Article pubs.acs.org/Biomac
Stimuli-Responsivity of Secondary Structures of Glycopolypeptides Derived from Poly(L‑glutamate-co-allylglycine) Kai-Steffen Krannig, Jing Sun,† and Helmut Schlaad* Max Planck Institute of Colloids and Interfaces, Department of Colloid Chemistry, Research Campus Golm, 14424 Potsdam, Germany ABSTRACT: Copolypeptides containing L-glutamate and various amounts of either D -/ DL-/L-allylglycine or D-/DL-/L-(3-(β-D glucopyranosyl)thio)propylglycine defect units were studied by circular dichroism (CD) and infrared (FT-IR) spectroscopy according to their secondary structures in dependence of pH and temperature. All samples adopt random coil conformation at high pH and α-helix at low pH without evidence for β-sheet formation. Folding into the α-helix structure is strongly affected by the number and configuration of allylglycine defects (which intrinsically stabilize β-sheets). Helix folding is facilitated upon the attachment of Dglucopyranose to the L- (but not the D-) allylglycine units, which is attributed to a different secondary structure preference of the L-(3-(βD-glucopyranosyl)thio)propylglycine (L: random coil; D: β-sheet) and a majority rule effect.
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INTRODUCTION Glycopolypeptides are an emerging class of materials, promoted by very recent advances in synthetic polypeptide chemistry1 and high potential usage, especially in life science applications, such as targeted drug delivery or diagnostics, or as model compounds or mimics to study the structural and functional properties of natural glycoproteins.2−5 Particularly interesting are stimuli-responsive glycopolypeptides, which can change their conformation or secondary structure in response to an external stimulus or change in environmental conditions. Recent examples include partially glycosylated poly(Lglutamate)s exhibiting helix-to-coil transitions triggered by pH,6,7 thermo-responsive poly(glyco-L-lysine)s,8 and oxidationresponsive poly(glyco-L-cysteine)s.9 Meanwhile, we have focused on the preparation of statistical copolypeptides combining the advantageous features of different functional amino acids.6,10 Particularly, glycosylated statistical copolypeptides of L-glutamate and allylglycine were found to undergo a transition from random coil to α-helix upon decreasing the aqueous solution pH, and the helices remained stable in acidic solution down to pH 3.5 (at which poly(Lglutamate) chains would long have precipitated).11 It was further recognized that the choice of the allylglycine enantiomer played a key role for the helicity and thus the quality of the secondary structure. Here we present a detailed analysis of the influence of different allylglycine enantiomers (D, DL , and L ), copolymer compositions (10−34 mol % allylglycine), and glycosylation on the helix formation of statistical copolypeptides with L-glutamate. Secondary structures of (non-) glycosylated copolypeptides were quantitatively analyzed by circular dichroism (CD) and infrared (FT-IR) spectroscopy. © 2014 American Chemical Society
EXPERIMENTAL METHODS
Materials. All chemicals were purchased from Sigma-Aldrich and used as received unless otherwise noted. γ-Benzyl-L-glutamate and trifluoroacetic acid (TFA) were supplied from Acros Organics (99+%), D-/DL-/L-allylglycine (>98%) from BoaoPharma, and triphosgene from Merck. Tetrahydrofuran (THF) was purchased from VWR and dried over sodium prior to use. Ethylacetate (EtOAc) was purchased from Th. Geyer GmbH and Co KG and dried over CaH2. Heptane was purchased from Roth (99%). Silica-gel came from Fluka and was dried at 150 °C for 48 h. Sodium salts of 1-thio-β-D-glucopyranose (>97%) and 1-thio-β-D-galactopyranose (>98%) were purchased from Carbosynth. 1-Hexylamine was supplied from Aldrich (99.5%) and distilled prior to use. N,N-Dimethylformamide (DMF, puriss.) and αpinene were used directly from the bottle under inert atmosphere. αMethyl-ω-amino-poly(ethylene oxide) (PEO-NH2) was received from Rapp Polymere GmbH. The NCAs of γ-benzyl-L-glutamate and allylglycine were made, as described earlier,6 following the Fuchs−Farthing method using triphosgene in THF solution at 50 °C. In the case of the allylglycine, α-pinene was used as a sacrificial HCl scavenger to strictly exclude any hydrochlorination of double bonds. The amino acid NCAs were purified by multiple precipitations from THF in heptane and isolated in good yields (50−60%). Copolypeptide Synthesis (General Procedure). Polymerization. γ-Benzyl-L-glutamate NCA and the respective amount of the (D-, DL-, or L-) allylglycine NCA were dissolved in little THF and transferred into a reactor. Upon removing the solvent by cryocondensation DMF was added to obtain a solution of 4% by weight. The reaction mixture was degassed by three consecutive freeze− pump−thaw cycles. The polymerization was initiated by addition of a Received: December 20, 2013 Revised: January 30, 2014 Published: February 3, 2014 978
