Synthesis of amphiphilic block copolymer consisting of glycopolymer and poly(l-lactide) and preparation of sugar-coated polymer aggregates

Article abstract of DOI:10.1002/pola.28401

The block glycopolymer, poly(2-(α-d-mannopyranosyloxy)ethyl methacrylate)-b-poly(l-lactide) (PManEMA-b-PLLA), was synthesized via a coupling approach. PLLA having an ethynyl group was successfully synthesized via ring-opening polymerization using 2-propyn-1-ol as an initiator. The ethynyl functionality of the resulting polymer was confirmed by MALDI-TOF mass spectroscopy. In contrast, PManEMA having an azide group was prepared via AGET ATRP using 2-azidopropyl 2-bromo-2-methylpropanoate as an initiator. The azide functionality of the resulting polymer was confirmed by IR spectroscopy. The Cu(I)-catalyzed 1,3-dipolar cycloaddition between PLLA and PManEMA was performed to afford PManEMA-b-PLLA. The block structure was confirmed by ¹H NMR spectroscopy and size exclusion chromatography. The aggregating properties of the block glycopolymer, PManEMA_16k-b-PLLA_6.4k (M_n,PManEMA = 16,000, M_n,PLLA = 6400) was examined by ¹H NMR spectroscopy, fluorometry using pyrene, and dynamic light scattering. The block glycopolymer formed complicated aggregates at concentrations above 21 mg·L^?1 in water. The d-mannose presenting property of the aggregates was also characterized by turbidimetric assay using concanavalin A.

Full text of DOI:10.1002/pola.28401

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Synthesis of Amphiphilic Block Copolymer consisting of Glycopolymer
and Poly(L-lactide) and Preparation of Sugar-coated Polymer Aggregates
Makoto Obata,1 Ryota Otobuchi,1 Tadao Kuroyanagi,1 Masaki Takahashi,1 Shiho Hirohara2
1Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-4-37 Takeda, Kofu, 400-8510, Japan
2Department of Chemical and Biological Engineering, National Institute of Technology, Ube College, 2-14-1 Tokiwadai, Ube,
755-8555, Japan
Correspondence to: M. Obata (E-mail: mobata@yamanashi.ac.jp)
Received 27 September 2016; accepted 15 October 2016; published online 00 Month 2016
DOI: 10.1002/pola.28401
ABSTRACT: The block glycopolymer, poly(2-(a-D-mannopyrano-
syloxy)ethyl methacrylate)-b-poly(L-lactide) (PManEMA-b-PLLA),
was synthesized via a coupling approach. PLLA having an ethy-
nyl group was successfully synthesized via ring-opening poly-
merization using 2-propyn-1-ol as an initiator. The ethynyl
functionality of the resulting polymer was confirmed by
MALDI-TOF mass spectroscopy. In contrast, PManEMA having
an azide group was prepared via AGET ATRP using 2-
azidopropyl 2-bromo-2-methylpropanoate as an initiator. The
azide functionality of the resulting polymer was confirmed by
IR spectroscopy. The Cu(I)-catalyzed 1,3-dipolar cycloaddition
between PLLA and PManEMA was performed to afford PMa-
nEMA-b-PLLA. The block structure was confirmed by 1H NMR
spectroscopy and size exclusion chromatography. The aggre-
gating properties of the block glycopolymer, PManEMA16k-b-
PLLA6.4k (Mn,PManEMA 5 16,000, Mn,PLLA 5 6400) was examined
by 1H NMR spectroscopy, fluorometry using pyrene, and
dynamic light scattering. The block glycopolymer formed com-
plicated aggregates at concentrations above 21 mgÁL21 in
water. The D-mannose presenting property of the aggregates
was also characterized by turbidimetric assay using concanava-
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2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A:
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KEYWORDS: atom transfer radical polymerization (ATRP); block
copolymers; ring-opening polymerization
been examined as a hydrophilic segment of amphiphilic
block copolymers for DDS applications.9–11 Thanks to the
recent development of a controlled radical polymerization
(CRP) technique, various block vinyl glycopolymers having
hydrophobic,12–23 hydrophilic,24–28 cationic,29–41 thermosen-
sitive,42,43 and other functional44,45 segments have been syn-
thesized by a chain extension strategy in atom transfer
radical polymerization (ATRP)14,20,22,23,28,36,38 and reversible
addition and fragmentation chain transfer (RAFT) polymeriza-
tion.12,13,15–19,21,24–35,37,39–45 For example, Su et al. synthesized
polystyrene-b-poly(4-(a-D-mannopyranosyloxymethyl)styrene)
via RAFT polymerization, followed by the deprotection of
the carbohydrate moieties.17 Their block glycopolymers
formed micelles, with glycopolymer segments located on
their outer surfaces (called glyco-outside micelles).
