The Science Of Polymers Biology Essay


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Synthetic polymers affect modern life so much that it's hard to imagine the world without them. From the last few decades, polymers are not only used in the automotive industry, semiconductor industry but are also widely used in the more advanced fields like nanotechnology, pharmaceutical industry in drug delivery and biomaterials (Hamerton 2002). Polymers or macromolecules are very large molecules with high molecular weight. Scientists are mainly focussing on the synthesis of polymers with desired structure and properties. Macromolecules are basically obtained by polymerisation of small molecules known as monomers which exhibits some specific properties. Due to these specific properties of monomers and by the help of initiator, solvent and catalyst, polymers of desired chain length, end groups, topology and side chain groups can be obtained. Well organised and controlled polymerisation techniques are needed for the controlled growth of the macromolecules (Morawetz 2002).

In 1956, Michael Szwarc discovered the living anionic polymerisation which has a great effect on the polymer science (Szwarc 1956, Szwarc et al. 1956). The work done by Michael Szwarc has leads to important development in the field of synthetic polymer science. Well-defined and organic polymers with good control on molar mass (Mn), polydispersity index (PDI) can be produced by using living radical polymerisation techniques. The basis of Szwarc's work was the elimination of termination and transfer reactions from the chain growth polymerisation (Syrett et al. 2010, Szwarc 1956). Synthetic chemists started focussing on the radical polymerisation because of the increasing demand of the ionic polymerisation, which leads in the development of controlled radical polymerisation (CRP) techniques in late 1990's. In other research fields also CRP attracted great attention.

CRP techniques works on the basis of equilibrium between the dormant and active species. To obtain well-defined macromolecules for a specific monomer or initiator, there is large variety of catalysts have been used. Therefore, there is a need of optimization reactions. Most of the chemical industries have established their own high-throughput experimentation laboratories. But there are very few laboratories which has the ability for the rapid screening and optimization of reactions by the help of automated parallel synthesizers. By using the parallel synthesis robots libraries of the compounds can be prepared under the similar experimental environment. In last decades, accurate and fast analysis of macromolecules is possible by the help of newly developed analytical tools.

CRP techniques help in the formation of polymers with specific and required functional groups at a specific position on the polymer chain with the help of initiators, monomers and catalysts. Block copolymers are also synthesised by using pre-synthesised macromolecules from post polymerisation modification reactions and their reaction with other small organic molecules like drugs and other macromolecules. Block copolymers also known as star-shaped copolymers has wide applications in nanotechnology and electronics.

The work done by Sharpless on the chirally catalysed oxidation reactions earned him to win Nobel Prize in 2001 (Syrett et al. 2010). In his work he also explained the concept of "click" chemistry. Click chemistry is the highly effective chemical reactions in between two easily approachable groups like azides and alkynes. Following the concept of click chemistry various reactions are carried out and are widely accepted in the field of biochemistry, medicinal chemistry and polymer science. The growing interest of "click" reactions among the scientists helps them to in the synthesis of macromolecules with future applications.

Controlled radical polymerisation techniques

To synthesise the well-defined polymers living radical polymerisation techniques always remain the first choice since 1956. In an anionic polymerisation reactions of styrene carried out by Szwarc showed that there is continuous increase in the polymer chain until all the monomer consumed in the reaction; with the addition of more monomer the polymer chain again started growing (Szwarc 1956). IUPAC stated that ionic polymerisation is a type of chain polymerisation reaction where ions or ion pairs acts as kinetic-chain carriers (IUPAC, goldbook). However, these techniques possess some limitations such as contrary between the monomers and reactive centres, need of highly pure chemicals and the limitation in the monomer selection due to specificity towards certain chemical groups.

These challenges in polymerisation reactions forced scientists to develop the new polymerisation techniques. Radical polymerisation is one of the alternative polymerisation techniques. Radical polymerisation techniques disfavour the polymerisation of vinyl monomers and found to be more progressive towards various functional groups. Free radical polymerisation is known as the most common route to obtain polymers with wide distribution in molar mass. But, in industries polymers having high polydispersity indices are advantageous for example, plasticizing effects is observed during processing of low molar mass polymers with small polymer chains and having high molar mass distributions. But, polymers with these properties are not suitable for future applications and also make the development of structure property relationships difficult for the polymer chemists.

In free radical polymerisation kinetics initiation reaction rates are extremely slower than the termination rates. Due to these reasons high molar mass chains formed during the initial stage but later with decrease in monomers concentration leads to the formation of low molar mass polymers are obtained, which also leads in the broad distributions in the molar mass of proteins. There are various recorded attempts which were carried out to gain improved control over free radical polymerisation (Moad and Rizzardo 1995). "Inferter" is the one of the technique used to control the free radical polymerisation. In these technique compounds perform their action as initiator, terminating and transferring agent (Ajayaghosh and Francis 1998, 1999, Otsu and Matsumoto 1998). In another technique bulky organic compounds like triarylmethyl derivatives were used (Borsig et al. 1967, Qin et al. 1999, Sebenik 1998). These techniques have various disadvantages like monomer reacts directly with the counter radicals, slow exchange and initiation and also thermal decomposition. Therefore a desired control on free radical polymerisation was not obtained from these techniques.

