Synthesis of Staudinger-type molecular probe for catch-and-release purification of the binding protein for potassium isolespedezate, a leaf-closing substance of leguminous plant

Article abstract of DOI:10.1246/cl.2008.52

We synthesized azide-containing photoaffinity probe 1 based on the structure of potassium isolespedezate. This probe can be used for catch-and-release-mechanism purification of binding protein for 1: photo-crosslinking with 1 gave azide-labeled receptor which can be captured by phosphane-linked gel matrix by the Staudinger ligation. After washing the gel, the caught binding protein can be released by the reductive cleavage of disulfide bond in 1. This process can be used as a convenient method for the purification of binding protein for bioactive natural product. Copyright

Full text of DOI:10.1246/cl.2008.52

52
Chemistry Letters Vol.37, No.1 (2008)
Synthesis of Staudinger-type Molecular Probe for Catch-and-release Purification of the Binding
Protein for Potassium Isolespedezate, a Leaf-closing Substance of Leguminous Plant
Tomohiko Fujii,1 Nobuki Kato,1 Izumi Iwakura,2 Yoshiyuki Manabe,1 and Minoru Uedaꢀ1
1Department of Chemistry, Tohoku University, Aoba-ku, Sendai 980-8578
2Department of Applied Physics and Chemistry and Institute for Laser Science, University of Electro-Communications,
1-5-1 Chofugaoka, Chofu, Tokyo 182-8585
(Received October 3, 2007; CL-071096; E-mail: ueda@mail.tains.tohoku.ac.jp)
We synthesized azide-containing photoaffinity probe 1
1)
Matrix
Ph2P
O
Ph2P
based on the structure of potassium isolespedezate. This probe
can be used for catch-and-release-mechanism purification of
binding protein for 1: photo-crosslinking with 1 gave azide-la-
beled receptor which can be captured by phosphane-linked gel
matrix by the Staudinger ligation. After washing the gel, the
caught binding protein can be released by the reductive cleavage
of disulfide bond in 1. This process can be used as a convenient
method for the purification of binding protein for bioactive
natural product.
Matrix
MeOOC
H
3
Linker
N
Linker N3
S
S
S
S
O
2) Washing
1) DTT, H2O
2) Elution
h
ν
O
OH
SH
O
S
HO
OH
H
H
N
BP
H2N
O
N
N
H
S
O
N3
O
O
O
OOC
Azide-containing phtoaffinity probe (1)
BP: Binding Protein
Circadian rhythmic leaf-closing and leaf-opening move-
ments called nyctinasty are widely observed in legumes. It is
well known that Charles Darwin is a pioneer in this field.1 We
identified chemical factors controlling this movement, and found
that their binding proteins are involved in the control of nycti-
nasty.2,3 The bioorganic studies on this binding protein needs
its supply. Thus, an efficient method for the purification of the
binding protein is required.
Figure 1. Catch-and-release purification of binding protein
using probe 1.
Table 1. Comparison of the reactivity of several azides in the
Staudinger ligation
H
N
Table 1
rt
+
COOMe
PPh2
N3
R
R
Ph2P
O
O
Azide (4–9)
(0.1 M)
95% CH3CNaq.
2
The Staudinger ligation is used as truly chemoselective reac-
