Supporting Information for:
Gelation Characteristics and Applications of
Poly(ethylene glycol) End-Capped with Hydrophobic
Biodegradable Dipeptides
G. Rajesh Krishnana, Yuan Yuana, Ayesha Arzumanda, Debanjan Sarkar*,a, b
a
b
Department of Biomedical Engineering, University at Buffalo, State University of New York,
Buffalo, NY 14260, USA
Department of Chemical and Biological Engineering, University at Buffalo, State University of
New York, Buffalo, NY 14260, USA
*Correspondence to: D. Sarkar.
Ph: +1-716-645-8497, Fax: +1-716-645-2207
Email: debanjan@buffalo.edu
Procedures for Synthesis of End-Capping Molecules and Polymers
S a. Synthesis of Hexyl Ester of Tyrosine (1) 1-3
NH2
HO
O
OH
NH2
1) Hexanol, SOCl2, 0 oC, 1h
2) 80 oC, overnight
HO
O
O
1
In a 200 mL round bottom flask L-tyrosine (5 g, 27.59 mmol) and hexanol (50 mL) were mixed
and cooled to 0 oC in an ice bath. Thionyl chloride (2.41 mL, 33.11 mmol) was added to the flask
and the reaction mixture was stirred at 0 oC for an hour. It was allowed to warm to room
temperature slowly and then kept in an oil bath heated to 80 oC with stirring. The tyrosine started
dissolving and the reaction mixture was stirred at 80 oC for overnight. It was cooled to room
temperature to precipitate both unreacted tyrosine and its hexyl ester as quaternary ammonium
salts. The white solid was filtered under vacuum to remove excess hexanol and washed with
ether cooled to 4 oC. The white color solid was dissolved in water and the pH was raised to 7.5
using sodium bicarbonate. The aqueous layer was extracted using ethyl acetate. The ethyl acetate
layer was washed with brine, dried using sodium carbonate and evaporated to get the hexyl ester
of tyrosine. Yield= 7.245 g (89%)
FTIR (KBr) max 3340, 3286, 2960, 2931, 2862, 1726, 1613, 1597, 1519, 1469, 1396, 1259,
1469, 1259, 1215, 1171, 1019, 948, 828, 783, 556, 523; 1HNMR (CDCl3, 500 MHz), : 7.00 (d,
2H, J=8.1983 Hz, aromatic), 6.68 (d, 2H, J=8.1183 Hz, aromatic), 4.14 (t, 2H, J= 6.6986 Hz, OCH2CH2), 3.74 (br, NH2), 3.74 (q, 1H, J=2.0745 Hz, CH2CHNH2 ), 3.05 (dd, 2H, CH2, tyrosine),
2.85 (dd, 2H, CH2), 1.64 (q, 2H, J=6.8985 Hz, OCH2CH2), 1.32 (m, 6H, CH2-CH2-CH2) 0.91 (t,
3H, J=6.8985 Hz, CH3); ESIMS= Found 266.1756 (Calculated 266.1751)
S b. Synthesis of Desamino Tyrosine-Tyrosyl Hexyl Ester (DTH, 2)1-3
OH
NH2
HO
O
COOH
1) EDC, 0 oC, 1h
2) r. t., 24 h
O
H
N
HO
O
O
O
2
1
OH
In a 100 mL round bottom flask tyrosine hexyl ester (2 g, 6.77 mmol) was dissolved in dry THF
(50 mL) and cooled to 0 oC in an ice bath. To it 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) (1.26 g, 8.11 mmol) was added followed by 3-(4-hydroxy) propionic acid (1.34 g, 8.14
mmol). The reaction mixture was stirred at 0 oC for 1 h and then it was slowly warmed to room
temperature and stirred at room temperature for 24 h. The solvent was removed under vacuum.
The residue was dissolved in ethyl acetate and transferred to separating funnel. The organic layer
was washed with saturated sodium bicarbonate (20 mL x 3), 0.1 M HCl (20 mL x 3) and brine.
