Improving the immunostimulatory potency of diethanolamine-containing lipid A mimics

Article abstract of DOI:10.1016/j.bmc.2013.02.024

Lipid A is the active principal of gram negative bacterial lipopolysaccharide (LPS) in the activation of Toll-like receptor 4 (TLR4). Given the important role TLR4 plays in innate immunity and the development of adaptive immune responses, ligands that can modulate TLR4-mediated signaling have great therapeutic potential. Recently, we have reported a series of monophosphorylated lipid A mimics as potential ligands of TLR4, in which a diethanolamine moiety is employed to replace the reducing end (d-glucosamine). In this paper, we describe the synthesis of two further diethanolamine- containing lipid A mimics, 3 and 4, in an effort to mimic more closely the di-phosphate nature of natural lipid A. Both mimic 3, with an additional phosphate on the diethanolamine acyclic scaffold, and mimic 4, with a terminal carboxylic acid moiety as a phosphate bioisostere, serve to increase the potency of the immunostimulatory response induced, as measured by the induction of the cytokines TNF-α, IL-6, and IL-1β in the human monocytic cell line THP-1. In addition, mechanistic studies involving the known TLR4 antagonist lipid IVa confirm TLR4 as the target of the diethanolamine-containing lipid A mimics.

Full text of DOI:10.1016/j.bmc.2013.02.024

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Improving the immunostimulatory potency of diethanolamine-
containing lipid A mimics
Jordan D. Lewicky a, Marina Ulanova b, Zi-Hua Jiang a,
⇑
a Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1
b Medical Sciences Division, Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipid A is the active principal of gram negative bacterial lipopolysaccharide (LPS) in the activation of
Toll-like receptor 4 (TLR4). Given the important role TLR4 plays in innate immunity and the development
of adaptive immune responses, ligands that can modulate TLR4-mediated signaling have great therapeu-
tic potential. Recently, we have reported a series of monophosphorylated lipid A mimics as potential
Received 27 December 2012
Revised 4 February 2013
Accepted 13 February 2013
Available online xxxx
ligands of TLR4, in which a diethanolamine moiety is employed to replace the reducing end (D-glucosa-
mine). In this paper, we describe the synthesis of two further diethanolamine-containing lipid A mimics,
3 and 4, in an effort to mimic more closely the di-phosphate nature of natural lipid A. Both mimic 3, with
an additional phosphate on the diethanolamine acyclic scaffold, and mimic 4, with a terminal carboxylic
acid moiety as a phosphate bioisostere, serve to increase the potency of the immunostimulatory response
Keywords:
Toll-like receptor 4
Lipid A
Glycolipid
Immunostimulant
induced, as measured by the induction of the cytokines TNF-a, IL-6, and IL-1b in the human monocytic
cell line THP-1. In addition, mechanistic studies involving the known TLR4 antagonist lipid IVa confirm
TLR4 as the target of the diethanolamine-containing lipid A mimics.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
groups are not a strict requirement, however, as other negatively
charged and acidic groups have been employed as bioisosteres of
Toll-like receptor (TLR) 4 plays an important role in the innate
immunity and the development of adaptive immune responses.
The activation of TLR4 by Gram-negative bacterial lipopolysaccha-
ride (LPS) has been extensively studied, and molecular mecha-
nisms post-activation delineated.1 The ability to regulate the
induction of an adaptive immune response has made TLR4 an
attractive target in terms of developing vaccine adjuvants.2,3 Sig-
nificant effort has thereby been directed towards the development
of ligands for TLR4 via synthetic analogs and mimics of lipid A (1,
Fig. 1), which is the active principal of LPS in triggering TLR4-med-
iated signaling.4,5 The end goal of these efforts has been the maxi-
mization of the beneficial immunostimulatory activity, while
minimizing the inherent endotoxic properties of LPS/lipid A.
TLR4 is expressed as a complex with the obligate accessory pro-
tein MD-2.1 The structural basis of the recognition of lipid A/LPS by
TLR4/MD-2 is highly complex, and has garnered considerable
attention. The structural features important for optimal immuno-
logic activity include the degree, pattern, and chain length of the
lipid acylation.6,7 The two phosphate groups in lipid A also greatly
affect the immunologic activity of lipid A/LPS. Deletion of either of
the phosphates reduces endotoxic activity ꢀ100-fold, while
elimination of both phosphates abolishes all activity.8 Phosphate
the phosphates, with only minor effects on activity noted.9,10 Of
note are the monophosphoryl lipid A (MPLA) derivatives, which
are devoid of the anomeric phosphate.11 These derivatives are
known to have reduced toxicity, yet retain potent immunostimula-
tory activity.11–13 Interestingly, MPLA has been shown to selec-
tively activate the TLR4-TRAM-TRIF signaling pathway, but not
the TLR4-MAL-MyD88 pathway, thus potentially accounting for
the reduced toxicity.14 These results, as well as structure–activity
relationship data of other lipid A analogs,15–18 have clearly shown
the significance of the anomeric phosphate group in modulating
the potency and the activity/endotoxicity profile of lipid A mole-
cules. Our understanding of the recognition of lipid A/LPS by
TLR4/MD-2 has improved as of late, thanks largely to a protein
crystal structure of the receptor complex bound to LPS.19 The
two phosphate groups of lipid A have been shown to play an
important role in receptor–ligand binding, and the subsequent
receptor dimerization and activation. They provide ionic interac-
tions with positively charged residues on both TLR4 and MD-2,
and the adjacent TLR4 within the dimer complex.
As part of our continuing work on the design and synthesis of
TLR4 ligands as immunostimulatory agents,20–23 we recently
reported a new class of lipid A mimics, as exemplified by 2
(Fig. 1), in which the reducing-end glucosamine residue of the
natural disaccharide scaffold has been replaced by an acylated
diethanolamine moiety.24 Mimic 2 was shown to possess potent
⇑
Corresponding author. Tel.: +1 807 766 7171; fax: +1 807 346 7775.
E-mail address: zjiang@lakeheadu.ca (Z.-H. Jiang).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/.2013.02.024
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

2
J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
OH
OH
O
O
O
O
O
(HO) PO
2
(HO) PO
2
O
O
O
O
HO
N
R
O
NH
O
NH
O
O
O
2
3
4
R = CH OH
2
O
NH
O
O
O
O
P(OH)
2
O
O
O
O
HO
O
O
O
O
R = CH OP(O)(OH)
2
O
HO
2
O
R = COO H
1 E. coli lipid A
Figure 1. Structure of E. coli lipid A (1) and diethanolamine-containing lipid A mimics (2–4).
immunostimulatory activity in the human monocytic cell line THP-
1, presumably stimulating TLR4. The therapeutic potential of com-
pound 2 as an immunostimulant is currently under further inves-
tigation. Given the key role the two phosphate groups of lipid A
play in the dimerization and activation of the TLR4/MD-2 receptor
complex,19 we have therefore designed and synthesized two more
diethanolamine-containing lipid A mimics, 3 and 4 (Fig. 1) with the
goal of further improving the potency of mimic 2. Mimic 3 includes
an additional phosphate on the diethanolamine scaffold while mi-
mic 4 contains a terminal carboxylic acid moiety as a phosphate
bioisostere. It is possible that mimics 3 and 4 may possess a higher
binding affinity for the TLR4/MD-2 receptor complex, thus poten-
tially increasing the potency of the induced immunostimulatory
response. Herein, we report the synthesis and preliminary biolog-
ical activity of lipid A mimics 3 and 4.
issues with acylation selectivity, and interesting side reactions in
which the solvent tetrahydrofuran molecule was ring-opened to
form 1-hydroxybutyl adducts.24 We therefore have chosen to keep
the terminal hydroxyl moiety of the acyclic scaffold protected until
the late stages of the synthesis. The amine moiety of diethanola-
mine was first protected as the 2,2,2-trichloroethoxy carbamate
(Troc) to furnish 5 in 78% yield, along with a 7% yield of 6, in which
one of the b-hydroxy moieties has reacted to form the Troc carbon-
ate. Attempts at the protection of one of the b-hydroxy moieties of
5 as the tert-butyldiphenyl silyl ether (TBDPS) yielded mono-sily-
lated 7 and di-silylated 8 in 54% and 6%, respectively, with a 35%
recovery of unreacted 5.
The trimethylsilyl trifluoromethanesulfonate catalyzed glyco-
sylation of 7 with known imidate donor 925 yielded glycoside 10
in 94% (Scheme 2). Similar to our previous work, compound 10 ex-
isted as a mixture of two rotational isomers in an approximate 1:1
ratio as a result of prohibited free rotation around the carbamate
C–N bond. The desired b-glycosidic linkage in 10 was confirmed
via NMR spectral data (1H NMR: d 4.53, d, J 8.5 Hz, H-1 from one
isomer & d 4.65, d, J 8.5 Hz, H-1 from the other isomer). Compound
10 was treated with guanidine nitrate–sodium methoxide at room
temperature to give the O-deacetylated product in 91% yield, leav-
ing the two N-Troc groups intact.26 The acid catalyzed installation
of the 4,6-di-O-benzylidene group furnished 12 in 87% yield. The
N,N-dimethylaminopyridine (DMAP) and N,N0-diisopropylcarbodi-
imide (DIC) promoted acylation of the 3-OH moiety in 12 with dil-
ipid acid 1427 yielded 13 in 93%. Regioselective benzylidene ring
opening of 13 was brought about via sodium cyanoborohydride
(NaBH3CN) and an HCl(g)-infused diethyl ether solution at 0 °C to
give 15, with the free 4-OH, in 87%. Finally, reaction of 15 with
5-phenyl tetrazole (5-Ph-tetrazole) and dibenzyl N,N-dii-
sopropylphosphoramidite [(BnO)2PN(iPr)2], followed by the
m-chloroperbenzoic acid (m-CPBA) promoted oxidation at 0 °C
furnished the desired protected phosphotriester functionality of
advanced intermediate 16 in 85%. Following the removal of both
N-Troc protection groups, the intermediate 16 would allow for
2. Results & discussion
2.1. Synthesis
There is significant evidence indicating that the degree, pattern,
and chain length of the lipid acylation in lipid A molecules are
important factors contributing to their overall biological activity.6,7
Our previous synthesis of lipid A mimic 2 installed a defined lipid
onto the diethanolamine acyclic scaffold at the early stages, thus
limiting the potential for incorporating variation in any of the
above mentioned acyl characteristics.17 We therefore chose to em-
ploy an alternate synthetic strategy for accessing lipid A mimics 3
and 4, in an effort to ultimately allow for facile acyl chain variation
in future structure–activity relationship investigations.
The synthesis began with the preparation of the diethanola-
mine-based glycosylation acceptor 7 (Scheme 1). In our previous
synthesis of mimic 2 (Fig. 1), the terminal hydroxyl moiety of the
diethanolamine acyclic scaffold was left free throughout the syn-
thesis. This proved problematic at many stages of the synthesis,
including a propensity to form undesired di-glycosylated products,
a
b
OR
RO
OTBDPS
HO
NH(CH CH OH)
2
2
2
N
N
Troc
Troc
5
6
R = H
7
8
R = H
R = TBDPS
R = Troc
Scheme 1. Reagents and conditions: (a) Troc-Cl, NaHCO3, H2O, 78% for 5 and 7% for 6; (b) TBDPS-Cl, Et3N, CH2Cl2, 0 °C, 54% for 7 and 6% for 8 (35% recovery of 5).
