Spacer-Mediated Control of Coumarin Uncaging for Photocaged Thymidine

Article abstract of DOI:10.1021/acs.joc.9b02617

Despite its importance in the design of photocaged molecules, less attention is focused on linker chemistry than the cage itself. Here, we describe unique uncaging properties displayed by two coumarin-caged thymidine compounds, each conjugated with (2) or without (1) an extended, self-immolative spacer. Photolysis of 1 using long-wavelength UVA (365 nm) or visible (420, 455 nm) light led to the release of free thymidine along with the competitive generation of a thymidine-bearing recombination product. The occurrence of this undesired side reaction, which is previously unreported, was not present with the photolysis of 2, which released thymidine exclusively with higher quantum efficiency. We propose that the spatial separation between the cage and the substrate molecule conferred by the extended linker can play a critical role in circumventing this unproductive reaction. This report reinforces the importance of linker selection in the design of coumarin-caged oligonucleosides and other conjugates.

Full text of DOI:10.1021/acs.joc.9b02617

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Article
Spacer-Mediated Control of Coumarin Uncaging for Photocaged
Thymidine
Shengzhuang Tang, Jayme Cannon, Kelly Yang, Matthew F. Krummel, James R. Baker, Jr.,
and Seok Ki Choi*
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ABSTRACT: Despite its importance in the design of photocaged
molecules, less attention is focused on linker chemistry than the cage
itself. Here, we describe unique uncaging properties displayed by two
coumarin-caged thymidine compounds, each conjugated with (2) or
without (1) an extended, self-immolative spacer. Photolysis of 1
using long-wavelength UVA (365 nm) or visible (420, 455 nm) light
led to the release of free thymidine along with the competitive
generation of a thymidine-bearing recombination product. The
occurrence of this undesired side reaction, which is previously
unreported, was not present with the photolysis of 2, which released
thymidine exclusively with higher quantum efficiency. We propose
that the spatial separation between the cage and the substrate
molecule conferred by the extended linker can play a critical role in
circumventing this unproductive reaction. This report reinforces the
importance of linker selection in the design of coumarin-caged oligonucleosides and other conjugates.
photocaging including coumarin,23,24,43,44 ortho-nitroben-
zene,24,45−47 and p-hydroxyphenylacyl.24,48 Of these, coumarin
displays distinct structural and photochemical proper-
ties.1,10,28,35,36 It shows several benefits such as high molar
absorptivity in the UV and visible light (320−440 nm), use for
wavelength-selective uncaging,6,24,28,29,49 and ability for two-
photon uncaging at the near-infrared wavelength (720 and 830
nm).7 Its flexible linkage chemistry at the C4 position allows
photocaging applications to a variety of substrates containing
functional moieties of carboxylate,10 amide,50 amine,51
alcohol,12,52,53 thiol,29,54 and phosphate.36,55,56 It also serves
as an effective protecting group for the carbonyl functionality
of a nucleotide base as in the synthesis of caged 2′-
deoxyguanosine22 and 2′-deoxythymidine.23,24 Access to such
caged compounds has led to diverse applications in biological
and biomedical areas by enabling spatiotemporal control in
fluorescence imaging,10,56 mapping of ligand-evoked channel
gating,6,7,10 therapeutic activation or delivery,51,57 signal
transduction,6,52,54,56 DNA hybridization,22−24 and gene
expression.12,25
INTRODUCTION
■
Photocaging refers to temporarily conjugating a functional
molecule with a photocleavable group1 as a strategy to prevent
its activity until temporally or spatially released by light.2−5
Since its first application to neurotransmitter ligands for
channel gating studies including those by Hoffman et al.2 and
Hess et al.,3 photocaging applications have expanded to various
fields including cellular signaling,6−9 imaging,10,11 optoge-
netics,12−15 photopharmacology,16,17 and drug delivery.11,18−20
This strategy also plays a key role in the design of
oligonucleotide probes that enable light-controlled DNA
hybridization21−24 and gene regulation.25,26
The efficiency of photon-mediated release of caged drugs or
biomolecules is largely dependent on the cage moiety because
this chromophore directly determines the wavelength
selectivity and mechanism associated with the release.1,27,28
However, the cage structure can also be responsible for the
occurrence of undesired recombination reactions12,29,30 that
can reduce the uncaging efficiency. In this current study, we
examine the concept that linker optimization can enhance the
uncaging efficiency while circumventing undesired side
reactions. We illustrate this concept using coumarin-caged
thymidine (Figure 1), the type of structure frequently used in
the synthesis of photocaged oligonucleotides.1,28,31
Received: September 26, 2019
Various cage molecules have been developed, which include
ortho-nitrobenzene,3,32−34 coumarin,1,10,28,35,36 carbazole,37,38
and quinoline.39−42 Some of these are used for thymidine
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Figure 1. Competing paths proposed in the radical-based photon uncaging of thymidine (dT) caged with coumarin through no spacer (A) or a
self-immolative benzyl (Bn) spacer (B). DEACM = 7-diethylamino-4-methylcoumarin.
a
Scheme 1. Synthesis of Coumarin-Caged Thymidines 1 and 2
a
Reagents and conditions: (i) SeO2, xylene, 150 °C; (ii) NaBH4, MeOH, 0 °C to rt; (iii) MsCl, Et3N, CH2Cl2, 0 °C; (iv) 4-hydroxybenzaldehyde,
K2CO3, THF, 45 °C, 60%; (v) NaBH4, MeOH, rt, 84%; (vi) tert-butyldimethylsilyl chloride (TBDMS-Cl), imidazole, DMF, 100%; (vii) POCl3,
1,2,4-1H-triazole, MeCN, 0 °C, 85%; (viii) 5, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), MeCN, rt, 97%; (ix) 7, DBU, MeCN, rt, 93%; (x)
Bu4NF, AcOH, THF, rt, 82% (for 1), 71% (for 2).
Compared to ortho-nitrobenzene uncaging which occurs
selectively via an intramolecular cyclization,1,58 coumarin
uncaging occurs via a homolytic and/or heterolytic bond
cleavage.27 Therefore, it involves the generation of a transient
radical (or ion) pair, which remains reactive (Figure 1).
However, because of their close spatial proximity, the radical
pair has the potential to undergo an undesired recombination
reaction (path b) via [1,3] photoisomerization (or more
broadly defined photo-Claisen rearrangement59) in lieu of the
release of an uncaged molecule (path a). Recently, a few
B
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studies29,30,60−62 including ours12 reported the occurrence of
undesired side products in the photolysis of quinoline-caged
tertiary amines,61,62 coumarin-caged peptides,29,30 capsaicin,60
and 4-hydroxytamoxifen.12 While the prevalence of these side
reactions varies with the structure and linkage type of an
individual caged molecule, we demonstrated that the spatial
separation of the coumarin cage through an extended spacer
prevents these side reactions, thus enhancing the uncaging
efficiency.12
In the present study, we investigate the occurrence of
undesired photo-catalyzed rearrangement in a coumarin ether-
caged thymidine and develop linker chemistry for blocking
these side reactions. First, we describe linker chemistries for
the synthesis of two thymidine (dT)-caged compounds
(Figure 1). The first utilizes a standard approach based on a
direct ether linkage.22−24 The second involves a novel
approach based on a 4-hydroxybenzyl spacer, which was
selected because it appeared to provide sufficient length for
spatial separation of the radical (or ion) pair as well as to
facilitate the self-immolative release63 of thymidine even from
the undesired byproduct (path b). We also present evidence
for the central role of these linker designs in determining the
release kinetics of free thymidine by irradiation at either UV
(365 nm) or visible light (420, 455 nm). This information
provides novel insights that help advance our understanding of
the role of linker chemistry in the photoactivation of caged
molecules.
