Two-photon uncaging of bioactive thiols in live cells at wavelengths above 800 nm

Article abstract of DOI:10.1039/d0ob01986k

Photoactivatable protecting groups (PPGs) are useful for a broad range of applications ranging from biology to materials science. In chemical biology, induction of biological processesviaphotoactivation is a powerful strategy for achieving spatiotemporal control. The importance of cysteine, glutathione, and other bioactive thiols in regulating protein structure/activity and cell redox homeostasis makes modulation of thiol activity particularly useful. One major objective for enhancing the utility of photoactivatable protecting groups (PPGs) in living systems is creating PPGs with longer wavelength absorption maxima and efficient two-photon (TP) absorption. Toward these objectives, we developed a carboxyl- and dimethylamine-functionalized nitrodibenzofuran PPG scaffold (cDMA-NDBF) for thiol photoactivation, which has a bathochromic shift in the one-photon absorption maximum fromλ_max= 315 nm with the unfunctionalized NDBF scaffold toλ_max= 445 nm. While cDMA-NDBF-protected thiols are stable in the presence of UV irradiation, they undergo efficient broad-spectrum TP photolysis at wavelengths as long as 900 nm. To demonstrate the wavelength orthogonality of cDMA-NDBF and NDBF photolysis in a biological setting, caged farnesyltransferase enzyme inhibitors (FTI) were prepared and selectively photoactivated in live cells using 850-900 nm TP light for cDMA-NDBF-FTI and 300 nm UV light for NDBF-FTI. These experiments represent the first demonstration of thiol photoactivation at wavelengths above 800 nm. Consequently, cDMA-NDBF-caged thiols should have broad applicability in a wide range of experiments in chemical biology and materials science.

Full text of DOI:10.1039/d0ob01986k

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Two-photon uncaging of bioactive thiols in live
cells at wavelengths above 800 nm†
Cite this: DOI: 10.1039/d0ob01986k
Matthew D. Hammers,a Michael H. Hodny,a Taysir K. Bader,a M. Mohsen Mahmoodi,a
Sifei Fang,a Alexander D. Fenton, a Kadiro Nurie,a Hallie O. Trial,b Feng Xu,a
a
Andrew T. Healy,a Zachary T. Ball, b David A. Blanka and Mark D. Distefano
*
Photoactivatable protecting groups (PPGs) are useful for a broad range of applications ranging from
biology to materials science. In chemical biology, induction of biological processes via photoactivation is
a powerful strategy for achieving spatiotemporal control. The importance of cysteine, glutathione, and
other bioactive thiols in regulating protein structure/activity and cell redox homeostasis makes modulation
of thiol activity particularly useful. One major objective for enhancing the utility of photoactivatable pro-
tecting groups (PPGs) in living systems is creating PPGs with longer wavelength absorption maxima and
efficient two-photon (TP) absorption. Toward these objectives, we developed a carboxyl- and dimethyl-
amine-functionalized nitrodibenzofuran PPG scaffold (cDMA-NDBF) for thiol photoactivation, which has
a bathochromic shift in the one-photon absorption maximum from λmax = 315 nm with the unfunctiona-
lized NDBF scaffold to λmax = 445 nm. While cDMA-NDBF-protected thiols are stable in the presence of
UV irradiation, they undergo efficient broad-spectrum TP photolysis at wavelengths as long as 900 nm.
To demonstrate the wavelength orthogonality of cDMA-NDBF and NDBF photolysis in a biological setting,
caged farnesyltransferase enzyme inhibitors (FTI) were prepared and selectively photoactivated in live
cells using 850–900 nm TP light for cDMA-NDBF-FTI and 300 nm UV light for NDBF-FTI. These experi-
ments represent the first demonstration of thiol photoactivation at wavelengths above 800 nm.
Consequently, cDMA-NDBF-caged thiols should have broad applicability in a wide range of experiments
in chemical biology and materials science.
Received 29th September 2020,
Accepted 23rd November 2020
DOI: 10.1039/d0ob01986k
rsc.li/obc
Considerable effort has been undertaken to enhance PPG
utility in live systems. The ortho-nitrobenzyl family of caging
Introduction
Photoactivatable protecting groups (PPGs) are useful for a
broad range of applications;1–5 examples include spatiotem-
poral control of biological processes6 and the creation of
hydrogels with tunable properties.7–9 In general, PPGs are
employed to protect or “cage” molecules that are hence stably
masked, until irradiation with an exogenous light source
exposes a functional group (typically a carboxylate, amine, or
thiol) critical for function. In biological studies, photolysis can
be used to generate ligands, substrates, or drugs in active
form. Since the first example of a caged biomolecule 40 years
ago (caged ATP),10 the breadth of photoactivatable substrates
has expanded beyond small molecules to include macromol-
ecular DNA/RNA,11,12 peptides, and even whole proteins.13–18
groups (ONB, Scheme 1) has classically been the most widely
utilized motif,4,19,20 but it requires high energy excitation for
photolysis (<350 nm) which is potentially damaging in cell
culture and living tissue. Furthermore, ONB has poor absorp-
tivity at more physiologically useful near-IR (NIR) wavelengths
aDepartment of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
E-mail: diste001@umn.edu
bDepartment of Chemistry, Rice University, Houston, TX, 77005, USA
†Electronic supplementary information (ESI) available: Supplemental figures,
NMR spectra, and crystallographic data (CIF) are provided. CCDC 1943530. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
d0ob01986k
Scheme 1 Photoactivatable protecting group families. LG = leaving
group.
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amenable for 650–950 nm two-photon (TP) photolysis.21 NIR tissue penetration, the availability of longer-wavelength-
light penetrates more deeply into tissue than UV light, with absorbing PPGs may allow for orthogonal photoactivation of
greater spatial precision,22 and causes less phototoxicity.23 different thiols within the same sample, for example in a
Thus, creating PPGs with longer wavelength and TP absorptiv- peptide containing several distinct cysteine residues. Toward
ity is a central driver of current research. More recent scaffolds this goal, we describe a new dimethylamine-functionalized
such as ortho-nitrobiphenyl (ONBP)24 and brominated hydroxy- nitrodibenzofuran
coumarin (Bhc)25 have contributed to this effort by displaying (cDMA-NDBF) for thiol photoactivation. We hypothesized that
improved TP uncaging efficiencies. this electron-withdrawing substituent may facilitate more
containing
an
α-carboxyl
ester
The development of long-wavelength caging groups for rapid photolysis based on observations with α-carboxy ONB
thiols is particularly desirable because of the importance of structures;4,34,35 in addition, the presence of an ester moiety
cysteine and other bioactive thiols in cellular processes.26 We could be used to increase the water solubility of this protecting
previously demonstrated that Bhc is generally not ideal for group by subsequent hydrolysis to the corresponding acid.
