A continuous bioreactor prepared via the immobilization of trypsin on aldehyde-functionalized, ring-opening metathesis polymerization-derived monoliths

Article abstract of DOI:10.1021/ma101922s

The ring-opening metathesis polymerization (ROMP) of norborn-2-ene (NBE) and cis-cyclo- octene (COE) was initiated with well-defined Grubbs-type initiators, i.e., RuCl₂(CHPh)(PCy₃)₂ (1), [RuCl₂(PCy₃)- (IMesH₂)(CHPh)] (2), and [RuCl₂(3-Br-Py)₂(IMesH₂)(CHPh)] (3)(MesH ₂ = 1, 3-bis(2, 4, 6-trimethylphenyl)- imidazolin-2-ylidene, PCy ₃ = tricyclohexylphosphine, 3-Br-Py = 3-bromopyridine). Reaction of the living polymers with O₂ (air) resulted in the formation of aldehyde-semitelechelic polymers in up to 80% yield, depending on the initiator and monomer used. To proof aldehyde formation, the terminal aldehyde groups were converted into the corresponding 2, 4-dinitrophenylhydrazine derivatives, and the structure of the hydrazones was confirmed by NMR and IR spectroscopy. This simple methodology was then used for the functionalization of ROMP-derived monoliths prepared from NBE, 1, 4, 4a, 5, 8, 8a-hexahydro-1, 4, 5, 8-exo-endo-dimethanonaphthalene (DMN-H6) and (NBE-CH₂O) ₃SiCH₃, to yield aldehyde-funtionalized monoliths. The extent of aldehyde formation was determined by hydrazone formation. Up to 8 μmol of aldehyde groups/g monolith could be generated by this approach. Finally, these aldehyde-functionalized monoliths were used for the immobilization of trypsin. Excellent proteolytic activity of the immobilized enzyme was found both under batch and continuous flow conditions.

Full text of DOI:10.1021/ma101922s

Macromolecules 2010, 43, 9601–9607 9601
DOI: 10.1021/ma101922s
A Continuous Bioreactor Prepared via the Immobilization
of Trypsin on Aldehyde-Functionalized, Ring-Opening
Metathesis Polymerization-Derived Monoliths
†
Mavila Sudheendran and Michael R. Buchmeiser*
,†,‡
†
Institut f u€ r Polymerchemie, Lehrstuhl f u€ r Makromolekulare Stoffe und Faserchemie, Universit a€ t Stuttgart,
‡
Pfaffenwaldring 55, D-70569 Stuttgart, Germany, and Institut f u€ r Textilchemie und Chemiefasern (ITCF),
K o€ rschtalstr. 26, D-73770 Denkendorf, Germany
Received August 23, 2010; Revised Manuscript Received November 3, 2010
ABSTRACT: The ring-opening metathesis polymerization (ROMP) of norborn-2-ene (NBE) and cis-cyclo-
octene (COE) was initiated with well-defined Grubbs-type initiators, i.e., RuCl
IMesH )(CHPh)] (2), and [RuCl (3-Br-Py) (IMesH )(CHPh)] (3) (MesH
imidazolin-2-ylidene, PCy =tricyclohexylphosphine, 3-Br-Py=3-bromopyridine). Reaction of the living
2
(CHPh)(PCy
3 2 2 3
) (1), [RuCl (PCy )-
(
2
2
2
2
2
=1,3-bis(2,4,6-trimethylphenyl)-
3
polymers with O (air) resulted in the formation of aldehyde-semitelechelic polymers in up to 80% yield,
2
depending on the initiator and monomer used. To proof aldehyde formation, the terminal aldehyde groups
were converted into the corresponding 2,4-dinitrophenylhydrazine derivatives, and the structure of the
hydrazones was confirmed by NMR and IR spectroscopy. This simple methodology was then used for the
functionalization of ROMP-derived monoliths prepared from NBE, 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo-endo-
2 3 3
dimethanonaphthalene (DMN-H6) and (NBE-CH O) SiCH , to yield aldehyde-funtionalized monoliths.
The extent of aldehyde formation was determined by hydrazone formation. Up to 8 μmol of aldehyde groups/g
monolith could be generated by this approach. Finally, these aldehyde-functionalized monoliths were used
for the immobilization of trypsin. Excellent proteolytic activity of the immobilized enzyme was found both
under batch and continuous flow conditions.
Introduction
1-3 (IMesH = 1,3-bis(2,4,6-trimethylphenyl)imidazolin-
2
2-ylidene, PCy =tricyclohexylphosphine, 3-Br-Py=3-bromo-
pyridine (Figure 1) with air (O ) in the absence of any
3
Ring-opening metathesis polymerization (ROMP) has become
1-4
2
a powerful tool for polymer synthesis.
