Binding properties and esterase activity of monoclonal antibodies elicited against sucrose 6-heptylphosphonate

Article abstract of DOI:10.1016/S0008-6215(01)00199-9

Various sugar phosphonates were prepared by a Mitsunobu condensation between phosphonic diacids and properly protected carbohydrates. 6′-O-p-Aminophenylsucrose 6-heptylphosphonate was coupled to Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KL

Full text of DOI:10.1016/S0008-6215(01)00199-9

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Carbohydrate Research 334 (2001) 295–307
Binding properties and esterase activity of monoclonal
antibodies elicited against sucrose 6-heptylphosphonate
Marie-Christine Scherrmann,* Aure´lia Boutboul, Bernard Estramareix,
Anne-Sophie Hoffmann, Andre´ Lubineau*
Laboratoire de Chimie Organique Multifonctionnelle, Baˆtiment 420, Uni6ersite´ de Paris XI, F-91405 Orsay, France
Received 18 May 2001; accepted 10 July 2001
Abstract
Various sugar phosphonates were prepared by a Mitsunobu condensation between phosphonic diacids and
properly protected carbohydrates. 6%-O-p-Aminophenylsucrose 6-heptylphosphonate was coupled to Bovine Serum
Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH) and the KLH conjugate was used for generation of
monoclonal antibodies. Binding properties of these antibodies were screened by competitive enzyme-linked immuno-
sorbent assay (ELISA) using the BSA conjugate. A monoclonal antibody with good binding properties showed a
regioselective esterase activity toward 6-octanoylsucrose compared with 6%-octanoylsucrose. © 2001 Elsevier Science
Ltd. All rights reserved.
Keywords: Sugar phosphonate; Sucrose; Monoclonal antibodies; Abzyme
1. Introduction
was shown that monoclonal antibodies gener-
ated against phosphonates, which are transi-
tion state analogues for the hydrolysis of
esters, can also catalyse transesterification re-
actions.14–20 Following this concept, we fo-
cused on the generation of catalytic antibodies
designed for the preparation of monoesters of
sucrose 1 (Scheme 1). Sucrose esters can be
used as surfactants in the food, detergent and
cosmetic industries.21 Usually, these fatty acid
esters are prepared as mixtures, either by es-
terification of sucrose with acyl chlorides or
acyl chloroformates, or by transesterification
between sucrose and fatty esters.22–26 Pure and
clearly defined esters in a primary position
have been obtained using tributylstannylox-
ide.27 Selective acylation of unprotected su-
crose has also been accomplished with
3-acylthiazolidine-2-thiones introduced by
Plusquellec and coworkers.28–30 Alternatively,
these esters can also be prepared through bio-
Since the first examples of catalytic antibod-
ies reported by the groups of Lerner1 and
Schultz,2 a remarkable number of papers re-
ported that antibodies with good binding spe-
cificity toward an haptenic transition state
analogue could provide powerful tools for
regio- and stereoselective organic synthesis.3–6
Among these reports, some have dealt with
carbohydrate chemistry, most of the time to
obtain antibodies with glycosidase activity7–11
while only one group has reported an anti-
body which hydrolysed regio- and stereoselec-
tively acylated carbohydrates.12,13 Since the
very beginning of the discovery of abzymes, it
* Corresponding authors. Tel.: +33-1-69154719; fax: +
33-1-69154715.
E-mail address: mcscherr@icmo.u-psud.fr (M.-C. Scher-
rmann).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00199-9

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M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
Scheme 1. Conversion of sucrose to 6-octanoylsucrose proceeding through a tetrahedral intermediate.
2. Results and discussion
chemical methods. However, lipase-catalysed
esterifications of sucrose in aqueous buffers
gave rise to complex mixtures of mono-, di-,
and polyacylsucrose.31 In contrast, monoesters
at the 1% position have been obtained in anhy-
drous DMF32,33 or in pyridine34 with a
protease which catalysed the transesterifica-
tion between sucrose and activated esters
while the use of a lipase in tert-butyl alcohol35
led to the formation of a mixture of 6 and 6%
monoesters. Therefore, selective reactions that
could provide esters at the 6 position would be
valuable.
We decided to prepare compound 2 as hap-
ten to generate monoclonal antibodies. The
p-aminophenyl group installed at the 6% posi-
tion was designed to couple the hapten to a
carrier protein to obtain an immune response
in immunised mice. In order to evaluate the
recognition properties of the monoclonal anti-
bodies, other sugar phosphonates (11-14) were
also synthesised and used as inhibitors in com-
petitive enzyme-linked immunosorbent assay
(ELISA). We hereby report these results as
well as the catalytic properties of one mono-
clonal antibody toward the hydrolysis of sev-
eral sucro esters.
Synthesis of sugar phosphonates.—Hapten 2
was prepared by coupling heptylphosphonic
acid with compound 8 which was obtained in
seven steps from 2,3,4,6,1%,3%,4%-hepta-O-
acetylsucrose (3)36,37 according to Scheme 2.
The p-nitrophenyl group was introduced by a
Mitsunobu reaction using triphenylphosphine
and diisopropyl azodicarboxylate (DIAD) in
97% yield. After Zemple´n deacetylation and
hydrogenation in the presence of 10% palla-
dium-on-carbon catalyst in a 9:1 t-BuOH–
water mixture, 6 was obtained in quantitative
yield. Then, treatment with thioethyl trifl-
uoroacetate in methanol afforded 7 in 92%
yield. Compound 8 was then prepared in 52%
overall yield from 7 through a three-step pro-
cedure without purification of the intermedi-
ates. First, the OH-6 of polyol 7 was
regioselectively protected by treatment with
dimethoxytrityl chloride (DMTrCl) in pyri-
Scheme 2. (i) p-Nitrophenol, PPh3, DIAD, THF; (ii) MeONa,
MeOH; (iii) H2, Pd/C, t-BuOH–H2O; (iv) CF3COSEt,
MeOH; (v) (1) DMTrCl, pyridine, (2) Ac2O, pyridine (3)
TsOH, CH2Cl2–MeOH.

