A structurally simple self-immolative reagent that provides three distinct, simultaneous responses per detection event

Article abstract of DOI:10.1021/jo2018763

A general design is presented for a stimulus-responsive small molecule that is capable of responding to a specific applied chemical or physical signal by releasing two different types of pendant small molecules and a colorimetric indicator simultaneously. A key aspect of this design is the ease with which these reagents are prepared: typically, only four synthetic steps are required. Moreover, the modular construction strategy provides access to stimuli-responsive reagents that are capable of (i) responding to a variety of applied signals and (ii) releasing a number of different small molecules that contain primary alcohols, secondary alcohols, or phenols. These stimuli-responsive reagents are stable under physiological conditions (neither hydrolysis nor thermal degradation of the reagent occurs in significant quantity), and when they are exposed to the appropriate applied signal, they release both pendant small molecules and the colorimetric indicator completely within hours. Finally, unlike other functional groups, such as carbonates, that are used to connect alcohol-bearing molecules to controlled-release reagents, the linkage described in this article increases in hydrolytic stability (rather than decreases) as the pK_a of the pendant alcohol decreases (Figure presented).

Full text of DOI:10.1021/jo2018763

Article
pubs.acs.org/joc
A Structurally Simple Self-Immolative Reagent That Provides Three
Distinct, Simultaneous Responses per Detection Event
Sean A. Nunez, Kimy Yeung, Nicole S. Fox, and Scott T. Phillips*
̃
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
S
* Supporting Information
ABSTRACT: A general design is presented for a stimulus-
responsive small molecule that is capable of responding to a
specific applied chemical or physical signal by releasing two
different types of pendant small molecules and a colorimetric
indicator simultaneously. A key aspect of this design is the ease
with which these reagents are prepared: typically, only four
synthetic steps are required. Moreover, the modular construction
strategy provides access to stimuli-responsive reagents that are
capable of (i) responding to a variety of applied signals and
(ii) releasing a number of different small molecules that contain
primary alcohols, secondary alcohols, or phenols. These stimuli-responsive reagents are stable under physiological conditions
(neither hydrolysis nor thermal degradation of the reagent occurs in significant quantity), and when they are exposed to the
appropriate applied signal, they release both pendant small molecules and the colorimetric indicator completely within hours.
Finally, unlike other functional groups, such as carbonates, that are used to connect alcohol-bearing molecules to controlled-
release reagents, the linkage described in this article increases in hydrolytic stability (rather than decreases) as the pKa of the
pendant alcohol decreases.
INTRODUCTION
■
Stimuli-responsive small molecules that are capable of performing
more than one type of function in response to a specific applied
signal1−6 are needed for a variety of applications. These
applications include certain types of therapeutics (e.g., pro-
drugs),1−4,7−10 diagnostics,2,3,11−16 “smart” packaging (e.g., for
indicating and possibly preventing/slowing the spoilage of food),17
corrosion-indicating or anticorrosion coatings,18 and reagents for
applications in the agriculture and cosmetics industries, among
others.17 However, the development and application of stimuli-
responsive reagents that are capable of performing multiple
functions simultaneously has been limited by the lengthy syntheses
that are required to prepare these types of reagents.1−6 This article
describes a new design for stimuli-responsive reagents that are
capable of releasing two different types of pendant small molecules
and revealing a separate yellow indicator molecule (Figure 1), all
in response to a single, specific chemical detection event. This new
Figure 1. Design of a small-molecule controlled-release reagent that is
design should enable others to easily and quickly prepare
analogous reagents for a variety of applications, including
stimuli-responsive materials.18
capable of responding to a specific chemical signal by releasing two
different types of alcohols and simultaneously indicating the release
event by forming a yellow color. This type of controlled-release
reagent is accessible in ≤4 synthetic steps.
Background Leading to the Design of the Controlled-
Release Reagents Described Herein. The design shown in
Figure 1 builds upon nearly 60 years of studies in the area of
small-molecule controlled-release reagents.1−4 This research
area began with initial demonstrations of reagents that
responded to a signaling molecule by releasing a single pendant
molecule,19 which led to Katzenellenbogen’s revised design20
that connects an activity-based detection unit with a pendant
molecule through a linker (the general formula for this design is
activity-based detection unitlinkerpendant small mole-
cule).1,4,20−23 More recently, efforts by De Groot,24 Shabat,25,26
McGrath,27 and others1−4 have resulted in systems that use a
linker to release more than one copy of a pendant small
Received: September 20, 2011
Published: November 17, 2011
© 2011 American Chemical Society
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molecule (typically two to four copies) per detection event.
The function of these latter reagents has been expanded even
further, such that now they are capable of releasing more than
We based this design on our previous studies of an
autocatalytic reagent.29 Our expectation from these previous
studies was that aldehyde byproduct 5 would provide a bright
yellow color, which would serve as a direct indicator of the
detection and controlled-release events.30
one type of pendant small molecule per detection event.5,6,28
Despite the exceptional progress in these seminal studies,5,6,28
access to reagents that release more than one type of pendant
small molecule from a single reagent has been limited by lengthy
synthetic routes, which often require 17 or more synthetic steps.5
Herein, we provide one solution to these synthetic challenges for
creating dual- and triple-release reagents that liberate alcohol-
bearing small molecules.
RESULTS AND DISCUSSION
■
Procedure for Synthesizing Model Reagent 9. We
tested the efficacy of the design depicted in Figure 1 by
preparing a model reagent (compound 9, Scheme 1), which
a
Scheme 1. Synthesis of Model Compound 9
Design of a Synthetically Accessible Stimulus-
Responsive Reagent That Releases Two Different
Alcohols and a Colorimetric Indicator. A number of pH-
responsive acetal reagents have been prepared for the
controlled release of pendant alcohols under acidic conditions
via acid-catalyzed hydrolysis.4 Our design (Figure 1) draws on
acetal chemistry but differs in the mechanism of the response.
We sought a reagent that is stable at neutral pH yet is capable
of releasing the pendant alcohols in response to a specific
chemical signal, such as an enzyme, through an azaquinone
methide release mechanism (Figure 2), rather than through an
a
Legend: (a) DPPA, Et3N, dioxane, 100 °C (93%); (b) TMSOMe,
TMSOTf, CH2Cl2, 78 °C → 25 °C (95%).
was designed to release 2 equiv of methanol in response to
hydrogen peroxide. As shown in Scheme 1, model compound 9
was prepared from commercially available starting materials in
two synthetic steps31 and 88% overall yield. The brevity of this
synthesis is a substantial improvement over current controlled-
release reagents that release two copies of the same pendant
small molecule; such reagents typically require more than six
steps to prepare.32
Stability of Model Reagent 9. Since acetals are sensitive
to the pH of the solution in which they are dissolved,33 we
tested the stability of reagent 9 in 1:1 CD3CN−buffered D2O at
various pH values and temperatures.34 Under these conditions,
less than 6% of reagent 9 (7 mM) decomposed after 6 h of
exposure to 1:1 CD3CN−buffered D2O (2.5 mM phosphate
buffer, pH 7.0) at 25 °C, as measured by integration of the
1
Figure 2. Proposed mechanism for the release of both equivalents of
benzyl alcohol (3) from acetal reagent 1. We anticipated that
byproduct 5 would provide a bright yellow color as an indicator of the
detection and controlled-release events.29
acetal methine hydrogen in the H NMR spectrum relative to
the CHO peak in compound 8, which was the only byproduct
(other than methanol) observed in the 1H NMR spectrum.35As
expected, as the pH of the solution decreased (Figure 3b), the
acid-catalyzed decomposition of 9 increased, and as the pH of
the solution increased to pH 8 or greater, reagent 9 showed no
signs of decomposition over the 6 h exposure period.
Moreover, Figure 3c reveals that moderate temperature
fluctuations away from 25 °C have little effect on the
nonspecific hydrolysis of reagent 9. For example, in 1:1
CD3CN−buffered D2O (2.5 mM phosphate buffer, pH 8.0),
reagent 9 (7 mM) shows no signs of hydrolysis (i.e., 100 ± 1%
of the acetal remained) even after 6 h of heating at 35 °C. At
45 °C, only ∼3% of reagent 9 decomposed after 6 h, and
significant decomposition (13 ± 2% decomposition) was
observed only after reagent 9 was heated for 6 h at 55 °C.
Controlled Release of Methanol from Reagent 9 in
Response to Hydrogen Peroxide. While reagent 9 is stable
for many hours at neutral pH, it responds quickly and completely
to a specific applied chemical signal (i.e., hydrogen peroxide). 1H
acid-catalyzed hydrolysis pathway. While the benzylic acetal
shown in Figure 1 certainly is sensitive to acidic conditions, we
anticipated that the reagent would be stable under the
physiological conditions that would be encountered in the
context of many detection scenarios.
Our general design is based on the hypothesis that reaction
of the activity-based detection unit with an analyte (e.g.,
hydrogen peroxide)12−14 will release an aniline intermediate (2,
Figure 2) via formation of quinone methide and loss of carbon
dioxide. We reasoned that 2 would not be stable and instead
would release one alcohol (e.g., 3 in Figure 2) from the benzylic
acetal via formation of azaquinone methide 4.24 Addition of
water to 4 would make an unstable hemiacetal, which would
form aldehyde 5 by releasing the second pendant alcohol.
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Figure 4. Rate of release of methanol from model acetal 9 and control
carbonate 10. (a) The release event was triggered in both cases by
addition of 10 equiv of hydrogen peroxide−urea to either 9 or 10
(7 mM) in 1:1 CD3CN−buffered D2O (2.5 mM phosphate buffer, pH
8.0) at 25 °C. (b) The rate of release of methanol was monitored using
1H NMR by quantifying either the disappearance of the acetal methine
(9) or the appearance of CH3OD (10) relative to 2,5-dimethylfuran
(DMFu) (an internal standard).36 The data were normalized for
the number of equivalents of methanol being released. The release
experiments were performed in triplicate; the data points reflect the
average values (black, 9; blue, 10), and the error bars reveal the
standard deviation from the average. The error bars for the black data
points are smaller than the dots. The dotted line reflects the point above
which a yellow color becomes apparent visually (see part c for example
photographs) when 9 responds to hydrogen peroxide. When 20% of
reagent 9 releases both equivalents of methanol, for example, the
solution will have turned a distinct yellow color. (c) Visual color that
appears when 9 responds to hydrogen peroxide. The color does not
develop in the absence of hydrogen peroxide or for the carbonate 10,
even when 10 reacts with hydrogen peroxide. The reaction conditions
are the same as described in (a). The clarity of the photographs was
enhanced using Adobe Photoshop using the “Auto Levels” function.
Figure 3. Measurements of the stability of model reagent 9 (7 mM) in
1:1 CD3CN−buffered D2O solutions ranging in pH from 6 to 10 and
ranging in temperature from 15 to 55 °C. The buffer was 2.5 mM
phosphate buffer for solutions with pH values of 6−8 and was 2.5 mM
bicarbonate buffer for solutions with pH values of 9 and 10.
(a) Aldehyde 8 and methanol were the only byproducts observed
during these stability studies, and therefore, ratios of the 1H NMR
integration values of the aldehyde hydrogen relative to the acetal
methine hydrogen were used to calculate the percent of acetal 9
remaining at the conclusion of the stability experiments. Graphs (b) and
(c) reveal the percent of 9 remaining (relative to aldehyde 8) after 6 h of
exposure to the specific conditions. The experiments were performed in
triplicate, and the error bars reveal the standard deviations from the
average values. The experiments were conducted at 25 °C for (b) and at
pH 8.0 for the experiments represented in (c).
to that of the acetal. In the presence of hydrogen peroxide, both
equivalents of methanol are released slightly faster from acetal 9 than
the single equivalent is released from carbonate 10.
NMR analysis shows that 9 (7 mM) releases both equivalents of
methanol within ∼2 h when exposed to excess hydrogen
peroxide−urea (10 equiv) in 1:1 CD3CN−buffered D2O (2.5 mM
phosphate buffer, pH 8.0) at 25 °C (Figure 4).
Because carbonate linkages are a standard strategy for appending
an alcohol to a controlled-release reagent,1−4 we prepared control
compound 10 (Figure 4a) to compare the behavior of the carbonate
As expected,29 acetal 9 also reveals a colored byproduct when
hydrogen peroxide is detected (Figure 4c), thus providing a
visual indication of the detection event. In contrast, the
byproduct (i.e., p-aminobenzyl alcohol) of the reaction between
10 and hydrogen peroxide is not colored.
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Rate of Nonspecific Release of Methanol from
Reagent 9. The rate of background release of methanol due
to nonspecific hydrolysis of the acetal is also an important
consideration when designing a controlled-release reagent.
