Nitroxygenation of quercetin by HNO

Article abstract of DOI:10.1016/j.tetlet.2015.12.040

The flavonol quercetin undergoes both enzymatic and non-enzymatic reactions with nitroxyl (HNO/NO^-), similar to analogous reactions with dioxygen, but in which N is regioselectively found in the ring-cleaved product. Here we report on kinetic and thermodynamic analysis of the non-enzymatic nitroxygenation reaction in water, which is orders of magnitude faster than the comparable dioxygenation. The second order rate constants were determined from variable temperature reactions, which allowed determination of the reaction activation enthalpy (ΔH^≠ = 9.4 kcal/mol), entropy (ΔS^≠ = -8.3 cal/mol K), and free energy (ΔG^≠ = 11.8 kcal/mol). The determined standard state energy (ΔG^o) and activation free energy, as well as the low entropic energy of reaction, are consistent with a proposed single electron transfer (SET) rate determining step.

Full text of DOI:10.1016/j.tetlet.2015.12.040

Tetrahedron Letters 57 (2016) 399–402
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Nitroxygenation of quercetin by HNO
⇑
Xiaozhen Han, Murugaeson R. Kumar, Patrick J. Farmer
Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798, United States
a r t i c l e i n f o
a b s t r a c t
ꢀ
Article history:
The flavonol quercetin undergoes both enzymatic and non-enzymatic reactions with nitroxyl (HNO/NO ),
similar to analogous reactions with dioxygen, but in which N is regioselectively found in the ring-cleaved
product. Here we report on kinetic and thermodynamic analysis of the non-enzymatic nitroxygenation
reaction in water, which is orders of magnitude faster than the comparable dioxygenation. The second
order rate constants were determined from variable temperature reactions, which allowed determination
Received 22 October 2015
Revised 4 December 2015
Accepted 8 December 2015
Available online 9 December 2015
–
–
of the reaction activation enthalpy (
DH
= 9.4 kcal/mol), entropy (
D
S
= ꢀ8.3 cal/mol K), and free energy
Keywords:
Quercetin
Nitroxyl/HNO
Nitroxygenation
Single electron transfer
–
o
(DG = 11.8 kcal/mol). The determined standard state energy (DG ) and activation free energy, as well as
the low entropic energy of reaction, are consistent with a proposed single electron transfer (SET) rate
determining step.
Ó 2015 Elsevier Ltd. All rights reserved.
Quercetin and other flavonoids are antioxidants found in fresh
fruits and vegetables that have been shown to play an important
preventative role in cardiovascular diseases and aging.1 A num-
ber of enzymes have been found which decompose flavonoids by
OH
OH
OH
OH
O2
O
C
HO
O
B
C
HO
CO
A
–3
A
O
CO2H
OH
OH
OH
O
reaction with dioxygen, Scheme 1.4 The same reaction occurs
,5
non-enzymatically, though typically at higher pH.4
Scheme 1.
In a series of mechanistic studies, the Speier group proposed
that in organic solvents the non-enzymatic reaction proceeds by
a way of a rate-limiting single electron transfer (SET) between
the deprotonated flavonol and oxygen, but in protic solvents a sec-
HO
HO
OH
OH
5
OH
ond slower bimolecular reaction competes. Metal complex mod-
HO
O
O
O
HO
OH
HO
OH
els for the enzymatic reactions have also been examined by
HNO
-CO
NH
O
+
O
6
OH
7
,8
Speier and others. In both the free or metal-bound non-enzy-
matic oxygenations, the reactions are typically observed at tem-
peratures above 70 °C, with reported activation free energies of
greater than 24.1 kcal/mol.5
NC
OH
OH OH
2
OH OH
3
1
4
a,6f
Scheme 2.
Nitroxyl, HNO, is the reduced and protonated congener of NO,
which is isoelectronic with singlet O . HNO displays biological
2
effects distinct from that of NO, for example, as an enzyme
decomposes to give the observed 2,4,6-trihydroxybenzoic acid
and 3,4-dihydroxybenzonitrile products. Importantly, like dioxy-
genation, a non-enzymatic nitroxygenation of quercetin with
HNO proceeds at high pH yielding the same regioselective prod-
ucts, again suggesting the deprotonated quercetinate anion is the
dominant reactant.
9
inhibitor and ionotropic agent that may be used in the treatment
1
0
of heart failure. Recently, we reported the unprecedented substi-
tution of HNO for dioxygen in the activity of Mn-substituted Quer-
cetin Dioxygenase, Mn-QDO, resulting in the incorporation of both
9
heteroatoms of HNO regioselectively into the product, Scheme 2.
In these reactions, HNO is generated in situ from a precursor, and
in the presence of enzyme and substrate, and like dioxygenation,
cleaves the central O-heterocyclic ring to release CO. The reaction
likely proceeds through an analogous depsidic product 2, which
The coupling of HNO to an enolic carbon center is similar to the
so-called nitroso aldol reactions, NA, in which nitroso compounds
couple with activated ketones and aldehydes yielding both
11
O- and N-bound adducts, Scheme 3. The early examples of these
aldol condensations utilized nitrosobenzene and strongly activated
enolates or silyl enol ethers, typically yielding N-bound
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.tetlet.2015.12.040
040-4039/Ó 2015 Elsevier Ltd. All rights reserved.
0

