Wavelength-Selective Uncaging of Two Different Photoresponsive Groups on One Effector Molecule for Light-Controlled Activation and Deactivation

Article abstract of DOI:10.1021/jacs.1c02817

Photocleavable protecting groups (PPGs) play a pivotal role in numerous studies. They enable controlled release of small effector molecules to induce biochemical function. The number of PPGs attached to a variety of effector molecules has grown rapidly in recent years satisfying the high demand for new applications. However, until now molecules carrying PPGs have been designed to activate function only in a single direction, namely the release of the effector molecule. Herein, we present the new approach Two-PPGs-One-Molecule (TPOM) that exploits the orthogonal photolysis of two photoprotecting groups to first release the effector molecule and then to modify it to suppress its induced effect. The moiety resembling the tyrosyl side chain of the translation inhibitor puromycin was synthetically modified to the photosensitive ortho-nitrophenylalanine that cyclizes upon near UV-irradiation to an inactive puromycin cinnoline derivative. Additionally, the modified puromycin analog was protected by the thio-coumarylmethyl group as the second PPG. This TPOM strategy allows an initial wavelength-selective activation followed by a second light-induced deactivation. Both photolysis processes were spectroscopically studied in the UV/vis- and IR-region. In combination with quantum-chemical calculations and time-resolved NMR spectroscopy, the photoproducts of both activation and deactivation steps upon illumination were characterized. We further probed the translation inhibition effect of the new synthesized puromycin analog upon light activation/deactivation in a cell-free GFP translation assay. TPOM as a new method for precise triggering activation/deactivation of effector molecules represents a valuable addition for the control of biological processes with light.

Full text of DOI:10.1021/jacs.1c02817

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Article
Wavelength-Selective Uncaging of Two Different Photoresponsive
Groups on One Effector Molecule for Light-Controlled Activation
and Deactivation
Isam Elamri,§ Chahinez Abdellaoui,§ Jasleen Kaur Bains, Katharina Felicitas Hohmann,
Santosh Lakshmi Gande, Elke Stirnal, Josef Wachtveitl,* and Harald Schwalbe*
Cite This: J. Am. Chem. Soc. 2021, 143, 10596−10603
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ABSTRACT: Photocleavable protecting groups (PPGs) play a pivotal
role in numerous studies. They enable controlled release of small
effector molecules to induce biochemical function. The number of PPGs
attached to a variety of effector molecules has grown rapidly in recent
years satisfying the high demand for new applications. However, until
now molecules carrying PPGs have been designed to activate function
only in a single direction, namely the release of the effector molecule.
Herein, we present the new approach Two-PPGs-One-Molecule
(TPOM) that exploits the orthogonal photolysis of two photoprotecting
groups to first release the effector molecule and then to modify it to
suppress its induced effect. The moiety resembling the tyrosyl side chain
of the translation inhibitor puromycin was synthetically modified to the
photosensitive ortho-nitrophenylalanine that cyclizes upon near UV-
irradiation to an inactive puromycin cinnoline derivative. Additionally, the modified puromycin analog was protected by the thio-
coumarylmethyl group as the second PPG. This TPOM strategy allows an initial wavelength-selective activation followed by a
second light-induced deactivation. Both photolysis processes were spectroscopically studied in the UV/vis- and IR-region. In
combination with quantum-chemical calculations and time-resolved NMR spectroscopy, the photoproducts of both activation and
deactivation steps upon illumination were characterized. We further probed the translation inhibition effect of the new synthesized
puromycin analog upon light activation/deactivation in a cell-free GFP translation assay. TPOM as a new method for precise
triggering activation/deactivation of effector molecules represents a valuable addition for the control of biological processes with
light.
