Substituents on quinone methides strongly modulate formation and stability of their nucleophilic adducts

Article abstract of DOI:10.1021/ja062948k

Electronic perturbation of quinone methides (QM) greatly influences their stability and in turn alters the kinetics and product profile of QM reaction with deoxynucleosides. Consistent with the electron-deficient nature of this reactive intermediate, electron-donating substituents are stabilizing and electron-withdrawing substituents are destabilizing. For example, a dC N3-QM adduct is made stable over the course of observation (7 days) by the presence of an electron-withdrawing ester group that inhibits QM regeneration. Conversely, a related adduct with an electron-donating methyl group is very labile and regenerates its QM with a half-life of approximately 5 h. The generality of these effects is demonstrated with a series of alternative quinone methide precursors (QMP) containing a variety of substituents attached at different positions with respect to the exocyclic methylene. The rates of nucleophilic addition to substituted QMs measured by laser flash photolysis similarly span 5 orders of magnitude with electron-rich species reacting most slowly and electron-deficient species reacting most quickly. The reversibility of QM reaction can now be predictably adjusted for any desired application.

Full text of DOI:10.1021/ja062948k

Published on Web 08/17/2006
Substituents on Quinone Methides Strongly Modulate
Formation and Stability of Their Nucleophilic Adducts
Emily E. Weinert,† Ruggero Dondi,‡ Stefano Colloredo-Melz,‡
Kristen N. Frankenfield,† Charles H. Mitchell,† Mauro Freccero,*,‡ and
Steven E. Rokita*,†
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Maryland,
College Park, Maryland 20742, and Dipartimento di Chimica Organica, UniVersita` di PaVia,
Via le Taramelli 10, 27100 PaVia, Italy
Received April 27, 2006; E-mail: mauro.freccero@unipv.it; rokita@umd.edu
Abstract: Electronic perturbation of quinone methides (QM) greatly influences their stability and in turn
alters the kinetics and product profile of QM reaction with deoxynucleosides. Consistent with the electron-
deficient nature of this reactive intermediate, electron-donating substituents are stabilizing and electron-
withdrawing substituents are destabilizing. For example, a dC N3-QM adduct is made stable over the course
of observation (7 days) by the presence of an electron-withdrawing ester group that inhibits QM regeneration.
Conversely, a related adduct with an electron-donating methyl group is very labile and regenerates its QM
with a half-life of approximately 5 h. The generality of these effects is demonstrated with a series of alternative
quinone methide precursors (QMP) containing a variety of substituents attached at different positions with
respect to the exocyclic methylene. The rates of nucleophilic addition to substituted QMs measured by
laser flash photolysis similarly span 5 orders of magnitude with electron-rich species reacting most slowly
and electron-deficient species reacting most quickly. The reversibility of QM reaction can now be predictably
adjusted for any desired application.
Scheme 1. Repair and Reformation of a DNA Adduct
Introduction
Developing reagents for selective reaction with cellular
components is often dismissed due to an unnecessary assumption
that covalent processes are limited to irreversible chemistry.
Reversible covalent reaction has the potential to overcome
kinetic traps that limit both potency and selectivity. Reactive
species may be regenerated repeatedly in a reversible process
to escape trapping with nontargeted materials and even over-
come cellular repair processes. The ability of the natural product
ecteinascidin 743 (Et-743) to bind covalently to duplex DNA
and yet still migrate between various nucleotide sequences, for
example, also allows Et-743 to reassociate with DNA after its
initial adduct has been removed by excision repair (Scheme 1).1
In contrast, reagents such as nitrogen mustards react irreversibly
with strong nucleophiles in DNA, most notably the N7 of
guanine (Scheme 2) and other cellular compounds.2-4 The most
significant effect of the mustards, DNA cross-linking, only
proceeds if a second nucleophile is proximal to the initial site
of attachment. However, this second nucleophile is not typically
present and reaction is quenched since the mustard cannot
migrate to a site in DNA that would support cross-linking.
Scheme 2. Nucleobase Structure
† University of Maryland.
‡ Universita` di Pavia.
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2971-2987.
Reversible alkylating agents may also react with a range of
cellular components, but after multiple regenerations they may
ultimately associate with their thermodynamically favorable
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10531-10549.
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9
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J. AM. CHEM. SOC. 2006, 128, 11940-11947
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Quinone Methide Sensitivity to Electronic Modulation
A R T I C L E S
Scheme 3. Reversible and Irreversible Formation of QM Adducts
targets (e.g., Et-743 with duplex DNA, Scheme 1). Reversibility
then extends the effective lifetime of electrophiles and conse-
quently enhances their potential for selective biological activity.
