Synthesis and characterization of a new two photon excitable acid sphingomyelinase FRET probe

Article abstract of DOI:10.1016/j.bmc.2021.116303

Recently, FRET probes for acid sphingomyelinase (ASM) have enabled the observation of enzyme activity in intact cells for the first time. Here we present an ASM FRET probe specifically optimized for 2-photon excitation. To facilitate probe characterization and comparison to the previous probe, we mixed the two intact probes with defined amounts of the probes' ceramide cleavage products and mounted them on lipid beads. Directly excited NBD FRET acceptor fluorescene proved to be a useful means of reference and showed that the new probe is brighter, albeit only moderately, than the previous one. The new probe was then used to detect inhibition by various ASM inhibitors microscopically for the first time. Also in cells, directly excited acceptor fluorescence proved to be a useful parameter in addition to FRET to visualize inhibition of ASM.

Full text of DOI:10.1016/j.bmc.2021.116303

Bioorg. Med. Chem. 44 (2021) 116303
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of a new two photon excitable acid
sphingomyelinase FRET probe
,
Zain H. Mohameda b, Cosima Rheinc, Benjamin Schmidd, Philipp Tripald,
,
*
Johannes Kornhuberc, Christoph Arenza
a
¨
Institute for Chemistry, Humboldt Universitat zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany
b Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt
c
¨
Department of Psychiatry and Psychotherapy, Friedrich-Alexander Universitat Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
d
¨
Optical Imaging Centre Erlangen (OICE), Friedrich-Alexander Universitat Erlangen-Nürnberg (FAU), Erlangen, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Recently, FRET probes for acid sphingomyelinase (ASM) have enabled the observation of enzyme activity in
intact cells for the first time. Here we present an ASM FRET probe specifically optimized for 2-photon excitation.
To facilitate probe characterization and comparison to the previous probe, we mixed the two intact probes with
defined amounts of the probes’ ceramide cleavage products and mounted them on lipid beads. Directly excited
NBD FRET acceptor fluorescene proved to be a useful means of reference and showed that the new probe is
brighter, albeit only moderately, than the previous one. The new probe was then used to detect inhibition by
various ASM inhibitors microscopically for the first time. Also in cells, directly excited acceptor fluorescence
proved to be a useful parameter in addition to FRET to visualize inhibition of ASM.
Sphingolipids
Two-photon microscopy
Enzyme inhibitors
Antidepressants
¨
Forster resonance energy transfer (FRET)
FRET probes
1. Introduction
lipids.6
ASM is constitutively active in the endo-lysosomal compartment and
Sphingolipids act as key regulators of many cellular functions
including cell proliferation, differentiation, and apoptosis.1 Sphingo-
myelin (SM) is the most abundant sphingolipid in mammalian cells and
has an essential role in eukaryotic cell viability.2 SM is found prefer-
entially in the outer leaflet of the plasma membrane and its high packing
density and affinity for cholesterol are key to the formation of micro-
domains also known as lipid rafts.3 These highly ordered and protein-
rich membrane sub-domains have been reported to play pivotal roles
in signal transduction. Some time ago, the existence of sphingolipid-
enriched microdomains < 20 nm in size and with lifetimes of up to
20 ms has been shown experimentally in living cells.4 Among a number
of isoenzymes, acid sphignomyelinase (ASM) is thought to play a para-
mount role in the cleavage of SM in the context of various diseases.5
According to one model, the ASM-mediated cleavage of sphingomyelin
within the plasma membrane affects number, size and properties of
sphingolipid-enriched microdomains and ultimately the activity of
membrane proteins. According to an alternative model, but not in strict
contradiction to the previous one, abundant sphingomyelin serves as a
reservoir for rapid ASM-mediated formation of downstream signaling
an inherited deficiency of its activity leads to the fatal Niemann-Pick
disease of types A or B.7 Upon stimulation, ASM is secreted to the
extracellular space and is found highly elevated e.g. in the urine of pa-
tients suffering from peritonitis.8 Inhibition of ASM is considered to be a
promising strategy for the treatment of diseases like acute lung injury,9
major depression10 or metastasis of melanoma.11 Various stresses like
UV irradiation or serum deprivation or inflammatory effectors like in-
terleukins or TNFa have been reported to trigger the translocation of
ASM from the lysosomes to extracellular space. Based on this evidence,
most investigators favour the model that the secreted form of ASM is the
crucial factor and actual mediator in many pathologies by acting on the
outer leaflet of the plasma membrane in an autocrine and paracrine
manner.5 However, in vitro tests have repeatedly shown the inactivity of
the enzyme at neutral pH, challenging the model of ASM-mediated
cleavage at the plasma membrane.12 Lipid species are highly mobile
within cells and current analytical methods for measuring ASM lack
spatio-temporal resolution, so for many pathologies the exact mecha-
nism for ASM remains unclear. To address the growing interest to
determine ASM activities in intact cells, we have developed two
* Corresponding author.
E-mail address: arenzchr@hu-berlin.de (C. Arenz).
https://doi.org/10.1016/j.bmc.2021.116303
Received 1 April 2021; Received in revised form 27 June 2021; Accepted 4 July 2021
Available online 13 July 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
different ASM FRET probes.13,14 Besides their high sensitivity and
spatio-temporal resolution, FRET probes have some distinct advantages
over other fluorescent probes. Unlike quenched probes or turn-on
probes, for which the observed fluorescence intensity is dependent on
many factors like cellular delivery or transport, FRET probes offer the
possibility of ratio imaging. From the relative ratio of FRET donor and
acceptor fluorescence, the cleavage status of the probe can be deduced,
independent of its concentration.15
moderate yields, but the desired 4 was finally collected in pure form, as
confirmed with 1H NMR and mass data.
