Note: Descriptions are shown in the official language in which they were submitted.
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LIVING RADICAL POLYMERIZATION INITIATOR COMPRISING A FUNCTIONAL GROUP CAPABLE
OF
REACTING WITH POLYEPTIDES OR THE LIKE, COMB POLYMER OBTAINED THEREP7ITH,
POLYPEPTIDE CONJUGATES ANL7 DRUGS OBTAINED THEREFROM.
The invention relates to processes of making comb polymers from monomers
comprising
alkoxy polyethers, such as polyalkylene glycol such as poly (ethylene glycol),
or
polytetrahydrofuran (PTHF). Such methods may include the use of an initiator
compound
which comprises a moiety which, when attached to the comb polymer, is capable
of
binding to a protein or polypeptide. The initiator compounds and finished comb
polymers,
and their uses, are also included within the invention.
The modification of proteins with polymers such as poly (ethylene glycol),
which is known
by the abbreviation PEG, is well-known in the art. PEG-derivatives are
manufactured, for
example, by Shearwater Corporation, Huntsville, AL., USA, and Enzon, Inc.,
Bridgewater,
NJ., USA. Uses of PEG are reviewed in catalogues from both of those companies,
and
indeed in the 2002 Enzon, Inc. Annual Report.
The attachment of PEG to proteins or polypeptides, known as PEGylation has
been found
to have a number of benefits. Firstly, this reduces the antigenicity and
immunogenicity of a
molecule to which PEG is attached. PEG also produces markably improved
circulating
half lives i~z vivo due to either evasion of renal clearance as a result of
the polymer
increasing the apparent size of the molecule to above the glomerular
filtration limit, and/or
through evasion of cellular clearance mechanisms. PEG can mark~.bly improve
the
solubility of proteins and polypeptides to which it is attached, for example
PEG has been
found to be soluble in many different solvents, 'ranging from water to many
organic
solvents such as toluene, methylene chloride, ethanol and acetone. An
application of this
has been to use PEG-modified antibodies, for example to phase partition target
molecules
or cells. PEGylation has also been found to enhance proteolytic resistance of
the
conjugated protein, and improve bioavailability via reduced losses at
subcutaneous
injection sites. PEGylation also has been observed to reduce the toxicity of
the proteins or
polypeptides to which it is attached, improve thermal and mechanical stability
of the
molecules and allow the improved formulation into materials used for some slow
release
administration strategies. These advantages are reviewed in, for example, the
articles by
Chapman A.P. (Advanced Drug Delivery Reviews, Vol. 54 (2002), pages 531-545).
The
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chemistry of polypeptide and protein PEGylation is further reviewed in the
article by
Roberts, M.J., et al. (Advanced Drug Delivery Reviews, Vol. 54 (2000, pages
459-476),
and the article by I~instler, O., et al. (Advanced Drug Delivery Reviews, Vol.
54 (2002),
pages 477-485).
A number of PEGylated drugs are on the market, For example, PEG-INTRONTM is an
a-interferon product produced by Schering-Plough and Enzon, Inc. which is used
to treat
hepatitis C and cancer. ProthecanTM is a PEG-enhanced version of camptothecin,
a
topoisomerase I inhibitor that is effective against some cancers. PEGylated
taxol and
several enzyme-based products have also been produced which show, for example,
better
uptake in tumours and reduced side-effects compared to non-PEGylated
compounds. As
discussed in the review by Roberts (Supra), polymers such as PEG may be
attached via a
number of reactive amino acids on protein or polypeptide molecules, including
lysine,
cysteine, histidine, arginine, aspartic acid, glutamic acid, serine,
threonine, tyrosine
N-terminal amino groups and C-terminal carboxylic acid groups. In the case of
glycoproteins, vicinal hydroxyl groups can be oxidised with periodate to form
two reactive
formyl moieties. A wide range of functional groups may be attached to
compounds such as
PEG to allow them to attach to lysine aW ine groups and N-terminal amine
groups. These
include succinimidyl succinate, hydroxysucciiiamide and hydroxysuccinamide
esters,
aldehyde derivatives such as propionaldehyde and acetaldehyde, propionate and
butanoate
derivatives of succinimidyl, benzotriazole carbonate, p-nitrophenyl carbonate,
trichlorophenyl carbonate and carbonylimidazole. Compounds such as tresylate
are known
to bind to proteins via nucleophilic attack. There are also a number of
compounds which
can react with cysteine residues on proteins or polypeptides. These include
rrialeimides,
v;,~inylsulphones, pyridyl sulphides and iodoacetamides. Furthermore,
succinimidyl
carbonate can also be used as a functionalised group to attach PEG or other
polymers to
alanine or histidine amino acids within a protein or polypeptide. As already
indicated, the
reaction of such functionalised groups is already well-characterised as
indicated in the
articles by Roberts, Kinsler and Chapman, and indeed as shown in, for example,
the
Shearwater Catalogue (2001).
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The PEG currently on the market is usually in the form of long poly (ethylene
glycol)
polymers or branched or star-shaped poly (ethylene glycols).
The Applicants have now identified that it is possible to produce comb
polymers which
allow the size of the polymer attached to biological substances, for example,
proteins and
polypeptides, nucleic acids (DNA and RNA), carbohydrates and fats, to be
varied and to be
controlled. This allows the possibility of producing a wide variety of
different polymers
for attaching to proteins and polypeptides, which may be varied in their size
and
hydrodynamic volume to vary the properties of the compound to which the
polymer is
attached. For example, this may be used to vary the stability, solubility,
toxicity and/or
drug retention time of a drug which has been covalently attached to such co-
polymers.
Such co-polymers are capable of being produced in a controlled manner by so-
called living
radical polymerisation.
Living radical polymerisation is subject of International Patent Application
No. WO
97/47661. Supported polymerisation catalysts and specific polymerisation
initiators are
also shown in WO 99/28352 and WO 01/94424. Basically, the system uses a
compound
complexed with a transition metal. This compound is preferably an
organodiimine,
although one of the nitrogens of the diimine is preferably not part of an
aromatic ring (e.g.
a 1,4-diaza-1,3-butadiene, a 2-pyridinecarbaldehyde imine, an oxazolidone or a
quinoline
carbaldehyde).
Living free radical systems, which involve the use of free radical initiators
are also known,
see for example WO 96/30421 and WO 97/18247. This is reviewed in I~amigaito,
et al.,
Chem. Rev. (2001), Vol. 12, pages 3689-3745.
A combination of the catalyst and the initiators has in the past been used to
polymerise
olefinically un-saturated monomers, such as vinylic monomers. The inventors
have now
realised that these systems may be used to produce comb polymers in a
controlled manner.
These comb polymers may have a ,functional group attached to them via
conventional
chemistry. However, the inventors have also realised that the initiators used
in living
radical polymerisation are attached to the comb polymer as a result of the
reaction of the
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initiator with the monomers. This means that it is possible to functionalise
the comb
polymer at the same time as producing the co-polymer, by using a
functionalised initiator.
Accordingly, the first aspect of the invention provides a method of producing
a comb
polymer comprising the steps of:
(a) Providing:
(i) a plurality of monomers which are linear, bxanched or
star-shaped, substituted or non-substituted, preferably containing 2,
especially from 3 to 10, carbon atoms, and have an olefinically unsaturated
moiety attached thereto, the olefinically unsaturated moiety being capable of
undergoing addition polymerisation;
(ii) an initiator compound; the initiator compound comprising a
homolytically cleavable bond;
(iii) a catalyst capable of catalysing the polymerisation of the monomer;
and
(b) Causing the catalyst to catalyse, in combination with the initiator, the
polymerisation of a plurality of the monomers to produce the comb polymer;
wherein the initiator compound (ii) comprises a moiety which, when attached to
the comb
polymer, is capable of binding to a biological substance.
The monomers in (i) are preferably alkoxy polyethers such as poly (alkylene
glycol) or
polytetrahydrofuran.
The comb polymer may have a moiety which, when attached to the comb polymer,
is
capable of binding e.g. a protein or polypeptide, attached to it using
conventional
chemistry. However, as already indicated, it is possible to produce initiator
compounds
hich have that moiety attached to them. Therefore, preferably the initiator
compound
comprises a moiety which, when attached to a comb polymer, is capable of
binding to a
biological substance, such as a protein or polypeptide, nucleic acid (DNA or
RNA),
carbohydrates or fats.
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Preferably, the poly (alkylene glycol) is a polymer of an alkylene glycol
containing from
2-10, especially at least 3, carbon atoms, most preferably poly (ethylene
glycol), poly
(propylene glycol) or poly (butylene glycol). For example, poly (ethylene
glycol) may be
used.
In its most common form, this is a linear or branched polyether terminated
with hydroxyl
groups. This is synthesised by anionic ring opening polymerisation of ethylene
oxide
initiated by nucleophilic attack of a hydroxide ion on the epoxide ring. It is
also possible to
modify polyethylene glycol, for example by placing a monomethoxy group on one
end to
produce monomethoxy PEG (mPEG). This is synthesised by an ionic ring opening
polymerisation initiated with methoxide ions and is commercially available.
However,
trace amounts of water present in the reaction mixture causes the production
of significant
~vantities of PEG which is terminated at both ends by hydroxy groups. This is
undesirable,
as the moiety capable of binding to proteins or peptides will then attach to
both ends of the
polymer chain, which will cause unwanted cross-linking of proteins in the
body.
A method intended to minimise the production of this impurity is to initiate
the ring
opening of ethylene oxide by nucleophilic attack of a benzoxy ion on the
epoxide ring. In a
similar manner to the above process, monobenzoxy PEG is produced, as well as
the PEG
chain terminated at both ends by hydroxy. This mixture is methylated,
producing one chain
terminated with Bz0 and OMe, and dimethoxy PEG. Hydrogenation of this mixture
eliminates the benzoxy group to yield mPEG and dimethoxy PEG. Dimethoxy PEG
remains present as an inert impurity. However, even using this process, the
product
obtained still contains 5-10% of the unwanted dihydroxy PEG according to its
certificate of
analysis.
The process of the present invention yields a product which is substantially
100% pure,
eliminating substantially all of the dihydroxy PEG impurity, thus avoiding the
disadvantages of the known processes, and removing the possibility of the
cross-linking of
proteins.
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Branched and star-shaped polymers such as PEG are available from a number of
commercial sources, such as Enzon and Shearwater. Polytetrahydrofurans may
also be
obtained from commercial sources, such as Aldrich (Gillingham, Dorset, UK.).
Preferably, the molecular weight of the PEGmethacrylate is 475, 1100, 2080,
5000 or
20,000.
The polyalkylene glycol and polytetrahydrofuran comprises an olefinically
unsaturated
moiety, for example at the end of the polymer chain. This olefinically
unsaturated moiety
is capable of undergoing additional polymerisation.
The olefmically unsaturated monomer may be a methacrylate, an acrylate, a
styrene,
methacrylonitrile or a dime such as butadiene.
Examples of olefinically unsaturated moieties that may be used include methyl
methacrylate, ethyl methacrylate, propyl methacrylate (all isomers), butyl
methacrylate (all
isomers), and other alkyl methacrylates; corresponding acrylates; also
functionalised
methacrylates and acrylates including glycidyl methacrylate, trimethoxysilyl
propyl
methacrylate, allyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl
methacrylate,
dialkylaminoalkyl methacrylates such as dimethylethylamino methacrylate;
fluoroalkyl
(meth)acrylates; methacrylic acid, acrylic acid; fumaric acid (and esters),
itaconic acid
(and esters), malefic anhydride: styrene, a,-methyl styrene; vinyl halides
such as vinyl
chloride and vinyl fluoride; acrylonitrile, methacrylonitrile; glycerol;
vinylidene halides
of formula CHz = C(Hal)z where each halogen is independently Cl or F;
optionally
substituted butadienes of the formula CHz = C(R'S) C(R'S) = CHz where R'S is
independently H, Cl to C10 alkyl, Cl, or F; sulphonic acids or derivatives
thereof of
formula CHz = CHSOzOM wherein M is Na, K, Li, N(R'6)d where each R'6 is
independently H or C, to Coo alkyl, COZ, ON, N(R'6)z or SOZOZ and Z is H, Li,
Na, K or
i'~1(R'~)a; acrylamide or derivatives thereof of formula CHz = CHCON(R'6)z and
methacrylamide or derivative thereof of formula CHz = C(CH3)CON(R'6)z.
Mixtures of such monomers may be used.
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Such unsaturated moieties may be attached, for example, at an end of the
polymer, by
conventional chemistry. Alternatively, such monomers may be obtained
commercially.
For example, PEGacrylate, diacrylate, methacrylate and dimethacrylate are
commercially
available from Aldrich (Gillingham, Dorset, UK.).
The unsaturated moiety may be attached to the polyalkylene glycol or
polytetrahydrofuran
by means of any suitable linkage groups, for example via a methyl ether
linkage. Hence, it
is possible to use poly (ethylene glycol) methyl ether methacrylate (available
from Aldrich
Chemicals). One advantage of using the living radical polymerisation technique
is that
commercially available compounds such as this, which have free-radical
inhibitors, such as
hydroquinones, may be used without further purification. With conventional
free-radical-based systems the presence of a free-radical inhibitor will
inhibit the addition
polymerisation reaction. This is not the case with living radical
polymerisation.
The initiator compound may comprise a homolytically cleavable bond with a
halogen atom.
This may contain a bond that breaks without integral charge formation on
either atom by
homolytic fission. As described in WO 97/01589, WO 99128352 and WO 01/94424,
it is
believed that true free-radicals do not appear to be formed using some
catalysts. It is
believed that this occurs in a concerted fashion whereby the monomer is
inserted into the
bond without formation of a discrete free-radical species in the system. That
is, during
propagation this results in the formation of a new carbon-carbon bond and a
new
~::arbon-halgen bond without free-radical formation. A free-radical which is
an atom or
group of atoms having an unpaired valance electron and which is a separate
entity without
interactions, is not produced by the interaction of the initiator compound
with the monomer
~~iiih which it interacts.
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g
Suitable initiatar compounds are described in, for example, WO 97/47661.
However, it is
preferable that the initiator compound also comprises a moiety which, when
attached to the
comb polymer, is capable of binding to a protein or polypeptide. These
moieties are known
in the art, as indeed described in Roberts, et crl. (Supra), ~Chapman (Supra)
and, for
example, in the catalogues of Enzon and Shearwater.
The initiator may be a thioester or xanthate. These are used in so-called RAFT
(Reversible
Addition Fragmentation chain transfer and nitric oxide mediated
polymerisation) and
MADIX catalysation. The initiators and their reactions are described in WO
99/31144,
WO 98/01478 and US 6,153,705.
Preferably, the initiator compound (ii) is selected from:
A-S-C(O)-R, A-S-C(S)-O-R, R-S-C(O)-A, R-S-C(S)-O-A, where R is C, to Coo
substituted
or non-substituted, straight chain, branched chain, cyclic, heterocyclic or
aromatic alkyl;
A-B-X
A
0\
A ~ ~.,/X 0 ~p~x A g
0 ~ 0
0
o x
A
A g
B
a si gA
A 8 "-----
B
o A
x
~zgA cnBA
X
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a- B A
BA
AB s x -o
ao ~Y
~ Q'_' B A
where: X = a halide, especially Cl or Br,
A = a moiety which, when attached to the comb polymer, is capable of binding
to a protein ar polypeptide,
B is a linker and may or may not be present.
A is preferably selected from succinimidyl succinate, N-hydroxy succimimide,
succinimidyl propionate, succinimidyl butanoate, propionaldehyde,
acetaldehyde, tresylate,
triazine, vinylsulfone, benzotriazole carbonate, maleimide, pyridyl sulfide,
iodoacetamide
and succinimidyl carbonate.
The linker is preferably selected from a C, to Czo substituted or non-
substituted, straight
chain, branched chain cyclic, heterocyclic or aromatic alkyl group; -.(CHZZ)a
CHI-,
-CH2ZCHz-, -(CH~CHZZ)~ R, -(CH~CH(CH~)Z)n R, -(CHZ)b-C(O)-NH-(CHz)~ ,
-(CHa)~-NH-C(O)-(CHz),; , -N(R)~-; -S-; N-R; or -O-R; where R = C, to Czo
substituted
or non-substituted, straight chain, branched chain cyclic, heterocyclic or
aromatic alkyl, Z
is O or S, and n, a, b and c are independently selectable integers between l
and 10.
Preferably, the linlcer contains I, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
Most preferably,
the linker is methyl, ethyl, propyl, butyl or pentyl.
Preferably, the moiety which is capable of reacting with the protein or
polypeptide has the
formula:
ct
N-i-
-O--~~ /N
N
ct
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-O-SO; CFi_CF3 ,
0 0
-O-C-O-N
O
0 N N
-O-C-O-N
O _
O C O ~ ~ N0.
C1
O
II
° °
0
-O-C-CH_CHi C-O-N
s
O
O
17
II
-O(Cj~i)"C-O-N
where n = integer of 0 to 10
O
0
O
-O(CFi,)mCEIC-O-:~' where m = integer of 0 to 10, Y is an aliphatic or
y 0 ~ aromatic moiety
O
II
-VFiCCH,I
-S-S
N ,
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O
0
,o' ~ N=N
0-N
and
t~~
1 ro 1 or bu 1 X is a
where R is H, methyl, ethy , p py ty ,
halide, especially Cl or Br.
Most preferably, the initiator (ii) has a formula:
0
~ ,~:'~,~ ~.
4
0
0
0
x
N~~ ~ where n is an integer of 0 to 10, and X is a halide,
especially Cl or Br.
O
The initiator has a compound selected from:
Ohle 0 0
_ O Hr
N~N / 0 9r \ ~N- . N.0 Br
\ ° s °
Cf N N J 0 p O
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O
Me HO
H
N-O O
-Br
O O , O
S
O
0
O Br
O
O
H
O Br
-O
Br
-O O O
O
O
O
N~\~O~ Br
O
O O
O
Boc NH~O
s
Br
O
N
~O
O
O Br
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The catalyst may be capable of catalysing the polymerisation reaction by
living radical
polymerisation (see e.g. WO 97!47661) or living free radical polymerisation
(see e.g. WO
96/30421, WO 97/18247 and Kamagaito M., et al., Chem. Rev. (2001), Vol. 101
(12),
pages 3689-3745).
Preferably the catalyst comprises a ligand which is any N-, O-, P- or S-
containing
compound which can coordinate in a ~-bond to a transition metal or any carbon-
containing
compound which can coordinate in a ~-bond to the transition metal, such that
direct bonds
between the transition metal and growing polymer radicals are not formed.
The catalyst may comprise a first compound
MY
where: M is a transition metal having an oxidation state which is capable of
being
oxidised by one formal oxidation state,
Y is a mono, divalent or polyvalent counterion.
The catalyst may also be defined by the formula:
LML ~n+ An
.1m
where: M = a transition metal having an oxidation state which is capable of
being oxidised by one formal oxidation state,
L - an organodiimine where at least one of the nitrogens of the diimine
is not part of an aromatic ring,
A - anion,
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n - integer of 1 to 3,
m = an integer of 1 to 2.
The metal ion may be attached to a coordinating ligand, such as (CH; CN)a. Y
may be
chosen from Cl, Br, F, I, NO,, PFD, BF4, SOø, CN, SPh, SCN, SePh or triflate
(CF:~ S03).
Copper (I) triflate may be used. This is available in the form of a
commercially available
benzene complex (CF~S03Cu)zC~Hb.
The especially preferred compound used~is CuBr.
A may be F, Cl, Br, I, N, Oi, S04 or CuXz (where X is a halogen).
The transition metal may be selected from Cu+, Cu2k, Fe2+, Fej+, RuZ+, Ru3+,
Crz+, C~+,
Mo2+, Mo'+, WZ+, W~+, Mn3+ Mn4+, Rh3+, Rh4+, Rea+, Re3+, Co+, Co2+, VZ+, V3~,
2n+, Znz+,
Au+, Auz+, Ag+ and Agz+.
Preferably the organodiimine has a formula selected from:
a 1.~4-~iiaza-l.a-butadiene
a ~-pyridine carbaldehl'de imine
a
N
R2
Ra
~s
R'
~N
R9
R10
R5
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an ~~azolidone.
o~~
~N ~N
r
Ri2
or a quinoline carbaldehvde
\
\ ~ /
'~ il
v
~ Eia
where R,, Rz, R,o, R",. R,z and R,~ may be varied independently and R,, Rz,
R,o, R", R,z and
R, ~ may be H, straight chain, branched chain or cyclic saturated alkyl,
hydroxyalkyl,
carboxyalkyl, aryl (such as phenyl or phenyl substituted where substitution is
as described
for R4 to R~) CHzAr (where Ar = aryl or substituted aryl) or a halogen.
Preferably R,, Rz,
R,o, R", R,z and R,3 may be a C, to Czo alkyl, hydroxyalkyl or carboxyallcyl,
in particular C,
to Cd alkyl, especially methyl or ethyl, n-propylisopropyl, n-butyl, sec-
butyl, tart butyl,
cyclohexyl, 2-ethylhexyl, octyl decyl or lauryl.
Pre~e:red li~ands include:
.,
'w ~ i :V
N
Et n'CsHu
Furmula ZS Formni~ ?9 ~-.--.---1~ '~
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16
w
~N . . N
~~H't
Formula 31 Formula 3~ Formula 33
11 W1
a
c
H N~ N
n-C~Ht3 n-C~$ts n'~sHt~
Forriula 3~ . Formula 35 Formula 3b
Iw . w w
E
I
tl=C9~ ~
n..C n~~~
Formv j ~ ~7 Formula 38
Fon~n~ta 3~
/N .
.
( R1 ~ (s7
Formula :~0 Fonnuia ~.1 Formula 42
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17
1 0 ~.
t R S7 'N (7~--
N
* L~.i
H ~ a-CøH~
r
Fonaula .43 ~ ~ Formula 44 Formula 45
0 0 . ,.,, w
i
cs_s~ ~ 1 ~t
N~,.
~l~u
Formula 46 Formula ~?
«.
_ 1'N
Formula A$ off
14
.W N
N
Formula 49
OH
.
V
7
Formula ~0
~, cooH
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1~
ana
N
~N
Formula ~ 1
where: ~ indicates a chirai centre
R14 - Hydrogen. C; to C;o branched chain alkyl. carbox~r- or
hydroxy- C ~ to C~ o alkyl.
Preferably the catalyst is
.. IV
with Cu Br
C31~~
Preferably the organodiimine is N-(n-propyl)-2-pyridylmethanimine (NMPI), N-
ethyl-2-
pyridyl methanimine ox N-(n-ethyl)-2-pyridylmethanimine.
Other catalysts are described in WO 96/30421 and WO 97/18247.
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19
Preferably the catalyst comprises a bipyridine group, such as 4,4'-di(5-nonyl)-
2.2'-bipyridyl
(dNbpy).
A plurality of different monomers as defined in part (i) of the invention may
be used. This
allows the production of statistical co-polymers.
Alternatively, or additionally, a bloclc co-polymer may be produced by
additionally
polymerising one or more different olefmically unsaturated monomers. For
example, the
olefinically unsaturated monomers may be selected from methyl methacrylate,
ethyl
~nethacrylate, propyl methacrylate (all isomers), butyl methacrylate (all
isomers), and other
alkyl methacrylates; corresponding acrylates; also functionalised
methacrylates and
acrylates including glycidyl methacrylate, trimethoxysilyl propyl
methacrylate, allyl
methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate,
dialkylaminoallcyl
methacrylates; fluoroalkyl (meth)acrylates; methacrylic acid, acrylic acid;
fumaric acid
(and esters), itaconic acid (and esters), malefic anhydride: styrene, cc-
methyl styrene; vinyl
halides such as vinyl chloride and vinyl fluoride; acrylonitrile,
methacrylonitrile;
vinylidene halides of formula CHz = C(Hal)z where each halogen is
independently Cl or F;
optionally substituted butadienes of the formula CHz = C(R'S) C(R'S) = CHz
where R'S is
independently H, C, to C,o alkyl, Cl, or F; sulphonic acids or derivatives
thereof of formula
CHz = CHSOzOM wherein M is Na, K, Li, N(R'~)4 where each R'6 is independently
H or
Cl to C10 alkyl, COZ, ON, N(R'~)z or S020Z and Z is H, Li, Na, K or N(R'6)4;
acrylamide or derivatives thereof of formula CHz = CHCON(R'6)z and
methacrylamide or
derivative thereof of formula CHz = C(CH~)CON(R'6)z.
The monomers may be polymerised prior to or after the polymerisation of the
monomers as
defined in part (I) of the invention.
The polymerisation reaction may be reactive in a number of different solvents,
such as
hydrophobic or hydrophilic solvents. These include water, propionitrile,
hexane, heptane,
dimethoxyethane, diethoxyethane, tetrahydrofuran, ethylacetate, diethylether,
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N,N-dimethylformamide, anisole, acetonitrile, diphenylether,
methylisobuiyrate,
butan-2-one, toluene and xylene.
