Synthesis and characterization of bichromophoric 1-deoxyceramides as FRET probes

Article abstract of DOI:10.1039/d1ob00113b

The suitability as FRET probes of two bichromophoric 1-deoxydihydroceramides containing a labelled spisulosine derivative as a sphingoid base and two differently ω-labelled fluorescent palmitic acids has been evaluated. The ceramide synthase (CerS) cataly

Full text of DOI:10.1039/d1ob00113b

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Synthesis and characterization of bichromophoric
1-deoxyceramides as FRET probes†
Cite this: Org. Biomol. Chem., 2021,
19, 2456
Eduardo Izquierdo,a,b Mireia Casasampere,b Gemma Fabriàs,
b,c
José Luís Abad, b Josefina Casas*b,c and Antonio Delgado
*
a,b
The suitability as FRET probes of two bichromophoric 1-deoxydihydroceramides containing a labelled spisulo-
sine derivative as a sphingoid base and two differently ω-labelled fluorescent palmitic acids has been evaluated.
The ceramide synthase (CerS) catalyzed metabolic incorporation of ω-azido palmitic acid into the above
labeled spisulosine to render the corresponding ω-azido 1-deoxyceramide has been studied in several cell
lines. In addition, the strain-promoted click reaction between this ω-azido 1-deoxyceramide and suitable fluor-
ophores has been optimized to render the target bichromophoric 1-deoxydihydroceramides. These results
pave the way for the development of FRET-based assays as a new tool to study sphingolipid metabolism.
Received 21st January 2021,
Accepted 22nd February 2021
DOI: 10.1039/d1ob00113b
rsc.li/obc
The intracellular levels of Cer are the result of the catabolic
processes from higher SLs (sphingomyelin, glycosphingolipids
Introduction
Sphingolipids (SLs) are one of the major classes of lipids in and ceramide-1-phosphate), together with biosynthesis de novo
eukaryotes. Canonical SLs derive from the sphingoid base dihy- by the N-acylation of dhSo with a variety of fatty acids (FA),
drosphingosine (dhSo) or (2R,3S) 2-amino-1,3-octadecanediol prior to their desaturation by a specific desaturase (Des1) that
(Scheme 1), which is metabolically modified to account for the introduces a C4(E) double bond into the sphingoid base. In
different families of SLs known to date.1 Among them, cera- this context, ceramide synthases (CerS) are a family of enzymes
mides (Cer) occupy a pivotal position in the metabolic pathways responsible for the N-acylation of dhSo (in the de novo
and they are considered as a metabolic hub.2 Apart from their pathway) or So (in the catabolic pathway) to form dhCer and
fundamental structural role in cell membranes,3 Cer are also Cer, respectively (Scheme 1).
important second messengers in several cellular processes. In
Six isoforms of CerS have been identified in mammals,
this regard, Cer have been reported to activate apoptosis in each one of them encoded by a unique gene (CerS1-6).10 CerS
response to a variety of cell stress inducing agents4–6 and, con- enzymes are expressed differently in various tissues11 and
sistent with their role in apoptosis, various types of cancer cells their levels of expression change during development,
have been shown to reduce their Cer levels as a survival strategy suggesting that populations of Cer with particular acyl chain
through the overexpression of CDase enzymes.7 Furthermore, lengths might be generated to meet the specific physiological
Cer also participate in the regulation of autophagy and stimu- needs of each tissue.12 Moreover, the nature of the acyl
late cell cycle arrest, cell differentiation,8 and senescence, the chains is a determinant of the biophysical properties of the
last by modifying telomerase activity.9
resulting Cer and also the signaling pathways in which they
participate.13 The development of modern lipidomic tech-
niques14 has allowed determination of the relative abundance
of the various Cer types in a range of biological contexts, and
has provided some insight into the effect of the acyl chain
composition on the physiological role of Cer.15 Given the
importance of CerS activity in cell fate, we became interested
in the development of new chemical probes16 towards this
end. In a previous work, we reported on the use of 1-deoxydi-
hydrosphingosine (doxdhSo, spisulosine or ES285) as a suit-
able probe for the profiling of CerS activity in intact cells.17
On the basis that 1-deoxysphingolipids (doxSLs) can be vir-
tually considered as “dead-end” metabolites, due to the lack
of the C1-OH group, we envisioned that a fluorescent probe
derived from spisulosine, together with a suitable FA ana-
aDepartment of Pharmacology, Toxicology and Medicinal Chemistry, Unit of
Pharmaceutical Chemistry (Associated Unit to CSIC). Faculty of Pharmacy and Food
Sciences. University of Barcelona (UB), Joan XXIII 27-31, 08028 Barcelona, Spain.
E-mail: antonio.delgado@ub.edu
bResearch Unit on BioActive Molecules, Department of Biological Chemistry, Institute
for Advanced Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18-26, 08034-
Barcelona, Spain. E-mail: fina.casas@iqac.csic.es
cLiver and Digestive Diseases Networking Biomedical Research Centre (CIBEREHD),
ISCIII, 28029 Madrid, Spain
†Electronic supplementary information (ESI) available: Calculated spectral
overlap integrals and Förster critical distances (Table S1), photophysical pro-
perties of the monochromophoric compounds (Table S2), photophysical pro-
perties of the bichromophoric compounds (Table S3), and the NMR spectra of
the synthesized compounds. See DOI: 10.1039/d1ob00113b
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Scheme 1 Metabolic routes leading to Cer.
logue, could be used to develop a FRET-based assay to cycloaddition (SPAAC) of the resulting doxdhCer RBM5-159 with a
monitor CerS activity.18
BCN-derived fluorescent dye (RBM5-142 (MCC) or RBM5-143
The affinity towards lipid phases, fluorescent properties and (NR)) should render the bichromophoric doxdhCer RBM5-160 or
sensitivity to the polarity of the surrounding environment of the RBM5-161, respectively, whose FRET emission should correlate
fluorophores NBD and Nile red (NR) has promoted the use of with CerS activity (Scheme 2).
these groups in several biological applications related to the study
In this paper, we wish to report on the synthesis and photo-
of the cell membrane.19,20 Due to the existing overlap between the chemical characterization of the above probes and reagents,
emission band of NBD and the absorption band of NR, these two their validation as CerS substrates and their suitability as
fluorophores have also been incorporated into lipids as a donor– FRET partners.18
acceptor fluorophore pair to perform FRET experiments.21–24
More recently, 7-methoxycoumarin-3-carboxylate (MCC) has also
been used as an alternative fluorophore partner for NBD in FRET
Synthesis of the probes
experiments.25 In this case, however, NBD played the role of the
acceptor fluorophore, whereas MCC was used as the donor. On
this basis, we considered the NBD-labelled spisulosine RBM5-155
and the ω-azido palmitic acid (ωN3PA) as suitable CerS substrates
for an ideal experiment design. A strained-promoted alkyne–azide
The synthesis of the NBD probe RBM5-155 was carried out as
depicted in Scheme 3. Starting from the anti-configured allylic
alcohol RBM5-084,26 a cross-metathesis reaction with the bro-
moalkene RBM5-149, using the second generation Grubbs’
Scheme 2 Conceptual design of the required probes for a FRET-based CerS assay.
