Mevalonate analogues as substrates of enzymes in the isoprenoid biosynthetic pathway of Streptococcus pneumoniae

Article abstract of DOI:10.1016/j.bmc.2009.12.050

Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway, which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this pathway appears to be regulated at the mevalonate kina

Full text of DOI:10.1016/j.bmc.2009.12.050

Bioorganic & Medicinal Chemistry 18 (2010) 1124–1134
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Mevalonate analogues as substrates of enzymes in the isoprenoid biosynthetic
pathway of Streptococcus pneumoniae
Takashi Kudoh a, Chan Sun Park a, Scott T. Lefurgy b, Meihao Sun b, Theodore Michels a, Thomas S. Leyh b,
,
*
Richard B. Silverman a,
*
a Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, Center for Molecular Innovation and Drug Discovery,
and Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208-3113, United States
b Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway,
which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this
pathway appears to be regulated at the mevalonate kinase (MK) step, which is strongly feedback-inhib-
ited by diphosphomevalonate (DPM), the penultimate compound in the pathway. The human mevalonate
pathway is not regulated by DPM, making the bacterial pathway an attractive antibiotic target. Since
DPM has poor drug characteristics, being highly charged, we propose to use unphosphorylated, cell-per-
meable prodrugs based on mevalonate that will be phosphorylated in turn by MK and phosphomevalo-
nate kinase (PMK) to generate the active compound in situ. To test the limits of this approach, we
synthesized a series of C3-substituted mevalonate analogues to probe the steric and electronic require-
ments of the MK and PMK active sites. MK and PMK accepted substrates with up to two additional car-
bons, showing a preference for small substituents. This result establishes the feasibility of using a prodrug
strategy for DPM-based antibiotics in S. pneumoniae and identified several analogues to be tested as
inhibitors of MK. Among the substrates accepted by both enzymes were cyclopropyl, vinyl, and ethynyl
mevalonate analogues that, when diphosphorylated, might be mechanism-based inactivators of the next
enzyme in the pathway, diphosphomevalonate decarboxylase.
Received 11 October 2009
Revised 15 December 2009
Accepted 16 December 2009
Available online 24 December 2009
Keywords:
Isoprenoid pathway
Mevalonic acid
Phosphomevalonic acid
Diphosphomevalonic acid
Mevalonate kinase
Phosphomevalonate kinase
Diphosphomevalonate decarboxylase
Prodrug
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
substantial antimicrobial resistance.3 Vancomycin, the antibiotic
of last resort, has shown signs of weakness in some strains of
Streptococcus pneumoniae kills over a million people a year,
mostly children and the elderly, and is the primary cause of com-
munity-acquired pneumonia and bacterial meningitis.1,2 Universal
vaccination programs in the US have led to a reduction in the tar-
geted serotypes of S. pneumoniae; however, this effort has resulted
in serotype replacement by nonvaccinated strains that show
S. pneumoniae,4,5 and widespread resistance to beta-lactams and
macrolides, including multiple-drug-resistance, makes treatment
difficult and expensive.6–9 The need for new strategies to combat
this deadly pathogen in the short term is unequivocal.
A presently unexploited antibiotic target in S. pneumoniae is the
mevalonate pathway (Fig. 1), which is required for the organism to
survive in lung and serum.10 This pathway converts mevalonate to
isopentenyl diphosphate (IPP), the building block of isoprenoids, in
three enzymatic steps, each carried out by a member of the GHMP
kinase family. We recently discovered that the first enzyme, mev-
alonate kinase (MK), is feedback-inhibited by diphosphomevalo-
nate (DPM) through binding at a high-affinity allosteric site at
Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; DPM-
DC, diphosphomevalonate decarboxylase; DTT, dithiothreitol; GHMP, galactoki-
nase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase; HEPES,
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; IPTG, isopropyl-1-thio-b-
D-galactopyranoside; LDH, lactate dehydrogenase; 2-ME, b-mercaptoethanol; Mev,
mevalonate; Pmev, phosphomevalonate, DPM, diphosphomevalonate; HMG-CoA,
3-hydroxy-3-methyl-glutaryl-CoA; IPP, isopentenyl diphosphate; MK, mevalonate
kinase; NADH, nicotinamide adenine dinucleotide, reduced form; Pi, inorganic
phosphate; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PMK, phosphomeval-
onate kinase; Tris, tris(hydroxymethyl)aminomethane; unit, 1
formed per minute at a saturating concentration of substrate(s).
* Corresponding authors. Tel.: +1 847 491 5653; fax: +1 847 491 7713 (R.B.S.).
E-mail addresses: thomas.leyh@einstein.yu.edu (T.S. Leyh), Agman@chem.
northwestern.edu (R.B. Silverman).
concentrations (Ki = 0.5 lM) where the human homolog of MK is
unaffected.11 The allosteric site is thought to lie at the MK dimer
interface and appears to be absent from the MKs of mammals12
and some bacteria,13 raising the possibility that the effects of
DPM inhibition would be limited to few species.
lmol of product
DPM is not expected to cross the cell membrane because of its
high negative charge (4-); therefore, we have focused on a prodrug
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.050

T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
1125
Figure 1. The mevalonate pathway. MK, mevalonate kinase; PMK, phosphomevalonate kinase; DPM-DC, diphosphomevalonate decarboxylase.
strategy in which mevalonate analogues are taken up by cells and
phosphorylated by the mevalonate pathway enzymes to generate
inhibitors in situ. An analogous strategy has been used to show
that 6-fluoromevalonate inhibits isoprenoid biosynthesis in
cultured human cells.14,15 This result is consistent with the com-
pound crossing the cell membrane and being converted in situ to
the phosphorylated form; the diphosphorylated compound (6-flu-
oro-5-diphosphomevalonate) is a potent inhibitor of the human
diphosphomevalonate decarboxylase (DPM-DC).16 The present
study seeks to expand the repertoire of DPM prodrugs with a series
of compounds elaborating the C3-methyl group with hydrocarbons
of variable length, branching, and saturation (Scheme 1). Several
mevalonate analogues were designed with an eye toward mecha-
nism-based inhibition of DPM-DC, in addition to binding to the
MK allosteric site, in a dual-acting antibiotic approach. In the pres-
ent view of the DPM-DC transition state, substantial positive
charge forms on C3 during turnover.17 To exploit this mechanism
for inactivation, we synthesized DPM analogues (vinyl-, ethynyl-,
and cyclopropyl-substituted) that delocalize this positive charge
through resonance, transiently forming a strong electrophile that
could undergo attack by a protein nucleophile in the vicinity of
the putative substrate-binding pocket of DPM-DC.18 An advantage
of this strategy is that the potency of the antibiotic is enhanced
because the inhibitor acts at two points in the same pathway. Be-
cause the human mevalonate pathway enzymes are closely related
to those from S. pneumoniae, the specificity of antibiotics directed
against DPM-DC is a concern. Whereas a functional mevalonate
pathway is required for survival of the bacterium, the human path-
way can be suppressed with minimal side-effects with the use of
cholesterol-lowering drugs (statins, HMG-CoA reductase) and anti-
proliferative agents (bisphosphonates, farnesyl transferase).19,20
Therapeutic benefits that arise by controlling flux through the hu-
man mevalonate pathway suggest that DPM-DC inhibitors may
have additional clinical uses. In the present study, we assess the
limitations on side chains for a prodrug strategy; binding of these
analogues to the MK allosteric site and DPM-DC inactivation will
be the subject of a future study.
TBDPSCl gave mono-protected diol 8 in 86% yield. Then, the cyclic
ketal group of 8 was readily cleaved using PPTS to give 9. The other
hydroxyl group of 9 was protected as a MOM ether to give bis-pro-
tected diol 10 in a quantitative yield. Diol 10 was treated with
21
Grignard reagents in the presence of CeCl3 to generate tertiary
alcohols 11a–e, 11g, 11i–k in good yields (77–99%). Selective
deprotection of MOM group in 11a–g, 11i–k, followed by an oxida-
tion of the resulting primary alcohol using IBX and NaClO2 pro-
vided the corresponding carboxylic acids 13a–e, 13g, 13i–k.
Finally, desilylation of the TBDPS group in 13a–e, 13g, 13i–k coin-
cidently led to an intramolecular cyclization to give the desired lac-
tones 1a–e, 1g, 1i–k.
The cyclopropyl analogue (1f) had to be prepared by a slightly
different route starting from the diprotected alcohol intermediate
(11f) because of problems with the MOM ether deprotection step
(Scheme 3).
The bulky isopropyl group could not be installed using standard
Grignard reaction conditions to make 11h, either with or without
CeCl3. Consequently, the 2-propenyl intermediate (12g) was hydro-
genated to the isopropyl analogue (12h), and the synthesis of 11h
was completed as usual (Scheme 4).
To carry out the enzyme assays it was necessary to hydrolyze
lactones 1a–k prior to incubation with the enzyme. Hydrolysis
was carried out smoothly in aqueous 2.0 M LiOH at room temper-
ature for 1 h. It was found that re-lactonization occurred in water
(t1/2 = 14 days), methanol (t1/2 = 7 days), or neat (t1/2 = 3 days),
but was suppressed in 50 mM Hepes/K+ buffer containing 1 mM
MgCl2 at pH 8.0 (stable at least one week), which is the buffer used
to assay MK and phosphomevalonate kinase (PMK).
3. Results
To assess their viability as substrates of the enzymes in the
mevalonate pathway, each of the mevalonate analogues was tested
as a substrate for both MK and PMK. These activities are imperative
for in vivo conversion of the alcohols to the corresponding diph-
osphorylated analogues. Reaction progress was monitored opti-
cally at 386 nm using a well-established pyruvate kinase/lactate
dehydrogenase coupled enzyme system in which the ADP pro-
duced by phosphorylation of the mevalonate analogue is linked
stoichiometrically (1:1) to NADH oxidation.22 Reaction progress
curves (Fig. 2) are the average of three assays done under identical
conditions. The primary enzyme was varied from analogue to ana-
logue to allow each progress curve to finish in a reasonable amount
of time (see Fig. 2 legend). To compare the activity of compounds,
2. Chemistry
The general synthetic route to the racemic vinyl, ethyl, n-propyl,
n-butyl, i-butyl, 2-propenyl, allyl, ethynyl, and 1-propynyl mevalo-
nate lactone analogues (1a–e, g, i–k) is shown in Scheme 2. Selec-
tive protection of the ketone functional group with ethylene glycol
in the presence of BF3–OEt2, followed by LAH reduction provided
diol 7 in a high yield. Next, selective protection of diol 7 using
the data were normalized to a 1.0
l
M enzyme concentration, since
HO
R
AT P ADP
AT P ADP
O
O
P
O
P
O-
HO
R
HO
R
HO
R
+H2O
-H2O
HOOC
HOOC
P
HOOC
OH
O
- O-
O
- O
O-
O
O
O
O
1
2
3
4
MK
PMK
R = vinyl, Et, n-Pr, i-Pr, isopropenyl, cyclopropyl,
n-Bu, i-Bu, allyl, ethynyl, 1-propynyl
Scheme 1.