The reaction temperature may be carried out from -20 to greater than
200°C, especially +5
to 130°C. WO 97147661, for example, shows examples of living radical
polymerisation
and the typical conditions that may be used.
Preferably, the ratio of organodiimine : transition metal is 0.01 to 1000,
preferably 0.1 to
10, and transition metal ion (as MY) : initiator is 0.0001 to 1000, preferably
0.1 to 10,
where the degree of polymerisation is controlled by the ratio of monomer to
initiator. All
ratios are given as weight : weight. Preferably the components are the
catalyst of formula:
[ML",]"+A"- (defined above) are at a ratio of catalyst : initiator of 3 : 1 to
1 : 100.
preferably, the amount of diimine : metal used in the system is between 100 :
1 and 1 : l,
preferably 5 : 1 to 1 : l, more preferably 3 : 1 to 1 : 1, by weight.
Preferably the concentration of monomer in a solvent used is 100% - 1 %,
preferably 100%
- 5%, vol. : vol.
Preferred ratios of initiator to catalyst or 1:100 - 100:1" typically 1:1.
Preferred ratios of monomer : initiator are 1:1 to 10,000:1, especially S:1 to
100:1.
The reaction may be undertaken under an inert atmosphere such as nitrogen or
argon, and
may be carried out in suspension, emulsion, mini-emulsion or in a dispersion.
Preferably, the catalyst is a supported catalyst, that is, at least a part of
the catalyst is
attached to a support. Such supported catalysts are shown in, for example, WO
99/28352.
The support may be inorganic, such as silica, especially silica gel.
Alternatively, the
support may be organic, especially an organic polymer, such as a cross-linked
organic
polymer, including poly (styrene-w-divinylbenzone). The support may be in the
form of
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21
beads. The advantage of using a supported catalyst is that it allows the
catalyst to be
removed from the system and recycled/reused.
The comb polymer may incorporate a fluorescently-labelled monomer. For
example, the
method may additionally comprise a step of copolymerising or block
polymerising with at
least one fluorescently-labelled monomer capable of undergoing addition
polymerisation.
This can be carried out simply by using a monomer which has a fluorescent
moiety, such as
fluorescein, or coumarin, attached to an olefinically unsaturated moiety. The
olefinically
unsaturated moiety may be selected from those unsaturated moieties defined
above.
Preferably, the fluorescent label is coumarin, especially coumarin 343.
Coumarin is
particularly advantageous because it allows the comb polymer to be used to
attach to
proteins and the attachment of the proteins to be visualised using a confocal
microscope.
This allows, for example, 'the detection of individual proteins or indeed the
visualisation of
whole bacterial or other cells. Indeed, initial results have indicated that
bacterial cells can
be readily visualised, using a comb polymer according to the invention, to
attach to E.coli
and Streptomyces cells.
A further aspect of the invention provides initiator compounds capable of
being used in a
living radical polymerisation reaction comprising a moiety which, when
attached to a
polymer, is capable of binding to a protein or polypeptide. Initiators for use
in a living
radical polymerisation reaction having the following formulae are also
provided:
A-S-C(O)-R, A-S-C(S)-O-R, R-S-C(O)-A, R-S-C(S)-O-A, where R is C, to Czo
substituted
or non-substituted, straight chain, branched chain, cyclic, heterocyclic or
aromatic alkyl;
A-B-X
cozBA cazBA
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22
o e--- 8 A
cad A
A B ~ :t
ac= ~x '~ ' ° =-X
0
~ BA
A
B A
B SBA
i
x A B ; ''
A ~ ~ ~ \o
0 0
° X
A
A B
BA
AB
8
A
x
where: X = a halide, especially Cl or Br,
A = a moiety which, when attached to the comb polymer, is capable of binding
to a protein or polypeptide,
B is a linlcer and may or may not be present.
Preferably, A is selected from succinimidyl succinate, N-hydroxy succimimide,
succinimidyl propionate, succinimidyl butanoate, propionaldehyde,
acetaldehyde, tresylate,
triazine, vinyl sulfone, benzotriazole carbonate, maleimide, pyridyl sulfide,
iodoacetamide
and succinimidyl carbonate.
Preferably, tile linker is selected from a C, to Cao substituted or non-
substituted, straight
chain, branched chain cyclic, heterocyclic or aromatic alkyl group; -(CHzZ)a
CHz-,
-~'HzZCH~-, -(CHzCHzZ)"-R, -(CHzCH(CH~)Z)n R, -(CH2)~-C(O)-NH-(CHz)~ ,
-(CH~)~-NH-C(O)-(CHa)Y , N(R)z-; -S-; -N-R; or -O-R; where R = C, to Czo
substituted
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23
or non-substituted, straight chain, branched chain cyclic, heterocyclic or
aromatic alkyl, Z
is O or S, and n, a, b and c are independently selectable integers between 1
and 10.
Preferably the moiety capable of reacting with a protein or polypeptide has a
formula:
ct
w
-°- y ~~y
cl
0-SO:'Cg~CF, ,
0 0
II
-O-C-p_y
n
0
O
II
-O-C-O_,~
O _
-°-
-°-~-° ~ / ct
O
_O_C_
i
O O O
-O-C-CH=CH:-C-0_
i
0
O
0
il
-O(CH2)~C-0-V where n = integer of 0 to 10
0
0 where m = integer of 0 to 10, Y is an aliphatic or
O
aromatic moiety
I
Y ~i ,
O
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24
0
-h'HC CEiiI
r~
_S_S
N-
O
-N I
i
O
4 N-N
-O-C-O-N
and
where R' is H, methyl, ethyl, propyl or butyl, X is a
N~N halide es eciall Cl or Br.
Y
X
N
preferably the initiator has a formula of
/O O ~n
~ - o c~
~ - L Jr; 0
O
,O O
,Fe
aC
O n
where n is an integer of 0 to 10, and X is a
halide, especially Cl or Br.
O
The iutiator especially has the formula:
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o~.te ~j /
v
0 ~'9r \ .l,\ ~ 0~ /Br Br
N P~ / ~ ~ N ~ N 0 I
o ~ ~-o° / \ /~/ l\
C;'~ri~N ~ ~ ! et- 1 p
0 4
O HO~
Me g ~O
N-O Br
~ O ~ O Br
i
S
O
r
O Br
O
O
H J~
p- / Br
-O
Br
_O o 0
O
0 0
OHO Br
O O
O
Boc NH~O
Br
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26
The terminal amine group may be protected by any suitable protecting group,
such as BOC.
Deprotection is achieved by addition of acid, such as trifluoroacetic acid.
Alternatively a
furan intermediate may be produced which can then be converted to maleimide.
Under normal conditions, the aldehyde-based initiators will tend to react non-
selectively
with proteins, i.e. they will react substantially equally with both terminal
nitrogen atoms
and, for example, a lysine NHz group, if the reaction conditions are not
controlled.
However, under the right reaction pKa for the particular aldehyde chosen, the
aldehyde can
be controlled to specifically target the terminal nitrogen.
A further aspect of the invention provides comb polymers capable of binding a
protein or
polypeptide obtainable by a method of the invention.
A further aspect provides a comb polymer having a general formula:
A-~)d-(E)e (F)f
where: A may or may not be present, and where present is a moiety capable of
binding to a
protein or a polypeptide,
D, where present, is obtainable by additional polymerisation of one or more
olefinically unsaturated monomers which are not as defined in E.
E is obtainable by additional polymerisation of a plurality of monomers which
are
linear, branched, or star-shaped, substituted or non-substituted, and have an
olefinically unsaturated moiety.
F, where present, is obtainable by additional polymerisation of one or more
olefinically unsaturated monomers which are not as defined in E.
d and f are an integer between 0 and 500, especially 0 to 300 or 0 to 100.
a is an integer of 0 to 1000, especially 0 to 10, 50, 100, 200, 300, 400, 500,
600,
700, 800 or 900
and wherein when A is present, at least one of D, E and F is present.
Preferred monomers used to obtain E are poly (alkylene glycol) or
polytetrahydrofuran.
.3r
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27
This includes both functionalised comb polymer and non-functionalised comb
polymer,
where the moiety capable of attaching to a protein or polypeptide may be
attached later by
other chemistry.
Preferably the comb polymer has an average total molecular weight of 2,000-
X0,000,
especially 20,000-40,000.
Examples of preferred comb polymers, obtainable according to the process of
the
invention, are:
O
O
N~ Br
O L Jx
O ~ O
1 p
O
~n
O
O
N~ Br
O L Jx
O ~ O
O
O
~n
,Me O
Br
L JX
O
3 O
H
O
~n
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2$
a
Br
HO ~.' o e~ ~ x
~O
la
0
~n
'. ° o
y Br
o x
a
0
0
~n
0
Br
i~O
~O
lO
O
~n
\ °
~-t 1 Br
C~ J~ Ix
O
, ~O
/ n
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29
a
B
-0 0 0 ~~ x r
~o
0
0
'f-_'_
0
/~ . o
N O~O Br
a L
O O
O
O
O
BocNH~~-''~O Br
O
O
O
O ~ O
N~
O Br
11 O
O
O
O
'''
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These polymers can be used either directly to react with useful biomolecules
or converted
simply into new macromolecules that will react with useful biomolecules.
The comb polymer may be fluorescently labelled, especially with a coumarin. A
still
further aspect of the invention provides a method of attaching a polymer to a
compound
comprising reacting a comb polymer according to the invention with said
compound. The
compound may be a protein or polypeptide or may indeed be any compound having
a
suitable free thiol or free amine group, depending on the initiator used. Such
compounds
include amines, such as benzylamines and ethylenediamine, amino acids and
carbohydrates
such as sugars.
Preferably such compounds are biologically-active compounds, such as drugs.
The
combination of such compounds in combination with a pharmaceutically
acceptable carrier
are also provided. The compounds may include cancer chemotherapeutic agents,
antibiotics, anti-fungal and/or immunosuppressants.
For example, Figures 23 and 24 show HPLC traces and SIBS-PAGE for the reaction
of
lysozyme with a polymer prepared according to the invention. These figures
clearly
illustrate the progress of the reaction as the polymer selectively conjugates
to only one of
lysozyme's seven amino groups.
A still further aspect of the invention provides a method of fluorescently
labelling a
compound, virus, microorganism or cell comprising the step of reacting the
compound,
virus, microorganism or cell with a fluorescently labelled comb polymer
according to the
invention. The use of a comb polymer as a fluorescent label is also provided.
The fluorescently labelled comb polymer may be used to attach antibodies which
in turn
nay be used to selectively bind to pre-defined antigens. This allows the
selective labelling
of the compounds to take place.
Methods of producing such antibodies are well-known in the art and indeed
monoclonal
antibodies may be produced by the well-known Kohler-Milstein method.
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31
Previously, when polymers have been used to bind to proteins, they have had to
be of a low
molecular weight, as a polymer with a molecular weight of e.g. 20,000 could
not be
excreted from the body by the liver. To combat this problem, four polymers of
approximately 5,000 molecular weight each were bound to the protein, and
eventually
excreted without problem. An advantage that is provided by the comb polymers
of the
invention is that they can possess molecular weights of 20,000 and still be
bound to the
proteins without the problems of excretion found with conventional polymers.
This is due
to an ester linkage which is found in each "finger" of the comb polymer.
Preliminary
results show that this ester linkage is readily hydrolysed by enzymes, causing
the forgers to
gradually break off the main polymer backbone. This enables a 20,000 molecular
weight
polymeer to become smaller over time until it reaches a molecular weight which
enables it
to be excreted by the liver. Conventional chain polymers cannot offer this
advantage but
remain in the bloodstream without being excreted.
Initial results indicate that the comb polymers of the invention are stable
over weeks in rat
serum, but slowly break down in the manner detailed above.
The invention will now be described by way of example only with reference to
the
following examples:
Figure 1 shows the evolution of molecular weight distribution and
polydispersity for the
LRP (living radical polymerisation) of methyl methacrylate initiated by a
N-hydroxysuccinimide (NHS) initiator.
Figure 2 shows SEC curves for NHS functionalised poly (MMA), solid curve, and
the
produce (N-benzylamide functionalised poly (MMA), dashed curve).
Figure 3. First order kinetic plot for the LRP of PEGMA initiated by NHS-Br,
[PEGMA]°/[CuBr]o/[NHSBr]°/jL]° = 10/1/1/2.1 in toluene
(33% v/v) at 30°C.
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32
Figure 4. Evolution of the molecular weight distribution and polydispersity
for the
LRP of PEGMA initiated by NHS-Br,
[PEGMA]°/[CuBr]°/[NHSBr]°/[L]° = 10/1/1/2.1 in
toluene (33% v/v) at 30°C.
Figure 5. Evolution of the molecular weight distribution and polydispeersity
for the
LRP of MPEG(395)MA initiated using initiator 8,
[MPEG(395)MA]o/[CuBr]o/[NHSBr]o =
10/1/1/2 in tuluene (50% v/v) at 30°C.
Figure 6. Selected region (2.7-4.3 ppm) of the 1H NMR spectrum of a NHS ester
functionalised poly(MPEG(395)MA) prepared from initiator 8 (M" = 6400 g.moln,
MW/M°
= 1.09).
Figure 7. First order kinetic plot for the LRP of MPEG(395)MA using initiator
7,
[MPEG(395)MA]o/[CuBr]o/[NHSBr]o/[Propyl Ligand]o = 10/1/1/2 ub toluene (50%
v/v) at
30°C.
Figure 8. Evolution of the molecular weight distribution and polydispeersity
for the
LRP of MPEG(395)MA using initiator 7, [MPEG(395)MA]o/[CuBr]o/[NHSBr]o/[Propyl
Ligand]o = 1011/1/2 in toluene at 30°C.
Figure 9. Rate plot for TMM-LRP of MPEG(1000)MA initiator 12,
[monomer] : [initiator] : [CuCI] : [L] = 5 :1:2:2, T = 70°C.
Figure 10. Dependence of M" on conversion for MPEG(1000)MA initiator 12,
[~~a.onomer] : [initiator] : [CuCI] : [L] = 5:1:1:2, T = 70°C.
Figure 11. Rate plot for TMM-LRP of MPEG(1000)MA initiator 12,
[monomer] : [initiator] : [CuBr] : [L] = 20:1:1:2, T = 50°C.
Figure 12. Dependence of Mn on conversion for TMM-LRP of MPEG(1000)MA
initiator 12, [monomer]:[initiator]:[CuBr]:[L] = 20:1:1:2, T = 50°C.
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Figure 13. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14,
[monomer] : [initiator] : [CuBr] : [L] = 6:1:1:2.
Figure 14. Dependence of Mn on conversion for TMM-LRP of MPEG(395)MA using
initiator 14, [monomer] : [initiator] : [CuBr] : [L] = 6:1:1:2.
Figure 15. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14,
[monomer] : [initiator] : [CuBr] : [L] = 28 :1:1:2. T = 40°C.
Figure 16. Dependence of Mn on conversion for TMM-LRP of MPEG(395)MA using
i~~.gtiator 14, [monomer] : [initiator] : [CuBr] : [L] = 6:1:1:2. T =
40°C.
Figure 17. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14,
[monomer] : [initiator] : [CuBr] : [L] = 28:1:1:2. T = 60°C.
~9igure 18. Dependence of Mn on conversion for TMM-LRP of MPEG(395)MA using
initiator 14, [monomer] : [initiator] : [CuBr] : [L] = 6:1:1:2. T =
60°C.
Figure 19. Online 'H NMR experiment: Rate plot for TMM-LRP of MPEG(395)MA
using initiator 14, [monomer] : [initiator] : [CuBr] : [L] = 10:1:1:2. T =
40°C.
Figure 20. Rate plot for TMM-LRP of MPEG(395)MA using initiator 15,
[monomer] : [initiator] : [CuBr] : [L] = 8:1:1:2. T = 30°C.
l~sigure 21. Dependence of M" on conversion for TMM-LRP of MPEG(395)MA using
initiator 15, [monomer] : [initiator] : [CuBr] : [L] = 8 :1:1:2. T = 3
0°C.
Figure 22. Kinetic plot for the hydrolysis of N-succinimidyl terminated
poly(MPEG(395)MA initiated by 8 in different buffers.
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34
Figure 23. HPLC traces for the reaction of succinimide terminated
poly(MPEG(395)MA) prepared from initiator 8 (M" = 6400 g.mol'', MW/M" = 1.11)
with
Lysozyme ([polymer] / [lysozyme] 20:1).
Figure 24. SDS-PAGE for the conjugation of lysozyme with succinimide
terminated
poly(MPEG(395)MA) prepared from initiator 8 (M" = 6400 g.mol'', MW/M" = 1.11)
(20
equivalents).
Figure 25. HPLC traces for the reaction of succinimide terminated
poly(MPEG(395)MA) prepared from initiator 8 (M" = 6400 g.mof', MW/Mn = 1.11)
with
~,ysozyme ([polymer] / [lysozyme] 5:1).
Figure 26. HPLC traces for the reaction of succinimide terminated
poly(MPEG(395)MA) prepared from initiator 8 (M" = 6400 g.mofl, MW/Mn = 1.11)
with
Lysozyme ([polymer] / [lysozyme] 2:1).
Figure 27. Comparison of the HPLC traces of various conjugates of lysozyme
obtained
with different ratios of polymer/lysozyme using succinimide terminated
poly(MPEG(395)MA) prepared from initiator 8.
Figure 28. Kinetic plot for the hydrolysis of the succinimide end group of
poly(MPEG(395)MA) polymer initiated by 7 in different buffers.
Figure 29. 'H NMR spectrum of a NHS ester functionalised (initiator 7)
poly(MPEG(395)MA) (M" = 2700 g.mol'', MW/M" =1.12).
Figure 30. 'H NMR spectrum of a N-benzylamide functionalised
poly(MPEG(395)MA) (M" = 2800 g.moY', MW/Mn = 1.15).
Figure 31. HPLC traces for the reaction of poly(MPEG(395)MA) prepared from
initiator 7 (M" = 2700 g.mof', MW/Mn = 1.12) with lyso~yme ([polymer] /
[lysozyme] 30:1).
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Figure 32. SDS-PAGE for the conjugation of poly(MPEG(395)MA) prepared from
initiator 7 with lysozyme at different reaction time and different ratio
polymer / protein (a)
5/1, (b) 10/1 and (c) 30/1.
Figure 33. SEC-HPLC chromatography of the conjugation reaction of Lysozyme
with
the aldehyde-terminated polymer (Mn~22,000, PDi 1.09).
Figure 34. Retro-Diels-Alder reaction: ( ~ _ "initiator" and ~ = maleimido
signals) a) t
=0; b)t=3.S h; c)t=7h.
synthesis of N [2-(2'-bromo-2'-methylpropionyloxy)-ethyl]phthalimide, 6.
N (2-hydroxyethyl)phthalimide (Aldrich, 99%) (19.12 g, O.lmol) was dissolved
in
anhydrous THF (250 mL) with triethylamine (28.1 mL, 0.2 mol) under nitrogen in
a 500
mL round-bottomed flask equipped with a magnetic stirrer. The flask was cooled
to 0°C
with an ice bath before the dropwise addition of 2-bromoisobutyryl bromide
(13.9 mL, 0.11
mol). The mixture was stirred for 45 minutes and allowed to reach room
temperature.
Subsequently the reaction mixture was poured into an excess of cold water and
extracted
with diethyl ether (3 x 50 mL). The organic layer was washed with a saturated
aqueous
solution of NazCO3 (3 x 50 mL), acidified water (pH = 4.5, 3 x 50 mL), and
again the
saturated aqueous solution of NazC03 (3 x 50 mL). The organic layer was dried
over
anhydrous MgS04 and filtered. Finally the solvent was removed under reduced
pressure by
using the rotary evaporator in order to isolate the title compound (30.6 g,
yield 90 %) as a
yellowish solid.
m.p. 63-65°C, IR (solid, ATR cell) v (crri') 1774 (C~,,~,=O), 1705
(C=O); 'H NMR (CDCl3,
X98 K, 300MHz) 8 1.81 (s, 6H, C(CH3)zBr), 3.95 (t, 2H, J-- 5.3 Hz, CHzN), 4.35
(t, 2H, J=
5.4 Hz, CH20), 7.67 (m, 2H, CH Ar), 7.78 (m, 2H, CH Ar). 13C NMR (CDCl3, 298
K, 75
MHz) 8 31.00 (2C, C(CH3)zBr), 37.12 (1C, CHzN), 55.92 (1C, C(CH3)zBr), 63.42
(1C,
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CH~O), 123.78 (2C, CH Ar), 132.35 (1C, CAr), 133.54 (2C, CH Ar), 168.40 (2C,
C~,,~,=O),
171.87 (1C, C=O).
Synthesis of N (2-bromo-2-methylpropionyloxy) succinimide, 7.
JJ
N-0 Br
O
This was prepared from N hydroxysuccinimide (NHS) using a similar procedure to
that
given above for the synthesis of compound 6. The solvent used in this case was
anhydrous
dichloromethane as NHS is insoluble in THF. The title compound was obtained in
85
yield as a white solid.
m.p. 72-74°C; IR (solid, ATR cell) v (cm') 1772 (C~y~i=O), 1728 (C=O);
'H NMR (CDCl3,
298 K, 300 MHz) ~ 2.08 (s, 6H, C(CH3)ZBr), 2.87 (s, 4H, CHZ). '~C NMR (CDCl3,
298 K,
75 MHz) ~ 26.03 (2C, CHZ), 31.09 (2C, C(CH3)ZBr), 51.60 (1C, C(CH~)ZBr),
167.89 (1C,'
C=O), 169.02 (2C, C~,,~,=O); MS (+EI], (mJz) 266, 265, 156, 151, 149, 123,
121, 116, 115,
91, 87, 70, 69. Anal. Calcd for C$H,oNOaBr: C = 36.39; H = 3.82; N = 5.30, Br
= 30.26.
Found: C = 36.35; H = 3.82; N = 5.03; Br = 30.17.
4-[(4-chloro-6-methoxy -1,3,5-triazin-2-yl)amino]phenol, 4.
A solution of 2,4-dichloro-6-methoxy-1,3,5-triazine Z9 (9.00 g, 50.0 mmol) in
100 mL of
acetone was cooled to 0 °C and, under stirring, solid 4-aminophenol
(5.46 g, 50.0 mmol)
was added in small portions over ca. 2 min. The white suspension was then left
to warm to
ambient temperature and stirred for further 1 h, whilst being neutralized with
a 2 M
aqueous solution of Na2COs. The mixture was then poured into 500 mL of
ice/water and
the resulting white precipitate was filtered and dried, to give 9.60 g (38.0
mmol, yield 76%)
of 4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenol that can be used for
the next
step without fiuther purifications. An analytical sample was obtained by flash
chromatography (CC, Si02, petroleum ether/ EtzO 1:1, Rf= 0.14). The NMR
analysis
~~G-DMSO) revealed the presence, in solution, of 2 rotational isomers (molar
ratio 7:3).
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37
m.p. 172 °C dec.; IR v~~~ 3476 cni'. v~oH> 3269 cm'.
Major isomer'H NMR (db-DMSO, 298K, 300 MHz) 8 3.94 (s, 3H, OCH3); 6.79 (d, J =
8.8
Hz, 2H, CH Ar), 7.48 (d, ,I = 8.8 Hz, 2H, CH Ar), 9.40 (s, 1 H, OH), 10.46 (s,
1 H, NH);
'3C{'H} NMR (d~-DMSO, 298K, 75 MHz) ~ 55.52 (1C, OCH3); 115.46 (2C, CH Ar),
123 .91 (2C, CH Ar), 129.49 ( 1 C, C Ar), 154.44 ( 1 C, C Ar), 164. 81 ( 1 C,
C Ar), 169.5 7
( 1 C, C Ar), ,171.23 ( 1 C, C Ar).
Minor isomer'H NMR (d6-DMSO, 298K, 300 MHz) 8 3.96 (s, 3H, OCH3); 6.79 (d, J =
8.9
Hz, 2H, CH Ar), 7.39 (d, .I = 8.9 Hz, 2H, CH Ar), 9.42 (bs, 1 H, OH), 10.10.32
(s, 1 H,
NH);'3C{'H} NMR (d6-DMSO, 298 K, 75 MHz) 8 55.10 (1C, OCH3); 115.46 (2C, CH
Ar), 123.03 (2C, CH Ar), 129.26 (1C, C Ar), 154.76 (1C, C Ar), 165.20 (1C, C
Ar), 170.48
(1C, C Ar), 170.64 (1C, C Ar); Anal. Calcd for C10H9C1N4O2: C = 47.54, H =
3.59, N =
22.18, Cl = 14.03, Found: C = 47.57, H = 3.55, N = 22.10, Cl = 14.8.
~i,-[a~~-ehloro-6-methoxy-1,3,5-triaziri-2-yl)amino]phenyl2-bromo-2-
methylpropionate, 5
OMe .
N- '_N / ~ Br
~ ( ~ .
