Selective and diagnostic labelling of serine hydrolases with reactive phosphonate inhibitors

Article abstract of DOI:10.1039/b717345h

Reactive phosphonates are important probes to target the active site of serine hydrolases, one of the largest and most diverse family of enzymes. Developing such inhibitory probes is of special importance in activity based protein profiling, a strategy that is increasingly used to gain information about a certain class of enzymes in complex proteosomes. Therefore, gaining detailed information about these inhibition events on the individual protein level is important since it affords information that can be used to fine-tune the probe for a specific task. Here, we report a novel and versatile synthesis protocol to access a variety of functionalised p-nitrophenyl phosphonate (PNPP) inhibitors from a common azide functionalised precursor using click chemistry. The obtained PNPPs were successfully used to covalently label serine hydrolases in their active sites with molecular tags. Furthermore, a model study is described in which we developed straightforward protocols that can be used to study protein inhibition events. Kinetic studies using UV-Vis and fluorescence spectroscopy techniques revealed that these PNPPs possess different inhibition rates for various proteins and were shown to be suitable probes to discriminate between various lipases. Additionally, we demonstrate that PNPPs are highly selective for serine hydrolases, making these probes very interesting as diagnostic or affinity probes for studying proteins in complex proteosomes. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b717345h

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Selective and diagnostic labelling of serine hydrolases with reactive
phosphonate inhibitors†
Harmen P. Dijkstra,*a Hein Sprong,b Bas N. H. Aerts,a Cornelis A. Kruithof,a Maarten R. Egmondb and
Robertus J. M. Klein Gebbink*a
Received 9th November 2007, Accepted 29th November 2007
First published as an Advance Article on the web 17th December 2007
DOI: 10.1039/b717345h
Reactive phosphonates are important probes to target the active site of serine hydrolases, one of the
largest and most diverse family of enzymes. Developing such inhibitory probes is of special importance
in activity based protein profiling, a strategy that is increasingly used to gain information about a
certain class of enzymes in complex proteosomes. Therefore, gaining detailed information about these
inhibition events on the individual protein level is important since it affords information that can be
used to fine-tune the probe for a specific task. Here, we report a novel and versatile synthesis protocol to
access a variety of functionalised p-nitrophenyl phosphonate (PNPP) inhibitors from a common azide
functionalised precursor using click chemistry. The obtained PNPPs were successfully used to
covalently label serine hydrolases in their active sites with molecular tags. Furthermore, a model study is
described in which we developed straightforward protocols that can be used to study protein inhibition
events. Kinetic studies using UV–Vis and fluorescence spectroscopy techniques revealed that these
PNPPs possess different inhibition rates for various proteins and were shown to be suitable probes to
discriminate between various lipases. Additionally, we demonstrate that PNPPs are highly selective for
serine hydrolases, making these probes very interesting as diagnostic or affinity probes for studying
proteins in complex proteosomes.
research field. Also alternative chemical biological methods that
Introduction
use small molecules for covalent linking to target proteins, thereby
Proteins play a crucial role in regulating many complex biological
creating the potential of studying these proteins in their natural
environment, were reported.7 A recent and highly interesting
strategy in this area is the covalent, selective modification of the
active site of proteins by small molecule inhibitors, a technique
called activity-based protein profiling (ABPP).8 This technique
not only labels proteins in an efficient and potentially selective
manner, it also knocks out the natural activity of proteins, an
event that can give valuable information about protein function.
Additionally, since the active site is targeted, this protocol can
potentially also discriminate between different classes of proteins
or even individual proteins. By decorating the inhibitors with
diagnostic or affinity tags, selective protein isolation and analysis
has become more straightforward.
Reactive phosphonate inhibitors (Fig. 1) have been successfully
applied in the field of ABPP to covalently modify members of the
serine hydrolase superfamily with molecular tags, e.g. fluorescent
groups and biotin.8–10 Cravatt and co-workers reported the use of
functionalised fluorophosphonates (LG = F) as potent inhibitors
for serine hydrolases. They demonstrated that ABPP is a suitable
technique to label specific proteins in complex proteomic mixtures
with diagnostic and/or affinity tags in order to gather information
about the active site and function of unexplored or even unknown
proteins.11 Hermetter and co-workers have reported on the use
of p-nitrophenyl (PNP) phosphonates (LG = p-nitrophenol) as
irreversible ABPP-inhibitors for lipases and esterases.10a,b They
extended this technology to develop microarrays for the iden-
tification and characterisation of novel and unknown lipolytic
enzymes.10c Recently, we reported on the selective introduction of
mechanisms. Using classical biological analysis techniques, e.g.
sequencing and crystallographic techniques, the structures of
many of these proteins have been elucidated. Despite these
achievements, studying the specific function of proteins in their
natural environment remains difficult due to the complex post-
translational events1 by which proteins are regulated. In search for
more direct methods to study proteins, the field of proteomics has
produced some valuable techniques, such as LC-MS/MS to study
protein modification2 and protein microarrays to study the natural
activities of proteins.3 Although these methods have provided
detailed information about protein expression and interactions
and in vitro functioning of proteins, they are not suitable for
studying protein function in cells or tissue. Therefore new methods
in which chemical and biological techniques are combined have
been developed in order to (chemoselectively) modify proteins
for the purpose of studying their function in complex biological
media.4 Especially native chemical ligation (NCL)5 and expressed
protein ligation (EPL)6 have been intensively applied in this
aChemical Biology & Organic Chemistry, Department of Chemistry, Faculty
of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Nether-
lands. E-mail: h.p.dijkstra@uu.nl, r.j.m.kleingebbink@uu.nl.; Fax: +31
(0)30 252 3615; Tel: +31 (0)30 253 1889/3120
bMembrane Enzymology, Department of Chemistry, Faculty of Science,
Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands
† Electronic supplementary information (ESI) available: ESI-MS data
of the various protein constructs and fluorescence spectroscopy data as
indicated in the text. See DOI: 10.1039/b717345h
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factors that play a role in these events. Here we demonstrate
how novel PNP phosphonate inhibitors can be used to selectively
and covalently modify serine hydrolases in an active site-directed
manner and how UV–Vis and fluorescence spectroscopy can be
used to efficiently monitor the inhibition event. Additionally, we
report a new straightforward synthesis protocol for functionalised
phosphonate inhibitors in which click chemistry is used to
conveniently attach various desired functionalities in the final step
to a PNP phosphonate building block.
