Neoglycoconjugates from synthetic tetra- and hexasaccharides that mimic the terminus of the O-PS of Vibrio cholerae O:1, serotype Inaba

Article abstract of DOI:10.1039/b211660j

A glycosyl acceptor and a glycosyl donor having the N-3-deoxy-L-glycero-tetronic acid side chain already attached have been prepared and used for the synthesis of the di- through to the hexasaccharide that mimic the upstream terminus of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Inaba. The target tetra- and the hexasaccharide, which were obtained in the form of 5-methoxycarbonylpentyl glycosides, were linked to BSA using squaric acid diester chemistry. The conjugation reactions were monitored by surface enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS). This allowed the progression of the conjugation of the synthetic oligosaccharides in a controlled way and termination of the reaction when the desired molar hapten/BSA ratio had been reached, yielding neoglycoconjugates with predetermined carbohydrate/carrier ratios. The ability to monitor the conjugation by the SELDI-TOF MS technique made it possible to prepare, from one hapten in a one-pot reaction, several neoglycoconjugates having different, predetermined carbohydrate/carrier ratios.

Full text of DOI:10.1039/b211660j

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Neoglycoconjugates from synthetic tetra- and hexasaccharides that
mimic the terminus of the O-PS of Vibrio cholerae O:1,
serotype Inaba
Xingquan Ma, Rina Saksena, Anatoly Chernyak and Pavol Kovácˇ*
NIDDK, LMC, National Institutes of Health, Bethesda, MD 20892, USA
Received 26th November 2002, Accepted 6th January 2003
First published as an Advance Article on the web 10th February 2003
A glycosyl acceptor and a glycosyl donor having the N-3-deoxy--glycero-tetronic acid side chain already attached
have been prepared and used for the synthesis of the di- through to the hexasaccharide that mimic the upstream
terminus of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Inaba. The target tetra- and the
hexasaccharide, which were obtained in the form of 5-methoxycarbonylpentyl glycosides, were linked to BSA using
squaric acid diester chemistry. The conjugation reactions were monitored by surface enhanced laser desorption
ionization-time of flight mass spectrometry (SELDI-TOF MS). This allowed the progression of the conjugation of
the synthetic oligosaccharides in a controlled way and termination of the reaction when the desired molar hapten/
BSA ratio had been reached, yielding neoglycoconjugates with predetermined carbohydrate/carrier ratios. The
ability to monitor the conjugation by the SELDI-TOF MS technique made it possible to prepare, from one hapten
in a one-pot reaction, several neoglycoconjugates having different, predetermined carbohydrate/carrier ratios.
Preparation of neoglycoconjugates from the analogous
Introduction
disaccharide has been reported.4
It has been generally accepted that the O-specific polysacchar-
ide (O-PS) domain of lipopolysaccharides (LPS) of gram
negative bacteria determines the serological specificity.1 It
should, therefore, be possible to use oligosaccharides that
mimic immunologically dominant epitopes of O-polysacchar-
ides as antigenic components of conjugate vaccines. Cholera is
a serious enteric disease that remains a persistent problem in the
developing and, occasionally, in the developed world.2 We are
currently interested in developing a conjugate vaccine for this
disease, and have synthesized a number of oligosaccharides
that mimic the structure of the O-PS of the two main causes
for cholera, namely Vibrio cholerae O:1, serotypes Inaba
and Ogawa. Structures of the two O-PSs are shown in Fig. 1.
The internal part of the two polysaccharides is identical;
they differ in the presence of a methoxyl group at C-2 of the
upstream, terminal 4-amino-4,6-dideoxy-α--mannopyranose
(perosamine) in the Ogawa O-PS. Neoglycoconjugates prepared
from the hexasaccharide that represents the upstream terminus
of the O-PS of Vibrio cholerae O:1, serotype Ogawa have shown
immunogenicity for the Ogawa LPS-specific antibodies.3
To enable further studies of specificity and cross-reactivity
involved in the Vibrio cholerae O:1 serotypes, we have now pre-
pared neoglycoconjugates from the tetra- and hexasaccharides
representing the upstream epitopes of the Inaba O-PS.
Results and discussion
In principle, one can envision two fundamentally different
strategies to assemble oligosaccharides composed of N-
acylated perosamine. In the original approach by Bundle et
al.,5–7 perosamine-containing oligosaccharides were assembled
using mono- or disaccharide building blocks containing azido
groups at position 4, that is intermediates lacking the requisite
N-acyl group. We have adopted the same strategy to synthesize
some methyl α-glycosides of fragments of O-PS of Vibrio
cholerae O:1.8–10 A different assembly strategy can be based on
intermediate building blocks having the N-acyl group already
in place. Also, the stage at which the linker (spacer) should be
attached is an important aspect to consider, when such oligo-
saccharides are to be used for conjugation to carriers. In our
initial synthesis11 of linker equipped N-acylated perosamine-
containing haptens we first prepared 2-(trimethylsilylethyl) (SE)
glycosides of the requisite oligosaccharides from building
blocks lacking the N-acyl group. When the targeted oligo-
saccharide was assembled, the azido groups were reduced to
amino groups and the resulting amines were N-acylated. Sub-
sequently, the SE functionality was exchanged for an aglycon
Fig. 1 Structure of the O-PS of Vibrio cholerae O1, serotypes Inaba and Ogawa.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
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(spacer), which made the haptens amenable to chemical linking
to a carrier. Generally, the drawback of such an approach is
that it requires many chemical manipulations to be performed
with the assembled oligosaccharide molecule. We have, in the
past, prepared an N-acyl-perosamine-containing disaccharide
from monosaccharide building blocks that had the requisite
N-acyl group already in place,12 and showed that such an
approach is feasible. However, since the key glycosyl donor used
in the cited work lacked a selectively removable protecting
group at the site of further extension of the oligosaccharide
chain, the overall reaction scheme was not applicable to syn-
thesizing higher oligosaccharides. Here we describe a new
approach to the synthesis of tetra- and hexasaccharide frag-
ments of the O-PS of Vibrio cholerae O:1, serotype Inaba, from
intermediates having the N-acyl side chain already in place, and
conjugation thereof to bovine serum albumin (BSA).
desired compound 5, was found when 1 was treated with 5-
methoxycarbonyl pentanol14 in the presence of SnCl4, as
described by Banoub and Bundle15 for similar conversions.
Subsequent reduction of the azido group in 5 with H2S,
followed by N-acylation of the resulting amine 6, as described
for 3, gave the fully protected compound 7, which upon con-
ventional deacetylation (Zemplén) gave the desired alcohol 8.
Having secured the aforementioned key intermediates 4 and 8
we could proceed with oligosaccharide syntheses in the stepwise
manner. We opted for the sequential approach, rather than the
blockwise approach because, in separate work, we intend to use
intermediates obtained in this way to make each of the mono-
through to the pentasaccharide–BSA constructs in the serotype
Ogawa series. Similar hexasaccharide constructs showed suffi-
cient immunogenicity and protective capacity to have potential
as a cholera vaccine.3 The lower oligosaccharide–BSA con-
structs will be used to identify the smallest-size hapten that
can be used as the antigenic component in the target synthetic
vaccine for cholera. Thus, the N-iodosuccinimide/silver tri-
fluoromethanesulfonate-mediated (NIS/AgOTf ) reaction of 4
with 8 gave the fully protected disaccharide 9. Deacetylation of
9, followed by reaction of the formed disaccharide glycosyl
acceptor 10 with the donor 4 afforded (Scheme 3) the fully
protected trisaccharide 11. Repetition of this sequence of
reaction three more times gave, eventually, the fully protected
tetra-, penta-, and the hexasaccharides, 13, 15, and 17, respec-
The starting point in the synthesis was the known5,10 di-
acetate 1, which was converted to the amine 3 through the
thioglycoside 2. N-Acylation of 3 with 2,4-O-benzylidene--
glycero-tetronic acid (29) gave the key glycosyl donor 4 having a
selectively removable protecting group at O-2 allowing exten-
sion of the oligosaccharide chain at that position. The acid 29,
previously obtained by saponification of the corresponding
t-butyl ester13 (28) has now been obtained directly from the triol
(27)13 by oxidation with pyridinium dichromate (Experimental,
Scheme 1).
tively. Deacetylation of the tetrasaccharide 13 (13
14) and
hydrogenolysis, to simultaneously remove the protecting benzyl
and benzylidene groups, gave the fully deprotected methyl ester
19. The analogous hexasaccharide 23, which showed charac-
teristics identical to the previously described substance,11 was
obtained in a similar way.
The deprotected tetra- and hexasaccharides 19 and 23 are
glycosides whose aglycon allows further conversions to sub-
stances amenable to conjugation to amine-containing mole-
cules by squaric acid chemistry.16 We have studied the efficiency
of this method in detail,4,17,18 and have concluded that, when
applied to synthetic oligosaccharides, it is superior to conju-
gation by reductive amination. Conjugation applying squaric
acid chemistry is based on the findings by Tietze et al.19,20 who
showed that squaric acid diesters react with amines at different
Scheme 1
To obtain the initial glycosyl acceptor (Scheme 2), the di-
acetate 1 was first converted to 5-methoxycarbonyl glycoside 5
via the thioglycoside 2. The yield (79%) was satisfactory but a
more direct route, which gave a comparable yield (75%) of the
Scheme 2
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
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Scheme 3
pH to give either symmetrical or unsymmetrical squaric acid
after 15 days there would have been only little of the monoester
diamides. Thus, the 5-methoxycarbonylpentyl glycosides 19
and 23 were first treated with ethylenediamine ( 20 and 24,
respectively), and then with 3,4-diethoxy-3-cyclobutene-1,2-
dione (squaric acid diethyl ester) at pH 7. The resulting squaric
acid monoamides 21 and 25, respectively, were isolated by
chromatography and treated with BSA at pH 9 to complete the
conjugation protocol.19,20
25 left in the reaction mixture, due to the hydrolysis of the
reagent.4
Based on the above observations, all further conjugations were
carried out at the hapten/BSA ratio of 100:1. Under these con-
ditions, reaction of the tetrasaccharide 21 gave conjugate 22B
(21/BSA=11.6, see Experimental) after a reaction time of 2 days.
