Functional and reactive polymethacrylates suitable for preparation of peptide/protein-polymer conjugates

Article abstract of DOI:10.1016/j.reactfunctpolym.2010.07.016

Three reactive polymers, poly(para-nitrophenoxycarbonyloxyethyl methacrylate-co-methyl methacrylate) P(p-NPCEMA-co-MMA) [rCoP1], poly(methyl methacrylate-co-butyl methacrylate-co-allyl methacrylate-co- phenoxycarbonyloxyethyl methacrylate) P(MMA-co-BMA-co

Full text of DOI:10.1016/j.reactfunctpolym.2010.07.016

Reactive & Functional Polymers 70 (2010) 767–774
Contents lists available at ScienceDirect
Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
Functional and reactive polymethacrylates suitable for preparation
of peptide/protein–polymer conjugates
*
**
Dragos Popescu, Helmut Keul , Martin Möller
Institute of Technical and Macromolecular Chemistry, RWTH Aachen and DWI an der RWTH Aachen e.V., Pauwelsstr. 8, D-52056 Aachen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Three reactive polymers, poly(para-nitrophenoxycarbonyloxyethyl methacrylate-co-methyl methacry-
late) P(p-NPCEMA-co-MMA) [rCoP1], poly(methyl methacrylate-co-butyl methacrylate-co-allyl methac-
rylate-co-phenoxycarbonyloxyethyl methacrylate) P(MMA-co-BMA-co-AMA-co-PCEMA) [rCoP2] and
poly(methyl methacrylate-co-dimethylaminopropyl methacrylate-co-dodecyl methacrylate-co-phenoxy-
carbonyloxyethyl methacrylate) P(MMA-co-DMAPMA-co-DMA-co-PCEMA) [rCoP3], respectively, were
prepared following two synthetic concepts. Following the first concept, p-NPCEMA and PCEMA were pre-
pared starting with commercially available HEMA and were copolymerized via ATRP with MMA, BMA
and AMA. According to the second concept phenoxycarbonyloxy decorated polymethacrylates were
obtained via polymer analogous reaction of a HEMA containing functional polymethacrylate, P(MMA-
co-DMAPMA-co-DMA-co-HEMA) [fCoP], obtained via a cascade reaction of enzymatic transacylation
and free radical polymerization. The highly reactive poly(methacrylate)s and silk peptides, hen egg-white
lysozyme and Candida antarctica lipase B were used for the preparation of protein/peptide-poly(methac-
rylate) bioconjugates.
Received 21 April 2010
Received in revised form 15 July 2010
Accepted 17 July 2010
Available online 22 July 2010
Keywords:
Reactive methacrylates
Atom transfer radical polymerization
Free radical polymerization
Functional poly(methacrylate)s
Bioconjugation
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
ing the pharmacokinetics of conjugates [7,8] discovered an
anticancer agent bearing both hydrophilic and hydrophobic parts
Peptide/protein–polymer conjugates are hybrid materials,
which covalently combine one or more copies of a peptide se-
quence or protein with a synthetic polymer [1,2] and are either de-
signed to benefit from the behaviour of both components or to
overcome shortcomings inherent to the components alone. To
date, the most well known representatives of this class of materials
are conjugates of peptides/proteins with poly(ethylene glycol)
(PEG). PEGylation of proteins is established as a powerful strategy
to improve their in vivo properties [3]. Over the past three decades
PEGylation has developed into a well-established technology to
modify therapeutic proteins and improve their stability and solu-
bility, enhance circulation half-life, reduce immunogenicity and
antigenicity, and prevent proteolytic degradation.
The first reports on peptide/polymer conjugates go back to the
early 1950s [4]. In the 1970s, Davis and Abuchowski found that
covalent attachment of PEG to bovine serum albumin (BSA) and
bovine liver catalase was a useful strategy to reduce or eliminate
the immunogenicity of these proteins and increase their blood cir-
culation times [5,6]. Further, in the mid-1980s, Maeda et al., study-
by combining the antitumor protein neocarzinostatin and poly
(styrene-co-maleic anhydride). Other early examples of peptide/
protein–polymer conjugates include a variety of hybrid di- and
triblock copolymers prepared by ring – opening polymerization
of
a–amino acid N-carboxyanhydrides (NCA’s) using appropriate
synthetic polymer macroinitiators [9].
Peptide/protein–polymer conjugates have attracted interest for
a number of reasons. First of all, the peptide/protein segment can
provide these materials with unique self-assembly properties and
can induce the formation of hierarchically organized nanoscale
structures, both in solution and in the solid state, with higher level
of complexity as compared to block copolymers. In several in-
stances, the sensitivity of the protein secondary structure to envi-
ronmental parameters such as temperature, pH or ion strength
allows to reversibly manipulate the nanoscale structure formation
of these hybrid materials. The literature on this subject is vast and
has been summarized in various review articles [10–12]. The prop-
erties and the multifunctionality of the peptide/polymer–conju-
gates makes them very attractive for usage in drug and gene
delivery applications [13–15], in modulation of the mechanical
properties of synthetic polymers [16], in hydrogels [17–19] as well
as therapeutic proteins [20–22].
* Corresponding author.
