M. Biagiottia,
G. Borghesea,
P. Francescatoa,
C. F. Morellia,
A. M. Albertinib,
T. Bavaroc,
D. Ubialicd,
R. Mendichie and
G. Speranza*ad
aDipartimento di Chimica, Università degli Studi di Milano, via C. Golgi 19, I-20133 Milano, Italy. E-mail: giovanna.speranza@unimi.it
bDipartimento di Biologia e Biotecnologie “L. Spallanzani”, Università degli Studi di Pavia, via A. Ferrata 9, I-27100 Pavia, Italy
cDipartimento di Scienze del Farmaco, Università degli Studi di Pavia, viale T. Taramelli 12, I-27100 Pavia, Italy
dISTM-CNR, via C. Golgi 19, I-20133 Milano, Italy
eISMAC-CNR, via E. Bassini 15, I-20133 Milano, Italy
First published on 26th April 2016
Poly(γ-glutamic acid) is a linear anionic biopolymer synthesized by bacterial fermentation from sustainable resources. Being water soluble, biodegradable, edible and non-toxic to humans and the environment, applications of γ-PGA are of interest in a broad range of industrial sectors. However, preparation of γ-PGA derivatives is plagued by several difficulties including its scarce solubility in organic solvents. We here report a γ-PGA derivatization procedure based on the use of its tetrabutylammonium salt. The modified solubility of γ-PGA provided by counterion exchange led to the synthesis of poly(α-ethyl-γ-glutamate), poly(α-benzyl-γ-glutamate) and poly(α-n-butyl-γ-glutamate) under smoother conditions and an almost complete functionalization degree.
Among others, γ-PGA has been proposed as flocculant and chelating agent in waste-water treatment, cryoprotectant and texture enhancer in food industry, moisturizer in cosmetic products, biological adhesive and drug carrier in medicine, and so on.3,4
Even more interesting perspectives arise from the possibility to chemically modify the polymer in order to modulate its chemical–physical properties and create new materials suitable for technological as well as biomedical applications.5
The synthesis of esters or amides from the free α-carboxylic groups is the most commonly used approach to attain chemically modified γ-PGA derivatives.5
However, this is a quite challenging task, given the peculiarities of γ-PGA, i.e. its scarce solubility, the high viscosity of its solutions and the inherent low chemical reactivity of the α-carboxylic side groups which are highly hindered.5 A further constraint is frequently represented by isolation and purification of the obtained products.
To date, the preparation of γ-PGA esters can be achieved by the procedure firstly reported by Kubota and coworkers.6,7 This protocol relies on the reaction of γ-PGA with alkyl halides in the presence of sodium hydrogencarbonate in an organic solvent such as DMF, DMSO or NMP at 60 °C (Scheme 1a). Under these conditions, esterification of γ-PGA with several linear alkyl halides, up to dodecyl and including dihalogenoalkanes, was carried out.5 The reaction outcome was found to be dependent on the size of the alkyl group, being better results obtained with short-chain alkyl halides.7 The derivatization step must be usually reiterated in order to achieve a high degree of functionalization. The possibility of a reduction in molecular weight upon prolonged reaction under these conditions has been also reported.8,9
To overcome these limitations, a two-step esterification method was developed,8 which was proved to be efficient also for the preparation of poly(γ-glutamate)s with medium or long alkyl chain (from 12 to 22 carbon atoms)10 as well as with mono-, di- and tri-ethylene glycols.11 The method consists in submitting poly(α-methyl-γ-glutamate) or poly(α-ethyl-γ-glutamate), easily obtained by the method already described, to transesterification reactions with the appropriate alcohol in the presence of Ti(OBu)4 (Scheme 1b).
However, the scarce solubility of γ-PGA in organic solvents strongly narrows the broader application of these methodologies.
Following an established strategy used in polysaccharide chemistry to prepare hyaluronic acid derivatives as well as organic solvent-soluble salts of cellulose sulfate,12,13 we here report an efficient method aimed at increasing the solubility of γ-PGA by exchanging its counterion by a quaternary ammonium salt. Such an exchange was proved to be beneficial on esterification reaction as a result of reactivity enhancement, also assisting γ-PGA manipulation.
All other reagents and resins were purchased from Sigma-Aldrich (Milan, Italy) and/or from VWR International (Milan, Italy) and were used without further purification. All solvents were of HPLC grade and, when necessary, they were dried on molecular sieves (3 Å). 1H NMR spectra were acquired at 400.13 MHz in D2O or in DMSO-d6 on a Bruker Advance 400 spectrometer (Bruker, Karlsruhe, Germany) interfaced with a workstation running Windows operating system and equipped with a TopSpin software package. Chemical shifts are given in ppm (δ) and are referenced to TSP [3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt, δMe 0.00 ppm] as external standard or to DMSO signal as internal standard (δH DMSO 2.50 ppm).