A more efficient conjugation protocol was designed next.
As described in the Experimental, two glycoconjugates with
predetermined saccharide/carrier ratio were made from the
tetrasaccharide 21 and hexasaccharide 25, each in a one-pot
reaction. To wit, for example, our aim was to obtain the hexa-
saccharide-based conjugates having molar carbohydrate/BSA
ratio of 6:1 and 20:1. The reaction was set up so that at the
onset of the reaction the molar 25/BSA ratio was 100:1. With
the 59 lysine groups in BSA22,23 (molecular mass 66,430 Da)
we deemed this excess to be sufficient to achieve the required
higher carbohydrate-onto-protein loading, while maintaining
a reasonable reaction rate. When the molar carbohydrate/BSA
ratio of ∼6:1 had been reached (3 h, as shown by SELDI-TOF
MS), a portion of the reaction mixture was withdrawn and
processed. The rest of the material was allowed to react further,
until SELDI-TOF MS showed that the molecular mass of the
conjugate that was being formed became close to 20 (22 h).
After processing and freeze-drying, the products 26A and 26C
were obtained as white solids whose molecular mass was virtu-
ally the same as that to the predetermined ones (see attached
formulae and Experimental). Neoglycoconjugates from the
tetrasaccharide monoamide 21 (22A and 22C; targeted ratios
hapten/BSA of 5 and 15, respectively) were obtained in a
similar way (see Experimental). In the conjugations described
above, both the tetra- and the hexasaccharide monoamides
appeared to be less reactive than the corresponding mono-
saccharide,17 but we can not explain the apparent higher
reactivity of the hexasaccharide 25, compared to that of the
analogous tetrasaccharide 21.
Monitoring the conjugation reaction by surface enhanced
laser desorption ionization-time of flight mass spectrometry
(SELDI-TOF MS) in combination with the ProteinChip®
System21 can furnish valuable data concerning the conjugation
process. We have shown21 that this technique provides virtually
real-time information about the increasing molecular weight of
the neoglycoconjugate formed, and allows the reaction to be
terminated when the desired carbohydrate/BSA ratio had been
reached. Thus, the ability to use the ProteinChip® System as a
tool to monitor conjugation of synthetic carbohydrates to
proteins has become a turning point in the preparation of neo-
glycoconjugates. During this work, it was interesting to com-
pare the efficiency of conjugation of the hexasaccharide 25 with
BSA with that of the corresponding monosaccharide. In the
latter case, under optimum conditions,17 using chicken serum
albumin (CSA) as a carrier (containing 46 lysine residues/CSA),
a neoglycoconjugate with 29 saccharide residues/CSA was
obtained after two days of reaction time. Our first conjugate
made from the hexasaccharide 25 was targeted to have the 25/
BSA ratio (loading) of 5:1. Based on the reaction rate observed
with the monosaccharide17 we presumed that the targeted load-
ing should be possible to achieve by conducting the conjugation
at the initial 25/BSA molar ratio of 10:1, namely with a 100%
excess of the hapten. However, the conjugation under these
conditions was much slower than in the case of the mono-
saccharide,17 and no increase in the degree of substitution
beyond the final 25/BSA ratio of 2.9 was noted after 15 days.
Further extension of the reaction time was deemed futile, since
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
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General methods
N-acylated -perosamine residues in the molecule. When
reporting assignments of NMR signals of oligosaccharides,
sugar residues in oligosaccharides are serially numbered,
beginning with the one bearing the aglycone, and are identified
by a Roman numeral superscript in listings of signal assign-
ments. Nuclei-assignments without a superscript notation
indicate that those signals have not been individually assigned.
Thus, for example, in a spectrum of a pentasaccharide, a
resonance denoted H-3 could be that of H-3 of either sugar
residue. When reporting NMR data, the nuclei belonging to the
3-deoxy--glycero-tetronic acid side chain are referred to as
primed (Ј), and those of the spacer aglycon as double primed
(Љ). Surface enhanced laser desorption ionization-time of flight
mass spectrometry (SELDI-TOF MS) was done using PBS-II
Mass Reader in combination with the ProteinChip® System
(Ciphergen Biosystems, Inc.), as previously described.21
Attempts have been made to obtain correct analytical data for
all new compounds. However, some compounds tenaciously
retained traces of solvents, despite exhaustive drying, and
analytical figures for carbon could not be obtained within
0.3%. The structure of these compounds is evident from
the mode of syntheses and m/z values found in their mass
spectra, and the purity was verified by TLC and NMR spectro-
scopy. These materials were obtained as white solids, which
showed mps within 2–3 ЊC. However, since we were unable to
Unless stated otherwise, optical rotations were measured at
ambient temperature for solutions in chloroform (c ∼1), with a
Perkin-Elmer automatic polarimeter, Model 341. All reactions
were monitored by thin-layer chromatography (TLC) on silica
gel 60 coated glass slides (Whatman or Analtech). Column
chromatography was performed by gradient elution from
columns of silica gel. Solvent mixtures less polar than those
used for TLC were used at the onset of development. NMR
spectra were measured at 300 MHz (1H) and 75 MHz
(13C), with a Varian Mercury spectrometer. Assignments of
NMR signals were made by first-order analysis of the spectra
and, when feasible, the assignments were supported by homo-
nuclear decoupling experiments or homonuclear and hetero-
nuclear 2-dimensional correlation spectroscopy, run with the
software supplied with the spectrometers. Spectra of higher
oligosaccharides were also analyzed by comparison with
spectra of related substances reported previously from this
laboratory8–11,24 or elsewhere.7 When the latter approach was
used, to aid in the 13C NMR signal–nuclei assignments, advan-
tage was taken of variations of line intensity expected for oligo-
saccharides belonging to the same homologous series.25,26 Thus,
spectra showed close similarity of chemical shifts of equivalent
carbon atoms of the internal residues, and an increase in the
relative intensity of these signals with the increasing number of
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
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induce these materials to crystallization from common organic
solvents, we describe them below as amorphous solids. Ethylene-
diamine was freshly distilled, bp 115–116 ЊC. Palladium-on-
charcoal catalyst (5%, ESCAT 103) was a product of Engelhard
([M ϩ NH4]ϩ). Anal. Calcd for C28H35NO7Sؒ0.5 H2O: C, 62.43;
H, 6.73; N, 2.59. Found: C, 62.39; H, 6.54; N, 2.56.
5-Methoxycarbonylpentyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-
dideoxy-ꢀ-D-mannopyranoside (5)
Industries.
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDAC), (S)-1,2,4-butanetriol, and 1-hydroxy-
benzotriazole (HOBT) were purchased from Aldrich Chemical
Co., and used as supplied. t-Butanol was dried with molecular
sieves 3 Å, and used without distillation. Sep-Pak C18 cart-
ridges were products of Waters Corporation, Inc. t-Butyl
2,4-O-benzylidene-3-deoxy--glycero-tetronate (28) was made,
essentially, as reported. However, the procedure given in the
Experimental is described in more detail than previously, and it
also lists complete assignment of lines in the 13C NMR spectrum
of 28. 2,4-O-Benzylidene-3-deoxy--glycero tetronic acid (29)
was prepared (Scheme 1) by saponification of the ester 2813 or
by oxidation of 1,2,4-(S)-butanetriol (27), as described below.
a. A mixture of ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-di-
deoxy-1-thio-α--mannopyranoside7 (2, 8.0 g, 21.9 mmol),
5-methoxycarbonylpentanol27 (5.0 g, 34.2 mmol) and 4 Å mole-
cular sieves (6 g)) in dry CH2Cl2 (30 mL) was stirred for 15 min
at room temperature. The mixture was cooled to Ϫ20 ЊC, and
NIS (6.9 g, 32.4 mmol) was added, followed by solid AgOTf
(2.1 g, 8.2 mmol, 0.37 equiv.). A few minutes later, the mixture
turned red and stirring was continued for another 15 min, when
the reaction was quenched by the addition of Et3N (2 mL). The
mixture was partitioned between a mixture of aq. Na2S2O3 and
NaHCO3, the organic layer was dried, and concentrated. Chro-
matography (7:1 hexane–EtOAc) gave 5 (7.8 g, 79%), which was
identical (TLC, NMR) with the compound described below.
b. SnCl4 (0.35 mL, 2.75 mmol) was added to a solution
of 1,2-di-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α--manno-
pyranose7 (1, 1 g, 2.75 mmol) in dry CH2Cl2 (30 mL) fol-
lowed by a solution of 5-methoxycarbonylpentanol27 (525 mg,
3.6 mmol) in dry CH2Cl2 (2 mL) and the solution was stirred at
room temperature until TLC (5:1 hexane–EtOAc) showed that
only a little starting material remained (5–7 h). One product,
whose chromatographic mobility was very close to that of
the starting 1, was formed. The mixture was neutralized with
aqueous NaHCO3, and the product was extracted with CH2Cl2.
The organic phase was dried, concentrated, and chromato-
graphy gave the amorphous 5 (930 mg, 75%), [α]D ϩ16Њ (c 0.95).
