Towards the identification of unknown neuropeptide precursor-processing enzymes: Design and synthesis of a new family of dipeptidyl phosphonate activity probes for substrate-based protease identification

Article abstract of DOI:10.1016/j.bmc.2010.09.066

Specific proteolytic processing of inactive precursors is an exquisite cellular mechanism that triggers the activation of numerous physiologic peptides and proteins. This process ensures the generation of biologically active peptides, such as many neurope

Full text of DOI:10.1016/j.bmc.2010.09.066

Bioorganic & Medicinal Chemistry 18 (2010) 8350–8355
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Towards the identification of unknown neuropeptide precursor-processing
enzymes: Design and synthesis of a new family of dipeptidyl phosphonate
activity probes for substrate-based protease identification
Eduard Sabidó a,b, Teresa Tarragó a, Ernest Giralt a,b,
⇑
a Institut de Recerca Biomèdica, Baldiri Reixach, 10-12, 08028 Barcelona, Spain
b Department de Química Orgànica, Universitat de Barcelona, Martí Franquès, 1-11, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Specific proteolytic processing of inactive precursors is an exquisite cellular mechanism that triggers the
activation of numerous physiologic peptides and proteins. This process ensures the generation of biolog-
ically active peptides, such as many neuropeptides and peptide hormones, in the appropriate cellular
compartments at the right time, and its failure leads to several pathological conditions. Identification
of the proteases involved in this limited proteolysis is, therefore, an essential step for the subsequent
establishment of new therapeutic targets. As a first effort along this line, we synthesized eight new dipep-
tidyl phosphonate activity-based probes and used them to explore the soluble proteome from mouse
brain and pituitary gland for substrate-based protease identification both by in-gel analysis and mass
spectrometry.
Received 22 March 2010
Revised 19 September 2010
Accepted 24 September 2010
Available online 7 October 2010
Keywords:
Neuropeptide
Proteases
Activity-based probes
Proteomics
Ó 2010 Elsevier Ltd. All rights reserved.
Phosphorus
1. Introduction
cleavage occurring at tryptophan, leucine and other amino acids.8
The neuropeptide-processing peptidases involved in this new
Many bioactive peptides and proteins (e.g., neuropeptides, cell
surface glycoproteins and proteolytic enzymes) are synthesized
as large inactive precursors, which require specific proteolytic pro-
cessing to yield the final active peptides.1,2 The synthesis of large
precursors and the need to undergo limited proteolysis to gain bio-
logical activity is a key cellular mechanism for the generation of
biologically active peptides in the appropriate cellular compart-
ments and at the pertinent moment. Many cellular processes, such
as cell–cell communication, immunity, and programmed cell
death, are regulated by the specific proteolysis of precursor pro-
teins, thus, converting proteases involved in these processes into
attractive targets for drug discovery.3
Neuropeptides and peptide hormones are among the most
intensively studied proteolytic-activated peptides, not only be-
cause of their involvement in a great number of physiological
processes,4,5 but also because many neuropeptide-processing pep-
tidases remain largely unknown. Classical biosynthetic cleavage of
neuropeptide precursors occurs at basic residues within the con-
sensus sequence Lys/Arg-(Xxx)n-Lys/Arg; with n = 0, 2, 4 or 6. This
cleavage is performed by a relatively small number of well-known
peptidases, including prohormone convertases and cathepsin L.6,7
However, the discovery and characterization of new neuropeptides
has led to the description of a new non-classical pathway with
non-classical pathway remain largely unknown, although a few
have recently been reported, such as proteins SKI-1/S1P, NARC-1,
PPE/ADAM-10 and endothelin-converting enzymes.9–13
Identification of these unknown proteases is an essential
requirement for the subsequent establishment of new therapeutic
targets that could restore physiological neuropeptide levels in
pathological situations. Several approaches have used broad-spec-
trum activity-based probes for the functional characterization of
serine and cysteine proteases.14,15 However, the development of
new highly selective probes16 based on natural substrates facili-
tates the identification of proteases and can, thus, lead to the
establishment of new therapeutic targets using substrate-based
protein identification. Recently, we demonstrated the possibility
of using substrate-based dipeptidyl phosphonates to selectively
monitor endogenous post-proline protease activity by mass spec-
trometry and in-gel analysis.17 Here, we expand on this initial ap-
proach by synthesizing eight new dipeptidyl phosphonate probes
and use them to explore mouse brain and pituitary gland proteo-
mes for substrate-based protease identification.
