Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphate analogues as chelation-based inhibitors of methylerythritol phosphate synthase

Article abstract of DOI:10.1021/jo0516786

A series of 1-deoxy-D-xylulose 5-phosphate (DXP) analogues were synthesized and evaluated as inhibitors of E. coli methylerythritol phosphate (MEP) synthase. In analogues 1-4, the methyl group in DXP was replaced by hydroxyl, hydroxylamino, methoxy, and a

Full text of DOI:10.1021/jo0516786

Synthesis and Evaluation of 1-Deoxy-D-xylulose 5-Phosphate
Analogues as Chelation-Based Inhibitors of Methylerythritol
Phosphate Synthase
Joel R. Walker and C. Dale Poulter*
315 South 1400 East RM 2020, Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
poulter@chemistry.utah.edu
Received August 9, 2005
A series of 1-deoxy-D-xylulose 5-phosphate (DXP) analogues were synthesized and evaluated as
inhibitors of E. coli methylerythritol phosphate (MEP) synthase. In analogues 1-4, the methyl
group in DXP was replaced by hydroxyl, hydroxylamino, methoxy, and amino moieties, respectively.
In analogues 5 and 6, the acetyl moiety in DXP was replaced by hydroxymethyl and aminomethyl
groups. These compounds were designed to coordinate to the active site divalent metal in MEP
synthase. The carboxylate (1), methyl ester (3), amide (4), and alcohol (5) analogues were inhibitors
with IC50’s ranging from 0.25 to 1.0 mM. The hydroxamic acid (2) and amino (6) analogues did not
inhibit the enzyme.
Introduction
Isoprenoid compounds are the largest class of natural
product metabolites with over 30 000 known members.1
Although diverse in structure and function, these mol-
ecules are constructed from two simple five-carbon build-
ing blockssisopentenyl diphosphate (IPP) and dimeth-
ylallyl diphosphate (DMAPP). Until the late 1980s, IPP
and DMAPP were thought to originate exclusively from
acetyl CoA through the mevalonate (MVA) pathway.
However, labeling patterns from isotope incorporation
experiments, which were inconsistent with the MVA
pathway,2-4 led to the discovery of a nonmevalonate route
to isoprenoid compounds from glyceraldehyde 3-phos-
phate and pyruvate. The first committed step of the new
route is the synthesis of 2-C-methyl-D-erythritol 4-phos-
phate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP)
by MEP synthase5 (EC 1.1.1.267) (Figure 1). Since the
FIGURE 1. Reactions catalyzed by MEP synthase.
discovery of MEP as a precursor for the isoprenoid
pathway in the early 1990s, the remaining intermediates
between MEP and IPP/DMAPP have been characterized
and the genes that encode the biosynthetic enzymes have
been identified.6
* Corresponding author. Phone: 801-581-6685. Fax: 801-581-4391.
(1) Sacchettini, J. C.; Poulter, C. D. Science 1997, 277, 1788-1789.
(2) Flesch, G.; Rohmer, M. Eur. J. Biochem. 1988, 175, 405-411.
(3) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H.
Biochem. J. 1993, 295, 517-524.
(4) Rohmer, M.; Seemann, M.; Horbach, S.; Bringer-Meyer, S.; Sahm,
H. J. Am. Chem. Soc. 1996, 118, 2564-2566.
(5) Proteau, P. J. Bioorg. Chem. 2004, 32, 483-493.
10.1021/jo0516786 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/12/2005
J. Org. Chem. 2005, 70, 9955-9959
9955

Walker and Poulter
SCHEME 1a
a Key: (a) 0.6 equiv of NaBH4, MeOH, 0 °C, 2 h, 32%; (b)
P(OBn)3, I2, pyridine, CH2Cl2, -30 °C to rt, 2 h, 79%; (c) KOH,
MeOH, rt, 16 h, 99%; (d) (i) H2, Pd/C, t-BuOH, rt, 2 h, (ii) H2O, 3
d, 85%; (e) (i) H2, Pd/C, MeOH, rt, 3 h, (ii) H2O, 2 d, 48%.
