Synthesis of amphiphilic A3B mikto-arm copolymers from a sugar core: Combination of hydrophobic PCL and hydrophilic glycopolymers for biocompatible nanovector preparation

Article abstract of DOI:10.1002/pola.24109

Amphiphilic A₃B mikto-arm copolymers have been synthesized using a t-butyl-diphenyl silyl-based methylglucoside derivative. The latter has been first used as initiator for the polymerization of ε-caprolacto ne leading to three-arm starshaped st

Full text of DOI:10.1002/pola.24109

Synthesis of Amphiphilic A3B Mikto-Arm Copolymers from a Sugar Core:
Combination of Hydrophobic PCL and Hydrophilic Glycopolymers for
Biocompatible Nanovector Preparation
FABIAN SURIANO, OLIVIER COULEMBIER, PHILIPPE DUBOIS
Laboratory of Polymeric and Composite Materials (LPCM), Center of Innovation and Research in MAterials and Polymers
(CIRMAP), University of Mons—UMONS, Place du Parc 23, 7000 Mons, Belgium
Received 5 March 2010; accepted 16 April 2010
DOI: 10.1002/pola.24109
Published online in Wiley InterScience (www.interscience.wiley.com).
ABSTRACT: Amphiphilic A3B mikto-arm copolymers have been
synthesized using a t-butyl-diphenyl silyl-based methylgluco-
side derivative. The latter has been first used as initiator for
the polymerization of e-caprolactone leading to three-arm star-
shaped structures followed by several postpolymerization
steps to obtain star-shaped poly(e-caprolactone) macroinitiator.
Atom transfer radical polymerization (ATRP) of diisopropyli-
dene galactose methacrylate in THF at 60 ꢀC using CuBr ligated
with 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) as
catalytic complex allowed the formation of A3B mikto-arm
copolymers with different compositions and molecular
weights. Selective deprotection of sugar protecting groups
finally generated amphiphilic mikto-arm copolymers. The mo-
lecular characterization of those copolymers was performed by
1H NMR spectroscopy and gel permeation chromatography
(GPC) analysis. The self-assembly of the copolymers into mi-
cellar aggregates and the related critical micellization concen-
tration (CMC) in aqueous media were determined by dynamic
light scattering (DLS) and UV-visible spectroscopy, respec-
C
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tively. 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym
Chem 48: 3271–3280, 2010
KEYWORDS: amphiphiles; glycopolymer; micelles; mikto-arm
copolymer; ring-opening polymerization
INTRODUCTION The use of polymeric micelles as nanometric
drug carrier systems has attracted much attention over the
past decades. Indeed, polymeric micelles offer the advantage
to trap drugs, such as anticancer agents, e.g., paclitaxel1 and
doxorubicin,2,3 in their hydrophobic core. Such drug-loaded
polymeric micelles have been exploited as therapeutic sys-
tems for anticancer treatment thanks to their nanometric
size allowing them to escape from reticuloendothelial system
(RES).4,5 Indeed, one of the most important features in
designing polymers for drug delivery applications relies
upon the size of the resulting micelles. The latter should be
in the range of 5 and 200 nm to allow long circulation time
in the bloodstream, selective tumor targeting due to
enhanced permeability and retention effect (EPR) in solid
tumors, and avoid RES uptake.6,7
radical polymerization (ATRP),11 nitroxide-mediated free rad-
ical polymerization (NMP),12,13 and reversible addition-frag-
mentation chain transfer (RAFT)14–16 have been widely stud-
ied and developed leading to countless possibilities taking
into account variations in the monomer nature, chain-end
functionality and polymer architecture along with the abil-
ities to associate radical processes to other polymerization
techniques such as ring-opening polymerization (ROP) of
various cyclic monomers. This last option is of particular
interest since a large range of functional materials can be
obtained. As an example, Matyjaszewski et al. prepared cylin-
drical core-shell brushes from poly((2-hydroxyethyl methac-
rylate)-graft-poly((e-caprolactone)-block-n-butyl
acrylate))
(PHEMA-g-(PCL-b-PBA)) using a combination of ROP and
ATRP processes.17 Later, Wiltshire and Qiao prepared core-
crosslinked star polymers by crosslinking hydroxyl 2-bromoiso-
butyrate telechelic PCL with ethylene glycol dimethacrylate fol-
lowed by polymerization of methyl methacrylate by ATRP.18
Crosslinking reaction was also extended to ATRP of N,N-
dimethyl amino-2-ethylmethacrylate (DMAEMA) and ax-di-
methacrylate PCL leading to the formation of hydrogels as
reported by Mespouille et al.19 Other ATRP/ROP combinations
have allowed the formation of different structures as versatile
as dendrimers,20,21 brush-22, stars23 or comb-like polymers24
Practically, polymeric micelles are mostly obtained from
amphiphilic random, block, or grafted copolymers.8 More-
over, controlling polymer molecular parameters and architec-
ture is of prime importance since a narrow dependence
exists between material properties and related structure.9 To
that end, efforts have been invested to reach polymerization
mechanisms allowing the control over the polymerization of
a wide variety of monomers. In this field, controlled radical
polymerization techniques (CRP),10 such as atom transfer
Additional Supporting Information may be found in the online version of this article. Correspondence to: O. Coulembier (E-mail: olivier.
coulembier@umons.ac.be)
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Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 3271–3280 (2010) 2010 Wiley Periodicals, Inc.
