Design of photocaged puromycin for nascent polypeptide release and spatiotemporal monitoring of translation

Article abstract of DOI:10.1002/anie.201410940

The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC-puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome-bound polypeptide chains was detected invitro. Upon illumination, cytotoxic activity, invitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC-puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2 % and the NVOC-puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two-photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons. On like a light: The antibiotic puromycin (green) is a translation inhibitor that triggers the release of the nascent polypeptide chain (red) from the ribosome (yellow) and it is used in a number of applications. A photocaged puromycin derivative, NVOC-puromycin, was synthesized and characterized. Both functional recovery upon UV illumination and biological inactivity invitro and invivo were demonstrated.

Full text of DOI:10.1002/anie.201410940

Angewandte
Chemie
DOI: 10.1002/anie.201410940
Photoactivation
Design of Photocaged Puromycin for Nascent Polypeptide Release and
Spatiotemporal Monitoring of Translation**
Florian Buhr, Jçrg Kohl-Landgraf, Susanne tom Dieck, Cyril Hanus, Deep Chatterjee,
Andreas Hegelein, Erin M. Schuman, Josef Wachtveitl, and Harald Schwalbe*
Abstract: The antibiotic puromycin, which inhibits protein
translation, is used in a broad range of biochemical applica-
tions. The synthesis, characterization, and biological applica-
tions of NVOC-puromycin, a photocaged derivative that is
activated by UV illumination, are presented. The caged
compound had no effect either on prokaryotic or eukaryotic
translation or on the viability of HEK 293 cells. Furthermore,
no significant release of ribosome-bound polypeptide chains
was detected in vitro. Upon illumination, cytotoxic activity,
in vitro translation inhibition, and polypeptide release trig-
gered by the uncaging of NVOC-puromycin were equivalent to
those of the commercial compound. The quantum yield of
photolysis was determined to be 1.1 Æ 0.2% and the NVOC-
puromycin was applied to the detection of newly translated
proteins with remarkable spatiotemporal resolution by using
two-photon laser excitation, puromycin immunohistochemis-
try, and imaging in rat hippocampal neurons.
formed peptidyl-puromycin is resistant to cleavage by the
PTC, thus resulting in premature chain termination and
subsequent release from the ribosome on a subsecond time-
scale.[7–9]
Puromycin and a number of synthetic derivatives have
been used as PTC substrate analogues to gain insights into
peptide bond formation.[8–15] Unlike other methods for
nascent polypeptide release, such as treatment with
RNase A/EDTA or NaOH, the use of puromycin does not
cause ribosomal particles to disintegrate and nor does it
induce the formation of insoluble aggregates.[7] In many
experiments, nascent polypeptide chain release is induced by
puromycin,[16] and at low concentrations, it can be used to
identify translating ribosomes,[17] to purify newly synthesized
polypeptide chains and associated chaperones,[18] or to
monitor translation. Chemically modified versions of puro-
mycin are common as well. In mRNA display, covalently
bound puromycin facilitates the attachment of an mRNA
progenitor to its respective peptide product[19] and in TRAP
display,[20] a puromycin-coupled DNA linker is hybridized
with mRNAs, while fluorescent puromycin conjugates have
been used to identify nascent polypeptide chains in vivo.[17]
More recently, biotinylated puromycin was used for puromy-
cin-associated nascent chain proteomics (PUNCH-P) to
monitor translation on a proteomic scale.[21]
In order to allow spatiotemporal control of the application
of puromycin for biophysical and cellular studies, a photo-
caged derivative of puromycin was synthesized and charac-
terized. The principal utility of photocaged antibiotics as
light-activated effectors has been demonstrated previously.
Photocaged rapamycin has been used to induce activity of the
small GTPase Rac in a cellular context[22] and photocaged
anisomycin has been used to locally inhibit protein syn-
thesis.[23] In the case of puromycin, however, the scope of its
biological applications is much wider owing to its unique
mode of action and would be further extended by biologically
compatible light-activation. The well-established 6-nitrover-
atryloxycarbonyl (NVOC) photolabile protecting group,[24–26]
which was used in this study, meets critical requirements for
compatibility with biological systems: sufficient water sol-
ubility, low cytotoxicity, high dark stability, and moderate
ambient light tolerance, as well as an uncaging wavelength
beyond the absorption range of common biomolecules.
