Synthesis of pseudo-disaccharide analogues of lipid A: Haptens for the generation of antibodies with glycosidase activity towards lipid A

Article abstract of DOI:10.1081/CAR-120004331

In order to develop a generic treatment of sepsis caused by infections with Gram-negative bacteria, a series of pseudo-disaccharide analogues of lipid A (1-5) was synthesized. These adducts not only harbor a 2-acylaminodideoxynojirimycin unit mimicking th

Full text of DOI:10.1081/CAR-120004331

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
SYNTHESIS OF PSEUDO-DISACCHARIDE
ANALOGUES OF LIPID A: HAPTENS FOR THE
GENERATION OF ANTIBODIES WITH GLYCOSIDASE
ACTIVITY TOWARDS LIPID A
Richard J.B.H.N. van den Berg , Daan Noort , Gijs A. van der Marel , Jacques
H. van Boom & Hendrik P. Benschop
To cite this article: Richard J.B.H.N. van den Berg , Daan Noort , Gijs A. van der Marel , Jacques
H. van Boom & Hendrik P. Benschop (2002) SYNTHESIS OF PSEUDO-DISACCHARIDE
ANALOGUES OF LIPID A: HAPTENS FOR THE GENERATION OF ANTIBODIES WITH
GLYCOSIDASE ACTIVITY TOWARDS LIPID A, Journal of Carbohydrate Chemistry, 21:3, 167-188
To link to this article: http://dx.doi.org/10.1081/CAR-120004331
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J. CARBOHYDRATE CHEMISTRY, 21(3), 167–188 (2002)
SYNTHESIS OF PSEUDO-DISACCHARIDE
ANALOGUES OF LIPID A: HAPTENS FOR
THE GENERATION OF ANTIBODIES
WITH GLYCOSIDASE ACTIVITY
TOWARDS LIPID A
Richard J. B. H. N. van den Berg,1 Daan Noort,1
Gijs A. van der Marel,2 Jacques H. van Boom,2,
*
and Hendrik P. Benschop1
1Department of Chemical Toxicology, TNO Prins Maurits Laboratory,
P.O. Box 45, NL-2280 AA Rijswijk, The Netherlands
2Leiden Institute of Chemistry, Gorlaeus Laboratories, University of
Leiden, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands
ABSTRACT
In order to develop a generic treatment of sepsis caused by infections
with Gram-negative bacteria, a series of pseudo-disaccharide analogues
of lipid A (1–5) was synthesized. These adducts not only harbor a 2-
acylaminodideoxynojirimycin unit mimicking the transition state of the
glycosidic hydrolysis, but also a 2-N, 3-O-diacylated glucosamine moiety
capable of generating catalytic antibodies with more selective glycosi-
dase properties towards lipid A.
INTRODUCTION
Endotoxins, the complex lipopolysaccharides (LPS) situated in the outer mem-
brane of Gram-negative bacteria, are extremely potent toxins.[1] Most of the biological
activities of LPS reside in the small terminal disaccharide phospholipid moiety known
*
Corresponding author. E-mail: j.boom@chem.leidenuniv.nl
167
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168
VAN DEN BERG ET AL.
as lipid A.[2] Therapeutic strategies under development aim at either preventing en-
dotoxin interaction with host effector cells or interrupting endotoxin mediated signal
transduction pathways. This objective can be attained in blocking the synthesis and
binding of endotoxin, thus neutralizing its activity.[3–5]
Our approach to suppress sepsis caused by Gram-negative bacteria is based on
catalytic antibodies capable of degrading lipid A via hydrolysis of the interglycosidic
bond resulting in the formation of non-toxic monosaccharides. Since catalytic anti-
bodies are supposed to have catalytic activities with tailor-made specificities, these
abzymes have many potential therapeutically applications. For example, Landry et al.
succeeded in the generation of a catalytic antibody that was effective in detoxifying
cocaine from the blood stream via hydrolysis of the benzoyl ester function.[6–9] Based
on this result it was envisioned that a generic treatment of sepsis caused by Gram-
negative bacteria might be feasible using specific and selective glycosidase antibodies
capable of degrading lipid A.
Several groups have reported the design and generation of antibodies with gly-
cosidase activity.[10–16] In general, haptens are based on iminocyclitol glycosidase
inhibitors such as deoxynojirimycin and isofagomine, which in terms of polarity and
shape resemble the transition state of the glycosidic cleavage reaction. The protonated
endocyclic nitrogen atom of iminocyclitols mimics the electronic charge developing in
the transition state formed during cleavage of the interglycosidic bond. In a previous
paper[17] we reported the preparation of 2-acylaminodideoxynojirimycin derivatives
mimicking the transition state of the hydrolysis of the interglycosidic bond at the non-
reducing end of lipid A. It turned out that monoclonal antibodies raised against these
haptens showed promising glycosidase activity (results to be published).
It was envisaged, based on the early studies by Dong[18] as well as Yu,[14] that
conjugation of 2-N, 3-O-diacylated glucosamine derivatives to 2-acylaminodideoxyno-
jirimycin units would afford haptens suitable to raise specific glycosidase antibodies
towards lipid A. The latter can be achieved by anchoring the 2-N, 3-O-diacylated
glucosamine units, which mimic the reducing part of lipid A, to the endocyclic nitrogen
atom of the iminoglucitol moieties via a flexible linker.
We here report the synthesis of several pseudo-disaccharide analogues of lipid A
(i.e. compounds 1–5, Figure 1) containing 2-acylaminodideoxynojirimycins as well as
2-N, 3-O-diacylated glucosamine units.
Figure 1.

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
RESULTS AND DISCUSSION
169
The easily accessible 3,4,6-tri-O-benzyl-2-N-benzyloxycarbonylamino-1,2,5-tri-
deoxy-1,5-iminoglucitol (6)[17] was used as a starting compound for the preparation of
iminocyclitols 11 and 12 (Scheme 1) mimicking the non-reducing part of lipid A. In
the first step, the carboxymethyl linker in 7 was introduced by alkylation of the
endocyclic nitrogen atom of 6 with tert-butyl bromoacetate under the agency of cesium
carbonate in dimethyl formamide[19] to give compound 7 in a yield of 95%. Selective
removal of the benzyloxycarbonyl (Z) protecting group in 7 proceeded smoothly by
hydrogenation over Degussa type palladium on carbon to give 8 in a quantitative yield.
PyBOP mediated coupling[20] of amine 8 with myristic acid and (R)-3-dodeca-
noyloxytetradecanoic acid[21] gave the N-acylated derivatives 9 and 10, respectively, in
good yield. Removal of the tert-butyl group in compounds 9 and 10 was readily
effected by treatment with neat trifluoroacetic acid, affording building blocks 11 and 12
in 100% and 83% yield, respectively.
The individual hapten units 34, 35 and 36, mimicking the reducing part of lipid
A, were obtained by subjecting D-glucosamine (13) to the sequence of reactions de-
picted in Scheme 2. Accordingly, D-glucosamine (13) was converted in two steps into N-
benzyloxycarbonyl protected glucosamine 15.[22] Acetonation of 15 with 2,2-dime-
thoxypropane and a catalytic amount of p-toluenesulfonic acid gave partially protected
derivative 16 in a quantitative yield. Condensation of the secondary hydroxyl group in
16 with myristic acid under the agency of DCC[23] afforded 17 in a yield of 86%.
Removal of the Z-protecting group in 17 by hydrogenation over palladium on carbon in
ethyl acetate gave amine 18 in a quantitative yield. PyBOP mediated condensation of
amine 18 with the individual acids 19–21, of which the primary amine function was
protected with the Z-group, afforded the corresponding derivatives 22–24. At this
stage, N-acylated compound 22 was transformed into the primary amino derivative 34
by following the four-step process as portrayed in Scheme 2. Thus, de-acetonation of
compound 22 with trifluoroacetic acid in aqueous tetrahydrofuran,[24] followed by
Scheme 1. Conditions: (i) tert-butyl bromoacetate, Cs2CO3, DMF, 95%; (ii) EtOAc, 5% Pd/C,
H2, 100%; (iii) myristic acid, PyBOP, DiPEA, DCM, 89%; (iv) (R)-3-HOOCCH2CHO
(COC11H23)C11H23, PyBOP, DiPEA, DCM, 70%; (v) TFA (11, 100%), (12, 83%).

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170
VAN DEN BERG ET AL.
Scheme 2. Conditions: (i) benzyl chloroformate, NaHCO3, H2O; (ii) HCl/MeOH (2%, w/w),
53%; (iii) 2,2-dimethoxypropane, toluene-4-sulfonic acid, acetone, DCM, 100%; (iv) myristic
acid, DCC, DMAP, DCM, 86%; (v) 5% Pd/C, H2, 100%; (vi) 19/20/21, PyBOP, DiPEA, DCM,
(22, 87%), (23, 96%), (24, 87%); (vii) TFA, THF/H2O, (4/1, v/v), (25, 99%), (26, 63%), (27,
87%); (viii) MsCl (TsCl), pyridine, (28, 80%), (29, 78%), (30, 79%); (ix) NaN3, DMF, 80°C, (31,
84%), (32, 72%), (33, 73%) (x) triphenylphosphine (1.5 equiv), H2O (1.2 equiv), THF, (34,
100%), (35, 78%), (36, 52%).

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
171
regio-selective sulfonylation of the primary hydroxyl group of diol 25 gave sulfonylate
28 in an overall yield of 80%. Treatment of the latter derivative with sodium azide in
dimethyl formamide at elevated temperature afforded the 6-azidoglucosamine com-
pound 31. Subsequent reduction of the resulting azido function in 31 under the agency
of triphenylphosphine in aqueous tetrahydrofuran[25] gave the requisite 6-amino de-
rivative 34 in yield of 84% based on sulfonylate 28. In a similar way compounds 35
and 36 were attained by subjecting acetonides 23 and 24 to the same four-step process
as described for the synthesis of 34.
Having the requisite building blocks 11, 12, 34–36 at hand, introduction of the
required amide bond of the fully protected compounds 37–41 could be readily accom-
plished (see Scheme 3) using PyBOP as the condensation agent. For example, PyBOP-
mediated condensation of acid 11 and amine 34 proceeded smoothly to afford the fully
protected hapten 37 in a yield of 96%. The identity and homogeneity of compound 37
Scheme 3. Conditions: (i) PyBOP, DiPEA, DCM, (37, 96%), (38, 96%), (39, 51%), (40, 88%),
(41, 96%); (ii) 10% Pd/C, DMF (1, 100%), (2, 72%), (3, 54%), (4, 88%), (5, 65%).