1126
T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
ethylene glycol
BF3-Et2O
TBDPSCl
NaH
O
LiAlH4
PPTS
O
O
O
O
O
O
EtO2C
CO2Et
EtO2C
CO2Et
THF
CH2Cl2
HO
R
OTBDPS
ZrCl4
aq. acetone
reflux
96%
HO
OH
THF, 0 °C - rt
2 steps 96%
5
6
7
0 °C - rt
86%
0 °C - rt
8
O
MOMCl
O
1)
HO
RMgX, CeCl3
DIPEA
CH2Cl2
100%
HO
OTBDPS
MOMO
OTBDPS
MOMO
OTBDPS
THF, -78 °C2)
i-PrOH
90 °C
9
10
R = vinyl, X = Br : 11a, 94%
R = Et, X = Br : 11b, 87%
R = n-Pr, X = Cl : 11c, 92%
R = n-Bu, X = Cl : 11d, quant.
R = i-Bu, X = Cl : 11e, 85%
R = cyclopropyl, X = Br, 11f, 89%
R = 2-propenyl, X = Br; 11g, 85%
R = allyl, X = Br; 11i, quant.
R = ethynyl, X = Br, 11j, 79%
R = 1-propynyl, X = Br, 11k, 77%
HO
R
HO
R
1) Oxidation3)
HO
R
HCl
aq. acetone
HOOC
HO
OTBDPS
OTBDPS
2) NaClO2, NaH2PO4
2-methyl-2-butene
aq. acetone
O
O
R = vinyl : 12a, 78%
R = Et : 12b, 68%
R = n-Pr : 12c, 67%
R = n-Bu : 12d, 90%
R = i-Bu : 12e, 78%
R = 2-propenyl : 12g, 80%
R = allyl, 12i, 86%
R = vinyl : 13a, 2 steps 76%
R = Et : 13b, 2 steps 78%
R = n-Pr : 13c, 2 steps 59%
R = n-Bu : 13d, 2 steps 79%
R = i-Bu : 13e, 2 steps 84%
R = 2-propenyl : 13g, 2 steps 73%
R = allyl, 13i, 2 steps 74%
R = vinyl : 1a, 85%
R = Et : 1b, 72%
R = n-Pr : 1c, 87%
R = n-Bu : 1d, 84%
R = i-Bu : 1e, 88%
R = 2-propenyl : 1g, 70%
R = allyl, 1i, 82%
R = ethynyl, 12j, 93%
R = 1-propynyl, 12k, 81%
R = ethynyl, 13j, 2 steps 64%
R = 1-propynyl, 13k, 2 steps 62%
R = ethynyl, 1j, 77%
R = 1-propynyl, 1k, 86%
1) CeCl3 was not used for the vinyl and allyl analogues.
2) The ethynyl and propynyl analogues did not react at -78 oC but did work at 0 °C - rt
3) Dess-Martin periodinane/CH2Cl2 0 °C to room temp. was used for the n-propyl analogue, and IBX/DMSO was used for the all other analogues.
Scheme 2.
HO
1) IBX, DMSO
2) NaClO2, NaH2PO4,
ZrCl4,
i-PrOH,
O
HO
HO
HO
TBAF
MOMO
OTBDPS
MOMO
OH
MOMO
O
90oC 18%
THF
86%
2-methyl-2-butene,
aq. acetone
O
O
11f
14
15
3) MeI, K2CO3, DMF
46%
1f
Scheme 3.
H2, Pd-C
i-PrOH
quant.
HO
HCl
1) IBX, DMSO
HO
HO
HO
HO2C
aq. acetone
80%
HO
OTBDPS
HO
OTBDPS
OTBDPS
2) NaClO2, NaH2PO4
2-methyl-2-butene
aq. acetone
O
O
12g
12h
13h
1h
2 steps 99%
Scheme 4.
activity was linear with enzyme concentration (data not shown).
MK reactions were initiated by addition of analogue (Fig. 2A).
The MK reactions were allowed to reach completion before adding
PMK, resulting in the progress curves shown in Figure 2B. The end-
point of the MK reaction occurred at 50% of the initial concentra-
tion of the racemic compound, as would be expected for
conversion of a single enantiomer.
the selection of protecting groups for the terminal alcohols. Ini-
tially, p-methoxybenzyl was selected as one of the alcohol protect-
ing groups, but it caused problems later in the synthesis when it
was removed; the methoxymethyl (MOM) protecting group was
much more suitable. Steric hindrance seems to be the main cause
for failure of the Grignard reaction; neither the isopropyl nor the
tert-butyl analogue could be made directly by this route. However,
the isopropyl analogue was prepared by catalytic hydrogenation of
the 2-propenyl analogue.
4. Discussion
4.2. Validation of the prodrug strategy
4.1. Synthetic approach
To evaluate their potential as prodrugs, eleven mevalonate
analogues were tested as substrates for MK and PMK. Seven of
The yields of all of the steps in the synthetic route were excel-
lent (see Schemes 2–4 for yields). The main complications came in

T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
1127
Figure 2. Mevalonate analogues as substrates for MK and PMK. Reaction progress curves for mevalonate kinase (A) and phosphomevalonate kinase (B). Color coding is
identical in panels A and B. Reaction conditions: MK (Mev, 0.050 M; 2a, 1 M; 2b, 1 M; 2c, 20 M; 2e, 20 M; 2f, 20 M; 2i, 20 M; 2j, 10 M) or PMK (Pmev, 0.005 M; 3a,
0.075 M; 3b, 0.150 M; 3c, 1.0 M; 3e, 2.0 M; 3f, 0.50 M; 3i, 2.0 M; 3j, 0.10 M), pyruvate kinase (10.0 units/ml), lactate dehydrogenase (20.0 units/ml), ATP (4.0 mM),
MgCl2 (5.0 mM), NADH (0.82 mM), phosphoenolpyruvate (1.0 mM), KCl (50 mM), b-mercaptoethanol (20.0 mM), compounds 2a–2k (200 M, racemic mixture) or 3a–c, e, f, i,
j (100
M), Hepes/K+ (50 mM, pH 8.0), T = 25 2 °C. The activities of compounds 2d, 2g, 2h and 2k with MK were indistinguishable from background. (C) Mevalonate
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
l
analogues (R-groups) listed in the order of their reaction rate with MK.
the analogues (a, b, c, e, f, i, and j) proved to be substrates for both
enzymes, and are, therefore, viable prodrug candidates. The
remaining four analogues (d, g, h, and k) were inactive with MK
(<0.0025% of the activity of mevalonate).
and mechanism-based inactivators of DPM-DC, the next enzyme in
the isoprenoid biosynthetic pathway.
6. Experimental procedures
6.1. General methods
The progress-curve based comparison of the reactivity of each
active analogue reveals an inverse relationship between reaction
rate and the size of the R-group (Fig. 2). For example, increasing
the length of the mevalonate C3 methyl group by a single methy-
lene (b) has only a small effect on the reaction rate with MK and
PMK. However, a two-carbon increase (c) significantly reduces
reactivity, and a three-carbon increase (e) profoundly decreases
the reactivity. The geometry of the R-group also has a dramatic ef-
fect on reactivity; branched compounds are excluded (cf., 2g vs 2i,
or 2h vs 2f). The similar trend in reactivity for both enzymes is not
surprising given their similar GHMP-kinase active-site architec-
tures.23,24 The changes in reaction rate may be due to alterations
in Vmax and/or Km, and a full kinetic analysis of seven compounds
with the three mevalonate pathway enzymes (as substrates, inhib-
itors, and allosteric regulators) will be reported in due course.
Flash chromatography was performed with 230–400 mesh sil-
ica gel. 1H NMR, 13C NMR and 31P NMR spectra were obtained using
Varian 300, 400 and 500 MHz spectrometers. NMR spectra were re-
corded in ppm (d) relative to tetramethylsilane (d = 0.00) as an
internal standard unless stated otherwise and are reported as fol-
lows; chemical shift, multiplicity (br = broad, s = singlet, t = triplet,
q = quartet, m = multiplet), coupling constant, and integration. Sol-
vents and liquid reagents were transferred using hypodermic
syringes. All other reagents and solvents used were reagent grade.
All glassware was dried in an oven at 150 °C prior to use. Tetrahy-
drofuran (THF) and diethyl ether (Et2O) were distilled from so-
dium/benzophenone. Methylene chloride and triethylamine were
dried over calcium hydride prior to use. Reactions were monitored
by thin layer chromatography (TLC) with 0.25 mm E. Merck pre-
coated silica gel plate (60 F254). Small and medium scale purifica-
tions were performed by flash chromatography. Pyruvate kinase
and lactate dehydrogenase were obtained from Roche Applied
Science (Indianapolis, IN). Kinetic measurements of enzymatic
reactions were made using a Cary 100 or 400 UV/Vis spectropho-
tometer (Varian Inc., Palo Alto, CA).
