Chiral peptide nucleic acids

Details Chiral peptide nucleic acids Chiral peptide nucleic acids

2001-08-17

2004-03-30

Marschel, Ardin H. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C435S006120, C530S333000

Reexamination Certificate

active

06713603

ABSTRACT:

INTRODUCTION
This invention describes the synthesis and properties of a novel class of chiral peptide nucleic acids (cPNAs) which hybridise strongly with complementary nucleic acids. As such they have potential as antigene and antisense agents and as tools in molecular biology.
Oligonucleotides are potentially useful for the regulation of genetic expression by binding with DNA or mRNA
1
. However, natural oligonucleotides are degraded by nucleases, consequently there is considerable interest in synthetic oligonucleotide analogues which are stable under physiological conditions. Recently, there has been interest in oligonucleotide analogues in which the sugar-phosphate backbone is replaced by a peptide chain
2
after the success of the so-called Peptide Nucleic Acids (PNA)
3
, but more correctly referred to as Polyamide Nucleic Acids
4
.
The sugar phosphate backbone of a nucleic acid consists of a repeating unit of six atoms, configurationally and conformationally constrained by the D-ribose or 2′-deoxy-D-ribose ring. If this could be replaced by a dipeptide unit the new backbone would be amenable to preparation by solid phase peptide synthesis. Molecular modelling by computer graphics suggested that a peptide chain consisting of an alternate sequence of a “nucteo-amino acid” derived from proline and a “spacer amino acid”, which could be any amino acid, should be a suitable structural analogue of the ribose phosphate backbone of nucleic acids as shown.
SUMMARY OF INVENTION
This invention provides compounds of formula (I)
where n is 1 or 2-200
B is a protected or unprotected heterocyclic base capable of Watson-Crick or of Hoogsteen pairing,
R is H, C1-C12 alkyl, C6-C12 aralkyl or C6-C12 heteroaryl which may carry one or more substituents preferably selected from hydroxyl, carboxyl, amine, amide, thiol, thioether or phenol,
X is OH or OR′ where R′ is a protecting group or an activating group or a lipophilic group or an amino acid or amino amide or nucleoside,
Y is H or a protecting group or a lipophilic group or an amino acyl group or nucleoside.
When n is 1, these compounds are peptide nucleotide analogues. When n is 2 to about 30 these compounds are peptide oligonucleotides, which are synthesised as described below and can be hybridised to ordinary oligo or polynucleotides. Typically the two strands are hybridised to one another in a 1:1 molar ratio by base-specific Watson-Crick base pairing.
B is a base capable of Watson-Crick or of Hoogsteen pairing. This may be a naturally occurring nucleobase selected from A, C, G, T and U; or a base analogue that may be base specific or degenerate, e.g. by having the ability to base pair with both pyrimidines (T/C) or both purines (A/G) or universal, by forming base pairs with each of the natural bases without discrimination. Many such base analogues are known, e.g. hypoxanthene, 3-nitropyrrole, 5-nitroindole, and those cited by Lin and Brown
5
and all are envisaged for use in the present invention.
The compounds of formula (I) contain proline of undefined stereochemistry. Although compounds with the trans-stereochemistry may have interesting properties, compounds with the cis-stereochemistry are preferred either with the D-configuration as shown in (II) or the L-configuration shown in structure (III). In these compounds both stereoisomers of the “spacer amino acid” NHCHRCO are envisaged.
Provided that it does not sterically hinder chain extension of hybridisation, the group R could have diverse structures. The group, however, can be chosen to confer desired hydrophobic, hydrophilic and/or electrostatic properties on the molecule. When the group R is other than H it generates a chiral centre and the two stereoisomers may allow for discrimination in the hybridisation of DNA and RNA. When the amino acid (—NH—CHR—CO—) is a naturally occurring amino acid, then the amino acid should be readily and cheaply available as a building block for compounds of this invention. Any of the natural or unnatural &agr;-amino acids could be used e.g. glycine or L- or D-serine or lysine. The nature of X can be varied from the negatively charged carboxylate ion (X═O
−
) to the incorporation of a positively charged lysine residue. Examples of the latter are provided in the experimental section and can be used to prevent aggregation and to assist hybridisation to the negatively charged oligonucleotides. Y will most commonly be H but could be any group which might be useful to improve the physical or biological properties of the material.
Any one of B, R, X and Y may include a signal moiety, which may be for example a radioisotope, an isotope detectable by mass spectrometry or NMR, a hapten, a fluorescent group or a component of a chemiluminescent or fluorescent or chromogenic enzyme system. The signal moiety may be joined to the peptide nucleotide analogue either directly or through a linker chain of up to 30 atoms as well known in the field.
In another aspect the invention provides a method of making the peptide nucleotide analogue of formula (I), comprising the steps of:
a) reacting an N-protected C-protected 4-hydroxy proline with a base selected from N
3
-protected thymine, N
6
-protected adenine, N
4
-protected cytosine, N
2
—O
6
-protected guanine and N
3
-protected uracil.
b) deprotecting the proline amino group of the product of a),
c) reacting the product of b) with an N-protected amino acid,
d) optionally removing protecting groups from the product of c).
In another aspect the invention provides a method of converting a peptide nucleotide analogue of formula (I) in which n is 1 into a peptide oligonucleotide of formula (I) in which n is 2-200, comprising the steps of:
i) providing a support carrying primary amine groups,
ii) coupling an N-protected peptide nucleotide analogue of formula (I) to the support,
iii) removing the N-terminal protecting group,
iv) coupling an N-protected nucleotide analogue of formula (I) to the thus-derivatised support,
v) repeating steps iii) and iv) one or more times, and
vi) optionally removing the resulting peptide oligonucleotide from the support.
The invention also provides a compound of formula (IV)
where R
2
is H or a protecting group,
R
3
is H or a protecting group compatible with R
2
, and
B is a protected or unprotected heterocyclic base.
The invention also provides a compound of formula (V)
where R
2
is diphenylmethyl, and
R
3
is t-butoxycarbonyl.
DETAILED DESCRIPTION
The (2R,4R) (“cis-D”)-proline was chosen since this is analogous to the stereochemistry of deoxyribonucleotides. The lack of negative charge on the peptide backbone would be expected to lead to a higher affinity for complementary oligonucleotide sequences in nucleic acids. Moreover these novel peptide nucleic acids can also be modified easily by using different “spacer amino acids” to affect physical and biological properties such as solubility, cell permeability, etc. in order to achieve higher therapeutic activity. Such peptide nucleic acids should be stable to proteases since they contain substituted D-proline residues at alternate sites. Since coupling to secondary amino acids can be slow and inefficient, it was decided to use dipeptide building blocks in which the amino-acyl-proline bond is formed in solution as in dipeptide (1). In the alternative arrangement, i.e. prolyl-amino acid, there is likely to be a serious problem of racemisation during the coupling if the amino acid is chiral, whereas such racemisation is not expected when the C-terminus of the activated fragment is proline because N-acylprolines can not racemise by the oxazolone mechanism
6
.
Because of the mild conditions used for the deprotection of the N-Fmoc group, the Fmoc/O
t
Bu strategy in solid phase peptide synthesis is favoured over the classical Boc/OBzl strategy
7
. Furthermore, most machine synthesisers capable of handling small scale synthesis (50 &mgr;mol or less) can accommodate only the Fmoc/O
t
Bu strategy. For these reasons, it was decided to use the Fmoc instead of Boc as the N-protecting group.
There were two possible synthet

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