This involves the nucleophilic reaction of each cage molecule
with triazolyl thymidine,65,66 resulting in an O-substitution at
the 4 position (Scheme 1). First, thymidine (dT) was fully
protected as O-TBDMS 9 and converted to triazolylpyrimidi-
none 10 according to the established protocol.65,66 Its structure
is in agreement with NMR data and HRMS (ESI) calculated
for 10 (C24H43N5O4Si2 [M + H]+, 522.2932; found, 522.2927,
Figure S4).
Compound 1 dTO‑DEACM was prepared in two steps that
began with the O-substitution of 10 with a coumarin cage 5
under a condition catalyzed by DBU. This led to 11 (93%;
HRMS calcd for C36H57N3O7Si2 [M + H]+, 700.3813; found,
700.3806), and its O-TBDMS was deprotected by treatment
with tetrabutylammonium fluoride (TBAF)/acetic acid in
tetrahydrofuran (THF), affording 1 (isolated yield 82%). It
was characterized for its structural identity by NMR (1H, 13C)
spectroscopy, HRMS (calcd for C24H29N3O7 [M + Na]+,
494.1903; found, 494.1898), and UV−vis spectrophotometry
(Figure 2). Its characterization data are fully consistent with
RESULTS AND DISCUSSION
■
Synthesis of Coumarin Cages. Scheme 1 summarizes the
synthesis of two coumarin cages, which include 7-diethylami-
nocoumarin-4-methyl (DEACM) alcohol (5)36 and 7.
Coumarin cage 5 was synthesized according to literature
protocols that involve selenium dioxide12,36,64 oxidation of 3 to
an aldehyde derivative 4 and subsequent reduction to 5 using
sodium borohydride.12,36 In our previous approach on linker
extension for 5,12 we employed a carbamate-based linkage
through N1,N2-dimethylethane-1,2-diamine as the self-immo-
lative spacer. However, this approach has not been applicable
for thymidine because of the lack of synthetic methodology
developed for the carbamate linkage at its O-4 position, the
preferred site for photocaging. Therefore, we designed a new
approach by derivatizing 5 to 7 for use as an ether-based self-
immolative linker, which is compatible at O-4.
Compound 7 is a new structure with a 4-hydroxybenzyl
spacer attached at its phenolic moiety to DEACM through an
ether linkage. Its end-to-end length (∼7.3 Å), which is
predicted in a stable extended conformation, is similar to the
distance (∼6.8 Å) of a spacer (N1,N2-dimethylethane-1,2-
diamine) that was shown to be effective in preventing the
undesired photo-Claisen rearrangement that occurs during
coumarin release.12 The synthesis of 7 was performed in two
steps: (i) O-alkylation of 4-hydroxybenzaldehyde with the O-
methanesulfonate12 derivative of 5 and (ii) sodium borohy-
dride reduction of the aldehyde to a hydroxymethyl derivative.
Its structural identity is consistent with the data from standard
analytical methods including high-resolution mass spectrome-
try (HRMS), showing agreement of the observed molecular
mass with the calculated theoretical value for 7 (C21H21NO4
[M + H]+, 352.1549; found, 352.1544).
Figure 2. UV−vis absorption spectra of caged thymidine compounds
1, 2 and their building blocks. Each arrow indicates the relative level
of absorption at the wavelength of light used in uncaging experiments.
the anticipated values as reported in the literature.23 Its purity
was determined as ≥95% by ultraperformance liquid
chromatography (UPLC) (Figure S12, Supporting Informa-
tion).
Compound 2 dTO‑Bn‑DEACM was prepared in the same
manner as for 1 except replacing 5 with 7. Its synthesis
involved O-substitution of 10 with a coumarin cage 7 under
the conditions catalyzed by DBU, leading to 12 (97%; HRMS
calcd for C43H64N3O8Si2 [M + H]+, 806.4232; found,
806.4223). The caged thymidine 2 was obtained upon
deprotection of O-TBDMS 12 by treatment with TBAF/acetic
acid in THF (isolated yield 71%). Its structural identity was
fully characterized by NMR (1H, 13C) spectroscopy (Figure
S9), MS, and UV−vis spectrophotometry (Figure 2). Its exact
molecular mass is in good agreement with an experimental
value measured by HRMS (calcd for C21H35N3O8 [M + H]+,
578.2502; found, 578.2496). Its purity was ≥95% by UPLC
analysis (Figure S12).
UV−Vis Absorption Property. We observed the
absorption properties of the coumarin (DEACM), a common
cage present in 1 and 2, as shown in their UV−vis spectra
(Figure 2). It shows a broad range of strong absorption (340−
440 nm) with λmax values at 393 nm (1, ε = 10,209 M−1 cm−1)
and 390 nm (2, ε = 11,603 M−1 cm−1), which remain almost
unchanged compared to the absorption of the cage alone (λmax
= 387 nm, ε = 22,687 M−1 cm−1). We selected three different
wavelengths for investigating uncaging kinetics of 1 and 2,
which include 365 nm (long-wavelength UVA) and 420 nm
Synthesis of Coumarin-Caged Thymidines. The syn-
thesis of each caged thymidine 123 and 2 was performed using
a conventional strategy long established for nucleoside bases.
C
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Figure 3. Uncaging kinetics of 1 (A) and 2 (B) by photolysis using long-wavelength UV (365 nm) or visible light (420, 455 nm). Representative
UPLC traces (left) refer to those at 420 nm acquired after the indicated period of photolysis of 1 and 2, respectively, at 0.1 mg/mL in 35% (v/v) aq
methanol. Each plot (right) shows the fraction (Fr) of thymidine (dT) and a byproduct (*; dTN‑DEACM) released as a function of irradiation time.
Each Fr is based on % area under curve (AUC) analysis of the released products from UPLC traces.
Table 1. Summary of Photouncaging Efficiency of Coumarin-Caged Thymidine Compounds
365 nm
420 nm
455 nm
b
b
b
Φ
Φ
Φ
a
a
a
caged thymidine
kdecay (s−1
)
dT
byproduct
0.005
kdecay (s−1
)
dT
byproduct
0.017
kdecay (s−1
)
dT
byproduct
0.023
1 dTO‑DEACM
2 dTO‑Bn‑DEACM
8.9 × 10−4
1.1 × 10−3
0.006
0.014
1.5 × 10−3
1.1 × 10−3
0.015
0.025
1.2 × 10−4
5.8 × 10−4
0.017
0.081
a
b
Rate constant (first order) for the decay of caged thymidine 1 or 2. Quantum efficiency (Φ) of release for thymidine (dT) or thymidine-retaining
byproduct dTN‑DEACM = [dc/dt]initial/[qn,p(1 − 10−A)], where qn,p = photon flux (qp/NA, mol s−1) measured by ferrioxalate actinometry.67,68 dc/dt =
initial rate of thymidine or byproduct release (mol s−1). A = absorbance of 1 or 2 at the wavelength of irradiated light.
Figure 4. Uncaging kinetics of dTN‑DEACM by photolysis at 420 nm. (A) Representative UPLC traces overlaid and (B) comparison with the traces
of 1 dTO‑DEACM. Each of the traces labeled as 1 + dTN‑DEACM (B) refers to the coinjection (1:1 mixture) of two indicated samples, each prepared
separately. Their photolysis was performed as described in Figure 3.
(visible light) as the preferred range and 455 nm as a
comparator that appears less effective for photoactivation given
weaker molar absorptivity (ε = 1115 (1) or 1653 (2) M−1
cm−1). The benzyl spacer present in 2 lacks any absorbance
above 300 nm, and it has no effect on the photoactivation at
any of these wavelengths.
