Radiolytic one-electron reduction characteristics of tyrosine derivative caged by 2-oxopropyl group

Article abstract of DOI:10.1016/j.bmcl.2008.10.018

We employed X-irradiation to activate a caged amino acid with a 2-oxoalkyl group. We designed and synthesized tyrosine derivative caged by a 2-oxoalkyl group (Tyr(Oxo)) to evaluate its radiolytic one-electron reduction characteristics in aqueous solution. Upon hypoxic X-irradiation, Tyr(Oxo) released a 2-oxopropyl group to form the corresponding uncaged tyrosine. In addition, radiolysis of dipeptides containing Tyr(Oxo) revealed that the efficiency of radiolytic removal of 2-oxopropyl group increased significantly by the presence of neighboring aromatic amino acids.

Full text of DOI:10.1016/j.bmcl.2008.10.018

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6126–6129
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radiolytic one-electron reduction characteristics of tyrosine derivative
caged by 2-oxopropyl group
*
*
Kazuhito Tanabe , Masahiko Ebihara, Nao Hirata, Sei-ichi Nishimoto
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Kyoto 615-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We employed X-irradiation to activate a caged amino acid with a 2-oxoalkyl group. We designed and syn-
thesized tyrosine derivative caged by a 2-oxoalkyl group (Tyr(Oxo)) to evaluate its radiolytic one-electron
reduction characteristics in aqueous solution. Upon hypoxic X-irradiation, Tyr(Oxo) released a 2-oxopro-
pyl group to form the corresponding uncaged tyrosine. In addition, radiolysis of dipeptides containing
Tyr(Oxo) revealed that the efficiency of radiolytic removal of 2-oxopropyl group increased significantly
by the presence of neighboring aromatic amino acids.
Received 18 July 2008
Revised 2 October 2008
Accepted 3 October 2008
Available online 7 October 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Caged tyrosine
2-oxopropyl group
X-irradiation
Hypoxic conditions
Caged amino acids and proteins, the functions of which can be
regulated by external triggers, are applied widely to biological re-
search.1–5 The activities of these molecules can be blocked by
chemical modification, while their intrinsic activities are recovered
by external stimulation such as photoirradiation6 and enzymatic
treatment.4 In view of a feature that their functions are easily reg-
ulated both temporally and spatially, these caged amino acids are
useful for studying protein chemistry in biological systems.
Recently we have proposed caged drugs (prodrugs) that are
activated by hypoxic X-irradiation.7–11 Our studies illustrated that
a 2-oxoalkyl group has an effective functionality for caging antitu-
mor drugs such as 5-fluorouracil (5-FU)8,9 and 5-fluorodeoxyuri-
dine (5-FdUrd),10 and for radiolytic activation of the resultant
caged drugs. This class of radiation activated caged drugs release
the 2-oxoalkyl group by reducing hydrated electrons, which are
generated as the major active species along with hydrogen atoms
and hydroxyl radicals upon radiolysis of an aqueous solution. An
activation mechanism has been proposed by which the 2-oxoalkyl
group undergoes one-electron reduction by hydrated electrons to
form corresponding p* anion radical, followed by thermal activa-
tion into the r* anion radical that has a weakened N–C bond be-
tween the 5-FU unit and the 2-oxoalkyl group and is readily
hydrolyzed to release the 2-oxoalkyl group. In this study, we de-
signed and synthesized an amino acid caged by a 2-oxopropyl
group on tyrosine (Tyr), which has a high affinity for hydrated elec-
trons.12 Under hypoxic conditions, radiolytic one-electron reduc-
tion of tyrosine derivative possessing 2-oxopropyl group
(Tyr(Oxo)) resulted exclusively in releasing of 2-oxopropyl group
to recover the corresponding uncaged Tyr. In the case of dipeptides
bearing Tyr(Oxo), the neighboring amino acid was identified to
have a strong influence on the overall efficiency of radiolytic
one-electron reductive release of the 2-oxopropyl group from
Tyr(Oxo).
The synthetic procedure of Tyr(Oxo) is outlined in Scheme 1.
