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The Role of Protecting Groups in Synthesis and Self-Assembly of Glycopolymers Yu Zhao, Yufei Zhang, Changchun Wang, Guosong Chen, and Ming Jiang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01716 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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The Role of Protecting Groups in Synthesis and Self-Assembly of Glycopolymers Yu Zhao, Yufei Zhang, Changchun Wang, Guosong Chen*, Ming Jiang

The State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Genetics and Development and Department of Macromolecular Science, Fudan University, Shanghai, 200433 China Email: [email protected], Tel: +86 21 55664275

ACS Paragon Plus Environment

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ABSTRACT Protection and deprotection are basic procedures in oligosaccharide synthesis. Taking advantages of the processes of attaching and removing the protecting groups, preparation of oligosaccharides with complex structures can be achieved with relatively high yields. However, the role of protecting groups in solution properties and self-assembly of synthetic glycopolymers has been overlooked in literature. In this paper, we focused on such effects for well-designed copolymers in which different numbers of benzyl (Bn) groups are installed regio-selectively in saccharide rings. Thus, three block copolymers P1, P2 and P3 composed of a common block of PNIPAm and a glycopolymer block with trisaccharide triMan side chains differing in the respective number of Bn (0, 2 and 6), were prepared. The solutions of these block copolymers in water were investigated by dynamic and static light scatting and VT-1H NMR. We found that all of the three copolymers P1, P2 and P3 formed association at room

temperature.

Particularly,

P1

associated

loosely

due

to

carbohydrate-carbohydrate interaction (CCI) while P3 formed tight aggregates due to hydrophobic interactions between Bn, and P2 behaved between those of P1 and P3. Moreover, upon heating, phase transition of PNIPAm block took place leading to micelle formation. Hydrodynamic radius of P1 and P2 increased significantly as expected, while P3 did not follow this trend, because during heating, collapse and accumulation of the PNIPAm chains would occur within the tight aggregates mainly, so it leads to a little change of the size.


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INTRODUCTION The indispensable role that carbohydrates and their derivatives play in some important biological processes including bacteria infection, signal transduction, immune response etc. have been studied for decades.1,2,3 The recent tremendous developments of such research in glycobiology largely depend on the sufficient usage of various chemical tools, especially carbohydrate chemistry. 4 , 5 Carbohydrate chemistry enables not only precise reproduction of natural carbohydrate structures but also design and preparation of unnatural carbohydrate derivatives. The latter helps mimicking a lot of biological structures aiming at revealing their functions, as well as creating biomimetic materials.6,7,8,9 Among these materials, glycopolymer normally bearing multiple numbers of saccharides as side chains is one of the most important building blocks.10,11,12,13,14,15 Because multiple reactive hydroxyl groups exist on the sugar backbone, their protection/deprotection are essential in the synthesis of oligosaccharides and glycopolymers. Very recently, distinguishable from the previous use of deprotection as a synthetic tool only, we proposed the concept of “deprotection induced self-assembly” based on the finding that at removal of protective groups, self-assembly of glycopolymers takes place generating micelles or vesicles. And the assembled morphology was found to be controllable by adjusting the rate of removal of the protective groups from the sugar species.16,17 In these works, we noticed that before deprotection, the protect groups being a part of the glycopolymer should have their own effect on the solution state of the glycopolymer. However, until now no work focused on exploring such effect either on the solution properties or the assembly behavior of glycopolymers. For performing such research, regio-selective protection of hydroxyl groups with given protective groups on glycopolymers which has not been achieved in literature, of course is a precondition. Herein,

glycopolymers

containing

branched

trimannopyranosaccharide

α-man-(1→2)/α-man-(1→6)-α-man (“triMan” for clarity, Figure 1) as side chain is synthesized. This high mannose structure was selected because of their widely existence on the surface of bacteria and high immunogenicity. 18 The short


