Post-polymerization modification of poly(L-glutamic acid) with D -(+)-glucosamine

Article abstract of DOI:10.3390/molecules191219751

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.

Full text of DOI:10.3390/molecules191219751

Molecules 2014, 19, 19751-19768; doi:10.3390/molecules191219751
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Post-Polymerization Modification of Poly(L-glutamic acid)
with D-(+)-Glucosamine
Peter Perdih, Sašo Čebašek, Alenka Možir and Ema Žagar *
National Institute of Chemistry, Laboratory for Polymer Chemistry and Technology, Hajdrihova 19,
SI-1000 Ljubljana, Slovenia
* Author to whom correspondence should be addressed; E-Mail: ema.zagar@ki.si;
Tel.: +386-1-4760-203; Fax: +386-1-4760-300.
External Editor: Atsushi Sudo
Received: 30 October 2014; in revised form: 20 November 2014 / Accepted: 24 November 2014 /
Published: 27 November 2014
Abstract: 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.
Keywords: poly(L-glutamic acid); glucosamine; glycopolypeptide
1. Introduction
Carbohydrate moieties attached to a polymer backbone make the resulting copolymers applicable in
various biomedical applications since carbohydrate moieties play a key role in many biological

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processes, e.g., cell–cell contacts, signal transmission, inflammation and recognition [1–3]. Thus,
glycopolymers have been employed as macromolecular drugs [4], carbohydrate-based vaccines [5],
carriers for drug [6,7] and gene [8] delivery, hydrogels [9], agents for MRI visualization of specific
cell types [10] and as matrices for cell cultures [11–13]. Furthermore, synthetic glycopolymers were
studied as antifreeze glycoprotein mimics [14].
Glycopolymers have been synthesized by polymerization of glycosylated monomers or by
post-polymerization glycosylation of the precursor polymers [15,16]. Polymerization of glycosylated
monomers was conducted by various techniques, i.e., the controlled radical polymerization
techniques [3,17–21] and ring-opening polymerization (ROP) of glycosylated α-amino acid
N-carboxyanhydride (NCA) monomers [22–31].
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 [2,36–46].
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 [38,53–55] 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

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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 1H-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.
2. 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
1
9.1 ppm in the 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
1
freeze-dried. The PBGlu was obtained in 94% yield and was characterized by H- and 13C-NMR

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spectroscopy, SEC-MALS and MALDI-TOF MS to confirm the proposed structure and to determine
the molar mass characteristics (Figure 1).
Scheme 1. Synthetic pathway for the preparation of P(Glu-GlcN) copolymers.
Reagents and conditions: (i) Triphosgene, THF, 55 °C; (ii) [NCA]0:[hexylamine]0 = 50:1, DMF,
0 °C; (iii) HBr/AcOH, TFA, r.t., dialysis; (iv) DMTMM, GlcN·HCl (in three portions), water,
pH 8, r.t., dialysis.
Figure 1. (a) 1H-NMR spectrum of PBGlu recorded in DMSO-d6. (b) 13C-NMR spectrum
of PBGlu recorded in DMSO-d6. (c) Enlarged SEC-MALS chromatogram of PBGlu (solid
line: RI response, dashed line: LS response at angle 90°) together with the molar mass vs.
elution volume. (d) Enlarged MALDI-TOF mass spectrum of PBGlu at the peak apex,
recorded in reflector positive ion mode. Molecular weights are annotated. Inset shows
wider mass range of PBGlu mass spectrum.
The number-average degree of polymerization (DPn) of PBGlu was 41, as determined by 1H-NMR
spectroscopy by comparing the integrals of the signals for the α-proton of BGlu moiety (signal g,
3.92 ppm) and the methyl group of hexylamine initiator moiety (signal a, 0.82 ppm), (Figure 1a).
SEC-MALS chromatogram shows narrow molar mass distribution of the PBGlu homopolymer with

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ĐM of 1.06 and the Mn of 8.4 kDa that corresponds to the DPn of 38 (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.
Figure 2. Structures of the species as assigned from the PBGlu MALDI-TOF mass
spectrum (Figure 1d).
Figure 3. (a) 1H-NMR spectrum of PGlu recorded in D2O. (b) 13C-NMR spectrum of PGlu
recorded in D2O. (c) Enlarged SEC-MALS chromatogram of PGlu (solid line: RI response,
dashed line: LS response at angle 90°) together with the molar mass vs. elution volume.
(d) Enlarged MALDI TOF mass spectrum of PGlu at the peak apex, recorded in reflector
negative ion mode. Monoisotopic molar masses are annotated. Inset shows wider mass
range of PGlu mass spectrum.

