Control of an Unusual Photo-Claisen Rearrangement in Coumarin Caged Tamoxifen through an Extended Spacer

Article abstract of DOI:10.1021/acschembio.6b00999

The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.

Full text of DOI:10.1021/acschembio.6b00999

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Article
Control of an Unusual Photo-Claisen Rearrangement in
Coumarin Caged Tamoxifen through an Extended Spacer
Pamela T. Wong, Edward W. Roberts, Shengzhuang Tang, Jhindan Mukherjee,
Jayme Cannon, Alyssa J. Nip, Kaitlin Corbin, Matthew F. Krummel, and Seok Ki Choi
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00999 • Publication Date (Web): 13 Feb 2017
Downloaded from http://pubs.acs.org on February 14, 2017
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Control of an Unusual PhotoꢀClaisen Rearrangement
in Coumarin Caged Tamoxifen through an Extended
Spacer
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Pamela T. Wong,‡ab Edward W. Roberts,‡c Shengzhuang Tang,ab Jhindan Mukherjee,ab Jayme
Cannon,ab Alyssa J. Nip,c Kaitlin Corbin,c Matthew F. Krummel,*c and Seok Ki Choi*ab
a Michigan Nanotechnology Institute for Medicine and Biological Sciences, b Department of
Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109, United
States
c Department of Pathology, University of California, San Francisco, 513 Parnassus Ave,
HSW512, San Francisco, California 94143, United States
*To whom correspondence should be addressed
Phone: (415) 514ꢀ3130; Email: matthew.krummel@ucsf.edu
Phone: (734) 647ꢀ0052; Email: skchoi@umich.edu
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The use of coumarin caged molecules has been well documented in numerous photocaging
applications including for the spatiotemporal control of Creꢀestrogen receptor (CreꢀERT2)
recombinase activity. In this article, we report that 4ꢀhydroxytamoxifen (4OHT) caged with
coumarin via a conventional ether linkage led to an unexpected photoꢀClaisen rearrangement
which significantly competed with the release of free 4OHT. The basis for this unwanted
reaction appears to be related to the coumarin structure and its radicalꢀbased mechanism of
uncaging as it did not occur in orthoꢀnitrobenzyl (ONB) caged 4OHT that was otherwise linked
in the same manner. In an effort to perform design optimization, we introduced a selfꢀimmolative
linker longer than the ether linkage, and identified an optimal linker which allowed rapid 4OHT
release by both singleꢀphoton and twoꢀphoton absorption mechanisms. The ability of this
construct to actively control CreꢀERT2 mediated gene modifications was investigated in mouse
embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP)
reporter dependent gene recombination was controlled by 4OHT release and measured by
confocal fluorescence microscopy and flow cytometry. In summary, we report the implications
of this photoꢀClaisen rearrangement in coumarin caged compounds, and demonstrate a rational
linker strategy for addressing this unwanted side reaction.
Key words: Tamoxifen, Coumarin Cage, PhotoꢀClaisen Rearrangement, TwoꢀPhoton Uncaging,
CreꢀERT2 Recombination, Green Fluorescent Protein
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The application of photocaging technology1ꢀ3 has yielded numerous novel photoprobes that
have enabled the active control of molecular activities and cellular processes by light. The
fundamental concept behind these strategies is the temporary inactivation of a biologically active
molecule through the covalent attachment of a photocleavable cage molecule until the caged
molecule is reactivated by light exposure.2 Several types of cage molecules, each distinct in their
structural and photochemical properties,4ꢀ6 have been reported, including orthoꢀnitrobenzene
(ONB),1, 7, 8 coumarin,9, 10 nitrobenzofuran,11 and quinoline12 chromophores. The contributions of
their applications have been well documented in numerous fields ranging from ion channels1, 13
and enzymes14, 15 to gene expression,16 photopharmacology,17, 18 and controlled drug transport or
delivery.19ꢀ21
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Recently, a number of laboratories22ꢀ24 including ours25, 26 have reported on caged forms of
ligands for the estrogen receptor (ER), such as tamoxifen (TAM),26 4ꢀhydroxytamoxifen
(4OHT)25, 27 and their homologues,22ꢀ24 and have validated their ability to control lightꢀinducible
reporter gene modifications in optogenetic models.23, 28 In our studies,25, 26 we used transgenic
mouse embryonic fibroblasts (MEFs) with a reporter cassette (mTmG29) which contains a gene
