Synthesis of biotin-containing phosphoramidite linker with polyether spacer arm.

A phosphoramidite linker unit, based on glycerol backbone and containing a biotin residue attached through a tetraethylene glycol spacer arm, was synthesized. DMTr-Glycidol and tetraethylene glycol were used as starting materials. After conversion of one of hydroxy groups in tetraethylene glycol into an amino group, the epoxy cycle in DMTr-glycidol was opened by this amino alcohol, resulting in the corresponding ether and some quantity of secondary amine. After attaching of biotin residue to the ether followed by phosphitylation, the desirable linker was obtained. The structure of the linker was confirmed by (1)H-(1)H COSY, (1)H-(13)C HSQC, (1)H-(13)C HMBC, (1)H-(15)N HSQC, and (1)H-(15)N HMBC spectra. The resulted phosphoramidite linker unit is suitable for use in common DNA synthesizers. This approach can be used for preparation of various modifiers containing reporter groups attached to the primary amino function using conventional procedures.

Improved synthesis of trinucleotide phosphoramidites and generation of randomized oligonucleotide libraries.

A new method to produce a set of 20 high quality trinucleotide phosphoramidites on a 5-10 g scale each was developed. The procedure starts with condensation reactions of P-components with N-acyl nucleosides, bearing the 3 '-hydroxyl function protected with 2-azidomethylbenzoyl, to give fully protected dinucleoside phosphates 13. Upon cleavage of dimethoxytrityl group from 13, dinucleoside phosphates 16 are initially transformed into trinucleoside diphosphates 19 and then the 2-azidomethylbenzoyl is selectively removed under neutral conditions to generate trinucleoside diphosphates 5 in excellent yield. Subsequent 3 '-phosphitylation affords target trinucleotide phosphoramidites 7. When mutagenic oligonucleotides are synthesized employing mixtures of building blocks 7 as well as following the new synthetic protocol, representative oligonucleotide libraries are generated in good yields.

HPLC electrospray mass spectrometric characterization of trimeric building blocks for oligonucleotide synthesis.

Trimeric nucleotide building blocks are valuable in synthesis of randomized oligonucleotides. In this study, we have developed HPLC-MS and HPLC-MS/MS methods for quality control of protected trinucleotides. C18 reversed-phase HPLC was used for purity evaluation, and base sequences were verified using negative ion electrospray ionization mass spectrometry (ESI-MS) and collision induced dissociation (CID) MS/MS. The principal dissociation pathway was formation of w-ions, which represent the 3'-5' direction. The other major fragments were d-ions, which are formed by cleavage of 5' C-O bonds of the sugars. The developed method was suitable for verification of purity and structure of the 29 trinucleotides studied.