DNA-templated homo- and heterodimerization of peptide nucleic acid encoded oligosaccharides that mimick the carbohydrate epitope of HIV

Full text of DOI:10.1002/anie.200903328

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Chemie
DOI: 10.1002/anie.200903328
Bioorganic Chemistry
DNA-Templated Homo- and Heterodimerization of Peptide Nucleic
Acid Encoded Oligosaccharides that Mimick the Carbohydrate
Epitope of HIV**
Katarzyna Gorska, Kuo-Ting Huang, Olivier Chaloin, and Nicolas Winssinger*
Dedicated to Professor Jean-Marie Lehn on the occasion of his 70th birthday
Homo- and heterooligomerization of receptors is a funda-
mental feature of cellular recognition and signal transduc-
tion.[1–3] Protein–carbohydrate interactions often achieve high
avidity by the cooperative interaction of multiple units.[4] An
example of therapeutic significance is the interaction of the
cholera toxin with cell-surface carbohydrates, for which high
avidity was achieved (106-fold enhancement over the mono-
mer) by using a synthetic oligomer with an architecture
matching the geometry of the toxin receptor.[5] While this
example illustrates the importance of accessing oligomeric
carbohydrate structures with controlled topology, the syn-
thetic challenges of accessing complex architectures have
hampered progress. Herein we report a simple method to
obtain oligomeric carbohydrates with controlled topology in a
combinatorial fashion by the self-assembly of oligosacchar-
ides tagged with peptide nucleic acids (PNAs) onto DNA
templates. The potential of this method is demonstrated with
different supramolecular architectures which mimic multiple
copies of the carbohydrates (high mannose nonasaccharide, 1,
Figure 1) that shield gp120 and are known to interact in a
multivalent fashion with 2G12, an antibody that shows broad-
spectrum activity against HIV.[6–8]
highly cooperative binding mode, but also for its selectivity
for gp120 bearing high-mannose carbohydrates compared to
the host. Indeed, the 2G12 antibody displays appropriately
Figure 1. Structure of the high-manose nonasaccharide present on
gp120. Carbohydrate units shown in red interact directly with 2G12.[9]
The crystal structure of 2G12 with the high-mannose sugar
1 showed that 2G12 assembled into an interlocked dimer,
thereby resulting in two additional binding sites at its
dimerization interface (Figure 2).[9,10] This observation not
only provided a rationale for the high affinity of the antibody
for its target (HIVꢀs glycoprotein 120–gp120) by virtue of the
[*] K. Gorska, K.-T. Huang, Dr. O. Chaloin, Prof. N. Winssinger
Institut de Science et Ingꢀnierie Supramolꢀculaires (ISIS)
Universitꢀ de Strasbourg—CNRS (UMR 7006)
8 allꢀe Gaspard Monge, 67000 Strasbourg (France)
Fax: (+33)3-9024-5112
E-mail: winssinger@isis.u-strasbg.fr
Homepage: http://www-isis.u-strasbg.fr/winssinger/
[**] K.G. and K.-T.H. contributed equally to this work. This work was
supported by a grant from the European Research Council (ERC
201749). A fellowship from Boehringer Inglheim Fonds (to K.G.)
and the French Ministry of Research and Education (K.-T.H.) is
gratefully acknowledged. 2G12 was obtained through the NIH AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH; and the HIV-1 gp120 monoclonal antibody (2G12) from Dr.
Hermann Katinger. We thank Laurence Oswald for her technical
assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200903328.
Figure 2. Proposed binding mode of 2G12 dimer with gp120.[9]
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spaced binding sites that match the spacing of these structures
cleavage was observed upon treatment with acid. Saccharides
labeled with a thioalkyl group at the anomeric position[26]
could also be efficiently coupled to the chloroacetamide-
substituted PNA, but using DBU rather than Hꢂnigꢀs base,
thus affording conjugate 5 after cleavage. Starting from PNA
2, a similar strategy was used to install two units of the
carbohydrate by treatment of the chloroacetamide product
with ethylenediamine. The resulting PNA–diamine conjugate
was then treated with chloroacetyl chloride followed by a
thiosaccharide to obtain, after cleavage, product 6, which
bears two copies of the carbohydrate. Assuming a trans or anti
conformation for all the bonds, the distance between the
anomeric centers in the two carbohydrate units is 11.5 ꢁ.[27]
Conversely, PNA 2 can be coupled to an orthogonally
protected lysine (Fmoc, Mtt). Cleavage of both the Fmoc
and Mtt[28] protecting groups followed by chloroacetylation
and coupling with thioglycosides II and III affords products 7
and 8, respectively, in which the same carbohydrate units are
separate by a maximum distance of 15 and 19 ꢁ repectively.
