Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

Proteolysis targeting chimeras (PROTACs) are a promising therapeutic strategy to selectively promote the degradation of protein targets by exploiting the ubiquitin-proteasome system. Among the limited number of E3 ligase ligands discovered for the PROTAC technology, ligands of cereblon (CRBN) E3 ligase, such as pomalidomide, thalidomide, or lenalidomide, are the most frequently used for the development of PROTACs. Our group previously reported that a phenyl group could be tolerated on the C4-position of lenalidomide as the ligand of CRBN to develop PROTACs. Herein, we report a modular chemistry platform for the efficient attachment of various ortho-, meta-, and para-substituted phenyls to the C4-position of the lenalidomide via Suzuki cross-coupling reaction, which allows the systematic investigation of the linker effect for the development of PROTACs against any target. We examined the substrate scope by preparing twelve lenalidomide-derived CRBN E3 ligase ligands with different linkers.

Nucleic acids are information-rich and readily available biomolecules, which can be used to template the polymerization of synthetic macromolecules. Here, we highlight the control over the size, composition, and sequence one can nowadays obtain by using this methodology. We also highlight how templated processes exploiting dynamic covalent polymerization can, in return, result in therapeutic nucleic acids fabricating their own dynamic delivery vector - a biomimicking concept that can provide original solutions for gene therapies.

Kelp is an abundant, farmable biomass-containing laminarin and alginate as major polysaccharides, providing an excellent model substrate to study their deconstruction by simple enzyme mixtures. Our previous study showed strong reactivity of the glycoside hydrolase family 55 during hydrolysis of purified laminarin, raising the question of its reactivity with intact kelp. In this study, we determined that a combination of a single glycoside hydrolase family 55 β-1,3-exoglucanase with a broad-specificity alginate lyase from the polysaccharide lyase family 18 gives efficient hydrolysis of untreated kelp to a mixture of simple sugars, that is, glucose, gentiobiose, mannitol-end glucose, and mannuronic and guluronic acids and their soluble oligomers. Quantitative assignments from nanostructure initiator mass spectrometry (NIMS) and 2D HSQC NMR spectroscopy and analysis of the reaction time-course are provided. The data suggest that binary combinations of enzymes targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay's potential for epigenetic evaluation of clinical samples, benefiting research and patient management.

Metabolites orchestrate cellular processes as either substrates, co-enzymes, inhibitors, or activators of cellular proteins such as enzymes and receptors. Although traditional biochemical and structural biology-based approaches have been successfully employed for the discovery of protein-metabolite interactions, they often fail to detect transient and low-affinity biomolecular relationships. Another limitation of these approaches is that they are performed under in vitro conditions lacking the physiological context. Recently developed mass spectrometry-based methodologies overcome both these shortcomings, and have resulted in the discovery of global protein-metabolite cellular interaction networks. Herein, we describe traditional and modern approaches for the discovery of protein-metabolite interactions, and discuss the impact of these discoveries on our understanding of cellular physiology and on drug development.

Artificial intelligence (AI) in the form of deep learning has promise for drug discovery and chemical biology, for example, to predict protein structure and molecular bioactivity, plan organic synthesis, and design molecules de novo. While most of the deep learning efforts in drug discovery have focused on ligand-based approaches, structure-based drug discovery has the potential to tackle unsolved challenges, such as affinity prediction for unexplored protein targets, binding-mechanism elucidation, and the rationalization of related chemical kinetic properties. Advances in deep-learning methodologies and the availability of accurate predictions for protein tertiary structure advocate for a renaissance in structure-based approaches for drug discovery guided by AI. This review summarizes the most prominent algorithmic concepts in structure-based deep learning for drug discovery, and forecasts opportunities, applications, and challenges ahead.

Molecular biology and biochemistry interpret microscopic processes in the living world in terms of molecular structures and their interactions, which are quantum mechanical by their very nature. Whereas the theoretical foundations of these interactions are well established, the computational solution of the relevant quantum mechanical equations is very hard. However, much of molecular function in biology can be understood in terms of classical mechanics, where the interactions of electrons and nuclei have been mapped onto effective classical surrogate potentials that model the interaction of atoms or even larger entities. The simple mathematical structure of these potentials offers huge computational advantages; however, this comes at the cost that all quantum correlations and the rigorous many-particle nature of the interactions are omitted. In this work, we discuss how quantum computation may advance the practical usefulness of the quantum foundations of molecular biology by offering computational advantages for simulations of biomolecules. We not only discuss typical quantum mechanical problems of the electronic structure of biomolecules in this context, but also consider the dominating classical problems (such as protein folding and drug design) as well as data-driven approaches of bioinformatics and the degree to which they might become amenable to quantum simulation and quantum computation.

