Design and Synthesis of Quenched Activity-based Probes for Diacylglycerol Lipase and α,β-Hydrolase Domain Containing Protein 6

Article abstract of DOI:10.1002/asia.201800452

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore–quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.

Full text of DOI:10.1002/asia.201800452

DOI: 10.1002/asia.201800452
Full Paper
Quenched Activity-based Probes
Design and Synthesis of Quenched Activity-based Probes for
Diacylglycerol Lipase and a,b-Hydrolase Domain Containing
Protein 6
E. J. van Rooden,[a] M. Kohsiek,[a] R. Kreekel,[a] A. C. M. van Esbroeck,[a]
A. M. C. H. van den Nieuwendijk,[b] A. P. A. Janssen,[a] R. J. B. H. N. van den Berg,[b]
H. S. Overkleeft,[b] and M. van der Stelt*[a]
Abstract: Diacylglycerol lipases (DAGL) are responsible for
the biosynthesis of the endocannabinoid 2-arachidonoylgly-
cerol. The fluorescent activity-based probes DH379 and HT-
01 have been previously shown to label DAGLs and to cross-
react with the serine hydrolase ABHD6. Here, we report the
synthesis and characterization of two new quenched activi-
ty-based probes 1 and 2, the design of which was based on
the structures of DH379 and HT-01, respectively. Probe 1
contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a flu-
orophore–quencher pair, whereas probe 2 employs a Cy5-
fluorophore and a cAB40-quencher. The fluorescence of
both probes was quenched with relative quantum yields of
0.34 and 0.0081, respectively. The probes showed target in-
hibition as characterized in activity-based protein profiling
assays using human cell- and mouse brain lysates, but were
unfortunately not active in living cells, presumably due to
limited cell permeability.
b (DAGLa and DAGLb).[5] The DAG lipases belong to the serine
hydrolase superfamily and selectively hydrolyze the sn-1 ester
within diacylglycerols. DAGLa is mainly responsible for the
generation of 2-AG in the central nervous system, whereas
DAGLb mostly acts in the periphery.[6] To date, isoform selective
inhibitors have not been reported. DAGLa inhibitors have po-
tential as drug candidates for obesity as well as neurodegener-
ative disorders.[7] Most of the reported covalent DAGL inhibi-
tors also target a,b-hydrolase domain containing protein 6
(ABHD6).[8,9] ABHD6 is thought to control 2-AG levels post-syn-
aptically.[10] The enzyme responsible for catalyzing the bulk 2-
AG in the brain, monoacylglycerol lipase (MAGL), acts at presy-
naptic sites. To gain a better understanding of the regulation
of 2-AG levels we need to study the activity of these enzymes
in their native environment.
Introduction
The endocannabinoid signaling system (ECS) consists of the
cannabinoid receptors, their endogenous ligands, that is, 2-
arachidonoylglycerol (2-AG) and anandamide (AEA), and the
enzymes regulating the levels of these ligands.[1] The endocan-
nabinoid system is involved in a wide array of neurophysiologi-
cal processes, including nociception, cognitive function and
appetite. Enzymes involved in the ECS are consequently poten-
tial therapeutic targets for an array of human disorders, includ-
ing pain, neurodegenerative diseases and obesity.[2–4] The en-
docannabinoid 2-AG is synthesized from diacylglycerol (DAG)
by hydrolysis catalyzed by either sn-1-diacylglycerol lipase a or
[a] E. J. van Rooden, M. Kohsiek, R. Kreekel, A. C. M. van Esbroeck,
A. P. A. Janssen, Prof. Dr. M. van der Stelt
Molecular Physiology
Activity-based protein profiling (ABPP) is a method to study
native enzyme activity.[11] Activity-based probes (ABPs) form a
covalent bond with active enzymes and thus report on the
amount of active enzyme present in a biological system at a
given time. Various fluorescent ABPs for DAGL and ABHD6,
such as DH379[12] and HT-01,[8] have been reported.
Leiden Institute of Chemistry
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: m.van.der.stelt@chem.leidenuniv.nl
[b] A. M. C. H. van den Nieuwendijk, Dr. R. J. B. H. N. van den Berg,
Prof. Dr. H. S. Overkleeft
Quenched ABPs (qABPs) have been developed to decrease
the fluorescent signal arising from an unbound fluorescent
probe.[13] A qABP consists of an electrophilic trap, a recognition
element, a fluorophore (F) and a complementary quencher (Q)
(Figure 1a). The quencher is part of the leaving group, which
disassociates after the formation of the enzyme-probe com-
plex. Bogyo and co-workers were the first to synthesize a qABP
and developed a set of qABPs for cathepsins.[14–18] To date vari-
Bio-organic Synthesis
Leiden Institute of Chemistry
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/asia.201800452.
This manuscript is part of a special issue on chemical tools and materials
for biological/medicinal applications. A link to the Table of Contents of the
special issue will appear here when the complete issue is published.
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Figure 1. qABP design for DAGL and ABHD6. (a) General design of a triazole urea as qABP for serine hydrolases. (F) is the fluorophore and (Q) is the quencher.
(b) Probe DH379 and probe 1. (c) Probe HT-01 and probe 2.
ous enzyme classes, including kinases,[19] serine proteases[20]
and cysteine proteases such as legumain[21] and caspases,[22]
have been targeted with qABPs. However, metabolic serine hy-
drolases have not yet been studied with qABPs. Here, we
report on the synthesis and characterization of qABPs for the
metabolic serine hydrolases DAGL and ABHD6.
acted with 6. The two regioisomers thus formed were separat-
ed by column chromatography yielding inhibitor 3. To obtain
probe 1, alkylation of alcohol 5 led to azide 8, which was re-
duced by treatment with triphenylphosphine and water to
give 9. A nucleophilic aromatic substitution reaction between
amine 9 and 2,4-dinitrofluorobenzene yielded the desired
alkyne 10, which contains dinitroaniline as a quencher. CuAAC
of alkyne 10 with pivaloyloxymethyl-azide (POM-N3) gave the
POM-protected triazole, which was deprotected in basic solu-
tion to give compound 11.[35] Similarly as described above for
triazole 6, triphosgene coupling of 11 with 7 gave intermedi-
ate 12 after separation of two regioisomers by column chro-
matography. A CuAAC reaction between azide 12 and acety-
lene-functionalized BODIPY-FL[25] furnished probe 1.