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
OAc
3
OR
O
a
O
AcO
AcO
RO
RO
+
7
O
OTBDPS
N
NH
NH
Troc
OC(NH)CCl 3
Troc
Troc
10 R = Ac
11 R = H
9
b
c
OBn
O
Ph
O
O
e
RO
O
O
O
OTBDPS
OTBDPS
O
RO
N
N
O
O
NH
NH
Troc
Troc
Troc
Troc
O
n-C11 H23
n-C13 H27
15 R = H
12 R = H
O
f
d
16 R = P(O)(OBn)2
13 R =
O
n-C 13H27
n-C11H23
CO
O
O
n-C 13H27
COOH
n-C11H23
14
Scheme 2. Reagents and conditions: (a) TMSOTf, CH2Cl2, 94%; (b) guanidinium nitrate, NaOMe/MeOH, CH2Cl2, 91%; (c) PhCH(OMe)2, p-TsOH, CH3CN, 87%; (d) 14, DIC, DMAP,
CH2Cl2, 93%; (e) NaBH3CN, HCl(g)-Et2O, THF, 0 °C; 87%; (f) (i) (BnO)2PN(iPr)2, 5-Ph-tetrazole, CH2Cl2, (ii) m-CPBA, 0 °C, 85%.
OBn
O
OBn
O
O
O
3
4
a
(BnO) PO
2
c or d
(BnO) PO
2
e
16
O
OR
O
O
O
N
N
R
O
O
O
O
NH
NH
O
O
O
O
O
O
O
O
O
n-C
H
11 23
n-C
H
11 23
O
O
O
n-C
H
11 23
n-C
H
11 23
n-C
H
13 27
n-C
H
13
27
O
O
n-C
H
n-C
H
11 23
11 23
n-C
H
13 27
n-C
H
13 27
n-C
H
27
n-C
H
13 27
13
19 R = CH OP(O)(OBn)
17 R = TBDPS
18 R = H
2
2
b
20 R = COOH
Scheme 3. Reagents and conditions: (a) (i) Zn dust, HOAc; (ii) 14, HBTU, (iPr)2NEt, DMF, 65%; (b) (Bu)4NF, CH2Cl2, 90%; (c) (i) (BnO)2PN(iPr)2, 5-Ph-tetrazole, CH2Cl2, (ii) m-
CPBA, 0 °C, to give 19, 71%; (d) TEMPO, BAIB, CH2Cl2, H2O, to give 20, 83%; (e) H2, Pd/C, THF, 70% for 3 and 77% for 4.
chemoselective acylation of the two amine moieties, since the pri-
mary amine is anticipated to be more reactive toward an acylation
reagent than the secondary amine due to the increased steric hin-
drance in the secondary amine.31,32 Thus, 16 could serve as a com-
mon precursor to access molecules with different acylation
patterns on the two amine groups.
Compound 16 was subjected to a zinc treatment in acetic acid
to furnish the di-amine, which was immediately coupled with dil-
ipid acid 1427 under the promotion of the peptide coupling reagent
O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluorophos-
phate (HBTU) to form 17 in 65% overall yield (Scheme 3). Deprotec-
tion of the acyclic scaffold hydroxyl via a tetrabutylammonium
fluoride (TBAF) treatment furnished our previously reported inter-
mediate 1817 in 90% yield. Compound 18 was subjected to a similar
phosphoramidite treatment as 15 (Scheme 2) to install the desired
phosphotriester functionality in 19 in a 71% yield. Compound 19
was found to be quite labile during chromatography purification
or under storage in the freezer. We speculate that the decomposi-
tion of 19 may involve an intramolecular substitution forming the
relatively stable oxazolinium ion 19-A24,28 (Scheme 4). Oxazolini-
um ions have been reported and structurally confirmed by NMR
as intermediates for b-hydroxy alkyl amides, which react readily
with a carboxylic acid to form esters under neutral condition.28
The oxazolinium ion 19-A may undergo nucleophilic attack or an
elimination reaction leading to further decomposed products such
as 19-B to 19-D. At this stage of the investigation, the structures of
these decomposed products have not been determined. Practically,
due to its relative instability, compound 19 had to be purified
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4
J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
O
O
R'O
O
P(OBn)2
O
P(OBn)2
R'O
Nu
N
N
Nu
19-B
R
19
R
B
O
O
H
N
R'O
Nu
R'O
R'O
N
N
O
B
O
R
R'O
O
P(OBn)2
O
R
R
N
H
O
19-A
B
R
19-D
19-C
O
Oxazolinium ion
Scheme 4. Proposed mechanism accounting for the ready decomposition of 19.
quickly and immediately subjected to global deprotection via cat-
alytic hydrogenation under atmospheric pressure in the presence
of palladium on charcoal to yield 3. The (2,2,6,6-tetramethylpiperi-
din-1-yl)oxyl (TEMPO) and bis(acetoxy)iodobenzene (BAIB) pro-
moted oxidation of the acyclic scaffold free hydroxyl group in 18
furnished acid 20, with which no stability issues were noted. A
similar catalytic hydrogenation to remove benzyl protecting
groups in acid 20, afforded 4. The structures of 3 and 4 were con-
firmed by 1H NMR and MALDI-MS data.
2.2. Biological evaluations
The binding of agonistic ligands to the TLR4–MD-2 receptor
complex leads to the release of various pro-inflammatory cyto-
kines, including tumour necrosis factor-a (TNF-a), interleukin-6
(IL-6), and interleukin-1b (IL-1b), as well as other immunomodu-
lating mediators including interferon-b (IFN-b) and nitric oxide.1
To evaluate the relative immunostimulatory activity of lipid A
mimics 2–4, we therefore measured the direct effects of the mim-
ics on TNF-a, IL-6, and IL-1b production in phorbol 12-myristate
13-acetate (PMA) differentiated human monocytic cell line THP-1.
Lipid A mimics (2–4) were tested over a wide range of concen-
trations (10ꢁ4
lM–10 lM), and the cytokine responses follow a
clear dose response relationship (Fig. 2). A maximum response le-
vel is achieved for all three cytokines at a stimulus concentration of
between 0.1
10 M stimulus concentration results in decreased responses. Sig-
nificant levels of TNF- and IL-1b are induced by all three mimics
at concentrations in the 10ꢁ3
M range. Measurable IL-1b is also
observed for mimic 4 at a concentration as low as 10ꢁ4
M. In gen-
lM and 1.0 lM, after which a further increase to a
l
a
l
l
eral, both mimics 3 and 4 show increased potency over mimic 2,
with mimic 4 showing the greatest potency for stimulating pro-
duction of all three cytokines. This trend is most evident in the
IL-6 response data, in which mimics 3 and 4, in comparison to 2,
exhibit an approximate 10-fold and 100-fold increase in potency,
respectively. The results indicate that the phosphate/phosphate
bioisostere group on the diethanolamine moiety plays an impor-
tant role in the immunostimulatory potency of these lipid A
mimics.
The suspected mode of action for lipid A mimics 2–4 is stimula-
tion of the TLR4–MD-2 receptor complex. In an effort to confirm
TLR4 as the target of our diethanolamine-containing lipid A mim-
ics, we performed a competitive inhibition study with lipid IVa. Li-
pid IVa is the tetra-acyl biosynthetic precursor of lipid A, which has
been shown to bind to the human TLR4/MD-2 receptor complex,
yet not induce any activation.29 It follows that lipid IVa is a potent
antagonist of LPS-induced TLR4 activation in the human monocytic
cell line THP-1.30 We therefore tested the potential of lipid IVa to
Figure 2. Cytokine production by THP-1 cells after stimulation with LPS and lipid A
mimics (2–4). THP-1 cells were incubated for 24 h with increasing concentrations of
2–4. TNF-a (A), IL-6 (B), and IL-1b (C) in cell supernatants were measured via
ELISAs. The results are shown as the average of two separate experiments.
inhibits the production of both TNF-
LPS and mimic 4. At a concentration of 0.1
point a maximum level of cytokine induction is expected, the pres-
a
and IL-1b induced by both
inhibit the TNF-a and IL-1b cytokine response of THP-1 cells to li-
lM of mimic 4, at which
pid A mimic 4 (Fig. 3). Co-treatment with lipid IVa significantly
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J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
Figure 3. Inhibited cytokine production by THP-1 cells after stimulation with an agonist (LPS or lipid A mimic 4) in the presence of lipid IVa. THP-1 cells were incubated for
24 h with either LPS or lipid A mimic 4 in the presence of increasing concentrations of lipid IVa. TNF-a (A) and IL-1b (B) in cell supernatants were measured via ELISAs. The
results are shown as the average of two separate experiments.
ence of lipid IVa at 0.5
the production of TNF-
ginally measurable. In contrast, the presence of lipid IVa at 0.1
exhibits only partial antagonism against the production of both
cytokines induced by 4 at a concentration of 0.1 M. It is worth
noting that complete inhibition of TNF- and IL-1b production in-
duced by 0.001 M of 4 in the presence of 0.1 M of lipid IVa (100-
fold of 4) is not observed, suggesting that mimic 4 may have a
higher binding affinity with TLR4/MD-2 than lipid IVa. Based on
these data, we conclude that compound 4 is an agonist of TLR4,
and other diethanolamine-containing lipid A mimics are likely li-
gands of TLR4 as well.
In conclusion we have successfully synthesized two new mem-
bers of diethanolamine-containing lipid A mimics, in which the
previously free terminal hydroxyl moiety has now been replaced
with either an additional phosphate, or a phosphate bioisostere
carboxylic acid functionality. Structurally speaking, both the phos-
phate group in lipid A mimic 3, and the acidic phosphate bioisoste-
l
M appears to have completely inhibited
m (multiplet), br (broad). Structural assignments were made using
standard gCOSY and gHSQC methodology. NMR peaks belonging to
primary lipid chains are denoted with an L subscript, whereas
those belonging to secondary lipid chains are denoted with an L0
subscript. MALDI mass spectra were measured on the Applied Bio-
systems Mariner 4700 System at the University of Western Ontar-
io. Optical rotations were measured with Perkin Elmer 343
Polarimeter at 22 °C.
a, while the production of IL-1b is still mar-
lM
l
a
l
l
3.2. N,N-Bis(2-hydroxyethyl)-2,2,2-
trichloroethoxymethanamide (5) & N-(2-|hydroxyethyl)-N-{2-
(2,2,2-trichloroethoxycarbonyloxy)-ethyl}-2,2,2-
trichloroethoxymethanamide (6)
To a solution of diethanolamine (1.10 g, 10.45 mmol) and
sodium bicarbonate (3.07 g, 36.58 mmol) in water (70 mL), 2,2,2-
trichloroethoxychloroformate (2.88 g, 13.59 mmol) was added
drop wise in about 3 min. As the mixture was stirred at room tem-
perature, a viscous insoluble oil formed and settled to the bottom
of the reaction vessel. After stirring for 3 h, the mixture was
extracted with EtOAc (3 ꢂ 135 mL), after which the combined
organic layers were dried over Na2SO4 and concentrated. Purifica-
tion via flash column chromatography (hexane/EtOAc/MeOH,
1:1:0.1) yielded 5 (2.29 g, 78%) and 6 (332 mg, 7%), both as color-
less syrups.
re in mimic
4
serve to increase the potency of the
immunostimulatory response induced when compared to the ter-
minal free hydroxyl in mimic 2. Interestingly, the terminal acidic
moiety of mimic 4 yields a higher potency than the phosphate moi-
ety of mimic 3. Competitive inhibition studies with lipid IVa, a
known human TLR4 antagonist, confirm TLR4 as the target of
diethanolamine-containing lipid A mimics. Further studies are
underway to investigate the application of this novel class of lipid
A mimics as immunostimulatory therapeutic agents.