M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
297
Scheme 3. (i) (1) PPh3, DEAD, pyridine (2) LiOH aq 1 M, CH3CN then Dowex H+; (ii) Ac2O, MeOH, Et3N.
Following the same strategy, phosphonates
11 and 12 were prepared by reacting
2,3,1%,3%,4%,6%-hexa-O-acetylsucrose45 with hep-
tyl- or methyl-phosphonic acid respectively.
After treatment with LiOH, 11 was obtained
in 68% yield after C-18 flash chromatography,
while 12 was isolated as its triethylammonium
salt in 66% yield after DEAE-Sephadex A25
ion-exchange chromatography. The regioselec-
tivity of the phosphonylation was confirmed
dine, then peracetylation in the same pot with
acetic anhydride gave the fully protected
derivative in which selective hydrolysis of the
methoxytrityl ether (p-TsOH in 2:1 CH2Cl2–
MeOH) gave, after flash chromatography,
pure compound 8.
Phosphonic acid monoesters are usually
prepared by selective cleavage of symmetrical
or non-symmetrical phosphonate diesters.38
Another strategy is based on the direct mo-
noesterification of phosphonic acid with alco-
hol in the presence of a condensing reagent
such as 1,3-dicyclohexylcarbodiimide39 or
trichloroacetonitrile.40 However these methods
require a large excess of alcohol. Alterna-
tively, the synthesis of phosphonate esters has
been reported from phosphonic monoacids
and alcohols using Mitsunobu coupling condi-
tions.41,42 This strategy has been applied to
achieve phosphonylation and phosphorylation
of a purine nucleoside43 and yields were im-
proved by performing the reaction in anhy-
drous pyridine. This solvent effect was
explained by the enhanced nucleophilic reac-
tivity of the phosphate and the phosphonate
anions in the presence of pyridine.44 Accord-
ingly, we found that the Mitsunobu reaction
can provide monophosphonates when con-
ducted in pyridine under alcohol limiting con-
ditions. Hence, compound 8 was reacted with
heptylphosphonic acid (3 equiv), triphenyl-
phosphine and diethyl azodicarboxylate
(DEAD) in pyridine at 120 °C for 4 h (Scheme
3). As the resulting phosphonate was difficult
to purify at this stage, it was deprotected
directly with LiOH added to the crude mix-
ture. Pure 2 was obtained, after C-18 flash
chromatography, in 65% yield from 8. Then,
N-acetylation with acetic anhydride in MeOH
in the presence of triethylamine afforded 10 in
quantitative yield.
1
by H NMR. The two H-6 protons of 11 and
12 appeared as two ddd with 6.5 and 7.5 Hz
proton-phosphorous coupling constants. The
signals were simplified upon phosphorous ir-
radiation.
2,3,4,6,1%,3%,4%-Hepta-O-acetyl-
sucrose40,41 and methyl 2,3,4-tri-O-acetyl-a-
-
D
glucopyranoside46 were similarly reacted with
heptylphosphonic acid. Deacetylation and
purification by C-18 flash chromatography af-
forded 13 and 14 in 67 and 70% yields,
respectively.
Hapten coupling to carrier proteins. Produc-
tion of monoclonal antibodies.—To produce
antibodies, animals must be immunised with
an hapten–protein conjugate. Two strategies
were tested to carry out the conjugation. In
the first one, maleimide-activated proteins47
were used. Coupling was performed by a
three-step procedure. At first, compound 2
was reacted with disuccinimido dithiobispropi-
onate 15 in DMF (Scheme 4). The disulfide 16
was then reduced with n-tributylphosphine in
MeOH–water to give the thioderivative 17,
which was coupled to the maleimide-activated
carrier proteins MA-KLH and MA-BSA to
give the conjugates MA-KLH-2 and MA-
BSA-2, respectively. Quantification of the
hapten–carrier ratio (see Section 4) afforded
ratios in the range 4:1.
In the second strategy, the conjugation was
carried out using the heterobifunctionnal glu-
taryl reagent 18,48 to afford KLH-2 and BSA-

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M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
A standard protocol for cell fusion was
adopted and the resulting hybridoma cells
were screened by an ELISA technique for
binding to the hapten-BSA conjugate BSA-2.
Cloning by limiting dilution gave seven mono-
2 (Scheme 5). This method proved to be more
efficient since the hapten-to-protein ratios
were higher (16:1).
Then, keyhole limpet hemocyanin conjugate
KLH-2 was used to immunise mice (Balb/c).
Scheme 4. (i) DMF; (ii) n-Bu3P, MeOH–water; (iii) phosphate buffer pH 7.3.
Scheme 5. (i) DMF, Et3N; (ii) pyrophosphate buffer pH 8, BSA of KLH.

M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
299
Table 1
The reaction was monitored by an enzymatic
determination of sucrose.50 The described pro-
tocol was adapted in order to carry out the
measurement in 96-well polyvinyl assay plate
(ELISA plate, see Section 4). An aliquot of
the reacting mixture was treated with b-fruc-
tosidase (from yeast) at pH 4.6. The sucrose
obtained in the abzymatic reaction gave fruc-
tose and glucose while the remaining oc-
tanoylsucrose gave fructose and 6-octanoyl-
glucose. Hexokinase (HK) was then added at
pH 7.6 to catalyse the phosphorylation of
glucose and fructose by adenosine-5%-triphos-
phate (ATP). The glucose 6-phosphate ob-
tained was oxidised in gluconate-6-phosphate
with nicotinamide-adenine-dinucleotide-phos-
phate (NADP) in the presence of glucose-6-
phosphate dehydrogenase (G6P-DH) with the
formation of an equimolar amount, relative to
sucrose, of reduced nicotinamide-adenine-din-
ucleotide-phosphate (NADPH). The amount
of NADPH was determined by its absorbance
at 340 nm. Only the antibody XA6 showed
hydrolytic activity. In the initial screening, the
hydrolysis was tested at pH 8 (Bicine buffer),
37 °C with 10% Me2SO with an initial concen-
tration of the sucro-ester of 0.1 mM and an
antibody concentration of 4 mM. Parallel ex-
periments were carried out in the presence of
phosphonate 11 (10−2 mM). Antibody XA6
proved to accelerate the reaction with respect
to the uncatalysed hydrolysis (in the absence
of antibody). Furthermore, the acceleration
was completely suppressed in the presence of
compound 11, demonstrating that catalysis
took place in the combining site of the anti-
body. For this antibody, experiments were
repeated in the same conditions (concentra-
tion and temperature) but at different pH
values, ranging from 4.5 to 9. The optimal pH
was found to be around 7. This antibody was
then further studied at pH 7.35. The antibody-
catalysed hydrolysis rates of 6-octanoylsucrose
in the presence of 9 mM antibody were mea-
sured as a function of substrate concentration
under the conditions used in the aforemen-
tioned screening. The ester concentrations
were varied from 0.11 to 1.1 mM and the
observed rates were corrected for the un-
catalysed rate of hydrolysis. Antibody XA6
IC50 in mM for phosphonates-antibodies binding, determined
by competitive ELISA
Antibody
Phosphonates
10
11
12
13
14
50
240
7
87
140
24
60
20
10A10
7E8
XA6
1D11
8C8
1
14
nd
nd
nd 10
nd 15
1
5
3
5
\500
\500
\500
\500
\500
\500
\500
\500
\500
\500
\500
70
\500
\500
100
9.5
\500
\500
\500
\500
3G10
7E11
15
15
200
nd: not determined.
clonal antibodies specific for hapten 2 (the
N-acetylated hapten 10 was used as inhibitor).
They were purified by chromatography on
protein A-coupled Sepharose 4B.
Binding properties of monoclonal antibod-
ies.—In order to evaluate the relative contri-
bution of the transition-state structural
elements to binding, the affinity for the vari-
ous phosphonates 10–14 as well as for the
methyl ester 2049 was determined by competi-
tive inhibition enzyme-linked immunoassay
and was expressed as IC50 in mM (Table 1).
All the antibodies revealed a binding
affinity for the sucrose-6-heptylphosphonate
(11) showing that the p-acetamidophenyl
group did not contribute to a great extent to
the recognition. Only XA6 bound to the re-
gioisomer 13. Phosphonate 12 without the
hydrophobic chain did not appear to bind up
to 500 mM, whereas binding of 20 without
sugar is weak or non existent; these results
suggested that both the fatty acid chain and
the sugar are important for the recognition
process. Furthermore, the binding properties
of the glucoside derivative 14 revealed a rela-
tive importance of the fructose unit in the
recognition.
Esterase acti6ity of antibodies.—All the an-
tibodies were initially tested for their esterase
properties toward 6-octanoylsucrose (1).26,30

