Gas-phase studies of substrates for the DNA mismatch repair enzyme MutY

Article abstract of DOI:10.1021/ja309082k

The gas-phase thermochemical properties (tautomeric energies, acidity, and proton affinity) have been measured and calculated for adenine and six adenine analogues that were designed to test features of the catalytic mechanism used by the adenine glycosyl

Full text of DOI:10.1021/ja309082k

Article
pubs.acs.org/JACS
Gas-Phase Studies of Substrates for the DNA Mismatch Repair
Enzyme MutY
Anna Zhachkina Michelson,‡ Aleksandr Rozenberg,‡ Yuan Tian,‡ Xuejun Sun,‡ Julianne Davis,‡
Anthony W. Francis,† Valerie L. O’Shea,† Mohan Halasyam,† Amelia H. Manlove,§ Sheila S. David,*,§,†
and Jeehiun K. Lee*,‡
‡Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901,
United States
†Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States
§Department of Chemistry, University of California, Davis, California 95616, United States
S
* Supporting Information
ABSTRACT: The gas-phase thermochemical properties (tautomeric energies,
acidity, and proton affinity) have been measured and calculated for adenine
and six adenine analogues that were designed to test features of the catalytic
mechanism used by the adenine glycosylase MutY. The gas-phase intrinsic
properties are correlated to possible excision mechanisms and MutY excision
rates to gain insight into the MutY mechanism. The data support a mechanism
involving protonation at N7 and hydrogen bonding to N3 of adenine. We also
explored the acid-catalyzed (non-enzymatic) depurination of these substrates,
which appears to follow a different mechanism than that employed by MutY, which we elucidate using calculations.
INTRODUCTION
■
Cellular DNA is inevitably damaged by both exogenous and
endogenous agents, resulting in a variety of chemical
modifications that are associated with mutagenesis, carcino-
genesis, and aging.1−4 Oxidative damage is extremely prevalent,
and one of the most common species formed by reactive
oxygen species is 8-oxo-7,8-dihydroguanine (OG).5−7 During
DNA replication, adenine (A) is usually inserted opposite OG
Figure 1. Adenine removal from DNA catalyzed by the MutY
glycosylase.
to form a relatively stable OG:A mismatch.8 Because
undamaged guanine (G) prefers to pair with cytosine (C),
not adenine, the oxidation, if not repaired, can result in
permanent G:C→T:A transversion mutations.
stearothermophilus structure with a fluorinated 2′-deoxyadeno-
sine, show multiple hydrogen-bonding contacts as well as
hydrophobic interactions between substrate and enzyme.14
Kinetic isotope effect studies imply an SN1-type reaction where
the nucleobase leaves (possibly protonated at N7) to yield an
oxocarbenium ion which is then attacked by water.14−16 Recent
computational simulations support N7 protonation as well as
an active site that organizes solvent to place water molecules
into key catalytic positions.17
The examination of thermochemical properties in the gas
phase, which provides the “ultimate” nonpolar environment,
reveals intrinsic reactivity that can be correlated to activity in
other media, such as hydrophobic active sites.18−24 In this
paper, we calculate and measure the gas-phase acidities and
proton affinities of a series of adenine analogues (not
heretofore studied in vacuo) and compare these results to the
In the face of the constant assault to DNA, organisms have
developed elaborate DNA repair pathways. In Escherichia coli,
oxidative damage is repaired by a “GO” repair pathway that
utilizes three enzymes: MutT, Fpg, and MutY.9−11 MutT
hydrolyzes the OG deoxynucleoside triphosphate to yield the
OG deoxynucleoside monophosphate and pyrophosphate,
preventing its incorporation into replicating DNA.12 Fpg
(also called MutM) catalyzes removal of OG from OG:C
base pairs, and an associated β- and δ-lyase activity at the
resultant abasic site, leading to strand scission.1,13 MutY is a
somewhat unusual glycosylase enzyme: rather than targeting a
damaged base, MutY catalyzes removal of adenine from OG:A
mispairs in DNA (Figure 1).1,12,13 MutY is remarkably specific
such that 2′-deoxyadenosine residues within A:T pairs are not
touched.
Because of the importance of damaged base repair to genome
integrity, the mechanisms of repair enzymes are of great
interest.11 MutY crystal structures, in particular a 2009 Bacillus
Received: September 12, 2012
Published: October 29, 2012
© 2012 American Chemical Society
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relative rates of MutY-catalyzed base excision of these
analogues within the context of duplex DNA paired with OG.
The acid-catalyzed non-enzymatic cleavage of these substrates
is also explored.
ln(k1/k2) = (1/RT )(ΔHBiH − ΔHAH
)
(2)
eff
METHODS
■
The relationship of these rate constants to ΔHacid is shown in eq 2,
where R is the gas constant and Teff is the effective temperature38 of
the activated dimer.33−36 The ratio of the intensities of the two
deprotonated products yields the relative acidity of the two
compounds of interest (eq 2), assuming that the dissociation has no
reverse activation energy barrier and that the dissociation transition
structure is late (and therefore indicative of the stability of the two
deprotonated products). These assumptions are generally true for
proton-bound systems.36,39,40
All of the nucleobase analogues (except Z1) and reference acids and
bases are commercially available and were used as received. Synthesis
of 1-deazaadenosine (Z1) and Z1-containing oligonucleotides was
similar to methods previously described; additional details are given in
the Supporting Information.25 Adenine glycosylase assays and acid-
catalyzed depurination reactions were also performed as described
previously; additional details are provided in the Supporting
Information.26
The bracketing method was used to measure the gas-phase acidity
and proton affinity values. A Fourier transform ion cyclotron
resonance (FT-ICR) mass spectrometer with dual cell setup
(described previously) was used.18,19,21,27,28 The magnetic field was
3.3 T; the baseline pressure was 1 × 10−9 Torr. The solid nucleobase
analogues were introduced into the cell via a heatable solids probe,
while liquid reference acids and bases were introduced via a system of
heatable batch inlets. Water was pulsed into the cell and ionized by an
electron beam (typically 8 eV (for HO−) or 20 eV (for H3O+), 6 μA,
0.5 s) to generate hydroxide and hydronium ions. Substrate ions were
generated by deprotonation or protonation of reference acids or bases
(with hydroxide or hydronium ions, respectively) and then selected
and transferred from one cubic cell to another via a 2-mm hole in the
middle trapping plate. Transferred ions were cooled with pulsed argon
gas that allowed the pressure to rise to 10−5 Torr. Experiments were
conducted at ambient temperature.
