Combined atom-transfer radical polymerization and ring-opening polymerization to design polymer-polypeptide copolymer conjugates toward self-aggregated hybrid micro/nanospheres for dye encapsulation

Article abstract of DOI:10.1002/pola.27713

A designed orthogonal dual initiator is employed to construct poly(methyl methacrylate)-block-polytyrosine copolymer conjugates via the combination of atom-transfer radical polymerization of methyl methacrylate, "click" chemistry and ring-opening polymerization of tyrosine-α-amino acid N-carboxyanhydride monomer. The polymer-polypeptide conjugate undergoes self-aggregation in dimethylformamide to produce hybrid micro/nanospheres owing to the formation of composite micelle as evidenced from field emission scanning electron microscopy and dynamic light scattering study. A simple solution-based approach is described to encapsulate an organic dye (Rhodamine-6G) into the aggregated hybrid micro/nanospheres.

Full text of DOI:10.1002/pola.27713

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Combined Atom-Transfer Radical Polymerization and Ring-Opening
Polymerization to Design Polymer–Polypeptide Copolymer Conjugates
Toward Self-Aggregated Hybrid Micro/Nanospheres for Dye
Encapsulation
Anupam Saha, Tapas K. Paira, Mrinmoy Biswas, Somdeb Jana, Sanjib Banerjee,
Tarun K. Mandal
Polymer Science Unit, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
Correspondence to: T. K. Mandal (E-mail: psutkm@iacs.res.in)
Received 8 April 2015; accepted 28 May 2015; published online 17 June 2015
DOI: 10.1002/pola.27713
KEYWORDS: aggregation; ATRP; bioconjuagte; biopolymers; diblock copolymers; micro/nanostructures; nanoparticles; polymer-
polypeptide; ROP
2
3,24
INTRODUCTION In present decades, interest in combining
polypeptides/peptides of precise chemical structure and
functionality with stabile and processable synthetic polymers
technique to synthesize different polypeptides.
In gen-
eral, polymer–peptide/polypeptide conjugates are synthe-
sized by “grafting from” technique instead of “grafting to”
involving the following two protocols: (1) it is the prepara-
tion of the sequence-defined peptide segment by solid/solu-
tion-phase route followed by its transformation into
macroinitiators for ATRP of various vinyl monomers to syn-
1
–3
has been growing rapidly.
The presence of the peptide
segment can introduce biocompatibility, bioactivity, and self-
4
–6
assembly property in such conjugate materials.
Although
the introduction of the synthetic polymeric component con-
trols the overall physical and chemical properties of the con-
2
5–27
thesize desired peptide–polymer conjuagtes,
(2) it is the
7
jugate materials. Such conjugate materials have potential
synthesis of amino acid NCAs followed by its ROP using
amino-terminated synthetic polymers to polypeptide–poly-
mer block copolymer conjugates. Thus, several different
techniques have been employed to synthesize polypeptide/
peptide–polymer conjugates of various polymers and
peptides and investigated their self-aggregation behavior in
8
9
wide-ranging biomedical, tissue engineering, biomaterial,
1
0
28
and drug-delivery applications. Furthermore, peptide–poly-
mer conjugates may enable them to self-assemble into func-
tional well-defined micro/nanostructured biomolecular
1
1,12
materials with great implications in both biological
and
1
3–15
29–31
31
nonbiological applications.
different solvents.
Nagai et al. used di-tert-butyltricar-
bonate to prepare NCAs and synthesized poly(a-amino
3
2
There have been tremendous developments in recent years
in the synthetic tool box available to polymer chemists with
the introduction of controlled radical polymerization (CRP),
which allows superior control over molecular weights,
acid)s. Lu et al. synthesized polytyrosine by ROP using pro-
pargyl amine as initiator and blended the polypeptide with
poly(4-vinylpyridine) in dimethylformamide (DMF) and
2
9
MeOH solutions. Klok et al.
synthesized (styrene) -b-
10
1
6–18
molecular weight distribution, and microstructure.