In mid 1990's new controlled radical polymerisation techniques were developed. These new techniques were developed by mainly focussing on the equilibrium between dormant and active species. Three main techniques Atom Transfer radical Polymerisation (ATRP) (Wang and Matyjaszewski 1995), Reversible Addition Fragmentation chain Transfer Polymerisation (RAFT) (Chiefari et al. 1998, Moad et al. 2008) and Nitroxide Mediated Radical Polymerisation (NMP) (Hawker et al. 2001, Moad and Rizzardo 1995) provide best control on the polymer growth. These three techniques gained most of the attention due to simplicity in the procedure and their ability to introduce stable chain end groups which can be reactivated by various post polymerisation changes.

Atom transfer radical polymerisation (ATRP)

In 1995, Sawamoto and Matyjaszewski was the first who reported ATRP technique (Wang and Matyjaszewski 1995). Among CRP techniques ATRP is the most widely used method. ATRP technique allows scientists to form polymers in piece-by-piece method and controlled manner just by putting together monomers. Polymers with specific functionalities and good polydispersity index (PDI) can be obtained using ATRP. ATRP allows the formation of complex polymer structures by using a specific catalyst that has capability to add one or more monomers to a growing polymer chain at a given time. By varying the temperature and other reaction conditions ATRP process can be shutdown and re-started. This provides a uniform and precise control on the architecture and composition of the polymer.

Nine international companies in Japan, USA and Europe are basically producing large variety of polymers based on the ATRP technique. ATRP is widely used in the preparation of pigments dispersants for cosmetics, adhesives, printing ink, chromatographic packing and sealants. ATRP technique has various other applications such as: preparation of coating material for cardiovascular stents, scaffoldings for bone regeneration, degradable plastics, and automobile industry and in drug delivery (Matyjaszewski et al. 2002).

The mechanism on which ATRP works is the reversible redox chemical reaction between transition metal complexes and alkyl halides. Metal complex leads to the reversible activation of the carbon-halogen terminals which helps the ATRP to proceed. Figure 1 shows the redox reaction between the halogen atom at the polymer terminal and metal centre.

Figure 1: General scheme for atom transfer radical polymerisation(Tang and Matyjaszewski 2006).

ATRP works similar to inner sphere electron transfer process, which contains homolytic halogen transfer between a lower oxidation state transition metal complex (Mtn/L) and dormant species (Pn-X) also known as initiator(Matyjaszewski 1998, Matyjaszewski et al. 2007, Matyjaszewski and Woodworth 1998). The transfer leads to the formation of radicals (Pn*) and higher oxidation state metal complex (X-Mtn+1/L). Halogen on the higher oxidation state metal complexes reacts with the free radicals to form Pn-X again or formation of oligomeric structures by the addition of monomer(Singleton et al. 2003). After sometime free radicals combine with the halogen form Mtn/L leads to the formation of dormant species, which usually depends on the deactivation rates. The metal complex activates the dormant species carbon-halogen bond and as a result to that a similar carbon-halogen bond formed at polymer terminal by various set of reactions. In a given time low concentration of free radicals but, there fast and reversible transformation into dormant species before addition to monomers are the key factors for ATRP.

Most ATRP requires four essential components which are needed to be added or to be formed in situ are:

Monomers which can radically polymerise.

An initiator with one transferrable group or atom preferably halogen atom.

A transition metal compound for one electron redox reaction.

A ligand to form a complex with transition metal compound.

Activation and deactivation constants define the rate for an ATRP reaction. Conversion of monomer (P), initiator concentration ([RX]), targeted degree of polymerisation (DPn), deactivator concentration X-CuIIY/Ln (denoted by [CuII]), and the ratio of propagation rate constant (kdeact) defines the PDI (Mw/Mn) for a polymer. It is more challenging to determine the kdeact directly but, if the values of kATRP and kact are known it can be calculated by using equation. The kATRP helps in determining the values for kp and the radical concentration ([Pm.]) and these values defines the values for rate of polymerisation. To determine all these values kATRP becomes more crucial. Catalyst CuIY/Ln e.g. [CuI] is used in less concentrations in the modern ATRP techniques(Qiu et al. 2000).

Ligands also play an important role in the ATRP procedure. Matyjaszewski et al. showed a comparison chart for the nitrogen based ligands (Tang and Matyjaszewski 2006). Figure 2 showing the EtBriB with activation rate constants (kact) for various ligands. Activation rate constant values are measured directly and converted in a logarithmic scale to compare the activities of copper complexes with various ligands. kact are sometimes underestimated for active complexes because of the extrapolation. Electrochemical studies states that Cu(II) catalyst will become more active when it is better stabilised by the ligand. In a general scheme the most stable complexes are formed by tetradentate ligands. Cyclam-B found to be most active ligands because Cu(II) catalyst is stabilised further by ethylene linkage. During the formation of Cu complex, cyclic ligands shows normal activity and shown in the middle of the scale. Left hand side of the scale shows most of the linear tetradentate ligands, except BPED. Moderately active complexes are formed by tridentate ligands e.g. BPMPA and PMDETA. Left hand side of the scale showing all the bidentate ligands which forms the slightest active complexes.