Results and discussion
Synthesis of PNP phosphonate probes
Reactive phosphonates contain a good leaving group (LG) at-
tached to the electrophilic P-atom, rendering these P–LG bonds
very reactive towards a nucleophilic serine in the active site of
proteins but also to other (even weak) nucleophiles. Therefore,
in the synthesis of these probes extra care has to be taken to
prevent cleavage of this bond, as it is crucial to its biological
activity. In our studies we focussed on PNP phosphonate (PNPP)
inhibitors since the PNP leaving group appeared to be an ideal
probe to study the interaction between a selected serine hydrolase
and a functionalised PNPP inhibitor (vide infra). Preferentially, a
general synthesis protocol for phosphonate inhibitors should fulfil
a few criteria: (1) the synthetic route should be straightforward
without tedious work-ups and consist of only a few steps; (2) the
desired tag (R, Fig. 1, e.g. diagnostic and affinity tags) should
be introduced in the final step in order to get fast access to
a variety of functionalised inhibitors and (3) the final coupling
step should be mild and tolerate many functionalities. Since
such protocols are lacking for PNPP inhibitors, we decided to
explore possible synthesis routes that fulfil these criteria. In this
study we decided to keep the ethyl group (Rꢀ = Et, Scheme 1)
attached to the phosphonate moiety constant whereas the group
R can be changed at will depending on the desired functionality
needed for protein study/isolation. For the final step in which
the tag-functionality will be introduced we have chosen to use
the well-known click reaction, a 3 + 2 cycloaddition reaction
between an azide and an acetylene in the presence of a copper(I)
catalyst affording a triazole heterocycle. This reaction tolerates
many sensitive functionalities and is thus expected to leave the
Fig. 1 Examples of reactive phosphonates.
organometallic pincer fragments into the active site of cutinase, a
20 kDa member of the lipase family, using a functionalised PNP
phosphonate.12 The organometallic probes have great potential as
diagnostic and affinity tags in protein profiling and as phasing
probes in protein crystal structure elucidation.13
Serine hydrolases constitute a large superfamily of enzymes
and are responsible for many hydrolysis reactions in nature,
e.g. esterifications, amidations and acyltransfer. As outlined in
Scheme 1 for lipases (a subfamily of serine hydrolases), reaction
of reactive phosphonates with an enzyme results in protein–
inhibitor complexes in which the phosphorous atom binds in
=
a tetrahedral manner, with the P O binding in the oxy-anion
hole. This tetrahedral orientation closely resembles the hydrolysis
transition state for the fatty-acid ester hydrolysis, a reaction readily
catalysed by lipases. Since these phosphonates react irreversibly
and stoichiometrically with serine hydrolases they are powerful
probes to isolate, characterise and study serine hydrolases.
We are particularly interested in studying in detail the interac-
tion between a protein and an inhibitor since this is the crucial
step in ABPP and little to no information is available about the
Scheme 1 Proposed mechanism for lipase inhibition by reactive phosphonates.
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P–LG bond intact. Moreover, the click reaction can also be used
to chemically modify proteins by decorating a protein first with
one of the click functionalities followed by a reaction with a tag
containing the complementary click functionality.14 This copper(I)
catalysed reaction even proceeds in complex proteomic mixtures.
Thus, with this click-chemistry strategy, the tag potentially can be
introduced prior to or after the protein inhibition event, making
this route very versatile and general.
Our new synthetic route for the functionalised PNPP inhibitors
is outlined in Scheme 2. In the first step commercially avail-
able 2-(1-bromoethyl)-diethylphosphonate is treated with azide
amberlite-resin in DMF giving a nucleophilic replacement of
the bromide for an azide functionality. Next, azide phosphonate
derivative 1 is treated with excess oxalyl chloride in diethyl ether
to afford the moisture sensitive chlorophosphonate 2. Due to
the sensitive nature of the P–Cl bond, this compound was only
analysed by NMR spectroscopy to determine full conversion
of 1 into 2. When full conversion was obtained all volatiles
were removed and 2 was immediately treated with p-nitrophenol
(PNP) in benzene in the presence of triethylamine, affording
common azide PNP phosphonate intermediate 3 in 41% overall
yield from 2-(1-bromoethyl)diethylphosphonate. Thus, compound
3 was synthesised in only 3 steps in gram scale quantities, is air
and moisture stable and is easy to handle.
Fig. 2 New functionalised PNPP inhibitors.
Monitoring protein inhibition
Azide intermediate 3 was explored in the click reaction with
various functionalised acetylenes and turned out to be an excellent
precursor to access a variety of functionalised phosphonate
inhibitors. In Fig. 2 three selected phosphonate inhibitors are
depicted that were prepared in this study. Two different tags are
depicted: a fluorescent dansyl tag (4a) that can potentially be
used to study the protein–inhibitor construct using fluorescent
spectroscopy, and a biotin moiety (4b) that may serve as an affinity
tag to isolate proteins using (strept)avidine columns. To illustrate
the versatility of this protocol, we also prepared bisphosphonate
inhibitor 4c that has the potential to cross-link proteins. All
products were obtained using standard click chemistry condi-
tions ([Cu(MeCN)4BF4], 2,6-lutidine in MeCN)15 in near quan-
titative yields using the appropriately propargyl-functionalised
dansyl, biotin and triethyleneglycol precursors, respectively. No
hydrolysis of the P–OPNP bond was observed, showing the
excellent functional group tolerance of the click reaction, mak-
ing this a convenient protocol to access a variety of reactive
phosphonates.
