Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1999-07-22
2003-04-08
Solola, T. A. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S312400, C548S312700, C548S313700, C548S314700, C548S334500, C548S557000, C536S022100, C536S023100, C536S025300, C536S025330, C536S025600, C536S026100
Reexamination Certificate
active
06545162
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of peptide chemistry. Specifically, this invention relates to a novel process for preparing polyamides and polyamide conjugates containing imidazole and pyrrole carboxamides using solid state chemistry. Also included in this invention is a simple and effective method for preparing analogs of the antiviral antibiotics Netropsin and Distamycin A.
BACKGROUND OF THE INVENTION
Proteins and peptides play a critical role in virtually all biological processes, functioning as enzymes, hormones, antibodies, growth factors, ion carriers. antibiotics, toxins. and neuropeptides. Biologically active proteins and peptides. therefore, have been a major target for chemical synthesis. Chemical synthesis is used to verify structure and to study the relationship between structure and function, with the goal of designing novel compounds for potential therapeutic use. Thus, modified or novel peptides may be synthesized which have improved therapeutic activity and/or reduced side effects.
There are two basic methods for synthesizing proteins and peptides: the chemistry is either carried out in solution (solution phase) or on a solid support (solid phase). A major disadvantage of solution phase synthesis of peptides is the poor solubility of the protected peptide intermediates in organic solvents. Additionally, solution phase synthesis requires extensive experience on the part of the scientist and the purifications are difficult and time consuming. Solid phase synthesis overcomes these problems and thus, has become the method of choice in synthesizing peptides and proteins.
The basic approach for solid phase peptide synthesis is illustrated in FIG.
1
. Briefly, the carboxy-terminal amino acid of the peptide to be synthesized is protected and covalently attached to a solid support, typically a resin. The subsequent amino acids (which have also been protected) are then sequentially added. When the synthesis is complete the peptide is deprotected, cleaved from the resin and purified. Because the molecules being synthesized are so large it is imperative that the steps proceed rapidly, in high yields and with minimal side reactions.
The most commonly used solid supports are cross-linked polystyrene and polydimethylacrylamide resins, which are both derivatives of polyethylene. In 1978, Merrifield and coworkers introduced the tert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenyl-acetamidomethyl-resin (PAM resin), a novel polystyrene resin for solid phase peptide synthesis (Mitchell et al. (1978) J. Org. Chem. 43:2845-2852). PAM resin has a preformed resin ester linkage which is stable to trifluoroacetic acid and can be cleaved under a variety of conditions including, liquid hydrogen fluoride. aminolysis, hydrolysis, hydrazinolysis, catalytic hydrogenation, or lithium borohydride to give a peptide acid, amide, hydrazide. or primary alcohol (Stewart and Young (1984) in
Solid Phase Peptide Synthesis,
sec. ed., Pierce Chemical Company, Illinois pp. 88-95).
Netropsin and Distamycin A (
FIGS. 2A and 2B
) are heterocyclic polyamides, containing imidazole (Im) and pyrrole (Py) carboxamides. These compounds are isolated from
Streptomyces distallicus
and exhibit antibiotic, antiviral and antitumor activity. Other members of this family of antibiotics include noformycin (Diana (1973) J. Med. Chem. 16:3774-3779), kikumycin B (Takaishi et al. (1972) Tetrahedron Lett. 1873), and anthelvencin (Probst et al. (1965) Antimicrob. Agents Chemother. 789). Netropsin and Distamycin A are two examples of the many small molecules (MW<2 kD) which can bind and/or cleave DNA with modest sequence specificity (Krugh (1994) Curr. Opin. Struct. 4:351-364). These drugs block template function by binding to specific nucleotides in the minor groove of double-stranded DNA.