Coupling of N-(tert-butoxycarbonyl)tyrosine methyl ester 1 with
a
-bromoacetone and deprotection of the terminal amino group
gave Tyr(Oxo) (3).13
We initially performed one-electron reduction of Tyr(Oxo) by
the X-radiolysis of an argon-purged aqueous solution containing
excess 2-methyl-2-propanol as the scavenger of oxidizing hydroxyl
radicals.14 Reducing hydrate electrons (eaqÀ) are generated as the
major active species under these radiolysis conditions, in which
the yield of reducing hydrogen atoms is much less than those of
hydrated electrons and oxidizing hydroxyl radicals.15 Figure 1
shows the reaction profiles of the radiolytic reduction of Tyr(Oxo)
under hypoxic conditions. The appearance of a single new peak in
Figure 1 is attributable to the formation of uncaged Tyr as con-
firmed by the overlap injection of authentic samples in the HPLC
analysis. The G values (the number of molecules produced or chan-
ged per 1 J of radiation energy absorbed by the reaction system)
were 223 nmol/J for the decomposition of caged Tyr(Oxo) and
130 nmol/J for the formation of the corresponding uncaged Tyr.16
These results clearly indicate that the 2-oxopropyl group on the
amino acid can be similarly removed as in the case of prodrugs.10
To assess the effect of molecular oxygen on the radiolysis of
Tyr(Oxo), we performed similar one-electron reduction under
* Corresponding authors. Tel.: +81 75 383 2500; fax: +81 75 383 2501.
E-mail addresses: tanabeka@scl.kyoto-u.ac.jp (K. Tanabe), nishimot@scl.kyoto-u.
ac.jp (S. Nishimoto).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.018

K. Tanabe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6126–6129
6127
Scheme 1. Reagents and conditions: (a) K2CO3, KI, bromoacetone, acetone, reflux, quant.; (b) HCl in Et2O, rt, 78%.
Figure 1. HPLC profiles for the one-electron reduction of Tyr(Oxo) (3) (95
lM) upon hypoxic X-radiolysis (0, 150, 400, and 700 Gy) of aqueous solution containing 10 mM 2-
methyl-2-propanol.
aerobic conditions. In contrast to the hypoxic X-radiolysis, the re-
moval of the 2-oxopropyl group to convert Tyr(Oxo) into Tyr be-
came considerably less efficient in the aerobic X-radiolysis
(Fig. 2). The G values were estimated as 81 nmol/J for the decom-
position of Tyr(Oxo) and 51 nmol/J for the formation of Tyr. In view
of well-documented evidence that molecular oxygenÀefficiently
inhibits radiolytic reduction due to the trapping of eaq to form a
superoxide anion radical,17 the one-electron reduction of Tyr(Oxo)
À
by eaq is likely to occur in a hypoxia-selective manner.
To identify the effect of the neighboring amino acid on the
radiolytic reduction of Tyr(Oxo), we compared one-electron reduc-
tion reactivity of dipeptides 7–13 comprising Tyr(Oxo) and seven
types of natural amino acids upon hypoxic or aerobic X-irradiation.
Dipeptides 7–13 were prepared from 3 (Ac-Tyr-OMe) as outlined
in Scheme 2. The phenol group of 4 was alkylated by a-bromoace-
tone, and subsequent hydrolysis gave acid 6. Coupling of 6 with the
corresponding amino acids furnished the synthesis of dipeptides
7–13.18 Figure 3 shows representative reaction profiles of the
radiolytic reduction of dipeptide Tyr(Oxo)-Gly under hypoxic con-
ditions. Similar to the radiolysis of monomeric Tyr(Oxo), reductive
Figure 2. Decomposition of Tyr(Oxo) (3) (open symbol) and release of Tyr (filled
symbol) in the hypoxic (circle) or aerobic (triangle) radiolysis of aqueous solution
containing 10 mM 2-methyl-2-propanol. Each error bar represents the SE calcu-
lated from three experimental results.
Scheme 2. Reagents and conditions: (a) K2CO3, KI, bromoacetone, acetone, reflux, 77%; (b) LiOH, MeOH–H2O, rt, 76%; (c) aminoacid methyl ester hydrochloride, HBTU, DIEA,
THF, rt, 7–34% (for 7–12); (d) 1-methylhistidine methyl ester hydrochloride, EDCI, triethylamine, DMF, rt, 28% (for 13).

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K. Tanabe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6126–6129
Figure 3. HPLC profiles for the one-electron reduction of Tyr(Oxo)-Gly (7) (100
lM) upon hypoxic X-radiolysis (0, 100, 300, and 500 Gy) of aqueous solution containing
10 mM 2-methyl-2-propanol.
References and notes
Table 1
G values (nmol/J) for the decomposition of Tyr(Oxo) (3) and dipeptides bearing
Tyr(Oxo) and the formation of corresponding uncaged Tyr and dipeptides upon X-
radiolysisa
1. Kaplan, J. H.; Forbush, B.; Hoffman, J. F., III Biochemistry 1978, 17, 1929.
2. Rock, R. S.; Chan, S. I. J. Org. Chem. 1996, 61, 1526.
3. Zou, K.; Cheley, S.; Givens, R. S.; Bayley, H. J. Am. Chem. Soc. 2002, 124, 8220.
4. Santos, S. D.; Chandravarkar, A.; Mandal, B.; Mimna, R.; Murat, K.; Saucède, L.;
Tella, P.; Tuchscherer, G.; Mutter, M. J. Am. Chem. Soc. 2005, 127, 11888.