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M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
3. Conclusions
was found to obey Michaelis–Menten kinetics
for the hydrolysis of 6-octanoylsucrose. The
We developed a straightforward access to
sugar phosphonates from phosphonic acids
using a Mitsunobu reaction. This allowed us
to prepare a hapten for the generation of
monoclonal antibodies toward sucrose-6-hep-
tylphosphonate and other structurally related
phosphonates to elicit binding properties of
the antibodies. Seven monoclonal antibodies
specific for hapten 2 were obtained. Competi-
tive inhibition ELISA revealed that both the
fatty acid chain and the sugar are important
for the recognition process. Among the seven
antibodies, one proved to be catalytic for the
hydrolysis of 6 and 6%-octanoylsucrose with a
regioselectivity for the ester at C-6. This
abzyme has now to be tested for ester synthe-
sis in thermodynamic (reverse of hydrolysis)
or kinetic (transesterification) conditions.
corrected rates were analysed by
a
Lineweaver–Burk plot to determine the
Michaelis–Menten parameters. The first-order
rate constant kcat was 6.5 h−1 and Michaelis
constant KM was 0.25 mM. The background
hydrolysis rate constant (kuncat) was 2.4×
10−3
h
−1. The hydrolytic properties of this
antibody were then examined toward 6%-
octanoylsucrose26,28 as well as toward methyl
6-octanoyl-a-
-glucopyranoside.51 In this
D
case, the abzymatic reaction was also moni-
tored by an enzymatic method. In the proto-
col described above, b-fructosidase was
replaced with a-glucosidase (maltase recombi-
nant from Saccharomyces cere6isiae). The es-
ters’ concentrations were varied from 0.1 to 1
mM and the observed rates were corrected for
the uncatalysed rate of hydrolysis. The
Michaelis–Menten parameters obtained by a
Lineweaver–Burk plot are shown in Table 2
as well as the kinetic parameters determined
for 6-octanoylsucrose as substrate.
4. Experimental
General methods.—All moisture-sensitive
reactions were performed under Ar atmo-
sphere using oven-dried glassware. All sol-
vents were dried over standard drying agents
and freshly distilled prior to use. Flash-
column chromatography was performed on
Silica Gel 60A C.C. (6–35 mm, SDS) or on
Lichroprep RP-18 (25–40 mm, E. Merck). Re-
actions were monitored by TLC on Silica Gel
(E. Merck) 60 F254 with detection by charring
with H2SO4. Melting points were determined
with a capillary apparatus and are uncor-
rected. Optical rotations were measured at
2692 °C. NMR spectra were recorded at rt
with Bruker AC 200, AC 250 or AM 400
spectrometers. 85% H3PO4 was used as exter-
XA6 showed a significant rate acceleration
for the sucrose esters hydrolysis with values of
kcat/kuncat of 2.6×103 and 1.2×103 whereas
the acceleration with methyl-6-octanoyl-a- -
D
glucopyranoside was only 529. Thus, abzyme
XA6 can distinguish regioisomers as shown by
the specific constants (kcat/KM) 4.5-fold higher
for 6-octanoylsucrose with respect to 6%-oc-
tanoylsucrose. This is mainly due to a varia-
tion of kcat since KM is similar for the two
esters. It is also interesting to note that the
rate enhancements and the specific constants
showed great differences for sucrose deriva-
tives and methyl 6-octanoyl-a- -glucopyran-
D
31
oside (Table 2).
nal standard for P NMR. Mass spectra were
Table 2
Michaelis–Menten parameters for XA6 toward various substrates (pH 7.35; 37 °C; 10% Me2SO)
6-Octanoylsucrose
6%-Octanoylsucrose
Methyl-6-octanoyl-a-D-glucopyranoside
kcat (h−1
)
6.50
0.25
1.70
0.9
1.0
KM (mM)
0.30
kuncat (h−1
)
2.4 10−3
26
1.4 10−3
5.7
1.7 10−3
0.9
k
k
cat/KM (h−1/mM−1
)
cat/kuncat
2.6 103
1.2 103
529

M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
301
(dd, 1 H, J2,3 9.5, J3,4 9.5 Hz, H-3), 3.50 (d, 1
H, J1%a,1%b 11.0 Hz, H-1%a), 3.49 (dd, 1 H, J5,6b
6.5 J6a,6b 12.0 Hz, H-6b), 3.44 (d, 1 H, H-1%b),
3.24 (dd, 1 H, H-2), 3.10 (dd, 1 H, H-4); 13C
NMR (CD3OD, 62.9 MHz): l 165.4 (CꢀNO2),
142.7 (ArCꢀO), 126.7 and 115.9 (4 ArC),
105.5 (C-2%), 93.4 (C-1), 81.3 (C-5%), 78.7 (C-
3%), 76.4 (C-4%), 74.7 (C-3), 74.4 (C-2), 73.2
(C-5), 71.6 (C-4), 71.5 (C-6%), 63.6 (C-1%), 62.7
(C-6). Anal. Calcd for C18H25NO13·0.5 H2O:
C, 45.77; H, 5.55; N, 2.96. Found: C, 45.74;
H, 5.63; N, 3.06.
recorded on a MAT 95S instrument. Elemen-
tal analyses were performed at the CNRS
Microanalytical Laboratory (Gif-sur-Yvette,
France).
1%,2,3,3%,4,4%,6-Hepta-O-acetyl-6%-O-p-nitro-
phenylsucrose (4).—To
a
solution of
2,3,4,6,1%,3%,4%-hepta-O-acetylsucrose (10.0 g,
15.7 mmol), p-nitrophenol (2.6 g, 1.2 equiv)
and triphenylphosphine (5.6 g, 1.5 equiv) in
toluene (150 mL) was added diisopropylazodi-
carboxylate (4.5 mL, 1.5 equiv) over a 5 min
period. After 12 h the mixture was concen-
trated and the residue was purified by flash
chromatography (1:2 petroleum ether–
EtOAc) to afford 4 (11.5 g, 97%); mp 95 °C
(from EtOAc–petroleum ether); [h]D 61° (c
1.02, CHCl3); IR (KBr): w 2963, 1751, 1594,
6%-O-p-Aminophenylsucrose (6).—A vigor-
ously stirred mixture of 5 (7.0 g, 15,1 mmol)
and 10% Pd on activated carbon (1.2 g) in 9:1
t-BuOH–water (140 mL) was degassed under
diminished pressure and saturated with hydro-
gen (by a H2-filled balloon) three times. The
suspension was stirred for an additional 3 h at
rt under a slightly positive pressure of H2,
then filtered through a pad of Celite, and
concentrated to afford 6 (6.5 g) as a white
solid in quantitative yield; mp 111 °C; [h]D 70°
(c 0.4, MeOH); IR (KBr): w 3380, 2924, 1635,
1
1517, 1371, 1344, 1224, 1038 cm−1; H NMR
(CDCl3, 250 MHz): l 8.19–8.27 (m, 2 H, Ar),
7.01–7.11 (m, 2 H, Ar), 5.71 (d, 1 H, J1,2 3.5
Hz, H-1), 5.55 (d, 1 H, J3%,4% 5.5 Hz, H-3%), 5.48
(dd, 1 H, J2,3 10.5, J3,4 9.6 Hz, H-3), 5.47 (dd,
1 H, J4%,5% 5.5 Hz, H-4%), 5.05 (t, 1 H, J4,5 9.6
Hz, H-4), 4.85 (dd, 1 H, H-2), 4.06–4.46 (m, 8
H, 2 H-1%, H-5, H-5%, 2 H-6, 2 H-6%), 2.18,
2.14, 2.13, 2.10, 2.08, 2.03 and 1.94 (7 s, 21 H,
7 COCH3); 13C NMR (CDCl3, 62.9 MHz): l
169.7–170.9 (7 COCH3), 163.0 (CꢀNO2),
141.9 (ArCO), 125.9 and 114.7 (4 ArC), 104.3
(C-2%), 90.2 (C-1), 79.4 (C-5%), 75.7 (C-3%), 75.2
(C-4%), 70.2 (C-2), 69.2 (C-5), 68.8 (C-6%), 68.4
and 68.1 (C-3 and C-4), 62.4 (C-1%), 61.6 (C-
6), 20.8–21.1 (7 COCH3). Anal. Calcd for
C32H39NO20: C, 50.73; H, 5.19; N, 1.85; O,
42.23. Found: C, 50.56; H, 5.48; N, 1.94; O,
42.11.
1
1512, 1456, 1236, 1137, 1059 cm−1; H NMR
(CD3OD, 250 MHz): l 6.5–6.7 (m, 4 H, Ar),
5.27 (d, 1 H, J1,2 3.5 Hz, H-1), 4.12 (dd, 1 H,
J5%,6%a 7.0, J6%a,6%b 12.0 Hz, H-6%a), 3.8–4.0 (m, 4
H, H-6%b, H-3%, H-5%, H-4%), 3.68 (ddd, 1 H,
J4,5 9.6, J5,6a 2.0, J5,6b 5.0 Hz, H-5), 3.64 (dd, 1
H, J6a,6b 11.5 Hz, H-6a), 3.55 (dd, 1 H, J2,3 9.6,
J3,4 9.6 Hz, H-3), 3.52 (dd, 1 H, H-6b), 3.47 (s,
2 H, 2 H-1%), 3.25 (dd, 1 H, H-2), 3.17 (dd 1
H, H-4); 13C NMR (CD3OD, 62.9 MHz): l
153.4 (CꢀNH2), 141.8 (ArCꢀO), 118.2 and
116.7 (4 ArC), 105.5 (C-2%), 93.4 (C-1), 81.5
(C-5%), 78.8 (C-3%), 76.5 (C-4%), 74.7 (C-3), 74.1
(C-2), 73.2 (C-5), 71.4 (C-4), 71.2 (C-6%), 63.7
(C-1%), 62.4 (C-6). Anal. Calcd for
C18H27NO11·0.5H2O: C, 48.87; H, 6.38; N,
3.17. Found: C, 48.26; H, 6.39; N, 3.23.
6% - O - p - Trifluoroacetamidophenylsucrose
(7).—S-Ethyltrifluorothioacetate (9.5 mL,
74.5 mmol, 5 equiv) was added to a solution
of 6 (6.2 g, 14.3 mmol) in MeOH (30 mL).
The resulting solution was kept at rt for 5 h,
then diluted with 60 mL of water and washed
with 2×40 mL portions of hexane. The
aqueous layer was concentrated to give 7 as a
white solid. (7.0 g, 92%); mp 108 °C; [h]D 55°
(c 0.5, MeOH); IR (KBr): w 3378, 2930, 1710,
6%-O-p-Nitrophenylsucrose (5).—A solution
of 4 (11.0 g, 14.5 mmol) in MeOH (150 mL)
was made basic by the addition of NaOMe
(80 mg, 1.4 mmol). After 2 h, the mixture was
neutralized with Dowex-50 (H+) resin,
filtered, and concentrated to give 5 as a white
solid (6.7 g, 100%); mp 115 °C; [h]D 62° (c 0.4,
MEOH); IR (KBr): w 3400, 2922, 1592, 1507,
1
1438, 1341, 1271, 1181, 1119, 1056 cm−1; H
NMR (CD3OD, 200 MHz): l 8.00–8.10 (m, 2
H, Ar), 6.95–7.05 (m, 2 H, Ar), 5.21 (d, 1 H,
J1,2 3.5 Hz, H-1), 4.47 (dd, 1 H, J5%,6%a 7.0,
J6%a,6%b 10.6 Hz, H-6%a), 4.1 (dd, 1 H, J5%,6%b 1.5
Hz, H-6%b), 3.9–4.02 (m, 3 H, H-3%, H-4%,
H-5%), 3.65–3.80 (m, 2 H, H-5, H-6a), 3.53