FIGURE 2. Structures of fosmidomycin and DXP analogues.
The MEP entry into the isoprenoid pathway is found
in most eubacteria, green algae, and the plastids of
higher plants, while the MVA entry is found in eukary-
otes, including the cytosol of higher plants, and archae-
bacteria.6,7 Furthermore, the MEP pathway has been
found in the vestigial plastids of several pathogenic
parasites, including the malarial parasite Plasmodium
falciparum.8 Common pathogenic bacteria that rely on
the MEP root include those responsible for tuberculosis,
bronchitis, and meningitis.9 Inhibition or disruption of
the enzymes in the MEP pathway is lethal and suggests
a novel drug target for chemotherapy of infectious
diseases.
Fosmidomycin (Figure 2) is a phosphonic acid metabo-
lite from Steptomyces rubellomurinus discovered in the
late 1970s.10,11 The compound is a broad spectrum
antibiotic that inhibits bacterial cell wall biosynthesis.
The mechanism of action of fosmidomycin remained a
mystery until after the discovery of the MEP pathway,
when it was found to be a slow, tight binding inhibitor
of MEP synthase with KI ) 21 nM.12,13 Fosmidomycin has
been used to treat malarial infections, although it has a
high ED50 because of its poor bioavailability.8 The hy-
droxamic acid isostere of fosmidomycin and its methyl
derivative are also potent inhibitors of MEP synthase.14
These results suggest that MEP synthase is a valid target
for small molecules. We now report the synthesis and
evaluation of six analogues of DXP.
Results
Synthesis. The structures of fosmidomycin and hy-
droxyaminooxobutyl phosphonic acid, known inhibitors
of MEP synthase, along with the six DXP analogues
reported in this study are shown in Figure 2. The
syntheses of carboxylate 1 and methyl ester 3 are given
in Scheme 1. (-)-Dimethyl 2,3-O-isopropylidene-D-tar-
trate (7) was treated with 0.6 equiv of sodium borohy-
dride to give monoester 8.15 The ester was phosphorylated
with dibenzyl phosphoroiodidate (DBPI) generated in
situ16 to give benzyl phosphate monoester 9,17 which was
then saponified to provide carboxylic acid 10. The car-
boxylate moiety in 10 was partially esterified when the
benzyl groups were removed by hydrogenation in metha-
nol. This side reaction was avoided by replacing methanol
with tert-butyl alcohol. The isopropylidene moiety was
removed to give 1 by allowing the debenzylated phos-
phate to stand in water for 2 days. Methyl ester analogue
3 was obtained from 9 by removing the benzyl groups in
methanol.
Analogues 2, 4, and 5 were obtained from methyl ester
9 as shown in Scheme 2. The methyl ester was treated
with hydroxylamine, and the resulting hydroxamic acid
(11) was deprotected as described previously to give 2.
Amide 4 was obtained by treating 9 with ammonia,
followed by deprotection, while alcohol 5 was generated
by reduction of 9 with lithium borohydride, followed by
deprotection.
(6) Kuzuyama, T.; Seto, H. Nat. Prod. Rep. 2003, 20, 171-183.
(7) Lichtenthaler, H. K. Annu. Rev. Plant Mol. Biol. 1999, 50, 47-
65.
(8) Jomaa, H.; Wiesner, J.; Sanderbrand, S.; Altincicek, B.; Weide-
meyer, C.; Hintz, M.; Turbackova, I.; Eberl, M.; Zeidler, J.; Lichtentha-
ler, H. K.; Soldati, D.; Beck, E. Science 1999, 285, 1573-1576.
(9) Testa, C. A.; Brown, M. J. Curr. Pharm. Biotechnol. 2003, 4,
248-259.
(10) Kamiya, T.; Hemmi, K.; Takeno, H.; Hashimoto, M. Tetrahedron
Lett. 1980, 21, 95-98.
(11) Kuemmerle, H.-P.; Muranaka, T.; Soneoka, K.; Konishi, T. Int.
