Biologically Active Branched Polysaccharide Mimetics: Synthesis via

Letter Cite This: ACS Macro Lett. 2018, 7, 772−777

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Biologically Active Branched Polysaccharide Mimetics: Synthesis via Ring-Opening Polymerization of a Maltose-Based β‑Lactam Ruiqing Xiao,† Jialiu Zeng,‡ and Mark W. Grinstaff*,†,‡ †

Department of Chemistry and ‡Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States S Supporting Information *

ABSTRACT: Stereoregular poly-amido-saccharides bearing α-glucopyranose branches (Mal-PASs) are synthesized by anionic ring-opening polymerization of a maltose-based βlactam monomer followed by debenzylation. The polymerization affords high molecular weight polymers (up to 31500 g/mol) with narrow dispersities (Đ < 1.1). Deprotected MalPASs are highly soluble in water and adopt a left-handed helical conformation in solution. Turbidimetric assay shows that Mal-PASs are multivalent ligands to lectin Concanavalin A.

P

> 1.5).10 Therefore, new polymerization approaches to welldefined, high molecular weight branched polysaccharides and their mimetics are highly sought after. Poly-amido-saccharides (PASs) are a new type of saccharide polymer in which the O-glycosidic linkages in natural polysaccharides is replaced with (1 → 2)-amide linkages.11 With saccharide moieties interconnected by an amide bond, these polymers exhibit characteristics of both polysaccharides and polypeptides, such as possessing pyranose-backbones and adopting helical conformations.12 However, due to lack of sufficient terminal saccharide residues, previously synthesized linear PASs display weak interactions with carbohydrate binding lectins.13 Herein, we report the first synthesis of novel branched polysaccharide mimetics with lectin binding activity, i.e., α-N-(1 → 2)-D-maltose poly-amido-saccharides (Mal-PASs). Specifically, we report: (1) the synthesis of a maltose β-lactam monomer (Mal-lactam) via [2 + 2] cycloaddition of 3,6,2′,3′,4′,6′-hexo-O-benzyl-D-maltal with chlorosulfonyl isocyanate (CSI); (2) the anionic ring-opening polymerization of Mal-lactam and subsequent deprotection; (3) characterization of Mal-PASs by nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), and circular dichroism (CD); and (4) recognition of Mal-PASs by Concanavalin A (Con A). Con A is a well-studied plant lectin that specifically binds nonreducing terminal α-D-mannopyranose and α-D-glucopyranose units.14 In order to synthesize a branched polysaccharide mimetic that binds Con A, we designed a new maltose-derived β-lactam monomer, namely, 2-carboxy-2-deoxy-3,6-di-O-benzyl-4-(2′,3′,4′,6′-tetra-O-benzyl-α-glucopyranosyl)-α-D-gluco-

rotein−carbohydrate interactions play essential roles in mediating a variety of intercellular recognition processes, such as cell adhesion, cell differentiation, and infection by viruses and bacteria.1 Despite their biological importance, monovalent protein−carbohydrate interactions are generally weak in affinity and broad in specificity.2 Nature overcomes these challenges through the use of multiple binding interactions between lectins and branched oligosaccharides and polysaccharides, i.e., the multivalent effect.3 However, due to the structural complexity of branched polysaccharides and the difficulty in isolation and purification, it is challenging to elucidate the structure−function relationship of proteincarbohydrate interactions. Advances in modern polymer chemistry are enabling the synthesis of various glycopolymers and glycopolypeptides where saccharide moieties are pendant groups along the polymer backbone.4 Although these polymers can mimic the lectin-binding function of branched polysaccharides, their main chains do not portray the compositions, structures, and conformations of the pyranose- or furanosebackbones of natural polysaccharides.5 Therefore, the synthesis of stereoregular branched polymers that compositionally, structurally, and functionally mimic complex natural polysaccharides are of interest and of importance. Cationic ring-opening polymerization of anhydrodisaccharides has been studied since the 1970s as a method to prepare branched polysaccharides.6 It offers precise regio- and stereoselective control over the branching structure at the monomer level.7 However, this method suffers from the low reactivity of anhydrodisaccharide monomers, thus it is difficult to obtain branched polysaccharides with high molecular weights.8 For example, the polymerization of a 1,6-anhydrocellobiose derivative gave benzylated polymers with number-average molecular weights (Mn) up to 7300 g/mol, which corresponds to a degree of polymerization (DP) of 8.5.9 Meanwhile, this method produces branched polymers with broad dispersities (Đ © XXXX American Chemical Society