SYNTHESIS OF AMPHIPHILIC A3B MIKTO-ARM COPOLYMERS, SURIANO, COULEMBIER, AND DUBOIS
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
sometimes dealing with ring-opening metathesis polymeriza-
tion (ROMP) as an additional technique. Besides, star-branched
polymers having arms of different structures, also referred to
as mikto-arm polymers recently attracted increasing interest
thanks to their ability to highly change the morphology of their
supramolecular structures,25–28 resulting in a wide number of
applications such as in nanotechnology, biological engineering,
and drug/gene delivery.29,30
tored by TLC during 16 h using a heptane/ethyl acetate (4:1
v:v) mixture as eluent and a sulfuric acid/ethanol (1:9 v:v)
mixture to reveal sugar derivatives. At the end of the reac-
tion, 50 mL of a diethyl ether and ethyl acetate mixture (1:1
v:v) were added and the organic layer was washed twice
with a 100 mL 1M HCl solution, once with 100 mL of a 0.1
M sodium carbonate aqueous solution and twice with 100
mL of deionized water. The product was recrystallized ꢀin
diethyl ether, filtered, and dried till constant weight at 40 C.
Herein we report the synthesis of three-arm star-shaped
poly(e-caprolactone) obtained by polymerization of (e-capro-
lactone) (CL) from the three equatorial hydroxyl groups of
an initially protected tert-butyldiphenylsilyl ether-based a-
methylglucoside derivative. Post-polymerization derivatiza-
tion of chain-end alcohols with acetic anhydride followed by
selective hydrolysis of tert-butyldiphenylsilyl ether led to the
formation of an acetylated star-shaped PCL bearing a single
primary alcohol. Synthesis of such star-shaped PCL allowed
the preparation of new star-shaped macroinitiator converting
the primary alcohol into a 2-bromoisobutyrate moiety. This
so-formed macroinitiator was used to initiate the ATRP poly-
merization of 1,2;3,4-di-O-isopropylidene-6-O-methacryloyl-D-
galactopyranose (MAIGP) to produce perfectly controlled
amphiphilic mikto-arm copolymers able to self-assemble in
aqueous medium (Scheme 1).
1
Yield ¼ 62%. H NMR (300 MHz, DMSO-d6, d ppm): 0.97 (s,
9 H, Hm), 3.5–3.65 (m, 9 H, He þ Hc þ Hb þ Hg þ Hf þ
Hd), 4.6–4.9 (m, 4 H, Ha þ Hj þ Hiþ Hh), 7.35–7.85 (m, 10
H, Hk þ Hl). 13C-NMR (300 MHz, DMSO-d6, d ppm): d 20,
28, 56, 72, 73, 74, 75, 101, 128–137.
Synthesis of a-Hydroxyl x-6-O-t-Butyl Diphenyl Silyl-a0-
methyl Glucoside Three-Arm Star Poly(e-caprolactone)
In a previously flamed and purged round-bottom flask
equipped with a three way stopcock and a rubber septum,
were introduced 1.3 g of 6-O-tert-butyl diphenylsilyl a-
methyl glucoside (3 mmol) previously dried by three succes-
sive azeotropic distillations by addition of THF. To the initia-
tor were successively added 25.6 mL of THF and 8 mL of
e-CL (72.2 mmol). The mixture was allowed to warm up to
80 ꢀC before adding a 0.31 M Sn(Oct)2 solution (1.16 mL,
0.36 mmol) by using a previously flamed and nitrogen
purged syringe. The polymerization reaction proceeded for
16 h and was stopped by the addition of 1M HCl aqueous
solution (0.8 mL). The polymer was selectively precipitated
in cold methanol, filtered, and dried till constant weight at
40ꢀC. Yield ¼ 77%. 1H NMR (300 MHz, CDCl3), d ppm: 1.05
(s, 9 H, Hn), 1.40 (m, 6 H, Hj), 1.65 (m, 12 H, Hi), 2.31 (t, 6
H, Hh), 3.35–3.45 (m, 6 H, Hg þ Hf þ Hb), 3.65 (t, 6 H, Hk0),
4.06 (t, 6 H, Hk), 4.7–5.5 (m, 4 H, Ha þ Hc þ Hd þ He), 7.4–
7.8 (m, 10 H, Hl þ Hm)). Mn NMR ¼ 2700 g molꢁ1.Mw/Mn
¼ 1.13.