For the coupling of the synthesized bromo-coumarin carboxylic acid
4 to a six-carbon atom linker, several routes were tested (not shown).
The optimal route to the intermediate 7 turned out to be the coupling
with the t-butyl ester of the aminohexanoic acid 5 to yield 6. The latter
was treated with trifluoracetic acid and after evaporation of the solvent
the pure product 7 was achieved in 60% yield over two steps without the
need for further purification. The protected sphingosine intermediate 8,
which was developed and described by us previously,14 was treated with
hydrazine to remove the phthalimide protection group and the free
amine 9 was reacted with NBD-chloride in the presence of DIPEA to
yield 10 in 85% yield (Scheme 2). Next, 10 was treated with hydro-
chloric acid in dioxane to remove the remaining protecting groups and
the crude intermediate 11 was reacted with the active ester of the
bromo-coumarin hexanoic acid 7 to afford the target FRET probe 12.
Incubation of 12 with recombinant human ASM led to a pronounced
and time-dependent change in fluorescence (Fig. 1A). The fluorescence
of the new coumarin increased significantly by about tenfold with a
concomitant decrease of the NBD acceptor fluorescence to roughly one
third. The limited NBD fluorescence decrease compared to that of the
previously reported FRET probe could be attributed to the counteracting
effect of the new coumarin derivate emission, which partially over-
lapped with the emission spectra of NBD. Indeed, the new coumarin is
somewhat red-shifted compared to the 1st generation coumarin.14,18,23
The Michaelis constant (KM) of the enzymatic cleavage of the probe 12 is
We previously succeeded in using our first-of-its-kind probe to
monitor cellular ASM activity in two-photon (2P) microscopy. This
technique uses the energy from two coincident photons to excite a
specific fluorophore. This effectively doubles the excitation wavelength
and prevents UV irradiation of cells and tissues. Another advantage is
that the extremely high photon density required for 2P excitation is
achieved in only a single focal point, resulting in excellent three-
dimensional resolution. 2P microscopy provides a particularly high
penetration depth and thus offers advantages in the examination of
intact tissues such as brain slices, embryos, whole organs, and live
animals.16,17
Our previous probe was not particularly bright in 2P applications, so
relatively high probe concentrations were required for microscopy. This
made complete cleavage of the probe slow and possibly reduced the
temporal resolution of the processes under investigation.14 Since we did
not use a 2P-optimized dye in our initial study, we saw considerable
room for improvement of our original probe. Here, we present a new
ASM FRET probe that is better suited for 2P excitation (2PE) due to the
incorporation of a recently reported bromocoumarin derivative with a
particularly high two-photon excitation cross-section.18
8
μ
M and the maximal velocity (Vmax) value is 36.9
μmol/(mg h)
compared to 8.9 M and 3 mol/(mg h) for our recently reported visible
μ
μ
range FRET probe, reflecting a rapid turnover of the new probe by ASM.
These observations confirm that low molecular weight dyes are
preferred with respect to catalytic efficiency and similarity to the native
substrate. The ability of the new FRET probe 12 to differentiate between
different inhibitor concentrations and different concentrations of ASM
was also tested and confirmed (Fig. 1B and C).
2. Results and discussion
The new coumarin derivative was synthesized with modifications to
the route reported previously (Scheme 1). Benzyl propiolate 1 was
synthesized through Steglich esterification of propiolic acid with benzyl
alcohol,19 which afforded 1 in high yield and purity.20,21 The ester 1 was
then reacted in a cyclic addition with the commercially available 6,7-
dihydroxy-coumarin22 to yield the intermediate 2 in almost quantitative
yield.
The incubation of the probe 12 with Hela cell lysate at pH 5 led to a
rapid change in probe fluorescence, while no change was observed at pH
7.4 (Fig. 1D), suggesting that neutral sphingomyelinases are not able to
efficiently cleave the probe. This was further confirmed by incubating
probe 12 with either the recombinant nSMase2 enzyme or ASM Ko MEF
lysate (Fig. S2).
Next, 2 was hydrogenated to achieve the carboxylic acid 3, which
was then brominated using N-bromo succinimide (NBS). Our original
effort to first brominate and then deprotect the carboxylic acid was
unsuccessful, as catalytic hydrogenation obviously led to partial sub-
stitution of the previously introduced bromine atom by hydrogen. This
reaction yielded in about 25% of the de-brominated compound as an
inseparable side product (Fig. S1). Due to this issue, we had to purify the
polar free acid through two consecutive steps, which resulted in only
After in vitro characterization of 12, the suitability of the probe in
live cell microscopy was tested. To this end, 12 was applied as BSA-
complex to Human H4 neuroglioma cells that were transiently trans-
fected with an ASM-cherryN1 construct and incubated for 2 h at a final
concentration of 1 µM, before the cells were visualized using 2P fluo-
rescence microscope. 2P excitation of the bromo-coumarin at 720 nm
Scheme 1. Synthesis of the bromo-coumrin derivative 7. a) DCC, DMAP, DCM,12 h, 80%; b) DMAP, dry DCM, 7 h, 97%; c) Pd/C, H2 (1 atm) , dry THF, 12 h,75%; d)
NBS, NaOAc, dry DMF,12 h, 50%. e) DMAP, NHS, EDCl , dry DCM, 0 ◦C then rt, 22 h, crude f) 6AHA, thionyl chloride, 2 h, NaHCO3 in t-BuOH, 2 h, 75%; g) 5, dry
DCM, EDCl, HOBt, rt, 20 h, 75%; h) TFA, dry DCM, rt, 3 h, 80%.
