Post-Polymerization Modification of Poly(l-glutamic acid) with d-(+)-Glucosamine

Carboxyl functional groups of poly(L-glutamic acid) (PGlu) were modified with a D-(+)-glucosamine (GlcN) by amidation using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling reagent. The coupling reaction was performed in aqueous medium without protection of hydroxyl functional groups of D-(+)-glucosamine. Poly(L-glutamic acid) and GlcN functionalized polyglutamates (P(Glu-GlcN)) were thoroughly characterized by 1D and 2D NMR spectroscopy and SEC-MALS to gain detailed information on their structure, composition and molar mass characteristics. The results reveal successful functionalization with GlcN through the amide bond and also to a minor extent through ester bond formation in position 1 of GlcN. In addition, a ratio between the α- and β-form of glucosamine substituent coupled to polyglutamate repeating units as well as the content of residual dimethoxy triazinyl active ester moiety in the samples were evaluated.

The synthesis of glycopolymers by the post-polymerization modification approaches avoids the complex and tedious procedures required for glycosylated monomer synthesis and purification [3]. On the other hand, the post-polymerization strategies often suffer from incomplete glycosylation [23]. To circumvent these issues Deming and Kramer [32] reported highly efficient post-polymerization modification procedure for preparation of glycosylated poly(L-methionine). More often the click reactions are applied for glycosylation of the propargyl-or the azide-functionalized polymers with complementary modified carbohydrates. Various polymethacrylates were thus click-glycosylated [3,33,34]. Wu et al. [35] expanded the applicability of the azide-alkyne click chemistry to quantitative glycosylation of alkyne peripheral groups of dendrimers with azide-bearing sugars. The azide-alkyne, thiol-ene and thiol-yne reactions have been successfully applied also for efficient modification of polypeptides with carbohydrates [2,[36][37][38][39][40][41][42][43][44][45][46].
If quantitative glycosylation is not the objective, the sugar functionality is often introduced into the polymer via amide bond formation [23]. The amine side-chain groups of polylysine were reacted with glycosyl lactones under basic conditions to attach the sugar moieties to polylysine via the amide bonds [47]. Various condensation agents were reported for modification of polymers with carbohydrate and other biologically interesting substituents. Activation of carboxyl functional group was achieved by applying carbodiimides [48,49], N-hydroxysuccinimide ester [50], pentafluorophenyl ester [34] or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) coupling reagent. The DMTMM was applied to attach aminosugars to poly(acrylic acid), poly(methacrylic acid) [51], poly(L-glutamic acid) [52] and protein [49] scaffolds in good yields and with controllable degrees of substitution. Amphiphilic glycopolymers were shown to self-assemble in water [38,[53][54][55] and were for this reason studied for the application in RNA [56] and drug [57] delivery, and for construction of synthetic viral capsids [58]. Nanoparticles were formed from glycopolymers also by using phenylboronic acid moiety as reversible crosslinking agent [59]. These glycopolymer based nanoparticles were loaded with insulin and studied in vitro [60] and in vivo [61] for insulin delivery.
Well defined glycosylated acrylate block copolymers synthesized in a sequence-controlled fashion without post-polymerization modifications were prepared and successfully tested as competitive substrates for a C-type lectin [21]. In another study, a series of glycosylated methacrylamides were prepared [34]. Glycopolymers were synthesized with varying degrees of carbohydrate moiety substitution and with variable linker lengths between the carbohydrates and the methacrylamide backbone [34]. By applying a post-polymerization modification strategy, a galactose derivative was incorporated into the poly(methacrylamide) scaffolds. These glycopolymers were tested in bacterial-toxin and peanut agglutin inhibition experiments to reveal the impact of the glycopolymer characteristics on their inhibition potency [34].
