Carbamate linker strategy in solid-phase synthesis of amino-functionalized glycoconjugates for attachment to solid surfaces and investigation of protein-carbohydrate interactions

Article abstract of DOI:10.1002/ejoc.200800670

Amino-functionalized serine-based galactose and glucose neoglycolipids were prepared by solid-phase synthesis using a carbamate strategy for anchoring amino functionalities to a (2-fluoro-4-hydroxymethylphenoxy)acetic acid linker resin. Key synthetic step

Full text of DOI:10.1002/ejoc.200800670

FULL PAPER
DOI: 10.1002/ejoc.200800670
Carbamate Linker Strategy in Solid-Phase Synthesis of Amino-Functionalized
Glycoconjugates for Attachment to Solid Surfaces and Investigation of
Protein-Carbohydrate Interactions
Sara Spjut,[a] Maciej Pudelko,[a][‡] Mirja Hartmann,[a][‡‡] and Mikael Elofsson*[a]
Keywords: Solid-phase synthesis / Glycoconjugates / Carbohydrates / Proteins / Carbohydrate protein interactions / Carba-
mate linker / Gel-phase 19F NMR spectroscopy / Microtiter plates
Amino-functionalized serine-based galactose and glucose
neoglycolipids were prepared by solid-phase synthesis using
a carbamate strategy for anchoring amino functionalities to
a (2-fluoro-4-hydroxymethylphenoxy)acetic acid linker resin.
Key synthetic steps were monitored with gel-phase 19F NMR
neoglycolipids was conjugated with didecyl squarate and
then immobilized in amino-functionalized microtiter plates
and the glycoconjugates were successfully probed with a ga-
lactose-binding lectin.
spectroscopy. Cleavage from the solid support was per- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
formed with trifluoroacetic acid. The terminal amine of the
Germany, 2009)
characterization of interactions. For glycoconjugates the
presentation on the array surface mimics the multivalent
presentation on cell surfaces. The number and type of gly-
coconjugates, the array surface material, and the method
used to attach the glycoconjugate to the surface can vary.
Recent studies have examined several attachment methods
on different types of surfaces.[2–6] A method where two dif-
ferent amines cross-react with dialkyl squarate under mildly
basic conditions is very efficient and can be used for both
primary and secondary amines.[10–13]
Because oligosaccharides and glycoconjugates are com-
plicated to synthesize and the isolation of pure and well-
defined material from natural sources is demanding, ef-
ficient methods for the preparation of these compounds are
needed. Glycosphingolipids, like β-galactosylceramide 1,
are abundant in nature and involved in numerous biological
recognition events.[14] In cell membranes the hydrophobic
part of the glycolipid is buried in the lipid layer and the
hydrophilic part, the carbohydrate portion, is exposed to
the surroundings for recognition. Because the lipid part of
1 contains two stereocenters and one defined double-bond
simplified analogs have been synthesized.[15] The serine-
based neoglycolipid 2 is such an analog.[16,17] The synthesis
of compound 2, subsequent conjugation to amino-function-
alized plates and evaluation with lectins have been de-
scribed.[16,17] In one report the synthesis was carried out on
solid phase using a linker and protective groups containing
fluorine that enable gel-phase 19F NMR spectroscopy to
measure yield and stereochemical outcome during synthe-
sis.[16] The carboxylic acid in compound 2 was coupled to
amino-functionalized microtiter plates and the resulting
neoglycolipid surface was subsequently probed with biotin-
Introduction
Carbohydrates are the most abundant of the four major
classes of biomolecules, which also include proteins, lipids,
and nucleic acids. While proteins and nucleic acids are gen-
erally linear polymers, carbohydrates can be highly
branched and have complex structures, a tendency that re-
sults in almost unlimited structural variation. In cells,
carbohydrates are found as glycoconjugates where the
carbohydrate is attached to other macromolecules forming
e.g. glycoproteins and glycolipids. Glycoconjugates are in-
volved in a wide variety of biological processes, for example
viral entry, signal transduction, inflammation, cell–cell in-
teractions, bacteria–host interactions, fertilization, and de-
velopment.[1] For that reason it is of great interest to further
investigate the role of glycoconjugates in biological systems.
Recently, carbohydrate microarray techniques have been
developed to study carbohydrate–protein interactions.[2–8]
In addition microarray techniques are applicable also to
study small molecules in general.[9] These techniques have
many advantages including high-throughput, minimal con-
sumption of analyte and immobilized molecules, and direct
[a] Department of Chemistry, Umeå University,
90187 Umeå, Sweden
E-mail: mikael.elofsson@chem.umu.se
[‡] Current address: Institut für Organische Chemie, Universität
Mainz,
Duesbergweg 10–14, 55128 Mainz, Germany
[‡‡]Current address: Otto Diels Institute of Organic Chemistry,
Christiana Albertina University of Kiel,
Otto-Hahn-Platz 4, 24098 Kiel, Germany
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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labeled RCA120 from Ricinus communis with affinity for α- e.g. trifluoroacetic acid (TFA).[18,19] The fluorinated linker
and β-Gal, followed by horseradish peroxidase conjugated enables monitoring of key reactions with gel-phase 19F
avidin (avidin-HRP) for detection.
NMR spectroscopy.[20]
In this paper we describe the solid-phase synthesis of the
amino-functionalized biotin model compounds 8a and 8b
and serine-based neoglycolipids 3 and 17 (Figure 1 and
Schemes 2 and 3) using a carbamate-linker strategy and gel-
phase 19F NMR spectroscopy as monitoring technique. The
amino-functionalized conjugates were immobilized in
amino-functionalized microtiter plates using squaric acid
chemistry to give small molecule surfaces that were probed
with labeled avidin and a galactose-binding lectin.
Synthesis of Model Compounds
To evaluate the carbamate-linker strategy we synthesized
the biotinylated model compounds 8a and 8b (Scheme 2)
that also can be used to optimize array conditions using
labeled avidin. Polystyrene resins grafted with polyethyl-
eneglycol e.g. TentagelTM are excellent for solid-phase gly-
coconjugate synthesis and gel-phase 19F NMR spec-
troscopy.[20–23] Resin 4[16] was converted into the carbonate
5 through base-catalyzed reaction with p-nitrophenyl chlo-
roformate (Scheme 1).[18,19] Under basic conditions N-
Fmoc-1,3-diaminopropane and N-Fmoc-1,6-diaminohex-
ane reacted with the carbonate yielding the carbamate resin
6a and 6b. The reactions were monitored with gel-phase 19
F
NMR spectroscopy and all conversions were complete as
illustrated for 6b (Scheme 1). Fmoc-Deprotection was per-
formed using piperidine (20% in DMF) and biotin was cou-
pled to the amine with N,NЈ-diisopropylcarbodiimide
(DIC) and 1-hydroxy-7-azabenzotriazole (HOAt) to give
resin 7a and 7b (Scheme 2). Cleavage from the resin with
trifluoroacetic acid (TFA 90% in water) at room tempera-
ture yielded 8a in quantitative yield and 8b in 70% yield
based on the initial loading of TentaGel-NH2. Functionali-
zation of 8a and 8b was performed with dimethyl squarate
in DMF and triethylamine giving 9a and 9b in quantitative
yields.