Received: April 23, 2018 Accepted: June 7, 2018

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DOI: 10.1021/acsmacrolett.8b00302 ACS Macro Lett. 2018, 7, 772−777

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ACS Macro Letters Scheme 1. Polymer Synthesis and Deprotection Reactions

pyranosyl β-aminolactam (Mal-lactam, Scheme 1), which upon ring-opening polymerization affords a novel saccharide polymer possessing a pendant α-D-glucopyranose branch at each repeating unit. The Mal-lactam monomer was obtained, on a multigram scale, by [2 + 2] cycloaddition of 3,6,2′,3′,4′,6′-hexoO-benzyl-D-maltal with CSI at −61 °C followed by in situ reductive removal of the sulfonyl group (Scheme S1). However, 1 H NMR analysis of this product reveals that, along with the desired monomer Mal-lactam, it also contains 7% of the diastereomer with the four-membered β-lactam ring cis-fused to the β face of the pyranose ring, indicating that the [2 + 2] cycloaddition reaction is not stereoselective (Figure S1). This is in contrast to the stereospecific cycloaddition of monosaccharide glycals with CSI, which occurs exclusively anti with respect to the C3 substituent of the sugar ring.15 The stereochemical ambiguity of this reaction is likely because the large 2′,3′,4′,6′tetra-O-benzyl-α-D-glucopyranose group at the C4 position exerts steric hindrance to the approach of CSI from the α face of the pyranose ring. To our delight, diastereopure Mal-lactam was isolated as a colorless semisolid after repeated flash silica chromatography (Figure S2). The structure of this new monomer was characterized and confirmed by 1H NMR, 13C NMR, COSY, HSQC, and HMBC combined with FT-IR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) analyses (SI). Mal-lactam polymerized readily in tetrahydrofuran (THF) at 0 °C, using lithium bis(trimethylsilyl)amide (LiHMDS) as the catalyst and tert-butylacetyl chloride as the initiator via anionic ring opening polymerization (Schemes 1 and S2).12 The reaction was quenched with saturated ammonium chloride solution, and the resultant polymers were isolated in high yields by reprecipitation from hexane and then methanol (83−91%). Polymer molecular weights were determined using GPC with THF as the eluent and polystyrene standards. Varying monomer-to-initiator ratios ([M]/[I]) from 10:1 to 30:1 gave benzylated polymers (P1′−P4′) with Mn up to 31500 g/mol, which is considerably higher than the polymers obtained from cationic ring-opening polymerization of benzylated 1,6anhydromaltose (13400 g/mol).16 The higher polymerizability of Mal-lactam compared to the 1,6-anhydromaltose monomer likely arises from the high ring strain of the four-membered βlactam cis-fused to the pyranose ring. The GPC chromatograms of polymers P1′−P4′ all display narrow unimodal peaks (Đ = 1.05−1.07), and the chain lengths increase linearly with stoichiometry, indicating formation of well-defined polymers (Figure 1). Polymers P1′−P4′ were debenzylated using sodium metal in liquid ammonia followed by extensive dialysis against water. After lyophilization, deprotected Mal-PASs (P1−P4) were isolated as fluffy white solids in high yields (76−92%). The

Figure 1. Linear dependence of Mn (filled circles) of polymer P1′− P4′ on [M]/[I] and dispersities (open circles). The inset shows GPC elution curves for P1′−P4′.