Figure 2: ATRP activation rate constants for various ligands with EtBriB in the presence of CuIY (Y) Br or Cl) in MeCN at 35 °C: N2, red; N3, black; N4, blue; amine/imine, solid; pyridine, open. Mixed, left-half solid; linear, ; branched, ; cyclic, (Tang and Matyjaszewski 2006).

Structure of the copper complexes also defines the activity and follows the following order: bidentate ligands < tetradentate (linear) < tridentate < tetradentate (cyclic) < tetradentate (branched) < tetradentate (cyclic-bridged). Nitrogen atom nature is also important and follows the order: imine < aliphatic amine ≤ pyridine. In case of linkage for nitrogen atoms ethylene is better than propylene. Ligand structure plays a wide role in the activity of copper complexes and large difference in activity will be observed for a very small change in structure.


Carbohydrates are known as natural saccharides which are widely used as biomass, raw material and in foods. At industrial scale also saccharides are modified chemically to develop materials such as surfactants, fibres and moisturizers (Miura 2007). In biology carbohydrates are involved in a number of crucial events including signal transmission, cellular recognition, etc. Carbohydrates can be used as ligands to improve the distribution of drugs in the biological systems.

Carbohydrates ligands binds to lectins on the cell surface that can act as a receptor with a strong affinity towards various drugs (Lee and Lee 2000). But, there are various parallel interactions that take place to get a strong interaction between the receptors on the cell surface and the drug targeting ligands (Lee and Lee 2000). A weak interaction is observed between the one carbohydrate molecule and one protein molecule. Therefore, to get a strong interaction carbohydrate molecules are placed along a polymer backbone which is known as glycocluster effect (Ting et al. 2010).

Polymers carrying carbohydrate functional groups are known as glycopolymers. Glycopolymers include linear glycopolymers, spherical glycopolymers and glycodendrimers in the form of nanoparticles and vesicles (Pieters 2009). These advanced materials have broad applications in the biological field such as: multivalent interactions with the lectins on the cell surface and the ability to bind mannose receptors. Miura studied the glycopolymers of poly (vinyl saccharide). She observed amplification in the interaction of protein-saccharide due to glycopolymers. She also suggested the use of glycopolymers to develop various biomaterials such as: in tissue engineering and pathogen inhibitor(Miura 2007). In another experiment Stenzel et al. carried out the synthesis of glycopolymers and studied there multivalent recognitions with plant, animal, bacteria and toxin lectins (Ting et al. 2010).

Polymer science and Click chemistry

In 2001, Sharpless and his co-workers introduced the concept of click chemistry defines a modular synthetic approach to generate new moieties by joining small units together using heteroatom linkage. Their main goal was to develop a chemical reaction which can be wide in scope and give very high yield and also generate few by-products(Kolb et al. 2001).

Click chemistry reactions have various characteristics:

No sensitivity towards water and oxygen.

Use of solvents and reagents that are easily available.

No need of solvent or use of solvent freely available like water.

Simple isolation of products without using any chromatographic techniques.


A variety of click chemistry reaction exists in organic chemistry with wide applications. From all the reactions which achieve "Click status", Huisgen 1, 3-dipolar cycloaddition (CuAAC) of azides and alkynes to give 1,2,3 triazoles is often regarded as the "Cream of the Crop" amongst such processes (Moses and Moorhouse 2007). There is some safety concerns related to reaction because of the azide moiety explosive nature. But, except this property azide moiety shows very classical properties like low susceptibility to hydrolysis. Figure 3 shows the general scheme for the CuAAC reaction.

Figure 3: General reaction scheme for Huisgen 1, 3-dipolar cycloaddition (CuAAC)(Nicolas et al. 2007).

In general azide and alkyne moieties can be regarded as biologically stable, inert, unique and very small. Catalysts such as transition metal ions greatly increase the reaction rate for the Huisgen 1, 3-dipolar cycloaddition of azides and alkynes. Presence of catalyst also provides stereospecifity to the reaction which makes this cycloaddition equivalent to click chemistry. Nitrogen based ligands and copper as catalyst helps to perform the reactions. Other transition metal ions (Pd, pt, Ru, Ni and Fe) and ligands (bipyridine based, terpyridine derivatives and PMDETA) are also examined to amplify the current field of copper-catalyzed cycloaddition reactions (Boren et al. 2008, Chassaing et al. 2008, Golas et al. 2006, Rodionov et al. 2005, Urbani et al. 2008)

The Interchange of both the moieties is possible like one moiety can tag the molecule and second moiety can be used for subsequent detection. Click chemistry has wide applications especially when the use of antibodies or direct labelling methods is not efficient. Molecules like nucleotides, amino acids and sugars can be easily tagged by using click chemistry label because of the small size of the. The building blocks act as effective enzymes substrates and with the help of enzymes they assemble to form biopolymers. Mild permeabilization of click chemistry detection molecules helps them to easily penetrate through complex structures like supercoiled DNA.

