Solution phase synthesis of oligonucleotides

Details Solution phase synthesis of oligonucleotides Solution phase synthesis of oligonucleotides

2000-04-26

2003-01-14

McGarry, Sean (Department: 1635)

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

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S023100

Reexamination Certificate

active

06506894

ABSTRACT:

The present invention provides a method of synthesising oligonucleotides and oligonucleotide phosphorothioates in solution based on H-phosphonate coupling and in situ sulfur transfer, carried out at low temperature. The invention further provides a process for the stepwise synthesis of oligonucleotides and oligonucleotide phosphorothioates in which one nucleoside residue is added at a time, and the block synthesis of oligonucleotides and oligonucleotide phosphorothioates in which two or more nucleotide residues are added at a time.
In the past 15 years or so, enormous progress has been made in the development of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides (RNA sequences) and their analogues ‘Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotides and Analogs’, Agrawal, S. Ed., Humana Press, Totowa, 1993. Much of the work has been carried out on a micromolar or even smaller scale, and automated solid phase synthesis involving monomeric phosphoramidite building blocks Beaucage, S. L.; Caruthers, M. H.
Tetrahedron Lett.,
1981, 22, 1859-1862 has proved to be the most convenient approach. Indeed, high molecular weight DNA and relatively high molecular weight RNA sequences can now be prepared routinely with commercially available synthesisers. These synthetic oligonucleotides have met a number of crucial needs in biology and biotechnology.
Following Zamecnik and Stephenson's seminal discovery that a synthetic oligonucleotide could selectively inhibit gene expression in Rous sarcoma virus, (Zamecnik, P.; Stephenson, M.
Proc. Natl. Acad. Sci. USA
1978, 75, 280-284), the idea that synthetic oligonucleotides or their analogues might well find application in chemotherapy has attracted a great deal of attention both in academic and industrial laboratories. For example, the possible use of oligonucleotides and their phosphorothioate analogues in chemotherapy has been highlighted in the report of Gura, T. Science, 1995, 270, 575-577. The so-called antisense and antigene approaches to chemotherapy (Oligonucleotides. Antisense Inhibitors of Gene Expression, Cohen. J. S., Ed., Macmillan, Basingstoke 1989 Moser, H. E.; Dervan, P. B.
Science
1987, 238, 645-649), have profoundly affected the requirements for synthetic oligonucleotides. Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials. Several oligonucleotide analogues that are potential antisense drugs are now in advanced clinical trials. If, as seems likely in the very near future, one of these sequences becomes approved, say, for the treatment of AIDS or a form of cancer, kilogram or more probably multikilogram quantities of a specific sequence or sequences will be required.
In the past few years, a great deal of work has been carried out on the scaling-up of oligonucleotide synthesis. Virtually all of this work has involved building larger and larger synthesisers and the same phosphoramidite chemistry on a solid support. The applicant is unaware of any recent improvement in the methodology of the phosphotriester approach to oligonucleotide synthesis in solution, which makes it more suitable for large- and even moderate-scale synthetic work than solid phase synthesis.
The main advantages that solid phase has over solution synthesis are (i) that it is much faster, (ii) that coupling yields are generally higher, (iii) that it is easily automated and (iv) that it is completely flexible with respect to sequence. Thus solid phase synthesis is particularly useful if relatively small quantities of a large number of oligonucleotides sequences are required for, say, combinatorial purposes. However, if a particular sequence of moderate size has been identified and approved as a drug and kilogram quantities are required, speed and flexibility become relatively unimportant, and synthesis in solution is likely to be highly advantageous. Solution synthesis also has the advantage over solid phase synthesis in that block coupling (i.e. the addition of two or more nucleotide residues at a time) is more feasible and scaling-up to any level is unlikely to present a problem. It is much easier and certainly much cheaper to increase the size of a reaction vessel than it is to produce larger and larger automatic synthesisers.
