Approach toward a generic treatment of gram-negative infections: Synthesis of haptens for catalytic antibody mediated cleavage of the interglycosidic bond in lipid A

Article abstract of DOI:10.1002/(sici)1099-0690(199910)1999:10<2593::aid-ejoc2593>3.0.co;2-c

In order to develop a generic treatment for infections with Gram- negative bacteria, we developed a synthesis of 2-acylamino-deoxynojirimycin derivatives (17, 18, 19 and 20), which will be used as haptens for raising catalytic antibodies capable of hydrol

Full text of DOI:10.1002/(sici)1099-0690(199910)1999:10<2593::aid-ejoc2593>3.0.co;2-c

FULL PAPER
Approach Toward a Generic Treatment of Gram-Negative Infections: Synthesis
of Haptens for Catalytic Antibody Mediated Cleavage of the Interglycosidic
Bond in Lipid A
Richard J. B. H. N. van den Berg,[a] Daan Noort,*[a] Elena S. Milder-Enacache,[a]
Gijs A. van der Marel,[b] Jacques H. van Boom,[b] and Hendrik P. Benschop[a]
Keywords: Endotoxin / Immunotherapy / Catalytic antibodies / Glycosidases / Azasugars
In order to develop a generic treatment for infections with [(benzyloxycarbonyl)amino]-2-deoxy-
Gram-negative bacteria, we developed a synthesis of 2- which was prepared from known 3,4,6-tri-O-benzyl-2-
acylamino-deoxynojirimycin derivatives (17, 18, 19 and 20), [(benzyloxycarbonyl)amino]-2-deoxy- -glucosamine (1) in four
steps in 47% overall yield. Antibodies were generated against
2-[(6-aminohexanoyl)amino]-2-deoxy- -glucono-δ-lactam (17)
D-glucono-δ-lactam (6),
D
which will be used as haptens for raising catalytic antibodies
capable of hydrolyzing the interglycosidic bond in the lipid A
D
moiety of endotoxins. A key intermediate in the preparation of coup-led to the carrier protein bovine serum albumin.
compounds 17, 18, 19 and 20 is 3,4,6-tri-O-benzyl-2-
Introduction
Endotoxins are complex lipopolysaccharides (LPS) situ-
ated in the outer membrane of the cell wall of Gram-nega-
tive bacteria.[1] High concentrations of LPS due to an invas-
ive infection with Gram-negative bacteria cause a strong
immune response in higher animals, leading to pathological
effects such as fever, shock, multiple organ failure and, in
the worst case, death. Most of the biological activities of
LPS are sited in the small terminal disaccharide phospholi-
pid moiety known as lipid A (Figure 1).[2] Lipid A has a
basic structure composed of a polyacylated β(1Ϫ6) disac-
charide of glucosamine 1,4Ј-bisphosphate.[3] The number
and structure of the fatty acid chains influence its endo-
toxic activity.[4]
Although sepsis by Gram-negative bacteria is commonly
accepted as a serious problem, no adequate drug has yet
been developed.[5] Despite treatment with antibiotics, bac-
terial sepsis has a high mortality rate (up to 80%)[6][7] be-
cause bacteria are capable of developing resistance to anti-
biotics. In addition, certain antibiotics induce an enhanced
release of endotoxin from bacteria.[8] Antibodies to LPS
Figure 1. Generalized structure of lipid A, R1 ϭ acyl group or
hydrogen; R2 ϭ O or NH
(either monoclonal or polyclonal) may be used as immuno- logues of lipid X, which were used as haptens for raising
therapeutic agents for the treatment of sepsis.[9] A major antibodies for enzymatic hydrolysis of the fatty acid chains
drawback of these antibodies is their specificity and lack in lipid A. Catalytic antibodies capable of hydrolyzing the
of cross-reactivity and cross-protectivity.[10] Our approach glycosidic bond in lipid A should produce the correspond-
towards a therapy for endotoxemia is based on catalytic ing nontoxic monosaccharides. Advantageously, a catalytic
antibodies capable of degrading lipid A. In this context Bal- antibody acts as an enzyme (in this case a glycosidase),
reddy et al.[11] reported the synthesis of phosphonate ana- which displays rate enhancement, specificity and turnover,
i.e., the ability to catalyze the reaction of many substrate
[a]
molecules. In contrast, antibodies that merely bind LPS
should be administered stoichiometrically.
Department of Chemical Toxicology, TNO Prins Maurits
Laboratory,
P. O. Box 45, NL-2280 AA Rijswijk, The Netherlands
Leiden Institute of Chemistry, Gorlaeus Laboratories,
University of Leiden,
P. O. Box 9502, NL-2300 RA Leiden,
The Netherlands.
Fax: (internat.) ϩ 31-15/284-3963
E-mail: noortd@pml.tno.nl
The active site of our catalytic antibodies should stabilize
the delocalized positive charge and the half-chair confor-
mation of the high-energy oxocarbenium ion formed during
cleavage of the glycosidic bond.[12] The first antibody glyco-
sidases were generated against haptens based on transition
[b]
Eur. J. Org. Chem. 1999, 2593Ϫ2600
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/1010Ϫ2593 $ 17.50ϩ.50/0
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R. J. B. H. N. van den Berg et al.
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state glycosidase inhibitors such as nojirimycin, isofagom- enzymatic synthesis using fructose 1,6-diphosphate aldolase
ine and castanospermine.[13] In these aminocyclitols the in combination with catalytic reductive amination.[16] Fur-
(protonated) nitrogen atom is at or near the anomeric neaux et al. described its preparation from N-acetyl--glu-
center and is anticipated to mimic the electronic charge de- cosamine in a six step synthesis in 10% overall yield.[17]
veloping in the transition state. Here we report the synthesis Furthermore, it has also been synthesized starting from 1-
of 2-acylamido-deoxynojirimycin derivatives, containing an deoxynojirimycin.[18][19] Recently, Vasella and co-workers
acyl chain function at the 2-amino function, which mimics reported a synthesis which is based on the facile transform-
the native lipid A and which is used for linking to a carrier ation of sugar lactones to azasugars,[20] as originally de-
protein. Currently we use these derivatives as haptens for scribed by Pandit and co-workers.[21] Our approach towards
the generation of antibodies with glycosidase activity the haptens is based on this transformation.
towards lipid A.
