N-Nitrosated N-hydroxyguanidines are nitric oxide-releasing diazeniumdiolates

Article abstract of DOI:10.1039/a801543k

N-Hydroxyguanidines can be nitrosatively converted to zwitterionic diazeniumdiolates of crystallographically-confirmed structure H₂N⁺=C[NHR][N(O)NO]^-, whose hydrolytic dissociation at physiological pH leads to both NO and N₂O; the results appear to account for the formation of the 'potential intercellular nitric oxide carrier' produced on exposing N^G- hydroxy-L-arginine (a metabolic intermediate in mammalian NO biosynthesis) to aerobic NO.

Full text of DOI:10.1039/a801543k

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N-Nitrosated N-hydroxyguanidines are nitric oxide-releasing diazeniumdiolates
Garry J. Southan,*a†‡ Aloka Srinivasan,b Clifford George,c Henry M. Falesd and Larry K. Keeferb
a Intramural Research Support Program, SAIC Frederick, NCI-FCRDC, Frederick, MD 21702, USA
b Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and
Development Center, Frederick, MD 21702, USA
c Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC 20375, USA
d Laboratory of Chemistry, National Heart, Lung and Blood Institute, Bethesda, MD 20892, USA
N-Hydroxyguanidines can be nitrosatively converted to
zwitterionic diazeniumdiolates of crystallographically-con-
firmed structure H2N+NC[NHR][N(O)NO]2, whose hydro-
lytic dissociation at physiological pH leads to both NO and
N2O; the results appear to account for the formation of the
‘potential intercellular nitric oxide carrier’ produced on
exposing NG- hydroxy-L-arginine (a metabolic intermediate
in mammalian NO biosynthesis) to aerobic NO.
introduced instead of air into anaerobic solutions of 1b and NO,
suggesting that N2O3, formed either on autoxidation of NO or
on radical coupling of NO with NO2, had reacted nitrosatively7
with 1b. When alternate nitrosation conditions were effected by
dissolving 0.1 g of 1b·HCl in 2 ml of 0.1 m aqueous AcOH and
adding 1 equiv. of sodium nitrite as a saturated aqueous solution
at 0 °C, a crystalline precipitate began to form that, when
collected after 20 min of total reaction time (yield of 2b 95%,
mp 136–147 °C with gradual decomposition), was suitable for
X-ray diffractometric investigation without further purification.
(Indeed, attempts to recrystallize this relatively unstable
substance have thus far led to partial decomposition.) The
molecular structure,§ refined to an R value of 0.034, is shown in
Fig. 1.
Nitric oxide (NO) is a recently discovered bioeffector molecule
that is critically involved in regulation of blood pressure,
neurotransmission, sexual function, immunity and an array of
other physiological phenomena.1 It is rapidly destroyed by
biochemical oxidants such as oxyhemoglobin, superoxide and
oxygen,2 leading to speculation that its regulatory properties
might depend, in part, on conversion to a biochemical storage
form3 that protects it from oxidation while in transit from the
site of biosynthesis in one cell to the target of its required
signalling action in another.
The crystallographic data show that nitrosation of 1b
occurred at the nitrogen bound to the oxygen atom to give a
diazeniumdiolate product 2b, as shown in Scheme 1. Note-
worthy structural features include the cisoid oxygens in the
Recently, the formation of such a ‘potential intercellular
nitric oxide carrier’ was reported to occur on exposing NG-
hydroxy-l-arginine 1a, an intermediate in NO biosynthesis, to
aerobic nitric oxide solutions.4 The product was ultraviolet-
active and longer lived as a vasodilator than molecular NO,5 but
its structure has yet to be elucidated.
We present evidence here based on work with model NA-
substituted N-hydroxyguanidines that this bioactive compound
may be a naturally occurring diazeniumdiolate (Scheme 1,
structure 2a). While there was no observable reaction when NO
was bubbled into degassed solutions of NA-(p-methoxybenzyl)-
N-hydroxyguanidine 1b in water, subsequent introduction of air
led to appearance of an ultraviolet maximum at 322 nm, a result
reminiscent of that seen with both N-hydroxyguanidine 1c5 and
1a itself.6 A similar outcome was observed when NO2 was
H2N
H2N
+ C
RHN
O–
NO/O2
C
NOH
N
or HONO
RHN
N
O
1a–d
2a–d
H
+
a R = H3N
–O2C
CH2CH2CH2
CH2
b R = MeO
c R = H
d R = O2N
CH2
Scheme 1
N(2)
O(2)
O(3)
O(1S)
N(3)
N(4′)
O(1′)
N(1)
O(1)
N(1′)
N(3′)
N(4)
O(3′)
O(2S)
N(2′)
O(2′)
N(1′)–N(2′) = 1.313(5)
N(1′)–O(1′) = 1.325(5)
N(2′)–O(2′) = 1.248(5)
N(1)–N(2) = 1.319(5)
N(1)–O(1) = 1.321(5)
N(2)–O(2) = 1.254(5)
Fig 1 Molecular structure of the asymmetric unit of 2b summarizing bond lengths within the diazeniumdiolate (ONNO) groups. Dotted atoms are symmetry
related. Dotted bonds are hydrogen bonds.
Chem. Commun., 1998
1191

