Article pubs.acs.org/Biomac
Hydrophobic Spacers Enhance the Helicity and Lectin Binding of Synthetic, pH-Responsive Glycopolypeptides Robert Mildner and Henning Menzel* Institute for Technical Chemistry, Braunschweig University of Technology, Hans-Sommer-Str. 10, 38106 Braunschweig, Germany S Supporting Information *
ABSTRACT: The influence of different hydrophobic spacers on the structural and lectin binding properties of well-defined glycopolypeptides decorated with galactose moieties was investigated. All glycopolypeptides were prepared from a poly(α,L-glutamic acid) (PGA) precursor via a polymeranalogous aqueous amide coupling reaction. Thereby, two alkyl spacers of different length (C6 and C11) as well as an aromatic spacer were introduced between the backbone and the galactose moieties, as confirmed by 1H NMR spectroscopy. The secondary structure was investigated as a function of the sugar density and the pH by circular dichroism (CD) spectroscopy. It was found that the helicity in acidic medium and thus the typical coil-to-helix transition is strongly enhanced by the hydrophobic spacers. Preliminary lectin binding tests via turbidimetric assay revealed that the spacers also significantly enhance the interaction of the glycopolypeptides with the lectin RCA120.
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INTRODUCTION Synthetic glycopolymers are important tools for the investigation and understanding of specific carbohydrate−protein interactions1 and promising materials for biomedical applications.2 Besides the variety of carbon backbone-based glycopolymers,3−5 glycopolypeptides have found recent interest due to their ability to form secondary structures and function as structural mimics of natural glycoproteins.6−8 A special focus has been laid on the synthesis and probing of glycopolypeptides that can respond to external stimuli, such as pH,9−11 temperature,11,12 or oxidation,13 by conformational changes. Our design of pH-responsive glycopolypeptides is based on the use of poly(α,L-glutamic acid) (PGA) as a scaffold, which is functionalized with sugars in a polymer-analogous reaction.10 The pH-responsive behavior of PGA (coil-to-helix transition in acidic medium) is well-known and has been investigated previously.14 Partially glycosylated polypeptides exhibit the same properties as long as the degree of functionalization is low. However, it was observed that the helicity at acidic pH significantly decreases and the transition becomes less distinct when the sugar density is increased. Apparently the glycosylated glutamate units disrupt the helical conformation in aqueous solution, probably due to the very hydrophilic groups near the polypeptide backbone.10 This behavior is unfavorable, because pH-sensitivity is an important feature in the design of responsive polymeric materials for biomedical applications, for example, for drug delivery.15 On the other hand, glycopolymers with high sugar content usually display a higher biological activity16−18 and therefore are interesting for use as multivalent ligands. By enhancing the helicity of © 2014 American Chemical Society
glycosylated PGAs in acidic medium it should be possible to create pH-responsive glycopolymers with high sugar content, that is, high biological activity. Efforts to enhance the helicity of polypeptides in aqueous medium have mainly been based on increasing the side-chain hydrophobicity,19 increasing the distance between the backbone and hydrophilic groups20 or incorporation of helix promoting amino acids in statistical copolypeptides.11 We decided to introduce hydrophobic spacers between backbone and carbohydrate moieties in order to increase both the sidechain hydrophobicity and the sugar-backbone distance, which should result in a higher helicity of the glycopolypeptides. Two alkyl spacers of different length (C6 and C11) and one aromatic spacer with similar length compared to the C6-spacer were chosen. The impact of this modification on the helix−coil transition was studied with PGA-based glycopolypeptides decorated with galactose moieties. Furthermore, it was checked if the change of the glycopolypeptide structure influences their interaction with lectins.
