Synthesis and Characterization of Photoactivatable Doxycycline Analogues Bearing Two-Photon-Sensitive Photoremovable Groups Suitable for Light-Induced Gene Expression

Article abstract of DOI:10.1002/cbic.201700628

We report the synthesis and photolytic properties of caged 9-aminodoxycycline derivatives modified with 2-{4′-bis-[2-(2methoxyethoxy)ethyl]-4-nitrobiphenyl-3-yl}prop-1-oxy (EANBP) and PEG7-ylated (7-diethylamino-2-oxo-2H-chromen-4-yl)methyl (PEG7-DEACM) groups. 9-Aminodoxycycline is a tetracycline analogue capable of activating transcription through the inducible TetOn transgene expression system and can be regioselectively coupled to two-photon-sensitive photo-removable protecting groups by carbamoylation. The EANBP-based caged 9-aminodoxycycline showed complex photochemical reactions but did release 10 % of 9-aminodoxycycline. However, 9-(PEG7-DEACMamino)doxycycline exhibited excellent photolysis efficiency at 405 nm with quantitative release of 9-aminodoxycycline and a 0.21 uncaging quantum yield. Thanks to the good two-photon sensitivity of the DEACM chromophore, 9-aminodoxycycline release by two-photon photolysis is possible, with calculated action cross-sections of up to 4.0 GM at 740 nm. Therefore, 9-(PEG7-DEACMamino)doxycycline represents a very attractive tool for the development of a light-induced gene expression method in living cells.

Full text of DOI:10.1002/cbic.201700628

Accepted Article
Title: Synthesis and Characterization of photoactivatable doxycycline
analogues bearing two-photon sensitive photoremovable groups
suitable for light induced gene expression
Authors: Alexandre Specht, Bastien Goegan, Firat terzi, Frédéric
Bolze, and Sidney Cambridge
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To be cited as: ChemBioChem 10.1002/cbic.201700628
Link to VoR: http://dx.doi.org/10.1002/cbic.201700628
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10.1002/cbic.201700628
ChemBioChem
FULL PAPER
system). Expansion of the biological space for TetR-based
expression systems has led to novel eukaryotic transcriptional
activators.[6] Random mutagenesis coupled with phenotypic
screening has given rise to variants termed rtTA (reverse
tetracycline-controlled transactivator), which exhibit a reversed
allosteric response and require the tetracycline agonists
anhydrotetracycline or doxycycline for DNA binding (TetOn
system).[7]
Synthesis and Characterization of
photoactivatable doxycycline analogues
bearing
two-photon
sensitive
photoremovable groups suitable for
light induced gene expression
Bastien Goegan,a Firat Terzi,b Frédéric Bolze,a
Sidney Cambridge,*b and Alexandre Specht *a
By rendering Tetracycline analogues (and in particular
doxycycline) photoactivatable, one has now the potential to
develop a method for high spatial and temporal control of gene
expression by irradiation with light. To do so, light controlled
transcriptional
activation
has
been
achieved
with
Abstract: We report herein the synthesis and the photolytic
photoremovable groups (e.g. caging groups).[1,4] 4,4-Dimethoxy-
2-nitrobenzyl-caged doxycycline derivatives have been
combined with the TetOn system in order to photoinduce
reporter gene expression in several eukaryotic systems
including plant leafs, brains slices, as well as living Xenopus
tadpoles, and living mouse embryos.[8] However, none of the
current photoactivatable doxycycline analogues could be
synthesized efficiently and the synthesis protocols were not
amenable to large-scale production of the compounds. Previous
synthetic strategies involved a coupling reaction without prior
protection of the various reactive functions of the doxycycline
molecule.[8] Consequently, published synthesis protocols report
poor yields and required a substantial effort for purification of the
final product. Another drawback of these light-triggered gene
expression systems is the use of UV sensitive photoremovable
protecting groups (PPG) and consequently low light penetration
depth and potential phototoxicity.[9] To overcome the dilemma
that only high-energy light can induce photochemical reactions
on PPG, two-photon (TP) photolysis was explored to shift from
UV-blue to red-IR excitation.[10] Indeed, this nonlinear optical
phenomenon allows the use of IR light for excitation leading to a
deeper light penetration in biological samples and low
phototoxicity. In addition, a higher intrinsic three-dimensional
spatial resolution is achieved since the two-photon absorption
phenomenon is limited to a very small volume at the focal point
of the optical system used (~ 1µm3). In this context, new
generations of PPGs with remarkably high two-photon
absorption cross-sections have been recently developed. Here,
we present the design, the efficient synthesis, and the
properties of EANBP and PEG7-DEACM caged 9-
aminodoxycycline. 9-Aminodoxycycline is
a
tetracycline
analogue able to activate transcription via the inducible TetOn
transgene expression system and can be regioselectively
coupled
by
carbamoylation
to
two-photon
sensitive
photoremovable protecting groups. The EANBP caged 9-
aminodoxycycline showed complex photochemical reactions but
did release 10% of 9-aminodoxycycline. However, the PEG7-
DEACM-9-aminodoxycycline exhibited excellent photolysis
efficiency at 405 nm with
a quantitative release of 9-
aminodoxycycline and a 0.21 uncaging quantum yield. Due to
the good two-photon sensitivity of the DEACM chromophore, 9-
aminodoxycyline release is possible by two-photon photolysis
with a calculated action cross-sections up to 4.0 GM at 740 nm.
