What are the differences between ascorbic acid and NADH as hydride and electron sources in vivo on thermodynamics, kinetics, and mechanism?

Article abstract of DOI:10.1021/jp2067974

Ascorbic acid (AscH₂) and dihydronicotinamide adenine dinucleotide (NADH) are two very important natural redox cofactors, which can be used as hydride, electron, and hydrogen atom sources to take part in many important bioreduction processes in vivo. The differences of the two natural reducing agents as hydride, hydrogen atom, and electron donors in thermodynamics, kinetics, and mechanisms were examined by using 5,6-isopropylidene ascorbate (iAscH^-) and β-d-glucopyranosyl-1, 4-dihydronicotinamide acetate (GluNAH) as their models, respectively. The results show that the hydride-donating ability of iAscH^- is smaller than that of GluNAH by 6.0 kcal/mol, but the electron-donating ability and hydrogen-donating ability of iAscH^- are larger than those of GluNAH by 20.8 and 8.4 kcal/mol, respectively, which indicates that iAscH^- is a good electron donor and a good hydrogen atom donor, but GluNAH is a good hydride donor. The kinetic intrinsic barrier energy of iAscH^- to release hydride anion in acetonitrile is larger than that of GluNAH to release hydride anion in acetonitrile by 6.9 kcal/mol. The mechanisms of hydride transfer from iAscH^- and GluNAH to phenylxanthium perchlorate (PhXn⁺), a well-known hydride acceptor, were examined, and the results show that hydride transfer from GluNAH adopted a one-step mechanism, but the hydride transfer from iAscH^- adopted a two-step mechanism (e-H?). The thermodynamic relation charts (TRC) of the iAscH^- family (including iAscH^-, iAscH?, iAsc?-, and iAsc) and of the GluNAH family (including GluNAH, GluNAH^?+, GluNA?, and GluNA⁺) in acetonitrile were constructed as Molecule ID Cards of iAscH^- and of GluNAH in acetonitrile. By using the Molecule ID Cards of iAscH^- and GluNAH, the character chemical properties not only of iAscH^- and GluNAH but also of the various reaction intermediates of iAscH^- and GluNAH all have been quantitatively diagnosed and compared. It is clear that these comparisons of the thermodynamics, kinetics, and mechanisms between iAscH^- and GluNAH as hydride and electron donors in acetonitrile should be quite important and valuable to diagnose and understand the different roles and functions of ascorbic acid and NADH as hydride, hydrogen atom, and electron sources in vivo.

Full text of DOI:10.1021/jp2067974

ARTICLE
pubs.acs.org/JPCB
What Are the Differences between Ascorbic Acid and NADH as
Hydride and Electron Sources in Vivo on Thermodynamics, Kinetics,
and Mechanism?
Xiao-Qing Zhu,* Yuan-Yuan Mu, and Xiu-Tao Li
State Key Laboratory of Elemento-Organic Chemistry, Department of Chemistry, Nankai University, Tianjin 300071, China
ABSTRACT: Ascorbic acid (AscH2) and dihydronicotinamide
adenine dinucleotide (NADH) are two very important natural
redox cofactors, which can be used as hydride, electron, and
hydrogen atom sources to take part in many important bio-
reduction processes in vivo. The differences of the two natural
reducing agents as hydride, hydrogen atom, and electron donors in thermodynamics, kinetics, and mechanisms were examined by
using 5,6-isopropylidene ascorbate (iAscHꢀ) and β-D-glucopyranosyl-1,4-dihydronicotinamide acetate (GluNAH) as their models,
respectively. The results show that the hydride-donating ability of iAscHꢀ is smaller than that of GluNAH by 6.0 kcal/mol, but the
electron-donating ability and hydrogen-donating ability of iAscHꢀ are larger than those of GluNAH by 20.8 and 8.4 kcal/mol,
respectively, which indicates that iAscHꢀ is a good electron donor and a good hydrogen atom donor, but GluNAH is a good hydride
donor. The kinetic intrinsic barrier energy of iAscHꢀ to release hydride anion in acetonitrile is larger than that of GluNAH to release
hydride anion in acetonitrile by 6.9 kcal/mol. The mechanisms of hydride transfer from iAscHꢀ and GluNAH to phenylxanthium
perchlorate (PhXn+), a well-known hydride acceptor, were examined, and the results show that hydride transfer from GluNAH
adopted a one-step mechanism, but the hydride transfer from iAscHꢀ adopted a two-step mechanism (eꢀH•). The thermodynamic
relation charts (TRC) of the iAscHꢀ family (including iAscHꢀ, iAscH•, iAsc•ꢀ, and iAsc) and of the GluNAH family (including
GluNAH, GluNAH•+, GluNA•, and GluNA+) in acetonitrile were constructed as Molecule ID Cards of iAscHꢀ and of GluNAH in
acetonitrile. By using the Molecule ID Cards of iAscHꢀ and GluNAH, the character chemical properties not only of iAscHꢀ and
GluNAH but also of the various reaction intermediates of iAscHꢀ and GluNAH all have been quantitatively diagnosed and
compared. It is clear that these comparisons of the thermodynamics, kinetics, and mechanisms between iAscHꢀ and GluNAH as
hydride and electron donors in acetonitrile should be quite important and valuable to diagnose and understand the different roles
and functions of ascorbic acid and NADH as hydride, hydrogen atom, and electron sources in vivo.
’ INTRODUCTION
acid.16 Just because ascorbic acid and NADH are two ubiquitous
natural organic reducing agents and play very important biochemical
roles, the chemistry of ascorbic acid and NADH have drawn
extensive interest and high attention of many researchers for a long
time.17ꢀ21 Especially, the structures, chemical properties, and
biofunctions of the two bioreducing agents in vivo have been the
key subjects of extensive studies.22ꢀ25 In order to discover the
detailed mechanism of ascorbic acid and NADH as hydride and
electron sources, various analogues of ascorbic acid and NADH
were designed and synthesized to mimic ascorbic acid and NADH
reductions.26ꢀ36 Examining the past publications on the chemistry
of ascorbic acid and NADH as hydride and electron sources shows
that, although the chemistry, especially the thermodynamics, ki-
netics, and mechanism of ascorbic acid and NADH reductions have
received extensive examination, most attention of chemists all
focused on the respective special chemical properties and roles of
ascorbic acid and NADH as bioreducing agents,37ꢀ45 no or very
little attention has be focused on the differences between ascorbic acid
and NADH as hydride and electron sources in thermodynamics,
Ascorbic acid (AscH2) (AscHꢀ exists as the predominant
form of AscH2 in physiological pH) and dihydronicotinamide
adenine dinucleotide (NADH) are two extremely important
natural redox cofactors (Scheme 1), which exist extensively
in vivo as effective one-electron (including hydrogen atom)
and/or two-electron (including hydride ion) donors to take part
in a wide range of biochemical processes.1,2 For example, as a
one-electron donor, ascorbate (AscHꢀ) can reduce dopamine-
β-hydroxylase;3 as a hydrogen atom (or proton-coupled electron)
donor, AscHꢀ can reduce α-tocopheroxyl radical,4 quinones,5
peroxynitrite,6 cytochrome b561,7 N-nitrosated tryptophan,8 and
glutathione peroxidase,9 etc. In addition, as a two-electron or
hydride ion donor, ascorbate (AscHꢀ) can transform into the
corresponding dehydroascorbic acid (Asc) in some plants by an
ascorbic acid oxidase.10 Similarly, for NADH, there exist more
than 400 enzymatic redox reactions which need NADH to offer
electrons or hydride ions in the living body.11 For example, in the
NADH respiratory chain, as a hydride anion (two electrons and a
proton) source, NADH can reduce molecular oxygen, flavins,
quionones, and cytochrome.12ꢀ15 In the citric acid cycle, as an
electron or hydride anion source, NADH can reduce oxaloacetic
acid, malic acid, pyruvic acid, oxalosuccinic acid, and α-ketoglutaric
Received: July 16, 2011
Revised:
October 17, 2011
Published: October 30, 2011
r
2011 American Chemical Society
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The Journal of Physical Chemistry B
ARTICLE
Scheme 1. Structures of Ascorbic Acid and NADH as Well as
Their Corresponding Core Structures
Scheme 2. Structures of iAscH2 and GluNAH to Mimic the
Core Structures of Ascorbic Acid and NADH Together with
Structure of (G)PhXn+ as a Hydride Acceptor
kinetics, and mechanism. In fact, it is quite difficult until now to safely
answer the following questions: (1) What are the differences between
ascorbic acid and NADH as hydride and electron sources in therm-
odynamics, kinetics, and mechanism? (2) Why must nature create
two water-soluble organic hydride donors, ascorbic acid and NADH,
to support life in vivo? (3) Why can ascorbic acid be used as an
antioxidant, but NADH cannot? (4) Whether or not the roles and
functions of ascorbic acid and NADH as hydride and electron sources
in vivo can exchange for each other? It is clear that these scientific
questions on the chemistry of ascorbic acid and NADH should be not
only fundamental but also very interesting and important, which, of
course, have attracted our high attention for a long time. Since most
biochemical processes with ascorbic acid and NADH as hydride and
electron sources in living body all take place in the polar organic regions
constructed with enzyme proteins rather than in the pure aqueous
solution, the chemical information of ascorbic acid and NADH as
hydride and electron sources in the polar organic regions constructed
with enzyme proteins should be more important and valuable than that
in the pure aqueous solution. In order to derive the characteristic
chemical information of ascorbic acid and NADH as hydride and
electron sources in the polar organic regions constructed with enzyme
proteins and to answer the questions proposed above, in this work, 5,6-
isopropylidene ascorbic acid (iAscH2) and β-D-glucopyranosyl-1,4-
dihydronicotinamide acetate (GluNAH) were chosen as the artificial
models of ascorbic acid and NADH, respectively (Scheme 2). Acet-
onitrile was chosen as a polar organic solvent to imitate the polar
organic regions constructed with enzyme proteins, because the polarity
of acetonitrile (ε = 37.5) is quite close to that of peptide bond in
proteins (ε = 37.0, 37.8, and 38.3 for HCONMe2, MeCONMe2 and
N,N-dimethylbenzamide, respectively).46
5.10 (m, 1H), 5.23 (m, 2H), 5.40 (s, 1H), 5.99 (d, 1H), 7.11 (s, 1H);
13C NMR (400 MHz, CDCl3):δ20.5, 20.6, 20.7, 20.8 22.8, 61.9, 68.0,
68.5, 73.1, 74.1, 89.6, 102.1, 104.3, 124.6, 137.5, 169.2, 169.4, 169.6,
180.1,170.7; IR (KBr): 3437, 3342, 3219, 1743, 1689, 1581,
1373 cmꢀ1; ESI-MS/M+ = 454.10.
The acetonitrile solutions of 5,6-isopropylidene ascrobate
(iAscHꢀ) were obtained by addition of 1 equiv of DBU to
solution of 5,6-isopropylidene ascorbic acid (iAscH2) under an
inert atmosphere,26c and it was characterized by its UVꢀvis
1
spectroscopy and H NMR. Substituted 9-phenylxanthylium
perchlorate [(G)PhXn+ClO4ꢀ] was synthesized according to
literature methods.49 Reagent grade acetonitrile was refluxed
over KMnO4 and K2CO3 for several hours and was distilled over
1
P2O5 under argon twice before use. H NMR spectra were
recorded in CDCl3 and DMSO-d6 on Bruker 400 or 300 MHz
NMR spectrometer. Chemical shifts are reported in ppm relative
to TMS by referencing to residual solvent.
Electrochemistry. The electrochemical experiments were
carried out by cyclic voltammetry (CV) and Osteryoung square
wave voltammetry (OSWV) using a BAS-100B electrochemical
apparatus in deaerated acetonitrile under argon atmosphere. The
electrodes used were as follows: working electrode, glassy carbon;
reference electrode, 0.01 M Ag/AgNO3 in (n-Bu)4NPF6/CH3CN
electrolyte solution; and auxiliary electrode, platinum wire.