J. Clin. Pharm. Ther. Toxic. 1985, 23, 515-520.
(12) Kuzuyama, T.; Shimizu, T.; Takahashi, S.; Seto, H. Tetrahedron
Lett. 1998, 39, 7913-7916.
(13) Koppisch, A. T.; Fox, D. T.; Blagg, B. S.; Poulter, C. D.
Biochemistry 2002, 41, 236-243.
(14) Kuntz, L.; Tritsch, D.; Grosdemange-Billiard, C.; Hemmerlin,
A.; Willem, A.; Bach, T. J.; Rohmer, M. Biochem. J. 2005, 386, 127-
135.
The synthesis of the amino analogue 6 is outlined in
Scheme 3. C2-symmetric (-)-2,3-O-isopropylidene-D-
threitol (14) was monoprotected with TIPSCl to give 15.18
(15) Batsanov, A. S.; Begley, M. J.; Fletcher, R. J.; Murphy, J. A.;
Sherburn, M. S. J. Chem. Soc., Perkin Trans. 1 1995, 1281-1294.
(16) Gefflaut, T.; Lemaire, M.; Valentin, M.-L.; Bolte, J. J. Org.
Chem. 1997, 62, 5920-5922.
(17) Fox, D. T.; Poulter, C. D. J. Org. Chem. 2005, 70, 1978-1985.
9956 J. Org. Chem., Vol. 70, No. 24, 2005

Synthesis and Evaluation of 1-Deoxy-D-xylulose 5-Phosphate Analogues
SCHEME 2a
TABLE 1. Inhibition MEP Synthase with DXP
Analoguesa
compd
IC50b (µM)
1
2
3
4
5
6
551 ( 133
>5000c
1024 ( 235
253 ( 27
310 ( 73
>5000c
a Reaction conditions: 50 mM HEPES buffer, pH 7.6, 3 mM
MgCl2, 1 mg/mL BSA, 8 nM MEP synthase, 150 µM NADPH, 100
µM DXP, 100-5000 µM analogue, 100 µL final volume, 37 °C, 2
min. b Value obtained by plotting specific activity versus concen-
tration (n ) 3). c 50% inhibition not observed at 5000 µM.
treated with TBAF to give primary alcohol 18. The
alcohol was phosphorylated and debenzylated as de-
scribed previously. The isopropylidene and BOC groups
were removed with HCl to give amino analogue 6.
MEP Synthase Assays. Recombinant E. coli MEP
synthase was obtained as previously reported.13,19 The
conversion of DXP to MEP was monitored spectrospcopi-
cally by measuring the change in absorbance at 340 nm
upon oxidation of NADPH to NADP+. Steady-state
a Key: (a) NaOCH3, NH2OH‚HCl, MeOH, rt, 16 h, 80%; (b) (i)
H2, Pd/C, MeOH, rt, 2 h, (ii) H2O, 3 d, 43%; (c) 7 N NH3, MeOH,
reflux, 2 h, 99%; (d) (i) H2, Pd/C, MeOH, rt, 2 h, (ii) H2O, 5 d, 99%;
(e) LiBH4, ether, 0 °C, 1 h, 82%; (f) (i) H2, Pd/C, MeOH, rt, 2 h, (ii)
H2O, 3 d, 94%.
kinetic parameters for the recombinant his6-tagged en-
DXP
zyme used in this study were kcat ) 33 s-1 and Km
)
SCHEME 3a
81 µM. These values agree with those previously reported
for the E. coli enzyme.19
IC50’s for the DXP analogues were determined using
the standard assay for samples where the enzyme had
been preincubated with the inhibitors for 2 min. The
reaction was initiated by adding DXP. The results are
shown in Table 1. Two of the inhibitors, 4 and 5, had
DXP
IC50’s that were slightly higher (3-4-fold) than Km
.
The methyl ester (3) and carboxylic acid (1) analogues
were less potent, and less than 50% inhibition was seen
at 5 mM concentrations of the hydroxamic acid (2) and
the amino (6) analogues. None of the DXP analogues were
as potent as fosmidomycin.
