New class of biodegradable polymers formed from reactions of an inorganic functional group

Article abstract of DOI:10.1021/ma300190b

Although numerous small molecules have been synthesized with sulfenamide bonds (R₂N-SR), this is the first report of the synthesis of polysulfenamides. These polymers are readily synthesized at room temperature using secondary diamines and dithiosuccinimides. The dithiosuccinimides were readily synthesized in one step by the reaction of dithiols such as HS(CH ₂)₆SH with N-chlorosuccinimide. The resulting dithiosuccinimides were either recrystallized or readily purified by chromatography on silica gel and required no special handling. The conversions of polymerization ranged from 95 to 98%, and the molecular weights of the polymer reached as high as 6300 g mol^-1. The sulfenamide bond was very stable in organic solvents, and no degradation was observed under atmospheric conditions in C₆D₆ for 30 days. In contrast, the sulfenamide bond readily decomposed in less than 12 h in D₂O. Polysulfenamides were fabricated into micrometer-sized particles loaded with dye and endocytosed into JAWSII immature dendritic and HEK293 cells. Polysulfenamides represent a new class of polymers that are readily synthesized, stable in aprotic solvents, and readily degrade in water.

Full text of DOI:10.1021/ma300190b

Article
pubs.acs.org/Macromolecules
New Class of Biodegradable Polymers Formed from Reactions of an
Inorganic Functional Group
Jun Yoo,† Denison J. Kuruvilla,‡ Sheetal R. D’Mello,‡ Aliasger K. Salem,‡ and Ned B. Bowden*,†
†Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
‡College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, United States
S
* Supporting Information
ABSTRACT: Although numerous small molecules have been
synthesized with sulfenamide bonds (R2N−SR), this is the first
report of the synthesis of polysulfenamides. These polymers are
readily synthesized at room temperature using secondary diamines
and dithiosuccinimides. The dithiosuccinimides were readily
synthesized in one step by the reaction of dithiols such as
HS(CH2)6SH with N-chlorosuccinimide. The resulting dithiosucci-
nimides were either recrystallized or readily purified by chromatog-
raphy on silica gel and required no special handling. The conversions
of polymerization ranged from 95 to 98%, and the molecular weights of the polymer reached as high as 6300 g mol−1. The
sulfenamide bond was very stable in organic solvents, and no degradation was observed under atmospheric conditions in C6D6
for 30 days. In contrast, the sulfenamide bond readily decomposed in less than 12 h in D2O. Polysulfenamides were fabricated
into micrometer-sized particles loaded with dye and endocytosed into JAWSII immature dendritic and HEK293 cells.
Polysulfenamides represent a new class of polymers that are readily synthesized, stable in aprotic solvents, and readily degrade in
water.
potential to open up new applications in this field and will allow
more complex, potentially “smart” drug delivery vehicles to be
synthesized. It is very challenging to design a polymer with a
new functional group along its backbone that renders it stable
at physiological pH but also allows it to be degraded at
reasonable time scales in the body. Recent work has shown the
promise of using biodegradable polymers linked with acetals or
disulfides in medical applications that provide alternatives to
functional groups based on carbonyls.21−24 In this paper, we
report the first synthesis of polymers with the sulfenamide
functional group within the backbone of macromolecules, how
these polymers degrade under different conditions, and how
they can be fabricated into micrometer-sized particles loaded
with fluorescent dye that are internalized into cells.
Polysulfenamides are a new class of polymers that we believe
will have real applications in medicine.
Sulfenamides are an understudied functional group with the
general formula of RS-NR′R″ that are used as protecting groups
for amines (typically as (Ph3C)S−NR2),25 vulcanizing agents in
the rubber industry,26−28 in the activation of C−H bonds,29 as
ligands on metals to promote O atom transfer,30,31 as
intermediates in the synthesis of sulfonamides,32 as oxidants
for alcohols,33,34 and as ligands in the synthesis of inorganic
coordination compounds.35−40 In a recent article we reported
on the synthesis of the first polydiaminosulfides based on the
INTRODUCTION
■
Biodegradable polymers have shown wide utility in a variety of
biomedical applications ranging from sutures, scaffolds for the
growth of cells, and polymeric depots that provided sustained
release of therapeutic agents.1−6 Optimal use of these materials
requires their molecular and macromolecular properties be
tailored to the specific application for which the material is to
be used.3,7−9 For instance, polymeric particles loaded with
drugs that are targeted to the endosomal compartments of cells
should ideally be stable at physiological pH in the bloodstream
but readily break down at a lower pH to release its drug cargo
in the endosome where the pH is approximately 5−6.10−13
Biodegradable polymers are attractive in drug delivery
applications because polymeric particles injected in vivo can
accumulate in several organs including the liver, spleen, lungs,
and heart and often result in toxic side effects if they do not
break apart into smaller, easily excretable side products.14,15
The degradation of these polymers is also the mechanism by
which controlled release of drugs and other therapeutics is
achieved. Most synthetic polymers in biomedical research are
polyesters, polyamides, polyanhydrides, or some combination
of these three functional groups. These polymers degrade to
short polymers or small molecules at reasonable times without
the use of enzymes.16−18 Thus, these polymers may be excreted
from the body before toxic side effects occur.
Integrating new functional groups into polymer chemistry
allows the fabrication of polymers with new properties and the
ability to tailor new materials.19,20 Specifically, the introduction
of a new functional group to synthesize biopolymers has the
Received: January 24, 2012
Revised: February 6, 2012
Published: February 21, 2012
© 2012 American Chemical Society
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Figure 1. Two different disulfenamides were synthesized in one pot from dithiols.
diaminosulfide functional group (R2N−S−NR2).41 The dia-
minosulfide functional group differs from the sulfenamide
functional group in its structure, reactivity, and reaction
products. A loose analogy to explain the differences between
diaminosulfides and sulfenamides is to compare this pair to
esters and anhydrides. Structurally, an anhydride is an ester
with an extra carbonyl. Similarly, a diaminosulfide is a
sulfenamide with an extra amine. Much as the structural
differences between an ester and anhydride lead to different
reactivities and products of reaction, the structural differences
between a sulfenamide and diaminosulfide lead to different
reactivities and products of reaction. A diaminosulfide should
not be confused for a sulfenamide any more than an ester is
confused for an anhydride.
chromatography on silica gel, the isolated yield was only 6%.
