Ponader et al. (2012) have firstly reported the synthesis of sequence-defined monodisperse glycopolymer segments by a combination of solid-phase synthesis and click chemistry. The triple bond functionalized building block 1-(Fluorenyl)-3,11-dioxo-7-(pent-4-ynoyl)-2-oxa-4,7,10-triazatetradecan-14-oic acid (TDS) and ethylenedioxy (EDS) building block from a commercially available diethylenetriamine and 2,2′ (Ethylenedioxy)bis (ethylamine) were prepared, respectively, in several steps. These building blocks having an fluorenylmethyloxycarbonyl (Fmoc)-protected amine to build block coupling on solid-phase according to standard Fmoc SPPS protocols allow for the sequence-defined positioning of alkyne moieties within monodisperse poly(amidoamine) PAA segments. Trityl-tentagel-OH resin modified with ethylenediamine linker used as a solid support for the coupling reaction of building blocks TDS and EDS on the resin (Figure 1).

In order to perform the click reaction, the alkyne group of the PAA backbone was deprotected by removal of the Fmoc protecting group with a 25% solution of piperidine in DMF. The click reaction between these alkyne groups and azidoethyl mannosides was performed using sodium ascorbate and CuSO₄ in the solvent mixture of DMF and water. After cleaving the short chain from the support resin, three different monodisperse glycopolymer segments were obtained with the same contour length but differ in their number and spacing of presented mannose ligands. Three different precision short glycopolymer chains having the same contour length but differ in their number and spacing of presented mannose ligands were obtained after cleaving from the support resin. As expected, trivalent glycopolymer segment showed higher binding affinity than others due to the number and position of mannose. Although this strategy provides to synthesis precision glycopolymers with the desired chain length, structure, and composition, it is still challenge to prepare long-chain glycopolymers with a good sequence control via this method.

An elegant approach to prepare precision glycopolymers in a convenient manner was reported by Baradel et al. (2013). They have utilized a combination of “living” radical polymerization technique and copper-catalyzed azide–alkyne cycloaddition (CuAAC). The NMP was performed to yield copolymers of styrene and maleimides (MIs) derivatives. Polymerization kinetics of these monomers showed a marked influence on macromolecular sequences and lead to efficient sequence controlled. The polymerization reaction was carried out in anisole at 120°C using alkoxyamine BlocBuilder as an initiator. The time-controlled addition of the protected MIs (three equivalents) during the polymerization of styrene, provided linear polystyrene chains presenting short but localized glycol functional regions (PDI = 1.23–1.24). Initially, triisopropylsilyl protected N-propargylmaleimide (TIPS-PMI) monomer was polymerized at the beginning of the reaction and then triethylsilyl protected N-propargylmaleimide (TES-PMI) was introduced after reaching full conversion of TIPS-PMI and further chain growth with styrene. Finally, trimethylsilyl protected N-propargylmaleimide (TMS-PMI) was added close to the end of the polymer chain due to their potential sensitivity to the reaction conditions (Figure 2). The precise chain positioning of all three reactive MIs was confirmed by the study of the kinetics of copolymerization.

Following the synthesis of precision polymers with controlled molecular weights and molecular weight distributions, the stepwise orthogonal deprotection and functionalization of the three distinct alkyne sites with different azide-functionalized sugars were studied. Firstly, the deprotection reaction for the removal of TMS group was carried out in a methanol/water/THF mixture with K₂CO₃ at 40°C for 7 h, and then the azidomannose was attached to the polymer via CuAAC click reaction. The TES group was then removed by treatment with K₂CO₃ in a methanol/water mixture at 60°C for 89 h, and then the alkyne was reacted with the azido-galactose derivative. Lastly, the last protecting TIPS group was removed in THF with tetrabutylammonium fluoride at room temperature for overnight and the clickable alkyne site reacted with the azido-N-acetylglucosamine derivative. The click reactions can be followed by the appearance of broad signals in the region δ = 3.1–5.0 ppm in ¹H NMR corresponding to the attachment of sugar groups. All click reactions were carried out in DMF at room temperature using of Cu(I)Br and 4,4′-di-n-nonyl-2,2′-bipyridine as the catalyst system.