Con-
poly(c-benzyl-L-glutamate) conjugates via ROP of c-benzyl-L-
glutamate NCA using primary amine-terminated oligo(styr-
ene) as the initiator and studied the self-assembly of diblock
sequently, there has been easy access to block copolymers
that has huge importance from the standpoint of creating
1
9,20
30
nanostructured polymeric materials.
In particular, sev-
copolymers. Huang and Chang reported the synthesis of
eral groups including our group put emphasis on the use of
CRP techniques such as atom-transfer radical polymerization
stimuli-responsive poly(N-isopropylacrylamide)-b-polylysine
copolymer and investigated their self-aggregation behavior. It
has been reported by many groups that polymer-block-pep-
tide/polypeptides conjugate can easily undergo self-
(
ATRP) to prepare polypeptide/peptide-based block copoly-
1
6,21,22
mer conjugates.
It is also known that ring-opening
3
3,34
35
polymerization (ROP) of a-amino acid N-carboxyanhydride
NCA) initiated by nucleophiles or bases is the most common
aggregation into micelles,
vesicles, and different micro-
3
6
(
structures in both aqueous and nonaqueous medium. In
Additional Supporting Information may be found in the online version of this article.
2015 Wiley Periodicals, Inc.
V
C
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SCHEME 1 Synthesis pathways for PMMA-b-Ptyr block copolymer conjugate.
this context, previously, we reported the synthesis of conju-
gates comprising sequence-defined short peptide and poly-
mer using ATRP/ROP techniques and their self-aggregation
into micro/nanospheres in different polar organic sol-
The effect of copolymer compositions on the self-aggregation
behavior is also investigated. PMMA-b-Ptyr conjugates of
shorter PMMA blocks are soluble in alkaline water of pH
12.5, which further permits the study of its aggregation in
water. The formation of composite micellar structures is jus-
tified by dye encapsulation study of rhodamine-6G (R6G), a
model dye, into the aggregated PMMA-b-Ptyr microspheres
via fluorescence microscopy and time-correlated single-
photon counting (TCSPC) technique.
2
6,37
vents.
We thought that it would be interesting to synthe-
size polymer–polypeptide conjugates and to study their self-
aggregation behavior in different solvents.
Herein, we report the synthesis of poly(methyl methacry-
late)-b-polytyrosine (PMMA-b-Ptyr) copolymer conjugate
from a designed orthogonal dual initiator via the combina-
tion of ATRP of methyl methacrylate (MMA), “click” chemis-
try and ROP of tyrosine–NCA. We further investigate the
self-aggregation behavior of the as-synthesized PMMA-b-Ptyr
bioconjugate in DMF, which exhibits the formation of
micron/nanometer-sized spherical aggregated structures as
examined through field emission scanning electron
microscopy (FESEM) and dynamic light scattering (DLS).
RESULTS AND DISCUSSION
Synthesis of PMMA-b-Ptyr Conjugate
Our approach to synthesize the copolymer consisting of
PMMA and Ptyr blocks involved the following steps: (1) syn-
thesis of azide end-functional orthogonal ATRP initiator fol-
lowed by the polymerization of MMA to synthesize azide-
terminated PMMA (PMMA-N ) (Step I of Scheme 1), (2)
3
2
314
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toward the ROP of tyrosine–NCA and to prepare the conjugates of
varying lengths of PMMA blocks. The average molecular weights
of the obtained PMMA-N -I, PMMA-N -II, and PMMA-N -III were
3
3
3
found to be 7.0, 4.2, and 3.0 kDa with low polydispersity indices
PDIs) of 1.35, 1.22, and 1.25, respectively (chromatograms; Sup-
(
porting Information Table S1 and Figure S3), indicating the con-
trolled nature of the polymerization. It should be noted that there
was a little deviation from the theoretically calculated molecular
weights and the experimentally observed molecular weights
(Supporting Information Table S1).
Furthermore, in the second step, PMMA-N s were coupled
3
with propargyl amine via “click” reaction to produce PMMA-
NH2 (Scheme 1), an active macroinitiator for growing poly-
tyrosine through ROP of tyrosine–NCA. As shown in Figure 1,
there was
a
complete disappearance of the band at
2
1
2104 cm for azide group in the FTIR spectrum of a repre-
sentative sample (PMMA-NH -I), indicating complete transfor-
2
mation of PMMA-N to PMMA-NH .
