Mixed alkyl aryl phosphonate esters as quenched fluorescent activity-based probes for serine proteases

Article abstract of DOI:10.1039/c4ob02444c

Activity-based probes (ABPs) are powerful tools for the analysis of active enzyme species in whole proteomes, cells or animals. Quenched fluorescent ABPs (qABPs) can be applied for real time imaging, allowing the visualization of dynamic enzyme activation

Full text of DOI:10.1039/c4ob02444c

Organic &
Biomolecular Chemistry
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PAPER
Mixed alkyl aryl phosphonate esters as quenched
fluorescent activity-based probes for serine
proteases†
Cite this: Org. Biomol. Chem., 2015,
3, 2293
1
a
a
a,b
Sevnur Serim, Philipp Baer and Steven H. L. Verhelst*
Activity-based probes (ABPs) are powerful tools for the analysis of active enzyme species in whole pro-
teomes, cells or animals. Quenched fluorescent ABPs (qABPs) can be applied for real time imaging, allow-
ing the visualization of dynamic enzyme activation by fluorescent microscopy. Unfortunately, qABPs are
only available for a few enzymes. We here describe the design and synthesis of qABPs for serine proteases
based on a phosphonate ester scaffold.
Received 20th November 2014,
Accepted 17th December 2014
DOI: 10.1039/c4ob02444c
www.rsc.org/obc
Introduction
During the last decade, chemical proteomics has increasingly
been used for the functional characterization of proteins
within the context of a whole proteome. In contrast to classical
proteomics approaches, the technique of activity-based protein
profiling (ABPP) can distinguish between active and inactive
enzyme species by using small molecule activity-based probes
1
–3
(ABPs).
ABPs rely on a reactive group or ‘warhead’ that co-
valently binds to the active site of a specific subset of enzymes,
often in a mechanism-based reaction with an active site
nucleophile. A reporter tag on the ABP, such as a biotin or a
fluorophore, enables subsequent detection of the covalent
4
complex (Fig. 1A). ABPs have been applied for various pur-
poses including the profiling of enzyme activities in disease
models and the screening of enzyme inhibitors in complex
5
–7
proteomes.
ABPs have proven especially useful for the study of pro-
teases, since these enzymes are generally synthesized as in-
active zymogens and are tightly regulated by post-translational
Fig. 1 Activity-based probes and their quenched fluorescent versions.
A) The general approach of activity-based protein profiling. The detec-
tion tag, here depicted as a light bulb, can range from a fluorophore,
8
(
processes. Fluorophore-labeled ABPs do not only allow detec- biotin or radiotracer to a bioorthogonal detection handle such as an
azide or alkyne. (B) Fluorescently quenched activity-based probes rely
on reactive warhead with a leaving group that contains a fluorescence
quencher. (C) A diphenyl phosphonate (R and Aryl = Ph) or alkyl, aryl
phosphonate (R = alkyl) and the covalent attachment to the active site
tion of active proteases after gel electrophoresis, but also
enable the direct visualization in cells, tissue sections or live
animals by microscopy techniques. However, the intrinsic fluo-
rescence of these ABPs requires extensive washing of cells in
culture or long clearance times in vivo in order to remove
unreacted probe and obtain sufficient contrast, preventing
real-time imaging applications. Quenched fluorescent ABPs
of a serine protease.
(
qABPs) have been developed to overcome this problem. They
rely on a reactive warhead that carries a leaving group to which
a fluorescence quencher is attached. This results in an overall
dark probe, which only lights up after reaction with the target
enzyme (Fig. 1B). The synthesis of qABPs is challenging and
thus far, qABPs have only been reported for the clan CA and
a
Lehrstuhl für Chemie der Biopolymere, Technische Universität München,
Weihenstephaner Berg 3, 85354 Freising, Germany. E-mail: steven.verhelst@isas.de
b
Leibniz Institute for Analytical Sciences, ISAS, e.V., Otto-Hahn-Str. 6b,
4
†
4227 Dortmund, Germany
Electronic supplementary information (ESI) available: Supporting figures, and
9
–13
copies of chromatograms and spectra. See DOI: 10.1039/c4ob02444c
clan CD cysteine proteases and for selected phosphatases.