The analyze of the binding ability of the modified glycopolymers to hexose-specific lectins, namely, concanavalin A (ConA), peanut agglutinin (PNA), and wheat-germ agglutinin (WGA), was done by using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. Interestingly, these lectins showed high-binding affinity with mannose, galactose, and N-acetylglucosamine groups, respectively. This work is critically important to understand the complexity of GlycoCode (Becer, 2012) and lead other research groups to the synthesis of complex precision glycopolymers as a glycan mimic.

A versatile method to prepare different types of precision multi-block glycopolymers was performed by Zhang et al. (2013) via controlled polymerization technique, namely, SET-LRP. Firstly, several types of glycomonomers were prepared by clicking of 3-azidopropylacrylate (APA) to alkylated mannose, glucose, and fucose, respectively, via the CuAAC click reaction with CuSO₄ and sodium ascorbate in methanol/water mixture. Initially, the polymerization of glucose monomer (GluA) was undertaken using Cu(0)/Cu(II)/tris[2(dimethylamino)ethyl]amine (Me₆TREN) as the catalyst system in DMF at 20°C and yielded high-chain end fidelity glycopolymer allowed for polymerization of more of the same or different glycomonomers in one pot. As depicted in Figure 3, chain extension was carried out by addition of mannose monomer (ManA) in 1 ml DMSO into the polymerization reaction mixture via cannula under nitrogen. After reaching full monomer conversion, fucose monomer (FucA) was added with a same way. This sequential addition of the subsequent monomers was repeated one more time and continued until the glycopolymer reached to six short blocks (DP = 2 for each block; Mn = 7.6 kDa; Mw/Mn = 1.13). Moreover, the polymerization of di(ethylene glycol) ethyl ether acrylate (DEGEEA) and ManA was developed for a model thermoresponsive sequence controlled multi-block glycopolymers in a relatively large scale until reaching six blocks (Mn = 69.6 kDa; Mw/Mn = 1.23).

This work was the first to report the investigation of the binding affinity of the highly sequence controlled glycopolymers with the human lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). Their lectin binding properties was measured via SPR. Despite of the fact that the binding ability of the resulting precision glycopolymers exhibited a clear dependence with the number-average degree of polymerization of each glycomonomer, the glycopolymers with different sugar units did not present enough binding affinity, as well.

As opposed to the direct polymerization of glycomonomers, an alternative route was employed to prepare for precision multi-block glycopolymers by using the combination of copper(0) mediated living radical polymerization [Cu(0)-LRP] with thiol-halogen, thiol-epoxy and CuAAC click chemistry (Zhang et al., 2014). Well-defined alkyne and epoxide side-chain polymeric scaffold were obtained by polymerization of glycidyl acrylate (GA) and acrylic acid 3-trimethylsilanyl-prop-2-ynyl ester (TMSPA) from Cu(0)-LRP using Cu(0) wire as the activator and additional CuBr₂ as the deactivator and Me₆TREN as the ligand in DMSO at ambient temperature, allowed for further sequential thiol-halogen, thiol-epoxy, and CuAAC reactions to build functional glycopolymers with a defined sequence and spatial orientation. The reactivity of these glycopolymers with appropriate lectins has not been demonstrated yet.

All these reports on the synthesis of precision glycopolymers prepared by different techniques are quite promising in mimicking structural and functional responsibilities of glycocalyx (Yilmaz and Becer, 2013). They are versatile examples to take this challenge in the polymer science one more step forward and lead other research groups to synthesis controlled folding polymers having a great potential to be utilized in biological applications. Although the polymerization of monomers bearing carbohydrate can be carried out by a range of polymerization techniques, it is not suitable for introducing some different functional groups along polymer backbones. It should be mentioned that these reports opened new avenues for the synthesis of complex precision glycopolymers. SET-LRP technique was used to build multi-block glycopolymers with short blocks of sugar monomers in one pot by sequential addition of the subsequent monomers in a relatively large scale (Zhang et al., 2013, 2014). Unfortunately, it was not possible to determine any effect of sequence on binding in the system due to low degree of polymerization of each glycomonomer (DP = 2).

