19 research outputs found
Discovery of a 29-Amino-Acid Reactive Abiotic Peptide for Selective Cysteine Arylation
The regio- and chemoselective modification
of proteins or peptides
with chemical reagents is often challenging. One approach to overcome
this problem involves identifying abiotic polypeptide sequences that
react with specific small molecules. Toward this goal, we profiled
∼5 × 10<sup>13</sup> randomized 30-mer peptides using
mRNA display and high-throughput sequencing in search of polypeptides
that can undergo cysteine arylation with a water-soluble perfluoroarene.
Within this vast chemical space, we discovered a cysteine-containing
sequence with a second-order rate constant of 0.29 M<sup>–1</sup> s<sup>–1</sup> for arylation. An N- and C-terminal truncation
reduced the reaction rate, as did the addition of denaturants. When
the reactive peptide was covalently fused to the enzyme Sortase A,
we observed regiospecific arylation at a single cysteine site, leaving
the enzyme’s active site cysteine unchanged. Taken together,
these results demonstrate that long polypeptides of defined sequence,
when matched with the appropriate reactive group, can be used for
selective arylation of cysteine in water
Water-Soluble Palladium Reagents for Cysteine <i>S</i>‑Arylation under Ambient Aqueous Conditions
We report the use of a sulfonated
biarylphosphine ligand (sSPhos)
to promote the chemoselective modification of cysteine containing
proteins and peptides with palladium reagents in aqueous medium. The
use of sSPhos allowed for the isolation of several air-stable and
water-soluble mono- and bis-palladium reagents, which were used in
an improved protocol for the rapid <i>S</i>-arylation of
cysteines under benign and physiologically relevant conditions. The
cosolvent-free aqueous conditions were applied to the conjugation
of a variety of biomolecules with affinity tags, heterocycles, fluorophores,
and functional handles. Additionally, bis-palladium reagents were
used to perform macrocyclization of peptides bearing two cysteine
residues
Palladium Oxidative Addition Complexes for Peptide and Protein Cross-linking
A new method for cysteine–lysine
cross-linking in peptides
and proteins using palladium oxidative addition complexes is presented.
First, a biarylphosphine-supported palladium reagent is used to transfer
an aryl group bearing an <i>O</i>-phenyl carbamate substituent
to a cysteine residue. Next, this carbamate undergoes chemoselective
acyl substitution by a proximal lysine to form a cross-link. The linkage
so formed is stable toward acid, base, oxygen, and external thiol
nucleophiles. This method was applied to cross-link cysteine with
nearby lysines in sortase A*. Furthermore, we used this method for
the intermolecular cross-linking between a peptide and a protein based
on the p53-MDM2 interaction. These studies demonstrate the potential
for palladium-mediated methods to serve as a platform for the development
of future cross-linking techniques for peptides and proteins with
natural amino acid residues
An Umpolung Approach for the Chemoselective Arylation of Selenocysteine in Unprotected Peptides
Herein
we report an umpolung strategy for the bioconjugation of
selenocysteine in unprotected peptides. This mild and operationally
simple approach takes advantage of the electrophilic character of
an oxidized selenocysteine (Se–S bond) to react with a nucleophilic
arylboronic acid to provide the arylated selenocysteine within hours.
This reaction is amenable to a wide range of boronic acids with different
biorelevant functional groups and is unique to selenocysteine. Experimental
evidence indicates that under oxidative conditions the arylated derivatives
are more stable than the corresponding alkylated selenocysteine
Macrocyclization of Unprotected Peptide Isocyanates
A chemistry
for the facile two-component macrocyclization of unprotected
peptide isocyanates is described. Starting from peptides containing
two glutamic acid γ-hydrazide residues, isocyanates can be readily
accessed and cyclized with hydrazides of dicarboxylic acids. The choice
of a nucleophilic linker allows for the facile modulation of biochemical
properties of a macrocyclic peptide. Four cyclic NYAD-1 analogues
were synthesized using the described method and displayed a range
of biological activities
Nitrogen Arylation for Macrocyclization of Unprotected Peptides
We describe an efficient and mild
method for the synthesis of macrocyclic
peptides via nitrogen arylation from unprotected precursors. Various
electrophiles and lysine-based nucleophiles were investigated and
showed high-yielding product formation, even for a macrocyclization
scan with 14 variants. We found that nitrogen-linked aryl products
were more stable to base and oxidation when compared to thiol arylated
species, thereby highlighting the utility of this methodology. Finally,
N-aryl macrocyclization was performed on a p53 peptide inhibitor of
MDM2 and resulted in identification of a nanomolar binder with improved
proteolytic stability and cell permeability
Protein Thioester Synthesis Enabled by Sortase
Proteins containing a C-terminal thioester are important
intermediates
in semisynthesis. Currently there is one main method for the synthesis
of protein thioesters that relies upon the use of engineered inteins.