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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
1
salt. PGlu was characterized by H- and 13C-NMR experiments, SEC-MALS and MALDI-TOF MS
(Figure 3).
According to the 1H-NMR spectrum, the PGlu is quantitatively deprotected. The DPn determined by
1H-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.
Figure 4. Structures of the species as assigned from the PGlu MALDI-TOF mass spectrum
(Figure 3d).
A post-polymerization modification to prepare the P(Glu-GlcN)s with different degree of GlcN
functionalization and most probably randomly distributed GlcN moieties along the PGlu backbone was
conducted in aqueous solution by portion-wise addition of DMTMM and GlcN.HCl reagents to the
PGlu aqueous solution (Schemes 1 and 2). The pH of the reaction mixture was adjusted after every
addition of the reagents to 8 by the addition of saturated NaHCO3 solution. The reaction mixture was
stirred for 24 h.
Scheme 2. Reaction scheme of DMTMM promoted amide coupling of GlcN and PGlu [64,68].
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)

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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
13C, COSY and gHSQCad NMR experiments in D2O.
Table 1. 1H-NMR and SEC-MALS results for the P(Glu-GlcN) copolymers.
Reaction Conditions
Glu:GlcN:DMTMM
1H-NMR
DS(triazinyl)
SEC MALS
(Mn)calc.
Yield
(%)
Sample Denotation
DG
(%)
-
Mn
Mw
(kDa)
ĐM
(%)
(kDa) (kDa)
PGlu
-
-
-
6.4
7.4
8.0
8.9
6.9
9.2
9.5
9.9
1.1
1.2
1.2
1.1
95
87
74
76
14% P(Glu-GlcN)
23% P(Glu-GlcN)
45% P(Glu-GlcN)
1:0.5:0.25
1:1:0.5
14
23
45
1
2
3
7.5
8.1
9.3
1:1.5:0.75
1
(Mn)calc. was calculated from the DS(GlcN) as determined from the H-NMR spectra of the P(Glu-GlcN)
copolymers and the PGlu DPn of 43. DG in mol % is the degree of carboxyl group modification with GlcN.
DS in mol % is the residual degree of substitution with triazinyl activated ester.
1
DG was determined by 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).
Figure 5. 1H-NMR spectra of P(Glu-GlcN) copolymers recorded in D2O.
The proton signals g and g' overlap, but the 13C- 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

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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).
13
Figure 6. (a) 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.
1
In the 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
13
dimethoxy triazinyl ester moiety in P(Glu-GlcN) copolymers further confirms the C- and gHSQCad
13
NMR spectra (Figure 6a,b) for the 45% P(Glu-GlcN)). In the 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
1
6.54 ppm) was observed in the 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

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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 [69–72].
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
1
bond was also estimated. Since the 1-β signal at ~4.7 ppm in the 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. For
1
comparison, the H NMR spectrum (recorded in D2O, 25 °C) of the starting GlcN.HCl material
(Figure 7) reveals the ratio between the α and β GlcN form of 3/1. The presence of the signal for the
GlcN 1-β anomeric proton is revealed in the gHSQCad (Figure 6b) and COSY (Figure 6c) spectra.
Figure 7. 1H-NMR spectrum of GlcN.HCl recorded in D2O.

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The number-average molar masses ((Mn)calc.) of the synthesized glycopolypeptides were calculated
1
from the P(Glu-GlcN) 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.
Figure 8. Enlarged SEC-MALS chromatograms (solid lines: RI responses, dashed lines:
LS responses at angle 90°) of P(Glu-GlcN) copolymers: 14% P(Glu-GlcN): blue curve,
23% P(Glu-GlcN): red curve and 45% P(Glu-GlcN): black curve, together with molar mass
vs. elution volume.
3. Experimental Section
3.1. Materials
All solvents and reagents were used as received. Dry tetrahydrofuran (THF, anhydrous, >99.9%),
dry N,N-dimethylformamide (DMF, anhydrous, 99.8%) and trifluoroacetic acid (TFA, 99%) were
obtained from Sigma-Aldrich (St. Louis, MO, USA). THF (p.a.), n-hexane (p.a.), diethyl ether (p.a.)
and NaHCO3 (>99.0%) were obtained from Merck (Darmstadt, Germany). γ-Benzyl-L-glutamate
(BGlu) (>99%), DMTMM (99+%) and hydrogen bromide (pure, 33 wt.% solution in glacial acetic
acid) (HBr/AcOH) were obtained from Acros Organics (Geel, Belgium). Triphosgene (>99.0%) and
n-hexylamine (99%) were obtained from Aldrich. D-(+)-Glucosamine hydrochloride (GlcN.HCl) (>99%,
BioReagent) was obtained from Sigma (Steinheim, Germany). NaCl (Ph Eur) was obtained from Fluka
(Buchs, Switzerland). Matrix and cationizing agent used for MALDI-TOF MS analysis, super DHB,
which is a 9/1 (w/w) mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid,
and sodium trifluoroacetate (NaTFA), respectively, were both obtained from Sigma-Aldrich.

