Development of photoaffinity probes for γ-secretase equipped with a nitrobenzenesulfonamide-type cleavable linker

Article abstract of DOI:10.1016/j.bmcl.2009.10.086

We have developed photoaffinity probes for γ-secretase with a nitrobenzenesulfonamide-type linker that can be cleaved with 2-mercaptoethanol under physiological conditions.

Full text of DOI:10.1016/j.bmcl.2009.10.086

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6869–6871
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of photoaffinity probes for c-secretase equipped
with a nitrobenzenesulfonamide-type cleavable linker
Satoshi Yokoshima a,b, Yuzo Abe a, Naoto Watanabe a, Yoichi Kita a, Toshiyuki Kan c, Takeshi Iwatsubo a,b,d
,
Taisuke Tomita a,b, Tohru Fukuyama a,b,
*
a Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
b Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
c School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
d Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed photoaffinity probes for
can be cleaved with 2-mercaptoethanol under physiological conditions.
c
-secretase with a nitrobenzenesulfonamide-type linker that
Received 18 September 2009
Revised 19 October 2009
Accepted 20 October 2009
Available online 23 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
c
-Secretase
Photoaffinity labeling
Probe
Cleavable linker
Photoaffinity labeling is an efficient method for studying the
interactions between biologically active compounds (ligands) and
their target molecules.1 The method facilitates the identification
of both the target molecule and the binding domain. A photo-
affinity probe is usually composed of a ligand, a photoreactive
labeling group, and an indicator. A biotin tag is widely employed
as an indicator because the biotinylated molecule can be easily
detected by several immunological methods and isolated through
binding to avidin beads.2 One drawback to this approach is that
harsh conditions are required to disrupt the strong interaction
between the biotin and the avidin. Thus, to liberate the biotinyla-
ted molecule, the beads are typically boiled with detergents to
denature the avidin. These conditions may result in degradation
of the target molecules as well as contamination of the other pro-
teins that bind to the beads with non-specific manner. To over-
come this drawback, a variety of cleavable linkers, which can be
cleaved under mild conditions to specifically release the labeled
target molecules, have been developed.3
fragment(PS1 CTF)(Fig. 1).5 However, further detailedexaminations
of the DAPT-PS1 CTF interaction could not be conducted because of
the low purity of the labeled proteins, which is caused by contami-
nated proteins bound to the avidin beads. We have prepared the
other photoaffinity probes with disulfide moieties as cleavable link-
ers. However, they failed to effectively probe the PS1 CTF (data not
shown). These results prompted us to develop photoaffinity probes
equipped with a novel cleavable linker.
We envisioned that 2-nitrobenzensulfonamide would be an
effective cleavable linker because this molecule can be cleaved un-
der mild conditions using thiolate to generate an amine unit and a
F
O
O
Me
Me
O
O
H
N
F
F
N
H
N
H
O
O
Ph
Ph
O
We have focused on the functional analysis of
is one of the important therapeutic targets for Alzheimer’s disease.4
During the course of investigation of the potent -secretase inhibitor
c-secretase, which
O
S
F
HN
NH
c
H
N
O
H
H
DAPT (1), we synthesized the photoaffinity probe DAP-BpB (2) and
found that the major target of DAPT is presenilin 1 C-terminal
N
H
Ot-Bu
( )8
( )4
N
H
N
H
DAPT (1)
DAP-BpB (2)
* Corresponding author. Tel.: +81 3 5841 4777; fax: +81 3 5802 8694.
E-mail address: fukuyama@mol.f.u-tokyo.ac.jp (T. Fukuyama).
Figure 1. Structures of DAPT and DAP-BpB.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.086

