Carbohydrates as potentially versatile core subcarriers for multivalent immunogens

Article abstract of DOI:10.1039/c6ra27181b

Synthetic multivalent glycoclusters that carry carbohydrate antigen epitopes have been recognized as promising candidates for the development of carbohydrate based vaccines. Here we describe a convergent strategy for the synthesis of conjugation-ready multivalent glycoclusters using sugars as versatile core subcarriers. d-Glucose and gentiobiose were converted into poly-alkyne functionalized cores which were then decorated with an azide bearing model ligand d-glucose using click chemistry, to form structurally well-defined tetra- and heptavalent glycoclusters. Each cluster was conjugated to a model protein bovine serum albumin (BSA) by squaric acid chemistry. Carbohydrate clusters can be prepared in a variety of sizes and spatial arrangements by altering the structure and configuration of the core, depending on the mono-, or oligosaccharides used for their assembly. It is suggested that the use of carbohydrate as core subcarriers provides an opportunity to tailor the size and topology of antigens and modify multivalent presentation of immunogens in a way to optimize cluster effect for stronger immunoreactivity.

Full text of DOI:10.1039/c6ra27181b

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Carbohydrates as potentially versatile core
subcarriers for multivalent immunogens†
Cite this: RSC Adv., 2017, 7, 7591
Divya Kushwaha, Peng Xu and Pavol Kovac*
´ˇ
Synthetic multivalent glycoclusters that carry carbohydrate antigen epitopes have been recognized as
promising candidates for the development of carbohydrate based vaccines. Here we describe
a convergent strategy for the synthesis of conjugation-ready multivalent glycoclusters using sugars as
versatile core subcarriers. D-Glucose and gentiobiose were converted into poly-alkyne functionalized
cores which were then decorated with an azide bearing model ligand D-glucose using click chemistry, to
form structurally well-defined tetra- and heptavalent glycoclusters. Each cluster was conjugated to
a model protein bovine serum albumin (BSA) by squaric acid chemistry. Carbohydrate clusters can be
prepared in a variety of sizes and spatial arrangements by altering the structure and configuration of the
core, depending on the mono-, or oligosaccharides used for their assembly. It is suggested that the use
of carbohydrate as core subcarriers provides an opportunity to tailor the size and topology of antigens
and modify multivalent presentation of immunogens in a way to optimize cluster effect for stronger
immunoreactivity.
Received 22nd November 2016
Accepted 6th January 2017
DOI: 10.1039/c6ra27181b
www.rsc.org/advances
synthetic oligosaccharide antigens and recombinant proteins.
Thus, neoglycoconjugate vaccines, i.e. a vaccine from the fully
Introduction
Binding of carbohydrates to host proteins is a frequent event in synthetic carbohydrate antigen, would be well dened, easy-to-
biology. Carbohydrate–protein interactions control diverse characterize and reproduce molecules with long shelf life. The
1
physiologically important biological processes. To these carbohydrate and the recombinant protein elds have pro-
belongs also the recognition of O-specic polysaccharides (O- gressed to the stage that we can make virtually any structure
SP), which are the protective antigens of Gram-negative bacte- that would be needed. Thus, making such molecules is not the
rial pathogens, by the elements of the immune system of bottleneck responsible for the lack of progress in the synthetic
vertebrates. The magnitude of the immune response and, in vaccine development and for the fact that to date only one
2
turn, the spectrum and level of antibodies produced upon neoglycoconjugate vaccine is known to be clinically useful.
infection depend on the strength of these interactions. Nature
Making vaccines from synthetic oligosaccharides is a very
and evolution prepared us to respond to many infections in young eld and structural/architectural requirements for
close to perfect way. Thus, were it not for some, oen tolerable a potent vaccine are not known. Optimizing such features is
level of toxicity and pyrogenicity, vaccines based on killed or difficult because the formation of a glycoconjugate offers
attenuated pathogens would be the best and cheapest means of virtually an innite number of congurational choices, as very
3
prevention of infectious diseases. Glycoconjugate vaccines, the ably demonstrated by Stein.
man-made constructs from detoxied lipopolysaccharides (LPS)
When monovalent carbohydrate ligands are involved in inter-
and carrier proteins, are oen, although still not entirely, free actions with receptors, the binding is oen weak. To magnify the
from the undesirable effects bacterial vaccines oen have. The recognition, biological systems have circumvented this binding
ideal vaccine would contain the pure protective antigen as the limitation through multivalency. The phenomenon of multi-
sole active antigenic ingredient. Lack of a reliable method for valency (cluster effect) was rst described in their seminal work by
4
preparation of pure O-SP from LPS led to the idea of using Lee and coworkers. They observed that not only the number
synthetic oligosaccharides that mimic structures of O-SPs as of ligands in the cluster but also the ligand's spatial arrange-
antigenic components of conjugate vaccines. For the clinical ment affected the ligand's binding affinity to receptors. These
use, such constructs would be made from pure components, i.e. phenomena have not been systematically studied in connection
with vaccine development from synthetic oligosaccharides.
We have been interested in developing a conjugate vaccine
NIDDK, LBC, National Institutes of Health, Bethesda, MD 20892-0815, USA. E-mail:
kpn@helix.nih.gov
Electronic supplementary information (ESI) available: Copies of H and
spectra of all new compounds. SELDI-TOF MS of the BSA conjugates. See DOI:
0.1039/c6ra27181b
for cholera from synthetic fragments of O-specic poly-
saccharides (O-SP) of Vibrio cholerae O1 for a number of years.
When looking at how some variables affect the immunogenicity
1
13
5
†
C
1
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of glycoconjugates, we have examined the size of the oligosac-
Using carbohydrates as core molecules for creating clusters
6
7
charide, the length of the spacer (linker) and the carbohy- as probes for many purposes in the life sciences, also as chiral
8
15
16–32
drate–protein ratio. The conjugates used in those works were scaffolds, is quite common
but to our knowledge making
9
made according to the single-ended conjugation model, which carbohydrate clusters by attaching fragments of O-SPs to cores
ensured that the vaccines were well dened, non cross-linked by click chemistry and attaching the resulting cluster of anti-
molecules. For making conjugates containing hapten in the gens to carrier proteins by squaric acid chemistry to make
10
11
form of clusters we designed our own core subcarrier. Its neoglycoconjugate vaccines for bacterial diseases has not been
attractive feature was that it allowed to vary the number of explored. Here, we propose carbohydrates as convenient start-
ligands in the cluster but had the drawback that its use gave rise ing materials to form tailor-made core subcarriers for making
to cross-linked, high molecular mass constructs which were clustered immunogens/vaccines. The synthetic strategy and the
difficult to reproduce and characterize. Generally, the cross- methods for characterization of the constructs obtained are
linking can be avoided only when the fully assembled clusters generally applicable to making clusters from virtually any
are conjugated to the carriers according to the single-ended appropriately functionalized ligand. Clusters can be obtained in
9
model. Thus, the synthetic scheme towards a vaccine where many sizes and shapes when the core results from a rational
the antigen is present in a form of clusters must include a core choice of carbohydrate(s). The newly formed constructs can be
subcarrier that allows installation of a reasonable and dened used as vaccines when the ligand is a mimic of a bacterial
number of antigens and be equipped with the spacer bearing carbohydrate antigen, or tailor-made probes in many branches
only a single reactive group compatible with the intended of the life sciences.
chemistry of conjugation to carriers. Towards this goal, here we
We herein report a straightforward and efficient strategy for
have devised a generally applicable route for the preparation of the synthesis of two carbohydrate-centered glycoclusters and
conjugation-ready multivalent glycoclusters utilizing sugars as their conjugation to a model carrier protein BSA by squaric acid
versatile core subcarriers. D-Glucose and gentiobiose have been chemistry. The latter has become our method of choice for
chosen as model sugars for making the poly-alkyne function- conjugation because of its unsurpassed efficiency and experi-
33
alized cores and 6-azidohexyl 2,3,4,6-tetra-O-acetyl-b-D-gluco- mental simplicity. For the preparation of poly-alkyne func-
pyranoside was used as model hapten to be attached to the tionalized carbohydrate cores 19 and 22, we chose simple
3
4
cores. Cu(I)-catalyzed azide–alkyne “click” cycloaddition monosaccharide per-O-acetylated b-D-glucose
1
and the
12
(
CuAAC) was employed for their coupling to form structurally disaccharide per-O-acetylated b-gentiobiose 10 as starting
35
well dened tetra- and heptavalent glycoclusters. Furthermore, materials. The synthesis began with the introduction of spacer
we show how the cluster thus formed can be conjugated to the arm to per-O-acetates 1 and 10 by BF $Et O catalyzed glyco-
model protein bovine serum albumin (BSA) using squaric sidation with 6-chlorohexanol, to obtain 2 and 11 in 36% and
3
2
13,14
chemistry.
When a bacterial antigen or a synthetic fragment 64% yield respectively (Scheme 1 and 2). The bromo analog of 2
thereof would be used as hapten in such conjugation, an was previously obtained in the same yield in a similar way by
24
experimental immunogen (vaccine) would be formed.
Dubber et al. However, the chloro derivatives are preferred
intermediates when conversion to the corresponding azides is
anticipated because 6-chlorohexanol is
commodity. The azidation of 2 and 11 was brought about in the
When searching for the class of substance that was to become subsequent step, by their treatment with NaN
a less expensive
Results and discussion
ꢀ
3
in DMF at 75 C
the core to carry our antigen in the future cluster-type immu- to afford corresponding azides 3 and 12 in almost theoretical
nogen (vaccine), we took several factors into consideration. We yields. The starting materials and the products of the afore-
wanted the core not only to carry a dened number of haptens mentioned conversions showed very similar chromatographic
but to also allow us to vary the size of the cluster and ne-tune mobility in each case. However, the formation of 3 and 12 could
the spatial arrangements of the haptens around it. Carbohy- be monitored by NMR spectroscopy. It showed condently that
drates are among few classes of compounds that can full these the conversion of chlorides was complete and that these were
1
requirements. They are inexpensive commodities, many occur one-product reactions. In both cases, compared to the
H
0
naturally, and methods for orthogonal functionalization of this spectra of 2 and 11, the 2-proton triplet for H-6 of 3 and 12 was
0
13
class of compounds are readily available. The rich structural shied upeld by ꢁ0.26 ppm, and the C-6 signal in C NMR
and congurational diversity of carbohydrates allows them to spectra was shied downeld by ꢁ6.4 ppm.
adopt many forms (different ring size and anomeric varieties) or
With the acetylated azide armed glycosides 3 and 12 at hand,
unusual conformations (into which sugars can be forced by we intended to convert them to the corresponding poly-
deoxygenation at various positions in the pyranose or furanose hydroxylated glycosides 7 and 16 carrying a protected amine-
ring form). When such structures are utilized as cores, a large functionalized linker. The route employed was de-O-acetyla-
number of multivalent glycomimetics with diverse spatial tion (Zempl ´e n) of azide 3 to obtain 4, followed by the reduction
2
arrangements, number of haptens and degree of freedoms can of 4 under catalytic hydrogenation conditions (H , 5% Pd/C,
be constructed. Therefore, depending on the nature of the experimental, preparation of 7, A). Careful analysis of the
receptor, the use of carbohydrates as core allows optimization reaction mixture of hydrogenation of 4 by TLC revealed the
of the glycoside-cluster effect by providing the opportunity to presence of two poorly resolved major products in ꢁ1 : 1 ratio. A
tailor the valency and topology of the clusters formed.
challenging chromatographic separation on a small scale led to
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Scheme 1 Synthesis of the intermediate 7.
products amenable to structural analysis (NMR, MS). The
Following the optimized reaction conditions for the prepa-
compounds isolated were identied as the expected amine 5 ration of 7, the synthesis of its disaccharide analogue 16 was
and a by-product. Structure of the by-product 6 was determined attempted from azide 12 (Scheme 2). De-O-acetylation of 12
39
on the basis of the spectral data (NMR, MS) and the literature (Zempl ´e n) gave azide 13 in virtually theoretical yield, but
36–38
precedence.
The same holds for deduction of structures of hydrogenation of the latter under the conditions applied to 4
compounds 9, 15, and 18 described below. Because the was unsuccessful: more than 95% (TLC) of 13 remained
proportion of side product 6 was unacceptably high, more unchanged aer several hours. The effort to reduce azide 13
favourable conditions for azide reduction were sought. under high pressure (200 psi) also failed. The reduction of 13
Compound 5 was formed in a signicantly improved amount (5/ could be completed when carried out in 5% AcOH in MeOH as
6
, ꢁ10 : 1) when the reduction of 4 was performed in presence solvent. Sadly, though, the desired amine 14 and the side
of NH OH in MeOH (experimental, preparation of 7, B). On product 15 (deduced from MS and analogy to formation of 6)
4
preparative scale, the difficult separation of 5 and 6 was omitted were formed in ꢁ1 : 1 ratio (TLC). Because the treatment of
and the crude mixture was directly submitted for NH-Boc such mixture with Boc anhydride furnished 16 in only 38% yield
protection with di-tert-butyl dicarbonate in MeOH. In this way (in 3 steps from 12), and neither the yield of 7 was considered
the key intermediate NH-Boc protected amine 7 was isolated in quite satisfactory, an alternative, more efficient synthetic route
67% yield (from 3), aer chromatography.
for 7 and 16 was sought.