Synthesis of probe 2. The qABP 2 is based on the fluores-
cent ABP HT-01 (Figure 1c). Reduction of commercially avail-
able 4-ethynylbenzaldehyde (13) with sodium borohydride
gave alcohol 14, which was subsequently mesylated to provide
15 (Scheme 2). Next, nucleophilic substitution of the mesylate
with phthalimide yielded compound 16. A CuAAC reaction be-
tween alkyne 16 and TMS-azide resulted in triazole 17. Sepa-
rately, N-Boc-cadaverine (18) was nosylated and alkylated
using phenethylbromide. Subsequent deprotection with ethyl-
enediamine resulted in amine 19. This amine was treated with
triphosgene and coupled to triazole 17, resulting in a mixture
of two regioisomers, which were separated by column chroma-
tography to give the N1 isomer 20. The Cy5-analogue of HT-01
21 was made by deprotecting 20 with TFA and coupling the
resulting amine to a Cy5 activated ester. To make the probe 2,
Results and Discussion
The design of our qABPs 1 and 2 is based on triazole ureas,
DH379 and KT-01, respectively, both of which are ABPs for the
serine hydrolases targeted in the here-reported studies. DH379
targets DAGLa, DAGLb and ABHD6, while KT-01 labels DAGLb
and ABHD6 (Figure 1).[8,23] Both ABPs contain a triazole urea as
an electrophilic trap, which is a commonly used warhead for
serine hydrolases (Figure 1a).[12,24] BODIPY-FL[25] and 2,4-
DNA[26–30] were chosen as a fluorophore-quencher pair for
probe 1,[19,31] while Cy5 and cAB40 were selected as a fluoro-
phore-quencher pair for qABP 2.[32]
Synthesis of probe 1. The synthesis of probe 1 and DAGL
inhibitor 3, an azide analogue of inhibitor DH376, started with
the nucleophilic addition of lithium TMS-acetylene onto 4,4’-di-
fluorophenyl ketone 4 (Scheme 1), followed by deprotection in
basic solution to give alcohol 5. Copper(I)-catalyzed [2+3]
azide alkyne cycloaddition (CuAAC) of 5 with hydrazoic acid,
formed in situ from methanol and TMS-N3 in DMF,[33,34] gave tri-
azole 6 in moderate yield. Next, piperidine 7, which was syn-
thesized as previously reported,[12] was deprotected with acid
and the resulting amine was treated with triphosgene and re-
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Scheme 1. Synthesis of probe 1. Reagents and conditions: (a) i. TMS-acetylene, nBuLi, THF, À10–08C; ii. KOH, MeOH/THF, 08C, 74%; (b) TMS-N3, NaAsc, CuSO4,
DMF/MeOH, 608C, 59%; (c) i. 7, 40% TFA in DCM; ii. Triphosgene, DIPEA, THF, 08C; iii. 6, DIPEA, THF, 608C; (d) NaH, 3-azidopropanol tosylate, DMF, 08C—rt,
64%; (e) PPh3, H2O/THF, 608C, 100%; (f) 2,4-dinitrofluorobenzene, NEt3, DMF, 95%; (g) i. POM-azide, CuBr, TBTA, THF/H2O; ii. KOH, MeOH, 67%; (h) i. 7, 40%
TFA in DCM; ii. Triphosgene, DIPEA, THF, 08C; iii. 11, DIPEA, THF, 608C; (i) NaAsc, CuSO4, DMF, MeOH, BODIPY-FL.[25]
Scheme 2. Synthesis of qABP 2 and control compound 21. Reagents and conditions: (a) NaBH4, EtOH, 99%; (b) DCM, Et3N, MsCl, 08C, 94%; (c) potassium
phthalimide, DMF, 92%; (d) TMS-N3, CuI, DMF/MeOH (5/1), reflux, 64%; (e) i. NsCl, Et3N, THF; ii. Ph(CH2)2Br, Cs2CO3, ACN, 808C; iii. PhSH, Cs2CO3, ACN, 71%;
(f) i. triphosgene, DIPEA, THF; ii. 17, DMAP, DIPEA, THF, 27%; (g) i. TFA/DCM. ii. Cy5-OSu, DIPEA, DMF, 44%; (h) ethylenediamine, EtOH, 50%; (i) HCTU, DIPEA,
cAB40, DMF, 53%; (j) i. 10% TFA/DCM; ii. Cy5-NHS, DIPEA, DMF, 10%.
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the phthalimide 20 was deprotected with ethylenediamine.
Amine 22 was coupled to the cAB40 quencher (for synthesis,
see Experimental) and amide product 23 was deprotected by
TFA, followed by coupling to Cy5, yielding the final product 2.
Spectroscopic characterization. The absorbance spectrum
of probe 1 was compared to BODIPY-FL. In ethanol, the probe
and the parent BODIPY have identical absorbance maxima
(Table 1). In aqueous solution the maximal absorbance of the
To investigate whether qABP 1 could covalently label human
DAGLa, we employed a gel-based activity-based protein profil-
ing (ABPP) assay for rapid and efficient visualization of endoge-
nous serine hydrolase activity in native biological samples. Re-
combinant human DAGLa was over-expressed in HEK293T
cells, which were lysed and treated with varying concentra-
tions of qABP 1. The proteins were resolved by SDS-PAGE and
labeled proteins were visualized by in-gel fluorescence scan-
ning. Human DAGLa was dose-dependently labeled by
probe 1 (Figure 2a) and could be out-competed by the selec-
tive DAGL inhibitor, LEI105 (Figure 2b).[39]
Table 1. lmax (absorbance maximum) and Frelative (relative quantum yield)
of probe 1 and 2.
Compound
lmax in EtOH [nm]
lmax in H2O [nm]
F
relative in EtOH
BODIPY-FL
Probe 1
Cy5
497
497
644
644
497
505
639
650
1.0[a]
0.34[a]
1.0[b]
Probe 2
0.081[b]
[a] Relative to BODIPY-FL. [b] Relative to Cy5.
probe is shifted towards the red (approximately 10 nm) while
the parent BODIPY does not display such a redshift (Table 1).
Similar shifts in absorbance profiles have been previously ob-
served in comparable systems[26,27,36] and are indicative of a
ground state complex. This suggests that the fluorescence of
the probe is quenched to a certain extent by ground state
complex formation. Probe 2 is also slightly redshifted in water
compared to ethanol. To determine the amount of quenching,
we determined the fluorescence quantum yield, which is de-
fined as the ratio of the number of photons emitted to the
number of photons absorbed by a fluorophore. Fluorescence
quantum yields of qABPs 1 and 2 were determined relative to
their parent fluorophores, that is, BODIPY-FL and Cy5 (Table 1).
Figure 2. Activity-based protein profiling with probe 1. (a) Lysate of recombi-
nant human DAGLa over-expressed in HEK293T cells, treated with probe 1
(30 min, rt). (b) Competition of human DAGLa labeling by probe 1 with
LEI105 (30 min pre-incubation with LEI105, followed by 20 min incubation
with probe, rt).
Probe 1 has
a
relative quantum yield of 0.34 (Æ3-fold
quenched). Probe 2 has a relative quantum yield of 0.081 (Æ
12-fold quenched).
Biological characterization of probe 2. To test whether
probe 2 labeled ABHD6, lysates from two cell lines were used:
human osteosarcoma U2OS cells stably overexpressing ABHD6
fused to green fluorescent protein (GFP) as well as wildtype
mouse neuroblastoma Neuro2A (N2A) with endogenous
ABHD6 expression. In the U2OS lysates, a strong signal from
probe 2 was visible at approximately 70 kDa, corresponding to
the MW of the ABHD6-GFP fusion protein (Figure 3a). Incuba-
tion of wildtype N2A lysates with probe 2 resulted in fluores-
cent labeling of several proteins, including a protein at 35 kDa,
which was also labeled by the well-characterized ABHD6 probe
DH379 (Figure 3b). These results suggest that probe 2 cova-
lently labels mouse and human ABHD6.
Biological characterization of probe 1. To determine
whether qABP 1 inhibits human DAGLa, we used a colorimet-
ric assay with para-nitrophenyl butyrate as surrogate substrate
and membranes of HEK293T cells overexpressing recombinant
human DAGLa.[37,38] Compound 3, an azide analogue of
DH376, was used as positive control and showed good inhibi-
tory activity (IC50 =5 nm), comparable to DH376 (Table 2). Inter-
mediate 12, containing only a quencher, retained high inhibito-
ry activity, whereas qABP 1 was approximately ten-fold less
active. Of note, probe 1 is about as active as the ABP DH379.
Finally, live U2OS-ABHD6-GFP cells were treated with 2,
lysed and remaining ABHD6 activity was visualized post-lysis
with DH379. Almost no fluorescent signal from 2 was detected
and no decrease in ABHD6 activity was observed with DH379
labeling (Figure 4a), which suggested that probe 2 has limited
cell permeability. Of note, control compound 21 was able to
reduce ABHD6 activity in situ (Figure 4b), which sug-
gested that the reduced cell permeability is caused by the
quencher.
Table 2. Activity of DAGLa inhibitors in surrogate substrate assay.
Compound
pIC50 DAGLa
3
8.3Æ0.07
8.7Æ0.1
8.4Æ0.04
7.4Æ0.2
7.4Æ0.05
DH376
12
1
DH379
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Experimental Section
Chemistry
General methods. Reagents were purchased from Sigma Aldrich,
Acros or Merck and used without further purification unless noted
otherwise. Reactions under dry conditions were performed using
oven or flame-dried glassware and dry solvents (dried for a mini-
mum of 24 h over activated molecular sieves of appropriate (3–
4 ꢁ) pore size). Traces of water were removed from starting com-
pounds by co-evaporation with toluene. All moisture sensitive re-
actions were performed under an argon or nitrogen atmosphere.