2. Results and discussion
2.1. Design and synthesis of activity-based probes
Probes were designed based on chromogranin B-derived neuro-
peptides originated by proteolytic cleavage at non-canonical
⇑
Corresponding author.
E-mail address: ernest.giralt@irbbarcelona.org (E. Giralt).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.066

E. Sabidó et al. / Bioorg. Med. Chem. 18 (2010) 8350–8355
8351
sites.18–20 Among the several neuropeptide sequences reported
from chromogranin B, we selected those sequences with an un-
known related protease and with a conserved cleavage site among
different mammalian species ( Fig. 1). Thus, the phosphonate
probes synthesized here included a dipeptidyl moiety containing
these amino acids placed in positions P1 and P2 of the selected
neuropeptide sequences (Fig. 2). The presence of this dipeptidyl
moiety should enrich samples with proteases that recognize a cer-
tain amino acid combination and this should facilitate the identifi-
cation of the proteases involved in the processing of the selected
sequences.
In addition to the dipeptidyl moiety, the synthesized probes
also included a phosphonate functional group in the C-terminus
to bind the active site of serine proteases, and an alkyne functional
group introduced in the N-terminus to allow fluorescent TriN3-tag
conjugation21 by Cu(I)-catalyzed Huisgen [3+2] cycloaddition.22,23
To synthesize the dipeptidyl phosphonate probes, leucine- and
tryptophan-derived diphenyl 1-aminoalkanephosphonates 4 and
9 were previously synthesized as outlined in Scheme 1. Briefly,
compound 4 was obtained by an
as described previously24 (Scheme 1A) while 9 was synthesized
through a Michaelis–Arbuzov reaction and an -carbon alkylation
a-amidoalkylation in acetic acid
a
(Scheme 1B). Initially, previously synthesized ethyl diphenyl phos-
phite25 was used in a 5-day Michaelis–Arbuzov reaction to afford 5
(35%). The yield was reduced with shorter reaction times and no
significant increase was detected with longer reaction times.
Deprotection of the amine-protecting group with hydrazine gave
6 in good yield (74%) and the subsequent imine formation afforded
7 in 45% yield. The tryptophan analog 9 was finally prepared by
alkylation of 7 with KHMDS, HMPA and 3-indolylmethyl bromide
(8, 27%) followed by a final
a-nitrogen deprotection with TFA
and H2O in DCM as a dark oil (9, 90%).
Probe 1d was synthesized from 4 by direct peptide elongation
in solution whereas a convergent strategy was used for synthesiz-
ing probes 1a–c (Scheme 2). In the synthesis of probes 1a–c, com-
pounds 12a–c were obtained by solid-phase peptide synthesis and
subsequently coupled in solution to 4 or 9 using a polymer-bound
N,N0-dicyclohexylcarbodiimide to give 13a–c. The removal of the
side chain protecting groups of compounds 13a–c resulted in
probes 1a–c whereas for the synthesis of probes 2a–c transesteri-
fication (14a–c) was introduced before deprotection. Probe 2d was
directly obtained by transesterification of probe 1d.
Figure 2. Structure of the synthesized dipeptidyl phosphonate probes Aha-Glu-
LeuP(OPh)2 (1a), Aha-Glu-LeuP(OEt)2 (2a), Aha-Arg-LeuP(OPh)2 (1b), Aha-Arg-Leu-
P(OEt)2 (2b), Aha-Asp-TrpP(OPh)2 (1c), Aha-Asp-TrpP(OEt)2 (2c), Aha-Ala-LeuP(OPh)2
(1d), and Aha-Ala-LeuP(OEt)2 (2d).
