Selective removal of chymotrypsin using diphenyl α- aminoalkylphosphonate immobilized on Sepharose gel

Article abstract of DOI:10.1271/bbb.66.1111

A diphenyl α-aminoalkylphosphonate derivative, which is an irreversible inhibitor of chymotrypsin-like serine proteases, was immobilized on cyanogen bromide-activated Sepharose, and the selective binding of chymotrypsin to the obtained inhibitor-gel was evaluated using batch and column methods. Complete removal of chymotrypsin in an aqueous solution was done using the column method, while partial removal was done using the batch method.

Full text of DOI:10.1271/bbb.66.1111

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Selective Removal of Chymotrypsin Using Diphenyl α-
Aminoalkylphosphonate Immobilized on Sepharose
Gel
a
a
a
a
a
Shin ONO , Makiko UMEZAKI , Hajime NABARI , Ryutaro NAKAYAMA , Kumi HASEGAWA ,
a
a
ab
a
Satomi SAKO , Takayoshi FUJII , Isao YAMAZAKI & Toshiaki YOSHIMURA
a
Department of System Engineering of Materials and Life Science, Faculty of
Engineering, Toyama University Gofuku 3190, Toyama 930-8555, Japan
b
Yayoi Kagaku Kogyo Co., Ltd. Fukuoka-machi, Nishitonami-gun, Toyama 939-0135,
Japan
Published online: 22 May 2014.
To cite this article: Shin ONO, Makiko UMEZAKI, Hajime NABARI, Ryutaro NAKAYAMA, Kumi HASEGAWA, Satomi SAKO,
Takayoshi FUJII, Isao YAMAZAKI & Toshiaki YOSHIMURA (2002) Selective Removal of Chymotrypsin Using Diphenyl α-
Aminoalkylphosphonate Immobilized on Sepharose Gel, Bioscience, Biotechnology, and Biochemistry, 66:5, 1111-1115,
DOI: 10.1271/bbb.66.1111
To link to this article: http://dx.doi.org/10.1271/bbb.66.1111
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Biosci. Biotechnol. Biochem., 66 (5), 1111–1115, 2002
Note
Selective Removal of Chymotrypsin Using Diphenyl
Immobilized on Sepharose Gel
a-Aminoalkylphosphonate
Shin ONO,† Makiko UMEZAKI, Hajime NABARI, Ryutaro NAKAYAMA, Kumi HASEGAWA
Satomi SAKO, Takayoshi FUJII, Isao YAMAZAKI,* and Toshiaki YOSHIMURA
,
Department of System Engineering of Materials and Life Science, Faculty of Engineering,
Toyama University, Gofuku 3190, Toyama 930-8555, Japan
*
Yayoi Kagaku Kogyo Co., Ltd., Fukuoka-machi, Nishitonami-gun, Toyama 939-0135, Japan
Received August 13, 2001; Accepted January 16, 2002
A
diphenyl
a
-aminoalkylphosphonate derivative,
can be easily synthesized and are fairly stable against
hydrolysis, phosphonate derivatives can be coupled
with amino acids and peptides to give the corre-
sponding peptidyl phosphonate derivatives, which
act as more potent and selective serine protease inhi-
bitors than do phoshonate derivatives. Such proper-
ties of phosphonate derivatives are suitable for their
immobilization on a chromatography gel. In fact, 2-
aminoethyl p-nitrophenyl methylphosphonate was
immobilized on Sepharose 4B (Pharmacia Biotech),
and the obtained covalent a‹nity gel could be used
for puriˆcation of acetylcholinesterase. In this
study, a phosphonate derivative corresponding to
phenylalanine (Ahe-Phe-phosphonate, Fig. 2) was
chosen as an irreversible inhibitor for chymotrypsin-