When this molecule was purified by chromatography on basic
alumina oxide, the isolated yield was 76%. This result was
consistent with prior reports that described the decomposition
of sulfenamides on acidic silica gel.32,46 All subsequent
purifications of sulfenamides were completed by vacuum
distillation or by chromatography on basic alumina oxide.
Simple bimolecular reactions were studied to measure the
kinetics of the transamination reaction shown in Figure 2 to
Some of the general properties of sulfenamides made them
attractive functional groups to pursue for the synthesis of
polymers. Prior work by others with small molecules containing
sulfenamide bonds demonstrated that they were readily
synthesized and possessed reasonable stabilities.35,42−44 Mole-
cules with sulfenamide bonds degraded into radicals at elevated
temperatures which led to their applications as vulcanizing
agents in the rubber industry.26−28 On the basis of their
properties as protecting groups for amines, it was known that
sulfenamides were stable under neutral and basic conditions but
could be cleaved under acidic conditions.35 Furthermore,
sulfenamides have been synthesized in high yields from
activated thiols and amines at room temperature.
These properties were desirable for the development of new
biodegradable polymers. We sought a functional group that was
stable in organic solvents, would rapidly break down in acidic
water, and would possess higher stability in water at neutral pH
than at acidic pH. Additionally, prior reports of the synthesis of
sulfenamides in high yields were promising because the
development of a step growth polymerization requires that
the yield of the reaction to form the polymer be high and
typically exceed 95%.45
Figure 2. Kinetics of the transamination reaction in (a) was studied by
1H NMR spectroscopy in CD3OD as shown in (b). The conversion in
(b) refers to the conversion of molecule B to the indicated product.
The NMR tube was sealed to prevent the release of N-ethylmethyl-
amine.
determine the best conditions for polymerizations. Molecule B
was synthesized and reacted with N-benzylmethylamine in
C6D6, DMSO-d6, and CD3OD at different temperatures. This
reaction was very slow in C6D6 with no conversion after 24 h at
room temperature and only 11% conversion after 24 h at 60 °C.
When DMSO-d6 was used as the solvent, the reaction was
faster and the conversion reached 7% after 24 h at room
temperature and 32% after 24 h at 50 °C. The reaction
proceeded rapidly in CD3OD and reached a conversion of 40%
after 3 h, and at longer periods of time it reached an
equilibrium mixture of 50% of each sulfenamide. The rate
constant for the reaction in CD3OD was 5.1 × 10−4 M−1 s−1.
This reaction was most rapid in a polar, protic solvent which
suggests a charged transition state that is stabilized by hydrogen
bonding. It is believed that the reaction proceeded through an
SN2 reaction mechanism when N-benzylmethylamine attacked
the sulfur of the sulfenamide. It was possible that the hydrogen
bond donating ability of CD3OD lowered the energy
requirement for N-ethylmethylamine to leave.
In this article we report the first synthesis of polysulfena-
mides. No prior examples of polymers containing this
functional group have been reported so their synthesis was
developed by us for the first time. Additionally, we report the
stability of this functional group to different conditions and
how they can be applied as new drug delivery vehicles.
RESULTS AND DISCUSSION
■
Synthesis of Polysulfenamides from Disulfenamides.
Because polysulfenamides were unknown prior to our work, the
first step in this project was to develop their synthesis. In one
approach two different dithiols were activated with sulfuryl
chloride to yield intermediates with S−Cl bonds (Figure 1).35 If
left overnight exposed to air, these molecules formed disulfides.
To prevent the formation of disulfides, these molecules were
reacted with an excess of N-ethylmethylamine immediately
after their formation. When molecule A was purified by
When the polymerization of a disulfenamide with a diamine
was attempted, the results were not promising (Figure 3).
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Figure 3. Polymerization of a disulfenamide with a secondary diamine
in benzene yielded a low molecular weight polymer.
Although the kinetics of reaction were rapid in methanol, the
resulting polymer was insoluble in methanol which made this
solvent a poor choice to conduct polymerizations. Polymer-
izations at high concentrations of monomers in solvent
mixtures of (1/1 v/v) CD3OD/CDCl3 or (1.2/1 v/v)
CD3OD/DMSO-d6 at 40 °C yielded oligomers even after 3
days. A polymerization was attempted in refluxing benzene for
24 h with the reaction vented to allow N-ethylmethylamine
(boiling point of 36−37 °C) to boil off resulted in a low yield
of a modest molecular weight polymer (Mw = 2840 g mol−1).
This method of polymerization was hindered by lack of
solubility of the polymer in methanol and the slow kinetics for
polymerization in aprotic solvents.
Figure 5. (a) Reaction of a thiosuccinimide with a secondary amine in
CD2Cl2 is shown. The yield of the reaction was >97% after extended
reaction times. (b) Kinetics of a typical polymerization to demonstrate
that the polymerizations were rapid and went to quantitative
conversions at reasonable concentrations. The starting materials in
(b) were 3 times more concentrated than the starting materials in (a).
test polymerization shown in Figure 5b went to >95%
conversion within 50 min in CDCl3 at room temperature.
This polymerization method was used in all subsequent
polymerizations.
A series of polymers were synthesized from dithiosuccini-
mides and secondary diamines (Table 1). The polymerizations
Synthesis of Polysulfenamides from Dithiosuccini-
mides. To increase the rate of transamination and to allow the
use of aprotic solvents in a polymerization, dithiols were
activated with N-chlorosuccinimide (Figure 4). This reaction
Table 1. Synthesis of Polysulfenamides from
Dithiosuccinimides
Figure 4. Dithiosuccinimides were readily synthesized from N-
chlorosuccinimide and triethylamine.
went to high yields at room temperature, and the
dithiosuccinimides were stable to chromatography on silica
gel. Furthermore, molecule C was readily purified by
crystallization. The dithiosuccinimides were stable at room
temperature under ambient atmosphere, and they required no
special handling.
