New Spin Probes
for Biochemical Applications
Elena Bagryanskaya
N. N.Voroztsov Novosibirsk Institute of Organic Chemistry SB RAS
International Tomography Center, SB RAS, Novosibirsk, Russia
Outline
- application of nitroxides
- pH-sesitive high stable sterically substituted
nitroxides
- new spirocyclohexane-substituted nitroxides
for PELDOR measurements
- nitronyl nitroxides as a spin probes for NO
Spin probes - nitroxide and trityl radicals
• Structure and function of proteins using EPR and
site-directed spin labeling
• pH-sensitive probes
• spin probes for nitric oxide
• oxymetry
• redox probes
• antioxidants, etc…
Trityl radicals Versus Nitroxide radicals
Trityl
• Sharp EPR Singlet
• Biostability: relatively stable – hours
• EPR resolution: high, LW < 100 mG
• Oxygen sensitivity: High
• Main uses for EPR, EPR oximetry
and Overhauser-enhanced MRI.
Nitroxide
• Moderately broad EPR triplet;
• Biostability: easily reduced
• EPR resolution: relatively low
• Oxygen sensitivity: relatively low;
• Multiple use as redox status, pH
and ROS probes as well as spin
labeling agents and antioxidant,
etc
What is the pH- sensitivity of nitroxides?
pKa
N
+
N
aN
NH
H+
pH 7.06
N
O
O
NRH+
pH 4.21
16.0
NR
15.8
pH 2.56
15.6
aN, G
15.4
15.2
349
15.0
350
351
352
Magnetic field/ mT
14.8
pKa
14.6
14.4
4
5
6
7
8
9
10
pH
Observed HFI constants (aN) are pH- dependent
Ref.: V.Khramtsov, L. Weiner, I. Grigorjev, Volodarsky, Chem. Phys. Lett. 1982
353
Spin probes
Main problem for in vivo application:
reduction of nitroxides to diamagnetic (EPR-silent)
compounds
The ways to overcome problem:
Synthesis of sterically substituted nitroxides with low reduction
rate
Incapsulation of nitroxide radicals into nanocapsules and
liposomes
6
Incapsulation of spin probes in liposomes
HOOC
O
N
N
N
NH
S
NH
H2N
O
NR2
O
COOH
Gramicidin А
NR2 in liposome
Free NR2
Reduction of nitroxide in rat homogenate of
heart tissue with addition of 10 мМ succinate
Woldman, Ya.Y.; Semenov, S.V., Bobko, A.A.;
Kirilyuk I.A.; Polienko, J.F.; Voinov, M.A.;
Bagryanskaya, E.G.; Khramtsov, V.V. The
Analyst, 2009, 134, 904 – 910.
Reduction of nitroxide in the presence of cucurbit[7]urile
AMP=0.5mM; [Asc ] = 2.5 mМ,
NR
+ CB 1:2
+ CB 1:4
AMP (kAMPH+= 0.320 ±0.020 M-1s-1;)
AMP/CB7 = 1:1 (kobs= 0.097 ±0.008 M-1s-1)
AMP/CB7 = 1:2 (kobs= 0.040 ±0.006 M-1s-1)
AMP/CB7 = 1:10 (kobs= 0.020 ±0.004 M-1s-1)