Scheme 3 Synthesis of doxSL probes. Reagents and conditions: (a) RBM5-149, Grubbs’ 2nd gen. catalyst, CH2Cl2, reflux, 2 h, 44%, E/Z = 95 : 5; (b)
H2, 5 wt% Rh on Al2O3, MeOH, rt, 3 h, 86%; (c) NaN3, DMF, 80 °C, 3 h, 95%; (d) TES, Pd–C, MeOH : CHCl3 (9 : 1), rt, 10 min, 85%; (e) NBD-Cl, DIPEA,
MeOH, 0 °C to rt, overnight, 84%; (f) AcCl, MeOH, 0 °C to rt, overnight, 80%; (g) ωN3PA, EDC, HOBt, EtN3, CH2Cl2, rt, 2 h, 74%.
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catalyst, afforded a highly E-enriched E : Z mixture of RBM5- respectively, with the p-nitrophenyl carbonate RBM5-141, obtained
150, in agreement with the literature.26 This mixture was sub- by the reaction of commercial trans-9-hydroxymethyl[6.1.0]bicyclo-
jected to catalytic hydrogenation on Rh/Al2O3 to give the non-4-yne (BCN-OH) with 4-nitrophenyl chloroformate.30 The
corresponding saturated ω-bromo intermediate RBM5-151. corresponding bichromophoric click adducts RBM5-160 and
Subsequent nucleophilic displacement with sodium azide, fol- RBM5-161 were prepared for their complete photochemical
lowed by the reduction of the azido group with the Pd/C-TES characterization and as standards for FRET studies (Scheme 5).
system,27 reaction of the intermediate amine hydrochloride
with NBD-Cl,24 and final N-Boc removal afforded RBM5-155,
which was isolated and further manipulated as the corres-
ponding hydrochloride, given its apparent sensitivity to the
FRET efficiency of the bichromophoric
probes RBM5-160 and RBM5-161
alkaline conditions required for the formation of the free
amine.28 Finally, the N-acylation of RBM5-155 with ωN3PA29
The suitability of the selected FRET partners was corroborated
by calculation of the spectral overlap integral and Förster
radius (R0) (Table S1†) from the spectral data of the monochro-
mophoric compounds RBM5-142, RBM5-143 and RBM5-154
afforded the doxdhCer RBM5-159, which was used as the stan-
dard for quantitative LC-MS in cell assays (see below).
The fluorophores required for the SPAAC click reactions
were obtained from the modified ω-aminoalkyl MCC and NR
(Table S2†). The normalized absorption and emission spectra
derivatives RBM5-135 and RBM5-136, respectively, obtained as
of the bichromophoric probes RBM5-160 and RBM5-161 in
DMSO, EtOH and PBS buffer presented two bands owing to
the presence of the two fluorescent labels (Fig. 1). Compound
RBM5-160 presented two maxima at around 350 nm (MCC
shown in Scheme 4.18
The clickable reagents RBM5-142 and RBM5-143 were syn-
thesized by the condensation of RBM5-135 and RBM5-136,
moiety) and 470 nm (NBD moiety), while the maxima for
RBM5-161 were located around 485 nm (NBD moiety) and
550 nm (NR moiety). The fluorescence intensity was strongly
affected by the solvent, the highest fluorescence intensities
being recorded in EtOH for both compounds. However, their
emission in PBS buffer was reduced as a result of their lower
aqueous solubility. The position and shape of the emission
spectra in the two probes were slightly modified in the
different solvents, due to the solvatochromic effects.
Furthermore, there were also considerable deviations in the
absorption spectra of compounds RBM5-160 and RBM5-161,
when compared with the spectra of the related monochromo-
phoric compounds, probably due to the intramolecular attrac-
Scheme 4 Synthesis of the MCC and NR fluorophores. (a) (1). N-Boc-
tive interactions between the two fluorophores in each com-
1,6-hexanediamine, EDC, HOBt, Et3N, CH2Cl2, rt,
2 h, 57%; (2).
pound.31 The molar extinction coefficients (ε) of compounds
RBM5-160 and RBM5-161, calculated at their corresponding
two absorption maxima in DMSO and EtOH are listed in
Table S3.†
TFA : CH2Cl2 (1 : 2), 0 °C to rt, 1 h, quantitative; (b) NaNO2, HCl (aq.),
0 °C, 5 h, 67%; (c) naphthalene-1,6-diol, DMF, 160 °C, 4 h, 19%; (d) (1).
N-Boc-6-bromohexanamine, K2CO3, DMF, 85 °C, overnight, 78%; (2).
TFA : CH2Cl2 (1 : 2), 0 °C to rt, 1 h, quantitative.
Scheme 5 Synthesis of clickable fluorophores. Reagents and conditions: (a) RBM5-135, Et3N, CH2Cl2, rt, overnight, 89%; (b) RBM5-136, Et3N,
CH2Cl2, rt, overnight (90%); (c) RBM5-159, CH2Cl2, rt, overnight, 84–93% (for assay-like conditions, see text).
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Fig. 1 Normalised absorption (left panels) and emission (right panels) spectra for the bichromophoric compounds RBM5-160 (top) and RBM5-161
(bottom) at 5 µM in DMSO (blue), EtOH (black) and PBS (red). Emission spectra upon excitation at 340 nm (MCC in RBM5-160) and 455 nm (NBD in
RBM5-161), respectively. The absorption and emission spectra of RBM5-160 in DMSO and PBS were normalised to those of the same compound in
EtOH. The absorption spectra of RBM5-161 in EtOH and PBS were normalised to those in DMSO. The emission spectra of RBM5-161 in DMSO and
PBS were normalised to those in EtOH.
The intramolecular FRET efficiencies of compounds
RBM5-160 and RBM5-161 were estimated in DMSO and
EtOH, based on the loss of donor fluorescence in the pres-
Optimization of the SPAAC reaction
from RBM5-159
ence of the acceptor. To this end, we compared the integrated
The SPAAC reaction between the azide-tagged doxdhCer
fluorescence intensities (I), within the donor-specific wave-
RBM5-159 and the fluorescent dyes RBM5-142 and RBM5-143
length interval, of the donor (D) compounds (RBM5-142 and
was studied under different conditions in order to determine
the best reagent ratios and reaction times at a concentration
scale compatible with that expected in the CerS activity assay
in intact cells. The reaction progress was first monitored in
DMSO by analyzing the changes in the fluorescence emission
of the mixture upon irradiation at the donor-specific excitation
wavelength. For RBM5-160 (donor: MCC, acceptor: NBD, see
RBM5-154) to those of the related donor + acceptor (DA) com-
pounds (RBM5-160 and RBM5-161, respectively). The calcu-
lated FRET efficiency of the NBD/NR pair in RBM5-161
(ENBD/NR = 0.88–0.96) was higher than that of the MCC/NBD
pair in RBM5-160 (EMCC/NBD = 0.56–0.88). For both fluoro-
phore pairs, the FRET process was more efficient in EtOH
than in DMSO and, in the case of the NBD/NR pair in RBM5-
Scheme 2), the fluorescence emission at λem = 535 (NBD emis-
161, the two studied excitation wavelengths gave the same E
sion) at two single concentrations of RBM5-159 (5 and 20 µM)
value in EtOH (Table 1).
and increasing concentrations of the BCN-MCC reagent RBM5-
142 (from 5 to 50 µM) was always lower than that of a standard
solution of the expected adduct RBM5-160 at 5 and 20 µM
(results not shown). However, a similar experiment using the
BCN-NR reagent RBM5-143 (from 5 to 50 µM) led to fluo-
rescence intensities at λem = 625 (NR emission) comparable to
those of a standard solution of the expected adduct RBM5-161
using a 2.5 to 4 fold excess of the BCN-NR reagent RBM5-143
(Fig. 2).