Since the biocompatibility and biodegradability of glycopolymers are important criteria for their applicability in biomedical applications, synthetic glycopolypeptides are expected to be more appropriate for biomedical applications than glycosylated polymers bearing robust non-degradable carbon-carbon bonds in the backbone [2]. Recently, Mildner and Menzel [52] reported on the synthetic procedure for facile post-polymerization modification of poly(L-glutamic acid) (PGlu). PGlu was modified in water by coupling the amino sugar to the PGlu carboxylic functional groups in the presence of DMTMM as a coupling reagent. The resulting set of amino sugar modified polypeptides was characterized by 1 H-NMR and FTIR and the secondary structures of the obtained glycopolypeptides in solution were investigated by circular dichroism spectroscopy in the media with different pH values.
DMTMM has been applied as a coupling reagent for the preparation of glycopolymers [49,51] as well as for modification of carboxyl group-containing polysaccharides [62] and poly(L-glutamic acid) (PGlu) [63] with various amines in aqueous solution. Earlier, DMTMM was applied to modify poly(acrylic acid) with a wide range of amines bearing alkyl, hydroxyl, sulfonic acid or perfluoroalkyl groups [64]. DMTMM was thus shown to selectively promote amide bond formation in aqueous solution in the presence of non-protected hydroxyl and some other functional groups [51,64].
Research in our group is oriented toward the synthesis of polymeric carriers for delivery of biopharmaceuticals in the form of nanoparticles prepared by polyelectrolyte complexation methods. Recently, we have reported the preparation of nanoparticles highly loaded with a granulocyte colony-stimulating factor (GCSF) protein. The applied polymers were either chitosan grafted with PGlu chains [65] or PGlu modified with octyl hydrophobic substituents [66]. Complementary to study the effect of hydrophobic substituent of PGlu [66], our interest is to evaluate the effect of saccharide substituents on the interactions with GCSF protein and, consequently, nanoparticle formation.
Herein, we report the modification of PGlu carboxyl side groups with glucosamine hydrochloride (GlcN·HCl) using DMTMM as a coupling reagent which does not require the use of protection/deprotection chemistry for the saccharide's hydroxyl groups. The resulting copolymers P(Glu-GlcN) have been characterized by 1D and 2D NMR techniques to elucidate their detailed structure and composition and by SEC-MALS to determine the copolymers' molar mass averages and molar mass distribution.

Results and Discussion
BGlu NCA monomer was synthesized from BGlu starting material using triphosgene in dry THF (Scheme 1). The BGlu NCA was purified by recrystallization. ROP of BGlu NCA was initiated by hexylamine in dry DMF and the reaction mixture was kept in an ice-bath (Scheme 1) [67]. The conversion of BGlu NCA was monitored by disappearance of the signal for the NCA -NH-group at 9.1 ppm in the 1 H-NMR spectra of reaction aliquots. When the NCA was consumed, the reaction mixture was precipitated in ice-cold deionized water, the product collected by centrifugation and freeze-dried. The PBGlu was obtained in 94% yield and was characterized by 1 H-and 13 C-NMR spectroscopy, SEC-MALS and MALDI-TOF MS to confirm the proposed structure and to determine the molar mass characteristics ( Figure 1).   Figure 1c). The peak apex in MALDI-TOF mass spectrum of the PBGlu homopolymer belongs to the 36-mer and shows, beside the desired species with unmodified N-terminus and hexylamine C-terminus (signals A and C; potassium and sodium adducts), the presence of the species with the pyrrolidone and hexylamine end groups (signal B; potassium adduct), which are formed by intramolecular cyclization of the amino end group with the adjacent benzyl ester (Figure 1d). The structures of the corresponding species identified from the PBGlu MALDI-TOF mass spectrum are presented in Figure 2.  PBGlu was deprotected by applying HBr/acetic acid in TFA (Scheme 1). The obtained product was dissolved in saturated NaHCO3, purified by dialysis, and finally freeze-dried to obtain PGlu as sodium salt. PGlu was characterized by 1 H-and 13 C-NMR experiments, SEC-MALS and MALDI-TOF MS ( Figure 3).

Scheme 1. Synthetic pathway for the preparation of P(Glu-GlcN) copolymers.
