Most popular non-nucleosidic phosphoramidites for modification and labeling are based on two structural types: 1,2-diols and 1,3-diols. Products based on a 1,2-diol backbone were first described to allow amino-modification and biotin labeling. Technically, the 1,2-diol backbone has some drawbacks relative to the 1,3-diol backbone. The 1,2-diol backbone can participate in a dephosphorylation reaction since the 1,2-diol can form a favored 5-membered cyclic phosphate intermediate. This reaction is competitive with simple hydrolysis of the protecting groups and leads to some loss of label. However, the degree of loss at the 3’ terminus can be limited by the removal of the cyanoethyl protecting group using DBU or diethylamine prior to the cleavage and deprotection steps. Similarly, loss at the 5’ terminus can be eliminated by retaining the DMT group until the oligo is fully deprotected. Fortunately, the elimination reaction is virtually non-existent in the 1,3-diol backbone since the cyclic intermediate would be a 6-membered ring which is not favored for a cyclic phosphate intermediate.
IVD customers have requested a new backbone based on a 1,3-diol that would overcome any technical or IP issues surrounding our current products. We now offer a line of products based on the serinol backbone, which have been developed in close collaboration between Glen Research and Nelson Biotechnologies. Protected Biotin Serinol Phosphoramidite and CPG are protected with a t-butylbenzoyl group on the biotin ring. This group is designed to stop any phosphoramidite reactions at this active position in biotin. This protection avoids branching when using nucleophilic activators like DCI. The protecting group is easily removed during oligonucleotide cleavage and deprotection. The BiotinLC versions are similarly protected and should be useful for the synthesis of highly sensitive biotinylated probes. 6-Fluorescein Serinol Phosphoramidite and CPG are designed to prepare oligonucleotides containing one or several 6-Fluorescein (6-FAM) residues. Amino-Modifier Serinol Phosphoramidite and CPG are used to add amino groups into one or several positions in oligonucleotides. The amino group is protected with Fmoc, which may be removed on the synthesis column prior to solid-phase conjugation to the amino groups, or which may be removed during deprotection for subsequent solution phase conjugation to the amino groups.
Combining lipoic acid and our patented serinol backbone, we now offer Dithiol Serinol Phosphoramidite and the related 3’-Dithiol Serinol CPG. This unique architecture incorporates a linker that moves the bulky dithiol away from the phosphate backbone. The long spacer arm of Dithiol Serinol also allows multiple consecutive incorporations of the modifier without the need for intermediate spacer phosphoramidite additions to achieve optimal stepwise coupling efficiency.
We offer three products for use in Click Chemistry based upon our 1,3-diol product portfolio with the serinol backbone - a phosphoramidite for adding an alkyne group at the 5’ terminus or within the sequence, a synthesis support for labeling the 3’ terminus of oligonucleotides with an alkyne group, and DBCO-Serinol phosphoramidite as a copper-free click reagent .
Serinol Reagents for Modification and Labeling are covered by US Patent No.: 8,394,948.