Supporting Information for “Controlling the morphology of self-assemble chitosan
through derivatization”
Pimsiri Deemark, Supason P. Wanichwecharungruang, Rutchanee Nonthabenjawan, and
Chotiros Jornjangjun
1. Synthesis of chitosan derivatives
1.1 Synthesis of 2,4,5-trimethoxycinnamic acid
pyridine, piperidine
OCH3
O
H3CO
OCH3
reflux 7 h
+ HOOC
COOH
H3CO
O
H
H3CO
H3CO
OH
2,4,5-Trimethoxybenzaldehyde (0.981 g, 0.005 mole) was dissolved in 43.80 mL of pyridine
followed by malonic acid (0.52 g, 0.005 mole), and then piperidine (2.50 mL, 25.25 mmole)
was added to the homogeneous solution. The solution was heated at 85 ˚C for 7 h and the
precipitate, formed in 10% aqueous HCl, was harvested by centrifugation and washed
thoroughly with ethanol to obtain 2,4,5-trimethoxycinnamic acid.
2,4,5-trimethoxycinnamic acid: yellow solid. Yield: 88%. Tm: 165 - 167 C. FT-IR (KBr):
3432 (HOOC), 3004, 3068 and 3129 (H-C=C stretching), 1682 (C=O carboxylic), 1600
(C=C) and 755, 1463 and 1600 cm-1 (aromatic ring). 1H-NMR (DMSO-d6): δ = 3.7, 3.8 and
3.9 (s, 9H, 3×O-CH3), 6.39 (d, J=16.0 Hz, 1H, Ar-HC=CH-COOH), 6.7 and 7.2 (s, 2H, ArH), 7.77 (d, J=16.0 Hz, 1H, Ar-HC=CH-COOH) and 11.9 (broad, 1H, COO-H). UV–vis
(DMSO) max, (): 282 (12000) and 350 nm (13000 M-1cm-1, for the monomeric units). MS
(m/z): calcd. for C12H14O5, 238; found, 238 [M]+.
1.2 Chitosan grafted with phthaloyl functionality (PC)
-1-
O
O
OH
O
*
HO
NH 2
chitosan
*
O
O
DMF/N 2
1) 120 oC, 6 h
2) 65 o C, 17 h
OH
*
O
*
O
HO
O
N
O
phthaloylchitosan (PC)
Chitosan (3 g; 0.01 mole equivalent of glucosamine unit) was added into a mixture of
phthalic anhydride (0.02 moles) and 20 mL of anhydrous DMF, under a N2 atmosphere, with
stirring. Then, the mixture was initially heated at 120 ˚C for 6 h, and subsequentially at 65 OC
for 8 h. The mixture was then precipitated in ice water and washed thoroughly with methanol
to obtain phthaloylchitosan, PC.
Phthaloylchitosan (PC): brown solid. Yield: 80%. C/N ratio: 12.481. FT-IR (KBr): 3470
(OH), 1771 and 1710 (C2O2N, phthalimido), 1710 (C=O, ester) and 720, 1388 and 1650 cm-1
(aromatic ring). 1H-NMR (DMSO-d6 with 0.05% TFA): δ = 2.0 (s, NCOCH3), 2.7-4.7 (m,
H2-H6 of pyranose ring), 5.1 (broad, H1 of pyranose ring) and 7.57-7.66 (m, Ar-H of the
phthaloyl groups). UV–vis (DMSO) max: 286 nm.
