C1'-Branched Acyclic Nucleoside Phosphonates as Inhibitors of Plasmodium Falciparum 6-Oxopurine Phosphoribosyltransferase.

Hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT] is an excellent target for the development of new drugs to treat parasitic and bacterial infections as well as MYC-dependent triple-negative breast cancer. Inhibitors include compounds that mimic the transition state of the catalytic reaction and analogs of the two products of the reaction, the nucleoside monophosphates and pyrophosphate. One type of chemistry explored here is the design of purine-based C1'-branched acyclic nucleoside phosphonates bearing diverse structural attachments (secondary linkers) on the C1' atom.

Polymerase Synthesis of Hypermodified DNA Displaying a Combination of Thiol, Hydroxyl, Carboxylate, and Imidazole Functional Groups in the Major Groove.

We designed and synthesized a set of six 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing functional groups mimicking amino acid side chains in enzyme active sites (OH, SH, COOH, and imidazole) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through different linkers. These modified dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using several DNA polymerases. In primer extension (PEX), all modified dNTPs provided DNA containing one, two, three, or, (all) four modified nucleotides each bearing a different modification, although the thiol-modified dNTPs were worse substrates compared to the others.

Synthesis of 2-Substituted Adenosine Triphosphate Derivatives and their use in Enzymatic Synthesis and Postsynthetic Labelling of RNA.

A series of adenosine triphosphate (ATP) derivatives bearing chloro, fluoro, amino, methyl, vinyl, and ethynyl groups at position 2 are synthesized and tested as substrates for RNA and DNA polymerases. The modified nucleotides work well in in vitro transcription with T7 RNA polymerase and primer extension (PEX) using engineered DNA polymerases (TGK, 2M) except for the bulkier 2-vinyl- and 2-ethynyl-ATP derivatives that give truncated products. However, in single nucleotide incorporation followed by PEX, they still can be used for site-specific incorporation of reactive modifications into RNA that can be further used for postsynthetic labeling through thiol-ene or Cu-catalyzed alkyne-azide cycloadditions reactions.

Zwitterionic DNA: enzymatic synthesis of hypermodified DNA bearing four different cationic substituents at all four nucleobases.

We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing cationic substituents (protonated amino, methylamino, dimethylamino and trimethylammonium groups) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through hex-1-ynyl or propargyl linker. These cationic dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase. In primer extension (PEX), we successfully obtained DNA containing one, two, three, or (all) four modified nucleotides, each bearing a different cationic modification.

Glyco-DNA: Enzymatic Synthesis of Base-Modified and Hypermodified DNA Displaying up to Four Different Monosaccharide Units in the Major Groove.

A portfolio of six modified 2'-deoxyribonucleoside triphosphate (dNTP) derivatives derived from 5-substituted pyrimidine or 7-substituted 7-deazapurine bearing different carbohydrate units (d-glucose, d-galactose, d-mannose, l-fucose, sialic acid and N-Ac-d-galactosamine) tethered through propargyl-glycoside linker was designed and synthesized via the Sonogashira reactions of halogenated dNTPs with the corresponding propargyl-glycosides. The nucleotides were found to be good substrates for DNA polymerases in enzymatic primer extension and PCR synthesis of modified and hypermodified DNA displaying up to four different sugars. Proof of concept binding study of sugar-modified oligonucleotides with concanavalin A showed positive effect of avidity and sugar units count.

Novel analogues of a nonnucleoside SARS-CoV-2 RdRp inhibitor as potential antivirotics.

The RNA-dependent RNA polymerase (RdRp) represents a prominent target in the discovery and development of new antivirotics against RNA viruses, inhibiting the replication process. One of the most targeted RNA viruses of the last years is, without doubt, SARS-CoV-2, the cause of the recent COVID-19 pandemic. HeE1-2Tyr, a known inhibitor of flaviviral RdRp, has been discovered to also have antiviral potency against this coronavirus.

Synthesis and Biological Profiling of Quinolino-Fused 7-Deazapurine Nucleosides.

