Synthesis of highly controlled carbohydrate-polymer based hybrid structures by combining heparin fragments and sialic acid derivatives, and solid phase polymer synthesis

Article abstract of DOI:10.1039/C8CC04898C

Heparin is a polymeric carbohydrate with a variety of biomedical applications that is particularly challenging from a synthetic point of view. Here, we present the synthesis of carbohydrate-polymer based hybrid structures by combining defined heparin fragments with monodisperse, sequence-controlled glycooligo(amidoamines) suitable as glycan mimetic model compounds of heparin as demonstrated by STD-NMR binding studies with viral capsids.

Full text of DOI:10.1039/C8CC04898C

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DOI: 10.1039/C8CC04898C
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Synthesis of highly controlled carbohydrate-polymer based hybrid
structures by combining heparin fragments, sialic acid derivatives,
and solid phase polymer synthesis
Received 00th January 20xx,
Accepted 00th January 20xx
Mischa Baier,a Jana L. Ruppertz,a Moritz M. Pfleiderer,b Bärbel S. Blaum*b and Laura Hartmann*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Heparin is a polymeric carbohydrate with a variety of biomedical scaffold for the multivalent presentation of carbohydrate
applications that is particularly challenging from a synthetic point motifs. While such glycan mimetics can be used to investigate
of view. Here, we present the synthesis of carbohydrate-polymer the effects of overall length and sulfation pattern of heparin-
based hybrid structures by combining defined heparin fragments mimicking systems,9 they also offer the opportunity to study the
with monodisperse, sequence-controlled glycooligo(amidoamines) interplay of heparin and other components of the glycocalyx,
suitable as glycan mimetic model compounds of heparin as such as sialic acid (Neu5Ac) containing oligosaccharides. An
demonstrated by STD-NMR binding studies with viral capsid.
interesting example is MCPyV as it was shown to utilize both
GAGs of the heparin/HS-type as well as sialylated glycans as
cellular attachment factors or entry receptors.10
Glycosaminoglycans (GAGs) are a class of long, unbranched
polydispersed glycooligomers consisting of repeating units of
selectively sulfated disaccharides, generally an amino sugar
alternating with an uronic acid.1 They are important
components of the extracellular matrix and the glycocalyx, a
dense layer of carbohydrates decorating almost all eukaryotic
cells.2 Heparin, which is a secreted GAG, shows the highest
degree of sulfation of all GAGs and is widely used as an
anticoagulant.3 Structurally, it is closely related to heparan
sulfate (HS), which occurs on proteoglycans and acts as co-
receptor for numerous viruses, among them the human tumor
viruses papillomavirus 16 (HPV16) and Merkel cell polyomavirus
(MCPyV). Thus, heparin/HS- containing compounds have the
Recently, we introduced so-called solid phase polymer synthesis
(SPPoS) to access structurally and sequentially defined
glycomacromolecules as a novel class of glycan mimetics. In
short, tailor-made building blocks carrying a carboxylic acid as
well as a Fmoc-protected amine terminus are assembled
stepwise on solid support using standard peptide coupling
chemistry. Different functional side chain or main chain motifs
can be positioned within the resulting oligo(amidoamine)
scaffold and used for conjugation of specific oligosaccharides.11
These precision glycomacromolecules have been successfully
applied as model compounds in various binding studies and
have shown their potential as glycan mimetic structures in
different biotechnological and biomedical applications.11a, 11c, 12
3c,
4
potential to act as antivirals3b,
for papilloma- and
polyomaviruses.5
Since natural GAGs are chemically
Here, we now extend on this concept by applying, for the first
time, heparin fragments as carbohydrate moieties,
demonstrating the potential of our synthetic approach to create
heterogeneous compounds, it is often difficult to study whether
specific structural requirements exist for individual virus-GAG
interactions, such as the GAG chain length or specific sulfation
patterns.6 While defined GAG fragments can be obtained e.g.
