Metallo-Organozymes with Specific Proteolytic Activity

Article abstract of DOI:10.1002/chem.201803666

Metallopeptides that show efficiency and selectivity in peptide bond cleavage in water at room temperature and neutral conditions are presented. These small and versatile organozymes take advantage of metal-coordinating building blocks that are strategica

Full text of DOI:10.1002/chem.201803666

A Journal of
Accepted Article
Title: Metallo-Organozymes with Specific Proteolytic Activity
Authors: Ahmed Embaby, Lianne P. W. M. Lelieveldt, Frederik Diness,
and Morten Meldal
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Metallo-Organozymes with Specific Proteolytic Activity
Ahmed M. Embaby,[a], [c] Lianne P. W. M. Lelieveldt,[a], [b], [c] Frederik Diness[a] and Morten Meldal[a]*
Abstract:
Metallopeptides that show efficiency and
of metal binding peptides that hydrolyse the very constrained
and labile ampinicillin amide bond[6] or 4-nitrophenyl acetate[6-10]
were developed by design[11] or combinatorial selection,[12] but
there are only few reports on synthetic catalysts which can
challenge the corresponding enzymes in hydrolysis of peptide
bonds of peptide substrates. Zinc, iron and copper are abundant
metals in biocatalysis.[13] Zinc is the catalytic metal found
naturally in metallo-proteases and dehydrogenase,[14] iron is
present in redox centers, and is involved in catalase activity[15]
while copper appears in oxidases. The metal selected for
peptide catalysts has mainly been copper, also known to
facilitate reaction with oxygen (ROS).[16-18] However more exotic
metals have also been shown to affect hydrolysis of peptide
bonds.[19;20] Strangely Zn2+ was rarely used in catalysis. Co3+-
binding DOTA and tetramine cryptants conjugated to targeting
peptides have been synthesized for DNA nicking and peptide
cleavage.[21;22] In these examples, targeting and catalyst function
are at separate locations.
selectivity in peptide bond cleavage in water at room
temperature and neutral conditions are presented. These small
and versatile organozymes take advantage of metal coordinating
building blocks that are strategically positioned centrally in a
peptide backbone or in a peptide macrocycle. This approach
provided peptide-metal complexes with scaffolds capable of
utilizing the peptide functionality for productive binding of
fluorogenic FRET peptide substrates, subsequently leading to
highly selective peptide bond cleavage. The ligand chemistry
has been optimized to provide an easy access to new metallo-
peptides with the ability to cleave previously inaccessible
peptide bonds. Evolutionary principles of stepwise selection and
variation offered by combinatorial methods were used and were
guided by molecular modelling to develop catalytic metallo-
peptides that mimic metalloproteases.
Enzymes are Nature’s most efficient catalysts that can
accelerate chemical reactions up to 1017 fold with high chemical,
regio- and stereo selectivity.[1] Enzyme catalysed chemical
transformations are recognized as practical alternatives to
conventional organic synthesis in facing many industrial
challenges. Hence, enzymes are now being used in numerous
industrial applications within fermentation, biotechnology and
chemical transformations e. g. for the production of the
sweetener aspartame and semi-synthesis of penicillin or
herbicides.[2] However, their instability, high cost, limited
availability and poor activity in organic solvents are still major
challenges for their broader application.[3] Moreover, target-
The aim of the present study was to combine molecular
recognition and catalyst function to develop metallo-catalytic
peptides that form productive substrate binding and hydrolyse
peptide bonds in substrates at room temperature and neutral pH.