Flash chromatography was performed using SiliCycle silica gel type
SilicaFlash P60 (230–400 mesh). HPLC purification was performed
on a preparative LC-MS system (Agilent 1200 series) with an Agi-
lent 6130 Quadruple MS detector. TLC analysis was performed on
Merck silica gel 60/Kieselguhr F254, 0.25 mm. Preparative TLC was
performed on UNIPLATE Alumina GF 1000 mm plates. Compounds
were visualized using UV-irradiation, a KMnO4 stain (K2CO3 (40 g),
KMnO4 (6 g), H2O (600 mL) and 10% NaOH (5 mL)). A stain for or-
ganic azides was used as follows: i. 10% PPh3 in toluene, heating;
ii. Ninhydrin in EtOH/AcOH, heating.[40] 1H and 13C NMR spectra
were recorded on a Bruker AV-400 MHz spectrometer at 400 (1H)
and 100 (13C) MHz using CDCl3, CD3OD or (CD3)2SO as solvent,
unless stated otherwise. Spectra were analyzed using MestReNova
11.0.3. Chemical shift values are reported in ppm with tetramethyl-
silane or solvent resonance as the internal standard (CDCl3, d 7.26
for 1H, d 77.16 for 13C; CD3OD, d 3.31 for 1H, d 49.00 for 13C;
Figure 3. Activity-based protein profiling with probe 2. (a) ABPP on U2OS-
ABHD6-GFP lysate with probe DH379 (Cy3 signal, 1 mm, 20 min, rt), with or
without pre-incubation with probe 2 (Cy5 signal, 10 mm, 30 min, 378C).
(b) ABPP on N2A lysate with probe DH379 (Cy3 signal, 1 mm, 20 min, rt), with
or without pre-incubation with probe 2 (Cy5 signal, 10 mm, 30 min, 378C).
Gels with coomassie staining (lower panels) are shown as protein loading
controls.
1
(CD3)2SO, d 2.50 for H, d 39.52 for 13C). Data are reported as fol-
lows: chemical shifts (d), multiplicity (s=singlet, d=doublet, dd=
double doublet, td=triple doublet, t=triplet, q=quartet, m=mul-
tiplet, br=broad), coupling constants J (Hz), and integration. LC-
MS analysis was performed on a Finnigan Surveyor HPLC system
with a Gemmi C18 50ꢂ4.60 mm column (detection at 200–
600 nm), coupled to a Finnigan LCQ Adantage Max mass spec-
trometer with ESI. The applied buffers were H2O, MeCN and 1.0%
TFA in H2O (0.1% TFA end concentration). General: High resolution
mass spectra (HRMS) were recorded by direct injection on a q-TOF
mass spectrometer (Synapt G2-Si) equipped with an electrospray
ion source in positive mode with leu-enkephalin (m/z=556.2771)
as an internal lock mass. The instrument was calibrated prior to
measurement using the MS/MS spectrum of glu-1-fibrinopeptide B.
IR spectra were recorded on a Shimadzu FTIR-8300 and are report-
ed in cmÀ1. Optical rotations were measured on a Propol automatic
polarimeter (Sodium d-line, l=589 nm). Molecules shown are
drawn using Chemdraw v16.0.
Figure 4. In situ treatment of U2OS-ABHD6-GFP. (a) Probe 2-treated cells
(Cy5 signal, 1 h, 378C), lysed and labeled with DH379 (Cy3 signal, 20 min,
1 mm, rt). (b) Probe 21-treated cells (Cy5 signal, 1 h, 378C), lysed and treated
with DH379 (Cy3 signal, 20 min, 1 mm, rt). Gels with coomassie staining
(lower panels) are shown as protein loading controls.
General procedure for the CuAAC reaction. To a solution of
1 equiv. of azide and 1.0 to 1.5 equiv. of BODIPY alkyne (red or
green) in minimal DMF was added a freshly prepared solution of
0.15 equiv. NaAsc and 0.05 equiv. of CuSO4 in H2O. The resulting so-
lution was stirred for 18 h. The reaction mixture was diluted with
EtOAc and H2O and extracted with EtOAc (3x). The combined or-
ganic layers were washed with H2O (5x), brine, dried (MgSO4), fil-
tered and concentrated.
Conclusions
The qABPs 1 and 2 were successfully synthesized. Probe 1
showed characteristics of static quenching in aqueous solution
and showed activity in a surrogate substrate assay using re-
combinant human DAGLa. Probe 1 could dose-dependently
label human DAGLa in a gel-based activity-based protein
profiling assay. Probe 2 did label endogenously expressed
mouse ABHD6, but was not able to label ABHD6 in live cells.
Further optimization of fluorescent properties and cell permea-
bility of the probes is required to apply them in biological
studies.
Pivaloyloxymethyl-azide (POM-N3, Scheme 1). This procedure was
adapted from literature.[41] To a solution of NaN3 (0.44 g, 6.6 mmol)
in H2O (3.5 mL) was added POM-Cl (0.85 mL, 6 mmol). The resulting
mixture was stirred vigorously at 908C for 18 h. The reaction mix-
ture was diluted with water and extracted with DCM (3x). The com-
bined organic layers were carefully concentrated to yield a color-
1
less liquid (0.92 g, 5.9 mmol, 98%). H NMR (400 MHz, CDCl3): d=
5.14 (s, 2H), 1.26 ppm (s, 9H). 13C NMR (100 MHz, CDCl3): d=
178.22, 74.52, 39.11, 27.13 ppm. IR: n˜ =2102.41 (-N3), 1737.86 cmÀ1
(C=O).
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3-Azidopropanol tosylate (Scheme 1). To a solution of 3-bromo-
propanol (1.2 mL, 13.8 mmol) in water (40 mL) was added NaN3
(1.8 g, 27.6 mmol). The resulting reaction mixture was stirred at
808C for 3 days, allowed to cool to rt and extracted with EtOAc
(5x). The combined organic layers were washed with brine, dried
(Na2SO4), filtered and carefully concentrated. The residue was dis-
solved in DCM and NEt3 (3.8 mL, 27.6 mmol) was added. The result-
ing solution was cooled to 08C before addition of tosyl chloride
(4.0 g, 21 mmol) and stirred for 18 h. The reaction mixture was di-
luted with H2O and extracted with DCM (3x). The combined organ-
ic layers were dried (Na2SO4), filtered and concentrated over celite.
Purification of the residue by silica gel column chromatography
(9:1 PE:DCM) yielded a colorless liquid (2.3 g, 9 mmol, 65%), which
discolored slightly upon storage at rt. 1= ~1.5 gmLÀ1. TLC: Rf =0.3
(1:9 DCM:pentane). IR: n˜ =2095 cmÀ1 (-N3). 1H NMR (400 MHz,
CDCl3): d=7.83 (d, J=8.2 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H), 4.14 (t,
J=5.9 Hz, 2H), 3.41 (t, J=6.5 Hz, 2H), 2.49 (s, 3H), 1.92 ppm (p, J=
6.2 Hz, 2H). 13C NMR (100 MHz, CDCl3): d=145.28, 132.90, 130.17,
128.14, 67.20, 47.48, 28.66, 21.91 ppm.
1.84 ppm (m, 2H). 13C NMR (100 MHz, CDCl3): d=162.44 (d, J=
247.1 Hz), 138.85, 128.50 (d, J=8.2 Hz), 115.27 (d, J=21.6 Hz),
82.82, 79.17, 78.23, 61.55, 48.74, 29.33 ppm. LC-MS m/z: 328
[M+H]+.