Chemists are working to develop new "Click" reactions which can work without the presence of any metal catalyst. Bertozzi was the one who first carried out the click chemistry for dynamic in-vivo imaging without using any copper catalyst (Baskin and Bertozzi 2007, Baskin et al. 2007). In 2008, Lutz presented an idea to perform azide-alkyne cycloaddition without any copper catalyst (Lutz 2008). Other, click chemistry reactions are also present which works in the absence of metal catalysts and follow the click chemistry requirements. Other click chemistry reactions are: radical addition, nucleophilic substitution, Diels-Alder and Retro-Diels Alder reactions. Potential toxicity due to metal catalysts plays a vital role when the synthesised products are to be used in biological applications (Wang et al. 2003). In case of copper-catalyzed azide-alkyne cycloaddition, a ppm amount of copper remains in the product even after the purification. Therefore, there is a significant need of alternative click reactions which don't need any metal catalyst.

Post-polymerisation modifications, curing reactions and certain polymerisation reactions are widely performed by free-radical addition of thiol to a double bond (David and Kornfield 2008, Dondoni 2008, Nilsson et al. 2008, Willcock and O'Reilly 2010). Schlaad et al. synthesised poly [2-(3-butyl)-2-oxazoline] using living/controlled cationic isomerisation polymerisation and also performed the thio-click modification of the synthesised polymer. Model reactions were also carried out using various mercaptans e.g. dihydroxy functionalized thiols, fluorinated thiols and acetylated glucose thiols. To perform the "thio-click" reaction mild conditions (UV light to generate radicals) are used and also the reaction was carried out in the absence of transition metal (Gress et al. 2007). In an another report Schlaad et al. described the "thio-ene" modification of 1,2-polybutadiene polymer. In this experiment they used sunlight to produce radicals. This method is well suited for the production of biohybrid polymers containing sugars or amino acids on its backbone (ten Brummelhuis et al. 2008). In 2008, Hawker et al. presented a divergent approach for the synthesis of Poly (thioether) dendrimers using thiol-ene "click" chemistry. They performed the thiol-ene reactions without using any metal catalyst and solvent. A UV lamp with wavelength of 365 nm was used for 30 minutes for the irradiation of reaction mixture. A small amount of photoinitiator was also used to increase the rate of reaction by increasing the radical concentration(Killops et al. 2008).

Figure 4: Showing the end group modification of the synthesised RAFT polymers(Willcock and O'Reilly 2010).

Click chemistry technique has wide applications in the production of glycopolymers for biomedical applications. Perrier et al. reported a new strategy for the synthesis of hyper-branched and highly functionalised glycopolymers. They used the combination of living radical polymerisation and click chemistry. RAFT copolymerisation was used to synthesise the highly branched clickable backbone of the TMS-protected alkyne acrylate monomer with EGDMA. A group of "click" chemistry reactions mainly CuAAC and also, thiol-yne and thiol-ene addition were carried out to click glucose and galactose moieties on the highly branched polymer chain(Semsarilar et al. 2010). Haddleton and Mantovani established the one-pot synthesis of the glycopolymers by concurrent ATRP of an alkynyl monomer and copper catalysed azide-alkyne cycloaddition with azido functionalised sugars (Geng et al. 2008).


This project is framed within a wider project which has the ultimate goal to develop the cancer vaccines. This part of the project was to synthesise protein-glycopolymers conjugates which we could use to start mannose receptor and cell internalisation experiments. During this project we carried out numerous experiments. In the first part of the project synthesis of sugar azides and monomer was carried out. Later, atom transfer radical polymerisation (ATRP) of the synthesised monomer was carried out to obtain our polymers.

Scheme 1: General synthetic strategy followed in this work.

Two types of polymers were synthesised one with large chain and second with small chain by using same techniques. After polymerisation synthesis of glycopolymers for both the synthesised polymers was carried out using "click" chemistry. In the end, conjugation of the glycopolymers was performed with the "miniOva" protein. (expand: why are we using this protein?)

Experimental procedure

2.1 Model ATRP experiment

Model ATRP reaction was carried out to understand the procedure for ATRP. To perform the synthesis 2-dimethyl aminoethyl methacrylate monomer (4.0 ml, 23.5 mmmol), (E)-N-(pyridine-2-yl methylene) propan-1-amine ligand (73 µl, 0.47 mmol) and Benzyl-2-Bromo-2-methyl-propanoate initiator (60.5 µl, 0.25 mmol) was added in a dry Schlenk tube along with 2.5 ml of toulene. The Schlenk tube was sealed with a rubber stopper and three freeze-pump-thaw cycles were carried out. Then in the frozen mixture copper bromide (33.5 mg, 0.25 mmol) was added and again three cycles of nitrogen-vacuum were carried out. The Schlenk tube was kept at 80oC temperature with continuous stirring. 1H NMR samples were taken for the reaction timely to check the conversion of the monomer with time. After 95% conversion reaction was stopped and the stopper was removed. The reaction mixture was filtered through alumina to remove the catalyst and toluene was added. The resulting solution was treated with rotavapour to reduce the volume and precipitated using petroleum ether. The polymer was sticking to the walls of the beaker which was removed by the help of DCM. DCM was removed using rotavapour and final product was stored carefully. 3.5 g of final product was obtained (Figure 7).

