Nucleobase adducts bind MR1 and stimulate MR1-restricted T cells.

MR1T cells are a recently found class of T cells that recognize antigens presented by the major histocompatibility complex-I-related molecule MR1 in the absence of microbial infection. The nature of the self-antigens that stimulate MR1T cells remains unclear, hampering our understanding of their physiological role and therapeutic potential. By combining genetic, pharmacological, and biochemical approaches, we found that carbonyl stress and changes in nucleobase metabolism in target cells promote MR1T cell activation. Stimulatory compounds formed by carbonyl adducts of nucleobases were detected within MR1 molecules produced by tumor cells, and their abundance and antigenicity were enhanced by drugs that induce carbonyl accumulation. Our data reveal carbonyl-nucleobase adducts as MR1T cell antigens. Recognizing cells under carbonyl stress allows MR1T cells to monitor cellular metabolic changes with physiological and therapeutic implications.

Controlling the Photophysical Properties of a Series of Isostructural d6 Complexes Based on Cr0, MnI, and FeII.

Development of first-row transition metal complexes with similar luminescence and photoredox properties as widely used RuII polypyridines is attractive because metals from the first transition series are comparatively abundant and inexpensive. The weaker ligand field experienced by the valence d-electrons of first-row transition metals challenges the installation of the same types of metal-to-ligand charge transfer (MLCT) excited states as in precious metal complexes, due to rapid population of energetically lower-lying metal-centered (MC) states. In a family of isostructural tris(diisocyanide) complexes of the 3d6 metals Cr0, MnI, and FeII, the increasing effective nuclear charge and ligand field strength allow us to control the energetic order between the 3MLCT and 3MC states, whereas pyrene decoration of the isocyanide ligand framework provides control over intraligand (ILPyr) states. The chromium(0) complex shows red 3MLCT phosphorescence because all other excited states are higher in energy. In the manganese(I) complex, a microsecond-lived dark 3ILPyr state, reminiscent of the types of electronic states encountered in many polyaromatic hydrocarbon compounds, is the lowest and becomes photoactive. In the iron(II) complex, the lowest MLCT state has shifted to so much higher energy that 1ILPyr fluorescence occurs, in parallel to other excited-state deactivation pathways. Our combined synthetic-spectroscopic-theoretical study provides unprecedented insights into how effective nuclear charge, ligand field strength, and ligand π-conjugation affect the energetic order between MLCT and ligand-based excited states, and under what circumstances these individual states become luminescent and exploitable in photochemistry. Such insights are the key to further developments of luminescent and photoredox-active first-row transition metal complexes.

Design principles for cyclin K molecular glue degraders.

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

Catalyst control over pentavalent stereocentres.

A monumental diversity of catalytic methods imparts the ability to select one of two configurations of tetravalent stereocentres. Conversely, catalyst control over pentavalent stereocentres, where a fifth moiety bound to the central atom encodes an expanded stereochemical space, remained a challenge to be accomplished. Herein, we report the feasibility of the catalytic tractability of pentavalent stereocentres. A bifunctional iminophosphorane thiourea catalyst enables enantio- and diastereocontrol over pentavalent phosphoranes to differentiate configurationally stable enantiomers and ensembles of diastereomers which emerge together from a single stereocentre. The desired dioxophosphorane stereoisomers are obtained with excellent yield and selectivity (up to 99% yield, 96:4 e.r. and 99:1 d.r.), while stereodivergent catalysis reroutes the reaction for selective access to each of the viable stereoisomeric states of pentavalent phosphoranes. Considering the diversity of high-valent main group species, it is expected that catalyst control over pentavalent stereocentres significantly increases the synthetically addressable stereochemical space.

Unexpected dynamics in femtomolar complexes of binding proteins with peptides.

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

Photoredox-active Cr(0) luminophores featuring photophysical properties competitive with Ru(II) and Os(II) complexes.