The vast majority of RNA splicing in today's organisms is achieved by the highly regulated and precise removal of introns from pre-mRNAs via the spliceosome. Here we present a model of how RNA splicing may have occurred in earlier life forms. We have designed a hairpin ribozyme derived spliceozyme that mediates two RNA cleavages and one ligation event at specific positions and thus cuts a segment (intron) out of a parent RNA and ligates the remaining fragments (exons). The cut-out intron then performs a downstream function, acting as a positive regulator of the activity of a bipartite DNAzyme. This simple scenario shows how small RNAs can perform complex RNA processing dynamics, involving the generation of new phenotypes by restructuring segments of given RNA species, as well as delivering small RNAs that may play a functional role in downstream processes.

Glycosphingolipid (GSL) and glycosylphosphatidylinositol (GPI) are the two major glycolipids expressed by eukaryotic cells, and their metabolisms share the same machineries. Moreover, both GSLs and GPI-anchored proteins (GPI-APs) are localized in the cholesterol-rich regions, namely the lipid rafts, of the cell membrane, where many other signaling molecules are compartmentalized as well. Therefore, the interaction between GSLs and GPI-APs and their interactions with other molecules in the lipid rafts are inevitable. This review is focused on the influences of GSLs and GPI-APs on each other's biosynthesis, trafficking, cell membrane distribution, and biological functions, such as signal transduction.

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

Tissue endothelial cells express ABC-transporter enzymes that change the concentration of small molecules within different tissue compartments. These "blood-tissue barriers" have been shown to directly affect the efficacy and toxicity of anticancer, antimicrobial, psychiatric, and anti-epileptic drugs. Currently this phenomenon is best studied for the blood-brain barrier, but remains enigmatic for most other tissues. In addition, canonical pharmacokinetic theory specifically assumes an equal concentration of free drug within all tissue compartments. Inspired by Lipinski's "rule of 5," we here clarify current knowledge on drug-tissue distribution by: 1) curating the in-vivo literature on 73 drugs across 23 tissues and 2) developing two graphical web-based applications to visually describe and interpret data. These curated in-vivo dataset and visualization tools enabled us to achieve new insights into the logic of the barrier-tissue organization and showed remarkable correspondence to whole-body imaging of radiolabeled molecules.

Although rarely used in nature, fluorine has emerged as an important elemental ingredient in the design of proteins with altered folding, stability, oligomerization propensities, and bioactivity. Adding to the molecular modification toolbox, here we report the ability of privileged perfluorinated amphiphiles to noncovalently decorate proteins to alter their conformational plasticity and potentiate their dispersion into fluorous phases. Employing a complementary suite of biophysical, in-silico and in-vitro approaches, we establish structure-activity relationships defining these phenomena and investigate their impact on protein structural dynamics and intracellular trafficking. Notably, we show that the lead compound, perfluorononanoic acid, is 106 times more potent in inducing non-native protein secondary structure in select proteins than is the well-known helix inducer trifluoroethanol, and also significantly enhances the cellular uptake of complexed proteins. These findings could advance the rational design of fluorinated proteins, inform on potential modes of toxicity for perfluoroalkyl substances, and guide the development of fluorine-modified biologics with desirable functional properties for drug discovery and delivery applications.

Oceans cover 71 % of Earth's surface and are home to hundreds of thousands of species, many of which are microbial. Knowledge about marine microbes has strongly increased in the past decades due to global sampling expeditions, and hundreds of detailed studies on marine microbial ecology, physiology, and biogeochemistry. However, the translation of this knowledge into biotechnological applications or synthetic biology approaches using marine microbes has been limited so far. This review highlights key examples of marine bacteria in synthetic biology and metabolic engineering, and outlines possible future work based on the emerging marine chassis organisms Vibrio natriegens and Halomonas bluephagenesis. Furthermore, the valorization of algal polysaccharides by genetically enhanced microbes is presented as an example of the opportunities and challenges associated with blue biotechnology. Finally, new roles for marine synthetic biology in tackling pressing global challenges, including climate change and marine pollution, are discussed.

Tyrosine nitration, a post-translational modification (PTM) that takes place under nitrosative stress conditions, occurs through a non-enzymatic peroxynitrite-mediated reaction. Although protein nitration has long been considered an irreversible PTM involved in nitrosative stress-associated diseases, it has also been suggested to be a regulatory mechanism of signal transduction. Therefore, the development of tools that help to understand this protein modification is of great interest. Herein, we explore a TbIII -chelating metallopeptide to monitor tyrosine nitration. The luminescence of this probe decreases significantly between its non-nitrated and nitrated states, and this reduction in the luminescence intensity is directly related to the degree of tyrosine nitration after treatment with peroxynitrite. Remarkably, the luminescence intensity changes after nitration are not affected in the presence of complex biological media, which makes it a promising tool for understanding this protein modification.

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α3 β1 (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α3 β1 .