CI N N
A solution of 2-bromoisobutyryl bromide (1.0 mL, 7.90 mmol) in 20 mL of THF
was
added dropwise to a solution of 4-[(4-chloro-6-methoxy-1,3x5-triazin-2-
yl)amino]phenol
(1.9 g, 7.52 mmol) and triethylamine in 100 mL of THF, at -10 °C.
During the addition (ca.
15 min) precipitation of triethylammonium bromide was observed. The reaction
was
monitored by TLC (Si02, petroleum ether/ Et20 1:1, 4-[(4-chloro-6-methoxy
-1,3,5-triazin-2-yl)amino]phenol (starting material) Rf= 0.14;
4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenyl 2-bromo-2-
methylpropionate
(final product) Rf = 0.26). After 1.5 h the white suspension was poured into a
conical flask
containing 150 mL of Et~O and the ammonium salt removed by filtration on a
sintered
glass frit. The solvent was then evaporated at reduced pressure to give a
white crude
residue that was suspended in 10 mL of pentane and filtered. We obtained 2.56
g (6.37
mmol, yield 85%) of 4-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)amino]phenyl
2-bromo-2-methylpropionate as a white solid. The'H NMR analysis (d~-DMSO)
revealed
the presence, in solution, of 2 rotational isomers (molar ratio 7:3).
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38
m.p. 107-108 °C; IR v~~ 3365 crri'. v~C=o~ 1747 cm '.
Major isomer: 'H NMR (d6-DMSO, 298 K., 400 MHz) 8 2.OS (s, 6H, C(CH3)zBr),
3.96 (s,
3H, OCH3), 7.17 (d, J = 8.9 Hz, 2H, CH Ar) , 7.77 (d, J = 8.9 Hz, 2H, CH Ar),
10.78 (s,
1H, NH);'3C{'H} NMR (d6-DMSO, 298 I~, 100.6 MHz) 8 30.42 (2C, CH3), SS.7S (bs,
1C,
OCH~), 57.29 (IC, C(CH3)zBr), 121.96 (2C, CH Ar), I22.12 (2C, CH Ar), 136.29
(1C, C
Ar), 146.61 (bs, 1 C, C Ar), 165.10 (bs, 1 C, C Ar), 169.89 (bs, 1 C, C Ar), ,
170.16 ( 1 C,
C=O), I 71.33 (bs, 1 C, C Ar).
Minor isomer: 'H NMR (d6-DMSO, 298 I~, 400 MHz) 8 2.OS (s, 6H, C(CH3)zBr),
3.96 (s,
3H, OCH3), 7.17 (d, J = 8.9 Hz, 2H, CH Ar) , 7.69 (d, J = 8.9 Hz, 2H, CH Ar),
10.66 (s,
1H, NH);'3C~'H} NMR (d6-DMSO, 298 I~, 100.6 MHz) 8 30.42 (2C, CH3), SS.7S (bs,
1C,
OCH3), 57.29 (1C, C(CH3)zBr), 121.96 (2C, CH Ar), 122.73 (2C, CH Ar), I36.29
(1C, C
Ar), 146.61 (bs, 1 C, C Ar), 165.10 (bs, 1 C, C Ar), 169.89 (bs, 1 C, C Ar), ,
170.16 ( 1 C,
C=O), 171.33 (bs, IC, C Ar).
Typical Polymerisation of MMA.
CuBr (0.134 g, 0.934 mmol) was placed in an oven-dried Schlenk tube. The tube
was fitted
with a rubber septum, evacuated and flushed with dry Nz three times. Methyl
methacrylate
(10 mL, 93.4 mmol) and xylene (20 mL) were transferred to the tube via
degassed syringe.
The mixture was stirred rapidly under nitrogen and N (n-propyl)-2-
pyridylmethanimine
(NMPI) (0.408 g, 1.86 mmol) was added which imparted a deep red/brown colour
to the
solution. Appropriate initiator (0.934 mmol) was added and the resulting
solution was
degassed by three freeze-pump-thaw cycles. The resulting mixture was placed in
a
thermostatically controlled oiI bath at 90 °C. Samples were taken
periodically for
conversion and molecular weight analysis. Conversion was measured by
gravimetry by
drying to constant weight in a vacuum oven at 70 °C. The catalyst was
removed from the
samples by passing through a column of activated basic alumina prior to SEC.
(see Figure
1).
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Table 1 Polymerisation of MMA in Xylene Solution (33°Jo v/v) at 90
°C
Initiator [MMA]/[Cu(I)Br].[Culll)Br_![iJ1.1P1 [Initiator] Time \I° PDi
Com~. I:,,[Pol"]'
\Im ~ mol'' °~o ' 10' c'
S 100/110~~.~1' DSO 3300 1.14 6G
6 IOO/l/O/ll2.l 600 . 5900 1.20 75 0Ø17
19000
7 37iIID/112.1 1?S .800 L0~ 89 0.32
~5800~
7 60/0.95/0.05!12.1 '-8S0 3200 1.04 37 p,22
13100")
EiBr 10011/0!'Jl 2SSU '_500 1.16 71
I_~i001
° l5,[Pol*] = rate constant of propagation x [active propagating
polymer chains] from first order kinetic plot.
6 determined by the'H NMR peak intensity ratio on a BrUker DPX 300 MHz
'N~(n-octyl)-2-pyridylmethanimine used as the ligand
mole % HEMAl90 mole % MMA
Typical Polymerisation of Styrene.
~'.;~Br (0.055 g, 0.38 mmol) was placed in an oven dried Schlenk tube. The
tube was fitted
with a rubber septum, evacuated and flushed three times with dry Nz. Styrene
(10 mL, 96
mmol) was transferred to the tube via degassed syringe. The mixture was
stirred rapidly
under nitrogen and 4,4'-di(5-nonyl)-2.2'-bipyridyl (dNbpy) (0.314 g, 0.768
mmol) was
added, imparting a deep red/brown colour to the solution. Initiator 7 (0.035
g, 0.048 mmol,
0.192 mmol of initiating sites) was added and the resulting solution was
degassed by three
freeze-pump-thaw cycles. The resulting mixture was placed in a
thermostatically controlled
oil bath at 110 °C for 4.5 hours. The catalyst was removed from the
samples by passing
through a column of activated basic alumina prior to SEC.
Kinetic studies for initiators 6 and 7.
Samples were removed periodically using degassed syringes and quenched in
liquid
nitrogen for conversion and molecular weight analysis. Conversion was
determined by
NMR on a Bruker DPX 300. For Living Radical Polymerisation initiated by 6,
samples
were passed over a basic alumina column and then filtered in a syringe
equipped with a
0.22 ~.m hydrophobic filter prior to molecular weight studies. In the case of
LRP initiated
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by 7, molecular weight was determined by diluting the sample with THF and
letting it
settle overnight to precipitate the catalyst residues. 'The upper liquid was
then filtered with
a 0.22 pm hydrophobic filter. This method was chosen for
N-hydroxysuccinimide-functionalised polymers as these polymers could not be
passed over
basic alumina.
Synthesis of a N-benzylamide functionalised poly(MMA).
Benzylamine was added to a solution of N-hydroxysuccinimide terminated
poly(methyl
methacrylate) in anhydrous THF. N-hydroxysuccinimide-terminated poly(methyl
methacrylate).(M" = 3200 g mol-', PDI =1.06) (1.00 g, 0.313 mmol) and three
equivalents
of ~~enzylamine (0.100 mL, 0.938 mmol) were dissolved in 10 mL of dry THF in a
dry
Schlenk and stirred at 50°C for 3 days under nitrogen. After reaction,
the polymer was
precipitated in cold petroleum ether (see Figure 2).
This shows that N-benzylamide functional groups may be added and can be used
to reach
with free amide groups of the sort found in proteins.
0
3Z
N-O t THF
~ 5D'Cf7dsys ( /
O O ~ / O
Scheme Coupling of a N-hydroxysuccinimide terminated poly(MMA)
with benzylamine.
O
O Br
O
N-hydroxysuccinimide initiator ('~ (NHS-Br)
Reagents.
Polyethylene glycol) methyl ether methacrylate (Mn = ca 475, Aldrich, 99%) and
anhydrous toluene was degassed by bubbling with dry nitrogen for 30 minutes
before use.
The ligand N-(~-propyl)-2-pyridylmethanimine was prepared as described
previously'.
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Copper(I) bromide (Avocado, 98°1°) was purified as necessary by
a method based on that of
Keller and Wycof~. Other reagents were all commercial products and used
without further
purification.
Typical procedure.
Polymerizations were carried out at 30°C mediated by copper(I)
bromide /
N-(n-propyl)-2-pyridylmethanimine. A typical polymerization recipe is based on
33°1° v/v
monomer in toluene. The ratio of initiator/Cu(I)Br/ligand is 1/1/2.1 on a
molar basis. A dry
Schlenk tube was charged with Cu(I)Br (0.3099 g, 2.16x10'3 mol), NHS-Br (7)
(0.5704 g,
2.16x10'3 mol) and a magnetic bar prior to being deoxygenated by cycling
between nitrogen
and vacuum three times. To the flask was then added PEGMA (10 ml, 2.27x10'2
mol) and
toluene (20 ml). The mixture was immediately subjected to three freeze-pump-
thaw
degassing cycles. Finally N-(n-propyl)-2-pyridylmethanimine (0.707 ml,
4.54x10'3 mol)
was added and the flask was placed in an oil bath thermostatted at
30°C.
Kinetic studies.
S~nples were removed periodically using degassed syringes and quenched in
liquid
nitrogen for conversion and molecular weight analysis. Conversion was
determined by
NMR on a Broker DPX 300 MHz. Molecular weight was determined by diluting the
sample with toluene and allowing it to settle down overnight to remove the
copper
complexes. The upper liquid was then filtered with a 0.22~,m hydrophobic
filter. This
method was chosen because of the difficulty encountered to pass the polymer
over a basic
a.lumina column. Number average molecular weights (Mn) were determined by Size
Exclusion Chromatography (SEC) in a system fitted with a 5 mm guard column,
two
Polymer Labs mixed E columns, a differential refractive index detector, and an
auto
sampler. The system was eluted with THF at a rate of 1 mL/min. Toluene was
used as the
m-low marker.
Purification.
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N-hydroxysuccinimide functionalised poly(PEGMA) were purified by two
consecutive
purifications from a Toluene solution in diethyl ether.
Table 1. Kinetic data for the polymerisation of PEGMA initiated by NHS-Br in
toluene solution
(33% v/v) at 30°C ([PEGMA]o/[CuBr]°/[NHSBr]°/[L]°=
10/1/1/2.1).
Time Conversion M~, exP MW/M~ ~ Mn, the b
(h) (%) (g.mol-') (g mof')
1 8.9 2350 1.10 450
2 18.4 2860 1.26 920
3 27.1 3100 1.20 1360
4 34.7 3600 1.13 1730
17 80.8 5670 1.06 4040
" determined by SEC analysis calibrated with Poly(MMA) standards - THF. "
M",",~" _ ([M]" l (I]" X M. W.MMn x Conv.) I 100.
Table 2. Characterisation of Poly(PEGMA) prepared by LRP
Kp[Pol*]a M~, exp b MW/M~ M~, theo b
(h-~) (g.mol-') (g mof')
NHS-Poly(PEGMA) 0.096 6200 1.05 4040
" Kp[Pol*] = rate constant of propagation. " determined by SEC calibrated with
Poly(MMA) standards - THF (stabilised with topanol).
M~,n~." _ ([M]n / [I]" x M.W.MMA x COnV.) / 100.
References
(a) D. M. Haddleton, M. C. Crossman, B. H. Dana, D. J. Duncalf, A. M. Henning,
D.
I~ukulj and A. J. Shooter, Mac~omolecules,1999, 32, 2110.
(b) R. N. Keller and W. D. Wycoff, luorg. Synth., 1947, 2, 1.
Polymerisation of methoxypolyethyleneglycol methacrylate (2080) using the
initiator
derived from N-hydroxy succinimide
~PEGJI~IJI~CuJI~LJ =19.2/1/1/2 in 80% toluene solution (AJ U2-27a) @30"C
N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Gu(I)Br (0.027 g, 0.189
mmol, 1
eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight
= 2080,
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43
7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried
Schlenk tube.
The Schlenk tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (28 mL) was added to the Schlenk tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.05
g, 0.38 mmol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 30°C (t=0) and samples were removed periodically for conversion and
molecular weight
analysis. Conversion was followed by 1H NMR spectrometry and molecular weight
analysis by SEC.
The polymer was purified by the dropwise addition of the reaction solution to
a vigorously
stirred solution of diethyl ether (400 mL). The resulting white powder was
filtered,
dissolved in toluene (20mL) and precipitated in diethyl ether (400 mL). This
procedure was
repeated three times.
Table 1: Data for the polymerization of methoxypolyethyleneglycol methacrylate
(2080)
with an initiator derived from N-hydroxy succinimide at 30 °C in 80%
toluene solution.
Sample Time Conversion° Mn'' PDi°
/ minutes l
gg 4 3380 1.04
291 9 9820 1.09
901 17 10030 1.07
1369 23 11080 1.07
2760 26 12610 1.07
3965 28 14830 1.04
" ~:onversion was determined using 1H NMR ~ Molecular mass determined by SEC
using
PMMA standards.
Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate)
was
freeze dried prior to use to remove all water.
[PEG]/[I]/[Cu]/[L] =19.2/1/1/2 in 80% toluene solution (AJ U2-27b)
@50°C
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N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189
mmol, 1
eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight
= 2080,
7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried
Schlenk tube.
The Schlenk .tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (28 mL) was added to the Schlenk tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.05
g, 0.38 mmol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 50°C (t=0) and samples were removed periodically for conversion and
molecular weight
analysis. Conversion was followed by 'H NMR spectrometry and molecular weight
analysis by SEC.
The polymer was purified by the dropwise addition of the reaction solution to
a vigorously
stirred solution of diethyl ether (400 mL). The resulting white powder was
filtered,
dissolved in toluene (20mL) and precipitated in diethyl ether (400 mL). This
procedure was
repeated three times.
Table 2: Data for the polymerization of methoxypolyethyleneglycol methacrylate
(2080)
with an initiator derived from N-hydroxy succinimide at 50 °C in 80%
toluene solution.
Sample Time Conversion° Mn'' POi''
/ minutes !
gg 7 8700 1.06
2gg 12 10920 1.07
ggg 24 14450 1.05
1367 33 15810 1.04
2758 45 20220 1.07
3962 53 23180 1.07
"Conversion was determined using 1H NMR h Molecular mass determined by SEC
using
PMMA standards.
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Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate)
was
freeze dried prior to use to remove all water.
[PEG]/(I]/[Cu]/[L] =19.211/1/2 in 80% toluene solution (AJ U2-27c)
@90°C
N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189
mmol, 1
eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight
= 2080,
7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried
Schlenk tube.
The Schlenk tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (28 mL) was added to the Schlenle tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.05
g, 0.38 mmol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 90°C (t=0) and samples were removed periodically for conversion and
molecular weight
analysis. Conversion was followed by 'H NMR spectrometry and molecular weight
analysis by SEC.
The polymer was purified by the dropwise addition of the reaction solution to
a vigorously
stirred solution of diethyl ether (400 mL). The resulting white powder was
filtered,
dissolved in toluene (20mL) and precipitated in diethyl ether (400 mL). This
procedure was
repeated three times.
Table 3: Data for the polymerization of methoxypolyethyleneglycol methacrylate
(2080)
with an initiator derived from N-hydroxy succinimide at 90 °C in 80%
toluene solution.
Sample Time Conversion" Mnn PDih
/ minutes /
86 18 11100 1.08
289 26 14870 1.08
899 31 17900 1.08
1367 35 18110 1.09
2758 38 18110 1.09
3962 39 18240 1.08
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"Conversion was determined using 1H NMR h Molecular mass determined by SEC
using
PMMA standards.
Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate)
was
freeze dried prior to use to remove all water.
~PEGJI~IJI~CuJI~LJ = 23.9/1/1/2 in 66% toluene solution (AJ U2-ll)
@90°C
N-hydroxy succinimide initiator, (2.5 g, 9.47 mmol), Cu(I)Br (1.35 g, 9.47
mmol, 1 eq) and
methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 628,
142.0 g,
0.226 mol), and a magnetic follower were placed in an oven dried Schlenk tube.
The
S chlenk tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (261 mL) was added to the Schlenk tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N propyl-2-
pyridylmethanimine (2.80
g, 0.019 mol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 90°C (t=0) and samples were removed periodically for conversion and
molecular weight
analysis. Conversion was followed by 'H NMR spectrometry and molecular weight
analysis by SEC.
The polymer was purified by the dropwise addition of the reaction solution to
a vigorously
stirred solution of diethyl ether (1000 mL). The resulting oil was washed with
diethyl ether
(3 x 1000 mL) and then dried in vacuo.
Table 4: Data for the polymerization of methoxypolyethyleneglycol methacrylate
(628)
with an initiator derived from N-hydroxy succinimide at 90 °C in 66%
toluene solution.
Sample Time Conversion° ' Mn° PDi''
/ minutes /
48 21 4449 1.11
132 40 7198 1.08
185 44 7779 1.07
245 46 8105 1.09
300 48 8331 1.09
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Conversion was determined using 1H NMR h Molecular mass determined by SEC
using
PMMA standards.
Bisomer MPEGSSOMA was used as provided.
Polymerisation of methoxypolyethyleneglycol methacrylate (1080) using the
N-hydroxy succinimide derived initiator
[PEG]/[I]/[Cu]/[L] =13.9/1/1/2 in 66% toluene solution (AJ U2-13) @90°C
N-hydroxy succinimide initiator, (0.526 g, 1.99 mmol), Cu(I)Br (0.29 g, 2.02
mmol, 1 eq)
and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight =
1080,
29.62 g, 0.027 mol), and a magnetic follower were placed in an oven dried
Schlenk tube.
The Schlenk tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (60 mL) was added to the Schlenk tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.51
g, 3.96 mol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 90°C (t=0) and samples were removed periodically for conversion and
molecular weight
analysis. Conversion was followed by 'H NMR spectrometry and molecular weight
analysis by SEC.
The polymer was purified by the dropwise addition of the reaction solution to
a vigorously
stirred solution of diethyl ether (1000 mL). The resulting oil was washed with
diethyl ether
(3 x 1000 mL) and then dried in vacuo.
Table 5: Data for the polymerization of methoxypolyethyleneglycol methacrylate
1080)
with an initiator derived from N-hydroxy succinimide at 90 °C in 66%
toluene solution.
Sample Time Conversion° Mnh PDih
/ minutes /
1250 47.3 12180 1.16
2460 50.4 12460 1.16
3890 52.8 12540 1.20
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"Conversion was determined using 1H NMR h Molecular mass determined by SEC
using
PMMA standards.
~PEGJI~IJI~CuJI~LJ = 9.3/1/1/2 ivy 66% toluene solution (AJ U2-I S)
@90°C
N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89
mmol, 1 eq) and
methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 1080,
18.90
g, 0.018 mol), and a magnetic follower were placed in an oven dried Schlenk
tube. The
Schlenk tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (35 mL) was added to the Schlenk tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.51
g, 3.79 mmol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 90°C (t=0) and samples were removed periodically for conversion and
molecular weight
analysis. Conversion was followed by 'H NMR spectrometry and molecular weight
analysis by SEC.
Table 6: Data for the polymerization of methoxypolyethyleneglycol methacrylate
(1080)
with an initiator derived from N-hydroxy succinimide at 90 °C in 66%
toluene solution
Sample Time Conversion° Mnh PDi"
!minutes /
4160 88.7 9870 1.22
" Conversion was dertimined using 1H NMR h Molecular mass determined by SEC
using
PMMA standards.
Bisomer S l OW (50% aqueous solution of methoxypolyethyleneglycol
methacrylate) was
freeze dried prior to use to remove all water.
Polymerisation of methoxypolyethyleneglycol methacrylate (628) using the N-
hydroxy
succinimide derived initiator
~PEGJI~IJI~CuJI~LJ = 6.4/1/1/2 in 66% toluene solution (A,J U2-31a)
@30°C
N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89
mmol, 1 eq) and
methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 628,
7.57 g,
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0.012 mol), and a magnetic follower were placed in an oven dried Schlenk tube.
The
Schlenlc tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (14 mL) was added to the Schlenk tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.51
g, 3.79 mmol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 30°C (t=0) and samples were removed periodically for conversion and
molecular weight
analysis. Conversion was followed by 'H NMR spectrometry and molecular weight
analysis by SEC.
Table 7: Data for the polymerization of methoxypolyethyleneglycol methacrylate
(628)
with an initiator derived from N-hydroxy succinimide at 30 °C in 66%
toluene solution.
Sample Time Conversion° Mnh PDi''
/ minutes /
60 19 2850 1.04
131 32 3230 1.10
199 45 3560 1.12
250 53 3760 1.12
298 56 3980 1.12
" Conversion was determined using 1H NMR h Molecular mass determined by SEC
using
PMMA standards.
Bisomer MPEGSSOMA was used as provided.
~PEGJI~IJI~CuJI~LJ = 6.4/1/1/2 in 66% toluene solution (AJ U~-31 b) @50"C
N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89
mmol, 1 eq) and
n~ntl~oxypolyethyleneglycol methacrylate (PEG) (average molecular weight =
628, 7.57 g,
0.012 mol), and a magnetic follower were placed in an oven dried Schlenk tube.
The
Schlei~lc tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (14 mL) was added to the Schlenle tube. The resulting solution was
deoxygenated
via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.51
g, 3.79 mmol) was added. The reaction was placed in a thermostatically
controlled oil bath
at 50°C (t=0) and samples were removed periodically for conversion and
molecular weight
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analysis. Conversion was followed by 'H NMR spectrometry and molecular weight
analysis by SEC.
Table 8: Data for the polymerization of methoxypolyethyleneglycol methacrylate
(628)
with an initiator derived from N-hydroxy succinimide at 50 °C in 66%
toluene solution.
Sample Time Conversion Mnh PDih
/ minutes /
59 39 3212 1.09
126 56 3958 1.11
195 69 4375 1.13
246 75 4649 1.13
295 82 4874 1.13
°Conversion was determined using 1 H NMR. hMolecular mass determined by
SEC using
PMMA standards.
Bisomer MPEGSSOMA was used as provided.
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Experimental
General experimental
For all following polymerisations conversion data was obtained by 1H NMR
spectroscopy and molecular weight data (Mn and PDi) by SEC using PMMA
standards.
Methoxypolyethyleneglycol methacrylates were obtained from Sigma-Aldrich or
Laporte Performance Chemicals and used either as received (IVIPEG(395)MA: Mn
475 g mol-1 and BISOMER MPEG(550)MA: Mn 628 g mol-1) or freeze dried prior to
use to remove all water (BISOMER S10W MPEG(1000)MA: Mn 1080 g mol-1 and
BISOMER S20W MPEG(2000)MA: Mn = 2080 g mol-1).
The ligands N-(h-alkyl)-2-pyridylinethanimine were prepared as described
previously.l Copper(I) bromide was purified as necessary by a method based on
that
of Kelley and Wycoff.~
All other reagents were obtained from either Sigma-Aldrich, Romil, Fisher or
Acros
and used as received.
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Functional initiators
The following table lists the functional initiators used to polymerise the
methoxypolyethyleneglycol methacrylates. .
Table 1: Functional initiators.
Initiator code Initiator structure
O
Me H
8 N-O
Br
O O
O
Me Me
7 N-O
Br
O O
,Me
Cl
HO~
O
9
f
O Br
O
6 ~ ~ N~O
O
O Br
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i
s
O
O Br
O
11
H
~s
O Br
-O
Br
1~ -O O O
O
O
O
13 N~~O~,O Br
O '' ~O
O
14 BocNH~~O
Br
~r
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Functional polymers
The following table lists the functional polymers prepared using
methoxypolyethyleneglycol methacrylates and the initiators shown in Table 1.
These
polymers can be used either directly to react with useful biomolecules or
converted
simply into new macromolecules that will react with useful biomolecules.
Table 2: Functional polymers.
Initiator used Polymer structure
O
NCO 3r
O O
8
O
O
N~ Br
O L Jx
O O
O
O
~n
O~Me O
~ O x Br
N- \ N
~ ~ O
C1 N N O
H
O
~n
O
~n
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O
HO~. Br
O ~~ ~x
~O
O
O
~r
O
O
"'- N. Br
x
O ~O
O
O
~n
O
O x Br
I ~ ~o
~o 0
0
~n
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0
Br
O
,11 ~O
O
O O
.-O O O ~ x
Br
12 ~ O
O
O
O
O Br
O
I3 O O
O
O
~n
O
BocNH~~O ~ Br
14
O
O
~n
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O
O O
NCO ~ Br
15 O ~ ~ ~O
O
O
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Preparation of initiators and intermediates
Preparation of initiator 8
N hydroxysuccinimide-2-bromopropiohate
O
Me g
N-O
Br
O O
g -.
