Compounds for the synthesis of nucleotide or oligonucleotide...

Details Compounds for the synthesis of nucleotide or oligonucleotide... Compounds for the synthesis of nucleotide or oligonucleotide...

2001-08-27

2002-10-01

Richter, Johann (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025340, C536S025500, C536S025600

Reexamination Certificate

active

06458941

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to novel methods for the preparation of phosphoramidites and oligophosphoramidites. The methods are useful, inter alia, for the preparation of phosphoramidites which are useful, in turn, in the synthesis of oligonucleotide diagnostic reagents, research reagents and therapeutics agents.
BACKGROUND OF THE INVENTION
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused on interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to affect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as “antisense” agents. The oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Prior attempts at antisense therapy have provided oligonucleotides or oligonucleotide analogs that are designed to bind in a specific fashion to a specific mRNA by hybridization (i.e., oligonucleotides that are specifically hybridizable with a target mRNA). Such oligonucleotides and oligonucleotide analogs are intended to inhibit the activity of the selected MRNA by any of a number of mechanisms, i.e. to interfere with translation reactions by which proteins coded by the mRNA are produced. The inhibition of the formation of the specific proteins that are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits; however there are still problems to be solved. See generally, Cook, P. D.
Anti
-
Cancer Drug Design
1991, 6,585; Cook, P. D.
Medicinal Chemistry Strategies for Antisense Research, in Antisense Research & Applications
, Crooke, et al., CRC Press, Inc.; Boca Raton, Fla., 1993; Uhlmann, et al.,
A. Chem. Rev
. 1990, 90, 543.
Oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutics and diagnostics methods. But applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents often require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides and oligonucleotide analogs toward nuclease degradation.
A serious deficiency of unmodified oligonucleotides for these purposes, particularly antisense therapeutics, is the enzymatic degradation of the administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes.
A number of chemical modifications have been introduced into antisense agents (i.e., oligonucleotides and oligonucleotide analogs) to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense agents, to stabilize the antisense agents from nucleases and other enzymes that degrade or interfere with their structure or activity in the body, to enhance the antisense agents' binding to targeted RNA, to provide a mode of disruption (terminating event) once the antisense agents are sequence-specifically bound to targeted RNA, and to improve the antisense agents' pharmacokinetic and pharmacodynamic properties. It is unlikely that unmodified, “wild type,” oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases.
Potential applications of these oligonucleotides and their modified derivatives as drugs have created new challenges in the large-scale synthesis of these compounds.
The solid phase synthesis of oligonucleotides is inherently wasteful in that more than one equivalent of nucleosidic phosphoramidite synthons is used presumably to drive the reaction to completion. Given the vast amounts of oligonucleotide syntheses performed for research use and for large scale manufacture pursuant to clinical trials, the waste of expensive nucleoside phosphoramidites is a significant economic and ecological problem. This problem becomes more acute if one has to synthesize the monomer through a multistep synthesis before reaching the phosphoramidite stage, as is the case where modified sugar or nucleobase containing synthons are used in oligonucleotide therapeutic agents.
Consequently, there remains a need in the art for synthetic methods which do not require the sacrifice of large amount of phosphoramidite reagent. The present invention addresses these, as well as other needs.
SUMMARY OF THE INVENTION
The present invention is directed to novel methods for the recovery of phosphoramidites from waste products of traditional phosphoramidite synthesis. In preferred embodiments, methods are provided for the preparation of phosphoramidites comprising the steps of:
a reacting compound of formula:
wherein:
Z is an internucleoside linkage;
R
1
is a hydroxyl protecting group;
R
2
is H, halogen, OH, O-alkyl, O-alkylamino, O-alkylalkoxy, a polyether of formula (O-alkyl)
m
where m is 1 to about 10, or a protected hydroxyl group;
R
3
is a phosphoryl protecting group;
B is a nucleobase; and
n is 0 to about 100;
with a compound of formula:
wherein:
X
1
is Br or Cl;
X
2
and X
3
are each, independently, H, Br or Cl;
x and y are each, independently, 2 or 3, and the sum of x and y is 5; and
contacting the product of the reaction with a compound of formula HN(Q)
2
to yield a phosphoramidite of formula:
In some preferred embodiments Q is alkyl, preferably isopropyl.
R
2
is preferably H, a protected hydroxyl group, O-alkyl or O-alkylalkoxy. In more preferred embodiments R
2
is methoxyethoxy.
In some preferred embodiments R
3
is cyanoethyl, 4-cyano-2-butenyl, or diphenylmethylsilylethyl. Preferably, x is 3 and y is 2.
In preferred embodiments the reaction is performed in a solvent, which is preferably acetonitrile.
In some preferred embodiments the nucleobase is adenine, guanine, cytosine, thymine, uracil, 5-methyl cytosine or a protected derivative thereof.
In further preferred embodiments Z is a phosphodiester linkage, a phosphorothioate linkage, a phosphorodithioate linkage; or a phosphonate linkage. In particularly preferred embodiments Z is a phosphodiester linkage or a phosphorothioate linkage.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention presents novel methods for the preparation of phosphoramidites. The methods are useful for the recovery of a wide variety of species produced as waste products in oligonucleotide synthesis. In preferred embodiments, methods are provided for the preparation of phosphoramidites of formula:
wherein Z is an internucleoside linkage; R
1
is a hydroxyl protecting group; R
2
is H, halogen, OH, O-alkyl, O-alkylamino, O-alkylalkoxy, a polyether of formu

Affiliated with

Also associated with

Rating

Compounds for the synthesis of nucleotide or oligonucleotide... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Compounds for the synthesis of nucleotide or oligonucleotide..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compounds for the synthesis of nucleotide or oligonucleotide... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2992599