Mechanistic studies on the reaction between R2N-NONOates and aquacobalamin: evidence for direct transfer of a nitroxyl group from R 2N-NONOates to cobalt(III) centers

Article abstract of DOI:10.1002/anie.200904360

Tales of the unexpected: Transfer of a nitroxyl group from R2N-NONOates to aquacobalamin to form nitroxylcobaiamin does not proceed via H+-catalyzed R2NNONOate decomposition, but instead occurs via a probable NONOate-cobalamin intermediate (see scheme; r.

Full text of DOI:10.1002/anie.200904360

Angewandte
Chemie
DOI: 10.1002/anie.200904360
NONOates as Nitroxyl Donors
Mechanistic Studies on the Reaction between R N-NONOates and
2
Aquacobalamin: Evidence for Direct Transfer of a Nitroxyl Group
from R N-NONOates to Cobalt(III) Centers**
2
Hanaa A. Hassanin, Luciana Hannibal, Donald W. Jacobsen, Mohamed F. El-Shahat,
Mohamed S. A. Hamza, and Nicola E. Brasch*
The gaseous radical nitric oxide (CNO, NO) is a signaling
molecule that plays a vital role in biology. It facilitates
vasodilation and inhibits platelet aggregation in the cardio-
vascular system, initiates the pro-inflammatory immune
[
1,2]
response, and regulates neurotransmission.
Impaired NO
bioavailability is associated with a wide variety of vascular
[3]
pathologies, including endothelial cell dysfunction. Conse-
quently, there is considerable interest in NO donor molecules,
such as 1-(N,N-dialkylamino)diazen-1-ium-1,2-diolates (R N-
2
NONOates; Figure 1), which spontaneously decompose by
first-order acid-catalyzed processes to release up to two NO
[
4–6]
molecules and the corresponding amine.
R N-NONOates
2
Figure 1. Structures of selected R N-NONOates. (For definitions of the
abbreviations, see the Supporting Information).
2
are widely used as NO precursors in studies of NO-dependent
[
5,7]
biological processes
and as NO prodrugs with applications
in NO-releasing biomaterials, wound healing, organ protec-
[
5,8]
tion, and chemotherapy.
R N-NONOates can also be
medium and unreactive with most biomolecules, including
2
[
9]
[5,8,18]
combined with anti-inflammatory drugs and incorporated
into dendrimers, nanoparticles, or microspheres to optimize
thiols.
Reports on the reactions between R N-NON-
2
Oates and transition metal complexes are rare. X-ray
[
10–12]
II
their delivery.
reacting amines with NO(g) at high pressures.
alkylated R N-NONOate conjugates with considerably
These species are typically synthesized by
structures of Cu /R N-NONOate complexes show that NON-
2
[
6,13,14]
O2-
Oate can coordinate to metal centers through one or both
[19–21]
oxygen atoms.
For a series of metal cations, it was also
2
[15–17]
enhanced stability have also been developed.
shown that the NO release rates from the NONOate adduct
of the polyamine spermine is relatively insensitive to a range
One important distinction between R N-NONOates and
the majority of other NO donor species is that they are
generally regarded as being insensitive to the reaction
2
3
+
3+
3+
2+
2+
2+
of metal cations (Fe , Al , La , Ca , Zn , Mg ; rate of
spontaneous NONOate decomposition up to 60% slower,
presumably via binding of the NONOates to the metal
[
4]
center ). However, by functionalizing one alkyl substituent
[
*] H. A. Hassanin, Dr. N. E. Brasch
Department of Chemistry and School of Biomedical Sciences, Kent
State University
of the R N-NONOate dialkylamine moiety to provide addi-
2
tional donor atoms to the metal center, the rate of NO release
Kent, OH 44242 (USA)
from the metal-coordinated R N-NONOate can be slowed by
2
E-mail: nbrasch@kent.edu
[21]
over an order of magnitude.