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determined by 1H NMR spectroscopy and size exclusion chromatography (SEC), are summarized in Table 1. Samples
0.1 M solution of freshly distilled 1-hexylamine in DMF. The vessel was evacuated (0.5 mbar), and the reaction-mixture stirred at roomtemperature for 7 d. Each day, the polymerization was degassed twice to remove CO2. The polymerization was terminated by precipitation into 10× MeOH. The product (1) was collected by centrifugation and dried at 65 °C in high vacuo. 1H NMR (400.1 MHz, TFA-d): δ/ppm = 7.5−7.0, 5.7−5.5, 5.3−4.9, 4.8−4.4, 3.3, 2.8−1.7, 1.5, 1.3, 0.8. Debenzylation. To an ice-cold solution of copolypeptide 1 (1.0 eq relative to γ-benzyl-L-glutamate) in trifluoroacetic acid (TFA, 30 equiv) was added methanesulfonic acid (MSA, 34 equiv) and anisole (5 equiv) under vigorous stirring. The reaction mixture was stirred for 20 min at this temperature and then for an additional 40 min at room temperature. The polymer was precipitated from 10× Et2O and collected by centrifugation. The centrifugate was dissolved in 10% NaHCO3, extensively dialyzed (RC 1000) against Millipore water for 2 d and finally freeze-dried (→ 2). 1H NMR (400.1 MHz, D2O): δ/ppm = 5.7−5.6, 5.2−5.0, 4.4−4.0, 3.1, 2.5−2.3, 2.4−1.7, 1.6, 1.3, 0.8. Glycosylation. Copolypeptide 2, 1-thio-β-D-glucopyranose (or galactopyranose) (1.5 equiv with respect to allylglycine) (obtained from an aqueous solution of the sodium salt with DOWEX 50 after filtration and freeze-drying), and Irgacure 2959 (0.1 equiv with respect to allylglycine) were dissolved in 0.1 M acetate buffer (1.0 wt % with respect to allylglycine units) and put under argon. The vessel was sealed and placed under an UV-lamp (ExoTerra ReptiGlo 5.0 UVB 26W terrarium lamp) for 16 h. The reaction mixture was diluted and extensively dialyzed (RC 1000) against Millipore water for 2 d and finally freeze-dried (→ 3). 1H NMR (400.1 MHz, D2O): δ/ppm = 4.4, 4.3−3.9, 3.9−3.2, 3.1, 2.8−1.5, 1.4, 1.3, 0.8. Poly(ethylene oxide)-block-Polyallylglycine Synthesis. Polymerization. A solution of PEO-NH2 (Mn = 11150 g/mol, SEC with PEO calibration) and (D- or L-) allylglycine NCA (2 wt %, [NCA]0/ [NH2]0 = 20) in DMF was stirred at room temperature for 2 d. The product was precipitated multiple times into Et2O and isolated by filtration (→ 4). 1H NMR (400.1 MHz, TFA-d): δ/ppm =5.8−5.6, 5.4−5.1, 4.8, 4.6, 4.3−3.7, 2.8−2.6. SEC (NMP, PEO calibration): Mnapp = 13340 g/mol, (Mw/Mn)app = 1.03 (4a, D), Mnapp = 12620 g/ mol, (Mw/Mn)app = 1.04 (4b, L). Glycosylation. A solution of PEO-polyallylglycine 4a,b (2 wt % with respect to AGly units), 1-thio-β-D-glucopyranose (8 equiv (4a) or 3 eq (4b)), and benzophenone (0.1 equiv) in TFA was irradiated with UV-light for 2 d. The reaction mixture was diluted with water and extensively dialyzed (RC 1000) against Millipore water for 2 d and finally freeze-dried (→ 5a,b). 1H NMR (400.1 MHz, D2O): δ/ppm = 4.8, 4.4−3.7, 3.2−3.0, 2.4−1.8. Analytical Instrumentation. 1H NMR spectroscopy measurements were conducted at room temperature using a Bruker DPX-400 spectrometer operating at 400.1 MHz. TFA-d and D2O (SigmaAldrich) were used as solvents, and signals were referenced to the solvent signals at TFA δ = 11.52 ppm and D2O δ = 4.79 ppm. Size exclusion chromatography (SEC) with simultaneous UV/RI detection was performed with N-methyl-2-pyrrolidone (NMP + 5 wt % LiBr) as the eluent at 70 °C using a set of two 300 × 8 mm2 PSS-GRAM columns with average particle sizes of 7 μm and porosities of 100 and 1000 Å. Calibration was done using poly(methyl methacrylate) (PMMA) or poly(ethylene oxide) (PEO) standards (PSS, Mainz). CD spectroscopy was performed on a JASCO J-715 spectrometer with ∼0.02 wt % solutions of the polymers in Millipore water. Spectra were normalized to the concentration of the respective solution. FT-IR spectroscopy was conducted on a Thermo Scientific Nicolet iS5 FT-IR Spectrometer equipped with an iD5 ATR Accessory. Freeze-dried solid samples were measured in air at room temperature.