Here we report a simple strategy, utilizing sortase A, for routine
preparation of recombinant proteins containing a C-terminal <sup>α</sup>thioester. We used our method to prepare two different anthrax toxin
cargo proteins: one containing an <sup>α</sup>thioester and
another containing a D-polypeptide segment situated between two protein
domains. We show that both variants can translocate through protective
antigen pore. This new method to synthesize a protein thioester allows
for interfacing of sortase-mediated ligation and native chemical ligation
A Perfluoroaryl-Cysteine S<sub>N</sub>Ar Chemistry Approach to Unprotected Peptide Stapling
We
report the discovery of a facile transformation between perfluoroaromatic
molecules and a cysteine thiolate, which is arylated at room temperature.
This new approach enabled us to selectively modify cysteine residues
in unprotected peptides, providing access to variants containing rigid
perfluoroaromatic staples. This stapling modification performed on
a peptide sequence designed to bind the C-terminal domain of an HIV-1
capsid assembly polyprotein (C-CA) showed enhancement in binding,
cell permeability, and proteolytic stability properties, as compared
to the unstapled analog. Importantly, chemical stability of the formed
staples allowed us to use this motif in the native chemical ligation-mediated
synthesis of a small protein affibody that is capable of binding the
human epidermal growth factor 2 receptor
Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase
Ras/Rap1-specific
endopeptidase (RRSP) is a cytotoxic effector
domain of the multifunctional autoprocessing repeats-in-toxin (MARTX)
toxin of highly virulent strains of <i>Vibrio vulnificus</i>. RRSP blocks RAS-MAPK kinase signaling by cleaving Ras and Rap1
within the switch I region between Y32 and D33. Although the RRSP
processing site is highly conserved among small GTPases, only Ras
and Rap1 have been identified as proteolytic substrates. Here we report
that residues Y32 and D33 at the scissile bond play an important role
in RRSP substrate recognition, while the nucleotide state of Ras has
an only minimal effect. In addition, substrate specificity is generated
by residues across the entire switch I region. Indeed, swapping the
Ras switch I region into either RalA or RhoA, GTPases that are not
recognized by RRSP, generated chimeras that are substrates of RRSP.
However, a difference in the processing efficiency of Ras switch I
in the context of Ras, RalA, or RhoA indicates that protein regions
outside Ras switch I also contribute to efficient RRSP substrate recognition.
Moreover, we show that synthetic peptides corresponding to the Ras
and Rap1, but not RalA, switch I regions are cleaved by RRSP, demonstrating
sequence-specific substrate recognition. In conclusion, this work
demonstrates that the GTPase recognition of RRSP is independent of
the nucleotide state and is mainly driven by the Ras and Rap1 switch I loop
and also influenced by additional protein–protein interactions,
increasing the substrate specificity of RRSP
Salt Effect Accelerates Site-Selective Cysteine Bioconjugation
Highly efficient
and selective chemical reactions are desired.
For small molecule chemistry, the reaction rate can be varied by changing
the concentration, temperature, and solvent used. In contrast for
large biomolecules, the reaction rate is difficult to modify by adjusting
these variables because stringent biocompatible reaction conditions
are required. Here we show that adding salts can change the <i>rate constant over 4 orders of magnitude</i> for an arylation
bioconjugation reaction between a cysteine residue within a four-residue
sequence (Ï€-clamp) and a perfluoroaryl electrophile. Biocompatible
ammonium sulfate significantly enhances the reaction rate without
influencing the site-specificity of π-clamp mediated arylation,
enabling the fast synthesis of two site-specific antibody–drug
conjugates that selectively kill HER2-positive breast cancer cells.
Computational and structure–reactivity studies indicate that
salts may tune the reaction rate through modulating the interactions
between the π-clamp hydrophobic side chains and the electrophile.
On the basis of this understanding, the salt effect is extended to
other bioconjugation chemistry, and a new regioselective alkylation
reaction at π-clamp cysteine is developed