For (5): Rf 0.34 (hexane/EtOAc/MeOH, 1:1:0.1); 1H NMR
(500 MHz, CDCl3): d 3.52–3.63 (br m, 6H, NCH2 ꢂ 2, OH ꢂ 2),
3.86–3.94 (m, 4H, OCH2 ꢂ 2), 4.78 (s, 2H, Troc-CH2); 13C NMR
(125 MHz, CDCl3): d 52.55 (NCH2), 53.26 (NCH2), 61.57 (OCH2),
62.04 (OCH2), 75.45 (Troc-CH2), 95.61 (Troc-CCl3), 155.15 (C@O);
ESI-MS (m/z) Calcd for C7H12Cl3NO4 [M+Na]+: 301.9, found: 302.0.
For (6): Rf 0.65 (hexane/EtOAc/MeOH, 1:1:0.1); 1H NMR
(500 MHz, CDCl3): d 1.82–1.95 (br s, 0.5H, OH from one isomer),
2.20–2.34 (br s, 0.5H, OH from one isomer), 3.58–3.61 (m, 2H,
HOCH2CH2N), 3.73–3.77 (m, 2H, NCH2CH2OTroc), 3.82–3.88 (br
m, 2H, HOCH2), 4.44–4.49 (m, 2H, CH2OTroc), 4.76–4.81 (m, 4H,
Troc-CH2 ꢂ 2); 13C NMR (125 MHz, CDCl3): d 47.45 (HOCH2CH2N
from one isomer), 48.24 (HOCH2CH2N from one isomer), 51.10
(NCH2CH2OTroc from one isomer), 51.86 (NCH2CH2OTroc from
one isomer), 61.22 (HOCH2 from one isomer), 61.27 (HOCH2 from
one isomer), 66.66 (CH2OTroc from one isomer), 66.91 (CH2OTroc
from one isomer), 75.20 (O-Troc-CH2 from one isomer), 75.34
(O-Troc-CH2 from one isomer), 76.86 (N-Troc-CH2), 94.24
(O-Troc-CCl3), 95.22 (N-Troc-CCl3), 153.82 (O-Troc-C@O), 154.52
(N-Troc-C@O from one isomer), 155.05 (N-Troc-C@O from one iso-
mer); ESI-MS (m/z) Calcd for C10H13Cl6NO6 [M+Na]+: 475.9, found:
475.9.
3. Experimental
3.1. General methods
All air and moisture sensitive reactions were performed under
nitrogen atmosphere. All commercial reagents were used as sup-
plied. Anhydrous dichloromethane was distilled over calcium hy-
dride, whereas anhydrous N,N-dimethylformamide (DMF) was
purchased from Aldrich. ACS grade solvents were purchased from
Fisher Scientific and used for chromatography without distillation.
TLC plates (silica gel 60 F254, thickness 0.25 mm) and silica gel 60
(40–63 lm) for flash column chromatography were purchased
from SILICYCLE INC., Canada. 1H and 13C NMR spectra were re-
corded on a Varian Unity Inova 500 MHz spectrometer. Tetrameth-
ylsilane (TMS, d 0.00 ppm) or solvent peaks were used as internal
standards for 1H and 13C NMR spectra. The chemical shifts were gi-
ven in ppm and coupling constants in Hz indicated to a resolution
of 0.5 Hz. Multiplicity of proton signals is indicated as follows: s
(singlet), d (doublet), dd (double doublet), t (triplet), q (quartet),
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

6
J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3.3. N-(2-Hydroxyethyl)-N-{2-(tert-
butyldiphenylsilyloxy)ethyl}-2,2,2-
trichloroethoxymethanamide (7) & N,N-Bis{2-(tert-
Butyldiphenylsilyloxy)ethyl}-2,2,2-
trichloroethoxymethanamide (8)
isomer), 4.28 (dd, 0.5H, J 12.0, 12.0 Hz, H-6a from one isomer),
4.53 (d, 0.5H, J 8.5 Hz, H-1 from one isomer), 4.55 (d, 0.5H, J
12.5 Hz, Troc), 4.60 (d, 0.5H, J 12.0 Hz, Troc), 4.65 (d, 0.5H, J
8.5 Hz, H-1 from one isomer), 4.68 (d, 0.5H, J 12.5 Hz, Troc), 4.72
(d, 0.5H, J 12.5 Hz, Troc), 4.76 (d, 1.0H, J 12.0 Hz, Troc), 4.89 (d,
1.0H, J 12.0 Hz, Troc), 4.94 (d, 0.5H, J 8.0 Hz, NH from one isomer),
5.05 (dd, 0.5H, J 9.5, 9.5 Hz, H-4 from one isomer), 5.08 (dd, 0.5H, J
9.5, 9.5 Hz, H-4 from one isomer), 5.22 (dd, 0.5H, J 9.5, 9.5 Hz, H-3
from one isomer), 5.24 (dd, 0.5H, J 9.5, 9.5 Hz, H-3 from one iso-
mer), 5.31 (d, 0.5H, J 8.0 Hz, NH from one isomer), 7.38–7.46 (m,
6H, Ar-H), 7.62–7.66 (m, 4H, Ar-H); 13C NMR (125 MHz, CDCl3): d
19.11 (C(CH3)3 from one isomer), 19.14 (C(CH3)3 from one isomer),
20.69 (C(CH3)3 from one isomer), 20.80 (C(CH3)3 from one isomer),
26.85 (COOCH3), 48.26 (NCH2), 48.33 (NCH2), 50.22 (NCH2), 51.30
(NCH2), 61.90 (OCH2), 61.94 (OCH2), 61.98 (C-6), 62.48 (OCH2),
66.89 (OCH2), 68.50 (C-4 from one isomer), 68.58 (C-4 from one
isomer), 71.79 (C-5 from one isomer), 71.84 (C-5 from one isomer),
71.89 (C-3 from one isomer), 72.39 (C-3 from one isomer), 74.32
(Troc-CH2), 74.35 (Troc-CH2), 75.01 (Troc-CH2), 75.10 (Troc-CH2),
95.38 (Troc-CCl3), 95.45 (Troc-CCl3), 95.51 (Troc-CCl3), 95.68
(Troc-CCl3), 99.61 (C-1 from one isomer), 100.99 (C-1 from one iso-
mer), 127.80 (CH-Ar), 127.83 (CH-Ar), 127.84 (CH-Ar), 129.83 (CH-
Ar), 129.88 (CH-Ar), 133.11 (C-Ar), 133.16 (C-Ar), 133.29 (C-Ar),
133.34 (C-Ar), 135.56 (CH-Ar), 135.57 (CH-Ar), 153.97 (C@O Troc),
153.99 (C@O Troc), 154.20 (C@O Troc), 154.67 (C@O Troc), 169.49
(C@O), 170.57 (C@O). 170.64 (C@O), 170.71 (C@O), 170.73 (C@O);
MALDI-MS (m/z) Calcd for C38H48Cl6N2O13Si [M+Na]+: 1001.09,
found: 1001.08.
To a cooled solution (ice water bath) of 5 (2.00 g, 7.13 mmol)
and Et3N (902 mg, 8.91 mmol) in CH2Cl2 (8 mL), tert-butyldiphen-
ylsilyl chloride (2.45 g, 8.91 mmol) was added. After stirring at
room temperature for 3 hours, the reaction was quenched with
MeOH and concentrated. Flash column chromatography purifica-
tion (hexane/acetone, 3.5: 1) yielded
7 (2.01 g, 54%) and 8
(321 mg, 6%), as well as recovered 5 (706 mg, 35%), all as colorless
syrups.
For (7): Rf 0.38 (hexane/acetone, 3:1); 1H NMR (500 MHz,
CDCl3): d 1.09 (s, 9H, C(CH3)3), 2.69 (br s, 0.4H, OH from one iso-
mer), 2.94 (br s, 0.6H, OH from one isomer), 3.56–3.64 (m, 4H,
NCH2 ꢂ 2), 3.83–3.90 (m, 4H, OCH2 ꢂ 2), 4.68 (s, 1.2H, Troc-CH2
from one isomer), 4.78 (s, 0.8H, Troc-CH2 from one isomer),
7.40–7.48 (m, 6H, Ar-H), 7.67–7.70 (m, 4H, Ar-H); 13C NMR
(125 MHz, CDCl3): d 19.10 (C(CH3)3 from one isomer), 19.13
(C(CH3)3
from
one
isomer),
26.82
(C(CH3)3),
50.77
(NCH2CH2OTBDPS from one isomer), 51.42 (NCH2CH2OTBDPS from
one isomer), 51.46 (HOCH2CH2N from one isomer), 52.44
(HOCH2CH2N from one isomer), 61.54 (CH2OTBDPS from one iso-
mer), 61.72 (CH2OTBDPS from one isomer), 62.18 (HOCH2 from
one isomer, 62.72 (HOCH2 from one isomer), 75.15 (Troc-CH2 from
one isomer), 75.19 (Troc-CH2 from one isomer), 95.30 (Troc-CCl3
from one isomer), 95.57 (Troc-CCl3 from one isomer), 127.84
(CH-Ar), 127.86 (CH-Ar), 129.93 (CH-Ar), 132.92 (C-Ar), 135.58
(CH-Ar), 154.40 (C@O from one isomer), 155.45 (C@O from one iso-
mer); ESI-MS (m/z) Calcd for C23H30Cl3NO4Si [MꢁC4H9]+: 460.0,
found: 460.4.
3.5. N-{2-(tert-Butyldiphenylsilyloxy)-ethyl}-N-{2-[2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyranosyloxy]-
ethyl}-2,2,2-trichloroethoxymethanamide (11)
For (8): Rf 0.67 (hexane/acetone, 3:1); 1H NMR (500 MHz,
CDCl3): d 1.05 (s, 18H, C(CH3)3 ꢂ 2), 3.61–3.69 (m, 4H, NCH2 ꢂ 2),
3.85–3.92 (m, 4H, OCH2 ꢂ 2), 4.75 (s, 2H, Troc-CH2), 7.43–7.52
(m, 12H, Ar-H), 7.72–7.79 (m, 8H, Ar-H); 13C NMR (125 MHz,
CDCl3): d 19.20 (C(CH3)3), 26.88 (C(CH3)3), 50.86 (NCH2), 51.32
(NCH2), 62.24 (OCH2), 62.65 (OCH2), 75.04 (Troc-CH2), 95.62
(Troc-CCl3), 127.78 (CH-Ar), 127.82 (CH-Ar), 127.85 (CH-Ar),
129.70 (CH-Ar), 129.82 (CH-Ar) 129.84 (CH-Ar), 133.35 (C-Ar),
133.44 (C-Ar), 133.59 (C-Ar), 135.59 (CH-Ar), 135.62 (CH-Ar),
154.27 (C@O); ESI-MS (m/z) Calcd for C39H48Cl3NO4Si2 [MꢁC4H9]+:
698.2, found: 698.5.
