Alcohol, Aldehyde, and Ketone Liberation and Intracellular Cargo Release through Peroxide-Mediated α-Boryl Ether Fragmentation

Article abstract of DOI:10.1021/jacs.6b07890

α-Boryl ethers, carbonates, and acetals, readily prepared from the corresponding alcohols that are accessed through ketone diboration, react rapidly with hydrogen peroxide to release alcohols, aldehydes, and ketones through the collapse of hemiacetal intermediates. Experiments with α-boryl acetals containing a latent fluorophore clearly demonstrate that cargo can be released inside cells in the presence of exogenous or endogenous hydrogen peroxide. These experiments show that this protocol can be used for drug activation in an oxidative environment without generating toxic byproducts.

Full text of DOI:10.1021/jacs.6b07890

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Article
Alcohol, Aldehyde, and Ketone Liberation and Intracellular Cargo
Release through Peroxide–Mediated #–Boryl Ether Fragmentation
Ramsey D. Hanna, Yuta Naro, Alexander Deiters, and Paul E. Floreancig
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b07890 • Publication Date (Web): 16 Sep 2016
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Journal of the American Chemical Society
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Alcohol, Aldehyde, and Ketone Liberation and Intracellular
Cargo Release through Peroxide-Mediated α-Boryl Ether
Fragmentation
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Ramsey D. Hanna, Yuta Naro, Alexander Deiters,* and Paul E. Floreancig*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
ABSTRACT: α-Boryl ethers, carbonates, and acetals, readily prepared from the corresponding alcohols that are ac-
cessed through ketone diboration, react rapidly with hydrogen peroxide to release alcohols, aldehydes, and ketones
through the collapse of hemiacetal intermediates. α-Boryl acetals containing a latent fluorophore readily allow the demon-
stration that cargo can be released inside cells in the presence of exogenous or endogenous hydrogen peroxide. These
experiments show that this protocol can be used for drug activation in an oxidative environment without generating toxic
by-products.
of H
2
O without generating toxic by-products would be
2
INTRODUCTIONꢀ
valuable for applications in oxidative drug release.
Reactive oxygen species (ROS), including hydrogen perox-
ide, are linked to a number of disparate medical conditions
We have initiated a program with the objective of design-
ing readily accessible structures that have the capacity to
localize toward a cellular target and decompose under
oxidative conditions to release a biological effector. Our
1
2
3
including neurological diseases, cancer, aging, and dia-
4
betes. ROS-rich environments are also created through
1
3b
exposure to ionizing radiation, as encountered in radio-
initial design for alcohol release (Scheme 1) employed
acyl aminal substrates (1) that are available through reduc-
tive multicomponent unions of nitriles, chloroformates, and
5
therapy. Hydrogen peroxide's unique reactivity properties
and importance in these conditions have resulted its utili-
zation to initiate a number of processes in biological and
materials chemistry. Initial studies from Chang's group
1
6
alcohols. Aryl or vinyl boronate oxidation with H O re-
2
2
leases a quinone methide or acrolein and CO to form an
2
demonstrated that aryl boronates can be converted to
unstable hemiaminal (2) that collapses to release the alco-
6
fluorescent phenols by cellular H
2
O
2
. This result, coupled
hol. We reasoned that oxidation of α-boryl ethers or car-
bonates (3) would provide a similar unstable hemiacetal (4)
that releases an alcohol directly or through carbonate
breakdown with less by-product generation. This ap-
proach allows for the selection of a non-toxic ketone by-
product to serve as a guide in substrate design.
with Lo's employment of the boronate to phenol conver-
7
sion to effect benzylic leaving group departure, led to the
development of numerous compounds that release fluoro-
phores and other diagnostic tools in oxidatively stressed
8
cells. Additional applications of oxidatively triggered self-
9
immolative spacers have been developed to promote
1
0
11
particle breakdown and signal amplification.