Molecules 2014, 19
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3.2. Methods
3.2.1. NMR
1
1
1H-, 13C-, H-1H Correlation Spectroscopy (COSY) and H-13C gradient Heteronuclear Single
Quantum Coherence adiabatic version (gHSQCad) spectra were recorded in DMSO-d6 or in D2O on a
Unity Inova 300 instrument (Varian, Palo Alto, CA, USA) in the pulse Fourier-transform mode
with a relaxation delay of 5 s, and an acquisition time of 3 s. Tetramethylsilane (TMS, δ = 0) and
3-trimethylsilyl-2,2',3,3'-d4-propanoic acid sodium salt (TMSPA, δ = 0) were used as an internal
chemical-shift standards in DMSO-d6 and D2O, respectively.
3.2.2. 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.
3.2.3. 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

Molecules 2014, 19
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standard II and Protein Calibration Standard I (Bruker Daltonik). The data were processed with Bruker
FlexAnalysis 3.3.80 software.
3.3. Synthesis
3.3.1. γ-Benzyl-L-glutamate NCA (BGlu NCA)
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%).
3.3.2. Poly(γ-benzyl-L-glutamate) (PBGlu)
BGlu NCA (5.0 g, 19 mmol) was dissolved in dry DMF (62 mL) under argon in an ice-bath. A
solution of hexylamine (0.38 mmol) in dry DMF (1 mL) was added and the reaction mixture was
stirred in ice-bath for 2 days. Then, the reaction mixture was precipitated into cold deionized water.
The product was collected by centrifugation, washed with water several times and freeze-dried to obtain
white powdery material. Yield: 3.9 g (94%). SEC-MALS: Mn = 8.4 kDa, Mw = 8.9 kDa, ĐM = 1.06.
3.3.3. Poly(sodium-L-glutamate) (PGlu)
PBGlu (1.0 g, 4.7 mmol) was dissolved in TFA (25.6 mL) under argon. HBr/AcOH (33 wt.%)
(3.24 mL, 18.7 mmol) was slowly added. Reaction mixture was stirred for 90 min at room temperature
under argon. The deprotected polymer was precipitated in cold diethyl ether and collected by
centrifugation. The product was dissolved in saturated NaHCO3, dialyzed (MWCO: 100–500 Da)
against deionized water and finally freeze-dried to obtain PGlu as sodium salt. Yield: 0.67 g (95%).
Debenzylation: quantitative. SEC-MALS: Mn = 6.4 kDa, Mw = 6.9 kDa, ĐM = 1.08.
3.3.4. 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:
14% P(Glu-GlcN): PGlu (1 mmol Glu units), DMTMM (0.25 mmol), GlcN.HCl (0.5 mmol). Yield:
161 mg (87%). SEC-MALS: Mn = 7.4 kDa, Mw = 9.2 kDa, ĐM = 1.2.

Molecules 2014, 19
19763
23% P(Glu-GlcN): PGlu (1 mmol Glu units), DMTMM (0.50 mmol), GlcN.HCl (1.0 mmol). Yield:
164 mg (74%). SEC-MALS: Mn = 8.0 kDa, Mw = 9.5 kDa, ĐM = 1.2.
45% P(Glu-GlcN): PGlu (1 mmol Glu units), DMTMM (0.75 mmol), GlcN.HCl (1.5 mmol). Yield:
193 mg (76%). SEC-MALS: Mn = 8.9 kDa, Mw = 9.9 kDa, ĐM = 1.1.
4. 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.
Acknowledgments
The authors gratefully acknowledge the financial support of the Ministry of Higher Education,
Science and Technology of the Republic of Slovenia through the Slovenian Research Agency
(Program P2-0145 and Project L2-4166) and the Centre of Excellence, Polymer Materials and
Technologies, for access to the MALDI-TOF MS.
Author Contributions
E. Žagar and P. Perdih designed research and synthetic approach, analyzed the data and wrote the
paper; P. Perdih performed synthesis and NMR, A. Možir SEC-MALS, S. Čebašek MALDI-TOF MS
analysis. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds PBGlu, PGlu and P(Glu-GlcN) are available from
the authors.
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