A New Photocaged Puromycin for an Efficient Labeling of Newly Translated Proteins in Living Neurons

Article abstract of DOI:10.1002/cbic.201800408

Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons.

Full text of DOI:10.1002/cbic.201800408

Accepted Article
Title: A new photocaged puromycin for an efficient labelling of newly
translated proteins in living neurons
Authors: Harald Schwalbe, Isam Elamri, Maximillian Heumüller, Lisa M
Herzig, Elke Stirnal, Josef Wachtveitl, and Erin Schuman
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201800408
Link to VoR: http://dx.doi.org/10.1002/cbic.201800408
A Journal of
www.chembiochem.org

10.1002/cbic.201800408
ChemBioChem
COMMUNICATION
A new photocaged puromycin for an efficient labeling of newly
translated proteins in living neurons**
Isam Elamri+,[a] Maximilian Heumüller+,[b] Lisa-M. Herzig,[c] Elke Stirnal,[a] Josef Wachtveitl,[c] Erin M.
Schuman,[b] and Harald Schwalbe*,[a]
acid-based labeling methods, puromycin is not RNA codon-
dependent. This codon-independence allows an unbiased
labeling of newly synthesized proteins. Due to its efficient
incorporation, no amino acid starvation needs to be performed
prior to labeling.[9]
Monitoring of newly synthesized proteins is becoming increasingly
important to characterize proteome composition in regulatory
networks. Puromycin is a peptidyl transfer inhibitor, widely used in
cell biology for tagging newly synthesized proteins. Here, we report
synthesis and application of an optimized puromycin carrying a
photolabile protecting group as a powerful tool for tagging nascent
proteins with high spatiotemporal resolution. The photocaged 7-N,N-
Diethylamino-cumarin-4-yl]-methoxycarbonyl-puromycin (DEACM-
puromycin) was synthesized and compared with the previously
developed 6-Nitroveratryloxycarbonyl puromycin (NVOC-puromycin).
The photo-chemical behaviour as well as the effectiveness in
controlling the puromycylation in living hippocampal neurons using
two-photon excitation is superior to the previously used NVOC-
puromycin. We further report on the application of light-controlled
puromycylation to visualize new translated proteins in neurons.
Protein synthesis and degradation are fundamental steps in gene
expression.[1] It is thus important to monitor newly synthesized
proteins. The aminonucleoside puromycin is a low molecular
weight analogue of the 3´-end of tyrosine-loaded tRNA (tRNAtyr).
Puromycin is nonspecifically incorporated into the growing
nascent paptide chain during translation and causes dissociation
of the nascent peptide chain from the ribosome. The
puromycilated nascent peptide can be detected by anti-
puromycin antibodies.[2][3] At low concentration, puromycin is not
able to compete with the aminoacyl tRNA but binds specifically
at the C-terminus of the full-length protein, stochastically labeling
newly synthesized proteins.[4][5][6][7][8] Thus, it provides a snapshot
of the translatome. Furthermore, in contrast to isotope amino
Scheme 1. a. Structural similarity between 3´-end of a tyrosyl-tRNA and
the aminonucleoside anibiotic puromycin b. Differences are shown in
violet. c. Photocaged NVOC- or DEACM-puromycin.
In addition, the commercial availability and the easy handling of
puromycin makes puromycylation a powerful method for tagging
newly translated proteins. Previously,[10][11] we developed a
puromycin protected by a 6-nitroveratryl-oxycarbonyl (NVOC)
group (Scheme 1), which is a derivative of the well-known
nitrobenzyl (oNb)-photolabile protecting group[12][13][14]
.
In
addition to the spectroscopic investigation of the uncaging
mechanism, we showed using two-photon uncaging irradiation-
dependent spatiotemporal puromycylation of nascent protein
chains in hippocampal neurons. However, the solubility of
NVOC-puromycin is modest preventing the use of high
concentrations of the caged compound in cell culture
experiments. Furthermore, the extinction coefficient (6500 M-
1.cm-1) and the quantum yield (1.1 ± 0.2%)[11] are low. We
demonstrated[10] that the low quantum yield is a result of a triplet
photodeactivation pathway allowing efficient relaxation to the
ground state after irradiation.
[a]
I. Elamri*, E, Stirnal, Prof. Dr. H. Schwalbe
Center for Biomolecular Magnetic Resonance
Institute of Organic Chemistry and Chemical Biology
Goethe-University Frankfurt am Main (Germany)
Max-von-Laue-Straße 7, 60438 Frankfurt am Main (Germany)
Email: schwalbe@nmr.uni-frankfurt.de
[b]
[c]
M. Heumüller*, Prof. Dr. E. M. Schuman
Department of Synaptic Plasticity, MPI for Brain Research
Goethe-University Frankfurt am Main (Germany)
Max-von-Laue-Straße 4, 60438 Frankfurt am Mail (Germany)
L.-M. Herzig, Prof. Dr. J. Wachtveitl
We therefore report here on the development and application of
a more suitable photocleavable puromycin. 7-N,N-Diethylamino-
Institute of Physical and Theoretical Chemistry
Goethe-University Frankfurt am Main (Germany)
Max-von-Laue-Straße 7, 60438 Frankfurt am Main (Germany)
4-hydroxymethylcoumarin (DEACM) is
a frequently used
photolabile group for application in living organisms. The
coumarin-protecting group fulfils most criteria for cellular
applications. The high extinction coefficient (2.5 x 104 M-1cm-1 at
λmax = 369 nm)[15] increases the uncaging efficiency, so that a
relatively low light dose is sufficient. Furthermore, DEACM can
be cleaved off with light at wavelengths higher than 350 nm and
exhibits a satisfactory two-photon excitation cross-sections
permitting two-photon uncaging.[16][17][18][19]
[*]
These authors contributed equally to this work.