After 48 h in 1:1 CD3CN−buffered D2O (2.5 mM phosphate
buffer, pH 8.0, 25 °C), acetal 9 (7 mM) decomposed only
2.7 ± 1.0%, which is nearly identical with the rate of nonspecific
hydrolysis of carbonate 10 (7 mM) under the same conditions
(2.8 ± 0.1%) (Tables S5 and S6, Supporting Information).
The rate of nonspecific hydrolysis, however, is highly
dependent on the structure of the alcohol: carbonates derived
from acidic alcohols (e.g., phenols) hydrolyze much more readily
than carbonates derived from alcohols with pKa values closer to
16. The same increase in sensitivity is not observed with acetals
of the type depicted in Figure 1. For example, Figure 5 shows an
25 °C, acetal 11 (4 mM) showed no signs of nonspecific
hydrolysis, whereas carbonate 12 (4 mM) had hydrolyzed
completely within ∼4 h. These results demonstrate the versatility
and stability of acetal-based controlled-release reagents and
suggest that in the context of releasing phenols (or other acidic
alcohols) under neutral or slightly basic conditions (i.e., typical
physiological conditions), acetals may be a better choice than
carbonates for linking a pendant alcohol to the parent reagent.
Postulated Mechanism for the Controlled Release of
Alcohols from Benzylic Acetals. We performed two
experiments to explore the validity of the mechanism proposed
in Figure 2. Both of these experiments used model acetal 1
because the benzyl alcohol that is released absorbs at 254 nm
and is easily identifiable using LC-MS. The first experiment
involved treating 1 (1.7 mM) with 10 equiv of hydrogen
peroxide in 1:1 CH3CN−buffered water (2.5 mM phosphate
buffer, pH 8.0) at 25 °C, followed by LC-MS analysis of the
products. The UV-active compounds identified in the LC-MS
spectrum included direct products of the controlled-release
reaction (benzyl alcohol 13, phenol 14, and aniline 15) (Figure 6),
Figure 6. UV-active byproducts released upon addition of 10 equiv of
hydrogen peroxide−urea to model compound 1 (1.7 mM) in 1:1
CH3CN−buffered water (2.5 mM phosphate buffer, pH 8.0) at 25 °C.
Compounds 15−17 are yellow, thereby providing a visual indication
that hydrogen peroxide reacted with 1.
Figure 5. Rate of nonspecific hydrolysis of acetal 11 versus carbonate 12.
(a) Each compound was dissolved in 3:1 CD3CN−buffered D2O (2.5
mM phosphate buffer, pH 8.0) at 25 °C with 4 mM 11 or 12. (b) The
rate of nonspecific release of phenol from acetal 11 was monitored using
1H NMR by integration of the acetal methine hydrogen relative to 2,5-
dimethylfuran (DMFu) (an internal standard). The rate of nonspecific
hydrolysis of carbonate 12 was monitored using HPLC by integration of
peak areas for the hydrolysis product of 12 (the benzyl alcohol) relative
to anisole (an internal standard). The only products arising from
hydrolysis of 12 were the expected benzyl alcohol (i.e., the benzyl alcohol
that arises after hydrolysis of the carbonate; this benzyl alcohol is
compound 46 in the Supporting Information) and phenol. HPLC was
used instead of 1H NMR for quantifying the hydrolysis of 12 because of
overlap in the 1H NMR spectrum of the benzylic peaks for the carbonate
(12) and the hydrolyzed product. The release experiments were
performed in triplicate; the data points reflect the average values (red, 11;
blue, 12), and the error bars reveal the standard deviation from the
average. In most cases, the error bars are smaller than the dots.
as well as products (16 and 17) that arose from reaction of aniline
15 with the presumed quinone methide intermediate formed in
the release process.29 Because we found that compounds 15−17
are yellow,29 this experiment also validated our strategy for
releasing a colorimetric indicator for real-time monitoring of the
detection event.30
These LC-MS results confirm the products that we expected
from the proposed tandem quinone methide−azaquinone
methide release mechanism (Figure 2). While we cannot say
conclusively that the mechanism shown in Figure 2 is operative,
a second release experiment using H218O water instead of
H216O helps support the mechanistic hypothesis. This second
experiment was performed using conditions analogous to those
shown in Figure 6; however, when H218O was used instead of
H216O, the mass spectra for aldehydes 15−17 revealed nearly
complete incorporation of 18O as the aldehyde oxygen (see the
Supporting Information for details). This result is consistent
acetal (compound 11) that releases phenol. After 12 h in 3:1
CD3CN−buffered D2O (2.5 mM phosphate buffer, pH 8.0) at
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with the notion that the azaquinone methide intermediate 4
(Figure 2) is intercepted by water to form a hemiacetal that
then releases the second alcohol.
Access to Diverse Activity-Based Detection Units and
Acetals. Having established that the design shown in Figure 1
provides stable reagents that are capable of releasing two pendant
primary alcohols, our next goal was to explore the scope of the
design by evaluating the compatibility of the synthesis procedure
with different activity-based detection units (which often are
called triggers in related literature) and a variety of pendant
alcohols.
(a). Activity-Based Detection Units. Figure 7 shows five
representative controlled-release reagents, each containing a
different activity-based detection unit. The chosen activity-
based detection units should enable the reagents to release
pendant alcohols in response to hydrogen peroxide (9), UV
light (19), palladium(0) (21), fluoride ion (23), and penicillin
G amidase (25). All reagents (with the exception of 25; see the
Supporting Information) were prepared using procedures
analogous to those shown in Scheme 1 and are designed to
release methanol as a model pendant alcohol. For example,
exposure of 9 to hydrogen peroxide, 19 to UV light, and 23 to
fluoride ion releases both equivalents of methanol from each
reagent and provides the expected byproducts 15−17 with
rates that depend on the dose of the signal and the rate of
reaction between the signal and the activity-based detection
unit (an example stacked plot of time-dependent LC-MS traces
for 23 when exposed to fluoride is provided in Figure S2 in the
Supporting Information). The uniformly high yields (82−96%)
for acetal formation in the presence of these activity-based
detection units highlight the mild reaction conditions used in
the preparation of these highly functionalized reagents.31
(b). Acetals. The procedure for attaching alcohols to the
controlled-release reagent is mild and effective for attaching
primary alcohols (9, 1, 26, 27), secondary alcohols (28, 29),
and phenols (11) (Figure 8). As expected, hindered alcohols
such as tertiary alcohols (30) are not easily prepared and
isolated.37 The procedure also provides direct access to mixed
acetals, such as 26. In response to hydrogen peroxide, mixed
acetal 26 releases both ethanol and benzyl alcohol, in addition
to producing colorimetric byproducts 15−17. Compound
26 was prepared in only two synthetic steps38 and 62%
overall yield, which is a substantial improvement over state-of-
the-art controlled-release reagents that have similar levels of
function.5
Controlled Release in Response to β-D-Glucuronidase:
Release of Two Types of Fragrances and a Colorimetric
Indicator. Our straightforward route to reagents that
simultaneously release two different types of alcohols and a
colorimetric indicator allowed us to prepare a demonstration
reagent that responds to β-D-glucuronidase, which is an enzyme
marker of Escherichia coli (E. coli).39 This demonstration
reagent was prepared in seven synthetic steps (Scheme 2),
three of which were needed to prepare the activity-based
detection unit. The demonstration reagent is capable of
responding to β-D-glucuronidase in 30 mM phosphate-buffered
water (pH 7.4) at 30 °C by releasing citronellol, 2-
phenylethanol, and yellow indicator molecules.
Figure 7. Synthesis of five model controlled-release reagents, each
containing a different activity-based detection unit. The reagents are
designed to release 2 equiv of methanol in response to the following
signals: hydrogen peroxide (9); UV light (19); palladium(0) (21);
fluoride (23); penicillin G amidase (25).
the anomeric position of the β-D-glucuronic acid unit. This first
step is complete less than 20 min after exposure of 36 to β-D-
glucuronidase. The amount of the phenol intermediate
decreases slowly over the next 378 min until it is barely
detectable by LC-MS. Peaks corresponding to citronellol (37),
2-phenylethanol (38), 4-aminobenzaldehyde (15), colored
products 16 and 17, and phenol 14 all increase in intensity
In the presence of 125 units of β-D-glucuronidase, time-dependent
LC-MS analysis of the reaction products reveals that reagent 36 (1.9
mM in 30 mM phosphate-buffered water (containing 18% MeOH),
pH 7.4, 30 °C) proceeds through a stepwise release mechanism
in which the enzyme catalyzes the hydrolysis of phenol from
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CONCLUSIONS
■
This article provides a new design for a stimulus-responsive
small molecule that is capable of releasing either two copies of a
pendant alcohol and a colorimetric indicator in response to an
applied signal or of releasing two different types of alcohols and
a colorimetric indicator simultaneously. The reagent is
accessible typically in four or fewer synthetic steps, which is
exceedingly efficient with respect to the function achieved for
the number of synthetic steps required to prepare the reagent.
In addition to being efficient, the procedure for preparing
these reagents is modular and thus provides access to a range of
controlled-release reagents that are capable of responding to a
variety of applied signals and releasing an assortment of
pendant alcohols, including phenols. Extension of the system to
the controlled release of phenol-based fluorescent dyes such as
7-hydroxycoumarin should provide straightforward access to
selective and sensitive sensor reagents that are capable of signal
amplification.
Despite the acid-sensitive acetal functionality, the reagents
described herein are stable for hours under physiological
conditions and are capable of responding to specific applied
signals, such as the enzyme β-D-glucuronidase. This combina-
tion of ready availability, stability, and rapid and diverse
response characteristics may now enable the preparation of new
controlled-release reagents with multiple, diverse functions.
Such reagents have the potential for use in stimuli-responsive
materials,40 including providing a possible starting point for
creating new stimuli-responsive shell walls in responsive
capsules.18,41−44
EXPERIMENTAL SECTION
■
General Experimental Methods. All reactions were performed
in flame-dried glassware under a positive pressure of nitrogen, unless
otherwise noted. Air- and moisture-sensitive liquids were transferred
by syringe or cannula. Organic solutions were concentrated by rotary
evaporation (1−20 mmHg; the pressure used depended on the boiling
point of the solvent) at ambient temperature, unless otherwise noted.
Benzyloxytrimethylsilane, ethoxytrimethylsilane, isopropoxytrimethyl-
silane, methoxytrimethylsilane, trimethyl(phenoxy)silane, and all other
reagents were purchased commercially and were used as received
unless otherwise noted. Acetonitrile, dichloromethane, 1,4-dioxane,
N,N-dimethylformamide, and tetrahydrofuran were purified by the
method of Pangborn et al.45 Flash column chromatography was
performed as described by Still, Kahn, and Mitre,46 employing silica gel
(60 Å pore size, 32−63 μm, standard grade, Dynamic Adsorbents).
Thin-layer chromatography was carried out on Dynamic Adsorbents
silica gel TLC (20 × 20 w/h, F-254, 250 μm). Deionized water was
purified using a Millipore purification system (Barnstead EASYpure II
UV/UF).
Figure 8. Synthesis of different types of acetals: (a) procedure used to
prepare the acetals; (b) eight different acetals, including mixed
acetal 26.
beginning ∼20 min after first exposure of 36 to β-D-
glucuronidase (Figure 9a and Figure S1 (Supporting
Information)), and at the end of the 378 min time period,
the release of citronellol (37) and 2-phenylethanol (38) from
36 is nearly complete (Figure 9b). In the absence of β-D-
glucuronidase (but under otherwise identical reaction con-
ditions), 36 releases only 3% of citronellol (37) and 1% of
2-phenylethanol (38) after 378 min.
In addition to the analytical LC-MS results, qualitative
confirmation of the detection event is provided both by the
yellow color that is produced when 36 reacts with β-D-
glucuronidase (Figure 9c) and by the fresh, floral smell that is
emitted (see the Supporting Information for details)
(citronellol is a lemon-scented compound, and 2-phenylethanol
has a floral odor), which may be useful in the context of
personal hygiene products.17 In contrast, in the absence of β-D-
glucuronidase, no color is observed and no scent is detected
over the course of 378 min.
