Design, synthesis, and screening of a library of peptidyl bis(boroxoles) as oligosaccharide receptors in water: Identification of a receptor for the tumor marker TF-antigen disaccharide

Article abstract of DOI:10.1002/anie.200906620

Chemical Equation Presented Mini lectins: A new class of oligosaccharide receptors (see example) was designed by exploiting several modes of molecular recognition, including the unique ability of benzoboroxoles to complex hexopyranosides. The synthesis is modular, thus well suited to targeting specific oligosaccharides using combinatorial libraries.

Full text of DOI:10.1002/anie.200906620

Communications
DOI: 10.1002/anie.200906620
Carbohydrate Recognition
Design, Synthesis, and Screening of a Library of Peptidyl
Bis(Boroxoles) as Oligosaccharide Receptors in Water: Identification
of a Receptor for the Tumor Marker TF-Antigen Disaccharide**
Arnab Pal, Marie Bꢀrubꢀ, and Dennis G. Hall*
The important challenge of carbohydrate recognition in water
presents several exciting opportunities in chemical biology
and medicine, with potential applications in the analysis,
purification, diagnostic, and physiological control of biolog-
ically important glycans. Although a number of synthetic
receptors have been described for the recognition of complex
carbohydrates in organic solvents,[1] it is notoriously difficult
to achieve the same efficiency under physiological conditions
(i.e., water at neutral pH).[2] The essence of the problem lies in
the competition between the multiple hydroxy groups on the
carbohydrates and the overwhelming ones from the bulk
solvent, water. Recently, Davis and co-workers described a
water-soluble, complex cage-like receptor that displayed low
millimolar affinity and high selectivity for a distinct disac-
charide in neutral water.[3]
To be general, however, a receptor approach must address
the structural diversity of oligosaccharides while overcoming
the difficulty of predicting their favored conformations.
Combinatorial strategies appear to be ideal to address this
problem. To this end, we sought to design a simple class of
small receptors that could be synthesized in a modular fashion
amenable to the preparation of libraries. Any approach to the
recognition of carbohydrates in water should take advantage
of the intrinsic orientation of the sugarꢀs hydroxy groups on
the rigid oxacarbocyclic skeleton. In this regard, boronic acids
have the ability to form boronic esters reversibly with polyols
and sugars in water.[4,5] While the use of boronic acids is
regarded as one of the most promising approaches for the
recognition of carbohydrates in water,[6] it is not without
limitations. First and foremost, as boronic acids display a
marked preference for binding furanose sugars,[7] cell-surface
glycoconjugates are unfavorable targets because they are
comprised of hexopyranosides.[8] To overcome this issue,
Wang and co-workers successfully targeted a glycoprotein
using a very large library of boronic acid-modified DNA
aptamers termed “boronolectins”.[9] To avoid selectivity
issues that complicate the optimization of a discrete receptor,
Lavigne and co-workers used arrays of unidentified peptide-
boronic acids from a large mixture library to detect glyco-
proteins according to response patterns.[10]
In contrast to these strategies, we favor an approach
targeting each glycan of interest with a single, low molecular
weight receptor of defined composition. This approach
exploits
benzoboroxoles
as
hexopyranoside-binding
agents,[11] and couples boronate formation with other modes
of molecular recognition inspired from natureꢀs carbohy-
drate-binding proteins, lectins. In this model study, a ration-
ally designed library of synthetic receptors[12] is targeted
against an important tumor-associated carbohydrate antigen,
the Thomsen–Friedenreich (TF) disaccharide (Gal-b-1,3-
GalNAc, Scheme 1).[13] Synthetic TF receptors are of partic-
ular interest because anti-TF antibodies have proven difficult
to optimize.[14]
Because the TF antigen possesses two of the 4,6-diol or
cis-3,4-diol units that bind preferentially with benzoborox-
oles,[11] two such units were included on the receptors
(Scheme 1). A peptide backbone was chosen for ease of
synthesis and also for providing hydrogen bonding donor and
[*] A. Pal, Dr. M. Bꢀrubꢀ, Prof. D. G. Hall
Department of Chemistry, Gunning-Lemieux Chemistry Centre
University of Alberta, Edmonton, Alberta, T6G 2G2 (Canada)
Fax: (+1)780-492-8231
E-mail: dennis.hall@ualberta.ca
Homepage: http://www.chem.ualberta.ca/~dhall/
[**] This work was funded by the Natural Sciences and Engineering
Research Council (NSERC) of Canada (E.W.R. Steacie Memorial
Fellowship to D.G.H.) and the University of Alberta. M.B. thanks the
Killam Foundation for a postdoctoral fellowship (University of
Alberta). We thank Eric Pelletier for support in library purification by
HPLC, and Prof. David Bundle and Joanna Sadowska (Alberta
Ingenuity Center for Carbohydrate Science) for help with the
screening assay and access to the Lemieux Collection of Oligosac-
charides.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200906620.
