new spin probes for biochemical applications elena bagryanskaya n. n.voroztsov novosibirsk institute...
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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
• Sharp EPR Singlet
• Biostability: relatively stable – hours
• EPR resolution: high, LW < 100 mG
• Oxygen sensitivity: High
• Main uses for EPR, EPR oximetryand 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
Trityl radicals Versus Nitroxide radicals
S
S S
S
D3CD3C
CD3
CD3
OO
C3
O
OH
H
N
NO
ATI
H2NNH
NO
H2N+ H+
– H+
ATIH+
What is the pH- sensitivity of nitroxides?
349 350 351 352 353Magnetic field/ mT
aN
NR
NRH+
pH 7.06
pH 4.21
pH 2.56
Observed HFI constants (aN) are pH- dependent
4 5 6 7 8 9 10
14.4
14.6
14.8
15.0
15.2
15.4
15.6
15.8
16.0
a N, G
pH
pKa
Ref.: V.Khramtsov, L. Weiner, I. Grigorjev, Volodarsky, Chem. Phys. Lett. 1982
N
N
O
N
NH
O
H++pKa
Main problem for in vivo application:
reduction of nitroxides to diamagnetic (EPR-silent) compounds
Spin probes
Synthesis of sterically substituted nitroxides with low reduction rate
Incapsulation of nitroxide radicals into nanocapsules and liposomes
6
The ways to overcome problem:
Incapsulation of spin probes in liposomes
N
N
N
O
SNH
NHO
HOOC
OH2N
COOH
NR2Gramicidin А
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 rat homogenate of heart tissue with addition of 10 мМ succinate
NR2 in liposome
Free NR2
Reduction of nitroxide in the presence of cucurbit[7]urile
AMP=0.5mM; [Asc ] = 2.5 mМ,
I. Kirilyuk, D. Polovyanenko, S. Semenov, I. Grigor’ev, O. Gerasko, V. Fedin, E. Bagryanskaya, J. Phys. Chem. B 2010, 114, 1719–1728.
N N
O
N
N
O
N
NN N
N
O
N
O
N N
O
N
O
N
N
O
N
O
N N
N N
O
N
O
N
N
O
N
O
N N
N N
O
O
NN
N N
O
O
n=5-8,10
=N
N
NH2
O
N
O
NH2
N
O
OH
AMP ATIHMP
N
N
O
MTI
N
N
NH2
OH
N
OH
NH2
AMP-H ATI-H
N
O
N
TAMP
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)
Nitroxides radicals with high stability towards reduction
N
N
ON
N
O
0.027 s-1 0.0009 s-1
L. Marx, R. Chiarelli, T. Guiberteau and A. Rassat,
J. Chem. Soc. Perkin Trans. 1, 2000, 1181-1182.
The reduction rates
0 5 10 15 20 25 30
10
20
30
40
50
[R],
M
Time, min
Nitroxide reduction in rat’s blood
N
N
H2N
O
N
N
N
OEt
Et N
N
N
OEt
Et
Et
Et
N
N
OEt
Et
Et
EtN
N
O
I.A.Kirilyuk, A.A.Bobko, I.A.Grigor’ev, V.V. Khramtsov,
Org.Biomol.Chem., 2004, 2, 1025
0,0
0,2
0,4
0,6
0,8
k,
M-1s-1
N
N
O
N
N
O
N
N
O
N
N
OEt
Et N
N
On-Bu
n-BuN
N
O
Et
Et
N
N
O
Et
Et
N
N
OEt
Et
Et
Et
Reduction rate constants imidazollidine nitroxides with acrobat
NR + Asc- → NR-H + Asc-•
KNR
0,0
0,5
5,5
k, M
-1s-1
0.0050.02
0,0
0,5
5,5
k, M
-1s-1
NO
COOHNO
COOH
N
HO
ON
HO
O
N
N
O
N
N
O
N
N
O
