A versatile approach has been developed for the multiple labeling of oligonucleotides. First, three linkers as a H-phosphonate monoester derivative were condensed on a solid-supported T12 to introduce H-phosphonate diester linkages which were oxidized in the presence of propargylamine. Second, three galactosyl azide derivatives were conjugated to the solid-supported three-alkyne-modified T12 by a 1,3-cycloaddition so-called "click chemistry" in the presence of Cu(I) assisted by microwaves.

The synthesis of cyclic, branched, and bicyclic oligonucleotides was performed by copper-catalyzed azide-alkyne cycloaddition assisted by microwaves in solution and on solid support. For that purpose, new phosphoramidite building blocks and new solid supports were designed to introduce alkyne and bromo functions into the same oligonucleotide by solid-phase synthesis on a DNA synthesizer. The bromine atom was then substituted by sodium azide to yield azide oligonucleotides. Cyclizations were found to be more efficient in solution than on solid support. This method allowed the efficient preparation of cyclic (6- to 20-mers), branched (with one or two dangling sequences), and bicyclic (2 x 10-mers) oligonucleotides.

Carbohydrates and glycoconjugates play a major role in key biological events such as cell-cell recognition, pathogenesis, and inflammation. [1,2] As a consequence, there is a need to understand the structural parameters governing the recognition of carbohydrates by their receptors. This knowledge will be of use for both fundamental research and potential applications in diagnostics or therapeutics. However, research in this field is slowed by the wide diversity of carbohydrate structures and by the minute amounts of materials available for experimentation. The design of sensitive and highthroughput technologies for the characterization of oligosaccharide/protein interactions [3] is therefore emerging as an attractive tool for chemists and biochemists. Available techniques such as isothermal calorimetry, enzyme-linked lectin assay, and even crystallographic studies provide data on carbohydrate/protein interactions, but they are often limited by the amount of available material.Carbohydrate microarray technology [4][5][6][7][8][9][10][11][12][13][14][15][16] is a promising approach for probing carbohydrate/protein interactions, and it permits the simultaneous screening of a number of biological interactions with only minute amounts of material. A large family of carbohydrate derivatives has been designed for immobilization on surfaces by various means. [5][6][7][8][9][10][11][12][13][14] However, this technology has various limitations. Relative surface densities of bound ligands are often not assessed. A careful optimization of the orientation and the distance separating the carbohydrate probe from the surface is often required.The interactions of oligosaccharides with lectins are usually weak (mm range) and can be enhanced using the "cluster effect" with multivalent ligands. [17][18][19][20] In the latter case, the distance between the residues should be optimized for binding. [21][22][23] Finally, the syntheses of functionalized oligosaccharide ligands are labor intensive.We report herein an original approach for the surface immobilization of oligosaccharides using glycoconjugate molecules that present a DNA sequence for anchoring onto DNA chips through hybridization. This approach has been used in the field of protein microarrays, [24,25] but to our knowledge this is the first time that such a strategy has been reported in the field of glycoarrays.Several syntheses of glycoconjugated oligonucleotides have been reported, but none are suitable for introducing different carbohydrate moieties. [26][27][28] We designed a conjugate that incorporates carbohydrate residue(s) for interacting with a lectin, an oligonucleotide sequence for anchoring on the surface, and a fluorescent tag at the 5'-end for the determination of relative surface densities (Figure 1). These moieties were assembled through a combination of automated oligonucleotide synthesis, and amidative oxidation and 1,3-dipolar cycloaddition ("click" chemistry) performed on a solid support (Scheme 1).[29] We introduced either one or three saccharide residue...

Glyco oligonucleotide conjugates, each exhibiting two mannose and two galactose residues, were efficiently synthesized by two successive 1,3-dipolar cycloadditions (click chemistry). Two phosphoramidite derivatives were used: one bearing a bromoalkyl group as a precursor to azide functionalization and another bearing a propargyl group. After a first cycloaddition with a mannosyl-azide derivative, the bromine atoms were substituted with NaN(3) and a second click reaction was performed with a 1'-O-propargyl galactose, affording the heteroglyco oligonucleotide conjugate.