** Corresponding author.
E-mail addresses: keul@dwi.rwth-aachen.de (H. Keul), moeller@dwi.rwth-aachen.
de (M. Möller).
The synthesis of the conjugates can be performed using (i) a
convergent [23–27] or (ii) a divergent synthetic strategy [27–29].
1381-5148/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.reactfunctpolym.2010.07.016

768
D. Popescu et al. / Reactive & Functional Polymers 70 (2010) 767–774
The convergent synthesis (i) involves the direct coupling of a func-
tionalized peptide/protein with a complementary functionalized
synthetic polymer. In the case of divergent synthesis (ii), two main
routes can be distinguished: polymerization of synthetic mono-
mers using peptide/protein macroinitiators or synthesis of a pep-
tide segment on a soluble or solid-supported synthetic polymer.
For the convergent synthesis of peptide/protein–polymer con-
jugates, either via selective modification of a suitable end-reactive
polymer or via functionalization of appropriate reactive side
chains, a broad range of reactions is available. A detailed overview
of these coupling conditions has been published in a recent review
[25]. Table 1 lists some of the functional groups potentially avail-
able in proteins and the functionalities of the required polymers
[30].
Side-chain functional polymers may be prepared by a variety of
(controlled) radical polymerization methods. Within this approach,
monomers bearing reactive and functional side-groups are poly-
merized both in their protected or unprotected form, and subse-
quently reacted with the functional groups in the peptide/
protein. As an example, p-nitrophenyl chloroformate activation of
the pendant hydroxyl groups of poly[(ethylene glycol) methacry-
late] and poly(2-hydroxyethyl methacrylates) has been used to
introduce peptide sequences [31–33]. In this concept attention
has to be paid on eventual conformational or functional changes
in the protein induced by the conjugation with the polymer [34].
In our previous work we described the syntheses of different
multifunctional polymethacrylates via a cascade of enzymatic
and chemical reactions. By this procedure hydrophilic, hydropho-
bic and ammonium groups were introduced in the polymer back-
bone besides reactive phenyl carbonate groups [35]. These
reactive groups are known to interact with nucleophiles such as
amines without side reactions [36–39]. In the present study we re-
port preliminary results obtained for the convergent synthesis of
the peptide/protein–polymer conjugates using the reaction of dif-
ferent proteins like: silk peptides, lysozyme and Candida antarctica
lipase B (CALB) with different multifunctional polymethacrylates
bearing p-nitrophenyl carbonate or phenyl carbonate groups in
the side-chain. These reactions were performed either in N,N-
dimethylformamide (DMF) solution or in the case of CALB also in
miniemulsion. By preparing particles of reactive copolymers, a
large surface of the polymer is exposed for the interactions with
the functional groups of proteins. Protein concentration of the con-
jugates was determined via the Bio-Rad DC protein assay which is
similar to the well-documented Lowry assay [40]. When CALB was
used, the activity of the enzyme–polymer conjugate was deter-
mined via active site titration using p-nitrophenyl laureate as a
substrate.
(96%, Aldrich), butyl acetate (BuAc, 99.5%, Fluka), azobisisobutyro-
nitrile (AIBN, 98+%, Fluka), ethyl 2-bromoisobutyrate (EBrIB, 98%,
Aldrich), CuBr (98%, Fluka), 2,20-bipyridine (Bpy, 99+%, Aldrich),
3-dimethylamino-1-propanol (DMAP, 99%, Aldrich), 1-dodecanol
(D-ol, 98.5+%, Fluka), pyridine (Py, 99.5%, KMF Laborchemie), 4-
dimethylamino pyridine (99%, Aldrich), dichlormethane (99.5%,
Fluka), hen egg-white lysozyme [lyophilized powder 105,000 U/
mg, 2.185 mg protein/4.1 mg powder (53% protein), Fluka], silk
peptide [1.676 mg protein/2.8 mg compound (60% protein)] were
used as received. C. antarctica lipase B (CALB, lyophilized powder
10,000 U/g, Fluka) was purified prior use on a Sephadex G25 chro-
matography column using a 25 mM natrium phosphate buffer
solution as eluent at a pH 7. A commercial lipase, Novozyme 435
(Lipase B from C. antarctica immobilized onto macroporous acrylic
resin, 10,000 U/g Novo Nordisk) was dried in vacuum at room tem-
perature for 24 h and stored under nitrogen before it was used as a
biocatalyst for the transacylation reactions.
All reactions were carried out under nitrogen atmosphere.
Nitrogen (Linde, 5.0) was passed over molecular sieves (4 Å) and fi-
nely distributed potassium on aluminium oxide before use.
2.2. Measurements
1H NMR and 13C NMR spectra were recorded on a Bruker DPX-
300 FT-NMR spectrometer at 300 MHz and 75 MHz, respectively,
using deuterated chloroform (CDCl3) and tetramethylsilane (TMS)
as internal standard.
Size exclusion chromatography analyses (SEC) were carried out
using THF (with addition of 250 mg/L 2,6-di-tert-butyl-4-methyl-
phenol) and DMAc (with 1 g/L LiCl) as eluting solvents. For THF
as eluting solvent a pump (ERC Model 6420), a refractive index vis-
co-detector (WGE Dr. Bures ETA-2020) and an UV detector (Jasco
UV-2075Plus) were used at 35 °C with a flow rate of 1.0 mL/min.