The typical protocol for bracketing experiments has been described
previously.18,19,27,29,30 Proton-transfer reactions were conducted in
both directions. For example, for Z3 acidity bracketing, hydroxide was
used to deprotonate neutral Z3. Deprotonated Z3 was transferred into
the adjoining cell, where it was allowed to react with the neutral
reference acid AH with known gas-phase acidity. In the opposite
direction, the deprotonated reference acid A− was generated and
transferred into the adjoining cell, where it was allowed to react with
neutral Z3. The occurrence of proton transfer (efficiency of reaction
greater than 10%) is regarded as evidence that the reaction is
exothermic (denoted as “+” in the tables). Bracketing experiments are
run under pseudo-first-order conditions with the neutral reactant in
excess, relative to the reactant ions. Reading the pressure of the neutral
compounds from the ion gauges is not always accurate; therefore, we
“back out” the neutral substrate pressure from fast control reactions
(described previously).19,22,23,29,31,32
In order to obtain the acidity of compound AH, the natural
logarithm of the relative intensity ratios was plotted versus the acidities
for a series of reference acids (BiH), where the slope is 1/RTeff and the
y-intercept is −ΔHAH/RTeff. The Teff was obtained from the slope. The
acidity of compound AH, ΔHAH, was calculated from either eq 2 or the
y-intercept. The same procedure was applied for proton affinity
measurements (via formation of positively charged proton-bound
dimers).
The gas-phase calculations were conducted at the B3LYP/6-
31+G(d) level using Gaussian03 and Gaussian09.41−45 All the
structures were fully optimized in the gas phase, and frequencies
were calculated (no imaginary frequencies were found). Acidity and
proton affinity values are reported as ΔH at 298 K.
Dielectric medium calculations were done using the conductor-like
polarizable continuum solvent model (CPCM, single-point calcu-
lations on B3LYP/6-31+G(d) gas-phase optimized structures; UAKS
cavity) at B3LYP/6-31+G(d) as implemented in Gaussian03.46−48 The
“total free energy in solution” values are reported, and the solvation
free energy of a proton (−264.0 kcal mol−1) is accounted for.49,50
RESULTS
■
Adenine and the synthetically derived analogues studied herein
are shown in Figure 2.26,51,52 7-Deazaadenine (Z, 1), 3-
We also utilized the Cooks kinetic method in a quadrupole (LCQ)
ion trap mass spectrometer33−36 to measure the acidities and proton
affinities of adenine analogues. The Cooks kinetic method involves the
formation of a proton-bound complex, or dimer, of the unknown and a
reference acid or base of known acidity or proton affinity.
The proton-bound dimer ions were generated by electrospray
ionization (ESI) of 250 μM solutions of an unknown and a reference
acid (or base, for proton affinity measurement). Water−methanol
(20%) solution was used as solvent.37 One drop of acetic acid (for
proton affinity measurements) or ammonium hydroxide (for acidity
measurements) was occasionally used to promote dimer formation. An
electrospray needle voltage of ∼4 kV and a flow rate of 25 μL/min
were applied. The proton-bound complex ions were isolated and then
dissociated by applying collision-induced dissociation (CID); the
complexes were activated for about 30 ms. Finally, the dissociation
product ions were detected to give the ratio of the deprotonated (or
protonated) analyte and deprotonated (or protonated) reference acid.
A total of 40 scans were averaged for the product ions.
Figure 2. Adenine and synthetic analogues studied herein.
deazaadenine (Z3, 2), and 1-deazaadenine (Z1, 3) are missing
nitrogen at the N7, N3, and N1 positions, respectively (as
compared to the parent adenine), and were designed to test the
importance of the nitrogen at those positions.51−53 Substrates
Q (4), M (5), and B (6) (with atoms numbered as for adenine,
to be consistent) are nonpolar isosteres of adenine.26,52
7-Deazaadenine (Z, 1). 1. Calculations: Z Tautomers,
Acidity, and Proton Affinity. In our experience, DFT methods
The dissociation of the proton-bound dimer [AHBi]− is depicted in
eq 1, where AH is the compound of unknown acidity and BiH
represents a series of reference acids of known acidity. The rate
constants k1 and k2 are for the two different dissociation pathways.
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Figure 3. The eight possible tautomeric structures of 7-deazaadenine
(Z, 1). Gas-phase acidities are in red; gas-phase proton affinities are in
blue. Relative stabilities are in parentheses. Calculations were
conducted at B3LYP/6-31+G(d); reported values are ΔH at 298 K.
Figure 4. The seven possible tautomeric structures of 3-deazaadenine
(Z3, 2). Gas-phase acidities are in red; gas-phase proton affinities are
in blue. Relative stabilities are in parentheses. Calculations were
conducted at B3LYP/6-31+G(d); reported values are ΔH at 298 K.
Table 1. Summary of Results for Acidity Bracketing of 7-
Deazaadenine (Z, 1)
Table 3. Summary of Results for Acidity Bracketing of 3-
Deazaadenine (Z3, 2)
b
b
proton transfer
proton transfer
a
a
ref compd
m-cresol
ΔHacid
ref acid
conj base
ref compd
ΔHacid
ref acid
conj base
349.5 2.1
347.4 0.5
346.8 2.0
346.0 0.5
344.1 2.9
−
−
+
+
+
+
+
+
methyl cyanoacetate
trifluoro-m-cresol
2-chloropropanoic acid
malononitrile
340.80 0.60
339.2 2.1
337.0 2.1
335.8 2.1
333.5 2.9
−
−
+
+
+
+
+
+
acetic acid
butyric acid
+
+
formic acid
−
−
−
−
−
−
methacrylic acid
methyl cyanoacetate
pyruvic acid
340.80 0.60
difluoroacetic acid
331.0 2.2
b
Acidities are in kcal mol−1.54,55 A “+” indicates the occurrence and a
a
b
Acidities are in kcal mol−1.54 A “+” indicates the occurrence and a
a
“−” the absence of proton transfer.
“−” the absence of proton transfer.