tion for the preparation of bioconjugates which can be used even
in the complex environment of living cell.4 The azide group re-
acts with phosphane to form phospha-aza-ylide, which is trapped
by ester to give a stable amide bond. The azide group is small
and can be introduced into a bioactive molecule without de-
crease in bioacitivity. And it is more important that an azide
group can be used in a highly chemoselective modification of
target protein because an azide group is absent in nature. We
designed azide-containing photoaffinity probe 1 for chemical
modification on the binding protein of potassium isolespedezate,
a leaf-opening factor of Cassia mimosoides L.2 An azide group is
smaller than the widely used biotin group which can be trapped
by streptoavidin, thus probe 1 is expected to have higher
bioactivity than biotin-labeled probes. After photo-crosslinking,
probe 1 covalently binds to the corresponding binding protein to
give azide-labeled binding protein which can be trapped by gel
matrix 3 by the Staudinger ligation (Figure 1). After washing,
a disulfide bond in the linker moiety of 1 can be dissociated
by mild reduction to release biding protein. This catch-and-
release process can be used as a convenient method for the
purification of binding protein.
(0.1 M)
Reaction
time/h
Reaction
time/h
Entry
1
Azide
Entry
Azide
O
N3
4:
5:
>26
4
5
7:
8:
2.5
N3
N
H
O
O
O
N3
N3
N
H
N3
O
2
3
9
11
O
N3
N
H
N
Me
6:
6
9:
2.5
9.5
action, whereas ꢀ-azideacetate 5, ꢁ-azideacetamide 8, and N-
methyl ꢀ-azideacetamide 9 were moderately reactive. ꢀ-Azi-
deacetamides 6 and 7 were found to be highly reactive in the
Staudinger ligation and quantitatively gave corresponding amide
at rt within 2.5 h. This result strongly suggested that hydrogen
atom in an amide would be important for the acceleration of
the Staudinger ligation.
DFT calculation (B3LYP/6-31Gꢀ) on the transition state of
6 strongly suggested this hypothesis (Figure 2).5 A five-mem-
bered ring system was formed between an amide hydrogen
and the azide nitrogen.
On the design of 1, we used an ꢀ-azideacetamide unit as a
highly reactive azide unit in the Staudinger ligation. Before de-
signing the probe, we compared the reactivities of several azide
compounds in the Staudinger ligation reaction. We compared the
time until the disappearance of each azide in the reaction mixture
for the comparison of reactivity of each azide. As shown in
Table 1, an alkyl azide 4 was almost inert in the Staudinger re-
Thus, we designed and synthesized probe 1 containing
an ꢀ-azideacetamide group at the terminal of linker moiety
(Scheme 1). Properly protected azide compound 103 was re-
duced and coupled with linker moiety containing an ꢀ-azideace-
tamide group at the terminal 11.6 Introduction of benzophenone
as a photoaffinity labeling group and successive deprotection
gave probe 1.7 Probe 1 have a leaf-opening activity against a leaf
Copyright Ó 2008 The Chemical Society of Japan