H. A. Hassanin, Dr. M. F. El-Shahat, Dr. M. S. A. Hamza
Department of Chemistry, Faculty of Science, Ain Shams University
Abbassia, Cairo (Egypt)
Cobalamins (Cbls, vitamin B12 derivatives) are octahe-
drally coordinated cobalt(III) macrocycles, and can incorpo-
rate a range of ligands at the upper (b) axial site. These
L. Hannibal, Dr. D. W. Jacobsen
School of Biomedical Sciences, Kent State University
+
+
include aqua/hydroxy (H OCbl /HOCbl; pK (H OCbl ) =
2
a
2
7
.76 ꢀ 0.02 at 25.08C, total ionic strength I = 0.50m
Kent, OH 44242 (USA)
and
[
22]
(KNO ) ), cyanide (CNCbl), methyl (MeCbl), 5’-deoxyade-
3
Department of Cell Biology, Lerner Research Institute, Cleveland
Clinic,
Cleveland, OH 44195 (USA)
nosyl (AdoCbl), nitro (NO Cbl), and nitrosyl (NOCbl)
2
ligands. In searching for an efficient method to synthesize
nitroxylcobalamin (also referred to as nitrosylcobalamin,
NOCbl) for X-ray diffraction studies, we found that it can be
synthesized in high yield and purity by reacting HOCbl with
diethylamine-NONOate (DEA-NONOate, Figure 1) under
[
**] We thank Prof. Rudi van Eldik, University of Erlangen-Nꢀrnberg
(
Germany) and Prof. Istvꢁn Fꢁbiꢁn, University of Debrecen,
Hungary for useful discussions. This research was funded by the
Egyptian Ministry of Higher Education (PhD scholarship to H.A.H.),
[
23]
anaerobic conditions. Like other R N-NONOates, DEA-
and the NSF (CHE-0848397, N.E.B.). R N-NONOates=1-(N,N-
2
2
ꢁ
dialkylamino)diazen-1-ium-1,2-diolates, nitroxyl=NO .
NONOate decomposes under anaerobic conditions by a
clean, first-order, acid-catalyzed process to release NO and
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200904360.
[
4,24,25]
the corresponding amine, namely diethylamine (DEA).
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Control experiments showed that DEA-NONOate decom-
Table 1: Apparent rate constants kapp for the reaction between HOCbl
and DEA-NONOate, and observed rate constants for the spontaneous
poses cleanly into DEA alone (+ NO; pD 9.50 and 10.40;
1
decomposition of HOCbl (kHOCbl) and DEA-NONOate (k ).
L
H NMR spectroscopy). It is, however, well established that
+
[26]
ꢁ1
ꢁ1
3
ꢁ1
3
ꢁ1
neither H OCbl nor HOCbl react with NO. We therefore
pH
k
app [Lmol min
]
10 kHOCbl [min
]
10 k [min ]
L
2
carried out kinetic and mechanistic studies on this intriguing
reaction.
Figure 2 gives typical UV/Vis spectra for the reaction
between HOCbl (0.050 mm) and excess DEA-NONOate
9
9
.50
.80
0.68ꢀ0.02
0.29ꢀ0.03
0.14ꢀ0.01
0.33ꢀ0.01
0.97ꢀ0.02
1.43ꢀ0.05
1.32ꢀ0.03
1.24ꢀ0.01
0.555ꢀ0.008
0.197ꢀ0.001
10.40
10.80
0.056ꢀ0.002
t
=
2
1
ꢂ1 week
(
10.0 mm) as a function of time under anaerobic conditions
at pH 10.80 (0.30m CAPS buffer, 25.08C, I = 1.0m
NaCF SO )). High buffer concentrations (0.30m) were
[
a] I=1.0m (NaCF SO ), 0.30m buffer, 258C.
3 3
(
3
3
directly with the DEA-NONOate complex to give NOCbl,
and that decomposition of DEA-NONOate to form NO is not
a prerequisite for the reaction to occur. The direct transfer of
ꢁ
a nitroxyl group (NO ) from R N-NONOate to a transition
2
metal center to yield the corresponding nitroxyl complex is, to
our knowledge, unprecedented.
