New method for the synthesis and purification of branched mPEG 2lys

Article abstract of DOI:10.1016/j.reactfunctpolym.2011.10.010

Branched PEG structures are particularly useful for the pegylation of different biomaterials. In this regard, the branched structure bearing a lysine core and two mPEG chains linked by urethane bonds (mPEG₂lys) has been used for the pegylation of several important biomolecules. In this work we describe the preparation of a new functionalized mPEG featuring an alkoxy-carbonylimidazolium iodide reactive group that can be used for the urethane bonds formation required in mPEG₂lys (20 kDa). The procedure for the preparation of the mPEG-alkoxy-carbonylimidazolium iodide involves the initial reaction of mPEG with N,N′-carbonylbismidazole (CDI) as phosgene equivalent, followed by alkylation with methyl iodide. The functionalized mPEG was reacted with a silylated lysine derivative affording mPEG₂lys in a single step. Therefore, harmful reagents (like phosgene derivatives) are not employed, making the process safe and straightforward. A 2-step purification procedure of mPEG₂lys is also presented. It is noteworthy that by following this method pure mPEG₂lys was obtained with 41% isolated yield. The mPEG₂lys was further converted to the N- hydroxysuccinimidyl ester by diimide activation and successfully used in the pegylation of interferon α-2a.

Full text of DOI:10.1016/j.reactfunctpolym.2011.10.010

Reactive & Functional Polymers 72 (2012) 107–113
Contents lists available at SciVerse ScienceDirect
Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
New method for the synthesis and purification of branched mPEG lys
2
1
⇑
Marianela González, Ricardo J.A. Grau , Santiago E. Vaillard
Laboratorio de Química Fina, INTEC (UNL-CONICET), Predio CCT-CONICET Santa Fe, Colect. Ruta Nacional 168, Km 472, Paraje ‘‘El Pozo’’, 3000 Santa Fe, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2011
Received in revised form 17 October 2011
Accepted 22 October 2011
Available online 15 November 2011
Branched PEG structures are particularly useful for the pegylation of different biomaterials. In this regard,
the branched structure bearing a lysine core and two mPEG chains linked by urethane bonds (mPEG lys)
2
has been used for the pegylation of several important biomolecules. In this work we describe the prep-
aration of a new functionalized mPEG featuring an alkoxy-carbonylimidazolium iodide reactive group
2
that can be used for the urethane bonds formation required in mPEG lys (20 kDa). The procedure for
the preparation of the mPEG-alkoxy-carbonylimidazolium iodide involves the initial reaction of mPEG
with N,N -carbonylbismidazole (CDI) as phosgene equivalent, followed by alkylation with methyl iodide.
Keywords:
0
Branched PEG
Reactive PEG
Pegylation
Imidazolium salt
Polyethylene glycol
The functionalized mPEG was reacted with a silylated lysine derivative affording mPEG
step. Therefore, harmful reagents (like phosgene derivatives) are not employed, making the process safe
and straightforward. A 2-step purification procedure of mPEG lys is also presented. It is noteworthy that
by following this method pure mPEG lys was obtained with 41% isolated yield. The mPEG lys was further
converted to the N-hydroxysuccinimidyl ester by diimide activation and successfully used in the pegyla-
tion of interferon -2a.
2
lys in a single
2
2
2
a
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
The covalent attachment of polyethylene glycol (PEG) chains
tive position of l-lysine. One usual strategy to achieve mPEG activa-
tion involves the initial reaction of mPEG with toxic phosgene or
triphosgene to obtain the corresponding alkoxycarbonyl chloride
or triphosgene derivative [18,20]. These compounds are rather
unstable and, instead of being purified, they are treated with active
alcohols to yield electrophilic carbonates. Although several alco-
hols have been used or suggested in the literature, the use of
N-hydroxysuccinimide to yield activated mPEG 2a (Scheme 1) is
by far the most preferred method. After reacting l-lysine ethyl ester
with the electrophilic mPEG derivative to yield compound 1b, the
ester is in turn converted to the branched mPEG 1 by hydrolysis
under acid or basic conditions. Another activated PEG derivative
that can be used for the preparation of 1 is mPEG 2b (Scheme 1),
which is obtained from mPEG and p-nitrophenylchloroformate,
the latter being a toxic compound [21]. It is also well known that
mPEG carbonylimidazole 2c (Scheme 1) is a mild pegylating re-
agent that has been conjugated to various proteins [22,23].
