Synthesis and qualitative analysis of BACy and Its self-polymer

Article abstract of DOI:10.1002/jccs.201200271

Many synthetic strategies of a reversible cross-linker N,N'-bis(acryloyl) cystamine (BACy) involve the typical condensation between the amino group of cystamine and the acyl group of acryloyl chloride in the mixed-phase solvent system. In this study, the synthesis of BACy was performed in pure organic phase during the whole process. The yield and purity of synthesized BACy were comparable to those from aqueous/ organic phase procedures. In addition, polymerization of BACy was also carried out by free radical reaction to prepare the self-polymer and hydrogel which were characterized with FT-IR, DSC and UV/ VIS spectrophotometer. Notably, the BACy and its self-polymer were both cleavable when exposed to the reducing agents, i.e. 1,4-dithiothreitol (DTT) and 2-mercaptoethanol (-ME). Interestingly, the reduced product of BACy contains vinyl and thiol groups, which could be further applied to the co-polymerization with other monomeric units. On the other hand, carefully controlled reduction of BACy self-polymer may be used to create the modified polymers with available thiol-end groups for further chemistry. Together, our study provides modified procedure for BACy synthesis and characteristics of BACy self-polymer and hydrogel. Further application of BACy and its self-polymer in developing polymers with additional functionality is anticipated.

Full text of DOI:10.1002/jccs.201200271

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis and Qualitative Analysis of BACy and Its Self-polymer
Yan-Su Lin,a Hsuan-Hsuan Lee,b Wen-Fu Leea and Chao-Hsiung Linc,*
aDepartment of Chemical Engineering, Tatung University, Taipei, Taiwan, Republic of China
bTsing Hua College, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
cDepartment of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan,
Republic of China
(Received: May 12, 2012; Accepted: Sept. 13, 2012; Published Online: Nov. 19, 2012; DOI: 10.1002/jccs.201200271)
Many synthetic strategies of a reversible cross-linker N,N¢-bis(acryloyl)cystamine (BACy) involve the
typical condensation between the amino group of cystamine and the acyl group of acryloyl chloride in the
mixed-phase solvent system. In this study, the synthesis of BACy was performed in pure organic phase
during the whole process. The yield and purity of synthesized BACy were comparable to those from aque-
ous/organic phase procedures. In addition, polymerization of BACy was also carried out by free radical
reaction to prepare the self-polymer and hydrogel which were characterized with FT-IR, DSC and UV/
VIS spectrophotometer. Notably, the BACy and its self-polymer were both cleavable when exposed to the
reducing agents, i.e. 1,4-dithiothreitol (DTT) and 2-mercaptoethanol (b-ME). Interestingly, the reduced
product of BACy contains vinyl and thiol groups, which could be further applied to the co-polymerization
with other monomeric units. On the other hand, carefully controlled reduction of BACy self-polymer may
be used to create the modified polymers with available thiol-end groups for further chemistry. Together,
our study provides modified procedure for BACy synthesis and characteristics of BACy self-polymer and
hydrogel. Further application of BACy and its self-polymer in developing polymers with additional func-
tionality is anticipated.
Keywords: BACy; Disulfide linkage; Self-polymer; Hydrogel.
INTRODUCTION
Synthetic polymer hydrogel is a type of developing
by endogenous thiols in physical cell environments, in-
cluding glutathione (GSH),7 free reduced homocysteine,8
and free cysteine.9
materials, which comprises a three-dimensional network of
hydrophilic polymer chains capable of absorbing signifi-
cant water content during polymerization. Many developed
hydrogels are designed for pharmaceutical and medical ap-
plications, such as drug-releasing, polymer-protein conju-
gates,1 or polymer-drug conjugates.2,3 These hydrogels can
be a carry system to deliver the drugs or proteins in physi-
cal environment. Once the hydrogels arrived at the target
location, the polymer can be degraded and release the en-
closed materials. Therefore, a physical (e.g. hydrogen
bonding) or chemical (covalent, atomic, ionic) cross-link-
ing in an intelligent hydrogel polymer system is required to
response to the stimuli of external environment, such as
pH,4 chemical environment,5 temperature,6 and etc.