Controlling the self-assembly of DNA nanostructures using rationally designed logic gates is a major goal of dynamic DNA nanotechnology, which could facilitate the development of biomedicine, molecular computation, et al. In previous works, the regulations mostly relied on either toehold-mediated strand displacement or stimuli-driven conformational switch, requiring elaborately-designed or specific DNA sequences. Herein, we reported a facile, base-sequence-independent strategy for logically controlling DNA self-assembly through external molecules. The INHIBIT and XOR logic controls over the assembly/disassembly of DNA polyhedra were realized through cystamine (Cyst) and ethylenediamine (EN) respectively, which were further integrated into a half subtractor circuit thanks to the sharing of the same inputs. Our work provides a sequence-independent strategy in logically controlling DNA self-assembly, which may open up new possibilities for dynamic DNA nanotechnology.

We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide-alkyne cycloaddition reaction. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes.

The fungal cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide, and PF1022 consist of alternating N-methylated l-amino and d-hydroxy acids. They are synthesized by non-ribosomal peptide synthetases (NRPS). The amino acid and hydroxy acid substrates are activated by adenylation (A) domains. Although various A domains have been characterized thus giving insights into the mechanism of substrate conversion, little is known about the utilization of hydroxy acids in NRPSs. Therefore, we used homology modelling and molecular docking of the A1 domain of enniatin synthetase (EnSyn) to gain insights into the mechanism of hydroxy acid activation. We introduced point mutations into the active site and used a photometric assay to study the substrate activation. The results suggest that the hydroxy acid is selected by interaction with backbone carbonyls rather than by a specific side chain. These insights enhance the understanding of non-amino acid substrate activation and could contribute to the engineering of depsipeptide synthetases.

Engineered cysteines are frequently used for site-specific conjugation in antibody-drug conjugate (ADC) development. When cysteine-engineered mAbs are produced in the cell culture process, the sulfhydryl groups on the engineered cysteines are mostly in an oxidized form. The oxidized cysteines require multiple steps (such as reduction, reoxidation, and buffer exchanges) to reactivate for bioconjugation, which complicates the ADC production process and reduces yields. In this study, we identified a Q166C mutation in the light chain that allows the presence of free sulfhydryl groups during cell culture and purification process. This mutation is in the constant region and away from sites involved in antigen binding or Fc-mediated functions. The free sulfhydryl reacts readily with maleimide in a mild solution at a high conjugation rate. This is only the second such site reported (the first one is Q124C in the light chain). Using the Q166C mutation, we conjugated an anti-angiopoietin-2 (Ang-2) peptide on bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, to construct a peptide antibody conjugate, Ava-Plus, which could block two pro-angiogenic factors simultaneously. Ava-Plus showed high affinity for both VEGF and Ang-2 and demonstrated higher activity than bevacizumab in in vitro cell migration and in vivo mouse xenograft models.

C-Jun N-terminal kinase (JNK) is a key mediator involved in a variety of physiological processes. JNK activation is regulated in a complex manner by upstream kinases and phosphatases, and plays an important role in physiological processes such as the immune response and neuronal function. Therefore, JNK has become a therapeutic target for neurodegenerative diseases, ankylosing spondylitis, psoriasis, arthritis and other diseases. Inhibition of JNK activation in mitochondria holds great potential for Parkinson's disease (PD) therapy. However, no specific mitochondrial-targeted JNK inhibitor has been reported. We have developed a mitochondrial-targeted JNK inhibitor, P2, by linking a mitochondrial-specific cell-penetrating peptide to SP600125 (SP), a commercialized specific inhibitor of JNK. We found that P2 specifically inhibited mitochondrial JNK phosphorylation instead of nuclear JNK signaling. Further studies showed that P2 effectively rescued PD phenotypes both in vitro and in vivo, thus indicating that it is a potential therapeutic for PD.

Photosynthetic organisms such as diatoms microalgae provide innovative routes to eco-friendly technologies for environmental pollution bioremediation. Living diatoms are capable to incorporate in vivo a wide variety of chemical species dispersed in seawater, thus being promising candidates for eco-friendly removal of toxic contaminants. However, their exploitation requires immobilization methods that allow to confine microalgae during water treatment. Here we demonstrate that a biofilm of Phaeodactylum tricornutum diatom cells grown on the surface of a glassy substrate bearing boronic acid protruding moieties is stably anchored to the substrate resisting mechanical stress and it is suitable for removal of up to 80 % metal ions (As, Cr, Cu, Zn, Sn, Pb, Sb) in a model polluted water sample. Control experiments also suggest that stabilization of the biofilm adhesion occurs by interaction of boronic acid surface groups of the substrate with the hydroxyl groups of diatoms extracellular polysaccharides.

