Amphiphilic p-tert-butylcalix[4]arene scaffolds containing exposed carbohydrate dendrons

Article abstract of DOI:10.1002/(SICI)1521-3773(19990201)38:3<369::AID-ANIE369>3.0.CO;2-1

The best of both worlds. Amphiphilic hybrid molecules, in which water- exposed glycodendrons are attached to a hydrophobic p-tert- butylcalix[4]arene scaffold, display strong affinities for both carbohydrate- binding proteins and polystyrene surfaces. The molecules can form monolayers useful in bioanalytical devices.

Full text of DOI:10.1002/(SICI)1521-3773(19990201)38:3<369::AID-ANIE369>3.0.CO;2-1

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[13] a) B. J. Coe, M. C. Chamberlain, J. P. Essex-Lopresti, S. Gaines, J. C.
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Oudar, J. Chem. Phys. 1977, 67, 446; c) J. Zyss, J. L. Oudar, Phys. Rev.
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substituents provide the driving force for stable assembly and/
or adhesion of a calixarene monolayer to a surface, while the
hydrophilic carbohydrate ligands mimic the cellꢁs saccharide-
rich surface. This new type of hybrid molecules can serve as
coating carbohydrate ligands in competitive solid-phase
immunoassays (Figure 1).
Â
[15] E. Hendrickx, C. Dehu, K. Clays, J. L. Bredas, A. Persoons, ACS
Symp. Ser. 1995, 601, 82.
[16] M. Stähelin, D. M. Burland, J. E. Rice, Chem. Phys. Lett. 1992, 191,
245.
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1989.
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[19] This was confirmed by the use of 100-fold concentrated solutions (ca.
10 3 m) which afforded larger HRS signals, allowing the derivation of
30
b1064 values of 20, 35, and 62 Â 10
esu for the oxidized forms of
Figure 1. Glycocalix[4]arene hybrids used as coating antigens on a hydro-
phobic polystyrene surface.
[1]Cl3, [2]Cl3, and [3]Cl3, respectively (referenced to pNA in
methanol). Excessive UV absorption by H2O2 prevents measurement
of the spectra of the RuIII forms, precluding the estimation of b0 values.
Our model carbohydrate is the TN antigen (GalNAca1 !O-
Ser/Thr) corresponding to one of the immunodominant
epitopes found in human adenocarcinomas mucins.[7] This
family of carbohydrate-associated tumor markers is usually
cryptic in normal cells. We have also recently shown that the
O-linked Ser/Thr residues in the analogous Tantigen [Gal(b1-
3)-GalNAc(a1 !O-Ser/Thr)] were not essential to generate
mouse monoclonal antibodies that recognize cancer tissues.[8]
Consequently, the a-linked GalNAc moieties described here-
in were deprived of the O-Ser/Thr aglycon.
Amphiphilic p-tert-Butylcalix[4]arene
Scaffolds Containing Exposed Carbohydrate
Dendrons**
Â
Rene Roy* and Jin Mi Kim
The synthetic strategy for the construction of glycocalix[4]-
arenes was to attach suitable spacer-substituted a-GalNAc
residues to the calix[4]arene core; both convergent and
divergent approaches were employed. The key a-d-GalNAc
derivative 3 was prepared in four steps from N-acetyl-d-
galactosamine (1) (Scheme 1). The required calix[4]arene
core 7 was prepared by transforming commercial p-tert-butyl-
calix[4]arene (6) into the known tetraethyl ester[9] followed by
hydrolysis and treatment with thionyl chloride (Scheme 2).
Direct amidation of 7 with amine 3 and subsequent de-O-
acetylation provided tetravalent glycocalix[4]arene 8.