N,N¢-bis(acryloyl)cystamine (BACy) was usually
used as cross-linker to make mixed hydrogel polymers
such as SS-PEI (polyethyleimine)10 and PAA-Cy1 (poly-
amidoamines).11 The disulfide linkage formed by BACy in
such hydrogel polymers could be reductively cleaved.10,11
BACy was synthesized by a typical condensation reaction
between the amino group of cystamine and the acid chlo-
ride of acryloyl chloride.10,11
In the present study, the synthetic condition of BACy
was modified for more convenient manipulation. Upon the
polymerization, physical characteristics of self-polymer
and self-hydrogel of BACy were examined. In addition, the
reductive degradation of BACy and self-polymer by DTT
and b-ME was investigated.
Adisulfide linkage-based hydrogel is a tunable chem-
ical cross-linking composition in biomedical system, where-
as the interchange between free thiols and disulfide linkage
can be controlled through oxidants and reductants. The
disulfide reduction in this kind of hydrogel can be triggered
EXPERIMENTAL
Materials
Cystamine dihydrochloride (Acros, 97%) and acrylo-
* Corresponding author. Tel: +886-2-28267280; Fax: +886-2-28202449; E-mail: chlin2@ym.edu.tw
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yl chloride (AC) (Alfa Aesar, 96%) was used for the syn-
thesis of BACy. Triethylamine (TEA) (Merck-Schuchardt,
> 9%) was used to neutralize cystamine dihydrochloride.
Tetrahydrofuran (THF) (Sigma, HPLC 99.9%) was used as
condensation reaction solvent. The 2,2¢-azobis-isobutyro-
nitrile (AIBN, Showa) and tetramethylethylene diamine
(TEMED, Sigma, 99.5%) were used as an initiator for free
radical reactions. Either 1,4-dithiothreitol (DTT) (USB,
99.5%) or 2-mercaptoethanol (b-ME) (Sigma, 14.3 mol/L)
was used as the reducing reagents for the conversion of
disulfide linkage into free thiols.
Preparation of BACy hydrogel
A1.3 g of BACy was dissolved in 5 mL of acetonitrile
and then mixed with 5 mL of dddH2O containing 0.1 mL of
10% ammonium persulfate (APS) and 0.004 mL of
N,N,N¢,N¢-tetramethyl ethylenediamine (TEMED). The re-
sulting solution was allowed to polymerize at room temper-
ature for 24 hours.
Physical properties of BACy self-polymer and
hydrogel
Differential Scanning Calorimetry (DSC, Perkin Elmer
pyris 1 DSC) was used for melting point. The BACy self-
polymer was mixed with KBr powder as matrix pellets for
functional group analysis by scanning between 4,000 and
400 cm-1 on Fourier Transform Infrared spectra (FT-IR,
Perkin Elmer Spectrum GX FT-IR spectrometer).
For SEM observation, the BACy self-polymer and
hydrogel were equilibrated in deionized water at 25 °C for
48 hours and the resulting swollen gels were freeze-dried
(-50 °C) for 48 hours. The gels were first immersed in liq-
uid nitrogen, fractured and dehydrated by freeze-dried, fol-
lowed by gold sputtering (JEOL, JFC-1200, Tokyo, Japan).
The specimens were coated on a gold layer for proper sur-
face conduction. Fractured specimens were observed for
morphological details by scanning electron microscope
(JEOL, JSM-5300, Tokyo, Japan).
Synthesis and physical characterizations of BACy
One equivalent of cystamine dihydrochloride in 80
mL of THF was added with 4.2 equivalent of TEA and the
resulting solution was stirred for 24 hours at room tempera-
ture. Next, 2.2 equivalent of acryloyl chloride in 20 mL of
THF was added dropwise into the above solution at a rate
of 1 mL/min. The resulting mixture was allowed to react at
40 °C under nitrogen atmosphere for 24 hours. After reac-
tion, the remaining acryloyl chloride and THF were evapo-
rated using a rotary evaporator under reduced pressure. Re-
action products were then separated by column chromatog-
raphy with a gradient mixture of hexane and ethyl acetate.