Oxidation of the readily available 3,4,6-tri-O-benzyl-N-
benzyloxycarbonyl-glucosamine[22] (1) (Scheme 1) with
DMSO/Ac2O afforded lactone 2 in almost quantitative
yield (99%). Subsequent reaction with methanolic ammonia
resulted in the hydroxy-amide 3 in high yield (97%). Oxi-
dation of 3 with DMSO/Ac2O resulted in a mixture of com-
Results and Discussion
Synthesis of Haptens
Several groups have reported the synthesis of haptens for pounds, from which the desired hydroxylactam 4 could be
the generation of antibodies capable of catalyzing the hy- isolated in 58% yield. The corresponding ido-isomer was
drolysis or formation of glycosidic bonds.[13] The design of formed in 3% yield. The other main reaction product (31%)
these haptens is based on cyclic amine glycosidase inhibi- turned out to be the alkene 5. It is interesting to note that
tors such as nojirimycin, isofagomine and castanospermine. ring closure of the keto-amide occurred without treatment
Analogously, our haptens are based on the known synthetic with methanolic ammonia, which is in contrast to the re-
hexosaminidase inhibitor 2-acetamido-1,2,5-trideoxy-1,5- sults obtained by Pandit and co-workers[21] for the corre-
imino--glucitol. Various syntheses of this compound have sponding glucono derivative. Reduction of 4 with sodium
been reported. Fleet et al. described a 17-step synthesis cyanoborohydride afforded the lactam 6 in 85% yield, in
starting from -glucose.[14] Kappes and Legler obtained which the gluco-configuration at C-5 was firmly established
this molecule in an 11-step synthesis starting from N-acetyl- by 1H NMR spectroscopy. The interesting intermediate lac-
-glucosamine.[15] Wong and co-workers described a semi- tam 6, having a highly polarized bond,[23] can be regarded
Scheme 1. Conditions: (i) DMSO, Ac2O, 99%; (ii) 4  NH3/MeOH, 97%; (iii) DMSO, Ac2O, 58%; (iv) NaCNBH3, HCO2H, CH3CN,
85%; (v) BH3 ·THF, 78%; (vi) isopropanol, EtOAc, H2O, formic acid, 5% Pd/C, H2, 74%; (vii) DCM, TEA, Boc2O, 99%; (viii) as (vi) 89%
2594
Eur. J. Org. Chem. 1999, 2593Ϫ2600

Haptens for Catalytic Antibody Mediated Cleavage of the Interglycosidic Bond in Lipid A
FULL PAPER
Scheme 2. Conditions: (i) 11/12, PyBOP, DiPEA, DCM; (13, 97%), (14, 76%), (15, 97%), (16, 93%); (ii) isopropanol, EtOAc, formic acid,
10% Pd/C, H2; (17, 88%), (18, quantitative); (iii) a) DCM/TFA, 1:1 (v/v); b) as (ii); (19, quantitative), (20, 73%)
as a transition state analogue for the hydrolysis of glycosidic diated coupling[26] of 11 with 8 afforded the protected
linkages. -Glucono-δ-lactam has been proposed by others hapten 13 in excellent yield. Analogously, the protected
for use as a hapten in order to raise catalytic anti- hapten 14 was obtained from 8, by using the fatty acid 12,
bodies.[21b,c] This makes 6, after further modification, an which resembles the acyl chain at C-2 in the nonreducing
attractive hapten for the generation of monoclonal anti- end of lipid A. Haptens 15 and 16 were obtained by coup-
bodies with glycosidase activity (vide infra). Reduction of ling of 10 with 11 and 12, respectively. Haptens 13 and 14
6 with LiAlH4, as described by Pandit and co-workers,[21] were deprotected in one step by catalytic hydrogenation on
resulted in a complex mixture of compounds. Fortunately, Pd/C, affording the desired haptens 17 and 18, respectively,
reduction with BH3·THF[24] afforded the desired 1,2-dide- which gave satisfactory analytical data. Compounds 15 and
oxynojirimycin derivative 7 in good yield (78%).
16 were deprotected in two steps: by removal of the Boc
Both the lactam 6 and the 1,2-dideoxynojirimycin deriva- group with trifluoroacetic acid, followed by catalytic hydro-
tive 7 were used for the preparation of haptens. The Z- genation, giving haptens 19 and 20, respectively.
group in 6 was selectively removed by hydrogenation over
5% Pd/C (Degussa type) in a mixture of isopropyl alcohol,
ethyl acetate, water and formic acid, giving the 2-amino-
lactam derivative 8 in 74% yield, which can be coupled with
Immunochemistry
fatty acids. Prior to removal of the Z-group, the endocyclic
Preliminary immunochemical experiments were carried
nitrogen in 7 was protected with a Boc-group,[25] giving 9 out with hapten 17. To this end, the compound was conju-
in excellent yield. After selective removal of the Z-group the gated to BSA (Scheme 3), through 5,5Ј-dithiobis(2-nitro-
2-amino-1,2-dideoxynojirimycin derivative 10 was obtained benzoic acid, succinimidyl diester) (21) as linker. Reaction
in 89% yield.
of 17 with 2 equivalents of 21 afforded the monosubstituted
As a first approach to introduce an acyl chain into lac- derivative 22 in 41% yield. Subsequent coupling of 22 with
tam 8, a relatively short acyl chain was introduced, derived BSA gave the desired immunoconjugate 23. The hapten
from the carboxylic acid 11 (Scheme 2), carrying a pro- density of the conjugate was determined to be 6 haptens
tected amino group for coupling to a carrier protein. Vari- per BSA (overall coupling efficiency: 10%, determined after
ous coupling conditions were evaluated. Coupling of 8 with reduction of the BSA conjugate with TCEP and spectro-
the N-hydroxysuccinimidyl ester of 11 did not result in the photometric quantification of the thionitrophenolate ion).
desired acyl derivative. Activation of 11 with dicyclohexyl-
Balb/c mice were immunized with 23, and hybridomas
carbodiimide was not successful either, but PyBOP me- were prepared from the spleen cells by using standard hy-
Eur. J. Org. Chem. 1999, 2593Ϫ2600
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R. J. B. H. N. van den Berg et al.
FULL PAPER
Scheme 3. Conditions: (i) 17, DMF, 16 h, 41%; (ii) BSA, PBS/DMF, 30 h, 25%, (hapten/BSA 6/1)
bridoma protocols.[27] We obtained a total of 24 stable hy-
brids, producing polyclonal antibodies that exhibited bind-
ing specificity for 23 over background binding for BSA in
ELISA assays. Preliminary screening experiments showed
several of the polyclonal antibodies to be active in the assay
of 4-nitrophenyl-N-acetyl-β--glucosamine hydrolysis. Cur-
rently, derived monoclonal antibodies are being investigated
for their catalytic properties.
internal standard. The purity of the compounds was established by
1H NMR spectroscopy: > 95% in all cases. Mass spectra were re-
corded with a VG Quattro II triple quadropole mass spectrometer
(Fisons Instruments, Altrincham, UK). Column chromatography
was performed on Silica gel 60 (220Ϫ440 mesh ASTM, Fluka).