Ferrocene/ferrocenium redox couple (Fc/Fc+) was used as an
internal reference for all measurements. Scans were taken at 100
mV sꢀ1. The concentration of the substrate is approximately
1 mM with 0.1 M (n-Bu)4NPF6 in acetonitrile. The estimated
errors are small than 5 mV.
’ EXPERIMENTAL SECTION
Isothermal Titration Calorimetry (ITC). The titration experi-
ments were performed on a CSC4200 isothermal titration
calorimeter in acetonitrile at 298 K. The performance of the
calorimeter was checked by measuring the standard heat of
neutralization of an aqueous solution of sodium hydroxide with
a standard aqueous HCl solution. Data points were collected
every 2 s. The heat of reaction was determined following 10
automatic injections from a 250 μL injection syringe containing a
standard solution (∼3 mM) into the reaction cell (1.30 mL)
containing 1 mL of other concentrated reactant (∼10 mM).
Injection volume (10 μL) was delivered in a 0.5 s time interval
with 300 s between every two injections. The reaction heat was
obtained by integration of each peak except the first.65
Materials. Solvents and reagents were obtained from com-
mercial sources and used as received unless otherwise noted. DBU
(1,8-diazabicyclo[5.4.0]undec-7-ene) was purchased from Alfa, pur-
ified under reduced pressure, and stored in an argon-filled glovebox.
Tetrabutylammonium hexafluorophosphate ((n-Bu)4NPF6, Aldrich)
was recrystallized 3 times from CH2Cl2/Et2O and dried in vacuo at
110 °C for 10 h before preparation of a supporting electrolyte solution.
5,6-Isopropylidene ascorbic acid (iAscH2)47 and GluNAH48 were
prepared according to the previously described procedures, and were
identified by NMR, IR, and MS, respectively. The results are listed: for
iAscH2, 1H NMR (400 MHz, DMSO): δ1.23 (s, 6H), 3.85 (m, 1H),
4.07 (m, 1H), 4.24 (m, 1H), 4.68 (d, 1H), 8.47 (s, 1H), 11.28 (s, 1H);
13C NMR (400 MHz, DMSO): δ 25.7, 64.8, 73.4, 74.1, 108.9, 118.1,
152.2, 161.8, 170.1; IR (KBr): 3042, 3076, 2993, 2906, 2742, 1755,
1662, 1433 cmꢀ1; ESI-MS/M+ = 216.06. For GluNAH, 1H NMR
(400 MHz, CDCl3): δ 2.02ꢀ2.06 (m, 12H), 3.10 (s, 2H), 3.76 (m,
1H), 4.12 (m, 1H), 4.25 (m, 1H), 4.39 (d, 1H), 4.88 (m, 1H),
Kinetic Measurements. Kinetic runs were performed on an
Applied Photophysics SX.18MV-R stopped-flow spectrophot-
ometer. The SX.18MV-R uses a novel cell cartridge system that
has a dead time of around 1 ms with very high sensitivity. All
solutions used for kinetics were prepared in an Ar-filled glovebox.
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ARTICLE
Figure 1. Cyclic voltammogram (CV) and Osteryong square wave voltammogram (OSWV) of iAscHꢀ and GluNAH in anhydrous deaerated
acetonitrile solution containing 0.1 M (n-Bu)4NPF6 as supporting electrolyte: CV graph (black line), OSWV graph (red line). The sweep rate is
100 mV/s.
These measurements were under pseudo-first-order conditions
for the reactions of GluNAH and iAscHꢀ with (G)PhXn+, the
initial concentrations of GluNAH and iAscHꢀ ([GluNAH]0 and
[iAscHꢀ]0 for GluNAH and iAscHꢀ, respectively) are more than
20 times over [(G)PhXn+]0. The pseudo-first-order rate con-
stants were calculated by Guggenheim’s method, and then
converted to second-order rate constants by linear correlation
of the pseudo-first-order rate constants against the concentra-
tions of the excessive reactants. But, for the hydrogen-transfer
step in the hydride transfer process from iAscHꢀ to (G)PhXn+,
the second-order rate constants were obtained from slope of the
plots of At versus (A0 ꢀ At)/t by using Origin Software. Eyring
activation parameters were derived from Eyring plots. The
reaction constant (F) of these reactions were obtained according
to the equation log k = Fσ, where σ is Hammett substituent
parameters.
Figure 2. Cyclic voltammogram (CV) and Osteryong square wave
voltammogram (OSWV) of GluNA+ in anhydrous deaerated acetoni-
trile solution containing 0.1 M (n-Bu)4NPF6 as supporting electrolyte:
CV graph (full line), OSWV graph (red line). The sweep rate is
100 mV/s.
’ RESULTS
5,6-Isopropylidene ascorbic acid (iAscH2),47 GluNAH,48 and
(G)PhXn+ cations49 were prepared in this work by following the
previously described procedure, and identified by 1H NMR and
MS. iAscHꢀ was generated in situ by addition of 1 equiv of DBU
(1,8-diazabicyclo [5.4.0] undec-7-ene) to iAscH2 in CH3CN
according to the literature,26c and was characterized by UVꢀvis
hydride transfer from iAscHꢀ and GluNAH to (G)PhXn+ in
acetonitrile at 298 K was determined by titration calorimetry on a
CSC 4200 isothermal titration calorimeter (Figure 6). The
detailed results are also listed in Table 1.
1
spectrophotometry and H NMR spectroscopy. The oxidation
potentials of iAscHꢀ, GluNAH, and (G)PhXnH (Figures 1 and 4)
as well as the reduction potentials of GluNA+ and (G)PhXn+ in
acetonitrile were measured by using CV and OSWV, respectively
(Figures 2 and 5). The reduction potential of iAsc in acetonitrile
was obtained from the second oxidation peak of iAsc2‑ in
acetonitrile (Figure 3), because the first oxidation peak of iAsc2ꢀ
is reversible. The detailed results are summarized in Table 1.
When iAscHꢀ and GluNAH were treated by (G)PhXn+ in
acetonitrile at room temperature, respectively, the final products
are iAsc or GluNA+ and (G)PhXnH and the stoichiometric
relationships for the two reactions are all 1 mol per 1 mol, which
means that the two reactions (eqs 1 and 2) were completed by
hydride transfers. The molar enthalpy change (ΔHrxn) of the
-
-
-Þ
ΔHH-DðiAscH Þ ¼ Hf ðiAscÞ þ Hf ðH Þ-Hf ðiAscH
ð2Þ
The reduction rates of (G)PhXn+ by iAscHꢀ and GluNAH in
acetonitrile were determined according to monitoring the spec-
tra change of (G)PhXn+ at 449 nm by using an Applied Photo-
physics SX.18MV-R stopped-flow spectrometer (Figure 7). The
second-order rate constants (k2) for the reactions at different
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Figure 5. Cyclic voltammogram (CV) and Osteryong square wave
voltammogram (OSWV) of PhXn+ in anhydrous deaerated acetoni-
trile solution containing 0.1 M (n-Bu)4NPF6 as supporting electro-
lyte: CV graph (black line), OSWV graph (red line). The sweep rate is
100 mv/s.
Figure 3. Cyclic voltammogram (CV) and Osteryong square wave
voltammogram (OSWV) of iAsc2‑ in anhydrous deaerated acetonitrile
solution containing 0.1 M (n-Bu)4NPF6 as supporting electrolyte: CV
graph (black line), OSWV graph (red line). The sweep rate is 100 mV/s.
Table 1. Oxidation Potentials of iAscHꢀ, GluNAH, and
(G)PhXnH, Reduction Potentials of iAsc, GluNA+ and
(G)PhXn+ as Well as Reaction Enthalpy Changes of iAscHꢀ
and GluNAH with (G)PhXn+ in Dry Acetonitrile
Eox(XH)a
CV OSWV
Ered(X+)a
CV
ΔHrxn
b
X+
iAsc
GluNA+
(G)PhXn+
p-CF3
OSWV iAscHꢀ GluNAH
ꢀ0.380 ꢀ0.425 ꢀ0.595 ꢀ0.623
0.503
0.475 ꢀ1.269 ꢀ1.248
1.287
1.274
1.257
1.249
1.235
1.227
1.215
1.206
1.190
1.257 ꢀ0.335 ꢀ0.336 ꢀ18.1
1.241 ꢀ0.343 ꢀ0.343 ꢀ17.7
1.220 ꢀ0.353 ꢀ0.355 ꢀ17.3
1.208 ꢀ0.361 ꢀ0.360 ꢀ17.0
1.201 ꢀ0.374 ꢀ0.373 ꢀ16.7
1.195 ꢀ0.394 ꢀ0.394 ꢀ16.2
1.188 ꢀ0.409 ꢀ0.408 ꢀ15.8
1.176 ꢀ0.412 ꢀ0.413 ꢀ15.4
1.160 ꢀ0.430 ꢀ0.430 ꢀ14.9
ꢀ24.1
ꢀ23.7
ꢀ23.3
ꢀ23.0
ꢀ22.7
ꢀ22.2
ꢀ21.8
ꢀ21.4
ꢀ20.9
m-CF3
p-Cl
p-Br
m-OCH3
p-H
Figure 4. Cyclic voltammogram (CV) and Osteryong square wave
voltammogram (OSWV) of PhXnH in anhydrous deaerated acetonitrile
solution containing 0.1 M (n-Bu)4NPF6 as supporting electrolyte: CV
graph (black line), OSWV graph (red line). The sweep rate is 100 mV/s.
m-CH3
p-CH3
p-OCH3
a Measured by CV and OSWV methods in dry acetonitrile at room
temperature, the unit in volts vs Fc+/0 and reproducible to 5 mV or
better. b ΔHrxn was obtained from the reaction heats of eqs 1 and 2 by
switching the sign; the latter were measured by titration calorimetry in
dry acetonitrile at 298 K. The data, given in kcal/mol, were average
values of at least three independent runs. The reproducibility is (0.5
kcal/mol.
temperatures between 288 and 308 K are listed in Tables 2 and 4,
respectively. Activation parameters activation enthalpy (ΔHq)
and activation entropy (ΔSq) of the two reactions were derived
from the slope and intercept of the Eyring plots of ln(k2/T)
versus the reciprocal of the absolute temperature (1/T), which
are listed in Tables 3 and 4, respectively. Due to formation of a
new reaction intermediate (λmax = 372 nm) during the hydride
transfer from iAscHꢀ to (G)PhXn+ in acetonitrile, the formation
and decay kinetics of the reaction intermediate in the reaction
was also examined according to the UVꢀvis absorption change
of the reaction intermediate. The experimental results showed
that the formation rate of the reaction intermediate was equal to
the decay rate of (G)PhXn+ (Figure 8), indicating that the
formation of the reaction intermediate was directly derived from
the transformation of iAscHꢀ or (G)PhXn+ by electron or
hydrogen atom transfer. But the decay rate of the reaction
intermediate is much slower than the formation rate and obeys
the second-order rather than the first-order kinetic law, meaning
that decay of the reaction intermediate was due to the reaction of
the two separated rather than bonded reaction partners. If the
initial concentration of the excess reactant (iAscHꢀ) was chan-
ged, the decay rate of the reaction intermediate has no change,
which means that the decay of the reaction intermediate is not
dependent on the remained initial reactant. The second-order
rate constants (k2) of the reaction intermediate decay at different
temperatures between 288 and 308 K and the activation param-
eters: activation enthalpy (ΔHq), activation entropy (ΔSq), and
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Figure 7. Time-resolved spectrum at 449 nm due to PhXn+ for the
reaction of iAscHꢀ (1 ꢂ 10ꢀ2 M) with PhXn+ (4 ꢂ 10ꢀ4 M) in dry
acetonitrile at 298 K.
Figure 6. Isothermal titration calorimetry (ITC) for the reaction heat of
iAscHꢀ with 9-phenylxanthium perchlorate (9-PhXn+ClO4ꢀ) in acetonꢀi-
trile at 298 K. Titration was conducted by adding 10 μL of 9-PhXn+ClO4
(5.26 mM) every 300s into the acetonitrile containing iAscHꢀ (ca. 20mM).