Four columns with MZ-SD Plus gel were applied. The length of each
column was 300 mm, the diameter 8 mm, the diameter of the gel
particles 5 l
m, and the nominal pore widths were 50, 102, 103
and 104 Å. Calibration was achieved by using poly(methyl methac-
rylate) (PMMA) standards. For DMAc as eluting solvent a high pres-
sure liquid chromatography pump (Bischoff HPLC) and a refractive
index detector (Waters 410 Millipore) were used at 80 °C with a
flow rate of 0.8 mL/min. Three columns with MZ-DVB gel were ap-
plied. The length of each column was 300 mm, the diameter 8 mm,
the diameter of the gel particles 5 lm and the nominal pore widths
were 102, 103 and 104 Å. Calibration was achieved using poly
(methyl methacrylate) (PMMA) standards.
The Bio-Rad DC protein assay was used in order to determine
the protein concentration of the isolated enzymes and of the pro-
tein–polymer conjugates using bovine serum albumin 99% pur-
chased from Sigma–Aldrich as
a standard. This assay is a
2. Materials and methods
colorimetric test for protein concentration similar to the well-doc-
umented Lowry assay. The assay is based on the reaction of protein
with an alkaline copper tartrate solution and Folin reagent. As with
the Lowry assay, there are two steps which lead to colour develop-
ment: the reaction between protein and copper in an alkaline med-
ium; and the subsequent reduction of Folin reagent by the copper-
treated protein. The blue colour development is primarily due to
the amino-acids tyrosine and tryptophan, and to a lesser extent
to, cysteine, cystine and histidine. Thus the protein concentration
was determined measuring the absorbance of the solution at
750 nm with a Varian Cary 100 Bio spectrophotometer after a cal-
ibration with bovine serum albumin was performed.
For the activity tests, the isolated enzymes as well as protein–
polymer conjugates were weighed into safe-lock tubes and
layered with a 0.1 M Tris–HCl buffer solution (1.9 mL, pH = 8)
and the substrate solution (0.1% para-nitrophenyl laurate in aceto-
nitrile, 20 mL) was added. After 20 min at 37 °C the amount of
2.1. Materials
Methyl methacrylate (MMA, 99+%, Fluka), butyl methacrylate
(BMA, 99+%, Merck) and allyl methacrylate (AMA, 98%, Aldrich)
were purified via column chromatography on Al2O3 (Fluka, type
5016A basic). 2-Hydroxyethyl methacrylate (HEMA, 95+%, Fluka),
phenyl chloroformate (97+%, Fluka), 4-nitrophenyl chloroformate
Table 1
Functional groups in proteins and functionalities of the required polymers (30).
Amino acids (functional groups) Polymers/materials/surfaces
Lys (–NH2)
Cys (–SH)
Asp and Glu (–COOH)
Ser and Thr (–OH)
Carboxilic acid, active ester, epoxy
Vinyl sulfone, maleimide, pyridil disulfide
Amine, epoxy
Epoxy

D. Popescu et al. / Reactive & Functional Polymers 70 (2010) 767–774
769
para-nitrophenol produced was determined by measuring the
extinction of the solution at 410 nm with a Varian Cary 100 Bio
spectrophotometer. The reported values are an average of three
measurements.
moisobutyrate (EBrIB, 39 mg, 0.2 mmol) and butyl acetate (BuAc,
4 mL). The polymerization was started by placing the Schlenk tube
in a thermostated oil bath at 90 °C. After a reaction time of 4 h the
mixture was cooled to room temperature and quenched by addi-
tion of CH2Cl2 followed by stirring in air until the copper complex
was completely oxidized. The copper complex was removed by
washing with a solution of 5% HCl. The polymer was isolated by
precipitation in pentane. Yield of the P(MMA-co-p-NPCEMA),
(rCoP1): 1.2 g (31%). SEC-THF: Mn = 12,000 g/mol and Mw/Mn = 1.5.
1H NMR (CDCl3): MMA repeating units: d = 0.88/1.05/1.26 (rr/
mr/mm, s, 3 H, CH3); 1.85 (br, 2 H, CH2-backbone); 3.55 (s, br, 3
H, –O–CH3); p-NPCEMA repeating units: d = 0.88/1.05/1.26 (rr/mr/
mm, s, 3 H, CH3); 1.85 (br, 2 H, CH2-backbone); 4.27/4.48 (s, br, 2
H/2 H, –O–CH2–CH2–O–); 7.4–8.28 (br, 4 H, aromatic).
2.3. Syntheses
2.3.1. First concept
2.3.1.1. Synthesis of para-nitrophenoxycarbonyloxyethyl methacrylate
(p-NPCEMA). To
a solution of para-nitrophenyl chloroformate
(4.65 g, 23 mmol) in dry CH2Cl2 (30 mL) was added a solution of
dry pyridine (1.823 g, 23 mmol) in dry CH2Cl2 (30 mL). To this mix-
ture, a solution of 2-hydroxyethyl methacrylate (HEMA, 3 g,
23 mmol) in dry CH2Cl2 (30 mL) was added dropwise at a temper-
ature of 5 °C. The reaction mixture was stirred for 40 h at room
temperature (rT). The product was washed once with distilled
water, twice with HCl 1 M, twice with brine and dried on Na2SO4.
