Novel photoreactive cinnamic acid analogues to elucidate phenylalanine ammonia-lyase

Article abstract of DOI:10.1016/S0960-894X(00)00499-6

4-[3-(Trifluoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine from cinna

Full text of DOI:10.1016/S0960-894X(00)00499-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2481±2483
Novel Photoreactive Cinnamic Acid Analogues to Elucidate
Phenylalanine Ammonia-lyase
Makoto Hashimoto,a,* Yasumaru Hatanakab and Kensuke Nabetaa
aDepartment of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro 080-8555, Hokkaido, Japan
bInstitute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2360 Sugitani, Toyama, 930-0194 Japan
Received 10 July 2000; accepted 25 August 2000
AbstractÐ4-[3-(Tri¯uoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine
ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine
from cinnamic acid. Speci®c photolabeling of the enzyme was detected using biotinylated derivatives without the use of radio-
isotopes. The results indicated that the 4-[3-(tri¯uoromethyl) diazirinyl] skeleton will be a suitable photoreactive compound to
elucidate regulation of phenylpropanoid biosynthesis. # 2000 Elsevier Science Ltd. All rights reserved.
Phenylalanine ammonia-lyase (PAL) is an important
enzyme that converts l-phenylalanine into trans-cin-
namic acid, which in turn is the precursor of various
phenylpropanoids in plants.1,2 The reverse reaction, the
PAL-catalyzed enantioselective addition of ammonia to
trans-cinnamic acid, can be used for the synthesis of l-
phenylalanine.3,4 PAL is potentially a key regulatory
enzyme, because it catalyzes the initial common step in
the biosynthesis of phenylpropanoids. PAL activity is
absent in mammalian cells. Therefore the introduction
of PAL into mammalian cells to overcome phenyl-
ketoneurea, could be of great bene®t. It could alleviate
associated hyperphenylalaninemia and mental retarda-
tion, by reducing the l-phenylalanine level in urine.5,6
There have been few papers that have reported on the
coordination of the enzyme±substrate complex at the
PAL active site. Photoanity labeling is one of the most
useful methods to obtain information about binding
sites. This is because labeling depends on the formation
of the enzyme±ligand complex and therefore upon the
location of residues near the active center. Photoanity
labeling with various chemical precursors, aryldiazirine,
arylazide and benzophenone, has been used for the deter-
mination of ligand±protein interactions.14 18 Compara-
tive studies of these three photophors suggested that 3-
(tri¯uoromethyl) aryldiazirine is the most promising.19 21
But the complicated synthesis of the diazirinyl three
membered ring has resulted in fewer applications of it in
biomolecular studies than for other photophors.
On the basis of chemical modi®cation experiments on
PAL over three decades ago, it was proposed that a
dehydroalanine residue could play an important role as
an electrophilic group that was required for activity.7,8
Recently, we found a versatile approach for the post-
functionalization of the phenyldiazirine photophor
using the Friedel±Crafts reaction as a key step.15 This
enabled us to introduce 3-phenyl-3-tri¯uoromethyldiazi-
rine photophor as a part of the phenylpropanoid skele-
ton via carbon±carbon linkage. In this paper, we
describe the synthesis of such novel diazirinyl cinnamic
acid derivatives. We have also evaluated their properties
in reactions with PAL from Rhodotorulia glutinis.
Recently
a serine residue, a precursor of dehy-
droalanine, has been identi®ed by mutational studies as
being important for activity.9 A similar reaction
mechanism, in which dehydroalanine could play a cru-
cial role for activity, has been suggested for the homo-
logous enzyme histidine ammonia-lyase.10 Isotopic
studies of these two ammonia-lyases revealed that their
rate-limiting step slightly di€ered.11 13 This could
indicate that the environment of the active site is not
identical in these two enzymes.
Three ortho-substituted photoreactive benzaldehyde
derivatives 1±322 were treated with triphenylcarbo-
ethoxymethylenephosphorane to generate a mixture
(7:3) of (E)- and (Z)-ethyl cinnamates23 4±6 with yields
of 80, 88 and 75%, respectively. The hydrolysis of these
ethyl cinnamates with methanolic sodium hydroxide
*Corresponding author. Tel.: +81-155-49-5543; fax: +81-155-49-
5577; e-mail: hasimoto@obihiro.ac.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00499-6