2.2. Proteome exploration
The synthesized phosphonate probes were used for the func-
tional characterization of mouse soluble brain and pituitary gland
proteomes to detect those serine proteases that specifically recog-
nize the dipeptidyl moiety of the probes. First, both active and
heat-denatured proteomes were incubated with probes 1a–d and
2a–d (25 lM and 100 lM, 1 h at 25 °C). Samples were labeled
using the trifunctional fluorescent TriN3-tag (Supplementary
Fig. 1), and were analyzed by SDS–PAGE and visualized with a fluo-
rescence scanner. This in-gel approach allowed the analysis of mul-
tiple tissue-probe combinations in short amounts of time and
revealed the detection of several activity-based bands for the dif-
ferent phosphonate probes (Fig. 3 and Supplementary Figs. 2–9).
The most promising tissue–probe combinations from the in-gel
analyses were therefore selected for further characterization by
MudPIT analysis in order to identify those proteins specifically la-
beled by the dipeptidyl phosphonate probes. After incubating (1 h
at 25 °C) brain and pituitary gland homogenates with each phos-
phonate probe (25 lM), probe-protein complexes were labeled
with the TriN3-tag, and removed from the sample by means of avi-
din beads. The pulled-down proteins from both replicates were
analyzed by reverse-phase chromatography and subsequently
identified by MS/MS. The resulting data set was analyzed with
the SEQUEST software26 using the mouse IPI database.27 Protein
abundance was estimated from spectral counts28 and the maxi-
mum false-positive rate was set to 1% using a random coiled data-
base and the DTASelect software.29 A heat-denatured sample was
Figure 1. Multiple alignment of diverse chromogranin B-derived peptides. The
black bars highlight the amino acids in positions P1 and P2 of the cleavage site
while numbers refer to amino acid residues in the mouse chromogranin B sequence.

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E. Sabidó et al. / Bioorg. Med. Chem. 18 (2010) 8350–8355
Scheme 1. Reagents and conditions: (A) synthesis of H-LeuP(OPh)2 (4): (a) acetic acid, 1 h at 25 °C and 16 h at 50 °C; (b) HBr in acetic acid, 1 h at 25 °C. Neutralized with
triethylamine. (B) Synthesis of H-TrpP(OPh)2 (9): (a) POEt(OPh)2, xylene, reflux, 5 days; (b) hydrazine in THF, 72 h at 70 °C; (c) benzophenone in toluene, 12 h, reflux (Dean
Stark); (d) HMDA, KHMDS in THF, 30 min at ꢀ78 °C; (e) t-butyl-3-bromomethylindole-1-carboxylate in THF, 4 h at ꢀ78 °C; and (f) H2O (1%), TFA (3%) in DCM, 1 h at 25 °C.
Scheme 2. Reagents and conditions: (a) HATU, DIEA and Boc-Ala-OH in DMF, 16 h at 25 °C; (b) 4 M HCl in 1,4-dioxane, 30 min from 0 to 25 °C. Neutralization with
triethylamine; (c) HATU, DIEA and 5-hexynoic acid in DMF, 16 h at 25 °C; (d) polymer-bound N,N0-dicyclohexylcarbodiimide in DMF, 24 h at 25 °C; (e) removal of side-chain-
protecting groups with TFA–H2O (19:1), 1.5 h at 25 °C; (f) transesterification with KF, 18-crown-6 ether in ethanol, 10 min at 80 °C, 16 h at 25 °C; and (g) Removal of side-
chain-protecting groups with TFA–H2O (19:1), 1.5 h at 25 °C.
used as negative control to identify non activity-dependent probe-
protein complexes. A second control consisting of samples incu-
bated without any probe was also performed.
lesterase N precursor was identified with ten peptides (>25% of the
sequence coverage), of which five were unique peptides, that is,
peptides belonging only to that protein: AISESGVVINTNVGK,
SFNTVPYIVGFNK, EGASEEETNLSK, HSLLPPVVDTTQGK, NPPETDP
TEHTEH (Supplementary Fig. 10). The 60 kDa serine hydrolase ob-
served cleaved both amide and ester bonds and has already been
described as a surfactant convertase and a carboxylesterase.30,31
However, the putative involvement of the identified serine hydro-
lase in neuropeptide processing needs to be experimentally con-
firmed. Further work is required for the identification of the
other bands observed during in-gel analyses.