for red fluorescent protein (RFP) TdTomato flanked by loxP sites (Figure 1). These MEFs also
constitutively express Cre recombinase fused to a modified ER that has a reduced affinity for
native estrogen (UbcCreꢀERT230). In the absence of ER ligand, CreꢀERT2 recombinase remains
bound to cytoplasmic Hsp70, and the MEFs constitutively express RFP. However, upon binding
uncaged ligand, CreꢀERT2 dissociates from Hsp70 and is translocated to the nucleus where it
can then excise the Loxpꢀflanked gene for TdTomato from the mTmG29 reporter construct,
resulting in the expression of a membraneꢀbound green fluorescence protein (GFP). We reported
that 1 ONBꢀL1ꢀ4OHT, which is highly water soluble (≥20 mg mL−1) and cell permeable, has the
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ability to induce reporter expression with a spatial resolution sufficient for selectively marking
cells.25
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(A)
TdTomato (-TAM)
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Lox (Stop)
Lox
Cage
Cre-ERT2
(cytosol)
Photocaged
(inactive)
TAM
4OHT
Cage
GFP (+TAM)
+
Nucleus
L1 ("ether")
(B)
HO
O
O
O
N
N
O2N
O
OMe
H2N
O
N
H
4-Hydroxytamoxifen (4OHT)
1 ONB-L1-4OHT
L1
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L1
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O
N
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6
Br
HO
3
Et2N
O
O
O
O
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3 COM-L1-4OHT
2 BHC-L1-4OHT
Figure 1. Photocontrol of CreꢀER mediated GFP expression. (A) Schematic for lightꢀtriggered
activation of photocaged tamoxifen (TAM) or 4ꢀhydroxytamoxifen (4OHT), and the control of
reporter gene expression mediated by the Cre recombinaseꢀestrogen receptor (ER) fusion protein.
(B) Structures of 4OHT and its caged compounds including a previously reported 1 ONBꢀL1ꢀ
4OHT,25 and two new compounds 2 BHCꢀL1ꢀ4OHT, 3 COMꢀL1ꢀ4OHT, each caged with BHC or
COM through an ether linkage (L1), respectively. Abbreviations: ONB = orthoꢀnitrobenzyl; BHC
= 6ꢀbromoꢀ7ꢀhydroxycoumarinꢀ4ꢀmethyl; COM = 6ꢀdiethylaminocoumarinꢀ4ꢀmethyl
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The uncaging efficiency of tamoxifen caged compounds reported in existing CreꢀER models
is based on a singleꢀphoton mechanism of activation which typically occurs by absorption of
light in the UV range. However, the efficiency of twoꢀphoton mechanisms of activation which
use longer wavelength, near infrared (NIR) light remains mostly unevaluated.24 Twoꢀphoton
uncaging of photocaged compounds occurs only on the focal plane with small crossꢀsections of
absorption,6 but it offers significant unique advantages over the singleꢀphoton UV activation
including the deeper penetration capabilities of NIR light in tissue, as well as reduced light
scattering and greater spatial resolution.6, 9 Subsequently, our current efforts focused on the
design and validation of new 4OHT caged compounds for their uncaging efficiency by both
singleꢀ and twoꢀphoton mechanisms in vitro.
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Here, we discuss the lightꢀcontrolled recombinase activity observed in the two series of
4OHTꢀcaged compounds, the first based on the ONB cage including 1 ONBꢀL1ꢀ4OHT, and the
second based on the coumarin cage which was selected for its susceptibility to oneꢀphoton
uncaging by UV–visible light (405–420 nm)7, 31, 32 as well as for its enhanced crossꢀsections for
twoꢀphoton uncaging (δuncaging/GM = 0.21–1.99) compared to ONB (δuncaging/GM = 0.01–0.23).6
The importance of linker selection in the design of 4OHT caged compounds was then examined.
Despite its important role in conjugation chemistry, the ‘linkage’ or ‘linker’ (linkage + spacer) is
generally less prioritized in the design of caged compounds than the cage itself because it has
hitherto been thought of as playing no direct role in triggering the uncaging reaction and having
no contribution to the wavelength selectivity of the cage. Here, we provide evidence that use of a
standard linker can lead to poor uncaging efficiency, as illustrated by the dramatically reduced
uncaging efficiency of a coumarin cage attached directly to the phenolic substrate, 4OHT
through an ether linkage. Specifically, use of this linkage led to the unanticipated occurrence of a
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photoꢀClaisen rearrangement33 as the major reaction path, and thus significantly reduced the
efficiency of free drug release. We demonstrate that the occurrence of such undesired reaction is
effectively blocked by application of alternative linker chemistry.