We thank Dr. J. Wirmer-Bartoschek for critical reading of the
manuscript. This work was supported by DFG in graduate college
CLIC and in the collaborative research center SFB902. Work at
BMRZ is supported by the state of Hessen.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800408
ChemBioChem
COMMUNICATION
which is in particular favorable for biological applications. Hence,
application of DEACM-puromycin prevents photodamage of the
tissue. In addition, the extinction coefficient of DEACM-puromycin
is more than three times larger than the extinction coefficient of
NVOC-puromycin.[11][21] Accordingly, uncaging of the coumarin
caged-compound can be induced by a less harmful light dose as
compared to NVOC-puromycin. To quantify the uncaging
Figure 1. Absorption spectra of NVOC-puromycin (gray) and DEACM-
puromycin (black) in DMSO, where ε is the molar extinction coefficient and
λ the wavelength.
Scheme
carbonyl-puromycin (DEACM-puromycin) and 6-nitroveratyl-oxycarbonyl
puromycin (NVOC-puromycin). Reagents and conditions (a) 7-
diethylamino -4 -methylcoumarin (1 equiv.), DMF-DMA (2 equiv.), DMF,
160 °C, 7h; (b) 2 (1 equiv.), NaIO4 (3 equiv.), THF/H2O 1:1, R.T., 5h; (c) 3
(1 equiv.), NaBH4 (0.5 equiv.), EtOH, RT, 4 h, 98%; (d) 4 ( 1 equiv.), DMAP
(2 equiv.), 4-NO2Ph-chloroformate (2 equiv.), CH2Cl2, RT, 7 h. (e)
Puromycin▪2HCl (1 equiv.), DIPEA ( 20 equiv.), 5 (1,2 equiv.), DMAP (2
equiv.), CH2Cl2, 40 °C, 24 h, 25%. (f) Puromycin.2HCl (1 equiv.), DIPEA
(20 equiv.), NVOC-Cl (1, 5 equiv.) CH2Cl2, RT, 24 h, 75%. Abbreviation:
SeO2 = selenium dioxide; NaBH4 = sodium borohydride; NaIO4 = sodium
periodate, NO2Ph = Nitrophenyl; DIPEA = N,N-diisopropylethylamine;
DMF-DMA = N,N-dimethylformamide dimethyl acetal; DMAP = N,N-
dimethylamino-pyridine. Characterization of the data is presented in the
2 Synthesis of 7-N,N-Diethylamino-cumarin-4-yl]-methoxy-
efficiency, the uncaging quantum yield has been determined.
Because the absorption of the photoproduct DEACM-OH is
indistinguishable of DEACM-puromycin, the quantum yield cannot
be measured in the UV/vis-range. Therefore, both compounds
have been irradiated with 365 nm and the absorption changes in
the IR-range have been recorded (Figure S1). Besides other
spectral changes, the decarboxylation process of the carbamate
linker in both molecules can be monitored at 2337 cm-1, which is
the characteristic absorption of dissolved CO2.[22]
:
Supporting Information (Figures S4-11).
We synthesized DEACM-puromycin in five steps. Starting from 7-
Amino-4-methylcoumarin, the allylic methyl group can be oxidized
via the Riley-reaction using selenium dioxide to generate the
aldehyde 3 with a yield of 29%. An alternative route avoiding the
use of toxic selenium dioxide and featuring a higher yield, is the
two step procedure reported by Weinrich et al.,[20] starting with the
condensation to enamine 2 using DMF-DMA followed by
treatment with the oxidizing agent sodium periodate. In both
Figure 2. Determination of quantum yield by absorption of photoreleased
CO2 at 2337 cm-1 during UV excitation of NVOC-puromycin (circles) and
DEACM-puromycin (squares) with corresponding fits. The concentrations of
the compounds have been considered for scaling. In the supporting
information detailed information for the determination of the quantum yield
and the IR absorption spectra evolving during excitation of cage puromycin
are given.