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded at 25 °C. Proton chemical shifts are expressed in parts per
million (ppm, δ scale) and are referenced to tetramethylsilane
((CH3)4Si, 0.00 ppm) or to residual protium in the solvent (CHCl3,
7.27 ppm; CD2HOD, 3.31 ppm; DHO, 4.67 ppm; CD2HCN, 1.97
ppm).47 Data are represented as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet and/or
multiple resonances, br s = broad singlet), integration, and coupling
constant (J) in hertz. Carbon nuclear magnetic resonance spectra (13C
NMR) were recorded at 25 °C. Carbon chemical shifts are expressed
in parts per million (ppm, δ scale) and are referenced to the carbon
resonances of the NMR solvent (CDCl3, δ 77.0). Photographs were
obtained using a Nikon digital camera (D40). Pictures of samples
indicating a colorimetric response (Figures 4c and 9c) were balanced
with the “Auto Levels” function in Adobe Photoshop. LC analyses
were performed using a reversed-phase phenyl-hexyl column (150 mm ×
2.1 mm, 5 μm particle size). The column was equilibrated at 1:9
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Scheme 2. Synthesis of Reagent 36, Which Releases Citronellol (37), 2-Phenylethanol (38), and a Yellow Indicator When
a
Exposed to β-D-Glucuronidase in 30 mM Phosphate-Buffered Water (pH 7.4) at 30 °C
a
Legend: (a) 4-hydroxybenzaldehyde, Ag2O, MeCN (52%); (b) NaBH4, 1:1 CH2Cl2−MeOH (100%); (c) (i) 4-formylbenzoic acid, (COCl)2,
CH2Cl2, DMF, (ii) NaN3, H2O, acetone, (iii) 110 °C, toluene, (iv) 33, CH2Cl2 (82%); (d) TMSO-citronellol, TMSO-(CH2)2Ph, molecular sieves,
TMSOTf, CH2Cl2 (33%); (e) LiOH, H2O, MeOH, THF, 0 °C.
acetonitrile−water at a 0.5 mL/min flow rate, and after injection of the
sample, the gradient was ramped as follows: 1:3, 3 min; 1:1, 6 min, 3:1,
9 min; 9:1, 12 min; 1:9, 17 min, over a 20 min combined elution time.
Tandem LC−MS analyses were performed using the same LC method
with a substitution of the mobile phase to solutions of acetonitrile and
water that each contained 5 mM ammonium formate. The MS (ES)
parameters were as follows: gas temperature of 350 °C, drying gas flow
of 10−13 L/min, nebulizer pressure of 40−60 psig, and a voltage of
3000 V.
(CDCl3): δ 0.25 (s, 6H), 1.06 (s, 9H), 4.46 (s, 2H), 6.85 (d, 2H, J =
8.4 Hz), 7.19 (d, 2H, J = 8.4 Hz). The 1H NMR data are in agreement
with literature values.49
General Procedure for the Curtius Rearrangement: Procedure A.
To a round-bottom flask were added sequentially 4-formylbenzoic acid
(6; 1 equiv), 1,4-dioxane (to make a 0.5 M solution), triethylamine
(1.1 equiv), diphenylphosphoryl azide (1.1 equiv), and the desired
alcohol (1.1 equiv). The flask was equipped with a cold-finger reflux
condenser, and the reaction mixture was heated to 105 °C. After it was
stirred for 2 h at 105 °C, the product mixture was cooled to 25 °C. 1,4-
Dioxane was removed by rotary evaporation, and the resulting residue
was dissolved in a minimum of dichloromethane and was adsorbed
directly onto silica by rotary evaporation. The product was isolated by
silica gel flash column chromatography.
Synthesis Procedures. 4-(tert-Butyldimethylsilyloxy)benzalde-
hyde (39). To a round-bottom flask was added 4-hydroxybenzalde-
hyde (1.0 g, 8.2 mmol, 1 equiv), tetrahydrofuran (41 mL), and
imidazole (1.1 g, 16 mmol, 2.0 equiv). The flask was cooled to 0 °C,
and tert-butyldimethylsilyl chloride was added. The flask was warmed
to 25 °C and was stirred for 12 h. The product mixture was
concentrated by rotary evaporation. The residue was dissolved in
dichloromethane and was adsorbed directly onto silica by rotary
evaporation. Purification by silica gel flash column chromatography
(5% ethyl acetate in hexanes, increasing to 20% ethyl acetate) afforded
4-(tert-butyldimethylsilyloxy)benzaldehyde (39; 1.8 g, 7.5 mmol, 92%)
(a). Allyl 4-Formylphenylcarbamate (20). Reagents: 4-formylben-
zoic acid (6; 0.50 g, 3.3 mmol, 1 equiv), 1,4-dioxane (6.6 mL),
triethylamine (0.46 mL, 3.6 mmol, 1.1 equiv), diphenylphosphoryl
azide (0.78 mL, 3.6 mmol, 1.1 equiv), and allyl alcohol (0.25 mL, 3.6
mmol, 1.1 equiv). The residue was purified by silica gel flash column
chromatography (10% ethyl acetate in hexanes, increasing to 40%
ethyl acetate) to afford allyl 4-formylphenylcarbamate (20; 0.54 g, 2.6
mmol, 80%) as a pale yellow solid. Mp: 116 °C. IR (cm−1): 3312,
1
as a yellow oil. H NMR (CDCl3): δ 0.26 (s, 6H), 1.01 (s, 9H), 6.94
1
(d, 2H, J = 8.9 Hz), 7.80 (d, 2H, J = 8.0 Hz), 9.90 (s, 1H). The H
NMR data are in agreement with literature values.48
1
1730, 1674, 1594, 1527. H NMR (CDCl3): δ 4.69 (d, 2H, J = 5.4
4-(tert-Butyldimethylsilyloxy)phenylmethanol (40). To a round-
bottom flask was added sequentially 4-(tert-butyldimethylsilyloxy)-
benzaldehyde (39; 0.78 g, 3.2 mmol, 1 equiv), methanol (32 mL), and
sodium borohydride (0.18 g, 4.9 mmol, 1.5 equiv). The reaction
mixture was stirred at 25 °C for 2 h. The product mixture was diluted
with saturated aqueous ammonium chloride (5 mL), and this solution
was extracted using dichloromethane (3 × 20 mL). The organic
extracts were combined, washed sequentially with water (1 × 20 mL)
and brine (1 × 20 mL), and dried over sodium sulfate. Sodium sulfate
was removed by filtration, and the solution was concentrated to
dryness by rotary evaporation to afford 4-(tert-butyldimethylsilyloxy)-
phenylmethanol (40; 0.49 g, 2.0 mmol, 62%) as a yellow oil. 1H NMR
Hz), 5.26 (dd, 1H, J = 0.8 Hz, J = 2.0 Hz), 5.29 (dd, 1 H, J = 1.0 Hz,
J = 2.0 Hz), 5.90−6.00 (m, 1H), 7.46 (br s, 1H), 7.62 (d, 2H, J = 8.4
Hz), 7.85 (d, 2H, J = 8.0 Hz), 9.90 (s, 1H). 13C NMR (CDCl3): δ
191.1, 152.7, 143.7, 131.8, 131.4, 130.4, 118.5, 117.8, 66.1. MS (TOF
MS ES+, m/z): (M + H)+, 206.1. HRMS (TOF MS ES+, m/z): calcd
for C11H12NO3 (M + H)+ 206.0817, found 206.0799.
(b). 2-Nitrobenzyl 4-Formylphenylcarbamate (16). Reagents:
4-formylbenzoic acid (6; 0.50 g, 3.3 mmol, 1 equiv), 1,4-dioxane
(6.6 mL), triethylamine (0.46 mL, 3.6 mmol, 1.1 equiv),
diphenylphosphoryl azide (0.78 mL, 3.6 mmol, 1.1 equiv), and 2-
nitrobenzyl alcohol (0.56 g, 3.6 mmol, 1.1 equiv). The residue was
purified by silica gel flash column chromatography (10% ethyl acetate
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7.64−7.72 (m, 2H), 7.87 (d, 2H, J = 8.7 Hz), 8.15 (d, 1H, J = 7.9 Hz),
9.93 (s, 1H). 13C NMR (CDCl3): δ 152.7, 147.4, 137.5, 133.7, 133.4,
132.4, 128.9, 128.7, 127.5, 125.0, 118.3, 102.6, 63.5. MS (MS ES+, m/
z): (M + H)+, 301.3. HRMS (TOF MS ES+, m/z): calcd for
C15H13N2O5 (M + H)+ 301.0824, found 301.0819.
(c). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-For-
mylphenylcarbamate (8). Reagents: 4-formylbenzoic acid (6; 1.0 g,
6.7 mmol, 1 equiv) 1,4-dioxane (13.4 mL), triethylamine (0.93 mL, 7.3
mmol, 1.1 equiv), diphenylphosphoryl azide (1.6 mL, 7.3 mmol, 1.1
equiv), and 4-(hydroxymethyl)phenylboronic acid pinacol ester (7; 1.7 g,
7.3 mmol, 1.1 equiv). The residue was purified by silica gel flash
column chromatography (10% ethyl acetate in hexanes, increasing to
40% ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl 4-formylphenylcarbamate (8; 2.4 g, 6.2 mmol, 93%) as a
pale yellow solid. Mp: 40 °C. IR (cm−1): 3274, 2977, 1733, 1684,
1593, 1530. 1H NMR (CDCl3): δ 1.34 (s, 12H), 5.21 (s, 2H), 7.36 (d,
2H, J = 7.9 Hz), 7.45 (br s, 1H), 7.57 (d, 2H, J = 8.5 Hz), 7.79−7.81
(m, 4H), 9.87 (s, 1H). 13C NMR (CDCl3): δ 191.9, 153.3, 144.1,
139.0, 135.5, 132.0, 131.7, 127.8, 127.6, 118.3, 84.4, 67.7, 25.3, 25.2.
+
MS (TOF MS ES+, m/z): (M + NH4 ). HRMS (TOF MS ES+, m/z):
+
calcd for C21H28BN2O5 (M + NH4 ) 399.2091, found 399.2099.
(d). 4-(tert-Butyldimethylsilyloxy)benzyl 4-Formylphenylcarba-
mate (22). Reagents: 4-formylbenzoic acid (6; 0.28 g, 1.8 mmol,
1 equiv), 1,4-dioxane (3.7 mL), triethylamine (0.26 mL, 2.0 mmol, 1.1
equiv), diphenylphosphoryl azide (0.44 mL, 2.0 mmol, 1.1 equiv), and
4-(tert-butyldimethylsilyloxy)phenylmethanol (40; 0.49, 2.0 mmol, 1.1
equiv). The residue was purified by silica gel flash column
chromatography (5% ethyl acetate in hexanes, increasing to 40%
ethyl acetate) to afford 4-(tert-butyldimethylsilyloxy)benzyl 4-
formylphenylcarbamate (22; 0.42 g, 0.78 mmol, 42%) as a pale yellow
solid. Mp: 122−123 °C. IR (cm−1): 3317, 2952, 2855, 1722, 1675,
1
1589, 1507. H NMR (CDCl3): δ 0.18 (s, 6H), 0.98 (s, 9H), 5.12 (s,
2H), 6.82 (d, 2H, J = 8.4 Hz), 7.25 (d, 2H, J = 8.3 Hz), 7.57 (d, 2H,
J = 8.5 Hz), 7.78 (2H, d, J = 8.6 Hz), 9.84 (s, 1H). 13C NMR
(CDCl3): δ 191.0, 156.1, 152.9, 143.7, 131.6, 131.3, 130.2, 128.2,
120.2, 118.0, 67.3, 25.6, 18.2, −4.4. MS (ES+, m/z): (M + H+), 386.4.
HRMS (TOF MS ES+, m/z): calcd for C21H28NO4Si (M + H+)
386.1788, found 386.1775.
N-(4-(Hydroxymethyl)phenyl)-2-phenylacetamide (41). To a
round-bottom flask was sequentially added 4-aminobenzyl alcohol
(1.0 g, 8.1 mmol, 1 equiv), potassium acetate (1.6 g, 16 mmol, 2.0
equiv), and N,N-dimethylformamide (81 mL). The white suspension
was cooled to −60 °C, and phenacetyl chloride (1.1 mL, 8.2 mmol, 1.0
equiv) was added dropwise to the suspension; each drop of phenacetyl
chloride caused a yellow discoloration of the reaction suspension, and
this color was allowed to dissipate before addition of the next drop of
phenacetyl chloride. The resulting suspension was warmed to 25 °C
and then was diluted with aqueous sodium hydroxide solution (20 mL,
1.0 M). The reaction solution was neutralized to pH 7 with aqueous
hydrochloric acid solution (1.0 M), and the product mixture was
extracted using dichloromethane (2 × 40 mL). The combined
dichloromethane extracts were washed sequentially with water (2 × 40
mL) and brine (1 × 40 mL) and then were dried over sodium sulfate.