Glycocalix[4]arenes of higher valencies were synthesized
by a semiconvergent approach. Divalent a-d-GalNAc pre-
cursor 5a and its deprotected form 5b were first obtained by
treatment of amine 3 with N-Boc-6-aminohexanoic acid
followed by trifluoroacetolysis and N-bromoacetylation to
give 4 in 85% yield (see Scheme 1). Double N-alkylation of
mono-N-Boc-1,4-diaminobutane with 4 gave dimer 5a in 73%
yield, which was deprotected to provide 5b. Treatment of acid
chloride 7 with mono-N-Boc-1,4-diaminobutane afforded 9 in
63% yield. Trifluoroacetolysis of 9 gave 10, which was N,N-
dialkylated with 4 to provide octavalent glycocalix[4]arene 11
in 64% yield after deprotection (Scheme 3). Octameric
tetraamine derivative 13 was also obtained by double
N-alkylation of 10 using 4-bromoacetamido-1-Boc-butanedi-
amine (51%, Scheme 4). Finally, hexadecameric glyco-
calix[4]arene 14 was prepared from octameric amine 13 and
bromoacetamido-GalNAc derivative 4 after de-O-acetyla-
tion.
Calixarenes are cyclic molecules containing a cavity useful
in host ± guest chemistry.[1] Their intrinsic amphiphilic archi-
tecture also makes them ideal candidates for the study of
water± monolayer surface interactions. In this respect, they
surpass their cyclodextrin counterparts.[2] In spite of these
interesting featuresÐand in addition possible variation of
conformational organization, substitution of the upper and
lower rims, and shape and sizeÐonly limited efforts have
been made to construct biologically relevant calixarenes
containing carbohydrate moieties.[3, 4] In line with this con-
cept, the synthesis of nondendritic galactose octamers attach-
ed to a calix[4]resorcarene scaffold possessing lipophilic side
chains has been described. However, as opposed to our work
presented herein, the hydrophilic carbohydrate residues were
used for polar attachment to a polar quartz surface.[5]
We describe here the first synthesis of dendritic,[6] water-
soluble, carbohydrate-containing p-tert-butylcalix[4]arenes
and their lectin-binding properties. These carbohydrate-con-
taining calix[4]arenes can serve as models to further inves-
tigate factors influencing multivalent carbohydrate ± protein
interactions at the molecular level. The lipophilic p-tert-butyl
[*] Prof. R. Roy, J. M. Kim
Department of Chemistry, University of Ottawa
10 Marie Curie, Ottawa, ON K1N6N5 (Canada)
Fax: (‡1)613-562-5170
E-mail: rroy@science.uottawa.ca
[**] We thank the National Science and Engineering Research Council of
Canada (NSERC) for financial support and Dr. P. Thibault, National
Research Council of Ottawa (NRC), for MALDI-TOF mass spec-
trometry experiments.
The ligands 5b, 8, 11, and 14 were purified by size-exclusion
1
chromatography (Sephadex LH20, MeOH). H NMR spec-
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Scheme 1. Synthesis of key monomer 3 and dimer 5b. a) HOCH2CH2Cl,
BF3 ´ OEt2, reflux for 4 h, 258C for 48 h; b) NaN3 (10 equiv), NaI (1 equiv),
CH3CN, reflux, 48 h, 85% (2 steps); c) Ac2O, pyridine, 258C, 16 h, 80%;
d) 1. H2-Pd/C, AcOH, MeOH, 16 h; 2. Amberlite IRA-400(Cl) resin,
MeOH, 16 h, quantitative; e) HO2C(CH2)5NHBoc (1.2 equiv), DIPEA
(2.5 equiv), TBTU (1.2 equiv), CH2Cl2, 08C, 30 min, 76%; f) 1. 20% TFA,
CH2Cl2, 258C, 2 h; 2. ClCOCH2Br (1.2 equiv), DIPEA (2.5 equiv), CH2Cl2,
08C, 30 min, 85%; g) H2N(CH2)4NHBoc (0.9 equiv), DIPEA (1.2 equiv),
CH3CN, reflux, 48 h, 73%; h) 1. 1m NaOMe, MeOH, pH 9, 258C, 3 h;
2. 20% TFA, CH2Cl2, 258C, 2 h, 83% (2 steps). Boc ˆ tert-butoxycarbonyl,
DIPEA ˆ diisopropylethylamine, TBTU ˆ O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluronium tetrafluoroborate, TFA ˆ trifluoroacetic acid.
