Convenient synthesis of photoaffinity probes and evaluation of their labeling abilities

Article abstract of DOI:10.1021/ol070376i

Convenient synthesis of a variety of photoaffinity probes was accomplished by utilizing our Ns strategy and novel resin. The synthetic probes were evaluated via the labeling ability with the preseniline 1 C-terminal fragments, which was identified as a therapeutic target for Alzheimer's disease.

Full text of DOI:10.1021/ol070376i

ORGANIC
LETTERS
2007
Vol. 9, No. 11
2055-2058
Convenient Synthesis of Photoaffinity
Probes and Evaluation of Their Labeling
Abilities
Toshiyuki Kan,*,† Yoichi Kita,‡ Yuichi Morohashi,‡ Yusuke Tominari,‡
Shinnosuke Hosoda,‡ Taisuke Tomita,‡ Hideaki Natsugari,‡
Takeshi Iwatsubo,‡ and Tohru Fukuyama*,‡
School of Pharmaceutical Sciences, UniVersity of Shizuoka, 52-1 Yada, Suruga-ku,
Shizuoka 422-8526, Japan, and Graduate School of Pharmaceutical Sciences,
UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan
fukuyama@mol.f.u-tokyo.ac.jp
Received February 20, 2007
ABSTRACT
Convenient synthesis of a variety of photoaffinity probes was accomplished by utilizing our Ns strategy and novel resin. The synthetic probes
were evaluated via the labeling ability with the preseniline 1 C-terminal fragments, which was identified as a therapeutic target for Alzheimer’s
disease.
Photoaffinity labeling has been demonstrated to be a remark-
ably efficient method for studying the interaction of biologi-
cally significant compounds (ligands) with their target
macromolecules.1 The method allows the identification of
the target and also the binding domain within the target
protein. An appropriate photoaffinity probe is usually
prepared by attachment of a photoreactive labeling group
and an indicator such as a biotin tag2 to the ligand molecule.
However, the incorporation of such a bulky group often
causes a significant decrease in the affinity with the target
molecule. Therefore, the search for a suitable probe for a
labeling group and/or a linker continues to be a compelling
need in photoaffinity labeling studies. Thus, a convenient
synthesis of diverse probes and screening of their labeling
abilities would be an attractive prospect, especially consider-
ing that the three-dimensional space of the binding site of
an enzyme or a receptor protein is generally not known.
Recently, we investigated the screening of a potent
inhibitor and the functional analysis of γ-secretase,3 which
was identified as an important therapeutic target for Alzhe-
imer’s disease (AD).4 During the course of our investigation
based on potent inhibitor DAPT (1),5 we developed a facile
synthetic method for a photoaffinity probe by utilizing the
polymer-bound reagent 3.6 Furthermore, the utility of 3 was
demonstrated by synthesis of the probe 4,6 and the photo-
affinity labeling experiments of 4 clarified that the major
direct target molecules for the DAPT are preseniline 1
† University of Shizuoka.
‡ University of Tokyo.
(1) For a review of photoaffinity labeling, see: (a) Kotozyba-Hilbert,
F.; Kapfer, I.; Goeldner, M. Angew. Chem., Int. Ed. Engl. 1995, 34, 1296.
(b) Fleming, S. A. Tetrahedron 1995, 51, 12479. (c) Dorma´n, G.; Prestwich,
G. D. Trends Biotechnol. 2000, 18, 64.
(2) For use of a biotin tag for catching the target protein, see: (a)
Hofmann, K.; Kiso, Y. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3516. (b)
Hofmann, K.; Finn, F. M.; Kiso, Y. J. Am. Chem. Soc. 1978, 100, 3585.
For use in photoaffinity labeling, see: (c) Gilbert, B. A.; Rando, R. R. J.
Am. Chem. Soc. 1995, 117, 8061. (d) Hatanaka, Y.; Hashimoto, H.; Kanaoka,
Y. J. Am. Chem. Soc. 1998, 120, 453.
(3) For a recent review on secretases, see: Wolfe, M. S. Nat. ReV. Drug
DiscoVery 2002, 1, 859 and references therein.
10.1021/ol070376i CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/05/2007