The synthesis of propargylated pentaerythrityl phosphodiester oligomers (PePOs) was achieved using a DNA synthesizer with a bis-propargylated pentaerythritol-based phosphoramidite. An azido fucose derivative was reacted under "click" chemistry conditions activated by microwaves to construct a series of glycosylated PePOs bearing 4, 6, 8, and 10 L-fucose residues. Binding to the fucose-specific bacterial lectin (PA-IIL) was determined for the fucosylated PePOs through an enzyme-linked lectin amplification competition assay. The IC50 values measured are 10-20 times better than for monovalent l-fucose and denotate for a "macromolecular" effect rather than a "cluster" effect.

A solid support bearing an azido linker was used to synthesize a 3'-azido-alkyl-oligonucleotide by phosphoramidite chemistry. The resulting oligonucleotide was either conjugated by 1,3-dipolar cycloaddition on solid support or in solution with mannose-propargyl derivative and in solution with dansyl propargyl. Besides, after introduction of an alkyne function at the 5'-end, the resulting oligonucleotide bearing both 3'-azide and 5'-alkyne functions was circularized.

Carbohydrates and glycoconjugates play a major role in key biological events such as cell-cell recognition, pathogenesis, and inflammation. [1,2] As a consequence, there is a need to understand the structural parameters governing the recognition of carbohydrates by their receptors. This knowledge will be of use for both fundamental research and potential applications in diagnostics or therapeutics. However, research in this field is slowed by the wide diversity of carbohydrate structures and by the minute amounts of materials available for experimentation. The design of sensitive and highthroughput technologies for the characterization of oligosaccharide/protein interactions [3] is therefore emerging as an attractive tool for chemists and biochemists. Available techniques such as isothermal calorimetry, enzyme-linked lectin assay, and even crystallographic studies provide data on carbohydrate/protein interactions, but they are often limited by the amount of available material.Carbohydrate microarray technology [4][5][6][7][8][9][10][11][12][13][14][15][16] is a promising approach for probing carbohydrate/protein interactions, and it permits the simultaneous screening of a number of biological interactions with only minute amounts of material. A large family of carbohydrate derivatives has been designed for immobilization on surfaces by various means. [5][6][7][8][9][10][11][12][13][14] However, this technology has various limitations. Relative surface densities of bound ligands are often not assessed. A careful optimization of the orientation and the distance separating the carbohydrate probe from the surface is often required.The interactions of oligosaccharides with lectins are usually weak (mm range) and can be enhanced using the "cluster effect" with multivalent ligands. [17][18][19][20] In the latter case, the distance between the residues should be optimized for binding. [21][22][23] Finally, the syntheses of functionalized oligosaccharide ligands are labor intensive.We report herein an original approach for the surface immobilization of oligosaccharides using glycoconjugate molecules that present a DNA sequence for anchoring onto DNA chips through hybridization. This approach has been used in the field of protein microarrays, [24,25] but to our knowledge this is the first time that such a strategy has been reported in the field of glycoarrays.Several syntheses of glycoconjugated oligonucleotides have been reported, but none are suitable for introducing different carbohydrate moieties. [26][27][28] We designed a conjugate that incorporates carbohydrate residue(s) for interacting with a lectin, an oligonucleotide sequence for anchoring on the surface, and a fluorescent tag at the 5'-end for the determination of relative surface densities (Figure 1). These moieties were assembled through a combination of automated oligonucleotide synthesis, and amidative oxidation and 1,3-dipolar cycloaddition ("click" chemistry) performed on a solid support (Scheme 1).[29] We introduced either one or three saccharide residue...

The 59 end of eukaryotic mRNA carries a N 7 -methylguanosine residue linked by a 59-59 triphosphate bond. This cap moiety ( 7m GpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 59 exonucleases and avoiding its recognition as ''nonself'' by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 59-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N 7 -methylguanosine, the synthesis of RNAs possessing a cap structure at the 59 end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 29-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 59-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN 29-Om -RNAs were purified before the N 7 -methyl group was added by enzymatic means using the human (guanine-N 7 )-methyl transferase to yield 7m GpppN-RNAs (cap-0) or 7m GpppN 29-Om -RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies.