Competitive formation of N-diazeniumdiolates and N-nitrosamines via anaerobic reactions of polyamines with nitric oxide

Article abstract of DOI:10.1021/ol902282y

"Chemical Equation Presented" Reactions of amines with nitric oxide (NO) at high pressures form diverse NO donor species, highly dependent on the precursor structure. While monoamine precursors favor the formation of N-diazeniumdiolates in high yield, pol

Full text of DOI:10.1021/ol902282y

ORGANIC
LETTERS
2009
Vol. 11, No. 23
5462-5465
Competitive Formation of
N-Diazeniumdiolates and N-Nitrosamines
via Anaerobic Reactions of Polyamines
with Nitric Oxide
Peter N. Coneski and Mark H. Schoenfisch*
Department of Chemistry, UniVersity of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-3290
schoenfisch@unc.edu
Received October 4, 2009
ABSTRACT
Reactions of amines with nitric oxide (NO) at high pressures form diverse NO donor species, highly dependent on the precursor structure.
While monoamine precursors favor the formation of N-diazeniumdiolates in high yield, polyamines exhibit competitive formation of N-nitrosamines
and diazeniumdiolates, resulting in mixed products containing significant percentages of undesired N-nitroso compounds.
The role of nitric oxide (NO) in physiological signaling
pathways, vasodilation, wound healing, and platelet aggrega-
tion has stimulated much research related to the molecular
storage of NO.1-5 Formulations of scaffolds modified with
functionalities capable of storing and releasing NO have been
developed as both tools for further elucidating NO’s role in
physiology and as potential therapeutics.6-12 Among the
numerous types of NO-donating compounds, N-diazenium-
diolates are among the most widely researched donors due
to their straightforward synthesis, long-term storage capabili-
ties, and ability to spontaneously release NO under physi-
ological conditions.6,8
Although the first report regarding the synthesis of
N-diazeniumdiolates occurred almost 50 years ago,13,14 it was
not until the identification of NO as a ubiquitous signaling
molecule in the early 1990s that synthetic strategies to
manipulate NO storage and release, and associated pharma-
cological effects were significantly accelerated.6,15 Two
primary hypotheses regarding the mechanism of diazenium-
diolate formation under anaerobic conditions have been
proposed differing only in the reactive state of NO. In the
first, NO reacts with an amine to form the nitrosamine radical
anion that subsequently reacts with another molecule of NO
to form the diazeniumdiolate.16-18 In contrast, others have
proposed that NO first dimerizes to N2O2 and then acts
electrophilically in coupling with the amine to form the
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10.1021/ol902282y CCC: $40.75
Published on Web 11/09/2009
 2009 American Chemical Society