As mentioned earlier, reactive phosphonates similar to 3 and 4
were reported to be covalent inhibitors for lipases and esterases.10,12
The nucleophilic serine-OH in the active site of these enzymes
attacks the phosphorus atom of the phosphonate inhibitor, a
process in which PNP is released (LG, Scheme 1). Since this
process blocks the natural activity of the corresponding protein,
this methodology is referred to as active-site directed inhibition
and is the crucial step in ABPP. Although it has been reported
that reactive phosphonates react at different rates with serine hy-
drolases (using fluorophosphonates),11b no studies were performed
on individual proteins that could shine light on the factors that are
responsible for the differences in inhibition rate. Especially in the
field of protein diagnosis and isolation from complex proteomic
mixtures using ABPP, gathering information about the inhibition
event of individual proteins is of great value since it ultimately
can be used to fine-tune the outcome of these experiments. In
this study we decided to study several individual enzymes in
our attempt to develop straightforward techniques to study the
Scheme 2 i. DMF, RT, 20 h, 74%; ii. oxalyl chloride, Et2O, RT, 24 h, 95%; iii. p-nitrophenol, Et3N, C6H6, RT, 3 h, 64%; iv. [Cu(MeCN)4BF4], 2,6-lutidine,
MeCN, RT, 12 h, >90%.
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interaction of these enzymes with our PNPP probes. For this
purpose, we initially selected three lipases, i.e. cutinase, Candida
antarctica lipase B (CALB) and Chromobacterium viscosum lipase
(CVL). We were particularly interested in the inhibition rates of
these enzymes when treated with PNPP inhibitors 4 since this
would provide valuable information about the accessibility of the
active sites of these proteins. The leaving group in this protocol,
i.e. PNP, is brightly yellow coloured in solution at the inhibition
conditions (50 mM tris, 0.1% triton buffer, pH 8.0) and therefore
the release of PNP in solution can be conveniently monitored
with UV–Vis spectroscopy. Since the reaction between a lipase
and a PNP phosphonate inhibitor is stoichiometric, the rate of
PNP release is a direct measure of the inhibition rate and thus of
the accessibility of the active site. We were also interested to see
whether the fluorescent properties of the dansyl group (4a) could
be used to gather more detailed information about the active site of
these proteins. Therefore, we set out a number of inhibition studies
with these lipases using various inhibitors and demonstrate how
UV–Vis and fluorescence spectroscopy techniques can be used
to get insight into the factors that influence the selectivity and
efficiency of protein inhibition.
Initially, we started with cutinase (the N172K mutant) since this
lipase is well studied and has some attractive features making it
an ideal candidate for proof-of-concept studies.16 Cutinase is a
lipase that is lacking the polypeptide lid, commonly observed in
the family of lipases, covering the active site. As a consequence
the active site of cutinase is readily solvent exposed. Due to these
features covalent inhibition of cutinase using reactive phosphonate
inhibitors should be straightforward. Indeed, cutinase (typically
25–125 lM) was conveniently labelled with 3, 4a and 4b in an active
site-directed manner by mixing cutinase in a tris–triton buffer
at pH 8.0 with an excess (typically 5–8 fold) of the appropriate
inhibitor and allowing the reaction to proceed to completion
within a certain time period. These inhibition times were deter-
mined by monitoring the PNP release using UV–Vis analysis.
In addition to the PNP release we also performed a control
experiment to verify full inhibition by adding aliquots of the
inhibition solution to a solution containing p-nitrophenyl butyrate
(Scheme 3). Lipases are known to hydrolyse the ester bond of
aliphatic PNP-esters, e.g. PNP butyrate, and thus monitoring the
residual lipase activity in this activity assay is a direct measure for
the amount of protein inhibition. These experiments demonstrated
that the inhibition rate greatly depended on the inhibitor with
3 > 4b > 4a. This behaviour is fully attributed to the increased
steric bulk at the phosphonate moiety of the inhibitors going
from 3 → 4b → 4a and is in agreement with our earlier reported
results.12 Fig. 3 shows the representative graphs for the reaction
of cutinase with 4a and clearly illustrates that the data obtained
from the inhibition experiment and from the activity control
assay result in the similar reaction profiles. The inhibition event
followed first order kinetics, giving an inhibition rate constant
Fig. 3 Typical inhibition and activity control curves.
(k) for cutinase with 4a of 0.13 min−1. After full inhibition
was obtained, the cutinase–inhibitor constructs were purified by
dialysis using 150 mM NH4OAc buffer. The purified cutinase–
inhibitor constructs (∼25 lM) were subsequently analysed by
ESI-MS spectrometry, revealing the formation of the desired
cutinase–inhibitor constructs; Mw(cutinase N172K) = 20619 Da,
Mw(cutinase-3) = 20781 Da, Mw(cutinase-4a) = 21070 Da and
Mw(cutinase-4b) = 21063 Da (see also ESI†).
After successful inhibition of cutinase with our PNPPs we
decided to extend this research to other lipases, i.e. Candida
antarctica lipase B (CALB) and Chromobacterium viscosum lipase
(CVL). CALB and CVL are two well-studied lipases with high
esterase activity in the hydrolysis of PNP-butyrate (Scheme 3).17
For these experiments we only used dansyl inhibitor 4a since
it allows us to perform additional analyses using fluorescence
spectroscopy. Treatment of CALB and CVL with 4a (8 equiv-
alents) resulted in full inhibition of both lipases according to
the PNP release and the activity control assay. Similar data as
shown for cutinase (Fig. 3) were obtained. It was found that
CALB (k = 0.66 min−1) possesses even higher first order inhibition
rates than cutinase (k = 0.13 min−1) with 4a whereas CVL (k ꢁ
0.01 min−1) inhibition was much slower. The remarkable difference
in inhibition rates, as found here within the subfamily of lipases,
can be explained by comparing reported crystal structures of all
three lipases.18 The crystal structures clearly reveal that the active
site of CVL is buried much deeper inside the protein than the active
sites of cutinase and CALB. As a consequence a considerable
amount of steric hindrance occurs during the inhibition reaction
of CVL. These experiments demonstrate how specially designed
reactive phosphonates in combination with a simple analysis
technique can be used to conveniently access information about
the active site of proteins, both qualitatively and quantitatively.
Fluorescence spectroscopy studies
Also for CALB-4a and CVL-4a we attempted to acquire ESI-
MS data of the protein–inhibitor constructs. Unfortunately, after
Scheme 3 Activity control test.