2

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
Scheme 2. Synthesis of the new probe 12. a) Hydrazine hydrate 64%, MeOH/CHCl3 (1:1), rt, 4.5 h, 82%, b) NBD-Cl, DIPEA, MeOH/CHCl3 (1:3), rt, 4 h, 85%; c) 4 M
HCl (in 1,4-dioxan), MeOH, 70 ◦C, 2 h, Used directly without further purification; d) dry DIPEA, dry DCM, active ester of 7, 24 h, 39%.
resulted in the detection of a fluorescence signal in both, the coumarin
and NBD channels. Notably, the ratio of fluorescence intensities (cou-
marine/NBD) was higher for the new probe 12, compared to the 1st
genereation probe (Figs. S3 and 4) and higher in the ASM overexpressing
cells compared to wild type cells (not shown). We therefore concluded
that the probe was taken up and metabolized by live cells.
directly acting inhibitor, for 1 h and 24 h respectively. Subsequently, 12
was added and cells were incubated for another 1 h or 24 h, leading to
total incubation times of 2 h, (2x) 25 h and 48 h, respectively. We ex-
pected stronger activity of Arc39 for short incubations and superiority of
fluoxetine for long incubations because of its mode of action as a slow-
acting lysosomotropic agent. A representative image of probe treated
cells is presented in Fig. 3. The quantitation for these experiments is
shown in Fig. 4.
In order to compare their brightness in cells, 12 or the previously
reported probe14 were incubated with cells and excited (2P) with
different laser intensities. It was hoped that this would allow for a simple
differentiation of the different probe brightnesses. However, when laser
power was reduced from 10 to 5 neither probe 12 nor the 1st generation
probe gave rise to a detectable fluorescence (Fig. S5). We therefore
concluded that any differences in probe excitability were more subtle
than expected.
Following our protocol developed for the beads, not only the
coumarin/NBD emission ratio after 2P coumarin excitation (FRET
mode) was monitored, but also fluorescence derived from direct NBD
excitation (Fig. 4). After 24 h probe incubation the FRET mode
coumarin/NBD ratio showed a significant reduction only for the 24 h
pre-incubation with fluoxetine. For the other experiments, only subtle
reductions were obtained, which we attributed to the fact that the ma-
jority of the probe was converted and differences in cleavage kinetics
were already marginalized (Fig. 4A). To our surprise, a similar picture
was seen for the 1 h probe incubation. Here, the obvious change in FRET
rate for 24 h fluoxetine treatment was more pronounced and also 1 h
incubation with fluoxetine showed significant reduction in the
coumarin/NBD emission rate. Again, only small change was observed
with Arc39 irrespective of incubation time. Moreover, the FRET ratio of
cells in the absence of inhibitors was almost the same after 1 h or 24 h of
incubation with the probe, which also supports the thesis that most of
the probe was already metabolized after 1 h, at least in absence of in-
hibitors. When normalized to direct NBD excitation/emission, the FRET-
derived NBD (FRET NBD/direct NBD), was markedly increased for all
inhibitor-treated samples (Fig. 4B). In the 1 h probe incubation setup, for
which these differences were again more pronounced, the effect for 24 h
pre-incubation fluoxetine was again greatest, followed by 1 h pre-
incubation with fluoxetine or Arc39. Interestingly, 24 h pre-incubation
with Arc39 was slightly less efficient than 1 h pre-incubation time,
suggesting that the Arc39 mediated inhibition is more transient than
that of fluoxetine. Interestingly, the alternative readout resulted in
highly significant differences between the individual experiments,
which were also visible before but only as minor trends. Also, the
We therefore decided to compare the two probes in a cell-free sys-
tem, immobilized on “lipid beads”. For this purpose, liposomes with a
defined amount of probe were prepared and mounted onto de-lipidated
silica beads. This excluded any differences due to different cleavage
kinetics or cellular penetration. The primary 2P microscope visualiza-
tion of the silica beads coated with 12 is displayed in Fig. 2. After an
initial experiment confirmed the feasibility and robustness of the
method, it was decided to compare lipid beads loaded with 1:1 mixtures
of the probes with their respective synthetic cleavage products
(coumarin labeled ceramides). As both types of beads contained the
same amount of NBD, the respective coumarine fluorescence intensities
obtained upon 2P excitation of coumarin at 720 nm were normalized to
the NBD fluorescence intensities after direct excitation of NBD. In this
setup, bright signals were obtained not only for laser power 10 but also
for 5 and 3. Interestingly, for each excitation intensity, the fluorescence
of 12 was found to be about 20% higher compared to the previous probe.
This was, however, lower than originally expected.