To obtain a greater distance between the carbohydrate units,
the Fmoc group can be selectively removed and a 10 ꢁ PEG
spacer added prior to the chloroacetylations to obtain 10 after
glycoside conjugation and release from the resin. Finally, to
obtain different carbohydrate units on each side of the lysine
residue, a first chloroacetylation/carbohydrate coupling is
performed after cleavage of the Fmoc group but prior to
removal of the Mtt group. A second chloroacetylation/
carbohydrate coupling can then be performed after cleavage
of the Mtt group to obtain heterodimeric conjugates such as
11. Similar sequences were utilized to conjugate carbohy-
drates at the C teminus of the PNA, starting from resin 12, to
afford conjugates 13–17 bearing a single unit of a-1,2-
mannose dimers or two units separated by distances ranging
from 11.5 to 25 ꢁ. The PNA–carbohydrate conjugates were
then assembled into dimers and oligomers by hybridization to
the appropriate DNA template. Considering the high persis-
tent length of the double helix and the fact that all the PNA
sequences are 10 mers, it can be anticipated that carbohydrate
units in architectures such as those in entries 2, 3, and 15 of
Figure 3 will be separated by 30, 60, and 90 ꢁ respectively.
However, the inclusion of a PEG linker between the PNA and
the carbohydrate allows for some degree of flexibility.
A pilot library of over 30 architectures was tested for their
affinity to 2G12 by surface plasmon resonance (SPR). The
antibody was immobilized accordingly to a previously de-
scribed protocol.[12] Under these conditions, no notable
binding was observed for nonasaccharide 1, as previously
reported by Danishefsky and co-workers,[12,29] and in agree-
ment with the binding mode reported by Wilson[9,10] which
involves four units of mannose disaccharide from multiple
units of 1. Significant binding (mm) was observed for
conjugates having the key a-1,2-mannose disaccharide units
(Figure 4). However, the distance between the two carbohy-
drate units of this disaccharide was critical for the binding,
thus attesting to the importance of their cooperativity for
avidity. Only conjugates bearing an 11 atom spacer between
the two disaccharide units (structure 7 and 15) displayed any
binding (entries 12–18, Figure 3). It is interesting to note that
the distance between the two mannose units involved in the
on the viral surface. Interestingly, the crystallographic infor-
mation suggests that only the terminal mannose moieties
shown in red (Figure 1) are involved in the interaction with
the antibody. Corroborating these results, it has been shown
that carbohydrate 1 or fragments thereof bearing the terminal
mannose presented at high density on a surface have a higher
affinity for 2G12 than their monomeric counterparts.[9,10]
While the nonasaccharide 1 has no notable affinity for
2G12, Danishefsky and co-workers have shown that a
trimer of 1 displayed on a rigid scaffold[11] binds 2G12 with
moderate affinity,[12] while Wang et al. showed that a tetramer
of a mannose tetrasaccharide on a similar scaffold also binds
2G12 with micromolar affinity.[13] The distance between the
two primary binding sites in 2G12 is 30 ꢁ (measured from
PDB 1OP5[9]), and while some level of cooperativity has been
achieved with previously reported oligomers, a more system-
atic investigation of the optimal spacing geometry between
the ligands to maximize the cooperativity has not been
reported. In fact Danishefsky and co-workers have shown
that the oligomer conjugated to an immunogenic protein is
able to elicit an antibody response that recognizes the
oligomer of 1 but fails to neutralize HIV, thereby suggesting
that it is not an optimal mimic of the epitope of gp120.[14]
Dendrons of 1 have also been shown to bind 2G12 cooper-
atively.[15]