C-terminal fragments (PS1 CTFs),7 which were discovered
from the genetic investigation of familial AD. Additionally,
in a competing photolabeling experiment of 4 against various
classes of inhibitors, we dissected the inhibitory mechanism
of 1.7 However, the more effective labeling probe for
obtaining a large enough quantity of proteins was strongly
required for an exact determination of the DAPT binding
site by mass spectrometry at the single amino acid level.
Solid-phase synthesis has the advantage of readily providing
the affinity probes by a simple operation without any tedious
purification steps.6 We envisioned an application of this
methodology to combine other labeling groups and/or linker
moieties to afford diverse photoaffinity probes which would
be efficient for labeling experiments. Herein, we report a
solid-phase synthesis of a variety of photoaffinity probes
(Scheme 1) and an evaluation of their labeling ability with
the PS1 CTFs.
romethylaryl diazirine was a reliable photophore, due to its
excellent stability. As shown in Scheme 2, synthesis of the
Scheme 2. Synthesis of Probes 12a and 12b
Scheme 1. Our Synthetic Strategy for Photoaffinity Probes by
Solid-Phase Synthesis
probes 12a,b began with the diazirine derivative 5, which
was reported by Hatanaka et al.8 For the alkyl (Alk) linker
probe 12a, alkylation of 5 with the alkyl bromide 6a9
promoted conversion of the aldehyde to the azide. Depro-
tection of the THP group of 7a and condensation with the
diamine derivative 8a9 were accomplished by the Mitsunobu
reaction of the Ns amide (Ns strategy).10,11 After removal of
(5) Dovey, H. F.; John, V.; Anderson, J. P.; Chen, L. Z.; de Saint Andrieu,
P.; Fang, L. Y.; Freedman, S. B.; Folmer, B.; Goldbach, E.; Holsztynska,
E. J.; Hu, K. L.; Johnson-Wood, K. L.; Kennedy, S. L.; Kholodenko, D.;
Knops, J. E.; Latimer, L. H.; Lee, M.; Liao, Z.; Lieberburg, I. M.; Motter,
R. N.; Mutter, L. C.; Nietz, J.; Quinn, K. P.; Sacchi, K. L.; Seubert, P. A.;
Shopp, G. M.; Thorsett, E. D.; Tung, J. S.; Wu, J.; Yang, S.; Yin, C. T.;
Schenk, D. B.; May, P. C.; Altstiel, L. D.; Bender, M. H.; Boggs, L. N.;
Britton, T. C.; Clemens, J. S.; Czilli, D. L.; Dieckman-MacGinty, D. K.;
Droste, J. J.; Fuson, K. S.; Gitter, B. D.; Hyslop, P. A.; Johnstone, E. M.;
Li, W. Y.; Little, S. P.; Mabry, T. E.; Miller, F. D.; Audia, J. E. J.
Neurochem. 2001, 76, 173.
(6) Kan, T.; Tominari, Y.; Morohashi, Y.; Natsugari, H.; Tomita, T.;
Iwatsubo, T.; Fukuyama, T. Chem. Commun. 2003, 2244.
(7) Morohashi, Y.; Kan, T.; Tominari, Y.; Fuwa, H.; Okamura, Y.;
Watanabe, N.; Natsugari, H.; Fukuyama, T.; Iwatsubo, T.; Tomita, T. J.
Biol. Chem. 2006, 281, 14670.
(8) (a) Hashimoto, M.; Kanaoka, Y.; Hatanaka, Y. Heterocycles 1997,
46, 119. (b) A one-pot incorporation of diazirine and the biotin tag was
reported. See: Hatanaka, Y.; Kempin, U.; Jong-Jip, P. J. Org. Chem. 2000,
65, 5639.
(9) Detailed synthetic procedures are described in the Supporting
Information.
(10) For a review of Ns strategy, see: (a) Kan, T.; Fukuyama, T. J. Syn.
Org. Chem. Jpn. 2001, 59, 779. (b) Kan, T.; Fukuyama, T. Chem. Commun.
2004, 353.
First, we planned to replace the diazirine labeling (Da)
group in the benzophenone (Bp) of the probe intermediate
3. Among several reactive diazirines, we found that trifluo-
(4) (a) Takahashi, Y.; Hayashi, I.; Tominari, Y.; Rikimaru, K.; Morohashi,
Y.; Kan, T.; Natsugari, H.; Fukuyama, T.; Tomita, T.; Iwatsubo, T. J. Biol.
Chem. 2003, 278, 18664. (b) Fuwa, H.; Okamura, Y.; Morohashi, Y.;
Tomita, T.; Iwatsubo, T.; Kan, T.; Fukuyama, T.; Natsugari, H. Tetrahedron
Lett. 2004, 45, 2323. (c) Kan, T.; Tominari, Y.; Rikimaru, K.; Morohashi,
Y.; Natsugari, H.; Tomita, T.; Iwatsubo, T.; Fukuyama, T. Bioorg. Med.
Chem. Lett. 2004, 14, 1983. (d) Takahashi, Y.; Fuwa, H.; Kaneko, A.;
Sasaki, M.; Yokoshima, S.; Koizumi, H.; Takebe, T.; Kan, T.; Iwatsubo,
T.; Tomita, T.; Natsugari, H.; Fukuyama, T. Bioorg. Med. Chem. Lett. 2006,
16, 3813. (e) Fuwa, H.; Hiromoto, K.; akahashi, Y.; Yokoshima, S.; Kan,
T.; Fukuyama, T.; Iwatsubo, T.; Tomita, T.; Natsugari, H. Bioorg. Med.
Chem. Lett. 2006, 16, 4184.
(11) (a) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995,
36, 6373. (b) Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai, Y.; Kan, T.
Tetrahedron Lett. 1997, 38, 5831. (c) Fukuyama, T.; Cheung, M.; Kan, T.
Synlett 1999, 1301. (d) Kurosawa, W.; Kan, T.; Fukuyama, T. Org. Synth.
2002, 79, 186.
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Org. Lett., Vol. 9, No. 11, 2007

