Marcos Pires

Promotion of cell adhesion on biomaterials is crucial for the long-term success of a titanium implant. Herein a novel concept is highlighted combining very stable and affine titanium surface adhesive properties with specific cell binding moieties in one molecule. A peptide containing l-3,4-dihydroxyphenylalanine was synthesized and affinity to titanium was investigated. Modification with a cyclic RGD peptide and a heparin binding peptide (HBP) was realized by an efficient on-resin combination of Diels–Alder reaction with inverse electron demand and Cu^I catalyzed azide–alkyne cycloaddition. The peptide was fluorescently labeled by thiol Michael addition. Conjugating the cyclic RGD and HBP in one peptide gave improved spreading, proliferation, viability, and the formation of well-developed actin cytoskeleton and focal contacts of osteoblast-like cells.

Let′s stick together: An efficient on-resin combination of a Diels–Alder reaction with inverse electron demand and a Cu^I catalyzed azide–alkyne cycloaddition is used to link a stable and affine TiO₂-binding peptide to two cell adhesive motifs. The multifunctional construct improved the cell adhesion of osteoblast-like cells through the cooperative action of the peptides. DOPA=l-3,4-dihydroxylphenylalanine.

“Stapled” peptides are typically designed to replace two non-interacting residues with a constraining, olefinic staple. To mimic interacting leucine and isoleucine residues, we have created new amino acids that incorporate a methyl group in the γ-position of the stapling amino acid S5. We have incorporated them into a sequence derived from steroid receptor coactivator 2, which interacts with estrogen receptor α. The best peptide (IC₅₀=89 nm) replaces isoleucine 689 with an S-γ-methyl stapled amino acid, and has significantly higher affinity than unsubstituted peptides (390 and 760 nm). Through X-ray crystallography and molecular dynamics studies, we show that the conformation taken up by the S-γ-methyl peptide minimizes the syn-pentane interactions between the α- and γ-methyl groups.

Branching out: γ-Branched stapling amino acids were synthesized and incorporated into peptides to produce high-affinity inhibitors of the estrogen receptor/steroid receptor coactivator interaction. Some branched stapled peptides were more effective than the unfunctionalized peptide. The influence of 1,5-interactions on peptide conformation was analyzed by circular dichroism, X-ray crystallography, and molecular dynamics.

Among the well-known phototriggers, the p-hydroxyphenacyl (pHP) group has consistently enabled the very fast, efficient, and high-conversion release of active molecules. Despite this unique behavior, the pHP group has been ignored as a delivery agent, particularly in the area of theranostics, because of two major limitations: Its excitation wavelength is below 400 nm, and it is nonfluorescent. We have overcome these limitations by incorporating a 2-(2′-hydroxyphenyl)benzothiazole (HBT) appendage capable of rapid excited-state intramolecular proton transfer (ESIPT). The ESIPT effect also provided two unique advantages: It assisted the deprotonation of the pHP group for faster release, and it was accompanied by a distinct fluorescence color change upon photorelease. In vitro studies showed that the p-hydroxyphenacyl–benzothiazole–chlorambucil conjugate presents excellent properties, such as real-time monitoring, photoregulated drug delivery, and biocompatibility.

Let it go… The two major limitations for theranostics of the p-hydroxyphenacyl (pHP) phototrigger were overcome by incorporating a benzothiazole appendage to enable rapid excited-state intramolecular proton transfer (ESIPT; see picture). The ESIPT effect had two key advantages: It assisted the deprotonation of pHP group for faster release of the anticancer drug chlorambucil and led to a fluorescence color change upon photorelease.

Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications.

Director′s cut: The Flexizyme technique is used to incorporate acetyl-lysine and the non-hydrolyzable thioacetyl-lysine into full-length proteins in vitro and site-specifically into human histone H3, either individually or in pairs. The thioacetyl group of the modified histone H3 could not be removed by the histone deacetylase sirtuin.

A new click bioorthogonal reaction system was devised to enable the fast ligation (k_ON≈340 m⁻¹ s⁻¹) of conjugatable derivatives of a rigid cyclic diol (nopoldiol) and a carefully optimized boronic acid partner, 2-methyl-5-carboxymethylphenylboronic acid. Using NMR and fluorescence spectroscopy studies, the corresponding boronates were found to form reversibly within minutes at low micromolar concentration in water, providing submicromolar equilibrium constant (K_eq≈10⁵–10⁶ m⁻¹). Efficient protein conjugation under physiological conditions was demonstrated with model proteins thioredoxin and albumin, and characterized by mass spectrometry and gel electrophoresis.

A click bioorthogonal reaction system was devised for the fast ligation of conjugatable derivatives of a rigid cyclic diol (nopoldiol) with an optimal boronic acid partner, 2-methyl-5-carboxymethylphenylboronic acid. The resulting boronates form reversibly within minutes at low concentrations in water. Efficient protein conjugation under physiological conditions was demonstrated with model proteins thioredoxin and albumin.