Peptide nucleic acids[16,17] are attractive tags for program-
ming self-assembled structures, since their chemistry is
significantly more permissive than that of natural oligonu-
cleotides. Furthermore, the higher affinity of PNAs for
natural oligonucleotides allows for shorter tags, which are
more specific and less sensitive to the ionic strength of the
solution.[18] For example, we have shown that they could be
used to encode combinatorial libraries of peptides.[19–22] In
fact, only monosaccharide–DNA conjugates have been
reported thus far for microarraying[23] and to study lectin
interactions,[24,25] whereas larger oligosaccharides with more
complex branching patterns have never been reported. As
shown in Scheme 1, carbohydrates can be efficiently coupled
to polymer-bound PNAs at the C terminus or the N terminus
by coupling a thiol (thioacetals or carbohydrates bearing a
thioalkyl group at the anomeric position) to a chloroaceta-
mide with a mild base (such as Hunig’s base or DBU). For the
purpose at hand, a 10 mer PNA was deemed appropriate as it
would provide a melting temperature (Tm) of greater than
508C and would present adjacent ligands on the same face of
the helix. Thus 10 mer PNA 2 obtained by standard Fmoc
chemistry and bearing a short polyethyleneglycol (PEG)
spacer (10 ꢁ) at the N terminus was coupled to chloroacetyl
chloride and treated with commercially available tetraacetyl
glucothiolactol I (Scheme 1) in the presence of Hꢂnigꢀs base.
Analysis of the cleavage product by LC–MS and MALDI MS
indicated complete conversion and showed a single signal for
product 3. The acetyl groups on the glucose moiety were
removed by treatment with ammonia in MeOH, which was
found to be equally efficient prior to or after cleavage from
the resin with TFA. The same procedure was applied for the
coupling of unprotected disaccharide II[26] to afford conjugate
4 after cleavage. To our gratification, no trace of glycosidic
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Scheme 1. Synthesis of PNA–oligosacharide conjugates. a) ClCH2COCl, 2,6-lutidine, DMF; b) saccharide (10 equiv), for I or II: EtiPr2N (15 equiv)
or DBU (10 equiv) for III and IV, DMF; c) TFA/H2O 95:5; d) 2m H3N in MeOH; e) HO-Lys(Mtt)Fmoc, EtiPr2N, HCTU, DMF; 20% piperidine in
DMF; f) HO2CCH2(O(CH2)2)2NH2, EtiPr2N, HCTU, DMF; 20% piperidine in DMF; g) ethylenediamine (2ꢁ10 equiv), DMF. DBU=1,8-
diazabicyclo[5.4.0]undec-7-ene, TFA=trifluoroacetic acid, Mtt=4-methyltrityl, Fmoc=9-fluorenylmethoxycarbonyl, HCTU=2-(6-chloro-1H-benzo-
triazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate, Boc=tert-butoxycarbonyl, dep=deprotection.
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Figure 3. Affinity of supramolecular complexes to 2G12 measured by SPR (ka (mÀ1 sÀ1) and kd (sÀ1)). Solutions were prepared by mixing
1 equivalent of each PNA with their respective template. Binding kinetics were obtained using a Langmuir 1:1 model (Rmax/c2 >50). * denotes the
concentration of the template.
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[1] J. P. Pin, R. Neubig, M. Bouvier, L. Devi, M. Filizola, J. A.
Javitch, M. J. Lohse, G. Milligan, K. Palczewski, M. Parmentier,
M. Spedding, Pharmacol. Rev. 2007, 59, 5 – 13.
[2] L. L. Kiessling, J. E. Gestwicki, L. E. Strong, Angew. Chem.
2006, 118, 2408 – 2429; Angew. Chem. Int. Ed. 2006, 45, 2348 –
2368.
[3] M. Mammen, S. Choi, G. Whitesides, Angew. Chem. 1998, 110,
2908 – 2953; Angew. Chem. Int. Ed. 1998, 37, 2754 – 2794.
[4] J. Lundquist, E. Toone, Chem. Rev. 2002, 102, 555 – 578.
[5] P. I. Kitov, J. M. Sadowska, G. Mulvey, G. D. Armstrong, H.
Ling, N. S. Pannu, R. J. Read, D. R. Bundle, Nature 2000, 403,
669 – 672.
Figure 4. Measurment of the affinity for the architecture shown in
entry 12 of Figure 3 by SPR (green: 18 mm, magenta: 9 mm, red:
4.5 mm).
