Studies towards neoglycoconjugates from the monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa using the diethyl squarate reagent

Article abstract of DOI:10.1016/S0008-6215(98)00261-4

The effect of reaction time, concentration and molar excess of hapten upon the efficiency of the conjugation of carbohydrates to proteins using the diethyl squarate reagent has been studied using chicken serum albumin (CSA) as the carrier protein and a li

Full text of DOI:10.1016/S0008-6215(98)00261-4

Carbohydrate Research 313 (1998) 15±20
Studies towards neoglycoconjugates from the
monosaccharide determinant of Vibrio cholerae
O:1, serotype Ogawa using the diethyl squarate
reagent
Jian Zhanga, Alfred Yergeyb, Je€rey Kowalakb, Pavol Kovaca,*
a National Institute of Health: NIDDK, Laboratory of Medicinal Chemistry, 8 Center Drive, Bethesda,
MD 20892-0815, USA
b National Institute of Health: NICHD, Laboratory of Cellular and Molecular Biophysics,
10 Center Drive, Bethesda, MD 20892-1580, USA
Received 21 July 1998; accepted 30 September 1998
Abstract
The e€ect of reaction time, concentration and molar excess of hapten upon the eciency of the
conjugation of carbohydrates to proteins using the diethyl squarate reagent has been studied using
chicken serum albumin (CSA) as the carrier protein and a linker-equipped d-glucose derivative as
the hapten. A high degree of incorporation of the latter into CSA was achieved with high e-
ciency, and the use of a large excess of the ligand was not necessary. Conjugation of the immu-
nodominant monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa, bearing the
same spacer, followed a similar pattern, showing that the nature of the carbohydrate does not
substantially a€ect the outcome of the conjugation and that a predicted degree of antigen-loading
onto carrier protein is possible to achieve. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: Vaccine; Dialkyl squarate; Immunogen; Antigen; Neoglycoconjugate; Vibrio cholerae
1. Introduction
vaccine research and development. Concerning
neoglycoconjugate immunogens, it has been shown
that conjugate vaccines made from antigenic O-
polysaccharides [1,2], as well as from fragments
thereof (e.g., ref [3].), can induce protective anti-
bodies. A large part of our e€ort is directed
towards developing (semi)synthetic vaccines for
infectious diseases caused by bacterial pathogens.
In a recent detailed study [4] done as part of our
attempts towards a vaccine against cholera [5±15],
we found 4-(3-deoxy-l-glycero-tetronamido)-4,6-
dideoxy-2-O-methyl-ꢀ-d-mannopyranose to con-
Traditional vaccines based on immunogens pre-
sent in killed or attenuated bacteria are often
pyrogenic or have other undesirable e€ects. It is
hoped that a new generation of vaccines prepared
by chemical conjugation of antigens to suitable
carriers will be free from these de®ciencies. Such
vaccines have already opened new horizons in
* Corresponding author. Tel.: +1-301-496-3569; fax:
+1-301-402-0589; e-mail: kpn@helix.nih.gov
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00261-4

16
J. Zhang et al./Carbohydrate Research 313 (1998) 15±20
tribute about 90% of the maximum binding energy
with a number of monoclonal antibodies for Vibrio
cholerae O:1, serotype Ogawa. We therefore
deemed it useful to include a neoglycoconjugate
prepared from this de®ning hapten in our future
evaluation of the immunogenicity of various con-
jugates made from V. cholerae O:1 antigens. We
previously reported on the conjugation of a
spacer-equipped monosaccharide determinant of
V. cholerae O:1, serotype Ogawa to chicken serum
albumin (CSA) by reductive amination [16]. Here
we describe synthesis of a neoglycoconjugate pre-
pared from that determinant monosaccharide
using the squaric acid diester chemistry [17,18].
(i.e., 46 [21]) upon the outcome of the conjugation.