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172
VAN DEN BERG ET AL.
was fully ascertained by NMR spectroscopy and mass spectrometry. Similar yields
were obtained by condensation of 11 with 35 and 12 with 34 as well as 35 to yield the
fully protected haptens 38, 40 and 41, respectively. In contrast, the coupling of acid 11
with amine 36 to give amide 39 was in terms of yield not fully satisfactory, and may be
1
ascribed to the increased lipophilicity of amine 36. H, 13C NMR and mass spectro-
metric data of the four conjugates 38–41 were in complete accordance with the pro-
posed structures. In the final stage, haptens 37–41 were deprotected by hydrogenolysis
over palladium on carbon in dimethyl formamide. Hydrogenolysis of compound 37
proceeded in near quantitative yield to give hapten 1, the identity and homogeneity of
which was fully ascertained by NMR spectroscopy and mass spectrometry. The same
results were obtained by hydrogenolysis of compounds 38 and 40 to yield the haptens
2 and 4, respectively. Unfortunately, unmasking of compounds 39 and 41 was rather
sluggish and led to the isolation of haptens 3 and 5 in moderate yields. The disap-
pointing outcome of the latter hydrogenolysis may also be due to the intrinsically high
lipophilic nature of compounds 39 and 41. The identity of compounds 2–5 could be
readily ascertained by mass spectrometry. Unfortunately, it turned out that the structure
assignment of haptens 2–5 was seriously hampered by the fact that NMR spectra[26]
could not be interpreted due to extensive line broadening.
CONCLUSION
In summary, we have synthesized five pseudo-lipid A analogues as potential
haptens for the generation of catalytic antibodies with glycosidase activity towards lipid
A. These haptens contain a primary amino function in the N-acyl chain of the 2-N, 3-O-
diacylated glucosamine units which will serve as a handle of anchoring haptens 1–5 to
a carboxylic acid terminus of a carrier protein. The immunochemical evaluation of the
haptens will be reported in due course.
EXPERIMENTAL
General Methods. Toluene (Merck) was distilled from P2O5 and stored over sodium
wire. Dichloromethane and N,N-dimethylformamide were purchased from Biosolve Ltd.
and freshly distilled from CaH2. N,N-Diisopropylethylamine (Acros Chimica) was
distilled from p-toluenesulfonyl chloride (60 g/L) and redistilled from potassium
hydroxide pellets (40 g/L). Benzyl chloroformate, tert-butyl bromoacetate, cesium
carbonate, N,N-dicyclohexylcarbodiimide, 4-(dimethylamino)pyridine, 2,2-dimethoxy-
propane, D-glucosamine hydrochloride, methanesulfonyl chloride, palladium on carbon
(5%, Degussa E101 NO/W), sodium azide, tetrahydrofuran, toluene-p-sulfonic acid,
toluene-p-sulfonyl chloride and triphenylphosphine were purchased form Fluka. PyBOP
was purchased from NovaBiochem. Trifluoroacetic acid was purchased from Acros
1
Chimica. H NMR and 13C NMR data were recorded with a Varian VXR-400S (399.9/
1
100.6 MHz). H and 13C chemical shifts are given in ppm (d) relative to tetramethyl-
silane (d= 0.00), DMSO-d5 (d =2.525), DMSO-d6 (d= 39.6) and CDCl3 (d= 77.00) as
internal standard. The purity of the compounds was established by 1H NMR spec-
troscopy: >95% in all cases. Mass spectra were recorded with a VG Quattro II triple

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
173
quadropole mass spectrometer (Fisons Instruments, Altrincham, UK). Column
chromatography was performed on Silica gel 60 (220–440 mesh ASTM, Fluka).
TLC analysis was performed with silica gel TLC plates (Fluka) with detection by UV
absorption (254 nm) where applicable and charring with 20% H2SO4 in MeOH or am-
monium molybdate (25 g/L) and ceric ammonium sulfate (10 g/L) in 20% H2SO4. Prior
to reactions that require anhydrous conditions, traces of water were removed by co-
evaporation with dry toluene. These reactions were conducted under dry argon atmos-
phere. Hydrogenations were executed at atmospheric pressure under an atmosphere of
hydrogen gas maintained by an inflated balloon. Polytetrafluoroethylene (PFTE) filters
were purchased from Alltech (Breda, The Netherlands).
3,4,6-Tri-O-benzyl-2-[(benzyloxycarbonyl)amino]-1,5-N-[(O-tert-butyl-carboxy-
methyl)imino]-1,2,5-trideoxy-D-glucitol (7). To a solution of 6 (1.20 g, 2.12 mmol)
in DMF (20 mL) cesium carbonate (700 mg, 2.14 mmol) and tert-butyl bromoacetate
(1.15 mL, 7.81 mmol) were added. The reaction mixture was stirred for 16 h at ambient
temperature, after which TLC analysis indicated complete conversion of starting ma-
terial into a compound with Rf = 0.92 (ethyl acetate/hexane, 1:1, v/v). The mixture was
diluted with DCM (100 mL) and washed with aqueous NaOH (1 M, 50 mL). After
drying over MgSO4, the organic layer was concentrated in vacuo. The crude product
was purified by silica gel column chromatography. Elution was performed with DCM/
1
MeOH (100:0! 96.5:3.5, v/v). Yield 1.37 g (95%). H NMR (CDCl3): d =1.37 (s, 9H,
CH3, tBu), 2.71 (dd, 1H, H-1ax, J1ax,1eq = 11.6 Hz, J1ax,2 =8.9 Hz), 3.04 (br. s, 1H, H-5),
3.10 (br. d, 1H, H-1eq), 3.27 (d, 1H, Ha-acetyl, J= 17.7 Hz), 3.35 (br. t, 1H, H-3), 3.52
(dd, 1H, H-6, J6,6’ =10.5 Hz, J5,6 = 2.8 Hz), 3.57 (d, 1H, Hb-acetyl, J= 17.7 Hz), 3.59 (t,
1H, H-4), 3.72 (dd, 1H, H-6’, J6,6’ =10.5 Hz, J5,6’ =3.9 Hz), 3.79 (m, 1H, H-2), 4.40–
4.76 (m, 6H, 3 ꢀ CH2 Bn), 4.89 (s, 1H, NH), 5.05 (dd, 2H, CH2 Z), 7.18–7.39 (m,
20H, CH-arom Bn/Z). 13C{1H} NMR (CDCl3): d =28.16 (CH3 tBu), 50.88 (C-2), 53.38
(C-1), 55.08 (CH2 tert-butyl acetate), 61.61 (C-5), 66.02 (C-6), 66.51 (CH2 Z), 73.39,
73.66, 73.97 (3 ꢀ CH2 Bn), 78.39 (C-4), 80.90 (Cq tBu), 81.43 (C-3), 127.58–128.50
(CH-arom Bn/Z), 156.00 (C= O Z), 170.63 (C =O tert-butyl acetyl). ES-MS; m/z:
681.5, [M +H]+; monoisotopic MW calculated for C41H48N2O7 = 680.35.
2-Amino-3,4,6-tri-O-benzyl-1,5-N-[(O-tert-butyl-carboxymethyl)imino]-1,2,5-tri-
deoxy-D-glucitol (8). Pd/C (5%, Degussa type E101 NO/W, 100 mg) was added to a
solution of 7 (109 mg, 0.160 mmol) in ethyl acetate (5 mL). Hydrogen was passed
through the stirred mixture for 1 h, after which TLC analysis indicated the complete
conversion of starting material into a compound with Rf =0.20 (MeOH/DCM, 5:95,
v/v). The mixture was passed over a short column containing a layer of glass wool and
a layer of hyflo1 and, finally, over a PTFE filter. Concentration of the filtrate in vacuo
1
yielded 8 as a white solid (94 mg; 100%). H NMR (CDCl3): d =1.48 (s, 9H, CH3,
tBu), 1.77 (br. s, 2H, NH2), 2.72 (t, 1H, H-1ax), 2.92 (m, 3H, H-1eq, H-2, H-5), 3.16
(t, 1H, H-3 J =8.9 Hz), 3.30 (d, 1H, Ha-acetyl, J= 17.6 Hz), 3.51 (t, 1H, H-4, J=9.1 Hz),
3.55 (dd, 1H, H-6), 3.59 (d, 1H, Hb-acetyl, J=17.6 Hz), 3.72 (dd, 1H, H-6’, J6,6’ = 10.6 Hz,
J
5,6’ = 3.2 Hz), 4.44–4.94 (m, 6H, 3 ꢀ CH2 Bn), 7.18–7.35 (m, 15H, CH-arom Bn).
13C{1H} NMR (CDCl3): d= 28.22 (CH3 tBu), 52.64 (C-2), 54.43 (CH2 tert-butyl
acetate), 57.78 (C-1), 62.35 (C-5), 65.90 (C-6), 73.50, 74.71, 75.10 (3 ꢀ CH2 Bn), 79.67
(C-4), 81.00 (Cq tBu), 88.52 (C-3), 127.59–128.52 (CH-arom Bn), 137.70, 138.50,

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VAN DEN BERG ET AL.
138.78 (3 ꢀ Cq Bn), 170.55 (C= O tert-butyl acetyl). ES-MS; m/z: 547.5, [M +H] +;
monoisotopic MW calculated for C33H42N2O5 = 546.31.
3,4,6-Tri-O-benzyl-1,5-N-[(O-tert-butyl-carboxymethyl)imino]-2-(tetradecanoyl)
amino-1,2,5-trideoxy-D-glucitol (9). To a stirred mixture of myristic acid (92 mg,
0.403 mmol), PyBOP (231 mg, 0.605 mmol) and DiPEA (76 mL, 0.443 mmol) in DCM
(10 mL) a solution of 8 (200 mg, 0.366 mmol) in DCM (10 mL) was added. After 30
min, TLC analysis indicated the complete conversion of starting material into a
compound with Rf = 0.89 (MeOH/DCM, 5:95, v/v). The reaction mixture was diluted
with DCM (100 mL) and washed with water (1 ꢀ 50 mL). After drying over MgSO4,
the organic layer was concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography. Elution was performed with hexane/
1
ethyl acetate (80:20 !60:40, v/v). Yield 228 mg (89%). H NMR (CDCl3): d =0.88 (t,
3H, CH3 myristyl), 1.25 (m, 20H, 10 ꢀ CH2 myristyl), 1.48 (s, 9H, 3 ꢀ CH3 tBu), 1.49
(m, 2H, CH2 myristyl), 1.94 (m, 2H, CH2 myristyl), 2.57 (dd, 1H, H-1ax, J1ax,1eq =11.8
Hz, J1ax,2 =7.0 Hz), 3.15 (m, 1H, H-5), 3.17 (dd, 1H, H-1eq, J1ax,1eq =11.8 Hz,
J
1eq,2 =3.8 Hz), 3.29 (d, 1H, Ha-acetyl, J= 17.7 Hz), 3.45 (t, 1H, H-3), 3.52 (dd, 1H, H-
6, J6,6’ =10.2 Hz, J5,6 = 4.9 Hz), 3.54 (d, 1H, Hb-acetyl, J= 17.7 Hz), 3.60 (t, 1H, H-4,
J=6.1), 3.77 (dd, 1H, H-6’, J6,6’ =10.3 Hz, J5,6’ =4.9 Hz), 4.00 (m, 1H, H-2), 4.41–4.70
(m, 6H, 3 ꢀ CH2 Bn), 5.85 (d, 1H, NH, J2,NH =7.1 Hz), 7.24–7.35 (m, 15H, CH-arom
Bn). 13C{1H} NMR (CDCl3): d= 14.15 (CH3 myristyl), 22.74–36.92 (CH2 myristyl),
28.21 (CH3 tBu), 48.30 (C-2), 51.15 (C-1), 55.67 (CH2 acetyl), 61.51 (C-5), 66.28 (C-
6), 72.93, 73.31, 73.39 (3 ꢀ CH2 Bn), 77.73 (C-4), 78.97(C-3), 80.80 (Cq tBu), 127.67–
128.59 (CH-arom Bn), 138.02, 138.41, 138.45 (3 ꢀ Cq Bn), 170.90 (C= O acetyl),
172.96 (C =O myristyl). ES-MS; m/z: 757.5, [M +H]+; monoisotopic MW calculated
for C47H68N2O6 =756.5.
3,4,6-Tri-O-benzyl-1,5-N-[(O-tert-butylcarboxymethyl)imino]-2-[(R)-3-(dodecanoy-
loxytetradecanoyl)]amino-1,2,5-trideoxy-D-glucitol (10). (R)-3-Dodecanoyloxytetra-
decanoic acid (73 mg, 0.171 mmol) was coupled with compound 8 (94 mg, 0.172
mmol) as described for the preparation of compound 9. The crude product was purified
by silica gel column chromatography; elution was performed with hexane/ethyl acetate
1
(80:20 ! 60:40, v/v). Rf = 0.66 (hexane/ethyl acetate, 2:1, v/v). Yield 115 mg (70%). H
NMR (CDCl3): d =0.88 (m, 6H, 2 ꢀ CH3 acyloxyacyl), 1.25 (m, 34H, CH2 acyl-
oxyacyl), 1.40 (s, 9H, 3 ꢀ CH3 tBu), 1.55 (m, 4H, 2 ꢀ CH2 acyloxyacyl), 2.21 (m, 4H,
2ꢀ CH2 acyloxyacyl), 2.56 (dd, 1H, H-1ax, J1ax,eq = 11.7 Hz, J1ax,2 = 7.3 Hz), 3.12 (m,
2H, H-1eq, H-5), 3.28 (d, 1H, Ha-acetyl, J= 17.8 Hz), 3.45 (t, 1H, H-3, J=6.5 Hz),
3.53 (dd, 1H, H-6, J6,6’ = 10.3 Hz, J5,6 =3.6 Hz), 3.55 (d, 1H, Hb-acetyl, J =17.8 Hz),
3.60 (t, 1H, H-4, J=6.3 Hz), 3.76 (dd, 1H, H-6’, J6,6’ =10.3 Hz, J5,6’ = 5.0 Hz), 4.00 (m,
1H, H-2), 4.40–4.71 (m, 6H, 3 ꢀ CH2 Bn), 5.11 (m, 1H, CHO acyloxyacyl), 6.06 (d,
1H, NH, J2,NH =7.5 Hz), 7.23–7.33 (m, 15H, CH-arom Bn). 13C{1H} NMR (CDCl3):
d= 14.12 (CH3 acyloxyacyl), 22.70–41.65 (CH2 acyloxyacyl), 28.17 (CH3 tBu), 48.48
(C-2), 51.30 (C-1), 55.52 (CH2 acetyl), 61.43 (C-5), 66.27 (C-6), 71.21 (CHO
acyloxyacyl), 72.92, 73.05, 73.34 (3 ꢀ CH2 Bn), 77.64 (C-4), 79.33 (C-3), 80.77 (Cq
tBu), 127.63–128.54 (CH-arom Bn), 137.98, 138.40, 138.45 (3 ꢀ Cq Bn), 169.43,
170.82, 173.12 (3 ꢀ C= O amide, ester). ES-MS; m/z: 954.67 [M +H]+, monoisotopic
MW calculated for C59H90N2O8 =955.63.

