RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

ABNOH-Linked Nucleotides and DNA for Bioconjugation and Cross-linking with Tryptophan-Containing Peptides and Proteins.

Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of ABNO-H-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable 3-fused hexahydropyrrolo-indole conjugates. Similarly modified ABNO-H-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNO-H-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.

Nucleotides Bearing Red Viscosity-Sensitive Dimethoxy-Bodipy Fluorophore for Enzymatic Incorporation and DNA Labeling.

Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 3,3'-dimethoxy-2,2'-diphenyl-6-(4-hydroxyphenyl)-bodipy fluorophore attached through a propargyl or propargyl-triethylene glycol linker to position 5 of 2'-deoxycytidine were designed and synthesized. They exerted bright red fluorescence and good sensitivity to viscosity changing their lifetime from 1.6 to 4.5 ns. The modifed dNTPs were substrates for DNA polymerases and were used in enzymatic synthesis of labeled DNA through primer extension. The modified DNA probes served as viscosity sensors responding to protein binding by changes of lifetime. The nucleotide with longer linker (dCpegMOBTP) was transported to live cells and incorporated into the genomic DNA, which can be useful for staining of DNA and imaging of DNA synthesis.

trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging.

A series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 2- or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO- and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels-Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.

Unlocking the Hydrolytic Mechanism of GH92 α-1,2-Mannosidases: Computation Inspires the use of C-Glycosides as Michaelis Complex Mimics.

The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via O S2 →B2,5 →1 S5 and 3 S1 →3 H4 →1 C4 . For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4 C1 →4 H5 /1 S5 →1 S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH's natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5 →B2,5 /1 S5 →1 S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.

Glucosylated 5-Hydroxymethylpyrimidines as Epigenetic DNA Bases Regulating Transcription and Restriction Cleavage.

5-(ß-d-Glucopyranosyloxymethyl)-2'-deoxyuridine and -cytidine 5'-O-triphosphates were prepared and used for polymerase-mediated (primer extension or PCR) synthesis of DNA containing glucosylated 5-hydroxymethyluracil (5hmU) or 5-hydroxymethyluracil (5hmC). The presence of any glucosylated pyrimidines fully protected DNA from cleavage by type II restriction endonucleases. On the other hand, while the presence of glucosylated 5hmU completely inhibited transcription by bacterial (Escherichia coli) RNA polymerase, the DNA containing the corresponding glucosylated 5hmC allowed a similar level of transcription as natural DNA. This suggests different roles of these hypermodified bases in the epigenetic regulation of transcription in bacteriophages or kinetoplastid parasites. Consequently, enzymatic glucosylation of 5hmC-containing DNA can be used for tuning of transcription activity.

Glyoxal-Linked Nucleotides and DNA for Bioconjugations and Crosslinking with Arginine-Containing Peptides and Proteins.

Glyoxal-linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of 4-azidophenylglyoxal or a Sonogashira reaction of 4-bromophenylglyoxal with 5-ethynyl-dUMP or -dUTP. The triphosphates were used as substrates for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The glyoxal-linked nucleotides reacted with arginine-containing peptides to form stable imizadolone-linked conjugates. This reactive glyoxal modification in DNA was used for efficient bioconjugations and crosslinking with Arg-containing peptides or proteins (e. g., histones) and was found to be more reactive than previously reported 1,3-diketone-linked DNA probes.

Silicon-bridged (1→1)-disaccharides: an umpoled glycomimetic scaffold.

Modification of the carbohydrate scaffold is an important theme in drug and vaccine discovery. Therefore, the preparation of novel types of glycomimetics is of interest in synthetic carbohydrate chemistry. In this manuscript, we present an early investigation of the synthesis, structure, and conformational behaviour of (1→1)-Si-disaccharides as a novel type of glycomimetics arising from the replacement of interglycosidic oxygen with a dimethyl-, methylpropyl-, or diisopropylsilyl linkage. We accomplished the preparation of this unusual group of umpoled compounds by the reaction of lithiated glycal or 2-oxyglycal units with dialkyldichlorosilanes. We demonstrated the good stability of the "Si-glycosidic" linkage under acidic conditions even at elevated temperatures. Next, we described the conformational landscape of these compounds by the combination of in silico modelling with spectroscopic and crystallographic methods. Finally, we explained the observed conformational flexibility of these compounds by the absence of gauche stabilizing effects that are typically at play in natural carbohydrates.