Sequence-based protein design approaches are being adopted to generate highly functional enzymes; however, screening the enzymes remains a time-consuming task. In this study, by analyzing the enzymatic properties of four ancestral meso-2,6-diaminopimelate dehydrogenases (AncDAPDHs), AncDAPDH-N1, -N2, -N3, and -N4, we attempted to define a new index parameter that is helpful for efficiently screening the enzymes. Biochemical and thermodynamic analyses indicated that only AncDAPDH-N4 exhibited greater thermal stability than and activity similar to those of native DAPDHs. Structural and sequence comparisons between DAPDH from Corynebacterium glutamicum (CgDAPDH) and the AncDAPDHs suggested that "quality of mutations" is a potential index parameter. In fact, the mutations introduced from CgDAPDH to AncDAPDH-N4 correlated highly with the mutations accumulated during the evolution process from mesophiles to thermophiles. These results suggest that, although there are several exceptions, the correlation coefficient can be used as an index parameter for screening high-functioning enzymes from sequence data.

The increased success of small metal-containing molecules as pharmaceutical agents has prompted investigations into the pharmacological activity of a different class of metal-based compounds; supramolecular coordination complexes (SCCs). Such complexes have been extensively investigated for their anticancer activity, with many displaying activities comparable or superior to available clinical chemotherapeutic drugs. Here, we evaluated a series of quinoline-containing binuclear complexes and metallarectangles for their in vitro anticancer activity in the hormone receptor positive MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. The preliminary cytotoxic screen, in the MCF-7 cell line, revealed that the ligand (7-chloro-4-(pyridin-4-yl)quinoline, L) and metallarectangle [{Ir(μ-Cl)(Cp*)}4 (μ-L)2 ](OTf)4 display superior activity to cisplatin, while [{Ru(p-cymene)}4 (μ-η2 -η2 -C2 O4 )2 (μ-L)2 ](OTf)4 was more potent than cisplatin in the triple-negative MDA-MD-231 cell line. Upon evaluation in a multidose screen, ligand L and metallarectangle [{Ir(μ-Cl)(Cp*)}4 (μ-L)2 ](OTf)4 displayed antiproliferative activity almost two-fold greater than cisplatin in the MCF-7 cell line, while [{Ru(p-cymene)}4 (μ-η2 -η2 -C2 O4 )2 (μ-L)2 ](OTf)4 was over two-times more active than cisplatin in the MDA-MB-231 cell line. Additionally, using the non-tumorigenic MCF-12 A breast epithelial cell line, the compounds demonstrate increased selectivity toward breast cancer cells over non-tumorigenic cells. Furthermore, investigations into the interactions of ligand L and selected complexes with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) indicate favourable binding.

The N2A segment of titin functions as a pivotal hub for signal transduction and interacts with various proteins involved in structural support, chaperone activities, and transcriptional regulation. Notably, the "unique N2A" (UN2A) subdomain has been shown to interact with the stress-regulated cardiac ankyrin repeat protein (CARP), which contributes to the regulation of sarcomeric stiffness. Previously, the UN2A domain's three-dimensional structure was modelled based on its secondary structure content identified by NMR spectroscopy, considering the domain in isolation. In this study, we report experimental long-range distance distributions by electron paramagnetic resonance (EPR) spectroscopy between the three helixes within the UN2A domain linked to the immunoglobulin domain I81 in the presence and absence of CARP. The data confirm the central three-helix bundle fold of UN2A and show that this adopts a compact and stable conformation in absence of CARP. After binding to CARP, no significant conformational change was observed, suggesting that the UN2A domain retains its structure upon binding to CARP thereby, mediating the interaction approximately as a rigid-body.

Alzheimer's disease (AD) is the most prevalent cause of dementia and has become a health concern worldwide urging for an effective therapeutic. The amyloid hypothesis, currently the most pursued basis of AD drug discovery, points the cause of AD to abnormal production and ineffective removal of pathogenic aggregated amyloid-β (Aβ). AD therapeutic research has been focused on targeting different species of Aβ in the amyloidogenic process to control Aβ content and recover cognitive decline. Among the different processes targeted, the clearance mechanism has been found to be the most effective, supported by the recent clinical approval of an Aβ-targeting immunotherapeutic drug which significantly slowed cognitive decline. Although the current AD drug discovery field is extensively researching immunotherapeutic drugs, there are numerous properties of immunotherapy in need of improvements that could be overcome by an equally performing chemical drug. Here, we review chemical and immunotherapy drug candidates, based on their mechanism of modulating the amyloid cascade, selected from the AlzForum database. Through this review, we aim to summarize and evaluate the prospect of Aβ-targeting chemical drugs.

For the development of delivery systems, the solubilization of hydrophobic guest molecules in water is an important yet challenging task. This can be achieved by preparing stable aqueous solutions with a high concentration of guest molecules using a natural product as a solubilizing agent and a mechanochemical high-speed vibration milling apparatus as a solubilizing method. Various solubilizing agent-guest molecule complexes can be obtained via the exchange between solubilizing agents, which enables the "on-off" switching of the properties of functional guest molecules, such as fluorescence intensity, and photodynamic activity. In the exchange method, guest molecules can transfer into cell membranes such as lysosomes and exosomes. Therefore, the exchange method of the solubilizing agents not only creates novel solubilizing agent-guest molecule complexes but also is applied to drug delivery systems.