4
00
X. Han et al. / Tetrahedron Letters 57 (2016) 399–402
O
Table 2
O addition
N addition
Ph
R'
NH
Temperature dependence of k3
N
O
R
O
O
O
ꢀ1 ꢀ1
)a ꢁ 104
R'
T (K)
k
3
(M
s
R
Ph
R'
HNO
DNO
R
N
OH
288
293
298
303
308
0.782 (±0.15)
1.10 (±0.05)
1.34 (±0.09)
1.85 (±0.07)
2.49 (±0.12)
0.418 (±0.13)
0.573 (±0.08)
0.852 (±0.07)
1.32 (±0.05)
2.06 (±0.09)
Ph
Scheme 3.
a
Experimental variance is within 5%, average of three trials (error in
parenthesis).
Table 3
Determined activation parameters at 20 °C for Eq. 3
–
–
–
–
D
(
H
D
S
ꢀT
D
S
DG
a
b
kcal/mol)
(cal/mol K)
(kcal/mol)
(kcal/mol )
HNO
9.38 (±0.04)
ꢀ8.27 (±0.03)
2.42
ꢀ1.48
11.80
12.13
DNO 13.61 (±0.02)
5.07 (±0.04)
a
From intercepts of the Eyring plots.
–
b
–
–
From the equation
D
G
=
D
H
ꢀ TDS .
Figure 1. Absorbance spectra obtained over the course of reaction of quercetin
0.04 mM) with HNO donor Angeli’s salt (1.00 mM) in pH 8.0 sodium phosphate
(
buffer at 298 K.
[
T.S.]
y
g
r
e
n
Q + HNO
1
E
Table 1
ET
1.8kcal/mol
Temperature dependence of AS decomposition
11.0kcal/mol
k
1
(s 1)a
ꢀ
T (K)
-
Q + HNO
HNO
DNO
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4
4
3
3
3
ꢀ4
ꢀ4
ꢀ3
ꢀ3
ꢀ3
288
293
298
303
308
5.0 ꢁ 10
7.0 ꢁ 10
1.8 ꢁ 10
2.6 ꢁ 10
3.4 ꢁ 10
4.0 ꢁ 10
6.0 ꢁ 10
1.5 ꢁ 10
2.3 ꢁ 10
2.8 ꢁ 10
a
Experimental variance within 5%.
Benzonitrile and benzoic acid
Figure 3. Reaction coordinate diagram for Eq. 3 showing determined activation
energy in red, and calculated value for initial outer-sphere electron transfer
mechanism.
hydroxyamino products.12–14 More recent work by Yamamoto and
15
coworkers have shown a much wider scope of NA reactivity, with
Lewis-acid catalysis yielding O-bound aminooxy adducts.1
These reactions are proposed to occur through bimolecular nucle-
ophilic attack, rather than outersphere SET.
6,17
In this Letter, we investigate the kinetics and thermodynamics
of the non-enzymatic reaction to address questions regarding the
mechanism of nitroxygenation. Reactions between quercetin and
HNO were monitored by the decay of the quercetin absorption
band with kmax of 400 nm, as shown in Figure 1. The majority of
experiments were run at pH 8.0 in phosphate buffer, conditions
1
8
a
at which quercetin (pK = 7.1) is almost 90% deprotonated and
compatible with the use of Angeli’s Salt (AS) as a stable source of
HNO. Under analogous conditions, no side reaction of quercetin
ꢀ
with the byproduct NO , was seen on the timescale of the mea-
2
sured reactivity.
ꢀ
! HNO þ NOꢀ2 ðk
HN
2
O
1
Þ
ð1Þ
ð2Þ
ð3Þ
3
HNO þ HNO ! N
2
O þ H
2
O
2
ðk Þ
Figure 2. Overlay of modeled (dot) and experimentally-derived (dash) concentra-
tions for a typical reaction, with residual plot above.
ꢀ
Q
þ HNO ! Product ðk Þ
3