like proteases as a model system for the selective
removal of serine proteases using a covalent chro-
matographic technique (Fig. 1).
which is an irreversible inhibitor of chymotrypsin-like
serine proteases, was immobilized on cyanogen
bromide-activated Sepharose, and the selective binding
of chymotrypsin to the obtained inhibitor-gel was eval-
uated using batch and column methods. Complete
removal of chymotrypsin in an aqueous solution was
done using the column method, while partial removal
was done using the batch method.
Key words: covalent chromatography; diphenyl
a-
1
2)
aminoalkylphosphonate; irreversible inhi-
bitor of serine protease; selective removal
of chymotrypsin
Isolation and puriˆcation of naturally occurring
proteins and peptides are essential for studies on their
structures and biological functions. In these proc-
esses, unexpected degradation of the target proteins
or peptides by contaminating proteases often results
in a decrease in yield. To prevent unexpected degra-
dation, a cocktail of protease inhibitors has been
used to inactivate contaminating proteases.
However, the inactivated proteases still remain in the
solution with the target products, and their separa-
tion from the target products is required in further
processes. A technique to selectively remove the con-
taminating proteases would be useful for isolation
and puriˆcation of the target products. This study
was therefore designed to examine whether a cova-
lent chromatographic technique using irreversible
protease inhibitors is useful for removing some con-
taminating proteases (Fig. 1).
The
synthesized as described previously.
diphenyl phosphonate analog of -benzyloxycar-
bonylphenylalanine, Z-Phe-phosphonate, was syn-
thesized by -amidoalkylation of triphenyl phosphite
Ahe-Phe-phosphonate
derivative
was
1
3)
First,
a
N
a
with benzyl carbamate and phenylacetaldehyde in
acetic acid. Consequent cleavage of the benzylox-
ycarbonyl group with 25
z HBr acetic acid gave the
W
corresponding hydrogen bromide salt of the Phe-
phosphonate. Then 6-aminohexanoic acid was in-
troduced into the Phe-phosphonate as a spacer, i.e.,
the desired spacer-binding derivative (Ahe-Phe-
phosphonate) was obtained as its tri‰uoroacetate salt
by coupling of
N-tert-butyloxycarbonylaminohex-
anoic acid to the Phe-phosphonate followed by
deprotection of the tert-butyloxycarbonyl group with
tri‰uoroacetic acid. The ˆnal product was puriˆed by
silica-gel chromatography and identiˆed by IR,
matrix-assisted laser desorption ionization time-of-
Diphenyl
a-aminoalkylphosphonate derivatives
have been reported to be selective and irreversible in-
1
–11)
hibitors of serine proteases.
The mechanism by
which phosphonate derivatives inhibit serine pro-
teases is similar to that proposed for diisopropyl
‰
ight mass spectroscopy (MALDI-TOF-MS), and
1
14)
4
)
H-NMR.
‰
uorophosphates. Since phosphonate derivatives
†
To whom correspondence should be addressed. Shin ONO, FAX: +81-76-445-6703; E-mail: shinono
＠
eng.toyama-u.ac.jp
Abbreviations: CNBr, cyanogen bromide; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-‰ight mass spectrometry