Polymer with Mw = 14.0 kDa, Mn = 11.0 kDa, Mw/Mn = 1.2

Synthesis of sugar azides

Synthesis of mannose azide

2'-bromoethyl--D-mannopyranoside (1)

Mannose (10 g, 55.5 mmol) was dissolved in bromoethanol (78.68 ml, 1,110 mmol) and then amberlite IR-125 (10 g) was added. Then mixture was heated at 90oC in 250 ml round bottom flask equipped with a refluxing unit. After 30 minutes reaction was stopped and 13C NMR was carried out using DMSO. The suspension was filtered using short cotton wool pad to remove the amberlite. The filtered solution was washed with water and DCM three times and water phase was collected. The water phase was then placed on the freeze drier to remove the water. After the drying finished product was removed from the freeze drier and dissolved in methanol and precipitated in diethyl ether. 13.5 g of 2'-bromoethyl--D-mannopyranoside (47 mmol, 85 %) was obtained and, as the 13C NMR spectrum was found to be identical to the one reported in the literature for the same derivative, was used for the next step without further purification.

2'-azidoethyl--D-mannopyranoside (2)

2'-bromoethyl--D-mannopyranoside (13.51 g, 27 mmol) and sodium azide (3.5 g, 54 mmol) were dissolved in 19.5 ml of water and 116.5 ml of acetone. When the solution became slightly turbid, it was heated till reflux (55oC) and stirred continuously. 13C NMR of the reaction mixture was carried out using D2O which confirmed the formation of 2'-azidoethyl--D-mannopyranoside. The acetone in the reaction mixture was removed using rotavapour and water phase was placed on freeze drier to get the final product. Column push chromatography was carried out to get the pure product(Geng et al. 2007). 5.5 g of final product was obtained (Figure 5).

IR (neat): = 3410, 2931, 2112, 1637, 1617, 1347, 1218, 1136, 1084 cm-1

1H NMR (400.03 MHz, D2O, 298 K) δ = 3.45 (m, 2H, CH2N3); 3.55-3.60 (m, 2H, CH2CH2N3); 3.61-3.67 (m, 2H, CH2OH); 3.67-3.91 (m, 4H, 4Ã-CH); 4.92 (d, J = 1. 5 Hz, 1H, CH).

13C{1H} NMR (100.59 MHz, D2O, 298 K) δ = 50.4 (1C, CH2N3); 61.3 (1C, CH2OH); 66.1 (1C, CH2CH2O); 67.2 (1C, CH); 70.5 (1C, CH); 71.2 (1C, CH); 73.9 (1C, CH); 100.2 (Canomeric);

Mass Spectrometry (+ESI-MS) m/z (%): 248 (100), 212 (60), 127(40).

Synthesis of galactose azide

2'-bromoethyl--D-galactopyranoside (3)

Galactose (6.5 g, 35 mmol) was dissolved in bromoethanol (50 ml, 705.5 mmol) and then amberlite IR-125 (6.35 g) was added. Then mixture was heated at 90oC in 250 ml round bottom flask equipped with a refluxing unit. After 30 minutes reaction was stopped and 13C NMR was carried out using DMSO. The suspension was filtered using short cotton wool pad to remove the amberlite. The filtered solution was washed with water and DCM three times and water phase was collected. The water phase was then placed on the freeze drier to remove the water. After the drying finished product was removed from the freeze drier and dissolved in methanol and precipitated in diethyl ether. 7.77 g of 2'-bromoethyl-D-galactopyranoside was obtained.

2'-azidoethyl--D-galactopyranoside (4)

2'-bromoethyl--D-galactopyranoside (7.77 g, 27 mmol) and sodium azide (3.5 g, 54 mmol) were dissolved in 34 ml of water and 116.5 ml of acetone. When the solution became slightly turbid, it was heated till reflux (55oC) and stirred continuously. 13C NMR of the reaction mixture was carried out using D2O which confirmed the formation of 2'-azidoethyl--D-galactopyranoside. The acetone in the reaction mixture was removed using rotavapour and water phase was placed on freeze drier to get the final product. Column push chromatography was carried out to get the pure product(Geng et al. 2007). 4.5 g of final product was obtained. The 1H/13C NMR analysis results obtained was similar to the previous recorded experiment (Figure 5).

IR (neat): =3322, 2953, 2098, 1644, 1303, 1265, 1121, 1061, 998, 910 cm-1

1H NMR (400.03 MHz, D2O, 298 K) δ = 3.57 (m, 2H, CH2N3); 3.60 (m, 1H, CH) 3.64-3.72 (m, 2H, CH2CH2N3); 3.76-3.80 (m, 2H, CH2OH); 3.83 (m, 1H, CH); 3.93 (m, 1H, CH); 4.05 (m, 1H, CH); 4.46 (d , J = 7.78Hz, 1H, CH). 13C{1H} NMR (100.59 MHz, D2O, 298 K) δ = 50.55 (1C, CH2N3); 60.95 (1C, CH2OH); 68.38 (1C, CH2CH2O); 68.63 (1C, CH); 70.69 (1C, CH); 72.71 (1C, CH); 75.18 (1C, CH); 102.89 (Canomeric); Anal. Calcd. for C8H15N3O6 C, 38.55; H, 6.07; N, 16.86; Found: C, 38.62; H, 6.00; N, 16.73; Mass Spectrometry (+ESI-MS) m/z (%): 102 (100), 118 (34), 172 (42), 217 (11), 272 [M+Na] (65) (Geng et al. 2007).