1.3 Phthaloylchitosan grafted with various cinnamoyl fuctionalities (CPC1, CPC2,
CPC3, 4CPC1, 4CPC2, 4CPC3, 245CPC1, 245CPC2, 245CPC3, 245CPC4 and
245CPC5)
Coupling agent method
-2-
R5
R5
R4
R3
O
R3
OH
R2
OH
EDCI/HOBt
*
O
HO
O
N
R2
O
O
*
O
R4
R6
R6
*
O
*
O
HO
O
O
PC
N
O
CPC: R 2-R6 = H
4CPC: R 2 , R 3, R 5 , R6 = H; R 4 = OCH 3
245CPC: R 3, R6 = H; R2 , R 4, R5 = OCH 3
PC (0.5 g, 1.85 × 10-3 moles glucosamine unit) was stirred with cinnamic acid (0.1097 g, 7.41
× 10-4 moles) in 20 mL of DMF solution containing HOBt (7.41 × 10-4 moles) at room
temperature. EDCI (7.41 × 10-4 moles) was added at 4 °C and the mixture was kept at 4 °C
for 1 h and then at room temperature for 12 h. The mixture was dialyzed against water and
washed thoroughly with methanol to obtain CPC2. CPC1 was derived in the same way except
that the amounts of cinnamic acid, EDCI and HOBt were ten folds reduced.
Likewise 4CPC1, 245CPC1, 245CPC2 and 245CPC3 were also prepared using a
similar procedure except that the type of acid was changed to 4-methoxycinnamic acid or
2,4,5-trimethoxycinnamic acid and the amounts added were 7.4 × 10-4, 1.0 × 10-4, 7.4 × 10-4
and 1.0 × 10-3 moles for 4CPC1, 245CPC1, 245CPC2 and 245CPC3, respectively. The
amount of EDCI and HOBt were also adjusted according to the amount of the added cinnamic
acid.
Acid chloride method
-3-
O
R5
Cl
R6
S
R5
R6
Cl
R4
O
R3
O
R4
OH
R2
R3
R2
Cl
R3
R4
R5
OH
*
O
*
O
HO
O
N
R6
R4
O
R3
R2
R2
R5
R6
O
Cl
O
O
*
O
HO
O
O
N
O
*
O
PC
CPC: R 2-R6 = H
4CPC: R2 , R 3, R5 , R 6 = H; R 4 = OCH 3
245CPC: R 3 , R 6 = H; R 2, R4 , R 5 = OCH 3
Cinnamic acid (0.5487 g, 3.70 × 10-3 mole) and freshly distilled thionyl chloride (2.70 mL,
3.70 × 10-2 mole) were refluxed for three h in a two-neck-round bottom-flask attached with a
condenser and a drying tube. After that, unreacted thionyl chloride was removed under
reduced pressure leaving cinnamoyl chloride in the flask. PC (0.5 g) and 30 mL of freshly
distilled DMF were then added to the flask and the mixture was heated for 24 h at 80 ˚C prior
to cooling to room temperature. The obtained CPC3 was then precipitated in ice water and
washed with methanol.
Similar procedure was used to prepare 4CPC2, 4CPC3, 245CPC4 and 245CPC5, only
that 4-methoxycinnamic acid or 2,4,5-trimethoxycinnamic acid, as appropriate, was used in
placed of cinnamic acid. Amounts of 4-methoxycinnamic acid for 4CPC2, 4CPC3 were 2.0 ×
10-3 and 3.7 × 10-3, respectively. Amounts of 2,4,5-trimethoxycinnamic acid for 245CPC4
and 245CPC5 were 2.0 × 10-3 and 3.7 × 10-3 mole, respectively.
-4-
Cinnamoylphthaloylchitosan (CPC): cream powder. Yield > 85% for CPC1, CPC2 and
CPC3. C/N ratio: 12.491, 13.510, 17.263 for CPC1, CPC and CPC3, respectively. FT-IR
(KBr): 3457 (OH), 1772 and 1714 (C2O2N, phthalimido), 1714 (C=O ester), 1135 (C-O-C)
and 726, 1391, 1455 and 1616 cm-1 (aromatic ring). 1H-NMR (DMSO-d6 with 0.05% TFA): δ
= 2.0 (s, NCOCH3), 2.7-4.7 (m, 2H2-H6 of pyranose ring), 5.1 (broad, H1 of pyranose ring),
6.57 (d, J=16.0 Hz, Ar-HC=CH-COOR), 7.48 and 7.60 (d, J=8.0 Hz, Ar-H of cinnamoyl
groups) and 7.64-7.73 (m, Ar-H of phthaloyl groups and Ar-HC=CH-COOR). UV–vis
(DMSO) max: 287 nm.