5. Conclusions
We have demonstrated the feasibility of using C3-substituted
mevalonates as prodrugs to generate DPM analogues in situ. By
knowing which analogues are suitable substrates for these
enzymes, we can synthesize the appropriate DPM analogues for
testing as allosteric inhibitors of MK as well as potential inhibitors

1128
T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
6.2. 1,5-Dihydroxy-3-pentanone-ethylideneacetal (7)
mixture of diethyl acetone-1,3-dicarboxylate (5, 4.04 g,
mixture was stirred for 10 min and evaporated. The residue was
partitioned between AcOEt and ice cooled aqueous 0.1 M HCl,
and the organic layer was washed with H2O, brine, dried with
Na2SO4, and evaporated. The residue was dried under vacuum to
give 10 (128 mg, 100%) as a colorless oil: 1H NMR (400 MHz, CDCl3)
d 7.37–7.66 (m, 10H, Ph  2), 4.60 (s, 2H, MOM-CH2), 3.79–3.97 (m,
4H, CH2OMOM, CH2OTBDPS), 3.34 (s, 3H, MOM-CH3), 2.66–2.76 (m,
4H, CH2CH2O Â 2), 1.03 (s, 9H, t-Bu); 13C NMR (100 MHz, CDCl3) d
207.60, 135.41, 133.25, 129.58, 127.58, 96.39, 62.33, 59.40, 55.10,
45.79, 43.38, 26.62, 18.99. HRMS (pos. ion ESI) m/z calcd for
(M+Na)+ C23H32NaO4Si: 423.19621. Found: 423.19487.
A
20.0 mmol), ethylene glycol (4.46 mL, 80.0 mmol), and BF3ÁEt2O
(3.80 mL, 30.0 mmol) in CH2Cl2 (40 mL) was stirred at 0 °C for
90 min and room temperature for 46 h. H2O was added to the mix-
ture at 0 °C, and the mixture was stirred at 0 °C for 30 min and ex-
tracted with AcOEt. The organic layer was washed with H2O, brine,
dried with Na2SO4, and evaporated. The residue was treated again
as mentioned above and worked up the same way. A mixture of the
crude product and LiAlH4 (1.0 M in THF, 40 mL, 40 mmol) in THF
(150 mL, HPLC grade) was stirred at 0 °C for 1 h and room temper-
ature for 15 h, and aqueous 2.0 M NaOH was added to the mixture
at 0 °C. The mixture was stirred at 0 °C for 30 min, and the resulting
gel was filtered off with Celite and washed with acetone. The com-
bined filtrate and washings were evaporated, and the residue was
co-evaporated with i-PrOH (Â2) and purified by silica gel column
chromatography (5% MeOH in AcOEt) to give 7 (3.13 g, 96%) as a
colorless oil: 1H NMR (300 MHz, CDCl3) d 4.06 (s, 4H, acetal-
CH2 Â 2), 3.70–3.77 (m, 4H, CH2OH Â 2), 2.56 (br s, 2H, OH Â 2),
1.97–2.05 (m, 4H, CH2CH2OH Â 2) 13C NMR (125 MHz, CDCl3) d
112.27, 64.78, 58.67, 38.32. HRMS (pos. ion ESI) m/z calcd for
(M+Na)+ C7H14NaO4: 185.07843. Found: 185.07876.
6.6. Grignard reaction
A mixture of ketone 10, Grignard reagent (2.0 equiv for 11i,
2.5 equiv for 11g, 3.0 equiv for 11a–d, f, j, k, 4.0 equiv for 11e)
and CeCl3 (1.5 equiv) in dry THF (as a 1 mM solution) was stirred
at À78 °C for 3–6 h (except for 11j and 11k) or at 0 °C for 2 h
and room temperature for 4 h (for 11j and 11k). Aqueous saturated
NH4Cl was added to the mixture at À78 °C (except for 11j and 11k)
or at room temperature (for 11j and 11k), and the mixture was
stirred at room temperature for 30 min and extracted with AcOEt.
The organic layer was washed with H2O, brine, dried with Na2SO4,
and evaporated. The residue was purified by silica gel column chro-
matography (5% AcOEt in hexane for 11g, 10% AcOEt in hexane ex-
cept for 11g) to give the corresponding Grignard products.
6.3. 1-O-(tert-Butyldiphenylsilyl)-1,5-dihydroxy-3-pentanone-
ethylideneacetal (8)
A
mixture of
7
(2.61 g, 16.1 mmol) and NaH (964 mg,
6.7. 1-O-(tert-Butyldiphenylsilyl)-5-O-methoxymethyl-3-vinylp-
24.1 mmol) in THF (150 mL, HPLC grade) was stirred at 0 °C for
1 h, and TBDPSCl (6.27 mL, 24.1 mmol) was added at 0 °C. The mix-
ture was stirred at 0 °C for 30 min and room temperature for 69 h,
entan-1,3,5-triol (11a)
Compound 11a (2.02 g, 94%) was obtained as a colorless oil
from 10 (2.00 g, 5.00 mmol): 1H NMR (300 MHz, CDCl3) d 7.36–
7.68 (m, 10H, Ph  2), 5.75–5.84 (m, 1H, CH2@CH), 5.41–5.47,
5.19–5.23 (each m, each 1H, CH2@CH), 4.60 (s, 2H, MOM-CH2),
4.19 (s, 1H, OH), 3.59–3.94 (m, 4H, CH2OMOM, CH2OTBDPS), 3.35
(s, 3H, MOM-CH3), 1.62–2.06 (m, 4H, CH2CH2O Â 2), 1.04 (s, 9H,
and AcOH (500 lL) was added at 0 °C. The mixture was stirred at
0 °C for 10 min, and solid NaHCO3 and MeOH were added. The mix-
ture was stirred at 0 °C for 30 min, and the suspension was filtered
through Celite and washed with acetone. The combined filtrate and
washing were evaporated, and the residue was purified by silica
gel column chromatography (25% AcOEt in hexane) to give 8
(5.57 g, 86%) as a colorless oil: 1H NMR (300 MHz, CDCl3) d 7.37–
7.68 (m, 10H, Ph  2), 3.69–3.98 (m, 8H, acetal-CH2  2, CH2OH,
CH2OTBDPS), 2.74–2.78 (m, 1H, OH), 1.91–1.95 (m, 4H,
CH2CH2O Â 2), 1.04 (s, 9H, t-Bu); 13C NMR (100 MHz, CDCl3) d
135.52, 133.55, 129.63, 127.65, 111.09, 64.50, 59.86, 58.76, 39.29,
38.61, 26.76, 19.04. HRMS (pos. ion ESI) m/z calcd for (M+Na)+
C23H32NaO4Si: 423.19621. Found: 423.19611.
t-Bu); 13C NMR (100 MHz, CDCl3)
d 142.51, 135.53, 135.51,
132.80, 129.81, 129.79, 127.75, 127.71, 113.80, 96.51, 75.26,
64.20, 61.49, 55.26, 41.54, 40.59, 26.72, 18.95. HRMS (pos. ion
ESI) m/z calcd for (M+Na)+ C25H36NaO4Si: 451.22751. Found:
451.22721.
6.8. 1-O-(tert-Butyldiphenylsilyl)-3-ethyl-5-O-methoxymeth-
ylpentan-1,3,5-triol (11b)
6.4. 1-O-(tert-Butyldiphenylsilyl)-1,5-dihydroxy-3-pentanone (9)
Compound 11b (1.14 g, 87%) was obtained as a colorless oil
from 10 (1.21 g, 3.02 mmol): 1H NMR (300 MHz, CDCl3) d 7.38–
7.69 (m, 10H, Ph  2), 4.61 (s, 2H, MOM-CH2), 3.65–3.88 (m, 5H,
CH2OMOM, CH2OTBDPS, OH), 3.36 (s, 3H, MOM-CH3), 1.76–1.85
(m, 4H, CH2CH2O Â 2), 1.52–1.58 (m, 2H, Et-CH2), 1.05 (s, 9H, t-
Bu), 0.86 (t, 3H, Et-CH3, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d
135.48, 132.92, 129.73, 127.68, 96.42, 73.81, 64.18, 61.09, 55.24,
39.32, 37.59, 31.86, 26.69, 18.92, 8.19. HRMS (pos. ion ESI) m/z
calcd for (M+Na)+ C25H38NaO4Si: 453.24316. Found: 453.24342.
A mixture of 8 (2.45 g, 6.11 mmol) and pyridinium p-toluene-
sulfonate (PPTS, 6.14 g, 24.4 mmol) in acetone (55 mL) and H2O
(5.5 mL) was stirred with reflux for 4 h. The mixture was diluted
with AcOEt and washed with H2O, brine, dried with Na2SO4, and
evaporated. The residue was purified by silica gel column chroma-
tography (30% AcOEt in hexane) to give 9 (2.09 g, 96%) as a color-
less oil: 1H NMR (300 MHz, CDCl3) d 7.37–7.66 (m, 10H, Ph  2),
3.84–3.97 (m, 4H, CH2OH, CH2OTBDPS), 2.63–2.75 (m, 4H,
CH2CH2O Â 2), 2.49 (br s, 1H, OH), 1.03 (s, 9H, t-Bu); 13C NMR
(100 MHz, CDCl3) d 210.61, 135.44, 133.15, 129.70, 127.66, 59.48,
57.71, 45.82, 45.26, 26.66, 19.03. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C21H28NaO3Si: 379.16999. Found: 379.16975.
6.9. 1-O-(tert-Butyldiphenylsilyl)-3-(2-methoxymethyloxyeth-
yl)hexan-1,3,-diol (11c)
Compound 11c (2.04 g, 92%) was obtained as a colorless oil
from 10 (2.00 g, 5.00 mmol): 1H NMR (300 MHz, CDCl3) d 7.38–
7.69 (m, 10H, Ph  2), 4.60 (s, 2H, MOM-CH2), 3.65–3.87 (m, 5H,
CH2OMOM, CH2OTBDPS, OH), 3.36 (s, 3H, MOM-CH3), 1.76–1.85
(m, 4H, CH2CH2O Â 2), 1.45–1.50 (m, 2H, CH3CH2CH2), 1.26–1.41
(m, 2H, CH3CH2CH2), 1.05 (s, 9H, t-Bu), 0.89 (t, 3H, CH3CH2CH2,
J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 135.54, 132.96, 132.93,
129.79, 127.73, 96.47, 73.66, 64.26, 61.17, 55.29, 41.93, 39.86,
6.5. 1-O-(tert-Butyldiphenylsilyl)-5-O-methoxymethyl-1,5-dihy-
droxy-3-pentanone (10)
A mixture of 9 (114 mg, 320
and DIPEA (223 l, 1.28 mmol) in CH2Cl2 (3 mL) was stirred at
room temperature for 90 min, and then MeOH was added. The
lmol), MOMCl (49 ll, 640 lmol),
l

T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
1129
38.24, 26.76, 18.96, 17.05, 14.69. HRMS (pos. ion ESI) m/z calcd for
7.38–7.69 (m, 10H, Ph  2), 5.81–5.83 (m, 1H, CH2@CH), 5.02–5.08
(m, 2H, CH2@CH), 4.60 (s, 2H, MOM-CH2), 3.83–3.91, 3.69–3.73 (each
m, each 2H, CH2OTBDPS or CH2OMOM), 3.35 (s, 3H, MOM-CH3),
2.31–2.32 (m, 2H, CH2@CHCH2), 1.75–1.87 (m, 4H, CH2CH2O Â 2),
1.05 (s, 9H, t-Bu); 13C NMR (100 MHz, CDCl3) d 135.42, 134.06,
132.80, 129.71, 127.65, 117.79, 96.32, 73.27, 63.93, 60.94, 55.14,
44.22, 39.81, 38.21, 26.67, 18.86. HRMS (pos. ion ESI) m/z calcd for
(M+Na)+ C26H38NaO4Si: 465.24316. Found: 465.24243.