Guanidinium nitrate (1.87 g, 15.32 mmol) was dissolved in
MeOH:CH2Cl2 (9:1, 150 mL) and sodium methoxide in methanol
solution (0.5 M, 6 mL) was added. 10 (1.86 g, 1.89 mmol) was dis-
solved in the above solution and stirred at room temperature for
3 h. The mixture was then neutralized by adding weak acidic
ion-exchange resin (Amberlite IRC-64). The resin was filtered and
the filtrate concentrated. The residue was purified via flash column
chromatography (CH2Cl2/MeOH, 13: 1) to afford 11 (1.48 g, 91%) as
a white fluffy solid. Rf 0.29 (hexane/acetone, 3:2); ½a D22
ꢁ12.5 (c 1.0,
ꢃ
CHCl3); 1H NMR (500 MHz, CDCl3): d 1.04 (s, 9H, C(CH3)3), 3.25–
3.67 (m, 8H, H-2, H-4, H-6b, H-6a, NCH2 ꢂ 2), 3.69–3.85 (m, 5H,
H-3, H-5, ROCHH, CH2OTBDPS), 3.87–3.99 (m, 1H, ROCHH), 4.43
(d, 0.4H, J 8.0 Hz, H-1 from one isomer), 4.54 (d, 0.6H, J 8.0 Hz,
H-1 from one isomer), 4.61 (d, 0.6H, J 12.0 Hz, Troc), 4.63 (d,
0.6H, J 12.0 Hz, Troc), 4.69 (d, 1H, J 12.0 Hz, Troc), 4.71 (d, 0.4H, J
12.0 Hz, Troc), 4.74 (d, 0.4H, J 12.0 Hz, Troc), 4.78 (d, 1H, J
12.0 Hz, Troc), 4.99 (br s, 3H, OH ꢂ 3), 6.17 (d, 0.4H, J 8.0 Hz, NH
from one isomer), 6.31 (d, 0.6H, J 8.0 Hz, NH from one isomer),
7.36–7.43 (m, 6H, Ar-H), 7.61–7.64 (m, 4H, Ar-H); 13C NMR
(125 MHz, CDCl3): d 19.14 (C(CH3)3 from one isomer), 19.17
(C(CH3)3 from one isomer), 26.90 (C(CH3)3), 48.22 (NCH2), 48.42
(NCH2), 49.93 (NCH2), 51.43 (NCH2), 57.63 (C-2 from one isomer),
57.78 (C-2 from one isomer), 61.35 (OCH2), 61.92 (OCH2), 62.40 (C-
6), 66.23 (OCH2), 68.32 (OCH2), 70.07 (C-4 from one isomer), 70.20
(C-4 from one isomer), 73.95 (C-5 from one isomer), 74.61
(Troc-CH2), 74.72 (C-5 from one isomer), 75.09 (Troc-CH2), 75.17
(Troc-CH2), 75.50 (C-3 from one isomer), 75.63 (Troc-CH2), 95.31
(Troc-CCl3), 95.61 (Troc-CCl3), 95.64 (Troc-CCl3), 100.03 (C-1 from
one isomer), 101.50 (C-1 from one isomer), 127.81 (CH-Ar),
127.87 (CH-Ar), 129.82 (CH-Ar), 129.90 (CH-Ar), 133.09 (C-Ar),
133.27 (C-Ar), 135.57 (CH-Ar), 154.46 (C@O Troc), 154.88 (C@O
Troc), 155.17 (C@O Troc), 155.57 (C@O Troc); MALDI-MS (m/z)
Calcd for C32H42Cl6N2O10Si [M+Na]+: 875.06, found: 875.05.
3.4. N-{2-(tert-Butyldiphenylsilyloxy)ethyl}-N-{2-[3,4,6-tri-O-
acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-
glucopyranosyloxy]-ethyl}-2,2,2-trichloroethoxymethanamide
(10)
A solution of 7 (997 mg, 1.92 mmol) and imidate 9 (1.26 g,
2.02 mmol) in dry CH2Cl2 (8 mL) in the presence of molecular
sieves (4Å, 4.0 g) was stirred under nitrogen at room temperature
for 30 min. A solution of TMSOTf (0.05 M in dry CH2Cl2, 0.8 mL)
was added drop wise in about 3 min. The mixture was stirred at
room temperature for 1 h before a saturated sodium bicarbonate
solution (15 mL) was added to quench the reaction. Solids were fil-
tered out, and the filtrate was extracted with CH2Cl2 (3 ꢂ 30 mL).
The combined organic phase was dried over Na2SO4, concentrated,
and purified via flash column chromatography (hexane/acetone,
2.5: 1) to yield 10 (1.77 g, 94%) as a fluffy white solid. Rf 0.42 (hex-
ane/acetone, 5:2); ½a D22
ꢃ
+ 0.9 (c 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3): d 1.04 (s, 9H, C(CH3)3), 2.01–2.09 (m, 9H, Ac ꢂ 3), 3.38–
3.47 (m, 2H, NCH2), 3.51–3.72 (m, 4H, H-2, H-5, NCH2), 3.75–3.86
(m, 3H, ROCHH, CH2OTBDPS), 3.94–4.04 (m, 1H, ROCHH), 4.08–
4.13 (m, 1H, H-6b), 4.26 (dd, 0.5H, J 12.0, 12.0 Hz, H-6a from one
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
3.6. N-{2-(tert-Butyldiphenylsilyloxy)-ethyl}-N-{2-[4,6-O-
benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
b-D-glucopyranosyloxy]-ethyl}-2,2,2-
trichloroethoxymethanamide (12)
4.69 (d, 0.6H, J 8.0 Hz, H-1 from one isomer), 4.72 (d, 0.4H, J
12.0 Hz, Troc), 4.74 (d, 0.6H, J 12.0 Hz, Troc), 4.78 (d, 0.6H, J
12.0 Hz, Troc), 4.80 (d, 0.4H, J 12.0 Hz, Troc), 4.87 (d, 0.4H, J
12.0 Hz, Troc), 5.18–5.26 (m, 1H, H-3L), 5.32–5.38 (m, 1.4H, H-3,
NH from one isomer), 5.50–5.54 (m, 1.6H, Ph-CH, NH from one iso-
mer), 7.34–7.49 (m, 11H, Ar-H), 7.58–7.69 (m, 4H, Ar-H); 13C NMR
(125 MHz, CDCl3): d 14.18 (CH3), 19.13 (C(CH3)3 from one isomer),
19.17 (C(CH3)3 from one isomer), 22.73 (CH2), 25.01 (CH2), 25.09
(CH2), 26.86 (C(CH3)3 from one isomer), 26.88 (C(CH3)3 from one
isomer), 29.16 (CH2), 29.33 (CH2), 29.35 (CH2), 29.39 (CH2),
29.400 (CH2), 29.56 (CH2), 29.58 (CH2), 29.68 (CH2), 29.70 (CH2),
29.71 (CH2), 29.73 (CH2), 33.87 (CH2), 33.92 (CH2), 34.37 (CH2),
39.22 (C-2L from one isomer), 39.32 (C-2L from one isomer),
48.35 (NCH2), 48.69 (NCH2), 50.42 (NCH2), 51.28 (NCH2), 56.85
(C-2), 61.96 (OCH2), 62.51 (OCH2), 66.30 (C-5 from one isomer),
66.35 (C-5 from one isomer), 67.57 (OCH2), 68.55 (C-6), 68.65
(OCH2), 69.96 (C-3L from one isomer), 69.99 (C-3L from one iso-
mer), 70.99 (C-3 from one isomer), 71.27 (C-3 from one isomer),
74.41 (Troc-CH2), 74.44 (Troc-CH2), 75.04 (Troc-CH2), 75.09
(Troc-CH2), 78.74 (C-4 from one isomer), 78.80 (C-4 from one iso-
mer), 95.42 (Troc-CCl3), 95.51 (Troc-CCl3), 95.69 (Troc-CCl3),
100.95 (C-1 from one isomer), 101.47 (Ph-CH), 101.84 (C-1 from
one isomer), 126.16 (CH-Ar), 127.80 (CH-Ar), 127.84 (CH-Ar),
128.27 (CH-Ar), 129.18 (CH-Ar), 129.82 (CH-Ar), 129.86 (CH-Ar),
133.17 (C-Ar), 133.20 (C-Ar), 133.32 (C-Ar), 133.36 (C-Ar), 135.58
(CH-Ar), 136.82 (C-Ar), 154.01 (C@O Troc), 154.19 (C@O Troc),
154.36 (C@O Troc), 154.50 (C@O Troc), 170.01 (C@O), 170.06
(C@O), 173.41 (C@O), 173.50 H (C@O); MALDI-MS (m/z) Calcd for
To a solution of 11 (1.35 g, 1.58 mmol) in CH3CN (8 mL), benz-
aldehyde dimethyl acetal (308 mg, 2.02 mmol) and p-toluene sul-
fonic acid (15 mg, 0.08 mmol) were added successively. The
mixture was stirred at room temperature for 2 h before being
quenched with Et3N (0.5 mL), and concentrated. The residue was
purified via flash column chromatography (hexane/acetone, 3:1)
to yield 12 (1.29 g, 87%) as a white fluffy solid. Rf 0.46 (hexane/ace-
tone, 5:2); ½a 2D2
ꢃ
ꢁ17.9 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d
1.05 (s, 9H, C(CH3)3), 3.33–3.66 (m, 8H, H-2, H-4, H-5, OH,
NCH2 ꢂ 2), 3.68–3.99 (m, 6H, H-3, H-6b, OCH2 ꢂ 2), 4.27 (dd,
0.4H, J 10.0, 10.0 Hz, H-6a from one isomer), 4.29 (dd, 0.6H, J
10.0, 10.0 Hz, H-6a from one isomer), 4.45 (d, 0.4H, J 8.0 Hz, H-1
from one isomer), 4.53 (d, 0.6H, J 8.0 Hz, H-1 from one isomer),
4.61 (d, 1.4H, J 12.0 Hz, Troc), 4.69 (0.6H, J 12.0 Hz, Troc), 4.72 (d,
0.6H, J 12.0 Hz, Troc), 4.74 (d, 1H, J 12.0 Hz, Troc), 4.81 (d, 0.4H, J
12.0 Hz, Troc), 5.23 (d, 0.4H, J 8.0 Hz, NH from one isomer), 5.50
(s, 0.4H, Ph-CH from one isomer), 5.52 (s, 0.6H, Ph-CH from one
isomer), 5.74 (d, 0.6H, J 8.0 Hz, NH from one isomer), 7.33–7.44
(m, 9H, Ar-H), 7.47–7.51 (m, 2H, Ar-H), 7.61–7.65 (m, 4H, Ar-H);
13C NMR (125 MHz, CDCl3): d 19.15 (C(CH3)3 from one isomer),
19.18 (C(CH3)3 from one isomer), 26.89 (C(CH3)3), 48.38 (NCH2),
48.45 (NCH2), 50.19 (NCH2), 51.35 (NCH2), 58.45 (C-2 from one iso-
mer), 58.62 (C-2 from one isomer), 61.97 (OCH2), 62.51 (OCH2),
66.03 (C-5 from one isomer), 66.17 (C-5 from one isomer), 66.83
(OCH2), 68.55 (C-6), 70.74 (C-3 from one isomer), 71.81 (C-3 from
one isomer), 74.66 (Troc-CH2), 75.06 (Troc-CH2), 75.15 (Troc-CH2),
81.40 (C-4), 95.38 (Troc-CCl3), 95.48 (Troc-CCl3), 95.70 (Troc-CCl3),
99.86 (C-1 from one isomer), 101.35 (C-1 from one isomer), 101.86
(Ph-CH from one isomer), 101.89 (Ph-CH from one isomer), 126.43
(CH-Ar), 126.45 (CH-Ar), 127.83 (CH-Ar), 127.87 (CH-Ar), 128.45
(CH-Ar), 128.49 (CH-Ar), 129.42 (CH-Ar), 129.46 (CH-Ar), 129.85
(CH-Ar), 129.91 (CH-Ar), 133.12 (C-Ar), 133.14 (C-Ar), 133.31 (C-
Ar), 133.33 (C-Ar), 135.58 (CH-Ar), 137.03 (C-Ar), 154.05 (C@O
Troc), 154.54 (C@O Troc), 154.85 (C@O Troc), 155.37 (C@O Troc);
C
67H98Cl6N2O13Si [M+Na]+: 1399.49, found: 1399.50.