Experiments in MeOH, EtOH and sodium acetate buffer
(NaOAc 250 mM, NaCl 200 mM, 0.1% Triton X-100) also
showed a remarkable enhancement of the emission at 625 nm
(acceptor emission, λexc = 455 nm) in comparison with RBM5-
159, together with an attenuation of the fluorescence emission
Table 1 Study of the intramolecular FRET efficiency of the bichromo-
phoric compounds RBM5-160 and RBM5-161
Compound
Solvent
λex (nm)
Ea
RBM5-160
DMSO
EtOH
DMSO
340
340
470
455
470
455
0.56
0.86
0.90
0.88
0.96
0.96
RBM5-161
EtOH
a FRET efficiencies (E) were calculated from the decrease of the donor
emission (see Experimental section).
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Fig. 2 Bar diagram representing the changes in fluorescence emission at 625 nm (bottom), resulting from the excitation at 455 nm, of mixtures
containing different ratios of compounds RBM5-159 and RBM5-143 in DMSO at various reaction times. Compound RBM5-159 was used as the nega-
tive control, equivalent to 0% conversion, whereas compound RBM5-161 was used as the positive control, equivalent to 100% conversion. The
results correspond to the mean standard deviation of at least two independent experiments with triplicates.
at 535 nm (donor emission, λexc = 455 nm), in agreement with blasts (MEF). Even though the total amount of doxdhCers,
the formation of the desired cycloadduct RBM5-161 (Fig. S1†). arising from acylation of RBM5-155 with endogenous FAs, was
The background emission of RBM5-143 at 625 nm observed at very similar in the two cell lines (∼120–130 pmol equiv./106
the donor excitation (λexc = 455 nm) is indicative of an exci- cells), the profile of the different doxdhCer species differed
tation cross-talk (or acceptor excitation bleed-through, AEB), a considerably. In A549 cells, there was a high proportion of
phenomenon related to the overlap of the donor and acceptor elongated C18N3 and C20N3 doxdhCer species (resulting from
absorption spectra. This phenomenon, together with the the action of the endogenous elongases),33 whereas the non-
related emission cross-talk (or donor emission bleed-through, elongated doxdhCer RBM5-159 accounted for less than a third
DEB), arising from the overlap of the donor and acceptor emis- of the total doxdhCers (∼28%). Conversely, in MEF cells, the
sion spectra32 must be carefully considered in the design of non-elongated RBM5-159 was the most abundant metabolite
ratiometric experiments based on the development of the (85% of the total doxdhCers), whereas the formation of
FRET effect. In contrast, ratiometric experiments based on elongated species was minimal. These results show that the
FRET attenuation21,22,24 may benefit from the disappearance elongation process is unpredictable and highly dependent on
of these interferences. The experimental AEB and DEB calcu- the cell line, although it appears to be independent on the
lated for RBM5-161 are listed in Table S4 and Fig. S2, S3.†
overall CerS activity. Attempts to reduce or avoid the acyl chain
elongation by using the FA biosynthesis inhibitors TOFA or
cerulenin were fruitless.34–36 Moreover, the incorporation of
ωN3C13 acid, as well as that of branched αN3PA and (ω-1)N3PA,
was also tested. The first two were deemed as poor CerS sub-
CerS catalyzed incorporation of
ω-azidopalmitic acid into RBM5-155 in strates since only modest amounts of the corresponding
doxdhCers were formed under the standard assay. Conversely,
cells
the branched (ω-1)N3PA was efficiently incorporated, although
The suitability of the probe RBM5-155 and ωN3PA as CerS sub- the high ratio of the elongated doxdhCer observed made us
strates was evaluated by examining the extent of their meta- disregard its use (results not shown). For practical purposes,
bolic conversion into the corresponding N-acylated doxdhCer the overexpression of the CerS under study may improve the
RBM5-159 by LC-MS. Initial experiments were carried out in signal-to-noise ratio of the assay. As a proof of concept, human
lung adenocarcinoma A549 cells and mouse embryonic fibro- embryonic kidney cells (HEK293T) were transfected with the
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Fig. 3 N-Acylation of the doxdhSo probe RBM5-155 with the endogenous natural FAs or ωN3PA. HEK293T cells transfected with the plasmid con-
taining human CerS5 (bars in orange) or simply treated with the transfection reagent PEI (bars in grey) were incubated for 90 min with RBM5-155
(5 µM) in the absence (left) or in the presence (right) of ωN3PA (0.5 mM) complexed in 0.5% acid-free BSA. After lipid extraction, the different
doxdhCer species were quantified by UPLC-TOF. Left: Amount of doxdhCers containing endogenous natural FAs; right: amount of doxdhCers con-
taining the administered ωN3PA and the corresponding elongated FAs. The results correspond to the mean standard deviation of at least two inde-
pendent experiments with triplicates.
human CerS5 gene using the cationic polymer polyethyl- a stain solution of phosphomolibdic acid (5.7% in EtOH).
enimine (PEI),37 and were subsequently incubated with the Usual work up refers to washing of the combined organic
doxdhSo probe RBM5-155 and ωN3PA. UPLC-TOF analysis of layers with brine (2 × 25 mL), drying over anhydrous MgSO4
the lipid extracts showed a higher ratio of the C16-doxdhCer and concentration to dryness. Compounds were purified by
species in the transfected cells, in agreement with the expected flash column chromatography, using silica gel (Chromatogel
preference of the CerS5 isoform for the C16 FAs38 (Fig. 3).