The functionalization degree was determined by 1H NMR analysis, in particular measuring the ratio of intensities of the backbone α-CH signal at about 4.2 ppm and of the side-chain OCH2 signal in the interval 4.04–5.1 ppm.
The SEC-MALS experimental conditions were selected on the basis of samples solubility. γ-PGA Na salt (2) and acid form (1): columns, 2 ultrahydrogel (2000–500 Å) from Waters; mobile phase, 0.1 M NaCl + 0.1 M phosphate buffer pH 7.0; temperature, 35 °C; flow rate, 0.8 mL min−1; injection volume, 150 μL; sample concentration, about 1 or 5 mg mL−1.
γ-PGA TBA salt (3): columns, 2 TSKGel (G4000–G3000) from Tosoh; mobile phase, 0.1 M NaCl + 0.1 M phosphate buffer pH 7.4; temperature, 35 °C; flow rate, 0.8 mL min−1; injection volume, 100 μL; sample concentration, about 2 mg mL−1. γ-PGA esters (4–6): columns, 2 PLgel Mixed C from Polymer Laboratories; mobile phase, DMF + 0.05 M LiBr; temperature, 50 °C; flow rate, 0.8 mL min−1; injection volume, 100 μL; sample concentration, about 4 mg mL−1.
1H NMR (400 MHz, DMSO-d6) δ 0.94 (t, J = 7.2 Hz, 12H, –NCH2CH2CH2CH3), 1.27–1.37 (m, 8H, –NCH2CH2CH2CH3), 1.55–1.62 (m, 8H, –NCH2CH2CH2CH3), 1.67–1.82 (broad m, 2H, –CH2CH2CO–), 1.86–1.97 (broad m, 1H, –CH2CH2CO–), 1.99–2.10 (broad m, 1H, –CH2CH2CO–), 3.18–3.24 (m, 8H, –NCH2CH2CH2CH3), 3.55–3.63 (broad m, 1H, –CHCOO), 6.99 (broad d, J = 4.8, 1H, –CONH).
13C NMR (100 MHz, DMSO-d6) δ 13.93 N(CH2CH2CH2CH3), 19.67 (N(CH2CH2CH2CH3)4), 23.55 (N(CH2CH2CH2CH3)4), 28.13 (CHCH2CH2CO), 32.42 (CHCH2CH2CO), 52.85 (CHCH2CH2CO), 58.07 (N(CH2CH2CH2CH3)4), 172.07 (CONH); 174.33 (COO).
A higher functionalization degree (72%) was achieved by reacting this material under the same conditions for further 2 days. 270 mg of 4 (26% yield, Mw 33.4 kg mol−1) were obtained. NMR data: in agreement with those previously reported.7
Once the additions were completed the solution was stirred at r.t. for 36 hours. The reaction mixture was diluted with EtOH (10 mL) and neutralized with a saturated NaHCO3 solution (15 mL). After removal of EtOH under reduced pressure, the residue was worked-up as above to give 30 mg of 4 as a white powder (70% yield, functionalization degree: 100%, Mw 21.8 kg mol−1). NMR data: in agreement with those previously reported.7
Solubility tests carried out on 3 as well as on γ-PGA (1) and γ-PGA sodium salt (2) (see Table 1) indicated that the presence of the TBA counterion improves the solubility of the biopolymer in organic solvent, particularly in the case of aprotic polar solvents such as DMF and DMSO, and short-chain alcohols (MeOH, EtOH, n-PrOH). However, γ-PGA TBA salt (3) still remains insoluble in ethers and branched alcohols (iso-PrOH and t-BuOH).