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purification by dialysis, no satisfactory ESI-MS data could be
obtained, probably due to (poly)glycosylation of the proteins as
only small glycol fragments were observed. Therefore, we decided
to study the protein–4a constructs in more detail by fluorescence
spectroscopy in order to confirm the presence of the dansyl group
in the protein–inhibitor construct. Typically, a 2.5 lM solution
of a dialysed protein–4a construct in 150 mM NH4OAc buffer
was excited at 340 nm (the maximum absorption wavelength of
the dansyl group) and the emission is measured from 400–560 nm
(Fig. 4). Free 4a in the same buffer was measured as a control.
In all fluorescence experiments also the native proteins without
inhibitors were measured as controls (data not shown) and in
all cases no emission between 400–560 nm was observed when
these solutions were excited from 290–370 nm. From Fig. 4 it
is clear that the fluorescence spectra of cutinase–4a and CALB–
4a (typical data shown for cutinase, dashed line) are similar to
that of the free inhibitor 4a (solid line) in solution, confirming
binding of the fluorescent dansyl probe to the proteins. The
emission maximum of cutinase–4a and CALB–4a (at 550 nm)
are slightly shifted to higher wavelength (15 nm) as compared to
free, unbound 4a. The curve for CVL–4a (dash–dot line) on the
other hand is completely different and shows no emission between
460–560 nm when excited at 340 nm. Earlier it was reported by
Whitesides et al. that the fluorescent signal of a dansyl inhibitor
disappeared by binding it in the active site of carbonic anhydrase
II. They suggested that surrounding tryptophan residues in the
interior of the protein quench the fluorescence of the dansyl
group.19 Therefore, we decided to excite the CVL–4a solution at a
higher energy wavelength (290 nm) and recorded the fluorescence
emission from 400–560 nm (Fig. 4, dotted line). Again free 4a
was measured as a control. Now a clear fluorescent signal was
observed with a maximum emission at 470 nm, a significant
shift when compared to the maximum emission wavelength of
4a, cutinase–4a and CALB–4a (max. em. ∼535–550 nm, also
when excited at 290 nm). Since we suspected that this enormous
shift was due to a reduced polarity surrounding the fluorescent
dansyl moiety, we decided to also measure 4a in various solvents
in order to determine how the emission maximum of 4a shifts
going from polar to non-polar solvents. We selected the following
solvents with decreasing polarities: 150 mM NH4OAc buffer,
MeCN, CH2Cl2 and hexanes. The fluorescent spectra (see ESI
for graph†) of 4a in these solvents clearly revealed that the
emission maximum when excited at 290 nm drastically shifts to
lower wavelengths when decreasing the polarity of the solvent.
Furthermore, a decrease in emission intensity is also observed, a
phenomenon similar to what we observed for the CVL–4a hybrid
as compared to, for example, cutinase–4a (Fig. 4). Accordingly,
the dansyl fluorescent group is located in the more hydrophobic
interior of the CVL protein, which is in agreement with the
deep active-site pocket of CVL (vide supra). Since the emission
maximum of the dansyl moiety in NH4OAc buffer is hardly
shifted for cutinase–4a and CALB–4a as compared to 4a, this
implies that for these constructs the dansyl group extends outside
the protein pocket into the aqueous solution. Again, this is in
agreement with the active sites of these proteins being on the
protein-surface and thus exposed to the aqueous buffer solution.
Clearly, this phenomenon is not unique for PNPPs and therefore
such straightforward protocols should be directly transferable to
other classes of inhibitors and proteins.
Comparing the fluorescent emission spectra of 4a and CVL–
4a at 290 nm excitation (Fig. 4) revealed that CVL–4a shows
considerable emission at 420–440 nm whereas 4a shows only
minimum emission in that wavelength range and this low emission
was found to be independent of the concentration of 4a (results
not shown). This means that the inhibition event of CVL with 4a
can be followed using fluorescence spectroscopy by monitoring the
emission increase at 440 nm; only CVL–4a shows emission at that
wavelength. Therefore, we performed the inhibition experiment as
described earlier using 8 equivalents of 4a with respect to CVL. At
time intervals, aliquots of this solution were taken, diluted
(10×, final concentration 2.5 lM) with NH4OAc buffer and the
fluorescent emission spectrum at 440 nm was measured (exciting
at 290 nm) (Fig. 5). After approximately 30 hours, complete
inhibition was obtained and this is in good agreement with the
kinetic data obtained from the esterase activity control (vide supra).
Given the high sensitivity of fluorescence spectroscopy, very low
chromophore concentrations can now be measured which allowed
us to obtain accurate data for the CVL inhibition event. It was
found that CVL inhibition with 4a followed first order kinetics
as well resulting in an accurate inhibition rate constant k =
0.002 min−1.
Fig. 4 Fluorescence spectroscopy study of 4a with various enzymes.
Fig. 5 Inhibition of CVL monitored by fluorescence spectroscopy.
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Click reaction on protein
mixture of all three lipases, only cutinase dimerisation is observed.
These results suggest that bifunctional probes such as 4c may be
of interest to investigate (selective) protein–protein interactions
in complex proteomic mixtures. This research is currently under
investigation.
Numerous groups have reported the use of click chemistry to
chemically modify biomolecules with special functionalities that
can be used for protein diagnosis and profiling.14 Therefore, we de-
cided to test whether cutinase–3, containing an azide functionality,
could be functionalised post-inhibition using click chemistry. Mix-
ing cutinase–3 with propargyl dansylamide in tris–triton buffer
pH 8.0 in the presence of CuSO4 (1 mM), tris(benzyltriazole)amine
(TBTA, 500 lM), tris(ethylcarboxy)phosphine hydrochloride
(TCEP, 1 mM) and sodium dodecylsulfate (SDS, 1%) and
incubating the resulting mixture overnight at room temperature,
indeed resulted in the formation of a fluorescently labelled
cutinase. Successful labelling was determined by analysing the
mixture with SDS-PAGE analysis and subsequently exposing the
gel to UV-light prior to coomassie blue staining (Fig. 6a + b,
respectively). This clearly revealed fluorescently labelled cutinase
whereas a control experiment in which CuSO4 was omitted gave
no fluorescent labelling of cutinase. This means that our strategy
allows both introduction of the desired functionality prior to and
after the protein inhibition event, giving an extra dimension to
our synthesis design (Scheme 2) and making this strategy very
versatile.