4-methoxycinnamoylphthaloylchitosan (4CPC): white powder. Yield > 80% for 4CPC1,
4CPC2 and 4CPC3. C/N ratio: 13.670, 15.024 and 19.889 for 4CPC1, 4CPC2 and 4CPC3,
respectively. FT-IR (KBr): 3458 (OH), 1775 and 1711 (C2O2N, phthalimido), 1711 (C=O
ester), 1141 (C-O-C) and 723, 1388, 1598 and 1619 cm-1 (aromatic ring). 1H-NMR (DMSOd6 with 0.05% TFA): δ = 2.0 (s, NCOCH3), 3.85 (s, O-CH3), 2.70-4.70 (m, H2-H6 of
pyranose ring), 5.10 (broad, H1 of pyranose ring), 6.43 (d, J=16.0 Hz, Ar-HC=CH-COOR),
7.02 and 7.68 (d, J=8.0 Hz, Ar-H of cinnamoyl groups) and 7.46-7.58 (m, Ar-H of phthaloyl
groups and Ar-HC=CH-COOR). UV–vis (DMSO) max: 287, 310 nm.
2,4,5-trimethoxycinnamoylphthaloylchitosan (245CPC): yellow powder. Yield > 80% for
245CPC1, 245CPC2, 245CPC3, 245CPC4 and 245CPC5. C/N ratio: 13.021, 13.562, 15.092,
16.266 and 21.622 for 245CPC1, 245CPC2, 245CPC3, 245CPC4 and 245CPC5, respectively.
FT-IR (KBr): 3455 (OH), 1770, 1715 (C2O2N, phthalimido), 1715 (C=O ester), 1131 (C-OC) and 720, 1385, 1458, 1513 and 1601 cm-1 (aromatic ring). 1H-NMR (DMSO-d6 with
0.05% TFA): δ = 2.0 (s, 3H, NCOCH3), 3.81, 3.90 and 3.91 (s, 3×O-CH3), 2.70-4.70 (m, H2H6 of pyranose ring), 5.10 (broad, H1 of pyranose ring), 6.48 (d, J=16.0 Hz, Ar-HC=CH-5-
COOR), 6.77 and 7.30 (s, Ar-H of cinnamoyl groups), 7.87 (d, J=16.0 Hz, Ar-HC=CHCOOR) and 7.38-7.52 (m, Ar-H of phthaloyl groups). UV–vis (DMSO) max: 286 and 350
nm.
1.4 Chitosan grafted with various cinnamoyl functionalities (CC1, CC2, CC3, 245CC1,
245CC2 and 245CC3)
O
R5
Cl
R6
R4
S
R5
Cl
O
R6
R4
O
OH
R3
R2
R3
R5
*
O
HO
O
*
O
R3
R2
Cl
OH
R6
R4
OH
R2
O
HO
O
OH
O
HO
NH
O
*
NH 2
Cl
*
O
R6
R5
NH 2
R2
chitosan
R4
R3
CC: R 2-R6 = H
4CC: R 2 , R3 , R 5, R6 = H; R 4 = OCH 3
245CC: R 3, R6 = H; R2 , R 4, R5 = OCH3
Cinnamic acid (7.0 × 10-4 moles) and freshly distilled thionyl chloride (3.00 mL) were
refluxed for six h in a two-neck-round bottom-flask attached with a condenser and a drying
tube. After that, unreacted thionyl chloride was removed under reduced pressure leaving
cinnamoyl chloride in the flask. Chitosan (2.0 g, 1.25 × 10-2 moles of glucosamine units) and
80 mL of freshly distilled DMF were then added to the flask and the mixture was heated for
24 h at 80 ˚C. The reaction mixture was then cooled to room temperature. The obtained
product, CC1, was precipitated in ice water and washed thoroughly with methanol.