disappearance of the aromatic proton peaks at 6.80−7.49 ppm in the 1H NMR spectra and the aromatic C−H stretch at 3000−3100 cm−1 in the FT-IR spectra confirmed complete removal of the benzyl protecting groups (Figure S5). The 1H NMR spectra of P1−P4 collected in D2O show well-resolved signals and couplings (Figure 2A). The small J-coupling between protons H1 and H2 (5.0 Hz) and large couplings between protons H2 and H3 (9.9 Hz) and between protons H3 and H4 (9.9 Hz) indicate a chair conformation for the backbone sugar repeating unit. The H1 signal of the Nterminus unit is visible as a doublet at 5.70 ppm and the H2 signal of the C-terminus unit is visible as a doublet of doublets centered at 2.73 ppm (SI). In the 13C NMR spectra, the carbon signals corresponding to the disaccharide repeating unit appear as 13 sharp peaks (Figure 2B), indicating that Mal-PASs are stereoregular and epimerization did not occur during the polymerization and deprotection reactions. Specific optical rotations ([α]25 D ) measured in water range from +208° for P1 to +214° for P4. These high positive [α]25 D values are consistent with the α configuration of glycosidic linkages on both the polymer backbone and side chains.16 The molecular weights of Mal-PASs were determined by aqueous GPC with dextran standards. Based on GPC analysis, P1−P4 possess Mn values ranging from 4800 to 11800 g/mol with narrow dispersities (Đ = 1.14−1.23, Figure S6). The DP measured by GPC and NMR are consistent with the corresponding values for the benzylated polymers (Table 1), indicating that the deprotection process proceeded efficiently without noticeable degradation of the polymer backbone. The MALDI-TOF spectrum of P1 contains only one population of peaks with a spacing of 351.6 m/z, equal to the molar mass of the expected repeating unit (Figure S7). In 773

DOI: 10.1021/acsmacrolett.8b00302 ACS Macro Lett. 2018, 7, 772−777

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ACS Macro Letters

Figure 2. (A) 1H NMR spectrum and (B) 13C NMR spectrum of P4 (only the repeating unit and N-terminus are shown in the inset structure).

addition, the m/z values of the observed molecular ion peaks correspond to the calculated molar mass of the potassiumcharged polymers having chain ends of the t-butylacetyl initiating group at the N-terminus and a maltose N-acyllactam terminating group at the C-terminus. For example, the observed value of 4001.7 m/z agrees with the calculated molar mass of 4001.6 g/mol for the K+ adduct Mal-PAS 10-mer (Figure S7). These data confirm not only the efficient incorporation of the initiator, but also a molecular weight range in good agreement with the Mn values estimated by GPC and 1H NMR analysis. Mal-PASs are highly soluble in water, and aqueous solutions of P1−P4 at concentrations up to 40.0 mg/mL were stable and remained clear for a week of observation at 25 °C (higher

concentrations were not investigated). This is in sharp contrast to the previous observation that glucose-based PASs (GlcPASs) have limited solubility and tend to precipitate from aqueous solutions over time,11 possibly because the pendant glucose branches in Mal-PASs prevent the aggregation of polymer chains. This increase in water solubility was also observed for branched polysaccharides prepared by glycosylation of linear polysaccharides.17 To investigate the solution conformation of Mal-PASs, CD spectra of P1−P4 were recorded in water. As shown in Figure 3, the CD spectra of P1−P4 display a minimum at 221 nm and a maximum at 189 nm, similar to the CD spectra of Glc-PASs, indicating that MalPASs adopt a similar left-handed helical conformation in 774

DOI: 10.1021/acsmacrolett.8b00302 ACS Macro Lett. 2018, 7, 772−777

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ACS Macro Letters Table 1. Polymer Characterization Using GPC, NMR, and Optical Rotation entry

