Polymers from amino acids: Development of dual ester-urethane melt condensation approach and mechanistic aspects

Article abstract of DOI:10.1021/bm300697h

A new dual ester-urethane melt condensation methodology for biological monomers-amino acids was developed to synthesize new classes of thermoplastic polymers under eco-friendly and solvent-free polymerization approach. Naturally abundant l-amino acids were converted into dual functional ester-urethane monomers by tailor-made synthetic approach. Direct polycondensation of these amino acid monomers with commercial diols under melt condition produced high molecular weight poly(ester-urethane)s. The occurrence of the dual ester-urethane process and the structure of the new poly(ester-urethane)s were confirmed by ¹H and ¹³C NMR. The new dual ester-urethane condensation approach was demonstrated for variety of amino acids: glycine, β-alanine, l-alanine, l-leucine, l-valine, and l-phenylalanine. MALDI-TOF-MS end group analysis confirmed that the amino acid monomers were thermally stable under the melt polymerization condition. The mechanism of melt process and the kinetics of the polycondensation were studied by model reactions and it was found that the amino acid monomer was very special in the sense that their ester and urethane functionality could be selectively reacted by polymerization temperature or catalyst. The new polymers were self-organized as β-sheet in aqueous or organic solvents and their thermal properties such as glass transition temperature and crystallinity could be readily varied using different l-amino acid monomers or diols in the feed. Thus, the current investigation opens up new platform of research activates for making thermally stable and renewable engineering thermoplastics from natural resource amino acids.

Full text of DOI:10.1021/bm300697h

Article
pubs.acs.org/Biomac
Polymers from Amino acids: Development of Dual Ester-Urethane
Melt Condensation Approach and Mechanistic Aspects
S. Anantharaj and M. Jayakannan*
Department of Chemistry, Indian Institute of Science Education and Research (IISER)-Pune, Dr. Homi Bhabha Road, Pune−411008,
Maharashtra, India
S
* Supporting Information
ABSTRACT: A new dual ester-urethane melt condensation
methodology for biological monomers−amino acids was
developed to synthesize new classes of thermoplastic polymers
under eco-friendly and solvent-free polymerization approach.
Naturally abundant L-amino acids were converted into dual
functional ester-urethane monomers by tailor-made synthetic
approach. Direct polycondensation of these amino acid
monomers with commercial diols under melt condition
produced high molecular weight poly(ester-urethane)s. The
occurrence of the dual ester-urethane process and the structure
of the new poly(ester-urethane)s were confirmed by 1H and 13
C
NMR. The new dual ester-urethane condensation approach was
demonstrated for variety of amino acids: glycine, β-alanine, L-
alanine, L-leucine, L-valine, and L-phenylalanine. MALDI-TOF-MS end group analysis confirmed that the amino acid monomers
were thermally stable under the melt polymerization condition. The mechanism of melt process and the kinetics of the
polycondensation were studied by model reactions and it was found that the amino acid monomer was very special in the sense
that their ester and urethane functionality could be selectively reacted by polymerization temperature or catalyst. The new
polymers were self-organized as β-sheet in aqueous or organic solvents and their thermal properties such as glass transition
temperature and crystallinity could be readily varied using different L-amino acid monomers or diols in the feed. Thus, the
current investigation opens up new platform of research activates for making thermally stable and renewable engineering
thermoplastics from natural resource amino acids.
limited range of amino acids.11 Other than the NCA route, so
INTRODUCTION
■
far no effort has been put to utilize amino acids as monomers
for making high molecular weight polymers for commercial
application. Amino acids are naturally available bioresources,
and therefore, developing new synthetic polymerization
strategies using them would open up a new direction of
research activities toward amino acid polymeric materials for
applications in biomedical components and thermoplastics.
The present work is emphasized to develop new eco-friendly
melt polycondensation approach for L-amino acid monomers.
Solvent-free melt polycondensation process is one of the most
widely employed synthetic approach for producing commercial
engineering thermoplastics such as polyesters, polycarbonates
and polyamides.12 In this process, raw materials were melted
and subjected to condensation reaction to produce high viscous
resins which could be directly processes into desired objects. In
1946, Whinfield developed first melt polymerization route
transesterification in which a diester monomer was poly-
condensed with diols to produce polyesters (see Scheme 1).12
Amino acids are biological monomers and their macro-
molecular peptide sequence and chain length played a crucial
role on the size, function, and secondary structure of
proteins.1,2 Synthetic polymers based on amino acids have
been of great interest in chemistry−biology interface due to
their potential application in therapeutics, cosmetics, and
biodegradable and biocompatible engineering thermoplastics.3,4
Peptide linkages (amide bonds) were routinely made by the
self-condensation of amino acid using water removal agents
such as DCC; however, this route was not capable of making
higher molecular weight polymers having more than 8−10
repeating units.5 In an indirect approach, amino acids were
converted into dicarboxylic acid derivatives and polymerized
with diols or diamines to produce poly(ester-amide) and their
random copolymers.6−8 Ring-opening polymerization of amino
acids via N-carboxyanhydride (NCA) intermediate was another
important approach to make linear, block, and star-shaped
polypeptides.9 Better control over the NCA intermediates was
achieved by Deming and co-workers via Ni(COD)bpy
transition metal catalyzed polymerization process.10 However,
the interference of functional groups in multifunctional amino
acids (like serine) with the catalyst restricted this approach to a
Received: May 4, 2012
Revised: June 14, 2012
Published: June 20, 2012
© 2012 American Chemical Society
2446
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
in CDCl3 containing TMS as internal Standard. High resolution mass
spectra were obtained from Micro Mass ESI-TOF MS spectrometer.