(M+Na)+ C26H40NaO4Si: 467.25881. Found: 467.25863.
6.10. 1-O-(tert-Butyldiphenylsilyl)-3-(2-methoxymethyloxyeth-
yl)heptan-1,3,-diol (11d)
Compound 11d (248 mg, quant.) was obtained as a colorless oil
from 10 (200 mg, 500 l
mol): 1H NMR (400 MHz, CDCl3) d 7.38–7.69
(m, 10H, Ph  2), 4.60 (s, 2H, MOM-CH2), 3.84–3.89, 3.63–3.70 (each
m, each 2H, CH2OTBDPS, CH2OMOM), 3.78 (s, 1H, OH), 3.36 (s, 3H,
MOM-CH3), 1.73–1.88 (m, 4H, CH2CH2O Â 2, CH3CH2CH2), 1.45–1.51
(m, 2H, CH3CH2CH2CH2), 1.21–1.29 (m, 4H, CH3CH2CH2CH2,
CH3CH2CH2CH2), 1.05 (s, 9H, t-Bu), 0.89 (t, 3H, CH3CH2CH2CH2,
J = 6.4 Hz); 13C NMR (100 MHz, CDCl3) d 135.54, 132.96, 132.94,
129.79, 127.74, 96.47, 73.68, 64.26, 61.18, 55.29, 39.87, 39.25,
38.23, 26.76, 25.99, 23.26, 18.96, 14.05. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C27H42NaO4Si: 481.27446. Found: 481.27322.
6.15. 1-O-(tert-Butyldiphenylsilyl)-3-ethynyl-5-O-methoxym-
ethylpentan-1,3,5-triol (11j)
Compound 11j (1.55 g, 79%) was obtained as a light yellow oil
from 10 (1.83 g, 4.58 mmol): 1H NMR (500 MHz, CDCl3) d 7.36–
7.73 (m, 10H, Ph  2), 4.80 (s, 1H, OH), 4.63 (s, 2H, MOM-CH2),
4.20–4.25, 3.81–3.97 (each m, 1H and 3H, CH2OTBDPS or CH2O-
MOM), 3.37 (s, 3H, MOM-CH3), 2.50 (s, 1H, ethynyl-H), 2.06–2.12,
1.97–2.02, 1.83–1.87 (each m, 2H, 1H, and 1H, CH2CH2O Â 2),
1.05 (s, 9H, t-Bu); 13C NMR (125 MHz, CDCl3) d 135.49, 135.42,
132.62, 129.73, 129.71, 127.66, 127.61, 96.36, 85.44, 73.04, 70.34,
64.43, 61.79, 55.19, 42.64, 41.43, 26.63, 18.87. HRMS (pos. ion
ESI) m/z calcd for (M+Na)+ C25H34NaO4Si: 449.21186. Found:
449.21144.
6.11. 1-O-(tert-Butyldiphenylsilyl)-3-(2-methoxymethyloxyeth-
yl)-5-methylhexan-1,3,-diol (11e)
Compound 11e (2.73 g, 85%) was obtained as a colorless oil
from 10 (2.80 g, 7.00 mmol): 1H NMR (400 MHz, CDCl3) d 7.38–
7.69 (m, 10H, Ph  2), 4.60 (s, 2H, MOM-CH2), 3.84–3.87, 3.63–
3.71 (each m, each 2H, CH2OTBDPS, CH2OMOM), 3.35 (s, 3H,
MOM-CH3), 1.71–1.89 (m, 5H, CH2CH2O Â 2, (CH3)2CHCH2), 1.39–
1.42 (m, 2H, (CH3)2CHCH2), 1.05 (s, 9H, t-Bu), 0.92–0.96 (m, 6H,
6.16. 1-O-(tert-Butyldiphenylsilyl)-3-(2-methoxymethyloxyeth-
yl)-4-hexyn-1,3,-diol (11k)
(CH3)2CHCH2); 13C NMR (100 MHz, CDCl3)
d
135.44, 132.83,
Compound 11k (1.36 g, 77%) was obtained as a colorless oil
from 10 (1.60 g, 4.00 mmol): 1H NMR (500 MHz, CDCl3) d 7.38–
7.74 (m, 10H, Ph  2), 4.63 (s, 2H, MOM-CH2), 4.18–4.23, 3.79–
3.95 (each m, 1H and 3H, CH2OTBDPS or CH2OMOM), 3.37 (s, 3H,
MOM-CH3), 2.03–2.08, 1.92–1.96, 1.78–1.83 (each m, 2H, 1H, and
1H, CH2CH2O Â 2), 1.85 (s, 3H, Me), 1.05 (s, 9H, t-Bu); 13C NMR
132.82, 129.69, 127.64, 96.36, 74.10, 64.30, 61.16, 55.18, 47.90,
40.32, 38.81, 26.66, 24.68, 23.79, 18.86. HRMS (pos. ion ESI) m/z
calcd for (M+Na)+ C27H42NaO4Si: 481.27446. Found: 481.27385.
6.12. 1-O-(tert-Butyldiphenylsilyl)-5-O-methoxymethyl-3-
cyclopropyl-pentan-1,3,5-triol (11f)
(100 MHz, CDCl3) d 135.52, 135.47, 132.80, 132.71, 129.72,
129.68, 127.67, 127.64, 96.37, 81.00, 80.82, 70.52, 64.72, 62.03,
55.12, 43.03, 41.83, 26.65, 18.89, 3.47. HRMS (pos. ion ESI) m/z
calcd for (M+Na)+ C26H36NaO4Si: 463.22751. Found: 463.22726.
Compound 11f (0.43 g, 89%) was obtained as a colorless oil from
10 (0.96 g, 3.90 mmol): 1H NMR (500 MHz, CDCl3) d 7.67–7.70 (m,
4H, Ph), 7.38–7.44 (m, 6H, Ph), 4.61 (s, 2H, MOM-CH2), 3.83–3.89,
3.75–3.81 (each m, each 2H, CH2OTBDPS, CH2OMOM), 3.76 (s, 1H,
OH), 3.36 (s, 3H, MOM-CH3), 1.93–1.87 (m, 2H, CH2CH2O), 1.78–
1.75 (m, 2H, CH2CH2O), 1.04 (s, 9H, t-Bu), 0.69 (m, 1H, CH of cyclo-
propyl), 0.52–0.43 (m, 2H, CH2 of cyclopropyl), 0.34–0.32 (d, 2H,
CH2 of cyclopropyl, J = 8.5 Hz): 13C NMR (125 MHz, CDCl3) d
135.74, 133.14, 129.99, 127. 93, 96.71, 71.69, 64.82, 61.86, 60.56,
55.49, 42.25, 41.30, 26.98, 19.13. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C26H38NaO4Si: 465.24316. Found: 465.24308.
6.17. Deprotection of the MOM group
A mixture of the Grignard products and ZrCl4 (0.5 equiv) in i-
PrOH (as a 1 mM solution) was stirred at 90 °C for 30 min. The mix-
ture was partitioned between AcOEt and H2O, and the organic layer
was washed with brine, dried with Na2SO4, and evaporated. The
residue was purified by silica gel column chromatography (25%
AcOEt in hexane) to give the corresponding alcohols.
6.13. 1-O-(tert-Butyldiphenylsilyl)-5-O-methoxymethyl-3-(1-
6.18. 1-O-(tert-Butyldiphenylsilyl)-3-vinylpentan-1,3,5-triol (12a)
methylvinyl)pentan-1,3,5-triol (11g)
Compound 12a (1.42 g, 78%) was obtained as a colorless oil
from 11a (2.02 g, 4.72 mmol): 1H NMR (300 MHz, CDCl3) d 7.39–
7.68 (m, 10H, Ph  2), 5.76 (dd, 1H, CH2@CH, Jtrans = 22.8 Hz,
Jcis = 13.6 Hz), 5.56 (dd, 1H, CH2@CH (trans), Jvicinal = 22.8 Hz,
Jgeminal = 1.6 Hz), 5.33 (dd, 1H, CH2@CH (cis), Jvicinal = 13.6 Hz,
Jgeminal = 1.6 Hz), 4.72 (s, 1H, OH), 3.73–3.96 (m, 4H, CH2OH,
CH2OTBDPS), 3.32 (br s, 1H, OH), 1.52–2.14 (m, 4H, CH2CH2O Â 2),
1.04 (s, 9H, t-Bu); 13C NMR (100 MHz, CDCl3) d 142.01, 135.45,
135.42, 132.26, 132.22, 129.91, 129.87, 127.79, 127.71, 114.72,
78.04, 61.90, 59.50, 41.96, 40.71, 26.60, 18.82. HRMS (pos. ion
ESI) m/z calcd for (M+Na)+ C23H32NaO3Si: 407.20129. Found:
407.20051.
Compound 11g (2.52 g, 85%) was obtained as a colorless oil
from 10 (2.67 g, 6.66 mmol): 1H NMR (400 MHz, CDCl3) d 7.37–
7.67 (m, 10H, Ph  2), 5.24, 4.99 (each s, each 1H, CH2@C), 4.60
(s, 2H, MOM-CH2), 4.28 (br s, 1H, OH), 3.53–3.87 (m, 4H,
CH2OTBDPS, CH2OMOM), 3.35 (s, 3H, MOM-CH3), 1.71–2.03 (m,
4H, CH2CH2O Â 2), 1.61 (s, 3H, Me), 1.03 (s, 9H, t-Bu); 13C NMR
(100 MHz, CDCl3)
d 147.11, 135.43, 135.41, 132.72, 132.70,
129.68, 129.65, 127.63, 127.58, 112.20, 96.46, 77.34, 64.06, 61.32,
55.13, 39.89, 38.72, 26.60, 19.53, 18.84. HRMS (pos. ion ESI) m/z
calcd for (M+Na)+ C26H38NaO4Si: 465.24316. Found: 465.24369.