H
2
2
O is an
Prior studies
attractive agent for initiating prodrug unraveling in many
cases because it is small and can access sterically hin-
dered sites in structures that are inaccessible to enzymes,
which are commonly utilized for this purpose. Cargo re-
O
H2O2
R'
NH2
B
O
ROH
H
N
R'
O
RO
12
lease from antibodies serves as an example of a process
O + CO2
RO
O
1
2
that can benefit from activation by a small molecule. Per-
oxide-mediated drug release has been explored to a lim-
ited extent. However substrates for these processes em-
This work
13
O
H2O2
HO OR
R'
ploy aryl or vinyl boronates as oxidative triggers to pro-
mote release from the benzylic or allylic position. Thera-
peutic applications of these systems, therefore, can be
complicated by the significant toxicity of the resultant qui-
none methide or acrolein by-products. Thus alternative
structural motifs that release compounds in the presence
ROH
O
B
OR
R'
O
3
4
R'
14
15
Scheme 1. Alcohol release through boronate oxi-
dation.
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This manuscript describes the realization of this approach
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through the release of several diverse structures via oxida-
tive fragmentation of α-boryl ethers, carbonates, and ace-
tals. Specific advances include 1) the development of ex-
perimentally facile conditions for the synthesis of α-boryl
alcohols through a variant of a known ketone diboration
protocol, 2) the preparation of α-boryl ethers and car-
bonates through conditions that avoid strong base, 3) the
demonstration that α-boryl ethers decompose rapidly and
N
BF4–
Cy
O
O
O
O
N
O
Cy
O
+
B B
CuCl, NaOtBu,MeOH
PhMe, 82%
O
B
OH
5
6
O
O
ClCH OMe
2
O
B
O
iPr NEt, CH Cl
2
2
2
92%
7
efficiently in the presence of H
2
2
O under mildly basic condi-
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
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9
0
tions while α-boryl carbonates decompose more slowly, 4)
the elaboration of several protocols for preparing cyclic
boryl-substituted acetals, 5) the observation that the ace-
tals can liberate aldehydes and ketones in the presence of
PhOC(O)Cl
pyridine, CH Cl
O
O
2
2
O
B
O
71%
O
H
2
2
O , 6) the application of the acetal breakdown to release
8
fluorophores at low substrate and peroxide concentra-
tions, 7) the validation of the capacity of the acetals to
release cargo in cells through stimulation with exogenous
Scheme 2. α-Boryl alcohol synthesis and func-
tionalization.
H
2
O
2
, and 8) the demonstration that cargo can be released
The oxidative breakdown of compounds 7 and 8 was
in cells by endogenous H resulting from chemically
2
O
2
achieved by subjecting them (~25 mM) to urea•H
mM) in a mixture of CD CN and aqueous (D O) buffer (pH
8.0). The buffer was selected to mimic the experimental-
2
O
2
(300
stimulated oxidative stress. These results clearly illustrate
that α-boryl ethers, carbonates, and acetals are viable
substrates for releasing biological effectors in cells in re-
sponse to oxidative conditions while avoiding the genera-
tion of toxic by-products.
3
2
=
2
4
ly determined pH of mitochondria. in consideration of
potential applications to mitigating neuronal oxidative
stress. Initial experiments were conducted in a 5:1 ratio of
CD CN and buffer (Scheme 3). Reaction progress was
3
1
RESULTSꢀANDꢀDISCUSSIONꢀ
monitored by H NMR through following the disappear-
ance of the signals for diastereotopic hydrogens from the
methylene group in the starting materials and the appear-
ance of the corresponding enantiotopic hydrogens in bu-
tanone. Conversions were calculated by comparison to
the internal standard 1,2-dimethoxyethane.
Ether, carbonate, and cyclic acetal substrate
synthesis and decomposition. The success of this
project was contingent upon identifying suitable approach
1
7
to α-boryl alcohol formation. We initially employed
1
8
Clark's ketone-relevant variation of the Sadighi carbonyl
1
9
diboration protocol for the conversion of 5 to boryl alco-
O
2
0
urea•H
2
O
2
, CD
3
CN, D
2
O
O
hol
6
(Scheme 2).