has been widely used to improve the biopharmaceutical properties
of several bioactive drugs [1–3]. In this regard, the use of branched
PEG structures is particularly attractive due to the numerous
advantages that branched PEG conjugates offer over the equivalent
linear forms [4]. Among the plethora of available pegylating re-
agents, the branched methoxy-PEG having a lysine core (mPEG lys,
2
compound 1, Fig. 1) has gained considerable attention as activat-
able precursor for pegylation [5–14]. In fact, the Y-shaped
mPEG lys 1 is capable of providing a high degree of shielding effec-
2
tiveness. Branched mPEG 1 is not reactive enough to be used di-
rectly for conjugation to biomaterials. Therefore, it is usually
converted to the more reactive N-hydroxysuccinimidyl ester by
means of diimide activation [15,16].
Few methodologies for the synthesis of 1 have been published
or patented [5,17–20]. A simple inspection of the l-lysine structure
suggests that the amino group at the
tive than the amino group at the position (the pKa of the
group is 1–2 pH units higher than the -amino group). Hence, a
It has been recently shown that related alkyl-carbamoylimidazo-
lium iodides 3 (Scheme 1), which can easily be obtained from
aliphatic amines and carbonylbisimidazole (CDI) by a two-step pro-
cedure, are useful intermediates for the preparation of ureas, amides
and carbamates, among other compounds [24]. Moreover, it has
been reported that alkyl-carbamoylimidazolium iodides are roughly
a
position might be less reac-
e
e
-amino
a
sufficiently strong activated mPEG derivative should be used to
accomplish the required urethane bond formation at the less reac-
3
00–400 times more reactive than their parent alkyl-carbamoylim-
idazoles [24]. Assuming that this reactivity difference might also be
maintained when moving from mPEG-alkoxycarbonylimidazole 2c
to the new mPEG-alkoxycarbonylimidazolium iodide 2d (Scheme
1), it seemed plausible that the mPEG-imidazolium salt 2d would
⇑
Corresponding author. Tel.: +54 342 4511370x1100; fax: +54 342 4511079.
E-mail addresses: mgonzal@intec.unl.edu.ar (M. González), cqfina@santafe-
conicet.gov.ar (R.J.A. Grau), svaillard@intec.unl.edu.ar (S.E. Vaillard).
1
Deceased.
1
381-5148/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.reactfunctpolym.2011.10.010

1
08
M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113
2
Fig. 1. Structure of mPEG lys 1.
be reactive enough to afford the two required urethane bonds pres-
ent in mPEG lys 1. With this idea in hand and challenged by the
disadvantages found in the methods currently available for the syn-
thesis of mPEG lys 1, we developed the new synthetic procedure
depicted in Scheme 2. The proposed synthetic sequence to branched
mPEG lys 1 involves only three mild reaction steps on which the
2
2
2
Scheme 2. New synthesis strategy for the preparation of mPEG lys 1.
2
new PEG derivative 2d proved to be a useful key intermediate. As
a distinctive feature, by this synthetic route, the use of toxic and dan-
gerous phosgene or triphosgene derivatives is avoided. To complete
Polyacrilamide gel electrophoresis (SDS–PAGE) was performed
according to the methods of Laemmli [26], with 8% of polyacrila-
mide, to evaluate PEG species in crude reaction mixtures. After
running, gels were rinsed with distilled water and placed in a 5%
barium chloride solution. The gels were maintained for 10 min
with gentle mixing, and then they were rinsed again, and placed
in a 0.1 N iodine solution for 5–10 min. In the case of proteins or
PEG-protein conjugates, 15% polyacrilamide gels were run, and
standard procedures with Coomasie brilliant blue were followed
to visualize them.
the preparation method of mPEG
fication procedure is presented. Finally, the purified mPEG
activated to N-hydroxysuccinimide ester and the pegylation of
interferon -2a was successfully achieved.
2
Lys 1, a convenient two-step puri-
2
lys 1 was
a
2
. Experimental
2.1. Materials
mPEG 20 kDa was purchased to JenKem Technology (Allem, USA).
