Nanoparticles Based on Novel Carbohydrate-Functionalized Polymers

Polymeric nanoparticles can be used for drug delivery systems in healthcare. For this purpose poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) offer an excellent polymeric matrix. In this work, PLGA and PEG polymers were functionalized with coumarin and carbohydrate moieties such as thymidine, glucose, galactose, and mannose that have high biological specificities. Using a single oil in water emulsion methodology, functionalized PLGA nanoparticles were prepared having a smooth surface and sizes ranging between 114–289 nm, a low polydispersity index and a zeta potential from −28.2 to −56.0 mV. However, for the corresponding PEG derivatives the polymers obtained were produced in the form of films due to the small size of the hydrophobic core.


Introduction
Described as the manipulation of atomic matter, nanotechnology was described theoretically in the 1960s by Richard Feynman, and the practice emerged a decade later. After Taniguchi's, Drexler's, and other scientist's valuable contributions, nanomedicine has developed [1,2] and recently, the three main applications of nanomedicine are in tissue engineering, nanoprobes, and nanoparticles for drug delivery.
Suitable nanoparticles are capable of transporting drugs in a targeted manner to a specific tissue, cell or organ, minimizing the toxic effects and high therapeutic doses inherent in most current pharmacological treatments [3][4][5]. Recent advances in drug encapsulation and/or delivery of nanoparticles have demonstrated the enormous potential that these nanomaterials can have in healthcare, and their ability to improve the pharmacokinetic and pharmacodynamic properties of an active ingredient, thereby increasing the effectiveness of treatment and reducing the toxicity to patients [6].
Depending on the application (diagnosis, imaging, or therapy), different types of nanoparticles have been proposed, and these can be divided into two main groups: organic and inorganic nanoparticles. In the first group are dendrimers, liposomes, and polymeric nanoparticles. The second group includes quantum dots, silica, gold, and silver nanoparticles. Due to the high rate of tissue accumulation, that can result in toxicity problems, nanoparticles that cannot be degraded by the body are not as popular and attractive as biodegradable and biocompatible polymeric nanoparticles (PNPs) [7,8].
PNPs play an important role in therapeutic applications, such as encapsulation and controlled drug release [9,10]. The PNP matrix, size [11][12][13], surface area [14], shape [15,16], and surface electrical Propargylation reactions of 1, and other commercially available coumarins, namely umbelliferone, 3-carboxylic acid coumarin, and 4-methyl-7-hydroxy coumarin, are presented in Scheme 2. The propargylation conditions used were the ones already reported in the literature [34,35]. Alkyne coumarins 2, 4, 5, and 6 were obtained in excellent yields, and pure enough for further reactions. NMR and FT-IR spectra (see Supplementary Materials for 1 H, 13 C and MS/MALDI-TOF spectra for novel synthesized compounds) were in accordance with those already reported in the literature. For propargylated coumarin 4, methylene protons were observed at 4.77 ppm, and the alkyne proton at 2.58 ppm. Alkyne stretch bands were observed at 3266 and 2119 cm −1 . Coumarin 5 presented the methylene proton signal at 4.95 ppm and the alkyne proton at 2.56 ppm. FT-IR spectrum presented the alkyne stretches at 3315 and 2117 cm −1 . Ester hydrolysis of 2 was accomplished with sodium hydroxide in ethanol at 80 • C, affording 3 in 95% yield.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 21 Alkyne coumarins 2, 4, 5, and 6 were obtained in excellent yields, and pure enough for further reactions. NMR and FT-IR spectra (see Supplementary Materials for 1 H, 13 C and MS/MALDI-TOF spectra for novel synthesized compounds) were in accordance with those already reported in the literature. For propargylated coumarin 4, methylene protons were observed at 4.77 ppm, and the alkyne proton at 2.58 ppm. Alkyne stretch bands were observed at 3266 and 2119 cm −1 . Coumarin 5 presented the methylene proton signal at 4.95 ppm and the alkyne proton at 2.56 ppm. FT-IR spectrum presented the alkyne stretches at 3315 and 2117 cm −1 . Ester hydrolysis of 2 was accomplished with sodium hydroxide in ethanol at 80 °C, affording 3 in 95% yield. In some cases, a linker was necessary in order to bond the coumarin, or the carbohydrate, moiety to the polymer and for these cases compound 7 was prepared. Due to its high volatility and reactivity, 7 was always maintained in diethyl ether, even for its quantification and characterization. Subsequent triazole ring formation with coumarin 6, afforded 8 in good yield (Scheme 3). Scheme 3. Triazole linker 8 was prepared by the substitution of bromide in 2-bromoethylamine hydrobromide using sodium azide, followed by cycloaddition to form the 1,2,3-triazole ring.

Ligand Preparations-Carbohydrates
Specific carbohydrate derivatization was challenging due to the presence of several hydroxyl groups having similar reactivities. However, depending on the reaction conditions, the anomeric hydroxyl can be preferential replaced. Furthermore, in order for a carbohydrate to be recognized by lectins, it often must be non-reducing.
Insertion of a triazole ring between the carbohydrate moiety and the coumarin/polymer moieties not only increases the linkage strength, but mimics the amide group, which can induce, or improve, the biological activity of the final compounds. Propargylation of glucose, galactose, and mannose (Scheme 4) using immobilized sulfuric acid on silica [36], afforded the necessary terminal alkynes, 9-11, for posterior cycloaddition reactions. The propargylation yields obtained were poor to moderate (38%-68%), and the products were mixtures of α and β anomers, with a 2:1 ratio. This ratio was calculated taking advantage of the vicinal coupling constants characteristics for hexoses (proton near 5 ppm as a broad singlet and  proton around 4.50 ppm, with 3 JH1-H2 = 7.9 Hz), and with 1 JH-C coupling constant for mannopyranose 11. Benzylation of α and β propargyl glucopyranosyl mixtures afforded the per-O-benzylated anomers, which could be separated by column chromatography, using 6:1 hexane/ethyl acetate as eluent. In some cases, a linker was necessary in order to bond the coumarin, or the carbohydrate, moiety to the polymer and for these cases compound 7 was prepared. Due to its high volatility and reactivity, 7 was always maintained in diethyl ether, even for its quantification and characterization. Subsequent triazole ring formation with coumarin 6, afforded 8 in good yield (Scheme 3). In some cases, a linker was necessary in order to bond the coumarin, or the carbohydrate, moiety to the polymer and for these cases compound 7 was prepared. Due to its high volatility and reactivity, 7 was always maintained in diethyl ether, even for its quantification and characterization. Subsequent triazole ring formation with coumarin 6, afforded 8 in good yield (Scheme 3). Scheme 3. Triazole linker 8 was prepared by the substitution of bromide in 2-bromoethylamine hydrobromide using sodium azide, followed by cycloaddition to form the 1,2,3-triazole ring.

