MeOH); 1H NMR (500 MHz,
C6D6) δ −0.01 (s, 6H), 0.89 (s, 9H), 2.45 (d, 1H, J = 2.8 Hz, OH,
Exch. D2O), 3.46 (t, 1H, J = 10.5 Hz), 3.53 (ddd, 1H, J = 9.0, 6.2, 5.1
Hz), 3.77−3.82 (m, 2H), 3.89 (dd, 1H, J = 10.5, 4.8 Hz), 4.22 (dd,
1H, J = 10.7, 5.4 Hz), 5.31 (s, 1H), 7.10 (t, 1H, J = 7.4 Hz), 7.18 (t,
2H, J = 7.6 Hz), 7.61 (d, 2H, J = 7.5 Hz); 13C NMR (126 MHz, C6D6)
δ −5.5, 18.3, 25.9, 65.3, 65.5, 71.0, 80.6, 101.2, 126.7, 128.9, 138.7; IR
3456 (OH), 2930, 2857, 1463, 1254, 1089, 837. No physical data were
reported for this known compound.26 However, the physical data were
similar to those reported by Fukumoto27 for the enantiomer (1H
NMR, CDCl3).
Figure 5. TLC plate showing the different products formed following
a 1 h incubation of MESP (500 μM), IspDF (4.8 μM), or
[H281S]IspDF, IspE (6.2 μM), unlabeled ATP or [γ-32P]ATP (150
μM, 320 μCi/μmol), and [α-32P]CTP (150 μM, 40 μCi/μmol). The
reactions were quenched with methanol. Lane 1: MESP, [32P]CTP,
[32P]ATP, IspDF, IspE; lane 2: MESP, [32P]CTP, [32P]ATP,
[H281S]IspDF; lane 3: MESP, [32P]CTP, ATP, IspDF, IspE.
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1,3-Benzylidene-D-erythritol (3). A solution of silyl ether 2 (0.97 g,
2.99 mmol) in THF (5 mL) was stirred and cooled at 0 °C as 1.0 M
Bu4NF (3.3 mL, 3.29 mmol) was added dropwise over 2 min. The
cooling bath was removed, and the reaction mixture was allowed to stir
at room temperature for 15 min. The reaction solution was diluted
with H2O (5 mL) and Et2O (70 mL). Following extraction of the
aqueous layer with Et2O (70 mL), the ethereal layers were combined,
dried (MgSO4), and evaporated to give 1.67 g of an oil. Column
purification (EtOAc:hexane 70:30) gave 0.50 g (79%) of a white solid:
TLC Rf = 0.47 (EtOAc); 1H NMR (500 MHz, C5H5N) δ 3.95 (t, 1H,
J = 10.4 Hz), 4.15 (ddd, 1H, J = 9.4, 5.4, 1.7 Hz), 4.33−4.39 (m, 2H),
4.49 (d, 1H, J = 11.8 Hz), 4.59 (dd, 1H, J = 10.7, 5.4 Hz), 5.82 (s, 1H),
6.69 (br s, 1H, OH, Exch. D2O), 7.22 (d, 1H, J = 5.8 Hz), 7.36 (m,
2H), 7.74 (m, 2H); 13C NMR (126 MHz, C5H5N) δ 61.9, 62.3, 72.3,
84.9, 101.5, 127.2, 128.4, 129.0, 139.5. The NMR data agree with
those reported by Pinto in the literature (1H NMR CD3OD).15
1,3-O-Benzylidene-D-erythritol 4-Dibenzylphosphate (4). The
phosphorylation was carried out as described by MacDonald et al.28
A solution of dibenzyl chlorophosphate was prepared by stirring a
mixture of N-chlorosuccinimide (76 mg, 0.57 mmol) and dibenzyl
phosphite (150 mg, 0.57 mmol) in benzene (2 mL) at room
temperature for 1 h. The precipitate was separated by centrifugation
and the supernatant added to a solution of 1,3-O-benzylidene-D-
erythritol 3 (100 mg, 0.48 mmol) in dry pyridine (4 mL) at 0 °C. The
resultant solution was stirred for 24 h at 0 °C and 24 h at room
temperature. Another portion of freshly prepared dibenzyl chlor-
ophosphate (0.57 mmol) in benzene (1 mL) was added, and stirring
was continued for 48 h. The reaction was quenched with ice (∼1 g),
and volatiles were removed under reduced pressure. The residue was
diluted with water (30 mL) and extracted with DCM (3 × 30 mL).
The combined organic layers were dried over Na2SO4, and the solvent
was evaporated. Purification of the residue by chromatography (silica
gel benzene/ethyl acetate 1:1) yielded 26 mg of a 1:1 mixture of 4 (18
mg, ca. 8%) and starting material (8 mg) and 27.4 mg (12%) of 4 as a
colorless oil: TLC Rf 0.24 (hexane/ethyl acetate 4:6), 0.26 (benzene/
2H); 13C NMR (126 MHz, C6D6) δ −5.8, −5.7, 18.1, 24.0, 25.8, 63.0,
68.1, 72.2, 82.2, 102.0, 126.7, 128.5, 128.9, 138.8.
1,3-O-Benzylidene-2-C-ethyl-D-erythritol (6). A solution of silyl
ether 5 (780 mg, 2.21 mmol) in anhydrous THF (8 mL) was stirred at
0 °C as Bu4NF (2.7 mL, 1 M in THF) was added. After 15 min at
room temperature, H2O (5 mL) was added; the product was extracted
with ether (80 mL); and the ethereal extracts was dried (MgSO4) and
evaporated. Purification by flash chromatography (silica gel, hexane/
ethyl acetate 4:6) afforded 486 mg (92%) of a viscous oil: TLC Rf 0.24
(hexane/ethyl acetate 4:6); 1H NMR (500 MHz, C6D6) δ 0.92 (t, 3H,
J = 7.5 Hz), 1.35 (app sextet, 1H, J = 7.5 Hz), 2.09 (app sextet, 1H, J =
7.5 Hz), 3.25 (d, 1H, J = 11 Hz), 3.58 (dd, 1 H, J = 6.5, 10.4 Hz), 3.67
(t, 1H, J = 6.5 Hz), 3.73 (dd, 1H, J = 5.9, 10.4 Hz), 4.08 (d, 1H, J = 11
Hz), 5.34 (s, 1H), 7.10−7.19 (m, 13H), 7.54 (m, 2H); 13C NMR (126
MHz, C6D6) δ 23.8, 61.4, 67.7, 72.3, 83.9, 102.1, 126.8, 138.7.