via enzymatic degradation of the natural polysaccharides7 or
chemical synthesis8 it is also highly interesting to create glycan
mimetic structures as model compounds for systematic
structure-property correlation studies. An important class of
such glycan mimetics are the glycopolymers, using a polymeric
a variety of GAG-mimetic structures. Heparin fragments can be
13
accessed via chemical synthesis,8,
but the challenging
synthesis and limited amounts available through such
approaches prompted us to choose enzymatic degradation of
unfractionated heparin, providing the defined heparin dp2
fragment in a suitable scale.7 A pentasaccharide fragment,
Fondaparinux®, used as anticoagulant and commercially
available, is applied here as well.14
a.Institute of Organic and Macromolecular Chemistry, Heinrich-Heine-University
Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany
E-mail: laura.hartmann@hhu.de
The conjugation of heparin fragments, in principle, can be
accomplished at the non-reducing end, at the reducing end,15
as well as at the uronic acid carboxylic acids and amino sugar
amines.16 Here, we chose the carboxylic acids of the uronic acids
b.Interfaculty Institute of Biochemistry, University of Tuebingen, Hoppe-Seyler-
Strasse 4, 72076 Tuebingen, Germany
†Electronic Supplementary Information (ESI) available: Materials, methods and
experimental data. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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as anchor points for straightforward combination with our buffer pH 6.5. Fully dp2-conjugated glycomacroVmieowlAercticuleleOnOlin2e
oligoamide-based scaffolds. It has been shown that DMTMM was obtained with 81 % purity after diaDlyOsIi:s10a.n10d39io/Cn8eCxCc0h4a8n98gCe
((4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium
as determined by UV integration of signals in RP-HPLC (see ESI).
chloride) is promising as a coupling reagent for the synthesis of Here, impurities can partially be attributed to different
17
GAG-adducts.16a,
In general, following our previously glucosamine pyranose forms and partial loss and re-addition of
introduced concept, conjugation of heparin fragments in the sulfates during synthesis and workup (see ESI).
side chains of oligo(amidoamine) scaffolds leads to multivalent
Following our second approach, we inversed the conjugation
glycan mimetics. Our toolbox of building blocks also gives us the
flexibility to use the heparin fragment itself as scaffold and
attach multiple copies of another ligand, e.g. Neu5Ac moieties
(Figure 1). This is the first example of a GAG fragment used as
scaffold to multivalently present glycan motifs and generate a
novel class of heteromultivalent glycan mimetics.
scheme and first synthesized carbohydrate-functionalized
oligo(amidoamines) that should then be conjugated onto a
larger heparin fragment, here now the pentasaccharide
Fondaparinux®. Such non-natural heteromultivalent glycan
mimetics can then be used as model structures investigating the
interplay of different GAG and non-GAG fragments e.g. on the
For both classes of molecules, Fmoc-Lys(Boc)-OH (
for the introduction of primary amine groups and subsequent scaffold introducing first a lysine followed by three EDS and an
conjugation to the uronic acid carboxylic acids of heparin azide functionalized building block12b
) was synthesized
1) was used adhesion of MCPyV. For this purpose, an oligo(amidoamine)
(3
fragments. Fmoc-based amino acids are easily combined with (Figure 1B). Following previously established protocols,
our tailor-made building blocks.18 Following the first approach, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) of
20, 21
oligomer 1
ethylene-glycol based building blocks EDS18a
on a TentaGel® S RAM resin. O1 was isolated after cleavage the propargyl-functionalized Neu5Ac
(
O1), consisting of two lysine spaced by three propargyl-functionalized Neu5Ac
(
4
)
or propargyl-
(2
) was synthesized functionalized 3'sialyllactose (3’SL,
5
) was performed. In case of
, instead of 0.1
4
from solid support also removing Boc protecting groups and ion equivalents of copper(II) sulfate, 2 equivalents were required
exchange to chloride as counter-ion (Figure 1A). DMTMM for full conjugation. Potentially, propargyl-functionalized
promoted coupling parameters were taken from test couplings Neu5Ac is capable of complexing copper ions to some degree
of an unprotected glucuronic acid derivative to a resin-bound and thereby reducing coupling efficiency. After cleavage from
oligo(amidoamine) following protocols from Falchi et al.19 (see support and in-situ deprotection of lysine side chains using,
ESI for experimental set-up and spectra). Although the ideal glycomacromolecules O3 and O4 were purified via preparative
conditions on the solid phase do not reflect the ideal conditions RP-HPLC and ion exchange to give the final structures as
1
in solution, attachment of two heparin dp2-fragments (
6
) to O1 hydrochloride salts in high purities as determined by H NMR,
proceeded smoothly in a 9:1 mixture of DMF and phosphate RP-HPLC-MS and HR-MS (see ESI). In the second step, O3 and
Figure 1: Synthesis of oligo(amidoamine)-heparin fragment conjugates. 1A) Homovalent dp2-adduct; 1B) Homomultivalent Fondaparinux-adducts; 1C) Applied building blocks.