3-((1,1-Bis(4-fluorophenyl)prop-2-yn-1-yl)oxy)propan-1-amine
(9). To a solution of 8 (0.25 g, 0.77 mmol) in 1:10 H2O:THF was
added PPh3 (0.45 g, 1.7 mmol). The reaction mixture was stirred at
608C for 18 h, concentrated, diluted with EtOAc and H2O, basified
with 1m NaOH and extracted with EtOAc (3x). The combined or-
ganic layers were washed with brine, dried (MgSO4), filtered and
concentrated. Purification of the residue by silica gel column chro-
matography (10% methanolic solution of 3% NH3 in DCM) yielded
a colorless film (234 mg, 0.77 mmol, 100%). TLC: Rf =0.27 (1:9
MeOH:DCM). 1H NMR (400 MHz, CDCl3): d=7.57–7.42 (m, 4H),
7.07–6.92 (m, 4H), 3.52 (t, J=6.1 Hz, 2H), 2.91 (s, 1H), 2.86 (t, J=
6.9 Hz, 2H), 1.86 (s, 2H), 1.84–1.74 ppm (m, 2H). 13C NMR (100 MHz,
CDCl3): d=162.35 (d, J=246.9 Hz), 139.02 (d, J=3.1 Hz), 128.45 (d,
J=8.2 Hz), 115.19 (d, J=21.6 Hz), 83.03, 79.03, 78.05, 62.55, 39.48,
33.35 ppm. LC-MS m/z: 301.6 [M+H]+.
1-Amino-4-((4-(carboxymethyl)phenyl)amino)-9,10-dioxo-9,10-di-
hydroanthracene-2-sulfonate (cAB40, Scheme 2). To a solution of
bromaminic acid (2 g, 5 mmol) and 4-aminophenyl acetic acid
(674 mg, 4.45 mmol) in water (75 mL) were added Na2CO3 (795 mg,
7.5 mmol) and CuSO4 (120 mg, 0.75 mmol). The reaction mixture
was refluxed for 16 h, washed with DCM (3ꢂ50 mL) and concen-
trated. MeOH was added to the residue and after filtration the fil-
trate was concentrated and purification of the residue by reversed
phase silica gel column chromatography (H2O >1:4 MeOH:H2O)
yielded cAB40 as a blue solid (650 mg, 1.4 mmol, 32%). 1H NMR
(400 MHz, MeOD): d=8.34–8.32 (m, 2H), 8.26 (s, 1H), 7.88–7.77 (m,
2H), 7.36 (d, J=8.4 Hz, 2H), 7.26 (d, J=8.4 Hz, 2H), 3.62 ppm (s,
2H). LC-MS m/z: 452.9 [M+2H]+.
N-(3-((1,1-Bis(4-fluorophenyl)prop-2-yn-1-yl)oxy)propyl)-2,4-dini-
troaniline (10). To a solution of 9 (234 mg, 0.77 mmol) in DMF
(5 mL) was added NEt3 (0.2 mL, 1.5 mmol) and 2,4-dinitrofluoroben-
zene (100 mL, 0.77 mmol). The reaction mixture was stirred for 18 h,
diluted with Et2O and brine and extracted with Et2O (3x). The com-
bined organic layers were washed with H2O (5x), brine, dried
(MgSO4), filtered and concentrated to yield a viscous yellow oil
(341 mg, 0.73 mmol, 95%). TLC: Rf =0.33 in (2:8 EtOAc:pentane).
1
IR: n˜ =3366 (N-H), 3292 cmÀ1 (CC-H). H NMR (400 MHz, CDCl3): d=
9.10 (d, J=2.7 Hz, 1H), 8.62 (t, J=5.5 Hz, 1H), 8.24 (dd, J=9.5,
2.7 Hz, 1H), 7.54–7.43 (m, 4H), 7.05–6.92 (m, 5H), 3.62 (m, 4H),
2.96 (s, 1H), 2.10 ppm (m, 2H). 13C NMR (100 MHz, CDCl3): d=
162.40 (d, J=247.4 Hz), 148.39, 138.52 (d, J=3.0 Hz), 136.01,
130.39, 128.46 (d, J=8.2 Hz), 124.38, 115.25 (d, J=21.6 Hz), 114.03,
82.62, 79.40, 78.55, 61.70, 40.91, 28.89 ppm.
1,1-Bis(4-fluorophenyl)prop-2-yn-1-ol (5). To a cooled (À108C) so-
lution of ethynyltrimethylsilane (1.55 mL, 11 mmol) in dry THF
(20 mL) was slowly added nBuLi (2.5m, 4.5 mL, 11 mmol) and
stirred for 1 h. Subsequently, a solution of 4,4’-difluorobenzophe-
none (4, 2.18 g, 10 mmol) in dry THF (16 mL) was added in 15 mi-
nutes. The resulting solution was stirred at À108C for 1 h and then
08C for 1 h. The reaction mixure was quenched by the addition of
KOH (2m in MeOH, large excess) and stirred for 18 h, poured into
H2O, adjusted to pH 6–7 with 1m HCl and extracted with EtOAc
(3x). The combined organic layers were dried (MgSO4), filtered and
concentrated. Purification of the residue by silica gel column chro-
matography (1:19 EtOAc:pentane) yielded a yellow oil (1.81 g,
7.4 mmol, 74%). TLC: Rf =0.42 (1:9 EtOAc:pentane). IR: n˜ =3298
Bis(4-fluorophenyl)(1H-1,2,3-triazol-4-yl)methanol (6). To a de-
gassed solution of alkyne 5 (1.2 g, 5 mmol) in 1:5 MeOH:DMF
(20 mL) was added sequentially trimethylsilyl azide (1 mL,
7.4 mmol), CuSO4 (1m in water, 0.5 mL, 0.5 mmol) and NaAsc (1m,
1.5 mL, 1.5 mmol). After prolonged heating (5 days) and several
new portions of TMS-N3 (3ꢂ1 mL), the reaction was quenched
with H2O. The reaction mixture was filtered over celite, concentrat-
ed, diluted with H2O and extracted with Et2O (3x). The combined
organic layers were washed with H2O (3x), brine, dried (Na2SO4), fil-
tered and concentrated. Purification of the residue by silica gel
column chromatography (10 >30% EtOAc in pentane) yielded a
white solid with yellow discoloration. This solid was recrystallized
from CHCl3 to yield the title compound (835 mg, 2.91 mmol, 59%).
TLC: Rf =0.54 (1:1 EtOAc:pentane). IR: n˜ =3192 (N-H), 1601,
1
(CC-H), 1601, 1504, 1221, 1157 cmÀ1. H NMR (400 MHz, CDCl3): d=
7.60–7.50 (m, 4H), 7.07–6.96 (m, 4H), 2.90 (s, 1H), 2.79 ppm (s, 1H).
13C NMR (100 MHz, CDCl3): d=163.76, 127.99 (d, J=8.3 Hz), 115.35
(d, J=21.7 Hz), 76.10 ppm.
1504 cmÀ1 1H NMR (400 MHz, MeOD): d=7.58 (s, 1H), 7.42–7.29
.
4,4’-(1-(3-Azidopropoxy)prop-2-yne-1,1-diyl)bis(fluorobenzene)
(8). To a cooled (08C) solution of alkyne 5 (0.29 g, 1.2 mmol) in dry
DMF (10 mL) was added NaH (60% dispersion in mineral oil,
55 mg, 1.3 mmol) and the reaction mixture was stirred for 30 mi-
nutes. Next, 3-azidopropanol tosylate (0.44 g, 1.8 mmol) was added
dropwise. The resulting yellow solution was stirred for 18 h at rt,
diluted with EtOAc and H2O and extracted with EtOAc (3x). The
combined organic layers were washed with H2O (3x), brine, dried
(MgSO4), filtered and concentrated over celite. Purification of the
residue by silica gel column chromatography (1% EtOAc in pen-
tane) yielded a colorless viscous oil (0.25 g, 0.77 mmol, 64%). TLC:
Rf =0.57 (1:19 EtOAc:pentane). IR: n˜ =3297 (CC-H), 2095 cmÀ1 (N3).