MALDI-TOF MS of the polymers was determined using Applied
Biosystems 4800 PLUS Analyzer. The polymer samples were dissolved
in tetrahydrofuran (THF) at 10 mg/mL and dihydroxybenzoic acid
(DHB) was used as matrix. The matrix solution was prepared by
dissolving 30 mg in 1.0 mL of THF. A 1−2 μL aliquot of the polymer/
matrix mixture was used of the MALDI-TOF MS analysis. FT-IR
spectra of all compounds were recorded using Bruker alphaT Fourier
transform infrared spectrophotometer. Gel permeation chromato-
graphic (GPC) analysis of the polymer samples were performed using
Viscotek VE 1122 pump, Viscotek VE 3580 RI detector and Viscotek
VE 3210 UV/vis detector in tetrahydrofuran (THF) or dimethylfor-
maamide (DMF) using polystyrene as standards. Thermal analysis of
the polymers was performed using TA Q20 Differential Scanning
Calorimeter. The instrument was calibrated with indium standards. All
the polymers were heated to melt before recording their thermograms
to remove their previous thermal history. Polymers were heated and
cooled at 10 °C/min under nitrogen atmosphere and their
thermograms were recorded. Thermal stability of the polymers was
determined using Perkin Elmer thermal analyzer STA 6000 model at a
heating rate of 10 °C/min in nitrogen atmosphere. Circular dichroism
(CD) analysis of the polymer samples were done using JASCO J-815
CD spectrometer at 25 °C in THF, methanol, and water, depending
up on their solubility.
Scheme 1. Development of New Dual Ester-Urethane Melt
Polycondensation for Amino Acids
This route is adopted even today for manufacturing a few
million tons of polyesters every year. Ramakrishnan and co-
workers had reported melt transetherification approach for
linear and hyperbranched polyethers based on fully protected
bis-benzyl ethers.13 From our research group, Deepa et al. had
reported eco-friendly melt transurethane process (see Scheme
1) for polyurethane and the approach was aimed to replace the
toxic and hazardous isocyanate chemical pathways.14 In this
process, commercially available diamines were converted into
diurethane monomers which were polymerized with diols to
make polyurethanes (see Scheme 1). It is reasonable to
consider amino acids as half an acid and half an amine; thus,
development of solvent-free melt condensation approach by
combining the melt transurethane process developed in our
laboratory with the whinfield one (transesterification) would
open up a new avenue of research opportunities for amino acid
based condensation polymers.
Herein, we developed a new dual ester-urethane melt
polycondensation approach for amino acid monomers as
shown in scheme-1. In this new process, amino acids were
readily converted into dual ester-urethane monomers and
polycondensed with diols under melt conditions to produce
high molecular weight polymers. The new synthetic process
was tested for more than half-dozen of amino acids and diols.
The mechanistic aspects of the new process were studied by
NMR and MALDI-TOF-MS, and control reactions were
carried out to understand the kinetics of the polymerization.
The role of the catalyst, polymerization temperature, and
repeating unit structure on the molecular weight of the
polymers and their thermal properties were also investigated.
The newly synthesized amino acid polymers were found to self-
organize as either β-sheet or polyproline type-II secondary
structures. The present synthetic polymer approach, dual ester-
urethane process, is expected to pave the way for new research
platforms for amino acids, which has huge potential in
thermoplastic and biomedical applications.
Synthesis of Methyl Esters of Amino Acids. Typical procedure
for carboxylic methyl esters of amino acids was described for L-
phenylalanine. To a suspension of L-phenylalanine (8.67 g, 0.052 mol)
in methanol (85 mL), thionylchloride (11.4 mL, 18.74 g, 0.157 mol)
was added dropwise at 5 °C under a nitrogen atmosphere. The
reaction was refluxed for 12 h by stirring under nitrogen. The solvent
and excess thionylchloride were removed by distillation. The residue
solid was washed with dry diethylether (120 mL) and dried to get
1
product as white solid. Yield = 10.4 g (93%). H NMR (400 MHz,
D2O) δ ppm: 7.41−7.24 (m, 5H, ArH), 4.39 (m, 1H, CHCH2Ar),
3.79 (s, 3H, COOCH3), 3.33−3.18 (d, 2H, CH2Ar). FT-IR (cm−1):
3926, 2852, 2618, 1742, 1581, 1491, 1443, 1232, 1145, and 1056. HR-
MS (ESI+): m/z [M + H+] Calcd for C10H13NO2 [M+], 180.1025;
found, 180.1025.
The methyl ester of other amino acids, glycine, β-alanine, L-valine, L-
leucine, and L-alanine, was prepared as described above and the details
are provided in the Supporting Information.
Synthesis Ester-Urethane Monomers of Amino Acids. Typical
procedure is described for L-phenylalanine monomer. Hydrochloride
salt of the above methyl ester of L-phenylalanine (7.10 g, 0.032 mol)
was stirred in sodium bicarbonate solution (25 wt %, 65 mL) +
dichloromethane (45 mL) at 5 °C under a nitrogen atmosphere. To
this ice cold solution, methyl chloroformate (5.1 mL, 0.066 mol) was
added dropwise and the reaction was continued for 12 h at 25 °C. The
reaction mixture was extracted with dichloromethane and the organic
layer was dried over anhydrous Na2SO4. The liquid was further
purified by passing through silica gel column using ethyl acetate and
1
pet ether (1:4 v/v) as eluent. Yield = 6.0 g (89%). H NMR (400
MHz, CDCl3) δ ppm: 7.32−7.24 (m, 3H, ArH), 7.12 (d, 2H, ArH),
5.12 (b, 1H, -NH), 4.64 (q, 1H, CHCH2Ar), 3.73 (s, 3H, COOCH3),
3.67 (s, 3H, NHCOOCH3), 3.11 (d, 2H, CH2Ar). 13C NMR (100
MHz, CDCl3) δ ppm: 172.95, 156.24, 135.69, 129.15 (2C), 128.52
(2C), 127.06, 54.69, 52.24 (2C), 38.13. FT-IR (cm−1): 3338, 2954,
1705, 1518, 1444, 1355, 1253, 1210, and 1057. HR-MS (ESI+): m/z
[M + Na+] Calcd for C12H15NO4 [M+], 260.0898; found, 260.0899.
Dual ester-urethane monomers of other amino acids, glycine, β-
alanine, L-valine, L-leucine, and L-alanine, were prepared as described
above and details are provided in the Supporting Information.