PhXn+ in acetonitrile, which can be available from our previous work
(ꢀ96.8 kcal/mol).51 The thermodynamic driving forces of iAscHꢀ
and GluNAH to release hydrogen atom in acetonitrile and the
thermodynamic driving forces of iAscH• and GluNAH•+ to release
hydrogen atom and proton can be derived from eqs 5ꢀ7,52 respec-
tively. The three eqs 5ꢀ7 can be derived from the three thermo-
dynamic cycles according to Hess’s law,53,54 respectively. The results
are all summarized in Table 5. In order to conveniently compare the
chemical properties of iAscHꢀ and GluNAH as well as their various
corresponding reaction intermediates, the thermodynamic relation
charts of iAscHꢀ family (including iAscHꢀ, iAscH•, iAsc•ꢀ, andiAsc)
and GluNAH family (including GluNAH, GluNAH•+, GluNA•, and
GluNA+) (Scheme 5) were constructed using the determined six
thermodynamic parameters listed in Table 5, which can be defined as
Molecule ID Cards of iAscHꢀ and GluNAH.52 From the Molecule
ID Cards of iAscHꢀ and GluNAH, three different character thermo-
dynamic parameters can be obtained for each member in the iAscHꢀ
family and GluNAH family, which can be used to quantitatively
describe the three different characteristic chemical properties of the
members in iAscHꢀ family and GluNAH family at the same time.
The results are listed in Tables 6 and 7, respectively.
activation free energy (ΔGq) are listed in Tables 2 and 3,
respectively.
’ DISCUSSION
Comparison of Chemical Properties of iAscHꢀ and Glu-
NAH as Well as Their Various Reaction Intermediates as
Hydride, Electron, Hydrogen Atom, and Proton Donors.
Since AscHꢀ and NADH can act not only as a hydride donor
but also as a hydrogen atom donor and an electron donor in vivo,
and the chemical processes of hydride transfer from AscHꢀ and
NADH often involve multistep mechanism besides the hydride
one-step transfer mechanism (Scheme 3), it is conceived that the
following chemical processes are all possible to take place in vivo: (1)
AscHꢀ and NADH release a hydride anion, a hydrogen atom and an
electron. (2) The intermediates AscH• and NADH•+ release a hydro-
gen atom and a proton. (3) The intermediates Asc•ꢀ and NAD•
release an electron. In order to quantitatively describe and compare the
characteristic chemical behaviors of AscHꢀ and NADH as well as their
corresponding various reaction intermediates in vivo, the thermody-
namic driving forces of iAscHꢀ and GluNAH as AscHꢀ and NADH
models to release hydride, hydrogen atom, and electron, the thermo-
dynamic driving forces of iAscH• and GluNAH•+ as AscH• and
NADH•+ models to release hydrogen atom and proton as well as the
thermodynamic driving forces of iAsc•ꢀ and GluNA• as Asc•ꢀ and
NAD• models to release electron in acetonitrile all need examination
in detail.
In this work, thermodynamic driving forces of iAscHꢀ and
GluNAH as well as iAsc•ꢀ and GluNA• to release electron can be
expressed by their oxidation potentials, but the thermodynamic
driving forces of iAscHꢀ and GluNAH as well as their reaction
intermediates iAscH• and GluNAH•+ to release hydride, hydro-
gen atom, and proton were defined as the enthalpy changes of
the corresponding chemical processes.50 The enthalpy changes
of iAscHꢀ and GluNAH to release hydride anion in acetoni-
-
ΔHH-DðiAscH Þ ¼ ΔHrxnðeq 3Þ ꢀ HH-AðPhXnþÞ
ð4Þ
ð5Þ
-
-
ΔHHDðiAscH Þ ¼ ΔHH-DðiAscH Þ-F½EoredðiAscÞ-EoðH0=-Þꢁ
ΔHPDðiAscH•Þ ¼ ΔHHDðiAscH Þ-F½EooxðiAscH Þ-EoðHþ=0Þꢁ ð6Þ
-
-
trile can be obtained from the reaction enthalpy change (ΔHrxn
)
of hydride transfer from iAscHꢀ and GluNAH to 9-phenyl-
xanthium perchlorate (PhXn+ClO4ꢀ) in acetonitrile (eqs 3 and 4).
In eq 4, ΔHrxn were measured by using titration calorimetry in
ΔHHDðiAscH•Þ ¼ ΔHH-DðiAscH Þ-F½EooxðiAscH Þ-EoðH0=-Þꢁ
-
-
acetonitrile (Figure 6), and ΔHH A(PhXn+) is the hydride affinity of
ð7Þ
ꢀ
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Table 2. Dependence of the Reaction Rates of the First Step
and the Second Step Hydride Transfer from iAscHꢀ to
(G)PhXn+ in Acetonitrile on the Reaction Temperature
Table 3. Activation Parameters for the First Step and Second
Step of the Hydride Transfer from iAscHꢀ to (G)PhXn+ in
Dry Acetonitrile
a
k2 for the first step (ꢂ10ꢀ4 Mꢀ1 sꢀ1
)
for the first step
for the second step
(G)PhXn+
288 K
293 K
298 K
303 K
308 K
(G)PhXn+ ΔHqa ΔSqb ꢀTΔSqc ΔGqd ΔHqa ΔSqb ꢀTΔSqc ΔGqd
p-CF3
m-CF3
p-Cl
5.71
5.10
4.15
4.02
3.69
3.45
3.19
2.88
2.42
7.84
7.09
6.04
5.56
5.15
4.38
4.24
3.90
3.36
9.37
8.49
7.21
6.29
6.43
5.50
5.22
4.73
4.29
12.03
10.48
9.29
8.85
7.70
7.13
6.78
5.73
5.23
13.84
12.41
10.41
9.88
9.12
8.14
7.66
7.19
6.02
p-CF3
m-CF3
p-Cl
7.17 ꢀ11.72 3.50 10.67 4.88 ꢀ33.04 9.85 14.73
7.07 ꢀ12.28 3.66 10.73 5.01 ꢀ32.67 9.74 14.75
7.44 ꢀ11.37 3.39 10.83 5.03 ꢀ32.68 9.74 14.77
7.40 ꢀ11.64 3.47 10.87 5.18 ꢀ32.23 9.60 14.78
p-Br
p-Br
m-OCH3
p-H
m-OCH3 7.24 ꢀ12.33 3.67 10.91 5.31 ꢀ31.81 9.48 14.80
p-H
7.18 ꢀ12.76 3.80 10.98 5.46 ꢀ31.36 9.34 14.81
7.27 ꢀ12.58 3.75 11.02 5.78 ꢀ30.36 9.05 14.83
7.22 ꢀ12.92 3.85 11.08 5.90 ꢀ30.01 8.94 14.85
m-CH3
p-CH3
p-OCH3
m-CH3
p-CH3
p-OCH3 7.43 ꢀ12.56 3.73 11.16 6.00 ꢀ29.73 8.86 14.87
a From the slopes of the Eyring plots, the unit is kcal molꢀ1, and the
uncertainty is smaller than 0.05 kcal/mol. b From the intercepts of the
Eyring plots, the unit is cal molꢀ1 Kꢀ1, the uncertainty is smaller than
0.04 cal molꢀ1 Kꢀ1. c The unit is kcal molꢀ1. d From the equation ΔGq =
k2 for the second step (ꢂ10ꢀ1 Mꢀ1 sꢀ1
)
b
(G)PhXn+
288 K
293 K
298 K
303 K
308 K
ΔHq ꢀ TΔSq, the unit is kcal molꢀ1
.
p-CF3
m-CF3
p-Cl
7.22
7.03
6.79
6.55
6.32
6.08
5.80
5.59
5.38
8.31
7.90
7.57
7.29
7.16
7.10
6.78
6.53
6.29
10.02
9.57
9.34
9.14
9.00
8.75
8.50
8.39
8.10
11.59
11.18
10.79
10.60
10.39
10.28
9.99
13.00
12.80
12.35
12.03
11.84
11.64
11.50
11.29
10.98
Table 4. Dependence of k2 for the Hydride Transfer from
GluNAH to (G)PhXn+ in Acetonitrile on the Reaction Tem-
perature Together with the Corresponding Activation
Parameters
p-Br
m-OCH3
p-H
m-CH3
p-CH3
p-OCH3
k2 ꢂ 10ꢀ4a
9.60
(G)PhXn+ 288 K 293 K 298 K 303 K 308 K ΔHqb ΔSqc ꢀTΔSqd ΔGqe
9.29
a Second-order rate constants k2 for the first step of the hydride transfer
from iAscHꢀ to (G)PhXn+ were obtained from the corresponding
pseudo-first-order rate constants by the linear correlation against the
concentration of the (G)PhXn+, the experimental error is within 5%.
b Second-order rate constants k2 for the second step of the hydride
transfer from iAscHꢀ to (G)PhXn+ were obtained by using second-
order kinetic fitting method for the decay lines of the initially formed
reaction intermediate iAscH•, the experimental error is within 5%.
p-CF3
m-CF3
p-Cl
10.51 10.99 11.38 11.72 12.02 0.53 34.16 10.01 10.54
9.10 9.39 9.78 10.02 10.42 0.54 34.47 10.10 10.64
5.97 6.28 6.62 6.86 7.06 0.85 34.23 10.03 10.88
5.75 5.98 6.15 6.33 6.61 0.54 35.39 10.37 10.91
p-Br
m-OCH3 4.44 4.65 4.94 5.40 5.66 1.54 32.42 9.50 11.04
p-H
3.22 3.45 3.78 3.95 4.16 1.60 32.80 9.61 11.22
2.56 2.87 3.06 3.26 3.45 1.87 32.29 9.46 11.33
1.78 1.95 2.14 2.31 2.45 2.14 32.12 9.41 11.55
m-CH3
p-CH3
Comparison of Reaction Mechanism of iAscHꢀ and Glu-
NAH as Hydride Sources. Although iAscHꢀ and GluNAH all
can be used as hydride ion donors, the mechanisms of the
hydride transfer from the two hydride donors are generally
different, because the difference of redox properties between
iAscHꢀ and GluNAH is quite large (ΔE = 0.900 V). In fact, the
mechanism of the hydride transfer from NADH and AscHꢀ to a
two-electron acceptor (i.e., a hydride acceptor) is still a disputed
question so far.5,15,32d,56,57 The controversy focus is whether the
mechanism of the hydride transfer is a one-step or multistep
sequence involving electron transfer as the initial step: electronꢀ
protonꢀelectron, electronꢀhydrogen, or hydrogenꢀelectron.
One of the main reasons resulting in the difficulty to elucidate the
hydride transfer mechanism is that no detailed energetic data of
each possible mechanistic step for the hydride transfer are
available. From this work, we not only have obtained the therm-
odynamic driving forces of iAscHꢀ and GluNAH as hydride
donors to release a hydride anion, to release a hydrogen atom,
and to release an electron, as well as the thermodynamic driving
forces of the various reaction intermediates of iAscHꢀ and
GluNAH to release a hydrogen atom, to release a proton, and
to release an electron, but also have obtained the thermodynamic
driving forces of (G)PhXn+ as hydride acceptors to obtain a
p-OCH3 1.41 1.57 1.67 1.81 1.95 2.10 32.76 9.60 11.70
a Second-order rate constants k2 for the hydride transfer from GluNAH
to (G)PhXn+ in acetonitrile were obtained from the corresponding
pseudo-first-order rate constants by the linear correlation against the
concentration of the (G)PhXn+, the experimental error is within 5%.
b From the slop of the Eyring plots, the unit is kcal molꢀ1, and the
uncertainty is smaller than 0.05 kcal molꢀ1. c From the intercept of the
Eyring plots, the unit is cal molꢀ1 ꢀ1, and the uncertainty is smaller
K
than 0.04 cal molꢀ1 Kꢀ1. d The unit is kcal molꢀ1. e From the equation
ΔGq = ΔHq ꢀ TΔSq, the unit is kcal molꢀ1
.
hydride anion, to obtain a hydrogen atom, and to obtain an
electron, as well as the thermodynamic driving forces of the
various reaction intermediates of (G)PhXn+ to obtain a hydro-
gen atom, to obtain a proton, and to obtain an electron. These
thermodynamic data may been used to construct the Molecule
ID Card of iAscHꢀ or GluNAH and (G)PhXn+.58 It is easy to
examine the thermodynamics of the detailed mechanistic steps of
the hydride transfer from iAscHꢀ and GluNAH to (G)PhXn+
according to the thermodynamic analytic platforms which con-
sist of the Molecule ID Cards of iAscHꢀ or GluNAH and
(G)PhXn+ (Schemes 6 and 7). The detailed results of the
standard-state free energy change of each elementary step for
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ARTICLE
Scheme 3. Possible Pathways of the Hydride Transfer from
iAscHꢀ
Figure 8. UVꢀvis spectrum plots of PhXn+ at λ = 449 nm and a
reaction intermediate at λ = 372 nm against the reaction time. The initial
reaction condition: the concentration of iAscHꢀ and PhXn+ is 1 ꢂ 10ꢀ2
and 4 ꢂ 10ꢀ4 M, respectively, in dry acetonitrile at 298 K.
the hydride transfer from iAscHꢀ and GluNAH to (G)PhXn+ are
summarized in Table 8.