To further probe for formation of intermediate(s), the
1
reaction was followed using H NMR spectroscopy. Cbl
complexes have five corrin and nucleotide protons that
resonate in the aromatic region with chemical shifts depen-
dent on the b-axial ligand. Observation of the reaction of
HOCbl (2.96 mm) with DEA-NONOate (4.44 mm) at pD
1
1
6
1.30 by H NMR spectroscopy showed that HOCbl (d = 7.17,
.70, 6.49, 6.23, and 6.04 ppm) was cleanly converted into
NOCbl (d = 7.44, 7.27, 6.80, 6.35, and 6.25, in agreement with
[
27,29]
literature values
), without any detectable Cbl intermedi-
ate (Supporting Information, Figure S1). Alkaline conditions
were used to ensure that the spontaneous decomposition of
DEA-NONOate is negligible. The reaction stoichiometry was
1
determined by recording H NMR spectra of the products of
the reaction between HOCbl and 0.55, 1.1, 1.2, 1.5, or 2.2 mol
equivalents of DEA-NONOate at pD 10.42. With 0.55 equiv-
alents of DEA-NONOate, a mixture of NOCbl (ca. 55%) and
unreacted HOCbl (approx. 45%) was observed, whereas with
1
.2 equivalents of DEA-NONOate, HOCbl was essentially
completely converted into NOCbl (Figure 3a). Because
approximately 1.2 equivalents of DEA-NONOate is required
for the reaction to proceed to completion, this observation
suggests that only one of the two nitric oxide moieties in the
parent NONOate reacts with the cobalamin to form NOCbl.
Figure 2. UV/Vis spectra for the reaction between HOCbl (0.050 mm)
and DEA-NONOate (10.0 mm) at pH 10.80. (Spectra taken every
1
0.0 min, 0.30m CAPS, I=1.0m (NaCF SO ), 25.08C.) Isosbestic
3 3
points occur at 341, 370, and 498 nm. Inset a) First and last spectra.
The final product is NOCbl. b) Fit of the absorbance data at 356 nm
(
A356) versus time to a first-order rate equation, giving
ꢁ3
ꢁ1
kobs =(1.91ꢀ0.01)ꢂ10 min
.
necessary to ensure a stable pH was maintained during the
reaction. Figure 2, Inset a, shows a comparison between the
initial and final spectrum of the reaction in which HOCbl is
cleanly converted into NOCbl (lmax = 256, 278 (shoulder),
2
3
89, 315, and 478 nm) with sharp isosbestic points observed at
41, 370, and 498 nm, which is in agreement with literature
[27,28]
values.
In Figure 2, Inset b, the best fit of the absorbance
1
Figure 3. H NMR spectrum of the products of the reaction between
data at 356 nm versus time to a first-order rate equation is
superimposed upon the experimental data, giving k
HOCbl and 1.2 equiv DEA-NONOate after 5 days (pD 10.42, 0.50m
CAPS). a) Aromatic region showing the 5 characteristic signals of
NOCbl at d=7.73, 7.27, 7.00, 6.48, and 6.31 ppm. The small peaks are
impurities arising from Cbl decomposition at the high pD conditions.
b) 4.3–3.5 ppm region, showing signals attributable to DEA-NO
=
obs
ꢁ
3
ꢁ1
(
1.91 ꢀ 0.01) ꢀ 10 min . The rate of spontaneous acid-
catalyzed decomposition of DEA-NONOate to NO and
DEA was found to be more than one order of magnitude
slower than the reaction of DEA-NONOate with HOCbl
(
d=4.20, 4.18, 4.16, 4.15, 3.74, 3.72, 3.71, and 3.69 ppm) overlapping
1
with NOCbl signals. These signals were not observed in the H NMR
spectrum of the reactant HOCbl. Inset: H NMR spectrum of authentic
1
(
half-life for the spontaneous decomposition t1/2 ꢂ 1 week,
pH 10.80; Table 1). This result suggests that HOCbl reacts
DEA-NO (0.50m CAPS, pD=10.42).
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Experiments were carried out to identify the non-coba-
lamin reaction product(s). It was determined that nitrite was
not a reaction product (Griess assay; see Supporting Infor-
mation). Two other potential non-Cbl reaction products,
diethylamine (DEA) and N-nitrosodiethylamine (DEA-NO),
of HOCbl in alkaline solution has been reported previ-
ously.
[32,33]
The dependence of kobs on DEA-NONOate concentration
(2.50–25.0 mm) was studied at three other pH conditions
(pH 9.50, 9.80 and 10.40). The data is plotted in Figure S2 in
the Supporting Information and the rate constants are
summarized in Table 1. It was necessary to take into account
spontaneous DEA-NONOate decomposition in the treat-
ment of the kinetic data at pH 9.50 and 9.80 at the lower
DEA-NONOate concentrations by fitting to Equation (1):
1
are individually distinguishable by H NMR spectroscopy.