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M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
1
1513, 1205, 1158 cm−1; H NMR (CD3OD,
250 MHz): l 7.3–7.4 (m, 2 H, Ar), 6.8–6.9
(m, 2 H, Ar), 5.27 (d, 1 H, J1,2 3.5 Hz, H-1),
4.26 (dd, 1 H, J5%,6%a 7.0, J6%a,6%b 10.6 Hz, H-6%a),
3.85–4.05 (m, 4 H, H-6%b, H-3%, H-5%, and
H-4%), 3.70 (ddd, 1 H, J4,5 9.6, J5,6a 2.0, J5,6b
5.5 Hz, H-5), 3.66 (dd, 1 H, J6a,6b 11.5 Hz,
H-6a), 3.55 (dd, 1 H, J2,3 9.6, J3,4 9.6 Hz, H-3),
3.51 (dd, 1 H, H-6b), 3.47 (s, 2 H, 2 H-1%),
3.25 (dd, 1 H, H-2), 3.17 (dd, 1 H, H-4); 13C
H-6a), 3.50 (ddd, 1 H, J6b,OH 5.0 Hz, H-6b),
2.44 (dd, 1 H, OH), 2.13, 2.12, 2.07, 2.03, and
13
1.97 (6 s, 6 COCH3). C NMR (CDCl3, 62.9
MHz): l 170.7–169.5 (6 COCH3), 156.0 (C-
NH), 154.6 (q, JC,F 37.0 Hz, COCF3), 129.0
(ArCꢀO), 124.3 (2 ArC), 115.7 (q, JC,F 288.0
Hz, CF3), 112.8 (2 ArC), 103.9 (C-2%), 89.8
(C-1), 79.4 (C-5%), 75.7 (C-3%), 74.8 (C-4%), 70.6
(C-3), 70.3 (C-2), 69.2 (C-5), 68.5 (C-4), 67.7
(C-6%), 62.6 (C-1%), 60.8 (C-6), 20.5–20.8 (6
COCH3). Anal. Calcd for C32H38F3NO18: C,
49.17; H, 4.90; N, 1.79, Found: C, 49.01; H,
4.97; N, 1.62.
NMR (CD3OD, 62.9 MHz):
l
158.1
(CꢀNHCOCF3), 130.6 (ArCꢀO), 156.5 (q, JC,F
37.0 Hz, COCF3), 123.8 (2 ArC), 117.5 (q,
JC,F 287.0 Hz, CF3), 115.9 (2 ArC), 105.5
(C-2%), 93.4 (C-1), 81.5 (C-5%), 78.8 (C-3%), 76.5
(C-4%), 74.7 (C-3), 74.2 (C-2), 73.2 (C-5), 71.5
(C-4), 70.8 (C-6%), 63.7 (C-1%), 62.5 (C-6).
Anal. Calcd for C20H26F3NO12·2H2O: C,
42.48; H, 5.35; N, 2.48. Found: C, 42.74; H,
5.32; N, 2.19.
6%-O-p-Aminophenyl-6-heptylphosphonatosu-
crose (2).—To a stirred solution of 8 (1.0 g,
1.3 mmol), heptylphosphonic acid 9 (702 mg,
3.9 mmol), and triphenylphosphine (928 mg,
3.9 mmol), in dry Py (2 mL) at 120 °C, was
added DEAD (640 mL, 3.9 mmol). Stirring
was continued at 120 °C for 4 h, then the
mixture was cooled to rt and concentrated.
The residue was dissolved in MeCN (3 mL),
then 1 M aq LiOH (3 mL) was added under
vigorous stirring. After 1 h, the mixture was
neutralised with Dowex-50 (H+) resin, filtered
and concentrated. The residue was triturated
in a 1:1 MeOH–water mixture (20 mL) and
filtered. The filtrate was concentrated and the
residue was purified by C18 flash chromatog-
raphy (step gradient from 9:1 water–MeOH
to pure MeOH) to afford 2 (502 mg, 65%) as
a colorless foam; [h]D 56° (c 0.35, MeOH); IR
(KBr): w 3374, 2928, 2361, 1635, 1512, 1456,
2,3,4,1%,3%,4% - Hexa - O - acetyl - 6% - O - p - tri-
fluoroacetamidophenylsucrose (8).—A solution
of 7 (5.8 g, 10.9 mmol) and dimethoxy-
tritylchloride (4.5 g, 13.2 mmol) in Py (25 mL)
was kept at rt for 40 h. The mixture was then
diluted with Py (15 mL) and Ac2O (15 mL)
was added. After 12 h, the mixture was con-
centrated to dryness. The residue was treated
with a solution of p-TsOH (5 g, 29 mmol) in
a 2:1 CH2Cl2–MeOH mixture (180 mL) at
0 °C under vigorous stirring. After an addi-
tional hour at 0 °C the mixture was neutral-
ized with 5% aq NaHCO3 and extracted with
CH2Cl2 (250 mL). The organic layer was
washed with water, dried (MgSO4) and con-
centrated. The residual syrup was purified by
flash chromatography (2:1 CH2Cl2–EtOAc) to
afford 8 as a white solid (4.4 g, 52%); mp
86 °C (from EtOAc–cyclohexane); [h]D 64° (c
0.9, CHCl3); IR (KBr): w 3480, 3320, 2970,
1252, 1143, 1059, 997 cm−1
.
1H NMR
(CD3OD, 250 MHz): l 7.0–7.1 (m, 2 H, Ar),
6.95-6.85 (m, 2 H, Ar), 5.28 (d, 1 H, J1,2 3.5
Hz, H-1), 4.34 (dd, 1 H, J5%,6%a 8.0, J6%a,6%b 11.0
Hz, H-6%a), 3.7-4.1 (m, 7 H, H-6%b, H-5, H-6a,
H-6b, H-3%, H-4%, H-5%), 3.53 (dd, 1 H, J2,3 9.6,
J3,4 9.6 Hz, H-3), 3.44 (s, 2 H, 2 H-1%), 3.22
(dd, 1 H, H-2), 3.11 (dd, 1 H, J4,5 9.6 Hz,
H-4), 1.4–1.5 (m, 4 H, 2 CH2), 1.00–1.15 (m,
8 H, 4 CH2), 0.67–0.77 (m, 3 H, CH3); 13C
NMR (CD3OD, 62.9 MHz): l 155.4, 137.1,
120.1, and 116.9 (6 ArC), 105.2 (C-2%), 92.9
(C-1), 81.5 (C-5%), 78.3 (C-3%), 75.7 (C-4%), 74.6
(C-3), 73.6 (d, JC,P 6.9 Hz, C-5), 73.4 (C-2),
71.5 (C-4), 71.3 (C-6%), 64.7 (C-6), 54.8 (C-1%),
33.0 (Pꢀ(CH2)3ꢀCH2ꢀ), 32.3 (d, JC,P 16.6 Hz,
Pꢀ(CH2)2ꢀCH2ꢀ), 30.2 (Pꢀ(CH2)4ꢀCH2ꢀ), 28.0
(d, JC,P 135 Hz, PꢀCH2ꢀ), 24.7 (d, JC,P 3.7 Hz,
1755, 1380, 1240, 1160, 1040 cm−1; H NMR
1
(CD3OD, 250 MHz): l 7.90 (s, 1 H, NH),
7.46–7.55 (m, 2 H, Ar), 6.92–7.01 (m, 2 H,
Ar), 5.79 (d, 1 H, J1,2 4.0 Hz, H-1), 5.55 (d, 1
H, J3%,4% 5.5 Hz, H-3%), 5.52 (dd, 1 H, J2,3 10.5,
J3,4 10.0 Hz, H-3), 5.50 (dd, 1 H, J4%,5% 5.5 Hz,
H-4%), 4.97 (dd, 1 H, J4,5 10.0 Hz, H-4), 4.82
(dd, 1 H, H-2), 4.40 (ddd, 1 H, J5%,6%a 4.0, J5%,6%b
6.0 Hz, H-5%), 4.20–4.31 (m, 4 H, 2 H-6 and 2
H-1%), 4.13 (ddd, 1 H, J5,6a 2.0, J5,6b 4.5 Hz,
H-5), 3.63 (ddd, 1 H, J6a,OH 8.0, J6a,6b 12.5 Hz,