Ligand Preparations-Carbohydrates
Specific carbohydrate derivatization was challenging due to the presence of several hydroxyl groups having similar reactivities. However, depending on the reaction conditions, the anomeric hydroxyl can be preferential replaced. Furthermore, in order for a carbohydrate to be recognized by lectins, it often must be non-reducing.
Insertion of a triazole ring between the carbohydrate moiety and the coumarin/polymer moieties not only increases the linkage strength, but mimics the amide group, which can induce, or improve, the biological activity of the final compounds. Propargylation of glucose, galactose, and mannose (Scheme 4) using immobilized sulfuric acid on silica [36], afforded the necessary terminal alkynes, 9-11, for posterior cycloaddition reactions. The propargylation yields obtained were poor to moderate (38%-68%), and the products were mixtures of α and β anomers, with a 2:1 ratio. This ratio was calculated taking advantage of the vicinal coupling constants characteristics for hexoses (proton near 5 ppm as a broad singlet and  proton around 4.50 ppm, with 3 JH1-H2 = 7.9 Hz), and with 1 JH-C coupling constant for mannopyranose 11. Benzylation of α and β propargyl glucopyranosyl mixtures afforded the per-O-benzylated anomers, which could be separated by column chromatography, using 6:1 hexane/ethyl acetate as eluent. Scheme 3. Triazole linker 8 was prepared by the substitution of bromide in 2-bromoethylamine hydrobromide using sodium azide, followed by cycloaddition to form the 1,2,3-triazole ring.

Ligand Preparations-Carbohydrates
Specific carbohydrate derivatization was challenging due to the presence of several hydroxyl groups having similar reactivities. However, depending on the reaction conditions, the anomeric hydroxyl can be preferential replaced. Furthermore, in order for a carbohydrate to be recognized by lectins, it often must be non-reducing.
Insertion of a triazole ring between the carbohydrate moiety and the coumarin/polymer moieties not only increases the linkage strength, but mimics the amide group, which can induce, or improve, the biological activity of the final compounds. Propargylation of glucose, galactose, and mannose (Scheme 4) using immobilized sulfuric acid on silica [36], afforded the necessary terminal alkynes, 9-11, for posterior cycloaddition reactions. The propargylation yields obtained were poor to moderate (38%-68%), and the products were mixtures of α and β anomers, with a 2:1 ratio. This ratio was calculated taking advantage of the vicinal coupling constants characteristics for hexoses (α proton near 5 ppm as a broad singlet and β proton around 4.50 ppm, with 3 J H1-H2 = 7.9 Hz), and with 1 J H-C coupling constant for mannopyranose 11. Benzylation of α and β propargyl glucopyranosyl mixtures afforded the per-O-benzylated anomers, which could be separated by column chromatography, using 6:1 hexane/ethyl acetate as eluent.

Ligand Preparations-Thymidine
Tosylation of the primary hydroxyl group of 2'-deoxythymidine was accomplished using tosyl chloride (15,Scheme 6). Subsequent reaction with sodium azide, either by conventional heating in dimethylformamide (DMF) using an oil bath (85 °C, 15 h, 87%), or using microwave irradiation (100 °C, 250 W, 2 min, 90%), afforded the corresponding azide 16. Although a yield improvement was not significant using microwave heating, the reaction time was drastically reduced. Scheme 6. Synthesis of thymidine azide 16 by regioselective tosylation, followed by azide substitution, either using microwave or conventional heating.
Having the ligands already prepared, they were used to prepare PEG and PLGA derivatives, as described in the following sections.

PEG Derivatives
PEG is a versatile polymer for pro-drug development due to its high solubility in water and available commercially at various molecular weights, and its stealth properties [37]. PEG pro-drugs often diminish collateral effects and can be specifically administered on targeted tissues [38]. Although the conjugation of pharmaceutical agents with PEG has brought significant improvements such as an increased solubility in water, prolonged liberation rates, and reduction of toxicity, problems such as the necessity for high doses and drug resistance are still awaiting resolution. Multi-Scheme 5. Preparation of glycosyl amine 14 from propargyl glucoside 9.

Ligand Preparations-Thymidine
Tosylation of the primary hydroxyl group of 2'-deoxythymidine was accomplished using tosyl chloride (15,Scheme 6). Subsequent reaction with sodium azide, either by conventional heating in dimethylformamide (DMF) using an oil bath (85 • C, 15 h, 87%), or using microwave irradiation (100 • C, 250 W, 2 min, 90%), afforded the corresponding azide 16. Although a yield improvement was not significant using microwave heating, the reaction time was drastically reduced.
Having the ligands already prepared, they were used to prepare PEG and PLGA derivatives, as described in the following sections. Acetylation of propargyl glucopyranose 9, afforded 12. Subsequent triazole formation with azide 7 afforded 13 in good yield. De-acetylation using sodium methoxide led to the corresponding product 14 (Scheme 5).

