Synthesis of multivalent Streptococcus suis adhesion inhibitors by enzymatic cleavage of polygalacturonic acid and 'click' conjugation

Article abstract of DOI:10.1039/b800283e

A galabiose disaccharide building block was synthesized by an efficient pectinase cleavage of polygalacturonic acid and subsequent chemical functional group transformations. Besides the disaccharide, the corresponding trisaccharide was also obtained and modified. The compounds were subsequently conjugated to dendrimers with up to eight end groups using 'click' chemistry. The compounds were evaluated as inhibitors of adhesion of the pathogen Streptococcus suis in a hemagglutination assay and strong inhibition was observed for the tetra- and octavalent galabiose compound with MIC values in the low nanomolar range. The corresponding octavalent trisaccharide was a ca. 20-fold weaker inhibitor. The Royal Society of Chemistry.

Full text of DOI:10.1039/b800283e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of multivalent Streptococcus suis adhesion inhibitors by enzymatic
cleavage of polygalacturonic acid and ‘click’ conjugation
Hilbert M. Branderhorst,a Raymond Kooij,a Annika Salminen,b Lieneke H. Jongeneel,a
Christopher J. Arnusch,a Rob M. J. Liskamp,a Jukka Finneb and Roland J. Pieters*a
Received 8th January 2008, Accepted 11th February 2008
First published as an Advance Article on the web 29th February 2008
DOI: 10.1039/b800283e
A galabiose disaccharide building block was synthesized by an efficient pectinase cleavage of
polygalacturonic acid and subsequent chemical functional group transformations. Besides the
disaccharide, the corresponding trisaccharide was also obtained and modified. The compounds were
subsequently conjugated to dendrimers with up to eight end groups using ‘click’ chemistry. The
compounds were evaluated as inhibitors of adhesion of the pathogen Streptococcus suis in a
hemagglutination assay and strong inhibition was observed for the tetra- and octavalent galabiose
compound with MIC values in the low nanomolar range. The corresponding octavalent trisaccharide
was a ca. 20-fold weaker inhibitor.
cleavage of polygalacturonic acid was previously reported.13 The
Introduction
present work represents an optimization and an extension de-
The preparation of carbohydrates and their conjugates has become
scribing the discovery of a widely available highly effective enzyme
an important field for the study of biological processes and the
development of therapeutic lead structures.1 Many methods for
selective protection schemes and for the stereoselective introduc-
and easy conjugation procedures. Furthermore, besides the desired
disaccharide also the a(1,4) linked galactopyranosyl trisaccharide
was obtained which would require an even lengthier multistep
tion of glycosidic bonds have been developed over the years.2
synthetic route to prepare than the galabiose disaccharide.
While a synthetic strategy is often the most desired preparation
method, a biocatalytic degradation of readily available natural
carbohydrates or carbohydrate polymers can be advantageous. We
Results and discussion
report here on a synthetic route to a galabiose (Gala1–4Gal) and
its subsequent ‘click chemistry’ conjugation to dendrimers. A route
was used involving enzymatic degradation of polygalacturonic
acid and subsequent chemical modification steps. In our research
program on bacterial anti-adhesion compounds,3,4 the galabiose
The procedure started with the enzymatic cleavage of commer-
cially available pectin derived polygalacturonic acid, a polymer
consisting of a(1,4)linked galacturonic acid moieties. The polymer
was treated with the pectinase from Rhizopus oryzae and sepa-
rately also with the pectinase from Aspergillus niger in sodium
derivative 1 (Chart 1a) became of interest as its azido function
allows convenient conjugation to dendrimers using our microwave
heated “click” protocol.5 The galabiose sequence shows strong
binding to the bacterial Gram-positive pathogen Streptococcus
suis6 that can cause meningitis, septicemia, and pneumonia in
pigs and also meningitis in humans.7,8 Multivalent presentation of
the disaccharide sequence on dendritic scaffolds greatly enhances
their binding potency.4b,9 Other important biological targets that
bind to the galabiose epitope are the uropathogenic E. coli via its
PapG adhesin,4d,10 the bacterium Pseudomonas aeruginosa via its
PA-IL lectin11 and the Shiga like Toxin, a toxin of the AB5 family
produced by E. coli.12 The triazole linkage between the dendritic
scaffold and the galabiose that is described here is very similar to
the previously used4b,d amide connection. (Chart 1b).
acetate buffer at pH 4.5 (Scheme 1). Both enzymes showed the
expected cleavage activity but in the case of the Rhizopus oryzae
pectinase the relative amount of the desired disaccharide 3 versus
monosaccharide 2 and trisaccharide 4 was much higher and
was estimated by TLC to be ca. 2:2:1 (for 3:2:4) while for the
Aspergillus niger pectinase this ratio was ca. 1:2:2. For this reason
the pectinase from Rhizopus oryzae was chosen for further use.
Optimal amounts of the disaccharide were obtained when the
reaction appeared to have finished and no further conversion
was taking place. Initial attempts were made to separate the
obtained mixture of mono-, di and trisaccharides by ion exchange
chromatography using a formic acid gradient,13a however on a
10 gram scale this became impractical. Better and faster separation
was achieved by standard silica gel chromatography converting
the free sugars 2–4 to their methyl acetals (a/b ratio ≈ 3/1)
and the methyl esters 5–7, using the crude mixture and Dowex-
H+ catalysis in MeOH. After separation, the disaccharide 6 was
reduced by NaBH4 and its hydroxyls were subsequently acetylated
(Ac2O, pyridine). The anomeric methoxy group was replaced
with an acetate by treatment with H2SO4 in acetic anhydride
to give 9. The disaccharide 9 was converted to glycosyl donor
10 by selective anomeric deacetylation using hydrazine acetate
followed by standard introduction of the trichloroacetimidate
For the synthesis of the galabiose unit a fully synthetic strategy
was previously applied.4b While the route did produce the target
product, its many steps make it less ideal for large scale synthesis.
An alternative route towards galabiose based on an enzymatic
aDepartment of Medicinal Chemistry and Chemical Biology, Utrecht Insti-
tute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508
TB Utrecht, The Netherlands. E-mail: R.J.Pieters@uu.nl
bDepartment of Medical Biochemistry and Molecular Biology, University of
Turku, FI-20520 Turku, Finland
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Chart 1 a) Desired galabiose building block for conjugation via “click” chemistry; b) top linkage to dendritic structures compared to previously used
linkage (ref. 4b,d).
Scheme 1 Reagents and conditions (a) pectinase, pH = 4.5, 40 ◦C; (b) MeOH, Dowex-H+, then separate on silica, 28% for 6, 12% for 7; (c) i. NaBH4,
MeOH, ii. Ac2O, pyridine, 75% (d) H2SO4, Ac2O, quant.; (e) i. N2H4·AcOH, DMF, ii. Cl3CCN, DBU, CH2Cl2, 64% for 10, 42% for 14; (f) HO(CH2)3Br,
TMSOTf, 58% for 11, 55% for 15 (g) NaN3, DMF, quant. for 1, 94% for 16.
moiety. Coupling of the 3-bromopropanol spacer was achieved by
TMSOTf catalysis to give 11 which was subjected to a nucleophilic
substitution by NaN3 in DMF to give the desired target compound
1. The described procedure was also applied to the galacturonic
acid trisaccharide 7 to give 16 in comparable yields.
After optimizing the synthesis of the galabiose building blocks,
they were conjugated to the dendritic scaffolds. For this purpose
the so-called “click” reaction, the copper(I) catalyzed [3 + 2]
cycloaddition between azides and alkynes,14 was used with our
previously reported conditions.5 The dendrimers 17a–20a15 were
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Scheme 2 Glycodendrimer synthesis. Reagents and conditions: (a) i. TFA/CH2Cl2; ii. 4-pentynoic acid, BOP, DiPEA, yields: 70–95%; (b) i. 1, CuSO4,
sodium ascorbate, DMF, 80 ◦C, 20 min., yields: 66–84%, ii: NaOMe, MeOH, 35–77%; (c) i. 16, CuSO4, sodium ascorbate, DMF, 80 ◦C, 20 min., 79%, ii.
NaOMe, MeOH, 57%.
Table 1 Inhibition of the S. suis D282 mediated hemagglutination of
subjected to Boc cleavage, followed by BOP mediated coupling
of 4-pentynoic acid (Scheme 2). Subsequent conjugation of 17–
20b was performed with the galabiose building block 1 using
Cu(I) catalysis and microwave irradiation at 80 ◦C. Tetravalent
dendrimer 19b was also conjugated with the trisaccharide 16 to
give 19d. The obtained dendrimers were deacetylated with NaOMe
conditions to give 17c–20c and 19d.
human erythrocytes by galabiose derivatives
Compound
Valency
MIC (nM)
Rel. pot.a (per sugar)b
17c
18c
19c
19e
20c
19d
1
2
4
4
8
4
400
8
2.4
2.5
3.9
48
1 (1)
50 (25)
167 (42)
160 (40)
100 (13)
Hemagglutination assay
a Relative potency = IC50(monovalent)/IC50(multivalent ligand). b Relative
potency per sugar = relative potency/valency.
The galabiose dendrimers were tested for their interactions with
the S. suis adhesin by evaluating their inhibitory capacity of
hemagglutination inhibition as previously described.4b,9 The min-
imal inhibitory concentration (MIC) values were determined and
are depicted in Table 1. From the results a clear multivalency effect
was observed. The results were comparable with the previously
reported data, with low nanomolar inhibition for the multivalent
galabiose conjugates.4b The tetravalent galabiose dendrimer 19c
showed practically the same MIC value as the tetravalent reference
compound 19e with the amide linkage as it was retested in the
present study, indicating no influence of the structure of the linking
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moiety. The tested tetravalent galactose trisaccharide 19d, showed
significantly reduced inhibitory potency in comparison to the
tetravalent galabiose compound 19c, i.e. 48 nM vs. 2.4 nM.
were performed on a Varian G-300 spectrometer. Chemical shifts
are given in ppm relative to TMS (1H, 0.00 ppm), CD3OD (1H, 3.31
ppm), CDCl3 (13C, 77.0 ppm), D2O (1H, 4.80 ppm), CD3OD (13C,
49.2 ppm) or acetone (215.94 ppm in D2O). Exact masses were
measured by nano electro spray time-of-flight mass spectrometry
on a Micromass LCToF mass spectrometer at a resolution of
5000 fwhm. Gold-coated capillaries were loaded with 1 lL of
sample (concentration 20 lM) dissolved in a 1:1 (v/v) mixture of
CH3CN/H2O with 0.1% formic acid. NaI or poly(ethylene glycol)
(PEG) was added as internal standard. The capillary voltage was
set between 1100 and 1350 V, and the cone voltage was set at
30 V. Matrix Assisted Laser Desorption Ionisation Time of Flight
(MALDI ToF) MS was recorded on a Shimadzu Axima-CFR
with a-cyano-4-hydroxycinnamic acid or sinapic acid as a matrix.
