Preparation of some glycosyl amino acid building blocks via click reaction and construction of a glycotetrapeptide library using spot synthesis

Article abstract of DOI:10.1080/07328300802419873

The copper-catalyzed 1,3-dipolar cycloaddition reaction between ethyl 2,3,4-tri-O-actetyl-6-azido-6-deoxy-1-thio- -d-glucopyranoside (2), ethyl 2,3,4-tri-O-actetyl-6-azido-6-deoxy-1-thio- -d-galactopyranoside (4), methyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy

Full text of DOI:10.1080/07328300802419873

Journal of Carbohydrate Chemistry, 27:446–463, 2008
ꢀC
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328300802419873
Preparation of Some Glycosyl
Amino Acid Building Blocks via
Click Reaction
and Construction
of a Glycotetrapeptide Library
Using Spot Synthesis
Kai Gu¨nther, Carsten Schips, and Thomas Ziegler
Institute of Organic Chemistry, University of Tuebingen, Germany
The copper-catalyzed 1,3-dipolar cycloaddition reaction between ethyl 2,3,4-tri-
O-actetyl-6-azido-6-deoxy-1-thio-β-D-glucopyranoside (2), ethyl 2,3,4-tri-O-actetyl-6-
azido-6-deoxy-1-thio-β-D-galactopyranoside (4), methyl 2,3,4-tri-O-acetyl-6-azido-6-
deoxy-α-D-mannopyranoside (7), and methyl 2,3,6-tri-O-acetyl-2-azido-2-deoxy-β-D-
glucopyranoside (9), and tert-butyl-protected Fmoc-asparaginic acid propargylamide
(10) gave the corresponding protected glycosyl amino acid building blocks 11, 13, 15,
and 17 in 67% to 95% yield. The latter were converted into the corresponding pentaflu-
orophenyl esters 12, 14, 16, and 18, which were used for a spot synthesis of a combi-
natorial library containing 256 glycotetrapeptides. The library was screened for lectin-
binding affinity with the lectins Concanavalin A (Con A), phaseolus vulgaris (PHA-E),
and galantus nivalis (GNA).
Keywords Glycosyl amino acids, Click reaction, Glycopeptides, Combinatorial spot
synthesis, Oligosaccharide mimics
INTRODUCTION
Specific interactions of proteins with complex carbohydrate structures asso-
ciated with cell surfaces play a major role in many biologically important
mechanisms such as, for example, cell–cell recognition, signal transduction,
Received June 24, 2008; accepted August 19, 2008.
Address correspondence to Thomas Ziegler, Institute of Organic Chemistry, Univer-
sity of Tuebingen, Auf der Morgenstelle 18, 72076 Tuebingen, Germany. E-mail:
thomas.ziegler@uni-tuebingen.de
446

Using Click Reaction and Spot Synthesis
447
infection and inflammation mechanisms, and immunological processes. Differ-
ent glycosylation patterns and posttranslational modifications of carbohydrate
structures of glycoproteins are also responsible for their heterogeneity and
biological properties.[1] Cells can be physically and biologically distinguished
through their surface carbohydrate patterns. This is an important medicinal
aspect with regard to specific tumor markers on cell surfaces, which often
consist of distinct complex oligosaccharide structures.[2] Therefore, studying
carbohydrate–protein interactions at a molecular level provides a deeper un-
derstanding of fundamental biological regulation mechanisms and opens the
gate for novel analytical tools or to manipulate such specific processes for ther-
apeutic purposes. Unfortunately, isolation of pure complex oligosaccharides
from natural sources in order to study carbohydrate–protein interaction in de-
tail is a rather difficult venture owing to the micro-heterogeneity of naturally
occurring saccharides. Synthetic oligosaccharides, on the other hand, provide
for sufficient amounts of pure material for this purpose. However, the chemi-
cal synthesis of complex oligosaccharides is still a laborious, sometimes even
tedious, and often difficult task, although significant achievements in this field
had been accomplished in the past decades. Thus, novel approaches for the
efficient preparation of well-defined saccharide-containing structures that can
mimic the interaction between a specific protein and its natural saccharide
ligand are highly desirable.
For the construction of mimics for complex oligosaccharides, we follow a
concept in which simple glycosyl amino acid building blocks are used for the ef-
ficient combinatorial preparation of fully glycosylated peptides (glycopeptoids),
which, in turn, can bind to carbohydrate-recognizing proteins.[3] Recently, we
prepared a series of glycosyl amino acid building blocks via click reaction (Cu-
catalyzed 1,3-dipolar cycloaddition between azides and alkynes)[4] of 6-azido-
6-deoxy-glycosides with suitably protected asparaginic acid propargylamide.[5]
Click reactions have been widely applied in carbohydrate chemistry already,
and allow for a highly efficient synthesis of glycosyl amino acid building
blocks.[6] The 1,2,3-triazole moiety also mimics amino acids and, thus, is well
suited for the construction of glycosylated peptides.[7]
Here, we extended our previous approach[5] to the highly efficient synthesis
of glycosylated amino acid building blocks to other glycosides. Furthermore, we
converted the latter into the corresponding pentafluorophenyl esters and used
these active esters for the construction of a combinatorial library consisting of
256 glycotetrapeptides.
RESULTS AND DISCUSSION
Previously, ethyl 1-thio-glucoside (2) was prepared from ethyl 2,3,4-tri-O-
acetyl-1-thio-6-O-tosyl-β-D-glucopyranoside by nucleophilic substitution of the

K. Gu¨nther et al.
448
tosyl group with azide. However, the reaction resulted in anomerization and
yielded 2 in 41% yield only.[5] In analogy to the known procedure for the syn-
thesis of this compound, we now prepared 2 from easily available 1,2,3,4-tetra-
O-acetyl-6-azido-6-deoxy-β-D-glucopyranose 1.[8,9] Reaction of the latter with
Me3SiSEt/ZnCl2afforded 2 in 84% yield.[8] However, using EtSH/BF3-etherate
instead of Me3SiSEt/ZnCl2 resulted in a significantly lower yield, giving 2
in 37% yield. Similarly, treatment of 1,2,3,4-tetra-O-acetyl-6-azido-6-deoxy-D-
galactopyranose 3[10] with EtSH/BF3-etherate afforded 1-thio-galactoside 4 in
35% yield. Methyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy-α-D-mannopyranoside (7)
was prepared from methyl α-D-mannopyranoside 5 via its 6-O-tosyl derivative
6 by a slight modification of the previously described procedure[11] (see the
experimental section for details). Methyl 2,4,6-tri-O-acetyl-2-azido-2-deoxy-β-
D-glucopyranoside 9 was prepared in 46% yield from methyl β-D-glucosamine
(8)[12] by a catalytic diazotransfer from triflyl azide (Sch. 1).[13]
Next, azides 2, 4, 7, and 9 were coupled with N-fluorenylmethoxycarbonyl-
aspartic-1-propargylamide-4-t-butyl ester (10)[5] under catalysis with
(EtO)3PCuI[14] in toluene and irradiation with microwave[10] to afford
the corresponding 1,2,3-triazole-linked glycosyl asparaginic acid derivatives
11, 13, 15, and 17 in 67% to 95% yield. In general, the glucoside 2 and the
mannoside 7 reacted faster and gave higher yields of the coupled products 12
and 16, respectively, than the corresponding galactoside 4 and glucosamine
9. Without microwave irradiation of the reaction mixture, only a very slow
cycloaddition reaction was observed. Subsequent deprotection of compounds
11, 13, 15, and 17 with trifluoroacetic acid in dichloromethane, followed by
esterification of the intermediate carboxylic acids with pentafluorophenol and
dicyclohexyl carbodiimide (DCC), gave the pentafluorophenyl esters 12, 14,
16, and 18 in 51% to 74% overall yield. The latter were used as active ester
building blocks for the preparation of β-peptides via spot synthesis because
they can be stored without decomposition for an extended period of time.
For the preparation of a combinatorial peptide library consisting of 256
tetra-glycopeptides derived from the four building blocks 12, 14, 16, and 18, an
automated spot synthesis on cellulose paper sheets was applied.[15] The build-
ing blocks were denominated as building blocks R (Glc), N (Gal), A (Man),
and D (GlcN) (Sch. 1). A format of a 16 × 16 spot array on 10 × 14 cm pa-
per sheets was chosen. The cellulose paper sheets were derivatized by a slight
modification of Wenschuh’s procedure[16] (Sch. 2). First, the paper sheet was
treated with epibromohydrin and a catalytic amount of HClO4 in dioxane, fol-
lowed by 4,7,10-trioxa-1,13-tridecanediamine in DMF and methanolic sodium
methanolate solution to give the “safety catch” modified cellulose sheet I. The
density of free amino groups was determined to be in the range of 500 to 600
nmol/cm2 by reacting the paper sheet with Fmoc-protected pentafluorophenyl
glycinate 20, cleaving off the Fmoc groups of the bound glycine and deter-
mining the amount of cleaved Fmoc groups photospectrometrically.[17] Next,