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trisaccharide side chain brings ten free hydroxyl groups to one repeating unit of the glycopolymers, which significantly magnifies the effects of monosaccharide carrying four hydroxyl groups. By using the chemical tools in our hand, a key building block of monosaccharide containing 2 benzyl groups (Bn) and 2 acyl groups (Ac) with regio-selectivity (4, Scheme 1) was generated, which was crucial to obtain the branched trisaccharide side chain with controlled number and position of protect groups. Consequently, three block copolymers with their common first block poly(N-isopropylacrylamide) (PNIPAm) and second block containing branched triMan with different number of Bn as pendent group were synthesized. Therefore, block copolymers with glyco-block featuring regio-selective protection are prepared. They

are

PNIPAm-b-PtriMan

(P1)

without

any

protect

groups,

PNIPAm-b-PPMAtriMan2Bn (P2) and PNIPAm-b-PPMAtriMan6Bn (P3) with 2 and 6 Bn groups on each of the triMan repeating unit, respectively (Figure 1). Among the three polymers, the glyco-block of P1 is expected to be hydrophilic with branched sugar as pendent group. After two or six Bn groups are installed, P2 and P3 will change to amphiphilic. With a higher number of Bn groups, more hydrophobicity of the block polymer is expected. By using Dynamic Light Scattering (DLS) and Static Light Scattering (SLS), the effects of these Bn groups on the association and phase transition behavior of these block copolymers were investigated.


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Figure 1. Chemical structures of native triMan α-man-(1→2)/α-man-(1→ 6)-α-man (triMan) and the corresponding diblock glycopolymers P1, P2 and P3.

EXPERIMENTAL SECTION Materials. D-Mannopyranose was purchased from Darui fine chemical Co., Ltd. and used without further purification. Ion-exchange resin Dowex® 50WX2 was supplied by Sigma-Aldrich Co. LLC. and washed by methanol three times before use. Peroxidase conjugated Con A (Con A-HRP) and mannan form Saccharomyces cerevisiae was purchased from Sigma-Aldrich and used without further purification. 2,2’-azobisisobutyronitrile (AIBN, CP) supplied by Sinopharm Chemical Reagent Co., was

recrystallized

from

ethanol

before

use.

Dichloromethane

(DCM),

N,N-dimethylformamide (DMF), methanol and toluene were distilled before use. Tetrabutylammonium fluoride (TBAF) was purchased from J&K Chemical and used as received. Unless specially mentioned, all other chemicals were purchased from Sinopharm Chemical Reagent Co. and used as received. The reactions were monitored and the Rf values were determined using analytical thin-layer chromatography (TLC). The TLC plates were visualized by immersion into 5% sulfuric acid solution in ethanol followed by heating using hot air heating generator. General method for glycosylation. Typically, to prepare 16, azide octanol (0.58 g,


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3.4 mmol) and the regio-selectively protected glyco-donor 4 (2.0 g, 3.4 mmol) were dissolved in anhydrous dichloromethane and cooled to -30 °C under N2 atmosphere. Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 123 µL, 0.68 mmol) was then added into the solution with a microsyringe. The reaction mixture was kept at -30°C and stirred for another several minutes until TLC (ethyl acetate/hexane; 1:2) indicated the complete consumption of the starting material. The reaction mixture was quenched by injection of triethylamine (TEA, 0.1 mL) and poured into H2O (50 mL). After being extracted with dichloromethane (50 mL ×3), the combined organic layers were washed with dilute HCl (1 M, 25 mL) and saturated NaHCO3 respectively, dried over Na2SO4, and filtered. The filtrate was evaporated, and then the obtained solid was purified by chromatography (ethyl acetate/hexanes; 1:2), affording the required product 16 in 90% yield. In preparation of the corresponding branched triMan 5, 16 was firstly deacetylated by treating with sodium methoxide (0.1 M) in methanol for more than 2 h. The reaction mixture was adjusted to neutral by ion-exchange resin and then dried by evaporation under vacuum. The obtained glyco-acceptor was carried on reacting with a per-acetylated glycodonor S2 (1.25 equiv.×2) through glycosylation method described above. The work-up of the reaction mixture was similar to that of 16, affording the required product 17 in 50% yield. The detailed synthetic routes for glyco-donor and azide-functionalized alkyl alcohol were described in Supporting Information. Preparation of the diblock copolymer precursors. The polymerization procedure of the common first block PNIPAm follows the general strategy of RAFT polymerization. NIPAm (4 g, 35.4 mmol, 200 equiv.), chain transfer agent EMP (0.18 mmol, 1.0 equiv.), AIBN (0.04 mmol, 0.2 equiv.) and 25 mL 1,4-dioxane were sealed in a flask equipped with a magnetic stir bar. The solution was degassed by three freeze-pump-thaw cycles and purged with Ar before sealing. The solution was stirred at 65°C for several hours and monitored by GPC-MALS until the conversion rate was over 70%. The polymerization was quenched by removing the reaction flask from oil bath followed by cooling in liquid nitrogen immediately. The polymer was precipitated into cold ethyl ether, filtrated and then dissolved in THF and