3
2
To afford the synthesis of PMMA-b-Ptyr conjugates, in the
third step, we first prepared tyrosine–NCA from ring closing
of tyrosine using triphosgene as shown in Step III of
Scheme 1. FTIR (Fig. 1) and ESI-mass spectrum (Supporting
Information Fig. S4) established the formation of tyrosine–
NCA. The ROP of tyrosine–NCA was then performed with
2
FIGURE 1 FTIR spectra of PMMA-NH -I, PMMA-b-Ptyr-I conju-
gate, and tyrosine–NCA.
conversion of azide-terminated PMMA to amine-end func-
tional PMMA (PMMA-NH ) via the Huisgen’s 1,3-dipolar cyclo-
2
PMMA-NH s of varying block lengths as macroinitiators to
2
addition (Step II of Scheme 1), (3) ROP of tyrosine–NCA from
the as-synthesized macroinitiator PMMA-NH2 to obtain
PMMA-b-Ptyr (Step III of Scheme 1) conjugate. First, we pre-
pared 2-azidoethanol by the reaction of 2-chloroethanol with
sodium azide followed by its conversion into 2-azidoethyl 2-
bromoisobutyrate (ABIB) via coupling reaction with a-
obtain PMMA-b-Ptyr conjugates of different molecular
weights. FTIR spectra (Fig. 1) showed that the band of amide
2
1
group of NCA ring at 1765 cm
1
was totally shifted to
656 cm (amide I for polypeptide segment), whereas the
21
2
1
band of ACOOCH group of PMMA at 1730 cm remained
3
unaltered in PMMA-b-Ptyr-I conjugate, which indicated the
formation of Ptyr block at the end of PMMA block.
bromoisobutyryl bromide (Step I of Scheme 1). The obtained
1
2
-azidoethanol and ABIB were characterized by FTIR and H-
1
NMR (Supporting Information Figs. S1 and S2). ATRP of MMA
was then performed using ABIB initiator to synthesize
H-NMR spectrum (Supporting Information Fig. S5) of
PMMA-b-Ptyr-I revealed signals at d 5 3.545 ppm for methyl
ester protons of PMMA, 6.62–6.68 and 7.02–7.07 ppm for
aromatic protons of tyrosine ring, 7.935 ppm for tyrosinate
proton, and 9.26–9.34 ppm for amide protons of Ptyr along
with all expected signals which confirmed the attachment of
Ptyr block with PMMA block. We also observed similar sig-
nals for other two conjugates (PMMA-b-Ptyr-II and PMMA-b-
Ptyr-III) (Supporting Information Fig. S6). Table 1 lists the
molecular masses of all three conjugates calculated
from proton integration value of tyrosine and MMA units
PMMA-N
istic bands at 2104 and 1730 cm
stretching frequencies of azide and ester groups, respectively
Supporting Information Fig. S1). We finally synthesized three
samples namely, PMMA-N -I, PMMA-N -II, and PMMA-N -III of
3
. The FTIR spectrum of PMMA-N
3
showed character-
21
corresponding to the
(
3
3
3
different molecular weights simply by varying the monomer-to-
initiator ([M] /[I] ) ratio as listed in Supporting Information
o
o
Table S1. Our intention was to check the relative reactivities of
these macroinitiators (after transforming into PMMA-NH2)
TABLE 1 ROP Conditions of Tyrosine–NCA Using PMMA-NH
2
Macroinitiator for PMMA-b-Ptyr Conjugates and Their Molecular
a
Weight Analysis Data
b
Sample Name
Macroinitiator
[M]
o
/[I]
o
Conv. (%)
Mn, theo. (kDa)
Mn, NMR (kDa)
Mn,GPC (kDa)
PDI
PMMA-b-Ptyr -I
PMMA-b-Ptyr -II
PMMA-b-Ptyr -III
PMMA-NH
PMMA-NH
PMMA-NH
2
2
2
-I
-II
-III
45
60
60
50
62
50
11.05
10.95
8.45
12.2
15.9
12.9
11.8
1.30
–
–
–
–
a
b
Conditions: [I]
o
5 [PMMA-NH
2
-I]
0
5 1 wt % (w/v); solvent 5 DMF; tem-
n
The calculation of M , NMR is given in page S6 of Supporting Informa-
perature 5 room temperature; reaction time 5 72 h.
tion material.