N-hydroxysuccinimide (4.51 g, 39.22 mmol) and 2-bromopropionic acid (2.9 mL,
32.68 mmol) were dissolved in anhydrous DCM (1000 ml) in a 2000 mL round-
bottomed flask under nitrogen equipped with a magnetic stirrer. The flask was
then
cooled to 0°C with an ice bath before the dropwise addition of a
solution of N,N'-
Dicyclohexylcarbodiimide (6.70 g, 32.68 mmol) in 50 mL of anhydrous DCM. After
addition, the mixture was stirred at room temperature overnight. The reaction
mixture
was then filtered and the solvent evaporated to give a yellow solid that was
purified
by flash chromatography (CC, Si02, Et2O, Rf ~esc°~~ = 0.31). Obtained
7.2 g (28.91
mmol, 74%) of product as a white solid. Melting point: 69-70°C. 1H NMR
(CDC13) 8
(ppm) 1.96 (d, 3H, CH(CH )Br, J = 6.78 Hz), 2.86 (s, 4H, H~Y~~), 4.61 (q, 1H,
CH(CH3)Br, J= 7.03 Hz). 13C NMR (CDC13) ~ (ppm) 21.67 (1C, CH(CH3)Br) 25.74
(2C, C~Y~i), 34.97 (1C, CH(CH3)Br), 166.17 (1C, C=O), 168.69 (2C, C~Y~~=O). IR
(solid, ATR cell) v (cm 1) 1808, 1781 (C~y~]=O), 1729 (C=O). Mass spectroscopy
(+EI, m/z) 248.964. Elem. Anal. Theoretical for C~H8N0øBr: C, 33.62; H, 3.22;
N,
5.60. Found: C, 33.47; H, 3.16; N, 5.46.
Preparation of initiator 7
N hydroxysuccinimide-~-bromo-2-metl:ylpropionate
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O
Me Me
N-O
Br
O O
7
N-Hydroxysuccinimide (11.51 g, 0.1 mol) was dissolved in anhydrous
dichloromethane (100m1) with triethylamine (28.1mL, 0.2 mol) under nitrogen in
a
250m1 round-bottomed flask equipped with a magnetic stirrer. The flask was
cooled
to 0°C with an ice bath before the dropwise addition of 2-bromo-2-
methylpropionyl
bromide (13.9 mL, 0.11 mol). Next the mixture was stirred for 45 minutes and
allowed to reach room temperature. After this the reaction mixture was poured
into an
excess of cold water and extracted with diethyl ether (3 x 50 mL). The organic
layer
was subsequently washed with a saturated aqueous solution of sodium carbonate
(3 x
50 mL), acidified water (pH= 4.5, 3 x 50 mLl), and again the saturated aqueous
solution of sodium carbonate (3 x 50 mL). The organic layer was dried over
anhydrous magnesium sulphate and filtered. Finally the solvent was removed
under
reduced pressure by using the rotary evaporator in order to isolate the title
compound
in quantitative yield as a white solid. 1H NMR (CDCl3) 8 (ppm) 2.08 (s, 6H,
C(CH )zBr), 2.87 (s, 4H, H~y~~). 13C NMR (CDCl3) 8 (ppm) 26.03 (2C, C~y~~),
31.09
(2C, C(CH3)ZBr), 51.60 (1C, C(CH3)2Br), 167.89 (1C, C=O), 169.02 (2C,
C~y~~=O).
IR (solid, ATR cell) v (cm 1) 1803, 1772 (C~y~~=O), 1728 (C=O), 1394, 1359,
1197,
1121, 1071, 996, 924, 856, 811, 731, 648. Mass spectroscopy (+EI, m/z) 266,
265,
156, 151, 149, 123, 121, 116, 115, 91, 87, 70, 69. Elem. Anal. Theoretical for
C$H1oN04Br: C, 36.39; H, 3.82; N, 5.30; Br, 30.26. Found: C, 36.35; H, 3.82;
N,
5.03; Br, 30.17. Melting point 72-74°C.
Preparation of initiator 5
2,4 Dicl:l~r~-6-metlaoacy-1,3,5-triazine3
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OMe
1~ N
~ i~
Cl' _N-
G1
To 200 ml of methanol and 2S ml of water were added 33.6 g. (0.4 mole) of
sodium
bicarbonate and 36.8 g. (0.2 mole) of cyanuric chloride. This mixture was
stirred at 30
°C for 30 minutes until the evolution of carbon dioxide had nearly
ceased, and water
was then added. The crystalline solid which separated was filtered, washed
with
water, and dried in a vacuum desiccator. The yield of crude 2,4-dichloro-6-
methoxy-
triazine was 10.5 g. (5~%), m.p. 87-89°C. After recrystallization from
heptane the
m.p. was 88-90°C. Elem. Anal. Calcd. for C4H3N3OC1~: C, 26.67; H, 1.67;
N,23.35;
C1, 39.44. Found: C, 26.96; H, 1.84; N, 23.25; Cl, 39.19.
4 ~(4-chloro-6 methoxy -1,3,5 triazin-2 yl)amihoJphenol
OH
Cl
A solution of 2,4-dichloro-6-methoxy-1,3,5-triazine (9.00 g, 50.0 mmol) in 100
mL of
acetone was cooled to 0 °C and, under stirring, solid 4-amino phenol
(5.46 g, 50.0
mmol) was added in small portion, over ca. 2 min. The white suspension was
then let
to warm to room temperature and stirred for further 1 h, while being
neutralized with
a 2 M aqueous solution of NaaCO3 during the reaction. The mixture was then
poured
into S00 mL of ice/water and the resulting white precipitate was filtered and
dried, to
give 9.6 g (38.0 mmol, yield 76%) of 4-[(4-chloro-6-methoxy -1,3,5-triazin-2-
yl)amino]phenol that can be used without further purifications. An analytical
sample
can be obtained by flash chromatography (CC, SiO2, petroleum ether/ EtaO 1:1,
Rf =
0.14). The NMR analysis (DMSO d6) reveals the presence, in solution, of 2
rotational
isomers (molar ratio 7:3). M.p was 172 °C. IR v~ NHS 3476 cm 1. v~ oH>
3269 cm 1.
Major isomer: 1H NMR (DMSO d6) 8 = 3.94 (s, 3H, OCH3); 6.79 (d, J = 8.8 Hz,
2H,
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CH Ar), 7.48 (d, J = 8.8 Hz, 2H, CH Ar), 9.40 (s, 1H, OH), 10.46 (s, 1H, NH).
'3C
NMR (DMSO d6) 8 = SS.S2 (1C, OCH3); 115.46 (2C, CH Ar), 123.91 (2C, CH Ar),
129.49 ( 1 C, C Ar), 154.44 ( 1 C, C Ar), 164.81 ( 1 C, C Ar), 169.57 ( 1 C, C
Ar), ,171.23
(1C, C Ar). Minor isomer: 1H NMR (DMSO d6) S = 3.96 (s, 3H, OCH3); 6.79 (d, J=
8.9 Hz, 2H, CH Ar), 7.39 (d, J= 8.9 Hz, 2H, CH Ar), 9.42 (bs, 1H, OH),
10.10.32 (s,
1H, NH). 13C NMR (DMSO d6) 8 = SS.10 (1C, OCH3); 115.46 (2C, CH Ar), 123.03
(2C, CH Ar), 129.26 (IC, C Ar), 154.76 (1C, C Ar), 165.20 (1C, C Ar), 170.48
(1C,
C Ar), 170.64 (1C, C Ar).
4 ~(4-chloro-6-methoxy-1,3,5 triazin-2 yl)aminoJphenyl-~-bromo-2-
~aethylpropionate
,Me
C1
H
A solution of 2-bromoisobutryl bromide (1.0 mL, 7.90 mmol) in 20 mL of THF
was added dropwise to a solution of 4-[(4-chloro-6-methoxy-1,3,5-triazin-2-
yl)amino]phenol BIW009 (1.9 g, 7.52 mmol) and triethylamine (1.1 mL, 8.0
mmol) in 100 mL of THF, at - 10 'C. During the dropping (ca. 1S min) the
precipitation of triethylammonium bromide was observed. The reaction was
monitored by TLC (SiO2 ,petroleum ether! Et20 1:1, BIW009 (starting material)
Rf = 0.14; BIWO10 (final product) Rf = 0.26). After 1.S h the white suspension
was poured into a conical flask containing 150 mL of Et20 and the the ammonium
salt removed by filtration on a sintered glass frit. The solvent was then
evaporated
at reduced pressure to give a white crude residue that was suspended in 10 ml
of
pentane and filtered. Obtained 2.56 g (6.37 mmol, yield 8S%) of BIW010 as
white
solid. The NMR analysis (DMSO d6) revealed the presence, in solution, of 2
rotational isomers (molar ratio 7:3). M.p. 107-108 °C. IR v~ NH> 3365
cm 1. v~ ~=o>
1747 cm 1. Major isomer: IH NMR (DMSO d6) & = 2.05 (s, 6H, C(CH3)aBr), 3.96
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(s, 3H, OCH3), 7.17 (d, J = 8.9 Hz, 2H, CH Ar) , 7.77 (d, J = 8.9 Hz, 2H, CH
Ar),
10.78 (s, 1H, NH). 13C f iH) NMR (DMSO d6) s = 30.42 (2C, CH3), 55.75 (bs,
1C, OCH3), 57.29 (1C, C(CH3)aBr), 121.96 (2C, CH Ar), 122.12 (2C, CH Ar),
136.29 ( 1 C, C Ar), 146.61 (bs, 1 C, C Ar), 165.10 (bs, 1 C, C Ar), 169.89
(bs, 1 C,
C Ar), , 170.16 (1C, OC(O)C(CH3)2Br), 171.33 (bs, 1C, C Ar). Minor isomer: 1H
NMR (DMSO d6) S = 2.05 (s, 6H, C(CH3)ZBr), 3.96 (s, 3H, OCH3), 7.17 (d, J =
8.9 Hz, 2H, CH Ar) , 7.69 (d, J= 8.9 Hz, 2H, CH Ar), 10.66 (s, 1H, NH).
13C NMR (DMSO d6) 8 = 30.42 (2C, CH3), 55.75 (bs, 1C, OCH3), 57.29 (1C,
C(CH3)2Br), 121.96 (2C, CH .Ar), 122.73 (2C, CH Ar), 136.29 (1C, C Ar), 146.61
(bs,
1 C, C Ar), 165.10 (bs, 1 C, C .Ar), 169. 89 (bs, 1 C, C Ar), , 170. I 6 ( 1
C,
OC(O)C(CH3)2Br), 171.33 (bs, 1 C, C Ar).
preparation of initiator 9
.~ ~~ydroxyethyl 2-bromo-2-methylpropionate
HO~
O
O Br ~
9
Ethylene glycol (279 g, 4500 mmol) and Et3N (3.34 g, 33.0 mmol) were poured in
a
2-necked round bottom flask. To this was added dropwise and a solution of 2-
bromoisobutryl bromide (6.90 g, 30.0 mrnol) in anhydrous THF (SO mL) at room
temperature over ca. 1 h. The colourless solution was stirred overnight, then
diluted in
500 mL of water and extracted with 3x200 mL of a mixture of Et20/CHZCl2 (4:1).
The organic layers, reunited, were washed with 2x200 mL of water and dried
over
MgSOa. Evaporation of the solvent at reduced pressure (rotavapor, without
heating)
gave a pale yellow liquid. The latter was dissolved in ca. 30 mL of CH2Cla
then 10 g
of SiOa were added and the solvent evaporated again until a white powder was
obtained. This was poured in a column packed with Si02, (ca 15 cm depth)
previously
eluted with petroleum ether/Et2O 5:1 and purified by column chromatography
(elute
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with petroleum ether/EtZO 5:1) to eliminate the impurities. The desired
product, using
this solvent mixture, has an Rf ~0 (stays on the bottom of the TLC plate).
When the
impurities have been eliminated, the column was eluted with 100% Et2O, to give
a
colourless liquid. Yield 82%. IR v~_~ 3388 cm 1 (broad); vac=o~ 1731 cm ~. 1H
NMR
(CDC13) 8 = 1.97 (s, 6H, CH3); 3.89 (t, J = 4.6 Hz; 2H, OCHZCH OH); 4.33 (t, J
=
4.6 Hz; 2H, OCH CHZOH). 13C NMR (CDC13) ~ = 30.45 (2C, CH3), 55.55 (1C,
C(CH3)2Br); 60.66 (1C, OCHaCHaOH); 65.90 (1C, OCH2CH20H); 171.69 (1C,
C=O).
Preparation of initiator 6
2-Phthalimidoethyl 2-bromo-2-methylpropiohate
v tir
6
N-(2-hydroxyethyl)phthalimide (19.12 g, 0.1 mol) was dissolved in anhydrous
THF
(250mL) with triethylamine (28.1 mL, 0.2 mol) under nitrogen in a 500 mL round
bottom flask equipped with a magnetic stirrer and dropping funnel. The flask
was
cooled to 0 °C with an ice/salt bath before the dropwise addition of 2-
bromo-2-
methylpropionyl bromide (13.9 mL, 0.11 mol). The mixture was stirred for 45
minutes and allowed to reach room temperature before the mixture was poured
into an
excess of cold water and the product extracted with diethyl ether (3x 100 mL).
The
organic layer was subsequently washed with a saturated aqueous solution of
sodium
carbonate (3x100 mL), acidified water (pH 4.6, 3x100 mL) and again with
saturated
aqueous solution of sodium carbonate (3x100 mL). The organic layer was dried
over
anhydrous magnesium sulphate and filtered. The product was isolated via
reduction
under reduced pressure to obtain a white solid (25.79 g, 75.8 % yield). 1H NMR
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(CDC13) S (ppm) 1.81 (s, 6H, C(CH )zBr), 3.95 (t, 2H, J = 5.3 Hz, CH -N), 4.35
(t,
2H, J = 5.4 Hz, CH -O), 7.67 (m, 2H, Haro), 7.78 (m, 2H, Haro). tsC NMR
(CDCl3) 8
(ppm) 31.00 (2C, C(CH3)aBr), 37.12 (1C, CHZ-N), 55.92 (1C, C(CH3)aBr), 63.42
(1C,
CHa-O), 123.78 (2C, Caro), 132.35 (1C, CIVar°), 133.54 (2C, Caro),
168.40 (2C,
C~y~~=O), 171.87 (1C, C=O). IR (solid, ATR cell) v (cm 1) 2975, 1774
(C~y~~=O), 1705
(C=O), 1417, 1392, 1321, 1276, 1158, 1105, 1063, 985, 763, 716, 632. Melting
point
63-65°C.
Preparation of initiator 10
~'r~itylthiolether propahol
Ph
Ph~--S~~OH
Ph
Sodium hydride (10.95 g, 0.273 mol, 60% in oil) was suspended in THF (750 mL)
at
0°C. Triphenylinethanethiol (75.5 g, 0.273 mol) in THF (600m1) was
added to the
suspension and stirred at 0°C for 10 minutes. 3-Bromo-1-propanol (24.75
mL, 0.273
mol) in THF (300 mLl) was added and the mixture stirred at 0 °C for 20
minutes.
After this time TLC showed mostly one major product (Rf approx 0.3 ethyl
acetate/hexane 1:9). Water was added and the product extracted into ethyl
acetate
(20>1000 mL) (an aqueous NaCI solution was used to break up the emulsion),
dried
with sodium sulfate, filtered and concentrated. The product was purified by
column
chromatography using ethyl acetate/hexane 1:9 to 1:4 as the eluent (Si02) to
give a
white solid, which was triturated with hexane and filtered to give 65.2 g of
product.
IH NMR (CDCl3) b ppm 7.6-7.1 (m, 15H Har°), 3.58 (t, 2H, CH OH), 2.29
(t, 2H,
SCHa), 1.65 (q, 2H, CH2CH CHa), 1.45 (OH).
3-Tritylthioletherpropyl 2-brotr:o-2-methylpropiohate
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S
O
O Br
1~
'fritylthiolether propanol (50 g, 0.150 mol), triethylamine (31.3 mL, 0.225
mol) and
anhydrous tetrahydrofizran (125 mL) were placed in a three-necked round bottom
flask containing a magnetic follower and fitted with a pressure equalizing
dropping
funnel. The flask was cooled with the use of an ice bath and 2-bromoisobutyrl
bromide (27.8 mL, 0.225 mol) was added to the dropping funnel. Whilst stirring
the
2-bromoisobutyrl bromide was added drop-wise to the cooled solution and the
solution left stirring over night. The mixture is then filtered to remove the
triethylamine hydrochloride salt before the addition of dichloromethane (500
mL) and
subsequently washed with dilute hydrochloric acid (2x300 mL), dilute sodium
l~~idroxide (2x300 mL) and finally distilled water (3x300 mL). The Organic
layer was
separated and the product isolated by flash evaporation of solvent, the
product was
then triturated with hexane, filtered and the product collected in
quantitative yield. 1H
NMR (CDCl3) 8 ppm 7.4-7.1 (m, 15H, Haro), 4.02 (t, 2H, CHZCOa), 2.15 (t, 2H,
SCH2), 1.78 (s, 3H, C(CH3)2Br), 1.63 (q, 2H, CH~CH CH2).
preparation of initiator 11
4-(2-bromo-2-methylpropionate) beazaldehyde
O
O
H
O Br
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4-Hydroxybenzaldehyde 12.21 g (0.1 moles), triethylamine, 15.3 mL (0.11 moles)
and anhydrous THF 400 mL were placed in a 3 neck round bottomed flask.
Bromoisobutyryl bromide 13.6 mL (0.11 moles) was added slowly with stirring. A
white precipitate of triethylammonium bromide was formed. The mixture was left
to
react for 6 hours with stirring. On completion of the reaction
triethylammonium
bromide was removed by filtration and the THF was removed by rotary
evaporation.
The resulting orange liquid was dissolved in dichloromethane and subsequently
washed with 2 x 200 mL portions of saturated Na2C03 (aq), dil. HCl (aq) and
distilled
water. The dichloromethane was dried using MgS04 and the solvent removed by
rotary evaporation to give a yellow oily liquid which crystallised upon
standing. This
~~rvas recrystalised from diethyl ether x 2.Yield =18.95g (69.9 %). 1H NMR
(CDCl3) 8
(ppm) 10.00 (s, 1H, CHO), 7.94 (d, J = 4.6 Hz, 2H, Hue.°), 7.31 (d, J =
4.8 Hz 2H,
Haro)~ 2.06 (s, 6H, C(CH )2Br). 13C NMR (CDC13) 8 (ppm) 190.59, 169.33,
155.08,
134.07, 131.02, 121.71, 54.94, 30.25. IR (Solid, ATR Cell) 2984, 2820, 2730
(O=C-
H), 1746 (C=O), 1693 (H-C=O), 1590, 1500, 1374, 1262, 1207, 1153, 1132, 1099,
1009, 932, 881, 808, 658: +EI MS (m/z) 273, 271 (mass peaks), 210, 193, 163,
151,
149, 140, 123, 121, 102. Elem. Anal. Theoretical for H11 O3Br: C = 48.73 , H =
4.09;
Found: C = 48.63 , H = 4.03.
p~°eparation of initiator 12
2-(2,2 Dimethoxy-ethoxy)-ethanol
-O
-O O OH
~~~:~.ssium hydroxide (30 g, 0.51 mol) was suspended in ethylene glycol (100
ml) and
the mixture was heated to 115 °C with stirnng. After the KOH dissolved
completely,
2-chloro-1,1-dimethoxy-ethane (30.0 mL, 0.263 mol) was added dropwise (ca. 30
minutes) and the solution was stirred at 11 S °C for 72 h. The
resulting suspension was
cooled to room temperature and 150 mL water was added. The solution was
extracted
with dichloromethane (3x100mL) and the organic layers combined was washed with
brine (2x100 mL) and dried with MgSO4. After filtration the solvent was
removed
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under reduced pressure to give the product as a yellow oil (Yield, 17.7 g,
44.9%). 1H
NMR (400.03 MHz, CDCl3, 298 K) S = 2.20 (s, 1H, OH), 3.40 (s, 6H, OCH ), 3.55
(d, J = 5.3 Hz, 2H, CHCH ), 3 .63-3.61 (m, J = 4.0 Hz, OCH ), 3.74-3.72 (m, J
= 4. 0
Hz, 2H, CH OH), 4.52 (t, 1H, CH(OCH3)Z. i3C fiH) NMR (100.59 MHz, CDC13, 298
K) 8 = 54.12 (2C, CH3), 61.82 ( 1 C, CH~OH), 70.78 ( 1 C, CHCHzO), 73.00 ( 1
C,
OCH2CH2), 102.73 (1C, CH). Anal. Calcd. for C6H14O4: C, 47.99; H, 9.40; Found
C,
45.02; H, 8.74
2 Bromo-2-methyl propionic acid 2-(2,2-dimethoxy ethoxy)-ethyl ester
-O
Br
-O O O
O
12
A solution of 2-(2,2-dimethoxy-ethoxy)-ethanol (llg, 0.073 mol) and
triethylamine
(12 mL, 0.088 mol) in dichloromethane (150 mL) was cooled to 0°C and a
solution of
2-bromo-2-methyl propionyl bromide (8.5 mL, 0.069 mol) m 50 mL of
dichloromethane was added dropwise in ca. 30 min. After stirring overnight at
room
temperature the resulting suspension was filtered and the yellow solution was
washed
with saturated NaHC03 aqueous solution (2 x 100 mL) and dried with MgS04.
After
filtration the solvent was removed under reduced pressure and the yellow oily
residue
was purified by distillation (b.p. 70°C/0.02mbar) to give 14.0 g
(0.06Imol, yield:
89%) of product as a colourless oil. 1H NMR (400.03 MHz, CDCl3, 298 K) S =
1.94
(s, 6H, (CH )aCBr), 3.39 (s, 6H, OCH3), 3.56 (d, J= 5.3 Hz, 2H, CHCH ), 3.76
(t, J=
4.8 Hz, CHaOCH ), 4.33 (t, J = 4.8 Hz, 2H, CH OCO), 4.50 (t, 1H, CH(OCH3)a,.
isC(1H} NMR (100.59 MHz, CDC13, 298 K) b = 30.90 (2C, C(CH3)2), 54.13 (2C,
CH30), 55.79 (1C, BrC(CH3)2), 65.24 (1C, CH20C(=O)), 69.21 (1C, CHCH2O),
71.18 (1C, OCH2CHa), 102.83 (1C, CH).
Preparation of initiator 13
3-(2,5-Dioxo-2,5-dihydro pyrrol 1 yl) propionic acid
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68
O
N O
O OH
A solution of malefic anhydride (5.00 g, 0.0561 mol) in acetic acid (70 ml)
was added
dropwise to a solution of (3-alanine (5.50 g, 0.0561 mol) in acetic acid (25
ml) and the
mixture was stirred at room temperature for three hours. 50 mL of acetic acid
was
added to the white suspension and the mixture heated up to 115°C. After
1 h a limpid
olourless solution was observed. The mixture was then stirred at this
temperature
overnight and the colour turned to orange. The solvent was then removed under
reduced pressure and 30 mL of toluene was then added to the resulting orange
oil.
This was then evaporated under reduced pressure and this operation was
repeated 3
times. The orange residue was then purified by flash chromatography (CC, SiO~,
CHaCI~Jethyl acetate 9:1) to give the product as a white solid (3.86 g, 0.0228
mol,
41%). m.p. 105-107°C IR (neat): 3092, 2883, 2537, 1695, 1445, 1411,
1373, 1337,
1305, 1230, 1151, 1081, 1043, 924, 830, 773, 694, 618 cm 1. 1H NMR (400.03
MHz,
CDC13, 298 K) b = 2.69 (t, J = 7.3 Hz, 2H, CH2), 3.82 (t, J = 7.3 Hz, 2H,
CH2), 6.71
(s, 2H, CH~iny), 10.07 (bs, 1H, COOH). 13C{1H} NMR (100.59 MHz, CDC13, 298 K)
b = 32.62 (1C, CHI), 33.36 (1C, CH2), 134.38 (2C, CH~;ny;), 170.48 (1C, C),
176.64
(2C, C). Anal. Calcd. for C7H7N0~: C, 49.71; H, 4.17; N, 8.28. Found C, 49.35;
H,
4.19; N, 7.95.
3-(2,S Dioxo-2,S-dihydro pyrrol 1 yl) propionyl chloride (3-
'rzaleimidopropionyl
chloride)
O
N O
O C1
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3-(2,5-Dioxo-2,S-dihydro-pyrrol-1-yl)-propionic acid (2.20 g, 0.0130 mol) was
dissolved in CH2Cla (100 mL). Oxalyl chloride (l.l mL, 0.0130 mol) was then
added,
at room temperature. 50 p,L of DMF were added dropwise and an intense
evolution of
gas was observed. The solution was colourless before the addition of DMF and
remained colourless for lh, then slowly turned to very pale yellow (but still
very
limpid). After 3 h the solvent was removed under reduced pressure to give an
off
white solid that became pale brown after standing under vacuum at room
temperature
for 1 h. The acid chloride product so obtained was used directly without
further
purifications. IR (neat): 3095, 1803 1698, 1446, 1410, 1387, 1360, 1307, 1230,
1148,
1131, 1083, 1011,948, 922, 833, 719, 689, cm I. 1H NMR (400.03 MHz, CDCl3, 298
1~.) ~ = 3.25 (t, J= 6.9 Hz, 2H, CH2), 3.86 (t, J= 6.9 Hz, 2H, CHa), 6.73 (s,
2H,
CH~;ny;). 13C{1H} NMR (100.59 MHz, CDCl3, 298 K) 8 = 33.21 (1C, CHa), 44.97
~'1C, CHa), 134.47 (2C, CH,,;"y;), 170.08 (2C, C), 171.50 (1C, C).
