Synthesis of CD-based glycocluster-smart star polymer hybrids via combination of

Scheme 4. 3. Schematic representation for the synthesis of CD-based glycocluster-

smart star polymer hybrids via combination of ATRP and CuAAC and their LCST

self-assembled behaviour.

CD-centered star responsive polymers have interesting self-assembling behaviours and could form different type vesicles by tuning solution pH or temperature, which

has potential application in controlled drug delivery.200 Star-shaped block copolymers

have also been applied as universal hydrophobic drug carriers using the host-guest

interaction.201,202 Thus this inspired us to synthesize such Janus-type star responsive

polymers with sugar moieties for conjugation and potential drug delivery, through

which the surface sugar intensity should also be increased via specific self-assembly

in water by tuning the temperature.

The strategy was to first use the azide functionalised CD-based initiator for ATRP of NIPAM or DEGEEA and to subsequently use CuAAC to attach the monosaccharide on the primary face of cyclodextrin, Scheme 4.3.

The polymerization of NIPAM was performed under the catalysis of CuCl/Me6TREN

[Cyclodextrin centered glycoconjugates] [Chapter 4]

enough Cu(I) in the system to initiate ATRP at ambient room temperature, in Scheme 4.4.

Scheme 4. 4. Schematic representation for the synthesis of cyclodextrin-based

PNIPAM via ATRP.

In a similar to a previous report203, the first-order kinetic plots (Figure 4.6) show

significant curvature and in that report this was attributed to the 2-propanol solvent, which was thought to hydrogen-bond to monomer and polymer and thus reduce the deactivation of the polymerization catalyst. However, low dispersities were obtained

during the reaction and GPC analysis of the final polymer showed Mn,SEC = 21.6 kDa

and Mw/Mn = 1.25.

Figure 4. 6. Semi–logarithmic kinetic plots (left) and molecular weight and PDi vs

reaction time plots for polymerization of NIPAM from (N3)7-β-CD-(Br)10.

The polymerization of diethylene glycol methyl ether methacrylate (DEGEEA) was

conducted in toluene under the catalysis of CuBr/ N-ethyl-2-pyridylmethanimine at 60

°C. The kinetic results showed the linear dependence of Ln[M]0/[M] versus time in

Figure 4.7, which indicated that the chain radical concentration remained constant during the polymerization. The molecular weight of obtained polymer increase linearly with time and the PDI remained low (~1.2) during the polymerization, which indicated the living nature for the polymerization of DEGMEMA.

[Cyclodextrin centered glycoconjugates] [Chapter 4]

Figure 4. 7. Semi–logarithmic kinetic plots (left) and molecular weight and PDI vs

reaction time plots (right) for ATRP of DEGMEMA from (N3)7-β-CD-(Br)10.

Subsequently, the CD-centered PNIPAM with azide groups on the primary face of the CD was used for the CuAAC with 1-(2′-propargyl) D-mannose in DMSO under the catalysis of CuBr/bpy. The success of the click reaction was demonstrated by the

disappearance of azide group absorbance at 2100 cm-1 in the FT-IR spectrum (Figure

4.8) and the LCST temperature shifting from 31.8 to 36.9 °C due to the very hydrophilic sugar hydroxyl groups (Figure 4.9).

The SEC and NMR analysis did not reveal significant change due to the low content of mannose moieties compared with PNIPAM. The elution traces only changed

slightly after click reaction, Figure 4.10. Mn calculated from PMMA standards

showed that it increased from 21600 to 21800 and the PDi kept the same at 1.25. Due to the low ratio of mannose compared with PNIPAM component and also the overlap

between N-H and tri-azole ring proton at 7 – 9 ppm, 1H NMR only revealed PNIPAM

components and did not show significant peaks from mannose, Figure 4.11.

Figure 4. 8. FT-IR spectra obtained for propargyl mannose, (N3)7-β-CD-(PNIPAM)10

[Cyclodextrin centered glycoconjugates] [Chapter 4]

Figure 4. 9. Cloud point measurements of (N3)7-β-CD-(PNIPAM)10, (Mannose)7-β-

CD-(PNIPAM)10 (left) and (N3)7-β-CD-(PDEGMEMA)10-(Br)10, (N3)7-β-CD-

(PDEGMEMA)10-(N3)10 and (Mannose)7-β-CD-(PNIPAM)10-(Mannose)10 (right) by

UV/Vis spectroscopy at 500 nm. The analytes concentrations in water are all 1 mg/mL.

Figure 4. 10. Normalized SEC elution traces for (N3)7-β-CD-(PNIPAM)10 and

(Mannose)7-β-CD-(PNIPAM)10 via RI detector. The two samples were injected in

sequence at the same time.