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precipitated again. The procedure was repeated for three times and the polymer was obtained as light yellow powder after drying under vacuum at room temperature for 12 h. Polymerization of the second block PPFPMA and PTMSPMA followed a similar procedure as above, except EMP was replaced by the macromolecular chain transfer agent PNIPAm. PNIPAm-b-PTMSPMA must be treated by further deprotection of TMS protecting group before post polymerization modification.19 The trimethylsilyl protected polymer (300 mg) and acetic acid (1.5 equiv. mol/mol to the alkyne-trimethylsilyl groups) were dissolved in THF (20 mL). N2 was bubbled to remove O2 and the colorless solution was cooled to -20°C. Solution of TBAF⋅3H2O in THF (1.5 equiv. mol/mol with respect to the alkyne-trimethylsilyl groups) was added slowly via syringe. The resulting turbid mixture was stirred at this temperature for 30 min and then warmed to room temperature. The deprotection was complete in less than 6 h. The reaction solution was passed through a short silica column in order to remove the excess of TBAF and the pad was subsequently washed with additional THF. The resulting solution was then concentrated under reduced pressure and the polymer was precipitated in petroleum ether. Post

polymerization

modification.

In

preparation

of

P1,

precursor

PNIPAm-b-PPFPMA S7 (94 mg, 0.09 mmol respect to the PFPMA repeating units), triMan 5 (100 mg, 0.18 mmol) and diisopropylethylamine (DIPEA, 30 µL, 0.18 mmol) was dissolved in anhydrous DMF. The solution was heated to 50°C and stirred overnight. The reaction mixture was purified through dialysis against water, and the obtained aqueous solution was concentrated by freeze-drying. As to the preparation of P2, diblock copolymer precursor PNIPAm-b-PPMA (70 mg, 0.072·mmol of “clickable” alkyne units), triMan 6 (81 mg, 0.108 mmol) and cuprous bromide (CuBr, 5 mg, 0.036 mmol) were dissolved in anhydrous DMF (10 mL). The solution was degassed by bubbling nitrogen for 30 min. N,N,N',N'',N''-pentamethyl diethylenetriamine (PMDETA, 7.5 µL, 0.036 mmol) was then added and N2 was bubbled into the resulting solution for a another 10 min. The solution was stirred at 70°C for 12 h and then passed through a short neutral alumina pad eluting with DMF. The resulting solution was dialyzed against water and concentrated under


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freeze-drying. P3 was prepared following the similar procedure as P2. Characterization. 1H NMR spectra were recorded with an AVANCE III HD 400 MHz spectrometer. Gel permeation chromatography (GPC) analysis was carried out with a Waters Breeze 1515 GPC analysis system with two columns, TSK-gel α-2500 and TSK-gel α-3000, using DMF with 0.5 M LiBr as eluents at the flow rate of 1 mL/min at 80°C and PMMA as calibration standard. dn/dc corresponds to the slope of the dependence of refractive index (n) of a polymer solution as a function of the polymer concentration (c), which was measured by differential refraction detector, Wyatt Optilab T-rEX. Molecular weight of glycopolymer was determined by Wyatt MALS (Multi-Angle Light Scattering) detector, DAWN HELEOS II, with pre-measured dn/dc value. Dynamic light scattering studies were conducted using ALV/5000E laser light scattering (LLS) spectrometers. CONTIN analysis was used for the extraction of data. In this paper, we used the result collected at 90°, because no obvious scattering angle dependence of has been observed. Protein binding test was monitored by UV-2550 UV-Vis Spectrophotometer, Shimadzo. The absorbance curve was measured under the kinetic mode with an excitation wavelength of 500 nm. Typical procedure for the self-assembly of glycopolymers monitored by DLS. P1, P2 or P3 of certain mass were first dissolved in DMF separately. The solutions were then dialyzed against water for 3 d (MWCO 7000 Da). The obtained aggregates in water were diluted to the concentration of 0.2 mg/mL in volumetric flasks. After stabilized for at least 1 d, the aggregates were filtered through Millipore membrane (pore size 800 nm), and then added into the quartz cells by syringe. dn/dc measurement. Refractive index (n) of each glycopolymer solutions were measured independently by differential refraction detector at different concentrations (c). Then, the linear curve of n as a function of c was plotted; the slope of this curve is the value of dn/dc (mL/g). Typical procedure for binding test with Con A monitored by UV-Vis Spectrophotometer. A cell holding 2 mL aqueous solution of P1, P2 or P3 (0.2 mg/mL) was adjusted to specific temperature in advance, then Con A (1.0 mg/mL, 50