Dual Ester-Urethane Melt Polycondensation Process. Typical
dual ester-urethane melt polymerization procedure is explained for L-
phenylalanine monomer with 1,12-dodecanediol. Equimolar amounts
of amino acid monomer, L-phenylalanine monomer (0.76 g, 3.0 mmol)
and 1,12-dodecanediol (0.65 g, 3.0 mmol) were taken in a test tube-
shaped polymerization vessel and melted by placing the tube in oil
bath at 100 °C (see reaction vessel in the Supporting Information, SF-
EXPERIMENTAL SECTION
■
Materials. Glycine, L-alanine, β-alanine, L-valine, L-leucine, L-
phenylalanine, 1,4-cyclohexanedimethanol (CHDM), 1,12-dodeca-
ndiol (DD), diethyleneglycol (DEG), triethyleneglycol (TREG),
tetraethylene glycol (TEG), and titanium tetrabutoxide (Ti(OBu)4)
were purchased from Aldrich chemicals and used without further
purification. Methylchloroformate, thionyl chloride, and other solvents
were purchased locally and purified prior to use.
1
General Procedures. H and 13C NMR were recorded using 400
MHz JEOL NMR spectrophotometer. All NMR spectra were recorded
2447
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
1). The polycondensation apparatus was made oxygen and moisture
free by purging with nitrogen and subsequent evacuation by vacuum
under constant stirring. Titanium tetrabutoxide (11.0 mg, 0.03 mmol,
1.0 mol % equivalent to monomer) was added as catalyst and the melt
polycondensation was carried out at 150 °C for 4 h with constant
stirring under nitrogen purge. During this stage, the methanol was
removed along with the purge gas and the polymerization mixture
became viscous. The viscous melt was further subjected to high
vacuum (0.01 mm of Hg) at 150 °C for 2 h under stirring. At the end
of the polycondensation, the polymer, poly(ester-urethane), was
obtained as a white mass (weight = 0.98 g (82%). It was purified by
dissolving in tetrahydrofuran, filtered, and precipitated into methanol
to obtain fibrous product. Yield = 0.72 g (59%). 1H NMR (400 MHz,
CDCl3) δ ppm: 7.31−7.12 (m, 5H, ArH), 5.13 (b, 1H, NH), 4.62 (m,
1H, CH), 4.10−4.04 (m, 4H, COOCH2, NHCOOCH2), 3.10 (t, 2H,
CH2Ar), 1.56−1.25 (m, 20H, CH2). 13C NMR (100 MHz, CDCl3) δ
ppm: 171.74, 155.93, 135.85, 129.25, 128.46, 126.98, 65.56, 65.29,
54.65, 38.35, 29.50, 29.16, 28.89, 28.38, and 25.76. FT-IR (cm−1):
3348, 2924, 2853, 1717, 1505, 1458, 1397, 1346, 1249, 1196, and
1057. Molecular weights are given in Table 1.
mol %) was added and the condensation was carried out at 120 °C
under a nitrogen purge for 3 h. Subsequently, the temperature was
increased to 150 °C under a nitrogen purge for 3 h. Further, controlled
vacuum (1 mm of Hg) was applied at 150 °C for 2 h. During the
nitrogen and vacuum stage, various aliquots were taken every half an
hour interval. At the end of the condensation reaction, the product was
1
obtained as a slight yellow liquid. H NMR (400 MHz, CDCl3) δ
ppm: 7.36 (m, 10H, ArH), 5.18 (b, 1H, -NH), 5.09 (s, 2H,
PhCH2COO), 5.03 (s, 2H, PhCH2NHCOO), 3.91 (d, 2H, CH2). 13
C
NMR (100 MHz, CDCl3) δ ppm: 170.19, 156.56, 136.44, 135.39,
128.91 (2C), 128.79 (2C), 128.66 (2C), 128.47, 128.37, 67.45, 67.38,
and 43.08. FT-IR (cm−1): 3359, 2923, 1712, 1524, 1361, 1161, and
1052. MALDI-TOF MS: m/z [M + Na+] Calcd for C17H17NO4 [M+],
322.1055; found, 322.0435.
The other model reactions, (i) stepwise in the absence of catalyst
and (ii) direct at 150 °C in the presence of catalyst, are provided in the
Supporting Information.
RESULTS AND DISCUSSION
■
Monomer Design and Dual Ester-Urethane Melt
Polycondensation. Ester-urethane monomers were synthe-
sized starting from naturally available L-amino acids, as shown
in Scheme 2. Amino acids were converted into carboxylic acid
Table 1. Monomers, Molecular Weights, % Conversion, and
Thermal Properties of Polymers
b
b
e
f
Mn
Mw
TD
Tg
a
c
d
monomer
glycine
diol
(g/mol) (g/mol) xn
P
(°C) (°C)
Scheme 2. Synthesis of Dual Ester-Urethane Monomer from
Amino Acids
DEG
4600
4900
5200
9300
5500
4800
3400
8600
6100
8600
5600
4400
5600
8400
5900
5900
13300
7000
4800
17900
6700
24
21
18
40
19
16
14
28
20
18
35
14
20
25
18
20
39
19
15
47
96
95
94
97
95
93
92
96
95
97
94
93
95
96
94
95
97
95
93
98
240 8.6
TREG
TEG
8800
240 −2.9
250 −13.3
280 58.3
275 14.8
250 −26.6
280 6.7
8600
CHDM
DD
10400
14100
7300
L-alanine
β-alanine
L-valine
TEG
CHDM
DD
7200
14200
10900
15300
11000
6700
250 6.0
TEG
250 −24.3
260 24.1
270 13.7
255 −29.6
260 43.5
CHDM
DD
TEG
CHDM
DD
11000
11000
9800
270
-
L-leucine
TEG
250 −15.7
260 50.4
300 −13.1
265 −0.1
300 −8.3
300 64.0
CHDM
DD
11900
17000
14000
8300
chlorides and subsequently reacted with methanol to yield
amino acid methyl ester hydrochloride salts. The amine salt was
converted into its free amine by washing with aqueous
NaHCO3, then reacted with methyl chloroformate to obtain
dual ester-urethane monomer. Monomers of glycine, L-alanine,
L-valine, L-phenylalanine, L-leucine, and β-alanine were
synthesized as described above and their structures were
characterized by NMR, FT-IR, and mass spectroscopic analysis
(see Supporting Information). The monomers were synthe-
sized in an average yield of 75−84%. A dual ester-urethane melt
condensation reaction was carried out in one pot and two steps,
as shown in Scheme 3. Equimolar amounts of amino acid
monomer and diols were polycondensed using Ti(OBu)4 as
catalyst (1 mol %) under nitrogen purge and high vacuum
(0.01 mm of Hg). During this process, the viscosity of the melt
increased very rapidly and the stirring stopped at the end of the
polycondensation. The white polymer mass was purified using
the precipitation technique and dried in a vacuum oven (0.1
mm of Hg) prior to further analysis. In the current process, the
diol reacted with both methyl ester and methyl urethane units
in the amino acid monomers; as a result, the new class of
polymers are named as poly(ester-urethane)s. The new
polycondensation process was tested for various diols of di-,
L-phenyl-
alanine
TEG
CHDM
DD
32700
a
DEG, TREG, and TEG refer di-, tri-, and tetra-ethylene glycols,
respectively. CHDM and DD refer 1,4-cyclohexanedimethanol and
1,2-dodecane diol, respectively. Number (Mn) and weight average
(Mw) molecular weights were determined by GPC using THF as
solvent. The degree of polymerization (xn) was calculated using the
formula Mn = nMo, where Mo is repeating unit mass. The percent
conversion (P) was calculated using Carothers eq xn = 1/(1 − P).