In the preliminary experiments we used the linker-
equipped d-glucose derivative 3, bearing the same
spacer as the V. cholerae O:1-related hapten to be
conjugated later. We show here that the nature of
the carbohydrate does not substantially a€ect the
outcome of the conjugation, and that incorpora-
tion of a predicted number of haptens in a carrier
protein is possible to achieve by applying condi-
tions listed in Tables 1±3. This is important in the
rational design of well-de®ned, synthetic glyco-
conjugate vaccines, which may require a de®ned,
narrow-range loading of the antigen onto carrier
proteins.
To explore optimum conditions for conjugation,
neoglycoconjugates were prepared, and their
molecular mass was determined by MALDI-TOF
mass spectrometry. Thus, 6-methoxycarbonylhexyl
ꢁ-d-glucopyranoside (3), prepared from 2,3,4,6-
tetra-O-benzoyl-ꢀ-d-glucopyranosyl bromide [22]
(1) and methyl 6-hydroxyhexanoate [23] (11) via
the per-O-benzoate 2, was subjected to aminolysis
with ethylenediamine to give 4. Treatment of the
latter with squaric acid diethyl ester (12) gave 5,
which was readily puri®ed by column chromato-
graphy on silica gel. NMR and TLC showed no
signs of decomposition of 5 when its aqueous
2. Results and discussion
3,4-Diethoxy-3-cyclobutene-1,2-dione [17,18] or
its dimethoxy analog (squaric acid diesters)
have recently been used for preparation of neo-
glycoconjugates [19,20], but protocols applied
varied. Therefore, before conjugating the precious,
spacer-equipped V. cholerae O:1 antigen 8, we have
studied the e€ect of reaction time, concentration,
and molar excess of hapten to be linked over the
number of amino groups present in the CSA carrier

J. Zhang et al./Carbohydrate Research 313 (1998) 15±20
17
Table 1
E€ect of the concentration of 5 used upon hapten incorpora-
tion in CSA
9 with CSA yielded a product containing ꢀ29 resi-
dues of 10/CSA. This is in a remarkably good
agreement with the expected incorporation based
on the data from the conjugation study involving
an analogous derivative of d-glucose (Table 3).
Preparation of related neoglycoconjugates from
larger fragments of the O-polysaccharide of V.
cholerae O:1 is in progress. Immunological studies
with neoglycoconjugates prepared from various
fragments of the O-PS of V. cholerae O:1 will be
published in separate communications.
a
Concentration of
hapten (mM)
m/z
Number of
hapten residues
incorporated/CSA
Eciency
(%)
5
75374
77446
79085
79387
78613
21
45.6
56.9
66.0
67.6
63.4
10
15
20
25
26.2
30.4
31.1
29.2
a
Reaction time, 48 h; Hapten:l-lysine [equiv], 1:1.
General methods.ÐUnless stated otherwise,
optical rotations were measured at ambient tem-
perature for solutions in chloroform (c ꢀ1), with a
Perkin±Elmer automatic polarimeter, Model 341.
All reactions were monitored by thin-layer chro-
matography (TLC) on Silica Gel 60 coated glass
slides (Whatman or Analtech). Detection was
e€ected by charring with 5% (v/v) sulfuric acid in
ethanol. Column chromatography was performed
by gradient elution from columns of silica gel. Sol-
vent mixtures slightly less polar than those used for
TLC were used at the onset of development. Mass
spectra were obtained using PerSeptive BioSystems
Voyager Elite DE-STR (PE-Biosystems, Framing-
ham, MA) MALDI-TOF instrument. The instru-
ment was operated in the linear mode with 25 kV
accelerating voltage and a 300 ns ion-extraction
delay time. Samples for analysis (ꢀ0.1 mg) were
dissolved in deionized water (50 ꢂL) and applied as
1 ꢂL droplets to separate positions in the center of
the multiple-sample plate. An equal volume of
matrix (saturated solution of sinapinic acid in 1:1
acetonitrile±0.5% tri¯uoroacetic acid) was applied
over each dried sample and redried before being
inserted into the mass spectrometer. CSA, pur-
chased from Sigma Chemical Company, was pur-
i®ed as described previously [24], freeze-dried, and
used also as a mass standard. Results from all
analyses yielded a molecular mass for this protein
of about 66.9 kDa. Molecular masses of the glyco-
conjugates were calculated using the molecular
mass of CSA of 66,973 Da, predicted from the gene
sequence [21]. NMR signals were recorded with a
Varian Mercury spectrometer at 300 and 75 MHz
Table 2
E€ect of the reaction time upon incorporation of 5 in CSA
a
Time (h)
m/z
Number of
hapten residues
incorporated/CSA
Eciency (%)
16
40
64
112
160
77071
81724
82573
81342
81699
25
37
37
36
37
27.1
40.2
40.2
39.1
40.2
a
Hapten concentration, 14.2 mM; Hapten:l-lysine used
[equiv], 2:1.