Table 2. Summary of Results for Proton Affinity Bracketing
of 7-Deazaadenine (Z, 1)
Table 4. Summary of Results for Proton Affinity Bracketing
of 3-Deazaadenine (Z3, 2)
b
b
proton transfer
proton transfer
a
a
ref compd
PA
ref base
conj acid
ref compd
PA
ref base
conj acid
1-methylpiperidine
1-methylpyrrolidine
piperidine
232.1 2.0
230.8 2.0
228.0 2.0
226.6 2.0
225.5 2.0
+
+
−
−
+
2,2,6,6-tetramethylpiperidine
N,N-dimethylcyclohexylamine
triethylamine
235.9 2.0
235.1 2.0
234.7 2.0
234.4 2.0
232.1 2.0
232.0 2.0
230.8 2.0
228.0 2.0
+
+
−
−
−
−
+
+
+
pyrrolidine
−
−
+
di-sec-butylamine
+
3-picoline
+
1-methylpiperidine
−
−
−
−
PAs are in kcal mol−1.54 A “+” indicates the occurrence and a “−”
the absence of proton transfer.
a
b
N,N-dimethylisopropylamine
1-methylpyrrolidine
+
+
piperidine
+
a
b
PAs are in kcal mol−1.54 A “+” indicates the occurrence and a “−”
generally yield accurate values for thermochemical properties of
nucleobases, so we utilized B3LYP/6-31+G(d) to calculate the
relative tautomeric stabilities, acidities (ΔHacid), and proton
affinities (PA) of 7-deazaadenine (Z, 1).18,22,23,29,30 (Through-
out the paper, acidity and basicity are calculated only for those
tautomers within 10 kcal mol−1 of the most stable tautomer.) Z
has eight possible tautomeric structures (Figure 3). The most
stable tautomer 1a is over 10 kcal mol−1 more stable than the
next most stable species. We also calculated the acidity and
basicity of the most stable tautomer. The most acidic site of 1a
is predicted to be N9−H (ΔHacid = 343.5 kcal mol−1). The
most basic site of tautomer 1a is N1 (PA = 228.4 kcal mol−1).
2. Experiments: Z Acidity. We measured the acidity of Z
using acidity bracketing (details in the Methods section). The
conjugate base of Z deprotonates butyric acid (ΔHacid = 346.8
2.0 kcal mol−1); the reaction in the opposite direction
(butyrate with Z) also occurs (Table 1). We therefore bracket
the ΔHacid of Z as 347 3 kcal mol−1.
the absence of proton transfer.
3. Experiments: Z Proton Affinity. In bracketing the PA of Z,
we find that piperidine (PA = 228.0
2.0 kcal mol−1)
deprotonates protonated Z; the opposite reaction (Z
deprotonating protonated piperidine) also occurs (Table 2).
We therefore bracket the PA of Z to be 228 3 kcal mol−1.
3-Deazaadenine (Z3, 2). 1. Calculations: Z3 Tautomers,
Acidity, and Proton Affinity. Z3 has seven possible tautomers,
of which the most stable is the canonical “N9H” structure 2a
(Figure 4). The “N7H” tautomer 2b is predicted to be 4 kcal
mol−1 less stable than 2a. The gas-phase acidity of 2a is 335.3
kcal mol−1 (corresponding to the N9−H). The most basic site
of that tautomer is the N1, with a calculated PA of 233.2 kcal
mol−1.
2. Experiments: Z3 Acidity. The acidity of Z3 was measured
using the bracketing method (Table 3). We find that
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deprotonated Z3 is able to deprotonate 2-chloropropanoic acid
(ΔHacid = 337.0 2.1 kcal mol−1). The reaction in the opposite
direction (2-chloropropanoate with Z3) also occurs; we
therefore bracket the ΔHacid of Z3 to be 337 3 kcal mol−1.
3. Experiments: Z3 Proton Affinity. The results for the
bracketing of the PA of Z3 are in Table 4. We find that di-sec-
butylamine (PA = 234.4
deprotonate protonated Z3, but that the opposite reaction
(protonated di-sec-butylamine with Z3) does not occur. 1-
Methylpiperidine (PA = 224.7
deprotonate protonated Z3, but Z3 can deprotonate proto-
nated 1-methylpiperidine. We therefore bracket the PA of Z3 to
be 233 3 kcal mol−1.
1-Deazaadenine (Z1, 3). 1. Calculations: Z1 Tautomers,
Acidity, and Proton Affinity. Z1 has seven possible tautomeric
structures (Figure 5). The most stable tautomer 3a is over 6
2.0 kcal mol−1) is able to
Figure 6. The three possible tautomeric structures of Q (4). Gas-
phase acidities are in red; gas-phase proton affinities are in blue.
Relative stabilities are in parentheses. Calculations were conducted at
B3LYP/6-31+G(d); reported values are ΔH at 298 K.
2.0 kcal mol−1) cannot
Table 5. Summary of Results for Acidity Bracketing of Q (4)
b
proton transfer
a
ref compd
formic acid
ΔHacid
ref acid
conj base
346.0 0.5
344.1 2.9
343.8 2.1
340.80 0.60
339.2 2.1
−
−
−
+
+
+
methacrylic acid
2,4-pentanedione
methyl cyanoacetate
trifluoro-m-cresol
2-chloropropanoic acid
+
+
+
−
−
337.0 2.1
+
Acidities are in kcal mol−1.54,55 A “+” indicates the occurrence and a
a
b
“−” the absence of proton transfer.
Table 6. Summary of Results for Proton Affinity Bracketing
of Q (4)
b
proton transfer
a
ref compd
PA
ref base
conj acid
1-methylpiperidine
1-methylpyrrolidine
piperidine
232.1 2.0
230.8 2.0
228.0 2.0
226.6 2.0
226.4 2.0
225.5 2.0
222.3 2.0
222.0 2.0
+
+
+
+
+
+
−
−
−
−
+
+
+
+
+
+
Figure 5. The seven possible tautomeric structures of 1-deazaadenine
(Z1, 3). Gas-phase acidities are in red; gas-phase proton affinities are
in blue. Relative stabilities are in parentheses. Calculations were
conducted at B3LYP/6-31+G(d); reported values are ΔH at 298 K.
pyrrolidine
4-picoline
3-picoline
kcal mol−1 more stable than the next most stable species. The
most acidic site of 3a is predicted to be the N9 proton (ΔHacid
= 340.6 kcal mol−1). The most basic site of tautomer 3a is the
N3 (PA = 230.0 kcal mol−1).
pyridine
n-octylamine
PAs are in kcal mol−1.54 A “+” indicates the occurrence and a “−”
the absence of proton transfer
a
b
2. Experiments: Z1 Acidity. Efforts to sublime Z1 into the
gas phase for acidity and proton affinity bracketing were
unsuccessful; we failed to see signal corresponding to the
substrate. We were, however, able to measure the acidity and
proton affinity using an alternative to bracketing: the Cooks
kinetic method (details in the Methods). For the acidity
measurement, five reference acids were used (2-fluorobenzoic
acid, ΔHacid = 338.0 2.2 kcal mol−1; 3-hydroxybenzoic acid,
ΔHacid = 338.6 2.1 kcal mol−1; benzoic acid, ΔHacid = 340.2
stable tautomer 4a is just over 4 kcal mol−1 more stable than
the next most stable structure. The most acidic site of 4a is
N9−H, with ΔHacid = 338.4 kcal mol−1. The most basic site is
N7, with a PA of 223.0 kcal mol−1.