Therefore, PEG7-DEACM-9-aminodoxycycline represents a very
attractive tool for the development of a light induced gene
expression method in living cells.
Introduction
The photochemical regulation of gene function is a rapidly
advancing research field in the functional genomics era since
the manipulation of complexes tissues, such as interfering with
pattern formation during tissue development, requires precise
spatial and temporal control of gene expression.[1] Besides fully
genetically encoded photoactivated gene expression methods,[2]
synthetic photoresponsive molecules are particularly attractive to
developmental biologists[3] and neurobiologists.[4]
[a]
Dr. B. Goegan, Dr. F. Bolze, and Dr. A. Specht
Laboratoire de Conception et Application de Molécules
Bioactives, UMR 7199, CNRS/UDS
Faculté de Pharmacie
One of the most commonly used ligand-inducible gene
expression systems is the Tet system which takes advantage of
an Escherichia coli antibiotic resistance mechanism. Because of
its nanomolar affinity, the tetracycline-responsive gene
regulation system is highly specific and efficient and is thus used
widely for transgene expression. Its main element is the
homodimeric Tet repressor (TetR).[5] TetR tightly binds its
effector tetracycline, or the more potent derivative doxycycline,
as a magnesium (II) chelate to a regulatory core domain. This
leads to an allosteric conformational change in TetR that results
in its dissociation from the operator DNA sequence (TetOff
74 Route du Rhin, 67400 ILLKIRCH, France
Fax: (+33) 368854306
E-mail: specht@unistra.fr
[b]
F. Terzi, Dr. S. Cambridge
University of Heidelberg
Department of Functional Neuroanatomy
Im Neuenheimer Feld 307
69120 Heidelberg, Germany
E-mail: cambridge@ana.uni-heidelberg.de
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ChemBioChem
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NO2
NO2
characterization of caged doxycycline analogues using the TP
OH
O
Cl
O
sensitive
2-(4'-((di(tris-ethoxy(methoxy))amino)-4-nitro-[1,1'-
i)
O
biphenyl]-3yl) propan-1-ol (EANBP)[11] as well as a soluble
version of the well-known 7-(Dialkylamino)coumarin-4-yl]methyl
(DEACM)[12] caging groups.
O
N
O
O
O
O
N
O
O
EANBP
4
ii)
Results and Discussion
OH
O
O
OH
O
NO2
H
N
O
NH2
OH
Design and synthesis of 9-aminodoxycycline
O
H
H
OH
N
Doxycycline has
a complex structure and thus the
EANBP-9-amino-Doxycycline
O
N
O
synthesis of caged doxycycline is difficult. In particular,
doxycycline has many nucleophilic functional groups (Scheme 1).
To be able to link a PPG to a specific site on doxycycline, we
decided to introduce an additional highly nucleophilic group on
the doxycycline skeleton. Of course, doxycycline analogues with
this new functionality should still be able to induce transgene
expression via the TetOn system. For this, we focused on
position 9 of the doxycycline ring system. This position was
selected based on the X-Ray structure of the doxycycline-Mg2+
complex bound to the repressor (TetR).[13] The three-
dimensional arrangement of doxycycline-Mg2+ complex bound to
TetR revealed that position 9 could be further modified, without
significantly interfering with the capacity of doxycycline to
O
O
Scheme 2: Synthesis of EANBP-9-aminodoxycycline: i) EANBP,
trisphosgene, DIPEA, THF, RT, 1,5 h ii) 3, NaHCO3, dioxane,
H2O, RT, 2 h, r = 50 %.
We decided to also synthesize a second TP sensitive
caged-doxycycline analogue, using the well-known DEACM
PPG. However, this photoremovable protecting group is very
hydrophobic. In order to improve its water solubility, we
introduced polyethylene glycol chains (PEG) at the benzylic
position of the DEACM caging group using copper catalyzed
azide-alkyne cycloaddition (CuAAC) reactions.[14] Therefore, two
synthetic pathways have been developed in order to incorporate
either a propargyl or an azide function at the benzylic position of
the DEACM chromophore (scheme 3). Starting from
commercially available 7-(diethylamino)-4-methyl-2H-chromen-
interact with TetR. Therefore, 9-aminodoxycycline
3 was
prepared in two steps starting from commercial doxycycline
hyclate 1 (Scheme 1). The later was first nitrated in position 9
using a mixture of concentrated nitric and sulfuric acids leading
to 9-nitro-doxycycline 2 with 97 % yield. Subsequently, 9-
aminodoxycycline 3 was obtained with 96 % yield after catalytic
reduction of 2 using Pd/C in degassed methanol.