solution was kept for several days at room tem-
perature. To obtain conjugates, compound 5 was
treated with CSA (see Experimental) under various
conditions. Table 1 shows the bene®t of conducting
conjugation at high hapten concentrations. Max-
imum loading of the carrier protein with the hap-
ten, which could not be increased by using more
than 100% molar excess of hapten (Table 3), was
reached after a reaction time of 1.5±2 days
(Table 2).
For conjugation of the V. cholerae O:1 antigen,
hapten 9 was prepared from the known [16] thio-
glycoside 6, following the same protocol as that for
4. At a concentration of 12.7 mM, and a ratio 2.2:1
of the hapten:lysine present in CSA, conjugation of
Table 3
E€ect of the amount of 5 used upon its incorporation in CSA a
Hapten:l-lysine
used [equiv]
m/z
Number of
hapten residues
incorporated/CSA
Eciency (%)
1
for H and 13C, respectively. The assignment of
signals was made by ®rst-order analysis of the
spectra, and the assignments were supported by
homonuclear decoupling experiments or homo-
nuclear and heteronuclear 2-dimensional correla-
tion spectroscopy run with the software supplied
with the spectrometers. When reporting NMR
1:4
1:2
1:1
2:1
3:1
71070
74251
77259
78756
79321
10.3
18.2
25.8
29.5
30.9
89.5
79.1
56.0
32.0
22.4
a
Hapten concentration, 8.3 mM; Reaction time, 48 h.

18
J. Zhang et al./Carbohydrate Research 313 (1998) 15±20
spectral assignments, the nuclei of the 3-deoxy-l-
glycero-teronamide side chain are noted as primed
and those of the linker aglycon are noted as dou-
ble-primed (even in the absence of the 3-deoxy-l-
glycero-teronamide side chain). Solutions in
organic solvents were concentrated at 40 ꢁC/2 kPa.
5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-ben-
zoyl-b-d-glucopyranoside (2).ÐA solution of
silver tri¯uoromethanesulfonate (tri¯ate, 1.31 g,
5.1 mmol) in toluene (20 mL) was added dropwise
(2-Aminoethylamido)carbonylpentyl b-d-gluco-
pyranoside (4).ÐA solution of 3 (270 mg) in ethy-
lenediamine (4 mL) was stirred at 70 ꢁC in an
atmosphere of argon until TLC (1:1:0.1 EtOAc±
MeOH±concd NH4OH) showed that the reaction
was complete (ꢀ30 h). The mixture was con-
centrated and coevaporated with water to remove
excess of the reagent. The residue was chromato-
1
graphed to give amorphous 4 (239 mg, 81%). H
NMR (D2O): ꢃ 4.37 (d, 1 H, J1,2 7.9 Hz, H-1),
3.89±3.81 (m, 2 H, H-6a,100a), 3.68±3.56 (m, 2 H,
H-6b,100b), 3.45±3.15 (m, 6 H, H-2,3,4,5,600a,b),
2.74 (t, 2 H, J 7.1 Hz, H-700a,b), 2.21 (t, 2 H, J
7.4 Hz, H-500a,b), 1.62±1.51 (m, 4 H, H-200a,b,
400a,b), 1.36±1.26 (m, 2 H, H-300a,b); 13C NMR
(D2O): ꢃ 102.32 (C-1), 76.05 (C-5), 75.97 (C-3),
73.28 (C-2), 70.34 (C-100), 69.82 (C-4), 60.90 (C-6),
40.62 (C-600), 39.76 (C-700), 35.80 (C-500), 28.55 (C-
200), 25.13 (C-400), 24.77 (C-3); CIMS: m/z 337
([M+1]+).
