Pedro M. S. D. Cal

Iminoboronates: a new strategy for reversible protein modification.

Protein modification has entered the limelight of chemical and biological sciences, since, by appending small molecules into proteins surfaces, fundamental biological and biophysical processes may be studied and even modulated in a physiological context. Herein we present a new strategy to modify the lysines ε-amino group and the proteins N-terminal, based on the formation of stable iminoboronates in aqueous media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addition of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the observed stability toward hydrolysis of the iminoboronate constructs.

NHC−Iron-Catalyzed Aerobic Oxidative Aromatic Esterification of Aldehydes using Boronic Acids

NHC-iron complexes prepared in situ very efficiently afforded benzoates via the aerobic oxidative aromatic esterification of aldehydes with boronic acids. This method uses equimolar amounts of both the aldehyde and the boronic acid allowing the preparation of benzoates in yields up to 97%.

Cysteine-Selective Reactions for Antibody Conjugation†

Moving tracks from maleimide: New site-selective protein modification reactions at cysteine have been developed. Unlike conventional maleimide conjugation, which results in a labile thioether succinimide, the new bioconjugation reactions result in stable conjugates and provide opportunities to develop a new generation of homogeneous, stable, and therapeutically useful conjugates.

Discovery of new heterocycles with activity against human neutrophile elastase based on a boron promoted one-pot assembly reaction

Herein we demonstrate for the first time that a boron promoted one-pot assembly reaction may be used to discover novel enzyme inhibitors. Inhibitors for HNE were simply assembled in excellent yields, high diastereoselectivities and IC50 up to 1.10 μM, based on components like salicylaldehyde, aryl boronic acids and amino acids. The combination of synthetic, biochemical, analytical and theoretical studies allowed the identification of the 4-methoxy or the 4-diethyl amino substituent of the salicylaldehyde as the most important recognition moiety and the imine alkylation, lactone ring opening as key events in the mechanism of inhibition.

Reversible lysine modification on proteins by using functionalized boronic acids.

Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC50  value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells.

Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications.

Nickel-Catalyzed Azide–Alkyne Cycloaddition To Access 1,5-Disubstituted 1,2,3-Triazoles in Air and Water

Transition-metal-catalyzed or metal-free azide-alkyne cycloadditions are methods to access 1,4- or 1,5-disubstituted 1,2,3-triazoles. Although the copper-catalyzed cycloaddition to access 1,4-disubstituted products has been applied to biomolecular reaction systems, the azide-alkyne cycloaddition to access the complementary 1,5-regioisomers under aqueous and ambient conditions remains a challenge due to limited substrate scope or moisture-/air-sensitive catalysts. Herein, we report a method to access 1,5-disubstituted 1,2,3-triazoles using a Cp2Ni/Xantphos catalytic system. The reaction proceeds both in water and organic solvents at room temperature. This protocol is simple and scalable with a broad substrate scope including both aliphatic and aromatic substrates. Moreover, triazoles attached with carbohydrates or amino acids are prepared via this cycloaddition.

Intramolecular C–H insertion catalyzed by dirhodium(II) complexes using CO2 as the reaction media

Abstract In this work, the intramolecular C–H insertion of diazoacetamides catalyzed by dirhodium(II) complexes and using CO2 as solvent is disclosed. The expected lactams were obtained in yields over 97%. The asymmetric intramolecular C–H insertion was also achieved and the β-lactam 14 was obtained in >97% yield and 65% ee using the chiral dirhodium(II) catalyst Rh2(S-PTTL)4. Finally, the dirhodium(II) complex Rh2(OAc)4 was used in two consecutive cycles in which complete conversion to the lactam was observed.

Unveiling (-)-Englerin A as a Modulator of L-Type Calcium Channels.

Abstract The voltage‐dependent L‐type Ca2+channel was identified as a macromolecular target for (−)‐englerin A. This finding was reached by using an unprecedented ligand‐based prediction platform and the natural product piperlongumine as a pharmacophore probe. (−)‐Englerin A features high substructure dissimilarity to known ligands for voltage‐dependent Ca2+ channels, selective binding affinity for the dihydropyridine site, and potent modulation of calcium signaling in muscle cells and vascular tissue. The observed activity was rationalized at the atomic level by molecular dynamics simulations. Experimental confirmation of this hitherto unknown macromolecular target expands the bioactivity space for this natural product and corroborates the effectiveness of chemocentric computational methods for prioritizing target‐based screens and identifying binding counterparts of complex natural products.

Chemo- and Regioselective Lysine Modification on Native Proteins

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.