2-one
5,
the
7-(diethylamino)-2-oxo-2H-chromene-4-
carbaldehyde 6 was synthesized with 47 % yield using selenium
dioxide as an oxidizing agent.[15] The aldehyde 6 was converted
to the epoxide 7 with 67 % yield using carboxymethyl-4-
cyanophenylmethylsulfonium trifluoromethanesulfonate in the
presence of cesium carbonate.[16] The azide containing DEACM
analogue 8 was obtained by the opening of the epoxide 7 with
47 % yield using sodium azide. In parallel, the alkyne containing
DEACM analogue 9 was obtained with 85 % yield by the
reaction of freshly prepared propargyl zincate with the aldehyde
6 as previously reported. [17]
OH
O
O
O
OH
O
O
O
OH
O
O
O
OH
OH
OH
O2
N
H2N
9
NH2
OH
NH2
OH
i)
ii)
NH2
OH
H
H
N
H
H
N
H
H
N
OH
OH
OH
1
2
3
Scheme 1: Synthesis of 9-aminodoxycycline : i) H2SO4 (conc.),
HNO3, 0°C, 1h, r = 97 %, ii) MeOH, Pd/C (cat.), H2, RT, 1h, r =
96 %.
Synthesis of Caged 9-aminodoxycycline derivatives
The azide containing DEACM analogue 8 was used in a
CuAAC reaction with the mPEG7-propyne (see supporting
information for the synthesis) leading to the PEG7-DEACM
alcohol 10 with 60 % yield. This later was converted to the
corresponding chloroformate 12 in order to regioselectively graft
For an efficient TP uncaging at 800 nm, the EANBP caging
group was first selected to produce highly TP-sensitive caged
doxycycline analogues. Of note, this later photoremovable group
is bearing oligoethylene glycol (OEG) chains in order to improve
its water solubility. First, the alcohol of the caging chromophore
EANBP was synthesized according to a previously described
procedure.[11c] With 3, the EANBP-9-aminodoxycycline analogue
could be synthesized by regioselectively grafting the EANBP-
chloformate 4 to the 9-aminodoxycycline 3 with 50 % yield
(scheme 2).
the 9-aminodoxycycline
3 to obtain the PEG7-DEACM-9-
aminodoxycycline with 40 % yield. In parallel, the alkyne
containing DEACM analogue 9 was coupled to the mOEG4-
azide (see supporting information for the synthesis) with 85 %
yield. Surprisingly, we were not able to synthesize the
chloroformate of 11 since the reaction of 11 with trisphosgene
lead to the formation of the chlorinated analogue 13 with 99 %
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yield. This indicates that the triazole orientation can influence the
reactivity of our soluble versions of the DEACM caging group.
EANBP-9-aminodoxycycline
and
PEG7-DEACM-9-
aminodoxycyline are 375 nm and 390 nm with molar absorption
coefficients of 9 000 M-1cm-1 and 13 000 M-1cm-1, respectively.
Figure 1 shows the evolution of the UV-vis spectrum of EANBP-
9-aminodoxycycline (A) and PEG7-DEACM-9-aminodoxycycline
(B) during photolysis in phosphate buffer. The isosbestic points
at 340 and 450 nm for EANBP-9-aminodoxycycline and at 345
and 445 nm for PEG7-DEACM-9-aminodoxycycline indicate that
a clean photochemical reaction occurred leading to stable
photoproducts.
O
i)
N
O
O
N
O
O
6
5
iii)
ii)
O
OH
N
O
O
N
O
O
7
9
v)
iv)
O
O
O
HO
N
O
N
N3
O
N
11
HO
N
O
8
O
O
N
v)
vi)
N
O
O
O
O
O
O
N
O
O
N
O
N
O
O
N
O
N
N
O
N
Cl
O
N
HO
vi)
O
Cl
O
O
N
O
O
O
N
O
O
O
O
O
O
N
O
O
13
10
12
vi)
O
O
N
OH
H
N
H
O
HO
N
O
N
H2
N
O
O
N
9
OH
H
OH
O
O
O
O
O
N
O
O
O
O
PEG7-DEACM-9-amino-doxycycline
Scheme 3: Synthesis of PEG7-DEACM-9-aminodoxycycline : i)
SeO2, Xylene, reflux, 16 h, 47 %, ii) CsCO3,
r
=
(carboxymethyl)(4-cyanophenyl)methylsulfonium, THF, 60°C, 2h,
r = 67 %, iii) Propargyl bromide, Zn, THF, RT, r = 85 %, iv)
NaN3, NH4Cl, EtOH, H2O, reflux, 16 h, r = 47%, iv) PEG7-
propyne or OEG4-azide, CuSO4, ascorbic acid, proline, DMF, t-
BuOH, H2O, 60°C, 24 h, r = 60-85 %. vi) Trisphosgene, DIPEA,
THF, RT, 1,5 h, then NaHCO3, dioxane, H2O, RT, 2 h, r = 40%.