I. Kirilyuk, D. Polovyanenko, S. Semenov, I. Grigor’ev, O. Gerasko, V. Fedin, E. Bagryanskaya,
J. Phys. Chem. B 2010, 114, 1719–1728.
Nitroxides radicals with high stability towards reduction
The reduction rates
N
N
N O
N O
0.027 s-1
L. Marx, R. Chiarelli, T. Guiberteau and A. Rassat,
J. Chem. Soc. Perkin Trans. 1, 2000, 1181-1182.
0.0009 s-1
Nitroxide reduction in rat’s blood
N
50
Et
N
40
[R], M
Et
O
H 2N
30
N
N
O
20
10
0
5
10
15
Time, min
20
25
30
N Et
N
O
Et
N
N
N Et
N
O
Et
N Et
Et
Et
N
Et
O
I.A.Kirilyuk, A.A.Bobko, I.A.Grigor’ev, V.V. Khramtsov,
Org.Biomol.Chem., 2004, 2, 1025
Reduction rate constants imidazollidine nitroxides with
acrobat
KNR
NR + Asc- → NR-H + Asc-•
0,6
k, M s
-1 -1
0,8
0,4
0,2
0,0
Et
Et
N Et
N
O
Et
Et
Et
N
N
O
Et
Et
N
N
O
N
N
O
N
N
N
O
N
O
N Et
N
O
Et
N n-Bu
N
O
n-Bu
Comparative reduction rate constants of imidazoline and
imidazolidine nitroxides with acrobat
k, M s
-1 -1
5,5
0,5
0.02
0,0
0.005
Imidazolidine nitroxides
ATI
aN, G
15,5
3450
3460
3470
3480
Field, G
3490
3500
N
15,0
N
14,5
pK = 6.1
kred = 0.04
O
CO2Na
pK = 6.3
3510
14,0
kred = 0.85
2
3
4
5
6
7
pH
8
9
10 11
EPR spectra of imidazolidine nitroxides
N
N
N
N
N
N
O
O
O
N
N
O
N
N
O
N
D2C
D2C
N
O
Quantum chemical calculation Gaussian-983 B3LYP/6-31G
A A Bobko, I A Kirilyuk, N P Gritsan, D N Polovyanenko, I A Grigor’ev, V V Khramtsov, E G Bagryanskaya
Applied Magnetic Resonance (2010) 39 (4), 437-451
CD2
CD2
High stable hydrophilic pH-sensitive spin probe with pK 6.3
D3C
N
O
H3CD2C
H3CD2C
H3CD2C
H3CD2C
NH
OH
N
CD3
N
H3CD2C
CD2CH2CO2H
H3CD2C
OD
N
CD3
CD2CH2CO2H
O
pH 6.4
N
N
OH
N H
H+
N
CO2-
OH
CO2-
3460
3470
3480
3490
Magnetic field, G
3500
3510
PELDOR measurements of distances in proteins
π/2
π
τ
π
PELDOR
measurements
are possible
only at T<77 K
V(τ)
V(T)
νA
τ
T
π/2
νB
π
τ
V(τ)
π
τ
τ1
T
V(T) νA
τ1
νB
15
Piperidine nitroxides with spirocyclic moiety in α-carbons
OH
O
NH2
N
HO
O
N
OH
O
Carbamoyl-PROXYL
hydroxy-DICPO
O
N
HO
O
OH
oxo-DICPO
Reduction in liver homogenate of mice
Okazaki, S.; Mannan, M. A.; Sawai, K.; Masumizu, T.; Miura, Y.; Takeshita,
K. Free Rad. Res., 2007, 41(10), 1069-1077.
Kinoshita, Yu.; Yamada, K.; Yamasaki, T.; Sadsue, H.; Sakai, K.; Utsumi, H.
Free Rad. Res. 2009, 43(6), 565-571.
Piperidine nitroxides with spirocyclic moiety as spin labels
6,6
–1
lg[1/Tm(s )]
6,4
6,2
MTS
6,0
5,8
oxo-DICPO
5,6
40
80
120
160 200
T, K
• Measurements of distances at nitrogen temperatures
• Higher stability in reduction media
Kathirvelu, V.; Smith, C.; Parks, C.; Mannan, M. A.; Miura, Y.; Takeshita, K.; Eaton S. S.;
G. Eaton, R. Chem. Commun. 2009, 454–456.
New 2,5-spirocyclohexane-substituted nitroxides
EPR spectra of spirocyclohexane-substituted nitroxides
√
4,5 Гс
1
2
3
5
6
7
8
9
3,3 Гс
1,1 Гс
3460
3470
3480
3490
g-фактор
(±0.001%)
2,00581
2,00586
2,00584
2,00584
2,00586
2,00590
2,00581
2,00598
aN, Гс
(±0.3%)
15,96
15,78
16,05
16,08
15,98
14,83
15,98
15,80
3500
Гс
3490
3500
3510
Гс
3520
3530
3540
19
Spin echo intensity
Comparison of electron spin relaxation times T1 (b) and Tm (a) of
spirocyclohexane-substituted nitroxide and MTSSL
O O
S
S
9
MTSSL
0
2
4
6
8
time (s)
10
12
7,0
N
O
4,5
a)
Log[1/T1(s )]
-1
-1
Log[1/Tm(s )]
MTSSL
4,0
6,5
6,0
5,5
b)
OH
3,5
N
O
3,0
9
2,5
5,0
2,0
50
100
150
T(K)
200
50
100
150
200
T(K)
T2 and T1 of new nitroxides allow measurements of distances in
proteins at nitrogen temperatures using PELDOR
I. Kirilyuk, Y.F.Polienko, O.Krumkacheva, R.Strizhakov, Y. Gatilov, I. Grigorjev,
E.Bagryanskaya, J.Org.Chem., 2012, doi org/10.1021/jo301235j.