These conditions (PinB–BPin,
O
+
MeOH
pH = 8, t1/2 < 2 min
89 - 94%
O
B
O
(
ICy)CuCl, NaOtBu, PhMe, 50 °C followed by borate pro-
todeboration on silica gel) provided 6, but were deemed to
be unacceptable due to the low reaction rate and because
of the technical difficulty associated with the need to initi-
ate the reaction in a glove box. We reasoned that the rele-
vant copper carbene catalyst could be prepared in situ by
deprotonating the imidazolium salt in the presence of
CuCl, thereby obviating the need to isolate this sensitive
species. Moreover adding MeOH to the reaction mixture
substantially increased the rate of the reaction, in accord
7
O
HO
urea•H
pH = 8, t1/2 = 12 - 22 min
4%
2
O
2
, CD
3
CN, D
2
O
O
O
B
O
+
O
O
8
8
Scheme 3. Oxidative alcohol release.
Methoxymethyl ether 7 fragmented quite rapidly in the
presence of hydrogen peroxide. Over 50% of the starting
material was consumed in less than 2 minutes (Figure 1A),
and complete conversion was observed within 20 min with
an 89% NMR yield of butanone. Changing the solvent to a
21
with Molander's observations. These changes resulted in
the conversion of 5 to 6 in 82% yield within 1 h and with-
out recourse to glove box or Schlenk line techniques. The
experimental facility of this protocol appreciably enhances
access to α-boryl alcohols. This is significant because
boronates and related species with α-heteroatom substi-
1
:1 ratio of CD CN to buffer did not slow the reaction and
3
resulted in a slightly increased NMR yield of 94%. Moreo-
ver lowering the pH to a cytosolic-relevant value of 7.2 had
only a minimal effect on the rate despite the diminished
peroxy anion concentration (Figure 1B), providing a 91%
NMR yield of butanone.
21
tution are useful as substrates for cross-coupling and
22
chain elongation reactions, and as surrogates of func-
tionalized carboxylic acids for applications in medicinal
23
chemistry. The hydroxy groups can be functionalized
readily, as demonstrated through the formation of meth-
oxymethyl ether 7 and phenyl carbonate 8.
Carbonate 8, however, broke down much more slowly
under the oxidative conditions. Consumption of 50% of
the starting material required 22 min when the reaction
2
5
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Journal of the American Chemical Society
aptitude resulting from the presence of an electron with-
1
A
2
drawing acyl group. No evidence of a persistent peroxy-
boronate intermediate, such as 10, was observed upon
3
4
5
6
7
8
9
11
monitoring the progress of the reaction with B NMR,
however. Regardless of the origin of the effect, the capaci-
ty to control the breakdown rate through a simple struc-
tural modification provides kinetic versatility in drug release
strategy.
OPh
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
OOH
O
B O
OPh
B
O
O
O
9
10
Figure 2. Structures 9 and 10 as potential origins for the
slow breakdown of 8.
Several additional substrates were prepared to define the
scope of the process (Table 1). Secondary alcohols such
as cyclohexanol (from the breakdown of 11) and the more
complex menthol (from the breakdown of 13) are released
smoothly. Although the formation and fragmentation of
alkoxymethyl ethers proceeds rapidly and smoothly, direct
release of alcohols would be desirable for avoiding the
1
00
8
0
6
0
29
generation of toxic formaldehyde, particularly if the cargo
is not intended to effect a cytotoxic response. Primary and
secondary alcohols can be released directly, as shown in
entries 3-5. The use of an aldehyde-derived boronate in
entry 5 facilitated the synthesis of the ether. The oxidative
cleavage of 15 and 20 (entries 3 and 6) are also signifi-
cant because they show that functionalized substrates
participate well in this process, providing potential handles
for incorporating tissue-, cell-, or organelle-targeting func-
tional groups. Boronate 20 releases the antioxidant pen-
tamethyl chromanol (21), showing that this method could
be applied to the release of radical scavengers in the
presence of environments that are rich in reactive oxygen
species, such as mitochondria. As previously discussed,
this release was predictably somewhat slow due to the
carbonate linker. The release of carboxylic acids (entry 7),
while possible, is substantially slower than the release of
alcohols or carbonates and is therefore not likely to be
useful. Compound 22 showed a chemical shift of 27.0
pH 7.20
pH 8.00
4
0
2
0
0
0
250
500
750
1000
1250
Time (sec)
B
3
0
Figure 1. Oxidative breakdown of 7. (A) Reaction progress as
determined by H NMR. (B) Reaction progress as a function
1
of pH.
was conducted in a 5:1 mixture of CD
Changing the solvent to a 1:1 mixture of CD
resulted in a slightly increased rate, with 50% of the start-
ing material being consumed within 12 min. The reactions
were quite efficient, with both providing an 84% yield of
the desired products.