INTRODUCTION
■
In recent years, photocleavable protecting groups (PPGs) have
become a powerful tool for light-induced release of effector
molecules in Chemical Biology.1−8 The list of novel “caged”
effector molecules is constantly growing. Besides their
biological function, they satisfy a large number of different
photophysical requirements9 including high quantum yield
φ,10 absorption at λ > 350 nm,11−14 high absorption coefficient
ε,15 rapid uncaging,16,17 applicability for two-photon uncag-
ing,18,19 and wavelength-selective photolysis20 as well as
biocompatibility,21,22 and solubility under physiological con-
ditions,23 cell permeability,24−26 to name a few important
design criteria of PPGs.
Figure 1. Schematic representation of a doubly caged effector
molecule. PPG 1 (orange) is attached to the functional group of the
molecule. A second protecting group is incorporated as a part of the
active structure (blue triangle). Both can be removed selectively upon
orthogonal irradiation (with λ1 and λ2), which activates or deactivates
the effector molecule.
Here, we describe a new strategy that enables the light-
controlled activation and subsequent deactivation of effector
molecules. Figure 1 schematically demonstrates our approach:
in addition to the conventional photocleavable protecting
group (marked in orange), whose photochemical release
activates an effector molecule, the compound contains a
second photoresponsive group (marked in blue), ideally
Received: March 15, 2021
Published: July 8, 2021
© 2021 The Authors. Published by
American Chemical Society
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without affecting the biological activity induced by the effector.
The “uncaged” effector molecule after the first irradiation (λ1)
can subsequently be deactivated by irradiation at a different
wavelength (λ2) as a response of the photoreaction induced by
absorption of the second photocleavable protecting group.
Achieving practical applicability of this process requires a
number of aspects: 1. The first protecting group is able to
suppress the biological activity of the effector molecule. 2. Both
involved PPGs need to have absorption spectra with little
spectral overlap enabling independent, wavelength-selective
photoprocesses. 3. The second PPG attached to the effector
induces a modification that should not induce a significant loss
of effector activity. After removing the second PPG, the
effector molecule has to be inactive; thus, the effector molecule
has to be chemically modified in the second step.
Scheme 1. Schematic Representation of the Strategy of a
Puromycin Analog with Two Photoactivatable Groups
a
To develop the new method TPOM, we selected puromycin
as a naturally occurring antibiotic with widespread application
in biochemical studies. The aminonucleoside puromycin
produced by Streptomyces alboniger is a universal translation
inhibitor of the peptidyl transferase reaction. Furthermore, it is
frequently utilized to visualize newly synthesized proteins.27
Puromycin mimics the phenylalanyl-tRNA (Phe-a-tRNA)
(Scheme 1). It enters the ribosome and incorporates itself
into the growing peptide chain. As amide, the puromycin rest
cannot be released during translation. Antibodies raised against
these conjugates carrying puromycin on the C-terminal are
used to detect nascent chains.28 Puromycin acts codon
independently, does not rely on soluble translation factors,
and does not induce EF-Tu-GTPase activity.29 Interestingly, at
low concentration (e.g., 0.04 μM) puromycin does not act as
an inhibitor, but binds specifically to newly synthesized full-
length proteins at their C-terminus.30 Puromycin acts non-
selectively and exhibits a high toxicity in humans. This high
toxicity has prevented its application as an antibiotic.
Nonetheless, the use of puromycin continues to be of vital
importance for mechanistic studies of gene expression
regulation under physiological and pathological conditions.31
Previously,16,17,32,33 we developed two photolabile puromy-
cin derivatives protected by a single PPG: either by 6-
nitroveratryl oxycarbonyl (NVOC-puromycin) or by 7-N,N-
(diethylaminocumarin-4-yl)-methoxycarbonyl (DEACM-puro-
mycin). Both photosensitive derivatives showed promising
potential for photocontrolled release of puromycin.
a
Top: Structural similarity between 3′-end of a phenylalanyl-tRNA
and the aminonucleoside antibiotic puromycin. Below: (Step 1)
Uncaging of thio-coumarin group and release of active o-nitro-
puromycin upon irradiation at 470 nm. (Step 2) Light-induced photo-
cyclization at 355−365 nm to the inactive cinnoline derivative.