The organic layer was separated, dried using sodium carbonate and filtered. The solvent was
removed under vacuum to obtain DTH as a yellow colored viscous liquid. Yield=2.603 g (93%)
FTIR (Film on NaCl) max 3336, 2951, 2930, 2854, 1723, 1647, 1614, 1515, 1449, 1364, 1224,
828; 1HNMR (CDCl3, 500 MHz), : 7.59 (br, 1H, CONH), 6.88 (d, 2 H, J=9.9979 Hz,
aromatic), 6.71 (m, 6H, aromatic), 6.26 (br, 2H, -OH), 4.82 (q, 1H, J=7.5984 Hz,
CH2CHNHCO), 4.10 (m, 2H, O-CH2), 2.98 (q, 1H, J=8.7982 Hz, CH2CHCONH), 2.90 (q, 1H,
J=6.3487 Hz, CH2CHCONH), 2.79 (m, 2H, CH2), 2.46 (m, 2H, CH2), 1.61 (t, 2H, J=6.9985 Hz,
CH2), 1.29 (m, 6H, CH2CH2CH2), 0.89 (t, 3H, J=6.8336 Hz, CH3); ESIMS=Found 414.227
(414.2275 calculated)
S c. Synthesis of 3-(4-hydroxy phenyl) propionic acid ethanoate (HPE, 3)
COOH
COOCH2CH3
1) EtOH, H2SO4
r.t., overnight
OH
OH
3
In a 50 mL round bottom flask 3(4-hydroxy phenyl) propionic acid (1 g, 6.01 mmol) was
dissolved in 30 mL ethanol. To it 3 drops of concentrated sulphuric acid was added and the
reaction mixture was stirred at room temperature for overnight. Ethanol was removed under
vacuum, the residue was dissolved in ethyl acetate and washed with saturated sodium
bicarbonate (3x25 mL). The organic layer was further washed with brine and separated from the
aqueous layer. It was dried using sodium carbonate, filtered and the solvent was removed under
vacuum to get the required product as a pale yellow viscous liquid which crystallize as white
needles on standing.
FTIR (KBr) max= 3379, 2981, 2940, 2906, 1885, 1713, 1694, 1681, 1613, 1596, 1514, 1471,
1444, 1372, 1302, 1222, 1100, 1056, 1021, 871, 833, 793, 635; 1HNMR (CDCl3, 500 MHz), :
7.05 (d, 2H, aromatic, J=8.4832 Hz), 6.78 (d, 2H, aromatic, J=3.4143 Hz), 6.2 (s, br, -OH), 4.15
(q, 2H, J=7.1385 Hz, OCH2), 2.90 (t, 2H, J=7.6284 Hz, CH2), 2.62 (t, 2H, J=1.0397 Hz, CH2 ),
1.26 (t, 3H, J=7.1385 Hz, CH3); ESIMS 217.0830 (found) 217.0835 (calculated)
S d. Synthesis of (R)-hexyl 2-(acetamido)-3-(4-hydroxyphenyl)propanoate (TAE, 4)
NH2
HO
O
1
O
H
N
o
CH3COCl
1) Et3N, DMF, 0 C, 2h
2) r. t., 5 days
HO
O
4
O
O
In a round bottom flask tyrosine hexyl ester (1 g, 3.76 mmol) was dissolved in dry DMF (20 mL)
and cooled to 0 oC in an ice bath. To it triethyl amine (0.578 mL, 4.14 mmol) was added
followed by acetyl chloride (0.275 mL, 3.87 mmol). The reaction mixture was stirred at 0 oC for
two hours and then allowed to warm to room temperature. The reaction mixture was stirred at
room temperature for five days. The reaction was followed by TLC on silica gel using hexaneethyl acetate (1:1) as the eluent. After the completion of the reaction mixture was poured into
water and was extracted using ethyl acetate. The organic layer was washed with dil HCl (15
mLx3) saturated sodium bicarbonate (15 mL x 3) and brine (15 mL x 3). The organic layer was
separated, dried with sodium carbonate and filtered. The solvent was removed under vacuum to
get the required product as light yellow needles. Yield 720 mg
FTIR (Film on NaCl) max = 3280, 2951, 2930, 2854, 1735, 1654, 1614, 1516, 1447, 1373, 1218,
1123, 825; 1HNMR (CDCl3, 500 MHz), : 7.77 (s, 1H, CONH), 6.95 (d, 2H, J=8.4982 Hz,
aromatic), 6.75 (d, 2H, J=8.4982 Hz, aromatic), 6.25 (s, br, 1H, -OH), 4.87 (q, 1H, J= 5.6988
Hz, CH2CHNH2), 4.13 (m, 2H, O-CH2) 3.08 (dd, 1H, J=8.2983 Hz, 5.7988 CH2, tyrosine), 2.98
(dd, 1H, J=6.3487 Hz, CH2, tyrosine), 1.64 (q, 2H, J= 6.9985 Hz, CH2), 1.31 (m, 9 H, CH2-CH2CH2+COCH2CH3) 0.90 (t, 3H, J= 6.7486 Hz, CH3); ESIMS=308.1862 (Found), 308.1856
