Telescoping a Prenyltransferase and a Diterpene Synthase to Transform Unnatural FPP Derivatives to Diterpenoids.

New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.

Why pyridoxal phosphate could be a functional predecessor of thiamine pyrophosphate and speculations on a primordial metabolism.

The account attempts to substantiate the hypothesis that, from an evolutionary perspective, the coenzyme couple pyridoxal phosphate and pyridoxamine phosphate preceded the coenzyme thiamine pyrophosphate and acted as its less efficient chemical analogue in some form of early metabolism. The analysis combines mechanism-based chemical reactivity with biosynthetic arguments and provides evidence that vestiges of "TPP-like reactivity" are still found for PLP today. From these thoughts, conclusions can be drawn about the key elements of a primordial form of metabolism, which includes the citric acid cycle, amino acid biosynthesis and the pentose phosphate pathway.

Sesquiterpene Cyclase BcBOT2 Promotes the Unprecedented Wagner-Meerwein Rearrangement of the Methoxy Group.

Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.

Evolution at the Origins of Life?

The role of evolutionary theory at the origin of life is an extensively debated topic. The origin and early development of life is usually separated into a prebiotic phase and a protocellular phase, ultimately leading to the Last Universal Common Ancestor. Most likely, the Last Universal Common Ancestor was subject to Darwinian evolution, but the question remains to what extent Darwinian evolution applies to the prebiotic and protocellular phases. In this review, we reflect on the current status of evolutionary theory in origins of life research by bringing together philosophy of science, evolutionary biology, and empirical research in the origins field. We explore the various ways in which evolutionary theory has been extended beyond biology; we look at how these extensions apply to the prebiotic development of (proto)metabolism; and we investigate how the terminology from evolutionary theory is currently being employed in state-of-the-art origins of life research. In doing so, we identify some of the current obstacles to an evolutionary account of the origins of life, as well as open up new avenues of research.

Dextrans, Pullulan and Lentinan, New Scaffold Materials for Use as Hydrogels in Tissue Engineering.

The development of hydrogels based on dextrans, pullulan and lentinan to be used in biomedical applications including tissue engineering is reported. Despite the fact that selected polysaccharides such as hyaluronic acid are well established, little is known, how these polysaccharides can be chemically modified to create hydrogels under controlled conditions. In this study we present a small library of chemically modified polysaccharides which are used for a divergent approach to achieve biomedical relevant hydrogels. In this case the crosslinking is based on thio ether formation between thiol modified donor and vinylsulfone or maleimide modified acceptor components. Successful synthesis of the linker systems and coupling at the polysaccharides, hydrogel formation takes place under physiological conditions. We extended the study by coupling small molecules like adhesion factors for increasing cell compatibility as well as a dye for further studies. The different hydrogels were studied to their rheological properties, water uptake, their permeability, biodegrability and their cytotoxicity.

Is Simultaneous Binding to DNA and Gyrase Important for the Antibacterial Activity of Cystobactamids?

Cystobactamids are aromatic oligoamides that exert their natural antibacterial properties by inhibition of bacterial gyrases. Such aromatic oligoamides were proposed to inhibit α-helix-mediated protein-protein interactions and may serve for specific recognition of DNA. Based on this suggestion, we designed new derivatives that have duplicated cystobactamid triarene units as model systems to decipher the specific binding mode of cystobactamids to double stranded DNA. Solution NMR analyses revealed that natural cystobactamids as well as their elongated analogues show an overall bent shape at their central aliphatic unit, with an average CX-CY-CZ angle of ~110 degrees. Our finding is corroborated by the target-bound structure of close analogues, as established by cryo-EM very recently. Cystobactamid CN-861-2 binds directly to the bacterial gyrase with an affinity of 9 µM, and also exhibits DNA-binding properties with specificity for AT-rich DNA. Elongation/dimerization of the triarene subunit of native cystobactamids is demonstrated to lead to an increase in DNA binding affinity. This implies that cystobactamids' gyrase inhibitory activity necessitates not just interaction with the gyrase itself, but also with DNA via their triarene unit.

Chemoenzymatic Synthesis of a New Germacrene Derivative Named Germacrene F.

The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6-C7 to C7-C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name "germacrene F". Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature-dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror-symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.