1H NMR (CDCl3): δ 5.32 (dd, 1 H, J1,2 1.8, J2,3 3.3 Hz, H-2),
4.71 (d, partially overlapped, H-1), 4.69, 4.53 (2 d, 1 H each,
2J 11.0 Hz, CH2Ph), 3.81 (dd, 1 H, J3,4 9.8 Hz, H-3), 3.68 (s,
partially overlapped, COOCH3), 3.68–3.48 (m, 2 H, H-5,1aЉ),
3.43–3.34 (m, 2 H, 1bЉ, incl 3.40, t, partially overlapped, H-4),
2.33 (t, 2 H, J 7.5 Hz, H-5Љ a,b), 2.12 (s 3 H, COCH3), 1,70–1.53
(m, 4 H, H-4Љa,b,2Љa,b in that order), 1.44–1.31 (m, 6 H,
H-3Љa,b, incl 1.32, d, J5,6 6.3 Hz, H-6); 13C NMR (CDCl3):
δ 97.60 (C-1), 76.13 (C-3), 71.56 (CH2Ph), 67.66 (C-1Љ), 67.43
(C-2), 66.83 (C-5), 63.96 (C-4), 51.47 (COOCH3), 33.85 (C-5Љ
28.95 (C-2Љ), 25.61 (C-3Љ), 24.59 (C-4Љ), 20.96 COCH3), 18.44
(C-6); FABMS: m/z 450 ([M ϩ 1]ϩ), 472 ([M ϩ Na]ϩ). Anal.
Calcd for C22H31N3O7S: C, 58.78; H, 6.95; N 9.35. Found: C,
58.98; H, 6.87; N, 9.34.
Ethyl 4-amino-2-O-acetyl-3-O-benzyl-4,6-dideoxy-1-thio-ꢀ-D-
mannopyranoside (3)
Hydrogen sulfide was passed, for 45 min at 40 ЊC, through a
solution of ethyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-1-
thio-α--mannopyranoside7 (2, 43 g, 117.6 mmol) in pyridine–
water (2:1, v/v, 750 mL). The mixture was stirred at 40 ЊC in a
flask, which was equipped with a rubber septum and a rubber
balloon. After 3 h, TLC (30:1 CH2Cl2–MeOH) showed that
all the starting material was consumed, and that a more polar
product was formed. Nitrogen gas was passed through the
brown solution until almost no discoloration was noticeable,
and the mixture was concentrated. The residue was triturated
with EtOAc and the solids were filtered off. The filtrate was
concentrated, and crystallization from EtOAc gave 3 (35 g), the
mother liquid was chromatographed, to give another crop of
3 (2.9 g), total yield 95%, mp 91–92Њ (from isopropyl ether),
1
[α]D ϩ59Њ (c 0.5); H NMR (CDCl3): δ 5.42 (dd, 1 H, J1,2 1.4,
J2,3 3.1 Hz, H-2), 5.25 (d, 1 H, H-1), 4.68, 4.38 (2 d, 1 H each,
2J 11.3 Hz, CH2Ph), 3.91 (m, 1 H, H-5), 3.53 (dd, 1 H, J3,4 9.8
Hz, H-3), 2.90 (t, 1 H, H-4), 2.62 (m, 2 H, SCH2), 1.43 (bs, 2 H,
NH2), 1.30 (d, partially overlapped, J5,6 ∼5.8 Hz, H-6), 1.28
(t, partially overlapped, CH2CH3); 13C NMR (CDCl3): δ 82.51
(C-1), 77.96 (C-3), 71.23 (CH2Ph), 69.96 (C-5), 68.86 (C-2),
54.19 (C-4), 25.45 (SCH2), 20.98 (COCH3), 17.98 (C-6), 14.87
(CH2CH3); FABMS: m/z 340 ([M ϩ 1]ϩ), 362 ([M ϩ Na]ϩ).
Anal. Calcd for C17H25NO4S: C, 60.15; H, 7.42; N 4.13. Found:
C, 59.90; H, 7.34; N, 4.07.
5-Methoxycarbonylpentyl 2-O-acetyl-4-amino-3-O-benzyl-4,6-
dideoxy-ꢀ-D-mannopyranoside (6)
Ethyl 2-O-acetyl-3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-
glycero-tetronamido)-4,6-dideoxy-1-thio-ꢀ-D-mannopyranoside
(4)
Compound 5 (8.0 g, 17.8 mmol) was treated as described for
the preparation of 3. After processing, as described above,
chromatography gave amorphous 6 (7.1 g, 94%), [α]D Ϫ2.4Њ
(c 0.7). 1H NMR (CDCl3): δ 5.33 (dd, 1 H, J1,2 1.8, J2,3 3.1 Hz,
A mixture of the foregoing amine 3 (12 g, 35.3 mmol), acid 29
(8.9 g, 42.4 mmol), EDAC (10.0 g, 51.4 mmol), and HOBT
(7.2 g, 51.4 mmol) in CH2Cl2 (800 mL) was stirred at room
temperature for 1 h. TLC (30:1 CH2Cl2–MeOH) showed that
the starting material was consumed, and that a less polar
product was formed. The reaction mixture was concentrated,
and the residue was chromatographed (2:1, hexane–acetone)
to give 4 (18.1 g, 97%), mp 52.5–53.5Њ (from isopropyl ether);
[α]D ϩ35.6Њ (c 0.5). 1H NMR (CDCl3): δ 6.22 (d, 1 H, J4,NH 8.8
Hz, NH), 5.55 (s, 1 H, CHPh), 5.44 (dd, 1 H, J1,2 1.6, J2,3 3.1 Hz,
H-2), 5.23 (d, 1 H, H-1), 4.64, 4.36 (2 d, partially overlapped,
2J 12.0 Hz, CH2Ph), 4.38–4.32 (m, partially overlapped,
H-2Ј,4Јa), 4.13–3.96 (m, 3 H, H-4,5,4Јb), 3.70 (dd, 1 H, J3,4 10.1
Hz, H-3), 2.69–2.50 (m, 2 H, SCH2), 2.15 (s, 3 H, COCH3),
2.06, 1.91 (2 m, 2 H, H-3Ј), 1.27 (t, partially overlapped, J 7.4
Hz, CH2CH3), 1.24 (d, partially overlapped, J5,6 5.8, H-6);
13C NMR (CDCl3): δ 101.1 (CHPh), 82.25 (C-1), 76.42 (C-2Ј),
74.15 (C-3), 71.02 (CH2Ph), 69.08 (C-2), 69.0 (C-5), 67.25
(C-4Ј), 52.36 (C-4), 28.53 (C-3Ј), 25.49 (SCH2), 21.06 (COCH3),
17.81 (C-6), 14.80 (CH2CH3); CIMS: m/z 530 ([M ϩ 1]ϩ), 547
2
H-1), 4.75 (d, H-1), 4.71, 4.39 (2 d, 1 H each, J 11.1 Hz,
CH2Ph), 3.69–3.55 (m, 6 H, H-3,5, 1aЉ incl 3.68, s, COOCH3),
3.40, 3.37 (2 t, partially overlapped, J 6.3 Hz, 1 H, 1bЉ), 2.88
(t, 1 H, J 10.1 Hz, H-4), 2.33 (t, 2 H, J 7.5 Hz, H-5Љa,b), 2.12 (s
3 H, COCH3), 1.70–1.52 (m, 4 H, H-4Љa,b,2Љa,b, in that order),
1.47–1.35 (m, 4 H, NH2, H-3Љa,b), 1.28 (d, J5,6 6.3 Hz, H-6); 13
C
NMR (CDCl3): δ 97.98 (C-1), 77.68 (C-3), 71.26 (CH2Ph),
69.42 (C-5), 67.42 (C-1Љ), 67.15 (C-2), 53.83 (C-4), 51.47
(COOCH3), 33.89 (C-5Љ), 29.04 (C-2Љ), 25.70 (C-3Љ), 24.64
(C-4Љ), 21.02 COCH3), 18.04 (C-6). CIMS: m/z 424 ([M ϩ 1]ϩ).
Anal. Calcd for C22H33NO7: C, 62.39; H, 7.85; N, 3.31. Found:
C, 62.25; H, 7.92; N, 3.31.