thiol photoactivation because of a non-productive photorear- Such a strategy has been frequently used in the design of nitro-
rangement reaction which occurs upon irradiation.27 benzyl- and coumarin-based caging groups.34,36,37 cDMA-NDBF
Fortunately nitrodibenzofuran (NDBF, Scheme 1), a promising absorption is red-shifted significantly from the parent NDBF
class of caging groups with a planar biphenyl-like structure from 315 nm to 445 nm. We compare the wavelength depen-
fused through a bridging furanyl ether,28 is more suitable for dence of OP and TP photolysis kinetics using caged cysteamine
thiols. NDBF-protected thiols exhibit rapid one-photon (OP) compounds and found that cDMA-NDBF and NDBF caging
and TP uncaging rates with no observable side reactions.27 groups can be selectively photoactivated using either
Dreuw and Heckel et al. suggested computationally and 850–900 nm TP irradiation or UV irradiation, respectively.
demonstrated experimentally that installing a dimethylamine Having established orthogonal irradiation conditions, we
group onto the NDBF core (DMA-NDBF) induces a significant demonstrate wavelength-selective photoactivation of a caged far-
bathochromic shift in the chromophore’s absorption profile nesyltransferase enzyme inhibitor drug in live cells.
because of enhanced push–pull dipolar character within the
molecule.29,30 A follow-up study explored several cyclic amine
sutstituents.31 DMA-NDBF was also used by Ball and co-
Results and discussion
workers for amide nitrogen protection.32 Similarly, Specht
et al. achieved longer wavelength absorption and enhanced TP The synthesis of cDMA-NDBF began with commercially avail-
sensitivity in the ONBP scaffold (CANBP/EANBP) using dialky- able dibenzofuran (Scheme 2), which was acylated using acetyl
lamine functionalization.33
chloride under Friedel–Crafts conditions to give 1. Oxidative
These encouraging discoveries led us to explore dimethyl- 1,2-aryl migration of the resulting ketone was achieved using
amine-substituted NDBF for long wavelength TP thiol photoac- hypervalent iodine, trimethyl orthoformate, and methanol,38
tivation. In addition to reduced phototoxicity and deeper which ultimately provided the α-methyl ester present in the
Scheme 2 Synthesis of cDMA-NDBF-Br and conditions for thiol alkylation/protection.
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final protecting group. Nitration of 2 with sodium nitrite was
not regioselective and generated an inseparable isomeric
mixture of 7- and 3-nitrated products in a 3 : 1 ratio; however,
the desired 7-amino isomer was chromatographically separ-
ated after iron-mediated nitro reduction to give pure 4 in mod-
erate yield over two steps. We confirmed the correct isomeric
identity using X-ray crystallography (Fig. S1†). Bis(Boc) protec-
tion of the amine allowed for subsequent α-CH2 allylic bromi-
nation to give intermediate 7. After Boc-deprotection, the
regenerated amine was subjected to reductive amination with
paraformaldehyde and sodium cyanoborohydride to give di-
methylamine 8. Finally, nitration of the NDBF core’s 3-position
produced key intermediate cDMA-NDBF-Br (9). Thiol alkylation
of cDMA-NDBF-Br was performed at the end of this synthetic
sequence, which allows for modular conjugation with a thiol
of choice. Reaction of cDMA-NDBF-Br with cysteamine (CA)
was performed in the presence of zinc acetate and mildly
acidic conditions to prevent sulfur oxidation39 to give the final
protected thiol, cDMA-NDBF-CA (10). Analogous NDBF-CA
(11a, Scheme 3) was prepared as previously reported.9
Cysteamine was chosen as a model thiol for comparative
photolysis kinetics experiments given its simplicity and resem-
blance to the amino acid cysteine.
Fig. 1 UV-vis absorption spectra of cDMA-NDBF-CA (red) and
NDBF-CA (blue) (100 µM, 20% CH3CN/50 mM PB, pH 7.4).
than previously reported O-caged NDBF compounds (∼18 000
M−1 cm−1), presumably due to substituent effects of the caged
sulfur atom.28,30 In an effort to compare the spectral properties
of cDMA-NDBF-CA with the corresponding compound incor-
porating a methyl group in lieu of a carboxymethyl moiety, we
prepared 11b (Scheme 3), a synthetic intermediate previously
reported by Becker et al., and recorded its spectrum in
aqueous solution (Fig. S2†). The λmax value obtained there,
418 nm, was comparable to the value previously reported,
measured in DMSO (λmax = 424 nm). Comparison of the spec-
trum of 11b with that obtained with cDMA-NDBF-CA (10),
showed that the latter manifested a λmax red-shifted by 23 nm
relative to the former. Since the origin of that spectral shift
could reflect either the presence of the carboxymethyl group or
the thioether substituent in 10, absorption spectra were calcu-
lated for 10 along with analogues 11c and 11d using the
Dalton quantum chemistry suite.40 Those calculated spectra
(Fig. S3†) suggest that the caged thiol is the major cause of the
red-shifted λmax of 10 relative to 11b and are consistent with
previously reported spectral data for simple nitrobenzyl deriva-
tives.34 Finally, simple calculations using ChemDraw indicate
that hydrolysis of the carboxymethyl group present in 10 to the
corresponding acid increases the total polar surface area by
40 Å2 and decreases CLogP by approximately 4 Log units
(Fig. S4†); thus, the presence of the carboxymethyl moiety in
the cDMA-NDBF protecting group offers a potentially simple
means for augmenting the water solubility of NDBF-based
caged compounds.
With cDMA-NDBF-CA in hand, we first characterized its OP
absorption and photolysis properties and compared them with
those of NDBF-CA. These optical properties are summarized in
Table 1. As predicted, dimethylamine functionalization of the
NDBF core resulted in a significant bathochromic shift in the
UV-vis absorption maximum from λmax(NDBF-CA) = 315 nm to
λmax(cDMA-NDBF-CA) = 445 nm (Fig. 1). The molar extinction
coefficients of these S-caged NDBF compounds were lower
To examine the OP photolysis kinetics of caged thiols
cDMA-NDBF-CA (10) and NDBF-CA (11a), samples (100 µM,
20% CH3CN/50 mM PB, pH 7.4) were irradiated using 350 nm
and 419 nm bulbs in a Rayonet photoreactor, an instrument
Scheme 3 Additional NDBF-based analogues examined in this study.
Table 1 One-photon absorption and photolysis properties of cDMA-NDBF-CA and NDBF-CA. Absorption properties were determined in 20%
CH3CN/50 mM PB, pH 7.4
λmax (nm)
ε (λmax) (M−1 cm−1
)
ε (350)
ε (428)
Qu (350)
Qu (428)
δu (800, GM)
NDBF-CA
cDMA-NDBF-CA
315
445
12 400
18 800
10 300
13 400
608
18 100
0.57
0.00068
0.70
0.00036
0.20
0.24
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commonly found in laboratories studying photochemistry. home-built LED system (Fig. S6†) with 350 nm and 428 nm
Photon flux was controlled to within 3% for each wavelength LEDs to determine OP uncaging quantum yields (Qu) of each
to directly compare photolysis rates. NDBF-CA underwent compound. Similar trends in photolysis rates were observed
rapid photolysis during both 350 and 419 nm UV irradiations, using the LED apparatus (Fig. S7†). Minimal cDMA-NDBF-CA
and complete disappearance of starting material was observed photolysis was observed during the time required to comple-
after 15 and 90 seconds, respectively (Fig. 2a, Fig. S5†). These tely photolyze NDBF-CA. Qu data obtained using the LED-
results highlight the excellent UV-sensitivity of the parent based apparatus are summarized in Table 1.