Uncaging Kinetics via Direct Ether Linkage. Photolysis
of 1 was performed in an aqueous medium (0.21 mM)
supplemented with methanol (35%, v/v) for improving its
poor aqueous solubility, and its release kinetics was determined
by monitoring exposed solutions using UV−vis spectroscopy
and UPLC (Figure 3). UV−vis spectra acquired after
photolysis at 420 nm (exposure time = 0−20 min) show
absorption changes in an exposure and time-dependent
manner (Figure S13), but these are not adequate for the
detection and quantitation of thymidine released. In contrast,
UPLC traces acquired after photolysis (Figure 3A) show the
disappearance of 1 with the concomitant growth of free
thymidine (dT) (tr = 3.1 min) along with coumarin cage
released 5 (tr = 6.5 min). The AUC analysis of these peaks
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a
Scheme 2. Synthesis of dTO‑Bn‑DEACM 2 Phosphoramidite
a
Reagents and conditions: (i) dimethoxytrityl chloride (DMT-Cl), pyridine, 61% and(ii) N,N-diisopropyl-N-ethylamine (DIPEA),
dichloromethane, 73%.
provided a decay curve for compound 1 (right, Figure 3A). Its
regression analysis demonstrated a rate constant for first-order
perhaps because of the direct ether linkage of coumarin to
thymidine.
decay (kdecay = 1.5 × 10−3 s−1), quantum efficiency (ΦdT
=
Uncaging Kinetics via the Extended Spacer. We
similarly evaluated the uncaging kinetics of 2 dTO‑Bn‑DEACM
(0.17 mM in 35% (v/v) aq methanol) by irradiation at 420 nm
(Figure 3B). Its photolysis led to smaller changes in UV−vis
spectra with the lack of a clear trend compared to 1 dTO‑DEACM
(Figure S13). Overlaid UPLC traces show time-dependent,
rapid uncaging of 2 with ∼65% release of thymidine achieved
after an initial exposure for only 5 min. However, we did not
observe the undesired species (tr = 7.8 min) from the
photolysis of 2 that was seen with 1. Regression analysis of its
decay curve provided a decay constant (kdecay = 1.1 × 10−3
s−1), which is slightly lower than that of 1 (Table 1). However,
its lack of byproduct formation enhances its quantum
efficiency and sensitivity for thymidine release (ΦdT = 0.025,
0.015), and sensitivity (ΦdT × ε = 108 M−1 cm−1) for
thymidine release (Tables 1 and S1).
It is important to note that the UPLC traces also indicate the
release of an unknown species (tr = 7.8 min) that grows during
exposure in a time-dependent manner. It accounts for a
significant fraction in terms of quantum efficiency (Φbyproduct
=
0.017) compared to free thymidine. In order to characterize
the unknown product, the photolysis continued up to 30 min
until 1 was fully consumed, and this exposed solution was
analyzed by HPLC−MS (Figure S15). The peak assigned to
the unknown species shows no difference in its molecular mass
(found 472.2082) relative to 1 (calcd for [M + H]+
=
472.2084).
Φ
dT × ε = 183 M−1 cm−1) compared to 1 (ΦdT = 0.015, ΦdT
×
We cautiously assigned this byproduct as a thymidine-
ε = 108 M−1 cm−1). The single product and efficient kinetics
demonstrated with compound 2 offer evidence of advantages
in thymidine release from a cage with an extended linker as
compared to the direct ether linkage.
bearing dTN‑DEACM and verified its identity by its independent
1
synthesis and characterization by H NMR, HRMS (Figure
S7), and UPLC (Figure S12), collectively supportive of its N-
isomeric relationship to 1. We hypothesize that it might result
from the recombination of the two detached species (i.e., a
coumarin and thymidine radical pair), which are retained in
close proximity in a solvent cage, via O- to N-photo-
isomerization or [1,3] photo-Claisen rearrangement,59,69 as
depicted in Figure 1A (path b). Once generated, this
dTN‑DEACM practically loses its ability for photon uncaging
because it remains stable even after sufficiently long irradiation
(420 nm, 30 min) as indicated by UPLC traces (Figure 4A).
Its lack of uncaging is consistent, in part, with a report that an
oligonucleotide containing dTN‑DEACM decays approximately 2
Similarly, photolysis of 2 was studied at 365 and 455 nm.
Photolysis at 365 nm occurred as rapidly as at 420 nm without
the release of the byproduct (Figure S14B). It led to greater
quantum efficiency for thymidine release (ΦdT = 0.014) than 1
(ΦdT = 0.006) under an identical condition (365 nm).
Photolysis at 455 nm provided a similar result (Figure S14B),
although it occurred at a rate approximately 2-fold lower than
at 420 nm. This slower decay is anticipated given lower molar
absorptivity at 455 nm (Figure 2). However, it is notable that 2
showed ∼5-fold faster decay than 1 at 455 nm (Table 1). This
suggests that the spatial separation conferred by the extended
spacer might be able to prevent an unwanted recombination
event involving the detached species back to the parent 2 as
well (Figure 1).
orders of magnitude more slowly than an equivalent
23
oligonucleotide containing 1 dTO‑DEACM
.
In addition, its
UPLC traces confirm that it is identical to the byproduct
released from 1 (Figure 4B).
Synthesis of dTO‑Bn‑DEACM Phosphoramidite. The new
caged thymidine 2 offers potential utility as a building block in
oligonucleotide synthesis. As summarized in Scheme 2, it is
readily applicable for use with a standard nucleoside chemistry
involved in the synthesis of DMT-protected phosphoramidite.
As fully detailed in the Experimental Section, 2 was converted
to its phosphoramidite 14 in two steps (overall 45%). These
include the regiospecific protection of its hydroxymethyl group
at C5 with DMT (13) and a subsequent derivatization to 14
phosphoramidite at C4.
In summary, we quantitatively measured the decay rate and
quantum efficiency of thymidine release from coumarin-caged
thymidine prepared via either a direct ether linkage or an
extended linker with a self-immolative spacer.63 The direct
linkage in 1 dTO‑DEACM allows a fast decay upon activation with
either visible (420 nm) or long-wavelength UVA (365 nm)
We also performed the photolysis of 1 at two other
wavelengths (365, 455 nm) to determine whether the
occurrence of such an undesired product is dependent on
light wavelength. Photolysis at 365 nm occurred at a rate
similar to 420 nm and showed a comparable decay rate (kdecay
= 8.9 × 10−4 s−1). It also displayed a similar pattern of product
distribution that includes the undesired product (Figure
S14A). Photolysis at 455 nm occurred 7−13-fold more slowly
(kdecay = 1.2 × 10−4 s−1) than at either 365 or 420 nm,
respectively (Table 1). Its photolysis products also included
the undesired species dTN‑DEACM (Φbyproduct = 0.023) in
addition to thymidine. Thus, although the wavelength of light
plays a role in determining the rate of decay, it does not alter
the distribution of the released products. In summary, the
photolysis of 1 produced a thymidine-bearing byproduct,
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Isotope Laboratories, Inc. Column chromatography was performed
using silica gel of 200−400 mesh, and TLC was performed with silica
plates with a 250 μm thickness (Merck).
irradiation. However, it induces a thymidine-bearing byproduct
dTN‑DEACM regardless of the light wavelength used. In contrast,
the presence of the extended spacer in 2 dTO‑Bn‑DEACM leads to
better results including a fast decay, lack of the byproduct
formed, and greater efficiency of thymidine release (max 94 vs
58% with compound 1). We also demonstrated the synthesis
of 14 phosphoramidite, a new caged building block applicable
to the synthesis of caged oligonucleotides.
Analytical Methods. Caged products and their intermediates
were characterized for their structural identity and homogeneity by
standard analytical methods including NMR (1H, 13C) spectroscopy,
HRMS, UV−vis spectrometry, and UPLC.12
NMR spectra were acquired using a spectrometer from Varian (500
MHz for 1H; 100 MHz for proton-decoupled 13C) at 297.3 K using a
1
standard default pulse sequence. Chemical shift values for H NMR
CONCLUSIONS
spectra are reported in parts per million relative to tetramethylsilane
(TMS) or sodium 2,2-dimethyl-2-silapentane-5-sulfonate, each
serving as an internal standard (δ = 0.00 ppm), or relative to
known residual signals from the deuterated solvent used.