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
175
3,4,6-Tri-O-benzyl-1,5-N-[carboxymethylimino]-2-(tetradecanoyl)amino-1,2,5-tri-
deoxy-D-glucitol (11). A solution of 9 (118 mg, 0.156 mmol) in TFA (5 mL) was
stirred for 2 h at ambient temperature, after which TLC analysis indicated complete
conversion of starting material into a compound with Rf =0.12 (MeOH/DCM, 5:95,
v/v). After concentration of the reaction mixture, the residue was coevaporated with
1
toluene (3 ꢀ 5 mL). Yield 125 mg (quantitative). H NMR (CDCl3): d= 0.88 (t, 3H,
CH3 myristyl), 1.25 (m, 20H, CH2 myristyl), 1.48 (br. s, 2H, CH2 myristyl), 2.04 (m,
2H, CH2 myristyl), 2.95 (d, 1H, H-1ax), 3.26 (br. s, 2H, H-1eq, H-5), 3.45 (br. s, 1H,
Ha-acetyl), 3.56 (br. s, 2H, H-3, H-6), 3.72 (m, 2H, Hb-acetyl, H-6’), 3.89 (m, 1H, H-
4), 4.26 (br. s, 1H, H-2), 4.39–4.62 (m, 6H, 3 ꢀ CH2 Bn), 7.09 (br. s, 1H, NH), 7.14–
7.27 (m, 15H, CH-arom Bn), 9.40 (br. s, 1H, COOH). 13C{1H} NMR (CDCl3):
d =14.15 (CH3 myristyl), 22.73–36.68 (CH2 myristyl), 46.32 (C-2), 50 (C-1), 54.46
(CH2 acetyl), 61.08 (C-5), 64.88 (C-6), 73.10, 73.33, 73.49 (3 ꢀ CH2 Bn), 75.38 (C-4),
76.05(C-3), 127.82–128.71 (CH-arom Bn), 136.42–137.77 (Cq Bn), 165.08 (C= O
carboxymethyl), 174.39 (C= O myristyl). ES-MS; m/z: 701.5, [M +H] +; monoisotopic
MW calculated for C43H60N2O6 = 700.45.
3,4,6-Tri-O-benzyl-1,5-N-[carboxymethylimino]-2-[(R)-3-(dodecanoyloxytetradeca-
noyl)]amino-1,2,5-trideoxy-D-glucitol (12). Compound 10 (115 mg, 0.120 mmol)
was treated with TFA as described for the preparation of compound 11. The crude
product was purified by silica gel column chromatography. Elution was performed
with MeOH/DCM (0:100 !5:95, v/v). Rf = 0.12 (hexane/ethyl acetate, 1:2, v/v). Yield
1
90 mg (83%). H NMR (CDCl3): d= 0.88 (m, 6H, 2ꢀ CH3 acyloxyacyl), 1.25 (br. s,
34H, CH2 acyloxyacyl), 1.56 (m, 4H, CH2 acyloxyacyl), 2.23 (m, 4H, CH2 acy-
loxyacyl), 2.60 (d, 1H, H-1ax), 3.21 (m, 2H, H-1eq, H-5), 3.40 (d, 1H, Ha-acetyl), 3.51
(m, 2H, H-3, H-6), 3.66 (m, 2H, Hb-acetyl, H-6’), 3.86 (m, 1H, H-4), 4.13 (br. s, 1H,
H-2), 4.39–4.62 (m, 6H, 3 ꢀ CH2 Bn), 5.05 (m, 1H, CHO acyloxyacyl), 6.70 (br. s, 1H,
NH), 7.21–7.35 (m, 15H, CH-arom Bn). 13C{1H} NMR (CDCl3): d=14.13 (CH3
acyloxyacyl), 22.71–41.86 (CH2 acyloxyacyl), 47.03 (C-2), 49.49 (C-1), 56.66 (CH2
acetyl), 61.56 (C-5), 65.94 (C-6), 71.09 (CHO acyloxyacyl), 72.72, 73.12, 73.45
(3 ꢀ CH2 Bn), 75.83 (C-4), 76.08 (C-3), 127.69–128.70 (CH-arom Bn), 137.27, 137.46,
137.68 (3 ꢀ Cq Bn), 169.49, 171.96, 173.45 (3 ꢀ C =O acid, amide, ester). ES-MS; m/z:
899.7, [M +H] +; monoisotopic MW calculated for C55H82N2O8 = 898.6.
Methyl 2-[(benzyloxycarbonyl)amino]-2-deoxy-a-D-glucopyranoside (15). To a
cooled (0°C) solution of glucosamine hydrochloride (13) (20 g, 93 mmol) in water
(400 mL) NaHCO3 (14.4 g, 171 mmol) and benzyl chloroformate (17.4 mL, 104 mmol)
were added. The mixture was stirred at ambient temperature for 16 h, after which the
white crystalline residue (14) was filtered off, washed with cold acetone ( ꢁ 20°C) and
dried. The white crystals were dissolved in acidic methanol (2% HCl, w/w) and
refluxed for 7 h after which the reaction mixture was concentrated. The resulting
residue was purified by silica gel column chromatography. Elution was performed with
MeOH/DCM (10:90 ! 15:85, v/v). Yield 16 g (53%). Rf = 0.70 (MeOH/DCM, 15:85,
1
v/v). H NMR (DMSO-d6): d= 3.16 (m, 2H, H-4, H-5), 3.27 (s, 3H, OMe), 3.46 (m,
3H, H-2, H2-6), 3.67 (m, 1H, H-3), 4.51 (t, 1H, OH-6), 4.61 (d, 1H, H-1, J1,2 =3.2 Hz),
4.76 (d, 1H, OH-3), 4.98 (d, 1H, OH-4), 5.04 (dd, 2H, CH2 Z), 7.07 (d, 1H, NH,
J
2,NH =7.7 Hz), 7.31–7.42 (m, 5H, CH-arom Z). 13C{1H} NMR (DMSO-d6): d =54.37

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VAN DEN BERG ET AL.
(OMe), 55.95 (C-2), 60.89 (C-6), 65.34 (CH2 Z), 70.65 (C-5), 70.81 (C-3), 72.72 (C-4),
98.09 (C-1), 127.78, 128.35 (CH-arom Z), 137.16 (Cq Z), 156.17 (C=O Z).
Methyl 2-[(benzyloxycarbonyl)amino]-2-deoxy-4,6-O-isopropylidene-a-D-glucopyra-
noside (16). To a mixture of 15 (16 g, 49 mmol) in dry acetone (200 mL) and DCM
(150 mL) was added 2,2-dimethoxypropane (25 mL, 204 mmol) and p-toluene sulfonic
acid (0.4 g, 2.1 mmol). The resulting mixture was stirred at ambient temperature and
after 16 h, TLC analysis showed complete conversion of starting material into a com-
pound with Rf =0.80 (MeOH/DCM, 5:95, v/v). TEA (5 mL) and DCM (100 mL) were
added and the mixture was washed with water (50 mL). The organic layer was dried
(MgSO4) and concentrated. Purification of the crude product by silica gel column
chromatography (elution with MeOH/DCM (0:100 !5:95, v/v)), yielded 18 g (quan-
1
titative) of a yellow oil. H NMR (CDCl3): d = 1.42 (s, 3H, CH3 isopropylidene), 1.51
(s, 3H, CH3 isopropylidene), 2.84 (s, 1H, OH-3), 3.33 (s, 3H, OMe), 3.59 (m, 2H, H-4,
H-5), 3.74 (m, 2H, H-3, H-6), 3.87 (m, 2H, H-2, H-6’), 4.68 (d, 1H, H-1, J1,2 =3.4 Hz),
5.11 (s, 2H, CH2 Z), 5.19 (d, 1H, NH, J2,NH = 8.7 Hz), 7.32–7.36 (m, 5H, CH-arom Z).
13C{1H} NMR (CDCl3): d = 19.17, 29.14 (2 ꢀ CH3 isopropylidene), 55.28 (OMe),
55.92 (C-2), 62.36 (C-6), 63.36 (C-5), 67.30 (CH2 Z), 70.88 (C-3), 74.63 (C-4), 99.23
(C-1), 99.90 (Cq isopropylidene), 128.09–128.60 (CH-arom Z), 136.22 (Cq Z), 156.87
(C = O Z).
Methyl 2-[(benzyloxycarbonyl)amino]-2-deoxy-4,6-O-isopropylidene-3-O-tetradeca-
noyl-a-D-glucopyranoside (17). To a solution of 16 (5.545 g, 15.10 mmol) in DCM
was added DMAP (2.03 g, 16.6 mmol), myristic acid (3.79 g, 16.6 mmol) and DCC
(3.43 g, 16.6 mmol). The reaction mixture was stirred for 18 h at ambient temperature.
TLC analysis (MeOH/DCM, 1:99, v/v) showed complete conversion of starting ma-
terial into a compound with Rf = 0.91. DCU was filtered off and the filter was washed
with DCM (3 ꢀ 25 mL). DCM was concentrated and the crude product was purified
by silica gel column chromatography. Elution was performed with MeOH/DCM
(0:100 ! 3:97, v/v). Yield: 7.49 g (86%) of a colorless oil. 1H NMR (CDCl3): d= 0.88 (t,
3H, CH3 myristyl), 1.25 (br. s, 20H, CH2 myristyl), 1.36 (s, 3H, CH3 isopropylidene),
1.46 (s, 3H, CH3 isopropylidene), 1.54 (m, 2H, CH2 myristyl), 2.22 (m, 2H, CH2
myristyl), 3.36 (s, 3H, OMe), 3.69 (m, 2H, H-4, H-5), 3.76 (t, 1H, H-6), 3.87 (dd, 1H, H-
6’, J5,6’ =4.7 Hz, J6,6’ =10.2 Hz), 3.97 (m, 1H, H-2, J1,2 =3.7 Hz, J= 10.2 Hz), 4.69 (d,
1H, H-1, J1,2 = 3.7 Hz), 5.06 (s, 2H, CH2 Z), 5.12 (m, 2H, H-3, NH), 7.26–7.36 (m, 5H,
CH-arom Z). 13C{1H} NMR (CDCl3): d=14.15 (CH3 myristyl), 19.11, 29.11 (2 ꢀ CH3
isopropylidene), 22.74–34.38 (CH2 myristyl), 54.60 (C-2), 55.32 (OMe), 62.51 (C-6),
63.86 (C-5), 66.93 (CH2 Z), 70.36 (C-3), 72.15 (C-4), 99.43 (C-1), 99.73 (Cq isopro-
pylidene), 128.03, 128.18, 128.55 (CH-arom Z), 136.40 (Cq Z), 156.01 (C =O Z),
173.83 (C =O ester).
Methyl 2-amino-2-deoxy-4,6-O-isopropylidene-3-O-tetradecanoyl-a-D-glucopyrano-
side (18). To a solution of 17 (6.69 g, 11.6 mmol) in EtOAc (100 mL) was added
Pd/C (10%, 1.0 g). Hydrogen was passed through the stirred mixture for 66 h. TLC
analysis showed complete conversion of starting material into a new product with
Rf = 0.26 (MeOH/DCM, 1:99, v/v). The mixture was filtered over a PTFE filter. The
filtrate was concentrated under reduced pressure. Yield: 5.19 g (quantitative) of a