EXPERIMENTAL
Materials
e-Caprolactone (CL, from Acros, 99%) was dried over cal-
cium hydride for 48 h at room temperature and distilled
under reduced pressure just before use. Stannous octoate
(Sn(Oct)2) has been distilled under vacuum, diluted in dry
toluene and stored in glass ampoules. N,N-dimethylamino-4-
pyridine (DMAP, from Acros, 99%), 2-bromoisobutyryl bro-
mide (from Aldrich, 98%), 1,1,4,7,10,10 hexamethylenetetr-
amine (HMTETA, from Aldrich, 97%), tetrabutylammonium
fluoride (TBAF, from Aldrich, 97%), t-butyl-diphenyl silyl
chloride (TBDPSCl, from Fluka, 97%), methyl a-D-glucopyran-
oside (from Aldrich, 99%), pyridine (from Aldrich, 99.8%),
formic acid (from Aldrich, 98%), acetic acid (from Aldrich,
99.7%), acetic anhydride (from Aldrich, 99%), and pyrene
(from Aldrich, 98%) were used as receivꢀed. Magnesium sul-
fate (from Acros, 99%) was dried at 100 C for 48 h prior to
use. Copper bromide (CuBr, from Fluka, 98%) was purified
in acetic acid for 24 h and recrystallized in ethanol under
inert atmosphere until a white powder was obtained. THF
from Biosolve and chloroform from Chemlab were dried on
a solvent purification system MB SPS-800 from MBraun.
1,2;3,4-Di-O-isopropylidene-6-O-methacryloyl-a-D-galactopyr-
anose (MAIGP) has been synthesized prior to use applying
previously reported method.31
Synthesis of a-Acetyl x-6-O-t-Butyl Diphneyl Silyl-a0-methyl
Glucoside Three-Arm Star Poly(e-caprolactone)
In a flamed and purged round-bottomed flask were introduced
6 g of three-arm star-PCL (Mn ¼ 2700 g molꢁ1, 2.22 mmol,
6.67 mmol OH), 30 mL of triethylamine (220 mmol) and 75
ꢀ
mL of THF. The mixture was heated to 50 C when 19 mL of
acetic anhydride (200 mmol) were added. After 48 h of reac-
tion, the solvent was removed and the as-obtained crude solid
was dissolved in 10 mL of THF. The polymer was selectively
precipitated in cold methanol, filtered and dried till constant
weight at 40 ꢀC. Yield ¼ 98%. 1H NMR (300 MHz, CDCl3, d
ppm): 1.05 (s, 9 H, Hn), 1.40 (m, 6 H, Hj), 1.65 (m, 12 H, Hi),
2.05 (s, 9 H, Hl), 2.31 (t, 6 H, Hh), 3.35–3.45 (m, 6 H, Hg þ Hf
þ Hb), 4.06 (t, 6 H, Hk), 4.7–5.5 (m, 4 H, Ha þ Hc þ Hd þ He),
7.4–7.8 (m, 10 H, Hm þ Hn). Mn NMR ¼ 2700 g molꢁ1, Mn SEC
¼ 2400 g molꢁ1, Mw/Mn ¼ 1.1.
Synthesis of 6-O-Tert-Butyl Diphenylsilyl
a-Methyl Glucoside
In a flamed purged round-bottom flask were introduced 2.12
g of a-D-methyl glucoside (10.93 mmol), 0.2 g of DMAP (1.64
mmol), and 50 mL of pyridine. The mixture was heated to
50 ꢀC and 2.6 mL of tert-butyl diphenylsilyl chloride
(10 mmol) were added. The reaction evolution was moni-
Selective Deprotection of tert-Butyl Diphenylsilyl Ether
Synthesis of a-Acetyl x-a0-Methyl Glucoside Three-Arm
Star Poly(e-caprolactone) (Star-PCL-OH)
In a PE container were introduced 4 g of a-acetyl x-6-O-t-
butyl diphneyl silyl-a0-methyl glucoside three-arm star
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SCHEME 1 General synthetic route to access mikto-arm architecture where
R
represents 1,2;3,4-Di-O-isopropylidene-D-
galactopyranose.
poly(e-caprolactone) (Mn ¼ 2700 g molꢁ1, 1.41 mmol) and
85 mL of a 1 M TBAF solution (THF/acetic acid 85:15 v:v).
After 24 h, the medium was concentrated before being dis-
solved in 10 mL of THF. The polymer was selectively precipi-
tated in cold methanol, filtered, and dried till constant
weight at 40 ꢀC. Yield ¼ 99%. 1H NMR (300 MHz, CDCl3,
d ppm): 1.40 (m, 6 H, Hj), 1.65 (m, 12 H, Hi), 2.05 (s, 9 H,
Hl), 2.31 (t, 6 H, Hh), 3.45 (s, 3 H, Hb), 3.65–3.75 (m, 3 H, Hf
SYNTHESIS OF AMPHIPHILIC A3B MIKTO-ARM COPOLYMERS, SURIANO, COULEMBIER, AND DUBOIS
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
þ Hg), 4.1 (t, 6 H, Hk), 4.7–5.5 (m, 4 H, Ha þ Hc þ Hd þ
in dimethylsulfoxide (1 mL), followed by progressive addi-
tion (1 drop/10 s) of 10 mL of deionized water under vigor-
ous stirring. Organic solvent was removed by transferring
the solution in a preswollen membrane (Spectra/Por, molec-
ular weight cutoff size, 3500 D), dialysis against water for
He). Mn NMR ¼ 2800 g molꢁ1, Mn SEC ¼ 2400 g molꢁ1
Mw/Mn ¼ 1.1.