Figure 1. β-Galactosylceramide 1 and the serine-based analogs 2
and 3.
Results and Discussion
In previous studies compound 2 (Figure 1) was coval-
ently linked to secondary amines in CovaLinkTM microtiter
plates through the addition of N-hydroxysuccinimide
(NHS) and 1-[3-(diethylamino)propyl]-3-ethylcarbodiimide
(EDC) in water.[16,17] This immobilization procedure re-
quired relatively large amounts of the valuable neoglyco-
lipid, m concentrations in the coupling buffer, and thus a
more efficient strategy would be beneficial. One strategy is
based on the fact that two different amines efficiently cross-
link through dialkyl squarate under mildly basic condi-
tions.[10–13] On the basis of this we designed the amino func-
tionalized neoglycolipid 3 (Figure 1). In order to enable so-
lid-phase synthesis a linker suitable for attachment of
amines was needed and we decided to use a carbamate-
linker strategy[18,19] based on the previously described linker
[2-fluoro-4-(hydroxymethyl)phenoxy]acetic acid.[16] The
carbamate linkage is expected to withstand conditions
throughout the glycoconjugate synthesis and subsequent
Scheme 1. Synthesis of the carbamate resin 6a and 6b and gel-phase
19F NMR spectra recorded for resin 4, 5, and 6b. a) N-methyl
morpholine, p-nitrophenyl chloroformate, CH2Cl2; b) N-Fmoc-1,3-
diaminopropane hydrochloride or N-Fmoc-1,6-diaminohexane hy-
cleavage can be performed with acid of moderate strength, drochloride, N,N-diisopropylethylamine, DMF.
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Carbamate-Linker Strategy for Amino-Functionalized Glycoconjugates
Immobilization and Detection of Compounds 9a and 9b in
Amino-Functionalized Microtiter Plates
Compounds 9a and 9b were dissolved in sodium hydro-
carbonate buffer (pH 9) and serially diluted (1.0 m to
0.24 n for 9a and 1.1 m to 2.2 n for 9b) in transparent
amino-functionalized microtiter plates and incubated for
18 h at room temperature. The immobilized model com-
pounds 9a and 9b were detected with avidin-HRP (4 µg/mL
in PBS containing 0.05% Tween 20, 100 µL) and subse-
quent addition of substrate solution. Optimization resulted
in conditions producing good signal-to-noise ratio allowing
efficient detection of the immobilized biotin (Figure 2, a
and b). Neither blocking with bovine serum albumin
(BSA)[16] nor blocking with acetic anhydride (20% in
water)[13] improved the results for the biotin avidin-HRP
combination. In all three cases unspecific binding of biotin
avidin-HRP was low. A second improvement was to replace
a washing step with Cova buffer by water. These results
indicate that the carbamate linker and squarate array strat-
egy are promising for more complex compounds.
To further explore the potential of the strategy the model
compounds 9a and 9b (2.5 m to 0.61 µ) were also immo-
bilized in white amino-functionalized microtiter plates and
subsequently probed with fluorescein isothiocyanate labeled
avidin (avidin-FITC) (Figure 2, c and d). During optimiza-
tion of this protocol it was found that blocking with BSA
gave less unspecific binding of avidin-FITC compared to no
blocking or blocking with acetic anhydride.
Scheme 2. Synthesis of model compounds 9a and 9b. a) i) Piperidine
(20% in DMF); ii) HOAt, DIC, -biotin, DMF; b) 3ϫTFA (90%
in H2O), quantitative yield and 70% yield, respectively; c) dimethyl
squarate, TEA, DMF, quantitative yield for both 9a and 9b.
Figure 2. a) and b) Binding of avidin-HRP to the immobilized model compounds 9a and 9b. c) and d) binding of avidin-FITC to the
immobilized model compounds 9a and 9b. The compounds were serially diluted and covalently immobilized in wells of clear (a and b)
or white (c and d) aminofunctionalized microtiter plates and probed with the given avidin conjugate. The concentrations of the model
compounds added to the well are given on the x-axis. The points represent the average of triplicate runs and error bars are set to Ϯ one
standard deviation. In the graphs, the average value of a triplicate of controls was subtracted from the observed values (triplicates).
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The data clearly show the efficiency of the immobiliza- cose donors 11 and 12 (Figure 3) were synthesized accord-
tion chemistry and that detection can be based on both ing to previously described methods.[20] The fluorinated
absorbance and fluorescence. Detection with avidin-HRP benzoyl groups and the fluorinated linker make it possible
was superior compared to avidin-FITC both in terms of to monitor the outcome of the glycosylations with gel-phase
background level and signal to noise. Binding of avidin to 19F NMR spectroscopy. N-Bromosuccinimide (NBS) and
the two model compounds appeared to be unaffected by tetrabutylammonium trifluoromethanesulfonate (QOTf)
the two spacer lengths used (see Figure 2, compare part a were used as the promoter system.[24] Glycosylation of 10
with b and c with d).
with 11 (3 equiv.), QOTf (0.65 equiv.), and NBS (4 equiv.)
resulted in 50% glycosylation according to gel-phase 19F
NMR spectroscopy. When the procedure was repeated the
yield increased to 65% yield. Glycosylation of 10 with 12
under similar conditions gave 35% glycosylation and when
the procedure was repeated, 57% yield according to gel-
phase 19F NMR spectroscopy. Although the 19F NMR
spectra allowed estimation of the glycosylation yields the
spectra were of relatively poor quality. The reason for the
strong broadening of the fluorine signals is not known.
Compound 13 and 14 were cleaved from the resin by ad-
dition of TFA/H2O, 9:1 giving the partially deprotected
neoglycolipids 15 and 16 in 24% and 12% yield, respec-
tively, based on the initial loading of TentaGel-NH2 resin.
The p-fluorobenzoate protecting groups were removed
using NaOMe in methanol to give the deprotected neogly-
colipids 3 and 17. Both products were problematic to purify
with flash-column chromatography. Preparative reversed-
phase LC-MS gave partially pure 3 and it was therefore
decided to carry out purification after functionalization
with didecyl squarate. Compound 3 and 17 were treated
with didecyl squarate (ca. 3.3 equiv.)[13] to give 18 and 19 in
20% and 27%, respectively after purification with reversed-
phase HPLC. Didecyl squarate gives a more lipophilic
product than dimethyl squarate, which was considered im-
portant from a purification point of view. The total yield
based on the initial loading of the resin was 4.8% for 18
and 2.4% for 19 over 13 steps corresponding to an average
yield of 75–80% in each step.
Synthesis of Neoglycolipids
For preparation of neoglycolipids the longer spacer, i.e.
resin 6b, was chosen as starting material. The serine-based
lipid part 10 was assembled from 6b under standard peptide
synthesis conditions (Scheme 3).[16] The galactose and glu-
Figure 3. Glycosyl donors.