Next, the probe’s suitability to monitor ASM activities in presence of
different inhibitors in live cells was tested. Accordingly, H4 human
neuroglioma cells were treated either with fluoxetine, a widely used
antidepressant and a nonspecific but highly effective functional inhibi-
tor of acid sphingomyelinase (FIASMA),24 or Arc39,25 a specific and
3

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
Fig. 1. A) Spectral change of probe 12 upon cleavage by ASM at different time intervals: Black = 0 min, blue = 600 min, red- brown = 1 h-7 h. B) Fluorescence
increase of the FRET probe 12 coumarin dye upon ASM cleavage in presence of 0 nM (black), 20 nM (red), 100 nM (green), 500 nM (blue) ASM inhibitor Arc39. C)
Time dependent fluorescence increase of the FRET probe 12 Coumarin dye in presence of ASM (green = 0.002 µg/ml, blue = 0.02 µg/ml, red = 0.20 µg/ml, black = 2
µg/ml) D) Probe 12 incubated with Hela cell lysates at different pH values: red = pH 5, blue = pH 7.4, black: Control experiment with the probe only. Default values:
Probe conc. = 1 μM, λ Ex. = 347 nm, λ Em. = 430 nm, conc. ASM = 0.2 µg/ml, pH = 5.
changes in coumarin fluorescence were significant for all samples, when
normalized to directly excited NBD (Fig. S6).
this study once more underscores for the strength of dual-labeled probes
compared to simpler turn-on and turn-off probes.
3. Conclusion
4. Experimental section
The exact mechanisms of acid sphingomyelinase in various pathol-
ogies can only be revealed by determining enzyme activity with high
spatial and temporal resolution. In our opinion, these conditions can
only be achieved at reasonable effort by our ASM FRET probes. Here we
demonstrate the synthesis of a new ASM FRET probe with moderately
increased sensitivity. For its accurate 2P characterization, the intact
probe in mixture with the synthetic cleavage product was applied to so-
called lipid beads and normalized using directly excited NBD fluores-
cence. This had the advantage of characterizing the cleavage of the
probe independent of FRET efficiency or partial spectral overlap of the
two dyes. In addition, the significantly reduced cleavage of the probe in
Arc39 or FIASMA-treated cells was demonstrated for the first time,
which can be seen as a clear indication that no other significant enzyme
activity exists that can cleave the ASM probe in cells. This thesis was
supported by in vitro experiments using cell lysates at neutral pH or with
recombinant neutral sphingomyelinase and with lysates of ASM knock-
out cells. The cell-based assays suggested that in absence of inhibitors a
major portion of the probe was already metabolized after 1 h of incu-
bation. In these experiments, the fluorescence obtained from direct
excitation of the FRET acceptor provided a very useful readout mode. In
this setup, the inhibitor activities were clearly highlighted. Therefore,
4.1. Life-cell microscopy
Human H4 neuroglioma cells (Promochem, Wesel, Germany) were
cultured in DMEM supplemented with 10% (v/v) FCS and 4 mM L-
glutamine. Cells were maintained at 37 ◦C in a humidified atmosphere
with 5% CO2 and monitored for potential mycoplasma infection using
the MycoAlert Mycoplasma Detection Kit (Lonza, Cologne, Germany).
All reagents used for cell culture were purchased from Biochrom (Berlin,
Germany). Transient transfections were carried out in 6-well trays with
1.5x10e5 H4 cells per well. Generation of the ASM-CherryN1 plasmid
was described earlier.26 24 h after seeding cells, the media was changed
and cells were transfected with 1 μg of plasmid DNA per well, using X-
tremeGENE DNA transfection reagent from Roche (Basel, Switzerland)
according to manufacturer’s instructions. Alternatively, H4 cells were
treated with fluoxetine (10 µM) (Sigma Aldrich, St. Louis, MO, USA) or
Arc39 (10 µM) for 1 h and 24 hrs, respectively. 1 h before analysis, the
BSA-complexed FRET probe was added to the media at a final concen-
tration of 1 µM. Cells were washed with PBS directly before live cell
microscopy. Lipid beads were diluted in PBS (1:20). Live cell microscopy
was performed on a Zeiss LSM 880 NLO equipped with a 680–1300 nm
tunable and fixed 1040 nm 2-photon laser from Newport SpectraPhysics
4

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
Fig. 2. Fluorescence microscopy graphs of the lipid beads coated with probe 12 labeled liposomes. A-C 100% intact FRET probe. A) After two-photon excitation of
coumarin at 720 nm, B) NBD after direct 2P excitation at 960 nm, and C) the merge of both graphs. D) 2P-excited coumarine fluorescence normalized to directly
excited NBD fluorescence of lipid beads loaded with 1:1 mixtures of intact probe and respective coumarine ceramide of 12 (blue bars) or the 1st generation FRET
probe (red bars) at different laser powers (LP 3, LP 5, LP 10).
and the ZEN 2010 software (Carl Zeiss MicroImaging). Two-photon
excitation at 720 nm resulted in the detection of the uncleaved probe
that indicates sphingomyelin (NBD signal, green) and the cleaved probe
that indicates ceramide (Coumarin signal, red). Signals were referenced
to the direct NBD signal obtained by two-photon excitation at 960 nm.
Images were quantified using Fiji / Image J. Figures were assembled
using OMERO.
4.3. Chemical Synthesis
4.3.1. Benzyl propiolate (1)
4.2. Preparation of recombinant human ASM
A solution of DMAP (30 mg, 0.24 mmol, 0.01 eq.) and DCC (5.16 g,
25 mmol, 1 eq.) in DCM (30 mL) was added slowly over 1.5 h to a so-
lution of propiolic acid (1.54 mL, 24.84 mmol, 1 eq.) and benzyl alcohol
(2.8 mL, 27.03 mmol, 1.1 eq.) in DCM (30 mL) at 0 ◦C. The suspension
was then stirred for overnight at room temperature, monitoring through
by TLC (EtOAc: Cyclohexan 1:2). Upon completion, the mixture was
filtered through a layer of Celite and the filtrate concentrated in vacuo.