Our new puromycin derivative developed here extends the
use of PPGs to two fundamental processes: the release of
puromycin as an effector by light and the subsequent
deactivation of this effector, again by light. In designing such
a molecule, we came across previous related approaches of
sequential photocleavage of different effectors from multi-
protected substrates. Klan et al. reported a monochromophoric
linker to orthogonally release two different effectors.34
Similarly, Singh et al. developed a novel approach based on
a synthesized o-hydroxycinnamate derivative that enables the
controlled release of alcohol and carboxylic acid.35 Sterner et
al. developed light-sensitive diarylethene-based competitive
inhibitors that upon irradiation with UV light were able to
undergo an electro-cyclization affecting their affinity for the
active site of an allosteric bienzyme complex.36 These previous
approaches could not be transferred to the controlled
activation and deactivation of puromycin, because the
introduced photolabile groups are incompatible for release of
a functional puromycin.
Our approach is based on work by Schultz et al., who
demonstrated a technique in which a protein containing
photosensitive 2-nitrophenylalanine (2-NPA) was expressed
and site-specifically photocleaved.37 The photochemistry of 2-
NPA involves a cinnoline-forming photocyclization after UV-
light illumination. The reported tolerance of puromycin to
different structural modifications38−42 inspired us to modify
the amino acid part O-methyl-L-tyrosine of conventional
puromycin to the photoactivable o-nitrophenylalanine. In
such a derivative, the new photosensitive puromycin analogue
(o-nitro-puromycin II) would structurally resemble the 3′end
of tRNAPhe and contain a photosensitive group (Scheme 1).
Additionally, as is the case with the conventional puromycin,
the photoprotection of the α-amino group of modified o-nitro-
puromycin (II) with a coumarylmethyl derivative as the
carbamate (I) allows control of the release of biologically
active o-nitro-puromycin (II). Simultaneously, the introduced
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o-nitrophenylalanine part enables a different photochemistry
initiating the photocyclization to the nonactive cinnoline
derivative III, due to the lack of the α-amino group.
presence of DEACM an energy transfer to the nitrophenyl part
takes place preventing a selective uncaging of DEACM during
the activation step. Additionally, we observed by light-coupled
NMR experiments that released DEACM-OH (with high ε =
20 800 M−1 cm−1) absorbs the incident light intended for the
photocyclization and undergoes a photo side-reaction to an
unknown byproduct (Figure S15). To circumvent this
disadvantage, we tested other PPG derivatives as the photolysis
efficiency depends strongly on the type of the substituents of
the coumarin scaffold (Figure 2).3,21,43−45 As is apparent from
RESULTS AND DISCUSSION
We synthesized thio-DEACM-nitro-puromycin (I) in nine
steps (Scheme 2). Starting from 7-amino-4-methyl coumarin,
■
Scheme 2. Synthesis of Caged thio-DEACM-ortho-nitro-
a
puromycin I
Figure 2. Main absorption bands of different coumarin derivatives in
1:1 acetonitrile/water mixtures. From left to right: BhCM (black, λmax
= 327 nm), DBMAC and BMAC (purple and blue, λmax = 353 nm),
DCMAC (cyan, λmax = 357 nm), DEACM (green, λmax = 380 nm),
BDEACM (orange, λmax = 406 nm), mc-DBMAC (red, λmax = 425
nm/441 nm), thio-DEACM (dark red, λmax = 470 nm). Correspond-
ing synthesis procedures and characterizations of 4-hydroxymethyl-
coumarin derivatives are presented in the Supporting Information (pp
S10−S11).