For efficient catalysis and strong metal binding, the design of
such catalyst requires a metal coordinating ligand, preferably
embedded in a peptide backbone. In Nature, metalloproteases
exploit the architecture of the protein to have the amino acid side
chains positioned to create the most suitable coordination
environment to the metal centre. Our catalyst design considered
flexibility, bite angle, metal affinity, ligand incorporation in the
peptide, and access of peptide functionality to the catalyst centre.
selective peptide catalysts are emerging as
a potential
alternative to conventional drugs. They are required in tiny
amounts and they may be identified without information about
the target protein.[4] In this perspective, protein-cleaving drugs
would be particularly advantageous. Non-catalysed hydrolysis of
peptide bonds is extremely slow, on average, it proceeds with a
half-life of about 500 and 600 years at neutral pH and 25 °C for
a C-terminal and an internal peptide bond, respectively.[5]
Natural proteases have been developed through extensive
molecular evolution to affect efficient peptide cleavage. An array
[a]
A. M. Embaby , L. P. W. M. Lelieveldt , Assoc. Prof. Dr. F. Diness,
Prof. Dr. M. Meldal
Center for Evolutionary Chemical Biology and Nano-Science center
Department of Chemistry, University of Copenhagen
Universitetsparken 5, DK2100 Copenhagen, Denmark
* E-mail: meldal@nano.ku.dk
Current address: Department of Biomolecular Chemistry, Radboud
University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The
Netherlands
Figure 1. Molecular modelling presents a conformationally stable peptide
macrocycle 4 with phenanthroline ligand, in orange, and coordinated Zn2+, 22,
in blue. This catalyst binds with electrostatic surface complementarity and high
specificity to the substrate Y'-Q-L-R-L↓H-K''-G-OH (35) and forms a stable
complex and a further stabilized transition state of hydrolysis of the L-H-
peptide bond.
[b]
[c]
A. M. Embaby and L.P.W.M Lelieveldt contributed equally to this
work.
We selected two metal coordinating heterocycles 2,2’-
bipyridine (Bpy) and 1,10-phenanthroline (Phen) for this study.
As previously described Bpy ligands can be attached to the N-
terminus of the peptide[23] or to a side chain of an unnatural
amino acid.[24] Bpy ligands have also previously been
Supporting information for this article is given via a link at the end of
the document.
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incorporated directly in the peptide chain.[25;26] Bpy ligands for
peptide incorporation were functionalized with both an amine
and a carboxylic acid group, however, often on the same
pyridine ring. The extended conformation induced by a Bpy
residue is favorable and similar to extended structures in
proteins.[27] Phen derivatives have been less prevalent in peptide
ligands.
Of great importance for catalyst development is the
introduction of amino acid residues that facilitate interaction
between the metal, the peptide ligand and the target substrate,
thereby providing binding and catalytic potential. Molecular
modelling (see supporting information) guided our design of
peptide sequences that could incorporate stable metal ligands in
the backbone and putatively recognize potential substrates
including a core-sequence of Alzheimer A-1-40 (Figure 2). Four
catalytic peptides containing either a Bpy or a Phen ligand
(Figure 1 and 2) were designed by MD-calculations starting from
the metal coordinating core and building the catalyst structure in
an iterative process of structure extension and annealing. For
instance, peptide 1 provided auxiliary carboxylate and imidazole
positioned for metal coordination (Figure 2A), while calculation
started from the crystal structure of the core sequence of
Alzheimer A 1-40 fibrils (Figure 2B and C) used as a template
for stepwise growth of catalyst peptide 2.
Scheme 1. A) Structure of peptide ligands 1, 2 and building block 5. B) SPPS
and incorporation of Phen 6 into peptide 3 and 4: a) Phen-derivative 6 in 2 %
acetic acid in DMF, NaBH(OAc)3; b) 0.1M NaOH, 1 h.
A)
The four catalyst ligands (1, 2, 3 and 4) were synthesized,
coordinated with selected transition metals, and screened
against
a FRET-based combinatorial substrate library that
contained approximately 5 x 105 peptide sequences. This
provided information on proteolytic activity and selectivity that
could not easily be identified by other methodologies.
B)
C)
Synthesis of bipyridyl building block (Ppy)
5
and
phenanthroline building block (Phen) 6[28] is described in the
supporting information and Scheme SI-1. Bpy containing
peptides 1 and 2 (Scheme 1) provided histidines positioned for
metal coordination (1) or was designed (2) to bind to A-1-40.