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In our continuous efforts to develop novel probes for serine S1 pocket of the target serine protease. The aldehyde was
proteases, we here describe the design and synthesis of the reacted with diethyl phosphite and benzyl carbamate in a
22
first qABPs for serine proteases based on mixed alkyl, aryl Mannich-type condensation,
forming the phosphonate
phosphonates.
scaffold (Scheme 1). In order to target elastase-like proteases,
isobutyraldehyde was used to form the valine-like phospho-
nate 1. For trypsin-like proteases, p-nitrobenzaldehyde was
chosen as a starting material, giving phosphonate 2 with a
p-nitrophenyl group. At a later stage, this group can be trans-
Results and discussion
In the past, ABPs for serine protease have utilized several types formed into a p-guanidino-phenyl substituent, a mimic of the
2
3
of electrophilic moieties as warheads, including fluoro- arginine side chain. The Cbz protecting group of 2 was
1
4,15
16
17–19
phosphonates,
sulfonyl fluorides and isocoumarins.
removed with HBr–AcOH and the free amine was coupled to
However, these do not contain leaving groups that can be used hexynoic acid. The terminal alkyne handle will allow the intro-
for the incorporation of a fluorescence quencher moiety. duction of a fluorophore by mild click chemistry conditions,
α-Amino diphenyl phosphonates (DPPs), however, contain a which are compatible with the base-labile aryl phosphonate
phenoxy leaving group, which is expulsed upon reaction with functional group. For the selective hydrolysis of one ethyl
the active site serine of a serine protease (Fig. 1C). Further- group, we initially used saponification with NaOH. However,
more, the selectivity of DPPs can be adjusted by varying the P1 this reaction resulted in a mixture of products. Gratifyingly, a
element as well as by elongation with a longer peptide. There- non-hydrolytic mono-dealkylation by S
N
2 substitution using
2
4
fore, DPPs have been used as potent and selective serine pro- LiBr in refluxing 2-butanone yielded the desired product. A
2
0,21
tease inhibitors and ABPs.
In the design for qABPs, it is of Boc-protected tyramine (7) was introduced by carbodiimide-
crucial importance that the reactive group contains one single mediated esterification. For the valine-like molecule, the trans-
leaving group, making diaryl phosphonates unsuitable. To formation to a mixed alkyl aryl phosphonate ester was more
overcome this problem, we synthesized qABPs as mixed alkyl efficient before introduction of the hexynoic acid group
aryl phosphonates, which we made from dialkyl phosphonates (Scheme 1). We reduced the nitro-group of compound 11 with
2
by selective removal of one alkyl group, subsequent introduc- SnCl and reacted the free amine with di-Boc-2-(trifluoro-
tion of the aryl leaving group, and finally introduction of the methylsulfonyl)guanidine, followed by Boc-deprotection yield-
fluorophore and the quencher.
ing building block 13.
The starting point of the synthesis consisted of an alde-
With the two alkyl aryl phosphonates 10 and 13 in hand,
hyde, which will form the P1 element that is recognized by the the stage was set to introduce the fluorophore and quencher
Scheme 1 Synthesis of the mixed ethyl aryl phosphonate building blocks 10 and 13. (a) Cbz-NH
3
2
2
, diethyl phosphite, AcCl, 0 °C, 1 h, 88–95%. (b)
3% HBr–AcOH, 1 h. (c) hexynoic acid, HATU, DIEA, DMF, overnight, 74% over 2 steps for compound 4, 26% over 2 steps for compound 10. (d) LiBr,
-butanone, 70 °C, overnight, 65–83%. (e) 7, DIC, DMAP, toluene, 70 °C, overnight, 96% (for compound 8). (f) H , Pd-C, overnight. (g) SnCl , EtOH,
0 °C, 1 h, then 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, TEA, CH Cl , overnight, 17% (for compound 12, 3 steps from 6). (h) TFA–CH Cl
/1, 1 h, 37%. (i) Boc O, Na CO , dioxane–H O, 1 h, 90%.
2
2
7
2
2
2
2
1
2
2
3
2
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Scheme 2 Synthesis of the qABPs 14 and 15. (a) TAMRA-NH-(CH
QSy7-OSu, DIEA, dmso, 3 h, 3–16% over 3 steps.
