Development of UV-responsive catch-and-release system of a cysteine protease model peptide

Article abstract of DOI:10.1016/j.tet.2011.09.062

Cysteine proteases are attractive drug targets due to their involvement in a wide variety of diseases. To evaluate the potential of a particular protease as a drug target, use of a reagent that controls activity of the protease is indispensable. In this context, we have developed a catch-and-release reagent that first forms a covalent bond with the active center thiol of a cysteine protease to suppress its activity and then is removed by UV-irradiation to release the parent active protease. In this paper, the design and synthesis of a catch-and-release reagent of thiols are described. Its application to caging (catch) and UV-induced uncaging (release) of a model peptide derived from an active site of caspase-9 and introduction of a recognition moiety on the reagent are also reported.

Full text of DOI:10.1016/j.tet.2011.09.062

Tetrahedron 67 (2011) 8879e8886
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Development of UV-responsive catch-and-release system of a cysteine protease
model peptide
,
a
a
a
a
a
Akira Shigenaga a , Ko Morishita , Keiko Yamaguchi , Hao Ding , Koji Ebisuno , Kohei Sato ,
*
Jun Yamamoto a, Kenichi Akaji b, Akira Otaka a
,
*
a Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
b Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 August 2011
Received in revised form 13 September 2011
Accepted 15 September 2011
Available online 22 September 2011
Cysteine proteases are attractive drug targets due to their involvement in a wide variety of diseases. To
evaluate the potential of a particular protease as a drug target, use of a reagent that controls activity of
the protease is indispensable. In this context, we have developed a catch-and-release reagent that first
forms a covalent bond with the active center thiol of a cysteine protease to suppress its activity and then
is removed by UV-irradiation to release the parent active protease. In this paper, the design and synthesis
of a catch-and-release reagent of thiols are described. Its application to caging (catch) and UV-induced
uncaging (release) of a model peptide derived from an active site of caspase-9 and introduction of
a recognition moiety on the reagent are also reported.
Keywords:
Aromatization
Caged compound
Cysteine protease
Protective group
UV-responsive
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
selectively caught by the reagent and is inactivated.4,5 If the catch-
and-release reagent can be irreversibly removed by exposure to UV
Cysteine proteases represent attractive drug targets due to their
involvement in a wide variety of conditions, such as cardiovascular,
inflammatory, viral and immunological disorders, and cancer.1
Methodology for controlling the activity of a disease-related cys-
teine protease may provide useful means for evaluating the pro-
tease as a drug target. In this context, caged cysteine proteases that
release the parent proteases after exposure to UV light have been
reported.2 Activity control of the proteases is enabled by UV-
induced conversion of the ‘inactive’ caged proteases to the ‘active’
parent proteases. Recently, an advanced methodology, which en-
ables three-step activity control, such as ‘active (intact enzyme)’ to
‘inactive (caging in living cells by addition of a caging reagent)’ to
‘active (uncaging by UV-irradiation),’ was developed for enzymes
other than cysteine proteases.3 To our knowledge, its application to
cysteine proteases has yet to be reported. Therefore, we designed
a catch-and-release system for the advanced activity control of
a cysteine protease as shown in Scheme 1. When a catch-and-
release reagent possessing a recognition moiety for selective
binding to the target protease and a warhead to react with an active
center thiol is added to a proteome, the target cysteine protease is
light, photo-induced re-activation of the protease is achieved.
Scheme 1. Catch-and-release strategy for controlling the activity of cysteine proteases.
In this article, the design and synthesis of the UV-responsive
catch-and-release reagent of thiols are described. Introduction of
the recognition moiety on the reagent is also discussed.
2. Results and discussion
2.1. Design of catch-and-release reagent
We previously reported a stimulus-responsive amino acid6 that
induced peptide bond cleavage after stimulus induced deprotection
followed by lactonization of the trimethyl lock moiety (Scheme 2).7
* Corresponding authors. E-mail addresses: ashige@ph.tokushima-u.ac.jp
(A. Shigenaga), aotaka@ph.tokushima-u.ac.jp (A. Otaka).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.09.062

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A. Shigenaga et al. / Tetrahedron 67 (2011) 8879e8886
With this in mind, UV-responsive catch-and-release reagent 1,
composed of a trimethyl lock-like moiety and the UV-removable o-
nitrobenzyl (o-NB) group, was designed (Scheme 3). Compound 1
possesses an electron-deficient olefin moiety and a recognition
moiety (R), therefore, it can recognize a target protease and react
via Michael addition to generate the corresponding inactivated
cysteine protease 2. After UV-induced removal of the o-nitrobenzyl
group followed by lactonization of the trimethyl lock-like moiety,
intermediate 3 should easily aromatize to release the parent cys-
teine protease.
crystallography (Scheme 5). Therefore, we assumed that the ge-
ometry of 5 was also (E).
Scheme 4. Reagents and conditions: (i) o-Nitrobenzyl bromide, K2CO3, CH2Cl2, 77%;
(ii) H2N-NHBoc, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate
(TBTU), N,N-diisopropylethylamine (DIEA), DMAP, CH2Cl2, 92%; (iii) OxoneÒ, MeOH/
H2O, 0 ꢀC to room temperature, 74%; (iv) Piperidine, AcOH, toluene, reflux, 85%. (o-NB:
o-nitrobenzyl).
Scheme 2. Stimulus-responsive amino acid based on trimethyl lock system (PG:
protective group removable by a stimulus).
Scheme 5. Reagents and conditions: (i) PhSO2CH2CO2Me, piperidine, AcOH, toluene,
reflux, 83%. All hydrogen atoms of the X-ray structure of 11 are omitted for clarity.
2.3. Catch-and-release experiment of compound 5
Scheme 3. Design of catch-and-release reagent for control of activity of cysteine
protease.