Selective protein labelling
The significant difference in inhibition rates between cutinase,
CALB and CVL with 4a implies that protein discrimination can
be achieved using PNPP probes. To test this hypothesis, we mixed
all three proteins in an approximately 1 : 1 : 1 ratio in tris–
triton buffer and treated this mixture with both an excess of
4a and with only 0.3 equivalents of 4a. After incubating these
solutions overnight at room temperature, the resulting mixtures
were analysed by SDS-PAGE analysis by exposing the gel to
UV-light (Fig. 6d) prior to coomassie blue staining (Fig. 6e) in
order to determine fluorescent labelling of proteins. Treating the
individual proteins with 4a resulted in a clear fluorescent spot
for all proteins (lanes 3, 4 and 5 for cutinase, CALB and CVL,
respectively). Additionally, treatment of the mixture with an excess
of 4a also resulted in the fluorescent labelling of all three proteins
(lane 7). However, addition of only 0.3 equivalents of 4a gave
only fluorescent labelling of cutinase and CALB spots (lane 8).
Again this is in good agreement with the earlier determined slow
inhibition rate of CVL, preventing CVL labelling with 4a in the
latter case. Thus, PNPP probes can discriminate between proteins,
even within a subfamily of proteins (lipases).
Moreover, in order to determine the specificity of PNP phospho-
nate probes for serine hydrolases, we selected a range of proteins
from different protein (sub)families possessing various modes of
action and incubated them with 4a as described earlier. Subtilisin,
PagP, PagL and patatin are all known serine hydrolases. PagL
and patatin are both reported to have esterase activity whereas
subtilisin is a hydrolase and PagP an acyl transferase. Treatment
of these proteins with 4a followed by SDS-PAGE analysis revealed
fluorescent labelling of all these proteins. Incubating 4a with
members of other protein (sub)families, i.e. papain (cysteine
hydrolases), bovine serum albumin (BSA), carbonic anhydrase
II (carbonate hydrolase), pepsin (aspartic acid protease) and
thermolysin (metalloprotease), revealed that PNP phosphonate
probes are highly selective for serine hydrolases as no fluorescent
labelling of these proteins occurred. These results strongly suggest
that PNP phosphonate inhibitors such as 4a are interesting can-
didates to selectively label serine hydrolases in complex proteomic
mixtures. Initial experiments revealed that 4a indeed selectively
labels proteins in living cells (HeLa cells). Currently, we are
optimising these results and are developing proteomic techniques
to isolate and analyse the labelled proteins.
Fig. 6 Protein labelling using reactive phosphonate probes; Click reaction
of cutinase–3 with propargyl dansylamide analysed with (a) UV–Vis and
(b) coomassie staining; (c) cutinase dimerisation using PNPP 4c; protein
discrimination using PNPP 4a and cutinase CALB and CBV analysed with
(d) UV–Vis (circle indicates CVL) and (e) coomassie staining.
Another proof of the versatility of our synthesis protocol is the
accessibility of bio-probes containing multiple PNP phosphonate
functionalities, e.g. 4c. Multivalent bioprobes hold great promise
in investigating protein cross-linking and may thus help to
understand protein–protein interaction in complex proteomic
mixtures.20 To test whether 4c can be used for this purpose, 4c was
first reacted with a slight excess of cutinase under the standard inhi-
bition conditions, resulting in cutinase dimer formation (Fig. 6c).
Surprisingly, with CALB and CVL no protein cross-linking was
observed which is probably due to steric hindrance of the protein;
if one of the phosphonate groups is already bound to a protein,
the reaction of the second phosphonate group is hampered due to
the steric bulk of the protein. Additionally, when 4c is added to a
Conclusions
We have described a novel and straightforward synthesis protocol
for reactive PNPP probes in which the desired tag was introduced
in the final step using click chemistry. Both diagnostic (4a) and
affinity (4b) tags could be introduced using this strategy whereas
inhibitors containing multiple PNP phosphonate moieties (4c)
are also available through this methodology. The products are
produced in only four steps and since we use a mild click coupling
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reaction in the final step it is relatively easy to fine-tune these
probes for selected (groups of) proteins by varying the length
and nature of the linkers. This strategy should by no means be
limited to reactive PNPP probes. The PNPP probes were shown
to be selective ABPP inhibitors for serine hydrolases and they can
discriminate between proteins, even within a subfamily of proteins
(lipases). Because the click reaction is bio-orthogonal we were
also able to alter the sequence of events by first inhibiting the
enzyme with an azide-functionalised PNP phosphonate inhibitor
(3) followed by introduction of the diagnostic tag (dansyl group)
using click chemistry. This emphasises the flexibility and versatility
of our approach. Additionally, we developed easy protocols using
UV–Vis and fluorescence spectroscopy to monitor the protein–
inhibition event, providing accurate inhibition rate constants
for various enzymes and valuable information about the active
site (accessibility) of serine hydrolases. Gathering such data is
of special importance in ABPP since it gives valuable insight
into the factors that influence the protein inhibition event, the
crucial step in ABPP. These results illustrate how small organic
molecules can serve as diagnostic probes to gather information
about the structure and location of the active site of (unknown)
proteins. Finally, the high selectivity of our PNP phosphonate
probes for serine hydrolases suggests that these probes are ideally
suited to selectively label proteins in complex proteomic mixtures,
e.g. living cells, making them highly interesting for the field of
proteomics.
cutinase were recorded on a Finnigan LC-Q ion-trap mass
spectrometer. All samples were introduced using a nanoflow
electrospray source (Protana, Odense, Denmark).
Synthesis of diethyl 2-azidoethylphosphonate (1)21. Azide ex-
change resin on amberlite (32.2 g, 122.36 mmol N3) was weighed
in a 250 mL round bottom flask. Next, DMF (70–80 mL)
was added until the suspension could be stirred conveniently.
Finally, diethyl-2-bromoethylphosphonate (2.36 mL, 12.0 mmol)
was added drowise by means of a syringe. 31P-NMR was used
to monitor the conversion of this reaction. After the reaction
had reached completion (20 h), the reaction mixture was filtered,
and the resin was washed with DMF. Subsequently, the combined
DMF-layer was concentrated in vacuo and Et2O (20–30 mL) was
added. The mixture was filtered once again and the solid washed
with Et2O. Removal of all volatiles in vacuo afforded a yellow oil
and this oil was distilled under reduced pressure; yield: 2.62 g, 74%.