Such activity has been investigated with carcinogens such as
malondialdehyde5-8 and a variety of anticancer drug candidates
based on cyclopropylpyrroloindole.9,10 For two closely related
drug candidates, duocarmycin SA (duoSA) is 20-fold more
cytotoxic than duocarmycin A (duoA). This result is likely a
function of the slower reaction of duoSA vs that of duoA with
nucleophiles and its faster release from adducts formed by
nucleophilic coupling.11,12 A direct correlation between revers-
ibility of DNA adducts and cytotoxicity was also extended to a
number of additional cyclopropylpyrroloindole derivatives as
well.13
Our laboratories and others have applied the reversibility of
quinone methide (QM) generation for efficient cross-linking and
target-promoted alkylation of DNA.14-18 Related QMs had
previously been implicated in biological activation of 2,6-di-
tert-butyl-4-methylphenol,19 tamoxifen,20 and a variety of natural
products including mitomycin21 and certain diterpenone cat-
echols.22 Initial model studies suggested a selectivity of QM
for weak nucleophiles and an ability of some adducts to form
reversibly.19,23-26 Further investigation revealed that this unusual
selectivity was a function of QM adduct stability rather than
initial product formation.27-29 In contrast to earlier assumptions,
the strongest nucleophiles of DNA reacted most quickly with a
simple o-QM model to form kinetic products. However, these
products are not stable and regenerate the o-QM for ultimate
transfer to weaker nucleophiles that react irreversibly and form
thermodynamic products (Scheme 3).27,30
Computational studies on this same o-QM model anticipated
the experimental results with remarkable accuracy.28,29 A low
value for ∆Gq was calculated for both adduct formation and
QM regeneration with strong nucleophiles such as dC N3, dA
N1, and dG N7 (Scheme 2), whereas ∆Gq values for addition
of the exo-amino groups (dG N2 and dA N6) to o-QM were
considerably higher and essentially irreversible. The free energy
of activation should also be sensitive to variations in the
electronic properties of the intermediate QMs. A relatively
electron-rich derivative may be more stable and hence more
readily generated than its electron-poor counterpart. Hence,
electron-withdrawing and -donating groups attached directly on
the QM should influence the rate of QM formation and adduct
stability. A series of such derivatives has now been prepared to
test this possibility and determine the sensitivity of the QMs to
a variety of substituents. A method for predictably manipulating
QM stability will be indispensable for optimizing the kinetics
and selectivity of target-promoted alkylation of DNA15 and will
additionally facilitate use of QMs for mechanism-based in-
activation of enzymes31 and drug release by self-immolative,
or cascade-release, dendrimers.32-34 Quinone methides are also
used as intermediates in organic synthesis35 and can be stabilized
by metal coordination.36-39
Results and Discussion
A limited number of studies previously began to explore
substituent effects on QM reactivity,40-43 but no data was
available to estimate structural effects influencing the kinetics
or product distribution of the deoxynucleoside reaction. Electron-
donating and -withdrawing groups have now been placed para
and meta to the QM methylene to avoid complications from
steric effects. The products of reversible reaction were expected
to be most susceptible to changes in the QM stability since their
presence depends on the rates of both nucleophilic addition to
the QM as well as subsequent elimination to regenerate the QM.
In contrast, the products formed irreversibly should depend on
only the relative strength of their nucleophilic components to
compete for initial addition to the QM.
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Formation and Decomposition of dC Adducts Generated
from QMs of Varying Reactivity. Our initial investigation to
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X.; Zhang, X. L.; Zhou, X.; Weng, L. J. Am. Chem. Soc. 2003, 125, 1116-
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Chem. Soc. 2004, 126, 13973-13979.
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Toxicol. 1996, 9, 1368-1374.
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(21) Tomasz, M. Chem. Biol. 1995, 2, 575-579.
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Chem. Soc. 1999, 121, 6773-6779.
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Soc. 2001, 123, 11126-11132.
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125, 3544-3553.
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A R T I C L E S
Weinert et al.
Scheme 4. QM Generation from Silyl-Protected Precursors
Table 1. Benzylic Proton Shifts of Substituted QM Adducts (ppm)
adducts
formed by
dC N3
dA N1
dA N6
dG N1
dG N2
guanine N7
QM1
QM2
QM3
4.96
5.03
5.11
5.26
ND
5.16
4.76
4.72
4.69
5.28
5.24
5.09
4.58
4.33
4.43
5.49
5.30
5.40
determine the extent to which QM reactivity can be controlled
by aromatic substituents was performed with dC since this
deoxynucleoside forms a single adduct upon incubation with
QM1.44 The quinone methide precursor (QMP1) originally used
to generate the unsubstituted QM1 was also modified alterna-
tively with a methyl (QMP2) and methyl ester (QMP3) group
to examine the effect of electron-donating and -withdrawing
groups, respectively (Scheme 4). As described previously,
fluoride was used to initiate deprotection of the QMPs.44
Subsequent elimination of acetate generated the QMs for dC
alkylation. Both QM2 and QM3 also generated only a single
adduct with dC under the same conditions used previously to
form the dC N3 adduct of QM1.44 1H NMR data for these new
derivatives are consistent in each case with linkage through the
N3 of dC. The chemical shifts of the benzylic methylene protons
are quite characteristic of QM adduct structure25,26 and benzylic
signals for the dC N3-QM adducts of QM1, QM2, and QM3
differ by no more than approximately 0.1 ppm (Table 1).