1
112
S. ONO et al.
Fig. 3. EŠects of Reaction Time on Changes in Enzymatic Activ-
ity of Chymotrypsin Caused by Treatment with Gel-I and the
Control Gel by the Batch Method.
The remaining activities of chymotrypsin in the supernatants
of the sample solutions after treatment with gel-I and the control
gel were estimated using Bz-Tyr-pNA as a substrate. The
amount of gel was 0.1 ml, and the reaction times were 10, 20, 30,
Fig. 1. Schematic Representation of Removal of Proteases by
Covalent Chromatographic Technique Using Irreversible Pro-
tease Inhibitors.
Immobilization of irreversible inhibitors on a solid support
for chromatography (step 1), reaction of the inhibitor-immobi-
lized solid support with a sample solution containing con-
taminating proteases (step 2), and selective binding of con-
taminating proteases to the inhibitor-immobilized solid support
and 40 min. Results are expressed as means
±
SD of independent
triplicate experiments. gel-I, control gel.


to give
step 3).
a target product without contaminating proteases
(
8
.0) was used. The volume of the obtained Phe-
phosphonate inhibitor-immobilized gel (gel-I) was
about 3.5 ml. The amount of the immobilized Phe-
phosphonate inhibitor was estimated to be
4
.8 mmol ml of gel from the reduced amount of the
W
Ahe-Phe-phosphonate derivative remaining in the
coupling buŠer after immobilization. Gel-I was
stored in 40 m
pH 7.8) at 4 C.
For estimation of the amount of chymotrypsin
binding to gel-I by the batch method, 0.5 ml of
chymotrypsin solution (40 ) was incubated with
gel-I (0.1–0.3 ml) in a micro test tube at 25 C for
0–40 min using a rotator RT-50 (Titec). After cen-
M
Tris-HCl containing 16 m
M
CaCl
2
(
9
Fig. 2. Spacer-Binding Phosphonate Inhibitor (Ahe-Phe-
phosphonate) of Chymotrypsin-Like Serine Proteases.
As a spacer for immobilization, a 6-aminohexanoyl group
Ahe) was introduced into the phosphonate inhibitor (Phe-
m
M
(
phosphonate).
9
1
trifugation, portions of the supernatant were meas-
ured for the remaining activity using Bz-Tyr-pNA as
a substrate. On the other hand, CNBr-activated
Separose 4B gel treated with only 2-ethanolamine
was put through the same binding experiment as the
reference (control gel). Figure 3 shows the eŠects of
reaction time (10–40 min) on the remaining activity
of chymotrypsin when 0.1 ml of each of the gels (gel-
I and control gel) was used. Regardless of the reac-
tion time, the remaining activity of chymotrypsin was
The inhibitory activities of the obtained Ahe-Phe-
phosphonate toward chymotrypsin and trypsin were
evaluated before its immobilization. The Ahe-Phe-
phosphonate irreversibly and time-dependently inac-
tivated chymotrypsin with an inactivation rate con-
„
1 „1
stant,
k
obs [I], of 43
±
5.0
M
s , while it showed
W
weaker inactivation ability toward trypsin with a
„
1
„1 15)
k
obs [I] of 0.42
±
0.03
M
s
.
These results suggest
W
that Ahe-Phe-phosphonate is able to selectively inac-
tivate chymotrypsin-like serine proteases.
40–44
incubation with the control gel. These results indicate
that about 40 of chymotrypsin in the sample solu-
z z
after incubation with gel-I and 78–81 after
For preparation of the phosphonate inhibitor-
immobilized gel, Ahe-Phe-phosphonate (5 mg) was
immobilized on a commercially available a‹nity
chromatography gel (cyanogen bromide (CNBr)-acti-
z
tion can bind to gel-I under these experimental condi-
tions. Therefore, we chose 10 min as the reaction
time for further experiments.
To determine the optimal amount of gel for the
removal of serine protease by the batch method, we
vated Sepharose 4B, 1 g) in 0.1
buŠer containing 0.5 NaCl at pH 8.3. For blocking
the remaining active group, 2-ethanolamine (1 , pH
M
NaHCO coupling
3
M
M

Selective Removal of Chymotrypsin Using Irreversible Inhibitor
1113
Fig. 4. EŠects of Amount of Gel on the Changes in Enzymatic
Activities of Chymotrypsin and Trypsin Caused by Treatment
with Gel-I and the Control Gel by the Batch Method.
The remaining activities of chymotrypsin in the supernatants
of the sample solutions after treatment with gel-I and the control
gel were estimated using Bz-Tyr-pNA as a substrate. The reac-
tion time was 10 min, and 0.1, 0.2, and 0.3 ml of gel-I or the
control gel were used. Similarly, the remaining activities of tryp-
sin were estimated using Bz-Arg-pNA as a substrate. Results are
Fig. 5. Comparison of the Contents in the Sample Solution of
Chymotrypsin before (A) and after (B) Treatment with a Gel-I-
Packed Column (5
A sample solution (12
a tripeptide, Gly-Gly-Gly (5
passed through the gel-I-packed column at a ‰ow rate of
×
50 mm).
m
M
, 50
m
m
l) of chymotrypsin containing
M), as an internal standard was
expressed as means
±
SD of independent triplicate experiments.

gel-I, control gel.