A metal-free living radical polymerization technique, NMP, was used for incorporation of the desired sugar units at the designed location of the chain. Although NMP represents an excellent avenue for the polymerization of styrene and its derivatives having bioinert and biocompatible properties, it does not show the tolerance to the flexible range of reaction conditions in terms of the solvents, monomers, and temperatures.

Last but not least, solid-phase synthesis allowed achieving an excellent sequence control with the low quantity of the isolated material after several steps. Despite these recent developments, it is still essential to advance precision sequence controlled synthesis techniques to encode glycopolymer–lectin binding code and to prepare more complex precision glycopolymers as a glycan mimic. These studies provided not only new routes to prepare precision glycopolymers that only differ from each other for the length and the nature linker connecting the sugar units to the macromolecular backbone but also to compare structurally similar ligands that only differ for the nature of the carbohydrate moieties.

Kempe et al. (2011) have employed the synthesis of a new sugar functionalized 2-oxazoline monomer (Figure 6). In order to prepare this glycomonomer, 4-bromobenzonitrile was reacted with 2-aminoethanol in the presence of a Lewis-acid catalyst. The alkyne groups were modified with a trimethylsilyl (TMS) protection group. The glycomonomer, 2-(azidoethyl)-1-β-d-glucopyranosetetraacetate (Ac₄Glc-YnOx), was obtained with high yield via CuAAC reaction using Cu(II) acetate and sodium ascorbate after the removing TMS groups.

The copolymerization of Ac₄Glc-YnOx with 2-ethyl-2-oxazoline and 2-(dec-9-enyl)-2-oxazoline was carried out via the microwave synthesizer using methyl tosylate as an initiator in acetonitrile at 120°C for 12 h. After that, the pendant alkene group in the side-chain of these well-defined glycopolymers was modified with different thiols, namely, 2,3,4,6-tetra-O-acetyl-1-thio-β-d-glucopyranose, dodecanethiol, and 3-mercaptopropionic acid via thiol-ene photoaddition reaction to analyze thermosensitivity and pH sensitivity of these glycopolymers in water and PBS. Finally, these glycopolymers showed strong binding ability with ConA.

A synthetic methodology based on three tandem post-polymerization and modifications to synthesize glycopolymers with high specificity and selectivity as mimicking glycan architecture due to secondary binding motif over the polymer side-chain has been reported (Jones et al., 2014). Initially, glycidyl methacrylate (GMA) was polymerized via Cu(I)-mediated polymerization and introduced with sodium azide in DMF at 50°C to yield the azide functional groups. The reaction produced a secondary alcohol that was followed by infrared spectroscopy (IR). Subsequently, acyl chlorides were reacted with alcohol to install secondary motifs due to the fact that aromatic groups can bind to the sialic acid site. Following to this modification, the azide groups were reacted with β-d-propargyl galactose by Cu(I)-catalyzed cycloaddition. By this route, a small library of glycopolymers was prepared and used to study their binding affinity to their corresponding lectins. The bacterial toxin (CTx), the causative agent of cholera infection, and PNA, a model galactose-binding non-pathogenic lectin, were used for the investigation of the binding affinity of these glycopolymers. According to the assay studies, the specificity and selectivity of the glycopolymers to CTx increased with a secondary binding pocket within CTx due to allosteric interactions of the neuraminic acid in the CTx. Generally, PNA showed higher binding ability than CTx. This work achieved to use glycan-mimetic branching to introduce specificity and selectivity as well as affinity into synthetic glycopolymers.

Wang et al. (2014a) developed RAFT-based one-step polymerizations to prepare tri-component statistical fluorescent glycopolymers as illustrated in Figure 7. Three different monomers including N-(2-hydroxyethyl) acrylamide (HEAA), N-(2-aminoethyl)methacrylamide (AEMA), and N-(2-glyconamidoethyl)-methacrylamides possessing varied pendant carbohydrates were used for the one-step tri-component RAFT-based copolymerization. Totally, five different glycomonomers were synthesized by using the same procedure. RAFT copolymerization was carried out in DMF using (4-cyanopentanoic acid)-4-dithiobenzoate as a RAFT agent and 4,4′-azobis-(4-cyanovaleric acid) as an initiator at 70°C for 24 h. Their subsequent modification with varied fluorescent labels due to functional primary amine groups on AEMA afforded a well-defined statistical tri-component glycopolymers with varied fluorescent labels demonstrating versatile reporters of specific lectins.