NDBF caging group for thiol photoactivation. Conversely, OP
Although the slow OP photolysis of cDMA-NDBF-CA is sur-
photolysis of cDMA-NDBF-CA was dramatically slower. prising, particularly at the longer wavelength more closely
Irradiation for 40 minutes at 350 nm produced only 65% de- aligned with its maximal light absorption, these results are
protection, and 419 nm photolysis was slower yet, with 15% consistent with previously reported data obtained using
uncaging occurring during the same period. Overall, NDBF-CA related DMA-NDBF-N-caged nucleotides. In that work, the
photolysis proceeded 700- (350 nm) and 345-fold (419 nm) authors reported quantum yields of 1.1% and 0.05% for
faster than cDMA-NDBF-CA.
irradiation at 340 nm and 420 nm.30 Since the cDMA-NDBF
The data obtained in the Rayonet reactor for NDBF-CA scaffold is also resistant to UV-mediated photolysis in the
showing efficient deprotection at 419 nm was somewhat sur- context of caged thiols, it appears that the 445 nm absorption
prising given the low extinction coefficient manifested by that HOMO–LUMO transition may not proceed toward photo-
compound at that longer wavelength; however, that can be induced bond cleavage. This phenomenon warrants further
attributed to the broad spectral bandwidth of the Rayonet investigation.
bulbs ( 50 nm). To circumvent that issue, we next used a
Relatively few examples of two-photon thiol photoactivation
exist in the literature, most of which use the Bhc protecting
group.41–43 However, a non-productive photorearrangement
reaction occurs during Bhc-thiol irradiation which limits
product conversion even after long irradiation times
(>2 hours).42 To circumvent this problem, a 3-methyl group
was added to Bhc (mBhc), and 800 nm TP photolysis of a
mBhc-cysteine containing peptide proceeded with a TP unca-
ging action cross section (δu) of 0.13 GM,43 where δu is the
product of the TP absorption and uncaging quantum yield (δu
= δaΦu). NDBF was then found to be even more efficient for
thiol uncaging, where photolysis experiments performed on a
similar NDBF-cysteine-caged peptide proceeded with δu = 0.20
GM.27 To study the TP uncaging efficiency of cDMA-NDBF-CA
within this context, δu was first determined at 800 nm using a
1 kHz Ti:sapphire laser to irradiate solutions in a microcuvette,
followed by subsequent HPLC analysis, a method used exten-
sively for the analysis of TP uncaging reactions.25,44–46 Bhc-
acetate uncaging (Bhc-OAc, 0.42 GM) was used as a reference.
Photolysis rates of cDMA-NDBF-CA and NDBF-CA at 800 nm
were similar (Fig. 2b), with ∼85% cleavage observed over
40 minutes for each compound. δu were determined to be 0.24
GM and 0.20 GM for cDMA-NDBF-CA and NDBF-CA, respect-
ively (Table 1). These results confirm that both cDMA-NDBF
and NDBF are more effective caging groups for TP thiol photo-
release compared with Bhc and mBhc-based systems. Since
Becker et al. did not report a TP uncaging action cross section
for DMA-NDBF-N-caged amine cleavage, a direct comparison
with that work is not possible. However, Ball and coworkers
did report uncaging data for a related DMA-NDBF-caged amine
(11e) using the same methodology described here to measure
Fig. 2 (a) One-photon photolysis of cDMA-NDBF-CA (red, 100 µM) and
●
▲
)
NDBF-CA (blue, 100 µM) after irradiation at 350 nm ( ) and 419 nm (
using a Rayonet photoreactor. Photon flux held to within 3% for each the TP uncaging cross-section of 10.32 Using that data, we esti-
wavelength. (b) Two-photon photolysis of cDMA-NDBF-CA (red,
mate a TP uncaging action cross-section of 0.21 GM for 11e (see
Fig. S8†). Thus, DMA-NDBF and cDMA-NDBF appear to have
100 µM), NDBF-CA (blue, 100 µM), and Bhc-OAc (black dashed, 100 μM)
after 800 nm irradiation using a 1 kHz Ti:Sapphire laser (125 mW, 80 fs
pulse width). Photolysis reactions were performed in photolysis buffer
(1 mM DTT, 20% CH3CN/50 mM PB, pH 7.4). Percent starting material
similar properties in terms of their TP cleavage characteristics.
Unlike several O-caged compounds,47,48 TP photolysis kine-
was quantified using HPLC analysis of individually photolyzed samples.
tics data for all previously reported caged thiols is limited to
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800 nm. Having established that cDMA-NDBF-CA undergoes wavelength-dependent release of a thiol-containing drug mole-
efficient photolysis with 800 nm irradiation, we sought to cule in live cells. Farnesyltransferase inhibitor (FTI) prevents a
probe the 750–900 nm spectral window in more detail with the posttranslational farnesylation modification of Ras-family pro-
objective of achieving longer-wavelength photolysis. Samples teins, which are potentially oncogenic small GTPase pro-
of cDMA-NDBF-CA and NDBF-CA were irradiated at 750, 800, teins.49 After several subsequent processing steps, farnesylated
850, and 900 nm using an 80 MHz tunable Ti:sapphire mode- Ras translocates to the plasma membrane where it interacts
locked oscillator laser system (Fig. S9†). This laser manifests with other key proteins involved in signal transduction.
different optical properties than the 1 kHz system used in Inhibition by farnesyltransferase causes unfarnesylated Ras to
experiments presented in Fig. 2b. Each pulse generated from accumulate in the cytosol and hence prevents key protein–
this system has ∼104 less energy than those generated with the protein interactions necessary for cell signaling. Using MDCK
1 kHz system; thus, longer irradiation times are required. Both cells stably expressing a GFP-H-Ras fusion protein, the pres-
compounds photolyzed at similar rates using 750 and 800 nm ence of FTI can be determined by observing cytosolic GFP-H-
irradiation (Fig. 3, Fig. S10†). However, NDBF-CA photolysis Ras fluorescence rather than normal membrane-localized fluo-
was dramatically slower at longer wavelengths, with essentially rescence (Fig. S11†).42
no uncaging observed using 850 nm and 900 nm light. In con-
trast, cDMA-NDBF-CA maintains its photolytic efficiency at cal for its inhibitory activity. Alkylation of this thiol to yield a
850 nm completely and at 900 nm to a moderate degree. thioether renders the drug essentially inactive. cDMA-NDBF-FTI
The chemical structure of FTI contains a thiol which is criti-
cDMA-NDBF-CA represents the first example of thiol photo- (12) and NDBF-FTI (13) compounds (Fig. S12†) were synthesized
activation at wavelengths above 800 nm. Two-photon excitation using the general thiol alkylation procedure outlined above.