0
7
.05 ml min, and 40 m
.8) was used as the eluent. The eluted peak fraction was collect-
M
Tris-HCl containing 16 m
2
M CaCl (pH
W
tested various amounts of gel-I (0.1–0.3 ml) and
measured the remaining activity of chymotrypsin
ed, and its composition was analyzed by a gel-ˆltration HPLC
Waters Protein-Pak60 2, 20 m phosphate buŠer (pH 7.0)
containing 100 m Na SO , 30 C, 0.5 ml min). The elution
(
×
M
(
Fig. 4). When the amount of gel was 0.1 ml, the
remaining activities of chymotrypsin after incubation
with gel-I and the control gel were 42 and 90 , re-
M
9
2
4
W
proˆles were expressed by monitoring at 280 nm
z
spectively. Based on these ˆndings, the net rate of
chymotrypsin bound to gel-I, which we called the
tide, Gly-Gly-Gly, was added to the chymotrypsin
solution as an internal standard. The ‰ow rate was
chymotrypsin removal rate, was estimated to be 48
z
by comparison of the remaining activities observed
after incubation with gel-I and the control gel. The
chymotrypsin removal rates by 0.2 and 0.3 ml of gel-I
0.05 ml min, and 40 m
M
Tris-HCl buŠer containing
W
16 m
M
CaCl
2
(pH 7.8) was used as an eluent. The
eluted fraction (30–50 min) was collected and ana-
lyzed using a gel-ˆltration HPLC system. Figure 5
shows the analytical gel-ˆltration HPLC proˆles for
the chymotrypsin solution before and after passing
through the gel-I-packed column. A peak corre-
sponding to chymotrypsin completely disappeared
after the chymotrypsin solution had been passed
through the gel-I-packed column, while a peak corre-
sponding to the standard Gly-Gly-Gly still remained.
Disappearance of the chymotrypsin peak was ob-
served in nine further experiments using the same gel-
I-packed column. In addition, enzymatic activity in
the eluted fraction was also evaluated under condi-
tions similar to those described in the legend of Fig.
4, and chymotryptic activity in the eluted fraction
was found to decrease to an undetectable level (data
not shown). These results demonstrated that
chymotrypsin in a sample solution can be completely
removed by using gel-I packed in a column.
were 57 and 58
z
, respectively, suggesting that an in-
crease in the amount of gel is not eŠective for increas-
ing the chymotrypsin removal rate. On the other
hand, the net rates of trypsin bound to 0.1, 0.2, and
0
.3 ml of gel-I, which we called trypsin removal rates,
were 2, 12, and 11 , respectively. A comparison of
z
the chymotrypsin and trypsin removal rates by gel-I
indicated that the most eŠective binding of
chymotrypsin to gel-I can be obtained when 0.1 ml of
gel-I is used. Under these experimental conditions,
about 90 nmol of chymotrypsin was estimated to be
removed by 1 ml of gel-I. All of these results indicate
that chymotrypsin can be selectively, although only
partially, removed by using gel-I and the batch
method.
To try to complete the removal of chymotrypsin,
gel-I was packed into a columm (5
×
50 mm), and a
) was
passed through the gel-I-packed column. A tripep-
portion (50
m
l) of chymotrypsin solution (12
m
M
To evaluate the selectivity in removal of