2
)
3
-N
3
, Cu(I), THF, overnight. (b) TFA–CH
2
Cl
2
1/1, 1 h (for compound 10 only). (c)
groups. First, a tetramethylrhodamine (TMR)-functionalized creatic elastase and neutrophil elastase which show selectivity
azido-propylamine was coupled by Cu(I)-catalyzed cyclo- for small hydrophobic P1 side chains. As expected, the reactiv-
addition. Subsequently, a succinimide derivative of QSy7, a ity matched the natural substrate specificity of the target pro-
powerful fluorescent quencher, was coupled to the amino- teases (Fig. 2A): probe 15 containing the P1 arginine mimic
function of the tyramine leaving group, yielding qABPs 14 and strongly labeled the trypsin-like proteases, whereas the P1
1
5 (Scheme 2).
valine probe 14 showed intense labeling of neutrophil elastase,
After the successful synthesis of the two serine protease which prefers a P1 valine residue. Probe 14 displayed weaker
qABPs, we aimed for a basic evaluation of their labeling and labeling of chymotrypsin and pancreatic elastase, which have
quenching properties. Activity-based labeling by qABPs 14 and preference for large hydrophobic and alanine residues,
1
5 was tested on purified serine proteases of different specifici- respectively. To show sensitivity of the alkyl, aryl phosphonate
ties: trypsin and urokinase plasminogen activator, which have qABPs, we labeled a decreasing amount of trypsin with probe
preference for basic P1 side chains, chymotrypsin and cathep- 15 and found that as little as 2 ng of this protease target could
sin G, which prefer large hydrophobic P1 side chains, and pan- be detected (Fig. S1†). Additionally, we performed labeling of
trypsin in the presence of a crude lysate of the human cancer
cell line HT-29. The detectable amount of trypsin was 0.025%
1
9
of total protein, comparable to other serine protease probes
Fig. S2†).
For imaging applications, it is of fundamental impor-
(
tance that there is a clear difference between the probe’s
fluorescence in the unbound/quenched state and the bound/
unquenched state. We tested this by incubation of probe 15 in
PBS with or without bovine trypsin as a target protease. Satisfy-
ingly, the signal of the probe was similar to the used buffer,
which indicates both the strongly quenched TAMRA fluo-
rescence and the probe stability at physiological pH. As
expected, a rapid increase in fluorescence occurred upon reac-
tion with the protease (Fig. 2B).
Conclusions
Fig. 2 Evaluation of serine protease qABPs. (A) Labeling of serine pro-
teases with qABPs 14 and 15 (Try = bovine trypsin, uPA = human uro-
kinase plasminogen activator, CatG = human cathepsin G, Ctry = bovine
chymotrypsin, PPE = porcine pancreatic elastase, NE = human neutro- proteases
phil elastase). Each sample was treated with dmso (−) or an inhibitor (+)
before incubation with the qABP. The used inhibitors included DCI for
trypsin and elastase, DFP for uPA, PMSF for cathepsin G and DAP22c for
chymotrypsin. (B) qABP 15 (2 μM) was incubated in PBS with or without
bovine trypsin (0.05 mg mL−1) and the fluorescence (ex: 544 nm, em:
Up to date, qABPs have only been developed for cysteine
9
–13
13
and for tyrosine phosphatases. In this paper,
we have described the first synthesis of qABPs for serine pro-
teases. They are based on a phosphonate reactive electrophile
that, in contrast to previously reported serine protease phos-
phonate ABPs, are mixed alkyl aryl phosphonates. The
biochemical evaluation shows that the phosphonate qABPs
590 nm) was followed in a 96-well plate reader for 1 h.
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covalently label their serine protease targets with expulsion of ether and redissolved in 1 M HCl. The water phase was
the fluorescent quencher. The selectivity is steered by the P1 extracted four times with EtOAc and the collected organic
4
element, and may be further influenced by introduction of layers were washed with brine, dried over MgSO and concen-
2
5
extended recognition elements in the P2–P4 positions As trated under reduced pressure.
elevated serine protease activities are present in several patho- General procedure for coupling of 5-hexynoic acid. 5-Hexy-
logies such as inflammation and tumorigenesis, we expect that noic acid (2 eq.) was preactivated with HATU (1.9 eq.) and
the mixed alkyl aryl phosphonate qABPs as synthesized here DIEA (4 eq.) in DMF (4 mL) for 20 min at rt. To this mixture,
will be useful tools for the future study of these processes. the phosphonate with a free α-amino group (1 eq.) and
Application of these probes in complex proteomes and DIEA (1.6 eq.) in DMF (3 mL) were added and stirred o/n at rt.
imaging studies by fluorescent microscopy are currently under The solvent was removed, and the oily product was redis-
investigation and will be reported in due course.
solved in EtOAc. The organic phase was washed twice with 1 M
HCl, twice with saturated NaHCO , and brine. The organic
and concentrated under reduced
3
layer was dried over MgSO
pressure.
