Monomeric building blocks for labeling peptide nucleic acids

Details Monomeric building blocks for labeling peptide nucleic acids Monomeric building blocks for labeling peptide nucleic acids

2000-01-14

2002-05-14

Riley, Jezia (Department: 1656)

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

Organic compounds

Carbohydrates or derivatives

C435S006120, C536S022100, C536S024100, C536S025300, C514S001000, C514S04400A

Reexamination Certificate

active

06388061

ABSTRACT:

DESCRIPTION
The invention relates to novel monomeric building blocks for labeling peptide nucleic acids and similarly constructed nucleic acid-binding oligomers possessing groups which are coupled to a nitrogen base and/or to the peptide backbone of the peptide nucleic acid. The invention furthermore relates to peptide nucleic acids which contain at least one labelled monomeric building block.
The chemical insertion of labeling groups, e.g. reporter molecules, into nucleic acids is of great importance for a large number of applications. The requirement here is for couplable groups, e.g. amino groups, to be incorporated into the nucleic acid in a specific manner. Specially modified monomeric nucleotide building blocks, which are compatible with the strategy for synthesizing nucleic acids which is subsequently employed, are prepared for this purpose. Thus, trifluoroacetyl-protected or Fmoc-protected aminoalkyloxy-phosphoramidites are, for example, used for 5′-end-labeling in nucleic acid chemistry (cf., e.g., EP-A-224 578, Coull et al., Tetrahedron Lett. 27 (1986), 3991-3994). Phosphoramidites which contain a trifluoroacetyl-protected or Fmoc-protected amino acid group and a DMTr ether have been used for labeling within a nucleic acid molecule, with it being possible for the parent substances to be of non-nucleotide (EP-A-0 313 219, Nelson et al., Nucleic Acids Res. 17 (1989, 7179-7186) or nucleotide (Ruth, DNA 4 (1985), 93, WO 84/03285) nature. In the case of a nucleotide parent substance, particular positions in the nitrogen base or the sugar are suitable for coupling reporter molecules (cf., e.g., Ruth (1991) Oligodeoxynucleotides with reporter groups attached to the base, in: Oligonucleotides and Analogs; A Practical Approach (F. Eckstein, HRSG), Oxford University Press, Oxford, UK, pp. 255-282 and Manoharan et al., Tetrahedron Lett. 36 (1995) 3647-3650).
Corresponding nucleoside triphosphates which are modified at the level of the nitrogen base or the ribose and which are suitable for enzymically synthesizing a nucleic acid are likewise known (EP-A-0063 879, EP-A-0286 898).
The functional groups which are able to couple reporter molecules can be blocked by a suitable protecting group such that the reporter group can only be coupled after the protecting group has been removed from the synthesized nucleic acid by means of so-called post-labeling. However, a reporter molecule can also be directly coupled onto the functional group provided that it is stable under the conditions pertaining during nucleic acid synthesis and protecting group removal. An amino side group which is able to couple reporter molecules can otherwise also be introduced at internucleotide phosphate groups, with the resulting oligonucleotide phosphite triesters or H-phosphonate being oxidized to the corresponding phosphoramidate using a mono-protected diamine (WO 92/08728 and Agrawal et al., Nucleic Acids Res. 18 (1990), 5419).
In the case of the hybridization of nucleic acids which are coupled to reporter molecules within the strand, the melting temperature, Tm, is generally found to be lower after a complementary nucleic acid counterstrand has been hybridized on. In some cases, this lowering of the melting temperature can lead to problems with regard to the specificity and sensitivity of nucleic acid hybridization methods.
