Synthesis of biotinylated multipodal glycoclusters on a solid support

Article abstract of DOI:10.1002/ejoc.201200926

Trivalent glycoconjugates, each bearing a biotin side arm in addition to three different sugar ligands, have been synthesized on a solid support. The conjugates were assembled on an orthogonally protected pentaerythrityl tetramine core that was anchored to the support through a backbone amide linker. Peptide coupling chemistry was applied to elongate three of the branches with β-alanine and a fully acylated glycosylacetic acid. The fourth branch was levulinoylated and oximated with aminooxy-derivatized D-biotin, followed by acidolytic release into solution. The acyl protecting groups were removed by methoxide-catalyzed transesterification in methanol.

Full text of DOI:10.1002/ejoc.201200926

FULL PAPER
DOI: 10.1002/ejoc.201200926
Synthesis of Biotinylated Multipodal Glycoclusters on a Solid Support
Marika Karskela,*[a] Miriam von Usedom,[a] Pasi Virta,[a] and Harri Lönnberg[a]
Keywords: Carbohydrates / Glycoconjugates / Glycoclusters / Solid-phase synthesis / Acylation
Trivalent glycoconjugates, each bearing a biotin side arm in
addition to three different sugar ligands, have been synthe-
sized on a solid support. The conjugates were assembled on
an orthogonally protected pentaerythrityl tetramine core that
was anchored to the support through a backbone amide
linker. Peptide coupling chemistry was applied to elongate
three of the branches with β-alanine and a fully acylated
glycosylacetic acid. The fourth branch was levulinoylated
and oximated with aminooxy-derivatized
D-biotin, followed
by acidolytic release into solution. The acyl protecting groups
were removed by methoxide-catalyzed transesterification in
methanol.
meric sugar clusters[29] have been prepared on a solid sup-
port. Conjugates bearing two different sugars and derived
from pentaerythritol[30] or a cyclic peptide[31] have been ob-
tained by modular synthesis in solution.
Introduction
Carbohydrate–protein interactions play a decisive role in
initiation of many human diseases. Multiantennary oligo-
saccharides anchored to cell membranes usually recognize
the proteins of extracellular pathogens,[1–3] although the op-
posite is also possible: carbohydrates can be recognized by
the cell membrane proteins[4–6]. The interactions between
carbohydrates and carbohydrate-binding proteins, called
lectins, are typically multipodal; high-affinity binding is
achieved through simultaneous binding of several sugar li-
gands to subunits of an oligomeric protein structure, a phe-
nomenon known as the “cluster glycoside effect”.[7–14] In
other words, a clustered arrangement both of the sugars
and of their binding sites is required for high affinity and
specificity. Accordingly, a plethora of multipodal glycocon-
jugates have been designed and synthesized as drug candi-
dates with the aim of blocking microbial infections at an
early stage.[15–20]
A lectin is usually specific for only one type of sugar,
so homoclusters are needed for recognition. Some recent
findings, however, lend support for the existence of a heter-
ocluster effect: the existence of synergic recognition of two
different sugars.[21–23] Preparation of glycoclusters contain-
ing two or even three different sugars is therefore of interest,
and some examples of the syntheses of such conjugates have
been reported.[24] Short linear,[25] branched,[26] and cyclic[27]
peptide conjugates containing up to three different sugars,
a lysine-branched structure containing two different sugars
and one nucleoside,[28] and an N,N-bis(2-aminoethyl)-
glycine-branched structure bearing two different homotri-
We now report on a new method for preparation of syn-
thetically challenging asymmetric glycoclusters on solid
supports. A branched peptide structure was used as the
scaffold because the aglycon moiety of the cluster, as well
as the saccharide ligands, can contribute to high-affinity
binding to lectins, and peptide–peptide interactions can well
be stabilizing. In other words, the diversity of the clusters
can in principle be based both on the sugar ligands and on
the composition of the peptide branches. The clusters were
further conjugated with a biotin ligand to allow convenient
immobilization of the conjugates (Figure 1) on avidin- or
streptavidin-coated surfaces. Besides biotin, the approach
should be extendable to conjugation of the glycoclusters
with any aminooxy-functionalized compound, such as
DOTA derivatives,[32] oligonucleotides,[33–35] and pept-
ides.[36] The advantage of solid-phase synthesis – the easy
removal of excess of reagents by simple washing – is a re-
markable benefit in multistep conjugate synthesis of this
kind, consisting of several removals of protecting groups
and couplings of building blocks.
[a] Department of Chemistry, University of Turku,
20014 Turku, Finland
Fax: +358-23336700
E-mail: marika.karskela@utu.fi
Homepage: http://www2.sci.utu.fi/kemia/en/research/orgchem/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200926.
Figure 1. General structure of the glycoconjugates.
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Multipodal Glycoclusters on a Solid Support
fluoroacetic acid (TFA) gave the free acids 9, 10, and 16.
Despite thorough evaporation after the acid treatment, each
product contained approximately 1 equiv. TFA. The TFA-
containing products could, however, be successfully used in
the subsequent reactions. Compounds 9 and 10 were fur-
ther converted into acid chlorides 11 and 12 with thionyl
chloride to ensure efficient coupling on assembly of the ste-
rically rather crowded clusters on a solid support. Acyl
chlorides are useful in peptide couplings that for steric
reasons proceed sluggishly with conventional activators.
Results and Discussion
Synthesis of Glycosyl Ligands
Scheme 1 outlines the preparation of fully acylated 1-O-
functionalized glycopyranosides used for the assembly of
glycoclusters. Allyl 2,3,4,6-tetra-O-toluoyl-β-d-glucopyr-
anoside (1),[27] allyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyran-
oside (2),[37] allyl 2,3,4,6-tetra-O-acetyl-β-d-galactopyran-
oside (3),[37] and allyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyr-
anoside (13)[37] used as starting materials were prepared as
described in the literature. Osmium tetroxide hydroxylation
of the allyl double bonds, followed by oxidative cleavage of
the diols and Jones oxidation of the obtained aldehydes,
gave the desired carboxymethyl derivatives 4, 5, 6, and 14
Synthesis of the Biotin Ligand 19
The biotin-derived ligand intended to allow the attach-
in satisfactory yields. RuIII-catalyzed oxidation of the glyco- ment of glycoclusters to streptavidin-coated surfaces was
sidic allyl groups to carboxymethyl groups[27,38] suffered prepared as depicted in Scheme 2. Besides biotin, the ligand
from cleavage of the toluoyl groups and hence gave rather contained a 1,4-phenylene chromophore and a Boc-pro-
modest yields (53% for 4). It should also be noted that the tected aminooxy function. The purpose of the chromo-
acetylated allyl α-d-mannopyranoside 13[37] was converted phore was to keep the cluster UV-active after removal of
into its toluoylated counterpart before the oxidation, be- the toluoyl protecting groups, whereas that of the aminooxy
cause toluoylation is known to improve the chromato- functionality was to allow conjugation to the keto group
graphic behavior of glycoclusters. The aromatic toluoyl on the levulinoyl branch by solid-supported oximation. In
groups increase the UV absorption and sharpen the RP principle, the carboxy function of biotin could be coupled
HPLC signals for the clusters relative to acetyl groups.
directly to the amino group of the scaffold, rather than to
To couple β-alanine tethers to the carboxy functions of levulinic acid. Conjugation to a sterically less hindered
4, 6, and 14, these compounds were converted into active site – that is, to the keto group of a levulinoyl side arm –
esters with N-hydroxysuccinimide (HOSu) and DCC. Acyl by oximation, however, should ensure higher efficiency of
substitution with β-alanine tert-butyl ester then afforded 7, conjugation. It should be noted that the high efficiency of
8, and 15, and removal of the tert-butyl groups with tri- solid-supported oximation has been demonstrated by the
Scheme 1. Synthesis of the sugar units. Reagents and conditions: i) OsO4, NaIO4, aq. 1,4-dioxane; ii) CrO3, H2SO4, aq. acetone; iii) 1) HOSu,
DCC, 1,4-dioxane, 2) H-β-Ala-OtBu·HCl, K2CO3, H2O; iv) TFA, CH2Cl2; v) SOCl2; vi) 1) NaOMe, MeOH, 2) 4-toluoyl chloride, Py.
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syntheses of numerous structurally complicated bioconju-
gates.[33–35,39,40] Accordingly, d-biotin and N-(4-methoxy-
trityl)-1,4-phenylenedimethanamine[41] (17) were first cou-
pled by 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetrameth-
yluronium hexafluorophosphate (HATU) activation to ob-
tain 18. Ligand 19 was then obtained by removal of the 4-
methoxytrityl protection with TFA and coupling of N-Boc-
aminooxyacetic acid to the exposed amino group by DCC
activation with use of 1-hydroxybenzotriazole (HOBt) as an
auxiliary nucleophile. A related tag bearing a primary
amino function has previously been used to facilitate isola-
tion and mass spectrometric characterization of oligosac-
charides.[42]
Assembly of Homotypic Gluco Cluster 27
The methodology for the synthesis of asymmetric hetero-
typic clusters was first studied by preparation of a homo-
typic gluco cluster. To improve the detection of intermedi-
Scheme 2. Synthesis of the biotin moiety. Reagents and conditions:
i) HATU, DIEA, DMF; ii) 1) TFA, CH2Cl2, MeOH,
2) BocAOAOH, HOBt·H2O, DCC, DIEA, DMF.
Scheme 3. Synthesis of the glucose cluster. Reagents and conditions: i) NaCNBH3, DMSO, NMP; ii) MeONH2·HCl, K2CO3, aq. THF;
iii) (Fmoc-β-Ala)2O, CH2Cl2, DMF; iv) Ac2O-capping; v) piperidine, DMF; vi) 4, HOBt, DCC, NMP; vii) Me3P, toluene, aq. 1,4-dioxane;
viii) Fmoc-β-Ala-OH, HBTU, DIEA, DMF; ix) 5, HBTU, DIEA, DMF; x) Pd(Ph3P)4, PhSiH3, CH2Cl2; xi) CH3CO(CH2)2COOH,
HBTU, DIEA, DMF.
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Multipodal Glycoclusters on a Solid Support
ates by RP HPLC, two of the attached glucose ligands car- Pd0 treatment, and levulinic acid was coupled to the ex-
ried toluoyl rather than acetyl protecting groups. Addition- posed branch by HBTU activation to conclude the synthe-
ally, the introduction of two identically protected glucose sis of the ketone-containing glucose cluster 27. Although it
units makes the spiro carbon of the cluster achiral and was not performed here, the method allows the branches to
hence avoids the existence of the cluster as a pair of dia- be further elongated with additional β-alanine units before
stereomers, which markedly facilitates analysis of the test attachment of the sugar residues.
reactions and NMR characterization of the final product.
