Phosphoramidite derivatives, their preparation and the use there

Details Phosphoramidite derivatives, their preparation and the use there Phosphoramidite derivatives, their preparation and the use there

1992-11-03

1996-10-22

Jones, W. Gary

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536 2532, 536 231, C07H 2100

Patent

active

055678114

DESCRIPTION:

BRIEF SUMMARY
This invention relates to certain novel phosphoramidite derivatives, particularly biotinyl and phosphotyrosinyl phosphoramidite derivatives, which are useful in the incorporation of single or multiple reporter groups on synthetic oligonucleotides. Processes for the preparation of these phosphoramidite derivatives are also disclosed.
Chemically labelled oligonucleotides are today commonly used as hybridisation probes for the detection of specific gene sequences, including those associated with human genetic diseases. Such probes may be labelled with biotin and this is highly detectable due to its affinity for binding to the proteins avidin and streptavidin. Biotin can thus in some circumstances provide a safer and more convenient form of labelling than would the use of a radioactive label (such as .sup.32 P). In addition to hybridisation probes, biotin-labelled synthetic DNA (particularly 5'-biotinylated oligonucleotides) has found uses in ligase-mediated gene detection, direct di-deoxy. sequencing following the polymerase chain reaction and the non-radioactive sequencing of DNA. However, the use of such materials has heretofore been restricted by the lack of an efficient and straightforward method for their production.
Numerous methods are known for the attachment of a single biotin moiety or other single reporter groups to the 5'-end of a synthetic oligodeoxyribonucleotide. Most of these involve the use of a linker phosphoramidite or H-phosphonate as the final coupling step in machine-aided assembly of the oligonucleotide .sup.1-4. After deprotection, an amino, thiol, or other functional group is generated at the 5'-end of the oligonucleotide and this group must then be reacted with an activated biotin derivative in a separate step.
For the attachment of multiple biotins and other labels, the most common procedures involve the preparation of a nucleoside derivative specially functionalised on the heterocyclic base to give a reactive functional group upon deprotection. The functionalised nucleoside is incorporated either enzymatically.sup.5,6 or chemically as a phosphoramidite derivative.sup.7,8. Once again an extra step is necessary in order to convert the functional groups into the appropriate polybiotinylated species. More recently, Roget et al.sup.9 have shown that it is possible to use 4-N-(6-N-biotinylaminohexyl)-2.sup.i -0-deoxycytidine (or -5-methyl-2'-deoxycytidine) derivatised as a phosphoramidite in machine-aided assembly of oligonucleotides to generate, upon deprotection, biotinylated nucleotide tails on the 5'-end of oligonucleotides. A 45-mer oligonucleotide tailed in this way is claimed to be more sensitive in in situ hybridisation using a streptavidin-alkaline phosphatase detection system than the same 45-mer tailed at the 3'-end with biotin dUTP by an enzymatic method .sup.10, although no quantitation was reported.
Cytidine derivatives functionalised on the heterocycle with biotin are not particularly conveniently prepared in that the 4-thiodeoxynucleoside starting materials are expensive. Moreover, the use of oligonucleotide tails may limit the stereochemical accessibility of the biotin moieties or alter the hybridisation properties of the oligonucleotide probe to which it is attached. Thus the use of a much simpler, non-nucleosidic linker phosphoramidite reagent capable of allowing the incorporation of multiple biotins or other reporter groups would be desirable.
Recently, Nelson et al. .sup.11 have reported that a 3-amino-1,2-propanediol unit can be functionalised to provide a phosphoramidite that can be used in oligonucleotide assembly. After deprotection, the oligonucleotide contains a 5'-tail of aliphatic primary amino groups on a repeating branched 3-carbon backbone. Whereas five such units can be efficiently assembled at the 5'-end of an oligonucleotide, only 65% of the amino groups could be functionalised subsequently with biotin, however. Very recently, Haralambidis et al..sup.12 have reported the incorporation of up to 10 biotin residues on the 3'-end of an oligonucleotide by me

REFERENCES:
Misiura et al., Nuc. Acids. Res., vol. 18, No. 15, (1990) pp. 4345-4354.
Nelson et al., Nuc. Acids Res., vol. 17, No. 18, (1989) pp. 7179-7186.

Affiliated with

Also associated with

Rating

Phosphoramidite derivatives, their preparation and the use there does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Phosphoramidite derivatives, their preparation and the use there, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Phosphoramidite derivatives, their preparation and the use there will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2359478