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µL) in HEPES buffer (pH 7.4) was added and stirred rapidly. Absorbance evolution at incident wavelength of 500 nm was then plotted as function of time. In order to minimize the interference of temperature change during mixing, Con A solution was preheated to the same temperature as that of glycopolymer. Typical procedure for enzyme-linked lectin assay (ELLA). Following the previous reports,20 96-well assay plates (Corning Costar®) were coated overnight with 100 µL of 10 µg/mL mannan (from Saccharomyces cerevisiae) in 10 mM HEPES buffer (containing 1 mM Ca2+ and 1 mM Mn2+) at room temperature (r.t.). The wells were then washed with PBST (containing 0.05% Tween 20) and blocked with 1% BSA for 1 h. The polymers P1, P2 and P3 diluted in HEPES buffer with a serial concentrations from 0.2 µM to 100 µM were firstly incubated with 5 µg/mL peroxidase conjugate Con A (ConA-HRP) at r.t. or 37°C for 1 h respectively. Then the mixtures were transferred into the mannan-coated plates for 1 h of incubation at each temperature. The plates were then washed and 100 µL of 3,3',5,5'-tetramethylbenzidine (TMB, eBioscience) was added into each well for color development. The reaction was stopped by adding 50 µL of 1 M H2SO4. The absorbance was measured at 450 nm by using a Microplate Reader (BioTek ELx800). Each sample was measured in triplicates. Data were presented as means ± SEM.

RESULTS AND DISCUSSIONS Synthesis of branched triMan with different numbers of Bn and their corresponding glycopolymers. In general, tailor-made glycopolymers with controlled structures can be prepared by either post-polymerization modification or polymerization of glycomonomers.19,

21 , 22 , 23

In this study, we followed the

post-polymerization modification, because the precious bulky triMan side chain was produced by multi-step synthesis first. To construct a protect group-adjustable trisaccharide with branched structure, a building block based on D-Mannopyranoside, donor 4 was prepared following a 9-step synthesis (Scheme 1a). The anomeric carbon was modified with a good leaving group trichloroacetimidate and the other four hydroxyl groups were selectively protected, i.e. the 2- and 6-hydroxyl by Ac, while 3-


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and 4-hydroxyl by Bn. The detailed synthetic route is shown in supporting information (Scheme S1). It is known that Ac can be easily removed under basic condition, while Bn remains stable in most conditions except strong reducing environment. This difference in deprotection enabled selective exposure of specific hydroxyl groups and then secures the synthesis of polymers P1, P2 and P3 with different numbers of Bn. Then branched triMan was constructed by glycosylation (Scheme 1b). In order to connect the oligosaccharide to the side chain of the polymers, 4 was attached to an azide-functionalized

alcohol

via

glycosylation

catalyzed

by

trimethylsilyl

trifluoromethanesulfonate (TMSOTf) (Scheme 1b, i, ii).24 In the following step, the 2- and 6-hydroxyl groups of the purified product (16, 18) were deprotected by treating with sodium methoxide in methanol (Scheme 1b, iii). The exposed hydroxyl groups could further react with two per-acetylated donors S2 (Scheme 1b, iv) or two selectively protected donors 4 (Scheme 1b, v), forming protected triMan precursors 17, 19 and 20. After the next round of deacetylation, triMan derivatives bearing two Bn (triMan2Bn, 6) and six Bn (triMan6Bn, 7) were prepared. TriMan without Bn (5) was prepared via an additional hydrogenation reduction catalyzed by palladium on carbon (Scheme 1b, vi) with the azide reduced to amine at the same time.