Scheme 2 Synthesis of the key intermediate 16.
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1
3
We have previously described a convenient conversion of with integral values of four and seven protons respectively. In
C
a sugar azide to the corresponding N-acetamido derivative by NMR spectra, the signals for two acetylenic carbons appeared at
conducting the catalytic hydrogenation (H , Pd/C) in presence d ꢁ80.0 ppm (C^C–H) and ꢁ74.0 ppm (C^C–H). The molecular
2
40
+
of Ac O using MeOH as solvent. Because in the present situ- ion peak [M + Na] at m/z 554.2731 for 19 and at 830.3727 for 22
2
ation neither the starting material nor the conversion products conrmed the complete alkylation in each case.
contained hydroxyl groups, the use of MeOH as solvent became
To demonstrate the feasibility of our synthetic strategy in
unimportant. Accordingly, hydrogenation (5% Pd/C) of azide 3 glycocluster formation, azidosugar 3 was chosen as click syn-
in presence of di-tert-butyl dicarbonate using DCM as solvent thon to decorate the carbohydrate-centered building blocks 19
41–44
effected the reduction to amine and simultaneous NH-Boc and 22 (Scheme 3). A Cu(I) catalyzed click reaction
was
protection in one pot (experimental, preparation of 7, C), to conducted between 19 (1.0 equiv.) and 3 (5.0 equiv.) in presence
give a mixture of desired amine 8 and side product 9 (Scheme 1). of CuI and diisopropylethylamine (DIPEA), which afforded 20
A simple chromatographic separation afforded amine 8 (76%), (77%). The synthesis of 23 (71%) was achieved in a similar
which was de-O-acetylated under Zempl ´e n transesterication manner through click coupling of 22 (1.0 equiv.) and 3 (8.0
1
conditions, to afford the key intermediate 7 in virtually theo- equiv.). The H NMR spectra of 20 and 23 showed presence of
retical yield. Synthesis of disaccharide 16 was achieved from 12 four (d 8.16, 7.94, 7.68 ppm in 1 : 2 : 1 ratio) and seven newly
(75%, overall in two steps) in a similar way (Scheme 2).
formed triazolyl proton singlets (d 8.15, 8.04, 7.94, 7.89,
To proceed towards the title cores, glycosides 7 and 16 were 7.77 ppm in 2 : 1 : 2 : 1 : 1 ratio), respectively, and the signals
ꢀ
treated with propargyl bromide and KOH at 0 C / rt, to obtain for the alkyne ^C–H protons were absent. This, along with
the corresponding alkyne functionalized, clickable intermediates the MS data, conrmed the successful multiple click reactions
1
3
1
9 and 22 in 75% and 58% yield, respectively. The structure in both cases. The C NMR spectra of 20 and 23 revealed
conrmation for 19 and 22 was provided by spectral analysis the presence of characteristic carbon signals at d ꢁ145–144
1
(
NMR, HRMS). In the H NMR spectrum of each of 19 and 22, the and d ꢁ124–123 ppm, respectively, for C-4 and C-5 of the
characteristic ^C–H protons appeared as multiplet at d 2.5 ppm 1,4-disubstituted 1,2,3-trizole ring. The complete structural
Scheme 3 Synthesis of tetra- and heptavalent glycoclusters 21 and 24 via click chemistry.
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Scheme 4 Conjugation of the glycoclusters 21 and 24 to BSA by squaric acid chemistry.
assignment of 20 and 23 was achieved with the aid of 2D NMR
experiments ( H COSY, TOCSY and H– C HSQC).
Each of the monomethyl ester 25 and 27 was next treated
with BSA in 0.5 M borate buffer at pH 9 for 2 days, according to
1
1
13
45
The fully protected clusters 20 and 23 were then treated with the protocol developed in our lab. The conjugations were
TFA in DCM to remove the Boc protecting group, and subse- carried out at a hapten/BSA ratio 6 : 1 and at a hapten
quent de-O-acetylation (Zempl ´e n) yielded the conjugation-ready concentration ꢁ4.0 mM. Aer freeze drying, the SELDI MS
clusters 21 and 24, respectively, in good yields aer purication analysis of the puried conjugates showed average molecular
by reverse-phase chromatography. The structures of 21 and 24 mass 73 700 Da for the conjugate 26 (molar ratio 25 : BSA
were conrmed by NMR (1D and 2D) and HRMS spectral data. ꢁ4.1 : 1) and 77 600 Da for the conjugate 28 (molar ratio
Conjugation of the synthetic glycoclusters to BSA carrier 27 : BSA ꢁ3.7 : 1).
13,14,45,46
protein was executed by squaric acid chemistry.
Thus,
clusters 21 and 24 were treated with 3,4-dimethoxy-3-
cyclobutene-1,2-dione (dimethyl squarate) at pH 7 (phosphate Conclusions
buffer) to give the squaric acid monoester derivatives 25 and 27,
respectively, which were readily puried by chromatography
We propose that carbohydrates can serve as versatile sub-
carriers for clusters of a variety of ligands whose valences and
spatial arrangement can be predetermined by judicious choice
of natural or chemically modied carbohydrates. Following that
rational, two structurally well dened tetra- and heptavalent
glycoclusters bearing amino-functionalized linker were synthe-
sized by the Cu(I)-catalyzed click coupling of polyalkyne-
functionalized D-glucose and gentiobiose cores and azide-
equipped ligand. Squaric acid chemistry was successfully
applied for conjugation of the obtained amino group-
containing, synthetic, multivalent glycoclusters to the carrier
4
6
(
Scheme 4). The formation of 25 and 27 was established by
1
HRMS and NMR spectroscopy. In the H NMR spectra of each of
5 and 27, the –OCH protons attached to the squaric acid
moiety resonated at d 4.36 ppm and their C NMR spectra
2
3
1
3
1
3
showed –OCH carbon as two signals at d ꢁ61 ppm. The
C
3
spectra of monoesters 25 and 27 exhibited typical splitting of
some signals due to the double bond nature of the vinylogous
amide group, which is characteristic of squaric acid amide
14
esters.
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protein BSA. The synthetic materials, many of which were ob-
6-Chlorohexyl 2,3,4,6-tetra-O-acetyl b-D-glucopyranoside (2).
tained in the analytically pure state, were characterized by To a solution of 1,2,3,4,6-penta-O-acetyl-b-D-glucopyranose
physical constants (mp, [a] ) and spectral data (NMR, HRMS). (8.0 g, 20.5 mmol) and 6-chlorohexanol (3.0 mL, 22.5 mmol)
D
3 2
in anhydrous DCM (200 mL), BF Et O (7.6 mL, 61.5 mmol) was
added drop wise and the mixture was stirred at room temper-
ature for 5 h. The mixture was diluted with DCM, neutralized
Experimental section
General methods
with aqueous NaHCO , and washed with brine (3 ꢃ 200 mL).
3
Melting points were determined with a Koer hot stage. Optical The organic layer was concentrated and the residue was crys-
rotations were measured at ambient temperature for solutions tallized from EtOH. The crystals were washed with cold EtOH
in CHCl
3
and MeOH with a Perkin–Elmer automatic polarim- (twice) and with hexane to afford 2 (3.44 g, 36%). Recrystallized
ꢀ
eter, Model 341. HPLC grade solvents were used, and reactions 2 (EtOH) showed mp 98–99 C, R 0.5 (2 : 1 hexane–acetone),
f
2
3
1
requiring anhydrous conditions were carried out under and [a] ꢄ17.8 (c 0.7, CHCl ). H NMR (CDCl , 600 MHz): d 5.20
D
3
3
nitrogen or argon. Reactions were monitored by thin-layer (t, 1H, J 9.5 Hz, H-3), 5.09 (t, 1H, J 9.7 Hz, H-4), 4.98 (dd, 1H, J1
,2
chromatography (TLC) on silica gel 60 glass slides. Spots were 8.0 Hz, J2,3 9.6 Hz, H-2), 4.49 (d, 1H, H-1), 4.26 (dd, 1H, J5,6a
visualized by charring with H SO in EtOH (5% v/v). Column 4.8 Hz, J6a,6b 12.3 Hz, H-6 ), 4.14 (dd, 1H, J
2.5 Hz, H-6 ), 3.87
2
4
a
5,6b
b
0
chromatography was performed with Biotage Isolera chro- (dt, 1H, J 6.3, 9.6 Hz, H-1 ), 3.69 (ddd, 1H, J 10.0 Hz, H-5), 3.53
a
5,4
0
0
matograph equipped with a Varian Evaporative Light-Scattering (t, 2H, J 6.7 Hz, H-6 ), 3.48 (dt, 1H, J 6.6 Hz, H-1 ), 2.09, 2.04,
Detector. Nuclear magnetic resonance (NMR) spectra were 2.03, 2.00 (4 s, 12H, 4 COCH ), 1.76 (m, 2H, H-5 ), 1.59 (m, 2H,
measured at 600 MHz for H, and 150 MHz for C for solutions H-2 a,b), 1.44 (m, 2H, H-4 a,b), 1.36 (m, 2H, H-3 a,b) ppm;
in CDCl , MeOD and D O with Bruker Avance spectrometer. NMR (CDCl , 150 MHz): d 170.67, 170.30, 169.34, 169.26 (4
Chemical shis were reported relative to TMS ( H: d 0.00 ppm), COCH ), 100.80 (C-1), 72.85 (C-3), 71.76 (C-5), 71.34 (C-2), 69.89
a,b
b
0
3
a,b
0
1
13
0
0
13
C
3
2
3
1
3
1
3
1
13
0
0
0
CHCl ( C: d 77.00 ppm), MeOH ( H: d 3.31 ppm; C: d 49.00 (C-1 ), 68.47 (C-4), 61.98 (C-6), 44.92 (C-6 ), 32.45 (C-5 ), 29.21 (C-
ppm) or H O (d 4.79 ppm). Assignments of NMR signals were 2 ), 26.48 (C-4 ), 25.11 (C-3 ), 20.72, 20.64, 20.60, 20.58 (4
3
0
0
0
2
aided by 1D and 2D experiments (APT, COSY, TOCSY, HSQC, COCH ) ppm. HRMS (ESI): m/z calcd for C H NO Cl [M +
3
20 35
10
+
HMQC, and HMBC). When reporting assignment of NMR NH ] : 484.1949; found: 484.1953; anal. calcd for C H ClO :
4
20 31
10
signals, nuclei associated with the subcarrier spacer (linker) are C, 51.45; H, 6.69; found: C, 51.35; H, 6.62.