The catch-and-release reaction of a thiol with compound 5 was
examined. First, benzyl mercaptan was used as a thiol for simplicity
(Scheme 6). Benzyl mercaptan was treated with compound 5 and
Et3N in CH2Cl2, affording the Michael addition product 12 in 82%
yield. After purification, caged thiol 12 was irradiated by UV
(>365 nm) for 5 min, and the reaction mixture was shaken after
addition of 2,20-dithiodipyridine9 and Et3N. The reaction progress
was monitored by HPLC (Fig. 1). After UV-irradiation, the o-nitro-
benzyl group of substrate 12 was completely removed to generate
phenol 13. By the addition of 2,20-dithiodipyridine and Et3N fol-
lowed by shaking for 2 h, intermediate 13 was successfully aro-
matized to release benzyl mercaptan derivative 14 and 4. The
products were identified by ESI-MS and comparison of retention
times with those of authentic samples.10 The synthesis of an au-
thentic sample of compound 4 is depicted in Scheme 7. Briefly, after
2.2. Synthesis of catch-and-release reagent
Catch-and-release reagent 5 possessing a Boc-protected acyl
hydrazine for facile introduction of the recognition moiety was
synthesized as shown in Scheme 4. Phenol 68 was alkylated with o-
nitrobenzyl bromide to afford ether 7. Knoevenagel condensation of
7 with sulfonylacetamide 10 derived from sulfanylacetic acid 8 was
performed and catch-and-release reagent 5 was obtained. Because
the geometry of compound 5 could not be determined using NMR
and X-ray methods, we synthesized easily crystallizable surrogate
11 and its geometry was determined to be (E) using X-ray

A. Shigenaga et al. / Tetrahedron 67 (2011) 8879e8886
8881
protection of phenol 6 with a methoxymethyl group followed by
Knoevenagel condensation with commercially available phenyl-
sulfonylacetic acid methyl ester, obtained product 16 was treated
with TFA to afford aromatized product 4.
Scheme 7. Reagents and conditions: (i) Chloromethyl methyl ether, DIEA, CH2Cl2, 81%;
(ii) PhSO2CH2CO2Me, AcOH, piperidine, toluene, reflux, 94%; (iii) TFA, CH2Cl2, 96%.
Scheme 6. Reagents and conditions: (i) Et3N, CH2Cl2, 82%; (ii) UV-irradiation
(>365 nm, 5 min) in MeOH followed by addition of 2,20-dithiodipyridine and Et3N.
and each step of the catch-and-release reaction was monitored by
HPLC (Scheme 8). In MeCN/phosphate buffer (pH 7.8), peptide 17
was treated with excess catch-and-release reagent 5, and caged
peptide 18 was obtained after 24 h of incubation at 37 ꢀC (Fig. 2(a),
(b)). Next purified peptide 18 was irradiated by UV (>365 nm,
5 min), and the o-nitrobenzyl group of 18 was removed to generate
phenol 19 (Fig. 2(c), (d)). After 2 h of incubation at 37 ꢀC in the
presence of tris(2-carboxyethyl)phosphine (TCEP), release of
caspase-9 model peptide 17 occurred along with generation of
aromatized product 4 as major products (Fig. 2(e)).13
These results suggest that compound 5 can potentially work as
a prototype of a catch-and-release reagent for controlling the ac-
tivity of cysteine proteases.
2.4. Introduction of recognition moiety onto catch-and-
release reagent
Introduction of a recognition moiety, such as a substrate peptide
sequence of a target protease to the catch-and-release reagent is of
value for selective caging of the target protease in a proteome.4,5
Previously, one of the authors (K.A.) reported that a peptidyl sub-
strate derivative of SARS cysteine protease (SARS 3CLpro) possessing
an aldehyde at the C-terminal position acted as a reversible in-
hibitor of SARS 3CL .
pro 14 Therefore, we decided to introduce catch-
and-release reagent 5 onto the C-terminus of peptidyl substrates to
control the activity of SARS 3CLpro (Scheme 9).15 Peptide conjugates
22 and 23 were synthesized as follows. After removal of the Boc
group of 5, the obtained product was coupled with Boc-protected
glutamine to generate compound 21. Then, the N-terminal Boc
group was removed by acid treatment, and the obtained product
was coupled with protected peptides 24 or 25, which were syn-
thesized using standard Boc-based solution phase peptide syn-
thesis. Finally, the obtained materials were subjected to global
deprotection to generate peptide conjugates 22 and 23. Application
of peptide conjugates 22 and 23 to control activity of SARS 3CLpro is
underway in our laboratory.
3. Conclusion
Fig. 1. HPLC profiles of photo-reaction of compound 12: (a) before UV-irradiation, (b)
after UV-irradiation (>365 nm, 5 min), (c) after UV-irradiation (>365 nm, 3 min)
followed by addition of 2,20-dithiodipyridine and Et3N and shaking for 2 h. Analytical
HPLC conditions: linear gradient of solvent B in solvent A, 50e80% over 30 min.
Compounds eluted at 3e6 min are derivatives of photo-cleaved o-nitrobenzyl group.
*2,20-Dithiodipyridine.
In conclusion, a catch-and-release reagent for thiols was de-
veloped. By using this reagent, catch and UV-induced release of
thiols including an active site model peptide of caspase-9 were
achieved. Introduction of a peptidyl recognition moiety onto the
reagent was also successful. We believe that these results represent
an important step toward the development of catch-and-release
systems of cysteine proteases for controlling their activities. Ap-
plication of the catch-and-release reagent to control activity of
SARS 3CLpro is underway.
Next, the catch-and-release reaction of a model peptide derived
from a cysteine protease was examined. In this experiment, model
peptide 1711 derived from the active site of caspase-912 was used,

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A. Shigenaga et al. / Tetrahedron 67 (2011) 8879e8886
Scheme 8. Reagents and conditions: (i) MeCN/phosphate buffer (pH 7.8)¼1:1 (v/v), 37 ꢀC; (ii) UV-irradiation (>365 nm, 5 min) in MeCN/phosphate buffer (pH 7.8)¼1:1 (v/v)
containing glutathione and EDTA followed by incubation at 37 ꢀC in the presence of TCEP. (A: alanine; C: cysteine; E: glutamic acid; F: phenylalanine; G: glycine; I: isoleucine; K:
lysine; Q: glutamine; Y: tyrosine).
