Mapping the active site of the bacterial enzyme LpxC using novel carbohydrate-based hydroxamic acid inhibitors

Article abstract of DOI:10.1081/CAR-200068781

LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase), an enzyme involved in the biosynthesis of lipid A, is crucial for the growth of Gram-negative bacteria. This enzyme has accordingly been identified as a potential target for the development of nove

Full text of DOI:10.1081/CAR-200068781

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Mapping the Active Site of the
Bacterial Enzyme LpxC Using Novel
Carbohydrate‐Based Hydroxamic Acid
Inhibitors
Xuechen Li a b , Amanda L. McClerren c , Christian R. H. Raetz c
Ole Hindsgaul a
&
a Department of Chemistry , University of Alberta , Edmonton, AB,
Canada
b Department of Chemistry and Chemical Biology , Harvard
University , Cambridge, MA, USA
c Department of Biochemistry , Duke University Medical Center ,
Durham, NC, USA
Published online: 16 Aug 2006.
To cite this article: Xuechen Li , Amanda L. McClerren , Christian R. H. Raetz & Ole Hindsgaul (2005)
Mapping the Active Site of the Bacterial Enzyme LpxC Using Novel Carbohydrate‐Based Hydroxamic
Acid Inhibitors , Journal of Carbohydrate Chemistry, 24:4-6, 583-609
To link to this article: http://dx.doi.org/10.1081/CAR-200068781
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Journal of Carbohydrate Chemistry, 24:583–609, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200068781
Mapping the Active
Site of the Bacterial Enzyme
LpxC Using Novel
Carbohydrate-Based
Hydroxamic Acid Inhibitors
Xuechen Li
Department of Chemistry, University of Alberta, Edmonton, AB, Canada and
Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
MA, USA
Amanda L. McClerren and Christian R. H. Raetz
Department of Biochemistry, Duke University Medical Center, Durham, NC, USA
Ole Hindsgaul
Department of Chemistry, University of Alberta, Edmonton, AB, Canada
LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase), an enzyme involved in
the biosynthesis of lipid A, is crucial for the growth of Gram-negative bacteria. This
enzyme has accordingly been identified as a potential target for the development of
novel antibiotics against Gram-negative bacteria. The carbohydrate-derived hydroxa-
mic acid 1 (1,5-anhydro-2-C-(carboxymethyl N-hydroxyamide)-2-deoxy-3-O-myristoyl-
D-glucitol) was previously shown to exhibit a wide spectrum of inhibitory activity
against LpxC enzymes. Here we describe the preparation of seven analogs of 1 and
their enzymatic evaluation. Two of the hydroxyl groups (OH-3 and 6) of the GlcNAc
residue were found to be involved in the binding interaction, and there is an important
hydrophobic interaction in the vicinity O-3 position with the enzyme that recognizes
aromatic as well as aliphatic substituents.
Keywords Carbohydrate analog, Enzyme inhibitor, Hydroxamic acid, Deacetylase
Received February 24, 2005; accepted May 5, 2005.
In memory of Jacques H. van Boom.
Address correspondence to Ole Hindsgaul, Carlsberg Laboratory, Gamle Carlsberg Vej 10,
DK-2500 Valby, Copenhagen, Denmark. E-mail: hindsgaul@crc.dk
583

X. Li et al.
584
INTRODUCTION
The discovery of compounds with novel modes of antibacterial action is
urgently needed to circumvent bacterial resistance. Lipopolysaccharide
(LPS), also known as endotoxin,[1] is the major structural constituent of the
outer membrane of Gram-negative bacteria protecting the sensitive inner
membrane. LPS, released from the dying bacteria after treatment with conven-
tional antibacterial agents, causes septic shock. The annual number of deaths
from septic shock is about 70,000 in the United States alone,[2] a figure compar-
able to AIDS. Therefore, the inhibition of the biosynthesis of LPS would have
multiple beneficial effects for the therapy of the Gram-negative infections by
killing the bacteria and reducing the amount of endotoxin released when
other antibiotics are administered simultaneously. Currently no marketed
antibiotics are directed against the LPS biosynthetic pathway. LPS consists
of lipid A, two KDO residues, the core oligosaccharide, and the O-antigen.
The lipid A and two KDO units have been shown to be minimally required
for the bacterial growth in general. Many Gram-negative bacteria, including
most pathogens, synthesized lipid A species resembling the one found in
Escherichia coli. Recently, lipid A has been recognized as a promising new
target for the development of antibiotics against Gram-negative bacteria.[3]
Of many enzymes involved in lipid A biosynthesis, the N-deacetylase
named LpxC, which catalyzes the first committed step in the pathway
(Sch. 1),[4] has been suggested to be a good pharmaceutical target[5] and has
attracted a lot of attention.[5–10] LpxC requires enzyme-bound Znþþ to effect
amide bond cleavage. Several classes of hydroxamic acid derivatives have
been reported to be potent inhibitors of LpxC (Fig. 1). Merck scientists first
reported phenyloxazoline hydroxamic acid derivatives that were potent
Scheme 1

Mapping the Active Site of LpxC
585
Figure 1: Structures of the substrate and inhibitors of LpxC.
inhibitors of LpxC.[5] Extensive series of analog failed to yield compounds that
were broad-spectrum inhibitors.[6] More recently, sulfonamide derivatives of
a-(R)-amino hydroxamic acid have been reported to show antibacterial activi-
ties against a wider panel of pathogens.[8]
Our laboratory conceived of the idea of incorporating the hydroxamic acid
directly into the carbohydrate substrate at the position of the acetamide group
undergoing enzymatic hydrolysis.[6] The rationale was that all of the enzymes
must recognize the natural substrate, so the more that an inhibitor had in
common with this substrate, the more likely it would be a broad-spectrum
inhibitor. Compound 1 (Fig. 1) was synthesized and indeed was shown to
exhibit a wide spectrum of inhibitory activity with an IC50 ¼ 7.0 + 0.5 mM
(A. aeolicus LpxC); 7.2 + 1.9 mM (E. coli LpxC).[6a] This class of LpxC inhibitor

X. Li et al.
586
contains the most potent compounds reported to date that inhibit the LpxCs of
the most evolutionarily diverse Gram-negative bacteria. To explore structure-
activity relationships, seven new analogs of 1, namely 2–8, have now been
synthesized and evaluated (Fig. 1) These compounds contain simple modifi-
cations in structure: replacement of OH groups by H or OMe, and variations
of the 14-carbon lipidic ester at O-3.
RESULTS AND DISCUSSION
Compound 1 is a 1,2-dideoxy-2-C-branched-monosaccharide. To the best of
our knowledge, there was no example reported in literature of the synthesis
of this type of compound starting from a carbohydrate substrate. We
recently, however, reported an efficient synthetic pathway for compound 1.[6b]
1,6-Anhydro-b-D-glucopyranose derivatives underwent reduction with Et3SiH
in the presence of a Lewis acid, providing rapid access to carbohydrate
synthons of this type. However, proper protecting groups were crucial for the
transformation: some protecting groups, like alkyl and silyl ethers, proved
effective, while the transformation was not successful with free hydroxyl or
acetyl groups (Sch. 2).[6b]
Synthesis of analogs of 1. The first attempt to obtain the 4-deoxy analog 2 is
shown in Scheme 3. Selective reductive ring opening of the benzylidene acetal
in 9[6b] using NaCNBH3/HCl in THF gave the 6-O-benzyl ether 10 in 82% yield.
However, attempted thiocarbonylation with either thiocarbonyldiimidazole or
pentafluorophenyl chlorothionoformate under standard conditions failed to
produce the desired products. It was assumed that the long chain at C-3 may
have blocked the reaction and that thiocarbonylation at the hydroxamic ester
nitrogen might be also occurring. To circumvent this problem, we O-acetylated
11, the precursor to 9,[6b] to produce 12, where the long-chain fatty acid ester
was no longer present. At the same time, the hydroxamic ester nitrogen was
Scheme 2

Mapping the Active Site of LpxC
587
Scheme 3: aReaction conditions: (a) NaCNBH3/HCl, THF, 82%; (b) Ac2O, pyridine;
(c) NaCNBH3/HCl, THF, 80%.
also protected with an acetyl group. Reductive ring opening of 12 now afforded
13 in good yield as expected (80%). Surprisingly, however, the attempted
thiocarbonylation was still not successful. Therefore, we reasoned that the
CONHOBn group may not be compatible with the thiocarbonylation
conditions.
Finally, the synthesis of the required 4-deoxy analog 2 was accomplished as
shown in Scheme 4, starting from 14,[6b] which allowed us to remove O-4 before
the labile CONHOBn group was introduced. Regioselective reductive ring
opening of the benzylidene acetal in 14 gave 15, which on thiocarbonylation
using thiocarbonyldiimidazole in refluxing toluene overnight gave 16 in 64%
over two steps. Treatment of 16 with Bu3SnH and AIBN gave 17 in 85%
yield. Ozonolysis of 17 followed by reduction with PPh3 gave aldehyde 18
(87%). Further oxidation to the carboxylic acid derivative using NaClO2 and
NaH2PO4 in t-BuOH/2-methyl-2-butene/H2O yielded 19 (100%). This
product was coupled with O-benzylhydroxylamine using EDC to afford the pro-
tected hydroxamic acid derivative 20 (70%). After hydrogenation (H2, Pd/C) in
AcOH, compound 2 was obtained (70%).
The conversion of the 4,6-O-benzylidene acetal in 14 to the 4-benzyl ether
was unsuccessful. This is because the standard conditions used in this

X. Li et al.
588
Scheme 4: aReaction conditions: (a) NaCNBH3/HCl, THF, 82%; (b) Im2C(S), toluene, 79%;
(c) AIBN, Bu3SnH, toluene, 85%; (d) O3, Ph3P, CH2Cl2, 87%; (e) NaClO2, NaH2PO4,
.
2-methyl-2-butene/H2O/t-BuOH, 100%; (f) BnONH2 HCl, TEA, EDC, CH2Cl2, 70%; (g) H2,
Pd/C. AcOH, 70%.
.
transformation, such as the combinations of LiAlH4/ACl3 or BH3 THF/(Lewis
acid), were not compatible with the presence of an ester group or a double bond.
Selective reductive ring opening of the benzylidene acetal could, however, be
[11]
.
achieved with Bu2BOTf and BH3 THF
from 9 (Sch. 5) to give 21 with
Scheme 5: aReaction condition: (a) Bu2BOTf, BH3 THF, 71%; (b) I2, Ph3P, imidazole, toluene,
80%; (c) Bu3SnH, AIBN, toluene, 82%; (d) H2, Pd/C, AcOH, 70%.
.