denoted with a prime and those associated with the ligand
6-Azidohexyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (3). A
spacer are denoted with a double prime. Sugar residues in mixture of acetate 2 (4.66 g, 10.0 mmol) and NaN (2.6 g, 40.0
3
ꢀ
oligosaccharides are serially numbered, beginning with the one mmol) in DMF (15 mL) was stirred at 75 C. For monitoring the
1
bearing the aglycone, and are identied by a Roman numeral progress of reaction by H NMR, the solvent was removed from
superscript in listings of signal assignments. Liquid a few drops of the reaction mixture with strong stream of air, the
Chromatography-Electron Spray-Ionization Mass Spectrometry residue was triturated with CDCl , and the extract was ltered
3
(ESI-MS) was performed with a Hewlett–Packard 1100 MSD directly into the NMR tube. When the spectrum showed
spectrometer. Conjugation of carbohydrates was monitored by complete disappearance of the starting material (ꢁ4 h), the
the BioRad Protein Chip SELDI system using NP-20 chip arrays. mixture was concentrated and the residue was partitioned
3
,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) was between CHCl and brine. Aer concentration of the organic
3
used as matrix. Attempts have been made to obtain correct phase, pure azide 3 was obtained by crystallization of the
combustion analysis data for all new compounds. However, residue from EtOH as described for 2. Two more crops were
some compounds tenaciously retained traces of solvents, obtained in the same way from the mother liquor (total yield,
despite exhaustive drying, and analytical gures for carbon 4.35 g, 92%). The recrystallized (EtOH) azide 3 showed mp 79–
ꢀ
23
could not be obtained within ꢂ0.3%. The palladium-on- 80 C, R 0.5 (2 : 1 hexane–acetone) and [a] ꢄ18.9 (c 0.7,
f
D
1
charcoal catalyst used was purchased from Engelhard Indus- CHCl ). H NMR (CDCl , 600 MHz): d 5.20 (t, 1H, J 9.6 Hz, H-3),
3
3
tries (Escat 103, Lot# FC96303). BSA was purchased from Sigma 5.09 (t, 1H, J 9.8 Hz, H-4), 4.98 (dd, J1,2 8.0 Hz, J2,3 9.6 Hz, H-2),
Chemical Company: A. Cat. Number A-0281; B. Fraction V, 4.49 (d, 1H, H-1), 4.26 (dd, 1H, J5,6a 4.8 Hz, J6a,6b 12.3 Hz, H-6 ),
a
0
0
Sigma Cat. no. A-4503, from which fatty acids were removed by 4.14 (dd, 1H, J5,6b 2.5 Hz, H-6 ), 3.87 (dt, 1H, J 6.3, 9.6 Hz, H-1 ),
b
a
47
charcoal treatment. Buffers used were as follows; A, BuffAR pH 3.69 (ddd, 1H, J5,4 10.0 Hz, H-5), 3.48 (dt, 1H, J 6.7 Hz, H-1 ),
b
0
7
.0 Reference solution (Macron, Cat. no. 0031-04), concentrated 3.26 (t, 2H, J 7.0, H-6 ), 2.09, 2.04, 2.03, 2.01 (4 s, 12H, 4
to 1/10 volume; B, 0.5 M borate buffer pH 9, made in house (1 L) COCH ), 1.59 (m, overlapped, 4H, H-2 a,b, 5 a,b), 1.37 (m, over-
from boric acid (30.9 g), KCl (26.1 g), and KOH (8.42 g), and nal lapped, 4H, H-3 a,b, 4 a,b) ppm; C NMR (CDCl , 150 MHz):
a,b
0
0
3
0
0
13
3
adjustment to pH 9.0 by addition of solid KOH. During squaric d 170.68, 170.31, 169.40, 169.26 (4 COCH ), 100.81 (C-1), 72.85
3
0
acid monoester formation reaction, pH of the reaction mixture (C-3), 71.77 (C-5), 71.35 (C-2), 69.89 (C-1 ), 68.48 (C-4), 61.99 (C-
0
0
0
0
0
was monitored intermittently with a Corning, Model 440 pH 6), 51.33 (C-6 ), 29.24, 28.75, 26.38, 25.40 (C-2 , C-3 , C-4 , C-5 ),
meter equipped with model MI-410 microelectrode (Micro- 20.73, 20.64, 20.61, 20.59 (4 COCH ) ppm. HRMS (ESI): m/z
3
+
electrodes, Inc. Bedford, NH, USA) and when necessary, pH was calcd for C H N O [M + NH ] : 491.2353; found: 491.2351;
2
0
35
4
10
4
maintained at 7 by addition of buffer salts, obtained by freeze- anal. calcd for C H N O : C, 50.74; H, 6.60; N, 8.87; found: C,
2
0
31 3 10
drying Buffer A. Solutions in organic solvents were dried with 50.63; H, 6.53; N, 8.85.
ꢀ
anhydrous Na
2
SO
4
, and concentrated at <40 C/2 kPa.
7596 | RSC Adv., 2017, 7, 7591–7603
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6
-N-(tert-Butyloxycarbonyl)hexyl b-D-glucopyranoside (7) of 5% Pd/C (60 mg) at room temperature overnight. Aer
A. To a solution of acetate 3 (500 mg, 1.05 mmol) in MeOH (6 conventional processing, the crude mixture of 5 and 6 (281 mg)
mL), a solution of NaOMe in MeOH was added until strong obtained was dissolved in MeOH (5 mL) and treated overnight
alkalinity and the mixture was kept at room temperature until with di-tert-butyl dicarbonate (330 mg, 1.50 mmol). Aer
the reaction was complete (ꢁ6 h). The mixture was diluted with concentration, the residue was chromatographed (8 : 1 CHCl
3
–
+
MeOH, neutralized with Amberlite IR 120H , ltered and MeOH) to yield 7 as white foam (310 mg, 67%, in three steps
2
3
concentrated. Chromatography (4 : 3 : 0.2 DCM–acetone– from 3); R
f
0.5 (6 : 1 CHCl
3
–MeOH); [a]
D
ꢄ30.7 (c 0.8, CHCl
3
).
1
MeOH) afforded 6-azidohexyl b-D-glucopyranoside 4 (310 mg,
6%) as colorless syrup. R
H NMR (MeOD, 600 MHz): d 4.24 (d, 1H, J1,2 7.8 Hz, H-1), 3.90
0
9
f
0.25 (4 : 3 : 0.2 DCM–acetone– (dt, 1H, J 6.8, 9.5 Hz, H-1
a
), 3.86 (dd, 1H, J5,6a 2.1 Hz, J6a,6b
), 3.54 (dt, 1H, H-
), 3.34 (t, 1H, J 8.8 Hz, H-3), 3.28 (t, overlapped, 1H, H-4), 3.25
), 3.55 (dt, 1H, (m, overlapped, 1H, H-5), 3.16 (dd, 1H, J2,3 9.0 Hz, H-2), 3.02 (t,
1
MeOH). H NMR (MeOD, 600 MHz): d 4.24 (d, 1H, J1,2 7.9 Hz, H- 11.9 Hz, H-6
), 3.66 (dd, 1H, J5,6b 5.4 Hz, H-6
b
a
0
0
b
1
), 3.91 (dt, 1H, J 6.7, 9.6 Hz, H-1
6a,6b 12.0 Hz, H-6 ), 3.66 (dd, 1H, J5,6b 5.3 Hz, H-6
), 3.34 (t, 1H, J 8.8 Hz, H-3), 3.29–3.24 (m, over- 2H, J 7.0 Hz, H-6 a,b), 1.63 (m, 2H, H-2 a,b), 1.49–1.38 (m, over-
a
), 3.86 (dd, 1H, J5,6a 2.2 Hz,
1
J
a
b
0
0
0
J 6.7 Hz, H-1
b
0
0
0
lapped, 4H, H-6 , H-4, H-5), 3.17 (dd, 1H, J 9.0 Hz, H-2), 1.62 lapped, H-5 a,b, H-3 a,b), 1.43 (s, overlapped, NHCOOC(CH
m, overlapped, 4H, H-2 a,b, 5 a,b), 1.42 (m, overlapped, 4H, H- 1.34 (m, 2H, H-4 a,b) ppm; C NMR (MeOD, 150 MHz): d 158.57
3
)
3
),
a,b
2,3
0
0
0
13
(
0
0
13
3
7
6
3
a,b, 4 a,b) ppm; C NMR (MeOD, 150 MHz): d 104.37 (C-1), (NHCOOC(CH
3
)
3
), 104.37 (C-1), 79.78 (NHCOOC(CH
3
)
3
),
0
0
8.14 (C-3), 77.92 (C-5), 75.14 (C-2), 71.69 (C-4), 70.67 (C-1 ), 78.14 (C-3), 77.92 (C-5), 75.13 (C-2), 71.68 (C-4), 70.75 (C-1 ),
0
0
0
0
0
0
2.79 (C-6), 52.40 (C-6 ), 30.63 (C-2 ), 29.86 (C-5 ), 27.62, 26.64 (C- 62.78 (C-6), 41.28 (C-6 ), 30.90 (C-5 ), 30.69 (C-2 ), 28.80
0
0
0 0
) ), 27.65 (C-4 ), 26.76 (C-3 ) ppm. HRMS (ESI): m/
3 3
, C-4 ) ppm. HRMS (ESI): m/z calcd for C12
Na] : 328.1485; found: 328.1479.
H
23
N
3
O
6
Na [M + (NHCOOC(CH
z calcd for C17
+
+
H
33NO
Na [M + Na] : 402.2104; found: 402.2111;
: C, 53.81; H, 8.77; N, 3.69; found: C,
8
A stirred solution of azido sugar 4 (310 mg, 1.01 mmol) in anal. calcd for C17
MeOH (ꢁ5 mL) was treated with hydrogen in presence of 5% Pd/ 53.73; H, 8.85; N, 3.65.
C (60 mg) at room temperature overnight. TLC (1 : 2 : 0.06
C. A suspension of 5% Pd/C (50 mg) in DCM (2 mL) was
DCM–MeOH–25% NH OH) showed that two major products added to the solution of azide 3 (250 mg, 0.53 mmol) and di-tert-
H33NO
8
4
were formed in an approximate ratio of 1 : 1 (R
overlapping). Aer ltration through Celite pad, the ltrate was mixture was stirred at room temperature under H
f
ꢁ0.1, partially butyl dicarbonate (230 mg, 1.05 mmol) in DCM (4 mL) and the
atmosphere
2
concentrated. For identication, a small amount of the mixture for 5 h, when TLC showed complete consumption of starting
was chromatographed to obtain the major products, which were material. Aer conventional processing, as above, and concen-
sufficiently pure for spectral identication. Eluted rst was tration, the residue was chromatographed (4 : 1 hexane–
1
material identied as 5 (white solid), H NMR (MeOD, 600 acetone) to give 6-N-(tert-butyloxycarbonyl)hexyl 2,3,4,6-tetra-O-
MHz): d 4.24 (d, 1H, J 7.8 Hz, H-1), 3.91 (dt, 1H, J 6.7, 9.6 Hz, acetyl-b-D-glucopyranoside 8 (218 mg, 76%), R
f
0.4 (2 : 1 hexane–
, 600 MHz): d 5.20 (t, 1H, J 9.5 Hz, H-3),
), 3.34 (m, 1H, H-3), 3.29– 5.07 (t, 1H, J 9.7 Hz, H-4), 4.97 (dd, 1H, J1,2 8.0 Hz, J2,3 9.6 Hz, H-
1
,2
0
1
H-1 ), 3.86 (m, 1H, 12.0 Hz, H-6 ), 3.66 (dd, 1H, J
12.0 Hz, acetone). H NMR (CDCl
3
a
a
6a,6b
0
J
5,6b 5.3 Hz, H-6
b
), 3.55 (dt, 1H, H-1
b
3
.24 (m, overlapped, 2H, H-4, H-5), 3.16 (m, 1H, H-2), 2.67 (t, 2H, 2), 4.57–4.48 (bs, NH), 4.49 (d, 1H, H-1), 4.26 (dd, 1H, J5,6a
0
J 7.2 Hz, H-6 a,b), 1.64 (m, 2H, CH
linker), 1.39 (m, overlapped, 4H, 2 ꢃ CH
2
linker), 1.50 (m, 2H, CH
2
4.8 Hz, J6a,6b 12.3 Hz, H-6 ), 4.14 (dd, 1H, J5,6b 2.5 Hz, H-6 ), 3.85
a b
1
3
0
linker) ppm; C NMR (dt, 1H, J 6.3, 9.6 Hz, H-1
), 3.69 (ddd, 1H, J5,4 9.9 Hz, H-5), 3.47
2
a
0
0
(
(
3
MeOD, 150 MHz): d 104.37 (C-1), 78.15 (C-3), 77.93 (C-5), 75.13 (dt, 1H, J 6.7 Hz, H-1
b
), 3.09 (t, 2H, J 6.9 Hz, H-6 a,b), 2.08, 2.03,
0
0
0
C-2), 71.68 (C-4), 70.73 (C-1 ), 62.77 (C-6), 42.19 (C-6 ), 32.92, 2.02, 2.00 (4 s, 12H, 4 COCH
3
), 1.56 (m, 2H, H-2 a,b), 1.46 (m,
0
0.64, 27.66, 26.86 (4 ꢃ CH linker) ppm. HRMS (ESI): m/z calcd overlapped, H-5 a,b), 1.44 (s, overlapped, NHCOOC(CH
3
)
3
), 1.32
, 150
3
), 155.97
2
+
0
0
13
for C H NO Na [M + Na] : 302.1580; found: 302.1584. Later (m, overlapped, 4H, H-3 a,b, 4 a,b) ppm; C NMR (CDCl
3
1
2
25
6
1
eluted was the side product 6 (white solid), H NMR (MeOD, 600 MHz): d 170.56, 170.20, 169.33, 169.19 (4 COCH
MHz): d 4.24 (d, 1H, J1,2 7.8 Hz, H-1), 3.91 (dt, 1H, J 6.7, 9.6 Hz, (NHCOOC(CH
)
), 100.82 (C-1), 79.03 (NHCOOC(CH
)
), 72.92
3
3
3
3
0
0
H-1
a
), 3.86 (m, 1H, H-6
a
), 3.66 (dd, 2H, J6a,6b 11.8 Hz, J5,6b (C-3), 71.82 (C-5), 71.44 (C-2), 69.95 (C-1 ), 68.61 (C-4), 62.05 (C-
0
0
0
0
5.2 Hz, H-6
b
), 3.55 (dt, 2H, H-1
b
), 3.34 (m, 2H, H-3), 3.29–3.24 6), 40.51 (C-6 ), 29.99 (C-5 ), 29.29 (C-2 ), 28.41
0
0
(
m, overlapped, 4H, H-4, H-5), 3.16 (t, 2H, J 8.2 Hz, H-2), 2.61 (t, (NHCOOC(CH
3
)
3
), 26.41 (C-4 ), 25.50 (C-3 ), 20.65, 20.58, 20.53,
0
4
H, J 7.6 Hz, H-6 ), 1.64 (m, 4H, 2 ꢃ CH linker), 1.54 (m, 4H, 20.51 (4 COCH
3
) ppm. HRMS (ESI): m/z calcd for C25H41NO12Na
a,b
2
+
2
ꢃ CH linker), 1.40 (m, overlapped, 8H, 4 ꢃ CH linker) ppm; [M + Na] : 570.2526; found: 570.2526. Eluted later was the side
2
2
1
3
1
C NMR (MeOD, 150 MHz): d 104.36 (C-1), 78.13 (C-3), 77.91 (C- product 9. H NMR (CDCl
3
, 600 MHz): d 5.20 (t, 2H, J 9.5 Hz, H-
), 75.12 (C-2), 71.67 (C-4), 70.74 (C-1 ), 62.77 (C-6), 50.43 (C-6 ), 3), 5.07 (t, 2H, J 9.7 Hz, H-4), 4.97 (dd, 2H, J1,2 8.0 Hz, J2,3 9.6 Hz,
linker) ppm. HRMS (ESI): m/ H-2), 4.49 (d, 2H, H-1), 4.26 (dd, 2H, J5,6a 4.8 Hz, J6a,6b 12.2 Hz,
0
0
5
3
0.63, 29.96, 28.11, 26.93 (4 ꢃ CH
2
+
z calcd for C24
H
48NO12 [M + H] : 542.3177; found: 542.3183. H-6
a
), 4.14 (dd, 2H, J5,6b 2.5 Hz, H-6
), 3.69 (ddd, 2H, J5,4 10.0 Hz, H-5), 3.47 (dt, 2H, J 6.7 Hz, H-
b
), 3.85 (dt, 2H, J 6.5, 9.6 Hz,
0
Because 5, the intended intermediate to 7, was not the major H-1
a
0
0
reaction product, this route to 7 was not pursued further.