60 Å, 35–75 μm) as the stationary phase. Mobile phases and
1
gradients are specified in each case. H and 13C nuclear mag-
netic resonance spectra were recorded on a Varian – Mercury
400 (1H NMR at 400 MHz and 13C NMR at 100.6 MHz) spectro-
meter using CDCl3 or CD3OD as solvent. Chemical shifts of
Conclusions
In summary, the NBD probe RBM5-155 and the modified fatty deuterated solvents were used as internal standards. Chemical
acid ωN3PA behave as suitable CerS substrates in cells. Despite shifts are given in parts per million (ppm) and coupling con-
the degree of incorporation and elongation of the fatty acid stants (J) in hertz (Hz). Splitting patterns have been described
being strongly dependent on the particular cell line, studies as a singlet (s), broad singlet (br s), doublet (d), triplet (t),
carried out with the doxdhCer RBM5-159 indicated its suit- quartet (q), quintuplet (p), multiplet (m), apparent (app) or
ability as a SPAAC reaction partner with the BCN modified NR combinations of these descriptive names. Specific optical
fluorophore RBM5-143 to render the bichromophoric rotations were recorded on a digital PerkinElmer 34 polari-
doxdhCer RBM5-161, exhibiting a high FRET efficiency in the meter at 25 °C in a 1 dm, 1 mL cell, using a sodium light lamp
solvent systems used. These results represent an interesting (λ = 589 nm). Specific optical rotation values ([α]D) are
addition to the available chemical toolbox of labelled sphingo- expressed in deg−1 cm3
g
−1, and concentrations (c) are
lipids for FRET-based studies in cells. Efforts along this line reported in g per 100 mL of solvent. High-resolution mass
are underway in our lab and will be reported in due course.
spectra (HRMS) were recorded Waters Aquity UPLC system con-
nected to a Waters LCT Premier Orthogonal Accelerated Time
of Flight Mass Spectrometer (Waters, Milford, MA, USA) oper-
ated in the positive electrospray ionisation mode. Samples
were analyzed by FIA (Flow Injection Analysis), using ACN/
water (70 : 30) as the mobile phase. Samples were analyzed
Experimental section
Synthesis
General procedures. All chemicals were purchased from using a 10 μL volume injection. The m/z ratios are reported as
commercial sources and used as received unless otherwise values in atomic mass units.
noted. Dry solvents (THF, DMF and CH2Cl2), obtained from a
((1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-yl)methyl (6-(7-methoxy-2-
PureSolv dispenser and subsequently degassed with an inert oxo-2H-chromene-3-carboxamido)hexyl)carbamate (RBM5-142).
gas, were used in most reactions. Synthesis grade (hexane, To an ice-cooled solution of N-Boc protected RBM5-135 18
EtOAc, Et2O and CH2Cl2) or HPLC-grade (MeOH) solvents were (93 mg, 0.22 mmol) in dry CH2Cl2 (6 mL) was added dropwise
used for extractions and purifications. Progression of the reac- neat TFA (1.5 mL). After stirring at rt for 2 h in the dark, the
tions was controlled by thin layer chromatography (TLC), using reaction mixture was concentrated to dryness to afford the
ALUGRAM® SIL G/UV254 (Macherey-Nagel) silica gel pre- corresponding crude amine trifluoroacetate (71 mg). This
coated aluminum sheets (layer: 0.2 mm, silica gel 60). crude was taken up in CH2Cl2 (10 mL), followed by the sequen-
Compounds were detected by using UV light (λ = 254 nm) and tial addition of Et3N (108 µL, 0.78 mmol) and p-nitrophenol
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activated carbonate ester RBM5-141 30 (70 mg, 0.22 mmol). 1.4 Hz, 1H), 3.41 (t, J = 6.9 Hz, 2H), 2.08–2.00 (m, 2H),
After stirring overnight at rt in the dark, the reaction mixture 1.85 (dt, J = 14.5, 7.0 Hz, 2H), 1.46–1.33 (m, 3H), 1.32–1.25
was evaporated in vacuo and the residue was flash chromato- (m, 16H).
graphed (from 0 to 20% EtOAc in CH2Cl2) to give the desired
carbamate RBM5-142 (98 mg, 89%) as an off-white solid.
1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.76 (br s, 1H),
13C NMR (101 MHz, CDCl3) δ 139.4, 114.2, 34.2, 34.0, 33.0,
29.8, 29.8, 29.7, 29.7, 29.6, 29.3, 29.1, 28.9, 28.3.
(2′S,3′R,4′EZ)-tert-Butyl (18-bromo-3-hydroxyoctadec-4-en-2-yl)
7.58 (d, J = 8.7 Hz, 1H), 6.93 (dd, J = 8.7, 2.4 Hz, 1H), 6.86 (d, J carbamate (RBM5-150). To a stirred solution of RBM5-084 26,40
= 2.4 Hz, 1H), 4.69 (br s, 1H), 4.13 (d, J = 8.1 Hz, 2H), 3.91 (s, (480 mg, 2.38 mmol) and olefin RBM5-149 (1.59 g, 5.49 mmol)
3H), 3.44 (td, J = 7.1, 5.8 Hz, 2H), 3.20–3.12 (m, 2H), 2.34–2.15 in degassed CH2Cl2 (20 mL), second generation Grubbs’ cata-
(m, 6H), 1.67–1.24 (m, 11H), 0.99–0.86 (m, 2H).
lyst (101 mg, 0.12 mmol) was added in one portion at rt. The
13C NMR (101 MHz, CDCl3) δ 164.9, 162.1, 162.1, 156.9, resulting mixture was refluxed in the dark for 2 h, cooled to rt
156.8, 148.3, 131.0, 115.0, 114.1, 112.6, 100.4, 99.0, 62.7, 56.2, and concentrated in vacuo to afford a crude, which was puri-
41.0, 39.7, 30.0, 29.5, 29.2, 26.7, 26.5, 21.6, 20.2, 18.0.
HRMS calcd for C28H35N2O6 ([M + H]+): 495.2490, found: MTBE in CH2Cl2). Compound RBM5-150 was obtained as an
495.2496. inseparable mixture of E/Z isomers (colourless thick oil,
((1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-yl)methyl (6-((9-(diethyl- 485 mg, 44%).
amino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)hexyl)carbamate
1H NMR (400 MHz, CDCl3) (E isomer) δ 5.71 (dt, J = 14.6,
fied by flash chromatography on silica gel (from 0 to 5%
(RBM5-143). To an ice-cooled solution of N-Boc protected 6.7 Hz, 1H), 5.42 (dt, J = 15.0, 7.5 Hz, 1H), 4.76 (br s, 1H), 4.11
RBM5-136 18 (100 mg, 0.19 mmol) in dry CH2Cl2 (6 mL) was (dd, J = 6.4, 3.0 Hz, 1H), 3.73–3.58 (m, 1H), 3.40 (t, J = 6.9 Hz,
added dropwise neat TFA (1.5 mL). After stirring at rt for 2 h in 2H), 2.09–1.96 (m, 2H), 1.85 (dt, J = 14.5, 6.9 Hz, 2H), 1.44 (s,
the dark, the reaction mixture was concentrated to dryness to 9H), 1.43–1.23 (m, 20H), 1.07 (d, J = 6.9 Hz, 3H).
afford the corresponding crude amine trifluoroacetate (81 mg).
13C NMR (101 MHz, CDCl3) (mixture of E/Z isomers) δ
This crude was taken up in CH2Cl2 (10 mL), followed by the 156.1, 155.7, 134.2, 133.5, 129.4, 128.7, 125.6, 124.9, 79.4, 79.2,
sequential addition of Et3N (93 µL, 0.67 mmol) and p-nitro- 75.4, 73.5, 51.0, 50.0, 37.3, 33.9, 33.4, 32.8, 32.6, 32.3, 29.6,
phenol activated carbonate ester RBM5-141 30 (60 mg, 29.5, 29.5, 29.4, 29.2, 28.7, 28.4, 28.1, 15.2, 14.3.