Using Click Reaction and Spot Synthesis
449
Scheme 1.
Fmoc-protected Rink linker 19 was coupled onto the modified cellulose sheet
(N,Nꢁ-diisopropyl carbodiimide, pentafluorophenol in N-methyl pyrrolidone),
followed by capping of the unreacted amino groups with acetic anhydride and
cleavage of the Fmoc groups with piperidine in DMF. Finally, glycinate 20 was
coupled to the Rink linker and deprotected to give the paper sheet II, ready for
attaching the building blocks (Sch. 2). The terminal glycine moiety was intro-
duced in order to avoid steric hindrance for the attachment of the first glycosyl
amino acid building block since the primary amino group of the glycine moiety
is better accessible than the secondary amino group of the Rink linker. Rink

K. Gu¨nther et al.
450
Scheme 2.
linker 19 and glycine derivative 20 were not coupled to the entire paper sheet
but, instead, only applied to the 256 spots where the glycopeptides were to be
synthesized. This strategy diminished the unwanted unspecific binding of pro-
tein to the entire paper sheet via interaction with the hydrophobic Rink linker
in the final lectin screening tests (see below). It also enabled the easy visual-
ization of the progress of the final cleavage of the glycopeptides from the paper
sheet with trifluoroacetic acid vapor, which gave dark red spots produced by
the cations formed by the remaining Rink linker moiety.[3d]
Next, the building blocks were spotted onto the paper sheet in a 16 × 16
matrix using an autospot robot (Sch. 3). On each of the 256 spots, 2 × 10 nmol
of building blocks R, N, A, and D were applied, resulting in spots of approxi-
mately 0.13 cm2. The building blocks were applied to the individual spots on
the paper sheet as a 0.1-M solution in N-methyl pyrrolidone (NMP) containing

Using Click Reaction and Spot Synthesis
451
Scheme 3.
0.06 M 3-hydroxy-1,2,4-benzotriazine-4-(3H)-one (HODhbt) for enhancing the
efficiency of the coupling steps.[16b] An overall amount of approximately 20.5
µmol of building blocks R, N, A, and D were needed for the synthesis of the en-
tire library of 256 individual glycotetrapeptides. A critical and time-consuming
operation in the spot synthesis of the glycotetrapeptide library was the wash-
ing cycles after the coupling and Fmoc deprotection steps, which had to be per-
formed thoroughly. The completeness of the coupling, Fmoc deprotection, and
washing steps was best controlled by staining the paper sheet with bromophe-
nol blue solution in DMF.[15a] The final deacetylation of the bound glycote-
trapeptides was effected by rinsing the cellulose membrane with a methanolic
solution of NH3 overnight followed by washing with ethanol.[3d] Detachment
of the glycotetrapeptides containing a N-terminal glycineamide from the mem-
brane for analytical purposes can be achieved by exposing the membrane to an
atmosphere of trifluroacetic acid in a desiccator as described previously.[3d]
The glycotetrapeptide library was screened for lectin-binding affinities
with horseradish peroxidise conjugates of Concanavalin A (Con A, canavalis
ensiformis lectin), phytohaemagglutinine (PHA-E, phaseolus vulgaris lectin),
and galantus nivalis lectin (GNA) as described previously.[3d] Figure 1 shows
the typical pattern of red spots for the binding of GNA after the peroxidase-
catalyzed formation of Wuster’s Red from 3-amino-9-ethyl-carbazole and
H2O2.[18a] The latter staining procedure was superior to other commonly ap-
plied staining methods using lectin-peroxidase conjugates[18b–d] because the
background staining of the cellulose membrane was kept to a minimum.

K. Gu¨nther et al.
452
Figure 1. Screening Results.
Compared to the binding of lectins to previous glycotetrapeptide libraries
obtained from similar building blocks in which the sugar moieties were gly-
cosidically linked to the β-peptide backbone via alkyl spacers,[3a,d] the tested
lectins showed a different binding affinity here. Specifically, Con A bound to all
spots with rather low selectivity. This can be attributed to the relatively low
selectivity of Con A for binding to mannose and glucose. Nevertheless, Con A
bound slightly stronger to those glycotetrapeptides that contained block R (glu-
cose) as the last attached building block. No significantly enhanced binding to
peptides rich in block A (mannose) could be observed though. GNA, which dis-
plays a selectivity for mannose-rich oligosaccharides, did not bind any stronger
to glycotetrapeptides rich in block A (mannose) here. Instead, GNA preferen-
tially bound to those peptides that contained blocks R (glucose) and N (galac-
tose) as the last attached blocks. Furthermore, the sequence RXNN, where X
is any other sugar moiety, preferentially bound GNA. Similarly, PHA-E lectin,
which selectively binds to the trisaccharide sequence Gal-β1,4-GlcNAc-β1,2-
Man, also preferentially bound to those glycotetrapeptides with blocks R (glu-
cose) or N (galactose) as the last attached block. Here, an enhanced affinity
of PHA-E for the sequences RXNN, RXNR, and RXRN was observed. In gen-
eral, those peptides that contain ethyl 1-thio-glycosides bound the lectins bet-
ter than those containing methyl glycosides.
CONCLUSION
It could be shown that glycotetrapeptides constructed out of β-peptidically
linked asparaginic acid having glycosyl 1,2,3-triazole moieties attached to each
α-carboxylic group of the peptide backbone can indeed mimic the binding
of lectins to their natural ligands. Thus, glycopeptoids of the type described
here may be suitable devices for studying protein–carbohydrate interactions
on a molecular level in order to gain a better insight in the various factors