ꢁ
at 30 C to a mixture of 1 (2.83 g, 4.3 mmol), 11
[23] (0.872 g, 7.5 mmol), 1,1,3,3-tetramethylurea
(0.35 g, 3.0 mmol) and 4 A molecular sieves (1 g),
which had been stirred for 15 min. After 30 min,
TLC (2:1 hexane±EtOAc) showed that all 1 was
consumed. The mixture was neutralized with Et3N,
®ltered, the ®ltrate was concentrated, and the resi-
due was chromatographed to give amorphous 2
(2.4 g, 77%). [ꢀ]d +11ꢁ (c 1.4); H NMR (CDCl3):
1
ꢃ 5.94 (t, 1 H, J 9.9 Hz, H-3), 5.71 (t, 1 H, J 9.9 Hz,
H-4), 5.55 (dd, 1 H, J1,2 7.9, J2,3 9.5 Hz, H-2), 4.87
(d, 1 H, H-1), 4.67 (dd, 1 H, J5,6a 3.1, J6a,6b 12.3 Hz,
H-6a), 4.53 (dd, 1 H, J5,6b 5.1 Hz, H-6b), 4.19 (m, 1
H, H-5), 3.93 (m, 1 H, H-100a), 3.60 (s, 3 H, OCH3),
3.55 (m, 1 H, H-100b), 2.05 (m, 1 H, H-500a,b), 1.53
(m, partially overlapped, H-200a,b), 1.45 (m, par-
tially overlapped, H-40a,b), 1.23 (m, 2 H, H-300a,b);
13C NMR (CDCl3): ꢃ 173.76 (CONH), 165.97,
165.66, 165.05, 164.90 (4 COPh), 101.16 (C-1),
72.80 (C-3), 72.01 (C-5), 71.78 (C-2), 69.76 (C-100),
69.70 (C-4), 63.04 (C-6), 51.20 (OCH3), 33.56 (C-
500), 28.90 (C-200), 25.17 (C-30), 24.26 (C-40); CIMS:
m/z 742 ([M+18]+). Anal. Calcd for C41H40O12:
C, 67.96; H, 5.52. Found: C, 68.05; H, 5.54.
1-[(2-Aminoethylamido)carbonylpentyl b-d-gluco-
pyranoside]-2-ethoxycyclobutene-3,4-dione (5).ÐA
solution of the foregoing amine
4 (80 mg,
0.24 mmol) in ethanol (10 mL) was treated at room
temperature with 12 (35 mL, 0.24 mmol) until TLC
(2:1 EtOAc±MeOH) showed that the reaction was
complete (ꢀ6 h). After concentration, the residue
was chromatographed to give amorphous 5 (85 mg,
78%). 1H NMR (D2O): ꢃ 4.79±4.65 (m, 2 H,
CH2CH3), 4.38 (bd, 1 H, J1,2 7.9 Hz, H-1), 3.88±
3.80 (m, 2 H, H-6a,100a), 3.70±3.64 (m, 2 H, H-
6b,100b), 3.61±3.52 (m, 2 H, H-700a,b), 3.46±3.16 (m,
6 H, 2,3,4,5,600a,b), 2.19 (t, 2 H, J 7.2 Hz, H-500),
1.60±1.48 (m, 4 H, H-200400), 1.40 (q, 3 H, J 7 Hz,
CH2CH3), 1.32±1.24 (m, 2 H, H-300a,b); 13C NMR
(D2O, typical splitting of some NMR signals due
to the double bond nature of the vinylogous amide
group, characteristic [18] of squaric acid amide
esters): ꢃ 102.34 (C-1), 76.03 (C-5), 75.97 (C-3),
73.30 (C-2), 70.88, 70.76 (CH2CH3), 70.31 (C-100),
69.82 (C-4), 60.93 (C-6), 44.17, 44.09 (C-700), 38.57,
38.37 (C-600), 35.89 (C-500), 28.59 (C-200), 25.28 (C-
400), 24.81 (C-300), 15.23, 15.16 (CH2CH3); CIMS:
m/z 478 ([M+18]+).
