Gold-Mediated Multiple Cysteine Arylation for the Construction of Highly Constrained Bicycle Peptides.

Bicycles are constrained bicyclic peptides formed through reaction of three cysteine residues within a linear sequence with a trivalent, symmetrical small molecule scaffold. Bicycles with high binding affinities to therapeutically important targets can be discovered using screening technologies such as phage display. Increasing the chemical diversity of Bicycles should improve the probability of finding hits to new targets and can be achieved by expanding the toolbox of Bicycle forming chemistries. Gold(III) S-arylation has recently been described as a method for the efficient bioconjugation of cysteine residues under conditions compatible with phage display. Herein, we explore the scope and generality of this methodology for Bicycle construction through the synthesis and evaluation of four novel tris-Gold complexes. These new scaffolds were systematically reacted with a variety of peptide sequences, varying in amino acid loop lengths. All four scaffolds proved to be capable and selective reactive partners for each peptide sequence and afforded the desired Bicycle products in 13-48% isolated yield. This work exemplifies Gold-mediated arylation as a general approach for construction of novel, highly constrained Bicycles.

Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents.

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody-drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the ß-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.

Short hydrogen bonds enhance nonaromatic protein-related fluorescence.

Fluorescence in biological systems is usually associated with the presence of aromatic groups. Here, by employing a combined experimental and computational approach, we show that specific hydrogen bond networks can significantly affect fluorescence. In particular, we reveal that the single amino acid L-glutamine, by undergoing a chemical transformation leading to the formation of a short hydrogen bond, displays optical properties that are significantly enhanced compared with L-glutamine itself. Ab initio molecular dynamics simulations highlight that these short hydrogen bonds prevent the appearance of a conical intersection between the excited and the ground states and thereby significantly decrease nonradiative transition probabilities. Our findings open the door to the design of new photoactive materials with biophotonic applications.

Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides.

The nature of the first genetic polymer is the subject of major debate1. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life2,3-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA4. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life5.

Use of a fluorinated probe to quantitatively monitor amino acid binding preferences of ruthenium(ii) arene complexes.

In order to address outstanding questions about ruthenium complexes in complex biological solutions, 19F NMR spectroscopy was used to follow the binding preferences between fluorinated RuII(η6-arene)(bipyridine) complexes and protected amino acids and glutathione. Reporting what ruthenium compounds bind to in complex environments has so far been restricted to relatively qualitative methods, such as mass spectrometry and X-ray spectroscopic methods; however, quantitative information on the species present in the solution phase cannot be inferred from these techniques. Furthermore, using 1H NMR, in water, to distinguish and monitor a number of different complex RuII(η6-arene) adducts forming is challenging. Incorporating an NMR active heteroatom into ruthenium organometallic complexes provides a quantitative, diagnostic 'fingerprint' to track solution-phase behaviour and allow for unambiguous assignment of any given adduct. The resulting 19F NMR spectra show for the first time the varied, dynamic behaviour of organoruthenium compounds when exposed to simple biomolecules in complex mixtures. The rates of formation of the different observed species are dramatically influenced by the electronic properties at the metal, even in a closely related series of complexes in which only the electron-donating properties of the arene ligand are altered. Preference for cysteine binding is absolute: the first quantitative solution-phase evidence of such behaviour.

Diverged Plant Terpene Synthases Reroute the Carbocation Cyclization Path towards the Formation of Unprecedented 6/11/5 and 6/6/7/5 Sesterterpene Scaffolds.

Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)-mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co-expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation.

Unearthing a sesterterpene biosynthetic repertoire in the Brassicaceae through genome mining reveals convergent evolution.

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (-)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (-)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.

Guaianolides and a seco-Eudesmane from the Resinous Exudates of Cushion Bush (Leucophyta brownii) and Evaluation of Their Cytostatic and Anti-inflammatory Activity.