2 | J. Name., 2012, 00, 1-3
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loops engages with compound O6. An optimizedVliienwkAertriclleeOnnglitnhe
DOI: 10.1039/C8CC04898C
may be necessary to bridge the Neu5Ac and GAG binding sites
on the capsid. Nevertheless, our NMR experiments
demonstrate that GAG and sialic acid units can, in principle,
engage in lectin binding in the context of the investigated
glycomacromolecules.
O4 were then used to functionalize the two carboxylic acid
moieties of Fondaparinux® (Figure 1B). This was accomplished
using 1.5 equivalents of O3 and 10 equivalents of DMTMM per
carboxylic acid; full conversion was accomplished as shown by
RP-HPLC-MS. The product was deprotected with lithium
hydroxide and isolated after dialysis and ion exchange. Figure 2
shows
the
1H NMR
spectra
of
Fondaparinux®,
In conclusion, we have demonstrated the synthesis of
glyomimetic conjugates of heparin fragments using our toolbox
of oligo(amidoamines), also introducing Neu5Ac and 3’SL
ligands. In the first approach we followed the concept of
conjugating carbohydrate ligands as pending side chains onto
an oligo(amidoamine) scaffold for the first time using heparin
dp2-fragments. Furthermore, we introduced an inverse
strategy towards oligo(amidoamine)-based glycan mimetics
where solid-phase derived glycomacromolecules are used as
side chains for conjugation onto larger heparin fragments. We
have used this strategy to create conjugates of multiple Neu5Ac
or 3’SL ligands bound to a heparin fragment. First binding
studies probing the interaction of glycomacromolecules with
the MCPyV capsid proved their potential as glycan mimetics
targeting papilloma- and polyomaviruses. In general, this
approach is applicable for the conjugation of oligo(amidoamine)
side chains onto various oligo- and polysaccharides, such as
hyaluronic or colominic acid giving access to novel
carbohydrate-polymer based hybrid materials with potential
applications in biotechnology and biomedicine.
glycomacromolecule O3 and the resulting adduct O5
,
demonstrating successful synthesis of the fully conjugated
heparin fragment. Characteristic signals of both carbohydrate
ligands as well as from the oligo(amidoamine) backbone are
clearly visible and allow for quantification of the degree of
conjugation. When coupling the 3’SL-functionalized
glycomacromolecule O4 even 1.15 equivalents per carboxylic
acid were sufficient to give the fully conjugated product in a
high yield of 86%. Mono-substituted product and excessive
reagents were easily removed by dialysis. Final conjugate O6
was obtained in high purity after deprotection, dialysis and ion
exchange as determined by RP-HPLC-MS, SAX-HPLC and
1H NMR (see ESI). All macromolecules are listed in table 2.
First insights into the applicability of the synthesized
compounds as glycan mimetics were gained in saturation
transfer difference NMR (STD-NMR) experiments with the
MCPyV capsid, which binds to unmodified heparin dp2,
Fondaparinux, and 3’SL. STD-NMR provides spectra from which
the proximity of individual ligand protons to protein protons in
the binding site can be delineated.11b, 22 For O2, we observed
saturation transfer to protons of the unsaturated uronic acid as
well as to the GlcNS anomeric proton (see ESI). Saturation
transfer was also detected to the aliphatic lysine chains and, to
a lesser degree, the succinic acid CH2-groups. For O6, we
observed saturation transfer to a set of the 3’SL protons
previously shown to constitute the MCPyV Neu5Ac binding
epitope (see ESI).23 However, in this compound, no signature
protons of the Fondaparinux scaffold were detected, suggesting
that only the exposed Neu5Ac binding site on the capsid’s apical
The authors thank the German Research Council (DFG) for
financial support through FOR2327 (Virocarb) (HA 5950/5-1; BL
1294/3-1) and INST 208/735-1. Moreover, we thank Prof. Dr.
Nicole L. Snyder for helpful discussions during sialic acid
synthesis as well as Dr. Vincent Truffault of the NMR facility of
the Max Planck Institute for Developmental Biology.
Conflicts of interest
There are no conflicts to declare.
Figure 2: 1H NMR (D2O) of Fondaparinux® (2A), compounds O3 (2B) and O5 (2C). Product O5 shows all the 1H-signals of Fondaparinux as well as the aromatic signals, the sialic acid
signals as well as the signals of the aliphatic lysine chain. Downfield shifting of the –ε-CH2- signal in front of the ζ-NH2 of the lysine, can be seen (see red arrow).
