Synthesis of a new water soluble 2,2-bifunctionalized spin label and its application to troponin C

Article abstract of DOI:10.1246/cl.2006.834

A new water soluble 2,2-bifunctionalized spin label (2,2E-BSL) having pyrrolidine nitroxide moiety was synthesized starting from a nitro compound with two hydroxymethyl group converted to the linkers of the 2,2E-BSL, and was applied to label troponin C (TnC). Labeled TnC through two linkages were successfully isolated, and 2,2E-BSL was proved to be immobilized on TnC by EPR measurement. Copyright

Full text of DOI:10.1246/cl.2006.834

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34
Chemistry Letters Vol.35, No.8 (2006)
Synthesis of a New Water Soluble 2,2-Bifunctionalized Spin Label
and Its Application to Troponin C
1
2
3
3
Ã1
Tasuku Hirayama, Masayasu Taki, Motoyoshi Nakamura, Toshiaki Arata, and Yukio Yamamoto
Graduate School of Human & Environmental Studies, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501
1
2
Graduate School of Grobal Environmental Studies, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501
3
Department of Biological Sciences, Graduate School of Science, Osaka University,
1-1 Machikaneyama, Toyonaka, Osaka 560-0043
(Received April 21, 2006; CL-060480; E-mail: yamamoto y@mbox.kudpc.kyoto-u.ac.jp)
A new water soluble 2,2-bifunctionalized spin label (2,2E-
hydroxymethyl groups which were converted to the linkers
of 2,2-BSL. The distance between each reaction center was
preliminarily examined to fit that of two cysteine residues on
TnC by molecular modeling.
BSL) having pyrrolidine nitroxide moiety was synthesized
starting from a nitro compound with two hydroxymethyl group
converted to the linkers of the 2,2E-BSL, and was applied to
label troponin C (TnC). Labeled TnC through two linkages
were successfully isolated, and 2,2E-BSL was proved to be
immobilized on TnC by EPR measurement.
In Scheme 1, 2-bromo-2-nitro-1,3-propanediol (1) was treat-
ed with 2,2-dimethoxypropane to yield bromonitrodioxane 2 in
82% yield. Reduction of 2 to remove bromine atom by NaBH4
provided nitrodioxane 3 in 90% yield. By Michael addition with
methyl vinyl ketone, nitroketone 4 was obtained in quantitative
yield. Nitrone 5 was obtained as a white powder in 56% yield by
treating 4 with zinc dust and acetic acid in ethanol. The high
purity of 5 is critical to the next reaction, that is, the Grignard
reaction was unsuccessful which nitrone 5 contained a small
amount of impurities. Reaction of nitrone 5 with MeMgI fol-
2
lowed by oxidation with MnO provided nitroxide radical 6 in
53% yield. In an acidic deprotection of nitroxide 6, decomposi-
tion of 6 was observed. After several unsuccessful attempts, we
finally obtained diol 7 in 72% yield when 6 was treated with 80%
AcOH. Diol 7 was converted to 8 by treating with NaH and THP
ether of 2-bromoethanol. After deprotection of THP by oxalic
For clarifying a physiological protein motion, spin labels
1
,2
can be employed as an orientational probe. Most of spin labels