Mapping the Active Site of LpxC
589
OH-6 free (71%). Treatment of 21 with I2, Ph3P and imidazole afforded 22 in
80% yield. Deiodination with Bu3SnH and AIBN successfully furnished 23
(82%). Hydrogenation (H2, Pd/C, AcOH) of 23 then gave 3 (70%).
The preparation of the 6-OMe derivative 4 would require an O-methylation
reaction at some point. We therefore decided to install the methyl ether in the
reaction sequence before introduction of the CONHOBn group to avoid possible
side reactions (Sch. 6). O-Myristoylation of 24[6b] gave 25 (84%), which on treat-
ment with SnCl4 and Et3SiH afforded 26 (46%). Methylation of 26 was easily
accomplished. Treatment with methyl trifluoromethanesulfonate and 2,6-
di(tert-butyl-4-methyl) pyridine gave 27 (98%). Finally, as in Scheme 4, the
reaction sequence was completed by ozonolysis, followed by reduction to the
aldehyde 28 (80%) and further oxidation to carboxylic acid 29 (100%).
.
Coupling with O-benzylhydroxylamine hydrochloride (BnONH2 HCl) gave
the hydroxamide 30 (71%), and upon hydrogenation (H2, Pd/C, AcOH),
compound 4 was obtained (69%).
The preparation of 4-O-methyl derivative 5 is shown in Scheme 7.
Compound 31, obtained from D-glucal according to the literature,[12] was sub-
jected to Birch reduction to afford 32 (85%). Cyclization of 32 gave 33 (89%).
Scheme 6: aReaction conditions: (a) myristoyl chloride, pyridine, 84%; (b) SnCl4, Et3SiH,
CH2Cl2, 46%; (c) MeOTf, DBMP, CH2Cl2, 98%; (d) O3, Ph3P, CH2Cl2, 80%; (e) NaClO2,
.
NaH2PO4, 2-methyl-2-butene/H2O/t-BuOH, 100%; (f) BnONH2 HCl, TEA, EDC, CH2Cl2, 71%;
(g) H2, Pd/C, AcOH, 69%.

X. Li et al.
590
Scheme 7: aReaction conditions: (a) Na, NH3, THF, 85%; (b) (Bu3Sn)2O, I2, CH3CN, 89%;
(c) Ac2O, pyridine, 100%; (d) allyltributyltin, AIBN, toluene, 69%; (e) MeOH-H2O-Et3 N, 100%;
(f) Et3SiH, SnCl4, CH2Cl2, 66%; (g) t-TBDPSCl, imidazole, DMF, 97%; (h) myristoyl chloride,
pyridine, 95%; (i) O3, Ph3P, CH2Cl2, 81%; (j) NaClO2, NaH2PO4, 2-methyl-2-butene/H2O/t-BuOH,
.
100%; (k) BnONH2 HCl, TEA, EDC, CH2Cl2, 68%; (l) TBAF, THF, 91%; (m) H2, Pd/C, AcOH, 64%.
Radical addition of allyltributylstannane with AIBN as the initiator followed
by deacetylation gave 34 in 69% yield. Reductive opening of the 1,6-anhydro
ring using our strategy gave 35 (66%). Selective t-butyldiphenylsilyation at
the primary alcohol of 35 gave 36 (97%). O-Acylation with myristoyl chloride
in pyridine then gave 37 (95%). Ozonolysis of 37 followed by reduction using
PPh3 gave aldehyde 38 (81%). Further oxidation using NaClO2 and
NaH2PO4 in t-BuOH/2-methyl-2-butene/H2O yielded 39 quantitatively. This
carboxylic acid coupled with O-benzylhydroxylamine using EDC to furnish
40 (68%). The t-butyldiphenylsilyl group was removed by TBAF to give 41
(91%). Hydrogenation of 41 gave 5 (64%).
Compounds 6, 7, and 8 were prepared according to the same synthetic
strategy as the preparation of 1,[6b] using 1-bromotetradecane, benzoyl
chloride, and palmitoyl chloride instead of myristoyl chloride (Sch. 8).

Mapping the Active Site of LpxC
591
Scheme 8: aReaction conditions: (a) 1-bromotetradecane, NaH, DMF, 70%; (b) H2, Pd/C,
AcOH, 70%; (c) benzoyl chloride, pyridine, 95%; O3, Ph3P, CH2Cl2, 87%; NaClO2, NaH2PO4,
.
2-methyl-2-butene/H2O/t-BuOH, 100%; BnONH2 HCl, TEA, EDC, CH2Cl2, 70%; H2, Pd/C, AcOH,
62%; (d) palmitoyl chloride, pyridine, 55%; H2, Pd/C, AcOH, 70%.
Inhibitor Evaluation. The seven new analogs of 1 were enzymatically eval-
uated as inhibitors of the E. coli LpxC in vitro. The results are presented in
Figure 2. A detailed picture of the topography of a substrate-based inhibitor
binding with the LpxC enzyme can be proposed based on these results. The
two hydroxyl groups of the GlcNAc residue appear to be involved in the
binding interaction, since replacement of either hydroxyl group with
hydrogen caused a loss of the inhibitory activity. The enzyme cannot tolerate
Figure 2: Inhibition of E. coli LpxC by compounds 1–8 (1 mg/mL).

X. Li et al.
592
increased steric bulk at OH-6, since the 6-O-methyl derivative had only barely
detectable inhibitory activity (3%), while the enzyme can better tolerate the
steric increase at OH-4 to some extent (30%). Our previous studies have
shown that the inhibitor with or without a shorter O3 fatty acid constituent
(C6) lost inhibitory activity dramatically,[6a] suggesting the enzyme may have
a narrow cleft exactly designed to fit the myristoyl chain of the substrate
(UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc), which contributes importantly to
binding interaction.[15]
Surprisingly, a decreased inhibitory activity was observed when adding
two more methylene groups to the parent 1 to produce 8. We reason that
with the terminal methyl group the myristoyl chain packed against the
enzyme, a longer acyl chain either cannot fit or is forced into solution that is
unfavorable. The replacement of the acyl chain with a corresponding ether
chain (6) resulted in the loss of some activity. The reason for this could be
that the carbonyl group of the acyl chain is also involved in interaction with
the protein. Therefore, certain long acyl chains, neither too short nor too
long, are crucial for potent inhibition.
The inhibition data collected in Figure 2 independently suggest the
binding mode presented schematically in Figure 3. This model is in
complete agreement with that derived by solution NMR spectroscopy of the
complex of LpxC with 1 and of the crystal structure of the native
protein.[15] The most promising position for further elaboration to produce
superior inhibitors should be at the 3-position, where clearly there is a hydro-
phobic interaction with the enzyme. Importantly, replacement of a myristoyl
ester chain with a benzoyl group (7) did not diminish the inhibitory activity,
Figure 3: Schematic of proposed binding mode of 1 with LpxC.

Mapping the Active Site of LpxC
593
and therefore this hydrophobic interaction is permissive to some structural
variation. This may explain the success of many aromatic hydroxamic
acids[5] in inhibiting the enzyme. We anticipate that this class of substrate-
based designed inhibitors may lead to effective antibiotic against Gram-
negative bacteria.
EXPERIMENTAL
General Procedure
Analytical TLC was performed on Silica Gel 60-F254 (E. Merck, Darmstadt)
with detection by quenching of fluorescence and/or by charring with 5%
sulfuric acid in EtOH. All commercial reagents were used as supplied. Column
chromatography was performed on Silica Gel 60 (E. Merck 40–60 mM,
Darmstadt). Iatrobeads (beaded silica gel 6RS-8060) were from Iatron Labo-
1
tatories (Tokyo). H NMR spectra were recorded on 300 MHz (Varian Inova
300), 400 MHz (Varian Inova 400), or 500 MHz (Varian Inova 400). The first-
order proton chemical shifts, dH, are referenced to either residual CHCl3 (dH
7.24, CDCl3) or CH3OD (dH 3.30, CD3OD). The 13C chemical shifts, dC, are
referenced to internal CDCl3 (dC 77.00), or CD3OD (dC 49.00). The diastereoto-
pic methylene protons of benzyl groups are reported as ABd, meaning second-
order doublets, with the chemical shifts reported as the midpoint between the
peaks. Organic solutions were dried prior to concentration under vacuum at
,408C (bath). Microanalyses and electrospray mass spectra were performed
by the analytical services of this department.
1,5-Anhydro-6-O-benzyl-2-deoxy-3-O-myristoyl-2-C-(2-propenyl)-D-
glucitol (15). To a solution of 14[6b] (160 mg, 0.33 mmol) and NaCNBH3
(207 mg, 3.29 mmol) in dry THF (6 mL), HCl in Et2O was added until bubbles
ceased. The mixture was diluted with CH2Cl2 (20 mL) and water (5 mL) and
poured into sat. NaHCO3 (5 mL). The organic layer was washed with brine
(5 mL), dried over Na2SO4 and then concentrated. The residue was purified
1
by chromatography (EtOAc/hexane, 1 : 5) to yield 15 (131 mg, 82%). H NMR
(400 MHz, CDCl3) d 7.38–7.25 (5H, m), 5.79 (1H, m, H-20), 5.10–4.90 (2H, m,
H-30), 4.72 (1H, dd, J ¼ 9.0, 10.6 Hz. H-3), 4.60 and 4.58 (2H, ABd,
J ¼ 12.0 Hz, OCH2Ph), 3.96 (1H, dd, J ¼ 4.7, 11.9 Hz, H-1q), 3.71 (2H, m,
H-6), 3.55 (1H, m, H-4), 3.39 (1H, m, H-5), 3.15 (1H, t, J ¼ 11.9 Hz, H-1 ax),
2.36 (2H, t, J ¼ 7.4 Hz, OC(O)CH2), 2.18 (1H, m, H-10), 1.95 (1H, m, H-2),
1.86 (1H, m, H-10), 1.63 (2H, m), 1.36–1.12 (20H, m), 0.90 (3H, t, J ¼ 6.7 Hz);
13C NMR (100 MHz, CDCl3) d 175.2, 137.8, 134.6, 128.4, 127.7, 117.1, 79.5,
78.4, 73.7, 71.7, 70.3, 69.8, 40.1, 34.4, 32.3, 31.9, 29.6, 29.4, 29.3, 29.2, 29.1,
24.9, 22.6, 14.1. HRMS calcd. for C30H48O4Na (M þ Naþ) 511.3399; found
511.3401.