Ligand Preparations-Thymidine
Tosylation of the primary hydroxyl group of 2'-deoxythymidine was accomplished using tosyl chloride (15,Scheme 6). Subsequent reaction with sodium azide, either by conventional heating in dimethylformamide (DMF) using an oil bath (85 °C, 15 h, 87%), or using microwave irradiation (100 °C, 250 W, 2 min, 90%), afforded the corresponding azide 16. Although a yield improvement was not significant using microwave heating, the reaction time was drastically reduced. Scheme 6. Synthesis of thymidine azide 16 by regioselective tosylation, followed by azide substitution, either using microwave or conventional heating.
Having the ligands already prepared, they were used to prepare PEG and PLGA derivatives, as described in the following sections.

PEG Derivatives
PEG is a versatile polymer for pro-drug development due to its high solubility in water and available commercially at various molecular weights, and its stealth properties [37]. PEG pro-drugs often diminish collateral effects and can be specifically administered on targeted tissues [38]. Although the conjugation of pharmaceutical agents with PEG has brought significant improvements such as an increased solubility in water, prolonged liberation rates, and reduction of toxicity, problems such as the necessity for high doses and drug resistance are still awaiting resolution. Multi-Scheme 6. Synthesis of thymidine azide 16 by regioselective tosylation, followed by azide substitution, either using microwave or conventional heating.

PEG Derivatives
PEG is a versatile polymer for pro-drug development due to its high solubility in water and available commercially at various molecular weights, and its stealth properties [37]. PEG pro-drugs often diminish collateral effects and can be specifically administered on targeted tissues [38]. Although the conjugation of pharmaceutical agents with PEG has brought significant improvements such as an increased solubility in water, prolonged liberation rates, and reduction of toxicity, problems such as the necessity for high doses and drug resistance are still awaiting resolution. Multi-therapy presents itself as a viable solution since polymeric pro-drugs are able to form nanoparticles that are able to encapsulate a different drug, allowing the reduction of administered doses, and consequently an increased therapeutic efficiency [39][40][41].
Esterification of coumarin 3-carboxylic acid with PEG using N,N-dicyclohexylcarbodiimide (DCC), and catalytic 4-dimethylaminepyridine (DMAP, Scheme 7) afforded the fluorescent polymer 17 in excellent yield. Product formation was observed by FT-IR, which confirmed the presence of the ester linkage at 1766 cm −1 . The proton NMR spectrum showed the presence of one coumarin moiety, determined by comparing the relative intensity of the coumarin signals to PEG signals, since PEG with an average molecular weight of 1000 g/mol was expected to present between 84 and 96 protons. Integration of aromatic signals between 8.57 and 7.32 ppm for one coumarin, resulted in 88 PEG protons (between 4.60 and 3.50 ppm), which is within the expected values. This is a very important result, since the other PEG hydroxyl should be free to link to the carbohydrate moiety. A 13 C NMR spectrum supports the esterification success through coumarin signals between 117 and 163 ppm. The terminal hydroxyl of ester 17 was then tosylated to provide ester 18, in good yield. A tolyl methyl proton signal was observed at 2.45 ppm and a corresponding carbon signal at 21.6 ppm. Aromatic proton signals where observed between 7.90 and 7.32 ppm, and between 136.5 and 115.5 ppm in the carbon spectrum. The tosyl group was substituted using sodium azide in DMF, affording the azide 19 in very good yield. Product formation was confirmed by the absence of tosyl signals both in the proton and carbon spectra, and by the observation of an azide stretch band in the FT-IR at 2104 cm −1 . Cycloaddition with the previously prepared propargyl galactopyranose 10 using catalytic CuI, and diisopropylethylamine (DIPEA) in freshly distilled tetrahydrofuran (THF) afforded the desired triazole-containing glycoconjugate 20 in only 18% yield. The poor yield obtained is believed to be due to copper complexation with the free hydroxyls of propargyl galactopyranose. Adding 2,2'-bipyridyl in excess to the reaction mixture, did not improve this result [42]. A Proton NMR spectrum showed the triazole proton signals shifted between 8.23 and 7.99 ppm, and the coumarin signals in the expected aromatic region, namely between 8.60 and 7.30 ppm. Galactopyranose signals were observed between 5.10 and 4.40 ppm, with PEG signals between 4.30 and 3.34 ppm. Carbon spectrum confirmed these results, mainly by the presence of the anomeric carbon at 98.8 ppm and triazole signal at 124.9 ppm. Matrix-Assisted Laser Desorption/Ionization with Time-of-Flight analyser (MALDI-TOF) mass spectrum presented a medium mass/charge of 1512, where the expected value was 1401 m/z., which result is within the range for (C 2 H 4 O) n H 2 O, with n = 21-24.
Molecules 2020, 25, x FOR PEER REVIEW 5 of 21 therapy presents itself as a viable solution since polymeric pro-drugs are able to form nanoparticles that are able to encapsulate a different drug, allowing the reduction of administered doses, and consequently an increased therapeutic efficiency [39][40][41]. Esterification of coumarin 3-carboxylic acid with PEG using N,N-dicyclohexylcarbodiimide (DCC), and catalytic 4-dimethylaminepyridine (DMAP, Scheme 7) afforded the fluorescent polymer 17 in excellent yield. Product formation was observed by FT-IR, which confirmed the presence of the ester linkage at 1766 cm −1 . The proton NMR spectrum showed the presence of one coumarin moiety, determined by comparing the relative intensity of the coumarin signals to PEG signals, since PEG with an average molecular weight of 1000 g/mol was expected to present between 84 and 96 protons. Integration of aromatic signals between 8.57 and 7.32 ppm for one coumarin, resulted in 88 PEG protons (between 4.60 and 3.50 ppm), which is within the expected values. This is a very important result, since the other PEG hydroxyl should be free to link to the carbohydrate moiety. A 13 C NMR spectrum supports the esterification success through coumarin signals between 117 and 163 ppm. The terminal hydroxyl of ester 17 was then tosylated to provide ester 18, in good yield. A tolyl methyl proton signal was observed at 2.45 ppm and a corresponding carbon signal at 21.6 ppm. Aromatic proton signals where observed between 7.90 and 7.32 ppm, and between 136.5 and 115.5 ppm in the carbon spectrum. The tosyl group was substituted using sodium azide in DMF, affording the azide 19 in very good yield. Product formation was confirmed by the absence of tosyl signals both in the proton and carbon spectra, and by the observation of an azide stretch band in the FT-IR at 2104 cm −1 . Cycloaddition with the previously prepared propargyl galactopyranose 10 using catalytic CuI, and diisopropylethylamine (DIPEA) in freshly distilled tetrahydrofuran (THF) afforded the desired triazole-containing glycoconjugate 20 in only 18% yield. The poor yield obtained is believed to be due to copper complexation with the free hydroxyls of propargyl galactopyranose. Adding 2,2'-bipyridyl in excess to the reaction mixture, did not improve this result [42]. A Proton NMR spectrum showed the triazole proton signals shifted between 8.23 and 7.99 ppm, and the coumarin signals in the expected aromatic region, namely between 8.60 and 7.30 ppm. Galactopyranose signals were observed between 5.10 and 4.40 ppm, with PEG signals between 4.30 and 3.34 ppm. Carbon spectrum confirmed these results, mainly by the presence of the anomeric carbon at 98.8 ppm and triazole signal at 124.9 ppm. Matrix-Assisted Laser Desorption/Ionization with Time-of-Flight analyser (MALDI-TOF) mass spectrum presented a medium mass/charge of 1512, where the expected value was 1401 m/z., which result is within the range for (C2H4O)nH2O, with n = 21-24.