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2
N2O2 group and the presence of two different conformers of
also detected by HPLC. At pH 7.4, 0.5 moles of N2O were
produced along with 0.3 moles of NO per mole of 2b
dissociated. These reactions are summarized in Scheme 2.
Comparable results were observed on hydrolysis of 2c and
2d.
2b along with two water molecules in the asymmetric unit that
forms a hydrogen bonded helix (Fig. 1). Similar results were
obtained on nitrosation of the p-nitrophenyl analogue 1d;
although the structure of the diazeniumdiolate product 2d did
not refine well (R = 0.125), this was due to disorder in the
crystal which could not be modelled in the refinement. The
atomic connectivity and atom assignments are, however, well
defined.¶
Since nitrosation of all four N-hydroxyguanidines 1a–d
yields hydrolytically unstable products with ultraviolet maxima
near 320 nm that generate significant quantities of NO in neutral
buffer, the crystallographic data for products 2b and 2d strongly
suggest that the ‘nitric oxide carrier’ seen by Hecker et al.4 on
exposing 1a to aerobic NO was diazeniumdiolate 2a.
We postulate that our data may be of two-fold biological
significance. First, if free 1a can be shown to encounter suitably
nitrosating conditions in vivo, the product 2a would constitute a
naturally occurring diazeniumdiolate that spontaneously re-
leases NO at physiological pH; two other natural products
containing the diazeniumdiolate functional group, dopastin and
alanosine, have been reported to release NO only on one-
electron oxidation.11 Second, the NO-generating properties of
compounds 2 may render them useful as prodrugs for treating
clinical disorders arising from deficiencies of biosynthetic NO.1
The possible medicinal value of 2 and analogous structures will
be investigated.
Rapid measurement of the ultraviolet spectrum on dissolution
of 2b in water permitted quantitative determination of the
characteristic chromophore’s extinction coefficient (2.5 mm21
cm21 at lmax = 322 nm). The NMR spectrum, run at 250 °C
in CD3OD–tetramethylsilane to minimize the rapid decomposi-
tion observed at room temperature, consisted of singlets for the
CH3O and CH2 protons at d 3.77 and 4.57, respectively, and an
aryl AAABBA pattern with shifts of d 6.93 and 7.30 (ortho and
meta to OCH3, respectively) and J = 8.6, 2.7, and 0.4 Hz for the
ortho, meta and para couplings, respectively. The mass spectra
of 2b and 2d were unusual (but not unprecedented) in their
failure to produce an observable MH+ ion in either the
electrospray or fast atom bombardment modes; strong peaks
due to the respective benzyl cations were seen in both cases, but
15N-labelling indicated that the terminal NO was rapidly lost
even under the mildest possible electrospray conditions.
Compound 2b was found to decompose with gas evolution on
dissolution in aqueous media. At pH 10, N-(p-methoxy-
benzyl)cyanamide 3b and N2O were each produced in !95%
yield in a relatively slow reaction (half-life at 37 °C variable but
estimated as 15–25 min) that is presumably initiated by
deprotonation of the NH2 group followed by loss of cis-
hyponitrite (2O–NNN–O2), a known progenitor of N2O.8
Compound 2b tended to disappear more rapidly as pH was
lowered, with a half-life at pH 3 of about 5 min; some