The MudPIT analysis resulting from incubation of the pituitary
gland homogenates with the phosphonate probe 1d led to the
identification of the liver carboxylesterase N precursor, a 60 kDa
serine hydrolase (IPI00138342; P23953; ESTN_MOUSE) and its
predicted counterparts (Table 1). In these assays, the liver carboxy-
lesterase N precursor entries were, together with a phosphoglycer-
ate kinase (IPI00230002, IPI00555069), the only proteins detected
with an over eight-fold sample/control ratio and present in both
sample replicates. All the other proteins detected were either pres-
ent in the sample and at least one control (<eight-fold) or had less
than five spectral counts (Supplementary Table). The liver carboxy-
These results constitute not only a further step towards the
identification of unknown neuropeptide-processing enzymes, but
also confirmation that dipeptidyl phosphonate probes are valuable

E. Sabidó et al. / Bioorg. Med. Chem. 18 (2010) 8350–8355
8353
Figure 3. (A) Specific bands detected in an activity-dependent manner when exploring the brain soluble proteome with probes 1a–d and 2a–d. Both active and heat-
denatured soluble proteomes were incubated with probes 1a–d and 2a–d (25 and 100 M, 1 h at 25 °C) and labeled using the trifunctional fluorescent TriN3-tag. Samples
l
were analyzed by SDS–PAGE and visualized with a fluorescence scanner. (B) Specific bands detected in an activity-dependent manner when exploring the pituitary gland
soluble proteome with probes 1a–d and 2a–d. Samples were prepared and analyzed as described for A).
Table 1
Spectral counts of proteins identified by MudPIT with an over eight-fold sample/control ratio and more than five
spectral counts
IPI numbera
(Mouse)
Sample
Control A
(Boiled sample)
Control B
(No. probe)
Sample/controls ratio
IPI00553586
IPI00677642
IPI00675322
IPI00678863
IPI00672511
IPI00671100
IPI00667966
IPI00664458
IPI00662318
IPI00138342
IPI00230002
IPI00555069
12.0 2.0
0.0 0.0
0.0 0.0
n.a.
8.0 1.0
6.0 1.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
n.a.
n.a.
a
IPI00138342: liver carboxylesterase
N precursor. IPI00555069, IPI00230002: phosphoglycerate kinase.
IPI00553586, IPI00677642, IPI00675322, IPI00678863, IPI00672511, IPI00671100, IPI00667966, IPI00664458,
IPI00662318: PREDICTED—Similar to liver carboxylesterase.
tools for substrate-based protease identification in complex sam-
ples and, eventually, for the identification of the proteases involved
in the proteolytic processing of chromogranin B-derived peptides.
der dipeptidyl phosphonate probes effective tools for substrate-
based protease identification, which can be used to screen complex
proteomes and to pinpoint unknown neuropeptide-processing
enzymes.
3. Conclusion
4. Experimental section
4.1. Synthesis
A new family of dipeptidyl phosphonate activity-based probes
for substrate-based identification of new proteases is reported.
These probes were designed based on chromogranin B-derived
neuropeptides originated by proteolytic cleavage at non-canonical
sites and used for exploring the soluble proteome from mouse
brain and pituitary gland. An efficient synthetic methodology for
the preparation of these probes was developed. The in-gel analysis
revealed several putative candidates that were detected by the
synthetic probes in an activity-dependent manner. Preliminary
mass spectrometry analysis of these candidates allowed the iden-
tification of a 60 kDa serine hydrolase (IPI00138342; P23953;
ESTN_MOUSE) as a protease putatively involved in the proteolytic
processing of chromogranin B-derived peptides. These results ren-
For detailed probe synthesis and characterization the reader is
referred to the Supplementary data.
4.2. In-gel analysis
4.2.1. Preparation of soluble brain and pituitary gland proteomes
Brain and pituitary gland homogenates were obtained from
adult male mice (BALB/c, 8 weeks old). Both tissues were frozen
after extraction and homogenized using 8 ml (brain) and 2 ml
(pituitary gland) of PBS (pH 7.4) and a tight douncer homogenizer

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E. Sabidó et al. / Bioorg. Med. Chem. 18 (2010) 8350–8355
from Wheaton Science Products (Millville, NJ USA). Homogenates
were centrifuged on a bench centrifuge (5 min, 4 °C, 2000 rpm)
and on an ultracentrifuge (1 h, 4 °C, 100,000g). Pellets were dis-
carded and the supernatant was collected to obtain the soluble
proteome. The total protein content was quantified with the Bio-
Rad Protein Assay (Bio-Rad, Hercules, CA, USA) using bovine serum
albumin as standard. Aliquots of the brain and pituitary gland
homogenates were immediately prepared and stored at ꢀ80 °C.
