A red-shifted two-photon-only caging group for three-dimensional photorelease

Article abstract of DOI:10.1039/c7sc05182d

Based on nitrodibenzofuran (NDBF) a new photocage with higher two-photon action cross section and red-shifted absorption was developed. Due to calculations, a dimethylamino functionality (DMA) was added at ring position 7. The uncaging of nucleobases afte

Full text of DOI:10.1039/c7sc05182d

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Unger, M. A. H. Fichte, D. A. Gacek, A. Dreuw, J. Wachtveitl, P. Walla and A. Heckel, Chem. Sci., 2018,
DOI: 10.1039/C7SC05182D.
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A red-shifted two-photon-only caging group for three-dimensional
photorelease
Received 00th January 20xx,
Accepted 00th January 20xx
Yvonne Becker,‡a Erik Unger,‡a Manuela A. H. Fichte,a Daniel A. Gacek,b Andreas Dreuw,c Josef
Wachtveitl,d Peter J. Wallab and Alexander Heckel*a
DOI: 10.1039/x0xx00000x
Based on nitrodibenzofuran (NDBF) a new photocage with higher two-photon action cross section and red-shifted
absorption was developed. Due to calculations, a dimethylamino functionality (DMA) was added at ring position 7. The
uncaging of nucleobases after two-photon excitation (2PE) could be visualized via double-strand displacement in a
hydrogel. With this assay we achieved three-dimensional photorelease of DMA-NDBF-protected DNA orthogonal to NDBF-
protected strands. While being an excellent 2PE-cage, DMA-NDBF is surprisingly stable under visible-light one-photon
excitation (1PE). This case of excitation-specific photochemistry enhances the scope of orthogonal photoregulation.
www.rsc.org/
efficiency ꢀꢁ can be described (analogous to the “quantum
product” in 1P-uncaging) by the product of absorption cross
Introduction
section ꢀꢂ and the uncaging quantum yield Φꢁ (ꢀꢁ ꢃ ꢀꢂ ∙
Over the last decades photolabile protecting groups (PPGs)
Φꢁꢄ.16 The ꢀꢂ of chromophores depends on the length and
planarity of the π-electron system and substituent effects (i.e.
push-pull-systems).16,23,24 The positions of the substituents are
crucial: a dipolar character as well as quadrupolar or octupolar
enhance the 2P-absorption.23
became
a frequently used tool to regulate bioactive
molecules1 such as neurotransmitter,2-5 hormones6,7 and even
macro-molecules like proteins8,9 and oligonucleotides.10-12
One crucial long-term goal is a red-shifting13 of the light-
induced photorelease (uncaging14) into the therapeutic
window (~650-950 nm15). It is less harmless for living cells and
deeper tissue penetration becomes possible in biological
applications due to less absorption and scattering of e.g.
blood.16 In the 1990s a promising (un)caging strategy for
higher wavelengths (> 650 nm) has emerged, based on two-
photon (2P) sensitive photolabile groups.17-20 PPGs with 2P-
absorption character are cleavable with femtosecond pulsed
lasers. This non-linear optical process can be seen as a
simultaneous absorption of two photons. In many cases – but
not all21 – the resulting electronically excited state is the same
when photons of half the energy are used. This process was
first described by Maria Göppert-Mayer.22 It can be used to
realize photochemistry with 3D spatial resolution since the
excitation depends on the squared intensity (p~I2). Excitation
volumes can be as small as a femtoliter.16 The 2P-uncaging
In 2006, Ellis-Davies et al. introduced the new chromophore
3-nitrodibenzofuran (NDBF) for 2P-photolysis of NDBF-
EGTA:Ca2+ with ꢀꢁ = 0.6 GM at 720 nm.25
In this paper, based on the NDBF core (see compound
1), we rationally designed and synthesized the dimethylamino
derivate DMA-NDBF-OH ( , Fig. 1). Due to calculations with the
1, Fig.
2
DFT/B3LYP method and a 6-31*G basis set for the ground state
equilibrium structures and TDDFT/BHLYP for exited states the
dipolar structure should be red-shifted and have an increased
26
ꢀꢂ
.