The decomposition temperature (TD) of the sample was determined
b
c
d
e
f
for 10% weight loss using TGA under nitrogen. The glass transition
temperature (Tg) was determined by DSC at 10°/min.
All other amino acid-based polymers with various diols: 1,4-
cyclohexanedimethanol (CHDM), 1,12-dodecandiol, diethylene glycol
(Di-EG), triethylene glycol (Tri-EG), and tetraethylene glycol (Tetra-
EG) are provided in the Supporting Information.
Model Reactions for Kinetic Studies. Typical reaction is
described for benzyl alcohol (Bz) with the glycine monomer. Benzyl
alcohol (2.21 g, 20.0 mol) and glycine monomer (1.50 g, 10.0 mol)
were taken in a test-tube shaped polymerization apparatus and melted
by placing in an oil bath at 100 °C with constant stirring. After
degassing, as described for the polymerization, Ti(OBu)4 (0.035 g, 1
2448
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
Scheme 3. Synthesis of Polymers via Dual Ester-Urethane
a
Melt Polycondenstaion
a
The sample in the vial showed the fibrous polymer obtained using
1,12-dodecane diol with glycine monomer.
1
Figure 1. H and 13C NMR spectra of glycine monomer and its 1,12-
dodecane diol polymer.
tri-, and tetraethylene glycols, 1,12-dodecanol (linear aliphatic
diols), and 1,4-cyclohexanedimethanol (cycloaliphatic diols)
and also various amino acid monomers (see Scheme 2). Both
diols and amino acid monomers were varied in the feed to
produce wide range of polymer structures. The structures of
poly(ester-urethane)s are provided in the Supporting Informa-
tion (SF-2).
NMR Spectroscopy and Molecular Weights. The
occurrence of a dual ester-urethane process and the structure
of the new poly(ester-urethane)s were confirmed by NMR
spectroscopy. NMR spectra of glycine monomer and its
corresponding polymer with 1,12-dodecane diol are shown in
Figure 1. The different types of the protons and carbon atoms
in the monomer and polymer structure were assigned by
alphabets.
polymers. 13C NMR spectra of the monomer and polymers also
confirmed the occurrence of the polycondensation. A similar
NMR spectral analysis was done for all amino acid monomers
in polycondensation with various diols and the details are
provided in the Supporting Information (SF-3−5). Similarly
FT-IR spectra of the polymers were recorded to confirm the
formation of ester and urethane linkages (see Supporting
Information, SF-6). The polymers showed peaks at 3317 and
1526 cm−1 with respect to N−H and C−N stretching
frequencies, respectively. The peaks for ester and urethane
linkages appeared at 1739 and 1687 cm−1, respectively,14 which
confirmed the formation of expected poly(ester-urethane)
structure.
In the 1H NMR spectrum of the monomer, the methyl ester
protons in the ester (a) and the urethane (b) appeared very
closely as a singlet at 3.67 and 3.62 ppm, respectively. The N−
H proton and the NH-CH2-COO (c) from the amino acid unit
appeared at 5.47 and 3.88 ppm, respectively. Upon polymer-
ization, two new triplets appeared at 4.12 ppm and 4.05 ppm
c o r r e s p o n d i n g t o − C H 2 C H 2 O O C ( a ′ ) a n d
−CH2CH2OOCNH (b′) protons in the ester and urethane
linkages. All other aliphatic protons in the dodecane diol unit
appeared below 2.00 ppm. During the polycondensation
process, the nucleophilic attack of −OH groups in the diols
at the carbonyl of ester and urethane linkages lead to the
removal of low boiling alcohol (as methanol in the present
case). The fast and efficient removal of methanol from the
equilibrium drove the polycondensation to produce higher
molecular weight chains. The comparison of NMR spectra
revealed that −OCH3 protons and carbon in the monomer had
completely vanished in the polymer spectra (at 3.70−3.64
ppm), which confirmed the occurrence of the dual ester-
urethane process as well as formation of high molecular weight
The molecular weight of the newly synthesized amino acid
poly(ester-urethane)s were determined by gel permeation
chromatography (GPC) in THF. The GPC chromatograms
are provided in the Supporting Information (SF-7) and their
molecular weights are summarized in Table 1. Almost all the
polymers showed monomodal distribution, indicating the
formation of uniform molecular weight chains by the melt
route (see SF-7). The molecular weight of the polymers were
obtained in the range of Mn = 5.0−19.2 × 103 and Mw = 10.1−
32.6 × 103 g/mol with the average polydispersity of 2.1. The
number average degrees of polymerization (xn) of the polymers
were determined from the equation, Mn = (Mo) × (xn), where
Mo is the repeating units mass.15 Based on the Carrothors
equation,15 xn = 1/(1 − P) for the step condensation process,
where “P” is the percent conversion (or extent of the reaction),
both xn and p were obtained as 20−40 units and 96−98% of
occurrence of reaction, respectively (see table 1). These values
are very good for newly developed laboratory scale (1.0−2.0 g)
melt polycondenstaion process. The molecular weights of the
polymer samples were also determined in dimethylformaamide
2449
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
Figure 2. TGA profiles (a) and GPC chromatograms (b) of glycine monomer and 1,12-dodecane diol polymerization aliquots. The Mn determined
by NMR and GPC technique were plotted for aliquots at various reaction times (c).