Scheme 1. Synthetic routes of triMan derivatives (5, 6 and 7) installed with different amounts of Bn.

(a) Synthesis of selectively protected glycodonor 4 through a nine-step strategy. (b) (i) 8-azide-1-octanol, TMSOTf, dichloromethane, -30 °C; (ii) 2-azidoethanol, TMSOTf, dichloromethane, -30 °C; (iii) sodium methoxide, methanol; (iv) S2, TMSOTf,


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dichloromethane, -30 °C; (v) 4, TMSOTf, dichloromethane, -30 °C; (vi) Palladium on activated carbon (Pd/C), H2 (5 MPa), methanol.

Meanwhile, the block copolymer precursors were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization (Scheme S4). The common block PNIPAm was prepared first. As a thermal sensitive polymer, PNIPAm block endowed the glycopolymers P1, P2 and P3 a lower critical solution temperature (LCST) at which phase transition of PNIPAm occurs. Through a combination of gel permeation chromatography and multi-angle light scattering (GPC-MALS), the molecular weight (Mw) of the first block was measured as 17 kDa (degree of polymerization (DP) 150) with a narrow polydispersity (PD.I) (Figure S2). The characterization data of the second block, as precursors to the sugar block, i.e. poly(trimethylsilyl propargyl methacrylate) (PTMSPMA) and poly(pentafluorophenol methacrylate) (PPFPMA) were calculated from the GPC-MALS measurements of the corresponding diblock copolymer (Table 1). The PTMSPMA block prepared according to literature was engaged for post-polymerization modification via click chemistry with azide-functionalized triMan 6 and 7.19 After removal of trimethylsilyl groups by tetrabutylammonium fluoride (TBAF), the exposed alkynyl groups reacted with azide of 6 and 7 by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), affording the desired glycopolymers P2 and P3 (Scheme 2a). To prepare P1 without Bn, hydrogenation to remove Bn also reduced the azide to amine. PPFPMA block was thus designed instead of PTMSPMA. The preparation of P1 by aminolysis of PNIPAm150-b-PPFPMA6 with 5 was performed in DMF (Scheme 2b). The longer alkyl chain of 5 was employed to increase the aminolysis efficiency.

Table 1. Mw, PD.I and molecular formula of glycopolymers and their precursors characterized by GPC-MALS and 1H NMR. 1

GPC-MALS Polymer

Mw

PD. I.

PNIPAm150

17.0 kDa

1.01

H NMR

Polymer

Mw

PD. I.

Code

Polymer

Mw

PNIPAm150-b-PPFPMA6

17.4 kDa

1.14

P1

PNIPAm150-b-PtriMan6

21.2 kDa

PNIPAm150-b-PTMSPMA35

23.8 kDa

1.16

P2

PNIPAm150-b-P(triMan2Bn17-co-PPMA18)

34.1 kDa


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P3

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PNIPAm150-b-P(triMan6Bn18-co-PPMA17)

41.4 kDa

Scheme 2. Preparation of glycopolymer P1, P2 and P3 through post-polymerization modification of block copolymer precursor.

(a): Ethyldiisopropylamine (DIPEA), DMF; (b) Copper(I) bromide (CuBr), Pentamethyldiethylenetriamine (PMDETA), DMF.

The success of each triMan modification in P1, P2 and P3 was confirmed by 1H NMR spectroscopy (Figure 2). Besides the strong peaks of isopropyl group at about 1.0 ppm (Figure 2, peak a), peaks representing protons from the pyranose ring of triMan appeared between 3.5 and 5.5 ppm (Figure 2, peak b). Meanwhile, the peaks between 7.0 and 7.5 ppm were attributed to Bn groups (Figure 2, peak d). Additionally, the spectra of P2 and P3 exhibited the tiny peaks at about 8.0 ppm representing the formation of triazole ring (Figure 2, peak e). By 1H NMR, the carbohydrate content of glycopolymer was estimated through comparing the integral area of pyranose peaks with internal standard tetramethylsilane. By using DP of


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PTMSPMA and PPFPMA calculated according to the result of GPC-MALS, the molecular formula as well as modification ratio of each glycopolymer was estimated (Table 1). The result indicated that after post-polymerization modification, the sugar contents of P2 and P3 were similar, while the amount of hydroxyl groups on P1 was similar to that of P2, which made the three polymers compatible for further assembly and agglutination studies.