Using Click Reaction and Spot Synthesis
453
determining the binding of proteins to carbohydrates. Further binding stud-
ies with additional lectins and with glycopeptide libraries constructed out of
building blocks containing glycosidically linked sugar moieties,[5] as well as
quantitative binding studies of lectins to such glycopeptides, are under way
now.
EXPERIMENTAL
All solvents were dried and distilled prior to their use. Reactions were per-
formed under Ar and monitored by TLC on Polygram Sil G/UV silica gel plates
from Machery & Nagel. Detection was affected by charring with H2SO4 (5% in
EtOH) or by inspection of the TLC plates under UV light. NMR spectra were
recorded on a Bruker ARX 250 spectrometer at 100 MHz for proton spectra and
62.9 MHz for carbon spectra, and on a Bruker Avance 400 spectrometer at 400
MHz for proton spectra and 100 MHz for carbon spectra. Tetramethylsilane
was used at the internal standard. FAB-MS was performed on a Finnigan MAT
TSQ 70 spectrometer. HRFD-MS was performed on a Bruker FT-ICR spectrom-
eter. Elemental analyses were performed on a Hekatech CHN analyzer. Optical
rotations were measured with a Perkin-Elmer Polarimeter 341. Preparative
chromatography was performed on silica gel (0.032–0.063 mm) from Machery
& Nagel using different mixtures of solvents as eluents. Microwave-assisted
reactions were performed with a CEM-focused microwave synthesis system,
type Discover. Spot syntheses were performed with an Intavis AG Bioanalysis
Instruments AutoSpot Robot ASP 222.
Ethyl 2,3,4-Tri-O-acetyl-6-azido-6-deoxy-1-thio-
β-D-glucopyranoside (2)
BF3 etherate (1.08 mL, 8.60 mmol) was added at 0◦C to a solution of com-
pound 1[8,9] (2.00 g, 5.36 mmol) and ethyl mercaptane (0.79 mL, 9.4 mmol) in
dichloromethane (100 mL). The mixture was stirred for 2 h at 0◦C, warmed
to rt, and stirred for additional 8 h. The mixture was washed with saturated
aqueous NaHCO3 solution, dried with Na2SO4, and filtered. Concentration of
the filtrate in vacuo and chromatography of the residue (toluene, ethyl acetate
3:1) afforded 2 (0.74 g, 37%) as a white amorphous foam. The compound was
identical to the one previously prepared.[5,8]
Ethyl 2,3,4-Tri-O-acetyl-6-azido-6-deoxy-1-thio-
β-D-galactopyranoside (4)
Treatment of compound 3[10] (2.00 g, 5.36 mmol), ethyl mercaptane (0.79
mL, 9.4 mmol), and BF3 etherate (1.08 mL, 8.60 mmol) in dichloromethane
(100 mL) as described for the preparation of compound 2 gave 4 (0.71 g, 35%)

K. Gu¨nther et al.
454
as a white amorphous foam. [α]D20 = –13.8 (c 1.00, CHCl3). FAB MS m/z: found
1
376.0, calcd. 376.1 [M+H]+. H NMR (CDCl3): δ (ppm) = 5.39 (d, 1 H, J1,2
=
3.5 Hz , H-1), 5.25 (t, 1 H, J3,4 = 9.9 Hz, H-3), 5.08 (dd, 1 H, J2,3 = 3.3 Hz, H-2),
4.56 (d, 1 H, J = 10.2 Hz, H-4), 3.84 (dd, 1 H, J = 3.8 Hz , H-5), 3.53 (m, 1 H,
H-6a), 3.14–3.19 (m, 1 H, H-6b), 2.71–2.82 (m, 2 H, SCH2), 2.18, 2.08, 1.99 (3s,
9 H, COCH3), 1.30 (t, 3 H, J = 7.4 Hz, SCH2CH3). 13C NMR (CDCl3): δ (ppm)
= 170.2, 169.9, 169.5 (C O), 83.8 (C-1), 76.2 (C-5), 71.8 (C-3), 68.1 (C-2), 67.2
(C-4), 50.7 (C-6), 24.3 (SCH2), 20.7, 20.6, 20.5, (COCH3), 14.7 (SCH2CH3).
Methyl 2,3,4-Tri-O-acetyl-6-O-p-toluolsulfonyl-
α-D-mannopyranoside (6)
p-Toluenesulfonyl chloride (5.90 g, 31.0 mmol) was added with stirring at 0◦C
to a solution of methyl α-D-mannopyranoside 5 (5.00 g, 25.8 mmol) in pyridine
(50 mL), and stirring was continued for 2 h. Acetic anhydride (50 mL, 0.53 mol)
was added and stirring at 0◦C was continued for 3 h. The solution was poured
onto crushed ice, and the mixture was extracted with chloroform (3 × 100 mL).
The combined extracts were washed with saturated aqueous NaHCO3 solu-
tion, dried over MgSO4,and filtered. Concentration of the filtrate in vacuo and
filtration of the residue over a short column of silica gel (toluene/ethyl acetate
3:1) gave crude 6 (7.01 g, 79%), which was sufficiently pure for the next step.
1H NMR (CDCl3): δ (ppm) = 7.77 (d, 2 H, J = 8.1 Hz, Tos-Ar-H), 7.31 (d, 2 H,
J = 8.1 Hz, Tos-Ar-H), 4.93–5.25 (m, 3 H, H-1,2,3), 4.65 (s, 1 H, H-5), 3.80–3.86
(m, 1 H, H-4), 3.33 (s, 3 H, OCH3), 3.15–3.32 (m, 2 H, H-6a,b), 2.01, 2.00, 1.93
(3s, 9 H, COCH3).13C NMR (CDCl3): δ (ppm) = 169.3, 169.3, 169.2 (C O),145.0,
132.4, 129.7, 128.3 (Tos-Ar-C), 97.8 (C-1), 69.2 (C-2), 68.8 (C-4), 68.2 (C-3), 66.5
(C-5), 54.6 (OCH3), 67.3 (C-6), 21.8 (Tos-CH3), 20.2, 20.1, 19.9, (COCH3).
Methyl 2,3,4-Tri-O-acetyl-6-azido-6-deoxy-α
-D-mannopyranoside (7)
A solution of compound 6 (7.00 g, 20.09 mmol) and NaN3(10.0 g, 0.154 mol)
in dry DMSO (100 mL) was stirred at 50◦C for 4 h followed by stirring at rt
for 16 h. The resulting slurry was poured onto crushed ice, and the mixture
was extracted with dichloromethane (4 × 50 mL). The combined extracts were
washed two times with water, dried over MgSO4, and filtered. Concentration
of the filtrate in vacuo and filtration of the residue over a short column of silica
gel (n-hexane/ethyl acetate 5:1) followed by crystallization from ethanol gave
7 (5.62 g, 81%). mp. 100◦C (mp.[11] 99–100◦C). [α]D20 = +65.7 (c 1.0, CHCl3). All
NMR data are identical to those reported in the literature.[11]