Comparative reduction rate constants of imidazoline and imidazolidine nitroxides with acrobat
N
N
O
NH
O
N
N
OHOH CO2H
N
N
O CO2Me
OC((CH2)2CO2H)2
CO2H CO2Me
NH4OAc
NaOH
2 3 4 5 6 7 8 9 10 11
14,0
14,5
15,0
15,5
a N, G
pH
pK = 6.1kred = 0.04
N
N
O CO2Na
CO2Na
N
N
O CO2Na
pK = 6.3kred = 0.85
3450 3460 3470 3480 3490 3500 3510
Field, G
Imidazolidine nitroxides
ATI
N
N
O
N
N
O
N
N
O
N
N
O
N
N
CD2
CD2
D2C
D2C
O
N
N
O
EPR spectra of imidazolidine nitroxides
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
Quantum chemical calculation Gaussian-983 B3LYP/6-31G
High stable hydrophilic pH-sensitive spin probe with pK 6.3
3460 3470 3480 3490 3500 3510
Magnetic field, G
pH 6.4
N
N
OH CO2-
N
N
OH CO2-
HH+
N
NH3CD2C
H3CD2C CD2CH2CO2H
D3C
OD
CD3
NH
OH3CD2C
H3CD2COH
N
NH3CD2C
H3CD2C CD2CH2CO2HO
CD3
15
PELDOR measurements
are possible only at T<77 K
τ τ
π/2 π
πT
νA
νB
V(τ)V(T)
τ1 τ1
τ τ
π/2 π
πT
V(τ)V(T) νA
νB
N
O
SS
O
O
N
O
SS
Protein
+ Protein-SH
Спиновая меткаMTS
Метка в боковой цепи
PELDOR measurements of distances in proteins
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 in α-carbons
N
O
hydroxy-DICPO
OH
HO OH
N
O
oxo-DICPO
O
HO OH
N
O
O
NH2
Carbamoyl-PROXYL
Reduction in liver homogenate of mice
Piperidine nitroxides with spirocyclic moiety as spin labels
N
O
SS
OO
MTSSL
Kathirvelu, V.; Smith, C.; Parks, C.; Mannan, M. A.; Miura, Y.; Takeshita, K.; Eaton S. S.; G. Eaton, R. Chem. Commun. 2009, 454–456.
40 80 120 160 200
5,6
5,8
6,0
6,2
6,4
6,6
oxo-DICPO
lg[1
/Tm(s
–1)]
T, K
MTS
N
O
O
HO OH
•Measurements of distances at nitrogen temperatures•Higher stability in reduction media
NO
COOH
1NO
CONH2
2NO
CH2OH
3NO
CN
4NO
COOH
5
NO
CONH2
6NO
CN
7
NC
NO
COOH
8
HOOC
NO
CONH2
9
HOOC
NO
CONH2
10
NO
COOH
1tNO
CONH2
2tNO
CH2OH
3tNO
CN
4tNO
COOH
5t
NO
CONH2
6tNO
CN
7t
NC
NO
COOH
8t
HOOC
NO
CONH2
9t
HOOC
New 2,5-spirocyclohexane-substituted nitroxides
3460 3470 3480 3490 3500
1,1 Гс
3,3 Гс
4,5 Гс
Гс
√g-фактор(±0.001%)
aN, Гс
(±0.3%)1 2,00581 15,962 2,00586 15,783 2,00584 16,055 2,00584 16,086 2,00586 15,987 2,00590 14,838 2,00581 15,989 2,00598 15,80
19
3490 3500 3510 3520 3530 3540Гс
NO
CONH2
6
NO
CONH2
10
NO
CONH2
6t
NO
COOH
1
EPR spectra of spirocyclohexane-substituted nitroxides
50 100 150 2005,0
5,5
6,0
6,5
7,0
50 100 150 2002,0
2,5
3,0
3,5
4,0
4,5
0 2 4 6 8 10 12
Lo
g[1
/Tm(s
-1)]
T(K)
a) b)
Lo
g[1
/T1(s
-1)]
T(K)
9
Spi
n ec
ho in
tens
ity
time (s)
MTSSL
N
S
O
SO O
MTSSL
N
OH
O9
Comparison of electron spin relaxation times T1 (b) and Tm (a) of spirocyclohexane-substituted nitroxide and MTSSL
I. Kirilyuk, Y.F.Polienko, O.Krumkacheva, R.Strizhakov, Y. Gatilov, I. Grigorjev, E.Bagryanskaya, J.Org.Chem., 2012, doi org/10.1021/jo301235j.