Discussion
Several crystal structures have been reported for MEP
synthase.20-24 Overall, the structure consists of three
domains: an N-terminal domain that binds NADPH; a
“connective” domain that contains the active site; and a
C-terminal domain also involved in cofactor binding. The
active site consists of phosphate and divalent metal
binding motifs separated by a hydrophobic region. The
enzyme is active in buffers containing Mg2+ or Mn2+ and
it is unclear which the physiologically active metal is. In
the fosmidomycin-Mn2+ ternary complex,22 the N-formyl
hydroxylamine moiety engages the metal by a bidentate
a Key: (a) NaH, TIPSCl, THF, 0 °C to rt, 3 h, 94%; (b)
phthalimide, Ph3P, DIAD, THF, 0 °C to rt, 16 h, 99%; (c) (i)
N2H4‚H2O, EtOH, 60 °C, 1 h, (ii) di-tert-butyl dicarbonate, Na2CO3,
IPA/H2O, 0 °C to rt, 16 h, 95%; (d) 1 M TBAF, THF, 0 °C to rt, 1
h, 91%; (e) P(OBn)3, I2, pyridine, CH2Cl2, -30 °C to rt, 4 h, 81%;
(f) (i) H2, Pd/C, MeOH, rt, 4 h, (ii) 1 M HCl, 24 h, 52%.
(19) Fox, D. T.; Poulter, C. D. Biochemistry 2005, 44, 8360-8368.
(20) Reuter, K.; Sanderbrand, S.; Jomaa, H.; Wiesner, J.; Stein-
bacher, S.; Beck, E.; Hintz, M.; Klebe, G.; Stubbs, M. T. J. Biol. Chem.
2002, 277, 5378-5384.
(21) Yajima, S.; Nonaka, T.; Kuzuyama, T.; Seto, H.; Ohsawa, K. J.
Biol. Chem. (Tokyo) 2002, 131, 313-317.
(22) Steinbacher, S.; Kaiser, J.; Eisenreich, W.; Huber, R.; Bacher,
A.; Rohdich, F. J. Biol. Chem. 2003, 278, 18401-18407.
(23) Yajima, S.; Hara, K.; Sanders, J. M.; Yin, F.; Ohsawa, K.;
Wiesner, J.; Jomaa, H.; Oldfield, E. J. Am. Chem. Soc. 2004, 126,
10824-10825.
(24) MacSweeney, A.; Lange, R.; Fernandes, R. P. M.; Schulz, H.;
Dale, G. E.; Douangamath, A.; Proteau, P. J.; Oefner, C. J. Mol. Biol.
2005, 345, 115-127.
The hydroxyl moiety was displaced with phthalimide
using Mitsunobu conditions to generate 16. The phthal-
imide moiety was replaced with t-BOC, and 17 was
(18) Blagg, B. S. J.; Poulter, C. D. J. Org. Chem. 1999, 64, 1508-
1511.
J. Org. Chem, Vol. 70, No. 24, 2005 9957

Walker and Poulter
chain length of 2 is a poor inhibitor for MEP synthase
with IC50 ) 6.2 mM.17
In conclusion, a series of DXP analogues were synthe-
sized and evaluated as inhibitors of MEP synthase. The
most potent members of the group closely approximated
the size of DXP and contained functional groups that
could bind to the divalent metal in the active site.
Changes that compromise either of these two features
result a substantial decrease in potency.
FIGURE 3. Chelation of fosmidomycin and analogues to the
active site metal in MEP synthase.
