Melt polycondensation approach for reduction degradable helical polyester based on l-cystine

Article abstract of DOI:10.1002/pola.28172

Melt polycondensation approach is developed for new classes of reduction responsive disulfide containing functional polyesters based on l-cystine amino acid resources under solvent free process. l-Cystine was converted into multi-functional ester-urethane monomer and subjected to thermoselective transesterification at 120 °C with commercial diols in the presence of Ti(OBu)₄ to produce polyesters with urethane side chains. The polymers were produced in moderate to high molecular weights and the polymers were found to be thermally stable up to 250 °C. The β-sheet hydrogen bonding interaction among the side chain urethane unit facilitated the self-assembly of the polyester into amyloid-like fibrils. The deprotection of urethane unit into amine functionality modified the polymers into water soluble cationic polyester spherical nanoparticles. The reduction degradation of disulfide bond was studied using DTT as a reducing agent and the high molecular weight polymers chains were found be chopped into low molecular weight oligomers. The cytotoxicity of cationic disulfide nanoparticle was studied in MCF-7 cells and they were found to be biocompatible and non-toxic to cells upto 50 μg/mL. The custom designed reduction degradable and highly biocompatible disulfide polyesters from l-cystine are useful for futuristic biomedical applications.

Full text of DOI:10.1002/pola.28172

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Melt Polycondensation Approach for Reduction Degradable Helical
Polyester Based on L-Cystine
Santhanaraj Anantharaj, Manickam Jayakannan
Department of Chemistry, Indian Institute of Science Education and Research (IISER)-Pune, Dr. Homi Bhabha Road, Pune,
Maharashtra 411008, India
Correspondence to: M. Jayakannan (E-mail: jayakannan@iiserpune.ac.in)
Received 18 March 2016; accepted 13 May 2016; published online 29 June 2016
DOI: 10.1002/pola.28172
ABSTRACT: Melt polycondensation approach is developed for
new classes of reduction responsive disulfide containing func-
tional polyesters based on L-cystine amino acid resources
under solvent free process. L-Cystine was converted into multi-
functional ester-urethane monomer and subjected to thermose-
lective transesterification at 120 8C with commercial diols in
the presence of Ti(OBu)4 to produce polyesters with urethane
side chains. The polymers were produced in moderate to high
molecular weights and the polymers were found to be ther-
mally stable up to 250 8C. The b-sheet hydrogen bonding inter-
action among the side chain urethane unit facilitated the self-
assembly of the polyester into amyloid-like fibrils. The depro-
tection of urethane unit into amine functionality modified the
polymers into water soluble cationic polyester spherical nano-
particles. The reduction degradation of disulfide bond was
studied using DTT as a reducing agent and the high molecular
weight polymers chains were found be chopped into low
molecular weight oligomers. The cytotoxicity of cationic disul-
fide nanoparticle was studied in MCF-7 cells and they were
found to be biocompatible and non-toxic to cells upto 50 lg/
mL. The custom designed reduction degradable and highly
biocompatible disulfide polyesters from L-cystine are useful for
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futuristic biomedical applications.
2016 Wiley Periodicals,
Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 2864–2875
KEYWORDS: amino acids; melt polycondensation; Polyconden-
sation; Polyesters; redox polymers; Self-assembly; stimuli-sen-
sitive polymers; transesterification
cerols,25 and self-healing polymers.14,26 Among the natural L-
amino acid resources, L-cysteine has thiol (-SH) functionality
and it readily oxidized into stable disulfide dimer L-cystine.
N-carboxyl anhydrides of L-cystine27–29 and L-methionine30
monomers were subjected to ring opening polymerization to
produce star shaped polymers and functional polypeptides,
respectively. Very recently, L-cystine diamine was polymer-
ized with fatty dicarboxylic acids in solution polycondensa-
tion route to make poly(disulfide amide) nanoparticles for
docetaxel delivery.31 These amino acid-based poly(disulfide)s
were used as nanoscaffolds for delivering anticancer
drugs32–39 and gene.40 These examples clearly envisage the
importance of the disulfide polymers as reduction degrad-
able thermoplastics and also as useful scaffold material for
biomedical applications.