Scheme 7. Synthesis of glycoconjugate 20.
In order to form symmetric or asymmetric PEG derivatives diazide 22 was prepared through the sodium azide reaction with ditosylate 21 (Scheme 8). Formation of di-tosylated product 21 was confirmed by proton NMR, where the relative intensity of the tosyl signals, compared to the PEG methylene signals, were as expected for the di-tosylation. As such, eight protons appear between 7.90 and 7.30 ppm corresponding to both tosyl groups. The two corresponding methyl protons presented as a singlet at 2.45 ppm. Compound 22 was confirmed by the absence of tosyl NMR signals, and the presence of a triplet at 3.39 ppm, corresponding to both methylene groups bonded to azide groups.
Equimolar cycloaddition of 22 with previously prepared propargyl coumarin 4 afforded 23 with very good yields. Proton spectrum presented in the aromatic region the protons belonging to the coumarin (between 7.70 and 6.20 ppm) and the triazole (8.00 ppm) moieties. The methylene of the coumarin moiety was observed at 5.29 ppm as a singlet, as expected. PEG proton signals were shifted in four different places, where the most deshielded triplet, at 4.60 ppm, corresponded to the In order to form symmetric or asymmetric PEG derivatives diazide 22 was prepared through the sodium azide reaction with ditosylate 21 (Scheme 8). Formation of di-tosylated product 21 was confirmed by proton NMR, where the relative intensity of the tosyl signals, compared to the PEG methylene signals, were as expected for the di-tosylation. As such, eight protons appear between 7.90 and 7.30 ppm corresponding to both tosyl groups. The two corresponding methyl protons presented as a singlet at 2.45 ppm. Compound 22 was confirmed by the absence of tosyl NMR signals, and the presence of a triplet at 3.39 ppm, corresponding to both methylene groups bonded to azide groups.
showed the azide band at 2106 cm −1 , indicating that unwanted di-cycloaddition was not obtained.
Subsequent cycloaddition on the other side of the polymer, either using propargylated galactopyranose 10 or propargylated mannopyranose 11, led to the formation of glycoconjugates 24 or 25, respectively, in 11% and 17% yields. Structure confirmation by proton NMR was difficult due to the overlap of PEG and carbohydrate signals, while MALDI results were in accordance with the expected estimated values (estimated medium value of 1426 m/z, obtained 1511 m/z for compound 24, and 1477 m/z for compound 25). Scheme 8. Synthesis of glycoconjugates 24 and 25 from poly(ethylene glycol) (PEG).
Diesterification of PEG with coumarin alkyne 3 using DCC afforded a new fluorescent polymer, 26 (Scheme 9). Product 26 was easily identified by proton NMR, where the relative intensity of coumarin signals, compared to PEG methylene signals, were as expected for the diester. As such, 4 aromatic protons at 7.47 and 6.98 ppm corresponded to both coumarins. PEG proton signals were identified at 4.23 and 3.66 ppm, with a relative intensity for 86 protons. Carbon spectrum analysis was also consistent with the proposed structure (see experimental part).
Subsequent double azide-alkyne Huisgen cycloaddition with thymidine azide 16 using a catalytic amount of CuI and DIPEA in THF, afforded the symmetric polymer 27. Structural characterization was observed to be in agreement with the proposed structure. More precisely, proton NMR spectrum showed new triazole protons at 7.77 ppm and coumarin signals in the expected aromatic region at 7.41 and 6.98 ppm. Thymidine signals were observed at 7.30, 6.24, and 4.47 ppm, and PEG signals at 4.20 and 3.60 ppm. All the relative intensity for the assigned protons were in agreement with bis-thymidine cycloaddition. The carbon spectrum confirmed these results, mainly by the presence of a triazole signal at 135.5 ppm, the anomeric carbon at 84.9 ppm and the PEG main carbon chain at 70.5 ppm. The MALDI-TOF expected medium value was 2132 m/z, and it was obtained 2153 m/z for compound 27, with sizes ranging from 1932 to 2461 m/z, in which the increment observed was due to ethylene glycol monomer.

Scheme 9.
Synthesis of a double thymidine-containing fluorescent PEG derivative, 27, starting by PEG di-esterification with coumarin 3, followed by double triazole ring formation with thymidine derivative 16.

