An allosteric HTRA1-calpain 2 complex with restricted activation profile.

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

C-C Coupling of Carbene Molecules on a Metal Surface in the Presence of Water.

A novel surface-confined C-C coupling reaction involving two carbene molecules and a water molecule was studied by scanning tunneling microscopy in real space. Carbene fluorenylidene was generated from diazofluorene in the presence of water on a silver surface. While in the absence of water, fluorenylidene covalently binds to the surface to form a surface metal carbene, and water can effectively compete with the silver surface in reacting with the carbene. Water molecules in direct contact with fluorenylidene protonate the carbene to form the fluorenyl cation before the carbene can bind to the surface. In contrast, the surface metal carbene does not react with water. The fluorenyl cation is highly electrophilic and draws electrons from the metal surface to generate the fluorenyl radical which is mobile on the surface at cryogenic temperatures. The final step in this reaction sequence is the reaction of the radical with a remaining fluorenylidene molecule or with diazofluorene to produce the C-C coupling product. Both a water molecule and the metal surface are essential for the consecutive proton and electron transfer followed by C-C coupling. This C-C coupling reaction is unprecedented in solution chemistry.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

PPI-Affinity: A Web Tool for the Prediction and Optimization of Protein-Peptide and Protein-Protein Binding Affinity.

Virtual screening of protein-protein and protein-peptide interactions is a challenging task that directly impacts the processes of hit identification and hit-to-lead optimization in drug design projects involving peptide-based pharmaceuticals. Although several screening tools designed to predict the binding affinity of protein-protein complexes have been proposed, methods specifically developed to predict protein-peptide binding affinity are comparatively scarce. Frequently, predictors trained to score the affinity of small molecules are used for peptides indistinctively, despite the larger complexity and heterogeneity of interactions rendered by peptide binders. To address this issue, we introduce PPI-Affinity, a tool that leverages support vector machine (SVM) predictors of binding affinity to screen datasets of protein-protein and protein-peptide complexes, as well as to generate and rank mutants of a given structure. The performance of the SVM models was assessed on four benchmark datasets, which include protein-protein and protein-peptide binding affinity data. In addition, we evaluated our model on a set of mutants of EPI-X4, an endogenous peptide inhibitor of the chemokine receptor CXCR4, and on complexes of the serine proteases HTRA1 and HTRA3 with peptides. PPI-Affinity is freely accessible at https://protdcal.zmb.uni-due.de/PPIAffinity.

Surface Diffusion Aided by a Chirality Change of Self-Assembled Oligomers under 2D Confinement.

Chirality switching of self-assembled molecular structures is of potential interest for designing functional materials but is restricted by the strong interaction between the embedded molecules. Here, we report on an unusual approach based on reversible chirality changes of self-assembled oligomers using variable-temperature scanning tunneling microscopy supported by quantum mechanical calculations. Six functionalized diazomethanes each self-assemble into chiral wheel-shaped oligomers on Ag(111). At 130 K, a temperature far lower than expected, the oligomers change their chirality even though the molecules reside in an embedded self-assembled structure. Each chirality change is accompanied by a slight center-of-mass shift. We show how the identical activation energies of the two processes result from the interplay of the chirality change with surface diffusion, findings that open the possibility of implementing various functional materials from self-assembled supramolecular structures.

Controlling Reactivity-Real-Space Imaging of a Surface Metal Carbene.

Metal carbenes are key intermediates in a plethora of homogeneous and heterogeneous catalytic processes. However, despite their importance to heterogeneous catalysis, the influence of surface attachment on carbene reactivity has not yet been explored. Here, we reveal the interactions of fluorenylidene (FY), an archetypical aromatic carbene of extreme reactivity, with a Ag(111) surface. For the first time, the interaction of a highly reactive carbene with a metal surface could be studied by scanning tunneling microscopy (STM). FY chemisorbs on Ag(111) with an estimated desorption energy of 3 eV, forming a surface bound silver-carbene complex. The surface interaction leads to a switching of the electronic ground state of FY from triplet to singlet, and to controlled chemical reactivity. This atomistic understanding of the interplay between carbenes and metal surfaces opens the way for the development of novel classes of catalytic systems based on surface metal carbenes.

Solvent-Enhanced Conformational Flexibility of Cyclic Tetrapeptides.

Solvent and temperature can affect the structural properties of cyclic peptides by controlling their flexibility. Here, we investigate two cyclic peptides, featuring beta turns. Using temperature-dependent NMR and FT-IR, we observed a pronounced temperature effect on the conformation of the cyclic peptide D-1 in CHCl3 but a much smaller effect in CH3 CN. Almost no effect was observed for its diastereomer L-1 within a similar temperature range and using the same solvents. With the aid of Replica Exchange Molecular Dynamics simulations and Quantum Mechanics/Molecular Mechanics calculations, we were able to explain this behavior based on the increased flexibility of D-1 (in CHCl3 ) in terms of intramolecular hydrogen bonding. The largest temperature dependence is observed for D-1 in CHCl3 , while the temperature effect is less pronounced for L-1 in CHCl3 and for both peptides in CH3 CN. This work provides new insights into the role of the environment and temperature on the conformations of cyclic peptides.