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EXPERIMENTAL SECTION
Materials. All chemicals were obtained from Sigma-Aldrich and used as received unless otherwise stated. γ-Benzyl-L-glutamate-Ncarboxyanhydride (BLG-NCA) was purchased from Isochem (France). β-D-Galactosylamine was obtained from Carbosynth (U.K.). Dialysis membranes (Spectra/Por 3, MWCO 3500) were purchased from Roth (Germany). Lectin from Ricinus communis Received: September 5, 2014 Revised: October 31, 2014 Published: November 3, 2014 4528
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Figure 1. Structures of the investigated glycopolypeptides without (1) and with hydrophobic spacer (2−4) (shown without hexylaminyl and acetyl end groups). (castor bean) Agglutinin RCA120 (buffered aqueous solution) was obtained from Sigma. DMF was obtained from Roth (Germany) and dried before use. DMSO was obtained from Acros Organics (Belgium). Instrumentation. 1H NMR spectra were recorded on a Bruker AV III-400 NMR-spectrometer (400 MHz). DMF-SEC was conducted on a set of PSS Gram columns (precolumn, 10 μm; 2× 1000 Å, 10 μm) with DMF containing LiBr (0.1 M) as the eluent at 60 °C and with a flow rate of 0.5 mL min−1. The injection volume was 100 μL with a sample concentration of 1 mg mL−1. Chromatograms were recorded with a refractive index detector (Shodex RI-101, 45 °C) and a 3-angle light scattering detector (Wyatt MiniDawn Tristar). Aqueous SEC was conducted on a set of PSS Suprema columns (precolumn, 10 μm; 100 Å, 10 μm; 10000 Å, 10 μm) with phosphate buffer (0.02 M, pH 7.4) containing NaCl (0.1 M) and 0.05% NaN3 as the eluent at 40 °C and with a flow rate of 1 mL min−1. The injection volume was 100 μL with a sample concentration of 1 mg mL−1. Chromatograms were recorded with a refractive index detector (Shodex RI-101, 35 °C) and an 18angle light scattering detector (Wyatt Dawn DSP). Synthesis of the Poly(α,L-glutamic acid) Precursor. Hexylamine initiated NCA-polymerization of BLG-NCA (M/I = 60) in DMF (5 d, N2, 0 °C) yielded poly(γ-benzyl-L-glutamate) (PBLG).21 The terminal amine groups were quenched with acetic anhydride. Yield: 95%. DMF-SEC [dn/dc = 0.122]: Mn = 13,200 g mol−1 [DP = 60], Đ ≤ 1.1. The PGA (sodium salt) precursor was obtained by deprotection of the PBLG with HBr (33% in AcOH) in TFA (90 min, RT)22 and subsequent deprotonation with NaHCO3. Yield: 91%. Aqueous SEC [dn/dc = 0.179]: Mn = 8100 g mol−1 [DP = 63], Đ ≤ 1.1. 1H NMR end group analysis: DP = 60. Synthesis of Glycopolypeptides. In a preceding reaction step hydrophobic spacers were attached to galactose by HBTU mediated amide coupling of the corresponding Fmoc-protected spacer to β-Dgalactosylamine and subsequent deprotection of the Fmoc-group according to a literature procedure.23 Glycopolypeptides were prepared by DMT-MM mediated aqueous amide coupling of the glycosyl compounds (β-D-galactosylamine, N-(6-aminohexanoyl)-β-Dgalactosylamine, N-(11-aminoundecanoyl)-β-D-galactosylamine, N-(4(aminomethyl)benzoyl)-β-D-galactosylamine) to the PGA (sodium salt) precursor according to a previously described protocol.10 For more details, see Supporting Information. CD Spectroscopy. CD spectra were recorded on a Jasco J-715 spectropolarimeter at ambient temperature. A quartz cell with 0.1 cm path length was used. For the pH-dependent measurements a stock solution of polypeptide (0.2 mg mL−1) in 0.01 M NaCl solution was titrated with aqueous HCl. Spectra were recorded between 250 and 190 nm. Each spectrum represents the average of five measurements. A baseline was taken from the pure solvent and subtracted from the
spectra. The spectra were smoothed using a Savitzky-Golay smoothing filter. Mean residue ellipticities were calculated using the equation: [Θ]MRW = (Θ × MMRW)/(10 × c′ × l) with experimental ellipticity Θ in mdeg, mean residue weight MMRW in g mol−1, mass concentration c′ in mg mL−1, and path length l in cm. Helicities fα were calculated from the mean residue ellipticities at λ = 222 nm using the following equation: fα = (−[Θ222]MRW + 3000)/39000.24 Lectin Binding Tests. Turbidimetric assays were conducted in HEPES buffered saline (HBS; pH 7.4) at room temperature on a Jasco V-630 UV/vis spectrophotometer using quartz cells with 10 mm path length according to the following protocol: Pure HBS is placed in the reference cell. 120 μL of a RCA120 solution (concn = 0.5 mg mL−1 = 3.8 μM) are placed in the sample cell and the absorbance is set to zero. Then the glycopolypeptide solution (concn = 0.5 mg/mL) is gradually added (1.5 μL) to the sample cell and the solution is mixed with a pipet. The turbidity is measured after 4 min at λ = 450 nm.