3
CN and buffer.
CN and buffer
3
1
1
ppm in the B NMR spectrum, indicating that coordination
between the boron and the carbonyl group is likely to play
a role in preventing oxidative cleavage through peroxide
attack.
The rate difference for alcohol release between acetal and
carbonate substrates indicates that the rate determining
step in these processes is boronate oxidation rather than
hemiacetal collapse. The slow breakdown of the car-
bonate could result from intramolecular coordination be-
tween the carbonyl oxygen and the boron, as illustrated by
(Figure 2) thereby inhibiting the approach of HOO to the
boron. Crystal structures show this type of coordination in
α-boryl amides, and intramolecular coordination has
been shown to confer stability to boronates. However the
B chemical shift of 8 (δ 32.2 ppm) is nearly identical to
the B chemical shift in 7 (δ 32.1 ppm), and is significantly
different from amido pinacolboronates, which show
chemical shifts of approximately 15 ppm.
the breakdown could be slowed by a diminished migratory
a
Table 1. Alcohol release scope.
–
9
b
entry substrate
product
t
1/2
yield
(%)
d
c
(min)
2
6
2
7
O
O
1
1
B
O
HO
1
O
<2
84
11
11
12
B
11
26,28
Alternatively
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Page 4 of 10
EtI, AgOTf,
,6-tBu2Py
CH2Cl2, 45%
O
HO
O
O
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
2
2
B
O
<2
85
B
OH
O
B
O
O
O
OTBS
OTBS
1
3
14
2
4
15
O
HO
3
4
O
B
O
<2
<2
89
89
O
O
B
BiBr3, Et3SiH
CH3CN, 77%
H
B
OTES
+
O
O
O
O
OTBS
16
1
5
25
17
HO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
Scheme 4. Etherification in the absence of strong
base.
O
B
O
1
8
17
The functional group tolerance of the process and the ca-
pacity for α-boryl alcohols to add into oxocarbenium ions
suggested that the scope could be expanded further to
promote aldehyde and ketone release. The preparation of
the substrates for these studies is illustrated in Scheme 5.
Cyclic acetal substrates can be prepared either through
oxidative or classical exchange reactions. Ketone 26,
available from commercially available 4-hydroxy-2-
O
<
2
83
O
B
OH
5
6
O
19
12
HO
O
O
O
B
O
O
O
90
79
16
O
3
4
OTBS
butanone,
underwent copper-catalyzed borylation
smoothly to yield alcohol 27. DDQ-mediated oxidative
2
1
2
0
3
5
cyclization provided acetal 28 in 78% yield. Removing
the PMB group from 27 under hydrogenolytic conditions
followed by acetal exchange with the dimethyl acetal of
benzophenone provided acetal 29 in 49% yield over two
steps.
O
HO
O
B
O
>936
7
O
O
22
23
a
Reactions run with 6 – 12 equiv H
O (5:1) at rt. See the Supporting Information
for the preparation of the substrates. As determined by
monitoring substrate consumption. Determined by H NMR
through comparing to the internal standard 1,2-
dimethoxyethane.
2
O
2
•urea at pH = 8.0 in
b
CD
3
CN and D
2
c
PinBBPin, CuCl
O
O
d
1
ICyBF , NaOtBu
4
B
OH
O
O
MeOH, PhMe, 84%
O
OMe
OMe
2
6
27
OMe
The synthesis of alkyl ethers is challenging in comparison
to the synthesis of alkoxymethyl ethers because direct
Williamson ether syntheses with α-boryl alcohols are
prone to undergo bora-Brook rearrangements that ren-
der the oxygen non-nucleophilic. Direct etherification re-
quires sufficiently potent electrophiles to subvert the need
for alkoxide generation. This can be achieved (Scheme 4)
by activating halide leaving groups with AgOTf, allowing
for hindered pyridines to be used as proton scavengers.