6.14. 1-O-(tert-Butyldiphenylsilyl)-3-(2-methoxymethyloxyeth-
yl)-5-hexen-1,3,-diol (11i)
6.19. 1-O-(tert-Butyldiphenylsilyl)-3-ethylpentan-1,3,5-triol (12b)
Compound 11i (1.78 g, quant.) was obtained as a light yellow oil
Compound 12b (1.09 g, 68%) was obtained as a light yellow oil
from 10 (1.60 g, 4.00 mmol): 1H NMR (400 MHz, CDCl3)
d
from 11b (1.79 g, 4.17 mmol): 1H NMR (400 MHz, CDCl3)
d

1130
T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
7.40–7.69 (m, 10H, Ph  2), 3.81–3.93 (m, 4H, CH2OTBDPS, CH2OH),
1.65–1.93 (m, 6H, CH2CH2O Â 2, Et-CH2), 1.05 (s, 9H, t-Bu), 0.84 (t,
3H, Et-CH3, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 135.52, 132.51,
132.43, 129.97, 129.96, 127.85, 127.84, 76.34, 61.41, 59.50, 38.74,
38.20, 31.68, 26.73, 18.92, 8.61. HRMS (pos. ion ESI) m/z calcd for
(M+Na)+ C23H34NaO3Si: 409.21694. Found: 409.21610.
hydrogen atmosphere. The mixture was filtered off, and the filtrate
was evaporated. The residue was purified by silica gel column
chromatography (33% AcOEt in hexane) to give 12h (1.51 g, quant.)
as a colorless oil: 1H NMR (400 MHz, CDCl3) d 7.40–7.69 (m, 10H,
Ph  2), 4.32 (br s, 1H, OH), 3.83–3.96 (m, 4H, CH2OTBDPS, CH2OH),
3.68 (br s, 1H, OH), 1.61–2.03 (m, 5H, CH2CH2O Â 2, (CH3)2CH), 1.05
(s, 9H, t-Bu), 0.95, 0.88 (each d, each 3H, (CH3)2CH, each J = 6.6 Hz);
13C NMR (125 MHz, CDCl3) d 135.53, 132.51, 132.42, 129.99,
129.96, 127.85, 127.83, 78.28, 61.32, 59.43, 35.40, 35.31, 34.68,
26.74, 17.25, 16.83. HRMS (pos. ion ESI) m/z calcd for (M+Na)+
C24H36NaO3Si: 421.23259. Found: 423.23154.
6.20. 1-O-(tert-Butyldiphenylsilyl)-3-(2-hydroxyethyl)hexan-
1,3,-diol (12c)
Compound 12c (1.20 g, 67%) was obtained as a light yellow oil
from 11c (1.99 g, 4.47 mmol): 1H NMR (400 MHz, CDCl3) d 7.40–
7.69 (m, 10H, Ph  2), 4.31 (s, 1H, OH), 3.82–3.93 (m, 4H,
CH2OTBDPS, CH2OH), 1.63–1.94 (m, 6H, CH2CH2O Â 2, CH3CH2CH2),
1.16–1.31 (m, 2H, CH3CH2CH2), 1.05 (s, 9H, t-Bu), 0.91 (t, 3H,
CH3CH2CH2, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 135.54,
132.49, 132.40, 129.98, 127.84, 76.39, 61.44, 59.57, 41.68, 39.39,
38.73, 26.75, 18.92, 17.50, 14.67. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C24H36NaO3Si: 423.23259. Found: 423.23204.
6.25. 1-O-(tert-Butyldiphenylsilyl)-3-(2-hydroxyethyl)-5-hex-
en-1,3,-diol (12i)
Compound 12i (1.34 g, 86%) was obtained as a colorless oil from
11i (1.74 g, 3.93 mmol): 1H NMR (500 MHz, CDCl3) d 7.38–7.69 (m,
10H, Ph  2), 5.69–5.77 (m, 1H, CH2@CH), 5.03–5.08 (m, 2H,
CH2@CH), 3.82–3.98, (m, 4H, CH2OTBDPS, CH2OMOM), 2.47, 2.41
(each dd, each 1H, CH2@CHCH2, Jvicinal = 7.5 Hz, Jgeminal = 14.0 Hz),
1.88–1.94, 1.65–1.83 (each m, 1H and 3H, CH2CH2O Â 2), 1.06 (s,
9H, t-Bu); 13C NMR (100 MHz, CDCl3) d 135.41, 133.70, 132.34,
132.24, 129.88, 127.76, 118.25, 75.83, 61.25, 59.32, 43.87, 39.45,
38.71, 26.64, 18.81. HRMS (pos. ion ESI) m/z calcd for (M+Na)+
C24H34NaO3Si: 421.21694. Found: 421.21837.
6.21. 1-O-(tert-Butyldiphenylsilyl)-3-(2-hydroxyethyl)heptan-
1,3,-diol (12d)
Compound 12d (1.70 g, 90%) was obtained as a colorless oil
from 11d (2.10 g, 4.57 mmol): 1H NMR (400 MHz, CDCl3) d 7.40–
7.70 (m, 10H, Ph  2), 3.83–3.93 (m, 4H, CH2OTBDPS, CH2OH),
1.58–1.95 (m, 6H, CH2CH2O Â 2, CH3CH2CH2CH2), 1.28–1.33 (m,
4H, CH3CH2CH2CH2, CH3CH2CH2CH2), 1.05 (s, 9H, t-Bu), 0.88–0.92
(m, 3H, CH3CH2CH2CH2); 13C NMR (100 MHz, CDCl3) d 135.52,
132.48, 132.37, 129.96, 127.85, 127.83, 76.42, 61.44, 59.55, 39.37,
38.96, 38.70, 26.73, 26.41, 23.23, 18.91, 14.00. HRMS (pos. ion
ESI) m/z calcd for (M+Na)+ C25H38NaO3Si: 437.24824. Found:
437.24760.
6.26. 1-O-(tert-Butyldiphenylsilyl)-3-ethynylpentan-1,3,5-triol
(12j)
Compound 12j (1.40 g, 93%) was obtained as a light yellow oil
from 11j (1.67 g, 3.92 mmol): 1H NMR (500 MHz, CDCl3) d 7.37–
7.74 (m, 10H, Ph  2), 5.19 (s, 1H, OH), 4.31–4.36, 4.10–4.18,
3.85–3.90 (each m, 1H, 1H, and 2H, CH2OTBDPS or CH2OMOM),
2.96 (br s, 1H, OH), 2.56 (s, 1H, ethynyl-H), 2.13–2.17, 2.03–2.08,
1.86–1.91, 1.69–1.74 (each m, each 1H, CH2CH2O Â 2), 1.05 (s,
9H, t-Bu); 13C NMR (100 MHz, CDCl3) d 135.51, 135.41, 132.16,
132.13, 129.90, 129.85, 127.76, 127.67, 85.37, 73.46, 72.57, 62.29,
59.89, 43.75, 41.95, 26.58, 18.82. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C23H30NaO3Si: 405.18564. Found: 405.18531.
6.22. 1-O-(tert-Butyldiphenylsilyl)-3-(2-hydroxyethyl)-5-meth-
ylhexan-1,3,-diol (12e)
Compound 12e (1.96 g, 82%) was obtained as a colorless oil
from 11e (2.65 g, 5.78 mmol): 1H NMR (400 MHz, CDCl3) d 7.40–
7.69 (m, 10H, Ph  2), 3.86–3.97 (m, 4H, CH2OTBDPS, CH2OH),
1.65–1.99 (m, 5H, CH2CH2O Â 2, (CH3)2CHCH2), 1.48–1.60 (m, 2H,
(CH3)2CHCH2), 1.05 (s, 9H, t-Bu), 0.97, 0.92 (each d, each 3H,
6.27. 1-O-(tert-Butyldiphenylsilyl)-3-(2-hydroxyethyl)-4-hex-
yn-1,3,-diol (12k)
(CH3)2CHCH2, each, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3)
d
135.55, 132.46, 132.37, 129.99, 127.87, 127.85, 76.86, 61.47,
59.63, 47.89, 40.07, 39.41, 26.74, 24.79, 24.75, 24.06, 18.92. HRMS
(pos. ion ESI) m/z calcd for (M+Na)+ C25H38NaO3Si: 437.24824.
Found: 437.24788.
Compound 12k (1.12 g, 81%) was obtained as a light yellow oil
from 11k (1.53 g, 3.47 mmol): 1H NMR (500 MHz, CDCl3) d 7.37–
7.75 (m, 10H, Ph  2), 5.01 (s, 1H, OH), 4.29–4.34, 4.11–4.15,
3.83–3.89 (each m, 1H, 1H, and 2H, CH2OTBDPS or CH2OMOM),
3.07 (br s, 1H, OH), 2.09–2.15, 1.99–2.04, 1.82–1.86, 1.65–1.71
(each m, each 1H, CH2CH2O Â 2), 1.88 (s, 3H, Me), 1.05 (s, 9H, t-
Bu); 13C NMR (100 MHz, CDCl3) d 135.60, 135.55, 132.45, 132.30,
129.96, 129.89, 127.84, 127.78, 81.64, 80.90, 72.83, 62.53, 60.17,
44.12, 42.44, 26.68, 18.90, 3.56. HRMS (pos. ion ESI) m/z calcd for
(M+Na)+ C24H32NaO3Si: 419.20129. Found: 419.20104.