The sodium sulfate was removed by filtration, and the resulting
solution was concentrated by rotary evaporation. N-(4-(Hydroxymethyl)-
phenyl)-2-phenylacetamide (41; 1.9 g, 7.9 mmol, 94%) was used without
Figure 9. A stimulus-responsive small molecule that simultaneously
releases 2-phenylethanol, citronellol, and yellow indicator molecules in
response to β-D-glucuronidase under physiological conditions. (a) The
stimulus-responsive reagent and the products arising from the release
reaction. Products 15−17 provide the yellow color. (b) Rate of release
of 2-phenylethanol (38) (blue) and citronellol (37) (red) when 36
(1.9 mM) is exposed to 125 units of β-D-glucuronidase in 4.6:1
phosphate buffer (30 mM, pH 7.4, containing 0.4% bovine serum
albumin (w/v))−methanol at 30 °C. The circular data points were
obtained in the presence of β-D-glucuronidase, and the square data
points were obtained in the absence of β-D-glucuronidase. The percent
release of citronellol and 2-phenylethanol was obtained by integration
of HPLC peak areas (210 nm) of citronellol and 2-phenylethanol
relative to anisole (an internal standard). No detectable change in
relative peak areas occurred beyond 378 min; therefore, the final data
point for each was set to 100%. (c) Time-dependent photographs of
36 in the absence (left) and presence (right) of β-D-glucuronidase
under the same conditions as described in (b). The yellow color
produced from the detection event is lighter in this figure than the
color shown in Figure 4c because the concentration of the stimulus-
responsive reagent (36) in this figure is 3.7-fold lower than the
concentration of the reagent in Figure 4c. The clarity of the
photographs was enhanced using the “Auto Levels” function in
Adobe Photoshop.
1
further purification. H NMR (CD3OD): δ 3.63 (s, 2H), 4.53 (s, 2H),
7.21−7.33 (m, 7H), 7.51−7.54 (m, 2H). The 1H NMR data are in
agreement with literature values.50
N-(4-Formylphenyl)-2-phenylacetamide (24). To a round-bottom
flask containing dimethyl sulfoxide (0.71 mL, 10 mmol, 5.0 equiv) at
25 °C was added a solution of oxalyl chloride (0.39 mL, 4.5 mmol,
2.3 equiv) in dichloromethane (25 mL) at −78 °C. The resulting
mixture was stirred for 10 min at −78 °C. To this cold reaction
mixture was added a −78 °C solution of N-(4-(hydroxymethyl)-
phenyl)-2-phenylacetamide (41; 0.50 g, 2.0 mmol, 1 equiv) dissolved
in THF (10.0 mL). The resulting reaction solution was stirred for 15
min at −78 °C. Triethylamine (1.4 mL, 10 mmol, 5.0 equiv) was
added dropwise. The reaction mixture was stirred for 15 min at −78
°C and then was warmed to 25 °C. Water (3 mL) was added to the
in hexanes, increasing to 50% ethyl acetate) to afford 2-nitrobenzyl
4-formylphenylcarbamate (16; 0.68 g, 2.2 mmol, 69%) as a pale yellow
1
solid. Mp: 177−179 °C. IR (cm−1): 3306, 1729, 1683, 1592, 1522. H
NMR (CDCl3): δ 5.66 (s, 2H), 7.09 (br s, 1H), 7.46−7.63 (m, 3H),
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product solution, followed by dichloromethane (15 mL). The layers
were separated, and the organic layer was washed sequentially with
water (2 × 25 mL) and brine (1 × 25 mL) and then was dried over
sodium sulfate. The sodium sulfate was removed by filtration, and the
filtrate was concentrated by rotary evaporation. The resulting residue
was purified by silica gel flash column chromatography (20% ethyl
acetate in hexanes, increasing to 60% ethyl acetate) to afford N-(4-
formylphenyl)-2-phenylacetamide (24; 0.49 g, 2.0 mmol, 100%) as a
yellow solid. Mp: 106 °C. IR (cm−1): 3257, 3191, 2843, 2745, 1694,
concentrated by rotary evaporation. The resulting residue was
dissolved in toluene (to make a 0.20 M solution), and the flask was
affixed with a cold-finger reflux condenser. The reaction mixture
was heated to 110 °C and held at that temperature until the azide was
converted to the isocyanate (as determined by thin-layer chromato-
graphy). The reaction flask was cooled to 25 °C, and a solution of the
desired alcohol (1.1 equiv) in dichloromethane (0.2 M) was added to
the isocyanate solution. The reaction mixture was stirred at 25 °C for
12 h or until the isocyanate was consumed (as determined by thin-
layer chromatography). Dichloromethane and toluene were removed
from the product mixture by rotary evaporation, and the residue was
dissolved in ethyl acetate. The organic solution was washed
sequentially with saturated aqueous sodium bicarbonate solution
(1×), water (1×), and brine (1×) and then was dried over sodium
sulfate. The sodium sulfate was removed by filtration, and the solution
was concentrated by rotary evaporation. The resulting residue was
purified by silica gel flash column chromatography.
1
1660, 1592, 1530. H NMR (CDCl3): δ 3.76 (s, 2H), 7.31−7.40 (m,
5H), 7.64 (d, 2H, J = 8.5 Hz), 7.80 (d, 2H, J = 8.6 Hz), 7.85 (br s,
1H), 9.89 (s, 1H). 13C NMR (CDCl3): δ 191.1, 169.7, 143.4, 133.9,
132.2, 131.0, 129.4, 129.3, 127.8, 119.3, 44.8. MS (TOF MS ES+, m/
z): (M + H+), 240.1. HRMS (TOF MS ES+, m/z): calcd for
C15H14NO2 (M + H+) 240.1025, found 240.1013.
Methyl 1-(4-Formylphenyl)-2,3,4-tri-O-acetyl-β-D-glucopyranuro-
nate (32). To a round-bottom flask was added sequentially methyl
2,3,4-tri-O-acetyl-D-glucopyranosyluronate bromide (31; 1.1 g, 2.7
mmol, 1 equiv), 4-hydroxybenzaldehyde (0.37 g, 3.05 mmol, 1.1
equiv), acetonitrile (30 mL), and silver(I) oxide (1.6 g, 6.9 mmol, 2.5
equiv). The flask was wrapped in aluminum foil, and the reaction
mixture was stirred for 18 h at 25 °C. The mixture was filtered through
a plug of Celite 545, with ethyl acetate as eluent (50 mL). The filtrate
was collected, and the solvent was removed by rotary evaporation. The
resulting residue was dissolved in ethyl acetate (75 mL), and the
solution was washed with saturated aqueous sodium bicarbonate (5 ×
50 mL). The organic layer was washed with brine (3 × 50 mL) and
then dried over sodium sulfate. Sodium sulfate was removed by
filtration, and the resulting solution was concentrated by rotary
evaporation. The resulting residue was purified by silica gel flash
column chromatography (25% acetone in hexanes) to afford methyl
1-(4-formylphenyl)-2,3,4-tri-O-acetyl-β-D-glucopyranuronate (32; 0.63
(a). (2S,3S,4S,5R,6S)-Methyl 3,4,5-Triacetoxy-6-(4-((4-
formylphenylcarbamoyloxy)methyl)phenoxy)-tetrahydro-2H-
pyran-2-carboxylate (34). Reagents: (i) 4-formylbenzoic acid (6;
0.25 g, 1.7 mmol, 1 equiv), oxalyl chloride (0.29 mL, 3.3 mmol, 2.0
equiv), N,N-dimethylformamide (1 drop), dichloromethane (8.4 mL),
acetone (8.4 mL); (ii) sodium azide (0.22 g, 3.3 mmol, 2.0 equiv),
water (8.4 mL); (iii) 1-(4-hydroxymethylphenyl)-2,3,4-tri-O-acetyl-β-
D-glucopyronuronate (35; 0.81 g, 1.8 mmol, 1.1 equiv), toluene (8.4
mL), dichloromethane (8.4 mL). The residue was purified by silica gel
flash column chromatography (10% ethyl acetate in hexanes,
increasing to 60% ethyl acetate) to afford (2S,3S,4S,5R,6S)-methyl
3,4,5-triacetoxy-6-(4-((4-formylphenylcarbamoyloxy)methyl)-
phenoxy)tetrahydro-2H-pyran-2-carboxylate (34; 0.80 g, 1.4 mmol,
82%) as a white solid. Mp: 75−78 °C. IR (cm−1): 3351, 2975, 1729,
1
1596, 1531. H NMR (CDCl3): δ 2.04−2.09 (m, 9H), 3.71 (s, 3H),
1
g, 1.4 mmol, 52%) as a white solid. H NMR (300 MHz, CDCl3): δ
4.22 (d, 2H, J = 11 Hz), 5.15−5.18 (m, 3H), 5.25−5.36 (m, 3H), 6.98
(d, 2H, J = 8.0 Hz), 7.34 (d, 2H, J = 8.0 Hz), 7.45 (br s, 1H), 7.58 (d,
2H, J = 8.0 Hz), 7.81 (d, 2H, J = 8.0 Hz), 9.88 (s, 1H). 13C NMR
(CDCl3): δ 190.9, 170.0, 169.3, 169.1, 166.8, 156.7, 152.7, 143.5,
131.6, 131.2, 130.7, 130.1, 118.0, 117.1, 98.8, 72.6, 71.7, 71.0, 69.0,
9.88 (s, 1H), 7.83 (d, 2 H, J = 8.8 Hz), 7.09 (d, 2 H, J = 8.7 Hz), 5.36
(m, 3H), 5.30 (m, 3H), 4.27 (m, 1H), 3.68 (s, 3H), 2.02 (s, 9H). The
1H NMR data are in agreement with literature values.51
Methyl 1-(4-Hydroxymethylphenyl)-2,3,4-tri-O-acetyl-β-D-gluco-
pyronuronate (33). To a round-bottom flask was added methyl
1-(4-formylphenyl)-2,3,4-tri-O-acetyl-β-D-glucopyranuronate (32; 0.59 g,
1.3 mmol, 1 equiv) and dichloromethane (14 mL), and the mixture
was cooled to 0 °C. A solution of sodium borohydride (56 mg, 49
mmol, 1.1 equiv) in methanol (13.5 mL) was added via syringe, and
the resulting reaction mixture was stirred at 0 °C for 30 min. The
mixture was diluted with saturated aqueous ammonium chloride
solution (75 mL), and the aqueous layer was extracted sequentially
using dichloromethane (3 × 25 mL) and ethyl acetate (3 × 25 mL).
The organic layers were combined and dried over sodium sulfate.
Sodium sulfate was removed by filtration, and the solution was
concentrated by rotary evaporation to afford methyl 1-(4-hydroxy-
methylphenyl)-2,3,4-tri-O-acetyl-β-D-glucopyronuronate (33; 0.45 g,
1.0 mmol, 76%) as a white foam. 1H NMR (300 MHz, CDCl3): δ 7.29
(d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J = 8.7 Hz), 5.33−5.22 (m, 3H), 4.60
(s, 1H), 4.18−4.15 (m, 1H), 3.70 (s, 1H), 2.09 (s, 1H), 2.03 (d, 9H,
J = 2.5 Hz). The 1H NMR data are in agreement with literature values.51
General Procedure for the Curtius Rearrangement: Procedure B.
To a round-bottom flask was added 4-formylbenzoic acid (6; 1 equiv),
N,N-dimethylformamide (1 drop), and dichloromethane (to make a
0.2 M solution). The reaction mixture was cooled to 0 °C, and oxalyl
chloride (2.0 equiv) was added dropwise to the reaction mixture. The
mixture was stirred for 20 min at 0 °C and then was warmed to 25 °C.
Dichloromethane was removed from the reaction mixture by rotary
evaporation. The resulting crude acid chloride solution was dissolved
in acetone (to make a 0.2 M solution), and the solution was cooled to
0 °C. To the acid chloride solution was added to a 5 °C solution of
sodium azide (2.0 equiv) in water to make a 0.4 M solution. The
reaction mixture was stirred until the acid chloride was consumed, as
determined by thin-layer chromatography. The reaction mixture was
diluted with water, and the biphasic solution was extracted twice using
ethyl acetate. The combined organic layers were dried over sodium
sulfate, the solids were removed by filtration, and the solution was
+
66.8, 52.9, 20.5 (double intensity), 20.4. MS (ES+, m/z): (M + NH4 ),
605.1. HRMS (TOF MS ES+, m/z): calcd for C28H33N2O13 (M +
+
NH4 ) 605.1983, found 605.1958.
General Procedure for TMSCl-Catalyzed HMDS Protection of
Alcohols. To a round-bottom flask was added the desired alcohol (1
equiv) and hexamethyldisilazane (0.6 equiv). Chlorotrimethylsilane
(0.05 equiv) was added dropwise via syringe. After being stirred
overnight at 25 °C, the suspension was purified directly using silica gel
flash column chromatography to afford the pure silyl ether.