As a result of the higher affinity and selectivity, as compared with customary nucleic acids, in their base pairing with a complementary nucleic acid counterstrand, peptide nucleic acids (PNAs) are gaining ever increasing importance for carrying out hybridization reactions (Egholm et al., J. Am. Chem. Soc. 114 (1992), 1895-1897, WO 92/20 702 and WO 92/20 703). In the PNAs, the sugar phosphate backbone of the nucleic acids is replaced with a peptide backbone, e.g. a 2-aminoethylglycine backbone. The nitrogen bases are coupled on at their central nitrogen atom, e.g. by way of a methylenecarbonyl group. The synthesis of a PNA therefore differs appreciably from that of a DNA since different protecting groups and coupling schemes are required. To date, functional groups for further derivatizations, e.g. for inserting reporter molecules, have been introduced at the N terminus of the last PNA building block either directly or after one or more &ohgr;-amino acids have been additionally coupled on. It has also been possible to insert functionalizable groups terminally by incorporating lysine residues at the C terminus and at the N terminus.
Since only a limited number of reporter molecules can be inserted into PNA by means of this terminal labeling, there is a great need for alternative methods for inserting labeling groups into PNA.
The present invention achieves this object by providing novel monomeric building blocks for PNA synthesis, which building blocks allow labeling groups to be inserted within the PNA molecule strand, with the labeling group being coupled to a nitrogen base and/or to a peptide backbone.
Surprisingly, it was observed, in this connection, that when PNAs are labeled on their nitrogen bases and on their peptide backbone there is then either an increase in the melting temperature of hybrids with a nucleic acid as compared with hybrids containing unlabeled PNA strands or else the destabilization is at least weaker than in the case of a corresponding DNA-DNA hybrid which contains a labeled DNA strand. This surprising increase in the melting point leads to an increased specificity in hybridization reactions and enables more stringent washing conditions and/or shorter probes to be used in detection methods.
A first embodiment of the present invention relates to monomeric building blocks for synthesizing nucleic acid-binding peptide oligomers which carry a labeling group, or a group which is able to couple with a labeling group, on the nitrogen base. Such monomers are preferably depicted by compounds of the formula (I):
in which:
B is a natural or unnatural nitrogen base which optionally carries a protecting group,
L is a labeling group which is preferably selected from signal-emitting groups, intercalators and pharmaceutically active groups or a group which is able to couple with a labeling group and which optionally carries a protecting group,
A, C and D are in each case, independently of each other, chemical bonds or organic radicals,
E is a group which is selected from N, R
1
N
+
or CH, where R
1
is an organic radical or hydrogen,
Q is a group NR
2
Y, where R
2
is an organic radical or hydrogen, and Y is a protecting group or a carrier,
I is a group which is selected from COX, CSX, SOX or SO
2
X, where X is OH, SH, OM, SM or a protecting group, and M is a cation, preferably a metal cation or an ammonium cation, and
n is an integer from 1 to 3, preferably 1.
A, C and D are preferably C
1
-C
10
-alkylene, alkenylene or alkynylene radicals which can optionally carry heteroatoms such as O, N, P or halogen and/or substituents.
A is particularly preferably a —(CH
2
)
1
—CO— radical, where 1 is an integer from 0 to 5. A is most preferably a —CH
2
CO— radical. C is particularly preferably a —(CH
2
)
k
—CHR′— radical, where k is an integer from 0 to 5 and R′ is hydrogen or the side chain of a naturally occurring amino acid. C is most preferably a —(CH
2
)
2
— radical. D is particularly preferably a (CH
2
)
m
—CHR″— radical, where m is an integer from 0 to 5 and R is hydrogen or the side chain of a naturally occurring amino acid. D Is most preferably a —CH
2
— radical. Alternatively, A and B or A and D can also be bridged with each other, i.e. form a common ring structure.
In another aspect, the present invention relates to monomeric building blocks for synthesizing nucliec acid-binding peptide oligomers which contain at least one signal-emitting group on the peptide backbone. These monomeric building blocks are preferably compounds of the formula (II):
in which:
B, A, C, D, E, Q and I are defined as in the case of the (I) compounds,
L is a labeling group which is preferably selected from signal-emitting groups, intercalators and pharmaceutically act

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