3.8. N-{2-(tert-Butyldiphenylsilyloxy)-ethyl}-N-{2-[6-O-benzyl-
2-deoxy-3-O-((R)-3-tetradecanoyloxytetradecanoyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranosyloxy]-ethyl}-
2,2,2-trichloroethoxymethanamide (15)
A solution of 13 (1.51 g, 1.09 mmol) in dry THF (10 mL) and
molecular sieves (4 Å, 4.0 g) was stirred at room temperature un-
der nitrogen for 30 min. Sodium cyanoborohydride (550 mg,
8.75 mmol) was added and the mixture cooled to 0 °C, followed
by the drop wise addition of a dry, saturated ethereal-HCl(g) solu-
tion until no further gas was evolved. The mixture was poured into
a saturated sodium bicarbonate solution (100 mL) and solids were
filtered out before removal of the THF in vacuo. The resulting solu-
tion was extracted with EtOAc (3 ꢂ 100 mL), with the combined
organic phase dried over Na2SO4 and concentrated. Flash column
chromatography of the residue (hexane/acetone, 4:1) afforded 15
(1.31 g, 87%) as a colorless syrup. Rf 0.31 (hexane/acetone, 4:1);
MALDI-MS (m/z) Calcd for
906.06, found: 906.12.
C
39H46Cl6N2O10Si [M+NaꢁC4H9]+:
3.7. N-{2-(tert-Butyldiphenylsilyloxy)-ethyl}-N-{2-[4,6-O-
benzylidene-2-deoxy-3-O-((R)-3-
tetradecanoyloxytetradecanoyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranosyloxy]-ethyl}-
2,2,2-trichloroethoxymethanamide (13)
½
a 2D2
ꢃ
ꢁ6.6 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.91 (t, 6H,
A mixture of 12 (1.16 g, 1.23 mmol), dilipid acid 14 (590 mg,
1.30 mmol), N,N-dimethylaminopyridine (15 mg, 0.12 mmol) and
N,N0-diisopropylcarbodiimide (235 mg, 1.85 mmol) in CH2Cl2
(7 mL) was stirred at room temperature for 4 h. Water (0.5 mL)
was added and the mixture stirred for a further 1 h. The solids were
then filtered through a scintered glass funnel with a bed of Na2SO4.
The filtrate was concentrated and the residue purified by flash col-
umn chromatography (hexane/acetone, 4.5:1) to afford 13 (1.58 g,
J 6.5 Hz, CH3 ꢂ 2), 1.07 (s, 9H, C(CH3)3), 1.24–1.38 (br m, 38H,
0
CH2 ꢂ 19), 1.54–1.68 (br m, 4H, H-4L, H-3L ), 2.31 (t, 2H, J 7.5 Hz,
0
H-2L ), 2.48–2.61 (m, 2H, H-2L), 3.46–3.72 (m, 8H, H-2, H-4, H-5,
OH, NCH2 ꢂ 2), 3.75–3.86 (m, 5H, H-6b, H-6a, CH2OTBDPS,
ROCHH), 3.94–4.05 (m, 1H, ROCHH), 4.42 (d, 0.4H, J 8.5 Hz, H-1
from one isomer), 4.48 (d, 0.6H, J 12.0 Hz, Troc), 4.52–4.65 (m,
2.6H, H-1 from one isomer, Ph-CH2), 4.68 (d, 1H, J 12.0 Hz, Troc),
4.72 (d, 0.6H, J 12.0 Hz, Troc), 4.76 (d, 0.4H, J 12.0 Hz, Troc), 4.80
(d, 0.4H, J 12.0 Hz, Troc), 4.89 (d, 1H, J 12.0 Hz, Troc), 5.00 (dd,
0.4H, J 10.0, 10.0 Hz, H-3 from one isomer). 5.02 (dd, 0.6H, J 10.0,
10.0 Hz, H-3 from one isomer), 5.13–5.20 (m, 1H, H-3L), 5.26 (d,
0.4H, J 8.0 Hz, NH from one isomer), 5.49 (d, 0.6H, J 8.0 Hz, NH from
one isomer), 7.30–7.47 (m, 11H, Ar-H), 7.63–7.68 (m, 4H, Ar-H);
13C NMR (125 MHz, CDCl3): d 14.14 (CH3), 19.11 (C(CH3)3 from
one isomer), 19.15 (C(CH3)3 from one isomer), 22.70 (CH2), 24.98
(CH2), 25.14 (CH2), 26.86 (C(CH3)3), 29.15 (CH2), 29.30 (CH2),
29.36 (CH2), 29.38 (CH2), 29.51 (CH2), 29.53 (CH2), 29.55
(CH2), 29.65 (CH2), 29.66 (CH2), 29.68 (CH2), 29.72 (CH2), 31.93
93%) as a colorless syrup. Rf 0.38 (hexane/acetone, 4:1); ½a D22
ꢁ14.1
ꢃ
(c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.91 (t, 6H, J 6.5 Hz,
CH3 ꢂ 2), 1.08 (s, 9H, C(CH3)3), 1.14–1.36 (br m, 38H, CH2 ꢂ 19),
0
0
1.53–162 (br m, 4H, H-4L, H-3L ), 2.16–2.23 (m, 2H, H-2L ), 2.53–
2.66 (m, 2H, H-2L), 3.46–3.68 (m, 7H, H-2, H-4, H-5, NCH2 ꢂ 2),
3.71–3.87 (m, 4H, H-6b, CH2OTBDPS, ROCHH), 3.94–4.04 (m, 1H,
ROCHH), 4.33 (dd, 0.4H, J 10.0, 10.0 Hz, H-6a from one isomer),
4.34 (dd, 0.6H, J 10.0, 10.0 Hz, H-6a from one isomer), 4.55 (d,
0.4H, J 12.0 Hz, Troc), 4.59 (d, 0.6H, J 12.0 Hz, Troc), 4.61 (d, 0.4H,
J 8.0 Hz, H-1 from one isomer), 4.63 (d, 0.6H, J 12.0 Hz, Troc),
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

8
J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
(CH2), 31.94 (CH2), 34.51 (CH2), 34.64 (CH2), 40.09 (C-2L), 48.29
(NCH2), 48.36 (NCH2), 50.22 (NCH2), 51.20 (NCH2), 55.70 (C-2),
61.89 (OCH2), 62.48 (OCH2), 66.86 (OCH2), 68.29 (OCH2), 66.71
(C-6 from one isomer), 66.73 (C-6 from one isomer), 70.10 (C-4),
71.00 (C-3L from one isomer), 71.03 (C-3L from one isomer),
73.69 (Ph-CH2), 74.36 (Troc-CH2), 74.38 (Troc-CH2), 74.51 (C-5
from one isomer), 74.63 (C-5 from one isomer), 75.03 (Troc-CH2),
75.11 (Troc-CH2), 75.65 (C-3 from one isomer), 76.12 (C-3 from
one isomer), 95.43 (Troc-CCl3), 95.58 (Troc-CCl3), 95.61 (Troc-
CCl3), 95.71 (Troc-CCl3), 100.17 (C-1 from one isomer), 101.44 (C-
1 from one isomer), 127.67 (CH-Ar), 127.69 (CH-Ar), 127.77 (CH-
Ar), 127.81 (CH-Ar), 128.46 (CH-Ar), 129.78 (CH-Ar), 129.82 (CH-
Ar), 133.20 (C-Ar), 133.36 (C-Ar), 135.57 (CH-Ar), 137.80 (C-Ar),
137.85 (C-Ar), 154.05 (C@O Troc), 154.20 (C@O Troc), 154.38
(C@O Troc), 154.58 (C@O Troc), 171.48 (C@O), 174.37 (C@O); MAL-
DI-MS (m/z) Calcd for C67H100Cl6N2O13Si [M+Na]+: 1401.50, found:
1401.45.
CCl3), 99.77 (C-1 from one isomer), 100.58 (C-1 from one isomer),
127.65 (CH-Ar), 127.80 (CH-Ar), 127.84 (CH-Ar), 128.04 (CH-Ar),
128.11 (CH-Ar), 128.14 (CH-Ar), 128.38 (CH-Ar), 128.63 (CH-Ar),
128.67 (CH-Ar), 129.81 (CH-Ar), 129.84 (CH-Ar), 133.19 (C-Ar),
133.21 (C-Ar), 133.34 (C-Ar), 133.37 (C-Ar), 135.59 (CH-Ar),
138.00 (C-Ar), 138.02 (C-Ar), 154.02 (C@O Troc), 154.22 (C@O
Troc), 154.49 (C@O Troc), 170.28 (C@O), 170.32 (C@O), 173.61
(C@O),
C
173.75
(C@O);
MALDI-MS
(m/z)
Calcd
for
81H113Cl6N2O16PSi [M+Na]+: 1661.56, found: 1661.48.
3.10. N-{2-(tert-Butyldiphenylsilyloxy)-ethyl}-N-{2-[6-O-
benzyl-2-deoxy-4-O-(di-O-benzylphosphono)-3-O-((R)-3-
tetradecanoyloxytetradecanoyl)-2-((R)-3-
tetradecanoyloxytetradecanamido)-b-D-glucopyranosyloxy]-
ethyl}-(R)-3-tetradecanoyloxytetradecanamide (17)
To a solution of 16 (440 mg, 0.27 mmol) in glacial acetic acid
(5 mL), zinc powder (analytical grade, <10 , 750 mg) was added
l
3.9. N-{2-(tert-Butyldiphenylsilyloxy)-ethyl}-N-{2-[6-O-benzyl-
2-deoxy-4-O-(di-O-benzylphosphono)-3-O-((R)-3-
tetradecanoyloxytetradecanoyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranosyloxy]-ethyl}-
2,2,2-trichloroethoxymethanamide (16)
and the mixture was stirred at room temperature for 45 min. The
mixture was then filtered, and the solids were washed with acetic
acid (10 mL). The filtrate was slowly poured into a saturated so-
dium bicarbonate solution (300 mL) and then extracted with
CH2Cl2 (3 ꢂ 150 mL). The combined organic phase was washed
with further saturated sodium bicarbonate solution (100 mL),
dried over Na2SO4, and concentrated to give the crude di-amine
(338 mg, 98%) as a colorless syrup.