PROTACs (Proteolysis-Targeting Chimeras) have emerged as a groundbreaking class of chemical tools that facilitate the degradation of target proteins by leveraging the ubiquitin-proteasome system (UPS). However, the effective utilization of PROTACs in chemical biology studies and therapeutics encounters significant challenges when it comes to achieving cell-selective protein degradation and in vivo applications. This review article aims to shed light on recent advancements in the development of Pro-PROTACs, which exhibit controlled protein degradation capabilities in response to external stimuli or disease-related endogenous biochemical signals. The article delves into the specific chemical strategies employed to regulate the interaction between PROTACs and E3 ubiquitin ligases or target proteins. These strategies enable spatial and temporal control over the protein degradation potential of Pro-PROTACs. Furthermore, the review summarizes recent investigations regarding the delivery of PROTACs using biodegradable nanoparticles for in vivo applications and targeted protein degradation. Such delivery systems hold great promise for enabling efficient and selective protein degradation in vivo. Lastly, the article provides a perspective on the future design of multifunctional PROTACs and their intracellular delivery mechanisms, with a particular focus on achieving cell-selective protein degradation.

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5-3 h) with high conversions (95-99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50-mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

Multicolor labeling for monitoring the intracellular localization of the same target type in the native environment using chemical fluorescent dyes is a challenging task. This approach requires both bioorthogonal and biocompatible ligations with an excellent fluorescence signal-to-noise ratio. Here, we present a metabolic glycan labeling technique that uses homemade fluorogenic dyes to investigate glycosylation patterns in live cells. These dyes allowed us to demonstrate rapid and efficient simultaneous multilabeling of glycoconjugates with minimum fluorescence noise. Our results demonstrate that this approach is capable of not only probing sialylation and GlcNAcylation in cells but also specifically labeling the cell-surface and intracellular sialylated glycoconjugates in live cells. In particular, we performed site-specific dual-channel fluorescence imaging of extra and intracellular sialylated glycans in HeLa and PC9 cancer cells as well as identified fluorescently labeled sialylated glycoproteins and glycans by a direct enrichment approach combined with an MS-based proteomic analysis in the same experiment. In conclusion, this study provides multilabeling tools in cellular systems for simultaneous site-specific glycan imaging and glycoproteomic analysis to study potential cancer- and disease-associated glycoconjugates.

β-Carboline alkaloids have a variety of pharmacological activities, such as antitumor, antibiosis and antidiabetes. Harmine and harmol are two structurally similar β-carbolines that occur in many medicinal plants. In this work, we chose harmine and harmol to impede the amyloid fibril formation of human islet amyloid polypeptide (hIAPP) associated with type 2 diabetes mellitus (T2DM), by a series of physicochemical and biochemical methods. The results indicate that harmine and harmol effectively prevent peptide fibril formation and alleviate toxic oligomer species. In addition, both small molecules exhibit strong binding affinities with hIAPP mainly through hydrophobic and hydrogen bonding interactions, thus reducing the cytotoxicity induced by hIAPP. Their distinct binding pattern with hIAPP is closely linked to the molecular configuration of the two small molecules, affecting their ability to impede peptide aggregation. The study is of great significance for the application and development of β-carboline alkaloids against T2DM.

Co-delivery of anticancer drugs and target agents by endogenous materials is an inevitable approach towards targeted and synergistic therapy. Employing DNA base pair complementarities, DNA nanotechnology exploits a unique nanostructuring method and has demonstrated its capacity for nanoscale positioning and templated assembly. Moreover, the water solubility, biocompatibility, and modifiability render DNA structure suitable candidate for drug delivery applications. We here report single-stranded DNA tail conjugated antitumor drug paclitaxel (PTX), and the co-delivery of PTX, doxorubicin and targeting agent mucin 1 (MUC-1) aptamer on a DNA nanobarrel carrier. We investigated the effect of tail lengths on drug release efficiencies and dual drug codelivery-enabled cytotoxicity. Owing to the rapidly developing field of structural DNA nanotechnology, functional DNA-based drug delivery is promising to achieve clinical therapeutic applications.

BODIPY photosensitizers have been integrated with a hypoxia-activated prodrug to achieve synergistic photodynamic therapy (PDT) and chemotherapy. A novel BODIPY derivative BDP-CN was designed and synthesized. It had two cyano groups to make it complex well with a water-soluble pillar[5]arene. Their association constant was calculated to be (6.8±0.9)×106  M-1 . After self-assembly in water, regular spherical nanocarriers can be formed; these were used to encapsulate the hypoxia-activated prodrug tirapazamine (TPZ). BDP-CN displayed excellent photodynamic activity to complete PDT. In this process, O2 can be continuously consumed to activate TPZ to allow it to be converted to a benzotriazinyl (BTZ) radical with high cytotoxicity to complete chemotherapy. As a result, the formed nanoparticles showed excellent synergistic photodynamic therapy and chemotherapy efficacy. The synergistic therapy mechanism is discussed in detail.