M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
303
(KBr): w 3387, 2926, 2856, 1652, 1467, 1178,
PꢀCH2ꢀCH2ꢀ), 23.7 (Pꢀ(CH2)5ꢀCH2ꢀ), 14.5
1
1041 cm−1; H NMR (CD3OD, 400 MHz): l
31
(CH3); P NMR (CD3OD, 100 MHz): l 28.7;
5.36 (d, 1 H, J1,2 3.5 Hz, H-1), 4.08 (ddd, 1 H,
J6a,6b 11.5, J6a,P 6.5, J5,6a 2.0 Hz, H-6a), 4.06–
4.09 (m, 2 H, H-3% and H-4%), 4.02 (ddd, 1 H,
J6b,P 7.5, J5,6b 4.5 Hz, H-6b), 3.93 (ddd, 1 H,
J4,5 9.5 Hz, H-5), 3.74–3.83 (m, 3 H, H-5%,
H-6%a, and H-6%b), 3.72 (dd, 1 H, J2,3 9.5, J3,4
9.5 Hz, H-3), 3.61 (d, 1 H, J1%a,1%b 12.0 Hz,
H-1%a), 3.57 (d, 1 H, H-1%b), 3.43 (dd, 1 H,
H-2), 3.40 (dd, 1 H, H-4), 1.52–1.65 (m, 4 H,
(CH2)2), 1.27–1.40 (m, 8 H, (CH2)4), 0.87–
ESI–HRMS Calcd for C25H42NO13PꢀH:
594.2315. Found 594.2312. Anal. Calcd for
C25H42NO13P·1.5 H2O: C, 48.23; H, 7.29; N,
2.25. Found: C, 48.29; H, 7.03; N, 2.02.
6%-p-Acetamidophenyl-6-heptylphosphonato-
sucrose (10).—To a solution of 2 (70 mg, 0.12
mmol) in MeOH (500 mL) was added Ac2O
(60 mL) and Et3N (50 mL, 0.36 mmol). The
solution was kept at rt for 2 h, then diluted
with water (500 mL) and concentrated to af-
ford quantitatively 10 (75 mg) as an amor-
phous solid; [h]D 60° (c 0.5, MeOH); IR
(KBr): w 3417, 2927, 1657, 1512, 1460, 1409,
13
0.93 (m, 3 H, CH3); C NMR (CD3OD, 62.9
MHz): l 105.0 (C-2%), 93.2 (C-1), 83.5 (C-5%),
79.0 (C-3%), 75.8 (C-4%), 74.3 (C-3), 73.5 (d, JC,P
6.9 Hz, C-5), 73.2 (C-2), 71.1 (C-4), 64.3 (C-6),
64.1 (C-6%), 63.7 (C-1%), 33.0 (Pꢀ(CH2)3ꢀ
CH2ꢀ), 2.3 (d, JC,P 16.6 Hz, Pꢀ(CH2)2ꢀCH2ꢀ),
30.2 (Pꢀ(CH2)4ꢀCH2ꢀ), 28.0 (d, JC,P 135 Hz,
PꢀCH2ꢀ), 24.7 (d, JC,P 3.7 Hz, PꢀCH2ꢀCH2ꢀ),
23.7 (Pꢀ(CH2)5ꢀCH2ꢀ), 14.5 (CH3); 31P NMR
(CD3OD, 100 MHz): l 32.0; ESI–HRMS
Calcd for C19H37O13PꢀH: 503.1893. Found
503.1882. Anal. Calcd for C19H37O13P.0.5
H2O: C, 44.44; H, 7.46; Found: C, 43.13; H,
7.47.
1
1243, 1143, 1060 cm−1; H NMR (CD3OD,
250 MHz): l 7.38–7.46 (m, 2 H, Ar), 6.90–
6.98 (m, 2 H, Ar), 5.38 (d, 1 H, J1,2 3.5 Hz,
H-1), 4.45 (dd, 1 H, J5%,6%a 8.0, J6%a,6%b 11.0 Hz,
H-6%a), 3.87–4.25 (m, 7 H, H-6%b, H-5, H-6a,
H-6b, H-3%, H-4%, and H-5%), 3.71 (dd, 1 H, J2,3
9.6, J3,4 9.6 Hz, H-3), 3.61 (s, 2 H, 2 H-1%),
3.42 (dd, 1 H, H-2), 3.32 (dd, 1 H, J4,5 9.6 Hz,
H-4), 1.50–1.60 (m, 4 H, 2 CH2), 1.20–1.32
(m, 8 H, 4 CH2), 0.82–0.91 (m, 3 H, CH3); 13C
NMR (CD3OD, 62.9 MHz): l 171.2 (CO),
156.9 133.0, 122.8, and 115.8 (6 ArC), 105.2
(C-2%), 92.9 (C-1), 81.7 (C-5%), 78.3 (C-3%), 75.6
(C-4%), 74.6 (C-3), 73.7 (d, JC,P 6.9 Hz, C-5),
73.4 (C-2), 71.5 (C-4), 70.9 (C-6%), 65.0 (C-6),
64.0 (C-1%), 32.9 (Pꢀ(CH2)3ꢀCH2ꢀ), 32.4 (d,
JC,P 16.6 Hz, Pꢀ(CH2)2ꢀCH2ꢀ), 30.2
(Pꢀ(CH2)4ꢀCH2ꢀ), 28.0 (d, JC,P 135 Hz,
PꢀCH2ꢀ), 24.8 (d, JC,P 3.7 Hz, PꢀCH2ꢀCH2ꢀ),
23.7 (Pꢀ(CH2)5ꢀCH2ꢀ), 23.5 (COCH3), 14.5
Methylphosphonatosucrose
triethylammo-
nium salt (12).—2,3,1%,3%,4%,6%-Hexa-O-acetyl-
sucrose (300 mg, 0.5 mmol) was treated with
methylphosphonic acid (96 mg, 1.5 mmol),
triphenylphosphine (360 mg, 1.5 mmol) and
DEAD (250 mL, 1.5 mmol) in Py (800 mL) as
described for the preparation of 2. After
LiOH treatment (see above) and DEAE-Sep-
hadex A 25 (HCO
form) chromatography
−
3
31
(0.2 M bicarbonate triethylammonium), 12
was obtained as a white amorphous solid (172
mg, 66%); [h]D 39° (c 0.69, MeOH); IR (KBr):
w 3414, 2937, 2738, 2678, 2491, 1652, 1476,
(CH3); P NMR (CD3OD, 100 MHz): l 28.7;
ESI–HRMS calcd for C27H44NO14P-H:
636.2421. Found 636.2422. Anal. Calcd for
C27H44NO14P·3 H2O: C, 46.89; H, 7.29; N,
2.03. Found: C, 46.81; H, 6.85; N, 2.08.
1
1171, 1036 cm−1; H NMR (CD3OD, 400
MHz): l 5.36 (d, 1 H, J1,2 3.5 Hz, H-1), 4.09
(ddd, 1 H, J6a,6b 11.5, J6a,P 6.5, J5,6a 2.0 Hz,
H-6a), 4.04–4.07 (m, 2 H, H-3% and H-4%),
4.01 (ddd, 1 H, J6b,P 7.5, J5,6b 5.0 Hz, H-6b),
3.94 (ddd, 1 H, J4,5,9.5 Hz, H-5), 3.74–3.84
(m, 3 H, H-6%a, H-6%b, and H-5%), 3.70 (dd, 1
H, J2,3 9.5, J3,4 9.5 Hz, H-3), 3.61 (d, 1 H,
J1%a,1%b 11.5 Hz, H-1%a), 3.57 (d, 1 H, H-1%b),
3.41 (dd, 1 H, H-2), 3.36 (dd, 1 H, H-4), 3.19
(q, 6 H, J 7.0 Hz, (CH3CH2)3N), 1.32 (t, 9 H,
(CH3CH2)3N), 1.23 (d, 3 H, JH,P 16.5 Hz,
6-Heptylphosphonatosucrose
(11).—
2,3,1%,3%,4%,6%-Hexa-O-acetylsucrose (400 mg,
0.67 mmol) was treated with heptylphospho-
nic acid (364 mg, 2.0 mmol), triphenylphos-
phine (480 mg, 2.0 mmol) and DEAD (330
mL, 2.0 mmol) in Py (1 mL) as described for
the preparation of 2. After LiOH treatment
(see above) and C-18 flash chromatography
(step gradient from 9:1 water–MeOH to pure
MeOH), 11 (230 mg, 68%) was obtained as a
white powder; [h]D 38° (c 0.7, MeOH); IR