Due to the pharmaceutical potential of this family of peptides a considerable amount of research has been devoted to the study of these compounds and their analogues. The x-ray crystal structure of a 1:1 complex of Netropsin with the B-DNA dodecamer 5′-CGCGAATTCGCG-3′ (SEQ ID NO:1) provides an understanding of how the sequence specificity is achieved, revealing that the amide hydrogens of the NV-methylpyrrolecarboxamides form bifurcated hydrogen bonds with adenine N3 and thymidine O2 atoms on the floor of the minor groove. (Koopka et al. (1985) Proc. Natl. Acad. Sci. 82:1376; Koopka et al. (1985) J. Mol. Biol. 183:553). The pyrrole rings completely fill the groove excluding the guanine amino group of a G, C base pair while making extensive van der Waals contacts with the walls of the groove, thereby affording specificity for A,T sequences. (Taylor et al. (1985) Tetrahedron 40:457; Schultz and Dervan (1984) J. Biomol. Struct. Dyn. 1:1133). Efforts to design ligands specific for G, C containing sequences, were largely unsuccessful (see e.g., Lown et al. (1986) Biochemistry 25:7408; Kssinger et al. (1987) Biochemistry 26:5590; Lee et al. (1987) Biochemistry 27:445; Lee et al. (1993) Biochemistry 32:4237), until the discovery that two polyamides combine side-by-side in the minor groove of DNA, forming a 2:1 complex with the DNA. (Pelton (1989) Proc. Natl. Acad. Sci., USA 86:5723-5727; Pelton (1990) J. Am. Chem. Soc. 112:1393-1399; Chen et al. (1994) M. Struct. Biol. Nat. 1:169-175; Wade et al. (1992) J. Am. Chem. Soc. 114:8783-8794; Mrksich et al (1992) Proc. Natl. Acad. Sci., USA 89:7586-7590; Wade (1993) Biochemistry 32:11385-11389; Mrksich et al. (1994) J. Am. Chem. Soc. 116:7983-7988). Each ligand interacts with one of the DNA strands in the minor groove, with the imidazole nitrogen making specific hydrogen bonds with one guanine amino group. Thus, both Distamycin A and imidazole containing ligands such as the designed polyamide imidazole-pyrrole-pyrrole-dimethylaminoproplyamine (IrmPyPy-Dp), 1-methylimidazole-2-carboxamide Netropsin, bind specifically in the minor groove as 2:1 polyamide/DNA complexes recognizing G, C sequences.
From studies of the 2:1 model it is now known that the combination of imidazole/pyrrole carboxamide recognize a G, C base pair, and the combination of pyrrole carboxamide/imidazole recognizes a C, G base pair, the pyrrole carboxamide/pyrrole carboxamide combination is partially degenerate for T, A and A, T. The utility of the 2:1 model as an aid in designing ligands with sequence specificity for DNA is illustrated by the designed polyamide imidazole-pyrrole-imidazole-pyrrole-dimethylaminopropylamine (ImPyImPy-Dp) which binds a four base pair core sequence 5′-GCGC-3′. This is a complete reversal of the natural specificity of Netropsin and Distamycin A.
The literature contains a number of reports of the total synthesis of various members of this family of polyamides and their analogues. All of the reported syntheses have been performed in the solution phase. The amide bond unit in these polyamides is formed from an aromatic carboxylic acid and an aromatic amine, both of which have proven problematic for solution phase coupling reactions. The aromatic acids are often unstable resulting in decarboxylation and the aromatic amines have been found to be highly air and light sensitive (Lown and Krowicki (1985) J. Org. Chem. 50:3774-3779). It was believed that the variable coupling yields, long (often >24 hour) reaction times, numerous side products, and wide scale use of acid chloride and trichloroketone intermediates in solution phase coupling reactions would make the synthesis of the aromatic carboxamides difficult, if not impossible by solid phase methods (He et al. (1993) J. Am. Chem. Soc. 115:7061-7071; Church et al. (1990) Biochemistry 29:6827-6838; Nishiwaki et al. (1988) Heterocycles 27:1945-1952). Thus, to date, there have been no reported attempts to synthesize this class of compounds using solid phase methodology.
The process of developing new ligands with novel sequence specificity generally involves four stages; design, synthesis, testing, and redesign of the model (Dervan (1986) Science 232:464). While exploring the limits of the 2:1 model, the synthet
Baird Eldon E
Dervan Peter B.
California Institute of Technology
Foley & Lardner
Solola T. A.
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