Reversible vesicles based on one and two head supramolecular cyclodextrin amphiphile induced by methanol

Carbohydrate Research 345 (2010) 914–921

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Reversible vesicles based on one and two head supramolecular cyclodextrin amphiphile induced by methanol Wei An  , Huacheng Zhang  , Lizhen Sun, Aiyou Hao *, Jingcheng Hao, Feifei Xin School of Chemistry and Chemical Engineering and Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, Shandong University, Jinan 250100, PR China

a r t i c l e

i n f o

Article history: Received 4 December 2009 Accepted 1 February 2010 Available online 6 February 2010 Keywords: Vesicles Cyclodextrin Ferrocene ‘One head’ supramolecular amphiphile ‘Two head’ supramolecular amphiphile

a b s t r a c t Reversible vesicles based on supramolecular inclusion of hydroxypropyl-b-CD (HPbCD) and N,N0 bis(ferrocenylmethylene)-diaminohexane (BFD) were prepared in water and methanol–water mixtures. The inclusion stoichiometry of HPbCD with BFD was in a molar ratio of 2:1, which could be named as ‘two head’ supramolecular amphiphile when the solvent was water. However, the inclusion stoichiometry of HPbCD with BFD would tend to be a molar ratio of 1:1 based on introduction of methanol to the solvent, especially when the volume ratio of methanol and water was more than 1:4, which could be named as ‘one head’ supramolecular amphiphile. The inclusion compounds could switch between ‘one head’ and ‘two head’ conformations by changing the methanol concentration of the solvents. The vesicles were also found to be responsive to the stimulus of external molecules. When the inclusion ability between HPbCD and an external guest was relatively stronger, the vesicles were easily destroyed. Furthermore, the vesicles disappeared after adding an oxidizing agent. NMR was used to confirm the conformation of the mixture of HPbCD and BFD in water. The structure and morphology of the vesicles were characterized by TEM and DLS. The vesicles may be used in smart materials, drug delivery and molecular recognition. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Vesicles with special cell-like structures are the general model systems for mimicking biological membranes,1 and have attracted much attention for their special properties as well as wide applications in drug delivery and materials.2–7 In order to find more useful vesicles, a number of vesicles have been prepared by synthetic amphiphilic cyclodextrins (CDs).8,9 CDs, consisting of six (a-), seven (b-), and eight (c-) glucose units linked by a-1?4-glycosidic linkages, which have a truncated cone form with a hydrophilic outer surface and a hydrophobic inner cavity,10 could include many molecules to form inclusion complexes11 based on various interactions, such as electrostatic, hydrogen bonding, hydrophobic interactions, and van der Waals.12 CDs have been concerned a lot for their combined properties of liposomes and macrocylic host molecules, which offers great potential to encapsulate and solubilize drugs or to recognize and bind specific types of guest molecules.13–16 Recently, our group17 reported a novel kind of redox-responsive vesicles prepared from CDs and ferrocene derivative in water with a complex stoichiometry of 1:1, which could be named as ‘one head’ supramolecular amphiphile. Here, we report a vesicle system

* Corresponding author. Tel.: +86 531 88363306; fax: +86 531 88564464. E-mail address: [email protected] (A. Hao).   These authors contributed equally to this work. 0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2010.02.001

based on supramolecular inclusion of hydroxypropyl-b-CD (HPbCD), and N, N0 -bis(ferrocenylmethylene)-diaminohexane (BFD) with a complex stoichiometry of 2:1 in water, which could be named as ‘two head’ supramolecular amphiphile compared with that of ‘one head’. When methanol was introduced to the solvent, the inclusion stoichiometry of HPbCD with BFD would tend to be, and even became a molar ratio of 1:1 when the volume ratio of methanol and water was more than 1:4. The inclusion compounds could switch between ‘one head’ and ‘two head’ conformations by changing the methanol concentration of the solvents. It is notable that the vesicles were stable for about one week in water at room temperature and became less stable with methanol concentration increasing in the solvents. On the other hand, the vesicles would disappear when the inclusion complexation of HPbCD with BFD was relatively weakened by external guests or an oxidizing agent. The structure and morphology of the vesicles were characterized by TEM and DLS. 2. Results and discussion 2.1. Vesicle formation and methanol effect Transmission electron microscopy (TEM) was employed to investigate the aggregates of the inclusion complex of HPbCD with BFD. Spherical vesicular structures with diameters about 200 nm were observed (Fig. 1a and b). No vesicles were detected by TEM