INTRODUCTION Polymer micelles are promising vehicles for
drug delivery system (DDS).1–3 Amphiphilic block copoly-
mers form nanoparticles with core-shell type microstruc-
tures. Polyethylene glycol (PEG) is the de facto standard, as a
non-ionic hydrophilic segment used in various types of
amphiphilic polymers.4 The PEG segment affords not only
steric stabilization of nanoparticles but also stealth behavior
from the reticuloendothelial system to give prolonged blood
circulation. However, some drawbacks and limitations of PEG
have been recognized in DDS applications.5 The PEG segment
dramatically decreases the interaction with blood compo-
nents. However, an immunological response to PEG segments
is frequently recognized, and the mechanism is not fully
understood. Non-biodegradability, a lack of homing ability,
and interference in cellular uptake are also drawbacks of
PEG segments in DDS applications. Therefore, PEG alterna-
tives have been intensively explored by many researchers.
In contrast, block glycopolymers consisting of non-vinyl poly-
mers, such as biodegradable polyester or polyamide seg-
ments, are relatively rare. In the synthesis of such block
glycopolymers, the synthetic strategies are roughly divided
into three categories: (1) macromolecular initiator or chain
transfer agent (CTA) approach; (2) (orthogonal) bifunctional
Synthetic polymers bearing carbohydrates on the side chain
are known as glycopolymers, which are an important class
of biomimetic materials.6–9 Recently, glycopolymers have
Additional Supporting Information may be found in the online version of this article.
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initiator for CRP and ring-opening polymerization (ROP)
approach, and (3) polymer-coupling approach. Block glycopoly-
mers having PEG,46,47 polypeptide,48 poly(e-caprolactone),49–52
and poly(ethylene-alt-propylene)53 segments were synthesized
by the first approach. In this approach, an initiating functional-
ity for ATRP or a CTA functionality for RAFT polymerization is
introduced to the chain end. However, it is frequently difficult
or impossible to confirm and quantify the extent of post-
polymerization modification by conventional spectroscopic and
chromatographic techniques, due to very low concentrations of
the end group. The second approach is a variation on the first
approach, but has the advantage of being able to eliminate
post-polymerization modification to install an initiator or CTA
functionality. This approach used molecules having two func-
tionalities to initiate two different types of polymerizations,
such as CRP and ROP. Ideally, these two functional groups do
not interfere in either polymerization; this feature is known as
“orthogonal.” If one functionality interferes with the other, one
side may be inactivated by protecting groups in the first poly-
merization. Block glycopolymers having poly(L-lactide)
(PLLA),54,55 poly(e-caprolactone),56 and polystyrene57 were
synthesized by using this strategy. For example, Ganda et al.
synthesized block glycopolymers having poly(e-caprolactone)
by using benzyl 2-hydroxyethyl carbonotrithioate as a bifunc-
tional initiator.56 The first and second approaches consist of
CRP followed by ROP. Hence, the polymer synthesized in the
first polymerization must dissolve in the second polymeriza-
tion medium and must not interfere with the second polymeri-
zation. These requirements are sometimes difficult to satisfy in
the synthesis of block glycopolymers consisting of two seg-
ments having very different solubilities, such as unprotected
glycopolymer and polyester or polyamide. Even though the
protected glycopolymer may have solubility similar to those of
polyether or polyamide, the deprotection of the glycopolymer
can degrade the biodegradable segments.
recrystallized from toluene just before use. 2-Propyn-1-ol
was purchased from Tokyo Chemical Industry and distilled
under reduced pressure just before use. Tin(II) 2-
ethylhexanoate (Sn(Oct)2) was purchased from Wako Pure
Chemical Industries, (Osaka, Japan) and used as received.
