α-Oxo Semicarbazone Peptide or Oligodeoxynucleotide Microarrays

Abstract

We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific alpha-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide alpha-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody.

Aryldithioethyloxycarbonyl (Ardec): A New Family of Amine Protecting Groups Removable under Mild Reducing Conditions and Their Applications to Peptide Synthesis

“…It should allow the synthesis of fully functional and sensitive post-translationally site-specific modified proteins (glyco-and lipoproteins) which play important roles in numerous biological processes. [54] Furthermore, in order to get functionalized biopolymers suitable for chemoselective ligation reactions, [55,56] the Ardec-based protection strategy might be extended to other chemical functional groups such as aldehydes and ketones, for which few protecting groups removable under mild conditions are available. [57] Experimental Section General methods: Column chromatographic purifications were performed on silica gel (40-63 mm) from SdS.…”

Chemical Micropatterning of Polycarbonate for Site‐Specific Peptide Immobilization and Biomolecular Interactions

“…For comparison, a ratio of about 10 was obtained for a similar experiment performed by using microscope glass slides derivatized by semicarbazide groups. [8] These data demonstrate that semicarbazide nanoparticles adsorb onto PC and that their ability to react chemoselectively with a glyoxylylpeptide is preserved.…”

“…[8] Peptide 11 was modified by a COCHO group at the C terminus and was thus expected to react chemoselectively with semicarbazide groups, whereas 12 was modified by an amide group so could only physisorb to the nanoparticles. The printed PC slides were incubated under cover-glass with 11 or 12 (0.1 mm in acetate buffer (0.1 m, pH 5.5)) for 1 h at 37 8C, washed with phosphate buffered saline (0.01 m, pH 7.2 containing 0.05 % Tween 20) to remove excess peptide, then analyzed with a standard fluorescence microarray scanner at 532 nm ( Figure 1).…”

“…The semicarbazide groups present on the micropatterns were site-specifically treated with unprotected peptides 3 derivatized by an a-oxoaldehyde group, to give substrate 4: peptides linked to the micropatterns through semicarbazone bonds. [7][8][9] The surface between the spots was left unchanged. The use of nanoparticles of different diameters (27 to 304 nm) permitted us to study the influence of surface curvature on signal strength and capture specificity.…”

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

“…Site-specific immobilization strategies were developed in order to avoid a blocking of binding sites resulting in a loss of activity of the probe molecules. Synthetic peptides can be modified with a variety of binding moieties such as maleimide [70], a cyclopentadien conjugate [71], N-terminal cystein [72,73], biotin [74,75], thiol [76], and by the oxidation of N-terminal serine and threonine [74,75] yielding a glyoxylyl group [77,78]. These groups react with surfaces presenting thiol (maleimide, bromoacetyl), quinones (cyclopentadien), hydrazides (oxidized serine and threonine), reactive -oxo-semicarbazone groups, cysteine (glyoxylyl), and streptavidin or neutravidin (biotin).…”