cases, the obtained aldehyde
3 is reduced via sodium
borohydride to give the alcohol 4 in 98%. Since amines are most
effectively photolabile-protected via a carbamate linker, it is
necessary to first modify the alcohol 4 to DEACM-4´-nitrophenyl
carbonate 5, which then reacts without further purification with
puromycin▪HCl in the presence of DIPEA and DMAP to yield the
final product 6 with a total yield of 25%. NVOC-puromycin was
synthesized according to the published procedure [7] using
NVOC-Cl in the presence of DIPEA (75%, yield). Uncaging of both
caged puromycin types was verified by using reversed HPLC and
a laser-coupled NMR setup. (See the Supporting Information
Figures S12-15). As shown in Figure 1, the absorption of NVOC-
and DEACM-puromycin share similar spectral features beyond
300 nm, which results from the puromycin moiety in both
compounds. However, the absorption maximum at 375 nm of
DEACM-puromycin is 30 nm red-shifted compared to NVOC-
puromycin. Moreover, the coumarin caged-compound shows
strong absorption characteristics above 400 nm. Therefore, the
uncaging of DEACM-puromycin can be induced with visible light,
As shown in Figure 2, the increase of the carbon dioxide
absorbance observed in the first 10 min of irradiation, can be
described with a linear function. On this timescale the assumption
that the caged compounds are the dominant absorbing species is
valid, because the photolysis has not yet led to a significant
product formation. Consequently, the absorbance change at
2337 cm-1 is only caused by product formation and therefore, is
directly related to the number of uncaging photoreactions taking
place. We determined an uncaging quantum yield of 2.5 ± 0.4%
for DEACM-puromycin and 1.2 ± 0.1% for NVOC-puromycin (for
calculation see Supporting Information Eq. 1-4). The uncaging
efficiency as the product of extinction coefficient and quantum
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800408
ChemBioChem
COMMUNICATION
Figure 3. a. Scheme of the experimental workflow. Primary cultured neurons are prepared from hippocampal brain tissue and used for three types of
experiments. Broad illumination at 365nm (1.) or partial illumination at 365nm using a mask (2.) For high spatial resolution two-photon uncaging at 720nm
(3.) was applied. A general time line for experiments is depicted at the bottom of b. Western blot showing the puromycin signal of 20ug of protein as well
as the ß-Actin signal as a loading control. c. Maximum intensity projections of immunocytochemically (ICC) stained primary hippocampal neurons.
Puromycylated protein is labeled (anti-puromycin antibody) and the intensity is depicted with a fire look-up table (LUT) (calibration bar indicates signal
intensity in %), Anti-Map2 labeling, highlighting neuronal dendrites,is shown gray. The specific conditions are indicated with the images: ‘No Puro’ means
no puromycin has been added to the medium, ‘+Light’ neurons were illuminated 30s at 365nm, ‘-Light’ cultures were treated the same way but were not
illuminated. Scale bar represents 10um. d. Statistic analysis (Mann-Whitney U test for NVOC-Puro) of (c.) with each column representing data from 52-
62 neurons and showing the average puromycin signal normalized to the puromycin intensity of the (uncaged) puromycin treated cells. The error bars
indicted SEM and significance was determined using a Mann-Whitney U test. e. Maximum intensity projections of immunocytochemically labeled primary
hippocampal neurons that were pre-incubated with 3µM DEACM-puromycin. Puromycin signal is highlighted by a fire-LUT (anti-puromycin antibody)
and GFP-transfected cells are visualized in green (GFP fluorescence), GFP transfected cells are marked in the puromycin channel by filled triangles
and un-transfected cells by empty triangles. Uncaging in transfected neurons was conducted (yellow, filled triangle) by 720nm two-photon illumination
on two 1um2 spots within the cell body. In control cells no illumination was (white, filled triangle). Scale bare is 20um. f. Same as (e.) but NVOC-
puromycin instead of DEACM-puromycin.
yield is for DEACM-puromycin more than six times larger than for
NVOC-puromycin. This is, in theory, beneficial for the use in
cellular application. Following the direct comparison of both
caging compounds using analytic chemistry, we set out to
evaluate their performance in a cellular environment. Protein
synthesis plays, for example, a crucial role in learning and
memory formation, thus there is a great demand for tools to
monitor translation with high temporal and spatial resolution in
neuroscience.[23][24] We used primary rat cultured hippocampal
neurons to investigate the uncaging properties. First, we
demonstrate that DEACM-puromycin is taken up by the cell as
previously shown for NVOC-puromycin (Figure S2). The
illumination dependence of both puromycin-derivaties was
compared by broad illumination of the whole culture dish, partial
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800408
ChemBioChem
COMMUNICATION
illumination using a physical mask, or precise single cell
illumination using two-photon uncaging (Figure 3a.).
Immunocytochemical staining with an anti-puromycin antibody,
revealed that native puromycin exhibits the strongest labeling,
followed by irradiated DEACM-puromycin which is more than 3
times more efficient than irradiated NVOC- puromycin (Figure 3.
c, d.). This observed difference in labeling correlates well with the
observed difference in uncaging efficiency of the compounds.
Similar results were obtained using Western blotting (Figure 3. b.),
with DEACM-caged puromycin showing significantly stronger
signal than its predecessor for both concentrations (3M/10M).