X. Li et al.
594
1,5-Anhydro-6-O-benzyl-2-deoxy-3-O-myristoyl-2-C-(2-propenyl)-4-
O-(1-thiocarbonylimidazol)-D-glucitol(16).
A
solution of 15 (91 mg,
0.19 mmol) and N,N-thiocarbonyldiimidazole (110 mg, 0.62 mmol) in dry toluene
(4 mL) was refluxed overnight. After cooling to rt, the mixture was concentrated
and the residue was purified directly by chromatography (EtOAc/hexane, 1 : 4)
1
to give 16 (85 mg, 79%). H NMR (400 MHz, CDCl3) d 8.21 (1H, s), 7.50 (1H, s),
7.24–7.18 (5H, m), 7.00 (1H, s), 5.79 (1H, t, J ¼ 9.5 Hz, H-4), 5.69 (1H, m, H-20),
5.16 (1H, dd, J ¼ 9.2, 10.7 Hz, H-3), 5.05–4.98 (2H, m, H-30), 4.46 (2H, s,
OCH2Ph), 4.06 (1H, dd, J ¼ 4.6, 11.5 Hz, H-1 eq), 3.70 (1H, m, H-5), 3.58 (1H,
dd, J ¼ 3.2, 10.7 Hz, H-6), 3.51 (1H, dd, J ¼ 5.1, 10.7 Hz), 3.38 (1H, t,
J ¼ 11.5 Hz, H-1 ax), 2.24–2.10 (4H, m, H-2, H-10, OC(O)CH2), 1.90 (1H, m,
H-10), 1.40 (2H, m), 1.38–1.00 (20H, m), 0.88 (3H, t, J ¼ 6.7 Hz); 13C NMR
(100 MHz, CDCl3) d 183.5, 173.2, 134.0, 130.9, 128.2, 127.8, 127.7, 117.5, 79.3,
77.7, 74.4, 73.7, 69.9, 69.2, 40.6, 34.1, 32.1, 31.9, 29.6, 29.5, 29.3, 28.9, 24.7,
22.6, 14.1. HRMS calcd. for C34H50N2O5SH (M þ Hþ) 599.3519; found 599.3518.
1,5-Anhydro-6-O-benzyl-2,4-dideoxy-3-O-myristoyl-2-C-(2-propenyl)-
D-glucitol (17). To a solution of 16 (63 mg, 0.11 mmol) and AIBN (5 mg,
0.04 mmol) in dry toluene (3 mL) was added Bu3SnH (85 mL, 0.33 mmol)
under argon. The mixture was refluxed for 3 hr. After cooling to rt, the
mixture was concentrated. Column chromatography (EtOAc/hexane, 1 : 6) of
the residue gave 17 (44 mg, 85%). 1H NMR (500 MHz, CDCl3) d 7.38–7.24
(5H, m), 5.70 (1H, m, H-20), 5.05–4.95 (2H, m, H-30), 4.70 (1H, m, H-3), 4.56
and 4.54 (2H, ABd, J ¼ 12.3 Hz, OCH2Ph), 4.04 (1H, dd, J ¼ 4.2, 11.8 Hz,
H-1 eq), 3.61 (1H, m, H- 5), 3.94–3.80 (2H, m, H-6), 3.38 (1H, t, J ¼ 11.8 Hz,
H-1 ax), 2.28 (2H, t, J ¼ 7.3 Hz, OC(O)CH2), 2.25 (1H, m, H-4), 1.98 (1H, m,
H-10), 1.86–1.78 (2H, m, H-2, H-10), 1.60 (2H, m), 1.39 (1H, q, J ¼ 11.6 Hz,
H-4), 1.34–1.18 (20H, m), 0.90 (3H, t, J ¼ 7.0 Hz); 13C NMR (125 MHz,
CDCl3) d 173.2, 137.9, 135.0, 128.3, 127.6, 116.7, 75.5, 73.5,73.4, 72.8, 70.3,
40.7, 34.6, 34.1, 32.7, 31.9, 29.7, 29.6, 29.4, 29.3, 29.2, 25.1, 22.8, 14.2. HRMS
calcd. for C30H48O4Na (M þ Naþ) 495.3450; found 495.3456.
2-C-(Aldehydomethyl)-1,5-anhydro-6-O-benzyl-2,4-dideoxy-3-O-myr-
istoyl-D-glucitol (18). Ozone was passed through a solution of 17 (48 mg,
0.10 mmol) in CH2Cl2 (10 mL) at 2788C until the solution turned blue. The
excess ozone was removed with a stream of oxygen for 10 min, followed by
addition of Ph3P (53 mg, 0.20 mmol). The mixture was allowed to warm to rt
for 2 hr and then concentrated. The residue was purified by chromatography
1
(EtOAc/hexane, 1 : 3) to afford 18 (42 mg, 87%). H NMR (400 MHz, CDCl3)
d 9.71 (1H, s), 7.38–7.20 (5H, m), 4.67 (1H, m, H-3), 4.56 and 4.52 (2H, ABd,
J ¼ 12.3 Hz, OCH2Ph), 4.04 (1H, dd, J ¼ 4.5, 11.8 Hz, H-1 eq), 3.63 (1H, m,
H-5), 3.50–3.40 (2H, m, 2H-6), 3.19 (1H, t, J ¼ 11.8 Hz, H-1 ax), 2.45–2.32
(2H, m, H-10, H-2), 2.23 (2H, t, J ¼ 7.4 Hz, OC(O)CH2), 2.14 (1H, m, H-4),
2.02 (1H, m, H-10), 1.60 (2H, m), 1.44 (1H, q, J ¼ 11.7 Hz, H-4), 1.34–1.18

Mapping the Active Site of LpxC
595
(20H, m), 0.86 (3H, t, J ¼ 7.0 Hz); 13C NMR (100 MHz, CDCl3) d 199.9, 173.1,
137.8, 128.3, 127.6, 75.5, 73.5, 73.4,72.6, 70.0, 42.9, 36.4, 34.4, 33.9, 31.9,
29.7, 29.6, 29.5, 29.4, 29.2, 24.9, 22.8, 14.2.
2-C-(Carboxymethyl)-1,5-anhydro-6-O-benzyl-2,4-dideoxy-3-O-myris-
toyl-D-glucitol (19). To a solution of 18 (38 mg, 0.08 mmol) in t-BuOH (2 mL)
and 2-methyl-2-butene (0.6 mL) was added a solution of NaClO2 (91 mg,
0.80 mmol) and NaH2PO4 (138 mg, 0.80 mmol) in water (1.5 mL) over 5 min.
The mixture was stirred for 1 hr, then diluted with ice water and extracted
with EtOAc. The combined organic extracts were washed with brine (5 mL),
dried over Na2SO4, evaporated, and further dried under oil vacuum overnight
to afford 19. The acid was used without purification. 1H NMR (400 MHz,
CDCl3) d 7.38–7.20 (5H, m), 4.67 (1H, m, H-3), 4.56 and 4.52 (2H, ABd,
J ¼ 12.3 Hz, OCH2Ph), 4.04 (1H, dd, J ¼ 4.5, 11.8 Hz, H-1 eq), 3.63 (1H, m,
H-5), 3.50–3.40 (2H, m, 2H-6), 3.19 (1H, t, J ¼ 11.8 Hz, H-1 ax), 2.45–2.32
(1H, m, H-10), 2.23 (3H, m, OC(O)CH2,H-2), 2.10–2.00 (2H, m, H-4, H-10),
1.60 (2H, m), 1.44 (1H, q, J ¼ 11.7 Hz, H-4), 1.34–1.18 (20H, m), 0.86 (3H, t,
J ¼ 7.0 Hz); 13C NMR (100 MHz, CDCl3) d 177.2, 173.4, 137.8, 129.7, 128.4,
127.7, 127.6, 75.5, 73.4, 73.0, 72.5, 69.9, 38.0, 34.3, 33.8, 32.9, 31.9, 29.7,
29.6, 29.5, 29.4, 29.2, 24.9, 22.8, 14.2.
1, 5-Anhydro-6-O-benzyl-2-C-(carboxymethyl N-benzyloxyamide)-2,4-
dideoxy-3-O-myristoyl-D-glucitol (20). To a solution of the acid 19 (40 mg,
.
0.08 mmol) and BnONH2 HCl (13 mg, 0.08 mmol) in CH2Cl2 (0.5 mL) were
added successively EDC (19 mg, 0.10 mmol) and triethylamine (13 mL,
0.10 mmol) at room temperature. The reaction mixture was stirred for 2 hr.
CH2Cl2 (10 mL) was then added and the solution was washed with water
(5 mL) and brine (5 mL), then dried over Na2SO4. The residue after concen-
tration was purified by chromatography (toluene/EtOAc, 4 : 1) to afford 20
1
(34 mg, 70%). H NMR (400 MHz, CD3OD) d 7.40–7.20 (10H, m), 4.84 and
4.80 (2H, ABd, J ¼ 11.2 Hz, OCH2Ph), 4.70 (1H, m, H-3), 4.53 (2H, s,
OCH2Ph), 3.93 (1H, dd, J ¼ 4.3, 11.5 Hz, H-1 eq), 3.62 (1H, m H-5), 3.52–
3.42 (2H, m, H-6), 3.18 (1H, t, J ¼ 11.3 Hz, H-1 ax), 2.30 (2H, m, OC(O)CH2),
2.20–2.10 (2H, m, H-2, H-10), 2.02 (1H, m, H-4), 1.80 (1H, dd, J ¼ 7.2,
13.8 Hz, H-10), 1.58 (2H, m), 1.35 (1H, q, J ¼ 11.5 Hz, H-4), 1.33–1.20 (20H,
m), 0.88 (3H, t, J ¼ 6.7 Hz); 13C NMR (100 MHz, CD3OD) d 174.9, 170.7,
139.5, 136.9, 130.3, 129.7, 129.5, 129.4, 128.9, 128.7, 79.0, 76.7, 74.8, 74.4,
73.9, 70.8, 39.7, 35.2, 33.1, 32.9, 30.8, 30.7, 30.6, 30.5, 30.4, 30.2, 25.9, 23.8,
14.5. HRMS calcd. for C36H53NO6Na (M þ Naþ) 618.3765; found 618.3762.
1, 5-Anhydro-2-C-(carboxymethyl N-hydroxyamide)-2,4-dideoxy-3-O-
myristoyl-D-glucitol (2). Pd/C (8 mg) was added to a solution of 20 (12 mg,
20 mmol) in AcOH (2 mL). The reaction mixture was stirred for 4 hr under a
balloon of H2. After filtration through Celite, the Celite pad was washed well

X. Li et al.
596
with MeOH. The solvent was evaporated and the residue was purified by
gravity chromatography on an Iatrobeads SiO2 column (CHCl3/MeOH, 15 : 1)
1
to give 2 (5.9 mg, 70%). H NMR (400 MHz, CD3OD) d 4.71 (1H, m, H-3), 4.00
(1H, dd, J ¼ 4.3, 11.5 Hz, H-1 eq), 3.52–3.44 (3H, m, 2H-6, H-5), 3.22 (1H, t,
J ¼ 11.5 Hz, H-1 ax), 2.33 (2H, OC(O)CH2), 2.22–2.15 (2H, H-2, H-10), 2.02
(1H, m, H-4), 1.83 (1H, dd, J ¼ 9.7, 15.7 Hz, H-10), 1.61 (2H, m), 1.38–1.26
(21H, m, H-4, C10H20), 0.90 (3H, t, J ¼ 6.8 Hz); 13C NMR (100 MHz, CD3OD)
d 175.0, 170.7, 78.3, 75.1, 70.9, 65.9, 39.8, 35.1, 34.8, 33.1, 32.9, 30.8, 30.7,
30.6, 30.5, 30.4, 30.2, 25.9, 23.7, 14.4. HRMS calcd. for C22H41NO6Na
(M þ Naþ) 438.2832; found 438.2829.
1,5-Anhydro-4-O-benzyl-2-C-(carboxymethyl
N-benzyloxyamide)-2-
deoxy-3-O-myristoyl-D-glucitol (21). A solution of 1M BH3 in THF
(0.2 mL) was added to 9 (12 mg, 20 mmol) at 08C, and then a solution of 1M
Bu2BOTf in CH2Cl2 (0.02 mL, 20 mmol) was added to the reaction slowly.
After 1 hr at 08C, triethylamine (0.1 mL) was then added, followed by careful
addition of methanol until the evolution of H2 ceased. The reaction mixture
was then concentrated and purified by chromatography (toluene/EtOAc,
1
2 : 1) to give 21 (8.5 mg, 71%). H NMR (500 MHz, CD3OD: CDCl3 ¼ 4 : 1) d
7.40–7.20 (10H, m), 4.92 (1H, dd, J ¼ 9.1, 10.9 Hz, H-3), 4.80 (2H, overlapped
with H2O, OCH2Ph), 4.64 and 4.58 (2H, 2d, J ¼ 11.3 Hz, OCH2Ph), 3.89 (1H,
dd, J ¼ 4.8, 11.6 Hz, H-1 eq), 3.80 (1H, dd, J ¼ 2.0, 12.0 Hz, H-6), 3.67 (1H,
dd, J ¼ 4.6, 12.0 Hz, H-6), 3.54 (1H, t, J ¼ 9.1 Hz, H-4), 3.36 (1H, m, H-5),
3.19 (1H, t, J ¼ 11.6 Hz, H-1 ax), 2.28 (2H, m, OC(O)CH2), 2.16 (1H, m, H-2),
2.06 (1H, dd, J ¼ 5.0, 14.8 Hz, H-10), 1.81 (1H, dd, J ¼ 8.7, 14.8 Hz, H-10),
1.52 (2H, m), 1.34–1.18 (20H, m), 0.88 (1H, t, J ¼ 6.8 Hz); 13C NMR
(125 MHz, CD3OD: CDCl3 ¼ 4 : 1) d 175.4, 170.2, 139.5, 136.8, 130.3, 129.7,
129.5, 129.4, 128.8, 128.7, 81.9, 79.4, 79.1, 78.5, 78.4, 75.7, 70.4, 62.4, 40.0,
35.2, 33.1, 32.5, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3, 25.8, 23.8, 14.6. HRMS
calcd. for C36H53NO7Na (M þ Naþ) 634.3720; found 634.3721.
1,5-Anhydro-4-O-benzyl-2-C-(carboxymethyl N-benzyloxyamide)-2,6-
dideoxy-6-iodo-3-O-myristoyl-D-glucitol (22). To a solution of 21 (12 mg,
20 mmol) in toluene (1 mL) were added Ph3P (10.3 mg, 40 mmol), I2 (10 mg,
0.040 mmol) and imidazole (5.3 mg, 78 mmol) successively. The mixture was
stirred at rt for 4 hr and was then directly purified by chromatography
1
(EtOAc/toluene, 1 : 2) to give 22 (11 mg, 80%). H NMR (400 MHz, CD3OD) d
7.40–7.20 (10H, m), 4.99 (1H, dd, J ¼ 8.9, 10.9 Hz, H-3), 4.83 (2H, overlapped
with H2O, OCH2Ph), 4.72 and 4.65 (2H, ABd, J ¼ 11.3 Hz, OCH2Ph), 3.86 (1H,
dd, J ¼ 4.8, 11.7 Hz, H-1 eq), 3.50 (1H, dd, J ¼ 2.8, 10.9 Hz, H-6), 3.42 (1H, t,
J ¼ 8.9 Hz, H-4), 3.40 (1H, dd, J ¼ 10.9, 5.2 Hz, H-6), 3.27 (1H, t, J ¼ 11.7 Hz,
H-1 ax), 3.01 (1H, m, H-5), 2.28 (2H, m, OC(O)CH2), 2.19 (1H, m, H-2), 2.06
(1H, dd, J ¼ 5.1, 14.8 Hz, H-10), 1.81 (1H, dd, J ¼ 8.5, 14.8 Hz, H-10), 1.52
(2H, m), 1.35–1.18 (20H, m), 0.90 (1H, t, J ¼ 6.8 Hz); 13C NMR (100 MHz,