The obtained BACy was further re-crystallized in solution
of hexane/ethyl acetate (30/70; v/v). Purity of BACy was
above 99%, which was measured by HPLC (Agilent HPLC
1100 dissolution analysis system).
Mechanical properties of hydrogel
1. Measurement of swelling ratio
1H and 13C nuclear magnetic resonance (NMR) was
used to analyze the synthesized BACy in CDCl3 or d6-ace-
tone and the NMR spectra were recorded on a 500 MHz
Bruker spectrometer. BACy was further examined using
Elemental Analysis (Heraeus Vario EL III) for the purity of
composition, and Differential Scanning Calorimetry (Perkin
Elmer pyris 1 DSC system) was used for measuring melting
point. For functional group analysis, BACy was mixed
with KBr powder as the matrix pellet and subsequently
scanned in a FT-IR (Perkin Elmer Spectrum GX FT-IR sys-
tem) with the scanning ranged from 4,000 to 400 cm-1.
Self-polymerization of BACy
The swelling ratio was measured at 25 °C until swell-
ing equilibrium. After wiping off water on the surface with
filter paper, the swelling ratio (Q) of the gel was recorded
during the course of swelling at regular time intervals.
Q = (Ww-Wd)/Wd
where Ww was the weight of the wet gel at different times
and Wd was the weight of the dry gel.
2. Measurement of mechanical properties
The gel strength was measured by uniaxial compres-
sion experiment with universal tester (LLOYD, LRX; J. J.
Lloyd, Poole, UK). The shear modulus was calculated from
equation:12,13
A 2.6 g of BACy was mixed with trace amount of
AIBN in THF at 60 °C for 24 hours under nitrogen. Result
of TLC analysis indicated that no BACy was present in the
reaction product. Precipitate of BACy self-polymer was
filtered, washed with THF and vacuum dried. The FT-IR
spectrum of the BACy self-polymer showed no presence of
vinyl absorption.
t = F/A = G (l-l-2)
where t was compression stress, F was the compression
load, A was the cross-sectional area of swollen gels, and l
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BACy Self-polymer
was compression strain (L/L0). At low strains, a plot of
shear stress versus (l-l-2) would yield a straight line whose
slope was shear modulus (G). The effective crosslink den-
sity (r) could be calculated from the shear modulus and
polymer volume fraction (n2) as follows:
ily removed by evaporation under reduced pressure. Sepa-
ration of BACy from the unreacted cystamine was achieved
using column chromatography with hexane/ethyl acetate
(50/50, v/v). The resulted BACy was further purified by
crystallization in hexane/ethyl acetate (30/70, v/v). This
modified procedure is relatively easy with a comparable
yield of 69%, considering the omitted use of aqueous/or-
ganic phase solvent system.
r = G/n21/3RT
where R was the gas constant and T was the absolute tem-
perature.
BACy: white crystal; Mp: 126-127 °C.; ELEM. Anal.
Calcd for C10H16N2O2S2: C, 46.15%; H, 6.15%; N, 10.77%;
S, 24.61%. Found: C, 45.82%; H, 6.13%; N, 10.29%; S,
23.62% (within ± 1% magnitude of error). Purity of the
synthetic BACy was higher than 99% based on High Per-
formance Liquid Chromatography (HPLC). FT-IR: 1,653
cm-1 for C=O bond in amide; 1,553 cm-1 for N-H bond in
amide; 1,620 cm-1 for C=C bond in vinyl group.
Reductive degradation of BACy and self-polymer
BACy or its self-polymer with different volumes of
38 mM DTT or b-ME in acetonitrile (ACN) was added into
a 1cc eppendorf tube and adjusted with dddH2O to final 1
mL in total volume. The mixture was incubated at 37 °C for
24 hours to allow the reductive degradation. For degrada-
tion of BACy, a TLC was used to monitor whether the start-
ing BACy remained in the solution. For degradation of
BACy self-polymer, the absence of white precipitates was
considered to be a complete reaction.