The product obtained after evaporation of the solvent was a yel-
lowish liquid. Recrystallization from hexane yielded p-NPCEMA
(3.135 g, 47%) as a white crystalline powder.
2.3.1.4. Synthesis of P(MMA-co-BMA-co-AMA-co-PCEMA) (rCoP2)
[39]. Typical copolymerization procedure: In a Schlenk tube were
mixed under nitrogen CuBr (0.49 g, 3.4 mmol), bipyridine (Bpy,
1.07 g, 6.8 mmol), PCEMA (10 g, 40 mmol), MMA (3 g, 30 mmol),
BMA (2.84 g, 20 mmol), AMA (1.26 g, 10 mmol), ethyl 2-bromo-
isobutyrate (EBrIB, 0.667 g, 3.42 mmol) and butyl acetate (17.1 mL).
The polymerization was started by placing the Schlenk tube in a
thermostated oil bath at 90 °C. After a reaction time of 1 h the mix-
ture was cooled to room temperature and quenched by addition of
THF followed by stirring in air until the copper complex was com-
pletely oxidized. The coppercomplex was removedvia column chro-
matography on aluminium oxide. The copolymer was isolated by
precipitation in MeOH/H2O (1/3 volume ratio). Yield of the copoly-
mer P(MMA-co-BMA-co-AMA-co-PCEMA), (rCoP2): 7.2 g (41%).
SEC-THF: Mn = 11,000 g/mol and Mw/Mn = 1.6.
NO2
3
11
O
6
8
O
10
4
2
7
O
O
1
5
9
O
1H NMR (CDCl3): d = 1.97 (s, H1); 4.49–4.54 (m, H5, H6); 5.64 (s, br,
H3’); 6.18 (s, br, H3); 7.4 (d, H9); 8.3 (d, H10) ppm.
13C NMR (CDCl3): d = 18.26 (C1); 61.88 (C5); 66.9 (C6); 121.78
(C10); 125.35 (C9); 126.46 (C3); 135.73 (C2); 145.5 (C11); 152.42
(C7) 155.41 (C8); 167.04 (C4) ppm.
1H NMR (CDCl3): MMA repeating units: d = 0.9/1.03/1.4 (rr/mr/
mm, s, –CH3); 1.85 (br, –CH2-backbone); 3.59 (s, –O–CH3); BMA
repeating units: d = 0.9/1.03/1.4 (rr/mr/mm, s, –CH3); 1.6 (br, –
CH2-backbone); 3.94 (s, br, –O–CH2–); AMA repeating units:
d = 0.9/1.04/1.26 (rr/mr/mm, s, –CH3); 1.9 (br, –CH2-backbone);
4.46 (s, br, –O–CH2–); 5.23–5.31 (m, –CH = CH2 allyl); 5.9 (broad
signal, –CH = CH2 allyl); PCEMA repeating units: d = 0.9/1.03/1.4
(rr/mr/mm, s, –CH3); 1.85 (br, –CH2-backbone); 4.2–4.4 (s, br, –O–
CH2–CH2–O–); 7.2–7.4 (br, phenyl).
2.3.1.2. Synthesis of phenoxycarbonyloxyethyl methacrylate (PCE-
MA)[39]. To a solution of phenyl chloroformate (12.03 g, 80 mmol)
in dry CH2Cl2 (60 mL) a solution of dry pyridine (Py, 9.11 g,
115 mmol) in dry CH2Cl2 (60 mL) was added. To this mixture, a
solution of 2-hydroxyethyl methacrylate (HEMA, 10 g, 80 mmol)
in dry CH2Cl2 (60 mL) was added dropwise at a temperature of
5 °C. The reaction mixture was stirred for 70 h at rT. The product
was dissolved in CH2Cl2 (100 mL), washed twice with a 5% HCl
solution and once with brine and dried on Na2SO4. The product ob-
tained after evaporation of the solvent was a yellowish liquid. Yield
of PCEMA: 17.22 g (90%).
2.3.2. Second concept
2.3.2.1. Procedure for lipase-catalyzed transacylation. Stock solutions
of DMA/MMA and DMAPMA/MMA were prepared from MMA, 1-
dodecanol and 3-dimethylamino-1-propanol with Novozyme 435
as catalyst according to Ref. [35].
10
11
9
2.3.2.2. PreparationofP(MMA-co-DMA-co-DMAPMA-co-PCEMA), (rCoP3)
[35]. P(MMA-co-DMA-co-DMAPMA-co-HEMA), (fCoP), SEC-DMAc:
Mn = 59,000 g/mol and Mw/Mn = 2 was reacted with phenyl chlorofor-
mate according to reference 35 producing P(MMA-co-DMA-co-DMAP-
MA-co-PCEMA), (rCoP3) with SEC-DMAc: Mn = 49,000 g/mol and Mw/
Mn = 2.5.
8
O
7
O
2,2'
3
6
O
O
4
5
1
2.4. Representative procedure for synthesis of polymer–protein
conjugates
O
1H NMR (CDCl3): d = 1.96 (s, H1); 4.46 (m, H5, H6); 5.59 (s, H2’); 6.16
(s, H2); 7.16–7.36 (m, phenyl and H9, H10 and H11).