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RESULTS AND DISCUSSION
Synthesis of Coumarin Caged 4OHT via Ether Linkage. Using a similar linker strategy
applied for ONBꢀbased compound 1,25 we designed two coumarin caged compounds of 4OHT, 2
BHCꢀL1ꢀ4OHT and 3 COMꢀL1ꢀ4OHT (Figure 1), each by tethering 4OHT (Z and E isomers) at
its phenolic moiety to a BHC (6ꢀbromoꢀ7ꢀhydroxycoumarinꢀ4ꢀmethyl)9 or COM (6ꢀ
diethylaminocoumarinꢀ4ꢀmethyl)7 cage molecule through an ether linkage. The synthesis of 2
and 3 was performed by direct Oꢀalkylation of 4OHT with the corresponding coumarinylꢀ4ꢀ
methyl methanesulfonate. Each of these caged compounds was obtained with a purity of ≥95%
(UPLC) without detectable free 4OHT (Figure S6, Supporting Information). Their structural
identity was fully characterized by a combination of standard analytical methods including NMR
(1H, 13C) spectroscopy (Figure S1–S5), mass spectrometry and UV–vis spectrophotometry
(Supporting Information). The exact molecualr masses of 2 and 3 are in good agreement with the
experimetal values measured by high resolution mass spectrometry (HRMS): calcd for 2
C36H34BrNO5 [M + H]+ 640.1693, found 640.1711; calcd for 3 C40H44N2O4 [M + H]+ 617.3374,
found 617.3373.
Unusual Release Kinetics of 2 and 3. We investigated the efficiency of lightꢀcontrolled
release of 4OHT from 2 by exposure to long wavelength UVA light (max intensity at 365 nm;
Figure 2). After photolysis, the exposed solutions were analyzed by thin layer chromatography
(TLC) (Figure S7), which demonstrated the rapid disappearance of 2 with the concomitant
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growth of a broad spot that migrated as far as the 4OHT reference molecule. However, while
subsequent quantitative analysis performed by ultrahigh performance liquid chromatography
(UPLC) also indicated the release of 4OHT, it revealed that it was only as a minor fraction of the
total products (Ф4OHT = 0.09). The majority of the product consisted of a pair of closely running,
unknown products (tr = 10 min) with the ~1:2 AUC (area under curve) ratio of the total of these
two peaks relative to the 4OHT peak (Figure 2C). The unknown products were isolated by flash
column chromatography, and their structural identity was assigned as 4 (Z/E isomers) based on
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the combination of data from H NMR spectroscopy and LCꢀMS mass spectrometry. Thus,
analysis of the LC–MS data indicated no change in the molecular mass ([M + H]+ = 640.1680)
1
compared to the parent caged molecule 2 (Figure S8). The H NMR spectral analysis shows
alterations broadly in 4OHT and BHC signals (Figure S9), in particular, in those from Cꢀ3 and
Cꢀ4 protons which are assigned in reference to C3ꢀ or C4ꢀsubstituted coumarin compounds
(Table S1).34, 35 On the basis of these data, we believe that 4 was formed via the photoꢀClaisen
rearrangement33 of 2 as proposed in Figure 2 with a quantum efficiency (ФPhotoꢀClaisen = 0.22)
greater than that of 4OHT release (Ф4OHT = 0.09). The occurrence of these unexpected products
from 2 is, in retrospect, explained by its [3,3] bond framework which is composed of an allyl
(BHC)ꢀtoꢀaryl (4OHT) ether, a unique structural feature required for the occurrence of photoꢀ
Claisen rearrangement.33, 36
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Figure 2. Mechanism of photoꢀClaisen rearrangement. (A) Two competing reaction paths of 2
BHCꢀL1ꢀ4OHT (Z and E isomers) that occur in response to light (365 nm); path A leading to free
4OHT, and path B towards the formation of a major byproduct 4 (Z/E isomers) via photoꢀClaisen
rearrangement. (B) Overlaid UVꢀvis spectra. Inset (B): a plot of absorbance at 328 nm against
UV exposure time. (C) UPLC traces acquired for references and photolyzed 2 (78 ꢀM in 20%
(v/v) aqueous methanol).