Kinetic measurement of dC addition to the QMs was
performed under conditions identical to those first used to follow
reaction of QM1.30 The maximum yield of adduct formed in
each case was similar, but their rates of formation and stability
varied greatly (Figure 1). The dC N3 adduct of the electron-
rich QM2 formed most rapidly, and a maximum yield was
achieved in less than 1 h. Decomposition of this adduct was
also very rapid with a half-life of approximately 5 h and resulted
in release of dC and presumably the quenching of QM2 with
water. In contrast, the unsubstituted QMP1 examined previously
generated a maximum yield of dC N3-QM1 adduct after 4-10
h. This product exhibited moderate stability under the reaction
conditions and decomposed with a half-life of approximately
24 h. Finally, the electron-poor QM3 formed very slowly over
40 h, but the dC N3 adduct that resulted from its reaction
persisted without change over a minimum of 72 h. No
decomposition of the dC N3-QM3 adduct was even detected
after an extended incubation of 7 days.
Figure 1. Formation and decomposition of quinone methide adducts of
dC N3. Reaction conditions and product analysis are described in the
Experimental Procedures. Each point represents the average of at least three
independent determinations and was fit to exponential processes for
highlighting the net trends of the data. The indicated error derives from the
standard deviations.
fold more rapidly than QM3 that contains an electron-
withdrawing group. This same trend is apparent for QM
regeneration from each of the dC N3 adducts. Thus, small
changes in the electronics of the QMs affect their reactivity and
adduct stability dramatically and now also predictably.
Deoxynucleoside Competition with an Electron-Rich QM.
The consequences of QM stability on the kinetics of forming
all deoxynucleoside adducts was next examined with a competi-
tive equimolar mixture of dA, dG, dC, and T in analogy to
earlier studies with the unsubstituted QM1.30 Product standards
were first isolated after reaction with individual deoxynucleo-
1
sides, and each product was characterized by H NMR, high-
resolution mass spectrometry (HRMS), and UV absorbance.
Once again, the chemical shift of the benzylic protons was
diagnostic of the linkage between the QMs and deoxynucleo-
sides (Table 1). Characteristic changes in the UV absorbance
maxima further confirmed the assignments (Supporting Infor-
mation). Reaction of QM2 with dA yielded two adducts linked
through its N1 and N6 positions. Only the N6 adduct was stable
enough for isolation and characterization. However, the UV
maximum of the second product was recorded during HPLC
analysis and is consistent with an N1 adduct (Supporting
Information). Three products were generated by QM2 and dG
and identified as adducts of its N1, N2, and N7 positions. Both
the N1 and N2 adducts were characterized as their dG deriva-
tives. The N7 adduct of dG was evident during chromatographic
separation but spontaneously deglycosylated to form its guanine
derivative. This secondary product was stable and subject to
standard analysis. Similar results had previously been observed
with reaction of dG and QM1.26,30 No adducts of T were
observed under the range of conditions examined in this work.
Monitoring the competitive reaction of dA, dC, dG, and T
with QMP2 over time resulted in the same trends observed
previously with QMP1 except the time frame of the evolving
products was significantly compressed (Figure 2). The initial 2
h were dominated by reaction of the strong nucleophiles, dA
N1, and dC N3. However, the resulting adducts decomposed
with half-lives of less than 4 h. The weaker nucleophiles in
The rate of adduct formation primarily reflects the rate of
initial QM formation by elimination of the benzylic acetate after
rapid desilylation. Electron donation of a single methyl sub-
stituent stabilizes QM2 sufficiently to promote its formation
by at least 4-fold relative to formation of QM1 (Figure 1 and
Supporting Information). Similarly, QM1 forms perhaps 10-
(44) Rokita, S. E.; Yang, J.; Pande, P.; Greenberg, W. A. J. Org. Chem. 1997,
62, 3010-3012.
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Quinone Methide Sensitivity to Electronic Modulation
A R T I C L E S
Figure 3. Time-dependent profile of adducts formed by the electron-poor
QM3 and deoxynucleosides. Reaction conditions and product analysis are
identical to those used for Figure 1. Again, each point represents the average
of at least three independent determinations and was fit to exponential
processes for highlighting the net trends of the data. The indicated error
derives from the standard deviations.
Figure 2. Time-dependent profile of adducts formed by the electron-rich
QM2 and deoxynucleosides. Reaction conditions and product analysis are
identical to those used for Figure 1. Again, each point represents the average
of at least three independent determinations and was fit to exponential
processes for highlighting the net trends of the data. The indicated error
derives from the standard deviations.
guanine N7 and dG N2. The dG N7 adduct did not accumulate
since its deglycosylation is likely faster than its formation due
to the slow generation of QM3. The product of dG N1 alkylation
was also not initially apparent but could later be formed when
the concentration of dG was raised from the standard 0.25 mM
to 5.0 mM. Computational studies for QM1 indicated that
reaction at dG N1 has an activation energy approximately 3
kcal/mol higher than that of dG N2 as modeled with 9-methyl-
guanine in water.29 This can be used to explain the greater yield
of the dG N2 adduct over the yield of the dG N1 adduct with
QM1 and QM3. Surprisingly, QM2 exhibited the opposite
selectivity. The origin of this difference is currently subject to
additional computational analysis. Once again, no reaction of
T was detected.
turn formed adducts more slowly over 8 h and most likely as a
result of QM2 regeneration from the adducts of dA N1 and dC
N3. QM2 generation from its initial QMP2 precursor is nearly
complete within the first 2 h (Supporting Information). This
again illustrates the ability of reversible adducts to extend the
effective lifetime of an otherwise highly transient intermediate.