Using Click Reaction and Spot Synthesis
455
Methyl 2,4,6-Tri-O-acetyl-2-azido-2-deoxy-
β-D-glucopyranoside (9)
According to reference [13a], a 9-M solution of triflyl azide in pyridine was
freshly prepared by dissolving NaN3 (0.59 g, 9.06 mmol) in pyridine (10 mL),
adding trifluorometanesulfonic anhydride (1.24 mL, 7.2 mmol) at 0◦C, stirring
the mixture at 0◦C for 2 h, and filtering off all precipitated salts. The solution of
triflyl azide was added dropwise at 0◦C to a solution of compound 8[12] (2.00 g,
6.26 mmol), triethylamine (1.75 mL, 12.52 mmol), and CuSO4× 5 H2O (16 mg,
0.0626 mmol) in pyridine (10 mL). After the addition of the triflyl azide solution
was completed, and the mixture was stirred at rt for 16 h and concentrated
in vacuo. Chromatography (toluene/ethyl acetate 3:1) of the residue afforded
9 (1.0 g, 46%). [α]2D0 = +5.0 (c 1.0, CHCl3). FAB MS m/z: found 346.0, calcd.
1
346.31 [M+H]+. H NMR (CDCl3): δ (ppm) = 4.90–4.97 (m, 2 H, H-1,5), 4.18–
4.25 (m, 2 H, H-3,4), 4.02–4.05 (m, 1 H, H-6a), 3.59–3.62 (m, 1 H, H-6b), 3.56
(s, 3 H, OCH3), 3.38–3.42 (m, 1 H, H-2), 2.00, 2.00, 1.93 (3s, 9 H, COCH3). 13
C
NMR (CDCl3): δ (ppm) = 170.6, 170.0, 169.5 (C O), 102.9 (C-1), 72.5 (C-3), 71.7
(C-5), 68.4 (C-4), 63.7 (C-6), 61.8 (OCH3), 57.4 (C-2), 21.2, 21.1, 20.9 (COCH3).
t-Butyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-{[1-(2,3,4-tri-O-
acetyl-6-deoxy-1-ethylthio-β-D-glucopyranos-6-yl)-1H-1,2,3-
triazol-4-yl]methyl}-L-α-asparaginate (11)
A solution of compound 2 (225 mg, 0.6 mmol), compound 10[5] (261 mg, 0.6
mmol), diisopropyl ethylamine (0.31 mL, 1.8 mmol), and (EtO)3PCuI (24 mg,
0.06 mmol) in toluene (5 mL) was stirred 80◦C under microwave irradiation
(30 W) for 30 min. Concentration of the solution in vacuo and chromatography
of the residue (ethyl acetate) afforded 11 (450 mg, 91%). HRFD MS m/z: found
846.29859, calcd. 846.29906 [M+Na]+. The compound was identical to the one
previously obtained.[5]
Pentafluorophenyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-{[1-
(2,3,4-tri-O-acetyl-6-deoxy-1-ethylthio-β-D-glucopyranos-6-
yl)-1H-1,2,3-triazol-4-yl]methyl}-L-α-asparaginate (12)
A solution of compound 11 (397 mg, 0.5 mmol) and trifluoroacetic acid
(1.0 mL, 13.1 mmol) in dichloromethane (3 mL) was stirred at rt for 3 h,
concentrated in vacuo, coevaporated with toluene (5 × 10 mL), and redissolved
in ethyl acetate (5 mL). Pentafluorophenol (92 mg, 0.5 mmol) and dicyclohexyl
carbodiimide (103 mg, 0.7 mmol) were added to the solution at 0◦C and the mix-
ture was stirred for 14 h at 0◦C. Filtration of the mixture, concentration of the
filtrate in vacuo, and chromatography of the residue (n-hexane/ethyl acetate
1:5) afforded 12 (337 mg, 72%). [α]2D0 = +2.5 (c 1.0, CHCl3). FAB MS m/z: found

K. Gu¨nther et al.
456
1
956.22218, calcd. 956.22065 [M+Na]+. H NMR (CDCl3): δ (ppm) = 7.71 (d, 2
H, J = 7.6 Hz, Ar-H), 7.60 (s , 1 H, triazole-H), 7.49 (d, 2 H, J = 7.4 Hz, Ar-H),
7.35 (t, 2 H, J = 7.6 Hz, Ar-H), 7.18–7.26 (m, 3 H, Ar-H, NH), 5.87 (s, 1 H, NH),
5.15 (t, 1 H, J1,2 = 9.4 Hz, H-1), 4.92 (t, 1 H, J3,4 = 9.9 Hz, H-3), 4.77 (t, 1 H,
J2,3 = 9.7 Hz, H-2), 4.69 (s, 1 H, 3ꢁ) 4.36–4.56 (m, 9 H, Fmoc, H-2, H-4, H-6a, H-
5, CH2), 3.79 (s, 1 H, H-6b), 3.08–3.26 (m, 2 H, H-2ꢁa,b), 2.48 (q, 2 H, J = 7.1 Hz,
SCH2) 2.00, 1.99, 1.95 (3s, 9 H, COCH3), 1.18 (t, 3 H, J = 7.1 Hz, SCH2CH3).13C
NMR (CDCl3): δ (ppm) = 170.0, 169.8, 169.7, 169.3, 167.5, 156.1 (C O), 144.2
(triazole C-4), 143.4, 141.3 (Fmoc), 136.6–139.5 (Pfp), 127.8, 127.0 124.9, 120.0,
(Fmoc), 124.2 (triazole C-5), 83.5 (C-1), 76.0 (C-3), 73.4 (C-2), 69.6 (C-4), 69.5
(C-5), 67.4 (Fmoc-CH2), 51.0 (C-6), 49.3 (Asp-CH), 47.0 (Fmoc-CH), 35.3 (Asp-
CH2), 35.0 (CH2), 24.3 (SCH2), 20.6, 20.5, 20.5, (COCH3), 14.8 (SCH2CH3).
t-Butyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-{[1-(2,3,4-tri-O-
acetyl-6-deoxy-1-ethylthio-β-D-galactopyranos-6-yl)-1H-
1,2,3-triazol-4-yl]methyl}-L-α-asparaginate (13)
A solution of compound 4 (225 mg, 0.6 mmol), compound 10[5] (261 mg, 0.6
mmol), diisopropyl ethylamine (0.31 mL, 1.8 mmol), and (EtO)3PCuI (24 mg,
0.06 mmol) in toluene (5 mL) was stirred 80◦C under microwave irradiation
(30 W) for 1 h. Concentration of the solution in vacuo and chromatography
of the residue (ethyl acetate) afforded 13 (331 mg, 67%). [α]2D0 = +23.4 (c 1.0,
CHCl3). HRFD MS m/z: found 846. 29917, calcd. 846.29906 [M+Na]+. 1H NMR
(CDCl3): δ (ppm) = 7.70 (d, 2 H, J = 7.4 Hz, Ar-H), 7.58 (s, 1 H, triazole- H),
7.50 (d, 2 H, J = 7.4 Hz, Ar-H), 7.42 (s, 1 H, NH), 7.33 (t, 2 H, J = 5.8 Hz, Ar-H),
7.22 (t, 2 H, J = 7.4 Hz, Ar-H), 6.16 (d, 1 H, J = 8.4 Hz, NH), 5.38 (s, 1 H, H-1),
5.16 (t, 1 H, J3,4 = 9.7 Hz, H-3), 5.03 (s, 1 H, H-3ꢁ), 4.25–4.52 (m, 8 H, Fmoc, H-
2, H-4, H-5, CH2), 3.97–4.11 (m, 2 H, H-6a,b), 2.80 (d, 1 H, J = 15.5 Hz, H-2ꢁa),
2.63 (dd, 1 H, J = 6.1, H-2ꢁb), 2.50 (t, 2 H, J = 6.1 Hz, SCH2) 2.13, 1.98, 1.92 (3s,
9 H, COCH3), 1.38 (s, 9 H, CH3), 1.08 (t, 3 H, J = 7.4 Hz, SCH2CH3). 13C NMR
(CDCl3): δ (ppm) = 170.9, 170.6, 170.0, 169.7, 169.3, 155.9 (C O), 144.5 (tria-
zole C-4), 143.4, 141.1 (Fmoc), 127.5, 126.9, 124.9, 119.8, (Fmoc), 123.6 (triazole
C-5), 83.7 (C-1), 81.4 (t-Bu C), 76.7 (C-3), 75.0 (C-2), 71.5 (C-4), 67.9 (C-5), 67.0
(Fmoc CH2), 51.2 (Asp CH), 51.1 (C-6), 46.9 (Fmoc-CH), 37.4 (Asp-CH2), 34.8
(CH2), 27.8 (CH3), 24.4 (SCH2), 20.5, 20.5, 20.3 (COCH3), 14.7 (SCH2CH3).
Pentafluorophenyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-[1-
(2,3,4-tri-O-acetyl-6-deoxy-1-ethylthio-β-D-galactopyranos-6-
yl)-1H-1,2,3-triazol-4-yl]methyl-L-α-asparaginate (14)
A solution of compound 13 (397 mg, 0.5 mmol) and trifluoroacetic acid
(1.0 mL, 13.1 mmol) in dichloromethane (3 mL) was stirred at rt for 3 h,