1,3-O-Benzylidene-2-C-ethyl-D-erythritol 4-Dibenzylphosphate
(7). A solution of diol 6 (245 mg, 1.03 mmol) in THF (7 mL) was
stirred and cooled at −78 °C as an aliquot of 1.6 M BuLi in hexane
(0.96 mL, 1.54 mmol) was added dropwise. After 5 min, a solution of
dibenzyl chlorophosphate (1.54 mmol; freshly prepared from dibenzyl
phosphite) in benzene (5 mL) prepared as described for 4 was added.
Stirring was continued for 10 min at −78 °C and 15 min at room
temp. Ether (60 mL) and water (2 mL) were added, and the ethereal
layer was dried (MgSO4) and evaporated to give 520 mg of crude
product. Purification by flash chromatography (silica gel, hexane/ethyl
acetate 4:6) afforded 450 mg (88%) of an oil (14): TLC Rf 0.3
(hexane/ethyl acetate 4:6); 1H NMR (500 MHz, C6D6) δ 0.99 (t, 3H,
J = 7.5 Hz), 1.37 (app sextet, 1H, J = 7.5 Hz), 2.14 (app sextet, 1H, J =
7.5 Hz), 3.55 (d, 1H, J = 11 Hz), 4.16 (s, 1 H), 4.26 (d, 1H, J = 11
Hz), 4.25−4.33 (m, 1H), 4.89 (d, 1H, J = 8.2 Hz), 4.86−4.90 (m, 4H),
5.48 (s, 1H), 6.99−7.18 (m, 13H), 7.57 (m, 2H); 13C NMR (126
MHz, C6D6) δ 23.6, 67.0, 69.6, 69.6, 72.5, 84.9, 102.0, 126.9, 128.5,
128.7, 128.9, 136.20, 138.7; 31P NMR (202 MHz, C6D6) δ 0.21.
2-C-Ethyl-D-erythritol 4-Phosphate, Ammonium Salt (EEP).
Compound 7 (410 mg, 0.82 mmol) was hydrogenated (1 atm, −10
°C) using 10% Pd/C (82 mg) in MeOH (10 mL) for 30 min. The
mixture was filtered and solvent evaporated to give 270 mg of an oil.
The oil was treated with 20% Pd(OH)2/C (130 mg) in MeOH (7
mL) at 1 atm of H2 for 24 h. The catalyst was removed by filtration;
solvent was evaporated; and the residue was purified by chromatog-
raphy (silica gel, acetonitrile/isopropyl alcohol/10% aq. NH4OH
1:1:1). Finally, lyophilization of the eluate afforded 205 mg (94%) of
an amorphous solid: TLC Rf 0.24 (acetonitrile/isopropyl alcohol/10%
aq. NH4OH 1:1:1); 1H NMR (500 MHz, D2O) δ 0.88 (t, 3H, J = 7.6
Hz), 1.61 (AB part of ABX, 2H), 3.59 and 3.60 (ABq, 2H, J = 11.9
Hz), 3.81−3.85 (m, 2H), 3.93−3.98 (m, 1H); 13C NMR (126 MHz,
D2O) δ 27.8, 65.9, 67.4, 76.1, 78.8; 31P NMR (202 MHz, D2O) δ 4.94.
(Z)-tert-Butyl-dimethyl-[2-methyl-4-(tetrahydro-pyran-2-yloxy)-
but-2-enyloxy]silane (9). 3,4-Dihydro-2H-pyran (126 mg, 1.5 mmol)
and PPTS (25 mg, 0.1 mmol) were added to a solution of alcohol 816
(217 mg, 1 mmol) in CH2Cl2 at rt. After stirring overnight, the
solution was washed with saturated NaHCO3 (10 mL) and extracted
with CH2Cl2 (3 × 10 mL). The combined organic extracts were dried
over Na2SO4. The solvent was removed at reduced pressure, and the
residue was chromatographed on silica gel with gradient elution by
hexanes/ethyl acetate (0% to 10% ethyl acetate) to give 295 mg (98%)
of a colorless oil: Rf 0.87 (hexanes:ethyl acetate 1:1); 1H NMR
(CDCl3, 300 MHz) 0.07 (6H, s), 0.90 (9H, s), 1.49−1.85 (9H, m),
3.47−3.54 (1H, m), 3.83−3.91 (1H, m), 3.99−4.06 (1H, s), 4.18 (2H,
s), 4.21−4.28 (1H, m), 4.61 (1H, t, J = 2.9 Hz), 5.42 (1H, m); 13C
NMR (CDCl3, 75 MHz) −5.3, 18.3, 19.5, 21.0, 25.5, 25.9, 30.6, 61.8,
62.2, 62.9, 97.9, 122.4, 139.7; HRMS (MALDI) calcd for
C16H32O3SiNa [M + Na+] 323.2013, found 323.2001.
1
ethyl acetate 1:1); H NMR (500 MHz, C6D6) δ 3.6 (t, 2H, J = 10.5
Hz), 4.01 (br, 1H), 4.30 (t, 1H, J = 10.5 Hz), 4.35 (dd, 1H, J = 5.4,
10.5 Hz), 4.47 (m, 1H), 4.74 (br s, 1H), 4.81−4.93 (m, 4H), 5.31 (s,
1H), 6.99−7.18 (m, 13H), 7.59 (m, 2H); 13C NMR (126 MHz, C6D6)
δ 29.2, 61.4, 67.1, 69.8, 71.3, 81.6, 101.4, 126.8, 136.1, 138.6; 31P NMR
(202 MHz, C6D6) δ 1.35.