ꢀTΔSq, which means that the main activation energy block for
the hydride transfer from GluNAH to (G)PhXn+ is entropy
change rather than enthalpy change. This comparison also
supports that the hydride transfer from GluNAH to (G)PhXn+
in acetonitrile really took the concerted one step.
From Scheme 7 and Table 8, it is also easy to find that for the
hydride transfer from iAscHꢀ to (G)PhXn+, the state energy
change scales of the three initial steps (steps a, b, and c) in the
four possible pathways (Scheme 7) range from ꢀ2.1 to 0.1 kcal
for the electron transfer (step a), from 25.2 to 26.4 kcal/mol for
the hydrogen transfer (step b), and from ꢀ18.1 to ꢀ14.9 kcal/
mol for the concerted hydride transfer (step c). Since the state
energy changes for the hydrogen-transfer (step b) all are quite
positive (more positive than 25.2 kcal/mol), it is reasonable to
suggest that the hydrogen atom transfer process may be ruled out
as the initial step for the hydride transfer. For step c, since the
state energy changes all are quite negative (more negative than
ꢀ14.9 kcal/mol), it is evident that the concerted hydride transfer
mechanism (step c) should be most likely. As to the initial
electron transfer (step a), the possibility of the initial electron
transfer cannot be ruled out, because the thermodynamic block
for the electron transfer is none or too small (ꢀ2.1 to 0.1 kcal/
mol). In addition, the intrinsic barrier energies of electron trans-
fers are generally much smaller than that of hydride transfers,60
which would also favor the electron transfer. It is evident that
from the view of thermodynamics there exist at least three
possible mechanisms for the hydride transfer from iAscHꢀ to
(G)PhXn+ in acetonitrile: the concerted hydride one-step trans-
fer (step c), the hydrogen transfer initiated by electron transfer
(steps a and d, i.e., eꢀH•), and the initial electron transfer
followed by proton and electron transfer separately (steps a, e,
and f, i.e., eꢀpꢀe). However, the mechanism of eꢀpꢀe three
steps may be ruled out, because the thermodynamic block for the
proton transfer from iAscH• to (G)PhXn• in acetonitrile (step e)
is too high (26.2ꢀ27.3 kcal/mol).
From Scheme 6 and Table 8 it is easy to find that for the
hydride transfer from GluNAH to (G)PhXn+, the state energy
change scales of the three initial steps (steps a, b, and c) in the
four possible pathways range from 18.7 to 20.9 kcal/mol for the
electron transfer (step a), from 33.6 to 34.8 kcal/mol for the
hydrogen transfer (step b), and from ꢀ24.1 to ꢀ20.9 kcal/mol
for the concerted hydride transfer (step c). Since the state energy
changes for the concerted hydride transfer (step c) are all quite
negative (more negative than ꢀ20.9 kcal molꢀ1), and the state
energy changes for the electron transfer and for hydrogen trans-
fer are all quite positive (more positive than 18.7 kcal molꢀ1), it is
reasonable to suggest that the electron-transfer process (step a)
and the hydrogen-transfer process (step b) all can be ruled out as
the initial step for the reaction of GluNAH with (G)PhXn+; as a
result, the remaining concerted hydride transfer step (step c)
should be merely the reasonable pathway for the reaction of
GluNAH with (G)PhXn+ in acetonitrile.
In order to further verify the reaction mechanism of GluNAH
with (G)PhXn+ in acetonitrile, the kinetics of hydride transfer
from GluNAH to (G)PhXn+ in acetonitrile was examined. From
Table 4, it is found that activation free energetic scales of the
hydride transfer from GluNAH to (G)PhXn+ range from 10.5 to
11.7 kcal molꢀ1, which are much smaller than the standard-state
energy changes of the initial electron transfer (step a) (18.7ꢀ
20.9 kcal/mol), and also much smaller than the standard-state
energy changes of the initial hydrogen-transfer (step b)
(33.6ꢀ34.8 kcal/mol), but larger than the standard-state energy
change of the concerted hydride transfer (step c) (ꢀ24.1 to
ꢀ20.9 kcal/mol). According to the transition-state theory that
the activation free energy change is always larger than or at least
equal to the corresponding standard-state free energy change for
any elemental reaction,59 it is evident that both of the reaction
steps a and b should be ruled out as the initial reaction in the
reactions of GluNAH with (G)PhXn+, and the only remaining
step c is suitable for the reaction law (Figure 10).
In order to further diagnose the real reaction mechanism of
hydride transfer from iAscHꢀ to (G)PhXn+, the intermediate
products of the hydride transfer from iAscHꢀ to (G)PhXn+ in
dry acetonitrile were detected. The results are shown in Figures 8
and 9. From Figure 8, it is clear that when PhXn+ (4 ꢂ 10ꢀ4 M)
was mixed with excess iAscHꢀ (1 ꢂ 10ꢀ2 M) in acetonitrile, the
UVꢀvis absorbance of PhXn+ (449 nm) was rapidly decreased
with the reaction time, and meanwhile a new absorbance at 372 nm
By comparing ΔHq and ꢀTΔSq of the hydride transfer from
GluNAH to (G)PhXn+ in Table 4, it is found that the values of
ΔHq are generally much smaller than those of the corresponding
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ARTICLE
Table 5. Molar Enthalpy Changes of iAscHꢀ and GluNAH as Well as (G)PhXnH (XH) To Release Hydride Anions and Hydrogen
Atoms, and the Molar Enthalpy Changes of XH•+ To Release Hydrogen Atoms and Protons (kcal molꢀ1) as Well as Oxidation
Potentials of XH and X• in Acetonitrile (V versus Fc+/0
)
ΔHH D(XH)a
ΔHHD(XH)b
Eox(XH)c
ΔHPD(XH•+)b
ΔHHD(XH•+)b
Eox(X•)c
ꢀ
XH
iAscHꢀ
GluNAH
(G)PhXnH
p-CF3
80.6
74.6
69.0
77.4
ꢀ0.425
25.8
13.5
64.4
37.6
ꢀ0.623
ꢀ1.248
0.475
98.7
98.3
97.9
97.6
97.3
96.8
96.4
96.0
95.5
80.4
80.2
80.1
79.9
79.9
79.9
79.8
79.5
79.4
1.257
1.241
1.220
1.208
1.201
1.195
1.188
1.176
1.164
ꢀ1.5
ꢀ1.4
ꢀ1.1
ꢀ1.0
ꢀ0.6
ꢀ0.7
ꢀ0.6
ꢀ0.6
ꢀ0.4
43.7
43.7
43.8
43.7
43.6
43.2
43.0
42.9
42.6
ꢀ0.336
ꢀ0.343
ꢀ0.355
ꢀ0.360
ꢀ0.381
ꢀ0.394
ꢀ0.408
ꢀ0.413
ꢀ0.430
m-CF3
p-Cl
p-Br
m-OCH3
p-H
m-CH3
p-CH3
p-OCH3
ꢀ
a ΔHH D(XH) values for XH = iAscHꢀ and GluNAH were derived from eq 4, take ΔHH-A(PhXn+) = ꢀ96.8 kcal/mol in acetonitrile from ref 51; the
ΔHHꢀD(XH) values for XH = (G)PhXnH were derived from the corresponding enthalpy change of (G)PhXn+ with iAscHꢀ in acetonitrile minus
ꢀ
ΔHH D(iAscHꢀ) (80.6 kcal/mol). The uncertainties are all smaller than 1 kcal/mol. b ΔHHD(XH), ΔHPD(XH•+), and ΔHHD(XH•+) were estimated
from eqs 5ꢀ7, respectively, taking Eo(H0/ꢀ) = ꢀ1.128 V vs Fc and Eo(H+/0) = ꢀ2.298 V vs Fc in acetonitrile from Zhang’s work.55 The relative
uncertainties were estimated to be smaller than or close to 1 kcal/mol in each case. c The standard oxidation potentials of XH and X• in acetonitrile were
derived from the experimental results by using CV method, but if the electrochemical oxidations of XH or X• are irreversible, the standard oxidation
potentials of XH and X• in acetonitrile were derived from the OSWV results, because OSWV has been verified to be more exact electrochemical method
for evaluating the standard one-electron redox potentials of analyte with irreversible electrochemical processes than CV.50
Scheme 4. Three Thermodynamic Cycles Constructed on the Basis of the Chemical Process of iAscHꢀ To Release Hydride Anion
in Acetonitrile
Scheme 5. Molecule ID Cards of iAscHꢀ and GluNAH in Acetonitrile
appeared, and the increase of the absorption at 372 nm was
synchronous with the decrease of the absorption at 449 nm, which
means that the species with absorption at 372 nm was directly
derived from PhXn• or iAscH•. That is, the species with absorption
at 372 nm may belong to either PhXn• or iAscH•. But when the
substituted PhXn+, such as (p-CH3O)PhXn+, was used to replace
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ARTICLE
Table 6. Diagnoses of Chemical Properties for the Members in AscHꢀ Family According to the Molecule ID Card of iAscHꢀ
(Scheme 5)
* Note: ΔHPA(iAsc•ꢀ), ΔHHA(iAsc•ꢀ), ΔHH-A(iAsc), and ΔHHA(iAsc) are defined as the enthalpy changes of iAsc•ꢀ to obtain proton and to obtain
hydrogen atom, and the enthalpy changes of iAsc to obtain hydride anion and to obtain hydrogen in acetonitrile, respectively. The values are equal to the
enthalpy changes of the corresponding opposite species to release proton, to release hydrogen atom, and to release hydride anion in acetonitrile by
switching the signs.
Table 7. Diagnoses of Chemical Properties for the Members in GluNAH Family According to the Molecule ID Card of GluNAH
(Scheme 5)
* Note: ΔHPA(GluNA•), ΔHHA(GluNA•), ΔHH-A(GluNA+), and ΔHHA(GluNA+) are defined as the enthalpy changes of GluNA• to obtain proton and
to obtain hydrogen atom, and the enthalpy changes of GluNA+ to obtain hydride anion and to obtain hydrogen in acetonitrile, respectively. The values
are equal to the enthalpy changes of the corresponding opposite species to release proton, to release hydrogen atom, and to release hydride anion in
acetonitrile by switching the signs.
PhXn+ to react with iAscHꢀ, the position of the new absorption
peak was found not to change at 372 nm, which indicates that the
species with absorption at 372 nm should be iAscH• rather than
(G)PhXn•. It is evident that the finding of the new absorption at
372 nm during the hydride transfer from iAscHꢀ to (G)PhXn+
clearly indicates that the real mechanism of the hydride transfer
should be hydrogen transfer initiated by electron transfer (eꢀH•
multistep mechanism) rather than the concerted hydride transfer in
one step (step c). Figure 11 gives the reaction coordinate diagram of
the hydride transfer from iAscHꢀ to PhXn+ in dry acetonitrile.