Despite the availability of sophisticated computational
methods the optimisation of 2P-chromophors remains
formidable challenge.
a
Experimental and Results
The simulation of various NDBF derivatives predicted
a
preferred substitution of ring-position 7 with a dimethylamino
(DMA) functionality as donor (Fig. 1). The expected uncaging
efficiency of DMA-NDBF based on computed 2P-absorption
spectra should be more than 20 times higher than the one of
NDBF at its respective red-shifted maximum.26
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Fig. 1 The caging group precursor NDBF-OH (1) and its new dimethylamino
derivative DMA-NDBF-OH (2).
Fig.
2 Comparison of the 1P-absorption spectra of NDBF-OH (1, cyan) and its
dimethylamino derivative DMA-NDBF-OH (2, magenta) in DMSO. The maximum is
shifted bathochromically for photocage 2 due to addition of the N(Me)2 moiety and the
absorption at 420 nm is 79 times higher.
Small Molecule Synthesis and Characterisation
The synthesis of the caging group precursor DMA-NDBF-NH2
(
9) started with the iodation of 3-dimethyl-aminophenol (
3)
using KI and KIO3, followed by an electrophilic aromatic
substitution to form 2-iodo-5-dimethylaminophenol (
4) as
summarised in Scheme 1. The unsymmetrical aryl ether
formed via coupling with 4-fluoro-2-nitrobenzaldehyde (
6
was
5) and
subsequently reduced with trimethylaluminum. Hydrolysis led
to alcohol , which was used for a palladium-catalysed
intramolecular Heck-like reaction to yield the closed-ring form
. The azide was synthesised under Mitsunobu conditions
with in situ generated HN3. 8-(1-aminoethyl)-N,N-dimethyl-7-
7
2
8
nitrodibenzo-[b,d]furan-3-amine
9 was finally obtained via
Staudinger reaction.
For 1PE characterisation of DMA-NDBF-OH (
spectrum was recorded (Fig. 2) in DMSO and compared to the
one of unsubstituted NDBF-OH ( ). The absorption maximum
of
2) an absorption
1
Fig. 3 Excitation spectra resulting from 2PE between 770 and 1060 nm of 1 and 2 in
DMSO. The excitation power was set to 1.0 mW and squared dependence tested (†ESI).
Axes labelling for the “zoom” is the same. Solid lines are averages of the raw data
(dots).
2
is shifted bathochromically from 312 to 424 nm. With
15947 L∙mol-1∙cm-1 at 424 nm, the molar absorption is 98 times
higher
than the one of compound
1
(at 420 nm it is 79 times higher).
and
Then, 2P-fluorescence excitation (TPE) spectra of
1
2
were determined. Fig. 3 shows the relative fluorescence
intensities observed in the visible spectral range (~400-700
nm) after 2PE of
wavelength range between 770 and 1060 nm. We observed a
generally higher responsiveness to 2PE for than for . The
resulting fluorescence intensity for at 840 nm e.g. is 40 times
higher and should be proportional to the absorption cross
section ꢀꢂ of compound . Of course, these 2P-fluorescence
1 (cyan) and 2 (magenta) in DMSO using a
2
1
2
2
excitation spectra do not directly reflect the 2P-uncaging
efficiency ꢀꢁ as there is generally no direct relationship
between a molecules fluorescence quantum yield Φꢅ and the
quantum yield for the uncaging photochemistry
the observation of high fluorescence intensities after two-
photon excitation is a strong indication that compound can
indeed be two-photon excited quite readily, which is obviously
decisive prerequisite for any subsequent uncaging
Φꢁ. However,
Scheme 1 Synthesis of photocage 9. (a) KI, KIO3, 1N H2SO4, H2O, 3 h, rt, 52%. (b) KOtBu,
DMSO, 63%. (c) Al(CH3)3, CH2Cl2, 30 min., 0 °C, 97%. (d) Cs2CO3, Pd(OAc)2, H2O, DMAc,
3 d, 80 °C, 49%. (e) PPh3, HN3 (in situ), DEAD, THF 12 h, 0 °C -> rt, 93%. (f) PPh3, H2O,
THF/MeCN (1:1 v/v), 20 h, 70 °C -> rt, 71%. DMAc = dimethylacetamide, DEAD = diethyl
azodicarboxylate.
2
a
photochemistry (for more details see the discussion below and
the †ESI).