D-Erythritol 4-Phosphate, Ammonium Salt (EP). Deprotection of
the dibenzyl phosphate (4, 24 mg, 0.05 mmol) was accomplished by
hydrogenation using 20% Pd(OH)2/C (12 mg) in MeOH (3 mL)
with magnetic stirring at 1 atm of H2 for 24 h. The catalyst was filtered
off, and the solvent was evaporated. The residue was dissolved in water
(3 mL) followed by dropwise addition of 10% NH4OH (1 mL).
Lyophilization gave 12 mg (100%) of a flocculent amorphous solid:
TLC Rf 0.2 (acetonitrile/isopropyl alcohol/10% aq. NH4OH 1:1:1);
1H NMR (500 MHz, D2O) δ 3.62 (dd, 1H, J = 6.9, 11.4 Hz), 3.67 (br,
1H), 3.74 (br t, 1H, J = 6.9 Hz), 3.81 (d, 1H, J = 6.2), 3.90 (br, 2H);
13C NMR (126 MHz, D2O) δ 32.3, 65.5, 67.5, 74.1; 31P NMR (202
MHz, D2O) δ 5.36. The data agree with those reported by Lillo et al.10
1,3-O-Benzylidene-2-C-ethyl-D-erythritol 4-(t-butyldimethylsilyl)
Ether (5). A solution of 1,3-O-benzylidene-D-erythrulose and 4-(t-
butyldimethylsilyl) ether (1, 900 mg, 2.79 mmol)14 in dry ether (20
mL) was stirred and cooled at −78 °C as EtMgBr (1.4 mL, 4.2 mmol,
3 M in Et2O) was added dropwise. After 45 min, the reaction was
quenched with MeOH (5 mL) and allowed to warm to room
temperature. Satd. aq. NH4Cl (15 mL) and water (15 mL) were
added, and the product was extracted with ether (3 × 30 mL). The
combined organic layers were washed with satd. NaHCO3, dried
(MgSO4), and evaporated to give 1.1 g of crude material. Purification
by flash chromatography (silica gel, hexane/ethyl acetate 95:5)
afforded 810 mg (82%) of a viscous colorless oil: TLC Rf 0.42
(Z)-2-Methyl-4-(tetrahydro-pyran-2-yloxy)-but-2-en-1-ol (10). A
solution of 1 M TBAF in THF (4.36 mL, 4.36 mmol) was added
dropwise to a solution of 9 (437 mg, 1.45 mmol) in THF (15 mL) at 0
°C. The temperature was allowed to rise to rt. After stirring for 3 h, the
reaction was quenched with brine (15 mL). The layers were separated,
and the aqueous layer was extracted with ether (3 × 10 mL). The
combined organic extracts were dried over Na2SO4. The solvent was
1
(hexane/ethyl acetate 8:2); H NMR (500 MHz, C6D6) δ −0.044 (s,
3H), −0.036 (s, 3H), 0.86 (s, 9H), 1.10 (t, 3H, J = 7.5 Hz), 1.60 (app
sextet, 1H, J = 7.5 Hz), 2.24 (app sextet, 1H, J = 7.5 Hz), 2.86 (s, 1H),
3.46 (d, 1H, J = 11 Hz), 3.78 (dd, 1H, J = 7.8, 4.0 Hz), 3.87 (m, 2H),
4.26 (d, 1H, J = 11 Hz), 5.40 (s, 1H), 7.11−7.20 (m, 3H), 7.60 (m,
9175
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removed at reduced pressure, and the residue was chromatographed
on silica gel with gradient elution by hexanes/ethyl acetate (0% to 30%
ethyl acetate) to give 315 mg (94%) of a colorless oil: Rf 0.25
156.2; HRMS (MALDI) calcd for C10H19NO3Na [M + Na+]
224.1263, found 224.1268.
(Z)-[4-(Bisbenzyloxyphosphoryloxy)-2-methylbut-2-enyl]-
carbamic Acid tert-Butyl Ester (15). A solution of I2 (609 mg, 2.4
mmol) in CH2Cl2 (30 mL) was added to a solution of P(OBn)3 in
CH2Cl2 (20 mL) at −40 °C. The mixture was stirred for 15 min, and
the cooling bath was removed. After 30 min the solution became
colorless and was cannulated to a mixture of alcohol 14 (200 mg, 1.2
mmol) and pyridine (570 mg, 7.2 mmol) in CH2Cl2 (30 mL) at −40
°C over 30 min. After the addition was complete, the mixture was
stirred for 1 h at rt, and the solvents were removed at reduced
pressure. The residue was dissolved in ether (50 mL) and washed with
0.3 M KHSO4 (30 mL), satd. NaHCO3 (30 mL), and brine (30 mL).
The combined organic extracts were dried over Na2SO4, and solvent
was removed at reduced pressure. The residue was chromatographed
on silica gel with gradient elution by hexanes/ethyl acetate (0% to 30%
ethyl acetate) to give 320 mg (70%) of a colorless oil: Rf 0.48
1
(hexanes:ethyl acetate 1:1); H NMR (CDCl3, 300 MHz) 1.52−1.86
(9H, m), 2.59 (1H, broad s), 3.50−3.57 (1H, m), 3.82−3.90 (1H, m),
4.00−4.27 (4H, s), 4.70 (1H, t, J = 2.9 Hz), 5.51 (1H, t, J = 7.2 Hz);
13C NMR (CDCl3, 75 MHz) 19.1, 21.9, 25.3, 30.4, 61.4, 62.0, 62.4,
97.0, 123.3, 141.0; HRMS (MALDI) calcd for C10H18O3Na [M + Na+]
209.1148, found 209.1158.