Cellular uptake of caged-puromycin enables precise spatial
puromycylation, which we demonstrated by placing a physical
mask between the light source and the dish allowing only
illumination of a small region. Using this partial masking we were
able to confine puromycilation to the illuminated area of the dish
(Figure S3) exhibiting the same spatial potential as its
predecessor. To evaluate any potential impact of the illumination
procedure on the cell health of our neuronal cultures, we
performed an apoptosis assay. As expected, this revealed that
our robust UV irradiation protocol did not induce cell death and
had no impact on DNA integrity (Figure S16). Furthermore, the
uptake efficiency for both cages was quantified by measuring the
remaining amount of puromycin in the supernatant after 1h of
incubation on culture dishes with neurons or empty dishes as a
control. The cells take up around 55% of the native Puromycin
present in the medium during 1h of incubation, while only 45% of
NVOC-puromycin and 30% of DEACM-puromycin are taken up
during the same time period (Figure S17). This indicates that the
improved uncaging efficiency of DEACM-puromycin is mainly
responsible for the enhanced labeling in cell culture, since
DEACM-cage uptake is slightly reduced compared to the NVOC-
cage but nonetheless yields a stronger labeling.
Experiments that require very brief labeling periods also benefit
from the elevated uncaging efficiency. The advantages conferred
by this new cage make existing experiments more amenable and
may allow for novel experimental designs including investigation
of local protein synthesis at the base of spines or in the distal parts
of the dendritic branch, which is believed to be a key mechanism
involved in the formation and maintenance of memories.[25][26]
Experimental Section
Synthesis of DEACM-puromycin (6)
Method
methoxycarbonyl-puromycin (DEACM-puromycin) (2): To a
solution of 7-Amino-4-methyl-coumarin (20.00 g,
A:
7-N,N-Diethylamino-cumarin-4-yl]-
86.48mmol, 1 equiv.) in dry DMF (150 mL), DMF-DMA
(23 mL, 0.173 mol, 2 equiv.) was added. The mixture was
heated to reflux for 48 h. The reaction was then quenched
with the addition of conc. NaHCO3 solution and extracted
twice with 1 L of CH2Cl2. The combined organic layers were
dried over NaSO4 and evaporated to yield a brown solid. The
product was then isolated by recrystallization from EtOAc/c-
hex 1:2 (19.2 g, 78%). Rf = 0.38 (CH2Cl2/EtOAc, 7:3).
1H NMR (500 MHz, CDCl3): δ = 7.53 (d, J = 9.1 Hz, 1 H, 5-
H
), 7.21 (d, J = 13.0 Hz, 1 H, CHC
Hz, 1 H, 6- ), 6.49 (d, J = 2.7 Hz, 1 H,
H), 5.22 (d, J = 13.0 Hz, CHC N), 3.40 (q, J = 7.1 Hz, 4 H,
2CH3), 2.98 (s, 6 H, N(C 3)2), 1.20 (t, J = 7.0 Hz, 6 H,
CH2C
3) ppm. 13C NMR (125.8 MHz, CDCl3): δ = 163.4,
H
N), 6.55 (dd, J = 9.0, 2.7
H
H
-8), 5.85 (s, 1 H, 3-
H
H
C
H
H
156.4, 152.2, 150.1, 146.7, 124.7, 108.1, 107.8, 98.0, 93.5,
87.7, 44.6, 12.5 ppm. MS (ESI): m/z = calcd. for C17H23N2O2
[M+ H+]: 287.18; found 287.24.
7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde
NaIO4 (5.60 g, 26.20 mmol, 3 equiv.) was added to a
suspension of enamine (5.00 g, 17.50 mmol, 1 equiv.) in a
(3)*:
In order to demonstrate the subcellular spatial resolution of
uncaging, two-photon illumination at 720 nm was performed
(Figure 3. e, f.). GFP-transfected neurons were selected and
uncaging of both caged puromycin-derivatives revealed elevated
puromycin levels specifically within the targeted cells (yellow
triangle), in close proximity to the uncaging spots. Neurons that
were GFP transfected but not targeted (white triangle) showed no
increased puromycilation similar to cells that were neither
transfected nor targeted (empty triangle). Again, using the same
2
mixture of THF and H2O (40 mL, 1:1). A cloudy red
precipitate immediately formed. The reaction was stirred at
room temperature for 1 h. The precipitate was removed by
filtration and extracted twice with EtOAc. Subsequently,
conc. NaHCO3 solution was added and the aqueous layer
was extracted twice with EtOAc. The combined organic
layers were dried over MgSO4 and concentrated in vacuo.
The crude product was purified by silica gel chromatography
wavelength and intensity, DEACM-puromycin exhibited
a
significantly stronger labeling and a comparable low background
signal in non-illuminated regions. If less strong labeling is
preferred the light intensity, duration or illumination region can be
adjusted to achieve the desired result.
