Functional in vivo EPR Spectroscopy and Imaging Using Nitroxide and Trityl Radicals - Stable Radicals: Fundamentals and Applied Aspects of Odd-Electron Compounds - page 561

S S SS SS

N N

N N

N N

15

15

N
O

N
O

N
O .

N
O

N
O

N
O

.

R 1 SSR 1 R 2 SSR 2

15 N-R 2 SSR 2

Scheme 16.9 Structures of the disulfide nitroxyl biradicals (DNB): bis(2,2,5,5-tetramethyl-3-imidazoline-1-
oxyl-4-il)-disulfide (R 1 SSR 1 ), 12 bis(2,2,3,5,5-pentamethyl-1-oxyl-imidazolidine-4-ylmethyl)-disulfide (R 2 SSR 2 ) 144
and its

15 N-substituted analog, bis(2,2,3,5,5-pentamethyl-1-oxyl-imidazolidine-4-ylmethyl)-disulfide 1 15 N

−

( 15 N-R 2 SSR 2 ) 19

GSH

I m

10 4

gain 5 × 10 4

(a) (c)

GSH

I m

10 4

6.3 × 10 3

gain 2.5

×

(b) (d)

Figure16.17 TheL-bandEPRspectraof0.1mMaqueoussolutionsofR 2 SSR 2 (a,c)and 15 N-R 2 SSR 2 (b,d)before
(a,b)andafter(c,d)additionof1mMGSH.TheEPRspectraoftheRSSRand 15 N-R 2 SSR 2 labelshaveadditional
''biradical'' components due to intramolecular spin exchange between two radical fragments of the DNB. The
splittingofthedisulfidebondsinthereactionofthiol-disulfideexchangewithGSHresultsinthedisappearance
of the ''biradical''components and corresponding increase of the peak intensity of the ''monoradical'' spectral
component,I m . The maximalincreaseof theI m correspondingtocompletesplittingofthe disulfidebond in the
excessofGSHisequalto4.6-and3.0-foldforR 2 SSR 2 and 15 N-R 2 SSR 2 ,respectively.

DNB labels, being paramagnetic analogs of widely used disulfide Ellman's reagent, 133 react with thiols,

BSH, via the reversible reaction of thiol - disulfide exchange (Equation 16.1):

RS - SR + BSH ⇐⇒ RSSB + RSH (16.1)

EPR spectra of the DNBs are significantly affected by intramolecular spin exchange between two radical
fragments, as shown in Figures 16.17a and 16.17b. Figures 16.17c and 16.17d demonstrate the typical
changes of the EPR spectra of the DNB labels upon addition of GSH. The decrease of biradical spectral