2,5-Dihydroxybenzoic acid (DHBA) and polyethylene glycol
monomethyl ether (MPEG) (Mn 5 2000) were purchased
from Sigma-Aldrich (MO) and used as received. 2-(a-D-Man-
nopyranosyloxy)ethyl methacrylate (ManEMA) was prepared
according to our previous paper.59 1H and 13C{1H} NMR
spectra were recorded using an AVANCE III HD instrument
(500 MHz; Bruker Biospin K.K., Yokohama, Japan). IR spectra
were recorded on an IRAffinity-1S instrument (Shimadzu,
Kyoto, Japan) using a KBr pellet for PManEMA or a NaCl
disk for 2-azidopropyl 2-bromo-2-methylpropanoate. Size
exclusion chromatography (SEC) was performed with an
HPLC system (pump, LC-20AT; refractive index detector, RID-
10A, Shimadzu Co.) using Styragel HR4 (7.8 3 300 mm)
(Waters, MA), Styragel HR3 (7.8 3 300 mm), and Styragel
HR1 (7.8 3 300 mm) columns as a stationary phase and
N,N-dimethylformamide (DMF) containing 50 mM LiBr as a
mobile phase at a flow rate of 0.5 mL min21. The SEC sys-
tem was calibrated with
5 poly(methyl methacrylate)
(PMMA) standards (Showa Denko, K.K., Tokyo, Japan) rang-
ing in molecular weight from 2.89 to 202 kgÁmol21. The
number average molecular weights (Mns) and polydisper-
sities (Mw/Mns) were calculated by PMMA calibration.
Matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectra were recorded on a Voyager-
DETM PRO (Applied Biosystems, CA) operated in reflection
mode with a 20 kV acceleration voltage. Sample solution for
the MALDI-TOF mass spectrometry were prepared by mixing
5 lL of tetrahydrofuran (THF) solution of polymer (10
mgÁmL21) and 5 lL of THF solution of DHBA (30 mgÁmL21).
MPEG (Mn 5 2000) was used as an internal standard. The
size distribution of block glycopolymer aggregates was deter-
mined by dynamic light scattering measurements (UPA-
UT151; NIKKISO, Tokyo, Japan).
The third approach is usually very inefficient because of the
low concentration of polymer end groups. However, recent
development of a highly efficient and selective coupling meth-
odology, known as “click chemistry,” opens this route to the
synthesis of block glycopolymers. For example, Glassner et al.
synthesized PEG-b-poly(3-O-acryloyl-D-glucose) by hetero Diels-
Alder between cyclopentene-functionalized PEG and poly(3-O-
acryloyl-D-glucose) having a 2-pyridylthiocarboylthio group at
the terminating end, prepared by RAFT polymerization.58 Their
coupling reaction was ultra-fast (the coupling was completed
within 15 min) and catalyst-free. In this article, an amphiphilic
block glycopolymer consisting of PLLA as a hydrophobic seg-
ment and poly(2-(a-D-mannopyranosyloxy)ethyl methacrylate)
(PManEMA) was synthesized by a polymer-coupling approach
using Cu(I)-catalyzed 1,3-dipolar cycloaddition. In addition, the
micellar formation and lectin-binding ability of the resulting
block glycopolymer were examined.
2-Azidopropyl 2-Bromo-2-Methylpropanoate
The initiator, 2-azidopropyl 2-bromo-2-methylpropanoate,
was prepared according to the literature.60 Briefly, 3-bromo-
1-propanol (6.21 g, 45 mmol), NaN3 (5.05 g, 78 mmol), and
water (80 mL) were placed into a 200 mL flask equipped
with a thermometer and a condenser. The solution was
stirred at 80 8C for 30 h. After the solution was cooled
down, it was concentrated to a volume of approximately
30 mL under reduced pressure. The solution was saturated
with NaCl, and then extracted with toluene (200 mL 3 4).
After the extract was dried over anhydrous Na2SO4, the tolu-
ene solution was concentrated to a volume of approximately
30 mL under reduced pressure to give a toluene solution
(31.5 g) of 3-azido-1-propanol. The concentration of 3-azido-
1-propanol was determined to be 9.2 wt % by 1H NMR
spectroscopy.