,
Synthesis of a-Acetyl x-6-O-Bromoisobutyrate-a0-methyl
Glucoside Three-Arm Star Poly(e-caprolactone)
(Star-PCL-Br)
R
V
24 h. Solutions were then filtered through 1.2 mm Acrodisk
filter.
In a flamed and purged round-bottom flask were introduced
2.5 g of star-PCL-OH (Mn ¼ 2800 g molꢁ1, 0.85 mmol), 20
mg of DMAP (0.16 mmol), 0.2 mL of triethylamine (1.45
mmol), and 15 mL of THF. The mixture was allowed to
warm up to 50 ꢀC before adding 0.15 mL of 2-bromoisobu-
tyryl bromide (1.21 mmol). After 48 h, the medium was con-
centrated, solubilized by 5 mL of THF, and the crude poly-
mer was selectively precipitated in cold methanol, filtered
and dried till constant weight at 40 ꢀC. Yield ¼ 99%. 1H
NMR (300 MHz, CDCl3, d ppm): 1.40 (m, 6 H, Hj), 1.65 (m,
12 H, Hi), 1.95 (s, 6 H, Hm), 2.05 (s, 9 H, Hl), 2.31 (t, 6 H,
Hh), 3.45 (s, 3 H, Hb), 3.75 (m, 1 H, Hf), 4.1 (t, 8 H, Hk þ
Hg), 4.7–5.5 (m, 4 H, Ha þ Hc þ Hd þ He). Mn NMR ¼ 2700
g molꢁ1, Mn SEC ¼ 2400 g molꢁ1, Mw/Mn ¼ 1.1.
Solutions for the determination of the critical micellization
concentration (CMC) of the polymers in deionized water
were prepared by the dissolution of the copolymers (1 mg)
in dimethylsulfoxide (1 mL), followed by progressive addi-
tion (1 drop/10 s) of 10 mL of deionized water under vigor-
ous stirring. Organic solvent was removed by transferring
the solution in a preswollen membrane (Spectra/Por, molec-
ular weight cutoff size, 3500 D), followed by a dialysis
against water for 24 h. The stock solution was then diluted
to produce several solutions with various polymer concen-
trations ranging from 10ꢁ5 g Lꢁ1 to 1 g Lꢁ1. To each solu-
tion (10 mL) was added 0.1 mL of a pyrene solution in ace-
tone (6 ꢂ 10ꢁ5 mol Lꢁ1) and the acetone was finally slowly
evaporated in mild conditions for 24 h. The final pyrene con-
centration was 6 ꢂ 10ꢁ7 mol Lꢁ1 in all solutions.
Synthesis of A3B Mikto-arm Copolymers
In a round-bottom flask A were introduced 5.7 mg of copper
bromide (CuBr, 0.04 mmol) and 18.4 mg of 1,1,4,7,10,10-hex-
amethyl triethylene tetramine (HMTETA, 0.08 mmol). Three
freezing/thawing cycles were performed to get rid of the
trapped O2. In a round-bottom flask B were introduced 0.11
g of star-PCL-Br (Mn ¼ 2700 g molꢁ1, 0.04 mmol), 0.66 g of
1,2;3,4-di-O-isopropylidene-6-O-methacryloyl-D-galactopyr-
anose (MAIGP, 2 mmol) and 2 mL of THF. Nitrogen was
bubbled through the solution before transferring it into flask
A. The polymerization has been led at 60 ꢀC for 4 h then
stopped in liquid nitrogen and the polymer was selectively
recovered after precipitation in heptane, filtered, and dried
Characterizations
1H NMR spectra were recorded using a Bruker AMX-300 ap-
paratus at r.t. in CDCl3 (30 mg/0.6 mL) except for glucose-
based initiator and deprotected galactose-based copolymers
that were recorded in DMSO-d6 (30 mg/0.6 mL). 13C NMR
spectra were recorded at higher concentrations (60 mg/0.6
ml). Size exclusion chromatography (SEC) was performed in
ꢀ
THF at 35 C using a Polymer Laboratories liquid chromato-
gram apparatus equipped with a PL-DG802 degasser, an iso-
cratic HPLC pump LC 1120 (flow rate ¼ 1 mL/min), a Mara-
thon autosampler (loop volume ¼ 200 ml, solution conc. ¼ 1
mg/mL), a PL-DRI refractive index detector and three col-
umns: a PL gel 10 mm guard column and two PL gel Mixed-B
10 mm columns (linear columns for separation of MWPS
ranging from 500 to 106 daltons). Polystyrene or poly
(methyl methacrylate) standards were used for calibration.
UV-visible absorption spectra have been recorded Cary-Win
UV 50 spectrophotometer (from Varian) from 300 to 400
nm. Dynamic light scattering measurements were carried out
using a BI-160 apparatus (Brookhaven Instruments) or a
ZEN 3600 from Malvern Intruments with a He-Ne laser
delivering a vertically polarized light (k ¼ 633 nm). The
particle sizes and size distributions were calculated using
CONTIN algorithms or NNLS algorithms. For all particle size
measurements 5 runs were recorded and the data were
reported as the average mean diameter for 5 runs.