0.19 mmol). After stirring overnight at rt in the dark, the reac-
tion mixture was evaporated in vacuo and the residue was flash mate (RBM5-151).
(2′S,3′R)-tert-Butyl (18-bromo-3-hydroxyoctadecan-2-yl)carba-
solution of RBM5-150 (450 mg,
A
chromatographed (from 0 to 20% EtOAc in CH2Cl2) to give the 0.97 mmol) in degassed MeOH (45 mL) was hydrogenated at 1
desired carbamate RBM5-143 (104 mg, 90%) as a shiny dark- atm and rt in the presence of Rh/Al2O3 (60 mg, 15% w/w).
red solid.
After stirring for 3 h, the catalyst was removed by filtration
1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 8.7 Hz, 1H), 8.03 through a Celite® pad, and the solid was rinsed with MeOH (3
(d, J = 2.6 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.15 (dd, J = 8.7, 2.6 × 10 mL). The combined filtrates were concentrated in vacuo,
Hz, 1H), 6.65 (dd, J = 9.1, 2.7 Hz, 1H), 6.45 (d, J = 2.7 Hz, 1H), and the residue was subjected to flash chromatography on
6.29 (s, 1H), 4.70 (br s, 1H), 4.19–4.11 (m, 4H), 3.46 (q, J = 7.1 silica gel (from 0 to 1% MeOH in CH2Cl2) to yield RBM5-151
Hz, 5H), 3.25–3.15 (m, 2H), 2.34–2.16 (m, 6H), 1.90–1.82 (m, as a white solid (395 mg, 87%).
2H), 1.67–1.30 (m, 10H), 1.26 (t, J = 7.0 Hz, 6H), 0.97–0.87 (m,
[α]2D0 = −3.65 (c 1, CHCl3).
2H).
1H NMR (400 MHz, CDCl3) δ 4.74 (br s, 1H), 3.70–3.67 (m,
13C NMR (101 MHz, CDCl3) δ 183.4, 161.9, 156.9, 152.2, 1H), 3.64 (td, J = 8.0, 7.2, 2.7 Hz, 1H), 3.41 (t, J = 6.9 Hz, 2H),
150.9, 147.0, 140.3, 134.2, 131.2, 127.9, 125.7, 124.8, 118.4, 1.85 (dt, J = 14.5, 7.0 Hz, 2H), 1.71 (br s, 1H), 1.44 (s, 9H),
109.6, 106.7, 105.5, 99.0, 96.5, 68.3, 62.8, 45.2, 41.1, 30.1, 29.3, 1.43–1.35 (m, 4H), 1.34–1.23 (m, 22H), 1.08 (d, J = 6.8 Hz, 3H).
29.2, 26.6, 25.9, 21.6, 20.2, 17.9, 12.8.
HRMS calcd for C37H44N3O5 ([M + H]+): 610.3275, found: 33.6, 33.0, 29.8, 29.7, 29.6, 28.9, 28.6, 28.3, 26.2, 14.5.
610.3279. HRMS calcd for C23H47BrNO3 ([M
H]+): 464.2734,
15-Bromopentadec-1-ene (RBM5-149). A stirred solution of 466.2713, found: 464.2729, 466.2718.
14-pentadecen-1-ol39 (2.77 g, 12.23 mmol) and PPh3 (3.53 g,
(2′S,3′R)-tert-Butyl (18-azido-3-hydroxyoctadecan-2-yl)carba-
13C NMR (101 MHz, CDCl3) δ 156.0, 79.6, 74.6, 50.8, 34.2,
+
13.46 mmol) in anhydrous CH2Cl2 (100 mL) was cooled to mate (RBM5-152). To a stirred solution of RBM5-151 (395 mg,
0 °C, and NBS (2.61 g, 14.68 mmol) was added in small por- 0.85 mmol) in anhydrous DMF (8 mL) was added NaN3
tions over 10 min. The resultant dark yellow solution was (166 mg, 2.55 mmol). The mixture was heated to 80 °C and
allowed to warm to rt and stirred for 1 h, after which the stirred for 3 h under Ar. Water (20 mL) was next added, and
solvent was removed by vacuum evaporation. The residue was the mixture was extracted with Et2O (3 × 20 mL). Usual workup
then diluted with water (50 mL), extracted with hexanes (3 × afforded a crude, which was purified by flash chromatography
50 mL) and worked-up as usual to give a crude, which was pur- (from 0 to 1% MeOH in CH2Cl2) to give RBM5-152 (off-white
ified by flash chromatography on silica gel (isocratic 100% wax, 346 mg, 95%).
hexanes) to afford RBM5-149 as a colorless oil, (2.62 g, 74%).
1H NMR (400 MHz, CDCl3) δ 5.81 (ddt, J = 16.9, 10.2, 6.7
[α]2D0 = −4.13 (c 1, CHCl3).
1H NMR (400 MHz, CDCl3) δ 4.75 (s, 1H), 3.71–3.67 (m,
Hz, 1H), 4.99 (dq, J = 17.1, 1.9 Hz, 1H), 4.93 (ddt, J = 10.2, 2.4, 1H), 3.66–3.61 (m, 1H), 3.25 (t, J = 7.0 Hz, 2H), 1.75 (br s, 1H),
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1.66–1.54 (m, 2H), 1.44 (s, 9H), 1.40–1.35 (m, 4H), 1.34–1.23
(m, 22H), 1.07 (d, J = 6.8 Hz, 3H).
1H NMR (400 MHz, CD3OD) δ 8.52 (d, J = 8.7 Hz, 1H), 6.35
(d, J = 8.9 Hz, 1H), 3.75–3.65 (m, 1H), 3.53 (br s, 2H), 3.31–3.22
13C NMR (101 MHz, CDCl3) δ 156.0, 79.6, 74.6, 51.6, 50.7, (m, 1H), 1.77 (app p, J = 7.4 Hz, 2H), 1.55–1.37 (m, 8H),
33.6, 29.8, 29.8, 29.7, 29.7, 29.7, 29.7, 29.6, 29.3, 29.0, 28.5, 1.36–1.25 (m, 18H), 1.22 (d, J = 6.8 Hz, 3H).
26.8, 26.2, 14.4.
HRMS calcd for C23H47N4O3 ([M + H]+): 427.3643, found: 122.7, 99.6, 71.7, 52.6, 44.8, 34.0, 30.7, 30.6, 30.3, 29.2, 28.0,
427.3636. 27.0, 12.1.
(2′S,3′R)-tert-Butyl (18-amino-3-hydroxyoctadecan-2-yl)carba-
HRMS calcd for C24H42N5O4 ([M + H]+): 464.3231, found:
mate (RBM5-153). To a solution of RBM5-152 (300 mg, 464.3228.