(3,7-Dimethyloct-6-enyloxy)trimethylsilane (42). Reagents:
(±)-β-citronellol (37; 3.0 g, 19 mmol, 1 equiv), hexamethyldisilazane
(2.4 mL, 11 mmol, 0.60 equiv), chlorotrimethylsilane (0.12 mL, 0.95
mmol, 0.05 equiv). The product was purified by silica gel flash column
chromatography (2% ethyl acetate in hexanes, increasing to 10% ethyl
acetate) to afford (3,7-dimethyloct-6-enyloxy)trimethylsilane (42; 4.3 g,
18 mmol, 98%, colorless oil) as a mixture of enantiomers. IR (cm−1):
1
2956, 2920. H NMR (CDCl3): δ 0.12 (s, 9H), 0.83−0.87 (m, 3H),
1.17−1.32 (m, 4H), 1.56−1.64 (m, 7H), 1.95−1.97 (m, 2H) 3.55−
3.68 (m, 2H), 5.07 (t, 1H, J = 7.0 Hz). 13C NMR (CDCl3): δ 130.7,
124.9, 60.7, 39.8, 37.5, 29.1, 25.2, 19.2, 19.1, 17.7, −0.6. MS (TOF MS
EI+, m/z): (M + H+), 229.2; HRMS (TOF MS EI+, m/z): calcd for
C13H29OSi (M + H+) 229.1909, found 229.1937.
Trimethyl(phenethoxy)silane (43). Reagents: 2-phenylethanol
(38; 1.9 mL, 16 mmol, 1 equiv), hexamethyldisilazane (1.8 mL, 9.8
mmol, 0.60 equiv), chlorotrimethylsilane (0.11 mL, 0.8 mmol, 0.05
equiv). The residue was purified by silica gel flash column
chromatography (2% ethyl acetate in hexanes, increasing to 10%
ethyl acetate) to afford trimethyl(phenethoxy)silane (43; 3.0 g, 15
1
mmol, 94%) as a clear oil. H NMR (CDCl3): δ 0.20 (s, 9H), 2.96 (t,
2H, J = 7.3 Hz), 3.91 (t, 2H, J = 7.3 Hz), 7.28−7.42 (m, 5H). The 1H
NMR data are in agreement with literature values.52
(S)-Methyl 2-(Trimethylsilyloxy)propanoate (44). Reagents:
(−)-methyl L-lactate (0.92 mL, 9.6 mmol, 1 equiv), hexamethyldisilazane
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(1.2 mL, 5.7 mmol, 0.60 equiv), chlorotrimethylsilane (61 μL, 0.48
mmol, 0.05 equiv). The residue was purified using silica gel flash
column chromatography (2% ethyl acetate in hexanes, increasing to
10% ethyl acetate) to afford (S)-methyl 2-(trimethylsilyloxy)-
propanoate (44; 1.4 g, 8.2 mmol, 85%) as a clear oil. IR (cm−1):
5.24−5.39 (m, 3H), 5.92−6.03 (m, 1H), 6.84 (br s, 1H), 7.39 (s, 4H).
13C NMR (CDCl3): δ 153.6, 138.3, 133.5, 132.7, 127.9, 118.7, 103.1,
66.2, 52.9 (double intensity). MS (TOF MS ES+, m/z): (M + Na+),
274.1. HRMS (TOF MS ES+, m/z): calcd for C13H17NO4Na (M +
Na+) 274.1055, found 274.1056.
1
2956, 1745. H NMR (CDCl3): δ −0.11 (s, 9H), 1.13 (d, 3H, J = 6.8
(c). 4-(tert-Butyldimethylsilyloxy)benzyl 4-(Dimethoxymethyl)-
phenylcarbamate (23). Reagents: 3 Å molecular sieves (0.10 g), 4-
(tert-butyldimethylsilyloxy)benzyl 4-formylphenylcarbamate (22; 0.10 g,
0.27 mmol, 1 equiv), methoxytrimethylsilane (0.15 mL, 1.0 mmol, 4.0
equiv), dichloromethane (1.3 mL), trimethylsilyl trifluoromethanesul-
fonate (10 μL, 44 μmol, 0.20 equiv). The residue was purified using
silica gel flash column chromatography (20% ethyl acetate in
petroleum ether, increasing to 50% ethyl acetate) to afford 4-(tert-
butyldimethylsilyloxy)benzyl 4-(dimethoxymethyl)phenylcarbamate
(23; 0.12 mg, 0.22 mmol, 82%) as a pale yellow film. IR (cm−1):
3332, 2980, 1734, 1607, 1531. 1H NMR (CDCl3): δ 0.19 (s, 6H), 0.99
(s, 9H), 3.30 (s, 6H), 5.08 (s, 2H), 5.35 (s, 1H), 6.74 (br s, 1H), 6.85
(d, 2H, J = 12 Hz), 7.26 (d, 2H, J = 12 Hz), 7.38 (s, 4H). 13C NMR
(CDCl3): δ 156.3, 153.7, 138.3, 133.5, 130.5, 129.0, 127.9, 120.5,
118.6, 103.1, 67.3, 52.9, 26.0, 18.6, −4.0.
Hz), 3.47 (s, 3H), 4.07 (q, 1H, J = 6.5 Hz). 13C NMR (CDCl3): δ
174.2, 68.2, 51.7, 21.4, −0.2. MS (MS AP+, m/z): (M + H+), 177.1.
2-Methyl-1-phenyl-2-trimethylsilyloxypropane (45). Reagents: 2-
methyl-1-phenyl-2-propanol (0.98 mL, 6.6 mmol, 1 equiv),
hexamethyldisilazane (0.84 mL, 4.0 mmol, 0.6 equiv), chlorotrime-
thylsilane (42 μL, 0.33 mmol, 0.05 equiv). The residue was purified
using silica gel flash column chromatography (2% ethyl acetate in
hexanes, increasing to 10% ethyl acetate) to afford 2-methyl-1-phenyl-
2-trimethylsilyloxypropane (45; 1.2 g, 5.2 mmol, 79%) as a clear oil.
1H NMR (CDCl3): δ 0.35 (s, 9H), 1.48 (s, 6H), 2.98 (s, 2H), 7.44−
1
7.53 (m, 5H). The H NMR data are in agreement with literature
values.52
General Procedure A for the TMSOTf-Catalyzed Acetal For-
mation. To a round-bottom flask containing flame-dried, finely
ground, activated 3 Å molecular sieves (100 wt % with respect to
aldehyde) was added the desired aldehyde (1 equiv), dichloromethane
(to make a 0.20 M solution), and the desired silyl ether (4.0 equiv).
The resulting suspension was cooled to −78 °C in an ice bath and
placed under vacuum (∼1 mmHg) for 1 h at −78 °C, presumably to
degas the solvent, as described in ref 11. (Note: we did not observe a
decrease in the volume of the solution during this 1 h period under
vacuum. We did, however, notice that the yields of the product from
the reaction were substantially higher when this vacuum procedure was
included in comparison to when this procedure was omitted.) The
flask was filled with N2, and trimethylsilyl trifluoromethanesulfonate
(0.20 equiv) was added dropwise to the −78 °C suspension. The
resulting suspension was stirred for 3−6 h at −78 °C. The resulting
opaque suspension was warmed to 25 °C, and pyridine was added
dropwise until a clear solution was formed. The solution was diluted
with dichloromethane, and the molecular sieves were removed by
filtration. The filtrate was collected and washed sequentially with
saturated aqueous sodium bicarbonate solution (1×), water (1×), and
brine (1×). The organic layer was dried over sodium sulfate, the
sodium sulfate was removed by filtration, and the solution was
concentrated by rotary evaporation. The resulting residue was purified
using silica gel flash column chromatography.
(d). N-(4-(Dimethoxymethyl)phenyl)-2-phenylacetamide (25). Reagents:
3 Å molecular sieves (0.10 g), N-(4-formylphenyl)-2-phenylacetamide
(24; 0.10 g, 0.41 mmol, 1 equiv), methoxytrimethylsilane (0.23 mL,
1.7 mmol, 4.0 equiv), dichloromethane (2.1 mL), trimethylsilyl
trifluoromethanesulfonate (15 μL, 83 μmol, 0.20 equiv). The residue
was purified using silica gel flash column chromatography (20% ethyl
acetate in petroleum ether, increasing to 50% ethyl acetate) to afford
N-(4-(dimethoxymethyl)phenyl)-2-phenylacetamide (25) as a pale
yellow solid (0.11 g, 0.39 mmol, 95%). Mp: 89 °C. IR (cm−1): 3261,
1
3194, 3119, 3058, 2921, 1663, 1596, 1530. H NMR (CDCl3): δ 3.29
(s, 6H), 3.73 (s, 2H), 5.34 (s, 1H), 7.17 (br s, 1H), 7.26−7.43
(m, 9H). 13C NMR (CDCl3): δ 169.0, 137.7, 134.3, 134.2, 129.5,
129.2, 127.7, 127.4, 119.4, 102.6, 52.5, 44.8. MS (TOF MS ES+, m/z):
(M + H+), 286.1. HRMS (TOF MS ES+, m/z): calcd for C17H19NO3
(M + H+): 286.1443, found 286.1437.
(e). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-(Bis-
(3,8-dimethylnon-7-enyloxy)methyl)phenylcarbamate (27). Re-
agents: 3 Å molecular sieves (0.10 g), 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl 4-formylphenylcarbamate (8; 0.10 g, 0.26
mmol, 1 equiv), dichloromethane (1.3 mL), (3,7-dimethyloct-6-enyloxy)-
trimethylsilane (42; 0.24 g, 1.0 mmol, 4.0 equiv), and trimethylsilyl
trifluoromethanesulfonate (10 μL, 0.2 mmol, 0.20 equiv). The residue
was purified using silica gel flash column chromatography (10% ethyl
acetate in petroleum ether, increasing to 40% ethyl acetate) to afford
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-(bis(3,8-dime-
thylnon-7-enyloxy)methyl)phenylcarbamate (27; 0.18 g, 0.24 mmol,
92%) as a pale yellow film. The NMR data are complicated by
(a). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
(Dimethoxymethyl)phenylcarbamate (9). Reagents: 3 Å molecular
sieves (0.50 g), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl
4-formylphenylcarbamate (8; 0.50 g, 1.3 mmol, 1 equiv), dichloro-
methane (6.6 mL), methoxytrimethylsilane (0.72 mL, 5.2 mmol, 4.0
equiv), and trimethylsilyl trifluoromethanesulfonate (47 μL, 0.20
mmol, 0.20 equiv). The residue was purified using silica gel flash
column chromatography (10% ethyl acetate in petroleum ether,
increasing to 40% ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl 4-(dimethoxymethyl)phenylcarbamate (9;
0.53 g, 1.2 mmol, 95%) as a pale yellow solid. Mp: 104 °C. IR
1
diastereomers. IR (cm−1): 3313, 2918, 1713, 1607, 1531. H NMR
(CDCl3): δ 0.86−0.88 (m, 9H), 1.13−1.42 (m, 20H), 1.55−1.67 (m,
12H), 1.94−1.99 (m, 6H), 3.43−3.56 (m, 6H), 5.06−5.10 (m, 2H),
5.20 (s, 2H), 5.45 (s, 1H), 6.76 (br s, 1H), 7.35−7.40 (m, 6H), 7.82
(d, 2H, J = 8.0 Hz). 13C NMR (CDCl3): δ 153.1, 138.9, 137.6, 135.0,
134.2, 131.2, 131.0, 127.4, 127.2, 124.8, 124.7, 118.2, 101.1, 83.8, 66.8,
63.4, 61.1, 39.8, 37.1, 36.7, 36.6, 29.5, 29.4, 29.1, 25.6, 25.4, 24.8, 19.5,
19.4, 17.6. MS (TOF MS ES+, m/z): (M + Na+), 698.5. HRMS (TOF
MS ES+, m/z): calcd for C41H62BNO6Na (M + Na+) 698.4568, found
698.4592.
(f). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-(Bis-
(benzyloxy)methyl)phenylcarbamate (1). Reagents: 3 Å molecular
sieves (0.10 g), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl
4-formylphenylcarbamate (8; 0.10 g, 0.41 mmol, 1 equiv), benzyl-
oxytrimethylsilane (0.20 mL, 1.0 mmol, 4.0 equiv), dichloromethane
(1.3 mL), trimethylsilyl trifluoromethanesulfonate (12 μL, 52 μmol,
0.20 equiv). The residue was purified using silica gel flash column
chromatography (10% ethyl acetate in petroleum ether, increasing to
50% ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl 4-(bis(benzyloxy)methyl)phenylcarbamate (1; 0.13 g, 0.22
mmol, 84%) as a clear film. IR (cm−1); 3311, 2978, 1714, 1607, 1528.
1H NMR (CDCl3): δ 1.34 (s, 12H), 4.58 (s, 4H), 5.21 (s, 2H), 5.73 (s,
1
(cm−1): 3291, 2975, 1718, 1599, 1531. H NMR (CDCl3): δ 1.35 (s,
12H), 3.31 (s, 6H), 5.20 (s, 2H), 5.36 (s, 1H), 6.97 (br s, 1H), 7.39−
7.37 (m, 6H), 7.83 (d, 2H, J = 6.3 Hz). 13C NMR (CDCl3): δ 153.2,
138.9, 137.8, 134.9, 133.1, 131.2, 127.4, 127.2, 118.2, 102.7, 83.8, 66.8,
+
52.5, 24.7. MS (TOF MS ES+, m/z): (M + NH4 ), 445.2. HRMS
+
(TOF MS ES+, m/z): calcd for C23H34BN2O6 (M + NH4 ) 445.2510,
found 445.2527.