Insulin and adrenocorticotropin fragment 18–39 (Acth) were used
for calibration.
Conclusion
The preparation of derivatives of galabiose and the corresponding
trisaccharide were described involving a biocatalytic polysaccha-
ride degradation step followed by chemical modification steps. The
key step in this procedure is the efficient enzymatic cleavage of
the cheap polygalacturonic acid by a readily available pectinase
enzyme. Methylation of the crude cleavage mixture allowed
efficient separation by standard silica gel chromatography. Both
disaccharide and trisaccharide were thus obtained and subse-
quently subjected to the range of chemical modifications leading
to the galabiose building block and a galactose trisaccharide.
Compared to previous reports, both the purification and ligation
were optimized and this route serves as a good alternative to
the fully chemical synthesis approach and it does not require
expensive chemical reagents. Besides the desired disaccharide also
the corresponding trisaccharide was obtained which would require
many chemical steps to synthesize. Ligation of the carbohydrate
building blocks to a series of alkyne functionalized dendrimers
was achieved by “click” chemistry and was found to give excel-
lent coupling yields. The newly prepared galabiose dendrimers
showed comparable S. suis inhibition capacities as reflected in
the MIC values. The tetravalent trisaccharide was found not to
be as effective an inhibitor as the galabiose disaccharide. This
revised partial enzymatic preparation method is attractive for the
preparation of larger quantities of anti-adhesion compounds with
relatively low costs and efforts. It is thus well-suited to further
pursue the anti-adhesion strategy with multivalent carbohydrates,3
as an alternative intervention method against microbial pathogens,
using animal models.
Enzymatic cleavage of polygalacturonic acid. A solution of
polygalacturonic acid (10.0 g, ≈ 57 mmol monosaccharide) in
aqueous NaOAc (0.41 M, 500 mL) was adjusted to pH = 4.5 with
NaOH (4M). Pectinase (Rhizopus oryzae, 250 mg or Aspergillus
niger, 250 mg) was added to the polymer suspension and the
resulting mixture was allowed to stir for 24 h at 40 ◦C. TLC
(formic acid/n-BuOH/H2O, 6/4/1) showed the formation of the
mono-, di- and tri-galacturonic acid (Rf values resp. 0.35, 0.28
and 0.23). The reaction was terminated by stirring for 5 min at
100 ◦C. The reaction mixture was concentrated and co-evaporated
with MeOH to dryness. The crude mixture was suspended in
MeOH (250 mL) and Dowex-H+ (30 g) was added. The mixture
was refluxed for 18 h, the Dowex resin was filtered off and
the crude mixture was concentrated. Silica gel chromatography
(EtOAc/MeOH/H2O, 7/2/1 → 3/2/1) was used to separate the
mono- di- and trigalacturonic acid methyl esters 5, 6 and 7.
Methyl [methyl(methyl a-D-galactopyranosyluronate)-(1→4)-
a/b-D-galactopyranoside] uronate (6). Disaccharide 6 was ob-
tained as white foam (3.26 g, 28%). 1H NMR (300 MHz, CD3OD):
d = 5.11 (1H, d, J = 1.8 Hz), 4.51 (0.66H, d, H-1 a, J1,2 = 0.9 Hz),
4.37 (1H, d, J = 3.0 Hz), 4.20 (1H, m), 3.90 (1H, dd), 3.81 and
3.75 (2 × s, 2 × 3H, C(O)OCH3), 3.59 (0.6H, s, OCH3 b) and
3.42 (2.4H, s, OCH3 a). 13C NMR (CD3OD, 75.5 MHz): d = 171.9
and 170.9 (C-6, C-6ꢀ), 105.9 (C-1 b), 102.4 and 101.9 (C-1ꢀ, C-1
a), 80.5, 79.6, 74.7, 73.5, 73.0, 72.0, 71.3, 70.9, 70.0 and 69.9 (C-2,
C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 58.0 and 56.6 (OCH3 a/b),
53.2 and 52.7 (C(O)OCH3). HRMS for C15H24O13 (M, 412.1217):
found [M + Na]+ 435.0811, calcd. 435.1115.
Experimental section
General Remarks
Unless stated otherwise, chemicals were obtained from commer-
cial sources and used without further purification. All solvents
were purchased from Biosolve (Valkenswaard, The Netherlands)
˚
and were stored on molecular sieves (4A). Acetic anhydride
and pyridine were purchased from Acros (Geel, Belgium). Poly-
galacturonic acid and both pectinases from Aspergillus niger and
Rhizopus oryzae were obtained from Fluka. TLC was performed
on Merck precoated Silica 60 plates. Spots were visualized by
dipping in 10% H2SO4 in MeOH followed by heating. Microwave
reactions were carried out in a dedicated microwave oven, i.e.
the Biotage Initiator. The microwave power was limited by
temperature control once the desired temperature was reached.
A sealed vessel of 2–5 mL was used. Preparative HPLC runs
were performed on an Applied Biosystems workstation. Elution
was effected using a gradient of 5% MeCN and 0.1% TFA in
H2O to 5% H2O and 0.1% TFA in MeCN. Analytical HPLC
runs were performed on a Shimadzu automated HPLC system
equipped with an evaporative light scattering detector (PL-ELS
1000, Polymer Laboratories) and a UV/VIS detector operating at
220 and 254 nm. 1H NMR (300 MHz) and 13C NMR (75.5 MHz)
Methyl [methyl(methyl a-D-galactopyranosyluronate)-(1→4)-
(methyl-a-D-galactopyranosyluronate)-(1→4)-a/b-D-galacto-
pyranoside] uronate (7). Trisaccharide 7 was obtained as an
1
amorphous solid (1.35 g, 12%). H NMR (300 MHz, CD3OD):
d = 5.15 (1H, d, J = 1.8 Hz), 5.10 (1H, dd, J = 1.2 Hz), 4.51
(0.70H, s, H-1 a), 4.39–4.36 (2H, m), 4.20–4.18 (1H, m), 3.90 (1H,
t), 3.87 (1H, t), 3.80, 3.78 and 3.75 (3 × 3H, 3 × s, C(O)OCH3).
3.51 (1.0H, s, OCH3 b) and 3.42 (2.5H, s, OCH3 a). 13C NMR
(CD3OD, 75.5 MHz): d= 171.9, 171.3 and 170.9 (C-6, C-6ꢀ, C-6ꢀꢀ),
105.9 (C-1 b), 102.2 and 102.0 (C-1 a, C-1ꢀ, C-1ꢀꢀ), 80.6, 80.3, 79.7,
74.7, 72.9, 72.2, 72.0, 71.3, 70.9 and 70.1 (C-2, C-2ꢀ, C-2ꢀꢀ, C-3,
C-3ꢀ, C-3ꢀꢀ, C-4, C-4ꢀ, C-4ꢀꢀ, C-5, C-5ꢀ, C-5ꢀꢀ), 58.0 and 56.6 (OCH3
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a/b), 53.1, 53.0 and 52.7 (C(O)OCH3). HRMS for C22H34O19 (M,
were dissolved in dry CH2Cl2 (50 mL) and the solution was
cooled to 0 ◦C. DBU (1,8-diazabicyclo[5.4.0]undec-7-ene, 301 lL,
2.0 mmol) was added and the solution was stirred at 0 ◦C for 1 h
followed by 20 h at r.t. The reaction mixture was concentrated and
product 10 was isolated by silica gel chromatography (hex/EtOAc,
1/1) as a slightly yellow foam (3.30 g, 64%). 1H NMR (300 MHz,
602.1694): found [M + Na]+ 625.1316, calcd. 625.1592.
Methyl (2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl)-(1→4)-
2,3,6-tri-O-acetyl-a/b-D-galactopyranoside (8). To a solution of
digalacturonic acid methyl ester 6 (3.26 g) in MeOH (150 mL)
was added NaBH4 (4.0 g). The solution was stirred for 78 h.
After neutralization with Dowex-H+ the mixture was filtered and
concentrated to dryness. Crude disaccharide was suspended in
pyridine (100 mL) and Ac2O (50 mL) was added. The reaction
mixture was stirred for 18 h and concentrated in vacuo at
60 ◦C. Crude product was taken up in EtOAc (250 mL) and
washed with aqueous NaOH (1M, 100 mL) aqueous HCl (1M,
100 mL), and brine (100 mL). The organic layer was dried over
Na2SO4, filtered and concentrated. Silica gel chromatography
(hexane/EtOAc, 1/1 → 1/2) was used to obtain pure disaccharide
6 as a white foam (3.85 g). 1H NMR (300 MHz, CDCl3): d = 5.56
CDCl3): d = 8.69 (1H, s, OCNHCCl3), 6.61 (1H, d, H-1a, J1,2
=
3.6 Hz), 5.57 (1H, d, H-4ꢀ, J3 ,4 = 3.0 Hz), 5.42 (1H, dd, H-2, J1,2
=
ꢀ
ꢀ
3.6 Hz, J2,3 = 11.3 Hz), 5.37–5.28 (2H, m, H-3, H-3ꢀ), 5.24 (1H, dd,
H-2ꢀ, J1 ,2 = 3.6 Hz, J2 ,3 = 11.0 Hz), 5.03 (1H, m, H-1ꢀ, J1 ,2 = 3.6
Hz), 4.53 (1H, t, H-5, J5,6 = 6.0 Hz), 4.38–4.28 (3H, m, H-4, 2 × H-
6ꢀ), 4.16–4.11 (3H, m, H-5ꢀ, 2 × H-6), 2.12, 2.11, 2.04 and 2.00 (7 ×
3H, 4 × s, C(O)CH3). 13C NMR (CDCl3, 75.5 MHz): d = 170.4,
170.2, 170.0, 169.8 and 168.6 (C(O)CH3), 160.7 (OC(NH)CCl3),
98.9 and 93.5 (C-1, C-1ꢀ), 90.8 (OCNHCCl3), 70.5, 69.3, 68.1, 67.7,
67.2, 67.1 and 66.7 (C-2, C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 61.8
and 60.6 (C-6, C-6ꢀ), 20.9, 20.6 and 20.4 (C(O)CH3).