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing.

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3'-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3'-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Facile Approach to C-Glucosides by Using a Protecting-Group-Free Hiyama Cross-Coupling Reaction: High-Yielding Dapagliflozin Synthesis.

Invited for the cover of this issue is Kamil Parkan and co-workers at University of Chemistry and Technology and Institute of Organic Chemistry and Biochemistry, Prague. The cover graphic depicts a schematic representation of the assembly of aryl-C-glycosides based on a protecting-group-free Hiyama reaction. Read the full text of the article at 10.1002/chem.202101052.

Facile Approach to C-Glucosides by Using a Protecting-Group-Free Hiyama Cross-Coupling Reaction: High-Yielding Dapagliflozin Synthesis.

Access to unprotected (hetero)aryl pseudo-C-glucosides via a mild Pd-catalysed Hiyama cross-coupling reaction of protecting-group-free 1-diisopropylsilyl-d-glucal with various (hetero)aryl halides has been developed. In addition, selected unprotected pseudo-C-glucosides were stereoselectively converted into the corresponding α- and ß-C-glucosides, as well as 2-deoxy-ß-C-glucosides. This methodology was applied to the efficient and high-yielding synthesis of dapagliflozin, a medicament used to treat type 2 diabetes mellitus. Finally, the versatility of our methodology was proved by the synthesis of other analogues of dapagliflozin.

First Total Synthesis of Phytoprostanes with Prostaglandin-Like Configuration, Evidence for Their Formation in Edible Vegetable Oils and Orienting Study of Their Biological Activity.

Phytoprostanes (PhytoP) are natural products, which form in plants under oxidative stress conditions from α-linolenic acid. However, their epimers with relative prostaglandin configuration termed phytoglandins (PhytoG) have never been detected in Nature, likely because of the lack of synthetic reference material. Here, the first asymmetric total synthesis of such compounds, namely of PhytoGF1α (9-epi-16-F1t -PhytoP) and its diastereomer ent-16-epi-PhytoGF1α (ent-9,16-diepi-16-F1t -PhytoP), has been accomplished. The synthetic strategy is based on radical anion oxidative cyclization, copper(I)-mediated alkyl-alkyl coupling and enantioselective reduction reactions. A UHPLC-MS/MS study using the synthesized compounds as standards indicates PhytoG formation at significant levels during autoxidation of α-linolenic acid in edible vegetable oils. Initial testing of synthetic PhytoGs together with F1 -PhytoP and 15-F2t -IsoP derivatives for potential interactions with the PGF2α (FP) receptor did not reveal significant activity. The notion that PUFA-derived oxidatively formed cyclic metabolites with prostaglandin configuration do not form to a significant extent in biological or food matrices has to be corrected. Strong evidence is provided that oxidatively formed PhytoG metabolites may be ingested with plant-derived food, which necessitates further investigation of their biological profile.

Synthesis and anti-trypanosomal activity of 3'-fluororibonucleosides derived from 7-deazapurine nucleosides.

Trypanosoma brucei parasites cause Human African Trypanosomiasis and the current drugs for its treatment are often inefficient and toxic. This urges the need to development of new antitrypanosomal agents. We report the synthesis and biological profiling of 3'-deoxy-3'-fluororibonucleosides derived from 7-deazaadenine nucleosides bearing diverse substituents at position 7. They were synthesized through glycosylation of 6-chloro-7-bromo- or -7-iodo-7-deazapurine with protected 3'-fluororibose followed by cross-coupling reactions at position 7 and/or deprotection. Most of the title nucleosides displayed micromolar or submicromolar activity against Trypanosoma brucei brucei. The most active were the 7-bromo- and 7-iododerivatives which exerted double-digit nanomolar activity against T. b. brucei and T. b. gambiense and no cytotoxicity and thus represent promising candidates for further development.

Nucleotides bearing aminophenyl- or aminonaphthyl-3-methoxychromone solvatochromic fluorophores for the enzymatic construction of DNA probes for the detection of protein-DNA binding.