Accordingly, the applicability of the previously[43] reported
pentaerythrityl tetramine precursor 21 (Scheme 3) to the
synthesis of sugar clusters was first tested with toluoyl- and
Assembly of Heterotypic Cluster 31
acetyl-protected glucopyranosides 4 and 5. 4-(4-Formyl-3,5-
To obtain a cluster with three different sugar ligands a
dimethoxyphenoxy)butyric acid was first attached by slightly modified protocol was used (Scheme 4). Sugar li-
HOBt/DIC-activated (DIC: diisopropylcarbodiimide) cou- gands 9, 10, and 16, each containing a β-alanine-elongated
pling to an aminomethyl polystyrene resin derivatized with side arm, were used in order to reduced the number of cou-
a β-alanine spacer to afford support 20. Scaffold 21 was pling steps on-support. The support 22, containing both
then attached to this support by reductive amination. Both an unprotected primary amino group and an unprotected
of the unprotected amino functions of support-bound 22 secondary amino group, underwent selective acylation of
were acylated with Fmoc-β-alanine in a single step by the the primary function when coupled with β-alanine, consist-
anhydride method to afford 23. The Fmoc protecting ently with previously[43] reported acylations with α-amino
groups were removed with piperidine, and toluoylated acids. However, this was not the case when a closely related
glucose ligand 4 was coupled to the exposed amino groups core structure containing a second azido group in place of
with the aid of HOBt/DCC activation. Coupling to β-al- the Alloc-amino group[43] was used (data not shown); no
anine branches proceeded smoothly and no unreacted selectivity between the acylation of the primary and second-
amino groups could be detected. The azido group was re- ary amino groups with β-alanine could be observed. The
duced to an amino group with trimethylphosphane and the selectivity introduced by the Alloc group on 22 was ex-
third branch was elongated with Fmoc-β-alanine by ploited to achieve the desired versatility. Toluoylated mann-
HBTU-promoted coupling to provide 25. After removal of ose ligand 16 was first coupled to the primary amino group
the Fmoc group with piperidine, the third sugar ligand, by selective HOBt/DCC activation to afford support 28.
compound 5, was coupled by HBTU activation. The Alloc The acylation of the secondary amino group with β-al-
protection was removed from the resulting support 26 by anine-tethered sugars turned out to be very difficult, al-
Scheme 4. Synthesis of the heterotypic cluster. Reagents and conditions: i) 16, HOBt, DCC, NMP; ii) [CH3CO(CH2)2CO]2O, CH2Cl2;
iii) Ac2O-capping; iv) Me3P, toluene, aq. 1,4-dioxane; v) 10, HATU, DIEA, DMF; vi) 12, DIEA, CH2Cl2; vii) Pd(Ph3P)4, PhSiH3, CH2Cl2;
viii) 11, DIEA, CH2Cl2.
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though several coupling reagents were tested (data not sponds to average yields of 82% (32, 14 steps) and 83% (33,
shown). The coupling order was therefore altered and the 11 steps) per solid-supported reaction, including couplings
secondary amino group was acylated with levulinic acid, and deprotections. Global deprotection with NaOMe/
with application of repeated anhydride couplings to pro- MeOH was used to remove the acetyl and toluoyl groups,
duce 29 in a satisfactory yield. The azido group of 29 was followed by RP HPLC purification (Figure 2) to conclude
then reduced to an amino group and the third branch was the synthesis of the homotypic conjugate 34 and the hetero-
subjected to HATU-promoted coupling with acetylated ga- typic conjugate 35. The authenticities of the conjugates 32–
lactose ligand 10. Despite repeated couplings, only modest 35 were verified by ESI-MS (Table 1) and the conjugates
1
progress was obtained. The presence of one β-alanine sugar were characterized by H NMR spectroscopy.
ligand seemed to retard the subsequent couplings markedly.
Use of acid chlorides in these difficult couplings solved the
Table 1. ESI-MS data for biotinylated glycoclusters 32–35.
Conjugate
[(M + 2H+)/2]found
[(M + 2H+)/2]req
problem, however. Accordingly, the support was treated
with galactose ligand 12 to afford 30. The Alloc group was
then removed from the last branch, and the exposed amino
group was treated with glucose ligand 11 to provide the
desired heterotypic cluster 31.
32
33
34
35
1317.5209
1317.5212
761.3320[a]
761.3337[a]
1317.5184
1317.5184
761.3298
761.3298
[a] E/Z isomers; both peaks exhibited the same molecular mass
(Figure 2).
Synthesis of the Biotin Conjugates 32–35
The biotin-derived ligand was attached to the support-
bound clusters by oximation. The Boc protection of 19 was
thus removed with TFA to expose the aminooxy group, and
Conclusions
A method for the synthesis of trivalent homo- and het-
oximation of support-bound clusters 27 and 31 was carried erotypic glycoclusters on a solid support has been devel-
out in pyridine (Scheme 5). The biotinylated clusters were oped. The branches of the glycoclusters can be selectively
acidolytically released from the support and purified by RP constructed through elongation with β-alanine and conju-
HPLC (Figure 2) to afford the protected clusters 32 and 33 gation with various carbohydrates containing carboxy-func-
in 6% and 13% overall yields, respectively. This corre- tionalized side arms. Conjugation with carbohydrates con-
Scheme 5. Synthesis of the biotin conjugates. Reagents and conditions: i) TFA, CH2Cl2, MeOH; ii) resin 27 or 31, Py; iii) TFA, CH2Cl2;
iv) NaOMe, MeOH.
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Multipodal Glycoclusters on a Solid Support
NMR methods (e.g., TOCSY, COSY, HSQC, and HMBC) were
used for peak assignment. Reactions on a solid support were fol-
lowed by acidolytic release of a small aliquot of the resin-bound
compound and subjection of this to LC-MS (ESI) analysis on a
Thermo Hypersil Hypurity C18 (150ϫ4.6 mm, 5 μm) analytical
column at a flow rate of 1 mLmin–1. A linear gradient from TFA
(0.1%) in H2O/MeCN (1:1, v/v) to TFA (0.1%) in MeCN over
15 min was applied. For semipreparative purifications and desalt-
ing, a Thermo Hypersil-Keystone column (250ϫ10 mm, 5 μm) was
used with a flow rate of 3 mLmin–1. Isocratic elution with TFA
(0.1%) and MeCN (85%) in H2O (protocol A) or a linear gradient
from TFA (0.1%) in H2O to TFA (0.1%) in MeCN over 30 min
(protocol B) was used for purifications. UV detection at the wave-
length of 215 nm was applied throughout the work. The mass spec-
tra were recorded by the ESI ionization method.
2-(2,3,4,6-Tetra-O-toluoyl-β-D-glucopyranosyloxy)acetic Acid (4):
Allyl 2,3,4,6-tetra-O-toluoyl-β-d-glucopyranoside (1,[27] 2.54 g,
3.67 mmol) was dissolved in a mixture (4:1, v/v) of 1,4-dioxane and
water (50 mL), and OsO4 (325 μL, 2.5 wt.-% in 2-methylpropanol,
25.9 μmol) was added. The solution was stirred for 1 h, after which
NaIO4 (1.57 g, 7.34 mmol) was added in portions over 1 h. Stirring
was continued for an additional 2.5 h, and the volatiles were then
removed under reduced pressure. The residue was dissolved in
EtOAc and washed with water. The organic phase was dried with
Na2SO4 and filtered, and the solvents were evaporated to dryness.
The obtained aldehyde was dissolved in acetone (100 mL) and co-
oled to 0 °C. CrO3 (367 mg, 3.67 mmol) was dissolved in the small-
est possible amount of water (0.7 mL) together with H2SO4
(204 μL, 3.67 mmol), and added dropwise to the reaction mixture.
After 5 min, the ice water bath was removed and the reaction was
allowed to proceed at ambient temperature for 80 min. The reac-
tion was quenched by addition of propan-2-ol (5 mL) and water
(10 mL) and the organic solvents were evaporated. The residue was
dissolved in CH2Cl2 and washed with water and saturated aqueous
NaCl. The organic phase was dried with Na2SO4 and filtered, and
the solvents were evaporated to dryness. The residue was purified
by silica gel chromatography [pyridine (2%) and MeOH (10%) in
Figure 2. RP HPLC profiles of the purified biotin conjugates in
protected (32 and 33, protocol A) and deprotected (34 and 35, pro-
tocol B) form. The deprotected conjugates each show two peaks
corresponding to the E/Z isomers formed upon oximation.
1
CH2Cl2] to afford 4 (1.93 g, 74%) as a white powder. The H and
taining β-alanine elongated side arms was also applied in
order to diminish the number of coupling steps on support.
However, this led to sterically more demanding couplings,
so these compounds were coupled as acid chlorides instead
of through activator/additive-promoted peptide coupling.
For conjugation of the clusters with d-biotin, an aminooxy-
derivatized biotin ligand was attached by on-support oxim-
ation. Removal of the acyl protecting groups in solution
gave the fully deprotected conjugates. The introduced
4-oxopentynoate (Lev) groups in the clusters can be utilized
in the preparation of various other glycoconjugates by ox-
imation as well.
13C NMR spectra were consistent with the published data.[27]
(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxy)acetic Acid (5):
Allyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside (2,[37] 1.90 g,
4.89 mmol) was subjected to oxidation as described above for allyl
2,3,4,6-tetra-O-toluoyl-β-d-glucopyranoside (1). The crude product
was purified by silica gel chromatography [pyridine (2%) and
MeOH (18%) in CH2Cl2] to afford 5 (1.39 g, 70%) as a white pow-
1
der. The H and 13C NMR spectra were consistent with the pub-
lished data.[44]
(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)acetic Acid (6):
Allyl 2,3,4,6-tetra-O-acetyl-β-d-galactopyranoside (3,[37] 1.56 g,
4.02 mmol) was subjected to oxidation as described above for allyl
2,3,4,6-tetra-O-toluoyl-β-d-glucopyranoside (1). The crude product
was purified by silica gel chromatography [pyridine (2%) and
MeOH (18%) in CH2Cl2] to afford 6 (0.89 g, 54%) as a white pow-
Experimental Section
1
der. H NMR (18% CD3OD in CDCl3, 400 MHz): δ = 5.41 [d, J
General Remarks: 1,4-Dioxane and MeOH were dried with molecu-
lar sieves (3 Å), and CH2Cl2, DMF, DMSO, NMP, and pyridine
over molecular sieves (4 Å). Solid reagents were dried with P2O5 in
a vacuum desiccator. For Ac2O-capping on a solid support, a mix-
ture (1:1, v/v) of capping solutions A [acetic anhydride/(2,6-luti-
dine)/THF 1:1:8, v/v] and B [(N-methylimidazole)/THF 4:21, v/v]
was used. The NMR spectra were recorded at 400 or 500 MHz.