(b). Allyl 4-(Dimethoxymethyl)phenylcarbamate (21). Reagents:
3 Å molecular sieves (0.10 g), allyl 4-formylphenylcarbamate (20; 0.10 g,
0.48 mmol, 1 equiv), methoxytrimethylsilane (0.26 mL, 1.9 mmol, 4.0
equiv), dichloromethane (2.4 mL), trimethylsilyl trifluoromethanesul-
fonate (17 μL, 97 μmol, 0.20 equiv). The residue was purified using
silica gel flash column chromatography (20% ethyl acetate in
petroleum ether, increasing to 50% ethyl acetate) to afford allyl
4-(dimethoxymethyl)phenylcarbamate (21; 0.12 g, 0.45 mmol, 94%)
as a pale yellow solid. Mp: 61 °C. IR (cm−1): 3278, 2949, 1723, 1596,
1
1525. H NMR (CDCl3): δ 3.31 (s, 6H), 4.65 (d, 2H, J = 8.1 Hz),
1H), 6.76 (br s, 1H), 7.23−7.40 (m, 14H), 7.49 (d, 2H, J = 8.0 Hz),
10108
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7.82 (d, 2H, J = 7.8 Hz). 13C NMR (CDCl3): δ 153.1, 138.9, 137.9,
134.9, 133.3, 128.4, 128.3, 127.7, 127.6, 127.5, 127.2, 126.8, 118.3,
99.9, 83.8, 66.8, 65.1, 24.8. MS (TOF MS ES+, m/z): (M + Na+),
602.3. HRMS (TOF MS ES+, m/z): calcd for C35H38BNO6Na (M +
Na+) 602.2690, found 602.2722.
chromatography (5% ethyl acetate in petroleum ether, increasing to
40% ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl 4-(diphenoxymethyl)phenylcarbamate (11; 0.12 g, 0.21
mmol, 84%) as a white solid. The 1H and 13C NMR spectra are
complicated by rotamers. Mp: 72 °C. IR (cm−1): 3326, 2976, 1732,
1605, 1526. 1H NMR (CDCl3): δ 1.33 (s, 12H), 5.19 (s, 2H), 6.62 (s,
1H), 6.82 (br s, 1H), 6.95−7.02 (m, 6H), 7.20−7.27 (m, 4H), 7.35−
7.43 (m, 4H), 7.55 (d, 2H, J = 8.7 Hz), 7.82 (d, 2H, J = 8.1 Hz). 13C
NMR (CDCl3): δ 156.1, 153.1, 138.9, 138.6, 135.1, 132.4, 129.5,
129.4, 127.6, 127.4, 127.3, 122.4, 118.5, 117.5, 117.3, 100.3, 100.1,
99.8, 83.9, 66.9, 25.1, 24.8, 24.8, 24.5. Anal. Calcd: C, 71.88; N, 2.54;
H, 6.21. Found: C, 71.87; N, 2.51; H, 6.23.
(g). 2-Nitrobenzyl 4-(Dimethoxymethyl)phenylcarbamate (19). Re-
agents: 3 Å molecular sieves (0.10 g), 2-nitrobenzyl 4-formylphe-
nylcarbamate (18; 0.10 g, 0.33 mmol, 1 equiv), methoxytrimethylsi-
lane (0.18 mL, 1.3 mmol, 4.0 equiv), dichloromethane (1.6 mL),
trimethylsilyl trifluoromethanesulfonate (12 μL, 66 μmol, 0.20 equiv).
The residue was purified using silica gel flash column chromatography
(20% ethyl acetate in petroleum ether, increasing to 50% ethyl acetate)
to afford 2-nitrobenzyl 4-(dimethoxymethyl)phenylcarbamate (19;
0.11 g, 0.31 mmol, 96%) as a pale yellow solid. Mp: 103 °C. IR
(cm−1): 3268, 3117, 2932, 2836, 1724, 1603, 1522. 1H NMR
(CDCl3): δ 3.30 (s, 6H), 5.36 (s, 1H), 5.60 (s, 2H), 7.02 (br s, 1H),
7.39 (s, 4H), 7.45−7.50 (m, 1H), 7.62−7.64 (m, 2H), 8.11 (d, 2H, J =
9.0 Hz). 13C NMR (CDCl3): δ 152.7, 147.3, 137.5, 133.7, 133.4, 132.4,
128.9, 128.7, 127.4, 125.0, 118.3, 102.6, 63.5, 52.5. MS (TOF MS ES+,
m/z): (M + Na+), 369.1. HRMS (TOF MS ES+, m/z): calcd for
C17H18N2O6Na (M + Na+) 369.1063, found 369.1052.
General Procedure B for the TMSOTf-Catalyzed Acetal For-
mation. To a round-bottom flask containing flame-dried, finely
ground, activated 3 Å molecular sieves (100 wt % with respect to the
aldehyde) was added the desired aldehyde (1 equiv), dichloromethane
(to make a 0.20 M solution), and the desired silyl ether (4.0 equiv).
The resulting suspension was cooled to −78 °C in an ice bath and
placed under vacuum (∼1 mmHg) for 1 h at −78 °C to degas the
suspension. (Note: please see the comments about this step in General
Procedure A.) The flask was filled with N2, and trimethylsilyl
trifluoromethanesulfonate (0.20 equiv) was added dropwise. The
suspension was stirred for 1 h at −78 °C and then was warmed to
25 °C. After the mixture was stirred for 12 h at 25 °C, pyridine was
added dropwise to the opaque suspension until the solution became
clear. The solution was diluted with dichloromethane, and the
molecular sieves were removed by filtration. The filtrate was collected
and was washed sequentially with saturated aqueous sodium
bicarbonate solution (1×), water (1×), and brine (1×). The organic
layer was dried over sodium sulfate, the sodium sulfate was removed
by filtration, and the solution was concentrated by rotary evaporation.
The crude product was purified using silica gel flash column
chromatography.
(c). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
(Diisopropoxymethyl)phenylcarbamate (28). Reagents: 3 Å mole-
cular sieves (0.10 g), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzyl 4-formylphenylcarbamate) (8; 0.10 g, 0.26 mmol, 1 equiv),
dichloromethane (1.3 mL), isopropoxytrimethylsilane (0.19 mL, 1.0
mmol, 4.0 equiv), and trimethylsilyl trifluoromethanesulfonate (9.4 μL,
52 μmol, 0.20 equiv). The residue was purified using silica gel flash
column chromatography (10% ethyl acetate in petroleum ether,
increasing to 30% ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl 4-(diisopropoxymethyl)phenylcarbamate
(28; 53 mg, 0.10 mmol, 42%) as a pale yellow solid. Mp: 117 °C.
IR (cm−1): 3279, 2974, 1732, 1600, 1528. 1H NMR (CDCl3): δ 1.14−
1.19 (m, 12H), 1.34 (s, 12H), 3.85−3.92 (m, 2H), 5.20 (s, 2H), 5.51
(s, 1H), 6.69 (br s, 1H), 7.34−7.42 (m, 6H), 7.82 (d, 2H, J = 7.7 Hz).
13C NMR δ 153.6, 139.4, 138.0, 136.2, 135.5, 127.9, 127.7, 118.7, 99.2,
84.2, 68.2, 67.3, 25.2, 23.5, 22.9. MS (TOF MS ES+, m/z): (M + Na+),
506.2. HRMS (TOF MS ES+, m/z): calcd for C27H38BNO6Na (M +
Na+) 506.2690, found 506.2690.
General Procedure for the TMSOTf-Catalyzed Mixed-Acetal
Formation. To a round-bottom flask containing flame-dried, finely
ground, activated 3 Å molecular sieves (100 wt % with respect to the
aldehyde) was added the desired aldehyde (1 equiv), dichloromethane
(to make a 0.20 M solution), and a 1:1 mixture of the desired silyl
ethers (4.0 equiv total, 2.0 equiv of each silyl ether). The resulting
suspension was cooled to −78 °C and was placed under vacuum (∼1
mmHg) for 1 h at −78 °C to degas the suspension. (Note: please see
the comments about this step in General Procedure A.) The flask was
filled with N2, and trimethylsilyl trifluoromethanesulfonate (0.20
equiv) was added dropwise. The suspension was stirred for 1 h at −78 °C
and then was warmed to 25 °C. After the mixture was stirred for 12 h at
25 °C, pyridine was added dropwise to the opaque suspension until a
clear solution was formed. The solution was diluted with dichloro-
methane, and the molecular sieves were removed by filtration. The
filtrate was collected and was washed sequentially with saturated
aqueous sodium bicarbonate solution (1×), water (1×), and brine
(1×). The organic solvent was dried over sodium sulfate, the sodium
sulfate was removed by filtration, and the solution was concentrated by
rotary evaporation. The crude product was purified using silica gel
flash column chromatography.
(a). (2S,2′S)-Dimethyl 2,2′-((4-((4-(4,4,5,5-Tetramethyl-1,3,2-diox-
aborolan-2-yl)benzyloxy)carbonylamino)phenyl)methylene)bis-
(oxy)dipropanoate (29). Reagents: 3 Å molecular sieves (0.20 g),
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-formylphenyl-
carbamate (8; 0.20 g, 0.52 mmol, 1 equiv), dichloromethane (2.3 mL),
(S)-methyl 2-(trimethylsilyloxy)propanoate (44; 0.37 mL, 2.0 mmol,
4.0 equiv), and trimethylsilyl trifluoromethanesulfonate (19 μL, 0.10
mmol, 0.20 equiv). The residue was purified using silica gel flash
column chromatography (20% ethyl acetate in petroleum ether,
increasing to 50% ethyl acetate) to afford (2S,2′S)-dimethyl 2,2′-((4-
((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)-
carbonylamino)phenyl)methylene)bis(oxy)dipropanoate (29; 0.26 g,
0.45 mmol, 87%) as a pale yellow solid. IR (cm−1): 3355, 2980, 1721,
(a). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
(Benzyloxy(ethoxy)methyl)phenylcarbamate (26). Reagents: 3 Å
molecular sieves (0.40 g), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl 4-formylphenylcarbamate (8; 0.40 g, 0.95 mmol, 1 equiv),
ethoxytrimethylsilane (0.29 mL, 1.9 mmol, 2.0 equiv), benzyloxy-
trimethylsilane (0.34 mL, 1.9 mmol, 2.0 equiv), dichloromethane (4.7
mL), and trimethylsilyl trifluoromethanesulfonate (51 μL, 0.28 mmol,
0.20 equiv). The product was purified using silica gel flash column
chromatography (5% ethyl acetate in petroleum ether, increasing to
40% ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl 4-(benzyloxy(ethoxy)methyl)phenylcarbamate (26; 0.33 g,
0.63 mmol, 67%) as a pale yellow oil. 1H NMR data for compound 26
indicated a rotameric mixture, and the 1H NMR spectrum taken at 60 °C
showed significant coalescing of peaks. The following data were
collected at 25 °C, and therefore, unassigned rotameric peaks are
1
1604, 1531. H NMR (CDCl3): δ 1.34 (s, 12H), 1.39 (d, 3H, J = 6.8
Hz), 1.44 (d, 2H, J = 6.8 Hz), 3.65 (s, 3H), 3.72 (s, 3H), 4.14 (q, 1H,
J = 14 Hz), 4.76 (q, 1H, J = 9.4), 5.20 (s, 2H), 5.59 (s, 1H), 6.82 (br s,
1H), 7.37−7.45 (m, 6H), 7.82 (d, 2H, J = 7.9 Hz). 13C NMR
(CDCl3): δ 174.0, 173.7, 139.3, 138.9, 135.4, 132.8, 128.2, 127.7,
118.8, 100.7, 84.2, 70.6, 69.8, 67.2, 52.3, 25.2, 19.2. MS (MS ES+, m/
+
z): (M + NH4 ), 589.4. HRMS (TOF MS ES+, m/z): calcd for
+
C29H42N2O10B (M + NH4 ) 589.2933, found 589.2929.