Gas-Phase Deprotonation of Benzhydryl Cations: Carbene Basicity, Multiplicity, and Rearrangements.

Many fundamental properties of carbenes, particularly basicity, remain poorly understood. Herein, an experimental and computational examination of the deprotonation of a series of benzhydryl cations has been undertaken. These studies represent the first attempt at providing experimental values for diarylcarbene basicities. Pathways to deprotonation, including whether the singlet or triplet carbene is formed, are probed. Because diarylcarbenes are expected to be among the strongest organic bases known, assessing the energetics of protonation of these species is of fundamental importance for a wide range of chemical processes.

Conformer-Specific Heavy-Atom Tunneling in the Rearrangement of Benzazirines to Ketenimines.

5-Methoxy-2H-benzazirine was prepared via irradiation of the corresponding phenyl azide, isolated in an argon matrix at cryogenic temperatures. It undergoes ring expansion to the corresponding ketenimine in the dark at T < 30 K despite a calculated activation barrier of 4.9 kcal mol-1 [B3LYP/6-311++G(d,p)]. Since this rearrangement proceeds with a rate constant in the order of 10-4 s-1, exhibiting only a shallow temperature dependence, the results are interpreted in terms of heavy-atom tunneling. Of the four isomeric benzazirines resulting from the initial photolysis, only one can be observed to rearrange; this conformer specificity is explained by the other potentially observable rearrangements being either too fast or too slow to be detected due to the differences in heights and widths of their respective activation barriers.

Persistent Organic High-Spin Trinitrenes.

The septet ground state trinitrenes 1,3,5-trichloro-2,4,6-trinitrenobenzene and 1,3,5-tribromo-2,4,6-trinitrenobenzene were isolated in inert (Ar, Ne, and Xe) as well as reactive matrices (H2 , O2 , and H2 O) at cryogenic temperatures. These trinitrenes were obtained in high yields by UV photolysis of the corresponding triazides and characterized by IR and UV/Vis spectroscopy. The trinitrenes, despite bearing six unpaired electrons, are remarkably unreactive towards molecular oxygen and hydrogen and are persistent in water ice up to 160 K where the water matrix starts to sublime off.

Switching the Spin State of Pentafluorophenylnitrene: Isolation of a Singlet Arylnitrene Complex.

The chemistry of arylnitrenes is dominated by their triplet ground states and excited open-shell singlet states. This results in radical-type reactions and unwanted rearrangements, which diminish the use of arylnitrenes as intermediates in organic synthesis. While the closed-shell singlet states of arylnitrenes are expected to undergo useful chemical transformations (comparable to the closed-shell singlet states of carbenes), these states are too high in energy to be chemically accessible. When triplet pentafluorophenylnitrene is interacting with the Lewis acid BF3 under the conditions of matrix isolation, a Lewis acid-base complex consisting of the closed-shell singlet state of the nitrene and two molecules of BF3 is formed. Although the closed-shell singlet state of pentafluorophenylnitrene is calculated (CCSD(T)) to lie more than 25 kcal/mol above its triplet ground state, the reaction with BF3 results in switching the spin state from triplet to singlet. The formation of the singlet complex was monitored by IR, UV-vis, and EPR spectroscopy. DFT, CCSD(T), and CASPT2 calculations confirm the experimental findings.

Activation of Molecular Hydrogen by Arylcarbenes.

The hydrogenation reactions of diphenylcarbene 1, fluorenylidene 2, and dibenzocycloheptadienylidene 3 were investigated in solid H2 and D2 matrices and in H2 - and D2 -doped argon matrices at cryogenic temperatures. The reactivity of the carbenes towards H2 increases in the order 1<3<2. Whereas 1 is stable in solid H2 , 2 and 3 react fast under the same conditions via quantum chemical tunneling. In D2 both 1 and 3 are stable, whereas 2 slowly reacts. The different reactivity of the three carbenes is rationalized in terms of differing carbene stabilization energies.

Reactions of Cyclopentadienylidenes with CF3I: Electron Bond Donation versus Halogen Bond Donation of the Iodine Atom.

The interaction of cyclopentadienylidene and tetrachlorocyclopentadienylidene with the halogen bond donor CF3I has been studied by matrix isolation spectroscopy. The carbenes were produced by photolysis of the corresponding diazo compounds, matrix-isolated in argon doped with 1% CF3I at 3 K. Bimolecular reactions between the carbenes and CF3I were induced by annealing these matrices to 25-30 K to allow for the diffusion of trapped species. Instead of classical halogen-bonded complexes, these carbenes form complexes in which the iodine atom is shared between the carbene center and the CF3 group. Photolysis of the complexes at 3 K yields radical pairs, which reversibly react back to the complexes when the matrices are warmed to 25-30 K.