Chemical shifts are given in ppm, with use of internal TMS or
solvent residual signal as a reference. Appropriate 1D and 2D
= 3.2 Hz (avg.), 1 H, 4-H], 5.20 [dd, J = 7.8 (avg.), 10.4 Hz, 1 H,
2-H], 5.08 [dd, J = 3.2 (avg.), 10.4 Hz, 1 H, 3-H], 4.65 [d, J =
7.8 Hz (avg.), 1 H, 1-H], 4.30 (d, J = 15.8 Hz, 1 H, OCHH), 4.20–
4.11 (m, 3 H, 6-H and OCHH), 3.97 (m, 1 H, 5-H), 2.16, 2.11,
2.06, and 2.00 (4ϫs, 4 ϫ 3 H, CH3CO) ppm. 13C NMR (CDCl3,
100 MHz): δ = 170.9, 170.8, 170.4, and 170.3 (CO), 100.4 (C-1),
70.7 (C-5 and C-3), 68.9 (C-2), 67.1 (C-4), 66.6 (OCH2), 61.2 (C-
6), 20.6, 20.4, and 20.3 (CH3CO) ppm. HRMS (ESI): calcd. for
C16H22NaO12 [M + Na]+ 429.1009; found 429.0990.
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tert-Butyl 3-[(2,3,4,6-Tetra-O-toluoyl-β-
D
-glucopyranosyloxy)acet-
(CDCl3, 500 MHz): δ = 10.83 (br, 1 H, OH), 7.89, 7.84, 7.78, and
7.72 (4ϫd, J = 8.2 Hz, 4ϫ 2 H, CH Tol), 7.31 (br. t, J = 6.1 Hz,
1 H, NH), 7.19 (overlapping doublets, 2ϫ 2 H, CH Tol), 7.13 (d,
J = 8.1 Hz, 2 H, CH Tol), 7.07 (d, J = 8.1 Hz, 2 H, CH Tol), 5.91
(dd, J = 9.6, 9.8 Hz, 1 H, 3-H), 5.66 (dd, J = 9.6, 9.9 Hz, 1 H, 4-
H), 5.51 (dd, J = 7.8, 9.8 Hz, 1 H, 2-H), 4.86 (d, J = 7.8 Hz, 1 H,
1-H), 4.64 (dd, J = 2.9, 12.2 Hz, 1 H, 6-Ha), 4.43 (dd, J = 5.6,
12.2 Hz, 1 H, 6-Hb), 4.40 and 4.24 (2ϫd, J = 15.6 Hz, 2ϫ 1 H,
OCH2), 4.16 (ddd, J = 2.9, 5.6, 9.9 Hz, 1 H, 5-H), 3.50 (m, 2 H,
NHCH2), 2.55 (m, 2 H, CH2COO), 2.39, 2.35, 2.34, and 2.28 (4ϫs,
4ϫ 3 H, CH3-Ar) ppm. 13C NMR (CDCl3, 125 MHz): δ = 176.1
(COOH), 170.8, 166.6, 165.9, 165.7, and 165.4 (CO), 144.8, 144.6,
144.4, and 144.3 (C Tol), 130.0, 130.0, 129.9, 129.4, 129.3, 129.3,
and 129.2 (CH Tol), 126.7, 126.0, 126.0, and 125.9 (C Tol), 101.7
amido]propanoate (7): Compound 4 (1.61 g, 2.27 mmol) was con-
verted into the active ester and coupled to β-alanine as described
below for the conversion of compound 6 into 8. The crude product
was purified by silica gel chromatography (EtOAc/PE 3:2), to af-
ford 7 in 84% yield (1.60 g). 1H NMR (CDCl3, 500 MHz): δ =
7.90, 7.86, 7.78, and 7.72 (4ϫd, J = 8.2 Hz, 4ϫ 2 H, CH Tol),
7.20 (overlapping doublets, 2ϫ 2 H, CH Tol), 7.14 (d, J = 8.2 Hz,
2 H, CH Tol), 7.08 (d, J = 8.2 Hz, 2 H, CH Tol), 6.83 (t, J =
6.0 Hz, 1 H, NH), 5.90 (dd, J = 9.6, 9.9 Hz, 1 H, 3-H), 5.63 (dd,
J = 9.6, 9.9 Hz, 1 H, 4-H), 5.50 (dd, J = 7.8, 9.9 Hz, 1 H, 2-H),
4.83 (d, J = 7.8 Hz, 1 H, 1-H), 4.62 (dd, J = 2.9, 12.2 Hz, 1 H, 6-
Ha), 4.44 (dd, J = 5.7, 12.2 Hz, 1 H, 6-Hb), 4.34 (d, J = 15.1 Hz,
1 H, OCHH), 4.15 (ddd, J = 2.9, 5.7, 9.9 Hz, 1 H, 5-H), 4.08 (d, J
= 15.1 Hz, 1 H, OCHH), 3.40 (m, 2 H, NHCH2), 2.40, 2.37, 2.34, (C-1), 72.9 (C-5), 72.2 (C-3), 72.0 (C-2), 69.2 (C-4), 68.8 (OCH2),
and 2.29 (4ϫs, 4 ϫ 3 H, CH3-Ar), 2.34 (m, 2 H, CH2COO), 1.43
[s, 9 H, C(CH3)3] ppm. 13C NMR (CDCl3, 125 MHz): δ = 170.9,
168.5, 166.3, 165.8, 165.5, and 165.3 (CO), 144.5, 144.4, 144.2, and
144.0 (C Tol), 130.0, 130.0, 129.9, 129.9, 129.4, 129.3, 129.3, and
129.2 (CH Tol), 126.8, 126.2, 126.1, and 126.1 (C Tol), 101.5 (C-
1), 81.0 (Cq tBu), 72.9 (C-5), 72.3 (C-3), 72.0 (C-2), 69.3 (C-4), 69.1
(OCH2), 62.9 (C-6), 35.2 (CH2COO), 34.9 (NHCH2), 28.2
[C(CH3)3], 21.8, 21.8, 21.8, and 21.7 (CH3-Ar) ppm. HRMS
(ESI): calcd. for C47H51NNaO13 [M + Na]+ 860.3258; found
860.3220.
62.9 (C-6), 34.9 (NHCH2), 33.4 (CH2COOH), 21.8 and 21.7 (CH3-
Ar) (peaks referring to TFA are not listed) ppm. HRMS (ESI):
calcd. for C43H43NNaO13 [M + Na]+ 804.2632; found 804.2622.
3-[(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)acetamido]-
propanoic Acid (10): Compound 8 (0.948 g, 1.78 mmol) was depro-
tected as described above for the conversion of compound 7 into
9. Compound 10 was obtained in quantitative yield, but only 79%
pure (1.08 g), being contaminated by TFA. The product was used
1
as such in the following reaction. H NMR (CDCl3, 500 MHz): δ
= 11.03 (br, 1 H, COOH), 7.28 (br. t, 1 H, NH), 5.44 (dd, J = 0.8,
3.4 Hz, 1 H, 4-H), 5.22 (dd, J = 7.9, 10.5 Hz, 1 H, 2-H), 5.06 (dd,
J = 3.4, 10.5 Hz, 1 H, 3-H), 4.54 (d, J = 7.9 Hz, 1 H, 1-H), 4.40
and 4.22 (2ϫd, J = 15.5 Hz, 2ϫ 1 H, OCH2), 4.15 (m, 2 H, 6-H),
3.97 (m, 1 H, 5-H), 3.62 (m, 2 H, NHCH2), 2.66 (m, 2 H,
CH2COO), 2.18, 2.10, 2.07, and 2.01 (4ϫs, 4 ϫ 3 H, CH3CO) ppm.
13C NMR (CDCl3, 125 MHz): δ = 176.3 (COOH), 171.1, 171.0,
170.6, 170.5, and 170.4 (CO), 101.1 (C-1), 71.2 (C-5), 70.5 (C-3),
69.1 (C-2), 68.1 (OCH2), 67.0 (C-4), 61.4 (C-6), 34.9 (NHCH2),
33.4 (CH2COO), 20.9, 20.7, 20.7, and 20.6 (CH3CO) (peaks refer-
ring to TFA are not listed) ppm. HRMS (ESI): calcd. for
C19H27NNaO13 [M + Na]+ 500.1380; found 500.1398.
tert-Butyl 3-[(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)acet-
amido]propanoate (8): Compound 6 (0.870 g, 2.14 mmol) was dried
with P2O5 overnight and dissolved in dry 1,4-dioxane (9 mL) to-
gether with N-hydroxysuccinimide (0.300 g, 2.61 mmol). The solu-
tion was cooled on a cold water bath, and DCC (0.536 g,
2.60 mmol) in dry 1,4-dioxane (2 mL) was added. The reaction was
allowed to proceed at room temperature for 4.5 h, followed by re-
moval of the urea byproduct by filtration and washing with dry
dioxane (4 mL). For the coupling reaction, K2CO3 (0.328 g,
2.38 mmol) and β-alanine tert-butyl ester hydrochloride (0.429 g,
2.36 mmol) were dissolved in water (0.6 mL) and added to the solu-
tion of the active ester. After overnight (18 h) reaction, the reaction
mixture was filtered, the filtrate was concentrated to dryness, and
the residue was dissolved in CH2Cl2. The organic phase was
washed with water, dried with Na2SO4, and filtered, and the sol-
vents were evaporated to dryness. The residue was purified by silica
gel chromatography (EtOAc/PE 7:3 to 9:1) to afford 8 (0.971 g,
Synthesis of Acid Chlorides: 3-[(2,3,4,6-Tetra-O-toluoyl-β-d-gluco-
pyranosyloxy)acetamido]propanoyl chloride (11) and 3-[(2,3,4,6-
tetra-O-acetyl-β-d-galactopyranosyloxy)acetamido]propanoyl
chloride (12) were prepared by treating the corresponding acids 9
(1.49 g, 1.64 mmol) and 10 (390 mg, 645 μmol), respectively, with
SOCl2 (50 equiv.). The reaction mixtures were incubated at 38 °C
for 1 h, after which the volatiles were evaporated. Residues were
co-evaporated three times with dry CH2Cl2 to afford the products
in quantitative yields (1.36 g of 11 and 350 mg of 12) as white
foams. The products were used in the subsequent reactions without
characterization.