TLC-analysis was performed with silica gel TLC-cards (Fluka)
with detection by UV-absorption (254 nm) where applicable and
charring with 20% H2SO4 in MeOH or ammonium 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 toluene. Polytetrafluoroethylene
(PFTE) filters were purchased from Alltech (Breda, The Nether-
lands). 4-Nitrophenyl-N-acetyl-β--glucosamine, and 5,5Ј-di-
thiobis(2-nitrobenzoic acid, succinimidyl diester) were purchased
Conclusion
In conclusion, we have synthesized four pseudo-sugar-
aza-glycosides as potential antigens for glycoside-hydrolyz- from Molecular Probes, Inc. (Leiden, The Netherlands).
ing antibodies. Future studies will aim at further develop-
3,4,6-Tri-O-benzyl-2-[(benzyloxycarbonyl)amino]-2-deoxy-D-glu-
cono-δ-lactone (2): Acetic anhydride (15 mL) was added under ni-
trogen atmosphere to a solution of 3,4,6-tri-O-benzyl-2-[(benzyl-
ment of such antibodies.
oxycarbonyl)amino]-2-deoxy--glucosamine
(1)
(4.00 g,
Experimental Section
6.86 mmol) in anhydrous DMSO (25 mL). The mixture was stirred
for 16 h at ambient temperature. TLC analysis showed complete
conversion of starting material into a compound with Rf ϭ 0.57
(ethyl acetate/hexane, 1:2, v/v). The reaction mixture was diluted
with diethyl ether (150 mL), washed thoroughly with aqueous 10%
NaHCO3 (3 ϫ 100 mL) and twice with saturated NaCl
(2 ϫ 200 mL) solution. After drying on Na2SO4, the organic layer
was concentrated in vacuo. The crude product was purified by silica
gel column chromatography. Elution was performed with hexane/
ethyl acetate (4:1 Ǟ 2:1, v/v). Yield 3.95 g (99%). Ϫ 1H NMR
General Remarks: Toluene (Merck) was distilled from P2O5. Di-
chloromethane (Biosolve Ltd.) and acetonitrile (Biosolve Ltd.)
were freshly distilled from CaH2. Methyl sulfoxide (DMSO, Fluka)
was stirred overnight with CaH2 and distilled under reduced press-
ure. Methanol (Merck) was dried by refluxing with magnesium me-
thoxide and then distilled. N,N-Diisopropylethylamine (DiPEA,
Acros Chimica NV) was distilled from p-toluenesulfonyl chloride
and redistilled from potassium hydroxide pellets. 1H and 13C NMR
spectra were recorded on a Varian VXR-400S (399.9/100.6 MHz).
1H and 13C NMR chemical shifts are given in ppm (δ) relative to (CDCl3): δ ϭ 3.69 (2 H, br. s, H2-6), 4.01 (2 H, br. s, H-3, H-4),
tetramethylsilane (δ ϭ 0.00), CD2HOD (δ ϭ 3.307), [D6]DMSO 4.09 (1 H, t, J2,NH ϭ 7.4 Hz, H-2), 4.37 (1 H, br. s, H-5), 4.50 (2
(δ ϭ 2.525), [D4]MeOH (δ ϭ 49.0) or [D6]DMSO (δ ϭ 39.6) as H, dd, J ϭ 12.0 Hz, CH2 Bn), 4.58Ϫ4.81 (4 H, m, J ϭ 11.2 Hz,
2596
Eur. J. Org. Chem. 1999, 2593Ϫ2600

Haptens for Catalytic Antibody Mediated Cleavage of the Interglycosidic Bond in Lipid A
J ϭ 11.8 Hz, CH2 Bn), 5.07 (2 H, dd, J ϭ 12.1 Hz, CH2Z), 5.46 (1 and extracted with EtOAc (3 ϫ 50 mL). The organic fractions were
FULL PAPER
H, d, J2,NH ϭ 7.4 Hz, NH), 7.15Ϫ7.40 (20 H, m, CH-arom Bn/Z).
Ϫ
combined and washed with saturated aqueous NaCl (50 mL) solu-
tion. After drying on Na2SO4, the organic layer was concentrated
13C{1H} NMR (CDCl3): δ ϭ 56.42 (C-2), 67.33 (CH2Z), 68.12
(C-6), 73.63, 74.51, 74.74 (3 ϫ CH2 Bn), 76.22 (C-3), 79.01 (C-5), in vacuo, and the crude product was purified by silica gel column
79.54 (C-4), 127.73Ϫ128.59 (CH-arom Bn/Z), 136.11, 137.53, chromatography. Elution was performed with hexane/ethyl acetate
137.57, 137.68 (4 ϫ Cq Bn/Z), 156.21 (CϭO Z), 168.91 (CϭO lac- (2:1 Ǟ 1:4, v/v). Yield 1.75 g (85%). Rf ϭ 0.39 (ethyl acetate/hex-
1
tone).
ane, 2:1, v/v). Ϫ H NMR ([D6]DMSO): δ ϭ 3.37 (1 H, m, J5,6
2.8 Hz, J5,6Ј ϭ 3.9 Hz, J4,5 ϭ 9.1 Hz, H-5), 3.56 (2 H, ddd, J5,6
2.8 Hz, J5,6Ј ϭ 3.5 Hz, J6,6Ј ϭ 9.8 Hz, H2-6), 3.83 (1 H, dd, J4,5
9.1 Hz, J3,4 ϭ 9.8 Hz, H-4), 3.89 (1 H, dd, J3,4 ϭ 9.8 Hz, J2,3
ϭ
ϭ
ϭ
ϭ
3,4,6-Tri-O-benzyl-[(2-benzyloxycarbonyl)amino]-2-deoxy-D-glu-
cono-amide (3): A solution of 2 (5.32 g, 9.16 mmol) in anhydrous
methanolic ammonia (4 , 75 mL) was stirred for 1 h after which
it was concentrated in vacuo. TLC analysis showed complete con-
version of starting material into a compound with Rf ϭ 0.58 (ethyl
acetate/hexane, 2:1, v/v). Traces of ammonia in the residue were
removed by co-evaporation with toluene (1 ϫ 10 mL). Purification
of the residue was accomplished by silica gel column chromatogra-
phy (hexane/ethyl acetate 2:1 Ǟ 1:1, v/v). Yield 5.30 g (97%). Melt-
9.1 Hz, H-3), 3.99 (1 H, t, J2,3 ϭ 9.1 Hz, H-2), 4.44Ϫ4.78 (6 H, m,
CH2 Bn), 5.08 (2 H, dd, CH2Z), 7.22Ϫ7.37 (20 H, m, CH-arom
Bn/Z), 7.64 (1 H, d, J2,NH ϭ 8.7 Hz, NH), 7.74 (1 H, s, NH lactam).
Ϫ
13C{1H} NMR ([D6]DMSO): δ ϭ 54.24 (C-5), 55.61 (C-2), 65.34
(CH2Z), 68.88 (C-6), 72.37, 73.49, 73.70 (3 ϫ CH2 Bn), 77.07 (C-
4), 80.43 (C-3), 127.41Ϫ128.66 (CH-arom Bn/Z), 147.21, 138.11,
138.14, 138.41 (4 ϫ Cq Bn/Z), 156.43 (CϭO Z), 168.59 (CϭO lac-
tam). Ϫ ES-MS; m/z: 581.2, [M ϩ H]ϩ; monoisotopic MW calcu-
lated for C35H36N2O6 ϭ 580.3.