(EtOAc/c-hexane, 1:1), which yielded the title compound
3
(6.05 g, 75%). The product was obtained as a red oil. Rf =
1
0.40 (CH2Cl2, 100%). H NMR (600 MHz, CDCl3): δ = 10.1
(s, 1 H, CO
2.6 Hz, 1 H, 6-
8- ), 3.46 (q, J = 7.1 Hz, 4 H, C
6 H, CH2C
3) ppm. 13C NMR (125.8 MHz, CDCl3): δ = 192.5,
H
), 8.22 (d, J = 9.2 Hz, 5-
), 6.65 (s, 1 H, 3- ), 6.61 (d, J = 2.6 Hz, 1 H,
2CH3), 1.15 (t, J = 7.1 Hz,
H), 6.78 (dd, J = 9.2,
In conclusion, we have developed and synthesized an improved
caged puromycin (DEACM-cage) that offers superior labeling
properties due to its higher cleavage efficiency of the cage with
no drawbacks compared to its predecessor (NVOC-cage). This
suggests that DEACM-puromycin likely the best molecule of
choice, since less harmful light doses can be used for the same
amount of uncaging. DEACM-caged puromycin also exhibits a
red-shifted excitation maximum compared to NVOC-caged
puromycin allowing the use of single-photon uncaging within the
visible range. This again exposes the cell to less stress during the
uncaging process compared to the ultraviolet illumination needed
for the NVOC-cage. The increased sensitivity also opens up new
possibilities for the use in fragile areas of the cell or compartments
with low protein synthesis-rates, e.g. distal dendrites.
H
H
H
H
H
161.8, 157.4, 151.0, 143.9, 126.2, 117.3, 109.5, 103.7, 98.2,
44.8, 12.4 ppm. MS (ESI): m/z = calcd. for C14H16NO3 [M+
H+]: 246.11; found 246.16.
*
Compound (3) was also synthesized based on the
procedure reported by Weinrich et al.,[20] where no purification
by silica gel chromatography needs to be performed.
7-(diethylamino)-4-(hydroxymethyl)-2H-chromen-2-one (4):
A
solution of aldehyde (1.12 g, 4.60 mmol, 1 equiv.) was
3
dissolved in THF (15 mL) and cooled to 0 °C. NaBH4 (0.35 g,
9.19 mmol, 2 equiv.) was added and the reaction mixture
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800408
ChemBioChem
COMMUNICATION
was stirred under argon for 2 h at room temperature,
quenched with saturated NaHCO3 and the organic layer was
separated. The aqueous layer was extracted with CH2Cl2
and the organic phase was dried over Na2SO4 and
concentrated in vacuo. The crude product was purified by
silica gel chromatography (CH2Cl2/acetone, 5:1), which
equiv.) was prepared and added dropwise to the puromycin
solution to avoid an additionally 5´-O-acylation. The reaction
mixture was stirred at room temperature under N2-
atmosphere for 20 h. Light exposure was minimized. The
brown solution was diluted with CH2Cl2 (25 mL) and washed
with conc. NaHCO3 solution, brine and H2O (2 x 50 mL). The
organic layers were dried over MgSO4 and concentrated in
vacuo. The crude product was obtained as yellow oil. Silica
gel chromatography (CH2Cl2/MeOH, 9:1), subsequently
reversed-phase HPLC (H2O/ACN) and lyophilization
yielded the compound
4 (1.12 g, quant.). Product was
obtained as a yellow solid.
Method B: Riley oxidation with Se2O and reduction with
NaBH4: 7-Amino-4-methyl-coumarin (4.63 g, 20.0 mmol, 1
equiv.) and selenium dioxide (3.33 g, 30.0 mmol, 1.5 equiv.)
were dissolved in p-xylene (120 mL). The reaction mixture
was heated to reflux with vigorous stirring for 24 h. The
precipitate was filtered off and concentrated under reduced
pressure. The obtained dark brown oil and NaBH4 (380 mg,
10.0 mmol, 0.5 equiv.) were dissolved in ethanol (130 mL)
and stirred for 4 h at ambient temperature. Subsequently 1 M