1
b
), 3.13 (t, 4H, J 7.1 Hz, H-6 a,b), 2.08, 2.03, 2.02, 2.00 (4 s, 24H,
B. Compound 3 (500 mg, 1.05 mmol) was deacetylated as 2 ꢃ 4 COCH
3
), 1.57 (m, 4H, 2 ꢃ CH
2
linker), 1.48 (m, 4H, 2 ꢃ
), 1.39–1.24 (m, over-
linker) ppm; C NMR (CDCl , 150 MHz):
), 155.58
described in A and a solution of azido sugar 4 formed was CH linker), 1.44 (s, 9H, NHCOOC(CH )
2
3 3
1
3
hydrogenated in 1 : 20 25% NH OH–MeOH (ꢁ5 mL) in presence lapped, 8H, 4 ꢃ CH
2
3
4
d 170.59, 170.24, 169.36, 169.19 (2 ꢃ 4 COCH
3
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(
NHCOOC(CH
3
)
3
), 100.85 (C-1), 78.98 (NHCOOC(CH
3
)
3
), 72.95 portion of 12 when recrystallized (EtOH) as described for azide 3
0
ꢀ
(C-3), 71.84 (C-5), 71.47 (C-2), 70.02 (C-1 ), 68.63 (C-4), 62.08 (C- showed mp 129–130 C; Rf 0.4 (4 : 1 toluene–acetone);
0
23
1
6
), 46.96 (C-6 ), 29.42 (CH2 linker), 28.50 (overlapped, [a]D ꢄ22.3 (c 0.9, CHCl ). H NMR (CDCl , 600 MHz): d 5.19 (t,
3
3
I
I
I
NHCOOC(CH ) , CH linker), 26.67 (CH2 linker), 25.63 (CH2 overlapped, J 9.5 Hz, H-3 ), 5.18 (t, overlapped, J 9.5 Hz, H-3 ),
linker), 20.65, 20.60, 20.56, 20.54 (4 COCH
3
3
2
II
) ppm. HRMS (ESI): 5.07 (t, 1H, J 9.7 Hz, H-4 ), 4.99 (dd, 1H, J
71NO22Na [M + Na] : 1000.4365; found: 9.6 Hz, H-2 ), 4.93 (dd, 1H, J
1
II,2II 8.0 Hz, J
2
II,3II
3
+
II
I
m/z calcd for C45
000.4359.
A solution of 8 (218 mg, 0.40 mmol) in MeOH (4 mL), was 1H, J
H
1
I
,2
I
8.0 Hz, J
2
I
,3
I
9.7 Hz, H-2 ), 4.88 (t,
I
II
I
1
1H, J 9.7 Hz, H-4 ), 4.59 (d, 1H, H-1 ), 4.45 (d, 1H, H-1 ), 4.27 (dd,
II
II,6aII 4.9 Hz, J6aII,6bII 12.3 Hz, H-6
), 4.13 (dd, 1H, J
II,6bII
5
a
5
II
0
treated with NaOMe in MeOH as described for the preparation 2.3 Hz, H-6
of 4. Chromatography of the residue (8 : 1 CHCl –MeOH) (dd, overlapped, J
afforded 7 (146 mg, 73% in two steps from 3) as white foam, (m, overlapped, H-5 , H-5 ), 3.62 (m, 1H, 6 ), 3.47 (dt, 1H, J
which was in all aspects identical with 7 described in B.
3
-Chlorohexyl 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-b- 2.00, 1.99 (s, overlapped, 7 COCH ), 1.59 (m, overlapped, 4H, H-
D-glucopyranosyl)-b-D-glucopyranoside [6-chlorohexyl 2,3,4,2 ,-
3
3
tion of 1,2,3,4,2 ,3 ,4 ,6 -hepta-O-acetyl-b-gentiobiose (10, 2.0 g, 169.39, 169.26, 169.18 (7 COCH ), 100.79 (C-1 ), 100.55 (C-1 ),
2
b
), 3.88 (dt, overlapped, J 6.3, 9.6 Hz, H-1
a
), 3.85
I
5I,6aI
II
2.0 Hz, J
6aI,6bI
11.0 Hz, H-6 ), 3.70–3.66
3
a
I
I
b
0 0
6.6 Hz, H-1 ), 3.27 (t, 2H, J 6.9 Hz, H-6 a,b), 2.09, 2.04, 2.03, 2.02,
b
6
0
0
0
0
0
13
2
a,b, H-5 a,b), 1.38 (m, overlapped, 4H, H-3 a,b, H-4 a,b) ppm;
C
0
0
0
,4 ,6 -hepta-O-acetyl-b-gentiobioside] (11). To a stirred solu- NMR (CDCl , 150 MHz): d 170.59, 170.21, 170.17, 169.60,
3
0
0
0
0
II
I
35
I
II
I
II
I
.95 mmol) and 6-chlorohexanol (0.48 mL, 3.54 mmol) in dry 73.34 (C-5 ), 72.78, 72.73 (C-3 , C-3 ), 71.96 (C-5 ), 71.36 (C-2 ),
II
0
I
I
II
DCM (40 mL), BF Et O (1.09 mL, 8.84 mmol) was added slowly 71.11 (C-2 ), 69.75 (C-1 ), 69.17 (C-4 ), 68.31 (C-6 ), 68.27 (C-4 ),
at room temperature and the mixture was stirred for 5 h. The 61.82 (C-6 ), 51.32 (C-6 ), 29.23, 28.74 (C-5 ,C-2 ), 26.40, 25.44 (C-
mixture was diluted with DCM, neutralized with aqueous 4 , C-3 ), 20.71, 20.60, 20.63, 20.60, 20.58, 20.57, 20.56 (over-
3
2
II
0
0
0
0
0
NaHCO
3
and washed with brine (3 ꢃ 75 mL). The organic layer lapped,
7
O
COCH
3
) ppm. HRMS (ESI): m/z calcd for
+
was dried, concentrated and chromatography (10 : 1 toluene–
C
32
H
47
N
3
18Na [M + Na] : 784.2752; found: 784.2765; anal.
acetone) of the residue furnished 11 (1.44 g, 64%) as white solid. calcd for C32
H
47
N
3
O
18: C, 50.46; H, 6.22; N, 5.52; found: C,
A portion of 11 was crystallized (EtOH) to give material, mp 148– 50.36; H, 6.15; N, 5.48.
ꢀ
23
1
49 C; R
f
0.4 (4 : 1 toluene–acetone); [a]
D
ꢄ23.5 (c 0.8, CHCl
3
).
6-N-(tert-Butyloxycarbonyl)hexyl 6-O-(b-D-glucopyranosyl)-b-
1
I
H NMR (CDCl , 600 MHz): d 5.19 (t, overlapped, J 9.5 Hz, H-3 ), D-glucopyranoside [6-N-(tert-butyloxycarbonyl)hexyl b-gentio-
.18 (t, overlapped, J 9.5 Hz, H-3 ), 5.07 (t, 1H, J 9.7 Hz, H-4 ), bioside] (16)
.99 (dd, 1H, J1
8.0 Hz, J II II 9.6 Hz, H-2 ), 4.93 (dd, 1H, J
,3
.0 Hz, J 9.7 Hz, H-2 ), 4.88 (t, 1H, J 9.7 Hz, H-4 ), 4.60 (d, 1H, with a solution of NaOMe in MeOH as described for preparation
3
II
II
5
4
8
II
II,2II
1I,2I
A. Acetate 12 (0.78 g, 1.02 mmol) in MeOH (5 mL), was treated
2 ,3
I
I
I
I
2
II
I
H-1 ), 4.46 (d, 1H, H-1 ), 4.27 (dd, 1H, J
5
II,6aII 4.8 Hz, J6aII,6bII of 4 to afford 6-azidohexyl b-gentiobioside 13 (0.46 g, 95%) as
II
II
1
1
2.3 Hz, H-6
overlapped, J 6.2, 9.6 Hz, H-1
.6 Hz, J6a
a
), 4.13 (dd, 1H, J
5
II,6bII 2.3 Hz, H-6
), 3.88 (dt, white foam. R
), 3.85 (dd, overlapped, J
), 3.70–3.65 (m, overlapped, 2H, H- H-1 ), 4.14 (dd, 1H, J
, H-5 ), 3.62 (m, 1H, 6 ), 3.53 (t, 2H, J 6.7 Hz, H-6 a,b), 3.47 (dt, overlapped, J 6.6, 9.6 Hz, H-1 ), 3.87 (dd, overlapped, H-6 ),
H, J 6.6 Hz, H-1 ), 2.09, 2.04, 2.03, 2.02, 2.00, 1.99 (s, over- 3.78 (dd, 1H, J
lapped, 21H, 7 COCH ), 1.77 (m, 2H, H-5 ), 1.59 (m, 2H, H-
a,b), 1.45 (m, 2H, H-4 a,b), 1.37 (m, 2H, H-3 a,b) ppm; C NMR H-5 ), 3.37–3.33 (m, overlapped, 6H, H-3 , H-3 , H-4 ), 3.30–3.25
3 2
CDCl , 150 MHz): d 170.59, 170.22, 170.17, 169.60, 169.39, (m, overlapped, 4H, H-4 , H-6 a,b, H-5 ), 3.22 (dd, 1H, J
3
69.26, 169.18 (7 COCH ), 100.79 (C-1 ), 100.55 (C-1 ), 73.36 (C- 9.1 Hz, H-2 ), 3.18 (m, 1H, H-2 ), 1.62 (m, overlapped, 4H, H-
), 72.78, 72.74 (C-3 , C-3 ), 71.96 (C-5 ), 71.37 (C-2 ), 71.11 (C-
), 69.75 (C-1 ), 69.17 (C-4 ), 68.30 (C-6 ), 68.27 (C-4 ), 61.81 (C- NMR (MeOD, 150 MHz): d 104.82 (C-1 ), 104.39 (C-1 ), 78.01,
), 44.92 (C-6 ), 32.46 (C-5 ), 29.21 (C-2 ), 26.52 (C-4 ), 25.16 (C- 77.97 (C-5 , C-3 , C-3 ), 77.01 (C-5 ), 75.06 (C-2 , C-2 ), 71.57 (C-
), 20.71, 20.64, 20.60, 20.59, 20.57 (overlapped, 7 COCH ) ppm. 4 ), 71.46 (C-4 ), 70.84 (C-1 ), 69.76 (C-6 ), 62.74 (C-6 ), 52.39 (C-
HRMS (ESI): m/z calcd for C H ClO Na [M + Na] : 777.2349, 6 ), 30.64, 29.85, 27.61, 26.64 (C-2 , C-5 , C-4 , C-3 ) ppm. HRMS
b
f
0.46 (1 : 1 DCM–MeOH); H NMR (MeOD, 600
0
II
I
I
MHz): d 4.38 (d, 1H, J
II,2II 7.8 Hz, H-1 ), 4.26 (d, 1H, J
I
I
7.8 Hz,
), 3.89 (dt,
a
5
,6a
1
1
,2
I
I
I
1
5
1
I
,6b
I
11.1 Hz, H-6
a
5
I
,6a
I
2.0 Hz, J6a
I
,6b
I
11.5 Hz, H-6
a
II
I
I
0
0
II
b
a
a
0
I
5I,6aI
5.6 Hz, H-6 ), 3.67 (dd, 1H, J
II II 11.8 Hz, H-6b ), 3.56 (dt, 1H, J 6.6 Hz, H-1 ), 3.44 (m, 1H,
5II,6aII
5.5 Hz,
b
b
0
II
0
J
3
0
a,b
6a ,6b
b
0
0
13
I
I
II
I
2
II
0
I
II
(
II,3II
II
I
II
1
5
2
6
3
I
II
I
II
I
0
0
0
0
13
2
a,b, H-5 a,b), 1.42 (m, overlapped, 4H, H-3 a,b, H-4 a,b) ppm;
C
II
II
0
0
I
I
II
II
I
0
0
0
0
I
I
II
I
I
II
II
I
0
I
II
3
+
0
0
0
0
0
3
2
47
18
+
found: 777.2344; anal. calcd for C32
found: C, 50.61; H, 6.18.
