PNA synthons

Details PNA synthons PNA synthons

2000-05-12

2001-07-24

Riley, Jezia (Department: 1656)

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

Organic compounds

Carbohydrates or derivatives

C536S025300, C536S025320, C544S276000, C544S277000, C544S317000

Reexamination Certificate

active

06265559

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the field of Peptide Nucleic Acid (PNA) synthesis. More particularly, this invention relates to PNA synthons suitable for the synthesis and deprotection of PNA-DNA chimeras.
2. Description of the Background Art
Peptide Nucleic Acids (PNAs) are synthetic polyamides which are promising candidates for the sequence-specific regulation of DNA expression and for the preparation of gene targeted drugs. See European Patent applications EP 92/01219 and 92/01220 which are herein incorporated by reference. PNAs are biopolymer hybrids which possess a peptide-like backbone to which the nucleobases of DNA are attached. Specifically, PNAs are synthetic polyamides comprised of repeating units of the amino acid, N-(2-aminoethyl)-glycine, to which the nucleobases adenine, cytosine, guanine, thymine and uracil are attached through a methylene carbonyl group. Other natural and unnatural nucleobases, such as pseudo isocytosine, 5-methyl cytosine, pseudouracil, isocytosine, hypoxanthine and 2,6-diaminopurine, among many others, also can be incorporated in PNA synthons (see FIG.
8
).
PNAs are now routinely synthesized from monomers (PNA synthons) protected according to the t-Boc/benzyl protection strategy, wherein the backbone amino group of the growing polymer is protected with the t-butyloxycarbonyl (t-Boc) group and the exocyclic amino groups of the nucleobases, if present, are protected with the benzyloxycarbonyl (benzyl) group. PNA synthons protected using the t-Boc/benzyl strategy are now commercially available but are inconvenient to use because, among other reasons, harsh acidic conditions are required to remove these protecting groups.
The t-Boc/benzyl protection strategy requires very strong acids to remove all of the benzyl side chain nucleobase protecting groups. Typically, PNA oligomers are exposed to hydrofluoric acid or trifluoromethane sulfonic acid for periods of time often exceeding one hour to completely remove the benzyl side chain protecting groups. This harsh acid treatment needed for final deprotection will often decompose, among other acid sensitive moieties, nucleic acids and carbohydrates which might be attached to the nucleic acid oligomer. Furthermore, the use of hazardous acids such as hydrofluoric acid or trifluoromethane sulfonic acid is not commercially embraced in view of safety concerns for the operators and the corrosive effect on automation equipment and lines. The above described harsh conditions are particularly unsuitable for the synthesis of nucleic acids since the strong acid deprotection conditions will at least partially decompose nucleic acids.
In addition, the t-Boc/benzyl protection strategy is not orthogonal but differential. A differential strategy is defined as a system of protecting groups wherein the protecting groups are removed by essentially the same type of reagent or condition, but rely on the different relative rates of reaction to remove one group over the other. For example, in the t-Boc/benzyl protecting strategy, both protecting groups are acid labile, with benzyl groups requiring a stronger acid for efficient removal. When acid is used to completely remove the more acid labile t-Boc protecting groups, there is a potential that a percentage of benzyl groups will also be removed contemporaneously. Specifically, the t-Boc protecting group must be removed from the amino group backbone during each synthetic cycle so the next monomer can be attached to the backbone at the free amino site thereby allowing the polymeric chain to grow. The deprotection of the t-Boc amino protected backbone is accomplished using a strong acid such as trifluoroacetic acid. During this deprotection and subsequent construction of the PNA oligomer, removal of the nucleobase side chain protecting groups, i.e., the benzyls, is undesirable. However, trifluoroacetic acid is potentially strong enough to prematurely deprotect a percentage of the side chain benzyl groups, thereby introducing the possibility of polymer branching and reducing the overall yield of desired product.
An orthogonal strategy, on the other hand, removes the protecting groups under mutually exclusive conditions, e.g., one group is removed with acid while the other group is removed with base. Breipohl et al. have described an orthogonally protected PNA synthon using 9-fluorenylmethyloxycarbonyl (Fmoc) as the backbone protecting group and a triphenylmethyl (trityl) group as the side chain nucleobase protecting group. Breipohl et al. 1st Australian Peptide Conference, Great Barrier Reef, Australia, Oct. 16-21, 1994. This protection methodology, however, is incompatible with standard nucleic acid synthesis methodology.
Christensen et al. have described orthogonal PNA synthons wherein the t-Boc amino backbone protecting group is removed in strong acid then reprotected with 9-fluorenylmethyloxycarbonyl (Fmoc), a base labile protecting group. Christensen, L. et al. “Innovation and Perspectives in Solid Phase Synthesis and Complementary Technologies-Biological and Biomedical Applications,” 3rd SPS Oxford Symposia (1994). Although this protection strategy eliminates the potential for premature deprotection of the exocyclic amino group of the side chain nucleobase, extra steps are involved in preparation of this monomer. Additionally, strong acids such as hydrofluoric acid or trifluoromethane sulfonic acid still are required to remove the benzyl side chain protecting groups.
Nucleic acids (DNA and RNA) are now routinely synthesized using automated machines, numerous synthesis supports and various protection chemistries. The following U.S. patents cover a broad range of differing supports and protection chemistries and are herein incorporated by reference. U.S. Pat. Nos. 5,262,530; 4,415,732; 4,458,066; 4,725,677 (RE 34,069); and 4,923,901. Automated equipment and reagents are commercially available from PerSeptive Biosystems, Perkin Elmer (Applied Biosystems Division) and Pharmacia. Special 5′-amino synthons are described in Smith et al., Nucleic Acids Res. (1985) 13:2399 and in Sproat et al., Nucleic Acids Res. (1987) 15:6181. Special 5′-thio synthons are described in Sproat et al., Nucleic Acids Res. (1987) 15:4837. The reagents described in the above references are suitable for use on standard DNA synthesis instruments.
The preferred commercial method for nucleic acid synthesis utilizes the above reagents and methods as generally described by Koester et al. in U.S. Pat. No. 4,725,677 (RE 34,069). Consequently, the preferred synthons are &bgr;-cyanoethyl phosphoramidites having acid labile protection of the backbone 5′ hydroxyl group and base labile acyl-type protection of the exocyclic amino groups of the nucleobases.
The preferred acid labile backbone protecting group is 4,4′-dimethoxytriphenylmethyl (DMT). DMT is typically chosen because it can be removed farily rapidly (1-3 mintues) during each synthetic cycle with solutions containing 14% dichloroacetic acid or trichloroacetic acid in dichloromethane. Protecting groups with increased acid lability compared to DMT are susceptible to premature deprotection during the acid catalyzed coupling reactions (tetrazole is typically the acidic species). Protecting groups with decreased acid lability compared to DMT require longer reaction times and/or harsher reaction conditions for complete removal. Generally, harsher acidic deprotection conditions are avoided since the purine nucleobases are particularly susceptible to decomposition in acid. Although the aforementioned problems with protecting groups and synthetic conditions may be minimal during each synthetic cycle, the cumulative effect can generate significant impurities in oligonucleotide synthesis. Accordingly, as the length of the oligonucleotide increases, its purity tends to decrease.
Generally, base labile protecting groups are utilized for protection of the exocyclic amino groups of the nucleobases so that an orthogonally protected nucleic acid synthon results. The base labile protecting groups typi

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