P.
Bojarová
*a,
P.
Chytil
*b,
B.
Mikulová
a,
L.
Bumba
a,
R.
Konefał
b,
H.
Pelantová
a,
J.
Krejzová
a,
K.
Slámová
a,
L.
Petrásková
a,
L.
Kotrchová
b,
J.
Cvačka
c,
T.
Etrych
b and
V.
Křen
a
aInstitute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic. E-mail: bojarova@biomed.cas.cz
bInstitute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovský Sq. 2, CZ-162 06 Prague 6, Czech Republic. E-mail: chytil@imc.cas.cz
cThe Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic
First published on 21st March 2017
Novel conjugates of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers tethered with chitooligosaccharidic epitopes of varying lengths were shown to be potent ligands of a model lectin, wheat germ agglutinin (WGA). The azide-functionalized oligosaccharidic epitopes were prepared by the action of Tyr470Asn mutant β-N-acetylhexosaminidase from Talaromyces flavus in a single reaction step and were conjugated to HPMA copolymer precursors in a defined pattern and density through Cu+-catalyzed azide–alkyne cycloaddition. The soluble, biocompatible, and structurally flexible synthetic glycopolymers were studied for their binding to WGA in a competitive enzyme-linked lectin assay (ELLA), and the kinetics of interaction were analyzed by surface plasmon resonance (SPR). To the best of our knowledge, this study presents the first HPMA copolymers derivatized with long oligosaccharides that demonstrate high affinity to a lectin target. The binding affinities in the low nanomolar and subnanomolar ranges place the prepared glycopolymers among the best WGA ligands reported to date. This study demonstrates the targeting potential of these glycopolymers for therapeutically relevant lectins.
Current glycomics can mimic nature-like multivalent sugar displays in the form of synthetic glycoconjugates based on a polymer, dendrimer or solid scaffolds with miscellaneous compositions, architectures, and flexibilities.3,4 The orientation and density of the carbohydrate epitopes as well as the type and length of the connecting spacers are crucial parameters to be considered for the appropriate multiplication of the weak monovalent glycan–lectin interaction. Selective and efficient lectin targeting results in useful glyco-therapeutics and diagnostic tools, agents for targeted drug delivery, cell imaging or for other biological and biomedical applications.4
Wheat germ agglutinin (WGA) is a conventional model lectin used for studying multivalent carbohydrate–lectin interactions and structure–affinity relationships in tailored multivalent glycoconjugates.5 It is abundantly present in the endosperm of wheat (Triticum vulgaris) and in other natural sources including some mammalian cell membranes. WGA binds N-acetyl-D-glucosamine (GlcNAc) residues and their β1,4-linked oligomers as well as sialic acid. In a solution at physiological pH, WGA exists mostly as a homodimer with the molecular weight of 36000 g mol−1. Each monomer comprises four hevein-like domains (A–D), each containing a unique binding site for GlcNAc, with the shortest mutual distance of 13–14 Å.6 Recent studies reported by Renaudet and coworkers presented several complex dendrimeric inhibitors of WGA based on cyclopeptides7 and polyester scaffolds containing up to 96 GlcNAc units;8 these inhibitors demonstrated binding affinities towards WGA in the nanomolar range.
For the synthesis of glycopolymer scaffolds, various strategies including living radical, cationic/anionic, and ring opening metathesis polymerizations of carbohydrate-bearing monomeric blocks have been adopted in the last ten years.9 An alternative approach, also applicable for more complex glycan epitopes, is to conjugate functionalized glycan chains with appropriate functional groups distributed ad arbitrium along the polymer backbone. Advantageously, this strategy can exploit the elegant click chemistry10 approach – Cu+-catalyzed azide–alkyne cycloaddition (CuAAC),11 yielding triazole from an azide and a terminal alkyne.
The synthetic polymer carriers based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers represent the most attractive agents for specific drug delivery and targeting12 due to their non-immunogenicity, non-toxicity, biocompatibility, and water solubility.