The click reaction of CD-centered PDEGMEMA with 1-(2′-propargyl) D-mannose was first conducted under the same conditions. However, after the reaction SEC (Figure 4.12) showed a significant star-star coupling shoulder peak at higher MW region, which is probably due to the similar click and ATRP reaction conditions.

[Cyclodextrin centered glycoconjugates] [Chapter 4]

Figure 4. 11. 1H NMR spectra recorded in D2O for (N3)7-β-CD-(PNIPAM)10 (left)

and (Mannose)7-β-CD-(PNIPAM)10 (right).

Figure 4. 12. Normalized DMF SEC traces of (N3)7-β-CD-(PDEGMEMA)10 and

(Mannose)7-β-CD-(PNIPAM)10.

Figure 4. 13. Normalized DMF SEC traces of (N3)7-β-CD-(PDEGMEMA)10-(Br)10,

(N3)7-β-CD-(PDEGMEMA)10-(N3)10 and (Mannose)7-β-CD-(PNIPAM)10-

[Cyclodextrin centered glycoconjugates] [Chapter 4]

Figure 4. 14. FT-IR spectra obtained for propargyl mannose, (N3)7-β-CD-

(PDEGMEMA)10-(Br)10, (N3)7-β-CD-(PDEGMEMA)10-(N3)10 and (mannose)7-β-CD-

(PNIPAM)10-(mannose)10.

Thus the terminal bromine groups were first transformed into azide groups to avoid the star-star coupling which will also attach more sugar on the star polymers. After

reaction, DMF SEC analysis (Figure 4.13) revealed Mn = 22.0 KDa and PDi = 1.23

for (N3)7-β-CD-(PDEGMEMA)10-(N3)10, which almost has no change compared with

(N3)7-β-CD-(PDEGMEMA)10-(Br)10. FT-IR analysis (Figure 4.14) confirmed the

disappearance of absorbance at ~630 cm-1 (C-Br). The absorbance peak at ~2100cm-1,

which is characteristic of azide moieties, became stronger than that of (N3)7-β-CD-

(PDEGMEMA)10-(Br)10, which may be attributed to the increase of azide moieties.

Following the click reaction, the coupling phenomenon disappeared and SEC (Figure

4.13) showed slight MW increase and 1H NMR spectroscopy (Figure 4.15) proved

slight triazole ring peak at ~ 8 ppm, indicating the successful addition of mannose.

FT-IR (Figure 4.14) revealed the absorbance increase at ~2100 cm-1 after reaction

with sodium azide and total disappearance of the azide peak after click reaction with a

new absorbance at ~3000-3600 cm-1 showing the existence of mannose sugar

hydroxyl groups. The LCST temperature increased from 23.2 to 32.6 °C (Figure 4.9), which further showed the success of the click reaction.

[Cyclodextrin centered glycoconjugates] [Chapter 4]

Figure 4. 15. 1H NMR spectra recorded in CDCl3 for (N3)7-β-CD-(PDEGMEMA)10-

(Br)10 (left) and (Mannose)7-β-CD-(PDEGMEMA)10-(Mannose)10 (right).

All the above results demonstrated that the CD-based glycocluster - star polymer hybrids have been successfully synthesized by the combination of ATRP and CuAAC. The obtained glycoclusters and glycocluster-star polymer hybrids were then tested for the binding with DC-SIGN.

DC-SIGN is a tetrameric transmembrane protein and has a cluster structure. We are interested in whether these CD-centered glycocluster structures could bind with this lectin. Interestingly, SPR characterization showed that the binding between the mannose glycocluster and DC-SIGN tends to be very weak, although this supermolecule bears seven mono mannose units in a cluster shape. SPR revealed a slight response up to 10 Riu after injection of the analytes with flowing over the gold chip bearing DC-SIGN lectin on the surface for 900 seconds, Figure 4.23; however, after a further buffer flush the response decreased to close to the buffer line, suggesting that the previous response was not from the binding of glycocluster to DC- SIGN. The competition test also revealed a very weak inhibition effect between gp120 and DC-SIGN even under very high concentrations (4096 nM), as shown in Figure 4.24. Similarly, the glycocluster-star polymer hybrids showed very weak binding with DC-SIGN (Figure 4.23 & 4.24), which further indicates that the special cluster structure is not good for binding with DC-SIGN. This phenomenon maybe related to the narrow and crowded cluster structure, which cannot block all or most of the binding sties of complex DC-SIGN, thus the bindings are seen to be weak.

[Cyclodextrin centered glycoconjugates] [Chapter 4]