1
ing point 193.1Ϫ195.8°C (dec.). Ϫ H NMR (CDCl3): δ ϭ 3.07 (1
H, br. s, OH-5), 3.56 (1 H, dd, J5,6 ϭ 5.0 Hz, J6,6Ј ϭ 10 Hz, H-6),
3.63 (1 H, dd, J5,6Ј ϭ 5.3 Hz, J6,6Ј ϭ 10 Hz, H-6Ј), 3.66 (1 H, dd,
J3,4 ϭ J4,5 ϭ 7.1 Hz, H-4), 3.95 (1 H, br. s, H-5), 4.39 (1 H, bd,
J2,3 ϭ J3,4 ϭ 7.0 Hz, H-3), 4.41Ϫ4.69 (6 H, m, CH2 Bn), 4.52 (1
H, bd, J2,NH ϭ 5.9 Hz, H-2), 5.03 (2 H, dd, CH2Z), 5.95 (1 H, br.
s, NH), 6.09 (1 H, d, J2,NH ϭ 7.0 Hz, NH), 6.38 (1 H, br. s, NH),
7.18Ϫ7.28 (20 H, m, CH-arom Bn/Z). Ϫ 13C{1H} NMR (CDCl3):
δ ϭ 54.95 (C-2), 67.08 (CH2Z), 70.60 (C-5), 70.90 (C-6), 70.34,
74.26, 74.67 (3 ϫ CH2 Bn), 79.15 (C-3), 79.20 (C-4), 127.53Ϫ128.40
(CH-arom Bn/Z), 136.03, 137.50, 137.66, 137.94 (4 ϫ Cq Bn/Z),
156.43 (CϭO Z), 173.03 (CϭO lactam). Ϫ ES-MS; m/z: 599.4, [M
ϩ H]ϩ; monoisotopic MW calculated for C35H38N2O7 ϭ 598.3.
3,4,6-Tri-O-benzyl-2-[(benzyloxycarbonyl)amino]-1,2,5-trideoxy-
1,5-imino-D-glucitol (7): To neat 6 (108 mg, 0.186 mmol) was added
BH3·THF (1  in THF, 1.4 mL). The resulting solution was stirred
for 16 h at ambient temperature under a stream of nitrogen. The
solution was cooled in ice and the reaction was quenched by the
addition of aqueous HCl (6 , 0.5 mL). After stirring for 30 min
at room temperature, the pH of the mixture was brought to 10.
The organic layer was separated and the water layer was extracted
six times with ethyl acetate. After drying on Na2SO4, the organic
layer was concentrated. The crude product was purified by silica
gel column chromatography. Elution was performed with DCM/
ethyl acetate (100:0 Ǟ 50:50, v/v). Yield 82 mg (78%). Rf ϭ 0.28
(DCM/ethyl acetate, 1:1, v/v). Ϫ 1H NMR (CDCl3): δ ϭ 1.78 (1
H, br. s, NH cyclic), 2.38 (1 H, dd, J1ax,2 ϭ 10 Hz, J1ax,1eq ϭ 12 Hz,
H-1ax), 2.71 (1 H, m, J5,6 ϭ 5 Hz, J5,6Ј ϭ 8 Hz, J4,5 ϭ 9 Hz, H-5),
3.26 (1 H, dd, J1eq,2 ϭ 4 Hz, J1ax,eq ϭ 12 Hz, H-1eq), 3.32 (1 H, t,
J2,3 ϭ J3,4 ϭ 9 Hz, H-3), 3.47 (1 H, t, J3,4 ϭ J4,5 ϭ 9 Hz, H-4),
3.62 (3 H, m, H-2, H2Ϫ6), 4.42Ϫ4.83 (7 H, m, 3 ϫ CH2 Bn, NH),
5.05 (2 H, dd, CH2Z), 7.20Ϫ7.37 (20 H, m, CH-arom Bn/Z). Ϫ
13C{1H} NMR (CDCl3): δ ϭ 48.77 (C-1), 54.11 (C-2), 59.80 (C-5),
66.78 (CH2Z), 69.81 (C-6) 73.49, 74.63, 74.93 (3 ϫ CH2 Bn), 80.60
(C-3), 83.61 (C-4), 127.85Ϫ128.62 (CH-arom Bn/Z), 136.61,
138.08, 138.29 (Cq Bn/Z), 156.08 (CϭO Z). Ϫ ES-MS; m/z: 567.3,
[M ϩ H]ϩ; monoisotopic MW calculated for C35H38N2O5 ϭ 566.3.
3,4,6-Tri-O-benzyl-2-[(benzyloxycarbonyl)amino]-5-dehydro-2-
deoxy-5-hydroxy-D-glucono-δ-lactam (4): Acetic anhydride (12 mL)
was added under nitrogen atmosphere to a solution of 3 (3.22 g,
5.39 mmol) in anhydrous DMSO (20 mL). The mixture was stirred
for 16 h at ambient temperature. TLC analysis showed complete
conversion of starting material into a mixture of products. The re-
action mixture was diluted with diethyl ether (150 mL), washed
thoroughly with aqueous 10% NaHCO3 (3 ϫ 50 mL) solution and
with saturated NaCl (1 ϫ 50 mL) solution. After drying on
Na2SO4, the organic layer was concentrated in vacuo. The crude
product was purified by silica gel column chromatography. Elution
was performed with hexane/ethyl acetate (2:1 Ǟ 1:2, v/v). Yield
3.14 g (58%). Rf ϭ 0.44 (ethyl acetate/hexane, 2:1, v/v). Melting
point 147.5°C (dec.). Ϫ 1H NMR (CDCl3): δ ϭ 3.34 (2 H, dd,
J5,6 ϭ 9.7 Hz, H2Ϫ6), 3.79 (1 H, d, J3,4 ϭ 9.5 Hz, H-4), 3.95 (1 H,
br. s, OH-5), 4.04 (1 H, dd, J2,NH ϭ 8.5 Hz, J2,3 ϭ 8.7 Hz, H-2),
4.21 (1 H, dd, J2,3 ϭ 9.0 Hz, J3,4 ϭ 9.1 Hz, H-3), 4.40Ϫ4.90 (6 H,
2-Amino-3,4,6-tri-O-benzyl-2-deoxy-D-glucono-δ-lactam (8): Pd/C
(5%, Degussa type E101 NO/W, 66 mg) was added to a solution of
6 (51 mg, 88 µmol) in a mixture of formic acid/water/ethyl acetate/
isopropyl alcohol (3 mL, 3/3/33/60, v/v/v/v). Hydrogen was passed
through the stirred mixture for 2 h. TLC analysis indicated the
complete conversion of the starting material into a compound with
Rf ϭ 0.25 (ethyl acetate/hexane, 4:1, v/v). The mixture was passed
over a short column containing a layer of glass wool and a layer
of hyflo. Concentration of the filtrate in vacuo yielded 29 mg of a
yellow syrup (74%). Ϫ 1H NMR (CDCl3): δ ϭ 3.30 (1 H, dd,
J6,6Ј ϭ 9.7 Hz, J5,6 ϭ 7.8 Hz, H-6), 3.53 (3 H, m, H-2, H-5, H-6Ј),
3.63 (1 H, t, J3,4 ϭ 9.2 Hz, J4,5 ϭ 8.6 Hz, H-4), 4.28 (3 H, br. s,
m, 3 ϫ CH2 Bn), 5.07 (2 H, dd, CH2Z), 5.50 (1 H, bd, J2,NH
ϭ
7.5 Hz, NH), 6.56 (1 H, s, NH cyclic), 7.20Ϫ7.35 (20 H, m, CH-
arom Bn/Z). Ϫ 13C{1H} NMR (CDCl3): δ ϭ 57.36 (C-2), 67.38
(CH2Z), 72.52 (C-6), 73.75, 75.18, 75.40 (3 ϫ CH2 Bn), 78.27 (C-
3), 78.39 (C-4), 82.17 (C-5), 128.07Ϫ128.63 (CH-arom Bn/Z),
156.84 (CϭO Z), 169.49 (CϭO lactam). Ϫ ES-MS; m/z: 597.3, [M
ϩ H]ϩ; monoisotopic MW calculated for C35H36N2O7 ϭ 596.3.