Scheme 2. Synthesis of tetramer 8. a) BrCH2CO2Et (20 equiv), K2CO3
(20 equiv), acetone, 4-Š molecular sieves, reflux, 24 h, 84%; b) 1m KOH,
EtOH (1/1.1 v/v), reflux, 9 h, 90%; c) SOCl2, reflux, 2 h, quantitative; d) 3
(6 equiv), Et3N (12 equiv), CH2Cl2, 0 !258C, 2 h, 74%; e) NaOMe,
MeOH, pH 9, 258C, 2 h, 94%.
troscopy (D2O) shows that water-soluble glycocalix[4]arenes
8, 11, and 14 exist in the cone conformation as judged from the
singlets in the aromatic region (d ˆ 6.8) and the tert-butyl
signals at d ˆ 1.1 (Table 1). The ratios for the signals attributed
to the anomeric (d ˆ 4.9) and tert-butyl protons (d ˆ 1.1) were
used to confirm complete glycosylation.
These ligands were then evaluated for their relative lectin-
binding properties against Vicia villosa agglutinin (VVA).
This plant lectin has been used previously for binding studies
against a-d-GalNAc derivatives.[10] The direct binding abil-
ities and cross-linking behavior of di- (5b), tetra- (8), octa-
(11), and hexadecavalent ligands (14) toward VVA were
initially determined by turbidimetric analysis. Hypervalent
glycocalix[4]arene dendrimers demonstrated direct binding to
VVA by the rapid formation of insoluble precipitates
(Figure 2). Allyl 2-acetamido-2-deoxy-a-d-galactopyranoside
(allyl a-d-GalNAc) was used as an inhibitor for the cross-
linking of octavalent 11 with VVA, thus demonstrating the
sugar specificity of the binding. The interaction between 11
and VVA was so strong that a 250-fold molar excess of allyl a-
d-GalNAc was necessary to disrupt the cross-linking inter-
action.
Scheme 3. Synthesis of octamer 11. a) BocHN(CH2)4NH2 (6 equiv),
DIPEA (12 equiv), CH2Cl2, 08C, 3 h, 63%; b) 20% TFA, CH2Cl2, 258C,
2 h; c) 4 (10 equiv), DIPEA, CH3CN, 608C, 48 h; d) 1m NaOMe, MeOH,
258C, 16 h, 64% (2 steps).
The efficiency of each glycocalixarene to inhibit the binding
of asialoglycophorin, a natural glycoprotein of human eryth-
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Scheme 4. Syntheses of hexadecamer 14.
a) BocHN(CH2)4NHCOCH2Br
(10 equiv), DIPEA (14 equiv), CH3CN,
608C, 48 h, 51%; b) 20% TFA, CH2Cl2,
258C, 2 h; c) 4 (20 equiv), DIPEA,
CH3CN, 608C, 48 h; d) 1m NaOMe,
MeOH, 258C, 16 h, 69% (2 steps).
rocytes, to VVA was measured by enzyme-linked lectin assay
(ELLA). Asialoglycophorin (MN) was used as a coating
antigen for the solid-phase competitive experiments (MN is a
Table 1. Selected physical data for representative compounds.