In summary, the 9-aminodoxycycline analogue was used
to efficiently and regioselectively graft two photoremovable
groups using carbamate linkages to generate EANBP-9-
aminodoxycyline and PEG7-DEACM-9-aminodoxycyline, two
new caged doxycycline analogues.
Figure 1 (A) Changes in the UV/visible spectrum of EANBP-9-
aminodoxycycline during photolysis (405 nm) in phosphate
buffer (25 μM, pH 7.4). (B) Changes in the UV/visible spectrum
of PEG7-DEACM-9-aminodoxycycline during photolysis (405
nm) in phosphate buffer (60 μM, pH 7.4).
Photophysical and Photochemical characterization of caged
9-aminodoxycycline derivatives
The photolytic release of 9-aminodoxycycline was
analysed quantitatively by HPLC after irradiation in neutral
buffered medium. Surprisingly, EANBP-9-aminodoxycycline
afforded only a 10 % yield of released 9-aminodoxycyline.
Previously, we achieved significantly higher yields with EANBP-
The one-photon properties of EANBP-9-aminodoxycycline
and PEG7-DEACM-9-aminodoxycycline were investigated by
UV-visible spectroscopy. The absorption maxima at pH 7.2 of
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caged GABA[11d] and EANBP-caged Phosphate (Pi).[11c] Thus,
these data suggest that uncaging of EANBP-9-aminodoxycycline
leads to a product that “hijacks” or blocks 9-aminodoxycycline
release with a 90 % probability. This is supported by the
observation that the absorption maximum of the EANBP-9-
aminodoxycyline shifted from 397 nm to 375 nm compared to
EANBP-caged GABA (or Pi). Also, the photochemistry of this
class of photoresponsive molecules (i.e. orthonitrophenethyl
derivatives) is strongly dependent on solvent and basicity.[18]
One could speculate that the doxycycline moiety increased the
hydrophobic environment near the EANBP caging group. This
could potentially cause a shift of the absorption maximum of the
4-amino-4’-nitro-biphenyl chromophore and, unfortunately, lead
very suitable caged doxycycline derivative for visible light
photolysis at neutral pH. Moreover, based on the previously
reported two-photon absorption cross-section (da) for the
DEACM chromophore of 2,3 GM at 800 nm[20] and the
calculated value of 19 GM at 740 nm (see supporting
information),[21] the two-photon uncaging action cross-section (du
= da. F) of PEG7-DEACM-9-aminodoxycycline is estimated to be
4.0 GM at 740 nm and 0.5 GM at 800 nm. This two-photon
uncaging
action
cross-section
of
PEG7-DEACM-9-
aminodoxycycline is, to our knowledge, not only the highest
value reported for a caged doxycycline analogue but the highest
for any transcriptionally active small molecule including
Tamoxifen and IPTG.
to
a
new
major
photochemical
pathway
and
a
To demonstrate the good two-photon sensitivity of PEG7-
DEACM-9-aminodoxycycline, 100 µL of a 100 µM solution in
PBS were irradiated for 90 min using a femtosecond laser
(Insight Spectra-Physics) at 740 nm. HPLC analysis of the
photorearrangement producing a hydroxy-nitroso product 14
(Scheme 4).
photolyzed sample indicated
underscores the TP-sensitivity
a
30
%
of
conversion. This
PEG7-DEACM-9-
aminodoxycycline and suggests that it is suitable for TP-
mediated uncaging in vivo.
NO
O
9-aminodoxycycline
OH
O
14
Transcriptional efficacy of 9-aminodoxycycline and
photoactivated 9-aminodoxycycline
nitroso pathway
O
N
O
O
O
NO2
O
9-aminodoxycycline
O
hυ
To test the transcriptional efficacy of 9-aminodoxycycline,
we used dissociated hippocampal neurons which were virally
transduced with two components of the TetOn system, i.e. rtTA
NO2
O
N
O
beta elimination
pathway
O
O
and
a
Tet-dependent GFP construct. One week after
+
CO2
+
9-aminodoxycycline
transduction with adeno-associated viruses (AAV), the
doxycycline derivatives were added to the neurons at different
concentrations. Compared to unmodified doxycycline, 9-
aminodoxcycline had a lower affinity to rtTA as it required
roughly five-fold higher concentrations to achieve similar levels
of GFP expression (Figure 2 A, B). This indicates that 9-
aminodoxycycline is an attractive alternative to doxycycline as it
possesses similarly high transcriptional activity while being much
more amenable to chemical modification.