~ Bromo-2-methyl propionic acid 2 j3-(~,S-dioaco-2,5-dihydro pyrr~l 1 yl)-
propionyloxyJ-ethyl ester
O
O
N O~O Br
O O
13
2-Hydroxyethyl-2-bromo-2-methylpropionate (initiator 9) (0.187 g, 0.887 mmol)
and
3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid (0.300 g, 1.77 mmol) were
dissolved in dichloromethane (10 ml) under nitrogen in a 25 ml round-bottomed
flask.
Then N,N'-Dicyclohexylcarbodiimide (DCC) (0.366 g, 1.77 mmol) was added to the
solution. After one day at room temperature, very low conversion was observed
and
0.5 ml (9.50x10-3 mmol) of a solution of 4-dirnethylaminopyridine (DMAP) in
dichloromethane (Conc.DMAr =19 mmol/1) was added. Total conversion was then
achieved in 12 hours and the solvent was removed under reduced pressure. T'he
solid
residue was extracted with 3 x 50 ml of petroleum ether and the petroleum
ether was
removed by evaporation under vacuum in order to isolate a colourless oil
(0.270g,
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0.745 mmol, 84%). The pale pink residue was extracted with 3 x 50 ml of
diethylether, but TLC (CHaCla / AcOEt 9:1) revealed that only traces of the
ester were
present. An analytical pure sample was obtained by flash chromatography (cc,
Si4a,
Pet. Ether / Et20 3:1).
1H NMR (CDC13) 8 (ppm) 1.88 (s, 6H, C(CH )2Br), 2.62 (t, 2H, CH -COO(CH2)a-O,
Jab=7.02 Hz), 3.78 (t, 2H, (CO)aN-CH , Jab= 7.07 Hz), 4.27-4.35 (m, 4H,0-(CH
)Z-O),
6.68 (s, 2H, OC-CH=CH-GO). 13C NMR (CDC13) S (ppm) 30.61 (2C, C(CH3)2Br),
32.75 (1C, CH2-COO(CHZ)a-O), 33.46 (1C, (CO)aN-CH2), 55.45 (1C, C(CH3)aBr),
62.04 (1C, CH2-COO-CH2), 63.35 (1C, CH2-OOC-C(CH3)2Br), 134.25 (2C, OC-
CH=CH-CO), _170.29 (1C, C=O ester), 170.40 (1C, C=O ester), 171.39 (2C, O=C-
N(CH2)-C=O). IR (solid, ATR cell) v (cxri 1) 1769 ( v 'C=o, imiae))a 1736 ( v
~~=o, aeia))~
1707 ( 1~ (C=o, imide)).
Preparation of initiator 14
2-promo-2-methyl propionic acid 3-tert butoxycarbonylamino propyl ester
O
Boc VH~O
i
Br
14
A solution of 3-amino propanol (3.00 mL, 0.0392 mol) in 100 mL of THF was
cooled
to 0 °C and Boc20 (8.56 g, 0.0322 mol) in THF (50 mL) was added
dropwise (ca. 20
rain.). The solution was then warmed up to room temperature and stirred.for 3
h. TLC
analysis (Si02, 100% Et20) revealed the complete disappearance of the amino
alcohol
starting material (Rt=0) and the presence of the expected N-Boc-protected
amino
alcohol intermediate (R~0.25). The mixture was then cooled to 0 °C and
Et3N (6.0
mL, 0.0431 mol) was added via syringe. A solution of 2-bromo isobutyryl
bromide
(4.85 mL, 0.0392 mol) in THF (50 mL) was added dropwise in ca. 30 min. and the
resulting white suspension stirred at 0 °C for 1 h and at room
temperature for further 2
h. The mixture was then diluted with Et20 (200 mL) and the ammonium salt was
filtered off and washed with 3x50 mL of EtzO. The colourless solution was
washed
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71
with 3x100 mL of water and dried over MgSO4. Removal of the solvent under
reduced pressure gave the product a colourless oil that was purified by flash
chromatography (CC, SiO~, Petroleum ether/Et2O 8:1). Obtained 10.42 g (0.0321
mol,
82%) of (1) as colourless oil. 1R (neat): 3295, 2976, 1734, 1713, 1695, 1517,
1463,
1391, 1366 1273, 1163, 1109, 1013, 633 ciri 1.1H NMR (400.03 MHz, CDC13, 298
K)
8 = 1.43 (s, 9H, CH3), 1.88 (quint., J = 6.3 Hz, 2H, CH2), 1.93 (s, 6H, CH3),
3.23 (q, J
= 6.0 Hz, 2H, CH2), 4.24 (q, J= 6.0 Hz, 2H, CHa), 4.77 (bs, 1H, NH). 13C{1H}
NMR
(100.59 MHz, CDC13, 298 K) 8 = 28.54 (3C, CH3), 28.99 (1C, CHZ), 30.88 (2C,
CH3),
37.57 (1C, CH2), 55.95 (1C, C), 63.86 (1C, CHa), 156.03 (1C, C), 171.89 (1C,
C).
Anal. Calcd. for Ci2Ha~BrN04: C, 44.46; H, 6.84; N, 4.32; Br, 24.65. Found C,
44.48;
H, 6.91; N, 4.33. Br, 24.91.
Preparation of initiator 15
4,10 Dioxa-tricyclo~5.2.1.02'6Jdec-8-eHe-3,5 diof:e
O
p O
O
Malefic anhydride (30.00 g, 0.306 mol) was suspended in 150 mL of toluene and
the
mixture warmed to 80°C. Furan (33.4 mL, 0.459 mol) was added via
syringe and the
turbid solution stirred for 6 h. The mixture was then cooled to room
temperature and
the stirnng was stopped. After 1 h the resulting white crystals were filtered
off and the
solid washed with 2 x 30 mL of petroleum ether. Obtained 44.40 g (0.267 mol,
87%
yield) of the desired product as small white needless. m.p. 124-127 °C
(dec.) 1R
(neat): 1857, 1780, 1309, 1282, 1211, 1145, 1083, 1019, 947, 920, 902, 877,
847,
800, 732, 690, 674, 633, 575 cm 1. 1H NMR (400.03 MHz, CDC13, 298 K) ~ = 3.17
(s,
2H, CH), 5.45 (t, .1--- 1.0 Hz, 2H, CHO); 6.57 (t, J-- 1.0 Hz, 2H, CH",nyi). '
3C {'H }
NMR (100.59 MHz, CDCl3, 298 K) 8 = 48.85 (2C, CH), 82.35 (2H, CHO), 137.12
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(2C, CH";ny~), 170.04 (2C, CO). Anal. Calcd. for CBHbOa: C, 57.84; H, 3.64;.
Found C,
57.74; H, 3.68.
~-(2 Hydroxy-ethyl)-10-oxa-4-aza-tricyclo(5.2.LOZ'6Jdec-8-ehe-3,5-dioae
O
I O -~
~OH
O
The anhydride, 4,10-dioxa-tricyclo[5.2.1.Oa'6]dec-8-ene-3,S-dione, (2.00 g,
I2.0 x IO-3
mol) was suspended in SO mL of MeOH and the mixture cooled to 0°C. A
solution of
ethanolamine (0.72 mL, 12.0 x 10-3 mol) in 20 mL of MeOH was added dropwise (I
O
min) and the resulting solution was stirred for S min at 0°C, then 30
min at room
temperature and finally refluxed for 4 h. After cooling to room temperature
the
solvent was removed under reduced pressure, the white residue was dissolved in
1S0
mL of CHZCIZ and washed with 3 x 100 mL of water. The organic layer was dried
over MgSOa and filtered. Removal of the solvent under reduced pressure
furnished
the desired product (1.04 g S.0 x 10'3 mol, 42 %yield) as white solid that was
used for
the next step without further purifications. An analytical sample was obtained
by flash
chromatography (CC, SiO2, I00% ethyl acetate, R;{6)=0.26). m.p. 139-141
°C (dec).
IR (neat): 3472, 1681, 1435, 1405, 1335, 1269, 1168, 1100, lOS3, 1013, 959,
916,
875, 850, 807, 722, 705, 6S4 cm 1. 1H NMR (400.03 MHz, CDC13, 298 K) 8 =1.90
(bs, 1H, OH); 2.90 (s, 2H, CH), 3.69-3.72 (m, 2H, CH2), 3.76-3.78 (m, 2H,
CH2),
5.28 (t, J= 0.9 Hz, 2H, CH), 6.52 (t, J= 0.9 Hz, 2H, CH";"y;). 13C{1H} NMR
(100.59
MHz, CDC13, 298 K) S = 41.77 (2C, NCH2), 60.18 (2C, OCHZ), 47.50 (2C, CH),
81.04 (2C, CH), 136.60 (2C, CHw"y~), 176.97 (2C, C). Anal. Calcd. for CioH;
INOa: C,
57.41; H, 5.30; N, 6.70. Found C, 57.16; H, 5.37; N, 6.62.
2 Bromo-2-methyl propioftic acid 2-(3,5-dioxo-10-oxa-4-aza-
tricyclo~5.2.1.02'6jdec-
8-ea-4 yl)-ethyl ester
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O
O N
~O
O
O Br
v5
A solution of the alcohol, 4-(2-hydroxy-ethyl)-10-oxa-4-aza-
tricyclo[5.2.1.0°6]dec-8-
ene-3,5-dione, (2.22 g, 10.6 x 10-3 mol) and Et3N (1.6 mL, 11.7 x 10-3 mol) in
120 mL
of THF (the solution remains slightly turbid) was cooled to 0°C and a
solution of 2-
bromo isobutiryl bromide (1.4 mL, 11.1 x 10'3 mol) in 40 mL of THF was added
dropwise (30 min). The white suspension was stirred for 3 h at 0°C,
then at room
temperature overnight. The ammonium salt was filtered off and the solvent
removed
under reduced pressure to give a pale-yellow residue that was purified by
flash
chromatography (CC, Si02, petroleum ether/ethyl acetate l:l, R~{7)=0.23).
Obtained
3.54 g (9.88 x 10-3 mol, 93% yield) of initiator 15 as a white solid. m.p. 83-
85°C IR
(neat): 1733, 1695, 1419, 1395, 1336, 1278, 1157, 1106, 1015, 874, 852, 824,
724,
706, 654, 603 cm 1. 1H NMR (400.03 MHz, CDCl3, 298 K) 8 =1.86 (s, 6H, CH3),
2.84 (s, 2H, CH), 3.78 (t, J= 5.3 Hz, 2H, NCH2); 4.30 (t, J= 5.3 Hz, 2H,
OCH2); 5.23
(t, J--1.0 Hz, 2H, CHO); 6.49 (t, J--1.0 Hz, 2H, CH";ny;). 13C{iH} NMR (100.59
MHz, CDC13, 298 K) 8 = 30.65 (2C, CHa), 37.65 (2C, NCHZ), 47.56 (2C, CH),
55.80
(1C, C(CH3)aBr), 62.26 (OCHa), 80.91 (2H, CHO), 136.62 (2C, CH~;ny), 171.46
(1C,
COester)~ 175.95 (2C, CO;m;ae). Anal. Calcd. for C;4H;6NO5: C, 46.95; H,4.50;
N,3.91;
Br, 22.31. Found C, 46.88; H, 4.55; N, 3.79; Br, 22.22
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Preparation of PoIyPEG polymers
Polymerisations using Initiator 8
O
Me g
N-O
Br
O O
8
Polymerisation of MPEG(39S7MA
(PEGJIIIJI~CuJI~LJ =10/1/1/2 ih 50 vlv% toluene solutioH at 30 °C
A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 x 10-3 mol),
initiator 8
(0.569 g, 2.27 x 10-3 mol) and a magnetic follower prior to being deoxygenated
by
yrcling between nitrogen and vacuum three times. To a second Schlenk tube was
added MPEG(395)MA (10 mL, 22.74 x 10-3 mol), N-(n-ethyl)-2-pyridyhnethanimine
(0.64 mL, 4.54 x 10-3 mol) and toluene (10 mL). The mixture was immediately
subjected to five freeze-pump-thaw degassing cycles. This solution was then
transferred to the Schlenk tube containing the initiator and Cu(I)Br via a
cannula. The
resulting brown solution was stirred at 30 °C. Samples were removed
periodically
using degassed syringes and quenched in liquid nitrogen for conversion and
molecular
weight analysis.
T able 3: Kinetic data for the polymerisation of MPEG(395)MA initiated by 8 in
toluene solution (50% v/v) at 30 °C
([MPEG(395)MA]o/[CuBr]o/[NHSBr]o/[ligandJo =
10/1/1/2).
Experiment Time Conversion ln([M)o/[M)) M", rhea Mn, sEC Mw~n
(h) (%) (g.mol-t) (g.mof')
Toluene 2 4.5 0.046 230 3040 1.07
Ethyl Ligand 4 17.3 0.190 870 3710 1.30
6 30.2 0.359 1510 4480 1.13
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8 40.2 0.514 2010 4950 1.11
IO 49.3 0.679 2470 5530 1.10
21 92.3 2.564 4620 7980 1.09
Polymerisation of MPEG(550)MA
~PEGJI~IJI~CuJIILJ = 6.37/1/1/2 ire 73 wlvJ toluene solution at SOl70
°C
Initiator 8 (0.10 g, 0.400 mmol), Cu(I)Br (0.057 g, 0.400 mmol, 1 ec~ and
MPEG(550)MA, (1.60 g, 2.55 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (5.90 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N propyl-2-pyridylmethanimine (0.114 g, 0.797 mmol) was added.
The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The temperature was increased to 70 °C after 5 hours 32 minutes.
Table 4: Data for the polymerization of MPEG(550)MA with initiator 8 at 50/70
°C in
73 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
385 15 4730 1.05
1357 42 10440 1.10
1663 45 10280 I .12
(PEGJI~IJI~Cu~I~LJ = 6.37/1/1/2 ih 73 fvlv% toluene solution at 90 °C
Initiator 8 (0.10 g, 0.400 mmol), Cu(I)Br (0.057 g, 0.400 mmol, 1 e~ and
MPEG(550)MA (1.60 g, 2.341 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (5.90 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N propyl-2-pyridylmethanimine (0.114 g, 0.797 mmol) was added.
The
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76
reaction was placed in a thermostatically controlled oil bath at 90 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 5: Data for the polymerization of MPEG(550)MA with initiator 8 at 90
°C in 73
w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
138 46 6950 1.09
240 46 7220 1.12
314 48 7370 1.12
1293 59 7690 1.14
~PEGJI~IJI~CuJI~LJ = 31.9/1/1/2 ih 67 v/v% toluene solutioh at SO °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and
MPEG(550)MA (8.0 g, 12.7 mmol), and a magnetic follower were placed in an oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (14.7 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The polymer was purified by removing the solvent in vaeuv and dialysising the
residue using acidic water (pH ~4). Subsequent freeze drying isolated the
product.
Table 6: Data for the polymerization of MPEG(550)MA with initiator 8 at 50
°C in 67
v/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
120 7 7189 1.089
240 15 8976 1.074
360 20 10477 1.074
1320 87 23051 1.147
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~1'EGJI~IjI~CuJI~LJ = 6.4/1/1/2 in 67 v/v% toluene solution at 50 °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and
MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 7: Data for the polymerization of MPEG(550)MA with initiator 8 at 50
°C in 67
v/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
180 13 4984 1.064
360 31 6628 1.110
1320 86 11282 1.104
~PEGJI~IJI~Cu(I)JI~Cu(II)JI~LJ = 6.4/1/0.95/0.05/2 in 67 v/v% toluene solution
at
SO °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0545 g, 0.38 mmol, 0.95 eq),
Cu(II)Br
(0.0045 g, 0.02 mmol, 0.05 eq), and MPEG(550)MA (1.60 g, 2.55 mmol), and a
magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube
was
evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (3.0
mL)
was added to the Schlenk tube. The resulting solution was deoxygenated via
three
freeze pump thaw cycles and then degassed N ethyl-2-pyridylmethanimine (0.107
g,
0.80 mmol) was added. The reaction was placed in a thermostatically controlled
oil
bath at 50 °C (t=0) and samples were removed periodically for
conversion and
molecular weight analysis.
Table 8: Data for the polymerization of MPEG(550)MA with initiator 8 at 50
°C in 67
v/v% toluene solution.
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78
Sample Time Conversion Mn PDi
/ minutes /
180 5 4743 4743
360 16 5904 5904
1320 65 11202 11202
1800 78 12245 12245
~PEGJI~l,I~CuJI~LJ = 6.4/1/1/2 in 67 v/v% toluene solution act SO °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(nBr (0.0574 g, 0.40 mmol, 1 ec~ and
MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an
oven
dried Schlenl~ tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N propyl-2-pyridylinethanimine (0.119 g, 0.80 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 9: Data for the polymerization of MPEG(550)MA with initiator 8 at 50
°C in 67
v/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
180 13 4875 1.041
360 22 5601 1.087
1320 66 9897 1.091
,(PEGJI~IJI~CuJIILj = 6.4/1/1/2 in 67 vlv6 toluene solution act 50 °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 e~ and
MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N octyl-2-pyridylinethanimine (0.175 g, 0.80 mmol) was added. The
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79
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 10: Data for the polymerization of MPEG(550)MA with initiator 8 at 50
°C in
67 v/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
180 19 5034 1.075
360 33 6636 1.101
1320 85 11294 1.097
~PEGJI~IjI~CuJI(LJ = 6.4/1/1/2 iu 67 v/v% t~luehe solutioh at 70 °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 e~ and
MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 70 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 11: Data for the polymerization of MPEG(550)MA with initiator 8 at 70
°C in
67 v/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
60 20 5455 1.096
120 52 7898 1.114
180 73 9544 1.086
240 80 10207 1.095
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~PEGJI~IJI~CuJI~LJ = 6.4/1/1/2 in 67 v/v% toluene solution at SO °C.
Initiator 8 (6.0 g, 24 mmol), Cu(I)Br (3.44 g, 24 mmol, 1 eq) and MPEG(550)MA
(96
g, 0.153 mol), and a magnetic follower were placed in an oven dried Schlenk
tube.
The Schlenlc tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated toluene (176 mL) was added to the Schlenk tube. The resulting
solution was deoxygenated by bubbling with nitrogen for 1 hour and then
degassed N
ethyl-2-pyridylmethanimine (6.44 g, 48 mmol) was added. The reaction was
placed in
a thermostatically controlled oil bath at 50 °C (t=0) and samples were
removed
periodically for conversion and molecular weight analysis. The polymer was
purified
by removing the solvent in vacuo and dialysising the residue using acidic
water (pH
~4). Subsequent freeze drying isolated the product.
Table 12: Data for the polymerization of MPEG(550)MA with initiator 8 at 50
°C in
67 v/v% toluene solution.
Sample Time Conversion Mn PDi
l minutes l
1200 66 8207 1.118
1500 75 9276 1.082
1680 81 9342 1.096
Polymerisation of MPEG(1000)MA
~PEGJI~IJIICuJI~LJ = 23.2/1/1/2 ire 80 w/v% toluehe solution at 50 °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and
M.PEG(1000)MA (10.0 g, 9.3 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (40 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and
then
degassed N ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The polymer was purified by removing the solvent in vacuo and dialysising the
residue using acidic water (pH ~4). Subsequent freeze drying isolated the
product.
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Table 13: Data for the polymerization of MPEG(1000)MA with initiator 8 at 50
°C in
80 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
300 3 9760 1.056
1260 41 19436 1.087
3000 79 29013 1.132
7020 87 30046 1.149
,~PEGJI~IJI~CuJI~LJ = 46.3/1/1/2 ih 80 wlv6 toluene solutioh at 50 °C
Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and
MPEG(1000)MA (20.0 g, 18.5 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (80 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridyhnethanimine (0.107 g, 0.80 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at SO °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
'the polymer was purified by removing the solvent in vacuo and dialysising the
residue using acidic water (pH ~4). Subsequent freeze drying isolated the
product.
Table 14: Data for the polymerization of MPEG(1000)MA with initiator 8 at 50
°C in
80 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
300 4 11014 1.070
1260 15 14388 1.080
3000 31 26378 1.096
7020 53 33388 1.154
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~PEGJI~IJIICuJI~LJ = 23.2/1/1/2 in 80 wlv~o toluehe solution at 50 °C
Initiator 8 (1.0 g, 4.0 mmol), Cu(I)Br (0.574 g, 4.0 mmol, 1 eq) and
MPEG(1000)MA
(100 g, 93.0 mmol), and a magnetic follower were placed in an oven dried
Schlenk
tube. The Schlenk tube was evacuated and flushed with dry nitrogen three
times.
Deoxygenated toluene (200 mL) was added to the Schlenk tube. The resulting
solution was deoxygenated by bubbling with nitrogen for 1 hour and then
degassed N
ethyl-2-pyridylmethanimine (1.07 g, 8.0 mmol) was added. The reaction was
placed
in a thermostatically controlled oil bath at 50 °C (t=0) and samples
were removed
periodically for conversion and molecular weight analysis. The polymer was
purified
by removing the solvent in vacuo and dialysising the residue using acidic
water (pH
~4). Subsequent freeze drying isolated the product.
Table 15: Data for the polymerization of MPEG(1000)MA with initiator 8 at 50
°C in
80 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
4320 89 37676 1.143
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Polymerisations using Initiator 7
O
Me Me
N-O
Br
O O
Polymerisation of MPEG(395)MA
~PEGJI~IJI~CujI~LJ =10/1/1/2 in SO v/v% toluene solution act 30 °C
A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), initiator 7
(0.601
g, 2.27 mmol) and a magnetic follower prior to being deoxygenated by cycling
between nitrogen and vacuum three times. To a second Schlenk tube was added
MPEG(395)MA (10 mL, 22.74 mmol), N-(n-propyl)-2-pyridylinethanimine (0.71 mL,
4.54 mmol) and toluene (10 mL). The mixture was immediately subjected to five
freeze-pump-thaw degassing cycles. This solution was then transferred to the
Schlenk
tube containing the initiator and Cu(I)Br via a cannula. The resulting brown
solution
was stirred at 30 °C.Samples were removed periodically using degassed
syringes and
quenched in liquid nitrogen for conversion and molecular weight analysis.
Table 16: Kinetic data for the polymerisation of MPEG(395)MA initiated by 7 in
toluene solution (50% v/v) at 30°C
([MPEG(395)MA]o/[CuBr]o/[NHSBr]o/[ligand]o =
10/1/1/2).
Solvent Time Conversionln([M]o/[M])Mn, Mn, sEC Mw~n
l then
Ligand (h) (%) (g.mol'1)(g.mol-1)
Toluene 1 8.9 0.0933 450 2350 1.10
Propylligand2 18.4 0.204 920 2860 1.26
3 27.1 0.316 1360 3100 1.20
4 34.7 0.4259 1740 3600 1.13
17 80.8 1.6510 4050 5670 1.06
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~PEGJI~IJI~CuJI~LJ =10/1/1/2 itt SO v/v% anisole solution at 30 °C
A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), initiator 7
(0.601
g, 2.27 mmol) and a magnetic follower prior to being deoxygenated by cycling
between nitrogen and vacuum three times. To a second Schlenk tube was added
MPEG(395)MA (10 mL, 22.74 mmol), N-(n-ethyl)-2-pyridylmethanimine (0.64 mL,
4.54 mmol) and anisole (10 mL). The mixture was immediately subjected to five
freeze-pump-thaw degassing cycles. This solution was then transferred to the
Schlenk
tube containing the initiator and Cu(I)Br via a cannula. The resulting brown
solution
was stirred at 30 °C. Samples were removed periodically using degassed
syringes and
quenched in liquid nitrogen for conversion and molecular weight analysis.
Table 17: Kinetic data for the polymerisation of MPEG(395)MA initiated by 7 in
anisole solution (50% v/v) at 30°C
([MPEG(395)MA]o/[CuBr]o/[NHSBr]o/[ligand]o =
10/1/1/2).
Solvent / Time Conversion ln([M]o/[M]) M", then Mn, sEC Mw~n
Ligand (h) (%) (g.mofl) (g.mofl)
Anisole 2 17.0 0.1861 850 2670 1.11
Ethylligand 4 26.8 0.3116 1340 3460 1.12
6 34.8 0.4277 1740 4260 1.11
22 81.4 1.6809 4080 6350 1.06
~PEGJI~IJI~CuJI~LJ =10/1/1/2 i~z 50 v/v% ahisole solution at 30 °C
A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), initiator 7
(0.601
g, 2.27 mmol) and a magnetic follower prior to being deoxygenated by cycling
between nitrogen and vacuum three times. To a second Schlenk tube was added
MPEG(395)MA (10 mL, 22.74 mmol), N-(n-propyl)-2-pyridylmethanimine (0.71 mL,
4.54 mmol) and anisole (10 mL). The mixture was immediately subjected to five
freeze-pump-thaw degassing cycles. This solution was then transferred to the
Schlenk
c.'~abe containing the initiator and Cu(I)Br via a cannula. The resulting
brown solution
was stirred at 30 °C. Samples were removed periodically using degassed
syringes and
quenched in liquid nitrogen for conversion and molecular weight analysis.