2. Experiments: Q Acidity. The acidity of Q was bracketed as
shown in Table 5. The reaction of deprotonated Q with methyl
cyanoacetate (ΔHacid = 340.80 0.60 kcal mol−1) occurs, as
does the reverse reaction (deprotonated methyl cyanoacetate
with Q). We therefore bracket the acidity of Q as 341 2 kcal
mol−1.
3. Experiments: Q Proton Affinity. The proton affinity of Q
was bracketed as shown in Table 6. The results are somewhat
unusual, in that reaction was found to occur in both directions
for reference bases with PAs from piperidine (228.0 2.0 kcal
mol−1) to 3-picoline (225.5 2.0 kcal mol−1). The implications
of this will be addressed in the Discussion.
“6-Methylated” 1,3,7-Deazaadenine (M, 5). 1. Calcu-
lations: M Tautomers, Acidity, and Proton Affinity. There are
two possible tautomeric structures for M, with the more stable
being so by over 7 kcal mol−1 (Figure 7). The more stable
tautomer 5a has a calculated acidity (at the most acidic site,
2.2 kcal mol−1; phenylacetic acid, ΔHacid = 341.5
2.1 kcal
mol−1; and glycine, ΔHacid = 342.7 2.2 kcal mol−1), yielding
an acidity (ΔHacid) of 341 3 kcal mol−1.
3. Experiments: Z1 Proton Affinity. We were unable to
measure Z1 PA by bracketing (vide supra), but we were able to
successfully utilize the Cooks kinetic method. Using guanine
(PA = 229.3
2.0 kcal mol−1), N-methylpyrrolidine (PA =
230.8 2.0 kcal mol−1), 2,4-lutidine (PA = 230.1 2.0 kcal
mol−1), dimethylisopropylamine (PA = 232.0
2.0 kcal
mol−1), N-methylpiperidine (PA = 232.1 2.0 kcal mol−1), and
triethylamine (PA = 234.7 2.0 kcal mol−1) as reference bases,
we measure a PA for Z1 of 232 3 kcal mol−1.
“6-Methylated” 1-Deazaadenine (Q, 4). 1. Calculations:
Q Tautomers, Acidity, and Proton Affinity. There are three
possible tautomeric structures for Q (Figure 6). The most
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N9−H) of 347.9 kcal mol−1. The PA of the most basic site, C7,
is 215.7 kcal mol−1.
Figure 8. The two possible tautomeric structures of B (6). Gas-phase
acidities are in red; gas-phase proton affinities are in blue. Relative
stabilities are in parentheses. Calculations were conducted at B3LYP/
6-31+G(d); reported values are ΔH at 298 K.
Figure 7. The two possible tautomeric structures of M (5). Gas-phase
acidities are in red; gas-phase proton affinities are in blue. Relative
stabilities are in parentheses. Calculations were conducted at B3LYP/
6-31+G(d); reported values are ΔH at 298 K.
Table 7. Summary of Results for Acidity Bracketing of B (6)
b
proton transfer
a
ref compd
ΔHacid
ref acid
conj base
2. Experiments: M Acidity. Bracketing experiments with M
were hindered by the inability to see any mass spectrometric
signal when solid M was heated. However, we were able to
vaporize (via electrospray) proton-bound dimers of M with
reference acids, allowing us to measure the acidity of M via the
Cooks kinetic method. Four reference acids were used
(resorcinol, ΔHacid = 346.6 2.1 kcal mol−1; propanoic acid,
methacrylic acid
2,4-pentanedione
methyl cyanoacetate
trifluoro-m-cresol
malononitrile
344.1 2.9
343.8 2.1
340.80 0.60
339.2 2.1
−
−
+
+
+
−
−
−
+
335.8 2.1
+
Acidities are in kcal mol−1.54,55 A “+” indicates the occurrence and a
“−” the absence of proton transfer.
a
b
347.4
2.2 kcal mol−1; imidazole, 349.9
0.7; and 3-
aminophenol, 350.5
2.1 kcal mol−1), yielding an acidity
(ΔHacid) of 349 3 kcal mol−1.
Table 8. Summary of Results for Proton Affinity Bracketing
of B (6)
3. Experiments: M Proton Affinity. As noted above,
bracketing experiments with M were unsuccessful. In addition,
measurement of the PA of M by the Cooks kinetic method also
failed, as we were unable to form robust signal for proton-
bound dimers with reference bases.
b
proton transfer
a
ref compd
PA
ref base
conj acid
1-methylpiperidine
1-methylpyrrolidine
piperidine
232.1 2.0
230.8 2.0
228.0 2.0
226.6 2.0
225.5 2.0
+
+
−
−
+
“6-Methylated” 1,3-Deazaadenine (B, 6). 1. Calcula-
tions: B Tautomers, Acidity, and Proton Affinity. B has two
possible tautomers, both of which are relatively close in stability
(Figure 8). The most acidic site of the more stable tautomer 6a
is N9−H (ΔHacid = 339.4 kcal mol−1). The most basic site is
N7, with a calculated PA of 228.2 kcal mol−1.
+
pyrrolidine
−
−
+
3-picoline
+
PAs are in kcal mol−1.54 A “+” indicates the occurrence and a “−”
the absence of proton transfer
a
b
2. Experiments: B Acidity. The acidity of B was bracketed as
shown in Table 7. The reaction of deprotonated B with methyl
of strand scission as a function of time after NaOH quenching
of reaction mixtures. Reaction rate constants (kg) were
measured under conditions of single turnover ([MutY] >
[DNA substrate]) to remove complications associated with
rate-limiting product release. In the case of the OG:Z1-
containing duplex, the measured rate constant at 37 °C is 0.3
0.1 min−1. This represents a 40-fold decrease under the same
conditions for the reaction of MutY with the corresponding
OG:A-containing substrate (kg = 12 2 min−1). For the series
of adenine analogue substrates, the MutY glycosylase activity
(kg), relative to A, is shown in Table 9 (in decreasing order).