(b). 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
(Diphenoxymethyl)phenylcarbamate (11). Reagents: 3 Å molecular
sieves (0.10 g), (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl
4-formylphenylcarbamate) (8; 0.10 g, 0.26 mmol, 1 equiv), dichloro-
methane (1.3 mL), trimethyl(phenoxy)silane (0.19 mL, 1.0 mmol, 4.0
equiv), and trimethylsilyl trifluoromethanesulfonate (9.3 μL, 52 μmol,
0.20 equiv). The residue was purified using silica gel flash column
1
reported as well. IR (cm−1): 3307, 2976, 1711, 1600, 1529. H NMR
(CDCl3): δ 1.19−1.33 (m, 3H), 1.34 (s, 12H), 3.55−3.62 (m, 2H),
4.56 (d, 2H, J = 11 Hz), 5.20 (s, 2H), 5.60 (t, 1H, J = 40 Hz), 6.81 (s,
1H), 7.14−7.48 (m, 12H), 7.81 (d, 2H, J = 6.7 Hz). 13C NMR
10109
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(CDCl3): δ 191.4, 153.6, 153.1, 143.9, 139.4, 138.9, 138.6, 138.4,
138.2, 135.5, 135.4, 134.3, 132.2, 131.7, 128.9, 128.7, 128.4, 128.1,
128.0, 127.9, 127.7, 118.5, 116.7, 114.5, 112.0, 100.9, 100.4, 84.4, 84.3,
67.8, 67.4, 61.4, 25.4, 25.3, 25.2, 25.1, 15.6. MS (TOF MS ES+, m/z):
(M + Na+), 540.3. HRMS (TOF MS ES+, m/z): calcd for
C30H36BNO6Na (M + Na+) 540.2533, found 540.2532.
0 °C, and sodium borohydride (0.16 g, 4.3 mmol, 1.3 equiv) was
added. The resulting mixture was warmed to 25 °C, and after being
stirred at 25 °C for 12 h, the solution mixture was diluted with
saturated aqueous ammonium chloride solution (20 mL). The biphasic
solution was extracted with dichloromethane (4 × 20 mL), and the
combined organic layers were washed with brine (1 × 20 mL) and
dried over sodium sulfate. The sodium sulfate was removed by
filtration, and the solution was concentrated by rotary evaporation.
The residue was purified using silica gel flash column chromatography
(3% methanol in benzene, increasing to 10% methanol) to afford 4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-(hydroxymethyl)-
phenylcarbamate (46; 1.0 g, 2.7 mmol, 84%) as a yellow solid. Mp:
130 °C. IR (cm−1): 3454, 3236, 1726, 1604, 1537. 1H NMR (CDCl3): δ
1.33 (s, 12H), 4.60 (s, 2H), 5.19 (s, 2H), 6.86 (br s, 1H), 7.27 (d, 2H, J =
8.7 Hz), 7.34−7.38 (m, 4H), 7.80 (d, 2H, J = 8.0 Hz). 13C NMR
(CDCl3): δ 153.3, 138.9, 137.1, 136.0, 135.0, 128.2, 127.8, 127.2, 118.8,
83.8, 66.8, 64.8, 24.8. MS (TOF MS ES+, m/z): (M + NH4+), 401.2.
HRMS (TOF MS ES+, m/z): calcd for C21H30BN2O5 (M + NH4+)
401.2248, found 401.2253.
(b). (2S,3S,4S,5R,6S)-Methyl 3,4,5-Triacetoxy-6-(4-((4-((3,7-dime-
thyloct-6- enyloxy)(phenethoxy)methyl)phenylcarbamoyloxy)-
methyl)phenoxy)tetrahydro-2H-pyran-2-carboxylate (35). Reagents:
3 Å molecular sieves (0.30 g), (2S,3S,4S,5R,6S)-methyl 3,4,5-triacetoxy-
6-(4-((4-formylphenylcarbamoyloxy)methyl)phenoxy)-tetrahydro-2H-
pyran-2-carboxylate (34; 30 mg, 52 μmol, 1 equiv), (3,7-dimethyloct-
6-enyloxy)trimethylsilane (42; 23 mg, 0.10 mmol, 2.0 equiv),
trimethyl(phenethoxy)silane (43; 20 mg, 0.10 mmol, 2.0 equiv),
dichloromethane (0.23 mL), and trimethylsilyl trifluoromethanesulfo-
nate (1.8 μL, 0.10 mmol, 0.20 equiv). The product was purified using
silica gel flash column chromatography (20% ethyl acetate in
petroleum ether, increasing to 60% ethyl acetate) to afford
(2S,3S,4S,5R,6S)-methyl 3,4,5-triacetoxy-6-(4-((4-((3,7-dimethyloct-
6-enyloxy)(phenethoxy)methyl)phenylcarbamoyloxy)methyl)-
phenoxy)-tetrahydro-2H-pyran-2-carboxylate (35; 15 mg, 17 μmol,
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
((Phenoxycarbonyloxy)methyl)phenylcarbamate (12). To a round-
bottom flask was added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl 4-(hydroxymethyl)phenylcarbamate (46; 0.20 g, 5.2
mmol, 1 equiv), dichloromethane (2.6 mL), N,N-diisopropylethyl-
amine (0.23 mL, 1.3 mmol, 2.5 equiv), phenyl chloroformate (0.13
mL, 1.0 mmol, 2.0 equiv), and 4-(dimethylamino)pyridine (6.3 mg, 52
μmol, 0.10 equiv). The mixture was stirred at 25 °C for 12 h. The
resulting solution was diluted with saturated aqueous ammonium
chloride solution (1 × 5 mL), and the aqueous layer was extracted
using dichloromethane (2 × 10 mL). The organic extracts were
combined and dried over sodium sulfate. The sodium sulfate was
removed by filtration, and the solution was concentrated by rotary
evaporation. The residue was purified by silica gel flash column
chromatography (10% ethyl acetate in hexanes, increasing to 40%
ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl 4-((phenoxycarbonyloxy)methyl)phenylcarbamate (12; 0.24
g, 0.48 mmol, 92%) as a white solid. Mp: 88−92 °C. IR (cm−1): 3352,
1
33%) as a waxy white film. The H NMR and 13C NMR spectra likely
contain signals for rotamers, as was observed for compound 26. The
spectra also contain signals for diastereomers. IR (cm−1): 2920, 1750,
1
1610, 1508. H NMR (CDCl3): δ 0.82−1.05 (m, 3H), 1.15−1.91 (m,
13H), 1.92−2.27 (m, 11H), 3.72 (s, 3H), 4.04−4.19 (m, 1H), 4.41 (d,
2H, J = 10 Hz), 4.57 (d, 2H, J = 12 Hz), 5.12−5.14 (m, 3H), 5.18−
5.38 (m, 4H), 5.79−5.90 (m, 1H), 6.63 (br s, 1H), 7.00 (d, 2H, J = 8.0
Hz), 7.13−7.35 (m, 11H). 13C NMR (CDCl3): δ 170.1, 169.3, 169.2,
166.8, 156.6, 153.2, 139.0, 137.6, 131.2, 131.1, 130.0, 128.9, 128.9,
128.2, 127.4, 126.1, 124.7, 118.1, 117.1, 101.0, 99.0, 77.2, 72.6, 71.8,
70.9, 69.0, 66.4, 65.8, 63.8, 53.0, 37.1, 36.6, 36.3, 29.5, 25.7, 25.4, 20.6,
+
20.5, 19.4, 17.6. MS (TOF MS ES+, m/z): (M + NH4 ), 865.4. HRMS
+
(TOF MS ES+, m/z): calcd for C46H61N2O14 (M + NH4 ) 865.4123,
found 865.4097.
Lithium (2S,3S,4S,5R,6S)-6-(4-((4-((3,7-Dimethyloct-6-enyloxy)-
(phenethoxy)methyl)phenylcarbamoyloxy)methyl)phenoxy)-3,4,5-
trihydroxytetrahydro-2H-pyran-2-carboxylate (36). To a round-
bottom flask, cooled to 0 °C, was added (2S,3S,4S,5R,6S)-methyl
3,4,5-triacetoxy-6-(4-((4-((3,7-dimethyloct-6-enyloxy)(phenethoxy)-
methyl)phenylcarbamoyloxy)methyl)phenoxy)tetrahydro-2H-pyran-2-
carboxylate (35; 30 mg, 35 μmol, 1 equiv) and a 0 °C solution of
lithium hydroxide hydrate (8.4 mg, 0.21 mmol, 6.0 equiv) in a mixture
of methanol (2.6 mL), water (1.0 mL), and tetrahydrofuran (0.53
mL). The reaction mixture was stirred for 1 h at 0 °C, at which point
an aliquot of the solution was removed. HPLC analysis was used to
confirm complete deprotection. Methanol and tetrahydrofuran were
removed from the solution by rotary evaporation, and water was
removed by lyophilization to afford lithium (2S,3S,4S,5R,6S)-6-(4-((4-
( ( 3 , 7 - d i m e t h y l o c t - 6 - e n y l o x y ) ( p h e n e t h o x y ) m e t h y l ) -
phenylcarbamoyloxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-
2H-pyran-2-carboxylate (36) as a white solid. The product was used
1
2976, 2359, 1727, 1600, 1532. H NMR (CDCl3): δ 1.37 (s, 12H),
5.22 (s, 2H), 5.23 (s, 2H), 6.88 (br s, 1H), 7.10−7.20 (m, 3H), 7.37−
7.42 (m, 8H), 7.85 (d, 2H, J = 7.2 Hz). 13C NMR (CDCl3): δ 153.6,
153.1, 151.0, 138.8, 138.3, 135.0, 129.8, 129.3, 127.4, 127.2, 125.9,
121.1, 120.9, 118.7, 83.8, 69.9, 66.9, 24.5. MS (MS ES+, m/z): (M +
+
NH4 ), 521.3. HRMS (TOF MS ES+, m/z): calcd for C28H34BN2O7
+
(M + NH4 ): 521.2459, found 521.2465.
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
((Methoxycarbonyloxy)methyl)phenylcarbamate (10). To a round-
bottom flask was added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl 4-(hydroxymethyl)phenylcarbamate (46; 0.83 g, 2.2 mmol, 1
equiv), dichloromethane (22 mL), N,N-diisopropylethylamine (0.60
mL, 3.4 mmol, 1.5 equiv), methyl chloroformate (0.25 mL, 3.3 mmol,
1.5 equiv), and 4-(dimethylamino)pyridine (26 mg, 0.22 mmol, 0.10
equiv). The solution was stirred at 25 °C for 12 h. The resulting
mixture was diluted using saturated aqueous ammonium chloride
solution (20 mL), and the aqueous layer was extracted using
dichloromethane (2 × 20 mL). The organic layers were combined
and were dried over sodium sulfate. The sodium sulfate was removed
by filtration, and the resulting solution was concentrated by rotary
evaporation. The residue was purified using silica gel flash column
chromatography (20% ethyl acetate in hexanes, increasing to 40%
ethyl acetate) to afford 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl 4-((methoxycarbonyloxy)methyl)phenylcarbamate (10; 0.24
g, 0.48 mmol, 92%) as a white solid. Mp: 96−98 °C. IR (cm−1): 3350,
1
without further purification; therefore, a yield is not reported. The H
NMR and 13C NMR spectra likely contain signals for rotamers, as was
observed for compound 26. The spectra also contain signals for
diastereomers. Mp: 138 °C dec. IR (cm−1): 3296, 2919, 2359, 2338,
1593, 1510. 1H NMR (CD3OD): δ 0.85−0.92 (m, 3H), 1.15−2.05 (m,
13H), 2.85 (t, 2H, J = 6.1 Hz), 3.2−3.75 (m, 12H), 4.92−5.13 (m,
4H), 5.40 (s, 1H), 7.04−7.30 (m, 9H), 7.30−7.40 (m, 4H). 13C NMR
(CD3OD): δ 179.1, 175.1, 157.8, 154.5, 139.2, 138.9, 133.2, 130.6,
130.5, 129.3, 128.7, 127.9, 126.9, 125.8, 124.4, 117.9, 116.5, 101.4,
101.0, 76.7, 75.3, 73.4, 72.2, 67.5, 66.0, 65.9, 63.4, 63.3, 36.8, 36.7,
36.3, 35.9, 29.2, 29.1, 25.0, 24.5, 22.8, 18.7, 18.6, 16.4. MS (MS ES-,
m/z): (M − H)−, 706.3. HRMS (TOF MS ES+, m/z): calcd for
C39H49NO11Li (M + Li)+ 714.3466, found 714.3472.
1
2972, 2358, 1714, 1610, 1532. H NMR (CDCl3): δ 1.34 (s, 12H),
3.76 (s, 3H), 5.08 (s, 2H), 5.16 (s, 2H), 7.19 (s, 1H), 7.27−7.40 (m,
6H), 7.79 (d, 2H, J = 8.1 Hz). 13C NMR (CDCl3): δ 156.2, 153.8,
139.5, 138.7, 135.5, 130.6, 129.9, 127.8, 119.1, 84.4, 69.9, 69.8, 67.3,
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
(Hydroxymethyl)phenylcarbamate (46). To a round-bottom flask
was added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl 4-
formylphenylcarbamate (8; 1.3 g, 3.3 mmol, 1 equiv), dichloro-
methane (16 mL), and methanol (16 mL). The mixture was cooled to
+
55.3, 25.3. MS (TOF MS ES+, m/z): (M + NH4 ), 459.2. HRMS
(TOF MS ES+, m/z): calcd for C23H32BN2O7 (M + NH4+) 459.2303,
found 459.2294.