INTRODUCTION Macromolecular architectures with disulfide
chemical linkages are emerging as important polymeric
materials for diverse application in thermoplastic and bio-
medical industry due to their excellent responsiveness to
light, heat, pH, redox reagents, and enzymes.1–4 The disulfide
linkage in the polymer chains can be introduced via ring-
opening polymerization of cyclic oligo-disulfides5–10 or oxida-
tive polymerization of thiol end-capped oligo-ethylene glycols
(or PEGs).11,12 Recently, photo-induced disulfide metathesis
polymerization was developed to make hybrid polymers and
photo-mendable polymeric materials.13–15 Telechelic poly(di-
sulfide)s were made by carefully controlling the stoichiomet-
ric balance between di-thiols and dithiopyridines.16,17 Non-
sulphur mediated or indirect methodologies were also
reported for disulfide containing macromolecular architec-
tures. Disulfide containing TEMPO initiator for polystyrene,18
hyperbranched polymers,19 and star copolymers via RAFT
route20,21 and ATRP for branched poly(t-butyl acrylate)s22,23
are some of the important examples. Ring opening polymer-
ization of disulfide cyclic derivatives produce reduction
degradable linear polycarbonate,24 hyperbranched polygly-
Melt polycondensation is an important synthetic methodol-
ogy that is largely used for the manufacture of thermoplastic
engineering polymers such as polyesters, polyamides, and
polycarbonates.41 Unfortunately, no effort has been taken
until now for making disulfide linkage polymers based on
the melt polycondensation chemistry in the literature. Hence,
Additional Supporting Information may be found in the online version of this article.
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FIGURE 1 Approaches to develop reduction-degradable helical disulfide containing polyesters based on L-cystine amino acid
resources. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the development of solvent free melt process for disulfide
linkage polymer would provide new opportunity for reduc-
tion degradable polymeric materials that are yet to be
reported. To accomplish this goal, the present investigation
is aimed to develop disulfide linkage containing polyesters
based on natural L-amino acid resources. By combining the
melt polycondensation (green process) and natural L-amino
acid (bioresources), the present approach becomes unique in
making polymeric materials with reduction degradability,
biocompatibility, and ability to produce supramolecular heli-
cal structures in a single polymeric system. This concept is
shown in Figure 1.
novelty of the approaches may be listed as: (i) the dicar-
boxylic esters of the L-cystine monomer underwent thermo-
selective transesterification without disturbing the disulfide
and urethane functionality to produce new classes of main
chain disulfide linear polyesters, (ii) the disulfide linkages
in the polyester backbone was readily susceptible to reduc-
tive degradation into small molecular species, (iii) the side
chain urethane functionality facilitated b-sheet hydrogen
bonding interaction among the polymer chains and seeded
them for helical amyloid-fibril morphology, and (iv) on
deprotection of the urethane into -NH13 X2, the polyester
turned into non-toxic, biocompatible, and water soluble cat-
ionic species. The reductive degradation behaviors of disul-
fide polyester were studied using DTT and the process was
monitored by 1H-NMR and Gel permeation chromatographic
(GPC) techniques. The self-assembly of the polymers were
tested by CD spectroscopy, dynamic light scattering (DLS),
atomic force, and electron microscopes. The cytotoxicity of
the polymers was studied in MCF-7 cell lines and the
results revealed that these cationic disulfide polymers are
highly biocompatible and non-toxic to cells up to 50 lg/
mL. The newly developed L-cystine polymers are com-
pletely degradable under reduction process and biocompat-
ible for many futuristic biomedical applications.
The present investigation is emphasized to design and
develop new classes of reduction responsive disulfide con-
taining functional polyesters based on L-cystine resources.