-6-
Derivatives with a different degree of substitution were prepared as above, except that
the amounts of cinnamic acid were changed to 1.3 × 10-3 and 1.3 × 10-2 moles, respectively
for CC2 and CC3.
Likewise the cinnamic acid was swapped to 2,4,5-trimethoxycinnamic acid for
245CC1, 245CC2 and 245CC3 production. Amounts of 2,4,5-trimethoxycinnamic acid used
were 1.3 × 10-3, 5.0 × 10-3 and 1.3 × 10-2 moles for 245CC1, 245CC2 and 245CC3,
respectively.
Cinnamoylchitosan (CC): white powder. Yield > 65% for CC1, CC2 and CC3. C/N ratio:
5.560, 6.482, 12.114 for CC1, CC2 and CC4, respectively. FT-IR (KBr) 3457 (OH), 1712
(C=O ester), 1125 (C-O-C) and 717, 1391 and 1628 cm-1 (aromatic ring). 1H-NMR (D2O with
0.05% (v/v) acetic acid): δ = 2.0 (s, NCOCH3 of chitosan and COCH3 of the added acetic
acid), 2.8 (s, H2 of GluN), 3.3-3.8 (m br, H3-H6 and H2 of GluNcinnamoyl), 4.4 (broad, H1
of GluNcinnamoyl), 4.55 (broad, H1 of GluN), 6.4 (d, J=16.0 Hz, 1H, Ar-HC=CH-COOR),
7.3 (m, Ar-HC=CH-CONHR, Ar-H), 7.4-7.5 (m, Ar-H of cinnamoyl groups and Ar-CH=CHCOOR). UV–vis (DMSO) max: 287 (3400) nm.
2,4,5-trimethoxycinnamoylphthaloylchitosan (245CC): light yellow powder. Yield > 66% for
245CC1, 245CC2 and 245CC3. C/N ratio: 6.200, 8.211 and 11.109 for 245CC1, 245CC2 and
245CC3, respectively. FT-IR (KBr): 3458 (OH), 1713 (C=O ester), 1130 (C-O-C) and 720,
1385, 1458, 1512 and 1600 cm-1 (aromatic ring). 1H-NMR (400 MHz, DMSO-d6 with 0.05%
(w/v) TFA): δ = 2.0 (s, 3H, NCOCH3), 3.81, 3.89 and 3.91 (s, 3×O-CH3), 2.70-4.70 (m, H2H6 of pyranose ring), 5.10 (broad, H1 of pyranose ring), 6.48 (d, J=16.0 Hz, Ar-HC=CHCOOR), 6.78 and 7.30 (s, Ar-H of cinnamoyl groups), 7.87 (d, J=16.0 Hz, Ar-HC=CHCOOR). UV–vis (DMSO) max: 286 and 350 nm.
-7-
1.5 Chitosan grafted with phthaloyl, PEO and various grafted cinnamoyl fuctionalities
113
(PEO-CPC3, PEO-4CPC2, PEO-245CPC4)
O
R3
O
R3
R4
R4
R2
O
HO
R2
O
O 113
R5
O
R6
R5
R6
EDCI/HOBt
O
O
O
O
* HO
O
O
O
O
O
N
O
*
O
* HO
O
CPC: R2 -R 6 = H
4CPC: R 2, R3 , R 5, R6 = H; R4 = OCH 3
245CPC: R 3, R6 = H; R2 , R 4, R5 = OCH3
O
O
N
O
HO
O
O
O
N
O
*
O
PEO-CPC: R 2-R6 = H
PEO-4CPC: R2 , R 3, R5 , R 6 = H; R 4 = OCH 3
PEO-245CPC: R3 , R 6 = H; R 2, R4 , R 5 = OCH 3
Synthesis of PEO-CPC3 was carried out by stirring HOBt (1.0 x 10-3 moles), PEO-COOH
(1.0 x 10-3 moles) with CPC3 (1.5 x 10-3 moles) in DMF (50 mL) until the solution became
clear when EDCI (1.0 x 10-3 moles) was then added into the reaction and maintained at 4 OC
for 1 h and then left at room temperature for 12 h. The mixture was poured into ice-cold
water, the precipitate collected by centrifugation, washed with excess methanol, and dried.