Immobilization and Detection of the Neoglycolipids in
Amino-Functionalized Microtiter Plates
The neoglycolipid 18 was immobilized in transparent Co-
vaLinkTM microtiter plates as described for the biotin deriv-
atives 9a and 9b. Compound 18 was dissolved in sodium
hydrocarbonate buffer (pH 9) and serially diluted (0.1 m–
48n) on the microtiter plates and incubated for 24 h at
room temperature. The immobilized neoglycolipid was de-
tected with biotin-conjugated lectin from Ricinus communis
(RCA120, 5 µg/mL in PBS containing 0.05% Tween 20,
Scheme 3. Synthesis of squaric amide ester neoglycolipid 18 and
19. aa) i) Piperidine (20% in DMF); ii) Fmoc-6-aminohexanoic
acid, HOBt, DIC, DMF, BFB; iii) Piperidine (20% in DMF); iv)
Fmoc--serine HOBt, DIC, DMF, BFB; v) piperidine (20% in
DMF); vi) hexanoic acid, HOBt, DIC, DMF, BFB; b) 11 or 12,
QOTf, NBS, MS 3 Å, CH2Cl2, 65% and 57% respectively; c) TFA/
H2O, 9:1, 24% and 12% yield respectively; d) NaOMe (0.20  in
MeOH), MeOH; e) didecyl squarate, TEA, DMF, 20% and 27%
yield respectively.
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Carbamate-Linker Strategy for Amino-Functionalized Glycoconjugates
Scheme 4. Immobilization of squaric amide ester neoglycolipid 18 and 19 in amino-functionalized microtiter plates. a) NaHCO3 buffer
(pH 9), CovaLinkTM (Nunc A/S, Denmark) microtiter plate.
to the ten-fold higher concentration required when 2 was
immobilized as an amide using the same lectin and type of
plate (Scheme 4).[16]
The lectin RCA120 is specific for α- and β-galactose and
will therefore not recognize glucose. To investigate whether
the lectin can distinguish galactose from glucose in this
setup, 18 and 19 were dissolved and diluted to 0.025 m in
NaHCO3 buffer (pH 9). This concentration gives a strong
signal as shown in part a of Figure 4. The compounds were
immobilized and detected as described for 18. No binding
of the lectin was observed in the wells reacted with the glu-
cose neoglycolipid 19 (Figure 4, b).
Conclusions
The carbamate-linker strategy proved to be sufficiently
effective to allow solid-phase synthesis of amino-function-
alized biotin model compounds and galactose and glucose
neoglycolipids. With didecyl squarate the molecules were
cross-linked to microtiter plate wells functionalized with
secondary amines. The biotin plates were successfully
probed with avidin labeled with both HRP and FITC. The
glycoconjugate plates were successfully probed with a galac-
tose specific lectin labeled with HRP. Our results indicate
that this strategy can be applied for solid-phase synthesis of
amino-functionalized small molecules for attachment to so-
lid surfaces and subsequent detection with proteins carrying
different labels.
Figure 4. a) Binding of galactose selective lectin from Ricinus com-
munis (RCA120) to the immobilized neoglycolipid 18. The com-
pound was serially diluted in the wells and covalently immobilized
to the amino-functionalized wells. The immobilized neoglycolipid
was detected with biotin-conjugated RCA120 and avidin-HRP. The
concentration of the neoglycolipid added to the well is given on the
x axis. The points represent the average of triplicate runs and error
bars are set to Ϯ one standard deviation. In the graphs, the average
value of a triplicate of controls was subtracted from the observed
absorbencies (triplicates). b) Binding of the galactose selective lec-
tin RCA120 to the immobilized neoglycolipids 18 and 19
(0.025 m). The average value of a triplicate of controls was sub-
tracted from the observed absorbencies (triplicates). No binding
of the lectin was observed in the wells reacted with the glucose
neoglycolipid 19.
Experimental Section
General: Solid-phase synthesis was performed on TentaGel HL-
NH2 resin (0.42 mmol/g) from Rapp Polymere. CH2Cl2 was dis-
tilled from calcium hydride and DMF was distilled under vacuum.
Solvent mixtures are reported as volume (v/v) ratios. TLC was run
on Silica Gel 60 F254 (Merck) and the spots were detected in UV-
light and stained with H2SO4 in ethanol and heat. Silica gel
(Matrex, 60 Å, 35–70 mm, Grace Amicon) and solvents of analyti-
1
cal grade were used for flash column chromatography. H and 13C
NMR spectra were recorded at 298 K with a Bruker DRX-400 at
400 and 100 MHz, respectively, with CDCl3 or [D4]MeOH as sol-
100 µL) and avidin-HRP (4 µg/mL in PBS containing
0.05% Tween 20, 100 µL). A clear dose-response is ob-
served and indicating that the immobilization method of
the neoglycolipid was effective (Figure 4, a). As low as
0.01 m concentration of glycoconjugate 18 in the immobi-
vent and residual CHCl3 (δH = 7.27 ppm) or [D4]MeOH (δH
=
3.30 ppm) as internal standard for 1H and CDCl3 (δC = 77.23 ppm)
or [D4]MeOH (δC = 49.15 ppm) as internal standard for 13C. Peaks
that could not be assigned are not reported. J values are given in
Hz. Gel-phase proton decoupled 19F NMR spectra were recorded
lization buffer resulted in maximum absorbance in contrast at 298 K with a Bruker DRX-400 at 376 MHz on resin suspensions
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S. Spjut, M. Pudelko, M. Hartmann, M. Elofsson
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in CDCl3 with CFCl3 (δF = 0.00 ppm) as internal standard. Two
peaks appear in the spectra around 0 ppm. One is originating from
CFCl3 inside the polymer and one from CFCl3 outside the polymer.
The peak with higher shift was used as internal standard. Prepara-
tive reversed-phase LCMS were performed with a Waters LC sys-
tem equipped with an XTerra C-18 column (50ϫ19 mm, 5 µm,
125 Å), eluted with a linear gradient of MeCN in water, both of
which contained formic acid (0.2%) with a flow rate of 25 mLmin–1
and detection at 214 and 254 nm. Positive and negative electrospray
mass analyses were carried out with a Waters Micromass ZG 2000.
Preparative HPLC separations were performed with a Beckman
System Gold HPLC, using a Supelco Discovery Biowide Pore C18
column (250ϫ212 mm, 5 µm) eluted with a linear gradient of
MeCN in water, both of which contained trifluoroacetic acid
(0.1%). The flow rate was 11 mLmin–1 and detection at 214 nm.
Analytical HPLC were performed with a Beckman System Gold
HPLC, using a Supelco Discovery Biowide Pore C18 column
(250ϫ46 mm, 5 µm) with a flow rate of 1.5 mLmin–1 and detection
at 214 nm. In arrays; the absorbance was measured with a Tecan
Infinite® 200 plate reader and fluorescence with a Wallac Victor2 
1420 Multilabel Counter. High-resolution mass spectra were re-
corded with a Jeol SX102 mass spectrometer. Ions were produced
by a beam of Xenon atoms (6 keV) from a matrix of 3-nitrobenzyl
alcohol.