[6] J. R. Mascola, M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott,
D. Hayes, M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel,
Y. Lu, M. L. Robb, H. Katinger, D. L. Birx, J. Virol. 1999, 73,
4009 – 4018.
[7] B. Etemad-Moghadam, Y. Sun, E. K. Nicholson, G. B. Karlsson,
D. Schenten, J. Sodroski, J. Virol. 1999, 73, 8873 – 8879.
[8] J. M. Crawford, P. L. Earl, B. Moss, K. A. Reimann, M. S.
Wyand, K. H. Manson, M. Bilska, J. T. Zhou, C. D. Pauza, P. W.
Parren, D. R. Burton, J. G. Sodroski, N. L. Letvin, D. C. Mon-
tefiori, J. Virol. 1999, 73, 10199 – 10207.
[9] D. A. Calarese, C. N. Scanlan, M. B. Zwick, S. Deechongkit, Y.
Mimura, R. Kunert, P. Zhu, M. R. Wormald, R. L. Stanfield,
K. H. Roux, J. W. Kelly, P. M. Rudd, R. A. Dwek, H. Katinger,
D. R. Burton, I. A. Wilson, Science 2003, 300, 2065 – 2071.
[10] D. A. Calarese, H. K. Lee, C. Y. Huang, M. D. Best, R. D.
Astronomo, R. L. Stanfield, H. Katinger, D. R. Burton, C. H.
Wong, I. A. Wilson, Proc. Natl. Acad. Sci. USA 2005, 102,
13372 – 13377.
[11] O. Renaudet, P. Dumy, Org. Lett. 2003, 5, 243 – 246.
[12] I. J. Krauss, J. G. Joyce, A. C. Finnefrock, H. C. Song, V. Y.
Dudkin, X. Geng, J. D. Warren, M. Chastain, J. W. Shiver, S. J.
Danishefsky, J. Am. Chem. Soc. 2007, 129, 11042 – 11044.
[13] J. Wang, H. Li, G. Zou, L. Wang, Org. Biomol. Chem. 2007, 5,
1529 – 1540.
binding of nonasaccharide 1 is also 11 atoms. Since the
through-space distance between the anomeric substituents is
10 ꢁ (measured from PDB 1OP5), the shorter spacer present
in structures 6 and 14 clearly do not adequately replicate this
geometry (entries 9–11), whereas longer spacers in structures
8, 10, 16, and 17 (entry 22–25, 30–32; Figure 3) do not provide
the adequate level of cooperativity. Following the same
argument, architectures based on the mannose trisaccharide
fail to provide significant binding (entries 26–28; Figure 3).
The topology of the supramolecular architecture also has a
significant impact on binding. While the PEG spacer between
the carbohydrate moiety and the PNA does provide a certain
level of flexibility, a clear trend emerges on comparison of
entries 12 to 15 of Figure 3, with the shortest distance
(entry 12) being best. Considering that a distance of about
30 ꢁ is required for cooperative binding to 2G12, the
architecture in entry 12 (the maximum distance between the
branch points joining the carbohydrate units is 38.5 ꢁ) would
be most suitable, whereas the architecture of entry 15 shows a
lower level of cooperative binding. The architecture in
entry 16 has complimentary “sticky ends” and is anticipated
to polymerize extensively.[30] The value of 6.39 mm reported in
Figure 3 is for the DNA template, and thus underestimates
the affinity of the oligomeric polymer.
[14] J. G. Joyce, I. J. Krauss, H. C. Song, D. W. Opalka, K. M. Grimm,
D. D. Nahas, M. T. Esser, R. Hrin, M. Feng, V. Y. Dudkin, M.
Chastain, J. W. Shiver, S. J. Danishefsky, Proc. Natl. Acad. Sci.
USA 2008, 105, 15684 – 15689.
[15] S. K. Wang, P. H. Liang, R. D. Astronomo, T. L. Hsu, S. L. Hsieh,
D. R. Burton, C. H. Wong, Proc. Natl. Acad. Sci. USA 2008, 105,
3690 – 3695.
[16] P. E. Nielsen, Mol. Biotechnol. 2004, 26, 233 – 248.
[17] M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M.
Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, P. E.