13C NMR (101 MHz, CD3OD) δ 146.7, 145.8, 145.5, 138.6,
0.70 mmol) in degassed MeOH (16 mL) containing Pd–C
(2′S,3′R)-16-Azido-N-[3-hydroxy-18-[(7-nitrobenzo[c][1,2,5]oxa-
(60 mg, 20% w/w) neat TES (1.1 mL, 7.03 mmol) was added diazol-4-yl)amino]octadecan-2-yl]hexadecanamide (RBM5-159).
dropwise, and the resultant suspension was stirred at rt under EDC·HCl (12 mg, 64 µmol) and HOBt (7 mg, 52 µmol) were
an Ar-filled balloon. After stirring for 1 h, the reaction mixture sequentially added to an ice-cooled solution of
was filtered through a pad of Celite® and the solid was rinsed ω-azidopalmitic acid29 (13 mg, 44 µmol) in anhydrous CH2Cl2
with MeOH (3 × 5 mL). The combined filtrates were concen- (6 mL). The resulting mixture was vigorously stirred at rt under
trated in vacuo and the residue was triturated with hexanes (4 × Ar for 15 min, and next added dropwise to a solution of RBM5-
2 mL) to give the desired amine hydrochloride, without the 155 (20 mg, 40 µmol) and Et3N (89 µL, 0.20 mmol) in anhy-
need of further purification.
drous CH2Cl2 (6 mL), and the reaction was stirred at rt for
1H NMR (400 MHz, CD3OD) δ 3.53–3.47 (m, 1H), 3.47–3.41 additional 2 h. The mixture was next diluted with CH2Cl2
(m, 1H), 2.67 (t, J = 7.1 Hz, 2H), 1.53–1.46 (m, 4H), 1.44 (s, 9H), (50 mL), washed with brine (2 × 25 mL) and worked-up as
1.30 (s, 24H), 1.08 (d, J = 6.6 Hz, 3H).
usual to afford a crude, which was flash chromatographed
13C NMR (101 MHz, CD3OD) δ 157.8, 79.9, 75.3, 51.8, 42.3, (from 0 to 14% EtOAc in CH2Cl2) to afford RBM5-159 as a
34.8, 33.0, 30.8, 30.7, 30.6, 28.8, 28.0, 27.1, 15.5.
HRMS calcd for C23H49N2O3 ([M + H]+): 401.3738, found:
401.3742.
shiny orange solid (22 mg, 74%).
1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 8.6 Hz, 1H), 6.49
(br s, 1H), 6.17 (d, J = 8.7 Hz, 1H), 5.81 (d, J = 7.9 Hz, 1H),
(2′S,3′R)-tert-Butyl [3-hydroxy-18-[(7-nitrobenzo[c][1,2,5]oxadia- 4.05–3.96 (m, 1H), 3.66–3.59 (m, 1H), 3.53–3.45 (m, 2H), 3.24
zol-4-yl)amino]octadecan-2-yl]carbamate (RBM5-154). To (t, J = 7.0 Hz, 2H), 2.51 (br s, 1H), 2.17 (t, J = 7.6 Hz, 2H), 1.80
a
stirred solution of 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole (app p, J = 7.4 Hz, 2H), 1.69–1.53 (m, 4H), 1.49–1.20 (m, 48H),
(NBD-Cl) (132 mg, 0.66 mmol) in MeOH (15 mL) containing 1.09 (d, J = 6.8 Hz, 3H).
DIPEA (522 µL, 3.00 mmol) at 0 °C was added dropwise a solu-
13C NMR (101 MHz, CDCl3) δ 173.4, 144.4, 144.1, 144.1,
tion of RBM5-153 (240 mg, 0.60 mmol) in MeOH (10 mL). 136.7, 123.9, 98.6, 74.5, 51.6, 49.6, 44.2, 37.0, 33.7, 29.7, 29.7,
After the addition was complete, the mixture was allowed to 29.5, 29.5, 29.4, 29.3, 29.0, 28.6, 27.1, 26.8, 26.1, 25.9, 14.3.
warm to rt and stirred overnight. Then, the volatiles were
removed under reduced pressure and the residue was directly 743.5547.
HRMS calcd for C40H71N8O5 ([M + H]+): 743.5542, found:
subjected to flash chromatography on silica gel (from 0 to 1%
Compound RBM5-160. Compound RBM5-142 (7 mg,
MeOH in CH2Cl2) to afford RBM5-154 as a shiny orange wax 15 µmol) was added to a stirred solution of RBM5-159 (9 mg,
(278 mg, 82%).
12 µmol) in CH2Cl2 (4 mL). After stirring overnight at rt in the
1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 8.6 Hz, 1H), 6.31 (br dark, the reaction mixture was concentrated to dryness and
s, 1H), 6.17 (d, J = 8.7 Hz, 1H), 4.75 (br s, 1H), 3.73–3.66 (m, 1H), the residue was flash chromatographed on silica gel (from 0 to
3.66–3.62 (m, 1H), 3.49 (ap q, J = 6.7 Hz, 2H), 1.86 (br s, 1H), 5% MeOH in CH2Cl2) to afford the desired SPAAC reaction
1.81 (dt, J = 14.9, 7.4 Hz, 2H), 1.53–1.44 (m, 2H), 1.44 (s, 9H), adduct RBM5-160 (14 mg, 93%, inseparable mixture of diaster-
1.41–1.35 (m, 4H), 1.33–1.23 (m, 20H), 1.07 (d, J = 6.8 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 156.0, 144.3, 144.2, 144.0,
eomers) as a dark-orange solid.
1H NMR (400 MHz, DMSO-d6) (mixture of diastereomers) δ
136.7, 123.7, 98.6, 79.6, 74.6, 50.8, 44.2, 33.6, 29.8, 29.7, 29.7, 9.54 (s, 1H), 8.81 (s, 1H), 8.63 (t, J = 5.7 Hz, 1H), 8.50 (d, J = 9.1
29.7, 29.6, 29.6, 29.5, 29.3, 28.6, 28.5, 27.0, 26.1, 14.5. Hz, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H),
HRMS calcd for C29H50N5O6 ([M + H]+): 564.3756, found: 7.14–7.04 (m, 2H), 7.04 (dd, J = 8.7, 2.3 Hz, 1H), 6.41 (d, J = 9.3
564.3761.
(2S,3R)-2-Amino-18-[(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)
Hz, 1H), 4.47 (d, J = 6.1 Hz, 1H), 4.18 (t, J = 7.0 Hz, 2H),
4.07–3.96 (m, 2H), 3.89 (s, 3H), 3.66–3.58 (m, 1H), 3.50–3.39
amino]octadecan-3-ol hydrochloride (RBM5-155). An ice-cooled (m, 2H), 3.34–3.26 (m, 2H), 3.26–3.17 (m, 1H), 2.99–2.88 (m,
solution of RBM5-154 (85 mg, 0.15 mmol) in MeOH (10 mL) 4H), 2.77–2.61 (m, 2H), 2.15–2.05 (m, 3H), 2.01 (t, J = 7.3 Hz,
was treated with neat AcCl (54 µL, 0.75 mmol) and the result- 2H), 1.73–1.60 (m, 4H), 1.56–1.05 (m, 60H), 0.97 (d, J = 6.7 Hz,
ing mixture was allowed to warm to rt and stirred overnight in 3H), 0.94–0.82 (m, 2H).
the dark. Then, the solvent was evaporated in vacuo and the
residue was purified by flash chromatography on silica gel found: 1237.7983.
