Resinas bulk fill

The presence of the -SNO group in a molecule can be readily determined from both infrared and UV-visible spectroscopy.

The broad and strong IR band at 1480-1530 cm“l has been assigned to the stretching vibration of the N=0 bond of the 5-nitrosothiol. N=0 vibrations of tertiary 5-nitrosothiols have been found at frequencies lower than those of primary ^-nitrosothiols. A second absorption band at 600-730 cm"^ is characteristic of the vibration of the C-S bond (see Figure 2. 2). %T »- a- it- INO IN m UN NO cm

Figure 2. 2: Infrared Fourier Transform spectrum of compound 2.

The UV-visible spectra of the 5-nitrosothiols display two bands at aiound 330-340 nm and 550-600 nm, which are responsible for their red or green colour in solution. These bands are shifted to lower energies by substitution at the a carbon atom. Tertiary 5-nitrosothiols are usually green compounds, whereas primary and secondary 5'-nitrosothiols aie red (see Figure 2. 3).

<0 CD X I

in

Wavelength (nm)

Figure 2. 3: UV-visible spectrum of compound 2 (5 x 10“^ mol dm~3).

From the literature sources, only few a ^-nitrosothiols have been previously characterised by or l^c-NMR spectroscopy. Both methods allowed a very convenient and reliable proof for nitrosation of thiol compounds. While thiols and disulphides had very similar NMR spectra, the a protons in the ^'-nitrosothiols were strongly shifted downfield (about

Ippm) (Roy et al, 1994). The P protons were also affected by the ^-nitrosation, to an extent that was dependent on the substitution at the a carbon. Almost no shift was observed for the primary 5-nitrosothiols, about 0.1 ppm for the secondary 5-nitrosothiols and about 0.5-0.8 ppm for the tertiary 5-nitrosothiols (Roy et al, 1994).

The l^C-NMR spectra were also changed by nitrosation of thiols. Resonances of a carbon atoms were shifted downfield, with stronger shifts observed for tertiary ^-nitrosothiols and resonances of p-carbon atoms were shifted upfield. The l^C-NMR chemical shifts for 5- nitrosothiols were in general found to be intermediate between those of the corresponding thiols and those of the disulphide (Roy et at., 1994).

As the A-acetyl-D,L“p,p-dimethylcysteine used was a racemic mixture the products were formed as a mixture of two diastereomers: e.g. V-acetyl-D-p,p-dimethylcysteinyl-L-amino acid methyl ester, and V*-acetyl-L-p,(3-dimethylcysteinyl-L-amino acid methyl ester. Due to this the compounds have complicated ^H-NMR spectra in most cases, but we were able to identify the -SH, CH3CONH, and the proton of the peptide bond (-CONH-) which

indicate that we have the correct dipeptide. After the nitrosation of the dipeptides, there are still two diastereomers: 5-nitroso-V-acetyl-D-p,p-dimethylcysteinyl-L-amino acid methyl ester, and •S-nitroso-A-acetyl-L-p,p-dimethylcysteinyl-L-amino acid methyl ester, and it was possible to identify the CH3CONH- and -CONH- signals and the disappearance of -SH

which indicates that nitrosation has occurred. Some decomposition occurred while the spectra were being run.

In l^C-NMR for both the thiols and the S-nitrosothiols there are two lines for each carbon due to the presence of the two diastereomers and usually DEPT 90“ and DEPT 135" enabled ÇH3-, -CH2-, and -CH- to be distinguished.

In the case of compound 2 the two isomers formed are enantiomers rather than diastereomers. Accordingly only one set of lines is seen in the l^C-NMR and the proton spectrum is also simpler.

Figure 2. 4: ^H-NMR spectrum of compound 2.

Figure 2. 5: l^C-NMR spectrum of compound 2.

181 léi ■ 171 lèi ■ là ■ iii |j| 12, lii t i i . ai ii ' it ù si ' « ' it ' ù ' l't ' 1

Figure 2.6: DEPT 90°-NMR spectrum of compound 2.

i « i ■ l i t ' i ) i ' l i t l i i l i t ' û , ' t i i ■ i l l ■ « * * ,j|8 8 ' 81 ■ ?'• ■ 11 . ’ » «1 ■ s* ' ù ' i'« ' r

Figure 2. 7: DEPT 135°-NMR spectrum of compound 2.

im

M+H

I M+Na

Lrk

Figure 2. 8: FAB-MS spectrum of compound 2.

Compound 2 is pure but the others are contaminated by small amounts of the corresponding thiol and/or disulphide. This may be due to steric hindrance by the R group in the other compounds interfering with the nitrosation reaction.

Microanalysis of the green solid or green sticky solid obtained from some 5-nitrosothiols showed that some disulphide had formed in its isolation as indicated by the high caihon and low nitrogen values (see elemental analyses; Experimental).

Samples of ^-nitrosothiols were characterised by a vaiiety of criteria, including elemental analysis, IR (e.g. Figure 2. 2), UV-visible (e.g. Figure 2. 3), ^H-(e.g. Figure 2. 4), l^C- (e.g. Figure 2. 5), DEPT 90° (e.g. Figure 2. 6), and DEPT 135°-NMR spectroscopy (e.g. Figure 2.1). Accurate mass determinations of the molecular ion peaks were obtained only by the use of high resolution FAB-MS but not by other techniques (e.g. Figure 2. 8). Data are displayed in the Experimental Section (next chapter).

A chemical and physiological comparison of the action of (a) 5-nitrosated amino acid (SNAP; 1) (b) 5-nitrosated dipeptides (2-12) and (c) S'-nitrosated tripeptide (GSNO; 13) is the subject of a large proportion of this thesis. Data have been compiled which give an insight into why these three types of 5-nitrosothiols (1-13) act differently and how S~