1
The H NMR spectrum of the products of the reaction of
HOCbl and 1.2 equivalents of DEA-NONOate in alkaline
solution (pD 10.42) in the 3.5–4.3 ppm region revealed DEA-
NO to be the non-Cbl reaction product (Figure 3b). There-
fore, DEA-NONOate reacts directly with HOCbl to produce
NOCbl and DEA-NO. Formation of the corresponding
nitrosamine is undesirable from a biological and pharmaceut-
ical view point, given that many of these species, including
k
app ½Lꢃ
0
ðeꢁkL tꢁ1ÞÞ
Aobs ¼ A
1
þ ðA
0
ꢁA
1
Þexpð
ð1Þ
k
L
[
30]
DEA-NO, are carcinogenic. Nitrosamines are also products
of R N-NONOate photolysis and R N-NONOate decompo-
where Aobs, A , and A are the observed, initial, and final
0 1
absorbances respectively, kapp is the (pH-dependent) rate
constant, kL is the observed rate constant for spontaneous
2
2
[
30,32]
sition under aerobic conditions.
Although it has been
previously suggested that DEA-NO rapidly decompose to
NONOate decomposition, and [L] is the initial NONOate
concentration. The derivation of this equation is given in the
Supporting Information.
Above pH 10.80, the rate of reaction between HOCbl and
DEA-NONOate is extremely slow, whereas at pH values
below 9.50, the spontaneous decomposition of DEA-NON-
Oate was found to be within one order of magnitude or even
faster than the reaction of interest. For example, at pH 9.30,
the observed rate constant for the reaction between DEA-
NONOate (2.5 mm) and HOCbl (0.050 mm) is 3.0 ꢀ
0
[
4,25]
DEA and NO,
To confirm that DEA-NONOate, rather than its decom-
in this study it was a stable species.
+
+
position products, react with H OCbl /HOCbl, H OCbl /
2
2
HOCbl was added to a solution of DEA-NONOate that had
fully decomposed to DEA and NO(g) at pH 7.40, 8.50, or
9
.80, and the reaction was followed by UV/Vis spectroscopy.
No reactions were observed after 16 h under any of the three
pH conditions.
If indeed HOCbl reacts directly with DEA-NONOate, the
observed rate constant should depend on the DEA-NON-
Oate concentration. The dependence of kobs on DEA-NON-
Oate concentration (2.5–25.0 mm) at pH 10.80 was therefore
determined (Figure 4). Measurements at higher DEA-NON-
Oate concentrations were not possible owing to the limited
solubility of DEA-NONOate. Fitting the data to a straight
ꢁ
3
ꢁ1
10 min , whereas that for spontaneous decomposition of
ꢁ
3
ꢁ1
DEA-NONOate is 1.6 ꢀ 10 min . At higher NONOate
concentrations, although the rate of the Cbl/NONOate
reaction is faster, considerable interference occurs from gas
evolution despite gentle stirring with stir bars at the bottom of
the cuvettes. The gas arises from acid-catalyzed spontaneous
DEA-NONOate decomposition to NO(g) and DEA, leading
to unreliable data. Furthermore, a second reaction was
observed below pH 10, which was subsequently shown to
arise from excess NO(g) from decomposed DEA-NONOate
line gives a second-order rate constant, k = (0.056 ꢀ
app
ꢁ1
ꢁ1
0
.002)Lmol min (= kK’; see below) at pH 10.80 with an
ꢁ3
ꢁ1
intercept of (1.31 ꢀ 0.02) ꢀ 10 min . A control experiment
showed that HOCbl itself slowly decomposes at pH 10.80
ꢁ3
ꢁ1
(
kHOCbl = (1.32 ꢀ 0.03) ꢀ 10 min ; Table 1, which, within
reacting with NOCbl to form nitrocobalamin (NO Cbl). This
2
experimental error, is the same as the intercept. Note that
HOCbl decomposition is not strictly first-order and does not
proceed to completion at lower pH values. The self-reduction
reaction becomes increasingly important at lower pH values
and higher DEA-NONOate conditions. Further details are
given in the Supporting Information.