(Z)-2-[2-Methyl-4-(tetrahydropyran-2-yloxy)but-2-enyl]isoindole-
1,3-dione (11). A solution of DIAD (223 mg, 1.1 mmol) in THF (1
mL) was added dropwise to a solution of an alcohol 10 (187 mg, 1
mmol), phthalimide (162 mg, 1.1 mmol), and PPh3 (289 mg, 1.1
mmol), in THF (4 mL) at rt. After stirring overnight, the solvent was
removed at reduced pressure. The residue was chromatographed on
silica with gradient elution by hexanes/ethyl acetate (0% to 25% ethyl
acetate) to give 241 mg (76%) of a white solid: Rf 0.63 (hexanes:ethyl
1
1
(hexanes:ethyl acetate 1:1); H NMR (CDCl3, 300 MHz) 1.44 (9H,
acetate 1:1); mp = 77−78 °C; H NMR (CD2Cl2, 300 MHz) 1.48−
s), 1.74 (3H, t, J = 0.6 Hz), 3.71 (2H, d, J = 6.2 Hz), 4.54 (2H, dd, J1 =
7.7 Hz, J2 = 2.3 Hz), 4.94 (1H, broad s), 5.02 (2H, dd, J1 = 6.5 Hz, J2 =
1.7 Hz), 5.43 (1H, t, J = 7 Hz), 7.35 (10H, s); 13C NMR (CDCl3, 75
MHz) 21.8, 28.4, 40.4, 63.4, 63.4, 69.1, 69.2, 121.8, 121.9, 127.9, 128.0,
128.5, 128.6, 128.7, 135.8, 135.9, 140.5, 156.1; 31P NMR (CDCl3, 125
MHz) 0.68; HRMS (MALDI) calcd for C24H32NO6PNa [M + Na+]
484.1865, found 484.1872.
1.83 (9H, m), 3.47−3.54 (1H, m), 3.83−3.91 (1H, m), 4.21−4.28
(1H, m), 4.32 (2H, s), 4.38−4.45 (1H, m), 4.65−4.67 (1H, m), 5.53−
5.59 (1H, m), 7.70−7.76 (2H, m), 7.80−7.84 (2H, m); 13C NMR
(CD2Cl2, 75 MHz) 20.1, 21.9, 26.1, 31.3, 38.8, 62.7, 63.5, 98.6, 123.6,
126.7, 132.6, 134.3, 134.5, 168.7; HRMS (MALDI) calcd. for
C18H21NO4Na [M + Na+] 338.1363, found 338.1357.
(Z)-2-Methyl-4-(tetrahydropyran-2-yloxy) But-2-enylamine (12).
To a solution of phthalimide 11 (318 mg, 1 mmol) in EtOH (9 mL)
was added hydrazine hydrate (150 mg, 3 mmol) in EtOH (1 mL). The
solution was warmed to 50 °C, stirred for 30 min, and heated at reflux
for 2 h. The solution was cooled and filtered. The filter cake was
washed with EtOH (3 × 10 mL), and solvent was removed at reduced
pressure. The residue was dissolved in ether (20 mL), and 1 M NaOH
(5 mL) was added to adjust the pH to 12. The aqueous layer was
saturated with NaCl and extracted with ether. The combined organic
extracts were dried over Na2SO4. The solvent was removed at reduced
pressure to give 144 mg (77%) of a yellow liquid, which was used in
1-Deoxy-1-boc-amino-4-(dibenzyloxyphosphoryloxy)-3-methyl-
D-erythritol (16). A solution of AD mix β (3.14 g), NaHCO3 (565 mg,
6.73 mmol), and CH3SO2NH2 (160 mg, 1.68 mmol) in tBuOH:H2O
(1:1 v/v, 12 mL) was cooled to 0 °C, and alkene 15 (250 mg, 0.56
mmol) was added. The mixture was stirred overnight at 0 °C before
Na2SO3 (3.36 g) was added. The temperature was allowed to rise to rt
as the solution was stirred for 1 h. The layers were separated, and the
aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined
organic extracts were dried over Na2SO4, and solvent was removed at
reduced pressure. The residue was chromatographed on silica with
gradient elution by hexanes/ethyl acetate (0% to 30% ethyl acetate) to
give 172 mg (64%) of a colorless oil: Rf 0.25 (hexanes:ethyl acetate
1
the next step without further purification: H NMR (CD2Cl2, 300
20
1
MHz) 1.1.40−1.81 (11H, m), 3.23 (2H, s), 3.44−3.50 (1H, m), 3.78−
3.86 (1H, m), 3.95−4.02 (1H, m), 4.15−4.21 (1H, m), 4.57−4.60
(1H, m), 5.34−5.41 (1H, m); 13C NMR (CD2Cl2, 75 MHz) 20.1,
22.1, 26.1, 31.2, 42.9, 62.6, 63.1, 98.2, 122.6, 142.4.
1:1); [α]D + 11.7 (c 0.75, CHCl3); H NMR (CDCl3, 300 MHz)
0.04 (3H, s), 1.43 (9H, s), 2.96−3.05 (2H, m), 3.33−3.40 (1H, m),
3.68−3.72 (1H, m), 3.98−4.07 (1H, m), 4.33 (1H, t, J = 10.5 Hz),
4.69 (2H, s), 4.98−5.09 (4H, m), 5.36 (1H, broad s), 7.34 (10H, s);
13C NMR (CDCl3, 75 MHz) 19.7, 28.3, 48.4, 69.3, 69.4, 69.6, 69.7,
(Z)-[2-Methyl-4-(tetrahydropyran-2-yloxy) but-2-enyl] Carbamic
Acid tert-Butyl Ester (13). Amine 12 (600 mg, 3.24 mmol) was
dissolved in iPrOH/H2O (3:1, v/v, 140 mL), and solid Na2CO3 (3.24
g) was added. The solution was cooled to 0 °C. A solution of di-tert-
butyl dicarbonate (1.46 g, 6.48 mmol) in iPrOH/H2O (3:1, v/v, 20
mL) was added. The mixture was stirred overnight at rt. iPrOH was
removed at reduced pressure, and the aqueous layer was extracted with
ether. The combined organic extracts were dried over Na2SO4. The
solvent was removed at reduced pressure, and the residue was
chromatographed on silica with gradient elution by hexanes/ethyl
acetate (0% to 15% ethyl acetate) to give 640 mg (69%) of a colorless
73.3, 73.8, 80.4, 128.1, 128.1, 128.6 135.2, 135.6, 158.3; 31P NMR
(CDCl3, 125 MHz) 0.72; HRMS (MALDI) calcd for C24H34NO8PNa
[M + Na+] 518.1909, found 518.1909.