(DMF) GPC columns and their molecular weights are given in
Table ST1 in the Supporting Information. The molecular
weights determined by DMF columns were much higher up to
Mw = 62000, which again confirmed that the new dual ester-
urethane methodology developed in the present investigation
very robust in producing high molecular weight polymers based
on amino acid monomers. Further, the comparison of
molecular weights in Table 1 indicated that oligoethylene
diols produced relatively low molecular weight polymer chains
compared to that of the linear aliphatic (1,12-dodecanol) and
cycloaliphatic diols (CHDM). Among the amino acid
monomers, phenylalanine produced very high molecular
polymers with fibrous products compared to that of other
counterparts.
synthesized polymers could be much higher than that
summarized in Table 1. Thus, the above studies confirmed
that the newly developed dual ester-urethane process is
thermally stable and is a very efficient synthetic methodology
for amino acid polymers under solvent-free melt conditions.
End Group Analysis by MALDI-TOF-MS. The MALDI-
TOF MS technique is a very powerful tool for studying the
polymerization reaction via end group analysis. In typical A−A-
and B−B-type melt polycondensation reactions, four different
types of end groups are possible: (i) chain with AA ends, (ii)
chain with BB ends, (iii) chain with AB ends, and (iv)
macrocycles having the mass of repeating units (see scheme in
SF-9).16 To trace the functional groups in the chain ends,
aliquots were collected at various intervals in the polymer-
ization of glycine monomer and 1,12-dodecanediol and they
were subjected to MALDI-TOF MS analysis. MALDI-TOF MS
spectra of the aliquots corresponding to a nitrogen purge stage
(1 h and end of the N2 purge stage) and a vacuum stage are
shown in Figure 3. Mass values corresponding to the polymer
chain with end groups: A-(P)n-A, B-(P)n-B and A-(P)n-B and
cyclic (P)n were theoretically calculated for n = 1−12 and the
Glycine monomer with 1,12-dodecane diol polymerization
was carried out and aliquots were collected at various intervals
to check the thermal stability and chain growing ability of the
newly developed dual ester-urethane process. The TGA profiles
(see Figure 2a) of the aliquots showed continuous enhance-
ment of the thermal stability of the polymers with formation of
higher molecular weight chains from 180 to 280 °C. The GPC
chromatograms (see Figure 2b) of the aliquots also showed
enhancement in the molecular weight and the polymer samples
shifted from multimodel distribution to monomodel distribu-
tion with respect to the formation of high molecular weight
chain lengths. The number average molecular weights (Mn) of
the aliquots were determined by comparing the intensities of
1
repeating unit peaks with end groups in the H NMR spectra
(see SF-8). From the expression, Mn = xn Mo, where Mo is the
repeating unit mass, the number average molecular weight (Mn)
of polymer chains in the aliquots were estimated based on
NMR. The molecular weight (Mn) obtained from GPC and
NMR techniques was plotted against the reaction time and
shown in Figure 2c. The molecular weight of the polymers
linearly increased with time up to Mn = 3000 amu and showed
sudden increase at larger reaction time (especially in the NMR
data). The increase in the molecular weight at higher
conversion was attributed to the rapid increase in the melt
viscosity of the polymerization during the condensation
process. The Mn based on NMR was much higher than that
obtained from GPC in the higher molecular weight range. This
suggested that the polystyrene standard used for the calibration
of the GPC column (especially in THF solvent) could be
underestimating the actual molecular weights of the polymers
at higher conversion. Therefore, the molecular weight of the
Figure 3. MALDI-TOF mass spectra of glycine monomer and 1,12-
dodecane diol polymer at 1 h (a), end of nitrogen purge at 4 h (b) and
at the vacuum stage at 6 h (c).
2450
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
1
Figure 4. Model reaction of benzyl alcohol with glycine monomer at 120 and 150 °C. H NMR spectra of the aliquots are given for various time
intervals.
values are given in Table SF9. After a 1 h reaction time (see
Figure 3a), the samples showed mass peaks in the range of m/z
= 300−1100 amu with respect to the formation of oligomer
species. The peaks were assigned (Na+ and K+ ions) to the
chain-AA, chain-BB, and chain-AB end groups (for values, see
Table SF-9). At the end of the nitrogen purge stage (in Figure
3b), the chain length increased up to five repeating units.
Subsequent condensation under vacuum increased the
molecular weight of the polymers and chain length with
repeating units up to 12 were clearly visible in Figure 3c.
Few observations from the MALDI-TOF MS analysis were
(i) the ester and urethane functional groups of the amino acid
monomers were stable under high temperature melt polymer-
ization process, (ii) the molecular weight of the polymers
increased with increase in the reaction time, (iii) all three
possible chain ends like AA, BB, and AB were involved in the
condensation route and (iv) there is no macrocycle formation
in the dual ester-urethane polycondensation process. The
absence of macrocycle formation in the present melt
condensation approach is a very crucial point. This is because
polycondensation chemistry for other naturally occurring
monomers like lactic acid17 or NCA mediated route9a for
amino acids were known to form macrocycles which hampered
their high molecular weight formation. Interestingly, the amino
acid monomers underwent linear chain formation rather than
cyclization in the dual ester-urethane process which is very
crucial for high molecular weight formation. Similarly, MALDI-
TOF MS analysis was done for the polymerization reaction of
1, 12-dodecane diol with alanine (see SF-10). Thus, it could be
concluded that the amino acid functional groups (urethane and
ester groups) were thermally stable in the newly developed dual
ester-urethane melt polymerization and produced high
molecular weight polymers.