6870
S. Yokoshima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6869–6871
Table 1
Cleavage of the linker
O2N
Cl
CO2Me
O2N
CO2Me
Me
N
O
BocHN
( )4
S
S
a,b
O2N
Me
( )4
N
NH2·HCl
O
O
O
O
3
5
H
HCl·H2N
N
( )4
S
O
O
O
O
6
( )4
O2N
( )4
NHAc
N
NH2·HCl
O2N
S
Me
N
H
N
HCl·H2N
c-e
H
( )4
1) HS(CH2)2OH (10 eq)
buffer, rt
S
AcO
O
O
6
7
2) Ac2O, pyridine, rt
Scheme 1. Reagents and conditions: (a) BocHN(CH2)4NH2 (4), Et3N, CH2Cl2, rt; (b)
MeI, Cs2CO3, DMF, reflux; (c) LiOHÁH2O, THF–MeOH, rt; (d) BocHN(CH2)4NH2 (4),
WSCDÁHCl, HOBt, DMF, rt, 68% (four steps); (e) SOCl2, MeOH, rt.
Me
AcHN
N
( )4
Ac
8
Buffer
pH
9.2
Concentration of 6 (mM)
Time (h)
Yield (%, 7)
3-nitorobenzene unit.6 It has been reported that 3-nitro-4-sul-
famoylbenzamides can be used as linkers for solid-phase synthe-
sis.7 To examine whether this unit can be used for photoaffinity
labeling, we synthesized a model compound bearing 3-nitro-4-sul-
famoylbenzamide and evaluated its cleavability under physiologi-
cal conditions.
NaHCO3–
Na2CO3
KH2PO4–
NaHPO4
3.0
1.5
3.0
1.5
7
12
24
33
93
97
93
100
7.4
To access the model compound, known sulfonyl chloride 37 was
treated with N-Boc-butanediamine (4) and the resulting sulfon-
amide unit was methylated to afford 5 (Scheme 1). Hydrolysis of
the methyl ester in 5, coupling of the resultant carboxylic acid with
amine 4, and final deprotection of the Boc groups furnished the
requisite model compound 6.
With model sulfonamide 6 in hand, we attempted to cleave the
sulfonamide unit under physiological conditions. Sulfonamide 6
was treated with 10 equiv of 2-mercaptoethanol in two buffers.
The reaction progress was determined by isolation of 7 after acet-
ylation of the reaction mixture. As shown in Table 1, the cleavage
reactions proceeded smoothly in aqueous media to afford 7 in
excellent yield. The reaction rates depended on the concentrations
of both the substrate and 2-mercaptoethanol. The cleavage reac-
tion proceeded even in almost neutral phosphate buffer (pH 7.4)
although the time needed for completion of the reaction was long-
er than the reaction time required at higher pH. Both of the reac-
tion conditions were much milder than the conditions required
for breaking the avidin–biotin interaction.
With a suitable cleavable unit in hand, we focused on incorpo-
ration of the linker unit into photoaffinity probes (Scheme 2). Two
variants of carboxylic acids 9 with different chain lengths (n = 4 or
8) were prepared following the same procedures described in the
synthesis of the model compound. Condensation of 9 with biotin
unit 10 proceeded smoothly. After removal of the Boc group with
methanolic hydrochloric acid, the resulting salt was liberated with
Amberlite IR-400 (OMe form). Condensation of the resulting
amines 11 (n = 4, 8) with polymer-bound carboxylic acid units 12
(m = 1, 5, 7, 10),8 followed by acidic treatment, afforded photo-
affinity probes 13a–h with different lengths.
Usinganinvitroc-secretaseassay, wefoundthattheseprobeshad
inhibitory activities at almost equal levels to that of DAPT (Table 2).
We next investigated the labeling abilities of probes 13a–h. CHA-
PSO-solubilized lysates of HeLa cells (ꢀ150
lg) mixed with the
O
HN
NH
O
O
H
H
( )4
(
)
HN
NH
H
HCl·H2N
N
4
S
O2N
Cl
CO2Me
O2N
CO2H
H
O
O
H
10
Me
N
( )4
BocHN
O2N
( )n
a-c
S
S
d-f
( )4
N
H
N
S
Me
N
H
O
O
O
O
H2N
3
9
( )n
S
11
n = 4, 8
F
O
O
O
Ph
O
H
N
( )m
OH
g,h
F
N
H
N
N
F
H
O
Me
O
O
Ph
O
12
H
N
O
F
N
N
H
H
O
O
Me
O
HN
NH
H
compound
m
n
O
O
H
1
1
5
5
7
7
3
7
3
7
3
7
3
7
( )4
13a
13b
13c
13d
13e
13f
O2N
( )4
N
H
S
H
N
Me
N
( )m
( )n
N
S
H
O
O
O
10
10
13g
13h
Scheme 2. Reagents and conditions: (a) BocHN(CH2)nNH2, Et3N, CH2Cl2, rt; (b) MeI, Cs2CO3, MeCN, reflux; (c) LiOHÁH2O, THF–MeOH–H2O, rt; (d) 9, PyBoP, HOBt, DMF, rt; (e)
SOCl2, MeOH, rt; (f) Amberlite IR-400 (OMe form), MeOH; (g) 11, WSCDÁHCl, HOBt, i-Pr2NEt, NMP, rt; (h) TFA, CH2Cl2, rt.

S. Yokoshima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6869–6871
6871
Table 2
After capturing the biotinylated proteins with probe 13e, the
streptavidin sepharose beads were treated in carbonate buffer
(pH 9.2) containing 2% SDS in the presence or absence of 3% 2-
mercaptoethanol. To reduce non-specific degradation and/or
aggregation of the peptide, the cleavage reaction was performed
for 4 h at 20 °C. The eluates were analyzed by western blotting
(E in Fig. 3). To evaluate the efficiency of the cleavage, the residual
beads were subsequently boiled and the biotinylated PS1 CTFs
remained on the beads was analyzed (B in Fig. 3). In the presence
of 2-mercaptoethanol, the linker was cleaved to elute PS1 CTF from
the beads, while no PS1 CTF was detected in the eluates without 2-
mercaptoethanol. However, some amount of the biotinylated
proteins still remained on the beads. Although the cleavage was
expected to occur more efficiently at higher pH, amount of the cap-
tured PS1 CTF was lower under these conditions (data not shown).
Efforts to optimize the cleavage reaction (i.e., the reaction times,
the type of buffer, and the linker length) are currently underway.
In summary, we found that a nitrobenzenesulfonamide-type
linker could be cleaved under physiological conditions in the pres-
ence of 2-mercaptoethanol. Using this linker unit, we successfully
Inhibitory activities of probes 13a–h for Ab40 and 42a
Compound
Ab40 (%)
Ab42 (%)
13a
13b
13c
13d
13e
13f
81.9
81.9
54.9
78.2
62.8
62.3
74.5
55.0
75.9
77.0
60.2
59.3
66.2
89.3
86.3
69.2
13g
13h
DAPT (1)
77.4
76.3
a
Percentages of inhibition of the Ab generation in the presence of 1 lM of
compounds (DMSO = 0%). Detailed experimental conditions are described in Ref. 3b.
developed photoaffinity probes for the c-secretase.
Acknowledgements
This work was financially supported in part by a Grant for the
21th Century COE Program, Grants-in-Aid for Young Scientists (S)
from Japan Society for the Promotion of Science (JSPS) (T.T.), by
the Targeted Proteins Research Program of the Japan Science and
Technology Corporation, and Scientific Research on Priority Areas
from The Ministry of Education, Culture, Sports, Science and Tech-
nology (MEXT), Japan. N.W. was a research fellow of JSPS.
Figure 2. Photoaffinity labeling with probes 13a–h.
References and notes
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Figure 3. Photoaffinity labeling with probe 13e. 2ME: 2-mercartoethanol. E and B
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