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Soc., 2009, 131, 17394-17405; cP. Liu, L. Chen, J. VKie.wC.ArTtioclhe,OYn.linLe.
Ang, J.-E. Jee, J. Lim, S. S. Lee and S.-G.DLOeeI:,1C0h.1e0m39./CSc8iC.,C20041859,86C,
450-456.
Table 2: Overview of the oligomers obtained, molecular weights,
purities determined via RP-HPLC and SAX-HPLC, and yield.
Purity a
(RP-
Purity a
(SAX-
HPLC)
[%]
9. aC. I. Gama, S. E. Tully, N. Sotogaku, P. M. Clark, M. Rawat, N.
Vaidehi, W. A. Goddard Iii, A. Nishi and L. C. Hsieh-Wilson, nat.
chem. biol., 2006, 2, 467; bG. J. Sheng, Y. I. Oh, S.-K. Chang and
L. C. Hsieh-Wilson, J. Am. Chem. Soc., 2013, 135, 10898-10901;
cY. I. Oh, G. J. Sheng, S.-K. Chang and L. C. Hsieh‐Wilson, Angew.
Chem. Int. Edit., 2013, 52, 11796-11799; dL. Fu, M. Suflita and R.
J. Linhardt, Adv. Drug Deliv. Rev., 2016, 97, 237-249.
MW
[Da]
Yield
[%]
#
Structure
HPLC)
[%]
b
O1
O2
1079
2257
97
-
36
21
Δ
Δ
81
85
10. R. M. Schowalter, D. V. Pastrana and C. B. Buck, PLOS Path.,
2011, 7, e1002161.
b
O3
O4
1561
2138
> 99
-
25
27
11. aD. Ponader, F. Wojcik, F. Beceren-Braun, J. Dernedde and L.
Hartmann, Biomacromolecules, 2012, 13, 1845-1852; bD.
Ponader, P. Maffre, J. Aretz, D. Pussak, N. M. Ninnemann, S.
Schmidt, P. H. Seeberger, C. Rademacher, G. U. Nienhaus and L.
Hartmann, J. Am. Chem. Soc., 2014, 136, 2008-2016; cS. Igde, S.
Röblitz, A. Müller, K. Kolbe, S. Boden, C. Fessele, T. K. Lindhorst,
M. Weber and L. Hartmann, Macromol. Biosci., 2017, 17,
1700198.
12. aF. Broecker, J. Hanske, C. E. Martin, J. Y. Baek, A. Wahlbrink, F.
Wojcik, L. Hartmann, C. Rademacher, C. Anish and P. H.
Seeberger, Nature Communications, 2016, 7; bM. Baier, M.
Giesler and L. Hartmann, Chem. Eur. J., 2018, 24, 1619-1630.
13. aC. Zong, A. Venot, O. Dhamale and G.-J. Boons, Organic Letters,
2013, 15, 342-345; bO. P. Dhamale, C. Zong, K. Al-Mafraji and G.-
J. Boons, Org. Biomol. Chem., 2014, 12, 2087-2098.
14. aJ. Choay, M. Petitou, J. C. Lormeau, P. Sinaÿ, B. Casu and G.
Gatti, Biochem. Biophys. Res. Commun., 1983, 116, 492-499; bH.
Bounameaux and T. Perneger, Lancet, 2002, 359, 1710-1711.
15. aS. Masuko, K. Higashi, Z. Wang, U. Bhaskar, A. M. Hickey, F.
Zhang, T. Toida, J. S. Dordick and R. J. Linhardt, Carbohyd. Res.,
2011, 346, 1962-1966; bZ. Q. Wang, C. Shi, X. R. Wu and Y. J.
Chen, Chem. Commun., 2014, 50, 7004-7006.
b
97 β
-
O5
O6
4377
5026
98
88
87
74
86
90 ββ,
6 αβ/
βα,
< 4 %
αα c
a Puritiesj determined by integration of UV-signals in RP-HPLC (see
ESI for spectra and conditions). Not determined. c Anomeric
b
mixtures were expected since an anomeric mixture of 5 was used.
References
16. aE. Gemma, A. N. Hulme, A. Jahnke, L. Jin, M. Lyon, R. M. Muller
and D. Uhrin, Chem. Commun., 2007, 2686-2688; bD. Tebbe, R.
Thull and U. Gbureck, Biomed. Eng. Online, 2007, 6, 31.
17. aP. Farkaš, A. Čížová, S. Bekešová and S. Bystrický, Int. J. Biol.