(Calculated)
S e. Synthesis of (R)-hexyl 2-(benzamido)-3-(4-hydroxyphenyl)propanoate (TBE, 5)
NH2
HO
O
1
O
O
Cl
H
N
1) Et3N, DMF, 0 oC, 2h
2) r. t., 5 days
HO
O
O
O
5
In a round bottom flask tyrosine hexyl ester (1 g, 3.76 mmol) was dissolved in dry DMF (20 mL)
and cooled to 0 oC in an ice bath. To it triethyl amine (0.578 mL, 4.14 mmol) was added
followed by benzoyl chloride (0.449 mL, 3.87 mmol). The reaction mixture was stirred at 0 oC
for two hours and then allowed to warmed to room temperature. The reaction mixture was stirred
at room temperature for five days. The reaction was followed by TLC on silica gel using hexaneethyl acetate (1:1) as the eluent. After the completion of the reaction mixture was poured into
water and was extracted using ethyl acetate. The organic layer was washed with dil HCl (15
mLx3) saturated sodium bicarbonate (15 mLx 3) and brine (15 mLx3). The organic layer was
separated, dried with sodium carbonate and filtered. The solvent was removed under vacuum to
get the required product as yellow needles. Yield 1 g
FTIR (Film on KBr) max =3461, 3301, 2956, 2930, 2852, 1737, 1643, 1613, 1540, 1517, 1443,
1356, 1275, 1209, 1182, 1086, 804; 1HNMR (CDCl3, 500 MHz), : 7.57 (d, 2H, aromatic,
benzoyl, J=5.9187), 7.43 (m, 3H, aromatic, benzoyl), 6.98 (d, 2 H, J=1.4047, aromatic), 6.75 (d,
2H, J=1.9546, aromatic), 6.25 (s, br, 1 H, OH), 5.07 (q, 1H, J=7.4434, CH2CHNH2), 4.17 (t, 2H,
OCH2CH2, J=6.7136), 3.16 (td,1H, J=5.1329, 8.8531 ,CHCH2NH), 1.67 (p, 2H, J=6.6486,
OCH2CH2CH2), 1.37-1.32 (m, 6H), 0.91 (t, 3H, J=1.0997, CH3); ESIMS=370.2009 (Found)
370.2013 (Calculated)
S f. Synthesis of the Polymers P2, P3 and P4
In a 100 mL round bottom flask PEG (0.1 mmol) was dissolved in dry DMF (40 mL) by heating
the flask to 80 oC in an oil bath. Hexamethylene diisocyanate (42.05 mg, 0.25 mmol) was added
to the solution followed by tin-2-ethyl hexanoate (10 drops). The reaction mixture was stirred at
80 oC for 6 h and then cooled to room temperature. DTH (103.37 mg, 0.25 mmol) was added
followed by a fresh batch of tin-2-ethyl hexanoate (5 more drops). The reaction mixture was
stirred at 50 oC overnight. After cooling to room temperature the reaction mixture was added in
small portions to diethyl ether (100 mL) cooled in an ice bath. The polymers precipitated as a
light yellow solid were filtered under vacuum and washed with an excess of ether followed by
2:1 ether-dichloromethane mixture (50 mL) and acetone (50 mL) to ensure complete removal of
remaining starting materials. To remove any trapped solvents or starting material the polymer
was stirred in ether for overnight. The light yellow polymer was dried under vacuum at room
temperature for three days and stored at room temperature in air tight containers. Yield=93-95%
for all polymers
S g. Synthesis of Polymer P5
DTH (103.37 mg, 0.25 mmol) was dissolved in DMF (40 mL). To it hexamethylene diisocyante
(84.10 mg, 0.5 mmol) was added followed by tin(II)2-ethyl hexanoate (10 drops) and the
reaction mixture was stirred at 50 oC for 6 h. The reaction mixture was cooled to room
temperature and then PEG20000 (10 g, 0.5 mmol) was added followed by a fresh batch of
tin(II)2-ethyl hexanoate (5 drops). The reaction mixture was stirred overnight at 80 oC. After
cooling the polymer was precipitated and purified as described above. The polymer is light
yellow in color. Yield=9.77 g (96%)
S h. Synthesis of Polymer P6, P7, P8 and P10
These polymers were prepared according to the same procedure used for making polymer P1.