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General Procedure for Measuring Rates of Nonspecific Hydrolysis
and Rates of Release Using 1H NMR Spectroscopy. The NMR
spectrometer was programmed to acquire 32 scans for each sample
with a relaxation time of 2 s to ensure accurate integration values.
Buffer solutions were prepared according to Gomori53 and Sorensen,54
with the exception that D2O was used instead of water.
Example Procedure for Sample Preparation. Reagent 9 (3
mg) was dissolved in CD3CN (0.49 mL), and to the resulting solution
were sequentially added D2O (0.48 mL), a 0.1 M solution of sodium
phosphate or sodium bicarbonate buffer in D2O (25 μL), and a 0.8 M
solution of 2,5-dimethylfuran (an internal standard) in CD3CN (10
μL). The vial was capped and vortexed for ∼15 s. A portion (600 μL)
of the solution was transferred into an NMR tube. The tube was
capped and sealed with PTFE tape.
1H NMR spectra were acquired ∼10 min after sample preparation
(this time was set as t = 0 min) and then again after 6 h. Relative
integration values of the H NMR peaks corresponding to the aldehyde
1
hydrogen in 8 and the acetal methine hydrogen in 9 were used to
calculate the percentage of acetal 9 remaining at the conclusion of
each experiment (Table S5, Supporting Information). Integration
1
of the H NMR peak corresponding to CH3OD (relative to 2,5-
dimethylfuran) was used to calculate the percentage of carbonate
10 remaining at the conclusion of each experiment (Table S6,
Supporting Information). Each experiment was performed in
triplicate.
Rate of Nonspecific Hydrolysis of Acetal 11 and Carbonate
12. The nonspecific hydrolysis of acetal 11 and carbonate 12 were
monitored by 1H NMR at 25 °C (11) or by HPLC (12) at 25 °C. The
buffers were prepared using procedures identical with those described
above.
pH Stability of Model Reagent 9. The nonspecific hydrolysis of
1
acetal 9 was monitored by H NMR at 25 °C. The buffer solutions
1H NMR spectra were acquired ∼10 min after preparation of the
sample and then again after 720 min. Integration of the 1H NMR peak
corresponding to the methine hydrogen of acetal 11 (relative to 2,5-
dimethylfuran) was used to calculate the percentage of acetal 11
remaining at the conclusion of each experiment (Table S7, Supporting
Information). Each experiment was performed in triplicate.
Carbonate 12 (2.0 mg) was dissolved in CD3CN (0.74 mL), and to
the resulting solution were added sequentially D2O (225 μL), a 0.1 M
solution of pH 8.0 sodium phosphate buffer (25 μL), and a 0.8 M
solution of anisole in CD3CN (10 μL). The vial was capped and
vortexed for ∼15 s. Samples were injected onto the HPLC at 39 min
intervals, with the last injection occurring 741 min after the first
injection. Integration values for the benzyl alcohol product (46, which
arises from hydrolysis of the carbonate functionality in 12; the peak
was obtained using an absorbance detector set at 254 nm) relative to
anisole (254 nm) were used to calculate the percentage of carbonate
12 remaining (Table S8, Supporting Information) at each time point.
Each experiment was performed in triplicate.
consisted of sodium phosphate for pH values 6.0, 7.0, and 8.0 and of
sodium bicarbonate for pH 9.0 and 10.0.
A 1H NMR spectrum was acquired 10 min after sample preparation
and then again after 6 h. Integration of the 1H NMR peaks
corresponding to the aldehyde hydrogen in 8 and the acetal methine
hydrogen in 9 were used to calculate the percentage of acetal 9
remaining at the conclusion of the experiment (Table S1, Supporting
Information). Aldehyde 8, acetal 9, and methanol (arising from
1
hydrolysis of 9) were the only compounds in the H NMR over the
course of the experiment. Each experiment was performed in triplicate.
Thermal Stability of Model Acetal 9. The nonspecific
1
hydrolysis of acetal 9 was monitored by H NMR at 25 °C. The
solutions were buffered using sodium phosphate, and the pH was
adjusted to 8.0.
1H NMR spectra were acquired immediately after sample
preparation and then again after 6 h. During the 6 h time period,
the samples were maintained either at 15 °C (by immersion in a water-
filled refrigerated recirculator) or at 35, 45, or 55 °C (by immersion in
hot water baths maintained at their respective temperatures).
Integration of the 1H NMR peaks corresponding to the aldehyde
hydrogen in 8 and the acetal methine hydrogen in 9 were used to
calculate the percentage of acetal 9 remaining at the conclusion of the
experiment (Table S2, Supporting Information). Aldehyde 8, acetal 9,
and methanol (arising from hydrolysis of 9) were the only compounds
Postulated Mechanism for the Controlled Release of
Alcohols from Benzylic Acetals. The products arising from
reaction of acetal 1 with hydrogen peroxide−urea were identified
using an LC-MS. The buffers were prepared using procedures identical
with those described above.
Acetal 1 (1.0 mg) was dissolved in CH3CN (0.49 mL), and to the
resulting solution were added sequentially water (0.48 mL), a 0.1 M
solution of pH 8.0 sodium phosphate buffer in H2O (25 μL), and a 0.8
M solution of anisole in CH3CN (25 μL). The vial was capped and
vortexed for ∼15 s. To the vial was added a 1.73 M solution of
hydrogen peroxide−urea in H2O (10 μL), and the vial was capped and
vortexed for an additional ∼15 s. The mixture was allowed to stand for
3 h at 25 °C. After this period, the sample was injected into the LC-
MS. The molecular weights were determined for all UV-active
compounds present, and these values were compared to standards of
authentic material, when possible (Table S9, Supporting Information).
Into a vial were added a 3.5 mM solution of acetal 1 in CH3CN (49
μL), H218O (49 μL), a 0.1 M solution of pH 8.0 sodium phosphate
buffer in H2O (2.5 μL), and a 0.8 M solution of anisole in CH3CN (1
μL). The vial was capped and vortexed for ∼15 s. Into the vial was
added a 1.73 M solution of hydrogen peroxide−urea in H2O (1 μL),
and the vial was capped and vortexed for an additional ∼15 s. The
mixture was allowed to stand for 3 h at 25 °C. After this period, the
sample was injected into the LC-MS. The molecular weights were
determined for all UV-active compounds present, and these values
were compared with the retention times and m/z values found in the
unlabeled H2O study (Table S9).
Release of Two Types of Fragrances and a Colorimetric Indicator
in Response to β-D-Glucuronidase: An LC-MS Study. The sample of
36 was heated and maintained at 30 °C throughout the experiment.
The buffer solution was prepared according to Gomori53 and
Sorensen.54 The solutions were buffered using sodium phosphate,
and the pH was adjusted to 8.0.
Into an LC-MS vial were added a 10.8 mM solution of acetal 36 in
methanol (142 μL), a 75 mM aqueous solution of sodium phosphate
buffer with 1% w/v bovine serum albumin (320 μL, pH 7.4), water
1
in the H NMR over the course of the experiment. Each experiment
was performed in triplicate.
Controlled Release of Methanol from Model Reagents 9 and
10 in Response to Hydrogen Peroxide. The release of methanol
1
from model acetal 9 and control carbonate 10 was monitored by H
NMR at 25 °C. The solutions were buffered using sodium phosphate,
and the pH was adjusted to 8.0. The time intervals shown in Tables S3
and S4 (Supporting Information) are reported at the median time for
each single acquisition (rather than at the start or conclusion of the
acquisition).
A 1H NMR spectrum was acquired to establish baseline integration
values relative to 2,5-dimethylfuran (an internal standard). A solution
of H2O2−urea in D2O (2.9 M, 10 equiv; 23.4 μL was added to the
solution of 9 and 22.6 μL was added to the solution of 10) was added
to the NMR sample. The NMR tube was agitated by inversion (3×)
and returned to the spectrometer. The acquisition was started
immediately and was monitored over regular preprogrammed
intervals.
The release of methanol from 9 was measured by monitoring the
disappearance of the acetal methine hydrogen signal in 9 relative to
2,5-dimethylfuran (Table S3). For compound 10, the release of
methanol was measured by monitoring the appearance of CH3OD
(measured relative to 2,5-dimethylfuran) (Table S4). The data were
normalized for the number of equivalents of methanol released. Each
experiment was performed in triplicate.
Rate of Nonspecific Release of Methanol from Reagents 9
and 10. The nonspecific hydrolysis of acetal 9 and control carbonate
10 was monitored by 1H NMR at 25 °C. The solutions were buffered
using sodium phosphate, and the pH was adjusted to 8.0.
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The Journal of Organic Chemistry
Article
(332 μL), and a 0.8 M solution of anisole in methanol (5.0 μL). The
vial was capped and vortexed for ∼15 s. A 10 μL sample was analyzed
by LC-MS to establish the retention times and number of peaks prior
to exposure to β-D-glucuronidase. One minute prior to the second
automated injection at 19 min, a 19.23 unit/mL solution of β-D-
glucuronidase (6.5 μL) was added to the vial. The vial was capped and
vortexed (∼2 s). The sample was immediately injected onto the LC-
MS. The peaks shown in Figure S1 (Supporting Information) were
identified by comparison with the retention times and m/z values of
known standards, when possible. The peak areas of 2-phenylethanol
(38) and citronellol (37) were compared to the peak area of an
internal standard (anisole) at 210 nm. The data are shown in Table
S10 (Supporting Information).
Preparation and Analysis of 36 in the Absence of β-D-
Glucuronidase. Into a LC-MS vial were added sequentially a 10.8
mM solution of acetal 36 in methanol (142 μL), a 75 mM aqueous
solution sodium phosphate buffer with 1% (w/v) bovine serum
albumin (320 μL, pH 7.4), water (338 μL), and a 0.8 M solution of
anisole in methanol (5.0 μL). The vial was capped and vortexed for
∼15 s. The sample was injected into the LC-MS immediately. The
peaks shown in Figure S1 were identified by comparison to the
retention times and m/z values of known standards, when possible.
The peak areas of 2-phenylethanol (38) and citronellol (37) were
referenced to the peak area of an internal standard (anisole) at 210
nm. The data are shown in Table S10.
Detection of a Fragrance from Reagent 36 on Exposure to
β-D-Glucuronidase. Acetal 36 was prepared both in the presence
and absence of β-D-glucuronidase in a manner identical with the
conditions described above. After the addition of β-D-glucuronidase to
the experimental sample (and preparation of the control in the
absence of β-D-glucuronidase), the samples were incubated at 30 °C.
After they were heated for 380 min at 30 °C, the samples were cooled
to 25 °C. The vials were wrapped in aluminum foil (to mask any
differences in color), uncapped, and placed on a benchtop. Five
volunteers (Kyle Schmid, Daniel Wendekier, Anthony DiLauro, Jessica
Robbins, and Michael Olah) were individually and separately asked to
choose a vial and describe the odor. All volunteers detected a fragrance
in the experimental sample containing β-D-glucuronidase (descriptions
included “citrus-like”, “lemony”, “rosy”, and/or “floral”) and detected
no odor from the control sample containing no added β-D-
glucuronidase.
Release of Two Types of Fragrances and a Colorimetric
Indicator in Response to β-D-Glucuronidase. Colorimetric
Study. The colorimetric response of reagent 36 in the presence and
absence of β-D-glucuronidase was monitored by capturing photo-
graphs. The samples were maintained at 30 °C between photographs.
A LC-MS vial (fitted with a 250 μL glass insert) was loaded
sequentially with a 5.4 mM solution of reagent 36 in a 1:1 mixture of
methanol and water (71 μL), a 75 mM aqueous solution of pH 7.4
sodium phosphate buffer with 1% (w/v) bovine serum albumin (80
μL), and water (47 μL). The vial was capped and vortexed for ∼15 s.
To the vial was added a 19.23 unit/μL aqueous solution of β-D-
glucuronidase in 75 mM sodium phosphate with 1% (w/v) bovine
serum albumin (1.6 μL). The vial was capped and vortexed for ∼15 s.
Photographs were acquired over time.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sphillips@psu.edu.
■
ACKNOWLEDGMENTS
■
This work was supported in part by the Bill and Melinda Gates
Foundation as a subcontract from Harvard University
(Subcontract No. 01270716-00), the Arnold and Mabel
Beckman Foundation, the Camille and Henry Dreyfus
Foundation, 3M, Mr. Louis Martarano, a Bunton−Waller
Assistantship (S.A.N.), and The Pennsylvania State University.
We thank Dr. Landy K. Blasdel for her contributions to the
preparation of the paper, as well as Kyle Schmid, Daniel
Wendekier, Anthony DiLauro, Jessica Robbins, and Michael
Olah for testing whether a scent was released when 36 was
exposed to β-D-glucuronidase.
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* Supporting Information
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