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
177
colorless oil. 1H NMR (CDCl3): d =0.88 (s, 3H, t, CH3 myristyl), 1.25 (br. s, 20H,
CH2 myristyl), 1.36 (s, 3H, CH3 isopropylidene), 1.45 (s, 3H, CH3 isopropylidene),
1.64 (m, 2H, CH2 myristyl), 1.76 (m, 2H, NH2), 2.34 (q, 2H, CH2 myristyl), 2.86
(dd, 1H, H-2, J1,2 =3.6 Hz, J2,3 =10.0 Hz), 3.39 (s, 3H, OMe), 3.56 (d, 1H, H-4), 3.72
(m, 2H, H-5, H-6), 3.87 (m, 1H, H-6’), 4.71 (d, 1H, H-1, J1,2 = 3.6 Hz), 5.05 (t, 1H,
H-3). 13C{1H} NMR (CDCl3): d =14.11 (CH3 myristyl), 19.09, 29.13 (2 ꢀ CH3
isopropylidene), 22.70–34.61 (CH2 myristyl), 55.29 (OMe), 55.54 (C-2), 62.64 (C-6),
63.83 (C-5), 72.50 (C-4), 73.76 (C-3), 99.52 (Cq isopropylidene), 101.37 (C-1), 173.63
(C =O ester).
Methyl 2-[(6-benzyloxycarbonylamino)hexanoylamino]-2-deoxy-4,6-O-isopropyli-
dene-3-O-tetradecanoyl-a-D-glucopyranoside (22). To a stirred mixture of 6-ben-
zyloxycarbonylaminohexanoic acid (19; 516 mg, 1.95 mmol), PyBOP (1.522 g, 2.93
mmol) and DiPEA (367 mL, 2.15 mmol) in DCM (50 mL) was added 18 (0.913 g, 2.06
mmol) in DCM (10 mL). After 1 h, TLC analysis indicated the complete conversion of
starting material into a product with Rf =0.57 (hexane/ethyl acetate, 1:2, v/v). The
reaction mixture was diluted with DCM (100 mL) and washed with water (3 ꢀ 50 mL).
The organic layer was dried (MgSO4) and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography. Elution was per-
1
formed with hexane/ethyl acetate (60:40 ! 40:60, v/v). Yield 1.172 g (87%). H NMR
(CDCl3): d= 0.88 (t, 3H, CH3 myristyl), 1.23–1.64 (m, 28H, CH2 hexanoyl, myristyl),
1.37 (s, 3H, CH3 isopropylidene), 1.47 (s, 3H, CH3 isopropylidene), 2.11–2.35 (m, 4H,
hexanoyl, myristyl), 3.17 (br. q, 2H, CH2 hexanoyl), 3.35 (s, 3H, OMe), 3.66–3.79 (m,
3H, H-4, H-5, H-6), 3.87 (dd, 1H, H-6’, J5,6’ = 5.0 Hz, J6,6’ = 10.5 Hz), 4.27 (m, 1H, H-2,
J
1,2 = 3.7 Hz, J2,3 = 9.5 Hz), 4.66 (d, 1H, H-1 J1,2 = 3.7 Hz), 4.95 (s, 1H, NH ami-
nohexanoyl), 5.09 (s, 2H, CH2 Z), 5.12 (t, 1H, H-3, J2,3 = 9.5 Hz, J3,4 =9.5 Hz), 5.88 (s,
1H, NH), 7.28–7.35 (m, 5H, CH-arom Z). 13C{1H} NMR (CDCl3): d=14.08 (CH3
myristyl), 22.66–40.82 (CH2 hexanoyl, myristyl), 19.06, 26.23 (2 ꢀ CH3 isopropyli-
dene), 52.51 (C-2), 55.23 (OMe), 62.43 (C-6), 63.73 (C-5), 66.55 (CH2 Z), 70.38 (C-3),
71.94 (C-4), 99.07 (C-1), 99.73 (Cq isopropylidene), 128.03, 128.48 (CH-arom Z),
136.72 (Cq Z), 156.21 (C= O Z), 172.82, 174.26 (2 ꢀ C= O amide, ester).
Methyl 2-[(12-benzyloxycarbonylamino)dodecanoylamino]-2-deoxy-4,6-O-isopropy-
lidene-3-O-tetradecanoyl-a-D-glucopyranoside (23). 12-Benzyloxycarbonylamino-
dodecanoic acid 20 (0.642 g, 1.84 mmol) was coupled with compound 18 (0.815 g,
1.84 mmol) as described for the preparation of compound 22. The crude product was
purified by silica gel column chromatography. Elution was performed with hexane/
1
ethyl acetate (90:10 !40:60, v/v). Yield 1.368 g (96%). H NMR (CDCl3): d =0.88 (t,
3H, CH3 myristyl), 1.25 (m, 34H, CH2 dodecanoyl, myristyl), 1.37 (s, 3H, CH3 iso-
propylidene), 1.46 (s, 3H, CH3 isopropylidene), 1.57 (m, 6H, CH2 dodecanoyl,
myristyl), 2.10–2.33 (m, 4H, dodecanoyl, myristyl), 3.17 (q, 2H, CH2 dodecanoyl),
3.36 (s, 3H, OMe), 3.69–3.79 (m, 3H, H-4, H-5, H-6), 3.87 (dd, 1H, H-6’), 4.27 (m,
1H, H-2), 4.67 (d, 1H, H-1, J1,2 =3.6 Hz), 4.82 (s, 1H, NH aminododecanoyl), 5.09 (s,
2H, CH2 Z), 5.13 (q, 1H, H-3), 5.82 (d, 1H, NH), 7.27–7.35 (m, 5H, CH-arom Z).
13C{1H} NMR (CDCl3): d= 14.11 (CH3 myristyl), 22.68–41.15 (CH2 dodecanoyl,
myristyl), 19.07, 29.07 (2 ꢀ CH3 isopropylidene), 52.49 (C-2), 55.25 (OMe), 62.46 (C-
6), 63.78 (C-5), 66.55 (CH2 Z), 70.28 (C-3), 71.97 (C-4), 99.13 (C-1), 99.74 (Cq

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178
VAN DEN BERG ET AL.
isopropylidene), 128.04, 128.49 (CH-arom Z), 136.5 (Cq Z), 156.5 (C =O Z), 173.14,
174.23 (2 ꢀ C = O amide, ester).
Methyl 2-[(R)-3-(6-benzyloxycarbonylamino)hexanoyloxytetradecanoylamino]-2-
deoxy-4,6-O-isopropylidene-3-O-tetradecanoyl-a-D-glucopyranoside (24). (R)-3-(6-
Benzyloxycarbonylamino)hexanoyloxytetradecanoic acid 21 (0.516 g, 1.95 mmol) was
coupled with compound 18 (0.913 g, 2.06 mmol) as described for the preparation of
compound 22. The crude product was purified by silica gel column chromatography.
Elution was performed with hexane/ethyl acetate (80:20 !30:70, v/v). Yield 1.172 g
1
(87%). H NMR (CDCl3): d =0.88 (t, 6H, CH3 acyloxyacyl, myristyl), 1.25–1.67 (m,
48H, CH2 acyloxyacyl, myristyl), 1.36 (s, 3H, CH3 isopropylidene), 1.46 (s, 3H, CH3
isopropylidene), 2.24–2.45 (m, 6H, acyloxyacyl, myristyl), 3.17 (q, 2H, CH2 hexanoyl),
3.34 (s, 3H, OMe), 3.66–3.78 (m, 3H, H-4, H-5, H-6), 3.87 (dd, 1H, H-6’), 4.24 (m,
1H, H-2), 4.66 (d, 1H, H-1, J1,2 = 3.7 Hz), 4.92 (s, 1H, NH aminohexanoyl), 5.09 (m,
4H, CH2 Z, H-3, CHO acyloxyacyl), 5.97 (d, 1H, NH), 7.27–7.35 (m, 5H, CH-arom
Z). 13C{1H} NMR (CDCl3): d=14.12 (CH3 myristyl), 22.71–41.29 (CH2 acyloxyacyl,
myristyl), 19.10, 29.09 (2 ꢀ CH3 isopropylidene), 52.59 (C-2), 55.22 (OMe), 62.47 (C-
6), 63.79 (C-5), 66.59 (CH2 Z), 70.27 (C-3), 71.09 (CHO acyloxyacyl), 72.05 (C-4),
98.98 (C-1), 99.77 (Cq isopropylidene), 128.05, 128.52 (CH-arom Z), 156.45 (C= O Z),
169.62, 172.82, 174.26 (3 ꢀ C = O amide, ester).
Methyl 2-[(6-benzyloxycarbonylamino)hexanoylamino]-2-deoxy-3-O-tetradecanoyl-
a-D-glucopyranoside (25). To a stirred solution of 22 (0.801 g, 1.16 mmol) in
THF/water (4:1, v/v; 25 mL) at 0°C was added TFA (1 mL). The resulting solution was
allowed to warm to room temperature and left overnight. TLC analysis (ethyl acetate/
hexane, 2:1, v/v) showed complete conversion of starting material into a compound
with Rf =0.13. The reaction mixture was concentrated under reduced pressure. The
residue was diluted with diethyl ether (100 mL) and washed with water (3 ꢀ 50 mL).
The organic layer was dried over Na2SO4, and the solvent was removed under reduced
pressure. Purification of the residue by silica gel column chromatography with DCM/
ethanol (100:0 ! 93:7, v/v) yielded the desired diol 25 as a colorless oil (0.747 g;
1
99%). H NMR (CDCl3): d= 0.88 (t, 3H, CH3 myristyl), 1.24 (m, 22H, CH2 hexanoyl,
myristyl), 1.54 (m, 6H, CH2 hexanoyl, myristyl), 2.13 (t, 2H, CH2 hexanoyl, myristyl),
2.30 (m, 2H, CH2 hexanoyl, myristyl), 3.17 (q, 2H, CH2 hexanoyl), 3.37 (s, 3H, OMe),
3.65 (m, 1H, H-5, J4,5 =9.7 Hz), 3.78 (t, 1H, H-4, J3,4 =9.5 Hz, J4,5 = 9.5 Hz), 3.86 (s,
2H, H2-6), 4.20 (m, 1H, H-2, J1,2 = 3.4 Hz, J2,NH = 9.5 Hz, J2,3 =10.5 Hz), 4.70 (d, 1H,
H-1, J1,2 =3.4 Hz), 4.99 (br. s, 1H, NH aminohexanoyl), 5.08 (br. s, 2H, CH2 Z), 5.10
(t, 1H, H-3, J2,3 =10.5 Hz, J3,4 = 9.5 Hz), 5.99 (d, 1H, NH, J2,NH =9.2 Hz), 7.27–7.36
(m, 5H, CH-arom Z). 13C{1H} NMR (CDCl3): d= 14.11 (CH3 myristyl), 22.71–40.83
(CH2 hexanoyl, myristyl), 52.04 (C-2), 55.27 (OMe), 61.98 (C-6), 66.66 (CH2 Z), 68.93
(C-4), 71.52 (C-5), 73.78 (C-3), 98.49 (C-1), 128.11, 128.53 (CH-arom Z), 136.68 (Cq
Z), 156.55 (C =O Z), 173.14, 175.22 (2 ꢀ C = O amide, ester).
Methyl 2-[(12-benzyloxycarbonylamino)dodecanoylamino]-2-deoxy-3-O-tetradeca-
noyl-a-D-glucopyranoside (26). Acetonide 23 (1.368 g, 1.77 mmol) was treated with
TFA as was described for the preparation of compound 25. Silica gel column chro-
matography with DCM/ethanol (100:0 ! 94:6, v/v) of the residue yielded the desired