Coordination complexes of precious metals with the d6 valence electron configuration such as Ru(II), Os(II) and Ir(III) are used for lighting applications, solar energy conversion and photocatalysis. Until now, d6 complexes made from abundant first-row transition metals with competitive photophysical and photochemical properties have been elusive. While previous research efforts focused mostly on Fe(II), we disclose that isoelectronic Cr(0) gives access to higher photoluminescence quantum yields and excited-state lifetimes when compared with any other first-row d6 metal complex reported so far. The luminescence behaviour of the metal-to-ligand charge transfer excited states of these Cr(0) complexes is competitive with Os(II) polypyridines. With these Cr(0) complexes, the metal-to-ligand charge transfer states of first-row d6 metal complexes become exploitable in photoredox catalysis, and benchmark chemical reductions proceed efficiently under low-energy red illumination. Here we demonstrate that appropriate molecular design strategies open up new perspectives for photophysics and photochemistry with abundant first-row d6 metals.

Nanobody GPS by PCS: An Efficient New NMR Analysis Method for G Protein Coupled Receptors and Other Large Proteins.

NMR chemical shift changes can report on the functional dynamics of biomacromolecules in solution with sizes >1 MDa. However, their interpretation requires chemical shift assignments to individual nuclei, which for large molecules often can only be obtained by tedious point mutations that may interfere with function. We present here an efficient pseudocontact shift NMR method to assign biomacromolecules using bound antibodies tagged with lanthanoid DOTA chelators. The stability of the antibody allows positioning the DOTA tag at many surface sites, providing triangulation of the macromolecule nuclei at distances >60 Å. The method provides complete assignments of valine and tyrosine 1H-15N resonances of the ß1-adrenergic receptor in various functional forms. The detected chemical shift changes reveal strong forces exerted onto the backbone of transmembrane helix 3 during signal transmission, which are absorbed by its electronic structure. The assignment method is applicable to any soluble biomacromolecule for which suitable complementary binders exist.

Characterization and Quantification of Particle-Bound Criegee Intermediates in Secondary Organic Aerosol.

The ozonolysis of alkenes contributes substantially to the formation of secondary organic aerosol (SOA), which are important modulators of air quality and the Earth's climate. Criegee intermediates (CIs) are abundantly formed through this reaction. However, their contributions to aerosol particle chemistry remain highly uncertain. In this work, we present the first application of a novel methodology, using spin traps, which simultaneously quantifies CIs produced from the ozonolysis of volatile organic compounds in the gas and particle phases. Only the smallest CI with one carbon atom was detected in the gas phase of a ß-caryophyllene ozonolysis reaction system. However, multiple particle-bound CIs were observed in ß-caryophyllene SOA. The concentration of the most abundant CI isomer in the particle phase was estimated to constitute ∼0.013% of the SOA mass under atmospherically relevant conditions. We also demonstrate that the lifetime of CIs in highly viscous SOA particles is at least on the order of minutes, substantially greater than their gas-phase lifetime. The confirmation of substantial concentrations of large CIs with elongated lifetimes in SOA raises new questions regarding their influence on the chemical evolution of viscous SOA particles, where CIs may be a previously underestimated source of reactive species.

Nonacethrene Unchained: A Cascade to Chiral Contorted Conjugated Hydrocarbon with Two sp3-Defects.

We demonstrate that structurally complex carbon nanostructures can be achieved via a synthetic approach that capitalizes on a π-radical reaction cascade. The cascade is triggered by oxidation of a dihydro precursor of helical diradicaloid nonacethrene to give a chiral contorted polycyclic aromatic hydrocarbon named hypercethrene. In this ten-electron oxidation process, four σ-bonds, one π-bond, and three six-membered rings are formed in a sequence of up to nine steps to yield a 72-carbon-atom warped framework, comprising two configurationally locked [7]helicene units, a fluorescent peropyrene unit, and two precisely installed sp3-defects. The key intermediate in this cascade is a closed nonacethrene derivative with one quaternary sp3-center, presumably formed via an electrocyclic ring closure of nonacethrene, which, when activated by oxidation, undergoes a reaction cascade analogous to the oxidative dimerization of phenalenyl to peropyrene. By controlling the amount of oxidant used, two intermediates and one side product could be isolated and fully characterized, including single-crystal X-ray diffraction analysis, and two intermediates were detected by electron paramagnetic resonance spectroscopy. In concert with density functional theory calculations, these intermediates support the proposed reaction mechanism. Compared to peropyrene, the absorption and emission of hypercethrene are slightly red-shifted on account of extended π-conjugation and the fluorescence quantum yield of 0.45 is decreased by a factor of ∼2. Enantiomerically enriched hypercethrene displays circularly polarized luminescence with a brightness value of 8.3 M-1 cm-1. Our results show that reactions of graphene-based π-radicals-typically considered an "undefined decomposition" of non-zero-spin materials-can be well-defined and selective, and have potential to be transformed into a step-economic synthetic method toward complex carbon nanostructures.