5a: [a]2D0 ˆ ‡59.1 (c ˆ 1.0 in CHCl3); 1H NMR (500 MHz, CDCl3): d ˆ
1.24 ± 1.32 (q, J ˆ 7.4 Hz, 4H; 2  CH2), 1.39 (s, 9H; tBu), 1.47 ± 1.53 (m,
8H; 4  CH2), 1.93, 1.96, 2.00, 2.11 (4 s, 24H; OAc), 2.13 ± 2.17 (t, J ˆ
7.4 Hz, 4H; CH2CONH), 3.02 ± 3.33 (m, 14H; CH2, CHH), 3.45 ± 3.52 (m,
2H; CHH), 3.55 ± 3.66 (m, 2H; CH2), 3.68 ± 3.75 (m, 2H; CHH), 4.02 ± 4.10
(m, 4H; CH2), 3.70 ± 3.76 (m, 2H; CHH), 4.02 ± 4.10 (m, 4H; H-6), 4.12 ±
4.16 (m, 2H; H-5), 4.54 (ddd, J2,3 ˆ 11.4 Hz, JNH,2 ˆ 9.5 Hz, 2H; H-2), 4.84
(d, J1,2 ˆ 3.6 Hz, 2H; H-1), 4.93 ± 4.99 (m, 1H; NHBoc), 5.10 (dd, J3,4
ˆ
3.3 Hz, 2H; H-3), 5.32 (m, 2H; H-4), 6.90 ± 7.05 (br, 4H; NH), 7.50 ± 7.65
(br, 2H; NH); 13C NMR (125.8 MHz, CDCl3): d ˆ 20.7 (CH3CO2), 23.0
(NHCOCH3), 25.0, 26.1, 27.0 (3 Â CH2), 28.4 (tBu), 29.0, 36.2, 38.6, 39.0,
39.7, 42.0 (6 Â CH2), 47.4 (C2), 58.8 (CH2), 62.0 (C6), 66.7 (C5), 67.3 (C4),
68.1 (CH2), 68.5 (C3), 98.4 (C1), 156.6 (Ar), 170.4, 170.5, 170.6, 170.7, 173.5
Figure 2. Turbidimetric analysis of the binding of glycocalix[4]arenes with
‡
ˆ
(C O); positive-ion FAB-MS: m/z (%): 1275.6 (10.2) [M ‡1].
&
&
*
^
*
VVA. : 5b (2-mer), : 8 (4-mer), : 11 (8-mer), : 14 (16-mer), : 11 with
allyl a-d-GalNAc as an inhibitor. OD ˆ optical density measured at
490 nm.
‡
8: positive-ion FAB-MS: m/z (%): 1865.8 (2.4) [M ‡1].
11: [a]2D0 ˆ ‡53.6 (c ˆ 0.5 in DMSO); MALDI-TOF MS calcd for
C212H352N32O72: 4498: found: 4499 [M‡1]. 12: positive-ion FAB-MS calcd
for C156H264N24O32:2985; found: 2986 (0.2%) [M‡1].
blood group serotype). Horseradish peroxidase labeled VVA
(VVA-HRP) was used for the quantitative detection of
inhibition by optical density. The results for the inhibition of
asialoglycophorin ± VVA binding are shown in Figure 3. The
best result was obtained from the hexadecavalent conjugate
14 (IC50 ˆ 13.4mm), which represents a 12-fold increase in
potency over that of the allyl a-d-GalNAc monomer (IC50 ˆ
158.3mm).
12: positive-ion FAB-MS caled for C156H264N24O32: 2985; found: 2986
(0.2%) [M‡1].
14: [a]2D0 ˆ ‡66.4 (c ˆ 1.0 in MeOH); 1H NMR (D2O): d ˆ 1.11 (brs, 9H,
tBu), 1.32 ± 1.43, 1.45 ± 1.70 (m, 32H; internal CH2), 2.11 (s, 48H; NAc),
2.25 ± 2.37 (m, 32H; CH2CONH), 2.50 ± 2.66 (m, 24H; CH2N), 3.14 ± 3.44
(m, 124H; CONHCH2, NCH2CO, ArCHH, CHHO), 3.54 ± 3.64 (m, 32H;
NCHH, CHHO), 3.76 ± 3.87 (m, 52H; H-6, 4 ArCHH, NCHH), 3.92 ± 4.00
(m, 32H; H-2, H-5), 4.04 (dd, J3,4 ˆ 2.8 Hz, 16H; H-4), 4.25 (dd, J2,3
ˆ
11.0 Hz, 16H; H-3), 4.94 (d, J1,2 ˆ 3.4 Hz, 16H; H-1), 6.92 (brs, 8H; Ar);
13C NMR (D2O): d ˆ 21.7 (NAc), 23.4, 24.7, 25.4, 27.9, 30.8 (tBu), 35.4, 38.5,
49.4 (C2), 54.8, 57.9, 60.7 (C6), 66.0, 67.4 (C3), 68.1 (C4), 70.6 (C5), 96.8
Our most important finding was the monolayer-forming
ability of the GalNAc-containing calix[4]arenes. Tetra- and
hexadecavalent glycocalix[4]arenes 8 and 14, respectively,
ˆ
(C1), 128.4 (Ar), 172.4, 173.7, 174.0, 175.9, 176.3 (C O).