4
Experimental
Materials and equipment
Diethyl
phosphonate (1). To a mixture of benzyl carbamate (1.51 g,
0 mmol), diethyl phosphite (1.28 mL; 10 mmol) and isobutyr-
1-(N-benzyloxycarbonyl-amino)-2-methyl-propane-
Acetic acid, acetyl chloride, human urokinase plasminogen
activator and porcine pancreatic elastase were purchased at
VWR (Ismaning, Germany), benzyl carbamate, Boc anhydride,
chymotrypsin from bovine pancreas type II, copper(I)bromide,
diethyl phosphite, diisopropylcarbodiimide (DIC), 4-(dimethyl-
amino)pyridine (DMAP), N-diisopropyl-N-ethylamine (DIEA),
HBr (33% in acetic acid), isobutyraldehyde. lithium bromide,
sodium ascorbate, trypsin from bovine pancreas type I, and
tyramine were obtained from Sigma-Aldrich (Taufkirchen,
Germany), QSy-7 succinimidyl ester was bought from Invitro-
gen/Life Technologies (Darmstadt, Germany), HATU was pur-
1
aldehyde (1.14 mL, 12.5 mmol) cooled at −5 °C was added
dropwise 15 mL acetyl chloride. The reaction was stirred for
1
h, warmed to room temperature and the mixture was concen-
trated under reduced pressure. EtOAc was added and the
organic phase was washed with water, saturated NaHCO (2×)
3
and brine, dried on MgSO4 and evaporated under reduced
pressure. Silica column chromatography (50–100% EtOAc in
petroleum ether) gave the title compound as a colorless oil
+
(
3.03 g, 88% yield). ESI-MS: m/z 344.1925, C H NO P
16 27 5
1
requires 344.1621. H NMR (400 MHz, CDCl
5H), 5.19–5.08 (m, 3H), 4.16–3.82 (m, 5H), 2.27–2.17 (m, 1H),
.34–1.22 (m, 6H), 1.03 (d, 3H, J = 6.8 Hz), 1.0 (d, 3H, J = 6.9
Hz).
Diethyl
methanephosphonate (2). To a mixture of benzyl carbamate
0.75 g, 5 mmol), diethyl phosphite (0.64 mL; 5 mmol) and
-nitrobenzaldehyde (0.75 g, 5 mmol) cooled at 0 °C was
3
) δ 7.38–7.30 (m,
chased at Creosalus (Louisville, KY, USA), MgSO was obtained
4
from Carl Roth (Karlsruhe, Germany), and sodium carbonate
decahydrate, 2-butanone and human neutrophil elastase were
received from Applichem (Darmstadt, Germany). TBTA was
1
1-(N-benzyloxycarbonyl-amino)-(4-nitro-phenyl)-
2
6
made as previously described.
Thin layer chromatography (TLC) was performed on
ALUGRAM sil G/UV254 TLC plates (Carl Roth, Germany). Sol-
vents used for reactions and purification via column chromato-
graphy were purchased from Applichem, Germany.
Compounds were separated over silica gel with a grain distri-
bution of 0.04–0.063 mm and a pore size of 60 Å (Carl Roth,
Germany).
LC-MS spectra were recorded by an Agilent 1100 Series LC
system coupled to an Agilent 6210 ESI TOF mass spectrometer,
with separation on a Zorbax SB C18 5 µm (0.5 × 150 mm)
(
4
added dropwise 7.5 mL acetyl chloride. The reaction was
stirred for 1 h, warmed to room temperature and the mixture
was concentrated under reduced pressure. EtOAc was added
and the organic phase was washed with water, saturated
NaHCO
under reduced pressure. Silica column chromatography
50–100% EtOAc in petroleum ether) gave the title compound
as a white solid (2.01 g, 95% yield). ESI-MS: m/z 423.1368
3 4
(2×) and brine, dried on MgSO and evaporated
(
+
1
column and elution by solvents A (5% MeCN–H O + 0.1%
2
13 24 2 7
(found), C H N O P requires 423.1316. H NMR (500 MHz,
2
formic acid) and B (95% MeCN–H O + 0.1% formic acid) at a
DMSO-d ): δ = 8.64 (d, 1H, J = 9.2 Hz,), 8.23 (d, 2H, J = 8.6 Hz),
6
−1
flow rate of 20 µl min . HPLC purifications were carried out
on Waters Xbridge BEH130 Prep C18 5 µm (19 × 150 mm)
7
2
.77 (2H, d, J = 7.0 Hz), 7.41–7.28 (m, 5H), 5.34 (dd, 1H, J =
3.1 Hz, J = 9.9 Hz), 5.15–5.00 (m, 2H), 4.03–3.87 (m, 4H), 1.16
1
column. H NMR spectra were measured on a Bruker 400 MHz
(
t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.0 Hz, 3H).