This is illustrated by the ethylation of α-boryl alcohol 24 to
yield 15. Alternatively, reductive etherification of α-boryl
1
. H2, Pd/C, MeOH
DDQ, CH2Cl2
0 °C, 78%
2. PhC(OMe) Ph
2
O
O
2
7
O
O
PPTs, CH2Cl2
O
B
3
1
O
49% (two steps)
B
O
O
28
29
Scheme 5. Synthesis of cyclic acetal substrates.
The boryl-substituted acetals release their cargo readily,
3
2
as shown in Scheme 6. Boronate 28 reacted with H
2
2
O at
pH = 8.0 to provide hemiacetal 30, which broke down to
form anisaldehyde and 1-hydroxy-3-butanone in 94%
yield. Over 50% of the starting material was consumed
within 90 sec, and complete conversion occurred in <15
min. Similarly boronate 29 reacted to form benzophenone
quickly and efficiently. Therefore this variation of the proto-
col significantly extends the range of structures that can
be released in the presence of hydrogen peroxide. Moreo-
ver this strategy illustrates a new approach to designing
prodrugs for aldehydes and ketones, as previous efforts
have largely centered on the use of oximes and deriva-
3
3
silyl ethers in the presence of BiBr
for preparing these substrates under non-basic conditions.
Thus silyl ether 25, readily available from 6, can be con-
3
is a versatile method
densed with isobutyraldehyde in the presence of Et SiH to
3
yield 17. An additional benefit of the reductive annulation
protocol lies in the enhanced stability of α-boryl silyl ethers
in comparison to α-boryl alcohols. This allows for the sub-
strate scope to be broadened to include aldehyde-derived
boronates such as 19.
3
6,37
tives.
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Journal of the American Chemical Society
OMe
OMe
mediated decomposition. We also prepared benzylic car-
OMe
1
2
3
4
5
6
7
8
9
bonate 35.This compound releases its fluorophore
through the common oxidative 1,6-elimination pathway
and was synthesized to compare the background stability
of 33 with a well-vetted latent fluorophore motif.
H2O2•urea, pH = 8.0
CD3CN, D2O, t1/2 < 2 min
94%
O
O
O
H
O
O
+
O
B
O
O
HO
Fluorophore release was studied at a concentration of 25
OH
OH
2
8
30
µM for 33 at pH = 7.4 with H
2
2
O concentrations of 100
µM and 200 µM. The concentration of 32 was monitored
by excitation at 448 nm and emission at 499 nm (a wave-
length where 33 shows only slight emission), with product
release being quantitated by comparison to a standard
curve. The fluorophore release experiments are summa-
rized in Figure 3. The breakdown of 33 was conducted
in 1% DMSO in aqueous phosphate buffer. Fluorophore
concentration increased steadily with time. The rate
and extent of fluorophore release showed the expected
dependency upon H O concentration. Lowering the
H2O2•urea, pH = 8.0
CD3CN, D2O, t1/2 < 2 min
O
O
O
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
81%
B
O
O
2
9
Scheme 6. Aldehyde and ketone release through
oxidative acetal cleavage.
2
2
Latent fluorophore synthesis and release. All stud-
ies to this point were conducted at relatively high concen-
trations of substrate and peroxide. Determining whether
these processes can proceed at biologically relevant con-
centrations requires an analytical technique that is more
H O concentration from 200 µM to 100 µM slowed
2
2
fluorophore release to a small but noteworthy extent.