a
Figure 1. Structure and inhibitory potencies of synthetic probes. IC50 value for inhibition of Aâ 40 generation. Detailed experimental
conditions are described in ref 7.
the Boc group, the biotin unit was incorporated by the active
ester method. Removal of the Ns group, loading on the resin
10,12 and subsequent reduction of the azide group gave the
probe intermediate 11a. Using a procedure similar to that in
the preparation of 4, we carried out a condensation of the
ligand (DAP-OH) 26 and acidic cleavage from the resin to
provide the probe 12a efficiently. The hydrophilic probe 12b9
was obtained by a sequence similar to that used for 12a by
using the polyethylene glycol (Peg) linkers 6b9 and 8b,9 as
shown in Scheme 2 (12a, 47% from 5; 12b, 22% from 5).
Next we turned our attention to the preparation of a
disulfide-bridged probe. Although avidin-biotin affinity for
finding a labeled protein is a fine technique, the release of
the target protein from the streptavidin complex often
requires harsh conditions. Thus, a disulfide linker (Cya)
would be an attractive and useful contribution since it would
cleave under mild conditions and readily enable isolation of
the target protein.13 Synthesis of the probes 18a,b started
from the benzophenone derivative 136 as shown in Scheme
3. Incorporation of the amine functionality into 13 was
performed by the Mitsunobu reaction of the Ns-protected
ammonia 14.14 After removal of the THP group, the primary
alcohol was subjected to the Mitsunobu reaction with the
Ns-protected amino acid derivatives 15a,b,9 respectively.
Deprotection of the Ns groups, conjugation with the
resin 10, and simultaneous removal of the alloc group and
the allyl ester gave the polymer-bound amino acid derivatives
16a,b. Stepwise incorporation of the ligand (DAP-OH, 2)
and the biotinyl cystamine amide 179 and acidic cleavage
from the resin provided the probes 18a,b, respectively (18a,
77% from 13; 18b, 65% from 13). Using a similar procedure
(12) (a) Hidai, Y.; Kan, T.; Fukuyama, T. Tetrahedron Lett. 1999, 41,
4711. (b) Hidai, Y.; Kan, T.; Fukuyama, T. Chem. Pharm. Bull. 2000, 48,
1570. (c) Kan, T.; Kobayashi, H.; Fukuyama, T. Synlett 2002, 1338.
(13) Li H.; Liu, Y.; Fang, K.; Nakanishi, K. Chem. Commun. 1999, 365.
(14) Fukuyama, T.; Cheung, M.; Kan, T. Synlett 1999, 1301.
Org. Lett., Vol. 9, No. 11, 2007
2057