H
33 3
47ClO18: C, 50.90; H, 6.27, (ESI): m/z calcd for C18H N O11Na [M + Na] : 490.2013, found:
490.2011.
6
-Azidohexyl 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-b-
A solution of the foregoing azido sugar 13 (0.46 g, 0.98 mmol)
0
D-glucopyranosyl)-b-D-glucopyranoside [6-azidohexyl 2,3,4,2 ,- in 5% AcOH in MeOH (4 mL) was treated overnight with
0
0
0
3
,4 ,6 -hepta-O-acetyl-b-gentiobioside] (12). A mixture of 11 hydrogen in presence of 5% Pd/C (90 mg). The mixture was
(
2.53 g, 3.35 mmol) and NaN (0.87 g, 13.4 mmol) in DMF (10 ltered through Celite pad, the pad was washed with MeOH,
3
ꢀ
1
mL) was stirred at 75 C until H NMR spectrum showed and the ltrate was concentrated. The residue was a mixture of
+
complete disappearance of the starting material (ꢁ4 h; for 14 and 15 (MALDI-TOF MS: 14, C H NO Na [M + Na] : calcd
1
8
35
11
+
monitoring of the conversion, see preparation of 3). The 464.21; found 464.22 and 15, C36H67NO22Na [M + Na] : calcd
mixture was concentrated and the residue was partitioned 888.40; found 888.39), analogs of 5 and 6.
between CHCl The foregoing mixture was dissolved in MeOH (3 mL) and
and brine (3 ꢃ 75 mL). Chromatography of the
residue obtained upon concentration of the organic phase treated overnight with di-tert-butyl dicarbonate (0.32 g, 1.47
10 : 1 toluene–acetone) gave 12 (2.44 g, 95%) as white solid. A mmol). Aer concentration, the residue was chromatographed
3
(
7598 | RSC Adv., 2017, 7, 7591–7603
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Paper
5 : 1 CHCl
RSC Advances
II
II
(
3
–MeOH) to yield 16 as white foam (0.22 g, 38% in 4 ), 4.99 (dd, 2H, J
1
II,2II 8.0 Hz, J
2
II,3II 9.6 Hz, H-2 ), 4.93 (dd, 2H,
I
2
3
I
three steps from 12). R 0.3 (5 : 1 CHCl –MeOH); [a] ꢄ32.9 (c
J
1I,2I
8.0 Hz, J
2I,3I
9.6 Hz, H-2 ), 4.88 (t, 2H, J 9.6 Hz, H-4 ), 4.50 (d,
f
3
D
1
II
I
0
7
2
.8, MeOH). H NMR (MeOD, 600 MHz): d 4.38 (d, 1H, J II II 2H, H-1 ), 4.45 (d, 2H, H-1 ), 4.27 (dd, 2H, J
.8 Hz, H-1 ), 4.25 (d, 1H, J
5II,6aII
4.9 Hz, J II II
1
,2
6a ,6b
II
I
II
II
1I,2I
7.8 Hz, H-1 ), 4.14 (dd, 1H, J
), 3.88 (dt, overlapped, H-1
5I,6aI
12.3 Hz, H-6a ), 4.13 (dd, 2H, J
), 3.87 overlapped, 4H, H-1
), 3.67 3.62 (m, 2H, 6
5II,6bII
2.3 Hz, H-6b ), 3.90–3.85 (m,
I
0
0
I
II
I
.0 Hz, J6a
I
,6b
I
11.5 Hz, H-6
a
a
I
a
, 6
a
), 3.68 (m, overlapped, 4H, H-5 , H-5 ),
II
a
I
0
(
(
dd, overlapped, H-6
dd, 1H, J
) 3.78 (dd, 1H, J
5
I
,6b
II
I
5.6 Hz, H-6
b
b
), 3.45 (dt, 2H, J 6.6, 9.5 Hz, H-1
b
), 3.13 (m, 4H,
0
II,6bII 5.4 Hz, J6aII,6bII 11.8 Hz, H-6
), 3.55 (dt, 1H, J 6.6, H-6 a,b), 2.09, 2.04, 2.03, 2.02, 2.00, 1.99 (7 s, overlapped, 42H, 7
5
b
0
I
0
0
9
3
1
.6 Hz, H-1
b
), 3.44 (m, 1H, H-5 ), 3.37–3.33 (m, overlapped, H- COCH
3
), 1.56 (m, 4H, H-2 a,b), 1.48 (m, overlapped, H-5 a,b), 1.44
, H-3 , H-4 ), 3.29–3.24 (m, overlapped, H-4 , H-5 ), 3.22 (dd, (s, overlapped, NHCOOC(CH linker),
), 1.33 (m, 4H, 2 ꢃ CH
9.1 Hz, H-2 ), 3.17 (m, 1H, H-2 ), 3.03 (t, 2H, J 7.1 Hz, 1.26 (m, 4H, 2 ꢃ CH linker) ppm; C NMR (CDCl , 150 MHz):
I
II
I
II
II
3
)
3
2
II
I
13
H, J
0
2II,3II
2
3
0
0
H-6 a,b), 1.63 (m, 2H, H-2 a,b), 1.49–1.38 (m, overlapped, H-3 a,b, d 170.57, 170.20, 170.15, 169.60, 169.38, 169.23, 169.18 (7
0
II
I
H-5 ), 1.43 (s, overlapped, NHCOOC(CH ) ), 1.35 (m, 2H, H- COCH ), 155.51 (NHCOOC(CH ) ), 100.75 (C-1 ), 100.53 (C-1 ),
a,b
3 3
3
3 3
0
13
I
II
I
4
a,b
)
ppm;
C
NMR (MeOD, 150 MHz):
d
I
158.55 78.94 (NHCOOC(CH
3
)
3
), 73.22 (C-5 ), 72.78, 72.73 (C-3 , C-3 ),
II
II
I
II
0
I
(NHCOOC(CH
3
)
)
3
), 104.81 (C-1 ), 104.38 (C-1 ), 79.79 71.93 (C-5 ), 71.35 (C-2 ), 71.07 (C-2 ), 69.90 (C-1 ), 69.18 (C-4 ),
I
I
II
I
I
II
II
0
(
7
NHCOOC(CH
3
3
), 78.00, 77.96 (C-5 , C-3 , C-3 ), 77.02 (C-5 ), 68.32 (C-6 ), 68.25 (C-4 ), 61.78 (C-6 ), 46.86 (C-6 ), 29.37 (CH
2
I
II
II
I
0
5.06 (C-2 , C-2 ), 71.57 (C-4 ), 71.46 (C-4 ), 70.93 (C-1 ), 69.74 linker), 28.46 (overlapped, NHCOOC(CH
3
)
3
, CH
2
linker), 26.67,
linker), 20.69, 20.62, 20.61, 20.59, 20.57, 20.55
I
II
0
0
0
(C-6 ), 62.74 (C-6 ), 41.29 (C-6 ), 30.90 (C-5 ), 30.70 (C-2 ), 28.80 25.67 (each CH
2
0
0
(NHCOOC(CH ) ), 27.65 (C-4 ), 26.76 (C-3 ) ppm. HRMS (ESI): m/ (overlapped, 7 COCH ) ppm. HRMS (ESI): m/z calcd for
3
3
3
+
+
z calcd for C H NO Na [M + Na] : 564.2641; found: 564.2632. C H NO Na [M + Na] : 1576.6056; found: 1576.6051.
2
3
43
13
69 103
38
B. A solution of azide 12 (1.0 g, 1.31 mmol) and di-tert-butyl
dicarbonate (0.57 g, 2.62 mmol) in DCM (4 mL) was mixed with NaOMe in MeOH (4 mL) as described above, followed by chro-
a suspension of 5% Pd/C (0.20 g) in DCM (3 mL) and the mixture matography (5 : 1 CHCl –MeOH) to afford 16 (0.28 g, 96%) as
was stirred at room temperature under H atmosphere until TLC white foam, which was in all aspects identical with the material
Deacetylation of 17 (0.45 g, 0.54 mmol) was performed with
3
2
showed complete consumption of the starting material (ꢁ6 h). described above.
