Backbone Modifications Research Articles

Grape color variation involves genetic and micro-environmental changes that alter berry phenolic and aromatic composition

Black- and white-berried grapevine cultivars are distinguished by their ability to accumulate anthocyanins in the berry skin. To assess possible side effects of color variation in berry development and composition in a near-isogenic background, we compared white-berried somatic variants (WV) to their black-berried ancestors in ‘Garnacha’ and ‘Tempranillo’ cultivars. Absence of anthocyanins correlated with lower berry temperature during daytime in WV. At the transcriptome level, besides genes related to anthocyanin accumulation, transcripts encoding enzymes involved in flavonoid backbone biosynthesis and modification were downregulated from veraison in WV skin. Genes mapping to hemizygous genome regions in WV were also downregulated irrespective of berry tissue or developmental stage. Light-responsive genes including flavonol and monoterpenoid biosynthesis genes were upregulated in WV from veraison. In agreement, flavonol partitioning was altered and tri-hydroxylated forms were practically absent in WV, whereas higher levels of specific volatile monoterpenoids and their soluble precursors were detected in WV pericarp. Amino acid precursors of phenolic compounds tended to be higher in WV pericarp. Collectively, greater differences were observed in ‘Tempranillo’ than in ‘Garnacha’, revealing effects of genetic background. These results indicate that the grape color locus directly controls the metabolism of colorless flavonoids, whereas additional alterations in grape quality compounds are established in a cultivar-dependent manner in response to alteration of the berry microclimate caused by the absence of anthocyanins.

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase

AbstractDuring the total chemical synthesis of the water‐soluble globular Haemophilus Influenzae DNA ligase (Hin‐Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance‐improving strategies, such as installation of a terminal/side‐chain Arg tag or O‐acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase.

During the total chemical synthesis of the water-soluble globular Haemophilus Influenzae DNA ligase (Hin-Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance-improving strategies, such as installation of a terminal/side-chain Arg tag or O-acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.

Phenothiazine-Based Redox Polymers As Cathode-Active Materials in Li/Organic Batteries

The rising demand for energy storage is increasing the research on so-called next generation or beyond lithium ion battery technologies.[1] The most promising candidates are lithium metal-based batteries (LMBs) with the Li/S battery as the most advanced example.[2] Even though LMBs show a high energy storage ability, the sluggish kinetics combined with safety concerns keeps the field open for alternative battery principles. One of these are organic-based materials which offer a great opportunity for the next generation of energy storage.[3] Their energy densities are mostly lower compared to their inorganic counterparts, but the ability of fast electron transfer and therefore high kinetics leads to an increased power density (e.g. 33.6 kW kg−1[4] vs. common battery materials, e.g. 4.75 kW kg−1[5]). Redox polymers are one of the most versatile organic-based materials for energy storage as they show decreased solubility and offer countless possibilities for modifications of the polymeric backbone and the side chains, while the polymer itself can be conjugated or non-conjugated.[6] Herein, we present a non-conjugated redox polymer with a substituted poly(vinylene) backbone.[7] The redox active side chain consists of a methyl phenothiazine moiety and undergoes an one-electron transfer process during oxidation and forms a radical cation. This oxidation is accompanied by an anion insertion into the polymeric structure. Due to a π-π-interaction between the side chains this polymer is able to enable long-term cycling with increased C-rates resulting in an ultra high cycling stability of 10,000 cycles with a capacity retention of 93%. In this work, we will describe these interactions and verify them with spectroscopic methods like EPR- and X-ray photoelectron spectroscopy (XPS). Additionally, density functional theory calculations (DFT) are shown which support the strong stabilizing effect of the π-π-interactions in this organic material.[7] Beside the mechanistic studies on molecular scale, we will comment further on the mechanism on the macroscopic electrode level. Here, we performed microscopic (SEM, LSM) and spectroscopic experiments (XPS, UV/Vis) to uncover the unique cycling behavior of PVMPT.[8] Finally, we will present an approach to increase the cycling capacity of the material and discuss the influence on the stabilizing interactions.[9]

Discovery of novel fungal RiPP biosynthetic pathways and their application for the development of peptide therapeutics.