Mapping the Active Site of LpxC
597
CD3OD) d 175.1, 170.1, 139.5, 130.3, 129.7, 129.5, 129.4, 128.9, 128.7, 82.5,
79.5, 78.9, 77.9, 76.1, 70.1, 40.1, 35.1, 33.1, 32.3, 30.7, 30.5, 30.4, 30.2,
25.7, 23.7, 7.6. HRMS calcd. for C36H52NO6INa (M þ Naþ) 744.2737; found
744.2730.
1,5-Anhydro-4-O-benzyl-2-C-(carboxymethyl N-benzyloxyamide)-2,6-
dideoxy-3-O-myristoyl-D-glucitol (23). A solution of 22 (17 mg, 24mmol)
and AIBN (1.5 mg, 9.0 mmol) in dry toluene (0.5 mL) was degassed with argon
for 10 min. Bu3SnH (25 mL, 96 mmol) was then added under argon. The
mixture was heated to 908C for 30 min and, after cooling to rt, was directly
purified by chromatography (EtOAc/toluene, 1 : 2) to give 23 (11.5 mg, 82%).
1H NMR (400 MHz, CD3OD) d 7.40–7.20 (10H, m), 4.89 (1H, dd, J ¼ 9.0,
10.9 Hz, H-3), 4.82 (2H, overlapped with H2O, OCH2Ph), 4.62 and 4.60 (2H,
ABd, J ¼ 11.4 Hz, OCH2Ph), 3.79 (1H, dd, J ¼ 4.8, 11.7 Hz, H-1 eq), 3.36–3.30
(2H, m, H-4, H-5), 3.17 (1H, t, J ¼ 11.7 Hz, H-1 ax), 2.28 (2H, m, OC(O)CH2),
2.18 (1H, m, H-2), 2.06 (1H, dd, J ¼ 5.1, 14.8 Hz, H-10), 1.81 (1H, dd, J ¼ 8.7,
14.8 Hz, H-10), 1.52 (2H, m), 1.35–1.18 (23H, m, H-6 and C10H20), 0.89 (1H, t,
J ¼ 6.8 Hz); 13C NMR (100MHz, CD3OD) d 175.1, 170.2, 139.6, 136.9, 130.8,
130.2, 129.6, 129.4, 129.3, 128.7, 128.6, 84.5, 79.0, 78.2, 77.6, 75.9, 70.3, 40.4,
35.2, 33.2, 32.6, 30.9, 30.8, 30.6, 30.5, 30.3, 25.9, 23.9, 18.9, 14.6. HRMS calcd.
for C36H53NO6Na (M þ Naþ) 618.3765; found 618.3761.
1, 5-Anhydro-2-C-(carboxymethyl N-hydroxyamide)-2,6-dideoxy-3-O-
myristoyl-D-glucitol (3). Pd/C (7 mg) was added to a solution of 23 (12 mg,
20 mmol) in AcOH (2 mL). The reaction mixture was stirred for 4 hr under a
balloon of H2. After filtration through Celite, the Celite pad was washed well
with MeOH. The solvent was evaporated and the residue was purified by
gravity chromatography on an Iatrobeads SiO2 column (CHCl3/MeOH, 15 : 1)
1
to give 3 (5.9 mg, 70%). H NMR (400 MHz, CD3OD) d 4.70 (1H, dd, J ¼ 9.0,
10.9 Hz, H-3), 4.00 (1H, dd, J ¼ 4.7, 11.6 Hz, H-1 eq), 3.25 (1H, m, H-5), 3.20
(1H, t, J ¼ 11.6 Hz, H-1 ax), 3.09 (1H, t, J ¼ 9.0 Hz, H-4), 2.39 (2H, t,
J ¼ 7.4 Hz, OC(O)CH2), 2.25 (H, m, H-2), 2.11 (1H, dd, J ¼ 4.9, 10.0 Hz H-10),
1.82 (1H, dd, J ¼ 10.0, 15.7 Hz, H-10) 1.65 (2H, m), 1.40–1.25 (20H, m,
C10H20), 1.24 (3H, d, J ¼ 6.1 Hz, 2H-6), 0.90 (3H, t, J ¼ 6.8 Hz).
After sitting overnight in the NMR machine, myristoyl migration was obser-
1
ved: H NMR (400 MHz, CD3OD) d 4.70 (1H, dd, J ¼ 9.0, 10.9 Hz, H-3), 4.54
(0.4 H, t, J ¼ 9.0 Hz, H-4), 4.00 (1H, dd, J ¼ 4.7, 11.6 Hz, H-1 eq), 3.25 (1H, m,
H-5), 3.20 (1H, t, J ¼ 11.6 Hz, H-1 ax), 3.09 (1H, t, J ¼ 9.0 Hz, H-4), 2.39 (2H,
t, J ¼ 7.4 Hz, OC(O)CH2), 2.25 (H, m, H-2), 2.11 (1H, dd, J ¼ 4.9, 10.0 Hz
H-10), 1.82 (1H, dd, J ¼ 10.0, 15.7 Hz, H-10) 1.65 (2H, m), 1.40–1.25 (20H, m,
C10H20), 1.24 (3H, d, J ¼ 6.1 Hz, 2H-6), 1.10 (d, J ¼ 6.1 Hz, 2H-6), 0.90 (3H, t,
J ¼ 6.8 Hz); HRMS calcd. for C22H41NO6Na (M þ Naþ) 438.2832; found
438.2829.

X. Li et al.
598
1,6-Anhydro-4-O-benzyl-2-deoxy-3-O-myristoyl-2-C-(2-propenyl)-b-D-
glucopyranose (25). To a solution of 24 (340 mg, 1.23 mmol) in pyridine
(8 mL), DMAP (70 mg, 0.57 mmol) and myristoyl chloride (0.86 mL,
3.16 mmol) were successively added at 08C and then stirred at rt for 20 hr.
EtOAc (20 mL) was added to the reaction mixture and the organic layer was
washed with 10% citric acid (5 mL), H2O (5 mL ꢀ 2), sat. NaHCO3 (5 mL),
and brine (5 mL). The organic layer was dried over Na2SO4 and concentrated.
The residue was purified by chromatography (toluene/EtOAc, 9 : 1) to afford 25
1
(503 mg, 84%). H NMR (400 MHz, CDCl3) d 7.40–7.22 (5H, m), 5.80 (1H, m,
H-20), 5.49 (1H, s, H-1), 5.14–5.08 (2H, m, H-30), 4.90 (1H, s, H-3), 4.80 (1H,
d, J ¼ 12.3 Hz, OCH2Ph), 4.65 (1H, d, J ¼ 12.3 Hz, OCH2Ph), 4.54 (1H, br d,
J ¼ 5.6 Hz, H-5), 3.96 (1H, d, J ¼ 7.0 Hz, H-6), 3.70 (1H, dd, J ¼ 5.6, 7.0 H,
H-6), 3.22 (1H, s, H-4), 2.40–2.26 (4H, m, 2H-10 and OC(O)CH2), 1.79 (1H, br
t, J ¼ 8.0 Hz, H-2), 1.38–1.20 (20H, m), 0.82 (3H, t, J ¼ 6.7 Hz); 13C NMR
(100 MHz, CDCl3) d 172.8, 137.7, 135.6, 128.4, 127.8, 127.7, 117.4, 102.4,
75.5, 74.7, 71.1, 69.1, 64.8, 42.6, 34.5, 33.9, 31.9, 29.6, 29.4, 29.3, 29.2, 24.8,
22.6, 14.1. HRMS calcd. for C30H46O5Na (M þ Naþ) 509.3243; found 509.3243.
1,5-Anhydro-4-O-benzyl-2-deoxy-3-O-myristoyl-2-C-(2-propenyl)-D-
glucitol (26). To a solution of 25 (100 mg, 0.21 mmol) in CH2Cl2 (20 mL), Et3SiH
(0.16 mL, 1.02 mmol) and SnCl4 (36 mL, 0.31 mmol) were successively added at
08C. After stirring at rt for 1 hr, the reaction mixture was poured into cold sat.
NaHCO3 (5mL) and the aqueous layer was extracted with CH2Cl2 (10 mL ꢀ 2)
at once. The combined organic layers were washed with brine (5mL) and dried
over Na2SO4 and concentrated. The residue was purified by chromatography
1
(toluene/EtOAc, 6 : 1) to give 26 (46 mg, 46%). H NMR (400 MHz, CDCl3) d
7.38–7.20 (5H, m), 5.70 (1H, m, H-20), 5.06–4.95 (3H, m, H-3, H-30), 4.61 and
4.59 (2H, ABd, J ¼ 11.3 Hz, OCH2Ph), 3.94 (1H, dd, J ¼ 4.3, 11.6 Hz, H-1 eq),
3.85 (1H, dd, J ¼ 2.6, 11.9 Hz, H-6), 3.70 (1H, dd, J ¼ 4.0, 11.9 Hz, H-6), 3.52
(1H, t, J ¼ 9.4 Hz, H-4), 3.30 (1H, m, H-5), 3.18 (1H, t, J ¼ 11.6 Hz, H-1 ax),
2.31–2.10 (3H, m, H-10, OC(O)CH2), 1.95–1.80 (2H, m, H-2, H-10), 2.50 (2H,
m), 1.38–1.20 (20H, m), 0.84 (3H, t, J ¼ 6.7 Hz); 13C NMR (100 MHz, CDCl3) d
173.3, 137.9, 134.8, 128.4, 127.7, 127.6, 116.9, 79.8, 77.1, 74.5, 69.7, 62.1, 41.2,
34.4, 32.2, 31.8, 29.6, 29.5, 29.4, 29.3, 29.2, 24.8, 22.6, 14.1. HRMS calcd. for
C30H48O5Na (M þ Naþ) 511.3399; found 511.3399.
1,5-Anhydro-4-O-benzyl-2-deoxy-6-O-methyl-3-O-myristoyl-2-C-(2-
propenyl)-D-glucitol (27). To a solution of 26 (70 mg, 0.14 mmol) and
2,6-di(tert-butyl-4-methyl) pyridine (882 mg, 4.30 mmol) was added methyl tri-
fluoromethanesulfonate (0.32 mL, 2.84 mmol). The mixture was stirred over-
night and quenched by an aqueous ammonia solution (5 mL) and extracted
with CH2Cl2 (10 mL ꢀ 3). The extracts were washed with 10% HCl (5 mL),
and brine (5 mL), dried over Na2SO4, and concentrated. The residue was
purified by chromatography (toluene/EtOAc, 8 : 1) to afford 27 (71 mg, 98%).