From Table 1, it can be seen that the second-order rate
constant kapp increases with decreasing pH. The pK of DEA-
a
[4]
1
NONOate is 5.0, and a H NMR titration experiment
showed no further deprotonation for DEA-NONOate in the
range pH 8.5–12.5. Control experiments showed that DEA-
NONOate does not react with methylcobalamin (MeCbl),
cyanocobalamin (CNCbl) or adenosylcobalamin (AdoCbl).
This result suggests that a labile b-axial ligand, such as the
+
aqua ligand of H OCbl , is required for the reaction between
2
Cbls and NONOates to proceed. It is well established that
[
22]
HOCbl is inert to substitution. Using the value of k at
app
[22]
ꢁ1
ꢁ1
+
pH 9.50 (0.68Lmol min ) and pK (H OCbl ) = 7.76,
second-order rate constant for the reaction between H OCbl
and DEA-NONOate of approximately 38Lmol min (ca.
.63Lmol s ) was estimated. However, rate constants for
ligand substitution of the b-axial ligand of H OCbl are
typically two to four orders of magnitude larger than this.
The simplest mechanism consistent with the experimental
a
a
2
+
2
ꢁ
1
ꢁ1
Figure 4. Plot of observed rate constant kobs versus NONOate
concentration for the reaction between HOCbl and DEA-NONOate
ꢁ1
ꢁ1
0
+
at pH 10.80. The best fit of the data gives k =(0.056ꢀ0.002)
2
app
[34]
ꢁ1
ꢁ1
ꢁ3
ꢁ1
Lmol min (slope) and kHOCbl =(1.32ꢀ0.03)ꢂ10 min (intercept,
0
.30m CAPS, I=1.0m (NaCF SO ), 25.08C).
3 3
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8911

Communications
HOCbl, although further studies are
required to confirm this. Sterically
demanding substituents also stabilize
R N-NONOates with respect to acid-cata-
2
[6,35]
lyzed decomposition (Table 2).
Kinetic studies on the reaction between
+
H OCbl /HOCbl and MAHMA-NON-
2
Oate were also carried out. As with
DEA-NONOate above, HOCbl reacts
with MAMHA-NONOate to produce
NOCbl (Supporting Information, Fig-
ure S3). Interestingly, although the plot of
kobs versus NONOate concentration was
+
Scheme 1. Proposed mechanism for the reaction of H OCbl /HOCbl with R N-NONOate.
2
2
data is given in Scheme 1, in which a rapid pre-equilibrium to
form the NONOate-Cbl complex precedes rate-determining
Table 2: pK values and rate constants for spontaneous, acid-catalyzed
a
decomposition of R N-NONOates (378C).
2
nitrogen–nitrogen bond cleavage to give NOCbl and R N-
2
NO. The corresponding rate equation is given in Equation (2)
+
+
+
where K’ = K[H ]/([H ] + K (H OCbl ):
a
2
0
kK ½NONOateꢃ
kobs ¼ 1
þ kHOCbl
ð2Þ
0
þ K ½NONOateꢃ
ꢁ
1
+
ꢁ
R2N-NONOate
k1 at 378C [s
]
pK (HN R -NONOate )
a
2
[a]
[a]
Equation (2) reduces to kobs = kK’[NONOate] + kHOCbl when
[a]
DEA-NONOate
MAHMA-NONOate
DPTA-NONOate
1.1ꢀ0.4
5.0ꢀ0.2
5.9ꢀ0.3
[
a]
K’ is small, which is consistent with the Cbl reactant being
0.52ꢀ0.39
0.23ꢀ0.01
+
[b]
[b]
predominantly HOCbl, not H OCbl , and a reaction inter-
3.96ꢀ0.13
2
[
c]
ꢁ2[a]
[a]
mediate not being observable. Although it was not possible to
DETA-NONOate
(3.3ꢀ3.1)ꢂ10
3.1ꢀ0.4
3.21ꢀ0.10
(1.12ꢀ0.03)ꢂ10ꢁ
2[b]
[b]
+
obtain rate data for the reaction between H OCbl /HOCbl at
2
+
pH values close to the pK value of H OCbl , and therefore
show that H OCbl (pK 7.76 ), not HOCbl, reacts with
[a] Reference [4]. [b] This work. [c] See Supporting Information for
details. A second decomposition pathway for DETA-NONOate at
a
2
+
[22]
2
a
[
4]
higher pH was not observed. UV spectra results provide evidence
that protonation occurs at the amine nitrogen.