Bioactive peptide natural products are an important source of therapeutics. Prominent examples are the antibiotic penicillin and the immunosuppressant cyclosporine which are both produced by fungi and have revolutionized modern medicine. Peptide biosynthesis can occur either non-ribosomally via large enzymes referred to as non-ribosomal peptide synthetases (NRPS) or ribosomally. Ribosomal peptides are synthesized as part of a larger precursor peptide where they are posttranslationally modified and subsequently proteolytically released. Such peptide natural products are referred to as ribosomally synthesized and posttranslationally modified peptides (RiPPs). Their biosynthetic pathways have recently received a lot of attention, both from a basic and applied research point of view, due to the discoveries of several novel posttranslational modifications of the peptide backbone. Some of these modifications were so far only known from NRPSs and significantly increase the chemical space covered by this class of peptide natural products. Latter feature, in combination with the promiscuity of the modifying enzymes and the genetic encoding of the peptide sequence, makes RiPP biosynthetic pathways attractive for synthetic biology approaches to identify novel peptide therapeutics via screening of de novo generated peptide libraries and, thus, exploit bioactive peptide natural products beyond their direct use as therapeutics. This review focuses on the recent discovery and characterization of novel RiPP biosynthetic pathways in fungi and their possible application for the development of novel peptide therapeutics.

Backbone modifications in peptidic inhibitors of flaviviral proteases.

The NS2B-NS3 protease is a promising target for the development of drugs against dengue virus (DENV), West Nile virus (WNV) and related flaviviruses. We report the systematic variation of the peptide backbone of the two lead compounds Bz-Arg-Lys-d-Phg-NH2 and Bz-Arg-Lys-d-Phg(OBn)-NH2. While inhibitory activity against WNV protease was generally decreased, the inhibitory potency against DENV protease could be conserved and increased in several peptidomimetics, particularly in those containing a (NMe)arginine fragment or an N-terminal α-keto amide. Methylation at the α-position of the C-terminal phenylglycine led to a 6-fold higher potency against DENV protease. Peptidomimetics with modified backbone showed increased resistance against hydrolytic attack by trypsin and α-chymotrypsin.

Expanding the Functional Scope of the Fmoc-Diphenylalanine Hydrogelator by Introducing a Rigidifying and Chemically Active Urea Backbone Modification.

Peptidomimetic low‐molecular‐weight hydrogelators, a class of peptide‐like molecules with various backbone amide modifications, typically give rise to hydrogels of diverse properties and increased stability compared to peptide hydrogelators. Here, a new peptidomimetic low‐molecular‐weight hydrogelator is designed based on the well‐studied N‐fluorenylmethoxycarbonyl diphenylalanine (Fmoc‐FF) peptide by replacing the amide bond with a frequently employed amide bond surrogate, the urea moiety, aiming to increase hydrogen bonding capabilities. This designed ureidopeptide, termed Fmoc—Phe—NHCONH—Phe—OH (Fmoc‐FuF), forms hydrogels with improved mechanical properties, as compared to those formed by the unmodified Fmoc‐FF. A combination of experimental and computational structural methods shows that hydrogen bonding and aromatic interactions facilitate Fmoc‐FuF gel formation. The Fmoc‐FuF hydrogel possesses properties favorable for biomedical applications, including shear thinning, self‐healing, and in vitro cellular biocompatibility. Additionally, the Fmoc‐FuF, but not Fmoc‐FF, hydrogel presents a range of functionalities useful for other applications, including antifouling, slow release of urea encapsulated in the gel at a high concentration, selective mechanical response to fluoride anions, and reduction of metal ions into catalytic nanoparticles. This study demonstrates how a simple backbone modification can enhance the mechanical properties and functional scope of a peptide hydrogel.

Minicircle DNA purification: Performance of chromatographic monoliths bearing lysine and cadaverine ligands

Minicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.