diazeniumdiolate.16 Using the mechanistic understanding
from the hypothesis of sequential NO addition, herein we
demonstrate experimentally that nitrosamines form competi-
tively with diazeniumdiolates in anaerobic reactions of NO
with polyamines.
Previous work with secondary amines indicates efficient
diazeniumdiolate formation in situ under high pressures (4-5
atm) of NO and basic conditions.19-21 According to the
mechanisms first proposed by Drago and investigated by
others,16-18 the rate limiting step of diazeniumdiolate forma-
tion is the second addition of NO to the intermediate
nitrosamine radical anion. As such, low NO concentrations
and/or slow rate determining steps (Scheme 1) may facilitate
reaction vessel in order to minimize nitrosamine formation
via alternative pathways mediated by NO2.22 Nitric oxide
release from the products was quantified under either
standard physiological conditions (pH 7.4 phosphate buffered
saline, 37 °C) or highly basic conditions (1.0 M NaOH) in
the presence of direct 200 W broad spectrum light. The
degradation of N-diazeniumdiolates to NO and the parent
amine in phosphate buffered saline,6,15 and the stability of
nitrosamines in aqueous solutions at physiological pH7 are
well-known. Similarly, direct light irradiation in basic
solutions was chosen for the detection of nitrosamine-derived
NO due to the known photodegradation of nitrosamines and
stability of diazeniumdiolates under basic conditions.7,15
Unexpectedly, more pronounced NO release was observed
with light irradiation compared to physiological conditions
for diethylenetriamine (4) and N,N′-diheptyl ethylenediamine
(5) (Figure 2), indicating a mixture of products, including
Scheme 1. Mechanisms of Diazeniumdiolate Formation:
(a) Sequential NO Addition, (b) Dimer Addition16-18
incomplete diazeniumdiolate formation from nitrosamine
intermediates, which despite their ability to controllably
release NO in the presence of light, are widely considered
to be carcinogenic.7
To assess the possibility of nitrosamine formation, a
number of monoamine and polyamine species (Figure 1)
Figure 2. Real-time and total (inset) NO release of NO-treated 5
analyzed in the presence of 1.0 M NaOH, light (black), and PBS,
pH 7.4 (gray).
both the N-nitroso and the N-diazeniumdiolated species.
When the monoamine compounds proline (1), pyrrolidine
(2), and piperidine (3) were exposed to similar conditions,
NO release in the presence of light was minimal, indicating
preferential formation of N-diazeniumdiolates over nitro-
samines (Table 1).
Absorption spectroscopy was used to confirm the forma-
tion of both N-nitroso and N-diazeniumdiolate products on
polyamine precursors (4, 5, N,N′-diheptyl- 1,4-butylenedi-
(17) Zhang, H.; Annich, G. M.; Miskulin, J.; Stankiewicz, K.; Oster-
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(18) Drago, R. S.; Ragsdale, R. O.; Eyman, D. P. J. Am. Chem. Soc.
1961, 83, 4337–4339.
Figure 1. Amine compounds investigated: (1) proline, (2) pyrro-
lidine, (3) piperidine, (4) diethylenetriamine (DETA), (5) N,N′-
diheptyl ethylenediamine (DHED), (6) N,N′-diheptyl-1,4-butylene-
diamine (DHBD), (7) N,N′-dibutyl decylenediamine (DBDD).
(19) Saavedra, J. E.; Southan, G. J.; Davies, K. M.; Lundell, A.; Markou,
C.; Hanson, S. R.; Adrie, C.; Hurford, W. E.; Zapol, W. M.; Keefer, L. K.
J. Med. Chem. 1996, 39, 4361–4365.
(20) Saavedra, J. E.; Billiar, T. R.; Williams, D. L.; Kim, Y.-M.; Watkins,
S. C.; Keefer, L. K. J. Med. Chem. 1997, 40, 1947–1954.
(21) Reynolds, M. M.; Zhou, Z.; Oh, B. K.; Meyerhoff, M. E. Org.
Lett. 2005, 7, 2813–2816.
were synthesized and treated with high pressures (4 atm) of
NO for 3 d. Care was taken to remove oxygen from the
(22) Shank, R. C. Toxicol. Appl. Pharmacol. 1975, 31, 361–368.
Org. Lett., Vol. 11, No. 23, 2009
5463

