A Staphylococcus aureus lipoteichoic acid (LTA) derived structural variant with two diacylglycerol residues

Based on 1,2-O-isopropylidene-sn-glycerol five chiral building blocks containing differently modified glycerol residues were required for the synthesis of the target molecule 2. One of these building blocks is diacylglyceryl beta-gentiobioside carrying a phosphite residue at 6b-O position. Ligation of these five building blocks led to the desired glycerol phosphate backbone to which d-alanyl residues were attached, thus generating after O-deprotection the target molecule 2, a bisamphiphilic structural variant of Staphylococcus aureus LTA. This compound displayed higher potency in terms of cytokine release by human blood leukocytes than the monoamphiphilic variant LTA.     

α-Hydroxybenzylphosphonate Modified Oligonucleotides: Synthesis,Properties, and a Novel Route via Monomer Building Blocks

Abstract

In general, α-hydroxybenzylphosphonate modified 2′-deoxyadenosine-thymidine dimer building blocks 1, 2 are utilized for the incorporation into α-hydroxybenzylphosphonate pro-oligonucleotides. For a universal application of our pro-oligonucleotide concept on biologically relevant oligonucleotides a route for the synthesis of modified monomer building blocks 3 was developed and is presented herein.     

Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2'-deoxyuridine moieties

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of DNA oligonucleotides that bear 5-(mercaptomethyl)-2'-deoxyuridine moieties. The building blocks were synthesized in a straightforward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the generation of modified oligonucleotides containing the thiolated building blocks.     

Biologically inspired strategy for programmed assembly of viral building blocks with controlled dimensions

Facile fabrication of building blocks with precisely controlled dimensions is imperative in the development of functional devices and materials. We demonstrate the assembly of nanoscale viral building blocks of controlled lengths using a biologically motivated strategy. To achieve this we exploit the simple self-assembly mechanism of Tobacco mosaic virus (TMV), whose length is solely governed by the length of its genomic mRNA. We synthesize viral mRNA of desired lengths using simple molecular biology techniques, and in vitro assemble the mRNA with viral coat proteins to yield viral building blocks of controlled lengths. The results indicate that the assembly of the viral building blocks is consistent and reproducible, and can be readily extended to assemble building blocks with genetically modified coat proteins (TMV1cys). Additionally, we confirm the potential utility of the TMV1cys viral building blocks with controlled dimensions via covalent and quantitative conjugation of fluorescent markers. We envision that our biologically inspired assembly strategy to design and construct viral building blocks of controlled dimensions could be employed to fabricate well-controlled nanoarchitectures and hybrid nanomaterials for a wide variety of applications including nanoelectronics and nanocatalysis.     

How to tailor non-ribosomal peptide products--new clues about the structures and mechanisms of modifying enzymes

Non-ribosomal peptide products often contain modified building blocks or post-assembly line alterations of their peptide scaffolds with some of them being crucial for biological activity. These reactions such as halogenation, hydroxylation or glycosylation are mostly catalyzed by individual enzymes associated with the respective biosynthesis cluster. The versatile nature of these chemical modifications gives rise to a high degree of structural and functional diversity. Recent progress in this area enhances our insight about the mechanisms of these enzymes. Biotechnological applications might include the synthesis of novel, non-ribosomal peptide products or modified amino acid building blocks for pharmaceutical research.     

Peptidomimetics, a synthetic tool of drug discovery

The demand for modified peptides with improved stability profiles and pharmacokinetic properties is driving extensive research effort in this field. Many structural modifications of peptides guided by rational design and molecular modeling have been established to develop novel synthetic approaches. Recent advances in the synthesis of conformationally restricted building blocks and peptide bond isosteres are discussed.     

Three new double-headed nucleotides with additional nucleobases connected to C-5 of pyrimidines; synthesis,duplex and triplex studies

In the search for double-coding DNA-systems, three new pyrimidine nucleosides, each coded with an additional nucleobase anchored to the major groove face, are synthesized. Two of these building blocks carry a thymine at the 5-position of 2′-deoxyuridine through a methylene linker and a triazolomethylene linker, respectively. The third building block carries an adenine at the 6-position of pyrrolo-2′-deoxycytidine through a methylene linker. These double-headed nucleosides are introduced into oligonucleotides and their effects on the thermal stabilities of duplexes are studied. All studied double-headed nucleotide monomers reduce the thermal stability of the modified duplexes, which is partially compensated by using consecutive incorporations of the modified monomers or by flanking the new double-headed analogs with members of our former series containing propyne linkers. Also their potential in triplex-forming oligonucleotides is studied for two of the new double-headed nucleotides as well as the series of analogs with propyne linkers. The most stable triplexes are obtained with single incorporations of additional pyrimidine nucleobases connected via the propyne linker.     

Synthesis of encoded modified oligonucleotide libraries. Part 1: orthogonal chemistry

The basis for further development of combinatorial libraries of modified oligonucleotides tagged by a codifying sequence is discussed. The chemistry involved in the orthogonal synthesis of both strands and some representative examples of building blocks are presented.     