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Figure 1. TEM images of the vesicles of HPbCD and BFD with a molar ratio of 2:1 using phosphotungstic acid as the negative staining agent. The solvent is (a) water; (b) water; or methanol–water mixtures with a volume ratio of (c) 1:2; (d) 1:1; (e) 2:1 or (f) methanol. Scale bars are 100 nm.

1=DA ¼ 1=a þ 1=aK ap ½HPbCDn0

ð1Þ

water methanol:water, 1:2 (v:v) methanol:water, 1:1 (v:v) methanol:water, 2:1 (v:v)

5

log differential Rh

in solutions of HPbCD or BFD alone. It is obvious that the combination of HPbCD and BFD is crucial for the vesicle formation. The sizes and the size distributions of the vesicles were measured by dynamic light scattering (DLS), giving an average hydrodynamic radius (Rh) of 150 nm. The sizes of the vesicles formed in this study observed by DLS are much larger than that by TEM. This would be related to that the samples for DLS are vesicle solutions, whereas the samples for TEM are dried vesicle aggregates. Vesicular structures were also obtained in methanol–water mixtures with a volume ratio of 1:2, 1:1, 2:1, and even in pure methanol (Fig. 1c–f). The DLS results showed that the average hydrodynamic radius (Rh) of the vesicles ranged from 75 to 130 nm (Fig. 2 and Table 1). However, it is notable that the stability of the vesicles was different with the solvent compositions changing. The vesicles prepared in the methanol–water mixtures, especially in pure methanol became less stable than that in water, which could stay no more than four days. This may be related to the weaker supramolecular interaction of HPbCD with BFD, and even the weaker assembling between the inclusion compounds of HPbCD with BFD, when the methanol concentration increased in the solvents. The proposals were indicated by the result that the stoichiometry of HPbCD with BFD would change to a molar ratio of 1:1 from 2:1, when the volume ratio of methanol and water was more than 1:4 (Figs. S1–S3). The proposals were also indicated by that the inclusion constant of HPbCD with BFD decreased with methanol concentration increasing in the solvents (Fig. 3 and Table 2). The stoichiometries and the inclusion constants (Table 2) of HPbCD with BFD were determined by UV double-reciprocal method based on the Eq. 1.18,19

4

methanol

3 2 1 0 50

75 100 125 150 175 200 225

Rh (nm) Figure 2. DLS size distributions of HPbCD and BFD sample with a molar ratio of 2:1 in water; methanol–water mixtures with a volume ratio of 1:2, 1:1, 2:1 and in methanol.

Table 1 The sizes (Rh, nm) of the vesicles obtained in different solvents Item

Solvent composition

DLS (Rh, nm)

1 2 3 4 5

Water Methanol–water, 1:2 (v:v) Methanol–water, 1:1 (v:v) Methanol–water, 2:1 (v:v) Methanol

150 ± 5.1 130 ± 3.4 75 ± 3.6 100 ± 4.2 94.3 ± 2.2

Where DA is the change of absorbance of BFD in the present of HPbCD, a is a constant, [HPbCD]0 is the initial concentration of

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W. An et al. / Carbohydrate Research 345 (2010) 914–921 20

6

16

5

12

4

1/ΔA

1/ΔA

a

8

maximum value at a molar fraction of 0.4 (Fig. 4a), corresponding to a 2:1 stoichiometry of HPbCD with BFD,22 which was opposite to the 1:1 stoichiometry formed by bCD with BFD in water.17 This may be related to the cavity of HPbCD that is relatively larger than that of bCD.23 However, a 1:1 stoichiometry for HPbCD with BFD in methanol–water mixture with a volume ratio of 1:1 was obtained since a maximum value was found at the molar fraction of 0.5 ( Fig. 4b). This may due to methanol that is more hydrophobic than water which makes BFD more difficult to include by HPbCD. The results were consistent with those obtained by the above-mentioned double reciprocal method. In all, the inclusion stoichiometries of HPbCD with BFD could switch between 1:1 and 2:1 by adjusting the concentration of methanol in the solvents.

