Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-24
2003-07-15
Barts, Samuel (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S025320, C536S124000, C536S123100, C536S026800
Reexamination Certificate
active
06593464
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to processes for the substantial deprotection or deblocking of labeled oligonucleotides by use of an amino reagent such as ammonia.
2. Related Art
A variety of solid phase oligonucleotide synthesis techniques are known to those skilled in the art. Such techniques include phosphoramidite, phosphotriester, phosphodiester, phosphite and H-phosphonate methods and the like, each of which is generally known in the field of molecular biology. For example, the b-cyanoethyl phosphoramidite method is described in U.S. Pat. No. 4,458,066 issued to Caruthers, et al., entitled “Process for Preparing Polynucleotides,” which is incorporated herein by reference.
The phosphoramidite based synthesis of oligonucleotides requires the protection of the exocyclic amino groups. The most commonly used protecting groups for this purpose are benzoyl for the 6-amino of adenine and 4-amino of cytosine and isobutyroyl for 2-amino of guanine. Oligonucleotides are synthesized on solid support using nucleoside phosphoramidites where the amino groups are protected as shown below.
After the synthesis is completed the oligonucleotide is cleaved from the support and these protecting groups are removed by hydrolysis at high temperatures using concentrated ammonium hydroxide. After hydrolysis, the ammonium hydroxide has to be evaporated in order to obtain the desired oligonucleotide.
The use of hot concentrated ammonium hydroxide for the removal of these protecting groups has restricted the modified bases that can be used to those that can withstand these harsh conditions. For modified oligos such as dye containing oligos, heating in ammonium hydroxide cannot be used since the dyes are not stable under these conditions. In general, dye labeled oligos are deprotected by treatment with ammonium hydroxide at room temperature for over 24 hours or require special reagents for this purpose. See U.S. Pat. No. 4,965,349. Alternatively, oligos may be prepared using phosphoramidite having easily removable protecting groups which do not require the use of hot concentrated ammonium hydroxide. See Boal, J. H. et al.,
Nucl. Acids Res
. 24:3115-3117 (1996).
The standard method where deprotection with concentrated ammonium hydroxide is done at reduced temperature results in incomplete deprotection and over all low quality of dye labeled oligonucleotides. Often this requires tedious purification which results in low yield.
U.S. Pat. No. 4,965,349 describes a method of hydrolyzing base-labile linking groups between a solid phase support and oligonucleotides with a reagent comprising a lower alcohol, water and a non-nucleophilic hindered alkylamine. According to this patent, this cleavage reagent preserves the fluorescent characteristics of rhodamine dyes during cleavage from the solid support.
U.S. Pat. No. 5,514,789 describes a method for the cleavage and deprotection of newly synthesized oligonucleotides from solid supports with a gaseous cleavage/deprotection reagent such as gaseous ammonia, ammonium hydroxide vapors, and methylamine.
U.S. Pat. No. 5,518,651 describes a method for the cleavage and deprotection of insolubilized and protected oligonucleotides using an alkyl amine, e.g. t-butylamine and methylamine. According to this patent, the deprotection and cleavage of the oligonucleotides occurs at room temperature and in less than about 90 min.
U.S. Pat. No. 5,738,829 describes a method for the cleavage and deprotection of oligonucleotides from solid supports which involves incubation of the immobilized oligonucleotides with gaseous ammonia or ammonium hydroxide vapors. According to this patent, the method lends itself to the use of supports such as microtiter plates that can be used to perform up to 96 individual synthetic processes.
Glenn Research of Sterling Virginia offers phenoxyacetyl protected dA, 4-isopropylphenoxylacetyl protected dG and acetyl protected dC which can be used to prepare oligonucleotides. According to Glenn Research's web site, these monomers can be used with sensitive labeling reagents such as TAMRA, Cy5® and HEX since cleavage and deprotection can be carried out in 2 hours at room temperature with ammonium hydroxide or 0.005 M potassium carbonate in anhydrous methanol. In addition, according to this web site, it is possible to deprotect oligonucleotides containing acetyl protected dC monomers by treatment with ammonium hydroxide/methylamine for 10 min at 65° C. or less.
We have now found that the use of nucleophilic amino compounds under pressure and high temperature is an effective way to deprotect dye labeled oligos. The dye labeled oligos deprotected in this manner are fully deprotected and are of high quality. In addition, the process is simple and saves time, reducing the deblocking (processing) time from approximately 28 hours to 1 hour.
BRIEF SUMMARY OF THE INVENTION
The invention relates to a process for deblocking a detectably labeled oligonucleotide comprising contacting the blocked detectably labeled oligonucleotide with an effective amount of a nucleophilic amino compound under conditions that result in the deblocking of the oligonucleotide, thereby giving the deblocked oligonucleotide.
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International Search Report for International Patent Application No. PCT/US00/14274,
Gebeyehu Gulilat
Pires Richard M.
Barts Samuel
Henry Michael C.
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
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