EXPERIMENTAL
Materials and Analytical Techniques
All chemicals were of analytical grade. L-Lactide was pur-
chased from Tokyo Chemical Industry (Tokyo, Japan) and
The toluene solution of 3-azido-1-propanol (9.2 wt %,
7.70 g, 7.0 mmol) and triethylamine (1.10 g, 10 mmol) were
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placed into a 50 mL flask, which was sealed tightly with a
rubber septum. The solution was cooled in an ice bath, and
then 2-bromopropionyl bromide (1.2 mL, 11 mmol) was
added dropwise. The solution was stirred at ambient tem-
perature overnight. To the solution was added a portion of
water to quench the reaction. The resulting mixture was
extracted with toluene (100 mL) and washed with 1 M
NaOH (aq.), 1 M HCl (aq.), sat. NaHCO3 (aq.), water, and
brine. After drying over anhydrous Na2SO4, the solvent was
removed under reduced pressure. The crude product was
purified by column chromatography (silica gel, ethyl acetate/
hexane 5 2/8, v/v), followed by preparative gel permeation
chromatography to give a colorless oil. Yield, 96.7 mg (14%).
crude product was purified by dialysis with deionized water
for more than 3 days. After lyophilization, PManEMA was
obtained as
a white powder. Yield, 62.1 mg (72%).
Mn 5 16,000, Mw/Mn 5 1.24 (PMMA std.).
Click Synthesis of PManEMA-b-PLLA
A typical procedure for the coupling reaction was as follows:
PManEMA (Mn 5 16,000) (29.2 mg, 1.83 lmol), PLLA
(Mn 5 4500) (10.8 mg, 2.40 lmol), pentamethyldiethylenetri-
amine (PMDETA) (11.0 mg, 63.5 lmol), and dry DMF (1 mL)
were placed into a Schlenk tube. The tube was degassed
with several freeze-pump-thaw cycles. CuBr (2.40 mg, 16.7
lmol) was added to the tube. The tube was immersed into a
water bath thermostated at 25 8C, and stirred for 24 h. After
removal of the solvent under reduced pressure, the residue
was dissolved in water and dialyzed with deionized water
for more than 3 days. After lyophilization, the coupling prod-
uct was obtained as a white powder. Yield, 18.5 mg (46%).
1H NMR (500.1 MHz, CDCl3, Si(CH3)4 5 0 ppm):
d
(ppm) 5 4.28 (t, 3J 5 6.0 Hz, 2H, CH2AOA), 3.45 (t, 3J 5 6.8
Hz, 2H, CH2AN3) 1.97 (m, 2H, CH2), 1.94 (s, 6H, CH3).
13C{1H} NMR (125.8 MHz, CDCl3, CDCl3 5 77 ppm):
d
(ppm) 5 171.5 (C@O), 62.7 (CH2AO), 55.6 (C), 48.0 (CH2),
30.7 (CH3), 27.9 (CH2AN3). IR (NaCl disk): mmax
(cm21) 5 3200–2850 (br), 2100 (s, N3), 1736 (s, C@O).
Critical Aggregation Concentration
The critical aggregation concentration (CAC) was determined
by using pyrene as a hydrophobic fluorescent probe. The
typical procedure was as follows: A stock solution of poly-
meric aggregates was diluted with distilled water in the con-
centration range from 0.5 to 5.12 3 1027 mgÁmL21. An
aqueous solution of pyrene containing 0.1 vol % acetone
(2.4 3 1026 M) (1.6 mL) was added to the diluted polymer
solution (1.6 mL), and thoroughly mixed. The solution was
kept at 25 8C for 1 h in the dark. The fluorescence intensity
at 390 nm was measured, with photoexcitation at 333 nm
(I333) and 338 nm (I338) at 25 8C. The fluorescence intensity
ratio (I338/I333) was plotted as a function of the logarithm of
the polymer concentration. The CAC was determined as the
intersection point of two tangents to the plot at higher and
lower concentrations.