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The GPC trace of PMMA-b-Ptyr-I was unimodal and sym-
metrical, the analysis of which gave a Mn of 11.8 kDa and
PDI of 1.30 (Fig. 2). Figure 2 clearly also reveals a shift of
GPC trace of PMMA-b-Ptyr-I toward higher molar mass
region which accounts for 4.8 kDa, compared to that of
PMMA-N -I after ROP of tyrosine–NCA with PMMA-NH -I
3
2
macroinitiator. This further indicated the formation of
PMMA-b-Ptyr block copolymer. For PMMA-b-Ptyr-I sample,
there was a quite good agreement between the Mn meas-
ured from GPC and calculated from the aforementioned
NMR (Table 1 and Table S2). However, we were unable to
calculate M s of PMMA-b-Ptyr-II and PMMA-b-Ptyr-III,
n
where the Ptyr block lengths are comparatively higher than
that in PMMA-b-Ptyr-I. This difficulty may arise owing to
the increase of interaction among Ptyr block of PMMA-b-
3
2
n
Ptyr conjugate. Therefore, instead of M , GPC, we pro-
vided their molecular weights as the measurement from
1
H-NMR analysis (Table 1).
Self-Aggregation Behavior of PMMA-b-Ptyr Conjugates
It is well known that polymer–peptide/polypeptide bioconju-
gates have very high tendency toward self-aggregation in
3
9,40
solution.
Thus, we explored the self-aggregation behavior
of the as-synthesized PMMA-b-Ptyr conjugate in DMF. The
FESEM images (Fig. 3) of PMMA-b-Ptyr-1 revealed the for-
mation of micro/nanospheres in DMF. To check the morphol-
ogy evolution with time, we also examined DMF solution of
PMMA-b-Ptyr-1 conjugate, drop-casted at different time
intervals of 30 min, 4 h, and 24 h, respectively, via FESEM.
We observed the formation of self-aggregated micro/nano-
spheres with an average diameter of 121, 345, and 750 nm
for the sample drop-casted after at 30 min, 4 h, and 24 h,
respectively (Fig. 3), indicating the increment of size with
time. The histogram of particle size distributions at different
time intervals is also shown in Supporting Information
Figure S7.
3
FIGURE 2 GPC traces of PMMA-N -I and PMMA-b-Ptyr-I
conjugate.
1
from H-NMR spectra using the equation given in the
3
8
Supporting Information material. For the calculation of
M , we determined the number of MMA units present in all
n
three PMMA-N3s from gel permeation chromatography
1
(
GPC) data instead of H-NMR data, which is the same as
number of MMA units in the macroinitiator (Supporting
Information material). Because of the reason that we were
unable to find any distinguished signal of PMMA macroini-
tiator in all the conjugates, the obtained molecular weight
To provide further support on self-aggregation of PMMA-b-
Ptyr conjugate, we examined the DMF solution of the PMMA-
b-Ptyr-I via DLS at different time intervals. The hydrody-
1
of the conjugate from H-NMR data is actually average
molecular weight which is correlated with the theoretically
calculated value (Table 1).
h
namic diameter (D ) of PMMA-b-Ptyr-I was found to be
FIGURE 3 FESEM images of the self-aggregated PMMA-b-Ptyr-I conjugates in DMF (1 mg/mL) after keeping the solution undis-
turbed for different times (A) 30 min, (B) 4 h, and (C) 24 h.