2
2
.3. Synthesis and purification
0
N,N -carbonylbisimidazole (CDI), l-lysine, N-hydroxysuccinimide,
N,N-diisopropylethyl amine, N,O-bis(trimethylsilyl) acetamide, and
.3.1. Synthesis of mPEG-carbonylimidazole (2c)
mPEG-OH 20 kDa (0.1 mmol) was dissolved in anhydrous THF
0
N,N -dicyclohexylcarbodiimide were supplied by Sigma Aldrich (St.
4
Louis, MO). Acetonitrile was distilled from anhydrous MgSO and
(
6 mL) at 60 °C. CDI (0.3 mmol) was added and the solution was
stored over molecular sieves (4 Å). THF and diethyl ether were freshly
distilled from sodium benzophenone ketyl. DMSO was distilled un-
der vacuum and stored over molecular sieves (4 Å). Dry dichloro-
methane was obtained by distillation from P O . Methyl iodide was
2 5
prepared according to a reported method [25]. All other reagents
and solvents were used as received from the suppliers. Interferon
-2a was obtained from Pablo Cassará Laboratory (Buenos Aires,
Argentina). Vivaflow 200 ultrafiltration cassettes were purchased
from Sartorius (Hannover, Germany). XK 50/20 and XK 26/20 col-
stirred at 60 °C for 18 h. The solvent was removed under vacuum.
The residue was dissolved in water and then 5-fold extracted with
chloroform (25 mL each). The organic phase was evaporated at re-
duced pressure and dried (5 mm Hg) until constant weight. A
white solid was obtained (1.98 g, 99%). H NMR (300 MHz – Cl
1
3
CD),
d: 3.35 (s, 3H, OMe); 3.60 (brs, mPEG chain); 4.43–4.52 (m, super-
imposed with mPEG chain, CH OC(O)); 7.04 (s, 1H, Im-H); 7.40 (s,
H, Im-H); 8.11 (s, 1H, Im-H) (see Fig. 2, in Section 3).
a
2
1
umns, and Phenyl Sepharose HP were obtained from GE Healthcare
ꢀ
2.3.2. Synthesis of mPEG-carbamoylimidazolium salt (2d)
.98 g of product 2c was dissolved in 4 mL of acetonitrile at
room temperature. Freshly prepared methyl iodide was added
1.0 mmol), and the solution was stirred at room temperature for
6 h. The solvent was removed under reduced pressure and the
(
Piscataway, NJ). Fractogel EMD COO (M) was supplied by Merck
1
Chemicals (Darmstadt, Germany).
(
1
2.2. Physico-chemical characterization and analyses
1H NMR experiments were performed in CDCl
resulting solid residue was dried (5 mm Hg) until constant weight.
A yellow solid was obtained (1.94 g, 98%). H NMR (300 MHz –
3
in a Bruker
1
Avance 300 MHz spectrometer and referenced to the residual sol-
vent signal. A Bruker Biflex MALDI TOF mass spectrometer was used
to determine molecular weights of different mPEG fractions. UV–Vis
analyses were performed on a Shimadzu UV–Vis spectrophotome-
ter. SE-HPLC (size exclusion-HPLC) analyses were performed in a
Shimadzu apparatus equipped with a TSK Gel 3000SW, 7.5 ꢁ
Cl
superimposed with mPEG chain peak, CH
CH
3
CD), d: 3.36 (s, 3H, OMe); 3.63 (brs, mPEG chain); 3.86 (m,
2
OC(O)); 4.06 (s, 3H,
3
); 7.51 (s, 2H, 2ꢁ Im-H); 9,96 (s, 1H, Im-H) (see Figure, in
Section 3).
6
00 mm column (Tosoh) with UV detection at 280 nm. The mobile
2.3.3. Synthesis of mPEG
2.3.3.1. Preparation of Me
2
lys (1)
2 4 3 3
SiNH(CH ) (COOSiMe )-NHSiMe solution
phase for the analyses of different species in the conjugation
reaction and the chromatographic purification of the conjugate
was 50 mM sodium acetate buffer pH = 5.2, 0.2 M NaCl, 10% ethanol.
3
(4). A mixture of l-lysine (0.5 mmol), N,O-bis(trimethylsilyl)-acet-
amide (26.2 mol) and acetonitrile (0.30 mL) was irradiated in an
Scheme 1. Key step reaction for the synthesis of 1 with different activated mPEGs.