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RESULTS AND DISCUSSION Synthesis of Glycopolypeptides. All glycopolypeptides used in this study (Figure 1) were prepared via an aqueous amide coupling approach using the coupling agent DMTMM.10 This method is straightforward, versatile, and applicable even to sensitive amino sugars (e.g., glucosamine). The reaction is carried out in water and there is no need for any additive or buffer. The sugar density can be adjusted by the molar ratio of the coupling agent DMT-MM to PGA units. Five molar ratios were chosen to synthesize glycopolypeptides with varying sugar densities (Table 1). The resulting degrees of substitution (DS) with β-D-galactosylamine (1a−e) are a bit lower compared to other amino sugars used before [D-glucosamine10 and Dgalactosamine (data not shown)]. This could be either due to a Table 1. Sugar Densities of the Glycopolypeptides Determined by 1H NMR sample
molar ratio (PGA units/ DMT-MM)
DS (1a−e)
DS (2a−e)
DS (3a−e)
DS (4a−e)
a b c d e
1:2 1:1 1:0.5 1:0.25 1:0.1
0.59 0.44 0.29 0.16 0.06
0.60 0.38 0.22 0.11 0.05
0.80a 0.49 0.22 0.10 0.03
0.80 0.65 0.42 0.22 0.09
a
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Figure 2. Mean residue ellipticities of glycopolypeptides without spacer 1a−e (I), with C6 alkyl spacer 2a−e (II), with C11 alkyl spacer 3a−e (III), and with aromatic spacer 4a−e (IV) at different pH values taken from the corresponding CD spectra at λ = 222 nm.
lower reactivity of the amine or due to steric reasons (Dglucosamine and D-galactosamine can be in the open chain conformation, β-D-galactosylamine is restricted to the pyranose form). The sugar densities of the polymers with alkyl spacers (2, 3) are quite similar to 1, except for 3a, which has a significantly higher DS than 1a. However, the DS determined for 3a has an uncertainty, because it could not be determined by 1H NMR but was estimated gravimetrically (applying the average yield of the coupling reactions = 90%). The polymers with aromatic spacer (4) show the highest sugar densities for each molar ratio. All glycopolypeptides have a narrow molar mass distribution comparable to the PGA precursor (chromatograms from aqueous SEC are shown in the Supporting Information), which indicates that the galactose moieties are evenly distributed among the polypeptide chains and that the chains do not aggregate. The only exception is 3a that shows a shoulder at lower elution volume, which can be assigned to some aggregates. Interestingly, the glycopolypeptides with high DS are eluted slightly later than the PGA precursor and those with low DS. Apparently the hydrodynamic volume of the polymers is decreased, although the molecular weight is increased. This can be assigned to be a result of the loss of charged side groups and subsequent contraction of the polymer chain.