2
316
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SCHEME 2 Schematic representation for the formation of composite micelle in DMF. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
3
31 nm after 30 min of aggregation (Supporting Information
cases of PMMA-b-Ptyr-II and PMMA-b-Ptyr-III conjugates,
they assembled to a greater extent probably through hydro-
gen bonds between the OH groups of Ptyr block and the
carbonyl groups of PMMA block that led to flake-like mor-
phology. Similar types of interaction were also highlighted in
the case of blend of Ptyr and poly(4-vinylpyridine), leading
to the b-sheet type of secondary structures reported by Lu
Fig. S8). But, the D s were unambiguously increased to 512
h
and 941 nm when we kept the conjugate MMF solution
undisturbed for 4 and 24 h, respectively (Supporting Infor-
mation Table S3 and Figure S8). Thus, DLS data showed a
good correlation with that obtained from FESEM analysis.
Both the results of FESEM and DLS showed the increase of
size of the formed micro/nanospheres upon increasing time
of self-aggregation. Based on this observation and also from
32
et al. Furthermore, at the optimum pH of ꢀ12.5, PMMA-b-
Ptyr-II and PMMA-b-Ptyr-III conjugates were soluble in
water owing to the presence of tyrosinate ion in the Ptyr
moiety, but remain strongly aggregated and exhibit indistin-
guishable aggregated morphology (Supporting Information
Figs. S9A and S9B). The exact reason for the formation of
such nanostructure may be owing to the formation of Na
salt of Ptyr block. This results in strong ionic interaction
among the Ptyr block of the conjugate as observed by Guo
2
6,41
the literature survey including our earlier study,
we can
assume that the bigger spheres are nothing but composite or
giant micelles, composed of initially formed unit micelles of
PMMA-b-Ptry block copolymer. The formation of such com-
posite structure is owing to the further secondary interac-
tions among unit micelles via H-bonding of amide groups
between polypeptide chains as shown in Scheme 2. Unfortu-
nately, we were unable to identify unit micelles experimen-
tally, but we believe that the formation unit micelle is the
first and necessary step for the generation of higher order
composite micellar structure. Such unit micelle was formed
because of the difference in polarity between PMMA and
Ptyr chain with respect to DMF where Ptyr chain stretches
toward the solvent front and PMMA chain remain inside the
core of micelle (Scheme 2). We assume that the high interac-
tion between the polytyrosine chains may be responsible for
the formation of composite micelle at a faster rate by the
combination of unit micelles. This is probably the reason for
not observing any unit micelles experimentally.
42
et al. in the case of poly(ethylene glycol)-b-poly(aspartic
acid) conjugate.
Dye Encapsulation Study of Hybrid Micro/Nanospheres
To further investigate the mechanism of aggregation process,
we studied the aggregation behavior in the presence of R6G
dye. The fluorescence microscopic image of dye-encapsulated
spherical composite micellar aggregates was compared with
that of neat R6G dye solution (Supporting Information Fig.
S10). It was very clear that there was a nice encapsulation
of R6G molecules into the aggregated composite micelles
during the time of aggregation of PMAA-b-Ptyr-I conjugate in
DMF. From this study, we can further claim that the Ptyr
blocks, which expose toward the solvent, eventually help the
formation of composite micelles via secondary interaction
and PMMA blocks reside in the core during aggregation
Interestingly, the other two conjugates (PMMA-b-Ptyr-II and
PMMA-b-Ptyr-III) containing shorter hydrophobic PMMA
block were soluble in alkaline water (pH 5 12.5) owing to
the formation of phenolate ion. This provided us the oppor-
tunity to study the aggregation of these conjugates in water
along with that study in DMF solution. The aggregated mor-
phology of PMMA-b-Ptyr-II and PMMA-b-Ptyr-III conjugates
in DMF was completely different to that of PMMA-b-Ptyr-I in
DMF (Supporting Information Fig. S9). These results evi-
denced that the composition of PMMA and Ptyr block in the
conjugate also affected the aggregation pattern in solution.
As the Ptyr segment length is comparatively higher in the
(
Scheme 2).
For further clarification on the encapsulation of R6G dye
molecules into the aggregated micro/nanospheres, we also
performed time-resolved fluorescence study of the R6G-
loaded spheres and neat R6G in DMF using TCSPC. The time-
resolved multiexponential fluorescence decay curves of neat
R6G and R6G-encapsulated spheres were fitted according to
the following equation and shown in Supporting Information
Figure S11.