3
O
N
O
O
O
15
Scheme 4. Proposed photochemical pathways for EANBP-9-
aminodoxycycline adapted from Woll et al. [18b]
The PEG7-DEACM-9-aminodoxycycline in turn exhibited a
simple photolysis, since it afforded a quantitative yield of 9-
aminodoxycyline. A one-photon quantum yield of 0.21 for
PEG7-DEACM-9-aminodoxycyline uncaging could therefore be
determined by comparison with the DEACM-Gly reference
molecule at 405 nm by HPLC analysis.[12c] This later value is in
agreement with the good quantum yields observed with ethyl
substituted coumarin-4-yl PPG.[19]
Thus, the high photolysis quantum yield and the significant
molar absorption coefficient in the blue (e.F = 2700 M-1cm-1 at
390 nm) together with the very efficient release of 9-
aminodoxycycline makes PEG7-DEACM-9-aminodoxycycline a
Adding PEG7-DEACM-9-aminodoxycycline to Tet system
competent neuronal cultures did not produce any appreciable
levels of background fluorescence (Figure 2 A, B). In turn,
quantitative
photoactivation
of
PEG7-DEACM-9-
aminodoxycycline before adding it to the cultures yielded levels
of GFP fluorescence that were very similar to those induced by
unmodified PEG7-DEACM-9-aminodoxycycline. This strongly
suggested that the side product of the photolytic reaction (the
coumarin derivative 10) did not exhibit any significant toxicity as
the sensitive gene expression as well as neuronal morphology
were not impaired.
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Conclusions
There is
a sizeable need for two-photon sensitive caged
doxycycline derivatives which can be used to induce transgene
expression in dense three-dimensional tissue with two-photon
irradiation. This lead us to synthesize a new doxycycline
analogue, 9-aminodoxycycline, to allow easy and efficient
grafting of visible-light and two-photon sensitive photoremovable
chromophores onto it (i.e. EANBP and PEG7-DEACM). The 9-
aminodoxycyline
expression levels as the ‘gold standard’ doxycycline, albeit at
higher concentrations. 9-aminodoxycyline can be
derivative
induces
similar
transgene
regioselectively coupled by carbamoylation to two-photon
sensitive photoremovable groups. While photoactivation of the
EANBP-9-aminodoxycycline derivative led to some 9-
aminodoxycycline release, most of the desired product is
probably consumed by other complex photochemical reactions.
Presumably, a major photochemical pathway leading to a
hydroxy-nitroso product “hijacks” 9-aminodoxycycline release.
More importantly however, PEG7-DEACM-9-aminodoxycycline
showed a very efficient 9-aminodoxycycline release using visible
light photolysis (e.f = 2700 M-1cm-1 at 390 nm), or 740 nm two-
photon excitation (with a calculated δu = 4.0 GM).
In summary, we produced a new caged doxycycline
derivative that can be used in combination with the TetOn
system for photoactivated gene expression. Our future studies
will focus on the use of two-photon microscopy for uncaging of
PEG7-DEACM-9-aminodoxycycline in highly scattering tissue
such as the brain to induce transgene expression with high
spatiotemporal resolution.
Experimental Section
Synthesis
General: All chemicals for caged 9-aminodoxycycline synthesis were
analytical grade and purchased from Sigma-Aldrich, Alfa Aesar, or Acros
organics. A phenomenex C18 column PolarRP (4.6, 250 mm) was used
for HPLC analysis and a phenomenex C18 column PolarRP (10, 250
mm) was used for HPLC purifications. For HPLC analysis or purification,
elutions were performed at a flow rate of 1 mL/min or 4 mL/min,
respectively, using a linear gradient of acetonitrile in H20 (0,1 % TFA)
from 0 to 100 % (v/v) over 30 min. THF was distilled over sodium under
argon; methylene chloride was distilled over calcium hydride under argon.