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
179
1
diol as a white solid (0.816 g; 63%). H NMR (CDCl3): d=0.88 (t, 3H, CH3 myristyl),
1.25 (m, 34H, CH2 dodecanoyl, myristyl), 1.48 (br. t, 2H, CH2 dodecanoyl, myristyl)
1.56 (m, 4H, CH2 dodecanoyl, myristyl), 2.12 (m, 2H, CH2 dodecanoyl, myristyl), 2.31
(m, 2H, CH2 dodecanoyl, myristyl), 2.95 (br. s, 2H, 2 ꢀ OH) 3.17 (q, 2H, CH2 dode-
canoyl), 3.38 (s, 3H, OMe), 3.67 (m, 1H, H-5, J4,5 = 9.7 Hz), 3.77 (t, 1H, H-4, J3,4 =9.3,
J
J
4,5 = 9.5 Hz), 3.86 (d, 2H, H2-6), 4.21 (m, 1H, H-2, J1,2 = 3.6 Hz, J2,NH =9.4 Hz,
2,3 = 9.4 Hz), 4.69 (d, 1H, H-1, J1,2 =3.6 Hz), 4.78 (br. s, 1H, NH aminohexanoyl), 5.09
(br. s, 3H, CH2 Z, H-3), 5.83 (d, 1H, NH, J2,NH =9.3 Hz), 7.27–7.36 (m, 5H, CH-arom
Z). 13C{1H} NMR (CDCl3): d= 14.17 (CH3 myristyl), 22.75–36.78 (CH2 dodecanoyl,
myristyl), 41.20 (NCH2) 51.92 (C-2), 55.31 (OMe), 62.22 (C-6), 66.66 (CH2 Z), 69.21
(C-4), 71.45 (C-5), 73.82 (C-3), 98.53 (C-1), 126.81, 128.13, 128.72 (CH-arom Z),
136.72 (Cq Z), 156.49 (C= O Z), 173.34, 175.23 (2 ꢀ C= O amide, ester).
Methyl 2-[(R)-3-(6-benzyloxycarbonylamino)hexanoyloxytetradecanoylamino]-2-
deoxy-3-O-tetradecanoyl-a-D-glucopyranoside (27). To a cooled (0°C) solution of
24 (138 mg, 0.151 mmol) in DCM (1 mL) was added TFA (0.5 mL). After stirring for
2 h, TLC analysis indicated the complete conversion of starting material into a product
with Rf = 0.27 (ethyl acetate/hexane, 2:1, v/v). The reaction mixture was concentrated
and coevaporated with toluene (2 ꢀ 2 mL). Silica gel column chromatography with
hexane/ethyl acetate (50:50 !85:15, v/v) of the residue yielded the desired diol as a
white solid (115 mg; 87%). 1H NMR (CDCl3): d= 0.88 (t, 6H, CH3 acyloxyacyl,
myristyl), 1.25–1.66 (m, 40H, CH2 acyloxyacyl, myristyl), 1.51–1.66 (m, 8H, CH2
acyloxyacyl, myristyl), 2.28–2.44 (m, 6H, CH2, acyloxyacyl, myristyl), 2.68 (br. s, 1H,
OH-6), 3.18 (q, 2H, CH2 hexanoyl), 3.35 (s, 3H, OMe), 3.49 (br. s, 1H, OH-4), 3.64
(m, 1H, H-5), 3.74 (t, 1H, H-4), 3.84 (d, 2H, H2-6), 4.16 (m, 1H, H-2), 4.67 (d, 1H, H-
1, J1,2 =3.5 Hz), 5.02 (s, 1H, NH aminohexanoyl), 5.09 (m, 4H, CH2 Z, H-3, CHO
acyloxyacyl), 6.12 (d, 1H, NH), 7.28–7.35 (m, 5H, CH-arom Z). 13C{1H} NMR
(CDCl3): d =14.09 (CH3 myristyl), 22.69–41.31 (CH2 acyloxyacyl, myristyl), 52.06 (C-
2), 55.20 (OMe), 62.08 (C-6), 66.63 (CH2 Z), 69.14 (C-4), 71.14 (CHO acyloxyacyl),
71.55 (C-5), 73.66 (C-3), 98.35 (C-1), 128.03, 128.06, 128.51 (CH-arom Z), 136.55 (Cq
Z), 156.55 (C= O Z), 169.80, 172.81, 175.13 (3 ꢀ C = O amide, ester).
Methyl 2-[(6-benzyloxycarbonylamino)hexanoylamino]-2,6-dideoxy-6-O-mesyl-3-O-
tetradecanoyl-a-D-glucopyranoside (28). To a stirred solution of 25 (301 mg, 0.463
mmol) in dry pyridine (10 mL) was added mesyl chloride (1.5 equivalents, 60 mL).
After 24 h, methanol (2 mL) was added and the mixture was stirred for 0.5 h, after
which the mixture was concentrated. The residue was dissolved in DCM (50 mL) and
washed with water (1 ꢀ 25 mL). The organic layer was dried (MgSO4) and con-
centrated under reduced pressure. The crude product was purified by silica gel column
chromatography. Elution was performed with hexane/ethyl acetate (70:30 ! 20:80, v/v).
1
Rf =0.43 (hexane/ethyl acetate, 1:2, v/v). Yield 269 mg (80%). H NMR (CDCl3):
d =0.88 (t, 3H, CH3 myristyl), 1.25 (m, 24H, CH2 hexanoyl, myristyl), 1.45–1.60 (m,
4H, CH2 hexanoyl, myristyl), 2.13 (t, 2H, CH2 hexanoyl, myristyl), 2.31 (m, 2H, CH2
hexanoyl, myristyl), 3.05 (s, 3H, CH3 Ms), 3.15 (q, 2H, NCH2 hexanoyl), 3.39 (s, 3H,
OMe), 3.69 (m, 1H, H-4), 3.81 (br. d, 1H, OH-4), 3.86 (m, 1H, H-5), 4.23 (m, 1H, H-
2), 4.50 (q, 2H, H2-6), 4.70 (d, 1H, H-1, J1,2 = 3.6 Hz), 5.08 (m, 4H, H-3, CH2 Z, NH
aminohexanoyl), 5.99 (d, 1H, NH), 7.28–7.36 (m, 5H, CH-arom Z). 13C{1H} NMR

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180
VAN DEN BERG ET AL.
(CDCl3): d =14.02 (CH3 myristyl), 22.58–36.16 (CH2 hexanoyl, myristyl), 37.41 (CH3
Ms), 40.70 (CH2N), 51.60 (C-2), 55.40 (OMe), 66.45 (CH2 Z), 68.14 (C-4), 68.59 (C-
6), 69.77 (C-5), 73.38 (C-3), 98.43 (C-1), 127.92–128.43 (CH-arom Z), 136.58 (Cq Z),
156.42 (C =O Z), 172.87, 174.92 (2 ꢀ C =O amide, ester).
Methyl 2-[(12-benzyloxycarbonylamino)dodecanoylamino]-2-deoxy-6-O-mesyl-3-O-
tetradecanoyl-a-D-glucopyranoside (29). Compound 29 (1.175 g, 1.60 mmol) was
prepared as described for compound 28. Rf =0.68 (hexane/ethyl acetate, 1:2, v/v). Yield
1
1.01 g (78%). H NMR (CDCl3): d= 0.88 (t, 3H, CH3 myristyl), 1.25 (m, 34H, CH2
dodecanoyl, myristyl), 1.42–1.65 (m, 6H, CH2 dodecanoyl, myristyl), 2.12 (t, 2H, CH2
dodecanoyl, myristyl), 2.32 (m, 2H, CH2 dodecanoyl, myristyl), 3.07 (s, 3H, CH3 Ms),
3.17 (q, 2H, NCH2 dodecanoyl), 3.31 (br. d, 1H, OH-4), 3.39 (s, 3H, OMe), 3.70 (m,
1H, H-4, J3,4 =9.3 Hz, J4,5 =9.7 Hz), 3.86 (m, 1H, H-5, J4,5 = 9.9 Hz, J5,6 =6.7 Hz,
J
5,6’ =3.3 Hz), 4.23 (m, 1H, H-2, J1,2 3.6 Hz, J2,3 =9.4 Hz), 4.51 (d, 2H, H2-6), 4.70 (d,
1H, H-1, J1,2 =3.6 Hz), 4.82 (br. s, 1H, NH aminohexanoyl) 5.09 (dd 3H, H-3, CH2 Z,),
5.81 (d, 1H, NH, J2,NH = 8.9 Hz), 7.27–7.35 (m, 5H, CH-arom Z). 13C{1H} NMR
(CDCl3): d= 14.12 (CH3 myristyl), 22.69–36.67 (CH2 dodecanoyl, myristyl), 37.59
(CH3 Ms), 41.14 (CH2N), 51.63 (C-2), 55.52 (OMe), 66.57 (CH2 Z), 68.29 (C-4), 68.45
(C-6), 69.87 (C-5), 73.44 (C-3), 98.60 (C-1), 128.06, 128.50 (CH-arom Z), 136.70 (Cq
Z), 156.43 (C =O Z), 173.12, 175.07 (2 ꢀ C = O amide, ester).
Methyl 2-[(R)-3-(6-benzyloxycarbonylamino)hexanoyloxytetradecanoylamino]-2-
deoxy-3-O-tetradecanoyl-6-O-tosyl-a-D-glucopyranoside (30). Compound 27 (115
mg, 0.131 mmol) was treated with tosyl chloride (138 mg, 0.724 mmol) in pyridine (5
mL). After 96 h, methanol (2 mL) was added and the mixture was concentrated.
Further work-up as described for compound 28. The crude product was purified by
silica gel column chromatography. Elution was performed with hexane/ethyl acetate
1
(60:40 ! 35:65, v/v). Rf = 0.84 (hexane/ethyl acetate, 1:2, v/v). Yield 107 mg (79%). H
NMR (CDCl3): d=0.88 (t, 6H, CH3 acyloxyacyl, myristyl), 1.25–1.49 (m, 40H, CH2
acyloxyacyl, myristyl), 1.53 (m, 8H, CH2 acyloxyacyl, myristyl), 2.34 (m, 6H, acyl-
oxyacyl, myristyl), 2.44 (s, 3H, CH3 Ts), 3.13 (s, 1H, OH-4), 3.18 (q, 2H, CH2
hexanoyl), 3.29 (s, 3H, OMe), 3.59 (m, 1H, H-4), 3.79 (m, 1H, H-5), 4.14 (m, 1H, H-
2), 4.30 (d, 2H, H2-6), 4.59 (d, 1H, H-1, J1,2 =3.6 Hz), 4.94 (t, 1H, NH aminohexa-
noyl), 5.04 (dd, 1H, H-3), 5.07 (s, 2H, CH2 Z) 5.09 (m, 1H, CHO acyloxyacyl), 5.99
(d, 1H, NH), 7.27–7.80 (m, 9H, CH-arom Ts/Z). 13C{1H} NMR (CDCl3): d= 14.11
(CH3 acyloxyacyl), 21.65 (CH3 Ts), 22.69–41.30 (CH2 acyloxyacyl, myristyl), 51.72
(C-2), 55.35 (OMe), 66.63 (CH2 Z), 68.60 (C-4), 68.80 (C-6), 69.84 (C-5), 71.13 (CHO
acyloxyacyl), 73.43 (C-3), 98.22 (C-1), 127.76–129.84 (CH-arom Z), 133.01, 136.68,
144.98 (Cq Ts, Z), 156.52 (C= O Z), 169.62, 172.76, 175.07 (3 ꢀ C= O amide, ester).
Methyl 6-azido-2-[(6-benzyloxycarbonylamino)hexanoylamino]-2,6-dideoxy-3-O-
tetradecanoyl-a-D-glucopyranoside (31). To a solution of 28 (269 mg, 0.369 mmol)
in DMF (10 mL) was added sodium azide (36 mg, 0.55 mmol). After stirring for 4 h at
70°C, TLC analysis indicated the complete conversion of starting material. The mixture
was concentrated, diluted with DCM (50 mL) and washed with water (20 mL). The
organic layer was dried (MgSO4) and concentrated under reduced pressure. The crude