HCl (20 mL) was added to the suspension, and diluted with
H2O and extracted three times with CH2Cl2. The organic
layers were washed with H2O and brine, dried over Na2SO4
and reduced to an oil by rotary evaporation. The crude
product was purified by silica gel chromatography
afforded the title compound
6 as white needles (8.50 mg,
25%). Rf = 0.70 (CH2Cl2/MeOH, 9:1), rp-HPLC: [Kromasil
RP18, 4.60 x 250 mm, gradient: (H2O/ACN, 30-100%), ACN
1
in 50 min, 3 mL/min, Rt = 29.90 min], H NMR (500 MHz,
DMSO-d6): δ = 8.48 (s, 1 H,
1 H, 3´-N CO),7.80 (d, J = 8.83 Hz, 1 H, N
J = 9.04 Hz, 1 H, Coum -5), 7.27 (d, J = 8.6 Hz, 2 H, m-
(ph(OMe)-2 ))), 6.87 (d, J = 8.6 Hz, 2 H, o-(ph(OMe)-2 ))),
6.69 (dd, J = 9.04, 2.5 Hz, 1 H, Coum -6), 6.56 (d, J = 2.5
Hz, 1 H, Coum -8), 6.15 (br, 1 H, 2´-OH), 6.03 (d, J = 2.67,
1 H, 1´ ), 5.98 (s, 1 H, Coum -3), 5.24 (t, J = 5.30, 1 H, 5`-
OH), 5.17 (d, J = 6.67, 2 H, CoumC 2), 4.5 (br, 2 H, 2- H´, 3-
H´), 4.38 (td, J = 9.62, 4.10 Hz, 1 H, OMeTyr- (α)), 3.99 (m,
1 H, 4´-H), 3.74 (s, 3 H, OMe), 3.70 (m, 1 H, 5´ 2-OH), 3.51
(m, 1 H, 5´ 2-OH), 3.45 (q, J = 7.2 Hz, 4 H, C
(s, 6 H, (NCH3
H
-8), 8.27 (s, 1 H,
H-2), 8.26 (br,
H
H
COO), 7.41 (d,
H
H
H
H
H
H
H
H
(CH2Cl2/acetone, 5:1) (1.28 g, 26%). Rf
hexane/EtOAc, 1:2). 1H NMR (600 MHz, DMSO-d6): 7.48 (d,
J = 9.0 Hz, 1 H, 5- ), 6.70 (dd, J = 9.0, 2.6 Hz, 1 H, 6- ),
6.56 (d, J = 2.6 Hz, 1 H, 8- ), 6.12 (s, 1 H, 3-H), 5.55 (t, J =
5.6 Hz, 1H, O ), 4.73 (dd, J = 5.6, 1.3 Hz, 2 H, C 2OH), 3.46
(q, J = 7.1 Hz, 4 H, C 2CH3), 1.17 (t, J = 7.1 Hz, 6 H,
CH2C
3) ppm. 13C NMR (125.8 MHz, DMSO-d6): δ = 161.1,
=
0.28 (c-
H
H
H
H
H
H
2CH3), 3.33
2+
H
)
water) 2.98 (dd, J = 13.66, 4.1 Hz, 1 H,
H
H
OMeTyr-H(β)), 2.75 (m, 1 H, OMeTyr-H(β)), 1.14 (t, J =
7.1 Hz, 6 H, CH2CH3) ppm. 1H NMR resonances were
assigned using 1H-13C-HSQC, COSY, HMBC spectra
recorded at 500 MHz in DMSO-d6. MS (ESI): m/z = calcd. for
C37H44N8O9 [M+ H+]: 745.22; found 745.20.
H
H
156.8, 155.6, 150.2, 125.0, 108.5, 105.7, 103.9, 96.8, 59.0,
43.9, 12.3 ppm. MS (ESI): m/z = calcd. for C14H18NO3 [M+
H+]: 248.13; found 248.19.
Synthesis of NVOC-puromycin (7) 4,5-dimethoxy-2-
7-(diethylamino)-4-[(ylmethyl-(4´-nitrophenyl)]-2H-chromen-
nitrobenzyl-oxycarbonyl-puromycin. To
a suspension of
4-ylcarbonate (5): A mixture of alcohol
4 (22.7 mg, 0.09 mmol,
Puromycin. 2HCl (50mg, 0.092 mmol, 1 equiv.) in 2 mL dried
Dichlormethan, DIPEA (160uL, 0.92 mmol, 10 equiv.) was added
and stirred 5 min under N2-atmosphere to give a clear solution.
To this was added NVOC-Cl (37.8 mg, 0.13 mmol, 1.5 equiv.) and
the yellow mixture was stirred overnight under N2-atmosphere
at room temperature. Light exposure was minimized. The
brown solution was diluted with CH2Cl2 (25 mL) and washed
with conc. NaHCO3 solution, brine and H2O (2 x 50 mL). The
organic layers were dried over MgSO4 and concentrated in
vacuo. The crude product was obtained as yellow oil. Silica
gel chromatography (CH2Cl2/MeOH, 9:1), subsequently
reversed-phase HPLC (H2O/ACN) and lyophilization
afforded the title compound 6 as white needles (49 mg,
75%). Rf = 0.66 (n-Hex/EE: 1/3), rp-HPLC: [Kromasil RP18,
10 x 250 mm, gradient: (H2O/ACN, 30-100%), ACN in 50
1 equiv.), DMAP (22.5 mg, 0.18 mmol, 2 equiv.) and 4-
nitrophenyl chloroformate (22.3 mg, 0.11 mmol, 1.2 equiv.)
in dry CH2Cl2 (3 mL) were stirred under argon atmosphere
and in the absence of light overnight at room temperature.