Fig. 2. HPLC profiles of catch-and-release reaction of compound 5. Michael addition of peptide 17 to compound 5: (a) 0 h, (b) 24 h. UV-irradiation experiment of compound 18: (c)
before UV-irradiation, (d) after UV-irradiation (>365 nm, 5 min), (e) after UV-irradiation (>365 nm, 5 min) followed by incubation at 37 ꢀC in the presence of TCEP for 2 h. Analytical
HPLC conditions: linear gradient of solvent B in solvent A, 10e95% over 30 min *Glutathione. **Non-peptidyl compound.
Scheme 9. Reagents and conditions: (i) TFA then HCl/AcOEt; (ii) Boc-Q-ONp, HOBt$H2O, Et3N, DMF, 85% (2 steps); (iii) TFA then HCl/AcOEt; (iv) 24 (for 22) or 25 (for 23), EDC$HCl,
HOBt$H2O, Et3N, DMF; (v) TFA, TMSBr, thioanisole, m-cresol, 0 ꢀC (A: alanine; L: leucine; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; Np¼p-nitrophenyl).
4. Experimental section
4.1. General methods
products were detected by UV at 220 nm. A solvent system con-
sisting of 0.1% (v/v) TFA aqueous solution (solvent A) and 0.1% (v/v)
TFA in MeCN (solvent B) was used for HPLC elution. Photolysis by
UV-irradiation was performed using a Moritex MUV-202U with the
filtered output (>365 nm) of a 3000 mW/cm2 HgeXe lamp. Optical
rotation was determined on a JASCO P-2200 polarimeter (concen-
tration in g/100 mL).
All reactions were carried out under a positive pressure of argon
except for UV-irradiation experiments and solid phase peptide
synthesis. For column chromatography, Silica Gel 60 N (Kanto
Chemical Co., Inc., spherical, neutral, particle size 63e210 mm) was
employed. Exact mass spectra were recorded on a Waters Micro-
massÒ LCT PremierÔ (ESI-TOF) or a Bruker Esquire200T (ESI-Ion
Trap) spectrometer. NMR spectra were measured using a Bruker
AV400N, a JEOL GSX400 or a JEOL GSX300 spectrometer. For HPLC
separation, a Cosmosil 5C18-AR-II analytical column (Nacalai Tes-
que, 4.6ꢁ250 mm, flow rate 1 mL/min), a Cosmosil 5C18-AR-II semi-
preparative column (Nacalai Tesque, 10ꢁ250 mm, flow rate 2.5 mL/
min) or a Cosmosil 5C18-AR-II preparative column (Nacalai Tesque,
20ꢁ250 mm, flow rate 10 mL/min) was employed, and eluting
4.2. Synthesis of catch-and-release reagent 5
4.2.1. 2,4-Dimethyl-6-(2-nitrobenzyloxy)benzaldehyde (7). To a stir-
red solution of phenol 68 (400 mg, 2.66 mmol) in DMF (13.0 mL)
were added K2CO3 (1.44 g, 10.4 mmol) and o-nitrobenzyl bromide
(1.00 g, 4.60 mmol), and the resulting suspension was stirred at
room temperature for 4 h. After addition of saturated aqueous so-
lution of NH4Cl followed by stirring for 1 h, the mixture was
extracted with AcOEt. The organic layer was washed with saturated

A. Shigenaga et al. / Tetrahedron 67 (2011) 8879e8886
8883
aqueous solution of NH4Cl and brine, dried over Na2SO4, and con-
centrated in vacuo. The crude product was purified by column
chromatography (hexane/AcOEt¼10:1 (v/v)) and 585 mg of benz-
aldehyde 7 (2.05 mmol, 77%) was obtained as pale yellow needles;
75 MHz)
d
¼20.1, 21.8, 27.9, 67.2, 81.8, 110.8, 118.1, 124.4, 124.8, 128.3,
128.4, 128.5, 129.2, 133.4, 133.7, 134.1, 136.7, 138.4, 139.4, 141.9,
142.8, 146.6, 154.1, 154.9, 159.7; HRMS (ESI-TOF) m/z calcd for
C29H31N3NaO8S ([MþNa]þ) 604.1730, found 604.1746.
mp: 129e130 ꢀC; 1H NMR (CDCl3, 400 MHz)
d
¼2.36 (3H, s), 2.58
(3H, s), 5.57 (2H, s), 6.71 (1H, s), 6.73 (1H, s), 7.54 (1H, t, J¼8.0 Hz),
4.2.5. 2-Benzenesulfonyl-3-[2,4-dimethyl-6-(2-nitrobenzyloxy)phe-
7.74 (1H, t, J¼8.0 Hz), 7.94 (1H, d, J¼8.0 Hz), 8.21 (1H, d, J¼8.0 Hz),
nyl]-(E)-acrylic acid methyl ester (11). Piperidine (5.0
and AcOH (5.0 L, 87 mol) were added to a solution of aldehyde 7
(110 mg, 0.396 mmol) and phenylsulfonylacetic acid methyl ester
(99.0 mg, 0.462 mol) in toluene (4.00 mL). The reaction mixture
mL, 40 mmol)
10.75 (1H, s); 13C NMR (CDCl3, 100 MHz)
d
¼21.5, 22.2, 67.3, 110.9,
m
m
121.1, 125.0, 125.8, 128.2, 128.5, 133.1, 134.2, 1423, 145.9, 146.6, 161.4,
190.9; HRMS (ESI-TOF) m/z calcd for C16H16NO4 ([MþH]þ) 286.1079,
found 286.1090. Anal. calcd for C16H15NO4 C, 67.36; H, 5.30; N, 4.91,
found C, 67.20; H, 5.24; N, 5.00.