1-Amino-3-methyl-D-erythritol-4-phosphate, Ammonium Salt
(NMEP). A suspension of 16 (100 mg, 0.2 mmol) and Pd/C (8 mg)
in CH3OH (5 mL) was flushed with H2 and was allowed to stir for 1 h
under a balloon of H2. The mixture was concentrated at reduced
pressure. An 1H NMR spectrum showed that no benzyl groups
remained. The residue was dissolved in CH3OH (4 mL), and 3 M HCl
(1 mL) was added. After 30 min, the mixture was concentrated at
reduced pressure, and the residue was chromatographed on silica
eluted with a iPrOH:H2O:NH4OH (6:0.5:2.5 v/v/v) mixture to give
41 mg (89%) of a white solid: Rf 0.38 (iPrOH:H2O:NH4OH (6:1:3 v/
1
oil: Rf 0.56 (hexanes:ethyl acetate 1:1); H NMR (CDCl3, 300 MHz)
1.26−1.89 (18H, m2), 3.49−3.56 (1H, m), 3.78 (2H, d, J = 5.9 Hz),
3.83−3.90 (1H, m), 4.00−4.07 (1H, m), 4.21−4.27 (1H, m), 4.63−
4.65 (1H, m), 4.75 (1H, broad s), 5.51 (1H, t, J = 7 Hz); 13C NMR
(CDCl3, 75 MHz) 19.4, 22.0, 25.34, 28.3, 30.5, 40.8, 62.2, 62.7, 79.1,
97.7, 124.1, 138.0, 156.0; HRMS (MALDI) calcd for C15H27NO4Na
[M + Na+] 308.1828, found 308.1828.
20
1
v/v)); [α]D + 15.4 (c 0.65, D2O); H NMR (D2O, 300 MHz) 1.26
(3H, s), 3.07 (1H, d, J = 13.2 Hz), 3.25 (1H, d, J = 13.2 Hz), 3.75−
3.77 (1H, m), 3.81−3.91 (1H, m), 3.97−4.04 (1H, m); 13C NMR
(D2O, 75 MHz) 20.4, 46.2, 64.5, 71.4, 75.8 (J = 7 Hz); 31P NMR
(D2O, 125 MHz) 4.20; HRMS (MALDI) calcd for C5H13NO6P [M −
H+] 214.0486, found 214.0489.
(Z)-(4-Hydroxy-2-methylbut-2-enyl)carbamic Acid tert-Butyl
Ester (14). PPTS (25 mg, 0.1 mmol) was added to the solution of
13 (286 mg, 1 mmol) in EtOH (10 mL). The solution was stirred at
55 °C for 1 h and allowed to cool to rt. The solvent was removed at
reduced pressure, and the residue was chromatographed on silica with
gradient elution by hexanes/ethyl acetate (0% to 40% ethyl acetate) to
give 198 mg (98%) of a colorless oil: Rf 0.56 (hexanes:ethyl acetate
(2S,3S)-4-(tert-Butyldimethylsilanyloxy)-2,3-dihydroxy-3-methyl-
butyric Acid Methyl Ester (18). Alkene 1716 (300 mg, 1.16 mmol) was
asymmetrically dihydroxylated following the procedure described for
16. The residue was chromatographed on silica with gradient elution
by hexanes/ethyl acetate (0% to 15% ethyl acetate) to give 250 mg
1
20
1:1); H NMR (CDCl3, 300 MHz) 1.43 (9H, s), 1.76 (3H, t, J = 0.7
(73%) of a colorless oil: Rf 0.14 (hexanes:ethyl acetate 4:1); [α]D
+26.2 (c 1.8, CHCl3); 1H NMR (CDCl3, 300 MHz) 0.09 (6H, s), 0.91
(9H, s), 1.17 (3H, d, J = 0.6 Hz), 3.10 (1H, s), 3.34−3.37 (1H, m),
3.48 (1H, d, J = 10 Hz), 3.68 (1H, d, J = 10 Hz), 3.81 (3H, s), 4.11
Hz), 3.19 (1H, broad s), 3.74 (2H, dd, J1 = 5.6 Hz, J2 = 0.7 Hz), 4.14
(2H, t, J = 6.5 Hz), 4.91 (1H, broad s), 5.67 (1H, t, J = 7.3 Hz); 13C
NMR (CDCl3, 75 MHz) 21.6, 28.3, 40.7, 57.5, 79.9, 127.2, 136.2,
9176
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(1H, d, J = 8 Hz); 13C NMR (CDCl3, 75 MHz) −5.6, −5.6, 18.3,
19.64, 25.8, 52.5, 68.0, 73.3, 75.0, 173.7; HRMS (MALDI) calcd for
C12H26O5SiNa [M + Na+] 301.1435, found 301.1435.
Hz), 7.81 (2H, d, J = 8 Hz); 13C NMR (CDCl3, 75 MHz) −5.6, 18.1,
19.0, 21.6, 25.8, 67.6, 72.0, 72.7, 72.9, 128.0, 129.9, 132.6, 145.0;
HRMS (FTMS) calcd. for C18H33O6SSi [M+H+] 405.17616, found
405.17603.
(4S,5S)-5-(tert-Butyldimethylsilanyloxymethyl)-2-methoxy-1,3-di-
oxolane-4-carboxylic Acid Methyl Ester (19). To a solution of
trimethyl orthoformate (291 mg, 2.74 mmol) and diol 18 (200 mg,
0.69 mmol) in CH2Cl2 (9 mL) was added CSA (16 mg, 0.07 mmol) in
CH2Cl2 (1 mL). After stirring overnight, the solution was washed with
saturated NaHCO3; the layers were separated; and the aqueous layer
was extracted with CH2Cl2 (3 × 20 mL). The combined organic
extracts were dried over Na2SO4. The solvent was removed at reduced
pressure to give 230 mg (100%) of a colorless oil. This material
partially decomposed when chromatographed on silica and was used in
the next reaction without purification. A small sample was isolated by
chromatography for characterization: Rf 0.63 (hexanes:ethyl acetate
(2S)-1-(tert-Butyl-dimethylsilanyloxy)-2-[(S)-1-oxiranyl]propan-2-
ol (23). To a solution of tosylate 22 (367 mg, 0.91 mmol) in THF (6
mL) at 0 °C was added tBuOK (112 mg, 1.0 mmol) in THF (4 mL).
