Photoregulation of deacylation rate of acyl trypsin derived from photoresponsive inverse substrate

Article abstract of DOI:10.1016/j.bmcl.2003.07.013

The acyl trypsin was prepared by use of an inverse substrate, which is comprise of a photoresponsive 4-phenylazobenzoyl moiety. The acyl group in acyl trypsin has been shown to isomerize from trans-form (4t-trypsin) to cis-form (4c-trypsin)/from cis-form

Full text of DOI:10.1016/j.bmcl.2003.07.013

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3809–3812
Photoregulation of Deacylation Rate of Acyl Trypsin Derived
from Photoresponsive Inverse Substrate
Haruo Sekizaki,* Asako Kumagai, Kunihiko Itoh, Eiko Toyota, Kiyoshi Horita,
Yukari Noguchi and Kazutaka Tanizawa
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Received 6May 2003; accepted 28 July 2003
Abstract—The acyl trypsin was prepared by use of an inverse substrate, which is comprise of a photoresponsive 4-phenylazobenzoyl
moiety. The acyl group in acyl trypsin has been shown to isomerize from trans-form (4t-trypsin) to cis-form (4c-trypsin)/from cis-
form to trans-form by irradiation of UV–vis light. The deacylation rate of the cis-form (4c-trypsin) has been shown to be 18.6times
faster than that of the trans-form (4t-trypsin).
# 2003 Elsevier Ltd. All rights reserved.
Development of useful methods for photoregulation of
the activity of naturally occurring proteins and enzymes
has been major target in the research field of recent
protein engineering.1,2 From the standpoint of clinical
application, regulation of enzyme activity will be a pro-
mising technique. For example, photoregulation of
trypsin-like enzymes such as thrombin and plasmin is
connected to the development of coagulants and anti-
coagulants.3,4
Previously, we reported that inverse substrates such as
p-amidino- and p-guanidinophenyl esters behave as
specific substrates for trypsin and trypsin-like enzymes
and allow the specific introduction of an acyl group
carrying a non-specific residue into the enzyme active
site.10,12 The esters provided a novel method for the
specific introduction of the non-specific structure into a
trypsin active site. In this respect, inverse substrates are
expected to be a useful tool as the carrier of a photo-
responsive molecule into trypsin and trypsin-like
enzymes. Herein, we report the synthesis and properties
of a photoresponsive inverse substrate.
The methods of modification of enzymes by covalent
attachment of photoresponsive components are classi-
fied into three categories. One of them is the specific
modification of the enzyme active site by the use of
photoisomerizable inhibitors. Pioneering work by
Erlanger is known to be classified in this category, but it
is very difficult to design such residues that are photo-
isomerizable and are involved in covalent modification
at the same time.5 The second one is the direct random
introduction of photoresponsive components into the
enzyme molecule. In this case, the multi-site modifi-
cation of enzynes will occur. Thus, the products are less
informative on the structure–function relationship of
the emzyme.6 The third method is the site-specific
incorporation of photoresponsive components into an
enzyme.7ꢀ9
We designed 40-phenylazobenzoic acid 4-guanidinophenyl
ester methanesulfonate (4) as an inverse substrate. Syn-
thetic procedures are shown in Scheme 1.
Condensation of 4-phenylazobenzoic acid (1) with 4-
[N0,N00bis(Z)guanidino]phenol (2)13 by using benzotri-
azol-1-yloxy-tris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) reagent in DMF afforded 40-
phenylazobenzoic acid 4-[N0,N00bis(Z)guanidino]phenyl
ester (3) in 84% yield. Deprotection was carried out by
methansulfonic acid to give 40-phenylazobenzoic acid
4-guanidinophenyl ester (trans-Azo-OGu) (4t) in 73%
yield. The structure was determined by IR, UV, 1H NMR
spectra and elemental analysis.14
Compound (4t) exhibits reversible photoisomerization.
Upon irradiation of UV light at around 365 nm, the trans-
form (4t) isomerizes to the cis-form (4c). Irradiation of 4c
*Corresponding author. Tel.: +81-1332-3-3791; fax: +81-1332-3-
1211; e-mail: sekizaki@hoku-iryo-u.ac.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.013