ꢀ
until constant weight at 40 C. Yield ¼ 99%. The copper cat-
alyst was removed by passing the polymer solution in THF
through a basic alumina column. The solvent was then
evaporated to yield the pure polymer. 1H NMR (300 MHz,
CDCl3, d ppm): 0.88–1.03 (m, 3 H, Hi), 1.23–1.8 (m, 32 H, Hv
þ Hj þ Hn þ Hi), 1.64 (m, 4 H, Hd), 1.95 (s, 6 H, Hm), 2.05
(s, 9 H, Hl), 2.31 (t, 6 H, Hh), 3.43 (s, 3 H, Hb), 3.9–4.1 (m,
10 H, Hf þ Hg þ Ht þ Hk), 4.37 (m, 4 H, Hq þ Hs þ Hu),
4.62 (s, 1 H, Hr), 4.8–5.55 (m, 5 H, Ha þ Hc þ Hd þ He þ
Hp). Mn NMR ¼ 20,000 g molꢁ1, Mw/Mn ¼ 1.28.
General Procedure for the Selective Removal of
Isopropylidene Protective Groups from Sugar Units
Copolymer (1g) was dissolved in 50 mL of HCOOH/H2O (4:1
v/v) and the solution was stirred at room temperature for
48 h. Formic acid was then removed by dialysis against
water using a preswollen membrane (Spectra/Por, molecular
weight cutoff size, 3500D) for 48 h replacing water every 4
h (when possible). Deprotected copolymer was finally recov-
ered by freeze-drying. Yield ¼ 75–90%.
Preparation of Aqueous Micellar Solutions for
Dynamic Light Scattering Measurements
Micellar aggregates of the amphiphilic copolymers in water
were prepared by the dissolution of the copolymers (1 mg)
RESULTS AND DISCUSSION
The synthesis of brush-like architectures based on saccha-
rides was investigated via a ‘‘grafting through’’ approach. In
the proposed strategy depicted in Scheme 1, the ring-open-
ing polymerization (ROP) process takes place prior to the
atom transfer radical polymerization (ATRP). The first step
has then consisted on the synthesis of a saccharidic residue
able to initiate the ROP reaction from some of dangling
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TABLE 1 Molecular Characterizations of Star-Shaped Poly(e-caprolactone)s
Yield
(%)a
Mn th
Mn exp NMR
(g molꢁ1 c
Mn exp NMR per chain
(g molꢁ1 c
Mn SEC
(g molꢁ1 b
)
)
)
(g molꢁ1 d
)
Mw/Mn
d
Entry
Time (h)
1
2
a
16
40
77
95
2,500
5,600
2,700
6,400
750
2,200
6,300
1.13
1.29
2,000
d
As determined by gravimetry.
As determined by size exclusion chromatography (SEC) in THF at 35
ꢀC using PS standards taking into account the Mark-Houwink-Sakurada
b
Theoretical molecular weight: Mn ¼ Mw
th
þ 114*DPth*conv. where
ini
Mw ini is the initiator molecular weight.
c
parameters.
Experimental molecular weight as determined by 1H-NMR (500 MHz)
in CDCl3.
hydroxyl groups while providing another alcohol able to
undergo adequate derivatization for further ATRP. To that
end, a-methyl glucoside is well-suited since the pyranose
sugar is composed of a primary alcohol and three secondary
equatorial alcohols of same conformation and probability to
initiate the lactone ROP. Finally, this sugar is blocked in its
pyranosidic form by the presence of the methyl ether on the
anomeric alcohol, preventing any mutarotation and degrada-
tion at common operating temperature during the polymer-
ization reactions.
