Design and synthesis of chiral peptidic nucleic acids

Due to the increasing interest in the use ofoligonucleotide analogues as antisense and antigenedrugs, we designed a chiral analogue constituted of apeptidic frame bearing nucleobases in suitablepositions (C-PNA). We recently reported the synthesisof four nonnatural -amino acids with the DNAbases in the lateral chain. In this paper we presentan improved synthesis of the Fmoc monomers and theirpolymerisation to polypeptidic oligonucleotideanalogues using a modification of the standardprotocol for solid phase peptide synthesis.     

Unrestricted hybridization of oligonucleotides to structure-free DNA

The existence of secondary structure in long single-stranded DNA and RNA is a serious obstacle to the practical use of short oligonucleotide probes (<20-mers). Here, we show that replication of a highly structured DNA in the presence of a unique set of dNTP analogues leads to synthesis of daughter DNA with a significantly reduced level of secondary structure. This replicated DNA, composed of 2-aminoadenine, 2-thiothymine, 7-deazaguanine, and cytosine bases, was readily accessible to tiled 8-mer LNA and 15-mer DNA probes, whereas an unmodified version of the same DNA was inaccessible. Importantly, while the base analogues enhanced probe-target stability, they did not significantly reduce the specificity of base pairing. The availability of structure-free DNA targets should facilitate the use of short oligonucleotide probes and promote development of generic oligonucleotide microarrays.     

Solid-phase synthesis of cyclic analogues related to the hypoglycaemic peptide hGH(6-13): comparison of two i-->i + 4 lactam cyclization procedures.

The use of 1,3-diisopropylcarbodiimide (DIC) for the synthesis of cyclic analogues of the hypoglycaemic peptide fragment derived from the N-terminus of human growth hormone (hGH), namely hGH[6-13], is described. Different strategies were examined to achieve improved yields for the on resin side-chain to side-chain cyclization of the corresponding linear peptides containing reverse beta-turn motifs. When compared with the more reactive Castro's reagent, the results confirm that DIC in the presence of HOBt can be successfully employed to minimize the formation of intermolecular oligomeric byproducts associated with the preparation of cyclic hGH[6-14] peptide analogues based on an i-->(i + 4)Lys-->Glu or Glu-->Lys cyclization strategy.     

Synthesis and utilization of 13C(8)-enriched purines

A new and more efficient method is presented for the synthesis of 13C(8)-enriched adenine. In addition, we present for the first time the synthesis of 13C(8)-enriched 2-aminopurine and purine. All three analogues have been converted to the corresponding ribonucleosides. The adenine analogue has been further converted to the 2'-deoxy-nucleoside and incorporated into a synthetic oligonucleotide. Data is presented demonstrating the utility of 13C-enrichment in heteronuclear isotope-edited NMR spectra.     

Preparation of 5′-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)

Antisense oligonucleotides (ASODNs) have been widely used as an important tool for regulating gene expression, and developed into therapeutics. Natural ODNs are susceptible to nuclease degradation, nucleic acid analogues, however, have less side effects, stronger stability and more potent activities. Large-scale de novo synthesis of a certain oligonucleotide has been very difficult and costly. In a previous preliminary study, we developed the polymerase-endonuclease amplification reaction (PEAR) for amplification and large-scale preparation of natural antisense ODNs. Here we extended the method in preparation of a widely used modified oligonucleotide with 5′-O-(1-Thiotriphosphate) modifications. Using electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection, the purity of the PEAR product was measured as high as 100.0%. Using PEAR a large amount of a specific oligonucleotide can be produced starting from a small amount of synthetic seeds. It is suggested that PEAR can be a useful tool for large-scale production of modified oligonucleotides.     

Evaluation of Sulfur-Transfer Reagents for Large Scale Synthesis of Oligonucleotide Phosphorothioate Analogues

Abstract

Several sulfur-transfer reagents have been evaluated for large scale synthesis of oligonucleotide phosphorothioate analogues, in which 3-ethoxy-1, 2-dithiazoline-5-one (EDITH, 5) shows potential as an alternative to Beaucage reagent.     

Synthesis and properties of phosphorylated 3'-O-beta-D-ribofuranosyl-2'-deoxythymidine

A strategy was developed for the synthesis of 3'-O-beta-D-ribofuranosyl 2'-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5'-O- and 5'-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy.     