Solvent | γ-PGA (1) | γ-PGA Na (2) | γ-PGA TBA (3) |
---|---|---|---|
a Each test was performed by weighting an amount of about 15 μmol of substance (corresponding to 2.0 mg, 2.3 mg and 5 mg of 1, 2 and 3, respectively) and adding stepwise 50 μL of the selected solvent till complete dissolution or up to a final volume of 2 mL, under magnetic stirring at room temperature for 8 hours. Legend: −, insoluble; +, soluble; ++, very soluble; ±, sparingly soluble. | |||
MeOH | − | − | + |
EtOH | − | − | + |
n-PrOH | − | − | + |
i-PrOH | − | − | − |
n-BuOH | − | − | ± |
t-BuOH | − | − | − |
Benzyl alcohol | − | − | + |
2,2,2-Trifluoroethanol | − | − | + |
Diethyl ether | − | − | − |
Dioxane | − | − | − |
Acetonitrile | − | − | − |
DMF | − | ± | ++ |
DMSO | ± | ± | ++ |
To evaluate the effect of the solubility enhancement on reactivity, we investigated the esterification of the biopolymer and, specifically, the preparation of poly(α-ethyl γ-glutamate) (4) as a reference reaction. Poly(α-alkyl-γ-glutamate)s are among the most promising derivatives of PGA e.g. alkyl esters of PGA display improved thermal and mechanical properties with respect to the polyacid and have been investigated for their capability to form biodegradable fibers and films that can replace currently used non-biodegradable polymers.2,5
Two methodologies were used. The former method (namely, A) is the one already described above, firstly reported by Kubota5–7 and based on carboxylate chemistry, which was found to be effective when γ-PGA sodium salt (2) is used as the starting material (see Scheme 3, Method A).
![]() | ||
Scheme 3 Alkylation of γ-PGA salts with ethyl bromide (Method A) and esterification with oxalyl chloride-catalytic DMF and alcohol (Method B). |
The latter route (namely, B) was set up in our laboratories and consists in the in situ formation of the acyl chloride of poly(γ-glutamic acid) by treatment of both 2 and 3 with oxalyl chloride in the presence of a catalytic amount of dimethylformamide (DMF),15 followed by direct esterification with the proper alcohol (see Scheme 3, Method B).
Using γ-PGA sodium salt (2) as the starting material our strategy appears advantageous with respect to the Kubota's methodology in terms of reaction time (120 hours vs. 36 hours) and final yield (26% vs. 80%). Nevertheless, the functionalization degree is far to be excellent (72% vs. 22%, see Table 2, entries A1 and B1).
Methoda | Producta | T (°C) | Time (h) | Yield (%) | Functionalization degreeb (%) |
---|---|---|---|---|---|
a See Scheme 3.b The functionalization degree was determined by 1H NMR analysis, in particular measuring the ratio of intensities of the backbone α-CH signal at about 4.2 ppm and of the side-chain OCH2 signal in the interval 4.04–5.1 ppm.c Recovery due to the presence of solvent residues. | |||||
A1 | 4 | 45 | 120 | 26 | 72 |
A2 | 4 | 45 | 72 | 99 | 100 |
B1 | 4 | r.t | 36 | 80 | 22 |
B2 | 4 | r.t. | 36 | 70 | 100 |
5 | r.t. | 36 | 63c | 100 | |
6 | r.t. | 36 | 45 | 99 |
Contrariwise, when γ-PGA TBA salt (3) was used as the starting material in both procedures, an overall improvement of the reaction outcome was observed in terms of reaction time as well as yield and functionalization degree. In the case of Method A, the reaction time was reduced from 120 to 72 hours and, mostly important, both the functionalization degree and the yield were almost complete (cf. entries A1 and A2 in Table 2).
On the other hand, the use of γ-PGA TBA salt (3) in the procedure B, under the same experimental conditions (r.t., 36 hours), afforded an almost complete functionalized product (4), although with a slightly lower yield (cf. Table 2, entries B1 and B2). It is also worth noting that Method B does not require any heating.
Since the acyl chloride-based approach gave promising results for the preparation of poly(α-ethyl γ-glutamate) (4), we extended our methodology to the synthesis of other poly(α-alkyl γ-glutamate)s, in particular benzyl and n-butyl derivatives (5 and 6, respectively). These esters were chosen taking into account the different solubility of the γ-PGA TBA (3) in benzyl and n-butyl alcohols (Table 1). For both 5 and 6 a very high functionalization degree was achieved even if accompanied by a moderate yield in the case of 6. The effect of the lower solubility of 3 in n-butyl alcohol cannot be ruled out.
Footnote |
† Electronic supplementary information (ESI) available: Molecular weight distribution (MWD) of γ-PGA (1) and γ-PGA derivatives (2–6), 1H and 13C NMR of γ-PGA TBA salt (3), 1H NMR data of poly(α-ethyl γ-glutamate) (4), poly(α-benzyl γ-glutamate) (5) and poly(α-n-butyl γ-glutamate) (6). See DOI: 10.1039/c6ra08567a |
This journal is © The Royal Society of Chemistry 2016 |