Mass spectral data were acquired using a Micromass AutoSpec
Ultima Magnetic sector mass spectrometer in an electrospray
ionization (ESI) mode. HRMS for measuring the exact mass was
performed using a VG 70-250-S mass spectrometer (magnetic sector,
EI) or Agilent 6230 TOF HPLC-MS with a jet stream ESI source.
Spectral data for UV−vis absorption were acquired in a
PerkinElmer Lamda 20 spectrophotometer.
UPLC analysis was performed for homogeneity analysis and
compound characterization in a Waters Acquity System combined
with a photodiode array (PDA) detector. The UPLC analysis involved
running a sample in a C4 BEH column (100 × 2.1 mm, 300 Å) at a
flow rate of 0.2 mL min−1. The elution occurred in a linear gradient
composed of two mobile solvents, water and acetonitrile, each
containing TFA (0.1% v/v) (eluents A and B, respectively). Its
method consisted of an initial phase 1% B (0−1.4 min), a linear
increase to 80% B (1.4−13.4 min), a decrease to 50% B (13.4−13.8
min), a decrease to 1% B (13.8−14.4 min), and finally an isocratic
elution at 1% B (14.4−18 min). This method was also applied in the
kinetic analysis of product release.
■
This study offers a number of insights that advance our
knowledge in the use of photocaging. First, it offers evidence
for the presence of photorearrangement59,69 in coumarin ether-
caged thymidine 1, with the side reaction reported here being
identified for the first time. Thus, this study adds another
example of photorearrangement to a growing list of substrates
where this phenomenon is observed.29,30,60 Second, this study
highlights the importance of validating the release efficiency
using an isolated caged unit because it is better suited for
accurate and sensitive analysis than when it is present in
oligomeric units or coupled to larger macromolecules. Here,
we were able to determine uncaging kinetics by testing a caged
thymidine instead of a caged oligonucleotide,21−24,53 while
photolysis analysis of the latter by HPLC21−24,53 appears to be
unable to detect structural alterations that could occur after
uncaging or rearrangement. Third, this study highlights the
importance of developing an optimal analytical method for
monitoring these structures, such as UPLC. Other methods
commonly employed for evaluating uncaging kinetics, such as
UV−vis spectrometry (Figure S13) and thin-layer chromatog-
raphy (TLC),12 lack sufficient product separation with high
resolution.
Photocaging continues to increase in popularity for light-
controlled activation and has been applied to an increasing
variety of substrates ranging from small-molecule ligands,3,7,10
therapeutic agents,12,19,33 peptides,29,30,32 proteins9 to oligo-
nucleotides.22−24 Factors important for controlling the
uncaging efficiency include light wavelength,6,10,28,43 exposure
time, and media pH58 as well as the type of the cage
molecule.12,24,34,38 Here, we present evidence that linker
chemistry also plays an important role in determining the
uncaging efficiency for certain cage types such as coumarin.
Importantly, this result is consistent with recent findings
performed with other classes of substrates based on thiols and
phenols.12,29,30 In summary, this study validates using a
hydroxy benzyl spacer strategy for preventing coumarin
recombination, which can be potentially applicable to a
broad range of substrates from nucleotides (cytidine,70
guanosine22) to therapeutic molecules including those tested
for light-controlled prodrug activation.12,71−73
Synthesis of Coumarin Cages 5 and 7 (Scheme 1). 7-
(Diethylamino)-4-(hydroxymethyl)-2H-1-benzopyran-2-one (5).
This compound was synthesized in two steps according to our earlier
protocol.12 These involve the oxidation of 7-(diethylamino)-4-methyl-
2H-chromen-2-one 3 to 7-(diethylamino)-2-oxo-2H-chromene-4-
carbaldehyde 4 with selenium dioxide12,64 and reduction of the
aldehyde functionality to a primary alcohol using sodium borohy-
dride.12,23 It was characterized by H NMR and MS, each of which
1
1
was consistent with the published data including its H NMR and
1
UV−vis spectral data as noted here. H NMR (500 MHz, CDCl3): δ
7.34−7.32 (d, J = 10 Hz, 1H), 6.60 (br, 1H), 6.55 (s, 1H), 6.27 (s,
1H), 4.84 (s, 2H), 3.43−3.39 (q, J = 7 Hz, 4H), 1.22−1.19 (t, J = 7
Hz, 6H) ppm. UV−vis spectroscopy ([5] = 5.05 × 10−5 M in 35% aq
methanol): λmax 387 nm (ε = 22,687 M−1 cm−1), 249 nm (ε = 17,014
M−1 cm−1) (see Figure 2).
4-((7-(Diethylamino)-2-oxo-2H-1-benzopyran-4-yl)methoxy])-
benzaldehyde (6). First, 5 was derivatized to its methanesulfonate
according to Wong et al.12 This was used immediately without further
purification. TLC analysis: Rf (2% methanol/dichloromethane) =
0.44. MS (ESI) m/z (relative intensity, %) = 326 (100%) [M + H]+.
Second, potassium carbonate (464 mg, 3.36 mmol) was added to
4-hydroxybenzaldehyde (164 g, 1.34 mmol) in anhydrous THF (12
mL). The mixture was stirred at 45 °C for 10 min prior to adding a
solution of 7-diethylaminocoumarin-4-hydroxylmethyl methanesulfo-
nate 5 (365 mg, 1.12 mmol) in anhydrous THF (3 mL). The mixture
was stirred continuously for 4 h, followed by filtration and
concentration in vacuo. The residue was purified by flash column
chromatography by eluting with a mixture of dichloromethane/ethyl
acetate/hexanes (1:1:2). The product 6 was obtained as a yellow solid
(235 mg, 60%). TLC analysis: Rf (1:1:2 dichloromethane/ethyl
acetate/hexanes) = 0.33. MS (ESI) m/z (relative intensity, %) =
352.1 (100) [M + H]+. HRMS (EI) [M + H]+ m/z: calcd for
EXPERIMENTAL SECTION
■
Materials and Methods. Unless otherwise noted, all reagents and
solvents were used as received from commercial suppliers. These
include thymidine (≥99%), 4-hydroxybenzaldehyde (98%), meth-
anesulfonyl chloride (99.7%), 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite, 4,4′-dimethoxytriphenylmethyl chloride (≥97%),
acetonitrile, dichloromethane, cyclohexanes, hexanes, and ethyl
acetate (all from Sigma-Aldrich), 7-(diethylamino)-4-methyl-2H-
chromen-2-one (98%, TCI America), tert-butyldimethylchlorosilane
(97%, Alfa Aesar), and 1,2,4-1H-triazole (99.5%, Acros Organics).
1H and 13C NMR spectral data were acquired in deuterium-labeled
solvents including CDCl3 (99.96% D), DMSO-d6 (99.9% D), CD3OD
(99.8% D), and D2O (99.9% D), each purchased from Cambridge
+
C21H22NO4 , 352.1549; found, 352.1544. 1H NMR (500 MHz,
DMSO-d6): δ 9.90 (s, 1H), 7.92−7.90 (d, J = 10 Hz, 2H), 7.60−7.58
(d, J = 10 Hz, 1H), 7.33−7.31 (d, J = 10 Hz, 2H), 6.73−6.71 (d, J =
10 Hz, 1H), 6.56 (s, 1H), 6.14 (s, 1H), 5.46 (s, 2H), 3.46−3.42 (q, J
= 7 Hz, 4H), 1.11−1.14 (t, J = 7 Hz, 6H) ppm. 13C{1H} NMR (100
F
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MHz, DMSO-d6): δ 191.4, 191.3, 162.5, 160.7, 155.8, 150.8, 150.5,
131.8, 131.8, 130.2, 125.8, 125.6, 115.5, 115.3, 108.7, 105.2, 105.1,
96.9, 96.8, 65.6, 44.1, 44.0, 43.9, 12.33, 12.25 ppm.