Using Click Reaction and Spot Synthesis
457
concentrated in vacuo, coevaporated with toluene (5 × 10 mL), and redissolved
in ethyl acetate (5 mL). Pentafluorophenol (92 mg, 0.5 mmol) and dicyclohexyl
carbodiimide (103 mg, 0.7 mmol) were added to the solution at 0◦C, and the
mixture was stirred for 14 h at 0◦C. Filtration of the mixture, concentration
of the filtrate in vacuo, and chromatography of the residue (n-hexane/ethyl ac-
etate 1:5) afforded 14 (238 mg, 51%). [α]2D0 = +14.6 (c 1.0, CHCl3). HRFD MS
m/z: found 956.21171, calcd. 956.22065 [M+Na]+. 1H NMR (CDCl3): δ (ppm) =
7.71 (d, 2 H, J = 7.4 Hz, Ar-H), 7.59 (s, 2 H, tziazole-H, NH), 7.50 (d, 2 H, J =
7.6 Hz, Ar-H), 7.35 (t, 2 H, J = 7.1 Hz, Ar-H), 7.23 (t, 2 H, J = 7.4 Hz, Ar-H),
6.22 (d, 1 H, J = 8.4 Hz, NH), 5.36 (s, 1 H, H-1), 5.16 (t, 1 H, J3,4 = 9.9 Hz, H-3),
5.00 (dd, 1 H, J = 2.8 Hz, H-3ꢁ), 4.25–4.56 (m, 9 H, Fmoc, H-2, H-4, H-6a,
H-5, CH2), 4.07–4.13 (m, 1 H, H-6b), 3.13–3.26 (m, 2 H, H-2ꢁa,b), 2.53 (t, 2
H, J = 7.4 Hz, SCH2) 2.00, 2.00, 1.93 (3s, 9 H, COCH3), 1.11 (t, 3 H, J = 7.4
Hz, SCH2CH3). 13C NMR (CDCl3): δ (ppm) = 170.3, 169.7, 169.6, 169.5, 167.2,
156.1 (C O), 144.2 (triazole C-4), 143.4, 141.1 (Fmoc), 135.8–139.5 (Pfp), 127.7,
127.0 124.9, 119.9, (Fmoc), 123.8 (triazole C-5), 83.9 (C-1), 75.1 (C-3), 71.6
(C-2), 67.9 (C-4), 67.3 (C-5), 66.9 (Fmoc-CH2), 50.2 (C-6), 49.5 (Asp-CH), 46.9
(Fmoc-CH), 35.6 (Asp-CH2), 34.9 (CH2), 24.4 (SCH2), 20.6, 20.5, 20.4, (COCH3),
14.7 (SCH2CH3).
t-Butyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-{[1-(2,3,4-tri-O-
acetyl-6-deoxy-1-methoxy-α-D-mannopyranos-6-yl)-1H-1,2,3-
triazol-4-yl]methyl}-L-α-asparaginate (15)
A solution of compound 7 (207 mg, 0.6 mmol), compound 10[5] (261 mg, 0.6
mmol), diisopropyl ethylamine (0.31 mL, 1.8 mmol), and (EtO)3PCuI (24 mg,
0.06 mmol) in toluene (5 mL) was stirred 80◦C under microwave irradiation
(30 W) for 30 min. Concentration of the solution in vacuo and chromatography
of the residue (ethyl acetate) afforded 15 (462 mg, 95%). [α]2D0 = +24.2 (c 1.0,
CHCl3). HRFD MS m/z: found 816.62200, calcd. 816.30626 [M+Na]+. 1H NMR
(CDCl3): δ (ppm) = 7.75 (d, 2 H, J = 7.6 Hz, Ar-H), 7.66 (s , 1 H, triazole-H),
7.39 (d, 2 H, J = 7.4 Hz, Ar-H), 7.39 (t, 2 H, J = 7.4 Hz, Ar-H, 7.30 (t, 2 H,
J = 7.4 Hz, Ar-H), 7.04 (s, 1 H, NH), 5.89 (d, 1 H, J = 8.1 Hz, NH), 5.30 (dd,
1 H, J1,2 = 3.6, H-2), 5.20 (s, 1 H, H-1), 5.15 (t, 1 H, J = 9.9 Hz, H-3ꢁ), 4.60 (s,
1 H, H-3), 4.41–4.55 (m, 6 H, Fmoc, H-4, CH2), 4.32 (dd, 1 H, J5,6a = 8.9 Hz,
H-5), 4.18 (t, 1 H, J = 6.6 Hz, H-6a), 4.07–4.13 (m, 1 H, H-6b), 3.05 (s, 3 H,
CH3), 2.88 (dd, 1 H, J = 3.8 Hz, H-2ꢁa), 2.59 (dd, 1 H, J = 5.3 Hz, H-2ꢁb), 2.13,
2.09, 1.86 (3s, 9 H, COCH3), 1.42 (s, 9 H, CH3). 13C NMR (CDCl3): δ (ppm) =
170.9, 170.5, 170.1, 169.8, 169.7, 156.0 (C O), 144.6 (triazole C-4), 143.6, 141.3
(Fmoc), 127.7, 127.1, 124.9, 120.0 (Fmoc), 123.9 (triazole C-5), (98.3 (C-1), 81.8
(t-Bu C), 69.3(C-2), 69.0 (C-4), 68.6 (C-3), 67.3 (C-5), 67.1 (Fmoc-CH2), 55.4
(CH3), 51.2 (Asp-CH), 50.5 (C-6), 47.1 (Fmoc-CH), 37.2 (Asp-CH2), 35.1 (CH2),
28.0 (CH3), 20.8, 20.7, 20.6 (COCH3).