Pseudomonas aeruginosa lectin (PA-IL) bacteria and Galanthus nivalis plant lectin (GNL) were used to analyze the binding ability of these tri-component glycopolymers. While the α-mannose-containing polymer showed very strong binding with GNL, β-d-galactose-containing polymer showed enhanced binding ability with PA-IL. This report presented a new way to prepare a wide range of tri-component glycopolymers via RAFT-based one-pot polymerization.

Shi et al. (2012) reported the synthesis pyridyldisulfide (PDS) functional well-defined glycopolymer by RAFT polymerization of 2-(2,3,4,6-tetra-O-acetyl-β-d-glucosyl (oxy)ethyl methacrylate) (AcGlcEMA) glycomonomer in the presence of 4-cyanopentanoic acid dithiobenzoate (CPADB) and 4,4′-Azobis(4-cyanopentanoic acid) (ACPA) in 1,4-dioxane at 70°C for 24 h. The molar ratio of chain transfer agent to initiator was chosen as 10:1 and the polymerization was stopped at low monomer conversion (50–60%) in order to obtain efficient di-thioseter end functionalities for the subsequent modification with PDS. End-group modification was performed by aminolysis in the presence of ethanolamine and subsequently reaction with excess 2,2′-dithiodipyridine (DTP). After the removal of acetyl protecting groups, GSH was conjugated to PGlcEMA-PDS through the thiol–disulfide exchange reaction in 5 ml of phosphate buffer at pH 7.0 for 24 h under N₂ at room temperature as shown in Figure 8. The reaction was followed via a UV–Vis spectrophotometer at 343 nm and the pure glycopolymer–peptide bioconjugate was confirmed by the disappearance of the significant signals of pyridyl ring protons at δ 7.4–8.5 ppm. The binding affinity of the peptide-glycopolymer bioconjugate with ConA was been studied via UV–Vis spectroscopy, fluorescence quenching titration, and transmission electron microscopy (TEM) indicating good level of sugar–lectin binding as expected. Furthermore, its antioxidant activity was investigated in vitro using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The obtained results showed that PGlcEM-GSH bioconjugates are promising for the development of antioxidant delivery system, biomimetics, and biodetection.

An elegant strategy, based on the work of Godula and Bertozzi (2012) regarding preparing a series of fluorescent mucin mimetics displaying a range of α-N-acetylgalactosamine (GalNAc) valencies, was developed for the construction of a mucin mimetic glycopolymer microarray. To synthesize a glycopolymer as mucin mimicking, methylvinyl ketone was polymerized via RAFT using biotin containing trithiocarbonate chain transfer agent and 4,4′-azobis(4-yanovaleric acid) (ACVA) as a radical initiator in 2-butonane at 65°C for 16.5 h. The obtained polymers had biotin terminal functional groups at their one end for further conjugation to streptavidin-coated microarray substrates. The opposite end of the polymer chains was functionalized with a maleimide-functionalized Cy3 dye because of their use as a fluorescent label. The last step was the synthesis of biotin-terminated glycopolymer with a Cy3 label and the reaction was carried out in the presence of α-aminooxy-GalNAc under acidic conditions at 50°C for 20 h. After the attachment of the biotinylated mucin mimetics to the array surface with ~40–60% efficiency, their binding ability with four lectins [soybean agglutinin (SBA), Wisteria floribunda lectin (WFL), Vicia villosa-B-4 agglutinin (VVA), and Helix pomatia agglutin (HPA)] was examined. Generally, while HPA showed stronger avidities than other lectins toward all the polymers irrespective of their GalNAc valency, SBA showed propensity to cross-link the high-valency mucin mimetics. Interestingly, increasing in surface density array did not show any significant enhancement for the binding affinity of all lectins.