13C NMR (CDCl3): d = 18.26 (C1); 62.15 (C5); 66.23 (C6); 120.98
(C9); 126.17 (C11); 126.34 (C2); 129.58 (C10); 135.82 (C3); 151.06
(C8) 153.54 (C7); 167.00 (C4).
2.4.1. Reaction with silk peptide, lysozyme and CALB
The rCoP3 (80 mg, with 0.09 mmol PCEMA repeating units), sol-
uble fraction from silk protein (104 mg) and 4-dimethylamino pyr-
idine (DMAP, 11 mg, 0.09 mmol) were stirred in DMF (1 mL) for
48 h at 60 °C. At certain time intervals aliquots (10 lL) were with-
drawn from this mixture and analyzed by SEC, using DMAc as elu-
ent. The product was isolated by precipitation in diethylether. All
other reactions were performed according to this procedure (Ta-
ble 2). For the reactions 2, 3 and 4 protein loading assays were
performed.
2.3.1.3. Synthesis of P(MMA-co-p-NPCEMA) (rCoP1). Typical copoly-
merization procedure: In a Schlenk tube were mixed under nitro-
gen CuBr (29 mg, 0.2 mmol), bipyridine (Bpy, 62 mg, 0.4 mmol),
p-NPCEMA (2.95 g, 10 mmol), MMA (1 g, 10 mmol), ethyl 2-bro-

770
D. Popescu et al. / Reactive & Functional Polymers 70 (2010) 767–774
Table 2
dried in vacuo for 24 h and then the protein concentration
(1.2 mg protein/10.5 mg bioconjugate) was determined.
Polymer–protein conjugates: starting materials and reaction conditions.
No.
Polymer
(mg)
Protein
(mg)
DMAP
(mg/mmol)
DMF
(mL)
Conditions
3. Results and discussion
1
rCoP3
(80)
Silk peptide
(104)
11/0.09
1
T = 60 °C
t = 48 h
The goal of the present work is to prepare bioconjugates start-
ing with polymethacrylates bearing phenyl carbonate reactive
groups and silk peptides, lysozyme and C. antarctica lipase B. From
the polymer perspective, the attachment of a protein may provide
a synthetic polymer with unique functional and structural proper-
ties. One of the strategies for covalent linkage of proteins/peptides
2
rCoP1
(80)
Silk peptide
(80)
25.67/0.21
1/0.008
3
T = 60 °C
t = 23 h
3
rCoP1
(14)
Lysozyme
(14)
1
T = 24 °C
t = 43 h
4a
rCoP1
(5)
CALB
(4.3)
1.3/0.01
0.8
rT
t = 4 h
to a synthetic polymer comprises reaction of the e-amino group of
lysine residues in the protein with phenyl carbonate groups in the
a
Enzymatic activity of the bioconjugate was determined.
side chains of the polymer (lysines can make up to 6% of the overall
amino acid sequence [41,42]). In general, the e-amino group of ly-
2.4.2. Reaction with CALB in miniemulsion
sine is known to react with electrophilic reagents such as activated
esters, aldehydes or ketones and isocyanates/isothiocyanates.
One of the approaches to synthesize functional and reactive
poly(methacrylate)s makes use of 2-hydroxyethyl methacrylate
(HEMA) which can be functionalized with p-nitrophenyl chlorofor-
mate or phenyl chloroformate. The obtained reactive monomers
para-nitro phenoxycarbonyloxy ethyl methacrylate (p-NPCEMA)
and phenoxycarbonyloxy ethyl methacrylate (PCEMA) were
copolymerized with different methacrylates via free radical poly-
merization or controlled radical polymerization techniques. The
atom transfer radical polymerization (ATRP) [43–45] of p-NPCEMA
The rCoP2 (0.2 g with 0.46 mmol PCEMA) and hexadecane
(100 mg) were dissolved in toluene (2.2 g). This mixture and a
1.0 wt.% Lutensol AT50 solution in water (10.0 g, surfactant solu-
tion) were vigorously stirred for 1 h at 45 °C. The miniemulsion
was prepared by ultrasonicating the mixture during 120 s at 90%
amplitude (Branson sonifierW450 digital). A solution of CALB
(3 mL with 19 mg/mL protein, 25 mM sodium phosphate buffer,
pH 7) was added to the miniemulsion and stirred at ambient tem-
perature. After 19 h the mixture was freeze dried, and the solid was
washed with ethanol and with water. The washed conjugate was
-co-
-co-
-co-
O
O
O
O
O
O
O
O
O
(c)
PEPTIDE / PROTEIN - POLYMER CONJUGATES
C11H23
O
O
N
(c)
(c)
(rCoP3)
(e)
-co-
-co-
-co-
-co-
O
O
O
O
O
O
O
O
O
O
O
O
-co-
-co-
-co-
O
O
O
O
O
O
O
O
O
O
O
O
(rCoP2)
(b)
(rCoP1)
O
O
C11H23
OH
N
NO2
NO2
( b)
O
(fCoP)
O
O
O
O
O
(d)
O
O
O
O
(PCEMA)
(pNPCEMA)
Rf OH
Rf is:
(a)
(a)
+
O
O
O
O
N
OH
(HEMA)
C11H23
2nd Concept
1st Concept
Fig. 1. Schematic representation for the preparation of peptide/protein–polymer conjugates. 1st Concept: (a) preparation of reactive monomers; (b) copolymerization via
ATRP to result in reactive polymers; (c) reaction with the peptide/protein. 2nd Concept: (d-1) enzyme-catalyzed transacylation; (d-2) copolymerization of the monomer
mixture by FRP obtaining functional polymers; (e) polymer analogous reaction to result in reactive polymers.