If drugs or therapeutics for in vivo applications are bound to these polymers (affording the so-called polymer prodrugs), the occurrence of possible adverse effects is diminished and/or retarded. A typical application is in the treatment of cancer, especially that manifested with solid tumors, in which the polymer-prodrugs accumulate through the enhanced permeability and retention (EPR) effect.13 However, the typical uptake efficiency barely exceeds 10% of the used dose. Conjugation of polymer prodrugs to ligands that are specific to selected tumor receptors is the main idea of the active targeting treatment approach. Lectin receptors constitute very promising targets;4 the conjugation of polymers to specific glycan epitopes results in enhanced selectivity of binding to these receptors, affording high-affinity multivalent ligands with potential biomedical uses.
In the past, several studies have been devoted to exploring the ability of HPMA copolymers decorated with simple sugars to serve as ligands of endogenous lectins14 such as the asialoglycoprotein receptor or galectin-3. The main problem in the construction of these glycopolymers has always been the synthesis of highly specific tailored carbohydrate epitopes, especially those of an oligosaccharidic nature, to be efficiently conjugated to the polymer carrier. To the best of our knowledge, the only known oligosaccharide-bearing HPMA-based copolymer, recently reported by Roy et al.,15 carried a trimannoside prepared through a complex multistep synthetic procedure.
This study describes the synthesis of thirteen HPMA copolymers decorated with chitooligosaccharide epitopes of varying lengths and their capacity to interact with WGA as a model lectin. Azide-functionalized chitooligomers of one to five GlcNAc units were prepared in a single transglycosylation reaction catalyzed by the Tyr470Asn mutant of β-N-acetylhexosaminidase from Talaromyces flavus;16 they were conjugated in a defined density to propargyl-functionalized HPMA copolymers by the CuAAC reaction. The glycopolymers exhibited extremely strong binding to WGA, as shown in the competitive ELLA assays and in SPR studies; the affinities of the best conjugates were in the subnanomolar range, which ranks them among the strongest ligands reported to date.8 The present structures show a promising path to achieve high-affinity biocompatible glycopolymer carriers targeted at lectin-displaying structures.
The 1H and 13C NMR spectra of chitooligomer standards 26–29 were compared with the previously reported data;17 very good agreement was found for the carbon chemical shifts. Due to the strong overlap of proton signals, the assignment of individual GlcNAc units in 5–9 was mainly achieved using HSQC-TOCSY experiments, supported by the information extracted from the HSQC and COSY spectra. The β-anomeric configuration of the sugar units was determined from the magnitudes of the JH-1,H-2 coupling constants. The up-field shifted resonances of all the C-2 carbons were in accordance with N-acetylation at this position. The glycosidic linkage position was deduced from the downfield glycosylation shifts of the involved C-4 carbons and unambiguously confirmed using the heteronuclear correlations of these carbons with the anomeric protons of subsequent GlcNAc units. The attachment of the azidoethyl group was also confirmed by HMBC correlations.
The glycopolymers and their polymer precursors were investigated via a Bruker Avance III 600 spectrometer operating at 600.2 MHz with DMSO-d6 or D2O as the solvent. The width of the 90° pulse was 10 μs, with a relaxation delay of 10 s. The acquisition time was 3.63 s with 200 (16 for kinetics) scans. The structure and purity of the monomers and the contents of the polymer-bound propargyl groups and carbohydrates were determined in DMSO-d6. For the calculations, integral intensities of δ (ppm) = 3.67 (1 H, br s, CH–OH) or δ (ppm) = 4.71 (1 H, br s, CH–OH) of the HPMA monomer unit were used. To calculate the contents of propargyl groups in the polymer precursors 12a and 12b, these signals were compared with the integral intensities of δ (ppm) = 3.85 (2 H, br, CH2–CC); to calculate the contents of carbohydrate 3 in the glycopolymers 16–18, the signals were compared with the integral intensity δ (ppm) ≈ 7.84 (1 H, s, CH of triazole). Moreover, to calculate the contents of carbohydrate 2 in the glycopolymers 13–15, carbohydrate 5 in glycopolymers 19–20, carbohydrate 6 in glycopolymers 21–22, carbohydrate 7 in glycopolymers 23–24, and carbohydrate 8 in glycopolymer 25, the signals were compared with the integral intensity of δ (ppm) ≈ 7.80 (1 H, br, CH of triazole and NH–acetyl).