A similar procedure was used for the preparation of PEO-4CPC2 and PEO-245CPC4.
PEO-4CPC2 was prepared from 4CPC2 (1.5 x 10-3 moles) using 1.0 x 10-3 moles of PEOCOOH. PEO-245CPC4 was prepared from 245CPC4 (1.5 x 10-3 moles) using 1.0 x 10-3
moles of PEO-COOH. Amounts of EDCI and HOBt were adjusted accordingly.
PEO-cinnamoylphthaloylchitosan (PEO-CPC3): pale brown powder. Yield 91%. FTIR (KBr):
1770 (C=O of ester) and 1618 cm-1 (C=C). 1H-NMR (DMSO-d6 with 0.05% TFA): δ = 2.7-
-8-
5.50 (PEG and pyranose ring), 6.4 (d, Ar-CH=), 6.94 and 7.59 (d, Ar-H of cinnamoyl group),
7.48-7.72 (m, Ar-H of cinnamoyl group, Ar-HC=CH-COOR and Ar-H of phthaloyl group).
PEO-4-methoxycinnamoylphthaloylchitosan (PEO-4CPC2): pale brown powder. Yield > 90%
for PEO-4CPC1, PEO-4CPC2 and PEO-4CPC3. FTIR (KBr): 1772 (C=O of ester) and 1618
cm-1 (C=C). 1H-NMR (DMSO-d6 with 0.05% TFA): δ = 2.7-5.50 (OCH3, PEG and pyranose
ring), 6.4 (d, Ar-CH=), 6.90 and 7.60 (d, Ar-H of cinnamoyl group), 7.48-7.72 (m, Ar-H of
cinnamoyl group, 1H, Ar-HC=CH-COOR and Ar-H of phthaloyl group).
PEO-2,4,5-trimethoxycinnamoylphthaloylchitosan (PEO-245CPC4): pale brown powder.
Yield 90%. FTIR (KBr): 1773 (C=O of ester) and 1619 cm-1 (C=C). 1H-NMR (DMSO-d6 with
0.05% TFA): δ = 2.75-5.50 (OCH3, PEG and pyranose ring), 6.42 (d, 1H, Ar-CH=), 6.96 and
7.60 (d, 4H, Ar-H of cinnamoyl group), 7.48-7.78 (m, Ar-H of cinnamoyl group, ArHC=CH-COOR and Ar-H of phthaloyl group).
2. NMR Data
1
H-NMR spectra (in DMSO-d6 with ~0.05% TFA) of the PC clearly indicated the presence of
phthaloyl moieties by the appearance of signals at 7.3-7.7 ppm (Ar-H of the phthaloyl
groups). A degree of substitution (DS) was determined from the integration of the phthaloyl
protons against protons of the glucosamine backbone (2.7-5.8 ppm, 1H-6H of glucosamine).
Interference from the HOD peak at 3.3 ppm could be avoided by shifting the HOD peak to >
9 ppm through the addition of a small amount of trifluoroacetic acid (TFA) into the sample
just prior to the NMR analysis. The 1H-NMR spectrum also shows an insignificant amount of
the N-acetyl signal (s, 2.0 ppm NCOCH3) indicating less than 1% substitution of the N-acetyl
functionality.