denitrosation was observed at very low pH, 1b being the most
abundant of the organic products seen in 0.1 m HCl. The major
gaseous product at low pH proved to be NO, reaching yields of
0.9 moles per mole of 2b at pH 3 compared with 0.25 moles of
N2O. While the mechanism of NO formation is not clear at this
time, there can be little doubt about its production; its identity
was confirmed both by the presence of its aqueous autoxidation
product, nitrite ion,9 in the reaction mixture and by a well-
established, highly selective chemiluminescence method.10 In
physiological buffer (10 mm phosphate, pH 7.4), N-(p-
methoxybenzyl)urea 4b was produced in 90% yield, with 8%
conversion to 3b and a small amount of an unidentified product
Notes and References
† Present address: Inotek Inc., 3rd Floor, 3130 Highland Avenue,
Cincinnati, OH 45219-2374, USA.
‡ E-mail: gjsouthan@aol.com
§ Crystal data for 2b. C9H12N4O3·H2O, Mr = 242.24, monoclinic space
group P21, a = 12.452(2), b = 7.156(1), c = 12.887(2) Å, b = 97.74(1)°,
V = 1137.8(3) Å3, Z = 4, Dc = 1.414 Mg m23, l(Mo- Ka) = 0.71073 Å,
m = 0.113 mm21, F(000) = 512, T = 223 K. A set of 1773 reflections was
collected, and 1388 were observed with Fo > 4s(Fo), R1 = 0.034 and wR2
= 0.083.
¯
¶ Crystals were triclinic, space group P1, a = 6.256(1), b = 7.120(3), c =
13.180(2) Å, a = 95.11(1), b = 92.33(1), g = 106.01(1)°. CCDC 182/839.
1 L. K. Keefer, C .F. Nathan and W. J. Payne, in Encyclopedia of
Molecular Biology and Molecular Medicine, ed. R .A. Meyers, VCH,
Weinheim, 1996, vol. 4, p. 200.
2 D. A. Wink, M. B. Grisham, J. B. Mitchell and P. C. Ford, Methods
Enzymol., 1996, 268, 12.
3 D. I. Simon, M. E. Mullins, L. Jia, B. Gaston, D. J. Singel and J. S.
Stamler, Proc. Natl. Acad. Sci. USA, 1996, 93, 4736.
4 M. Hecker, H. Macarthur, W. C. Sessa, G. J. Southan, T. A. Swierkosz,
D. T. Walsh, A. Zembowicz and J. R. Vane, in The Biology of Nitric
Oxide. 2. Enzymology, Biochemistry and Immunology, ed. S. Moncada,
M. A. Marletta, J. B. Hibbs, Jr. and E. A. Higgs, Portland Press, London,
1992, p. 128.
RHN CN
5 A. Zembowicz, T. A. Swierkosz, G. J. Southan, M. Hecker and J. R.
Vane, Br. J. Pharmacol., 1992, 107, 1001.
3b
6 M. Hecker, M. Boese, V. B. Schini-Kerth, A. Mu¨lsch and R. Busse,
Proc. Natl. Acad. Sci. USA, 1995, 92, 4671.
7 D. L. H. Williams, Nitrosation, Cambridge University Press, Cam-
bridge, 1988.
8 J. Yoo and J. M. Fukuto, Biochem. Pharmacol., 1995, 50, 1995. These
authors have proposed 2 as a possible intermediate (postulated to form
on coupling of an oxidatively generated N-hydroxyguanidine radical
cation with NO) in the slow reaction of hydroxyguanidines with
anaerobic NO.
O–
2H+
N2O + H2O
N
O–
pH 10
N
H2N
+ C
RHN
O–
N
H2N
pH 7.4
N
+ N2O + NO
O
C
O
RHN
2b
4b
9 L. J. Ignarro, J. M. Fukuto, J. M. Griscavage, N. E. Rogers and R. E.
Byrns, Proc. Natl. Acad. Sci. USA, 1993, 90, 8103.
0.1 M HCl
H2N
10 M. Feelisch and E. A. Noack, Eur. J. Pharmacol., 1987, 142, 465; C. M.
Maragos, D. Morley, D. A. Wink, T. M. Dunams, J. E. Saavedra, A.
Hoffman, A. A. Bove, L. Isaac, J. A. Hrabie and L. K. Keefer, J. Med.
Chem., 1991, 34, 3242.
C
NOH
+ HONO
RHN
1b
11 T. A. Alston, D. J. T. Porter and H. J. Bright, J. Biol. Chem., 1985, 260,
4069.
R =
MeO
CH2
Scheme 2
Received in Corvallis, OR, USA, 23rd February 1998; 8/01543K
1192
Chem. Commun., 1998