Scheme 1. Design of peptidyl bis(boroxoles) library 1{1–20,21–40}.
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acceptor capabilities. The central amino acid position, flanked
by two diaminopropionic acid (Dpr) residues for attachment
of the benzoboroxole, was randomized with 20 natural and
nonnatural amino acids 1–20 offering functional and geo-
metrical diversity (spacer length and rigidity; Scheme 2).
Scheme 3. Capping groups 21–40 for library of receptors 1{1–20,21–
40}.
Scheme 2. Spacers 1–20 for library of receptors 1{1–20,21–40}.
Fmoc=9-fluorenylmethoxycarbonyl.
The acyl capping group consisted in a selection of 20
carboxylic acids (Scheme 3). Several library components for
both the spacer and terminal positions include aromatic
subunits because they are known in carbohydrate-binding
proteins to promote hydrophobic CH–p interactions with the
nonpolar face of saccharides (Scheme 1).[15] The length and
flexibility of the carboxybenzoboroxole-functionalized Dpr
side arms should allow for effective interactions of these
subunits with either faces of the disaccharide. The library was
assembled by solid-phase peptide synthesis using trityl resin,
and includes a short triethyleneglycol spacer and an anchor-
ing primary amine for increased aqueous solubility and for
eventual conjugation purposes (Scheme 4). The library of 400
receptors was prepared expeditiously by split–pool synthesis
using the IRORI radio-frequency encoding technology with
MicroKan reactors.[16] Key to this strategy is a late-stage
coupling of the diaminopropionic acid side chain amines with
5-carboxybenzoboroxole (2).[17] Compared to benzoboroxole,
an amide model of 2 provides a significant increase in
hexopyranoside-binding affinity, which is attributable to the
enhanced acidity gained through the electron-withdraw-
ing carboxamide (Scheme 5). Once completed, library
1{1–20,21–40} was cleaved with dilute trifluoroacetic acid,
Scheme 4. Preparation of library 1{1–20,21–40}. HBTU=O-(benzotria-
zol-1-yl)-tetramethyluronium hexafluorophosphate; HOAt=hydroxy-7-
azabenzotriazole; DIPEA=diisopropylethylamine; Alloc=allyloxycar-
bonyl; TFA=trifluoroacetic acid.
and all 400 members were purified by semipreparative
HPLC.[17,18]
Screening of the library of 400 peptidyl bis(boroxole)
receptors for binding to the TF antigen disaccharide
was performed using a competitive ELISA in 96-well
plates coated with Gal-b-1,3-GalNAc-O(CH2)8CO-BSA
(ca. 5.5 units/protein).[17] The competing protein receptor in
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Scheme 5. Pyranoside-binding with a 5-carboxybenzoboroxole amide.
this assay is the Arachis hypogaea (peanut) agglutinin lectin
(PNA), a tetrameric protein known to bind to Gal-b-1,3-
GalNAc with a dissociation constant Kd of 1 ꢁ 10À7 m.[19] The
peroxidase-labeled PNA lectin (1.0 ꢁ 10À9 m) was incubated
with each library member at a high concentration of 400 mm.
Following washing operations and addition of the chromo-
genic substrate (3,3’,5,5’-tetramethylbenzidine), the absorb-
ance was measured at 450 nm.[20] A total of 17 hits were
confirmed reproducibly, and IC50 values were measured on
the four most promising peptidyl bis(boroxoles).[17] The
most potent receptor, 1{17,29}, showed an IC50 of 20 mm
(Scheme 6). The surprising disparity between receptors
1{15,23} and 1{16,23}, which differ only by the stereochemical
configuration of the spacer, provided an early hint that
selective and subtle molecular recognition is taking place with
the TF antigen disaccharide.
Figure 1. Competition experiments to assess the selectivity of receptor
1{17,29} for saccharides. TF-antigen: Gal-b-1,3-GalNAc-O(CH2)8CO2Et.
be expected given the “Gal-1,3-Gal like” polyol pattern
offered by this saccharide.