Synthesis of PLLA Bearing an Ethynyl Group at the
Initiating End
A typical procedure for the ring-opening polymerization of L-
lactide was as follows: L-Lactide (1.02 g, 7.0 mmol), 2-
propyn-1-ol (10.2 mg, 0.18 mmol), Sn(Oct)2 (72 mg, 0.18
mmol), and dry toluene (5 mL) were placed into a 10 mL
flask, which was sealed tightly with a rubber septum. The
flask was immersed in an oil bath preheated at 90 8C, and
stirred for 20 h. After the resulting mixture was cooled
down, a small amount of the polymerization mixture was
taken to determine the monomer conversion by 1H NMR
spectroscopy. The solvent was evaporated under reduced
pressure, then the residue was poured into excess methanol.
The resulting precipitate was collected by centrifugation and
washed with methanol. After drying under vacuum, a white
powder was obtained. Yield, 905 mg (89%). Mn 5 10,000,
Mw/Mn 5 1.13 (PMMA std.).
Turbidimetric Assay
The binding behavior of PManEMA16k and PManEMA16k-b-
PLLA6.4k with concanavalin A (ConA) was examined by tur-
bidity assay according to a previous paper, with minor modi-
fications. ConA was diluted to 1 lM (based on ConA
tetramer) with 10 mM HEPES-buffered (pH 5 7.4) saline con-
taining 150 mM NaCl and 1 mM CaCl2 (referred to HBS).
The ConA solution (450 lL) was transferred to a 1 cm micro
cuvette, which was settled into a thermostated cell holder at
25 8C. Into the cuvette was rapidly injected 50 lL of distilled
Synthesis of PManEMA Having an Azide Group at the
Initiating End
A typical procedure for an aqueous activator generated by
electron transfer (AGET) ATRP was as follows: Ascorbic acid
(6.50 mg, 36.9 lmol) was dissolved in a mixture of 2-
propanol and water (2/8, v/v) (10 mL), and nitrogen gas
was bubbled through the solution for 1 h to prepare a deaer-
ated stock solution of ascorbic acid. ManEMA (86.5 mg, 296
lmol), 2-azidoethyl 2-bromo-2-methylpropanoate (0.64 mg,
2.5 lmol), CuCl2 (0.19 mg, 1.4 lmol), tris(2-pyridylmethyl)-
amine (0.38 mg, 1.3 lmol), and a mixture of 2-propanol and
water (2/8, v/v) (0.4 mL) were charged in a 1.5 mL auto
sampler vial, which was sealed with a rubber septum and
cap. Nitrogen gas was bubbled through the solution for 2
min. The vial was immersed in a preheated water bath at 30
8C. A deaerated stock solution of ascorbic acid (0.1 mL) was
injected into the vial to initiate the polymerization. After 1 h,
the polymerization mixture was exposed to air and a small
amount of water was added to quench the reaction. The
water solution of PManEMA16k or PManEMA16k-b-PLLA6.4k
.
The final concentration of the ManEMA unit in PManEMA16k
was varied from 6.3 to 100 lM. The final concentration of
PManEMA16k-b-PLLA6.4k was varied from 5 to 80 mgÁL21
.
RESULTS AND DISCUSSION
Synthesis of PLLA Having an Ethynyl Group at the
Initiating End
Ring-opening polymerization of L-lactide was carried out
using 2-propyn-1-ol as an initiator and tin(II) 2-
ethylhexanoate as a catalyst in dry toluene at 90 8C (Scheme
1). Table 1 summarizes the polymerization conditions and
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SCHEME 1 Synthesis of PManEMA-b-PLLA.
results. Figure 1 shows the SEC traces and MALDI-TOF mass
spectrum of the resulting PLLA. The mass spectrum shows
peaks corresponding to the sodium and potassium adducts
of the PLLA bearing an ethynyl group and hydrogen at the
initiating and terminating ends, respectively. However, minor
peaks are also found and are plausibly attributable to PLLA
initiated with water impurities. All SEC traces show relative-
ly narrow molecular weight distributions in which the poly-
dispersities fall between 1.05 and 1.12. The number average
molecular weight (Mn) of PLLA increased from 4500 to
10,000 with an increase in the monomer-to-initiator ratio. It
should be noted that the Mn values were obviously overesti-
mated due to the PMMA calibration. Unfortunately, no tech-
nique was available to determine the absolute Mn values of
the resulting PLLA.