Scheme 3. Synthesis of Probes 18a and 18b
Figure 2. Results of SDS-PAGE of the photoaffinity labeling
experiment by synthetic probes (4, 12a, 12b, 18a, 18b, and 21).
b
a-, absence of DAPT (1); +, presence of DAPT (1). Detailed
experimental conditions of labelings are described in ref 7.
in labeling ability or inhibition activities was observed
between the benzophenone and the diazirine photophores.
However, the hydrophobic (alkyl) linker appeared to be more
suitable than the hydrophilic (Peg) linker for this protease
compared with 12a and 12b. Although detection of the
labeled protein was achieved by utilizing probe 18a, the
similar disulfide-bridged probes 18b and 21 were unsuc-
cessful in labeling, even upon treatment with excess thiol.
Furthermore, the longer probe 21 exhibited no inhibitory
activity of γ-secretase at 10 mM. Presumably, the disulfide
bond and/or the biotin group of 18b and 21 blocks the
binding with γ-secretase. Unfortunately, we could not
discover the probes more effective than 4 in this work.
However, these results helped confirm the importance of
combinatorial synthesis of photoaffinity probes, which readily
provide various probes.
for the preparation of 15, we also obtained the diazirine-
type probe 219 efficiently as shown in Scheme 4 (21, 86%
from 5).
Scheme 4. Synthesis of Probe 21
In conclusion, we accomplished a convenient synthetic
method for photoaffinity probes and evaluated their utilities.
To our knowledge, there have been few or no such systematic
structure and photoaffinity labeling ability relationship (SAR)
studies of photoaffinity probes. The present investigation was
carried out by utilizing the combination of our Ns strategy10,11
and the novel resin 10.12 Additionally, this synthetic strategy
would be applicable not only to photoaffinity probes17 but
also to fluorescein probes, thus making a great contribution
to an area of chemical biology. Further investigation is
underway in our laboratories.
Since biochemical analysis of a membrane-bound protease
is known to be challenging, photoaffinity labeling is one of
the more effective methods for γ-secretase.15 Thus, the
photoaffinity labeling experiments were performed by the
following sequence. After mixing a CHAPSO (3-[(3-chola-
midopropyl)dimethylammonio]-2-hydroxypropanesulfonate)-
solubilized lysate of the HeLa S3 cell with probes as shown
in Figure 2 in the absence or presence of an excess amount
of DAPT (1), the solution was irradiated with long-wave
near-UV light. Subsequently, the biotinylated proteins were
captured with streptavidin sepharose beads, separated by
SDS-PAGE, and detected by an anti-PS1 CTF antibody.
Structures of the synthetic probes and inhibition activities16
(IC50) of Aâ 40 are shown in Figure 1. The results of
photolabeling (Western-blot analysis) are also shown in
Figure 2. Compared with 4 and 12a, no distinct difference
Acknowledgment. This work was financially supported by
SUNBOR Foundation, Uehara Memorial Foundation, 21st
Century COE Program, and a Grant-in-Aid for Scientific Re-
search on Priority Areas 17025011 from The Ministry of Edu-
cation, Culture, Sports, Science and Technology (MEXT),
Japan.
Supporting Information Available: Detailed experimen-
tal procedures and spectroscopic data (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
OL070376I
(17) Synthetic procedures for a similar probe intermediate 22, which
possessing reactive carboxylic acid functionality was also described in the
Supporting Information.
(15) Beher, D.; Fricker, M.; Nadin, A.; Clarke, E. E.; Wrigley, J. D. J.;
Li, Y.-M.; Culvenor, J. G.; Masters, C. L.; Harrison, T.; Shearman, M. S.
Biochemistry 2003, 42, 8133.
(16) Inhibitory potencies of probes on γ-secretase activity were analyzed
by cell-based assays. For detailed procedures, see ref 17.
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Org. Lett., Vol. 9, No. 11, 2007