1H NMR (400 MHz, CDCl3): d=7.54–7.43 (m, 4H), 7.09–6.94 (m, 4H),
3.54 (d, J=5.9 Hz, 2H), 3.46 (t, J=6.8 Hz, 2H), 2.92 (s, 1H), 1.99–
(m, 4H), 7.18–6.79 ppm (m, 4H). 13C NMR (100 MHz, DMSO): d=
161.11 (d, J=243.3 Hz),143.12, 128.96 (d, J=8.2 Hz), 114.38 (d, J=
21.3 Hz), 75.14 ppm. LC-MS m/z: 287.8 [M+H]+.
N-(3-(Bis(4-fluorophenyl)(1H-1,2,3-triazol-4-yl)methoxy)propyl)-
2,4-dinitroaniline (11). To a degassed solution of 10 (110 mg,
0.24 mmol) and POM-azide (60 mL, 0.35 mmol) in THF:H2O 10:3
(6.5 mL) was added CuBr (6.1 mg, 42 mmol) and the resulting sus-
pension was sonicated shortly before being stirred at 608C for 4
days. The reaction mixture was allowed to cool to rt before KOH
(2m in MeOH) was added and the resulting solution was stirred for
30 minutes, concentrated, diluted with EtOAc and H2O and extract-
ed with EtOAc (3x). The combined organic layers were washed
with a basic solution of 1m EDTA (basified with NH3(aq.), 2x), brine,
dried (MgSO4), filtered and concentrated. Purification of the residue
&
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by silica gel column chromatography (1:4 EtOAc: pentane) yielded
a yellow film (81 mg, 0.167 mmol, 67%). TLC: Rf =0.33 (1:1 EtOAc:-
pentane). IR: n˜ =3362 (N-H aniline), 3100 cmÀ1 (N-H triazole).
1H NMR (400 MHz, DMSO): d=15.03 (s, 1H), 8.94–8.74 (m, 2H), 8.22
(dd, J=9.7, 2.8 Hz, 1H), 7.50 (s, 1H), 7.39 (m, 4H), 7.21 (d, J=
9.5 Hz, 1H), 7.12 (m, 4H), 3.64–3.49 (m, 2H), 3.26 (m, 2H), 2.00–
1.81 ppm (m, 2H). 13C NMR (100 MHz, DMSO): d=161.18 (d, J=
245.2 Hz), 148.10, 140.21, 134.71, 130.00, 129.4 (d, J=8 Hz), 123.72,
115.28, 114.73 (d, J=21.3 Hz), 61.24, 40.30, 28.50 ppm. LC-MS m/z:
510.8 [M+H]+.
(3:7 EtOAc:pentane). HRMS
: 610.2349, found: 610.2350.
calculated for C31H31F2N7O3 [M+Na]+
m/z
tertButyl
(2R,5R)-5-(3-azidopropoxy)-2-benzylpiperidine-1-car-
((2R,5R)-5-(3-Azidopropoxy)-2-benzylpiperidin-1-yl)(4-((3-((2,4-dini-
trophenyl)amino)propoxy)bis(4-fluorophenyl)methyl)-1H-1,2,3-tria-
zol-1-yl)methanone (12) and ((2R,5R)-5-(3-azidopropoxy)-2-benzylpi-
peridin-1-yl)(4-((3-((2,4-dinitrophenyl)amino)propoxy)bis(4-fluoro-
phenyl)methyl)-2H-1,2,3-triazol-2-yl)methanone (25). Following the
procedure for the preparation of 3, but from triazole 11 at
100 mmol scale. Purification by silica gel column chromatography
(20 >25% EtOAc in PE). Yellow film, 74% yield as a mixture of N1
and N2-isomers. N1, apolar fractions, 12: TLC: Rf =0.38 (3:7 EtOAc:-
pentane). HRMS m/z calculated for C40H40F2N10O7 [M+Na]+:
833.2942, found: 833.2947. N2, polar fractions, 25: TLF: Rf =0.26
(3:7 EtOAc:pentane). HRMS m/z calculated for C40H40F2N10O7
[M+Na]+: 833.2942, found: 833.2956.
boxylate (7). To a cooled (08C) solution of tert-butyl (2R,5R)-2-
benzyl-5-hydroxypiperidine-1-carboxylate[23] (80 mg, 0.27 mmol) in
dry DMF was added NaH (33 mg, 0.82 mmol) and stirred for 30 mi-
nutes. Subsequently, 3-azidopropanol tosylate (280 mg, 1.1 mmol)
was added dropwise and the solution stirred at rt for 18 h. The
mixture was diluted with Et2O, poured into water and extracted
with Et2O (3x). The combined organic layers were washed with H2O
(2x), brine, dried (Na2SO4), filtered and concentrated. Purification of
the residue by silica gel column chromatography (7.5% Et2O in PE)
yielded a colorless oil (66 mg, 0.18 mmol or 65%). TLC: Rf =0.44
1
(1:9 Et2O:pentane). [a]D =À3.5. IR: 2096 (-N3), 1689 (Boc). H NMR
(400 MHz, CDCl3): d=7.37–7.07 (m, 5H), 4.57–4.05 (m, 2H), 3.62
(m, 2H), 3.41 (m, 2H), 3.26 (m, 1H), 3.04–2.81 (m, 1H), 2.69 (m,
2H), 1.94 (m, 1H), 1.85 (m, 2H), 1.60 (m, 3H), 1.36 ppm (m, 9H).
13C NMR (100 MHz, CDCl3): d=129.29, 128.55, 126.37, 79.72, 74.87,
65.35, 52.26, 48.56, 42.51, 36.03, 29.63, 28.31, 26.18 ppm. LC-MS m/
z: 274.7 (M-Boc), 374.5 [M+H]+.
((2R,5R)-2-Benzyl-5-(3-(4-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-
4l4,5l4-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-
1,2,3-triazol-1-yl)propoxy)piperidin-1-yl)(4-((3-((2,4-dinitrophenyl)a-
mino)propoxy)bis(4-fluorophenyl)methyl)-1H-1,2,3-triazol-1-yl)me-
thanone (1). This compound was obtained by the general proce-
dure for the CuAAC reaction on a 38 mmol scale. Purification of the
residue by silica gel column chromatography (50>60% EtOAc in
PE). TLC: Rf =0.25 (3:2 EtOAc:pentane). N1 isomer, 1, HRMS m/z cal-
culated for C59H63BF4N12O7 [M+H]+: 1139.5045, found: 1139.5076.
((2R,5R)-2-Benzyl-5-(3-(4-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-
4l4,5l4-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinin-10-yl)butyl)-1H-
1,2,3-triazol-1-yl)propoxy)piperidin-1-yl)(4-((3-((2,4-dinitrophenyl)a-
mino)propoxy)bis(4-fluorophenyl)methyl)-2H-1,2,3-triazol-2-yl)me-
thanone (26). This compound was obtained by the general proce-
dure for the CuAAC reaction on a 35 mmol scale. Purification of the
residue by silica gel column chromatography (50>60% EtOAc in
PE). TLC: Rf =0.25 (3:2 EtOAc:pentane). N2 isomer, 26, HRMS m/z
calculated for C59H63BF4N12O7 [M+H]+: 1139.5045, found:
1139.5067.
((2R,5R)-5-(3-Azidopropoxy)-2-benzylpiperidin-1-yl)(4-(bis(4-fluoro-
phenyl)(hydroxy)methyl)-1H-1,2,3-triazol-1-yl)methanone (3) and
((2R,5R)-5-(3-azidopropoxy)-2-benzylpiperidin-1-yl)(4-(bis(4-fluoro-
phenyl)(hydroxy)methyl)-2H-1,2,3-triazol-2-yl)methanone (24).
7
(4-Ethynylphenyl)methanol (14). To a solution of 4-ethynylbenzal-
dehyde (13, 390 mg, 3 mmol) in EtOH (6 mL) was added NaBH4
(390 mg, 10.3 mmol). The reaction mixture was stirred for 10 mi-
nutes, quenched with water and extracted with DCM. The organic
layer was washed with brine, dried (MgSO4), filtered, and concen-
trated to yield 14 (389 mg, 3 mmol, 99%) as a yellow oil. TLC: Rf =
(46 mg, 0.12 mmol) was dissolved in 40% TFA in DCM and stirred
for 15 minutes before the volatiles were removed under reduced
pressure and coevaporated with toluene. The thus obtained TFA
salt was dissolved in THF, treated with DIPEA (0.1 mL, 0.6 mmol)
and cooled to 08C before triphosgene (18 mg, 0.06 mmol) was
added. The resulting solution was stirred for 30 minutes at 08C.