Geländer Molecules with Orthogonal Joints: Synthesis of Macrocyclic Dimers.

Orthogonal joints, understood as connections with an angle of 90°, were introduced in the design of the "Geländer" model compounds 1 and 2. The banister, consisting of a conjugated carbazole dimer linked by either 1,3-butadiyne (2) or a single thiophene (1), wraps around an axis composed of a phthalimide dimer due to the dimensional mismatch of both subunits, which are interconnected by phenylene rungs. The "Geländer" structure was assembled from a monomer comprising the 1,4-diaminobenzene rung with one amino substituent as part of a 4-bromo phthalimide subunit forming the orthogonal junction to the axis, and the other as part of a masked 2-ethynyl carbazole as orthogonal joint to the banister. The macrocycle was obtained by two sequential homocoupling steps. A first dimerization by a reductive homocoupling assembled the axis, while an oxidative acetylene coupling served as ring-closing reaction. The formed butadiyne was further derivatized to a thiophene, rendering all carbons of the model compound sp2 hybridized. Both helical structures were fully characterized and chirally resolved. Assignment of the enantiomers was achieved by simulation of chiroptical properties and enantiopure synthesis.

Circularly Polarized Luminescence in a Möbius Helicene Carbon Nanohoop.

We present the first helicene carbon nanoohop that integrates a [6]helicene into [7]cycloparaphenylene. The [6]helicene endows the helicene carbon nanohoop with chiroptical properties and configurational stability typical for higher helicenes, while the radially conjugated seven para-phenylenes largely determine the optoelectronic properties. The structure of the helicene carbon nanoohop was unambiguously characterized by NMR, MS and X-ray analysis that revealed that it possesses a topology of a Möbius strip in the solid state and in solution. The chirality transfers from the [6]helicene to the para-phenylenes and leads to a pronounced circular dichroism and bright circularly polarized luminescence, which is affected by the structural topology of the nanohoop.

An Artificial Metalloenzyme Based on a Copper Heteroscorpionate Enables sp3 C-H Functionalization via Intramolecular Carbene Insertion.

The selective functionalization of sp3 C-H bonds is a versatile tool for the diversification of organic compounds. Combining attractive features of homogeneous and enzymatic catalysts, artificial metalloenzymes offer an ideal means to selectively modify these inert motifs. Herein, we report on a copper(I) heteroscorpionate complex embedded within streptavidin that catalyzes the intramolecular insertion of a carbene into sp3 C-H bonds. Target residues for genetic optimization of the artificial metalloenzyme were identified by quantum mechanics/molecular mechanics simulations. Double-saturation mutagenesis yielded detailed insight on the contribution of individual amino acids on the activity and the selectivity of the artificial metalloenzyme. Mutagenesis at a third position afforded a set of artificial metalloenzymes that catalyze the enantio- and regioselective formation of ß- and γ-lactams with high turnovers and promising enantioselectivities.

Manganese(I) Complex with Monodentate Arylisocyanide Ligands Shows Photodissociation Instead of Luminescence.