Overall, these preliminary control experiments demon-
strate that receptor 1{17,29} is specifically targeting the Gal-
b-1,3-GalNAc disaccharide and does so with a marked
selectivity. An approximate dissociation constant between
1{17,29} and Gal-b-1,3-GalNAc-O(CH2)8CO2Et can be
extracted from the IC50 value relative to the concentration
of PNA lectin used in the assay and the known Kd for the
complex between PNA and Gal-b-1,3-GalNAc.[19] The result-
ing estimated value of 0.5 mm is close to that of 0.9 mm
obtained by induced circular dichroism observed on the
peptidyl bis(boroxole) 1{17,29} (measured in CH3OH for
solubility at high concentrations) and calculated according to
an excellent fit to a 1:1 binding model.[17,21]
Receptor 1{17,29} may bind its target disaccharide using
several possible interactions, including boronate formation,
hydrogen bonding, hydrophobic packing, and CH–p inter-
actions. The fact that it contains the electron-rich p-methoxy-
phenylalanine and a furan as p donor components may be
indicative of CH–p interactions with the electron-deficient
hydrogens on the sugar rings. Because they are an important
element of our receptor design, we assessed the role of the
two boroxole units of 1{17,29} by comparison with the
corresponding bis(arylboronic acid) 3 and bis(phenylamide) 4
(Scheme 7), which were synthesized in a similar manner as in
Scheme 4.[17] With respective IC50 values of 54 and 100 mm, it is
not surprising to confirm that boroxoles are more favorable
than normal boronic acids for complexing hexopyranosides.
With a five-fold difference between 4 and 1{17,29}, it seems
unlikely, however, that both boroxole units of receptor
1{17,29} are involved in strong covalent interactions with
the two accessible diols of Gal-b-1,3-GalNAc. Other inter-
actions from the peptide backbone such as hydrogen bonding
or hydrophobic packing resulting from the aromatic R1 and
R2 components, must contribute significantly. These issues
Scheme 6. Most efficient receptors identified in the competitive ELISA
screening of library 1{1–20,21–40}.
The selectivity of receptor 1{17,29} for the Gal-b-1,3-
GalNAc disaccharide was assessed by monitoring the effect of
various concentrations of added carbohydrates incubated
under similar assay conditions in the presence of a fixed 20 mm
concentration of 1{17,29}, followed by washing and addition
of the PNA lectin. As depicted in Figure 1, soluble Gal-b-1,3-
GalNAc-O(CH2)8CO2Et[17] interfered very strongly with the
binding of 1{17,29} to the Gal-b-1,3-GalNAc-O(CH2)8CO-
BSA coated plates. The individual Gal and GalNAc glyco-
sides as well as the Gal-containing Lewis B tetrasaccharide[17]
competed only to a small extent at high concentrations, while
the structurally unrelated oligosaccharides trehalose and
cellobiose had no effect on the binding of 1{17,29} to the
Gal-b-1,3-GalNAc-O(CH2)8CO-BSA coated plates. The
blood group B trisaccharide [Gal-a-1,3-(Fuc-a-1,2)Gal-b]-
O(CH2)8CO2Et competed to a significant extent, which is to
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Scheme 7. Control compounds 3 and 4: analogues of 1{17,29} with
modified benzamide side chains.
[8] A notable exception would be boronate formation with the sialyl
unit of sialosides such as SLex: B. Wang, W. Yang, H. Fan, X.
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Anslyn, J. Am. Chem. Soc. 2007, 129, 13575 – 13583; b) S.
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will be addressed in future studies on the structure of the
complex.
In summary, we described a receptor design strategy that
exploits several modes of molecular recognition, including the
unique ability of benzoboroxoles to complex hexopyrano-
sides. The synthesis is modular, thus well suited to targeting
specific oligosaccharides using combinatorial receptor libra-
ries. This approach was successful in identifying a low
molecular weight receptor effective in neutral water and
selective for the TF-antigen disaccharide, a pivotal cancer
marker. Although it is remarkable that the moderate binding
affinity of this receptor matches the efficiency of some lectins,
it is still unsuitable for many applications. To this end, further
studies will aim to exploit multivalency effects with oligo-
meric receptors and assess their efficiency in the labeling of
TF-specific tumor cell lines.
Received: November 24, 2009
Published online: January 18, 2010
Keywords: benzoboroxoles · boronic acids ·
.
carbohydrate recognition · combinatorial chemistry · glycosides
[17] See the Supporting Information for details.
[18] Phenylglycine spacers 13 and 14 epimerize partly in the peptide
coupling.
[19] K. J. Neurohr, N. M. Young, H. H. Mantsch, J. Biol. Chem. 1980,
255, 9205 – 9209.
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[20] M.-G. Baek, R. Roy, Bioorg. Med. Chem. 2002, 10, 11 – 17.
[21] It was not possible to measure a Kd value by NMR spectroscopy
due to extensive peak broadening, which can likely be attributed
to slow exchange of boronate complexation on the NMR
timescale.
[2] For recent examples, see: a) R. D. Hubbard, S. L. Horner, B. J.
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