5-Methoxycarbonylpentyl 2-O-acetyl-3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranoside (7)
A mixture of the foregoing amine 6 (4.5 g, 10.6 mmol), 3-deoxy-
-glycero-tetronic acid derivative 29 (3.4 g, 16.2 mmol), EDAC
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
779

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(4.1 g, 32.4 mmol), and HOBT (2.9 g, 32.4 mmol) in CH2Cl2
(300 mL) was stirred at room temperature for 1 h. TLC (20:1
CH2Cl2–MeOH) showed that the reaction was complete,
and that a less polar product was formed. The reaction mixture
was concentrated, and the residue was chromatographed, to
86%), mp 70.5–71.5 ЊC (from EtOAc–hexane), [α]D Ϫ23Њ (c 0.6);
1H NMR (CDCl3): δ 6.37, 6.17 (2 d, 1 H each, J4,NH 8.3 and 9.7,
respectively, 2 NH), 5.55, 5.54 (2 s, 1 H each, CHPh), 5.49
(dd, 1 H, J1,2 1.8, J2,3 3.1 Hz, H-2II), 4.90 (d, 1 H, H-1II), 4.70–
4.40 (4 d, partially overlapped, 2 CH2Ph), 4.66 (d, partially
1
give amorphous 7 (5.4 g, 83%); [α]D ϩ 6.0Њ (c 0.6). H NMR
overlapped, J1,2 ∼1.7 Hz, H-1I), 4.38–4.30 (m, 4 H, H-2ЈI,II
,
(CDCl3): δ 6.33 (d, 1 H, J4,NH 9.7 Hz, NH), 5.55 (s, 1 H, CHPh),
5.33 (dd, 1 H, J1,2 1.8, J2,3 3.1 Hz, H-2), 4.74 (d, 1 H, H-1), 4.64
(d, 1 H, J 11.6 Hz, CHaPh), 4.39 (d, partially overlapped,
4ЈaI,II), 4.10 (q, partially overlapped, H-4II), 4.00, 3.99 (2 t,
partially overlapped, J 11.0 Hz, H-4ЈbI,II), 3.90–3.71 (m, 6 H,
H-2I,3I,4I,5I,II, incl dd at 3.73, J2,3 ∼3.1, J3,4 ∼10.7 Hz, H-3II),
3.62–3.54 (m, partially overlapped, H-1Љa), 3.60 (s, partially
overlapped, COOCH3), 3.32–3.29 (m, 1 H, H-1Љb), 2.27 (t,
2 H, J 7.4 Hz, H-5Љa,b), 2.09 (s, partially overlapped, COCH3),
2.11–1.82 (m, partially overlapped, H-3Јa,bI,II), 1.66–1.50 (m, 4
H, H-4Љa,b,2Љa,b in that order) 1.37–1.29 (m, 2 H, H-3Љa,b),
1.20, 1.17 (2 d, 6 H, J5,6 6.2 and 6.0 Hz, H-6I,II). 13C NMR
(CDCl3): δ 101.35, 101.11 (2 CHPh), 99.81 (C-1II), 98.82 (C-1I),
76.58 (2 C, C-2ЈI,II), 75.51 (C-3I), 74.43 (C-2I), 73.43 (C-3II),
71.81, 70.99 (2 CH2Ph), 68.52 (C-5), 67.28 (4 C, C-5,4ЈI,II,1Љ),
67.15 (C-2II), 52.66 (C-4I), 51.54 (C-4II), 51.40 (COOCH3),
33.83 (C-5Љ), 28.81 (C-2Љ), 28.54 (C-3Ј), 25.51 (C-3Љ), 24.50
(C-4Љ), 21.03 (COCH3), 17.96 (2C, C-6I,II). FABMS: m/z 1039.5
([M ϩ 1]ϩ), 1062.5 ([M ϩ Na]ϩ). Anal. Calcd for C57H70N2O16ؒ
0.5H2O: C, 65.31; H, 6.82; N, 2.67. Found: C, 65.01; H, 6.69;
N, 2.65.
2
CHbPh), 4.39–4.10 (m, partially overlapped, H-2Ј,4Јa), 4.05 (t,
partially overlapped, J4,5 ∼10.5 Hz, H-4), 4.05–3.98 (m, partially
overlapped, H-4Јb), 3.79 (dd, partially overlapped, H-3), 3.82–
3.74 (m, partially overlapped, H-5), 3.64, 3.62 (2 t, partially
overlapped, J 6.6 Hz, H-1Љa), 3.60 (s, partially overlapped,
COOCH3), 3.38, 3.35 (2 t, 1 H, H-1Љb), 2.29 (t, 2 H, J 7.9 Hz,
H-5Љa,b), 2.14 (s, 3 H, COCH3), 2.08–1.87 (m, 2 H, H-3Јa,b),
1.68–1.53 (m, 4 H, H-4Љa,b,2Љa,b), 1.39–1.30 (m, 2 H, H-3Љa,b),
1.24 (d, 3 H, J5,6 6.3 Hz, H-6); 13C NMR (CDCl3): δ 101.20
(CHPh), 97.73 (C-1), 76.57 (C-2Ј), 74.09 (C-3), 71.12 (CH2Ph),
67.55 (C-1Љ), 67.57, 67.52 (C-2,5), 67.30 C-4Ј), 52.16 (C-4),
51.42 (COOCH3), 33.87 (C-5Љ), 28.84 C-2Љ), 28.56 (C-3Ј), 24.55
(C-4Љ), (COCH3) 17.85 (C-6). CIMS: m/z 614 ([M ϩ 1]ϩ), 631
([M ϩ NH4]ϩ). Anal. Calcd for C33H43NO10: C, 64.58; H, 7.06;
N, 2.28. Found: C, 64.64; H, 6.91; N, 2.29.
5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-manno-
pyranoside (8)
5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-manno-
pyranosyl-(1 2)-(3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-
L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranoside (10)
Freshly prepared 1 M methanolic NaOMe (2.5 mL) was added
to a solution of 7 (4.6 g, 7.5 mmol) in dry MeOH (100 mL).
After 30 min, TLC (2:1 hexane-acetone) showed that no
starting material was present, and that a more polar compound
was formed. The solution was neutralized with Amberlite IR
120 (Hϩ) resin, the mixture was filtered, the filtrate was concen-
trated, and the residue was chromatographed, to give 8 (4.27 g,
∼100%), mp 87–87.5 ЊC, (from EtOAc–hexane), [α]D Ϫ20.4Њ
The foregoing fully protected compound 9 (4.6 g) was treated
with NaOMe as described for the preparation of 8. TLC
showed complete conversion of the starting material and form-
ation of a more polar product. After processing, as described
above, and chromatography, compound 10 (4.4 g, ∼100%) was
1
obtained as a white solid showing [α]D Ϫ9Њ (c 0.5); H NMR
(CDCl3): 6.34, 6.24 (2 d, 1 H each, J4,NH 9.6 Hz, NH), 5.55, 5.54
(2 s, 1 H each, CHPh), 5.07 (d, 1 H, J1,2 1.5 Hz, H-1II), 4.70–4.44
(4 d, partially overlapped, 2 CH2Ph), 4.68 (d, partially over-
lapped, J1,2 ∼1.8 Hz, H-1I), 4.39–4.29 (m, 4 H, H-2ЈI,II, 4ЈaI,II),
4.22 (bdd, 1 H, H-2II), 4.16 (q, partially overlapped, H-4II),
1
(c 0.7). H NMR (CDCl3): δ 6.37 (d, 1 H, J4,NH 9.7 Hz, NH),
5.56 (s, 1 H, CHPh), 4.82 (d, 1 H, J1,2 1.8 Hz, H-1), 4.67, 4.52
(2 d, 1 H each, 2J 12.0 Hz, CH2Ph), 4.40–4.31 (m, 2 H,
H-2Ј,4Јa), 4.07 (t, partially overlapped, J4,5 ∼10.2 Hz, H-4),
4.05–4.00 (m, partially overlapped, H-4Јb), 3.98 (dd, partially
overlapped, H-2), 3.78 (m, partially overlapped, H-5), 3.71
(dd, partially overlapped, H-3), 3.68–3.58 (m, 4 H, H-1Љa, incl s
at 3.60, COOCH3), 3.40, 3.37 (2 t, J 6.5 Hz, 1 H, H-1Љb), 2.58
(bs, 1 H, OH), 2.29 (t, 2 H, J 7.9 Hz, H-5Љa,b), 2.08–1.85 (m,
2 H, H-3Јa,b), 1.68–1.53 (m, 4 H, H-2Љa,b,4Љa,b), 1.39–1.28
(m, 2 H, H-3Љa,b), 1.22 (d, 3 H, J5,6 6.2 Hz, H-6); 13C NMR
(CDCl3): δ 101.13 (CHPh), 99.02 (C-1), 76.52 (C-2Ј), 76.47
(C-3), 71.42 (CH2Ph), 67.38 (C-1Љ), 67.25 C-4Ј), 67.23 (C-2),
66.93 (C-5), 51.69 (C-4), 51.40 (COOCH3), 33.89 (C-5Љ), 28.87
(C-2Љ), 28.57 (C-3Ј), 25.59 (C-3Љ), 24.56 (C-4Љ), 17.85 (C-6).
CIMS: m/z 572 ([M ϩ 1]ϩ), 589 ([M ϩ NH4]ϩ). Anal. Calcd for
C31H41NO9: C, 65.13; H, 7.23; N, 2.45. Found: C, 64.91; H,
7.20; N, 2.40.
4.03–3.95 (m, 4 H, H-2I,4I,H-4ЈbI,II), 3.77–3.66 (m, 4 H, 5I,II
,
incl dd at ∼3.75, J2,3 ∼2.7, J3,4 ∼10.6 Hz for H-3I, and dd at
∼3.68, J2,3 ∼3.3, J3,4 10.6 Hz for H-3II), 3.62–3.54 (m, partially
overlapped, H-1Љa), 3.58 (s, partially overlapped, COOCH3),
3.35–3.28 (m, 1 H, H-1Љb), 2.60 (bs, 1 H, OH), 2.27 (t, 2 H, J 7.4
Hz, H-5Љa,b), 2.06, 1.90 (2 m, H-3Јa,bI,II), 1.66–1.50 (m, 4 H,
H-4Љa,b,2Љa,b in that order), 1.37–1.28 (m, 2 H, H-3Љa,b), 1.20,
1.18 (2 d, 6 H, J5,6 6.4 Hz, H-6I,II); 13C NMR (CDCl3): δ 101.24,
101.07 (2 CHPh), 101.92 (C-1II), 98.93 (C-1I), 76.49, 76.40
(C-2ЈI,II), 75.94 (C-3I), 75.64 (C-3II), 73.41 (C-2I), 71.78, 71.03
(2 CH2Ph), 67.84, 67.58 (C-5I,II), 67.21 (C-1Љ), 67.18 (2 C,
C-4ЈI,II), 66.57 (C-2II), 52.05 (C-4I), 51.33 (COOCH3), 51.00
(C-4II), 33.75 (C-5Љ), 28.72 (C-2Љ), 28.47 (C-3ЈI,II), 25.44 (C-3Љ),
24.42 (C-4Љ), 17.88, 17.79 (C-6I,II); FABMS: m/z 997.5 ([M ϩ
1]ϩ), 1019.5 ([M ϩ Na]ϩ). Anal. Calcd for C55H68N2O15: C,
66.25; H, 6.87; N, 2.81. Found: C, 65.79; H, 6.85; N, 2.81.