Figure 2. 1H NMR spectra of P1, P2 and P3 in DMSO-d6. Protons of (a) isopropyl group of PNIPAm, (b) pyranose ring of oligosaccharide, (c) (CH3)2CHNHCO of PNIPAm, (d) Bn protecting group, (e) triazole ring are shown in the spectra. Assignment of other peaks: δ (ppm): 1.15 ~ 1.75 (CH2CHCO), 1.75 ~ 2.20 (CH2CHCO), 3.70 ~ 4.00 (CH3CHCH3 of NIPAm), 2.50 (DMSO), 3.34 (water).

Phase transition of glycopolymers differing in Bn unit number. The three glycopolymers were dissolved in DMF at 1 mg/mL, which were then dialyzed against water separately at 25ºC first. These aqueous solutions were diluted to 0.2 mg/mL. Their

thermo-induced

phase

transition

behavior

was

monitored

by

various-temperature dynamic light scatting (VT-DLS) and SLS. As shown in Figure 3,


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the evolution of scattered light intensity (Is/I0) versus temperature showed the classical phase transition behavior of PNIPAm chains (Figure 3a). The LCST of P1 without Bn was about 42 °C, almost 10 °C higher than that of PNIPAm homopolymer. Similarly, P2 with additional two Bn exhibited a lower LCST of about 40 °C, while the LCST of P3 with as much as six Bn groups decreased to about 36 °C. The result is generally in accordance with the known rational that decreasing hydrophilicity of the counter block leads to lower LCST of PNIPAm block.25

Table 2. SLS results of glycopolymers P1, P2 and P3 at 25 °C and 50 °C. Code P1 P2 P3

Ass1 Agg1 Ass2 Agg2 Ass3 Agg3

Temperature /˚C 25 50 25 50 25 50

Mwa /10 g·mol-1 1.2 89.2 1.7 4.9 17.6 15.1 8

Naggb /103 5.9 421.6 5.0 14.4 42.5 36.6

a

Apparent Mw of the aggregates calculated by Zimm plot at specific temperature.

b

calculated by comparing the Mw of aggregates and that of glycopolymers measured

by GPC-MALS.

Figure 3. Evolution of (a) Is/I0 and (b) of P1, P2 and P3 (0.2 mg/mL), measured by VT-DLS. Is represents absolute intensity of scattered light, while I0


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represents absolute intensity of incident laser light.

It was clearly observed that Is/I0 and of the three glycopolymers at 25 °C did not show the behavior of molecularly dispersed solutions, although in general both PNIPAm and glycopolymer blocks are thought to be water-soluble. As shown in Figure 3a, Is/I0 of P3 was much higher than those of P1 and P2, which could be attributed to the hydrophobic association between the extra Bn groups. However, the large values of P1, P2 and P3, i.e. 78 nm, 49 nm and 96 nm respectively revealed the association of the glycopolymers (Figure 3b) existing for all the three polymers at room temperature. Particularly, P1 without Bn groups did not show much difference compared to that of P2. Therefore, the association of the glycopolymer chains in this state should not be attributed to Bn exclusively. This association of P1 might be caused by carbohydrate-carbohydrate interaction (CCI), which was mainly contributed by hydrogen bonding between the hydroxyl groups and hydrophobic interaction between pyranose rings.26,27,28,29 The latter comes from the relatively non-polar face of the ring, that is on one mannopyranose ring, the hydroxyl groups at 2, 3 and 6 positions are on the top of the ring, resulting in a relatively hydrophobic face on the opposite side. Although CCI is relatively weak, for the current case, we suppose that the high density of the sugar units in triMan strengthens this interaction. 30 Meanwhile, the multivalent interactions along the polymer chain enhanced the overall CCI. This hypothesis was confirmed by the fact that the homopolymer with pendent triMan side groups as a control was found to form aggregates with around 85 nm by DLS (Figure S6). For clarity reasons, the associated states of P1, P2 and P3 at 25°C as discussed above are named Ass1, Ass2 and Ass3, respectively. To quantitatively demonstrate the effect of Bn on Ass1, Ass2 and Ass3 before heating, SLS was employed to characterize the association at 25°C. Here the apparent Mw of the three samples was measured to reach 108 g/mol (Table 2, Figure S7), confirming the existence of aggregation in solution. Comparing to Ass3, Ass1 and Ass2 gave a one manifold lower Mw and consequently much lower aggregation number