methylpropanoate as an initiator, CuCl2 and PMDETA as a
copper source and a ligand, respectively, and ascorbic acid
as a reducing agent in a mixture of water and 2-propanol
(8/2, v/v) at 30 8C for 1 h. Figure 2 shows an SEC trace and
IR spectrum of the resulting polymer, PManEMA. The Mn and
Mw/Mn values were determined to be 16,000 and 1.24,
respectively, on the basis of PMMA calibration. The relatively
narrow molecular weight distribution indicated that the
polymerization proceeded in a controlled fashion. The IR
spectrum shows the characteristic peak at approximately
Synthesis of PManEMA Having Azide Group at the
Initiating End
ManEMA and 2-azidopropyl 2-bromo-2-methylpropanoate
were prepared according to the literature.59,60Aqueous poly-
merization of ManEMA was hard to control by means of the
normal ATRP technique due to the high polymerization rate
in aqueous media. Hence, the AGET technique was applied to
the aqueous ATRP of ManEMA. The AGET ATRP of ManEMA
was carried out using 2-azidopropyl 2-bromo-2-
TABLE 1 Ring-opening polymerization of L-lactide in toluene at
90 8C
Conv.b
Yieldc
Mn (Mw/Mn)d
a
Run no.
[M]0/[I]0
Time
(h)
3
(%)
93
(%)
55
1
2
3
10
20
40
4,500 (1.05)
6,400 (1.10)
10,000 (1.12)
7
93
85
20
98
88
a
M, L-lactide; I, 2-propyn-1-ol; [M]0 5 1.4 M; [I]0/[Sn(Oct)2] 5 1.
Monomer conversion determined by 1H NMR spectroscopy.
Isolated yield.
b
c
d
Number average molecular weights (Mns) and polydispersities (Mw
/
FIGURE 1 SEC traces of PLLAs (a) and MALDI-TOF mass spec-
Mns) were determined by SEC using PMMA calibration.
trum of PLLA4.5k (run no. 1 in Table 1) (b).
4
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PManEMA14.2k segments. Figure 4 shows the SEC traces of
PLLA10k, PManEMA14.2k and the coupling product. The SEC
trace of the coupling product clearly shifted toward a shorter
retention time compared to those of PLLA10k and PManE-
MA14.2k. This indicates that the resulting polymer is a block
copolymer consisting of PManEMA14.2k and PLLA10k, namely,
PManEMA14.2k-b-PLLA10k. In the SEC trace of the coupling
products, however, a significant amount of impurities was
found on both sides of the main peak. The impurity appear-
ing in the higher retention region is unreacted PLLA, because
excess PLLA was used, as mentioned above. The impurity
found in the lower retention region is still unknown. The
main peak was fitted with an empirically transformed Gauss-
ian function,62 and the area percent of the main peak was
estimated to be 92% (see Supporting Information). The Mn
and Mw/Mn values were calculated using the best-fit curve
and PMMA calibration, and are listed in Table 2.
Sugar-Coated Aggregate Formation and Lectin-Binding
Properties
The water solubility of the resulting block glycopolymer
depended on the balance of the hydrophobic and the hydro-
philic segments. PManEMA16k-b-PLLA4.5k and PManEMA16k
-
FIGURE 2 SEC trace (a) and IR spectra (b) of PManEMA16k
bearing an azido group at the initiating end (gray and black
solid lines) and 3-azidopropyl 2-bromo-2-methylpropanoate
(dotted line).
2100 cm21, which is attributable to the stretching mode of
the azide group. This experimental evidence indicates that
the resulting PManEMA has an azido group at the initiating
end.