a
(a) 7-Diethylamino-4-methylcoumarin 1 (1 equiv), DMF−DMA (2
equiv), DMF, 160°C, 7 h; (b) 2 (1 equiv), NaIO4 (3 equiv), THF/
H2O 1:1, rt, 5 h; (c) 3 (1 equiv), NaBH4 (1.5 equiv), EtOH, rt, 4 h,
98%; (d) 4 acetic acid, DCC, DMAP, CH2Cl2, reflux, 4 h; (e) 5
Lawesson′s reagent, toluene, reflux, 24 h, (f) 6 1.25 M HCl in ethanol,
reflux, 15 h; (g) 7 (1 equiv), DMAP (2 equiv), 4-NO2Ph-
chloroformate (2 equiv), CH2Cl2, rt, 7 h; (h) 8 (1 equiv) 2-nitro-
phenylalanine.HCl (1 equiv), DMAP (2 equiv), DIPEA (20 equiv),
CH2Cl2, rt, 24 h; (i) 9 (1 equiv), EDCI (1 equiv), NHS (1 equiv),
Puromycin aminonucleoside (1 equiv), DMF, 0°−rt, 24 h. Synthesis
procedure and characterization of thio-DEACM-nitro-puromycin
(I,10) are presented in the Supporting Information (pp S2−S20).
the UV spectra, diethylamino-thiocoumarylmethyl (thio-
DEACM) combines the advantages of the different derivatives
as it features a diethylamino group on position 7 and a strong
electron-withdrawing group at position 2. Incorporation of
thio-DEACM would thus enable an excitation of the first PPG
to yield an activation of the thio-DEACM-nitro-puromycin (I)
with visible light. Of particular importance for the on/off
functionality is its absorption gap around 370 nm (Figure 2,
red area), which is advantageous in view of the deactivation
step.
Therefore, thio-coumarin alcohol 7 was synthesized as
follows (Scheme 2): Alcohol 4 was converted to ester 5 with
acetic acid catalyzed by DCC and subsequently thionylated by
Lawesson’s reagent.46 6 was hydrolyzed providing the desired
alcohol 7. Alcohol 7 was further activated by conversion to the
nitro-phenyl carbonate 8 as an intermediate, which then reacts
without further purification with the commercially available 2-
nitrophenylalanine to yield carbamate 9. In the last step, the
obtained carbamate 9 was treated with puromycin-amino-
nucleoside in the presence of the coupling reagents EDCI and
we prepared the alcohol DEACM-OH 4 by condensation of
coumarin 1 to the enamine 2 followed by treatment with
sodium periodate to the aldehyde 3 which was subjected to the
reduction using sodium borohydride to yield DEACM-alcohol
4.17 Using DEACM as one of the two photolabile groups in the
TPOM strategy posed, however, a challenging obstacle exists
for the intended photocyclization due to the high absorption of
4 that is released as a byproduct together with the effector
molecule o-nitro-puromycin (II) (Figures S24 and S25). The
attempt to generate the selectivity by applying single- and two-
photon-induced uncaging of the nitrophenyl part and
DEACM, respectively, remained unsuccessful, despite the
large difference in the two-photon cross section. In the
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NHS to yield doubly caged thio-DEACM-nitro-puromycin (I)
in 43% yield after reversed-phase chromatography.
355 nm, as the only photolabile fragment of o-nitro-puromycin
(II), led also to a cyclized cinnoline without a carboxylic acid
group. Contrary, the same laser irradiation of conventional
phenylalanine without the nitro group resulted in no structural
changes confirming the photoreaction of the 2-nitrophenyla-
lanine part, excluding any photodegradation of the amino acid
itself (Figure S12).
The photophysical properties of the thio-coumarylmethyl
and 2-nitrophenylalanine groups of the respective photocages
were examined. In search of a deactivation wavelength that is
within the absorption gap of thio-DEACM, several wavelengths
between 280 and 450 nm were tested for the photocyclization
of o-nitro-puromycin (II) in separate illumination experiments,
although o-nitro-puromycin (II) seems to barely absorb
beyond 300 nm (Figure 4, green line). DFT-calculated UV/
Since the modified puromycin analogue (II) varies from
different reported nitrobenzyl scaffolds,47,48 we decided to first
verify the photosensitivity of the o-nitro-puromycin (II) by
examining the proposed cyclization reaction by real-time laser-
NMR.49 We synthesized o-nitro-puromycin (II) as described
in the Supporting Information (pp S7−S9). The laser-coupled
1H NMR experiment showed that irradiation at 488 nm does
not affect o-nitro-puromycin (II), which is favorable for the
planned wavelength-selective uncaging of thio-DEACM
(Figure S12) that should occur before the cyclization step.