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(Nagg). In addition, Is/I0 of Ass1 and Ass2 was much lower than that of Ass3. These results indicated that P1 and P2 performed much looser association than P3 did;31,32 while these association was mainly due to CCI. It implies that only in P3 (Ass3) with six Bn groups per triMan unit, the hydrophobic interaction of Bn groups becomes strong enough leading to high association and then dense aggregates with high Mw and Is/I0. When the temperature was raised gradually to 50 °C, both Is/I0 and of P1 and P2 were increased. It is of cause owning to the formation of much more compact aggregated states (Agg1, Agg2) due to the collapse of PNIPAm, stabilized by the hydrated sugar blocks. These aggregates are kinds of micelles, similar to the micelles formed by ordinary diblock copolymers containing PNIPAm block after heating. As the temperature increased, P1 performed the most significant increase of the evolution of to about 240 nm, which was about three times higher than that below LCST. Meanwhile, of P2 increased from 50 nm to 90 nm. The result of was in accordance with Nagg of aggregates calculated from the result of SLS (Table 2). Starting from similar Nagg at 25 °C (Ass1: 5.9 × 103, Ass2: 5.0 × 103), P1 gave much higher Nagg than P2 (Agg1: 421.6 × 103, Agg2: 14.4 × 103). The phase transition behavior of P3 was very different from that of P1 and P2. Upon heating, its did not exhibit any increase, but even a decrease from 96 nm to 86 nm. Meanwhile, the Nagg calculated from SLS of P3 before and after heating showed a slight increase, i.e. from 36.6 × 103 to 42.5 × 103 (Table 2). This unusual behavior is understandable, as for P3, relatively compact aggregates already existed at 25ºC, so during heating collapse and accumulation of the PNIPAm chains would occur within the aggregates leading to a little change of the size.

Solvated moieties varying on aggregated P1 and P2 analyzed by VT-NMR and proposed aggregation mechanism. In order to confirm the possible mechanism above and gain insight information, various-temperature

1

H NMR (VT-NMR)

spectroscopy was utilized. To establish quantitative analysis, water soluble sodium 2,2-dimethyl-2-silapentane-5-sulfonate hydrate (DSS) was used as an interior


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standard, i.e. the amount of each functional groups was calculated by the ratio of integral area to that of DSS at 0 ppm. The concentration of glycocopolymers was adjusted to 1.0 mg/mL in order to obtain enough signal intensity for quantification. Concentration higher than 1.0 mg/mL led to obvious precipitation, which reduced the signal strength. According to the assignment of proton peaks mentioned above (Figure 2), the integral area of the single peak at about 1.0 ppm was used to estimate the content of PNIPAm block, while that of peaks between 3.2 and 3.8 ppm stand for the content of glyco-block (Figure S8). As shown in Figure 4a of the above two integral areas against temperature, the signal of the solvated PNIPAm of P1 decreased dramatically as the temperature was raised to above LCST, which was of cause attributed to the collapse of the thermal sensitive block (Figure S9). Meanwhile, the signal intensity of glyco-block stayed almost unchanged during heating. Besides, P2 behaved similarly to that of P1 (Figure 4b, S10, S11). The negligible drop might be attributed to the decreased concentration caused by slightly precipitation of P2. For P3, its VT-NMR experiment was performed at a lower concentration due to its poor solubility caused by too many Bn groups. The similar evolution of the NIPAm block and sugar block to that of P1 and P2 was obtained (Figure S12-14).

Figure 4. Normalized solvation of PNIPAm and sugar block of (a) P1 and (b) P2 (1.0 mg/mL) against temperature measured by VT-1H NMR. DSS is utilized as an internal standard.