According to gel-phase 19F NMR the conversion was quantitative.
19F NMR (CDCl3): δ = –134.0 ppm.
Resin 7b: Resin 6b (0.075 mmol) was treated with piperidine (20%
in DMF) for 5 min and washed with DMF (3ϫ5 mL) and distilled
DMF (3ϫ5 mL). 1-Hydroxy-7-azabenzotriazole (HOAt) (0.065 g,
0.474 mmol) and -biotin (0.077 g, 0.315 mmol) were dissolved in
distilled DMF (5 mL), N,NЈ-diisopropylcarbodiimide (DIC)
(0.049 mL, 0.316 mmol) was added and the mixture was stirred for
15 min at room temperature. The solution was added to the resin
followed by bromophenolblue (BFB) (20 µL, 2 m in DMF). The
mixture was agitated at room temperature for 16 h and washed with
CH2Cl2, DMF, THF, MeOH, DMF and CH2Cl2 (10 mL of each).
According to gel-phase 19F NMR the conversion was quantitative.
19F NMR (CDCl3): δ = –134.0 ppm.
1-Amino-3-(biotinoylamino)propane (8a): TFA (90% in H2O, 6 mL)
was added to resin 7a (0.075 mmol) and shaken at room tempera-
ture for 2 h. This procedure was repeated twice, the resin was fil-
tered and washed thoroughly with MeOH, CH2Cl2 and MeOH.
The solvents were combined and concentrated in vacuo. The prod-
uct was purified by preparative LC-MS (100% H2O to 100%
MeCN, 10 min) yielding 8a (60 mg, quantitative yield based on
resin loading). 1H NMR ([D4]MeOH): δ = 4.52–4.49 [m, 1 H,
CH(NH)CH2], 4.33–4.30 [m, 1 H, CH(NH)CH], 3.23–3.19 (m, 1
H, CHS), 3.15–3.12 (m, 2 H, CH2NHCO), 2.95–2.91 [m, 3 H,
NH2CH2, CHH(S)CH], 2.72–2.69 [m, 1 H, CHH(S)CH], 2.26–2.22
(m, 2 H, NHCOCH2), 1.88–1.83 (m, 2 H, COCH2CH2CH2CH2CH),
1.81–1.75 (m, 2 H, NH2CH2CH2CH2NH), 1.73–1.54 (m, 2 H,
COCH2CH2CH2CH2CH), 1.49–1.43 (m, 2 H, COCH2CH2CH2-
CH2CH) ppm. MS (ES+) calcd. for C13H25N4O2S 301.17 m/z [M +
H]+, observed 301.08.
Resin 5: The linker resin 4[16] (0.424 mmol) was allowed to swell in
4 mL of dry CH2Cl2, N-methylmorpholine (0.100 mL, 0.920 mmol)
was added and the mixture was cooled to 0 °C. p-Nitrophenyl chlo-
roformate (0.178 g, 0.883 mmol) was dissolved in 1 mL of dry
CH2Cl2 and added slowly under cooling. The mixture was agitated
overnight at room temperature and finally the resin was washed
with CH2Cl2, DMF and CH2Cl2 (5ϫ5 mL) yielding 5. According
to gel-phase 19F NMR the conversion was quantitative. 19F NMR
(CDCl3): δ = –133.5 ppm.
1-Amino-6-(biotinoylamino)hexane (8b): TFA (90% in H2O, 6 mL)
was added to resin 7b (0.075 mmol) and shaken at room tempera-
ture for 2 h. This procedure was repeated twice, the resin was fil-
tered and washed thoroughly with MeOH, CH2Cl2 and MeOH.
The solvents were combined and concentrated in vacuo. The prod-
uct was purified by flash column chromatography (four gradient
steps: CHCl3, CHCl3/MeOH, 1:1, CHCl3/MeOH/H2O, 65:35:7,
MeOH/H2O, 1:1) yielding 8b (18 mg, 70% yield based on resin
loading). Data for 8b in agreement with those previously pub-
lished.[25]
Resin 6a: The carbonate linker resin 5 (0.350 mmol) was washed
with dry DMF (2ϫ5 mL) and then allowed to swell in dry DMF
(5 mL). N-Fmoc-1,3-Diaminopropane hydrochloride (0.300 g,
1.01 mmol) and N,N-diisopropylethylamine (0.180 mL, 1.01 mmol)
were added and agitated at room temperature for 16 h. The resin
was washed with DMF (10ϫ5 mL), agitated with DMF for
10 min, washed with DMF, THF and CH2Cl2 (3ϫ5 mL) to give 6.
According to gel-phase 19F NMR the conversion was quantitative.
19F NMR (CDCl3): δ = –134.2 ppm.
3-[3-(Biotinoylamino)propylamino]-4-methoxy-3-cyclobuten-1,2-dione
(9a): Compound 8a (0.030 g, 0.100 mmol) was dissolved in distilled
DMF (7 mL), dimethyl squarate (0.057 g, 0.400 mmol) and trieth-
ylamine (56 µL, 0.402 mmol) were added and the mixture was
stirred at room temperature for 19 h and followed by TLC. The
reaction mixture was lyophilized and purified by flash column
chromatography (five gradient steps: heptane, heptane/CHCl3, 1:1,
CHCl3, CHCl3/MeOH, 1:1, MeOH) giving 9a (59 mg, quantitative
Resin 6b: The carbonate linker resin 5 (0.424 mmol) was washed
with dry DMF (2ϫ5 mL) and then allowed to swell in dry DMF
(5 mL). N-Fmoc-1,6-Diaminohexane hydrochloride (0.394 g,
1.05 mmol) and N,N-diisopropylethylamine (0.190 mL, 1.09 mmol)
were added and agitated at room temperature for 22 h. The resin
was washed with DMF (10ϫ5 mL), agitated with DMF for
10 min, washed with DMF, THF and CH2Cl2 (3ϫ5 mL) to give 6.
According to gel-phase 19F NMR the conversion was quantitative.
19F NMR (CDCl3): δ = –134.2 ppm.