One can further observe a changed solution behavior after glycosylation. Unlike the PGA precursor, which precipitates below pH 3, most of the glycopolypeptides (1a−e, 2a−e, and 4a−e) remain soluble until at least pH 2.5 because of the hydrophilic sugars. However, 3a−e have a precipitation point, that is dependent on the amount of functional groups. Compound 3e (low DS) precipitates below pH 3, comparable to the PGA precursor. Compounds 3d, 3c, and 3b (medium DS) precipitate below pH 3.5 and 3a (high DS) below pH 4. The long alkyl spacer seems to overcome the hydrophilicity of the galactose moieties, so that the polypeptides become more hydrophobic after functionalization and precipitate when uncharged. Secondary Structure of Glycopolypeptides. The secondary structures of the PGA precursor and all glycopolypeptides were elucidated in aqueous medium by CDspectroscopy. For each polymer, CD-spectra were recorded at different pH values between pH 7 and pH 2.5 (all CD-spectra can be found in the Supporting Information). For the PGA precursor the typical pH-responsive transition from a coil at pH 7 (helicity = 0%) to a helix at lower pH (helicity = 91%) is observed. The isodichroic point at 204 nm indicates that the transition is a pure helix−coil transition without the presence of β-sheets.25 This isodichroic point is observed as well for all glycopolypeptides, whose CD-spectra significantly change during pH-titration (1b−e, 2a−e, 3b−e, 4b−e; see Supporting 4530
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Information). Thus, all transitions are helix−coil transitions. They occur within the pH range of 5.5 to 3.5 as can be seen from the mean residual ellipticities at λ = 222 nm, which are characteristic for the secondary structure (Figure 2). Transition points are shifted to lower pH values, when the sugar density is increased. At neutral pH all polymers (except for 3a, 4a, and 4b) adopt a coil conformation with the characteristic Cotton effects at ∼197 nm (negative) and ∼217 nm (positive). In the case of 4a and 4b, a partially helical conformation is observed (helicities of 42% and 19%, respectively). The spectrum of 3a cannot be clearly assigned to a certain conformation. Either another secondary structure element (e.g., β-sheet) is involved or the spectra are distorted due to the observed aggregation. At acidic pH the polymers (except for 1a and 3a) have a partially helical structure (Cotton effects at ∼190 nm (positive), ∼208 nm (negative), and ∼222 nm (negative)). Galactose functionalized PGA without spacer (sugar-backbone distance: 5 Å) displays a helix−coil transition in the sugar density range from 0.06 to 0.44 (1b−e). But the helicity at acidic pH decreases with increasing DS with the result that no pH-sensitivity is observed at a sugar density of 0.59 (1a). The highly glycosylated polypeptide has a coil conformation over the whole pH range investigated. Upon introduction of the C6 alkyl spacer (sugar-backbone distance: 14 Å) the transition is observed in the whole DS range investigated from 0.05 to 0.60 (2a−e). Even at the highest sugar density (2a), at which the glycosylated PGA without spacer (1a) is not helical at all, a significant transition takes place with a resulting maximum helicity of 42%. Furthermore, for each DS a higher helicity in acidic medium is observed when comparing 1 and 2 (see Figure 3). Remarkably, elongation of the hydrophobic alkyl spacer (C11, sugar-backbone distance: 20 Å) does not lead to a further enhanced helicity. Helix−coil transitions are observed from DS 0.03 to 0.49 (3b−e), but the helicities at acidic pH are very similar to 2. Compound 3a does not show a transition. Obviously, at an amount of only 20% remaining carboxylate groups the polypeptide secondary structure is not affected by changes in pH. Increasing the side-chain hydrophobicity by introduction of the aromatic spacer (sugar-backbone distance: 13 Å), which has about the same length as the C6 alkyl spacer but is more hydrophobic, has a significant impact on the polypeptide helicity. Glycopolypeptides 4 show transitions in the DS range from 0.09 to 0.65 (4b−e). At a higher sugar density no transition takes place (4a; only 20% remaining carboxylate groups, compare to 3a). The highest helicities at acidic pH are observed compared to the other glycopolypeptides at each DS (see Figure 3) and the polypeptide with the highest DS (4a) is still 59% helical. Even at neutral pH the polypeptides with high DS (4a and 4b) adopt a partially helical conformation, although there is still a significant amount of charged carboxyl groups. So, the α-helix is strongly promoted by the glycosylated benzyl glutamine units. In summary, the helicity of glycopolypeptides can be significantly enhanced by increasing the distance between the very hydrophilic sugars and the backbone. However, above a certain distance no further increase in helicity occurs. An additional enhancement can be achieved by increasing the hydrophobicity of the spacer. The general trend of a decreasing helicity with increasing DS is observed as well for
Figure 3. Helicity at pH 7 (I) and maximum helicity in acidic medium (II) of glycopolypeptides (without spacer [1], with C6 alkyl spacer [2], with C11 alkyl spacer [3], with aromatic spacer [4]) and PGA precursor as a function of sugar density (*secondary structure not clear).