Alvárez-Paino et al. (2014) reported the synthesis of different amphiphilic glycopolymers as illustrated in Figure 9. Poly(ethylene glycol) methacrylate (PEGMA) was used to prepare a glycomonomer for further copolymerization methyl acrylate (MA) via free radical polymerization varying the initial feed composition. Firstly, PEGMA was activated with p-nitrophenylchloroformate in the presence of TEA in THF at 0°C for 24 h. After purification, the modified monomer with p-nitrophenyl carbonate groups was reacted with the glucosamine using TEA and hydroquinone in THF at room temperature to afford the glycomonomer. The copolymerization of MA and the obtained glycomonomer was carried out with azodiisobutyronitrile (AIBN) as an initiator in DMSO at 70°C. The different amphiphilic glycopolymers with the different ratio of the monomers were prepared by using this approach. The emulsion polymerization of MA was done with these glycopolymers as surfactants without the addition of any co-surfactant and the formation of colloidal stability, spherical, monodisperse, and small latex particles with low efficiency were achieved. The hydrophobic and insoluble part of these glycopolymers provided enhanced attachment of copolymer surfactant to the particles. Fluorescence microscopy was used to analyze the binding activity of these polymer-coated particles with ConA. The results showed the number of glycounits in the glycopolymer stabilizers affected the binding ability directly and significantly. Moreover, the films containing PEGMAGl-based copolymers showed higher binding affinity with ConA due to their flexibility.

In a similar approach, Sun et al. (2013) reported the effects of sugar regioisomerism (glycosidic linkage on different hydroxyl groups of the same sugar) on the binding ability of the self-assembled nanoparticles with lectins in a multivalent manner and the subsequent cell-uptake pathways as well. Basically, three self-assembled nanoparticles were prepared from triblock copolymers with the same polymeric backbone but different sugar regioisomers as pendant groups. Galactose was used as well-defined constitutional isomers due to its anomeric position (1-Gal) or 6-position (6-Gal) and a mannopyranoside (1-Man) as a control. Firstly, rod block of poly(9,9-dioctylfluorene) macroinitiator was conjugated with the bromine-functionalized polyfluorene (PF) initiator from the two ends of it for further occurring the middle block of the glycopolymers. Poly(glycidyl methacrylate) (PGMA) was synthesized via ATRP in the presence of the synthesized macroinitiator to yield triblock copolymer (PGMA-b-PF-b-PGMA). The pendent epoxide groups of the PGMA block were reacted with NaN₃ for the sugars modified with alkynes click targeting. 1-(2′-Propargyl)-d-galactose (α:β = 10:3) and 1-(2′-propargyl)-d-mannose were prepared and used for the click chemistry that yielded PF-1-Gal, PF-6-Gal, and PF-1-Man (Figure 10).

These glycopolymers contain the same middle rod block and side coil blocks with similar DP were performed for their self-assembly in water. Well-dispersed spheres investigated from TEM image took a shape from the polymer self-assembled into nanoparticles with the glyco block as the shell and the rod block as the core. These nanoparticles had the similar hydrodynamic radius between 19 and 38 nm with PDI around 0.21. These self-assembled nano-objects containing glycopolymers with well-defined isomerism on the surface showed some crucial properties such as high-water solubility, high stability during their binding to the specific lectins and the sustainability of multivalent binding tendency. The binding ability of these nanoparticles was tested with PNA and Erythrina crista-galli agglutinin (ECA). According to the results, even though NP-1-Man and NP-6-Gal did not show any binding ability with both PNA and ECA, NP-1-Gal showed strong binding with both lectins. Moreover, the asialoglycoprotein receptor (ASGPR) was used as a model of human lectin and its binding affinity with nanoparticles was examined by a quartz crystal microbalance (QCM). Expectedly, all nanoparticles showed similar binding ability with ASGPR due to previous investigations. This work is a very important proof to reveal how the effects of sugar regioisomersim in glycopolymers on their biological functions.

