OPERACIONALIZACION DE VARIABLES
DISCUSIÓN DE RESULTADOS
The fast rates of reaction of common thiols with peroxynitrite at room temperature and above (Table 2.8) preclude any observation of nuclear polarisation in the NMR spectrum.
Table 2.9 Second order rate constants for the reaction of alkaline peroxynitrite (final concentration 3,11 mM ) with a range of thiols (final concentration 31.1 mM ).
Thiol W M'* s'* -SH pÆ,^ tyjs DL-Penicillamine 1,740 ± 1.7^ 7.95" 4.5 77-Acetyl-DL-penicillamine 152 ±0.3^ 8.0 47 Glutathione 13,060 ±34" 8.72^^ 0.10 7/-Acetyl-L-cysteine 7.4 ± 0.05" 9.76" 20 L-Cysteine 5,900 ±160^ 8.21"*'/ 0.03 "ref. 129/ref.40(measm-edat37 °C),"ref. 1 3 0 /ref. 131, "ref. 132,^ref. 133.
The pH dependence of the second order rate constant as determined by Radi et
for L-cysteine suggests that the undissociated form of the thiol reacts with peroxynitrite anion. The reaction can be described by eqn. (11).
, - , & ( „ )
*[•*•])
where ^obsd is the observed rate constant at a given pH, kj is the second-order rate constant for the reaction of thiols with peroxynitrite anion, is the dissociation constant of peroxynitrite (6.8 at 37 °C) and is the dissociation constant of the thiol.
In order to try to detect nuclear polarisation that may arise from the [*^N]peroxynitrite/thiol reaction due to contributions from a radical pathway, the technique of freezing the solutions was employed as in the previous experiments with
aromatic substrates. Fig, 2.14 shows the spectral changes during the reaction of [^^N] peroxynitrite with #-acetyl-DL-penicillamine.
The signal for the nitrate ions shows some signs of enhanced absorption, but there is no emission signal for the nitrite ions; however, the rapid growth of the signal for nitrite ions suggests that only the latter stages of the reaction are being observed, i.e.
just after the point when the signal for the nitrite ions has passed from an emission signal to a conventional absorption signal. The reaction path is therefore probably the same as that previously described when L-tyrosine is the substrate.
(«) ref.
nitrate
nitrite 10
t/min 15 20
Fig. 2.14 Time course of the NMR signals in the reaction of alkaline peroxynitrite (approx. 0.10 m) with #-acetyl-DL-penicillamine (0.0191 g, 0.05 M). in
frozen phosphate buffer. [NaOH] = 0.15 M. (a) Spectra phased consistent with
previous figures with individual signals displayed stacked horizontally, (b) Raw spectral arrays.
(6) [’^ ] n itro b e n z e n e S u = 3 7 0 .3 p pm n itra te Sn = 3 7 5 .9 p pm I k I p e ro x y n itrite nitrite Sn = 6 0 9.1 p pm ^N = 5 5 4.5 p pm 10 15 //m in 20 2 5 3 0
Table 2.10 Summaiy of the characteristics of the NMR spectra illustrated in Fig. 2.14.
Signal 4i/ppm Enhancement Phase Change NO- 238.9 (609.2) Not observed Not observed
ONOO' 184.2 (554.5) No No
Therefore the most appropriate question to address is by what mechanism(s) may peroxynitrite effect nitrosation of thiols? Williams^^'^ has wiitten a short review on this topic. In principle, peroxynitrite could react with thiols via transfer of NO^ in an Sn2 reaction displacing the peroxide ion (Scheme 2.14).
A 0 = N Q- — © ► A— N = 0 + A 0 = N Q— OH © A 5 ► A— N = 0 + H0|> Scheme 2.14
However this pathway is highly unlikely as both species are very poor leaving groups therefore the reaction would be highly disfavoured on both thermodynamic and kinetic grounds. Williams draws an analogy between peroxynitrite and nitrous acid in that the latter species is not itself a nitrosating agent unless protonated to give the nitrous acidium ion such that the leaving group may be water.
The analogy ends there as protonated peroxynitrous acid, ONO^(H)OH, would not exist due to the rapid isomérisation of peroxynitrous acid to nitrate at physiological pH. More likely mechanisms are illustrated in Scheme 2.15.