1
yield). H NMR ([D4]MeOH, mixture of two rotamers): δ = 4.57–
4.50 [m, 1 H, CH(NH)CH2], 4.38–4.36 (m, 3 H, CH3OC), 4.33–
4.28 [m, 1 H, CH(NH)CH], 3.71–3.66 (m, 1 H, CH2NHC), 3.51–
3.45 (m, 1 H, CH2NHC), 3.25–3.19 [m, 3 H, CH2(NH)CO, CHS],
2.97 [dd, J = 12.7 and 4.9 Hz, 1 H, CH2(S)CH], 2.74 [d, J =
12.6 Hz, 1 H, CH2(S)CH], 2.26 (t, J = 7.4 Hz, 2 H, CH2CONH)
Resin 7a: Resin 6a (0.173 mmol) was treated with piperidine (20%
in DMF) for 5 min and washed with DMF (3ϫ5 mL) and distilled
DMF (3ϫ5 mL). 1-Hydroxy-7-azabenzotriazole (HOAt) (0.141 g,
1.04 mmol) and -biotin (0.170 g, 0.696 mmol) were dissolved in 1.83–1.75 (m, 2 H, NHCH2CH2CH2NH), 1.75–1.54 (m, 2 H,
distilled DMF (5 mL), N,NЈ-diisopropylcarbodiimide (DIC)
(0.107 mL, 0.691 mmol) was added and the mixture was stirred for
15 min at room temperature. The solution was added to the resin
followed by bromophenolblue (BFB) (20 µL, 2 m in DMF). The
mixture was agitated at room temperature for 16 h and washed with
COCH2CH2CH2CH2CH, COCH2CH2CH2CH2CH), 1.49–1.42 (m,
2 H, COCH2CH2CH2CH2CH) ppm. 13C NMR ([D4]MeOH): δ =
207.2, 193.4, 191.6, 176.2, 174.5, 166.0, 63.3, 61.6, 61.2, 57.0, 43.2,
42.7, 41.0, 37.3, 37.1, 36.8, 31.5, 31.2, 29.8, 29.5, 26.8 ppm. [α]2D5
=
+30 (MeOH). HRMS (FAB) calcd. for C18H26N4NaO5S 433.1522
CH2Cl2, DMF, THF, MeOH, DMF and CH2Cl2 (10 mL of each). [M + Na]+, found 433.1531.
354
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 349–357

Carbamate-Linker Strategy for Amino-Functionalized Glycoconjugates
3-[6-(Biotinoylamino)hexylamino]-4-methoxy-3-cyclobuten-1,2-dione 8.1 Hz, 2 H, ArH), 7.41–7.32 (m, 5 H, ArH), 7.11–7.04 (m, 4 H,
(9b): Compound 8b (0.012 g, 0.034 mmol) was dissolved in distilled
DMF (5 mL), dimethyl squarate (0.029 g, 0.205 mmol) and trieth-
ylamine (20 µL, 0.143 mmol) were added and the mixture was
stirred at room temperature for 91 h and followed by TLC. The
reaction mixture was lyophilized and purified by flash column
chromatography (CHCl3 to CHCl3/MeOH/H2O, 65:35:7) giving 9b
ArH), 6.99 (t, J = 8.7 Hz, 2 H, ArH), 5.70 (t, J = 9.8 Hz, 1 H, 2-
H), 5.49 (s, 1 H, PhCH), 5.31 (dd, J = 3.3 and 10.0 Hz, 1 H, 3-H),
4.88 (d, J = 9.8 Hz, 1 H, 1-H), 4.55 (d, J = 3.0 Hz, 1 H, 4-H), 4.44
(dd, J = 1.3 and 12.4 Hz, 1 H, 6-H), 4.08 (dd, J = 1.5 and 12.4 Hz,
1 H, 6-H), 3.74 (s, 1 H, 5-H), 2.35 (s, 3 H, CH3) ppm. 19F NMR
(CDCl3): δ = –105.2 and –105.6 ppm. 13C NMR (CDCl3): δ =
167.4, 167.3, 165.2, 164.8, 164.7, 164.1, 138.7, 137.6, 134.6, 132.7,
132.6, 132.5, 132.4, 129.7, 129.2, 128.2, 126.9, 126.6, 125.9, 125.8,
1
(22 mg, quantitative yield). H NMR ([D4]MeOH): δ = 4.51 [dd, J
= 7.8 and 4.3 Hz, 1 H, CH(NH)CH2], 4.38 (s, 3 H, CH3OC), 4.35–
4.31 [m, 1 H, CH(NH)CH], 3.62 (t, J = 6.9 Hz, 1 H, CH2NHC), 125.4, 125.3, 115.9, 115.8, 115.6, 115.5, 101.1, 85.3, 74.3, 73.7, 69.9,
3.42 (t, J = 6.9 Hz, 1 H, CH2NHC), 3.26–3.16 [m, 3 H, CH2(NH)
CO, CHS], 2.95 [dd, J = 12.7 and 5.0 Hz, 1 H, CH2(S)CH], 2.71
[d, J = 12.7 Hz, 1 H, CH2(S)CH], 2.22 (t, J = 7.3 Hz, 2 H, 83 °C; 12: H NMR (CDCl3): δ = 8.00–7.91 (m, 4 H, ArH), 7.40–
CH2CONH) 1.85–1.29 (m, 14 H, NHCH2CH2CH2CH2CH2CH2, 7.29 (m, 7 H, Ar), 7.14–6.99 (m, 6 H, ArH), 5.73 (t, J = 9.4 Hz, 1
69.2, 67.5, 21.4 ppm. HRMS (FAB) calcd. for C34H28F2NaO7S
641.1421 [M + Na]+ found 641.1423. [α]2D5 = +31 (MeOH); m.p.
1
COCH2CH2CH2CH2CH) ppm. 13C NMR ([D4]MeOH): δ = 206.5,
196.9, 193.5, 191.6, 175.9, 166.0, 63.3, 61.6, 61.1, 59.8, 57.0, 45.5,
45.2, 41.0, 40.1, 36.8, 29.7, 29.5, 26.9, 24.2 ppm. [α]2D5 = +27
(MeOH). HRMS (FAB) calcd. for C21H32N4NaO5S 475.1991 [M
+ Na]+, found 475.1996.
H, 3-H), 5.53 (s, 1 H, PhCH-), 5.39 (dd, J = 9.27 and 0.65 Hz, 1
H, 2-H), 4.94 (d, J = 9.98 Hz, 1 H, 1-H), 4.46 (dd, J = 5.0 and
5.76 Hz, 1 H, 5-H), 3.89–3.84 (m, 2 H, 4-H, 6-Ha), 3.75–3.70 (m,
1 H, 6-Hb), 2.35 (s, 3 H, CH3) ppm. 19F NMR (CDCl3): δ = –105.2
and –105.6 ppm. 13C NMR (CDCl3): δ = 101.5, 87.1, 78.5, 73.6,
7 1 . 3 , 7 0 . 9 , 6 8 . 5 , 2 1 . 2 p p m . H R M S ( FA B ) c a l c d . fo r
Resin 10: Resin 6 (0.424 mmol) was treated with piperidine (20%
in DMF, 2ϫ5 mL, 10 min) and washed with DMF (3ϫ5 mL) and
distilled DMF (2ϫ5 mL). Fmoc-6-aminohexanoic acid (0.597 g,
1.69 mmol), HOBt (0.346 g, 2.56 mmol) and DIC (0.264 mL,
1.69 mmol) were dissolved in distilled DMF (5 mL) and stirred at
room temperature for 10 min. The solution was transferred to the
resin followed by addition of BFB (20 µL, 2 m in DMF). The
suspension was agitated at room temperature for 4 h until the resin
turned yellow. The resin was washed with DMF and CH2Cl2
(5ϫ5 mL). The resin (0.424 mmol) was treated with 20% piperi-
dine in DMF (5 mL) for 2ϫ10 min. Washed with DMF (3ϫ5 mL)
and distilled DMF (2 ϫ 5 mL). Fmoc--serine (0.566 g,
1.729 mmol), HOBt (0.342 g, 2.53 mmol) and DIC (0.264 mL,
1.69 mmol) were dissolved in distilled DMF and stirred at room
temperature for 10 min. The solution was transferred to the resin
followed by addition of BFB (20 µL, 2 m in DMF). The suspen-
sion was agitated at room temperature overnight until the resin
turned yellow. The resin was washed with DMF and CH2Cl2
(5ϫ5 mL). The resin (0.424 mmol) was treated with 20% piperi-
dine in DMF (5 mL) for 2ϫ10 min. Washed with DMF (3ϫ5 mL)
and distilled DMF (2 ϫ 5 mL). Hexanoic acid (0.212 mL,
1.692 mmol), HOBt (0.349 g, 2.583 mmol) and DIC (0.264 mL,
1.696 mmol) were dissolved in distilled DMF and stirred at room
temperature for 10 min. The solution was transferred to the resin
followed by addition of BFB (20 µL, 2 m in DMF). The suspen-
sion was agitated at room temperature overnight until the resin
turned yellow. The resin was washed with DMF and CH2Cl2
(5ϫ5 mL) to give resin 10. 19F NMR (CDCl3): δ = –134.1 ppm.