1
85%) as a colorless solid. H NMR (CDCl3, 500 MHz): δ = 6.88
(t, J = 5.8 Hz, 1 H, NH), 5.40 (dd, J = 0.8, 3.4 Hz, 1 H, 4-H), 5.24
(dd, J = 7.9, 10.5 Hz, 1 H, 2-H), 5.03 (dd, J = 3.4, 10.5 Hz, 1 H,
3-H), 4.51 (d, J = 7.9 Hz, 1 H, 1-H), 4.30 (d, J = 15.0 Hz, 1 H,
OCHH), 4.15 (m, 2 H, 6-H), 4.08 (d, J = 15.0 Hz, 1 H, OCHH),
3.94 (m, 1 H, 5-H), 3.52 (m, 2 H, NHCH2), 2.46 (m, 2 H,
CH2COO), 2.17, 2.12, 2.05, and 2.00 (4ϫs, 4 ϫ 3 H, CH3CO), 1.46
[s, 9 H, C(CH3)3] ppm. 13C NMR (CDCl3, 125 MHz): δ = 171.3,
170.5, 170.2, 170.1, 169.8, and 168.4 (CO), 101.0 (C-1), 81.1 (Cq
tBu), 71.2 (C-5), 70.7 (C-3), 68.9 (C-2), 68.6 (OCH2), 67.0 (C-4),
61.3 (C-6), 35.3 (CH2COO), 34.8 (NHCH2), 28.2 [C(CH3)3], 20.9,
20.8, 20.7, and 20.7 (CH3CO) ppm. HRMS (ESI): calcd. for
C23H35NNaO13 [M + Na]+ 556.2006; found 556.2018.
(2,3,4,6-Tetra-O-toluoyl-α-D-mannopyranosyloxy)acetic Acid (14):
Allyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside[37] (13, 3.2 g,
8.2 mmol) was dissolved in NaOMe/MeOH solution (0.1 molL–1).
The mixture was stirred for 1 h, neutralized by addition of a strong
cation-exchange resin (Dowex 50WX8–200, H+-form), and filtered,
and the solvents were evaporated to dryness. The residue was dis-
solved in dry pyridine, and toluoyl chloride (13 mL, 98 mmol) was
added. The reaction mixture was stirred at 50 °C overnight and
diluted with CH2Cl2. The organic phase was washed with water
and then with saturated aqueous NaHCO3. The organic phase was
dried with Na2SO4 and filtered, and the solvents were evaporated
to dryness. The residue was purified by silica gel chromatography
(CH2Cl2) to afford allyl 2,3,4,6-tetra-O-toluoyl-α-d-mannopyran-
oside (4.3 g, 75%) as a white foam. The toluoylated compound
(3.7 g, 5.3 mmol) was dissolved in a mixture (4:1, v/v) of 1,4-diox-
3-[(2,3,4,6-Tetra-O-toluoyl-β-D-glucopyranosyloxy)acetamido]-
propanoic Acid (9): Compound 7 (1.38 g, 1.65 mmol) was dissolved
in dry CH2Cl2 (4.5 mL) and TFA (1.5 mL) was added. After 2 h,
the solution was concentrated to dryness and the residue was stored
overnight over KOH in a vacuum desiccator and then dried with
P2O5. The crude product still contained approximately 1 equiv.
TFA (1.49 g of 86-percent pure product, quantitative yield). The
product was used as such in the following reaction. 1H NMR
6600
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 6594–6605

Multipodal Glycoclusters on a Solid Support
ane and water (40 mL), and OsO4 (502 μL, 2.5 wt.-% in 2-methyl-
propanol, 40 μmol) was added. The solution was stirred for 1 h,
after which NaIO4 (2.3 g, 10.8 mmol) was added. Stirring was con-
tinued for an additional 1.5 h and the solution was then diluted
with CH2Cl2 and washed with water. The organic phase was dried
with Na2SO4 and filtered, and the solvents were evaporated to dry-
ness. The residue was purified by silica gel chromatography [EtOAc
in petroleum ether (20 Ǟ 40%), then MeOH in CH2Cl2 (5%)] to
afford (2,3,4,6-tetra-O-toluoyl-α-d-mannopyranosyloxy)acetalde-
hyde (1.1 g, 29%). The aldehyde (0.94 g, 1.4 mmol) was dissolved
3-[(2,3,4,6-Tetra-O-toluoyl-α-D-mannopyranosyloxy)acetamido]-
propanoic Acid (16): Compound 15 (0.795 g, 0.948 mmol) was de-
protected as described for the conversion of compound 7 into 9 to
afford 16 in quantitative yield as an 82% pure compound (0.905 g),
contaminated with TFA. The product was used as such in the next
reaction. 1H NMR (CDCl3, 500 MHz): δ = 10.65 (br, 1 H, COOH),
7.95, 7.88, 7.82, and 7.71 (4ϫd, J = 8.1 Hz, 4ϫ 2 H, CH Tol),
7.74 (br. t, 1 H, NH), 7.20 (d, J = 8.1 Hz, 2 H, CH Tol), 7.16
(overlapping doublets, 2ϫ 2 H, CH Tol), 7.08 (d, J = 8.1 Hz, 2 H,
CH Tol), 6.07 (dd, J = 10.1, 10.1 Hz, 1 H, 4-H), 5.84 (dd, J = 3.3,
in acetone (25 mL) and cooled to 0 °C. CrO3 (135 mg, 1.4 mmol) 10.1 Hz, 1 H, 3-H), 5.74 [dd, J = 1.5 (avg.), 3.3 Hz, 1 H, 2-H], 5.15
was dissolved in the smallest possible amount of water (0.3 mL)
together with H2SO4 (75 μL, 1.4 mmol) and added dropwise to the
reaction mixture. After 5 min, the ice water bath was removed and
the reaction was allowed to proceed at ambient temperature for
70 min. The reaction was quenched by addition of 2-propanol
(5 mL) and water (10 mL) and the organic solvents were evapo-
rated. The residue was dissolved in CH2Cl2 and washed with water
and saturated aqueous NaCl. The combined organic phases were
dried with Na2SO4 and filtered, and the solvents were evaporated
to dryness. The residue was purified by silica gel chromatography
[d, J = 1.5 (avg.) Hz, 1 H, 1-H], 4.70 (dd, J = 2.4, 12.2 Hz, 1 H, 6-
Ha), 4.48 (dd, J = 4.8, 12.2 Hz, 1 H, 6-Hb), 4.44 (d, J = 15.6 Hz,
1 H, OCH2), 4.41 (m, 1 H, 5-H), 4.23 (d, J = 15.6 Hz, 1 H, OCH2),
3.64 (m, 2 H, NHCH2), 2.64 (t, J = 6.1 Hz, 2 H, CH2COO), 2.42,
2.42, 2.34, and 2.30 (4 ϫ s, 4 ϫ 3 H, CH3-Ar) ppm. 13C NMR
(CDCl3, 125 MHz): δ = 175.9 (COOH), 170.3 (CONH), 166.7,
166.5, 165.8, and 165.7 (CO), 144.8, 144.8, 144.7, and 144.1 (C
Tol), 130.1, 130.0, 129.9, 129.5, 129.4, 129.3, and 129.3 (CH Tol),
126.8, 126.1, 126.0, and 125.9 (C Tol), 97.9 (C-1), 70.1 and 70.0
(C-3 and C-5), 69.8 (C-2), 66.5 (OCH2), 66.3 (C-4), 62.7 (C-6), 34.8
[pyridine (2%) and MeOH (10%) in CH2Cl2] to afford 14 (0.73 g, (NHCH2), 33.4 (CH2COO), 21.9, 21.8, and 21.8 (CH3-Ar) (peaks
75 %) as a white foam. 1H NMR (10 % CD3OD in CDCl3,
500 MHz): δ = 7.97, 7.90, 7.83, and 7.70 (4ϫd, J = 8.1 Hz, 4ϫ 2
H, CH Tol), 7.21 (d, J = 8.1 Hz, 2 H, CH Tol), 7.15 (d, J = 8.1 Hz,
4 H, CH Tol), 7.05 (d, J = 8.1 Hz, 2 H, CH Tol), 6.10 [dd, J = 10.0
(avg.), 10.1 Hz, 1 H, 4-H], 5.93 (dd, J = 3.2, 10.1 Hz, 1 H, 3-H),
5.80 (br. s, 1 H, 2-H), 5.24 (br. s, 1 H, 1-H), 4.68 (dd, J = 2.4,
12.2 Hz, 1 H, 6-Ha), 4.56 [ddd, J = 2.4, 4.0, 10.0 Hz (avg.), 1 H,
5-H], 4.43 (dd, J = 4.0, 12.2 Hz, 1 H, 6-Hb), 4.32 and 4.28 (2ϫd,
J = 16.5 Hz, 2ϫ 1 H, OCH2), 2.43, 2.42, 2.34, and 2.28 (4ϫs, 4 ϫ
3 H, CH3-Ar) ppm. 13C NMR (10% CD3OD in CDCl3, 125 MHz):
δ = 166.5, 165.7, 165.7, and 165.6 (CO), 144.3, 144.0, and 143.7 (C
Tol), 129.9, 129.8, 129.8, 129.7, 129.3, 129.2, 129.1, and 129.0 (CH
Tol), 127.0, 126.4, 126.3, and 126.2 (C Tol), 97.7 (C-1), 70.3 (C-2),
69.9 (C-3), 69.3 (C-5), 66.6 (C-4), 65.3 (OCH2), 62.6 (C-6), 21.7,
21.6, 21.5, and 21.5 (CH3-Ar) ppm. HRMS (ESI): calcd. for
C40H38NaO12 [M + Na]+ 733.2261; found 733.2270.
referring to TFA are not listed) ppm. HRMS (ESI): calcd. for
C43H43NNaO13 [M + Na]+ 804.2632; found 804.2649.