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(1H, d, H-4ꢀ, J3 ,4 = 3.0 Hz), 5.39 (1H, dd, H-3ꢀ, J2 ,3 = 11.7 Hz,
ꢀ
ꢀ
ꢀ ꢀ
J3 ,4 = 3.3 Hz), 5.25–5.18 (3H, m, H-3, H-2, H-2ꢀ), 4.98 (2H, m,
H-1, H-1ꢀ), 4.54 (1H, t, H-5, J5,6 = 6.6 Hz), 4.17–4.06 (6H, m, H-4,
H-5ꢀ, 2 × H-6, 2 × H-6ꢀ), 3.52 (0.6H, s, OCH3 b), 3.42 (2.4H, s,
OCH3 a), 2.14, 2.13, 2.09, 2.08, 2.07, 2.03 and 1.99 (7 × 3H, 7 × s,
C(O)CH3). 13C NMR (CDCl3, 75.5 MHz): d= 170.7, 170.4, 170.2,
170.1 169.9 and 169.9 (C(O)CH3), 101.7 (C-1 b), 99.0 (C-1ꢀ), 97.1
(C-1 a), 72.7, 71.8, 69.4, 68.5, 68.2, 67.7, 67.3 and 66.9 (C-2, C-2ꢀ,
C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 62.3, 61.9, 60.6 and 60.4 (C-6,
C-6ꢀ), 55.4 and 55.2 (OCH3 a/b), and 20.6 (C(O)CH3). HRMS
for C27H38O18 (M, 650.2058): found [M + Na]+ 673.1956, calcd.
672.9821.
3-Bromopropyl (2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-acetyl-b-D-galactopyranoside (11). A solu-
tion of 10 (1.22 g, 1.56 mmol) and 3-bromopropanol (0.68 mL,
7.8 mmol) in CH2Cl2 (30 mL) was stirred under N2 at 0 ◦C. TM-
SOTf (91 lL, 0.47 mmol) was added and the mixture was stirred
at 0 ◦C for 1 h. The mixture was warmed to r.t. and after 1 h
the reaction was quenched with Et3N and concentrated in vacuo.
Silica gel chromatography (hex/EtOAc, 3/2 → 1/1) afforded pure
11 (0.77 g, 65%). 1H NMR (300 MHz, CDCl3): d = 5.57 (1H, dd,
ꢀ
ꢀ
H-4ꢀ, J3 ,4 = 3.0 Hz, J4 ,5 = 1.1 Hz), 5.39 (1H, dd, H-3ꢀ, J2 ,3
=
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
11.0 Hz, J3 ,4 = 3.3 Hz), 5.22–5.14 (2H, m, H-2, H-2ꢀ), 4.93 (1H,
ꢀ
ꢀ
d, H-1ꢀ, J1 ,2 = 3.6 Hz), 4.82 (1H, dd, H-3, J2,3 = 10.7 Hz, J3,4
=
ꢀ
ꢀ
10.7 Hz), 4.48 (1H, d, H-1, J1,2 = 8.3 Hz), 4.54–4.43 (2H, m, H-5ꢀ,
Acetyl (2,3,4,6-tri-O-acetyl-a-D-galactopyranosyl)-(1→4)-2,3,
6-tri-O-acetyl-a/b-D-galactopyranoside (9). To a cooled solu-
tion of 8 (3.80 g, 5.84 mmol) in Ac2O (45 mL) was added
concentrated H2SO4 (150 lL). The solution was stirred at 0 ◦C for
2 h, diluted with CH2Cl2 (150 mL) and quenched with aqueous
NaHCO3 (5%, 10 mL). The reaction mixture was concentrated to
dryness at 60 ◦C. The residue was taken up in EtOAc (200 mL) and
washed with aqueous NaOH (1M, 75 mL) and brine (75 mL). The
organic phase was dried over Na2SO4, filtered and concentrated
to yield white foam 9 (3.83 g, 97%). 1H NMR (300 MHz, CDCl3):
H-6a), 4.20–4.10 (3H, H-6b and 2 × H-6ꢀ), 4.07 (1H, d, H-4, J3,4
=
2.8 Hz), 4.03–4.96 and 3.73–3.65 (each 1H, 2 × m, CH2OGal), 3.79
(1H, t, H-5, J5,6 = 5.0 Hz), 3.50 (2H, dd, CH2Br, J = 5.5 Hz, J = 7.4
Hz), 2.20–2.02 (2H, m, OCH2CH2CH2Br), 2.13, 2.11, 2.08, 2.07,
2.04 and 1.99 (7 × 3H, 7 × s, C(O)CH3). 13C NMR (75.5 MHz,
CDCl3): d= 170.5, 170.1, 169.8 and 169.2 (C(O)CH3), 101.4 (C-1),
99.4 (C-1ꢀ), 72.6, 71.9, 68.7, 68.5, 67.8, 67.3 and 67.0 (C-2, C-2ꢀ,
C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 67.1 (OCH2CH2CH2Br), 61.9 and
60.5 (C-6, C-6ꢀ), 32.3 (CH2Br), 30.3 (OCH2CH2CH2Br) and 20.6
(C(O)CH3). HRMS for C29H41BrO18 (M, 756.1476): found [M +
Na]+ 779.1374, calcd. 779.0682.
d = 6.37 (1H, d, H-1a, J1,2 = 3.6 Hz), 5.57 (1H, dd, H-4ꢀ, J3 ,4
=
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3.0 Hz, J4 ,5 = 3.3 Hz), 5.43–5.35 (2H, m, H-3 and H-3), 5.26–5.19
(2H, m, H-2ꢀ, H-2), 5.02 (H, d, H-1ꢀ, J1 ,2 = 3.6 Hz), 4.52 (1H, t,
H-5, J5,6 = 7.8 Hz), 4.38–4.05 (6H, m, H-4, H-5ꢀ, 2 × H-6ꢀ, 2 ×
H-6), 2.16, 2.14, 2.11, 2.11, 2.07, 2.03, 2.03 and 1.99 (8 × 3H, 8
× s, C(O)CH3). 13C NMR (CDCl3, 75.5 MHz): d = 170.5, 170.5,
170.3, 170.1, 169.8, 169.4, 169.0 and 168.8 (C(O)CH3), 99.1 and
89.8 (C-1 and C-1ꢀ), 70.3, 69.2, 68.4, 68.2, 67.7, 67.2, 67.1 and 66.1
(C-2, C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 61.5 and 60.5 (C-6,
C-6ꢀ), 20.9, 20.7 and 20.6 (C(O)CH3). HRMS for C28H38O19 (M,
678.2007): found [M + Na]+ 701.1905, calcd. 701.1530.
ꢀ
ꢀ
3-Azidopropyl (2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-acetyl-b-D-galactopyranoside (1). A solution
of 11 (1.21 g, 1.60 mmol) and NaN3 (0.52 g, 8 mmol) in dry
◦
DMF (20 mL) was stirred at 100 C for 20 h. The solution was
concentrated in vacuo at 60 ◦C, taken up in EtOAc (150 mL)
and washed with brine (75 mL). The organic phase was dried
(Na2SO4), filtered and concentrated. Product 1 was lyophilized
from MeCN/H2O and was obtained as white powder (1.09 g,
95%). 1H NMR (300 MHz, CDCl3): d = 5.50 (1H, dd, H-4ꢀ,
J3 ,4 = 3.0 Hz, J4 ,5 = 1.1 Hz), 5.32 (1H, dd, H-3ꢀ, J2 ,3 = 11.0 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
(2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl)-(1→4)-(2,3,6-
J3 ,4 = 3.3 Hz), 5.15–5.08 (2H, m, H-2, H-2ꢀ), 4.93 (1H, d, H-1ꢀ,
ꢀ
ꢀ
tri-O-acetyl-a-D-galactopyranosyl) trichloroacetimidate (10).
A
ꢀ
ꢀ
solution of 9 (4.50 g, 6.64 mmol) and N2H4·HOAc (672 mg,
J1 ,2 = 3.6 Hz), 4.75 (1H, dd, H-3, J2,3 = 10.7 Hz, J3,4 = 2.8
Hz), 4.42 (1H, d, H-1, J1,2 = 7.8 Hz), 3.91–3.88 and 3.57–3.54
(each 1H, 2 × m, CH2OGal), 3.79 (1H, t, H-5, J5,6 = 6.6 Hz),
3.34 (2H, t, CH2N3, J = 6.6 Hz), 2.06, 2.04, 2.01, 2.00, 1.99,
1.97 and 1.92 (7 × 3H, 7 × s, C(O)CH3) and 1.90–1.77 (2H, m,
OCH2CH2CH2N3). 13C NMR (75.5 MHz, CDCl3): d = 170.4,
7.29 mmol) in dry DMF (50 mL) was stirred at r.t. for 70 h. The
◦
mixture was concentrated in vacuo at 60 C, taken up in EtOAc
(100 mL) and washed twice with brine (50 mL). The organic phase
was dried over Na2SO4, filtered and concentrated. The white foam
(4.24 g, quantitative) and trichloroacetonitrile (2.0 mL, 20 mmol)
This journal is
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Org. Biomol. Chem., 2008, 6, 1425–1434 | 1429
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View Article Online
170.1, 169.8 and 169.1 (C(O)CH3), 101.1 (C-1), 99.4 (C-1ꢀ), 72.6,
71.9, 68.6, 67.8, 67.3 and 67.0 (C-2, C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ,
C-5, C-5ꢀ), 66.2 (OCH2CH2CH2N3), 61.9 and 60.4 (C-6, C-6ꢀ),
48.0 (OCH2), 28.9 (CH2N3) and 20.8 (C(O)CH3). HRMS for
C29H41N3O18 (M, 719.2385): found [M + Na]+ 742.2283, calcd.