For this purpose,
monomer in benzene for 24 h. In accordance with the results
Ru-based initiators have attracted significant attention. Their
ease of use, enhanced stability, high catalytic activity, and
their excellent functional group tolerance relative to the other
catalysts have broadened their synthetic utility beyond olefin
13
of Grubbs et al. on the decomposition products of 1, this
reaction afforded a mixture of benzaldehyde and cis/trans-
stilbene (Scheme 1, Figure S1, Supporting Information)
along with several Ru byproducts, which were not further
characterized. The yields of benzaldehyde and the stilbenes
were determined by GC-MS with the aid of an internal
standard and reference compounds and are summarized in
Table 1.
5-7
metathesis.
Interestingly, there exist diverging reports on the
stability of Grubbs’ type initiators vs oxygen, water, nitriles,
8
-13
amines, etc.
Gibson et al. were the first to use this reaction for
14,15
the preparation of semitelchelic ROMP-derived polymers.
Recent reports show that on exposure to oxygen these initiators
can afford Ru-hydride complexes along with organic byprod-
As can be seen, the benzaldehyde yield increased in the
order 1 > 2 ∼ 3 while the yield of the stilbenes decreased in
the order 2 > 1 > 3. Particularly the low stilbene yields
obtained with 3 clearly illustrate the reduced propensity of 3
to react with an aldehyde to give the corresponding stilbene.
10,16,17
ucts.
Thus, reaction of RuCl (CHPh)(PCy ) (1) with air
2 3 2
in benzene has been reported to result in the formation of
benzaldehyde as well as cis- and trans-stilbene along with various
1
3,18
Ru-containing byproducts (Scheme 1).
For the past decades,
several approaches toward the synthesis of telechelic or semi-
Reactivity of Living ROMP-Derived Polymers vs O . Next,
2
19-41
telechelic polymers have been reported.
In view of the
starting with a high-ring strain monomer, i.e., norborn-
2
synthetic potenial of the metathesis reaction of Ru-alkylidenes
with oxygen, we decided to employ this reaction for the prepara-
tion of aldehyde-functionalized, ROMP-derived polymeric monoliths.
This study describes as to what extent ROMP-derived polymers
-ene, we synthesized aldehyde-telechelic polymers via the
reaction of the living Ru-alkylidene of the ROMP-derived
poly(NBE) with O as outlined in Scheme 2. With the three
2
different initiators 1-3 used in this study, the desired alde-
hyde telechelic polymers were formed to a different extent.
Thus, in case a ROMP-derived poly(NBE) initiated by 1 was
reacted with air for 16 h, an aldehyde-semitelechelic poly-
mer with one polymer end bearing an aldehyde group was
obtained in 80% yield (Table 2) while the termination of
living poly(NBE) prepared by the action of 2 or 3 with O2
gave only 47% and 29% of aldehyde-semitelechelic polymers.
These data clearly reflect the different chemical stabilities of
can be functionalized upon termination with O and in which form
2
this reaction can be useful to material science and biocatalysis.
Results and Discussion
Reactivity of Ru-Based Initiators vs Air (O ). In order to
2
obtain a mechanistic understanding, we first reacted initiators
*Corresponding author. E-mail: michael.buchmeiser@ipoc.uni-stuttgart.de.
r 2010 American Chemical Society
Published on Web 11/16/2010
pubs.acs.org/Macromolecules

9
602 Macromolecules, Vol. 43, No. 23, 2010
Sudheendran and Buchmeiser
Scheme 1. Proposed Pathway for the Formation Benzaldehyde and of the Stilbenes
conditions showed only 15% and 55% of the aldehyde
functionality (Table 2). As observed for the poly(NBE)s,
SEC revealed a monomodal yet broad molecular weight
distribution (1.46 < PDI < 1.82).
To further proof the formation of the aldehyde termini for
both poly(NBE) and poly(COE), we converted the aldehyde
termini into the corresponding 2,4-dinitrophenylhydrazine
43,44
derivatives.