N-[4-(4-Methoxytritylaminomethyl)benzyl]biotinylamide (18): N-(4-
Methoxytrityl)-1,4-phenylenedimethanamine[41] (17, 720 mg,
1.76 mmol) was dissolved in dry DMF (4 mL). d-Biotin (384 mg,
1.57 mmol) in dry DMF (8 mL) was added, together with 2-(7-aza-
1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HATU, 607 mg, 1.60 mmol) in dry DMF (3 mL) and N,N-
diisopropylethylamine (DIEA, 330 μL, 1.89 mmol). The reaction
was allowed to proceed overnight (19 h) and the mixture was then
concentrated to dryness. The residue was purified by silica gel
chromatography (MeOH in CH2Cl2, 5 Ǟ 10%) to afford 18 (0.86 g,
1
76%). H NMR (CDCl3, 400 MHz): δ = 7.52 (d, J = 7.3 Hz, 4 H,
MMTr), 7.43 (d, J = 8.9 Hz, 2 H, MMTr), 7.34–7.16 (m, 10 H, Ar
and MMTr), 6.82 (d, J = 8.9 Hz, 2 H, MMTr), 6.46 (t, J = 5.7 Hz,
1 H, NHamide), 6.12 and 5.25 (2ϫbr. s, 2ϫ 1 H, 2ϫNHurea), 4.42
(br. dd, 1 H, 3Ј-H), 4.37 (d, J = 5.7 Hz, 2 H, 6-H), 4.24 (br. dd, 1
H, 2Ј-H), 3.77 (s, 3 H, CH3O), 3.30 (br. s, 2 H, 7-H), 3.08 (m, 1 H,
1Ј-H), 2.83 (dd, J = 4.9, 12.9 Hz, 1 H, 4-HaЈ), 2.64 (d, J = 12.9 Hz,
1 H, 4-HbЈ), 2.22 (br. t, 2 H, 4-H), 1.82 (br, 1 H, NH), 1.74–1.55
(m, 4 H, 1-H and 3-H), 1.41 (m, 2 H, 2-H) ppm. 13C NMR (CDCl3,
100 MHz): δ = 173.3 (C-5), 163.9 (C-5Ј), 158.0, 140.4, 138.2, and
137.2 (MMTr), 129.9, 128.6, 128.3, 128.0, 128.0, and 126.4 (MMTr
and Ar), 113.3 (MMTr), 70.6 (Cq MMTr), 61.9 (C-2Ј), 60.3 (C-3Ј),
55.7 (C-1Ј), 55.3 (CH3O), 47.7 (C-7), 43.3 (C-6), 40.6 (C-4Ј), 36.0
(C-4), 28.3 and 28.1 (C-1 and C-2), 25.8 (C-3) ppm. HRMS (ESI):
calcd. for C38H43N4O3S [M + H]+ 635.3056; found 635.3024.
tert-Butyl 3-[(2,3,4,6-Tetra-O-toluoyl-α-D-mannopyranosyloxy)acet-
amido]propanoate (15): Compound 14 (0.705 g, 0.992 mmol) was
converted into the active ester and coupled to β-alanine as de-
scribed above for the conversion of compound 6 into 8. Silica gel
chromatography of the crude product (EtOAc/PE 1:1) afforded
compound 15 in 96% yield (0.797 g). 1H NMR (CDCl3, 500 MHz):
δ = 7.96 and 7.92 (2ϫd, J = 8.1 Hz, 2ϫ 2 H, CH Tol), 7.85 and
7.73 (2ϫd, J = 8.1 Hz, 2ϫ 2 H, CH Tol), 7.21 (d, J = 8.1 Hz, 2
H, CH Tol), 7.18 and 7.15 (2ϫd, J = 8.1 Hz, 2ϫ 2 H, CH Tol),
7.17–7.14 (overlapping signals, 1 H, NH), 7.07 (d, J = 8.1 Hz, 2 H,
CH Tol), 6.08 [dd, J = 9.9 (avg.), 10.1 Hz, 1 H, 4-H], 5.88 (dd, J =
3.3, 10.1 Hz, 1 H, 3-H), 5.75 (dd, J = 3.3, 1.5 Hz, 1 H, 2-H), 5.14
(d, J = 1.5 Hz, 1 H, 1-H), 4.66 (dd, J = 2.1, 12.2 Hz, 1 H, 6-Ha),
4.47 (dd, J = 4.8, 12.2 Hz, 1 H, 6-Hb), 4.41 [ddd, J = 2.1, 4.8, 9.9
tert-Butyl 2-{[4-(Biotinylaminomethyl)benzyl]amino}-2-oxoethoxy-
carbamate (19): Compound 18 (1.90 g, 2.99 mmol) was dissolved
in dry CH2Cl2 (33 mL) containing TFA (1 %). After 5 min, dry
Hz (avg.), 1 H, 5-H], 4.33 and 4.14 (2ϫd, J = 15.2 Hz, 2ϫ 1 H, MeOH (2.2 mL) was added as a scavenger and the mixture was
OCH2), 3.62 (m, 2 H, NHCH2), 2.56 (t, J = 6.3 Hz, 2 H,
CH2COO), 2.43, 2.42, 2.35, and 2.30 (4ϫs, 4 ϫ 3 H, CH3-Ar), 1.45
[s, 9 H, C(CH3)3] ppm. 13C NMR (CDCl3, 125 MHz): δ = 171.6,
stirred for 1.5 h, followed by concentration to dryness. The free
amine and DIEA (0.575 mL, 3.30 mmol) were dissolved in dry
DMF (8 mL). N-(tert-Butoxycarbonyl)aminooxyacetic acid
168.0, 166.3, 165.5, and 165.5 (CO), 144.4, 144.4, 144.1, and 143.8 (0.687 g, 3.59 mmol) was dissolved in dry DMF (11 mL) together
(C Tol), 130.0, 130.0, 129.9, 129.4, 129.3, 129.2, and 129.2 (CH
Tol), 127.1, 126.5, 126.4, and 126.3 (C Tol), 98.0 (C-1), 81.3 (Cq
tBu), 70.0 (C-2), 69.9 (C-5), 69.7 (C-3), 67.3 (OCH2), 66.5 (C-4),
with 1-hydroxybenzotriazole monohydrate (HOBt·H2O, 0.551 g,
3.60 mmol) and DCC (0.743 g, 3.60 mmol). After 40 min, the pre-
cipitated dicyclohexylurea (DCU) was removed and the activated
62.7 (C-6), 35.1 (CH2COO), 34.9 (NHCH2), 28.2 [C(CH3)3], 21.9, acid was added to the solution containing the free amine. The reac-
21.8, 21.8, and 21.7 (CH3-Ar) ppm. HRMS (ESI): calcd. for tion was allowed to proceed for 19 h, the solution was concentrated
C47H51NNaO13 [M + Na]+ 860.3258; found 860.3240.
to dryness, and the residue was purified by silica gel chromatog-
Eur. J. Org. Chem. 2012, 6594–6605
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6601

M. Karskela, M. von Usedom, P. Virta, H. Lönnberg
FULL PAPER
raphy (MeOH in CH2Cl2, 13Ǟ20%). Despite attempted purifica-
tion, approx. 1 equiv. of DCU (determined by the integral ratios of
1H NMR) remained in the product, giving 1.78 g of 66% pure 19
in 73% yield. 1H NMR (17% CD3OD in CDCl3, 400 MHz): δ =
7.46 (t, J = 5.6 Hz, 1 H, NH), 7.24 (m, 4 H, Ar), 4.50 (dd, J = 4.9,
7.8 Hz, 1 H, 3Ј-H), 4.45 and 4.36 (2ϫbr. d, 2ϫ 2 H, 6-H and 7-
H), 4.32 (s, 2 H, 9-H), 4.27 (dd, J = 4.5, 7.8 Hz, 1 H, 2Ј-H), 3.17–
3.11 (m, 1 H, 1Ј-H), 2.91 (dd, J = 4.9, 12.9 Hz, 1 H, 4-HaЈ), 2.72
(d, J = 12.9 Hz, 1 H, 4-HbЈ), 2.22 (t, J = 7.4 Hz, 2 H, 4-H), 1.75–
1.56 (m, 4 H, 1-H and 3-H), 1.47 (m, 2 H, 2-H), 1.43 [s, 9 H,
C(CH3)3] ppm. 13C NMR (CDCl3, 100 MHz): δ = 173.9 (C-5),
169.7 (C-8), 164.0 (C-5Ј), 158.2 (CO Boc), 137.4, 136.9, 127.9,
127.8, 127.8, and 127.7 (Ar), 82.5 (Cq Boc), 75.4 (C-9), 61.8 (C-2Ј),
60.0 (C-3Ј), 55.4 (C-1Ј), 42.9 and 42.4 (C-6 and C-7), 40.2 (C-4Ј),
35.6 (C-4), 28.2 (C-1 and C-2), 27.9 [C(CH3)3], 25.4 (C-3) (peaks
referring to DCU are not listed) ppm. HRMS (ESI): calcd. for
C25H37N5NaO6S [M + Na]+ 558.2362; found 558.2353.
amino groups were acetylated with capping solutions A and B to
afford resin 23.
The Fmoc groups were removed from resin 23 by treatment with
piperidine in DMF (20%) for 30 min, followed by filtration, wash-
ing with DMF, CH2Cl2, and MeOH, and drying of the resin. To
conjugate the first two glycosyl groups, 2-(2,3,4,6-tetra-O-toluoyl-
β-d-glucopyranosyloxy)acetic acid (4, 220 mg, 309 μmol, 10 equiv.)
was dissolved in dry NMP (1 mL). HOBt in NMP (1 mol L–1
,
308 μL, 308 μmol, 10 equiv.) and DCC in NMP (1 molL–1, 306 μL,
306 μmol, 10 equiv.) were added and the solution was shaken for
30 min. The resin (196 mg, loading of 0.15 mmol g–1) was pre-
swelled in dry NMP (1 mL) and mixed with the solution of 4. After
2 h shaking, the resin was filtered, washed with DMF, CH2Cl2, and
MeOH, and dried. The unreacted amino groups were acetylated
with capping solutions A and B to afford resin 24.
Resin 24 (211 mg, theoretical loading of 0.15 mmolg–1) was sus-
pended in a mixture (1:4, v/v) of water and 1,4-dioxane (3.8 mL).
The azido group was reduced to an amino group by addition of a
solution of Me3P in toluene (1 mol L–1, 760 μL, 760 μmol,
24 equiv.) under nitrogen and shaking for 2.5 h. The resin was then
filtered, washed with dioxane, DMF, CH2Cl2, and MeOH, and
dried. β-Alanine was coupled by dissolving Fmoc-β-Ala (202 mg,
648 μmol, 20 equiv.) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate (HBTU, 245 mg, 645 μmol,
20 equiv.) in dry DMF (1.2 mL) and mixing the solution with the
pre-swelled resin in DMF (1 mL). DIEA (224 μL, 1.29 mmol,
40 equiv.) was added and the mixture was shaken overnight (17 h).