column (C18 Symmetry 300, 5 lm, Waters) and analyzed in a
10 cm Biphasic PicoFrit column (PF7515-04CMSCX-06CMPP2300,
New Objective) packed with C18 reverse-phase material and
strong cation exchange material. An automated five-step chroma-
tography was performed for each sample. The first step of each
run consisted of a 55 min gradient from 0% to 45% buffer B,
10 min gradient from 45% to 100% buffer B and a 20 min hold at
100% buffer B. The following steps were 112 min each with the fol-
lowing profile: 3 min of 100% buffer A, 2 min of X% buffer C, 1 min
of 95% buffer A, a 10 min gradient from 5% to 15% buffer B, a 45 min
gradient from 15% to 25% buffer B, and a 52 min gradient from 25%
to 55% buffer B. The 2 min buffer C percentages (X) in steps 2–5
were as follows: 25%, 50%, 80% and 100%. Buffer A: 95% H2O, 5%
MeCN and 0.1% formic acid. Buffer B: 20% H2O, 80% MeCN and
0.1% formic acid. Buffer C: 500 mM ammonium acetate, 95% H2O,
5% MeCN and 0.1% formic acid.
4.2.2. Activity-dependent labeling experiments
Probes 1a–d and 2a–d (25 and 100
either pituitary gland (0.5 mg mlꢀ1
(1 mg mlꢀ1) or a mixture of both, and samples were gently mixed
during 1 h at room temperature (total volume = 50 l). One micro-
liter of the TriN3-tag (5 mM in DMSO) was then added, followed by
l of freshly prepared tris-(2-carboxyethyl)-phosphine (50 mM
in H2O), 3 l of tris(triazolyl)amine (1.7 mM in DMSO/t-butanol
(1:4)) and 1 l of CuSO4 (50 mM in H2O). Samples were incubated
lM final) were added to
)
or brain homogenates
l
1
l
l
l
MS/MS data were analyzed with the SEQUEST software26 using
the mouse IPI database version 3.2327 from the European Bioinfor-
matics Institute and protein abundance was estimated from spec-
tral counts.28 The maximum false-positive rate was set to 1% using
a random coiled database and the DTASelect software.29
at 25 °C for 1 h and were intermittently vortex-mixed. Reaction
products were analyzed by SDS–PAGE (20 ꢁ 20 cm, 1.5 mm, Dual
Gel P10DS-1 Emperor Penguin from Thermo Fisher Scientific Inc.,
Waltham, MA USA) and visualized on a fluorescence scanner.
Heat-denatured samples (D) were inactivated (5 min at 95 °C) be-
fore adding the phosphonate probe to be used as a negative
Acknowledgments
control.
This work was supported by MCYT-FEDER (Bio2008-00799 and
Generalitat de Catalunya (XRB and Grup Consolidat)). E. Sabidó
was supported by a Grant from the ‘‘Ministerio de Educación
y Ciencia”, Spain.
4.3. Mass spectrometry assays
4.3.1. Sample preparation
Brain homogenates (1 mg mlꢀ1) were incubated (1 h at 25 °C)
Supplementary data
with peptidyl phosphonate probes 1a–d and 2a–d (25
volume = 500 l). TriN3-tag (11.6 l, 5 mM in DMSO) was subse-
quently added, followed by freshly prepared tris-(2-carboxy-
ethyl)-phosphine (11.6 l, 50 mM in H2O), tris-(triazolyl)amine
(35 l, 1.7 mM in DMSO/t-butanol (1:4)) and CuSO4 (11.6 l,
lM) (total
l
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.066.
l
l
l
References and notes
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methanol was repeated twice. 1.2% SDS in PBS (1 ml) were added
to the pellet and samples were sonicated briefly prior to sample
heating (5 min at 95 °C) and dilution with 0.2% SDS in PBS (5 ml).
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