initially accesses different electronically excited states than
MDCK cells expressing GFP-H-Ras were treated with either
one-photon excitation as a result of different optical selection cDMA-NDBF-FTI (12) or NDBF-FTI (13) and exposed to
rules. However, in the absence of directly measured two- different OP and TP irradiation conditions. FTI photoactiva-
photon absorption spectra, it is common to assume correlated tion was determined by observing the resulting fluorescence
energetic shifts in the optically accessible states and to use the distribution using a confocal microscope. In the absence of
one-photon absorption spectrum as a qualitative estimate, or irradiation, normal membrane-localized GFP-H-Ras was
surrogate, for the two-photon spectrum. Within these assump- observed for both compounds (Fig. S13†), indicating that the
tions our results showing efficient uncaging at 850 and caged FTIs were stable in cellular milieu. Consistent with our
900 nm are consistent with the red-shifted absorption of the in vitro UV photolysis kinetics, cells treated with
cDMA-NDBF chromophore. Correspondingly, NDBF-CA photo- cDMA-NDBF-FTI and irradiated with 300 nm UV light exhibi-
lysis rapidly declines at wavelengths longer than twice its OP ted membrane-localized GFP-H-Ras fluorescence (Fig. 4a),
absorption. Finally, it is interesting that the broad dynamic indicating that FTI was not photoactivated under these con-
range of cDMA-NDBF allows uncaging over a wide range of ditions. In contrast, the fluorescence became cytosolic in
wavelengths extending from 750 to 900 nm, suggesting the NDBF-FTI-treated cells after the same 300 nm exposure
aforementioned assumption may not always be definitive.
(Fig. 4b), indicating that FTI was efficiently photoactivated
The OP and TP photolysis experiments above identify two using UV light.
possible modes for orthogonal photoactivation in living
Cells were then irradiated with NIR TP light using a Nikon
systems, UV irradiation to photoactivate NDBF-protected thiols A1RMP confocal microscope. At all four wavelengths tested
and 850–900 nm TP irradiation to photoactivate cDMA-NDBF- between 750–900 nm, cytosolic localization of GFP-H-Ras was
protected thiols. To test this hypothesis, we examined the observed in cDMA-NDBF-FTI-treated cells (Fig. 4c top row).
This broad-spectrum cDMA-NDBF-FTI photoactivation is con-
sistent with the in vitro data presented above. In comparison,
NDBF-FTI uncaged efficiently at 750 nm and 800 nm, but
longer-wavelength 850 nm and 900 nm irradiation failed to
release the caged drug resulting in a membrane-bound localiz-
ation for GFP-H-Ras (Fig. 4d bottom row).
These in cellulo results highlight the utility of the
cDMA-NDBF caging group for modulating cellular processes
with long-wavelength light. To the best of our knowledge,
these experiments are the first demonstration of photoactiva-
tion of a bioactive thiol in live cells at wavelengths longer than
800 nm. The parent NDBF scaffold is an efficient tool for thiol
photoactivation, but it has significant limitations at longer
NIR wavelengths. Although 750 nm light is less phototoxic
than UV light, we noticed considerably more GFP photobleach-
Fig. 3 Comparison of TP photolysis rates for cDMA-NDBF-CA (red) and
NDBF-CA (blue) using 750–900 nm irradiation. Observed rate constants,
ing at this wavelength than we did while using 850 or 900 nm
kobs, were determined from linear fits to ln(fraction starting material) vs.
time.
irradiation (Fig. S14†), indicating that phototoxicity at 750 nm
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Fig. 4 MDCK cells expressing GFP-H-Ras fusion protein after treatment with cDMA-NDBF-FTI (15 μM, top row) or NDBF-FTI (15 μM, bottom row)
and (a and b) 300 nm irradiation for three minutes, or (c and d) 750 nm (500 mW), 800 nm (500 mW), 850 nm (1000 mW), or 900 nm (1000 mW)
irradiation. TP conditions: 32 seconds irradiation scan, 4 scan repeats. Cells were imaged 17 hours after treatment. Scale bar = 50 μm.
is not completely negligible; similar improvements in biologi- formance liquid chromatography (HPLC) analysis and purifi-
cal results have been previously noted with other PPGs acti- cation were performed using a Beckman model 125/166 instru-
vated at 900 nm.50 Thus, extending the excitation wavelength ment equipped with a UV detector and C18 columns (Agilent
>800 nm accesses a more benign region of the optical spec- Microsorb-MV 100-5, 4.6 × 250 mm and ZORBAX 300SB
trum. Finally, modern microscope technology provides for PrepHT, 21.2 × 250 mm). LC-MS analysis was performed using
easily tunable TP excitation wavelength and laser power to an Agilent 1200 series LCMSD SL single quadrupole system
match desired experimental parameters.
equipped with a C3 column (Agilent ZORBAX 300SB-C3, 5 μM,
4.6 × 250 mm) and a variable wavelength detector. An H2O/
CH3CN solvent system containing 0.1% HCO2H was used, con-
sisting of solvent A (H2O with 0.1% HCO2H) and solvent B
(CH3CN with 0.1% HCO2H). UV-visible spectra were recorded
using a Cary 50 Bio spectrometer. UV photolysis experiments
were performed either using a Rayonet reactor (8 × 419 nm or
16 × 350 nm bulbs) or a home-built LED reactor (Fig. S6†)
equipped with 8 × 350 nm LEDs (FoxUV, 5.5 mm) or 8 ×
428 nm LEDs (VISHAY, 5 mm) evenly spaced in a radial
arrangement with cuvette positioned at the center of the
reactor. 1H and 13C{1H} NMR data of synthetic compounds
were recorded on a Bruker 500 MHz instrument. Chemical
shifts are reported in ppm relative to residual protic solvent
resonances. Live-cell images were acquired using a Nikon
A1Rsi inverted confocal microscope equipped with SIM super
resolution, GaAsP 32-channel spectral detectors, and a Nikon S
PlanFluor 40×/0.60NA air objective. Two-photon irradiation of
live cell culture samples was performed using a Nikon A1RHD
MP upright confocal microscope equipped with a Nikon Fluor
40×/0.80NA water dipping objective.