Click Synthesis of PManEMA-b-PLLA
Synthesis of PManEMA-b-PLLA was performed by Cu(I)-cata-
lyzed 1,3-dipolar cycloaddition of PLLA bearing an ethynyl
group and PManEMA bearing an azido group at the initiating
end. Usually, the amount of the two components should be
adjusted, or an excess of one component, which is easily
removed after reaction, may be applied for successful poly-
mer coupling. However, as no technique was available for us
to determine the absolute Mn values, we used the Mn values
determined by SEC, calibrated by PMMA standards, to adjust
the apparent molarities of PManEMA and PLLA.61 Because
the Mn values of PLLA were obviously overestimated, excess
PLLA was applied in the coupling reaction. The coupling
reaction was carried out using CuBr and PMDETA as a cata-
lyst and a ligand, respectively, in DMF at 25 8C for 24 h. The
crude product was dialyzed against water, followed by lyoph-
ilization to afford a white powder. Figure 3 shows the 1H
NMR spectra of PLLA10k, PManEMA14.2k, and the coupling
product in DMSO-d6. The 1H NMR spectrum of the coupling
product shows peaks attributable to both PLLA10k and
FIGURE 3 1H NMR spectra of PLLA10kDa (a), PManEMA14.2kDa
(b), and the coupling product (c) in DMSO-d6. Symbols * and
** represent solvent and water peaks, respectively.
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FIGURE
5
1H NMR spectra of PManEMA16k-b-PLLA6.4k in
DMSO-d6 (a) and D2O (b). Symbols * and ** represent solvent
and water peaks, respectively.
FIGURE 4 SEC traces of PLLA10k (broken line), PManEMA14.2k
(dotted line) and the coupling product (PManEMA14.2k-b-
PLLA10k, solid line) (c) in DMF containing 50 mM LiBr.
tangent at the inflection point. Suriano et al. synthesized a
block glycopolymer, poly(e-caprolactone)-b-poly[(polyethy-
lene glycol mono methyl ether methacrylate)-co-(6-O-metha-
cryloyl-D-galactose)], in which the Mn values of the PLLA
segments and the overall block glycopolymer were 5700 and
25,300, respectively, via a macromolecular initiator approach
using the ATRP technique.49 The CAC value of their block
glycopolymer was estimated to be 8.4 mgÁL–1, which is
slightly lower than that of PManEMA16k-b-PLLA6.4k. Figure 7
shows the size distribution of PManEMA16k-b-PLLA6.4k aggre-
gates measured in aqueous solution at a concentration of 1
mgÁmL21. The main peak was found at approximately
30 nm, and the median radius was determined to be 41 nm.
However, minor peaks were found at 340 and 1400 nm. The
multimodal size distribution suggests a non-uniform struc-
ture for the aggregates, but details of these remain to be
solved.
b-PLLA6.4k were soluble in water, while PManEMA14k-b-
PLLA10k was insoluble in water. Figure 5 shows the H NMR
1
spectra of PManEMA16k-b-PLLA6.4k in DMSO-d6 and D2O. The
peaks at 1.45 and 5.20 ppm, attributable to the PLLA seg-
ment, completely disappeared in the 1H NMR spectrum
recorded in D2O, indicating that the PLLA segments con-
densed to form a core region. The critical aggregate concen-
tration (CAC) was determined by fluorometry, using pyrene
as a hydrophobic fluorescent probe. Figure 6 shows the fluo-
rescence intensity ratio between 333 and 338 nm in the
excitation spectrum, in which the detection wavelength was
390 nm, as a function of the concentration of PManEMA16k
-
b-PLLA6.4k. The plots were fitted with a sigmoid function and
the CAC was determined to be 21 mgÁL21 for PManEMA16k
-
b-PLLA6.4k from the crossing point of the base line and the
TABLE 2 Click synthesis of PManEMA-b-PLLA in DMF at 25 8Ca
PManEMA
PLLA
PManEMA-b-PLLA
b
b
Run no.
Mn
Mn
Yield (%)c
Mn (Mw/Mn)d
Water-solubility
1
2
3
16,000
16,000
14,200
4,500
6,400
46
63
68
19,400 (1.66)
28,300 (1.28)
27,100 (1.34)
᭺
᭺
3
10,000
a
c
[PManEMA]0/[PLLA]0/[CuBr]0/[PMDETA]0 5 1/1/10/25; [PManEMA]0 5
approximately 2 mM (based on the Mn value calculated using PMMA
calibration); time, 24 h.
Isolated yield.
d
Number average molecular weights (Mns) and polydispersities (Mw
/
Mns) were calculated on the basis of the best-fitted curve to the SEC
traces using PMMA calibration.
b
Number average molecular weights (Mns) determined by SEC using
PMMA standards.