1
114
S. ONO et al.
Table 1. Comparison of the Enzymatic Activities in the Eluted
Fractions through the Gel-I-Packed and Control Gel-Packed
Columns
a
Relative Activity (
z
)
Chymotrypsin
Trypsin
b
mixed sample
100e
ND
±
5.7 0.9
100
c
gel-I
6.2
7.7
±
0.6
0.3
d
control gel
±
a
Chymotryptic and tryptic activities in 100
through the gel-I-packed and control gel-packed columns were com-
pared to those in 100 l of the mixed sample solution. Each sample
100 l) was mixed with 890 l of 40 m Tris-HCl buŠer containing
6 m CaCl (pH 7.8), and then 10 l of DMSO solution of Bz-L-Tyr-
pNA or Bz-L-Arg-pNA was added. Final concentrations of Bz-L-Tyr-
ml of the eluted fractions
m
(
1
m
M
m
M
2
m
pNA and Bz-L-Arg-pNA were 10 mM. Relative activities are means
SD of triplicate experiments.
±
b
Chymotrypsin and trypsin were dissolved in 40 m
the ˆnal concentrations (15 ) of chymotrypsin and trypsin were meas-
ured by the UV absorption spectra (
280 nm), respectively).
M
Tris-HCl buŠer, and
m
M
1
z
1
z
A
＝
20.5 (278 nm) and
A
＝
15.0
(
c
The sample solution (50 ml) was passed through the gel-I-packed column
at 25 C and the elution was monitored at 220 nm.
The sample solution (50 ml) was passed through the control gel-packed
column. Other conditions were the same as those for the gel-I-packed
column experiment.
9
d
Fig. 6. Reversed-Phase HPLC Proˆles of the Protein Composi-
tions in the Eluted Fractions Passing through the Gel-I-Packed
and Control Gel-I Packed Columns.
e
Not detectable.
Reversed-phase HPLC analysis was done using an ODS-100s
column (3.0
×
150 mm, Tosoh) with an acetonitrile-tri‰uoroa-
cetic acid system as the mobile phase. The elution proˆles were
expressed by monitoring at 280 nm, and the ‰ow rate was
However, chymotryptic activity in the eluted fraction
from the gel-I-packed column decreased to an un-
detectable level, while tryptic activity was detected at
0
.3 ml min.
W
a level (6.2
z) similar to that in case of the control
chymotrypsin by the column method, a portion
50 l) of mixed sample solution containing
gel-packed column. These results for the protein
composition and enzymatic activity analyses demon-
strated that chymotrypsin can be completely and
selectively removed by the column method.
In this work, we prepared a Phe phoshonate
derivative that is an irreversible and selective inhibi-
tor of chymotrypsin and immobilized it on Sepharose
gel. The obtained inhibitor-immobilized gel was
demonstrated to be useful for removal of chymotryp-
sin in an aqueous solution by both batch and column
methods. In conclusion, we demonstrated that a
covalent chromatographic technique using diphenyl
(
m
chymotrypsin and trypsin was passed through the
gel-I-packed column under the same conditions as
those described in the legend of Fig. 5. The eluted
fraction was collected, and then the protein composi-
tion and enzymatic activity in the fraction were ana-
lyzed. The control gel was also packed into the
column (5
×
50 mm), and the mixed sample solution
was put through the obtained control gel-packed
column as a reference. Figure 6 shows the reversed-
phase HPLC proˆles of the protein compositions in
the eluted fractions passing through the gel-I-packed
and control gel-packed columns. Two peaks corre-
sponding to trypsin (19.6 min) and chymotrypsin
a
-aminoalkylphosphonate derivatives is useful for
selective removal of serine proteases.
(
20.9 min) were observed in the eluted fraction from
References
the control gel-packed column. On the other hand,
the chymotrypsin peak disappeared but the trypsin
peak still remained in the case of the gel-I-packed
column. This result indicated that chymotrypsin was
selectively removed by using the gel-I-packed
column. Similar selective removal of chymotrypsin
was also proved by comparison of the enzymatic
activities in both eluted fractions (Table 1). After
having been passed through the control gel-packed
column, chymotrypsin and trypsin were eluted as a
diluted fraction, but both chymotryptic and tryptic
1
2
)
)
Bartlett, P. A. and Lamden, L. A., Inhibition of
chymotrypsin by phosphonate and phosphonamide
peptide analogues. Bioorg. Chem., 14, 356–377
(
1986).
Lamden, L. A. and Bartlet, P. A., Aminoalkyl-
phosphono‰uoridate derivatives: Rapid and poten-
tially selective inactivators of serine peptidases.
Biochem. Biophys. Res. Commun., 112, 1085–1090
(
1983).
3) Oleksyszyn, J. and Powers, J. C., Irreveresible inhi-
bition of serine proteases by peptidyl derivatives of
a-
activities (5.7 and 7.7
z
compared to each activity in
aminoalkylphosphonate diphenyl esters. Biochem.
Biophys. Res. Commun., 161, 143–149 (1989).
the mixed sample, respectively) still remained.