By comparing ΔHq and ꢀTΔSq of the hydride transfer from
iAscHꢀ to (G)PhXn+ in step a and step d (Table 3), it is found
that the values of ΔHq are generally much larger than those of the
corresponding ꢀTΔSq for the hydride transfer in step a, indicating
the transition state in step a does not involve chemical bond
formation. This result also supports that step a of the hydride
transfer should belong to electron transfer, since electron transfer
does not involve chemical bond formation, and the entropy change
is generally quite small. But, for the reaction in step d, the values of
ΔHq are generally much smaller than those of the corresponding
ꢀTΔSq, indicating the transition state in step d involves formation
of a new chemical bond.
Comparison of Kinetic Intrinsic Barrier of iAscHꢀ and
GluNAH as Hydride, Electron, and Hydrogen Atom Donors
in Acetonitrile. From the mechanism analysis described above
on the hydride transfer from iAscHꢀ and GluNAH to (G)PhXn+
in acetonitrile, it is clear that the hydride transfer from GluNAH
to (G)PhXn+ is a one-step hydride transfer mechanism, but the
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Scheme 6. Thermodynamic Analytic Platform on the Mechanism of Hydride Transfer from GluNAH to PhXn+ in Acetonitrile
ARTICLE
hydride transfer from iAscHꢀ to (G)PhXn+ is multistep rather
than a one-step mechanism. For the hydride transfer from GluNAH
to (G)PhXn+, the one-step hydride transfer mechanism is not
difficult to comprehend, since the thermodynamic driving force of
the one-step hydride transfer is much larger than those of the initial
electron transfer and the initial hydrogen atom transfer. But, for the
hydride transfer from iAscHꢀ to (G)PhXn+, there are at least two
questions which are worthy of asking: (1) Why cannot the one step
hydride transfer from iAscHꢀ to (G)PhXn+ take place, although the
thermodynamic driving force of the one-step hydride transfer is
much larger than that of the initial electron transfer and the
hydrogen atom transfer? (2) Why is the rate of the hydrogen atom
transfer in the second step much lower than that of the initial
electron transfer, although the thermodynamic driving force of the
hydrogen atom transfer is much larger than that of the initial
electron transfer? In order to answer those questions, the kinetic
intrinsic barrier energy of the related chemical processes needs
examination, since a chemical reaction rate is not only dependent on
the thermodynamic driving force of the reaction but also dependent
on the intrinsic barrier energy of the reaction. In order to estimate
the intrinsic barrier energy of the each reaction step of hydride
transfer from iAscHꢀ and GluNAH to (G)PhXn+ in acetonitrile,
BrønstedꢀEvansꢀPolanyi relations on the each step reaction of the
hydride transfer from iAscHꢀ and GluNAH to (G)PhXn+ in
acetonitrile were examined (Figures 12ꢀ15).
step c is linear, and the line slope is 0.360 and the line intercept is
19.4 kcal/mol. The line intercept in Figure 12 is the value of the
activation free energy (ΔGq) of the hydride transfer from
GluNAH to (G)PhXn+ when the thermodynamic driving force
(ΔHrxn) is equal to zero. According to the definition of intrinsic
barrier energy of a reaction that the intrinsic barrier energy of a
reaction is defined as the activation free energy (ΔGq) of the
reaction when the thermodynamic driving force of the reaction is
zero,61 it is clear that the line intercept value (19.4 kcal/mol) in
q
Figure 12 is just the kinetic intrinsic barrier energy (ΔG0 ) of the
hydride transfer from GluNAH to (G)PhXn+ in acetonitrile.
However, for the kinetic intrinsic barrier energy of the hydride
transfer from iAscHꢀ to (G)PhXn+ in acetonitrile, the detailed
value cannot be directly derived from the BrønstedꢀEvansꢀ
Polanyi relation for the hydride transfer from iAscHꢀ to
(G)PhXn+ in one step like the case of the hydride transfer from
GluNAH to (G)PhXn+, since the real mechanism of the hydride
transfer from iAscHꢀ to (G)PhXn+ is not a hydride one-step
transfer. In order to infer the intrinsic barrier energy of the
hydride transfer from iAscHꢀ to (G)PhXn+ in acetonitrile, the
hydride transfer from iAscHꢀ to (G)PhXn+ in acetonitrile might
be considered as the pseudoconcerted hydride transfer in one
step as shown in Figure 11 (red line). It is clear that, when the
BrønstedꢀEvansꢀPolanyi relations on the pseudoconcerted
hydride transfer from iAscHꢀ to (G)PhXn+ in acetonitrile were
examined, a good linear plot of the activation free energy against
thermodynamic driving force ΔHrxn for the pseudoconcerted
From Figure 12, it is clear that the BrønstedꢀEvansꢀPolanyi
relation for the hydride transfer from GluNAH to (G)PhXn+ in
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Scheme 7. Thermodynamic Analytic Platform on the Mechanism of Hydride Transfer from iAscHꢀ to PhXn+ in Acetonitrile
ARTICLE
hydride transfer (i.e., step c) was obtained (Figure 13) and the
line slope is 0.760 and the line intercept is 26.3 kcal/mol. The
intrinsic barrier energy of the formal (pseudoconcerted) hydride
transfer from iAscHꢀ to (G)PhXn+ in acetonitrile is 26.3 kcal/
mol, which is larger than of the hydride transfer from GluNAH to
PhXn+ in acetonitrile (19.4 kcal/mol) by 6.9 kcal/mol. Since the
intrinsic barrier energy of the formal hydride transfer from
iAscHꢀ to (G)PhXn+ in acetonitrile is larger than that of the
hydride transfer from GluNAH to (G)PhXn+ by 6.9 kcal/mol
and thermodynamic driving force of the hydride transfer from
iAscHꢀ to (G)PhXn+ in acetonitrile is smaller than that of the
hydride transfer from GluNAH to (G)PhXn+ by 6.0 kcal/mol,
we can make a conclusion that GluNAH is a good hydride donor,
but iAscHꢀ is not a good hydride donor; the mechanism of
hydride transfer from GluNAH generally is a one step, but the
mechanism of hydride transfer from iAscHꢀ generally needs
multiple steps; i.e., in most cases, the concerted hydride transfer
from iAscHꢀ could be forbidden.
iAscHꢀ to (G)PhXn+ in step a (11.1 kcal/mol) and the followed
hydrogen atom transfer from iAscH• to (G)PhXn• (17.0 kcal/
mol), it is found that the intrinsic barrier energy of the initial
electron transfer in step a (11.1 kcal/mol) is smaller than that of
the following hydrogen transfer (17.0 kcal/mol) by 5.9 kcal/mol,
which can be used to understand why the hydrogen-transfer
reaction in the second step is the rate determined, although the
thermodynamic driving force of the hydrogen transfer is much
larger than that of the initial electron transfer. In the same way, it
is not difficult to understand why the hydride transfer from
iAscHꢀ to (G)PhXn+ chose the electron transfer initiated multi-
step mechanism rather than the concerted hydride transfer one-
step mechanism; the reason is that the intrinsic barrier energies of
the concerted hydride transfer from iAscHꢀ to (G)PhXn+ in
acetonitrile (26.3 kcal/mol) are larger than those of the corre-
sponding electron transfer from iAscHꢀ to (G)PhXn+ in acet-
onitrile (11.1 kcal/mol) by 15.2 kcal/mol, although the
thermodynamic driving force of the concerted hydride transfer
is much larger than that of the electron transfer.
In the same way, the intrinsic barrier energy of the initial
electron transfer from iAscHꢀ to (G)PhXn+ in step a and the
intrinsic barrier energy of the followed hydrogen atom transfer
from iAscH• to (G)PhXn• in step d all can be obtained from the
line intercept (11.1 kcal/mol) in Figure 14 and from the line
intercept (17.0 kcal/mol) in Figure 15. By comparing the
intrinsic barrier energies of the initial electron transfer from
Comparison of the Character of Transition States of the
Related Reactions with GluNAH and iAscHꢀ as Hydride,
Electron and Hydrogen Atom Source. Although the thermo-
dynamic diving force, intrinsic barrier energy, and mechanism of
GluNAH and iAscHꢀ as hydride, hydrogen atom, and electron
donors have been compared in detail, the transition structures of
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Table 8. Energetics of Each Mechanistic Step of Hydride
Transfer from iAscHꢀ and GluNAH to (G)PhXn+ in Acet-
onitrile Shown in Schemes 6 and 7 (kcal/mol)
ΔG (or ΔH) (kcal/mol)
(G)PhXn+ step aa
step bb
step cc
step dd
step ee
step f f
For Hydride Transfer from GluNAH to (G)PhXn+
p-CF3
m-CF3
p-Cl
18.7
18.9
19.1
19.3
19.7
20.0
20.4
20.5
20.9
33.7
33.7
33.6
33.7
33.8
34.2
34.4
34.5
34.8
ꢀ24.1
ꢀ23.7
ꢀ23.3
ꢀ23.0
ꢀ22.7
ꢀ22.2
ꢀ21.8
ꢀ21.4
ꢀ20.9
ꢀ42.8
ꢀ42.6
ꢀ42.5
ꢀ42.3
ꢀ42.5
ꢀ42.3
ꢀ42.2
ꢀ41.9
ꢀ41.8
14.9
14.8
14.5
14.4
14.2
14.2
14.0
14.0
13.8
ꢀ57.8
ꢀ57.4
ꢀ56.9
ꢀ56.6
ꢀ56.5
ꢀ56.3
ꢀ56.2
ꢀ55.9
ꢀ55.6
p-Br
m-OCH3
p-H
m-CH3
p-CH3
p-OCH3
Figure 9. UVꢀvis spectra change at λ = 372 nm for the reaction of
iAscHꢀ (1 ꢂ 10ꢀ2 M) with PhXn+ (4 ꢂ 10ꢀ4 M) in dry acetonitrile at
298 K over the course of 5 s.
For Hydride Transfer from iAscHꢀ to (G)PhXn+
p-CF3
m-CF3
p-Cl
ꢀ2.1
ꢀ1.9
ꢀ1.6
ꢀ1.5
ꢀ1.2
ꢀ0.7
ꢀ0.4
ꢀ0.3
0.1
25.3
25.3
25.2
25.3
25.4
25.8
26.0
26.1
26.4
ꢀ18.1
ꢀ17.7
ꢀ17.3
ꢀ17.0
ꢀ16.7
ꢀ16.2
ꢀ15.8
ꢀ15.4
ꢀ14.9
ꢀ16.0
ꢀ15.8
ꢀ15.7
ꢀ15.6
ꢀ15.5
ꢀ15.5
ꢀ15.4
ꢀ15.1
ꢀ15.0
27.3
27.2
26.9
26.8
26.4
26.5
26.4
26.4
26.2
ꢀ43.4
ꢀ43.0
ꢀ42.5
ꢀ42.2
ꢀ42.1
ꢀ41.9
ꢀ41.8
ꢀ41.5
ꢀ41.2
p-Br
m-OCH3
p-H
m-CH3
p-CH3
p-OCH3
a Derived from the equation ΔG(step a) = ꢀ23.06{Ered[(G)PhXn+] ꢀ
Eox(XH)}, XH = iAscHꢀ and GluNAH. b Derived from the equation
ΔH(step b) = ΔHHD(XH) ꢀ ΔHHD[(G)PhXnH•]. c Derived from the
equation: ΔH(step c) = ΔHH-D(XH) ꢀ ΔHH-D[(G)PhXnH]. d Derived
from the equation: ΔH(step d) = ΔHHD(XH•+) ꢀΔHHD[(G)PhXnH].
e Derived from the equation: ΔH(step e) = ΔHPD(XH•+) ꢀ ΔHPD
[(G)PhXn•+]. Derived from the equation: ΔG(step f) = ꢀ23.06{Eox
f
[(G)PhXnH] ꢀ Eox(X•) }.
the related reactions are not clear. To quantitatively elucidate the
structural characters of the transition states (such as bond
energies and bond lengths of the forming or breaking chemical
bonds), the effective charge changes on the reaction center atoms
(C4 for GluNAH and C9 for PhXn+) in the transition states were
estimated.