b

3 2

4 1 5

5

6

6

0.0 4.0x10 8.0x10 1.2x10 1.6x10

1/ [HPβ CD]

2

0

2 -2 L mol

500 1000 1500 2000 2500

1/ [HPβ CD]

L mol

-1

6

5

c

d

5

2.2. The properties of the vesicles

4 4

1/ΔA

1/ΔA

3

2

3 2 1

1 0

500 1000 1500 2000 2500

1/ [HPβ CD]

L mol

0

-1

500 1000 1500 2000 2500

1/ [HPβ CD]

L mol

-1

Figure 3. Double reciprocal plots of HPbCD with BFD inclusion compound in water and methanol–water mixtures at 206 nm. The solvent is: (a) water; methanol– water with a volume ratio of (b) 1:2; (c) 1:1; (d) 2:1.

HPbCD. Kap is the constant for the formation of the n:1 (host:guest) inclusion complex, which could be calculated from a plot of 1/DA versus 1=½HPbCDn0 . The complex stoichiometries of HPbCD with BFD in water and in the methanol–water mixture with a molar ratio of 1:1 were also measured by the continuous variation method (Job’s method).20,21 The Job’s plot for the binding of HPbCD with BFD in water showed a

2.2.1. The effects of external guests on the vesicles For the abilities of the vesicles, the influence of external guests (Scheme 1) on the vesicles was investigated by TEM. When methyl orange (MO) or piroxicam (equimolar with BFD) was used as the external guest, the vesicles disappeared. However, the vesicles could still be observed when bromophenol blue (BPB) or ampicillin (equimolar with BFD) was used (Fig. 6). The results indicate that the responsive behavior of the vesicles to external stimuli may be related to the inclusion ability between HPbCD and the external guest. The inclusion constants between HPbCD and the external guests were obtained based on Eq. 1 (Table 3). It is obvious that inclusion ability with MO or piroxicam is much stronger than that with BPB or ampicillin, which is consistent with our anticipation that the stronger inclusion ability of HPbCD with an external guest may bring in an unstable inclusion complex between HPbCD and BFD, even resulting in the disintegration of the vesicles (Scheme 2). This vesicle system has potential application in molecular recognition. The 1:1 stoichiometry for the complexation of HPbCD with MO, or ampicillin is coincident with those previously reported.24,25 However, the 2:1 stoichiometry for the complexation of HPbCD with piroxicam (Fig. 5) in this work is opposite to that previously

Table 2 The inclusion constants of HPbCD with BFD in the solvents at 206 nm Item

Solvent composition

Complex stoichiometry

Kap

R

1 2 3 4

Water Methanol–water, 1:2 (v:v) Methanol–water, 1:1 (v:v) Methanol–water, 2:1 (v:v)

2:1 1:1 1:1 1:1

3.32  105 L2 mol2 372 L mol1 359 L mol1 289 L mol1

0.9940 0.9995 0.9979 0.9994

0.25

a

0.5

b

0.20 0.4

ΔA

ΔA

0.15 0.10

0.3 0.2

0.05

0.1

0.00

0.0

0.0

0.2

0.4

0.6

r=[G]/([G]+[H])

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

r=[G]/([G]+[H])

Figure 4. Job’s plots by UV for binding of HPbCD with BFD in the solvents: (a) water, and (b) mixture of methanol–water with a volume ratio of 1:1.

W. An et al. / Carbohydrate Research 345 (2010) 914–921

OH

Br O S O

N N

N

ONa

Br O S O

O

Br OH

Br Bromophenol Blue (BPB)

Methyl Orange (MO)

NH2 O N

N H

OH

N O

O S

H N O

S

N OH

O

O

Piroxicam

H

Ampicillin

Scheme 1. The chemical structures of the selected external guests.