The leaving-group abilities of dA N,6 dG N1, and dG N2 are
not sufficient to regenerate the QM, and thus, their adducts
remained stable over the course of observation.
Initial reaction at dG N7 is less than might be expected from
its strong nucleophilicity. However, its adduct has the unique
ability to deglycosylate as well as eliminate QM2. At the
deoxynucleoside level, partitioning between deglycosylation and
QM regeneration had previously been observed to occur at
equivalent rates.30 The deglycosylation product from reaction
with QM2 accumulated over approximately 15 h and remained
stable since guanine N7 does not share the strong leaving-group
ability of dG N7. Although the kinetics of alkylation with QM2
were significantly faster than with QM1, all of the irreversible
adducts were stable for at least 6 days beyond the observations
of Figure 2. The nucleophilic sites used to create these adducts
therefore still lacked the leaving-group ability to eliminate from
a QM with the added electron density provided by a pendent
methyl group. This group was, however, sufficient to accelerate
QM regeneration from adducts that had previously exhibited
lability with QM1.30 Finally, the overall yields of stable adducts
were similar for QM1 and QM2, and only minor differences
in their relative formation was detected.
Reaction of QMP3 with dNs was significantly slower than
reaction with QMP1 due to electronic destabilization of QM3
from the attached methyl ester (Figure 3). Maximum accumula-
tion of the dC N3 adduct required more than 24 h and reflected
a slow generation of QM3 from its desilylated derivative of
QMP3 (Supporting Information). Additionally, this adduct was
stable well beyond the 120 h period of observation (see above).
The leaving-group ability of dC N3 was thus insufficient to
overcome the barrier to regenerate QM3 in contrast to its
behavior with the more electron-rich QM1 and QM2 deriva-
tives. The change in electronics between QM2 and QM3 did
not greatly affect the maximum yield of the dC N3 adduct
despite its conversion from a reversible to an irreversible adduct,
respectively.
Deoxynucleoside Competition with an Electron-Poor QM.
Individual deoxynucleosides were also treated with the electron-
poor QMP3 in order to generate synthetic standards for
structural and chromatographic markers. Incubation of dA with
QMP3 and fluoride resulted in adducts formed by its N1 and
N6 positions. This is consistent with reactions of both QM127
Formation of the dA N1 adduct similarly reflected the slow
generation of QM3 (Figure 3). A maximum yield for this adduct
required approximately 10 h with QMP3, whereas less than
0.5 h was necessary with the electron-rich QMP2. Reversibility
of the dA N1 adduct persisted despite the electron deficiency
of QM3, although regeneration of QM3 (half-life > 40 h, Figure
3) was greatly suppressed relative to comparable regeneration
of QM2 (half-life < 2 h, Figure 2). Behavior of the dC N3 and
dA N1 adducts is consistent with the relative strength of dC
N3 and dA N1 as leaving groups.30 The slow formation of the
1
and QM2. Additionally, the H NMR signals for the benzylic
positions were again diagnostic of their structures (Table 1).
Comparable reaction of QMP3 with dG yielded only two
products. These were isolated and identified as adducts of
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A R T I C L E S
Weinert et al.
Table 2.
dA N1 adduct combined with its remaining lability prevented
its accumulation to the same level as the dC N3 adduct or related
adducts with QM2. The guanine N7 adduct resulting from QM
alkylation and deglycosylation also formed slowly with QMP3,
but its yield was similar with both QM2 and QM3.
Destabilization of the QM affects both the kinetics of its
formation and the resulting product profile as evident from the
stability of the dC N3-QM3 adduct and the complete absence
of a dG N1 adduct. Adducts of dG N2 and dA N6 were still
generated by QM3 in similar yields and approach the levels of
accumulation measured with QM2 (Figure 2). However, these
QM3 adducts continue to increase for more than 120 h (rather
than 10 h for QM2). The original source of QM3 is mostly
consumed by 30 h, and thus continued generation of the dG N2
and dA N6 adducts likely derives from the regeneration of QM3
from its dA N1 adduct.
Effects of Aromatic Substituents on QM Generation. A
methoxy group was originally attached to the model QM1 in
order to examine a broad range of substituent effects on
deoxynucleoside adduct stability. Preliminary study of the adduct
formed between this electron-rich intermediate and dC N3
indicated that it was too labile for convenient analysis. Thus,
the weaker electron-donating methyl group was used in place
of the MeO group as described above. As a complement to the
range of derivatives available for deoxynucleoside study, a series
of N-morpholino derivatives was prepared to extend correlations
between QM stability and its rate of (re)generation. The
morpholino-QM adducts were chosen to replace deoxy-
nucleoside-QM adducts based on the pKa of morpholine’s
conjugate acid. This property reflects the nucleophile’s leaving-
group ability that, along with QM stability (see above),
determines the rate of adduct collapse and QM regeneration
(Scheme 3).30,45 For nucleophiles with conjugate acids exhibiting
pKa’s between approximately 4 and 8, QM regeneration has
been detected over hours under physiological conditions.30
Morpholine has a conjugate acid pKa of 8.4 which is at the high
end of this acidity range and should consequently provide some
stability to even highly electron-rich QM precursors. Accord-
ingly, morpholino adducts provided a basis for determining
electronic effects of QM reversibility over a greater range of
electronic perturbations than those available with the deoxy-
nucleosides.