Figure 10. Comparison of state energy changes for the three possible
initial steps of the hydride transfer from GluNAH to PhXn+ and the
activation free energy of the hydride transfer.
For the effective charge change on the C9 atom in (G)PhXn+
from the (G)PhXn+ in the reaction initial state to the “H--PhXn”
in the transition state, the effective charge on C9 in the transition
state can be estimated by using Hammett-type linear free energy
analysis method, since the Hammett linear free-energy relation-
ship analysis can provide a very efficient access to estimate the
effective charge distribution.34b Figure 16 shows the plots of the
activation Gibbs free energy change (ΔGq) and the thermo-
dynamic driving force (ΔHrxn) of the hydride transfer from
GluNAH to (G)PhXn+ against the Hammett substituent con-
stant σ. From Figure 16, two excellent straight lines with the line
slopes of ꢀ1.43 ( 0.05 (equivalent to F of 0.25)62 for the process
from the reactants to the transition state, and ꢀ3.91 ( 0.14
(equivalent to F of 0.69)62 for the hydride transfer from GluNAH
to (G)PhXn+ to form GluNA+ and (G)PhXnH are observed,
which means that Hammett linear free energy relationship holds in
the two chemical processes. According to the nature of Hammett
substituent effect, it is conceived that the sign of the line slope values
reflects increase or decrease of the effective charge on the carbon
(C9) atom in the pyran ring, and the magnitude of the line slope
values is a measurement of the effective charge change on the C9
atom during the corresponding reaction processes.63 In order to
quantitatively evaluate the relative effective charge changes on the
C9 atom in (G)PhXn+ from the (G)PhXn+ in the reaction initial
state to the (G)PhXn+ in the transition state, we defined that the
effective charges on the C9 atom in the free (G)PhXnH and in the
free (G)PhXn+ are zero and positive one (+1) unit, respectively.
This definition indicates that the negative line slope of ꢀ3.91 for the
hydride transfer from GluNAH to (G)PhXn+ to form GluNA+ and
(G)PhXnH is equivalent to the negative effective charge increase of
one unit (ꢀ1.000) on the C9 atom from (G)PhXn+ to (G)PhXnH.
According to this relationship, it is easy to deduce that the line slope
of ꢀ1.43 for the formation of the transition state is equivalent to the
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Figure 13. BrønstedꢀEvansꢀPolanyi relation of the formal hydride
transfer from iAscHꢀ to (G)PhXn+ in step c.
Figure 11. Reaction coordinate diagram of hydride transfer from
iAscHꢀ to PhXn+ in dry acetonitrile: (1) real pathway of hydride
transfer from iAscHꢀ to PhXn+ (green volcano curve); (2) hypothetical
one-step mechanism hydride transfer from iAscHꢀ to PhXn+ (red
volcano curve).
Figure 14. BrønstedꢀEvansꢀPolanyi relation of electron transfer from
iAscHꢀ to (G)PhXn+ (step a) in acetonitrile.
Figure 12. BrønstedꢀEvansꢀPolanyi relation for hydride transfer
from GluNAH to (G)PhXn+ in step c.
For the effective charge change on the C4 atom in GluNAH in
going from GluNAH to the “GluNA--H” in the transition state,
the following strategy can be used to estimate it. According to
Eyring theory about the transition state, the Gibbs activation free
energy of hydride transfer from GluNAH to PhXn+ (11.2 kcal/
mol) is equal to the sum of the formation enthalpy of “H--PhXn
bond” in the transition state [GluNA--H--PhXn]q and the
activation enthalpy of “GluNA--H bond” in the transition state.
Since the formation enthalpy of “H--C9 bond” in the transition
state (ꢀ35.4 kcal/mol) can be estimated according to the effective
charge change (ꢀ0.366) on the C9 atom in (G)PhXn+ going from
the reaction initial state to the transition state, the activation energy
of “C4--H bond” in the transition state should be 46.6 kcal/mol, i.e.,
the heterolytic dissociation energy of C4--H in the transition state is
74.6 ꢀ 46.6 = 28.0 kcal/mol. Since the effective charge increase of
one unit (+1.000) on the C4 atominGluNAHgoingfromGluNAH
to GluNA+ is equivalent to the C4ꢀH bond heterolytic dissociation
energy (74.6 kcal/mol), the effective positive charge increase on the
C4 atom in GluNAH going from the reaction initial state to the
transition state should be +0.625. On the basis of the definition of
the effective charge (0.000) on the C4 atom of GluNAH in the
reaction initial state, the effective positive charge on the C4 atom in
negative effective charge increase of 0.366 on the C9 atom from
the free (G)PhXn+ as the reactant to the (G)PhXn+ moiety in the
transition state. Since the effective charge on the C9 atom in free
(G)PhXn+ has been defined as positive one (+1.000) unit, the effec-
tive charge on the C9 atom in the transition state should be +0.634
unit (eq 12), which means that (G)PhXn+ gets negative charge
of ꢀ0.366 unit in going from the reaction initial state to the
transition state. Since the C9ꢀH bond formation energy of PhXnH
is ꢀ96.8 kcal/mol in acetonitrile and corresponds to the effective
charge increase of ꢀ1.000 unit on the C9 atom in the C9ꢀH bond
formation process, PhXn+ + Hꢀ f PhXnH, it is conceived that the
formation energy of “C9--H bond” in the transition state should
be ꢀ35.4 kcal/mol. In addition, from Hooke’s law we derive the
relationship of C9ꢀH bond length and C9ꢀH bond energy: Δr =
(2ΔΔH/k)1/2, in which Δr is the bond length change of C9ꢀH,
ΔΔH is bond energy change of C9ꢀH, and k is bond harmonic
ꢀ1 ꢀꢀ2
force constant of C9ꢀH, the value is about 513.2 kcal mol
Å
(in
acetonitrile).64 According to the formula, the C9--H bond length in
the transition state can be estimated. The result is 0.159 nm in
acetonitrile.
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Figure 15. BrønstedꢀEvansꢀPolanyi relation of the hydrogen atom
transfer from iAscH• to (G)PhXn• in step d.
Figure 17. Hammett plots of ΔH(step c) (9), ΔH(step d)(2),
ΔG(step a) (b), ΔGq(step c) (ƒ), ΔGq(step d) ([), and ΔGq(step
a) (1) for hydride transfer from iAscHꢀ to (G)PhXn+ in acetonitrile vs σ.
hydrogen atom transfer, and the formal hydride transfer against
the Hammett substituent constant σ (Figure 17); the results also
are shown in eqs 13, 14, and 15, respectively. The bond energies
of O3ꢀH and C9ꢀH in the related transition state are estimated
according to the effective charge change on the reaction center
atoms (O3 for iAscHꢀ and C9 for PhXnH) and the correspond-
ing O3ꢀH and C9ꢀH bond energies for iAscHꢀ and PhXnH at
ground states in acetonitrile, and the results also are given in
eqs 13ꢀ15, respectively.
For the reaction of hydride transfer from GluNAH to PhXn+
in CH3CN:
For the reaction of electron transfer from iAscHꢀ to PhXn+ in
CH3CN:
Figure 16. Hammett plots of ΔGq (9) and ΔHrxn (b) for hydride
transfer from GluNAH to (G)PhXn+ in acetonitrile vs σ.
the transition state can be easily estimated (+0.625). From the
effective charges on the C4 atom (+0.625) and the C9 atom
(+0.634) in the transition state, it is not difficult to get that the
transferring hydride anion in the transition state carries an effective
charge of ꢀ0.259 unit. Since the bond energy of C4ꢀHinthetransi-
tion state is 28.0 kcal/mol, the bond energy of C4ꢀH in the
transition state can be evaluated according to the same method as
used for C9ꢀH bond length in the transition state. The result is
0.143 nm. The effective charges on the reaction center atoms (C4
and C9), bond energies, and bond length of C4ꢀH and C9ꢀH in
the transition state for the hydride transfer from GluNAH to PhXn+
in acetonitrile are given in eq 12.
For the reaction of hydrogen atom transfer from iAscH• to
PhXn• in CH3CN:
For the reaction of the formal hydride transfer from iAscHꢀ to
PhXn+ in CH3CN:
By using the same strategy, the effective charges on the O3 in
iAscHꢀ and on the C9 in PhXn+ in the transition states for the
electron transfer from iAscHꢀ to PhXn+ in the initial step and for
the hydrogen atom transfer from iAscH• to PhXn• in the second
step as well as for the formal hydride one-step transfer from
iAscHꢀ to PhXn+ all can be estimated according to the related
line slopes in the plots of activation Gibbs free energy change
(ΔGq) and thermodynamic driving force of the electron transfer,
From the transition state of hydride transfer from GluNAH to
PhXn+ in eq 12, the following results can be found: (1) The
distance between two reaction center atoms is 0.302 nm, which is
much larger than the sum of the two CꢀH bond length
(0.220 nm), but smaller than the sum of the van der Waals
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Scheme 8. Thermodynamic Analytic Platform on the Possible Reactions of the Members between GluNAH Family and iAsc
Family in Acetonitrile
radius of the two reaction center carbon atoms (0.340 nm),
indicating that the valence bond orbital of the two reaction center
atoms could slightly overlap; i.e., the transition state of the
hydride transfer could be triangular rather than linear. (2) The
C4ꢀH bond length (0.143 nm) is shorter than that of C9ꢀH
bond length (0.159 nm), meaning that the transition state is
more similar to the reactants than to the products. (3) The
transferred hydrogen atom carried the charge of ꢀ0.259, meaning
that the dissociation of C4ꢀH bond and the formation of C9ꢀH
bond is not synchro percentage conversion before reaching the
transition state, and the degree of the C4ꢀH bond dissociation is
larger than that of the C9ꢀH bond formation. From the transition
state of the formal hydride transfer from iAscHꢀ to PhXn+ (eq 15),
it is clear that the effective charge of the center carbon atom is
positive (+0.088), which is evidently unreasonable. This result also
further indicates that the hydride transfer from iAscHꢀ to PhXn+
actually is not completed by one step.
Diagnoses of Possible Reactions of the Members between
the iAscHꢀ Family and the GluNAH Family. Since AscHꢀ and
NADH are two important bioreductants and the hydride transfer
from AscHꢀ and NADH could involve multistep mechanism, it is
conceived that AscHꢀ and NADH as well as their various
reaction intermediates all could coexist in living body. In order
to infer the possible reactions between AscHꢀ and NADH as well as
their various reaction intermediates in vivo, a mimic thermodynamic
analysis platform on the likely reactions among them was constructed
according to Molecule ID Cards of iAsc and GluNAH in acetonitrile
(Scheme 8). From Scheme 8, the following predictions on the
reactions between the members of iAscHꢀ family and GluNAH family
can be made: (i) When iAsc and GluNAH are mixed in acetonitrile,
hydride and hydrogen transfers are allowed from GluNAH to iAsc to
yield GluNA+ and iAscHꢀ or GluNA• and iAscH•, respectively, but
electron transfer is forbidden. (ii) When neutral radical iAscH• and
cation radical GluNAH•+ contacted each other in acetonitrile, iAscHꢀ
and GluNA+, iAsc•ꢀ and GluNA•, and iAsc and GluNAH could be for-
med by hydrogen, proton, and electron transfers, respectively. (iii)
When iAsc•ꢀ and GluNA• met together in acetonitrile, iAscHꢀ and
GluNAH as well as iAsc and GluNAH could be formed by hydrogen
and electron transfers, respectively, but proton transfer is forbidden. (iv)
When iAscHꢀ and GluNA+ were mixed in acetonitrile, no reaction was
allowed. Among the four couples of the reaction partners, the couple of
iAscHꢀ and GluNA+ isthemoststablereactionpartner, butthecouple
of iAscH• and GluNAH•+ is the most active reaction partner. It is
evident that these diagnoses should be useful for our understanding and
interpretation of the real reduction mechanism of dehydroascorbic acid
by NADH in vivo.
