5480 J. Am. Chem. Soc., Vol. 123, No. 23, 2001
DaVies et al.
Scheme 2. Possible Monoprotonation Products of 1
diolate functional group as well. Such effects must be largely
responsible for the remarkable sensitivity to parent amine
structure and the wide range of NO dissociation rates that
diazeniumdiolate substrates display. For 5 in particular, the
surprising degree of dependence on concentration of substrate
as well as of certain metal ions may help to explain the reported
variability in this zwitterion’s half-life from one medium to
1
0,25
another.
Experimental Section
Compound 2 was synthesized as previously described, as were
9
,26
,27 4, 4a, 5, 6, and MeN(OMe)NO.
28
29
27
27
30
3
Kinetic Studies. Rate constants were measured spectrophotometri-
cally by monitoring the decrease in absorbance of the diazeniumdiolate
chromophore with Hewlett-Packard 8451 and 8452A Diode Array UV-
visible spectrophotometers. In a typical experiment, reaction was
initiated by adding a 10-µL aliquot of a stock solution (ca. 10 mM in
10 mM NaOH) to 1.0 mL of buffer in a thermostated UV cell or, for
runs with short half-lives requiring rapid mixing, by delivering 1.0-
mL aliquots of the buffer, previously thermostated at 37 °C, into 10
µL of the basic diazeniumdiolate stock solution in the cell. With a
microprocessor-controlled diode array spectrophotometer, the latter
technique enabled the first data points to be collected within ca. 3 s of
mixing. Absorbance-time (A - t) data obtained from the HP 8451
spectrophotometer were imported into a spreadsheet and first-order rate
to 230-nm shift to be accompanied by appearance of the n f
π* peak between ∼330 and ∼400 nm that normally character-
izes such structures22 [including MeN(OMe)-NdO, Table 9].
Increases in absorbance at 340-360 nm were seen on acidifying
10 mM solutions of 6, but these were too slow to be attributable
to proton transfer; we assume that these slow increases in
absorbance reflect formation of R2N-NdO species on recom-
bination of autoxidized NO with the secondary amine coproduct
7
of NO release, rather than protonation of tautomer A.
Theoretical calculations of Taylor et al.20 have shown the
charge densities on the O(1) and O(2) oxygens in anions
constants were calculated from a linear regression of the ln(A
t
∞
- A )
-
X[N(O)NO] to be virtually the same, suggesting that either
2
plots, which generally showed excellent linearity (R ) 0.999) over at
least 3 half-lives. For measurements involving 5 at 1 mM concentration
levels, 100-µL aliquots of reaction mixtures, thermostated at 37 °C,
were taken at timed intervals and diluted 10-fold with buffer prior to
absorbance measurement. When absorbance changes were examined
repetitively over the wavelength range 200-400 nm, isosbestic points
were maintained for the duration of the reactions. Data were collected
at 37 °C except where indicated otherwise.
oxygen would be susceptible to protonation. A comparison of
the energies for the optimized geometries of the tautomeric
structures X[N(O)NO]H, generated on protonation of the two
oxygen sites, however, was reported to show a clear preference
-
for the geometry having the [N(O)NO] group protonated at
the terminal O2 site, except when strong electron withdrawing
groups are present (a situation that exists for 6 when the R2N
nitrogen has already been protonated). With electron density
partially delocalized over the diazeniumdiolate group, and with
proton shifts between sites being facile and subject to minor
No differences were noted in rate constants obtained for 2-6 with
the HP 8451 and HP 8452A spectrophotometers, despite the more
extensive sample irradiation with the former instrument. This speaks
against any photochemically induced dissociation pathways with 2-6,
although enhanced light-assisted decomposition has been noted with
other diazeniumdiolate substrates.31
-
polarizations within the [N(O)NO] group, a distinction between
protonation at O1, O2, or O1 and O2 (i.e., bridging or rapidly
equilibrating O-protonation sites) cannot be made with confi-
dence on the basis of the currently available data.
Buffers of 0.10 M phosphate were prepared by weight from Na
2
-
HPO ‚7H O and NaH PO ‚H O, while 0.10 M acetic acid/sodium
4
2
2
4
2
acetate and 0.050 M glycine/HCl buffers were prepared by partial
neutralization of the acid or base with NaOH and HCl, respectively.
Tris buffers (0.050 M) were prepared from Tris base and Tris
hydrochloride. In all cases, the pH of reaction mixtures was checked
by using a JENCO Electronics Microcomputer pH-Vision 6071 pH
meter.
UV Spectral Analysis. Measurement of spectral shifts resulting from
protonation of the diazeniumdiolate functional group was carried out
by adding 10 µL of a freshly prepared 10 mM solution of diazenium-
Conclusion
Detailed kinetic studies have shown that diazeniumdiolates
release NO with reaction rates that vary, often dramatically, with
both pH and structure. Dual protonation states and reaction
pathways have been identified for the zwitterionic polyamine
substrate 6. Protonation of the secondary amine nitrogen would
appear to be the event responsible for initiating dissociation of
NO for compounds 2-6 and other diazeniumdiolate substrates
at physiological pH.
(
25) Gow, A. J.; Thom, S. R.; Brass, C.; Ischiropoulos, H. Microchem.
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(
From comparison of the present data with those for the
diazeniumdiolates K[O3SN(O)NO]K and Na[ON(O)NO]Na,
1
23
24
(
whose acid-catalyzed decompositions have been shown to yield
N2O rather than NO as their principal gaseous product, it is
apparent that the decomposition mechanism adopted is also a
sensitive function of the heteroatom to which the diazenium-
diolate functional group is attached.
For amine-derived diazeniumdiolates R2N[N(O)NO] , elec-
tronic and geometric constraints imposed by R appear to have
competing influences on the decomposition of the diazenium-
(
(
L. K. J. Org. Chem. 1999, 64, 5124-5131.
(30) Boese, A. B., Jr.; Jones, L. W.; Major, R. T. J. Am. Chem. Soc.
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(31) Srinivasan, A.; Kebede, N.; Saavedra, J. E.; Nikolaitchik, A. V.;
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(
22) Lijinsky, W. Chemistry and Biology of N-Nitroso Compounds;
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(
1
(