interaction (Figure 3). This structure is believed to mimic
substrate binding of the C2 carbonyl oxygen and the C3
hydroxyl group in DXP. These two functionalities are
proposed to be in the inner coordination sphere of the
metal ion. A more recent structure, where MEP synthase
was cocrystallized with NADPH and fosmidomycin,
revealed a large conformational change in the “connec-
tive” domain24 to give a compact, solvent free, relatively
hydrophobic active site that tightly embraces the inhibi-
tor. The restricted volume of the pocket in the E‚
fosmidomycin‚NADPH complex explains why larger ana-
logues of DXP or fosmidomycin are not effective inhib-
itors.25
In the X-ray structure of the MEP synthase‚fos-
midomycin complex,22 the formyl oxygen and the N-
hydroxyl groups in fosmidomycin are coordinated to the
active site Mn2+. Presumably similar interactions occur
between the metal ion and the corresponding carbonyl
oxygens/hydroxyl groups in DXP and the rearranged
aldehyde (see Figure 1). DXP analogues 1-6 were
synthesized to explore the ability of a diol, â-amino
hydroxyl, and other R-hydroxycarbonyl moieties to inhibit
MEP synthase. The phosphate and diol portions of DXP
were preserved in all of the analogues. In 1-4, the methyl
group in DXP was replaced by hydroxyl, hydroxylamino,
methoxy, and amino moieties, respectively. In 5 and 6,
the acetyl moiety in DXP was replaced by hydroxymethyl
and aminomethyl groups. All of these compounds should,
in theory, be capable of coordinating with the active site
divalent metal.
The compounds that inhibit MEP synthase have R-hy-
droxycarboxylate (1), R-hydroxyester (3), R-hydroxyamide
(4), and 1,2-diol (5) moieties available for metal ion
binding. Analogue 4 was recently synthesized and shown
to inhibit (KI ) 90 µM) MEP synthase from Synechocys-
tis.26 Inhibition by 1, 3, and 4 in buffers containing Mg2+
suggests that negatively charged or neutral donor atoms
are accepted as chelators of the active site Mg2+. Inhibi-
tion by analogue 5 demonstrates that a carbonyl group
is not required for inhibition. Interestingly, amine 6 does
not inhibit the enzyme. Since Mg2+ forms complexes with
amines, the lack of inhibition may reflect that the amino
group in 6 is protonated. Also hydroxylamine analogue
2 is not an inhibitor. The lack of inhibition in this case
might be a result of the extremely confined volume of
the active site after the enzyme binds its substrates. The
chain of analogue 2 is one atom longer than that of the
potent hydroxamic acid isostere of fosmidomycin (Figure
2). In addition, a DXP analogue, where the methyl moiety
is replaced with an ethyl group, that approximates the
Experimental Section
(2S,3R)-5-(Bis-benzyloxyphosphoryloxymethyl)-2,2-
dimethyl[1,3]dioxolane-4-carboxylic Acid (10). To a solu-
tion of 9 (203 mg, 0.45 mmol) in methanol (60 mL) was added
1 M potassium hydroxide (2.3 mL, 2.3 mmol). After 16 h, the
solution was acidified to pH 3 with 1 M HCl and diluted with
ethyl acetate. The aqueous layer was extracted with ethyl
acetate, and the combined organic layers were dried (MgSO4)
and concentrated to give 195 mg (99%) of an oil: [R]D +8.66 (c
1
5.8, acetone); H NMR (CDCl3) δ 1.43 (s, 6H), 4.13-4.20 (m,
1H), 4.31-4.37 (m, 3H), 5.06 (d, 2H, J ) 3.9 Hz), 5.09 (d, 2H,
J ) 3.9 Hz), 7.33 (s, 10H); 13C NMR (CDCl3) δ 25.7, 26.7, 66.6,
69.9 (d, J ) 6.0 Hz), 74.5, 77.4 (d, J ) 6.0 Hz), 111.9, 128.1,
128.1, 128.6, 128.6, 128.7, 128.7, 135.3, 171.5; 31P NMR (CDCl3)
δ -0.32; HRMS (CI) calcd for C21H25O8P (M + H) 437.1365,
found 437.1363.
(2S,3R)-2,3-Dihydroxy-4-phosphonooxybutyric Acid (1).