Table 1. Molecular Characteristics of Poly(γ-benzyl-Lglutamate) 1′ and Poly[(γ-benzyl-L-glutamate)-coallylglycine]s 1a−e
1′ 1a 1b 1c 1d 1e
composition (monomers)
xa
yb
Mnappc (kg/mol)
(Mw/Mn)appd
BGlu BGlu/D-AGly BGlu/DL-AGly BGlu/L-AGly BGlu/L-AGly BGlu/L-AGly
0 0.10 0.10 0.10 0.15 0.34
51 50 49 50 52 50
8.3 9.5 9.0 9.1 9.3 9.7
1.15 1.11 1.10 1.09 1.11 1.09
a Mole fraction of AGly (1H NMR). bAverage number of repeat units (1H NMR). cApparent number-average molar mass (SEC; PMMA calibration). dApparent ratio of weight- over number-average molar mass (= dispersity index) (SEC; PMMA calibration).
were quantitatively (>95%) debenzylated to yield poly(Lglutamate) 2′ and the poly[(L-glutamate)-co-allylglycine]s 2a− e, which were afterward glucosylated to yield the poly[Lglutamate-co-(3-(β-D-glucopyranosyl)thio)propylglycine]s 3a− e (Scheme 1, chemical structures confirmed by 1H NMR). Secondary Structures in Dependence of Solution pH. All copolypeptides 2a−e and 3a−e were readily soluble in water (neutral or basic pH) at a concentration of 0.02 wt % (no formation of aggregates of glycopolypeptide).6 Conformations or secondary structures of the samples in dependence of solution pH were analyzed by circular dichroism (CD) spectroscopy. Interpretation of the results is based on the assumption that the formation of secondary structures is governed by the L-glutamate units as the major component (see Table 1), and the glycine units are like defects along the copolypeptide chain. The poly(L-glutamate) 2′ (as a reference) undergoes reversible transition from α-helix to random coil in dependence of solution pH or, within a restricted pH range, temperature.12 At pH 6.5 or higher, poly(L-glutamate) adopts random coil conformation, which in CD is recognized by a positive Cotton effect with a maximum at λ = 218 nm. At pH 6 or lower, α-helices are formed as indicated by two minima at λ = 208 and 222 nm.13 The helicity of the peptide chains can be estimated by: α − helix(%) = ( −[θ ]222 + 3000)/39 000) × 100%
(1)
with [θ]222 as the value of the mean residual ellipticity at λ = 222 nm14 (not considering any chain length dependence of helicity).15 Notably, β-sheet contributions can be excluded as indicated by the occurrence of an isodichroistic point at λ = 204 nm in the CD spectra of 2′ at different pH.16 Figure 1 shows the CD spectra of 2a (D-AGly), 2b (DLAGly), and 2c (L-AGly), exhibiting the same composition and chain length but different configuration of allylglycine defect units, in a pH range between 7.5 and 4.75. The spectra confirm that the chains are in random coil conformation at high pH and fold into α-helices at low pH; the transition occurs at around pH 6 (like for the homopolymer). Below pH 4.75, the copolypeptides (as well as 2′) start to precipitate out of solution, due to the protonation of the glutamate units (pKa 4.3)13 rendering the chains hydrophobic. The maximum helicities at pH 4.75 were calculated to be 45% (2a), 42% (2b), and 59% (2c) (71% (2′)). Already the presence of 10%
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RESULTS AND DISCUSSION One homopolypeptide and five different copolypeptides, having virtually the same chain length but varying in composition and stereochemistry, were synthesized by statistical copolymerization of mixtures of γ-benzyl-L-glutamate (BGlu) and D-/DL-/Lallylglycine (AGly) N-carboxyanhydrides (NCAs).6 The molecular characteristics of the samples 1′ and 1a−e, as 979
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Scheme 1. Debenzylation and Glucosylation of Poly[(γ-benzyl-L-glutamate)-co-allylglycine]a
Reaction conditions: (a) MSA/anisole/TFA, 0-25 °C, 60 min; (b) 1-thio-β-D-glucopyranose, Irgacure 2959, hν, 0.1 M aqueous acetate buffer, 25 °C, 16 h. a
Figure 1. CD spectra of ∼0.02 wt % aqueous solutions of copolypeptides 2a−c (10 mol % allylglycine, different configuration) in dependence of solution pH.
