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. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.

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.

Synthesis of sulfur karrikin bioisosteres as potential neuroprotectives.

The only known sulfur-containing karrikin, 3-methyl-2H-thiopyrano[3,4-b]furan-2-one, has been recently identified as an extremely efficient neuroprotective butenolide. Herein, we report the targeted synthesis of this compound as well as new synthetic protocols toward a class of compounds derived from 2H-furo[2,3-c]pyran-2-ones (karrikins) via bioisosteric exchange of oxygen with sulfur. In particular, we present synthetic procedures toward bioisosteres of karrikins with one or two sulfur heteroatoms incorporated into the core backbone together with evaluation of their biological activity in inhibition of acetylcholinesterase.

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.

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 N-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. The procedure exhibits a broad substrate scope of N-heterocycles and acetals, and, in the case of purine derivatives, also excellent regioselectivity, giving almost exclusively N-9 isomers.

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

2-Formyl-2'-deoxyadenosine triphosphate (dCHO 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 dCHO ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHO ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31-mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single-nucleotide extension followed by PEX was used for site-specific incorporation of one aldehyde-linked adenosine into a longer 49-mer sequence. The reactive formyl group was used for cross-linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647-linked hydroxylamine.

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. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

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. However, both photocaged building blocks can be used in polymerase synthesis and the photorelease of 5hmC in DNA.

Protected 5-(hydroxymethyl)uracil nucleotides bearing visible-light photocleavable groups as building blocks for polymerase synthesis of photocaged DNA.

Nucleosides, nucleotides and 2'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside monophosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).

C-H Imidation of 7-Deazapurines.

We developed and presented here a ferrocene-catalyzed C-H imidation of 7-deazapurines (pyrrolo[2,3-d]pyrimidines) with N-imidyl peroxyesters. The reactions occur regioselectively at position 8 in 7-deazapurines, leading to a series of 8-succinimido-, phtalimido-, or naphthalimido-7-deazapurine derivatives. Attempted hydrazinolysis of resulting 8-imidyl-7-deazapurines led to corresponding 8-amino-7-deazapurine, which was very unstable and quickly decomposed.

Acyclic Nucleoside Phosphonates Containing 9-Deazahypoxanthine and a Five-Membered Heterocycle as Selective Inhibitors of Plasmodial 6-Oxopurine Phosphoribosyltransferases.

Acyclic nucleoside phosphonates (ANPs) are an important class of therapeutic drugs that act as antiviral agents by inhibiting viral DNA polymerases and reverse transcriptases. ANPs containing a 6-oxopurine unit instead of a 6-aminopurine or pyrimidine base are inhibitors of the purine salvage enzyme, hypoxanthine-guanine-[xanthine] phosphoribosyltransferase (HG[X]PRT). Such compounds, and their prodrugs, are able to arrest the growth of Plasmodium falciparum (Pf) in cell culture. A new series of ANPs were synthesized and tested as inhibitors of human HGPRT, PfHGXPRT, and Plasmodium vivax (Pv) HGPRT. The novelty of these compounds is that they contain a five-membered heterocycle (imidazoline, imidazole, or triazole) inserted between the acyclic linker(s) and the nucleobase, namely, 9-deazahypoxanthine. Five of the compounds were found to be micromolar inhibitors of PfHGXPRT and PvHGPRT, but no inhibition of human HGPRT was observed under the same assay conditions. This demonstrates selectivity of these types of compounds for the two parasitic enzymes compared to the human counterpart and confirms the importance of the chemical nature of the acyclic moiety in conferring affinity/selectivity for these three enzymes.

C-H Phosphonation of Pyrrolopyrimidines: Synthesis of Substituted 7- and 9-Deazapurine-8-phosphonate Derivatives.

The Mn(OAc)3-promoted C-H phosphonation of 7-deazapurines (pyrrolo[2,3-d]pyrimidines) and 9-deazapurines (pyrrolo[3,2-d]pyrimidines) with diethylphosphite was developed. The reactions occur regioselectively at position 8 both in 7 and 9-deazapurines, leading to new deazapurine-8-phosphonate derivatives, which can be further modified and transformed to 6-(het)aryl-deazapurine derivatives or deprotected to free phosphonic acids.

Synthesis and biological properties of prodrugs of (S)-3-(adenin-9-yl)-2-(phosphonomethoxy)propanoic acid.