was purified by column chromatography by eluting with ethyl acetate/
hexanes/dichloromethane (1:1:1), yielding 10 as a white solid (0.94
g, 85%). TLC analysis: Rf (ethyl acetate/hexanes/dichloromethane;
1:1:1) = 0.17. MS (ESI) m/z (relative intensity, %) = 522.2 (20) [M
+ H]+, 544.2 (17) [M + Na]+, 1043.5 (75) [2M + H]+, 1065.5 (100)
[2M + Na]+. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
7-(Diethylamino)-4-[(4-(hydroxymethyl)phenoxy)methyl]-2H-1-
benzopyran-2-one (7). To a cold solution of 6 (200 mg, 0.626
mmol) in methanol (4 mL) in an ice bath was added sodium
borohydride (23 mg, 0.626 mmol). The mixture was stirred at the
same temperature for 30 min, and the reaction was quenched by
adding 0.1 M sodium hydroxide solution (1 mL). The solution was
concentrated in vacuo, and the residue was dissolved in dichloro-
methane (10 mL). After washing with water, the organic layer was
dried over sodium sulfate and concentrated in vacuo, yielding a
residue, which was purified by flash column chromatography by
eluting with a mixture of dichloromethane/ethyl acetate/hexanes
(1:1:1). The desired product 7 was obtained as a yellow solid (168
mg, 84%). TLC analysis: Rf (1:1:1 dichloromethane/ethyl acetate/
hexanes) = 0.39. MS (ESI) m/z (relative intensity, %) = 354.1 (100).
+
C24H44N5O4Si2 , 522.2932; found, 522.2927; [M + Na]+ calcd for
+
C24H43N5NaO4Si2 , 544.2751; found, 544.2745. 1H NMR (500 MHz,
CD3OD): 9.33 (s, 1H), 8.38 (s, 1H), 8.24 (s, 1H), 6.22−6.20 (t, J = 5
Hz, 1H), 4.49−4.47 (m, 1H), 4.13−4.11 (m, 1H), 3.98−3.85 (m,
2H), 2.63−2.58 (m, 1H), 2.45 (s, 3H), 2.21−2.16 (m, 1H), 0.93−
0.91 (s, 18H), 0.14−0.13 (s, 12H) ppm. 13C{1H} NMR (100 MHz,
DMSO-d6): 157.9, 153.41, 153.35, 152.9, 147.1, 145.3, 145.2, 104.4,
88.1, 87.9, 87.2, 87.1, 71.9, 71.8, 62.4, 40.9, 25.72, 25.65, 25.6, 17.9,
17.7, 16.2, 16.1, −4.77, −4.82, −4.97, −5.02, −5.6 ppm.
4-((7-(Diethylamino)-2-oxo-2H-1-benzopyran-4-yl)methoxy)-1-
((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyri-
midin-2(1H)-one (11). To a solution of 5 (420 mg, 1.70 mmol) and
10 (970 mg, 1.86 mmol) dissolved in anhydrous acetonitrile (14 mL)
was added 1,8-diazabicyclo[5,4,0]undec-7-ene (0.28 mL, 1.86 mmol).
The mixture was stirred at room temperature overnight and then
concentrated in vacuo. The residue was purified by column
chromatography by eluting with ethyl acetate/hexanes/dichloro-
methane (1:2:1), yielding 11 as a pale yellow foam (1.11 g, 97%).
TLC analysis: Rf (ethyl acetate/hexanes/dichloromethane; 1:2:1) =
0.62. MS (ESI) m/z (relative intensity, %) = 700.38 (55) [M + H]+,
1400.75 (45) [2M + H]+. HRMS (ESI-TOF) m/z: [M + H]+ calcd
+
HRMS (ESI-TOF) [M + H]+ m/z: calcd for C21H24NO4 , 354.1705;
1
found, 354.1711. H NMR (500 MHz, CD3OD): 7.58−7.56 (d, J =
10 Hz, 1H), 7.32−7.30 (d, J = 10 Hz, 2H), 7.04−7.02 (d, J = 10 Hz,
2H), 6.77−6.75 (dd, J1 = 5 Hz, J2 = 10 Hz, 1H), 6.57−6.56 (d, J = 5
Hz, 1H), 6.20 (s, 1H), 5.29 (s, 2H), 4.53 (s, 2H), 3.50−3.46 (q, J = 7
Hz, 4H), 1.23−1.20 (t, J = 7 Hz, 6H) ppm. 13C{1H} NMR (100
MHz, DMSO-d6): 160.7, 156.5, 155.8, 151.7, 150.4, 135.4, 128.0,
127.9, 125.7, 125.6, 114.5, 108.7, 105.4, 105.2, 105.1, 96.9, 96.8, 65.2,
62.5, 44.0, 43.9, 12.33, 12.26 ppm.
Synthesis of 1 dTO‑DEACM (Scheme 1). Synthesis of 1 was
performed according to Rodrigues-Correia et al.23 Its characterization
data are consistent with those values reported therein or as
anticipated, confirming its structural identity.
+
for C36H58N3O7Si2 , 700.3813; found, 700.3806. 1H NMR (500
MHz, DMSO-d6: 7.82 (s, 1H), 7.51−7.49 (d, J = 10 Hz, 1H), 6.72−
6.70 (d, J = 10 Hz, 1H), 6.55 (s, 1H), 6.15−6.12 (t, J = 5 Hz, 1H),
5.97 (s, 1H), 5.59−5.52 (m, 2H), 4.36 (s, 1H), 3.89 (s, 1H), 3.88−
3.72 (m, 2H), 3.45−3.41 (q, J = 7 Hz, 4H), 2.24−2.22 (m, 1H),
2.15−2.10 (m, 1H), 1.98 (s, 3H), 1.13−1.10 (t, J = 7 Hz, 6H), 0.87
(s, 18H), 0.08 (s, 12H) ppm. 13C{1H} NMR (500 MHz, DMSO-d6):
168.8, 160.6, 155.8, 154.2, 150.53, 150.47, 141.1, 125.4, 108.8, 105.2,
104.9, 102.9, 96.8, 87.3, 85.7, 72.0, 63.4, 62.6, 44.0, 40.5, 26.3, 25.7,
25.6, 17.9, 17.7, 12.3, 11.8, −4.8, −5.0, −5.5 ppm.
1-((2R,4S,5R)-4-(tert-Butyldimethylsilyl)oxy-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyri-
midine-2,4(1H,3H)-dione (9). Protection of thymidine through O-
TBDMS was performed according to an established protocol in the
literature.23,43 To a solution of thymidine (0.60 g, 2.48 mmol) in
dimethylformamide (DMF; 2.2 mL) were added tert-butyldimethyl-
silyl chloride (TBDMS-Cl) (1.49 g, 9.91 mmol) and imidazole (1.18
g, 17.34 mmol). The mixture was stirred at room temperature
overnight, and its reaction was quenched by adding ethanol (2 mL),
followed by stirring for 30 min. It was diluted with ethyl acetate (50
mL), and the white solid, which was precipitated, was filtered off and
washed with ethyl acetate (10 mL). The organic solutions were
combined, washed with water (3 × 50 mL) and brine (50 mL), and
then dried over sodium sulfate. Concentration of the solution in vacuo
afforded the product 9 as a white solid (1.19 g, 100%). TLC analysis:
Rf (1:10 methanol/dichloromethane) = 0.45. MS (ESI) m/z (relative
intensity, %) = 493.2 (100) [M + Na]+, 963.5 (50) [2M + Na]+.