1H-NMR (400 MHz, CDCl3): d 4.09 (m, 4H, P-(O–CH2–CH3)2),
3.53 (m, 2H, P–CH2–CH2–N3), 2.03 (m, 2H, P–CH2–CH2–N3),
1.34 (q, 6H, P-(O–CH2–CH3)2). 31P-NMR (162 MHz, CDCl3): d
27.97 (s).
Synthesis of ethyl 2-azidoethylphosphonochloridate (2). Com-
pound 1 (2.0 g, 9.65 mmol) was weighed in a dry Schlenk tube and
Et2O (30 mL) was added. Next, freshly distilled oxalyl chloride
(18.5 mL, 193.0 mmol) was added by means of a syringe. After 10 h
at room temperature the reaction was monitored with 31P-NMR,
showing full conversion. The reaction mixture was concentrated in
vacuo to yield a dark brown oil. Due to the sensitivity and toxicity
of this compound, the compound was used immediately; yield:
Experimental section
General comments
1
1.81 g, 95%. H-NMR (300 MHz, CDCl3): d 4.30 (m, 2H, P–O–
Organic solvents were dried over appropriate materials and
distilled prior to use. All reagents were obtained commercially
and used without further purification unless otherwise stated.
Triton X-100 was purchased from Serva and tris(hydroxymethyl)-
aminomethane from J. T. Baker. Buffer solutions were prepared
using Milli-Q grade water. Purification of water (18.2 MXcm) was
performed with the Milli-Q Synthesis system (Millipore; Quantum
CH2–CH3), 3.64 (m, 2H, P–CH2–CH2–N3), 2.44 (m, 2H, P–CH2–
CH3–N3), 1.40 (t, 3H, P–O–CH2–CH3). 13C–NMR (100 MHz,
CDCl3): d 16.13 (d, 3JP,C = 6.74 Hz, OCH2CH3), 33.77 (d, 1JP,C
=
124.91 Hz, PCH2), 45.10 (s, PCH2CH2), 64.01 (d, 2JP,C = 8.25 Hz,
OCH2). 31P-NMR (162 MHz, CDCl3): d 38.7 (s).
1
1
Ultrapure). H, 31P and 13C{ H} NMR spectra were recorded at
Synthesis of ethyl-4-nitrophenyl-2-azidoethylphosphonate (3).
Compound 2 was dissolved in dry benzene (40 mL) and a
previously prepared solution of 4-nitrophenol (1.27 g, 9.162 mmol)
and triethylamine (6.44 mL, 45.81 mmol) in benzene (40 mL)
was added dropwise at room temperature. After 3 h of stirring
at room temperature, the reaction mixture had turned black. The
mixture was concentrated in vacuo and subsequently dissolved in
Et2O (150 mL). This solution was washed with 1 M K2CO3 (aq)
(2 × 100 mL), water (3 × 100 mL), and brine (3 × 100 mL).
The organic layer was dried over MgSO4 and concentrated in
vacuo to yield a yellowish oil. The product was purified by column
chromatography (silica gel, Et2O–hexanes 1 : 4, Rf = 0,16); yield:
298 K on a Varian 400 Spectrometer at 300/400, 75/100 MHz and
162 MHz, respectively, and 31P{ H} NMR spectra were recorded
1
at 298 K on a Bruker AC200 at 81 MHz. All NMR chemical
shifts are in ppm referenced to residual solvents (31P{ H} NMR
1
shifts to H3PO4). The MALDI-TOF mass spectra were acquired
using a Voyager-DE Biospectrometry Workstation (PerSeptive
Biosystems Inc., Framingham, Ma, USA) mass spectrometer.
Sample solutions with an approximate concentration of 20–
30 mg mL−1 in CH2Cl2 or THF were prepared. The matrix was 3,5-
dihydroxybenzoic acid or 9-nitroanthracene with an approximate
concentration of 20–30 mg mL−1. A 0.2 lL of the sample and
0.2 lL of the matrix solution were combined and placed on a
golden MALDI target plate and analyzed after evaporation of
the solvents. Microanalyses were performed by Kolbe, Mikroana-
lystisches Laboratorium (Mu¨llheim a/d Ruhr, Germany). UV–Vis
experiments were performed at room temperature using a Carey-
100 spectrometer. Fluorescence spectroscopy was measured on a
Spex Fluorolog instrument. Infrared spectra were recorded with
a Perkin Elmer Spectrum 1 FT-IR spectrometer. Electrospray
ionization mass spectra of the wild-type cutinase and modified
1
1.75 g, 64%. H-NMR (300 MHz, CDCl3): d 8.26 (d, 2H, P–O–
ArH–NO2), 7.41 (d, 2H, P–O–ArH–NO2), 4.20 (m, 2H, P–O–
CH2–CH3), 3.65 (m, 2H, P–CH2–CH2–N3), 2.26 (m, 2H, P–CH2–
CH3–N3), 1.34 (t, 3H, P–O–CH2–CH3). 13C-NMR (100 MHz,
CDCl3): d 16.55 (d, 3JP,C = 6.03 Hz, OCH2CH3), 26.61 (d, 1JP,C
=
142.10 Hz, PCH2), 45.26 (d, 2JP,C = 3.02 Hz, PCH2CH2), 63.79 (d,
2JP,C = 6.74 Hz, OCH2), 121.23 (d, 3JP,C = 4.53 Hz, ArC), 125.95 (s,
ArC), 144.98 (s, ArC–NO2), 155.41 (d, 2JP,C = 8.95 Hz, ArC–O).
31P-NMR (162 MHz, CDCl3) d 25.95 (s). IR (cm−1): 2102 (N3);
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3
anal. calcd. for C10H13N4O5P: C, 42.26; H, 4.61; N, 19.71; found:
C, 42.08; H, 4.55; N, 19.53%.