For that purpose, both aldehyde-semitelechelic
poly(NBE) and poly(COE) were treated with a freshly
prepared acidic solution of 2,4-dinitrophenylhydrazine to
afford yellow precipitates of the corresponding hydrazones
Figure 1. Structures of the first- (1), second- (2), and third-generation
(
3) Grubbs initiator. PCy
3
= tricyclohexylphosphine, Mes = mesityl.
a
2
1
13
Table 1. Results of the Reaction of Initiators 1, 2, and 3 with O
(Scheme 3). The H NMR, C NMR, and IR data strongly
support the aldehyde functionality in the polymer chain. In
b
initiator
benzaldehyde (%)
stilbenes (%)
cis:trans
1
13
the H and C NMR, the peaks at δ=9.61 and 203.6 ppm
corresponding to the aldehyde group disappeared, and
new signals corresponding to the -HCdN- moiety at δ=
1
2
3
a
44
24
29
7
40
1
1:4
0:1
0:1
1
13
7
.49 and 155.9 ppm appeared in the H and C NMR,
b
Benzene, room temperature, 24 h. Determined by GC-MS analysis
using n-dodecane as internal standard.
respectively.
Application: Surface Functionalization of ROMP-Derived
Monolithic Supports. The above-described methodology
was applied to the functionalization of ROMP-derived
the initiators vs O . Thus, the stability of the living polymer
2
increases in the order 1 < 2 < 3.
In the polymers obtained with 1-3, the peaks at δ=9.61
monoliths by taking advantage of the truly living char-
acter of ROMP.
1
13
45,46
and 203.6 ppm in the H and C NMR correspond to the
aldehyde proton and carbon, respectively (Figures S2 and S3,
Supporting Information). Size exclusion chromatog-
raphy (SEC) revealed monomodal distributions (1.82 <
PDI < 1.98).
Monoliths were prepared from NBE,
1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo-endo-dimethano-
naphthalene (DMN-H6), 2-propanol, toluene, and 1 within
45,47
stainless steel columns according to published procedures.
Then the initiator was subjected to a metathesis reaction with
O (Scheme 3) by flushing the monolith consecutively with
2
In principle, this functionalization procedure may be
expected to produce a mixture of the aldehyde-semitelechelic
toluene and air. Since a rigid polymer is formed, side reac-
tions as described in Scheme 2 may well be expected to occur
to a very minor extent, if at all. In fact, as evidenced by
FT-IR, aldehyde groups formed at the surface of the structure-
forming microgobules. To further proof aldehyde formation
and to quantify the amount of aldehyde groups, these were
again converted into the corresponding hydrazones via
reaction with 2,4-dinitrophenylhydrazine. Both the signal
polymer via metathesis of the Ru-alkylidene with O as well
2
as a diphenyl-terminated polymer, e.g., via a coupling reac-
tion between a living polymer and an aldehyde-telechelic
one (Scheme 2). In such case, the latter polymer should
have a molecular weight twice as high as the parent polymer
chain. Using 3 as initiator, a comparison of the size exclusion
chromatography (SEC) results obtained for an ethyl vinyl
-
1
ether-terminated poly(NBE) (M =6800 g/mol, PDI=1.69)
n
at 1725 cm in the IR spectrum and the nitrogen content
(up to 30 μmol/g) as determined by elemental analysis confirmed
the successful functionalization sequence (up to 8 mmol/g).
Immobilization of Trypsin. The use of monoliths for the
immobilization of transition-metal-based catalysts or enzymes
and an O -terminated poly(NBE) (M =7100 g/mol, PDI=
2
n
1
.92) revealed that there was no significant bimolecular
coupling among the Ru-alkylidenes of the polymer. An
overlay over the SEC traces for these two polymers is shown
in Figure 2.
4
8-51
has been reported by our group
as well as by Svec and
5
2-54
Obviously, the by far less oxophilic Ru-alkylidenes are
Fr ꢀe chet.
(NBE-CH
The reason for the use of (NBE-CH
DMN-H6 is that it provides monoliths with better flow-
Monoliths were prepared from NBE and from
55
less prone to O -triggered bimolecular coupling than the
-O)
2
3
SiCH
3
according to a published protocol.
-O) SiCH instead of
2
4
2
Mo-based Schrock initiators, where almost quantitative
bimolecular coupling can be observed in the presence of O2.
In contrast to the initiators, where such a coupling occurs to
a significant extent producing substantial amounts of stil-
benes, this reaction is less favored (probable) with entangled
polymers, where the chain ends have a lower probability to react.
We then extended our investigation toward less strained
monomers such as cis-cyclooctene (COE). Again, reaction
2
3
3
55
through characteristics. Introduction of the aldehyde groups
was carried out as described above (Scheme 3). The aldehyde-
functionalized monolithic support was then used for the
5
6,57
immobilization of trypsin.
Because of the steric demands
of trypsin, we introduced tetraethylenepentamine (TEPA,
Figure 3) as a spacer between the aldehyde-functionalized
surface and the protein (Scheme 4).