Mapping the Active Site of LpxC
599
1H NMR (400 MHz, CDCl3) d 7.38–7.20 (5H, m), 5.70 (1H, m, H-20), 5.06–4.95
(3H, m, H-3, H-30), 4.61 and 4.59 (2H, ABd, J ¼ 11.3 Hz, OCH2Ph), 3.94 (1H, dd,
J ¼ 4.7, 11.7 Hz, H-1 eq), 3.58–3.53 (3H, m, 2H-6, H-4), 3.40–3.35 (4H, m, H-5,
OMe), 3.25 (1H, t, J ¼ 11.7 Hz, H-1 ax), 2.31–2.10 (3H, m, H-10, OC(O)CH2),
2.00–1.80 (2H, m, H-2, H-10), 2.50 (2H, m), 1.38–1.20 (20H, m), 0.84 (3H, t,
J ¼ 6.7 Hz); 13C NMR (100 MHz, CDCl3) d 173.3, 138.1, 134.9, 128.3, 127.6,
127.5, 116.8, 79.2, 77.2, 74.5, 71.4, 69.9. 59.2, 41.1, 34.4, 32.2, 31.8, 29.6,
29.4, 29.3, 29.2, 24.8, 22.6, 14.1. HRMS calcd. for C31H50O5Na (M þ Naþ)
525.3556; found 525.3554.
2-C-(Aldehydomethyl)-1,5-anhydro-4-O-benzyl-2-deoxy-6-O-methyl-3-
O-myristoyl-D-glucitol (28). Ozone was passed through a solution of
27 (30 mg, 60mmol) in CH2Cl2 at –788C until the solution turned blue. The
excess ozone was removed with a stream of oxygen for 10 min, followed by
addition of Ph3P (23 mg, 88 mmol). The mixture was allowed to warm to rt for
2 hr, concentrated, and purified by chromatography (EtOAc/hexane, 1 : 3) to
1
afford 28 (24 mg, 80%). H NMR (400 MHz, CDCl3) d 9.64 (1H, s), 7.38–7.20
(5H, m), 5.00 (1H, dd, J ¼ 9.1, 10.7 Hz, H-3), 4.61 and 4.59 (2H, ABd,
J ¼ 14.4 Hz, OCH2Ph), 3.98 (1H, dd, J ¼ 4.5, 11.4 Hz, H-1 eq), 3.64–3.56 (3H,
m, 2H-6, H-4), 3.44–3.34 (4H, m, H-5, OMe), 3.21 (1H, t, J ¼ 11.4 Hz, H-1 ax),
2.56–2.40 (2H, m, H-2, H-10), 2.31–2.10 (3H, m, H-10, OC(O)CH2), 2.00–1.80
(2H, m, H-2,), 2.50 (2H, m), 1.38–1.20 (20H, m), 0.84 (3H, t, J ¼ 6.7 Hz);
13C NMR (100 MHz, CDCl3) d 199.4, 173.4, 137.9, 128.4, 127.7, 127.5, 79.4,
76.9, 74.6, 71.3, 69.6, 59.2, 42.2, 36.4, 34.2, 31.9, 29.6, 29.5, 29.3, 29.2, 29.1,
24.7, 22.6, 14.1. HRMS calcd. for C30H48O6Na (M þ Naþ) 527.3349; found
527.3349.
2-C-(Carboxymethyl)-1,5-anhydro-4-O-benzyl-2-deoxy-6-O-methyl-3-O-
myristoyl-D-glucitol (29). To a solution of 52 (24 mg, 48 mmol) in t-BuOH
(1.5 mL) and 2-methyl-2-butene (0.5 mL) was added a solution of NaClO2
(43 mg, 0.48 mmol) and NaH2PO4 (66 mg, 0.48 mmol) in water (1 mL). The
mixture was stirred for 1 hr, then diluted with ice-water (5 mL), and extracted
with EtOAc (10 mL ꢀ 3). The combined organic extracts were washed with
brine (5 mL), dried over Na2SO4, concentrated, and dried under high vacuum
overnight to give 28. The acid was used without purification. 1H NMR
(400 MHz, CDCl3) d 7.38–7.20 (5H, m), 4.94 (1H, dd, J ¼ 9.0, 10.6 Hz, H-3),
4.61 and 4.59 (2H, ABd, J ¼ 14.4 Hz, OCH2Ph), 3.98 (1H, dd, J ¼ 4.7,
11.5 Hz, H-1 eq), 3.64–3.56 (3H, m, 2H-6, H-4), 3.40–3.32 (4H, m, H-5,
OMe), 3.21 (1H, t, J ¼ 11.5 Hz), 2.56–2.40 (2H, m, H-2, H-10), 2.31–2.10 (3H,
m, H-10, OC(O)CH2), 2.50 (2H, m), 1.38–1.20 (20H, m), 0.84 (3H, t,
J ¼ 6.7 Hz); 13C NMR (100 MHz, CDCl3) d 176.4, 173.5, 137.9, 128.5, 128.4,
127.7, 127.6, 79.3, 76.9, 76.5, 74.6, 71.3, 69.6, 59.2, 38.4, 34.3, 32.4, 31.9,
29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 24.7, 22.6, 14.1.

X. Li et al.
600
1,5-Anhydro-4-O-benzyl-2-deoxy-2-C-(carboxymethyl N-hydroxyamide)-
6-O-methyl-3-O-myristoyl-D-glucitol (30). To a solution of the acid 29
.
(24 mg, 50 mmol) and BnONH2 HCl (7.6 mg, 50 mmol) in CH2Cl2 (0.3 mL) were
added successively EDC (12.8 mg, 70mmol) and triethylamine (9mL, 70mmol)
at rt. The reaction mixture was stirred for 2 hr. CH2Cl2 (10 mL) was then added
and the solution was washed with water (5mL) and brine (5mL), then dried
over Na2SO4. After concentration, the residue was purified by chromatography
1
(toluene/EtOAc, 4 : 1) to afford 30 (21 mg, 71%). H NMR (400 MHz, CD3OD) d
7.40–7.20 (10H, m), 4.92 (1H, dd, J ¼ 9.0, 10.9 Hz, H-3), 4.93 and 4.80 (2H,
ABd, J ¼ 11.3 Hz, OCH2Ph), 4.60 and 4.58 (1H, dd, J ¼ 4.8, 11.5 Hz, H-1 eq),
3.62–3.54 (2H, m, 2H-6), 3.53 (1H, t, J ¼ 9.0 Hz, H-4), 3.36 (1H, m, H-5), 3.34
(3H, s, OMe), 3.29 (1H, t, J ¼ 11.5 Hz, H-1 ax), 2.28 (2H, m, OC(O)CH2), 2.17
(1H, m, H-2), 2.06 (1H, dd, J ¼ 5.1, 14.8 Hz, H-10), 1.82 (1H, dd, J ¼ 8.6,
14.8 Hz, H-10), 1.51 (2H, m), 1.34–1.18 (20H, m), 0.89 (3H, t, J ¼ 6.7 Hz); 13C
NMR (100MHz, CD3OD) d 175.2, 170.2, 139.6, 136.9, 130.3, 129.7, 129.5,
129.4, 126.7, 80.8, 79.0, 78.6, 78.3, 75.7, 72.7, 70.4, 59.5, 40.0, 35.1, 33.1, 32.4,
30.8, 30.7, 30.6, 30.5, 30.4, 30.2, 25.7, 23.7, 14.1. HRMS calcd. for
C37H55NO7Na (M þ Naþ) 648.3876; found 648.3874.
1,5-Anhydro-2-C-(carboxymethyl N-hydroxyamide)-2-deoxy-6-O-methyl-
3-O-myristoyl-D-glucitol (4). Pd/C (8 mg) was added to a solution of 30
(12 mg, 19 mmol) in AcOH (2 mL), The reaction mixture was stirred for 3 hr
under a balloon of H2. After filtration through Celite, the Celite pad was
washed well with MeOH. The solvent was evaporated and the residue was
purified by gravity chromatography on an Iatrobeads SiO2 column (CHCl3/
MeOH, 15 : 1) to give 4 (5.9 mg, 69%). 1H NMR (400 MHz, CD3OD) d 4.73
(1H, dd, J ¼ 8.7, 10.9 Hz, H-3), 3.91 (1H, dd, J ¼ 4.8, 11.7 Hz, H-1 eq), 3.64
(1H, dd, J ¼ 2.0, 10.8 Hz, H-6), 3.53 (1H, dd, J ¼ 5.4, 10.8 Hz, H-6), 3.40–
3.30 (2H, m, H-4, H-5), 3.20 (1H, t, J ¼ 11.7 Hz, H-1 ax), 2.38 (2H, t, J ¼ 7.3,
OC(O)CH2), 2.24 (1H, m, H-2), 2.09 (1H, dd, J ¼ 4.9, 14.5 Hz, H-10), 1.81 (1H,
dd, J ¼ 9.0, 14.5 Hz H-10), 1.51 (2H, m), 1.40–1.20 (20H, m), 0.9 (3H, t,
J ¼ 7.4 Hz); 13C NMR (100 MHz, CD3OD) d 175.7, 170.3, 81.4, 78.9, 73.3,
70.7, 70.4, 59.5, 39.8, 35.1, 33.1, 32.6, 30.8, 30.4, 30.6, 30.4, 30.2, 25.9, 23.7,
14.4. HRMS calcd. for C23H43NO7 (M þ Naþ) 468.2937; found 468.2936.
4-O-Methyl-D-glucal (32). A solution of sodium metal in 500 mL ammonia was
stirred for 1 hr at 2788C and then distilled to another three-neck round bottom
flask. Small pieces of sodium metal were added until a deep blue color persisted
at –788C. A solution of 31 (1.16 g, 3.40 mmol) and MeOH (0.276 mL, 6.80 mmol)
in THF (50 mL) was added dropwise to the above solution. The reaction mixture
was stirred for 2 hr, and then quenched with MeOH until the solution turned
clear. Ammonia was then evaporated and coevaporated with MeOH and AcOH.
The residue was purified by chromatography (EtOAc/acetone, 1 : 1) to give 32
(464 mg, 85%). The NMR data were the same as those described in the literature.[13]