K. Gu¨nther et al.
458
Pentafluorophenyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-{[1-
(2,3,4-tri-O-acetyl-6-deoxy-1-methoxy-α-D-mannopyranos-6-
yl)-1H-1,2,3-triazol-4-yl]methyl}-L-α-asparaginate (16)
A solution of compound 13 (397 mg, 0.5 mmol) and trifluoroacetic acid (1.0
mL, 13.1 mmol) in dichloromethane (3 mL) was stirred at rt for 3 h, concen-
trated in vacuo, coevaporated with toluene (5 × 10 mL), and redissolved in
ethyl acetate (5 mL). Pentafluorophenol (92 mg, 0.5 mmol) and dicyclohexyl
carbodiimide (103 mg, 0.7 mmol) were added to the solution at 0◦C, and the
mixture was stirred for 14 h at 0◦C. Filtration of the mixture, concentration
of the filtrate in vacuo, and chromatography of the residue (n-hexane/ethyl ac-
etate 1:5) afforded 14 (298 mg, 66%). [α]2D0 = +15.5 (c 1.0, CHCl3). HRFD MS
m/z: found 926.22785, calcd. 926.22785 [M+Na]+. 1H NMR (CDCl3): δ (ppm) =
7.71–7.74 (m, 3 H, J = 7.6 Hz, Ar-H, triazole-H), 7.53 (d, 2 H, J = 7.4 Hz, Ar-
H), 7.26–7.38 (m, 5 H, Ar-H, NH), 6.07–6.12 (m, 1 H, NH), 5.29 (d, 1 H, J1,2
=
3.6, H-2), 5.21 (s, 1 H, H-1), 5.10 (t, 1 H, J = 9.9 Hz, H-3ꢁ), 4.72 (s, 1 H, H-3),
4.30–4.60 (m, 7 H, Fmoc, H-4, H-5, CH2), 4.08–4.15 (m, 2 H, H-6a,b), 3.13–2.27
(m, 2 H, H-2ꢁa,b), 3.06 (s, 3 H, COCH3), 2.08, 2.03, 1.98 (3s, 9 H, Acetyl-CH3).
13C NMR (CDCl3): δ (ppm) = 171.3, 170.0, 169.8, 169.7, 167.3, 156.1 (C O),
144.0 (triazole C-4), 143.4, 141.2 (Fmoc), 136.6–139.5 (Pfp), 128.6, 127.7, 124.8,
119.9, (Fmoc), 124.0 (triazole C-5), 98.3 (C-1), 70.8 (C-2), 69.2 (C-4), 68.9 (C-3),
68.6 (C-5), 67.3 (Fmoc-CH2), 55.1 (CH3), 50.9 (C-6), 49.1 (Asp-CH), 46.9 (Fmoc-
CH), 35.3 (Asp-CH2), 34.9 (CH2), 20.6, 20.5, 20.5 (COCH3).
t-Butyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-{[1-(3,4,6-tri-O-
acetyl-2-deoxy-1-methoxy-β-D-glucopyranos-2-yl)-1H-1,2,3-
triazol-4-yl]methyl}-L-α-asparaginate (17)
A solution of compound 9 (207 mg, 0.6 mmol), compound 10[5] (261 mg, 0.6
mmol), diisopropyl ethylamine (0.31 mL, 1.8 mmol), and (EtO)3PCuI (24 mg,
0.06 mmol) in toluene (5 mL) was stirred 80◦C under microwave irradiation (30
W) for 1 h. Concentration of the solution in vacuo and chromatography of the
residue (ethyl acetate) afforded 15 (400 mg, 84%). [α]2D0 = +38.7 (c 1.0, CHCl3).
HRFD MS m/z: found 816.30618, calcd. 816.30626 [M+Na]+. 1H NMR (CDCl3):
δ (ppm) = 7.69 (d, 2 H, J = 7.6 Hz, Ar-H), 7.50–7.53 (m , 3 H, triazole-H, Ar-H),
7.33 (t, 2 H, J = 7.4 Hz, Ar-H), 7.21–7.26 (m, 3 H, Ar-H, NH), 6.07 (d, 1 H, J =
6.9 Hz, NH), 5.72 (t, 1 H, J = 9.9 Hz, H-3ꢁ), 5.09 (t, 1 H, J1,2 = 9.7 Hz, H-1), 4.92
(d, 1 H, J2,3 = 8.4 Hz, H-2), 4.36–4.51 (m, 4 H, Fmoc, H-5,), 4.42–4.36 (m, 4 H,
CH2, H-3), 4.10–4.17 (m, 2 H, H-4,6a), 3.83–3.86 (m, 1 H, H-6b), 3.32 (s, 3 H,
CH3), 2.81 (d, 1 H, J = 12.2 Hz, H-2ꢁa), 2.62 (dd, 1 H, J = 5.3 Hz, H-2ꢁb), 2.04,
1.98, 1.95 (3s, 9 H, COCH3), 1.39 (s, 9 H, CH3). 13C NMR (CDCl3): δ (ppm)
= 170.9, 170.6, 170.3, 169.4, 168.9, 155.9 (C O), 144.2 (triazole C-4), 143.5,
141.1 (Fmoc), 127.6, 126.9, 124.8, 119.8, (Fmoc), 123.7 (triazole C-5), 101.1

Using Click Reaction and Spot Synthesis
459
(C-1), 81.5 (t-Bu C), 72.0 (C-3), 71.6 (C-5), 68.5 (C-4), 66.9 (Fmoc-CH2), 63.8
(C-6), 61.6 (CH3), 57.2 (C-2), 51.2 (Asp-CH), 46.9 (Fmoc-CH), 37.4 (Asp-CH2),
34.9 (CH2), 27.8 (CH3), 20.5, 20.3, 20.0, (COCH3).
Pentafluorophenyl N2-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-{[1-
(3,4,6-tri-O-acetyl-2-deoxy-1-methoxy-β-D-glucopyranos-2-
yl)-1H-1,2,3-triazol-4-yl]methyl}-L-α-asparaginate (18)
A solution of compound 13 (397 mg, 0.5 mmol) and trifluoroacetic acid
(1.0 mL, 13.1 mmol) in dichloromethane (3 mL) was stirred at rt for 3 h,
concentrated in vacuo, coevaporated with toluene (5 × 10 mL), and redissolved
in ethyl acetate (5 mL). Pentafluorophenol (92 mg, 0.5 mmol) and dicyclohexyl
carbodiimide (103 mg, 0.7 mmol) were added to the solution at 0◦C, and the
mixture was stirred for 14 h at 0◦C. Filtration of the mixture, concentration
of the filtrate in vacuo, and chromatography of the residue (n-hexane/ethyl ac-
etate 1:5) afforded 18 (336 mg, 74%). [α]2D0 = +19.2 (c 1.0, CHCl3). HRFD MS
1
m/z: found 926.22858, calcd. 926.22785 [M+Na]+. H NMR (CDCl3): δ (ppm)
= 7.69 (d, 2 H, J = 7.4 Hz, Ar CH), 7.57–7.61 (m , 2 H, triazole-H, NH), 7.50
(t, 2 H, J = 7.1 Hz, Ar-H), 7.33 (t, 2 H, J = 7.4 Hz, Ar-H), 7.21 (t, 2 H, J =
7.4 Hz, Ar-H), 6.26 (d, 1 H, J = 8.9 Hz, NH), 5.70 (t, 1 H, J = 9.9 Hz, H-3ꢁ),
5.10 (t, 1 H, J1,2 = 9.7 Hz, H-1), 4.90 (d, 1 H, J2,3 = 8.1 Hz, H-2), 4.76 (d,
1 H, J3,4 = 6.6 Hz, H-3), 4.27–4.47 (m, 6 H, Fmoc, H-5, CH2), 4.04–4.16 (m,
2 H, H-4,6a), 3.82 (d, 1 H, J5,6b = 8.4 Hz, H-6b), 3.28 (s, 3 H, CH3), 2.19 (s,
2 H, H-2ꢁa,b), 2.01, 1.99, 1.95 (3s, 9 H, COCH3). 13C NMR (CDCl3): δ (ppm)
= 170.5, 169.8, 164.6, 169.1, 167.1, 156.0 (C O), 144.1 (triazole C-4), 143.8,
141.1 (Fmoc), 136.4–139.5 (Pfp), 127.6, 126.9 124.8, 119.8, (Fmoc), 124.0 (tria-
zole C-5), 101.0 (C-1), 72.0 (C-3), 71.6 (C-5), 68.4 (C-4), 67.1 (Fmoc-CH2), 63.9
(C-6), 61.6 (CH3), 57.0 (C-2), 51.0 (Asp -H), 46.9 (Fmoc-CH), 35.5 (Asp-CH2),
34.8 (CH2), 20.4, 20.2, 19.8, (COCH3).
Derivatized Cellulose Membrane I
Rectangular cellulose paper sheets (10 × 14 cm) were rinsed in dioxane (3
× 20 mL) and methanol (3 × 20 mL) for 10 min each and dried in a desiccator
in vacuo. Each membrane was soaked with dioxane (3 mL) containing 10 vol-
% epibromohydrin (0.3 mL) and 1 vol-% perchloric acid (30 µL) at rt for 2 h
followed by rinsing with dioxane and methanol and drying the membrane as
described above. Each membrane was then rinsed in DMF (20 mL) containing
20 vol-% 4,7,10-trioxa-1,13-tridecanediamine (4 mL) at rt for 2 h followed by
rinsing in a 5-M solution of NaOMe in methanol (20 mL) for 30 min, washing
the membrane with methanol (7 × 20 mL) for 2 min each, and drying it in
vacuo in a desiccator.