The reaction mixture was quenched with ice cold H2O and extract-
ed with EtOAc (3x). The combined organic layers were washed
with H2O, brine, dried (Na2SO4), filtered and concentrated. The
crude carbamoyl chloride was dissolved in THF, treated with DIPEA
1
0.61 (6:4 pentane:EtOAc). H NMR (400 MHz, CDCl3): d=7.48 (d, J=
8.2 Hz, 2H), 7.31 (d, J=8.1 Hz, 2H), 4.68 (s, 2H), 3.08 (s, 1H),
1.90 ppm (s, 1H). 13C NMR (100 MHz, CDCl3): d=141.67, 132.42,
126.85, 121.38, 83.59, 77.36, 64.94 ppm.
4-Ethynylbenzyl methanesulfonate (15). To a cooled (08C) solu-
tion of 14 (373 mg, 2.82 mmol) in dry DCM (15 mL) were added
Et3N (0.59 mL, 4.23 mmol) and MsCl (262 mL, 3.38 mmol). The reac-
tion mixture was stirred for 45 minutes, washed with water, ex-
tracted with DCM, dried (MgSO4), filtered and concentrated to
yield 15 (558 mg, 2.7 mmol, 94%). TLC: Rf =0.66 (6:4 pentane:E-
tOAc). 1H NMR (400 MHz, CDCl3): d=7.53 (d, J=8.3 Hz, 2H), 7.38
(d, J=8.3 Hz, 2H), 5.23 (s, 2H), 4.57 (s, 1H), 3.14 (s, 1H), 2.94 ppm
(s, 3H). 13C NMR (100 MHz, CDCl3): d=134.07, 132.70, 128.74,
123.37, 82.93, 78.57, 70.80, 38.47 ppm. LC-MS m/z: 211.0 [M+H]+.
(0.1 mL, 0.6 mmol), DMAP (16 mg, 0.12 mmol),
6
(35 mg,
0.12 mmol) and stirred at 608C for 2 h. The reaction mixture was
quenched with sat. aq. NH4Cl and extracted with EtOAc (3x). The
combined organic layers were washed with H2O (2x), brine, dried
(Na2SO4), filtered and concentrated. Purification of the residue by
silica gel column chromatography (15 >20% EtOAc in PE), to yield
two regioisomers. N1 isomer, apolar fractions, 3: TLC: Rf =0.60 (3:7
EtOAc:pentane). HRMS m/z calculated for C31H31F2N7O3 [M+Na]+:
610.2349, found: 610.2358. N2 isomer, polar fractions, 24: Rf =0.43
Chem. Asian J. 2018, 00, 0 – 0
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2-(4-Ethynylbenzyl)isoindoline-1,3-dione (16). To a cooled (08C)
solution of 15 (546 mg, 2.6 mmol) in dry DMF (10 mL) was added
phthalimide potassium salt (722 mg, 3.9 mmol). The reaction mix-
ture was stirred on ice for 2 h and at rt for 18 h. After addition of
water, the product precipitated. The suspension was filtered and
the solid was dissolved in DCM, washed with HCl (0.1m), brine,
dried (MgSO4), filtered and concentrated to yield 16 (625 mg,
2.4 mmol, 92%). 1H NMR (400 MHz, CDCl3): d=7.85 (m, 2H), 7.72
(m, 2H), 7.44 (d, J=8.4 Hz, 2H), 7.38 (d, J=8.4 Hz, 2H), 4.84 (s, 2H),
3.06 ppm (s, 1H). 13C NMR (100 MHz, CDCl3): d=137.10, 134.25,
132.58, 132.15, 128.69, 123.58, 77.63, 41.43 ppm.
combined organic layers were washed with brine, dried (MgSO4),
filtered and concentrated. The residue was dissolved in dry THF
(3 mL) and DMAP (39 mg, 0.32 mmol), DIPEA (167 mL, 0.96 mmol)
and 17 (97 mg, 0.32 mmol) were added and stirred at 608C for 3 h.
The reaction was quenched by the addition of NH4Cl (sat. aq.), ex-
tracted with EtOAc (3ꢂ15 mL), washed with water, brine, dried
(MgSO4), filtered and concentrated. Purification of the residue by
silica gel column chromatography (7:3 pentane:EtOAc) yielded 20
1
(55 mg, 86 mmol, 27%). H NMR (400 MHz, CDCl3): d=8.39 (s, 1H),
7.86 (m, 2H), 7.80 (m, 2H), 7.71 (m, 2H), 7.51 (m, 2H), 7.31–7.07 (m,
5H), 4.88 (s, 2H), 4.63 (s, 1H), 3.96 (m, 1H), 3.73 (m, 1H), 3.63–3.45
(m, 2H), 3.23–2.90 (m, 3H), 1.84–1.66 (m, 2H), 1.66–1.53 (m, 1H),
1.43 ppm (s, 9H). 13C NMR (100 MHz, CDCl3): d=156.15, 146.11,
136.85, 134.17, 132.15, 129.32, 128.99, 128.81, 126.80, 126.70,
126.51, 126.26, 123.52, 121.17, 120.95, 51.35, 49.21, 41.42, 40.38,
35.14, 33.58, 29.86, 28.51, 26.99, 24.06, 23.71 ppm. LC-MS m/z:
637.2 [M+H]+. HRMS m/z calculated for C36H40N6O5 [M+H]+:
637.3133, found: 637.3134.
2-(4-(1H-1,2,3-Triazol-4-yl)benzyl)isoindoline-1,3-dione (17). To a
degassed solution of 16 (102 mg, 0.4 mmol) and TMS-azide (79 mL,
0.6 mmol) in DMF:MeOH (4:0.8 mL) was added CuI (5 mg,
25 mmol). The reaction mixture was refluxed for 18 h, concentrated
and purification of the residue by silica gel column chromatogra-
phy (6:4 pentane:EtOAc) yielded 17 (78 mg, 0.26 mmol, 64%).
1H NMR (400 MHz, CDCl3): d=8.05–7.66 (m, 6H), 7.52 (m, 2H), 7.00
(s, 1H), 4.89 ppm (s, 2H). LC-MS m/z: 305.2 [M+H]+.
tertButyl (5-(4-(4-(aminomethyl)phenyl)-N-phenethyl-1H-1,2,3-tri-
azole-1-carboxamido)pentyl)carbamate (22). To a solution of 20
(27 mg, 0.04 mmol) in EtOH (1 mL) was added ethylene diamine
(4.3 mL, 0.06 mmol). The reaction mixture was stirred for 18 h, con-
centrated and purification of the residue by silica gel column chro-
matography (1:9 MeOH:DCM >1:9 MeOH:DCM+1% Et3N) yielded
22 (10 mg, 20 mmol, 50%). A side reaction was nucleophilic addi-
tion on the urea by the ethylenediamine, requiring the deprotec-
tion to be stopped before full conversion was reached. 1H NMR
(400 MHz, CDCl3): d=8.38 (s, 1H), 7.71 (m, 2H), 7.30 (m, 2H), 7.29–
7.12 (m, 5H), 4.60 (br s, 1H), 3.99 (s, 2H), 3.72–3.39 (m, 4H), 3.16–
3.00 (m, 4H), 1.75 (br s, 2H), 1.55–1.43 (m, 11H), 1.34–1.23 ppm (m,
2H). LC-MS m/z: 507.0 [M+H]+.
tertButyl (5-((2-nitro-N-phenethylphenyl)sulfonamido)pentyl)car-
bamate. To a solution of N-Boc-cadaverine (372 mg, 1.84 mmol) in
THF (8 mL) were added 2-nitrobenzenesulfonyl chloride (408 mg,
1.84 mmol) and Et3N (0.38 mL, 2.76 mmol). The cloudy reaction
mixture was stirred for 75 minutes, quenched with water (40 mL)
and extracted with EtOAc (3ꢂ20 mL). The combined organic layers
were washed with water (60 mL), brine (60 mL), dried (MgSO4), fil-
tered and concentrated. The residue was dissolved in CH3CN
(16 mL) and Cs2CO3 (1798 mg, 5.52 mmol) and phenethylbromide
(0.38 mL, 2.76 mmol) were added. The solution was stirred at 808C
for 6 h. Another equivalent of phenethylbromide (0.38 mL,
2.76 mmol) was added and stirred at 808C for 18 h. The mixture
was poured into water (50 mL) and extracted with EtOAc (3ꢂ
25 mL). The combined organic layers were washed with water
(50 mL), brine (50 mL), dried (MgSO4), filtered, and concentrated.