amine precursor. Intramolecular hydrogen bonding is
possible on the nitrosamine radical anion intermediate
formed during diazeniumdiolate formation for polyamine
compounds, but not monoamines. Specifically, neighboring
amines may act to stabilize the nitrosamine intermediate
in compounds derived from 4 and 5 in the form of
N-H···OdN, which has recently been shown to be more
stable than N-H···NdO.23 The lack of multiple amines
on other compounds such as 1-3 do not allow for this
type of stabilization. While fully formed diazeniumdiolates
and nitrosamines may both experience similar hydrogen
bond stabilization, interruption of the reaction progression
at the intermediate stage due to hydrogen bonding impedes
the sequential addition of NO and thus the complete
conversion to the diazeniumdiolate species. Although the
influence of hydrogen bonding is decreased in polar
solvents, collapse of the hydrophobic alkyl segments of
the molecules may further facilitate such stabilizing
interactions due to proximity of amines after chain
collapse. Representative NO release curves for the monoam-
ine 1 after treatment with NO indicate a strong preference
of diazeniumdiolation, as NO release due to light is
insignificant compared to that occurring in buffer immer-
sion alone (Figure 4).
Table 1. NO Release Characteristics of Monoamine and
Polyamine Compounds Exposed to 4 atm NO for 72 h
amine
precursor
NO initiation
method
NO release
(µmol/mg)a
nitrosamine/
diazeniumdiolateb
1
2
3
4
5
6
7
Light
Proton
Light
Proton
Light
Proton
Light
Proton
Light
Proton
Light
Proton
Light
Proton
0.04 ( 0.01
3.98 ( 0.49
0.06 ( 0.01
11.67 ( 0.65
0.06 ( 0.01
5.74 ( 0.22
1.14 ( 0.16
0.86 ( 0.09
2.16 ( 0.17
0.04 ( 0.01
1.58 ( 0.44
4.56 ( 0.51
1.47 ( 0.08
4.21 ( 0.31
1.00/49.75
1.00/97.09
1.00/47.85
1.00/0.38
1.00/0.01
1.00/1.44
1.00/1.43
a Total after 32 min analysis for monoamine compounds and 16 h
analysis for polyamine compounds based on duration of NO release.
b Calculated from total NO release assuming one and two molecules of
NO for nitrosamine and diazeniumdiolates respectively.
amine (6), and N,N′-dibutyl-1,10-decylenediamine (7)). Ab-
sorbance at 252 nm, indicative of N-diazeniumdiolates,6 was
most intense for monoamine compounds 1-3, but visible
for all species treated with NO except for 5, which exhibited
almost 100-fold greater preference for nitrosamine formation
(Table 1). All NO-treated polyamine compounds showed
supplementary absorbance values between 330-350 nm
associated with the nfπ* transition of N-nitroso compounds
(Figure 3).7 Of note, the molar absorptivities of these two
Figure 4. Real-time NO release of NO-treated 1 analyzed in 1.0
M NaOH with direct light (black), and PBS pH 7.4 (gray). [Inset:
enlarged view of NO flux in 1.0 M NaOH with direct light.]
Additional evidence supporting the hydrogen-bonding
dependence on the observed nitrosamine products is con-
firmed with other polyamine compounds. As the amine
spacing of polyamine compounds was increased from eth-
ylene for 5 to butylene for 6 (as well as ethylene (5) to
decylene (7)) the calculated nitrosamine:diazeniumdiolate
ratio decreased (NO-release analysis, Table 1). Additionally,
the UV absorbance due to nitrosamines decreased for 6 and
7 relative to 5, with a concomitant increase in the diazeni-
umdiolate absorbance (Figure 3). The shift from nitrosamine
to diazeniumdiolate formation may be attributed to the
Figure 3. UV/vis spectra for NO treated 2 (dashed black), 5 (solid
black), and 6 (solid gray) in MeOH. [Inset: Expanded view of
N-nitrosamine absorbance region.]
functional groups spanned several orders of magnitude, from
40-90 M-1 cm-1 for the nitrosamine peaks of the synthe-
sized polyamines to 7.2-9.4 × 103 M-1 cm-1 for diazeni-
umdiolates.6
The preference for diazeniumdiolate versus nitrosamine
formation seems to be influenced by the structure of the
(23) Roohi, H.; Gholipour, Y. Int. J. Quantum Chem. 2008, 108, 462–
471.
5464
Org. Lett., Vol. 11, No. 23, 2009

increased strain associated with the formation of larger ring
systems necessary for the intramolecular hydrogen bond
stabilitization on the nitrosamine intermediate. The decreased
ability for nitrosamine stabilization may facilitate the second
NO addition, thus increasing the efficiency of diazeniumdi-
olate formation.
To address the incomplete diazeniumdiolate formation,
alterations of the NO addition reactions were made. Experi-
ments were performed by increasing both the total reaction
time of the NO addition from 72 to 120 h and the amount
of NO available for reaction by increasing the pressure of
the NO reactor from 4 to 10 atm (maintaining a reaction
time of 72 h). In both instances, the presence of nitrosamines
was still observed for the polyamine compounds of interest
(data not shown).
Spectroscopic and NO-release analysis indicates that both
diazeniumdiolates and nitrosamines form competitively with
exposure of polyamines to high pressures of NO. The lack
of stabilizing intramolecular hydrogen bonds for monamine
compounds (e.g., 1-3) facilitates the conversion of nitro-
samine intermediates to the intended diazeniumdiolates.
However, molecules capable of forming intramolecular
hydrogen bonds display an impeded level of diazeniumdiolate
formation due to the increased stability of the nitrosamine
intermediate. As the effects of NO derived from diazeni-
umdiolate degradation has been reported to be beneficial by
numerous researchers, the results presented here indicate that
the increased incidence of nitrosamine formation on polyamine
scaffolds may pose concern for some applications of these
NO donors. In contrast, the suitability of monoamine species
as diazeniumdiolate donors is supported by minimal nitrosamine
formation. Overall, the results suggest that current synthetic
methods may require revision to minimize nitrosamine forma-
tion on polyamine compounds, or at least stringent characteriza-
tion to confirm their absence.
Acknowledgment. This work was supported by the
National Institutes of Health (NIH EB000708). We thank
Dr. Matthew Crowe and Dr. Sohrab Habibi, Department of
Chemistry, University of North Carolina at Chapel Hill, for
assistance with product analysis.
Supporting Information Available: Synthetic and ex-
perimental procedures and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
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