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Table 18: for the polymerisation
Kinetic of MPEG(395)MA initiated
data by 7 in
anisole
solution
(50% v/v)
at 30C
([MPEG(395)MA]o/[CuBr]o/[NHSBr]o/[ligand]o
=
10/1/1/2).
Solvent Time Conversion ln([M]o/[M])Mn, Mn, sEC Mw~n
/ then
Ligand (h) (%) (g.mol-1)(g.mol-1)
Anisole 2 9.8 0.1030 490 2070 1.10
Propylligand4 16.8 0.1837 842 2480 1.12
6 28.7 0.3378 1440 2870 1.13
27 83.4 1.7985 4180 6280 1.06
Polymerisation of MPEG(550)MA
~PEGJI~IJI~CuJI~L, = 6.4/1/1/2 ih 66 wlv% toluene solution at 30 °C
Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 ec~ and
MPEG(550)MA
(7.57 g, 0.012 mol), and a magnetic follower were placed in an oven dried
Schlenk
tube. The Schlenk tube was evacuated and flushed with dry nitrogen three
times.
Deoxygenated toluene (14 mL) was added to the Schlenk tube. The resulting
solution
was deoxygenated via three freeze pump thaw cycles and then degassed N ethyl-2-
pyridylmethanimine (0.51 g, 3.79 mrnol) was added. The reaction was placed in
a
thermostatically controlled oil bath at 30 °C (t=0) and samples were
removed
periodically for conversion and molecular weight analysis.
Fable 19: Data for the polymerization of MPEG(550)MA with initiator 7 at 30
°C in
66 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
60 19 2850 1.04
131 32 3230 1.10
199 45 3560 1.12
250 53 3760 1.12
298 56 3980 1.12
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~PEGJI~IJI~CuJI~LJ = 6.4/1/1/2 in 66 wlv~ toluene solution at SO °C
Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and
MPEG(550)MA) (7.57 g, 0.012 mol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (15 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The
reaction
was placed in a thermostatically controlled oil bath at 50 °C (t=0) and
samples were
removed periodically for conversion and molecular weight analysis.
Table 20: Data for the polymerization of MPEG(550)MA with initiator 7 at 50
°C in
66 w/v~ toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
59 39 3212 1.09
126 56 3958 1.11
195 69 4375 1.13
246 75 4649 1.13
295 82 4874 1.13
(PEGJI(IJI~CuJI~LJ = 23.9/1/1/2 iu 66 ~v/v% toluene solution at 90 °C
Initiator 7 (2.5 g, 9.47 mmol), Cu(I)Br (1.35 g, 9.47 mmol, 1 eq) and
MPEG(550)MA
(142.0 g, 0.226 mol), and a magnetic follower were placed in an oven dried
Schlenk
tube. The Schlenk tube was evacuated and flushed with dry nitrogen three
times.
Deoxygenated toluene (261 mL) was added to the Schlenk tube. The resulting
solution was deoxygenated via three freeze pump thaw cycles and then degassed
N
propyl-2-pyridylmethanimine (2.80 g, 0.019 mol) was added. The reaction was
placed
in a thermostatically controlled oil bath at 90 °C (t=0) and samples
were removed
periodically for conversion and molecular weight analysis
The polymer was purified by the dropwise addition of the reaction solution to
a
vigorously stirred solution of diethyl ether (1000 mL). The resulting oil was
washed
with diethyl ether (3 x 1000 mL) and then dried ira vacuo.
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Table 21: Data for the polymerization of MPEG(550)MA with initiator 7 at 90
°C in
66 w/v% toluene solution.
Sample Time Conversion Mn PDi
l minutes /
48 21 4449 1.11
132 40 7198 1.08
185 44 7779 1.07
245 46 8105 1.09
300 48 8331 1.09
~PEGJIIIJI~CuJI~LJ = 6.4/1/1/2 ih 66 wlv% toluene solution at SO °C
Initiator 7 (10.0 g, 0.038 mol), Cu(I)Br (5.41 g, 0.038 mol, 1 ec~ and
MPEG(550)MA
(151.0 g, 0.240 mol), and a magnetic follower were placed in an oven dried
Schlenk
tube. The Schlenk tube was evacuated and flushed with dry nitrogen three
times.
Deoxygenated toluene (302 mL) was added to the Schlenk tube. The resulting
solution was deoxygenated by bubbling with nitrogen for 1 hour and then
degassed N
ethyl-2-pyridylinethanimine (10.2 g, 0.0761 mol) was added. The reaction was
placed
in a thermostatically controlled oil bath at 50 °C (t=0). Conversion
was followed by
1H NMR spectrometry and molecular weight analysis by SEC.
Table 22: Data for the polymerization of MPEG(550)MA with initiator 7 at SO
°C in
66 w/v% toluene solution.
Sample Time Conversion Mn pD
/ minutes /
235 86.4 5140 1.13
~PEGJI~IJI~CuJI~LJ = 6.46/1/1/2 iu 62 w/v% toluene at SO °C
Initiator 7 (2.95 g, 1.119 ~ 10-Z mol), Cu(I)Br (1.60 g, 1.119 x 10'a mol) and
MPEG(550)MA (45.42 g, 7.23 X 10-2 mol) and a magnetic follower were placed in
an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Toluene (73 mL) was then added to the Schlenk tube and
the
mixture degassed via three consecutive freeze, pump, thaw cycles. On
completion
deoxygenated N-ethyl-2-pyridylinethanimine (3.16 mL, 2.24 x 10-a mol) was
added
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and the Schlenk placed in a thermostatically controlled oil bath at 50
°C (t=0) and
sampled for conversion and molecular weight analysis. The polymer was isolated
by
washing with diethyl ether and subsequently dialysed in acidified water (pH
~4)
Table 23: Data for the polymerization of MPEG(550)MA with initiator 7 at 50
°C in
62 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
300 84.9 4590 1.22
rPEGJI~IJI~~'uJI~LJ = 23.9/1/1/2 in 67 wlv°fo toluene solution at SO
°C
Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq), 2,2'-
bipyridyl
(0.059 g, 0.378 mmol), MPEG(550)MA, (2.84 g, 4.52 mmol) and a magnetic
follower
were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and
flushed with dry nitrogen three times. Deoxygenated toluene (5.68 mL) was
added to
the Schlenk tube and the resulting solution was deoxygenated via three freeze
pump
thaw cycles. The reaction was placed in a thermostatically controlled oil bath
at 50 °C
(t=0) and samples were removed periodically for conversion and molecular
weight
analysis.
Table 24: Data for the polymerization of MPEG(550)MA with initiator 7 at 50
°C in
67 w/v% toluene solution using 2,2'-bipyridyl ligand.
Sample Time Conversion Mn PDi
/ minutes /
240 85 15443 1.11
~PEGJI~IJI~CuJI~LJ = 23.9/1/1/2 in 67 w/v% toluene solution at 50 °C'
Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq), 4,4'-
dinonyl-
2,2'-dipyridyl (0.1545 g, 0.378 mmol), MPEG(550)MA, (2.84 g, 4.52 mmol) and a
magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube
was
evacuated and flushed with dry nitrogen three times. Deoxygenated toluene
(5.68 mL)
was added to the Schlenk tube and the resulting solution was deoxygenated via
three
freeze pump thaw cycles. The reaction was placed in a thermostatically
controlled oil
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bath at 50 °C (t=0) and samples were removed periodically for
conversion and
molecular weight analysis.
Table 25: Data for the polymerization of MPEG(550)MA with initiator 7 at 50
°C in
67 w/v% toluene solution using 4,4'-dinonyl-2,2'-dipyridyl ligand.
Sample Time Conversion Mn PDi
/ minutes /
240 79 15936 1.16
~PEGJI~IJI~CuJI~LJ = 23.9/1/1/1 in 67 w/v% toluene solution at SO °C
Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 ec~,
1,1,4,7,10,10-
hexamethyltriethylenetetramine (0.0435 g, 0.189 mmol), MPEG(550)MA, (2.84 g,
4.52 mmol) and a magnetic follower were placed in an oven dried Schlenlc tube.
The
Schlenk tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (5.68 mL) was added to the Schlenk tube and the resulting solution was
deoxygenated via three freeze pump thaw cycles. The reaction was placed in a
thermostatically controlled oil bath at 50 °C (t=0) and samples were
removed
periodically for conversion and molecular weight analysis.
Table 26: Data for the polymerization of MPEG(550)MA with initiator 7 at 50
°C in
67 w/v% toluene solution using l,1,4,7,10,10-hexamethyltriethylenetetramine
ligand.
Sample Time Conversion Mn PDi
/ minutes /
240 86 19060 1.16
~~'~EGJI~IJI~CuJIILJ = X3.9/1/1/1 in 67 wlv% toluene solution at SO °C
Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 ec~,
N,N,N',N",N"-
pentamethyldiethylenetriamine (0.0328 g, 0.189 mmol), MPEG(550)MA, (2.84 g,
4.52 mmol) and a magnetic follower were placed in an oven dried Schlenk tube.
The
Schlenk tube was evacuated and flushed with dry nitrogen three times.
Deoxygenated
toluene (5.68 mL) was added to the Schlenk tube and the resulting solution was
deoxygenated via three freeze pump thaw cycles. The reaction was placed in a
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thermostatically controlled oil bath at 50 °C (t=0) and samples were
removed
periodically for conversion and molecular weight analysis.
Table 27: Data for the polymerization of MPEG(550)MA with initiator 7 at 50
°C in
67 w/v% toluene solution using N,N,N',N",N"-pentamethyldiethylenetriamine
ligand.
Sample Time Conversion Mn PDi
/ minutes /
240 95 19019 1.20
Polymerisation of MPEG(1000)MA
~PEGJI~IJI~CuJI~LJ =13.9/1/1/2 iu 66 w/v% toluene solution act 90 °C
Initiator 7 (0.526 g, 1.99 mmol), Cu(I)Br (0.29 g, 2.02 mmol, 1 ec~ and
MPEG(1000)MA (29.62 g, 0.027 mol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (60 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated via three freeze pump thaw cycles and
then
degassed N ethyl-2-pyridylinethanimine (0.51 g, 3.96 mol) was added. The
reaction
was placed in a thermostatically controlled oil bath at 90 °C (t=0) and
samples were
removed periodically for conversion and molecular weight analysis.
The polymer was purified by the dropwise addition of the reaction solution to
a
vigorously stirred solution of diethyl ether (1000 mL). The resulting oil was
washed
with diethyl ether (3 x 1000 mL) and then dried in vacuo.
Table 28: Data for the polymerization of MPEG(1000)MA with initiator 7 at 90
°C in
66 w/v~o toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
1250 47.3 12180 1.16
2460 50.4 12460 1.16
3890 52.8 12540 1.20
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~PEGJI~IJI~CuJI~LJ = 9.0/1/0.24/0.24 in 75 w/v% toluene solution at SO
°C.
Initiator 7 (5.0 g, 0.019 mol), Cu(I)Br (0.66 g, 4.61 mmol, 0.24 eq) and
MPEG(1000)MA (185.0 g, 0.171 mol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (740 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridyhnethanimine (1.24 g, 9.24 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 29: Data for the polymerization of MPEG(1000)MA with initiator 7 at 50
°C in
75 w/v% toluene solution.
sample Time Conversion Mn PDi
/ minutes /
60 7 5650 0.93
120 11 5595 . 0.97
285 20 6315 1.02
1340 43 7993 1.08
7476 63 9543 1.06
~FEGJI~IjI~CuJI~LJ =18.5/1/1/2 in 75 wlv% toluene solution at SO °C
Initiator 7 (1.0 g, 3.79 mmol), Cu(I)Br (0.54 g, 3.79 mmol, 1 eq) and
MPEG(1000)MA (151.4 g, 0.140 mol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (608 mL) was added to the Schlenk
tube.
'The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridylmethanimine (1.02 g, 7.57 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 30: Data for the polymerization of MPEG(1000)MA with initiator 7 at 50
°C in
75 w/v% toluene solution.
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Sample Time Conversion Mn PDi
/ minutes /
4005 100 8607 1.32
~PEGJI~IJI~CuJI~LJ =18.5/1/1/2 iu 75 w/v% toluene solution at 50 °C
Initiator 7 (2.0 g, 7.57 mmol), Cu(I)Br (1.08 g, 7.57 mmol, 1 ec~ MPEG(1000)MA
(151.47 g, 0.140 mol), and a magnetic follower were placed in an oven dried
Schlenk
tube. The Schlenk tube was evacuated and flushed with dry nitrogen three
times.
Deoxygenated toluene (606 mL) was added to the Schlenk tube. The resulting
solution was deoxygenated by bubbling with nitrogen for 1 hour and then
degassed N
ethyl-2-pyridylmethanimine (2.03 g, 0.015 mol) was added. The reaction was
placed
in a thermostatically controlled oil bath at 50 °C (t=0) and samples
were removed
periodically for conversion and molecular weight analysis.
Table 31: Data for the polymerization of MPEG(1000)MA with initiator 7 at 50
°C in
75 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
195 20 7270 1.04
1380 53 11964 1.08
2735 73 13945 1.08
Polymerisation of MPEG(2000)MA
~PEGJI~IJI~CuJI~LJ =19.2/1/1/2 iu 80 wlv6 toluene solution at 30 °C
Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 e~ and
(MPEG(2000)MA) (7.55 g, 3.63 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated via three freeze pump thaw cycles and
then
degassed N ethyl-2-pyridylxnethanimine (0.05 g, 0.38 mmol) was added. The
reaction
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was placed in a thermostatically controlled oil bath at 30 °C (t=0) and
samples were
removed periodically for conversion and molecular weight analysis.
The polymer was purified by the dropwise addition of the reaction solution to
a
vigorously stirred solution of diethyl ether (400 mL). The resulting white
powder was
filtered, dissolved in toluene (20mL) and precipitated in diethyl ether (400
mL). This
procedure was repeated three times.
Table 32: Data for the polymerization of MPEG(2000)MA with initiator 7 at 30
°C in
80 w/vJ toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
89 4 3380 1.04
291 9 9820 1.09
901 17 10030 1.07
1369 23 11080 1.07
2760 26 12610 1.07
3965 28 14830 1.04
~PEGJI~IJI~CuJI~LJ =19.11/1/2 in 80 fv/v% toluene solutio~e at 50 °C
Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq) and
MPEG(2000)MA (7.55 g, 3.63 mmol), and a magnetic follower were placed in ari
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated via three freeze pump thaw cycles and
then
degassed N ethyl-2-pyridylinethanimine (0.05 g, 0.38 mmol) was added. The
reaction
was placed in a thermostatically controlled oil bath at 50 °C (t=0) and
samples were
removed periodically for conversion and molecular weight analysis.
The polymer was purified by the dropwise addition of the reaction solution to
a
vigorously stirred solution of diethyl ether (400 mL). The resulting white
powder was
filtered, dissolved in toluene (20mL) and precipitated in diethyl ether (400
mL). This
procedure was repeated three times.
Table 33: Data for the polymerization of MPEG(2000)MA with initiator 7 at 50
°C in
80 w/v% toluene solution.
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Sample Time Conversion Mn PDi
/ minutes /
86 7 8700 1.06
289 12 10920 1.07 ,
899 24 14450 1.05
1367 33 15810 1.04
2758 45 20220 1.07
3962 53 23180 1.07
~PEGJI~IJI~CuJIILJ =19.2/1/1/2 in 80 wlv! toluene solution at 90 °C
Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 e~ and
MPEG(2000)MA (7.55 g, 3.63 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated via three freeze pump thaw cycles and
then
degassed N ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The
reaction
was placed in a thermostatically controlled oil bath at 90 °C (t=0) and
samples were
removed periodically for conversion and molecular weight analysis.
The polymer was purified by the dropwise addition of the reaction solution to
a
vigorously stirred solution of diethyl ether (400 mL). The resulting white
powder was
filtered, dissolved in toluene (20mL) and precipitated in diethyl ether (400
mL). this
procedure was repeated three times.
Table 34: Data for the polymerization of MPEG(2000)MA with initiator 7 at 90
°C in
80 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
86 18 11100 1.08
289 26 14870 1.08
899 31 17900 1.08
1367 35 18110 1.09
2758 38 18110 1.09
3962 39 18240 1.08
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~PEGJI~IJI(CuJI~LJ = 9.3/1/1/2 in 66 fv/v.°/ toluene solution at 90
°C
Initiator 7 (O.S g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and
MPEG(1000)MA (18.90 g, 0.018 mol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (35 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated via three freeze pump thaw cycles and
then
degassed N ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The
reaction
was placed in a thermostatically controlled oil bath at 90 °C (t=0) and
samples were
removed periodically for conversion and molecular weight analysis.
Table 35: Data for the polymerization of MPEG(1000)MA with initiator 7 at 90
°C in
66 w/v% toluene solution.
Sample Time Conversion Mn PDi
/minutes /
4160 88.7 9870 1.22
~PEGJIIIJI~CuJI,(LJ =19.2/1/1/ in 80 w/v% toluene solution at 50/70 °C
Initiator 7 (0.67 g, 2.53 mmol), Cu(I)Br (0.36 g, 2.53 mmol, 1 eq) and
MPEG(2000)MA (101.24 g, 0.049 mol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (405 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated via three freeze pump thaw cycles and
then
degassed N ethyl-2-pyridylmethanimine (0.68 g, 5.07 mmol) was added. The
reaction
was placed in a thermostatically controlled oil bath at 50 °C (t=0) and
samples were
removed periodically for conversion and molecular weight analysis. The
temperature
was increased to 70 °C after 45 hours 15 minutes.
Table 36: Data for the polymerization of MPEG(2000)MA with initiator 7 at
50/70 °C
in 80 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes l
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4030 47 24,600 1.06
~PEGJI~IJI~Cu~I~LJ =19.2/1/1/2 ih 75 wlv% toluene at 50/70 °C
Initiator 7 (0.66 g, 2.5 ~ 10'3 moI), Cu(I)Br (0.36 g, Z,5 X 10'3 mol) and
MPEG(2000)MA (100.0 g, 4.81 x 10-2 mol) and a magnetic follower were placed in
an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with
dry
nitrogen three times. Toluene (300 mL) was then added to the Schlenk tube and
the
mixture deoxygenated by purging with nitrogen for 1 hour. Deoxygenated N-ethyl-
2-
pyridylinethanimine (0.706 mL, 5.0 X 10-3 mol) was then added and the Schlenk
placed in a thermostatically controlled oil bath at SO °C (t=0). The
temperature was
increased to 70°C after 24 hours. The polymer was isolated by washing
with diethyl
ether and subsequently dialysed in acidified water (pH ~4).
Table 37: Data for the polymerization of MPEG(2000)MA with initiator 7 at
50/70 °C
in 75 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
2880 94.2 . 21900 1.21
~~~Gjl~Ijl~CuJI~LJ = 28.8/1/1/2 in 75 wlv% toluene at 50/70 °C.
Initiator 7 (0.44 g, 1.67 ~ 10'3 mol), Cu(I)Br (0.24 g, 1.67 x 10'3 mol) and
MPEG(2000)MA (100.0 g, 4.81 X 10'2 mol) and a magnetic follower were placed in
an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with
dry
nitrogen three times. Toluene (300 mL) was then added to the Schlenk tube and
the
mixture deoxygenated by purging with nitrogen for 1 hour. Deoxygenated N-ethyl-
2-
pyridylmethanimine (0.47 mL, 3.33 ~ 10'3 mol) was then added and the Schlenk
placed in a thermostatically controlled oil bath at 50 °C C (t=0). The
temperature was
increased to 70°C after 24 hours. The polymer was isolated by washing
with diethyl
ether and subsequently dialysed in acidified water (pH ~4).
Table 38: Data for the polymerization of MPEG(2000)MA with initiator 7 at
50/70 °C
in 7S w/v% toluene solution.
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Sample Time Conversion Mn PDi
/ minutes /
2~~0 92.~ 26370 1.26
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Polymerisations using Initiator 5
,Me
Me Me
'Br
Cl
H
Polymerisation of MPEG(550)MA
~PEGJI~IJI~CuJI~LJ = 64/1/1/2 in 75 w/v% toluene solution at 50/90°x'
Initiator 5 (0.25 g, 0.622 mmol), Cu(I)Br (0.09 g, 0.622 mmol, 1 ec~ and
MPEG(S50)MA (24.90 g, 0.040 mol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (100 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridylinethanimine (0.167 g, 1.245 mmol) was added.
The
reaction was placed in a thermostatically controlled oil bath at SO °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The temperature was increased to 90°C after 3 hours 1 S minutes.
Table 39: Data for the polymerization of MPEG(SSO)MA with initiator 5 at 50/90
°C
in 7S w/v% toluene solution.
Sample Time Conversion Mn PDi
l minutes /
1S0 3 5376 1.07
353 9 10390 1.09
1750 27 1390 1.16
Polymerisation of MPEG(1000)MA
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jPEGJI~I~I~CuJI~LJ = 37/1/1/2 in 75 w/v~o toluene solution at 50 °C
Initiator 5 (0.125 g, 0.031 mmol), Cu(I)Br (0.044 g, 0.031 mmol, 1 ec~ and
MPEG(1000)MA (12.45 g, 0.012 mol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (50 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridylmethanimine (0.083 g, 0.062 mmol) was added.
The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 40: Data for the polymerization of MPEG(1000)MA with initiator 5 at 50
°C in
75 w/v°/~ toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
62 0 0 0
352 10 9713 1.06
1716 15 10924 1.08
2725 16 11240 1.08
4142 15 12800 1.11
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Y~olymerisations using Initiator 9
HO~
O
O Br \
9
Polymerisation of MPEG(2000)MA
~PEGjI~IJI~CuJI~LJ = 28.8:1/1/2 in 67 wlv°fo acetone at SO °C
~r~itiator 9 (0.035 g, 1.667 x 10~ mol), Cu(I)Br (0.024 g, 1.667 X 10'~ mol)
and
MPEG(2000)MA (10 g, 4.g1 X10-3 mol) and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Acetone (20 mL) was then added to the Schlenk tube and
the
mixture degassed via three consecutive freeze, pump, thaw cycles. On
completion
deoxygenated N-ethyl-2-pyridylinethanimine (0.05 mL, 3.54 ~ 10~ mol) was added
and the Schlenk placed in a thermostatically controlled oil bath at 50
°C (t=0) and
samples removed periodically for conversion and molecular weight analysis.
Table 41: Data for the polymerization of MPEG(2000)MA with initiator 9 at 50
°C in
67 w/v% acetone solution.
Sample Time Conversion Mn PDi
l minutes /
60 3 25270 1.06
360 19 22690 1.08
340 74 3200 1.14
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holymerisations using Initiator 6
N
\ ~ ~~
-o
Br
6
Polymerisation of MPEG(2000)MA
~PEGJI~IJI~CujIjLJ =14.4/1/1/2 iu 67 wlv% toluene at 30 °C
Initiator 6 (0.119 g, 3.333 ~ 10~ mol), Cu(I)Br (0.048 g, 3.333 ~ 10'4 mol)
and
MPEG(2000)MA ( 10 g, 4.81 ~ 10-3 mol) and a magnetic follower were placed in
an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Toluene (20 mL) was then added to the Schlenk tube and
the
mixture degassed via three consecutive freeze, pump, thaw cycles. On
completion
deoxygenated N-n-propyl-2-pyridylmethanimine (0.10 mL, 6.667 ~ 10~ mol) was
added and the Schlenk placed in a thermostatically controlled oil bath at 30
°C (t=0)
and sampled for conversion and molecular weight analysis.
Table 42: Data for the polymerization of MPEG(2000)MA with initiator 6 at 30
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
3900 29.5 24120 1.08
~PEGJI(IjI~CuJI~LJ = 21.63/1/1/2 in 67 wlv% toluehe at 30 °C
Initiator 6 (0.079 g, 2.222 X 10'4 mol), Cu(I)Br (0.031 g, 2.222 X 10~ mol)
and
PEG(2000)MA (10 g, 4.81 X10-3 mol) and a magnetic follower were placed in an
oven dried Schlenlc tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Toluene (20 mL) was then added to the Schlenk tube and
the
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mixture degassed via three consecutive freeze, pump, thaw cycles. On
completion
deoxygenated N-n-propyl-2-pyridylmethanimine (0.066 mL, 4.444 ~ 10~ mol) was
added and the Schlenk placed in a thermostatically controlled oil bath at 30
°C (t=0)
and sampled for conversion and molecular weight analysis.