5. Kuner, T.; Li, Y.; Gee, K. R.; Bonewald, L. F.; Augustine, G. J. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 347.
6. Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900.
7. Shibamoto, Y.; Zhou, L.; Hatta, H.; Mori, M.; Nishimoto, S. Jpn. J. Cancer Res.
2000, 91, 433.
8. Mori, M.; Hatta, H.; Nishimoto, S. J. Org. Chem. 2000, 65, 4641.
9. Shibamoto, Y.; Zhou, L.; Hatta, H.; Mori, M.; Nishimoto, S. Int. J. Radiat. Oncol.
Biol. Phys. 2001, 49, 407.
10. Tanabe, K.; Mimasu, Y.; Eto, Y.; Tachi, Y.; Sakakibara, S.; Mori, M.; Hatta, H.;
Nishimoto, S. Bioorg. Med. Chem. 2003, 11, 4551.
Hypoxic conditions
Aerobic conditions
Formation Decomposition Formation Decomposition
Tyr(Oxo) (3)
130
55
96
223
212
232
209
299
258
295
282
51
11
20
18
30
37
24
42
81
52
72
83
45
52
55
96
Tyr(Oxo)-Gly (7)
Tyr(Oxo)-Ala (8)
Tyr(Oxo)-Val (9)
Tyr(Oxo)-Phe (10)
Tyr(Oxo)-Tyr (11)
Tyr(Oxo)-Trp (12)
Tyr(Oxo)-His(1-Me)
(13)
72
155
196
131
158
11. Mori, M.; Ito, T.; Teshima, S.; Hatta, H.; Fujita, S.; Nishimoto, S. J. Phys. Chem.
2006, 110, 12198.
12. Rate constants for the reactions of hydrated electrons in aqueous solution
a
Aqueous solution of Tyr(Oxo) (3) and dipeptides (95–320 lM) containing
excess amount of 2-methyl-2-propanol19 were irradiated at ambient temperature
with X-ray source (5 Gy minÀ1).
around pH
7
was estimated as 8.8 Â 106, 4.2 Â 106, 5.0 Â 106, 1.4 Â 108,
3.4 Â 108, 2.9 Â 108, 6.0 Â 107, and 1.8 Â 1010 L molÀ1 s–1 for Gly, Ala, Val, Phe,
Tyr, Trp, His, and O2, respectively, see: Buxton, G. V.; Greenstock, C. V.; Helman,
W. P.; Ross, A. B. J. Phys. Chem. Ref. Data 1988, 17, 513.
13. Tyr(Oxo) (3): Mp 159–160 °C; 1H NMR (DMSO-d6, 300 MHz) d 8.56 (s, 3H), 7.13
(d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 4.79 (s, 2H), 4.22 (t, J = 6.0 Hz, 1H),
3.68 (s, 3H), 3.07 (m, 2H), 2.15 (s, 3H); 13C NMR (DMSO-d6, 75.5 MHz) d 204.1,
169.3, 157.0, 130.5, 126.8, 114.5, 72.0, 53.3, 52.5, 34.9, 26.2; FAB-MS: m/e 252
[(M+H)+]; HRMS Calcd for C13H18NO4 [(M+H)+] 252.1236, found 252.1228.
14. General procedure for radiolytic reduction: To establish hypoxia, aqueous
removal of the 2-oxopropyl group occurred commonly for all dipep-
tides in a hypoxia-selective manner. The dipeptides 10–13, in which
Tyr(Oxo) is linked to aromatic amino acids, showed higher G values
than dipeptides 7–9 with a linkage between Tyr(Oxo) and aliphatic
amino acids, as listed in Table 1. Thus, the neighboring amino acid
has a marked effect on the one-electron reduction of Tyr(Oxo) to
generate uncaged Tyr. It has been reported that aromatic amino
acids are reduced by eaqÀ faster than aliphatic amino acids.12 These
reduction characteristics of eaqÀ could be responsible for the acceler-
ated reduction of dipeptides bearing aromatic amino acids. It is most
likely that capturing of eaqÀ by the aromatic amino acid residues in
the caged dipeptides occurred more efficiently followed by rapid
intramolecular electron transfer to Tyr(Oxo), thereby resulting in
the efficient formation of an uncaged dipeptide.