Figure 2. CD spectra of ∼0.02 wt % aqueous solutions of copolypeptides 2c−e (10−34 mol % allylglycine, L-configuration) in dependence of solution pH.
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Figure 3. CD spectra of ∼0.02 wt % aqueous solutions of copolypeptides 3a−c (10 mol % (3-(β-D-glucopyranosyl)thio)propylglycine, different configuration) in dependence of solution pH.
Figure 4. CD spectra of ∼0.02 wt % aqueous solutions of copolypeptides 3c−e (10−34 mol % (3-(β-D-glucopyranosyl)thio)propylglycine, Lconfiguration) in dependence of solution pH.
Amazingly, 2e can still fold into a helix, albeit a disordered helix, despite the fact that in average every third unit along the poly(L-glutamate) chain presents an L-allylglycine defect, i.e., one defect per turn. Comparing all results for 2a−e seems to indicate that helicity is less affected by the number of allylglycine defects than by their configuration. The CD spectra of the glucosylated copolypeptides 3a−c (derived from 2a−c, respectively) at different pH are shown in Figure 3. These glucosylated samples exhibit a similar conformational behavior as poly(L-glutamate) (2′) and poly[(L-glutamate)-co-allylglycine] (2a−c), which is the formation of random coil at high pH 6 and α-helix at low pH (no β-sheet) with the transition occurring at pH 5.9−6.0. Due to the stabilizing effect of the hydrophilic glucose moieties, the pH can be lowered down to pH 3.6 without having collapse and precipitation of the helical chains.6 However, helicity reaches a constant or limiting value at pH 4 or below (see bottom CD
mole percent L-AGly units, i.e., every tenth L-glutamate unit being replaced by L-allylglycine, has noticeable impact on the comformation of the poly(L-glutamate) chain and reduces the helical content by 12%. Helicity further decreases, as expected, upon incorporation of either D-AGly (−26%) or DL-AGly (−29%). The presence of the AGly defects along the chain, on the other hand, does not promote the formation of β-sheets as indicated by the isodichroistic point at λ = 204 nm. The impact of the number of defects along the polypeptide chain on secondary structure and helicity is seen in the CD spectra of the copolypeptides 2c−e, which have the same chain length and all units L-configuration but different compositions (2c 10 mol %, 2d 15 mol %, and 2e 34 mol % L-AGly) (Figure 2). The maximum helicities were found decrease in the order of increasing number of L-allylglycine defects, i.e., 71% (2′), 59% (2c), 55% (2d), 47% (2e), as expected, which can be explained by a decreasing average length of L-glutamate segments. 981
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with the CD curve of a poly(L-amino acid) random coil). The presence of 4a,b β-sheets is also suggested by the CO (amide I) infrared absorption bands at ∼1626 cm−1 (solid state, spectra not shown).20 However, it should be considered that the conjugated PEO block may stabilize the secondary structure of the polypeptide, as has for instance been recognized for the αhelical structure of PEO-poly(L-lysine) block copolymers.21 The CD spectrum of 5b shows a curve with a minimum at λ = 200 nm and a maximum at λ = 218 nm, indicative of a random coil conformation (the relative intensity of the minimum is considerably lower than expected for a polypeptide random coil,16 due to the positive Cotton effect of D-glucose in the far-UV region). FT-IR analysis of the solid sample, however, suggests the presence of random coil/α-helix (broad amide I band at ∼1650−1680 cm−1, Figure 5).22 Evidently, the D-glucosylation of L-allylglycine units changes the preference toward formation of random coils (and α-helices) rather than β-sheets, which may be attributed to different spatial and solvated environments of the amino acid (glucose units are rather bulky and can bind considerable amounts of water). The situation appears to be, however, different in the case of 5a. Here, the CD curve shows two maxima at λ = 200 and 222 nm. Considering the contribution of the D-glucose, it is the second maximum that seems to indicate that 5a adopts a β-sheet conformation like its precursor 4a (FT-IR: β-sheet/α-helix, see Figure 5). The presence of a pendant D-glucose, however, may change the secondary structure preference of a poly(L-amino acid) chain but not, or to a lesser extent, that of a poly(D-amino acid) chain. (In this context, it would be interesting to see