Aer conventional work-up, as described above, chromatog-
6-N-(tert-Butyloxycarbonyl)hexyl 2,3,4,6-tetra-O-[{1-(2,3,4,6-
raphy (2 : 1 toluene–EtOAc) afforded 6-N-(tert-butyloxycarbonyl) tetra-O-acetyl-b-D-glucopyranosyloxyhexyl)-1,2,3-triazol-4-yl}
hexyl 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-b-D-glucopyr- methyl]-b-D-glucopyranoside (20). Propargyl bromide (0.40 mL,
ꢀ
anosyl)-b-D-glucopyranoside [6-N-(tert-butyloxycarbonyl) hexyl 3.64 mmol) was added drop wise, at 0 C under inert atmo-
0
0
0
0
2
,3,4,2 ,3 ,4 ,6 -hepta-O-acetyl-b-gentiobioside] 17 (0.867 g, 78%) sphere, to a stirred suspension of 7 (115 mg, 0.30 mmol) and
ꢀ
as white solid. Crystalline 17 showed mp 126–127 C (EtOH), R
.4 (4 : 1 toluene–acetone) and [a]
NMR (CDCl , 600 MHz): d 5.18 (t, overlapped, J 9.5 Hz, H-3 ), concentration the residue was partitioned between EtOAc and
.17 (t, overlapped, J 9.5 Hz, H-3 ), 5.07 (t, 1H, J 9.6 Hz, H-4 ), brine. The organic layer was dried with Na
f
KOH (136 mg, 2.42 mmol) in DMF (5 mL). The mixture was
). H allowed to attain room temperature and stirred overnight. Aer
2
3
1
0
D
ꢄ16.2 (c 0.7, CHCl
3
I
3
II
II
5
4
8
2
SO
4
, concentrated
II
.99 (dd, 1H, J1
II,2II
8.0 Hz, J II II 9.6 Hz, H-2 ), 4.93 (dd, 1H, J
1I,2I
and the residue was chromatographed (15 : 1 toluene–EtOAc) to
2 ,3
I
I
.0 Hz, J2 9.6 Hz, H-2 ), 4.88 (t, 1H, J 9.6 Hz, H-4 ), 4.60 (d, give 6-N-(tert-butyloxycarbonyl)hexyl 2,3,4,6-tetra-O-propargyl-b-
overlapped, H-1 ), 4.58 (broad s, overlapped, NH), 4.45 (d, 1H, D-glucopyranoside 19 (120 mg, 75%) as colorless syrup, R 0.3
H-1 ), 4.27 (dd, 1H, J
dd, 1H, J
dd, overlapped, 6 ), 3.68 (m, overlapped, 2H, H-5 , H-5 ), 3.62 1), 4.26–4.18 (m, overlapped, OCH
a 2
m, overlapped, 6
H, H-6 a,b), 2.09, 2.04, 2.03, 2.02, 2.00, 1.99 (7 s, overlapped, (dd, 1H, J5,6b 4.9 Hz, H-6
1H, 7 COCH ), 1.57 (m, 2H, H-2 a,b), 1.45 (m, overlapped, H- overlapped, H-1 ), 3.44 (m, overlapped, H-4), 3.38 (ddd, 1H, J
5,4
), 1.44 (s, overlapped, NHCOOC(CH ) ), 1.33 (m, overlapped, 9.8 Hz, H-5), 3.31 (dd, 1H, J 7.9 Hz, J2,3 8.9 Hz, H-2), 3.10 (m,
H, H-3 a,b, H-4 a,b) ppm; C NMR (CDCl , 150 MHz): d 170.60, 2H, J 6.0 Hz, H-6 ), 2.46 (m, 4H, 4 OCH C^CH), 1.60 (m, 2H,
70.21, 170.20, 169.61, 169.39, 169.29, 169.20 (7 COCH
NHCOOC(CH
I,3I
II
f
I
II
1
II,6aII 4.9 Hz, J6aII,6bII 12.3 Hz, H-6
), 4.13 (7 : 1 toluene–EtOAc). H NMR (CDCl
, 600 MHz): d 4.55–4.36
C^CH, NH), 4.26 (d, overlapped, J1,2 7.9 Hz, H-
C^CH), 3.88 (dt, 1H, J 6.5,
), 3.84 (dd, 1H, J5,6a 1.9 Hz, J6a,6b 10.8 Hz, H-6 ), 3.76
), 3.54 (t, 1H, J 8.9 Hz, H-3) 3.47 (m,
5
a
3
II
0
(
(
(
5
II,6bII 2.3 Hz, H-6
b
), 3.86 (dt, overlapped, H-1
a
I
), 3.85 (m, 7H, 3 OCH
2
I
II
I
0
0
b
), 3.46 (dt, 1H, J 6.6, 9.5 Hz, H-1
b
), 3.10 (m, 9.5 Hz, H-1
a
a
0
2
2
5
4
1
b
0
0
3
b
0
a,b
3 3
1,2
0
0
13
0
3
a,b
2
0
0
3
), 155.96 H-2 a,b), 1.49–1.44 (m, overlapped, H-5 a,b), 1.44 (s, overlapped,
II
I
0
(
(
(
(
(
2
(
8
3
)
)
3
3
), 100.78 (C-1 ), 100.54 (C-1 ), 79.01 NHCOOC(CH
), 73.34 (C-5 ), 72.78, 72.75 (C-3 , C-3 ), 71.94 lapped, H-4 a,b) ppm; C NMR (CDCl
3
)
3
), 1.37 (m, overlapped, H-3 a,b), 1.33 (m, over-
I
II
I
0
13
NHCOOC(CH
3
3
, 150 MHz): d 155.92
), 102.98 (C-1), 83.31 (C-3), 81.33 (C-2), 80.02,
C-6 ), 68.27 (C-4 ), 61.81 (C-6 ), 40.46 (C-6 ), 29.98 (C-5 ), 29.27 79.93, 79.80, 79.52 (4 OCH C^CH), 78.98 (NHCOOC(CH ),
C-2 ), 28.41 (NHCOOC(CH ) ), 26.48, 25.57 (C-4 , C-3 ), 20.71, 76.14 (C-4), 74.62, 74.33, 74.24, 74.23 (4 OCH C^CH), 74.05 (C-
II
I
II
0
0
I
C-5 ), 71.36 (C-2 ), 71.10 (C-2 ), 69.89 (C-1 ), 69.18 (C-4 ), 68.30 (NHCOOC(CH
3
)
3
I
II
II
0
2
3 3
)
0
0
0
3
3
2
0
0.64, 20.60, 20.58, 20.57 (overlapped, 7 COCH ) ppm. HRMS 5), 69.90 (C-1 ), 68.47 (C-6), 60.20, 59.94, 59.29, 58.61 (4 OCH -
3 2
ESI): m/z calcd for C H NO Na [M + Na] : 858.3372; found: C^CH), 40.47 (C-6 ), 29.95 (C-5 ), 29.46 (C-2 ), 28.38
58.3373; anal. calcd for C32
+
0
0
0
3
2
57
20
0
0
H
57NO20: C, 53.17; H, 6.87; N, 1.68; (NHCOOC(CH ) ), 26.47 (C-4 ), 25.67 (C-3 ) ppm. HRMS (ESI): m/
3 3
+
found: C, 53.24; H, 6.79; N, 1.72. The side product 18 was z calcd for C29
subsequently eluted from the column and obtained as white To a solution of sugar alkyne 19 (120 mg, 0.23 mmol) and
, 600 MHz): d 5.18 (t, overlapped, J 9.5 Hz, sugar azide 3 (534 mg, 1.13 mmol) in dry DCM (6.0 mL), CuI
8
H41NO Na [M + Na] : 554.2730; found: 554.2731.
1
solid. H NMR (CDCl
3
I
II
H-3 ), 5.17 (t, overlapped, J 9.5 Hz, H-3 ), 5.07 (t, 2H, J 9.6 Hz, H- (43 mg, 0.23 mmol) and DIPEA (39 mL, 0.23 mmol) were added
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0
00
and the mixture was stirred at room temperature under inert (m, overlapped, H-1
b
I
), 3.76–3.72 (m, overlapped, 4 H-1
b
), 3.74
I
atmosphere overnight. The mixture was concentrated and the (m, overlapped, H-3 ), 3.66 (m, overlapped, H-5 ), 3.64 (m,
residue was partitioned between brine and EtOAc. The organic overlapped, H-4 ), 3.62 (m, overlapped, 4 H-3 ), 3.57 (m, 4H, 4
layer was dried with NaSO , concentrated and chromatography H-5 ), 3.51 (m, overlapped, 4 H-4 ), 3.48 (m, overlapped, H-2 ),
I
II
II
II
I
4
II
0
(
2 : 1 CHCl
3
–acetone) followed by freeze-drying yielded 20 as 3.39 (m, 4H, 4 H-2 ), 3.13 (t, 2H, J 7.6 Hz, H-6 a,b), 1.98 (m, 8H,
0
0
0
white solid (426 mg, 77%), R
f
0.2 (2 : 1 CHCl
3
–acetone); H-5 a,b), 1.79 (m, overlapped, H-5 a,b), 1.74 (m, overlapped, H-
2
3
1
0
0
0
[a]
D
ꢄ18.0 (c 0.7, CHCl
3
). H NMR (CDCl
3
, 600 MHz): d 8.10 (s,
2 a,b), 1.68 (m, overlapped, 4 H-2 a,b), 1.53–1.43 (m, overlapped,
0
0
00
00
13
1
H, H-triazole), 7.90 (s, overlapped, 2H, H-triazole), 7.67 (s, 1H, 12H, H-4 a,b, H-3 a,b, 4 H-3 a,b), 1.36 (m, 8H, 4 H-4 a,b) ppm;
H-triazole), 5.20 (t, 4H, J 9.5 Hz, 4 H-3 ), 5.08 (t, 4H, J 9.7 Hz, 4 NMR (D O, 150 MHz): d 144.84, 144.34 (4 triazole C-4), 125.73,
2
H-4 ), 5.04–4.89 (m, overlapped, OCH -triazole), 4.97 (t, over- 125.42, 125.31, 125.28 (4 triazole C-5), 103.07 (C-1 ), 102.88 (C-
lapped, J 8.8 Hz, 4 H-2 ), 4.84–4.67 (m, overlapped, OCH -tri- 1 ), 83.54 (C-3 ), 81.70 (C-2 ), 77.53 (C-4 ), 76.57, 76.55 (over-
lapped, C-3 , C-5 ), 74.04 (C-5 ), 73.84 (C-2 ), 71.26 (C-1 ),
.0 Hz, 4 H-1 ), 4.35 (m, overlapped, 4 H-6 a,b), 4.32 (d, over- 70.92–70.89 (C-1 ), 70.38 (C-4 ), 68.39 (C-6 ), 66.09, 65.60, 65.32,
C
II
II
I
2
II
II
I
I
I
2
II
II
I
II
0
azole), 4.69 (bs, overlapped, NH), 4.48 (d, overlapped, 4H, J
1II,2II
II
00
00
II
I
8
I
II
00
lapped, J
1
I
,2
I
7.9 Hz, H-1 ), 4.27 (dd, 4H, J
5
II,6aII 4.7 Hz, J6aII,6bII 63.75 (4 OCH
2
-triazole), 61.50 (C-6 ), 50.98–50.93 (C-6 ), 40.10
), 3.91 (dt, (C-6 ), 29.94–29.85 (C-5 ), 29.21–29.16 (C-2 , C-2 ), 27.24 (C-5 ),
), 3.79 (m, 2H, 25.96–25.89, 25.35 (C-4 , C-3 , C-4 ) 25.11–25.04 (C-3 ) ppm;
II,5II 9.9 Hz, 4 H-5 ), 3.51 (m, overlapped, HRMS (ESI): m/z calcd for C72
II
II
0
00
00
0
0
1
1
2.2 Hz, 4 H-6
H, J 6.6, 9.4 Hz, H-1
a
), 4.13 (dd, 4H, J
5
II,6bII 2.2 Hz, 4 H-6
b
0
00
00
0
0
00
a
), 3.87–3.82 (m, 4H, 4 H-1
a
I
II
+
H-6a,b ), 3.69 (ddd, 4H, J
H-3 , H-4 , H-1 ), 3.49–3.44 (m, 4H, 4 H-1 ), 3.37 (m, 1H, H-5 ), found: 1652.8705.
4
H
126
N
13
O
30 [M + H] : 1652.8734;
I
I
0
00
I
b
b
I
0
0 0 0 0
3.33 (m, 1H, H-2 ), 3.10 (m, 2H, H-6 a,b), 2.08 (s, 12H, 4 COCH3),
6-N-(tert-Butyloxycarbonyl)hexyl 2,3,4,2 ,3 ,4 ,6 -hepta-O-[{1-
2
.03, 2.02 (s, overlapped, 24H, 8 COCH ), 2.00 (s, 12H, 4 (2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyloxyhexyl)-1,2,3-triazol-
3
0
0
0
COCH
3
), 1.91 (m, 8H, 4 H-5 a,b), 1.65 (m, overlapped, H-2 a,b), 4-yl}methyl]-b-gentiobioside (23). To a stirred suspension of 16
0
0
0
1
1
.56 (m, overlapped, 4 H-2 a,b), 1.49 (m, overlapped, H-5 a,b), (135 mg, 0.25 mmol) and KOH (196 mg, 3.49 mmol) in DMF (5
.43 (s, overlapped, NHCOOC(CH
0
0
3
13
)
3
), 1.42–1.30 (m, 4 H-3 a,b, 4 mL), propargyl bromide (0.45 mL, 3.98 mmol) was added drop
00
0
0
ꢀ
H-4 a,b, H-3 a,b, H-4 a,b) ppm; C NMR (CDCl
170.61, 170.23, 169.37, 169.23, (4 COCH
NHCOOC(CH ) ), 144.78, 144.72, 144.53, 144.31 (4 triazole C- tration, the residue was dissolved in EtOAc and washed with
3
, 150 MHz): wise at 0 C under inert atmosphere. The mixture was allowed to
d
3
), 156.01 attain room temperature and stirred overnight. Aer concen-
(
3
3
I
4
1
7
6
6
5
), 124.30, 123.73, 123.35, 122.84 (4 triazole C-5), 103.42 (C-1 ), brine (3 ꢃ 75 mL), the organic layer was dried (Na SO ) and
2
4
II I I
00.77 (C-1 ), 83.85 (C-3 ), 81.67 (C-2 ), 78.91 (NHCOOC(CH ) ), concentrated. The residue was chromatographed (10 : 1
7.49 (C-4 ), 74.45 (C-5 ), 72.80 (C-3 ), 71.73 (C-5 ), 71.30 (C-2 ), toluene–EtOAc) to obtain 6-N-(tert-butyloxycarbonyl)hexyl
9.94 (C-1 ), 69.84–69.81 (C-1 ), 69.05 (C-6 ), 68.44 (C-4 ), 66.28, 2,3,4,2 ,3 ,4 ,6 -hepta-O-propargyl-b-gentiobioside 22 (120 mg,
5.76, 65.72, 64.91 (4 OCH
3
3
II
I
I
II
II
0
00
I
II
0
0
0
0
II
1
2
-triazole), 61.93 (C-6 ), 50.43, 50.30, 58%) as colorless syrup; R
f
0.4 (5 : 1 toluene–EtOAc). H NMR
0
00
0
0
00
0
0.22, 50.19 (C-6 ), 40.47 (C-6 ), 30.13 (C-5 ), 29.94 (C-5 ), 29.56 (CDCl
), 26.49 (C-4 ), 26.20– C^CH), 4.40 (d, overlapped, H-1 ), 4.27 (d, overlapped, J
00 0 00
6.14 (C-4 ), 25.72 (C-3 ), 25.28 (C-3 ), 20.71, 20.66, 20.58, 20.56 7.9 Hz, H-1 ), 4.26–4.18 (m, overlapped, OCH C^CH), 4.23 (m,
2
3
, 600 MHz): d 4.55–4.35 (m, overlapped, NH, 6 OCH
2
-
0
0
II
(C-2 ), 29.15 (C-2 ), 28.41 (NHCOOC(CH
3
)
3
1
I
,2
I
I
2
I
0
(4 COCH ) ppm; HRMS (ESI): m/z calcd for C109H166N O [M + overlapped, H-6 ), 3.87 (dt, overlapped, 1H, J 6.4, 9.6 Hz, H-1 ),
3 13 48 a
a
+
II
H] : 2425.0948; found: 2425.0972; anal. calcd for 3.84 (dd, overlapped, J5
48: C, 53.98; H, 6.86; N, 7.51; found: C, 53.85; H, (dd, 1H, J
11.4 Hz, H-6
-Aminohexyl 2,3,4,6-tetra-O-[{1-(b-D-glucopyranosyloxyhexyl)- overlapped, J 8.8 Hz, H-3 ), 3.48–3.42 (m, overlapped, H-4 , H-
II,6aII
1.9 Hz, J II II 10.8 Hz, H-6a ), 3.76
6a ,6b
II
C
6
109
H
165
N
13
O
5
II,6bII 4.9 Hz, H-6
b
), 3.66 (dd, 1H, J
b
), 3.54 (t, overlapped, J 9.0 Hz, H-3 ), 3.51 (t,
5
I
,6b
I
7.5 Hz, J6a
I
,6b
I
I
I
.87; N, 7.40.