A series of quinolino-fused 7-deazapurine (pyrimido[5',4':4,5]pyrrolo[3,2-]quinoline) ribonucleosides were designed and synthesized. The synthesis of the key 11-chloro-pyrimido[5',4':4,5]pyrrolo[3,2-]quinoline was based on the Negishi cross-coupling of iodoquinoline with zincated 4,6-dichloropyrimidine followed by azidation and thermal or photochemical cyclization. Vorbrüggen glycosylation of the tetracyclic heterocycle followed by cross-coupling or substitution reactions at position 11 gave the desired set of final nucleosides that showed moderate to weak cytostatic activity and fluorescent properties.

Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases.

Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal.

Arylethynyl- or Alkynyl-Linked Pyrimidine and 7-Deazapurine 2'-Deoxyribonucleoside 3'-Phosphoramidites for Chemical Synthesis of Hypermodified Hydrophobic Oligonucleotides.

We designed and synthesized a set of 2'-deoxyribonucleoside 3'-phosphoramidites derived from 5-phenylethynyluracil, 5-(pentyn-1-yl)cytosine, 7-(indol-3-yl)ethynyl-7-deazaadenine, and 7-isopropylethynyl-7-deazaguanine. These nucleoside phosphoramidites were successfully used for automated solid-phase synthesis of oligonucleotides containing one or several modifications, including fully modified sequences where every nucleobase was displaying a modification, and their hybridization was studied. The phosphoramidite building blocks have potential for synthesis of hypermodified aptamers and other functional nucleic acid-based polymers, which sequence-specifically display amino acid-like hydrophobic substituents.

Superanionic DNA: enzymatic synthesis of hypermodified DNA bearing four different anionic substituents at all four nucleobases.

We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) derived from 5-substituted pyrimidines and 7-substituted 7-deazapurines bearing anionic substituents (carboxylate, sulfonate, phosphonate, and phosphate). The anion-linked dNTPs were used for enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase containing one, two, three, or four modified nucleotides. The polymerase was able to synthesize even long sequences of >100 modified nucleotides in a row by primer extension (PEX).

Synthesis of Polycyclic Hetero-Fused 7-Deazapurine Heterocycles and Nucleosides through C-H Dibenzothiophenation and Negishi Coupling.

A new approach for synthesizing polycyclic heterofused 7-deazapurine heterocycles and the corresponding nucleosides was developed based on C-H functionalization of diverse (hetero)aromatics with dibenzothiophene--oxide followed by the Negishi cross-cooupling with bis(4,6-dichloropyrimidin-5-yl)zinc. This cross-coupling afforded a series of (het)aryl-pyrimidines that were converted to fused deazapurine heterocycles through azidation and thermal cyclization. The fused heterocycles were glycosylated to the corresponding 2'-deoxy- and ribonucleosides, and a series of derivatives were prepared by nucleophilic substitutions at position 4.

C1'-Branched acyclic nucleoside phosphonates mimicking adenosine monophosphate: Potent inhibitors of Trypanosoma brucei adenine phosphoribosyltransferase.

Some pathogens, including parasites of the genus Trypanosoma causing Human and Animal African Trypanosomiases, cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Thus, their PSP enzymes are considered as promising drug targets, sparsely explored so far. Recently, a significant role of acyclic nucleoside phosphonates (ANPs) as inhibitors of key enzymes of PSP, namely of 6-oxopurine phosphoribosyltransferases (PRTs), has been discovered.

Structure-activity relationship and biochemical evaluation of novel fibroblast activation protein and prolyl endopeptidase inhibitors with α-ketoamide warheads.

Peptidomimetic inhibitors of fibroblast activation protein (FAP) are regarded as promising tools for tumor targeting in vivo. Even though several peptidomimetic compounds with nanomolar potency have been described, broad chemical space for further modification remained unexplored. Therefore, we set to analyze the structure-activity relationship (SAR) of pseudopeptide compound series with α-ketoamide warheads in order to explore the contributions of the P1' and P2' moieties to the inhibitory potency.