D. Popescu et al. / Reactive & Functional Polymers 70 (2010) 767–774
771
with MMA as well as of PCEMA with MMA, BMA and AMA are pre-
sented (Fig. 1, 1st concept).
mined by NMR spectroscopy and SEC chromatography can be ex-
plained by the calibration protocol based on PMMA standards, by
the accuracy of 1H NMR measurement and by poor signal resolu-
tion. Moreover, the symmetry of the SEC traces using UV and RI
detectors confirm a random distribution of the UV active com-
pound within the copolymer, Fig. 3.
Further, a multifunctional copolymer, rCoP2, was prepared via
ATRP using MMA, BMA, AMA and PCEMA in a 30/20/10/40 mol%
ratio as described elsewhere [39]. While the ratio of MMA and
BMA defines the bulk properties, the percentage of PCEMA defines
the degree of potential functionalization of the copolymer, the con-
centration of the allyl group was 10 mol% since such a concentra-
tion is sufficient for cross-linking and hence for surface
structuring via cross-linking [46]. The SEC-THF analysis shows a
Mn = 11,000 g/mol and a monomodal molecular weight distribu-
tion with a polydispersity of Mw/Mn = 1.6.
The PDI value is relatively high for a controlled polymerization
and might be due to the side reaction caused by AMA and by the
carbonate side-groups, since it is known that phenyl carbonates
and p-nitrophenyl carbonates are activated in the presence of ter-
tiary amines for nucleophilic substitution, and the –C,C– double
bonds in the allyl side chains at high monomer conversions leads
to branched and cross-linked structures. In addition, the symmetry
of the SEC-UV/RI traces reveals a random distribution of the aro-
matic side chains in the copolymer illustrated by Fig. 4.
An alternative to the chemical monomer synthesis was previ-
ously reported and makes use of a cascade reaction comprising a
sequence of enzymatic transacylation and free radical polymeriza-
tion [35]. Thus, highly functional poly(methacrylate)s presenting
besides hydrophilic, hydrophobic and ammonium groups, also
reactive groups exemplified by phenyl carbonate, were prepared
(Fig. 1, 2nd concept).
3.1. Synthesis of the reactive poly(methacrylate)s
3.1.1. First concept
The synthesis of the monomer PCEMA was described in a previ-
ous work [39], and implies the commercially available 2-hydroxy-
ethyl methacrylate (HEMA) as starting material. During this work
we used dry pyridine as acid scavenger and the yield of the product
was enhanced from 75% (when triethyl amine was used) to 90%. The
commercially available HEMA was used as starting material also for
the synthesis of the monomer p-NPCEMA. A solution of HEMA in
dry CH2Cl2 was allowed to react with p-nitrophenyl chloroformate
in the presence of dry pyridine as acid scavenger, at a temperature
not higher than 25 °C. The whole system has to be free of water, be-
cause p-nitrophenyl chloroformate reacts with water. All character-
istic signals of the monomer are observed in the 1H and 13C NMR
spectra with no impurities detected (Fig. 2A). Both reactive mono-
mers were stored under nitrogen in the dark in order to avoid
adventitious polymerization.
In the first polymerization experiments, p-NPCEMA was copoly-
merized with MMA in a molar ratio of 1:1, via ATRP. 1H NMR anal-
ysis of the resulting copolymer revealed that the two monomers
are incorporated into the polymer in a ratio of 1:1 as was expected
from the ratio in the feed (Fig. 2B) (compare intensity of signal A
and signals 5 and 6). Based on the integration of the repeating unit
signals and the integration of the methylene group of the initiator
(a), a molecular weight of 16,000 g/mol was calculated for rCoP1.
From SEC analysis using THF as the eluting solvent a molecular
weight of Mn = 12,000 and a polydispersity of Mw/Mn = 1.5 was
determined. The discrepancy between the molecular weight deter-
3.1.2. Second concept
In order to prepare functional methacrylates we investigated
the transacylation of MMA as substrate and different functional
alcohols (DMAP and D-ol) as reagents with Novozyme 435 as cat-
alyst. Under the applied conditions this transacylation leads to the
formation of a mixture of two methacrylates (DMAPMA or DMA
and MMA) in two alcohols (DMAP or D-ol), and CH3OH, respec-
tively. It is worth mentioning that all the transacylation reactions
were performed in bulk. An exhaustive study of these transacyla-
tions has been recently presented [35]. 1H NMR spectroscopy
served as tool for determination of the concentration of dimethyl-
amino propyl methacrylate (DMAPMA) and dodecyl methacrylate
(DMA) using the signals of the protons attached to sp2 hybridized
(A)
(B)
Fig. 2. 1H NMR spectrum of (A) p-NPCEMA and (B) of the P(MMA-co-p-NPCEMA) (rCoP1).