m
was refluxed for 17 h and was quenched by addition of H2O at room
temperature. The resulting mixture was extracted with AcOEt, and
the organic layer was washed with H2O and brine, dried over
Na2SO4, and concentrated in vacuo. The crude product was purified
by column chromatography (hexane/AcOEt¼4:1 (v/v)) and 152 mg
of compound 11 (0.320 mmol, 83%) was obtained as colorless
prisms. Recrystallized material from Et2O was brought to X-ray
4.2.2. N0-(2-Phenylsulfanylacetyl)hydrazinecarboxylic acid tert-butyl
ester (9). To
a solution of phenylthioacetic acid 8 (7.00 g,
41.6 mmol) in CH2Cl2 (100 mL) were added DMAP (460 mg,
3.70 mmol), HBTU (15.0 g, 39.5 mmol), and DIEA (6.88 mL,
39.5 mmol) at 0 ꢀC. The resulting mixture was stirred at room
temperature for 20 min, and tert-butyl carbazate (2.50 g,
37.8 mmol) was added to the reaction mixture at 0 ꢀC. After being
stirred at room temperature for 11 h, the resulting mixture was
quenched by addition of H2O and extracted with CH2Cl2. The or-
ganic layer was washed with 5% (w/v) aqueous solution of citric
acid, 5% (w/v) aqueous solution of NaHCO3 and brine, and was dried
over Na2SO4. After concentration in vacuo, the crude product was
purified by column chromatography (hexane/AcOEt¼2:1 (v/v)) and
9.83 g of sulfide 9 (34.8 mmol, 92%) was obtained as colorless oil: 1H
analysis; mp: 129e130 ꢀC; 1H NMR (CDCl3, 400 MHz)
d
¼2.32 (6H,
s), 3.40 (3H, s), 5.33 (2H, s), 6.63 (1H, s), 6.75 (1H, s), 7.30 (1H, t,
J¼7.6 Hz), 7.40 (1H, t, J¼7.6 Hz), 7.49 (1H, d, J¼7.6 Hz), 7.51 (2H, t,
J¼7.6 Hz), 7.65 (1H, t, J¼7.6 Hz), 8.01 (2H, d, J¼7.6 Hz), 8.10 (1H, d,
J¼7.6 Hz), 8.33 (1H, s); 13C NMR (CDCl3, 100 MHz)
¼20.1, 21.8, 52.0,
d
67.4, 110.9, 118.7, 124.5, 124.7, 128.1, 128.2, 128.5, 128.8, 132.7, 133.2,
133.9, 136.1, 138.6, 140.5, 1419, 143.5, 146.2, 155.0, 162.3; HRMS (ESI-
TOF) m/z calcd for C25H24NO7S ([MþH]þ) 482.1273, found 482.1273.
Anal. calcd for C25H23NO7S C, 62.36; H, 4.81; N, 2.91, found C, 62.13;
H, 4.79; N, 2.99.
NMR (CDCl3, 400 MHz)
7.22e7.26 (1H, m), 7.29e7.35 (2H, m), 7.35e7.40 (2H, m), 8.27 (1H,
br s); 13C NMR (CDCl3, 75 MHz)
d
¼1.43 (9H, s), 3.69 (2H, s), 6.40 (1H, br s),
4.3. Catch-and-release experiment of compound 5
d¼28.1, 36.2, 82.0, 127.0, 129.0,
129.3, 134.4, 155.1, 167.5; HRMS (ESI-TOF) m/z calcd for
4.3.1. Michael addition of benzyl mercaptan. To a solution of catch-
C13H18N2NaO3S ([MþNa]þ) 305.0936, found 305.0945.
and-release reagent 5 (40.0 mg, 69.7
added benzyl mercaptan (60.0 L, 510
510 mol) with additional stirring at room temperature for 6 h.
mmol) in CH2Cl2 (700
m
L) were
m
mmol) and Et3N (71.0
mL,
4.2.3. N0-(2-Benzenesulfonylacetyl)hydrazinecarboxylic acid tert-bu-
tyl ester (10). To a solution of sulfide 9 (2.13 g, 7.56 mmol) in MeOH/
H2O (1:1 (v/v), 4.0 mL) was slowly added OxoneÒ (5.11 g,
8.31 mmol) at 0 ꢀC, and the resulting mixture was stirred at room
temperature for 15 h. The reaction mixture was extracted with
CH2Cl2, and the organic layer was washed with brine, dried over
Na2SO4 and concentrated in vacuo. The crude product was purified
by column chromatography (hexane/AcOEt¼1:2 (v/v)) and 1.75 g of
sulfone 10 (5.57 mmol, 74%) was obtained as white powder; 1H
m
After addition of H2O, the mixture was extracted with CH2Cl2. The
organic layer was dried over Na2SO4 and concentrated in vacuo. The
crude product was purified by column chromatography (hexane/
AcOEt¼4:1 (v/v)) and 40.0 mg of diastereomeric mixture 12
(56.6 m
mol, 82%) was obtained as colorless amorphousness. 1H NMR
(CDCl3, 400 MHz, peaks derived form a major isomer was depicted)
¼1.50 (9H, s), 1.83 (3H, s), 2.09 (3H, s), 3.78 (1H, d, J¼13.1 Hz), 3.94
d
(1H, d, J¼13.1 Hz), 4.74 (1H, d, J¼11.2 Hz), 5.02 (2H, s), 5.30 (1H, d,
J¼11.2 Hz), 5.76 (1H, s), 6.46 (1H, s), 7.00 (1H, br s), 7.13e7.20 (2H,
m), 7.20e7.30 (6H, m), 7.38 (2H, d, J¼7.3 Hz), 7.48 (1H, tt, J¼7.6 and
1.2 Hz), 7.72 (1H, t, J¼7.6 Hz), 7.95 (1H, d, J¼7.6 Hz), 8.15 (1H, dd,
J¼8.0 and 1.0 Hz), 8.25 (1H, br s); HRMS (ESI-TOF) m/z calcd for
C36H39N3NaO8S2 ([MþNa]þ) 728.2076, found 728.2053.