NVOC-puromycin 2 (Figure 1) was obtained in 59% yield
in a one-step synthesis followed by reversed-phase HPLC
purification. Dissolved in DMSO or H2O, the caged com-
pound is stable in the dark for at least 48 h at room
temperature or for at least 6 months at À208C. Although
the protection of its protonatable amino group dramatically
T
he antibiotic agent puromycin (1), which was first isolated
from the gram-positive bacterium Streptomyces alboniger, is
a well-studied inhibitor of translation with a wide variety of
biochemical applications.[1] Puromycin enters the ribosomal
A site during translation, where it acts as a codon-unspecific
competitor of aminoacyl-tRNA as a result of its structural
similarity to the 3’ end of tyrosyl-tRNA.[2,3] Upon nucleophilic
attack, the C terminus of the nascent polypeptide chain is
transferred from the 3’ end of the peptidyl-tRNA located at
the ribosomal P-site near the peptidyl transferase center
(PTC) to the amino group of puromycin.[4–6] The newly
[*] F. Buhr, D. Chatterjee, A. Hegelein, Prof. Dr. H. Schwalbe
Center for Biomolecular Magnetic Resonance
Institute of Organic Chemistry and Chemical Biology
Goethe-Universitꢀt Frankfurt am Main
Max-von-Laue-Straße 7, 60438 Frankfurt am Main (Germany)
E-mail: schwalbe@nmr.uni-frankfurt.de
J. Kohl-Landgraf, Prof. Dr. J. Wachtveitl
Institute of Physical and Theoretical Chemistry
Goethe-Universitꢀt Frankfurt am Main
Max-von-Laue-Straße 7, 60438 Frankfurt am Main (Germany)
S. tom Dieck, C. Hanus, Prof. Dr. E. M. Schuman
Department of Synaptic Plasticity, MPI for Brain Research
Max-von-Laue-Straße 4, 60438 Frankfurt am Main (Germany)
[**] We thank E. Stirnal, B. Fꢁrtig, F. Sochor, M. Strumpf, R. Silvers and
C. Richter for support. This work was funded by the DFG in
Collaborative Research Center SFB807 and the HFSP. H.S. and J.W.
are members of the cluster of excellence: Macromolecular Com-
plexes.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410940.
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Figure 1. Constitution and uncaging of NVOC-puromycin. The nitro-
veratryloxycarbonyl protecting group is shown in red. During uncaging,
puromycin is released by decarboxylation, with formation of the final
nitroso caging remnant 3.
reduces polarity, the remaining water solubility of 2.5 mm
(208C) exceeds the required concentrations in cell culture
selection media by three orders of magnitude.[27,28] Upon UV
illumination above 320 nm, puromycin was recovered as
1
confirmed by H NMR spectroscopy and mass spectrometry.
Optimization for biochemical applications requires the pho-
tochemical characterization of the uncaging reaction, in which
quantum yield is the most critical parameter since it reflects
the expected amount of released puromycin after a given
illumination time. To determine the quantum yield, we
monitored the development of the nitroso absorption under
continuous UV irradiation (Figure 1, Figure 2a). To simplify
analysis, we only took into account the first 120 seconds of this
experiment, during which the exponential behavior can be
approximated by a linear function (Figure 2b). In this case,
we can assume that all photons are absorbed only by the
reactant and not by the product since the amount of product
formed within the investigated time range is negligible.
Furthermore, the absorbance change around 410 nm is
caused by the product only and can thus be directly used to
determine the number of total photoreactions. With these
considerations, we obtained a quantum yield of 1.1 Æ 0.2%.
The results of a second approach, which was based on the IR
absorption of soluble CO2, are presented in the Supporting
Information, along with the derivation of both methods.
Although a number of different caging groups are
conceivable, we decided to introduce the 6-nitroveratryloxy-
carbonyl group. For NVOC-puromycin, the electron-donating
substitutions necessary to shift the absorption to a biologically
compatible wavelength result in a reduction in quantum
efficiency via a nonreactive triplet-state trap in the uncaging
mechanism.[29] Despite this drawback, the NVOC group has
notable advantages over more recently established photo-
cages with superior photochemical characteristics. NVOC
chloride is commercially available and can be used directly in
a one-pot synthesis, whereas there is currently no commercial
access to the widely used coumarin-based chloroformiates.