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Scheme 2 Synthesis scheme of phosphoramidite 11 with new caging group 9. (g)
4-DMAP, DIPEA, DMF, 2 d, 90 °C, 62%. (h) TBAF, THF, 2 h, rt, 98%. (i) DMTrCl, pyridine,
12 h, rt, 51%. (j) DIPEA, 2-cyanoethyl-N,N’-diisopropylchlorophosphoramidite, CH2Cl2,
2 h, rt, 55%. DMAP = (dimethylamino)pyridine, DIPEA = N,N-diisopropylethylamine,
TBAF = tetrabutylammonium fluoride.
Fig. 4 (a) Decrease of absorption between approximately 350 and 540 nm due to
irradiation of DNA2 with dADMA-NDBF at position 8 and resulting uncaging (0.9 nmol, 45
μl, 365 nm, 0.6 mW). (b) HPLC analysis shows the reaction of the caged diastereomers
(tR = 21 min. and 23 min.) and increase of the uncaged species (tR = 13 min.) (260 pmol,
13 μl, 365 nm, 0.42 mW). With tR = retention time on chromatography column.
Caged Oligonucleotides Synthesis and Characterisation
Scheme 2 summarises the synthesis of phosphoramidite 11
with the new DMA-NDBF caging group for oligonucleotide
solid phase synthesis. Inosine was activated with 2,4,6-
triisopropyl-benzenesulfonyl chloride at the nucleobase and 3’
and 5’-OH protected with tert-butyldimethylsilyl (TBDMS)
before it was reacted with 9, 4-DMAP and DIPEA in DMF. After
a nearly quantitative TBAF-deprotection of the alcohols in THF
the 5’-OH was protected with 4,4’-dimethoxytrityl (DMTr).
Phosphoramidite 11 was obtained in a reaction with DIPEA and
2-cyanoethyl-N,N’-diisopropylchlorophosphoramidite
in
CH2Cl2.
Photocages NDBF-NH2 and DMA-NDBF-NH2
(
9)
were
introduced in three different sequences – resulting in five
different DNA strands (see Tab. 1).
Fig. 5 Comparison of 1P-photolysis of DNA1 and DNA2 at various wavelengths but with
the same number of photons per time. Conversion was determined by HPLC analysis.
(260 pmol, 13 μl, 0.42 mW in case of 365 nm irradiation).
Test-sequences DNA1 and DNA2, 15-mers with the cage at
position 8, were used for first investigations of 1P-absorption
behaviour after irradiation (Fig. 4) and 1P-photolysis (Fig. 5).
All irradiation-tests were performed with a concentration of
20 µM in PBS.
standard (uracil) the amount of starting material could be
determined – here demonstrated after irradiation (365 nm,
0.60 mW) for 0, 200 and 1200 seconds. The 1P-photolysis
behaviour was tested for DNA1 and DNA2 at various
wavelengths as shown in Fig. 5. The samples were irradiated
for up to 1200 seconds at 365 (0.42 mW), 385 (0.40 mW), 405
(0.38 mW) or 420 nm (0.37 mW). For comparison, the number
of photons per time was kept constant.
Figure 4a illustrates the absorption decrease of DNA2 with a
dADMA-NDBF residue between 350 and 540 nm after irradiation
with 365 nm. The absorption of the nucleobases at around 260
nm remained unaffected. Quantification of caged and uncaged
species could be performed subsequently via high-
performance liquid chromatography (HPLC; Fig. 4b). With an
internal
The 1P-photolysis of DNA1 was found to be significantly
faster at every tested wavelength compared to DNA2. For
example, after 1200 s irradiation at 385 nm DNA1 was
quantitatively uncaged, whereas 77% starting material
remained in case of DNA2. After the same time of irradiation
at 420 nm we observed 87% remaining starting material for
DNA1 and 97% of DNA2, even though DMA-NDBF has its
absorption maximum at 424 nm. Further experiments revealed
that with wavelengths >420 nm (i.e. 455 nm) no 1P-photolysis
could be detected at all for DNA2 – regardless of the power we
Table 1 The five synthesised DNA strands used in this investigation.