The desired morpholino adducts of substituted QMs were
synthesized by a Mannich reaction of 4-methylenemorpholinium
halide and the appropriate hydroxylated aromatic prescursor.
Substituents influencing QM stability ranged from the strongly
electron-donating -OMe group to the electron-withdrawing
-COOMe group (Table 2). These groups were placed at the
5-position like QMP2 and QMP3 as well as at the 4-position
to compare positional differences. The overall trend of adduct
stability (QMP4-QMP8) was monitored by conversion of the
morpholino adducts to their water adducts (benzylic alcohols,
QMA). The range of reactivities observed for this series was
even more dramatic than that observed for the deoxynucleoside
adducts above. An aqueous solution of the electron-poor adduct
QMP4 was fully stable after incubation at 100 °C for 3 h (Table
2). No consumption of QMP4 or formation of QMA3 was
detected by HPLC after this harsh treatment. This suggests that
T
t1 2 of QMP (min)
/
QMP
X
Y
QM
QMA
°
C
in water
QMP4 COOMe
H
QM3 QMA3 100
COOMe QM5 QMA5 100
stablea
149
115
11
QMP5
QMP6
QMP7
H
H
H
H
QM1 QMA1
QM7 QMA7
QM8 QMA8
50
22
22
OMe
H
QMP8 OMe
4
a No benzylic alcohol QMA3 was detected by HPLC.
QM3 was not generated under these conditions nor was QMP4
subject to direct substitution through a SN2 mechanism. This
alternative process should not be significantly influenced by
remote aromatic substitution, and thus QMA formation from
the other adducts is indicative of QM generation as detected in
a number of related systems14,15,25,45-47 rather than direct
substitution. Similarly, an alternative SN1 mechanism is not
consistent with the lack of reactivity of O-silyl QMPs and their
O-methyl derivatives compared to that of their parent phe-
nols14,48 since these substituents should all promote such a
process almost equally.49
An electron-withdrawing -COOMe group placed at the
4-position was not as effective at destabilizing its QMP (QMP5)
as the same group placed at the 5-position (QMP4, Table 2).
QMP5 was slowly converted to its benzylic alcohol QMA5,
albeit heating to 100 °C was still necessary. The unsubstituted
parent QMP6 was somewhat more labile. Formation of QMA1
from trapping of the transient QM1 was observed after heating
to only 50 °C. In sharp contrast, the electron-rich derivatives
containing methoxy substituents (QMP7, QMP8) were very
labile with half-lives of only minutes at 22 °C. The sensitivity
of QM generation to electronic effects that was first observed
for deoxynucleoside adducts is consequently quite general and
remarkable for other QM-producing adducts as well. Further-
more, the site of substitution within the QM adduct also plays
an important role in its stability. The o-QMs characterized in
this study were more strongly influenced by substitution at the
5- than at the 4-position (QMP4 vs QMP5 and QMP8 vs
QMP7). Inductive and resonance effects from both positions
apparently modulate QM stability, but only the 5-position can
participate directly in resonance with the nascent exocyclic
methylene.
Substituent Effects on Nucleophilic Addition to Quinone
Methides. Substituents used to tune the stability of QM adducts
should concurrently affect their electrophilicity and lifetime. The
magnitude of these perturbations has now been quantified by
laser flash photolysis (LFP). Photochemical activation of
hydroxybenzyl alcohols and phenolic Mannich bases (Scheme
5, L ) OH and NMe2, respectively) has been well described as
(46) Zhou, Q.; Pande, P.; Johnson, A. E.; Rokita, S. E. Bioorg. Med. Chem.
2001, 9, 2347-2354.
(47) Kumar, D.; Veldhuyzen, W. F.; Zhou, Q.; Rokita, S. E. Bioconj. Chem.
2004, 15, 915-922.
(45) Modica, E.; Zanaletti, R.; Freccero, M.; Mella, M. J. Org. Chem. 2001,
66, 41-52.
(48) Zeng, Q.; Rokita, S. E. J. Org. Chem. 1996, 61, 9080-9081.
(49) Hansch, C.; Leo, A.; Taft, R. W. Chem. ReV. 1991, 91, 165-195.
9
11944 J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006

Quinone Methide Sensitivity to Electronic Modulation
A R T I C L E S
Scheme 5. Photogeneration of QM
Table 3.
quaternary
ammonium
salts
quinone λ max
/
k2(H2O)/
k2(morpholine)a/
k2(RSH)/
105 M-1 s-
1
1
1
Y
methide
nm
M-1 s-
×
105 M-1 s-
×
QMP9
QMP10
QMP11 Cl
QMP12 COOMe QM5
QMP13 CN
QMP14 NO2
OMe
H
QM7
QM1
QM11 400
420
QM13 415
410
400
1.9
7.8
22.8
363
5.5
23
36
160
505
b
0.40
1.9
4.4
59
501
b
a mild source of QMs by Wan,50-52 Kresge53-55 and Saito.56
Recently, one of our laboratories has developed an alternative
photochemical precursor to QMs, benzylammonium salts of
phenolic Mannich bases (Scheme 5, L ) +NMe3I-).45 In contrast
to Mannich bases, these salts are not nucleophilic and do not
react with the transient QM. Also, these salts display higher
photochemical quantum yields than the corresponding alcohols
for QM generation.45 The quaternary ammonium salts are
therefore ideal for generating a series of o-QMs and monitoring
their consumption over time in the presence of various nucleo-
philes.