2482
M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2481±2483
generated a complicated mixture. By replacing the sol-
vent with ethanol, cinnamic acid derivatives 7±924 were
obtained in good yields (ꢀ90%). The diazirine photo-
phor was stable under these reaction conditions. Thus
we have developed a very simple and convenient route
for systematic synthesis of photoreactive phenylpropa-
noid analogues and their biotinyl derivatives starting
from easily available aldehydes 1±3 (Fig. 1).
Previous systematic studies indicated that alkoxy sub-
stitution at the ortho position and alkyl substitution at
the para position of cinnamic acid caused less inhibition
of PAL from Rhodotorulia glutinis.26 Furthermore,
ortho-hydroxy cinnamic acid was one of the strongest
inhibitors. But our hydroxy compound 7 contains 3-
(tri¯uoromethyl)diazirinyl group at the 4-position of
cinnamic acid. It seems that 3-(tri¯uoromethyl)diaz-
irinyl group on a benzene ring alters substrate speci®city
for PAL. It is reported that para-nitro substituted l-
phenylalanine is a good substrate for PAL from parsley.27
The 3-(tri¯uoromethyl)diazirinyl group should have simi-
lar characteristics as a nitro-group.
The biological properties of cinnamic acid derivatives 7±
9 were investigated as substrates for the reverse reaction
of PAL. The condition for l-phenylalanine forma-
tion3,25 did not generate l-phenylalanine analogues
from 7±9. But compounds 8 and 9 inhibited l-phenyl-
alanine formation in the presence of trans-cinnamic
acid, whereas compound 7 did not. This inhibition was
concentration dependent. The phenylalanine determi-
nation was performed by the method of Renard et al.25
Dixon plots revealed that 8 and 9 are competitive inhi-
bitors for PAL and Ki values were determined as
0.24Æ0.05 and 0.52Æ0.03 mM, respectively (Fig. 2).
Compound 7 is a precursor for radiolabeled 8 by
methylation, as has been established.28 The combination
of photoanity labeling and avidin-biotin technology
(photoanity biotinylation) would also be a powerful
tool to investigate the interaction of biomolecules and
substrates. We have reported that photoanity biotin-
ylation is a useful non-radioisotopic method to detect
and retrieve the labeled components from a complicated
mixture.29 31 PAL (85 pmol) and the same amount of
compound 9 were incubated at room temperature for 10
min in 9 M NH4OH (pH 10.5) or 0.1 M Tris (pH 8.5)
for phenylalanine formation or degradation, respec-
tively. As indicated in the legend to Figure 3, trans-cin-
namic acid and l-phenylalanine were included for
competitive inhibition. All mixtures were irradiated with
black light (15 W) for 10 min at 0 ꢁC. The samples were
directly subjected to electrophoresis by SDS-PAGE,
and the protein bands were transferred onto PVDF
[poly(vinylidene di¯uoride)] membrane. The blotted
membranes were treated as previously described for the
detection of biotinylated protein.29 Chemiluminescence
was detected at 78 kDa, corresponding with the PAL
subunit,32 in both the synthesis and degradation reac-
tions (Fig. 3, lane 1 and 3).
Figure 1. Synthesis of 4-[3-(tri¯uoromethyl)diazirinyl] cinnamic acid
derivatives. (i) Ph3PˆCHCOOC2H5, benzene, room temperature, 2 h;
(ii) 1 N NaOH, ethanol, room temperature, overnight.
Figure 3. Photoanity labeling of PAL with compound 9 in phenyl-
alanine formation (Tris pH 8.3, lane 1 and 2) and degradation
(ammonium hydroxide pH 10.5, lane 3 and 4). Details of labeling
conditions are described in the text. l-Phenylalanine and trans-cin-
namic acid (0.1 M) were included as competitive inhibitors (lane 2 and
4, respectively). Each sample (1 mg) was subjected to SDS-PAGE (10%
acrylamide gel) and transferred to PVDF membrane. The membrane
was blocked for 1 h in 5% skim milk in 0.1% Tween 20, phosphate-
bu€ered saline pH 7.4 (T-PBS), washed twice with T-PBS for 5 min
and immersed in streptavidin±peroxidase conjugate (1:1500 dilution of
the manufacturer's solution) in T-PBS for 1 h. After washing six times
with T-PBS for 5 min, the membrane was immersed in chemilumines-
cence detection reagents for 1 min and exposed to ®lm for 0.5 min.
Figure 2. Inhibition by compound 9 for phenylalanine formation at
di€erent concentrations of cinnamic acid. ~ 3 mM, * 2 mM, & 1
mM. The assay mixture contained various concentrations of 9, trans-
cinnamic acid and enzyme in 9 M NH4OH pH 10.5. It was incubated
at 30 ꢁC for 30 min. The values were determined in triplicate at each
concentration.