3,4,6-Tri-O-benzyl-2-[(benzyloxycarbonyl)amino]-2-deoxy-
cono-δ-lactam (6): NaCNBH3 (0.218 g, 3.44 mmol) was added to a
D-glu-
solution of 4 (2.11 g, 3.54 mmol) in anhydrous acetonitrile (36 mL) NH3ϩ), 4.35Ϫ4.90 (6 H, m, CH2 Bn), 6.62 (1 H, s, NH lactam),
and formic acid (4 mL). The reaction mixture was refluxed for 2 h.
7.17Ϫ7.34 (15 H, m, CH-arom Bn). Ϫ 13C{1H} NMR (CDCl3):
TLC analysis indicated the complete disappearance of the starting δ ϭ 54.79 (C-2), 55.80 (C-5), 70.67 (C-6), 73.45, 74.87, 75.25
material. After cooling to 0°C, the mixture was quenched by the
addition of aqueous HCl (1.0 , 3.4 mL). After stirring for 15 min,
the mixture was poured into a stirred mixture of EtOAc/10% aque-
ous NaHCO3 (200 mL, 1:1, v/v). The aqueous layer was separated
(3 ϫ CH2 Bn), 77.34 (C-4), 83.67 (C-3), 127.66Ϫ128.60 (CH-arom
Bn), 137.39, 137.51, 137.57 (3 ϫ Cq Bn), 171.24 (CϭO lactam). Ϫ
ES-MS; m/z: 447.2, [M ϩ H]ϩ; monoisotopic MW calculated for
C27H30N2O4 ϭ 446.2.
Eur. J. Org. Chem. 1999, 2593Ϫ2600
2597

R. J. B. H. N. van den Berg et al.
FULL PAPER
3,4,6-Tri-O-benzyl-2-[(benzyloxycarbonyl)amino]-1,5-[(tert-butyl- 3,4,6-Tri-O-benzyl-2-[(R)-3-(6-benzyloxycarbonylamino)-
oxycarbonyl)imino]-1,2,5-trideoxy- -glucitol (9): Triethylamine (36 hexanoyloxytetradecanoylamino]-2-deoxy- -glucono-δ-lactam
µL, 258 µmol) and BOC anhydride (45 mg, 206 µmol) were added (14): Compound 12 (11 mg, 22 µmol) was coupled with compound
to a solution of 7 (98 mg, 173 µmol) in anhydrous DCM (1.0 mL). 8 (10 mg, 22 µmol) as described for the preparation of compound
D
D
The solution was stirred for 46 h. TLC analysis showed complete
conversion of starting material into a compound with Rf ϭ 0.47
(ethyl acetate/hexane, 1:4, v/v). The reaction mixture was concen-
trated and the crude product was purified by silica gel column chro-
13. Yield 15.7 mg (76%). Rf ϭ 0.51 (MeOH/DCM, 5:95, v/v). Ϫ
1H NMR ([D5]pyridine): δ ϭ 0.86 (3 H, t, CH3 acyloxyacyl), 1.29,
1.56, 1.65, 1.87, 2.37, 2.90, 3.33 (35 H, m, CH2 acyloxyacyl), 3.68
(1 H, dd, J5,6 ϭ 5.7 Hz, J6,6Ј ϭ 9.7 Hz, H-6), 3.82 (1 H, dd, J5,6Ј
ϭ
matography. Elution was performed with DCM/MeOH (100:0 Ǟ 3.2 Hz, J6,6Ј ϭ 9.7 Hz, H-6Ј), 3.90 (1 H, br. s, H-5), 4.06 (1 H, t,
96.5:3.5, v/v). Yield 115 mg (99%). Ϫ 1H NMR ([D5]pyridine): δ ϭ
1.48 (9 H, s, tBu), 3.43 (1 H, d, H-1ax), 3.88 (3 H, m, H2-6, H-3),
4.05 (1 H, br. s, H-4), 4.21 (1 H, br. s, H-2), 4.45 (1 H, d, H-1eq),
4.52Ϫ4.80 (6 H, m, 3 ϫ CH2 Bn), 5.25 (3 H, m, H-5, CH2Z), 6.85
J4,5 ϭ 9.0 Hz, J3,4 ϭ 8.2 Hz, H-4), 4.55 (1 H, t, J2,3 ϭ 8.8 Hz, J3,4 ϭ
9.0 Hz, H-3), 4.82 (1 H, t, J2,3 ϭ 8.8 Hz, J2,NH ϭ 8.0 Hz, H-2),
4.47Ϫ4.51 (6 H, m, 3 ϫ CH2 Bn), 5.33 (2 H, s, CH2Z), 5.81 (1 H,
m, CHO acyloxyacyl), 7.31Ϫ7.38 (20 H, m, CH-arom Bn/Z), 7.85
(1 H, d, NH), 7.24Ϫ7.39 (20 H, CH-arom Bn/Z). Ϫ 13C{1H} NMR (1 H, bt, NH aminohexanoyl), 8.72 (1 H, s, NH lactam), 9.63 (1
([D5]pyridine): δ ϭ 27.67 (CH3tBu), 39.8 (C-1), 48.16 (C-2), 51.5 H, d, J2,NH ϭ 8.0 Hz, NH amide). Ϫ 13C{1H} NMR ([D5]pyridine):
(C-5), 65.91 (CH2Z), 67.22 (C-6), 70.94, 71.53, 72.34 (3 ϫ CH2 δ ϭ 13.57 (CH3 acyloxyacyl), 22.23Ϫ40.82 (CH2 acyloxyacyl),
Bn), 73.29 (C-4), 74.98 (C-3), 79.16 (Cq tBu), 127.28Ϫ128.24 (CH-
arom Bn/Z), 135.38, 137.68, 137.88, 138.49 (Cq Bn/Z), 155.45,
155.55 (CϭO Boc/Z). Ϫ ES-MS; m/z: 667.3, [M ϩ H]ϩ; monoiso-
topic MW calculated for C40H46N2O7 ϭ 666.3.