However, it is noteworthy that 1 ONBꢀL1ꢀ4OHT, which similarly meets a structural
requirement by having an allyl (ONB)ꢀtoꢀaryl ether framework, did not show such byproduct
formation but instead, released 4OHT predominantly as a single product (Ф4OHT = 0.13) in our
earlier study.25 We further investigated whether such differences in the product distribution
between 1 and 2 are specifically inherent to the coumarin cage by performing the release study
with 3 COMꢀL1ꢀ4OHT, a close analogue of 2. Its product distribution was consistent with that of
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2, demonstrating 4OHT release as a minor fraction of the products (Ф4OHT = 0.03) along with a
larger fraction of isomeric products (ФPhotoꢀClaisen = 0.05) which are identical to 3 in their
molecular masses as characterized by LCꢀMS mass spectrometry ([M + H]+ = 617.3369; Figure
S10). These results suggest that photoꢀClaisen products might occur selectively for coumarin
caged molecules, while the differences observed in quantum efficiency between BHC and COM
might be determined by the nature of the aromatic ring substituents on each coumarin cage.
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Use of coumarin caged molecules has gained strong popularity due to their high quantum
efficiency of uncaging and broad applicability to various functional substrates.6 However, the
photochemical properties of the coumarin cage which are responsible for leading to a deadꢀend
side product instead of the desired uncaging reaction have rarely been noted except for the recent
identification of the photoisomerization of BHC caged cysteineꢀcontaining peptides (C4ꢀCH2S to
C3ꢀS) by Distefano, et al.37 and Hagen, et al.,31 and the photorearrangement of 4ꢀ
coumarinylmethyl phenyl ethers by Hagen and their coworkers.34 Thus, our study offers strong
evidence for the broader occurrence of photorearrangement specifically in the class of coumarin
caged compounds designed with a direct ether linkage to a target molecule. It also shows that
this rearrangement reaction significantly reduces the uncaging efficiency, and suggests the need
for improving the design features in coumarin caged compounds.
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Figure 3. Design of extended spacers. (A) Release mechanism of extended linkers for coumarinꢀ
caged phenolic compounds in which photoꢀClaisen [1,3] rearrangement is forbidden due to lack
of close proximity and selfꢀimmolation occurs instead. (B) Two linker classes used in coumarin
or ONBꢀcaged 4OHT compounds 5–8. 4OHT is tethered to each cage molecule through a
carbonate (L2) or carbamate (L3) linker.
Coumarin Caged 4OHT via Extended Linker. We hypothesize that the selective occurrence
of photoꢀClaisen rearrangement in the etherꢀlinked coumarin caged compounds is attributable to
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the radical mechanism of CꢀO bond cleavage in combination with physical proximity between
the two radical species formed that allows recombination before they escape from their solvent
cage (Figure 2).33 To circumvent such proximityꢀpromoted photoꢀClaisen rearrangement, we
focused our design approach on extension of the linkage through either a selfꢀimmolative38, 39
carbonate (L2) or carbamate linker (L3) that provides an effective physical barrier to separate two
radical species to be formed far enough for their escape and release. Use of these extended
linkers was previously reported for photocaged compounds including BHCꢀcaged ceramides
(carbonate linkage),40 ONBꢀcaged rapamycin (carbonate),15 and ONBꢀcaged 4OHT
(carbamate).25 Application of these two linkers led to the synthesis of two classes of caged
compounds 5–8 as shown in Figure 3: (i) the carbonateꢀlinked 5 BHCꢀL2ꢀ4OHT; (ii) the
carbamateꢀlinked 6 ONBꢀL3ꢀ4OHT,25 7 BHCꢀL3ꢀ4OHT and 8 COMꢀL3ꢀ4OHT. Compound
homogeneity was ≥95% for each of these caged compounds as determined by UPLC analysis
(Figure S6), and their structural identity was fully supported by data collected by standard
analytical methods as described above (Supporting Information).
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Release Kinetics of Extended Linkers. We next evaluated the release kinetics of 5 BHCꢀL2ꢀ
4OHT (125 ꢀM in 20% (v/v) aqueous methanol) upon exposure to UVA light (Figure S11).
UPLC analysis of the photolyzed solution indicated rapid release of 4OHT (73% after 2 min).
However, its 4OHT release was also observed in the dark (9% at 2 h), pointing to the hydrolytic
instability of the carbonate linkage in 5 in the aqueous solution.