derivative. Tin octoate (Sn(Oct)2) has been chosen as catalyst
since its activity at 80 C is well-suited to the thermal stabil-
ꢀ
ity of the sugar-functionalized initiator. Indeed, as the
anomeric alcohol is blocked by a methyl ether group, poly-
merizations under those conditions are not likely to undergo
side reactions or degradation of the sugar moiety. As the
TBDPS sugar-based initiator proved to be poorly soluble in
toluene, polymerizations were conducted in THF under nitro-
gen pressure. The monomer concentration was set to 2 mol
Lꢁ1 while [Sn]0/[OH]0 molar ratio fixed to 0.04 to get a cata-
lytic amount of in situ generated tin alkoxide active species
as elsewhere explained.34 Two different sets of reactions
were performed to get star-shaped PCL structures character-
ized by overall degree of polymerizations (DPs) of about 24
(Table 1, entry 1) and 48 (Table 1, entry 2). As expected and
highlighted in Table 1, both polymerizations proceeded in a
controlled manner with an exquisite correlation between
theoretical and experimental molecular weights. As expected,
both CL ROPs seemed to proceed from all three secondary
alcohol groups of the glucosidic initiator since a complex set
of peaks [Fig. 1(Ha,c,d, and e)] appears between 4.6 and 5.6
ppm as theoretically predicted, principally attributed to the
three methine protons in the glucosidic ring (Hc,d, and e)
undergoing a downfield shift upon ester formation due to
the polymerization of CL. Quantitative evidence can be
obtained from the comparison of the hydroxymethylene end-
To promote the ROP from the secondary equatorial hydroxy
groups, a first derivatization reaction of the primary alcohol
has been realized. Purposely, tert-butyldiphenyl silyl (TBDPS)
ether is perfectly adapted since it is known to selectively
protect primary alcohols of pyranosidic sugars due to the
bulky substituents on the silicon atom and to increase the
relative solubility of the as-obtained compounds in organic
solvents, thus being particularly useful in the case of poly-
hydroxylated sugars.32,33
The protection of the a-methyl glucoside molecule has been
realized in pyridine with excess of tert-butyldiphenyl silyl
chloride at 50 ꢀC. After completion of the reaction (deter-
mined by thin layer chromatography, TLC), the medium is
concentrated and purified by both multiple extractions in
ethyl acetate and a recrystallization in diethyl ether leading
1
to an overall yield of 62%. The H NMR spectrum of the as-
0
group protons signal (Ik ) and the ones corresponding to the
obtained 6-O-tert-butyldiphenyl silyl a-methyl glucoside high-
lights the presence of a new signal centered at about 1 ppm
assigned to the methyl protons of the tert-butyl group (Hm)
while peaks appearing between 7 and 8 ppm (Hk, Hl) are
attributed to the phenyl protons giving credit to the expected
ether formation (See Supporting Information Fig. S1). It is
worth pointing out that integrations ratios between each
functional group fitted the expected composition taking into
account the inherent error of the NMR technique. The selec-
tivity of this reaction was also attested by 13C NMR spectros-
copy highlighting a clear downfield shift from 61 (Hg0) to 64
ppm (Hg) upon silyl ether formation from the primary alco-
hol group (Supporting Information Fig. S2), which is more
likely due to the strong electron-withdrawing effect caused
by both diphenyl substituents.
aromatic phenyl protons of the initiator residue (Il þ Im).
Normalization of the respective signals allowed for the calcu-
0
lation of the number of PCL chains per sugar as: (Ik /2)/((Il
þ Im)/10). In very good agreement and close to an expected
value of 3, experimental calculations of 3.3 and 3.2 were
obtained for respective entries 1 and 2 in Table 1. Using
0
both Ik and Ik attributed to repeating CL methylene protons
and end-groups ACH2AOH, respectively, allows for the calcu-
lation of the average molecular weight of each single PCL
0
0
chain (Mnchain¼ Mw CLꢃ(Ik þ Ik )/Ik ). An excellent correlation
between theoretical and experimental molecular weights
along with low polydispersity indices attests for a good con-
trol over the ROP of CL from the sugar-based multihydroxy-
lated initiator. Polydispersity indices also attest for the
homogeneity of all the single chains grafted on the sugar core.
The so-formed silyl ether glucoside has then been used as
initiator for the CL ROP, promoting polymerization from all
three secondary equatorial alcohol groups of the glucoside
In a next step, the functionalization of the three hydroxy
methylene end-groups of both star-shaped PCL by acetyla-
tion reaction coupled with the selective deprotection of the
SYNTHESIS OF AMPHIPHILIC A3B MIKTO-ARM COPOLYMERS, SURIANO, COULEMBIER, AND DUBOIS
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FIGURE 1 1H NMR spectrum of
three-arm star-shaped PCL in CDCl3
(entry 2, Table 1) (signal k0 is attrib-
uted to the ACH2AOH end-group).
[Color figure can be viewed in the
online issue, which is available at
www.interscience.wiley.com.]
silyl ether functions has been realized. The acetylation reac-
tion has been conducted in THF with a large excess of acetic
anhydride ([Ac2O]0/[OH]0 ¼ 30) in the presence of N,N0-
dimethylaminopyridine (DMAP) and triethylamine as catalyst
and acidic quencher, respectively. Reactions proceeded at 50
ꢀC for 48 h, then poured in a large excess of methanol to
selectively precipitate the end-capped polymers. The effec-
tiveness of the reaction has been evidenced by 1H NMR by
disappearance of the triplet initially present at 3.6 ppm cor-
responding to the a-hydroxymethylene end-group protons
along with the appearance of a new signal at 2 ppm corre-
sponding to the methyl protons (Hl) of the acetyl groups
(Supporting Information Fig. S3). As far as SEC analyses are
concerned, they have been performed using both RI and UV
(260 nm) detectors. To that end the three-arm star-shaped
PCL samples have been previously treated with phenyl iso-
cyanate in THF. Phenyl isocyanate is known to readily react
with any alcohol group if present, resulting in a phenyl car-
bamate formation.35 The covalently linked phenyl groups are
readily probed at 260 nm. Interestingly, UV detection did not
show any absorption peak on the SEC polymer traces, then
attesting for quantitative acetylation reactions.
initially present at about 1 ppm and between 7.3 and 7.8
ppm corresponding to the tert-butyl and the phenyl protons
of the TBDPS group, respectively (Supporting Information
Fig. S3). Interestingly, SEC traces present narrow molecular
weight distributions without any trace of degradation
(Mn app ¼ 2400 g molꢁ1; PDI ¼ 1.1; Mn app ¼ 6400 g molꢁ1
;
PDI ¼ 1.23 for entries 1 and 2, respectively, in Table 1).