glycopolypeptides with hydrophobic spacer, but much less pronounced than without spacer. Interaction with Lectin RCA120. The impact of sugar density and spacers on lectin recognition was estimated via turbidity measurements. The galactose decorated polypeptides are expected to recognize RCA120, a sugar binding protein consisting of two heterodimers connected by a disulfide bond, each having one binding site for galactose.26 A single glycopolypeptide chain (maximum linear dimension of PGA precursor: ∼22 nm, calculated with distance between two residues: 3.63 Å27) cannot span the distance between the two binding sites (∼100 nm28), but should be able to cluster and precipitate several RCA120 molecules. Since all polymers are obtained from the same precursor, different behavior in the turbidimetric assay can be directly related to the sugar density and the spacers. Therefore, the concentration-dependent change in turbidity was monitored over multiple additions of glycopolypeptide to an RCA120 solution.29,30 It was found that only glycopolypeptides with a sugar density higher than ∼0.40 precipitate the lectin. Probably, the interaction between glycopolypeptides with low DS and the RCA120 is too weak to induce clustering and precipitation, since carbohydrate− protein binding events are reversible. For glycopolypeptides 4531
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Figure 4. Turbidity after multiple additions of glycopolypeptides to an RCA120 solution (concn = 0.5 mg/mL in HBS, 3.8 μM; average of three independent measurements per polymer).
with a DS higher than ∼0.40, the concentration-dependent turbidimetric assays are shown in Figure 4. The turbidity increases after each addition of glycopolypeptide to the RCA120 solution until a plateau is reached where turbidity does not further increase with polymer concentration. Interestingly, in the case of 1a (DS 0.60) there is no turbidity observed below a concentration of 2 μM and no plateau was reached within the examined concentration range, whereas for glycopolypeptides with spacer and similar DS (2a, 4b), the plateau is already reached at this concentration. Apparently, lectin binding is enhanced by the presence of spacers, which probably lead to a better accessibility of carbohydrate moieties toward the lectin binding site. The optimum concentration for RCA120 binding of the different glycopolypeptides with spacer increases in the following order: 4a/4b (∼1.8 μM) < 2a (∼1.9 μM) < 4c (∼2.2 μM) < 3b (∼2.5 μM) < 3a (∼3.6 μM). Compounds 4a and 4b, both having a high sugar density, require the lowest concentration to bind the lectin. Compound 4c having a medium sugar density requires a higher concentration. So, as expected the glycopolypeptides with high sugar density are able to bind more lectin. It is possible that the partially helical secondary structure of glycopolypeptides 4a and 4b (helicity of 42% and 22%, respectively, at 0.2 mg/mL in HBS) has an impact on the turbidimetric assay. However, this cannot be verified by the experiments carried out with the set of glycopolypeptides available and therefore will be subject of future investigations. The behavior of 3a is a bit different. Although having the same sugar density as 4a, a much higher concentration is needed to precipitate the lectin. Probably there are less sugar moieties available for lectin binding because of the self-aggregation of 3a.
hydrophilic sugars and the polypeptide backbone. Aromatic spacers have a stronger impact than alkyl spacers of the same length, because of their higher hydrophobicity. The lectin binding is influenced by both sugar density and the presence of spacers. The glycopolypeptides with hydrophobic spacers show an enhanced interaction with the lectin RCA120 compared to glycopolypeptides without spacer. An increasing sugar density improves the interaction as well. These insights can be used for the design of pH-sensitive and biologically active glycoconjugates of more complex architectures.
CONCLUSIONS The introduction of hydrophobic spacers has a strong influence on both secondary structure and lectin binding of galactose decorated poly(α,L-glutamic acid)s. The helix−coil transition becomes more pronounced when spacers are present, especially at sugar densities above 40%, where glycopolypeptides prepared without spacer do not show a transition. The reason is an enhanced helicity in acidic medium. Hydrophobic spacers promote the helix formation by increasing the distance between
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ASSOCIATED CONTENT
S Supporting Information *
Experimental details for synthesis of glycosyl compounds and glycopolypeptides (incl. 1H NMR spectra and DS determination), SEC refractive index traces, and CD spectra of all polypeptides. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the IUPAC Transnational Call in Polymer Chemistry and the German Research Foundation (DFG; Grant: Me1057/17-1).
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