The resin was filtered, washed with DMF, CH2Cl2, and MeOH,
and dried. The coupling was repeated to ensure the best possible
loading. The unreacted amino groups were acetylated with capping
solutions A and B to afford resin 25.
Immobilization of the Pentaerythrityl Tetraamine Scaffold on an
Aminomethyl Polystyrene Support – Resin 22: N-(Fluoren-9-yl-
methoxycarbonyl)-β-alanine (N-Fmoc-β-Ala, 781 mg, 2.51 mmol)
and HOBt·H2O (395 mg, 2.58 mmol) were dissolved in dry DMF
(3 mL) and diisopropylcarbodiimide (DIC, 388 μL, 2.52 mmol) was
added. The mixture was shaken for 30 min, added to pre-swelled
aminomethyl polystyrene resin (1.00 g, loading 0.5 mmolg–1), and
shaken for 4 h. The resin was filtered, washed with DMF, CH2Cl2,
and MeOH, and dried. The loading of the resin, determined by the
release of benzofulvene, was 0.44 mmolg–1. The unreacted amino
groups were capped by a 50 min treatment with capping solu-
tions A and B (see General Remarks). The resin was filtered,
washed with DMF, CH2Cl2, and MeOH, and dried. The Fmoc
groups were then removed with piperidine in DMF (20%) over
50 min and the resin was filtered, washed with DMF, CH2Cl2, and
MeOH, and dried. To couple the 4-(4-formyl-3,5-dimethoxyphen-
oxy)butyric acid linker to the exposed amino groups, this acid
(0.485 g, 1.81 mmol) and HOBt·H2O (0.276 g, 1.80 mmol) were dis-
solved in dry DMF (3 mL), DIC (0.276 mL, 1.79 mmol) was
added, and the mixture was shaken for 20 min and then added to
pre-swelled (7 mL of dry DMF) resin. After 6 h shaking, the resin
was filtered, washed with DMF, CH2Cl2, and MeOH, and dried.
The pentaerythrityl tetramine precursor was then attached to a
sample of the derivatized resin 20. For this purpose, 2-[N-(allyloxy-
carbonyl)aminomethyl]-2-(azidomethyl)propane-1,3-diamine[43]
(21, 230 mg, 489 μmol, 5 equiv.) and NaCNBH3 (85 %, 36 mg,
490 μmol, 5 equiv.) were dissolved in a mixture (1:4, v/v) of DMSO
and NMP (1.0 mL) and added to the resin (222 mg, theoretical
loading of 0.44 mmolg–1). After 3 h shaking, the resin was filtered,
washed with DMF, CH2Cl2, and MeOH, and dried. The reaction
was repeated to ensure high loading. The unreacted formyl groups
were capped with a mixture of MeONH2·HCl (82 mg, 977 μmol,
10 equiv.) and K2CO3 (149 mg, 1.08 mmol, 11 equiv.) in a mixture
(1:9, v/v) of water and THF (3.1 mL). After 1 h shaking, the resin
was filtered, washed with aq. THF, THF, DMF, CH2Cl2, and
MeOH, and dried.
The Fmoc group was removed by treating the resin 25 (210 mg,
theoretical loading of 0.15 mmol g–1) with piperidine in DMF
(20 %) for 30 min, followed by filtration, washing with DMF,
CH2Cl2, and MeOH, and drying of the resin. (2,3,4,6-Tetra-O-
acetyl-β-d-glucopyranosyloxy)acetic acid (5, 128 mg, 316 μmol,
10 equiv.) and HBTU (121 mg, 319 μmol, 10 equiv.) were dissolved
in dry DMF (1 mL) and the solution was mixed with the pre-
swelled resin in DMF (1 mL). DIEA (110 μL, 631 μmol, 20 equiv.)
was added and the mixture was shaken for 3 h. The resin was fil-
tered, washed with DMF, CH2Cl2, and MeOH, and dried. The un-
reacted amino groups were acetylated with capping solutions A and
B to afford resin 26.
Resin 26 (212 mg, theoretical loading of 0.15 mmolg–1) was sus-
pended in dry CH2Cl2 (2 mL) and the allyloxycarbonyl group was
removed by addition of PhSiH3 (94 μL, 760 μmol, 24 equiv.) and a
catalytic amount of Pd(Ph3P)4 (approx. 0.5 equiv.). The suspension
was shaken under nitrogen for 1 h and the resin was then filtered,
washed with DMF, CH2Cl2, and MeOH, and dried. The palladium
treatment was repeated. The resin was pre-swelled in dry DMF
(1 mL), and levulinic acid (73.0 mg, 629 μmol, 21 equiv.) together
with HBTU (230 mg, 606 μmol, 20 equiv.) in dry DMF (1 mL)
were added. DIEA (209 μL, 1.20 mmol, 40 equiv.) was added and
the mixture was shaken for 3 h. The resin was filtered, washed with
DMF, CH2Cl2, and MeOH, and dried. The reaction was repeated
twice. The unreacted amino groups were acetylated with capping
solutions A and B to afford resin 27.
Assembly of the Homotypic Glucose Cluster – Resin 27: N-Fmoc-β-
Ala (484 mg, 1.56 mmol, 40 equiv.) was suspended in dry CH2Cl2
(3 mL), and DIC (122 μL, 0.78 mmol, 20 equiv.) was added. After
40 min shaking, the obtained anhydride was added to the pre-
swelled (1 mL of dry DMF) resin 22 (205 mg, theoretical loading
of 0.19 mmol g–1) to acylate the primary and secondary amino
groups. The mixture was shaken for 5 h, filtered, washed with
DMF, NMP, CH2Cl2, and MeOH, and dried. The reaction was
repeated twice to ensure highest possible loading (0.15 mmolg–1,
determined by the release of benzofulvene) and the unreacted
Assembly of the Heterotypic Cluster – Resin 31: Compound 16
(269 mg, 282 μmol, 4 equiv.) was combined with HOBt solution
(285 μL, 285 μmol, 4 equiv., 1 molL–1 in NMP), and DCC was
added (285 μL, 285 μmol, 4 equiv., 1 molL–1 in NMP). The mixture
was shaken for 30 min and added to resin 22 (185 mg, theoretical
6602
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 6594–6605

Multipodal Glycoclusters on a Solid Support
loading of 0.44 mmolg–1) with dry NMP (0.55 mL) to acylate the
primary amino groups. The mixture was shaken for 18 h, filtered,
washed with DMF, CH2Cl2, and MeOH, and dried. The reaction
was repeated to afford resin 28.
solved in aq. MeCN (80%). RP HPLC purification (protocol A)
yielded 2.5 mg [6% overall yield; see Equation (1)] of product 32.
1H NMR (CDCl3, 500 MHz): δ = 7.86 (d, J = 8.1 Hz, 4 H, CH
Tol), 7.83 (d, J = 8.1 Hz, 4 H, CH Tol), 7.77 (d, J = 8.1 Hz, 4 H,
CH Tol), 7.70 (d, J = 8.1 Hz, 4 H, CH Tol), 7.24–7.17 (m, 12 H,
CH Tol and Ar), 7.13 (d, J = 8.1 Hz, 4 H, CH Tol), 7.07 (d, J =
8.1 Hz, 4 H, CH Tol), 5.88 (dd, J = 9.6, 9.7 Hz, 2 H, 2 ϫ 3-H
GlcTol), 5.61 (dd, J = 9.6, 9.8 Hz, 2 H, 2ϫ4-H GlcTol), 5.47 (dd, J
= 7.8, 9.7 Hz, 2 H, 2ϫ2-H GlcTol), 5.21 (dd, J = 8.7, 10.3 Hz, 1
H, 3-H GlcAc), 5.04 (dd, J = 8.7, 10.7 Hz, 1 H, 4-H GlcAc), 5.00
(dd, J = 7.2, 10.3 Hz, 1 H, 2-H GlcAc), 4.84 (d, J = 7.8 Hz, 2 H,
2ϫ1-H GlcTol), 4.60–4.53 (m, 6 H, 2ϫ6-Ha GlcTol, 3ϫ16-Ha, and
1-H GlcAc), 4.45–4.42 (m, 5 H, 6-H or 7, 2ϫ6-Hb GlcTol, and 3Ј-
H), 4.33–4.23 (m, 6 H, 6-H or 7, 9-H, 6-Ha GlcAc, and 2Ј-H), 4.16
(m, 2 H, 2ϫ5-H GlcTol), 4.13–4.08 (m, 4 H, 6-Hb GlcAc and 3ϫ16-
Hb), 3.72 (m, 1 H, 5-H GlcAc), 3.57–3.38 (m, 6 H, 3ϫ15-H), 3.13
(m, 1 H, 1Ј-H), 2.92–2.67 (m, 10 H, 4-HaЈ, 12-H, 3ϫ13-H, and 4-
HbЈ), 2.53 (m, 4 H, 2ϫ14-H), 2.45–2.35 (m, 6 H, 14-H, 10-H, and
11-H), 2.38 (s, 6 H, 2ϫCH3-Ar), 2.34 (s, 12 H, 4ϫCH3-Ar), 2.30–
2.21 (m, 2 H, 4-H), 2.28 (s, 6 H, 2ϫCH3-Ar), 2.05, 2.04, 2.02, and
1.98 (4ϫs, 4 ϫ 3 H, 4ϫCH3 Ac), 1.87 and 1.84 (2ϫs, 3 H, CH3
E/Z isomers), 1.69 (m, 2 H, 3-H), 1.62 (m, 2 H, 1-H), 1.42 (m, 2
H, 2-H) (peaks referring to NH are not listed) ppm. HRMS (ESI):
see Table 1.
Levulinic acid anhydride was prepared as follows: levulinic acid
(343 mg, 2.95 mmol, 40 equiv.) was dissolved in dry CH2Cl2
(0.4 mL), and DIC (225 μL, 1.44 mmol, 20 equiv.) was added. After
20 min, the mixture (except for precipitated DCU) was added to
pre-swelled (0.8 mL of dry CH2Cl2) resin 28 (162 mg, theoretical
loading of 0.44 mmolg–1). The mixture was shaken for 4 h and then
filtered, washed with NMP, DMF, CH2Cl2, and MeOH, and dried.
Washing with DIEA in DMF (2%) and then with DMF, NMP,
CH2Cl2, and MeOH was performed to ensure that the amino
groups were in unprotonated form. The reaction was repeated until
no marked amount of free amino groups remained.The resin was
then subjected to acetic anhydride treatment with the capping solu-
tions A and B to afford resin 29.