While Wan,50-52 Kresge,53-55,57 Richard58-60 and our labo-
ratories30,45 have investigated the kinetic behavior of the parent
o-QM (QM1), its para isomer (p-QM), and a few of their
derivatives under aqueous conditions, no systematic analysis
of substituent effects on QM reactivity has yet been published
to our knowledge. Consequently, QMP9-QMP14 were syn-
thesized to examine a diverse range of electronic perturbations,
and QM reaction was tested with three prototypical nucleophiles.
1999
QM14 435 25400
a Keeping pH 9.0 with Na2CO3. b The transient absorbance of QM
bearing the strongest NO2 electron-withdrawing substituent in the presence
of morpholine and thiol were completely quenched. This could be due to
an electron-transfer mechanism from the nucleophile to the QM (work in
progress).
The presence of an increasingly strong electron-withdrawing
group (5-Cl, 5-COOMe, 5-CN, and 5-NO2) progressively
decreased QM lifetime as evident by the increasing values of
k2. The 5-NO2 (QM14) in particular resulted in a k2 for the
hydration reaction that was several orders of magnitude greater
than that of the parent, QM1. In contrast, the electron-donating
ability of the 5-MeO group decreased the rate of its QM (QM7)
consumption vs that of the parent QM1 by 4-5-fold. Although
the rates of QM decay were much faster in the added presence
of strong nucleophiles represented here by morpholine and
2-mercaptoethanol, their general sensitivity to electron-
withdrawing and -donating substituents remains nearly equiva-
lent. Still, the relative selectivity for the individual nucleophiles
decreases with increasing reactivity of the QM. Such data is
consistent with the much maligned reactivity-selectivity prin-
ciple despite the lack of reaction controlled by diffusion.61
The sensitivity of QM reaction to substituents was also
examined by its free energy relationship [ln(ky/kH)] for addi-
tion of morpholine (Figure 4a,a′), water (Figure 4b,b′), and
2-mercaptoethanol (Figure 4c,c′). Linear correlations were
slightly better with Hammett σp- constants (Figure 4a,b,c) than
with Hammett σp constants (Figure 4a′,b′,c′). The σp- constants
were originally set by changes in the pKa of para substituted
phenols and are now used for systems with strong resonance
interaction between substituent and reaction center. Such a case
is proposed for reactive QMs that appear to develop a degree
of aromaticity in the transition-state (TS) with negative charge
efficiently stabilized by delocalization over the former oxygen
carbonyl group and Y substituent (Scheme 6). Such resonance
contributions are likely even greater for substituents at the
5-position since they may influence the exocyclic methylene
of the QM more directly. Preliminary support for this prediction
is evident from the greater influence of substituents at the 5- vs
4-position as summarized in Table 2.
Each QM intermediate was generated from its corresponding
QMP (0.2 mM) by flash photolysis at 266 nm. QM consumption
was then monitored at its λmax under aqueous conditions (pH
7) in the absence and presence of 0.2-50 mM morpholine
(buffered at pH 9 with Na2CO3) and 2-mercaptoethanol (buff-
ered at pH 7 with sodium phosphate), alternatively (Table 3).
Second-order decay rate constants (k2) were calculated from the
concentration dependence of pseudo-first-order kinetic measure-
ments. The rate constants determined under each of the three
conditions exhibit parallel trends that are consistent with the
behavior of the deoxynucleoside and morpholino adducts.
Electron-donating groups significantly stabilize QM. Such
groups facilitate QM regeneration from their adducts (Figures
1-3, Table 2) and are now shown to slow nucleophilic addition
to the nascent QM as well (Table 3). In a complementary
manner, electron-withdrawing groups suppress QM regeneration
and strongly promote nucleophilic addition to the nascent QMs.
(50) Wan, P.; Barker, B.; Diao, L.; Fischer, M.; Shi, Y.; Yang, C. Can. J. Chem.
1996, 74, 465-475.
(51) Diao, L.; Yang, C.; Wan, P. J. Am. Chem. Soc. 1995, 117, 5396-5370.
(52) Brousmiche, D. W.; Xu, M.; Lukerman, M.; Wan, P. J. Am. Chem. Soc.
2003, 125, 12961-12970.
(53) Chiang, Y.; Kresge, A. J.; Zhu, Y. J. Am. Chem. Soc. 2001, 123, 8089-
8094.
(54) Chiang, Y.; Kresge, A. J.; Zhu, Y. J. Am. Chem. Soc. 2002, 124, 717-
722.