The reaction of a thiosuccinimide (molecule D in Figure 5)
with N-benzylmethylamine was much faster in aprotic solvents
than the same reaction between molecule B and N-
benzylmethylamine. In CD2Cl2 at room temperature the
reaction between molecule D and N-benzylmethylamine had
a rate constant of 8.2 × 10−4 M−1 s−1 and reached 35%
conversion after 4 h. This transamination reaction was ∼2 times
faster than the reaction between molecule B and N-
benzylmethylamine in CD3OD. The difference in solvent was
critically important because methylene chloride and chloroform
were excellent solvents for both monomers and polymers in
contrast to CD3OD where the polymer precipitated. In
addition, the reaction of a thiosuccinimide with N-benzylme-
thylamine went to >97% conversion at room temperature. The
a
The absolute Mw and PDI were measured with multiangle laser light
b
scattering and refractive index detectors unless otherwise noted. The
conversion was calculated based on the molecular weight of the
polymer and monomers according to the equation Xn = 1/(1 − P).
c
The Mw and PDI were measured versus polystyrene standards using a
calibration curve.
were completed in methylene chloride or chloroform at room
temperature for 24 h. The polymers had molecular weights up
to 6300 g mol−1, degrees of polymerization that ranged from
95% to 98%, and they were isolated in >80% yields by
precipitation in methanol. Secondary diamines were chosen for
this work because the synthesis of polymers using primary
diamines (such as NH2CH2CH2NH2) resulted in cross-linked,
insoluble polymers. The cross-links were possibly due to the
reaction of an amine with 2 equiv of a thiosuccinimide to yield
cross-links of the form RN(SR′)2.
These polymers were well characterized by size exclusion
chromatography (SEC). Entries 1 and 4 of Table 1 had strong
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peaks in both the multiangle laser light scattering detector and
the refractive index detector so absolute molecular weights were
calculated. For entries 2, 3, and 5 the light scattering signal was
too weak so the molecular weights were found by calibration
with polystyrene standards. In all examples the polymers were
molecule slowly degraded in CD3OD; after 33 days 15% of it
degraded at room temperature and only 27% degraded when
heated to 40 °C for the same period of time. These conditions
indicated that sulfenamides possessed reasonable stabilities
under neutral conditions in organic solvents. It should be noted
that the polysulfenamides were synthesized in organic solvents
under ambient conditions without any special handling.
The kinetics and products of degradation of molecule E were
studied under acidic conditions. First, molecule E was added to
CD3OD with 5 equiv of benzoic acid. In contrast to the
degradation of molecule E without benzoic acid (33 days to
reach 15% degradation), the degradation of molecule E in the
presence of benzoic acid reached 51% degradation within 4
min. When molecule E was exposed to 2 mol equiv of p-
toluenesulfonic acid (pKa < −2) or trifluoroacetic acid (pKa =
0.3) in CD3OD, it degraded to >90% within 4 min. Clearly, the
decomposition of sulfenamides was catalyzed by acid as
observed by others in prior work.32,46
1
analyzed by H NMR spectroscopy to study the end groups.
The peaks corresponding to the end groups integrated to very
low values which supported the SEC data for the molecular
weights and high conversions. An example of a SEC micrograph
is shown in Figure 6.
The products of degradation of molecule E were studied
under acidic conditions in D2O to understand how
polysulfenamides would break down under these conditions.
Molecule E was added to D2O with 7 mol equiv of acetic acid.
Because molecule E was insoluble in D2O, the reaction mixture
was vigorously stirred. After 41 h the organics were extracted
and initially studied by 1H NMR spectroscopy prior to isolation
Figure 6. SEC micrograph of the polymer from entry 1 in Table 1.
Degradation of Sulfenamides in Aprotic and Protic
Solvents. Little data were available on the stabilities of
sulfenamides in various solvents that could be used to anticipate
the stabilities of polysulfenamides. To address this limitation, a
sulfenamide was synthesized and its stability was studied in
1
by column chromatography. The H NMR spectrum of the
extract before chromatography indicated that ∼75% of the
product was the thiosulfinate (molecule F in Figure 8a) with
the remainder mostly composed of molecules G and H. The
initial reaction of molecule E with water yielded a sulfenic acid,
but sulfenic acids are transient intermediates that very rapidly
condense as shown in Figure 8b.40,47−49 On the basis of
literature precedent, molecule F decomposed via multiple
pathways to yield molecule G, molecule H, and a sulfinic acid
(molecule I).48,50−52 Thiosulfinate esters such as molecule F are
known oxidation states for cysteine residues and found in
nature products such as allicin (a component of garlic; Figure
8c). As such, thiosulfinates are already present in the body and
are known to decompose at reasonable ratesthe half-life of
the decomposition of allicin at pH = 7.5 at 37 °C is ∼2 days.8,50
To further study the degradation of sulfenamides in D2O, a
water-soluble sulfenamide was synthesized (Figure 9). The
Figure 7. Stability of molecule E was studied under three different
conditions.
NMR tubes in CD3OD and C6D6 (Figure 7). After 30 days, the
1H NMR spectrum of the sulfenamide in C6D6 was unchanged
which demonstrated its stability in aprotic solvents. This
Figure 8. (a) The first degradation product of the decomposition of a sulfenamide in D2O was molecule F. This molecule further decomposed to
yield molecules G and H. Molecules F, G, and H were observed by 1H NMR spectroscopy. Molecule I and hexanethiol were not observed but were
predicted by literature precedent. (b) Sulfenic acids were the first product of degradation of molecule E but they were known to rapidly react to yield
thiosulfinates as shown. (c) The structure of allicin.