Additionally, since the uncaging reaction proceeds through
a singlet pathway,[29] the absence of radical intermediates
might be beneficial for applications in vivo, where the
quenching of reactive triplet states by cellular radicals
should be avoided. To determine the available concentration
Figure 2. Quantum yield and uncaging kinetics determined by absorp-
tion of the nitroso caging remnant. a) Lower panel: Absorption spectra
of the caged compound (black curve) and after various irradiation
intervals with an intensity of 7 mW at 365 nm (gray curves). Upper
panel: Difference spectrum of the sample after 3000 s of continuous
irradiation. b) Absorbance at 410 nm during continuous UV irradiation
on a logarithmic time scale. The inset shows the linear time range that
was used for the fit. In both graphs, the thin grey curve shows the
linear fit curve.
of uncaged puromycin after increasing periods of illumination
in a practical setup of interest, an interval series of laser-
coupled 1H NMR experiments[30] was performed, in which
a molar excess of free puromycin with respect to common
antibiotic concentrations or concentrated prokaryotic ribo-
somes was reached on a low second timescale (see the
Supporting Information).
To assess the deactivation and recovery of the cytotoxic
effect, cultures of human embryonic kidney (HEK) 293 cells
were incubated with caged, uncaged, or commercial puromy-
cin. Uncaging was achieved by illumination with a 365 nm
LED, operated at 200 mW. After 24 h of incubation at 378C,
an XTT cell viability assay was performed to evaluate the
number of viable cells. The assay exploits the activity of
mitochondrial dehydrogenase since this correlates with the
number of metabolically active cells. The results for the caged
compound did not differ from those obtained with negative
controls, thus showing that NVOC-puromycin has no signifi-
cant toxicity and remains stable under cell culture conditions
(Figure 3a). However, the effect of previously illuminated
NVOC-puromycin was equivalent to that of the commercial
compound, corresponding to a complete recovery of cytotoxic
function after uncaging. In a further experiment, cell culture
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Figure 3. XTT cell viability assays with treated HEK 293 cells. a) HEK
293 cells treated with 6.3 mM NVOC-puromycin (NP), uncaged pur-
omycin (UP), or commercial puromycin (Puro) were incubated at 378C
for 24 h. Control experiments contained no additive or 0.1% DMSO.
Error bars represent one standard deviation and were calculated from
ten parallel experiments performed for each data point. The p-value
refers to all pairs of Control/DMSO/CP vs. UP/Puro. b) Filled circles:
HEK 293 cells were treated with 6.3 mM NVOC-puromycin and aliquots
were illuminated at 365 nm for increasing periods of time. All samples
were incubated at 378C for 24 h before the XTT assay was performed.
Empty circles: Negative control without NVOC-puromycin. All illumi-
nation experiments were performed in triplicate.
Figure 4. Firefly luciferase expression assays. a) Expression in two
different in vitro systems, where luciferase activity after 1 h of incuba-
tion with 50 mm caged, uncaged, or commercial puromycin was
compared. b) Expression in a reconstituted E. coli system lacking
release factors (black) or with a DNA template lacking a stop codon in
a rabbit reticulocyte system (gray). c) In a time-dependent interval
experiment, samples were incubated with NVOC-puromycin (black and
pale gray) or with no added compound (dark grey) as a control.
Luciferase activity was detected in samples taken every 5 min until
30 min, at which point the cell-free reactions treated with NVOC-
puromycin were illuminated at 365 nm for 1 min (black arrow) and
puromycin was added to the control samples. Error bars represent one
standard deviation and were calculated from triplicate measurements
for each data point.
aliquots with and without previous addition of the caged
compound were illuminated for increasing periods of time. As
shown in Figure 3b, the addition of NVOC-puromycin causes
cell viability measured after 24 h to decay exponentially with
illumination time. While no net decay caused by UVexposure
alone was detected after up to 30 s (Figure 3), 95 Æ 1% of the
maximum effect was achieved in the same period with
NVOC-puromycin. A time constant (t) of 9.7 Æ 0.6 s was
obtained under the given experimental conditions, which
corresponds to 25 Æ 1% of the maximum effect observed in
less than 3 s. This result implies that with an easily accessible
tenfold increase in initial concentration (63 mm), full cytotoxic
efficiency is achieved after 1 s of illumination. Since an initial
increase in cell viability is observed in the negative control,
UV illumination seems to have multiple effects. Cytotoxicity
however, was shown to be negligible in the given time frame.