4c. 1H-NMR (400 MHz, acetone-d6): d 8.28 (dd, JH,H
=
5
9.20 Hz, JH,P = 0.80 Hz, 4H, ArH), 8.02 (s, 2H, triazole-H),
3
4
7.49 (dd, JH,H = 9.20 Hz, JH,P = 1.20 Hz, 4H, ArH), 4.76–4.82
(m, 4H, PCH2CH2), 4.59 (s, 4H, OCH2-triazole), 4.20–4.26 (m,
4H, CH2CH3), 3.56–3.63 (m, 12H, 3 × CH2CH2O), 2.73–2.82 (m,
Typical procedure for the click reaction for the synthesis of
dansyl PNPP 4a. This reaction was carried out in a N2 atmo-
sphere. Azido phosphonate 3 (0.48 g, 1.6 mmol) was dissolved
in degassed acetonitrile (5 mL). Next, 2,6-lutidine (0.19 mL,
1.6 mmol) was added, followed by 5-(dimethylamino)-N-(prop-
2-ynyl)naphthalene-1-sulfonamide22 (0.46 g, 1.6 mmol). Finally,
[CuI(CH3CN)4PF6] (7.7 mg, 0.0021 mmol) was added. After
stirring this solution at room temperature for 24 h, the mixture
was concentrated in vacuo. The crude product was dissolved in
CH2Cl2 (10 mL) and this layer was washed with saturated NH4Cl
(aq) (20 mL), H2 (20 mL) and brine (15 mL). The organic layer was
dried (MgSO4) and concentrated giving 4a as a yellow solid. This
solid was washed with hexanes and dried in vacuo. Yield: 0.88 g,
93%.
4H, PCH2), 1.28 (t, JH,H = 7.60 Hz, 2H, CH2CH3); 13C-NMR
3
3
(100 MHz, CDCl3): d 15.91 (d, JP,C = 5.83 Hz, OCH2CH3),
1
27.11 (d, JP,C = 140.51 Hz, PCH2), 44.04 (s, PCH2CH2), 63.42
(d, 2JP,C = 6.64 Hz, OCH2), 64.35 (s, CH2), 69.69 (s, CH2), 70.52
(s, CH2), 70.57 (s, CH2), 121.56 (d, 3JP,C = 4.63 Hz, ArC), 123.80
(s, ArC), 125.80 (s, ArC), 144.91 (s, ArC), 145.07 (s, ArC), 155.76
(d, 2JP,C = 7.85 Hz, ArC–O); 31P-NMR (162 MHz, acetone-d6) d
25.36 (s). MALDI-TOF (m/z): 827.31 (calcd. M + H+: 827.25),
849.31 (calcd. M + Na+: 849.24), 865.27 (M + K+: 865.35); anal.
calcd. for C32H44N8O14P2: C, 46.49; H, 5.36; N, 13.55; found: C,
46.33; H, 5.30; N, 13.43%.
3
4a. 1H-NMR (400 MHz, acetone-d6): d 8.54 (d, JH,H
=
3
8.40 Hz, 1H, ArH), 8.39 (d, JH,H = 8.40 Hz, 1H, ArH), 8.30
General protein inhibition experiment
3
3
(d, JH,H = 9.20 Hz, 2H, ArH), 8.23 (d, JH,H = 7.20 Hz, 1H,
Typically, 10–30 mg of the appropriate enzyme is dissolved in
4.0 mL 0.1% triton, 50 mM Tris buffer at pH 8.0 (acidified with
conc. HCl). To 1.0 mL of the protein solution is added 10 lL of a
50 mM solution of 4a in MeCN and the p-nitrophenolate release
is monitored by UV–Vis analysis at 410 nm. Alternatively, at
certain time intervals, aliquots (typically 100 lL) of the inhibition
solution are taken, diluted 10× with 150 mM NH4OAc buffer
and the emission spectra of the resulting solutions at 440 nm
(290 nm excitation) are recorded on a fluorescence spectrometer.
The rate constants k were obtained by plotting −ln(C/C0) against
time, where C is concentration residual active (non-inhibited)
protein and C0 is total concentration protein. Activity assay:
at certain time intervals (depending on the inhibition rate of
the protein), 0.5 lL samples of the protein inhibition solution
were taken and added to a 0.25 mM solution of p-nitrophenyl
butyrate in tris–triton buffer pH 8.0. The rate of p-nitrophenolate
release was measured using UV–Vis analysis at 410 nm (against a
reference sample without protein). Analysing the data revealed a
first order inhibition processes. The slow inhibition rate for CVL
resulted in a considerable error in the UV–Vis data since the PNP
concentration in solution is initially below the detection limit of
UV–Vis spectroscopy and gave a high noise to signal ration. Thus
no accurate inhibition rate constant could be determined using
UV–Vis analysis.
After full inhibition, the protein mixtures were analysed by SDS-
PAGE-UV–Vis analysis, fluorescence spectroscopy and/or mass
spectrometry. For fluorescence spectroscopy and mass spectrom-
etry, the protein–inhibitor construct was first purified by dialysis
against 150 mM NH4OAc buffer using dialysis cassettes with a
Mw cut-off of 10 kDa. Next, the purified protein–4a solutions
were analysed by fluorescence spectroscopy by exciting the sample
at 290 nm and measuring the fluorescent emission from 400–
650 nm. For cutinase–4a and CALB–4a and free 4a in NH4Ac
(150 mM) buffer, the emission maxima are typically between 550–
560 nm whereas for CVL–4a the emission maximum lies at 475 nm.