Peptide Modifications: Versatile Tools in Peptide and Natural Product Syntheses

Peptide modifications via C–C bond formation have emerged as valuable tools for the preparation and alteration of non-proteinogenic amino acids and the corresponding peptides. Modification of glycine subunits in peptides allows for the incorporation of unusual side chains, often in a highly stereoselective manner, orchestrated by the chiral peptide backbone. Moreover, modifications of peptides are not limited to the peptidic backbone. Many side-chain modifications, not only by variation of existing functional groups, but also by C–H functionalization, have been developed over the past decade. This account highlights the synthetic contributions made by our group and others to the field of peptide modifications and their application in natural product syntheses.1 Introduction2 Peptide Backbone Modifications via Peptide Enolates2.1 Chelate Enolate Claisen Rearrangements2.2 Allylic Alkylations2.3 Miscellaneous Modifications3 Side-Chain Modifications3.1 C–H Activation3.1.1 Functionalization via Csp3–H Bond Activation3.2.2 Functionalization via Csp2–H Bond Activation3.2 On Peptide Tryptophan Syntheses4 Conclusion

Enhancement of the Electrical Properties of DNA Molecular Wires through Incorporation of Perylenediimide DNA Base Surrogates.

DNA has long been viewed as a promising material for nanoscale electronics, in part due to its well-ordered arrangement of stacked, pi-conjugated base pairs. Within this context, a number of studies have investigated how structural changes, backbone modifications, or artificial base substitutions affect the conductivity of DNA. Herein, we present a comparative study of the electrical properties of both well-matched and perylene-3,4,9,10-tetracarboxylic diimide (PTCDI)-containing DNA molecular wires that bridge nanoscale gold electrodes. By performing current-voltage measurements for such devices, we find that the incorporation of PTCDI DNA base surrogates within our macromolecular constructs leads to an approximately 6-fold enhancement in the observed current levels. Together, these findings suggest that PTCDI DNA base surrogates may enable the preparation of designer DNA-based nanoscale electronic components.

Quantitative mapping of DNA phosphorothioatome reveals phosphorothioate heterogeneity of low modification frequency.

Phosphorothioate (PT) modifications of the DNA backbone, widespread in prokaryotes, are first identified in bacterial enteropathogens Escherichia coli B7A more than a decade ago. However, methods for high resolution mapping of PT modification level are still lacking. Here, we developed the PT-IC-seq technique, based on iodine-induced selective cleavage at PT sites and high-throughput next generation sequencing, as a mean to quantitatively characterizing the genomic landscape of PT modifications. Using PT-IC-seq we foud that most PT sites are partially modified at a lower PT frequency (< 5%) in E. coli B7A and Salmonella enterica serovar Cerro 87, and both show a heterogeneity pattern of PT modification similar to those of the typical methylation modification. Combining the iodine-induced cleavage and absolute quantification by droplet digital PCR, we developed the PT-IC-ddPCR technique to further measure the PT modification level. Consistent with the PT-IC-seq measurements, PT-IC-ddPCR analysis confirmed the lower PT frequency in E. coli B7A. Our study has demonstrated the heterogeneity of PT modification in the bacterial population and we also established general tools for rigorous mapping and characterization of PT modification events at whole genome level. We describe to our knowledge the first genome-wide quantitative characterization of PT landscape and provides appropriate strategies for further functional studies of PT modification.

Protein Substrates for Reaction Discovery: Site-Selective Modification with Boronic Acid Reagents.