To a solution of 10 (156 mg, 0.36 mmol) in tert-butyl alcohol
(20 mL) was added 10% Pd/C (5 mg). After 2 h under hydrogen
(1 atm) the suspension was filtered. The filtrate was concen-
trated and dissolved in water (10 mL). The mixture was
allowed to stir for 3 days at rt. Lyophilization gave 66 mg (85%)
1
of foam: [R]D -2.27 (c 1.1, H2O); H NMR (D2O) δ 3.88-4.01
(m, 2H), 4.25 (td, 1H, J ) 2.3, 6.5 Hz), 4.41 (d, 1H, J ) 2.3
Hz); 13C NMR (D2O) δ 65.8 (d, J ) 5.0 Hz), 70.3, 70.7 (d, J )
8.0 Hz), 175.8; 31P NMR (D2O) δ 1.00; HRMS (FAB) calcd for
C4H9O8P (M - H) 214.9915, found 214.9957.
(2S,3R)-Methyl 2,3-Dihydroxy-4-phosphonooxybutyrate
(3). To a solution of 9 (200 mg, 0.44 mmol) in methanol (15
mL) was added 10% Pd/C (20 mg). After 3 h under hydrogen
(1 atm), the suspension was filtered. The filtrate was concen-
trated and dissolved in water (10 mL). The mixture was
allowed to stir for 2 days at rt. Lyophilization, followed by
cellulose chromatography (7:3 2-propanol/water, 50 mM NH4-
HCO3), gave 53 mg (48%) of glassy foam: [R]D -9.13 (c 1.9,
H2O); 1H NMR (D2O) δ 3.77 (s, 3H), 3.86 (td, 2H, J ) 2.1, 6.8
Hz), 4.19 (td, 1H, J ) 2.1, 6.8 Hz), 4.47 (d, 1H, J ) 2.1 Hz);
13C NMR (D2O) δ 64.5 (d, J ) 4.6 Hz), 70.8, 71.3 (d, J ) 8.0
Hz), 174.7; 31P NMR (D2O) δ 3.05; HRMS (FAB) calcd for
C5H11O8P (M - H) 229.0113, found 229.0094.
Dibenzyl (2S,3R)-5-Hydroxycarbamoyl-2,2-dimethyl-
[1,3]dioxolan-4-ylmethyl Phosphate (11). To a solution of
hydroxylamine hydrochloride (62 mg, 0.88 mmol) in methanol
(25 mL) was added 30 wt % sodium methoxide (246 mg, 1.3
mmol). A solution of 9 (200 mg, 0.44 mmol) in methanol (5
mL) was cannulated into the reaction. After 16 h, the solution
was acidified to pH 3 using 0.1 M HCl, diluted with ethyl
acetate, and then washed with water and brine. The aqueous
layer was extracted with ethyl acetate, and the combined
organic layers were dried (MgSO4) and concentrated. The
residue was chromatographed (95:5 CH2Cl2/MeOH) to give 159
1
mg (80%) of oil: [R]D +2.48 (c 5.9, CH2Cl2); H NMR (CDCl3)
δ 1.42 (s, 6H), 4.14-4.40 (m, 4H), 5.06 (s, 2H), 5.09 (s, 2H),
7.35 (s, 10H); 13C NMR (CDCl3) δ 25.9, 26.7, 66.4 (d, J ) 5.5
Hz), 69.5 (d, J ) 4.0 Hz), 74.3, 77.8 (d, J ) 7.0 Hz), 111.7,
128.0, 128.6, 135.5 (d, J ) 5.0 Hz), 167.2; 31P NMR (CDCl3) δ
-0.12; HRMS (CI) calcd for C21H26NO8P (M + H) 452.1474,
found 452.1465.
(25) Silber, K.; Heidler, P.; Kurz, T.; Klebe, G. J. Med. Chem. 2005,
48, 3547-3563.
Dibenzyl(2S,3R)-5-Carbamoyl-2,2-dimethyl[1,3]dioxolan-
4-ylmethyl Phosphate (12). To a solution of 7 N ammonia
(26) Phaosiri, C.; Proteau, P. J. Bioorg. Med. Chem. Lett. 2004, 21,
5309-5312.