5-Methoxycarbonylpentyl 2-O-acetyl-3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranosyl-(1 2)-3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
side (9)
5-Methoxycarbonylpentyl 2-O-acetyl-3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranosyl-(1 2)-(3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl-
(1 2)-3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranoside (11)
To a solution of 4 (9.12 g, 17.2 mmol) and 8 (8.20 g, 14.36
mmol) in dry CH2Cl2 (30 mL), were added 4 Å molecular sieves
(13 g), and the mixture was stirred for 15 min at room tem-
perature. The mixture was cooled to Ϫ20 ЊC, and NIS (4.5 g,
20.3 mmol) was added, followed by solid AgOTf (1.4 g,
5.6 mmol). When the mixture turned dark red, the mixture was
stirred for another 15 min, and the reaction was processed as
described for the preparation of 5 (procedure a). Chromato-
graphy (3:1 1:1 hexane–acetone) gave crystalline 9 (12.8 g,
Compound 4 (6.5 g, 12.3 mmol) when treated with 10 (10.2 g,
10.2 mmol) as described for the preparation of 9 gave, after
chromatography, amorphous 11 (11.9 g, 80%), [α]D Ϫ23Њ (c 0.5).
1H NMR (CDCl3): δ 6.40–6.29 (m, 3 H, incl 2 d at 6.35 and
6.31, partially overlapped, J ∼9.5 Hz, 3 NH), 5.54, 5.52, 5.51
(3 CHPh), 5.47 (dd, 1 H, J1,2 1.9, J2,3 3.0 Hz, H-2III), 4.99 (d,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
780

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1 H, J1,2 1.8 Hz, H-1II), 4.81 (d, 1 H, J1,2 1.8 Hz, H-1III), 4.67–
4.52 (m, 6 H, 5 CHPh, benzylic, incl. d at ∼4.64, partially over-
lapped, J1,2 ∼1.8 Hz, H-1I), 4.41–4.22 (m, 7 H, CHPh, benzylic,
H-2ЈI–III,4ЈaI–III), 4.13–3.68 (m, 14 H, H-2II,3I–III,4I–III,5I–III,4Ј-
bI–III, incl bdd at 3.87, H-2I), 3.60–3.51 (m, 4 H, H-1Љa, incl s at
3.53, COOCH3), 3.32–3.25 (m, 1 H, H-1Љb), 2.23 (t, 2 H, J 7.5 Hz,
H-5Љ), 2.08–1.78 (m, 9 H, H-3Јa,b, incl s at 2.06, COCH3), 1.63–
1.47 (m, 4 H, H-2Љa,b,4Љa,b), 1.34–1.22 (m, 2 H, H-3ЈI–IIIa,b),
1.18, 1.17, 1.12 (3 d, partially overlapped, 9 H, J ∼6.1 Hz,
H-6I–III); 13C NMR (CDCl3): δ 100.96, 100.91, 100.77 (3 CH-
Ph), 101.57 (C-1II), 98.96 (C-1III), 98.67 (C-1I), 76.41, 76.31,
76.20 (C-2ЈI–III), 75.41, 74.34, 73.33 (C-3I–III), 74.02 (C-2I), 73.46
(C-2II), 71.36, 71.07, 70.72 (3 CH2Ph), 68.28, 68.14, 67.31
(C-5I–III),, 67.06 (C-1Љ), 66.94 (4 C, C-2III,C-4ЈI–III), 51.97, 51.69,
51.45 (C-4I–III), 51.08 (COOCH3), 33.53 (C-5Љ), 28.50 (C-2Љ),
28.33 (3 C, C-3ЈI–III), 25.24 (C-3Љ), 24.20 (C-4Љ), 20.75 (COCH3),
17.90, 17.70, 17.65 (C-6I–III). FABMS: m/z 1464.6 ([M ϩ 1]ϩ),
1486.6 ([M ϩ Na]ϩ). Anal. Calcd for C81H97N3O22: C, 66.42;
H, 6.68; N, 2.87. Found: C, 66.20; H, 6.70; N, 2.89.
H-2Љa,b,4Љa,b), 1.33–1.21 (m, 2 H, H-3Љa,b), 1.13, 1.12. 1.08.
1.02 (4 d, partially overlapped, J5,6 6.2 Hz, H-6I–IV); 13C NMR
(CDCl3): δ 101.28, 101.21, 101.47 (2 C, C, C, 4 CHPh), 101.03,
100.25 (C-1II,III), 99.25 (C-1IV), 98.83 (C-1I), 76.64, 76.58, 76.53
(2 C, C, C, C-2ЈI–IV), 75.27, 74.97, 74.71 (C-3I–III), 74.14 (C-2I),
73.78 (2 C, C-2,3), 72.83 (C-2), 71.34, 71.06 (3 C, C, 4 CH2Ph),
68.57, 68.30, 67.52 (2 C, C, C, 5I–IV), 67.33 (C-1Љ), 67.26 (5 C,
C-2IV,C-4ЈI–IV), 52.18, 52.11, 51.69, 51.62 (C-4I–IV), 51.41
(COOCH3), 33.82 (C-5Љ), 28.79 (C-2Љ), 28.52 (4 C, C-3ЈI–IV),
25.49 (C-3Љ), 24.49 (C-4Љ), 21.05 (COCH3), 18.01, 17.92, 17.87
(2 C, C, C, C-6I–IV); FABMS: m/z 1890.81 ([M ϩ 1]ϩ), 1912.81
([M ϩ Na]ϩ). HRMS: m/z 2021.7383. C105H124N4O28Cs requires
2021.7456.
5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl-(1 2)-bis[3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-
glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl]-(1 2)-
3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-glycero-tetron-
amido)-4,6-dideoxy-ꢀ-D-mannopyranoside (14)
5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl-(1 2)-3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-glycero-
-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl-(1 2)-3-O-
benzyl-4-(2,4-O-benzylidene-3-deoxy-L-glycero-tetronamido)-
4,6-dideoxy-ꢀ-D-mannopyranoside (12)
Conventional deacetylation of the tetrasaccharide 13 gave 14 as
a white solid in virtually theoretical yield, [α]D Ϫ26.4Њ (c 0.4).
1
Definite signals in the H NMR spectrum (CDCl3) were at:
δ 6.46–6.16 (m, 4 H, 4 NH), 5.53 (s, 2 H, 2 CHPh), 5.52, 5.50
(2 s, 2 H, 2 CHPh), 5.02 (bs, 2 H, H-1IV, H-1), 4.92 (d, 1 H, J1,2
1.7 Hz, H-1), 4.67–4.27 (m, incl d at ∼4.63, partially overlapped,
H-1I), 3.83 (bt, 1 H, H-2I), 3.59–3.52 (m, 4 H, H-1Љa, incl s at
3.58 COOCH3), 3.32–3.24 (m, 1 H, H-1Љb), 2.51 (bd, 1 H, OH),
2.25 (t, 2 H, J 7.1 Hz, H-5Љ), 2.05–1.80 (m, 8 H, H-3ЈI–IVa,b),
1.80–1.64 (m, 4 H, H-2Љa,b,4Љa,b), 1.34–1.23 (m, 2 H, H-3Љa,b),
1.14, 1.10, 1.04 (3 d, 12 H, J5,6 6.3 Hz, H-6I–IV); 13C NMR
(CDCl3): δ 101.23, 101.13, 101.10, 101.08 (4 CHPh), 100.88
(C-1), 100.28 (2 C, C-1,1IV), 98.71 (C-1I), 76.57, 76.46, 76.41
(C, C, 2 C, C-2ЈI–IV), 75.87, 75.19, 75.05, 74.60 (C-3I–IV), 74.04
(C-2I), 72.80, 72.67 (C-2II,III), 71.26 (3 C), 71.07 (4 CH2Ph),
68.52, 68.45 (C-5II,III), 67.87. 67.43 (C-5I,IV), 67.15 (5 C,
C-4I–IV,1Љ), 66.72 (C-2IV), 52.07, 51.65, 51.52, 51.14 (C-4I–IV),
51.31 (COOCH3), 33.72 (C-5Љ), 28.69 (C-2Љ), 28.45 (C-3ЈI–IV),
25.41 (C-3Љ), 24.39 (C-4Љ), 18.00, 17.92, 17.85. 17.70 (C-6I–IV);
FABMS: m/z 1847.85 ([M ϩ 1]ϩ), 1869.85 ([M ϩ Na]ϩ). HRMS:
m/z 1979.7346. C103H122N4O27Cs requires 1979.7351.