Diethyl 1-(N-hex-5-ynoyl-amino)-(4-nitro-phenyl)methane-
DRX or a Bruker 500 MHz Avance III. Chemical shifts (δ) are
given in parts per million (ppm) relative to tetramethylsilane
as an internal standard.
phosphonate (4). Compound 1 was treated with 33% HBr–
AcOH solution until all the starting material was consumed as
indicated by TLC. Next, the solvent was removed under reduced
pressure with coevaporation from toluene, giving the product as
Methods
General procedure for dealkylation with LiBr. The diethyl bright orange solid, which was used in the next step without
phosphonate (1 eq.) and LiBr (1.2 eq.) were stirred overnight in purification. 5-Hexynoic acid was coupled as described under the
−1
butanone (5 mL mmol ) under reflux at 70 °C. The formed general procedure. Silica column chromatography (60–80%
precipitate was collected by filtration, washed with diethyl EtOAc in petroleum ether) gave the product in 74% yield as
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yellowish
crystals.
ESI-MS:
m/z
383.1385
(found), 3H), 4.54 (bs, 1H), 4.25–4.05 (m, 3H), 3.33 (t, 2H, J = 6.6 Hz),
+
1
C H N O P requires 383.1366. H NMR (500 MHz, DMSO- 2.78–2.71 (m, 2H), 2.35–2.26 (m, 1H), 1.44 (s, 9H), 1.32–1.21
1
7
24 2 6
d
6
): δ = 9.09 (dd, 1H, J = 9.6 Hz, J = 3.0 Hz), 8.24 (d, 2H, J = 8.5 (m, 3H), 1.15–1.03 (m, 6H).
Hz), 7.74 (dd, 2H, J = 8.8 Hz, J = 1.9 Hz), 5.63 (dd, 1H. J = 22.5 4-(N-Boc-2-aminoethyl)-phenyl, ethyl 1-(N-hex-5-ynylamino)-
Hz, J = 9.6 Hz), 4.08–3.84 (m, 4H), 2.78 (t 1H, J = 2.6 Hz), 2-methyl-propane-phosphonate (10). Compound 8 (650 mg,
.43–2.28 (m, 2H), 2.19–2.13 (m, 2H), 1.71–1.64 (m, 2H), 1.20 1.22 mmol) was dissolved in EtOH (4 mL). Pd/C (65 mg) and
2
(t, 3H, J = 7.0 Hz), 1.12 (t, 3H, J = 7.0 Hz).
AcOH (143 μL; 2.5 eq.) were added and the mixture was stirred
Ethyl 1-(N-benzyloxycarbonyl-amino)-2-methyl-propanephos- under 1 atm. hydrogen pressure until all starting material had
phonate (5). Compound 4 (686 mg, 2.0 mmol) was dealkylated disappeared. The catalyst was filtered off and the solvent was
with LiBr according to the general procedure affording the title evaporated, giving compound 9, which was used directly in
+
compound as a white solid (526 mg, 83% yield). ESI-MS: m/z the next step. ESI-MS: m/z 401.2218 (found), C H N O P
3
1
9
34 2 5
+
1
16.1322 (found), C14
400 MHz, CDCl ) δ 8.72 (s, 1H), 7.36–7.29 (m, 5H), 5.23–5.12 under the general procedure. The crude product was purified
m, 3H), 4.12–3.97 (m, 3H), 2.24–2.16 (m, 1H), 1.26 (t, 3H, J = by silica column chromatography (50–100% EtOAc in pet-
.0 Hz), 1.01 (dd, 3H, J = 6.9 Hz, J = 0.9 Hz), 0.98 (d, 3H, J = 6.9 roleum ether), giving the title compound as a slightly yellowish
Hz). oil, which partly solidified upon standing (26% over 2 steps).