M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113
109
Fig. 2. 1H NMR spectrum of 2c.
ultrasound sonicator at room temperature until complete dissolu-
tion of the reagents (5 min).
(0.826 g, 41.3% isolated yield starting from mPEG 20 kDa) was ana-
lyzed by SDS–PAGE (BaCl /I stain, Fig. 5) RP-HPLC-ELSD and char-
2
acterized by MALDI-TOF, H NMR (Figs. 6 and 7 in Section 3). 1
NMR (300 MHz – Cl CD), d: 0.90–0.95 (m, 2H, lysine skeleton);
.2–1.4 (m, 6H, lysine skeleton); 3.09 (s, 6H, OMe); 4.14 (m, 2H,
CH OC(O)); 7.49 (s, 1H, NH); 7.65 (s,1H, NH) (see Fig. 7 in
Section 3).
1
H
3
2
.3.3.2. Synthesis of mPEG
um iodide 2d (1.94 g) was dissolved in 8 mL of 1:1 acetoni-
trile:DMSO mixture. 81 L of the solution obtained previously
2
lys (1). mPEG-alkoxycarbonylimidazoli-
1
2
l
and N,N-diisopropylethylamine (0.18 mmol) were added. The reac-
tion mixture was stirred at 85 °C overnight and allowed reaching
room temperature. Brine solution was added and the aqueous
phase was 5-fold extracted with dichloromethane (20 mL each).
The combined organic extract was evaporated and dried at reduced
pressure (5 mm Hg) until constant weight. A white solid was ob-
tained (1.90 g, 98%). SDS–PAGE analyses of the crude reaction mix-
2
2
.4. Conjugation of IFN
a
-2a
.4.1. Activation of mPEG
to the N-hydroxysuccinimidyl ester (mPEG
of 1 was accomplished by a slight modification of a known method
6,27] as follows: N-hydroxysuccinimide (0.062 mmol) was
2
lys
1
2
lys-OSu): activation
2
ture indicated the formation of mPEG lys 1 (see Fig. 5, lane 4 in
[
Section 3). Under identical reaction conditions, but using alkoxy-
carbonylimidazole 2c instead of alkoxycarbonylimidazolium io-
dide 2d, product 1 was not obtained, as shown by SDS–PAGE
analysis of the crude reaction mixture (Fig. 5, lane 3).
dissolved in 2 mL of anhydrous dichloromethane and 2 mL of
anhydrous THF under nitrogen atmosphere and was kept under
stirring in an ice bath. mPEG lys (0.0206 mmol) and dicyclohexyl-
2
carbodiimide (0.041 mmol) were added under nitrogen. The solu-
tion was stirred for 2 h in the ice bath. A new aliquot of
dicyclohexylcarbodiimide (0.02 mmol) was then added and the
reaction mixture was stirred for 30 min in the ice bath and then
maintained for 16 h at 4 °C without stirring. Dry diethyl ether
was added to the reaction tube (30 mL), and the precipitated prod-
uct was separated by centrifugation. The solid was washed with
dry diethyl ether (30 mL), dried and re-dissolved in 2 mL of aceto-
2
2
.3.4. Purification of mPEG
.3.4.1. Pre-purification step. An aqueous solution containing 1.90 g
lys 1 was diafiltered through a 50,000 MW cutoff
PES membrane using a 0.2 M NaCl solution, at a flow rate of
2
Lys (1)
of crude mPEG
2
ꢀ
2
ꢀ1
1
20 L m
h . After one ultrafiltration cycle with 4 L of 0.2 M NaCl,
the solution was 5-fold extracted with dichloromethane. The com-
bined organic extract was evaporated and dried under vacuum
nitrile. Acetic acid (35 lL) was added and the solution was kept 1 h
under stirring at room temperature. The upper phase was sepa-
rated by centrifugation and the product was precipitated with
dry diethyl ether, separated by centrifugation and once more
washed and precipitated. The obtained solid was dried at reduced
pressure (5 mm Hg) until a constant weight (0.735 g, 89%). The
activation was achieved in 98% yield, as shown by a known spec-
trophotometric assay [28].
