10644 ten Cate et al.
Macromolecules, Vol. 38, No. 26, 2005
of material due to nonspecific adsorption to the station-
ary phase is often unavoidable.
cleavage mixture (2% (v/v) trifluoroacetic acid (TFA) in dichlo-
romethane), followed by isolation via diethyl ether precipita-
tion and lyophilization from acetonitrile/water (1:1). This
With this contribution, we present straightforward,
solid-phase-supported synthesis routes to obtain oli-
gopeptide-based RAFT agents that were subsequently
utilized for the polymerization of n-butyl acrylate (nBA).
For the synthesis of the oligopeptide-based RAFT
agents, two different strategies have been evaluated
avoiding the usual chromatographic purification proce-
dures, thereby providing a versatile route to fragile,
multifunctional, or complex RAFT agents. The first
approach included the coupling of a preformed carboxyl-
functionalized RAFT agent to the N-terminus of a resin-
bound peptide. The second synthesis route comprises a
functionality switch of a solid-phase-supported oligopep-
tide ATRP macroinitiator into an oligopeptide transfer
agent. The GDGFD peptide sequence was utilized to
demonstrate the process, making the investigation
directly comparable to our previous ATRP study.2
resulted in a mixture of 3 (76%) with a thioamide side product
1
(
(
24%) as a pink powder. H NMR (DMSO-d
6
): δ ) 8.34-8.17
2
m, 3H, NH), 8.13-7.98 (m, 2H, NH), 7.91 (d, JHH ) 8.2 ppm,
ArHortho), 7.69 (m, ArHpara), 7.69 (m, ArHmeta), 7.3-7.1 (m, 5H,
ArH), 4.7-4.4 (m, 4H, CH), 3.83-3.55 (m, 4H, CH ), 3.37 (m,
2H, CH ), 3.31 (m, 4H, CH ), 3.10 (t, J ) 5.9 Hz, CH ), 3.02
2
2
2
2
HH
2
2
2
(dd, 1H, JHH ) 4.6, 14 Hz, CH
2
), 2.78 (dd, 1H, JHH ) 9.4, 14
), 2.66 (dd, 2H, JHH ) 6.1, 16 Hz, CH ), 2.52-2.35
+ DMSO), 1.90 (s, CH ), 1.37 (s, 18H, tBu). ESI-MS:
m/z (%) ) 729 (4) [M2 - tBu + H] , 785 (5) [M2 + H] , 791
2
Hz, CH
m, CH
2
2
(
2
3
+
+
+
+
(
9
2
26) [M2 - 2tBu + TFA - H O + Na] , 807 (9) [M2 + Na] ,
+
+
48 (100) [M1 + Na] , 964 (12) [M1 + K] . M1 corresponds
with the mass of 3, and M2 corresponds with the mass of the
thioamide side product.
Synthesis of the Oligopeptide Transfer Agent (Ph)
C(S))S)CH(CH ))C(O))Gly-Asp(tBu)-Gly-Phe-Asp-
3
2 2
(tBu))NHCH CH OH) (5). Under argon atmosphere, a
solution of phenylmagnesium bromide, prepared from bro-
mobenzene (1.0 mL, 9.5 mmol) and magnesium turnings (220
mg, 9.05 mmol) in THF (6 mL), was filtered into a round-
bottom flask, containing anhydrous carbon disulfide (0.8 mL,
Experimental Section
Materials. 2-Bromopropionic acid (Aldrich, 99+%), n-butyl
acrylate (nBA, Aldrich, 99%), and N,N-dimethylformamide
1
3.3 mmol) while cooling with an ice-bath. The reaction
mixture was stirred for 2 h at room temperature. Then 2 mL
of water was added, and the THF was removed in vacuo. Water
(
DMF; Aldrich, 99+%) were distilled and stored at -15 °C.
THF was dried over Na/benzophenone and distilled prior to
use. All other reagents were used as received from Aldrich.
Fmoc-amino acid derivatives (Fmoc-Asp(tBu)OH, Fmoc-
GlyOH, Fmoc-PheOH), polystyrene-(2-aminoethanol-2-chlo-
rotrityl) resin (loading: 0.3 mmol/g) and 2-(1H-benzotriazole-
(
20 mL) and Et
acidified with HCl (1 M, 15 mL), and the product from the
aqueous layer was extracted twice with Et O (50 mL). The
combined organic fractions were dried over Na SO and
2
O (50 mL) were added, the aqueous layer was
2
2
4
concentrated in vacuo. The dithiobenzoic acid (310 mg, 2.0
mmol) was added to the oligopeptide precursor resin 4 (0.1
mmol) pre-swollen in THF (4 mL). After the addition of
pyridine (160 µL, 2.0 mmol) the reaction was stirred at 60 °C
for 15 h. The reaction mixture was cooled to room temperature,
1
-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
(
IRIS Biotech GmbH, Germany) were used as received. The
synthesis of the oligopeptide precursors 1 and 4 was described
2
previously. 4-cyano-4-((thiobenzoyl)sulfanyl)pentane carboxy-
lic acid (2) was synthesized according to literature procedures.