6
II
II
I
0
II
1
,2,3-triazol-4-yl}methyl]-b-D-glucopyranoside (21). TFA (320 mL) 5 , H-1
b
), 3.38 (dd, 1H, J
4
II,5II 9.8 Hz, H-5 ), 3.34 (dd, overlapped,
II,3II 9.0 Hz, H-2 ), 3.32–3.29 (m, overlapped, H-2 ,
ꢀ
II
I
was added drop wise at 0 C to a solution of 20 (180 mg, 0.074
mmol) in dry DCM (4.0 mL). The cooling was removed, and the H-4 ), 3.10 (m, 2H, H-6 ), 2.46 (m, 7H, 7 OCH C^CH), 1.60 (m,
mixture was stirred at room temperature under inert atmo- 2H, H-2 a,b), 1.46 (m, overlapped, H-5 ), 1.44 (s, overlapped,
sphere for 4 h, when TLC (8 : 1 CHCl –MeOH) showed complete NHCOOC(CH ) ), 1.35 (m, overlapped, 4H, H-3 a,b, H-4 a,b) ppm;
disappearance of 20. Aer concentration, the residue was
deacetylated under conditions described above, and the crude (C-1 ), 102.82 (C-1 ), 83.42 (C-3 ), 83.35 (C-3 ), 81.41 (C-2 ), 81.23
2
product was subjected to solid-phase extraction (C18 5 g Waters (C-2 ), 80.04, 79.99, 79.96, 79.91, 79.86, 79.80, 79.62 (7 OCH -
SepPack cartridge, 1 : 2 / 4 : 1 MeOH–water) to afford 21 C^CH), 79.03 (NHCOOC(CH
J
1
II,2II 7.9 Hz, J
2
I
0
a,b
2
0
0
a,b
0
0
3
3 3
1
3
3 3 3
C NMR (CDCl , 150 MHz): d 155.94 (NHCOOC(CH ) ), 103.32
II
I
I
II
I
II
I
II
3
)
3
), 76.38 (C-4 ), 76.04 (C-4 ),
C^CH),
(92 mg, 75%) as white foam, aer concentration and freeze 74.77, 74.68, 74.44, 74.37, 74.33, 74.31, 74.27 (7 OCH
2
1
I
II
0
I
II
drying. R 0.3 (C18-silica, 1 : 1 MeOH–water); H NMR (D O, 600 74.39 (C-5 ), 74.13 (C-5 ), 69.97 (C-1 ), 69.05 (C-6 ), 68.44 (C-6 ),
f
2
MHz): d 8.17, 8.12, 8.04, 8.01 (4 s, 4H, 4 H-triazole), 5.06 (m, 1H, 60.23, 60.20, 59.99, 59.86, 59.26, 58.68 (7 OCH C^CH), 40.53
2
0
0
0
OCH -triazole), 4.96–4.74 (m, overlapped, 6H, 3 OCH -triazole), (C-6 ), 30.02 (C-5 ), 29.54 (C-2 ), 28.42 (NHCOOC(CH ) ), 26.57,
2
2
3 3
I
0
0
4
.63 (d, overlapped, J
1
I
,2
I
7.6 Hz, H-1 ), 4.62 (m, overlapped, 25.79 (C-4 , C-3 ) ppm. HRMS (ESI): m/z calcd for C44
-triazole), 4.56–4.47 (m, overlapped, 12H, 4 H-1 , 4 H- [M + Na] : 830.3728; found: 830.3727.
H57NO13Na
II
+
OCH
2
0
0
II
a
0
6
4
3
a,b), 4.04 (m, overlapped, 4 H-6
), 4.01 (m, overlapped, H-1 ),
a
To a solution of the foregoing sugar alkyne 22 (120 mg, 0.14
), mmol) and sugar azide 3 (560 mg, 1.19 mmol) in dry DCM, CuI
), 3.77 (28 mg, 0.14 mmol) and DIPEA (26 mL, 0.14 mmol) were added
0
0
I
.00–3.95 (m, overlapped, 4 H-1
a
), 3.87 (m, overlapped, H-6
a
II
I
.85 (m, overlapped, 4 H-6
b
), 3.82 (m, overlapped, H-6
b
7600 | RSC Adv., 2017, 7, 7591–7603
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I
II
II
and the mixture was stirred at room temperature overnight H-5 ), 3.75 (m, overlapped, H-5 , H-4 ), 3.74 (m, overlapped, H-
0
III
I
III
under inert atmosphere. The mixture was concentrated and the 1 ), 3.71 (m, 7 H-3 , H-4 ), 3.65 (m, overlapped, 7 H-5 ), 3.63
residue was partitioned between brine and DCM. Concentration (m, overlapped, 7 H-4 ), 3.62 (m, overlapped, H-2 ), 3.60 (m,
of the organic phase, chromatography (1 : 1 CHCl –acetone) overlapped, H-2 ), 3.49 (m, 7 H-2 ), 3.11 (t, J 7.4 Hz, 2H, H-6 a,b),
and freeze-drying (benzene) afforded 23 (430 mg, 71%) as white 2.06 (m, 14H, H-5 a,b), 1.83–1.72 (m, 16H, H-5 a,b, 7 H-2 a,b), 1.70
b
III
II
I
III
0
3
0
0
0
00
2
3
1
0
0
0
solid; R
NMR (CDCl
triazole), 5.20 (7 t, overlapped, 7H, J 9.5 Hz, 7 H-3 ), 5.08 (7 t, 144.92, 144.90, 144.57, 144.45 (7 triazole C-4), 125.57, 125.33,
f
0.3 (1 : 1 CHCl
3
–acetone); [a]
D
ꢄ18.9 (c 0.8, CHCl
3
). H (m, overlapped, H-2 a,b), 1.62–1.39 (m, 32H, H-3 a,b, H-4 a,b, 7 H-
00 00
13
3
, 600 MHz): d 8.15, 8.04, 7.94, 7.89, 7.77 (s, 7H, H-
3 a,b, 7 H-4 a,b) ppm; C NMR (D O, 150 MHz): d 145.02, 144.96,
2
III
III
II
I
overlapped, 7H, J 9.7 Hz, 7 H-4 ), 5.06–4.66 (m, overlapped, 125.29, 125.24 (7 triazole C-5), 103.40 (C-1 ), 103.07 (C-1 ),
4H, 7 OCH -triazole), 4.97 (m, overlapped, 7 H-2 ), 4.49 (7 d, 102.95 (C-1 ), 83.82 (C-3 ), 83.72 (C-3 ), 81.84 (C-2 ), 81.57 (C-
III
III
I
II
I
1
2
III
II
II
I
II
III
III
overlapped, J1
III,2III
8.0 Hz, 7 H-1 ), 4.47 (m, overlapped, H-1 ), 2 ), 78.25 (C-4 ), 77.55 (C-4 ), 76.63, 76.61 (C-3 , C-5 ), 74.36,
00 0 00
.38–4.31 (m, overlapped, 7 H-6 ), 4.36 (m, overlapped, H-1 ), 74.32 (C-5 , C-5 ), 73.90 (C-2 ), 71.11 (C-1 ), 70.88 (C-1 ), 70.46
a,b
I
I
II
III
4
4
6
3
6
III
I
II
.27 (7 dd, overlapped, 7H, J
5
III,6aIII 4.5 Hz, J6aIII,6bIII 12.2 Hz, 7 H- (C-4 ), 69.09 (C-6 ), 68.73 (C-6 ), 66.22, 66.16, 65.69, 65.65,
III
I
III
III
a
), 4.21 (m, 1H, H-6
a
b 2
), 4.13 (7 dd, overlapped, 7H, 7 H-6 ), 65.62, 65.36, 64.11 (7 OCH -triazole), 61.59 (C-6 ), 50.99–50.94
0
00 00 0 00 00
a
), 3.80 (m, 2H, H- (C-6 ), 40.32 (C-6 ), 30.01–29.91 (C-5 ), 29.28–29.24 (C-2 ), 28.19
.89–3.82 (m, overlapped, 8H, H-1
a,b ), 3.75 (m, 1H, H-6
a
, 7 H-1
II
I
III
00
00
00
b
), 3.70 (m, 7H, 7 H-5 ), 3.54 (m, over- (C-5 ), 26.10–25.97 (C-4 ) 25.44, 25.14 (C-3 ). HRMS (ESI): m/z
I
I
+
lapped, H-3 ), 3.51 (m, overlapped, H-5 ), 3.50 (m, overlapped, H- calcd for C123
H
210
N
22
O
53 [M] : 2843.4414; found: 2843.4429.
6-(2-Methoxycyclobutene-3,4-dione)aminohexyl 2,3,4,6-tetra-
lapped, H-4 ), 3.38 (m, overlapped, H-5 ), 3.35 (m, overlapped, O-[{1-(b-D-glucopyranosyloxyhexyl)-1,2,3-triazol-4-yl}methyl]-b-
II
II
0 00
, H-4 ), 3.46 (m, overlapped, H-1 , 7 H-1 b), 3.39 (m, over-
b
3
I
II
II
I
0
H-2 ), 3.33 (m, overlapped, H-2 ), 3.06 (m, 2H, H-6 ), 2.08–2.00 D-glucopyranoside (25). 3,4-Dimethoxy-3-cyclobutene-1,2-dione
m, overlapped, 84H, 28 COCH ), 1.95–1.86 (m, 7 H-5 a,b), 1.61– (13 mg, 0.090 mmol) was added to the solution of 21 (30 mg,
a,b
00
(
1
1
3
0
0
00
00
.51 (m, overlapped, 7 H-2 a,b), 1.46–1.27 (m, 7 H-3 a,b, 7 H-4 a,b
.42 (s, overlapped, NHCOOC(CH ) ) ppm; C NMR (CDCl
3 3 3
)
0.018 mmol) in 1.13 mL of pH 7 phosphate buffer (0.5 M) and the
, 150 solution was kept with gentle motion at room temperature for
), 156.05 18 h. The mixture was concentrated and the residue was chro-
), 144.76, 144.70, 144.56, 144.52, 144.50, matographed (2 : 1 CHCl –MeOH) to give 25 (17 mg, 53%); R
1
3
MHz): d 170.63, 170.24, 169.40, 169.25 (28 COCH
NHCOOC(CH
3
(
3
)
3
3
f
1
1
1
1
44.31, 144.25 (7 triazole C-4), 124.29, 123.80, 123.72, 123.35, 0.13 (2 : 1 CHCl –MeOH). H NMR (MeOD, 600 MHz): d 8.04,
3
23.14 (7 triazole C-5), 103.71 (C-1 ), 103.23 (C-1 ), 100.77 (C- 8.02, 8.00, 7.97 (s, 4H, 4 H-triazole), 4.98 (m, 1H, OCH -triazole),
), 83.96 (C-3 ), 83.90 (C-3 ), 81.74 (C-2 ), 81.40 (C-2 ), 78.83 4.89–4.78 (m, overlapped, 4H, OCH -triazole), 4.68–4.58 (m,
II
I
2
III
I
II
I
II
2
I
II
I
(
3 3 2
NHCOOC(CH ) ), 78.18 (C-4 ), 77.36 (C-4 ), 74.54 (C-5 ), 74.47 overlapped, 4H, OCH -triazole, NH), 4.41–4.35 (m, overlapped, 4
II
III
III
III
00
I
(
C-5 ), 72.81 (7 C-3 ), 71.73 (7 C-5 ), 71.30 (7 C-2 ), 69.86 (7 C- H-6 a,b, OCH ), 4.38 (m, overlapped, H-1 ), 4.24 (4 d, overlapped,
3
0
0
II
I
III
II
0
II,2II 7.8 Hz, 4H, 4 H–1 ), 3.88 (m, overlapped, H-1
a
1
6
4
2
2
4
5
), 68.99 (C-6 ), 68.73 (C-6 ), 68.44 (7 C-4 ), 66.34, 65.92, 65.70,
J
1
), 3.87 (m,
), 3.71 (m, 2H,
III
00
00
00
II
a
4.83 (7 OCH
2
-triazole), 61.94 (7 C-6 ), 50.39–50.23 (7 C-6 ), overlapped, 4 H-1
0.44 (C-6 ), 30.20 (7 C-5 ), 29.88, 29.66, 29.50, 29.18 (7 C-2 ), H-6a,b ), 3.66 (dd, 4H, J
8.43 (NHCOOC(CH ) ), 26.47, 26.24, 26,21, 26.18, 25.70, (m, overlapped, H-6 ), 3.55 (m, overlapped, H-1 ), 3.54 (m,
5.29 ppm. HRMS (ESI): m/z calcd for C184H274N O [M] : overlapped, H-3 ), 3.52 (m, overlapped, 4 H-1 b), 3.46 (m, over-
a
), 3.86 (m, overlapped, 4 H-6
0
00
I
II
b
II,6bII 5.2 Hz, J6aII,6bII 11.9 Hz, 4 H-6
), 3.58
5
0
0
3
3
a
b
+
I
00
22
83
I
I
119.7896; found: 4119.7900; anal. calcd for C184H274N O : C, lapped, H-4 ), 3.40 (m, overlapped, H-5 ), 3.39 (m, overlapped, H-
22
83
0
II
3.61; H, 6.70; N, 7.48; found: C, 53.84; H, 6.81; N, 7.37.