In summary, we have demonstrated the activation of caged Tyr
possessing a 2-oxoalkyl group by X-radiolytic one-electron reduc-
tion. Hypoxic X-irradiation caused the efficient removal of the 2-
oxopropyl group on Tyr(Oxo), whereas the reaction efficiency de-
creased dramatically upon aerobic irradiation. More remarkable
was that the neighboring aromatic amino acids increased the
radiolytic reduction efficiency of Tyr(Oxo), presumably due to their
higher electron affinity. These results strongly indicate that func-
tions of amino acids and proteins may be regulated by means of
radiolytic reduction of 2-oxoalkyl group. Further exploration of
the one-electron reduction of longer and higher-order peptides
bearing Tyr(Oxo) by X-radiolysis is in progress.
solutions of Tyr(Oxo) (3) (95 lM) containing 10 mM 2-methyl-2-propanol
(20% 2-methyl-2-propanol in the case of Tyr(Oxo)-Phe, Tyr(Oxo)-Tyr and
Tyr(Oxo)-Trp for their insolublities) were purged with argon for 15 min and
then irradiated in a sealed Pyrex sample tube at ambient temperature with X-
ray source (Rigaku RADIOFLEX-350, 5.0 Gy minÀ1). After the irradiation, the
solution was subjected to HPLC analysis.
15. Radiolysis of a diluted aqueous solution at around pH 7.0 produces primary
water radicals such as oxidizing hydroxyl radical (ÅOH), reducing hydrated
À
electrons (eaq
) G values of
and reducing hydrogen atoms (ÅH) with the
G(ÅOH) = 280 nmol/J, G(eaqÀ) = 280 nmol/J, and G(ÅH) = 60 nmol/J, respectively.
16. Reductive degradation of peptide mainchain may lead to a small G values for
the formation of uncaged Tyr. See: Garrison, W. M. Chem. Rev. 1987, 87, 381.
17. Similar effect of oxygen was observed for the activation of 5-FdUrd prodrugs.
See: Tanabe, K.; Makimura, Y.; Tachi, Y.; Imagawa-Sato, A.; Nishimoto, S.
Bioorg. Med. Chem. Lett. 2005, 15, 2321. and Ref. 10.
18. Tyr(Oxo)-Gly (7): 1H NMR (DMSO-d6, 300 MHz) d8.43 (t, J = 6.0 Hz, 1H), 8.08 (d,
J = 8.4 Hz, 1H), 7.14 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 4.75 (s, 2H), 4.47
(m, 1H), 3.85 (d, J = 6.1 Hz, 2H), 3.63 (s, 3H), 2.94 (m, 1H), 2.65 (m, 1H), 2.14 (s,
3H), 1.75 (s, 3H); 13C NMR (DMSO-d6, 75.5 MHz) d 204.3, 172.0, 170.2, 169.0,
156.2, 130.4, 130.0, 114.0, 72.1, 53.9, 51.7, 38.2, 36.7, 26.2, 22.5; FAB-MS: m/e
351 [(M+H)+]; HRMS Calcd for C17H23N2O6 [(M+H)+] 351.1556, found 351.1545.
Tyr(Oxo)-Ala (8): Mp 158–159 °C; 1H NMR (DMSO-d6, 300 MHz) d8.45 (d,
J = 7.1 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz,
2H), 4.75 (s, 2H), 4.47 (m, 1H), 4.27 (m, 1H), 3.61 (s, 3H), 2.92 (m, 1H), 2.63 (m,
1H), 2.14 (s, 3H), 1.73 (s, 3H), 1.29 (d, J = 7.3 Hz, 3H); 13C NMR (DMSO-d6,
75.5 MHz) d 204.3, 172.9, 171.5, 169.0, 156.2, 130.3, 130.1, 114.0, 72.1, 53.7,
51.8, 47.5, 36.9, 26.2, 22.4, 16.8; FAB-MS: m/e 365 [(M+H)+]; HRMS Calcd for
C18H25N2O6 [(M+H)+] 365.1713, found 365.1726.
Tyr(Oxo)-Val (9): Mp 142–143 °C; 1H NMR (DMSO-d6, 300 MHz) d 8.21 (d,
J = 8.0 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz,
2H), 4.75 (s, 2H), 4.56 (m, 1H), 4.17 (m, 1H), 3.62 (s, 3H), 2.89 (m, 1H), 2.64 (m,
1H), 2.14 (s, 3H), 2.04 (m, 1H), 1.74 (s, 3H), 0.88 (t, J = 6.9 Hz, 6H); 13C NMR
(DMSO-d6, 75.5 MHz) d204.3, 171.9, 171.8, 169.1, 156.2, 130.3, 130.1, 114.0,
72.1, 57.4, 53.7, 51.6, 36.6, 29.9, 26.2, 22.4, 18.9, 18.2; FAB-MS: m/e 393
[(M+H)+]; HRMS Calcd for C20H29N2O6 [(M+H)+] 393.2026, found 393.2027.