Strand
Sequence (dA caged with x = NDBF, y = DMA-
NDBF)
DNA1
DNA2
DNA3
DNA4
DNA5
5‘-GCATAAA
A
A
xAAAGGTG-3‘
yAAAGGTG-3‘
5‘-GCATAAA
5‘-SH-(CH2)6-GC
5‘-SH-(CH2)6-GC
5‘-SH-(CH2)6-AG
A
A
A
xTAA
yTAA
xTAC
A
A
A
xTAA
yTAA
A
xGAT xCGCA-3‘
A
A
xGGTG-3‘
yGGTG-3‘
used! The 1P-quantum yield Φ 420
’
for DNA1 with a dANDBF
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residue was found to be 13.6% and for DNA2 with dADMA-NDBF 1PE appears to have a low uncaging quantum yield, i.e. this
0.05%.27 The respective quantum yields
340 were 24.05% and caging group can be considered as “one-photon-stable” (at
Φ
’
1.10%, showing that irradiation into the transition of DMA- least in the visible range) whereas after 2PE the intended
NDBF at around 340 nm results in some extent of uncaging uncaging reaction readily occurs with irradiation conditions
whereas the lower-energy transition does not (see also †ESI that have been shown to be compatible with living cells.30
for a more detailed analysis).
Because of its low 1P-photolysis rates, we decided to use
Compound 2 shows some similarity to an amino-substituted DMA-NDBF as a “2P-only-cage” which should be applicable for
ortho-nitrobenzyl caging group which has been investigated by complex orthogonal uncaging together with various 1P-cages –
Bochet et al.28 In their case only prior protonation of the amino especially also red-shifted ones that are currently the focus of
group led to
a
state with productive photolysis while much attention. We used DNA4 with dADMA-NDBF residues and
irradiation into the unprotonated state yielded a charge DNA5 with a different sequence and dANDBF residues for
transfer transition instead of uncaging. Investigations individual addressing together in the same hydrogel. For a
addressing the charge transfer character in the two-colour read-out ATTO565 and ATTORho14 were used as
photochemistry of compound
However, even at pH 2 there was no conversion of DNA2 upon matching quencher pairs (for an overview see scheme S2 in
irradiation at 455 nm.29
the †ESI). Fig. 7 provides an overview of optimised irradiation-
2 can be found in the †ESI. fluorophores F1 and F2 and BHQ2 (Q1) and BBQ-650III (Q2) as
For the 2-photon-characterisation of the photocages in DNA conditions, tested for the triply caged strands. With 420 nm
strands we used our recently published hydrogel-fluorescence-
assay on a confocal microscope and laser setup (†ESI).30,31
DNA3
Scheme 3 Hydrogel-fluorescence-assay to monitor uncaging. After irradiation with a
Fig. 6 (a) 2P-activation of fluorescence (ATTO565) with NDBF and DMA-NDBF, based on
defined wavelength the triply caged antisense strand becomes uncaged and is able to
(b) intensity measurements in a hydrogel after uncaging at the indicated wavelengths.
bind the 5’-fluorophore-labelled strand. By competition, the counter strand with the
DMA-NDBF has a red-shifted local maximum at 840 nm.
quencher is removed, the fluorescence increases.
and DNA4 with either dANDBF or dADMA-NDBF residues,
respectively, were immobilised via thiol-linkers in a hydrogel
(PVA-PEG), as illustrated in Scheme 3. Then the gel was soaked
with a buffered solution containing a duplex of a 15mer-strand
with a 5’-terminally attached fluorophore (ATTO565) and a 3’-
quencher (BHQ2) 11-mer strand. The quencher strand
displacement after uncaging of DNA3 or DNA4 led to increased
fluorescence in the focal plane. The uncaging-wavelengths
between 720 and 980 nm were generated with a pulsed
Ti:sapphire laser for 2P-excitation.
For each of the
Fig.
7 Tests with the triply caged DNA4 and DNA5 for optimised uncaging-
wavelengths investigated one line was written into the
hydrogel (Fig. 6b) and the resulting fluorescence was
quantified (Fig. 6a). The power was kept constant for every
line. The form of the pink spectrum in Fig. 6a (with a maximum
at 840 nm) resembles very much the one of the pink spectrum
in Fig. 2 (with a maximum at 424 nm). However, 1P-irradiation
at 420 nm results in a poor conversion whereas 2P-irradiation
at 2x420 nm efficiently produces the desired uncaged product
strand.
orthogonality. The best results were obtained with 420 nm with 780 nW and 2 scan
repeats, 730 nm and 840 nm with 15 mW and 5 scans.