After 20 h silica gel TLC (c-hexane/EtOAc, 1:1 (Rf = 0.80))
showed the reaction to be complete. To remove the chloride
salt of dimethylaminopyridinium, the mixture was washed
twice with H2O (2 x 100 mL). The organic layers were
evaporated to a brown solid. NMR-based estimated yield of
compound
5 was ca. 60%. The crude product was used in
the next step without further purification. An analytical
sample was purified by silica gel chromatography
(CH2Cl2/MeOH, 49:1). Rf = 0.80 (c-hexane/EtOAc, 1:1). H
1
NMR (600 MHz, CDCl3): δ = 8.23 (d, J = 9.15 Hz, 2 H, Ar
to –NO2), 7.35 (d, J = 9.15 Hz, 2 H, Ar m to –NO2), 7.26 (d,
J = 9.0 Hz, 1 H, 5- ), 6.59 (dd, J = 9.0, 2.4 Hz, 1 H, 6- ),
6.50 (d, J = 2.4 Hz, 1 H, 8-H), 6.17 (s, 1 H, 3-H), 5.33 (d, J =
1.1 Hz, 2 H, C 2OH), 3.36 (q, J = 7.2 Hz, 4 H, C 2CH3), 1.15
3) ppm. MS (ESI): m/z = calcd. for
H o
H
1
min, 3 mL/min, Rt = 20.5 min], H NMR (500 MHz, DMSO-
H
H
d6): δ = 8.44 (s, 1 H, H-8), 8.26 (br, 1 H, 3´-NHCO), 8.25 (s,
1 H, H-2), 7.78 (d, J = 8.76 Hz, 1 H, NHCOO), 7.69 (s, 1 H,
NO2Ar-H), 7.24 (d, J = 8.3 Hz, 2 H, m-(ph(OMe)-2H))), 7.13
(s, 1 H, NO2Ar-H), 6.81 (d, J = 8.4 Hz, 2 H, o-(ph(OMe)-2H))),
6.10 (d, J = 4.5 Hz, 1 H, 1´H), 6.00 (d, J = 2.3 Hz, 1 H, 2´-
OH), 5.28 (s, , 2 H, NVOC-CH2), 5.18 (t, J = 5.4 Hz, 1 H, 5`-
OH), 4.9 (m, 2 H, 2- H´), 4.36 (td, J = 9.62, 4.10 Hz, 1 H,
OMeTyr-H(α)), 3.94 (m, 1 H, 4´-H), 3.86 (s, 6 H, (OCH3)2),
3.70 (s, 3 H, OMe), 3.68 (m, 1 H, 5´H2-OH), 3.47 (m, 1 H,
5´H2-OH), 3, 30 (6H, (NCH3)2+water), 2.94 (dd, 3J = 13.8 Hz,
4J = 4.1 Hz, 1 H, OMeTyr-H (β)), 2.72 (m, 1 H, OMeTyr-H(β)),
H
H
(t, J = 7.1 Hz, 6 H, CH2C
H
C21H20N2O7 [M+ H+]: 413.08; found 413.06.
7-N,N-Diethylamino-cumarin-4-yl]-methoxycarbonyl-
puromycin (DEACM-puromycin) (6): To a suspension of
puromycin∙2HCl (25mg, 0.046 mmol, 1 equiv.) in dry CH2Cl2
(1 mL), DIPEA (175 µL, 0.92 mmol, 20 equiv.) was added
under stirring to give a clear solution and kept under N2-
atmosphere. A solution of scrude product
0.054 mmol, 1.2 equiv.) and DMAP (13.2 mg, 0.11 mmol, 2.4
5 (ca. 23 mg,
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800408
ChemBioChem
COMMUNICATION
ppm. 1H NMR resonances were assigned using 1H-13C-
HSQC, COSY, HMBC spectra recorded at 500 MHz in
DMSO-d6. 13C NMR (125.8 MHz, DMSO-d6): δ = 50.8, 55.4,
56.5, 56.8, 61.4, 62.8, 73.5, 83.9, 88.9, 108.6, 110.5, 113.9,
120.1, 128.7, 130.3, 130.8, 138.4, 139.4, 148.1, 150.2,
152.5, 154.0, 154.8, 155.9, 158.3, 172.4 ppm. MS (ESI): m/z
= calcd. for C32H38N8O11 [M+ H+]: 711.69; found 711.15.
[1]
R. Aviner, T. Geiger, O. Elroy-Stein, Nat. Protoc. 2014, 9, 751–
760.
W. Traub, Nature 1963, 198, 1165–1166.
D. Nathans, Pnas 1964, 51, 585–592.
E. Miyamoto-Sato, N. Nemoto, K. Kobayashi, H. Yanagawa,
Nucleic Acids Res. 2000, 28, 1176–1182.
W. J. Hansen, V. R. Lingappa, W. J. Welch, J. Biol. Chem. 1994,
269, 26610–26613.
N. Nemoto, E. Miyamoto-Sato, H. Yanagawa, FEBS Lett. 1999,
462, 43–46.
E. K. Schmidt, G. Clavarino, M. Ceppi, P. Pierre, Nat. Methods
2009, 6, 275–277.
S. Tom Dieck, L. Kochen, C. Hanus, M. Heumüller, I. Bartnik, B.
Nassim-Assir, K. Merk, T. Mosler, S. Garg, S. Bunse, et al., Nat.
Methods 2015, 12, 411–414.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Characterization data for all synthesized compounds can be
found in the Supporting Information.
UV/vis spectroscopy. UV/vis spectra were recorded with a
Specord S600 spectrophotometer (Analytik Jena).
[9]
J. Ge, C. W. Zhang, X. W. Ng, B. Peng, S. Pan, S. Du, D. Wang,
L. Li, K. L. Lim, T. Wohland, et al., Angew. Chemie - Int. Ed.
2016, 55, 4933–4937.
J. Kohl-Landgraf, F. Buhr, D. Lefrancois, J.-M. Mewes, H.
Schwalbe, A. Dreuw, J. Wachtveitl, J. Am. Chem. Soc. 2014, 136,
3430–3438.
F. Buhr, J. Kohl-Landgraf, S. Tomdieck, C. Hanus, D. Chatterjee,
A. Hegelein, E. M. Schuman, J. Wachtveitl, H. Schwalbe, Angew.
Chemie - Int. Ed. 2015, 54, 3717–3721.