A detailed phytochemical investigation of a dichloromethane extract of the resinous exudates of the cushion bush plant (Leucophyta brownii) resulted in the isolation of the new 8,12-guaianolides leucophytalins A (5) and B (6), the new 1,10-seco-eudesmane leucophytalin C (10), six rare 8,12-guaianolides (1-4, 7, and 8), and the xanthanolide tomentosin (9). The structures of all isolated compounds were elucidated on the basis of spectroscopic and spectrometric analyses. The structures of compounds isolated in crystalline form, including leucophytalins A and C, were further confirmed by X-ray crystallography. The crude extract exhibited moderate cytostatic activity against a breast cancer (MCF-7) and human colon cancer (HT-29) cell line with IC50 values of 9.3 and 18 µg/mL, respectively, and anti-inflammatory activity against the macrophage-like cell line RAW 264.7 with IC50 values of 3.9 and 6.1 µg/mL for thromboxane B2 and prostaglandin E2 production, respectively. The isolated compounds were evaluated for their cytostatic activity against MCF-7 and HT-29 cells (1, 3-10) and their anti-inflammatory activity against RAW 264.7 cells (1-10). All isolated compounds are most likely derived from (+)-germacrene A, and a biosynthetic pathway is proposed for these sesquiterpenoids.

(2,2'-Bipyridine-4,4'-dicarb-oxy-lic acid-κN,N')chlorido(2,2':6',2''-terpyridyl-κN,N',N'')ruthenium(II) perchlorate ethanol monosolvate monohydrate.

In the title compound, [RuCl(C(15)H(11)N(3))(C(12)H(8)N(2)O(4))]ClO(4)·C(2)H(5)OH·H(2)O, the geometry of the ClN(5) coordination set around the Ru(II) atom is close to octa-hedral, but distorted on account of the limited bite angles of the polypyridyl ligands. The complexes are linked by O-H⋯O hydrogen bonds between the carboxyl groups and the crystal lattice water mol-ecules into chains along [110]. Face-to-face stacking inter-actions are formed between terpyridine ligands, with inter-planar separations of 3.66 (1) and 3.42 (1) Å, and between bipyridine-4,4'-dicarb-oxy-lic acid ligands, with inter-planar separations of 3.65 (1) and 3.72 (1) Å. Three O atoms of the perchlorate ion are each disordered equally over two positions. The hy-droxy group of the ethanol mol-ecule is also disordered over two sites with refined occupancies of 0.794 (9) and 0.206 (9).

Trichlorido[(meth-yl{2-[meth-yl(2-pyridyl-meth-yl)amino]eth-yl}amino)acetonitrile]iron(III) methanol hemisolvate.

The title compound, [FeCl(3)(C(12)H(18)N(4))]·0.5CH(3)OH, contains an Fe(III) ion in a distorted octa-hedral coordination environment. The neutral N,N',N''-tridentate ligand adopts a fac coordination mode, and chloride ligands lie trans to each of the three coordinated N atoms. In the crystal, the complexes form columns extending parallel to the approximate local threefold axes of the FeN(3)Cl(3) octa-hedra, and the columns are arranged so that the uncoordinated nitrile groups align in an anti-parallel manner and the pyridyl rings form offset face-to-face arrangements [inter-planar separations = 2.95 (1) and 3.11 (1) Å; centroid-centroid distances = 5.31 (1) and 4.92 (1) Å]. The methanol solvent mol-ecule is disordered about a twofold rotation axis.

4-Fluoro-N-methyl-N-(1,2,3,4-tetra-hydro-carbazol-3-yl)benzene-sulfonamide.

In the title compound, C(19)H(19)FN(2)O(2)S, the hydrogenated six-membered ring of the carbazole unit adopts a half-chair conformation and the plane of the fluoro-phenyl ring forms a dihedral angle of 41.5 (1)° with respect to the carbazole mean plane. The crystal structure is segregated into layers containing the carbazole units and fluoro-phenyl rings in alternate (200) planes. The carbazole units form centrosymmetric face-to-face inter-actions [inter-planar separation = 4.06 (1) Å] and edge-to-face inter-actions in which the N-H group is directed towards an adjacent carbazole face, with a shortest H⋯C contact of 2.53 Å. The fluoro-phenyl rings form face-to-face contacts with an approximate inter-planar separation of 3.75 Šand a centroid-centroid distance of 4.73 (1) Å.