Synthesis and Antitrypanosomal Activity of 6-Substituted 7-Methyl-7-deazapurine Nucleosides.

Human African Trypanosomiasis caused by species is one of the most damaging neglected tropical diseases. While the number of newly diagnosed cases per year is record low, there is still high interest in the development of new antitrypanosomal agents in case of resistance to currently used drugs and their combinations, and to replace drugs with serious side effects. We report a series of 7-methyl-7-deazapurine (5-methyl-pyrrolo[2,3-]pyrimidine) ribonucleosides bearing alkyl, methylsulfanyl, methylamino, or diverse alkoxy groups at position 6 that was prepared through glycosylation of 6-chloro-7-methyl-7-deazapurine followed by nucleophilic substitutions or cross-coupling reactions at position 6 and deprotection.

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

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 N-protected phosphonic acids.

Efficient Synthesis of α-Branched Purine-Based Acyclic Nucleosides: Scopes and Limitations of the Method.

An efficient route to acylated acyclic nucleosides containing a branched hemiaminal ether moiety is reported via three-component alkylation of -heterocycle (purine nucleobase) with acetal (cyclic or acyclic, variously branched) and anhydride (preferentially acetic anhydride). The procedure employs cheap and easily available acetals, acetic anhydride, and trimethylsilyl trifluoromethanesulfonate (TMSOTf). The multi-component reaction is carried out in acetonitrile at room temperature for 15 min and provides moderate to high yields (up to 88%) of diverse acyclonucleosides branched at the aliphatic side chain.

Synthesis and Biological Profiling of Pyrazolo-Fused 7-Deazapurine Nucleosides.

A series of 8-substituted 1-methyl-1,4-dihydropyrazolo[3',4':4,5]pyrrolo[2,3-]pyrimidine (methylpyrazolo-fused 7-deazapurine) ribonucleosides have been designed and synthesized. Two synthetic approaches to the key heterocyclic aglycon , (i) a six-step classical heterocyclization starting from 5-chloro-1-methyl-4-nitropyrazole and (ii) a three-step cross-coupling and cyclization approach starting from the zincated 4,6-dichloropyrimidine, gave comparable total yields of 18% vs 13%. The glycosylation of was attempted by three different methods but only the Vorbrüggen silyl-base protocol was efficient and stereoselective to give desired β-anomeric nucleoside intermediate .

2-Formyl-dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove.

2-Formyl-2'-deoxyadenosine triphosphate (d ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of d ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The d ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX).

Synthesis and Cytotoxic and Antiviral Profiling of Pyrrolo- and Furo-Fused 7-Deazapurine Ribonucleosides.

Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3- d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3- d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2',4,5]pyrrolo[2,3- d]pyrimidine nucleosides were inactive.

Protected 2'-deoxyribonucleoside triphosphate building blocks for the photocaging of epigenetic 5-(hydroxymethyl)cytosine in DNA.

2'-Deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)cytosine (5hmC) protected with photocleavable groups (2-nitrobenzyl or 6-nitropiperonyl) were prepared and studied as substrates for the enzymatic synthesis of oligonucleotides and DNA containing a photocaged epigenetic 5hmC base. DNA probes containing photocaged or free 5hmC in the recognition sequence of restriction endonucleases were prepared and used for the study of the photorelease of caged DNA by UV or visible light at different wavelengths. The nitrobenzyl-protected dNTP was a slightly better substrate for DNA polymerases in primer extension or PCR, whereas the nitropiperonyl-protected nucleotide underwent slightly faster photorelease at 400 nm.

Identification and characterization of potential bioactive compounds from the leaves of Leucosidea sericea.

Ethnopharmacological Relevance: Leucosidea sericea is a southern African tree used for treating different diseases including microbial infections and inflammatory-related conditions.

Aim Of The Study: To isolate and identify the chemicals in Leucosidea sericea which possibly account for the diverse therapeutic effects of the species.

Materials And Methods: Leaf material was extracted using 20% methanol and subsequently partitioned with different solvents (hexane, dichloromethane, ethyl acetate and butanol).