PMe3 solution (1.37 mL, 1.37 mmol, 24 equiv., 1 molL–1 in tolu-
ene) was added under N2 to the resin 29 (130 mg, theoretical load-
ing of 0.44 mmol g–1). 1,4-Dioxane (1.2 mL) and water (0.2 mL)
were added and the mixture was shaken for 3.5 h. The resin was
filtered, washed with 1,4-dioxane, MeOH, DMF, CH2Cl2, and
MeOH, and dried. The reaction was repeated. Compound 10
(174 mg, 288 μmol, 5 equiv.) and HATU (109 mg, 287 μmol,
5 equiv.) were dissolved in dry DMF (680 μL) and added to the
resin. DIEA (99 μL, 568 μmol, 10 equiv.) was added and the mix-
ture was shaken for 4 h. The resin was filtered, washed with DMF,
CH2Cl2, and MeOH, and dried. The reaction was repeated twice
without significant progress. Instead, coupling with acid chloride
turned out to work well; the resin was pre-swelled with dry CH2Cl2
(0.70 mL) and DIEA (71 μL, 408 μmol) was added. Compound 12
(168 mg, 330 μmol, 7 equiv.) was dissolved in CH2Cl2 (0.90 mL)
and added to the resin. The reaction mixture was shaken for 18 h,
filtered, washed with CH2Cl2, DMF, CH2Cl2, and MeOH, and
dried. The coupling was repeated and then the resin was treated
with capping solutions A and B to afford resin 30.
Overall yields of the conjugates were calculated by taking the
change in the molecular mass of the product into account. The
final theoretical loading can be calculated by Equation (1):[45]
Lfinal = Linitial/(ΔMϫLinitial + 1) (1)
where Lfinal stands for the final theoretical loading [molg–1], Linitial
for the initial measured loading [molg–1], and ΔM for the change
in the molecular mass of the product.
Biotinylation of the Heterotypic Cluster – Conjugate 33: Compound
19 (851 mg, 1.05 mmol, 30 equiv.) was dissolved in CH2Cl2
(3.0 mL), and MeOH (0.30 mL) and TFA (3.0 mL) were added.
After 3 h stirring the solution was concentrated to dryness and the
residue was dissolved in pyridine (3.0 mL). Half of the solution
(15 equiv.) was added to pre-swelled (1.0 mL of pyridine) resin 31
(79 mg, theoretical loading of 0.44 mmolg–1) and the mixture was
then shaken overnight (21 h). The resin was filtered, washed with
aq. pyridine, pyridine, CH2Cl2, and MeOH, and dried. The reac-
tion was repeated with the rest of the biotin solution (15 equiv.,
stored at –20 °C). The product was released from the support with
TFA in CH2Cl2 (50%, 2 mL for 30 min), the crude product mixture
was filtered, and the filtrate was concentrated to dryness and dis-
solved in aq. MeCN (80%). RP HPLC purification (protocol A)
yielded product 33 [5.5 mg, 13% overall yield, see Equation (1)].
1H NMR (CDCl3, 500 MHz): δ = 7.99 (d, J = 8.1 Hz, 2 H, CH
Tol), 7.90 (d, J = 8.1 Hz, 2 H, CH Tol), 7.88 (d, J = 8.1 Hz, 2 H,
CH Tol), 7.84 (d, J = 8.1 Hz, 4 H, CH Tol), 7.79 (d, J = 8.1 Hz, 2
H, CH Tol), 7.72 (d, J = 8.1 Hz, 2 H, CH Tol), 7.69 (d, J = 8.1 Hz,
2 H, CH Tol), 7.25–7.14 (m, 16 H, CH Tol and Ar), 7.10 (d, J =
8.1 Hz, 2 H, CH Tol), 7.07 (d, J = 8.1 Hz, 2 H, CH Tol), 6.08 (m,
1 H, 4-H Man), 5.91–5.85 (m, 2 H, 3-H Glc and 3-H Man), 5.79
(br. s, 1 H, 2-H Man), 5.58 (br. dd, 1 H, 4-H Glc), 5.44 (m, 1 H,
2-H Glc), 5.42 (d, J = 2.8 Hz, 1 H, 4-H Gal), 5.21 (dd, J = 7.9,
10.3 Hz, 1 H, 2-H Gal), 5.12 (br. s, 1 H, 1-H Man), 5.06 (dd, J =
2.8, 10.3 Hz, 1 H, 3-H Gal), 4.85 (d, J = 7.7 Hz, 1 H, 1-H Glc),
4.70 (br. d, 1 H, 6-Ha Man), 4.61–4.52 (m, 5 H, 6-Ha Glc, 1-H
Gal, and 3ϫ16-Ha), 4.48–4.42 (m, 5 H, 6-Hb Glc, 3Ј-H, 6-H or 7,
and 5-H Man), 4.37–4.30 (m, 6 H, 6-H or 7, 9-H, 6-Hb Man, and
2Ј-H), 4.18–4.07 (m, 6 H, 6-H Gal, 5-H Glc, and 3ϫ16-Hb), 3.96
(br. dd, 1 H, 5-H Gal), 3.72–3.44 (m, 6 H, 3ϫ15-H), 3.18 (m, 1 H,
Resin 30 (90 mg, theoretical loading of 0.44 mmol g–1) was sus-
pended in dry CH2Cl2 (1.60 mL), and the allyloxycarbonyl groups
were removed by addition of PhSiH3 (118 μL, 956 μmol, 24 equiv.)
and a catalytic amount of Pd(Ph3P)4 (approx. 0.5 equiv.). The sus-
pension was shaken under nitrogen for 1 h and the resin was then
filtered, washed with CH2Cl2, DMF, CH2Cl2, and MeOH, and
dried. The resin was pre-swelled with dry CH2Cl2 (0.50 mL). Acid
chloride 11 (179 mg, 220 μmol, 6 equiv.) was dissolved in CH2Cl2
(0.60 mL) together with DIEA (47 μL, 270 μmol), and added to the
resin. The reaction mixture was shaken for 18 h, filtered, washed
with CH2Cl2, DMF, CH2Cl2, and MeOH, and dried. The coupling
was repeated three times and then the resin was treated with cap-
ping solutions A and B to afford resin 31.
Biotinylation of the Homotypic Cluster – Conjugate 32: Compound
19 (447 mg, 0.551 mmol, 30 equiv.) was dissolved in CH2Cl2
(1.5 mL), and MeOH (0.15 mL) and TFA (1.5 mL) were added.
After having been stirred for 3 h, the solution was concentrated to
dryness and the residue was dissolved in pyridine (2.5 mL). Half of
the solution (15 equiv.) was added to pre-swelled (0.80 mL of pyr-
idine) resin 27 (122 mg, theoretical loading of 0.15 mmolg–1), and
the mixture was then shaken overnight (21 h). The resin was filtered
off, washed with aq. pyridine, pyridine, CH2Cl2, and MeOH, and
dried. The reaction was repeated with the rest of the biotin solution
(15 equiv., stored at –20 °C). The product was released from the
support with TFA in CH2Cl2 (50%, 2 mL for 30 min) and the crude
product mixture was filtered, concentrated to dryness, and dis-
Eur. J. Org. Chem. 2012, 6594–6605
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6603

M. Karskela, M. von Usedom, P. Virta, H. Lönnberg
FULL PAPER
[3] B. Ernst, J. L. Magnani, Nat. Rev. Drug Discovery 2009, 8, 661–
677.
1Ј-H), 2.94–2.76 (m, 10 H, 4-HaЈ, 12-H, 3ϫ13-H, and 4-HbЈ), 2.63
(m, 2 H, 14-H), 2.51 (m, 6 H, 10-H, 11-H, and 14-H), 2.46, 2.45
(2ϫs, 2 ϫ 3 H, 2ϫCH3-Ar), 2.40 (s, 5 H, 14-H and CH3-Ar), 2.36
(s, 9 H, 3ϫCH3-Ar), 2.31–2.30 (m, 8 H, 2ϫCH3-Ar and 4-H),
2.15, 2.08, 2.05, and 2.01 (4ϫs, 4 ϫ 3 H, 4ϫCH3 Ac), 1.86 and
1.83 (2ϫs, 3 H, CH3 E/Z isomers), 1.72 (m, 2 H, 3-H), 1.65 (m, 2
H, 1-H), 1.44 (m, 2 H, 2-H) (peaks referring to NH are not
listed) ppm. HRMS (ESI): see Table 1.
[4] O. Srinivas, P. Larrieu, E. Duverger, C. Boccaccio, M.-T.
Bousser, M. Monsigny, J.-F. Fonteneau, F. Jotereau, A.-C. Ro-
che, Bioconjugate Chem. 2007, 18, 1547–1554.
[5] H. Yan, K. Tram, Glycoconjugate J. 2007, 24, 107–123.
[6] L. Zhu, Z. Ye, K. Cheng, D. D. Miller, R. I. Mahato, Biocon-
jugate Chem. 2008, 19, 290–298.
[7] Y. C. Lee, R. T. Lee, Acc. Chem. Res. 1995, 28, 321–327.
[8] M. Mammen, S.-K. Choi, G. M. Whitesides, Angew. Chem.
1998, 110, 2908; Angew. Chem. Int. Ed. 1998, 37, 2754–2794.
[9] J. J. Lundquist, E. J. Toone, Chem. Rev. 2002, 102, 555–578.
[10] B. E. Collins, J. C. Paulson, Curr. Opin. Chem. Biol. 2004, 8,
617–625.
[11] E. M. Munoz, J. Correa, E. Fernandez-Megia, R. Riguera, J.
Am. Chem. Soc. 2009, 131, 17765–17767.
[12] R. J. Pieters, Org. Biomol. Chem. 2009, 7, 2013–2025.
[13] T. K. Dam, C. F. Brewer, Adv. Carbohydr. Chem. Biochem.
2010, 63, 139–164.
Deprotected Conjugates 34 and 35: The protected conjugate (2.5 mg
of 32 or 5.5 mg of 33) was treated with NaOMe in MeOH
(20 mmolL–1, 0.5 mL) for 18 h, and then strong cation-exchange
resin (Dowex 50WX8–200) was added to neutralize the mixture.