9958 J. Org. Chem., Vol. 70, No. 24, 2005

Synthesis and Evaluation of 1-Deoxy-D-xylulose 5-Phosphate Analogues
in methanol (50 mL) was added 9 (150 mg, 0.33 mmol) in
methanol (5 mL). After 2 h at 50 °C, the solvent was
evaporated to give 147 mg (quant) of an oil: [R]D +0.68 (c 5.6,
After being warmed to 60 °C for 1 h, the reaction mixture was
cooled and the precipitate filtered. The filtrate was concen-
trated, dissolved in CH2Cl2, and washed with brine. The
aqueous layer was extracted with CH2Cl2, and the organic
layers were concentrated. The residue was dissolved in 3:1
IPA/water (40 mL). After the mixture was cooled to 0 °C,
sodium carbonate (1.0 g) was added, followed by addition of
di-tert-butyl dicarbonate (0.73 g, 3.35 mmol) in IPA (10 mL).
The reaction mixture warmed to rt overnight, acidified to pH
2 with 1 M HCl, diluted with ethyl acetate, and washed with
brine. The aqueous layers were extracted with ethyl acetate,
and the combined organic layers were dried (MgSO4) and
concentrated. The residue was chromatographed (9:1 hexanes/
ethyl acetate) to give 0.90 g (95%) of an oil: [R]D +5.46 (c 4.3,
acetone); 1H NMR (CDCl3) δ 1.05-1.07 (m, 21H), 1.38 (s, 3H),
1.40 (s, 3H), 1.43 (s, 9H), 3.37-3.41 (m, 2H), 3.75-3.86 (m,
2H), 3.88-3.90 (m, 1H), 4.03 (dd, 1H, J ) 5.1, 12.3 Hz), 5.04
(br s, 1H); 13C NMR (CDCl3) δ 11.8, 17.9, 26.9, 27.1, 27.4, 28.3,
42.3, 63.6, 77.9, 79.3, 94.1, 108.9, 156.4; HRMS (CI) calcd for
C21H43NO5Si (M + H) 418.2989, found 448.2953.
1
acetone); H NMR (CDCl3) δ 1.40 (s, 3H), 1.41 (s, 3H), 4.13-
4.24 (m, 3H), 4.36-4.42 (m, 1H), 5.04 (d, 2H, J ) 1.3 Hz), 5.07
(d, 2H, J ) 1.3 Hz), 6.57 (s, 1H), 6.64 (s, 1H), 7.33 (s, 10H);
13C NMR (CDCl3) δ 25.9, 26.7, 66.7 (d, J ) 5.5 Hz), 69.2 (d, J
) 5.1 Hz), 75.0, 77.7 (d, J ) 7.5 Hz), 111.2, 127.8, 127.8, 128.4,
128.4, 135.6 (d, J ) 7.0 Hz), 173.0; 31P NMR (CDCl3) δ -0.03;
HRMS (CI) calcd for C21H26NO7P (M + H) 436.1525, found
436.1542.
Dibenzyl (2R,3R)-5-hydroxymethyl-2,2-dimethyl[1,3]-
dioxolan-4-ylmethyl Phosphate (13). A solution of 9 (500
mg, 1.11 mmol) in ether (100 mL) was cooled to 0 °C before 2
M lithium borohydride (1.1 mL, 2.2 mmol) was added. After 1
h, the solution was quenched with NH4Cl (satd), diluted with
ether, and washed with water and brine. The aqueous layer
was extracted with ether, and the combined organic layers
were dried (MgSO4) and concentrated. The residue was chro-
matographed (ethyl acetate) to give 384 mg (82%) of an oil:
[R]D +6.67 (c 4.8, acetone); 1H NMR (CDCl3) δ 1.38 (s, 3H),
1.41 (s, 3H), 3.60 (dd, 1H, J ) 4.2, 12.0 Hz), 3.74 (dd, 1H, J )
3.9, 12.0 Hz), 3.91-3.95 (m, 1H), 4.04-4.14 (m, 3H), 5.04-
5.08 (m, 4H), 7.36 (s, 10H); 13C NMR (CDCl3) δ 26.8, 27.0, 61.9,
66.9 (d, J ) 6.0 Hz), 69.4 (d, J ) 5.6 Hz), 75.9 (d, J ) 8.0 Hz),
77.8, 109.7, 127.9, 128.5, 135.5 (d, J ) 6.5 Hz); 31P NMR
(CDCl3) δ -0.27; HRMS (CI) calcd for C21H27O7P (M + H)
423.1573, found 423.1558.