Conclusions
Motivated to access long-wavelength NIR thiol photoactivation,
we installed a dimethylamine group into the NDBF scaffold,
thereby achieving a significant bathochromic shift in the
chromophore’s absorption spectrum. The cDMA-NDBF caging
group is surprisingly resistant to cleavage upon UV irradiation
compared to the parent compound (NDBF), offering a window
for orthogonal uncaging. The cDMA-NDBF two-photon unca-
ging profile is particularly useful at wavelengths longer than
800 nm where NDBF is stable, and we were able to photoacti-
vate a thiol-containing enzyme inhibitor in live cells at wave-
lengths as long as 900 nm. Given those results, we expect the
cDMA-NDBF caging group will have broad applicability for
other biological studies involving bioactive thiols. Finally,
given the orthogonality manifested by cDMA-NDBF, it is likely
that this new protecting group will be useful for experiments
where selective thiol uncaging is required.
General procedure for calculation of one-photon spectra
Experimental
Materials & methods
Molecular structures were optimized in Gaussian 1651 at the
DFT level using the B3LYP52–54 functional and the 6-31G
Solvents and reagents used for chemical synthesis were pur- (d)55,56 basis set. An IEFPCM57 model was used to model the
chased from Sigma Aldrich and used as received. High-per- impact of the water and DMSO solvents on the states. Using
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the optimized structures, one-photon transition energies and 0.1% HCO2H/CH3CN, 1%-B 5 min and ramping to 100%-B
one-photon oscillator strengths were calculated with time over 10 min. Photolysis rates were determined based on a
dependent DFT (TD-DFT) using the Dalton quantum chemistry single exponential decay curve calculated in Origin software
suite.40 The same functional and basis set used for the optim- (OriginLab Corporation). Quantum efficiencies (Qu) were deter-
ization was used for calculating the transitions. For compari- mined using the following equation: Qu = (Iσt90%)
−1, where I is
son to experimental spectra the calculated transitions were the irradiation intensity in Einstein per cm2 per s as measured
convoluted with a Gaussian broadening function that had a by potassium ferrioxalate actinomoetry (see ESI†),59 σ is the
FWHM of 0.4 eV.
decadic extinction coefficient (1000 × ε, molar extinction coeffi-
cient) in M−1 cm−1, and t90% is the time required in seconds to
reach 90% completion.
Laser apparatus for two-photon (TP) irradiation
Two different laser apparatuses were used in two-photon kine-
tics experiments. A home-built regeneratively amplified Ti:sap-
General procedure for TP photolysis of caged molecules
phire laser operating at 1 kHz with an output power of A substrate solution (100 µM, 30 µL) was prepared in photo-
125 mW produced 80 fs (full-width-at half-max) Gaussian lysis buffer (1 mM DTT, 20% CH3CN/50 mM phosphate buffer
pulses centered around 800 nm. These pulses were used to (PB), pH 7.4) and transferred to a quartz microcuvette (Starna
collect 800 nm photolysis kinetics shown in Fig. 2b. This 16.10-Q-10/Z15, 10 × 1 × 1 mm, illuminated volume). The
system is described in detail elsewhere.58 The beam was sent sample was then irradiated using either a 1 kHz or 80 MHz Ti:
through a 35 cm focusing lens and then through the sample. sapphire laser (see above). After irradiation for the allotted
Samples (30 μL) were irradiated in a quartz microcuvette time, an aliquot (20 µL) was analyzed by HPLC in a similar
(Starna 16.10-Q-10/Z15, 1 mm × 1 mm sample window, 10 mm manner to UV photolysis experiments.
path length) 15 cm after the focal plane of the lens.
The tunable source used for the TP irradiation at multiple
wavelengths in Fig. 3 was a Spectra-Physics Tsunami mode-
Determination of TP uncaging action cross sections (δu, where
δu = δaΦu) at 800 nm
locked Ti:sapphire laser operating at 80 MHz with a power of Caged substrates were irradiated using the general procedure
450 mW. Each pulse had a Gaussian profile with a full width at at 800 nm using
a 1 kHz laser. Photolysis rates of
half maximum of 80 fs. The beam was sent through a 30 cm cDMA-NDBF-CA, NDBF-CA, and Bhc-OAc were determined
focusing lens and then the sample. Sample (30 μL) were irra- from HPLC experiments in a similar manner to UV photolysis
diated in a quartz microcuvette (Starna 16.10-Q-10/Z15, 1 mm experiments. Determination of δu values for cDMA-NDBF-CA
× 1 mm sample window, 10 mm path length) 12 mm behind and NDBF-CA was calculated by rate comparison with Bhc-OAc
the focal plane of the lens. After measuring the beam spot size reference standard (δaΦu = 0.42 GM) using the following
at this cuvette position, it was determined the beam fills the formula: δaΦu(NDBF) = δaΦu(ref) × kobs(NDBF)/kobs(ref).
sample volume.
NIR photolysis of caged thiols at multiple wavelengths
General procedure for UV photolysis of caged thiols using
Rayonet reactor
Caged substrates were irradiated using the general procedure
at 740 nm, 800 nm, 850 nm, and 900 nm using an 80 MHz
A substrate solution (100 µM, 150 µL) was prepared in photo- laser, and reaction progression were determined from HPLC.
lysis buffer (1 mM DTT, 20% CH3CN/50 mM phosphate buffer Photolysis rates, kobs, were calculated as linear fits to ln(frac-
(PB), pH 7.4) and transferred to a quartz cuvette. The sample tion starting material) vs. time.
was then irradiated on a rotating platform using a Rayonet
Cell culture and two-photon photoactivation of caged
farnesyltransferase inhibitor (FTI)
lyzed by HPLC, and integration of 260 nm absorption for the MDCK cells stably expressing GFP-H-Ras42 were cultured (10%
starting material was used to measure the reaction progression FBS/DMEM without Phenol Red indicator, 37 °C, 5% CO2
(solvent A: 0.1%TFA/H2O, solvent B: 0.1%TFA/CH3CN, 0%-B atmosphere) into Ibidi® 35 mm coverslip-bottomed dishes
photoreactor with either 350 nm or 419 nm UV light. After
irradiation for the allotted time, an aliquot (60 µL) was ana-
5 min and ramping to 100%-B over 25 min).
etched with a 500 μm-grid layout for monitoring condition-
specific cell groups during the experiment (Cat. no. 81168).