L-Cystine was suitably converted into multipurpose mono-
mer with dicarboxylic esters and urethane functional
groups. Earlier, we have reported dual ester-urethane con-
densation approach for L-amino acid based linear poly(es-
ter-urethane)s, hyperbranched poly(ester-urethane)s, and
functional polyesters.42–45 Based on this experience, in the
present investigation, melt polycondensation is successfully
developed for multi-functional L-cystine monomer to make
new disulfide containing polyesters (see Fig. 1). The
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EXPERIMENTAL METHODS
obtain white solid as product. It was further purified by
passing through silica gel column using ethyl acetate and pet
ether (1:4 v/v) as eluent. Yield 5 25.1 g (65%). 1H-NMR
(400 MHz, CDCl3) d ppm: 5.41 (b, 2H, -NH), 4.60 (m, 2H,
CH), 3.77 (s, 6H, CHCOOCH3), 3.17 (m, 4H, CH2S), and 1.45
(s, 18H, -NHCOO (CH3)3). 13C-NMR (100 MHz, CDCl3) d ppm:
171.15, 155.04, 80.30, 52.82, 52.69, 52.62, 41.33, 41.26, and
28.30. FT-IR (cm21): 3743, 3364, 2938, 1749, 1682, 1509,
1363, 1216, 1163, 1057, and 1017. HRMS (ESI1): m/z
[M 1 Na1] calcd. for C18H32N2O8S2Na [M1]: 491.1498; found:
491.1577.
Materials
L-Cystine, 1, 12-dodecandiol, 1, 10-decanediol, 1, 8-
octanediol, dithiothreitol (DTT), 1-decanol and titanium tet-
rabutoxide (Ti(OBu)4, tetrazolium salt: 3-4,5 dimethylthiazol-
2,5diphenyltetrazolium bromide (MTT), DMSO, hoechst, and
4% paraformaldehyde purchased from Aldrich chemicals and
used without further purification. Methyl chloroformate,
Ditertbutyldicarbonate, thionyl chloride, and other solvents
were purchased locally and purified prior to use.
General Procedures
Synthesis of Disulfide Containing Polyester (P-X) by Melt
POLYCONDENSATION PROCESS
1H and 13C-NMR were recorded using 400-MHz JEOL NMR
spectrophotometer. All NMR spectra were recorded in CDCl3
containing TMS as internal standard. FT-IR spectra of all
compounds were recorded using Bruker alphaT Fourier
transform infrared spectrophotometer. The mass of the
monomers were analzsed using a HRMS-ESI-Q-time-of-flight
LCMS (SynaptG2, Waters). GPC analysis which was per-
formed using Viscotek VE 1122 pump, Viscotek colum
T6000M General mixed org 300 3 8.0 mm (THF), Viscotek
VE 3580 RI detector and Viscotek VE 3210 UV/Vis detector
in tetrahydrofuran (THF) using polystyrene as standards at
25 8C. Thermal stability of the polymers was determined
using Perkin Elmer thermal analyzer STA 6000 model at a
heating rate of 10 8C/min in nitrogen atmosphere. Thermal
analysis of all polymers was performed using TA Q20 Differ-
ential 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 8C/min under nitrogen atmosphere and their thermo
grams were recorded. Circular dichroism (CD) analysis of the
polymer samples was done using JASCO J-815 CD spectrome-
ter at 20 8C in THF and water. FE-SEM images were recorded
using Zeiss Ultra Plus scanning electron microscope. For FE-
SEM analysis, the samples were prepared by drop casting on
silicon wafers and coated with gold. Atomic force microscope
(AFM) images were recorded by drop casting the samples on
freshly cleaved mica surface, using Veeco Nanoscope IV
instrument. The experiment was done in tapping mode.