According to the 1 H-NMR spectrum, the PGlu is quantitatively deprotected. The DPn determined by 1 H-NMR was calculated by comparing the integrals of the signals for the α-proton of the Glu moiety (signal g, 4.33 ppm) and the methyl group of the hexylamine initiator moiety (signal a, 0.86 ppm) (Figure 3a). Thus obtained PGlu DPn is 43 and is in good agreement with the DPn value of 42 determined by SEC-MALS (Figure 3c). Similar as MALDI-TOF mass spectrum of PBGlu, the mass spectrum of PGlu (Figure 3d) reveals the species corresponding to the PGlu chains with the unmodified amine N-terminus and the hexylamine C-terminus (signal D) as well as the PGlu species with the pyrrolidone and the hexylamine end groups (signal E). The peak apex belongs to the 36-mer as that in the mass spectrum of the benzyl-protected form (PBGlu). The structures of the corresponding species identified from the PGlu MALDI-TOF mass spectrum are presented in Figure 4.  The degree of substitution with GlcN (DG) was altered by the amounts of GlcN·HCl and DMTMM coupling reagent added. The GlcN·HCl was added in two-times molar excess relative to the DMTMM reagent ( Table 1). The obtained P(Glu-GlcN) copolymers were purified by dialysis. The P(Glu-GlcN) copolymers were characterized by NMR experiments in D2O and SEC-MALS using alkaline aqueous mobile phase (pH 10). The 45% P(Glu-GlcN) copolymer was additionally characterized in-depth by 13 C, COSY and gHSQCad NMR experiments in D2O. DG was determined by 1 H-NMR ( Figure 5) by normalizing the integral value of the signal for the PGlu α-CH proton to one (signal g, assigned to the α-CH proton of the unmodified Glu repeat units, and signal g', assigned to the α-CH proton of the modified Glu repeat units; both integrated for one proton at 4.32 ppm). The proton signals g and g' overlap, but the 13 C-and gHSQCad NMR data ( Figure 6) distinctly show the presence of two different signals for the corresponding carbon atoms. Then, a set of the signals belonging to GlcN substituents (signals 2-6, six protons between 3.48-3.94 ppm) was integrated and the obtained integral value was divided by six to determine the molar ratio between the GlcN substituent and the Glu backbone units. All three P(Glu-GlcN) samples show the DG of approximately one half of the targeted value (Table 1). Figure 6. (a) 13 C-NMR spectrum of 45% P(Glu-GlcN). (b) gHSQCad NMR spectrum of 45% P (Glu-GlcN). (c) COSY NMR spectrum of 45% P(Glu-GlcN). All NMR spectra were recorded in D2O.
In the 1 H-NMR spectra of the P(Glu-GlcN) copolymers ( Figure 5) a singlet j was observed at 4.03 ppm that corresponds to the OMe groups of the 4,6-dimethoxy-1,3,5-triazin-2-yl-activated ester moiety (Scheme 2), which were not aminolyzed by GlcN. Residual degree of substitution with triazinyl activated ester (DS(triazinyl)) was determined by dividing the integral value of the methoxy signal (signal j, six protons at 4.03 ppm) by six. The percent of residual triazinyl active ester moiety present in P(Glu-GlcN) copolymers was determined to be 1, 2 and 3 mol % for the 14% P(Glu-GlcN), 23% P(Glu-GlcN) and 45% P(Glu-GlcN) copolymers, respectively ( Table 1). The presence of the dimethoxy triazinyl ester moiety in P(Glu-GlcN) copolymers further confirms the 13 C-and gHSQCad NMR spectra (Figure 6a,b) for the 45% P (Glu-GlcN)). In the 13 C-NMR spectrum the signals at 58.8, 174.5 and 176.1 ppm are assigned to the methoxy carbon j, the triazine ring carbon n and the triazine ring carbons o, respectively (Figure 6a) [68], whereas the gHSQCad spectrum confirms the correlation between the methoxy protons signal j at 4.03 ppm and the corresponding carbon signal at 58.82 ppm (Figure 6b).