K. Gu¨nther et al.
460
Pentafluorophenyl 4ꢀ-{(R,S)-α-[1-(9-Fluorenyl)
methoxycarbonylamino]-2,4-dimethoxybenzyl}-
phenoxyacetate (19)
A
solution of 4ꢁ-{(R,S)-α-[1-(9-fluorenyl)methoxycarbonylamino]-2,4-
dimethoxybenzyl}-phenoxyacetatic acid (Rink amide linker) (135 mg, 0.25
mmol), pentafluorophenol (46 mg, 0.25 mmol), and diisopropyl carbodiimide
(40 µL, 0.26 mmol) in N-methylpyrrolidone (NMP) (1 mL) was stirred at rt for
30 min to give a ca. 0.25-M solution of 19 in NMP, ready for spotting the Rink
Amide Liner onto the derivatized cellulose membrane I.
Derivatized Cellulose Membrane II
A. Derivatization with Rink amide linker. A derivatized cellulose mem-
brane I was placed in the autospot robot and 256 spots in a 16 × 16 matrix
with a distance of 4.8 mm between the spots were created on the membrane
by automated pipetting 0.1 µL of the above solution of 19 in NMP onto each
spot, and repeating the spotting cycle for all spots after 30 min. The membrane
was then rinsed two times for 2 min with a solution of acetic anhydride (2 mL)
and diisopropyl ethylamine (4 mL) in DMF (20 mL), and washed with DMF (2
× 20 mL) for 2 min each. Next, the membrane was rinsed with a solution of
piperidine (5 mL) in DMF (20 mL) for 30 min at rt, rinsed with DMF (4 × 20
mL) and ethanol (4 × 20 mL) for 2 min each, and dried in vacuo in a desiccator.
B. Derivatization with Glycine. The membrane was placed back in the
autospot robot and 0.1 µL of a 0.25-M solution of pentafluorphenyl (9-
fluorenyl)methoxycarbonylaminoacetate (Fmoc-Gly-OPfp) 20 in NMP was ap-
plied on each spot. After 20 min at rt the cycle was repeated and after an
additional 20 min, the membrane was rinsed, and the Fmoc group was cleaved
off as described above under A.
Glycotetrapeptide Library
A derivatized cellulose sheet II was placed in the autospot robot for the
successive coupling of building blocks A (16), R (12), N (14), and D (18). For
each building block the following sequence was applied. A: 0.1 µL of a solution
of each building block (0.1 mmol) and HODhbt (60 µmol) in NMP (1 mL) was
pipetted on the Rink Amid Linker/glycine derivatized spots. After 30 min the
entire cycle was repeated. B: The membrane was rinsed two times for 2 min
at rt with a solution of acetic anhydride (0.8 mL) in DMF (20 mL). C: The
membrane was rinsed four times for 2 min at rt with DMF (20 mL). D: The
membrane was rinsed for 30 min at rt with a solution of piperidine (5 mL) in
DMF (20 mL). E: The membrane was rinsed as described under C followed by
rinsing four times at rt with ethanol (20 mL). F: The cycles A through E were
repeated three times until all 256 combinations of building blocks A, R, N, and

Using Click Reaction and Spot Synthesis
461
D were spotted onto the paper sheet. G: The membrane was dried in vacuo in
a desiccator after termination of the final step E.
Deblocking of the Glycotetrapeptide Library on the Cellulose
Sheet
The cellulose membrane containing the 256 glycotetrapeptides was rinsed
with a 5-M solution of NH3 in methanol (50 mL) in a Petri dish and shaking
the dish at rt for 16 h, followed by rinsing the membrane with ethanol (2 × 20
mL) for 2 min each and drying the membrane in vacuo.
Lectin Screening
A. Binding of lectins. The membrane was rinsed with 20 mM tris-
hydroxymethyl aminoethane (TRIS) buffer pH 7.5 (3 × 25 mL) for 5 min each,
followed by rinsing with 20 mM TRIS buffer pH 7.5 containing 50 mg TWEEN
per liter for 5 min. The wet membrane was then rinsed under shaking in a Petri
dish for 1 h at rt with a solution of the respective peroxidase-lectin conjugate of
Con A, PHA-E, and GNA (5 nmol) in 20 mM tris-hydroxymethyl aminoethane
buffer pH 7.5 (100 mL) containing 10 µL of a 0.1-M aqueous solution of CaCl2
and 10µL of a 0.1-M aqueous solution of MnCl2, followed by rinsing the mem-
brane successively with 20 mM TRIS buffer pH 7.5 (3 × 25 mL) for 5 min each
and 20 mM TRIS/TWEEN buffer pH 7.5 (25 mL) for 5 min. B. Staining of bound
lectins. The membrane containing the bound peroxidase-lectin conjugate was
rinsed in staining buffer, freshly prepared by mixing 20 mM acetate buffer pH
5.0 (47.5 mL), a solution of 3-amino-9-ethylcarbazole (20 mg, 90 mmol) in DMF
(2.5 mL), and 30% aqueous H2O2 solution (25 µL) until a visible red color of
the spots developed (5–15 min). The membrane is then rinsed with water and
scanned for documentary purposes.
ACKNOWLEDGMENT
We thank the Fonds der Chemischen Industrie for financial support of this
work and the groups of Profs. Albert and Zeller for measuring the NMR and
MS spectra.
REFERENCES
1. a) Sharon, N.; Lis, H. Lectins as cell recognition molecules. Science 1989, 246,
227–234; (b) Seitz, O. Glycopeptide synthesis and the effects of glycosylation on pro-
tein structure and activity. ChemBioChem 2000, 1, 214–246; (c) Sampson, N.S.; Mrk-
sich, M.; Bertozzi, R.C. Surface molecular recognition. Proc. Natl. Acad. Sci. USA 2001,
98, 12870–12871; (d) Rudd, P.M.; Elliott, T.; Cresswell, P.; Wilson, I.A. & Dwek, R.A.
Glycosylation and the immune system. Science 2001, 291, 2370–2376; (e) Crocker, P.R.
Mammalian Carbohydrate Recognition Systems, Springer, Heidelberg, 2001, 1–249.