The substrates are cleaved by MutY in the order A > Q > Z1 >
Z3 > B ≫ M = Z.
cyanoacetate (ΔHacid = 340.80
0.60 kcal mol−1) occurs;
however, deprotonated methyl cyanoacetate cannot deproto-
nate B. Deprotonated B cannot deprotonate 2,4-pentadione
(ΔHacid = 343.8
2.1 kcal mol−1), but deprotonated 2,4-
pentadione does deprotonate B. We therefore bracket the
acidity of B to be 343 3 kcal mol−1.
3. Experiments: B Proton Affinity. The PA of B was
measured using bracketing (Table 8). We find that the reaction
with piperidine (PA = 228.0 2.0 kcal mol−1) occurs in both
directions (piperidine deprotonates protonated B, and B
deprotonates protonated piperidine). We therefore measure
the PA of B as 228 3 kcal mol−1.
MutY-Catalyzed Base Excision. Most of the unnatural
adenine analogues studied herein (Z, Z3, Q, B, M) have
previously been examined as substrates for MutY in DNA
duplexes base-paired opposite OG, in order to probe enzymatic
mechanistic features.26,51−53 However, the modified Z1 duplex
substrate had not been previously examined and is not
commercially available. The appropriate monomer for auto-
mated DNA synthesis was synthesized and incorporated into a
30-nucleotide strand (Supporting Information). The glyco-
sylase activity of MutY on a 30-bp duplex containing a central
OG:X (X = adenine or adenine analogue) was evaluated as
previously reported.56 Briefly, this involves analyzing the extent
Acid-Catalyzed Depurination of Adenine Analogue-
Containing DNA. Previous work has shown that comparing
MutY rates to the susceptibility for acid-catalyzed depurination
can reveal insight into features of the enzyme-catalyzed rate.
Indeed, previous work with the hydrophobic analogues, B and
Q, showed that these analogues are more susceptible to
depurination than A.26 The relative acid-catalyzed depurination
of Z1 and Z3 relative to A in the 30-nt oligonucleotide was
evaluated using a modified Maxam−Gilbert G+A reaction
(Supporting Information). The extent of depurination was
quantitated using autoradiography (Figure S1, Supporting
Information), and the relative extents of depurination of Z1,
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Table 9. MutY Excision of Nucleobase Analogues Opposite
OG within 30 bp DNA Substrates
Table 11. Calculated (B3LYP/6-31+G(d); 298 K) and
Experimental Data
b
nucleobase
analogue
fold reduced relative
to OG:A substrate
substrate
calcd value
exptl value
a
kg (min−1
)
a
ΔHacid
Z (1)
343.5
335.3
340.6
338.4
347.9
339.4
347
c
A
12
2
−
10×
40×
100×
6000×
>24 000×
>24 000×
Z3 (2)
Z1 (3)
Q (4)
M (5)
B (6)
337
c
Q (4)
Z1 (3)
Z3 (2)
B (6)
M (5)
Z (1)
1.2 0.2
(341)
341
0.31 0.06
0.10 0.05
b
(349)
343
c
<0.002
de
,
c
c
NC
NC
adenine
334.8
333 (335)
e
a
a
Rates were measured at 37 °C. Errors represent standard deviation
PA
Z (1)
228.4
233.2
230.0
223.0
215.7
228.2
228
from the average. Value previously reported.51 Value previously
b
c
Z3 (2)
Z1 (3)
Q (4)
M (5)
B (6)
233
reported.26 Not cleaved above detection limit (<0.0005 min−1).
d
(232)
e
f
Value previously reported.52 Value previously reported.53
225.5−228.0
N/A
Z3, and A were normalized to depurination of another A
(position 11) within the DNA sequence (Figure S2, Supporting
Information). These results show that A, Z1, and Z3 at position
15 are depurinated at fractions of 0.9, 6.3, and 0.3 relative to
A11 in the same DNA sequence. Interestingly, this shows that,
in contrast to the enzyme-catalyzed excision rates, in acidic
water Z1 is depurinated 7 times more than A, whereas
depurination of Z3 is 3 times less efficient (Table 10). The
trend for acid-catalyzed depurination is Q = Z1 > B >A > Z3.
228
c
c
adenine
223.7
224 (225)
a
b
ΔHacid and PA values are in kcal mol−1
.
Non-parenthetical
experimental value is from bracketing measurement; Cooks kinetic
c
method value is in parentheses. Error is 3−4 kcal mol−1. Values
previously reported.21,22,28.
stable than 4a but perhaps not by as much as the calculations
indicate, such that there is some 4b present in our experiments.
For the PA bracketing experiment, the solid 4 is sublimed
into the gas phase via a solids probe (typical pressure in the
instrument is 10−7−10−8 Torr, and the probe is heated to a
temperature of roughly 400 K). If only 4a were present, one
would expect a bracketed PA of around 223 kcal mol−1, based
on the calculations (Figure 6, Table 11). The bracketing table
would have a “crossover point” near pyridine (Table 12).
Table 10. Relative Cleavage of Nucleobase Analogues in
Acidic Aqueous Solution
extent of acid-catalyzed depurination
nucleobase
(normalized to A)
A
1
a
Z3
Z1
B
0.3
a
7
b
Table 12. Hypothetical Bracketing Table if Only 4a Were
Present
2
b
Q
7
a
b
This work. Values reported previously.26
proton transfer
ref compd
PA
ref base
conj acid
1-methylpiperidine
1-methylpyrrolidine
piperidine
232.1 2.0
230.8 2.0
228.0 2.0
226.6 2.0
226.4 2.0
225.5 2.0
222.3 2.0
222.0 2.0
+
+
+
+
+
+
−
−
−
−
−
−
−
−
+
DISCUSSION
■
Calculated versus Experimental Values. The calculated
pyrrolidine
acidity and proton affinity values for all the substrates studied
herein are summarized in Table 11. (The acidity and proton
affinity of adenine were previously measured and calculated by
us and also appear in the table.) Generally, B3LYP/6-31+G(d)
appears to provide fairly accurate predictions for the
thermochemical values. The one instance where the calculated
and experimental data are quite disparate is for the proton
affinity of Q: the calculated value for the most stable tautomer
is 223.0 kcal mol−1, yet the bracketing experiment yields a wide
range where proton transfer occurs in both directions (PAs
from 225.5 to 228.0 kcal mol−1, Table 6). This is a fairly
significant discrepancy.