Additionally, a weak signal at relatively high chemical shift (1-ester signal, one proton at 6.54 ppm) was observed in the 1 H-NMR spectra of P(Glu-GlcN), which indicates a small degree of a side-product formation, i.e., the 1-ester signal due to the anomeric proton of GlcN substituent which is coupled to a Glu residue by an ester bond in position 1. The percent of the ester-coupled GlcN side-product relative to the Glu backbone residues was determined to be 1, 2 and 2 mol % for the 14% P(Glu-GlcN), 23% P(Glu-GlcN) and 45% P(Glu-GlcN) copolymers, respectively. The presence of the ester coupled GlcN substituents is further confirmed by the gHSQCad (Figure 6b) and COSY (Figure 6c) NMR spectra. In COSY spectrum a cross-peak (at 6.54 ppm, 4.13 ppm) is noticed and in gHSQCad the 1-ester anomeric proton signal (6.54 ppm) is correlated to the carbon signal at 97.2 ppm. This 1-ester carbon signal at 97.2 ppm lies between 1-α and 1-β carbon signals of the amide coupled GlcN substituents. Large downfield shift of the 1-ester anomeric proton signal is a consequence of coupling reaction with 1-OH of GlcN rather than -OH groups in other position (3, 4 or 6) since anomeric hydroxyl group is more reactive due to its lower pKa value [69][70][71][72].
It should be noted that complete hydrolysis of the active ester moiety residues of P(Glu-GlcN) copolymers can be achieved by treatment of the samples with 10% Na2CO3 aqueous solution, however, in addition to accompanying hydrolysis of the ester-conjugated GlcN substituents, the resulting P(Glu-GlcN) copolymers suffered from severe polypeptide backbone degradation as indicated by SEC-MALS (results not shown). The signals of the activated triazinyl ester moiety (signal j, 4.03 ppm) and the 1-ester signal at 6.54 ppm) can be observed also in the NMR spectrum of the analogous product reported by Mildner and Menzel [52].
The ratio between the α-and β-form of GlcN substituent coupled to Glu repeat units by the amide bond was also estimated. Since the 1-β signal at ~4.7 ppm in the 1 H-NMR spectra of P(Glu-GlcN) copolymers ( Figure 5) overlaps with the solvent signal, the proportion of the β-form was determined indirectly from the DG value (determined from the integral values of the 2-6 signals as described before), which was diminished by the integral values of 1-ester and 1-α signals. The 1-α integral value was then divided by the calculated 1-β value, and finally, the α/β ratios of 1.6/1, 1.3/1 and 1.7/1 for the 14% P(Glu-GlcN), 23% P(Glu-GlcN) and 45% P(Glu-GlcN), respectively, were determined.  The number-average molar masses ((Mn)calc.) of the synthesized glycopolypeptides were calculated from the P(Glu-GlcN) 1 H-NMR spectra based on the actual DG determined from the proton NMR spectra and the PGlu DPn of 43. The (Mn)calc. values are in good agreement with the Mn values determined by SEC-MALS (Table 1, Figure 8), indicating that under the applied experimental conditions the DMTMM coupling is a mild synthetic procedure which negligible impacts the polypeptide backbone degradation. In SEC-MALS chromatograms of P(Glu-GlcN) copolymers the peak slightly shifts toward larger elution volume with increasing molar mass, whereas the relative intensity of the light-scattering signal increases in the same order (Figure 8). These results indicate a change in P(Glu-GlcN) hydrodynamic volume with increasing the DG.