The primary as well as secondary amino groups were then
reacted with glutaraldehyde (GA, Figure 3) to produce a
large number of aldehyde functional groups, which were
then further used for the reaction with trypsin. The amount
of trypsin bound to the monolithic support was quantified by
elemental analysis. There, a nitrogen content of 86 μmol/g of
with O (air) resulted in the formation of aldehyde-teleche-
2
lic polymers. A maximum aldehyde formation of 61% was
obtained with 2-derived poly(COE). The peaks at δ=9.77
1
13
and 203.0 ppm in the H and C NMR support the forma-
tion of the aldehyde semitelechelic polymer (Figures S4 and S5,
Supporting Information). In contrast, end-group anal-
ysis of 1- and 3-derived poly(COE) prepared under the same

Article
Macromolecules, Vol. 43, No. 23, 2010 9603
Scheme 2. Synthetic Route to Aldehyde-Semitelechelic ROMP-Derived Polymers and Possible Side Reactions (Exemplified for Poly(NBE))
Table 2. Summary of ROMP-Derived Poly(NBE) Prepared by the
Action of 1, 2, and 3
For this purpose, aliquots were collected at different flow
rates and analyzed by RP-HPLC at 310 nm. As expected,
lower hydrolysis yields were obtained at higher flow rates,
i.e., shorter on column times due to a reduced contact time
between substrate and trypsin. A plot for the rate of hydro-
lysis against the flow rate is shown in Figure 7.
a
b
c
initiator
Mn,found (g/mol) PDI yield (%) CHO-terminated (%)
1
2
3
7500
8600
8900
1.98
1.93
1.82
91
86
62
80
47
29
a
0
Polymerizations conditions: benzene, [monomer] =1 M, monomer:
c
b
initiator=30:1; T = 23 °C, t = 1 h. Isolated yield. Determined by
H NMR spectroscopy.
Conclusion
1
A novel and convenient method for the synthesis of ROMP-
derived aldehyde-semitelechelic polymers via the metathesis of
the living Ru-alkylidenes of ROMP-derived polymers with O2
has been developed. The method was checked for all three
Grubbs-type initiators. Up to 80% of aldehyde-semitelechelic
poly(NBE) were observed when NBE was polymerized by 1 and
terminated with O . Though this simple reaction does not quanti-
2
tatively yield aldehyde-semitelechelic polymers and therefore
appears less suitable for the selective functionalization of linear
and soluble polymers, it is of significant synthetic utility for the
surface functionalization of insoluble (cross-linked) polymeric
materials, e.g., polymeric monoliths, where a significant degree of
surface functionalization up to 8 μmol can be achieved. This
amount of aldehyde groups allows for the generation of surface-
bound (branched) oligoaldehydes which finally offer access to the
permanent immobilization of enzymes, e.g., trypsin, and for
use of the thus-prepared supported enzymes in continuous flow
biocatalysis.
Figure 2. Comparison of the SEC curves between EVE- and O
poly(NBE) prepared by the action of 3.
2
-terminated
Experimental Section
monolith was found. Since the 8 μmol of aldehyde typically
found in such monoliths translates upon reaction with TEPA
into 40 μmol of N, the additional 46 μmol of N found by
elemental analysis is indicative of the successful immobiliza-
tion of trypsin on the ROMP-derived monolith.
General Remarks. All manipulations were performed under a
dinitrogen atmosphere in a glovebox (LabMaster 130, MBraun
Garching, Germany) or by standard Schlenk techniques unless
specified otherwise. RuCl
(IMesH )(CHPh)] (2) (PCy
,3-dimesitylimidazolin-2-ylidene), norborn-2-ene (NBE),
-norbornene-2-methanol, methyltrichorosilane, tetraethylene-
(CHPh)(PCy
) (1), [RuCl (PCy )-
2
3 2 2 3
After its immobilization, the proteolytic activity of the
immobilized protein was quantified by using N-R-benzoyl-
DL-arginine-p-nitroanilide hydrochloride (BAPNA) as sub-
strate, which upon hydrolysis produces N-R-benzoyl-DL-arginine
2
3
=tricyclohexylphosphine; IMesH =
2
1
5
pentamine (TEPA), trypsin from bovine pancreas (EC 3.4.21.4),
N-R-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA),
phosphate buffered saline tablets, Tween20, and deionized
water were obtained from Aldrich Chemical Co. (Germany).
Glutaraldehyde (GA, 25% in aqueous solution) was purchased
from ABCR GmbH and Co. KG (Karlsruhe, Germany).