We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores showed solvatochromic fluorescence with strong fluorescence at 433-457 nm in non-polar solvents and very weak fluorescence at 567 nm in alcohols. The nucleosides and nucleotides also showed only negligible fluorescence in alcohols or water. The triphosphates were substrates for DNA polymerase in the enzymatic synthesis of modified DNA probes that showed only very weak fluorescence in aqueous buffer but a significant light-up and blue shift were observed when they interacted with proteins (histone H3.1 or p53 for double-stranded DNA probes or single-strand binding protein for single-stranded oligonucleotide probes). Hence, nucleotides have good potential in the construction of DNA sensors for studying protein-DNA interactions. The modified dNTPs were also transported into cells using a cyclodextrin-based transporter but they were not incorporated into the genomic DNA.

1,2,4-Thiadiazole acyclic nucleoside phosphonates as inhibitors of cysteine dependent enzymes cathepsin K and GSK-3ß.

In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the position 2 of the heterocyclic moiety has been synthesized. Diisopropyl esters of PME- and HPMP-amines have been converted to the N-substituted ureas and then reacted with benzoyl, ethoxycarbonyl, and Fmoc isothiocyanates to give the corresponding thiobiurets, which were oxidatively cyclized to diisopropyl esters of 5-amino-3-oxo-2-PME- or 2-HPMP- 1,2,4-thiadiazol-3(2H)-ones. The phosphonate ester groups were cleaved with bromotrimethylsilane, yielding N5-protected phosphonic acids. The subsequent attempts to remove the protecting group from N5 under alkaline conditions resulted in the cleavage of the 1,2,4-thiadiazole ring. Similarly, compounds with a previously unprotected 5-amino-1,2,4-thiadiazolone base moiety were stable only in the form of phosphonate esters. The series of twenty-one newly prepared 1,2,4-thiadiazol-3(2H)-ones were explored as potential inhibitors of cysteine-dependent enzymes - human cathepsin K (CatK) and glycogen synthase kinase 3ß (GSK-3ß). Several compounds exhibited an inhibitory activity toward both enzymes in the low micromolar range. The inhibitory potency of some of them toward GSK-3ß was similar to that of the thiadiazole GSK-3ß inhibitor tideglusib, whereas others exhibited more favorable toxicity profile while retaining good inhibitory activity.

Multipodal insulin mimetics built on adamantane or proline scaffolds.

Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (Kd value of 0.5 µM) than proline-derived compounds (Kd values of 15-38 µM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics.

1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins.

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

α,γ-Dioxygenated amides via tandem Brook rearrangement/radical oxygenation reactions and their application to syntheses of γ-lactams.

Pyrrolidones are common heterocyclic fragments in various biologically active compounds. Here, a two-step radical-based approach to γ-lactams bearing three to four stereocenters starting from epoxides, N-allylic silylacetamides and TEMPO is reported. The sequence starts with a new tandem nucleophilic substitution/Brook rearrangement/single electron transfer-induced radical oxygenation furnishing orthogonally protected α,γ-dioxygenated N-allylamides with wide scope, mostly good yields, and partly good diastereo- and enantioselectivity for defined combinations of chiral epoxides and chiral amides. This represents a very rare example of an oxidative geminal C-C/C-O difunctionalization next to carbonyl groups. The resulting dioxygenated allylic amides are subsequently subjected to persistent radical effect-based 5-exo-trig radical cyclization reactions providing functionalized pyrrolidones in high yields as diastereomeric mixtures. They converge to 3,4-trans-γ-lactams by base-mediated equilibration, which can be easily further diversified. Stereochemical models for both reaction types were developed.

Ligand-dependent stereoselective Suzuki-Miyaura cross-coupling reactions of ß-enamido triflates.

The stereoselective Suzuki-Miyaura cross-coupling of (Z)-ß-enamido triflates is demonstrated. Depending on the nature of the ligand in the palladium catalyst, either retention or inversion of the configuration during the synthesis of ß,ß-diaryl-substituted enamides is observed. Thus, the method provides synthetic access to both isomers of the target enamides from (Z)-ß-enamido triflates.