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New lipid-linked 2′-deoxyribonucleoside triphosphates were synthesized and used for enzymatic construction of various lipid–oligonucleotide conjugates capable of efficient anchoring to plasma membranes.

Fully aromatic helicenes are attractive building blocks for the construction of inherently chiral π-conjugated macrocyclic nanocarbons. These hitherto rare molecular architectures are envisaged to exhibit remarkable (chir)optical properties, self-assembly, charge/spin transport, induced ring current or a fascinating Möbius topology. Here the synthesis of helically chiral macrocycles that combine angular dibenzo[5]helicene units as corners and linear

The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-LPPOs, loosely based on LPPOs, consisting of a central linker module with two attached connector modules on either side. The connector modules are then decorated with polar and hydrophobic modules. We performed an extensive structure-activity relationship study by varying the length of the linker and hydrophobic modules. The best compounds were active against both Gram-negative and Gram-positive species including multiresistant strains and persisters. LEGO-LPPOs act by first depleting the membrane potential and then creating pores in the cytoplasmic membrane. Importantly, their efficacy is not affected by the presence of serum albumins. Low cytotoxicity and low propensity for resistance development demonstrate their potential for therapeutic use.

Direct N-acylation of 1,2,3-NH-triazoles aimed at obtaining elusive N-acyl-1,2,3-triazoles was investigated. A preference for the formation of thermodynamically favoured N2 isomers was established and an influence of the hard/soft character of the electrophile on the regioselectivity of acylation was found. Although N-acylated 1,2,3-triazoles are hydrolytically unstable compounds, they were isolated and fully characterized, including crystal structure determination of examples of N1 and N2 acylated products by X-ray difraction. N1- and N2-acyltriazoles interconvert in the presence of Bronsted or Lewis acids, which explained the efficiency of triazole cleavage transformations proceeding via N1-acylated triazoles. Efficient synthesis of enamido triflates from NH-triazoles proceeding via the intermediacy of N2-acyl-1,2,3-triazoles was developed.

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)

The stereoselective Suzuki–Miyaura cross-coupling of (Z)-β-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 β,β-diaryl-substituted enamides is observed. Thus, the method provides synthetic access to both isomers of the target enamides from (Z)-β-enamido triflates.

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 (

We designed and synthesized a series of 2’-deoxyribonucleoside triphosphates (dNTPs) bearing various lipid moieties. Fatty acid- and cholesterol-modified dNTPs proved to be substrates for KOD XL DNA polymerase in primer extension reactions. They were also mutually compatible for simultaneous multiple incorporations into the DNA strand. The methodology of enzymatic synthesis opened a pathway to diverse structurally unique lipid ON probes containing one or more lipid units. We studied interactions of such probes with the plasma membranes of live cells. Employing rational design, we found a series of lipid ONs with enhanced efficiency of the membrane anchoring. In membrane stability of multiply modified ONs was superior to that of commonly studied ON analogues, in which a single cholesterol molecule is typically tethered to the thread end. Notably, some of the probes were detected at the cell surface even after 24 h upon removal of the probe solution. Such effect was general to several studied cell lines.

DNA-binding fluorescent probes are useful tools in cell biology for various applications enabling visualisation and quantification of DNA. Herein, we report on HeliDye1, a novel cationic helicene-like DNA probe, and its versatile usability. HeliDye1 exhibits fluorescence exclusively in the presence of DNA duplexes containing AT base pairs and (contrary to any recent DNA probe) it remains non-fluorescent in the presence of single-stranded DNA and RNA. Favourable chemical properties of HeliDye1 allowed its use in karyotyping and super-resolution microscopy. We present five more applications of HeliDye1. Viscosimetry showed that HeliDye1 is a minor groove binder. HeliDye1 interacts with double-stranded DNA as an induced (P)-enantiomer, which we confirmed by ECD spectroscopy and DFT calculations. ECD spectroscopy suggested the formation of HeliDye1 dimers. MD simulations predicted the structures of (P)-HeliDye1 dimers inside DNA and showed their stability in AT-rich sequences and instability in GC-rich sequences.

While alarmone nucleotides guanosine-3′,5′-bisdiphosphate (ppGpp) and guanosine-5′-triphosphate-3′-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3′-azido-3′-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate., SCOPUS: ar.j, info:eu-repo/semantics/published

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)., Arginine‐specific 1,3‐diketone‐linked reactive nucleotides are reported for the enzymatic construction of DNA probes and cross‐linking with peptides and proteins.

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.

A reaction of N-sulfonyl-1,2,3-triazole with boron trifluoride etherate afforded a (Z)-β-ensulfonylamido fluoride instead of the previously erroneously assigned E isomer. The correction of the stereochemistry was based on a ge-1D ROESY NMR experiment and X-ray crystal structure analyses. Application of the reaction to N-fluoroalkyl-1,2,3-triazoles afforded new (Z)-β-enamido fluorides in a stereoselective manner. A mechanism involving coordination of BF3 with the triazole ring and vinyl diazonium and vinyl cation intermediates was proposed.

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 μM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.