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ꢀ
ꢁ
N
X
EXPERIMENTAL
2
t
IðtÞ5
Aiexp
si
i51
Materials
MMA,
a-bromoisobutyryl
bromide,
propargylamine,
where A and s are the relative amplitude and lifetime of the
0
00 00
i
i
N,N,N ,N ,N -pentamethyldiethylenetriamine (PMDETA), R6G,
anhydrous copper(I) bromide (CuBr), and anhydrous cop-
per(I) chloride were purchased from Sigma-Aldrich. 2-Chloro
i-th fluorescence component, respectively. N is the number of
the fluorescence exponentials required for best nonlinear
least squares fitting of the fluorescence decay curves. Aver-
age fluorescence lifetime (s) was calculated from the decay
ethanol and sodium azide (NaN ) were purchased from
3
Merck, India. L-Tyrosine was received from SRL. All the
chemicals were used as received unless otherwise men-
tioned. MMA was distilled under reduced pressure after
washing with 5 wt % of aqueous NaOH prior to use. All the
solvents were distilled and dried prior to use. All aqueous
solutions were prepared with Milli-Q water.
times (s ) and the relative amplitudes (a ) using the following
i
i
relationship
<
s > 5s1a11s2a21s3a3
where s , s , and s are the component lifetimes and a , a ,
1
2
3
1
2
and a are relative amplitudes, respectively.
3
Synthesis of Azide End-Functional PMMA (PMMA-N3)
In a typical procedure, the molar ratio of the reactants is as
follows: Monomer:Initiator:CuCl:PMDETA 5 40:1:1:1. For
Multiexponential fluorescence decay curves showed the aver-
age life time of 3.7 ns for neat R6G, but an average life time
of 3.978 ns in the case of R6G-encapsulated micro/nano-
spheres (Supporting Information Table S4). Increase in aver-
age life time for R6G-encapsulated spheres may arise owing
to the interaction between dye molecules and conjugate mol-
ecules present in hybrid spheres. Such type of interaction
may be ascribed from either H-bonding or dipole–dipole
interaction between dye molecules and amide groups of Ptyr
segment which stretch toward the periphery of micelles in
solution.
PMMA-N
3
synthesis, typically, ABIB initiator (110.78 mg,
0.51 mmol) (for synthesis, see page S1 of Supporting Infor-
mation ESI) and copper(I) chloride (50.56 mg, 0.51 mmol)
were added to 3 mL of dry xylene in an oven-dried 25-mL
long-necked round-bottomed flask. The reaction mixture was
stirred and purged with N gas for 30 min. PMDETA (106.6
2
mL, 0.51 mmol) was then added to the round-bottomed flask
and again purged with N gas for 15 min until the solution
2
became green in color. The mouth of the long-necked round-
bottomed flask was carefully sealed with rubber septum
after purging the reaction mixture and MMA (2.17 mL, 20.4
mmol) was injected via syringe through rubber septum. The
reaction is schematically represented in Scheme 1. The
whole reaction mixture was shifted to an oil-bath preheated
to 90 8C and stirred for 48 h. After 48 h, the polymerization
reaction was quenched by adding excess of THF and copper
catalyst was carefully removed by passing the reaction mix-
ture through basic alumina column using THF as the eluent.
After being concentrated at rotary evaporator, the solution
was precipitated into large amount of n-hexane and dried in
vacuum oven at 50 8C for overnight to yield white product of
CONCLUSIONS
We demonstrated the synthesis of PMMA-b-Ptyr copolymer
conjugates of varying lengths of PMMA and Ptyr blocks via
the combination of ATRP of MMA, “click” chemistry and ROP
of tyrosine–NCA monomer. The GPC traces of azide end-
functional PMMAs showed unimodal distribution with nar-
row polydispersities. The single peak in the GPC trace of
PMMA-b-Ptyr-I conjugate confirmed that there was no any
macroinitiator (PMAM-NH 21) remained after the comple-
2
tion of ROP of tyrosine–NCA. The relative compositions of
MMA and Tyr units were determined from H-NMR analysis.