Size-Exclusion Chromatography Coupled to a Multi-Angle Light-Scattering Detector (SEC-MALS)
The SEC-MALS measurements were performed using an isocratic pump with an online vacuum degasser and an autosampler (all Agilent 1260 type, Agilent Technologies, Santa Clara, CA, USA). The separations of samples were carried out on a PolarGel-L column (Agilent Technologies, molar mass range: up to 30 kDa). Two different eluents were used: (i) 0.1 M sodium nitrate (NaNO3) solution with sodium azide (NaN3) (0.02% w/v) (both from Sigma-Aldrich), prepared with MiliQ water (18.2 MΩ/cm) at pH 10 for PGlu and P(Glu-GlcN) samples; and (ii) N,N-dimethylacetamide (DMAc) with 0.05 M lithium bromide (LiBr) (both from Sigma-Aldrich) for PBGlu sample. The detection was done through a system of successively on-line connected detectors: a multi-angle light-scattering DAWN-HELEOS detector (MALS with 18 angles), operating at a wavelength of 658 nm, and an interferometric refractive index (RI) detector Optilab rEX (both instruments from Wyatt Technology Corporation, Santa Barbara, CA, USA), operating at the same wavelength as the MALS detector. The nominal eluents flow rate was 1.0 mL/min. The mass of the samples injected onto the column was typically 1 × 10 −4 g, whereas the solution concentration was 1 × 10 −3 g/mL. The determination of absolute Mw and the calculation of Mn values from MALS detector require a sample-specific refractive-index increment (dn/dc), which was determined from the RI response assuming 100% of sample mass recovery from the column. For the data acquisition and evaluation Astra 5.3.4 software (Wyatt Technology Corporation) was utilized.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
The MALDI-TOF MS measurements were performed with an UltrafleXtreme MALDI-TOF-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with a 337 nm nitrogen laser, capable of executing reflector mode analysis. The reflector positive ion mode was used for acquiring the mass spectra of PBGlu and the reflector negative ion mode for the mass spectra of the PGlu. Dried-droplet method was used to spot the samples on a MALDI plate. The solutions of PBGlu (2 mg/mL), super DHB (20 mg/mL) and NaTFA (5 mM) in THF were mixed in a volume ratio of 1/10/1. PGlu was dissolved in water (2 mg/mL) and mixed with a solution of super DHB (20 mg/mL) in 30/70 (v/v) mixture of acetonitrile/aqueous 0.1% TFA (volume ratio: 1/10). In all cases 0.5 µL of the mixture was deposited on a MALDI target and allowed to dry on air. The mass spectra were acquired by summing spectra from ~500 selected laser shots. The calibration was made externally with a Peptide calibration standard II and Protein Calibration Standard I (Bruker Daltonik). The data were processed with Bruker FlexAnalysis 3.3.80 software.

Synthesis
A 250 mL flame-dried round-bottom flask was charged with BGlu (6.2 g, 26 mmol) and triphosgene (3.7 g, 13 mmol) under argon. Then, dry THF (130 mL) was added and the reaction mixture was stirred at 55 °C for 90 min. The clear reaction mixture was then concentrated under vacuum followed by precipitation in hexane. The product was crystallized three times from THF/hexane. Yield: 6.1 g (89%).

General Procedure for Modification of PGlu with GlcN (P(Glu-GlcN))
PGlu (1 mmol Glu units) was dissolved in deionized water (10 mL). The DMTMM and GlcN.HCl were added in three portions (one portion on three hours). The pH of the reaction mixture was adjusted to 8 by addition of saturated NaHCO3 after every addition of GlcN.HCl. The reaction mixture was stirred at room temperature for another 18 h. Then, it was transferred to dialysis bag (MWCO: 100-500 Da) and dialyzed against 0.1 M NaCl and against deionized water. After dialysis, the product was freeze-dried to obtain white fluffy solid. The ratio between PGlu, DMTMM and GlcN.HCl for the synthesis of differently substituted P(Glu-GlcN), the reaction yields and the molar mass characteristics of the products as determined by SEC-MALS are given below:

Conclusions
DMTMM was evaluated as a coupling reagent in post-polymerization modification of PGlu with GlcN to prepare P(Glu-GlcN) copolymers. Detailed characterization of the obtained P(Glu-GlcN) copolymers by 1D and 2D NMR experiments has shown successful functionalization of PGlu with GlcN via amide bond formation. The GlcN substituents were coupled to PGlu by the ester bond in position 1 to negligible extent. Additionally, up to 3 mol % of residual dimethoxytriazinyl active ester moiety were found to be present in all the obtained P(Glu-GlcN) copolymers. SEC-MALS results indicate negligible degradation of the polypeptide backbone during the glycosylation reaction, proving that DMTMM is a mild and effective amidation reagent.