742.1694.
and 20.6 (C(O)CH3). HRMS for C40H54O27 (M, 966.2852): found
[M + Na]+ 989.2119, calcd. 989.2750.
(2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl)-(1→4)-(2,3,6-
tri-O-acetyl-a,b-D-galactopyranosy)l-(1→4)-(2,3,6-tri-O-acetyl-
a-D-galactopyranosyl) trichloroacetimidate (14). A solution of
13 (2.50 g, 2.60 mmol) and N2H4.HOAc (262 mg, 2.85 mmol) in
dry DMF (20 mL) was stirred at r.t. for 70 h. The mixture was
concentrated in vacuo at 60 ◦C, taken up in EtOAc (150 mL) and
washed twice with brine (75 mL). The organic phase was dried
over Na2SO4, filtered and concentrated. The obtained white foam
(2.22 g, 93%) and trichloroacetonitrile (0.72 mL, 7.2 mmol) were
dissolved in dry CH2Cl2 (50 mL) and the solution was cooled
Methyl (2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl)-(1→4)-
(2,3,6-tetra-O-acetyl-a-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-
acetyl-a/b-D-galactopyranoside (12). To a solution of trigalac-
turonic acid methyl ester 7 (1.35 g) in MeOH (100 mL) was added
NaBH4 (2.0 g). The solution was stirred for 78 h. After neutraliza-
tion with Dowex-H+ the mixture was filtered and concentrated to
dryness. Crude trisaccharide was suspended in pyridine (50 mL)
and Ac2O (25 mL) was added. The reaction mixture was stirred for
18 h and concentrated in vacuo at 60 ◦C. Crude product was taken
up in EtOAc (250 mL) and washed with aqueous NaOH (1M,
100 mL) aqueous HCl (1M, 100 mL), and brine (100 mL). The
organic layer was dried over Na2SO4, filtered and concentrated.
Silica gel chromatography (hexane/EtOAc, 1/2 → 1/4) was used
to obtain pure trisaccharide 12 as a white foam (2.30 g). 1H NMR
◦
to 0 C under a N2. 1,8-Diazabicyclo[5.4.0]undec-7-ene (108 lL,
0.72 mmol) was added and the solution was stirred at 0 ◦C for 1 h
followed by 2 h at r.t.. The reaction mixture was concentrated and
product 14 was isolated by silica gel chromatography (hex/EtOAc,
2/3 → 1/3) as a white amorphous solid (1.17 g, 42%). 1H NMR
(300 MHz, CDCl3): d = 8.69 (1H, s, OCNHCCl3), 6.60 (1H, d,
H-1a, J1,2 = 3.3 Hz), 5.56 (1H, d, H-4ꢀꢀ, J3 = 2.8 Hz), 5.42–5.20
ꢀꢀ ꢀꢀ
,4
(6H, m, H-2, H-2ꢀ, H-2ꢀꢀ, H-3, H-3ꢀ, H-3ꢀꢀ), 5.00 and 4.99 (2 × 1H,
(300 MHz, CDCl3): d= 5.56 (1H, dd, H-4ꢀꢀ, J3 = 3.0 Hz, J4
=
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,4
,5
2 × d, H-1ꢀ, H-1ꢀꢀ, J1 ,2 = 3.0 Hz, J1 = 2.8 Hz), 4.53 (1H, t, H-5,
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
,2
1.4 Hz), 5.40 (1H, dd, H-3ꢀꢀ, J2 = 11.3 Hz, J3 = 3.3 Hz), 5.33
ꢀꢀ ꢀꢀ
,3
ꢀꢀ ꢀꢀ
,4
J5,6 = 7.2 Hz), 4.46–4.02 (10H, m, H-4, H-4ꢀ, H-5ꢀ, H-5ꢀꢀ, 2 × H-6,
2 × H-6ꢀ, 2 × H-6ꢀꢀ), 2.14, 2.12, 2.10, 2.09, 2.06, 2.05, 2.04, 2.04,
2.02 and 1.99 (10 × 3H, 10 × s, C(O)CH3). 13C NMR (CDCl3,
75.5 MHz): d = 170.4, 170.2, 170.1, 169.9 and 169.6 (C(O)CH3),
160.7 (OC(NH)CCl3), 99.3 and 99.1 (C-1ꢀ, C-1ꢀꢀ), 93.5 (C-1), 90.7
(OCNHCCl3), 70.6, 69.2, 68.9, 68.2, 67.7, 67.3, 66.9 and 66.7 (C-2,
C-2ꢀ, C-2ꢀꢀ, C-3, C-3ꢀ, C-3ꢀꢀ, C-4, C-4ꢀ, C-4ꢀꢀ, C-5, C-5ꢀ, C-5ꢀꢀ), 62.0,
61.2 and 60.3 (C-6, C-6ꢀ, C-6ꢀꢀ), 20.9 and 20.5 (C(O)CH3).
(1H, dd, H-3ꢀ, J2 ,3 = 11.3 Hz, J3 ,4 = 3.3 Hz), 5.25 (1H, t, H-2ꢀꢀ,
J = 2.8 Hz), 5.21–5.16 (3H, m, H-2, H-2ꢀ, H-3), 5.00–4.98 (2H,
m, H-1ꢀ, H-1ꢀꢀ), 4.95 (1H, d, H-1, J1,2 = 3.6 Hz), 4.54 (1H, t, H-5,
J5,6 = 6.6 Hz), 4.46–3.99 (10 H, m, H-4, H-4ꢀ, H-5ꢀ, H-5ꢀꢀ, 2 × H-6,
2 × H-6ꢀ, 2 × H-6ꢀꢀ), 3.51 (0.6H, s, OCH3 b), 3.41 (2.2H, s, OCH3
a), 2.14, 2.13, 2.10, 2.10, 2.09, 2.08, 2.07, 2.05, 2.04 and 1.99 (10 ×
3H, 10 × s, C(O)CH3). 13C NMR (CDCl3, 75.5 MHz): d = 170.5,
170.2, 170.1 and 170.0 (C(O)CH3), 101.8 (C-1 b), 99.2 and 99.1
(C-1ꢀ, C-1ꢀꢀ), 97.1 (C-1 a), 72.5, 72.0, 69.3, 69.2, 68.8, 68.5, 68.3,
67.8, 67.7, 67.3, and 66.9 (C-2, C-2ꢀ, C-2ꢀꢀ, C-3, C-3ꢀ, C-3ꢀꢀ, C-4,
C-4ꢀ, C-4ꢀꢀ, C-5, C-5ꢀ, C-5ꢀꢀ), 62.5, 62.1, 61.1 and 60.3 (C-6, C-6ꢀ,
C-6ꢀꢀ), 56.6 and 55.3 (OCH3 a/b), 20.9, 20.7 and 20.6 (C(O)CH3).
HRMS for C39H54O26 (M, 938.2903): found [M + Na]+ 961.2786,
calcd. 961.2801.
ꢀ
ꢀ
ꢀ ꢀ
3-Bromopropyl (2,3,4,6-tri-O-acetyl-a-D-galactopyranosyl)-
(1→4)-(2,3,6-tri-O-acetyl-a-D-galactopyranosyl)-(1→4)-2,3,6-
tri-O-acetyl-b-D-galactopyranoside (15).
A solution of 14
(1.17 g, 1.09 mmol) and 3-bromopropanol (0.48 mL, 5.5 mmol) in
◦
CH2Cl2 (25 mL) was stirred under N2 at 0 C. TMSOTf (63 lL,
0.33 mmol) was added and the mixture was stirred at 0 ◦C for
1 h. The mixture was warmed to r.t. and after 1 h the reaction
was quenched with Et3N and concentrated in vacuo. Silica gel
chromatography (hex/EtOAc, 2/3 → 1/3) afforded pure 15 as
Acetyl (2,3,4,6-tri-O-acetyl-a-D-galactopyranosyl)-(1→4)-(2,3,
6-tri-O-acetyl-a-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-
a/b-D-galactopyranoside (13). To a cooled solution of 12 (2.25 g,
2.40 mmol) in Ac2O (20 mL) was added concentrated H2SO4
(60 lL). The solution was stirred at 0 ◦C for 2 hours, diluted
with CH2Cl2 (100 mL) and quenched with aqueous NaHCO3
(5%, 5 mL). The reaction mixture was concentrated to dryness
at 60 ◦C. Crude product was taken up in EtOAc (200 mL) and
washed with NaOH (1M, 75 mL) and brine (75 mL). The organic
layer was dried over Na2SO4, filtered and concentrated. After
lyophilization 13 was obtained as white powder (2.08 g, 90%)
1H NMR (300 MHz, CDCl3): d = 6.37 (1H, d, H-1a, J1,2 = 3.6
ꢀꢀ ꢀꢀ
1
white foam (607 mg, 55%). H NMR (300 MHz, CDCl3): d =
5.56 (1H, dd, H-4ꢀꢀ, J3
= 3.3 Hz, J4
= 1.1 Hz), 5.39 (1H,
ꢀꢀ ꢀꢀ
,4
ꢀꢀ ꢀꢀ
,5
dd, H-3ꢀꢀ, J2
= 11.0 Hz, J3
= 3.3 Hz), 5.33–5.21 (3H, m,
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,3
,4
H-2ꢀ, H-2ꢀꢀ, H-3ꢀ), 5.00 and 4.98 (each 1H, 2 × d, H-1ꢀ and H-1ꢀꢀ,
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
J1 ,2 = 3.6 Hz, J1 = 3.3 Hz), 4.86 (1H, dd, H-3, J2,3 = 10.7 Hz,
,2
J3,4 = 2.8 Hz), 4.49 (1H, d, H-1, J1,2 = 7.7 Hz), 4.54–4.05 (9H,
m, H-4, H-5ꢀ, H-5ꢀꢀ, 2 × H-6, 2 × H-6ꢀ, 2 × H-6ꢀꢀ), 4.31 (1H, d,
H-4ꢀ, J3 ,4 = 1.9 Hz), 4.02–3.96 and 3.75–3.66 (2 × 1H, 2 × m,
CH2OGal), 3.82 (1H, t, H-5, J5,6 = 6.0 Hz), 3.50 (2H, dd, CH2Br,
J = 5.5 Hz, J = 6.6 Hz), 2.14, 2.11, 2.11, 2.10, 2.09, 2.07, 2.07,
2.06, 2.03 and 1.99 (10 × 3H, 10 × s, C(O)CH3) and 2.20–2.05 (2H,
m, OCH2CH2CH2N3). 13C NMR (75.5 MHz, CDCl3): d = 170.4,
170.1, 170.0, 169.9, 169.8 and 168.9 (C(O)CH3), 101.1 (C-1), 99.1
and 99.0 (C-1ꢀ, C-1ꢀꢀ), 72.2, 71.8, 69.1, 68.7, 68.4, 68.0, 67.8, 67.5,
67.1 and 66.6 (C-2, C-2ꢀ, C-2ꢀꢀ, C-3, C-3ꢀ, C-3ꢀꢀ, C-4, C-4ꢀ, C-4ꢀꢀ, C-
5, C-5ꢀ, C-5ꢀꢀ), 66.8 (OCH2CH2CH2Br), 62.0, 60.9 and 60.2 (C-6,
C-6ꢀ, C-6ꢀꢀ), 32.1 (CH2Br), 30.1 (OCH2CH2CH2Br), 20.7, 20.6 and
20.4 (C(O)CH3). HRMS for C41H57BrO26 (M, 1044.2321): found
[M + Na]+ 1067.1964, calcd. 1067.2159.