The lack of antiviral activity of recently described (S)-3-(adenin-9-yl)-2-(phosphonomethoxy)propanoic acid, or (S)-CPMEA in brief, has been speculated to possibly be due to the increased hydrophilicity of the molecule and, thus, by its limited cellular permeability. Efficient syntheses of novel lipophilic prodrugs of (S)-CPMEA masking either the carboxylic group or preferably both the phosphonate and carboxylic moieties, have been developed in order to increase bioavailability of the parent compound. Two prodrugs of (S)-CPMEA, namely phosphonate bis-amidate 15 and phenyloxy amidate 16, exhibited pan-genotypic anti-HCV activity at submicromolar concentrations.

Synthesis and biological profiling of 6- or 7-(het)aryl-7-deazapurine 4'-C-methylribonucleosides.

The synthesis and biological activity profiling of a large series of diverse pyrrolo[2,3-d]pyrimidine 4'-C-methylribonucleosides bearing an (het)aryl group at position 4 or 5 is reported as well as the synthesis of several phosphoramidate prodrugs. These compounds are 4'-C-methyl derivatives of previously reported cytostatic hetaryl-7-deazapurine ribonucleosides. The synthesis is based on glycosylation of halogenated 7-deazapurine bases with 1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-methyl-ß-d-ribofuranose followed by cross-coupling and nucleophilic substitution reactions. The final compounds showed low cytotoxicity and several derivatives exerted antiviral activity against HCV or Dengue viruses at micromolar concentrations.

Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7-Substituted 7-Deazaguanine Nucleobases.

Previous studies of polymerase synthesis of base-modified DNAs and their cleavage by restriction enzymes have mostly related only to 5-substituted pyrimidine and 7-substituted 7-deazaadenine nucleotides. Here we report the synthesis of a series of 7-substituted 7-deazaguanine 2'-deoxyribonucleoside 5'-O-triphosphates (dG(R) TPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dG(R) TPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7-substituted 7-deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage.

Synthesis and Evaluation of Novel Acyclic Nucleoside Phosphonates as Inhibitors of Plasmodium falciparum and Human 6-Oxopurine Phosphoribosyltransferases.

Acyclic nucleoside phosphonates (ANPs) are a promising class of antimalarial therapeutic drug leads that exhibit a wide variety of Ki values for Plasmodium falciparum (Pf) and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases [HG(X)PRTs]. A novel series of ANPs, analogues of previously reported 2-(phosphonoethoxy)ethyl (PEE) and (R,S)-3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) derivatives, were designed and synthesized to evaluate their ability to act as inhibitors of these enzymes and to extend our ongoing antimalarial structure-activity relationship studies. In this series, (S)-3-hydroxy-2-(phosphonoethoxy)propyl (HPEP), (S)-2-(phosphonomethoxy)propanoic acid (CPME), or (S)-2-(phosphonoethoxy)propanoic acid (CPEE) are the acyclic moieties. Of this group, (S)-3-hydroxy-2-(phosphonoethoxy)propylguanine (HPEPG) exhibits the highest potency for PfHGXPRT, with a Ki value of 0.1 µM and a Ki value for human HGPRT of 0.6 µM. The crystal structures of HPEPG and HPEPHx (where Hx=hypoxanthine) in complex with human HGPRT were obtained, showing specific interactions with active site residues. Prodrugs for the HPEP and CPEE analogues were synthesized and tested for in vitro antimalarial activity. The lowest IC50 value (22 µM) in a chloroquine-resistant strain was observed for the bis-amidate prodrug of HPEPG.

Synthesis, conformational studies, and biological properties of phosphonomethoxyethyl derivatives of nucleobases with a locked conformation via a pyrrolidine ring.

Systematic structure-activity studies on a diverse family of nucleoside phosphonic acids has led to the development of potent antiviral drugs such as HPMPC (CidofovirTM), PMEA (AdefovirTM), and PMPA (TenofovirTM), which are used in the treatment of CMV-induced retinitis, hepatitis B, and HIV, respectively. Here, we present the synthesis of a novel class of acyclic phosphonate nucleotides that have a locked conformation via a pyrrolidine ring. NMR analysis of these compounds revealed that the pyrrolidine ring has a constrained conformation when in the cis-form at pD < 10 via hydrogen bonding. Four of these compounds were tested as inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases. The most potent has a Ki of 0.6 µM for Plasmodium falciparum HGXPRT.

Pyrrolidine nucleotide analogs with a tunable conformation.

Conformational preferences of the pyrrolidine ring in nucleotide analogs 7-14 were investigated by means of NMR and molecular modeling. The effect of the relative configuration of hydroxy and nucleobase substituents as well as the effect of the alkylation or acylation of the pyrrolidine nitrogen atom on the conformation of the pyrrolidine ring were studied. The results of a conformational analysis show that the alkylation/acylation can be effectively used for tuning the pyrrolidine conformation over the whole pseudorotation cycle.

Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery.

Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.