To a solution of 15 (1.25 g, 0.90 mmol) in dry CH2Cl2 (8 mL), 5-
phenyltetrazole (265 mg, 1.80 mmol) and N,N-diisopropylphosph-
oramidite (0.62 mL, 1.80 mmol) were added. The mixture was stir-
red at room temperature for 1 h and then cooled to 0 °C before the
addition of m-chloroperbenzoic acid (505 mg, 77% purity,
2.25 mmol). The mixture was stirred at 0 °C for 1 h and then
poured into a saturated sodium bicarbonate solution (50 mL) and
extracted with CH2Cl2 (3 ꢂ 50 mL). The combined organic phase
was dried over Na2SO4, concentrated, and purified by flash column
chromatography (hexane/acetone, 4:1) to give 16 (1.26 g, 85%) as a
A solution of the crude di-amine (337 mg) in DMF (2 mL) was
added to a mixture of dilipid acid 14 (283 mg, 0.62 mmol), HBTU
(352 mg, 0.94 mmol) and DIPEA (0.16 mL, 0.94 mmol) in DMF
(5 mL). The resulting mixture was stirred at room temperature
for 18 h before being poured into a separatory funnel with water
(50 mL), and then extracted with Et2O (3 ꢂ 75 mL). The combined
organic phase was washed with a cold saturated sodium chloride
solution (3 ꢂ 8 mL), dried over Na2SO4, and concentrated. Flash
column chromatography of the residue (hexane/EtOAc, 2.5:1)
afforded 17 (381 mg, 67%) as a colorless syrup. Rf 0.35 (hexane/ace-
colorless syrup. Rf 0.40 (hexane/EtOAc, 5:2); ½a D22
ꢃ
ꢁ2.9 (c 1.0,
CHCl3); 1H NMR (500 MHz, CDCl3):
d 0.88 (t, 6H, J 6.5 Hz,
CH3 ꢂ 2), 1.04 (s, 9H, C(CH3)3), 1.17–1.32 (br m, 38H, CH2 ꢂ 19),
tone, 4:1); ½a 2D2
ꢃ
ꢁ1.5 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d
0
1.43–1.56 (br m, 4H, H-4L, H-3L ), 2.18–2.25 (t, 2H, J 7.5 Hz, H-
0.88 (t, 18H, J 6.5 Hz, CH3 ꢂ 6), 1.03 (s, 9H, C(CH3)3), 1.16–1.38
(br m, 114H, CH2 ꢂ 57), 1.52–1.68 (br m, 12H, H-4L ꢂ 6, H-
0
2L ), 2.38–2.47 (m, 2H, H-2L), 3.39–3.63 (m, 7H, H-2, H-5, H-6b,
0
0
NCH2 ꢂ 2), 3.68–3.82 (m, 4H, H-6a, CH2OTBDPS, ROCHH), 3.94–
4.02 (m, 1H, ROCHH), 4.40–4.54 (m, 4H, H-4, Ph-CH2, Troc), 4.57
(d, 0.4H, J 12.0 Hz, Troc), 4.59 (d, 0.6H, J 12.0 Hz, Troc), 4.64 (d,
0.6H, J 12.0 Hz, Troc), 4.68 (d, 0.4H, J 12.0 Hz, Troc), 4.71 (d, 0.4H,
J 8.0 Hz, H-1 from one isomer), 4.73 (d, 0.4H, J 12.0 Hz, Troc) 4.75
(d, 0.6H, J 8.0 Hz, H-1 from one isomer), 4.80 (d, 0.6H, J 12.0 Hz,
Troc), 4.86–4.93 (m, 4H, (PhCH2O)2P), 5.14–5.23 (m, 1H, H-3L),
5.34–5.40 (m, 1H, H-3), 5.43 (d, 0.4H, J 8.0 Hz, NH from one iso-
mer), 5.55 (d, 0.6H, J 8.0 Hz, NH from one isomer), 7.22–7.32 (m,
13H, Ar-H), 7.35–7.44 (m, 8H, Ar-H), 7.60–7.66 (m, 4H, Ar-H); 13C
NMR (125 MHz, CDCl3): d 14.20 (CH3), 19.13 (C(CH3)3 from one iso-
mer), 19.16 (C(CH3)3 from one isomer), 22.75 (CH2), 25.05 (CH2),
25.16 (CH2), 26.88 (C(CH3)3 from one isomer), 26.90 (C(CH3)3 from
one isomer), 29.21 (CH2), 29.37 (CH2), 29.41 (CH2), 29.58 (CH2),
29.59 (CH2), 29.62 (CH2), 29.68 (CH2), 29.70 (CH2), 29.72 (CH2),
29.74 (CH2), 31.97 (CH2), 34.34 (CH2), 34.49 (CH2), 39.37 (C-2L from
one isomer), 39.61 (C-2L from one isomer), 48.27 (NCH2), 48.62
(NCH2), 50.30 (NCH2), 51.17 (NCH2), 56.51 (C-2 from one isomer),
56.54 (C-2 from one isomer), 61.92 (OCH2ROCH2CH2NCH2-
CH2OTBDPS from one isomer), 62.49 (OCH2), 67.44 (OCH2), 68.46
(C-6), 68.58 (OCH2), 69.61–69.73 (m, (PhCH2O)2P), 69.99 (C-3L
from one isomer), 70.12 (C-3L from one isomer), 72.27 (C-3 from
one isomer), 72.62 (C-3 from one isomer), 73.46 (Ph-CH2), 74.00
(d, J 5.5 Hz, C-4 from one isomer), 74.05 (d, J 5.5 Hz, C-4 from
one isomer), 74.14 (C-5 from one isomer), 74.19 (C-5 from one iso-
mer), 74.44 (Troc-CH2), 75.01 (Troc-CH2), 75.06 (Troc-CH2), 95.46
(Troc-CCl3), 95.48 (Troc-CCl3), 95.50 (Troc-CCl3), 95.69 (Troc-
3L ꢂ 6), 2.17–2.51 (m, 11H, H-2L ꢂ 5, H-2L ꢂ 6), 2.75 (dd, 0.4H, J
15.5, 6.5 Hz, H-2LB from one isomer), 2.87 (dd, 0.6H, J 15.5,
5.0 Hz, H-2LA from one isomer), 3.07–3.18 (m, 0.4H, H-2 from
one isomer), 3.41–3.79 (m, 10.6H, H-2 from one isomer, H-5, H-
6b, H-6a, NCH2 ꢂ 2, CH2OTBDPS, ROCHH), 3.82–3.91 (m, 1H,
ROCHH), 4.36–4.51 (m, 3H, H-4, Ph-CH2), 4.66 (d, 0.4H, J 8.0 Hz,
H-1 from one isomer), 4.84–4.92 (m, 4H, (PhCH2O)2P), 5.04–5.22
(m, 3.6H, H-1 from one isomer, H-3L ꢂ 3), 5.29 (dd, 0.6H, J 10.0,
10.0 Hz, H-3 from one isomer), 5.73 (dd, 0.4H, J 10.0, 10.0 Hz, H-
3 from one isomer), 6.15 (d, 0.4H, J 8.0 Hz, NH from one isomer),
6.73 (d, 0.6H, J 8.0 Hz, NH from one isomer), 7.22–7.32 (m, 13H,
Ar-H), 7.34–7.44 (m, 8H, Ar-H), 7.58–7.64 (m, 4H, Ar-H); 13C
NMR (125 MHz, CDCl3): d 14.15 (CH3), 19.08 (C(CH3)3 from one iso-
mer), 19.15 (C(CH3)3 from one isomer), 22.73 (CH2), 25.06 (CH2),
25.09 (CH2), 25.15 (CH2), 25.18 (CH2), 25.25 (CH2), 25.33 (CH2),
25.42 (CH2), 25.83 (CH2), 26.85 (C(CH3)3 from one isomer), 26.90
(C(CH3)3 from one isomer), 29.17 (CH2), 29.23 (CH2), 29.27 (CH2),
29.31 (CH2), 29.35 (CH2), 29.41 (CH2), 29.43 (CH2), 29.49 (CH2),
29.54 (CH2), 29.56 (CH2), 29.61 (CH2), 29.64 (CH2), 29.68 (CH2),
29.73 (CH2), 29.76 (CH2), 31.97 (CH2), 34.04 (CH2), 34.12 (CH2),
34.33 (CH2), 34.43 (CH2), 34.49 (CH2), 34.60 (CH2), 34.700(CH2),
38.04 (C-2L), 38.13 (C-2L), 39.11 (C-2L), 39.26 (C-2L), 40.74 (C-2L),
41.25 (C-2L), 45.65 (NCH2), 47.09 (NCH2), 48.29 (NCH2), 50.45
(NCH2), 54.92 (C-2 from one isomer), 56.65 (C-2 from one isomer),
61.61 (OCH2), 62.64 (OCH2), 63.42 (C-6 from one isomer), 67.22 (C-
6 from one isomer), 68.21 (OCH2), 68.43 (OCH2), 69.46–69.67 (m,
(PhCH2O)2P), 69.99 (C-3L), 70.07 (C-3L), 70.15 (C-3L), 70.68 (C-3L),
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
71.22 (C-3L), 71.70 (C-3 from one isomer), 72.35 (C-3L), 72.87 (C-3
from one isomer), 73.30 (Ph-CH2 from one isomer), 73.32 (Ph-CH2
from one isomer), 73.73 (C-5 from one isomer), 73.98 (C-5 from
one isomer), 74.09 (d, J 5.5 Hz, C-4 from one isomer), 74.49 (d, J
5.5 Hz, C-4 from one isomer), 99.43 (C-1 from one isomer),
100.77 (C-1 from one isomer), 127.56 (CH-Ar), 127.60 (CH-Ar),
127.74 (CH-Ar), 127.77 (CH-Ar), 127.87 (CH-Ar), 127.90 (CH-Ar),
127.99 (CH-Ar), 128.10 (CH-Ar), 128.12 (CH-Ar), 128.31 (CH-Ar),
128.53 (CH-Ar), 128.56 (CH-Ar), 129.74 (CH-Ar), 129.77
(CH-Ar), 129.88 (CH-Ar), 129.90 (CH-Ar), 132.94 (C-Ar), 133.36
(C-Ar), 133.45 (C-Ar), 135.51 (CH-Ar), 135.54 (CH-Ar), 138.08 (C-
Ar), 169.70 (C@O), 170.03 (C@O), 170.24 (C@O), 170.41 (C@O),
170.50 (C@O), 170.74 (C@O), 173.14 (C@O), 173.29 (C@O),
173.40 (C@O), 173.45 (C@O), 173.52 (C@O), 174.18 (C@O); MAL-
DI-MS (m/z) Calcd for C131H215N2O18PSi [M+Na]+: 2186.54, found:
2186.48.
7.42 (m, 25H, Ar-H); 13C NMR (125 MHz, CDCl3): d 14.15 (CH3),
22.71 (CH2), 25.05 (CH2), 25.08 (CH2), 25.15 (CH2), 25.17 (CH2),
25.24 (CH2), 25.35 (CH2), 25.43 (CH2), 25.81 (CH2), 24.84 (CH2),
25.88 (CH2), 29.15 (CH2), 29.21 (CH2), 29.25 (CH2), 29.301
(CH2), 29.33 (CH2), 29.40 (CH2), 29.43 (CH2), 29.47 (CH2), 29.52
(CH2), 29.56 (CH2), 29.62 (CH2), 29.64 (CH2), 29.68 (CH2), 29.73
(CH2), 29.77 (CH2), 31.96 (CH2), 34.02 (CH2), 34.12 (CH2), 34.35
(CH2), 34.43 (CH2), 34.49 (CH2), 34.61 (CH2), 34.70 (CH2), 37.89
(C-2L), 38.15 (C-2L), 39.04 (C-2L), 39.16 (C-2L), 40.80 (C-2L), 41.20
(C-2L), 46.15 (NCH2), 46.22 (NCH2) 46.52 (NCH2), 48.63 (NCH2),
55.12 (C-2 from one isomer), 56.16 (C-2 from one isomer), 64.59
(d, J 5.5 Hz, CH2OP(O)(OBn)2 from one isomer), 65.87 (d, J 5.5 Hz,
CH2OP(O)(OBn)2 from one isomer), 67.34 (C-6), 68.48 (ROCH2),
68.52 (ROCH2), 69.41–69.66 (m, PhCH2O)2P, CH2OP(O)(OCH2Ph)2),
69.96 (C-3L), 70.04 (C-3L), 70.16 (C-3L), 70.68 (C-3L), 71.26 (C-3L),
71.83 (C-3L), 71.93 (C-3 from one isomer), 72.71 (C-3 from one iso-
mer), 73.32 (Ph-CH2), 73.81 (C-5 from one isomer), 73.83 (C-5 from
one isomer), 74.19 (d, J 5.5 Hz, C-4 from one isomer), 74.37 (d, J
5.5 Hz, C-4 from one isomer), 99.58 (C-1 from one isomer),
100.07 (C-1 from one isomer), 127.55 (CH-Ar), 127.58 (CH-Ar),
127.72 (CH-Ar), 127.75 (CH-Ar), 127.85 (CH-Ar), 127.90 (CH-Ar),
127.97 (CH-Ar), 128.11 (CH-Ar), 128.12 (CH-Ar), 128.31 (CH-Ar),
128.52 (CH-Ar), 128.54 (CH-Ar), 129.75 (CH-Ar), 129.78 (CH-Ar),
129.87 (CH-Ar), 129.90 (CH-Ar), 132.94 (C-Ar), 133.36 (C-Ar),
133.47 (C-Ar), 135.52 (CH-Ar), 135.54 (CH-Ar), 138.08 (C-Ar),
169.72 (C@O), 170.05 (C@O), 170.27 (C@O), 170.44 (C@O),
170.51 (C@O), 170.79 (C@O), 173.18 (C@O), 173.35 (C@O),
173.45 (C@O), 173.48 (C@O), 173.59 (C@O), 174.18 (C@O). Decom-
position issues prevented the acquiring of optical rotation and
mass spectral data.