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into the microparticles by mixing them in the polymer/
chloroform mixture prior to emulsification. The average size of
the microparticle was 2.6 μm with a PDI of 0.62. They were
spherical in shape, had a smooth surface, and had a negative
charge of −41 mV in PBS buffer at pH = 7.4 (Figure 10). The
reason for the negative charge was understood in terms of prior
experiments that demonstrated that sulfenamides slowly
degraded to yield sulfinic acids that deprotonated and led to
a negatively charged surface.
Microparticle degradation was studied at both physiological
pH (7.4) and acidic pH (4.0) to learn whether these particles
would decompose over time. Figure 10 shows the percentage
weight loss of the samples at different points in time. After 1
week, a weight loss of 24% was observed for microparticles in
acetate buffer as compared to a weight loss of 13% for particles
in PBS. On day 35, the weight loss in acidic buffer was ∼33% as
compared to ∼17% at physiological pH. Weight loss was
significantly higher for particles in acetate buffer as compared to
particles in PBS, indicating faster degradation of the micro-
particles in acidic environments. SEM images of the acidic pH-
degraded particles revealed a rough morphology of the polymer
surface that was consistent with polymer degradation (Figure
10c). The differences in the rates of degradation of micro-
particles at pH 7.4 and 4.0 were expected based on the rapid
degradation of molecule J under acidic conditions in D2O and
its slower degradation in neutral D2O.
The overall rate of degradation of a microparticle depends on
many factors other than the rate of degradation of the
functional group along the backbone. For instance, micro-
particles fabricated from polycaprolactone lose less than 1% of
their weight after 26 weeks in aqueous buffer,54 but
microparticles fabricated from poly(lactic-co-glycolic acid)
with 50% lactic acid and 50% glycolic acid lose approximately
half of their weight in 2 weeks in aqueous buffer.55 This
difference in rates of degradation of two similar polyesters is
due to factors such as differences in crystallinity, molecular
weight, molecular weight distribution, porosity, and hydro-
phobicity of the polymers.56 Thus, although the degradation of
the sulfenamide bond is rapid in water, microparticles
fabricated from polysulfenamides will exhibit a wide range of
degradation profiles based on the monomers used in their
synthesis.
Figure 9. Kinetics of decomposition of a sulfenamide was studied by
1H NMR spectroscopy in D2O with either 9 mol equiv of NaOH, 8
mol equiv of acetic acid, or no added acid or base.
decomposition of this molecule was studied in D2O with 8 mol
equiv of acetic acid, 9 mol equiv of KOH, or under neutral
conditions without the addition of acid or base. The rates of
decomposition were found under neutral (k = 4.7 × 10−4 s−1)
and basic conditions (k = 8.0 × 10−5 s−1), but the
decomposition was too rapid under acidic conditions to
measure a rate constant. Because the sulfenamide completely
decomposed within 4 min after addition to D2O with acetic
acid, only a lower limit for its rate of decomposition was found
(1.6 × 10−2 s−1). It is important to note that these rate
constants are several orders of magnitude higher than the
neutral and acidic degradation of esters.53 Because of the rapid
decomposition, we were unable to determine a value for the
pKa of a protonated sulfenamide.
Fabrication and Degradation of Microparticles of
Polysulfenamides. Microparticles were fabricated from the
polymer in entry 2 in Table 1 to study whether these polymers
could form the basis of drug delivery vehicles. The micro-
particles were prepared using an oil-in-water single emulsion
solvent evaporation methodology. In this approach, the
polymer was dissolved in chloroform and then emulsified in
surfactant containing water phase. The emulsion was stirred
overnight until the chloroform evaporated. Drugs and
fluorescent molecules such as rhodamine can be incorporated
The uptake of these microparticles was studied in JAWSII
and human embryonic kidney (HEK293) cells using confocal
microscopy. JAWSII cells are immature dendritic cells derived
from mouse bone marrow and are commonly targeted by
Figure 10. Microparticles with the structure of entry 2 in Table 1 were studied for their degradation and surface reactions. (a) Time-dependent
weight loss of the polymer incubated at acidic pH (4.0) and physiological pH (7.4). Data represent the percentage weight loss as mean of three
samples. (b) Scanning electron microscopy (SEM) image of microparticles as prepared. (c) SEM image of microparticles after incubation in acetate
buffer (pH 4.0) for 14 days. Microparticles at various stages of degradation can be observed.
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Figure 11. Uptake of microparticles fabricated from entry 2 in Table 1 and loaded with rhodamine and studied using confocal microscopy. (a−e)
The cells are JAWSII immature dendritic cells, and in (f−j) they are HEK293 cells. The nucleus of the cell was stained with DAPI to appear blue and
the rhodamine appeared red. In (d) and (i) the transmitted light images indicate the periphery of the cells and the location of the microparticles. In
(e) and (j) merged images clearly outline the periphery of the cell, the location of the nucleus, and the microparticles to clearly demonstrate that the
microparticles were found on the interior of the cells. The images were taken at different sections to ensure that the microparticles were in the same
focal plane as the planes taken through the cells. The microparticle in the far left of images (g−j) was not found in a cell and was used to show a free
microparticle.
Figure 12. (a) Cytotoxicity of polysulfenamide microparticles fabricated from entry 2 in Table 1 toward HEK293 cells. Cell viability was determined
by MTS assay and the expressed as percentage of control without microparticles. (b, c) These graphs show the in vivo ALT and AST levels in BALB/
c mice following i.p. injection of microparticles. There are five sets of data for each day in the graphs in (b) and (c). The data for each day are
presented in order of mice injected with PBS buffer, 10 mg of PLGA microparticles, 5 mg of polysulfenamide microparticles, 10 mg of
polysulfenamide microparticles, and 15 mg of polysulfenamide microparticles.
particle based vaccines for generation of therapeutic immune
responses. HEK293 cells are one of the most common cell lines
used to evaluate novel gene therapies. Microparticles were
loaded with rhodamine B during fabrication and were then
exposed to the JAWSII and HEK293 cells. The microparticles
were found to be located near the cytoplasm of the cell,
indicating the phagocytosis of the microparticles by cells
(Figure 11; see also the Supporting Information for an
additional image). The uptake of microparticles by both cell
lines suggests that this system has potential in immunological
applications, especially in vaccine delivery and potential for
gene therapy applications.