(BA) and p-nitroaniline (PNA). The product formation and
the rate of hydrolysis were monitored by RP-HPLC (Figure 4).
The BA that formed was analyzed at λ=214 nm, whereas
the remaining amount of BAPNA was quantified at λ =
3
10 nm. The hydrolysis yield of BAPNA was determined by
HEPES was purchased from Carl Roth GmbH (Germany).
(
comparing the area of BAPNA after hydrolysis to the area of
a standard BAPNA solution (0.25 mM). The plot of conver-
sion of BAPNA vs time is shown in Figure 5. As can be seen,
a conversion of 65% was accomplished after 60 min. We then
switched to continuous flow conditions and monitored the
hydrolysis rate of BAPNA for 3 h using an on column
55
NBE-CH O) SiCH was prepared according to the literature.
2 3 3
CDCl
˚
3 2
was distilled from CaH and stored over molecular sieves
(
system (SPS). [RuCl (3-Br-Py) (IMesH )(CHPh)] (3)
4 A). Toluene was dried by an MBraun solvent purification
58,59
and
,4,4a,5,8,8a-hexahydro-1,4,5,8-exo-endo-dimethanonaphthalene
2
2
2
1
6
0
DMN-H6) were prepared according to the literature. Pur-
(
(
residence) time of 10 min. As can be seen (Figure 6), a slow
chased starting materials and other chemicals or reagents were
used without further purification. NMR spectra were recorded
on a Bruker Avance 600 and Bruker Spectrospin 250 spectrom-
eter in the indicated solvent at 25 °C and are listed in parts per
decrease in the conversion of BAPNA with a total loss in
activity of <20% was observed. Finally, the influence of
the flow rate on BAPNA conversion was examined at 37 °C.
IIþ

9
604 Macromolecules, Vol. 43, No. 23, 2010
Sudheendran and Buchmeiser
Scheme 3. Reaction of Aldehyde-Semitelechelic ROMP-Derived Polymers with 2,4-Dinitrophenylhydrazine and Schematic Illustration of the
Synthesis of Aldehyde- and 2,4-Dinitrophenylhydrazone-Functionalized Monoliths
1
:9 mixture of CH
filtration and dried in vacuo. CHO-semitelechelic poly(NBE):
0% aldehyde formation; SEC: M =7500 g/mol, PDI=1.98.
2 2
Cl and methanol. It was collected by
8
n
1
H NMR (CDCl
3
): δ=9.61 (s, 1H, CHO), 7.34-7.17 (m, 5H, J=
.2 Hz, phenyl), 6.35 (d, 1H, J=15.6 Hz, Ph-CHdCH),
.21-6.17 (dd, 1H, J=15.6, 7.8 Hz, Ph-CHdCH), 5.35 (bm,
CHdCH trans), 5.21 (bm, -CHdCH cis), 2.78 (bs, -CH cis),
.43 (bs, -CH trans), 1.87-1.85 (bm, -CH ), 1.76 (bm, -CH
7
6
-
2
2
2
)
1
3
1
.35 (bm, -CH
2
2 3
), 1.08-1.02 (bm, -CH ). C NMR (CDCl ):
δ=203.6 (CHO), 138.3, 137.9, 135.6, 134.0, 133.1, 128.6, 126.5,
126.0, 52.0, 43.5, 43.2, 42.2, 41.5, 38.5, 33.0, 32.3. IR (ATR
mode): 2940 (m), 2862 (m), 1712 (vs), 1444 (w), 1260 (m),
Figure 3. Reagents used for the immobilization of trypsin on a mono-
lith support.