(
5 mm Hg) until constant weight (1.425 g, 75%).
2
.3.4.2. Purification of 1 by hydrophobic interaction chromatogra-
phy. 1.425 g of the pre-purified sample of 1 was dissolved in 4 M
NaCl solution (concentration of 15 mg/mL), and loaded on XK 50/
2
(
0 column packed with Phenyl Sepharose HP (GE Healthcare)
CV = 250 mL). The purification process was conducted under con-
ditions of stepwise gradient elution by ionic strength reduction.
Eluting samples containing pure mPEG lys were identified by
2
2.4.2. Conjugation of (mPEG
mPEG lys-OSu (250 mg) was dissolved in 2.3 mL of 1 mM HCl
previously cooled to 4 °C, and stirred in an ice bath. When the re-
agent was dissolved IFN -2a (45 mg, 3.25 mg/mL, 50 mM sodium
2
lys-OSu) with IFN a-2a
SDS–PAGE analysis, pooled and 4-fold extracted with dichloro-
methane (30 mL each). The combined extract was evaporated
and dried at reduced pressure (5 mm Hg). The solid obtained
2
a

1
10
M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113
borate buffer pH 8.0) was quickly added, and the reaction allowed
to proceed for 3 h at 4 °C under gentle stirring [6,27]. The solution
was quenched by the addition of acetic acid to pH 4.5, then diluted
eight times with 10 mM ammonium acetate pH 4.5 and loaded on
an ion exchange chromatography column (matrix: Fractogel EMD
compound 2c is presented in Fig. 3a. This spectrum shows a peak at
k
max at 228 nm, in good agreement with published data. Moreover,
ꢀ1
ꢀ1
we determined a
e value for compound 2c of 3760 M cm
(water, 231 nm, 25 °C) suggesting that the reaction of CDI with
mPEG (20 KDa) was complete.
ꢀ
COO (M); column: XK 26/20, GE Healthcare; CV = 60 mL; 4 °C).
After the seminal work of Salvensen and Rapoport, carbamoylim-
idazolium salts have received little attention in organic synthesis.
However, in last few years Batey has shown that carbamoylimidazo-
lium iodides, which can easily be obtained from amines and CDI fol-
lowed by alkylation with methyl iodide, are useful intermediates for
the preparation of ureas among other compounds [24,30,31]. With
the aim of taking advantage of the aforementioned reactivity differ-
ence between carbamoylimdazoles and carbamoylimidazolium io-
dides, compound 2c was reacted with methyl iodide under
extremely mild conditions to afford alkoxycarbonylimidazolium
iodide 2d with good yield (98%, room temperature, 16 h) [24]. After
The purification process was conducted under conditions of
stepwise gradient elution with the following buffers: 40 mM
ammonium acetate pH 4.5; 0.12 M NaCl in 40 mM ammonium
acetate pH 4.5; 0.5 M NaCl in 40 mM ammonium acetate pH 4.5
and 1 M NaCl in 40 mM ammonium acetate pH 4.5. Eluting sam-
ples were monitored by UV absorbance at 280 nm. Fractions con-
taining conjugate IFN
concentrated using Amicon Ultra centrifugal filters (regenerated
2
a-2a – mPEG lys were pooled and
Ò
cellulose, 10,000 MW CO). SE-HPLC and SDS–PAGE gels (stained
with Coomasie brilliant blue, and BaCl /I; using a commercially
2
1
available Ref. [29]) were used to evaluate the conjugation reaction
and the chromatographic purification (Fig. 8 in Section 3). Concen-
tration of proteins was determined by UV absorbance at 280 nm,
and by Lowry protein assays. The isolated final yield of the conju-
gation procedure was 26%.
removal of the reaction solvent and volatile products, the H NMR
analysis of the crude product (Fig. 4) indicated the formation of
the product, and the ratio of Im-CH
azole signals suggested that the akylation reaction was complete in
16 h at room temperature (Scheme 2).
3
to the OCH
3
and 3 ꢁ 1 H imid-
Imidazolium salt 2d is not stable in aqueous solutions. The
UV–Vis spectrum of compound 2d is presented in Fig. 3b. Compound
3
. Results and discussion
.1. Preparation and purification of mPEG
The complete process for the synthesis of branched mPEG
2
d presents peaks at kmax at 360 and 291 nm (acetonitrile, 25 °C).