The application of enzymes in synthetic organic chemistry has emerged as a powerful means to generate molecular complexity in a highly selective, efficient, and sustainable manner. While enzymes have increasingly been incorporated into synthetic sequences for numerous academic and industrial applications on their own and in sequential processes, their utility in cooperative catalysis with small molecule catalytic platforms has recently drawn increased attention across the field of organic synthesis. In this review, we present a selection of notable accomplishments in cooperative chemoenzymatic catalysis and provide a perspective on its future directions.

Clinical imaging techniques are widely used to detect, locate, and track the growth or shrinkage of cancerous tumors. Although these techniques have shown impressive results, they often come with health risks due to the use of toxic contrast agents or ionizing radiation. To address these limitations, research efforts have been focused on the development of new imaging techniques. Among the emerging medicinal methods, photoacoustic imaging is receiving much attention. This method effectively combines the most important benefits of both ultrasound and fluorescence imaging, while minimizing their respective drawbacks via a light-in and ultrasound-out approach. This review article focuses on the fundamental concept, recent advances, and strategies for novel contrast agents based on molecular metal complexes or metallic nanoparticles for use in photoacoustic imaging.

The surface proteome or "surfaceome" is a critical mediator of cellular biology, facilitating cell-to-cell interactions and communication with extracellular biomolecules. Constituents of the surfaceome can serve as biomarkers for changing cell states and as targets for pharmacological intervention. While some pathways of cell surface trafficking are well characterized to allow prediction of surface localization, some non-canonical trafficking mechanisms do not. Basigin (Bsg), a cell surface glycoprotein, has been shown to chaperone protein clients to the cell surface. However, understanding which proteins are served by Bsg is not always straightforward. To accelerate such identification, we applied a surfaceome proximity labeling method that is integrated with quantitative mass spectrometry-based proteomics to discern changes in the surfaceome of hepatic stellate cells that occur in response to the genetic loss of Bsg. Using this strategy, we observed that the loss of Bsg leads to corresponding reductions in the cell surface expression of monocarboxylate transporters MCT1 and MCT4. We also found that these relationships were unique to Bsg and not found in neuroplastin (Nptn), a related family member. These results establish the utility of the surfaceome proximity labeling method to determine clients of cell surface chaperone proteins.

Cyclic peptides are important building blocks for forming functional structures and have been applied in various fields. Considering the significant structural and functional roles of cyclic peptides in materials science and the attributed biophysical advantages, we provide an overview of cyclic peptide types that can self-assemble to form nanotubes, recent progress in stimuli-triggered cyclic peptide assembly, and methods to construct peptide and polymer conjugates based on cyclic peptides with alternative chirality. Specifically, we highlight the roles that stimuli-triggered cyclic peptides and their conjugates play in biomedical applications. Recent progress in other cyclic peptides acting as gelators in drug delivery and biomedicine are also summarized. These cyclic peptides with self-assembly properties are expected to act as adaptive systems for drug delivery and selective disease targeting.

Protein lipidation is a widespread modification that regulates protein subcellular localization, structure and function. Dysregulation of protein lipidation has been implicated in various human diseases, including neurological disorders, infectious diseases and cancers. Thus lipid-modifying enzymes and their substrate proteins are emerging as attractive drug targets. The development of small-molecule modulators of protein lipidation has remarkably impacted our understanding of lipid-modification biology and potential therapeutics. In this review, we summarize recent progress in small-molecule targeting of protein lipidation and highlight therapeutic opportunities.

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products - Rv1915 and Rv1916 - due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and 1 H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.

Adenosylcobalamin (AdoCbl), or coenzyme B12 , is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes that generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co-C) bond for catalysis. How do the enzymes activate and cleave the Co-C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of the adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies, which aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase, established the crucial importance of the steric strain of the Co-C bond and conformational stabilization of the adenosyl radical for coenzyme B12 catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B12 enzymes.

The early-career researchers showcased in this ChemBioTalents special collection, and many others who have established their independent scientific careers over the last three years, have experienced a unique set of circumstances. The Covid-19 pandemic necessitated new forms of communication and interpersonal interactions: From online interviews and virtual networking to relocating and establishing labs during a pandemic, we faced many challenges, but also unexpected opportunities. In this perspective, we reflect on this unique and formative time through personal anecdotes and viewpoints, trying to capture diverse experiences from the Chemical Biology community and beyond. We have tried to get a broad and varied set of perspectives, however, the selection is biased towards researchers who were able to start their independent careers.1.