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2.2.2. Redox-responsive properties of the vesicles As is known, CD/ferrocene inclusion complex has a lower diffusion coefficient than free ferrocene,27 which leads to the decreased peak currents observed in the cyclic voltammogram. Compared with the cyclic voltammogram of BFD, in the presence of HPbCD, a small but significant decrease in the anodic peak current (Ip, Fig. 7 and Table 4) was observed. This change is owing to the fact that BFD is more difficult to oxidize in the presence of HPbCD and it is more strongly bonded than its oxidized form.28 It can be concluded that the apolar ferrocene moiety of BFD was included in the cavity of HPbCD. The aggregates may be responsive to redox signals. However, the micro-morphologies are difficult to investigate by using the electrochemical redox.29 To mimic the first step in electrochemical oxidation, the chemical oxidizing agent, hydrogen peroxide,30 was used in our vesicle system. As anticipated, no vesicles were observed by TEM after addition of excess hydrogen peroxide (Scheme 2). This results that the vesicles responsive to an oxidizing agent might be of great value in materials and molecular recognition. 2.3. Possible mechanism for the formation of the vesicles

Table 3 The inclusion constant of complex between HPbCD and MO (BPB, ampicillin, piroxicam) Inclusion complex

Complex stoichiometry

Kap

R

HPbCD-MO HPbCD-BPB HPbCD-ampicillin HPbCD-piroxicam

1:1 1:1 1:1 2:1

5.6  103 L mol1 867 L mol1 520 L mol1 3.9  105 L2 mol2

0.9633 0.9789 0.9873 0.9990

reported.26 This may be due to the two hydrophobic heads of the relatively linear molecule of piroxicam, as well as the different substitution degree for the HPbCD used.

2.3.1. Possible conformation of the supramolecular inclusion of HPbCD with BFD According to the data above, we could hold that the combination of HPbCD and BFD played a key role in the vesicle formation. The HPbCD with BFD sample in water was studied by 1H NMR, which is one of the most powerful tools for realizing supramolecular assemblies in solution.31 In the presence of HPbCD, almost all the hydrogen resonances of BFD showed chemical shifts (Dd, Fig. 8 and Table 5). The largest downfield shifts were observed for Ha (4.297?4.335), Hb (4.237?4.271), Hc (4.165?4.184), indicating that the ferrocene side groups have been encapsulated within HPbCD cavities (Table 5). It is known that CD molecules adopt the conformation of a torus where the H-3 and H-5 protons are

Scheme 2. Schematic illustration of the vesicles: (a) the vesicles formed in water; (b1) methanol was introduced; (b2) when the volume ratio of methanol and water was more than 1:4; (c) adding an external guest, whose binding ability with HPbCD was relatively weaker; (d) adding an external guest, whose binding ability with HPbCD was relatively stronger; or adding excess hydrogen peroxide to the system.

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W. An et al. / Carbohydrate Research 345 (2010) 914–921 Table 4 The differences of Ep and Ip in the absence and presence of HPbCD

120

X

1/ΔA

80

BFD Compound DX (Xcompound  XBFD)

40

Ep (V)

Ip (A)

0.378 0.385 0.007

1.328  107 4.817  108 8.463  108

0 0.0

2.0x10 6

4.0x10 6

6.0x10 6

1/ [HPβ CD] 2 ( L2 mol -2)

8.0x10

Figure 5. Double reciprocal plot of HPbCD with piroxicam inclusion complex at 206 nm.

2.3.2. Possible formation mechanism of the vesicles No aggregates were observed by TEM in the aqueous solutions of HPbCD or BFD alone. Therefore, the combination of HPbCD and BFD would play a key role in the formation of the vesicles. According to the CV, UV and NMR data mentioned above, the inclusion complex formed by the inclusion compound of HPbCD with BFD was proposed, as shown in Scheme 2. The inclusion model resembles a conventional amphiphile, where HPbCD serves as the hydrophilic head facing the water and the chain as the hydrophobic tail, thereby enclosing an aqueous interior. According to the formation mechanisms of the vesicles formed by conventional amphiphiles, the possible mechanism of the vesicles formed in water is suggested in Scheme 2a. The inclusion stoichiometries of HPbCD with BFD would be reversible between 1:1 and 2:1 by adjusting the concentration of methanol in the methanol–water solvents. Switches between ‘one head’ and ‘two head’ formation mechanisms could be accomplished, when the volume ratio of methanol and water