5-(Methoxycarbonyl)pentyl b-d-glucopyranoside
(3).ÐConventional debenzoylation of 2 (Zemplen)
gave 3 in virtually theoretical yield. The crystalline
ꢁ
material showed mp 56±58 C (from ethyl acetate)
and [ꢀ]d 22ꢁ (c 0.9, H2O). H NMR (D2O): 4.42
1
(d, 1 H, J1,2 7.9 Hz, H-1), 3.94±3.86 (m, 2 H, H-
6a,100a), 3.74±3.60 (m, 5 H, H-6b,100b, incl s, 3.67,
OCH3), 3.45 (t, partially overlapped, J 9.2 Hz, H-
3), ꢀ3.41 (m, partially overlapped, H-5), 3.34 (bt,
partially overlapped, H-4), 3.32 (dd, 1 H, J2,3
9.1 Hz, H-2), 2.38 (t, 1 H, J 7.4 Hz, H-500a,b), 1.66±
1.56 (m, 4 H, H-200a,b,400a,b), 1.42±1.31 (m, 2 H, H-
300a,b); 13C NMR (D2O): ꢃ 102.27 (C-1), 76.00 (C-
5), 75.93 (C-3), 73.52 (C-2), 70.35 (C-100), 69.78 (C-
4), 60.90 (C-6), 52.19 (COOCH3), 33.71 (C-500),
5-Methoxycarbonylpentyl 3-O-acetyl-4-(2,4-di-
O-acetyl-3-deoxy-l-glycero-tetronamido)-4,6-di-
deoxy-2-O-methyl-a-d-mannopyranoside (7).ÐA
mixture of 6 [16], 970 mg, 2.16 mmol), 11 [23] (473 mg,
3.24 mmol) and 4A molecular sieves (1 g) in
CH2Cl2 (30 mL) was stirred for 15 min. Solid NIS
(680 mg, 3.02 mmol) was added, followed by a
solution of AgOTf (222 mg, 0.86 mmol) in toluene
28.51 (C-200), 24.75 (C-300), 24.51 (C-400); CIMS: m/z
+
.
326 ([M+18] ). Anal. Calcd for C13H24O8 0.5
H2O: C, 49.20; H, 7.94. Found: C, 49.22; H, 7.93.

J. Zhang et al./Carbohydrate Research 313 (1998) 15±20
19
H-1), 4.25 (dd, 1 H, J2 ,3 a 3.8, J2 ,3 b 8.6 Hz, H-20),
3.93 (dd, 1 H, J2,3 3.4, J3,4 10.2 Hz, H-3), 3.85±3.64
(m, 5 H, H-4,5,400a,b,100a), 3.56±3.47 (m, 2 H, H-
100b, incl bdd, 3.53, H-2), 3.45 (s, 3 H, OCH3), 3.26
(t, 2 H, J 6.3 Hz, H-600a,b), 2.77 (t, 2 H, H-700a,b),
2.24 (t, 2 H, J 7.3 Hz, H-500a,b), 2.06±1.94, 1.86±
1.76 (2 m, H-30a,b), 1.62±1.54 (m, 4 H, H-
200a,b,400a,b), 1.39±1.28 (m, 2 H, H-300a,b), 1.13 (d,
3 H, J5,6 5.7 Hz, H-6); 13C NMR (D2O): ꢃ 177.37,
177.26 (2 CO), 96.73 (C-1), 79.41 (C-2), 69.09 (C-
20), 67.97 (C-100), 67.84 (C-3), 67.31 (C-5), 58.92
(OCH3), 57.94 (C-40), 53.45 (C-4), 40.67 (C-600),
39.82 (C-700), 36.10 (C-30), 35.78 (C-500), 28.35 (C-
200), 25.11 (C-400), 25.02 (C-300), 16.90 (C-6); CIMS:
m/z 436 ([M+1]+), 453 ([M+18]+).
0
0
0
0
(8 mL). TLC (1:1 hexane±EtOAc) showed that the
reaction was complete in ꢀ5 min. After neutraliza-
tion with Et3N, the mixture was washed with aqu-
eous NaHCO3, water, dried, and concentrated.