Purification of the residue by silica gel column chromatography
(2:8 >3:7 EtOAc:pentane) yielded the title compound (781 mg,
1.6 mmol, 87%) as a yellow oil. TLC: Rf =0.75 (1:1 pentane:EtOAc).
1H NMR (400 MHz, CDCl3): d=7.95 (d, J=7.6 Hz, 1H), 7.67–7.58 (m,
3H), 7.26–7.15 (m, 5H), 4.52 (br s, 1H), 3.50 (t, J=8.0 Hz, 2H), 3.33
(t, J=7.6 Hz, 2H), 3.07–3.06 (m, 2H), 2.84 (t, J=8.0 Hz, 2H), 1.57
(m, 2H), 1.49–1.43 (m, 11H), 1.27 ppm (m, 2H). 13C NMR (100 MHz,
CDCl3) 156.06, 148.10, 138.11, 133.67, 133.48, 131.69, 130.73,
128.84, 128.69, 126.75, 124.24, 79.07, 48.87, 47.68, 40.35, 35.19,
29.69, 28.51, 27.82, 23.75 ppm. LC-MS m/z: 492.1 [M+H]+.
1-Amino-4-((4-(2-((4-(1-((5-((tert-butoxycarbonyl)amino)pentyl)(phe-
nethyl)carbamoyl)-1H-1,2,3-triazol-4-yl)benzyl)amino)-2-oxoethyl)-
phenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (23).
To a solution of cAB40 (8.5 mg, 0.02 mmol) and HCTU (7.7 mg,
0.02 mmol) in dry DMF (1 mL) was added DIPEA (3.6 mL,
0.02 mmol). The reaction mixture was stirred for 15 minutes before
addition of 22 (10 mg in 1 mL DMF, 0.02 mmol). The reaction mix-
ture was stirred for 3 h, concentrated and purification of the resi-
due by silica gel column chromatography (5:5:1 DCM:pentane:-
MeOH+1% AcOH) yielded 23 (10 mg, 11 mmol, 53%) as a blue
solid. 1H NMR (500 MHz, CDCl3/MeOD): d=8.31 (t, J=9.0 Hz, 2H)
8.27 (s, 1H), 7.84–7.74 (m, 4H), 7.35–7.13 (m, 12H), 4.46 (br s, 2H),
3.67–3.57 (m, 4H), 3.37 (s, 2H), 3.16–2.99 (m, 6H), 1.77 (br s, 2H),
1.43–1.33 (m, 11H), 1.31–1.26 ppm (m, 2H). 13C NMR (125 MHz,
CDCl3/MeOD) 183.97, 172.03, 140.92, 138.67, 134.53, 134.07, 133.09,
132.80, 131.04, 130.46, 128.90, 128.76, 128.13, 126.44, 126.31,
126.15, 124.29, 123.43, 54.73, 51.37, 46.77, 43.18, 42.86, 42.64,
29.58, 28.34 ppm. LC-MS m/z: 940.93 [M+H]+.
tertButyl (5-(phenethylamino)pentyl)carbamate (19). To a solu-
tion of tert-butyl (5-((2-nitro-N-phenethylphenyl)sulfonamido)pen-
tyl)carbamate (781 mg, 1.59 mmol) in CH3CN (15 mL) were added
Cs2CO3 (1.57 g, 4.77 mmol) and PhSH (244 mL, 2.38 mmol). The re-
action mixture was stirred for 18 h, poured into water (100 mL)
and extracted with DCM (3ꢂ50 mL). The combined organic layers
were dried (MgSO4), filtered and concentrated. Purification of the
residue by silica gel column chromatography (1:9 MeOH:DCM
>1:9 MeOH:DCM + 1% Et3N) yielded 19 (400 mg, 1.3 mmol, 82%)
1-Amino-4-((4-(2-((4-(1-((5-(6-(3,3-dimethyl-2-((1E,3E)-5-((Z)-1,3,3-tri-
methylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium-1-yl)-
hexanamido)pentyl)(phenethyl)carbamoyl)-1H-1,2,3-triazol-4-yl)ben-
zyl)amino)-2-oxoethyl)phenyl)amino)-9,10-dioxo-9,10-dihydroan-
thracene-2-sulfonate (2). A solution of 23 (9 mg, 9.6 mmol) in DCM
(9 mL) and TFA (1 mL) was stirred for 45 minutes. The solvent was
evaporated and co-evaporated with toluene. The crude was dis-
solved in dry DMF (5 mL), Cy5-OSu ester (5.5 mg, 9.6 mmol) and
DIPEA (5.2 mL, 0.03 mmol) were added and the reaction mixture
was stirred for 4 h, concentrated and purified by semi-preparative
HPLC to yield 2 (1.3 mg, 1.0 mmol, 10%) as a blue solid. HRMS m/z
calculated for C77H80N10O8S [M+H]+: 1305.5954, found: 1305.5941.
1
as a clear oil. H NMR (400 MHz, CDCl3): d=7.33–7.27 (m, 2H), 7.21
(dt, J=5.9, 1.4 Hz, 3H), 4.66 (br s, 1H), 3.74 (br s, 1H), 3.09 (m, 2H),
3.00–2.84 (m, 4H), 2.68 (m, 2H), 1.56 (m, 2H), 1.44 (s, 11H), 1.40–
1.23 ppm (m, 2H). LC-MS m/z: 307.2 [M+H]+.
tertButyl
(5-(4-(4-((1,3-dioxoisoindolin-2-yl)methyl)phenyl)-N-phe-
nethyl-1H-1,2,3-triazole-1-carboxamido)pentyl)carbamate (20). To a
cooled (08C) solution of 19 (98 mg, 0.32 mmol) in dry THF (3 mL)
were added DIPEA (167 mL, 0.96 mmol) and triphosgene (47 mg,
0.16 mmol). The reaction mixture was stirred on ice for 1 h,
quenched with water and extracted with EtOAc (3ꢂ15 mL). The
1-(6-((5-(4-(4-((1,3-Dioxoisoindolin-2-yl)methyl)phenyl)-N-phenethyl-
1H-1,2,3-triazole-1-carboxamido)pentyl)amino)-6-oxohexyl)-3,3-di-
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methyl-2-((1E,3E)-5-((Z)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-
dien-1-yl)-3H-indol-1-ium (21). solution of 20 (21 mg,
nol butyrate was determined from the absorbance at 420 nm after
30 min incubation at rt. All measurements were performed with
A
0.035 mmol) in DCM (0.9 mL) and TFA (0.1 mL) and stirred for 1 h.
The solvent was evaporated and co-evaporated with toluene. The
crude was dissolved in dry DMF (11 mL), Cy5-OSu ester (19 mg,
0.035 mmol) and DIPEA (11.9 mL, 0.09 mmol) were added and the
reaction mixture was stirred for 18 h, poured into H2O and extract-
ed with EtOAc (3x). The combined organic layers were washed
with water, brine, dried (Na2SO4), filtered and concentrated. Purifi-
cation of the residue by silica gel column chromatography (EtOAc
>1:19 MeOH:DCM) yielded the title compound (15 mg, 15 mmol,
44%) as a blue solid. 1H NMR (500 MHz, CDCl3): d=8.40 (s, 1H),
7.98–7.65 (m, 7H), 7.50 (m, 2H), 7.45–6.93 (m, 15H), 6.66 (s, 1H),
6.28 (s, 1H), 4.88 (s, 2H), 4.10 (s, 2H), 3.94 (s, 1H), 3.86–3.62 (m,
2H), 3.56 (s, 4H), 3.40–3.12 (m, 2H), 3.12–2.91 (m, 2H), 2.49 (s, 1H),
2.00–1.50 (m, 22H), 1.50–1.20 ppm (m, 7H).13C NMR (125 MHz,
CDCl3): d=173.51, 172.29, 168.17, 142.96, 142.00, 141.27, 140.69,
134.21, 132.20, 129.31, 129.15, 128.79, 128.76, 126.28, 125.60,
124.89, 123.56, 122.18, 111.20, 110.19, 52.23, 51.33, 49.49, 49.33,
48.95, 44.87, 41.46, 29.83, 28.18, 27.04, 26.50, 25.43, 25.22 ppm.