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H. Sekizaki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3809–3812
Scheme 1. Synthesis of inverse substrate.
at longer wavelength than 400 nm results in the reverse
isomerization. This isomerization rate is considerably
slow. Figure 1 shows the spectral changes associated
with the photoisomerization of 5 as a model of acyl
trypsin from inverse substrate 4. Yields of the cis-form
(5c) from the trans-form (5t) by irradiation at around
365 nm were determined by the absorption spectra. The
data are in good agreement with the yield calculated on
placement method,11,15 and the values are listed in
Table 1. Compound 4t was shown to have strong binding
affinity and to promote efficient acylation. The values
for k2/Ks (specificity index) are nearly equal within 4t
and 4c. The difference between 4t and 4c is emphasized
at their deacylation steps. These data suggest that both
isomers are a specific substrate of trypsin to give acyl
trypsin, and the acyl trypsin from 4t is more stable.
1
the basis of the peak intensity of HPLC and H NMR
spectra, respectively.
The kinetic parameters for 4t were directly determined
from the rate assay and those for 4c were calculated
from the apparent data of the mixture of 4t and 4c.
Figure 2 exemplifies the apparent enhancement of deacyl-
ation rates constant which were proportional to the
ratio of the cis-form (4c-trypsin). The other parameters
of 4c were also calculated from the apparent data
resulted from the cis–trans mixture.
The kinetic constants for the trypsin-catalyzed hydro-
lysis were analyzed on the basis of the following scheme.
In this scheme, the following symbols are used: E,
enzyme; S, substrate; ES, enzyme–substrate complex; EA,
acyl enzyme; P1, alcohol component of the substrate; P2,
acid component of the substrate; Ks, dissociation
constant of enzyme–substrate complex; k2, rate constant
of acylation step; k3, rate constant of deacylation step.
Analysis of kinetic parameters for trypsin-catalyzed
hydrolysis of 4t was carried out by the thionine dis-
Acyl trypsin was successfully obtained according to our
previous paper as follows.16 Trypsin was incubated with a
Table 1. Kinetic parameters for trypsin-catalyzed hydrolysis of
inverse substrates
Substrate
Ks
(M)
k2
(sꢀ1
k3
(sꢀ1
k2/Ks
(Mꢀ1 sꢀ1
)
)
)
trans-form (4t)
cis-form (4c)
1.06ꢁ10ꢀ5
4.73ꢁ10ꢀ5
1.063.78
7.75
ꢁ10ꢀ4
1.51ꢁ105
1.64ꢁ105
7.02ꢁ10ꢀ3
Figure 1. Photochromic attribute of acyl enzyme model containing
azobenzene group 5 at 4ꢁ10ꢀ5 M in CHCl3: (a) 100% 5t (no irradia-
tion of 5t, and or irradiation of 5c at longer wavelength than 400 nm
for 10 h); (b) 20% 5c (irradiation of 5t at around 365 nm for 30 s); (c)
50% 5c (irradiation of 5t: 60 s); (d) 90% 5c (irradiation of 5t: 120 s);
(e) 100% 5c (irradiation of 5t: 240 s).
Figure 2. Apparent deacylation rate constants.

H. Sekizaki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3809–3812
3811
Scheme 2. Photoisomerization.
15–20 molar excess of trans-Azo-OGu (4t), or a mixture of
trans-4t (45%) and cis-Azo-OGu (4c) (55%) for 2 min
in 50 mM Tris–HCl buffer (containing 25 mM CaCl2,
pH 8.0) at 25 ꢂC. After the pH was adjusted to 3.0 by
addition of 1 M HCl, the reaction mixture was gel-filtered
and lyophilized. Both resulted preparations were com-
pletely inactive as a result of acylation at the active site,
while the enzymatic activity was completely recovered
after incubation for 2.5 h at pH 8.0 as a result of deacyl-
ation (Scheme 2).
the cis-form (4c-trypsin). Irradiation of 4c-trypsin at longer
wavelength than 400 nm resulted in formation of 4t-
trypsin. Figure 3 illustrates the spectral changes associated
with the photoisomerization of 4-trypsin. This behavior
was almost the same as that of 5 shown in Figure 1.
As shown in Table 1, it is evident that the deacylation
rate of the photoisomerized cis-form (4c-trypsin) is 18.6
times faster than that of the trans-form (4t-trypsin),
although Ks and k2 values of 4c are 4.46- and 7.31-fold
larger than those of 4t. The difference in deacylation
rates of 4t and 4c enables photoregulation of the
enzyme activity by an external light source. Figure 4
Acyl enzyme (4t-trypsin) obtained using 4t exhibited rever-
sible photoisomerizable properties. Irradiation of the trans-
form (4t-trypsin) at around 365 nm resulted in formation of
Figure 3. Photochromic attribute of acyl enzyme (4-trypsin) at
5.6ꢁ10ꢀ5 M: (a) 100% 4t-trypsin (no irradiation of 4t-trypsin); (b)
10% 4c-trypsin (irradiation of 4t-trypsin at around 365 nm for 30 s);
(c) 30% 4c-trypsin (irradiation of 4t-trypsin: 60 s); (d) 55% 4c-trypsin
(irradiation of 4t-trypsin: 120 s); (e) 65% 4c-trypsin (irradiation of 4t-
trypsin: 120 s). Absorption spectra were obtained in a medium com-
posed of I mM HCl.
Figure 4. Photoresponsive on–off experiment using acyl trypsin (4t-
trypsin) (5.6ꢁ10ꢀ5 M in 1 mM HCl). Alternate irradiation at around
365 nm (l1) and at longer wavelength than 400 nm (l2). The value in
parentheses is irradiation time (s).