Current cancer treatments damage healthy cells and tissues, causing short-term and long-term side effects. New treatments are desired that show greater selectivity toward cancer cells and evade the common mechanisms of multidrug resistance. Membranolytic anticancer peptides (mACPs) hold promise against cancer and multidrug resistance. Amphipathicity, hydrophobicity, and net charge of mACPs participate in their respective interactions with cell membranes and their overall inhibition of cancer cells. To support the design of cell-line selective mACPs, we investigated the relationships that amino acid composition, physicochemical properties, sequence motifs, and sequence homology could have with their potency and selectivity towards several healthy and cancer cell lines. Sequence length and net charge are known to affect the selectivity of mACPs between cancer and healthy cell lines. Our study reveals that increasing the net charge or flexibility (i. e., small and aliphatic residues) influences their selectivity between cancer cell lines with comparable lipid compositions.

Mononuclear nonheme iron enzymes catalyze a large variety of oxidative transformations responsible for various biosynthesis and metabolism processes. Unlike their P450 counterparts, non-heme enzymes generally possess flexible and variable coordination architecture, which can endow rich reactivity for non-heme enzymes. This Concept highlights that the coordination dynamics of iron can be a key player in controlling the activity and selectivity of non-heme enzymes. In ergothioneine synthase EgtB, the coordination switch of the sulfoxide radical species enables the efficient and selective C-S coupling reaction. In iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases, the conformational flip of ferryl-oxo intermediate can be extensively involved in selective oxidation reactions. Especially, the five-coordinate ferryl-oxo species may allow the substrate coordination via O or N atom, which may facilitate the C-O or C-N coupling reactions via stabilizing the transition states and inhibiting the unwanted hydroxylation reactions.

We report the synthesis of a series of hydrazonyl sultones (HS) containing an ortho-CF3 group, a five- or six-membered sultone ring, and a varying N-aryl substituent, and characterization of their aqueous stability and reactivity toward bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) in a 1,3-dipolar cycloaddition reaction. To avoid purification of highly polar intermediates, we employed two protecting groups in our synthetic schemes. Most HS were obtained in moderate to good yields under optimized reaction conditions. The X-ray crystal structure analysis of two HS revealed that the partially negative-charged fluorine atoms in CF3 electrostatically shield the electrophilic nitrile imine (NI) center from a nucleophilic attack, underpinning their extraordinary aqueous stability. In addition, the N-aryl substituents further modulate HS reactivity and stability, with the electron-rich six-membered HS displaying excellent aqueous stability and increased cycloaddition reactivity. The utility of these improved HS reagents was demonstrated through fast and selective modification of a BCNK-encoded nanobody with second-order rate constants as high as 1500 M-1  s-1 in phosphate-buffered saline-ethanol (9 : 1), representing the fastest HS-BCN ligation reported in the literature.

Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A : T→G : C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G : C→A : T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Collimonas sp. (D-25), found in the soil of Akita Prefecture, is a gram-negative bacterium with the ability to synthesize gold nanoparticles (AuNPs). During the synthesis of AuNPs, one specific protein (DP-1) was found to have disappeared in the sonicated solution of the bacterium. Recombinant DP-1 (rDP-1) from Escherichia coli BL21 (DE3) was used to study the effect of DP-1 on the synthesis of AuNPs. AuNPs synthesized with rDP-1 result in small, stabilized nanoparticles. AuNPs synthesized by DP-1 retained the stability of both the dispersion and nano-size particles under high salt concentrations. Isothermal titration calorimetry was employed to investigate the bonding ratio of rDP-1 to AuNPs. Several thousand rDP-1 proteins are attached to the surface of an AuNP to form a protein corona containing multiple layers. These results suggest that DP-1 obtained from D-25 has a size and stability control function during AuNP synthesis.

Lipids are key constituents of numerous biomedical drug delivery technologies. Here, we present the design, synthesis and biophysical characterizations of a library of cationic lipids containing an acetal residue in their linker region. These cationic acetal lipids (CALs) were conveniently prepared through a trans-acetalization protocol from commercially available precursors. NMR studies highlighted the conformational rigidity at the acetal residue and the high hydrolytic stability of these CALs. Fluorescence anisotropy studies revealed that the CAL with a pyridinium headgroup (CAL1) formed highly cohesive vesicular aggregates in water. These structural and self-assembly features of the CAL1 allowed up to 196 % w/w loading of curcumin (Cur) as a representative hydrophobic drug. A reconstitutable formulation of Cur was obtained as a result, which could deliver the drug inside mammalian cells with very high efficiency. The hemocompatibility and cytocompatibility of CAL1 was significantly enhanced by creating a coating of polydopamine (PDA) onto its vesicular assemblies to produce hybrid lipid-polymer nanocapsules. This work demonstrates rapid access to the useful synthetic lipid formulations with high potential in drug and gene delivery applications.

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.