Ethyl
phosphonate (6). 3 (918 mg, 2.4 mmol) was dealkylated with 495.2618. H NMR (400 MHz, CDCl
LiBr according to the general procedure affording the title 7.11 (d, 2H, J = 9.3 Hz), 5.94 (dd, 1H, J = 19.0 Hz, J = 8.1 Hz),
5
H23NO P requires 316.1308. H NMR requires 401.2200. 5-Hexynoic acid was coupled as described
(
(
3
7
+
1-(N-hex-5-ynylamino)-(4-nitro-phenyl)-methane- ESI-MS: m/z 495.2809 (found), C H N O P
requires
) δ 7.16 (d, 2H, J = 9.3 Hz),
2
5
40 2 6
1
3
+
compound as a white powder in 65% yield. ESI-MS: [M + H]
4.66–4.51 (m, 2H), 4.28–4.16 (m, 2H), 3.37–3.38 (m, 2H),
2.79–2.72 (m, 2H), 2.47–2.22 (m, 5H), 2.07–1.98 (m, 1H),
+
1
m/z 355.1071 (found), C15
NMR (400 MHz, CDCl
20 2 6
H N O P requires 355.1053. H
3
) δ 8.20 (d, 2H, J = 8.6 Hz), 7.60–7.55 (m, 1.93–1.77 (m, 2H), 1.44 (s, 9H), 1.34–1.26 (m, 3H), 1.08–1.01
H), 7.19–7.14 (m, 1H), 5.59 (dd, 1H, J = 22.3 Hz, J = 8.8 Hz), (m, 6H).
.04–3.96 (m, 2H), 2.46 (t, 2H, J = 7.2 Hz), 2.28–2.18 (m, 2H), 4-(N-Boc-2-aminoethyl)-phenyl, ethyl 1-(N-hex-5-ynylamino)-
.99 (t, 1H, J = 2.6 Hz), 1.84 (t, 2H, J = 7.1 Hz), 1.24 (t, 3H, J = (4-N,N-di-Boc-guanidino-phenyl)-methanephosphonate (12).
.1 Hz). Compound 6 (1.0 eq.) and 7 (1.1 eq.) were dissolved in DMF,
N-tert-Butyloxycarbonyl-tyramine (7). To tyramine (274 mg; and DIC (8 eq.) and DMAP (0.1 eq.) were added. The reaction
.0 mmol), dissolved in 10 mL dioxane–water (1/1; v/v) was was set under N and heated overnight at 70 °C. The solvent
2
4
1
7
2
2
added sodium carbonate decahydrate (572 mg; 2 mmol). The was removed and the oily product was redissolved in EtOAc.
mixture was cooled in an icebath and Boc anhydride (480 mg; The organic layer was then washed 1× with saturated NaHCO
.1 eq.) was added. After 1 h, the reaction was allowed to warm 1× with 1 M HCl and brine, subsequently dried over MgSO
3
,
.
1
4
up to room temperature, after which EtOAc was added. The The solvent was removed under reduced pressure, giving com-
mixture was subsequently washed with 1 M KHSO (2×), water, pound 11 as a white powder, which was used without further
saturated NaHCO and brine. The organic phase was dried on purification. ESI-MS: m/z 574.2351 (found), C28
MgSO , filtered and concentrated under reduced pressure. requires 574.2313. Compound 11 (302 mg, 0.57 mmol) and
4
+
3
37 3 8
H N O P
4
2
Silica column chromatography (20–50% EtOAc in petroleum SnCl (536 mg, 5 eq.) were mixed in EtOH (2 mL) and refluxed
ether) yielded the title compound (425 mg; 1.80 mmol; 90%) at 70 °C for 1 h. The solvent was removed and the oily product
as a slightly yellowish oil which solidified upon standing. was redissolved in saturated NaHCO . The water phase was
3
+
+
ESI-MS: m/z 238.1447 [M + H] , 475.2838 [2M + H] (found), then extracted 3× with EtOAc. The combined organic phases
H N O requires 475.2803. were washed with brine, dried over MgSO and concentrated
39 2 6 4
H NMR (400 MHz, CDCl ) δ 7.01 (d, 2H, J = 8.3 Hz), 6.79 (d, under reduced pressure. The product was directly used for the
+
+
C
13
H
20NO
3
requires 238.1438; C26
1
3
2
H, J = 8.5 Hz), 6.26 (bs, 1H), 4.62 (bs, 1H), 3.37–3.28 (m, 2H), next reaction without further purification. ESI-MS: m/z
+
2
.70 (t, 2H, J = 7.0 Hz), 1.44 (s, 9H).