The yield of 32 was 88% with 200 µM H O and 78%
2
2
with 100 µM H O . Fluorophore release was minimal in
2
2
the absence of H O . A similar study of carbonate 35
1
2
2
sensitive than H NMR. Therefore we explored the poten-
showed that ratio of fluorophore release in the pres-
ence and absence of H O was nearly equivalent to the
tial for the release of a fluorophore at low substrate and
peroxide concentrations. The synthesis of a latent fluoro-
phore is shown in Scheme 7. Silyl ether 31, which was
prepared from the TBS ether of 4-hydroxy-2-butanone,
coupled with aldehyde 32 (prepared from commercially
2
2
2
5
ratio that was observed for 33. This indicates that the
inherent stability of the α-boryl ether moiety is compa-
rable to a well established oxidative trigger. Fluorophore
38
release in the absence of H O was studied at pH = 6.0
available materials in two steps) in the presence of
TMSOTf to yield acetal 33. This acetal was formed as a
2 2
39
and 4.5 to determine the stability of the acetal toward
acid. Fluorophore release was slightly inhibited at lower
pH values (see the Supporting Information for a graphic
with an expanded y-axis), which is significant for biolog-
ical applications in consideration of the capacity of en-
single stereoisomer, with the relative configuration being
determined through a NOESY experiment. The Noyori
acetalization conditions were significantly superior to
Brønsted acid-mediated protocols due to the absence of
protodeboration as a prominent competing reaction. Ac-
etalization induces significantly different fluorescence
properties relative to the aldehyde, with λex values of 448
nm and 402 nm and λem values of 510 nm and 452 nm for
32 and 33, respectively, thereby facilitating the monitoring
of oxidative breakdown. Acetal 34 was prepared through
a similar protocol to serve as a control compound in eval-
uating the importance of the oxidative trigger in peroxide-
40
dosomes to achieve pH values as low as 4.9.
3000
2
000
000
0
33 (+) 200 µM H O
2
2
3
3 (+) 100 µM H O
2
2
3
3 pH 4.5 (-) H O
2
2
1
33 pH 6.0 (-) H O
2
2
3
3 pH 7.4 (-) H O
2
2
NEt2
0
50
100
150
200
250
Time (min)
NEt2
HO
B
OTBS
O
urea•H2O2
CH3CN, H2O
pH = 8.0
Figure 3. Fluorophore release at low substrate and per-
oxide concentrations and pH stability studies.
TMSOTf
O
O
+
O
O
32
O
O
CH Cl , 73%
2
2
O
B
O
O
H
O
Acetal 34 did not release 32 at any H O concentration
2
2
25
over the time span of the experiment, thereby validat-
ing the importance of boronate oxidation in cargo re-
lease. Separate studies in the presence of a large ex-
cess of H O (10 mM) allowed for the determination of a
3
1
32
33
NEt2
O
O
O
2
2
O
O
O
O
-
3
-1
O
B
pseudo-first order rate constant of 1.47x10 sec for
2
5
O
the decomposition of 33. This rate compares favora-
bly to the peroxide-mediated decomposition of boryl-
substituted benzylic carbamates to generate quinone
O
O
3
4
35
8
a
Scheme 7. Synthesis of a latent fluorophore.
methides via 1,6-elimination. The 1,6-elimination pro-
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Journal of the American Chemical Society
Page 6 of 10
tocol is likely to be significantly slower for releasing ali-
phatic alcohols, however, in consideration of their lower
A
B
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13b
nucleofugacity and our prior observation
that the
rates of these processes are strongly correlated with
41
the rate of benzylic C–O bond cleavage.
Exposing 33 to a number of reactive oxygen species
showed that the breakdown is selective for H
2
O
2
(Figure 4).
500 µm
500 µm
Solutions of H , NaOCl, KO , and tBuOOH were pre-
2
O
2
2
pared by diluting commercially available material. Hydroxyl
and t-butoxyl radicals were prepared by mixing the corre-
C
D
7
6
5
4
3
2
1
0
*
**
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
sponding peroxide with FeSO
to consume residual peroxide. The chart shows the ratio
of fluorescence intensity after 30 min to the initial value.
4
•5H O and adding catalase
2
8
a
Aside from H
2
O
2
only hydroxyl radical showed a notable
fluorophore release, albeit significantly lower in magnitude
5
00 µm
compared to H
2
2
O -mediated release.