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
181
product was purified by silica gel column chromatography. Elution was performed with
hexane/ethyl acetate (55:45 ! 30:70, v/v). Rf = 0.76 (hexane/ethyl acetate, 1:2, v/v).
1
Yield 211 mg (84%). H NMR (CDCl3): d= 0.88 (t, 3H, CH3 myristyl), 1.25 (m, 22H,
CH2 hexanoyl, myristyl), 1.54 (m, 6H, CH2 hexanoyl, myristyl), 2.12 (t, 2H, CH2
hexanoyl, myristyl), 2.29 (m, 2H, CH2 hexanoyl, myristyl), 3.17 (q, 2H, CH2 hex-
anoyl), 3.41 (s, 3H, OMe), 3.46 (dd, 1H, H-6, J5,6 =6.2 Hz, J6,6’ = 13.2 Hz), 3.57 (dd,
1H, H-6’, J5,6’ =2.5 Hz, J6,6’ =13.2 Hz), 3.63 (t, 1H, H-4, J3,4 =9.3 Hz, J4,5 = 9.4 Hz),
3.79 (m, 1H, H-5, J4,5 =9.4 Hz, J5,6 = 6.2 Hz, J5,6’ =2.5 Hz), 4.25 (m, 1H, H-2, J1,2 =3.6
Hz, J2,3 =10.7 Hz, J2,NH = 9.5 Hz), 4.70 (d, 1H, H-1 J1,2 = 3.6 Hz), 4.92 (br. s, 1H, NH
aminohexanoyl), 5.04 (dd, 1H, H-3, J2,3 = 10.8 Hz, J3,4 =9.1 Hz), 5.08 (s, 2H, CH2 Z),
5.87 (d, 1H, NH, J2,NH = 9.4 Hz), 7.27–7.35 (m, 5H, CH-arom Z). 13C{1H} NMR
(CDCl3): d =14.10 (CH3 myristyl), 22.68–40.83 (CH2 hexanoyl, myristyl), 51.42 (C-6),
51.64 (C-2), 55.40 (OMe), 66.63 (CH2 Z), 69.76 (C-4), 71.15 (C-5), 73.97 (C-3), 98.44
(C-1), 128.05, 128.09, 128.52 (CH-arom Z), 136.67 (Cq Z), 156.49 (C=O Z), 172.73,
175.28 (2 ꢀ C =O amide, ester).
Methyl 6-azido-2-[(12-benzyloxycarbonylamino)dodecanoylamino]-2,6-dideoxy-3-
O-tetradecanoyl-a-D-glucopyranoside (32). Compound 29 (601 mg, 0.738 mmol)
was treated with sodium azide as described for the preparation of compound 31.
Rf =0.60 (hexane/ethyl acetate, 1:2, v/v). Yield 404 mg (72%). 1H NMR (CDCl3):
d =0.88 (t, 3H, CH3 myristyl), 1.25 (m, 34H, CH2 dodecanoyl, myristyl), 1.51 (m, 6H,
CH2 dodecanoyl, myristyl), 2.12 (m, 2H, CH2 dodecanoyl, myristyl), 2.30 (m, 2H, CH2
dodecanoyl, myristyl), 3.16 (q, 2H, NCH2 dodecanoyl), 3.42 (s, 3H, OMe), 3.46 (m,
2H, H-6, OH-4), 3.57 (dd, 1H, H-6’), 3.62 (m, 1H, H-4), 3.80 (m, 1H, H-5), 4.25 (m,
1H, H-2), 4.71 (d, 1H, H-1, J1,2 = 3.7 Hz), 4.89 (br. s, 1H, NH dodecanoylamino), 5.05
(dd, 1H, H-3), 5.08 (br. s, 2H, CH2 Z), 5.87 (d, 1H, NH J2,NH = 9.4 Hz), 7.28–7.35 (m,
5H, CH-arom Z). 13C{1H} NMR (CDCl3): d =14.07 (CH3), 22.64–41.08 (CH2
dodecanoyl, myristyl), 51.37 (C-6), 51.57 (C-2), 55.32 (OMe), 66.51 (CH2 Z), 69.62
(C-4), 71.16 (C-5), 73.75 (C-3), 98.40 (C-1), 127.99, 128.44 (CH-arom Z), 136.64 (Cq
Z), 156.42 (C= O Z), 173.06, 175.21 (2 ꢀ C =O amide, ester).
Methyl 6-azido-2-[(R)-3-(6-benzyloxycarbonylamino)hexanoyloxytetra-decanoyla-
mino]-2,6-dideoxy-3-O-tetradecanoyl-a-D-glucopyranoside (33). Compound 30
(107 mg, 0.103 mmol) was treated with sodium azide as described for the preparation
of compound 31. Rf =0.26 (hexane/ethyl acetate, 2:1, v/v). Yield 68 mg (73%). 1H
NMR (CDCl3): d =0.88 (t, 6H, CH3 acyloxyacyl, myristyl), 1.25 (m, 40H, CH2
acyloxyacyl, myristyl), 1.56 (m, 8H, CH2 acyloxyacyl, myristyl), 2.35 (m, 6H, CH2
acyloxyacyl, myristyl), 3.05 (d, 1H, OH-4), 3.20 (t, 2H, hexanoyl), 3.39 (s, 3H, OMe),
3.45 (dd, 1H, H-6), 3.56 (dd, 1H, H-6’), 3.61 (t, 1H, H-4), 3.79 (m, 1H, H-5), 4.22 (m,
1H, H-2), 4.70 (d, 1H, H-1, J1,2 =3.6 Hz), 4.90 (br. s, 1H, NH aminohexanoyl), 5.08
(m, 4H, CH2 Z, H-3, CHO acyloxyacyl), 6.02 (d, 1H, NH), 7.27–7.35 (m, 5H, CH-
arom Z). 13C{1H} NMR (CDCl3): d=14.10 (CH3 acyloxyacyl), 22.71–41.31 (CH2
acyloxyacyl, myristyl), 51.40 (C-6), 51.72 (C-2), 55.39 (OMe), 66.66 (CH2 Z), 69.78
(C-4), 69.89 (C-5), 71.15 (CHO acyloxyacyl), 73.75 (C-3), 98.28 (C-1), 128.06, 128.12,
128.54 (CH-arom Z), 136.62 (Cq Z), 156.5 (C =O Z), 169.66, 172.82, 175.26
(3 ꢀ C =O amide, ester).

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182
VAN DEN BERG ET AL.
Methyl 6-amino-2-[(6-benzyloxycarbonylamino)hexanoylamino]-2,6-dideoxy-3-O-
tetradecanoyl-a-D-glucopyranoside (34). To a solution of 31 (491 mg, 0.727 mmol)
in THF (73 mL) was added triphenylphosphine (286 mg, 1.09 mmol) and water (16 mL,
0.871 mmol). The mixture was refluxed for 3.5 h, after which TLC analysis indicated
the complete conversion of starting material into baseline material (hexane/ethyl ace-
tate, 1:2, v/v). The reaction mixture was concentrated and the crude product was
purified by silica gel column chromatography. Elution was performed with MeOH/
DCM/TEA (10:89:1 !14:85:1, v/v/v). Yield 497 mg (100%). 1H NMR (CDCl3):
d= 0.88 (t, 3H, CH3 myristyl), 1.25 (m, 22H, CH2 hexanoyl, myristyl), 1.54 (m, 6H,
CH2 hexanoyl, myristyl), 2.12 (t, 2H, CH2 hexanoyl, myristyl), 2.30 (m, 2H, CH2
hexanoyl, myristyl), 2.77 (br. s, 3H, NH2-6, OH-4), 3.03 (br. s, 2H, H2-6), 3.17 (q, 2H,
CH2 hexanoyl), 3.36 (s, 3H, OMe), 3.60 (m, 1H, H-5), 3.64 (t, 1H, H-4, J3,4 =8.6 Hz,
J
4,5 =8.6 Hz), 4.18 (m, 1H, H-2, J1,2 =3.6 Hz, J2,3 = 10.8 Hz, J2,NH =9.3 Hz), 4.65 (d,
1H, H-1 J1,2 =3.6 Hz), 4.95 (br. s, 1H, NH aminohexanoyl), 5.08 (m, 3H, CH2 Z, H-3,
2,3 =9.7 Hz, J3,4 =9.7 Hz), 5.87 (d, 1H, NH, J2,NH =9.3 Hz), 7.28–7.36 (m, 5H, CH-
J
arom Z). 13C{1H} NMR (CDCl3): d=14.11 (CH3 myristyl), 22.69–40.86 (CH2 hexa-
noyl, myristyl), 43.74 (C-6), 51.93 (C-2), 55.17 (OMe), 66.59 (CH2 Z), 71.09 (C-4),
71.34 (C-5), 73.71 (C-3), 98.43 (C-1), 128.06–128.60 (CH-arom Z), 136.74 (Cq Z),
156.45 (C =O Z), 172.69, 174.99 (2 ꢀ C =O amide, ester).
Methyl 6-amino-2-[(12-benzyloxycarbonylamino)dodecanoylamino]-2,6-dideoxy-3-
O-tetradecanoyl-a-D-glucopyranoside (35). Compound 32 (914 mg, 1.20 mmol)
was treated with triphenylphosphine as described for the preparation of compound
34. Rf =0.43 (MeOH/DCM, 1:9, v/v). The crude product was purified by silica gel
column chromatography. Elution was performed with MeOH/DCM/TEA (0:99.5:0.5 !
10:89.5:0.5, v/v/v). Yield 698 mg (78%). 1H NMR (CDCl3): d= 0.88 (t, 3H, CH3
myristyl), 1.25 (m, 34H, CH2 hexanoyl, myristyl), 1.52 (m, 6H, CH2 hexanoyl,
myristyl), 2.09 (m, 2H, CH2 hexanoyl, myristyl), 2.31 (m, 2H, CH2 hexanoyl, myristyl),
2.80 (br. s, 3H, NH2-6, OH-4), 3.03 (br. d, 2H, H2-6), 3.17 (q, 2H, CH2 hexanoyl), 3.37
(s, 3H, OMe), 3.55–3.67 (m, 2H, H-4, H-5), 4.19 (m, 1H, H-2), 4.66 (d, 1H, H-1,
J
1,2 =3.6 Hz), 4.83 (s, 1H, NH aminohexanoyl), 5.09 (br. t, 3H, H-3, CH2 Z), 5.81 (d,
1H, NH), 7.28–7.36 (m, 5H, CH-arom Z). 13C{1H} NMR (CDCl3): d =14.12 (CH3
myristyl), 22.70–41.14 (CH2 hexanoyl, myristyl), 43.59 (C-6), 51.86 (C-2), 55.19
(OMe), 66.55 (CH2 Z), 71.00 (C-4), 71.17 (C-5), 73.55 (C-3), 98.45 (C-1), 128.05,
128.50 (CH-arom Z), 136.74 (Cq Z), 156.43 (C= O Z), 173.05, 174.95 (2 ꢀ C = O
amide, ester).
Methyl 6-amino-2-[(R)-3-(6-benzyloxycarbonylamino)hexanoyloxy-tetradecanoyl-
amino-2,6-dideoxy-3-O-tetradecanoyl-a-D-glucopyranoside (36). Compound 36
was prepared as described for compound 34. The crude product was purified by silica
gel column chromatography. Elution was performed with MeOH/DCM/TEA (0:99:1 !
9:90:1, v/v/v). Yield 35 mg (52%). 1H NMR (CDCl3): d =0.88 (t, 6H, CH3 acyl-
oxyacyl, myristyl), 1.25 (m, 40H, CH2 acyloxyacyl, myristyl), 1.55 (m, 8H, CH2 acyl-
oxyacyl, myristyl), 2.35 (m, 6H, CH2 acyloxyacyl, myristyl), 3.04–3.33 (m, 7H, OH-4,
NH2-6, H2-6, NCH2 hexanoyl), 3.35 (s, 3H, OMe), 3.62–3.66 (m, 2H, H-4, H-5), 4.18
(m, 1H, H-2), 4.65 (d, 1H, H-1, J1,2 =3.5 Hz), 4.95 (br. s, 1H, NH aminohexanoyl),
5.08 (m, 4H, CH2 Z, H-3, CHO acyloxyacyl), 6.00 (d, 1H, NH), 7.27–7.36 (m, 5H,