Table 43: Data for the polymerization of MPEG(2000)MA with initiator 6 at 30
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
6600 27 27480 1.10
~PEGJI~IJI~CuJIILJ = 28.8/1/1/2 iu 67 rv/v% toluene at 30 °C
Initiator 6 (0.059 g, 1.667 x 10~ mol), Cu(I)Br (0.024 g, 1.667 X 10-4 mol)
and
MPEG(2000)MA (10 g, 4.81 ~ 10-3 mol) and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Toluene (20 mL) was then added to the Schlenk tube and
the
mixture degassed via three consecutive freeze, pump, thaw cycles. On
completion
deoxygenated N-n-propyl-2-pyridylmethanimine (0.049 mL, 1.667 ~ 104 mot) was
added and the Schlenk placed in a thermostatically controlled oil bath at 30
°C (t=0)
and sampled for conversion and molecular weight analysis.
rl'able 44: Data for the polymerization of MPEG(2000)MA with initiator 6 at 30
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
6600 18.3 25290 ~ 1.09
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Polymerisations using Initiator 10
S
O
G Br
Polymerisation of MPEG(395)MA
[PEG]/[I]/[Cu]/[L] = 25/1/1/2 in 67 w/v% toluene at 50 °C
Initiator 10 (0.81 g, 1.68 X 10-3 mol), Cu(I)Br (0.24 g, 1.68 ~ 10-3 mol) and
MPEG(395)MA (20.0 g, 4.21 ~ 10-a mol) and a magnetic follower were placed in
an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Toluene (41 mL) was then added to the Schlenk tube and
the
mixture deoxygenated by purging with nitrogen for 1 hour. Deoxygenated N-~c-
propyl-2-pyridylmethanimine (0.53 mL, 3.37 X 10-3 mol) was then added and the
Schlenk placed in a thermostatically controlled oil bath at 50 °C (t=0)
and sampled for
conversion and molecular weight analysis.
Table 45: Data for the polymerization of MPEG(395) with initiator 10 at 50
°C in 67
~~r/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
900 52.9 6300 I .11
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Polymerisations using Initiator 11
O
O
H
0
O Br
Polymerisation of MPEG(550)MA
~PEG,I~IJI~CuJI~LJ = 6.4/1/1/2 in 67 vlv% toluene solution at SO °C
Initiator 11 (0.103 g, 0.380 mmol), Cu(I)Br (0.054 g, 0.380 mmol, 1 ec~ and
T~1PEG(550)MA (1.51 g, 2.41 mmol), and a magnetic follower were placed in an
even
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (2.78 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 46: Data for the polymerization of MPEG(550)MA with initiator 11 at 50
°C in
67 v/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
60 24 3545 1.080
120 41 4002 1.102
180 53 4243 1.104
240 64 4506 1.109
300 70 4677 1.108
~PEGJI~IJI~CuJI~LJ = 6.4/1/1/2 in 67 vlvJ toluene solution at 50 °C
Initiator 11 (3.0 g, 11.1 mmol), Cu(I)Br (1.584 g, 11.1 mmol, 1 ec~ and
MPEG(550)MA (44.27 g, 70.5 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
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nitrogen three times. Deoxygenated toluene (81.3 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridylinethanimine (2.97 g, 22.2 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The polymer was purified by removing the solvent in vacuo and dialysising the
residue using acidic water (pH ~4). Subsequent freeze drying isolated the
product.
Table 47: Data for the polymerization of MPEG(550)MA with initiator 11 at 50
°C in
67 v/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes l
60 27 4800 1.096
120 45 5427 1.115
180 60 5895 1.127
240 67 6215 1.133
300 72 6343 1.125
Polymerisation of MPEG(2000)MA
~PEGJI~IJI~CuJI~LJ =12/1/1/2 iu 80 wlv~ toluene solutioh at 50/70 °C
Initiator 11 (0.1 g, 0.369 mmol), Cu(I)Br (0.053 g, 0.369 mmol, 1 eq) and
MPEG(2000)MA (9.24 g, 4.44 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (37 mL) was added to the Schlenk
tube.
'fhe resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The temperature was increased to 70 °C after 113 hours. The polymer was
purified by
removing the solvent in vacuo and dialysising the residue using acidic water
(pH ~4).
Subsequent freeze drying isolated the product.
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Table 48: Data for the polymerization of MPEG(2000)MA with initiator 11 at
50/70
°C in 80 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
1020 21 - -
1440 23 - -
6780 40 - -
9660 62 20333 1.117
~PEGJI~IJI~CuJI~LJ = 24/1/1/2 ih 80 wlv6 toluehe solution at SOl70 °C
Initiator 11 (0.1 g, 0.369 mmol), Cu(I)Br (0.053 g, 0.369 mmol, 1 eq) and
MPEG(2000)MA (18.48 g, 8.88 mmol), and a magnetic follower were placed in an
oven dried Schlenk tube. The Schlenlc tube was evacuated and flushed with dry
nitrogen three times. Deoxygenated toluene (74 mL) was added to the Schlenk
tube.
The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour
and
then degassed N ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The temperature was increased to 70 °C after 113 hours. The polymer was
purified by
removing the solvent in vacu~ and dialysising the residue using acidic water
(pH ~4).
Subsequent freeze drying isolated the product.
Table 49: Data for the polymerization of MPEG(2000)MA with initiator 11 at
50/70
°C in 80 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
1020 16 - -
2580 19
6780 22 - -
8220 53 - -
9660 0.57 22262 1.140
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Polymerisations using Initiator 12
-O
Br
-O O O
O
12
Polymerisation of MPEG(1000)MA
[PEG]/[I]/[Cu]/[L] = 5/1/1/2 in 66 v/v% toluene solution at 70 °C
IrT-(ethyl)-2-pyridylinethanimine ligand (1.41 mL, 10.92 mmol), initiator 12
(1.633 g,
5.46 mmol), and MPEG(1000)MA (27.3 mL, 30 g, 27.3 mmol) were charged to a dry
Schlenk tube along with toluene (60 mL) as the solvent and mesitylene (1 mL)
as an
internal standard. The tube was sealed with a rubber septum and subjected to
three
freeze pump thaw cycles. This solution was then cannulated under nitrogen into
another Schlenk tube, previously evacuated and filled with nitrogen,
containing
Cu(I)Cl (0.543 g, 5.46 mmol) and a magnetic follower. The brown solution was
subsequently heated to 70 °C with constant stirring (t = 0). Samples
were removed
periodically using a degassed syringe for molecular weight and conversion
analysis.
After 48 h the mixture was diluted with 50 mL of toluene, air was bubbled for
6 h and
the green suspension was kept at 0° C overnight. After filtration
through a short
neutral alumina column to remove the copper salt, the polymer was precipitated
from
diethyl ether. The polymer was collected by filtration and dried in vacuum
oven
(40°C) overnight.
Table 50: Data for the polymerization of MPEG(1000)MA with initiator 12 at 70
°C
in 66 v/v% toluene solution.
Sample Time Conversion Mn PDi
/ hours /
1 17.~ 1370 1.23
2.5 56.6 10700 1.09
4 73.5 11600 1.15
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78.9 11600 1.11
6 81.3 11600 1.19
7 85.9 12600 1.15
~PEGJI~I~I~CuJI~LJ = 20/1/1/2 in 66 v/v% toluene solution at 50 °C
N-(ethyl)-2-pyridylinethanimine ligand (0.35 mL, 2.73 mmol), initiator 12
(0.41 g,
1.37 mmol), PEG(1000)MA (27.3 mL, 30 g, 27.3 mmol) were charged to a dry
Schlenk tube along with toluene (60 mL) as the solvent and mesitylene (1 mL)
as
internal standard. The tube was sealed with a rubber septum and subjected to
three
freeze pump thaw cycles. This solution was then cannulated under nitrogen into
another Schlenk tube, previously evacuated and filled with nitrogen,
containing
Cu(I)Br (0.197 g, 1.37 mmol) and a magnetic follower. The brown solution was
subsequently heated to 50 °C with constant stirring (t = 0). Samples
were removed
l.~eriodically using a degassed syringe for molecular weight and conversion
analysis.
Half the reaction solution was removed with a dry cannula when conversion was
at
66%, bubbled for 6 h with air, and passed over a short neutral alumina column
to
removed copper salt. The solvent was removed under vacuum and the unreacted
monomer was removed by dialysis to give the polymer as a white powder. After
48 h
the remaining reaction mixture was diluted with 50 mL of toluene, air was
bubbled
for 6 h and the green suspension was kept at 0° C overnight. After
filtration through a
short neutral alumina column to remove the copper salt, the polymer was
precipitated
from diethyl ether. The polymer was collected by filtration and dried in
vacuum oven
(40 °C) overnight.
Table 51: Data for the polymerization of MPEG(1000)MA with initiator 12 at 50
°C
in 66 vlv% toluene solution.
Sample Time . Conversion Mn PDi
l hours /
1 5 1566 1.14
3 11 11400 1.06
6 26 13000 1.07
8 31 12700 1.09
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21 61 13600 1.13
24 66 13800 1.14
28 73 14500 1.2
31 76 14900 1.21
46 87 14600 1.18
50.5 89 16000 1.2
54 0.9 17600 1.19
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Polymerisations using Initiator 13
O
O
N~~O~O Br
O O I
t3
Polymerisation of MPEG(550)MA
~PEGJI~IJI~CuJI~LJ =15.9/1/1/2 ih 67 tv/v% toluehe solution at 30 °C
Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 ec~ and
MPEG(SSO)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (S.S mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylmethanimine (0.074 g, O.S6 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 30 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 52: Data for the polymerization of MPEG(S50)MA. with initiator 13 at 30
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
1440 7.8 5626 1.08
4260 14.0 61 S3 1.11
~PEGJI~IJI~CujI~LJ = 8/1/1/2 ih 67 wlvJ toluehe solutioh at SO °C
Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 e~ and
MPEG(SSO)MA (1.38 g, 2.20 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (2.75 mL) was added to the Schlenk tube. The
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resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylinethanimine (0.074 g, 0.56 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 53: Data for the polymerization of MPEG(550)MA with initiator 13 at 50
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
7200 44.8 7880 1.16
~PEGjI~IJI~CuJI~LJ =15.9/1/1/2 in 67 w/v% toluene solution at 50 °C
Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 rnmol, 1 e~ and
MPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (5.5 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylinethanimine (0.074 g, 0.56 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
'fable 54: Data for the polymerization of MPEG(550)MA with initiator 13 at 50
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
1440 16.4 7971 1.12
8640 27.2 8378 1.14
~PEGJI~IJI~CuJI~LJ = 31.11/1/2 in 67 w/v% toluene solution at 50 °C
Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 e~ and
MPEG(550)MA (5.51 g, 8.77 mmol), and a magnetic follower were placed in an
oven
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dried Schlenk tube. The Schlenle tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (11.0 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylinethanimine (0.074 g, 0.56 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 55: Data for the polymerization of MPEG(550)MA with initiator 13 at 50
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
1440 9.6 8504 1.11
5760 16.8 9999 1.14
8640 17.8 10208 1.13
~PEGJI~IJI~CuJI~LJ =15.9/1/1/2 in 67 w/v% toluene solution at 70 °C
Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 ec~ and
MPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (5.5 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylinethanimine (0.074 g, 0.56 mmol) was added. The
~°eaction was placed in a thermostatically controlled oil bath at 70
°C (t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
Table 56: Data for the polymerization of MPEG(550)MA with initiator 13 at 70
°C in
67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
120 13.6 5768 1.09
300 21.3 6814 1.10
4260 41.0 8444 1.15
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~PEGJI~IJI~CuJI~LJ =15.9/1/1/2 in 67 fvlv% toluene solution ut SOl90 °C
Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Cl (0.0273 g, 0.28 mmol, 1 ec~ and
MPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an
oven
dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry
nitrogen
three times. Deoxygenated toluene (5.5 mL) was added to the Schlenk tube. The
resulting solution was deoxygenated via three freeze pump thaw cycles and then
degassed N ethyl-2-pyridylmethanimine (0.074 g, 0.56 mmol) was added. The
reaction was placed in a thermostatically controlled oil bath at 50 °C
(t=0) and
samples were removed periodically for conversion and molecular weight
analysis.
The temperature was increased to 90 °C after 163 hours
Table 57: Data for the polymerization of MPEG(550)MA with initiator 13 at
50/90 °C
in 67 w/v% toluene solution.
Sample Time Conversion Mn PDi
/ minutes /
9780 3.8 4090 1.09
12660 81.0 16504 1.31
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Polymerisations using Initiator 14
O
Boc VH~~O
Br
14
Polymerisation of MPEG(395)MA
~PEGJI~IJI~CuJI~LJ = 6/1/1/2 iu SO v/v% toluene solution at 40 °C
N-(ethyl)-2-pyridylinethanimine ligand (1.07 mL, 1.017 g, 7.58 x 10'3 mol),
initiator
14 (1.229 g, 3.79 x 10'3 mol) and MPEG(395)MA (10.80 g, 22.70 x 10'3 mol) were
charged to a dry Schlenk tube along with toluene (10 mL) as the solvent (50%
v/v).
The tube was sealed with a rubber septum and subjected to three freeze-pump-
thaw
cycles. This solution was then cannulated under nitrogen into another Schlenk
tube,
previously evacuated and filled with nitrogen, containing Cu(I)Br (0.544 g,
3.79 x 10'
a ~~101) and a magnetic follower. The brown solution was subsequently heated
to 40 °C
with constant stirring (t = 0). Samples were removed periodically using a
degassed
syringe for molecular weight and conversion analysis. After 48 h the mixture
was
diluted with 50 mL of toluene, air was bubbled for 6 h and the green
suspension was
kept at 0 ° C overnight. After filtration through a celite~ pad, the
solvent was
removed under reduced pressure to give a yellow-brown oil which was dissolved
in
water (250 mL) and purified by dialysis (Millipore, regenerated cellulose,
MWCO 1
kDa, filtration area 0.23 m2) to give the expected polymer as a pale yellow
oil.
~°able 58: Polymerisation data for TMM-LRP of MPEG(395)MA using
initiator 14,
[monomer] : [initiator] : [CuBr] : [L] = 6:1:1:2.
Monomer/ Time Conv. ln([M]o/[M]) M" PDI
Initiator (wins) (%)
6:1 (40 °C) 60 34.76 0.478 5876 1.03
120 49.43 0.667 7250 1.08
180 62.05 0.889 8089 1.08
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240 71.54 1.118 9013 1.10
300 76.33 1.359 9262 1.10
360 80.18 1.556 9561 1.07
420 86.93 1.904 9932 1.10
480 88.72 2.216 10195 1.10
~PEGJI~IJIICuJI~LJ = 28/1/1/2 in 50 vlvs toluehe solution at 40 °C
N-(ethyl)-2-pyridylinethanimine ligand (1.07 mL, 1.017 g, 7.58 x 10-3 mol),
initiator
14 (0.263 g, 0.812 x 10'3 mol) and MPEG(395)MA (10.80 g, 22.70 x 10-3 mol)
were
charged to a dry Schlenk tube along with toluene (10 mL) as the solvent (50%
v/v).
The tube was sealed with a rubber septum and subjected to three freeze-pump-
thaw
cycles. This solution was then cannulated under nitrogen into another Schlenk
tube,
previously evacuated and filled with nitrogen, containing Cu(1)Br (0.116 g,
0.812 x
10-3 mol) and a magnetic follower. The brown solution was subsequently heated
to 40
°C with constant stirring (t = 0). Samples were removed periodically
using a degassed
syringe for molecular weight and conversion analysis. After 48 h the mixture
was
diluted with 50 mL of toluene, air was bubbled for 6 h and the green
suspension was
kept at 0° C overnight. After filtration through a celite~ pad, the
solvent was removed
under reduced pressure to give a yellow-brown oil which was dissolved in water
(250
mL) and purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa,
filtration area 0.23 m2) to give the expected polymer as a pale yellow oil.
Table 59: Polymerisation data for TMM-LRP of MPEG(395)MA using initiator 14,
[monomer]:[initiator]:[CuBr]:[L] = 28:1:1:2. T= 40 °C.
Monomer/ Time Conv. ln([M]o/[M])M" PDI
Initiator (mins) (%)
28:1 (40 C) 60 21.8 0.246 5500 1.05
120 24.7 0.284 5798 1.06
180 37.4 0.468 7001 1.09
240 41.5 0.536 7202 1.08
300 46.2 0.620 7633 1.07
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360 49.3 0.680 7733 1.09
420 50.7 0.707 7899 1.09
480 55.4 0.808 8099 1.08
~PEGJI~IJI~CuJI~LJ = 28/1/1/2 in 50 vlv% toluehe solution at 60 °C
N-(ethyl)-2-pyridylinethanimine ligand (1.07 mL, 1.02 g, 7.58 x 10-3 mol),
initiator 14
(0.263 g, 0.812 x 10-3 mol) and MPEG(395)MA (10.80 g, 22.70 x 10-3 mol) were
charged to a dry Schlenk tube along with toluene (10 mL) as the solvent (50%
v/v).
The tube was sealed with a rubber septum and subjected to three freeze-pump-
thaw
cycles. This solution was then cannulated under nitrogen into another Schlenk
tube,
previously evacuated and filled with nitrogen, containing Cu(I)Br (0.116 g,
0.812 x
10-3 mol) and a magnetic follower. The brown solution was subsequently heated
to 60
°C with constant stirring (t = 0). Samples were removed periodically
using a degassed
uyringe for molecular weight and conversion analysis. After 48 h the mixture
was
diluted with 50 mL of toluene, air was bubbled for 6 h and the green
suspension was
kept at 0° C overnight. After filtration through a celite~ pad, the
solvent was removed
under reduced pressure to give a yellow-brown oil which was dissolved in water
(250
mL) and purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa,
filtration area 0.23 m2) to give the expected polymer as a pale yellow oil.
Table 60: Polymerisation data for TMM-LRP of MPEG(395)MA using initiator 14,
[monomer] : [initiator] : [CuBr] : [L] = 28:1:1:2. T = 60 ° C.
Monomer/ Time Conv. ln([M]o/[M])M" PDI
Initiator (mins) (%
28:1 (60 C) 60 38.0 0.427 5233 1.06
120 48.7 0.682 5656 1.07
180 58.9 0.969 6116 1.06
240 67.3 1.257 6185 1.08
300 74.3 1.441 6416 1.07
360 78.9 1.618 6284 1.08
420 85.1 2.035 6291 1.08
480 89.1 2.182 6610 1.08
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~'PEGJI~IJI~C'uJI~LJ =10/1/1/2 in 50 v/v% d8-toluene solution at 40 °C
N-(n-octyl)-2-pyridylmethanimine ligand (0.052 mL, 0.050 g, 0.228 x 10-3 mol),
initiator 14 (0.037 g, 0.114 x 10-3 mol) and MPEG(395)MA (0.050 rnL, 0.540 g,
1.14
x 10-3 mol) were charged to a dry Schlenk tube along with ds-toluene (0.50 mL)
as the
solvent (50% v/v). The tube was sealed with a rubber septum and subjected to
three
freeze-pump-thaw cycles. This solution was then cannulated under nitrogen into
an
NMR tube, previously evacuated and filled with nitrogen, containing Cu(I)Br
(0.016
g, 0.114 x 103 mol). The tube was then heated to 40 °C and 1H NMR
spectra were
recorded every 15 minutes.
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Polymerisations using Initiator 15
O
IO
O
O
O Br
i5
Polymerisation of MPEG(395)MA
~PEGJI~IJI~CuJI~L, = 8/1/1/2 in 50 vlve toluehe solutioh at 30 °C
N-(ethyl)-2-pyridylinethanimine ligand (0.80 mL, 0.76 g, 5.68 x 10-3 mol),
initiator 15
(2.03 g, 5.68 x 10-3 mol) MPEG(395)MA (20.0 mL, 21.6 g, 45.50 x 10'3 moI) were
charged to a dry Schlenk tube along with toluene (20 mL) as the solvent (50%
v/v).
The tube was sealed with a rubber septum and subjected to three freeze-pump-
thaw
;;~cles. This solution was then cannulated under nitrogen into another Schlenk
tube,
previously evacuated and filled with nitrogen, containing Cu(I)Br (0.4I g,
2.84 x 10-3
mol) and a magnetic follower (t = 0). The brown solution was subsequently
stirred at
30°C. Samples were removed periodically using a degassed syringe for
molecular
weight and conversion analysis. After 7 h the mixture was diluted with 50 mL
of
toluene, air was bubbled for 6 h and the green suspension was kept at
0° C overnight.
After filtration through a celite~ pad, the solvent was removed under reduced
pressure to give a yellow-brown oil which was dissolved in water (250 mL) and
purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa, filtration
area
0.23 m2) to give the expected polymer as a pale yellow oil.
Table 61: Polymerisation data for the TMM-LItP of MPECr(395)MA using initiator
15, [monomer]:[initiator]:[CuBrJ:[LJ = 8:1:1:2. T = 30 °C.
Monomer/ Time Conv. ln([MJo/[MJ) M" PDI
Initiator (mins) (%)
8:1 (30 °C) 60 22.90 0.478 4532 1.07
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120 34.64 0.667 5008 1.11
180 45.66 0.889 5379 1.11
246 50.97 1.118 5662 1.09
420 67.14 1.359 6165 1.08
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Reactions of PoIyPEG Polymers
Reactions of PoIyPEG Polymers prepared from initiator 8
Hydrolytic stability of the succihimide ehd group of PoIyPEG polymer initiated
by 8
~n-line 1H NMR experiments was carried out, each using a different buffer. N-
succinimidyl (initiat~r 8) terminated poly(MPEG(39S)MA (Mn= 6400g/mol, PDT=
1.09) (SOmg, 0.00781 x 10'3 mol) were introduced in an NMR tube and dissolved
in
0.5 mL of the appropriate phosphate buffer (pH = 8, C=100 mM or 200mM). NMR
spectra were recorded regularly.
Table 62: Kinetic data for the hydrolysis of N-succinimidyl terminated
poly(MPEG(39S)MA initiated by 8 in different buffers.
100 mM phosphate 200 mM phosphate
buffer H = 8 buffer ( H = 8
Time Conversion Time Conversion
0 0 0 0
O.S 4.8 O.S 8.6
1 9.S 1 13.5
1.S 10.1 1.S 17.5
2 15.8 2 19.8
2.S 14.2 2.5 22.7
3 15.1 3 2S.S
3.S 18.5 3.S 26.7
4 22.4 4 28.9
4.S ~ 23.2 4.S 33.7
S 26.0 S 33.2
S.S 30.1 S.S 37.1
6 28.7 6 38.9
6.S 30.7 6.S 39.6
7 30.7 7 44.1
7.S 30.1 7.S 43.0
8 37.3 8 44.0
8.S 35.9 8.S 45.2
9 36.7 9 45.8
9.S 40.7 9.S 46.5
45.2 10 50.6
10.5 40.8 10.5 49.7
11 43.2 11 52.2
11.5 42.5 11.5 S2.S
12 44.8 12 SS.6
12.5 45.4 12.5 SS.S
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13 45.1 13 56.9
13.5 45.0 13.5 53.7
14.5 48.9
15 48.4
1S.S 50.1
16 49.6
16.5 50.1
17 52.6
17.5 50.2
18 53.2
18.5 52.0
19 54.9
19.5 SS.l
20 53.9
20.5 SS.S
21 SS.9
21.5 53.0
22 S4.S
22.5 SS.9
23 SS.3
23.5 57.6
24 58.9
Bioconjuctiou of succiuimide temihated PoIyPEG polymer initiated by 8
A set of three experiments was carried out, each containing a different ratio
polymer /
lysozyme. Low molecular weight succinimidyl ester terminated
poly(MPEG(39S)MA) prepared from initiator 8 (Mn = 6400 g/mol, PDI= 1.11) (8.9
mg, 1.39x10'6 mol) for a ratio 2/l, (22.6 mg, 3.SOx10'6 mol) for a ratio S/1
and (89.5
~~g, 13.99x10'6 mol) for a ratio 20/1 and lysozyme (10 mg, 0.699x10'6 mol) was
dissolved in 10 ml of anhydrous DMSQ and O.S mL of anhydrous TEA and stirred
at
room temperature under nitrogen. Samples were taken periodically and analyzed
by
HPLC. The HPLC system was fitted with a guard column, a BioSep-SEC-53000
column and a LTV detector continuously measuring the relative absorbance of
the
mobile phase at 21 S nm. The system was eluted with 0.1 % v/v trifluoroacetic
acid
solution in water and acetonitrile (69/31 v/v) at a rate of 0.5 mL/min. In the
case of a
ratio 30:1, the crude was analysed by SDS-PAGE (polyacrylamide resolving gel
cross-linking: 1 S%, running buffer: 25 mM TRIS base, 2S0 mM glycine, 0.1 %
SDS,
pH 8.7).