Tyr(Oxo)-Phe (10): Mp 107–108 °C; 1H NMR (DMSO-d6, 400 MHz) d 8.40 (d,
Acknowledgment
This research was partially supported by Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Science, Sports and
Culture, Japan to K.T.

K. Tanabe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6126–6129
6129
J = 7.6 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.24 (m, 5H), 7.12 (d, J = 8.8 Hz, 2H), 6.78
(d, J = 8.8 Hz, 2H), 4.73 (s, 2H), 4.47 (m, 2H), 3.58 (s, 3H), 2.95 (m, 3H), 2.61 (m,
1H), 2.14 (s, 3H), 1.72 (s, 3H); 13C NMR (DMSO-d6, 75.5 MHz) d 204.3, 171.7,
171.6, 168.9, 137.0, 130.2, 130.1, 129.0, 128.2, 128.1, 126.5, 114.0, 72.1, 53.6,
53.5, 51.8, 36.5, 26.2, 22.4; FAB-MS: m/e 441 [(M+H)+]; HRMS Calcd for
C24H29N2O6 [(M+H)+] 441.2026, found 441.2012.
J = 7.7 Hz, 1H), 7.17 to 6.94 (m, 5H), 6.77 (d, J = 8.6 Hz, 2H), 4.74 (s, 2H), 4.51
(m, 2H), 3.56 (s, 3H), 3.12 (m, 2H), 2.91 (m, 1H), 2.63 (m, 1H), 2.14 (s, 3H), 1.73
(s, 3H); 13C NMR (DMSO-d6, 75.5 MHz) d204.3, 172.1, 171.6, 169.0, 156.2,
136.0, 130.3, 130.1, 127.0, 123.7, 120.9, 118.4, 117.9, 113.9, 111.4, 109.2, 72.1,
53.7, 53.0, 51.8, 36.6, 26.9, 26.2, 22.4; FAB-MS: m/e 480 [(M+H)+]; HRMS Calcd
for C26H30N3O6 [(M+H)+] 480.2135, found 480.2126.
Tyr(Oxo)-Tyr (11): Mp 77–78 °C; 1H NMR (DMSO-d6, 300 MHz) d 9.21 (s, 1H),
8.32 (d, J = 7.5 Hz, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.98 (d,
J = 8.4 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 6.65 (d, J = 8.6 Hz, 2H), 4.74 (s, 2H), 4.42
(m, 2H), 3.57 (s, 3H), 2.89 to 2.27 (m, 4H), 2.14 (s, 3H), 1.72 (s, 3H); 13C NMR
(DMSO-d6, 75.5 MHz) d 204.3, 171.8, 171.5, 168.9, 156.2, 156.0, 130.3, 130.1,
130.0, 126.9, 115.0, 114.0, 72.1, 53.9, 53.6, 51.7, 36.6, 35.9, 26.2, 22.4; FAB-MS:
m/e 457 [(M+H)+]; HRMS Calcd for C24H29N2O7 [(M+H)+] 457.1975, found
457.1981.
Tyr(Oxo)-His(1-Me) (13): 1H NMR (DMSO-d6, 400 MHz) d 8.42 (d, J = 7.3 Hz,
1H), 8.07 (d, J = 7.3 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 8.3 Hz, 2H), 4.73
(s, 2H), 4.47 to 4.38 (2H), 3.58 (s, 3H), 3.56 (s, 3H), 2.96 to 2.53 (6H), 2.13 (s,
3H), 1.74 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) d 204.3, 171.8, 171.5, 169.0,
156.2, 137.3, 136.7, 130.1, 117.9, 114.0, 79.1, 72.1, 53.8, 52.3, 51.8, 36.4, 32.7,
29.7, 26.2, 22.4; FAB-MS: m/e 445 [(M+H)+]; HRMS Calcd for C22H29N4O6
[(M+H)+] 445.2087, found 445.2089.
19. We carried out one-electron reduction of 13 in aqueous solution containing
10% or 20% 2-methyl-2-propanol. We compared one-electron reactivity and
confirmed that both reactions showed similar profiles.
Tyr(Oxo)-Trp (12): Mp 80–81 °C; 1H NMR (DMSO-d6, 300 MHz) d 10.89 (s, 1H),
8.38 (d, J = 7.3 Hz, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.33 (d,