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
183
CH-arom Z). 13C{1H} NMR (CDCl3): d =14.17 (CH3 acyloxyacyl, myristyl), 22.74–
41.34 (CH2 acyloxyacyl, myristyl), 43.52 (C-6), 52.00 (C-2), 55.29 (OMe), 66.64 (CH2
Z), 70.43 (C-4), 71.15 (CHO acyloxyacyl), 71.49 (C-5), 73.32 (C-3), 98.31 (C-1),
128.11, 128.56 (CH-arom Z), 136.74 (Cq Z), 156.5 (C =O Z), 169.71, 172.79, 174.98
(3 ꢀ C =O amide, ester).
Compound 37. To a stirred mixture of 11 (30 mg, 0.428 mmol), PyBOP (25 mg,
0.480 mmol) and DiPEA (8.1 mL, 0.480 mmol) in DCM (3.6 mL) a solution of amino
sugar 34 (28 mg, 0.431 mmol) in DCM (2.8 mL) was added. After 1 h, TLC analysis
indicated the complete conversion of starting material into a compound with Rf =0.62
(MeOH/DCM, 7:93, v/v). The reaction mixture was diluted with DCM (50 mL) and
washed with water (1 ꢀ 20 mL). After drying over MgSO4, the organic layer was
concentrated under reduced pressure. The crude product was purified by silica gel
column chromatography. Elution was performed with hexane/ethyl acetate (40:60 !
100:0, v/v). Yield 55 mg (96%). 1H NMR (CDCl3): d= 0.88 (m, 6H, CH3 my-
ristyl), 1.25 (br. s, 42H, CH2 hexanoyl, myristyl), 1.52 (m, 8H, CH2 hexanoyl, my-
ristyl), 1.94 (m, 3H, CH2 hexanoyl, OH-4), 2.11 (t, 2H, CH2 hexanoyl), 2.27 (m, 2H,
OCOCH2), 2.41 (dd, 1H, H-1’ax, J1’ax,1’eq = 12.5 Hz, J1’ax,2’ =5.5 Hz), 2.90 (m, 1H, H-
6a), 3.04 (br. s, 1H, H-5’), 3.10 (dd, 1H, H-1’eq, J1’ax,1’eq =12.5 Hz, J1’eq,2’ =3.0 Hz),
3.17 (q, 2H, NCH2 hexanoyl), 3.29 (s, 3H, OMe), 3.39 (m, 3H, H-4, NCH2 acetyl),
3.52 (m, 1H, H-6’a), 3.55 (m, 1H, H-3’), 3.59 (m, 1H, H-5), 3.67 (t, 1H, H-4’), 3.83 (dd,
1H, H-6’b), 4.02 (m, 2H, H-2’, H-6b), 4.12 (m, 1H, H-2), 4.45–4.67 (m, 7H, 3 ꢀ CH2
Bn, H-1), 4.82 (br. s, 1H, NHCOO), 5.09 (s, 2H, CH2 Z), 5.11 (t, 1H, H-3), 5.77 (d,
1H, NH-2), 6.35 (br. d, 1H, NH-2’), 7.25–7.35 (m, 20H, CH-arom Bn/Z), 7.82 (m, 1H,
NH-6). 13C{1H} NMR (CDCl3): d =14.14 (CH3 2ꢀ myristyl), 22.72–38.84 (CH2
hexanoyl, myristyl), 39.85 (C-6), 40.91 (NCH2 hexanoyl), 47.56 (C-2’), 50.78 (C-1’)
52.55 (C-2), 55.19 (OMe), 57.69 (CH2 acetyl), 62.22 (C-5’), 66.44 (C-6’), 66.62 (CH2
Z), 69.23 (C-4), 70.84 (C-5), 72.10 (C-3), 72.82, 73.35, 73.50 (3 ꢀ CH2 Bn), 76.93 (C-
3’), 77.28 (C-4’), 98.62 (C-1), 127.80–128.64 (CH-arom Bn, Z), 136.75, 137.66,
137.83, 137.86 (4 ꢀ Cq Bn, Z), 156.75 (C= O Z), 172.59, 172.76, 173.18, 174.65
(4 ꢀ C =O amide, ester). ES-MS; m/z: 1332.9, [M +H]+; monoisotopic MW calculated
for C78H117N5O13 =1331.86.
Compound 38. Compound 11 (22 mg, 31.4 mmol) was coupled with compound 35
(24 mg, 32.7 mmol) as described for the preparation of compound 37. Rf =0.53 (MeOH/
DCM, 5:95, v/v). Yield 42.8 mg (96%). 1H NMR (CDCl3): d =0.88 (m, 6H, CH3
myristyl), 1.25 (br. s, 52H, CH2 dodecanoyl, myristyl), 1.46 (m, 4H, CH2 dodecanoyl,
myristyl), 1.55 (m, 8H, CH2 dodecanoyl, myristyl), 1.94 (q, 1H, OH-4), 2.10 (m, 2H,
CH2 dodecanoyl), 2.28 (m, 2H, OCOCH2), 2.44 (dd, 1H, H-1’ax, J1’ax,1’eq = 12.4 Hz,
J
1’ax,2’ =5.2 Hz), 2.90 (m, 1H, H-6a, J6a,6b =12.4 Hz), 3.05 (br. s, 1H, H-5’), 3.10 (dd,
1H, H-1’eq, J1’ax,1’eq = 12.5 Hz, J1’eq,2’ =3.1 Hz), 3.18 (q, 2H, NCH2 dodecanoyl), 3.29
(s, 3H, OMe), 3.38 (m, 3H, H-4, NCH2 acetyl), 3.52 (s, 1H, H-3’), 3.55 (m, 1H, H-6’a,
J
5’,6’a =3.3 Hz, J6a’6b’ =10.3 Hz), 3.60 (br. d, 1H, H-5, J4,5 = 9.6 Hz), 3.68 (t, 1H, H-4’),
3.84 (dd, 1H, H-6’b, J5,6’b =6.2 Hz, J6’a,6’b = 10.2 Hz), 4.02 (m, 1H, H-6b), 4.07 (m, 1H,
H-2’), 4.11 (m, 1H, H-2), 4.45–4.71 (m, 8H, H-1, NHCOO, 3 ꢀ CH2 Bn, H-1), 5.09 (s,
2H, CH2 Z), 5.12 (t, 1H, H-3, J= 10.4 Hz), 5.73 (d, 1H, NH-2), 6.36 (br. d, 1H, NH-2’),
7.22–7.35 (m, 20H, CH-arom Bn, Z), 7.85 (m, 1H, NH-6). 13C{1H} NMR (CDCl3):