M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2481±2483
2483
This labeling was inhibited by the presence of phenyl-
alanine (lane 2) and cinnamic acid (lane 4). The results
indicate that the photoreactive cinnamic acid derivatives
competed with natural substrates, suggesting that 4-[3-
(tri¯uoromethyl)diazirinyl] aryl diazirine would ®t to
the substrate binding site of PAL.
13. Klee, C. B.; Kirk, K. L.; McPhie, P.; Cohen, L. A. J. Biol.
Chem. 1975, 250, 5033.
14. Bayley, H. In Laboratory Techniques in Biochemistry and
Molecular Biology: Photogenerated Reagents in Biochemistry
and Molecular Biology; Work, T. S., Burdon, R. H., Eds.;
Elsevier: Amsterdam, 1983; pp 25±44.
15. Hatanaka, Y.; Nakayama, H.; Kanaoka, Y. Rev. Hetero-
atom Chem. 1996, 14, 213.
This paper describes for the ®rst time that the 4-[3-(tri-
¯uoromethyl)]diazirinyl substituted cinnamic acid deriva-
tives works as a competitive inhibitor and photoreactive
reagent for PAL. These results should promote the use
of 4-[3-(tri¯uoromethyl)] aryldiazirinyl based photo-
anity labeling in studies of phenylpropanoid bio-
synthesis.
16. Brunner, J. Annu. Rev. Biochem. 1993, 62, 483.
17. Dorm, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18,
64.
18. Kotzyba-Hilbert, F.; Kapfer, I.; Goeldner, M. Angew.
Chem., Int. Ed. Engl. 1995, 34, 1296.
19. Koumanov, F.; Yang, J.; Jones, A. E.; Hatanaka, Y.;
Holman, G. D. Biochem. J. 1998, 330, 1209.
20. Holman, G. D.; Parkar, B. A.; Midgley, P. J. W. Biochim.
Biophys. Acta. 1986, 855, 115.
21. Holman, G. D.; Karim, A. R.; Karim, B. Biochim. Bio-
phys. Acta. 1988, 946, 75.
Acknowledgements
22. Hashimoto, M.; Kanaoka, Y.; Hatanaka, Y. Heterocycles
1997, 46, 119.
23. Uwe, K.; Kanaoka, Y.; Hatanaka, Y. Heterocycles 1998,
49, 465.
This research was partially supported by the Ministry of
Education, Science, Sports and Culture, Grant-in-Aid
for Encouragement of Young Scientists, 12780433
(M.H.) and for Scienti®c Research 12470504 (Y.H.).
24. Compound
7 HR-FABMS calcd for C11H8F3N2O3
([M+H]+) 273.0487, found 273.0495; 1H NMR (CDCl3) d
7.95 (1H, d, J=16.2 Hz), 7.63 (1H, d, J=8.2 Hz), 6.76 (1H, s),
6.68 (1H, d, J=8.2 Hz), 6.65 (1H, d, J=16.2 Hz). Compound
8 HR-FABMS calcd for C12H10F3N2O3 ([M+H]+) 287.0644,
found 287.0665; 1H NMR (CDCl3) d 8.01 (1H, d, J=16.2 Hz),
7.53 (1H, d, J=8.2 Hz), 6.81 (1H, d, J=8.2 Hz), 6.62 (1H, s),
6.55 (1H, d, J=16.2 Hz), 3.84 (3H, s). Compound 9 HR-
FABMS calcd for C27H35F3N5O7S ([M+H]+) 630.2209,
found 630.2222; 1H NMR (CDCl3) d 7.94 (1H, d, J=16.2 Hz),
7.50 (1H, d, J=8.9 Hz), 6.81 (1H, d, J=8.9 Hz), 6.62 (1H, s),
6.63 (1H, d, J=16.2 Hz), 4.53 (1H, m), 4.36 (1H, m), 4.19 (2H,
m), 3.99 (2H, m), 3.75 (2H, m), 3.64 (2H, m), 3.57 (2H, m),
3.44 (2H, m), 3.16 (1H, m), 2.92 (1H, m), 2.75 (1H, m), 2.23
(2H, m), 1.70 (4H, m), 1.45 (2H, m).
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