Chromatography of the residue gave the desired,
amorphous glycoside 7 (1.14 g, ꢀ100%), [ꢀ]d
ꢁ
1
+46 ); H NMR (CDCl3): ꢃ 6.30 (d, 1 H, J4,NH
9.5 Hz, NH), 5.19 (dd, 1 H, J2,3 3.1, J3,4 11.0 Hz,
H-3), 5.07 (dd, 1 H, J2 ,3 a 4,6, J2 ,3 b 8.0 Hz, H-20),
4.82 (d, 1 H, J1,2 1.9 Hz, H-1), 4.31±4.21 (m, 1 H,
H-4), 4.19±4.06 (m, 2 H, H-40a,b), 3.76±3.62 (m, 5
H, H-5, H-100a, incl s at 3.69 for COOCH3), 3.50 (s,
3 H, OCH3), 3.49 (dd, 1 H, H-2), 3.42, 3.39 (2 t, 1
H, J 6.1 Hz, H-100b), 2.34 (t, 2 H, J 7.3 Hz, H-
500a,b), 2.22±2.04 (m, 11 H, overlapping H-30a,b
and 3 s at 2.15, 2.12, 2.05 for 3 COCH3), 1.73±1.54
(m, 4 H, H-200a,b, H-400a,b), 1.46±1.35 (m, 2 H, H-
300a,b), 1.21 (d, 3 H, J5,6 6.3 Hz, H-6); 13C NMR
(CDCl3): ꢃ 97.49 (C-1, JC,H 168.4 Hz), 77.93 (C-2),
71.13 (C-3), 70.91 (C-20), 68.18 (C-5), 67.11 (C-100),
59.87 (C-40), 59.51 (OCH3-2), 51.48 (COOCH3),
51.41 (C-4), 33.82 (C-500), 30.57 (C-30), 28.77 (C-200),
25.49 (C-300), 24.35 (C-400), 20.19, 20.70, 20.61 (3
COCH3), 17.76 (C-6); CIMS: m/z 551 ([M+18]+).
Anal. Calcd for C24H39NO12: C, 54.03; H,7.32; N,
2.63; Found: C, 53.93; H,7.33; N, 2.68.
0
0
0
0
1-[(2-Aminoethylamido)carbonylpentyl 4-(3-de-
oxy-l-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-
a-d -mannopyranoside]-2-ethoxycyclobutene-3,4-
dione (10).ÐThe foregoing amine 9 (32 mg) was
treated with diethyl squarate as described for the
preparation of 5 to give 10 (33 mg, 80%). 1H NMR
(D2O): ꢃ 4.96 (bs, 1 H, H-1), 4.76±4.65 (m, 1 H,
CH2CH3), 4.26 (dd, 1 H, J2,3a 4.0, J2,3b 8.7 Hz, H-
20), 3.95 (bd, 1 H, H-3), 3.83±3.77 (m, 2 H, H-4,5),
3.73±3.56 (m, 6 H, H-40a,b,100a,b,600a,b), 3.54 (bdd,
1 H, H-2), 3.46 (s, 3 H, OCH3), 3.38 (m, 2 H, H-
700a,b), 2.24±2.16 (m, 2 H, H-500a,b), 2.07±1.95 (m,
1 H, H-30a), 1.88±1.77 (m, 1 H, H-3b), 1.60±1.49
(m, 3 H, H-200a,b,400a,b), 1.46±1.39 (m, 3 H,
CH3CH2), 1.36±1.20 (m, 2 H, H-300a,b), 1.15 (d, 3
H, J5,6 5.6 Hz, H-6); 13C NMR (D2O): ꢃ 96.75 (C-
1), 79.38 (C-2), 70.88 (CH2CH3), 69.12 (C-20),
67.86 (C-3), 67.33 (C-5), 58.94 (OCH3), 57.96 (C-
40), 53.46 (C-4), 44.22 (C-700), 39.32 (C-600), 36.06
(C-30), 35.85 (C-500), 28.45 (C-200), 25.28 (C-400),
25.03 (C-300), 16.89 (C-6), 15.25 (CH2CH3); CIMS:
m/z 560 ([M+1]+), 577 ([M+18]+).