6 ), 3.35 (m, overlapped, 4 H-3 ), 3.29–3.24 (m, overlapped, 9H, 4
b
0 0 0 0
II
II
I
II
6
-Aminohexyl 2,3,4,2 ,3 ,4 ,6 -hepta-O-[{1-(b-D-glucopyranosy- H-4 , 4 H-5 , H-2 ), 3.17 (4 t, overlapped, J 8.5 Hz, 4H, 4 H-2 ),
0
0
00
loxyhexyl)-1,2,3-triazol-4-yl}methyl]-b-gentiobioside (24). TFA 1.89 (m, 8H, 4 H-5 a,b), 1.65–1.55 (m, overlapped, 12H, 4 H-2 a,b),
ꢀ
0
0
(
0
300 mL) was added drop wise at 0 C to a solution of 23 (150 mg, 1.46–1.37 (m, overlapped, 12H, 4 H-3 a,b), 1.31 (m, overlapped,
00
13
.036 mmol) in dry DCM (3.0 mL) and the mixture was stirred at 8H, 4 H-4 a,b) ppm; C NMR (MeOD, 150 MHz): d 190.03 (d, C]
room temperature under inert atmosphere for 4 h, when TLC O), 184.82 (d, C]O), 177.80 (d, NHC]COCH ), 174.49 (d,
3
(
8 : 1 CHCl –MeOH) showed complete disappearance of 23. NHC]COCH ), 146.29, 146.20, 145.87, 145.79 (4 triazole C-4),
3
3
I
Aer concentration, the residue (145 mg, 0.036 mmol) was 125.38, 125.29, 125.22, 125.17 (4 triazole C-5), 102.61 (C-1 ),
deacetylated (Zempl ´e n condition) and solid-phase extraction 102.35 (C-1 ), 84.88 (C-3 ), 82.97 (C-2 ), 78.48 (C-4 ), 78.12 (C-3 ),
II
I
I
I
II
II
I
II
II
0
(C18 5 g Waters SepPack cartridge, 1 : 2 / 4 : 1 MeOH/water) 77.91 (C-5 ), 75.62 (C-5 ), 75.13 (C-2 ), 71.68 (C-4 ), 70.79 (C-1 ),
00
I
afforded 24 (72 mg, 70%) as white foam, aer freeze drying. R
0
8
triazole), 5.17 (m, 2H, OCH -triazole), 5.07–4.75 (m, 12H, 6 27.22 (C-4 ), 27.08, 26.77, 26.44 (C-3 ) ppm. HRMS (ESI): m/z
OCH -triazole), 4.76 (m, overlapped, H-1 ), 4.69 (d, 1H, J
f
70.57 (C-1 ), 70.07 (C-6 ), 67.08, 66.34, 65.32 (overlapped, 4 OCH -
2
1
II
.3 (C18, 1 : 1 MeOH–water); H NMR (D
2
O, 600 MHz): d 8.24, triazole), 62.79 (C-6 ), 61.19, 61.07 (two signals, OCH
.21, 8.20, 8.18, 8.15, 8.13, 8.12 (7 s, overlapped, 7H, 7 H- 6 ), 45.58–45.28 (d, C-6 ), 31.39, 31.21 (C-5 ), 30.60, 30.50 (C-2 ),
3
), 51.30 (C-
0
0
0
00
00
0
0
00
2
II
+
1I,2I
calcd for C H N O [M + H] : 1762.8738; found: 1762.8719.
2
77 128 13 33
I
III
8
7
.0 Hz, H-1 ), 4.63 (m, overlapped, 7 H-1 ), 4.60 (m, overlapped,
Conjugation of 25 to BSA. BSA (18.86 mg, 0.00028 mmol) and
), 4.10–4.03 methyl squarate derivative 25 (3.00 mg, 0.0017 mmol) were
00
I
a
III
H-6 a,b), 4.33 (m, 1H, H-6
), 4.13 (m, 7H, 7 H-6
), 3.99 (m, overlapped, H-6
), 3.95 (m, overlapped, 7 H-6
), 3.93 (m, overlapped, H-6
a
00
II
(m, 7H, 7 H-1
a
a
), 3.97 (m, over- weighed into a 1 mL V-shaped reaction vessel and 425 mL of
), 3.94 (m, over- 0.5 M pH 9 borate buffer was added (to form ꢁ4.0 mM solution
0
III
lapped, H-1
lapped, H-6
a
I
b
II
b
b
), 3.89 (m, overlapped, with respect to cluster 25, cluster–BSA 6 : 1). The mixture was
I
II
00
H-3 ), 3.85 (m, overlapped, H-3 ), 3.83 (m, overlapped, 7 H-1
b
,
gently stirred at room temperature for 48 h, when SELDI-TOF
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Paper
showed the ratio of hapten–BSA to be 4.1 : 1 (conjugation effi-
ciency, 68%). The mixture was transferred into an Amicon Ultra
References
(
0.5 mL, 30 K cutoff) centrifuge tube and dialyzed (centrifuga-
1 A. Varki, Glycobiology, 1993, 3, 97–130.
ꢀ
tion at 4 C, 7 min, 8 times) against 10 mM aqueous (NH ) CO .
2 V. Verez-Bencomo, V. Fern ´a ndez-Santana, E. Hardy,
M. E. Toledo, M. C. Rodrıguez, L. Heynngnezz,
A. Rodriguez, A. Baly, L. Herrera, M. Izquierdo, A. Villar,
Y. Vald ´e s, K. Cosme, M. L. Deler, M. Montane, E. Garcia,
A. Ramos, A. Aguilar, E. Medina, G. Torano, I. Sosa,
I. Hernandez, R. Martınez, A. Muzachio, A. Carmenates,
L. Costa, F. Cardoso, C. Campa, M. Diaz and R. Roy,
Science, 2004, 305, 522–525.
4
2
3
Aer lyophilization, 19.87 mg (94%, based on BSA) of conjugate
6 was obtained as white solid.
-(2-Methoxycyclobutene-3,4-dione)aminohexyl 2,3,4,2 ,3 ,4 ,6 -
2
0
0
0
0
6
hepta-O-[{1-(b-D-glucopyranosyloxyhexyl)-1,2,3-triazol-4-yl}methyl]-
b-gentiobioside (27). 3,4-Dimethoxy-3-cyclobutene-1,2-dione
(
13 mg, 0.090 mmol) was added to the solution of 24 (25 mg,
.009 mmol) in 2.2 mL of pH 7 phosphate buffer (0.5 M) and
0
stirred at room temperature for 18 h. The mixture was concen-
3 K. E. Stein, Int. J. Tech. Assess. Health Care, 1994, 10, 167–176.
4 Y. C. Lee, R. R. Townsend, M. R. Hardy, J. Lonngren,
J. Arnarp, M. Haraldsson and H. Lonn, J. Biol. Chem., 1983,
258, 199–202.
5 P. Kov ´a ˇc , in Protein-Carbohydrate Interactions in Infectious
Diseases, ed. C. Bewley, Royal Society of Chemistry,
London, 2006, pp. 175–220.
6 M. D. Meeks, R. Saksena, X. Ma, T. K. Wade, R. K. Taylor,
P. Kov ´a ˇc and W. F. Wade, Infect. Immun., 2004, 72, 4090–
4101.
7 R. Saksena, X. Ma, T. K. Wade, P. Kov ´a ˇc and W. F. Wade,
FEMS Immunol. Med. Microbiol., 2006, 46, 116–128.
8 A. Chernyak, S. Kondo, T. K. Wade, M. D. Meeks, P. M. Alzari,
J. M. Fournier, R. K. Taylor, P. Kov ´a ˇc and W. F. Wade, J. Infect.
Dis., 2002, 185, 950–962.
trated and the residue was chromatographed (1 : 1 EtOAc–
1
MeOH) to yield 27 (13 mg, 50%); R
f
0.2 (1 : 1 EtOAc–MeOH). H
NMR (MeOH, 600 MHz): d 8.07, 8.05, 8.04, 8.03, 7.99 (s, 7H, H-
triazole), 5.02–4.79 (m, overlapped, 10H, 5 OCH -triazole), 4.69–
2
II
4
4
2
.61 (m, overlapped, 4H, 2 OCH -triazole), 4.47 (m, 1H, H-1 ),
.40–4.34 (m, overlapped, 7 H-6 , OCH ), 4.39 (m, overlapped,
H-1 ), 4.24 (7 d, overlapped, 7H, 7 H-1 ), 4.07 (m, 1H, H-6 ),
.90–3.84 (m, overlapped, 7 H-6 , 7 H-1 ), 3.81 (m, overlapped,
0
0
a,b
3
I
III
I
a
III
00
3
H-1
5
3
a
a
0
II
a
), 3.73 (m, 2H, H-6a,b ), 3.66 (7 dd, overlapped, 2H, J
5
III,6aIII
I
III
b
.0 Hz, J6aIII,6bIII 12.0 Hz, 7 H-6
), 3.63 (m, overlapped, H-6
),
b
0
I
0
II
00
I
.60–3.45 (m, overlapped, H-3 , H-6
a
, H-3 , 7 H-1
b
, H-5 , H-1 ,
b
II
I
II
H-4 ), 3.45–3.38 (m, overlapped, H-4 , H-5 ), 3.37 (m, overlapped,
0
III
I
H-6
.30 (m, overlapped, H-2 ), 3.28 (m, overlapped, 7 H-4 ), 3.26 (m,
b
), 3.35 (m, overlapped, 7 H-3 ), 3.32 (m, overlapped, H-2 ),
II
III
3
III
III
overlapped, 7 H-5 ), 3.17 (7 t, overlapped, 7 H-2 ), 1.89 (m, 14H,
9 W. E. Dick Jr and M. Beurret, in Conjugate Vaccines, ed. J. M.
Cruse and R. E. Lewis Jr, Krager, Basel, 1989, pp. 48–114.
00
H-5 ), 1.63–1.53 (m, 18H, 7 H-2 ), 1.46–1.38 (m, 14H, 7 H-
a,b
00
7
3
a,b
0
0
00
13
a,b), 1.35–1.27 (m, 18H, 7 H-4 a,b) ppm; C NMR (MeOH, 150 10 J. Zhang and P. Kov ´a ˇc , Carbohydr. Res., 1999, 321, 157–167.
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1
(
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+
) ppm. HRMS (ESI): m/z calcd for C128
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212
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8
SELDI-TOF showed the ratio of hapten–BSA to be 3.7 : 1
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1 M. L. Gening, D. V. Titov, S. Cecioni, A. Audfray, A. G. Gerbst,
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This research was supported by the Intramural Research
Program of the National Institutes of Health (NIH) and National
Institute of Diabetes and Digestive and Kidney Diseases.
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