-(N-Boc-2-aminoethyl)-phenyl, ethyl 1-(N-benzyloxycarbo- product from the previous reaction (255 mg, 0.47 mmol) was
nyl-amino)-2-methyl-propane-phosphonate (8). Compound 5 dissolved in DCM and 1,3-di-Boc-2-(trifluoromethylsulfonyl)-
200 mg, 0.63 mmol) and 7 (165 mg; 0.70 mmol), coevaporated guanidine (1.6 eq.) and TEA (2 eq.) were added. The mixture
with toluene, were dissolved in toluene (3.5 mL) and stirred at was refluxed overnight. The reaction was diluted with DCM
0 °C. Subsequently, DIC (450 μL) and DMAP (8 mg) were and the organic phase was washed with twice with 1 M KHSO
added, and the solution was stirred overnight. The solvent was once with saturated NaHCO and brine and subsequently
evaporated under reduced pressure, the residue dissolved in dried over MgSO . The solvent was removed under reduced
39 3 6
544.2681 (found), C28H N O P requires 544.2571. The crude
4
(
7
4
,
3
4
EtOAc and subsequently washed with 1 M KHSO
rated NaHCO and brine. Silica column chromatography chromatography (isocratic elution with EtOAc–petroleum ether
20–70% EtOAc in petroleum ether) gave the title compound as 1/1). The target compound was isolated as white crystals in
a white solid (323 mg; 0.60 mmol, 96%). ESI-MS: m/z 535.2596 17% yield over 3 steps. ESI-MS: m/z 786.4120 (found),
4
, water, satu- pressure and the crude product was purified by silica column
3
(
+
1
+
1
(
found), C27
H
40
N
2
O
7
P requires 535.2568. H NMR (400 MHz,
39 57 5
C H N O10P requires 786.3838. H NMR (500 MHz, DMSO-
CDCl ) δ 7.40–7.30 (m, 5H), 7.14–7.05 (m, 4H), 5.17–5.06 (m, d6): δ 11.4 (s, 1H), 10.02 (s, 1H), 9.03 (dd, 1H J = 9.8 Hz, J =
3
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1
2
5
3
7
2
.7 Hz,), 7.58–7.52 (m, 2H), 7.48 (d, 2H, J = 7.1 Hz), 7.17 (d, 5 μL bovine trypsin (1 mg mL− in PBS) were mixed to observe
H, J = 8.5 Hz), 7.02 (d, 2H, J = 8.2 Hz), 6.85 (t, 1H, J = 5.4 Hz), the unquenching. The reactions were followed for 60 min in a
.65 (dd, 1H, J = 21.5 Hz, J = 9.8 Hz), 4.02 (q, 2H, J = 7.2 Hz), fluorimeter (FLUOstar OPTIMA, BMG labtech) with an exci-
.14–3.08 (m, 2H), 2.77 (t, J = 2.6 Hz, 1H), 2.66 (t, 2H, J = tation filter of 544 nm and emission filter of 590 nm).
.5 Hz), 2.32–2.26 (m, 2H), 2.16–211 (m, 2H), 1.68–1.61 (quint,
H, J = 7.3 Hz), 1.51 (s, 9H), 1.41 (s, 9H), 1.37 (s, 9H), 1.18 (d,
J = 7.1 Hz, 3H).
-(N-QSy7-2-aminoethyl)-phenyl, ethyl 1-(3-(3-(N-tetramethyl-
rhodaminylamino)propyltriazolyl)butanoylamino)-2-methyl-
propane-phosphonate (14). Tetramethylrhodaminylamino-
propylazide (TAMRA-propyl-N ) (1.1 eq.) and compound 10
Acknowledgements
4
We thank Dr Oliver Frank for measuring NMR samples,
Prof. Dr Dieter Langosch for general support. We acknowledge
funding by the Emmy Noether Program of the German
Research Foundation and the TUM Junior fellow fund.
3
(2.0 μmol, 1.0 eq.) were mixed with CuBr (1 mM), TBTA (2 mM)
and sodium ascorbate (10 mM) in t-BuOH–water 1/1 (50 μL).