Synthesis of monomer (5)

A solution of 3-(trimethylsilyl) prop-2-yn-1-ol (12.5 mg, 97.5 mmol) and Et3N (17.25 mg, 127 mmol) was made in 125 ml of Et2O and kept at -20oC with continuous stirring. Another solution of methacryloyl chloride (11 ml, 116 mmol) was prepared in 62.5 ml of Et2O and added dropwise in the above prepared solution. The resulting solution was stirred overnight at room temperature. 1H NMR confirmed the finishing of the reaction. Triethyl ammonium salt was removed from the solution by filtration and the remaining solution was treated with rotavapour to reduce the volume.

The obtained product was dissolved in petroleum ether to form a suspension and treated with rotavapour to reduce the volume. The final product obtained was purified using column push chromatography. 1H NMR using chloroform confirms the final product 2-Methyl-acrylic acid 3-trimethylsilanyl-prop-2-ynyl ester(Geng et al. 2007) (Figure 6).

The 1H NMR (400 MHz, CDCl3, 298K) δ = 0.16 (s, 9H, Si(CH3)3); 1.93-1.94 (m, 3H, CH3C=CH2); 4.73 (s, 2H, OCH2); 5.58-5.59 (m, 1H, C=CHH); 6.14 (m, 1H, C=CHH)

13C {1H} NMR (400 MHz, CDCl3, 298K) δ = -0.2 (3C, Si(CH3)3); 18.4 (1C, CH3C=CH2); 53.0 (1C, OCH2); 92.0 (1C, C≡CSi(CH3)3); 99.2 (1C, C≡C Si(CH3)3); 126.5 (1C, CH3C=CH2); 135.8 (1C, CH3C=CH2); 166.6 (1C, COester).

IR (neat): á¹½ = 2960, 2186, 1727, 1638, 1452, 1366, 1314, 1292, 1251, 1147, 1035, 971, 942, 842, 813, 761 cm-1)

Mass Spectrometry (+ESI-MS) m/z (%): 245, 180 REPEAT.

Synthesis of polymer (8a, 8b)

Two types of polymers was synthesised with different chain length using the same below defined procedure and conditions. To perform the synthesis monomer (5) (5 mg, 24 mmmol), [2,2']Bipyridyl ligand (187 mg, 2 mmol) and 2-Bromo-2-methyl-propionic acid 3-(pyridin-2-yldisulfanyl)-propyl ester initiator (6) (419 mg, 2 mmol) was added in a dry Schlenk tube along with 12.2 ml of anisole. The Schlenk tube was sealed with a rubber stopper and three freeze-pump-thaw cycles were carried out. Then in the frozen mixture copper bromide (57 mg, 0.40 mmol) was added and again three cycles of nitrogen-vacuum were carried out. The Schlenk tube was kept at room temperature with continuous stirring. 1H NMR samples were taken for the reaction timely to check the conversion of the monomer with time. After 73% conversion reaction was stopped and the stopper was removed. The reaction mixture was filtered through alumina to remove the catalyst and toluene was added. In the end, polymer with trimethylsilyl group (7) was obtained.

Removal of Si(CH3)3 protecting group (8a, 8b)

For the deprotection of the polymer, acetic acid (2.1 ml, 36 mmol) was added and placed in the ice-acetone bath to maintain temperature -20oC. TBAF 1.0M (29.0 ml, 29.0 mmol) in THF was then added dropwise and the reaction mixture was allowed to stir overnight. Deprotection was checked timely by performing 1H NMR. After the deprotection finished amberlite IR-20 ion exchange resin was added (32.6 g, 72 mmol) and stirred continuously. After 4 hours of stirring amberlite was filtered off using cotton wool pad and the polymer solution was washed with water. The remaining solution was treated with rotavapour and precipitated in petroleum ether. Gel permeation chromatography was performed to calculate the molecular weight and PDI (Figure 8).

Polymer with D.P. = 72, Mw = 12.0 kDa, Mn = 8.0 kDa, Mw/Mn = 1.5

Synthesis of glycopolymers

Glycopolymers were synthesised for both the polymers (8a, 8b).In the synthesis of glycopolymers a fluorescent dye (9) "Oregon" was added to facilitate the characterisation of the glycopolymers in the further studies. The glycopolymers were synthesised using different concentrations of sugar azides; mannose azide and galactose azide.

For the synthesis of glycopolymers containing 100% mannose units (10a, 10b) 100 mg (0.509 mmol) of polymer, mannose azide (254 mg, 1.0 mmol), Oregon dye (3 mg, 0.01 mmol) and [2,2']Bipyridyl (15.9 mg, 0.109 mmol) was charged in Schlenk tube along with DMF and three cycles of freeze-pump-thaw were carried out. To the frozen solution copper bromide (7.3 mg, 0.051 mmol) was added and three nitrogen-vacuum cycles were performed. The tube was kept at room temperature with continuous stirring.