NMR (CDCl3, 400 MHz)
d
¼1.47 (9H, s), 4.12 (2H, s), 6.73 (1H, br s),
7.58 (2H, dd, J¼7.6 and 7.5 Hz), 7.69 (1H, tt, J¼7.5 and 1.1 Hz), 7.99
(2H, dd, J¼7.6 and 1.1 Hz), 8.71 (1H, br s); 13C NMR (CDCl3, 75 MHz)
d
¼28.1, 60.5, 82.4, 128.5, 129.4, 134.5, 138.0, 155.1,160.7; HRMS (ESI-
TOF) m/z calcd for C13H18N2NaO5S ([MþNa]þ) 337.0834, found
337.0833. Anal. calcd for C13H18N2O5S C, 49.67; H, 5.77; N, 8.91,
found C, 49.39; H, 5.55; N, 8.85.
4.3.2. UV-irradiation experiment of compound 12. Compound 12
(33
(>365 nm) for 5 min. To the reaction mixture were added 2,20-
dithiodipyridine (200 g, 0.90 mol) and Et3N (5.0 L, 35 mol) at
mg, 0.47 mmol) in MeOH (100 mL) was irradiated by UV
4.2.4. N0-{2-Benzenesulfonyl-3-[2,4-dimethyl-6-(2-nitrobenzyloxy)
phenyl]-(E)-acryloyl}hydrazinecarboxylic acid tert-butyl ester
m
m
m
m
(5). Piperidine (52.0
m
L, 531
m
mol) and AcOH (60.0
m
L, 1.06 mmol)
0 ꢀC, and the obtained mixture was shaken at room temperature for
2 h. Progress of the reaction was monitored by analytical HPLC.
Analytical HPLC conditions: linear gradient of solvent B in solvent
A, 50e80% over 30 min. Retention time: 12, 29.3 and 29.8 min; 13,
16.8 and 17.1 min; 4, 13.4 min; 14, 12.5 min. Intermediate 13 was
identified by LRMS (ESI-TOF) m/z calcd for [MþH]þ 571.2, found
571.2 (diastereomeric mixture). Compounds 4 and 14 were iden-
tified by RLMS and comparison of retention time with that of au-
thentic samples. Compound 4: LRMS (ESI-TOF) m/z calcd for
[MþH]þ 315.1, found 315.2. Compound 14:10 LRMS (ESI-TOF) m/z
calcd for [MþH]þ 234.0, found 234.2.
were added to a solution of sulfone 10 (1.84 g, 5.85 mmol) and
aldehyde 7 (1.50 g, 5.31 mmol) in toluene (60.0 mL). After refluxing
for 18 h, H2O was added to the reaction mixture at room temper-
ature. The mixture was extracted with AcOEt, and the organic layer
was washed with H2O and brine, dried over Na2SO4, and concen-
trated in vacuo. The crude product was purified by column chro-
matography (hexane/AcOEt¼3:1 (v/v)) and 2.62 g of catch-and-
release reagent 5 (4.50 mmol, 85%) was obtained as pale yellow
amorphousness; 1H NMR (CDCl3, 400 MHz)
d¼1.35 (9H, s), 2.30
(3H, s), 2.78 (3H, s), 5.45 (2H, s), 6.30 (1H, br s), 6.60 (1H, s), 6.71
(1H, s), 7.45 (1H, td, J¼7.6 and 1.2 Hz), 7.49e7.59 (3H, m), 7.62 (1H,
tt, J¼7.6 and 1.2 Hz), 7.71 (1H, d, J¼7.6 Hz), 8.03 (2H, dd, J¼7.6 and
1.2 Hz), 8.23 (1H, s), 8.27 (1H, d, J¼4.0 Hz); 13C NMR (CDCl3,
4.3.3. Caspase-9 model peptide (17). The protected peptidyl resin
was manually constructed using Fmoc-based solid phase peptide

8884
A. Shigenaga et al. / Tetrahedron 67 (2011) 8879e8886
synthesis on NovaSynÒ TGR resin (loading: 0.25 mmol/g). Fmoc-
protected amino acid derivatives (3 equiv) were successively con-
densed using N,N0-diisopropylcarbodiimide (3 equiv) in the pres-
ence of HOBt$H2O (3 equiv) in DMF for 2 h. The following side-
chain protective groups were used: Trt for cysteine and gluta-
resulting mixture was refluxed for 22 h and was quenched by ad-
dition of H2O at room temperature. The mixture was extracted with
AcOEt, and the organic layer was washed with H2O and brine, dried
over Na2SO4, and concentrated in vacuo. The crude product was
purified by column chromatography (hexane/AcOEt¼4:1 (v/v)) and
t
mine, Bu for glutamic acid, and Boc for lysine. Fmoc group was
170 mg of acrylate 16 (435
mmol, 94%) was obtained as pale yellow
removed by treatment of the resin with 20% (v/v) piperidine in DMF
for 10 min. The completed resin (41.0 mg) was treated with TFA/
thioanisole/m-cresol/1,2-ethanedithiol/Et3SiH/H2O (80:5:5:2.5:2.5:
5 (v/v), 2.0 mL) at room temperature for 2 h. After filtration, cooled
Et2O was added to the filtrate, and the resulting precipitate was
collected by centrifugation. The obtained precipitate was washed
with Et2O and purified by preparative HPLC to give model peptide
17 (1.0 mg, 10%). Analytical HPLC conditions: linear gradient of
solvent B in solvent A,1e80% over 30 min. Retention time: 13.0 min.