3
today are monofunctionalized spin labels (MSL) by which pro-
tein can be modified through only one linkage. In this case, their
free mobility on the protein interferes with accurate investiga-
tion of the conformational change of a protein. On the other
hand, bifunctionalized spin labels (BSL) can elucidate the pro-
tein motion more exactly because of their rigid binding through
two linkages. Some types of BSL which are composed of pyrro-
4
,5
lidine nitroxide bearing 3,4-substituents (3,4-BSL) or 2,2-sub-
stituents (2,2-BSL) have been synthesized and utilized to EPR
measurement of protein. Recently, we have reported the synthe-
5
sis of C2-chiral 3,4-BSL, (R,R)-BSL and (S,S)-BSL, and the la-
O
O
O
O
beling of troponin C with them. EPR spectra of the protein la-
beled with the C2-chiral 3,4-BSL revealed that these spin labels
were definitely immobilized on the ꢀ-helix. However, EPR sig-
nals would not reflect the small motion of the protein because an
unpaired electron orbital on nitrogen atom of the 3,4-BSL lo-
cates almost perpendicularly to the ꢀ-helix. On the other hand,
a
Br
b
HO
OH
NO2
O N
2
Br
c
O N
2
1
2
3
O
O
O
O
O
d
f
N
O
O N
2
2,2-BSLs can bind to a protein with its unpaired electron orbital
4
5
parallel to ꢀ-helix and should be more sensitive for structural
changes of the protein. Hideg et al. have reported a probe
O
O
OH
OH
e
g
2
,2-BSL (2,2H-BSL) containing two aromatic rings between ac-
6
N
N
O
tive thiol group and pyrrolidine nitroxide. However, when we
applied it for labeling TnC, most of it precipitated in the aqueous
solution of the protein and the labeling was unsuccesful. In the
course of investigating dynamics of TnC, which is the key
O
6
7
7
X
X
O
O
8
9
OTHP
8
N
h
i
protein regulating muscle contraction and relaxation, BSL
OH
I
O
which can bind to TnC at two cysteine residues is essential to
determination of its orientation on actin filament. In this context,
we designed a new 2,2-BSL having 2,2-substituents on pyrroli-
dine nitroxide and high water solubility for more accurate inves-
tigation of TnC dynamics. This is composed of 2,2-substituted
pyrrolidine nitroxide having ether bonds in their two linkers.
Although 2,2-disubstitued nitroxides are generally prepared by
the addition of the Grignard reagent to nitrones, several reagents
bearing protected functional groups were examined and found
to yield the desired products in poor yields. Now, we designed
a synthetic route starting from a nitro compound having two
X
10
j
2
,2E-BSL SSO CH
2 3
Scheme 1. Reagents and Conditions; (a) 2,2-dimethoxypro-
pane, CSA, room temp, 82%; (b) NaBH4, MeOH, rt, 90%; (c)
methyl vinyl ketone, tetramethylguanidine, MeOH, rt, quant;
(
d) Zn, AcOH, EtOH, rt, 56%; (e) MeMgI, ether, rt, and then
ꢀ
O2, MnO2, CHCl3, rt, 53%; (f) 80% AcOH, 60 C, 72%;
ꢀ
(
g) NaH, Br(CH2)2OTHP, DMF, 80 C, 17%; (h) oxalic acid,
dioxane, water, rt, quant; (i) MsCl, pyridine, rt, and then NaI,
acetone, reflux, 90%; (j) NaSSO CH , DMSO, rt.
2
3
Copyright ꢀ 2006 The Chemical Society of Japan

