Extendable stapling of unprotected peptides by crosslinking two amines with o-phthalaldehyde

Peptide modification methods that do not rely on the cysteine residue are underdeveloped, and their development could greatly expand the current toolbox for peptide chemistry. During the course of preliminary investigations into the classical ortho-phthalaldehyde (OPA)-amine-thiol condensation reaction, we found that in the absence of thiol, OPA readily condenses with two primary alkyl amines to form a class of underexplored isoindolin-1-imine compounds under mild aqueous conditions. From the intramolecular version of this OPA-2amines reaction, an efficient and selective methodology using mild reaction conditions has been developed for stapling unprotected peptides via crosslinking of two amino groups in both an end-to-side and side-to-side fashion. The stapling method is superfast and broadly applicable for various peptide substrates with the reacting amino groups separated by a wide range of different amino acid units. The macrocyclization reactions of selected substrates are completed within 10 seconds at 5 mM concentration and within 2 minutes at 50 μM concentration. Importantly, the resulting cyclized peptides with an isoindolinimine linkage can be extended in a one-pot sequential addition manner with several different electron-deficient π electrophiles, thereby generating more complex structures.

Analysis of Fragmentation Pathways of Peptide Modified with Quaternary Ammonium and Phosphonium Group as Ionization Enhancers

Peptide modification by a quaternary ammonium group containing a permanent positive charge is a promising method of increasing the ionization efficiency of the analyzed compounds, making ultra-sensitive detection even at the attomolar level possible. Charge-derivatized peptides may undergo both charge remote (ChR) and charge-directed (ChD) fragmentation. A series of model peptide conjugates derivatized with N,N,N-triethyloammonium (TEA), 1-azoniabicyclo[2.2.2]octane (ABCO), 2,4,6-triphenylopyridinium (TPP) and tris(2,4,6-trimetoxyphenylo)phosphonium (TMPP) groups were analyzed by their fragmentation pathways both in collision-induced dissociation (CID) and electron-capture dissociation (ECD) mode. The effect of the fixed-charge tag type and peptide sequence on the fragmentation pathways was investigated. We found that the aspartic acid effect plays a crucial role in the CID fragmentation of TPP and TEA peptide conjugates whereas it was not resolved for the peptides derivatized with the phosphonium group. ECD spectra are mostly dominated by cn ions. ECD fragmentation of TMPP-modified peptides results in the formation of intense fragments derived from this fixed-charge tag, which may serve as reporter ion.

Aqueous Solutions of Peptides and Trialkylamines Lead to Unexpected Peptide Modification

When trialkylamines are added to buffered solutions of peptides, unexpected adducts can be formed. These adducts correspond to Schiff base products. The source of the reaction is the unexpected presence of aldehydes in amines. The aldehydes can be detected in a few ways. Most importantly, they can lead to unanticipated results in proteomics experiments. Their undesirable effects can be minimized through the addition of other amines.

Experimental and Computational Investigations into Trivalent Phosphorous-Mediated Radical Thiol Desulfurization

Radical-mediated thiol desulfurization processes using tricoordinate phosphorous reagents are used in a range of applications from small molecule synthesis to peptide modification. A combined experimental and computational examination of the mechanism and kinetics of the radical desulfurization of alkyl thiyl radicals using trivalent phosphorus reagents was performed. Primary alkyl thiols undergo desulfurization between 10^6 to 10^9 M-1s-1 depending on the phosphorus component with either an H-atom transfer step or β-fragmentation of the thiophosphoranyl intermediate may be rate-controlling. While the desulfurization of primary aliphatic thiols showed a marked dependence on the identity of phosphorous reagent used with either a rate-controlling H-atom transfer or -fragmentation, thiols yielding stabilized C-centered radicals showed much less sensitivity. Support for a stepwise S-atom transfer process progressing via a distorted trigonal bipyramidal thiophosphoranyl radical intermediate was obtained from DFT calculated energetics and hyperfine splitting values.

Direct and Indirect Biomimetic Peptide Modification of Alginate: Efficiency, Side Reactions, and Cell Response

In the fast-developing field of tissue engineering there is a constant demand for new materials as scaffolds for cell seeding, which can better mimic a natural extracellular matrix as well as control cell behavior. Among other materials, polysaccharides are widely used for this purpose. One of the main candidates for scaffold fabrication is alginate. However, it lacks sites for cell adhesion. That is why one of the steps toward the development of suitable scaffolds for cells is the introduction of the biofunctionality to the alginate structure. In this work we focused on bone-sialoprotein derived peptide (TYRAY) conjugation to the molecule of alginate. Here the comparison study on four different approaches of peptide conjugation was performed including traditional and novel modification methods, based on 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM), thiol-Michael addition and Cu-catalyzed azide–alkyne cycloaddition reactions. It was shown that the combination of the alginate amidation with the use of and subsequent Cu-catalyzed azide–alkyne cycloaddition led to efficient peptide conjugation, which was proven with both NMR and XPS methods. Moreover, the cell culture experiment proved the positive effect of peptide presence on the adhesion of human embryonic stem cells.

Biomechanical and Biological Performances of Diels-Alder Crosslinked Thermogelling Bioink

Hydrogels are key components in bioink formulations to ensure printability and stability in biofabrication. In this study a well-known post-polymerization modification approach is introduced into thermogelling diblock copolymers, comprising poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine). While the thermogelling and shear-thinning properties allow excellent printability, trigger-less cell-friendly Diels-Alder click-chemistry yields long-term shape-fidelity. The introduced platform enables easy incorporation of cell-binding moieties (RGD-peptide) for cellular interaction. The hydrogel was functionalized with RGD-peptides using thiol-maleimide chemistry and growth as well as morphology of fibroblast seeded on top of the hydrogels confirmed the cell adhesion facilitated by the peptides. Finally, bioink formulations were tested for biocompatibility by incorporating fibroblasts homogenously inside polymer solution pre-printing and exhibited good cytocompatibility after the printing process and crosslinking. The established bioink system combining a two-step approach by physical precursor gelation followed by additional chemical stabilization offers a broad versatility for further biomechanical adaptation or bioresponsive peptide modification.

Biomechanical and Biological Performances of Diels-Alder Crosslinked Thermogelling Bioink

Hydrogels are key components in bioink formulations to ensure printability and stability in biofabrication. In this study a well-known post-polymerization modification approach is introduced into thermogelling diblock copolymers, comprising poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine). While the thermogelling and shear-thinning properties allow excellent printability, trigger-less cell-friendly Diels-Alder click-chemistry yields long-term shape-fidelity. The introduced platform enables easy incorporation of cell-binding moieties (RGD-peptide) for cellular interaction. The hydrogel was functionalized with RGD-peptides using thiol-maleimide chemistry and growth as well as morphology of fibroblast seeded on top of the hydrogels confirmed the cell adhesion facilitated by the peptides. Finally, bioink formulations were tested for biocompatibility by incorporating fibroblasts homogenously inside polymer solution pre-printing and exhibited good cytocompatibility after the printing process and crosslinking. The established bioink system combining a two-step approach by physical precursor gelation followed by additional chemical stabilization offers a broad versatility for further biomechanical adaptation or bioresponsive peptide modification.