The course of azide–alkyne cycloaddition was monitored in situ by 1H NMR. Polymer 12a (20 mg, 13.8 μmol of propargyl groups), carbohydrate 3 (4.00 mg, 13.8 μmol), and sodium ascorbate (1.32 mg, 6.9 μmol) were dissolved in D2O (0.6 mL); the solution was then placed in a NMR cuvette and bubbled with argon. After this, a solution of the catalyst CuSO4·5H2O (1.72 mg, 6.9 μmol) in D2O (50 μL), bubbled with argon, was added to the reaction mixture. The 1H NMR spectra were obtained before the reaction and 10, 30, 45, and 60 min after the addition of Cu+ catalyst.
The purity of the monomers for polymer synthesis was determined using a C18 reverse-phase Chromolith Performance RP-18e column (4.6 × 100 mm, Merck Millipore) with diode array detection. The mobile phase was water/acetonitrile/0.1% TFA with a gradient of 5–95% v/v acetonitrile and a flow rate of 5 mL min−1.
The molecular weights and dispersities of the polymers were determined using a Shimadzu HPLC system equipped with a gel permeation chromatography (GPC) column (TSKgel G3000SWxl, 300 × 7.8 mm; 5 μm), connected to refractive index (RI) Optilab®-rEX and multiangle light scattering (MALS) detectors (DAWN HELEOS II, Wyatt Technology Co., USA). A mixture of methanol/0.3 M sodium acetate buffer, pH 6.5 (4:1, v/v) at the flow rate of 0.5 mL min−1 was used as the mobile phase.
For the preparative reaction (Scheme 1), donor 4 (51 mg, 0.15 mmol), acceptor 3 (87 mg, 0.30 mmol), and the Tyr470 Asn mutant of the β-N-acetylhexosaminidase from T. flavus (4.73 U, 1.4 mg, 167 μL) were incubated in 50 mM citrate-phosphate buffer (pH 5.0, 3.0 mL) at 35 °C and 1000 rpm under monitoring by HPLC and TLC as described above. After 2.5 h (ca. 90% conversion), another portion of 4 (51 mg, 0.15 mmol) was added. After 8 h, the reaction was stopped by boiling for 2 min, and the mixture was centrifuged (13500 rpm; 10 min) after cooling down to room temperature. The supernatant was concentrated in vacuo to ca. 2 mL volume and loaded onto a Biogel P-2 column (2.6 × 100 cm, Bio-Rad, USA). Water was used as the mobile phase at the elution rate of 10 mL h−1. The fractions containing the products were obtained, traces of remaining p-nitrophenol were extracted with ethyl acetate (3 × 10 mL), and the samples were lyophilized; products 5–9 were obtained as white solids. Pure acceptor 3 was partially recovered (35 mg). For the respective NMR data, see the ESI, Tables S2–S6 and Fig. S2–S6.† 2-Azidoethyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (5), 32 mg, 0.065 mmol; HRMS (ESI−): found m/z 492.19479 (expected 492.19473 for [M − H], C18H30O11N5); [α]20589 −34.7 (c 0.190 in H2O). 2-Azidoethyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (6) 19 mg, 0.028 mmol; HRMS (ESI−): found m/z 695.27420 (expected 695.27410 for [M − H]−, C26H43O16N6); [α]20589 −21.9 (c 0.187 in H2O). 2-Azidoethyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (7), 10 mg, 0.011 mmol; HRMS (ESI−): found m/z 898.35309 (expected 898.35347 for [M − H], C34H56O21N7); [α]20589 −22.3 (c 0.175 in H2O). 2-Azidoethyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (8), 2.0 mg, 1.81 μmol; HRMS (ESI−): found m/z 1101.43077 (expected 1101.43285 for [M − H]−, C42H69O26N8); [α]20589 −12.3 (c 0.089 in H2O). 2-Azidoethyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (9), 2.7 mg, 2.07 μmol; HRMS (ESI−): found m/z 1304.51088 (expected 1304.51222 for [M − H]−, C50H82O31N9). The total isolated yield of the functionalized oligosaccharides 5–9 was 60% (based on the consumed acceptor 3).