In the case of 355 nm laser irradiation, most signals stemming
from the nitrophenyl ring and the amino nucleoside part of the
illuminated compound (II) decayed exponentially (Figure 3a).
Figure 4. Absorbance spectra of o-nitro-puromycin (II) during
continuous illumination at λd = 340 nm in 1:1 acetonitrile/water
mixture before (green), after 5 min (gray line), and after 2.8 h (red)
of illumination resulting in cinnoline product III. The arrows
underline the direction of absorption change during the first 5 min
of illumination in gray and during the following 2.7 h in red.
vis absorption spectra of o-nitro-puromycin (II) and its
cinnoline photoproduct III show that successful uncaging is
achievable with wavelengths up to 420 nm (Figure S20a). An
activation wavelength λa = 470 nm was applied to remove the
thio-DEACM, while deactivation was performed at λd
=
365 nm. Both the activation and deactivation wavelengths are
within the spectral window suitable for biological applications.
The cyclization of o-nitro-puromycin (II) without thio-
DEACM attached is shown in Figure 4. Based on DFT
calculations the emerging absorption band at 240 nm can be
assigned as the main absorption band of the cinnoline
photoproduct (Figure S20a). The deactivation step of o-
nitro-puromycin (II) successfully proceeds via an intermediate
to a cinnoline photoproduct. Considering the NMR results,
further decomposition reactions can be explained by the higher
illumination power compared to the UV/vis experiments.
During the activation step, the absorption bands of the thio-
DEACM 7 part of I at 474 and 250 nm disappear (Figure 5a,
blue spectrum compared to green spectrum) (Figure S16). A
residual band at 271 nm (Figure 5a, green solid line) can be
assigned to the desired uncaging product o-nitro-puromycin
(II) (Figure 5a, green area), indicating a successful activation
of the compound. Upon thio-DEACM cleavage, the alcohol
derivative thio-DEACM-OH is formed, in agreement with a
previous report.50 This particular thio-coumarin chromophore
undergoes further photolysis upon 470 nm illumination, which
yields DEACM-OH, but additional photoproducts are
possible.46 Irradiating pure thio-DEACM-OH at the activation
wavelength in a separate experiment (Figures S17 and S18a)
confirms the presence of DEACM-OH and another photolysis
product absorbing around 370 nm. The latter was identified by
Figure 3. Laser-coupled NMR experiment for tracking the photolysis
reaction of o-nitro-puromycin (II). Estimated Light power: 1 W.
NMR-sample: 500 μM in HEPES/D2O (10%), 355 nm. 1H-spectrum
1
measured after every 0.5 s irradiation. (a and b) Time-resolved H-
spectra and normalized peak integrals of H1′ and H2 corresponding
to the two compounds showing the decay of o-nitro-puromycin (II)
and the accumulation of the photoproducts III and IV. (c) Spectra
after HPLC purification of irradiated sample of o-nitro-puromycin II
showing: (d) photocyclized puromycin-cinnoline III and the release
of aminonucleoside puromycin IV as final photolysis step in
comparison with the initial structure before irradiation of o-nitro-
puromycin (II).
This decay was accompanied by a corresponding rise of new
photoproduct signals. As an example, Figure 3b shows the
exponential signal decay at 5.82 and 8.12 ppm and a signal
increase at 6.10 and 8.30 ppm, respectively. The new signals
could be assigned to the expected cinnoline derivative III.