Current / A

located inside the cavity, while H-2 and H-4 protons are outside of the cavity. Therefore, inclusion complex is more sensitive to the shift of H-3 and H-5 proton peaks.32 The clear shifts of H-3 and H-5 suggested that BFD was included by HPbCD (Table 6). On the other hand, the H-1 and H-6 are also used for the conformation recognition of CDs with guests in supramolecular chemistry for their clearly separated signs from others in 1H NMR. The shift of H-1 in HPbCD was much larger than that of H-6, which demonstrated that the ferrocene moieties should be included by HPbCD through the secondary face.33 The conformation of the inclusion complex was confirmed by 2D NMR ROESY which has a maximal observation limit at spatial proximity of 5 Å.31 The selected region of 2D NMR ROESY (600 MHz) of HPbCD and BFD sample with a molar ratio of 2:1 in D2O was shown in Figure 9. The ferrocene moieties on BFD could be included by the cavity of HPbCD, since correlations between Ha, Hb, Hc and H3, H5 which located inside the cavity of HPbCD, could be observed.

4.0x10

-7

-7

0.0

-4.0x10

-8.0x10

-7

-7

BFD mixture of BFD and HP β CD 0.0

0.2

0.4

0.6

0.8

E / V ( vs .SCE ) Figure 7. The cyclic voltammogram changes of the aqueous solution of BFD with 0.05 mol L1 NaCl as a supporting electrolyte in the absence (—) and presence of HPbCD (  ) at a scan rate of 50 mV s1, respectively.

was more than 1:4 (Scheme 2). The possible mechanism of the responses of the vesicles to external guests, and an oxidizing agent are also shown in Scheme 2. 3. Conclusions Reversible vesicles based on supramolecular inclusion of HPbCD and BFD were prepared in water and methanol–water mixtures. HPbCD and BFD could form a ‘two head’ supramolecular amphiphile with a molar ratio of 2:1 in water, which would tend to form a ‘one head’ supramolecular amphiphile with a molar ratio of 1:1, when methanol was introduced and the volume ratio of methanol and water was more than 1:4. The inclusion compounds could switch between ‘one head’ and ‘two head’ conformations by changing the methanol concentration of the solvents. The vesicles were also found to be responsive to the stimulus of external molecules. When the inclusion ability between HPbCD and an external guest was relatively stronger, the vesicles were easily destroyed. Furthermore, the vesicles disappeared after adding an oxidizing agent. The reversible vesicles induced by methanol have potential application in smart materials, drug delivery and molecular recognition.

Figure 6. TEM images of HPbCD with BFD sample with a molar ratio of 2:1 affected by external guests. (a) With the addition of ampicillin; (b) with the addition of BPB.

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Figure 8. 1H NMR spectra (300 MHz) of the mixture of HPbCD and BFD in comparison with HPbCD and BFD at ambient temperature with the solvent peak (d = 4.69 ppm) as the reference.

Table 5 1 H NMR: chemical shifts and chemical shift differences of BFD and its complex with HPbCD

d (BFD) d (complex) Dd (d(complex)  d(BFD))

Ha

Hb

Hc

Hd

He

Hf

Hg

4.297 4.335 0.064

4.237 4.271 0.034

4.165 4.184 0.019

3.959 3.961 0.002

2.826 2.826 0

1.483 1.485 0.002

1.182 1.182 0

Table 6 1 H NMR: chemical shifts and chemical shift differences of HPbCD and its complex with BFD

d (HPbCD)a d (complex) Dd (d(complex)  d(HPbCD))

H1

H3

H5

H6

5.061 5.047 0.014

3.908 3.913 0.005

3.746 3.720 0.026

3.621 3.626 0.005

a The H2 and H4 hydrogens could not be assigned due to signal overlapping in the H NMR spectrum.