To demonstrate the inhibition of in vitro translation,
firefly luciferase expression assays were performed by using
an E. coli extract based expression system and a rabbit
reticulocyte cell-free expression system. The addition of
50 mm of uncaged NVOC-puromycin or commercial puromy-
cin completely inhibits translation (Figure 4a), while the
caged compound shows no inhibitory effect compared to the
DMSO control. In the early days of co-translational protein
folding research, it was concluded that nascent polypeptide
chains of firefly luciferase had to be partially folded, but were
catalytically inactive while ribosome-bound.[34] This feature
was exploited in a modified luciferase assay, in which arrested
ribosomes with inactive luciferase chains covalently attached
to the tRNA at the P-site were generated. Puromycin-
mediated release of the nascent chain thus induces an
amount of enzyme activity equivalent to a single round of
translation with the excess puromycin preventing further
translation. Experiments were performed using two different
expression systems. First, a PCR-generated run-off template
was expressed in an E. coli system reconstituted from purified
components and lacking release factors. Second, a truncated
template without a stop codon was expressed in a rabbit
reticulocyte system. Figure 4b shows normalized luciferase
activity of the expressed constructs before (control) and after
the addition of caged, uncaged, or commercial puromycin.
The base level of activity caused by leak release is exceeded
four- to five-fold by puromycin-mediated release of the
ribosome-bound population. The difference observed
between the effects of commercial versus uncaged puromycin
are within experimental error, although it is conceivable that
for the caged compound, adjusted starting concentrations
may be required to recover an equivalent effect by uncaging,
especially if illumination time is limiting. In a further experi-
ment, luciferase activity was measured every 5 min during the
expression of ribosome-bound luciferase with added NVOC-
puromycin. After 30 min, reactions were illuminated for
1 min and three more data points were measured. In a control
reaction with no added compound, commercial puromycin
(50 mm) was introduced at the 30 min time point. A minor but
steady increase in enzyme activity is observed before
illumination because of leak release (Figure 4c). In all three
cases, the luciferase-activating effect of puromycin-mediated
release from the ribosome after illumination or puromycin
addition at 30 min was comparable, with the differences being
within experimental error. A significant difference between
the uncaging of the 50 mm and 200 mm NVOC-puromycin
reaction was only detected at the first data point after
uncaging (35 min), thus suggesting slow release kinetics as
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a result of insufficient concentra-
tions of the free compound. Since
the concentration of ribosomes in
cell-free extracts does not exceed
the lower mm range, a molar
excess of uncaged puromycin
accumulates rapidly upon illumi-
nation, given sufficient starting
concentrations and illumination
conditions.
At low concentrations, puro-
mycin has previously been used
to monitor protein transla-
tion.[17,32,33] In the next series of
experiments, we aimed to intro-
duce spatiotemporal control to
the puromycin-mediated moni-
toring of newly translated pro-
teins. First, we showed illumina-
tion dependence of nascent chain
puromycylation in cultures of rat
hippocampal neurons when incu-
bated with 3 mm NVOC-puromy-
cin, both by Western blotting
(Figure 5a) and immunocyto-
Figure 5. Puromycylation of nascent protein chains in rat hippocampal neurons. a) Western blot of lysed
cells probed with a primary anti-puromycin antibody. NVOC Pre-60/30: preincubated with 3 mm NVOC-
puromycin for 60/30 min followed by 3 washing steps. NVOC-Puro: 3 mm NVOC-puromycin. Puromycin:
3 mm commercial puromycin. Treated cells were subjected to illumination (+) or no illumination (À) for
30 s followed by a 10 min incorporation period. Loading control: Anti-GluA2. Negative control: Cells
incubated with DMSO (not shown). Lane intensity analysis in Figure S4 in the Supporting Information.
b) Microscopic immunocytochemistry images of hippocampal neurons stained with an anti-puromycin
antibody (red), DAPI (nucleus, blue), and an anti-MAP2 antibody (dendrites, green). Culture dishes were
incubated as described for the Western Blot and cells with or without illumination were processed for
puromycin immunocytochemistry. The preincubation time was 30 min and the illumination time was
60 s. c) Simple spatial control experiment using aperture illumination with a circular mask. Experimental
conditions were otherwise identical to the pre-incubated sample in (b). The white boxes represent the
zoomed micrographs on the right. The position of the illumination mask on the culture dish was circled
using a black marker (dark trail). d) Cells after two-photon uncaging of NVOC-puromycin at two spots
in a single, transfected neuron per dish. The whole dish was processed for puromycin immunohisto-
chemistry and the illuminated cell and other cells in the dish were identified based on the transfection
marker (GFP) and shape (lower right insets). Right panels: non-illuminated transfected control cells
after incubation (transfected cells, filled arrowheads; non-transfected cells, line arrows). Neither non-
illuminated, transfected cells nor the surrounding non-transfected cells show significant puromycin
incorporation. Left panel: puromycin signal following two-photon uncaging inside the cell body of an
illuminated neuron. The localization of the two illumination regions is indicated by white squares (top
left inset).
chemistry
(Figure 5b).