-
(55) Chiang, Y.; Kresge, A. J.; Zhu, Y. J. Am. Chem. Soc. 2002, 124, 6349-
Hansch had previously noted that σp constants often yield
6356.
better correlations than σp when resonance interaction develops
between the substituent and the reaction center, and the resulting
magnitude of F has provided insight into reaction mechanisms.49
For QM addition, the linear free energy correlations are positive
(56) Nakatani, K.; Higashida, N.; Saito, I. Tetrahedron Lett. 1997, 38, 5005-
5008.
(57) Chiang, Y.; Kresge, A. J. Org. Biomol. Chem. 2004, 2, 1090-1092.
(58) Toteva, M. M.; Moran, M.; Amyes, T. L.; Richard, J. P. J. Am. Chem.
Soc. 2003, 125, 8814-8819.
(59) Richard, J. P.; Toteva, M. M.; Crugeiras, J. J. Am. Chem. Soc. 2000, 122,
1664-1674.
(60) Toteva, M. M.; Richard, J. P. J. Am. Chem. Soc. 2000, 122, 11073-11083.
(61) Mayr, H.; Ofial, A. R. Angew. Chem., Int. Ed. 2006, 45, 1844-1854.
9
J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006 11945

A R T I C L E S
Weinert et al.
Figure 4. Free energy relationships ln(ky/kH) vs Hammet σp- (a,b,c) and Hammet σp (a′,b′,c′) for the addition reactions of morpholine (a, a′), water (b, b′),
and thiol (c, c′).
Scheme 6. Aromaticity Develops in the Transition State of QM
Addition
anionic character in the TS. The lower value for the phos-
phonium salt vs toluene was considered by Bordwell to indicate
a diminished charge delocalization on the salt due to Coulombic
and polarization contributions by the triphenylphosphonium
group.64 The smaller F value of morpholine vs those of water
and 2-mercaptoethanol may also be explained by the electrostat-
ics of the developing positive charge on the nitrogen nucleophile.
Conclusions
and large. The values of F equal 3.0, 5.9, and 5.4 for morpholine,
The formation of QMs had previously demonstrated a
water, and 2-mercaptoethanol, respectively (Figure 4a,b,c). In
dependence on the strength of the leaving group attached to
comparison, F values for the equilibrium acidity of phenol in
the benzylic position of its precursor when studying a model
water and DMSO are 2.1 and 5.3, respectively.62,63 The equi-
o-QM in the presence of biological nucleophiles (Scheme 3).30,45
librium acidity of para-substituted benzyltriphenylphosphonium
Now, both the formation and subsequent reaction of QMs have
cations to form the corresponding ylide in DMSO exhibited a
also been shown to be highly responsive to the presence of
F of 4.78.64 Much larger values of F (12 and 10) were
electron-withdrawing and -donating groups. All trends in
determined for substituent effects on the acidity of a benzylic
reactivity are consistent with the electron-deficient nature of
hydrogen in toluene and 10-substituted-9-methylanthracene
the QM intermediate. Electron-donating groups greatly facilitate
derivatives in DMSO, respectively.65,66 The intermediate values
initial QM generation and its subsequent regeneration from
of F for QM addition in water are then suggestive of significant
adducts formed by the nucleophiles of deoxynucleosides that
also function as good leaving groups. Conversely, electron-
withdrawing groups greatly suppress initial formation of QM
and suppress its regeneration from the reversible deoxynucleo-
side adducts. These properties similarly extend to nucleophiles
and leaving groups beyond those of DNA and provide a
foundation for predicting the general behavior of QM stability
and selectivity. In a complementary manner, electron-rich QMs
(62) Hammett, L. P. Physical Organic Chemistry; McGraw-Hill: New York,
1940; Chapter 7.
(63) Bordwell, F. G.; McCallum, R. J.; Olmstead, W. N. J. Org. Chem. 1984,
49, 1424-1427.
(64) Zhang, X.-M.; Fry, A. J.; Bordwell, F. G. J. Org. Chem. 1996, 61, 4101-
4106.
(65) Zhang, X.-M.; Bordwell, F. G.; Bares, J. E.; Cheng, J.-P.; Petrie, B. J.
Org. Chem. 1993, 58, 3051-3059.
(66) Bordwell, F. G.; Zhang, X.-M.; Cheng, J.-P. J. Org. Chem. 1993, 58, 6410-
6416.
9
11946 J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006

Quinone Methide Sensitivity to Electronic Modulation
A R T I C L E S
Laser Flash Photolysis (LFP) to Determine the Second-Order
Rate Constants for Addition of Water, Morpholine, and 2-Mer-
captoethanol to a Range of Quinone Methides. Kinetic studies of
nucleophilic addition to QMs were carried out at 25 °C, in pure water
for the hydration reaction and in the added presence of 2-mercapto-
ethanol (pH 7) and morpholine (adjusted to pH 9 by addition of
Na2CO3) alternatively. The pH of each solution was measured using
an Orion SA520 pH meter with a 8102 Ross electrode. QM1, QM5,
QM8, QM11, QM13, and QM14 were generated by flashing dilute
solutions of QMP9-QMP14 ((2-5) × 10-4 M). Disappearance of
the QMs was followed under pseudo-first-order conditions by monitor-
ing the absorbance decrease at the maximum wavelength, reported in
Table 3. Pseudo-first-order rate constants (kobsd) were obtained from
the fitting the absorbance data to a single-exponential function and were
reproducible to (5%. The second-order rate constants k2 (M-1 s-1) for
addition of nucleophiles to the QMs were determined as the least-
squares linear slopes of kobsd vs total concentration of the nucleophile
varied between 5 × 10-2 to 2 × 10-4 M.