1
The H and 13C NMR spectra of BACy in CDCl3
1
agreed with the expected structure. H NMR (500 MHz,
CDCl3): d = 5.7, 6.2 and 6.3 ppm for the acryl group
(CH2=CH-); d = 2.9 and 3.7 ppm for the methylene group
(-CH2-CH2-); d = 6.8 ppm for amide (Fig. 2a). As shown in
Fig. 2b, 13C NMR (500 MHz, CDCl3): d = 126.9 and 130.6
ppm for Ca and Cb in the acryl group; d = 166.0 ppm for Cc
in carboxyl group; d = 38.6 ppm for Cd; d = 37.8 ppm for
Ce.
RESULTS AND DISCUSSION
Synthesis and physical characteristics of BACy
BACy were synthesized mainly according to a classic
reaction procedure involved N-acylation of the amino
group of cystamine with acryloyl chloride as shown in Fig.
1. Whereas aqueous sodium chloride was usually used to
neutralize the cystamine dihydrochloride in the most pub-
lished reactions, a triethylamine (TEA) in THF was used
instead in this study, which allowed the resulting (C2H5)3N·
HCl to precipitate from reaction and be cleared through fil-
tration. The remaining acryloyl chloride and THF was eas-
Notably, d = 78 ppm were previously assigned as Cd
by Han et al. and Emilitri et al. respectively.10,11 However,
our expended spectrum between 35 and 80 ppm clearly
showed that peak at d = 78 ppm should belong to chemical
shifts of solvent CDCl3 (Fig. 2c). To clarify this assign-
Fig. 2. NMR spectra of synthesized BACy: (a) 1H
NMR in CDCl3, (b) 13C NMR in CDCl3, (c) ex-
pended region of 35-80 ppm in spectrum of (b),
(d) 13C NMR in d6-acetone.
Fig. 1. Synthetic scheme of BACy.
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ment, we have carried out the 13C NMR of BACy in d6-ace-
tone. Our results showed that the Cd peak at 38.6 ppm and
Ce peak at 37.8 ppm remained in the spectrum whereas the
peak at 78 ppm had disappeared due to exchange of sol-
vent, as shown in Fig. 2d.
puffy network and results more cavities in polymeric struc-
ture. Furthermore, the self-polymerization in THF at 60 °C
results more compact assembly of polymeric structure into
particle, as shown in right panel of Fig. 4. Notably, such
distinct assembly of polymeric structure between BACy
self-polymer and hydrogel was reflected on their dissimilar
melting points, 223.6 ± 1.0 °C for BACy self-polymer and
125.0 ± 1.0 °C for BACy hydrogel.
Preparation and physical characteristics of BACy
self-Polymer and hydrogel
BACy self-polymer was prepared by the radical-in-
duced polymerization in THF at 60 °C for 24 hours. The re-
action was nearly complete, as indicated by the absence of
vinyl group of BACy in IR spectrum (1,620 cm-1), as
shown in Fig. 3. Similarly, BACy hydrogel was prepared
by radical-induced polymerization in a mixed solution of
acetonitrile and water at room temperature for 24 hours.
The resulting hydrogel is a white non-transparent gel
which formed white solid after removal of water by ly-
pholyzer.
Whereas BACy self-polymer could not form solid
and large gel, the BACy hydrogel was able to be further
analyzed for its mechanical properties of polymeric gel.
Shear modulus: 158.82 g/cm2; Crosslinking density: 1.58
× 10-5 mole/cm3 (wet gel) and 2.53 × 10-4 mole/cm3 (dry
gel).