Mapping the Active Site of LpxC
601
3-O-Acetyl-1,6-anhydro-2-deoxy-2-iodo-4-O-methy-b-D-glucopyranose
(33). The glucal 32 (60 mg, 0.38 mmol) was treated with bis-(tributyltin)oxide
˚
(0.15 mL, 0.29 mmol) and actived powdered 3A molecular sieves (200 mg) in
refluxing dry acetonitrile (5 mL) for 3 hr. The mixture was cooled to 08C
under argon, and iodine (143 mg, 0.563 mmol) was added in one portion.
The dark-brown mixture was stirred at 08C for 15 min, then 3 hr at rt. The
mixture was filtered through Celite and concentrated. To the residue was
added aq. sodium thiosulfate (10 mL) and it was extracted with EtOAc
(20 mL ꢀ 3). The combined organic layers were dried over Na2SO4 and concen-
trated, and further dried under oil vacuum overnight. The residue was then
treated with Ac2O (3 mL) and pyridine (3 mL) overnight. After removing the
solvent, the residue was purified by chromatography (EtOAc/hexane, 1 : 3) to
1
afford 33 (110 mg, 89%). H NMR (500 MHz, CDCl3) d 5.65 (1H, s, H-1), 5.39
(1H, s, H-3), 4.54 (1H, d, J ¼ 5.8 Hz, H-5), 4.08 (1H, d, J ¼ 7.6 Hz, H-6), 3.90
(1H, s, H-2), 3.76 (1H, dd, J ¼ 5.8, 7.6 Hz, H-6), 3.50 (3H, s, OMe), 3.16 (1H,
s, H-4), 2.08 (3H, s); 13C NMR (125 MHz, CDCl3) d 169.2, 102.5, 78.2, 74.1,
71.3, 65.5, 57.4, 21.5, 21.0. HRMS calcd. for C9H13O5INa (M þ Naþ)
350.9700; found 350.9697.
1,6-Anhydro-2-deoxy-4-O-methyl -2-C- (2-propenyl)-b-D-glucopyranose
(34). A solution of 33 (110 mg, 0.34 mmol) and AIBN (20 mg, 0.12 mmol) in
dry toluene (6 mL) was degassed with argon for 15 min. Allyltributylstannane
(0.21 mL, 0.68 mmol) was added under argon, and the mixture was heated to
808C for 3 hr. TLC (EtOAc/hexane, 1 : 1) showed complete conversion. The
solvent was evaporated and a solution of the residue in acetonitrile (10 mL)
was washed with hexane (10 mL), and then concentrated. Chromatography
1
of the residue gave 56 mg (69%). H NMR (300 MHz, CDCl3) d 5.78 (1H, m,
H-20), 5.36 (1H, s, H-1), 5.18–5.04 (2H, m, H-30), 4.80 (1H, s, H-3), 4.55 (1H,
d, J ¼ 5.8 Hz, H-5), 4.00 (1H, d, J ¼ 8.2 Hz, H-6), 3.75 (1H, dd, J ¼ 5.9,
7.5 Hz, H-6), 3.46 (3H, s, OMe), 3.08 (1H, br s, H-4), 2.28 (2H, t, J ¼ 7.8 Hz,
H-10), 2.05 (3H, s, OAc), 1.76 (1H, br t, J ¼ 8.0 Hz, H-2).
A solution of above compound in 10 : 10 : 1 MeOH-H2O-Et3 N (5 mL) was
left overnight at rt and then concentrated, dried under oil vacuum overnight
1
to give 34. H NMR (400 MHz, CDCl3) d 5.80 (1H, m, H-20), 5.42 (1H, s, H-1),
5.10–5.06 (2H, m, H-30), 4.60 (1H, d, J ¼ 5.3 Hz, H-5), 4.10 (1H, d,
J ¼ 7.5 Hz, H-6), 3.72 (1H, dd, J ¼ 5.3, 7.5 Hz, H-6), 3.66 (1H, br s, H-3), 3.44
(3H, s, OMe), 3.20 (1H, br s, H-4), 2.28–2.20 (2H, m, H-10), 1.78 (1H, br t,
J ¼ 8.4, H-2); 13C NMR(100 MHz, CDCl3) d 135.8, 117.2, 103.4, 81.5, 74.4,
68.5, 65.4, 57.4, 45.7, 33.5. HRMS calcd. for C10H16O4Na (M þ Naþ)
223.0946; found 223.0947.
1,5-Anhydro-2-deoxy-4-O-methyl-2-C-(2-propenyl)-D-glucitol (35). To a
solution of 34 (196 mg, 0.98 mmol) and Et3SiH (0.82 mL, 5.15 mmol) in
CH2Cl2 (50 mL), SnCl4 (1.55 mL, 1M in CH2Cl2, 1.55 mmol) was added

X. Li et al.
602
dropwise at 08C. The reaction mixture was stirred at rt for 40 min. The reaction
was then poured into cold sat. NaHCO3 (5 mL) and extracted with CH2Cl2
(20 mL ꢀ 2). The combined organic layers were washed with brine (5 mL)
and dried with Na2SO4. The residue was purified by chromatography
1
(acetone/toluene, 1 : 6) to give 35 (130 mg, 66%). H NMR (400 MHz, CDCl3)
d 5.81–5.68 (1H, m, H-20), 5.05–4.98 (2H, m, H-30), 3.90–3.80 (2H, m, H-6,
H-1 eq), 3.68 (1H, dd, J ¼ 4.4, 11.8 Hz, H-6), 3.54 (2H, s, OMe), 3.34 (1H, dd,
J ¼ 8.7, 9.9 Hz, H-3), 3.15 (1H, m, H-5), 3.10–3.02 (2H, m, H-1 ax, H-4), 2.48
(1H, m, H-10), 1.89 (1H, m, H-2), 1.78 (1H, m, H-10); 13C NMR (100 MHz,
CDCl3) d 135.5, 116.9, 81.6, 79.7, 76.5, 69.7, 62.3, 60.8, 42.1, 32.4. HRMS
calcd. for C10H18O4Na (M þ Naþ) 225.1103, found 225.1103.
1,5-Anhydro-6-O-tert-butyldiphenylsily-2-deoxy-4-O-methyl-2-C-(2-
propenyl)-D-glucitol (36). To a solution of 35 (72 mg, 0.36 mmol) and
imidazole (48 mg, 0.71 mmol) in DMF (2 mL) was added tert-butyldiphenylsilyl
chloride (0.14 mL, 0.54 mmol). The mixture was stirred at rt for 3 hr,
then diluted with Et2O (30 mL), washed with H2O (5 mL ꢀ 2) and brine
(5 mL), dried over Na2SO4, and concentrated, and the residue was purified by
1
chromatography (EtOAc/hexane, 1 : 5) to afford 36 (152 mg, 97%). H NMR
(400 MHz, CDCl3) d 7.78-7.34 (10H, m), 5.80 (1H, m, H-20), 5.10–5.00 (2H, m,
H-30), 3.90 (1H, dd, J ¼ 4.7, 11.7 Hz, H-1 eq), 3.88–3.82 (2H, m, H-6),
3.54 (3H, s, OMe), 3.38–3.26 (2H, m, H-3, H-4), 3.09 (1H, m, H-5), 3.04 (1H, t,
J ¼ 11.7 Hz, H-1 ax), 2.48 (1H, m, H-10), 1.89 (1H, m, H-2), 1.78 (1H, m, H-10),
1.04 (9H, s); 13C NMR (100 MHz, CDCl3) d 135.8, 135.5, 133.8, 133.3, 129.5,
129.4, 127.5, 127.4, 116.6, 81.2, 80.4, 77.3, 69.8, 63.5, 60.7, 42.2, 32.7, 26.9,
19.5. HRMS calcd. for C26H36O4SiNa (M þ Naþ) 463.2281; found 463.2281.
1,5-Anhydro-6-O-tert-butyldiphenylsily-2-deoxy-4-O-methyl-3-O-myr-
istoyl-2-C-(2-propenyl)-D-glucitol (37). To a solution of 36 (147 mg,
0.33 mmol) in pyridine (5 mL), myristoyl chloride (0.27 mL, 0.99 mmol) was
added at 08C, and the mixture was stirred at rt for 3 hr. EtOAc (20 mL) was
added to the reaction mixture. The mixture was washed with H2O (5 mL ꢀ 2)
and brine (5 mL) and dried over Na2SO4. The residue was purified by chromato-
graphy to give 37 (206 mg, 95%). 1H NMR (400 MHz, CDCl3) d 7.78–7.34 (10H,
m), 5.74 (1H, m, H-20), 5.02–4.98 (2H, m, H-30), 4.80 (1H, dd, J ¼ 9.2, 10.4 Hz,
H-3), 3.93 (1H, dd, J ¼ 4.3, 11.6 Hz, H-1 eq), 3.88–3.84 (2H, m, H-6), 3.41 (3H,
s, OMe), 3.09 (1H, t, J ¼ 9.2 Hz, H-4), 3.20 (1H, m, H-5), 3.10 (1H, t,
J ¼ 11.6 Hz, H-1 ax), 2.38 (2H, t, J ¼ 7.9 Hz, OC(O)CH2), 2.35 (1H, m, H-2),
1.88 (2H, m, H-10), 1.66 (2H, m), 1.40–1.20 (20H, m), 1.06 (9H, s), 0.88 (3H, t,
J ¼ 6.7 Hz); 13C NMR (100 MHz, CDCl3) d 173.4, 135.8, 135.6, 135.2, 133.3,
129.5, 127.6, 127.4, 80.6, 78.6, 77.3, 69.6, 63.3, 60.1, 41.2, 34.6, 32.4, 31.9,
29.6, 29.5, 29.3, 29.2, 26.8, 24.9, 22.4, 19.4, 14.1. HRMS calcd. for C40H62O5Si
(M þ Naþ) 673.4262; found 673.4262.