Determining the structure of saccharides in their native environment is crucial to understanding their function and more accurately targeting their utilization. Nuclear magnetic resonance observables such as the nuclear Overhauser effect or spin-spin coupling constants are routinely utilized to study saccharides in their native water environment. However, while highly sensitive to the local environment, chemical shifts are mostly overlooked, despite being commonly measured for compounds identification. Although chemical shifts carry considerable structural information, their direct association with structure is notoriously difficult. This is mostly due to the similarity in the chemical nature of most saccharides causing similar physicochemical environments close to sugar C and H atoms, resulting in comparable chemical shifts. The rise of computational power allows one to compute reliable chemical shifts and use them to determine atomistic details of these sugars in solution. However, any prediction is severely limited by the computational protocol used and its accuracy. In this work, we studied a set of 31 saccharides on which we evaluated various computational protocols to calculate the total number of 375

Homologues of natural epigenetic pyrimidine nucleosides and nucleotides were designed and synthesized. They included 5-ethyl-, 5-propyl-, 5-(1-hydroxyethyl)-, 5-(1-hydroxypropyl)- and 5-acetyl- and 5-propionylcytosine and -uracil 2'-deoxyribonucleosides and their corresponding 5'

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.

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.

5-Hydroxymethylcytosine and uracil are epigenetic nucleobases, but their biological roles are still unclear. We present the synthesis of 2-nitrobenzyl photocaged 5-hydroxymethyl-2'-deoxycytidine and uridine 3'-O-phosphoramidites and their use in automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as primers for PCR to construct DNA templates modified in the promoter region that allowed switching of transcription through photochemical uncaging.

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.

Two isomeric sets of 4-substituted pyridopyrrolopyrimidine nucleobases were prepared through nucleophilic substitutions or cross-coupling reactions of 4-chloropyridopyrrolopyrimidines. The corresponding 4-amino-pyridopyrrolopyrimidines were glycosylated with 5-O-tritylribose using the modified Mitsunobu protocol. Several examples of the title heterocycles showed blue or green fluorescence. Testing of the pyridopyrrolopyrimidine nucleobases for the cytotoxic effect revealed micromolar activity of 4-benzofuryl derivatives in both series, preferentially in multidrug-resistant cancers.

5-(β-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 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.

All four isomeric series of novel 4-substituted pyrido-fused 7-deazapurine ribonucleosides possessing the pyridine nitrogen atom at different positions were designed and synthesized. The total synthesis of each isomeric fused heterocycle through multistep heterocyclization was followed by glycosylation and derivatization at position 4 by cross-coupling reactions or nucleophilic substitutions. All compounds were tested for cytostatic and antiviral activity. The most active were pyrido[4',3':4,5]pyrimidine nucleosides bearing MeO, NH2 , MeS, or CH3 groups at position 4, which showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism involved activation by phosphorylation and incorporation to DNA where the presence of the modified ribonucleosides causes double-strand breaks and apoptosis.

Bis(benzothienyl)ethene sulfones are very interesting molecules for super-resolution microscopy due to their photoswitching properties. However, functionalization of the 'classical' bis(benzothienyl)ethene sulfones with a five-membered central ring leads to significant decrease of quantum yields of photoconversion of the fluorescent closed form of the dye to the non-fluorescent open form that limits their application in microscopy. Here, we designed and synthesized diarylethenes with a fluorinated four-membered central ring that adds extra strain to the closed form of the dye. The reaction mechanism of their formation was studied, and byproducts formed upon structural rearrangement of the benzothiophene fragment were characterized. The photochromic properties of the new molecules were investigated by NMR and absorption spectroscopy. Some of these compounds show enhanced tendency to ring opening and have quantum yields of the ring-opening reaction in the range of 0.2-0.5.

Thymidine triphosphate bearing benzylidene-tetrahydroxanthylium near-IR fluorophore linked to the 5-methyl group via triazole was synthesized through the CuAAC reaction and was used for polymerase synthesis of labelled DNA probes. The fluorophore lights up upon incorporation to DNA (up to 348-times) presumably due to interactions in major groove and the fluorescence further increases in the single-stranded oligonucleotide. The labelled dsDNA senses binding of small molecules and proteins by a strong decrease of fluorescence. The nucleotide was used as a light-up building block in real-time PCR for detection of SARS-CoV-2 virus.

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 OS2  B2,5  1S5 and 3S  3H4  1C4. 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 4C1  4H5/1S5  1S5 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  B2,5/1S5  1S5 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.

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 β-C-glucosides, as well as 2-deoxy-β-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.

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A mild, atom-economic, and metal-free α-C-H amination of ethers using relatively stable nonafluorobutanesulfonyl (nonaflyl, Nf) azide as the aminating reagent to give N-sulfonyl hemiaminals is reported. This enables unprecedented C(sp3 ) difunctionalization reactions, leading to diverse functionalized amino group containing compounds starting from simple ethers in one pot.

Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.

Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.

We synthesized 2′-deoxycytidine and the corresponding nucleoside triphosphate bearing (indol-3-yl)methylene-2-methyl-5-oxo-4,5-dihydroimidazol-1-yl group (a tryptophan-based fluorophore from cyan fluorescent protein) linked through a propargyl group at position 5. The fluorophore is weakly solvatochromic, sensitive to pH, and, as a molecular rotor, it is highly sensitive to viscosity. In low viscosity solvents, the fluorescence is very weak, whereas in more viscous environment it lights up. Primer extension or PCR using the modified dCTrpTP and KOD XL DNA polymerase was used for construction of labelled oligonucleotides and DNA. Preliminary study showed a 2-fold increase of fluorescence of labelled ON probe in presence of single strand-binding protein indicating a potential of this label for sensing of protein-DNA interactions.