Preparative HPLC conditions: linear gradient of solvent B in solvent
A, 0e30% over 60 min. LRMS (ESI-Ion Trap) m/z calcd for [MþH]þ
1242.6, found 1242.8.
oil; 1H NMR (CDCl3, 400 MHz)
s), 3.57 (3H, s), 4.93 (2H, s), 6.73 (1H, s), 6.74 (1H, s), 7.54 (2H, t,
d
¼2.31 (3H, s), 2.32 (3H, s), 3.19 (3H,
J¼7.2 Hz), 7.61 (1H, t, J¼7.2 Hz), 8.01 (2H, d, J¼7.2 Hz), 8.17 (1H, s);
13C NMR (CDCl3, 100 MHz)
124.7, 128.2, 128.6, 133.1, 135.8, 138.6, 140.4, 141.8, 142.3, 154.1,
162.7; HRMS (ESI-TOF) m/z calcd for C20H22NaO6S ([MþNa]þ)
413.1035, found 413.1049.
d
¼20.1, 21.6, 51.9, 56.1, 94.5, 112.8, 119.1,
4.3.8. 3-Benzenesulfonyl-5,7-dimethylchromen-2-one (4). To a solu-
tion of acrylate 16 (49.0 mg, 125 mmol) in CH2Cl2 (1.00 mL) was
added TFA (1.00 mL), and the resulting mixture was stirred at room
temperature for 2 h. After addition of H2O, the mixture was
extracted with CH2Cl2. The organic layer was dried over MgSO4 and
4.3.4. Michael addition of model peptide 17. To a solution of model
concentrated in vacuo, and 39.0 mg of compound 4 (120
m
mol, 96%)
peptide 17 (2.00 mg,1.60
50 mM phosphate, 2.0 mL) was added catch-and-release reagent 5
(4.00 mg, 6.87
mol) in MeCN (2.0 mL). After incubation at 37 ꢀC
mmol) in sodium phosphate buffer (pH 7.8,
was obtained as yellow solid; 1H NMR (CDCl3, 400 MHz)
d¼2.43
(3H, s), 2.61 (3H, s), 6.99 (1H, s), 7.03 (1H, s), 7.55 (2H, t, J¼7.6 Hz),
7.63 (1H, t, J¼7.6 Hz), 8.14 (2H, d, J¼7.6 Hz), 8.93 (1H, s); 13C NMR
m
followed by HPLC purification, peptide conjugate 18 (0.78 mg, 28%)
was obtained as a diastereomeric mixture. Analytical HPLC condi-
tions: linear gradient of solvent B in solvent A, 10e95% over 30 min.
Retention time: 16.9, 17.2 or 17.4 min, respectively, for each di-
astereomer. Semi-preparative HPLC conditions: linear gradient of
solvent B in solvent A, 29e39% over 30 min. LRMS (ESI-TOF) m/z
calcd for [Mþ2H]2þ 912.4, found 912.1, 912.1 or 912.1, respectively.
(CDCl3, 75 MHz)
d
¼18.4, 22.1, 114.1, 115.1, 128.0, 128.9, 129.0, 129.3,
134.0, 138.8, 144.3, 144.4, 147.3, 155.1, 156.4; HRMS (ESI-TOF) m/z
calcd for C17H15O4S ([MþH]þ) 315.0691, found 315.0698.
4.4. Introduction of recognition moiety
4.4.1. General procedures for Boc-based solution phase peptide syn-
thesis. Deprotection procedure A: A substrate was treated with TFA
(5e10 mL/g substrate) for 1.5 h at 0 ꢀC. After evaporation, HCl/AcOEt
(4 M, 4 equiv) was added to the residues and the mixture was
stirred for 3 min and concentrated in vacuo. The obtained residue
was dried over KOH pellets under reduced pressure.
Deprotection procedure B: A substrate was treated with TFA
(5e10 mL/g substrate) for 1.5 h at 0 ꢀC. After evaporation, the ob-
tained residue was dried over KOH pellets under reduced pressure.
Purification procedure A: A material soluble in AcOEt was
extracted with AcOEt and the extract was washed successively with
5% (w/v) aqueous solution of NaHCO3 and brine, dried over Na2SO4,
and concentrated under reduced pressure.
4.3.5. UV-irradiation experiment of compound 18. A solution of
glutathione (reduced form, 0.25 M, 8.0
added to compound 18 (0.10 mg, 10 mol) in sodium phosphate
buffer (pH 7.8, 50 mM phosphate, 2 mM EDTA, 92 L), and the
resulting mixture was irradiated by UV (>365 nm) for 5 min. After
addition of an aqueous solution of TCEP$HCl (0.10 M, 30 L), the
mL) in MeCN (100 mL) was
m
m
m
reaction solution was incubated at 37 ꢀC and the reaction progress
was monitored by analytical HPLC. Analytical HPLC conditions:
linear gradient of solvent B in solvent A, 10e95% over 30 min. Re-
tention time: 19, 17.1 or 17.3 min, respectively, for each di-
astereomer; 4, 25.0 min; 17, 10.9 min. LRMS (ESI-Ion Trap) of
diastereomeric mixture of intermediate 19: m/z calcd for [MþH]þ
1688.7, found 1688.5.
Purification procedure B: A material insoluble in AcOEt was
precipitated by the addition of an appropriate solvent. The resulting
solid was filtered, washed successively with 5% (w/v) aqueous so-
lution of citric acid, 5% (w/v) aqueous solution of NaHCO3 and H2O,
and dried in vacuo.