Mapping the Active Site of LpxC
603
2-C-(Aldehydomethyl)-1,5-anhydro-6-O-tert-butyldiphenylsily-2-deoxy-4-
O-methyl-3-O-myristoyl-2-C-(2-propenyl)-D-glucitol (38). Ozone was
passed through a solution of 37 (150 mg, 0.23 mmol) in CH2Cl2 (20 mL) at
2788C until the solution turned blue. The excess ozone was removed with a
stream of oxygen for 10 min, followed by addition of Ph3P (121 mg,
0.46 mmol). The mixture was allowed to warm to rt for 3 hr, and then concen-
trated and purified by chromatography (EtOAc/hexane, 1 : 9) to afford 38
1
(122 mg, 81%). H NMR (400 MHz, CDCl3) d 9.70 (1H, s), 7.78–7.34 (10H, m),
4.87 (1H, dd, J ¼ 9.2, 10.4 Hz, H-3), 3.96 (1H, dd, J ¼ 4.5, 11.5 Hz, H-1 eq),
3.90–3.84 (2H, m, H-6), 3.44 (1H, t, J ¼ 9.2 Hz, H-4), 3.42 (3H, s, OMe), 3.20
(1H, m, H-5), 3.14 (1H, t, J ¼ 11.5 Hz, H-1 ax), 2.50–2.40 (2H, m, H-2, H-10),
2.38 (2H, t, J ¼ 7.4 Hz, OC(O)CH2), 2.20 (1H, m, H-10), 1.66 (2H, m), 1.40–
1.20 (20H, m), 1.06 (9H, s), 0.88 (3H, t, J ¼ 6.7 Hz); 13C NMR (100 MHz,
CDCl3) d 199.7, 173.5, 135.8, 135.6, 133.7, 133.2, 129.6, 127.6, 127.5, 80.6,
78.2, 77.1, 69.0, 60.2, 42.4, 36.4, 34.4, 31.9, 29.6, 29.4, 29.3, 29.2, 26.8, 24.9,
22.6, 19.4, 14.1.
2-C-(Carboxymethyl)-1, 5-anhydro-6-O-tert-butyldiphenylsily-2-deoxy-4-
O-methyl-3-O-myristoyl- 2-C-(2-propenyl)-D-glucitol (39). To a solution
of 38 (80 mg, 0.12 mmol) in t-BuOH (3 mL) and 2-methyl-2-butene (1 mL) was
added a solution of NaClO2 (110 mg, 1.23 mmol) and NaH2PO4 (169 mg,
1.23 mmol) in water (2 mL) slowly. The mixture was stirred for 1 hr, then
diluted with ice water (5 mL) and extracted with EtOAc (10 mL ꢀ 3). The
combined organic extracts were washed with brine (5 mL), dried over
Na2SO4, evaporated, and further dried under high vacuum overnight to
1
afford 39. The acid was used in the next step without purification. H NMR
(400 MHz, CDCl3) d 7.78–7.34 (10H, m), 4.90 (1H, dd, J ¼ 9.2, 10.6 Hz, H-3),
4.05 (1H, dd, J ¼ 4.6, 11.5 Hz, H-1 eq), 3.90–3.86 (2H, m, H-6), 3.44 (1H, t,
J ¼ 9.2 Hz, H-4), 3.42 (3H, s, OMe), 3.22 (1H, m, H-5), 3.18 (1H, t,
J ¼ 11.5 Hz, H-1 ax), 2.40–2.30 (4H, m, H-2, H-10, OC(O)CH2), 2.18 (1H, m,
H-10), 1.66 (2H, m), 1.40–1.20 (20H, m), 1.06 (9H, s), 0.88 (3H, t, J ¼ 6.7 Hz);
13C NMR (100 MHz, CDCl3) d 177.1, 173.5, 135.7, 135.5, 133.6, 133.2, 129.5,
127.6, 127.5, 80.6, 78.3, 76.8, 69.3, 63.1, 60.3, 38.6, 34.5, 32.7, 31.9, 29.7,
29.6, 29.4, 29.3, 26.9, 25.0, 22.8, 19.5, 14.2. HRMS calcd. C39H60O7SiNa
(M þ Naþ) 691.4006; found 691.4002.
1,5-Anhydro-2-C-(carboxymethyl N-hydroxyamide)-6-O-tert-butyldi-
phenylsily-2-deoxy-4-O-methyl-3-O-myristoyl-D-glucitol (40). To
a
.
solution of the acid 39 (25 mg, 37 mmol) and BnONH2 HCl (7.2 mg,
0.04 mmol) in CH2Cl2 (0.3 mL) were added successively EDC (9.5 mg,
0.05 mmol) and triethylamine (10 mL, 0.08 mmol) at rt. The reaction mixture
was stirred for 2 hr. CH2Cl2 (10 mL) was then added and the solution
was washed with water (5 mL) and brine (5 mL), and then dried over
Na2SO4. The residue after concentration was purified by chromatography

X. Li et al.
604
(toluene/EtOAc, 4 : 1) to afford 65 (19 mg, 68%).1H NMR (400 MHz, CD3OD) d
7.78–7.34 (10H, m), 4.80 (3H, overlapped with H2O, H-3, OCH2Ph), 3.86 (2H,
m, H-6), 3.84 (1H, dd, J ¼ 4.5, 11.5 Hz, H-1 eq), 3.43 (1H, t, J ¼ 9.4 Hz, H-4),
3.40 (3H, s, OMe), 3.20 (1H, m, H-5), 3.14 (1H, t, J ¼ 11.5 Hz, H-1 ax), 2.42–
2.34 (2H, m, OC(O)CH2), 2.25 (1H, m, H-2), 2.06 (1H, dd, J ¼ 5.1, 14.8 Hz,
H-10), 1.82 (1H, dd, J ¼ 8.7, 14.8 Hz, H-10), 1.63 (2H, m), 1.40–1.20 (20H, m),
1.06 (9H, s), 0.88 (3H, t, J ¼ 6.7 Hz); 13C NMR (100 MHz, CD3OD) d 175.1,
170.2, 136.9, 136.7, 134.4, 130.8, 130.3, 129.6, 129.4, 128.7, 128.6, 81.9, 79.8,
79.0, 78.6, 70.2, 64.4, 60.8, 40.2, 35.3, 33.2, 32.6, 30.9, 30.8, 30.7, 30.6, 30.5,
30.4, 27.5, 26.0, 23.9, 20.3, 14.6. HRMS calcd. C46H67NO7SiNa (M þ Naþ)
796.4584; found 796.4583.
1,5-Anhydro-2-deoxy-2-C-(carboxymethyl N-hydroxyamide)-4-O-methyl-
3-O-myristoyl-D-glucitol (41). A solution of 40 (16 mg, 21 mmol) in THF
(0.3 mL) was treated with TBAF (1.0 M solution in THF, 0.10 mL, 0.10 mmol) at
rt under argon. After 2.5 hr, the reaction was concentrated. The residue was dis-
solved in EtOAc (10 mL) and then washed with water (5 mL ꢀ 2) and brine, dried
over Na2SO4, and evaporated. The residue was purified by chromatography
1
(toluene/EtOAc, 1 : 1) to afford 41 (10 mg, 91%). H NMR (400 MHz, CD3OD:
CDCl3 ¼ 3 :1) d 7.40–7.34 (5H, m), 4.80 (3H, overlapped with H2O, H-3,
OCH2Ph), 3.86 (1H, dd, J ¼ 4.8, 11.7 Hz, H-1 eq), 3.77 (1H, dd, J ¼ 1.8, 11.9 Hz,
H-6), 3.63 (1H, dd, J ¼ 4.3, 11.9 Hz, H-6), 3.41 (3H, s, OMe), 3.25–3.11 (3H, m,
H-5, H-4, H-1 ax), 2.42-2.34 (2H, m, OC(O)CH2), 2.26 (1H, m, H-2), 2.05 (1H,
dd, J ¼ 4.9, 14.8 Hz, H-10), 1.80 (1H, dd, J ¼ 8.9, 14.8 Hz, H-10), 1.63 (2H, m),
1.40–1.20 (20H, m), 0.88 (3H, t, J ¼ 6.7 Hz); 13C NMR (100 MHz, CD3OD:
CDCl3 ¼ 3 :1) d 175.3, 170.2, 136.8, 136.0, 130.5, 130.3, 129.9, 129.7, 129.5,
128.6, 81.8, 79.9, 79.1, 78.5, 70.3, 62.4, 60.9, 39.9, 35.4, 33.1, 32.5, 30.8, 30.7,
30.6, 30.5, 30.4, 30.3, 27.3, 25.9, 23.8, 14.6. HRMS calcd. for C30H49NO7Na
(M þ Naþ) 558.3407; found 558.3408.
1,5-Anhydro-2-C-(carboxymethyl N-hydroxyamide)-2-deoxy-4-O-methyl-
3-O-myristoyl-D-glucitol (5). Pd/C (8 mg) was added to a solution of 41
(10 mg, 19 mmol) in AcOH (2 mL). The reaction mixture was stirred for 2.5 hr
under a balloon of H2. The reaction mixture was then filtered through Celite
and the Celite pad was washed well with MeOH. The solvent was evaporated
and the residue was purified by gravity chromatography on an Iatrobeads SiO2
column (CHCl3/MeOH, 15 : 1) to give 5 (5.3 mg, 64%). 1H NMR (400 MHz,
CD3OD) d 4.87 (1H, dd, J ¼ 8.7, 10.8 Hz, H-3), 3.93 (1H, dd, J ¼ 4.8, 11.6 Hz,
H-1 eq), 3.79 (1H, dd, J ¼ 2.0, 11.9 Hz, H-6), 3.65 (1H, dd, J ¼ 4.2, 11.9 Hz,
H-6), 3.41 (3H, s, OMe), 3.28 (3H, m, H-4, H-5, H-1 ax), 2.48–2.36 (2H, m,
OC(O)CH2), 2.28 (1H, m, H-2), 2.10 (1H, J ¼ 5.0, 14.5 Hz, H-10), 1.84 (1H,
dd, J ¼ 9.0, 14.5 Hz, H-10), 1.70 (2H, m), 1.40–1.30 (20H, m), 0.90 (3H, t,
J ¼ 6.7 Hz); 13C NMR (100 MHz, CD3OD) d 175.3, 170.3, 81.9, 79.9, 78.5,