ꢀ
ꢀ
Hz), 5.56 (1H, d, H-4ꢀꢀ, J3
= 2.2 Hz), 5.42–5.20 (6H, m, H-3,
,4
H-3ꢀ, H-3ꢀꢀ, H-2, H-2ꢀ, H-2ꢀꢀ), 5.00–4.98 (2H, 2 × d, H-1ꢀ, H-1ꢀꢀ,
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
,2
J1 ,2 = 3.6 Hz, J1
= 3.6 Hz), 4.53 (1H, t, H-5, J5,6 = 6.6 Hz),
4.46–4.00 (10 H, m, H-4, H-4ꢀ, H-5ꢀ, H-5ꢀꢀ, 2 × H-6, 2 × H-6ꢀ, 2 ×
H-6ꢀꢀ), 2.15, 2.14, 2.13, 2.11, 2.10, 2.08, 2.07, 2.05, 2.04, 2.01 and
1.99 (11 × 3H, 11 × s, C(O)CH3). 13C NMR (CDCl3, 75.5 MHz):
d = 170.7, 170.5, 170.3, 170.2, 170.1, 169.6 and 168.9 (C(O)CH3),
99.3 and 89.8 (C-1, C-1ꢀ, C-1ꢀꢀ), 70.5, 69.3, 69.1, 68.9, 68.3, 67.8,
67.4, 66.9 and 66.3 (C-2, C-2ꢀ, C-2ꢀꢀ, C-3, C-3ꢀ, C-3ꢀꢀ, C-4, C-4ꢀ,
C-4ꢀꢀ, C-5, C-5ꢀ, C-5ꢀꢀ), 61.9, 61.0 and 60.4 (C-6, C-6ꢀ, C-6ꢀꢀ) 20.9
1430 | Org. Biomol. Chem., 2008, 6, 1425–1434
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©

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3-Azidopropyl (2,3,4,6-tri-O-acetyl-a-D-galactopyranosyl)-
(1→4)-(2,3,6-tri-O-acetyl-a-D-galactopyranosyl)-(1→4)-2,3,6-
18b was obtained after silica gel chromatography (EtOAc) as a
white solid (380 mg, 92%). 1H NMR (300 MHz, CDCl3): d = 7.19
(2H, d, CHarom-2,6), 6.65 (1H, s, CHarom-4), 6.05 (2H, bs, C(O)NH),
4.09–4.06 (4H, m, OCH2CH2NH), 3.91 (3H, s, OCH3), 3.73–3.67
(4H, m, OCH2CH2NH), 2.57–2.51 and 2.46–2.40 (2 × 4H, 2 ×
m, C(O)CH2CH2CCH) and 1.95 (2H, t, CH2CH2CCH, J = 2.1
Hz). 13C NMR (75.5 MHz, CDCl3): d = 171.4 (C(O)NH), 166.7
(C(O)OCH3), 159.7 (Carom-3,5), 132.5 (Carom-1), 108.4 (Carom-2,6),
106.6 (Carom-4), 69.7 (CH2CH2CCH), 67.4 (OCH2CH2NH), 52.6
(C(O)OCH3), 39.1 (OCH2CH2NH), 35.5 (C(O)CH2CH2CCH)
and 15.1 (C(O)CH2CH2CCH).
tri-O-acetyl-b-D-galactopyranoside (16).
A solution of 15
(607 mg, 0.60 mmol) and NaN3 (195 mg, 3 mmol) in dry DMF
(20 mL) was stirred at 100 ◦C for 20 h. The solution was
◦
concentrated in vacuo at 60 C, taken up in EtOAc (50 mL) and
washed twice with brine (25 mL). The organic phase was dried
(Na2SO4), filtered and concentrated. After lyophilization 16 was
1
obtained as white powder (548 mg, 94%). H NMR (300 MHz,
CDCl3): d = 5.56 (1H, dd, H-4ꢀꢀ, J3
= 3.3 Hz), 5.39 (1H, dd,
ꢀꢀ ꢀꢀ
,4
H-3ꢀꢀ, J2
= 11.0 Hz, J3
= 3.3 Hz), 5.32–5.22 (3H, m, H-2ꢀ,
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,4
,3
H-2ꢀꢀ, H-3ꢀ), 5.17 (1H, dd, H-2, J1,2 = 7.7 Hz, J2,3 = 10.7 Hz), 4.99
and 4.98 (each 1H, 2 × d, H-1ꢀ, H-1ꢀꢀ, J1 ,2 = 3.6 Hz, J1 = 3.3
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
,2
Tetravalent pentynoic acid dendrimer (19b). Tetravalent den-
drimer 19a (1099 mg, 1.0 mmol) was stirred in a mixture of
CH2Cl2/TFA (1/1, v/v, 20 mL) with a trace of H2O for 1 h.
The reaction mixture was then concentrated to dryness and
taken up in CH2Cl2 (10 mL), BOP (2.65 g, 6.0 mmol) and 4-
pentynoic acid (589 mg, 6.0 mmol) were added followed by
DiPEA (1.98 mL, 12.0 mmol). The reaction mixture was stirred
for 18 h and concentrated at 60 ◦C. Dendrimer 19b was obtained
after silica gel chromatography (CH2Cl2/MeOH/DMF, 94/5/1
→ 85/10/5) as white solid (826 mg, 81%, small contamination).
1H NMR (300 MHz, DMSO): d = 8.66 (2H, s, C(O)NH), 8.15
Hz), 4.83 (1H, dd, H-3, J2,3 = 10.7 Hz, J3,4 = 2.8 Hz), 4.46 (1H, d,
H-1, J1,2 = 7.7 Hz), 4.56–4.02 (9H, m, H-4, H-5ꢀ, H-5ꢀꢀ, 2 × H-6,
2 × H-6ꢀ, 2 × H-6ꢀꢀ), 4.30 (1H, d, H-4ꢀ, J3,4 = 1.7 Hz), 3.98–3.92
and 3.66–3.64 (2 × 1H, 2 × m, CH2OGal), 3.79 (1H, t, H-5, J5,6
=
6.6 Hz), 3.38 (2H, t, CH2N3, J = 6.6 Hz), 2.14, 2.11, 2.10, 2.09,
2.08, 2.07, 2.06, 2.05, 2.03 and 1.99 (10 × 3H, 10 × s, C(O)CH3)
and 1.90–1.82 (2H, m, OCH2CH2CH2N3). 13C NMR (75.5 MHz,
CDCl3): d= 170.6, 170.2, 170.0 and 169.0 (C(O)CH3), 101.0 (C-1),
99.3 and 99.2 (C-1ꢀ, C-1ꢀꢀ), 72.4, 72.0, 69.2, 68.9, 68.5, 68.2, 68.0,
67.7, 67.3 and 66.8 (C-2, C-2ꢀ, C-2ꢀꢀ, C-3, C-3ꢀ, C-3ꢀꢀ, C-4, C-4ꢀ, C-
4ꢀꢀ, C-5, C-5ꢀ, C-5ꢀꢀ), 66.0 (OCH2CH2CH2N3), 62.1, 61.0 and 60.3
(C-6, C-6ꢀ, C-6ꢀꢀ), 47.9 (CH2N3), 28.9 (OCH2CH2CH2N3) and 20.5
(C(O)CH3). HRMS for C41H57N3O26 (M, 1007.3230): found [M +
Na]+ 1030.2924, calcd. 1030.3128.
(4H, t, C(O)NH), 7.09 (2H, m, CHarom-2,6), 7.04 (4H, s, CHarom
-
2ꢀ,6ꢀ), 6.84 (1H, t, CHarom-4), 6.65 (2H, t, CHarom-4ꢀ), 4.16 (4H,
t, OCH2CH2NH, J = 5.4 Hz), 4.01 (8H, t, OCH2CH2NH, J =
5.4 Hz), 3.83 (3H, s, OCH3), 3.64–3.58 (4H, m, OCH2CH2NH),
3.45–3.39 (8H, m, OCH2CH2NH), 2.73 (4H, t, CH2CH2CCH, J =
2.1 Hz) and 2.39–2.27 (16H, m, C(O)CH2CH2CCH). 13C NMR
(75.5 MHz, DMSO): d = 170.6 (C(O)NH), 165.9 (C(O)OCH3),
Monovalent pentynoic acid dendrimer (17b). Compound 17a
(295 mg, 1.0 mmol) was stirred in a mixture of CH2Cl2/TFA
(1/1, v/v, 20 mL) with a trace of water for 3 h. The reaction
mixture was then concentrated to dryness and taken up in
CH2Cl2 (10 mL). BOP (487 mg, 1.1 mmol) and 4-pentynoic acid
(108 mg, 1.1 mmol) were added followed by DiPEA (0.55 mL,
3.3 mmol). The mixture was stirred for 72 h and concentrated
in vacuo. The crude mixture was taken up in EtOAc (50 mL)
and washed twice with KHSO4 (1M, 15 mL), twice with NaOH
(1M, 15 mL) and with brine (15 mL). Pure product was obtained
159.7 (Carom-3,5), 159.4 (Carom-3ꢀ,5ꢀ), 136.2 (Carom-1ꢀ), 131.6 (Carom
-
1), 107.7 (Carom-2,6), 106.0 (Carom-2ꢀ,6ꢀ), 104.1 (Carom-4ꢀ), 83.7
(CH2CH2CCH), 71.2 (CH2CH2CCH), 66.6 (OCH2CH2NH), 52.3
(C(O)OCH3), 38.2 (OCH2CH2NH), 34.0 (C(O)CH2CH2CCH)
and 14.2 (C(O)CH2CH2CCH).