PLGA Derivatives.
PLGA is a hydrophobic, linear and biocompatible polymer, approved by the FDA and EMA for biological applications [43]. Upon hydrolysis, PLGA is able to release pharmaceutical agents, and the Equimolar cycloaddition of 22 with previously prepared propargyl coumarin 4 afforded 23 with very good yields. Proton spectrum presented in the aromatic region the protons belonging to the coumarin (between 7.70 and 6.20 ppm) and the triazole (8.00 ppm) moieties. The methylene of the coumarin moiety was observed at 5.29 ppm as a singlet, as expected. PEG proton signals were shifted in four different places, where the most deshielded triplet, at 4.60 ppm, corresponded to the methylene bonded to the triazole moiety, and the more shielded one, at 3.39 ppm corresponded to the methylene bonded to the azide moiety. Carbon spectrum sustained these results. FT-IR spectrum showed the azide band at 2106 cm −1 , indicating that unwanted di-cycloaddition was not obtained.
Subsequent cycloaddition on the other side of the polymer, either using propargylated galactopyranose 10 or propargylated mannopyranose 11, led to the formation of glycoconjugates 24 or 25, respectively, in 11% and 17% yields. Structure confirmation by proton NMR was difficult due to the overlap of PEG and carbohydrate signals, while MALDI results were in accordance with the expected estimated values (estimated medium value of 1426 m/z, obtained 1511 m/z for compound 24, and 1477 m/z for compound 25).
Diesterification of PEG with coumarin alkyne 3 using DCC afforded a new fluorescent polymer, 26 (Scheme 9). Product 26 was easily identified by proton NMR, where the relative intensity of coumarin signals, compared to PEG methylene signals, were as expected for the diester. As such, 4 aromatic protons at 7.47 and 6.98 ppm corresponded to both coumarins. PEG proton signals were identified at 4.23 and 3.66 ppm, with a relative intensity for 86 protons. Carbon spectrum analysis was also consistent with the proposed structure (see experimental part). methylene bonded to the triazole moiety, and the more shielded one, at 3.39 ppm corresponded to the methylene bonded to the azide moiety. Carbon spectrum sustained these results. FT-IR spectrum showed the azide band at 2106 cm −1 , indicating that unwanted di-cycloaddition was not obtained.
Subsequent cycloaddition on the other side of the polymer, either using propargylated galactopyranose 10 or propargylated mannopyranose 11, led to the formation of glycoconjugates 24 or 25, respectively, in 11% and 17% yields. Structure confirmation by proton NMR was difficult due to the overlap of PEG and carbohydrate signals, while MALDI results were in accordance with the expected estimated values (estimated medium value of 1426 m/z, obtained 1511 m/z for compound 24, and 1477 m/z for compound 25). Scheme 8. Synthesis of glycoconjugates 24 and 25 from poly(ethylene glycol) (PEG).
Diesterification of PEG with coumarin alkyne 3 using DCC afforded a new fluorescent polymer, 26 (Scheme 9). Product 26 was easily identified by proton NMR, where the relative intensity of coumarin signals, compared to PEG methylene signals, were as expected for the diester. As such, 4 aromatic protons at 7.47 and 6.98 ppm corresponded to both coumarins. PEG proton signals were identified at 4.23 and 3.66 ppm, with a relative intensity for 86 protons. Carbon spectrum analysis was also consistent with the proposed structure (see experimental part).
Subsequent double azide-alkyne Huisgen cycloaddition with thymidine azide 16 using a catalytic amount of CuI and DIPEA in THF, afforded the symmetric polymer 27. Structural characterization was observed to be in agreement with the proposed structure. More precisely, proton NMR spectrum showed new triazole protons at 7.77 ppm and coumarin signals in the expected aromatic region at 7.41 and 6.98 ppm. Thymidine signals were observed at 7.30, 6.24, and 4.47 ppm, and PEG signals at 4.20 and 3.60 ppm. All the relative intensity for the assigned protons were in agreement with bis-thymidine cycloaddition. The carbon spectrum confirmed these results, mainly by the presence of a triazole signal at 135.5 ppm, the anomeric carbon at 84.9 ppm and the PEG main carbon chain at 70.5 ppm. The MALDI-TOF expected medium value was 2132 m/z, and it was obtained 2153 m/z for compound 27, with sizes ranging from 1932 to 2461 m/z, in which the increment observed was due to ethylene glycol monomer.

Scheme 9.
Synthesis of a double thymidine-containing fluorescent PEG derivative, 27, starting by PEG di-esterification with coumarin 3, followed by double triazole ring formation with thymidine derivative 16.

PLGA Derivatives.
PLGA is a hydrophobic, linear and biocompatible polymer, approved by the FDA and EMA for biological applications [43]. Upon hydrolysis, PLGA is able to release pharmaceutical agents, and the Scheme 9. Synthesis of a double thymidine-containing fluorescent PEG derivative, 27, starting by PEG di-esterification with coumarin 3, followed by double triazole ring formation with thymidine derivative 16.
Subsequent double azide-alkyne Huisgen cycloaddition with thymidine azide 16 using a catalytic amount of CuI and DIPEA in THF, afforded the symmetric polymer 27. Structural characterization was observed to be in agreement with the proposed structure. More precisely, proton NMR spectrum showed new triazole protons at 7.77 ppm and coumarin signals in the expected aromatic region at 7.41 and 6.98 ppm. Thymidine signals were observed at 7.30, 6.24, and 4.47 ppm, and PEG signals at 4.20 and 3.60 ppm. All the relative intensity for the assigned protons were in agreement with bis-thymidine cycloaddition. The carbon spectrum confirmed these results, mainly by the presence of a triazole signal at 135.5 ppm, the anomeric carbon at 84.9 ppm and the PEG main carbon chain at 70.5 ppm. The MALDI-TOF expected medium value was 2132 m/z, and it was obtained 2153 m/z for compound 27, with sizes ranging from 1932 to 2461 m/z, in which the increment observed was due to ethylene glycol monomer.