The wide range of proton transfer in both directions for the
PA bracketing of Q (Table 6) raises the possibility that, under
our conditions, we have a mixture of the two most stable Q
tautomers (4a and 4b), and the more basic 4b (calculated PA
of 232.6 kcal mol−1) influences the experimentally observed
value. Although the B3LYP/6-31+G(d) calculations indicate
that 4b is nearly 5 kcal mol−1 less stable than 4a, prior studies
show that accurate calculations of nucleobase tautomer
stabilities can be elusive.57−61 It is possible that 4b is less
4-picoline
3-picoline
pyridine
n-octylamine
+
If only 4b were present, one would expect a similar table,
except the “crossover point” would be near the PA of the most
basic site of 4b, which is calculated to be 232.6 kcal mol−1
(Figure 6). Instead, as can be seen in Table 6, there is not a
clean crossover point, but rather a range in which the
deprotonation reaction occurs in both directions. We suspect
that the reason for this is that both tautomers 4a and 4b are
present.
In the reactions of a reference base with protonated 4, if both
tautomers were present, then the protonated substrate should
be a mixture of 4aH+ and 4bH+ (Figure 9). Under these
conditions, any reference base with a proton affinity greater
than or equal to 223.0 kcal mol−1 should deprotonate 4a.
Consistent with this expectation, we do observe proton transfer
for all the reference bases from 3-picoline (PA = 225.5 kcal
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Figure 11. Structures of 4a and 4b and calculated acidity of the most
acidic site. Relative stabilities of the two neutral tautomers are shown
in parentheses. Calculations were conducted at B3LYP/6-31+G(d)
[ΔH at 298 K in kcal mol−1].
acid with ΔHacid ≈ 338 kcal mol−1 or less. Experimentally, we
do see proton transfer “turn on” in this region, starting with
methyl cyanoacetate (ΔHacid = 340.8 kcal mol−1, Table 5, “ref
acid” column).
Figure 9. Structures of 4a, 4b, 4aH+, and 4bH+ and calculated proton
affinities. Relative stabilities of the two neutral tautomers are shown in
parentheses. Calculations were conducted at B3LYP/6-31+G(d) [ΔH
at 298 K in kcal mol−1].
In the opposite direction, deprotonated reference acids
would be allowed to react with 4, which is presumably a
mixture of 4a and 4b (Figure 11). In this direction, one would
expect to see reaction with any deprotonated reference acid
whose ΔHacid is 334 kcal mol−1 or higher (since 4b is present).
Instead, however, we do not see proton transfer “turn on” until
340.8 kcal mol−1 (Table 5, “conj base” column). We believe
that two factors are at play: one, as we saw with the PA
experiments, we have less 4b present, so reactions with 4b will
appear slower than they are. Second, we cannot preclude base-
catalyzed tautomerization of 4b to 4a taking place during the
bracketing experiment (Figure 12; brackets indicate ion−
molecule complexes; A− is the deprotonated reference acid). In
Figure 12, we show the reaction of a deprotonated reference
mol−1) to 1-methylpiperidine (PA = 232.1 kcal mol−1, Table 6,
“ref base” column).
In the opposite direction, the protonated reference bases are
allowed to react with 4. If both 4a and 4b are present, we would
expect reaction with any protonated reference base with a PA of
about 232 kcal mol−1 or less, because 4b has a calculated PA of
232.6 kcal mol−1 (Figure 9). We actually see proton transfer
“turn on” at a slightly lower value, 228.0 kcal mol−1 (at
piperidine; Table 6, “conj acid” column). Still, this value is
much higher than the calculated PA of 4a (223.0 kcal mol−1),
pointing to the probable presence of 4b.
We suspect that the proton transfer “turning on” at a slightly
lower value than calculated may be due to a mixture in which
4a predominates, with less of 4b. In this bracketing experiment,
in order to ascertain whether proton transfer occurs, we
measured the kinetics of the proton transfer. We tracked the
disappearance of the protonated reference base signal under
pseudo-first-order conditions (excess of 4). We can measure
the pressure of 4, but do not know what percentage of this
pressure corresponds to 4b versus 4a. Therefore, for those
protonated reference bases that only react with 4b, we measure
a rate constant for proton transfer that is less than the actual
rate constant, because we can only measure the overall pressure
of 4, but only 4b reacts. Our PA bracketing results are therefore
consistent with a mixture of 4a and 4b, with 4a predominating.
We should also address the bracketed acidity measurement of
Q. Tautomer 4a has a calculated acidity of 338.4 kcal mol−1;
tautomer 4b, 333.8 kcal mol−1. The measured value is 341 kcal
mol−1, which implies the presence of tautomer 4a but not 4b.
However, as we discuss above, we believe both tautomers are
present. So why do we measure an acidity consistent with 4a
only?
acid A− with 4b. In this Figure, the reference acid has ΔHacid
=
336 kcal mol−1, which is a higher value than the ΔHacid of 4b, so
proton transfer occurs to form deprotonated 4. However, if a
subsequent proton transfer takes place (whereby the N9− of
deprotonated 4, whose conjugate acid has an acidity of 338.4
kcal mol−1, deprotonates AH), then 4a and A− are formed as
products (Figure 12). Proton transfer between 4b and A− has
occurred, but since we only track the m/z ratio of A−, we would
have no way of knowing that proton transfer occurred. The
exothermic scenario shown in Figure 12 would look, by mass
spectrometry, as if no proton transfer has taken place: one
would only see A− signal when following the reaction progress.
This would therefore be marked as a “−” in the last column of
Table 5, even though proton transfer has occurred. Essentially,
therefore, the “−” entries in the rightmost column of Table 5
may actually be incorrect. Thus, the bracketed ΔHacid value of
341 kcal mol−1 does not necessarily mean that 4b is not
present.
Given the wide range of proton transfer in both directions for
the PA experiment (Table 6) and the ambiguity associated with
knowing whether proton transfer occurred in the acidity
experiment, we therefore believe that we most likely have a
mixture of 4a and 4b present under our experimental
conditions, with 4a predominating.
The deprotonation of both 4a and 4b results in the same
anion, which is allowed to react with reference acids (Figure
10). This anion should be able to deprotonate any reference
Biological Implications. MutY is a glycosylase that cleaves
adenine when it is base-paired to OG. The unnatural substrates
studied herein (Z, Z3, Z1, Q, B, M) were synthesized and
examined as substrates for MutY, in order to probe enzymatic
mechanistic features (most in prior work).26,51−53 The rates of
excision, relative to A, are shown in Table 9 (in decreasing
order). The substrates are cleaved by MutY in the order A > Q
> Z1 > Z3 > B ≫ M = Z.