Chemistry Letters Vol.35, No.8 (2006)
835
5
29 gauss
BSL (ca. 50 G). The decrease of 2Azz value was attributed to
3
flexibility of sp atoms of which the linkers of 2,2E-BSL consist.
(
a)
Additionally, the distance between two reactive groups is a little
longer than that of two cysteine residues of TnC. These two
factors increase the flexibility of spin lobe of 2,2E-BSL on
TnC. After more than a few hours storage, the signal intensity
of the labeled TnC became weak and the signals derived from
the free BSL were observed although excess BSL and incom-
plete labeled TnC were removed by gel filtration and HPLC.
Methanethiosulfonyl groups bind to cysteine residue through
disulfide bond, and therefore, it is possible that a dissociation
of the disulfide bonds between 2,2E-BSL and TnC might occur
during storage. BSL which binds through more stable bond is
necessary for more sensitive measurement and under investiga-
tion. For example, iodoacetamide group and maleimide group
are preferable.
(b)
45 gauss
In conclusion, we synthesized a new 2,2-substituted bifunc-
tionalized spin label 2,2E-BSL in reasonable overall yield and
applied it for spin labeling of TnC. In our synthetic route, two
hydroxymethyl groups of the commercially available starting
material were converted to the 2,2-substituents of pyrrolidine
nitroxide. Moreover, BSLs with variable linker length can be
synthesized by using other alkyl halides for diol 7. In labeling
experiment, 2,2E-BSL was found to have suitable water solubil-
ity. In EPR measurement, it was confirmed that 2,2E-BSL bound
to TnC with two linkages, and that completely labeled TnC was
isolated by anion exchange HPLC.
3
380
3400
3420
3440
3460
3480
[G]
Figure 1. EPR spectra of (a) 2,2E-BSL and (b) 2,2E-BSL-
labeled TnC.
acid, successive mesylation and substitution gave diiodide 10 in
an overall yield of 90%. Finally, diiodide 10 was treated with
NaSSO2CH3 to yield 2,2E-BSL which was directly employed
for the labeling experiment.
A chicken skeletal TnC mutant (S94C) having two cysteine
residues (94cys, 101cys) was expressed in E. coli cells. This
TnC mutant was employed for the label experiment with 2,2E-
BSL. The two cysteine residues were located on central E-helix
of TnC and the distance between the two S atoms is calculated to
We thank Dr Y. Maeda for giving us opportunity to perform
this collaborative work. This work is supported by Special
Coordination Funds for promoting Science and Technology
from Japanese Government.
˚
be 11 A.
The TnC mutant was purified with Sepharose Q anion ex-
change chromatography (2:0 Â 10 cm, Amersham Biosciences).
The purified TnC was treated with 5 mM of DTT in order to
reduce disulfide bonds and oxidized thiol, and then, DTT was
removed by Sephadex G-25 desalting column (1:5 Â 20 cm,
Amersham Biosciences). To the TnC solution, 2,2E-BSL was
added (final concentration of 100 mM). In contrast with labeling
with 2,2H-BSL, no precipitation appeared with 2,2E-BSL, ow-
References and Notes
1
T. Arata, in Current Methods in Muscle Physiology, ed. by
H. Sugi, Oxford University Press, 1998, p. 223.
2
a) L. J. Berliner, Spin Labeling Theory and Applications,
Academic Press, 1976. b) W. L. Hubbell, D. S. Cafiso, C.
Altenbach, Nat. Struct. Biol. 2000, 7, 735. c) L. Columbus,
W. L. Hubbell, Trends Biochem. Sci. 2002, 27, 288.
a) L. Coumbus, T. K a´ lai, J. Jek o¨ , K. Hideg, W. L. Hubbel,
Biochemistry 2001, 40, 3828. b) B. A. J. Baumann, B. D.
Hambly, K. Hideg, P. G. Fajer, Biochemistry 2001, 40, 7868.
a) T. K a´ lai, B. Rozsnayai, G. Jerkovich, K. Hideg, Synthesis
1994, 1079. b) R. M. L o¨ sel, R. Philipp, T. K a´ lai, K. Hideg,
W. E. Trommer, Bioconjugate Chem. 1999, 10, 578. c)
M. D. Wilcox, J. W. Parce, M. J. Thomas, D. S. Lyles, Bio-
chemistry 1990, 29, 5734. d) T. K a´ lai, M. Balog, J. Jek o¨ , W.
L. Hubbel, K. Hideg, Synthesis 2002, 2365.
ꢀ
ing to high water solubility. After the incubation at 4 C for
48 h, the reaction was stopped, and then unlabeled spin label
3
4
was removed by using Sephadex G-25. It is important for accu-
rate measurement to remove the mono-labeled TnC from the
mixture of labeled TnCs. For this purpose, Activated Thiol
Sepharose 4B (Amersham Bioscience) was employed in our
5
previous work. In the present experiment, labeled TnC through
two linkages was isolated successfully by applying anion ex-
change HPLC (UNO-Q, Bio-Rad). For EPR measurement, the
ꢀ
samples were concentrated to 50 mM and stored at 4 C.
The EPR spectrum of 2,2E-BSL itself showed a typical
sharp triplet signal for nitroxide radical (Figure 1a) from which
5
6
7
S. Chatani, M. Nakamura, H. Akahane, N. Kohyama, M.
Taki, T. Arata, Y. Yamamoto, Chem. Commun. 2005, 1880.
T. K a´ lai, J. Jek o¨ , W. L. Hubbel, K. Hideg, Synthesis 2003,
2084.
Although labeling experiment using 2,2H-BSL has not been
reported, we have demonstrated. After treated with DTT,
2,2H-BSL was added to a solution of the mutant TnC
(S94C) in 10 mM MOPS buffer (pH 8.5). Immediately, a
lot of white precipitation appeared.
2
Azz value was calculated to be 29 G. On the other hand, three
peaks of EPR spectrum for 2,2E-BSL labeled TnC was broader
than that of 2,2E-BSL, as shown in Figure 1b, indicating that the
free rotation of 2,2E-BSL axis restricted by two point binding.
From the broadened spectrum, the 2Azz value was calculated to
be 45 G that was much greater than that of commercially avail-
able MSL-labeled TnC (34 G). Unfortunately, the 2Azz value
of 2,2E-BSL labeled TnC was slightly smaller than that of 3,4-
8
S. Ebashi, M. Endo, I. Otsuki, Q. Rev. Biophys. 1969, 4, 351.
Published on the web (Advance View) June 27, 2006; doi:10.1246/cl.2006.834