4.3.6. 2-Methoxymethoxy-4,6-dimethylbenzaldehyde (15). To a stir-
red solution of phenol 68 (95.0 mg, 0.633 mmol) in CH2Cl2
(5.00 mL) were added chloromethyl methyl ether (127 mg,
1.58 mmol) and DIEA (0.550 mL, 3.15 mmol), and the resulting
mixture was stirred at room temperature for 23 h. The reaction was
quenched with 5% (w/v) aqueous solution of NaHCO3 at 0 ꢀC and
the resulting mixture was extracted with Et2O. The organic layer
was washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified by column chromatography
(hexane/AcOEt¼4:1 (v/v)) and 99.0 mg of aldehyde 15 (0.510 mmol,
81%) was obtained as colorless oil. 1H NMR (CDCl3, 400 MHz)
4.4.2. [(1S)-1-(N0-{2-Benzenesulfonyl-3-[2,4-dimethyl-6-(2-
nitrobenzyloxy)phenyl]-(E)-acryloyl}hydrazinocarbonyl)-3-
carbamoylpropyl]carbamic acid tert-butyl ester (21). Compound 5
(2.00 g, 3.43 mmol) was subjected to deprotection procedure A. To
solution of the resulting residue in DMF (10 mL), Et3N (947
mL,
6.80 mmol), Boc-Q-ONp (1.25 g, 3.43 mmol), and HOBt$H2O
(701 mg, 3.43 mmol) were added at 0 ꢀC. The reaction mixture was
stirred at room temperature for 4.5 h. After concentration in vacuo,
the resulting residue was subjected to purification procedure A and
the obtained material was purified by column chromatography
(CHCl3/MeOH¼19:1 (v/v)) and 2.06 g of compound 21 (2.90 mmol,
d
¼2.34 (3H, s), 2.55 (3H, s), 3.52 (3H, s), 5.26 (2H, s), 6.69 (1H, s),
6.86 (1H, s), 10.61 (1H, s); 13C NMR (CDCl3, 100 MHz)
d
¼21.4, 22.0,
56.3, 94.5, 112.8, 121.4, 126.0, 141.6, 145.5, 160.9, 191.4; HRMS (ESI-
TOF) m/z calcd for C11H14NaO3 ([MþNa]þ) 217.0841, found 217.0835.
85%) was obtained as colorless amorphousness; ½a D17
ꢃ16.7 (c 1.02,
ꢂ
CHCl3); 1H NMR (CDCl3, 400 MHz)
d
¼1.37 (9H, s),1.70e1.90 (2H, m),
4. 3 . 7 . 2 - B e n z e n es ul fo nyl- 3- ( 2- me t h ox y me t h ox y- 4, 6 -
2.06e2.18 (2H, m), 2.67 (6H, s), 4.02e4.16 (1H, br m), 5.38 (1H, d,
J¼15.4 Hz), 5.42 (1H, d, J¼15.4 Hz), 5.50 (1H, d, J¼7.6 Hz), 6.00 (1H,
br s), 6.37 (1H, br s), 6.60 (1H, s), 6.68 (1H, s), 7.44 (1H, t, J¼8.0 Hz),
7.46e7.55 (3H, m), 7.58 (1H, tt, J¼7.2 and 1.2 Hz), 7.67 (1H, d,
J¼7.6 Hz), 7.97 (2H, d, J¼7.6 Hz), 8.08 (1H, dd, J¼8.0 and 1.2 Hz), 8.21
dimethylphenyl)acrylic acid methyl ester (16). Piperidine (2.0
200 mol) and AcOH (2.0 L, 35 mol) were added to a solution of
aldehyde 15 (90.0 mg, 463 mol) and phenylsulfonylacetic acid
methyl ester (97.0 mg, 453 mol) in toluene (5.00 mL). The
mL,
m
m
m
m
m

A. Shigenaga et al. / Tetrahedron 67 (2011) 8879e8886
8885
(1H, s), 8.74 (1H, br s), 9.96 (1H, br s); 13C NMR (CDCl3, 75 MHz)
followed by filtration, the filtrate was concentrated in vacuo. The
residue was subjected to purification procedure B and reprecipi-
d
¼20.0, 21.6, 28.2, 29.2, 31.3, 51.7, 66.9, 80.0, 110.7, 117.8, 124.2,
124.6, 128.1, 128.3, 128.4, 129.1, 133.2, 133.6, 133.9, 136.4, 138.6,
139.4, 142.0, 146.6, 155.1, 155.7, 159.1, 169.6, 175.6; HRMS (ESI-TOF)
tation form DMF/AcOEt to give 170 mg of peptide 24 (238 mmol,
88%) as white powder; LRMS (ESI-Ion Trap) m/z calcd for [MþH]þ
m/z calcd for C34H39N5NaO10
S
([MþNa]þ) 732.2315, found
712.4, found 712.4.
732.2339.
4.4.5. Synthesis of protected peptide 25. To a solution of Boc-
R(Mts)-OH (3.15 g, 6.29 mmol) in THF (20 mL) were added isobutyl
chloroformate (816 mL, 6.29 mmol) and Et3N (1.67 mL, 12.0 mmol)
4.4.3. Synthesis of peptide conjugate 22 and 23. Typical procedure.
Compound 21 (94.3 mg, 133 mol) was treated with TFA (1 mL) at
m
0 ꢀC for 1.5 h. After evaporation, HCl/AcOEt (4 M, 200
mL) was added
at ꢃ21 ꢀC, and the reaction mixture was stirred at same tempera-
ture for 20 min. The resulting mixture was added to a solution of
TFA$H-L-OPac (prepared from Boc-L-OPac16 (2.00 g, 5.72 mmol)
according to deprotection procedure B) in DMF at 0 ꢀC. After
overnight reaction at room temperature, the solution was con-
centrated in vacuo. The product was subjected to purification
procedure A and reprecipitation from Et2O to give 2.51 g of Boc-
R(Mts)L-OPac (3.64 mmol, 64%).
to the residue at 0 ꢀC. The mixture was stirred at same temperature
for 3 min, and then evaporated. The obtained residue was dissolved
in DMF (1 mL), and to the solution were added Et3N (38
m
L,
mol), HOBt$H2O (29 mg,
m
mol) at 0 ꢀC. The reaction
273
140
m
mol), peptide 24 (100 mg, 140
m
mmol), and EDC$HCl (27 mg, 140
mixture was stirred at room temperature overnight. After con-
centration in vacuo, the resulting residue was subjected to purifi-
cation procedure B and reprecipitation from DMF/AcOEt to give
119 mg of the protected catch-and-release reagent (91.2 mmol,
Boc protected amino acids were coupled onto Boc-R(Mts)L-OPac
(2.40 g, 3.49 mmol) as similar to that described in Section 4.4.4.
Procedure and yield are summarized below.
70%) as white powder. Then, the product (50.0 mg, 383
treated with TMSBr (215 L), thioanisole (192 L), m-cresol
(81.1
L), and TFA (1.21 mL) at 0 ꢀC for 1.5 h. To the resulting mixture
mmol) was
m
m
Boc-V-OH: deprotection of substrate¼procedure A; purifica-
tion¼procedure A; reprecipitation form hexane; 97% yield.