2
vehicle H O2
Figure 5. Fluorophore release in HeLa cells treated with
exogenous H . Cells were incubated with 33 (10 µM) in
DPBS buffer for 45 min at 37°C, followed by replacement
with fresh DPBS containing (A) vehicle or (B) H (100 µM)
After 30 min, fluorescence was imaged (Zeiss Axio Observer
Z1, 20x objective, GFP filter (Set 38 HE; ex. 470 nm; em. 525
nm)). (C) Bright-field image of cells in (B) stained with Hoechst
3258 (1 µM) and imaged using a DAPI filter (Set 68; ex. 377
nm; em. 464 nm). (D) Mean fluorescence intensities were
calculated from three individual HeLa cells and set relative to
the mean fluorescence intensity prior to treatments (F/F). Er-
6
4
2
0
2
O
2
2
O
2
.
3
i
ror bars denote standard deviations, *** P < 0.001.
Blank H2O2 OCl-
O2- t-BuOOH ⋅OH ⋅OOt-Bu
differentiation between cell lines is possible. HeLa cells,
directly derived from cancerous cervical tissue, are ex-
prected to contain slightly elevated levels of endogenous
4
3
Figure 4. Comparison of fluorophore release by different
oxidants. [33] = 40 µM, [oxidant] = 200 µM, pH = 7.4.
H
2
O
2
and, therefore show higher background emission.
Quantitation of fluorophore release in the absence and
presence of exogenous H (Figure 6) indeed showed
that the background signal was significantly reduced in the
absence of exogenous H . These results further validate
0
0
2
O
2
Cellular fluorophore release. These results led us to
study the release of the fluorophore in cells to provide an
easily visualized demonstration of these compounds' ca-
pacity to release cargo in a biologically relevant environ-
ment. This was demonstrated in accord with Chang's pro-
2
O
2
the stability of α-boryl acetals in the absence of oxidants,
as required for selective applications to drug release in
4
2
tocol, whereby HeLa cells were incubated with 33 (10
µM) for 45 min and fluorophore release was imaged in the
A
B
absence and presence of exogenous 100 µM H
2
2
O . The
results are shown in Figure 5. Very little fluorophore re-
lease occurred within 30 min in the absence of external
H
2
2
O with the small response most likely being attributable
to the endogenous peroxide that is present in cancer
43
cells. Significant fluorophore release was observed in the
500 µm
500 µm
presence of H
2
2
O , however. This demonstrates that α-
boryl acetals are cell-permeable and can release cargo
within cells. Conducting these studies with control acetal
C
D
***
25
7
6
5
4
3
2
1
0
3
4 resulted in no fluorophore release, thereby providing
further evidence for the proposed release mechanism.
We repeated this experiment with HEK293T cells, derived
from the transformation of non-cancerous embryonic kid-
ney tissue with sheared adenovirus DNA and stable trans-
4
4
fection with the SV40 T antigen, with 33 to test whether
500 µm
vehicle H O2
2
ACS Paragon Plus Environment

Page 7 of 10
Journal of the American Chemical Society
Figure 6. Fluorophore release in HEK293T cells through
treatment with exogenous H . Cells were treated with 33
(10 µM) in DPBS buffer for 45 min at 37 °C, followed by re-
placement with fresh DPBS containing (A) vehicle or (B) H
(100 µM). After 30 min incubation, cellular fluorescence was
imaged on a Zeiss Axio Observer Z1 microscope using a 20x
objective and a GFP (Set 38 HE) filter (ex. 470 nm, em. 525
nm). (C) Bright-field image of cells in (B) stained with Hoechst
individual HeLa cells and set relative to the mean fluores-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
2
2
cence intensity prior to treatments (F/F). Error bars denote
i
standard deviations, ** P < 0.01.
2
O
2
Conclusionsꢀ
We have shown that α-boryl ethers and related structures
are excellent vehicles for releasing molecular cargo in an
oxidative environment. These compounds are accessed
from α-boryl alcohols that can be prepared by operational-
ly facile ketone or aldehyde borylation reactions. Although
these alcohols cannot be functionalized via their alkoxides,
they can be alkylated or acylated in the presence of weak
amine bases. Reductive alkylation provides an attractive
alternative to boryl ether formation under acidic conditions.