EPR spectra of spirocyclohexane-substituted spin labels
12
13
12
13
Q-band
X-band
342
343
344
345
346
347
348
1215
1216
Magnetic field / mT
O
O
O
S
S
NH
S
O
HN
N
O
12
1218
1219
Magnetic field / mT
S
N
O
1217
COOH
COOH
13
H2N
1220
1221
Reduction of 2,5-spirocyclohexasubstituted nitroxides
NR + AscH– → HA + Asc–
√N
1
5
8
3
9
6
2
√Nt
7
7,4
7,7
9
13
18
22
1t
5t
8t
3t
9t
6t
2t
мМ
22
9,7
20
25
33
30
58
125
10–2 M–1·s–1
pH 7.2
0,1
0,01
0
1
2
Time, min
3
4
5
Время, мин
6
0,1 М carbonate buffer
[NR] - 0,5–0,75 mM
[AscH–] =100 mМ; [GSH]= 50 mМ
22
Reduction rate constant of nitroxides by ascorbic acid
120
100
kII 10–2 M–1s–1
OH
R+R=(CH2)5
R=Me
80
N
O
60
40
20
0
HOOC
HO
CONH2
R
R
N
O
R
R
R
R
COOH
R
R
N
O
R
R
N
O
COOH
COOH
R
R
R
R
CONH2
R
R
N
O
R
R
N
O
R
R
R
R
H2NOC
R
R
N
O
R
R
COOH
N
O
R
R
The stability of 2,5-spirocyclohexane –substituted nitroxides is
more that three times higher that their tetramethylated analogs
and ~10–15 higher than 2,5-spirocyclohexane piperidine
I. Kirilyuk, Y.F.Polienko, O.Krumkacheva, R.Strizhakov, Y. Gatilov, I. Grigorjev,
E.Bagryanskaya, J.Org.Chem., 2012, doi org/10.1021/jo301235j.
Role of nitric oxide (II).
Vasolidation of blood vessels
Neuron mediator
cytotoxic activity
Etc…
Nitric oxide detection using EPR of nitronyl nitroxide
O
Akaike T. et al.
Biochemistry, 1993, 32, 827.
N
NO
N
N
–NO2
N
PTIO O
Problems: toxicity and fast reduction in vivo
PTI
O
Nitric oxide detection using NMR
O
F
F
N
N
NO
N
NN•
NNH
NNH
N
O
F
[O]
[H]
O
F
F
IN•
O
F
[O]
[H]
INH
F
F
N
N
N
N
OH F
INH
19F
OH F
-109.6
-109.8
-111.0
-111.2
-111.4
Chemical shift / ррм
NMR
Bobko A.A., Bagryanskaya E.G. Reznikov V, Kolosova
N. Khramtsov V.V. Free Rad. Biol. & Med., 2004, 36 (2),
248–258 , BBRC, 330 (2005) 367–370.
New low toxic hydrophilic nitronyl nitroxides
NN
IN
• Ration EPR signal intensities s of NN and IN should reflect nitric oxide
concentration in vivo.
• EPR tomography could give informnation about NN and IN distribution
If it is possible to use NN1 and NN2 in vivo as nitric oxide
spin probes using EPR tomography?
Stability of nitronyl nitroxides in model conditions
NN1 + AscH– (pH 7,1)
10
8
0,01 + 0,002 мM
6
M
4
2
0
0,01 + 0,01 мM
0
4
8
12
16
20 24
Время, мин Time, min
NN1
NN2
k = (1,2±0,1)·103 M–1·с–1
k = (1,4±0,1)·103 M–1·с–1
The reduction rate constants of NN1 and NN2 by
ascorbic acid are high and are close to the same for
other NNR
Stability of nitronyl nitroxides in blood of rats
NN1–2 в цельной крови
1
мM
NN1
0,1
NN2
0,01
0,0 0,4 0,8 1,2 1,6 2,0 2,4 2,8 3,2
Время, мин Time, min
NN1
NN2
kobs = (4±1)·103 M–1·s–1
kobs = (14,3±0,3)·103 M–1·s–1
The reduction rate constants of NN1 and NN2 in
blood are high at low NNR concentration and are
close to the same for other NNR
29
Penetration of NNR into cells
1,0
0,8
mМ
0,6
0,4
Plasma
Eritrocites
(1:4 dilution)
Blood
NN1 reduction in blood and it’s
component: plasma and
erytrocytes
0,2
0,0
0 2 4 6 8 10 12 14 16 18
Time, min
Coefficient of
distribution
octanol/water
kкр = 4·10–3 с–1
kэр = 1.1·10–3 с–1 ≈ 4kкр
octanol
P(NN1) = 0,85
water
NN1 penetrate into cells and are reduced in
erythrocytes
30
EPR tomography of mouse
mМ
0,3
0,2
0,1
0,0
0
15
30
45
60
Time, min
75
90 105
Pharmacokinetics of NN1
in vivo (mice)
350 360 370 380 390
Typical EPR spectrum
detected during EPR
tomography
measurements
Fast accumulation of NN1 in mice bladder
31
Nitric oxide detection using EPR tomography of mouse
0,3
mM
0,2
NNR
INR
0,1
0,0
0 15 30 45 60 75 90 105
Time, min
10 Гс
Only EPR spectra of
NNR were detected,
no contribution of
INR
Comparison of
pharmacokinetics of NN1
in control mice ( )
and with injection of
nitroglycerole 0,83mg/kg
Nitric oxide expression in vivo decreases NNR concentration,
which can be determined by reaction of NNR with NO as well as
other physiological processes.