A. Patchornik, B. Amit, R. B. Woodward, J. Am. Chem. Soc.
1970, 92, 6333–6335.
J. F. Cameron, J. M. J. Fréchet, J. Am. Chem. Soc. 1991, 113,
4303–4313.
C. G. Bochet, J. Chem. Soc. Perkin Trans. 1 2002, 125–142.
N. Gagey, P. Neveu, L. Jullien, Angew. Chemie - Int. Ed. 2007,
46, 2467–2469.
B. G. Ralph O. Schönleber, Jürgen Bendig, Volker Hagen,
Bioorganic Med. Chem. 2002, 10, 97–101.
C. Brieke, F. Rohrbach, A. Gottschalk, G. Mayer, A. Heckel,
Angew. Chemie 2012, 124, 8572–8604.
FTIR measurements. IR spectra were recorded with a Vertex 80
FTIR spectrometer (Bruker, Ettlingen). The spectrometer was
continuously purged with N2. Photolysis experiments were carried
out using a 365 nm LED (Thorlabs GmbH).
[10]
[11]
Cell Culture & transfection. Primary cultures were prepared
from rat hippocampus tissue and seeded at 40k cells on a 12mm
glas coverslip (Mattek dish) for imaging experiments or 600k for
western blot analysis. The cells were cultured at 37C and 5% CO2
in Neuro Basal A medium for 11 days (DIV11). At DIV 11 the cells
(1 dish contains 40k cells) were transfected with 1ug of the
calcium indicator GFP for 25 min using a combination of
Magnetofecatim (OZ Bioscience) and Lipofectamin 2000 (Thermo
Fisher). Subsequently the medium was replaced and cells were
allowed to express the GFP for 12h.
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
In vitro experimental flow. Primary hippocampal cultures were
pre-incubated in the dark for 30 or 60min with 3 or 10uM of either
NVOC- or DEACM-caged puromycin, followed by 30s of broad
whole dish illumination with 365 nm and a 10 min post-illumination
incubation to allow for incorporation of the uncaged puromycin. A
control dish was incubated with 3uM native puromycin for 10 min.
For ICC cells were briefly washed with pre-warmed PBS (pH 7.4),
fixed for 20 min with 4% PFA in PBS (pH 7.4) and permeabilized
for 15 min with 0.5% Triton X-100 in blocking buffer (4% goat
serum in PBS pH 7.4). Primary antibodies used for Map2
(188004, SYSY)) were used 1:1000 and for Puromycin (EQ0001,
Kerafast) 1:2000 for 12h in blocking buffer at 4C. Common
secondary Alexa-antibodies were used at 1:1000 for detection (45
min at RT). For westernblot cells were treated the same, but
instead of fixation they were harvested in lysis buffer (1% SDS,
1% Triton X-100 in PBS pH7.4). The same puromycin antibody
was used and bActin-antibody (Ab8227, Abcam) was used at
1:5000 for 12h at 4C. Primary antibodies were detected using
Licor IRDye antibodies (IRDye 800 & IRDye680) at 1:5000.
S. Weis, Z. Shafiq, R. a. Gropeanu, A. del Campo, J. Photochem.
Photobiol. A Chem. 2012, 241, 52–57.
C. A. Hammer, K. Falahati, A. Jakob, R. Klimek, I. Burghardt, A.
Heckel, J. Wachtveitl, J. Phys. Chem. Lett. 2018, 9, 1448–1453.
T. Weinrich, M. Gränz, C. Grünewald, T. F. Prisner, M. W.
Göbel, European J. Org. Chem. 2017, 2017, 491–496.
L.-M. Herzig, I. Elamri, H. Schwalbe, J. Wachtveitl, Phys. Chem.
Chem. Phys. 2017, 19, 14835–14844.
M. Falk, A. G. Miller, Vib. Spectrosc. 1992, 4, 105–108.
M. A. Sutton, E. M. Schuman, Cell 2006, 127, 49–58.
B. W. Agranoff, Sci. Am. 1967, 216, 115–122.
G. Aakalu, W. B. Smith, N. Nguyen, C. Jiang, E. M. Schuman,
Neuron 2001, 30, 489–502.
[22]
[23]
[24]
[25]
[26]
C. E. Holt, E. M. Schuman, Neuron 2013, 80, 648–657.
Imaging & uncaging.Broad illumination (full dish) was achieved
by placing the cell culture dish (with or without light impermeable
mask) on a UV-light table and illuminating the dish for 30s with
365nm. Precise subcellular illumination was achieved using a
Spinning disk system from Zeiss/3i in combination with a 2p laser
from Coherent. Uncaging was performed on two 1um2 sized spots
with 10 pulses (10ms) at a 60s interval. Fixed and stained cells
were imaged a confocal laser scanning microscope from Zeiss
(LSM 780).
Apoptosis Assay (TUNEL). Primary hippocampal cultures and
broad UV uncaging were performed as described above. The
“Click-iT™ TUNEL Alexa Fluor™ 647 Imaging Assay” from
Thermo Scientific (C10247) was used according to the manual to
detect DNA-strand breaks and evaluate cell health. An antibody
co-staining with Map2 (188004, SYSY, 1:1000) was performed.
This article is protected by copyright. All rights reserved.