Halogenated Rocaglate Derivatives: Pan-antiviral Agents against Hepatitis E Virus and Emerging Viruses.

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4', 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

Sesquiterpene Backbones Generated by Sesquiterpene Cyclases: Formation of iso-Caryolan-1-ol and an Isoclovane.

New sesquiterpene backbones are accessible after incubation of caryolan-synthase (GcoA) and presilphiperfolan-8-ß-ol synthase (BcBOT2) with a non-natural farnesyldiphosphate in which the central olefinic double bond is isomerized toward the methyl group. Two newly formed sesquiterpenoids are reported, a constitutional isomer of caryolan-1-ol (3), which we name iso-caryolan-1-ol (17), and the first terpenoid based on the isoclovane ring skeleton generated enzymatically thus far.

Heinz-Günter Floss (1934-2022).

Heinz-Günter Floss, a world-renowned biosynthetic chemist, passed away on December 19, 2022. He was one of the most influential scientists in expanding the range of methods for deciphering the biosynthetic pathways of complex natural products. He was a mentor to many collaborators who later followed in his footsteps, creating a global "scientific school of natural products".

Taming Bromine Azide for Use in Organic Solvents─Radical Bromoazidations and Alcohol Oxidations.

The formation of bromine azide from the bisazidobromate(I) anion or alternatively from Zhdankin's reagent, using a phosphonium bromide salt as a common starting point, is reported. After homolytic cleavage in the presence of alkenes or alcohols either 1,2-functionalization or alternatively the selective oxidation of secondary alcohols in the presence of primary alcohols occur. The scopes and limitations of the use of BrN3 are covered.

Ligand-free Ullmann-type arylation of oxazolidinones by diaryliodonium salts.

The arylation of azaheterocycles can be considered as one of the most important processes for the preparation of various biologically active compounds. In the present work, we describe a method for the copper-catalyzed N-arylation of hindered oxazolidinones using diaryliodonium salts. The method succeeds in good to excellent yields for the arylation of 4-alkyloxazolidinones, including sterically hindered isopropyl- and tert-butyl-substituted. The efficiency of the method was demonstrated for a wide range of diaryliodonium salts - symmetric and unsymmetric as well as ortho-substituted derivatives. The developed approach will provide an important contribution in the development and preparation of novel drugs and bioactive molecules containing oxazolidinone moieties.

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition.

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

On the evolution of coenzyme biosynthesis.

Covering: up to 2022The report provides a broad approach to deciphering the evolution of coenzyme biosynthetic pathways. Here, these various pathways are analyzed with respect to the coenzymes required for this purpose. Coenzymes whose biosynthesis relies on a large number of coenzyme-mediated reactions probably appeared on the scene at a later stage of biological evolution, whereas the biosyntheses of pyridoxal phosphate (PLP) and nicotinamide (NAD+) require little additional coenzymatic support and are therefore most likely very ancient biosynthetic pathways.

Cyclopropylmethyldiphosphates are substrates for sesquiterpene synthases: experimental and theoretical results.

New homo-sesquiterpenes are accessible after conversion of presilphiperfolan-8ß-ol synthase (BcBOT2) with cyclopropylmethyl analogs of farnesyl diphosphate, and this biotransformation is dependent on subtle structural refinements. Two of the three cyclisation products are homo variants of germacrene D and germacrene D-4-ol while the third product reported contains a new bicyclic backbone for which no analogue in nature has been described so far. The findings on diphosphate activation are discussed and rationalised by relaxed force constants and dissociation energies computed at the DFT level of theory.

Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies.

Drug-inducible suicide systems may help to minimize risks of human induced pluripotent stem cell (hiPSC) therapies. Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed. We have introduced a drug-inducible Caspase 9 suicide system (iCASP9) into the AAVS1 safe-harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. After transplantation into mice, drug treatment generally led to rapid elimination of teratomas, but single animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ∼ 3 × 10-8. Besides transgene elimination, presumably via loss of heterozygosity (LoH), silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, no escapees from biallelic iCASP9 cells were observed after treatment of up to 0.8 billion hiPSCs. The highly increased safety level provided by biallelic integration of the iCASP9 system may substantially contribute to the safety level of iPSC-based therapies.