Phen containing peptides 3 and 4 (Scheme 1) were also
designed using molecular modelling for the formation of an
appropriately sized Phen macrocycle with a cavity for metal
binding. The peptide contains two non-natural Dap–residues
spaced apart by a glutamine. Both Phen and Bpy coordinate
transition metals, while Phen presents significantly stronger
coordination than Bpy. However, Phen derivatives are inherently
insoluble, so incorporation of Phen in SPPS required a suitable
synthetic approach. Syn-orientation of coordinating nitrogen
atoms in the 2,9 bisaldehyde Phen 6, allowed incorporation into
peptides by reductive alkylation using two diaminopropionic acid
(Dap) side chains of a peptide to provide a macrocycle and a
cavity suitable for binding and stabilizing a metal centre.
Figure 2. Design of catalyst ligand 1 and 2. A) Compound
1 has an
octrahedral coordination sphere filled with Bpy and four carboxylates. B) The
crystal structure of A-1-40, with the core sequence 14-25 in yellow. C) A
FRET based A 14-25 substrate (Y’’HQKLVFFAEDVK´G) presenting the
important Phe residues in interaction with compound 2. The substrate
maintains the original conformation from the crystals structure during MD-
calculations (SI).
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Peptide ligands 1, 2, 3 and 4 were synthesized (Scheme 1
indication that catalyst 20 has some preference for arginine at
and SI-2)
on hydroxymethylbenzoic
amide (HMBA)
the P1´site.
functionalized PEGA800 solid support 7 by standard TBTU / Fmoc
protocol.[29;30] The resin was esterified with Fmoc-glycine using
MSNT N-MeIm conditions. In peptides 1 and 2, The azide of the
Bpy building block was reduced to an amine using dithiotreitol
(DTT) and N,N-diisopropylethylamine (DIPEA).[31;32] The
peptides were deprotected with a mixture of TFA, H2O and TIPS.
Subsequently it was cleaved off the resin with aqueous sodium
hydroxide. In peptide 3 and 4, resin-bound and Fmoc protected
8 and 9 provided two primary amines for reductive alkylation. By
A)
portion-wise addition of
6 and NaBH(OAc)3, macrocyclic
peptides 3 and 4 were obtained upon cleavage with NaOH.
Metal coordination of Zn(Ac)2·2H2O, CuBr and FeCl2·4H2O
with the purified peptides 1, 2, 3 and 4 was performed in EtOH
solution at 50 ºC for 2 h and products were confirmed by MALDI
FT-ICR mass spectrometry (Complexes 10-23, Table SI-2). The
ability of catalysts 11, 13, 14, 15, 20 and 21 towards cleavage of
peptides was confirmed with a FRET substrate solid phase
library 24 (Scheme SI-3).[33] Prior to these studies, any beads
showing background fluorescence were removed. Subsequent
inspection under a fluorescence microscope before hydrolysis
showed that all beads were dark (Figure 3A), indicating that
peptide sequences were intact. Upon addition of the catalysts
(250 µM) initiation of substrate cleavage was observed after only
30 minutes. The activities of these catalysts are illustrated in
Figure 3A, showing strongly fluorescent beads after 96 hours of
cleavage. The brightest beads were isolated at 16, 48 and 96 h
from each catalyst screen and the residual peptide substrates
and cleavage products were cleaved off using 5 % triethyl amine
in water and characterized by MS (Figure 4 and SI-03 -29).
B)
To access selectivity of cleavage in the remaining library the
progress of hydrolysis was monitored by fluorescence
microscopy over a period of 1 year at 4 oC. Interestingly, most of
the library (~90%) remained dark indicating that cleavage was
very sequence dependent and that peptide bond cleavage was
quite selective (Figure 3B). The untreated control substrate
library was unchanged during this period. Eleven substrate
sequences and their cleavage sites were identified by de novo
sequencing using ultra high resolution mass spectrometry (Table
1 and SI-MSMS spectra). The MS spectra clearly showed the
presence of at least two major peaks that correspond to the
intact and the cleaved peptides (Figure 4). These major peaks
were isolated in the MS-quadrupole and fragmented by MS-MS
with CID up to 50 eV for sequencing.