Finally, primary alcohol groups of the star-PCL-OH have been
converted into a 2-bromoisobutyrate moiety by reaction with
an excess of 2-bromo-2-methylpropionyl bromide (1.5 eq.) in
THF at 50 ꢀC for 48 h in presence of triethylamine and
DMAP as quencher and catalyst, respectively. At the end of
the reaction, triethylammonium bromide salts were filtered
off and the functionalized polymers (star-PCL-Br) were selec-
tively precipitated in cold methanol and dried until constant
1
weight. Completion of the reaction has been evidenced by H
NMR in CDCl3 from the disappearance of the hydroxymethy-
lene protons at 3.6 ppm (Supporting Information Fig. S3, Hg)
to the benefit of the new methyl protons appearing at 1.9
ppm (data not shown here).
The synthesis of mikto-arm copolymers has been performed
by conducting the ATRP of the noncommercially available
1,2;3,4-di-O-isopropylidene-6-O-methacryloyl-D-galactopyranose
(MAIGP) monomer using star-PCL-Br as macroinitiator and a
catalytic complex of copper bromide (CuBr) ligated with
The fourth step has consisted on the selective deprotection
of all silyl ethers to regenerate the primary alcohol group on
the glucoside moiety. Practically, the deprotection has been
performed accordingly to a literature procedure, using a tet-
rabutyl ammonium fluoride (TBAF) solution in THF in the
presence of acetic acid.36 After 21 hours, polymers were
selectively precipitated in cold methanol, filtered off and
dried at 40 ꢀC till constant weight. Recovering yields were
between 85 to 95% while deprotection proved quantitative
as attested by 1H NMR with the disappearance of the signal
1,1,4,7,10,10-hexamethyltriethylene
tetramine
(HMTETA)
([star-PCL-Br]0/[CuBr]0/[HMTETA]0 ¼ 1/1/2). Polymerizations
have been promoted in THF at 60 ꢀC for different reaction
times and three different polymerization degrees (DPs) in
MAIGP (25, 50, and 100) (Table 2). As evidenced, the poly-
merization of MAIGP successfully proceeded from both star-
PCL-Br macroinitiators with conversions higher than 75%.
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TABLE 2 Molecular Characteristics of the Mikto-arm Copolymers as Obtained by ATRP of MAIGP from Star-PCL-Br
Mn star-PCL-Br
Time
(h)
Targeted
Conv. in
MAIGP (%)a
Mn th
Mn exp
(g molꢁ1
)
DP MAIGP
(g molꢁ1 b
)
(g molꢁ1 c
)
Mw/Mn
d
Entry
1
2
3
4
a
2,700
2,700
6,400
6,400
2
4
4
8
25
50
75
99
91
86
c
8,600
18,700
21,200
34,500
8,700
20,000
20,900
35,100
1.19
1.28
1.34
1.22
50
100
As determined by gravimetry. Conv. (%) ¼ (Mrec – MS-PCL-Br – Mcata)/
MMAIGP x 100 where Mrec, Mstar-PCL-Br, Mcata are the masses of recovered
crude copolymer, star-PCL-Br macroinitiator and catalytic complex,
Experimental molecular weight as determined by 1H-NMR (see text).
As determined by size exclusion chromatography (SEC) in THF at 35
d
ꢀC using PS standards.
respectively.
b
Theoretical molecular weight: Mn
conv. x MwMAIGP).
¼
Mn
þ (DPtargeted
x
th
star-PCL-Br
Polymerization reactions took place in a well-controlled man-
ner as attested for by the excellent correlation between theo-
retical and experimental number average molecular weights.
1H NMR analysis allowed for determining the experimental Mn
of the four samples by comparing the anomeric protons of the
MAIGP repeating units at 5.5 ppm (Fig. 2, Hp) to the methyl-
ether protons of the a-methylglucoside core clearly appearing
at 3.4 ppm (Hb). Experimental average number molar masses
have then been determined as:
SEC traces characterized by low polydispersity indices (from
1.19 to 1.34) and a clear shift to lower elution volumes
attesting for the mikto-arm formation from both macroinitia-
tors (Figs. 3 and 4).
The ultimate step has consisted in the complete and selective
removal of the isopropylidene protective groups from the
pendant sugars all along the polymethacrylate backbone to
generate the amphiphilicity of the mikto-arm copolymers. Prac-
tically, mikto-arm copolymers were treated by an aqueous
solution of formic acid (80%) for 48 hours and dialyzed
against water. The white fluffy samples were recovered in
good yields (75 to 90%) and characterized by 1H NMR spec-
troscopy where the spectra showed the complete
Mn exp ¼ Ip=ðIb=3Þ ꢃ Mw MAIGP þ Mn starꢁPCL-Br
Moreover, the control over the ATRP reactions using star-
PCL-Br as macroinitiator is fully confirmed by monomodal
FIGURE 2 1H NMR spectrum (CDCl3) of
a mikto-arm copolymer based on three-
arm star-shaped PCL and MAIGP (see
entry 2, Table 2).