Temperature-Dependent Reactivity and Kinetics. To
study the mechanism and the role of the melt temperature on
the dual ester-urethane process; model reactions were carried as
shown in Figure 4. Typically, temperature and catalyst are the
two important parameters that drove the polycondensation
synthetic process. The thermogravimetric analysis of the amino
acid monomers showed that the ester and urethane linkages
were thermally stable only up to 150−180 °C depending on
their structures (see SF-11). Therefore, the melt reaction was
performed at 80, 100, 120, and 150 °C and it was found that
both ester and urethane units in the amino acid monomer were
only active above 120 °C. Based on these finding, two
temperatures were chosen for the model reaction at 120 and
150 °C. Benzylalcohol was selected as suitable alcohol for the
condensation because it provided possibility for identification
and quantification of ester and urethane products in the dual
1
ester-urethane reaction by H NMR. Glycine monomer was
condensed with twice the amount of benzyl alcohol and the
following approaches were adopted: (i) stepwise increase of the
temperature at 120 and 150 °C in the presence of catalyst, (ii)
stepwise increase of the temperature at 120 and 150 °C in the
absence of catalyst and (iii) direct condensation at 150 °C in
the presence of catalyst. Aliquots were taken at regular interval
1
and they were subjected to H NMR analysis to quantify the
1
unreacted monomers and products. H NMR spectra of the
aliquots for the model reaction (i) are shown in Figure 4 [for
model reactions (ii) and (iii), see SF-12 and SF-13].
In the initial mixture, peaks corresponding to the protons
C6H5CH2OH, gly-CH2COOCH3 (ester), and gly-HNCOO-
CH3(urethane) appeared at 4.59, 3.67, and 3.62 ppm,
respectively (the chemical shift region above 5.5 not shown
for simplicity). At 120 °C, only the carboxylic ester of the
amino acid monomer underwent reaction with benzyl alcohol;
2451
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
Figure 5. Plots of percent conversion vs reaction time in the presence of Ti-catalyst (a) and in the absence of catalyst (b) at stepwise 120−150 °C
reaction, and direct condensation at 150 °C in the presence of Ti-catalyst (c). First order kinetic plots vs reaction time (d) and the rate constant (k)
were determined from the slopes of linear fitting, and the values are summarized in the table.
as a result, the protons corresponding to ester part (at 3.67
ppm) disappeared and new peak with respect to protons in the
product PhCH2OOCCH2-gly appeared at 5.10 ppm. During
this ester-exchange process, the urethane part in the amino acid
monomer was completely inert. Upon subsequent increase in
the reaction temperature to 150 °C, the urethane functionality
in the monomer became active and underwent reaction with
benzyl alcohol to produce new urethane linkage
PhCH2OOCNH-gly. The disappearance of protons at 3.62
ppm of gly-HNCOOCH3 and appearance of new peak at 5.05
ppm confirmed the formation of PhCH2OOCNHCH2-gly.
Thus, it is clearly evident that the ester and urethane reactivity
could be easily varied by adjusting the appropriate con-
densation temperature in the present dual ester-urethane
process. The extent of the ester or urethane exchange reaction
at particular temperature (either at 120° and 150 °C) over a
period of reaction time was determined by comparing relative
peak intensities in the gly monomer at 3.67 or 3.62 ppm. The
extent of the reaction (or percent conversion) monomer was
plotted against the reaction time in Figure 5. In the presence of
catalyst (see Figure 5a), at 120 °C, the ester-exchange reaction
occurred more than 98% within 90 min. Upon increasing the
temperature of the same vessel to 150 °C (180 min was
corresponding to the initial time for the reaction at 150 °C),
the urethane unit became active and gave 98% conversion at
the end of reaction. In the absence of catalyst, the ester part of
the amino acid monomer underwent slow exchange with benzyl
alcohol to produce 80% of product at the end of the reaction
(see Figure 5b). However, the urethane unit was completely
inert in the absence of catalyst throughout the reaction. Direct
polycondensation at 150 °C (see Figure 5c), in the presence of
catalyst, both ester and urethane exchange reaction occurred
simultaneously. The ester exchange was very rapid (95% within
30 min) compared to the urethane exchange reaction which
occurred over a period of 360 min.
These percent conversion data indirectly reflected on the
composition and the amount of unreacted monomer at any
given time. Therefore, these data could be directly utilized for
determination of kinetic rate constant for the condensation
process. The data were fitted in the first order ester exchange
reaction using the logarithmic equation ln{[A]/[Ao]} = −kt,
where [A] is the concentration of unreacted monomer at time
“t”, [Ao] is the initial concentration of the monomer, and “k” is
the rate constant. The plots of ln{[A]/[Ao]} versus “t” for all
model reactions were given in Figure 5d. The rate constant for
the model reaction were obtained from the slope of the plots
and the values are summarized in the table in Figure 5. At 120
°C, the rate constant for ester-exchange reaction was obtained
as 2.7 × 10−2min−1, which was in accordance with the literature
values.18 In the absence of the Ti-catalyst, the ester-exchange
process was one order magnitude lower. The rate constants for
the urethane-exchange reaction were obtained as of 0.9−1.2 ×
10−2 min−1, which was almost half compared to its ester
counterpart. The comparison of the rate constant values
revealed that the ester exchange process was almost twice as
fast as the urethane-exchange in the dual ester-urethane process
of amino acid based monomers. The rate constant for the
polymerization reaction was also determined using the aliquots
data of glycine monomer with 1,12-dodecanol at 150 °C (see
Figure 2b). Based on the procedure explained for model
reactions, the rate constant for the polycondensation was
obtained as 1.4 × 10−2 min−1 (see plot in Figure 5d). The rate
2452
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
Figure 6. CD spectra of polymers in tetrahydrofuran (a) and in water (b) at 25 °C. DSC thermograms of the semicrystalline (c) and amorphous (d)
polymers at 10°/min heating/cooling rates.
constants for the polymerization were almost comparable to
that of the model reaction, hence, it may be concluded that
both model reaction and polycondensation followed similar
kinetics in the dual ester-urethane process. The above model
reactions revealed that the amino acid monomers were very
special for the condensation approach. Unlike the ester units,
the urethane linkage is very selective to the temperature of the
reaction (at 150 °C) and also need Ti-catalyst for the exchange
reaction to occur. Hence, using appropriate reaction conditions,
one could easily fine-tune the dual ester-exchange process for
making bis- or triurethane small molecules as well as block
copolymers based on naturally available amino acid starting
materials. Currently, the dual ester-urethane synthetic method-
ology is continued in these directions.