-9-
1
H-NMR (in DMSO-d6) spectra of CPC, 4CPC and 245CPC derivatives clearly
indicated the presence of the grafted cinnamoyl moieties through the appearance of the
resolved sharp doublet signals at 6.5 ppm (1H, J=16, 400Hz, Ar-CH=CH-COOR of the
cinnamoyl group). 4CPC and 245CPC derivatives also gave sharp singlet signal at 3.9 ppm
for the methoxy groups. The degrees of cinnamoyl substitution were estimated by integrating
the peak area at 6.4 ppm, which represented one proton (Ar-CH=CH-COOR) from each
cinnamoyl group, against the H1 peak of the glucosamine at 5.1 ppm.
1
H-NMR spectra of CC (in D2O with acetic acid) show resonances of aromatic and
methylene protons from cinnamoyl group (6.4-8.0 ppm, Ar-H and Ar-CH=CH-COOR).
Substitution degree of cinnamoyl group in CC could be determined through integration of
these peaks against integration of peaks at 2.8-4.0 ppm which represent H1-H6 of the
glucosamine moieties.
The 1H-NMR (in DMSO-d6) spectra of PEO-CPC3, PEO-4CPC2 and PEO-245CPC4
indicated the presence of the grafted cinnamoyl moieties through the appearance of the
resolved sharp doublet signals at 6.5 ppm (1H, J=16 Hz, Ar-CH=CH-COOR of the cinnamoyl
group). The degree of substitution of PEO and cinnamoyl were estimated by integrating the
peak area at 6.4 ppm, which represented one proton (Ar-CH=CH-COOR) from each
cinnamoyl group, against the glucosamine H1 peak at 5.1 ppm. The degree of substitution of
PEO could be estimated using the peak area at 2.7 - 4.50 ppm (OCH3 from methoxy groups
on the 4-methoxycinnamoyl moieties and 2,4,5-trimethoxycinnamoyl moities, -OCH2CH2Ofrom PEO and the H2 - H7 of glucosamine). Since the DS of cinnamoyl group is known, and
the glucosamine H1 peak is well resolved (5.1 ppm), the contribution of protons from the
OCH3 group from 4-methoxycinnamoyl (or 2,4,5-trimethoxycinnamoyl) moieties and H2 H7 of glucosamine could be subtracted out. Thus, the peak area representing only OCH2CH2O- from PEO could be obtained and compared with the area from the glucosamine
- 10 -
H1 peak at 5.1 ppm to obtain the DS of PEO. Interference from the HOD peak was avoided
through TFA addition, as mentioned earlier.
Substitution degree obtained from 1H NMR data for each derivative was confirmed
with C/N ratio (result from elemental analysis) except that of 245CC1, 245CC2 and 245CC3
in which their substitution degrees were based only on elemental analysis.
2.1 PC (chitosan grafted with phthaloyl moiety)
- 11 -
2.2
CPC (chitosan grafted with phthaloyl and cinnamoyl moieties)
- 12 -
2.3
4CPC (chitosan grafted with phthaloyl and 4-methoxycinnamoyl moieties)
- 13 -
2.4
245CPC (chitosan grafted with phthaloyl, and 2,4,5-trimethoxycinnamoyl moieties)
- 14 -
2.5
CC (chitosan grafted with cinnamoyl functionalities)
2.6
PEO-CPC3 (chitosan grafted wih poly(ethylene oxide), phthaloyl moieties and
cinnamoyl groups)
- 15 -
2.7
PEO-4CPC2 (chitosan grafted wih poly(ethylene oxide), phthaloyl moieties and 4-
methoxy-cinnamoyl groups)
- 16 -
2.8
PEO-245CPC4 (chitosan grafted wih poly(ethylene oxide), phthaloyl moieties and
2,4,5-trimethoxycinnamoyl groups)
- 17 -