For CVL, the inhibition event was monitored by fluorescence
spectroscopy by sampling 100 lL aliquots, diluting them 10× with
NH4OAc (150 mM) buffer and measuring the emission intensities
at 440 nm (290 nm excitation). The obtained inhibition curve
ArH), 7.65 (s, 1H, triazole-H), 7.55–7.62 (m, 2H, ArH), 7.49 (d,
3JH,H = 9.20 Hz, 2H, ArH), 7.26 (d, JH,H = 7.60 Hz, 1H, ArH),
3
7.20 (br. s, 1H, NH), 4.61 (m, 2H, PCH2CH2), 4.18–4.24 (m, 4H,
CH2N + OCH2), 2.88 (s, 6H, N(CH3)2), 2.55–2.66 (m, 2H, PCH2),
3
1.28 (t, JH,H = 7.20 Hz, 3H, OCH2CH3); 13C-NMR (100 MHz,
CDCl3): d 15.89 (d, 3JP,C = 5.83 Hz, OCH2CH3), 26.80 (d, 1JP,C
=
141.41 Hz, PCH2), 37.40 (s, CH2NH), 43.93 (s, PCH2CH2), 45.01
2
(s, N(CH3)2), 63.91 (d, JP,C = 6.64 Hz, OCH2), 115.41 (s, ArC),
119.63 (s, ArC), 121.64 (d, 3JP,C = 4.53 Hz, ArC), 121.88 (s, ArC),
123.63 (s, ArC), 125.91 (s, ArC), 126.21 (s, ArC), 128.26 (s, ArC),
129.21 (s, ArC), 129.80 (d, 3JP,C = 6.64 Hz, ArC), 130.22 (s, ArC),
130.32 (s, ArC), 136.32 (s, ArC), 144.99 (s, ArC), 151.99 (s, ArC),
155.41 (d, 2JP,C = 7.95 Hz, ArC–O); 31P-NMR (162 MHz, CDCl3) d
25.00 (s). MALDI-TOF (m/z): 588.44 (calcd. M+ : 588.57), 611.36
(calcd. M + Na+ : 611.57), 627.32 (calcd. M + K+ : 627.66); anal.
calcd. for C25H29N6O7PS: C, 51.02; H, 5.24; N, 12.62; found: C,
51.10; H, 5.28; N, 12.67%.
3
4b. 1H-NMR (400 MHz, acetone-d6): d 8.28 (d, JH,H
=
8.40 Hz, 2H, ArH), 8.13 (br m, 1H, NH–CH2), 7.98 (s, 1H,
triazole-H), 7.47 (d, 3JH,H = 8.40 Hz, 2H, ArH), 6.24 (br s, 1H, NH-
biotin), 6.12 (br s, 1H, NH-biotin), 4.77 (m, 2H, PCH2CH2), 4.50
(m, 1H, biotin-H), 4.39 (s, 2H, CH2NH), 4.31 (s, 1H, biotin-H),
4.20 (q, 3JH,H = 6.80 Hz, 2H, CH2CH3), 3.17 (m, 1H, CHS), 2.68–
2.92 (m, 4H, CH2P + CH2S), 2.20 (t, 3JH,H = 6.80 Hz, 2H, CH2CO),
1.70 (m, 2H, CH2), 1.61 (m, 2H, CH2), 1.37 (m, 2H, CH2), 1.25
(t, 3JH,H = 6.80 Hz, 2H, CH2CH3); 13C-NMR (100 MHz, CDCl3):
3
d 17.14 (d, JP,C = 5.73 Hz, OCH2CH3), 26.79 (s, CH2-biotin),
28.10 (d, 1JP,C = 141.01 Hz, PCH2), 29.51 (s, CH2), 29.69 (s, CH2),
35.84 (s, CH2CO), 36.69 (s, CH2NH), 41.49 (s, CH2S), 45.29 (s,
PCH2CH2), 57.05 (s, CHS), 61.57 (S, CH-biotin), 63.09 (S, CH-
biotin), 65.20 (d, 2JP,C = 7.04 Hz, OCH2), 122.95 (d, 3JP,C = 4.53 Hz,
ArC), 124.74 (s, ArC), 127.16 (s, ArC), 127.45 (s, ArC), 146.52 (s,
ArC), 156.67 (d, 2JP,C = 9.15 Hz, ArC–O), 165.81 (s, NHC(O)NH),
174.98 (s, C(O)NH); 31P-NMR (162 MHz, acetone-d6) d 27.16
(s). MALDI-TOF (m/z): 582.54 (calcd. M + H+: 582.18), 604.47
(calcd. M + Na+ : 604.17), 620.43 (calcd. M + K+ : 620.28); anal.
calcd. for C23H32N7O7PS·(H2O)0.5: C, 46.77; H, 5.63; N, 16.60;
found: C, 46.94; H, 5.43; N, 16.03%.
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exhibited first order kinetics, providing an accurate inhibition rate
constant for the reaction of CVL with 4a.
The molecular weights of cutinase–3, cutinase–4a and cutinase–
4b in 150 mM NH4OAc buffer were determined with ESI-MS
spectrometry (see ESI†).
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Fluorescence spectroscopy of PNP phosphonate probe 4a in
various solvents
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5 lM solutions of 4a in various solvents were prepared and their
emission spectra from 400–580 nm at 290 nm excitation were
measured (see ESI for spectra†). The following solvents were used
in decreasing order of polarity: 150 mM NH4OAc, MeCN, CH2Cl2
and hexanes.
13 A first example in which an organometallic moiety was used as a
phasing tool to facilitate the elucidation of a protein crystal structure
was recently obtained. These results will be published elsewhere: L.
Rutten, B. Wieczorek, J.-P. B. A. Mannie, C. A. Kruithof, H. P. Dijkstra,
M. R. Egmond, M. Lutz, A. L. Spek, G. van Koten, P. Gros, R. J. M.
Klein Gebbink, submitted.
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Click reaction on cutinase–3 with propargyl dansylamide
To a 125 lM solution of cutinase–3 (1.0 mL) in 0.1% triton, 50 mM
tris buffer at pH 8.0 was subsequently added a 50 mM propargyl
dansylamide solution in DMSO (10 lL), a 1.0 mM TCEP solution
in H2O (20 lL), a 100 lL TBTA (tris(benzyltriazolemethyl)amine)
solution in DMSO (2.0 lL), a 1% SDS solution in H2O (10 lM)
and a 1.0 mM CuSO4 solution in H2O (20 lL). The resulting solu-
tion was incubated for 12 h at room temperature and the mixture
was subsequently analysed by SDS-PAGE and UV–Vis analysis.
Finally, the modified protein was purified by dialysis against
150 mM NH4OAc buffer and analysis by ESI-MS spectrometry
revealed the formation of cutinase–4a (Mw = 21070 Da).
15 T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin, Org. Lett.,
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The authors kindly wish to thank C. Versluis and Prof.
A. J. R. Heck of the Biomolecular Mass Spectrometry group in
the Department of Pharmaceutical Sciences at Utrecht University
for measuring the ESI-MS spectra of various protein structures.
NRSC-Catalysis is kindly acknowledged for financial support
(HPD).
Notes and references
18 The crystal structure data was downloaded from the RSCB Protein
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