Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-06-06
2004-06-29
Ponnaluri, Padmashri (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091500, C436S518000, C530S300000, C536S022100
Reexamination Certificate
active
06756199
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to improved synthetic processes for forming oligomeric peptide nucleic:, acids and combinatorial libraries of these peptide nucleic acids. The invention further includes new peptide nucleic acid chimeric structures. The processes of the invention utilize both monomeric and sub-monomeric synthons to form the oligomeric peptide nucleic acids having either random or predefined sequences of monomeric units. Each of the monomeric units includes a chemical moiety thereon for binding of the oligomeric structures to proteins, nucleic acids, and other biological targets. In preferred embodiments, compounds prepared via the processes of the invention act as inhibitors of enzymes such as phospholipase A
2
and are useful for the treatment of inflammatory diseases including atopic dermatitis and inflammatory bowel disease.
BACKGROUND OF THE INVENTION
Traditional processes of drug discovery involve the screening of complex fermentation broths and plant extracts for a desired biological activity or the chemical synthesis of many new compounds for evaluation as potential drugs. The advantage of screening mixtures from biological sources is that a large number of compounds are screened simultaneously, in some cases leading to the discovery of novel and complex natural products with activity that could not have been predicted otherwise. The disadvantages are that many different samples must be screened and numerous purifications must be carried out to identify the active component, often present only in trace amounts. On the other hand, laboratory syntheses give unambiguous products, but the preparation of each new structure requires significant amounts of resources. Generally, the de novo design of active compounds based on high resolution structures of enzymes has not been successful.
In order to maximize the advantages of each classical approach, new strategies for combinatorial unrandomization have been developed independently by several groups. Selection techniques have been used with libraries of peptides (see Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G. & Schoofs, P. G.,
J. Immun. Meth
. 1987, 102, 259-274, Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T. & Cuervo, J. H.,
Nature
, 1991, 354, 84-86; Owens, R. A., Gesellchen, P. D., Houchins, B. J. & DiMarchi, R. D.,
Biochem. Biophys. Res. Commun
., 1991, 181, 402-40B), nucleic acids (see Wyatt, J.,R., et al.,
Proc. Natl. Acad. Sci. USA
, (in press); Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. & Anderson, K.,
Nucleic Acids Res
., 1993, 21, 1853-1856) and nonpeptides (see Simon, R. J., et al.,
Proc. Natl. Acad. Sci. USA
, 1992, 89, 9367-9371; Zuckermann, R. N., et al.,
J. Amer. Chem. Soc
., 1992, 114, 10646-10647; Bartlett, Santi, Simon, PCT WO91/19735; and Ohlmeyer, M. H., et al.,
Proc. Natl. Acad. Sci. USA
, 1993, 90, 10922-10926). The techniques involve iterative synthesis and screening of increasingly simplified subsets of oligomers. Monomers or sub-monomers that have been utilized include amino acids and nucleotides, both of which are bifunctional. Utilizing these techniques, libraries have been assayed for activity in cell-based assays, in binding or inhibition of purified protein targets or otherwise.
A technique, called SURF (Synthetic Unrandomization of Randomized Fragments), involves the synthesis of subsets of oligomers containing a known residue at one fixed position and equimolar mixtures of residues at all other positions. For a library of oligomers four residues long containing three monomers (A, B, C), three subsets would be synthesized (NNAN, NNBN, NNCN, where N represents equal incorporation of each of the three monomers). Each subset is then screened in a functional assay and the best subset is identified .(e.g. NNAN) A second set of libraries is synthesized and screened, each containing the fixed residue from the previous round, and a second fixed residue (e.g. ANAN, BNAN, CNAN). Through successive rounds of screening and synthesis, a unique sequence with activity in the assay can be identified. The SURFs technique is described in Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. & Anderson, K.,
Nucleic Acids Res
., 1993, 21, 1853-1856. The SURF method is further described in PCT patent application WO 93/04204, the entire contents of which is herein incorporated by reference.
Peptide nucleic acids have been demonstrated to be useful surrogates for oligonucleotide in binding to both DNA and RNA nucleic acids (see Egholm et al.,
Nature
, 1993, 365, 566-568 and reference cited therein and PCT applications WO 92/20702, WO 92/20703 and WO 93/12129). Additionally peptide nucleic acids have demonstrated the ability to effect strand displacement of double stranded DNA (see Patel, D. J.,
Nature
, 1993, 365, 490-492 and references cited therein). It is not known to prepare peptide nucleic acid libraries however or to use peptide nucleic acid monomers in combinatorial techniques.
OBJECTS OF THE INVENTION
It is an object of this invention to provide new methods for the synthesis of peptide nucleic acid oligomeric structures.
It is a further object of this invention to provide sub-monomer methods for preparing peptide nucleic acid oligomeric structures.
It is a further object of this invention to provide methods of generating libraries of random sequence peptide nucleic acid oligomeric structures.
It is a further object of this invention to provide new chimeric oligomeric compounds formed of peptide nucleic acid units and amino acids units.
It is a still further object of this invention to provide new combinatorial libraries comprising oligomeric compounds formed of peptide nucleic acid units and amino acid units.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the above objects and other objects as will become evident from the remainder of this specification, there are provided novel processes for the synthesis of peptide nucleic acid oligomers. There is further provided chimeric compounds of the peptide nucleic acids and normal amino acids and process for making the same. Combinatorial libraries comprising plurality of member compounds are provided by the invention. In addition there are provided certain novel process for the preparation of libraries of peptide nucleic acid oligomers having random sequences and libraries of peptide nucleic acid oligomers having both random and fixed positions.
In a first process of the invention there is provided a method of adding further peptide nucleic acid units to an amine terminated peptide nucleic acid oligomer on a solid phase synthesis resin. The method includes treating the amine terminated oligomer on the solid phase synthesis resin with a bifunctional acetyl synthon to react a first reactive site of the bifunctional acetyl synthon with the terminal amine of the oligomer to form a resin bound oligomer having a monofunctional acetyl moiety thereon. The method further includes selecting an alkyldiamine synthon having the first of its amino functional groups in the form of a protected amino group and the other of its amino functions groups as a free amine. The method further includes treating the resin bound oligomer having the monofunctional acetyl moiety thereon with the alkyldiamine synthon to covalently bond the acetyl moiety and the free amine group of the alkyldiamine synthon forming a resin bound oligomer having an extension thereon where the extension includes a secondary amine and a protected amino group. The method further includes treating the oligomer having the extension thereon with an acetylnucleobase synthon to form an amide bond between the acetylnucleobase synthon and the secondary amine of the extension forming a new protected is amine terminated resin bound peptide nucleic acid oligomer. The method further includes deprotecting the protected amino group of the resin bound extended oligomer and repeating further iteration of the method to further extend the oligomer. Upon completion of a product of the desired length, the synthesis is terminated.
In a f
Cook Phillip Dan
Kiely John
Sprankle Kelly
ISIS Pharmaceuticals Inc.
Ponnaluri Padmashri
Woodcock & Washburn LLP
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