Pathway {a) in Scheme 2.15 is essentially analogous to that proposed earlier with ArO” as the substrate. Sulfenyl radicals may be generated by an electron transfer either between HO’ or NO; and thiolate ion. Williams concludes that it is reasonable to
( a ) Caged Radicals Hemolysis ---
ONOOH ^ NOg + HO' Cage Return Free Radicals NOg + HO' R S ‘ RS® H® + N O f R S ' + N O f R S ' + HO® ib) 2 NOg ^ Ng04 (c) R SH + NOg --- ► O RS®-^ f P ” 4®
0=N o®
R SN O + N O f /OH ^ RS—N n S i-H RS- id) R S ' + NOg (e) O 'P
R S ~ N ® O© Isom. -► R S— N -H,o► R SN O COH R"®"NO R—S = 0 RS'\_y RS“|^ = 0 N =0 R S - S R %R R Scheme 2.15A less likely, but nevertheless possible alternative mechanism is that proposed by Pryor et alP^ Initial attack by NO^ (derived from homolysis of peroxynitrite) on RSH forms an (alkylthio)(hydroxy)aminoxyl radical. This reacts with a further
molecule of thiol to generate an i\yV-dihydroxyalkanesulfenamide and a sulfenyl radical. Loss of water yields iS-nitrosothiol. Presumably formation of the kinetically stable disulfide shown in pathway (e) provides the driving force for release of NO. An alternative route to release of NO is illustrated in pathway {d). This involves isomérisation of the alkyl(nitro)sulfane to an alkyl(nitrosooxy)sulfane and then to an alkyl(nitroso)oxo->U-sulfane. This may then undergo homolysis to an alkyl(oxo)-,#- sulfanyl radical. Dimérisation would form an (S,5'-dialkyl-iS',5'“dioxodi-A4-sulfane.
2.2.10.2 Decomposition of ^'-nitrosothiols
The decomposition of 5'-nitrosothiols has been shown to give different nitrogen- containing products according to the concentration of thiol present. At low or zero [RSH], nitric oxide is produced together with the disulfide, RSSH. However at high [RSH], the R-SNO is also reduced to RSSH, but the principal nitrogen-containing product becomes ammonia together with some N2O. Clearly this has important
ramifications for the toxicity of 5'-nitrosothiols such as glutathione and SNAP, currently undergoing clinical trials as NO-donor drugs, as the intracellular concentration of free and protein-boimd RSH in blood plasma can reach millimolar levels. Although the intermediates in the high [RSH] mechanism have not been resolved, it is possible that NO" (nitroxyl) is produced and may exist as trioxodinitrate (also known as oxyhyponitrite, nitrohydroxylamate, hyponitrate and Angeli’s salt). This can account for the formation of both ammonia and N2O (scheme
4.11). The *^N chemical shift of trioxodinitrate has been determined by Garber et alP^ to be ^ - 274 ppm relative to nitrite ( ^ - 34.1 ppm relative to '^N nitrobenzene or ^ 335.1 ppm relative to liquid ammonia at 25 °C). Samples of *^N 5"-nitroso-A- heptanoyl-DL-penicillamine (SNHP) and ^^N SNAP were prepared in order to ascertain if any intermediates in their decomposition could be identified by their ^^N NMR signals. The signal due to the -SNO group appeared at ^ 466 ppm but suffered
approximately fourfold linewidth broadening in both DMSO-de (SNHP) and a 1:1
mixture of methanol and pH 7.4 phosphate buffer (SNAP). A typical value for the linewidth was 20 Hz as compared with 5 Hz for the reference. Since an increase in the width of the signal is accompanied by a decrease in the height by the same factor it is unsurprising that no CIDNP effects could ever be detected for the 5'-nitrosothiol
RSNO + HgO ==F © RSNO + NOg RSNO + R’SH ^ RSH + HNOg RS® + NgOg RSH + R’SNO Scheme 2.16 ^ = 240-300 min t = 180-240 min t - 480-540 min t ~ 420-480 min = 120-180 min r V W ^ J V V j / = 360-420 min ? = 60-120 min t = 300-360 min linewidth 20 Hz — '— r ~ — '— r ~
460
/ ppm
460
Fig. 2.15 Decomposition of ['^N] S’-nitroso-A^-acetyl-DL-penicillamine (0.1 g, 0.09 m) in the presence of 7V-acetyl-DL-penicillamine (0.15 g, 0.157 M). Solvent
CHsOH/phosphate buffer, pH 7.0.
Fig. 2.15 shows the time course of the signal for the -SNO group in the decomposition of ^^N SNAP in the presence of an excess of the parent thiol. No signals other than that for the nitrosothiol could be detected, a problem which was undoubtedly exacerbated by the occurrence of extraneous noise during the acquisition periods. Furthermore, a significant build up of pressure inside the NMR tube caused
the top to be ejected from the machine providing somewhat anecdotal evidence for the formation of gas in this reaction.