Mapping the Active Site of LpxC
605
70.3, 62.3, 60.7, 40.0, 35.2, 33.1, 32.5, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3, 25.9,
23.7, 14.4. HRMS calcd. for C23H43NO7Na (M þ Naþ) 468.2937; found
468.2934.
1,5-Anhydro-4,6-O-benzylidene-2-C-(carboxymethyl N-benzyloxyamide)-
2-deoxy-3-O-tetradecane-D-glucitol (42). Compound 11 (40 mg, 0.10 mmol)
was dissolved in DMF (1 mL); sodium hydride (60% in oil, 10 mg, 0.25 mmol)
and 1-bromotetradecane (89 mL, 0.30 mmol) were then added. The reaction
mixture was stirred at rt for 5 hr. (TLC: EtOAc/toluene, 1 : 1 showed some
starting material was still left). The mixture was diluted with CH2Cl2 (10 mL),
washed with diluted HCl (5 mL), and sat. NaHCO3, dried over Na2SO4, and
concentrated, and the residue was purified by chromatography (EtOA/toluene,
1
3 : 1) to give 42 (40 mg, 70%). H NMR (400 MHz, CDCl3) d 8.41 (1H, br s, NH),
7.50–7.30 (10H, m), 5.54 (1H, s, CHPh), 4.88 (2H, br s, OCH2Ph), 4.27 (1H, dd,
J ¼ 4.9, 10.4 Hz, H-6), 4.01 (1H, dd, J ¼ 4.9, 11.5 Hz, H-1 eq), 3.90 (1H, dt,
J ¼ 6.7, 9.3 Hz, OCH2), 3.70 (1H, t, J ¼ 10.4 Hz, H-6), 3.56 (1H, t, J ¼ 8.9 Hz,
H-4), 3.48 (1H, m, OCH2), 3.36–3.30 (2H, m, H-5, H-1 ax), 3.26 (1H, dd,
J ¼ 8.9, 10.3 Hz, H-3), 2.40 (1H, br s, H-10), 2.18 (1H, br s, H-2), 1.95 (1H, br s,
H-10), 1.50 (2H, m), 1.33–1.18 (20H, m), 0.89 (3H, t, J ¼ 6.7 Hz); 13C NMR
(100 MHz, CDCl3) d 169.1, 137.5, 129.1, 128.8, 128.6, 128.1, 125.9, 101.1, 83.8,
80.5, 72.9, 71.7, 70.5, 68.9, 31.9, 30.2, 29.7, 29.6, 29.4, 29.3, 26.1, 22.6, 14.1.
HRMS calcd. for C36H54NO6 (M þ Hþ) 596.3951; found 596.3953.
1,5-Anhydro-2-C-(carboxymethyl N-hydroxyamide)-2-deoxy-3-O-tetra-
decane-D-glucitol (6). Pd-C (20 mg) was added to a solution of 42 (30 mg,
52 mmol) in AcOH (2 mL). The reaction mixture was stirred for 6 hr under a
balloon of H2. After the filtration, the solvent was evaporated and the
residue was purified on an Iatrobeads SiO2 column (CHCl3/MeOH, 15 : 1) to
1
give 6 (13 mg, 70%). H NMR (400 MHz, CD3OD) d 3.92–3.85 (2H, m, H-1 eq,
OCH2), 3.80 (1H, dd, J ¼ 2.3, 11.8 Hz, H-6), 3.59 (1H, dd, J ¼ 6.1, 11.8 Hz,
H-6), 3.53 (1H, m, OCH2), 3.33 (1H, d, J ¼ 10.0 Hz, H-4), 3.14 (1H, t, J ¼ 11.4
H-1 ax), 3.12 (1H, m, H-5), 3.00 (1H, dd, J ¼ 8.7, 10.0 Hz, H-3), 2.46 (1H, dd,
J ¼ 4.0, 14.3 Hz, H-10), 2.05 (1H, m, H-2), 1.84 (H, dd, J ¼ 9.5, 14.3 Hz, H-10),
1.56 (2H, m), 1.40–1.20 (20H, m), 0.85 (3H, t, J ¼ 6.7 Hz); 13C NMR
(100 MHz, CD3OD) d 171.2, 85.9, 82.9, 73.8, 73.1, 70.5, 63.1, 33.1, 32.6, 31.4,
30.8, 30.7, 30.5, 27.2, 23.7, 14.4. HRMS calcd. for C22H43NO6Na (M þ Naþ)
440.2988; found 440.2990.
1,5-Anhydro-3-O-benzyol-2-C-(carboxymethylN-hydroxyamide)-2-deoxy-
D-glucitol (7). To a solution of 1,5-Anhydro-4, 6-O-benzylidene-2-deoxy-2-C-(2-
propenyl)-D-glucitol[6b] (Sch. 8, 50mg, 0.18mmol) in pyridine (2mL), benzoyl
chloride (0.13 mL, 1.08 mmol) was added at 08C, and the mixture was stirred for
3 hr at rt. EtOAc (30 mL) was added to the reaction mixture and the solution was
washed with diluted HCl (5 mL), sat. NaHCO3 (5 mL), and brine (5 mL), and

X. Li et al.
606
dried over Na2SO4 and the residue was purified by chromatography (EtOAc/
hexane, 1 : 7) to give 63 mg (95%).
Ozone was passed through a solution of above compound (30 mg, 82 mmol)
in CH2Cl2 (10 mL) at 2788C until the solution turned blue. The excess ozone
was removed with a stream of oxygen for 10 min, followed by addition of
Ph3P (53 mg, 0.20 mmol). The mixture was allowed to warm to rt for 2 hr,
and then concentrated. The residue was purified by chromatography
(EtOAc/hexane, 1 : 3) to afford the aldehyde (26 mg, 87%).
To a solution of above compound (26 mg, 70 mmol) in t-BuOH (2 mL) and
2-methyl-2-butene (0.6 mL) was added
a solution of NaClO2 (91 mg,
0.80 mmol) and NaH2PO4 (138 mg, 0.80 mmol) in water (1.5 mL) over 5 min.
The mixture was stirred for 1 hr, then diluted with ice water and extracted
with EtOAc. The combined organic extracts were washed with brine (5 mL),
dried over Na2SO4, evaporated, and further dried under high vacuum over-
night to afford the acid (27 mg, 100%).
.
To a solution of the acid (27 mg, 70 mmol) and BnONH2 HCl (13 mg,
80 mmol) in CH2Cl2 (0.5 mL) were added successively EDC (19 mg, 0.10 mmol)
and triethylamine (13 mL, 0.10 mmol) at rt. The reaction mixture was stirred
for 2 hr. CH2Cl2 (10 mL) was then added and the solution was washed with
water (5 mL) and brine (5 mL), and then dried over Na2SO4. The residue
after concentration was purified by chromatography (toluene/EtOAc, 4 : 1) to
afford hydroxamide 24 mg (70%).
Pd-C (10 mg) was added to a solution of the above hydroxyamide (10 mg,
52 mmol) in AcOH (2 mL). The reaction mixture was stirred for 6 hr under a
balloon of H2. After the filtration, the solvent was evaporated and the
residue was purified on an Iatrobeads SiO2 column (CHCl3/MeOH, 15 : 1) to
1
give 7 (4 mg, 62%). H NMR (500 MHz, CD3OD) d 8.10–7.48 (5H, COC6H5),
5.02 (1H, dd, J ¼ 9.0, 10.8 Hz, H-3), 4.03 (1H, dd, J ¼ 4.7, 11.6 Hz, H-1 eq),
3.85 (1H, dd, J ¼ 2.4, 11.9 Hz, H-6), 3.67 (1H, dd, J ¼ 5.7, 11.9 Hz, H-6), 3.58
(1H, t, J ¼ 9.0 Hz, H-4), 3.30 (2H, m, H-5 and H-1 ax), 2.42 (1H, m, H-2),
2.20 (1H, dd, J ¼ 4.2, 14.6 Hz, H-10), 1.91 (1H, dd, J ¼ 9.8, 14.6 Hz, H-10).
HRMS calcd. for C15H19NO7Na 348.1059; found 348.1057.
1,5-Anhydro-2-C-(carboxymethyl N-hydroxyamide)-2-deoxy-3-O-pal-
mitoyl-D-glucitol (8). To a solution of 11 (20 mg, 50 mmol) in pyridine
(0.5 mL), DMAP (3 mg, 30 mmol) and palmitoyl chloride (30 mL, 0.10 mmol)
were successively added at 08C and stirred for 20 hr at rt. EtOAc (30 mL) was
then added to the reaction mixture and the solution was washed with 10%
citric acid (5 mL ꢀ 3), H2O (5 mL), sat. NaHCO3 (5 mL), and brine (5 mL). The
organic layer was dried over Na2SO4 and concentrated. The residue was
purified by chromatography (toluene/EtOAc, 4 : 1) to afford 18 mg (55%).
Pd-C (10 mg) was added to a solution of above compound (10 mg, 16 mmol)
in AcOH (2 mL). The reaction mixture was stirred for 6 hr under a balloon of

Mapping the Active Site of LpxC
607
H2. After the filtration through Celite, the Celite pad was washed well with
MeOH. The solvent was evaporated and the residue was purified by gravity
chromatography on an Iatrobeads SiO2 column (CHCl3/MeOH, 15 : 1) to give
1
8 (5 mg, 70%). H NMR (400 MHz, CD3OD) d 4.75 (1H, dd, J ¼ 9.0, 10.8 Hz,
H-3), 3.94 (1H, dd, J ¼ 4.7, 11.6 Hz, H-1 eq), 3.80 (1H, dd, J ¼ 2.3, 11.8 Hz,
H-6), 3.62 (1H, dd, J ¼ 5.7, 11.8 Hz, H-6), 3.38 (1H, t, J ¼ 9.0 Hz, H-4), 3.25–
3.15 (2H, m, H-5 and H-1 ax), 2.38 (1H, t, J ¼ 7.5 Hz, OC(O)CH2), 2.25 (1H,
m, H-2), 2.10 (1H, dd, J ¼ 4.9, 14.5 Hz, H-10), 1.82 (1H, dd, J ¼ 9.1, 14.5 Hz,
H-10), 1.60 (2H, m), 1.40–1.20 (24H, m), 0.88 (3H, t, J ¼ 6.7 Hz); 13C NMR
(100 MHz, CD3OD) d 175.7, 170.3, 82.7, 79.1, 70.7, 70.3, 62.9, 39.8, 35.1, 33.1,
32.7, 30.8, 30.7, 30.6, 30.4, 30.2, 25.9, 23.7, 14.4. HRMS calcd. for
C24H45NO7Na 482.3094; found 482.3097.
Inhibitor Assays
LpxC activity assay. The E. coli LpxC substrate, [a-32P]-UDP-3-O-(R-3-
hydroxymyristoyl)-GlcNAc was prepared and purified as described previously
by acylation of [a-32P] UDP-GlcNAc using purified E. coli LpxA (provided by T.
J. O. Wyckoff, Duke University).[14] The assays were performed at 308C and
contained 4 mM UDP-3-O-(R-3-hydroxymyristoyl)GlcNAc, 1 mg/mL BSA in
25 mM sodium phosphate, pH 7.4, and 0.1 nM purified E. coil LpxC. The
activity assays were performed in plastic microcentrifuge tubes in a reaction
volume of 20 mL. At each time point (chosen so that the total conversion to
product was less than 10%), 5 mL portions of each reaction mixture were
removed and added to 1 mL of 1.25 M NaOH to stop the reaction. The
alkaline samples were incubated for an additional 10 min at 308C to ensure
complete hydrolysis of the ester-linked acyl chains from the LpxC substrate
and product, and then were neutralized by the addition of 1 mL of 1.25 M
acetic acid and 1 mL of 5% trichloroacetic acid. The neutralized samples were
incubated on ice for 5 min and centrifuged for 2 min in a microcentrifuge.
Portions of the supernatants (1 mL) were spotted onto PEI-cellulose TLC
plates for separation of the remaining substrate (detected as [a-32P]-UDP-
GlcNAc) from the product (detected as [a-32P]-UDP-GlcN). After air drying,
the plates were soaked for 10 min in methanol to improve resolution before
chromatography. The plates were developed with 0.2 M aqueous guanidine-
HCl as the solvent system. The radioactive spots on the plates were analyzed
using a PhosphorImager equipped with ImageQuant software (Molecular
Dynamics, Inc.) to determine the yields of product produced in each reaction
mixture.
Inhibition of LpxC activity. Stock solutions (10 mg/mL) and any further
dilutions of each inhibitor were made in 100% dimethylsulfoxide (DMSO).
Compounds were added to a final concentration of 1 mg/mL to an assay
mixture containing 4 mM UDP-3-O-(R-3-hydroxymyristoyl)GlcNAc in 25 mM

X. Li et al.
608
sodium phosphate buffer, pH 7.4. Additional DMSO was added to maintain
compound solubility at a final assay concentration of 10% in DMSO. Purified
E. coli LpxC was incubated with 1 mg/mL BSA in buffer and diluted into the
final assay mixture to 0.1 nM at 308C. The initial velocities were plotted as a
function of inhibitor concentration for each compound. The percent inhibition
of each compound at 1 mg/mL was calculated based on comparison of the
specific activity of a reaction containing inhibitor to a wild-type reaction con-
taining no inhibitor and substituting DMSO instead.
ACKNOWLEDGEMENT
This work is supported by the Natural Science and Engineering Research
Council of Canada (O.H) and the NIH (grant GM-51310 to C.R.H.R). X.L. is
the recipient of a graduate scholarship in Carbohydrate Chemistry from the
Alberta Research Council. The authors thank Prof. Pei Zhou for helpful
discussions.
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