the impact of L-glucose side chains on the secondary structure of poly(D-/L-amino acid) chains; this is the subject of ongoing research.) This behavior is also reflected in the secondary structures and helicities of the copolypeptides 2a/3a (helicity: 45%/44%) and 2c/3c (59%/76%). Folding of the copoly(L-glutamate) chains into the α-helix structure is disturbed by the D-AGly and D-(3(β-D-glucopyranosyl)thio)propylglycine defects, to about the same extent, because both glycine units have a preference for βsheet (2a/3a). The D-glucosylation of L-allylglycine defects along a copoly(L-glutamate) chain, on the other hand, seems to change their secondary structure preference and facilitate folding of the polypeptide chain into a more ordered α-helical structure (2c/3c). The observation that L -(3-(β- D glucopyranosyl)thio)propylglycine units, despite their preference for random coil, are incorporated into the poly(Lglutamate) α-helix points to a majority rule effect.23 According to the majority rule, contrary to the sergeants and soldier principle,24 the conformation of the polypeptide chain is dominated by the majority component, here L-glutamate, over the minority component. This principle also seems to apply to the copoly(L-glutamate)s 3c−e, which have increasing amounts of L-(3-(β-D-glucopyranosyl)thio)propylglycine defects (10−34 mol %) but all fold into α-helices with virtually the same helicity (75−77%). Nevertheless, it cannot explain the limitation of the helical content to less than 80% (which is based on the assumption that the calculation of helical contents according to eq 1 is appropriate and correct). It is assumed that the limitation in helicity comes with the relatively short length of the chains (see above). It may be expected that copolypeptide samples with L-(3-(βD-glucopyranosyl)thio)propylglycine as majority component and L-glutamate as minority component would form random coils instead of helices. Unfortunately, this hypothesis could not
curves in Figure 3). Maximum helicities are found to be 44% (3a), 59% (3b), and 76% (3c) (at pH 4), and thus are similar to or considerably higher than the ones of 2a and 2b,c (+17%), respectively. Notably, 3c with all-L repeat units reaches a higher helical content (76% at pH 4.5) than the poly(L-glutamate) 2′ (71% at pH 4.75). It is further worth mentioning that the maximum helicity is barely affected by the nature of the sugar, galactose or glucose, as indicated by CD analysis of galactosylated samples (derived from 2a−c and thus directly comparable to 3a−c); helicities were found to be 41%, 55%, and 73%, respectively (data not shown).17 The observation that the helicity of 3c is higher than that of 2′ seems to suggest that the helical content increases with increasing number of L -(3-(β- D -glucopyranosyl)thio)propylglycine units. The results of CD analysis of the respective series 3c−e are shown in Figure 4. In fact, the glucosylation of L-allylglycine units leads to an increase of the helical content, i.e., +17% (2c → 3c), +20% (2d → 3d), and +30% (2e → 3e); however, all three samples 3c−e exhibit virtually the same maximum helicity of 75−77% (at pH 4) irrespective of composition. It appears that the helicity of the copolypeptide chains (built of ∼50 amino acid units) is limited to a value of ∼77% (71% for polyglutamate); higher helicities up to 100% may be achieved with longer chains (>100 units).7,8,15 The CD results for series 2a−e and 3a−e indicate that the incorporation of the allylglycine units interferes with the preferred folding of the poly(L-glutamate) chain into an α-helix. The glucosylated allylglycine units, on the other hand, seem to intrinsically stabilize an α-helical conformation of the polypeptide chain, especially when all amino acid units have the same configuration (see below). In order to examine the preferred secondary structure of the substituted glycine units, two poly(D-/L-allylglycine)s 4a (D, average number of repeat units, y = 16) 4b (L, y = 18, by 1H NMR), conjugated to PEO for better solubility, have been prepared and subsequently glucosylated to yield the poly[D-/L(3-(β-D-glucopyranosyl)thio)propylglycine]s 5a and 5b, respectively (Scheme 2).18 The glucosylated samples 5a,b were Scheme 2. Glucosylation of Poly(ethylene oxide)-blockPolyallylglycinea
a
Reaction conditions: (a) 1-thio-β-D-glucopyranose, benzophenone, hν, TFA, 25 °C, 2d.
completely soluble in water (