Scheme 2 Synthesis of glycopolymers 13–25. AIBN, 2,2′-azobisisobutyronitrile; DIPEA, N,N-diisopropylethylamine. |
Glycopolymers 13–25 were prepared by the reaction of propargyl groups of polymer precursor 12a or 12b with the respective azido-functionalized GlcNAc (2 and 3) or functionalized chitooligosaccharides 5–8 by Cu+-catalyzed azide–alkyne cycloaddition in water (Scheme 2). Glycopolymers 15 and 18 were synthesized from the polymer carrier 12b, containing a higher amount of propargyl groups (21.0 mol%). The other glycopolymers were synthesized from 12a. The sample preparation of glycopolymer 17 is as follows. A solution of CuSO4·5H2O (2.15 mg, 8.6 μmol) in water (25 μL) was added to a mixture of copolymer 12a (25 mg, 17.2 μmol of propargyl groups), sodium ascorbate (1.71 mg, 8.6 μmol), and carbohydrate 3 (5.00 mg, 17.2 μmol) dissolved in water (225 μL). The reaction mixture was bubbled with argon before and after the addition of Cu+ to the reaction mixture and was vortexed. After 30 min, PBS buffer with 5% EDTA disodium salt (750 μL) was added, and the polymer was purified by gel filtration via a PD10 desalting column containing Sephadex G-25 resin (GE Healthcare Life Sciences) using water as the mobile phase. The polymer fraction was lyophilized to obtain glycopolymer 17 (28.8 mg; 96% yield). The yields of glycopolymers 13–25 ranged approximately from 90 to 95%. For a detailed description of glycoconjugates, see Table 1.
Compound | M na (g mol−1) | Đ | Sugar motif | n | IC50 (nM) | r pc | r p/nd |
---|---|---|---|---|---|---|---|
a The molecular weights (Mn) and dispersities (Đ) of the polymers were determined using GPC with MALS and RI detection. b Average number of glycans per polymer chain (glycan content, mol%); n = 1, monovalent standard. c Relative potency, i.e. IC50 (monovalent standard)/IC50 (multivalent glycopolymer). d Relative potency per glycan. | |||||||
1 | GlcNAc | 1 | (17400 ± 2800) × 103 | 1 | 1 | ||
26 | (GlcNAc)2 | 1 | (800 ± 170) × 103 | 1 | 1 | ||
27 | (GlcNAc)3 | 1 | (107 ± 39) × 103 | 1 | 1 | ||
28 | (GlcNAc)4 | 1 | 70000 ± 3000 | 1 | 1 | ||
29 | (GlcNAc)5 | 1 | 41000 ± 9000 | 1 | 1 | ||
13 | 21000 | 1.13 | GlcNAc-N | 8.1 (6.3%) | 49600 ± 9900 | 351 | 43 |
14 | 22900 | 1.12 | GlcNAc-N | 12.3 (9.1%) | 10100 ± 1400 | 1723 | 140 |
15 | 27300 | 1.11 | GlcNAc-N | 21.9 (15.1%) | 360 ± 100 | 48333 | 2207 |
16 | 24600 | 1.05 | GlcNAc-O | 9.3 (6.2%) | 5100 ± 2300 | 3412 | 367 |
17 | 25200 | 1.06 | GlcNAc-O | 13.6 (9.4%) | 350 ± 120 | 49714 | 3655 |
18 | 29100 | 1.07 | GlcNAc-O | 24.0 (16.4%) | 7.6 ± 2.3 | 2289474 | 95395 |
19 | 22800 | 1.12 | (GlcNAc)2-O | 7.1 (5.4%) | 3.4 ± 1.1 | 235294 | 33140 |
20 | 24600 | 1.13 | (GlcNAc)2-O | 11.8 (9.4%) | 1.88 ± 0.47 | 425532 | 36062 |
21 | 23800 | 1.12 | (GlcNAc)3-O | 6.9 (5.3%) | 3.68 ± 0.98 | 29076 | 4214 |
22 | 26600 | 1.14 | (GlcNAc)3-O | 10.6 (8.2%) | 1.34 ± 0.64 | 79851 | 7533 |
