S-Nitrosothiols are now recognised as an important class of nitric oxide (NO) donor drugs as they decompose to give NO and the corresponding disulphide only:
RSNO 1/2RSSR + NO
If the RS- moiety is a thiol-containing amino acid, S-nitrosothioIs should have therapeutic potential as the disulphide produced on NO release should have no harmful side effects. The major requirements for a good NO generator are the following: first, the compound has to be easy to prepare in a stable and pure form, preferably as a solid; second, it has to release NO in aqueous solution, preferably without the need for redox activation; third, it has to generate NO quantitatively at a predictable rate; and fourth, the by-products should be nontoxic. Such compounds are rare.
5-Nitrosothiols are prepared by nitrosation of thiols and no purification procedure is possible because of the ease of decomposition. S-Nitrosothiols have been previously prepared by several methods, including reaction of thiols with nitrous acid under acidic conditions, and with dinitrogen tetroxide in carbon tetrachloride or ether (Williams, 1988). The nitrous acid method has limited applicability, and the use of dinitrogen tetroxide is complicated by the requirement for controlled stoichiometry and low reaction temperatures (Williams, 1988). Use of f-butyl nitrite was expected to be advantageous for the synthesis of a broad range of 5-nitrosothiols without the restrictions normally encountered with the use of dinitrogen tetroxide (Doyle et al., 1983). f-Butyl nitrite has the requisite chemical and physical properties of a useful synthetic reagent. It has a relatively low boiling point, and the corresponding alcohol can be easily removed from higher boiling or water- insoluble products (Doyle et at., 1983). Doyle et al. report that .S-nitrosothiois were conveniently formed by rapid nitrosyl exchange reactions with f-butyl nitrite (Doyle et al., 1983). In our work, f-butyl nitrite was used to form 5-nitrosothiols from the corresponding dipeptides according to the following Scheme (Scheme 2. 3):
1. f-butyl nitrite
2. CH2CI2
3. -78 “C
SNO
o R
Scheme 2. 3; The nitrosation reaction of the dipeptides to the corresponding ^-nitrosated dipeptides.
The dipeptides were converted into the corresponding 5-nitrosothiols (2-12) in high yields (more than 80%) by this procedure described in detail in the Experimental Section to give solids or sticky solid (Butler et al, 1996). Compound 1, 2, 4, 7, 10, 11, and 13 were obtained in a pure state but the others (3, 5, 6, 8, 9, and 12) were contaminated by small amounts of the unreacted thiol and/or by small amounts of the disulphide formed during nitrosation (see element analysis; Experimental Section).
For comparison purposes we also prepared 5-nitrosated /V-acetyl-D,L-(3,(l-dimethylcysteine (SNAP) and glutathione (GSNO).
(a) S-Nitrosated Amino-acid
5-Nitroso-N-acetyl-D,L-p,p-dimethylcysteine (SNAP; 1) was synthesised, characterised and its X-ray crystal structure obtained by Field et al in 1978. In the crystalline state, it is the most stable 5-nitrosothiol known to date, although in solution it decomposes to release NO and form its disulphide. It has been shown to be a potent relaxation agent for vascular smooth muscle and consequently a fast acting vasodilator (Ignarro et al, 1981a&b). Along with other 5-nitrosothiols, it possesses anticoagulant properties, as it inhibits the aggregation of blood platelets and their adhesion to the sub-endothelium (Radomski et al,
1992). In this work SNAP was synthesised as described by Doyle et a l in 1983: SNO
OH
(SNAP; 1)
(b) 5-Nitrosated Dipeptides
The following 5-nitrosated dipeptides were prepared by reaction of the thiol with r-butyl nitrite:
I, 5-nitroso-A-acetyl-D,L-p,p-dimethylcysteinylglycine methyl ester (2):
II. S-nitroso-iV-acetyl-D,L-p,p-dimethylcysteinyl-L-alanine methyl ester (3):