The fully protected compound 11 (11 g) was deacetylated, as
described for the preparation of 10, to give pure (TLC, NMR)
12 (10.6 g, ∼100%) as a white solid, [α]D Ϫ19.4Њ (c 0.4); 1H NMR
(CDCl3): δ 6.36–6.27 (bs, partially overlapped, NH), 6.30 (d,
partially overlapped, J 9.3 Hz, NH), 6.2 (d, 1 H, J 9.3 Hz, NH),
5.54, 5.52, 5.51 (3 s, 3 CHPh), 5.03 (d, 1 H, J1,2 1.6 Hz, H-1III),
4.98 (d, 1 H, J1,2 1.9 Hz, H-1II), 4.68–4.47 (m, 6 H, 5 CHPh, incl.
d at ∼4.64, partially overlapped, H-1I), 4.41–4.25 (m, 7 H,
CHPh, H-2ЈI–III,4ЈaI–III), 4.20–4.07 (4 H, H-2II,III,3I–III,4,4),
4.05–3.84 (m, 5 H, 4,4ЈbI–III incl bt at 3.87, H-2I), 3.76–3.66
(m, 6 H, H-3I–III,5I–III), 3.60–3.52 (m, 4 H, H-1Љa, incl s at 3.58,
COOCH3), 3.34–3.25 (m, 1 H, H-1Љb), 2.50 (d, 1 H, J 1.5 Hz,
OH), 2.26 (t, 2 H, J 7.4 Hz, H-5Љ), 2.09–1.79 (m, 6 H,
H-3ЈI–IIIa,b), 1.65–1.48 (m, 4 H, H-2Љa,b,4Љa,b), 1.35–1.23 (m,
2 H, H-3Љa,b), 1.17, 1.14, 1.07 (3 d, 9 H, J 6.2 Hz, H-6I–III); 13
C
NMR (CDCl3): δ 101.28, 101.20 (2 CHPh), 101.03 (2 C, CHPh,
C-1II), 100.35 (C-1III), 98.83 (C-1I), 76.51, 76.42 (2 C, C, C-2ЈI–III),
75.78 (C-3III), 75.27, 75.07 (C-3I,II), 74.33 (C-2I), 72.61 (C-2II),
71.46, 71.41, 71.06 (3 CH2Ph), 66.52, 67.84, 67.38 (5I–III), 67.20
(3 C, C-4ЈI–III,1Љ), 66.67, (C-2III), 52.24, 51.53, 51.14 (C-4I–III),
51.33 (COOCH3), 33.75 (C-5Љ), 28.72 (C-2Љ), 28.49 (C-3ЉI–III),
25.42 (C-3Љ), 24.42 (C-4Љ), 18.04, 17.87, 17.70 (C-6I–III).
FABMS: m/z 1422.6 ([M ϩ 1]ϩ), 1444.6 ([M ϩ Na]ϩ). HRMS:
m/z 1554.5477. C79H95N3O21Cs requires 1554.5512.
5-Methoxycarbonylpentyl 2-O-acetyl-3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranosyl-(1 2)-tris[3-O-benzyl-4-(2,4-O-benzylidene-
3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl]-(1 2)-3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranoside (15)
Glycosylation of 14 (5.0 g, 2.70 mmol) with 4 (2.5 g, 4.72
mmol), as described above for similar reactions gave, after
chromatography, the pentasaccharide 15 (5 g, ∼80%) as a white
5-Methoxycarbonylpentyl 2-O-acetyl-3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranosyl-(1 2)-bis[3-O-benzyl-4-(2,4-O-benzylidene-
3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-manno-
pyranosyl]-(1 2)-3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-
L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranoside (13)
1
solid, [α]D Ϫ35Њ (c 0.7). Definite signals in the H NMR spec-
trum (CDCl3) were at δ: 6.40–6.07 (m, 5 H, 5 NH), 5.54, 5.53,
5.52, 5.51, 5.48 (5 s, 5 H, 5 CHPh), 5.45 (bt, 1 H, H-2V), 5.01,
4.94, 4.89 (3 d, J1,2 ∼1.6 Hz, H-1II–IV), 4.80 (d, 1 H, J1,2 1.6 Hz,
H-1V), 4.61 (bd, partially overlapped, part of a larger m, H-1I),
4.13 (bt, partially overlapped, H-2), 3.82 (bt, partially over-
lapped, H-2I), 3.59–3.50 (m, 4 H, m, H-1ЉaЈ incl s at 3.59,
COOCH3), 3.30 (m, 1 H, H-1Љb), 2.25 (t, 2 H, J 7.5 Hz,
H-5Љa,b), 2.07–1.81 (m, 13 H, H-3ЈI–V, incl s at 2.07, COCH3),
1.64–1.46 (m, 4 H, H-2Љa,b,4Љa,b), 1.35–1.24 (m, 2 H, H-3Љa,b),
1.13–1.03 (5 d, partially overlapped, 15 H, H-6I–V); 13C NMR
(CDCl3): δ 101.28, 101.20, 101.15 (C, 3 C, C, 5 CHPh), 101.02,
100.45, 100.15 (C-1II–IV), 99.18 (C-1V), 98.80 (C-1I), 76.62, 76.54
(2 C, 3 C, C-2ЈI–Va,b), 75.18, 74.96, 74.81, 74.56, 74.29, 73.81,
73.60, 72.80, 72.68 (C-2I–IV,3I–V), 71.36, 71.27, 71.08, 71.04 (C,
2 C, C, C, 5 CH2Ph), 68.63, 68.57, 68.44, 67.57 (2 C, C, C, C,
C-5I–V), 67.31 (2 C, C-2V,1Љ), 67.26 (5 C, C-4ЈI–V), 52.11, 52.07,
Reaction of the glycosyl donor 4 (3.67 g, 6.94 mmol) and the
glycosyl acceptor 12 (8.2 g, 5.77 mmol), as described above for
similar glycosylations, afforded 13 (8.71 g, 80%), [α]D Ϫ30Њ
(c 0.8). Definite signals in the 1H NMR spectrum (CDCl3) were
at: δ 6.42–6.17 (m, 4 H, 4 NH), 5.53, 5.52, 5.51, 5.50 (4 s, 4 H,
4 CHPh), 5.44 (dd, 1 H, J1,2 1.8, J2,3 3.0, H-2IV), 5.01, 4.90 (2 d,
J1,2 1.7 Hz, H-1II,III), 4.79 (d, 1 H, J1,2 1.6 Hz, H-1IV), 4.65–4.49
(m, 8 H, 3.5 CH2Ph, incl d at ∼4.61, partially overlapped, H-1I),
4.12–4.08 (m, part of a larger m, H-2II,III), ∼3.83 (m, part of a
larger m, H-2IV), 3.59–3.50 (m, 4 H, H-1Љa, incl s at 3.58,
COOCH3), 3.30–3.23 (m, 1 H, H-1Љb), 2.25 (t, 2 H, J 7.6 Hz,
H-5Љa,b), 2.05–1.81 (m, 8 H, H-3ЈI–IVa,b), 1.63–1.46 (m, 4 H,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
781

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51.77, 51.64, 51.50 (C-4I–V), 51.41 (COOCH3), 33.82 (C-5Љ),
28.78 (C-2Љ), 28.56 (5 C, C-3ЈI–V), 24.48 (C-4Љ), 21.07 (COCH3),
18.06, 17.97, 17.92 (2 C, 2 C, C, C-6I–V); FABMS: m/z 2316.17
([M ϩ 1]ϩ), 2338.16 ([M ϩ Na]ϩ). HRMS: m/z 2446.9199.
C129H151N5O34Cs requires 2446.9295.
5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl-(1 2)-tetrakis[3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-
glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl]-(1 2)-
3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-glycero-tetron-
amido)-4,6-dideoxy-ꢀ-D-mannopyranoside (18)
5-Methoxycarbonylpentyl 3-O-benzyl-4-(2,4-O-benzylidene-3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl-(1 2)-tris[3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-
glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl]-(1 2)-
3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-L-glycero-tetron-
amido)-4,6-dideoxy-ꢀ-D-mannopyranoside (16)
Deacetylation of 17 (Zemplén) gave 18 in virtually theoretical
yield as an amorphous solid, [α]D Ϫ29.4 Њ (c 0.6). Definite
signals in the H NMR spectrum (CDCl3) were at δ: 6.47–6.12
1
(m, 6 H, 6 NH), 5.44, 5.53, 5.52, 5.51, 5.48 (5 s, 6 H, 6 CHPh),
5.00, 4.92, 4.87 (m, m, bd, 5 H total, H-1II–VI), 4.61 (bd, partially
overlapped, H-1I), 3.80 (bt, 1 H, H-2I), 3.58 (s, partially over-
lapped, COOCH3), 3.58–3.30 (m, partially overlapped, H-1Љa),
3.28, 3.24 (2 t, 1 H, J 6.3 Hz, H-1Љb), 2.25 (t, 2 H, J 7.5 Hz,
H-5Љa,b), 2.06–1.82 (m, partially overlapped, H-3Јa,bI–VI), 1.63–
1.46 (m, 4 H, H-2Љa,b,4Љa,b), 1.33–1.22 (m, 2 H, H-3Љa,b), 1.14–
1.00 (6 d, partially overlapped, H-6I–VI); 13C NMR (CDCl3):
δ 101.22, 101.09 (5 C, C, 6 CHPh), 100.96, 100.29 (C, 4 C,
C-1II–V), 98.76 (C-1I), 76.56 (6 C, C-2ЈI–VI), 76.02, 75.26, 75.11,
74.77, 74.51, 74.33 (2 C), 73.04, 72.89, 72.81 72.75 (C-2I–V,3I–VI),
71.43,71.25,71.20,70.97,(6Ctotal,CH2Ph),68.58,68.53,67.93,
67.55 (2 C, 2 C, C, C, C-5I–VI), 67.23 (7 C, C-1Љ,4ЈI–VI), 66.82
(H-2VI), 52.08, 51.78, 51.66 (3 C), 51.18 (C-4I–VI), 51.38
(COOCH3), 33.80 (C-5Љ), 28.77 (C-2Љ), 28.51 (C-3ЈI–VI), 25.48
(C-3Љ), 24.47 (C-4Љ), 17.97, 17.96, 17.92, 17.77 (6 C total,
C-6I–VI). FABMS: m/z 2699.38 ([M ϩ 1]ϩ), 2721.73 ([M ϩ Na]ϩ);
HRMS: m/z 2830.1067. C151H176N6O39Cs requires 2830.0760.