C
34H28F2NaO7S (641.6): 641.1421 [M + Na]+ found 641.1410.
[α]2D5 = +289 (CHCl3); m.p. 149–151 °C.
Resins 13 and 14: Resin 10, NBS, QOTf and glycosyl donors 11 or
12 were dried separately under vacuum overnight. Dried molecular
sieves 3 Å (0.080 g), dry CH2Cl2 (3 mL), 11 (3 equiv.) or 12
(3.4 equiv.) and tetrabutylammonium trifluoromethanesulfonate
(QOTf) (0.65 equiv. or 0.82 equiv.) were added to the resin 10
(0.106 mmol) or (0.120 mmol). The resins were agitated for 10 min.
N-bromosuccinimide (NBS) (4 eq or 5 equiv.) was added and the
mixtures were agitated at room temperature in absence of light for
5 h or 4.5 h. The resins were washed with CH2Cl2, 20% piperidine
in DMF, DMF and CH2Cl2 (5ϫ5 mL). According to gel-phase
19F NMR the yield were approximately 50% and 35% respectively.
The procedure was repeated to give the resins 13 and 14 in approxi-
mately 65% and 57% total yield according to gel-phase 19F NMR
spectroscopy. 13: 19F NMR (CDCl3): δ = –104.9 and –105.3 (2ϫ4-
FPhCO2) and –134.1 (linker) ppm; 14: 19F NMR (CDCl3): δ =
–104.7 and –105.4 (2ϫ4-FPhCO2) and –134.0 (linker) ppm.
{N-Hexanoyl-3-O-[2,3-di-O-(4-fluorobenzoyl)-β-D-galactopyranosyl]-
L
-seryl}-6-aminohexanoyl-6-aminohexylamine (15) and {N-Hexa-
noyl-3-O-[2,3-di-O-(4-fluorobenzoyl)-β-D-glucopyranosyl]-L-seryl}-
6-aminohexanoyl-6-aminohexylamine (16): The partially protected
neoglycolipid 15 and 16 were obtained from the resins 13 and 14
by addition of TFA/H2O, 9:1 (35 mL or 47 mL). The resins were
agitated at room temperature for 4 h and then thoroughly washed
with TFA/H2O, 9:1, CH2Cl2, THF and CH2Cl2. The filtrates were
collected, combined and concentrated to give 88 mg or 120 mg of
crude product in total. Purification by preparative LC-MS gave 15
(21 mg, 24 % yield) and 16 (8 mg, 12 % yield). 15: 19F NMR
(CDCl3): δ = –104.7, –105.1. MS (ES–) calcd. for C41H57F2N4O11
819.40 m/z (M – H)–, observed 819.70; 16: MS (ES+) calcd. for
C41H59F2N4O11 821.42 m/z [M + H]+, observed 821.47.
4-Methylphenyl 2,3-Di-O-(4-fluorobenzoyl)-4,6-O-benzylidene-1-
thio-β-
D
-galactopyranoside (11) and 4-Methylphenyl 2,3-Di-O-(4-
-glucopyranoside (12):
fluorobenzoyl)-4,6-O-benzylidene-1-thio-β-
D
4-Fluorobenzoyl chloride (2.5 equiv.) was added dropwise to a
solution of 4,6-O-benzylidene-1-thio-β--galactopyranoside
(3.53 g, 9.42 mmol) or 4,6-O-benzylidene-1-thio-β--glucopyrano-
side[26] (0.700 g, 1.87 mmol) in pyridine (40 mL or 12 mL). The
solution was stirred for 20 or 3 h, respectively, at room temperature
and then diluted with CH2Cl2, washed with saturated aqueous
[N-Hexanoyl-3-O-(β-
6-aminohexylamine (3) and [N-Hexanoyl-3-O-(β-
-seryl]-6-aminohexanoyl-6-aminohexylamine (17): The partially
D
-galactopyranosyl)-
L
-seryl]-6-aminohexanoyl-
D-glucopyranosyl)-
L
NaHCO3 (3ϫ) and H2O. The organic phase was dried with protected neoglycolipid 15 (0.021 g, 0.026 mmol) or 16 (0.008 mg,
Na2SO4, and concentrated. Residual pyridine was removed by co-
evaporation with toluene. The residue was purified by flash column
chromatography with gradient elution of heptane/ethyl acetate
(5:1Ǟ4:1 for 11 and 8:1Ǟ4:1 for 12) giving 11 and 12 as a color-
less solid (6.00 g and 1.13 g, respectively) in quantitative yields. 11:
1H NMR (CDCl3): δ = 8.02–7.90 (m, 4 H, ArH), 7.50 (d, J =
0.010 mmol) was dissolved in dry MeOH (15 mL) and NaOMe in
MeOH (0.20 , 10 equiv.) was added dropwise. The solutions were
stirred at room temperature for 1 h or 2 h. The reactions were
quenched by addition of AcOH and ice to pH 4 and the mixtures
were concentrated giving 46 mg of 3 and 16 mg of 17. Crude 3 was
dissolved in MeOH and purified by preparative LC-MS
Eur. J. Org. Chem. 2009, 349–357
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
355

S. Spjut, M. Pudelko, M. Hartmann, M. Elofsson
FULL PAPER
chromatography and lyophilized to give 0.024 g. From the 1H
NMR a peak at 9 ppm could be detected, which was identified as
formic acid salt formed during purification. Therefore 17 was not
purified and an exact yield could not be calculated. 3: 1H NMR
(Vector Laboratories, 4 µg/mL in PBS containing 0.05% Tween 20,
100 µL) for 45 min. The wells were washed with Milli-Q water
(2ϫ150 µL, 1 min and 1ϫ150 µL, 10 min) and citric acid buffer
(150 µL, 10 min), incubated with substrate solution [6 mg of O-
([D4]MeOH): δ = 4.55–4.50 [m, 1 H, OCH2CH(NH)CO], 4.26 (d, phenylenediamine and 5 µL H2O2 (30% aq.) in phosphate-citric
J = 6.9 Hz, 1 H, 1-H), 4.18–4.11 [m, 1 H, OCHHCH(NH)CO], acid buffer (0.1 , pH 5, 10 mL), 100 µL] for 20 min in the dark
3.86 (s, 1 H, 4-H) 3.81–3.68 [m, 3 H, 6-H, OCHHCH(NH)CO], and the substrate reaction was stopped with H2SO2 (1  aq.)