Chemical modification of natural proteins must navigate difficult selectivity questions in a complex polyfunctional aqueous environment, within a narrow window of acceptable conditions. Limits on solvent mixtures, pH, and temperature create challenges for most synthetic methods. While a protein's complex polyfunctional environment undoubtedly creates challenges for traditional reactions, we wondered if it also might create opportunities for pursuing new bioconjugation reactivity directly on protein substrates. This Account describes our efforts to date to discover and develop new and useful reactivity for protein modification by starting from an open-ended screen of potential transition-metal catalysts for boronic acid reactivity with a model protein substrate. By starting from a broad screen, we were hoping to take advantage of the very many potential reactive sites on even a small model protein. And perhaps more importantly, whole proteins as reaction screening substrates might exhibit uniquely reactive local environments, the results of a dense combination of functional groups that would be nearly impossible to mimic in a small-molecule context. This effort has resulted in the discovery of four new protein modification reactions with boronic acid reagents, including a remarkable modification of specific backbone N-H bonds. This histidine-directed Chan-Lam coupling, based on specific proximity of an imidazole and two amide groups, is one important example of powerful reactivity that depends on a combination of functional groups that proteins make possible. Other bioconjugation reactions uncovered include a three-component tyrosine metalation with rhodium(III), a nickel-catalyzed cysteine arylation, and an unusual ascorbate-mediated oxidative process for N-terminal modification. The remarkably broad scope of reactivity types encountered in this work is a testament to the breadth of boronic acid reactivity. It is also a demonstration of the diverse reactivities that are possible by the combined alteration of boronic acid structure and metal promoter. The discovery of specific backbone modification chemistry has been a broadly empowering reactivity. Pyroglutamate, a naturally occurring posttranslational modification, exhibits remarkably high reactivity in histidine-directed backbone modification, which allows us to treat pyroglutamate as a reactive bioorthogonal handle that is readily incorporated into proteins of interest by natural machinery. In another research direction, the development of a vinylogous photocleavage system has allowed us to view backbone modification as a photocaging modification which is released by exposure to light.

Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2′-deoxy-2′-fluoro (2′-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2′-F-monomer metabolites was low and transient in rats and humans. In vitro, 2′-F-nucleoside 5′-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2′-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2′-F-monomers were observed after weekly administration of two GalNAc–siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2′-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc–siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Analyzing Change in Protein Stability Associated with Single Point Deletions in a Newly Defined Protein Structure Database.

Protein backbone alternation due to insertion/deletion or mutation operation often results in a change of fundamental biophysical properties of proteins. The proposed work intends to encode the protein stability changes associated with single point deletions (SPDs) of amino acids in proteins. The encoding will help in the primary screening of detrimental backbone modifications before opting for expensive in vitro experimentations. In the absence of any benchmark database documenting SPDs, we curate a data set containing SPDs that lead to both folded conformations and unfolded state. We differentiate these SPD instances with the help of simple structural and physicochemical features and eventually classify the foldability resulting out of SPDs using a Random Forest classifier and an Elliptic Envelope based outlier detector. Adhering to leave one out cross validation, the accuracy of the Random Forest classifier and the Elliptic Envelope is of 99.4% and 98.1%, respectively. The newly defined database and the delineation of SPD instances based on its resulting foldability provide a head start toward finding a solution to the given problem.

Influence of backbone modification of difluoroquinoxaline-based copolymers on the interchain packing, blend morphology and photovoltaic properties of nonfullerene organic solar cells

The impact of the crystalline orientation of donor and acceptor molecules on the photovoltaic properties with modification of polymer backbone structures.

Receptor selectivity from minimal backbone modification of a polypeptide agonist

Human parathyroid hormone (PTH) and N-terminal fragments thereof activate two receptors, hPTHR1 and hPTHR2, which share ∼51% sequence similarity. A peptide comprising the first 34 residues of PTH is fully active at both receptors and is used to treat osteoporosis. We have used this system to explore the hypothesis that backbone modification of a promiscuous peptidic agonist can provide novel receptor-selective agonists. We tested this hypothesis by preparing a set of variants of PTH(1-34)-NH2 that contained a single β-amino-acid residue replacement at each of the first eight positions. These homologs, each containing one additional backbone methylene unit relative to PTH(1-34)-NH2 itself, displayed a wide range of potencies in cell-based assays for PTHR1 or PTHR2 activation. The β-scan series allowed us to identify two homologs, each containing two α→β replacements, that were highly selective, one for PTHR1 and the other for PTHR2. These findings suggest that backbone modification of peptides may provide a general strategy for achieving activation selectivity among polypeptide-modulated receptors, and that success requires consideration of both β2- and β3-residues, which differ in terms of side-chain location.

Enhanced Stability of DNA Oligonucleotides with Partially Zwitterionic Backbone Structures in Biological Media.