-1
1
026 (w), 965 (s), 908 (s), 732 (s), 699 (w), 648 cm (w). CHO-
semitelechelic poly(COE): 15% aldehyde formation; SEC: M =
million downfield from TMS as an internal standard for proton
and carbon. FT-IR spectra were recorded on a Bruker Vector22
spectrometer using ATR technology. Quantification of the
functional groups in the monolith was accomplished by ele-
mental analysis on a Vario EL (Elementar Analysensysteme
GmbH, Hanau, Germany). Molecular weights and polydisper-
sity indexes (PDIs) of the polymers were determined by size
exclusion chromatography (SEC) on Polymer Laboratories
columns (PLgel 10 mm MIXEDB, 7.5ꢀ300 mm) in tetrahy-
drofuran (THF) at 25 °C vs PS using an autosampler and a
n
1
8
400 g/mol, PDI=1.77. H NMR (CDCl
CHO), 7.35-7.17 (m, 5H, J=7.2 Hz, phenyl), 6.37 (d, 1H, J=
6.2 Hz, Ph-CHdCH), 6.25-6.20 (dd, 1H, J=13.8, 6.6 Hz,
3
): δ=9.77 (s, 1H,
1
Ph-CHdCH), 5.39 (bm, -CHdCH trans), 5.35 (bm, -CHdCH
cis), 2.42 (bm, -CH), 2.21 (bm, -CH), 2.02-1.97 (bm,
-
CH), 1.65-1.62 (bm, -CH), 1.48-1.45 (bm, -CH), 1.34
13
(
bm, -CH
CHO), 138.0, 131.5, 130.5, 130.0, 129.8, 128.6, 126.8, 126.0,
14.2, 44.0, 33.2, 32.7, 29.8, 27.3. IR (ATR mode): 2916 (m),
2849 (m), 2360 (vs), 2341 (vs) 1727 (vs), 1467 (vs), 1437 (w), 1284
2
), 1.28 (bm, -CH
2 3
). C NMR (CDCl ): δ=203.0
(
1
4
84 UV detector (254 nm, all Waters Corp.). The flow rate was
-1
set to 0.7 mL min . Narrow polystyrene (PS) standards were
purchased from Waters Corp. Prior to measurements, polymer
solutions were filtered through Millipore 0.20 μm filters. The
trypsin activity was monitored and analyzed by an Agilent
Technology HPLC system (Germany). The system consisted
of a binary HPLC pump, a diode array UV-vis detector, an
autosampler, a column oven, and a sample thermostat. The
products were analyzed by RP-HPLC using an Agilent-Eclipse
XDB-C18 column (5 μm, 4.6 ꢀ 150 mm i.d., Zorbax RX-SIL).
Preparation of Aldehyde-Semitelechelic Polymers. A 5 mL
Schlenk flask was charged with 1 (29 mg, 0.035 mmol) in 1 mL of
benzene and a stir bar. In a separate vial, NBE (100 mg, 1.06 mmol)
was dissolved in benzene (1 M solution). The solution was
then added to the catalyst solution via a syringe under vigorous
stirring. After 1 h, the solution was purged with air under stirring
for 16 h. The reaction mixture was then poured into a large
excess of methanol (25 mL), and the precipitated polymer was
filtered off. The polymer obtained was then precipitated from a
-
1
(
m), 1070 (w), 965 (s), 717 (s), 691 cm (w).
General Procedure for the Preparation of 2,4-Dinitrophenyl-
hydrazine Derivatives. 0.25 g of 2,4-dinitrophenylhydrazine was
suspended in 5 mL of ethanol, and then 0.5 mL of concentrated
sulfuric acid was added cautiously. The warm solution (0.5 mL)
was then added to a solution of aldehyde-functionalized poly-
mer suspended in 0.5 mL of a mixture of CH
2 2
Cl and methanol
(1:1). The mixture was stirred for 15 min and poured into
methanol (25 mL). The precipitate was filtered and washed with
methanol to afford the corresponding hydrazones. Poly(NBE)-
): δ=11.00 (s, 1H), 9.13 (m, 1H),
1
hydrazone: H NMR (CDCl
3
8.29 (m, 1H), 7.92 (m, 1H), 7.49 (d, 1H) 7.35-7.17 (m, 5H, J=
7.2 Hz, phenyl), 6.37 (d, 1H, J = 15.6 Hz, Ph-CHdCH),
6.22-6.18 (dd, 1H, J=15.6, 7.8 Hz, Ph-CHdCH), 5.35 (bm,
-CHdCH trans), 5.21 (bm, -CHdCH cis), 2.78 (bs, -CH cis),
2.43 (bs, -CH trans), 1.88-1.77 (bm, -CH
) 1.36 (bm, -CH
),
2
2
13
2 3
1.09-1.03 (bm, -CH ). CNMR(CDCl ):δ=155.9 (-HCdN-),

Article
Macromolecules, Vol. 43, No. 23, 2010 9605
Scheme 4. Schematic Illustration for the Immobilization of Trypsin on a ROMP-Derived Monolithic Support
Figure 5. Plot of conversion vs time for the tryptic digest of 0.25 mM
BAPNA at different reaction times. Conditions: T = 37 °C; mobile
phase of the trypsin monolith was 50 mM HEPES buffer.