Conjugation of the squaric acid derivative 5 with
CSA. In a typical experiment, a solution of 5
(13 mg, 0.028 mmol) and CSA (20.6 mg,
3.08Â10 4 mmol) in a KHCO3±Na2B4O7 bu€er
(pH 9.0, 2 mL, corresponding to a ꢀ2:1 hapten:
lysine ratio at a hapten concentration of 14.1 mM)
was stirred at room temperature. Samples
(200 ꢂL), periodically withdrawn at time intervals
listed in Table 2, were centrifuged using 3 K Cen-
tricon ®lters (cut-o€, 3 K), washed with deionized
water (three times), and the material retained was
freeze-dried Alternatively, samples containing neo-
glycoconjugates were dilayzed for 48 h against six
changes of deionized water (2 L each), followed by
freeze-drying. The products, obtained in virtually
5-Methoxycarbonylpentyl 4-(3-deoxy-l-glycero-
tetronamido)-4,6-dideoxy-2-O-methyl-a-d-manno-
pyranoside (8).ÐConventional deacetylation (Zem-
plen) of 7 (1.2 g) gave 8 as an amorphous hygro-
scopic solid; [ꢀ]d +8ꢁ (c 1.1, H2O). 1H NMR
(D2O): ꢃ 4.97 (d, 1 H, J1,2 1.7 Hz, H-1), 4.26 (dd, 1
H, J2 ,3 a 3.8, J2 ,3b 8.7 Hz, H-20), 3.96 (dd, 1 H, J2,3
3.4, J4,5 10.3 Hz, 3.86±3.77 (m, 2 H, H-4,5), 3.74±
3.66 (m, 6 H, H-40a,b,100a, incl s, 3.68, OCH3-7),
3.57±3.47 (m, 5 H, H-2,100b, incl s, 3.47 OCH3-2),
2.39 (t, 2 H, J 7.4 Hz, H-500a,b), 2.08±1.96, 1.89±
1.77 (2 m, 1 H each, H-30a,b), 1.67±1.56 (m, 4 H,
H-200a,b,400a,b), 1.42±1.32 (m, 2 H, H-300a,b), 1.15
(d, 3 H, J5,6 5.8 Hz, H-6); 13C NMR (D2O): ꢃ 96.75
(C-1), 79.41 (C-2), 69.08 (C-20), 67.94 (C-100), 67.85
(C-3), 67.32 (C-5), 58.94 (OCH3-2), 57.94 (C-40),
53.46 (C-4), 52.19 (OCH3-7), 36.08 (C-30), 33.70
(C-500), 28.30 (C-200), 25.02 (C-200), 24.13 (C-400),
16.90 (C-6); CIMS: m/z 408 ([M+1]+), 425
([M+18]+).
0
0
0
(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-
l-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-a-
d-mannopyranoside (9).ÐCompound 8 (240 mg)
was treated with ethylenediamine as described for
the preparation of 4 to give amorphous 9 (250 mg,
1
98%). H NMR (D2O): ꢃ 4.95 (d, 1 H, J1,2 1.5 Hz,

20
J. Zhang et al./Carbohydrate Research 313 (1998) 15±20
theoretical yields, were analyzed by MALDI-TOF
mass spectrometry.
[8] P.-S. Lei, Y. Ogawa, J.L. Flippen-Anderson, and
P. Kovac, Carbohydr. Res., 275 (1995) 117±129.
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Conjugation of the squaric acid derivative 10 with
CSA. Compound 10 (14 mg, 0.025 mmol) and
CSA (16.7 mg, 2.5Â10 4 mmol) was stirred in a
KHCO3±Na2B4O7 bu€er (pH 9.0, 2 mL, corre-
sponding to a 2.2:1 hapten:lysine ratio at a hapten
concentration of 12.7 mM) for 2 days. After dia-
lysis as described above and freeze drying, the
MALDI-TOF analysis of the product obtained
(17 mg, ꢀ100%, m/z 82,003) showed that the con-
jugate contained ꢀ29 hapten residues/CSA.
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