Secondary Structures and Thermal Properties of
Amino Acid Polymers. The newly synthesized polymers
were derived from repeating units of L-amino acid monomers,
and therefore, circular dichroism (CD) spectroscopic analysis
was carried out for the polymers to investigate their ability to
form secondary structures. Generally, CD measurements are
based on the difference in the absorbance between right and left
circularly polarized light of optically active molecules. In the
present case, the ester-urethane chemical linkage of the chains
absorbed in the far-UV region with three electronic transitions:
(i) n−π* transition at ∼220 nm polarized along the carbonyl
bond, (ii) π−π* transition at 185−200 nm polarized in the
direction of the C−N bond, and (iii) π−π* transition at 140
nm polarized approximately perpendicular to the C−N bond
direction.19 Typically, peptides showed a positive band at 192
nm and two negative bands at 208 and 222 nm with respect to
α-helical conformation and the β-sheet structure exhibited a
positive CD band at 195−198 nm and a negative CD band at
218 nm.20a Random coil structures are expected to show single
negative CD band at 195 nm.20a
L-phenylalanine are optically active, and therefore, their
polymers could be expected to have tendency to form self-
assembled secondary structures like those observed in peptides
and proteins. Polymers belonging to these amino acid
monomers were subjected to CD studies in tetrahydrofuran,
water, and methanol depending on their solubility. CD spectra
of representative polymers in THF and water are shown in
Figure 6 (for methanol, SF-14). In THF, all the polymers
(except phenylalanine), showed one positive CD band at 210−
215 nm for π−π* and a negative CD band around 230 nm for
n−π* transition corresponding to the secondary structure in β-
sheet conformation.20a The water-soluble polymers also
showed CD bands, as observed in THF solvent, which
confirmed that the β-sheet secondary structure was retained
even in the aqueous medium. Phenylalanine-based polymers
showed positive CD band at 230 nm, which was attributed to
the aromatic side chains, and their spectral features resembled
the polyproline type II helical coil conformation.20b The above
results confirmed that the newly synthesized amino acid
poly(ester-urethanes) are very efficient in forming secondary
structures similar to that of polypeptides. Hence, these new
polymers could be very useful for applications in biological
systems.
TGA analysis of the polymers revealed that these newly
synthesized polymers were thermally stable up to 280−300 °C
(see SF-15).The thermal properties of the newly synthesized
polymers were analyzed by DSC and few representative
thermograms are shown in Figure 6c (see SF-16). Most of
the poly(ester-urethanes) were sluggish to crystallize and
showed only glass transition temperatures (Tg; see Table 1).
Interestingly, polymers of 1,12-DD with glycine, alanine, and β-
alanine showed clear melting and crystallization peaks under
the heating/cooling cycles (see Figure 6c). Their transition
temperatures and enthalpy values are given in Table ST-2 in the
Supporting Information. The crystalline nature was attributed
to the less steric hindrance provided by these amino acid units
Among the amino acid monomers chosen for the present
investigation, the monomers L-alanine, L-valine, L-leucine, and
2453
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
in the polymer backbone. Glycine-based polymer with PEGs
showed a decrease in Tg with an increase in the length of the
ethylene oxide units (see Figure 6d). The polymers synthesized
from phenylalanine were found to have maximum Tg values,
which is attributed to the high rigidity contributed by the amino
acid repeating units. The thermal analysis suggested that by
choosing appropriate amino acid based monomers and diols,
the thermal properties of the poly(ester-urethane)s could be
easily fine-tuned for various thermoplastic applications.
condensation approach will open up new platform of research
activates based on amino acids in polymer synthesis literature.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR data, FTIR spectra, HR-MS data, GPC chromatograms,
MALDI-TOF mass spectra, TGA profile, circular dichorism
(CD), and DSC profile, photographs of the reaction vessel, and
structure of the polymers are provided. This material is
available free of charge via the Internet at http://pubs.acs.org.
CONCLUSION
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: jayakannan@iiserpune.ac.in. Fax: +91-20-2590 8186.
A dual ester-urethane condensation approach was successfully
developed for biological monomers-amino acids to make new
classes of thermoplastic polymers under solvent free polymer-
ization methodology. L-aminoacids were converted into their
corresponding ester-urethane monomers by simple tailor-made
approach. These monomers were condensed with various
commercial diols under melt polycondensation process in one-
pot in two stages. In this process, oligomers of 1−4 repeating
units were initially obtained under nitrogen purge which upon
further polycondensation under reduced vacuum produced
high molecular weight polymers. The occurrence of the melt
dual ester-urethane process and the structure of the new
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are thankful for the research grant from
Department of Science and Technology (DST), New Delhi,
India, under the Project SR/S1/OC-54/2009. S.A. thanks CSIR
New Delhi for a Ph.D. research fellowship.
1
poly(ester-urethane)s were confirmed by H and 13C NMR
REFERENCES
■
spectroscopies. The new dual ester-urethane condensation
approach was demonstrated for a variety of amino acids:
glycine, β-alanine, L-alanine, L-leucine, L-phenylalanine, and L-
valine, along with commercial diols di-, tri-, and tetraethylene
glycols, 1,12-dodecane diol (linear aliphatic diols), and 1,4-
cyclohexanedimethanol (cycloaliphatic diols). The molecular
weights of the polymers were obtained in the range of
moderate to high with polydispersity ∼2.1. The percentage
conversion of the new process was found to be more than 97-
98% with respect to degree of polymerization, xn = 30−40
units. The end group analysis by MALDI-TOF MS revealed
that the amino acid functional groups (urethane and ester
groups) were very much thermally stable in the dual ester-
urethane melt polymerization process and produced high
molecular weight polymers. The mechanism of melt dual ester-
urethane process and the kinetics of the polycondensation were
studied by model reactions using benzyl alcohol. It was found
that the amino acid monomer was very special in the sense that
their ester and urethane reaction selectivity could be tuned
either by the catalyst or the polymerization temperature. The
presence of Ti-catalyst and 150 °C are essential for the
urethane-exchange reaction, whereas the ester-exchange could
occur at much lower temperatures at 120 °C. The rate constant
values revealed that the ester exchange process was almost
twice as fast as the urethane-exchange in the amino acid based
monomers. The thermal properties of the newly synthesized
polymers obtained using 1,12-dodecane diol along with glycine,
alanine, and β-alanine were found to be semicrystalline solid
and showed melting and crystallization peaks. CD analysis of
the synthesized polymers confirmed that these new poly(ester-
urethane)s were efficient structures to produce self-organized β-
sheets like polypeptides in water or organic solvents. In a nut
shell, the new dual ester-urethane process is very robust in
producing a thermally stable, high molecular weight β-sheet
structure forming poly(ester-urethanes) from largely abundant
naturally occurring biological monomers-amino acids. Further,
the temperature-selective reactivity of the current process could
be exploited for making tailor-made block copolymers and
oligomers in amino acid chemistry. Thus, the new melt
(1) Deming, T. J. Adv. Mater. 1997, 9, 299−311.