Figure 3. Hydrolysis of peptides by catalysts (compound numbers indicated)
in a fluorescently quenched peptide substrate library, 24. A) Active beads with
cleaved substrates after 96 h of incubation at 20 oC. B) The increase in
number of cleavages observed after 360 days at 4 oC. The library with catalyst
14 in B is shown with magnification of both 1.4 and 10 x indicating the extent
of library hydrolysis.
Figure 4 shows a typical MS analysis of an active hit, 32 in
Table 1, collected from the screening well of catalyst 20. The MS
spectrum showed
a minor peak at m/z 1179.6213 that
corresponds to the intact peptide with the sequence Y'H-K-A-R-
R-K''G and a major peak at m/z 479.2747 that corresponds to
the cleaved peptide with the sequence R-K''G. In addition,
another very minor peak at m/z 635.37 corresponds to a second
cleaved peptide with the sequence R-R-K''G which gives the
Figure 4. Typical MS analysis of substrate 32 collected from screening well of
catalyst 20. The substrate 32 (m/z 1179.62) presents major selective cleavage
between the two arginines (m/z 479.27). The insert shows the the library with
the fluorescent bead containing substrate 32.
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substrate 32 with zinc complex 22 was also followed over 3
weeks by LC-MS confirming kinetics by appearance of the
cleaved products and the complete hydrolysis of the substrate
(FT-ICR-MALDI and LC-MS in supporting information). This was
observed in comparison to a control sample of the substrate 32
in buffer without addition of catalyst 22 showing no sign of
hydrolysis.
Other active hits, 34 and 35, collected from the screening
well of catalyst 21 after 3 days and also cleaved by 22 (Figure 1),
both showed two major peaks. Substrate 34 presented m/z
1057.5491 (Y'-A-Q-T-L-L-K''-G) and m/z 778.4361 (Q-T-L-L-K''-
G). Substrate 35 was cleaved by 22 and presented m/z
1177.5992 (Y'-Q-L-R-L-H-K''-G-OH) and m/z 737.3995 (Y'-Q-L-
R-L-OH) while Bpy-catalysts 11-15 mainly removed the
nitrotyrosine. Molecular modelling (Figure 1, SI-1 and SI-2) of
the catalyst, 22 in complex with the substrates 32, 34 and 35
indicated stable complexes with excellent recognition due to
The hydrolysis fragments of substrates 34 and 35 were also
identified by FT-ICR-MALDI after 60 h. Although hydrolysis with
subtilisin was much faster (complete hydrolysis in few minutes),
it seemed less specific than that of the Zn2+ complex 22
according to MS of the cleavage products. Catalysts 23
provided only insignificant cleavage during the time course of
the assay.
Table 1. Cleavage of peptide substrates with catalysts. The substrates in an
octapeptide library with five varied positions were cleaved by catalysts, 11, 13,
14, 15, 20 and 21 and cleavage products identified by MS-MS. The cleavage
site is indicated and entries 8, 10 and 11 were performed in solution.
Entry
Catalyst
Substrate
1
2
11
13
14
14
14
14
15
20
20
21
21
Bpy-1 Fe2+
25
26
27
28
29
30
31
32
33
34
35
Y'↓H-L-K-K-R-K''-G-OH
Y'-V-Y↓L-D-Y-K''-G-OH
Y'↓L-P-V-F-H-K''-G-OH
Y’↓W-S-T-R-K-K’’-G-OH
Y’-K↓L-R-A-F-K’’-G-OH
Y'-R-M↓L-Q-K-K''-G-OH
Y‘↓F-R-Y-D-H-K''-G-OH
Y’-H-K-A-R↓R-K’’-G-OH
Y’-L-S-K↓K-A-K’’-G-OH
Y'-A↓Q-T-L-L-K''-G-OH
Y'-Q-L-R-L↓H-K''-G-OH
Bpy-1 Zn2+
Bpy-2 Cu2+
Bpy-2 Cu+
Bpy-2 Cu+
Bpy-2 Cu+
Bpy-2 Fe2+
Phen-4 Cu +
Phen-4 Cu +
Phen-4 Fe2+
Phen-4 Fe2+
3
4
5
6
7
8
9
10
11
Figure 5. Hydrolysis of substrate 34 by catalysts 20 (Cu+), 21 (Fe2+), 22 (Zn2+),
and 23 (Co3+). Complete hydrolysis with subtilisin (2 * 10-6 M,) result in 63000
AU. Substrate 34 background is also shown. Data were corrected for
bleaching effects. Similar hydrolysis curves for substrates 32 and 35 are
presented in Figure SI-30&31.