4-((7-(Diethylamino)-2-oxo-2H-1-benzopyran-4-yl)methoxy)-1-
((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-
methylpyrimidin-2(1H)-one (1). To a solution of 11 (1.09 g, 1.56
mmol) in THF (3.6 mL) was added acetic acid (0.88 mL). The
mixture was stirred at room temperature for 10 min, followed by the
addition of 1 M TBAF in THF (4.7 mL). The mixture was stirred
overnight and evaporated in vacuo. The residue was triturated and
washed with saturated sodium bicarbonate solution (3 × 10 mL),
water (5 × 10 mL), and ethyl acetate/hexane (1:1; 3 × 10 mL). The
desired product 1 was obtained as an orange white solid (0.6 g, 82%).
TLC analysis: Rf (cyclohexane/acetone; 1:1) = 0.1. UPLC: tr = 8.72
min (homogeneity ≥ 95%). MS (ESI) m/z (relative intensity, %) =
494.1 (30) [M + Na]+, 943.4 (16) [2M + H]+, 965.3 (30) [2M +
+
HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C22H42N2NaO5Si2 ,
1
493.2530; found, 493.2523. H NMR (500 MHz, CD3OD): 7.54 (s,
1H), 6.25−6.23 (t, J = 5 Hz, 1H), 4.48−4.46 (m, 1H), 3.93−3.91 (m,
1H), 3.87−3.79 (m, 2H), 2.23−2.10 (m, 2H), 1.87 (s, 3H), 0.93−
0.91 (s, 18H), 0.12−0.11 (s, 12H) ppm.
+
Na]+. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C24H29N3NaO7 ,
494.1903; found, 494.1898. 1H NMR (500 MHz, DMSO-d6): 8.13 (s,
1H), 7.51−7.49 (d, J = 10 Hz, 1H), 6.73−6.71 (dd, J1 = 3 Hz, J2 = 10
Hz, 1H), 6.56−6.55 (d, J = 3 Hz, 1H), 6.15−6.12 (t, J = 5 Hz, 1H),
5.98 (s, 1H), 5.56 (s, 2H), 4.24 (m, 1H), 3.83−3.81 (m, 1H), 3.66−
3.56 (m, 2H), 3.46−3.42 (q, J = 7 Hz, 2H), 2.24−2.19 (m, 1H),
2.06−2.00 (m, 1H), 1.98 (s, 3H), 1.14−1.11 (t, J = 7 Hz, 6H) ppm.
13C{1H} NMR (100 MHz, DMSO-d6): 168.7, 160.6, 155.8, 154.3,
1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methyl-4-
(1H-1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (10). Substitution of O-
TBDMS protected thymidine with 1,2,4-1H-triazole at the C4
position was performed according to a protocol reported in the
literature.23,43 To a cold solution of 1,2,4-1H-triazole (10.4 g, 150.8
mmol) in anhydrous acetonitrile (43.5 mL) in an ice bath were added
phosphoryl chloride (3.1 mL, 33.28 mmol) and then triethylamine
(22.9 mL, 164.2 mmol), each in a dropwise manner. After stirring for
15 min, a solution of 9 (1.0 g, 2.12 mmol) in anhydrous acetonitrile
(16.9 mL) was added to the mixture. The final mixture was heated to
room temperature and stirred overnight. To quench the reaction, a
saturated solution of sodium bicarbonate (10 mL) was added to the
mixture, and it was stirred for 30 min. Volatile solvents were removed
by concentration in vacuo, and the residue was extracted with
dichloromethane (45 mL). The organic solution was washed with
saturated sodium bicarbonate (30 mL), water (30 mL), and brine (30
mL), dried over sodium sulfate, and evaporated in vacuo. The residue
150.6, 150.5, 141.8, 125.5, 125.4, 108.81, 108.78, 105.2, 104.9, 104.7,
102.8, 96.9, 96.8, 87.7, 87.6, 85.7, 85.5, 69.93, 69.85, 63.3, 60.9, 44.1,
44.0, 43.9, 40.6, 12.32, 12.25, 11.82, 11.78 ppm. UV−vis spectroscopy
([1] = 1.06 × 10−4 M in 35% aq methanol): λmax = 393 nm (ε =
10,209 M−1 cm−1), 282 nm (ε = 5926 M−1 cm−1), 254 nm (ε = 8721
M−1 cm−1) (see Figure 2).
Synthesis of 2 dTO‑Bn‑DEACM (Scheme 1). 4-((4-((7-(Diethylami-
no)-2-oxo-2H-1-benzopyran-4-yl)methoxy)benzyl)oxy)-1-
((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyri-
midin-2(1H)-one (12). To a solution of 7 (290 mg, 0.82 mmol) and
10 (471 mg, 0.90 mmol) dissolved in acetonitrile (6 mL) was added
G
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1,8-diazabicyclo[5,4,0]undec-7-ene (0.135 mL, 0.90 mmol). The
mixture was stirred at room temperature overnight and then
concentrated in vacuo. The residue was purified by column
chromatography by eluting with ethyl acetate/hexanes/dichloro-
methane (1:3:1), yielding 12 as a pale yellow foam (615 mg, 93%).
TLC analysis: Rf (ethyl acetate/hexanes/dichloromethane; 1:2:1) =
bromide.74 This reaction mixture was diluted with acetone (5 mL),
followed by the addition of thymidine (236 mg, 0.98 mmol) and
potassium carbonate (270 mg, 1.95 mmol). The mixture was stirred at
55 °C for 48 h and concentrated in vacuo. The residue was purified by
silica column chromatography by eluting with a mixture of acetone
and cyclohexanes (1:1), affording a brown solid. It was rinsed with
dichloromethane (3 × 1 mL) and then dried under high vacuum,
yielding dTN‑DEACM as a pale yellow solid (140 mg, 37%). TLC
analysis: Rf (1:1 acetone/cyclohexanes) = 0.36. UPLC traces acquired
for dTN‑DEACM show that it elutes at tr = 8.57 min slightly faster than 1
at tr = 8.72 min (Figure S12). Its structural identity was confirmed
through spectroscopic characterization. MS (ESI) m/z (relative
intensity, %) = 472.20 (100) [M + H]+, 494.19 (10) [M + Na]+.
+
0.40. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C43H64N3O8Si2 ,
1
806.4232; found, 806.4223. H NMR (500 MHz, DMSO-d6): δ 7.74
(s, 1H), 7.59−7.57 (d, J = 10 Hz, 1H), 7.43−7.41 (d, J = 10 Hz, 2H),
7.13−7.11 (d, J = 10 Hz, 2H), 6.72−6.70 (d, J = 10 Hz, 1H), 6.55 (s,
1H), 6.16−6.14 (t, J = 5 Hz, 1H), 6.12 (s, 1H), 5.33 (s, 2H), 5.28 (s,
2H), 4.36 (s, 1H), 3.86 (s, 1H), 3.80−3.72 (m, 2H), 3.45−3.41 (q, J
= 7 Hz, 4H), 2.22−2.20 (m, 1H), 2.13−2.07 (m, 1H), 1.88 (s, 3H),
1.13−1.10 (t, J = 7 Hz, 6H), 0.87−0.86 (s, 18H), 0.08−0.07 (s, 12H)
ppm. 13C{1H} NMR (100 MHz, DMSO-d6): 169.4, 160.7, 157.6,
155.8, 154.4, 151.4, 150.4, 140.4, 129.9, 128.8, 125.7, 114.8, 108.7,
105.4, 105.2, 103.1, 96.8, 87.2, 85.5, 72.0, 67.6, 65.3, 62.6, 44.0, 40.4,
25.7, 25.6, 17.9, 17.7, 12.3, 11.9, −4.8, −5.0, −5.5 ppm.