Boc-T(Bzl)-OH: deprotection of substrate¼procedure A; purifi-
cation¼procedure B; reprecipitation from AcOEt/hexane; 84% yield.
Boc-A-OH: deprotection of substrate¼procedure A; purifica-
tion¼procedure B; reprecipitation form Et2O; 95% yield.
Acetyl capping and removal of Pac group were performed as
similar to that described in Section 4.4.4. Acetyl cap: deprotection
procedure B; purification procedure B; reprecipitation from DMF/
AcOEt; 93% yield. Removal of Pac group: purification procedure B;
reprecipitation from DMF/AcOEt; 82% yield; LRMS (ESI-Ion Trap) m/
z calcd for [MþH]þ 873.5, found 873.5.
m
was added cooled Et2O and the generated precipitate was collected
by centrifugation. The precipitate was washed with Et2O and pu-
rified by preparative HPLC to give 12.3 mg of peptide conjugate 22
(10.9 mmol, 29%). Protected precursor of compound 22: LRMS (ESI-
Ion Trap) m/z calcd for [MþK]þ 1341.5, found 1341.6. Protected
precursor of compound 23: LRMS (ESI-Ion Trap) m/z calcd for
[MþH]þ 1464.6, found 1464.6. Compound 22: analytical HPLC
conditions: linear gradient of solvent B in solvent A, 30e80% over
30 min. Preparative HPLC conditions: linear gradient of solvent B in
solvent A, 40e50%. LRMS (ESI-Ion Trap) m/z calcd for [MþH]þ
1123.5, found 1123.8. Compound 23: analytical HPLC conditions:
linear gradient of solvent B in solvent A, 30e80% over 30 min.
Preparative HPLC conditions: linear gradient of solvent B in solvent
A, 37e47%. LRMS (ESI-Ion Trap) m/z calcd for [Mþ2H]þ 596.8, found
597.0.
Acknowledgements
We thank Prof. Dr. K. Miyamoto, The University of Tokushima,
for his support in X-ray analysis. This research was supported in
part by a Grant-in-Aid for Scientific Research on Innovative Areas
‘Fusion Materials: Creative Development of Materials and Explo-
ration of Their Function through Molecular Control’ (No. 2206)
from the Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan (MEXT), Young Scientists (B), Scientific Research (B),
and Challenging Exploratory Research from the Japanese Society
for the Promotion of Science (JSPS). The Takeda Science Foundation
and the Astellas Foundation for Research on Metabolic Disorders
are also acknowledged. J.Y. is grateful for a SUNBOR Scholarship.
4.4.4. Synthesis of protected peptide 24. To a solution of H-L-OPac
(prepared from Boc-L-OPac16 (2.00 g, 5.72 mmol) according to
deprotection procedure A. Pac: phenacyl), Et3N (1.67 mL,
12.0 mmol), Boc-V-OH (1.37 g, 6.29 mmol) and HOBt$H2O (1.30 g,
6.29 mmol) in DMF (20 mL), EDC$HCl (1.21 g, 6.29 mmol) was
added at 0 ꢀC. The reaction mixture was stirred at room tempera-
ture overnight. After concentration in vacuo, the resulting residue
was subjected to purification procedure A, and reprecipitation form
hexane to give 1.30 g of Boc-VL-OPac (2.89 mmo, 46%).
Boc protected amino acids were coupled onto Boc-VL-OPac
(1.30 g, 2.89 mmol) as similar to that described above. Procedure
and yield are summarized below.
Boc-A-OH: Deprotection of substrate¼procedure A; purifica-
tion¼procedure B; reprecipitation form AcOEt; 85% yield.
Boc-S(Bzl)-OH: deprotection of substrate¼procedure A; purifi-
cation¼procedure B; reprecipitation from CHCl3/Et2O; 89% yield.
Boc-T(Bzl)-OH: deprotection of substrate¼procedure A; purifi-
cation¼procedure B; reprecipitation form CHCl3/Et2O; 80% yield.
To a solution of TFA$H-T(Bzl)S(Bzl)AVL-OPac (prepared from
Supplementary data
Crystallographic data of 11 has been deposited with the Cam-
bridge Crystallographic Data Center as supplementary publication
number CCDC 839726.
Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2011.09.062.
References and notes
Boc-T(Bzl)S(Bzl)AVL-OPac (350 mg, 338
deprotection procedure B) in pyridine/DMF (1:1 (v/v), 4 mL) was
added Ac2O (319 L, 3.38 mmol). The reaction mixture was stirred
at room temperature overnight. After concentration in vacuo, the
resulting residue was subjected to purification procedure B and
reprecipitation from DMF/AcOEt to give 226 mg of Ac-T(Bzl)S(Bzl)
AVL-OPac (272 mmol, 81%). It was suspended in AcOH/DMF (9:1 (v/
v), 3.50 mL), and Zn dust (356 mg, 5.44 mmol) was added to the
suspension. After stirring the mixture at room temperature for 3 h
mmol) according to
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m

8886
A. Shigenaga et al. / Tetrahedron 67 (2011) 8879e8886
4. Reviews including target enzyme selective S-alkylation in living cells, see: (a)
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see: (a) Perez, D. I.; Palomo, V.; Perez, C.; Gil, C.; Dans, P. D.; Luque, F. J.; Conde, S.;
Marinez, A. J. Med. Chem. 2011, 54, 4042e4056; (b) Lee, H.-M.; Xu, W.; Lawrence,
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Bhatia, M.; Pellechia, P. J.; Thompson, P. R. J. Am. Chem. Soc. 2006, 128,
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synthesis.
12. Earnshaw, W. C.; Martins, L. M.; Kaufmann, S. H. Annu. Rev. Biochem. 1999, 68,
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13. Whereas a glutathione was added to sequester photo-generated derivatives of
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added to reduce them.
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