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H. Sekizaki et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3809–3812
exemplifies the interconversion of 4t-trypsin to 4c-tryp-
sin by the sequential on–off and off–on switching of 4t-
trypsin.17
5. Kaufman, H.; Vratsanos, S. M.; Erlanger, B. F. Science
1968, 162, 1487.
6. Willner, I.; Rubin, S.; Riklin, A. J. Am. Chem. Soc. 1991,
113, 3321.
7. Hamachi, I.; Hiraoka, T.; Yamada, Y.; Shinkai, S. Chem.
Lett. 1998, 537.
In conclusion, chemical modification of the active site of
the trypsin by use of an inverse substrate carrying azo-
benzene photoresponsive groups have been applied to
photoregulation of the enzyme activity.
8. Willner, I.; Blonder, R.; Katz, E.; Stocker, A.; Buckmann,
A. F. J. Am. Chem. Soc. 1996, 118, 5310.
9. Blonder, R.; Katz, E.; Willner, I.; Wray, V.; Buckmann,
A. F. J. Am. Chem. Soc. 1997, 119, 11747.
10. Tanizawa, K.; Kasaba, Y.; Kanaoka, Y. J. Am. Chem.
Soc. 1977, 99, 4485.
Thus, inverse substrates will provide a useful means to
regulate the activity of trypsin and trypsin-like enzymes.
11. Nozawa, M.; Tanizawa, K.; Kanaoka, Y. J. Pharmacobio-
Dyn. 1980, 3, 213.
12. Itoh, K.; Sekizaki, H.; Toyota, E.; Tanizawa, K. Chem.
Pharm. Bull. 1995, 43, 2082.
Acknowledgements
13. Sekizaki, H.; Itoh, K.; Toyota, E.; Tanizawa, K. Chem.
Pharm. Bull. 1996, 44, 1577.
This work was supported in part by a Grant-in-Aid for
High Technology Research Program from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan, and by a grant from the Japan Private School
Promotion Foundation.
14. Selected data for trans-Azo-OGu (4t): mp 237–240 ꢂC
(recrystallization from MeOH/Et2O); IR (KBr) g 1728 cmꢀ1
;
UV l (e) 328 (27634), 448 (933) nm; 1H NMR (400MHz,
(CD3)2SO) d 2.34 (s, 3H), 7.37 (d, J=9.3 Hz, 2H), 7.42 (d, J=8.7
Hz, 2H), 7.44 (bs, 4H), 7.65–7.75 (m, 3H), 7.96–8.00 (m, 2H),
8.08 (d, J=8.5 Hz, 2H), 8.35 (d, J=8.5 Hz, 2H), 9.66 (s, 1H);
elemental analysis (%) calcd for (C21H21N5O5S): C 55.37, H
4.65, N 15.38, S 7.04; found: C 54.95, H 4.69, N 14.97, S 7.08.
15. Brandt, K. G.; Himoe, A.; Hess, G. P. J. Biol. Chem.
1967, 242, 3973.
References and Notes
1. Willner, I. Acc. Chem. Res. 1997, 30, 347.
2. Willner, I.; Katz, E. Angew. Chem., Int. Ed. Engl. 2000, 39,
1180.
3. Smith, R. A. G.; Dupe, R. J.; English, P. D.; Green, J.
Nature (London) 1981, 290, 505.
4. Ikram, S.; Lewis, S.; Bucknall, C.; Sram, I.; Thomas, N.;
Vincent, R.; Chamberlain, D. Br. Med. J. (Clin. Res. Ed.)
1986, 293, 786.
16. Tanizawa, K.; Nakano, M.; Kanaoka, Y. Bioorg. Chem.
1987, 15, 50.
17. Photoisomerization of cis-form (4c-trypsin) to trans-form
(4t-trypsin) needs a very long time. In irradiation at longer
wavelength than 400 nm, change of absorption was not
observed in 30 s.