772
D. Popescu et al. / Reactive & Functional Polymers 70 (2010) 767–774
RI detector
UV detector
RI detector
UV detector
rCoP1
rCoP2
20
25
30
35
20
25
30
35
Elution volume (mL)
Elution volume (mL)
Fig. 3. SEC traces (solvent THF) using UV/RI detectors for the rCoP1.
Fig. 4. SEC traces (solvent THF) using UV/RI detectors for the rCoP2.
carbon atoms of the product and MMA at d = 5.5–6.1 ppm as a ref-
erence, and the signal corresponding to the methylene group of
DMA at d = 4.13 ppm and of d = 4.20 ppm for DMAPMA.
DMAPMA d = 4.01 (signal 2), 2.2 (signal 5) and 2.3 ppm (signal
4); for DMA d = 3.9 (signal 6) and 1.27 ppm (signal 8) and for HEMA
d = 3.8 ppm (signal 10). Based on the integration of the signals of
protons of the repeating units the composition of the fCoP was
determined (MMA)51-co-(DMAPMA)17-co-(DMA)12-co-(HEMA)20
with the indices representing the molar ratio of repeating units.
Via cascade reaction we obtained multifunctional polymers
with hydrophobic, hydrophilic, tertiary amine and hydroxy groups
(fCoP). The hydroxy groups can be esterified with phenyl chloro-
formate and the tertiary amine groups are prone for quaterniza-
tion. The preparation of the reactive copolymer rCoP3 will be
discussed as an example. fCoP was successfully converted in meth-
ylene chloride solution in the presence of pyridine as acid scaven-
ger into the respective ester rCoP3. The course of reaction was
followed by means of 1H NMR spectroscopy (Fig. 5B). The conver-
sion of HEMA repeating units into PCEMA repeating units is no-
ticed by the characteristic signals of the protons of the phenyl
The concept of cascade reactions was applied for the synthesis
of multifunctional polymers: starting with MMA and a functional
alcohol ROH, in the first step a transacylation was performed
resulting a mixture of two monomers (MMA and RMA), which, in
the second step, after the removal of the enzyme by filtration
and after addition of 2-hydroxyethyl methacrylate (HEMA) – was
subjected to free radical polymerization. The ratio of monomers
was determined by means of 1H NMR spectroscopy. Usually the
signals adjacent to the functional group were selected to quantify
the ratio of repeating units. 1H NMR analyses show characteristic
signals for each repeating unit, and based on the integration of
these signals the composition of copolymers was determined.
Fig. 5A depicts the NMR peaks used for the determination of the
copolymer composition: for MMA d = 3.59 ppm (signal 1); for
O
11
OH
O
9
O
O
O
O
10
6
O
7
8
O
11
9
O
O
5
N
12
O
3
10
4
14
13
O
O
2
1
8
back-bone
1
9
H2O
CD3OD
5
10
6
CDCl3
11
3
7
2
4
(A)
(B)
8
1
9
12, 13, 14
5
3
2,6
4.0
11 10
7
4
7.0
6.0
5.0
3.0
2.0
1.0
ppm
Fig. 5. 1H NMR spectrum of (A) fCoP, the copolymer P(MMA-co-DMAPMA-co-DMA-co-HEMA) and (B) rCoP3 bearing the reactive carbonate group.

D. Popescu et al. / Reactive & Functional Polymers 70 (2010) 767–774
773
fCoP
rCoP3
0.06
conjugate
M =142000
n
43h
19h
0.05
M =130000
n
0.04
M =109000
n
M = 49000
0.03
n
rCoP3
0.02
silk
peptide
0.01
15
20
25
30
Elution volume (mL)
15
20
25
Fig. 7. SEC traces (solvent DMAc) of the silk peptide, of the reactive copolymer
rCoP3 and of different samples taken from the reaction between rCoP3 and the silk
peptide.
Elution volume (mL)
Fig. 6. SEC traces (solvent DMAc) of the functional copolymer (fCoP) and the
reactive copolymer (rCoP3).
the DMAc-SEC eluograms for starting materials: the silk peptide
and the functional copolymer (rCoP3) as well as the samples taken
at different times of the reaction. The bioconjugate was isolated by
carbonate groups which appear in the aromatic region of the spec-
tra at d = 7.2–7.4 ppm as well as from the shift of the signals of the
ethylidene protons from d = 3.9–4.0 ppm in HEMA repeating units
to d = 4.2–4.4 ppm in PCEMA repeating units.
Fig. 6 depicts the SEC traces of the functional copolymer (fCoP)
as well as of the reactive copolymer (rCoP3) using DMAc as eluent.
The decrease of the molecular weight from 59,000 g/mol of the
functional copolymer to 49,000 g/mol for the reactive one might
be explained by the differences of the hydrodynamic radius of
the copolymers in DMAc solvent, while the increase of the polydis-
persity from 2 (fCoP) to 2.5 (rCoP3) can be ascribed to the interac-
tions of the polymer with the column leading to a bimodal
distribution.
precipitation in diethyl ether and SEC analysis revealed
a
Mn = 142,000 g/mol. It can clearly be seen that part of the silk pro-
tein was preferentially linked to the polymer since the molecular
weight of the hybrid fraction is different from that of the polymer
fraction.