PLGA Derivatives
PLGA is a hydrophobic, linear and biocompatible polymer, approved by the FDA and EMA for biological applications [43]. Upon hydrolysis, PLGA is able to release pharmaceutical agents, and the resulting lactic and glycolic acids are metabolized on the Krebs cycle [44]. A polymer with a molecular weight range between 7000 and 17,000 g/mol was chosen with a 50:50 glycolic/lactic acid ratio, which translates into a more amorphous final structure.
PLGA glycoconjugate 28 was prepared by amide formation between 14 and PLGA using methanesulfonic acid (Scheme 10). Due to the fact that amines are more reactive than alcohols, it was assumed that an amide was the major product of this reaction, although esterification may have also occurred.
Molecules 2020, 25, x FOR PEER REVIEW 7 of 21 resulting lactic and glycolic acids are metabolized on the Krebs cycle [44]. A polymer with a molecular weight range between 7000 and 17,000 g/mol was chosen with a 50:50 glycolic/lactic acid ratio, which translates into a more amorphous final structure. PLGA glycoconjugate 28 was prepared by amide formation between 14 and PLGA using methanesulfonic acid (Scheme 10). Due to the fact that amines are more reactive than alcohols, it was assumed that an amide was the major product of this reaction, although esterification may have also occurred.

Scheme 10.
Amidation reaction between poly(lactic-co-glycolic acid) (PLGA) and previously prepared glycoside 14 to afford the PLGA glycoconjugate 28. Proton NMR analysis of compound 28 identified the methyl, methylene, and methine groups that form the polymer at 1.58, 4.83, and 5.22 ppm, respectively. The characteristic signal from the triazole moiety can be identified at 7.01 ppm, and a few sugar signals between 4.43 and 4.11 ppm. For polymer 29 it was also possible to identify the methyl, methylene, and methine groups from the polymer chain at 1.58, 4.83, and 5.24 ppm, respectively, plus the coumarin and triazole characteristic protons at 8.29, 7.80, and 7.54 ppm. Identification of these ligands was very difficult, due to high NMR signal accumulation from the polymer structure. Nevertheless, it was possible to identify very distinctive signals for both polymers. MALDI-TOF analysis could not be made for these polymers due to their very high polydispersity. Correlation between zeta potentials of their respective nanoparticles, and these observed NMR signals allowed us to conclude that the functionalization had occurred.

Nanoparticles Preparation and Characterization
Polymeric glycoconjugates were transformed into nanoparticles with the glycosides towards their exterior. Oil in water emulsification/solvent evaporation technique is an easy and highly reproducible method for nanoparticle preparation, and it was our choice for the transformation of the prepared polymers into nanoparticles.
Nanoparticles from glycoconjugate 20 were prepared and analyzed by Scanning Electron Scheme 10. Amidation reaction between poly(lactic-co-glycolic acid) (PLGA) and previously prepared glycoside 14 to afford the PLGA glycoconjugate 28.
Coumarin 8 reacted with PLGA in the presence of methanesulfonic acid (Scheme 11) to afford the fluorescent amide, 29.
Molecules 2020, 25, x FOR PEER REVIEW 7 of 21 resulting lactic and glycolic acids are metabolized on the Krebs cycle [44]. A polymer with a molecular weight range between 7000 and 17,000 g/mol was chosen with a 50:50 glycolic/lactic acid ratio, which translates into a more amorphous final structure. PLGA glycoconjugate 28 was prepared by amide formation between 14 and PLGA using methanesulfonic acid (Scheme 10). Due to the fact that amines are more reactive than alcohols, it was assumed that an amide was the major product of this reaction, although esterification may have also occurred.

Scheme 10.
Amidation reaction between poly(lactic-co-glycolic acid) (PLGA) and previously prepared glycoside 14 to afford the PLGA glycoconjugate 28. Proton NMR analysis of compound 28 identified the methyl, methylene, and methine groups that form the polymer at 1.58, 4.83, and 5.22 ppm, respectively. The characteristic signal from the triazole moiety can be identified at 7.01 ppm, and a few sugar signals between 4.43 and 4.11 ppm. For polymer 29 it was also possible to identify the methyl, methylene, and methine groups from the polymer chain at 1.58, 4.83, and 5.24 ppm, respectively, plus the coumarin and triazole characteristic protons at 8.29, 7.80, and 7.54 ppm. Identification of these ligands was very difficult, due to high NMR signal accumulation from the polymer structure. Nevertheless, it was possible to identify very distinctive signals for both polymers. MALDI-TOF analysis could not be made for these polymers due to their very high polydispersity. Correlation between zeta potentials of their respective nanoparticles, and these observed NMR signals allowed us to conclude that the functionalization had occurred.

Nanoparticles Preparation and Characterization
Polymeric glycoconjugates were transformed into nanoparticles with the glycosides towards their exterior. Oil in water emulsification/solvent evaporation technique is an easy and highly reproducible method for nanoparticle preparation, and it was our choice for the transformation of the prepared polymers into nanoparticles.
Nanoparticles from glycoconjugate 20 were prepared and analyzed by Scanning Electron Microscopy (SEM) and the results are presented in Figure 1 (A) and (B). This glycoconjugate formed mostly a polymeric film upon deposition, with some dispersed agglomerates. The size of the Scheme 11. Synthesis of fluorescent PLGA derivative, 29 by direct PLGA amidation with coumarin 8 in acidic conditions. Proton NMR analysis of compound 28 identified the methyl, methylene, and methine groups that form the polymer at 1.58, 4.83, and 5.22 ppm, respectively. The characteristic signal from the triazole moiety can be identified at 7.01 ppm, and a few sugar signals between 4.43 and 4.11 ppm. For polymer 29 it was also possible to identify the methyl, methylene, and methine groups from the polymer chain at 1.58, 4.83, and 5.24 ppm, respectively, plus the coumarin and triazole characteristic protons at 8.29, 7.80, and 7.54 ppm. Identification of these ligands was very difficult, due to high NMR signal accumulation from the polymer structure. Nevertheless, it was possible to identify very distinctive signals for both polymers. MALDI-TOF analysis could not be made for these polymers due to their very high polydispersity. Correlation between zeta potentials of their respective nanoparticles, and these observed NMR signals allowed us to conclude that the functionalization had occurred.