Figure 4. RP-HPLC chromatogram obtained for the tryptic digestion
of BAPNA: (a) standard BAPNA, (b) standard PNA, (c) on-column
tryptic digestof 0.25 mM BAPNA in 10 min at 37 °C. Peak 1 is BA, peak
2
is PNA, and peak 3 is undigested BAPNA. Conditions: flow rate 1 mL
1
-
min ; 25 °C; mobile phases: A: 95% water þ 5% acetonitrile (ACN) þ
(
m, 5H, J=7.2 Hz, phenyl), 6.37 (d, 1H, J=15.6 Hz, Ph-CHd
0
.1% trifluoroacetic acid (TFA); B: 95% ACN þ 5% water þ 0.1%
CH), 6.25-6.20 (m, 1H, J=15.6, 7.2 Hz, Ph-CHdCH), 5.38
TFA; gradient: 0 min 4% B, 9 min 50% B, 10 min 50% B; UV (214 nm);
(
(
bm, -CHdCH trans), 5.35 (bm, -CHdCH cis), 2.02-1.96
injection volume 20 μL.
13
bm, -CH), 1.33 (bs, -CH ), 1.28 (bm, -CH ). C NMR
2
2
1
1
2
1
45.2, 138.0, 135.6, 134.0, 133.1, 130.1, 128.0, 126.9, 126.0,
23.7, 43.5, 43.2, 42.2, 41.5, 38.5, 33.0, 32.3. IR (ATR mode):
941 (m), 2862 (m), 1616 (vs), 1591 (vs), 1518 (vs), 1464 (vs),
(CDCl ): δ = 152.7 (-HCdN-), 138.1, 130.5, 130.0, 128.6,
3
126.9, 126.0, 116.6, 37.7, 32.8, 29.8, 29.2, 27.4. IR (ATR mode):
2917 (m), 2849 (m), 1617 (vs), 1593 (vs), 1509 (vs), 1466 (vs),
1333 (w), 1214 (w), 1069 (w), 965 (s), 756 (s), 719 cm (w).
-1
-1
445 (s), 1335 (vs), 1215 (s), 1041 (s), 964 (s), 832 (w), 755 cm
1
(
s). Poly(COE)-hydrazone: H NMR (CDCl ): δ=11.02 (s, 1H),
Synthesis of Aldehyde-Functionalized Monoliths. Monoliths
were prepared in stainless steel columns (250 ꢀ 3 mm) according
3
9
.13 (m, 1H), 8.29 (m, 1H), 7.92 (m, 1H), 7.52 (t, 1H), 7.34-7.17

9
606 Macromolecules, Vol. 43, No. 23, 2010
Sudheendran and Buchmeiser
Figure 6. Tryptic activity in a continuous flow experiment. Conditions:
on column time 10 min; T = 37 °C; mobile phase: 0.25 mM BAPNA in
Figure 7. Influence of the flow rate on the hydrolysis of BAPNA.
Conditions: T=37 °C; mobile phase: 0.25 mM BAPNA in 50 mM
HEPES buffer.
5
0 mM HEPES buffer.
4
5
to a previously published protocol. Two solutions A and B
were prepared and cooled to -20 °C. Solution A (NBE:DMN-
ethanol to remove any unattached TEPA. A 2 wt % solution of
glutaraldehyde in Na CO buffer (20 vol % THF) at pH 9.2 was
2 3
6
H :2-propanol=25:25:40 and solution B (toluene:1=10:1 (all in
wt %) were mixed and rapidly transferred to the stainless steel
column. The column was kept at 0 °C for 30 min, and then the
polymerization was allowed to complete at room temperature
for 1 h. After 1 h, the monolith was flushed with toluene for
then introduced into the monolith at a flow rate of 0.05 mL/min.
The monolith was then sealed and kept at room temperature for
another 2 h. Finally, the monolith was flushed with 10 vol %
THF in deionized water followed by absolute ethanol to remove
any unattached GA. The glutaraldehyde-functionalized mono-
lith was then flushed with 50 mM HEPES buffer solution at
pH 8 to remove any organic solvent in the monolith that might
interfere with the trypsin derivatization. A fresh solution of
trypsin (3 mg/mL) in 50 mM HEPES buffer (100 mM NaCl,
1
5 min at a flow rate of 0.2 mL/min to remove any unattached
catalyst. The monolith was then flushed consecutively with
toluene and air for 16 h and then kept at room temperature.
After 24 h, the monolith was flushed with THF to remove
any Ru byproducts. IR (ATR mode): 2938 (m), 2861 (m),
1
8
725 (νCO, m), 1649 (m), 1448 (s), 1341 (m), 1148 (w), 963 (s),
31 (w), 731 cm (s).