L-Cystine monomer
1
(0.67 g, 0.001 mol), 1, 12-
dodecanediol (0.29 g, 0.001 mol, for polymer P-12), and ben-
zoquinone (catalytic amount, 2.0 mg) were taken in a test
tube-shaped polymerization vessel and melted in oil bath at
100 8C. The polycondensation apparatus was made oxygen
and moisture free by purging with nitrogen and consequent
evacuation by vacuum under constant stirring. Titanium tet-
rabutoxide (0.005 g, 0.01 mmol, 1.0 mol %) was added as
catalyst and the melt polycondensation was carried out at
120 8C 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 120 8C for 2 h under stirring. At the end
of the polycondensation, the polymer was obtained as trans-
parent resin. Yield 50.67 g (82%). 1H-NMR (400 MHz,
CDCl3) d ppm: 5.43 (b, 2H, NH), 4.60 (m, 2H, CH), 4.17 (t,
4H, CH2COOCH2), 3.15 (m, 4H, CH2S), 1.70 (m, 4H, CH2),
1.47 (s, 18H, -NHCOO (CH3)3), and 1.30 (m, 16H, CH2). 13C-
NMR (100 MHz, CDCl3) d ppm: 170.72, 155.06, 80.19, 65.97,
53.08, 41.47, 29.43, 29.19, 28.49, 28.33, and 25.82. FT-IR
(cm21): 3743, 3377, 2925, 2859, 1709, 1503, 1363, 1163,
and 1057.
Similarly, L-cystine monomer 1 was polymerized with 1,8-
octanediol and 1,10-decanediol to produce P-8 and P-10,
respectively. NMR data and the molecular weights for these
polymers are provided in the Supporting Information.
Synthesis of Dimethyl 3, 30-Disulfanediylbis (2-((tert-
butoxycarbonyl) amino) propanoate) (Monomer 1)
Model Reaction Studies
1-decanol (0.35 g, 0.002 mol) and L-cystine monomer
(0.33 g, 0.001 mol) were taken in a test tube shaped poly-
merization apparatus and melted by placing in an oil bath at
100 8C with constant stirring. After degassing, Ti(OBu)4
(0.003 g, 1 mol%) was added and the condensation was car-
ried out at 120 8C under nitrogen purge for 4 h. At the end
of the condensation reaction, the product was obtained as
To a suspension of L-cystine (19.8 g, 0.082 mol) in methanol
(200 mL), thionylchloride (20.4 mL, 33.4 g, 0.283 mol) was
added drop wise at 0 8C under nitrogen atmosphere.The
above reaction mixture was brought to room temperature
and refluxed for 12 h under nitrogen. Methanol and the
unreacted thionylchloride were removed by distillation fol-
lowing which the solid mass was dissolved in triethylamine
(46 mL, 33.7 g, 0.333 mol) and dichloromethane (200 mL)
mixture at 0 8C. To the reaction mixture, Boc anhydride
(41.0 mL, 41.1 g, 0.191 mol) was added drop wise at 0 8C. It
was brought to 25 8C and stirring was continued for 12 h.
The reaction mixture was poured into water and then
extracted with dichloromethane. The organic layer was dried
over anhydrous Na2SO4 and the solvent was removed to
1
viscous liquid. H-NMR (400MHz, CDCl3) d ppm: 5.43 (b, 2H,
-NH), 4.61 (m, 2H, CH), 4.17 (m, 4H, COOCH2), 3.18 (d, 2H,
CH2), 1.68 (m, 4H, CH2), 1.47 (s, 18H, -NHCOO (CH3)3), 1.28
(m, 28H, CH2), and 0.88 (t, 6H, CH3). 13C-NMR (100 MHz,
CDCl3) d ppm: 170.71, 155.05, 80.19, 53.07, 41.53, 31.88,
29.53, 29.49, 29.29, 29.21, 28.48, 28.33, 25.85, 22.67, and
14.09. FT-IR (cm21): 3324, 2928, 2856, 1709, 1515, 1459,
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SCHEME 1 Synthesis of disulfide functional monomer 1, model compound, and polymers P-X. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
1451, 1193, and 1047. MALDI-TOF MS: m/z [M 1 Na1] calcd.
for C36H68N2O8S2Na[M1]5 743.4315; Found 5 743.5544.