Nanoparticles Preparation and Characterization
Polymeric glycoconjugates were transformed into nanoparticles with the glycosides towards their exterior. Oil in water emulsification/solvent evaporation technique is an easy and highly reproducible method for nanoparticle preparation, and it was our choice for the transformation of the prepared polymers into nanoparticles.
Nanoparticles from glycoconjugate 20 were prepared and analyzed by Scanning Electron Microscopy (SEM) and the results are presented in Figure 1A,B. This glycoconjugate formed mostly a polymeric film upon deposition, with some dispersed agglomerates. The size of the agglomerates ranged between 220 and 580 nm.
Glycoconjugates 24 and 25 were also transformed into nanoparticles and analyzed by SEM, and are presented in Figure 1 (C)-(F). Galactoconjugate 24 formed more agglomerates than glycoconjugate 20, and a film was still present. For the mannoconjugate 25, it was observed that it formed irregular films, with multiple cracks, and a high degree of convolution of neighboring agglomerates either by the polymer's preferential formation of films, or due to film deposition on top of those agglomerates. Dynamic Light Scattering (DLS) technique (results presented in Figure 2), where the particles are suspended in water in a colloidal manner, showed average particle size of 253 (20), 118 (24), and 193 nm (25), peak size of 219 (20), 105 (24), and 171 nm (25) and high polydispersity indexes of 5.828 (20), 0.440 (24), and 0.682 (25). These results confirmed that PEG derivatives formed polydisperse samples, with aggregates.  Glycoconjugates 24 and 25 were also transformed into nanoparticles and analyzed by SEM, and are presented in Figure 1C-F. Galactoconjugate 24 formed more agglomerates than glycoconjugate 20, and a film was still present. For the mannoconjugate 25, it was observed that it formed irregular films, with multiple cracks, and a high degree of convolution of neighboring agglomerates either by the polymer's preferential formation of films, or due to film deposition on top of those agglomerates. Dynamic Light Scattering (DLS) technique (results presented in Figure 2), where the particles are suspended in water in a colloidal manner, showed average particle size of 253 (20), 118 (24), and 193 nm (25), peak size of 219 (20), 105 (24), and 171 nm (25) and high polydispersity indexes of 5.828 (20), 0.440 (24), and 0.682 (25). These results confirmed that PEG derivatives formed polydisperse samples, with aggregates. PLGA glucoconjugate 28 was capable of forming spherical nanoparticles, with a smooth surface and with practically no aggregation as seen in Figure 3 (A) and (B). The particles presented a size range of 114-234 nm. DLS gave similar results, with an average particle size of 189 nm, peak size 171 nm (Figure 4(A)) and a low polydispersity index of 0.174. The zeta potential was also measured, with a value corresponding to −28.2 mV.
Nanoparticles from the coumarin-containing PLGA derivative, 29, presented a spherical and smooth surface, with no aggregation (Figure 3 (C) and (D)) and size range of 174-289 nm. DLS results showed an average particle size of 273 nm, peak size 219 nm (Figure 4(B)), a polydispersity value of 0.146, and a zeta potential of −56.0 mV. This high zeta potential value may be explained by the presence of both the triazole ring and amide bonds towards the exterior of the particles, as expected by the technique employed for their formation. PLGA glucoconjugate 28 was capable of forming spherical nanoparticles, with a smooth surface and with practically no aggregation as seen in Figure 3A,B. The particles presented a size range of 114-234 nm. DLS gave similar results, with an average particle size of 189 nm, peak size 171 nm ( Figure 4A) and a low polydispersity index of 0.174. The zeta potential was also measured, with a value corresponding to −28.2 mV.

Materials and Methods
All reagents used were purchased from Sigma-Aldrich/Merck, Carbosynth or Fluka. The solvents used as reaction media were dried according to the procedures described in the literature [45]. Briefly, Nanoparticles from the coumarin-containing PLGA derivative, 29, presented a spherical and smooth surface, with no aggregation (Figure 3C,D) and size range of 174-289 nm. DLS results showed an average particle size of 273 nm, peak size 219 nm ( Figure 4B), a polydispersity value of 0.146, and a zeta potential of −56.0 mV. This high zeta potential value may be explained by the presence of both the triazole ring and amide bonds towards the exterior of the particles, as expected by the technique employed for their formation.

Materials and Methods
All reagents used were purchased from Sigma-Aldrich/Merck, Carbosynth or Fluka. The solvents used as reaction media were dried according to the procedures described in the literature [45]. Briefly, DMF was dried with barium oxide, filtered, distilled and stored with 3Å molecular sieves. Dichloromethane (DCM) was distilled from calcium hydride under an argon atmosphere and used immediately. Propargyl alcohol was dried overnight with potassium carbonate, distilled and used immediately. PEG was purchased from Sigma-Aldrich, with an average molecular weight of 1000. D,L-poly(lactic-glycolic acid) (PLGA) 50:50, with a molecular weight range of 7000-17,000 was purchased from Sigma-Aldrich. Column chromatography was performed using Carlo Erba's silica gel, 40-63 µm mesh. Preparative TLC was performed using silica gel 60GF DGF254 purchased from Macherey-Nagel.

Nanoparticles Preparation
Polymer matrix (50 mg) was dissolved in 5 mL of DCM and poured into 8.0 mL of aqueous PVA 2%. The resulting oil-in-water preparation was sonicated at 60 W for a minute, in periods of 10 s. The resulting emulsion was magnetically stirred overnight. The resulting suspension was centrifuged, and the pellet was washed three times with deionized water.