The product was purified using column chromatography (7% EtOAc in
Cyclohexan) to yield a colorless oil (3.17 g, 80%).
Human acid sphingomyelinase (ASM) was expressed in insect Sf9
cells and purified to homogeneity, as described previously.27,28 The re-
combinant enzyme purified in the presence of Zn2+ (0.1 mM) had an
activity of 23 nmol hꢀ 1 mLꢀ 1 as determined in a micellar assay system.
1H NMR (500 MHz, CDCl3) δ : 7.41–7.33 (m, 5H, CHar), 5.23 (s, 2H,
CH2), 2.89 (s, 1H, CH).
5

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
Fig. 3. Exemplary fluorescence microscopy of H4 cells treated with fluoxetine before adding probe 12 for 1 h. Indicated are control conditions and stimulations with
fluoxetine for 1 h and 24 hrs. From left to right: After two-photon excitation of coumarin at 720 nm, NBD after two-photon excitation at 870 nm and the merge of
both graphs. Scale bars indicate 10 µm.
13
13
–
–
–
–
C NMR (126 MHz, CDCl ) δ : 152.43 (C O), 134.35 (C ), 128.74
C NMR (126 MHz, CDCl3) δ : 167.70 (C O), 161.32 (C O),
–
–
3
ar
(CHar), 128.70 (CHar), 128.59 (CHar), 75.05(≡CH) , 74.56(≡C), 67.93
152.51(C-Me), 150.69(Ccou), 150.59 (Ccou), 144.63 (Ccou), 135.20 (Cph),
128.77 (CHph), 128.71 (CHph), 128.59 (CHph), 114.07 (Ccou), 112.48 (C
= CH), 109.59 (CHcou), 102.37 (CHcou), 98.61 (OCH), 67.29 (CH2),
40.22 (CH2), 19.28 (CH3).
(CH2).
UPLC-MS (ESI): C10H8O2 Na [M + Na]+: m/z Calc.: 183.0, found:
183.1
UPLC-MS (ESI): C20H17O6 [M + H]+: m/z Calc.: 353.1, found: 353.1
4.3.2. Benzyl 2-(8-methyl-6-oxo-6H-[1,3]dioxolo[4,5-g]chromen-2-yl)
acetate (2)
4.3.3. 2-(8-Methyl-6-oxo-6H-[1,3]dioxolo[4,5-g]chromen-2-yl)acetic acid
(3)
A suspension of 2 (1.1 g, 3.1 mmol, 1 eq.) and Pd/C (10%, 35.9 mg,
0.012 mmol, 0.01 eq.) in dry THF (375 mL) was stirred under hydrogen
atmosphere (p(H2) = 1 atm) at room temperature overnight. After
complete conversion TLC (10% MeOH in DCM) of 2 the mixture was
filtered through Celite and washed with MeOH. The solvent was
removed under reduced pressure and the residue was purified by column
chromatography (gradient from 1.5% MeOH to 8% MeOH in DCM)
yielding (532 mg, 65%) of 3 as an orange solid.
To a solution of 6,7-dihydroxy-coumarin (500 mg, 2.6 mmol, 1 eq.)
in dry DCM (25 mL), benzyl propiolate 1 (500 mg, 3.1 mmol, 1.2 eq.)
and DMAP (507 mg, 4.15 mmol, 1.6 eq.) were added under argon at-
mosphere at room temperature. After being stirred for 7 h, the solvent
was removed under reduced pressure. The product was purified using
column chromatography (25% EtOAc in Cyclohexan) afforded 2 (892
mg, 97%) as a white crystalline solid.
1H NMR (500 MHz, CDCl3) δ : 7.39–7.31 (m, 5H, CHph), 6.91 (s, 1H,
CHcou), 6.79 (s, 1H, CHcou), 6.64 (t, J = 5.1 Hz, 1H, OCH), 6.16 (d, J =
1.2 Hz, 1H, C = CH), 5.19 (s, 2H, CH2), 3.07 (d, J = 5.1 Hz, 2H, CH2),
2.35 (d, J = 1.2 Hz, 3H, CH3).
1H NMR (300 MHz, DMSO‑d6) δ 7.25 (s, 1H , CHcou), 7.07 (s, 1H,
CHcou), 6.66 (t, J = 5.2 Hz, 1H, OCH), 6.22 (d, J = 1.1 Hz, 1H, C = CH),
3.04 (d, J = 5.2 Hz, 2H, CH2), 2.35 (d, J = 1.1 Hz, 3H, CH3).
13
–
–
–
C NMR (75 MHz, DMSO) δ 169.75 (C O), 160.63 (C O), 154.07
–
6

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
mixture was stirred (2d) till complete conversion of 3 monitored by TLC
(0.5% MeOH in DCM). The mixture was extracted three times with DCM.
The combined organic layers were dried over MgSO4 and concentrated
under reduced pressure. The resulting residue was purified by column
chromatography (gradient from 0.5% MeOH to 5% MeOH in DCM)
yielding 7 (163 mg, 50%.)
1H NMR (500 MHz, (CD3)2CO) δ 7.23 (s, 1H, CHcou), 6.88 (s, 1H,
CHcou), 6.68 (t, J = 5.2 Hz, 1H, OCH), 3.10 (d, J = 5.2 Hz, 2H,CH2), 2.58
(s, 3H, CH3).