Other anhydrous solvents were purchased from Sigma-Aldrich. All
Figure 2. (Photoactivated) transgene induction with
doxycycline (Dox) derivatives. (A) Representative images of
GFP expression (24 h after induction) with different doxycycline
derivatives. One-photon irradiation (430 nm) was performed
prior to administration to cells. (Scale bar: 20 µm). (B)
Quantification of somatic GFP fluorescence levels in rat
hippocampal cultures 24 h after induction. The total fluorescence
values within an equal sized region of interest were assessed
using NIH ImageJ (n = 20-40 neurons for each condition; error
bars: standard deviation; PEG7-DEACM-9-aminodoxycycline =
caged NH2-Dox)
aqueous solutions were prepared with deionized water from
commercial water purifier with a conductivity of 18 MΩ or higher.
a
1H and 13C spectra were recorded with a 400 MHz bruker Avance 400
instrument in CDCl3 (internal standard 7.24 ppm for 1H and 77 ppm,
middle of the three peaks, for 13C spectra) or [D4] MeOD (internal
standard 3.31 ppm for 1H and 49 ppm for 13C spectra). An agilent MM-
ESI-ACI-SQ MSD 1200 SL spectrometer or an agilent LC-MS RRLC
1200SL/ESI Qtof 6520 spectrometer was used for ESI analysis. TLCs
were run on Merck precoated aluminium plates (Si 60 F254). Column
chromatography was performed Merck silica gels (60-120 mesh).
Absorption spectra were recorded with a UVIKON XS spectrometer.
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EANBP-chloroformate (4): To
a
stirred solution of EANBP (see
produce the targeted compound with 47 % yield (125.5 mg) as a clear
red oil. 1H NMR (400 MHz, CDCl3): δ = 7.3 (d, 1H, J = 9.0 Hz), 6.5 (dd,
1H, J = 9.0 Hz, 2.4 Hz), 6.48 (d, 1H, J = 2.4 Hz), 6.3 (s, 1H), 5.1 (m, 1H),
3.5 (m, 2H), 3.4 (q, 4H, J = 7.0 Hz), 2.7 (d, 1H, J = 3.6 Hz), 1.2 (t, 6H, J =
7.0 Hz) ppm; 13C NMR (400 MHz, CDCl3): δ = 162.6, 156.7, 154.4, 150.7,
148.3, 124.5, 108.9, 106.3, 105.8, 98.3, 69.6, 56.6, 45.1, 12.5 ppm; MS
(ESI): calculated for C15H18N4O3 : 302.138, found 302.042.
supporting information) (63.5 mg, 0,13 mmol) in 1.5 mL of anhydrous
THF, triphosgene (39.54 mg, 0,13 mmol) and DIPEA (22.2 µL, 0.13
mmol) were slowly added at 0 °C. The reaction mixture was stirred at
room temperature for 90 min. Then the solvent was evaporated to yield
the EANBP-chloroformate as a pale yellow oil which was used in the next
step without further purification.
PEG7-DEACM (10): mPEG7-propyne (see supporting information) (150
mg, 0.40 mmol), compound 8 (126.1 mg, 0.42 mmol), ascorbic acid (34.9
mg, 0.19 mmol), and proline (9.1 mg, 0.08 mmol) were dissolved in a
mixture of DMF/t-butanol/water (1:15:1 in vol.). The mixture was
degassed by three freeze–pump–thaw cycles, then copper sulfate (12.7
mg, 0.08 mmol) was added. The reaction mixture was stirred at 60 °C
and monitored by TLC/HPLC until all starting material disappeared
(approximatively 24h). After completion of the reaction, the mixture was
diluted with water and extracted with dichloromethane (3x). The organic
layer was dried over MgSO4 and evaporated under reduced pressure.
The reaction products were purified using silica gel column
chromatography (CH2Cl2/CH3OH: 1/0 to 96/4 in vol.) and revealed with
phosphomolybdic acid (PMA) TLC stain to give the targeted compound
with 69 % yield (185 mg) as an orange oil. Analytical HPLC : tR : 16.2
EANBP-9-aminodoxycycline: 9-Aminodoxycycline
3 (47.2 mg, 0.10
mmol) and NaHCO3 (21.6 mg, 0.26 mmol) were dissolved in water (5 mL).
After the gas development stopped, EANBP-Chloroformate 4 (36 mg,
0.07 mmol) dissolved in dioxane (5 mL) was added at 0°C. The reaction
mixture was stirred at room temperature for 2 h. The completion of the
reaction was monitored by HPLC analysis. The solvents were removed
under vacuum and the residue was purified by RP-HPLC (C18; using a
mixture of solvent A: TFA (0.1 %) in H2O and solvent B: acetonitrile, 91-
9 %, FR: 4 mL/min-1, tR: 10 min) to afford 25 mg (50 % yield) as a yellow
powder. Analytical HPLC : tR : 19.2 min, purity : > 99 % ; 1H NMR (400
MHz, CDCl3): δ = 7.86 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 2.2 Hz, 1H), 7.46