3.56–3.45 (m, 3 H, 2-H, 3-H, 5-H), 3.21–3.12 [m, 4 H,
(100 µL). The optical density was measured at 490 nm against a
2ϫCO(NH)-CH2], 0.94 (t, J = 6.7 Hz, 3 H, CH3) ppm. MS (ES+) reference at 650 nm. All concentrations and controls were per-
calcd. for C27H53N4O9 577.38 m/z [M + H]+, observed 577.43; 17:
1H NMR ([D4]MeOH): δ = 4.54 [t, J = 4.85, 1 H, OCH2CH(NH)
CO], 4.30 (d, J = 7.6 Hz, 1 H, 1-H), 4.15 [dd, J = 5.5 Hz and
10.3 Hz, 1 H, OCHHCH(NH)CO], 3.88 (d, J = 11.5 Hz, 1 H, 4-
H), 3.73 [dd, J = 5.6 and 10.3 Hz, 1 H, OCHHCH(NH)CO], 3.22–
3.12 [m, 5 H, 2-H, 3-H, 5-H, CO(NH)-CH2CH2], 2.91 [t, J =
7.4 Hz, 2 H, CO(NH)-CH2], 0.92 (t, J = 6.4 Hz, 3 H, CH3) ppm.
MS (ES+) calcd. for C27H53N4O9 577.38 m/z [M + H]+, observed
577.72.
formed in triplicate samples.
Detection with Avidin-FITC: Compound 9a and 9b in Na2HCO3
buffer (aq., pH 9) were serially diluted (2.5 m to 0.61 µ) in white
microtiter plate wells (CovaLinkTM, Nunc A/S, Denmark) and in-
cubated with shaking for 18 h at room temperature. Control wells
were incubated with buffer alone. All wells were washed with Milli-
Q water (1ϫ150 µL, 1 min and 2ϫ150 µL, 10 min) and blocked
by incubation overnight with BSA (1% w/v in PBS, 200 µL) at 4 °C.
The wells were washed with PBS containing BSA (1% w/v) and
Triton X-100 (0.05%) (2ϫ200 µL, 1 min) and PBS (1ϫ200 µL,
1 min). Avidin-FITC (from egg white, Sigma, 50 µg/mL in PBS,
100 µL) were added to the wells and incubated with shaking for
3 h. The wells were washed with Milli-Q water (2ϫ150 µL, 1 min
and 1ϫ150 µL, 10 min) and phosphate-citric acid buffer (0.1 ,
pH 5, 100 µL) was added to each well. The fluorescence was mea-
sured at 485/535 (ex/em).
3-{[N-Hexanoyl-3-O-(β-
noyl-6-aminohexylamino}-4-decyloxy-3-cyclobutene-1,2-dione (18)
and 3-{[N-Hexanoyl-3-O-(β- -glucopyranosyl)- -seryl]-6-amino-
D-galactopyranosyl)-L-seryl]-6-aminohexa-
D
L
hexanoyl-6-aminohexylamino}-4-decyloxy-3-cyclobutene-1,2-dione
(19): Compound 3 (0.015 g, 0.026 mmol) or 17 (0.008 g crude prod-
uct) and 3,4-didecyloxy-cyclobut-3-ene-1,2-dione[13] (3.3 equiv.)
were dissolved in distilled DMF (5 mL and 2 mL, respectively).
TEA (1.65 equiv.) was added and the solutions were stirred at room
temperature for 20 h. The DMF was removed under vacuum giving
0.069 g of 18 and 0.020 g of 19. Purification by preparative HPLC
gave 18 (4.2 mg, 20% yield) and 19 (2.2 mg, 27% yield). 18: 1H
NMR ([D4]MeOH): δ = 4.77–4.68 (m, 2 H, COCH2C9H19), 4.56
[t, J = 5.3 Hz, 1 H, CH2CH(NH)CO], 4.27 (d, J = 7.4 Hz, 1 H, 1-
H), 4.14 [dd, J = 4.7 and 10.4 Hz, 1 H, OCHHCH(NH)CO], 3.95–
3.69 [m, 4 H, 4-H, OCHHCH(NH)CO, 6-H], 3.67–3.48 (m, 3 H,
2-H, 3-H, 5-H), 3.44 (t, J = 6.9 Hz, 2 H, COCH2), 3.27–3.14 (m,
Formation of Neoglycolipid Microtiter Plates: A stock solution
(0.5 m) of neoglycolipid 18 in NaHCO3 buffer (pH 9) was pre-
pared and serially diluted in transparent CovaLinkTM (Nunc A/S,
Denmark) microtiter plate wells, resulting in 100 µL neoglycolipid
solution in each well in the concentration range of 48 nM to
0.1 m. Control wells were incubated with buffer alone. The plate
was shaken at room temperature for 24 h, emptied, washed with
Cova buffer (2ϫ150 µL, 1 min and 1ϫ150 µL, 10 min) and water
(1ϫ150 µL, 1 min). Any remaining amino-groups were blocked
with acetic anhydride (20% in water) (100 µL, 2 h), emptied and
washed with water (2ϫ150 µL, 1 min and 1ϫ150 µL, 10 min).
Biotin-conjugated lectin from Ricinus communis (RCA120, Vector
Laboratories, 5 µg/mL in PBS containing 0.05% Tween 20, 100 µL)
was added to the wells. The plate was shaken for 70 min at room
temperature and the wells were emptied and washed with PBS with
0.05% Tween 20 (2ϫ150 µL, 1 min). Avidin-HRP (Vector
Laboratories, 4 µg/mL in PBS containing 0.05% Tween 20, 100 µL)
was added to the wells. The plate was shaken at room temperature
for 45 min, emptied and washed with Cova buffer (2ϫ150 µL,
1 min and 1ϫ150 µL, 10 min) and phosphate-citric acid buffer
(0.1 , pH 5) (1ϫ150 µL, 1 min and 1ϫ150 µL, 10 min). Substrate
solution [6 mg of O-phenylenediamine and 5 µL H2O2 (30% aq.)
in phosphate-citric acid buffer (0.1 , pH 5, 10 mL), 100 µL] was
added to the wells. The plate was incubated with shaking in the
dark at room temperature for 15 min and absorbance was mea-
sured at 490 nm against a reference at 650 nm. All concentrations
and controls were performed in triplicate samples.