Toxicity of Polysulfenamide Microparticles. The in vitro
toxicity of these microparticles was evaluated with HEK293
cells. In initial work, these cells were incubated with the
microparticles at a range of concentrations of 1−1000 μg per
mL of growth medium for 4 h. The cell viability was measured
by standard techniques of measuring the metabolic activity of
cells in a control without microparticles and the metabolic
activity of cells that were exposed to the microparticles. The
ratio of these concentrations was plotted as the “cell viability” in
the y-axis of Figure 12a. No toxicity was observed at even the
highest concentrations after 4 h, so a longer incubation time of
24 h was studied and shown in Figure 12a. At the expected
working concentrations of microparticles minimal toxicity was
observed. Interestingly, at the highest concentration of 1 mg of
microparticles per mL of solution where the wells containing
HEK293 cells were beginning to become saturated with
microparticles, the cell viability was still 57%. This suggests
that polysulfenamides have low toxicity and are therefore
suitable for drug, gene, and vaccine delivery applications.
The in vivo toxicity of the polysulfenamide microparticles
were evaluated in BALB/c mice. Microparticles were
administered via intraperitoneal (i.p.) injection because this
route of administration is commonly utilized for evaluating
microparticle based vaccines which are an intended application
for this novel polymer and because the i.p. route allows
particles to enter the systemic circulation allowing for a better
gauge of systemic toxicity than subcutaneous or intramuscular
injection. The mice were divided into five groups and were
injected with either 5, 10, or 15 mg of polysulfenamide
microparticles from entry 2 in Table 1; 10 mg of microparticles
of poly(D,L-lactide-co-glycolide) as a control group; or a sterile
PBS buffer solution. The mass of particles injected in these
experiments was significantly higher than the typical therapeutic
mass of microparticles injected for vaccine based application.
The mice were followed for 23 days, and blood was drawn at 0,
2, 9, 16, and 23 days to test for the levels of liver enzymes.
Specifically, alanine aminotranferease (ALT) and aspartate
aminotransferase (AST) are liver enzymes (transaminases) that
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Paraformaldehyde (16% solution, EM grade) was obtained from
electron microscopy services (Hatfield, PA), and Vectashield
mounting medium for fluorescence with DAPI was a product of
Vector laboratories (Burlingame, CA). 1-Hexanethiol, 1,6-hexanedi-
thiol, 2-mercaptoethyl ether, N-benzylmethylamine, N-ethylmethyl-
amine, N,N′-dimethyl-1,6-hexanediamine, N-chlorosuccinimide, trie-
thylamine, ethylenediamine, benzoic acid, p-toluenesulfonic acid, acetic
acid, and sulfuryl chloride were purchased from Aldrich or Acros
Organics at their highest purity and used as received. HPLC grade
chloroform purchased from Acros Organics was used as the GPC
solvent after filtering it through a glass frit. All other solvents were
reagent grade and purchased from Acros Organics. Piperazine (99%)
was purchased from Aldrich. It was purified by sublimation under
vacuum at 130 °C. N,N’-Diethylethylenediamine (95%) was purchased
from Aldrich and distilled under vacuum at 30 °C. Genduran silica gel
60 (230−400 mesh) and basic alumina Brockman activity I (60−325
mesh) were purchased from Fisher Scientific and used for column
chromatography.
Characterization of Small Molecules and Polysulfenamides.
1H and 13C NMR spectra were recorded on a Bruker DPX 300 at 300
and 75 MHz, respectively. CDCl3 was used as NMR solvent with
tetramethylsilane (TMS) as an internal standard. SEC was performed
using chloroform as the mobile phase (1.00 mL min−1) at 40 °C. A
Waters 515 HPLC pump and two Waters columns (Styragel HR4 and
HR5E) were used in series. A DAWN EOS 18 angle laser light
scattering detector from Wyatt Corp. to measure light scattering and a
Wyatt Optilab DSP to measure changes in refractive index were used
to measure absolute molecular weights of polymers. Polystyrene
standards (1260, 3790, 13 000, and 30 300 g mol−1) were used to
generate a calibration curve to measure relative molecular weights of
some polysulfenamides as indicated in Table 1.
can be detected systemically as a result of hepatocellular
damage (liver inflammation, necrosis, cellular degeneration, and
increased membrane permeability) and are approved FDA
assays for determination of preclinical toxicity in new chemical
entities.
The in vivo study showed that the polysulfenamide
microparticles did not result in any significant increase in the
serum ALT and AST levels over the study period, relative to
either day 0 or poly(D,L-lactide-co-glycolide) microparticles,
indicating lack of any liver damage (Figure 12b,c). In addition,
mice were monitored daily and remained in good health as
determined by the body condition scoring technique. The
vertebrae and dorsal pelvis were not prominent but palpable
with slight pressure, indicating a state of good health in the
mice. No adverse injection site reactions such as infection,
redness, or wounding were observed. Furthermore, no mice
died as a result of injections of polysulfenamide microparticles
over the entire study period. In summary, the in vivo and in vitro
studies showed little or no adverse effects from the
polysulfenamides. Although these results are promising for an
initial demonstration of toxicity, the toxicity of polysulfena-
mides will require a more thorough investigation in future
studies to fully probe any toxicity resulting from the polymers
or their degradation products (such as the sulfenic acids).