X. Han et al. / Tetrahedron Letters 57 (2016) 399–402
401
OH
OH
OH
OH
+
OH
OH
HO
HO
OH
O
HO
O
O
O
O
-
H+
HNO
O
O
OH
OH
O
OH
OH
OH
OH
H
OH
N
HO
O
O
HO
O
O
ET
+
HN O
O
O
OH
O
OH
OH
OH
H
N
OH
OH
HO
HO
O
O
O
Nucleophilic attack
NH
O
O
OH
O
OH
O
Scheme 4.
The reaction sequence in Eqs. 1–3 was used to model the kinetic
data. These reactions are complicated by the slow release of HNO
anion, at an estimated reduction potential of 0.52 V NHE,24 as com-
, which would be
required for dioxygenation by SET. Thus the accelerated rate of
nitroxygenation versus oxygenation can be attributed to the differ-
ence in driving force in the SET rate-determining step.
pared to that of superoxide at ꢀ0.33 V from O
2
19
25
from AS, and its competitive dimerization forming N
perature dependence under our conditions for the first-order rate
constant k for Eq. 1 was obtained following the loss of absorbance
of Angeli’s salt at 250 nm, in both H
literature value for the bimolecular rate of Eq. 2, k
2
O. The tem-
1
2
O and D
2
O solutions, Table 1. A
These results are consistent with the proposed mechanism
shown in Scheme 4. In this sequence, quercetin is first deproto-
nated, generating an anion with substantial electron density
located on C2 of the central ring. The rate-limiting electron transfer
between quercetinate and HNO produces a quercetin radical and
6
ꢀ1 ꢀ1
2
= 8 ꢁ 10 M
s ,
9,19
was assumed unchanged under these conditions.
The temperature dependence of the second-order rate constant
of Eq. 3, k , was obtained by converting absorbance data to concen-
tration versus time and then fitting the data to the reaction
3
ꢀ
the aminoxyl radical anion, HNO . Radical–radical coupling gener-
2
0
sequence using REACT for Windows, Version 1.2, Figure 2. Table 2
gives the determined rate constants for analogous reactions of
ates an N-oxyamino anion; this key intermediate may also be gen-
erated by nucleophilic attack of a C2-based carbanion on HNO,
analogous to the nitroso aldol reactions. But the relatively small
entropy of activation suggests no bond formation in the rate-deter-
2
HNO and DNO, the latter being the assumed reactant in D O
solution.
–
26,27
Using the data in Table 2, the activation enthalpy (
D
H ) and
mining step, thus supporting the outersphere SET mechanism.
–
activation entropy (
plots of ln(k /T) versus the reciprocal of the absolute temperature
1/T), respectively, and are given in Table 3 for reactions in both
O and D O. Analysis of the variable temperature kinetic data
on this reaction obtains an activation free energy
1.8 kcal/mol, shown in Table 3. The kinetic isotope effect, KIE,
derived from rate constants obtained in H O and D O solutions is
.92 at 293 K. This value suggests, as well as the relatively small
D
S ) for the reaction were derived from Eyring
The incipient N-oxyamino anion then undergoes an intramolecular
nucleophilic attack at the C4 carbonyl, releasing CO and forming
the proposed depsidic intermediate which decomposes to the
observed products.
2
(
H
2
2
–
D
G
of
1
Conclusion
2
2
1
The nitroxygenation of quercetin with HNO is much more facile
than the comparable dioxygenation, some 1000-fold faster at room
temperature than an analogous dioxygenation at 70 °C. Thermody-
namic analysis yields an activation barrier very similar to that pre-
dicted for a rate-determining SET step; the difference in rates may
be attributed to a large difference in SET driving force. The unique
regioselectivity of nitroxygenation may be of use in organic syn-
thesis, as well as to provide mechanistic insight in comparison to
analogous dioxygenation reactions.
entropic energies of activation, suggest that no bonds are made
or broken in the rate-determining step.
ꢀ
Q
þ O
2
! Product ðk
4
Þ
ð4Þ
For comparison, the bimolecular rate constant of dioxygenation,
ꢀ1 ꢀ1
s
, ca. 104 slower than that of nitroxy-
4
k , at 293 K is 0.46 M
genation. Likewise, the reported enzymatic dioxygenation reaction
4
of quercetin by Mn-QDO was ca. 10 slower than the analogous
9
,4a
nitroxygenation.
Thus nitroxygenation appears to be funda-
mentally and significantly more facile than dioxygenation in these
reactions.
Acknowledgments
This research was supported by the National Science Founda-
tion (PJF CHE-1057942).
G ¼ F½E ðQH ꢀ E ðHNO0=ꢀÞꢃ
ꢂ
o
þ=0
o
ð5Þ
D
Further insight is obtained by application of the Nernst relation-
ship, Eq. 5, utilizing the reported potentials versus NHE for querce-
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