+
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C24H30N3O7 , 472.2084;
found, 472.2079. 1H NMR (500 MHz, DMSO-d6): 7.91 (s, 1H),
7.68−7.66 (d, J = 10 Hz, 1H), 6.75−6.72 (dd, J1 = 5 Hz, J2 = 10 Hz,
1H), 6.56−6.55 (d, J = 5 Hz, 1H), 6.22−6.19 (t, J = 7.5 Hz, 1H), 5.43
(s, 1H), 5.25−5.24 (d, J = 5 Hz, 1H), 5.12 (s, 2H), 5.08−5.06 (t, J =
5 Hz, 1H), 4.27−4.26 (m, 1H), 3.80−3.78 (m, 1H), 3.63−3.58 (m,
2H), 3.46−3.43 (q, J = 5Hz, 4H), 2.20−2.13 (m, 2H), 1.88 (s, 3H),
1.14−1.12 (t, J = 5 Hz, 6H) ppm. 13C{1H} NMR (100 MHz, DMSO-
d6): 162.5, 160.6, 155.7, 151.1, 150.5, 150.3, 135.6, 125.3, 108.8,
108.4, 105.8, 103.0, 96.8, 87.4, 85.1, 70.1, 61.1, 44.0, 40.6, 39.6, 12.9,
12.3 ppm.
4-((4-((7-(Diethylamino)-2-oxo-2H-1-benzopyran-4-yl)methoxy)-
benzyl)oxy)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-5-methylpyrimidin-2(1H)-one (2
dTO‑Bn‑DEACM). Acetic acid (0.46 mL) was added to a solution of 12
(530 mg, 0.66 mmol) in THF (2.3 mL). The mixture was stirred at
room temperature for 10 min, followed by the addition of 1 M TBAF
in THF (2 mL). The mixture was stirred overnight, and the mixture
was diluted with diethyl ether (20 mL), which led to precipitation of
the product as a pale yellow solid. It was collected, triturated, and
washed with saturated sodium bicarbonate (3 × 10 mL), water (5 ×
10 mL), ethyl acetate/hexane (1:1; 5 × 10 mL), and cyclohexane/
dichloromethane/acetone (5:1:1; 10 mL). The solid was dried in
vacuo, yielding 2 as a pale yellow solid (270 mg, 71%). A TLC
analysis showed only one spot with an Rf (cyclohexane/acetone; 1:2)
value of 0.3. UPLC: tr = 9.32 min (homogeneity ≥ 95%). MS (ESI)
m/z (relative intensity, %) = 230.1 and 336.1 (100), 578.2 (30) [M +
H]+, 600.2 (15) [M + Na]+. HRMS (ESI-TOF) m/z: [M + H]+ calcd
Synthesis of 2 dTO‑Bn‑DEACM Phosphoramidite (Scheme 2). 1-
((2R,4S,5R)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-
hydroxytetrahydrofuran-2-yl)-4-((4-((7-(diethylamino)-2-oxo-2H-1-
benzopyran-4-yl)methoxy)benzyl)oxy)-5-methylpyrimidin-2(1H)-
one (13). O-Protection with a 4,4′-dimethoxytrityl (DMT) group was
performed by adding 4,4′-dimethoxytrityl chloride (182 mg, 0.54
mmol) to 2 (248 mg, 0.43 mmol) in pyridine (6.5 mL) cooled at 0
°C. The mixture was stirred overnight while allowing it to warm up to
room temperature. The reaction was quenched by adding ethanol (0.2
mL), and the reaction mixture was evaporated in vacuo and then
coevaporated with toluene (3 × 10 mL) to remove any residual
pyridine. The crude product was purified by column chromatography
by eluting with cyclohexane/acetone (1:1), yielding 13 as a pale
yellow solid (230 mg, 61%). TLC analysis: Rf (cyclohexane/acetone;
1:2) = 0.50. UPLC: tr = 12.15 min (homogeneity ≥ 95%). MS (ESI)
m/z (relative intensity, %) = 902.3 (90) [M + Na]+. HRMS (ESI-
TOF) m/z: [M + Na]+ calcd for C52H53N3NaO10+, 902.3629; found,
+
1
for C21H35N3O8 , 578.2502; found, 578.2496. H NMR (500 MHz,
CD3OD): 8.15 (s, 1H), 7.58−7.56 (d, J = 10 Hz, 1H), 7.46−7.44 (d,
J = 10 Hz, 2H), 7.06−7.08 (d, J = 10 Hz, 2H), 6.76−6.74 (dd, J1 = 3
Hz, J2 = 10 Hz, 1H), 6.57−6.56 (d, J = 3 Hz, 1H), 6.26−6.24 (t, J = 5
Hz, 1H), 6.21 (s, 1H), 5.37 (s, 2H), 5.30 (s, 2H), 4.38−4.36 (m, 1H),
3.95−3.94 (m, 1H), 3.84−3.72 (m, 2H), 3.50−3.46 (q, J = 7 Hz, 4H),
2.41−2.38 (m, 1H), 2.17−2.13 (m, 1H), 1.96 (s, 3H), 1.23−1.20 (t, J
= 7 Hz, 6H) ppm. 13C{1H} NMR (100 MHz, DMSO-d6): 169.3,
160.7, 157.6, 155.8, 154.5, 151.5, 150.4, 141.11, 141.05, 130.0, 129.8,
128.9, 125.8, 125.6, 114.9, 114.7, 108.7, 105.6, 105.3, 105.1, 102.9,
96.9, 96.8, 87.6, 87.5, 85.5, 85.3, 70.0, 69.9, 67.4, 65.3, 61.0, 44.0,
43.9, 40.6, 12.34, 12.25, 11.9 ppm. UV−vis spectroscopy ([2] = 8.67
× 10−5 M in 35% aq methanol): λmax = 390 nm (ε = 11,603 M−1
cm−1), 277 nm (ε = 8133 M−1 cm−1), 252 nm (ε = 11,773 M−1 cm−1)
(see Figure 2).
1
902.3618. H NMR (500 MHz, CD3OD): 8.02 (s, 1H), 7.57−7.55
(d, J = 10 Hz, 1H), 7.45−7.43 (d, J = 10 Hz, 2H), 7.40−7.39 (d, J = 5
Hz, 2H), 7.30−7.25 (m, 6H), 7.22−7.19 (m, 1H), 7.08−7.06 (d, J =
10 Hz, 2H), 6.85−6.84 (d, J = 5 Hz, 4H), 6.76−6.74 (dd, J1 = 3 Hz, J2
= 10 Hz, 1H), 6.56−6.55 (d, J = 3 Hz, 1H), 6.28−6.25 (t, J = 5 Hz,
1H), 6.21 (s, 1H), 5.40−5.33 (m, 2H), 5.30 (s, 2H), 4.51−4.50 (m,
1H), 4.06−4.05 (m, 1H), 3.76 (s, 6H), 3.50−3.47 (m, 4H), 3.45−
3.34 (m, 2H), 2.51−2.48 (m, 1H), 2.33−2.29 (m, 1H), 1.50 (s, 3H),
1.22−1.19 (t, J = 7 Hz, 6H) ppm. 13C{1H} NMR (100 MHz, DMSO-
d6): 169.4, 160.7, 158.1, 157.6, 155.8, 151.4, 150.4, 144.7, 140.5,
135.4, 135.2, 129.9, 129.7, 128.8, 127.9, 127.6, 126.8, 125.7, 114.8,
113.2, 108.7, 105.4, 105.2, 103.2, 96.8, 85.9, 85.7, 85.4, 70.1, 67.5,
65.3, 63.4, 55.0, 44.0, 40.7, 12.3, 11.4 ppm.
Synthesis of dTN‑DEACM (Scheme S1). 3-((7-(Diethylamino)-2-
oxo-2H-1-benzopyran-4-yl)methyl)-1-((2R,4S,5R)-4-hydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4-
(1H,3H)-dione (dTN‑DEACM). Synthesis of dTN‑DEACM was performed by
N-alkylation of thymidine (dT) at the 3 position with 7-
diethylaminocoumarinyl-4-methyl bromide74 according to the con-
dition reported by Yoshida et al.44
To a cold solution of 5 (160 mg, 0.65 mmol) and triethylamine
(272 μL, 1.95 mmol) in dichloromethane (4 mL) in an ice water bath
was added methanesulfonyl chloride (182 μL, 2.34 mmol). This
reaction mixture was stirred in an ice bath for 1 h prior to dilution
with dichloromethane (4 mL). The solution was washed several times
using 0.5 N HCl (2 × 5 mL), saturated NaHCO3 solution (5 mL),
water (5 mL), and brine (5 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated in vacuo, yielding 7-
diethylaminocoumarinyl-4-methyl methanesulfonate, which was used
immediately.