Cells were treated with either cDMA-NDBF-FTI or NDBF-FTI
(1 mL, 15 μM) and incubated for 2 h. After washing the cells
General procedure for UV photolysis of caged thiols using LED
reactor and determination of quantum efficiencies
A substrate solution (10 μM, 200 μL) was prepared in photo- with PBS to remove excess caged FTIs and replacing with fresh
lysis buffer (15 mM DTT, 20% CH3CN/50 mM phosphate media, groups of cells were exposed to various TP irradiation
buffer (PB), pH 7.4) and transferred to a round quartz tube (10 conditions. A ∼300 × 300 μm selected region of interest was
× 50 mm, 2 mm inner wall thickness). After irradiation for the irradiated using either 750 nm (500 mW), 800 nm (500 mW),
allotted time, an aliquot (100 µL) was analyzed by LC-MS for 850 nm (1000 mW), or 900 nm (1000 mW) light for 32 s/frame
determination of remaining starting material via integration of repeated four times. The cells were then incubated for a period
330 nm (NDBF-CA) or 435 nm (cDMA-NDBF-CA) of peaks dis- of 17 hours to allow for GFP-H-Ras protein turnover. After
playing the correct m/z (solvent A: 0.1%HCO2H/H2O, solvent B: incubation, the same irradiated cell groups were imaged using
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confocal fluorescence microscopy. The fluorescence signal 4.40 g, 78.9 mmol) was added portionwise whilst vigorously
from GFP-H-Ras (λex = 488 nm, λem = 505–515 nm) and localiz- stirring, and the reaction was stirred at reflux overnight. The
ation were acquired. Whereas plasma membrane H-Ras localiz- reaction mixture was filtered through a pad of Celite, and the
ation indicated normal, prenylated H-Ras, cytosolic H-Ras remaining iron residue was thoroughly rinsed with EtOAc. The
accumulation indicated FTI photoactivation and inhibition of combined crude product solution was concentrated under
farnesyltransferase.
vacuum, dissolved in EtOAc, washed with 10% NaHCO3, brine,
and dried using Na2SO4. Pure compound 4 was obtained after
column chromatography purification (3 : 2 Hex/EtOAc) as a
Synthesis and characterization
2-Acetyldibenzofuran (1). Compound 1 was synthesized as red-brown solid (3.22 g, 38% over two steps). 1H NMR
previously reported.27
(500 MHz, DMSO) δ 7.71 (s, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.44
Methyl dibenzofuran acetate (2). Compound 1 (9.00 g, (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 6.74 (s, 1H), 6.63 (d,
42.9 mmol) was dissolved in CH2Cl2 (5 mL) and then added to J = 8.3 Hz, 1H), 5.59 (s, 2H), 3.77 (s, 2H), 3.62 (s, 3H). 13C NMR
CH3OH (140 mL). While stirring, trimethyl orthoformate (126 MHz, DMSO) δ 171.95, 158.00, 153.86, 149.74, 128.72,
(35.2 mL, 344 mmol) was added to the solution, followed by 125.70, 124.91, 121.21, 119.77, 119.69, 111.93, 110.86, 110.56,
slow addition of conc. H2SO4 (18.3 mL, 344 mmol) in an ice 95.21, 51.69. HRMS (m/z): [M + Na]+ calcd for [C15H13NO3Na]+
bath. PhI(OAc)2 (14.5 g, 46.2 mmol) was added, and the reac- 278.0788, found 278.0798.
tion was stirred at rt for 3 h. Water (100 mL) was added to the
Methyl
2-(7-((Boc)amino)dibenzofuran-2-yl)acetate
(5).
reaction flask, and the CH3OH was removed under vacuum. Compound 4 (2.20 g, 8.62 mmol) was dissolved in DMF
After pH neutralization with NaHCO3, the crude product was (10 mL). DIPEA (3.00 mL, 17.2 mmol) and Boc anhydride
extracted into CH2Cl2, washed with brine, and dried using (11.28 g, 51.7 mmol) were then added, and the reaction was
Na2SO4. Pure compound 2 was obtained after column chrom- stirred at room temperature overnight. After addition of H2O,
atography purification (Hex/EtOAc gradient, 19 : 1 to 1 : 1) as a the crude product was extracted into EtOAc. The combined
1
yellow oil (9.77 g, 95%). H NMR (500 MHz, CDCl3) δ 7.94 (d, J organic fractions were washed with 5% LiCl, brine, and dried
= 7.8 Hz, 1H), 7.87 (s, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.52 (d, J = using Na2SO4. Pure compound 5 was obtained after column
8.4 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.37 (d, J = 10.3 Hz, 1H), chromatography purification (Hex/EtOAc gradient, 19 : 1 to
7.33 (t, J = 7.6 Hz, 1H), 3.79 (s, 2H), 3.73 (s, 3H). 13C{1H} 38 1 : 1) as a yellow solid (2.50 g, 82%). 1H NMR (500 MHz, CDCl3)
NMR (126 MHz, CDCl3) δ 172.41, 156.65, 155.50, 128.56, δ 7.81 (s, 1H), 7.75 (m, 2H), 7.46 (d, J = 8.2 Hz, 1H), 7.29 (d, J =
128.45, 127.39, 124.67, 124.11, 122.85, 121.47, 120.84, 111.81, 8.3 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 6.79 (s, 1H), 3.76 (s, 2H),
111.77, 52.26, 41.17. HRMS (m/z): [M + Na]+ calcd for 3.72 (s, 3H), 1.55 (s, 9H). 13C{1H} NMR (126 MHz, CDCl3) δ
[C15H12O3Na]+ 263.0679, found 263.0691.
172.51, 157.44, 155.77, 152.75, 138.24, 128.51, 127.50, 124.66,
Methyl 2-(nitrodibenzofuran-2-yl)acetate (3). Compound 2 120.81, 119.18, 113.97, 111.58, 102.01, 80.92, 52.26, 41.17,
(8.00 g, 33.2 mmol) was dissolved in TFA (100 mL). While stir- 28.48. HRMS (m/z): [M + Na]+ calcd for [C20H21NO5Na]+
ring, NaNO3 (3.52 g, 41.4 mmol) was slowly added over 378.1312, found 378.1313.
5 minutes, and the reaction was stirred at rt for 3 h. H2O
Methyl 2-(7-( bis (Boc)amino)dibenzofuran-2-yl)acetate (6).
(100 mL) was added to the reaction flask, and the crude Compound 5 (2.50 g, 7.03 mmol) was dissolved in CH3CN
product was extracted by EtOAc, washed with 10% NaHCO3, (75 mL). Boc anhydride (3.06 g, 14.1 mmol) and DMAP
brine, and dried using Na2SO4. After removal of solvent, the (0.086 g, 0.703 mmol) were added, and the reaction mixture
nitrated material was obtained as a 3 : 1 isomeric mixture of 7- was stirred at rt overnight. After removal of CH3CN under
and 3-nitrodibenzofuran (3a and 3b respectively). The desired vacuum, water was added, and the crude product was extracted
7-nitro isomer was obtained after purification in the following into EtOAc. The combined organics were washed with brine,
step, and this crude mixture was used without further purifi- and dried using Na2SO4. Pure compound 6 was obtained after
cation. 1H NMR (500 MHz, CDCl3) δ 8.44 (3a, s, 1H), 8.39 (3b, s column chromatography purification (Hex/EtOAc gradient,
1H), 8.28 (3a, d, J = 8.5 Hz, 1H), 8.03 (3a, d, J = 8.5 Hz, 1H), 19 : 1 to 1 : 1) as a yellow solid (3.20 g, >99%). 1H NMR
7.99 (3b, d, J = 7.8 Hz, 1H), 7.96 (3a, s, 1H), 7.89 (3b, s, 1H), (500 MHz, CDCl3) δ 7.88 (m, 2H), 7.52 (d, J = 8.4 Hz, 1H), 7.38
7.64 (3b, d, J = 8.3 Hz, 1H), 7.61 (3a + 3b, m, 2H), 7.51 (3a, d, J (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 7.13 (d, J = 8.2 Hz, 1H), 3.79 (s,
= 8.5 Hz, 1H), 7.43 (3b, t, J = 7.5 Hz, 1H), 4.18 (3b, s, 2H), 4.81 2H), 3.72 (s, 3H), 1.41 (s, 18H). 13C{1H} NMR (126 MHz, CDCl3)
(3a, s, 2H), 3.74 (3a + 3b, s, 6H). 13C NMR (126 MHz, CDCl3) δ δ 172.37, 156.45, 156.21, 151.89, 138.68, 128.88, 128.68,
172.04, 170.99, 158.55, 157.68, 155.54, 154.51, 147.15, 147.06, 124.30, 123.37, 123.22, 121.56, 120.43, 111.89, 111.78, 83.05,
146.99, 131.02, 130.12, 129.91, 129.74, 129.33, 124.93, 124.75, 52.32, 41.16, 28.06. HRMS (m/z): [M
+
Na]+ calcd for
123.96, 122.98, 122.58, 122.44, 121.87, 120.79, 118.67, 112.48, [C25H29NO7Na]+ 478.1836, found 478.1954.