Recently reported manganese(I) complexes with chelating arylisocyanide ligands exhibit luminescent metal-to-ligand charge-transfer (MLCT) excited states, similar to ruthenium(II) polypyridine complexes with the same d6 valence electron configuration used for many different applications in photophysics and photochemistry. However, chelating arylisocyanide ligands require substantial synthetic effort, and therefore it seemed attractive to explore the possibility of using more readily accessible monodentate arylisocyanides instead. Here, we synthesized the new Mn(I) complex [Mn(CNdippPhOMe2)6]PF6 with the known ligand CNdippPhOMe2 = 4-(3,5-dimethoxyphenyl)-2,6-diisopropylphenylisocyanide. This complex was investigated by NMR spectroscopy, single-crystal structure analysis, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) measurements, IR spectroscopy supported by density functional theory (DFT) calculations, cyclic voltammetry, and time-resolved as well as steady-state UV-vis absorption spectroscopy. The key finding is that the new Mn(I) complex is nonluminescent and instead undergoes arylisocyanide ligand loss during continuous visible laser irradiation into ligand-centered and charge-transfer absorption bands, presumably owed to the population of dissociative d-d excited states. Thus, it seems that chelating bi- or tridentate binding motifs are essential for obtaining emissive MLCT excited states in manganese(I) arylisocyanides. Our work contributes to understanding the basic properties of photoactive first-row transition metal complexes and could help advance the search for alternatives to precious metal-based luminophores, photocatalysts, and sensors.

Structure determination of high-energy states in a dynamic protein ensemble.

Macromolecular function frequently requires that proteins change conformation into high-energy states1-4. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here we develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts5 (PCSs) with Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion6 (PCS-CPMG). Our approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, we find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5 angström). Applying our methodology to Adk during catalysis, we find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, our solution PCS-CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60 kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.

Pseudocontact Shifts in Biomolecular NMR Spectroscopy.

Paramagnetic centers in biomolecules, such as specific metal ions that are bound to a protein, affect the nuclei in their surrounding in various ways. One of these effects is the pseudocontact shift (PCS), which leads to strong chemical shift perturbations of nuclear spins, with a remarkably long range of 50 Å and beyond. The PCS in solution NMR is an effect originating from the anisotropic part of the dipole-dipole interaction between the magnetic momentum of unpaired electrons and nuclear spins. The PCS contains spatial information that can be exploited in multiple ways to characterize structure, function, and dynamics of biomacromolecules. It can be used to refine structures, magnify effects of dynamics, help resonance assignments, allows for an intermolecular positioning system, and gives structural information in sensitivity-limited situations where all other methods fail. Here, we review applications of the PCS in biomolecular solution NMR spectroscopy, starting from early works on natural metalloproteins, following the development of non-natural tags to chelate and attach lanthanoid ions to any biomolecular target to advanced applications on large biomolecular complexes and inside living cells. We thus hope to not only highlight past applications but also shed light on the tremendous potential the PCS has in structural biology.

Pyrene-Decoration of a Chromium(0) Tris(diisocyanide) Enhances Excited State Delocalization: A Strategy to Improve the Photoluminescence of 3d6 Metal Complexes.

There is a long-standing interest in iron(II) complexes that emit from metal-to-ligand charge transfer (MLCT) excited states, analogous to ruthenium(II) polypyridines. The 3d6 electrons of iron(II) are exposed to a relatively weak ligand field, rendering nonradiative relaxation of MLCT states via metal-centered excited states undesirably efficient. For isoelectronic chromium(0), chelating diisocyanide ligands recently provided access to very weak MLCT emission in solution at room temperature. Here, we present a concept that boosts the luminescence quantum yield of a chromium(0) isocyanide complex by nearly 2 orders of magnitude, accompanied by a significant increase of the MLCT lifetime. Pyrene units in the diisocyanide ligand backbone lead to an enlarged π-conjugation system and to a strongly delocalized MLCT state, from which nonradiative relaxation is less dominant despite a sizable redshift of the emission. While the pyrene moiety is electronically coupled to the core of the chromium(0) complex in the excited state, UV-vis absorption and 2D NMR spectroscopy show that this is not the case in the ground state. Luminescence lifetimes and quantum yields for our pyrenyl-decorated chromium(0) complex exhibit an unusual bell-shaped dependence on solvent polarity, indicative of two counteracting effects governing the MLCT deactivation. These two effects are identified as predominant deactivation either through an energetically nearby lying metal-centered state in the most apolar solvents, or alternatively via direct nonradiative relaxation to the ground state following the energy gap law in more polar solvents. This is the first example of a 3d6 MLCT emitter to benefit from an increased π-conjugation network.

Manganese(I) complexes with metal-to-ligand charge transfer luminescence and photoreactivity.