K. Gu¨nther et al.
462
2. Danishefsky, J.S.; Allen, J.R. From the laboratory to the clinic: a retrospective on
fully synthetic carbohydrate-based anticancer vaccines. Angew. Chem. Int. Ed. Engl.
2000, 39, 836–863.
3. a) Ziegler, T.; Ro¨seling D.; Subramanian, L.R. Preparation of glycosyl amino acids
as building blocks for the combinatorial synthesis of neoglycoconjugates. Tetrahedron:
Asymm. 2002, 13, 911–914; (b) Schips, C.; Ziegler, T. A practical one-pot synthesis of
new S-glycosyl amino acid building blocks for combinatorial neoglycopeptide synthe-
sis. J. Carbohydr. Chem. 2005, 24, 773–788; (c) Ziegler, T.; Schips, C. A process for
the Mitsunobu coupling of 1-thiohexoses and aminoaclohols. Ger. Patent 2005, DE
102004046010. Chem. Abstr. 2005, 144, 36465; (d) Ziegler, T.; Schips, C. An efficient
Mitsunobu protocol for the one-pot synthesis of S-glycosyl amino acid building blocks
and their use in combinatorial spot synthesis of glycopeptide libraries. Nature Proto-
cols, 2006, 1, 1987–1994.
4. a) Huisgen, R.; Knorr, R.; Mo¨bius, L.; Szeimies, G. Einige Beobachtungen zur Ad-
dition organischer Azide an CC-Dreifachbindungen. Ber. Dtsch. Chem.. Ges. 1965, 98,
4014–4021; (b) Sharpless, K.B.; Fokin, V.V.; Green, L.G.; Rostovtsev, V.V. A stepwise
Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ ligation” of azides
and terminal alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596–2599; (c) Meldal, M.;
Christensen, C.; Tornoe, C.W. Peptidotriazoles on solid phase: [1,2,3]-triazoles by re-
giospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.
J. Org. Chem. 2002, 67, 3057–3064; (d) Bock, V.D.; Hiemstra, H.; van Maarseveen, J.H.
Cu(I)-catalyzed alkyne-azide “Click” cycloadditions from mechanism and synthetic per-
spective. Eur. J. Org. Chem. 2006, 51–68.
5. Pietrzik, N.; Schips, C.; Ziegler, T. Efficient synthesis of glycosylated asparaginic
acid building blocks via click chemistry. Synthesis 2008, 519–526.
6. a) Dedola, S.; Nepogodiev, S.A.; Field, R.A. Recent applications of the Cu(I)-
catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in carbohydrate
chemistry. Org. Biomol. Chem. 2007, 5, 1006–1017; (b) Winkinson, B.L.; Bornaghi,
L.F.; Poulson, A.-A.; Houston, T.A. Synthetic utility of glycosyl triazoles in carbohy-
drate chemistry. Tetrahedron 2006, 62, 8115–8125; (c) Fernandez-Megia, E.; Correa, J.;
Rodriguez-Meizoso, I.; Riguera, R. A click approach to unprotected glycodendtrimers.
Macromol. 2006, 39, 2113–2120; (d) Perion, R.; Ferrieres, V.; Garcia-Moreno, M.I.;
Mellet, C.O.; Duval, R.; Fernandez, J.M.G.; Plusquellec, D. 1,2,3-Triazoles and related
glycoconjugates as new glycosidase inhibitors. Tetrahedron 2005, 61, 9118–9128; (e)
Bodine, K.D.; Gin, D.Y.; Gin, M.S. Highly convergent synthesis of C3- or C2-symmetrical
carbohydrate macrocycles. Org. Lett. 2005, 7, 4479–4482; (f) Do¨rner, S.; Westermann,
B. A short route for the synthesis of sweet macrocycles via a click-dimerization-ring-
closing metathesis approach. Chem. Commun. 2005, 2852–2854; (g) Hoffmann, B.;
Bernet, B.; Vasella, A. Oligosaccharide analogues of polysaccharides. Part 24. Syn-
thesis of cyclodextrin analogues containing a substituted buta-1,3-diyne or a 1,2,3-
triazole unit and analysis of intramolecular hydrogen bonds. Helv. Chim. Acta 2002,
85, 265–287; (h) Dos Anjos, J.V.; Sinou, D.; de Melo, S.J.; Srivastava, R.M. Synthe-
sis of triazole-linked manno- and glucopyranosyl amino acids. Synthesis 2007, 2647–
2652.
7. Angell, Y.L.; Burgess, K. Peptidomimetics via copper-catalyzed azide-alkyne cy-
cloadditions. Chem. Soc. Rev. 2007, 36, 1674–1689.
8. Jiang, J.; Biggins, J.B.; Thorson, J.S. Expanding the pyrimidine diphosphosugar
repertoire: the chemoenzymatic synthesis of amino- and acetamidoglucopyranosyl
derivatives. Angew. Chem. Int. Ed. Engl. 2001, 40, 1502–1505.
9. Maunier, V.; Boullanger, P.; Lafont, D.; Chevalier, Y. Synthesis and surface-active
properties of amphiphilic 6-aminocarbonyl derivatives of D-glucose. Carbohydr. Res.
1997, 299, 49–57.

Using Click Reaction and Spot Synthesis
463
10. Joosten, J.A.F.; Evers, B.; van Summeren, R.P.; Kamerling, J.P.; Vliegenthart,
J.F.G. Synthesis of β-D-Galp-(1-4)-β-D-GlcpNAc-(1-2)-α-D-Manp-(1-O)(CH2)7CH3 mim-
ics to explore the substrate specificity of sialyltransferases and trans-sialidases. Eur. J.
Org. Chem. 2003, 3569–3586.
11. Cottaz, S.; Brimacombe, J.S.; Ferguson, M.A.J. An imino-linked carba-disaccharide
α-D-mannosidase inhibitor. Carbohydr. Res. 1993, 247, 341–345.
12. Billing, J.F.; Nilsson, U.J. Cyclic peptides containing a δ-sugar amino acid—
synthesis and evaluation as artificial receptors. Tetrahedron 2005, 61, 863–874.
13. a) Yan, R.-B.; Yang, F.; Wu, Y; Zhang, L.-H.; Ye, X.-S. An efficient and improved
procedure for preparation of triflyl azide and application in catalytic diazotransfer re-
action. Tetrahedron Lett. 2005, 46, 8993–8995; (b) Bai, Y.; Boons, G.J.; Burton, A.;
Johnson, M.; Haller, M. Vinyl glycosides in oligosaccharide synthesis (part 6): 3-buten-
2-YL 2-azido-2-deoxy glycosides and 3-buten-2-YL 2-phthalimido-2-deoxy glycosides as
novel glycosyl donors. J. Carbohydr. Chem. 2000, 19, 939–958.
14. Perez-Balderas, F.; Ortega-Munoz, M.; Morales-Sanfrutos, J.; Hernandez-Mateo,
F.; Calvo-Flores, F.G.; Calvo-Asin, J.A.; Isac-Garcia, J.; Santoyo-Gonzales, F. Multiva-
lent neoglycoconjugates by regiospecific cycloaddition of alkynes and azides using or-
ganic soluble copper catalysts. Org. Lett. 2003, 5, 1951–1954.
15. a) Frank, R. Spot-synthesis: an easy technique for the positionally addressable,
parallel chemical synthesis on a membrane support. Tetrahedron 1992, 48, 9217–9232;
(b) Frank, R. The SPOT-synthesis technique: synthetic peptide arrays on membrane
supports—principles and applications. J. Immunol. Meth. 2002, 267, 13–26.
16. (a) Ast, T.; Heine, N.; Germeroth, L.; Schneider-Mergener, J.; Wenschuh, H. Effi-
cient assembly of peptomers on continuous surfaces. Tetrahedron Lett. 1999, 40, 4317–
4318; (b) Ast, T.; Heine, N.; Germeroth, L.; Schneider-Mergener, J.; Wenschuh, H. Syn-
thesis and screening of peptoid arrays on cellulose membranes. Tetrahedron 2003, 59,
9919–9930.
17. Czarnik, A.W. Solid-Phase Organic Synthesis. John Wiley & Sons, USA, 2001,
1, 17.
18. a) Koretz, K.; Leman, J.; Brandt, I.; Mo¨ller, P. Metachromasia of 3-amino-9-
ethylcarbazole (AEC) and its prevention in immunoperoxidase techniques. Histochem.
1987, 86, 471–478; (b) Frey, A.; Meckelein, B.; Externest, D.; Schmidt, M.A. A sta-
ble and highly sensitive 3,3X,5,5X-tetramethylbenzidine-based substrate reagent for
enzyme-linked immunosorbent assays. J. Immunol. Meth. 2000, 233, 47–56; (c) Poor,
M.L.; Santa, P.F.; Sittampalam, G.S. Visualization of multiple protein bands on the
same nitrocellulose membrane by double immunoblotting. Analytical Biochem. 1988,
175, 191–195; (d) Kobayashi, R.; Tashima, Y. Visualization of antigen on nitrocellulose
membrane by the oxidative coupling reaction of A/,Nꢁ-dimethyl-p-phenylendiamine and
4-chloro-1–naphthol. Anal. Biochem. 1989, 183, 9–12.