3.11. N-(2-Hydroxyethyl)-N-{2-[6-O-benzyl-2-deoxy-4-O-(di-O-
benzylphosphono)-3-O-((R)-3-tetradecanoyloxytetradecanoyl)-
2-((R)-3-tetradecanoyloxytetradecanamido)-b-
glucopyranosyloxy]-ethyl}-(R)-3-
D-
tetradecanoyloxytetradecanamide (18)
To a solution of 17 (890 mg, 0.41 mmol) in CH2Cl2 (3 mL) and
glacial acetic acid (0.1 mL), a tetrabutylammonium fluoride solu-
tion (1 M in THF, 0.62 mL) was added, and the mixture was stirred
at room temperature for 18 h. The mixture was the poured into a
saturated sodium bicarbonate solution (30 mL), extracted with
CH2Cl2 (3 ꢂ 50 mL), dried over Na2SO4, and concentrated. Flash col-
umn chromatography of the residue (hexane/EtOAc/MeOH,
3:1:0.1) yielded 18 (715 mg, 90%) as a colorless syrup. Spectro-
scopic data are identical with those reported previously.17
3.13. N-{Carboxymethyl}-N-{2-[6-O-benzyl-2-deoxy-4-O-(di-O-
benzylphosphono)-3-O-((R)-3-tetradecanoyloxytetradecanoyl)-
3.12. N-{2-(di-O-Benzylphosphono)-ethyl}-N-{2-[6-O-benzyl-2-
deoxy-4-O-(di-O-benzylphosphono)-3-O-((R)-3-
tetradecanoyloxytetradecanoyl)-2-((R)-3-
2-((R)-3-tetradecanoyloxytetradecanamido)-b-
glucopyranosyloxy]-ethyl}-(R)-3-
tetradecanoyloxytetradecanamide (20)
D-
tetradecanoyloxytetradecanamido)-b-D-glucopyranosyloxy]-
ethyl}-(R)-3-tetradecanoyloxytetradecanamide (19)
To a solution of 18 (80 mg, 0.042 mmol) in CH2Cl2 (3 mL) and
water (0.5 mL), (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO,
3 mg, 0.017 mmol) and bis(acetoxy)iodobenzene (54 mg,
0.168 mmol) were added. The mixture was stirred at room temper-
ature for 2 h before being poured into a 10% sodium thiosulphate
solution (10 mL), and extracted with CH2Cl2 (3 ꢂ 15 mL). The com-
bined organic phases were dried over Na2SO4, concentrated, and
purified by repeated flash chromatography (hexane/EtOAc/MeOH,
3:1:0.2) to afford 20 (67 mg, 83%) as a colorless syrup. Rf 0.35 (hex-
To a solution of 18 (85 mg, 0.044 mmol) in dry CH2Cl2 (1.5 mL),
5-phenyltetrazole
(13 mg,
0.088 mmol)
and
N,N-dii-
sopropylphosphoramidite (30
l
L, 0.088 mmol) were added. The
mixture was stirred at room temperature for 30 min and then
cooled to 0 °C before the addition of m-chloroperbenzoic acid
(22 mg, 77% purity, 0.099 mmol). The mixture was stirred at 0 °C
for 30 min before being poured into a 10% sodium thiosulphate
solution (10 mL) and then extracted with CH2Cl2 (3 ꢂ 20 mL). The
combined organic phase was washed with a saturated sodium
bicarbonate solution (15 mL), dried over Na2SO4, concentrated,
and purified by repeated flash chromatography (hexane/EtOAc,
3:2 ? 1:1) to give 19 (69 mg, 71%) as a colorless syrup. Rf 0.28
(hexane/EtOAc, 3:1; 1H NMR (500 MHz, CDCl3): d 0.86 (t, 18H, J
6.5 Hz, CH3 ꢂ 6), 1.14–1.39 (br m, 114H, CH2 ꢂ 57), 1.50–1.69 (br
ane/EtOAc, 3:1); ½a D22
ꢃ
ꢁ0.9 (c 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3): d 0.88 (t, 18H, J 6.5 Hz, CH3 ꢂ 6), 1.16–1.39 (br m, 114H,
0
CH2 ꢂ 57), 1.48–1.64 (br m, 12H, H-4L ꢂ 6, H-3L ꢂ 6), 2.19–2.52
0
(m, 11H, H-2L ꢂ 5, H-2L ꢂ 6), 2.63 (dd, 0.4H, J 15.5, 6.5 Hz, H-2LB
from one isomer), 2.86 (dd, 0.6H, J 15.5, 5.0 Hz, H-2LA from one iso-
mer), 3.27–3.34 (m, 0.6H, H-2 from one isomer), 3.44–3.68 (m, 5H,
H-5, H-6b, ROCH2CH2N, ROCHH), 3.70–3.78 (m, 1.4H, H-2 from one
isomer, H-6a), 3.87–3.98 (m, 1H, ROCHH), 4.00–4.21 (m, 2H,
NCH2COOH), 4.40–4.53 (m, 3H, H-4, Ph-CH2), 4.59 (d, 0.4H, J
8.0 Hz, H-1 from one isomer), 4.83–4.92 (m, 4H, (PhCH2O)2P),
4.98 (d, 0.6H, J 8.0 Hz, H-1 from one isomer), 5.09–5.24 (m, 3H,
H-3L ꢂ 3), 5.28 (dd, 0.4H, J 10.0, 10.0 Hz, H-3 from one isomer),
5.61 (dd, 0.6H, J 10.0, 10.0 Hz, H-3 from one isomer), 6.32 (d,
0.4H, J 8.0 Hz, NH from one isomer), 6.67 (d, 0.6H, J 8.0 Hz, NH from
one isomer), 7.21–7.34 (m, 15H, Ar-H); 13C NMR (125 MHz, CDCl3):
d 14.12 (CH3), 22.71 (CH2), 25.05 (CH2), 25.14 (CH2), 25.22 (CH2),
25.32 (CH2), 25.38 (CH2), 25.58 (CH2), 29.21 (CH2), 29.24 (CH2),
29.28 (CH2), 29.32 (CH2), 29.39 (CH2), 29.40 (CH2), 29.42
(CH2), 29.45 (CH2), 29.47 (CH2), 29.49 (CH2), 29.52 (CH2), 29.57
(CH2), 29.60 (CH2), 29.62 (CH2), 29.66 (CH2), 29.68 (CH2), 29.71
0
0
m, 12H, H-4L ꢂ 6, H-3L ꢂ 6), 2.18–2.57 (m, 11H, H-2L ꢂ 5, H-2L
ꢂ
ꢂ 6), 2.68 (dd, 0.4H, J 15.5, 6.5 Hz, H-2LB from one isomer), 2.78
(dd, 0.6H, J 15.5, 5.0 Hz, H-2LA from one isomer), 3.20–3.37 (m,
2.6H, H-2 from one isomer, NCH2), 3.43–3.48 (m, 1H, H-6b),
3.51–3.70 (m, 4.4H, H-2 from one isomer, H-5, NCH2, ROCHH),
3.75–3.87 (m, 2H, H-6a, ROCHH), 4.06–4.16 (m, 2H, CH2O-
P(O)(OBn)2), 4.40–4.52 (m, 3H, H-4, Ph-CH2), 4.72 (d, 0.4H, J
8.0 Hz, H-1 from one isomer), 4.89–4.94 (m, 4H, (PhCH2O)2P),
4.97–5.04 (m, 4H, CH2OP(O)(OCH2Ph)2), 5.07 (d, 0.6H, J 8.0 Hz, H-
1 from one isomer), 5.09–5.23 (m, 3H, H-3L ꢂ 3), 5.39 (dd, 0.4H, J
10.0, 10.0 Hz, H-3 from one isomer), 5.67 (dd, 0.6H, J 10.0,
10.0 Hz, H-3 from one isomer), 6.59 (d, 0.4H. J 8.0 Hz, NH from
one isomer), 6.88 (d, 0.6H, J 8.0 Hz, NH from one isomer), 7.18–
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

10
J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
(CH2), 29.73 (CH2), 29.75 (CH2), 31.95 (CH2), 31.97 (CH2), 34.27
(CH2), 34.31 (CH2), 34.44 (CH2), 34.50 (CH2), 34.54 (CH2), 34.62
(CH2), 37.86 (C-2L), 37.94 (C-2L), 39.19 (C-2L), 39.40 (C-2L), 40.87
(C-2L), 41.45 (C-2L), 47.59 (NCH2), 48.94 (NCH2), 49.20 (NCH2),
51.54 (NCH2), 55.05 (C-2 from one isomer), 55.97 (C-2 from one
isomer), 67.63 (OCH2), 68.40 (C-6), 69.25 (OCH2), 69.52–69.63
(m, PhCH2O)2P), 70.15 (C-3L), 70.18 (C-3L), 70.44 (C-3L), 70.87 (C-
3L), 71.16 (C-3L), 71.80 (C-3L), 72.09 (C-3 from one isomer), 72.62
(C-3L), 73.35 (Ph-CH2 from one isomer), 73.44 (Ph-CH2 from one
isomer), 73.70 (C-5 from one isomer), 73.74 (C-5 from one isomer),
74.22 (d, J 5.5 Hz, C-4 from one isomer), 74.30 (d, J 5.5 Hz, C-4 from
one isomer), 99.76 (C-1 from one isomer), 100.50 (C-1 from one
isomer), 127.59 (CH-Ar), 127.64 (CH-Ar), 127.72 (CH-Ar), 127.80
(CH-Ar), 127.99 (CH-Ar), 128.04 (CH-Ar), 128.10 (CH-Ar), 128.13
(CH-Ar), 128.33 (CH-Ar), 128.38 (CH-Ar), 128.54 (CH-Ar), 128.56
(CH-Ar), 128.59 (CH-Ar), 128.60 (CH-Ar), 128.64 (CH-Ar), 128.66
(CH-Ar), 135.55 (C-Ar), 135.61 (C-Ar), 137.67 (C-Ar), 137.98 (C-
Ar), 169.90 (C@O), 170.35 (C@O), 170.79 (C@O), 170.93 (C@O),
171.00 (C@O), 171.25 (C@O), 173.23 (C@O), 173.39 (C@O),
173.75 (C@O), 173.78 (C@O), 173.91 (C@O); MALDI-MS (m/z) Calcd
for C115H195N2O19P [M+Na]+: 1962.39, found: 1962.30.
3.16. Reagents for biological experiments
Escherichia coli LPS 011:B4 was obtained from Sigma. Each of
synthetic lipid A mimics 2–4 were reconstituted in 20% DMSO in
phosphate buffered saline (PBS) with brief sonication, aliquoted
and stored at ꢁ80 °C. A fresh aliquot was used for each individual
experiment. Solution concentrations were set such that the total
addition of DMSO never exceeded 0.5% to avoid toxic effects.