2
and the resin was extensively washed with THF, H O, THF,
2
5-27
Therefore, dithiobenzoic acid
was synthesized, oxidized to
and subsequently reacted with
and DCM. Liberation from the support and isolation of the
2
7,28
bis(thiobenzoyl) disulfide,
final oligopeptide RAFT agent was accomplished as described
4
,4′-azobis(4-cyanovaleric acid).29
1
above. H NMR(DMSO-d
6
): δ ) 8.59 (m, 1H, NH), 8.27-8.23
Instrumentation. Mass spectrometry was performed on a
(br m, 2H, NH), 8.12 (m, 1H, NH), 8.06 (d, 1H, NH), 7.92 (d,
2
high performance liquid chromatograph electron spray ioniza-
tion mass spectrometer (LC-ESI-MS) (Shimadzu, qp8000R,
Duisburg, Germany). Nuclear magnetic resonance spectra
2H, J ) 8.4 Hz, ArHortho), 7.64 (m, 1H, ArHpara), 7.53 (m,
HH
2
1H, NH), 7.48 (t, 2H, J ) 7.5 Hz, ArH
HH
meta
), 7.3-7.1 (m, 5H,
2
ArH), 4.73 (q, 1H, J ) 7.0 Hz, CH), 4.63-4.42 (m, 3H, CH),
HH
2
(
NMR) were recorded on a Bruker DPX-400 spectrometer at
00.1 MHz. Samples to determine the monomer conversion
were taken directly from the polymerization mixture and
diluted with CDCl . The conversion was determined relative
3.78 (m, 2H, CH ), 3.66 (dd, 1H, J ) 3.6 Hz, CH ), 3.61
2
HH
2
2
4
(dd, 1H, J ) 3.6 Hz, CH ), 3.8-3.3 (br m, H O), 3.37 (t,
HH 2 2
2
2
2H, J ) 6.2 Hz, CH ), 3.10 (m, 2H, CH ), 3.01 (dd, 1H, J
HH
HH
2
2
3
) 4.5, 14 Hz, CH ), 2.77 (m, 1H, CH ), 2.66 (m, 2H, CH ), 2.41
2 2 2
2
to DMF as internal standard by comparing the integral
intensity of the resonance of vinylic protons of the monomer
with the formamide proton of the DMF. Resonances used: (δ
2 3
(m, 2H, CH ), 1.54 (d, 3H, J ) 7.0 Hz, CH ), 1.37 (s, 18H,
+
tBu) ppm. ESI-MS: m/z (%) ) 761 (18) [M - 2tBu + H] , 799
+
+
(3) [M - 2tBu + K] , 817 (20) [M - tBu + H] , 873 (13) [M +
+
+
+
)
6.60-5.70 ppm, 3H, H
2
CdCH, monomer) and (δ ) 8.01 ppm,
H] , 895 (100) [M + Na] , 911 (18) [M + K] , 967 (6) [M +
+
s, DMF). GPC measurements were carried out in THF as
eluent using three 5-Å MZ-SDV columns with pore sizes of
TFA - H O + H] .
2
General RAFT Polymerization Procedure. The oli-
gopeptide RAFT agent (10 mg, 10.5 µmol) was dissolved in
DMF (2.12 mL). After the addition of nBA (11.1 mmol) and
AIBN (0.091 mg, 0.55 µmol), the reaction mixture was carefully
degassed and heated to 60 °C. Samples of 0.2 mL were taken
for kinetic analysis (GPC, NMR).
For further characterization purposes, the polymer peptide
conjugate was precipitated multiple times from DMF and THF
2
in MeOH/H O (1:1) and freeze-dried from acetonitrile/benzene
3
5
6
10 , 10 , and 10 Å (flow rate 1 mL/min). The detection was
performed with an RI (Shodex RI-71) and a UV detector (TSP
UV 1000; 260 nm). Linear PS-standards (PSS, Germany) were
used for calibration. Samples were taken from the polymeri-
zation mixture, diluted with THF and used for Mn,app. and M
determination.
Synthesis of the oligopeptide transfer agent (Ph)C-
S))S)C(CH )(CN))CH )CH )C(O))Gly-Asp(tBu)-
Gly-Phe-Asp(tBu))NHCH CH OH) (3). 4-Cyano-4-((thioben-
w
/
M
n
(
3
2
2
2
2
(1:1) to extract, eventually, the remaining peptide or peptide
RAFT agent.
zoyl)sulfanyl)pentane carboxylic acid (167 mg, 0.6 mmol) was
dissolved in 5 mL of dichloromethane (DCM). After the
addition of DCC (62 mg, 0.3 mmol), the reaction mixture was
stirred for 30 min at room temperature. The resulting anhy-
dride solution was filtered, transferred to 2-5 mL of NMP,
and added to the pre-swollen oligopeptide precursor resin 1
Results and Discussion
Combining the structural and functional control of
peptides with the diversity and stability of polymers in
polymer-peptide conjugates can result in biohybrid
materials that have the potential to interact with
biological systems. In particular, the combination of
solid-phase-supported peptide synthesis and controlled
radical polymerization is highly attractive to obtain such
materials. Therefore, oligopeptide transfer agents were
(
0.1 mmol) in NMP, and the mixture was stirred for 4 h at
room temperature under argon atmosphere (Kaiser’s test
results indicated that no free amine groups were left). The
resin was washed thoroughly with NMP, DCM, NMP, THF,
and DCM. The liberation of the macroinitiator from the
support was accomplished by 5-30 min treatment with a