Figure 10. Structure of deprotonated 4a and 4b and calculated
acidities. Calculations were conducted at B3LYP/6-31+G(d) [ΔH at
298 K in kcal mol−1].
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Figure 12. Reaction coordinate for the base-catalyzed tautomerization of 4b to 4a. “A−” represents deprotonated reference acid. Values in red are
B3LYP/6-31+G(d) calculated ΔHacid values (298 K).
Scheme 1
As a key step in initiating DNA repair, BER glycosylases,
such as MutY, cleave the N-glycosidic bond (Figure 1) to
release the damaged or inappropriate base. MutY is a
monofunctional glycosylase that hydrolyzes the N-glycosidic
bond on 2′-deoxyadenosine to yield an abasic site−DNA
product and free adenine base. We are interested in whether
the intrinsic properties that we study herein can lend insight
into the features used by MutY to catalyze N-glycosidic bond
breakage. Presumably, the better a leaving group the nucleobase
is, the more easily it is cleaved. Since acidity and leaving group
ability are generally correlated, we would expect more easily
cleaved bases to be more acidic. By examining the intrinsic
N9−H acidity for various possible mechanisms, we can lend
insight into the operative mechanism. Furthermore, based on
our studies with other glycosylases, we postulate that MutY
may provide a nonpolar active site that serves to enhance the
differences in acidity among nucleobases, and in doing so, aids
in the discrimination of substrate bases over nonsubstrate
bases.18,19,21,23,62 Thus, studies in the gas phase, which is the
ultimate nonpolar environment, are relevant.
Mechanistic studies of MutY, including crystal structure, KIE
studies, and computer (molecular dynamics and QM/MM)
simulations point to protonation at N7 to facilitate
cleavage.14−17,63 A recent crystal structure of Bacillus
stearothermophilus MutY bound to a DNA duplex containing
a fluorinated 2′-deoxyadenosine paired with OG shows multiple
hydrogen bonding contacts as well as hydrophobic interactions
between substrate and enzyme.14,16,63 Glu-43 and Tyr-126
coordinately contact N7; the position of the glutamate indicates
that it is probably protonated (the carboxylic acid as opposed to
the carboxylate). Glu-43 is expected to be quite acidic, allowing
partial or full bonding of its proton to N7. A hydrogen bond
from Arg-26 to water to N3 is observed, as well as a hydrogen
bond from Arg-31 to N1. Various salt bridges exclude water
and create a hydrophobic environment.14,16,26,51,52
Acidity: N9−H Acidity of Neutral Nucleobase Ana-
logues. First we considered a mechanism where the N-
glycosidic bond is simply cleaved without any pre-protonation;
in such a scenario, the leaving group is a deprotonated anion
(Mechanism A, Scheme 1). In this case, the acidities of the
neutral substrates will correlate to their leaving group abilities.
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The calculated (B3LYP/6-31+G(d)) values for the N9−H
acidity for the neutral nucleobase analogues are shown in
Figure 13. Lower values are more acidic, so adenine is the most
Q > B > Z1 ≫ Z > M. (The acidities of Z and M without
protonation are shown since they lack an N7.)
The acidity trend, at first glance, does not correlate to the
known excision rates for these substrates by MutY (Table 9, A
> Q > Z1 > Z3 > B > M = Z). However, there are some
consistencies. M and Z, because of the inability to protonate at
N7, are the least acidic by far and would not be expected to be
prone to cleavage, which is what is observed experimentally. Q
and Z1 are more easily cleaved than would be predicted by the
acidities in Figure 14, but both have one feature in common: a
nitrogen at N3, which has been proposed to be important for
MutY excision.14,16,26,51
In order to model the effect of acidity by hydrogen bonding
at N3, we used HF as a simple hydrogen-bonding donor.70 The
hydrogen bond at N3 increases the acidity at N9−H of the N7-
protonated substrates by roughly 2−3 kcal mol−1. In Figure 15,
Figure 13. Calculated (B3LYP/6-31+G(d)) gas-phase acidities (ΔH,
kcal mol−1) of N9−H for neutral adenine analogues. Substrates are
ordered in decreasing acidity (increasing ΔHacid value).
acidic substrate (ΔHacid = 334.8 kcal mol−1).21,22,61,64−69 The
next most acidic substrate is Z3 (ΔHacid = 335.3 kcal mol−1).
The trend from most to least acidic in the gas phase is A > Z3 >
Q > B > Z1 > Z > M (where A is most acidic). This trend does
not agree with the known excision rates for these substrates by
MutY (Table 9), which is A > Q > Z1 > Z3 > B ≫ M = Z.
Furthermore, the acidity values for Z and M do not seem high
enough (relative to the other nucleobases) to explain the
experimentally observed lack of excision. Thus, a mechanism
where the nucleobases are simply cleaved as anions seems
unlikely, at least based on the intrinsic acidity of these
substrates. This is not surprising, since most experimental
evidence points to pre-protonation at N7 (Mechanism B,
Scheme 1).
Acidity: N9−H Acidity of N7-Protonated Substrates.
The N9−H acidity values for the N7-protonated nucleobase
analogues are shown in Figure 14 (listed in order of increasing
acidity). N7-Protonation greatly increases the N9−H acidity
(by more than 100 kcal mol−1). The acidity trend is A ∼ Z3 >
Figure 15. Calculated (B3LYP/6-31+G(d)) gas-phase acidities (kcal
mol−1) of N9−H for N7-protonated, N3-hydrogen-bonded adenine
analogues. Substrates are ordered in decreasing acidity (increasing
ΔHacid values).
we show the gas-phase acidities of the substrates protonated at
N7 and hydrogen-bonded to N3 (when N is present at the 7
and 3 positions). The resultant acidity trend is A > Z3 ∼ Q >
Z1 > B ≫ Z > M.
This trend compares quite favorably to the actual MutY
excision rates (Table 9, A > Q > Z1 > Z3 > B > M = Z). The
only substrate “out of place” is Z3, whose gas-phase acidity
when protonated at N7 is quite high and implies that Z3 should
be prone to cleavage. The reduced ability of MutY to remove
Z3 is somewhat of a mystery from the experimental point of
view as well; in earlier work one of us postulated that the lack of
a N at the 3-position might render N7 more difficult to
protonate, which could reduce excision rate. Our gas-phase PA
calculations do indicate that Z3 is less basic at N7 than Q and
Z1 (Figure 16), so this may explain the observed excision
trend.26,51−53
Figure 14. Calculated (B3LYP/6-31+G(d)) gas-phase acidities (kcal
mol−1) of N9−H for N7-protonated nucleobase analogues. (Z and M
have no N7 to protonate; acidities are shown for comparison.)
