Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2003-04-24
2004-10-26
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S026600
Reexamination Certificate
active
06809190
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a novel method for producing a functional peptide nucleic acid monomer, a functional peptide nucleic acid oligomer produced by that method, and its intermediates. More particularly, the present invention relates to a production method comprising introducing one type or two or more types of a functional molecule post-synthetically following introduction of a precursor PIVA monomer unit into a PNA oligomer.
Nucleic acids consist of DNA and RNA that govern the genetic information of living organisms. In contrast, peptide nucleic acids (PNA) refers to modified nucleic acids in which the sugar phosphate skeleton of a nucleic acid has been converted to an N-(2-aminoethyl)glycine skeleton (FIG.
1
). Although the sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge under neutral conditions resulting in electrostatic repulsion between complementary chains, the backbone structure of PNA does not inherently have a charge. Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences. Moreover, since PNA is extremely stable with respect to nucleases and proteases in the living body and is not decomposed by them, studies are being conducted on its application to gene therapy as an antisense molecule.
As a result of using PNA in technology that conventionally used DNA as a medium, it has become possible to compensate for those shortcomings of DNA that were heretofore unable to be overcome. For example, PNA can be applied to “DNA microarray technology” for rapid and large-volume systematic analysis of genetic information, as well as recently developed “molecular beacons” used a probes capable of detecting that a base sequence has been specifically recognized using emission of fluorescent light. Since both of these use DNA lacking enzyme resistance as the medium, strict sampling is required when using these technologies. The satisfying of this requirement is the key to achieving greater sophistication of these technologies.
On the other hand, since PNA is completely resistant to enzymes, by substituting the use of DNA for PNA in DNA microarray technology and molecular beacons, the previously mentioned technical shortcomings can be overcome, leading to expectations of being able to take further advantage of the merits of these technologies.
Although there are many other fields in which the use of PNA is expected to lead to further advancements in addition to DNA microarray technology and molecular beacons, in these fields it will be necessary to design novel PNA monomers by enabling PNA to function efficiently, namely by realizing the efficient introduction of functional molecules into PNA monomers.
Since ordinary solid-phase peptide synthesis methods are used for PNA oligomer synthesis methods, classification of PNA monomer units according to PNA backbone structure yields the two types consisting of Fmoc type PNA monomer units and tBoc type PNA monomer units (FIG.
2
).
Methods for synthesizing Fmoc type PNA monomer units have already been established, and since their oligomer synthesis can be carried out using an ordinary DNA automated synthesizer, synthesis can be carried out on a small scale by the following route:
(wherein X represents guanine, thymine, cytosine or adenine).
Initially, tBoc type PNA monomer units like those shown below:
were used and this was followed by the establishment of more efficient synthesis methods.
However, since the previously mentioned Fmoc type was developed that offered easier handling, the frequency of use of the tBoc type is decreasing.
However, when introducing a functional molecule other than the four types of nucleic acid bases of guanine, thymine, cytosine and adenine, such as when introducing a photofunctional molecule, there are many cases in which the functional molecule to be introduced is unstable under alkaline conditions, and thus a tBoc type of PNA backbone structure that is not used under alkaline conditions is highly useful. A patent application for a “method for producing t-butoxycarbonyl-aminoethylamine and amino acid derivatives” has already been made by the inventors of the present invention as Japanese Patent Application No. 2000-268638.
In addition, there are also five examples of synthesis of monomer units of photofunctional oligo PNA in the prior art. Although all of these use the above route, their yields are either not described or are extremely low (Peter E. Nielsen, Gerald Haaiman, Anne B. Eldrup PCT Int. Appl. (1998) WO 985295 A1 19981126, T. A. Tran, R.-H. Mattern, B. A. Morgan (1999) J. Pept. Res, 53, 134-145, Jesper Lohse et al. (1997) Bioconjugate Chem., 8, 503-509, Hans-georg BAtz, Henrik Frydenlund Hansen, et al. Pct Int. Appl. (1998) WO 9837232 A2 19980827, Bruce Armitage, Troels Koch, et al. (1998) Nucleic Acid Res., 26, 715-720). In addition, since the structures of the compounds used have the characteristic of being comparatively stable under alkaline conditions, they are expected to be uable to be produced in good yield using a method similar to the above-mentioned methods of the prior art, namely the following route A, if an unstable chromophore attaches under alkaline conditions.
Thus, since there are typically many cases in which photofunctional molecules or other functional molecules are expensive, methods for synthesizing more pertinent functional PNA, namely methods for extremely rapidly introducing these functional molecules for (1) efficient introduction of functional molecules into a PNA backbone structure in the design of functional PNA monomer units, (2) synthesis routes in consideration of cost performance, and (3) adaptation to applications as gene diagnostic drugs, have been sought.
In consideration of the above problems, the inventors of the present invention found a novel method for producing functional PNA monomers consisting of synthesizing a photofunctional PNA monomer 4 nearly quantitatively by using a t-butoxycarbonylaminoethylamine derivative 6 for the PNA backbone structure, and condensing with an active ester form 5 containing the pentafluorophenyl group of 1 as indicated in the following route B.
In addition, the inventors of the present invention found a different method for synthesizing functional PNA monomers by using a benzyloxycarbonyl-&ohgr;-amino acid derivative instead of the above t-butoxycarbonylaminoethylamine derivative 6 for the PNA backbone structure (route C). Patent applications have already been made for these methods.
Thus, methods for ultimately synthesizing functional PNA are being established industrially that consist of synthesizing functional PNA monomers according to methods using either of the above routes B or C, followed by polymerization of those monomers. Namely, it is becoming possible to industrially synthesize large volumes of functional PNA used as PNA probes using existing functional PNA synthesis methods.
On the other hand, improvements are also being made on methods for producing functional PNA for the purpose of improving cost performance and allowing ultra-high-speed introduction of functional molecules. For example, a method has been reported in which functional molecules are introduced into PNA oligomers post-synthetically by using the following precursor PNA monomer unit as a different approach from the method described above using functional PNA monomer units (Oliver Seitz: Tetrahedron Letters 1999, 40, 4161-4164).
In this method, after introducing the above precursor PNA monomer unit into a PNA oligomer, functional PNA is synthesized by additionally introducing a functional molecule.
However, this method has the disadvantage of there being limitations on the types of functional molecules that can be introduced.
For example, as indicated below, the commercially available photofunctional molecule, succinimide ester, is unable to be introduced. Although it is necessary to first introduce a linker such as Fmoc-Gly in order to in
Ikeda Hisafumi
Tonosaki Madoka
Credia Japan Co., Ltd.
Goldberg Richard M.
Riley Jezia
LandOfFree
Functional peptide nucleic acid and its production method does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Functional peptide nucleic acid and its production method, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Functional peptide nucleic acid and its production method will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3273275