HRMS calcd for C68H105N10O11 ([M + H]+): 1237.7959,
(from 0 to 20% MeOH in CH2Cl2) to afford RBM5-155 as a
Compound RBM5-161. Compound RBM5-143 (10 mg,
shiny orange wax (65 mg, 86%).
15 µmol) was added to a stirred solution of RBM5-159 (10 mg,
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13 µmol) in CH2Cl2 (4 mL). After stirring overnight at rt in the the origin using GraphPad Prism version 7.00 for Windows
dark, the reaction mixture was concentrated to dryness and (GraphPad Software Inc., La Jolla, CA, USA). Then, ΦF was cal-
the residue was flash chromatographed on silica gel (from 0 to culated by using eqn (1), where the subscripts x and Std.
5% MeOH in CH2Cl2) to afford the desired SPAAC reaction denote sample and standard, respectively, Grad equals the
adduct RBM5-161 (16 mg, 88%, inseparable mixture of diaster- slope of the plot of the integrated fluorescence intensity vs.
eomers) as a dark-red solid.
absorbance at the λex and η is the refractive index of the
1H NMR (400 MHz, DMSO-d6) (mixture of diastereomers) δ solvent.
9.53 (br s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H),
7.94 (d, J = 2.5 Hz, 1H), 7.64–7.59 (m, 1H), 7.49 (d, J = 8.6 Hz,
1H), 7.25 (dd, J = 8.7, 2.5 Hz, 1H), 7.10 (t, J = 5.7 Hz, 1H), 6.81
ꢀ
ꢁ
ꢀ
ꢁ
2
Gradx
GradStd:
ηx
ΦF;x ¼ ΦF;Std:
ꢀ
ꢀ
ð1Þ
2
ηStd:
(d, J = 9.2 Hz, 1H), 6.64 (d, J = 2.4 Hz, 1H), 6.38 (d, J = 9.0 Hz,
1H), 6.18 (s, 1H), 4.47 (d, J = 6.1 Hz, 1H), 4.21–4.10 (m, 4H),
4.02 (d, J = 7.8 Hz, 2H), 3.67–3.57 (m, 1H), 3.50 (q, J = 7.0 Hz,
4H), 3.46–3.39 (m, 2H), 3.25–3.18 (m, 1H), 3.15–3.04 (m, 4H),
3.05–2.95 (m, 2H), 2.94–2.86 (m, 2H), 2.78–2.59 (m, 2H),
2.13–1.90 (m, 7H), 1.83–1.73 (m, 2H), 1.70–1.59 (m, 4H),
1.54–1.10 (m, 58H), 0.96 (d, J = 6.7 Hz, 3H), 0.93–0.82 (m, 2H).
HRMS calcd for C77H114N11O10 ([M + H]+): 1352.8745,
found: 1352.8760.
Spectral overlap integrals (J(λ)) of the donor–acceptor pairs
were calculated using the specific J(λ) calculator tool from a|e
UV-Vis-IR Spectral Software version 2.2 for Windows from
FluorTools. To calculate J(λ), a|e UV-Vis-IR Spectral Software
uses eqn (2), where FD is the normalised donor emission spec-
trum, εA is the extinction coefficient spectrum of the acceptor,
and λ is the wavelength.
ð1
JðλÞ ¼
FDðλÞ ꢀ εAðλÞ ꢀ λ4dλ
ð2Þ
0
Spectroscopic studies
Förster radius (R0) of the two different donor–acceptor pairs
were calculated by using eqn (3), where ΦD is the quantum
yield of the donor, J(λ) is the spectral overlap integral, η is the
refractive index of the solvent and κ is a constant that reflects
the relative orientation of the excited donor’s electric field and
the acceptor’s absorption dipole. For molecules in which the
rotational diffusion of the dyes is faster than the donor’s
fluorescence lifetime, κ takes a value of 2/3.42 R0 was expressed
in Å.
Absorption spectra were recorded on a Jasco V-730 UV-Vis
spectrophotometer using a spectral bandwidth of 1 nm, a
response time of 0.24 s, a data interval of 1 nm (except for
quinine sulphate and compound RBM5-142, in which case the
data interval was of 0.2 nm) and a scan rate of 200 nm min−1
.
Measurements were carried under an inert atmosphere (continu-
ous flow of nitrogen gas) at a constant temperature of 20 °C.
Fluorescence emission spectra were recorded on a Photon
Technology International (PTI) QuantaMaster fluorometer at
room temperature. The excitation and emission monochroma-
tors were set at 0.5 mm, giving a spectral bandwidth of 2 nm
(except for fluorescein and compounds RBM5-155 and RBM5-
159, in which case the monochromators were set at 0.35 mm,
giving a spectral bandwidth of 1.4 nm). The data interval was
1 nm and the integration time was 1 s. All measurements were
carried out using a Hellma 1.5 mL PTFE-stoppered fluorescence
quartz cuvette (4 clear windows) with a 1 cm path length.
1=6
R0 ¼ 0:211 ꢀ ½κ2 ꢀ ΦD ꢀ JðλÞ ꢀ ηꢁ4
ꢂ
ð3Þ
Composite spectra deconvolution
The absorption and emission spectra of RBM5-161 were
decomposed into their individual components using the
Composite Spectrum Regression App for OriginPro version
2018 (OriginLab Corporation, Northampton, MA, USA). The
spectra of RBM5-154 (donor) and RBM5-143 (acceptor) were
used as the reference for x1 and x2, respectively, to express the
spectra of RBM5-161 as the linear combination of the spectra
of their individual components, according to the general eqn
(4), where x1 and x2 are the spectra of the donor and the accep-
tor, respectively, multiplied for an appropriate coefficient (a1
and a2).
Molar extinction coefficients (ε) were calculated according
to Lambert–Beer’s law from solutions at concentrations
ranging between 0.25 and 25 µM in the appropriate solvent
system (spectrophotometric grade solvents). The absorbance
Abs
max
values at the λ
were then plotted against the corresponding
concentrations and adjusted to a linear regression function
forced through the origin (i.e. the line was forced to intercept
(0,0)) using GraphPad Prism version 7.00 for Windows
(GraphPad Software Inc., La Jolla, USA). Only the absorbance
values in the range between 0.05 and 1 were used. Since we
y ¼ a1x1 þ a2x2
ð4Þ
The deconvolution regression was carried out forcing an
used a 1 cm path length cuvette, ε equals the slope of the intercept with the origin, thereby obtaining a fitted curve (xi,yi
graph. values), the values for the a1 and a2 coefficients and a fitting
Fluorescence quantum yields were measured (ΦF) following score (R-square and SE); the reference spectra of the individual
the comparative method described by Resch-Genger and components (x1 and x2) were then multiplied by the a1 and a2
Rurack41 (IUPAC technical report). The integrated fluorescence coefficients to obtain the calculated spectra.
intensity (i.e. the area under the curve of the emission spec-
FRET efficiency. A series of solutions of the D compounds
trum) was plotted against the corresponding Abs value at the (RBM5-142 and RBM5-154) and the DA compounds (RBM5-160
λex and adjusted to a linear regression function forced through and RBM5-161) was prepared such that the Abs value at the
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corresponding λex was approximately between 0.01 and 0.1. and ω-azidopalmitic acid (500 μM final concentration) for 2 h.