Mn(theo)a

Mn(GPC)b

DP(GPC)b

Đc

DP(NMR)d

e [α]25 D

yieldf

P1′ P2′ P3′ P4′ P1 P2 P3 P4

9020 13480 17940 26860 3750 5290 7540 12450

9400 13300 19000 31500 4800 6500 8700 11800

9.4 13.8 20.2 34.2 12.4 17.3 23.4 32.3

1.05 1.05 1.06 1.07 1.17 1.18 1.23 1.15

N.D. N.D. N.D. N.D. 12.3 19.6 26.4 37.5

+70.8 +72.0 +69.7 +71.4 +208.6 +211.1 +212.4 +214.9

91 94 88 83 76 82 85 92

a

For P1′−P4′: calculated based on [M]/[I] ratios g/mol; for P1−P4: calculated based on the DP of benzylated polymers. bDetermined by THF GPC against polystyrene standards for P1′−P4′ or aqueous GPC against dextran standards for P1−P4, g/mol. cMw/Mn. dDetermined by NMR end group analysis. eMeasured by polarimeter in chloroform for P1′−P4′ or water for P1−P4, °. fIsolated yield, %; N.D., not determined.

Figure 4. Turbidimetric assay of Mal-PASs with Con A in HEPES buffer. [Con A]: 1 μM; [Mal-PASs]: 50 μM based on the maltose repeating unit.

Figure 3. CD spectra of Mal-PASs in water at 25 °C.

aqueous solutions.18 The mean residue ellipticity (MRE) of Mal-PASs at 220 nm ([θ]220) is approximately −12700 deg· cm2·dmol−1, which is slightly smaller than the [θ]220 value of the Glc-PAS (P5, −14600 deg·cm2·dmol−1), indicating that the introduction of glucopyranose branches to the Glc-PAS backbone may lead to a slight destabilization of the helical conformation.19 The CD spectra of polymer P4 remain unchanged over a broad pH range from 2.0 to 12.0, or in the presence of high ionic strength (2.0 M NaCl), or with protein denaturant (4.0 M urea), showing that the conformation of Mal-PASs is stable in aqueous solutions (Figure S8). At physiological pH, Con A exists in a tetrameric form with each subunit containing a sugar binding site.20 Thus, Con A tends to aggregate and eventually precipitate in the presence of multivalent ligands.21 The affinity of a multivalent ligand to Con A can be evaluated from the clustering rate of aggregation using an established turbidimetric assay.22 Con A was quickly mixed with a 10-fold excess of Mal-PASs in HEPES buffer at pH 7.4 at 25 °C, and the solution turbidity was monitored by UV−vis absorbance at 420 nm over a period of 20 min. The initial rate of Con A clustering (ki) was determined by linear fit to the initial portion of the data. The results for the turbidimetric assay are summarized in Figure 4 and Table S1. The interactions of Mal-PASs with Con A are highly dependent on the DP. Upon increasing the DP(NMR) from 12.3 to 37.5, the clustering becomes faster and more pronounced (Table S1). It has previously been shown that glycopolymers and glycodendrimers that span multiple binding sites of Con A (separated by about 6.5 nm) exhibit higher binding affinities because of multivalent interactions via the chelate effect.23 Molecular dynamics (MD) simulation of an all-atom (AA) model

produced using a modified CHARMM force field revealed that a 10 mer of Glc-PAS has a length of approximately 2.8 nm.18 Since Mal-PASs possess the same backbone and adopt a similar conformation as Glc-PASs, the average lengths of polymers P1, P2, P3, and P4 were calculated to be about 3.7, 5.8, 7.7, and 10.8 nm, respectively, according to DP(NMR) (Table S1). Therefore, P3 and P4 are sufficiently long to span the distance between two binding sites of Con A (6.5 nm). However, it should be noted that Mal-PASs are polydispersed macromolecules consisting of species of different length, and those lower molecular weight polymers (P1 and P2) also contain a fraction of longer species. As a result, polymers P1 and P2 also show the ability to cluster Con A to some extent. As expected, altrose-derived PAS (Alt-PAS, P6, Mn = 8400 g/mol) does not induce turbidity as altropyanose is not a ligand for Con A (Figure 4). The recognition of linear Glc-PAS (P5, Mn = 9200 g/mol) by Con A is also negligible, probably due to the low concentrations used in this assay along with the minimal terminal sugar residues to which the lectin can bind.11 It is noteworthy that control polymer P5 and P6 have a DP(NMR) of 47.4 and 52.7, respectively, which are long enough to span the distance of two binding sites of Con A. Addition of an excess of methyl α-D-mannopyranose (5.0 mM), a stronger ligand to Con A than α-D-glucopyranose, leads to instantaneous dissociation of the lectin-polymer conjugates and loss of solution turbidity, confirming that Mal-PASs bind Con A at the same site as natural carbohydrates. Turbidimetric assays are used intensively to study the recognition of Con A with glycopolymers with various polymer backbones.24 For comparison, a glycopolymer with pendant α775