electrostatic surface and charge complementarity.
This tight binding allowed the metal to coordinate with the target
carbonyl group and a water molecule to be located in proximity
for hydrolysis. Moreover, the transition state of the substrate
Y'AQTLLK''G is more tightly bound to catalyst 22 than the
substrate itself with two water molecules, stabilizing the
attacking hydroxyl group and the leaving amino group,
respectively (Figure 6). In contrast, catalysts 13 and 14 cleaved
as an endo-peptidase and the cleavage was slow and in many
instances they only removed the N-terminal nitrotyrosine.
In-solution hydrolysis of substrates 32, 34 and 35 with peptide 4
derived catalysts 20 (Cu+) 21 (Fe2+) 22 (Zn2+) 23 (Co3+) was
investigated. Treatment of the three Abz / Y(NO2) substrates
o
(1mM) with 22 (250 M) in 0.1 M tris buffer at pH=8 and 37 C
led to gradual increase in fluorescence (Figure 5 and SI-30&31)
clearly indicating hydrolysis as compared to the fast complete
substrate hydrolysis by subtilisin (2 M). Particularly the zinc
complex 22 showed excellent activity. The hydrolysis of
Figure 6. Molecular modelling presenting the tetrahedral intermediate of
substrate 34, Y'-A↓Q-T-L-L-K''-G-OH tightly bound to catalyst 22.
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In conclusion, we have developed novel peptide bond
cleaving catalysts via a synthetic strategy for Bpy and Phen
building blocks suitable for incorporation by SPPS. The
compounds were readily incorporated (5) or attached (6) to a
designed peptide chain. After metal coordination and
subsequent addition to a FRET-based combinatorial library, the
catalysts showed selective proteolytic activity within few hours.
Using this approach novel metal coordinated peptides, or
metallo-organozymes, could be synthesized. The principles
outlined here can be used for optimization of selective catalysts
drug candidates to combat pathogens or catalysts for
bioprocessing. In addition to peptide cleavage, such catalysts
may have activity in a wide range of other reactions including
those of organic chemistry, as revealed through further studies
of these unique compounds. The present results open many
opportunities which we are currently pursuing.
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Detailed description of the synthetic procedures, LC-MS
1
chromatograms and H- and 13C-NMR and HRMS are provided
in the supporting information.
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Acknowledgements The authors would like to kindly
acknowledge Dr. Sanne Schoffelen and Dr. Ming Li for assistance with mass
spectrometry.
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Keywords: Organometallic catalysis
• Combinatorial chemistry • N-
Heterocyclic ligands • Peptide mimetics • Catalytic drugs.
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Metallopeptides that cleave other
Ahmed M. Embaby, Lianne P.W.M.
peptides
combinatorial
were
identified
by
Lelieveldt , Frederik Diness and Morten
Meldal*
on-resin
selection.
These “organozymes”, containing
phenanthroline metal binding sites,
like proteases, cleave FRET peptide
Page No. – Page No.
Metallo-organozymes with specific
proteolytic activity
((Insert TOC Graphic here))
substrates
with
high
substrate
specificity. The cleavage involves
formation of a tight binding transition
state of hydrolysis. The principles
outlined for organozymes design is a
platform for development of catalytic
drugs, for processing pathogens.
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