THP-1 cells were obtained from American Type Culture Collection
(ATCC). RPMI-1640 media, fetal bovine serum, and antibiotic-anti-
mycotic 100ꢂ were obtained from Gibco BRL. Phorbol 12-myris-
tate 13 acetate (PMA) was purchased from Sigma, dissolved in
DMSO, aliquoted and stored at ꢁ80 °C. Lipid IVa was purchased
from Peptide Institute, Inc. (Osaka, Japan), and was dissolved in
DMSO.
3.17. Cell maintenance
THP-1 cells were maintained at 37 °C and 5% CO2 atmosphere in
RPMI-1640 media supplemented with 10% heat-inactivated fetal
bovine serum and 1% antibiotic-antimycotic 100ꢂ. Cell counting
was performed using a Beckman Coulter ViCell X-R instrument,
with viability being determined through the trypan blue cellular
exclusion method.
3.14. N-{2-Phosphonoethyl}-N-{2-[2-deoxy-4-O-phosphono-3-
O-((R)-3-tetradecanoyloxytetradecanoyl)-2-((R)-3-
tetradecanoyloxytetradecanamido)-b-D-glucopyranosyloxy]-
ethyl}-(R)-3-tetradecanoyloxytetradecanamide (3)
3.18. Cytokine induction and measurement
To a solution of 19 (53 mg, 0.027 mmol) in freshly distilled THF
(45 mL), palladium on charcoal (5%, 26 mg) was added and the
mixture was stirred at room temperature under a hydrogen atmo-
sphere (balloon) for 24 h. The mixture was filtered, and the filtrate
concentrated. The residue was purified by flash column chroma-
tography (CHCl3/MeOH, 9:1 and then CHCl3/MeOH/H2O, 3:1:0.2)
to afford 3 (28 mg, 70%) as white fluffy solid after being freeze
dried from a dioxane–CHCl3 mixture (95:5). Rf 0.37 (CHCl3/
THP-1 pre-monocytic cells were plated at 0.5 ꢂ 106 cells wellꢁ1
in 6-well tissue culture plates containing the RPMI media further
supplemented with 25 ng mLꢁ1 of PMA. After 48 h, the media
was removed and the now adhered monocytic THP-1 cells were
washed with PBS. The wells were then refilled with serum-free
RPMI media, incubated for 3 h, and then exposed to stimuli. After
24 h stimulation, culture supernatants were collected and stored
frozen (ꢁ80 °C) until assayed for cytokine production.
MeOH/H2O/NH4OH, 3:2:0.2:0.2); ½a D22
ꢃ
ꢁ0.1 (c 1.0, CHCl3); 1H
All cytokine ELISAs were performed in 96-well MaxiSorp plates.
Ready-Set-Go! ELISA kits (eBioscience) were used for cytokine
NMR (500 MHz, CDCl3): d 0.89 (t, 18H, J 6.5 Hz, CH3 ꢂ 6), 1.22–
1.43 (br m, 114H, CH2 ꢂ 57), 1.54–1.68 (br m, 12H, H-4L ꢂ 6, H-
quantification of human TNF-a, IL-6, and IL-1b according to the
0
0
3L ꢂ 6), 2.20–2.82 (br m, 12H, H-2L ꢂ 6, H-2L ꢂ 6), 3.68–4.16 (br
m, 12H, H-2, H-5, H-6b, H-6a, NCH2 ꢂ 2, OCH2 ꢂ ꢂ 2), 4.48–4.70
(br m, 2H, H-1, H-4), 5.06–5.38 (br m, 4H, H-3, H-3L ꢂ 3); MAL-
DI-MS (m/z) Calcd for C94H180N2O21P2 [M+Na]+: 1758.24, found:
1758.20.
manufacturer’s instructions. The absorbance was measured at
450 nm with wavelength correction set to 540 nm using a micro-
plate reader (BMG Labtech). All cytokine values were measured
in duplicate, and are presented as the mean SD of two separate
experiments.
3.15. N-{Carboxymethyl}-N-{2-[2-deoxy-4-O-phosphono-3-O-
((R)-3-tetradecanoyloxytetradecanoyl)-2-((R)-3-
Supplementary data
tetradecanoyloxytetradecanamido)-b-D-glucopyranosyloxy]-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.024.
ethyl}-(R)-3-tetradecanoyloxytetradecanamide (4)
In a similar manner as described for preparation of 3, a solution
of 20 (35 mg, 0.018 mmol) and palladium on charcoal (5%, 20 mg)
in freshly distilled THF (40 mL) was stirred under a hydrogen
atmosphere (balloon) at room temperature for 24 h. The mixture
was filtered, the filtrate concentrated, and the resulting residue
was purified by flash column chromatography (CHCl3/MeOH, 9:1
and then CHCl3/MeOH/H2O, 3:1:0.1) to yield 4 (23 mg, 77%) as a
white fluffy solid after being freeze-dried from a dioxane–CHCl3
References and notes
1. Akira, S.; Yamamoto, M. Adv. Exp. Med. Biol. 2010, 667, 59.
2. Jiang, Z.-H.; Koganty, R. R. Curr. Med. Chem. 2003, 10, 1423.
3. Ulevitch, R. J. Nat. Rev. Immunol. 2004, 4, 512.
4. Westphal, O.; Lüdertitz, O. Z. Naturforsch. 1952, 7, 148.
5. Miyake, K. J. Endotoxin Res. 2006, 12, 195.
6. Hajjar, A. M.; Ernst, R. K.; Tsai, J. H.; Wilson, C. B.; Miller, S. I. Nat. Immunol.
2002, 3, 354.
7. Kusumoto, S.; Fukase, K.; Fukase, Y.; Kataoka, M.; Yoshizaki, H.; Sato, K.;
Oikawa, M.; Suda, Y. J. Endotoxin Res. 2003, 9, 361.
8. Rietschel, E. T.; Kirikae, T.; Schade, F. U.; Mamat, U.; Schmidt, G.; Loppnow, H.;
Ulmer, A. J.; Zahringer, U.; Seydel, U.; Di Padova, F.; Schreier, M.; Brade, H.
FASEB J. 1994, 8, 217.
9. Ulmer, A. J.; Heine, H.; Feist, W.; Kusumoto, S.; Kusama, T.; Brade, H.; Schade,
U.; Rietschel, E. T.; Flad, H. D. Infect. Immun. 1992, 60, 3309.
10. Kataoka, M.; Hashimoto, M.; Suda, Y.; Kusumoto, S.; Fukase, S. Heterocycles
2006, 69, 395.
mixture (95:5). Rf 0.41 (CHCl3/MeOH/H2O, 3:1:0.1); ½a D22
ꢁ0.1 (c
ꢃ
1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.80 (t, 18H, J 6.5 Hz,
CH3 ꢂ 6), 1.12–1.33 (br m, 114H, CH2 ꢂ 57), 1.46–1.62 (br m,
0
0
12H, H-4L ꢂ 6, H-3L ꢂ 6), 2.16–2.31 (m, 9H, H-2L ꢂ 3, H-2L ꢂ 6),
2.33–2.65 (m, 3H, H-2L ꢂ 3), 3.45–3.72 (br m, 7H, H-2, H-5, H-6b,
H-6a, ROCH2CH2N, ROCHH), 3.75–3.98 (br m, 3H, NCH2COOH,
ROCHH), 4.41–4.55 (br m, 2H, H-1, H-4), 5.04–5.20 (br m, 4H,
11. Johnson, D. A.; Keegan, D. S.; Sowell, C. G.; Livesay, M. T.; Johnson, C. L.;
Taubner, L. M.; Harris, A.; Myers, K. R.; Thompson, J. D.; Gustafson, G. L.;
H-3, H-3L ꢂ 3); MALDI-MS (m/z) Calcd for C94H177N2O19
P
[M+Na]+: 1692.25, found: 1692.17.
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024

J. D. Lewicky et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
11
Rhodes, M. J.; Ulrich, J. T.; Ward, J. R.; Yorgensen, Y. M.; Cantrell, J. L.;
Brookshire, V. G. J. Med. Chem. 1999, 42, 4640.
20. Jiang, Z.-H.; Budzynski, W. A.; Skeels, L. N.; Krantz, M. J.; Koganty, R. R.
Tetrahedron 2002, 58, 8833.
12. Myers, K. R.; Truchot, A. T.; Ward, J.; Hudson, Y.; Ulrich, J. T. In Cellular and
Molecular Aspects of Endotoxin Reactions; Nowotny, A., Spitzer, J. J., Ziegler, E. J.,
Eds.; Endotoxin Research Series; Elsevier: Amsterdam, 1990; Vol. 1, pp 145–
156.
21. Jiang, Z.-H.; Bach, M. V.; Budzynski, W. A.; Krantz, M. J.; Koganty, R. R.;
Longenecker, B. M. Bioorg. Med. Chem. Lett. 2002, 12, 2193.
22. Jiang, Z.-H.; Budzynski, W. A.; Qiu, D.; Yalamati, D.; Koganty, R. R. Carbohydr.
Res. 2007, 342, 784.
13. Ulrich, J. T., Myers, K. R., Eds.Vaccine Design: The Subunit and Adjuvant
Approach; Powell, M. F., Newman, M. J., Eds.; Plenum: New York, 1995; pp
495–524.
14. Mata-Haro, V.; Cekic, C.; Martin, M.; Chitton, D. M.; Casella, C. R.; Mitchell, T. C.
Science 2007, 316, 1628.
23. Lewicky, J. D.; Ulanova, M.; Jiang, Z.-H. Carbohydr. Res. 2011, 346, 1705.
24. Lewicky, J. D.; Ulanova, M.; Jiang, Z.-H. RSC Adv. 2012, 2, 1917.
25. Dullenkopf, W.; Castro-Palomino, J. C.; Manzoni, L.; Schmidt, R. R. Cabohydr.
Res. 1996, 296, 135.
26. Ellervik, U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627.
27. Kiso, M.; Tanaka, S.; Fujita, M.; Fujishima, Y.; Ogawa, Y.; Hasagawa, A.
Carbohydr. Res. 1987, 162, 247.
15. Gaekwad, J.; Zhang, Y.; Zhang, W.; Reeves, J.; Wolfert, M. A.; Boons, G. J. J. Biol.
Chem. 2010, 285, 29375.
16. Zhang, Y.; Gaekwad, J.; Wolfer, M. A.; Boons, G. J. J. Am. Chem. Soc. 2007, 129,
5200.
28. Stanssens, D.; Hermanns, R.; Wories, H. Prog. Org. Coat. 1993, 22, 379.
29. Saitoh, S.; Akashi, S.; Yamada, T.; Tanimura, N.; Kobayashi, M.; Konno, K.;
Fukase, K.; Kusumoto, Y.; Kosagi, A.; Miyake, K. Int. Immunol. 2004, 16, 961.
30. Golenbock, D. T.; Hampton, R. Y.; Qureshi, N.; Takayama, K.; Raetz, C. R. H. J.
Biol. Chem. 1991, 266, 19490.
17. Kusumoto, S.; Fukase, K. Chem. Rec. 2006, 6, 333.
18. Fujimoto, Y.; Adachi, Y.; Akamatsu, M.; Fukase, Y.; Kataoka, M.; Suda, Y.;
Fukase, K.; Kusumoto, S. J. Endotoxin. Res. 2005, 11, 341.
19. Park, B. S.; Song, D. H.; Kim, H. M.; Choi, B. S.; Lee, J. O. Nature 2009, 458,
1191.
31. O’Sullivan, M. C.; Dalrymple, D. M. Tetrahedron Lett. 1995, 36, 3451.
32. Kim, T. H.; Yang, G.-Y. Tetrahedron Lett. 2002, 43, 9553.
Please cite this article in press as: Lewicky, J. D.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/.2013.02.024