Octavalent pentynoic acid dendrimer (20b). Octavalent den-
drimer 20a (597 mg, 0.25 mmol) was stirred in a mixture of
CH2Cl2/TFA (1/1, v/v, 10 mL) with a trace of H2O for 1 h.
The reaction mixture was then concentrated to dryness and
taken up in dry DMF (5 mL), BOP (1.33 g, 3 mmol) and
4-pentynoic acid (295 mg, 3 mmol) were added followed by
DiPEA (0.99 mL, 6 mmol). The reaction mixture was stirred
for 18 h and concentrated at 60 ◦C. Silica gel chromatography
(CH2Cl2/MeOH/DMF, 94/5/1 → 85/10/5) was used for pu-
rification although a small impurity remains present (621 mg, ≈
1
as a white solid (192 mg, 70%). H NMR (300 MHz, CDCl3):
d = 7.64 (1H, m, CHarom-6), 7.55 (1H, s, CHarom-2), 7.35 (1H,
t, CHarom-5), 7.11 (1H, dd, CHarom-4), 6.12 (1H, bs, C(O)NH),
4.10 (2H, t, OCH2CH2NH, J = 5.4 Hz), 3.92 (3H, s, OCH3),
3.74–3.68 (2H, m, OCH2CH2NH), 2.59–2.53 and 2.48–2.42 (2 ×
2H, 2 × m, C(O)CH2CH2CCH) and 1.94 (1H, t, CH2CH2CCH).
13C NMR (75.5 MHz, CDCl3): d = 171.3 (C(O)NH), 166.8
(C(O)OCH3), 158.6 (Carom-3), 131.8 (Carom-1), 129.8 (Carom-5), 122.7
(Carom-6), 119.8 (Carom-4), 115.1 (Carom-2), 69.6 (CH2CH2CCH), 67.3
(OCH2CH2NH), 52.4 (C(O)OCH3), 39.2 (OCH2CH2NH), 35.5
(C(O)CH2CH2CCH) and 15.1 (C(O)CH2CH2CCH).
1
quantitative). H NMR (300 MHz, DMSO): d = 8.69 (6H, s,
C(O)NH), 8.16 (8H, t, C(O)NH), 7.07–7.04 (14H, m, CHarom-2,6,
2ꢀ,6ꢀ, 2ꢀꢀ,6ꢀꢀ), 6.82 (1H, s, CHarom-4), 6.70 (2H, bs, CHarom-4ꢀ), 6.64
(4H, d, CHarom-4ꢀꢀ), 4.14 (12H, bs, OCH2CH2NH), 4.01 (16H, s,
OCH2CH2NH), 3.80 (3H, s, OCH3), 3.61 (12H, d, OCH2CH2NH),
3.42 (16H, d, OCH2CH2NH), 2.72 (8H, bs, CH2CH2CCH) and
2.35–2.29 (32H, m, C(O)CH2CH2CCH). 13C NMR (75.5 MHz,
Divalent pentynoic acid dendrimer (18b). Divalent dendrimer
18a (454 mg, 1.0 mmol) was stirred in a mixture of CH2Cl2/TFA
(1/1, v/v, 16 mL) with a trace of water for 3 h. The reaction
mixture was then concentrated to dryness and taken up in CH2Cl2
(5 mL), BOP (0.97 g, 2.2 mmol) and 4-pentynoic acid (215 mg,
2.2 mmol) were added followed by DiPEA (1.10 mL, 6.6 mmol).
The reaction mixture was stirred for 18 h and concentrated. Crude
product was taken up in EtOAc (100 mL) and washed with KHSO4
(1M, 50 mL), NaOH (1M, 50 mL) and brine (50 mL). Dendrimer
DMSO): d = 170.6 (C(O)NH), 165.9 (C(O)OCH3), 159.4 (Carom
-
3ꢀꢀ,5ꢀꢀ), 136.2 (Carom-1ꢀꢀ), 106.0 (Carom-2ꢀꢀ,6ꢀꢀ), 104.1 (Carom-4ꢀꢀ), 83.7
(CH2CH2CCH), 71.2 (CH2CH2CCH), 66.6 (OCH2CH2NH), 53.4
(C(O)OCH3), 40.3 (OCH2CH2NH), 34.0 (C(O)CH2CH2CCH)
and 14.2 (C(O)CH2CH2CCH).
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 1425–1434 | 1431
©

View Article Online
1
General “click” procedure
obtained as a white foam (155 mg, 84%). H NMR (300 MHz,
CDCl3): d = 7.44 (2H, bs, CHtriazole), 7.15 (2H, s, CHarom-2,6),
6.67 (1H, s, CHarom-4), 6.62 (2H, bs, NHCO), 5.58 (2H, dd, H-
Dendrimer was mixed with the galabiose azide (1.5 eq per
alkyne), CuSO4·5H2O (0.15 eq per alkyne) and sodium ascorbate
(0.3 eq per alkyne) in 1% H2O/DMF. The mixture was heated
under microwave irradiation to 80 ◦C for 20 min. The reaction
mixture was concentrated in vacuo at 60 ◦C and after silica gel
chromatography the dendrimer was obtained.
4ꢀ, J3 ,4 = 2.5 Hz), 5.41 (2H, dd, H-3ꢀ, J2 ,3 = 11.3 Hz, J3 ,4
=
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ ꢀ
3.3 Hz), 5.23–5.16 (4H, m, H-2, H-2ꢀ), 5.00 (2H, d, H-1ꢀ, J1 ,2
=
ꢀ
ꢀ
3.6 Hz), 4.81 (2H, dd, H-3, J2,3 = 10.7 Hz, J3,4 = 2.5 Hz),
4.47 (2H, d, H-1, J1,2 = 7.70 Hz), 4.55–4.35, 4.17–4.00 and
3.87–3.75 (10H, 12H, 4H, 3 × m, H-1, H-4, H-5ꢀ, H-6, H-6ꢀ,
OCH2CH2NH, CH2Ntriazole, CH2OGal), 3.90 (3H, s, C(O)OCH3),
3.65 (4H, bs, OCH2CH2NH), 3.50–3.42 (1H, m, H-5), 3.06 and
2.67 (2 × bs, each 4H, C(O)CH2CH2Ctriazole), 2.20–2.02 (4H, m,
OCH2CH2CH2Ntriazole), 2.14, 2.10, 2.09, 2.08, 2.06, 2.04 and 1.99
(7 × 6H, 7 × s, C(O)CH3). 13C NMR (75.5 MHz, CDCl3): d =
170.2, 170.1, 170.0, 169.8, 169.6 and 168.9 (C(O)CH3), 166.1
(C(O)OCH3), 159.2 (Carom-3,5), 131.7 (Carom-1), 107.7 (Carom-2,6),
106.4 (Carom-4), 100.7 (C-1), 99.1 (C-1ꢀ), 72.3, 71.6, 68.3, 67.5,
67.0 and 66.8 (C-2, C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 66.6
(OCH2CH2CH2Ntriazole), 65.4 (OCH2CH2NH), 61.7 and 60.2 (C-6,
C-6ꢀ), 51.9 (C(O)OCH3), 38.5 and 29.7 (C(O)CH2CH2Ctriazole), 20.5
and 20.3 (C(O)CH3). HRMS for C80H108N8O42 (M, 1852.6561):
found [M + Na]+ 1875.6348, calcd. 1875.6459. The general
deacetylation procedure yielded compound 18c as a white foam
General deacetylation procedure
A solution of the dendrimer in MeOH (5 mL) was treated
with NaOMe in MeOH (30% wt solution, 50 lL) for 5 h. The
solution was neutralized by Dowex-H+, filtered and concentrated
in vacuo. Deprotected dendrimers were purified using preparative
HPLC. Pure products were obtained after lyophilisation.