The reaction was heated overnight at 40 °C under nitrogen, when
ESI-MS showed the formation of the triazole adduct. ESI-MS:
2
+
m/z 504.2381 (found), C53
H
69
N
8
O
10
P
requires 504.2432. The
Notes and references
solvent was removed under reduced pressure and the crude
compound was treated with TFA–DCM (1/1) + 2.5% water
1 B. F. Cravatt, A. T. Wright and J. W. Kozarich, Annu. Rev.
Biochem., 2008, 77, 383–414.
(50 μL) for 1 h. The solvent was removed in vacuo and the
product was detected by ESI-MS: m/z 454.2139 (found),
2 W. P. Heal, T. H. T. Dang and E. W. Tate, Chem. Soc. Rev.,
2011, 40, 246–257.
3 S. H. L. Verhelst and M. Bogyo, QSAR Comb. Sci., 2005, 24,
261–269.
2
+
C
48
H
61
N
8
O
8
P
requires 454.2169. The resulting free amine
was coupled to QSy7 succinimidyl ester (1.1 eq.) in dmso
50 μL) with DIEA (1.8 eq.). After 3 h, the reaction mixture was
(
purified by RP-HPLC. Product containing fractions were
pooled and lyophilized, giving probe 14 as a purple powder
4 A. M. Sadaghiani, S. H. L. Verhelst and M. Bogyo, Curr.
Opin. Chem. Biol., 2007, 11, 20–28.
(
C
16% yield over three steps). ESI-MS: m/z 1546.6544 (found),
5 W. P. Heal, S. R. Wickramasinghe and E. W. Tate, Curr.
Drug Discovery Technol., 2008, 5, 200–212.
6 M. J. Niphakis and B. F. Cravatt, Annu. Rev. Biochem., 2014,
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7 A. B. Berger, P. M. Vitorino and M. Bogyo,
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8 S. Serim, U. Haedke and S. H. L. Verhelst, ChemMedChem,
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+
87
H
93
N
11
O
12PS requires 1546.6458.
4
-(N-QSy7-2-aminoethyl)-phenyl, ethyl 1-(3-(3-(N-tetramethyl-
rhodaminylamino)propyltriazolyl)butanoylamino)-(4-guanidino-
phenyl)-methanephosphonate (15). Compound 12 was Boc-
deprotected in TFA–DCM (1/1) for 30 min. The solvent was
removed and the oily product was purified by HPLC, giving a
white power in 37% yield. ESI-MS: m/z 486.2244 (found),
C
+
24
H
33
N
5
O
4
P requires 486.2265. The product was coupled by
9 G. Blum, S. R. Mullins, K. Keren, M. Fonovic, C. Jedeszko,
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click chemistry to TAMRA-propyl-N as described above, puri-
fied by HPLC and isolated as a pink powder after lyophilization
3
(
16%). The QSy-7 succinimidyl ester was coupled as described 10 G. Blum, G. von Degenfeld, M. J. Merchant, H. M. Blau and
above and the final product 15 was isolated after HPLC purifi- M. Bogyo, Nat. Chem. Biol., 2007, 3, 668–677.
cation in 18% yield as a purple powder. ESI-MS: m/z 1637.6591 11 L. E. Edgington, A. B. Berger, G. Blum, V. E. Albrow,
+
(
found), C91
H
94
N
14
O
12PS requires 1637.6628.
M. G. Paulick, N. Lineberry and M. Bogyo, Nat. Med., 2009,
15, 967–U177.
Labeling of proteases. Each protease (375 ng) in PBS was
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3
0 min with 1% dmso or an inhibitor (1 mM DCI for trypsin
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2 μM), which were diluted from a 100× stock in dmso. The
Q.-H. Xu and S. Q. Yao, J. Am. Chem. Soc., 2011, 133,
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(
proteins were resolved on a 15% SDS-PAGE gel (30% of the 14 Y. S. Liu, M. P. Patricelli and B. F. Cravatt, Proc. Natl. Acad.
sample volume was loaded) and scanned with a typhoon
TRIO+ fluorescent scanner.
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1
00 μL. As a blank control 98 μL PBS and 2 μL dmso were
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used. Compound 15 (2 μL from a 100 μM dmso stock) and
9
8 μL PBS constituted the no protease control, whereas com- 17 C. M. Kam, A. S. Abuelyaman, Z. Z. Li, D. Hudig and
pound 15 (2 μL from a 100 μM dmso stock), 93 μL PBS and
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