Interestingly, an HPLC-purification of an irradiated sample
containing all photoproducts showed a subsequent photo-
induced cleavage of the cinnoline derivative III to the
nucleoside and amino acid moieties as a consecutive reaction
of derivative III (Figure 3c and 3d). Noteworthy, controlled
irradiation of the amino acid part 2-nitrophenylalanine at
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Figure 5. (a) Absorption spectra of activation in 1:1 acetonitrile/
water mixture. Thio-DEACM-nitro-puromycin (I) before (blue line)
and after 14 min illumination at 470 nm (green line), which is
identical to the (2x(7):1x(II)) weighted overlay (black dotted line) of
the synthesized o-nitro-puromycin (II) (green area) and the
illuminated thio-DEACM (7) (gray area). (b) Difference IR-spectra
measured every 60 s in DMSO during continuous illumination of (I)
at the activation wavelength (470 nm) evolving from the black (zero
line: no illumination of the starting compound (I)) to the green line
(photoproduct of the activation step: o-nitro-puromycin (II)). DFT-
calculated vibrational modes as sticks of thio-DEACM-nitro-
puromycin (I) (circles) and o-nitro-puromycin (II) (square). (c)
Determination of quantum yield by light induced absorption changes
monitoring the photoinduced release of CO2 upon irradiation of (I)
at 2337 cm−1 with a fit through the first 8 data points (500 s of
illumination) (p S30).
Figure 6. (a) UV/vis spectroscopic study of deactivation in 1:1
acetonitrile/water mixture. Starting material is the activated sample
including side products (green line). After illumination at 365 nm for
1 h a spectrum (red line) arises, which can be reconstructed by the
overlay (black dotted line) of illuminated o-nitro-puromycin (II) (red
area) and the illuminated thio-DEACM with 365 nm for 1 h (gray
area). (b) Difference IR-spectra measured every 60 s in DMSO of o-
nitro-puromycin (II) including side product with illumination at
365 nm evolving from the black (zero line: no illumination of the
sample) to the red line (photoproducts of the deactivation step:
including cinnoline product III). DFT-calculated vibrational modes as
sticks of o-nitro-puromycin (II) (squares) and cinnoline product III
(triangles).
that the thio-DEACM side products absorb at the deactivation
wavelength (λd), the UV/vis experiments unambiguously show
that the deactivation step is not inhibited. Comparing the two
mass spectrometry (Figure S14) and NMR spectroscopy. The
results indicate the formation of a coumarylmethyl hydrogen
uncaging quantum yields (φo‑nitro‑puro/cinnoline = 2.5%,
−
−
sulfate derivative DEACM-OSO3 as the major photoproduct
φDEACM‑OSO3
= 0.1%) reveals that the deactivation
/DEACM−OH
of photolyzed thio-DEACM-OH.
of nitro-puromycin is favored over the photolysis of DEACM-
OSO3 (Figures S18b and S20b, approximately 33%
−
Since the absorption spectrum of the doubly caged
compound (I) is in principle an additive superposition of the
individual components, with slightly red-shifted absorption
bands (Figure S16), the uncaging products are hardly
distinguishable in the UV/vis region. IR-experiments after
illumination reveal, however, absorption changes that clearly
confirm uncaging (Figure 5b). The newly appearing positive
absorption band at 2337 cm−1 in combination with the
vanishing band at 1728 cm−1 demonstrate the release of CO2
from the carbamate linker during illumination taking place in
the last step of uncaging.37 Two other prominent absorption
changes can be observed for IR bands at 1579 and 1522 cm−1
characteristic for ring vibrations of the coumarin moiety,
confirming its photolysis. Compared to the reported DEACM-
puromycin17 the uncaging wavelength could successfully be
red-shifted by about 100 nm without a significant loss of
quantum yield (1.7%, Figure S18; 69% conversion within
2 min of light exposure: 2 × 1016 photons/s). In combination
with the high extinction coefficient ε474 nm = 17464 M−1 cm−1
this results in a reasonable uncaging cross section φ·ε =
297 M−1 cm−1.
deactivated within 3 min of illumination: 5 × 1015 photons/
s). Further changes of the absorption around 370 nm confirm a
subsequent side reaction of the thio-coumarin photoproduct.
The remaining band is assigned to DEACM-OH (Scheme 4).