1

4. Experimental section 4.1. Materials and instruments Ferrocene, 1,6-diaminohexane, sodium borohydride and other chemicals were all obtained from Country Medicine Reagent Co. Ltd, PR China. The HPbCD (substituted degree is 6.8 on average) was obtained from Huantai Xinda Co. Ltd, PR China. Methanol orange was obtained from East China Normal University Chemical and bromophenol blue was bought from Shanghai SSS Reagent Co, Ltd, PR China. Ampicillin and piroxicam were purchased from Shandong Chengchuang Pharmaceutical Co. Ltd, PR China. All organic reagents were of analytical purity and used as received without further purification. Water was triply distilled. BFD was synthesized by reaction of 1,6-diaminohexane with ferrocenecarboxaldehyde according to the method described.17

The sonication was performed on KQ116 ultrasonic cleaners, Kushan ultrasonic apparatus Co. Ltd, PR China; TEM was performed on a JEM-100CX II transmission electron microscope operated at an acceleration voltage of 100 KV; DLS experiment was performed on a Wyatt QELS Technology DAWN HELEOS instrument using a 12-angle-replaced detector (99°) and a 50 mW solid-state laser (658.0 nm); CV was performed in a conventional three-electrode cell with a model CH1650 electrochemical workstation. A glassy carbon electrode (GCE) was used as the working electrode, with a platinum plate as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode; UV spectra were recorded with TU-1800pc UV–vis Spectrophotometer at 298 K; 1H NMR spectra of HPbCD and BFD in comparison with HPbCD and BFD were obtained using the API Bruker Avance 300 M NMR at 298 K with the solvent peak (d = 4.69 ppm, D2O) as the standard reference. 2D 1H–1H ROESY spectra for the inclusion compound of HPbCD with BFD was recorded using an INOVA-600 (600 MHz) spectrometer at ambient temperature. A mixing time of 0.200 s, a relaxation delay time of 1.000 s, and an acquisition time of 0.228 s were used. All pulse sequences were set according to the company standards. 4.2. Preparation of vesicles Two molar amounts of HPbCD were added to the aqueous solution of BFD (104 mol L1, 50 mL) and the sample solution

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W. An et al. / Carbohydrate Research 345 (2010) 914–921

Figure 9. Selected region of 2D NMR ROESY (600 MHz) spectrum of HPbCD and BFD sample with a molar ratio of 2:1 in D2O at room temperature.

was sonicated for 30 min at room temperature before detection. The solutions with different volume ratio of methanol–water were prepared by the same method. The effect of external stimuli was studied by adding an external guest (such as piroxicam, equimolar with BFD), or excess hydrogen peroxide to the sample solution. 4.3. TEM measurement A drop of the sample solution was placed onto a TEM copper grid covered by a polymer support film. The samples were stained by depositing a drop of 0.2 wt % phosphotungstic acid aqueous solution onto the surface of the sample loaded grid. 4.4. DLS measurement In this study, all the solutions were filtered through 450 nm Millipore filters and all DLS measurements were performed at room temperature. The CONTIN program was used to calculate the mean hydrodynamic diameter with Astra software.

and water, finally washed with triply water. The solutions were deoxygenated by N2 bubbling for 10 min prior to the experiments, and a blanket of N2 was maintained through the experiments. 4.6. UV measurement To examine the inclusion effect, the concentration of BFD (2  105 mol L1) was kept constant with the concentration of HPbCD ranging from 4  104 mol L1 to 6.4  103 mol L1. The complex stability constants between HPbCD and other guests were obtained using the same method. The complex stoichiometries of HPbCD with BFD in water and methanol–water mixture with a molar ratio of 1:1 were also determined using Job’s continuous variation method. A set of working solutions were obtained by mixing Vg mL of the stock BFD solution (1  104 mol L1) with (Vt  Vg) mL of the stock HPbCD solution (1  104 mol L1), where Vt is a fixed total volume and Vg is a variable value (from 0 to 10 mL, 0 6 Vg 6 Vt).

4.5. CV measurement

Acknowledgements

Prior to each experiment, the glassy carbon electrode was polished with a-alumina power, then rinsed thoroughly with triply distilled water and sonicated in a 1:1 (v:v) mixture of nitric acid

This work was supported by the NSFC (Grant No. 2062537) and National Basic Research Program of China (973 Program, 2009CB930103).

W. An et al. / Carbohydrate Research 345 (2010) 914–921

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