CONCLUSIONS
■
We report the first synthesis and characterization of
polysulfenamides. These polymers are unique because they
integrate a new functional group into polymer chemistry that
has found some applications in small molecule chemistry. Two
methods to synthesize polysulfenamides were reported, and
both polymerization reactions went to high conversions (up to
98%). It is notable that the synthesis of polysulfenamides from
dithiosuccinimides proceeded at room temperature in methyl-
ene chloride; these reaction conditions are mild and suggest
that more complex monomers can be used. Although the
molecular weights of the polysulfenamides reported here did
not reach 10 000 g mol−1, higher molecular weights are possible
if the conversions of polymerization are increased or if higher
molecular weight monomers are used. These polymers were
stable in aprotic solvents and only slowly degraded in protic,
organic solvents.
Hexanesulfenyl Chloride. Alkylsulfenyl chlorides were prepared
according to a literature procedure.57 Since they quickly returned to
thiols after being exposed to air, alkylsulfenyl chlorides were used in
situ for the preparation of sulfenamides. Sulfuryl chloride (6.28 g,
0.047 mol) was added dropwise to a solution of hexanethiol (5 g,
0.042 mol) in CH2Cl2 (30 mL) at 0 °C under nitrogen and stirred for
1
2 h 45 min. H NMR (CDCl3): δ 0.90 (t, 3H, J = 6.8 Hz), 1.39 (m,
6H), 1.78 (m, 2H), 3.11 (t, 2H, J = 7.1 Hz). 13C NMR (CDCl3): δ
14.03, 22.52, 27.83, 28.02, 31.31, 41.52.
2,2′-Oxydiethanesulfenyl Dichloride. 2-Mercaptoethyl ether (3
g, 0.22 mol) was reacted with a solution of sulfuryl chloride (6.15 g,
1
0.046 mol) in CH2Cl2 (30 mL) following the previous procedure. H
NMR (CDCl3): δ 3.33 (t, 4H, J = 6.3 Hz), 3.88 (t, 4H, J = 6.2 Hz).
13C NMR (CDCl3): δ 40.92, 68.10.
N-Ethylmethylhexanesulfenamide (Molecule B). A solution of
hexanesulfenyl chloride (6.45 g, 0.042 mol) in CH2Cl2 (30 mL) was
slowly added to a solution of N-ethylmethylamine (7.50 g, 0.13 mol)
in CH2Cl2 (50 mL) at 0 °C under nitrogen and stirred for 7 h. The
reaction was diluted with anhydrous Et2O (130 mL) and washed with
saturated NaCl solution in water (3 × 50 mL). The organic phase was
dried over anhydrous magnesium sulfate and evaporated to give a
brown oil. The product was isolated by vacuum distillation at 30 °C to
yield a colorless oil (3.93 g, 53% yield). 1H NMR (CDCl3): δ 0.89 (t,
3H, J = 6.8 Hz), 1.14 (t, 3H, J = 7.2 Hz), 1.35 (m, 6H), 1.54 (m, 2H),
2.65 (t, 2H, J = 7.2 Hz), 2.75 (s, 3H), 2.82 (q, 2H, J = 7.1 Hz). 13C
NMR (CDCl3): δ 13.79, 14.09, 22.58, 28.27, 28.89, 31.59, 31.86,
46.82, 53.93. HRMS calcd for C9H21NS: 175.1395. Found: 175.1392.
N,N’-Ethylmethylbis(2-mercaptoethyl)disulfenamide (Mole-
cule A). A solution of 2,2′-oxidiethanesulfenyl dichloride (4.49 g,
0.022 mol) in CH2Cl2 (30 mL) was reacted with a solution of N-
ethylmethylamine (7.70 g, 0.13 mol) in CH2Cl2 (50 mL) following the
previous procedure. The extract was filtered through basic alumina to
yield a colorless oil (3.29 g, 60% yield). 1H NMR (CDCl3): δ 1.13 (t,
6H, J = 7.1 Hz), 2.74 (s, 6H), 2.82 (m, 8H), 3.67 (t, 4H, J = 7.1 Hz).
13C NMR (CDCl3): δ 13.70, 32.68, 46.89, 54.13, 70.01. HRMS calcd
Polysulfenamides present new opportunities in macro-
molecules because they possess a functional group that was
previously unknown in polymer chemistry. In this article we
describe how polysulfenamides possess many of the right
characteristics for applications in the delivery of pharmaceut-
icals. For instance, they were stable in organic solvents but
degraded in water and degraded more rapidly in acidic water.
Microparticles were fabricated from a polysulfenamide, and
these microparticles were readily internalized by two different
cell lines. Other applications of polysulfenamides are envisioned
that take advantage of the thermal lability of sulfenamide bonds,
oxidation of the sulfurs, or their rapid degradation under acidic
medium. Because many new opportunities for macromolecules
arise when a new functional group is integrated into polymer
science, we anticipate new applications for polysulfenamides.
EXPERIMENTAL PROCEDURES
■
Materials. Phosphate buffer saline (PBS) is a product of Sigma-
Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle’s Medium
(DMEM) was obtained from Gibco BRL (Grand Island, NY).
LysoTracker Green was obtained from Invitrogen (Eugene, OR).
for C10H24N2OS2: 252.1321. Found: 252.1324.
N-(Hexanethio)succinimide (Molecule D).58 Hexanethiol (3 g,
25.37 mmol) was added dropwise to a solution of N-chlorosuccini-
mide (3.36 g, 26.64 mmol) in CH2Cl2 (70 mL) at 0 °C under nitrogen
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and stirred for 20 min. The solution changed to a yellow-green color
and immediately became cloudy. A solution of triethylamine (2.82 g,
27.91 mmol) in CH2Cl2 (10 mL) was added dropwise to the reaction.
After the mixture was stirred for 2.5 h, it was washed with a saturated
NaCl solution in water (4 × 30 mL). The organic layer was dried over
anhydrous magnesium sulfate and evaporated to give a colorless oil.