Additionally, the loss of the nitro-functionality of the oNB
moiety at 1390 cm−1 and the symmetric stretch mode of the
amino group at 3516 cm−1 confirm the deactivation of the
central functional group of puromycin (Figure 6b). The
formation of the cyclization product can be verified by the
positive bands at 1139 and 1165 cm−1 that can be assigned to
vibrational modes of the cinnoline moiety including the NN
double bond in accordance to DFT calculated modes (Figure
6b). For the presumed deactivation mechanism, water has to
be eliminated in the last step, which is supported by the
positive absorption change at 3569 cm−1. A double uncaging
can be carried out directly with 365 nm (Figures S22 and S23).
Analysis of the absorption changes during the first 8 min
reveals that both the activation and deactivation step occur
simultaneously to a certain extent.
We used the newly developed TPOM puromycin to control
protein synthesis in a cell-free coupled transcription−trans-
lation system (Figure 7). The expression inhibition induced by
the released o-nitro-derivative of puromycin was monitored
using shifted green fluorescent protein (sGFP) in a microplate
reader. We monitored the sGFP expression as a function of
increased concentrations of puromycin (Figure S27a) and o-
Figure 6 shows the deactivation step of a sample previously
activated with 470 nm light. It contains the side products of the
first uncaging step. Upon exposure to 365 nm the absorption
band in the UV-region undergoes a bathochromic shift, which
is indicative for the formation of a cinnoline product as known
from DFT-calculations (Figure S20a). Regardless of the fact
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a
Scheme 4. Reaction Sequence Summary
a
Doubly caged Puromycin (thio-DEACM-nitro-puromycin I) is inactivated by thio-DEACM. After illumination at 470 nm the coumarin cage is
removed, while the biologically active o-nitro-puromycin (II) is regenerated (activation step: green area). In a parallel pathway, two other
−
photoproducts are produced as byproducts after photolysis of thio-DEACM: DEACM-OH and DEACM-OSO3 . In the deactivation pathway, o-
nitro-puromycin (II) undergoes a photocyclization, resulting in a cinnoline product III, which is inactive due to the missing amino group
(deactivation step: red area). Furthermore, the cinnoline moiety splits from the remaining puromycin scaffold. In this reaction route, the byproduct
−
DEACM-OSO3 from the first step vanishes, leaving only the DEACM-OH behind.
since sGFP expression levels were comparable to those without
any inhibitor addition. This and the effect of illumination at
488 and 365 nm were examined by using native PAGE analysis
(Figure 7b), since thio-DEACM and sGFP absorption curves
overlap justifying the fluorescence decay of this sample (Figure
S27b).
In the next step, upon laser irradiation at 488 nm thio-
DEACM was cleaved releasing o-nitro-puromycin (II) that
inhibits almost 80% of the initial sGFP expression. In contrast,
thio-DEACM-nitro-puromycin (I) and o-nitro-puromycin (II)
which were deactivated by a 365 nm and 488/365 nm
irradiation, respectively, did not show inhibition of the sGFP
expression (Figures 7b and S27b). These results are in
accordance with our above-mentioned spectroscopic and
analytical investigations. Thus, thio-DEACM-nitro-puromycin
(I) shows no inhibition effect and can be activated through
488 nm irradiation releasing the active o-nitro-puromycin (II).
This compound can be again deactivated by a subsequent
irradiation at 365 nm releasing the photocyclized cinnoline
puromycin derivative. The same observation was made, when
thio-DEACM-nitro-puromycin I was illuminated at 365 nm
directly (Figure 7b and c). An obvious direct interpretation is
that at 365 nm the cleavage of thio-DEACM and the
cyclization to the cinnoline ring are simultaneously occurring
events.
Figure 7. (a) Inhibition of sGFP expression with increasing o-nitro-
puromycin (II) concentration. 1 mM o-nitro-puromycin (II) results
in a 3-fold inhibition of sGFP expression. (b and c) thio-DEACM-
nitro-puromycin (I) was illuminated, respectively with LEDs at
365 nm, 365/488 nm, and 488 nm, and the effects of the illumination
wavelength were tested by native PAGE analysis of sGFP expression
and compared with non-illuminated thio-DEACM-nitro-puromycin
(I) (w/o illumination) and sGFP control sample without addition of
inhibitor (sGFP control).