6.23. 1-O-(tert-Butyldiphenylsilyl)-3-(1-methylvinyl)pentan-
1,3,5-triol (12g)
Compound 12g (251 mg, 80%) was obtained as a light yellow oil
from 11g (349 mg, 790 l
mol): 1H NMR (400 MHz, CDCl3) d 7.38–
7.68 (m, 10H, Ph  2), 5.41, 5.13 (each s, each 1H, CH2@C), 4.84 (br
s, 1H, OH), 3.71–3.87 (m, 4H, CH2OTBDPS, CH2OH), 3.37 (br s, 1H,
OH), 1.70–2.11 (m, 4H, CH2CH2O Â 2), 1.66 (s, 3H, Me), 1.04 (s, 9H,
t-Bu); 13C NMR (100 MHz, CDCl3) d 146.83, 135.41, 135.38, 132.23,
132.17, 129.86, 129.81, 127.74, 127.70, 113.13, 80.41, 61.90, 59.56,
40.16, 38.96, 26.55, 19.50, 18.78. HRMS (pos. ion ESI) m/z calcd for
(M+Na)+ C24H34NaO3Si: 421.21694. Found: 437.21606.
6.28. Oxidation from the alcohol to the carboxylic acid
A mixture of the alcohol and IBX (1.5 equiv) in DMSO (as a 0.1 M
solution, except for 12c) or the alcohol and Dess–Martin periodinane
(3.0 equiv)inCH2Cl2 (asa0.1 Msolution, for12c)wasstirredat room
temperature for 15 h. Aqueous 0.1 M Na2S2O3 was added to the mix-
ture at room temperature, and the mixture was stirred for 10 min
and extracted with AcOEt. The organic layer was washed with H2O,
brine, dried with Na2SO4, and evaporated. A mixture of the crude
product, NaClO2 (3.0 equiv), NaH2PO4ÁH2O (1.0 equiv), and 2-
methyl-2-butene (4.0 equiv) in acetone/H2O = 3:1 (as a 0.1 M solu-
tion) was stirred at room temperature for 4 h. The mixture was
6.24. 1-O-(tert-Butyldiphenylsilyl)-3-hydroxy-3-(1-methyleth-
yl)pentan-1,5-diol (12h)
A mixture of 12g (1.47 g, 3.68 mmol) and Pd–C (400 mg) in i-
PrOH (35 mL) was stirred at room temperature for 18 h under a

T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
1131
diluted with AcOEt, washed with ice cooled aqueous 0.1 M HCl, H2O,
and brine, dried with Na2SO4, and evaporated. The residue was puri-
fied by silica gel column chromatography (25–75% AcOEt in hexane)
to give the corresponding carboxylic acids.
7.68 (m, 10H, Ph  2), 5.50 (br s, 1H, OH), 3.87–3.99 (m, 2H,
CH2OTBDPS), 2.54–2.62 (m, 2H, CH2CO2H), 1.73–1.92 (m, 3H,
CH2CH2O, (CH3)2CHCH2), 1.51–1.63 (m, 2H, (CH3)2CHCH2), 1.06 (s,
9H, t-Bu), 0.99, 0.94 (each d, each 3H, (CH3)2CHCH2, each
J = 6.6 Hz); 13C NMR (100 MHz, CDCl3) d 173.00, 135.37, 135.32,
131.80, 131.66, 130.12, 130.10, 127.90, 127.86, 74.77, 61.29,
47.50, 43.96, 38.42, 26.62, 24.49, 24.37, 23.87, 18.79. HRMS (pos.
ion ESI) m/z calcd for (M+Na)+ C25H36NaO4Si: 451.22751. Found:
451.22784.
6.29. 5-O-(tert-Butyldiphenylsilyl)-3,5-dihydroxy-3-vinylpenta-
noic acid (13a)
Compound 13a (1.12 g, 76%) was obtained as a colorless oil
from 12a (1.42 g, 3.70 mmol): 1H NMR (400 MHz, CDCl3) d 7.41–
7.65 (m, 10H, Ph  2), 5.83 (dd, 1H, CH2@CH, Jtrans = 16.8 Hz,
Jcis = 10.4 Hz), 5.55 (d, 1H, CH2@CH (trans), J = 16.8 Hz), 5.33 (d,
1H, CH2@CH (cis), J = 10.4 Hz), 3.91–3.97, 3.78–3.80 (each m, each
1H, CH2OTBDPS), 2.67, 2.58 (each d, each 1H, CH2CO2H, J = 15.6 Hz),
2.05–2.13, 1.66–1.70 (each m, each 1H, CH2CH2O), 1.05 (s, 9H, t-
Bu); 13C NMR (100 MHz, CDCl3) d 172.58, 140.04, 135.45, 135.42,
131.96, 131.90, 130.18, 130.11, 127.97, 127.89, 115.52, 74.96,
61.70, 45.64, 39.48, 26.69, 18.89. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C23H30NaO4Si: 421.18056. Found: 421.18084.
6.34. 5-O-(tert-Butyldiphenylsilyl)-3,5-dihydroxy-3-(1-methyl-
vinyl)pentanoic acid (13g)
Compound 13g (1.35 g, 73%) was obtained as a colorless oil
from 12g (1.79 g, 4.48 mmol): 1H NMR (400 MHz, CDCl3) d 7.39–
7.65 (m, 10H, Ph  2), 5.32, 5.09 (each s, each 1H, CH2@C), 3.84–
3.89, 3.73–3.78 (each m, each 1H, CH2OTBDPS), 2.72, 2.67 (each
d, each 1H, CH2CO2H, J = 15.4 Hz), 2.01–2.08, 1.82–1.86 (each m,
each 1H, CH2CH2O), 1.72 (s, 3H, Me), 1.05 (s, 9H, t-Bu); 13C NMR
(100 MHz, CDCl3)
d 172.30, 145.36, 135.35, 135.32, 131.86,
6.30. 5-O-(tert-Butyldiphenylsilyl)-3,5-dihydroxy-3-ethylpenta-
noic acid (13b)
131.79, 130.05, 129.99, 127.86, 127.77, 113.32, 77.27, 61.58,
44.41, 37.80, 26.57, 19.13, 18.80. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C24H32NaO4Si: 435.19621. Found: 435.19619.
Compound 13b (700 mg, 78%) was obtained as a colorless oil
from 12b (871 mg, 2.25 mmol): 1H NMR (400 MHz, CDCl3) d
7.39–7.67 (m, 10H, Ph  2), 3.85–3.98 (m, 2H, CH2OTBDPS), 2.59,
2.53 (each d, each 1H, CH2CO2H, J = 15.6 Hz), 1.66–1.88 (m, 4H,
CH2CH2O, Et-CH2), 1.06 (s, 9H, t-Bu), 0.91 (t, 3H, Et-CH3,
J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 174.85, 135.46, 135.42,
132.29, 132.22, 130.00, 129.98, 127.85, 127.82, 74.16, 61.09,
42.80, 38.15, 32.00, 26.69, 18.87, 8.08. HRMS (pos. ion ESI) m/z
calcd for (M+Na)+ C23H32NaO4Si: 423.19621. Found: 423.19677.
6.35. 5-O-(tert-Butyldiphenylsilyl)-3,5-dihydroxy-3-(1-methyl-
ethyl)pentanoic acid (13h)
Compound 13h (1.51 g, 99%) was obtained as a light yellow oil
from 12h (1.48 g, 3.70 mmol): 1H NMR (500 MHz, CDCl3) d 7.39–
7.67 (m, 10H, Ph  2), 3.95–3.99, 3.88–3.92 (each m, each 1H,
CH2OTBDPS), 2.58, 2.51 (each d, each 1H, CH2CO2H, J = 15.8 Hz),
2.01–2.06 (m, 1H, (CH3)2CH), 1.88–1.93, 1.78–1.83 (each m, each
1H, CH2CH2O), 1.07 (s, 9H, t-Bu), 0.99, 0.90 (each d, each 3H,
(CH3)2CH, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) d 170.53, 135.35,
135.32, 131.88, 131.84, 130.11, 130.07, 127.89, 127.84, 76.72,
61.12, 39.90, 35.01, 34.89, 26.63, 18.81, 17.00, 16.80. HRMS (pos.
ion ESI) m/z calcd for (M+Na)+ C24H34NaO4Si: 437.21186. Found:
437.21238.
6.31. 3-{2-(tert-Butyldiphenylsilyloxy)ethyl}-3-hydroxyhexa-
noic acid (13c)
Compound13c (447 mg, 59%) was obtained as a colorless oilfrom
12c (739 mg, 1.84 mmol): 1H NMR (400 MHz, CDCl3) d7.41–7.67 (m,
10H, Ph  2), 3.85–3.96 (m, 2H, CH2OTBDPS), 2.59, 2.52 (each d, each
1H, CH2CO2H, J = 15.6 Hz), 1.76–1.92 (m, 2H, CH2CH2O), 1.57–1.67
(m, 2H, CH3CH2CH2), 1.31–1.39 (m, 2H, CH3CH2CH2), 1.06 (s, 9H, t-
Bu), 0.92 (t, 3H, CH3CH2CH2, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3)
d 173.31, 135.36, 135.33, 131.91, 131.79, 130.08, 130.05, 127.87,
127.83, 74.24, 61.23, 43.38, 41.64, 37.95, 26.63, 18.80, 17.02,
14.33. HRMS (pos. ion ESI) m/z calcd for (M+Na)+ C24H34NaO4Si:
437.21186. Found: 437.21247.
6.36. 3-{2-(tert-Butyldiphenylsilyloxy)ethyl}-3-hydroxy-5-hex-
enoic acid (13i)
Compound 13i (995 mg, 74%) was obtained as an yellow oil
from 12i (1.30 g, 3.27 mmol): 1H NMR (500 MHz, CDCl3) d 7.39–
7.67 (m, 10H, Ph  2), 5.74–5.80 (m, 1H, CH2@CH), 5.08–5.15 (m,
2H, CH2@CH), 3.95–4.00, 3.86–3.91, (each m, each 2H, CH2OTBDPS
or CH2OMOM), 2.60, 2.56 (each d, each 1H, CH2CO2H, J = 15.8 Hz),
6.32. 3-{2-(tert-Butyldiphenylsilyloxy)ethyl}-3-hydroxyhepta-
noic acid (13d)
2.47, 2.41 (each dd, each 1H, CH2@CHCH2, Jvicinal = 7.5 Hz, Jgeminal
=
14.0 Hz), 1.88–1.93, 1.78–1.83 (each m, each 1H, CH2CH2O), 1.06 (s,
9H, t-Bu); 13C NMR (100 MHz, CDCl3) d 175.51, 135.29, 132.89,
132.38, 132.36, 129.76, 127.65, 118.79, 73.12, 60.69, 43.86, 42.80,
39.15, 26.57, 18.74. HRMS (pos. ion ESI) m/z calcd for (M+Na)+
C24H32NaO4Si: 435.19621. Found: 435.19639.