In the past, oligonucleotide synthesis in solution has been carried out mainly by the conventional phosphotriester approach that was developed in the 1970s (Reese, C. B.,
Tetrahedron
1978, 34, 3143-3179; Kaplan, B. E.; Itakura, K. in ‘Synthesis and Applications of DNA and RNA’, Narang, S. A., Ed., Academic Press, Orlando, 1987, pp. 9-45). This approach can also be used in solid phase synthesis but coupling reactions are somewhat faster and coupling yields are somewhat greater when phosphoramidite monomers are used. This is why automated solid phase synthesis has been based largely on the use of phosphoramidite building blocks; it is perhaps also why workers requiring relatively large quantities of synthetic oligonucleotides have decided to attempt the scaling-up of phosphoramidite-based solid phase synthesis.
Three main methods, namely the phosphotriester (Reese, Tetrahedron, 1978), phosphoramidite (Beaucage, S. L. in
Methods in Molecular Biology
, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 33-61) and H-phosphonate (Froehler, B. C. in
Methods in Molecular Biology
, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 63-80; see also WO94/15946 and Dreef, C. E. in Rec. Trav. Chim. Pays-Bas, 1987, 106, p512) approaches have proved to be effective for the chemical synthesis of oligonucleotides. While the phosphotriester approach has been used most widely for synthesis in solution, the phosphoramidite and H-phosphonate approaches have been used almost exclusively in solid phase synthesis.
Two distinct synthetic strategies have been applied to the phosphotriester approach in solution.
Perhaps the most widely used strategy for the synthesis of oligodeoxyribonucleotides in solution involves a coupling reaction between a protected nucleoside or oligonucleotide 3′-(2-chlorophenyl)phosphate (Chattopadhyaya, J. B.; Reese, C. B.
Nucleic Acids Res.,
1980, 8, 2039-2054) and a protected nucleoside or oligonucleotide with a free 5′-hydroxy function to give a phosphotriester. A coupling agent such as 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-1H-triazole (MSNT) (Reese, C. B.; Titmas, R. C.; Yau, L.
Tetrahedron Lett.,
1978, 2727-2730) is required. This strategy has also been used in the synthesis of phosphorothioate analogues. Coupling is then effected in the same way between a protected nucleoside or oligonucleotide 3′-S-(2-cyanoethyl or, for example, 4nitrobenzyl)phosphorothioate (Liu, X.; Reese, C. B.
J. Chem. Soc., Perkin Trans.
1, 1995, 1685-1695) and a protected nucleoside or oligonucleotide with a free 5′-hydroxy function. The main disadvantages of this conventional phosphotriester approach are that some concomitant 5′-sulfonation of the second component occurs (Reese, C. B.; Zhang, P.-Z.
J. Chem. Soc., Perkin Trans.
1, 1995, 2291-2301) and that coupling reactions generally proceed relatively slowly. The sulfonation side-reaction both leads to lower yields and impedes the purification of the desired products.
The second strategy for the synthesis of oligodeoxyribonucleotides in solution involves the use of a bifunctional reagent derived from an aryl (usually 2-chlorophenyl) phosphorodichloridate and two molecular equivalents of an additive such as 1-hydroxybenzotriazole (van der Marel, et al,
Tetrahedron Lett.,
1981, 22, 3887-3890). A related bifunctional reagent, derived from 2,5-dichlorophenyl phosphorodichloridothioate (Scheme 1b), has similarly been used (Kemal, O et al,
J. Chem. Soc., Chem. Commun.,
1983, 591-593) in the preparation of oligonucleotide phosphorothioates.
The main disadvantages of the second strategy result directly from the involvement of a bifunctional reagent. Thus the possibility exists of symmetric

Affiliated with

Also associated with

Rating

Solution phase synthesis of oligonucleotides does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Solution phase synthesis of oligonucleotides, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Solution phase synthesis of oligonucleotides will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3035014