’ CONCLUSIONS
In this work, 5,6-isopropylidene ascorbate (iAscHꢀ) and β-D-
glucopyranosyl-1,4-dihydro- nicotinamide acetate (GluNAH) were
synthesized as ascorbate (AscHꢀ) and dihydronicotinamide adenine
dinucleotide (NADH) models. After the thermodynamics, kinetics,
and mechanism of iAscHꢀ and GluNAH as hydride, hydrogen atom,
and electron sources to react with the corresponding form of the
substituted phenylxanthium perchlorate (G)PhXn+ as the reaction
partners in acetonitrile were examined and compared in detail, the
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following conclusions can be made: (1) iAscHꢀ is a quite weak
hydride donor, but a good one-electron donor. GluNAH is a weak
one electron donor, but a good hydride donor. The electron-donating
ability of iAscHꢀ in acetonitrile is larger than that of GluNAH by
0.900 V (equivalent to 20.8 kcal/mol), but the hydride-donating
ability of iAscHꢀ in acetonitrile is smaller than that of GluNAH by
6.0 kcal/mol, which means that GluNAH is a good two-electron
(or hydride anion) donor, and iAscHꢀ is a good single electron
donor. (2) iAscHꢀ is a good hydrogen atom donor, but GluNAH is a
weak hydrogen atom donor; the hydrogen-donating ability of iAscHꢀ
in acetonitrile is larger than that of GluNAH by 8.4 kcal/mol, which
suggests that iAscHꢀ is a good antioxidant, but GluNAH cannot be
used as antioxidant. (3) iAscHꢀ as hydride source generally adopts
multistep hydride-transfer mechanism initiated by electron transfer,
but GluNAH as hydride source generally adopts one-step hydride-
transfer mechanism. (4) Intrinsic energy of iAscHꢀ as hydride source
is much larger than that of GluNAH in acetonitrile; the difference is
6.9 kcal/mol, which also further suggests iAscHꢀ is not a good
hydride donor. (5) As hydrogen atom donor or as antioxidant, the
mechanism of iAscHꢀ to release hydrogen atom is generally multi-
step, and in the most cases, the hydrogen atom transfer was initiated
by electron transfer. Since the structure of GluNAH is quite close to
the redox active center structure of NADH, and the structure of
iAscHꢀ is quite close to the redox active center structure of AscHꢀ, as
well as the polarity of acetonitrile is quite close to that of peptide bond
in proteins, it is evident that these important experimental results can
be used to well answer the title question: what are the differences of
ascorbic acid and NADH as electron and hydride sources in vivo on
thermodynamics, kinetics, and mechanism?
(7) (a) Nakanishi, N.; Takeuchi, F.; Tsubaki, M. J. Biochem. 2007,
142, 553–560. (b) Njus, D.; Wigle, M.; Kelley, P. M.; Kipp, B. H.;
Schlegel, H. B. Biochem. 2001, 40, 11905–11911.
(8) Kytzia, A.; Korth, H.-G.; Sustmann, R.; de Groot, H.; Kirsch, M.
Chem.—Eur. J. 2006, 12, 8786–8797.
(9) May, J. M.; Qu, Z. C.; Li, X. Biochem. Pharmacol. 2001,
62, 873–881.
(10) Nishiyama, I.; Yamashita, Y.; Yamanaka, M.; Shimohashi, A.;
Fukuda, T.; Oota, T. J. Agr. Food. Chem. 2004, 52, 5472–5475.
(11) Gebicki, J.; Marcinek, A.; Zielonka, J. Acc. Chem. Res. 2004,
37, 379–386.
(12) (a) Styer, L. Biochemistry, 3rd ed; Freeman: New York, 1988;
Chapter 17. (b) Walsh, C. Enzymatic Reaction Mechanism; W. H.
Freeman: San Francisco, 1979; Chapter 10. (c) Westheimer, F. H.
Pyridine Nucleotide Coenzyme; Dolphin, D., Poulson, R., Avramovic, O.,
Eds.; Wiley-Interscience: New York, 1988; Part A, p 253.
(13) Birrell, J. A.; Yakovlev, G.; Hirst, J. Biochemistry 2009,
48, 12005–12013.
(14) Yamazaki, S.; Tsai, L. J. Biol. Chem. 1980, 255, 6462–6465.
(15) Carlson, B. W.; Miller, L. L. J. Am. Chem. Soc. 1985, 107,
479–485.
(16) (a) Biochemistry; Campbell, M. K., Ed.; Saunders College
Publishing: London, 1995; pp 285ꢀ290. (b) Nicotinamide Nucleotide
Transhydrogenase in the Enzymes; Rydstorm, J., Hoeck, L., Ernster, Eds.;
New York, 1976; pp 76ꢀ98.
(17) (a) Silverblatt, E.; Robinson, A. L.; King, C. G. J. Am. Chem. Soc.
1943, 65, 137–141. (b) Weissberger, A.; LuValle, J. E.; Thomas, D. S.
J. Am. Chem. Soc. 1943, 65, 1934–1939. (c) Weissberger, A.; LuValle,
J. E. J. Am. Chem. Soc. 1944, 66, 700–705.
(18) (a) Fukuzumi, S.; Tanaka, T. Photoinduced Electron Transfer;
Fox, M. A., Chanon, M., Eds.; Elsevier: Amsterdam, The Netherlands,
1988; Part C, Chapter 10. (b) Fukuzumi, S. Advances in Electron Transfer
Chemistry; Mariano, P. S., Ed.; JAI Press: Greenwich, CT, 1992; pp
67ꢀ175.
’ AUTHOR INFORMATION
(19) Zhu, X.-Q.; Yang, Y.; Zhang, M.; Cheng, J.-P. J. Am. Chem. Soc.
2003, 125, 15298–15299.
Corresponding Author
*E-mail: xqzhu@nankai.edu.cn.
(20) (a) Zielonka, J.; Marcinek, A.; Adamus, J.; Gebicki, J. J. Phys.
Chem. A 2003, 107, 9860–9864. (b) Bartlett, P. N.; Simon, E. J. Am.
Chem. Soc. 2003, 125, 4014–4015.
(21) (a) Gassner, G.; Wang, L.; Batie, C.; Ballou, D. P. Biochemistry
1994, 33, 12184–12193. (b) Gassner, G. T.; Lippard, S. J. Biochemistry
1999, 38, 12768–12785. (c) Niviꢁere, V.; Vanoni, M. A.; Zanetti, G.;
Fontecave, M. Biochemistry 1998, 37, 11879–11887.
(22) (a) Choi, H.-I.; Kim, H.-J.; Park, J.-I.; Shin, E.-H.; Kim, D.-W.; Kim,
S.-S. Bioorg. Med. Chem. Lett. 2009, 19, 2079–2082. (b) Spickenreither, M.;
Braun, S.; Bernhardt, G.; Dove, S.; Buschauer, A. Bioorg. Med. Chem. Lett.
2006, 16, 5313–5316.
’ ACKNOWLEDGMENT
Financial support from the National Natural Science Foundation
of China (Grant Nos. 21072104, 20921120403, and 20832004),
the Ministry of Science and Technology of China (Grant No.
2004CB719905), and the 111 Project (B06005) is gratefully
acknowledged.
(23) (a) Pang, J.; Hay, S.; Scrutton, N. S.; Sutcliffe, M. J. J. Am. Chem.
Soc. 2008, 130, 7092–7097. (b) Brondijk, T. H. C.; van Boxel, G. I.;
Mather, O. C.; Quirk, P. G.; White, S. A.; Jackson, J. B. J. Biol. Chem.
2006, 281, 13345–13354.
(24) (a) Bruice, T. C. Acc. Chem. Res. 1980, 13, 256–262. (b) Walsh,
C. Acc. Chem. Res. 1980, 13, 148–155.
(25) (a) Nisimoto, Y.; Jackson, H. M.; Ogawa, H.; Kawahara, T.;
Lambeth, J. D. Biochemistry 2010, 49, 2433–2442. (b) Afanasyeva, M. S.;
Taraban, M. B.; Purtov, P. A.; Leshina, T. V.; Grissom, C. B. J. Am. Chem.
Soc. 2006, 128, 8651–8658. (c) Juarez, O.; Morgan, J. E.; Barquera, B.
J. Biol. Chem. 2009, 284, 8963–8972.
(26) (a) Warren, J. J.; Mayer, J. M. J. Am. Chem. Soc. 2010,
132, 7784–7793. (b) Warren, J. J.; Mayer, J. M. J. Am. Chem. Soc.
2008, 130, 2774–2776. (c) Warren, J. J.; Mayer, J. M. J. Am. Chem. Soc.
2008, 130, 7546–7547.
’ REFERENCES
(1) (a) Vitamin C in Health and Disease; Packer, L., Fuchs, J., Eds.;
Marcel Dekker: New York, 1997; Vol. 5. (b) Kroneck, P. M. H.;
Armstrong, F. A.; Merkle, H.; Marchesini, A. Ascorbate oxidase:
molecular properties and catalytic activity. In Ascorbic Acid: Chemistry,
Metabolism and Uses; Seib, P. A., Tolbert, B. M., Eds.; Advances in
Chemistry Series 200; American Chemical Society: Washington, DC,
1982; pp 223ꢀ248. (c) Neta, P. Chem. Rev. 1972, 72, 533–543.
(d) Karrer, P. Chem. Rev. 1934, 14, 17–30.
(2) (a) Stout, D. M.; Meyers, A. I. Chem. Rev. 1982, 82, 223–243.
(b) Murakami, Y.; Kikuchi, J.-I.; Hisaeda, Y.; Hayashida, O. Chem. Rev.
1996, 96, 721–758. (c) Eisner, U.; Kuthan, J. Chem. Rev. 1972, 72, 1–42.
(3) Harnadek, G. J.; Ries, E. A.; Farhat, A.; Njus, D. J. Biol. Chem.
1990, 265, 18135–18141.
(4) Bisby, R. H.; Parker, A. W. Arch. Biochem. Biophys. 1995,
317, 170–178.
(5) Isaacs, N. S.; Eldik, R. J. Chem. Soc., Perkin Trans. 2 1997, No. No.
8, 1465–1467.
(6) Bartlett, D.; Church, D.; Bounds, P.; Koppenol, W. H. Free
Radical Biol. Med. 1995, 18, 85.
(27) (a) Sajenko, I.; Pilepic, V.; Brala, C. J.; Ursic, S. J. Phys. Chem. A
2010, 114, 3423–3430. (b) Vuina, D.; Pilepic, V.; Ljubas, D.; Sankovic,
K.; Sajenko, I.; Ursic, S. Tetrahedron Lett. 2007, 48, 3633–3637.
(28) De Biase, P. M.; Turjanski, A. G.; Estrin, D. A.; Doctorovich, F.
J. Org. Chem. 2005, 70, 5790–5798.
14809
dx.doi.org/10.1021/jp2067974 |J. Phys. Chem. B 2011, 115, 14794–14811

The Journal of Physical Chemistry B
ARTICLE
(29) Chatterjee, D.; Sengupta, A.; Mitra, A.; Basak, S. Inorg. Chem.
Commun. 2006, 9, 1219–1222.
(30) Prieto, F.; Coles, B. A.; Compton, R. G. J. Phys. Chem. B 1998,
102, 7442–7447.
(40) (a) Fukuzumi, S.; Suenobu, T.; Kawamura, S.; Ishida, A.;
Mikami, K. Chem. Commun. 1997, No. 3, 291–292. (b) Fukuzumi, S.;
Tokuda, Y. Bull. Chem. Soc. Jpn. 1992, 65, 831–836.
(41) Chikashita, H.; Ide, H.; Itoh, K. J. Org. Chem. 1986, 51,
5400–5405.
(42) Sigala, P. A.; Kraut, D. A.; Caaveiro, J. M. M.; Pybus, B.; Ruben,
E. A.; Ringe, D.; Petsko, G. A.; Herschlag, D. J. Am. Chem. Soc. 2008,
130, 13696–13708.