13
–
–
–
C NMR (126 MHz, (CD ) CO) δ 169.20 (C O), 157.15 (C O),
–
3 2
152.31 (C-Me), 151.87 (Ccou), 149.87 (Ccou), 146.10 (Ccou), 114.44
(Ccou), 111.44 (CHcou), 110.42 (C-Br), 103.98 (CHcou), 98.44 (OCH),
40.00 (CH2), 20.21 (CH3).
UPLC-MS (ESI): C13H10BrO6 [M + H]+: m/z Calc.: 341.0, found:
341.0
4.3.5. Tert-butyl 6-aminohexanoate (5)
Aminohexanoic acid (2.0 g, 15.25 mmol, 1 eq.) was dissolved in
thionyl chloride (5.0 mL, 68.6 mmol, 4.5 eq.). This solution was stirred
at rt for 2 h and concentrated in vacuo to dryness. To the reddish semi-
solid product, a slurry of sodium bicarbonate (2.6 g, 30.4 mmol, 2.0 eq.)
in t-BuOH (9.0 mL; 86.6 mmol; 5.7 eq.) was added and the slurry was
stirred at rt for another 2 h. The residual t-BuOH was removed in vacuo
at 40 0C. The thick white slurry was diluted with ethyl acetate and
washed with of 1 M NaOH (2x), H2O (2x) and brine (1x). The organic
layer was dried over Na2SO4 and concentrated to afford 5 (2.14 g, 75%)
as a colorless oil.
1H NMR (500 MHz, CDCl3) δ 2.75 (s, 2H, NH2), 2.20 (t, J = 7.5 Hz,
2H, CH2), 1.58 (dt, J = 15.2, 7.5 Hz, 2H, CH2), 1.46 (dd, J = 14.4, 7.1
Hz, 2H, CH2), 1.42 (s, 9H, C(CH3)3), 1.33 (ddd, J = 15.7, 9.1, 6.1 Hz, 2H,
CH2).
13
–
C NMR (126 MHz, CDCl3) δ 173.28 (C O), 80.12 (C(Me) ), 35.64
–
3
(CH2), 28.24 (CH3), 26.51 (CH2), 25.03 (CH2).
UPLC-MS (ESI): C10H21NO2 [M + H]+: m/z Calc.: 188.2, found:
188.2
4.3.6. Tert-butyl 6-(2-(7-bromo-8-methyl-6-oxo-6H-[1,3]dioxolo[4,5-g]
chromen-2-yl) acetamido)hexanoate (6)
Fig. 4. Different modes of probe analysis in presence of validated ASM in-
hibitors. A conventional ratio coumarin/NBD fluorescence after 2P-excitation
of coumarin, only (after 24 h or 1 h incubation with 12. B Alternative evalu-
ation by ratio fluorescence of NBD (after 2P excitation of coumarin)/fluores-
cence of NBD after direct excitation of NBD. Fluox = fluoxetine, ArC = Arc39.
The times given for the inhibitors are pre-incubation times before
probe addition.
(C-Me), 150.86 (Ccou), 150.41 (Ccou), 144.74 (Ccou), 113.83 (Ccou),
111.67 (C = CH), 110.64 (CHcou), 103.29 (CHcou), 98.24 (OCH), 19.11
(CH3).
To a solution of 4 (175 mg, 0.51 mmol, 1 eq.) and tert-butyl 6-amino-
hexanoate 5 (192.2 mg, 1.02 mmol, 2 eq.) in dry DCM (25 mL) were
added EDCl (118 mg, 0.61 mmol, 1.2 eq.) and HOBt (81 mg, 0.61 mmol,
1.2 eq.) under argon atmosphere at room temperature. The reaction was
monitored by TLC. After completion of the reaction (20 h), it was
washed with 1 N HCl and saturated NaCl solution, dried over Na2SO4
and concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography (gradient from 0.5%
MeOH to 1% MeOH in DCM) yielding 6 (192 mg, 75%) as an off white
solid.
UPLC-MS (ESI): C13H11O6 [M + H]+: m/z Calc.: 263.1, found: 263.1
4.3.4. 2-(7-Bromo-8-methyl-6-oxo-6H-[1,3]dioxolo[4,5-g]chromen-2-yl)
acetic acid (4)
1H NMR (300 MHz, CDCl3) δ 6.90 (s, 1H, CHcou), 6.66 (s, 1H, CHcou),
6.62 (t, J = 5.3 Hz, 1H, OCH), 6.43 (t, J = 5.5 Hz, 1H, NH), 3.28 (dd, J =
13.1, 6.7 Hz, 2H, CH2), 2.90 (d, J = 5.3 Hz, 2H, CH2), 2.48 (s, 3H, CH3),
2.20 (t, J = 7.3 Hz, 2H, CH2), 1.66–1.47 (m, 4H, 2x CH2), 1.40 (s, 9H, C
(CH3)3), 1.38–1.28 (m, 2H, CH2).
To a solution of 3 (250 mg, 0.95 mmol, 1 eq.) in dry DMF (10 mL)
were added NBS (508 mg, 2.85 mmol, 3 eq.) and NaOAc (15.5 mg, 0.19
mmol, 0.1 eq.) under argon atmosphere at room temperature. The
7

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
13
was stirred for 1 h at 0 ◦C then at rt for overnight with protection from
light. Then, TLC and UPLC-MS indicated complete conversion of the
starting material. The reaction mixture was diluted with DCM (25 mL)
and washed with 0.5 M KHSO4 (2x 20 mL) and brine (20 mL). The
organic layer was dried (Na2SO4), filtered and evaporated to dryness.