Chemistry Letters Vol.37, No.1 (2008)
53
O
O
Ph3P
O
3
gel
N
H
N
H
1
+
MeOOC
N
N
P
H
N
1) H2O, rt
2) Washing
N
OH
C
C
O
Ph2P
O
O
HO
H
N
H
N
O
S
N
H
O
S
N
H
O
O
NH2
H
N
HO
O
O
gel
COO
O
Figure 2. Proposed transition state of 6 in the Staudinger reac-
tion at the B3LYP/6-31Gꢀ level.
O
15
1) 1 mM DTTaq, 95 °C, 5 min
(a)
2) Eluted by H2O
Azide unit
OH
OH
1) Pd-CaCO3 / H2,
MeOH
OH
N3
NH
O
HO
HO
HO
HO
O
O
HO
2) 11, DCC, HOBt,
DMF
O
HS
O
N
H
O
O
NPhth
HO
COOtBu
NPhth
COOtBu
61% in 2 steps
NH2
Detected by LC-(+)-ESI-MS
at m/z 609
COO
O
10
12
1) TFA (neat)
16
1
2) H2N-NH2/H2O, EtOH
3) 4-bromomethyl-
benzophenone,
NEt3, DMF
Scheme 2. Model experiments of catch-and-release.
11% in 3 steps
1 ꢁ 10ꢂ5 M of 1 and gel 3 can proceed in high yield in an aque-
ous solution, and probe 1 can be released by mild reductive con-
ditions. Thus, probe 1 would be a powerful tool for the purifica-
tion of binding protein of 1, and a similar method can be widely
applicable for the cases of other bioactive natural products.
O
H
N
H
N
S
:
Azide unit
N3
O
S
O
O
(b)
N3CH2COOH,
DCC, HOBt
O
O
H2N
NHBoc
N3
O
N
NHBoc
COOH
CH2Cl2
41%
H
13
14
This work was supported by Grant-in-Aid for Scientific
Research on Priority Area No. 16073216 from MEXT for
MU, Grant-in-Aid from JSPS for TF and II, ITC of UEC for
DFT calculation.
1) TFA:CH2Cl2
= 1:19
O
O
N3
O
S
N
N
S
H
H
2)
HOOC
S
COOH
S
11
DCC, HOBt, DMF
52% in 2 steps
References and Notes
1
C. Darwin, The Power of Movement in Plants. Third Thou-
sand., John Murray, London, 1882.
Scheme 1. (a) Synthesis of azide-containing photoaffinity
probe 1; (b) Synthesis of the azide unit containing an ꢀ-azidea-
cetamide group.
2
a) M. Ueda, S. Yamamura, Angew. Chem., Int. Ed. 2000, 39,
1400. b) M. Ueda, Y. Nakamura, Nat. Prod. Rep. 2006, 23,
548. c) M. Ueda, Y. Nakamura, Plant Cell Physiol. 2007,
48, 900.
of Cassia mimosoides at 5 ꢁ 10ꢂ5 M which is twice as strong as
the previously reported biotin-linked photoaffinity probe.3
We carried out a model examination of catch-and-release
process using 1 (Scheme 2). We synthesized gel matrix 3 using
corresponding phosphane and activated agarose gel (Affi-gel 10,
Bio-Rad Co., Ltd.). This gel matrix 3 (1 ꢁ 10ꢂ3 M equivalence)
was mixed with aqueous solution of 1 (1 ꢁ 10ꢂ5 M) at rt. Time
course change in the level of remaining probe 1 was quantita-
tively analyzed by LC-ESI MS (Bruker esquire 4000, Bruker
Daltonics Co., Ltd.). About 82% of probe 1 was consumed
and would be linked with gel 3 within 4 h under these conditions,
and almost all of 1 was consumed within 8 h. No degradation
product of probe 1 was detected by LC-MS/MS analyses of re-
action mixture. And we also confirmed that probe 1 which is co-
valently linked to gel 3 can be released by 1 mM DTT(aq) treat-
ment (95 ꢃC, 5 min) to give thiol 16. This result clearly demon-
strated the success in catch-and-release process using the
Staudinger ligation and successive reductive cleavage of disul-
fide bond.
3
4
5
6
T. Fujii, Y. Manabe, T. Sugimoto, M. Ueda Tetrahedron
2005, 61, 7874.
a) E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007. b) M.
Kohn, R. Breinbauer, Angew. Chem., Int. Ed. 2004, 43, 3106.
¨
Gaussian 03, Revision D.02, Gaussian, Inc., Wallingford CT,
2004.
1
1: H NMR (300 MHz, CD3OD, rt): ꢂ 7.81–7.59 (9H, m),
7.48 (2H, d, J ¼ 8:8 Hz), 7.18 (1H, s), 6.73 (2H, d,
J ¼ 8:8 Hz), 5.19 (1H, d, J ¼ 8:4 Hz), 4.74 (2H, m), 4.51
(1H, d, J ¼ 13:2 Hz), 4.02 (1H. dd, J ¼ 3:2, 11.0 Hz), 3.86
(3H, s), 3.69 (1H, m), 3.62 (2H, s), 3.60–3.39 (14H, m)
13C NMR (150 MHz, CD3OD, rt): ꢂ198.5, 172.5, 172.0,
171.3, 161.2, 140.5, 139.2, 138.0, 135.0, 134.8, 132.3,
131.7, 130.4, 128.9, 126.2, 117.1, 102.4, 76.0, 72.3, 72.2,
71.1, 70.5, 62.5, 53.8, 44.0, 43.4, 42.3, 41.6, 41.1. HR-
ESI-MS (positive) m=z: [M + Na]þ calcd for C39H45O12N7-
NaS2, 890.2460; found 890.2464, IR (film) ꢃ: 3297, 2113,
D
Our results showed that the Staudinger ligation between
1656, 1606 cmꢂ1; ½ꢀꢄ23 ꢂ6:8ꢃ (c 0.5, MeOH).
Published on the web (Advance View) December 1, 2007; doi:10.1246/cl.2008.52