The solution was allowed to stir for 1 h at 0 °C; the solvent was
removed at reduced pressure; and the residue was chromatographed
on silica with gradient elution by hexanes/ethyl acetate to give 196 mg
20
(93%) of a colorless oil: Rf 0.36 (hexanes:ethyl acetate 1:1); [α]D
+15.0 (c 2.6, CHCl3); 1H NMR (CDCl3, 300 MHz) 0.08 (6H, s), 0.91
(9H, s), 1.18 (3H, s), 2.36 (1H, s), 2.74 (1H, dd, J1 = 4.0 Hz, J2 = 1.0
Hz), 2.83 (1H, dd, J1 = 2.8 Hz, J2 = 2.2 Hz), 3.01 (1H, dd, J1 = 2.8 Hz,
J2 = 1.0 Hz), 3.51 (1H, d, J = 9.8 Hz), 3.61 (1H, d, J = 9.8 Hz); 13C
NMR (CDCl3, 75 MHz) −5.6, 18.2, 20.9, 25.8, 44.0, 55.7, 68.4, 70.2;
HRMS (FTMS) calcd for C11H25O3Si [M + H+] 233.15675, found
233.15712.
1
3:2); dr 87:13 (by H NMR analysis of the orthoester); for major
diastereomer 1H NMR (CDCl3, 300 MHz) 0.02 (6H, s), 0.86 (9H, s),
1.49 (3H, s), 3.34 (3H, s), 3.56 (2H, dd, J1 = 18 Hz, J2 = 10.6 Hz),
4.40 (1H, s), 5.88 (1H, s); 13C NMR (CDCl3, 75 MHz) −5.7, −5.6,
18.3, 22.4, 25.7, 51.6, 52.1, 65.6, 79.0, 84.7, 116.0, 168.5; HRMS
(MALDI) calcd for C14H28O6SiNa [M + Na+] 343.1547, found
343.1558.
(2S)-2-[(S)-1-Oxiranyl]propane-1,2-diol (24). The complex Et3N−
3HF (1.1 g, 6.9 mmol) in THF (2 mL) was added dropwise to a
solution of silyl ether 23 (160 mg, 0.69 mmol) in THF (8 mL) at rt.
After stirring overnight, Et3N (5 mL) was added; solvent was removed
at reduced pressure, and the residue was chromatographed on silica
with gradient elution by hexanes/ethyl acetate (0% to 30% ethyl
acetate) to give 68 mg (83%) of a colorless oil: Rf 0.29 (hexanes:ethyl
acetate 1:1); er 17:1 (as determined by 19F NMR analysis of the
Mosher’s ester); [α]D20 +17.4 (c 0.65, CHCl3); 1H NMR (CDCl3, 300
MHz) 1.26 (3H, s), 2.36 (1H, broad), 2.50 (1H, s), 2.81−2.88 (2H,
m), 3.04 (1H, dd, J1 = 2.9 Hz, J2 = 1.1 Hz), 3.49−3.62 (2H, m); 13C
NMR (CDCl3, 75 MHz) 22.3, 44.5, 56.6, 67.2, 70.3; HRMS (FTMS)
calcd for C5H9O2 [M − OH] 101.05971, found 101.06000.
(4S,5S)-5-[tert-Butyldimethylsilanyloxymethyl)-2-methoxy-5-
methyl[1,3]dio-xolan-4-yl]methanol (20). A solution of 2 M LiBH4
in THF (0.6 mL, 1.2 mmol) was added dropwise by a syringe to a
solution of 19 (200 mg, 0.6 mmol) in ether (20 mL) at rt. The
resulting solution was stirred for 1 h before EtOH (5 mL) and brine (5
mL) were added. The organic layer was separated, and the aqueous
layer was extracted with ether (3 × 10 mL). The combined organic
extracts were dried over Na2SO4. The solvent was removed at reduced
pressure to give 175 mg (quant) of a colorless oil. This material
partially decomposed when chromatographed on silica and was used in
the next reaction without purification. A small sample was isolated by
chromatography for characterization: Rf 0.63 (hexanes:ethyl acetate
3-Methyl-D-erythritol-4-thiolophosphate, Ammonium Salt
(MESP). To epoxide 50 (34 mg, 0.29 mmol) was added trisodium
thiophosphate (126 mg, 0.32 mmol) in H2O (0.6 mL) at rt. The
solution was allowed to stir overnight and then lyophilized. The
residue was chromatographed on cellulose to give 25 mg of a white
1
3:2); dr 87:13 (by H NMR analysis of the orthoester); for major
diastereomer 1H NMR (CDCl3, 300 MHz) 0.11 (6H, s), 0.91 (9H, s),
1.45 (3H, s), 3.32 (3H, s), 3.71−4.09 (5H, m), 5.67 (1H, s); 13C NMR
(CDCl3, 75 MHz) −5.7, −5.7, 18.1, 22.8, 25.7, 51.7, 60.3, 65.0, 81.3,
82.6, 114.8; HRMS (FTMS) calcd for C12H25O4Si [M − OCH3]
261.1517, found 261.15164.
20
powder (32%): Rf 0.42 (iPrOH:H2O:NH4OH 6/1/3 v/v/v); [α]D
1
+16.3 (c 0.53, D2O); H NMR (D2O, 300 MHz) 1.09 (3H, s), 2.67−
2.78 (1H, m), 2.96−3.05 (1H, m), 3.51 (2H, dd, J1 = 17.2 Hz, J2 =
11.8 Hz), 3.72 (1H, dd, J1 = 8.4 Hz, J2 = 2.2 Hz); 13C NMR (D2O, 75
MHz) 17.7, 31.5 (J = 3 Hz), 66.7, 75.0, 75.5 (J = 3 Hz); 31P NMR
(D2O, 125 MHz) 17.82; HRMS (FTMS) calcd for C5H14O6PS [M +
H+] 233.02432, found 233.02482.