Substrates are ordered in decreasing acidity (increasing ΔHacid values).
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be more quickly cleaved than Z1, but experimentally the
opposite is observed. However, the overall trend of which bases
should be cleaved more quickly than A and which less quickly is
consistent between calculations and experiment, supporting a
mechanism in which acid pre-protonates the most basic site
prior to cleavage.
1,3-Deazaadenine (Z13) Prediction. One additional
substrate we studied computationally is Z13 (Figure 18).
Figure 16. Calculated (B3LYP/6-31+G(d)) gas-phase PAs (ΔH, kcal
mol−1) of N7 for adenine and analogues. Substrates are ordered in
increasing PA.
Figure 18. Calculated (B3LYP/6-31+G(d)) gas-phase and (aqueous)
acidity (kcal mol−1) of N9−H for N7-protonated nucleobase analogue
Z13.
Thus, the gas-phase N9−H acidities of these substrates can
be compared to known excision rates to lend insight into the
MutY mechanism. The gas-phase acidities of the substrates
track with excision rates when N7 is protonated and a hydrogen
bond is formed to N3, implying that these are features that
MutY may provide to enable nucleobase cleavage.
Acid-Catalyzed Non-enzymatic Depurination. We have
also studied the nonenzymatic excision of some of these
nucleobase analogues in acidic aqueous solution and find a
different trend than that for MutY-catalyzed excision. B, Q, and
Z1 are depurinated more quickly than A while Z3 is
depurinated more slowly (Q = Z1 > B > A > Z3, Table
10).26 Because this excision trend is different from that
catalyzed by MutY, we postulate that the mechanism in acidic
aqueous solution is different. Presumably, under acidic
conditions, the most basic site of a given nucleobase analogue
is protonated, which favors cleavage of the nucleobase. Since
the most basic site is not always N7, the acidic aqueous
mechanism is different from that of MutY.
This is a logical extension of the various analogues already
studied (Figure 2); this particular derivative is missing an N at
both the 1 and 3 positions. Z13 could be a substrate for MutY,
although the only nitrogen available for hydrogen bonding and/
or protonation is N7. Compared to the other nucleobase
analogues, the gas-phase acidity at N9−H when that N7 is
protonated is fairly poor (ΔHacid = 230.1 kcal mol−1). Given
our hypothesis that N7 is protonated and N3 is hydrogen
bonded, the acidity of N9−H of Z13 is even less than that of B
(Figure 15). We would therefore expect Z13 to be cleaved by
MutY quite slowly, even more slowly than B (but still faster
than Z or M).
We also calculated the N9−H acidity for the N7-protonated
Z13 in a water dielectric, to predict the ease of acid-catalyzed
non-enzymatic depurination. That acidity is 23.6 kcal mol−1,
which is more acidic (by 4.6 kcal mol−1) than adenine. We
would therefore expect Z13, like Q, B, and Z1, to be cleaved
more quickly than adenine under acidic aqueous conditions.
To mimic these conditions, relevant calculations should
involve pre-protonation of the most basic site and a polar
environment. To that end, we calculated the N9−H acidity for
the nucleobase analogues when the most basic site is
protonated, in a water dielectric (Figure 17). The acidity
CONCLUSIONS
■
The heretofore unknown thermochemical properties of adenine
and six adenine analogues have been calculated and measured
herein. Gas-phase measurements benchmark our calculations.
Comparison of the stability of the N9−H bond (in terms of
acidity) versus known MutY excision rates point to a MutY-
catalyzed mechanism involving protonation at N7, with
hydrogen bonding at N3. This conclusion is consistent with
other MutY mechanistic studies (crystal structures, kinetic
isotope effects). We also find that our calculations for the N9−
H acidity when the most basic site is protonated are consistent
with experimental data for acid-catalyzed depurination in water.
Our work shows that fundamental studies of biological species
are valuable for lending insight into mechanisms for which
these species are substrates.
Figure 17. Aqueous N9−H acidities (kcal mol−1) of nucleobase
analogues with the most basic site protonated.
values are much lower than those in the gas phase, as would be
expected in water. These calculations indicate an N9−H acidity
trend of Q > B > Z1 > A > Z3. We would therefore expect Q,
B, and Z1 to be cleaved more quickly than adenine, and Z3 to
be cleaved more slowly, which is consistent with the
experimental results (Table 10). The calculations, which do
not include specific solvation, are not perfect; B is predicted to
ASSOCIATED CONTENT
■
S
* Supporting Information
Cartesian coordinates for all calculated species, experimental
details, acid-catalyzed depurination assays (Figures S1 and S2),
and complete refs 44 and 45. This information is available free
of charge via the Internet at http://pubs.acs.org.
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(34) McLuckey, S. A.; Cameron, D.; Cooks, R. G. J. Am. Chem. Soc.
1981, 103, 1313−1317.
AUTHOR INFORMATION
■
Corresponding Author
ssdavid@ucdavis.edu; jee.lee@rutgers.edu
(35) McLuckey, S. A.; Cooks, R. G.; Fulford, J. E. Int. J. Mass
Spectrom. Ion Phys. 1983, 52, 165−174.
(36) Green-Church, K. B.; Limbach, P. A. J. Am. Soc. Mass Spectrom.
2000, 11, 24−32.
Notes
The authors declare no competing financial interest.
(37) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse,
C. M. Science 1989, 246, 64−71.
ACKNOWLEDGMENTS
́
(38) Drahos, L.; Vekey, K. J. Mass Spectrom. 1999, 34, 79−84.
■
(39) Ervin, K. M. Chem. Rev. 2001, 101, 391−444.
(40) Gronert, S.; Feng, W. Y.; Chew, F.; Wu, W. Int. J. Mass Spectrom.
2000, 195/196, 251−258.
We gratefully thank the NSF, ACS-PRF, NCI/NIH
(CA67985), and the National Center for Supercomputer
Applications for support. Also, thanks to Mu Chen for valuable
discussions and help with the work.
(41) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652.
(42) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785−789.
(43) Kohn, W.; Becke, A. D.; Parr, R. G. J. Chem. Phys. 1996, 100,
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