ꢀ1
ꢀ1
The estimated
respectively.
evalues for these peaks are 1907 and 3808 M cm ,
3
2
Lys 1
The conversion to the TMS derivatives is a well-established
methodology for the derivatization of amino acids. Under ultra-
sound irradiation, l-lysine was converted to the TMS derivative 4
using excess of N,O-bis(trimethylsilyl)acetamide and acetonitrile
as solvent (Scheme 2) [32]. Compound 4 is completely soluble in
organic solvents making unnecessary the use l-lysine ethyl ester.
It addition, since the TMS group can easily be removed by standard
aqueous work-up, an additional hydrolysis step after the coupling
of the activated mPEG reagent should not be needed.
2
lys 1
is depicted in Scheme 2. The strategy involves only few steps usually
under mild reaction conditions. Noteworthy, none of these steps
require inert atmosphere or special experimental setup. Given that
compound 1 bearing two 20 kDa mPEG chains is one of the most
useful branched mPEGs for the pegylation of bioactive molecules,
we decided to explore the envisaged strategy using mPEG 20 kDa
as starting material. The first step of the synthesis strategy involves
the well-known reaction of mPEG with CDI to afford mPEG-alkoxi-
carbonylimidazole 2c under mild reaction conditions (Scheme 2).
Other similar methods [22,23] for the preparation of activated poly-
We found that heating a mixture of 2d and 4 at 85 °C for 16 h in
1
:1 acetonitrile:DMSO using diisopropylethylamine as base, fol-
lowed by standard aqueous work-up, constituted the optimal reac-
tion conditions for the synthesis of mPEG lys 1. It is noteworthy
that only 10 mol% (5 eq.%) were enough to obtain mPEG lys with
1
mer 2c have also been published. The H NMR analysis of the crude
2c clearly indicated the formation of the product, and the ratio of
OCH
2
3
to the 3 ꢁ 1 H imidazole signals suggested the reaction pro-
2
good yield. The SDS–PAGE analysis of the crude product showed
the required branched structure 1 (40 kDa) and unreacted mPEG
or monomeric mPEGlys (both 20 kDa) in an estimated ratio of
ceeded to completion; hence the crude product was used in the next
step without further purification (Fig. 2).
ꢀ1
ꢀ1
Looze and coworkers determined a
e value of 3590 M cm
6
0:40 (by visual inspection of SDS–PAGE; see Fig. 5, lane 4). As re-
for t-butyloxycarbonylimidazole (water, 231 nm, 25 °C), and it
was indicated that this value can be used with confidence for com-
pound 2c with a mPEG chain of 5 kDa [23]. The UV–Vis spectrum of
ported for PEG-proteins conjugates [6,27,33] bearing this kind of
branched PEG structure, the electrophoretic mobility is consider-
Fig. 3. (a) UV–Vis spectrum of 2c and (b) UV–Vis spectrum of 2d.

M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113
111
Fig. 4. 1H NMR spectrum of 2d.
tion and large column volumes were employed in ion exchange
chromatography. Ultrafiltration methods have been specially stud-
ied to separate pegylated proteins from unreacted materials based
on differences in their size [35–38]. It has been reported that the
number, length and structure of the polymer chain attached to
proteins influence the sieving coefficients, not allowing their esti-
mation with only the hydrodynamic volume [38]. Moreover, at
high filtrate flow rates an elongation on the PEG chain is produced,
which increases when longer PEG chains are attached [39].
With the aim of using chromatographic techniques without
compromising the capacity of the matrix, we performed a pre-puri-
fication of mPEG
ultrafiltration in
2
lys 1 by economic and robust tangential flow
diafiltration mode (PES membrane and
lys 1
sample (estimated by visual inspection of SDS–PAGE gels, see
Fig. 5, lane 5) was purified by HIC, eluting mPEG lys 1 in the pure
a
5
0,000 MW cutoff, see Section 2). An 80% enriched mPEG
2
Fig. 5. SDS–PAGE analysis from different samples: lane 1: protein molecular weight
markers; lane 2: mPEG (20 kDa); lane 3: crude product mixture of the reaction of
mPEG derivative 2c with 4; lane 4: crude product mixture of the reaction of mPEG
derivative 2d with 4: lane 5: pre-purified sample of 1 after being diafiltered; lane 6:
2
1
form. The product was characterized by H NMR, MALDI-TOF and
RP-HPLC-ELSD (Figs. 6 and 7) [5]. To the best of our knowledge,
2
purified mPEG lys by HIC.