The obtained material was directly used without further purification.
–
–
–
C NMR (75 MHz, CDCl ) δ 173.08 (C O), 166.45 (C O), 157.26
–
3
–
(C O), 151.10 (C-Me), 150.82 (Ccou), 148.62 (Ccou), 145.23 (Ccou),
–
113.49 (Ccou), 111.21 (CHcou), 109.98 (C-Br), 102.49 (CHcou), 97.97
(OCH), 80.18 (C(Me)3) , 42.06 (CH2), 39.43 (CH2), 35.30 (CH2), 28.96
(CH2), 28.10 (CH3), 26.22 (CH2), 24.49 (CH2), 20.01 (CH3).
UPLC-MS (ESI): C23H28BrNO7Na [M + Na]+: m/z Calc.: 532.1, found:
532.1
4.3.9. tert-butyl ((2S,3R,E)-1-(((2-aminoethoxy)(hydroxy)phosphoryl)
oxy)-3-(methoxy methoxy)octadec-4-en-2-yl)carbamate (9)
4.3.7. 6-(2-(7-bromo-8-methyl-6-oxo-6H-[1,3]dioxolo[4,5-g]chromen-2-
yl)acetamido)hexanoic acid (7)
Compound 814 (110 mg, 0.16 mmol, 1 eq.) was dissolved in 6 mL
MeOH/CHCl3 (1:1). Hydrazine hydrate (64%, 30 μL, 0.63 mmol, 4 eq.)
was added and the mixture stirred at rt for 4.5 h when TLC indicated
complete consumption of the starting material. The solvent was evap-
orated and the crude material was dried under high vacuum. The ma-
terial was adsorbed on Celite® and purified using short flash column
chromatography (CHCl3: MeOH: H2O = 100:15:1 → 65:25:3). The title
compound was isolated as a colorless solid (73.4 mg, 82%).
1H NMR (400 MHz, Pyr-d5) δ 7.74 (s, 1H,NH), 5.82 (d, J = 15.1 Hz,
1H, =CHCH2), 5.76–5.61 (m, 1H, =CHCH), 4.94 (d, J = 5.9 Hz, 1H,
OCHHO), 4.76 (d, J = 5.9 Hz, 1H, OCHHO), 4.67–4.44 (m, 4H, 2 ×
CH2OP), 4.39 (s, 1H, CHNH), 3.51 (s, 5H, CH2NH2, OCH3), 2.08 (s, 2H,
CH2), 1.53 (d, J = 37.2 Hz, 9H, OC(CH3)3), 1.36 (d, J = 39.4 Hz, 20H, 10
× CH2), 0.90 (t, J = 6.7 Hz,3H, CH3).
To a solution of 6 (279 mg, 0.55 mmol, 1 eq.) in dry DCM (3 mL) was
added dropwise at RT 1 mL TFA. After stirring (3 h) at RT, TLC and
UPLC-MS indicated complete deprotection of the t-butyl ester. the sol-
vent and TFA residues were evaporated under reduced pressure to give
the free acid 7 as a white pure solid (198.5 mg, 80% yield).
1H NMR (500 MHz, DMSO‑d6) δ 8.08 (t, J = 5.6 Hz, 1H, NH), 7.39 (s,
1H, CHcou), 7.13 (s, 1H, CHcou), 6.67 (t, J = 5.4 Hz, 1H, OCH), 3.06 (dd,
J = 12.7, 6.8 Hz, 2H, CH2), 2.85 (d, J = 5.4 Hz, 2H, CH2), 2.54 (s, 3H,
CH3), 2.19 (t, J = 7.4 Hz, 2H, CH2), 1.55–1.44 (m, 2H, CH2), 1.44–1.35
(m,123H, CH2), 1.33–1.21 (m, 2H, CH2).
13C NMR (126 MHz, Pyr-d5) δ 157.16 (COO), 137.40 (=CHCH2),
128.15 (=CHCH), 94.38 (OCH2O), 78.79(OC(CH3)3), 77.44 (CHO),
65.81 (CH2OP), 63.25 (CH2OP), 56.11 (OCH3), 55.77 (NCHCH2OP),
33.17 (CH2CH = ), 32.60 (CH2), 30.47 (CH2), 30.43 (CH2), 30.41 (CH2),
(CH2), 30.10 (CH2), 30.06 (CH2), 29.94 (CH2), 29.15 (CH2), 23.42
(CH2), 14.76 (CH3).