54.51 (C-5), 55.24 (C-2), 65.42 (CH2Z), 69.74 (C-6), 71.02 (CHO
acyloxyacyl), 72.66, 73.76, 73.95 (3 ϫ CH2 Bn), 77.49 (C-4), 81.04
(C-3), 127.26Ϫ128.10 (CH-arom Bn/Z), 137.55, 137.91, 138.28,
138.72 (4 ϫ Cq Bn/Z), 168.78 (CϭO amide), 170.11 (CϭO lac-
tam), 172.43 (CϭO ester). Ϫ ES-MS; m/z: 920.6, [M ϩ H]ϩ; mono-
isotopic MW calculated for C55H73N3O9 ϭ 919.5.
2-Amino-3,4,6-tri-O-benzyl-1,5-[(tert-butyloxycarbonyl)imino]-
1,2,5-trideoxy-D-glucitol (10): Compound 9 (210 mg, 315 µmol) was
deprotected as described for compound 8. Yield 135 mg (89%).
Rf ϭ 0.40 (MeOH/DCM, 5:95, v/v). Ϫ 1H NMR (CDCl3): δ ϭ
1.45 (9 H, s, tBu), 1.79 (2 H, br. s, NH2), 3.02 (1 H, br. s, H-2),
3.27 (1 H, d, H-1ax), 3.60 (1 H, br. s, H-3), 3.69 (2 H, dd, H2-6),
3.77 (1 H, s, H-4), 3.82 (1 H, bd, H-1eq), 4.42Ϫ4.62 (6 H, m,
3 ϫ CH2 Bn), 4.65 (1 H, br. s, H-5), 7.19Ϫ7.33 (15 H, m, CH-arom
Bn). Ϫ 13C{1H} NMR (CDCl3): δ ϭ 28.47 (CH3 tBu), 43.09 (C-
1), 49.36 (C-2), 52.91 (C-5), 67.73 (C-6), 71.43, 72.03, 72.81
(3 ϫ CH2 Bn), 73.56 (C-4), 77.66 (C-3), 79.83 (Cq tBu),
127.49Ϫ128.44 (CH-arom Bn), 137.96, 138.14, 138.47 (3 ϫ Cq Bn),
156.20 (CϭO Boc). Ϫ ES-MS; m/z: 533.4, [M ϩ H]ϩ; monoisotopic
MW calculated for C32H40N2O5 ϭ 532.3.
3,4,6-Tri-O-benzyl-2-[(6-benzyloxycarbonyl)aminohexanoyl-
amino]-1,5-[(tert-butyloxycarbonyl)imino]-1,2,5-trideoxy-
D-glucitol (15): Compound 11 (4.7 mg, 19 µmol) was coupled with
compound 10 (10 mg, 19 µmol) as described for the preparation of
compound 13. Yield 14.2 mg (97%). Rf ϭ 0.88 (MeOH/DCM, 5:95,
v/v). Ϫ 1H NMR ([D5]pyridine): δ ϭ 1.36 (2 H, m, CH2 hexanoyl),
1.51 (9 H, s, tBu), 1.55 (2 H, m, CH2 hexanoyl), 1.63 (2 H, m, CH2
hexanoyl), 2.14 (2 H, t, CH2 hexanoyl), 3.35 (2 H, m, CH2 hexa-
noyl), 3.46 (1 H, bd, H-1ax), 3.95 (2 H, m, H2Ϫ6), 3.97 (1 H, t, H-
3), 4.13 (1 H, br. s, H-4), 4.42 (1 H, br. s, H-1eq), 4.51Ϫ4.83 (6 H,
m, 3 ϫ CH2 Bn), 4.58 (1 H, br. s, H-2), 7.22Ϫ7.53 (20 H, m, CH-
arom Bn/Z), 7.91 (1 H, br. s, NH aminohexanoyl). Ϫ 13C{1H}
NMR ([D5]pyridine): δ ϭ 24.92, 26.18, 29.43, 36.18, 40.61
(5 ϫ CH2 hexanoyl), 27.70 (CH3tBu), 39.2 (C-1), 45.83 (C-2), 52.5
(C-5), 65.46 (CH2Z), 67.12 (C-6), 70.91, 71.38, 72.28 (3 ϫ CH2 Bn),
73.43 (C-4), 74.72 (C-3), 78.96 (Cq tBu), 127.19Ϫ128.22 (CH-arom
Bn), 137.55, 137.73, 137.91, 138.47 (Cq Bn/Z), 155.43, 156.52 (Cϭ
O Boc/Z), 171.15 (CϭO amide). Ϫ ES-MS; m/z: 780.4, [M ϩ H]ϩ;
monoisotopic MW calculated for C46H57N3O8 ϭ 779.4.
3,4,6-Tri-O-benzyl-2-[(6-benzyloxycarbonyl)aminohexa-
noylamino]-deoxy-D-glucono-δ-lactam (13): To a stirred mixture of
6-(benzyloxycarbonylamino)hexanoic acid (11) (7 mg, 28 µmol),
PyBOP (14.3 mg, 27 µmol) and DiPEA (5 µL, 29 µmol) in DCM
(260 µL) was added a solution of 8 (12 mg, 27 µmol) in DCM (260
µL). After 30 min, TLC analysis indicated the complete conversion
of starting material into a compound with Rf ϭ 0.36 (MeOH/
DCM, 5:95, v/v). The reaction mixture was diluted with DCM
(1 mL) and washed with water (1 mL). After drying over MgSO4,
the organic layer was concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography.
3,4,6-Tri-O-benzyl-2-[(R)-3-(6-benzyloxycarbonylamino)-
hexanoyloxytetradecanoylamino]-1,5-[(tert-butyloxycarbonyl)imino]-
1,2,5-trideoxy-D-glucitol (16): Compound 12 (9.2 mg, 19 µmol) was
Elution was performed with MeOH/DCM, (0:100 Ǟ 6.5:93.5, v/v).