(m, 3H), 7.15 (d, J = 6.9 Hz), 6.8 (d, J = 6.9 Hz, 1H), 6.78 (d, J = 8.5 Hz,
2H), 3.93 (m, 2H), 3.6 (m, 16H), 3.37 (s, 6H), 3.15 (s, 6H), 1.57 (d, J =
6.9 Hz, 3H), 1.43 (d, J = 7.2 Hz, 3H) ppm.
1
min ; UV : 390 nm ; H NMR (400 MHz, CDCl3): δ = 7.88 (s, 1H), 7.53 (d,
1H, J = 8.8 Hz), 6.61 (dd, 1H, J = 8.8 Hz, 2.4 Hz), 6.5 (s, 1H), 6.34 (s,
1H), 5.35 (d, 1H, J = 9.1 Hz), 4.8 (d, 1H, J = 13.5 Hz), 4.69 (s, 2H), 4.29
4-carbaldehyde-7-diethylamino-coumarin (6): Selenium dioxide (960
mg, 8.6 mmol) was added to
a solution of 7-Diethylamino-4-
(m, 1H), 3.3-3.7 (m, 32H), 3.32 (s, 3H), 1.19 (t, 6H, J = 7.0 Hz) ppm; 13
C
methylcoumarin (1 g, 4.3 mmol) in p-xylene (42 mL). The resulting
mixture was heated at reflux for 16 h until complete consumption of the
starting material and was then filtered while hot to remove black selenium.
The filtrate was concentrated under reduced pressure and the crude
product was purified by column chromatography on silica gel
(heptane/ethylacetate (AcOEt) 8/2 in vol.) to yield 4-carbaldehyde-7-
diethylamino-coumarin (500 mg, 47 %) as an orange oil. 1H NMR (400
MHz, CDCl3): δ = 10 (s, 1H), 8.3 (d, 1H, J = 9.0 Hz), 6.6 (dd, 1H, J = 9.0
Hz, 2.4 Hz), 6.51 (d, 1H, J = 2.4 Hz), 6.43 (s, 1H), 3.4 (q, 4H, J = 7.0 Hz),
1.2 (t, 6H, J = 7.0 Hz) ppm; 13C NMR (400 MHz, CDCl3): δ = 192.5, 161.8,
157.4, 151.0, 143.9, 126.7, 117.2, 109.7, 103.7, 98.3, 44.8, 12.4 ppm.
NMR (400 MHz, CDCl3): δ = 162.7, 156.6, 154.8, 150.9, 145, 124.7,
109.1, 106.2, 105.9, 97.9, 72.1, 70.6, 69.8, 68.9, 64.6, 59.2, 56.1, 44.9,
12.6 ppm; MS (ESI): calculated for C33H52N4O11 : 680.363, found 680.275.
PEG7-DEACM-chloroformiate (12): To
a stirred solution of PEG7-
DEACM 10 (56 mg, 0.08 mmol) in 1.5 mL of anhydrous THF, triphosgene
(26.9 mg, 0.09 mmol) and DIPEA (15 μL, 0.06 mmol) were slowly added
at 0 °C. The reaction mixture was stirred at room temperature for 90 min,
then the solvent was removed by rotary evaporation to yield the targeted
compound as a pale yellow oil. It was used for the next step without
further purification to minimize hydrolytic processes.
7-(diethylamino)-4-(oxiran-2-yl)-2H-chromen-2-one
(7):
4-
carbaldehyde-7-diethylamino-coumarin (236 mg, 0.96 mmol) and
6
PEG7-DEACM-9-aminodoxycycline: 9-Aminodoxycycline 3 (34.8 mg,
0.07 mmol) and NaHCO3 (15.9 mg, 0.07 mmol) were dissolved in water
(1 mL). After gas development stopped, PEG7-DEACM-chloroformiate 11
(56.2 mg, 0.07 mmol) dissolved in dioxane (2 mL) was added at 0 °C.
The reaction mixture was stirred at room temperature for 2 h. The
completion of the reaction was monitored by HPLC analysis. The
solvents were removed under vacuum and the residue was purified by
RP-HPLC (C18 using a mixture of solvent A: TFA (0.1 %) in H2O and
solvent B: acetonitrile, 91-9 %, FR: 4 mL/min-1, tR: 9 min) to afford 35 mg
(40 % yield) as a yellow powder. Analytical HPLC : tR : 17.3 min ; UV :
345, 390 nm ; 1H NMR (400 MHz, CDCl3): δ = 7.8 (s, 1H), 7.49 (d, 1H, J
= 8.5 Hz), 6.72 (d, 1H, J = 8.2 Hz), 6.59 (dd, 1H, J = 8.5 Hz, 2.5 Hz), 6.4
(s, 1H), 6.25 (d, 1H, J = 8.5 Hz),, 5.97 (s, 1H), 4.79 (d, 1H, J = 13.5 Hz),
4.64 (m, 1H), 4.54 (s, 2H), 3.44-3.56 (m, 28H), 3.32 (q, 4H, J = 7.0 Hz),
2.85 (s, 6H), 1.12 (t, 6H, J = 7.0 Hz) ppm; MS (ESI): calculated for
C56H75N7O20: 1165.506, found 1165.328.