1-((2R,4S,5R)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)-
methyl)-4-(((2-cyanoethyl)-N,N-bis(1-methylethyl)phosphino)oxy)-
tetrahydrofuran-2-yl)-4-((4-((7-(diethylamino)-2-oxo-2H-1-benzo-
pyran-4-yl)methoxy)benzyl)oxy)-5-methylpyrimidin-2(1H)-one
(14). To a solution of 13 (140 mg, 0.16 mmol) dissolved in
anhydrous dichloromethane (5 mL) were added in sequence DIPEA
(0.14 mL, 0.8 mmol) and then 2-cyanoethyl N,N-diisopropylchlor-
ophosphoramidite (71 μL, 0.32 mmol). The mixture was stirred for 2
h, diluted with dichloromethane (5 mL), and washed extensively with
saturated sodium bicarbonate (2 × 5 mL), water (5 mL), and brine (5
mL). The organic layer was evaporated in vacuo, and the residue was
purified by column chromatography by eluting with cyclohexane/
dichloromethane/acetone (2:1:1). The desired product 14 was
obtained as a yellow solid (126 mg, 73%). UPLC is not applicable
for its analysis because of the aqueous instability of its
phosphoramidite moiety. TLC analysis: a single spot with Rf
(cyclohexane/dichloromethane/acetone; 2:1:1) of 0.39. MS (ESI)
To this derivative dissolved in THF (5 mL) was added lithium
bromide (113 mg, 1.3 mmol). This mixture was stirred at room
temperature for 3 h to produce 7-diethylaminocoumarinyl-4-methyl
H
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J. Org. Chem. XXXX, XXX, XXX−XXX

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Article
m/z (relative intensity, %) = 1080.4 (20) [M + H]+, 1102.4 (30) [M
+ Na]+. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for
C61H70N5NaO11P+, 1102.4707; found, 1102.4696. 1H NMR (500
MHz, CD3OD): 7.85 (s, 1H), 7.59−7.57 (d, J = 10 Hz, 1H), 7.43−
7.41 (d, J = 10 Hz, 2H), 7.38−7.37 (d, J = 5 Hz, 2H), 7.30−7.21 (m,
7H), 7.13−7.11 (d, J = 10 Hz, 2H), 6.88−6.86 (dd, J1 = 3 Hz, J2 = 10
Hz, 4H), 6.73−6.71 (dd, J1 = 3 Hz, J2 = 10 Hz, 1H), 6.56−6.55 (d, J =
3 Hz, 1H), 6.22−6.20 (t, J = 10 Hz, 1H), 6.13 (s, 1H), 5.34 (s, 2H),
5.29 (s, 2H), 4.54−4.53 (m, 1H), 4.05−4.06 (m, 1H), 3.72 (s, 6H),
3.74−3.68 (m, 2H), 3.52−3.47 (m, 2H), 3.46−3.42 (q, J = 10 Hz,
4H), 2.77−2.75 (t, J = 10 Hz, 2H), 2.48−2.43 (m), 2.33−2.29 (m,
1H), 1.57 (s, 3H), 1.13−1.10 (m, 12H), 0.98−0.97 (d, J = 5 Hz, 6H)
ppm. 13C{1H} NMR (100 MHz, DMSO-d6): 169.4, 160.7, 158.2,
157.6, 155.8, 154.3, 151.4, 150.4, 144.5, 140.6, 135.2, 135.1, 129.9,
129.7, 128.8, 127.9, 127.6, 126.8, 125.7, 118.9, 114.8, 113.2, 108.7,
105.4, 105.2, 103.4, 96.8, 86.0, 85.4, 84.4, 67.6, 65.3, 62.8, 58.3, 58.2,
55.0, 46.2, 44.0, 42.6, 42.5, 29.6, 26.3, 24.3, 24.2, 24.1, 22.2, 20.7,
19.8, 19.2, 18.8, 12.3, 11.4 ppm.
48109, United States; orcid.org/0000-0001-5633-4817;
Email: skchoi@umich.edu
Authors
Shengzhuang Tang − Michigan Nanotechnology Institute for
Medicine and Biological Sciences and Department of Internal
Medicine, University of Michigan Medical School, Ann Arbor,
Michigan 48109, United States
Jayme Cannon − Michigan Nanotechnology Institute for
Medicine and Biological Sciences and Department of Internal
Medicine, University of Michigan Medical School, Ann Arbor,
Michigan 48109, United States
Kelly Yang − Michigan Nanotechnology Institute for Medicine
and Biological Sciences, University of Michigan Medical School,
Ann Arbor, Michigan 48109, United States
Matthew F. Krummel − Department of Pathology, University of
California, San Francisco, San Francisco, California 94143,
United States
Photon Uncaging Experiments. Light Sources. Photolytic
studies for thymidine release were performed with three light sources
(Luzchem Research). These include (i) a UVA lamp with a maximal
intensity at 365 nm; (ii) a visible light lamp at 420 nm (LZC-420);
and (iii) a light-emitting diode lamp for blue light 445−465 nm
(LZC-LBL). Photon flux density (qn,p = qp/NA, mol × s−1) of each
lamp was determined by ferrioxalate actinometry according to a
standard protocol.67,68 Photon flux density (qn,p = qp/NA, mol ×
James R. Baker, Jr. − Michigan Nanotechnology Institute for
Medicine and Biological Sciences and Department of Internal
Medicine, University of Michigan Medical School, Ann Arbor,
Michigan 48109, United States; orcid.org/0000-0002-
4478-1932
−1) determined for each lamp by ferrioxalate actinometry is
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.9b02617
s
summarized in Table S2 (Supporting Information).
Uncaging Kinetics and Analysis. Typically, a solution of caged
thymidine 1 or 2 (dTcaged: 0.05−0.1 mg/mL) in aq methanol (30−
35% v/v) was prepared and exposed to the light at a distance of ∼5
cm. After each exposure period, the solution was aliquoted out as a
function of irradiation time (0−30 min), and each aliquot was
analyzed by UPLC and UV−vis absorption spectrometry to
determine the kinetics of thymidine release and byproduct formation.
Certain aliquots were further analyzed by HPLC-MS (ESI) to validate
the identity of thymidine and released molecular species in the
photolyzed solution.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the National Cancer
Institute, National Institutes of Health under award
1R21CA191428. S.K.C. acknowledges support from the
Michigan Nanotechnology Institute for Medicine and Bio-
logical Sciences, University of Michigan Medical School.
Quantum efficiency (Φ) for the release of thymidine (dT) is
defined in eq 1, and its value was calculated according to a standard
method as noted in eq 268
ABBREVIATIONS
dT, thymidine; DEACM, 7-diethylamino-4-methylcoumarin;
UPLC, ultraperformance liquid chromatography
■
Φ
= [number of thymidine released]
/[number of photon absorbed]
release
(1)
(2)
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= (ΔdT/Δt)/(qn,p(1 − 10−A))
■
Φ
release
̌
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.9b02617.
■
sı
Summary of photo physicochemical properties 1 and 2;
summary of photon flux density; synthetic scheme of
dTN‑DEACM; copies of spectral data; UPLC traces for
photouncaging; and HPLC-MS analysis of photolysis
products from 1 (PDF)
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AUTHOR INFORMATION
Corresponding Author
Seok Ki Choi − Michigan Nanotechnology Institute for Medicine
and Biological Sciences and Department of Internal Medicine,
University of Michigan Medical School, Ann Arbor, Michigan
■
I
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