CONCLUSION
■
Here, we designed and probed TPOM as a new method to
release and then destroy the function of a low molecular weight
effector molecule in a biological system. A part of the active
structure of the target molecule is synthetically modified to a
second photoresponsive scaffold that undergoes a structural
change to one or more inactive molecules upon wavelength
selective irradiation. To demonstrate this dual function, we
synthesized thio-DEACM-nitro-puromycin (I), a double
photoresponsive analog of the frequently used aminonucleo-
side antibiotic puromycin. To achieve the required chromatic
nitro-puromycin analog (II) concentration, resulting in a 5-
fold and 3-fold inhibition, respectively (Figure 7a). Apparently,
the structural modification of conventional puromycin by
introducing the NO2-group and by removing the para-methoxy
group diminishes the inhibition activity of (sGFP) translation.
However, the deactivation by protecting the primary α-amino
group with thio-DEACM to the carbamate (I) was successful,
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J. Am. Chem. Soc. 2021, 143, 10596−10603

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
orthogonality, several 4-hydroxycoumarin derivatives were
synthesized and screened. To report the successfully photo-
cyclization of o-nitro-puromycin II, a laser-coupled H NMR
Elke Stirnal − Institute of Organic Chemistry and Chemical
Biology, Goethe-University Frankfurt, Frankfurt am Main
60438, Germany
1
experiment was performed showing a light-induced formation
of the cinnoline derivative followed by the release of
puromycin aminonucleoside (Scheme 4). The successful
activation/deactivation upon blue light uncaging of thio-
DEACM and subsequent near-UV-induced photocyclization
of o-nitro-puromycin analog II were confirmed by IR-
measurements and DFT calculations. Furthermore, steady-
state spectroscopy revealed a side path of coumarin by-
products. Finally, with in vitro transcription−translation
experiments we examined the controlling ability of the
translation inhibition effect of thio-DEACM-nitro-puromycin
I. The expression inhibition was monitored using shifted green
fluorescent protein (sGFP). The cleavage of thio-DEACM
released the active o-nitro-puromycin II, which can be in turn
deactivated by irradiating with λd. Moreover, direct deactiva-
tion of thio-DEACM-nitro-puromycin I was also observed. The
introduction of the Two-PPGs-One-Molecule method for
restricting the activity of target biomolecules is a promising
strategy and a significant addition to the continuously growing
toolbox of spatiotemporally photocontrolled elements for
biological applications.
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c02817
Author Contributions
§I.E. and C.A. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Anna Wacker, Dr. Jan Ferner and Christoph
Kaiser for helpful discussions. This work has been supported
by GRK 1986 “CLiC” and by the DFG large scale equipment
programme for the purchase of lasers coupled to NMR
spectrometers (project number: 280251937). Work at BMRZ
is supported by the state of Hesse.
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c02817.
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Corresponding Authors
Josef Wachtveitl − Institute of Physical and Theoretical
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Email: wveitl@theochem.uni-frankfurt.de
Harald Schwalbe − Institute of Organic Chemistry and
Chemical Biology, Goethe-University Frankfurt, Frankfurt
am Main 60438, Germany; orcid.org/0000-0001-5693-
7909; Email: schwalbe@nmr.uni-frankfurt.de
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Isam Elamri − Institute of Organic Chemistry and Chemical
Biology, Goethe-University Frankfurt, Frankfurt am Main
60438, Germany
Chahinez Abdellaoui − Institute of Physical and Theoretical
Chemistry, Goethe-University Frankfurt, Frankfurt am Main
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Jasleen Kaur Bains − Institute of Organic Chemistry and
Chemical Biology, Goethe-University Frankfurt, Frankfurt
am Main 60438, Germany
Katharina Felicitas Hohmann − Institute of Organic
Chemistry and Chemical Biology, Goethe-University
Frankfurt, Frankfurt am Main 60438, Germany
Santosh Lakshmi Gande − Institute of Organic Chemistry
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