In the synthesis of glycopolymers containing 66% mannose (11a, 11b) same procedure was followed but, 161.6 mg (0.70 mmol) of mannose azide and 86.3 mg (0.35 mmol) of galactose azide was used. For the synthesis of glycopolymers containing 34% mannose (12a, 12b), mannose azide (86.3 mg, 0.35 mmol) and galactose azide (167.6 mg, 0.70 mmol) was used and same procedure was followed. Glycopolymers containing 0% mannose (13a, 13b) were also synthesised using galactose azide (254 mg, 1.0 mmol) and same other compounds and same procedure as followed for other glycopolymers.

The progress of all the reactions was monitored timely by carrying out 1H NMR. After the completion of reaction, the reaction mixture was transferred in a solution of 30 ml distilled water and 2 g of sodium sulphide and stirred for 30 minutes. The solution was filtered to remove the sodium sulphide salt and then dialysis was performed for the water solution containing glycopolymers. After dialysis water was removed using freeze drier and also P2O5. Same procedure was performed for all the glycopolymers. Final products were stored in a closed container at room temperature. Figure 9 shows the reaction sequence for the production of glycopolymers (10a, 10b, 11a, 11b, 12a, 12b, 13a, 13b). Molecular weight for the glycopolymers was found to be 27.2 kDa.

Results and discussion

3.1 Synthesis of the required sugar azides (2) and (4)

The required mannose and galactose sugar azides were prepared as described in Figure 5. Briefly either D-mannose or D-galactose was suspended in an excess (10 equivalents) of 2-bromoethanol and amberlite IR-125(H+ form) acid catalyst was added at 90°C. Condensation occurred with elimination of water and was monitored by 13C NMR by following the disappearance of the sugar anomeric carbon (~93 ppm) and the appearance of a anomeric glycoside signal at ~104 ppm. Particular care was taken in making sure that after purification (water/dichloromethane washings followed by freeze-drying) no residual bromoethanol was left. This because we wanted to avoid the formation of potentially explosive 2-azidoethanol in the following step. Nucleophilic substitution of the obtained products was further carried out with sodium azide, water and acetone to obtain the final product 2'-azidoethyl--D-mannopyranoside (2) and 2'-azidoethyl--D-galactopyranoside (4) (Figure 5).

Figure 5: Reagents and conditions: (a) bromoethanol, amberlite IR-125, 90oC; (b) sodium azide, water, acetone, 90oC.

The synthesis of mannose azide was a simple process but, special care was taken to avoid any explosion due to the highly explosive nature of the azide moiety. The final product obtained was not pure enough that's why a column push chromatography was carried out to purify it further. Need NMR spectra

3.2 Monomer (5)

Synthesis of monomer 2-Methyl-acrylic acid 3-trimethylsilanyl-prop-2-ynyl ester (5) protected by trimethylsilyl group was carried out. The synthesis was performed using 3-(trimethylsilyl) prop-2-yn-1-ol and methacryloyl chloride in the presence of Et3N and Et2O at a temperature of -20oC.

Figure 6: Reagents and conditions: Et3N, Et2O and methacryloyl chloride.

Traces of the starting material were observed even after the reaction finished. To remove the traces column push chromatography was carried out. Monomer (5) was found to be volatile so, proper care was taken during the treatment with rotavapour to avoid the loss of the product (Figure 6). 1H NMR of the product shows a shift in the peek from 4.2 ppm to 4.7 ppm which confirms the formation of the final product (Figure 7).

Figure 7: 1H NMR spectra for the monomer (5) showing various peeks.

3.3 Polymerisation experiments

3.3.1 Model ATRP experiment: controlled polymerisation of 2-dimethyl aminoethyl methacrylate

In order to familiarise with controlled polymerisation techniques and getting the required skills for working under inert atmosphere using Schlenk lines an ATRP of methyl methacrylate model monomer was carried out. During model ATRP, controlled polymerisation of 2-dimethyl aminoethyl methacrylate was carried out in toluene. The polymerisation was carried out using (E)-N-(pyridine-2-yl methylene) propan-1-amine/copper bromide as the catalyst and benzyl-2-bromo-2-methyl propanoate as the initiator at 80oC (Figure 8).

Figure 8: Reagents and conditions: (a) (E)-N-(pyridine-2-yl methylene) propan-1-amine (b) copper bromide, 80oC.

1H NMR of the polymer shows a presence of a new peek at ~4.2 ppm which is the confirmation for the formation of final product. A gradual increase in polymer peek was observed with time which means more conversion of monomer to polymer (Figure 9).

Figure 9: 1H NMR spectra for the model ATRP polymer showing the peeks for monomer and the polymer.

Some deviation from the linearity was observed in first-order kinetic plots during the polymerisation. But, overall polymerisation was occurred in a control manner because of the linear increase in Mn with the conversion of monomer as well as slight molecular weight distributions. Graph 1 shows the (a) processed conversion Vs time (b) Mn Vs conversion (c) PDI Vs conversion first order kinetic plots for the model ATRP.