MCF-7 cell lines. 1 3 103 cells were seeded in each well of a
96- well plate (Corning) having 100 lL of DMEM with 10%
FBS (fetal bovine serum) and the cells were incubated at 37
8C under CO2 environment for 16 h. Media was aspirated
from each well and various concentrations of polymer PA-8
and PA-10 were prepared in 100 lL of DMEM with FBS and
were given to the cells. Along with polymer samples blank
control of media alone was maintained in triplicates. The
cells were further incubated for 72 h without changing the
media. A fresh sample of MTT in sterile PBS (5 mg/mL) was
prepared and diluted to 50 lg/mL in 100 lL of DMEM with
FBS and this was added to each well. Cells were then incu-
bated with MTT for 4 h at 37 8C. The media was aspirated
and purple formazan crystals were obtained. These crystals
were generated as a result of reduction of MTT by mitochon-
drial dehydrogenase enzyme from live cells. These were dis-
solved in 100 lL DMSO. The 96-well plate was shook for 2
min and the absorbance from formazan crystals (dissolved
in DMSO) was measured using microplate reader at 570 nm
(Varioskan Flash). The absorbance obtained from each well
was representative of the number of cells viable per well.
The mean of absorbance values were calculated for the cor-
responding polymer and control and that of blank control
samples was subtracted from the average of treated samples.
The values obtained from the control samples were taken as
100% and relative percentage values for the samples were
calculated accordingly.
Deprotection of Boc polymer (PA-X)
The disulfide polymer P-12 (0.81 g, 0.001 mol) was taken in
a 50 mL single neck RB flask and then 5 mL of dry DCM
was added into it. At 0 8C trifluoroacetic acid (TFA) (1.33 g,
1.1 mL, 0.012 mol) was added dropwise, the content was
brought to 25 8C and the stirring was continued for 1 h. The
solvent and TFA was removed by rotavapor and the product
was purified by precipitation in cold diethyl ether.
Yield 5 0.59 g (89%). 1H-NMR (400 MHz, CD3OD) d ppm:
4.38 (m, 2H, CH), 4.19 (m, 4H, COOCH2), 3.26 (d, 4H, CH2),
1.63 (m, 4H, CH2), and 1.28 (m, 16H, CH2).13C-NMR (100
MHz, CDCl3) d ppm: 168.43, 66.58, 61.20, 51.52, 40.22,
32.81, 29.41, 29.11, 28.24, 27.91, 27.75, 27.62, 25.86, 25.65,
and 25.23. FT-IR (cm21): 2927, 2856, 1750, 1661, 1532, and
1137.
Similarly, the deprotection of P-10 and P-8 polymers and
their details are provided in the Supporting Information.
Degradation study
The disulfide polymer (P-12) (0.10 g, 0.0001 mol) and DTT
(0.25 g, 0.001 mol) were taken in dry THF (10 mL) in
50 mL two neck round bottom flask. The polymer solution
was refluxed at 70 8C and aliquots were taken periodically.
1
The aliquots were analyzed by GPC and H-NMR to estimate
the extent of degradation.
RESULTS AND DISCUSSION
Cell Viability Assay (MTT Assay)
Synthesis of Disulfide Functional Polyester
Tetrazolium salt 3-(4,5 dimethylthiazol)22,5-diphenyl tetra-
zolium bromide (MTT) was used in an assay to determine
the cytotoxic effects of the polymer PA-8 and PA-10 in
The L-cystine amino acid contains two carboxylic acids and
an amine functional group with the disulfide bond. L-Cystine
amino acid carboxylic acid functional groups were converted
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