DOI: 10.1021/acsmacrolett.8b00302 ACS Macro Lett. 2018, 7, 772−777

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ACS Macro Letters D-mannopyranoses (DP = 38), synthesized by ring-opening metathesis polymerization, showed a ki of about 0.49 au/min, which is 5.8 times larger than the ki value of P4 (0.085 au/ min).25 The decreased initial clustering rate of P4 compared to the mannose glycopolymer may be ascribed to two reasons. First, Con A exhibits a higher binding constant with α-Dmannopyranose than with α-D-glucopyranose (Kb(Man)/Kb(Glc) ≈ 4:1), and this binding selectivity can be further enhanced for glycopolymers due to multivalent effect.26 Second, it was suggested that glycopolymers with flexible backbones tend to show stronger binding ability with lectins as they allow better adjustment of sugar ligands to the lectin geometries.27 According to CD analysis, Mal-PASs adopt a rigid helical conformation, while the mannose glycopolymer reported by Kiessling et al. possessed a more flexible polynorbornene backbone.25 However, the influence of polymer backbone rigidity on lectin binding is still under investigation, as the work of Kiick et al. showed that glycopolypeptides with helical backbone can be superior as multivalent ligands to glycopolypeptides with random-coiled structures.28 Overall, the lectin binding study shows that, although possessing a rigid helical polymer backbone, Mal-PASs are multivalent ligands for Con A. The cytotoxicity of Mal-PASs at different concentrations was evaluated using an MTS cell proliferation assay in three cell lines: human embryonic kidney (HEK293), human cervix adenocarcinoma (HeLa), and mouse embryonic fibroblast (NIH3T3) cells in vitro. As shown in Figure S9, polymers P1−P4 did not decrease cell viability at concentrations up to 1.0 mg/mL after 24 h of incubation in comparison to the media-treated control cells, indicating no cytotoxicity. In summary, stereoregular branched polysaccharide mimetics are synthesized via anionic ring-opening polymerization of a maltose-based β-lactam (Mal-lactam) monomer. This method ensures polymer stereoregularity as the polymerization proceeds without affecting the stereochemistry of the anomeric carbon. Compared to the ring-opening polymerization of anhydrodisaccharides, this method affords well-defined branched polymers with higher molecular weights and narrower dispersities. The deprotection reaction proceeds smoothly to give water-soluble stereoregular Mal-PASs, as confirmed by NMR, FT-IR, optical rotation, and MALDI-TOF mass spectrometry. CD analysis indicates that in aqueous solution Mal-PASs adopt a stable left-handed helical conformation. Finally, turbidimetric assays reveal that these rigid Mal-PASs are able to cluster and aggregate Con A, in a length-dependent manner, due to the existence of multiple α-D-glucose branches. Mal-PASs are promising biologically active polymers for biomedical applications, and studies are ongoing to further investigate the dependence of lectin recognition on epitope density by synthesizing copolymers with various branching frequencies. The anionic ring-opening polymerization of disaccharide-based β-lactams expands the current repertory of approaches available to complex saccharide polymers with biological activities.

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Experimental procedures and data for all the compounds, NMR, IR, and CD spectra of the polymers, as well as cell viability data (PDF).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Mark W. Grinstaff: 0000-0002-5453-3668 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Boston University for support. REFERENCES

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DOI: 10.1021/acsmacrolett.8b00302 ACS Macro Lett. 2018, 7, 772−777

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ACS Macro Letters

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DOI: 10.1021/acsmacrolett.8b00302 ACS Macro Lett. 2018, 7, 772−777