In order to increase the reaction rate instead of phenyl carbon-
ate group, p-nitro phenyl carbonate was chosen as leaving group as
described in the literature [32].
The reactive copolymer, rCoP1, with a concentration of 54 mol%
p-NPCEMA was used for the reactions with silk peptide, lysozyme
and CALB. All these reactions were performed in DMF as solvent
and are presented in Table 4.
In the reaction the polymer rCoP1, silk peptide and DMAP were
dissolved in DMF and stirred at 60 °C for 23 h. Due to solubility
problems SEC analysis was not performed. At the end of the reac-
tion the bioconjugate was isolated by precipitation in diethyl ether
and dried under vacuum (10ꢀ3 mbar). The protein content in the
isolated conjugate was determined as described in Section 2 and
compared with the pure silk peptide’s protein content. Thus, for
the reaction between rCoP1 and silk peptide, for 7 mg conjugate
a protein content of 1.373 mg was determined using the Bio-Rad
DC protein assay. The efficiency of bioconjugation was determined
from the values obtained from the protein content test reported to
the amount of the polymer and protein in the feed. The same
procedure was applied for the reaction 3, between the reactive
copolymer rCoP1 and lysozyme.
3.2. Synthesis of polymer–protein conjugates
All the reactive copolymers (Table 3) prepared via the two con-
cepts presented in this paper were purified and were reacted with
different proteins in order to investigate the potential application
of these materials for synthesizing bioconjugates or even for en-
zyme immobilization.
The first experiment between a polymer and a protein was per-
formed using the reactive copolymer, rCoP3 and the soluble frac-
tion of silk peptide. The soluble fraction of silk peptide contains
different amino-acids suitable for covalent linking. Lysine, histi-
dine and arginine are convenient for conversion of the free amino
group with the reactive side chains of the copolymer.
The polymer dissolved in dimethylformamide (DMF) was added
to the peptide solution in DMF. 4-Dimethylaminopyridine (DMAP)
was used in a catalytic amount, since the reaction requires the
presence of a base. The mixture was stirred at 60 °C for various
times, and samples were analyzed via size exclusion chromatogra-
phy (SEC) using dimethyl acetamide (DMAc) as eluent. It was ob-
served that at the beginning of the reaction the peptide was not
soluble; after 19 h a viscose suspension is obtained. Fig. 7 shows
Table 4
Peptide/protein–polymer bioconjugates. Protein content and activity results.
No. Bioconjugate Mass
bioconjugatea
Mass
proteina (mg) (%)
Yieldb
CALB
activityc
(mg)
1d
2
3
rCoP3-Silk
rCoP1-Silk
rCoP1-Lyz
rCoP1-CALB
rCoP2-CALB
n.d.
7
4.5
3
n.d.
n.d.
65%
89%
22%
51%
n.d.
n.d.
n.d.
0.437
1.253
1.373 (20%)
1.07 (24%)
0.31 (10%)
1.2 (11%)
4
Table 3
5e
10.5
Overview of the reactive poly(methacrylate)s used.
a
Determined using Bio-Rad DC protein assay, as described in Section 2.
Efficiency of bioconjugation was determined from the values obtained from the
Name
Repeating units in the copolymer (mol%)
b
MMA
p-NPCEMA/PCEMA
BMA/DMAPMA
AMA/DMA
protein content test reported to the amount of the polymer and protein in the feed.
c
CALB activity determined as described in Section 2. All the values represent the
rCoP1
rCoP2
rCoP3
46
30
50
p-NPCEMA 54
PCEMA 39
PCEMA 18
–
–
extinction at 410 nm. The pure enzyme has an extinction of 1.859 for 60
For reaction 4 the extinction was determined for 6 g CALB, while for reaction 5 the
extinction was determined for 60 g CALB.
lg CALB.
BMA 22
DMAPMA 16
AMA 9
DMA 11
l
l
The composition (mol%) was calculated from the 1H NMR spectrum using the
characteristic signals of each repeating unit as described in Section 3 of this paper.
Protein content and activity tests not determined. Reaction followed by SEC.
Reaction performed in miniemulsion.
d
e

774
D. Popescu et al. / Reactive & Functional Polymers 70 (2010) 767–774
For reaction (4) besides the protein content in the bioconjugate,
Functional Materials” (BIOPRODUCTION/NMP-2-T-2007-026515)
funded by the European Commission. The authors are grateful to
Claudia Formen for performing the protein content and enzyme
activity tests.
activity tests were also performed using 0.1 wt.% p-nitrophenyl
laurate (pNPL) as substrate. The samples were incubated for
20 min at 37 °C, and then the extinction was determined at a wave
length of 410 nm measuring the formation of p-nitrophenol. For
the pure CALB (60
while for the bioconjugate rCoP1-CALB was calculated an extinc-
tion of 0.437 (6 g CALB, due to the low amount of bioconjugate).
lg CALB) an extinction of 1.859 was determined
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Acknowledgments
This work was carried out in the framework of the IP-project
(Sustainable Microbial and Biocatalytic Production of Advanced