The absorption and emission spectra of each solution were The medium was removed, the cells were washed with 400 μL
recorded using a 1 cm path length quartz cuvette, as described PBS and harvested with 400 μL of trypsin–EDTA and 600 μL of
above. The absorption and emission spectra of the DA com- medium. UPLC-MS analysis was performed as mentioned
pounds were subjected to deconvolution regression using the below. To overexpress CerS5, 24
h before transfection,
Composite Spectrum Regression App for OriginPro version HEK293T cells were seeded in 6-well plates (2 × 105 cells per
2018 (OriginLab Corporation, Northampton, MA, USA), to well) and transfected with 2.5 μg per well of plasmid harboring
isolate the spectra of the donors. The integrated fluorescence the human CerS5 gene using 0.01 mg mL−1 of PEI in Opti-
intensity was then plotted against the Abs value at the λex and MEM for 6 h. Complete DMEM medium supplemented with
adjusted to a linear regression function forced through the 10% FBS was added and cells were incubated for 48 h. After
origin using GraphPad Prism version 7.00 for Windows transfection, cells were treated with RBM5-155 and ωN3PA as
(GraphPad Software Inc., La Jolla, CA, USA). The FRET indicated above.
efficiency was calculated by using eqn (5), where GradD and
Lipid extraction. Cell pellets were suspended with 100 μL of
GradDA are the slopes of the plots of the integrated fluo- H2O and mixed with 750 μL of methanol : chloroform, 2 : 1.
rescence intensity vs. absorbance at the λex of the donor alone Samples were heated at 48 °C overnight and next day, 75 μL of
and the donor component (upon spectral deconvolution) in 1 M KOH in methanol was added, followed by 2 h of incu-
the presence of the acceptor, respectively.
bation at 37 °C. Afterwards, the saponification was neutralised
with 75 μL of 1 M acetic acid and solvent was removed using a
Speed Vac Savant SPD131DDA (Thermo Scientific).
GradDA
E ¼ 1 ꢁ
ð5Þ
GradD
UPLC-MS analysis. Lipid extracts, fortified with internal
Donor emission bleed-through (DEB). The DEB was calcu- standards (N-dodecanoylsphingosine, N-dodecanoylglucosyl-
lated as the ratio, in the form of a percentage, between the sphingosine, N-dodecanoylsphingosylphosphorylcoline and
slope of the plot of absorbance at the λex vs. the integrated C17-sphinganine, 0.2 nmol each) were solubilised in 150 μL of
fluorescence intensity of the donor component upon spectral methanol. Samples were then centrifuged at 9300g for 3 min
deconvolution (GradDDA), and that of the original non-deconvo- and 130 μL of the supernatant was injected into a Waters
luted spectrum (GradTDoAt) (see eqn (6)). In both cases, the fluo- Aquity UPLC system connected to a Waters LCT Premier
rescence intensity was integrated for the wavelength interval Orthogonal Accelerated Time of Flight Mass Spectrometer
corresponding to the emission of the acceptor. The data and (Waters, Milford, MA, USA) operated in the positive electro-
the method used to generate the plots were the same as for the spray ionisation mode. Full scan spectra from 50 to 1500 Da
calculation of FRET efficiencies
were acquired and individual spectra were summed to produce
data points every 0.2 s. Mass accuracy and reproducibility were
maintained by using an independent reference from
LockSpray. The analytical column was a 100 mm × 2.1 mm i.
d., 1.7 μm C8 Acquity UPLC BEH (Waters). The two mobile
phases were phase A: methanol/water/formic acid (74/25/1 v/v/
v) and phase B: methanol/formic acid (99/1 v/v), and both also
contained 5 mM ammonium formate. A linear gradient was
programmed—0.0 min: 80% B; 3 min: 90% B; 6 min: 90% B;
15 min: 99% B; 18 min: 99% B; 20 min: 80% B. The flow rate
GradDDA
DEB ¼
Tot ꢀ 100
GradDA
ð6Þ
Acceptor emission bleed-through (AEB). This parameter was
estimated following a reported protocol.32 The AEB for the
different donor–acceptor pairs were calculated as the percen-
tage between the slopes of the plots of absorbance at the λex vs.
integrated fluorescence intensity corresponding to the calcu-
lated AEB spectra and to those corresponding to FdDAðTotÞ (inte-
grated fluorescence of the non-deconvoluted spectra, being
(eqn (7)).
was 0.3 mL min−1
.
Gradd
GraddDAðTotÞ
DAðAEBÞ ꢀ 100
ð7Þ
Conflicts of interest
AEB ¼
There are no conflicts to declare.
Cell studies
General procedures. Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS), penicillin/streptomycin
Acknowledgements
solution, Opti-MEM and polyethyleneimine (PEI) were from Partial financial support from the Project CTQ2017-85378-R
Invitrogen. Cells were cultured at 37 °C in 5% CO2 in DMEM (AEI/FEDER, UE) of the Spanish Ministry of Science,
supplemented with 10% foetal bovine serum and 100 ng mL−1 Innovation and Universities is acknowledged. We are grateful
each of penicillin and streptomycin.
to Prof. Anthony H. Futerman (Weizmann Institute of Science,
CerS catalyzed incorporation of ωN3PA into RBM5-155. A549 Rehovot, Israel) for providing the CerS5 plasmid and to Prof.
and MEF cells were seeded in 6-well plates (2 × 105 cells per Vicente Marchán (UB) for technical support. Experimental con-
well) and treated with RBM5-155 (10 μM final concentration) tributions from Mr Alexandre García and Mr Pedro Rayo are
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Organic & Biomolecular Chemistry
also acknowledged. About the source of the cells, we pur- 15 S. Grösch, S. Schiffmann and G. Geisslinger, Chain Length-
chased from ATCC (https://www.lgcstandards-atcc.org/en.aspx).
We acknowledge support of the publication fee by the CSIC
Specific Properties of Ceramides, Prog. Lipid Res., 2012,
51(1), 50–62.
Open Access Publication Support Initiative through its Unit of 16 I. Nieves, P. Sanllehí, J.-L. L. Abad, G. Fabriàs, J. Casas and
Information Resources for Research (URICI).
A. Delgado, Chemical Probes of Sphingolipid Metabolizing
Enzymes, in Bioactive Sphingolipids in Cancer Biology and
Therapy, ed. Y. Hannun, C. Luberto, L. Obeid and C. Mao,
Springer International Publishing, Cham, 2015, pp.
437–469.
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