4
H, 2ϫCONHCH2), 1.89–1.78 (m,
4
H, OCH2CH2,
COCH2CH2), 1.44–1.33 (m, 4 H, 2ϫCH2-CH3), 0.98–0.91 (m, 6
H, 2ϫCH3). 13C NMR ([D4]MeOH): δ = 176.3, 174.8, 172.1,
105.3, 77.1, 74.9, 74.7, 72.5, 70.6, 70.4, 62.7, 54.9, 45.5, 45.2, 40.3,
40.2, 37.1, 36.8, 33.1, 32.6, 31.9, 31.6, 31.2, 30.6, 30.4, 30.3, 30.2,
30.0, 27.6, 27.5, 27.4, 27.2, 27.0, 26.7, 26.6, 26.4, 23.7, 23.7, 23.5.
[α]2D5 = +19 (MeOH). HRMS (FAB) calcd. for C41H72N4NaO12
835.5044 [M + Na]+, observed 835.5045; 19: 1H NMR ([D4]-
MeOH): δ = 4.71–4.65 [m, 2 H, CO(C)OCH2C9H19], 4.54 [t, J =
4.9 Hz, 1 H, OCH2CH(NH)CO], 4.29 (d, J = 7.7 Hz, 1 H, 1-H),
4.12 [dd, J = 5.5 and 10.3 Hz, 1 H, OCHHCH(NH)CO], 3.82 (d,
J = 11.5 Hz, 1 H, 4-H), 3.76 [dd, J = 5.6 and 10.3 Hz, 1 H,
OCHHCH(NH)CO], 3.70–3.65 (m, 1 H, 5-H), 3.42 (t, J = 7.8 Hz,
2 H, COCH2) 3.38–3.12 [m, 10 H, 2-H, 2ϫ6-H, 3-H, 2ϫCO(NH)-
CH2CH2, COCH2], 1.87–1.75 [m, 2 H, CO(C)OCH2CH2C8H17]
0.93–0.88 (m, 6 H, 2 Me). 13C NMR ([D4]MeOH): δ = 185.5, 184.8,
176.4, 176.1, 172.6, 172.1, 104.2, 77.9, 77.7, 74.9, 74.4, 71.4, 71.0,
70.3, 62.7, 62.4, 47.7, 45.4, 40.0, 36.8, 36.5, 32.8, 32.6, 30.2, 29.9,
27.0, 26.3, 23.3, 14.1. [α]2D5 = –13.4 (MeOH). HRMS (FAB) calcd.
for C41H72N4NaO12 835.5044 [M + Na]+, observed 835.5024.
Supporting Information (see also the footnote on the first page of
1
this article): H NMR spectra for compounds 9a, 9b, 18, and 19.
Formation of Model Compound Microtiter Plates
Detection with Avidin-HRP: Compound 9a and 9b in Na2HCO3
buffer (aq., pH 9) were serially diluted (1.0 m to 0.24 n for 9a
and 1.1 m to 2.2 n for 9b) in transparent microtiter plate wells
(CovaLinkTM, Nunc A/S, Denmark) and incubated with shaking
for 18 h at room temperature. Control wells were incubated with
buffer alone. All wells were washed with Milli-Q water (1ϫ150 µL,
1 min and 2ϫ150 µL, 10 min) and incubated with avidin-HRP
Acknowledgments
This work was supported by the Swedish Research Council and the
J. C. Kempe Foundation.
[1] R. A. Dwek, Chem. Rev. 1996, 96, 683–720.
356
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 349–357

Carbamate-Linker Strategy for Amino-Functionalized Glycoconjugates
[2] K. R. Love, P. H. Seeberger, Angew. Chem. Int. Ed. 2002, 41,
3583–3586.
[3] T. Feizi, F. Fazio, W. Chai, C.-H. Wong, Curr. Opin. Struct.
Biol. 2003, 13, 637–645.
[4] D. M. Ratner, E. W. Adams, M. D. Disney, P. H. Seeberger,
ChemBioChem 2004, 5, 1375–1383.
[5] K. Larsen, M. B. Thygesen, F. Guillaumie, W. G. T. Willats,
K. J. Jensen, Carbohydr. Res. 2006, 341, 1209–1234.
[6] M. S. Timmer, B. L. Stocker, P. H. Seeberger, Curr. Opin. Chem.
Biol. 2007, 11, 59–65.
[7] M. M. Lan Ban, Angew. Chem. Int. Ed. 2008, 47, 3396–3399.
[8] S. Park, M.-R. Lee, I. Shin, Chem. Soc. Rev. 2008, 37, 1579–
1591.
[9] M. Uttamchandani, D. P. Walsh, S. Q. Yao, Y. T. Chang, Curr.
Opin. Chem. Biol. 2005, 9, 4–13.
[10] L. Tietze, M. Arlt, M. Beller, K. Glusenkamp, E. Jahde, M.
Rajewsky, Chem. Ber. 1991, 124, 1215–1221.
[11] L. F. Tietze, C. Schroeter, S. Gabius, U. Brinck, A. Goerlach-
Graw, H. J. Gabius, Bioconjugate Chem. 1991, 2, 148–153.
[12] V. P. Kamath, P. Diedrich, O. Hindsgaul, Glycoconjugate J.
1996, 13, 315–321.
[15]
[16]
G. Magnusson, Adv. Drug Delivery Rev. 1994, 13, 267–284.
F. K. Wallner, H. A. Norberg, A. I. Johansson, M. Mogemark,
M. Elofsson, Org. Biomol. Chem. 2005, 3, 309–315.
M. Elofsson, J. Broddefalk, T. Ekberg, J. Kihlberg, Carbohydr.
Res. 1994, 258, 123–133.
J. R. Hauske, P. Dorff, Tetrahedron Lett. 1995, 36, 1589–1592.
B. A. Dressman, L. A. Spangle, S. W. Kaldor, Tetrahedron Lett.
1996, 37, 937–940.
M. Mogemark, F. Gårdmo, T. Tengel, J. Kihlberg, M. Elofs-
son, Org. Biomol. Chem. 2004, 2, 1770–1776.
M. Mogemark, M. Elofsson, J. Kihlberg, Org. Lett. 2001, 3,
1463–1466.
M. Mogemark, M. Elofsson, J. Kihlberg, J. Org. Chem. 2003,
68, 7281–7288.
F. K. Wallner, S. Spjut, D. Boström, M. Elofsson, Org. Biomol.
Chem. 2007, 5, 2464–2471.
K. Egusa, K. Fukase, Y. Nakai, S. Kusumoto, Synlett 2000,
27–32.
R. Kottani, R. A. Valiulin, A. G. Kutateladze, Proc. Natl.
Acad. Sci. USA 2006, 103, 13917–13921.
U. Ellervik, H. Grundberg, G. Magnusson, J. Org. Chem. 1998,
63, 9323–9338.
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[13] A. Bergh, B.-G. Magnusson, J. Ohlsson, U. Wellmar, U. J. Nils-
son, Glycoconjugate J. 2001, 18, 615–621.
[14] S. Brodesser, P. Sawatzki, T. Kolter, Eur. J. Org. Chem. 2003,
2021–2034.
Received: July 8, 2008
Published Online: December 4, 2008
Eur. J. Org. Chem. 2009, 349–357
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