As stated earlier, Reszka et al reported a novel spin trap for nitric oxide i.e. the anion generated on treatment of nitromethane (CH3NO2) with base in aqueous
solution. Compounds of types RCH2N O 2 and RR CHNO2 were examined where R,R' are electron acceptor or alkyl groups.
2.2.3.1. Nitromethane.
The reaction of nitromethane with NO in basic conditions was investigated, and after several attempts using the same experimental conditions, the following spectrum was obtained, essentially the same as that of Reszka et al This was satisfactorily simulated (figure 2.1) using the followi^^ parameters: a(N)= 11.56G (NO2), a(N)= 6.5G (NO), and a(H)= 2.83G. When the sanïple was left overnight and re-examined by EPR spectroscopy the following morning, no signal was observed, even when the basicity was increased by addition of 2M NaOH.
:
I ■ ' t Fig. 2.1 2.2.3.2. Nitroethane.
Nitric oxide was bubbled through a 50mM solution of degassed nitroethane for a period of 24 hours. Samples were taken after 0.5, 3, and 20 hours. The only sample to show any sign of spin trap activity was that taken at 20h. A spectrum of greater than 10 lines was observed as shown. By analogy with nitromethane, the act
form of nitroethane is CH3C H =N 0 2 % and thus the following, scheme 2.3, was proposed:
C H 3C H = N 0 2 ‘ + N O ---^-CHaCH---- NOg'" NO CH3CH NO2'" CH;^---NO2' NO NO GH3C — NO2’
I
NO CHoG NOc O N Scheme 2.3Thus, from the above it can be seen that there would be two triplet splittings from the non-equivalent nitrogens, further split into quartets by the methyl hydrogens. As can be seen from the spectrum shown below, figure 2.2, this is not the case. Also shown is a simulation of the nitroethane spectrum, using the following values; a(lN )= 16.5G, a(lH )= 6.8G, and a(lH)= 4.2G. When compared to the literature, these splittings are consistent with those exhibited by a dialkyl nitroxide in an aqueous environment. The structure of this nitroxide has not yet been determined.
ImT _
Fig. 2.2
The intensification of the signal on increasing the pH with 2M NaOH could be due to precursors formed at lower pH from the primary adduct CH3CH(N0 )N02*~ by addition of a second molecule of NO to the nitroso moiety, itself a spin trap. Analysis by GC- MS showed that no identifiable products were present.
2.2.3.3. Methyl nitroacetate.
A solution of methyl nitroacetate was prepared in the same concentration as that used for nitromethane, i.e. 50mM. The solvent was 0.5M NaOH. The solution was degassed and NO was bubbled through for 5 minutes. No EPR signal was observed. The pH was raised by addition of 5cm^ 2M NaOH but the expected spectrum was absent. Analysis by GC-MS did not reveal any trace of the expected spin adduct 9,
H3CO 2C -C H -N O 2 '
NQ- 9
or its degradation products. The reason for this could be that there is some interference from the -CO2- group, but this is unlikely as this functionality should promote anion formation. Alternatively, it could be that the radical is too transient to be detected, or the time taken for observation was too great.
2.2.3.4. Diethyl malonate.
A 50mM solution of diethyl malonate (C2H5O2CCH 2CO 2C2H 5) in 0.5M NaOH was degassed for 1 hour. Nitric oxide was bubbled through for 1 hour and a sample taken for EPR. No paramagnetic species were present. Analysis by GC-MS indicated that no reaction had taken place.
2.2.3.5. Malononitrile.
A 50mM solution of malononitrile in 0.5M NaOH was degassed for 1 hour. Nitric oxide was bubbled through for 2 minutes, and a sample was taken. No EPR signal was observed . The basicity was increased by addition of 2M NaOH (5cm3) and NO bubbled for a further 2 minutes. There was still no signal observed. After 3 hours of gassing, no EPR spectrum was obtained. On removal of the reaction vessel, white smoke was emitted and identified as ammonia. This must have been produced from the hydrolysis of the malononitrile. Nitriles can be hydrolysed to the acid or the amide, but in basic conditions acid formation is favoured. When samples were analysed by GC- MS, no products corresponding to the following were observed:
H O — Il---Il— O H ---H g N — |j---|j— N H g
0
0
O
O
2.2.3.6. 2~Nitropropane.
A 50mM solution of 2-nitropropane in 0.5M NaOH was degassed and NO bubbled through for 24 hours. There was no reaction during this period, and analysis by EPR spectroscopy revealed no formation of a radical adduct. 2-Nitropropane does not act as a trap for NO.
2.2.3.7. Ethyl nitroacetate.
In line with MeN0 2 , a 50mM solution of ethyl nitroacetate was prepared in 0.5M NaOH. The solution was degassed and NO bubbled through. No EPR spectra were recorded after 0.5, 1.5, and 4 hours. There was a colour change from colourless to pale yellow, but it was concluded that ethyl nitroacetate does not act as a spin trap for NO.
2.2.3.8. General conclusions.
From the above investigations it can be seen that only the nitromethane anion will successfully form an adduct with NO. The trapping of NO by nitroalkanes is obviously highly specific. It appears that a high pH is required to promote the aci form of the compound, and this will be difficult to adapt for trapping in a physiological medium. The complexity of the splitting pattern may cause some problems for interpretation, but may also be beneficial in that they will be easily identified as characteristic nitric oxide adducts.