(1PE, 780 nW, 2 scans) it was possible to only uncage the
NDBF, with 730 nm (2PE, 15 mW, 5 scans) both – however
DMA-NDBF much more efficiently than NDBF – and 840 nm
(2PE, 15 mW, 5 scans) only DMA-NDBF, while leaving the other
cage intact in the same hydrogel. Based on these results, seven
rectangular shapes (steps) were written into the hydrogel with
840 nm (Fig. 8), as well as a circular shape with 420 nm. The
laser beam direction followed the z-axis. The 2P-steps with
Apparently, DMA-NDBF is one of the few cases where 1PE
and 2PE with twice the wavelength do not result in the same
photochemical behaviour. In our case, the excited state after
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spatial resolution in z had a distance of 25 µm between them. substantial one-photon oscillator strength as well as two-
The circular 1P-irradiation resulted in the cylindrical staircase photon absorption cross section.26 However, different
core shown in Fig. 8. A z-stack (Fig. S4) with two detection photochemical behaviour is induced, because 1PE and 2PE
channels (Ch. 1: fluorescence excitation 543 nm, detection at electronic excitations couple to different molecular
557-612 nm; Ch. 2: excitation 633 nm, detection 671-721 nm) vibrations.33 This unusual “two-photon-only” behaviour offers
was imaged at laser setup 2 (†ESI). Figure 8a shows the interesting applications for light-regulation scenarios with
magenta-channel 1, cyan-channel
2 and the overlaid increased complexity. We had recently presented orthogonal
combination in the x/y-plane. For Figures 8b and c the two-colour, two-photon uncaging30 where we had to carefully
staircase was rotated in the z-/y-plane. The colours in Figure 8c control the two-photon irradiation conditions (especially the
demonstrate the height in the hydrogel. The laser powers used power). With DMA-NDBF, we can now perform efficient two-
were in a range compatible with living cells. For instance, 18- photon uncaging with red light leaving a broad spectral
24 mW were found to be tolerated by HEK cells, dorsal root window open for orthogonal 1P-uncaging in the red part of the
ganglia and liver cells for 10-20 scans.32
spectrum. In addition, 1PE can now be performed before 2PE
with DMA-DNBF, which is surprisingly “one-photon-stable”.
With previous two-photon caging groups, the 2PE had to be
performed as first photochemical operation. This adds another
degree of freedom to ever more complex scenarios of complex
light control.34,35 Its one-photon-stability and yet easy and
selective photolysis under 2PE, its red-shifted 2P absorption
which lies perfectly in the therapeutic window and its perfect
stability to regular ambient light make the DMA-NDBF a very
interesting caging group for future biological applications.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
This research was funded by the Deutsche Forschungs-
gemeinschaft (GRK 1986—CLiC). We gratefully acknowledge
the help of the Heilemann group (Goethe-University
Frankfurt).
Notes and references
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Fig. 8 3D picture of a winding staircase with a one-photon uncaging (420 nm) core and
two-photon (840 nm) steps. (a) Channel1, Channel 2 and combination in x-/y-plane and
(b) z-/y-plane. (c) Colour-coded height profile of the z-/y-plane (0 – 160 µm).
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Conclusion
In summary we designed, synthesised, and characterised a
new dimethylamino derivative of the NDBF photolabile
protecting group (PPG). It shows a red-shifted one- and two-
photon absorption compared to the NDBF group – which is
important for biological applications. As predicted by
theoretical calculations the DMA-NDBF PPG shows a better
two-photon photolysis behaviour compared to NDBF.
However, to our surprise it turned out that the one-photon
photolysis efficiency of DMA-NDBF is rather poor, especially
for wavelengths beyond 400 nm. Based on our calculations we
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6 | J. Name., 2012, 00, 1-3
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Chemical Science
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DOI: 10.1039/C7SC05182D
With a new photolabile protecting group - exclusively cleavable by two-photon-excitation - complex light
scenarios for three-dimensional uncaging are possible.