α-Boryl ethers release alcohols extremely rapidly in the
3
3258 (1 µM) and imaged using a DAPI (Set 68) filter (ex. 377
nm, em. 464 nm). (D) Mean fluorescence intensities were
calculated from individual ROIs (n = 3) and set relative to the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mean fluorescence intensity prior to treatments (F/F
bars denote standard deviations.
i
). Error
oxidatively stressed environments. Conducting these stud-
ies with the control acetal 34 again resulted in no fluoro-
2
5
phore release.
presence of H
2
2
O while α-boryl carbonates decompose
While these studies provide compelling evidence for the
capacity of α-boryl acetals to release cargo in cells, the
results would have significantly more impact if fluorophore
somewhat more slowly, providing a predictable mecha-
nism for controlling the rate of alcohol release. The capaci-
ty to functionalize α-boryl alcohols under acidic conditions
provides a pathway to generate α-boryl acetals. These
acid-stable structures readily release aldehydes and ke-
release could be achieved through endogenous H
2
O
2
gen-
eration. Phorbol myristate acetate (PMA) promotes intra-
4
5
cellular H
2
O generation. Therefore HeLa cells were incu-
2
tones upon exposure to H
2
2
O . The ability to liberate cargo
bated with PMA (1 µM) for 60 min followed by the addition
of 33 (10 µM). Fluorophore release in cells that were treat-
ed with PMA was evidenced by a significant increase of
fluorescence (Figure 7), in contrast to the lack of fluoro-
phore release in cells that were not treated with PMA.
These results clearly show the capacity of α-boryl acetals
to release compounds inside of cells in response to en-
at low substrate and peroxide concentrations was validat-
ed through the release of a fluorescent aldehyde. Fluoro-
phore release can also be achieved inside cells with exog-
enous H
2
O
2
or with endogenous, chemically stimulated
H
2
O
2
generation. The presence of the boronate group is
essential to these processes, in support of the proposed
pathway for the breakdown. The capacity to release mole-
cules inside cells with a sterically non-demanding oxidant
while generating non-toxic by-products indicates that the-
se compounds will be valuable for drug release in oxida-
tively stressed cells.
dogenous concentrations of H
2
2
O .
A
B
ASSOCIATEDꢀCONTENTꢀꢀ
Supporting Information
500 µm
500 µm
The Supporting Information is available free of charge on the
ACS Publications website.
**
C
D
4
3
2
1
0
Schemes for compound synthesis, characterization of all
compounds, general protocols for compound release, proce-
dures for fluorophore release in cells.
5
00 µm
AUTHOR INFORMATION
DMSO PMA
Corresponding Authors
Figure 7. Cellular fluorophore release in HeLa cells by en-
dogenous, PMA-stimulated H generation. Cells were pre-
* florean@pitt.edu, deiters@pitt.edu
2
O
2
Notes
treated in DMEM containing (A) DMSO or (B) PMA (1 uM) and
incubated at 37°C for 60 minutes. Media was replaced with
fresh DPBS containing 33 (10 uM) and cells were incubated
for an additional 60 min at 37°C before fluorescence was
imaged (Zeiss Axio Observer Z1, 20x objective, GFP filter (Set
The authors have applied for a provisional patent that covers
the drug delivery applications of this work.
ACKNOWLEDGMENT
3
8 HE; ex. 470 nm; em. 525 nm)). (C) Bright-field image of
We thank the National Institutes of Health (AI068021 for P. E.
F. and GM112728 for A. D.) and the National Science Foun-
dation (CHE-1362396 for P. E. F.) for generous support of
this work. Y. N. was supported in part by a Mellon Graduate
cells in (B) stained with Hoechst 33258 (1 µM) and imaged
using a DAPI filter (Set 68; ex. 377 nm; em. 464 nm). (D)
Mean fluorescence intensities were calculated from three
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 10
Fellowship. We thank Viswanathan Elumalai for assistance
with the NMR experiments.
(12) For a review, see: Böhme, D.; Beck-Sickinger, A. G. J.
Pept. Sci. 2015, 21, 186.
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37) Although ketone and aldehyde release under endogenous
ski, D.; Michałowski, B.; Dybala-Defratyka, A.; Wójcik, T.; Michal-
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conditions is limited to oximes and derivatives, light-mediated
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F.; Tao, S.-L.; Zhang, X.-H.; Wu, S.-K.; Lee, S.-T. Org. Lett.
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(41) A recent report has detailed the influence of leaving
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