(2) Yu, M.; Nowak, A. P.; Deming, T. J. J. Am. Chem. Soc. 1999, 121,
12210−12211.
(3) Katz, J. S.; Zhong, S.; Ricart, B. G.; Pochan, D. J.; Hammer, D. A.;
Burdick, J. A. J. Am. Chem. Soc. 2010, 132, 3654−3655.
(4) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M.
Chem. Rev. 1999, 99, 3181−3198.
(5) Wadhvani, P.; Afonin, S.; Leronimo, M.; Buerck, J.; Ulrich, A. S. J.
Org. Chem. 2006, 71, 55−61.
(6) Sun, H.; Meng, F.; Dias, A. A.; Hendriks, M.; Feijen, J.; Zhong, Z.
Biomacromolecules 2011, 12, 1937−1955.
(7) (a) Guo, K.; Chu, C. C. Biomacromolecules 2007, 8, 2851−2861.
(b) Guo, K.; Chu, C. C. J. Polym. Sci., Part A: Polym. Chem. 2007, 45,
1595−1606. (c) Deng, M..; Wu, J.; Reinhart-King, C. A.; Chu, C. C.
Biomacromolecules 2009, 10, 3037−3047. (d) De Wit, M. A.; Wang, Z.;
Atkins, K. M.; Mequanint, K.; Gillies, E. R. J. Polym. Sci., Part A: Polym.
Chem. 2008, 46, 6376−6392.
(8) (a) Paredes, N.; Rodriguez-Galan, A.; Puiggali, J. J. Polym. Sci.,
Part A: Polym. Chem. 1998, 36, 1271−1282. (b) Asin, L.; Armelin, E.;
Montane, J.; Rodriguez-Galan, A.; Puiggali, J. J. Polym. Sci., Part A:
Polym. Chem. 2001, 39, 4283−4293.
(9) (a) Kricheldorf, H. R. Angew. Chem., Int. Ed. 2006, 45, 5752−
5784. (b) Zhong, B.; Fischer, K.; Schmidt, M. Macromol. Chem. Phys.
2005, 206, 157−162. (c) Inoue, K.; Sakai, H.; Ochi, S.; Itaya, T.;
Tanigaki, T. J. Am. Chem. Soc. 1994, 116, 10783−10784.
(10) Deming, T. J. J. Am. Chem. Soc. 1998, 120, 4240−4241.
(11) Deming, T. J. Nature 1997, 390, 386−389.
(12) Whinfield, J. R. Nature 1946, 158, 930.
(13) (a) Jayakannan, M.; Ramakrishnan, S. Chem. Commun. 2000, 19,
1967−1968. (b) Jayakannan, M.; Ramakrishnan, S. Macromol. Chem.
Phys. 2000, 201, 759−767. (c) Jayakannan, M.; Ramakrishnan, S.
Macromol. Rapid Commun. 2001, 22, 1463−1473. (d) Behera, G. C.;
Ramakrishnan, S. Macromolecules 2004, 37, 9814−9820. (e) Roy, R.
K.; Ramakrishnan, S. Macromolecules 2011, 44, 8398−8406.
(14) (a) Deepa, P.; Jayakannan, M. U.S. Patent 2007/0117950 A1,
2007. (b) Deepa, P.; Jayakannan, M. J. Polym. Sci., Part A: Polym.
Chem. 2008, 46, 2445−2458. (c) Deepa, P.; Jayakannan, M. J. Polym.
Sci., Part A: Polym. Chem. 2007, 45, 2351−2366.
(15) Odian, G. Principles of Polymerization, 3rd ed.; John Wiley and
Sons, Inc.: New York, 1991; Chapter 2, p 53.
(16) Jayakannan, M.; van Dongen, J. L. J.; Janssen, R. A. J.
Macromolecules 2001, 34, 5386−5393.
2454
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455

Biomacromolecules
Article
(17) Chmura, A. J.; Chuck, C. J.; Davidson, M. G.; Jones, M. D.;
Lunn, M. D.; Bull, S. D.; Mahon, M. F. Angew. Chem., Int. Ed. 2007, 46,
2280−2283.
(18) (a) Collins, S.; Kenwright, A. M.; Pawson, C.; Peace, S. K.;
Richards, R. W.; MacDonald, W. A.; Mills, P. Macromolecules 2000, 33,
2974−2980. (b) Yang, H.; He, J.; Liang, B. J. Polym. Sci., Part B: Polym.
Chem. 2001, 39, 2607−2614.
(19) Violette, A.; Averlant-Petit, M. C.; Semetey, V.; Hemmerlin, C.;
Casimir, R.; Graff, R.; Marraud, M.; Braiand, J. P.; Rongnan, D.;
Guichard, G. J. Am. Chem. Soc. 2005, 127, 2156−2164.
(20) (a) Greenfield, N.; Davidson, B.; Fasman, G. D. Biochemistry
1967, 6, 1630−1637. (b) Shi, Z.; Olson, C. A.; Rose, G. D.; Baldwin,
R. L.; Kallenbach, N. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9190−
9195.
2455
dx.doi.org/10.1021/bm300697h | Biomacromolecules 2012, 13, 2446−2455