The melleolides are a family of structurally and functionally diverse sesquiterpenoids with potential applications as fungicides, antimicrobials, and cancer therapeutics. The initial and terminal steps of the biosynthesis pathway in Armillaria spp. have been characterized, but the intermediate steps are unclear. Biosynthetic gene clusters in A. mellea and A. gallica were shown to encode a terpene cyclase, a polyketide synthase, and four CYP450 monooxygenases. We have characterized CYPArm3, which is responsible for the hydroxylation of Δ-6-protoilludene, but the functions of the other CYP450s remain to be determined. Here we describe CYPArm2, which accepts Δ-6-protoilludene and 8α-hydroxy-6-protoilludene as substrates. To investigate the products in more detail, we generated recombinant Saccharomyces cerevisiae strains overexpressing CYPArm2 in combination with the previously characterized protoilludene synthase and 8α-hydroxylase. Using this total biosynthesis approach, sufficient quantities of product were obtained for NMR spectroscopy. This allowed the identification of 8α,13-dihydroxy-protoilludene, confirming that CYPArm2 is a protoilludene 13-hydroxylase.

Chitin, the most abundant amino polysaccharide in Nature, has many applications in different fields. However, processing of this recalcitrant biopolymer in an environmentally friendly manner remains a major challenge. In this context, lytic polysaccharide monooxygenases (LPMOs) are of interest, as they can act on the most recalcitrant parts of chitin and related insoluble biopolymers such as cellulose. Efficient LPMO catalysis can be achieved by feeding reactions with H2 O2 , but careful control of H2 O2 is required to avoid autocatalytic enzyme inactivation. Herein, we present a coupled enzyme system in which a choline oxidase from Arthrobacter globiformis is employed for controlled in situ generation of H2 O2 that fuels LPMO-catalyzed oxidative degradation of chitin. We show that the rate, stability and extent of the LPMO reaction can be manipulated by varying the amount of choline oxidase and/or its substrate, choline chloride, and that efficient peroxygenase reactions may be achieved using sub-μM concentrations of the H2 O2 -generating enzyme. This coupled system requires only sub-stoichiometric amounts of the reductant that is needed to keep the LPMO in its active, reduced state. It is conceivable that this enzyme system may be used for bioprocessing of chitin in choline-based natural deep eutectic solvents.

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.

Horse Liver Alcohol Dehydrogenase (HLADH) has been immobilized on calcium-alginate beads and used for both oxidation and reduction reactions. To avoid swelling of the beads and their subsequent breakage, calcium ions were added to both reaction and storage solutions, allowing the beads to maintain the initial structural features. The techniques used for this purpose revealed that 2 mM Ca2+ is the optimal concentration, which does not significantly change the weight of the beads, the amount of water in them, and their external and internal structure. The optimized experimental procedure has been used to verify the properties of the enzyme in terms of reusability, storage, and thermal stability. The addition of calcium ions allows the enzyme to retain more than 80 % of its initial activity for fourteen cycles and approximately 50 % at the twentieth cycle. Moreover, when the biocatalyst has been stored in a buffer solution containing 2 mM Ca2+ , the retention of enzyme activity after 30 days was 100 %, compared to that measured before incubation. The encapsulated enzyme exhibits greater thermal stability than free HLADH up to at least 60 °C, preventing dimer dissociation into the two subunits.

Pyrazines are ubiquitous in nature - biosynthesized by microorganisms, insects, and plants. Due to their great structural diversity, they own manifold biological functions. Alkyl- and alkoxypyrazines for instance play a key role as semiochemicals, but also as important aroma compounds in foods. Especially 3-alkyl-2-methoxypyrazines (MPs) have been of great research interest. MPs are associated with green and earthy attributes. They are responsible for the distinctive aroma properties of numerous vegetables. Moreover, they have a strong influence on the aroma of wines, in which they are primarily grape-derived. Over the years various methods have been developed and implemented to analyse the distribution of MPs in plants. In addition, the biosynthetic pathway of MPs has always been of particular interest. Different pathways and precursors have been proposed and controversially discussed in the literature. While the identification of genes encoding O-methyltransferases gave important insights into the last step of MP-biosynthesis, earlier biosynthetic steps and precursors remained unknown. It was not until 2022 that in vivo feeding experiments with stable isotope labeled compounds revealed l-leucine and l-serine as important precursors for IBMP. This discovery gave evidence for a metabolic interface between the MP-biosynthesis and photorespiration.

With an increasing demand for macromolecular biotherapeutics, the issue of their poor cell-penetrating abilities requires viable and relevant solutions. Herein, we report tripeptides bearing an amino acid with a perfluoroalkyl (RF ) group adjacent to the α-carbon. RF -containing tripeptides were synthesized and evaluated for their ability to transport a conjugated hydrophilic dye (Alexa Fluor 647) into the cells. RF -containing tripeptides with the fluorophore showed high cellular uptake efficiency and none of them were cytotoxic. Interestingly, we demonstrated that the absolute configuration of perfluoroalkylated amino acids (RF -AAs) affects not only nanoparticle formation but also the cell permeability of the tripeptides. These novel RF -containing tripeptides are potentially useful as short and noncationic cell-penetrating peptides (CPPs).