III. 5~nitroso-A^-acetyl-D,L-p,p-dimethylcysteinyl-L-valine methyl ester (4):
IV. 5-nitroso-V-acetyl-D,L-p,p-dimethylcysteinyl-L-leucine methyl ester (5): SNO
O I O
/
X
(5)
V. 5'-nitroso-V-acetyl-D,L-p,p-dimethylcysteinyl-L-phenylalanine methyl ester (6): SNO O
A
O(6)
55VI. 5^-nitroso-V-acetyl-D,L-p,p-dimethylcysteinyl-L“isoleucine methyl ester (7):
Vn. 5'-nitroso-V-acetyl-D,L-p,p-dimethylcysteinyl-L-methionine methyl ester (8): SNO
O
/
/
(8)
VIII. i'-nitroso-V-acetyl-DjL-Pjp-dimethylcysteinyl-L-Ve-CBZ-L-lysine methyl ester (9): SNO o
o
o
NH O(9)
56IX. 5-nitroso-A^-acetyl“D,L-3,p-dimethylcysteinyl-L-proline methyl ester (10): SNO
(10)
X. 5-nitroso-A^-acetyl-D,L-p,p-dimethylcysteinyl-L-aspartic acid dimethyl ester (11); SNO
XI. 5'-nitroso-A/^-acetyl-D,L-(I,p“dimethylcysteinyl-L-glutamic acid dimethyl ester (12): SNO
(c) s-Nitrosated Tripeptide
5-Nitroso-L-glutathione (GSNO; 13) was first synthesised and characterised in 1985 by Hart using a modification of a method designed by Saville (1958). GSNO is a derivative of the tripeptide L-glutathione (L-y-glutamyl-L-cysteinylglycine), which is found in numerous cellular systems and is considered an essential constituent of all living cells. L-Glutathione is generally the most abundant intracellular non-protein thiol (Meister and Anderson, 1983), with a concentration approaching 2-3 mM in erythrocytes. Consequently, the discovery of EDRF and the suggestion that it is an S-nitrosothiol, together with evidence that the physiological half-life of NO can be stabilised by thiols (Ignarro et al, 1980a,b&c; Stamler et al., 1992a,b,c,d&e; Feelisch et at., 1994), raises the possibility that S~ nitrosoglutathione could be the most important ^-nitrosothiol synthesised in vivo and involved in physiological processes.
Since 1985, GSNO has been synthesised by other researchers using different methods (Park and Means, 1989; Clancy and Abramson, 1992).
GSNO has been shown to be a hypotensive drug as effective as sodium nitroprusside (Means and Park, US patent 1990) and a potent inhibitor of platelet aggregation (Radomski et al, 1992). Along with SNAP, it is one of the few stable 5-nitrosothiols in the crystalline state, and GSNO kept at 4 °C or below is stable over a number of months (Park and Means, 1989; Butler et al, 1996). In this work GSNO was prepared as described in the literature by Hart in 1985.
SNO
NHg
(GSNO; 13)
5-Nitrosothiols 1, 2, 4, 7,11, and 13 were obtained as stable green solids in the pure form. 5-Nitrosothiols 3, 5, 6, and 12 were also green solids and were obtained together with a small amount of the starting material (the corresponding thiols) and/or the corresponding disulphide. ^-Nitrosothiol 10 was obtained as a green sticky solid in the pure form. ^-Nitrosothiols 8 and 9 were obtained as green sticky solids, and were contaminated with a small amount of the starting material (the coixesponding thiols) and/or the corresponding disulphide.
Dipeptides and their derivatives are sometimes difficult to characterise by elemental analysis as small amounts of the disulphide or thiol are present and we were unable to remove these impurities. In most instances FAB-MS was used to obtain a molecular ion peak and this gave the required characterisation, which was confirmed by a close examination of the I^C- NMR spectrum to ensure that it contained no additional, unassigned peaks.
In order to see how 5-nitrosothiols might bind to metal ions, which is the main factor affecting their stability and perhaps their biological activity, a representation of the three- dimensional structure was produced using the computer programme Chem 3D and is shown in Figure 2. 1.
m
S-nitrosated amino acid (SNAP; 1) S-nitrosated dipeptides ( 2-12)
S-nitrosated tripeptide (GSNO; 13)
Figure 2.1: The three-dimensional nature of SNAP (1), 5-nitrosated dipeptides (2-12), and GSNO (13).