6
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FIGURE 6 The I338nm/I333nm values as a function of the concen-
tration of PManEMA16k-b-PLLA6.4k
.
Even though the structure of the aggregates is unclear, the
highly water-soluble PManEMA segments must be located on
the outer surface of the aggregates. The functionality of the
D-mannose residue presented on the aggregate surfaces was
evaluated by the binding ability to concanavalin A (ConA),
which is a lectin recognizing D-mannose. ConA forms a
homotetramer at neutral pH, and each unit has one carbohy-
drate recognition domain. Hence, a material presenting mul-
tiple D-mannose residues forms aggregates by mixing with
ConA. In order to evaluate the kinetics of the aggregate for-
mation, we employed a turbidimetric assay in HEPES buffer
(pH 5 7.4) containing 150 mM NaCl and 1 mM CaCl2.59 Fig-
ure 8 shows the time course of the optical density at
420 nm in a mixture of ConA with PManEMA16k and
FIGURE 8 Time course of optical density at 420 nm in a mix-
ture of ConA and PManEMA16k (a) and PManEMA16k-b-PLLA6.4k
aggregate solution (b) at 25 8C. The concentration of ConA
homotetramer was 1 lM. The concentration of the ManEMA
unit in PManEMA16k was varied from 6.3 to 100 lM. The
concent ration of PManEMA16k-b-PLLA6.4k was varied from 5 to
80 mgÁL21
.
PManEMA16k-b-PLLA6.4k at 25 8C. The resulting curve for
PManEMA16k at a concentration of ManEMA units of 50 lM
was quite similar to that reported previously,59 indicating
the good reproducibility of this technique. The initial incre-
ment rate of the optical density was increased with an
increase in ManEMA units from 6.3 to 25 lM, and was
almost constant above 25 lM. For PManEMA16k-b-PLLA6.4k
,
the initial increment rate of the optical density increased
when the polymer concentration increased from 5 to 20
mgÁL21, and was almost constant above a polymer concen-
tration of 20 mgÁL21. Unfortunately, the effect of aggregate
formation on ConA binding cannot be separated from the
contribution of the increase in the concentration of ManEMA
units.63 However, the increment rate of the optical density
did not decrease even above a polymer concentration of 20
mgÁL21, suggesting that the aggregate structure did not
interfere with the interaction between D-mannose residue
and ConA. These results indicate that PManEMA16k-b-
FIGURE 7 Size distribution of PManEMA16k-b-PLLA6.4k aggre-
gates in aqueous solution. The concentration of the block
glycopolymer was 1 mgÁmL21
.
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16 X. Wu, L. Su, G. Chen, M. Jiang, Macromolecules 2015, 48,
3705–3712.
PLLA6.4k forms aggregates that efficiently represent D-man-
nose residues.
17 L. Su, C. Wang, F. Polzer, Y. Lu, G. Chen, M. Jiang, ACS
Macro Lett. 2014, 3, 534–539.
CONCLUSIONS
18 J. Lu, C. Fu, S. Wang, L. Tao, L. Yan, D. M. Haddleton, G.
Chen, Y. Wei, Macromolecules 2014, 47, 4676–4683.
The block glycopolymer, PManEMA-b-PLLA, was synthesized
by Cu(I)-catalyzed 1,3-dipolar cycloaddition between PLLA
bearing an ethynyl group and PManEMA bearing an azide
group at the initiating end. The block architecture was con-
firmed by SEC and 1H NMR spectroscopy. The water-
solubility of the block glycopolymer depends on the balance
of the two segments. The aggregate formation of the block
glycopolymer, PManEMA16k-b-PLLA6.4k, was examined by 1H
NMR spectroscopy using selective solvents, fluorometry
using pyrene as a hydrophobic probe, and DLS measurement.
The block glycopolymer forms a complicated aggregate in
water at concentrations above 21 mgÁmL21. The aggregates
present D-mannose units on their outer surfaces, and can
interact with the lectin, concanavalin A.
19 V. Ladmiral, M. Semsarilar, I. Canton, S. P. Armes, J. Am.
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The authors wish to thank Prof. Hidenori Okuzaki, University of
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