tert-Butyl{(4R,5R)-5-Hydroxymethyl-2,2-dimethyl[1,3]-
dioxolan-4-ylmethyl}carbamate (18). A solution of 17 (5.32
g, 12.74 mmol) in THF (250 mL) was cooled to 0 °C before 1
M TBAF (15.3 mL, 15.3 mmol) was added via syringe. After
30 min, the reaction mixture was stirred at rt for 30 min. The
mixture was diluted with ethyl acetate and washed with water
and brine. The aqueous layers were extracted with ethyl
acetate, dried (MgSO4), and concentrated. The residue was
chromatographed (6:4 hexanes/ethyl acetate) to give 3.00 g
(91%) of an oil, which solidified upon standing: mp 69-71 °C;
[R]D +0.94 (c 2.9, acetone); 1H NMR (acetone-d6) δ 1.33 (s, 3H),
1.34 (s, 3H), 1.41 (s, 9H), 3.31 (dd, 2H, J ) 4.8, 11.3 Hz), 3.66
(d, 2H, J ) 4.8 Hz), 3.79-3.85 (m, 1H), 3.90-3.96 (m, 1H),
6.10 (br s, 1H); 13C NMR (acetone-d6) δ 27.4, 27.5, 28.7, 43.3,
63.0, 78.3, 79.0, 80.5, 109.3, 156.9, 206.5; HRMS (CI) calcd for
C12H23NO5 (M + H) 262.1654, found 262.1630.
2-{(4R,5R)-2,2-Dimethyl-5-triisopropylsilanyloxymethyl-
[1,3]dioxolan-4-ylmethyl}isoindole-1,3-dione (16). To a
solution of 15 (5.30 g, 16.7 mmol) in THF (250 mL) were
sequentially added phthalimide (2.69 g, 18.3 mmol) and
triphenylphosphine (4.81 g, 18.3 mmol). Upon cooling 0 °C,
diisopropyl azodicarboxylate (3.6 mL, 18.3 mmol) was added
dropwise. After the mixture was warmed to rt overnight, the
solvent was evaporated and resuspended in ether. The solid
was filtered and triturated with ether. The filtrate was
concentrated and chromatographed (8:2 hexanes/ethyl acetate)
to give 7.43 g (99%) of an oil, which solidified upon standing:
Acknowledgment. We thank Dr. D. Fox for sup-
plying recombinant enzyme. This work was supported
by NIH Grant No. GM22521.
1
mp 46-47 °C; [R]D +6.95 (c 3.8, acetone); H NMR (CDCl3) δ
1.03 (s, 18H), 1.35 (s, 3H), 1.40 (s, 3H), 3.76-4.02 (m, 5H),
4.38 (dd, 1H, J ) 6.0, 12.6 Hz), 7.71-7.72 (m, 2H), 7.84-7.85
(m, 2H); 13C NMR (CDCl3) δ 11.8, 17.9, 27.1, 27.2, 40.7, 63.8,
76.2, 79.7, 109.5, 123.3, 132.1, 133.9, 168.1; HRMS (CI) calcd
for C24H37NO5Si (M + H) 448.2519, found 448.2511.
Supporting Information Available: General methods;
experimental protocols for the syntheses of 2, 4-6, 8, 9, 15,
and 19; 1H, 13C, and 31P NMR spectra for compounds 1-6; 1H
and 13C NMR spectra for compounds 10-13 and 16-19; and
protocols for enzyme assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
tert-Butyl{(4R,5R)-2,2-Dimethyl-5-triisopropylsilanyloxy-
methyl[1,3]dioxolan-4-ylmethyl}carbamate (17). To a so-
lution of 16 (1.0 g, 2.23 mmol) in absolute ethanol (50 mL)
was added hydrazine hydrate (1.4 mL, 45 mmol) via syringe.
JO0516786
J. Org. Chem, Vol. 70, No. 24, 2005 9959