23 | 25900 | 1.33 | (GlcNAc)4-O | 6.6 (4.8%) | 2.58 ± 0.46 | 27131 | 4111 |
24 | 29200 | 1.12 | (GlcNAc)4-O | 9.9 (7.2%) | 2.78 ± 0.19 | 25180 | 2543 |
25 | 22800 | 1.09 | (GlcNAc)5-O | 2.9 (2.2%) | 11.6 ± 1.6 | 3534 | 1219 |
In total, thirteen conjugates of functionalized oligosaccharides with HPMA copolymers were prepared by means of the efficient CuAAC click reaction. Fig. 1 shows the reaction progress during the synthesis of glycopolymer 17, as monitored by 1H NMR. Within 10 min after adding the Cu+ catalyst, compound 3 was completely bound to polymer precursor 12a, as demonstrated by the disappearance of peaks 8 and 9, belonging to the propargyl moiety, and by the formation of peaks 10 and 11, related to the triazole moiety (Fig. 1).
GlcNAc azide 1 yielded glycoconjugates 13–15 (containing 6.3, 9.1, and 15.1 mol% of 1, respectively). 2-Azidoethyl GlcNAc 3 afforded glycoconjugates 16–18 (containing 6.2, 9.4, and 16.4 mol% of 3, respectively). Glycopolymers 19 and 20 originated from dimer 5 (with 5.4 and 9.4 mol% of 5, respectively), glycopolymers 21 and 22 from trimer 6 (with 5.3 and 8.2 mol% of 6, respectively), and glycopolymers 23 and 24 from tetramer 7 (4.8 and 7.2 mol% of 7, respectively). Pentamer 8 yielded glycopolymer 25 (2.2 mol%). In general, lower amounts and simpler structures of saccharides were nearly quantitatively bound to the polymer precursors, in contrast to the longer chitooligomer chains. This was probably caused by steric hindrance decreasing the content of bound saccharide, especially in the case of pentamer 8. The molecular weights of the glycopolymers slightly increased as compared to those of their respective polymer precursors. This increase may be ascribed to the presence of the saccharide moieties in the polymer structure. As expected, the attachment of saccharides did not influence the polymer dispersity.
The results clearly show a vast difference between the two linkers tested – whereas the GlcNAc-HPMA conjugates containing the shorter and more rigid azido linker (13–15) demonstrated IC50 values around the μM range (from 49 μM to 360 nM, depending on the carbohydrate content), the more flexible 2-azidoethyl linker brought a significant improvement in the inhibition potency, with an IC50 as low as 7.6 nM for 18 (16 mol% GlcNAc). In the case of both linkers, a significant decrease in IC50 of roughly one order of magnitude was observed with the increasing GlcNAc content from 6 to 9 mol% and again from 9 to 15 mol%. This reflects an easier structural orientation of the GlcNAc units attached to the polymer chain towards the lectin binding sites. The relative inhibitory potency per glycan (rp/n, Table 1) is a measure of potency enhancement induced by the cluster glycoside effect. In this respect, glycopolymer 18 proved to be the best compound in the series, demonstrating a multivalency factor of over 95000 compared to that of the monovalent GlcNAc standard.