Reductive degradation of BACy self-polymer
To investigate the cleavage potential of disulfide
bond in BACy self-polymer, we further carried out the re-
duction of BACy and BACy self-polymer using either 1,4-
dithiothreitol (DTT) or 2-mercaptoethanol (b-ME). Pro-
gression of disulfide bond reduction in BACy could be
monitored by the RF change of reactant and product on thin
layer chromatography (TLC). Our results showed that one
equivalent of DTT could completely cleave the disulfide
bond of BACy after 24 hours at 37 °C whereas some start-
ing BACy remained when using one equivalent of b-ME
under same reaction conditions.
BACy self-polymer: white powder; Mp: 223.6 ± 1.0
°C. FT-IR: 1,642 cm-1 for C=O bond in amide and 1.527
cm-1 for N-H bond in amide. BACy hydrogel: non-trans-
parent white gel; white powder after dehydration and
grinding, Mp: 125.0 ± 1.0 °C.
Swelling capacity of BACy self-polymer and hydro-
gel in water was further investigated. The resulting swell-
ing ratio (SR or Q) was 10.23 for BACy hydrogel, which is
better than the 1.97 of BACy self-polymer. It is anticipated
that BACy self-polymer was produced majorly in powder
form and may not be able to form a porous network with
large space inside of cavities. On the other hand, the BACy
hydrogel was shown to contain bigger pore size than the
self-polymer, demonstrated by scanning electron micro-
scope (SEM) in Fig. 4. This result indicated that polymer-
ization in the presence of water at room temperature allows
more space to be occupied by solvent molecules to form
Further reduction of BACy self-polymer with DTT or
b-ME in acetonitrile was carried out at 37 °C for 48 hours.
As shown in Fig. 5a, the solution containing the reaction
mixture became completely clear for reaction with DTT but
white precipitates remained in the reaction with b-ME.
This data indicates that DTT can efficiently cleave the
disulfide bond of BACy polymer and increase the solubil-
Fig. 4. Morphology of polymer and hydrogel of SEM
photographs.
Fig. 3. IR spectrum of BACy and its polymer.
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BACy Self-polymer
ity of BACy self-polymer in acetonitrile. After reductive
degradation of BACy-self-polymer, the resulting product
becomes a less cross-linked polymer with thiol-end groups
on the linear polymer backbone, as shown in Fig. 5b. It is
anticipated that the reductively cleavable feature of BACy
self-polymer will expand its application in the develop-
ment of degradable polymer-drug conjugates as well as gel
materials transformable into solution upon reduction. More-
over, the produced polymer from reductive degradation of
BACy self-polymer has the thiol-end groups that could aid
in adsorption to certain surfaces like gold particles.
CONCLUSIONS
In this study, the BACy along with its self-polymer
and hydrogel were successfully prepared by condensation
polymerization in THF and free radical polymerization in
different solvent, respectively. Further investigation to-
wards the polymer characteristics for water adsorption, the
BACy self-polymer formed powder and exhibited a rela-
tively low swelling ratio than BACy hydrogel (1.97 vs.
10.23), which is likely due to the less extent of cavity
formed in 3D structure. Furthermore, both disulfide link-
ages in BACy and self-polymers could be easily reduced to
thiols by DTT. Although less frequently discussed in litera-
ture, the reduced product from BACy, N-(2-mercaptoeth-
yl)acrylamide, contains thiol and vinyl group, which can be
further conjugated with other monomer to form biomedical
materials, i.e. the SDS-PAGE for protein separation.14
More importantly, the product from DTT reduction of
BACy self-polymer remains as polymer with less degree of
disulfide cross-linking, which exhibited more flexibility
and better solubility. In addition, the new thiol-end group
on the polymeric backbone provides further potential to
couple with other thiol-specific surfaces like gold particle
or maleimide-immobilized plate, which would greatly in-
crease the biomedical and biotechnical application of BACy
self-polymer.
ACKNOWLEDGEMENTS
This work was supported by the National Science
Council of Taiwan, R.O.C. (NSC 97-2113-M-010-001 and
NSC 98-2311-B-010-003-MY3) and the Ministry of Edu-
cation for “Grant of Aim for Top University Project”.
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