As
expected, labelling of cells
treated with commercial puro-
mycin was roughly equal in the
presence or absence of illumina-
tion. By contrast, in cells exposed
to NVOC-puromycin, puromycy-
lation was only observed after
illumination (Figure 5a). Inter-
estingly, illumination-dependent
puromycylation, albeit to a lower
extent, was also observed after
preincubation (30 min/60 min) of
the cells with NVOC-puromycin,
followed by washes to remove
extracellular NVOC-puromycin before illumination (Æ) and
a 10 min incorporation period, thus indicating that the caged
compound is also taken up, albeit with slower kinetics than
puromycin. Nonetheless this fact allowed us to spatially
restrict puromycylation by simply adding an illumination
mask. The first spatial control experiment was performed by
using an aperture that allowed partial illumination of the cell
culture dish (Figure 5c) after preincubation of the entire dish
with 3 mm NVOC-puromycin for 30 min and washing before
a 60 s illumination period. Puromycylation inside the illumi-
nated inner area compared to the non-illuminated outer area
was equivalent to the results for NVOC-puromycin with and
without illumination, thus demonstrating that simple spatial
control can be achieved through selective illumination within
a single cell culture dish.
(8 ꢀ 8 pixels, 5 ms illumination per pixel, 10 times in one
minute intervals, Figure 5d, upper left inset). To detect
puromycylation, dishes were processed for puromycin immu-
nocytochemistry 10 min after the first laser pulse. Imaging of
illuminated versus non-illuminated transfected and non-
transfected cells in the same dishes revealed that puromycy-
lation of newly translated proteins was detected with surpris-
ing spatial precision. Puromycin signal above background was
only detected in the illuminated cell and the intensity was
highest within a narrow strip around the illumination spots,
whereas only a faint background signal was detected in non-
illuminated cells.
In summary, we present the synthesis of a photocaged
puromycin derivative and its biophysical and biochemical
characterization. By using NVOC as photolabile protecting
group, we demonstrated photoinducible toxicity to eukaryotic
cells, as well as the inhibition of protein translation in vitro in
prokaryotic and eukaryotic cell-free expression systems. We
further showed that triggered polypeptide chain release can
To dramatically increase spatial resolution, GFP-trans-
fected neurons were incubated with 3 mm NVOC-puromycin
for 30 min and a single cell was illuminated by using a two-
photon laser at 720 nm in two 1.6 ꢀ 1.6 mm regions of interest
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[13] S. R. Starck, X. Qi, B. N. Olsen, R. W. Roberts, J. Am. Chem.
be achieved in vitro. Lastly, we demonstrated the use of
NVOC-puromycin to spatially control the release and incor-
poration of puromycin into living cells. Since local synthesis of
proteins near neuronal synapses is thought to be one of the
key mechanisms involved in the cellular expression of
learning and memory,[34] the spatiotemporal monitoring of
protein synthesis and the identification of these newly
synthesized proteins is a task of utmost importance. Puromy-
cylation is a powerful mechanism for tagging nascent proteins.
The demonstrated illumination-dependent spatiotemporal
control of puromycin application in combination with living
neurons promises an exciting approach to accomplish this
task. Localized translation plays a much wider role than in
just learning and memory and has been shown in a multitude
of systems.[34,35] We conclude that photocaged puromycin will
be of great use for numerous existing and new biological
applications.
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Communications
Photoactivation
On like a light: The antibiotic puromycin
(green) is a translation inhibitor that
triggers the release of the nascent poly-
peptide chain (red) from the ribosome
(yellow) and it is used in a number of
applications. A photocaged puromycin
derivative, NVOC-puromycin, was syn-
thesized and characterized. Both func-
tional recovery upon UV illumination and
biological inactivity in vitro and in vivo
were demonstrated.
F. Buhr, J. Kohl-Landgraf, S. tom Dieck,
C. Hanus, D. Chatterjee, A. Hegelein,
E. M. Schuman, J. Wachtveitl,
H. Schwalbe*
&&& — &&&
Design of Photocaged Puromycin for
Nascent Polypeptide Release and
Spatiotemporal Monitoring of Translation
6
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Angew. Chem. Int. Ed. 2015, 54, 1 – 6
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