New synthetic routes to protected purine 2''-O-methylriboside-3''-O-phosphoramidites using a novel alkylation procedure.

A highly selective alkylation procedure has been developed enabling new synthetic routes to be established for protected purine 2'-O-methylriboside-3'-O-phosphoramidites; building blocks for the assembly of 2'-O-methyloligoribonucleotides. The new procedure avoids the use of the highly toxic and potentially explosive reagent diazomethane and is far superior to the use of silver oxide/methyl iodide. Moreover, the use of highly versatile key intermediates will enable the synthesis of a wide variety of base modified analogues as well as other 2'-O-alkylriboside derivatives.     

Evaluation of oligonucleotides containing two novel 2'-O-methyl modified nucleotide monomers: a 3'-C-allyl and a 2'-O,3'-C-linked bicyclic derivative.

The two ribo-configured nucleosides 1-(3-C-allyl-2-O-methyl-beta-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl )- 2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.     

Synthesis of oligonucleotide building blocks of 2'-deoxyguanosine bearing a C8-arylamine modification

C8-Arylamine-dG adducts were synthesized by palladium-catalyzed cross-coupling reactions. The corresponding 5'-O-DMTr-3'-O-phosphoramidite-C8-arylamine-dG adducts were synthesized as potential building blocks for the automated synthesis of site-specifically modified oligonucleotides.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

Convergent synthesis of an inner core GPI of sperm CD52

An inner core of the GPI anchor of sperm CD52 antigens was synthesized by a highly convergent process using specially modified inositol, glucosamine and phospholipid as key building blocks. This paper also presents a new and efficient procedure to prepare 1,2,6-differentiated derivatives of inositol for GPI syntheses.     

Surprising duplex stabilisation upon mismatch introduction within triply modified duplexes

Three different modified phosphoramidite nucleoside building blocks equipped with additional protected imidazole, masked alcohol and masked carboxylate functionality are synthesized and incorporated into oligonucleotides. Based on the serine-protease active site model, doubly and triply modified duplexes are created and tested for stability. Analysis of different spatial distributions of the extra functionalities shows that careful positioning can even overcome duplex destabilisation caused by the introduction of mismatches.     

2'-Azido RNA, a versatile tool for chemical biology: synthesis, X-ray structure, siRNA applications, click labeling

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2'-azido cytidine and 2'-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2'-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2'-azido uridine and 2'-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2'-azido group supports the C3'-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2'-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2'-azido modifications are compatible with 2'-fluoro and/or 2'-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2'-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2'-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.     

Convenient strategies for the synthesis of 1,4-phenylene spaced sugars

Two synthetic strategies have been developed for the synthesis of spaced sugars with lipophilic 1,4-phenylene core. A building block combining the usefulness of Weinreb amide functionality and modified Julia olefination reaction has been explored towards this objective. This building block offers complete flexibility in attaching any desired sugar derivative across phenylene spacer via C-C bond formation. The other strategy uses carbohydrate based building blocks for the synthesis of symmetrical as well as unsymmetrical 1,4-phenylene spaced sugars. This is the first report for the synthesis of 1,4-phenylene spaced sugars via C-C bond formation.     

Synthesis of 2'-O-modified adenosine building blocks and application for RNA interference

RNA-interference has been recognized as a powerful tool to control gene function and has been used for gene silencing by knocking down mRNA. Chemically modified RNAs, especially 2'-O-modification, successfully improved their physicochemical and pharmaceutical properties such as stability, nuclease resistance and delivery. Here, we report the synthesis of adenosine building blocks with different 2'-tethered modifications like aminoethyl and guanidinoethyl and show that they are compatible with RNAi function. They enhance the half life of the siRNA in serum suggesting that these modifications can enhance the pharmacokinetic properties and knock down activity of siRNAs in vivo.     

Determination of the absolute P-configuration of a phthalidyl-phosphonate thymidine-thymidine dimer

Phthalidyl modified oligonucleotide thymidine-thymidine dimer building blocks were synthesized via the H-phosphonate-method. The compounds which are diastereomeric at the phosphorus atom were separated by chromatography and the absolute configuration at the phosphorus atoms was determined using ROE-experiments using the corresponding methyl-phosphonates.     

DNA containing non-nucleosidic phenanthrene building blocks with asymmetrical linkers

The synthesis and hybridization properties of oligonucleotides containing phenanthrene building blocks with non-nucleosidic linkers of different length are described. It was found that the length of the linkers, as well as the combination of unequal linkers can have a substantial influence on the thermal stability of the modified DNA.     

Hairpin mimics with phenanthroline- and bipyridine-derived linkers

The synthesis and structural stabilities of modified oligonucleotide hairpins containing phenanthroline- and bipyridine-modified loops is reported. Phenanthroline (phen) and bipyridine (bipy) building blocks were synthesized, incorporated into DNA-oligonucleotides, and analyzed by thermal denaturation experiments. The so modified oligomers were found to form stable hairpin structures. Tm values were not affected by divalent transition metals.