Deacetylation of the fully protected pentasaccharide 15 gave
the glycosyl acceptor 16 in virtually theoretical yield as a
white solid, [α]D Ϫ24.8Њ (c 0.5). Definite signals in the 1H NMR
spectrum (CDCl3) were at δ: 6.42, 6.32, 6.27, 6.20, 6.16 (5 d,
partially overlapped, 5 H, J4,NH ∼9.4 Hz, 5 NH), 5.54, 5.52, 5.49
(3 s, 5 H, 5 CHPh), 5.01, 4.93, 4.89 (bs, 2 bd, 4 H, J1,2 ∼1.6 Hz,
H-1II–V), ∼4.61 (bd, partially overlapped, H-1I), 3.81 (bt, 1 H, H-
2I), 3.59 (s, partially overlapped, COOCH3), 3.56 (m, partially
overlapped, H-1Јa), 3.29–3.23 (m, 1 H, H-1Љb), 2.25 (t, J 7.5 Hz,
2 H, H-5Љa,b), 2.05–1.80 (m, 2 H, H-3ЈI–Va,b), 1.64–1.46 (m,
4 H, H-2Љa,b,4Љa,b), 1.32–1.24 (m, 2 H, H-3Љa,b), 1.13–1.01 (5 d,
partially overlapped, H-6I–V); 13C NMR (CDCl3): δ 101.18,
101.21, 101.26 (C, 3 C, C, 5 CHPh), 100.96, 100.30 (C, 3 C,
C-1II–V), 98.77 (C-1I), 76.58, 76.54 (2 C, 3 C, C-2ЈI–V), 75.98,
75.21, 75.15, 74.69, 74.50, 72.91 (2 C), 72.69 (C-2II–IV,3II–IV),
74.26 (C-2I), 71.40, 71.22, 71.06 (C, 3 C, C, 5 CH2Ph), 68.62,
68.50, 67.95, 67.55 (2 C, C, C, C, C-5I–V), 67.33 (C-1Љ), 67.26
(C-4ЈI–V), 66.84 (C-2V), 52.08, 51.81, 51.76, 51.21 (C, C, 2 C, C,
C, C-4I–V), 51.40 (COOCH3), 33.81 (C-5Љ), 28.78 (C-2Љ), 28.54
(C-3ЈI–V), 25.49 (C-3Љ), 24.48 (C-4Љ), 18.02, 17.96, 17.92, 17.79
(2 C, C, C, C, C-6I–V). HRMS: m/z 2404.9253. C127H149CsN5O23
requires 2404.9189.
5-Methoxycarbonylpentyl 4-(3-deoxy-L-glycero-tetronamido)-
4,6-dideoxy-ꢀ-D-mannopyranosyl-(1 2)-bis[4-(3-deoxy-L-
glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl]-(1 2)-
4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-manno-
pyranoside (19)
A mixture of 18 (500 mg) and 5% palladium-on-carbon catalyst
(200 mg) in methanol was stirred under hydrogen overnight,
when TLC (1:2:0.1 CH2Cl2–MeOH–NH4OH) showed that the
reaction was complete. Conventional processing and elution
from a small column of silica gel gave amorphous 19 (230 mg,
5-Methoxycarbonylpentyl 2-O-acetyl-3-O-benzyl-4-(2,4-O-
benzylidene-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranosyl-(1 2)-tetrakis[3-O-benzyl-4-(2,4-O-benzyl-
idene-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-manno-
pyranosyl]-(1 2)-3-O-benzyl-4-(2,4-O-benzylidene-3-deoxy-
L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranoside (17)
1
77%). Definite signals in the H NMR (D2O) spectrum were
at: δ 5.15, 5.11 (2 d, 1 H each, J1,2 1.7 Hz, H-1II,III), 4.99 (d, 1 H,
J1,2 1.9 Hz, H-1IV), 4.82 (d, 1 H, J1,2 1.7 Hz, H-1I), 4.22–4.16
(m, 4 H, H-2ЈI–IV), 3.66 (s, partially overlapped, COOCH3), 2.34
(t, 2 H, J 7.5 Hz, H-5Љa,b), 2.09–1.76 (m, 8 H, H-3Јa,bI–IV),
1.70–1.57 (m, 4 H, H-4Љa,b,2Љa,b), 1.47–1.37 (m, 2 H, H-3Љa,b),
1.18–1.15 (4 d, partially overlapped, H-6I–IV); 13C NMR (D2O):
δ 103.83 (C-1IV), 102.73, 102.45 (C-1II,III), 100.20 (C-1I), 79.90
(C-2I), 79.09, 78.95 (C-2II,III), 70.96 (C-2IV), 70.67 (4 C, C-2ЈI–IV),
70.02, 69.63, 69.46, 69.35, 68.74 (C, C, 3 C, 2 C, C (C-3I–IV,5I–IV),
68.42 (C-1Љ), 59.42 (4 C, C-4Ј), 54.76, 54.58, 54.51, 54.14
(C-4I–IV), 52.10 (COOCH3), 38.24 (4 C, C-3ЈI–IV), 34.63 (C-5Љ),
30.03 (C-2Љ), 26.71 (C-3Љ), 25.60 (C-4Љ), 18.32 (2 C), 18.26, 18.19
(C-6I–IV); FABMS: m/z 1135.6 ([M ϩ 1]ϩ).
Reaction of 4 (1.4 g, 2.64 mmol) and 16 (3.0 g, 1.32 mmol), as
described above for similar reactions, followed by isolation
of the main product by chromatography (5:1 3:1 toluene–
acetone), gave amorphous 17 (2.7 g, 75%), [α]D Ϫ36.4Њ (c 0.66).
1
Definite signals in the H NMR spectrum (CDCl3) were at
δ: 6.42–6.14 (m, 6 H, 6 NH), 5.53, 5.52, 5.51, 5.47 (4 s, partially
overlapped, 6 CHPh), 5.45 (bt, partially overlapped, H-2VI),
5.02, 4.93, 4.88, (3 bs, 4 H, H-1II–V), 4.80 (bs 1 H, H-1VI), ∼4.60
(bd, partially overlapped, H-1I), 3.57 (COOCH3), 3.54 (m, par-
tially overlapped, H-1Љa), 3.32–3.23 (m, 1 H, H-1Љb), 2.24 (t,
2 H, J 7.5 Hz, H-5Љa,b), 2.07 (s, partially overlapped, COCH3),
2.10–1.78 (m, partially overlapped, H-3Јa,bI–VI), 1.63–1.46 (m,
4 H, H-2Љa,b,4Љa,b), 1.33–1.21 (m, 2 H, H-3Љa,b), 1.14–1.01 (6 d,
partially overlapped, H-6I–VI); 13C NMR (CDCl3): δ 101.17,
101.10, 101.06, 101.02, (2 C, 2 C, C, C, 6 CHPh), 100.87,
100.30, 100.07 (C, 2 C, C, C-1II–V), 99.11 (C-1VI), 98.68 (C-1I),
76.51, 76.45 (3 C, 3 C, C-2ЈI–VI), 75.08, 74.91, 74.74, 74.48,
74.28, 73.71, 73.65, 72.89, 72.78, 72.71 (C, C, C, C, 2 C, C, C, C,
C, C, C-2I–V,3I–VI), 71.27, 71.23, 71.20, 70.96, 70.92 (C, C, C, C,
2 C, 6 CH2Ph), 68.57, 68.53, 68.48, 68.37, 67.49 (C, C, C, C, 2
C, C-5I–VI), 67.24 (C-1Љ), 67.17 (7 C, C-2VI,4ЈI–VI), 52.99, 51.93,
51.70, 51.56, 51.37, 51.31 (C, C, C, C, C, 2 C, C-4I–VI,-
COOCH3), 33.73 (C-5Љ), 28.69 (C-2Љ), 28.48 (6 C, C-3ЈI–VI), 25.41
(C-3Љ), 24.40 (C-4Љ), 20.98 (COCH3), 18.79, 17.98, 17.91, 17.86
(6 C, C-6I–VI). FABMS: m/z 2741.09 ([M ϩ 1]ϩ), 2747.3 ([M ϩ
Li]ϩ); HRMS: m/z 2872.1184. C153H178N6O40Cs requires
2872.1133.
5-Methoxycarbonylpentyl 4-(3-deoxy-L-glycero-tetronamido)-
4,6-dideoxy-ꢀ-D-mannopyranosyl-(1 2)-tetrakis[4-(3-deoxy-L-
glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl]-(1 2)-
4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-manno-
pyranoside (23)
This compound was obtained (∼90%) by treatment of 18, as
described for tetrasaccharide 14. The NMR characteristics
agreed with those reported previously for the independently
synthesized substance.11
(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-L-glycero-tetro-
namido)-4,6-dideoxy-ꢀ-D-mannopyranosyl-(1 2)-bis[4-(3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl]-(1 2)-4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranoside (20)
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
782

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In a closed round bottomed flask, a solution of compound 19
(240 mg) in ethylenediamine (3 mL) was heated at 60–70 ЊC
overnight, when TLC (1:2:0.1 CH2Cl2–MeOH–NH4OH)
showed that the reaction was complete. After concentration
(70Њ C/133 Pa), the residue was purified by elution from a
series of Sep-Pak C18 cartridges. The material was applied as
a solution in water, and elution was effected with a gradient of
H2O 50% MeOH. Concentration of the relevant fractions,
filtration and freeze-drying gave amorphous, hygroscopic amine
20 (140 mg, ∼58%). The 1H NMR spectrum of the amine 20 was
very similar to that of the ester 19, and contained 2-proton
triplets at δ 3.48 and 3.06 for H-6Љa,b and H-7Љa,b, respectively.