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Figure 4. Release kinetics of coumarinꢀcaged 4OHT. (A) A mechanism for the photoactivation
of 7 BHCꢀL3ꢀ4OHT and 8 COMꢀL3ꢀ4OHT that involves cleavage to 9 spacerꢀ4OHT, and
subsequent selfꢀimmolative cyclization of 9, resulting in release of 4OHT. (B, C) UV–vis spectra
and UPLC traces obtained after the photolysis of 7 (110 ꢀM, 20% (v/v) aqueous methanol) as a
function of exposure time. (D) A plot of the photochemical release kinetics of products (4OHT +
9; %AUC from UPLC traces), each from 7 or 8, respectively.
We then investigated the release kinetics of the carbamateꢀlinked caged compounds 7 and 8.
Exposure to UVA light triggered the release of free 4OHT through formation of its carbamate
derivative 9 (Figure 4, Figure S12). UPLC analysis indicates that drug release occurred in an
exposure timeꢀdependent manner and resulted in ~70% release of 4OHT and its direct precursor
9 after exposure for 5 min (7) and 10 min (8). 4OHT release was also confirmed by LCꢀMS mass
spectrometric analysis which showed the appearance of a single peak corresponding to free
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4OHT ([M + H]+ = 388.2269; Figure S13). Regression analysis of the decay curve of 7 or 8
(%AUC, Figure 4C) over the exposure time suggests that 7 is consumed with a rate constant
(firstꢀorder decay, k) of 0.343 min−1 which is faster than that of 8 (k = 0.192 min−1) at a
comparable concentration. The greater decay rate of 7 is positively correlated with its higher rate
of 4OHT release (Figure 4D), suggesting that its initial cleavage to 9 which is triggered by light
serves as the rate determining step for the release of drug. As a consequence, the quantum
efficiency of uncaging (Ф[4OHT + 9]) calculated for 7 (0.21) is higher than those of 6 (0.05) and 8
(0.07) as summarized in Table 1. This greater release efficiency suggests an advantage of using 7
over 8 as the more promising probe for twoꢀphoton activation and reporter gene expression in
cells as discussed below.
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Table 1. Summary of the photochemical properties of caged 4ꢀhydroxytamoxifen (4OHT)
compounds.
CageꢀLinker
4OHT
(Ln)ꢀ Linkage
Фa
Photoꢀ
λ
max, nm (ε,
M−1 cm−1)
4OHT
4OHT + 9
Claisen 4
1 ONBꢀL1ꢀ4OHTref 25 Ether
340 (390)
0.13
0.09
nd
ꢀ
2 BHCꢀL1ꢀ4OHT
3 COMꢀL1ꢀ4OHT
5 BHCꢀL2ꢀ4OHT
Ether
328 (8,787)
0.22
0.05
nd
ꢀ
Ether
394 (11,357) 0.03
ꢀ
Carbonate
340 (6,154)
0.11b
ꢀ
6 ONBꢀL3ꢀ4OHTref 25 Carbamate 340 (317)
ꢀ
ꢀ
ꢀ
nd
0.05
0.21
0.07
7 BHCꢀL3ꢀ4OHT
Carbamate 330 (9,990)
Carbamate 394 (13,500)
nd
8 COMꢀL3ꢀ4OHT
nd
a
Singleꢀphoton quantum efficiency Ф = [dc/dt]initial/[qn,p(1−10−A)] where qn.p = photon flux
(qp/NA = 11.65 × 10−8 mol s−1) measured by ferrioxalate actinometry (Ф = 1.26);41, 42 A =
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absorbance at 365 nm; dc/dt = initial rate of 4OHT release or Claisen product (mol s−1).42 b
Contribution from nonꢀphotochemical (preexisting) hydrolysis excluded. nd = not detectable
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In summary, the two extended linker classes investigated here are distinguished not only by the
aqueous stability of their linkages but also by their modes of achieving drug release. First, the
carbonate linker used in 5 shows excellent photochemical ability to release free 4OHT with a
decay rate greater than either of the carbamateꢀlinked compounds, 7 or 8. However, it lacks
sufficient chemical stability in aqueous solution, which is needed for precise active control. Such
instability is anticipated to be worse in cell media and in in vivo environments that contain
substantially higher concentrations (mM) of various nucleophilic species such as amino acids,
amines and protein molecules. This insufficient stability of the carbonate linkage as observed in
5 was not noted previously in other caged compounds linked through a carbonate moiety to the
ONB15 or BHC40 cage, indicating perhaps the involvement of other additional factors such as the
steric effect of the substrate molecules themselves. In contrast, the carbamateꢀlinked compounds
7 or 8 show good stability in aqueous solution with no decomposition detected for either
compound, at least during the entire incubation period of up to 24 h as determined by UPLC
analysis.