(43) (a) Zhang, B.-L.; Zhu, X.-Q.; Lu, J.-Y.; He, J.-Q.; Wang,
P. G.; Cheng, J.-P. J. Org. Chem. 2003, 68, 3295–3298. (b) Zhu,
X.-Q.; Wang, H.-Y.; Wang, J.-S.; Liu, Y.-C. J. Org. Chem. 2001, 66,
344–347.
(44) Kimura, M.; Tanaka, K. Angew. Chem., Int. Ed. 2008, 47,
9768–9771.
(45) Ohno, A.; Oda, S.; Ishikawa, Y.; Yamazaki, N. J. Org. Chem.
2000, 65, 6381–6387.
(46) Isaacs, N. S. Physical Organic Chemistry, 2nd ed.; Addison
Wesley Longman: London, 1995.
(47) Cho, B. H.; Kim, J. H.; Jeon, H. B.; Kim, K. S. Tetrahedron 2005,
61, 4341–4346.
(48) Viscontini, M.; Hochreuter, R.; Karrer, P. Helv. Chim. Acta
1953, 36, 1777–1783.
(31) (a) Fukuzumi, S.; Fujioka, N.; Kotani, H.; Ohkubo, K.; Lee,
Y.-M.; Nam, W. J. Am. Chem. Soc. 2009, 131, 17127–17134. (b) Yuasa, J.;
Yamada, S.; Fukuzumi, S. J. Am. Chem. Soc. 2008, 130, 5808–5820.
(c) Fukuzumi, S.; Kotani, H.; Lee, Y.-M.; Nam, W. J. Am. Chem. Soc.
2008, 130, 15134–15142. (d) Yuasa, J.; Yamada, S.; Fukuzumi, S. J. Am.
Chem. Soc. 2006, 128, 14938–14948. (e) Yuasa, J.; Fukuzumi, S. J. Am.
Chem. Soc. 2006, 128, 14281–14292.
(32) (a) Fukuzumi, S.; Yuasa, J.; Suenobu, T. J. Am. Chem. Soc. 2002,
124, 12566–12573. (b) Fukuzumi, S.; Ohkubo, K.; Okamoto, T. J. Am.
Chem. Soc. 2002, 124, 14147–14155. (c) Fukuzumi, S.; Fujii, Y.;
Suenobu, T. J. Am. Chem. Soc. 2001, 123, 10191–10199. (d) Fukuzumi,
S.; Koumitsu, S.; Hironaka, K.; Tanaka, T. J. Am. Chem. Soc. 1987,
109, 305–316.
(33) Matsuo, T.; Mayer, J. M. Inorg. Chem. 2005, 44, 2150–2158.
(34) (a) Zhu, X.-Q.; Zhang, J.-Y.; Cheng, J.-P. J. Org. Chem. 2006,
71, 7007–7015. (b) Zhu, X.-Q.; Cao, L.; Liu, Y.; Yang, Y.; Lu, J.-Y.;
Wang, J.-S.; Cheng, J.-P. Chem.—Eur. J. 2003, 9, 3937–3945. (c) Zhu,
X.-Q.; Li, H.-R.; Li, Q.; Ai, T.; Lu, J.-Y.; Yang, Y.; Cheng, J.-P. Chem.—
Eur. J. 2003, 9, 871–880. (d) Zhu, X.-Q.; Liu, Y.; Zhao, B.-J.; Cheng, J.-P.
J. Org. Chem. 2001, 66, 370–375. (e) Zhu, X.-Q.; Zou, H.-L.; Yuan,
P.-W.; Liu, Y.; Cao, L.; Cheng, J.-P. J. Chem. Soc., Perkin Trans. 2. 2000,
No. No. 9, 1857–1861. (f) Zhu, X.-Q.; Zhao, B.-J.; Cheng, J.-P. J. Org.
Chem. 2000, 65, 8158–8163. (g) Zhao, B.; Zhu, X.; Lu, Y.; Xia, C.-Z.;
Cheng, J.-P. Tetrahedron Lett. 2000, 41, 257–260. (h) Cheng, J.-P.; Lu,
Y.; Zhu, X.-Q.; Sun, Y.-K.; Bi, F.; He, J.-Q. J. Org. Chem. 2000, 65,
3853–3857. (i) Zhu, X.-Q.; Liu, Y.-C.; Cheng, J.-P. J. Org. Chem. 1999,
64, 8980–8981.
(49) Dauben, J. H.; Honnen, L.; Harmon, K. J. Org. Chem. 1960,
25, 1442–1445.
(50) Zhu, X.-Q.; Zhang, M.-T.; Yu, A.; Wang, C.-H.; Cheng, J.-P.
J. Am. Chem. Soc. 2008, 130, 2501–2516.
(51) Zhu, X.-Q.; Liang, H.; Zhu, Y.; Cheng, J.-P. J. Org. Chem. 2008,
73, 8403–8410.
(52) Zhu, X.-Q.; Tan, Y.; Cao, C.-T. J. Phys. Chem. B 2010,
114, 2058–2075.
(53) Equations 5ꢀ7 were constructed from the thermodynamic
cycles in Scheme 4. Note that we used the term free-energy change ΔGet
to replace the enthalpy change ΔHet in eqs 5ꢀ7 for the electron transfer
processes. The justification of this treatment is that entropies associated
with electron transfer are negligible, as verified by Arnett et al: Arnett,
E. M.; Amarnath, K.; Harvey, N. G.; Cheng, J.-P. J. Am. Chem. Soc. 1990,
112, 344–355.
(54) Wayner, D. D. M.; Parker, V. D. Acc. Chem. Res. 1993,
26, 287–294.
(55) Zhang, X.-M.; Bruno, J. W.; Enynnaya, E. J. Org. Chem. 1998,
63, 4671.
(56) Warren, J. J.; Tronic, T. A.; Mayer, J. M. Chem. Rev. 2010,
110, 6961–7001.
(57) Miller, L. L.; Valentine, J. R. J. Am. Chem. Soc. 1988, 110,
3982–3989.
(58) Zhu, X.-Q.; Liu, Q.-Y.; Chen, Q.; Mei, L.-R. J. Org. Chem. 2010,
75, 789–808.
(35) (a) Kil, H. J.; Lee, I. S. H. J. Phys. Chem. A 2009,
113, 10704–10709. (b) Lee, I. S. H.; Kil, H. J.; Ji, Y. R. J. Phys. Org.
Chem. 2007, 20, 484–490. (c) Lee, I. S. H.; Ji, Y. R.; Jeoung, E. H. J. Phys.
Chem. A 2006, 110, 3875–3881. (d) Lee, I. S. H.; Chow, K. H.; Kreevoy,
M. M. J. Am. Chem. Soc. 2002, 124, 7755–7761. (e) Lee, I. S. H.; Jeoung,
E. H.; Kreevoy, M. M. J. Am. Chem. Soc. 2001, 123, 7492–7496. (f) Lee,
I. S. H.; Jeoung, E. H. J. Org. Chem. 1998, 63, 7275–7279. (g) Kreevoy,
M. M.; Kotchevar, A. T. J. Am. Chem. Soc. 1990, 112, 3579–3583.
(h) Ostovic, D.; Lee, I. S. H.; Roberts, R. M. G.; Kreevoy, M. M. J. Org.
Chem. 1985, 50, 4206–4211.
(36) (a) Aizpurua, J. M.; Palomo, C.; Fratila, R. M.; Ferron, P.;
Benito, A.; Gomez-Bengoa, E.; Miranda, J. I.; Santos, J. I. J. Org. Chem.
2009, 74, 6691–6702. (b) Hapiot, P.; Moiroux, J.; Saveant, J. M. J. Am.
Chem. Soc. 1990, 112, 1337–1343. (c) Xu, H.-J.; Dai, D.-M.; Liu, Y.-C.;
Li, J.; Luo, S.-W.; Wu, Y.-D. Tetrahedron Lett. 2005, 46, 5739–5742.
(d) Ellis, W. W.; Raebiger, J. W.; Curtis, C. J.; Bruno, J. W.; DuBois, D. L.
J. Am. Chem. Soc. 2004, 126, 2738–2743.
(37) (a) Ogata, K.; Murayama, H.; Sugasawa, J.; Suzuki, N.; Fukuzawa,
S.-i. J. Am. Chem. Soc. 2009, 131, 3176–3177. (b) Srogl, J.; Voltrova, S. Org.
Lett. 2009, 11, 843–845. (c) Dzygiel, P.; Reeve, T. B.; Piarulli, U.;
Krupicka, M.; Tvaroska, I.; Gennari, C. Eur. J. Org. Chem. 2008, No. 7,
1253–1264. (d) Sharma, R. K.; Nethaji, M.; Samuelson, A. G. Tetrahedron:
Asym. 2008, 19, 655–663. (e) Ram, R. N.; Singh, V. Tetrahedron Lett. 2006,
47, 7625–7628.
(59) (a) Page, M.; Williams, A. Organic and Bio-organic Mechanism;
Longman: Harlow, UK, 1997; Chapter 3, pp 52ꢀ79. (b) Williams, A.
Acc. Chem. Res. 1984, 17, 425–430. (c) Williams, A. Acc. Chem. Res. 1989,
22, 387–392. (d) Williams, A. Adv. Phys. Org. Chem. 1991, 27, 1.
(e) Williams, A. J. Am. Chem. Soc. 1985, 107, 6335–6339.
(60) Eberson, L. Electron Transfer Reactions in Organic Chemistry;
Springer: Berlin, 1987.
(38) (a) Xu, H.-J.; Liu, Y.-C.; Fu, Y.; Wu, Y.-D. Org. Lett. 2006,
8, 3449–3451. (b) Lo, H. C.; Fish, R. H. Angew. Chem., Int. Ed. 2002,
41, 478–481.
(61) (a) Marcus, R. A. J. Phys. Chem. 1968, 72, 891–899. (b) Albery,
W. J. Annu. Rev. Phys. Chem. 1980, 31, 227–263. (c) Breugst, M.;
Zipse, H.; Guthrie, J. P.; Mayr, H. Angew. Chem., Int. Ed. 2010, 49,
5165–5169.
(39) (a) Fang, X. Q.; Xu, H. J.; Jiang, H.; Liu, Y. C.; Fu, Y.; Wu, Y. D.
Tetrahedron Lett. 2009, 50, 312–315. (b) Akagawa, K.; Akabane, H.;
Sakamoto, S.; Kudo, K. Org. Lett. 2008, 10, 2035–2037. (c) Kang, Q.;
Zhao, Z.-A.; You, S.-L. Org. Lett. 2008, 10, 2031–2034. (d) Martin,
N. J. A.; Ozores, L.; List, B. J. Am. Chem. Soc. 2007, 129, 8976–8977.
(e) Yang, J. W.; List, B. Org. Lett. 2006, 8, 5653–5655. (f) Martin,
N. J. A.; List, B. J. Am. Chem. Soc. 2006, 128, 13368–13369. (g) Zhang,
Z.; Gao, J.; Xia, J.-J.; Wang, G.-W. Org. Biomol. Chem. 2005, 3,
1617–1619.
(62) According to Hammett equation log K = Fσ and ΔH ≈ ꢀRT
log K, the line slope of ΔH against σ should be approximately equal to
ꢀ5.7F.
(63) Zhu, X.-Q.; Li, Q.; Hao, W.-F.; Cheng, J.-P. J. Am. Chem. Soc.
2002, 124, 9887–9893.
(64) The value is derived from the k-value of Csp3ꢀH in gas (674.6
kcal Å2 molꢀ1 from the following book: Cottrell, T. L. The strengths of
Chemical Bonds; Butterworths Scientific Publication: London, 1954)
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after revision by using solvent (acetonitrile) effect on the Csp3ꢀH bond
dissociation energy.
(65) Typically, the first injection shows less heat than expected. This
is often due to diffusion across the tip of the needle or to difficulties in
positioning the buret drive.
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