DEA-NONOate, the increase in kapp as the pH is decreased
[
4]
(
Table 1) is consistent with this interpretation.
+
The reactions between H OCbl /HOCbl and three other
2
R N-NONOates were also investigated. DETA-NONOate
2
(
Figure 1) was chosen for studies at lower pH conditions to
linear at pH 10.80, curvature was observed at lower pH values
(Supporting Information, Figure S4). Additional studies
revealed that the most likely explanation for the curvature
is a nitrite impurity (approx. 10%) in commercial MAHMA-
NONOate, which is not easily removed. Nitrite reacts rapidly
further probe whether the increase in the apparent rate
constant kapp with decreasing pH arises from H OCbl /
HOCbl speciation, because DETA-NONOate is remarkably
stable to acid-catalyzed decomposition when compared with
other R N-NONOates (Table 2). However, at pH 7.4, the
+
2
+
[36,37]
with H OCbl to form nitrocobalamin (NO Cbl),
and the
2
2
2
+
reaction between H OCbl /HOCbl and DETA-NONOate
formation of NO Cbl slows down the apparent rate of the
2
2
was extremely slow and incomplete, with a rate constant of
the same order as that for the spontaneous rate of decom-
reaction between HOCbl and MAHMA-NONOate. Further
details are given in the Supporting Information.
ꢁ
4
ꢁ1
position of the NONOate (k ꢄ 8 ꢀ 10 min , k = 1.6 ꢀ
Finally, if a labile axial ligand is required for the transfer of
obs
L
ꢁ4
ꢁ1
ꢁ
1
0
min ; [DETA-NONOate] = 7.5 mm, ca. 70% of
HOCbl/H OCbl converted into NOCbl over 2.5 days).
NO from R N-NONOates to cobalt(III) corrinoids to
2
+
produce the corresponding nitroxyl complex, then R N-
2
2
III
Although the reaction was faster at pH 5.88, once again
NONOates should also react with the closely related Co
+
H OCbl was only partially converted into NOCbl and k and
cobinamide derivatives (Cbi) in which the nucleotide is
2
L
[
38]
kobs were within a factor of two of each other. Similar results
cleaved at the phosphodiester. In support of this, aquahy-
droxycobinamide was found to react rapidly with DEA-
+
were obtained for the reaction of H OCbl /HOCbl with
2
DPTA-NONOate (pH 9.30, 8.00, 7.40, and 6.80), with the
NONOate to produce the corresponding NOCbi complex
+
ꢁ1
extent of H OCbl /HOCbl conversion into NOCbl decreas-
(kobs = 0.5 min ,
[DEA-NONOate] = 1.0 mm,
pH 9.30,
2
ing with decreasing pH. At pH 6.80, practically no reaction
25.08C). Detailed kinetic studies on this system are currently
+
occurred between H OCbl and DPTA-NONOate, with the
underway.
2
1
latter species apparently decomposing before it could react
To summarize, UV/Vis and H NMR spectroscopy studies
+
with H OCbl . Therefore, it appears that having two sterically
on the reaction between R N-NONOates and HOCbl/
2
2
+
+
bulky substituents on the secondary amine of R N-NON-
H OCbl suggest that H OCbl reacts directly and essentially
2 2
2
Oates leads to both thermodynamically and kinetically
stoichiometrically with DEA-NONOate to give NOCbl and
the corresponding toxic nitrosoamine DEA-NO. Decompo-
+
unfavorable reactions of these species with H OCbl /
2
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sition of the NONOate to produce NO is not a prerequisite
for the reaction to occur. To our knowledge, a direct reaction
Parrish, S. El-Gayar, U. Schleicher, C. Bogdan, L. K. Keefer, J.
Med. Chem. 2008, 51, 3961 – 3970.
16] H. Chakrapani, T. C. Wilde, M. L. Citro, M. M. Goodblatt, L. K.
Keefer, J. E. Saavedra, Bioorg. Med. Chem. 2008, 16, 2657 –
[
[
between R N-NONOate and a transition metal complex to
2
produce a nitroxyl complex is unprecedented. Given the
2664.
widespread use of R N-NONOates as NO donors and the
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