(4S,5S)-5-[tert-Butyldimethylsilanyloxymethyl)-2-methoxy-5-
methyl[1,3]dio-xolan-4-yl]methyl p-Toluenesulfonate (21). Alcohol
20 (60 mg, 0.21 mmol) and DMAP (75 mg, 0.62 mmol) were
dissolved in CH2Cl2 (5 mL), and TsCl (78 mg, 0.41 mmol) in CH2Cl2
(2 mL) was added by syringe. The mixture was allowed to stir
overnight. The solvents were removed at reduced pressure, and the
residue was chromatographed on silica gel with gradient elution by
hexanes/ethyl acetate (0% to 10% ethyl acetate) to give 90 mg (98%)
of a colorless oil: Rf 0.59 (hexanes:ethyl acetate 3:2); dr 87/13 (by 1H
NMR analysis of the orthoester); [α]D20 +10.2 (c 0.9, CHCl3); for the
Product Studies. TLC Analysis. Enzymatic reactions were carried
out at 37 °C in 0.1 mM Tris·HCl buffer, pH 7.6, containing 5 mM
DTT, 10 mM MgCl2, 150 μM CTP, 150 μM ATP, 500 μM MEP
(racemic), D-EP or D-MTP or D-EEP, 4.8 μM IspDF, 6.2 μM IspE
(where applicable) in a final volume of 50 μL. [32P]NTPs were diluted
from 5 mM stock solutions of 40 μCi/μmol [α-32P]CTP and 320 μCi/
μmol [γ-32P]ATP. Reactions were initiated by addition of CTP. After
1 h, the reactions were quenched with 60 μL of methanol and were put
on ice. TLC analysis (Polygram Sil N-HR; Macherey and Nagel) was
performed by spotting 4.5 μL of the reaction mixture and developing
the plates with n-propanol/ethyl acetate/H2O (6:1:3, v/v/v).
Radioactivity was quantified by phosphorimaging.
LC−MS Analysis. LC−MS analyses for incubations with EP, EEP,
NMEP, and MESP similar to those described in the TLC analyses
were carried out on a 1 mM scale using unlabeled CTP (0.75 mM),
unlabeled ATP (0.75 mM), IspDF (4.8 μM), and IspE (6.2 μM). The
reactions were incubated for 1 h and were centrifuged through a
membrane (10 kDa cutoff) to remove the enzymes. Products were
detected by negative-ion electrospray LC−MS using a Phenomex
Prodigy 5 μ ODS(3) 100A (250 × 4.60 mm 5 μM) column eluted
with 20 mM N,N′-dimethylhexylamine in 10% methanol, pH 7.0,
adjusted with formic acid (Buffer A) and 2 mM N,N′-dimethylhexyl-
amine in 50% methanol, pH 7.0 (Buffer B), as previously described.4
Time Course Studies. A solution of 500 μM EP (or EEP), 4.8 μM
IspDF, and 6.2 μM IspE in 0.1 M Tris·HCl buffer, pH 7.6 (37 °C), 10
mM MgCl2, containing 5 mM DTT, in a final volume of 100 μL was
preincubated for 10 min at 37 °C. γ-[32P]ATP (150 μM) and α-[32P]
1
major diastereomer H NMR (CDCl3, 300 MHz) 0.82 (9H, s), 1.34
(3H, s), 2.45 (3H, s), 3.29 (3H, s), 3.32 (1H, d, J = 10.5 Hz), 3.50
(1H, d, J = 10.5 Hz), 4.10−4.18 (2H, m), 4.33−4.41 (1H, m), 5.68
(1H, s), 7.13 (2H, d, J = 8 Hz), 7.80 (2H, d, J = 8 Hz); 13C NMR
(CDCl3, 75 MHz) −5.9, −5.7, 17.9, 21.6, 25.6, 51.5, 65.1, 68.4, 79.7,
82.5, 115.4, 128.0, 129.9, 132.7, 144.9; HRMS (FTMS) calcd for
C19H31O6SSi [M − OCH3] 415.1605, found 415.16047.
(2R,3S)-4-(tert-Butyldimethylsilanyloxy)-2,3-dihydroxy-3-methyl-
butyl Tosylate (22). A solution of 21 (230 mg, 0.54 mmol) in THF (6
mL, 3 M HCl:THF, 1:25) was allowed to stir for 30 min. The mixture
was cooled to 0 °C; 6 mL of ammonia in MeOH were added; and the
solution was allowed to stir for 15 min at rt. The solvent was removed
at reduced pressure, and the residue was chromatographed on silica
with gradient elution by hexanes/ethyl acetate (0% to 30% ethyl
acetate) to give 160 mg (76%) of a colorless oil; Rf 0.36 (hexanes:ethyl
20
1
acetate 1:1); [α]D +18.9 (c 1.7, CHCl3); H NMR (CDCl3, 300
MHz) 0.06 (6H, s), 0.88 (9H, s), 1.07 (3H, s), 2.45 (3H, d, J = 0.3
Hz), 2.71−2.74 (2H, broad), 3.38 (1H, d, J = 10 Hz), 3.67 (1H, d, J =
10 Hz), 3.82 (1H, dd, J1 = 6 Hz, J2 = 2.5 Hz), 4.09 (1H, dd, J1 = 8 Hz,
J2 = 2.0 Hz), 4.32 (1H, dd, J1 = 8.0 Hz, J2 = 2.5 Hz), 7.36 (2H, d, J = 8
9177
dx.doi.org/10.1021/jo501529k | J. Org. Chem. 2014, 79, 9170−9178

The Journal of Organic Chemistry
Article
CTP (150 μM, 40 μCi/μmol) were added sequentially to initiate the
reaction. At various times, 6 μL portions of the mixture were removed
and quenched with 6 μL of methanol. After 61 min, an additional 2 μg
portions of each enzyme were added to the reaction mixture.
Inhibition Studies with MTP. A solution of 500 μM MEP and 0.36
μM IspDF in 0.1 M Tris·HCl buffer, pH 7.6 (37 °C), containing 5
mM DTT and 10 mM MgCl2 was preincubated for 10 min. α-[32P]
CTP (150 μM) was added to initiate the reaction. At various times, 6
μL portions of the mixture were removed and quenched with 6 μL of
methanol. The samples were analyzed by TLC.4 The same experiment
was performed except MTP was added to 5 mM. No change was seen
in the rate of formation of CDP−ME.