The mechanism of 4OHT release by 5 involves photocleavage of the CH2–OC(=O) bond
followed by loss of CO2 to yield free 4OHT (Figure 3). Similarly, both 7 and 8 rely on the same
type of photocleavage reaction, but instead yield release of 9, a 4OHT precursor. However, this
39
derivative subsequently undergoes intramolecular selfꢀimmolation38,
through its spacer
methyl(2ꢀ(methylamino)ethyl)carbamate25 to release free 4OHT. Our observation that 7 shows
both a greater decay rate and a higher quantum efficiency of 4OHT release than 8 (Table 1,
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Figure 4) clearly indicates that BHC in 7 is more effective in triggering the first photocleavage
reaction than COM in 8.
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Figure 5. Confocal fluorescence microscopy analysis of the photocontrol of CreꢀERT2 mediated
GFP expression in UbcCreERT2 mTmG MEF cells. As controls, MEFs were treated with (A)
media alone, or (B) 4OHT without UVA exposure. MEFs were treated with 2 BHCꢀL1ꢀ4OHT
(C) without or (D) with UVA exposure. MEFs were treated with 7 BHCꢀL3ꢀ4OHT (E) without or
(F) with UVA exposure. [4OHT or caged compound] = 250 nM. UVA exposure time = 3 min.
tdTomato fluorescence (red), and GFP fluorescence (green) are shown. Nuclei were labeled with
DAPI (blue).
Cre-ERT2 mediated GFP expression in vitro. After validation of 4OHT release kinetics in
solution, we selected two coumarin caged compounds 2 BHCꢀL1ꢀ4OHT and 7 BHCꢀL3ꢀ4OHT to
compare their ability to control GFP expression in UbcCreERT2 mTmG MEF reporter cells
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(Figure 5). This cell line constitutively expresses CreꢀERT2 from the ubiquitin promoter and
contains an mTmG reporter cassette which contains a loxp flanked gene for membrane bound red
fluorescent protein (RFP), tdTomato.25, 26 TdTomato is constitutively expressed. However, upon
CreꢀERT2 binding of free 4OHT, the loxPꢀflanked RFP gene is deleted by the translocated Cre
recombinase, and a membrane bound GFP protein is expressed instead.
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We first examined the photocontrol of the 4OHT constructs in the modulation of such switches
in the expression of the reporter gene using confocal fluorescence microscopy. As similar to a
published protol,25 MEFs were treated with 4OHT, 2 or 7, each at a concentration of 250 nM in
media and exposed to UVA light for 3 min as this exposure time falls within the middle of the
optimal exposure range for uncaging of all of the compounds tested (Figure 4). The cells were
subsequently incubated at 37°C for 24 h prior to being processed for confocal imaging. Cells
treated with media alone and cells treated with each caged compound in the absence of UVA
exposure demonstrated a lack in detectable GFP fluorescence (Figure 5). Furthermore, untreated
cells exposed to UVA for 3 min also showed no GFP fluorescence (Figure S14). In contrast, as a
positive control, MEFs were treated with free 4OHT, which led to a detectable increase in GFP
fluorescence even in the absence of UVA.
Cells treated with 2 or 7 with UVA exposure (+UVA; right), resulted in a substantial increase
in GFP fluorescence intensity compared to the treated cells without UV exposure (left),
validating UVA mediated photouncaging. However, GFP fluorescence induced by 2 appeared to
be weaker than that induced by 7. While these differences between the activities of the two
compounds as observed by confocal microscopy are only qualitative, these results are consistent
with the release kinetics observed in solution where the release of free 4OHT upon UVA
exposure was more efficient for 7 than for 2, the etherꢀlinked compound that suffered from the
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photoꢀClaisen rearrangement as the major pathway for photocleavage. In contrast, a similar
experiment with 5 BHCꢀL2ꢀ4OHT (+/−UVA) indicates its potent activity in inducing GFP
expression, but with lack of UVꢀmediated control (Figure S14), which is consistent with its
hydrolytic instability in aqueous solutions (Figure S11). In summary, these confocal results are
supportive of the ability of 4OHT caged compounds to control the induction of GFP expression
through CreꢀERT2 recombinase activity in response to light. Our ongoing and future efforts are
focused on developing and validating an efficient method of applying the probe 7 for
spatiotemporal reporter activation in vivo as illustrated in Figure S15.