109.78, 108.19, 52.46, 52.41, 40.99, 40.23. HRMS (m/z): [M +
Methyl 2-bromo-2-(7-( bis (Boc)amino)dibenzofuran-2-yl)
acetate (7). Compound 6 (0.126 g, 0.277 mmol) was dissolved
Na]+ calcd for [C15H11NO5Na]+ 308.0529, found 308.0517.
Methyl 2-(7-aminodibenzofuran-2-yl)acetate (4). The iso- in CCl4 (6 mL). Benzoyl peroxide (3 mg, 0.01 mmol) and
meric mixture of nitrated products 3a and 3b (7.52 g, N-bromosuccinimide (49.0 mg, 0.275 mmol) were added, and
26.3 mmol) and CaCl2 (2.92, 26.3 mmol) and were added to the reaction was stirred at reflux for 5 h. The reaction was
hot ethanol : H2O (150 mL, 20 : 1). Iron powder (300 mesh, diluted with CH2Cl2 and washed with 5% NaHCO3, brine, and
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dried using Na2SO4. Pure compound 7 was obtained after mixture was stirred at rt for 24 h. The reaction mixture was
column chromatography purification (3 : 2 Hex : EtOAc) as a diluted with H2O, and the pH was made slightly basic with
1
yellow solid (0.149 g, >99%). H NMR (500 MHz, CDCl3) δ 8.17 K2CO3. The product was extracted into CH2Cl2, washed with
(s, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.55 (d, brine, and dried using Na2SO4. The final product was purified
J = 8.5 Hz, 1H), 7.37 (s, 1H), 7.16 (d, J = 8.2 Hz, 1H), 5.55 (s, using preparative HPLC (solvent A: H2O-0.1%TFA, solvent B:
1H), 3.82 (s, 3H), 1.40 (s, 18H). 13C{1H} NMR (126 MHz, CDCl3) CH3CN-0.1% TFA, solvent gradient: 0%B-5 min followed by
δ 169.07, 157.17, 156.63, 151.84, 139.00, 130.77, 128.15, 0–100%B over 60 min) to afford pure cDMA-NDBF-CA (10) as
124.53, 123.55, 123.00, 121.44, 120.65, 112.24, 111.88, 83.23, the TFA salt (3 mg, 7 μmol, 30%). 1H NMR (500 MHz, DMSO) δ
53.62, 46.63, 28.02. HRMS (m/z): [M
[C25H28BrNO7Na]+ 556.0941, found 556.0944.
Methyl
+
Na]+ calcd for 8.40 (s, 1H), 8.16 (s, 1H), 8.02 (d, 1H), 7.77 (s, 2H), 6.97 (s, 1H),
6.94 (d, 1H), 5.49 (s, 1H), 3.67 (s, 3H), 3.08 (s, 6H), 3.01 (t, 2H),
2-bromo-2-(7-(dimethylamino)dibenzofuran-2-yl) 2.84 (t, 2H). 13C NMR (126 MHz, DMSO) δ 170.44, 161.30,
acetate (8). Compound 7 (0.180 g, 0.337 mmol) was dissolved 153.51, 153.02, 143.57, 130.39, 128.49, 123.26, 121.83, 115.60,
in CH2Cl2 (1 mL), and TFA (2 mL) was added to the reaction 110.62, 110.26, 109.29, 93.88, 93.67, 53.50, 50.25, 40.71, 38.79,
flask. The reaction stirred at rt for 15 min, after which TFA was 29.90. HRMS (m/z): [M
+
H]+ calcd for [C19H22N3O5S]+
removed under vacuum. The product residue was dissolved in 404.1275, found 404.1297.
a 3 : 1 mixture AcOH : H2O (4 mL). After addition of parafor-
maldehyde (0.505 g, 1.68 mmol), the reaction was stirred for
NDBF-CA (11a) was prepared as previously reported.9
cDMA-NDBF-FTI (12). cDMA-NDBF-FTI was prepared accord-
10 min, followed by addition of NaBH3CN (0.106 g, ing to the general procedure described above from reaction
1.68 mmol). The reaction was stirred at rt for 5 h. The reaction with cDMA-NDBF-Br and FTI (20%). 1H NMR (500 MHz,
mixture was diluted with H2O, and the pH made slightly basic CDCl3) δ 8.41 (s, 1H), 8.17 (s, 1H), 7.29–7.18 (m, 7H), 6.98 (s,
with K2CO3. The aqueous solution was extracted into CH2Cl2, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.49 (s 1H), 4.90 (m,
and the combined organics were washed with brine and dried 1H), 4.39 (m, 1H), 3.68 (s, 3H), 3.09 (s, 6H), 3.07 (d, J = 3.3 Hz,
using Na2SO4. After equilibrating a silica gel column with 3H), 3.00 (d, J = 3.3 Hz, 6H), 2.19 (m, 1H), 2.03 (m, 1H), 1.36
0.1% triethylamine (in 5% EtOAc : Hex), the product was puri- (m, 1H), 1.24 (m, 3H), 1.19–1.15 (m, 10H) 0.89–0.71 (m, 8H).
fied using a EtOAc : Hex gradient (5–50%) to afford pure 8 as a HRMS (m/z): [M + H]+ calcd for [C43H60N5O11S2]+ 886.3725,
1
yellow solid (0.030 g, 0.082 mmol, 24%). H NMR (500 MHz, found 886.3717.