T2 and T1 of new nitroxides allow measurements of distances in proteins at nitrogen temperatures using PELDOR
342 343 344 345 346 347 348 1215 1216 1217 1218 1219 1220 1221
X-band
Magnetic field / mT
1213
Magnetic field / mT
Q-band13
12
N
O
SS
OO
12
N
S
O
S
HN
NH
COOH
O
O
COOHH2N13
EPR spectra of spirocyclohexane-substituted spin labels
0,1 М carbonate buffer [NR] - 0,5–0,75 mM [AscH–] =100 mМ; [GSH]= 50 mМ
22
√N √Nt
1 7 1t 225 7,4 5t 9,78 7,7 8t 203 9 3t 259 13 9t 336 18 6t 302 22 2t 58
12510–2 M–1·s–1 0 1 2 3 4 5 6
0,01
0,1
мМ
Время, мин
pH 7.2
NO
COOH
1
NO
CONH2HOOC
9
NO
CONH2
10NO
DICPO
Time, min
Reduction of 2,5-spirocyclohexasubstituted nitroxides
N
COOH
RR
RR
O
HOOC
N
COOH
RR
RR
O
H2NOC
N
COOH
RR
RR
O
N
CONH2
RR
RR
O
N
CONH2
RR
RR
O
N
COOH
RR
RR
O
N RR
RR
O
HO0
20
40
60
80
100
120k II
10–
2 M–
1 s–1
R+R=(CH2)
5
R=Me
N
OH
O
Reduction rate constant of nitroxides by ascorbic acid
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).
Akaike T. et al.Biochemistry, 1993, 32,
827.
Nitric oxide detection using EPR of nitronyl nitroxide
N
N
O
O
N
N
OPTIO PTI
NO
–NO2
Problems: toxicity and fast reduction in vivo
Nitric oxide detection using NMR
N
N
O
O
N
N
O
F
F
F
F
N
N
O
OH
F
F
N
N
OH
F
F
[H][O] [H][O]
NN• IN•
NNH INH
NO
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.
N
N
O
O
N
N
O
F
F
F
F
N
N
O
OH
F
FN
N
OH
F
F
[H][O] [H][O]
NN• IN•
NNH INH
NO
19F NMR
-109.6 -109.8 -111.0 -111.2 -111.4
Chemical shift / ррм
NNIN
•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
New low toxic hydrophilic nitronyl nitroxides
N
N
NH
NO
ON
N
NH
NO
ONN1 NN2
If it is possible to use NN1 and NN2 in vivo as nitric oxide spin probes using EPR tomography?
N
NO
ON
N
O
+ NO
– NO2NH
N
NH
N
0 4 8 12 16 20 240
2
4
6
8
10 NN1 + AscH– (pH 7,1)
0,01 + 0,01 мM
M
Время, мин
0,01 + 0,002 мM
Stability of nitronyl nitroxides in model conditions
NN1 k = (1,2±0,1)·103 M–1·с–1
NN2 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
N
N
NH
NO
ON
N
NH
NO
ONN1 NN2
Time, min
0,0 0,4 0,8 1,2 1,6 2,0 2,4 2,8 3,20,01
0,1
1
NN2
мM
Время, мин
NN1–2 в цельной крови
NN1
29
NN1 kobs = (4±1)·103 M–1·s–1
NN2 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
N
N
NH
NO
ON
N
NH
NO
ONN1 NN2
Stability of nitronyl nitroxides in blood of rats
Time, min
0 2 4 6 8 10 12 14 16 180,0
0,2
0,4
0,6
0,8
1,0 Plasma Eritrocites
(1:4 dilution) Blood
mМ
Time, min
P(NN1) = 0,85
30
NN1 reduction in blood and it’s component: plasma and
erytrocytes
kкр = 4·10–3 с–1
kэр = 1.1·10–3 с–1 ≈ 4kкр
NN1 penetrate into cells and are reduced in erythrocytes
Coefficient of distribution
octanol/waterwater
octanol
N
N
NH
NO
O
Penetration of NNR into cells
0 15 30 45 60 75 90 1050,0
0,1
0,2
0,3mМ
Time, min
350 360 370 380 390
31
Fast accumulation of NN1 in mice bladder
Typical EPR spectrum detected during EPR
tomography measurements
Pharmacokinetics of NN1in vivo (mice)
EPR tomography of mouse
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.