INR was not detected, probably due to fast reduction.
Conclusions
• Nitroxides are the unique and very promising organic compounds with
high potential for biomedical applications in therapy and diagnostics
• Sterically substituted imidazoline and imidazolidine nitroxides
combine
high pH-sensitivity and high stability in reduction media
• The new spin labels and spin probes of 7-azadispiro[5.1.5.2]-pendecane
and 7-azadispiro[5.1.5.2]pentadeca-14-ene series were synthesized and
demonstrated clear advantages over tetramethylpyrroline nitroxides with
respect to electron relaxation rates allowing PELROR distance
measurements at liquid nitrogen temperature range and higher stability.
•
The new low toxic hydrophylic nitronyl nitroxides were used as a spin
probes for nitric oxide in vivo EPR tomography. Nitric oxide expression
in vivo decreases NNR concentration, which can be determined by
reaction of NNR with NO as well as other physiological processes.
33
Acknowledgement:
N.N.Voroztsov Novosibirsk Institute of Organic Chemistry SB RAS
Igor Kirilyuk
Igor Grigor’ev
Julia Polienko
Denis Komarov
International Tomography Center SB RAS
Olesya Krumkacheva
Sergey Semenov
Rodion Strishakov
Dmitrii Polovyanenko
Victor Ovcharenko
Elena Fursova
Institute of Cytology and Genetics, Novosibirsk
N. Kolosova
Ohio State University, Medical Center, USA
V. Khramtsov,
A. Bobko
Laboratory of Magnetic Resonance
International Tomography Center SB RAS, Novosibirsk, Russia
D. Polovyanenko
S. Semenov
O. Krumkacheva
M. Fedin
Trityl radicals Versus Nitroxide radicals
Trityl
• Sharp EPR Singlet
• Biostability: relatively stable – hours
• EPR resolution: high, LW < 100 mG
• Oxygen sensitivity: High
• Main uses for EPR, EPR oximetry
and Overhauser-enhanced MRI.
Nitroxide
• Moderately broad EPR triplet;
• Biostability: easily reduced
• EPR resolution: relatively low
• Oxygen sensitivity: relatively low;
• Multiple use as redox status, pH
and ROS probes as well as spin
labeling agents and antioxidant,
etc
Oxidative properties of nitroxides
O
O
K
N
N
O
15
N
OH
N
Dikanov, S.A.; Grigor’ev, I.A.;
Volodarsky, L.B.; Tsvetkov, Yu.D.;
Russ. J. Phys. Chem. A, 1982
N
OH
15
N
O
 ID 
K   
 IT 
1 mT
2
R
N
N
O
R
N
N
N
R
N
O
O
O
N
O
R
N
R
N
O
N
O
0.001
O
R
O N
0.01
0.1
R
1
K
10
Nitroxide-hydroxylamine(15N) equilibrium
K
K
15
NR + CPH- N
1 :
1
27
 ID 
K   
 IT 
C OOH
26
15
NR-H + CP-15N
N
2
OH
CPH-15N
NR
1 mT
4
3
CP-15N
2
1
0
C OOH
HO
N
N
N
N
O
O
N
N
O
N
N
N
O
N
O
N
N
O
N
N
O
N
N
O
N
O
N
O
Reduction by Ascorbate
O
O
N
N
R
+ Asc
R
N
N
O
NN
 DHA = dehydroascorbate
 DGA = diketogulonic acid
 Asc·– = ascorbate radical
OH
Hydroxylamine
NN1
k1 = (1.2±0.1)·103 M–1·s–1
k–2 = (3.0±0.5)·103 M–1·s–1
NN2
k1 = (1.4±0.1)·103 M–1·s–1
k–2 = (3.5±0.5)·103 M–1·s–1
39
Обратимость восстановления НР аскорбатом
HO
N
O
BBO
EPR signal, %
100
80
500 mM Ascorbate
250 mM Ascorbate
125 mM Ascorbate
60
0
200
400
Time, s
600
[BBO] 0.1 mM, pH 7.4
A.A.Bobko, I.A.Kirilyuk, I.A.Grigor’ev, J.L.Zweier,
V.V.Khramtsov. Free Radic. Biol. Med., 2007, V.
42, P. 404-412.