–
–
–
C NMR (126 MHz, DMSO‑d ) δ 174.43 (C O), 165.99 (C O),
–
6
–
–
31P NMR (202 MHz, pyr-d5) δ = 2.82
156.49 (C O), 152.04 (C-Me), 150.67 (Ccou), 148.41 (Ccou), 144.85
UPLC-MS (ESI): C27H56N2O8P [M + H]+: m/z Calc.: 567.4, found:
567.4
(Ccou), 113.20 (Ccou) , 111.46 (CHcou), 108.93 (C-Br), 103.45 (CHcou),
97.70 (OCH), 40.77 (CH2), 40.02 (CH2), 38.36 (CH2), 33.60 (CH2),
28.70 (CH2), 25.92 (CH2), 24.18 (CH2), 19.98 (CH3),
UPLC-MS (ESI): C19H21BrNO7 [M + H]+: m/z Calc.: 454.1, found:
454.1
4.3.10. 4-((2S,3R,E)-2-(tert-butyloxycarbonylamino)-3-
(methoxymethoxy)octadec-4-en-1 phosphoethylamino)-7-nitrobenzo-2-oxa-
1,3-diazol (10)
4.3.8. 2,5-dioxopyrrolidin-1-yl 6-(2-(7-bromo-8-methyl-6-oxo-6H-
[1,3]dioxolo[4,5-g]chromen-2-yl)acetamido)hexanoate (Active ester
of 7)
The primary amine 9 (250 g, 0.44 mmol, 1 eq.) was dissolved in 10
mL MeOH/CHCl3 (1:3). DIPEA (300 μL, 1.76 mmol, 4 eq.) and. NBDCl
(264 mg, 1.32 mmol, 3 eq.) were added and stirring was continued at rt
away from light. After 4 h, all volatiles were evaporated under a stream
of air and the residue was dissolved in 20 mL CHCl3. The solution was
washed twice with 0.1 M aq. HCl (25 mL each) with CHCl3 backwash.
The organic fractions were collected and evaporated in vacuo. The or-
ange crude obtained was subjected to column chromatography (CHCl3:
MeOH:H2O = 100:15:1 → 66:10:1 to afford (270 mg , 85%) of the title
product as a yellowish brown solid.
Under argon atmosphere, 7 (0.050 g, 0.11 mmol, 1 eq.) was dis-
solved in dry DCM (10 mL) and DMAP (26.9 mg, 0.22 mmol, 2 eq.), as
well as N-hydroxysuccinimide (25.3 mg, 0.22 mmol, 2 eq) were added.
At 0 ◦C, EDCl (46.4 mg, 0.242 mmol, 2.2 eq.) was added and the mixture
1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.6 Hz, 1H), 6.05 (d, J =
8.0 Hz, 1H), 5.59–5.23 (m, 1H), 5.02 (dd, J = 14.8, 8.0 Hz, 1H), 4.38 (d,
8

Z.H. Mohamed et al.
Bioorganic & Medicinal Chemistry 44 (2021) 116303
J = 6.4 Hz, 1H), 4.30–4.18 (m, 6H), 3.94 (s, 1H), 3.81–3.71 (m, 2H),
3.52 (s, 2H), 3.07 (s, J = 6.4 Hz, 3H), 1.77 (d, J = 6.6 Hz, 2H), 1.14 (s,
9H), 0.99 (s, 22H), 0.62 (t, J = 6.6 Hz, 3H).
1019.1
Declaration of Competing Interest
13C NMR (101 MHz, CDCl3) δ 156.14, 144.12, 137.46, 136.93,
125.58, 98.50, 93.28, 79.24, 76.35, 64.39, 62.64, 55.09, 53.74, 32.05,
31.58, 29.32, 29.14, 29.01, 28.93, 28.79, 27.83, 22.31, 13.56.
31P NMR (162 MHz, CDCl3) δ ꢀ 1.42.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
UPLC-MS (ESI): C33H55N5O11P [Mꢀ H]-: m/z Calc.: 728.4, found:
728.2
Acknowledgement
4.3.11. (2S,3R,E)-2-(6-(2-(7-bromo-8-methyl-6-oxo-6H-[1,3]
dioxolo[4,5-g]chromen-2-yl) acetamido)hexanamido)-3-
hydroxyoctadec-4-en-1-yl(2-((7-nitrobenzo[c][1,2,5] oxadiazol-4-
yl)amino)ethyl) hydrogen phosphate (12)
The authors are grateful for funding by the DFG to C.A. (AR 376/12-
2). Z.H.M is grateful for fellowship provided by the DFG graduate school
(SALSA). Two photon microscopy was performed on a Zeiss LSM 880
NLO, funded by Deutsche Forschungsgemeinschaft (DFG, German
Research Fundation) - project 261193037. We thank Juliana Monti for
excellent technical assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116303.
In an argon atmosphere, 10 (46 mg, 0.063 mmol, 1 eq.) was dis-
solved in MeOH (6 mL). At rt, 4 M HCl in 1,4-dioxan (081.5 µL, 1.56
mmol, 25 eq.) was added and the mixture was stirred at 70 ◦C for 2 h
with exclusion of light. Then, UPLC-MS and TLC indicated complete
conversion of the starting material. The mixture was concentrated in a
stream of air and dried under high vacuum. The orange residue 11 was
dissolved in dry DCM (4 mL) and dry DIPEA (85 µL, 0.49 mmol, 8 eq.).
Active ester of 7 (0.045 g, 0.08 mmol, 1.3 eq.) was dissolved in dry DCM
(3 mL) and added dropwise to the first solution at 0 ◦C. After 24 h
stirring at RT, UPLC-MS and TLC showed complete conversion. MeOH
(1 mL) was added to quench the reaction and left to stir for 1 h. The
reaction mixture was diluted with CH2Cl2 (75 mL) and washed twice
with 10 mL 0.1 M aq. HCl. The organic layer was dried (Na2SO4), and the
volatiles were removed in vacuo. The material obtained was purified
using preparative TLC (CHCl3:MeOH:H2O = 65:25:3) to give the final
probe 12 as an orange solid (25 mg, 39%).
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111.54, 109.92, 102.92, 98.19, 71.78, 64.67, 54.30, 41.66, 39.46,
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UPLC-MS (ESI): C45H61BrN6O14P [Mꢀ H]-: m/z Calc.: 1019.3, found:
9