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Figure 6. Twoꢀphoton activation of caged 4OHT compounds 1, 2, 3, 6 and 7. (A, C, D) Hanging
drops of 1, 3 and 6 were exposed to IR light for 10 min on the order of 5 × 1010 pulses (80 fs, 80
MHz) at different wavelengths at various powers. The uncaged compound solution was then
diluted and added to UbcCreꢀERT2 mTmG MEFs. At 24 h, the percent of GFP+ cells was
determined by flow cytometry. Values of %GFP+ cells reported here refer to those obtained
values after background correction by subtraction of the signal from cells exposed to a vehicle
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(DMSO) which was set to 0. (B) The above process was repeated with 1 for various irradiation
times at 750 nm at 240 mW. (E, F) Compounds 2 and 7 were irradiated at different wavelengths
for 10 min at 120 mW and these were similarly used to convert reporter MEFs. The percent
GFP+ cells was measured by flow cytometry. [1] = 100 nM, [2] = 2.5 ꢀM, [3] = 50 nM, [6] =
200 nM, [7] = 250 nM. * P <0.05
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Two-photon uncaging in vitro. After validation of release kinetics by singleꢀphoton exposure,
we determined the efficiency by which these compounds were uncaged by twoꢀphoton excitation
(Figure 6). ONB groups have been reported to have narrow twoꢀphoton cross sections of
absorption in the 750 nm range while coumarin (COM, BHC) cages have broader twoꢀphoton
cross sections in the 800 nm range.6 This means that coumarin cages uncage more efficiently at
wavelengths more useful for use in tissue. As such we tested the uncaging efficiency of 1 ONBꢀ
L1ꢀ4OHT and 6 ONBꢀL3ꢀ4OHT by twoꢀphoton illumination at 730 nm and 750 nm, and 3 COMꢀ
L1ꢀ4OHT by twoꢀphoton illumination at each of three different wavelengths (780 nm, 800 nm,
820 nm) at various laser powers. To get significant uncaging (≥2% GFP+) of 1, 240 mW
illumination was required at 750 nm for 10 min while %GFP+ was positively correlated with the
irradiation time with a maximum value of ~8% (Figure 6A–6B). However even at this high
power 3 and 6 showed no appreciable uncaging (Figure 6C–6D). The efficiency of uncaging of 1
could be improved by increasing the time of the exposure (Figure 6B); however at 240 mW, twoꢀ
photon illumination using a pulsed laser at 750 nm would damage tissues which would be
exacerbated by these long exposures. 1 was more efficiently activated than 6 by two photon
illumination consistent with the increased decay rate seen with single photon illumination.
Since 3 COMꢀL1ꢀ4OHT undergoes such a significant side reaction, it is likely this lack of
uncaging is due to this inefficient uncaging. As such we compared the efficiency of uncaging of
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BHC caged compounds 2 BHCꢀL1ꢀ4OHT and 7 BHCꢀL3ꢀ4OHT at wavelengths between 800 nm
and 840 nm (Figure 6E–6F). These uncaging experiments were performed at the concentration
which is either identical to that used in CreꢀERT2 mediated GFP expression (250 nM for 7;
Figure 5) or higher (2.5 ꢀM for 2) due to its relatively low efficiency of 4OHT release. Hanging
drops of 2 and 7 were illuminated for 10 min at 120 mW laser power. Both 2 and 7 showed
uncaging activity at 800 nm, and similar to the observations made for singleꢀphoton uncaging, 7
was also uncaged most efficiently by twoꢀphoton excitation. Indeed at 120 mW power for 10
min, 7 was 6 times more active in inducing GFP expression than 1 (2.7% GFP+ compared to
0.4% GFP+). The carbonate linked compound 5 was not tested for two photon uncaging as it is
unstable in aqueous solutions and so was as potent at inducing GFP expression with or without
UVA illumination (Figure S14).
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CONCLUSION
In summary, we designed a series of new 4OHT probes caged with coumarin through the
extended spacer for light controlled GFP gene expression in UbcCreERT2 mTmG MEFs, and
validated their effective uncaging activity in vitro by both singleꢀ and twoꢀphoton mechanisms.