2
HIC has not previously been employed to separate mPEG lys from
unreacted PEG. We found that this procedure is quite successful for
this purpose being able to efficiently separate with high resolution
more than 2 g of pre-purified sample using a column volume of
ably slowed owing to the large hydrodynamic volume of PEG (each
ethylene oxide subunit of PEG can form an adduct with 3 mole-
cules of water) [34]. As shown in Fig. 5, in the electrophoresis pro-
2
50 mL.
The synthesis strategy and the purification procedure described
herein allowed us to obtain pure mPEG lys 1 with 41% isolated
2
cedures, branched mPEG lys run as a globular protein of 75 kDa. In
2
agreement with its expected lower reactivity, mPEG-alkoxycarb-
onylimidazole 2c reacted with 4 affording 1 in trace amounts, as
shown by the SDS PAGE analysis (Fig. 5, lane 3).
yield. Unfortunately, we have not been able to properly compare
this overall yield with those obtained in previously published
methods because the synthesis and purification procedures for
preparing this branched PEG derivative have been described in de-
tail, but the final yields of the procedures have not been clearly
reported.
The development of adequate techniques for the purification
and characterization of PEGs and PEG derivatives has been a criti-
cal issue for the progress on the synthesis of new and interesting
linear and branched PEGs. Given the fact that PEG is UV invisible
and that, in high molecular weight PEGs, the changes introduced
upon modification of the reactive hydroxyl end group are almost
undetectable when compared with polyethylene glycol chain,
more sophisticated methods are required to separate them.
2
3.2. Activation of mPEG lys and pegylation of IFN a-2a
2
Branched mPEG lys 1 has been separated from the monomeric
forms (native mPEG or mPEGlys) by gel permeation and ion ex-
change chromatography [5]. Although being useful, limited
With the aim of evaluating the performance of mPEG
tained as previously indicated, branched mPEG 1 was converted to
the N-hydroxysuccinimidyl ester (mPEG lys-OSu) under reaction
conditions similar to those indicated in the literature (diimide acti-
2
lys 1 ob-
2
2
amounts of mPEG lys were processed in gel permeation purifica-

1
12
M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113
2
Fig. 6. (a) MALDI-TOF spectrum of pre-purified crude mixture by diafiltration; peaks of 20 kDa PEGs species, mPEG lys (40 kDa) and oligomer structures can be seen. (b) RP-
HPLC-ELSD chromatogram of the same sample.
Fig. 7. 1H NMR spectrum of 1.
Fig. 8. (a) Chromatogram of SE-HPLC analysis of crude pegylation reaction, showing the presence of the different species, (b) Coomasie brilliant blue stained gel and (c)
Ò
barium/iodine stained gel to evaluate pegylation reaction. Lane 1: protein molecular weight markers; lane 2: IFN
lane 5: purified mPEG lys-IFN -2a.
a-2a; lane 3: PEGASYS ; lane 4: crude pegylation reaction;
2
a

M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113
113
vation at 0 °C) [6,27]. Using a reported assay, the yield of the
activation reaction was 98% [28].
acknowledges having been granted a fellowship from CONICET-
CARBONFE.
IFN
tion conditions (sodium borate buffer, pH 8, at 4 °C using 5-fold
excess of mPEG lys-OSu) [6,27]. SE-HPLC analysis of the crude
pegylation reaction indicated 35% of monopegylated protein, 2.5%
of oligomer species, and 62.5% of unmodified interferon (see
2
a-2a was pegylated with mPEG lys-OSu under known reac-
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excellent resolution.
2
The performance of mPEG lys 1 was evaluated by its conversion
to mPEG
lation of IFN
2
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The authors wish to express their gratitude to Agencia Nacional
de Promoción Científica y Tecnológica (ANPCyT), to Consejo Nacion-
al de Investigaciones Científicas y Técnicas (CONICET), to Universi-
dad Nacional del Litoral (UNL) of Argentina and to CARBONFE, for
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[
2