Similarly, the 13C NMR spectrum contained additional signals
at δ 42.14 and 41.80 for C-6Љ and C-7Љ, respectively. FABMS:
m/z 1163.51 ([M ϩ 1]ϩ), 1185.51 ([M ϩ Na]ϩ).
for 10 min. With continued stirring, a mixture of CH2Cl2–DMF
(v/v, 4:1, 1 L) was added and, after 30 min, Ac2O (78 mL,
8 equiv.) was added portionwise, followed by dry t-butanol
(197.2 mL, 20 equiv.). A solution of the 2,4-O-benzylidene-(S)-
1,2,4-butanetriol13 (27, 20 g, 103 mmol) in CH2Cl2–DMF (v/v,
4:1, 250 mL) was then added dropwise over a period of 1.5 h,
and the mixture was stirred until TLC showed that all starting
material was consumed (∼3 h). One major, fast moving, strongly
UV positive product was formed, as shown by TLC. When a
silica gel plate was sprayed with 5% H2SO4 in EtOH and heated,
the spot corresponding to the product turned first red, the color
faded on continued heating, and the material charred only very
weakly. A number of very minor, more polar by-products were
also formed. After cooling (∼ 10 ЊC), solid NaHCO3 (140 g) was
added, followed by EtOH (110 mL), and the stirring was con-
tinued for 2 h. The mixture was concentrated, and the residue
was co-evaporated with pyridine and toluene to remove DMF.
The residue was suspended in EtOAc and, after filtration, the
solid was washed with EtOAc, and the combined filtrates were
concentrated. The residue was purified by chromatography
(6:1 to 4:1, hexane–EtOAc) to give the desired product 27 as
white solid (22.6 g, 83%), which was identical (1H NMR) to the
previously described13 material. 13C NMR (CDCl3): δ 168.93
(CO), 100.95 (CHPh), 81.66 [C(CH3)3], 75.48 (C-2), 66.66
(C-4), 26.15 (C-3), 27.91 [C(CH3)3].
1-{(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl-(1 2)-bis[4-(3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl]-(1 2)-4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranoside}-2-ethoxycyclobutene-3,4-dione (21)
A solution of amine 20 (60 mg, 0.05 mmol) in buffer pH 7
(7 mL) was treated with squaric acid diethylester (13 µL, 0.08
mmol), and kept at room temperature until TLC (1:3 CH2Cl2–
MeOH) showed complete conversion of the starting material
into a less polar UV positive product. After concentration, the
residue was eluted from a series of Sep-Pak C18 cartridges to
give, after freeze-drying, the squaric acid monoamide 21 (44
mg, 73%). The 1H NMR spectrum of 21 was very similar to that
of the amine 20, and contained multiplets at δ 4.76–4.69
(CH3CH2O) and 1.49–1.43 (CH3CH2O). The 13C NMR spec-
trum was very similar to that of the amine 20. Signals for the
OCH2CH3 (δ 70.85 and 70.74) and OCH2CH3 (16.23 and
16.16) each appeared as doublets, the splitting of these signals
being due to the vinylogous amide group, characteristic of
squaric acid amide esters.19 FABMS: m/z 1287.5 ([M ϩ 1]ϩ),
1309.5 ([M ϩ Na]ϩ).
2,4-O-Benzylidene-3-deoxy-L-glycero-tetronic acid (29)
Pyridinium dichromate (216 g, 4 equiv.) was added at room
temperature to a solution of crystalline13,28 2,4-O-benzylidene-
(S)-1,2,4-butanetriol (27, 30 g, 154.6 mmol) in DMF (400 mL),
and the mixture was stirred overnight, when TLC (10:1
CH2Cl2–EtOAc) showed absence of the starting material. Water
(450 mL) was added, and the mixture was extracted with
EtOAc (5×600 mL). The organic layer was washed with water,
to remove some colored material, and concentrated to remove
EtOAc. DMF was removed by co-evaporation with pyridine or
xylene to give acid 29 (28 g, 85%) as a white solid, which showed
1
the same H and 13C NMR characteristics as the previously
(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-L-glycero-tetro-
namido)-4,6-dideoxy-ꢀ-D-mannopyranosyl-(1 2)-tetrakis[4-(3-
deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-mannopyrano-
syl]-(1 2)-4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-
mannopyranoside (24)
described substance.13
Conjugation of squaric acid monoamides to BSA
A. Preparation of the neoglycoconjugate 26A. Hexasaccharide
monoamide 25 (8 mg, 4.5 µmol) was transferred from its
weighing container, using 200 µL of phosphate buffer pH 9
divided into three portions (100, 50 and 50 µL), into a suspen-
sion of BSA (30 mg, 0.45 µmol) in the same buffer (100 µL),
which had been homogenized with the aid of a vortexer, and the
mixture was stirred at room temperature. SELDI-TOF analysis
after 1, 21, 96 h and 14 d showed that the hapten/BSA ratio was,
0.32, 1.4, 1.5 and 2.8, respectively, which did not increase after 3
more days. The mixture was diluted with phosphate buffer pH 7
(1 mL), and the solution was subjected to ultrafiltration using
the Amicon cell equipped with PM-10 membrane (Millipore
Copporation, Bedford, MA). The retained material was
freeze-dried to give the title conjugate 26A (25 mg, 83%) having
average m/z 71,464, corresponding to 26A/BSA ratio of 2.9,
as shown by SELDI-TOF MS.
B. Preparation of neoglycoconjugates 26B and 26C. Solid
hexasaccharide monoamide 25 (27 mg, 15.2 µmol) was trans-
ferred from its weighing container (using 600 µL of phosphate
buffer pH 9 divided into three 200 µL portions) into a suspen-
sion of BSA (10 mg, 0.15 µmol) in the same buffer (400 µL),
which had been homogenized with the aid of a vortexer. The
mixture was stirred at room temperature and, after 3 h, when
SELDI-TOF MS analysis showed that the 25/BSA ratio in the
conjugate being formed was ∼6 a portion (500 µL) was with-
drawn and processed as described in A. After freeze-drying,
SELDI-TOF MS analysis showed that the molecular mass of
the product 26B (5.5 mg, 94%) was 78,080 Da (25/BSA = 6.6).
Treatment of ester 23 as described for 19 gave the amorphous
amine 24 (80%). The 1H NMR spectrum of 24 was very similar
to that of the ester 23, and contained 2-proton triplets at δ 3.26
and 2.74 for H-6Љa,b and H-7Љa,b, respectively. Similarly, the 13
C
NMR spectrum contained additional signals at δ 42.58 and
41.91 for C-6Љ and C-7Љ, respectively. FABMS: m/z 1657.88
([M ϩ 1]ϩ), 1679.86 ([M ϩ Na]ϩ).
1-{(2-Aminoethylamido)carbonylpentyl 4-(3-deoxy-L-glycero-
tetronamido)-4,6-dideoxy-ꢀ-D-mannopyranosyl-(1 2)-tetrakis-
[4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-ꢀ-D-manno-
pyranosyl]-(1 2)-4-(3-deoxy-L-glycero-tetronamido)-4,6-dide-
oxy-ꢀ-D-mannopyranoside}-2-ethoxycyclobutene-3,4-dione (25)
Treatment of amine 24 with squaric acid diethylester, as
described for the preparation of 21, gave the monoamide 25
(73%). The NMR spectra of the compound were similar to
those of the starting amine 24 and showed signals characteristic
of the presence of the squaric acid O-ethyl ester group, as in the
spectrum of the analogous tetrasaccharide 21. FABMS:
m/z 1781.74 ([M ϩ 1]ϩ), 1803.74 ([M ϩ Na]ϩ).
t-Butyl 2,4-O-benzylidene-3-deoxy-L-glycero-tetronate (28)
CrO3 (41.2 g, 4 equiv.) was added, portionwise with stirring and
exclusion of moisture, to pyridine (66.8 mL, 8 equiv.) contained
in a 3 L round-bottomed flask, and the stirring was continued
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 7 7 5 – 7 8 4
783

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3 A. Chernyak, S. Kondo, T. K. Wade, M. D. Meeks, P. M. Alzari,
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The material remaining in the reaction vessel was allowed to
react for a total of 22 h, when SELDI-TOF MS analysis
showed that the 25/BSA ratio in the conjugate formed was ∼19.
The mixture was processed as described above and, after freeze-
drying, the molecular mass of the product 26C (6.9 mg, 92%)
was 99,740 Da, corresponding to 25/BSA = 19.
C. Preparation of the neoglycoconjugate 22B. BSA (2.5 mg,
∼0.04 µmol) was treated with the tetrasaccharide monoamide
21 (5 mg, ∼4 µmol) as described above (A), and the mixture was
stirred at room temperature until the molecular mass of the
conjugate being formed remained essentially unchanged, as
shown by periodical SELDI-TOF analysis (2 d). The mixture
was processed as described above in A and, after freeze drying,
SELDI-TOF MS analysis showed a peak centered at m/z 80,850
Da corresponding to the 21/BSA ratio in the conjugate 22B
thus obtained (2.5 mg, 82%) of 11.6.
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D. Preparation of neoglycoconjugates 22A and 22C. Solid
tetrasaccharide monoamide 21 (20 mg, 15.5 µmol) and a
solution of BSA (10 mg, 0.15 µmol) were allowed to react in
phosphate buffer pH 9 (1 mL) at room temperature, as
described above in B. After 4.5 h, when SELDI-TOF MS
analysis showed that the 21/BSA ratio in the conjugate being
formed was 4.9, a portion (500 µL) was withdrawn and pro-
cessed as described above. After freeze drying, SELDI-TOF
MS analysis showed that the molecular mass of the product
22A (4.8 mg, 89%) was 72,028 Da, corresponding to 21/BSA
ratio of 4.5. The remaining reaction mixture was worked-up
after 3.5 d, when SELDI-TOF MS analysis showed that
molecular mass of the conjugate being formed was 84,588 Da.
After processing, as described above, repeated SELDI-TOF MS
analysis showed that the 21/BSA ratio in the product 22C (5.8
mg, 90%) was 14.6.
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