Quantification of the Aldehyde Group. The chemically acces-
-
1
10 mM CaCl ) was prepared and injected into the monolith at a
2
flow rate of 0.05 mL/min. The monolith was then sealed and
kept for 2 h at room temperature. Finally, the monolith was
flushed with 0.1 M phosphate buffer solution containing 0.01 wt %
Tween 20 and then with phosphate buffered saline (PBS) solution.
After the immobilization the monolith was stored at 4 °C.
sible aldehyde groups in the monolith were quantified via
conversion into the corresponding 2,4-dinitrophenylhydra-
zones. For this purpose, the monolith was taken out of the
column, ground up and dried in vacuo overnight. 100 mg of the
sample were suspended in 2 mL of methanol and stirred with
Proteolytic Activity of the Immobilized Trypsin (Batch Experiment).
The proteolytic activity of the immobilized trypsin was monitored
by measuring the hydrolysis products of a standard solution of
N-R-benzoyl-DL-arginine-p-nitroanilide (BAPNA), i.e., N-R-benzoyl-
DL-arginine (BA) and p-nitroaniline (PNA). A 0.25 mM standard
solution of BAPNA in 50 mM HEPES buffer solution at pH 8 was
introduced into the monolith at 37 °C. After 10 min, the reaction
mixture was eluted at a flow rate of 0.05 mL/min for 10 min. The
conversion of BAPNA was calculated by comparing the area of
BAPNA after proteolysis to the area of the standard BAPNA.
Proteolytic Activity of the Immobilized Trypsin (Continuous
Flow). The activity of immobilized trypsin was monitored under
continuous flow conditions for 3 h as follows. A standard solu-
tion of BAPNA (0.25 mM) in 50 mM HEPES buffer at pH 8 was
pumped through the bioreactor at a flow rate of 0.05 mL/min at
2
mL freshly prepared 2,4-dinitrophenylhydrazine reagent (vide
supra). After 1 h, the monolith was filtered, washed with
methanol and then dried in vacuo. Quantification of the hydra-
zone groups was accomplished by elemental analysis. IR (ATR mode):
2
936 (m), 2862 (m), 2165 (m), 1648 (m), 1617 (m), 1592 (m), 1447
s), 1408 (m), 1337 (m), 1143 (w), 964 (s), 837 (w), 733 (s), 688 (m)
(
cm . Elemental analysis: C, 88.3; H, 9.51; N, 0.06.
-1
Monoliths for Trypsin Immobilization. Monoliths were pre-
pared in stainless steel columns (100 ꢀ 4.6 mm i.d.) according to
55
a previously published protocol. Briefly, two solutions A and B
were prepared. Solution A consisted of NBE:(NBE-CH
SiCH :2-propanol (20:20:45.7 wt %), solution B consisted of
.4 wt % of 1 in 13.9 wt % of toluene. Both solutions were cooled
2 3
-O) -
3
0
to -20 °C, mixed for ∼1 min, and then rapidly transferred into
3
7 °C. Aliquots were collected in 10 min intervals and analyzed
the stainless steel column. The column was kept at 0 °C for
by RP-HPLC at 310 nm. For the separation conditions refer to
Figure 4. The extent of hydrolysis of BAPNA was calculated by
comparing the area of BAPNA of each 10 min aliquot to the
area of a standard BAPNA. No perceptible loss in activity was
observed for at least 3 h.
3
3
0 min, and then the polymerization was allowed for another
0 min at room temperature. The monolith was flushed with
toluene for 30 min at a flow rate of 0.2 mL/min to remove any
nonreacted monomers. Then the monolith was consecutively
flushed with toluene and air at room temperature for 24 h.
Finally, the support was washed with THF to remove any Ru
byproducts.
Acknowledgment. Financial support provided by the Bundes-
ministerium f u€ r Bildung und Forschung (BMBF, Contract
Modification of Aldehyde-Functionalized Monoliths with Tetra-
5
6
3
1P5555), the Graduate School of Excellence “BuildMona” at
ethylenepentamine (TEPA) and Immobilization of Trypsin. A2wt%
solution of TEPA in 0.1 M Na CO buffer solution (pH 9.2) and
0 vol % of THF was introduced into the aldehyde functional-
the University of Leipzig, Germany, and the University of
Stuttgart, Germany, is gratefully acknowledged.
2
3
1
ized monolith at a flow rate of 0.05 mL/min. Then the reaction
was allowed for 2 h at room temperature. The monolith was
flushed with 10% THF in deionized water followed by absolute
Supporting Information Available: GC-MS of stilbenes
2
formed via the reaction of O with a Grubbs-type initiator;

Article
Macromolecules, Vol. 43, No. 23, 2010 9607
1
13
H and C NMR spectra of the semitelechelic polymers. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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