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Yteactions of PoIyPEG Polymers prepared from initiator 7
Hydrolytic stability of the succit~imide ehd group of PoIyPEG polymer
initiated by 7
On-line 1H NMR experiments was carried out, each using a different buffer. N-
succinimidyl (initiator 7) terminated Poly(MPEG(395)MA (Mn= 2700g1mo1, PDI=
1.12) (SOmg, 0.0185 x 10-3 mol) were introduced in an NMR tube and dissolved
in
0.5 mL of the appropriate buffer (200 mM phosphate buffer (pH = 6 and pH = 8),
100
mM phosphate buffer (pH = 8) or 200 mM borate buffer (pH = 9.2)). NMR spectra
were recorded regularly.
Table 63: Kinetic data for the hydrolysis of the succinimide end group of
Poly(MPEG(395)MA polymer initiated by 7 in different buffers.
200 100 200 200
mM mM mM mM
phosphate phosphate phosphate borate
buffer buffer buffer buffer
H= ( H=8 (pH
H=8 =
9.2)
Time ConversionTime ConversionTimeConversion Time Conversion
h % (h) (% h %) h
0.03 5.0 0.5 2.5 0.5 3 48 0.8
0.06715.0 1 4.8 1 5.48 192 3
0.10 25.0 1.5 6.4 1.5 7.6 336 5.2
0.13 35.0 2 9.1 2 9.13 504 9.6
0.17 43.8 2.5 10.2 3 11.56
0.20 48.5 3 11.2 4 14.33
0.23 55.0 3.5 12.4 5 16.5
0.27 60.0 4 14.1 21 31.7
0.30 64.0 4.5 16.1 22 31.32
0.33 66.0 5 17.4 23 33.86
0.37 69.0 5.5 19.6 24 34.04
0.40 70.0 6 20.6 25 33.35 '
0.43 72.0 6.5 24 39 42.73
0.47 73.0 7 26.2 57 49.2
0.50 74.5 7.5 26.9 77 54.11
0.67 81.4 8 27.7 99 57.3 ',
0.83 87.2 8.5 29.4 125 63
1.00 92.9 9 28.9 146 65.24
1.17 96.7 9.5 30 171 67.35
1.33 100 10 32.7 195 69.7
12.5 35.7 220 72.22
15 38.9 257 75
17.5 41.9 270 75.47
20 46 299 78.07
22.5 49
25 50.7
27.5 53.4
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30 54.4
32.5 57.6
35 59
37.5 61.6
40 62.9
42.5 65.1
45 66.3
47.5 68.4
50 69.1
52.5 70
55 72
59.5 73.3
Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (22.4 mL, 0.333 mol) and a magnetic follower were placed into
a
three necked round bottom flask fitted with a pressure equalising dropping
funnel.
The system was flushed with nitrogen and placed under positive pressure. A
solution
of succinimide terminated poly(MI'EG(550)MA) [Mn 4590 PDi 1.22] (3.0 g, 9.38
~ 10-4 mol) dissolved in anhydrous dichloromethane (12 mL) was added to the
dropping funnel and the solution added drop-wise to the ethylenediamine. The
solution was left stirnng for 16 hours before dialysing and subsequently
freeze drying
to isolate the product. IH NMR spectra shows the reduction of the succinimide
O=C-
CH -CH -C=O resonance at 2.75 ppm.
Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (14.85 mL, 0.222 mol) and a magnetic follower were placed into
a
three necked round bottom flask fitted with a pressure equalising dropping
funnel.
The system was flushed with nitrogen and placed under positive pressure. A
solution
of succinimide terminated poly(MPEG(S50)MA) [Mn 4590 PDi 1.22] (2.0 g, 6.25
X 10-4 mol) dissolved in water (20 mL) was added to the dropping funnel and
the
solution added drop-wise to the ethylenediamine. The solution was left
stirring for 16
hours before dialysing and subsequently freeze drying to isolate the product.
1H NMR
spectra shows reduction of the succinimide O=C-CH -CH -C=O resonance at 2.75
ppm.
Conversion of succinimide end group of PoIyPEG to aanine group
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Ethylenediamine (20.0 mL, 0.299 mol), water (20 mL) and a magnetic follower
were
placed into a three necked round bottom flask fitted with a pressure
equalising
dropping funnel and the solution cooled by placing in an ice bath. The system
was
flushed with nitrogen and placed under positive pressure. A solution of
succinimide
terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (5.0 g, 1.56 ~ 10-3 mol)
dissolved in water (50 mL) was added to the dropping funnel and the solution
added
drop-wise to the ethylenediamine. The solution was left stirnng for 24 hours
before
dialysing and subsequently freeze drying to isolate the product. 1H NMR
spectra
shows reduction of the succinimide O=C-CH -CH -C=O resonance at 2.75 ppm.
Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (1.35 mL, 0.02 mol), water (5 mL) and a magnetic follower were
placed into a three necked round bottom flask fitted with a pressure
equalising
dropping funnel and the solution cooled by placing in an ice bath. The system
was
flushed with nitrogen and placed under positive pressure. A solution of
succinimide
terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (1.0 g, 3.13 ~10'~ mol)
dissolved in water (25 mL) was added to the dropping funnel and the solution
added
drop-wise to the ethylenediamine. The solution was left stirring for 3 hours
before
adding the solution to 2 L of water and dialysing. Subsequently the dialysed
solution
was freeze dried to isolate the product. 1H NMR spectra shows reduction of the
succinimide O=C-CH -CH -C=O resonance at 2.75 ppm.
Cohversi~n of succihimide end group of PoIyPEG to amihe group
Ethylenediamine (1.35 mL, 0.02 mol), anhydrous dichloromethane (5 mL) and a
magnetic follower were placed into a three necked round bottom flask fitted
with a
pressure equalising dropping funnel and the solution cooled by placing in an
ice bath.
The system was flushed with nitrogen and placed under positive pressure. A
solution
of succinimide terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (1.0 g, 3.13
~ 10~ mol) dissolved in anhydrous dichloromethane (25 mL) was added to the
dropping funnel and the solution added drop-wise to the ethylenediamine. The
solution was left stirring for 3 hours before adding the solution to 2 L of
water and
dialysing. Subsequently the dialysed solution was freeze dried to isolate the
product.
1H NMR spectra shows reduction of the succinimide O=C-CH -CH -C=O resonance
at 2.75 ppm.
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Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (2.68 mL, 0.04 mol), water (10 mL) and a magnetic follower
were
placed into a three necked round bottom flask fitted with a pressure
equalising
dropping funnel and the solution cooled by placing in an ice bath. The system
was
flushed with nitrogen and placed under positive pressure. A solution of
succinimide
terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (2.0 g, 6.25 x 10~ mol)
dissolved in water (50 mL) was added to the dropping funnel and the solution
added
drop-wise to the ethylenediamine. The solution was left stirnng for 5.5 hours
before
adding the solution to 2 L of water and dialysing. Subsequently the dialysed
solution
was freeze dried to isolate the product. 1H NMR spectra shows reduction of the
succinimide O=C-CH -CH -C=O resonance at 2.75 ppm.
Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (2.67 mL, 0.04 mol), water (10 mL) and a magnetic follower
were
placed into a three necked round bottom flask fitted with a pressure
equalising
dropping funnel and the solution cooled by placing in an ice bath. The system
was
flushed with nitrogen and placed under positive pressure. A solution of
succinimide
terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (2.0 g, 6.25 ~ 10-4 mol)
~?issolved in water (50 mL) was added to the dropping funnel and the solution
added
drop-wise to the ethylenediamine. The solution was stirred for 4 hours before
neutralising the solution with 2M HCl and the water subsequently removed using
high
vacuum. The polymer was dialysed and then freeze dried to isolate the product.
1H
NMR spectra shows reduction of the succinimide O=C-CH -CH -C=O resonance at
2.75 ppm.
Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (0.36 mL, 0.013 mol), water (1 mL) and a magnetic follower
were
placed into a three necked round bottom flask fitted with a pressure
equalising
dropping fiu~nel and the solution cooled by placing in an ice bath. The system
was
flushed with nitrogen and placed under positive pressure. A solution of
succinimide
terminated poly(MPEG(2000)MA) [Mn 24600 PDi 1.06] (5.0 g, 2.03 x 10-4 mol)
diss~lved in water (100 mL) was added to the dropping funnel and the solution
added
drop-wise to the ethylenediamine. The solution was stirred for 4 hours before
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neutralising the solution with 2M HC1 and the water subsequently removed using
high
vacuum. The polymer was dialysed and then freeze dried to isolate the product.
1H
NMR spectra shows reduction of the succinimide O=C-CH -CH -C=O resonance at
2.75 ppm.
Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (2.5 mL, 0.03 mol), water (10 mL) and a magnetic follower were
placed into a three necked round bottom flask fitted with a pressure
equalising
dropping funnel and the solution cooled by placing in an ice bath. The system
was
flushed with nitrogen and placed under positive pressure. A solution of
succinimide
terminated poly(MPEG(2000)MA) [Mn 24600 PDi 1.06] (15.0 g, 7.5 ~ 10-4 mol)
dissolved in water (400 mL) was added to the dropping funnel and the solution
added
drop-wise to the ethylenediamine. The solution was stirred for 4 hours before
neutralising the solution with 2M HCl then adding NaCI (140 g) before
extracting into
dichloromethane (4 x 150 mL). The organic layers were combined and dried over
NaaS04 filtered and then evaporated to dryness before being washed with
diethyl
ether. The polymer was dialysed and then freeze dried to isolate the product.
1H NMR
spectra shows reduction of the succinimide O=C-CH -CH -C=O resonance at 2.75
ppm.
Conversion of succinimide end group of PoIyPEG to amine group
Ethylenediamine (5.60 mL, 0.0~ mol), water (lO.mL) and a magnetic follower
were
placed into a three necked round bottom flask fitted with a pressure
equalising
dropping funnel and the solution cooled by placing in an ice bath. The system
was
flushed with nitrogen and placed under positive pressure. A solution of
succinimide
terminated poly(MPEG(2000)MA) [Mn 21900 PDi 1.21] (33.5 g, 1.53 ~ 10-3 mol)
dissolved in water (500 mL) was added to the dropping funnel and the solution
added
drop-wise to the ethylenediamine. The solution was stirred for 4 hours before
neutralising the solution with 2M HCl then adding NaCI (140 g) before
extracting into
dichloromethane (4 x 150 mL). The organic layers were combined and dried over
NaaSO4 filtered and then evaporated to dryness before being washed with
diethyl
ether. The polymer was dialysed and then freeze dried to isolate the product.
1H NMR
spectra shows reduction of the succinimide O=C-CH -CH -C=O resonance at 2.75
ppm.
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Conversion of amine end group of PoIyPEG to maleimide group
Amine terminated poly(MPEG(550)MA) [Mn 3200] (0.5 g, 1.56 ~ 10~ mol),
saturated
sodium hydrogen carbonate (2.5 mL) and a magnetic follower were placed into a
three necked round bottom flask and cooled by placing in an ice bath. The
system was
flushed with nitrogen and placed under an inert atmosphere. To this solution
N-methoxycarbonylmaleimide (0.1 g, 6.45 x 10~ mol) was added with vigorous
stirring. After ten minutes water (5 mL) was added and the reaction left
stirring for a
further 45 minutes. The pH was then adjusted to 3 with O.SN sulfuric acid and
NaCI
(0.15 g) was added. The polymer was then extracted in to dichloromethane (3 x
50
mL), the extracts were combined and dried over Na2S04 before being filtered
and
evaporated to dryness. The polymer was then washed with diethyl ether and
dried
under vacuum at room temperature. 1H NMR spectra shows appearance of the
maleimide resonances at ~5.9-6.4 and ~6.7 ppm.
Conversion of amine end group of PoIyPEG to maleimide group
Amine terminated poly(MPEG(550)MA) [Mn 3200] (1.0 g, 3.13 ~ 10'4 mol),
saturated
sodium hydrogen carbonate (5 mL) and a magnetic follower were placed into a
three
necked round bottom flask and cooled by placing in an ice bath. The system was
flushed with nitrogen and placed under an inert atmosphere. To this solution
N-methoxycarbonylinaleimide (0.2 g, 1.29 X 10-3 mol) was added with vigorous
stirring. After ten minutes water (10 mL) was added and the reaction left
stirring for a
further 45 minutes. The pH was then adjusted to 3 with O.SN sulfuric acid and
NaCI
(0.30 g) was added. The polymer was then extracted in to dichloromethane (3 x
50
mL), the extracts were combined and dried over Na2S04 before being filtered
and
evaporated to dryness. The polymer was then washed with diethyl ether and
dried
under vacuum at room temperature. 1H NMR spectra shows appearance of the
maleimide resonances at ~5.9-6.4 and ~6.7 ppm.
Conversion of amine end group of PoIyPEG to maleirraide group
Amine terminated poly(MPEG(550)MA) [Mn 3200] (1.0 g, 3.13 X10-4 mol),
saturated
sodium hydrogen carbonate (5 mL) and a magnetic follower were placed into a
three
necked round bottom flask and cooled by placing in an ice bath. The system was
flushed with nitrogen and placed under an inert atmosphere. To this solution
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N-methoxycarbonylinaleimide (0.20 g, 1.29 ~ 10'3 mol) was added with vigorous
stirring. After ten minutes water (10 mL) was added and the reaction left
stirring for a
further 45 minutes. The pH was then adjusted to 3 with O.SN sulfuric acid and
NaCI
(3.75 g) was added. The polymer was then extracted in to dichloromethane (3 x
50
mL), the extracts were combined and dried over Na2S04 before being filtered
and
evaporated to dryness. The polymer was then washed with diethyl ether and
dried
under vacuum at room temperature. 1H NMR spectra shows appearance of the
maleimide resonances at ~5.9-6.4 and ~6.7 ppm.
Conversion of amine end group of PoIyPEG to maleimide group
Amine terminated poly(MPEG(2000)MA) [Mn 24600] (5.0 g, 2.03 ~ 10'4 mol),
saturated sodium hydrogen carbonate (15 mL) and a magnetic follower were
placed
into a three necked round bottom flask and cooled by placing in an ice bath.
The
system was flushed with nitrogen and placed under an inert atmosphere. To this
solution N-methoxycarbonylmaleimide (0.13 g, 8.13 ~ 10'4 mol) was added with
vigorous stirring. After ten minutes water (15 mL) was added and the reaction
left
stirring for a further 45 minutes. The pH was then adjusted to 3 with O.SN
sulfuric
acid and NaCI (7.5 g) was added. The polymer was then extracted in to
dichloromethane (4 x 50 mL), the extracts were combined and dried over Na2SO4
before being filtered and evaporated to dryness. The polymer was then washed
with
diethyl ether and dried under vacuum at room temperature. 1H NMR spectra shows
appearance of the maleimide resonances at ~5.9-6.4 and ~6.7 ppm.
Conversion of amine end group of PvIyPEG to maleimide group
Amine terminated poly(MPEG(2000)MA) [Mn 24600 PDi 1.06] (1 S.0 g, 6.1 X 10'4
mol), saturated sodium hydrogen carbonate (45 mL) and a magnetic follower were
placed into a three necked round bottom flask and cooled by placing in an ice
bath.
The system was flushed with nitrogen and placed under an inert atmosphere. To
this
>>p~~ution N-methoxycarbonylmaleimide (0.38 g, 2.44 ~ 10'3 mol) was added with
vigorous stirring. After ten minutes water (15 mL) was added and the reaction
left
stirnng for a further 45 minutes. The pH was then adjusted to 3 with O.SN
sulfuric
acid and NaCI (7.5 g) was added. The polymer was then extracted in to
dichloromethane (3 x 50 mL), the extracts were combined and dried over Na2SO4
before being filtered and evaporated to dryness. The polymer was then washed
with
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diethyl ether and dried under vacuum at room temperature before final
purification
through dialysis and isolation by freeze drying. 1H NMR spectra shows
appearance of
the maleimide resonances at ~5.9-6.4 and ~6.7 ppm.
Couplihg of succiuimide temiuated PoIyPEG to beuzylamiue
0
N gr \ NHZ CHZCI2orwater ~N Br
y 5 .~,. ~ U H 5
~ ~~ o
0 0~0~0~ o o'L
m-6
Two different experiments were carried out, each using a different solvent.
Low
~~olecular weight poly(MI'EG(395)MA) (Mn= 2700g/mol, PDI= 1.12) (lg, 0.370x10'
3 mol) prepared from initiator 7 (i.e. succinimide terminated) and benzylamine
(0.40
ml, 3.7x10-3 mol) was dissolved in 10 ml of dry chloroform or distilled water
and
stirred at room temperature for 20 hours under nitrogen. After reaction, the
solvent
was removed under vacuum by using a rotary evaporator. The crude was purified
by
preparative GPC before being precipitated of the polymer in cold Petroleum
Ether
(40-60°C Fraction).
Biocotzjuctiou of succihimide temiuated PoIyPEG polymer
A set of three experiments was carried out, each containing a different ratio
polymer /
lysozyme. Moreover each set of experiments was left to react for either 4
hours or 20
hours. Low molecular weight poly(MPEG(395)MA) prepared from initiator 7 (i.e.
succinimide terminated) (Mn= 2700 g/mol, PDI= 1.12) (41.6 mg, 15.4x10-3 mol)
for a
ratio 5/l, (83.2 mg, 30.8x10-3 mol) for a ratio 10/1 and (249.5 mg, 92.4x10-3
mol) for
a ratio 30/1 and lysozyme (50 mg, 3.08x10-3 mol) was dissolved in 10 ml of 200
mM
phosphate buffer (pH = 8) and stirred at 4 °C for 4 hours or 20 hours
under nitrogen.
The reaction was followed by HPLC in the case of a ratio polymer l lysozyme
30/1.
The HPLC system was fitted with a guard column, a BioSep-SEC-53000 column and
a UV detector continuously measuring the relative absorbance of the mobile
phase at
215 nm. The system was eluted with 0.1 % vlv trifluoroacetic acid solution in
water
and acetonitrile (69/31 v/v) at a rate of 0.5 mL/min. In each case, the crude
was
purified in dialysis bag (Spectra/Porl, MWCO = 6-8000 g/mol) and analysed by
SDS-
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130
PAGE (polyacrylamide resolving gel cross-linking: 15%, running buffer: 25 mM
TRIS base, 250 mM glycine, 0.1% SDS, pH ~.7).
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Reactions of PoIyPEG Polymers prepared from initiator 12
Conversion of acetal end group of PvIyPEG to aldehyde group
Acetal-terminated polymer (Mn 11000, PDi 1.15, 3.0 g, 0.27 mmol) was dissolved
in
a 1:1 trifluoroacetic acid (TFA)/Ha0 solution (100 mL) and the solution was
stirred at
room temperature for 48 hours. Most of the acid was removed under reduced
pressure
and the crude was dissolved in water and purified by dialysis. The aqueous
solution
was then freeze-dried to give the desired aldehyde terminal polymer (2.8 g,
0.25
mmol, 93 %) as an off white solid. (M"~11,000, PDi 1.13)
Conversion of acetal end group of PoIyPEG to aldehyde group
~~cetal-terminated polymer (Mn 22,000, PDi 1.09, 3.0 g, 0.14 mmol) was
dissolved in
a 1:1 trifluoroacetic acid (TFA)/H20 solution (100 mL) and the solution was
stirred at
room temperature for 48 hours. Most of the acid was removed under reduced
pressure
and the crude was dissolved in water and purified by dialysis. The aqueous
solution
was then freeze-dried to give the desired aldehyde terminal polymer (2.8 g,
1.3 mmol,
°~3 %) as an off white solid. (M"~22,000, PDi 1.09)
Conversion of acetal end group of PoIyPEG to aldehyde group
Acetal-terminated polymer (Mn = 32,000, PDi = 1.09, 3.0 g, 0.094 mmol) was
dissolved in a l:l trifluoroacetic acid (TFA)/HzQ s~lution (100 mL) and the
solution
was stirred at room temperature for 48 hours. Most of the acid was removed
under
~T~,~.~uced pressure and the crude was dissolved in water and purified by
dialysis. The
aqueous solution was then freeze-dried to give the desired aldehyde terminal
polymer
(2.7 g, 0.084 mmol, 90 %) as an off white solid. (M"~32,000, PDi 1.11)
Bioconjugation of deprotected PoIyPEG polymers prepared from initiator 12
Bioconjuction of aldehyde-terminated PoIyPEG polymer
Lysozyme (6 mg, 4.2x 10-4 mmol) and aldehyde-terminated polymer (Mn~22,000,
PDi
1.09, 110 mg, 0.01 mmol) was dissolved in 5 mL of acetate/acetic acid buffer
(pH =
5) and 0.15 mL of NaCNBH3 (0.25 mM in water) was added dropwise. The solution
was stirred at room temperature and samples were taken at regular intervals.
The
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reaction was monitored by HPLC fitted with a guard column, a bioSep-SEC-53000
column and an UV detector.
Bioconjuctioh of aldehyde-terminated PoIyPEGpolytner
Lysozyme (6 mg, 4.2x 10'~ mmol) and aldehyde terminated polymer (Mn~22,000,
PDi
1.09, 110 mg, 0.01 mmol) was dissolved in 5 mL of phosphate buffer (pH = 6)
and
0.15 mL of NaCNBH3 (0.25 mM solution in water) was added dropwise. The
solution
was stirred at room temperature and samples were taken at regular intervals.
The
reaction was monitored by HPLC fitted with a guard column, a bioSep-SEC-53000
column and an UV detector.
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Reactions of PoIyPEG Polymers prepared from initiator 14
Conversion of BOC eud group of PoIyPEG to amine group
O O~O~m CF3C00' O O~O~m
BocHN~O ~ / CF3COOH H3N~ ~0
Br ~ Br
p n CH~CI2, rt O n
BOC terminated polymer (Mn=6400 g mol-1, 3.2 g, 1.0 mmol) was dissolved in
CHZCI2 (25 mL), trifluoroacetic acid (3.9 mL, 50 mmol) was added via syringe
and
the resulting solution was stirred at room temperature for 16 h. The solvent
was then
removed under reduced pressure and the resulting orange-brown oil was
dissolved in
~eionized water and dialyzed. The polymer solution was freeze-dried. Toluene
(50
mL) was then added and the solvent was removed under reduced pressure. This
procedure was repeated three times and the expected amine terminated polymer
as the
trifluoroacetic acid salt (2.5 g, 0.~1 mmol, ~1 % yield) was obtained as a
yellow-
orange oil. 1H NMR revealed the complete disappearance of the singlet relative
to the
Boc group at 1.4 ppm. MW/Mn (GPC)=1.11
Co~aversioh of amine end group of PoIyPEG to maleimde group
0
CF3COO' O O~p~ N O O~O
HsN~O Br m --1 ~ NCO ' Jm
Br
O n O O n
3-Maleimidopropionyl chloride (13.0 mmol) was dissolved in 100 mL of CH~C12,
diisopropylethylamine (D1PEA, 2.3 mL, 13.0 mmol) was added via syringe and the
solution was cooled to 0°C. A solution of amine terminated polymer as
the
trifluoracetic acid salt (1.5 g, 0.47 mmol) in 30 mL of CHZCl2 was added
dropwise
(ca. 15 min) and the mixture was stirred at 0°C for lh, then at room
temperature for 2
days. The solvent was then removed under reduced pressure and 200 mL of water
were added to the brown residue. The suspension was centrifugate and purified
by
dialysis (Millipore, regenerated cellulose, MWCO 1 lcDa, filtration area 0.23
m2). The
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polymer solution was freeze-dried. Toluene (50 mL) was then added and the
solvent
was removed under reduced pressure. This procedure was repeated three times
and
the expected maleimde terminated polymer was obtained as a pale yellow oil. A
conversion of ~0 % can be calculated by iH NMR, comparing the integration of
the
vinylic protons of the maleimide moiety and that of the terminal OCH3 of the
PEG
side-chains. Mw/Mn (GPC) = 1.06.
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135
Reactions of PoIyPEG Polymers prepared from initiator 15
Conversion of furan end group of PoIyPEG to maleimde group
O '~ ~'
O O O~O~m O O O O~O~m
N-~ Br ~ N~ ~~~~~Br
O n toluene reflux O
O 7h p
A solution of polymer prepared from initiator 15 (3.Og, 0.36 mmol) in toluene
(25
mL) was warmed to reflux and the reaction was monitored by 1H NMR analysis on
samples taken at regular intervals of time. After 7 h the solvent was removed
under
reduced pressure to give the maleimide terminated polymer as a pale orange
oil.
Comparison of the integration of the maleimide vinyl signals and the terminal
OCH3
of the PEG side-chains confirmed that the maleimide function did not decompose
during the deprotection step.
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References
1. D. M. Haddleton, M. C. Crossman, B. H. Dane, D. J. Duncalf, A. M. Henning,
D. Kul~ulj and A. J. Shooter, Macromolecules,1999, 32, 2110.
2. R. N. Keller and W. D. Wycoff, Inorg. Synth., 1947, 2, 1.
3. James R. Dudley, Jaclc T. Thurston, Frederic C. Schaefer, Dagfrid Holm-
Hansen, Clarence J. Hull, and Pierrepont Adams, J. Am. Chem. Soc.,1951, 73,
2986.