Yield 18 mg (97%). Ϫ H NMR ([D6]DMSO): δ ϭ 1.27, 1.39, 1.49 preparation of compound 13. Yield 17.6 mg (93%). Rf ϭ 0.63
(6 H, m, 3 ϫ CH2 hexanoyl), 2.10 (2 H, t, CH2 hexanoyl), 2.97 (2
coupled with compound 10 (10 mg, 19 µmol) as described for the
1
1
(MeOH/DCM, 2:98, v/v). Ϫ H NMR ([D5]pyridine): δ ϭ 0.87 (3
H, m, CH2 hexanoyl) 3.39 (1 H, m, J4,5 ϭ 7.7 Hz, J5,6 ϭ 4.0 Hz, H, t, CH3 acyloxyacyl), 1.24 (20 H, br. s, CH2 acyloxyacyl), 1.52
J5,6Ј ϭ 3.3 Hz, H-5), 3.55 (1 H, dd, J5,6 ϭ 4.0 Hz, J6,6Ј ϭ 9.9 Hz, (9 H, s, tBu), 1.67, 2.32Ϫ2.66, 3.37 (12 H, m, 6 ϫ CH2 acyloxya-
H-6), 3.59 (1 H, dd, J5,6Ј ϭ 3.3 Hz, J6,6Ј ϭ 9.9 Hz, H-6Ј), 3.79 (1 cyl), 3.50 (1 H, d, H-1ax), 3.93 (2 H, m, H2Ϫ6), 4.00 (1 H, s, H-
H, t, J3,4 ϭ 8.9 Hz, J4,5 ϭ 7.7 Hz, H-4), 3.87 (1 H, t, J2,3 ϭ 9.5 Hz, 3), 4.14 (1 H, s, H-4), 4.42 (1 H, br. s, H-1eq), 4.50Ϫ4.84 (6 H, m,
J3,4 ϭ 8.9 Hz, H-3), 4.13 (1 H, t, J2,3 ϭ 9.5 Hz, J2,NH ϭ 8.7 Hz, 3 ϫ CH2 Bn), 4.65 (1 H, t, H-2), 5.33 (2 H, br. s, CH2Z), 5.63 (1
H-2), 4.44Ϫ4.76 (6 H, m, 3 ϫ CH2 Bn), 5.01 (2 H, s, CH2Z), 7.19 H, m, CHO acyloxyacyl), 7.32Ϫ7.50 (20 H, m, CH-arom Bn/Z),
(1 H, t, NH aminohexanoyl), 7.26Ϫ7.38 (20 H, m, CH-arom Bn/
7.65 (1 H, dd, NH amide), 7.89 (1 H, s, NH aminohexanoyl). Ϫ
13.80 (CH3 acyloxyacyl),
Z), 7.68 (1 H, s, NH lactam), 8.19 (1 H, d, NH amide). Ϫ 13C{1H} 13C{1H} NMR (CDCl3):
δ
ϭ
NMR (CDCl3): δ ϭ 24.84, 26.05, 29.50, 35.90, 40.81 (5 ϫ CH2 22.10Ϫ40.96 (CH2 acyloxyacyl), 27.72 (CH3tBu), 39 (C-1), 46.05
hexanoyl), 54.32 (C-5), 55.23 (C-2), 66.56 (CH2Z), 70.26 (C-6), (C-2), 53 (C-5), 65.48 (CH2Z), 67.19 (C-6), 70.65 (CHO acyloxy-
73.39, 74.59, 74.69 (3 ϫ CH2 Bn), 77.46 (C-4), 80.11 (C-3),
127.76Ϫ128.72 (CH-arom Bn/Z), 136.88, 137.43, 137.60, 138.16
acyl), 70.90, 71.43, 72.31 (3 ϫ CH2 Bn), 73.34 (C-4), 74.77 (C-3),
79.07 (Cq tBu), 127.20Ϫ128.25 (CH-arom Bn/Z), 137.56, 137.86,
(Cq Bn/Z), 156.53 (CϭO Z), 168.79 (CϭO lactam), 173.47 (Cϭ 137.92, 138.47 (Cq Bn/Z), 155.45, 156.55 (CϭO Boc/Z), 168.40,
O amide). Ϫ ES-MS: m/z: 694.3, [M ϩ H]ϩ; monoisotopic MW
calculated for C41H47N3O7 ϭ 693.3.
172.32 (CϭO amide/ester). Ϫ ES-MS; m/z: 1006.6, [M ϩ H]ϩ;
monoisotopic MW calculated for C60H83N3O10 ϭ 1005.6.
2598
Eur. J. Org. Chem. 1999, 2593Ϫ2600

Haptens for Catalytic Antibody Mediated Cleavage of the Interglycosidic Bond in Lipid A
FULL PAPER
2-[(6-Aminohexanoyl)amino]-2-deoxy-
mic acid (20 µL) and Pd/C (10%, 20 mg) were added to a mixture
D
-glucono-δ-lactam (17): For-
Compound 22: To a stirred and cooled (0°C) solution of 5,5Ј-di-
thiobis(2-nitrobenzoic acid, succinimidyl ester) (21) (3.76 mg, 6.37
of 13 (13 mg, 18 µmol) in isopropyl alcohol/EtOAc/H2O (1.5 mL, µmol) in DMF (0.40 mL) was added dropwise a solution of 17
2/3/1, v/v/v). Hydrogen was passed through the stirred mixture for (0.92 mg, 3.18 µmol) in DMF (0.1 mL). The reaction mixture was
2 h. After filtrating of the mixture over a PTFE filter, the filtrate stirred for 16 h at 4°C. The crude product was purified by FPLC
was concentrated under reduced pressure. Yield 4.6 mg (88%). Ϫ
(PepRPCTM HR 10/10 column) to give pure 22 (1.0 mg, 1.3 µmol,
1H NMR ([D4]MeOH): δ ϭ 1.46 (2 H, t, CH2 hexanoyl), 1.69 (4
41%). Ϫ ES-MS; m/z: 765.0, [M ϩ H]ϩ; monoisotopic MW calcu-
H, m, 2 ϫ CH2 hexanoyl), 2.31 (2 H, t, CH2 hexanoyl), 2.93 (2 H, lated for C30H32N6O14S2 ϭ 764.1.
t, CH2 hexanoyl), 3.25 (1 H, m, H-5), 3.58 (1 H, t, J4,5 ϭ 9.3 Hz,
BSA Conjugate 23: To a stirred and cooled (0°C) solution of BSA
J3,4 ϭ 9.4 Hz, H-4), 3.63 (1 H, dd, J6,6Ј ϭ 11.4 Hz, J5,6 ϭ 5.8 Hz,
H-6), 3.82 (1 H, t, J2,3 ϭ J3,4 ϭ 9.8 Hz, H-3), 3.84 (1 H, dd, J6,6Ј
(5 mg) in PBS (0.6 mL) was added dropwise a mixture of 22
(1.0 mg, 1.3 µmol) in DMF/PBS (0.1 mL, 7:3, v/v). The reaction
mixture was stirred for 30 h at 4°C and subsequently applied to
a centrifuge ultrafilter (Centrex UF-2, MW cutoff 10 kD). After
filtration (1 h, 5000 g), the retentate containing BSA conjugate 23
was washed with PBS (2 ϫ 1 mL), followed by filtration (2 ϫ 1 h,
5000 g). Hapten density (6:1, hapten/BSA) was determined by mea-
suring the release of thionitrophenolate ion (absorption at 412 nm)
after reduction of the BSA conjugate 23 with TCEP.