Compound 13d (1.31 g, 79%) was obtained as a colorless oil
from 12d (1.60 g, 3.86 mmol): 1H NMR (400 MHz, CDCl3) d 7.41–
7.67 (m, 10H, Ph  2), 5.38 (br s, 1H, OH), 3.85–3.97 (m, 2H,
CH2OTBDPS), 2.59, 2.53 (each d, each 1H, CH2CO2H, J = 15.2 Hz),
1.77–1.92 (m, 2H, CH2CH2O), 1.64–1.67 (m, 2H, CH3CH2CH2CH2),
1.28–1.31 (m, 4H, CH3CH2CH2CH2, CH3CH2CH2CH2), 1.06 (s, 9H, t-
Bu), 0.90 (t, 3H, CH3CH2CH2CH2, J = 6.8 Hz); 13C NMR (100 MHz,
CDCl3) d 174.14, 135.45, 135.43, 132.14, 132.04, 130.10, 130.08,
127.92, 127.89, 74.15, 61.24, 43.43, 39.15, 38.30, 26.71, 25.87,
23.00, 18.89, 13.91. HRMS (pos. ion ESI) m/z calcd for (M+H)+
C29H33O4Si: 429.24242. Found: 429.24286.
6.37. 5-O-(tert-Butyldiphenylsilyl)-3,5-dihydroxy-3-ethynylpen-
tanoic acid (13j)
Compound 13j (908 mg, 64%) was obtained as a light yellow oil
from 12j (1.36 g, 3.55 mmol): 1H NMR (500 MHz, CDCl3) d 7.39–
7.72 (m, 10H, Ph  2), 4.22–4.27, 3.89–3.93 (each m, each 1H,
CH2OTBDPS), 2.89, 2.84 (each d, each 1H, CH2CO2H, J = 15.8 Hz),
2.55 (s, 1H, ethynyl-H), 2.14–2.16, 1.90–1.94 (each m, each 1H,
CH2CH2O), 1.06 (s, 9H, t-Bu); 13C NMR (100 MHz, CDCl3) d 174.71,
135.33, 135.28, 132.33, 129.73, 129.71, 127.72, 127.62, 84.08,
73.27, 68.17, 61.29, 45.96, 41.65, 26.54, 18.74. HRMS (pos. ion ESI)
m/z calcd for (M+Na)+ C23H28NaO4Si: 419.16491. Found: 419.16488.
6.33. 3-{2-(tert-Butyldiphenylsilyloxy)ethyl}-3-hydroxy-5-me-
thylhexanoic acid (13e)
Compound 13e (1.63 g, 84%) was obtained as a colorless oil
from 12e (1.87 g, 4.51 mmol): 1H NMR (400 MHz, CDCl3) d 7.42–

1132
T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
6.38. 3-{2-(tert-Butyldiphenylsilyloxy)ethyl}-3-hydroxy-4-hexy-
6.44. 4-Hydroxy-4-(2-methylpropyl)-2-oxotetrahydropyrane (1e)
noic acid (13k)
Compound 1e (149 mg 88%) was obtained as a light yellow oil
Compound 13k (686 g, 62%) was obtained as a light yellow oil
from 12k (1.07 g, 2.71 mmol): 1H NMR (500 MHz, CDCl3) d 7.40–
7.73 (m, 10H, Ph  2), 4.25–4.30, 3.86–3.90 (each m, each 1H,
CH2OTBDPS), 2.89, 2.84 (each d, each 1H, CH2CO2H, J = 15.5 Hz),
2.11–2.15, 1.77–1.81 (each m, each 1H, CH2CH2O), 1.85 (s, 3H,
Me), 1.06 (s, 9H, t-Bu); 13C NMR (100 MHz, CDCl3) d 174.61,
135.41, 135.37, 132.42, 132.33, 129.79, 129.75, 127.68, 127.65,
81.60, 79.41, 68.67, 61.72, 46.52, 41.89, 26.57, 18.80, 3.41. HRMS
(pos. ion ESI) m/z calcd for (M+Na)+ C24H30NaO4Si: 433.18056.
Found: 433.18014.
from 13e (421 mg, 982 l
mol): 1H NMR (400 MHz, CDCl3) d 4.58–
4.64, 4.32–4.37 (each m, each 1H, CH2O), 2.66, 2.52 (each d, each
1H, CH2COO, J = 17.2 Hz), 1.83–1.90 (m, 3H, CH2CH2O,
(CH3)2CHCH2), 1.48–1.58 (m, 2H, (CH3)2CHCH2), 1.02, 1.00 (each
s, each 3H, (CH3)2CHCH2); 13C NMR (100 MHz, CDCl3) d 171.45,
70.25, 65.93, 50.63, 43.46, 34.28, 24.48, 24.44, 23.35. HRMS (pos.
ion ESI) m/z calcd for (M+Na)+ C9H16NaO3: 195.09917. Found:
195.09929.
6.45. 4-Hydroxy-4-(1-methylvinyl)-2-oxotetrahydropyrane (1g)
6.39. Deprotection of the TBDPS group
Compound 1g (99 mg 70%) was obtained as an yellow oil from
13g (371 mg, 899 l
mol): 1H NMR (500 MHz, CDCl3) d 5.04, 4.96
A solution of the carboxylic acid in acetone/aqueous 1.0 M
HCl = 1:1 (as a 1 mM solution) was stirred at room temperature
for 24 h and evaporated. The residue was purified by silica gel col-
umn chromatography (75% AcOEt in hexane) to give the corre-
sponding lactones.
(each s, each 1H, CH2@C), 4.63 (ddd, 1H, CH2O, J = 0.8, 3.8,
9.0 Hz), 4.36 (ddd, 1H, CH2O, J = 4.0, 4.0, 5.5 Hz), 2.75, 2.68 (each
d, each 1H, CH2COO, J = 17.0 Hz), 2.08–2.14, 1.91–1.96 (each m,
each 1H, CH2CH2O), 1.84 (s, 3H, Me); 13C NMR (100 MHz, CDCl3)
d 171.18, 147.58, 111.20, 71.73, 65.94, 42.01, 32.65, 18.09. HRMS
(pos. ion ESI) m/z calcd for (M+Na)+ C8H12NaO3: 179.06787. Found:
179.06769.
6.40. 4-Hydroxy-2-oxo-4-vinyltetrahydropyrane (1a)
Compound 1a (32 mg 85%) was obtained as a colorless oil from
6.46. 4-Hydroxy-4-(1-methylethyl)-2-oxotetrahydropyrane (1h)
13a (104 mg, 261 l
mol): 1H NMR (400 MHz, CDCl3) d 5.97 (dd, 1H,
CH2@CH, Jtrans = 17.6 Hz, Jcis = 10.4 Hz), 5.33 (d, 1H, CH2@CH (trans),
J = 17.6 Hz), 5.22 (d, 1H, CH2@CH (cis), J = 10.4 Hz), 4.61–4.68, 4.35–
4.40 (each m, each 1H, CH2O), 2.66 (s, 2H, CH2CO2), 1.93–2.06, 1.89–
1.92 (each m, each 1H, CH2CH2O); 13C NMR (100 MHz, CDCl3) d
170.50, 141.83, 114.03, 70.01, 65.88, 42.52, 33.93. Anal. Calcd for
C7H10O3: C, 59.14; H, 7.09. Found: C, 59.33; H, 6.99.
Compound 1h (157 mg 80%) was obtained as a light yellow oil
from 13h (520 mg, 1.25 mmol): 1H NMR (500 MHz, CDCl3) d 4.60
(ddd, 1H, CH2O, J = 4.0, 11.0, 11.0 Hz), 4.35 (ddd, 1H, CH2O, J = 3.5,
3.5, 10.5 Hz), 2.60, 2.55 (each d, each 1H, CH2COO, J = 17.0 Hz),
1.93(ddd, 1H, CH2CH2O, J = 4.0, 5.5, 10.5 Hz), 1.79–1.84 (m, 1H,
(CH3)2CH), 1.70–1.77 (m, 1H, CH2CH2O), 0.98, 0.96 (each s, each
3H, (CH3)2CH); 13C NMR (100 MHz, CDCl3) d 172.11, 72.37, 65.92,
40.52, 37.46, 31.30, 16.37, 16.13. HRMS (pos. ion ESI) m/z calcd
for (M+Na)+ C8H14NaO3: 181.08352. Found: 181.08354.
6.41. 4-Ethyl-4-hydroxy-2-oxotetrahydropyrane (1b)
Compound 1b (92 mg 72%) was obtained as a colorless oil from
13b (354 mg, 883
l
mol): 1H NMR (400 MHz, CDCl3) d 4.58–4.64,
6.47. 4-Hydroxy-2-oxo-4-(2-propenyl)tetrahydropyrane (1i)
4.33–4.38 (each m, each 1H, CH2O), 2.61, 2.52 (each d, each 1H,
CH2CO2H, J = 17.6 Hz), 1.83–1.91 (m, 2H, CH2CH2O), 1.59–1.66 (m,
2H, Et-CH2), 0.98 (t, 3H, Et-CH3, J = 7.2 Hz); 13C NMR (100 MHz,
CDCl3) d 171.45, 70.18, 66.01, 42.61, 34.79, 33.41, 7.12. HRMS
(pos. ion ESI) m/z calcd for (M+Na)+ C7H12NaO3: 167.06787. Found:
167.06792.
Compound 1i (129 mg 82%) was obtained as a light yellow oil
from 13i (413 mg, 1.00 mmol): 1H NMR (500 MHz, CDCl3) d 5.81–
5.89 (m, 1H, CH2@CH), 5.17–5.34 (m, 2H, CH2@CH), 4.57–4.65 (m,
1H, CH2O), 4.35 (ddd, 1H, CH2O, J = 3.5, 3.5, 5.5 Hz), 2.61, 2.55 (each
d, each 1H, CH2COO, J = 17.5 Hz), 2.31–2.49 (m, 1H, CH2@CHCH2),
1.91–1.97, 1.83–1.88 (each m, each 1H, CH2CH2O); 13C NMR
(100 MHz, CDCl3) d 171.26, 131.57, 120.13, 69.31, 65.85, 46.35,
42.49, 33.49. HRMS (pos. ion ESI) m/z calcd for (M+Na)+
C8H12NaO3: 179.06787. Found: 179.06769.