1
43
PMMA-N (yield 5 1.988 g, 97%). Finally, PMMA-N of dif-
3
3
The self-aggregation of PMMA-b-Ptyr conjugate molecules in
DMF resulted in the formation of micro/nanospheres as
examined by FESEM and DLS study. The hybrid micro/nano-
spheres were actually the composite micelles formed by the
aggregation of unit micelles of PMMA-b-Ptyr conjugate mole-
cules owing to the difference in polarity of PMMA and Ptyr
block, resulting in difference in solubility in DMF. The
PMMA-b-Ptyr conjugates of longer Ptyr block lengths were
soluble in alkaline water (pH 5 12.5), which, upon standing,
formed aggregated structures of indistinguishable morphol-
ogy. The addition of an organic dye, R6G, during the aggrega-
tion of conjugates in DMF resulted in the formation of dye-
encapsulated hybrid micro/nanospheres, which further
ensured the aggregation of conjugate molecules. The dye
encapsulation into micro/nanospheres was observed though
fluorescence microscopy and average life-time measurement
from TCSPC experiment. These polymer–polypeptide hybrid
micro/nanospheres can be utilized in the field of delivery of
dyes and drugs.
ferent molecular weights (Supporting Information Table S1)
were successfully synthesized by the variation of ratio of
[
M ]/[I ] using same protocol.
0 0
Synthesis of Amino End-functional PMMA (PMMA-NH2)
The PMMA-NH was synthesized via copper-catalyzed azide–
2
alkyne click reaction (CuAAC) with propargyl amine and
PMMA-N3 as shown in Scheme 1. In typical experiment,
PMMA-N -I (300 mg, 0.05 mmol) and CuBr (21.52 mg, 0.15
3
mmol) were dissolved in 5 mL of dry THF with slow stirring
in a two-necked round-bottomed flask whose mouth was
attached with N gas balloon. The solution was purged with
2
N2 gas for 15 min, PMDETA (32 mL, 0.15 mmol) was then
added to solution. The round-bottomed flask containing the
reaction mixture was carefully degassed via three freeze–
pump–thaw cycles to remove any traces of oxygen and was
tightly sealed with a rubber septum. Propargyl amine (11.5
mL, 0.18 mmol) was then injected into the solution by a
syringe and stirred for 24 h at room temperature. The dark
2
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green copper complex was removed by passing the reaction
mixture through basic alumina column and the brown prod-
uct was isolated by precipitation into hexane (yield 5 75%).
15 A. K. Shaytan, E.-K. Schillinger, P. G. Khalatur, E. Mena-
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Synthesis of PMMA-b-Ptyr Conjugate
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Typically, tyrosine–NCA (0.16 g, 0.775 mmol) (Supporting
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neck round-bottomed flask. Sequentially, PMMA-NH2-I
1
8 S. Y. Kim, C. F. Zukoski, Macromolecules 2013, 46, 6634–
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643.
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9 T. Smart, H. Lomas, M. Massignani, M. V. Flores-Merino, L.
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120.6 mg) was dissolved in 2 mL of dry DMF and was
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The reaction mixture was degassed via three freeze–pump–
thaw cycles and stirred for 3 days at room temperature. At
the end of complete reaction, the brown product was iso-
lated by precipitation into diethyl ether and dried in vacuum
oven at 35 8C (yield 5 50%). Two other PMMA-b-Ptyr conju-
gates were also prepared by varying PMMA and Ptyr compo-
sition as summarized in Table 1. The synthesized and
purified PMMA-b-Ptyr conjugates were characterized by
NMR, FTIR, and GPC analyses.
2
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ACKNOWLEDGMENTS
Silberring, A. Dworak, B. Trzebicka, J. Polym. Sci. Part A:
Polym. Chem. 2012, 50, 3104–3115.
A. Saha sincerely thanks CSIR, India, for providing fellowship.
This research is supported by grants from the CSIR, New Delhi,
India. Thanks are also owing to the partial financial support
from BRNS, India.
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