SYNTHESIS OF AMPHIPHILIC A3B MIKTO-ARM COPOLYMERS, SURIANO, COULEMBIER, AND DUBOIS
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
FIGURE 4 SEC traces of the initial three-arm star-shaped PCL
macroinitiator (Mn ¼ 6300 g molꢁ1; blue curve) and two mikto-
arm copolymers obtained via ATRP (red curve ¼ entry 3, Table
2; green curve ¼ entry 4, Table 2).
FIGURE 3 SEC traces of the star-shaped PCL-Br macroinitiator
(Mn ¼ 2700 g molꢁ1; blue curve) and two corresponding mikto-
arm copolymers obtained after MAIGP ATRP (red curve ¼ entry
1, Table 2; green curve ¼ entry 2, Table 2).
and 2 focus on the CMC determination of samples based on
the star-PCL-Br with the lower molar mass (Mn ¼ 2700 g
molꢁ1; DP ¼ 18) while entries 3 and 4 concern samples
disappearance of the isopropylidene protons along with a gen-
eral upfield shift of sugar ring protons without any modifica-
tion of the other integrated signal values (data not shown
here). Under such prevailing conditions FTIR spectroscopy evi-
denced for the selective deprotection of the isopropylidene
protecting groups. FTIR spectrum of the deprotected mikto-
arm copolymers confirms the presence of AOH groups as evi-
denced by a broad absorption around 3400 cmꢁ1 along with
isopropylidene ether signals disappearance (m ¼ 1160 cmꢁ1),
while ester linkages (m ¼ 1730 cmꢁ1) remained untouched
with no evidence of carboxylate signals (data not shown here).
based on star-PCL-Br with the higher molar mass (Mn
¼
6400 g molꢁ1; DP ¼ 51). In both cases, increasing the length
of the polymethacrylate backbone bearing the pendant
hydrophilic sugars while maintaining a same PCL length
resulted in an increase of the observed CMCs. This can be
readily explained by a higher solubility of the resulting unim-
ers in solution due to higher sugar content in the
copolymers.
Copolymers (entries 2 and 3) are both composed by a poly-
methacrylate backbone with quite similar length (DPpolymeth
The as-obtained amphiphilic mikto-arm copolymers were
finally studied in aqueous solution to determine whether
they self-organize or not as stable micelles with dimensions
low enough to be potentially used as drug delivery systems.
To that end the critical micellization concentrations (CMC)
along with the size of the micelles have been investigated by
UV analysis and dynamic light scattering (DLS) experiments.
To determine the CMC of the amphiphilic copolymers, pyrene
has been used as molecular probe.37 Table 3 summarizes the
CMC values obtained for amphiphilic copolymers along with
their hydrophilic-lipophilic balance (HLB) as calculated by
the Griffin relationship.38 Although all CMCs are determined
in the range of some mg/L, variations in compositions may
be highlighted for the different samples. In Table 3, entries 1
¼
50 and 46, respectively), while the overall MnPCL
increases. A higher PCL content thus reduces the solubility
of the unimers in water, promoting self-organization and for-
mation of micelles at lower concentration. Finally, copoly-
mers with different compositions but having roughly the
same HLB may also be compared. Comparison of samples in
entries 1 and 3 highlights a lower CMC value for the second
copolymer, thus with the higher overall molecular weight.
Here we might consider that for the second sample, PCL
chains are longer and thus less soluble in water. Moreover,
longer PCL chains are also more likely to crystallize in the
micelle core thus diminishing the CMC as well.
TABLE 3 Dependence of HLB, CMC, and Micelle Mean Diameter as a Function of Amphiphilic Mikto-arm Copolymer Composition
Entry
MnPCL (g molꢁ1
)
MnPolymeth (g molꢁ1
)
HLBa
CMC (mg/L)b
Dh (nm)c
1
2
3
4
a
2,700
2,700
6,400
6,400
4,700
12,400
11,400
21,300
9.2
11.9
9.3
3.7
5.4
1.6
2.4
73 6 0.19
61 6 0.36
56 6 0.26
46 6 0.36
11.2
b
As determined by the Griffin relationship: HLB ¼ 20 x MH/(MH þ ML)
where MH and ML are the total molecular weights of the hydrophilic
and lipophilic segments, respectively.
As determined by pyrene encapsulation (Supporting Information Figs.
S4 and S5).
c
As determined by Dynamic Light Scattering with [Copolymer] ¼ 50 mg/L.
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ARTICLE
low CMCs along with low and narrow average sizes when
forming micelles in solution.
This work was partially supported by both the Re´gion Wal-
lonne and Fonds Social Europe´en in the frame of Objectif 1-Hai-
naut: Materia Nova program. O.C. is Research Associate and F.S.
is ‘‘aspirant’’ for the Belgian F.N.R.S. LPCM thanks the ‘‘Belgian
Federal Government Office Policy of Science (SSTC)’’ for general
support in the frame of the PAI-6/27.
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