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VAN DEN BERG ET AL.
d= 14.20 (CH3 dodecanoyl, myristyl), 22.78–36.88 (CH2 dodecanoyl, myristyl), 39.85
(C-6), 41.24 (NCH2 dodecanoyl), 47 (C-2’), 50 (C-1’) 52.56 (C-2), 55.25 (OMe), 57.78
(CH2 acetyl), 62 (C-5’), 66.47 (C-6’), 66.66 (CH2 Z), 69.20 (C-4), 70.94 (C-5), 71.93
(C-3), 72.86, 73.39, 73.52 (3 ꢀ CH2 Bn), 77.30 (C-3’, C-4’), 98.69 (C-1), 127.86–
128.71 (CH-arom Bn/Z), 136.80, 137.87 (Cq Bn, Z), 156 (C= O Z), 173.04, 173.30,
174.69, (C =O amide, ester). ES-MS; m/z: 1416.85, [M + H]+; monoisotopic MW
calculated for C84H129-N5O13 =1415.96.
Compound 39. Compound 11 (10 mg, 14.3 mmol) was coupled with compound 36
(13.7 mg, 15,7 mmol) as described for the preparation of compound 37. Rf =0.86
1
(MeOH/DCM, 5:95, v/v). Yield 11.4 mg (51%). H NMR (CDCl3): d =0.88 (t, 9H,
CH3 acyloxyacyl, myristyl), 1.25 (m, 60H, CH2 acyloxyacyl, myristyl), 1.52 (m, 10H,
CH2 acyloxyacyl, myristyl), 1.94 (m, 3H, OH-4, CH2 myristyl), 2.30 (m, 6H, NCH2
hexanoyl, OCOCH2), 2.42 (dd, 1H, H-1’ax), 2.88 (m, 1H, H-6a), 3.06 (br. s, 1H, H-5’),
3.11 (dd, 1H, H-1’eq), 3.18 (q, 2H, NCH2 hexanoyl), 3.27 (s, 3H, OMe), 3.35 (m, 1H,
H-4), 3.40 (s, 2H, NCH2 acetyl), 3.53 (m, 2H, H-3’, H-6’a), 3.58 (m, 1H, H-5), 3.68 (t,
1H, H-4’), 3.83 (dd, 1H, H-6’b), 4.03 (m, 2H, H-2’, H-6b), 4.10 (m, 1H, H-2), 4.45–
4.67 (m, 7H, 3 ꢀ CH2 Bn, H-1), 4.85 (br. s, 1H, NHCOO), 5.10 (m, 4H, CH2 Z, H-3,
CHO acyloxyacyl), 5.93 (d, 1H, NH-2), 6.32 (br. d, 1H, NH-2’), 7.25–7.35 (m, 20H,
CH-arom Bn, Z), 7.86 (m, 1H, NH-6). 13C{1H} NMR (CDCl3): d =14.20 (CH3
myristyl), 22.78–36.89 (CH2 acyloxyacyl, myristyl), 39.85 (C-6), 40.94 (C-1’), 41.35
(NCH2 hexanoyl), 47.47 (C-2’), 52.61 (C-2), 55.19 (OMe), 57.81 (CH2 acetyl), 62.16
(C-5’), 66.48 (C-6’), 66.67 (CH2 Z), 69.20 (C-4), 70.87 (C-5), 71.16 (CHO acyl-
oxyacyl), 71.89 (C-3), 72.81, 73.40, 73.48 (3 ꢀ CH2 Bn), 76 (C-3’), 77.30 (C-4’), 98.48
(C-1), 127.85–128.71 (CH-arom Bn, Z), 136.77, 137.64, 137.85 (Cq Bn, Z), 156.49
(C = O Z), 169.64, 172.80, 173.36, 174.67 (4 ꢀ C= O amide, ester). ES-MS; m/z:
1559.00, [M + H]+; monoisotopic MW calculated for C92H143N5O15 =1558.05.
Compound 40. Compound 12 (30 mg, 0.334 mmol) was coupled with compound 34
(23 mg, 0.334 mmol) as described for the preparation of compound 37. Rf =0.48
1
(hexane/ethyl acetate, 1:4, v/v). Yield 45 mg (88%). H NMR (CDCl3): d 0.88 = (dt,
9H, CH3 acyloxycyl, myristyl), 1.25 (br. s, 56H, CH2 acyloxyacyl, hexanoyl, myristyl),
1.52 (m, 10H, CH2 acyloxyacyl, hexanoyl, myristyl), 1.86 (s, 1H, OH-4), 2.11 (t, 2H,
NCH2 hexanoyl), 2.16–2.33 (m, 6H, CH2 acyloxyacyl, hexanoyl, myristyl), 2.40 (dd,
1H, H-1’ax, J1’ax,1’eq =12.5 Hz, J1’ax,2’ = 5.8 Hz), 2.96 (m, 1H, H-6a), 3.05 (br. s, 1H, H-
5’), 3.10 (dd, 1H, H-1’eq, J1’ax,1’eq =12.5 Hz, J1’eq,2’ = 3.4 Hz), 3.17 (q, 2H, CH2 he-
xanoyl), 3.29 (s, 3H, OMe), 3.37 (m, 1H, H-4), 3.40 (s, 2H, NCH2 acetyl), 3.54 (m, 2H,
H-6a’, H-3’), 3.59 (m, 1H, H-5), 3.67 (t, 1H, H-4’), 3.83 (dd, 1H, H-6b’), 4.03 (m, 2H,
H-2’, H-6b), 4.12 (m, 1H, H-2), 4.44–4.67 (m, 6H, 3 ꢀ CH2 Bn), 4.50 (d, 1H, H-1),
4.81 (s, 1H, NHZ), 5.09 (m, 4H, CH2 Z, H-3, CHO acyloxyacyl), 5.79 (d, 1H, NH-2),
6.55 (d, 1H, NH-2’), 7.25–7.35 (m, 20H, CH-arom Bn, Z), 7.87 (m, 1H, NH-6).
13C{1H} NMR (CDCl3): d= 14.16 (CH3, dodecanoyl, myristyl), 22.74–36.49 (CH2
dodecanoyl, hexanoyl, myristyl), 39.78 (C-6), 40.91, 41.80, 41.92 (CH2 myristyl,
hexanoyl, dodecanoyl), 47.65 (C-2’), 50.93 (C-1’), 52.56 (C-2), 55.19 (OMe), 57.69
(CH2 acetyl), 62.20 (C-5’), 66.43 (C-6’), 66.66 (CH2 Z), 69.21 (C-4), 70.95 (C-5), 71.15
(CHO acyloxyacyl), 72.12 (C-3), 72.83, 72.99, 73.35 (3 ꢀ CH2 Bn), 76 (C-3’), 77 (C-
4’), 98.61 (C-1), 127.73–128.65 (CH-arom Bn, Z), 136.76, 137.80, 137.86, 137.89

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PSEUDO-DISACCHARIDE ANALOGUES OF LIPID A
185
(4 ꢀ Cq Bn, Z), 156.46 (C= O Z), 169.34, 172.64, 173.18, 173.32, 174.68 (5 ꢀ C= O
amide, ester). ES-MS; m/z: 1531.1, [M + H]+; monoisotopic MW calculated for
C90H139N5O15 = 1530.0.
Compound 41. Compound 12 (20 mg, 22.2 mmol) was coupled with compound 35
(18 mg, 24.5 mmol) as described for the preparation of compound 37. Rf =0.62 (MeOH/
DCM, 5:95, v/v). Yield 34.5 mg (96%). 1H NMR (CDCl3): d =0.88 (m, 9H, CH3
dodecanoyl, myristyl), 1.25 (m, 68H, CH2 acyloxyacyl, dodecanoyl, myristyl), 1.54 (m,
10H, CH2 acyloxyacyl, dodecanoyl, myristyl), 2.10 (m, 2H, NCH2 dodecanoyl), 2.23
(m, 6H, CH2 acyloxyacyl, dodecanoyl, myristyl), 2.43 (dd, 1H, H-1’ax, J1’ax,1’eq =12.4
Hz, J1’ax,2’ =5.1 Hz), 2.97 (m, 1H, H-6a, J6a,6b = 12.6 Hz), 3.08 (br. s, 1H, H-5’), 3.11
(dd, 1H, H-1’eq, J1’ax,1’eq = 12.6 Hz, J1’eq,2’ =2.8 Hz), 3.18 (q, 2H, NCH2 acyl), 3.29 (s,
3H, OMe), 3.39 (m, 3H, H-4, NCH2 acetyl), 3.55 (m, 2H, H-6a’, H-3’), 3.60 (m, 1H, H-
5, J4,5 =9.8 Hz), 3.67 (t, 1H, H-4’), 3.84 (dd, 1H, H-6b’, J5’,6b’ = 6.3 Hz, J6a’,6b’ =10.5
Hz), 4.03 (m, 1H, H-6b, J5,6b =4.2 Hz, J6a,6b =12.6 Hz) 4.06 (m, 1H, H-2’), 4.12 (m,
1H, H-2, J1,2 = 3.5 Hz, J2,3 =9.8 Hz), 4.42–4.78 (m, 8H, H-1, NHZ, 3 ꢀ CH2 Bn), 5.08
(m, 4H, CH2 Z, H-3, CHO acyloxyacyl), 5.74 (d, 1H, NH-2, J2,NH = 9.1 Hz), 6.59 (d,
1H, NH-2’, J2’NH = 6.3 Hz), 7.25–7.36 (m, 20H, CH-arom Bn, Z), 7.87 (m, 1H, NH-6).
13C{1H} NMR (CDCl3): d =14.20 (CH3 3ꢀ dodecanoyl, myristyl), 22.78–36.87 (CH2
acyloxyacyl, dodecanoyl, myristyl), 39.78 (C-6), 41.23, 41.79 (CH2 acyloxyacyl,
dodecanoyl, myristyl), 47.79 (C-2’), (50, C-1’), 52.53 (C-2), 55.21 (OMe), 57.69 (CH2
acetyl), 62.20 (C-5’), 66.41 (C-6’), 66.66 (CH2 Z), 69.18 (C-4), 71.03 (C-5), 71.13
(CHO acyloxyacyl), 71.95 (C-3), 72.83, 73.30, 73.35 (3 ꢀ CH2 Bn), 77.09 (C-3’), 77.31
(C-4’), 98.61 (C-1), 127.74–128.68 (CH-arom Bn, Z), 136.80, 137.88, (Cq Bn, Z),
156.46 (C=O Z), 169.31, 173.02, 173.18, 173.25, 174.66 (4 ꢀ C =O amide, ester). ES-
MS; m/z: 1615.00, [M +H]+; monoisotopic MW calculated for C96H151N5O15 =1614.12.
Compound 1. To a solution of 37 (10.4 mg, 7.54 mmol) in DMF (0.5 mL), Pd/C
(10%, 5 mg) was added. Hydrogen was passed through the stirred mixture for 46 h.
After filtration of the mixture over a PTFE filter, the filtrate was concentrated under
1
reduced pressure. Yield 7 mg quantitative. H NMR (pyridine-d5): d =0.87 (m, 6H,
2 ꢀ CH3 acyl), 1.24 (m, 43H, CH2 acyl), 1.39 (m, 2H, CH2 acyl), 1.53 (m, 2H, CH2
acyl), 1.65 (m, 2H, CH2 acyl), 1.82 (m, 4H, 2 ꢀ CH2 acyl), 2.06 (m, 2H, CH2 acyl),
2.18 (t, 1H, H-1’ax), 2.57–2.29 (m, 8H, CH2 acyl), 2.65 (m, 1H, H-5’), 3.05 (d, 1H, H-
acetyl), 3.26 (t, 2H, CH2 acyl), 3.38 (dd, 1H, H-1’eq), 3.41 (s, 3H, OMe), 3.75 (m, 3H,
H-acetyl, H-4’, H-6a), 4.04 (dd, 1H, H-6b), 4.10 (dd, 1H, H-3’), 4.26 (t, 1H, H-4), 4.35
(t, 1H, H-6’a), 4.57 (m, 1H, H-5), 4.62 (m, 1H, H-2’), 4.86 (m, 1H, H-2), 4.97 (dd, 1H,
H-6’b), 5.11 (d, 1H, H-1), 5.84 (dd, 1H, H-3), 8.65 (d, 1H, NH), 8.89 (d, 1H, NH’).
13C{1H} NMR (pyridine-d5): d= 13.65 (CH3 myristyl), 22.29–38.90 (CH2 acyl), 40.62
(C-1’), 50.75 (C-2’), 51.76 (C-2), 54.69 (C-6), 55.16 (OMe), 57.14 (CH2 acetyl), 59.73
(C-5’), 69.02 (C-5), 69.96 (C-4), 72.36 (C-6’), 73.07 (C-3), 73.56 (C-4’), 75.78 (C-3’),
99.12 (C-1), 166.63 (C= O ester), 172.71, 173.24, 173.31 (3 ꢀ C =O amide). ES-MS;
m/z: 928.77, [M + H]+; monoisotopic MW calculated for C49H93N5O11 =927.69.
Compound 2. Compound 38 (12.9 mg, 9.11 mmol) was treated as described for the
preparation of compound 1. Yield 6.6 mg (72%) of a white solid. ES-MS; m/z:
1012.54, [M +H]+; monoisotopic MW calculated for C55H105N5O11 = 1011.78.

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VAN DEN BERG ET AL.
Compound 3. Compound 39 (1.9 mg, 1.2 mmol) was treated as described for the
preparation of compound 1. Yield 0.7 mg (54%) of a white solid. ES-MS; m/z:
1154.98, [M + H]+; monoisotopic MW calculated for C63H119N5O13 =1153.88.
Compound 4. Compound 40 (6.7 mg, 4.38 mmol) was treated as described for the
preparation of compound 1. Yield 4.3 mg (88%) of a white solid. ES-MS; m/z: 1126.9,
[M +H]+; monoisotopic MW calculated for C61H115N5O13 = 1125.85.
Compound 5. Compound 41 (8.2 mg, 5.1 mmol) was treated as described for the
preparation of compound 1. Yield 4.0 mg (65%) of a white solid. ES-MS; m/z:
1211.18, [M + H]+; monoisotopic MW calculated for C67H127N5O13 =1209.94.
ACKNOWLEDGMENTS
We thank S.H. van Krimpen for recording the NMR spectra and A.G. Hulst for
performing electrospray MS analysis.
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Received November 5, 2001
Accepted January 28, 2002