Monovalent galabiose dendrimer (17c). The general “click”
procedure was applied. Monovalent galabiose derivative was
obtained as a white foam (179 mg, 72%). H NMR (300 MHz,
1
CDCl3): d = 7.65 (1H, d, CHarom-6), 7.53 (1H, t, CHarom-2), 7,35
(1H, t, CHarom-5), 7.09 (1H, dd, CHarom-4), 6.34 (1H, bs, NHCO),
5.58 (1H, dd, H-4ꢀ, J3 ,4 = 3.0 Hz, J4 ,5 = 1.1 Hz), 5.41 (1H,
ꢀ
ꢀꢀ
ꢀ ꢀ
1
dd, H-3ꢀ, J2 ,3 = 11.0 Hz, J3 ,4 = 3.3 Hz), 5.23–5.16 (2H, m, H-
(66.3 mg, 49%). H NMR (300 MHz, D2O): d = 7.75 (2H, bs,
ꢀ
ꢀ
ꢀ ꢀ
2, H-2ꢀ), 5.00 (1H, d, H-1ꢀ, J1 ,2 = 3.9 Hz), 4.80 (1H, dd, H-3,
J2,3 = 10.7 Hz, J3,4 = 2.7 Hz), 4.45 (1H, d, H-1, J1,2 = 7.70 Hz),
4.55–4.35, 4.21–4.04 and 3.90–3.76 (5H, 6H, 2H, 3 × m, H-1, H-4,
H-5ꢀ, H-6, H-6ꢀ, OCH2CH2NH, CH2Ntriazole, CH2OGal), 3.91 (3H, s,
OCH3), 3.69–3.63 (2H, bs, OCH2CH2NH), 3.49–3.40 (1H, m, H-
5), 3.06 and 2.67 (2 × bs, each 2H, C(O)CH2CH2Ctriazole), 2.20–
2.02 (2H, m, OCH2CH2CH2Ntriazole), 2.14, 2.10, 2.09, 2.08, 2.06,
2.04 and 1.99 (7 × 3H, 7 × s, C(O)CH3). 13C NMR (75.5 MHz,
CDCl3): d = 170.5, 170.4, 170.1, 169.9 and 169.2 (C(O)CH3),
CHtriazole), 7.05 (2H, s, CHarom-2,6), 6.62 (1H, s, CHarom-4), 4.97
(2H, s, H-1ꢀ), 4.39–4.27 (6H, m), 4.02–3.70 (25H, m), 3.56–3.53
(8H, m), 2.97 and 2.60 (2 × 4H, 2 × bs, C(O)CH2CH2Ctriazole) and
2.02 (4H, bs, OCH2CH2CH2Ntriazole). 13C NMR (75.5 MHz, D2O):
d = 175.7 (C(O)NH), 168.9 (C(O)OCH3), 160.1 (Carom-3,5), 132.3
(Carom-1), 108.9 (Carom-2,6), 107.5 (Carom-4), 103.7 (C-1), 101.0 (C-
1ꢀ), 77.9, 77.7, 75.9, 75.6, 73.2, 73.0, 71.5, 69.9 and 69.5 (C-2, C-2ꢀ,
C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 67.7 (OCH2CH2CH2Ntriazole), 67.1
(OCH2CH2NH), 61.3 and 60.8 (C-6, C-6ꢀ), 47.6 (CH2Ntriazole), 39.4
and 35.8 (C(O)CH2CH2Ctriazole) and 21.9 (OCH2CH2CH2Ntriazole).
HRMS for C52H80N8O28 (M, 1264.5082): found [M + Na]+
1287.1271, calcd. 1287.4980.
ꢀ
ꢀ
166.6 (C(O)OCH3), 158.3 (Carom-3), 131.4 (Carom-1), 129.4 (Carom
-
5), 122.3 (Carom-6), 119.6 (Carom-4), 114.7 (Carom-2), 101.0 (C-1),
99.4 (C-1ꢀ), 72.6, 71.9, 68.5, 67.7, 67.2 and 67.0 (C-2, C-2ꢀ, C-
3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 66.8 (OCH2CH2CH2Ntriazole), 65.6
(OCH2CH2NH), 61.9 and 60.4 (C-6, C-6ꢀ), 52.1 (C(O)OCH3),
46.6 (CH2Ntriazole), 38.8 and 30.0 (C(O)CH2CH2Ctriazole), 20.8 and
20.6 (C(O)CH3). HRMS for C44H58N4O22 (M, 994.3543): found
[M + H]+ 995.3600, calcd. 995.3621. The general deacetylation
procedure yielded compound 17c as a clear oil (30.7 mg, 35%). 1H
NMR (300 MHz, D2O): d = 7.72 (1H, bs, CHtriazole), 7.61 (2H, s,
CHarom-6), 7.46–7.40 (2H, m, CHarom-2,5), 7.14 (1H, d, CHarom-4),
Tetravalent galabiose dendrimer (19c). The general “click”
procedure was applied. Protected tetravalent galabiose dendrimer
19c was obtained as a white foam (151 mg, 78%). 1H NMR
(300 MHz, CDCl3): d = 7.44 (4H, bs, CHtriazole), 7.36 (2H, bs,
C(O)NH), 7.15 (2H, s, CHarom-2,6), 6.94 (8H, bs, CHarom-2ꢀ,6ꢀ
and C(O)NH), 6.73 (1H, bs, CHarom-4), 6.52 (2H, bs, CHarom
-
4ꢀ), 5.57 (4H, dd, H-4ꢀ, J3 ,4 = 2.2 Hz), 5.41 (4H, dd, H-3ꢀ,
ꢀ
ꢀ
J2 ,3 = 11.0 Hz, J3 ,4 = 3.3 Hz), 5.23–5.15 (8H, m, H-2, H-2ꢀ),
4.97 (1H, d, H-1ꢀ, J1 ,2 = 3.6 Hz), 4.39–4.27 (6H, m), 4.39–4.31 (2H,
m), 4.22 (2H, t), 4.02–3.97 (4H, m), 3.94–3.67 (14H, m), 3.56–3.42
(5H, m), 2.98 and 2.60 (2 × 2H, 2 × bt, C(O)CH2CH2Ctriazole) and
2.00 (2H, bt, OCH2CH2CH2Ntriazole). 13C NMR (75.5 MHz, D2O):
d = 175.6 (C(O)NH), 169.4 (C(O)OCH3), 158.9 (Carom-3), 131.5
(Carom-1), 130.8 (Carom-5), 123.1 (Carom-6), 121.0 (Carom-4), 115.6
(Carom-2), 103.7 (C-1), 101.0 (C-1ꢀ), 77.9, 77.7, 75.7, 73.1, 71.6,
71.4, 69.8 and 69.6 (C-2, C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ),
67.5 (OCH2CH2CH2Ntriazole), 67.0 (OCH2CH2NH), 61.2 and 60.7
(C-6, C-6ꢀ), 53.4 (C(O)OCH3), 47.7 (CH2Ntriazole), 39.4 and 35.7
(C(O)CH2CH2Ctriazole) and 21.7 (OCH2CH2CH2Ntriazole). HRMS
for C30H44N4O15 (M, 700.2803): found [M + H]+ 701.0662, calcd.
701.2881.
ꢀ
ꢀ
ꢀ ꢀ
ꢀ
ꢀ
5.00 (4H, d, H-1ꢀ, J1 ,2 = 3.7 Hz), 4.81 (4H, dd, H-3, J2,3
=
ꢀ
ꢀ
10.7 Hz, J3,4 = 2.4 Hz), 4.54–4.35, 4.21–4.08, 3.98 and 3.87–3.80
(22H, 22H, 8H, 12H, 3 × m, 1 × bs, H-1, H-4, H-5ꢀ, H-6, H-6ꢀ,
OCH2CH2NH, OCH2CH2NH, CH2Ntriazole, CH2OGal), 3.87 (3H, s,
OCH3), 3.57 (6H, bs, OCH2CH2NH), 3.45 (4H, bs, H-5) 3.03 and
2.66 (2 × bs, 2 × 8H, C(O)CH2CH2Ctriazole), 2.20–2.00 (8H, m,
OCH2CH2CH2Ntriazole), 2.13, 2.10, 2.08, 2.07, 2.05, 2.04 and 1.99
(7 × 12H, 7 × s, C(O)CH3). 13C NMR (75.5 MHz, CDCl3): d =
170.5, 170.4, 170.0, 169.9 and 169.2 (C(O)CH3), 167.9 (C(O)NH),
166.4 (C(O)OCH3), 159.5 (Carom-3,5 and Carom-3ꢀ,5ꢀ), 136.3 (Carom
-
-
1ꢀ), 131.9 (Carom-1), 108.1 (Carom-2,6), 106.5 (Carom-4), 106.0 (Carom
2ꢀ,6ꢀ), 104.8 (Carom-4ꢀ), 100.9 (C-1), 99.4 (C-1ꢀ), 72.5, 71.8, 68.6,
68.4, 67.7, 67.2 and 67.0 (C-2, C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-
5ꢀ), 66.7 (OCH2CH2CH2Ntriazole), 65.6 (OCH2CH2NH), 61.8 and
60.4 (C-6, C-6ꢀ), 52.2 (C(O)OCH3), 46.7 (CH2Ntriazole), 39.5 and
Divalent galabiose dendrimer (18c). The general “click” pro-
cedure was applied. Protected divalent galabiose dendrimer was
1432 | Org. Biomol. Chem., 2008, 6, 1425–1434
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©

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38.6 (OCH2CH2NH), 38.6 and 30.0 (C(O)CH2CH2Ctriazole), 20.8
and 20.6 (C(O)CH3). HRMS for C170H226N18O86 (M, 3895.3864):
found [M + 3Na]3+ 1321.7095, calcd. 1321.4519. The general
deacetylation procedure yielded compound 19c as a white foam
(1H, bs, CHarom-4), 6.48 (2H, bs, CHarom-4ꢀ), 5.56 (4H, d, H-4ꢀꢀ,
J3
= 2.7 Hz), 5.40 (4H, dd, H-3ꢀꢀ, J2
= 11.1 Hz, J3 ,4
=
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀ
ꢀ
,4
,3
3.3 Hz), 5.31–5.15 (12H, m, H-2, H-2ꢀ, H-2ꢀꢀ), 5.00 (4H, d, H-
1ꢀꢀ, J1
= 3.6 Hz), 4.97 (4H, d, H-1ꢀ, J1 ,2 = 1.8 Hz), 4.81 (4H,
ꢀꢀ ꢀꢀ
ꢀ ꢀ
,2
1
(64.3 mg, 74%). H NMR (300 MHz, D2O): d = 7.65 (4H, s,
d, H-3, J2,3 = 9.9 Hz), 4.56–4.30, 4.22–3.98 and 3.82–3.75 (25H,
24H, 8H, 3 × m, H-1, H-4, H-4ꢀ, H-5ꢀ, H-5ꢀꢀ, H-6, H-6ꢀ, H-6ꢀꢀ,
OCH2CH2NH, OCH2CH2NH, CH2Ntriazole, CH2OGal), 3.87 (3H, s,
OCH3), 3.57 (6H, bs, OCH2CH2NH), 3.44 (4H, bs, H-5), 3.01 and
2.65 (2 × 8H, 2 × bs, C(O)CH2CH2Ctriazole), 2.20–2.00 (8H, m,
OCH2CH2CH2Ntriazole), 2.14, 2.12, 2.09, 2.07, 2.06, 2.03 and 1.99
(10 × 12H, 10 × s, C(O)CH3). 13C NMR (75.5 MHz, CDCl3):
d = 170.6, 170.4, 170.2, 170.1, 169.9 and 169.2 (C(O)CH3), 167.2
CHtriazole), 6.79 (6H, s, CHarom-2,6, 2ꢀ,6ꢀ), 6.48 (3H, t, CHarom-4,
4ꢀ), 4.95 (4H, s, H-1ꢀ), 4.37–4.27 (16H, m), 4.01–3.45 (84H, m),
2.89 and 2.52 (2 × 8H, 2 × bs, C(O)CH2CH2Ctriazole) and 2.00
(8H, m, OCH2CH2CH2Ntriazole). 13C NMR (75.5 MHz, D2O): d =
175.4 and 169.6 (C(O)NH), 168.2 (C(O)OCH3), 160.0 (Carom-3ꢀ,5ꢀ),
159.8 (Carom-3,5), 133.0 (Carom-1), 131.7 (Carom-1ꢀ), 108.6 (Carom
-
2,6), 106.9 (Carom-2ꢀ,6ꢀ), 105.3 (Carom-4ꢀ), 103.6 (C-1), 100.9 (C-
1ꢀ), 77.8, 77.7, 73.0, 71.5, 71.3, 69.7 and 69.5 (C-2, C-2ꢀ, C-
3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 67.5 (OCH2CH2CH2Ntriazole), 67.0
(OCH2CH2NH), 61.2 and 60.7 (C-6, C-6ꢀ), 47.5 (CH2Ntriazole), 39.2
and 35.7 (C(O)CH2CH2Ctriazole) and 21.7 (OCH2CH2CH2Ntriazole).