Graph 1: (a) Processed conversion Vs time (b) Mn Vs conversion (c) PDI Vs conversion, first order kinetic plots for the model ATRP.

3.3.2 ATRP to obtain clickable alkyne polymers (8a, 8b)

Polymers with different chain length were synthesised depending on the ability of monomer (5) to act as a versatile "clickable" polymeric scaffold (Figure 10). Polymerisation reaction was performed by using initiator (6) and copper bromide/ [2,2'] Bipyridyl as a catalyst in the presence of anisole. Both polymers were synthesised using same procedure and under the same conditions.

Figure 10: Reagents and conditions: (a) 5, [2,2']Bipyridyl, anisole, copper bromide, room temperature; (b) acetic acid, TBAF 1.0M in THF, amberlite IR-20, -20oC.

Retro-Diels-Alder deprotection was carried out for both the polymers using TBAF in THF, acetic acid and amberlite IR-20 to remove the trimethylsilyl protective group. In a previous experiment by Ladmiral et al, TBAF mediated deprotection was performed but lower terminal alkyne content was observed (Ladmiral et al. 2006). So, in this new case acetic acid was used as a buffering agent to obtain terminal alkyne polymers in virtually 100% yield. For both the polymers some deviation from the linearity was observed in first-order kinetic plots during the polymerisation. But, overall polymerisation was occurred in a control manner because of the linear increase in Mn with the conversion of monomer as well as slight molecular weight distributions. The polymer (8a) was carrying 72 monomer units and polymer (8b) was carrying 40 monomer units (Figure 8). The final products obtained were pure enough which leads to no further purification of the products. 1H NMR shows a peek at ~2.52 ppm which confirms the formation of terminal alkyne polymer (Figure 11).

Figure 11: 1H NMR spectra for the polymers (8a, 8b) showing various peeks.

3.4 CuAAC experiments: synthesis of libraries of glycopolymers

Glycopolymers containing varying sugar azide units were synthesised for both the polymers (8a, 8b). The glycopolymers were tagged with a fluorescent dye "Oregon" to ease the characterisation during the various in vitro studies like Lectin recognition analysis, PEGlyation chemistry, L-selectin binding and also in spermatozoa stability studies. Glycopolymers were synthesised using copper bromide/ [2,2'] Bipyridyl as a catalytic system.

Figure 12: Reagents and conditions: (a) [2,2']Bipyridyl, DMF, ambient temperature; (b) H2O, sodium sulphide, ambient temperature.

The products obtained by click chemistry were pure enough which leads to no further purification. Water present in the glycopolymers was removed using freeze drier and P2O5 as desiccator. To check how well the fluorescent dye was tagged with the glycopolymers, glycopolymers were studied under fluorimeter. Results obtained from the fluorimeter shows that glycopolymers are tagged with dye accurately (= 465 nm). The molecular weight of glycopolymers was observed to be 27.2 kDa. 1H NMR of the glycopolymers shows the formation of triazole (8.3 ppm) and also a peek observed at ~ 5.2 ppm which confirmed the completion of reaction (Figure 13). Libraries of the glycopolymers were almost similar in the macromolecular features (macromolecular architecture, Mn, PDI).

100% mannose with assignments.png

Figure 13: 1H NMR spectra for the glycopolymers showing various peeks.

3.5 Protein conjugation experiments

3.5.1 Lectin ELISA in order to analyse the binding of Glycopolymers with MR constructs (CRD4-7)

Recognition of mannose receptors which were obtained from the Chinese Hamster cells was observed for the libraries of the glycopolymers using Lectin ELISA and Inhibition-ELISA. SDS-PAGE and Western Blot analysis was also carried out. (Need further information)

ELISA procedure was carried out in collaboration with University of Nottingham Medical School. Mannose receptor belongs to class of C-type lectins molecules which binds to the carbohydrates in a calcium-dependent manner. Mannose receptor specificity depends on the eight CRD containing C-type lectin domains (McGreal et al. 2006). Out of eight domains CRD4-7 construct has a strong affinity of binding with carbohydrates and rest of the domains don't have this affinity. We used CRD1-3 and CRD4-7 mannose receptor constructs to check the binding ability of the glycopolymers with mannose receptor (MR), this will be useful in the cell internalisation experiments.

Lectin ELISA was performed by using different concentration solution of glycopolymers and following the standard ELISA procedure and the results were recorded. The binding affinity of the CRD4-7 MR construct was observed to be dependent on the density of the sugar epitopes present in the glycopolymers. There was no binding observed for the CRD1-3 MR construct for any of the glycopolymer. The binding of the glycopolymers to CRD4-7 follows the following order 66% mannose (11a) > 100% mannose (10a) > 34% mannose (12a) > 0% mannose (13a) as shown in Graph 2.





Graph 2: (a) 100% mannose binding affinity (b) 66% mannose binding affinity (c) 34% mannose binding affinity (d) 0% mannose binding affinity with CRD4-7 MR construct.

3.5.2 Inhibition ELISA

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