Solid-Phase Synthesis of Surfactin,a Powerful Biosurfactant Produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>, and of Four Analogues

Using the Fmoc methodology, we report the chemical synthesis of surfactin and of four of its analogues, by stepwise solid-phase peptide synthesis (SPPS) on Sasrin resin. Formation of depsipeptide bond was performed with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In developing our strategy, surfactin was used as a model and we synthesized both its racemic mixture and its R isoform. (R) 3-hydroxy fatty acid was obtained using Candida antarctica lipase from the racemic fatty acid, allowing a further identification of both R and S isoforms in the racemic mixture. Analogues were synthesized as racemic linear lipopeptides. Then, both enantiomers were separated and purified by adsorption chromatography on silicic acid, following cleavage from the resin. Linear R lipoheptapeptides were identified by TLC. They exhibit, in all cases, higher R_f values than those of the corresponding S isoforms. Cyclization was then performed independently for each enantiomer, using a HATU/DIEA coupling in solution. The yields were highly dependent on the position and on the nature of the modified amino acids.     

Synthesis of 1′,2′-methano-2′,3′-dideoxynucleosides as potential antivirals

The synthesis of constrained nucleosides has become an important tool to understand the SAR in the interaction between biological and synthetic nucleotides in the context of antisense oligonucleotide therapy. The incorporation of a cyclopropane into a furanose ring of a nucleoside induces some degree of constrain without affecting significantly the steric environment of a nucleoside. Here, we report a new, short and stereocontrolled synthesis of two constrained nucleosides analogues, 1′,2′- methano-2′,3′-dideoxyuridine 9, and the corresponding cytidine analog 12. X-ray crystallography revealed that the furanose ring in the constrained uridine and cytidine analogues was flattened with virtual loss of pseudorotation. The phosphoramidate esters of the novel constrained uridine and cytidine nucleosides, intended as prodrugs, were tested in cell-based assays for viral replication across the herpes virus family and HIV inhibition courtesy of Merck laboratories, Rahway. They were also tested in antiproliferative assays against colorectal and melanoma cell lines. Unfortunately, none of the compounds showed activity in these assays.     

Achievements and prospects of the directed search for antiviral agents in the nucleoside series and their derivatives]

Recent advances of antiviral drug design among nucleosides and their derivatives have been summarized. The first chapter deals with the history of nucleic acids components and further developments in this area. Next part discusses the mechanism of action of biologically active nucleosides: 2',3'-dideoxynucleosides, acyclic analogues, phosphonate derivatives and nucleoside antibiotics. The third chapter describes planning of complicated synthesis of nucleoside analogues from branched-chain sugars and stereo-specific formation of glycosidic bond upon synthesis of ribonucleoside and 2'-deoxyribonucleoside. The last part outlines further perspectives, i. e. preparation of antiviral compounds and use of nucleoside analogues in oligonucleotide synthesis.     

Hydrolytic stability of a phosphate-branched oligonucleotide incorporating a ribonucleoside 3'-phosphotriester unit

A phosphate-branched oligonucleotide has been prepared by using an appropriately protected trinucleoside phosphotriester building block in conventional solid-phase synthesis. Hydrolysis of the branched oligonucleotide has been followed over a wide pH range. Comparison of the present results with those previously obtained for simpler analogues indicates that a trinucleoside 3',3',5'-monophosphate, when embedded in an oligonucleotide structure, is stabilized toward hydroxide-ion catalyzed cleavage by more than one order of magnitude, lending some support to the feasibility of existence of phosphate-branched RNA X in biological systems.     

Nucleoside 3′-O-(2-Oxo-“Spiro”-4.4-Pentamethylene-1.3.2-Oxathiaphospholane)S: Monomers For Stereocontrolled Synthesis Of Oligo(Nucleoside Phosphorothioate/Phosphate)S

Abstract

Attempts at synthesis of “chimeric” oligonucleotide constructs (PO/PS-Oligos) possessing phosphate and P-stereodefined phosphorothioate internucleotide linkages via combined phosphoramidite/oxathiaphospholane methods were unsuccessful. Therefore, novel monomers for oxathiaphospholane method, namely 5′-O-DMT-deoxyribonucleoside 3′-O(2-oxo-.spiro-4.4-pentamethylene-1.3.2-oxathiaphospholane)s, were prepared and used together with their diastereomerically pure 2-thio analogues for the stereocontrolled synthesis of “chimeric” oligonucleotide constructs (PO/PS-Oligos).     

Progress toward the synthesis of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages.