MALDI ToF MS for C114H170N18O58 (M, 2719.0906): found [M +
Na]+ 2743.20, calcd. 2743.65.
(C(O)NH), 166.4 (C(O)OCH3), 159.5 (Carom-3ꢀꢀ,5ꢀꢀ), 136.4 (Carom
-
1ꢀꢀ), 131.7 (Carom-1), 108.3, 108.0, 106.3, 106.1 and 104.7 (Carom),
101.0 (C-1), 99.6 and 99.3 (C-1ꢀ, C-1ꢀꢀ), 72.5, 72.3, 72.1, 71.9,
69.0, 68.7, 68.4, 67.8, 67.5 and 66.8 (C-2, C-2ꢀ, C-2ꢀꢀ, C-3, C-
3ꢀ, C-3ꢀꢀ C-4, C-4ꢀ, C-4ꢀꢀ, C-5, C-5ꢀ, C-5ꢀꢀ), 65.6 (OCH2CH2NH),
62.1, 61.0 and 60.3 (C-6, C-6ꢀ, C-6ꢀꢀ), 52.1 (C(O)OCH3), 46.5
(CH2Ntriazole), 39.5 and 38.6 (OCH2CH2NH), 35.3 and 30.0
(C(O)CH2CH2Ctriazole), 20.6 and 20.5 (C(O)CH3). HRMS for
C218H290N18O118 (M, 5047.7245): found [M + Na]+ 5072.99, calcd.
5073.6748. General deacetylation procedure yielded compound
19d as a white foam (28.4 mg, 57%). 1H NMR (300 MHz, D2O):
d = 7.70 (4H, s, CHtriazole), 6.84 (2H, s, CHarom-2,6), 6.79 (4H, s,
CHarom-2ꢀ,6ꢀ), 6.51 (3H, t, CHarom-4, 4ꢀ), 5.01–4.97 (8H, m, H-1ꢀ,
H-1ꢀꢀ), 4.41 (4H, t), 4.29 (16H, m), 4.11–3.60 (86H, m), 3.55–3.40
Octavalent galabiose dendrimer (20c). The general “click”
procedure was applied. Protected octavalent galabiose dendrimer
was obtained as a white foam (131 mg, 66%). 1H NMR (300 MHz,
CDCl3): d = 7.68 (8H, bs, CHtriazole), 7.46 and 7.30 (8H and 6H,
2 × bs, C(O)NH), 7.07 (CHarom-2,6), 6.89 (12H, s, CHarom-2ꢀ,6ꢀ,
2ꢀꢀ,6ꢀꢀ), 6.69 (1H, s, CHarom-4), 6.40 (6H, bs, CHarom-4ꢀ, CHarom-4ꢀꢀ),
5.57 (4H, d, H-4ꢀ, J3 ,4 = 2.7 Hz), 5.41 (4H, dd, H-3ꢀ, J2 ,3
=
ꢀ
ꢀ
ꢀ ꢀ
11.0 Hz, J3 ,4 = 3.3 Hz), 5.23–5.15 (8H, m, H-2, H-2ꢀ), 5.00
ꢀ
ꢀ
(18H, m), 2.91 and 2.54 (2 × 8H, 2 × bs, C(O)CH2CH2Ctriazole
)
(4H, d, H-1ꢀ, J1 ,2 = 3.7 Hz), 4.83 (4H, d, H-3, J2,3 = 10.8 Hz),
4.55–4.30, 4.20–4.00 and 3.90–3.70 (40H, 45H, 46H, 3 × m, H-1,
H-4, H-5ꢀ, H-6, H-6ꢀ, CH2Ntriazole, OCH2CH2NH, OCH2CH2NH,
CH2OGal, C(O)OCH3), 3.53 (16H, bs, OCH2CH2NH), 3.45 (8H,
m, H-5), 2.19–2.01 (16H, m, OCH2CH2CH2Ntriazole), 2.14, 2.13,
2.10, 2.08, 2.05, 2.04 and 1.99 (7 × 12H, 7 × s, C(O)CH3). 13C
NMR (75.5 MHz, CDCl3): d = 170.5, 170.3, 170.0, 169.9 and
169.2 (C(O)CH3), 167.6 (C(O)NH), 166.4 (C(O)OCH3), 159.4
(Carom-3,5, 3ꢀ,5ꢀ, 3ꢀꢀ,5ꢀꢀ), 136.3 (Carom-1ꢀ, 1ꢀꢀ), 131.9 (Carom-1), 108.2,
106.0 and 104.4 (Carom-4, 4ꢀ, 4ꢀꢀ, Carom-2,6, 2ꢀ,6ꢀ, 2ꢀꢀ,6ꢀꢀ), 100.9 (C-1),
99.4 (C-1ꢀ), 72.5, 71.8, 68.6, 68.4, 67.7, 67.2 and 67.0 (C-2, C-
2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 66.7 (OCH2CH2CH2Ntriazole),
65.7 (OCH2CH2NH), 61.8 and 60.4 (C-6, C-6ꢀ), 30.0 and 29.5
(C(O)CH2CH2Ctriazole) and 20.5 (C(O)CH3). The general deacety-
lation procedure yielded compound 20c as a white foam (54.5 mg,
77%). 1H NMR (300 MHz, D2O): d = 7.67 (8H, s, CHtriazole), 6.73
(10H, s, CHarom-2,6, 2ꢀ,6ꢀ, 2ꢀꢀ,6ꢀꢀ), 6.42 (6H, t, CHarom-4, 4ꢀ, 4ꢀꢀ), 4.95
(8H, d, H-1ꢀ, J1,2 = 3.6 Hz), 4.37–4.30 (25H, m), 4.01–3.43 (140H,
m), 2.87 and 2.51 (2 × 16H, 2 × bs, C(O)CH2CH2Ctriazole) and
2.01 (16H, m, OCH2CH2CH2Ntriazole). 13C NMR (75.5 MHz, D2O):
d = 175.2, 169.3 and 169.1 (C(O)NH), 167.9 (C(O)OCH3), 159.9
(Carom-3ꢀꢀ,5ꢀꢀ), 103.6 (C-1), 100.9 (C-1ꢀ), 77.8, 77.7, 75.7, 73.0, 71.5,
71.3, 69.7 and 69.5 (C-2, C-2ꢀ, C-3, C-3ꢀ, C-4, C-4ꢀ, C-5, C-5ꢀ), 67.5
(OCH2CH2CH2Ntriazole), 67.0 (OCH2CH2NH), 61.2 and 60.7 (C-6,
C-6ꢀ), 47.5 (CH2Ntriazole), 39.2 and 35.5 (C(O)CH2CH2Ctriazole) and
21.7 (OCH2CH2CH2Ntriazole). MALDI ToF MS for C238H350N38O118
(M, 5628.2555): found [M + Na]+ 5654.46, calcd. 5654.50.
ꢀ
ꢀ
and 2.00 (8H, m, OCH2CH2CH2Ntriazole). 13C NMR (75.5 MHz,
D2O): d = 175.3 and 169.7 (C(O)NH), 168.3 (C(O)OCH3), 160.0
(Carom-3ꢀ,5ꢀ), 159.8 (Carom-3,5), 136.2 (Carom-1ꢀ), 106.8 (Carom), 103.5
(C-1), 101.1 and 100.8 (C-1ꢀ, C-1ꢀꢀ), 79.6, 77.5, 75.6, 72.8, 71.6,
71.4, 69.8, 69.5 and 69.2 (C-2, C-2ꢀ, C-2ꢀꢀ, C-3, C-3ꢀ, C-3ꢀꢀ, C-
4, C-4ꢀ, C-4ꢀꢀ, C-5, C-5ꢀ, C-5ꢀꢀ), 67.4 (OCH2CH2CH2Ntriazole), 66.9
(OCH2CH2NH), 61.2 and 60.5 (C-6, C-6ꢀ, C-6ꢀꢀ), 47.7 (CH2Ntriazole),
39.2 and 35.4 (C(O)CH2CH2Ctriazole), 30.1 (OCH2CH2NH) and
21.5 (OCH2CH2CH2Ntriazole). MALDI ToF MS for C138H210N18O78
(M, 3367.3019): found [M + Na]+ 3391.87, calcd. 3392.21.
Hemagglutination
Hemagglutination assays were performed as described
previously.4b Briefly, equal volumes of bacteria and 5% sialidase-
treated human erythrocytes were mixed and hemagglutination was
visually recorded after 1 h incubation on ice. For inhibition assays,
2-fold dilutions of galabiose compounds (25 lL) were mixed with
bacteria (25 lL). After 5 min of incubation at room temperature,
50 lL of the erythrocytes was added. The hemagglutination
was recorded as described and the MIC values (the lowest
concentration completely inhibiting the hemagglutination) were
recorded.
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