Muñoz-Bonilla et al. (2013) developed a very efficient approach to prepare a variety of amphiphilic block glycopolymers based on 2-{[(d-glucosamin-2-N-yl)carbonyl]oxy}ethylacrylate (HEAGI) and n-butyl acrylate (BA) or methyl methacrylate (MMA) by ATRP. They have reported the use of acrylic-based monomers and methacrylic glycomonomers on well-defined glycopolymers and presented an easy methodology to prepare amphiphilic glycopolymer. Firstly, unprotected glycomonomer (HEAGI) was synthesized according to procedure described previously. The monofunctional and difunctional macroinitiators of poly(BA) and poly(MMA) were prepared to use for the homopolymerization reaction of the glycomonomer carried out in DMF at 90°C by using N,N,N′,N′,N′-pentamethyldiethylenetriamine (PMDETA) and CuCl as a catalyst system. In this way, a small library of the well-defined amphiphilic block glycopolymers having di- and triblock glycopolymers with different hydrophobic blocks and varying the hydrophilic block lengths was demonstrated. These amphiphilic block glycopolymers showed the self-assembly ability to form micelles in aqueous solution. The dynamic light scattering (DLS) was used to determine the size of micelles at a copolymer concentration of 1 mg/ml at 25°C in distilled water or adding NaCl solutions. The DLS measurements revealed that the average diameters values are between 150 and 160 nm due to strong hydrogen bond interactions between hydroxyl groups and these polymers.

Moreover, their binding affinity with ConA was analyzed by using the turbidimetry. Interestingly, the results confirmed that the architecture of the block glycopolymers, diblock, or triblock glycopolymer did not show a significant influence on the lectin recognition process. It means that the glucose density over the polymer chain affects the molecular recognition independently in this study.

An elegant one-pot system was developed by Lu et al. (2014a) for the synthesis of novel porphyrin–glycopolymer conjugates. RAFT polymerization and one-pot conjugation reaction were used together to combine multi-reactions. Firstly, methacrylamido glucopyranose (MAG) was polymerized via RAFT using 2-cyanoprop-2-yl-a-dithionaphthalate (CPDN) as the RAFT agent and AIBN as the initiator. Then, the obtained glycopolymers were conjugated with protoporphyrin (PpIX) via one-pot reaction combining the multi-step reactions including reduction of RAFT end group to thiols, converting protoporphyrin to protoporphyrinogen, thiol-ene reaction of protoporphyrinogen with thiol-terminal glycopolymer, and the oxidation of protoporphyrinogen to yield the porphyrin–glycopolymer conjugate, respectively. Sodium mercury amalgam was used as a catalyst during the one-pot reaction (Figure 11). All these following reactions were confirmed by NMR, UV–Vis, and fluorescence spectroscopy.

The obtained porphyrin-PMAG conjugates showed the amphiphilic behavior in the water due to hydrophobic porphyrin in the middle and hydrophilic glycopolymer at both ends. Scanning electron microscopy (SEM) and DLS were used to characterize the self-assembled micelles. Their binding abilities with ConA as well as anti-cancer effect for cancer cells (K562) were studied. Glycomicelles showed high and specific binding ability with ConA. In in vitro studies, the cytotoxic test of glycoparticles against K562 cells in low doses revealed that these self-assembled micelles killed the cancer cells under light irradiation and light treatment length dependent manners. Therefore, this report is quite promising for the development of applications for cancer imaging and therapy.

Lou et al. (2014) achieved to produce novel galactose functionalized thermoresponsive injectable microgels. Poly(N-isopropylacrylamideco-6-O-vinyladipoyl-d-galactose) P(NIPA Am-co-VAGA) was synthesized via a combination of enzymatic transesterification and emulsion copolymerization. Firstly, 6-O-vinyladipoyl-d-galactose (VAGA) was prepared by controllable regioselective enzymatic transesterification with alkaline protease as a catalyst in anhydrous pyridine at 50°C for 4 days. The purification was performed by gel column chromatography. Subsequently, the free radical precipitation emulsion polymerization of NIPAAm with VAGA was performed in the presence of the crosslinker N,N-methylenebis(acrylamide) (BIS) and sodium dodecyl sulfate (SDS) in deionized water at 70°C using ammonium persulfate (APS) as an initiator to yield a series of galactose functionalized thermoresponsive crosslinked microgels. Field emission SEM and DLS were used to show the porous structure of microgels and a dramatic reduction in particle size when they were heated above their critical solution temperatures. The microgels became more viscous liquids that flowed easily and were injectable when they were heated up to body temperature. In in vitro study, the microgels were loaded with bovine serum albumin (BSA) and its release studied at 25 and 37°C (Figure 12). These results showed that the faster release rate of BSA was obtained below the LCST of the polymers.