But the linker or end groups were changed according to table 3 in the manuscript. The molar
ratio of PEG, linkers and the end groups were kept same as in the synthesis of P1. Reaction
conditions and product isolation were same as that described in the synthesis of P1. Yield=9396% for all polymers
S i. Synthesis of Polymer P9
In a 100 mL round bottom flask PEG (0.1 mmol) was dissolved in dry DMF (40 mL) by heating
a mixture of PEG and DMF to 80 oC in an oil bath. To that solution hexamthylene diisocyanate
(42.05 mg, 0.25 mmol) was added followed by tin(II)2-ethyl hexanoate (10 drops). The reaction
mixture was stirred at 80 oC for 6 h. The reaction mixture was cooled to room temperature;
methanol (5 mL) was added and stirred for 2 h to convert the macromolecular diisocyanate into
the macromolecular amine. After cooling the polymer was precipitated and purified as described
above. The polymer is light yellow in color. Yield= 3.490g (96.2%)
References:
1. Pulapura S, Li C, Kohn J. Biomaterials 1990; 11(9):666–678.
2. Ertel SI, Kohn J. Polym Mater Sci Engng 1992; 66:224–225.
3. Kimberly A. H.; Kohn, J. J. Bio. Compat. Polym. 1995; 10:327-340.
Figure S1. 1HNMR spectrum of polymer P1. Ratio of integrals for PEG H to phenolic H is 1606
(3181 PEG H and 1.98 phenol H), ratio of integral of PEG H to CH3 protons is 530.16 (3181
PEG H and 6 CH3 protons), ratio of integral of PEG H to aromatic protons is 318.1 (3181 PEG H
and 13 aromatic H). Few solvent peaks are found in high field of the spectrum.
Figure S2. 1HNMR spectrum (A) and 13CNMR spectrum (B) of DTH with spectral interpretation
Figure S3. 1H NMR spectra of end
groups used to verify the specificity
of DTH A) HPE; 3-(4-hydroxy
phenyl) propionic acid ethanoate B)
TAE; (R)-hexyl 2-acetamido-3-(4hydroxyphenyl)propanoate C) TBE;
(R)-hexyl-2-(benzamido)-3-(4hydroxyphenyl)propanoate
Figure S4. 1HNMR spectrum of polymer P3. Ratio of integrals for PEG H to phenolic H is 42.06
(90.44 PEG H and 2.15 phenol H), ratio of integral of PEG H to CH3 protons is 15.78 (90.44
PEG H and 5.73 CH3 protons), ratio of integral of PEG H to aromatic protons is 5.19 (90.44 PEG
H and 17.41 aromatic H). Few solvent peaks are found in high field of the spectrum.
Figure S5. 1HNMR spectrum of polymer P5. Only PEG peaks are visible along with few alkyl
peaks.
Figure S6. 1HNMR spectrum of polymer P7. Ratio of integral of PEG H to aromatic H 396.6
(3181 PEG H to 8.021 aromatic H)
Figure S7. 1HNMR spectrum of polymer P8. Ratio of integral of PEG H to aromatic to alkyl H is
167.42 (3181 PEG H to 19 aromatic H)
Figure S8. 1HNMR spectrum of polymer P9. Ratio of integrals of PEG H to aromatic H shows
509.243 (3636 PEG H to 7.14 aromatic H)
Figure S9. 1HNMR spectrum of linear segmented polyurethane formed from PEG, HDI and
DTH with spectral interpretation. Spectra shows the absence of phenolic proton in the polymer
Figure S10. Gel formation by P1. Images comparing the solvent interaction of P1 and chain
extended linear polyurethanes (linear-PU). A) 50 mg each of P1 (pale yellow solid) and linear
PU (yellow solid) in 1 mL water. P1 forms gel while linear-PU dissolves B) After 5 minutes of
heating. C) After 20 minutes of shaking
Figure S11. P1 gels exhibits shear thinning behavior with decrease in viscosity with increasing
shear rate (A). P1 hydrogel retains gel-like character after being injected through 19 gauge
needle (0.6 mm diameter) (B)
Figure S12. SEM image of hydrogel obtained by solvent exchange of organogel (DCM was
exchanged with water). Morphology of organogel changed to more porous one (in comparison to
Figure 5D). Inset shows higher magnification.