All chitooligosaccharide-HPMA conjugates (19–25, containing glycans with two to five GlcNAc moieties) exhibited excellent inhibition potencies in the low nanomolar range: glycopolymer 22, derivatized with 8.2 mol% of trimer 6, had an IC50 of 1.34 nM. Note that no significant improvement of inhibition potency was observed with the increasing molar content of glycans in the oligosaccharide-presenting series, in contrast to the GlcNAc conjugates. This may be because the longer oligosaccharide chains, combined with the high flexibility of the polymer backbone, may structurally adapt to lectin binding requirements even at low carbohydrate contents (7–10 glycans per polymer chain). This is especially outstanding in the case of N,N′,N′′,N′′′,N′′′′-pentaacetylchitopentaose-HPMA conjugate 25, where only three glycans per polymer molecule resulted in an IC50 of 11.6 nM – a similar result to that of conjugate 18, carrying twenty-four GlcNAc units. This is also an excellent result compared to that of the previously reported dendrimers, which generally required several dozens of GlcNAc units per molecule to reach comparable inhibition potencies.8 It may be concluded that the derivatization of HPMA copolymer with longer chitooligomer chains results in highly efficient WGA inhibitors, even with a strikingly low carbohydrate content in the molecule. This parameter is especially important due to the fact that a high molar content of the carbohydrate portion may result in altered physicochemical properties and pharmacokinetics of the glycopolymer compared to that of the sugar-void analogue.
Glycopolymera | k a1 × 105 (M−1 s−1) | k d1 × 10−5 (s−1) | k a2 (RU s−1) | k d2 (s−1) | K Db (nM) | |
---|---|---|---|---|---|---|
a Compound number, type of glycan attached, and its content in the glycopolymer (mol%). b Apparent equilibrium dissociation constant (KD) for the bivalent analyte binding model was calculated as follows: c Estimated dissociation constant (KD) derived from the steady state (Req) values of the association phase of the sensograms. | ||||||
13 | GlcNAc-N (6.3%) | N.D. | N.D. | N.D. | N.D. | >10000c |
15 | GlcNAc-N (15.1%) | N.D. | N.D. | N.D. | N.D. | >100c |
17 | GlcNAc-O (9.4%) | 1.6 ± 1.5 | 2528 ± 850 | 140 ± 95 | 272 ± 94 | 104 ± 35 |
18 | GlcNAc-O (16.4%) | 3.5 ± 1.2 | 610 ± 290 | 0.9 ± 0.7 | 2.5 ± 1.1 | 13 ± 5 |
19 | (GlcNAc)2-O (5.4%) | 6.1 ± 2.4 | 71 ± 35 | 0.005 ± 0.004 | 0.1 ± 0.1 | 1.1 ± 0.6 |
20 | (GlcNAc)2-O (9.4%) | 5.7 ± 1.8 | 34 ± 21 | 26 ± 12 | 603 ± 213 | 0.6 ± 0.3 |
21 | (GlcNAc)3-O (5.3%) | 3.6 ± 1.5 | 45 ± 26 | 0.003 ± 0.02 | 0.1 ± 0.1 | 1.2 ± 0.5 |
22 | (GlcNAc)3-O (8.2%) | 4.1 ± 1.8 | 21 ± 13 | 6.8 ± 2.8 | 43 ± 24 | 0.4 ± 0.2 |
23 | (GlcNAc)4-O (4.8%) | 3.5 ± 1.3 | 72 ± 36 | 0.5 ± 0.3 | 2.4 ± 1.2 | 1.7 ± 0.8 |
24 | (GlcNAc)4-O (7.2%) | 3.1 ± 2.1 | 9.5 ± 0.4 | 23 ± 14 | 51 ± 21 | 0.2 ± 0.1 |
25 | (GlcNAc)5-O (2.2%) | 1.3 ± 0.9 | 102 ± 35 | 1.9 ± 1.1 | 52 ± 18 | 7.6 ± 3.1 |
The kinetics of interaction of the O-linked glycopolymers with WGA did not fit a simple 1:1 Langmuir binding model; however, the data were described well using a bivalent analyte model (Fig. 3). This model describes the interaction of an immobilized ligand (WGA) with an analyte (glycopolymer) that carries two identical and independent binding sites. The bivalent analyte model results in two sets of rate constants, one for each binding step, where the second binding step is directed by the first binding step. This cooperative binding is called avidity and usually reflects the polyvalent nature of a glycopolymer. The calculated association and dissociation rate constants (ka1, ka2, kd1, and kd2) for the binding of the O-linked glycopolymers to WGA are listed in Table 2. The data show that the O-linked glycopolymers bind WGA with KD in the nanomolar and subnanomolar ranges, which ranks them among the best ligands of WGA ever reported.8 The overall