The product was isolated by column chromatography using silica gel
and 20% ethyl acetate in hexanes to yield a colorless oil (4.73 g, 87%
precipitated into methanol (10 mL) and washed with methanol (15
mL). The polymer was dried under vacuum to yield a yellowish oil. 1H
NMR (CDCl3): δ 1.14 (t, 6H, J = 6.9 Hz), 2.77 (t, 4H, J = 7.1 Hz),
2.90 (q, 4H, J = 7.0 Hz), 3.04 (s, 4H), 3.65 (t, 4H, J = 7.2 Hz). 13C
NMR (CDCl3): δ 14.27, 35.14, 53.66, 56.79, 69.98.
Polymer from Entry 5 in Table 1. N,N′-(Hexanethio)-
disuccinimide (1 g, 2.90 mmol) in CHCl3 (6 mL) was reacted with
N,N′-dimethyl-1,6-hexanediamine (0.42 g, 2.90 mmol) following the
previous procedure. The polymer was precipitated into methanol (10
mL). The polymer was redissolved in a minimal amount of CHCl3 and
precipitated into methanol (7 mL) to yield a yellowish oil after drying
under vacuum. 1H NMR (CDCl3): δ 1.31 (m, 4H), 1.41 (m, 4H), 1.55
(m, 8H), 2.64 (t, 4H, J = 7.4), 2.76 (m, 10H). 13C NMR (CDCl3): δ
26.87, 28.18, 28.40, 28.89, 31.68, 47.12, 59.87.
1
yield). H NMR (CDCl3): δ 0.88 (t, 3H, J = 6.6 Hz), 1.28 (m, 4H),
1.40 (m, 2H), 1.54 (m, 2H), 2.85 (m, 6H). 13C NMR (CDCl3): δ
13.99, 22.45, 27.90, 28.11, 28.61, 31.28, 37.62, 177.23. HRMS calcd for
C10H17NO2S: 215.0980. Found: 215.0979.
(2,2′-Oxydiethenthio)disuccinimide. 2-Mercaptoethyl ether (10
g, 0.072 mol) was reacted with a solution of N-chlorosuccinimide
(20.28 g, 0.15 mol) in CH2Cl2 (300 mL) and a solution of
triethylamine (16.10 g, 0.16 mol) in CH2Cl2 (55 mL) following the
previous procedure. The mixture was purified by column chromatog-
raphy using 95% ethyl acetate in hexanes to yield a white solid (11.42
Polymer from
a Disulfenamide (Figure 3). N,N′-
Ethylmethylbis(2-mercaptoethyl)disulfenamide (1.34 g, 5.32 mmol)
was reacted with N,N′-diethylethylenediamine (0.62 g, 5.32 mmol) in
refluxing benzene (5 mL) for 24 h. After evaporating the solvent, the
polymer was precipitated into methanol (2 × 15 mL). The polymer
1
g, 48% yield). H NMR (CDCl3): δ 2.78 (s, 8H), 2.95 (t, 4H, J = 5.9
Hz), 3.68 (t, 4H, J = 5.9 Hz). 13C NMR (CDCl3): δ 28.68, 36.93,
70.90, 177.29. HRMS calcd for C12H16N2O5S2 + Na+: 355.0398.
Found: 355.0387.
was dried under vacuum to yield a yellowish oil (0.12 g). H NMR
1
(CDCl3): δ 1.14 (t, 6H, J = 6.9 Hz), 2.77 (t, 4H, J = 7.1 Hz), 2.90 (q,
4H, J = 7.0 Hz), 3.04 (s, 4H), 3.65 (t, 4H, J = 7.2 Hz). 13C NMR
(CDCl3): δ 14.27, 35.14, 53.66, 56.79, 69.98.
N,N’-(Hexanethio)disuccinimide (Molecule C). 1,6-Hexanedi-
thiol (7 g, 0.047 mol) was reacted with a solution of N-
chlorosuccinimide (13.06 g, 0.098 mol) in CH2Cl2 (230 mL) and a
solution of triethylamine (10.37 g, 0.102 mol) in CH2Cl2 (35 mL)
following the previous procedure to give a brown solid. The solid was
recrystallized from methanol (350 mL) 3 times to yield a white solid
Preparation of Microparticles. Blank microparticles were
prepared by oil-in-water (o/w) single emulsion, solvent evaporation
technique. Briefly, 300 mg of polymer was dissolved in 3.5 mL of
chloroform in a heated water bath (55 °C). This organic phase was
then emulsified with 35 mL of 1% (w/v) Mowiol at 13 500 rpm for 1
min using an IKA Ultra-Turrax T25 basic homogenizer (IKA,
Wilmington, NC). The mixture was stirred overnight to evaporate
the organic phase. The microparticles were then washed three times
with deionized water (5000 rpm for 10 min) and lyophilized
(Labconco FreeZone 4.5, Kansas City, MO). Microparticles were
stored at −20 °C until use.
Surface Morphology. Microparticle morphology was assessed by
scanning electron microscopy (SEM, Hitachi S-4000). Air-dried
microparticles were placed on silicon wafers mounted on SEM
specimen stubs. The stubs were coated with ∼5 nm of gold by ion
beam evaporation followed by imaging using SEM operated at 3 kV
accelerating voltage.
Particle Size and Zeta Potential. Microparticle size and zeta
potential was measured using the Zetasizer Nano ZS (Malvern,
Southborough, MA). The microparticles were suspended in deionized
water at a concentration of 1 mg mL−1. The zeta potential was
measured using folded capillary cell in automatic mode, and the size
was measured using a disposable sizing cuvette (DTS0012). The size
was measured at 25 °C at a 173° scattering angle.
In Vitro Analysis of Microparticle Degradation. For in vitro
analysis, ∼85 mg of the microparticles was suspended in either
phosphate buffer saline (PBS, pH 7.4) or acetate buffer (pH 4). The
samples were agitated at 37 °C, 150 rpm in a horizontal shaker (C24
incubator shaker, New Brunswick Scientific, Edison, NJ). At specific
time points, the samples were centrifuged for 10 min at 7000 rpm. The
pellet was washed three times with deionized water and lyophilized.