This elegant report achieved the designing novel microgel drug delivery system that was the combination of themoresponsive and hepatocellular carcinoma targeting attributes into a single polymer. These novel thermoresponsive injectable microgels seem to have a potential for a wide range of biomaterials applications.

The achievement for the preparation of the modified gold nanorods (GNRs) with glycopolymeric coatings was employed by Lu et al. (2014b) The Cu(0)-catalyzed one-pot reaction combining SET-RAFT for the synthesis of glycopolymers was investigated for the first time in this study. Side-chain functionalized glycopolymers were prepared via one-pot and one-step technique. The polymerization and click reaction were carried out in situ using 2-cyanoprop-2-yl-a-dithionaphthalate (CPDN) as the RAFT agent and EBBr as the initiator in DMSO at 25°C. Subsequently, PMDETA was added and the reaction mixture was kept for 4 h. The polymerization kinetics revealed that the relationship between the molecular weight and the monomer conversion was linear with narrow polydispersity (Mw/Mn = 1.1–1.3). Therefore, this approach provided a design of polymers with special side-chain functionality. Moreover, the rate of click reaction was significantly higher than the polymerization rate. In order to make the glycopolymers being grafted to gold nanrods easily, the end-group reduction of the glycopolymers was undertaken in the presence of hexylamine/triethylamine as reductant at 50°C for 24 h. The thiol-terminal groups were confirmed by UV–Vis spectroscopy after the end-group modification. Then, these thiol-terminated glycopolymers covered the surface of gold nanorods to form a self-assembled monolayer on the GNPs surface due to the interaction of Au–S bond (Figure 13). The obtained glyco-nanorods were examined via TEM and DLS. According to the selective binding ability of PNA with galactosyl residues, PNA was used a model lectin to study the recognition ability of the nanoparticles with lectins. The results demonstrated that the glycopolymer substituted GNRs have a great binding affinity with PNA due to the sufficient numbers of galactose groups on the surface of the nanoparticles. This work opened new avenue for polymer chemists to prepare well-defined glycoparticles by using this one-pot/one-step technique.

Lu et al. (2014c) have recently been able to prepare glycopolymer-functionalized Ag nanoclusters (Gly–Ag NCs) that were fluorescent and presented high-binding ability and cytotoxity. RAFT polymerization was employed for the synthesis of sugar- and acid-containing polymers in the presence CPDN as the RAFT agent and AIBN as the initiator in DMF at 70°C. Then, these glycopolymers were mixed with AgNO₃ and then placed into the CEM instruments. Silver nanoclusters that are decorated with glycopolymers (PMAG-Ag NCs) were fabricated in the absence of reducing agents under microwave irradiation. Silver nanoclusters were chosen to decorate the glycopolymers due to their fluorescent and cytotoxic properties providing advantages in both cancer imaging and therapy. These nanoclusters were characterized using SEM, TEM, AAS, and fluorescence spectroscopy. These fluorescent nanoparticles could significantly bind to and show high cytotoxicity against GLUT over-expressing cancer cells K562. Moreover, they inhibited their viability possibly through the enhancement of reactive oxygen species (ROS) production. Therefore, the obtained nanoclusters are promising to treat cancers, such as multidrug-resistant tumors and eye-related neo-vascular diseases.

Kurtulus et al. (2014) developed a gene delivery system via the synthesis of spermine-like glycopolymer using RAFT. They reported for the first time to synthesize a new methacrylate monomer, namely, 2-((tert-butoxycarbonyl) (2-((tert-butoxycarbonyl)amino)-ethyl)amino)ethylmethacrylate (BocAEAEMA). After the RAFT polymerization of BocAEAEMA, P(BocAEAEMA) was used as a macro-RAFT agent to make chain extension with mannose acrylate (ManAc) synthesized in according to the procedure reported by Roy and Mukhopadhyay (2007) This chain extension reaction was undertaken in DMF at 70°C for 12 h using AIBN. After the deprotection of amine groups by removing BOC groups, P(AEAEMA)-b-P(ManAc) was obtained to analyze the binding ability of this well-defined glycopolymer with DNA. Even though P(AEAEMA) showed high-proton sponge capacity in comparison to polyethyleneimine (PEI) to form polyplexes with DNA because of electrostatic force between polymer and DNA, the glycopolymer block decreased this interaction between P(AEAEMA) and DNA regardless of their specific binding affinity with proteins. Besides, the size of P(AEAEMA)-b-P(ManAc) and DNA formed polyplex particles are found to be smaller than the size of P(AEAEMA) and DNA formed particles in according to DLS measurements due to the hydrophilic property of the glycopolymer. These synthesized spermine-like glycopolymers can be used as endosomal escaping agents for the applications of several treatments, such as gene therapies.