Precious metal complexes with the d6 valence electron configuration often exhibit luminescent metal-to-ligand charge transfer (MLCT) excited states, which form the basis for many applications in lighting, sensing, solar cells and synthetic photochemistry. Iron(II) has received much attention as a possible Earth-abundant alternative, but to date no iron(II) complex has been reported to show MLCT emission upon continuous-wave excitation. Manganese(I) has the same electron configuration as that of iron(II), but until now has typically been overlooked in the search for cheap MLCT luminophores. Here we report that isocyanide chelate ligands give access to air-stable manganese(I) complexes that exhibit MLCT luminescence in solution at room temperature. These compounds were successfully used as photosensitizers for energy- and electron-transfer reactions and were shown to promote the photoisomerization of trans-stilbene. The observable electron transfer photoreactivity occurred from the emissive MLCT state, whereas the triplet energy transfer photoreactivity originated from a ligand-centred 3π-π* state.

Human T cells engineered with a leukemia lipid-specific TCR enables donor-unrestricted recognition of CD1c-expressing leukemia.

Acute leukemia relapsing after chemotherapy plus allogeneic hematopoietic stem cell transplantation can be treated with donor-derived T cells, but this is hampered by the need for donor/recipient MHC-matching and often results in graft-versus-host disease, prompting the search for new donor-unrestricted strategies targeting malignant cells. Leukemia blasts express CD1c antigen-presenting molecules, which are identical in all individuals and expressed only by mature leukocytes, and are recognized by T cell clones specific for the CD1c-restricted leukemia-associated methyl-lysophosphatidic acid (mLPA) lipid antigen. Here, we show that human T cells engineered to express an mLPA-specific TCR, target diverse CD1c-expressing leukemia blasts in vitro and significantly delay the progression of three models of leukemia xenograft in NSG mice, an effect that is boosted by mLPA-cellular immunization. These results highlight a strategy to redirect T cells against leukemia via transfer of a lipid-specific TCR that could be used across MHC barriers with reduced risk of graft-versus-host disease.

An Ortho-Tetraphenylene-Based "Geländer" Architecture Consisting Exclusively of 52 sp2 -Hybridized C Atoms.

A new type of "Geländer" molecule based on a ortho-tetraphenylene core is presented. The central para-quaterphenyl backbone is wrapped by a 4,4'-di((Z)-styryl)-1,1'-biphenyl banister, with its aryl rings covalently attached to all four phenyl rings of the backbone. The resulting helical chiral bicyclic architecture consists exclusively of sp2 -hybridized carbon atoms. The target structure was assembled by expanding the central ortho-tetraphenylene subunit with the required additional phenyl rings followed by a twofold macrocyclization. The first macrocyclization attempts based on a twofold McMurry coupling were successful but low yielding; the second strategy, profiting from olefin metathesis, provided satisfying yields. Hydrogenation of the olefins resulted in a saturated derivative of similar topology, thereby allowing the interdependence between saturation and physico-chemical properties to be studied. The target structures, including their solid-state structures, were fully characterized. The helical chiral bicycle was synthesized as a racemate and separated into pure enantiomers by HPLC on a chiral stationary phase. Comparison of recorded and simulated chiroptical properties allowed the enantiomers to be assigned.

Sulfone "Geländer" Helices: Revealing Unexpected Parameters Controlling the Enantiomerization Process.

The two sulfonyl-bridged Geländer helices 1a and 2a are obtained by oxidation of the corresponding sulfide bridged precursors 1b and 2b. Both Geländer structures are fully characterized by NMR, high-resolution mass spectrometry, and optical spectroscopies. X-ray diffraction with a single crystal of 2a provides its solid-state structure. Both Geländer helices 1a and 2a are separated into enantiomers, and their racemizations are monitored by circular dichroism. For 1a, consisting of two equally sized macrocycles, a substantial increase in the enantiomerization barrier is observed upon going from the sulfide to the sulfone, and only a subtle rise is detected for the constitutional isomer 2a with two macrocycles of different size during the same transformation. This results not only in 1a with the highest configurational stability in the series of hitherto investigated Geländer structures but also challenges the so far hypothesized correlations between bridging structures and the Gibbs free energy of enantiomerization. The simulation of the enantiomerization process in the macrocyclic subunits suggests the proximity of the endotopic hydrogens as parameter responsible for the heights of the enantiomerization barrier.