Macromol., 2013, 60, 325-327; bM. D’Este, D. Eglin and M. Alini,
Carbohydr. Polym., 2014, 108, 239-246.
18. aM. F. Ebbesen, C. Gerke, P. Hartwig and L. Hartmann, Polym.
Chem., 2016, 7, 7086-7093; bC. Gerke, M. F. Ebbesen, D. Jansen,
S. Boden, T. Freichel and L. Hartmann, Biomacromolecules,
2017, 18, 787-796; cS. Boden, K. Wagner, M. Karg and L.
Hartmann, Polymers, 2017, 9, 716; dT. Freichel, S. Eierhoff, N. L.
Snyder and L. Hartmann, J. Org. Chem., 2017, 82, 9400-9409.
19. A. Falchi, G. Giacomelli, A. Porcheddu and M. Taddei, Synlett,
2000, 2, 275-277.
20. R. Šardzík, G. T. Noble, M. J. Weissenborn, A. Martin, S. J. Webb
and S. L. Flitsch, Beilstein J. Org. Chem., 2010, 6, 699-703.
21. M. F. Ebbesen, D. Itskalov, M. Baier and L. Hartmann, ACS Macro
Lett., 2017, 6, 399-403.
22. aM. Mayer and B. Meyer, Angew. Chem. Int. Edit., 1999, 38,
1784-1788; bM. Mayer and B. Meyer, J. Am. Chem. Soc., 2001,
123, 6108-6117; cB. S. Blaum, U. Neu, T. Peters and T. Stehle,
Acta Cryst. F., 2018, 74, 451-462; dK. S. Bücher, H. Yan, R.
Creutznacher et. al., Biomacromolecules, 2018, DOI:
10.1021/acs.biomac.8b00829.
1. J. Esko, K. Kimata and U. Lindahl, in Essentials of Glycobiology.,
ed. A. Varki, 2nd. edn., 2009.
2. aA. Varki, Glycobiology, 1993, 3, 97-130; bK. Kato and A. Ishiwa,
Trop. Med. Health, 2015, 43, 41-52.
3. aB. Mulloy, E. Gray and T. W. Barrowcliffe, Thromb. Heamost.,
2000, 84, 1052-1056; bI. Capila and R. J. Linhardt, Angew. Chem.
Int. Edit., 2002, 41, 391-412; cI. Capila and R. J. Linhardt, Angew.
Chem., 2002, 114, 426-450; dR. J. Linhardt, J. Med. Chem., 2003,
46, 2551-2564.
4. aR. S. Aquino, E. S. Lee and P. W. Park, in Glycosaminoglycans in
Development, Health and Disease, ed. L. Zhang, 1st edn., 2010;
bR. S. Aquino and P. W. Park, Front. Biosci., 2016, 21, 1260-1277.
5. aP. M. Day and M. Schelhaas, Curr. Opin. Virol., 2014, 4, 24-31;
bJ. Guan, S. M. Bywaters, S. A. Brendle, R. E. Ashley, A. M.
Makhov, J. F. Conway, N. D. Christensen and S. Hafenstein,
Structure, 2017, 25, 253-263.
6. aJ. Dasgupta, M. Bienkowska-Haba, M. E. Ortega, H. D. Patel, S.
Bodevin, D. Spillmann, B. Bishop, M. Sapp and X. S. Chen, J. Biol.
Chem., 2011, 286, 2617-2624; bC. Cerqueira, Y. Liu, L. Kuhling,
W. G. Chai, W. Hafezi, T. H. van Kuppevelt, J. E. Kuhn, T. Feizi and
M. Schelhaas, Cell. Microbiol., 2013, 15, 1818-1836.
7. aR. J. Linhardt, K. G. Rice, Y. S. Kim, D. L. Lohse, H. M. Wang and
D. Loganathan, Biochem. J., 1988, 254, 781-787; bD. Ruhela, K.
Riviere and F. C. Szoka, Bioconjugate Chem., 2006, 17, 1360-
1363.
23. U. Neu, H. Hengel, B. S. Blaum et. al., PLOS Path., 2012, 8.
8. aH. Michael F. and B. Geert‐Jan, Eur. J. Org. Chem., 2002, 2002,
2033-2038; bS. Arungundram, K. Al-Mafraji, J. Asong, F. E. Leach,
I. J. Amster, A. Venot, J. E. Turnbull and G.-J. Boons, J. Am. Chem.
4 | J. Name., 2012, 00, 1-3
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