Zhou et al. (2012) reported a strategy for gene delivery system based on the synthesis of well-defined glycopolymers grafted onto Poly(l-lysine) (PLL). Despite of the significant condensation capacity with plasmid DNA due to positively charged hydrophilic amino group in water, PLL is not generally chosen as gene delivery vectors because of high cytotoxicity and low-transfection efficiency. In this study, to reduce the cytotoxicity and increase the transfection efficiency of PLL significantly, PLL was modified by well-defined saccharide-containing polymers. Firstly, glycomonomer, 2-O-meth-acryloyloxyethoxyl-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-(1–4)-2,3,6-tri-O-acetyl-β-d-gluco pyranoside, (MAEL) was polymerized via RAFT in the presence of CPADB and AIBN in chloroform at 70°C for 24 h. After the deprotection of the acetyl groups, the obtained glycopolymers were grafted onto PLL through the amidation reaction between the carboxyl group of the dithioester residue on the glycopolymer and the amino group on PLL mediated NHS and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to yield four different DPMAEL-g-PLL due to their different polyelectrolyte properties because of the N/P (charge ratio of amine to phosphate) ratios. The zeta potentials of these DPMAEL-g-PLL complexes were measured by DLS and an increase of the N/P ratio turned the zeta potentials to positive charge. The glycopolymer modified PLL showed lower cytotoxicity than PLL in according to the MTT assays with the mouse embryonic fibroblast cell line (NIH3T3) and human hepatoma cell line (HepG2). Moreover, these obtained complexes enhanced the transfection efficiencies of PLL, as well. As depicted in Figure 14, pDNA condensation by the DPMAEL-g-PLL complexes was investigated by agarose gel electrophoresis. The results demonstrated that low SD (substitution degree) on PLL increased the pDNA condensation capacity. This work allowed PLL to be more useful and applicable for gene delivery with enhanced biological properties.

Wang et al. (2014b) employed to synthesize the thermoresponsive glycopolymers poly(N-isopropyl-acrylamide-co-6-O-vinyladipoyl-d-glucose) (poly-NIPAM-co-OVDG; PND) and poly(N-isopropylacrylamide-co-6-O-vinylazelaicoyl-d-glucose) (poly-NIPAM-co-OVZG; PNZ) via free radical polymerization process. The polymerization reaction was undertaken in anhydrous ethanol at 60°C for 8 h with the presence of AIBN as an initiator. In total, a set of six copolymers was prepared with different ratios of NIPAM. Then, electrospinning process was used to prepare nanofibers comprising blends of poly-NIPAM-co-OVDG/poly-NIPAM-co-OVZG with poly-l-lactide-co-ε-caprolactone (PLCL). Before electrospinning process, the spinning solutions were prepared by dissolving of PND/PNZ and PLCL in a mixture of dichloromethane and acetone (2:1 v/v) at ambient temperature and stirring until forming a homogeneous solution. An electrical potential of 12 kV was applied while the spinning solution was flowing through a stainless steel capillary needle to a collector plate coated in Al foil. As depicted in Figure 15, the obtained nanofibers were characterized by using SEM. Additionally, MTT assay confirmed that these nanofibers have generally good biocompatibility with HeLa cells and minimum cytotoxicity, as well. Even though these nanofibers did not have sufficient ability to inhibit non-specific adsorption of bovine serum albumin onto their surfaces, they showed significant interaction with ConA. The results revealed that when these glycopolymer nanofibers are loaded with ConA, the adsorbed ConA can easily be desorbed with a glucose solution and used to induce death in the cell population. This work can be utilized easily into other glycosylated polymers given their temperature sensitive properties and have the sensitive and specific recognition with lectins.