0 15 30 45 60 75 90 1050,0
0,1
0,2
0,3
mM
Time, min
10 ГсOnly EPR spectra of NNR were detected, no contribution of INR
N
N
O
ON
N
O
+ NO
– NO2NH
N
NH
N
Comparison of pharmacokinetics of NN1 in control mice ( ) and with injection of nitroglycerole 0,83mg/kg
Nitric oxide detection using EPR tomography of mouse
NNRINR
33
•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.
Conclusions
Acknowledgement:N.N.Voroztsov Novosibirsk Institute of Organic Chemistry SB RASIgor KirilyukIgor Grigor’evJulia PolienkoDenis KomarovInternational Tomography Center SB RASOlesya KrumkachevaSergey SemenovRodion StrishakovDmitrii PolovyanenkoVictor OvcharenkoElena FursovaInstitute of Cytology and Genetics, NovosibirskN. Kolosova
Ohio State University, Medical Center, USAV. Khramtsov, A. Bobko
Laboratory of Magnetic ResonanceInternational Tomography Center SB RAS, Novosibirsk, RussiaD. Polovyanenko S. SemenovO. Krumkacheva M. Fedin
Trityl
• Sharp EPR Singlet
• Biostability: relatively stable – hours
• EPR resolution: high, LW < 100 mG
• Oxygen sensitivity: High
• Main uses for EPR, EPR oximetryand 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
Trityl radicals Versus Nitroxide radicals
S
S S
S
D3CD3C
CD3
CD3
OO
C3
O
OH
H
N
NO
ATI
H2NNH
NO
H2N+ H+
– H+
ATIH+
Oxidative properties of nitroxides
N
R
O
N
R
O
N
N
R
O
N
N
R
O
NO R
N
N
R
O
O
N
N
O
R
O
0.001 0.01 0.1 1 10K
1 mT
2
T
D
I
IK
N
15N
OH
O
N
O
N
15N
O
O
N
OH
KDikanov, S.A.; Grigor’ev, I.A.; Volodarsky, L.B.; Tsvetkov, Yu.D.;Russ. J. Phys. Chem. A, 1982
0
1
2
3
4
26
27
K
N
N
O
N
N
O
N
N
O
N
N
O
N
N
O
N
N
O
N
N
O
N
N
O
N
HO
O
N
C
O
OOH
NR + CPH-15N NR-H + CP-15NK
1 : 1
1 mT
CP-15N
NR2
T
D
I
IK
15N
C
OH
OOH
CPH-15N
Nitroxide-hydroxylamine(15N) equilibrium
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
N
N
R
O
O
+ Asc
NN
N
N
R
O
OH
Hydroxylamine
DHA = dehydroascorbate
DGA = diketogulonic acid
Asc·– = ascorbate radical
O
HO O
OHOH2CHOHC O
O O
OHOH2CHOHCO
O O
OHOH2CHOHC
HOOH
O
O
O
OH
OH
AscH– Asc – DGADHA 39
0 200 400 600
60
80
100
EPR
sig
nal,
%
Time, s
500 mM Ascorbate 250 mM Ascorbate 125 mM Ascorbate
N
HO
O
BBO
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.
Обратимость восстановления НР аскорбатом
[BBO] 0.1 mM, pH 7.4
O O
OO
HO
HOO O
O-HO
HO
HO
O O
O-HO
HO
HOO O
O-O
HO
HOO O
OO
HO
HON
ON
O
N
OH
N
OH
HO
HO
OH
O
O
OH
O
H2O
N
O
Продуктыокисления
H+
-H+
GSH