Deficient stability towards nuclease-mediated degradation is one of the most relevant tasks in the development of oligonucleotide-derived biomedical agents. This hurdle can be overcome through modifications to the native oligonucleotide backbone structure, with the goal of simultaneously retaining the unique hybridization properties of nucleic acids. The nucleosyl amino acid (NAA)-modification is a recently introduced artificial cationic backbone linkage. Partially zwitterionic NAA-modified oligonucleotides had previously shown hybridization with DNA strands with retained base-pairing fidelity. In this study, we report the significantly enhanced stability of NAA-modified oligonucleotides towards 3′- and 5′-exonuclease-mediated degradation as well as in complex biological media such as human plasma and whole cell lysate. This demonstrates the potential versatility of the NAA-motif as a backbone modification for the development of biomedically active oligonucleotide analogues.

Expanding the Material Space of Biosustainable Poly(sophorolipids) by Modular Functionalization.

A general strategy to modify the structurally interesting poly(lactonic sophorolipid) (Poly(LSL)), a polymer derived from the biobased sophorolipid monomer, is presented. Effective backbone modification is achieved via a triazolinedione (TAD)-ene-reaction. This enables the straightforward introduction of various functionalities to the double bond in the fatty acid segment of the Poly(LSL). The reaction occurs quantitatively in stoichiometric ratios up to a targeted functionalization degree of 50% and complete functionalization of all double bonds is feasible when three equivalents excess of the TAD moiety with respect to the double bonds are used. It is shown that the thermal and mechanical properties of the modified polymers can be tailored via type and degree of functionalization. The exploited TAD ligation fulfills the criteria of an economic and efficient reaction, making the presented modification strategy straightforward to fine-tune the material properties and extending the applicability of Poly(LSL) as a material.

Metabolism of Peptide Drugs and Strategies to Improve their Metabolic Stability.

Despite the therapeutic use of peptides is limited because of their metabolism in vivo, there are no systematic reviews explaining degradation of peptides by peptidases. This review summarizes peptidases present in the tissues and metabolic characteristics of peptides, and provides recent strategies for improving the metabolic stability of peptides. We reviewed a number of peptidases including their functional groups, tissue localization and cleavage specificity. Given the broad distribution of peptidases in the body, several tissues, such as the liver, kidney, lung, blood, nasal epithelial cells, placenta and skin, have the capacity to metabolize peptides. We compared the metabolic characteristics of peptides in these tissues and then summarized strategies for improving peptide stability. In addition to the primary organs including liver, kidney, gastrointestinal tract and blood involved in peptide metabolism, other organs such as the lung, skin, placenta and nasal mucosa may also play a role in peptide degradation. At present, the main measures to improve the stability of the peptide include N- and/or C-terminal modification or substitution, D-amino acid or unnatural amino acid substitution, cyclization, backbone modification, nanoparticle formulations and increased molecular mass. This review summarized the key in vivo peptidases and their tissue distribution characteristics, and presented strategies to improve the metabolic stability and bioavailability of peptide drugs. These viewpoints will benefit the further development and utilization of peptide drugs.

Large-Stokes-shift-based folded DNA probing systems targeting DNA and miRNA 21 with signal amplification

Large-Stokes-shift based simple folded DNA probing system (LSFP) had a simple folded DNA structure and exhibited a large Stokes-shifted (194 nm) fluorescence signal upon excitation at a single wavelength (386 nm). This Stokes shift was achieved through a simple combination of donor and acceptor fluorophores and employing multi-FRET systematically. This unique large Stokes-shifted fluorescence signal was used to detect target DNA with large increases in the fluorescence signal (9.7–14.2 fold). This LSFP exhibited enough selectivity, distinguishing a perfectly matched sequence from the probe itself and mismatched sequences. Surprisingly, when DSN was used for signal amplification with miR21P probing system whose target is miRNA 21, it showed high sensitivity (13.7 aM) and selectivity (one base mismatch discrimination). This system has several advantages over other molecular beacons (MBs): (i) it is easy to design and synthesize the probing system that does not require the construction of a finely designed stem and loop, as in most MBs (this can prevent the degradation of miR21P itself by DSN enzyme without special backbone modification); (ii) it can control unique fluorescence, such as large Stokes-shifted fluorescence through a simple combination of donor and acceptor fluorophores; and (iii) through signal amplification using DSN, it can efficiently detect extremely small amounts of target miRNA with high sensitivity (13.7 aM).