Inductive heating and flow chemistry - a perfect synergy of emerging enabling technologies.

Inductive heating has developed into a powerful and rapid indirect heating technique used in various fields of chemistry, but also in medicine. Traditionally, inductive heating is used in industry, e.g., for heating large metallic objects including bending, bonding, and welding pipes. In addition, inductive heating has emerged as a partner for flow chemistry, both of which are enabling technologies for organic synthesis. This report reviews the combination of flow chemistry and inductive heating in industrial settings as well as academic research and demonstrates that the two technologies ideally complement each other.

On the Evolutionary History of the Twenty Encoded Amino Acids.

α-Amino acids are essential molecular constituents of life, twenty of which are privileged because they are encoded by the ribosomal machinery. The question remains open as to why this number and why this 20 in particular, an almost philosophical question that cannot be conclusively resolved. They are closely related to the evolution of the genetic code and whether nucleic acids, amino acids, and peptides appeared simultaneously and were available under prebiotic conditions when the first self-sufficient complex molecular system emerged on Earth. This report focuses on prebiotic and metabolic aspects of amino acids and proteins starting with meteorites, followed by their formation, including peptides, under plausible prebiotic conditions, and the major biosynthetic pathways in the various kingdoms of life. Coenzymes play a key role in the present analysis in that amino acid metabolism is linked to glycolysis and different variants of the tricarboxylic acid cycle (TCA, rTCA, and the incomplete horseshoe version) as well as the biosynthesis of the most important coenzymes. Thus, the report opens additional perspectives and facets on the molecular evolution of primary metabolism.

Identification of structurally re-engineered rocaglates as inhibitors against hepatitis E virus replication.

Hepatitis E virus (HEV) infections are a leading cause of acute viral hepatitis in humans and pose a considerable threat to public health. Current standard of care treatment is limited to the off-label use of nucleoside-analog ribavirin (RBV) and PEGylated interferon-α, both of which are associated with significant side effects and provide limited efficacy. In the past few years, a promising natural product compound class of eukaryotic initiation factor 4A (eIF4A) inhibitors (translation initiation inhibitors), called rocaglates, were identified as antiviral agents against RNA virus infections. In the present study, we evaluated a total of 205 synthetic rocaglate derivatives from the BU-CMD compound library for their antiviral properties against HEV. At least eleven compounds showed inhibitory activities against the HEV genotype 3 (HEV-3) subgenomic replicon below 30 nM (EC50 value) as determined by Gaussia luciferase assay. Three amidino-rocaglates (ADRs) (CMLD012073, CMLD012118, and CMLD012612) possessed antiviral activity against HEV with EC50 values between 1 and 9 nM. In addition, these three selected compounds inhibited subgenomic replicons of different genotypes (HEV-1 [Sar55], wild boar HEV-3 [83-2] and human HEV-3 [p6]) in a dose-dependent manner and at low nanomolar concentrations. Furthermore, tested ADRs tend to be better tolerated in primary hepatocytes than hepatoma cancer cell lines and combination treatment of CMLD012118 with RBV and interferon-α (IFN-α) showed that CMLD012118 acts additive to RBV and IFN-α treatment. In conclusion, our results indicate that ADRs, especially CMLD012073, CMLD012118, and CMLD012612 may prove to be potential therapeutic candidates for the treatment of HEV infections and may contribute to the discovery of pan-genotypic inhibitors in the future.

Asymmetric Total Synthesis of Antibiotic Elansolid A.

Elansolid A is a structurally complex polyketide macrolactone natural product that exhibits promising antibacterial properties. Its challenging asymmetric total synthesis was achieved by a convergent strategy, in which the tetrahydroindane core of the molecule and an eastern vinyl iodide moiety were combined as the main fragments. The central tetrahydroindane motif was constructed with high stereoselectivity by a bioinspired intramolecular Diels-Alder cycloaddition, generating four stereogenic centers in a single step. The stereocontrol of this key step could be achieved by virtue of a 1,3-allylic strain generated by the temporary introduction of a steric-directing iodine substituent on the substrate. The formation of the macrolactone motif that completes the synthesis was achieved via two different retrosynthetic disconnections, namely, a Suzuki-Miyaura cross-coupling or an alternative Mukaiyama esterification reaction.