304
M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
CH3); 13C NMR (CD3OD, 62.9 MHz): l 105.0
(C-2%), 93.1 (C-1), 83.7 (C-5%), 78.9 (C-3%), 75.8
(C-4%), 74.4 (C-3), 73.4 (d, JC,P 9.5 Hz, C-5),
73.3 (C-2), 71.2 (C-4), 64.5 (d, JC,P 5.0 Hz,
C-6), 64.0 and 63.9 (C-1% and C-6%), 47.5
((CH3CH2)3N), 12.3 (d, JC,P 138 Hz, PꢀCH3),
9.12 ((CH3CH2)3N). 31P NMR (CD3OD, 100
chromatography, 14 (249 mg, 70%) was ob-
tained as a white amorphous solid. [h]D 67° (c
0.53, MeOH); IR (KBr): w 3414, 2927, 2854,
1
1186, 1030 cm−1; H NMR (CD3OD, 400
MHz): l 4.64 (d, 1 H, J1,2 3.6 Hz, H-1), 3.77
(ddd, 1 H, J5,6 4.5, J6,P 7.5, J6,6% 11.5 Hz, H-6),
3.70 (ddd, 1 H, J5,6% 2.0, J6%,P 6.5 Hz, H-6%),
3.62 (dd, 1 H, J2,3 9.0, J3,4 9.0 Hz, H-3), 3.57
(ddd, 1 H, J4,5 9.5 Hz, H-5), 3.44 (dd, 1 H, J3,4
9.0 Hz, H-4), 3.40 (dd, 1 H, H-2), 3.39 (s, 3 H,
OCH3), 1.51–1.65 (m, 4 H, 2 CH2), 1.26–1.41
(m, 8 H, (CH2)4), 0.09 (t, 3 H, J 7.0 Hz, CH3);
13C NMR (CD3OD, 62.9 MHz): l 101.3 (C-1),
72.8 (d, JC,P 5.7 Hz, C-5), 74.7 (C-4), 73.6
(C-2), 71.1 (C-4), 64.5 (d, JC,P 5.2 Hz, C-6),
55.6 (OCH3), 32.9 (P(CH2)3CH2), 32.3 (d, JC,P
16.7 Hz, P(CH2)2CH2), 30.1 (P(CH2)4CH2),
28.1 (d, JC,P 136.4 Hz, PCH2), 24.7 (d, JC,P 4.7
Hz, PCH2CH2), 23.7 (P(CH2)5CH2), 14.4
MHz):
l
26.4. ESI–HRMS Calcd for
C13H25O13PꢀH: 419.0954. Found 419.0949.
6%-Heptylphosphonatosucrose
(13).—
2,3,4,6,1%,3%,4%-Hepta-O-acetylsucrose (636 mg,
1 mmol) was treated with heptylphosphonic
acid (540 mg, 3 mmol), triphenylphosphine
(714 mg, 3 mmol) and DEAD (500 mL, 3
mmol) in Py (1.5 mL) as described for the
preparation of 2. After LiOH treatment (see
above) and C-18 flash chromatography, 13
(337 mg, 67%) was obtained as a white pow-
der; [h]D 42° (c 0.5, MeOH); IR (KBr): w 3248,
2990, 2931, 1753, 1696, 1533, 1251, 1059
31
(CH3); P NMR (CD3OD, 100 MHz): l 27.5;
1
cm−1; H NMR (CD3OD, 400 MHz): l 5.36
ESI–HRMS Calcd for C14H28O8P -H:
355.1521; Found 355.1519. Anal. Calcd for
C14H29O8P: C, 47.19, H, 8.20, Found: C, 47.41
H, 8.32.
(d, 1 H, J1,2 3.5 Hz, H-1), 4.07–4.16 (m, 3 H,
H-6a, H-6b, and H-6%a), 4.02 (ddd, 1 H, J6%a,6%b
11.5, J6%b,P 5.5, J5,6%b 4.0 Hz, H-6%b), 3.95 (ddd,
1 H, J4,5 10.5, J5,6b 5.5, J5,6a 2.5 Hz, H-5),
3.80–3.86 (m, 2 H, H-5% and H-3%), 3.75 (dd, 1
H, J2,3 9.5, J3,4 9.0 Hz, H-3), 3.70 (dd, 1 H, J
12.0, J 5.5 Hz, H-4%), 3.68 (d, 1 H, J1%a,1%b 12.0
Hz, H-1%a), 3.64 (d, 1 H, H-1%b), 3.41 (dd, 1 H,
H-2), 3.26 (dd, 1 H, H-4); 13C NMR (CD3OD,
62.9 MHz): l 105.3 (C-2%), 93.5 (C-1), 82.3 (d,
JC,P 7.1 Hz, C-5%), 78.8 (C-3), 76.3 (C-4%), 74.6
(C-3), 74.2 (C-5), 73.4 (C-2), 71.9 (C-4), 65.6
(d, JC,P 5.0 Hz, C-6%), 63.5 (C-1%), 62.6 (C-6),
32.9 (P(CH2)3CH2), 32.4 (d, JC,P 17.1 Hz,
P(CH2)2CH2), 30.1 (P(CH2)4CH2), 28.1 (d,
JC,P 135.4 Hz, PCH2), 24.7 (d, JC,P 4.7 Hz,
PCH2CH2), 23.7 (P(CH2)5CH2), 14.4 (CH3);
31P NMR (CD3OD, 100 MHz): l 28.5. ESI–
HRMS Calcd for C19H37O13PꢀH: 503.1893.
General procedure for the coupling of 2 to
MA-proteins: Preparation of MA-KLH-2 and
MA-BSA-2 conjugates.—A solution of 2 (150
mg, 0.25 mmol) and 3,3%-dithiodipropionic
acid bis(succinimido)ester (33 mg, 0.08 mmol)
in DMF (450 mL) was heated at 60 °C for 8 h.
The resulting solution was directly purified by
LH-20 chromatography (85×2 cm, MeOH)
to afford 16 (56 mg, 51%) as an amorphous
1
solid. H NMR (CD3OD, 250 MHz) selected
data: l 7.15–7.35 (m, 2 H, Ar), 6.70–6.85 (m,
2 H, Ar), 5.22 (d, 1 H, J1,2 3.5 Hz, H-1), 2.89
(t, 2 H, J 7.0 Hz, CH2ꢀS), 2.62 (t, 2 H,
CH2ꢀCH2ꢀS), 1.30–1.50 (m, 4 H, Pꢀ(CH2)2ꢀ),
1.03–1.20 (m, 8 H, Pꢀ(CH2)2ꢀ(CH2)4ꢀ), 0.71
(t, 3 H, J 5.5 Hz, Pꢀ(CH2)6ꢀCH3); ESI–MS:
m/z 681.1 (M/2−H), 1363.4 (M), 1385.4
(M−H+Na), 1386.3 (M+Na). To a solu-
tion of 16 (23 mg, 16.8 mmol) in a 9:1 MeOH–
water mixture (1 mL) was added n-Bu3P (10
ml, 33.6 mmol) at rt, and the mixture was
stirred for 1 h then concentrated. Chromatog-
raphy of the residue (C 18, 1:1 MeOH–water)
gave 17 (18 mg, 78%) as a white amorphous
Found
503.1889.
Anal.
Calcd
for
C19H37O13P·0.5 H2O: C, 44.44; H, 7.46;
Found: C, 44.32; H, 7.62.
Methyl-6-heptylphosphonato-h- -glucopy-
D
ranoside (14).—Methyl-2,3,4-tri-O-acetyl-a- -
D
glucopyranoside (320 mg, 1 mmol) was treated
with heptylphosphonic acid (540 mg, 3 mmol),
triphenylphosphine (714 mg, 3 mmol) and
DEAD (500 mL, 3 mmol) in Py (1.5 mL) as
described for the preparation of 2. After
LiOH treatment (see above) and C-18 flash
1
solid. H NMR (CD3OD, 200 MHz) selected
data: l 7.35–7.20 (m, 2 H, Ar), 6.70–6.80 (m,
2 H, Ar), 5.24 (d, 1 H, J1,2 3.5 Hz, H-1), 4.28