Selective Removal of Chymotrypsin Using Irreversible Inhibitor
1115
4
5
)
)
Oleksyszyn, J. and Powers, J. C., Irreversible inhibi-
tion of serine proteases by peptide derivatives of (
aminoalkyl)phosphonate diphenyl esters. Biochemis-
try, 30, 485–493 (1991).
C.-M., Winkler, U., Johnson, D. A., Froelich, C. J.,
Hudig, D., and Powers, J. C., Synthesis and evalua-
tion of diphenyl phosphonate esters as inhibitors of
the trypsin-like granzymes A and K and mast cell
tryptase. J. Med. Chem., 41, 2289–2301 (1998).
a
-
Woodard, S. L., Jackson, D. S., Abuelyaman, A. S.,
Powers, J. C., Winkler, U., and Hudig, D.,
Chymase-directed serine protease inhibitor that reacts
with a single 30-kDa granzyme and blocks NK-
mediated cytotoxicity. J. Immunol, 153, 5016–5025
12) Ashani, Y. and Wilson, I. B., A covalent a‹nity
column for the puriˆcation of acetylcholinesterase.
Biochim. Biophys. Acta, 276, 317–322 (1972).
13) Oleksyszyn, J., Subotkowska, L., and Mastalerz, P.,
(
1994).
Diphenyl 1-aminoalkanephosphonates. Synthesis,
6
7
8
9
)
)
)
)
Fastrez, J., Jespers, L., Lison, D., Renard, M., and
Sonveaux, E., Synthesis of new phosphonate inhibi-
tors of serine proteases. Tetrahedron Lett., 30,
985–986 (1979).
p
14) TFA
・
Ahe-Phe (OPh)
2
; white solid; mp 223–225
9
C;
1593, 1545, 1491, 1261, 1205,
OD)
H
„
1
1
IR
n
max (KBr) cm
6
861–6864 (1989).
1186, 1137, 947; H-NMR (400 MHz, CD
3
d
J
＝
Hamilton, R., Walker, B. J., and Walker, B., A con-
venient synthesis of N-protected diphenyl phoshonate
esters analogues of ornithine, lysine, and homoserine.
Tetrahedron Lett., 34, 2847–2850 (1993).
0.96–1.09 (m, 2H), 1.29–1.45 (m, 4H), 2.01 (t,
7.0, 2H), 2.65 (t, 7.8, 2H), 2.92–3.01 (m, 1H),
J
＝
4.93–5.01 (m, 1H), 7.07–7.30 (m, 15H); MALDI-
+
TOF-MS (m z) 467.7 (M+1) .
W
Hamilton, R., Kay, G., Shute, R. E., Travers, J.,
Walker, B. J., and Walker, B., The synthesis of phos-
phonate analogues of amino acids and peptides.
Phosphorus Sulfur Silicon, 76, 127–130 (1993).
Oleksyszyn, J., Boduszek, B., Kam, C.-M., and
Powers, J. C., Novel amidine-containing peptidyl
phosphonates as irreversible inhibitors for blood
coagulation and related serine proteases. J. Med.
Chem., 37, 226–231 (1994).
15) Inactivation rate constants (kobs [I]) were measured
W
using the incubation method under pseudo-ˆrst-order
reaction conditions. For chymotrypsin, the Ahe-Phe-
phosphonate derivative (5
chymotrypsin (0.5 ) in 40 m
taining 16 m CaCl (pH 7.8) at 25
of 300 l, were withdrawn at 0.5, 5, 20, and 40 min
after the start of incubation and mixed with the
Tris-HCl buŠer (2670 l) and Bz-L-Tyr-pNA (30 l,
60 ) in DMSO to measure the remaining activities.
For trypsin, the enzyme and Ahe-Phe-phosphonate
concentrations were 0.4 and 320 , respectively,
and Bz-Arg-pNA (100 ) in DMSO was used as a
substrate. Pseudo-ˆrst-order inactivation rate con-
stants ( obs) were obtained from plots of ln vs
time (triplicate experiments).
m
M
) was incubated with
Tris-HCl buŠer con-
C. Portions, each
mM
M
M
2
9
m
m
m
1
1
0) Abuelyaman, A. S., Hudig, D., Woodard, S. L., and
Powers, J. C., Fluorescent derivatives of diphenyl [1-
m
M
(
N
-peptidylamino)alkyl]phosphonate esters: Synthe-
m
M
m
M
m
M
sis and use in the inhibition and cellular localization
of serine proteases. Bioconjugate Chem., 5, 400–405
(
1994).
k
v
t
v
0
.
W
1) Jackson, D. S., Fraser, S. A., Ni, L.-M., Kam,