7-(Prop-2-yn-1-yloxy)-2H-Chromen-2-One (4)
Potassium iodide (2.454 g, 14.7 mmol) and potassium carbonate (2.045 g, 14.7 mmol) were added to a solution of umbelliferone (2.011 g, 12.4 mmol) in DMF (30 mL) under magnetic stirring and argon atmosphere. Then, propargyl bromide 80% in toluene (1.7 mL, 14.8 mmol) was added drop-wise and the reaction was heated at 80 • C. After the completion of the reaction (2 h 30 min), dichloromethane was added (40 mL) and the mixture was transferred to a separating funnel and washed three times with water (3 × 20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to afford 7-(prop-2-yn-1-yloxy)-2H-chromen-2-one, 4, as a light brown solid (2.382 g, 96% without purification) and was used, as such, for further reactions. m.p.  (6) 7-Hydroxy-4-methyl coumarin (3.532 g, 20.1 mmol) was dissolved in dry acetone (30 mL) and anhydrous potassium carbonate (3.209 g, 20.2 mmol) was added. The reaction was magnetically stirred for 30 min, and propargyl bromide (2.7 mL, 24.1 mmol) was added. The reaction flask was heated at 50 • C and reacted for 18 h. The solvent was evaporated, and the residue was dissolved in DCM (20 mL) and washed four times with water (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated, to afford product 6 (3.786 g, 88%) as a beige solid. m.p. D-glucose (4.149 g, 23.0 mmol) was dissolved in propargyl alcohol (7.8 mL, 132.0 mmol), and sulfuric acid immobilized in silica gel (150.4 mg) was added. The reaction mixture was heated at 65 • C overnight. After cool down to room temperature, the mixture was purified by silica column chromatography using an ethyl acetate/acetone/water (10:10:1) mixture, affording 9 as a mixture of α and β anomers, 2:1 ratio. Sulfuric acid supported on silica was prepared accordingly with the procedure described by Roy and Mukhopadhyay [36]. For detailed characterization, the α, β mixture 9 was per-O-acetylated using acetic anhydride to give the α, β-propargylated per-acetylated glucopyranose 12. (10) D-galactose (2.000 g, 11.1 mmol), propargyl alcohol (3.2 mL, 55.6 mmol) and sulfuric acid supported on silica (72.5 mg) were mixed together under magnetic stirring and an argon atmosphere. The reaction flask was heated at 65 • C overnight, and after cooling the mixture was chromatographed [ethyl acetate (AcOEt), then 10:10:1 AcOEt/acetone/water] affording 45% of propargylated galactopyranose 10 as a yellowish oil (1.099 g, 0.5 mmol, 2:1 α/β ratio). Sulfuric acid supported on silica was prepared accordingly with the procedure described by Roy and Mukhopadhyay [36]. D-mannose (2.000 g, 11.1 mmol), propargyl alcohol (3.2 mL, 55.6 mmol) and sulfuric acid supported on sílica (58 mg) were mixed together under magnetic stirring and argon atmosphere. The reaction flask was heated at 65 • C overnight, and after cooling the mixture was purified by column chromatography (AcOEt, then 10:10:1 AcOEt/acetone/water) affording propargylated mannopyranose 11 as a yellowish oil (0.928 g, 38%, 2:1 α/β ratio). Sulfuric acid supported on silica was prepared accordingly to the procedure described by Roy and Mukhopadhyay [36]. Compound 13 (0.110 g, 0.2 mmol) was dissolved in dry methanol at ice-bath temperature, and sodium methoxide was added (0.012 g, 0.2 mmol). The reaction was allowed to warm to room temperature and stirred for a further 3 h. Pre-activated and thoroughly washed acidic Dowex was added until the pH changed from basic to acidic. The solution was filtered and the solvent evaporated to give compound 14 (0.071 g, 0.2 mmol, 100%) as a white foam. Due to the complex anomeric mixture this compound's characterization was based on the spectra of per-O-acetyl compound 13. (15) In a round bottom flask 2'-deoxythymidine (1.569 g, 6.5 mmol) was dissolved in dry pyridine (7.5 mL) and the flask cooled in an ice bath for 1 h. Tosyl chloride (1.236 g, 6.5 mmol) dissolved in dry pyridine (2.5 mL) was added dropwise and the reaction allowed to attain room temperature during 17 h. The solvent was then evaporated, and the resulting crude product was dissolved in ethanol at 75 • C and then cooled down to −10 • C for 12 h. The resulting crystals of 15 were filtered and washed with cold ethanol (3 × 1.0 mL, 1.889 g, 54%). m.p. 3.3.16. 5'-Azide-5'-Deoxythymidine (16) Method I-conventional heating: compound 15 (0.562 g, 1.4 mmol) was dissolved in DMF (5.0 mL), and sodium azide was added (0.407 g, 6.4 mmol). The reaction mixture was heated to 85 • C on an oil bath for 15 h. The solvent was then evaporated under reduced pressure and the mixture was purified by silica gel column chromatography using chloroform/methanol (9:1) as eluent, affording 16 (0.325 g, 87%) as a beige solid.

Conclusions
The use of these two polymeric building blocks (PEG and PLGA) has allowed us to prepare a new type of macromolecule functionalized with carbohydrate and coumarin moieties. The polymer PEG was selectively functionalized making possible the production of asymmetric macromolecules that contain at one terminal the carbohydrate unit and at the other the coumarin molecule. This asymmetry provides the ability to obtain a self-assembling control during the nanoparticles oil in water transformation. This kind of macromolecules showed a tendency to form aggregates with sizes ranging between 220 and 580 nm, respectively, but the majority of the sample presented a polymeric film after the medium evaporation. This film formation can suggest that the hydrophobic core is not big enough to provide the desired self-assembling effect.
PLGA has been successfully functionalized and transformed into stable and spherical nanoparticles with a smooth surface, with practically no aggregation. The size of these different particles ranges between 114-289 nm with a zeta potential value of −28.2 mV for the glucoconjugate and −56.0 mV for the coumarin-containing derivative. Using a single oil in water emulsion technique, it was possible to obtain low polydispersity indexes for all PLGA nanoparticles.