Nielsen, Nature 1993, 365, 566 – 568.
[18] Z. L. Pianowski, N. Winssinger, Chem. Soc. Rev. 2008, 37, 1330 –
1336.
[19] F. Debaene, J. Da Silva, Z. Pianowski, F. Duran, N. Winssinger,
Tetrahedron 2007, 63, 6577 – 6586.
[20] J. L. Harris, N. Winssinger, Chem. Eur. J. 2005, 11, 6792 – 6801.
[21] J. Harris, D. E. Mason, J. Li, K. W. Burdick, B. J. Backes, T. Chen,
A. Shipway, G. Van Heeke, L. Gough, A. Ghaemmaghami, F.
Shakib, F. Debaene, N. Winssinger, Chem. Biol. 2004, 11, 1361 –
1372.
[22] N. Winssinger, R. Damoiseaux, D. C. Tully, B. H. Geierstanger,
K. Burdick, J. L. Harris, Chem. Biol. 2004, 11, 1351 – 1360.
[23] Y. Chevolot, C. Bouillon, S. Vidal, F. Morvan, A. Meyer, J. P.
Cloarec, A. Jochum, J. P. Praly, J. J. Vasseur, E. Souteyrand,
Angew. Chem. 2007, 119, 2450 – 2454; Angew. Chem. Int. Ed.
2007, 46, 2398 – 2402.
The novelty of the approach reported herein is that it
exploits the programmability of hybridization to generate a
library of architectures which emulate the topologies of
complex carbohydrates. The gp120 epitope has stimulated
tremendous efforts towards the production of vac-
cines.[12,15,31–33] While this example illustrates the importance
of multimeric recognition with controlled topology, the
generality of this concept extends far beyond pathogen
recognition[34] since it is a recurrent feature in cellular
recognition and communication, with many signal trans-
duction pathways also being regulated by multimeric inter-
actions.[2] Better insight into the mechanisms that govern
these two hierarchical levels requires methods to prepare
well-organized oligomers with controlled topology. Last but
not least, these principles should be applicable beyond
carbohydrate recognition to generate small molecules or
drug fragments in a combinatorial fashion and provide an
encoding strategy for dynamic combinatorial libraries.[35,36]
Received: June 19, 2009
[24] K. Matsuura, M. Hibino, Y. Yamada, K. Kobayashi, J. Am Chem.
Soc. 2001, 123, 357 – 358.
[25] K. Matsuura, M. Hibino, T. Ikeda, Y. Yamada, K. Kobayashi,
Chem. Eur. J. 2004, 10, 352 – 359.
Keywords: bioorganic chemistry · carbohydrates · DNA · HIV ·
.
peptide nucleic acids
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[26] See the Supporting Information for synthetic procedures.
[31] X. Geng, V. Y. Dudkin, M. Mandal, S. J. Danishefsky, Angew.
Chem. 2004, 116, 2616 – 2619; Angew. Chem. Int. Ed. 2004, 43,
2562 – 2565.
[32] J. D. Warren, X. Geng, S. J. Danishefsky, Top. Curr. Chem. 2007,
267, 109 – 141.
[27] Calculated assuming a 2.5 ꢁ through-space distance between
three atoms in an anti configuration and 1.5 ꢁ for a single bond.
[28] S. Pothukanuri, Z. Pianowski, N. Winssinger, Eur. J. Org. Chem.
2008, 3141 – 3148.
[33] S. Sirois, M. Touaibia, K. C. Chou, R. Roy, Curr. Med. Chem.
2007, 14, 3232 – 3242.
[34] A. Imberty, Y. M. Chabre, R. Roy, Chem. Eur. J. 2008, 14, 7490 –
7499.
[35] J. M. Lehn, Science 2002, 295, 2400 – 2403.
[36] J. M. Lehn, Chem. Eur. J. 1999, 5, 2455 – 2463.
[29] V. Y. Dudkin, M. Orlova, X. Geng, M. Mandal, W. C. Olson, S. J.
Danishefsky, J. Am. Chem. Soc. 2004, 126, 9560 – 9562.
[30] For a recent review on glycodendrimers, see Y. M. Chabre, R.
Roy, Curr. Top. Med. Chem. 2008, 8, 1237 – 1285.
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