5% DMSO present and a final protein concentration of
0.2 mgmLÀ1. Negative controls consisted of mock transfected
membranes or 10 mm Orlistat inhibited hDAGLa membranes. All
measurements were performed in duplo (N=2, n=2).
Cell culture. Cells were cultured at 378C under 7% CO2 in DMEM
containing phenol red, GlutaMax, 10% (v/v) New Born Calf Serum
(Thermo Fisher), penicillin and streptomycin (200 mgmLÀ1 each;
Duchefa). For selection and maintenance of stable expression cell
lines, complete DMEM was supplemented with G418 (0.4 mgmLÀ1).
Medium was refreshed every 2–3 days and cells were passaged
twice a week at 80–90% confluence by resuspension in fresh
medium (Neuro2A, HEK293T) or by trypsinization (U2OS).
Plasmids. The hDAGLa plasmid was constructed as described
before.[38] Briefly, full length human cDNA of hDAGL-a was pur-
chased from Biosource and cloned into mammalian expression
vector pcDNA3.1, containing genes for ampicillin and neomycin re-
sistance. A FLAG-linker was made from primers and cloned into
the vector at the C-terminus of hDAGL-a. The plasmid was grown
in XL-10 Z-competent cells and prepped (Maxi Prep, Qiagen). The
sequences were confirmed by sequence analysis at the Leiden
Genome Technology Centre.
HRMS m/z calculated for C63H69N8O4 [M]+: 1001.5436, found:
+
1001.5449.
Transfection. HEK293T cells were grown to ꢀ70% confluency in
15 cm dishes. Prior to transfection, culture medium was refreshed
(15 mL). A 3:1 (m:m) mixture of polyethyleneimine (PEI, 60 mg/well)
and plasmid DNA (20 mg/well) was prepared in serum free culture
medium and incubated for 10 min at rt. Transfection was per-
formed by dropwise addition of the PEI/DNA mixture (2 mL/well)
to the cells. 24 h post-transfection, the medium was refreshed and
after 48 h cells were harvested.
U2OS_ABHD6-GFP stable expression. Full-length human cDNA of
ABHD6 (Source Bioscience) was cloned into mammalian expression
vector pcDNA3.1, containing genes for ampicillin and neomycin re-
sistance. The inserts were cloned in frame with a C-terminal FLAG-
and GFP-tag. Plasmids were isolated from transformed XL-10 Z-
competent cells (Maxi Prep kit: QiaGen) and sequenced at the
Leiden Genome Technology Center. Sequences were analyzed and
verified (CLC Main Workbench). One day prior to transfection U2OS
cells were seeded to a 6 wells plates (ꢀ0.5 million cells/well). Prior
to transfection, culture medium was aspirated and a minimal
amount of medium was added. A 3:1 (mmÀ1) mixture of polyethy-
leneimine (PEI) (3.75 mg/well) and plasmid DNA (11.25 mg/well) was
prepared in serum free culture medium and incubated (15 min,
RT). Transfection was performed by dropwise addition of the PEI/
DNA mixture to the cells. After 24 h, transfection efficiency was de-
termined by fluorescence microscopy and transfection medium
was exchanged for selection medium containing 800 mgmLÀ1
G418. 48 h Post-transfection single cells were seeded to 96 wells
plates in 100 mL selection medium. After 14 days, plates were in-
spected for cell growth, clones were checked for ABHD6-GFP ex-
pression by fluorescence microscopy (GFP channel). From here on,
cells were grown in maintenance medium containing 400 mgmLÀ1
G418, and expanded in 12- and 6-wells plates and 10 cm dishes.
Spectroscopic characterization
UV-VIS spectra (400–700 nm) were recorded on a Shimadzu Phar-
maSpec UV-1700 UV/Visible spectrophotometer in a cuvette with
1 cm light path. Fluorescence spectra were recorded on a Shimad-
zu RF-5301PC spectrofluorophotometer in a quartz cuvette with
four polished windows with a light path of 1 cm. The spectrofluor-
ophotometer was set at high sensitivity and to record spectra with
a 1 nm sampling interval. For BODIPY-FL and probe 1, the wave-
length of excitation was set to 497 nm and emission spectra were
recorded from 500–700 nm with a slit width of 1.5 nm. For Cy5
and probe 12, the wavelength of excitation was set to 646 nm and
emission spectra were recorded from 650–800 nm with a slit width
of 3 nm. Absorbance and fluorescence spectra were recorded at
five concentrations in 2 mL of 96% EtOH. The slopes of the absorb-
ance versus fluorescence plot were used to determine the relative
fluorescence quantum yields (gradient probe divided by gradient
parent fluorophore).
Biological assays
Surrogate substrate assay. The surrogate substrate assay was per-
formed as published previously,[38 ]with the following adaptions:
100 mL per well as final volume and an endpoint measurement of
the absorbance. Briefly, the assay was performed in a transparent
flat bottomed 96 wells plate. The membranes used in these experi-
ments were derived from HEK293T cells overexpressing human
DAGLa. The assay was performed in 50 mm HEPES buffered to
pH 7.0. Inhibitors were incubated for 20 min at rt, followed by ad-
dition of p-nitrophenol butyrate. The final p-nitrophenol butyrate
concentration was 300 mm. The amount of hydrolysis of p-nitrophe-
Inhibitor treatment. The medium was aspirated and 0.5 mL
serum-free medium containing the inhibitor was added. After incu-
bation for 1 h at 378C, medium was removed and PBS added, re-
moved, trypsin buffer was added, quenched with 1 mL medium
and the cells were harvested by pipetting. After centrifugation
(5 min, 1000 g), the medium was removed, the cells were resus-
pended in PBS, centrifuged again (5 min, 1000 g) and the pellets
flash frozen with N2 (l) and stored at À808C.
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Whole cell lysate preparation. Cell pellets were thawed on ice, re-
suspended in cold lysis buffer (20 mm HEPES pH 7.2, 2 mm DTT,
250 mm sucrose, 1 mm MgCl2, 2.5 UmLÀ1 benzonase) and incubat-
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trations for the inhibitors are indicated in the main text and Figure
legends. Reactions were quenched with 4x Laemmli buffer (5 mL,
240 mm Tris (pH 6.8), 8% (w/v) SDS, 40% (v/v) glycerol, 5% (v/v) b-
mercaptoethanol, 0.04% (v/v) bromophenol blue). 10 or 20 mg per
reaction was resolved on a 10% acrylamide SDS-PAGE gel (180 V,
75 min). Gels were scanned using Cy2, Cy3 and Cy5 multichannel
settings on a ChemiDoc MP (Bio-Rad) and stained with Coomassie
after scanning. Fluorescence was normalized to Coomassie staining
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Conflict of interest
[28] H. Li, J. Kang, L. Ding, F. Lꢅ, Y. Fang, J. Photochem. Photobiol. A 2008,
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The authors declare no conflict of interest.
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Keywords: activity-based
endocannabinoid · enzymes · fluorescent probes · quenched
activity-based probes
protein
profiling
·
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Version of record online: && &&, 0000
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FULL PAPER
Quenched Activity-based Probes
E. J. van Rooden, M. Kohsiek, R. Kreekel,
A. C. M. van Esbroeck,
A. M. C. H. van den Nieuwendijk,
A. P. A. Janssen, R. J. B. H. N. van den Berg,
H. S. Overkleeft, M. van der Stelt*
Pairing up: The synthesis and character-
ization of two new quenched activity-
based probes for diacylglycerol lipase
and a,b-hydrolase domain containing
protein 6 with BODIPY-FL and 2,4-dini-
troaniline or Cy5-fluorophore and a
cAB40-quencher as a fluorophore–
quencher pair is reported. The probes
showed target inhibition as character-
ized in activity-based protein profiling
assays using human cell and mouse
brain lysates, but not in living cells.
&& – &&
Design and Synthesis of Quenched
Activity-based Probes for
Diacylglycerol Lipase and a,b-
Hydrolase Domain Containing Protein
6
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ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