react much more slowly but more selectively with nucleophiles
than do the electron-poor QMs. These fundamental character-
istics provide the basis for explaining the product profiles
observed after DNA has been exposed to various QMs. In the
future, these same characteristics may be used to fine-tune the
stability and reactivity of QM conjugates for target-promoted
and gene-specific alkylation.15 An equivalent approach may also
be used to optimize the kinetics and sensitivity of polymers
based on QMs to disassemble for drug release.32-34
Experimental Procedures for Quinone Methide
Reaction
Kinetic Studies with Individual Deoxynucleosides. To an aqueous
solution (70 µL) of 4 mM phenol, 0.5 mM deoxynucleoside, 10 mM
potassium phosphate (pH 7), and 500 mM KF was added the quinone
methide precursor (QMP1, QMP2, or QMP3, alternatively) in DMF
(30 µL), yielding a final concentration of 25 mM. The reactions were
incubated at 37 °C and, at the indicated times, analyzed directly by
reverse phase HPLC (C-18, Varian, Microsorb-MV 300, 5 µm particle
size, 250 mm × 4.6 mm) using a gradient of 3% CH3CN, 9.7 mM
TEAA, to 25% CH3CN, 7.5 mM TEAA at 1 mL/min over 66 min. For
QMP1 reaction, TEAA was adjusted to pH 4, and for QMP2 and
QMP3, the TEAA was adjusted to pH 6. Product formation was
monitored by integrating the elution profile at 260 nm generated by a
diode array detector and normalizing its signal by the relative ꢀ260 of
each product and absorbance of an internal standard (phenol).
Competition Studies in the Presence of all Deoxynucleosides. To
an aqueous solution (70 µL) of 4 mM phenol, 0.25 mM of dA, dC,
dG, and T (1 mM total dN), 10 mM potassium phosphate (pH 7), and
500 mM KF, was added the quinone methide precursor (QMP1, QMP2,
or QMP3, alternatively) in DMF (30 µL), yielding a final concentration
of 25 mM. Reactions were incubated at 37 °C and analyzed as described
above.
Kinetics of Quinone Methide Regeneration and Benzylic Alcohol
Formation from the Morpholino Quinone Methide Adducts. A stock
solution of QMP6 in acetonitrile was diluted with water/acetonitrile
(1:1) to yield a final concentration of 1.0 mM, and anisole (0.6 mM)
was added as internal standard. The resulting solution was incubated
for 240 min at 50 °C. Every 5 min, an aliquot (200 µL) was removed
and cooled at -20 °C in a ice-salt bath prior to analysis by reverse
phase HPLC (C-18, Intersil ODS-2, 5 µm, column dimension: φ )
4.6 mm, length ) 250 mm, φ ) 10.0 mm, length ) 250 mm) with
50% aqueous CH3CN (1 mL/min). The benzylic alcohol (QMA1) was
detected with a variable-wavelength detector and compared to an
authentic sample. Reactions of the adducts (QMP4, QMP5, QMP7,
and QMP8) were monitored similarly although reaction time and
temperature were varied as indicated in Table 2 to detect the benzylic
alcohols QMA3, QMA5, QMA7, and QMA8.
Laser Flash Photolysis Apparatus. The laser pulse photolysis
apparatus consisted of a Nd:YAG laser used at the fourth harmonic of
its fundamental wavelength. It delivers a maximum power of 10 mJ at
266 nm with 10 ns pulse duration. The monitoring system was arranged
in a cross-beam configuration and consisted of a 275 W Xe arc lamp,
an F/3.4 monochromator, and a five-stage photomultiplier supplied by
Applied Photophysics. Signals were captured by a Hewlett-Packard
54510A digitizing oscilloscope, and the data were processed on a 486-
based computer system using software developed in-house. Solutions
for analysis were placed in a fluorescence cuvette (d ) 10 mm) and
adjusted to an absorbance of 1.5.
Acknowledgment. This research was supported in part by
the National Institutes of Health (CA81571, S.E.R.), the Howard
Hughes Medical Institute through the Undergraduate Biological
Sciences Education Program (K.N.F., C.H.M.) and Pavia
University (FAR 2004, M.F.). We thank Dr. Yui-fai Lam for
help with NMR characterization of the deoxynucleoside adducts.
Supporting Information Available: All synthetic protocols
and product characterization; extinction coefficients and λmax
values for deoxynucleoside-QM adducts; time-dependent con-
sumption of the deprotected derivatives of QMP1, QMP2, and
QMP3 to form QM1, QM2, and QM3, respectively; sample
HPLC chromatograms used to monitor deoxynucleoside adducts
formed by QM2 and QM3. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA062948K
9
J. AM. CHEM. SOC. VOL. 128, NO. 36, 2006 11947