M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
305
tion on Sephadex G25 in PBS. The concentra-
tion (2.8 mg/mL for BSA-2 and 1.3 mg/mL
for KLH-2) and the number of haptens was
determined as described above; 14 hapten/
KLH and 11 hapten/BSA were obtained.
Antibody production.—Four Balb/c mice
each received a subcutaneous injection of 100
mg of KLH-2 (75 mL of the solution obtained
as described above) emulsified in complete
Freund’s adjuvant (75 mL) on day 1. On day 7
and 14 mice received a new subcutaneous
injection of KLH-2 (75 mL) emulsified in in-
complete Freund’s adjuvant. On day 21,
serum was taken from the mice and the titers
determined by ELISA. On day 42 the mice
with the highest titer receive a final boost
intraperitoneally with 100 mg of KLH-2 in 75
mL of PBS. Three days after the last boost, the
spleens were taken from the mice and the cells
(108) were fused with 2×107 X63/HGPRT−
myeloma cells in the presence of PEG 4000.
Cells were plated into eight 96-well cell culture
plates, each well containing 100 mL of spleno-
cyte feeders. After 9 days, the plates were
screened by ELISA for binding to BSA-2 con-
jugate and the cells that displayed positive
response to binding test were subcloned and
expanded to produce large quantities of mon-
oclonal antibodies.
Purification of antibodies.—The tissue cul-
ture supernatant (150 mL) was spun at 1000g
for 30 min. NaCl (26.3 g) and glycine (11.2 g)
were added and the pH was adjusted to 8.9
with 0.5 M NaOH. The resulting solution was
filtered on a 0.22 mm nylon membrane and
loaded on a 5 mL HiTrap protein A-column
(Pharmacia Biotech). The column was washed
with 30 mL of binding buffer (Biorad) and the
elution was realised with 100 mM glycine–
HCl buffer pH 3. Each fraction was neu-
tralised with 1 M tris buffer pH 8. The
fractions containing the IgG were concen-
trated on a Centricon 30 (Amicon) and were
stored at −20 °C in sterile PBS pH 7.35 at a
concentration of ꢀ20 mg/mL. The concentra-
tion was determined by measurement of the
absorbance at 280 nm. Generally 7–15 mg of
antibodies were obtained.
(dd, 1 H, J5%,6%a 7.5, J6%a,6b 11.5 Hz, H-6%a), 3.45
(s, 2 H, 2 H-1%), 3.26 (dd, 1 H, J2,3 9.5 Hz,
H-2), 2.89 (t, 2 H, J 7.0 Hz, CH2ꢀSH), 2.61 (t,
2 H, CH2ꢀCH2ꢀSH), 1.31–1.50 (m, 4 H,
Pꢀ(CH2)2), 1.10–1.25 (m, 8 H, (CH2)2ꢀ
(CH2)4), 0.73 (t, 3 H, J 5.5 Hz, Pꢀ(CH2)6ꢀ
CH3). To a solution of maleimide–BSA or
maleimide–KLH (ꢀ5 mg) in 0.5 mL of 10
mM phosphate buffer saline (PBS, pH 7.3)
was added a solution of 17 (6 mg, 8.8 mmol)
in PBS (1 mL) and the mixture was gently
stirred overnight at 4 °C. Unreacted 12 was
separated from the modified proteins by gel-
filtration on Sephadex G25 in PBS. The con-
centration of MA-KLH-2 and MA-BSA-2
was determined by bicinchoninic acid assay;52
3 and 0.9 mg/mL were obtained, respectively,
for the KLH and the BSA conjugates. The
number of hapten molecules per BSA or KLH
was estimated by amine titration and by mea-
suring the absorbance at 271 nm (umax of 2).
3.3 hapten/KLH (calcd for a molecular weight
of 100,000) and 3.5 hapten/BSA were
obtained.
General procedure for the coupling of 2 to
nati6e proteins: preparation of BSA-2 and
KLH-2 conjugates.—To a solution of 2 (117
mg, 0.2 mmol) in a mixture of DMF (2 mL)
and Et3N (35 mL, 1.25 equiv) was added 5-
[(2,5 - dioxo - 1 - pyrrolidinyl)oxy] - 5 - oxopenta-
noyl chloride (62 mg, 1.2 equiv). The mixture
was stirred for 90 min at rt, then concentrated.
Flash chromatography of the residue (1:1
CH2Cl2–MeOH) gave 19 (122 mg, 75%) as an
1
amorphous solid; H NMR (CD3OD, 250
MHz): l 7.30–7.40 (m, 2 H, Ar), 6.80–6.90
(m, 2 H, Ar), 5.38 (d, 1 H, J1,2 3.5 Hz, H-1),
4.35 (dd, 1 H, J5%,6%a 7.5, J6%a,6%b 11.5 Hz, H-6%a),
3.60–4.10 (m, 7 H, H-3%, H-4%, H-5%, H-6%b,
H-5, and 2 H-6), 3.58 (dd, 1 H, J2,3 9.6, J3,4 9.6
Hz, H-3), 3.48 (s, 2 H, 2 H-1%), 3.30 (dd, 1 H,
H-2), 2.70 (s, 4 H, CO(CH2)2CO), 2.60 (t, J
7.0 Hz, CH2(CH2)2), 2.36 (t, J 7.0 Hz,
(CH2)2CH2), 1.95 (q, 1 H, J 7.0 Hz,
CH2CH2CH2), 1.30–1.50 (m, 4 H, (CH2)2),
1.05–1.30 (m, 8 H, (CH2)4), 0.70–0.85 (m, 3
H, CH3). To a solution of BSA or KLH (10
mg) in 50 mM pyrophosphate buffer pH 8 (2
mL) was added a solution of 19 (60 mg, 74
mmol) in DMF (100 mL). The mixture was
stirred for 2 h at rt then purified by gel-filtra-
Titration of antibodies in animal serum.—
Each well of a 96-well polyvinyl assay plate
(ELISA plate) was coated with 50 mL of the