The dry weight of the pellet was recorded, and percentage weight loss
was calculated for each sample. In addition, the samples were also
analyzed by scanning electron microscopy to observe change in surface
morphology of microparticles.
1
(9.90 g, 62% yield). H NMR (CDCl3): δ 1.47 (m, 8H), 2.83 (m,
12H). 13C NMR (CDCl3): δ 27.62, 27.65, 28.62, 37.48, 177.26.
HRMS calcd for C14H20N2O4S2 + Na+: 367.0762. Found: 367.0757.
N-Benzylmethylhexanesulfenamide. N-Benzylmethylamine
(1.11 g, 9.19 mmol) was added to a solution of molecule B (1.94 g,
9.01 mmol) in CH2Cl2 (2.5 mL) and stirred at room temperature for
19 h. The reaction was diluted with anhydrous Et2O (30 mL) and
washed with water (4 × 20 mL). The organic phase was dried over
anhydrous magnesium sulfate and evaporated to give a yellowish oil
(1.7 g, 81% yield). 1H NMR (CDCl3): δ 0.89 (t, 3H, J = 6.8 Hz), 1.34
(m, 6H), 1.56 (m 2H), 2.69 (m 5H), 4.01 (s, 2H), 7.28 (m, 5H). 13C
NMR (CDCl3): δ 14.07, 22.56, 28.26, 28.80, 31.56, 32.88, 45.97,
65.47, 127.25, 128.23, 128.68, 138.90. HRMS calcd for C14H23NS:
237.1551. Found: 237.1541.
Polymer from Entry 1 in Table 1. Piperazine (0.176 g, 2.05
mmol) was added to a stirred solution of (2,2′-oxidiethenthio)-
disuccinimide (0.71 g, 2.05 mmol) in CHCl3 (3.5 mL) and reacted at
room temperature for 24 h. The polymer was precipitated into
methanol (35 mL) and washed with methanol 3 times. The polymer
1
was dried under vacuum to yield a white powder. H NMR (CDCl3):
δ 2.86 (t, 4H, J = 6.8 Hz), 2.97 (s, 8H), 3.65 (t, 4H, J = 6.9 Hz). 13C
NMR (CDCl3): δ 32.92, 57.28, 70.10.
Polymer from Entry 2 in Table 1. N,N’-(Hexanethio)-
disuccinimide (3 g, 8.71 mmol) in CHCl3 (19 mL) was reacted
with piperazine (0.75 g, 8.71 mmol) following the previous procedure
to yield a white powder. 1H NMR (CDCl3): δ 1.40 (m, 4H), 1.56 (m,
4H), 2.69 (t, 4H, J = 7.5 Hz), 2.99 (s, 8H). 13C NMR (CDCl3): δ
28.62, 28.82, 32.17, 57.30.
Polymer from Entry 3 in Table 1. N,N’-(Hexanethio)-
disuccinimide (1 g, 2.90 mmol) in CHCl3 (5 mL) was reacted with
N,N′-diethylethylenediamine (0.34 g, 2.90 mmol) following the
previous procedure. The polymer was precipitated into methanol
(10 mL). The polymer was redissolved in a minimal amount of CHCl3
and precipitated into methanol (2 mL) to yield a yellowish oil after
Particle Uptake. Uptake of rhodamine labeled microparticles was
studied in JAWSII cells and HEK293 cells. Chambers were initially
coated with 300 μL of poly(L-lysine) (0.1% w/v) overnight. Following
coating, ∼1 × 104 cells were seeded per well and incubated overnight
at 37 °C in a humidified 5% CO2-containing atmosphere. Rhodamine-
labeled particles were added to the media at a concentration of 50 μg
per well. The particles were incubated with the cells for ∼18 h. Cells
were washed three times with sterile PBS and fixed with 4%
paraformaldehyde. The nucleus was stained with DAPI. Vectashield
mounting medium was added onto slide and sealed with coverslip. The
samples were imaged using LSM710 confocal microscope (Carl Zeiss
MicroImaging).
1
drying under vacuum. H NMR (CDCl3): δ 1.14 (t, 6H, J = 7.1 Hz),
1.40 (m, 4H), 1.54 (m, 4H), 2.60 (t, 4H, J = 7.4 Hz), 2.90 (q, 4H, J =
7.1 Hz), 3.05 (s, 4H). 13C NMR (CDCl3): δ13.99, 27.92, 28.91, 34.27,
53.23, 56.52.
Polymer from Entry 4 in Table 1. (2,2′-Oxydiethenthio)-
disuccinimide (0.625 g, 1.77 mmol) in CH2Cl2 (1.5 mL) was reacted
with N,N′-diethylethylenediamine (0.21 g, 1.77 mmol) following the
previous procedure. After evaporating the solvent, the polymer was
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of the water-soluble sulfenamide in Figure 9, kinetic
experiments, and degradation of sulfenamides. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail ned-bowden@uiowa.edu, Tel (319) 335-1198.
Notes
(30) Huynh, M. H. V.; White, P. S.; Meyer, T. J. Angew. Chem., Int.
Ed. 2000, 39, 4101.
The authors declare no competing financial interest.
(31) Huynh, M. H. V.; Morris, D. E.; White, P. S.; Meyer, T. J.
Angew. Chem., Int. Ed. 2002, 41, 2330.
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ACKNOWLEDGMENTS
■
N.B.B. gratefully thanks the NSF for generous funding (CHE-
0848162). A.K.S. gratefully acknowledges support from the
American Cancer Society (RSG-09-015-01-CDD), the National
Cancer Institute at the National Institutes of Health
(1R21CA13345-01/1R21CA128414-01A2/UI Mayo Clinic
Lymphoma SPORE), and the Pharmaceutical Research and
Manufacturers(PhRMA) Foundation. A.K.S. thanks the Core
Microscopy Research Facility for use of imaging equipment.
(37) Guarino, V. R.; Karunaratne, V.; Stella, V. J. Bioorg. Med. Chem.
Lett. 2007, 17, 4910.
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