ϭ
11.4 Hz, J5,6Ј ϭ 2.8 Hz, H-6Ј), 4.02 (1 H, d, J2,3 ϭ 10 Hz, H-2),
4.82 (3 H, br. s, 3 ϫ OH), 8.58 (2 H, br. s, NH2). Ϫ 13C{1H} NMR
([D4]MeOH): δ ϭ 25.69, 26.55, 28.03, 36.40, 43.43 (5 ϫ CH2 hexa-
noyl), 56.76 (C-2), 58.46 (C-5), 62.69 (C-6), 70.76 (C-4), 73.33 (C-
3), 176.40 (CϭO). Ϫ ES-MS; m/z: 290.2, [M ϩ H]ϩ; monoisotopic
MW calculated for C12H23N3O5 ϭ 289.2.
2-[(R)-3-(6-Amino)hexanoyloxytetradecanoylamino]-2-deoxy-D-glu-
cono-δ-lactam (18): Compound 14 (7.8 mg, 8.5 µmol) was treated
as described for the preparation of compound 17. Yield 5.0 mg
(quantitative). Ϫ 1H NMR ([D4]MeOH): δ ϭ 0.89 (3 H, t, CH3
acyl), 1.28, 1.42, 1.65, 2.35, 2.50 (31 H, m, CH2 acyloxyacyl), 2.77
(1 H, br. s, H-1ax), 2.93 (2 H, t, CH2 acyloxyacyl), 2.96 (1 H, br.
s, H-5), 3.28 (1 H, br. s, H-1eq), 3.48 (2 H, br. s, H-3, H-4), 3.85
(2 H, dd, H2Ϫ6), 3.90 (1 H, br. d, H-2), 5.23 (1 H, m, CHO acyloxy-
acyl). Ϫ 13C{1H} NMR ([D4]MeOH): δ ϭ 14.37 (CH3 acyl),
23.68Ϫ42.00 (CH2 acyloxyacyl), 46.47 (C-1), 50.90 (C-2), 59.87 (C-
6), 62.15 (C-5), 71.20 (C-4), 72.70 (CHO acyloxyacyl), 75.60 (C-3),
174.68 (CϭO). Ϫ ES-MS; m/z: 516.5, [M ϩ H]ϩ; monoisotopic
MW calculated for C26H49N3O7 ϭ 515.4.
Acknowledgments
We thank S. H. van Krimpen for recording the NMR spectra and
A. G. Hulst for performing electrospray MS analyses. R. H. Mars-
Groenendijk and Dr G. P. van der Schans are acknowledged for
the immunizations of mice and for other immunotechnical contri-
butions. Dr. H. S. Overkleeft is acknowledged for stimulating dis-
cussions.
[1] [1a]
C. R. H. Raetz, Annu. Rev. Biochem. 1990, 59, 129Ϫ170. Ϫ
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2-(6-Aminohexanoyl)amino-1,2,5-trideoxy-1,5-imino-D-glucitol
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(19): To a cooled (0°C) and stirred solution of 15 (7.1 mg, 9.1
µmol) in DCM (0.5 mL) was added TFA (0.5 mL). After 30 min,
TLC analysis showed complete conversion of the starting material
into a compound with Rf ϭ 0.56 (DCM/MeOH, 92/8, v/v). The
mixture was concentrated in vacuo. Subsequently, the residue was
treated as described for the preparation of compound 17. Yield
5.5 mg (quantitative). Ϫ 1H NMR ([D4]MeOH): δ ϭ 1.42, 1.67,
2.28 (8 H, m, 4 ϫ CH2 hexanoyl), 2.87 (1 H, t, J1ax,1eq ϭ 12.7 Hz,
J1ax,2 ϭ 12.3 Hz, H-1ax), 2.92 (2 H, m, CH2 hexanoyl), 3.05 (1 H,
m, J4,5 ϭ 9.1 Hz, J5,6 ϭ 5.3 Hz, J5,6Ј ϭ 3.1 Hz, H-5), 3.38 (1 H,
dd, J1eq,2 ϭ 5.1 Hz, J1ax,1eq ϭ 12.7 Hz, H-1eq), 3.52 (2 H, m, J2,3 ϭ
9.6 Hz, J4,5 ϭ 9.1 Hz, H-3, H-4), 3.83 (1 H, dd, J5,6 ϭ 5.3 Hz,
J6,6Ј ϭ 11.8 Hz, H-6), 3.90 (1 H, dd, J5,6Ј ϭ 3.1 Hz, J6,6Ј ϭ 11.8 Hz,
H-6Ј), 3.99 (1 H, m, J1eq,2 ϭ 5.1 Hz, J2,3 ϭ 9.6 Hz, H-2). Ϫ
13C{1H} NMR ([D4]MeOH): δ ϭ 25.99, 26.88, 28.24, 36.47, 40.56
(5 ϫ CH2 hexanoyl), 45.85 (C-1), 50.01 (C-2), 59.17 (C-6), 62.03
(C-5), 70.56 (C-4), 75.11 (C-3). Ϫ ES-MS; m/z: 276.3, [M ϩ H]ϩ;
monoisotopic MW calculated for C12H25N3O4 ϭ 275.2.
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[13c] J. Yu, L. C. Hsieh, L. Koch-
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was treated as described for the preparation of compound 19. Yield
3.2 mg (73%). Ϫ 1H NMR ([D4]MeOH): δ ϭ 0.89 (3 H, t, CH3
acyloxyacyl), 1.28, 1.42, 1.65 (27 H, m, CH2 acyloxyacyl), 2.35 (2
H, t, CH2 acyloxyacyl), 2.50 (2 H, d, CH2 acyloxyacyl), 2.77 (1 H,
br. s, H-1ax), 2.93 (2 H, t, CH2 acyloxyacyl), 2.96 (1 H, br. s, H-
5), 3.28 (1 H, br. s, H-1eq), 3.48 (2 H, br. s, H-3, H-4), 3.85 (2 H,
m, H2Ϫ6), 3.90 (1 H, br. d, H-2), 5.23 (1 H, m, CHO acyloxyacyl).
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[13e]
116, 11197Ϫ11198. Ϫ
W. Dong, K. W. McCabe, M. Bols,
[13f]
M. R. Sierks, Adv. Gene Technol. 1995, 58. Ϫ
D. Shabat, S.
C. Sinha, J.-L. Reymond, E. Keinan, Angew. Chem. Int. Ed.
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[13h]
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J. Yu, S. Y. Choi, S. Lee, H.
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13C{1H} NMR ([D4]MeOH): δ ϭ 14.37 (CH3), 23.68Ϫ42.00
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[13i]
mun. 1997, 1957Ϫ1958. Ϫ
K. D. Janda, L.-C. Lo, C.-H. L.
(CH2 acyloxyacyl), 46.47 (C-1), 50.90 (C-2), 59.87 (C-6), 62.15 (C-
5), 71.20 (C-4), 72.70 (CHO acyloxyacyl), 75.60 (C-3), 174.68 (Cϭ
O). Ϫ ES-MS; m/z: 502.6, [M ϩ H]ϩ; monoisotopic MW calculated
for C26H51N3O6 ϭ 501.4.
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