CDCl3) δ 7.99 (s, 1H), 7.74 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 8.4
NDBF-FTI (13). NDBF-FTI was prepared according to the
Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 6.81 (s, 1H), 6.77 (d, J = 8.6 general procedure described above from reaction with
1
Hz, 1H), 5.53 (s, 1H), 3.81 (s, 3H), 3.06 (s, 6H). 13C NMR NDBF-Br27 and FTI (20%). H NMR (500 MHz, CDCl3) δ 8.66
(126 MHz, CDCl3) δ 169.22, 159.24, 156.33, 151.43, 130.08, (d, J = 7.5 Hz, 1H), 8.43 (d, J = 13.4 Hz, 1H), 8.36 (m, 2H), 7.83
125.94, 125.47, 121.25, 119.74, 112.89, 111.37, 109.18, 94.60, (d, J = 8.5 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.53 (d, J = 7.6 Hz,
53.55, 47.38, 41.01. HRMS (m/z): [M + Na]+ calcd for [C17H16 1H), 7.27–7.19 (m, 4H), 4.91 (q, J = 6.0 Hz, 1H), 4.75 (q, J = 7.1
BrNO3Na]+ 384.0206, found 384.0216.
Hz, 1H), 4.40 (q, J = 6.3 Hz, 1H), 4.11 (s, 1H), 4.06 (d, J = 8.6
Methyl 2-bromo-2-(7-(dimethylamino)-3-nitrodibenzofuran- Hz, 1H), 3.94 (d, J = 10.9 Hz, 1H), 3.83 (m, 1H), 3.78 (m, 2H),
2-yl)acetate (cDMA-NDBF-Br, 9). Compound (30 mg, 3.72 (d, J = 10.5 Hz, 2H), 3.42 (m, 1H), 3.36 (d, J = 5.2 Hz, 1H),
8
82 μmol) was dissolved in 1 mL THF. Conc. H2SO4 (4 mL) was 3.31 (d, J = 5.7 Hz, 1H), 3.14 (m, 2H), 3.03 (m, 1H), 3.00 (d, J =
added to the reaction flask, and it was set in an ice bath. 5.7 Hz, 3H), 2.87 (m, 1H), 2.72 (m, 1H), 2.19 (m, 1H), 2.04 (m,
While stirring, 68% HNO3 (3.5 µL, 80 μmol) was added, and 1H), 1.76 (d, J = 6.4 Hz, 3H), 1.48 (s, 1H), 1.36 (m, 1H), 1.24 (m,
the reaction was stirred at 0 °C for 3 h. Water was then added, 1H), 1.18 (d, J = 5.8 Hz, 6H), 1.09 (s, 1H), 0.85–0.66 (m, 6H).
and the pH was made slightly basic with K2CO3. The product HRMS (m/z): [M + H]+ calcd for [C40H55N4O9S2]+ 799.3405,
was extracted into EtOAc, washed with brine, and dried using found 799.3422.
Na2SO4. After equilibrating a silica gel column with 0.1% tri-
ethylamine (in 5% EtOAc : Hex), the product was purified
using
a EtOAc : Hex gradient (5–50%) to afford pure
Author contributions
cDMA-NDBF-Br as a yellow solid (20 mg, 59%). 1H NMR
(500 MHz, CDCl3) δ 8.29 (s, 1H), 8.20 (s, 1H), 7.84 (d, 1H), 6.82 M. D. H. and M. M. designed the molecules and carried out
(d, 1H), 6.80 (s, 1H), 6.31 (s, 1H), 3.84 (s, 3H), 3.12 (s, 6H). 13C the major experiments reported here. M. H. H., T. K. B., S. F.,
NMR (126 MHz, CDCl3) δ 168.91, 161.59, 154.47, 131.06, A. F., K. N., H. T., F. X. and A. H. provided important assistance
130.76, 126.18, 122.65, 122.56, 121.50, 110.10, 108.58, 93.80, in synthesis, measurements and calculations. Z. B., D. B. and
53.76, 45.10, 43.97, 40.81. HRMS (m/z): [M + H]+ calcd for M. D. supervised the work. M. D. H. and M. D. wrote the
[C17H16BrN2O5]+ 407.0237, found 407.2229.
manuscript.
General procedure for thiol alkylation with cDMA-NDBF-Br
and NDBF-Br. cDMA-NDBF-Br (10 mg, 25 μmol) and cystea-
mine (2 mg, 36 μmol) were dissolved in DMF (2 mL). A solu-
tion of zinc acetate (27 mg, 120 μmol) in 1 : 1 CH3CN : H2O
Conflicts of interest
containing 0.1% TFA (2 mL) was added, and the reaction The authors report no competing financial interests.
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16 E. J. Petersson, G. S. Brandt, N. M. Zacharias,
D. A. Dougherty and H. A. Lester, in Methods in Enzymology,
Academic Press, 2003, vol. 360, pp. 258–273.
Acknowledgements
We thank Dr Guillermo Marques for valuable microscopy
insights and support. We thank Dr Viktor Young for solving 17 S. Loudwig and H. Bayley, in Dynamic Studies in Biology:
the X-ray crystal structure of intermediate 6. Microscopy was
performed at the University of Minnesota Imaging Center, a
Phototriggers, Photoswitches and Caged Biomolecules, Wiley-
VCH, 2005, pp. 253–304.
Nikon Center of Excellence for confocal microscopy. X-Ray 18 D. S. Lawrence, Curr. Opin. Chem. Biol., 2005, 9, 570–
crystallography was performed at the University of Minnesota 575.
X-Ray Crystallographic Laboratory supported by The National 19 A. Patchornik, B. Amit and R. B. Woodward, J. Am. Chem.
Science Foundation (CHE-1229400 Mass spectrometry analysis Soc., 1970, 92, 6333–6335.
was performed at the University of Minnesota Department of 20 S. R. Adams and R. Y. Tsien, Annu. Rev. Physiol., 1993, 55,
Chemistry Mass Spectrometry Laboratory (MSL), supported by 755–784.
the Office of the Vice President of Research, College of Science 21 K. König, J. Microsc., 2000, 200, 83–104.
and Engineering, and the Department of Chemistry at the 22 L. Shi, L. A. Sordillo, A. Rodriguez-Contreras and R. Alfano,
University of Minnesota, as well as The National Science
J. Biophotonics, 2016, 9, 38–43.
Foundation (CHE-1336940). This work was supported by the 23 G. C. R. Ellis-Davies, Nat. Methods, 2007, 4, 619–628.
National Institute of General Medical Sciences, including F32 24 G. Sylvestre, C. Sébastien, S. Alexandre, A. Karine, O. David,
GM126862 to M. H. and R01 GM084152 and R21 CA185783
to M. D.
Z. Burkhard, L. Jérémie, H. Stefan, B. Frédéric, N. Jean-
François and G. Maurice, ChemBioChem, 2008, 9, 1303–
1307.
25 T. Furuta, S. S. H. Wang, J. L. Dantzker, T. M. Dore,
W. J. Bybee, E. M. Callaway, W. Denk and R. Y. Tsien, Proc.
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