The synthesis of building blocks for the preparation of nonionic oligonucleotide analogues with sulfonate and sulfonamide internucleotide linkages is described. Coupling conditions for the conversion of several of these monomers to dimers are also described.     

Synthesis And Binding Study Of Phosphonate Analogues Of Pnas And Their Hybrids With Pnas

Abstract

The preparation of monomers for the synthesis of phosphonate analogues of peptide nucleic acids containing the four natural nucleobases: thymine, cytosine, adenine and guanine, has been ccomplished. The monomers obtained were used for the automated online solid phase synthesis of pure phosphono-PNA oligomers as well as chimeras consisting of phosphono-PNA and PNA resudies. The hybridization properties of these oligonucleotide mimics to complementary DNA and RNA fragments were studied.     

New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleotide phosphorothioate analogues.

A set of new sulfurizing agents representing disulfides of arylsulfonic acids has been developed for the automated synthesis of phosphorothioate oligonucleotide analogues via the phosphoramidite method. These reagents, such as bis(benzenesulfonyl)disulfide, bis(p-toluenesulfonyl)disulfide, bis(p-methoxybenzensulfonyl)disulfide, and bis (p-chlorobenzenesulfonyl) disulfide, are easily prepared crystalline solid compounds. They are relatively inexpensive, easy to handle, and efficiently convert internucleotide cyanoethyl phosphite to the phosphorothioate triester within 1-2 min. The efficiency of phosphorothioate oligonucleotide synthesis with the use of these reagents is comparable to that of phosphodiester oligonucleotides.     

Synthesis and Properties of Phosphorylated 3′-O-β-D-Ribofuranosyl-2′-deoxythymidine

Abstract

A strategy was developed for the synthesis of 3′-O-β-D-ribofuranosyl 2′-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5′-O- and 5″-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy.     

Unexpected Shift to a 4-Imino Tautomer Upon N4-Acylation of 5-Methylcytosin-1-yl Nucleoside Analogues

Abstract

In contrast with the behaviour of 5-unsubstituted cytosine nucleoside analogues, 5-methylcytosine derivatives show upon N⁴-benzoylation, commonly used as base protection in oligonucleotide synthesis, a tautomeric change of the base moiety from a 4-amino- into a 4-imino isomer. In the latter form, which is easily diagnosticized by ¹³C NMR and confirmed by X-ray data, the compounds seem to be hydrolytically less stable.     

A convenient solid-phase method for synthesis of 3'-conjugates of oligonucleotides.

We present a new procedure for the preparation of 3'-conjugates of oligonucleotides through solid-phase synthesis. A suitable universal solid support was readily prepared using a series of peptide-like coupling reactions to incorporate first a spacer and then an L-homoserine branching unit. The N-alpha-position of the homoserine carries an Fmoc protecting group that is removed by treatment with piperidine to liberate an amino group suitable for attachment of the conjugate (e.g., small organic molecule, fluorescent group, cholesterol, biotin, amino acid, etc.) or for assembly of a short peptide. The side-chain hydroxyl group of the homoserine carries a trityl protecting group. After TFA deprotection, the hydroxyl group acts as the site for oligonucleotide assembly. An additional spacer, such as aminohexanoyl, may be incorporated easily between the conjugate molecule and the oligonucleotide. A number of examples of synthesis of 3'-conjugates of oligonucleotides and their analogues are described that involve standard automated oligonucleotide assembly and use of commercially available materials. The linkage between oligonucleotide and 3'-conjugate is chirally pure and is stable to conventional ammonia treatment used for oligonucleotide deprotection and release from the solid support. The homoserine-functionalized solid support system represents a simple and universal route to 3'-conjugates of oligonucleotides and their derivatives.     

Oligonucleotides containing pyrazolo[3,4-d]pyrimidines: 8-Aza-7-deazaadenines with bulky substituents in the 2- or 7-position

The synthesis of the 2'-deoxyadenosine analogues 1b, 2b, and 3c modified at the 7- and/or 2-position is described. The effect of 7-chloro and 2-methylthio groups on the duplex stability is evaluated. For that, the nucleosides 1b, 2b, and 3c were converted to the corresponding phosphoramidites 15, 19, and 22, which were employed in the solid-phase oligonucleotide synthesis. In oligonucleotide duplexes, compound 1b forms stable base pairs with dT, of which the separated 1b-dT base pairs contribute stronger than that of the consecutive base pairs. Compound 2b shows universal base pairing properties while its N8 isomer 3c forms duplexes with lower stability.