6.42. 4-Hydroxy-2-oxo-4-propyltetrahydropyrane (1c)
Compound 1c (124 mg 87%) was obtained as a light yellow oil
from 13c (372 mg, 897 l
mol): 1H NMR (400 MHz, CDCl3) d 4.57–
4.64, 4.32–4.37 (each m, each 1H, CH2O), 2.62, 2.53 (each d, each
1H, CH2CO2, J = 17.6 Hz), 1.83–1.96 (m, 2H, CH2CH2O), 1.52–1.60
(m, 2H, CH3CH2CH2), 1.38–1.43 (m, 2H, CH3CH2CH2), 0.98 (t, 3H,
CH3CH2CH2, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d 171.36,
70.06, 65.97, 44.60, 43.02, 33.89, 16.11, 14.24. Anal. Calcd for
C8H14O3: C, 60.74; H, 8.92. Found: C, 60.32; H, 9.00.
6.48. 4-Ethynyl-4-hydroxy-2-oxotetrahydropyrane (1j)
Compound 1j (109 mg 77%) was obtained as a colorless oil from
13j (397 mg, 1.00 mmol): 1H NMR (500 MHz, CDCl3) d 7.39–7.72
(m, 10H, Ph  2), 4.55–4.61, 4.40–4.46 (each m, each 1H, CH2O),
2.95, 2.86 (each d, each 1H, CH2COO, J = 17.5 Hz), 2.64 (s, 1H, ethy-
nyl-H), 2.21–2.27, 2.14–2.19 (each m, each 1H, CH2CH2O); 13C NMR
(100 MHz, CDCl3) d 169.33, 84.18, 73.54, 65.57, 63.58, 44.35, 35.42.
HRMS (pos. ion ESI) m/z calcd for (M+Na)+ C7H8NaO3: 179.03657.
Found: 163.03694.
6.43. 4-Butyl-4-hydroxy-2-oxotetrahydropyrane (1d)
Compound 1d (144 mg 84%) was obtained as a light yellow oil
from 13d (429 mg, 1.00 mmol): 1H NMR (400 MHz, CDCl3) d
4.57–4.64, 4.32–4.37 (each m, each 1H, CH2O), 2.62, 2.52 (each d,
each 1H, CH2COO, J = 17.2 Hz), 1.84–1.95 (m, 2H, CH2CH2O), 1.57–
1.59 (m, 2H, CH3CH2CH2CH2), 1.35–1.37 (m, 4H, CH3CH2CH2CH2,
CH3CH2CH2CH2), 0.93 (t, 3H, CH3CH2CH2CH2, J = 6.6 Hz); 13C NMR
(100 MHz, CDCl3) d 171.54, 69.80, 65.97, 42.88, 41.88, 33.73,
24.82, 22.73, 13.80. Anal. Calcd for C9H16O3: C, 62.77; H, 9.36.
Found: C, 62.86; H, 9.50.
6.49. 4-Hydroxy-2-oxo-4-(2-propynyl)tetrahydropyrane (1k)
Compound 1k (32 mg 85%) was obtained as a colorless oil from
13k (104 mg, 261 l
mol): 1H NMR (500 MHz, CDCl3) d 4.52–4.57,
4.40–4.44 (each m, each 1H, CH2O), 2.91, 2.80 (each d, each 1H,
CH2COO, J = 17.5 Hz), 2.08–2.20 (m, 2H, CH2CH2O), 1.86 (s, 3H,
Me); 13C NMR (100 MHz, CDCl3) d 169.61, 81.67, 79.98, 65.84,

T. Kudoh et al. / Bioorg. Med. Chem. 18 (2010) 1124–1134
1133
63.89, 44.98, 35.97, 3.25. HRMS (pos. ion ESI) m/z calcd for (M+Na)+
6.53. Recyclization analysis
C8H10NaO3: 177.05222. Found: 177.05199.
A solution of the lactone in aqueous 2.0 M LiOH (1 mL) was stir-
red at room temperature for 1 h, and evaporated. The residue was
purified by silica gel column chromatography (25% MeOH in
CH2Cl2) to give the corresponding hydrolyzed carboxylic acids.
They were kept as a dry form or a solution of MeOH, H2O, or a buf-
fer (50 mM HEPES and 1 mM MgCl2, pH 8.0) at room temperature.
Only the buffer solution resulted in no recyclization after a week.
6.50. 3-Cyclopropyl-1-O-(methoxymethyl)pentan-1,3,5-triol (14)
A mixture of 11f (885 mg, 2.00 mmol) and TBAF (1.0 M in
THF, 2.40 mL, 2.40 mmol) in THF (10 mL) was stirred at room
temperature for 5 h and evaporated. The residue was purified
by silica gel column chromatography (75% AcOEt in hexane) to
give 14 (350 mg, 86%) as a colorless oil: 1H NMR (400 MHz,
CDCl3) d 4.63 (s, 2H, MOM-CH2), 3.80–4.02 (m, 4H, CH2OH,
CH2OMOM), 3.49 (s, 1H, OH), 3.39 (s, 3H, MOM-CH3), 3.21 (br
s, 1H, OH), 1.75–2.10 (m, 4H, CH2CH2O Â 2), 0.40–0.75 (m, 5H,
cyclopropyl). 13C NMR (125 MHz, CDCl3) d 96.40, 72.72, 64.77,
59.44, 55.39, 41.86, 39.95, 18.68, À0.56, À0.60. HRMS (pos. ion
ESI) m/z calcd for (M+Na)+ C10H20NaO4: 227.12538. Found:
227.12558.
6.54. Preparation of linearized mevalonate analogues
Lactones 1a–1h were dissolved in 2.0 M aqueous LiOH and
incubated for 1 h at 37 °C. Each solution was titrated to pH 7.0
using concentrated HCl to give 2a–2h. Compounds were diluted
to a final concentration of 2 mM for storage at À80 °C.
6.55. In vitro enzyme assay
6.51. Methyl 3-cyclopropyl-3-hydroxy-5-(methoxymethoxy)-
pentanoate (15)
Recombinant S. pneumoniae MK (Uniprot: Q8DR51) and PMK
(Uniprot: Q8DR49) were overexpressed in Escherichia coli as
dual-tagged His6-GST fusion proteins. The purification and re-
moval of affinity tags was carried out as previously described.11
The phosphorylation of substrates generates ADP, which can be
monitored continuously using the well-established pyruvate ki-
nase/lactate dehydrogenase coupled assay system11 (see Fig. 2 leg-
end). MK reactions were initiated by the addition of racemic
A
mixture of 14 (490 mg, 2.40 mmol) and IBX (1.01 g,
3.60 mmol) in DMSO (24 mL) was stirred at room temperature
for 15 h. Aqueous 0.1 M Na2S2O3 was added to the mixture at room
temperature, and the mixture was stirred for 10 min and extracted
with AcOEt. The organic layer was washed with H2O, brine, dried
with Na2SO4, and evaporated. A mixture of the crude product, Na-
ClO2 (651 mg, 7.20 mmol), NaH2PO4ÁH2O (331 mg, 2.40 mmol), and
2-methyl-2-butene (1.00 mL, 9.60 mmol) in acetone (18 mL) and
H2O (6 mL) was stirred at room temperature for 3 h. The mixture
was diluted with AcOEt, and washed with ice cooled aqueous
0.1 M HCl, H2O, and brine. The water layer was extracted with
AcOEt/acetone = 1:1 (three times), and the combined organic lay-
ers were dried with Na2SO4, and evaporated. The crude mixture
and K2CO3 (719 mg, 5.20 mmol) in anhydrous DMF (8.7 mL) was
stirred at 0 °C for 20 min. MeI (0.16 mL, 2.60 mmol) was added to
the mixture at 0 °C, and the mixture was stirred at room tempera-
ture for 8 h. MeOH (1 mL) was added to the mixture at 0 °C, and the
mixture was stirred at room temperature for 10 min. The mixture
was partitioned between AcOEt and H2O, and the organic layer was
washed with brine, dried with Na2SO4, and evaporated. The residue
was purified by silica gel column chromatography (10% AcOEt in
hexane) to give 15 (257 mg, 46%) as a colorless oil: 1H NMR
(500 MHz, CDCl3) d 4.62 (s, 2H, MOM-CH2), 3.82–3.86, 3.75–3.80
(each m, each 1H, CH2OMOM), 3.70 (s, 3H, CO2CH3), 3.54 (br, 1H,
OH) 3.37 (s, 3H, MOM-CH3), 2.61 (s, 2H, CH2CO2CH3), 1.93–2.02
(m, 2H, CH2CH2O), 0.85–0.90 (m, 1H, cycloproyl), 0.31–0.49 (m,
4H, cyclopropyl). 13C NMR (125 MHz, CDCl3) d 172.71, 96.37,
70.12, 64.28, 55.25, 51.45, 45.02, 39.92, 18.89, 0.09, À0.57. HRMS
(pos. ion ESI) m/z calcd for (M+Na)+ C11H20NaO5: 255.12029.
Found: 255.12039.
compound (2a–2k) to 200
reaction progress was monitored at 386 nm (
l
M (100
l
M in each enantiomer), and
e
386 = 0.61 mMÀ1).
Following completion of the MK reaction (0.2–4 h), PMK was
added to the cuvette and phosphorylation of compounds 3a–c,
e, f, i, j was monitored. Three progress curves were obtained for
each compound and were averaged to produce the traces shown
in Figure 2. The reproducibility of the replicate curves was as-
sessed statistically using the pooled standard deviation (Mev:
5.41, 243 points; 2a: 6.23,1286 points; 2b: 5.03, 1847 points;
2c: 4.33, 484 points; 2e: 1.82, 310 points; 2f: 0.41, 3642 points;
2i: 5.20, 242 points; 2j: 2.51, 2894 points; Pmev: 2.05, 76 points;
3a: 4.94, 493 points; 3b: 0.83, 284 points; 3c: 3.70, 301 points; 3e:
3.26, 561 points; 3f: 1.69, 508 points; 3i: 2.58, 218 points; 3j:
1.51, 847 points).
Acknowledgment
The authors are grateful to the National Institutes of Health (AI
068989) for financial support of this research.
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