We believe that this study represents a significant advancement and gives new insights into the
development of photoprobes that enable precise control of cell labeling based on a CreꢀER
reporter system. Uncaging by singleꢀphoton irradiation occurred rapidly, in as short of an
exposure time as 2 min for both BHC and COM caged compounds, with quantum efficiency (Ф)
as high as 0.21 (Ф, Table 1). BHC caged compounds exhibited more effective uncaging than
their COM analogues. A representative compound, 7, was activated on MEFs by UVA,
demonstrating its ability to temporally induce GFP reporter expression.
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Uncaging by twoꢀphoton irradiation induced an increase in the %GFP+ cells as high as 8% for
certain caged compounds (Figure 6), though this was less efficient than that observed by singleꢀ
photon irradiation (UVA)25. This result was anticipated due to the generally lower efficiency of
uncaging by twoꢀphoton.5, 6 The twoꢀphoton efficiency was dependent on the structural type of
the cage, with BHC (7) showing greater GFP expression than ONB (1). Such higher efficiency
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by BHC is attributable primarily to its higher twoꢀphoton crossꢀsection of absorption (δabsorption
)
rather than its quantum efficiency of uncaging (Ф = 0.21) which is not significantly higher than
that of ONB (Ф = 0.13, Table 1). In addition, twoꢀphoton factors including irradiation
wavelength, intensity and exposure time made a significant contribution to the uncaging
efficiency. However, due to inability for direct correlation between the twoꢀphoton and singleꢀ
photon factors, systematic screening of these parameters for each compound was needed for the
identification of the optimal uncaging conditions as done in this study.
We believe that this study offers rare insights into the mechanism of the photouncaging
process, and has broad implications in the field of photocaging technology. Despite the extensive
use of coumarin caged compounds,3, 6, 37 only few previous studies have indicated the occurrence
of photorearrangement in caged thiols and phenols.31, 34, 37 Our present study reports on the
prevalence of photoꢀClaisen rearrangement33, 36 in coumarin ether caged compounds, but no
evidence is observed for the implication of such rearrangement in ONB uncaging which occurs
exclusively via an intramolecular cyclization of its nitro group.6 We attribute the basis of this
photorearrangement to the radicalꢀbased mechanism of coumarin uncaging in combination with
the presence of an ether linkage which, due to its shorter length in nature brings the transient
radical species generated by light exposure into close proximity, thus promoting their
recombination.33 We suggest the incorporation of an extended linker as an effective strategy to
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circumvent its negative effect on uncaging efficiency. Use of this strategy enabled us to block
this rearrangement possibly by sufficiently separating the two radical species within their solvent
cages and thus preventing their recombination.
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The photoꢀClaisen byproduct formed was not detectable by methods commonly used in this
field such as UV–vis spectrometry (Figure 2B) or by TLC analysis which had insufficient
resolution of separation (Figure S7). Instead, its detection and quantitation required the
development of a specific UPLC method that provides sufficient product resolution. Thus, this
study highlights the importance of linker development in the design of 4OHT caged compounds,
as well as in the design of other photocaged compounds, and underscores the importance of
analytical method choice for evaluating the formed products. It is notable that lack of such
precise analysis can result in inaccurate interpretation of biological activities in vitro and in vivo
as some of byproductꢀlike Cꢀ4 substituted coumarin compounds are associated with promiscuous
activities such as acting as ligands of estrogen receptors,43 antioxidants,44 and antiviral agents.45
MATERIALS AND METHODS
Methods for the synthesis of caged 4OHT compounds, and analytical methods (1H and 13C
NMR spectroscopy, UV–vis spectrometry, mass spectrometry, UPLC) are described in detail in
the Supporting Information and the references cited therein. Full details for release kinetics25, 26
and cell studies in vitro (singleꢀphoton25 and twoꢀphoton13 uncaging for induction of GFP
expression) are also provided in the Supporting Information.
Supporting Information Available: This material is available free of charge via the Internet at
http://pubs.acs.org.
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Author Contributions. These authors contributed equally: Pamela T. Wong and Edward W.
5
6
Roberts.
7
8
9
Acknowledgment. This work was supported in part by the National Cancer Institute, National
Institutes of Health under award 1R21CA191428. SKC acknowledges support from Michigan
Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan Medical
School.
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