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The direct evidence provided by VT-NMR analysis deepened our understanding on the microstructure of the aggregates during heating. Combined with the result of VT-DLS, transformation process of the glycopolymers in aqueous solution can be deduced (Figure 5). Below LCST, both NIPAm and triMan moieties of P1 and P2 disperse randomly in the amorphous association region. The association was so loose that the weak shielding of one block has little effect on NMR signal intensity of the other. When the temperature was raised to above LCST, PNIPAm block collapsed and formed the core of a more compact micelle while the glyco-block formed the corona. However, P3 at room temperature formed rather condensed aggregates due to the strong association between the Bn groups, which would prevent further inter-aggregate accumulation upon heating resulting in smaller micelles. Combining with the results from DLS and SLS, aggregation states of P1, P2 and P3 before and after heating are drawn in Figure 5.

Figure 5. Proposed evolution of P1, P2 and P3 in solution upon heating and their possible binding to Con A.

Binding to lectin analyzed by UV-vis spectra. To further compare the behavior and functions of the triMan side chains of P1, P2 and P3, their binding ability to model lectin Concanavalin A (Con A) and Peanut agglutinin (PNA) was investigated. Con A


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exists as a tetramer in neutral condition with each of its monomer bearing one mannose-binding site, which means Con A acts as a crosslinker between glycopolymers or glyco-nanoparticles33 in solution resulting in turbidity increase. On the contrary, PNA preforms no specific recognition with mannose residues. Herein we monitored the binding of P1, P2 and P3 at both 25 °C and 50 °C by UV-vis spectroscopy. The test was performed in HEPES buffer with calcium and manganese ions (20 mM, pH 7.4). After addition of Con A, the absorbance of the micelles at 50°C generally increased faster than those of the loose associates at 25 °C (Figure 6), which can be attributed to the relative higher sugar density on the surface of micelles than the associated chains. Among the three micelles at 50 °C, it was unexpected that Agg2 performed much more significant binding than the other two. We speculate that, for the relatively small and dense Agg2, inter-micellar crosslinking became primary leading to a large scale of aggregation. The strong binding ability of Agg2 to ConA was supported by enzyme-linked lectin assay (ELLA) with its IC50 of about 4 µM, indicating higher binding affinity of Agg2 to Con A than small molecular mannosyl ligands (Figure S15).20 As far as Agg3, on the contrary, it could hardly bind to Con A, because the 3,4 diols of the exposed mannose residues were blocked by Bn, which made the sugar unrecognizable to the carbohydrate recognition domain of Con A. Moreover, as a control, the binding experiment of P1, P2 and P3 to PNA did not show any significant turbidity increase at 25 °C and 50 °C (Figure S16).


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Figure 6. Absorbance evolution vs time of Ass1, Ass2, Ass3 at 25 °C and Agg1, Agg2, and Agg3 at 50 °C after addition of ConA. The concentration of glycopolymers in aqueous solution is 0.2 mg/mL, while that of Con A solution in HEPES buffer is 1.0 mg/mL.

CONCLUSIONS In this paper, the effect of protective group on solution properties and self-assembly of glycopolymer-containing block copolymers has been explored. For the first time, different numbers of Bn groups (0, 2, and 6) were introduced on the synthetic trisaccharide side chain as desired in the way of regio-selectivity. This success in synthesis made such study possible. It was found that at room temperature the hydrophobic interactions in the copolymer with 6 Bn led to strong association forming compact aggregates while other copolymers with less or without Bn formed loose association due to mainly CCI. Such difference induced diverse behavior in the process of the subsequent thermo-induced micellization, i.e. the copolymer with the high content of Bn showed much less change in the size of the aggregates than the other two copolymers because in this case the collapse and accumulation of the thermo-sensitive block occur mainly within the existing compact aggregates.

Supporting information Synthesis and characterization of glycodonor 4, branched triMan with different


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amounts of Bn 4, 5, 6, glycopolymers P1, P2 and P3, including 1H NMR as well as details of GPC-MALS, VT-DLS, VT-NMR and UV spectra are all available in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author *Email: [email protected]

ACKNOWLEDGMENT National Natural Science Foundation of China (Nos. 21474020, 91227203, 51322306, 91527305 and GZ962) is acknowledged for financial support.


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The Role of Protecting Groups in Synthesis and Self-Assembly of Glycopolymers Yu Zhao, Yufei Zhang, Changchun Wang, Guosong Chen*, Ming Jiang The State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Genetics and Development and Department of Macromolecular Science, Fudan University, Shanghai, 200433 China Email: [email protected], Tel: +86 21 55664275


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Role of Protecting Groups in Synthesis and Self ... - ACS Publications

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