(12) Witschel, M. C.; Hcf
̧
fken, H. W.; Seet, M.; Parra, L.; Mietzner,
T.; Thater, F.; Niggeweg, R.; Rcḩ l, F.; Illarionov, B.; Rohdich, F.;
Kaiser, J.; Fischer, M.; Bacher, A.; Diederich, F. Angew. Chem., Int. Ed.
2011, 50, 7931−7935.
(13) Fox, D. T.; Poulter, C. D. J. Org. Chem. 2005, 70, 1978−1985.
(14) Lagisetti, C.; Urbansky, M.; Coates, R. M. J. Org. Chem. 2007,
72, 9886−9895.
(15) Ghavami, A.; Johnston, B. D.; Pinto, B. M. J. Org. Chem. 2001,
66, 2312−2317.
(16) Koppisch, A. T.; Blagg, B. S. J.; Poulter, C. D. Org. Lett. 2000, 2,
215−217.
(17) Gefflaut, T.; Lemaire, M.; Valentin, M.-L.; Bolte, J. J. Org. Chem.
1997, 62, 5920−5922.
(18) Mitsunobu, O. Synthesis 1981, 1, 1−28.
ASSOCIATED CONTENT
■
(19) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev.
1994, 94, 2483−547.
S
* Supporting Information
Additional figures for time course TLC and LC−MS
chromatograms. NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org/
(20) Hoye, T. R.; Jeffrey, C. D.; Shao, F. Nat. Protoc. 2007, 2, 2451−
2458.
(21) Nishino, T.; Ogura, K.; Seto, S. Biochim. Biophys. Acta 1971,
235, 322−325.
(22) Poulter, C. D.; Rilling, H. C. Prenyl Transferases and Isomerase.
In Biosynthesis of Isoprenoid Compounds; Porter, J. W., Ed.; John Wiley
& Sons: New York, 1981; Vol. 1, Chapter 4, pp 162−224.
(23) Poulter, C. D.; Argyle, J. C.; Mash, E. A.; Laskovics, G. M.;
Wiggins, P. L.; King, C. R. Inhibitors of Isoprenoid Biosynthesis. In
Regulation of Insect Development and Behavior; Chemistry and
Biochemistry of Juvenile Hormones; Wroclaw Technical University
Press: Wroclaw, Poland, 1981; Chapter 3, pp 149−162.
(24) Janthawornpong, K.; Krasutsky, S.; Chaignon, P.; Rohmer, M.;
Poulter, C. D.; Seemann, M. J. Am. Chem. Soc. 2013, 135, 1816−1822.
(25) Phan, R. M.; Poulter, C. D. J. Org. Chem. 2001, 66, 6705−6710.
(26) Barker, S. A.; Foster, A. B.; Haines, A. H.; Lehmann, J.; Webber,
J. M.; Zweifel, G. J. Chem. Soc. 1963, 4161−4167.
(27) Ihara, M.; Takino, Y.; Tomotake, M.; Fukumoto, K. J. Chem.
Soc., Perkin Trans. 1 1990, 8, 2287−2292.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: poulter@chemistry.utah.edu.
*E-mail: rmcoates@illinois.edu.
Present Addresses
§(S.G.K.) Twenty-six Corporate Circle, Albany Molecular
Research Inc., Albany, N.Y. 12212.
⊥(C.E.D.) Goodwin Procters’s Technology and Life Sciences
Group, Exchange Place, 53 State St., Boston, MA 01209.
#(C.L.) Universite
synthes
LSPCMIB, 118 route de Narbonne F-31062, Toulouse cedex 9,
France.
́
de Toulouse, UPS, CNRS, Laboratoire de
e et physio-Chimie de Molecules d’Interet Biologique,
̀
́
́
̂
(28) MacDonald, D. L.; Fischer, H. O. L.; Ballou, C. E. J. Am. Chem.
Soc. 1956, 78, 3720−3722.
Notes
The authors declare no competing financial interest.
∥(M.U.) Deceased.
ACKNOWLEDGMENTS
■
We are grateful to the National Institute of General Medical
Sciences (GM 25521 to CDP and GM 13956 to RMC) for
financial support of this research.
REFERENCES
■
(1) Bochar, D. A.; Freisen, J. A.; Stauffacher, C. V.; Rodwell, V. W.
Biosynthesis of Mevalonic acid from Acetyl Co-A). In Comprehensive
Natural Products Chemistry; Cane, D., Ed.; Pergamon Press: Oxford,
1999; Vol. 2, pp 15−44.
(2) Miziorko, H. M. Arch. Biochem. Biophys. 2012, 505, 131−143.
(3) Zhao, L.; Chang, W.-C.; Xiao, Y.; Liu, H.-W.; Liu, P. Annu. Rev.
Biochem. 2013, 82, 497−530.
(4) Testa, C. A.; Lherbet, C.; Pojer, F.; Noel, J. P.; Poulter, C. D.
Biochim. Biophys. Acta 2006, 1764, 85−96.
(5) Lherbet, C.; Pojer, F.; Richard, S. B.; Noel, J. P.; Poulter, C. D.
Biochemistry 2006, 45, 3548−3553.
(6) Hunter, W. N. Curr. Top. Med. Chem. 2011, 11, 2048−2059.
(7) Morris, F.; Vierling, R.; Boucher, L.; Bosch, J.; Freel Meyers, C. L.
ChemBioChem. 2013, 14, 1309−1315.
(8) Ponaire, S.; Zingle, C.; Tritsch, D.; Grosdemange-Billiard, C.;
Rohmer, M. Eur. J. Med. Chem. 2012, 51, 277−285.
(9) Majumdar, A.; Shah, M. H.; Bitok, J. K.; Hassis-LeBeau, M. E.;
Meyers, C. L. F. Mol. Biosys. 2009, 5, 935−944.
(10) Lillo, A. M.; Tetzlaff, C. N.; Sangari, F. J.; Cane, D. E. Bioorg.
Med. Chem. Lett. 2003, 13, 737−739.
(11) Wang, W.; Oldfield, E. Angew. Chem., Int. Ed. 2014, 53, 4294−
4310.
9178
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