The mixture was filtered, the filtrate was concentrated to dryness,
and the resulting residue was dissolved in a biphasic mixture of
Et2O and water (1:1, v/v, 1.0 mL). The organic layer was removed,
the water phase was concentrated to remove the remaining Et2O,
and the crude mixture was desalted and purified by RP HPLC
(protocol B) to give the globally deprotected conjugate in 34% (34,
0.5 mg) or 82% (35, 2.6 mg) isolated yield.
Conjugate 34: 1H NMR (D2O, 500 MHz): δ = 7.21 (m, 4 H, CH
Ar), 4.51–4.46 (m, 3 H, 3Ј-H and 9-H), 4.38 (d, J = 7.9 Hz, 3 H,
3ϫ1-H Glc), 4.35 (br. s, 2 H, 6-H or 7), 4.30–4.22 (m, 6 H, 3ϫ16-
Ha, 6-H or 7, and 2Ј-H), 4.13 (d, J = 15.7 Hz, 3 H, 3ϫ16-Hb),
3.81 (dd, J = 2.2, 12.3 Hz, 3 H, 3ϫ6-Ha Glc), 3.62 (dd, J = 5.6,
12.3 Hz, 3 H, 3ϫ6-Hb Glc), 3.51–3.40 (m, 6 H, 3ϫ15-H), 3.40
[dd, J = 8.8, 9.4 Hz (avg.), 3 H, 3ϫ3-H Glc], 3.37–3.30 (m, 6 H,
3ϫ5-H Glc and 3ϫ4-H Glc), 3.25 [dd, J = 7.9, 9.4 Hz (avg.), 3
H, 3ϫ2-H Glc], 3.18 (m, 1 H, 1Ј-H), 2.98–2.82 (m, 9 H, 12-H,
3ϫ13-H, and 4-HaЈ), 2.69 (d, J = 13.0 Hz, 1 H, 4-HbЈ), 2.48–2.43
(m, 10 H, 3ϫ14-H, 10-H, and 11-H), 2.22 (br. t, 2 H, 4-H), 1.87
and 1.83 (2ϫs, 3 H, CH3 E/Z isomers), 1.61–1.43 (m, 4 H, 3-H
and 1-H), 1.27 (m, 2 H, 2-H) (peaks referring to NH are not
listed) ppm. HRMS (ESI): see Table 1.
Conjugate 35: 1H NMR (D2O, 500 MHz): δ = 7.21 (m, 4 H, CH
Ar), 4.78 (br. d, 1 H, 1-H Man), 4.51–4.45 (m, 3 H, 3Ј-H and 9-
H), 4.38 (d, J = 8.0 Hz, 1 H, 1-H Glc), 4.34 (br. s, 2 H, 6-H or 7),
4.32 (d, J = 7.7 Hz, 1 H, 1-H Gal), 4.29–4.22 (m, 6 H, 3ϫ16-Ha,
6-H or 7, and 2Ј-H), 4.16–4.10 (m, 4 H, 3ϫ16-Hb and 6-Ha Gal),
3.98 (dd, J = 3.1, 15.3 Hz, 1 H, 6-Hb Gal), 3.92 (br. dd, 1 H, 2-H
Man), 3.83 (br. d, 1 H, 4-H Gal), 3.81 (dd, J = 2.0, 12.3 Hz, 1 H,
6-Ha Glc), 3.79–3.74 (m, 2 H, 6-Ha Man and 3-H Man), 3.71–3.54
(m, 5 H, 6-Hb Man, 6-Hb Glc, 5-H Gal, 4-H Man, and 3-H Gal),
3.51–3.39 (m, 9 H, 2-H Gal, 5-H Man, 3ϫ15-H, and 3-H Glc),
3.37–3.30 (m, 2 H, 5-H Glc and 4-H Glc), 3.25 (dd, J = 8.0, 9.3 Hz,
1 H, 2-H Glc), 3.18 (m, 1 H, 1Ј-H), 2.96–2.84 (m, 9 H, 12-H, 3ϫ13-
H, and 4-HaЈ), 2.68 (d, J = 13.0 Hz, 1 H, 4-HbЈ), 2.48–2.44 (m, 10
H, 3ϫ14-H, 10-H, and 11-H), 2.22 (br. t, 2 H, 4-H), 1.87 and 1.83
(2ϫs, 3 H, CH3 E/Z isomers), 1.61–1.43 (m, 4 H, 3-H and 1-H),
1.27 (m, 2 H, 2-H) (peaks referring to NH are not listed) ppm.
HRMS (ESI): see Table 1.
[14] S. Cecioni, S. Faure, U. Darbost, I. Bonnamour, H. Parrot-
Lopez, O. Roy, C. Taillefumier, M. Wimmerov, J.-P. Praly, A.
Imberty, S. Vidal, Chem. Eur. J. 2011, 17, 2146–2159.
[15] A. Imberty, Y. M. Chabre, R. Roy, Chem. Eur. J. 2008, 14,
7490–7499.
[16] B. Ernst, J. L. Magnani, Nat. Rev. Drug Discovery 2009, 8, 661–
677.
[17] Y. M. Chabre, D. Giguere, B. Blanchard, J. Rodrigue, S. Roch-
eleau, M. Neault, S. Rauthu, A. Papadopoulos, A. A. Arnold,
A. Imberty, R. Roy, Chem. Eur. J. 2011, 17, 6545–6562.
[18] M. Almant, V. Moreau, J. Kovensky, J. Bouckaert, S. G. Gouin,
Chem. Eur. J. 2011, 17, 10029–10038.
[19] Z. H. Soomro, S. Cecioni, H. Blanchard, J.-P. Praly, A. Im-
berty, S. Vidal, S. E. Matthews, Org. Biomol. Chem. 2011, 9,
6587–6597.
[20] E. Galante, C. Geraci, S. Sciuto, V. L. Campo, I. Carvalho, R.
Sesti-Costa, P. M. M. Guedes, J. S. Silva, L. Hill, S. A. Nepogo-
diev, R. A. Field, Tetrahedron 2011, 67, 5902–5912.
[21] M. Ambrosi, N. R. Cameron, B. G. Davis, Org. Biomol. Chem.
2005, 3, 1593–1608.
[22] M. Gomez-Garcia, J. M. Benito, D. Rodriguez-Lucena, J.-X.
Yu, K. Chmurski, C. Ortiz Mellet, R. Gutierrez Gallego, A.
Maestre, J. Defaye, J. M. Garcia Fernandez, J. Am. Chem. Soc.
2005, 127, 7970–7971.
[23] M. Gomez-Garcia, J. M. Benito, A. P. Butera, C. Ortiz Mellet,
J. M. Garcia Fernandez, J. L. Jimenez Blanco, J. Org. Chem.
2012, 77, 1273–1288.
[24] H. Lönnberg, Curr. Org. Synth. 2009, 6, 400–425.
[25] P. Arya, A. Barkley, K. D. Randell, J. Comb. Chem. 2002, 4,
193–198.
[26] J. Katajisto, T. Karskela, P. Heinonen, H. Lönnberg, J. Org.
Chem. 2002, 67, 7995–8001.
[27] P. Virta, M. Karskela, H. Lönnberg, J. Org. Chem. 2006, 71,
1989–1999.
[28]
D. V. Filippov, H. van den Elst, C. M. Tromp, G. A.
van der Marel, C. A. A. van Boeckel, H. S. Overkleeft, J. H.
van Boom, Synlett 2004, 5, 773–778.
[29]
[30]
A. Diaz-Moscoso, J. M. Benito, C. Ortiz Mellet, J. M. Gar-
cia Fernandez, J. Comb. Chem. 2007, 9, 339–342.
M. Ortega-Muñoz, F. Perez-Balderas, J. Morales-Sanfrutos, F.
Hernandez-Mateo, J. Isac-Garcia, F. Santoyo-Gonzalez, Eur. J.
Org. Chem. 2009, 15, 2454–2473.
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectra of 6–10, 14–16, 18, 19, and 32–35, and mass
spectra of 34 and 35.
[31]
B. Thomas, M. Fiore, I. Bossu, P. Dumy, O. Renaudet, Beilstein
J. Org. Chem. 2012, 8, 421–427.
Acknowledgments
[32]
[33]
J. Hovinen, Chem. Biodiversity 2006, 3, 296–303.
J. Katajisto, P. Virta, H. Lönnberg, Bioconjugate Chem. 2004,
15, 890–896.
The authors thank Dr. Johanna Katajisto and Emilia Kiuru M.Sc.
for preparing some of the starting materials.
[34]
[35]
M. Karskela, P. Virta, M. Malinen, A. Urtti, H. Lönnberg,
Bioconjugate Chem. 2008, 19, 2549–2558.
M. Karskela, M. Helkearo, P. Virta, H. Lönnberg, Bioconjugate
Chem. 2010, 21, 748–755.
[1] R. A. Dwek, Chem. Rev. 1996, 96, 683–720.
[2] H.-J. Gabius, H.-C. Siebert, S. André, J. Jiménez-Barbero, H.
Rüdiger, ChemBioChem 2004, 5, 740–764.
6604
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 6594–6605

Multipodal Glycoclusters on a Solid Support
[36]
[37]
[41] A. Kiviniemi, P. Virta, H. Lönnberg, Bioconjugate Chem. 2010,
21, 1890–1901.
[42] J. Hsu, S. J. Chang, A. H. Franz, J. Am. Soc. Mass Spectrom.
2006, 17, 194–204.
[43] P. Virta, M. Leppänen, H. Lönnberg, J. Org. Chem. 2004, 69,
2008–2016.
[44] W. H. Binder, W. Schmid, Monatsh. Chem. 1995, 126, 923–931.
[45] T. Karskela, P. Heinonen, P. Virta, H. Lönnberg, Eur. J. Org.
Chem. 2003, 1687–1691.
D. Forget, D. Boturyn, E. Defrancq, J. Lhomme, P. Dumy,
Chem. Eur. J. 2001, 7, 3976–3984.
M. Schmidt, S. K. Chatterjee, B. Dobner, P. Nuhn, Chem. Phys.
Lipids 2002, 114, 139–147.
[38] M. Ghosh, R. G. Dulina, R. Kakarla, M. J. Sofia, J. Org.
Chem. 2000, 65, 8387–8390.
[39] O. P. Edupuganti, Y. Singh, E. Defrancq, P. Dumy, Chem. Eur.
J. 2004, 10, 5988–5995.
[40] A. Meyer, N. Spinelli, P. Dumy, J.-J. Vasseur, F. Morvan, E.
Defrancq, J. Org. Chem. 2010, 75, 3927–3930.
Received: July 13, 2012
Published Online: October 8, 2012
Eur. J. Org. Chem. 2012, 6594–6605
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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