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Received: June 24, 1998
Revised version: October 13, 1998 [Z12049IE]
German version: Angew. Chem. 1999, 111, 380 ± 384
Keywords: calixarenes ´ dendrimers ´ glycosides ´ lectins
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Figure 3. Enzyme-linked lectin assay of the inhibition of the binding of
asialoglycophorin to horseradish peroxidase labeled VVA B4 by allyl d-
GalNAc and by ligands 5b, 8, 11, and 14 (VVA B4 is the isolectin made up
of four B subunits).
[6] a) R. Roy, Curr. Opin. Struct. Biol. 1996, 6, 692 ± 702; b) R. Roy,
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were incubated at various concentrations (0.1 ± 2.5 mg per
well) on polystyrene microtiter plates in phosphate-buffered
saline, and VVA-HRP was used to detect the direct binding
properties. As shown in Figure 4, hexadecavalent glyco-
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Organically Templated Mixed-Valent
TiIII/TiIV Phosphate with an Octahedral ±
Tetrahedral Open Framework**
Sambandan Ekambaram and Slavi C. Sevov*
The zeolitic aluminum silicates and the more recently
discovered microporous aluminum phosphates have attracted
tremendous interest due to their application as catalysts, ion-
exchangers, and molecular sieves in many technologically
important processes. Nevertheless, due to the presence of
main group elements only, these materials have no potential
for redox reactions and redox catalysis. It is highly desirable,
therefore, to build microporous compounds containing
d-block metals as an integral part of the framework and in
close proximity to the voids. Titanium has been of particular
interest as a potential substitute for silicon owing to the
available oxidation state of four and appropriate size.
Substitutions of the tetrahedral silicon have been achieved
at the doping level,[1] and despite the small amounts of
titanium, the resulting materials have shown highly improved
Figure 4. Enzyme-linked lectin assay (VVA-HRP) showing the hydro-
phobic adsorption of glycocalix[4]arenes onto the surface of a polystyrene
&
*
microtiter plate. : 14 (16-mer), : 8 (4-mer). OD ˆ optical density
measured at 410 nm.
calix[4]arene 14 was hydrophobically adsorbed onto the
microtiter plate surface with as little as 0.2 mg of material
per well. Moreover, the interaction could be again inhibited
by adding excess allyl a-d-GalNAc monomer (data not
shown).
Amphiphilic a-GalNAc-containing p-tert-butylcalix[4]
arenes with up to 16 carbohydrate units were efficiently
synthesized by a novel double N-alkylation strategy. In these
novel biomaterials the carbohydrate residues are well ex-
posed to aqueous environments as shown by their lectin-
binding properties. Moreover, the materials can be directly
adsorbed onto the lipophilic surface of polystyrene microtiter
plates and thus should be useful in bioanalytical devices.
[*] Prof. S. C. Sevov, S. Ekambaram
Department of Chemistry and Biochemistry
University of Notre Dame
Notre Dame, IN 46556 (USA)
Fax: (‡1)219-631-6652
E-mail: ssevov@nd.edu
[**] We thank the National Science Foundation (DMR-9701550 and
DMR-9703732) and the Bayer Corporation (Postdoctoral Fellowship)
for the financial support.
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