Isolation of an Antiaromatic Singlet Cyclopentadienyl Zwitterion.

The reaction of triplet tetrachlorocyclopentadienylidene with BF3 in rare gas matrices yields a zwitterion consisting of a cyclopentadienyl cation bearing a positive charge and a negatively charged BF3 unit. IR and UV-vis spectra as well as the absence of EPR signals demonstrate a singlet ground state of the zwitterion, and its calculated geometry and magnetic properties clearly reveal a strong antiaromatic character. The zwitterion is highly labile and by visible or IR irradiation rearranges via a 1,2-fluorine migration from boron to carbon. Interaction with a second molecule of BF3 stabilizes the zwitterion and suppresses the fluorine migration, thus providing a convenient and efficient synthesis of an antiaromatic molecule under very mild conditions.

Photoinduced Reversible Electron Transfer Between the Benzhydryl Radical and Benzhydryl Cation in Amorphous Water-Ice.

The benzhydryl radical is generated in high yields by flash-vacuum thermolysis of 1,1,2,2-tetraphenylethane with subsequent trapping of the product in argon or amorphous water at 3-4 K. Photoionization of the radical with various UV lights and electron sources produces the benzhydryl cation, which was identified by IR and UV-vis spectroscopy. In solid argon, the formation of the benzhydryl cation is irreversible, whereas in amorphous water-ice the electron transfer is reversible, and irradiation into the major absorption band at 443 nm of the cation leads back to the radical by electron attachment. Applications of ionization of organic matter trapped in water-ice to icy environments in astrophysics and planetary sciences, including Earth, are discussed.

Activation of the B-F Bond by Diphenylcarbene: A Reversible 1,2-Fluorine Migration between Boron and Carbon.

Experiments in low-temperature matrices reveal that triplet diphenylcarbene inserts into the very strong B-F bond of BF3 in a two-step reaction. The first step is the formation of a strongly bound Lewis acid-base complex between the singlet state of diphenylcarbene and BF3 . This step involves an inversion of the spin state of the carbene from triplet to singlet. The second step requires visible-light photochemical activation to induce a 1,2-F migration from boron to the adjacent carbon atom under formation of the formal insertion product of the carbene center into BF3 . The 1,2-F migration is reversible under short-wavelength UV irradiation, thus leading back to the Lewis acid-base adduct.

The Cope Rearrangement of 1,5-Dimethylsemibullvalene-2(4)-d1 : Experimental Evidence for Heavy-Atom Tunneling.

As an experimental test of the theoretical prediction that heavy-atom tunneling is involved in the degenerate Cope rearrangement of semibullvalenes at cryogenic temperatures, monodeuterated 1,5-dimethylsemibullvalene isotopomers were prepared and investigated by IR spectroscopy using the matrix isolation technique. As predicted, the less thermodynamically stable isotopomer rearranges at cryogenic temperatures in the dark to the more stable one, while broadband IR irradiation above 2000 cm-1 results in an equilibration of the isotopomeric ratio. Since this reaction proceeds with a rate constant in the order of 10-4  s-1 despite an experimental barrier of Ea =4.8 kcal mol-1 and with only a shallow temperature dependence, the results are interpreted in terms of heavy-atom tunneling.

Reaction of Triplet Phenylnitrene with Molecular Oxygen.

Triplet carbenes react with molecular oxygen with rates that approach diffusion control to carbonyl O-oxides, whereas triplet nitrenes react much slower. For investigating the reaction of phenylnitrene with O2, the nitrene was generated by flash vacuum thermolysis (FVT) of phenylazide and subsequently isolated in O2-doped matrices. FVT of the azide produces the nitrene in high yield and with only minor contaminations of the rearranged products that are frequently observed if the nitrene is produced by photolysis. The phenylnitrene was isolated in solid Ar, Xe, mixtures of these rare gases with O2, and even in pure solid O2. At temperatures between 30 and 35 K an extremely slow thermal reaction between the nitrene and O2 was observed, whereas at higher temperatures, solid Ar and O2 rapidly evaporate. Only O2-doped Xe matrices allowed us to anneal at temperatures above 40 K, and at these temperatures, the nitrene reacts with O2 to produce nitroso O-oxide mainly in its syn conformation. Upon visible light irradiation (450 nm), the nitroso oxide rapidly rearranges to nitrobenzene.

Chirality control of a single carbene molecule by tip-induced van der Waals interactions.

Non-covalent interactions such as van der Waals interactions and hydrogen bonds are crucial for the chiral induction and control of molecules, but it remains difficult to study them at the single-molecule level. Here, we report a carbene molecule on a copper surface as a prototype of an anchored molecule with a facile chirality change. We examine the influence of the attractive van der Waals interactions on the chirality change by regulating the tip-molecule distance, resulting in an excess of a carbene enantiomer. Our model study provides insight into the change of molecular chirality controlled by van der Waals interactions, which is fundamental for understanding the mechanisms of chiral induction and amplification.