306
M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
dilution in BSA 1% (the IgG dilution was
adapted in order to obtain a significant ab-
sorbance in the well without inhibitors). The
ELISA protocol was then followed as already
described. The IC50 were deduced from the
analysis of the curves obtained by plotting the
absorbance at 490 nm vs the inhibitors’
concentrations.
conjugate BSA-2 (30 mg/mL) in phosphate
buffer salt (PBS 10 mM, pH 7.2 phosphate,
150 mM NaCl) and incubated at rt overnight.
The plate was washed twice with PBS. A
solution of BSA (3% in PBS, 300 mL) was
added to the wells and the plate was incubated
at 37 °C for 2 h. The plate was washed twice
with PBS and serum was added at different
dilutions (1/50 to 1/400,000). The plate was
incubated for 2 h at 37 °C. Washing with PBS
was repeated seven times and a solution of
goat anti-mouse IgG-horseradish peroxidase
in BSA 1.5% was added to each well. The
plate was incubated for 1 h at 37 °C and
washed seven times with PBS. To each well
was added 50 mL of a solution of o-phenylene-
diamine (OPD, 0.5 mg/mL) in 0.1 M sodium
citrate (pH 5.5) containing 0.1% hydrogen
peroxide. The colour was allowed to develop
for 10–20 min at rt in the dark and 100 mL of
2 N H2SO4 containing 0.5% Na2SO3 were
added to stop the reaction. The absorbance at
490 nm was measured using a microtiter plate
reader. The titer was defined as the serum
dilution at which the absorbance became that
of the background. Titers between 1/105 and
1/4×105 were obtained.
Enzyme-linked immunosorbent assay.—Pos-
itive hybridoma clones producing IgG which
specifically bind to the hapten were identified
by ELISA. ELISA plates were prepared as
described above (treatment with BSA-2, incu-
bation, washing and saturation with 3% BSA).
The hybridoma supernatant from the each
wells of the culture plates was added to the
ELISA plates and incubated for 2 h at 37 °C.
The plates were washed with PBS and the
above protocol was followed. Positive clones
Kinetics measurements.—Stock solutions of
the esters were prepared in Me2SO with vary-
ing concentrations from 5.3 to 1.1 mM. To
600 mL of phosphate buffer (pH 7.35) were
added 80 mL of the above dilutions and 26 mL
of a 0.13 mM antibody solution or 26 mL of
buffer (uncatalysed reactions). The reactions
were incubated in a controlled-temperature
shaker at 37 °C and 140 rpm. Periodically, 30
mL aliquots were withdrawn and placed in an
ELISA plate. A solution of b-fructosidase (20
mL, 72 u/mL in 0.32 M citrate pH 4.6) for the
6-octanoylsucrose hydrolysis kinetics, or 20
mL of a solution of a-glucosidase (60 u/mL in
0.32 M citrate pH 4.6) for the 6%-octanoylsu-
crose or the methyl-6-octanoyl-a- -glucopy-
D
ranoside hydrolysis, were added and the plate
was incubated at 25 °C for 15 min. NADP
(2.4 mg/mL, 90 mL) and ATP (5.7 mg/mL)
solutions in triethanolamine buffer pH 7.6
were added. The plate was incubated 3 min at
25 °C and the absorbance A1 was read at 340
nm. A suspension of HK (5 mL, 300 u/mL)
and G6P-DH (140 u/mL) were added and the
plate was incubated for 15 min at 25 °C. The
absorbance A2 was measured at 340 nM. The
glucose concentrations (amount of hydrolysed
ester) were deduced from the differences A2−
A1 by comparison with a standard curve ob-
tained from dilutions of sucrose or methyl-
were
identified
by
comparison
with
a- -glucopyranoside prepared in phosphate
D
background.
buffer containing 10% Me2SO coated on the
same plate.
Competiti6e enzyme-linked immunosorbent
assay.—ELISA plates were prepared as de-
scribed above (treatment with BSA-2, incuba-
tion, washing and saturation with 3% BSA).
Dilutions of the appropriate inhibitor (10–14
and 20) were prepared from DMF stock solu-
tion in BSA 1% from concentration ranging
from 10−3 to 1 mM. The proportion of DMF
was adjusted to 2% (v/v) in each dilution. In
each well of the ELISA plates were added 25
mL of these dilutions and 25 mL of an IgG
Acknowledgements
This work was supported by Eridania
Be´ghin-Say and the Ministe`re de l’Education
Nationale, de l’Enseignement Supe´rieur et de
la Recherche (programme Biotechnologie no.
375). We thank Dr P. Lesot (Laboratoire de
Chimie Structurale Organique, Orsay, France)

M.-C. Scherrmann et al. / Carbohydrate Research 334 (2001) 295–307
307
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