comparison of the KD values reveals that the binding properties of the O-linked glycopolymers carrying glycans consisting of two (19, 20), three (21, 22), and four (23, 24) carbohydrate units are relatively similar, with binding affinities approximately 2 to 8 times higher for the glycopolymers with higher glycan molar contents (20, 22, 24) than for those with lower glycan contents (19, 21, 23). In particular, the KD for the high-content (7.2 mol%) glycopolymer 24 derivatized with chitotetraose reached the value of 0.2 nM, which is the highest binding affinity reported in our study. This high binding affinity can be attributed to the very slow dissociation rate of the 24-WGA complex because the association rates of the O-linked glycopolymers remained comparable. The O-linked glycopolymer 25 carrying chitopentaose was characterized by a faster dissociation rate of the encounter complex, yielding a decrease of the KD value to 7.4 nM, which is very likely due to the relatively low glycan content (2.2 mol%) in 25. The comparatively lowest binding affinities of the O-linked glycopolymers were established for those decorated with single GlcNAc units (17 and 18). The predominant mechanism contributing to their lower binding capability was the very fast dissociation rate of the encounter complex; this was especially pronounced in glycopolymer 17, which has a lower GlcNAc content (9.4 mol%; cf. 18 with 16.4 mol% GlcNAc).
Overall, these results clearly show that high-affinity binding of the chitooligosaccharide-modified HPMA copolymers to WGA is caused by the avidity effect of multiple sugar moieties in the glycopolymers. The first association rate constants ka1, representing the initial stage of the binding process, appear to be similar for all the glycopolymer–WGA interactions, indicating that the single GlcNAc unit is sufficient to elicit the interaction of the HPMA copolymer with the lectin surface. On the other hand, single GlcNAc units are not sufficient to maintain a stable complex due to a much faster dissociation (kd1) of the glycopolymers derivatized with single GlcNAc units as compared to those with di-, tri or tetrasaccharide units, suggesting significant beneficial effects of additional sugar units for the stability of the glycopolymer–WGA interaction. The density of glycans on the polymer carrier (given by their molar content) much more strongly influences the glycopolymer binding affinity in the case of derivatization with single GlcNAc units than with longer chitooligomer chains, probably due to the high flexibility and spatial adaptability of longer glycans.
The most outstanding improvement in the binding of the glycopolymers to WGA was brought about by the increased length of the glycan chain, especially when comparing the monomeric and dimeric GlcNAc: glycopolymer 19 (with dimeric GlcNAc) binds more than three orders of magnitude (IC50) more strongly to WGA than its counterpart 16 (with mono-GlcNAc). As suggested by the analysis of the binding kinetics, longer glycan chains stabilize the glycopolymer–WGA complex better and slow down its dissociation. The binding parameters KD acquired for oligosaccharide-decorated glycopolymers 20, 22, and 24 are in the subnanomolar range (0.6, 0.4, and 0.2 nM, respectively) and rank these glycopolymers among the best WGA ligands ever reported. Note that just ten glycans per polymer molecule are sufficient to achieve this impressive binding efficiency.
Footnote |
† Electronic supplementary information (ESI) available: Structural characterization of functionalized chitooligomers 3, 5–9 (NMR data and spectra, MS spectra, HPLC chromatograms); structural identification of chitooligomer standards 26–29; synthesis of polymer precursors 12a and 12b; structural characterization of glycopolymers 13–25 (NMR spectra). See DOI: 10.1039/c7py00271h |
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