cesium carbonate (470 mg, 1.44 mmol) were dissolved in THF (4 mL)
and heated at 60 °C for 10 min. Then a suspension of (carboxymethyl)(4-
cyanophenyl)methylsulfonium (300 mg, 1.44 mmol) (as prepared by
Forbes et al. [16]) in THF (6 mL) was slowly added to the reaction mixture
and the reaction was heated at 60°C for 2 h. The solution was cooled at
room temperature, filtered through celite, concentrated under reduced
pressure, dried over Mg2SO4, and purified by column chromatography on
silica gel (heptane/AcOEt: 7/3 in vol.) to give the targeted compound with
67 % yield (173 mg) as an orange-red oil. 1H NMR (400 MHz, CDCl3): δ
= 7.5 (d, 1H, J = 9.0 Hz), 6.6 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 6.51 (d, 1H, J =
2.4 Hz), 6.02 (s, 1H), 4.04 (m, 1H), 3.4 (q, 4H, J = 7.0 Hz), 3.21 (m, 1H),
2.7 (m, 2H), 1.2 (t, 6H, J = 7.0 Hz) ppm; 13C NMR (400 MHz, CDCl3): δ =
162, 156.2, 151.9, 150.8, 124.6, 108.7, 106.9, 103.9, 97.8, 49.9, 48.6,
44.8, 12.5 ppm; MS (ESI): calculated for C15H17NO3 : 259.121, found
259.013.
4-(2-azido-1-hydroxyethyl)-7-(diethylamino)-2H-chromen-2-one (8):
To a mixture of 7 (173.2 mg, 0.67 mmol) in Ethanol (2 mL) was added
NaN3 (52.11 mg, 0.8 mmol), NH4Cl (36.8 mg, 0.69 mmol), and water (2
mL). The mixture was stirred at reflux for 16 h, then the crude product
was filtrated to remove NaN3 excess and EtOH was removed by rotary
evaporation. H2O (10 mL) was added and the solution was extracted with
Diethyl Ether (Et2O) 3×15 mL). The organic layer was dried over MgSO4
and evaporated under reduced pressure. The resulting oil was purified by
column chromatography on silica gel (Heptane/AcOEt : 3/7 in vol.) to
One photon photolysis
A
solution of EANBP-9-aminodoxycycline or PEG7-DEACM-9-
aminodoxycycline (respectively at 25 and 60 µM) in 10 mM phosphate
buffer (4 mL), pH 7.4 was exposed to a LUMOS 43 LED source (Atlas
Photonics Inc.) at 405 nm (Typical optical output: 200 mW/cm2). The
reaction was monitored by UV and aliquots of samples (100 µL) were
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analyzed by HPLC to determine the percentage of released 9-
aminodoxycycline using a calibration curve (see supporting information).
The quantum yield for the photoconversion was determined in phosphate
buffer (10 mM, pH 7.4) at 25°C by comparison with the photolysis of
DEACM-Gly (F = 0.11)[12c] of the same concentration (50 µM) as
reference. Light (405 ± 0.2 nm) from a 1000 W Hg lamp from Hanovia
was focused on the entrance slit of a monochromator for photolysis.
Aliquots (100 µL) were subjected to reversed-phase HPLC to determine
the extent of the photolytic conversions. Quantum yields were calculated
by considering conversions up to 20 %, to limit as much as possible
errors due to undesired light absorption during photolysis.
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Acknowledgements
This work was supported by the Université de Strasbourg (IDEX
Grant for A.S.) the CNRS and by a Grant from the Agence
Nationale de la Recherche (Contract No. ANR-13-JSJV-0009-01
to A.S. and S. C.)
Keywords: Uncaging • Gene expression • Optopharmacology •
Tet-on system • Two-photon uncaging
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Entry for the Table of Contents
FULL PAPER
Bastien Goegan, Firat Terzi, Frédéric
Bolze, Sidney Cambridge,* and
Alexandre Specht*
Page No. – Page No.
Synthesis and Characterization
of photoactivatable
doxycycline analogues bearing
two-photon sensitive
photoremovable groups
suitable for light induced gene
expression
The development of photolabile, i.e. caged versions of 9-aminodoxycycline, which
photolyse after visible light irradiation or 740 nm two-photon excitation, are reported
herein. The 9-aminodoxycycline is a tetracycline analogue that can be efficiently
coupled by carbamoylation to photoremovable protecting groups, leading to new
caged doxycycline derivatives that can be used in combination with the TetOn
system for photoactivated gene expression.
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