Compositions and Methods Pertaining to Guanylation of PNA Oligomers

1 . A method comprising:

a) reacting one or more primary or secondary amine groups of a PNA oligomer comprising a backbone with a reagent of formula I under basic conditions:

 to thereby form a protected guanidinium group from each of said one or more reacted amine groups;

wherein,

each Pg is the same or a different amine protecting group;

LG is a leaving group that can be displaced by reaction with the one or more amine groups of the PNA oligomer; and

wherein each protected guanidinium group has the formula I′:

 wherein, R 1 is hydrogen or an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl or heteroarylalkyl group.

2 . The method of claim 1 , further comprising deprotecting the one or more amine groups prior to its/their reaction with the reagent of formula I.

3 . The method of claim 1 , further comprising deprotecting one or both of the amine protecting groups, Pg, of the guanylated PNA oligomer.

4 . The method of claim 3 , wherein the deprotection of one or both of the amine protecting groups, Pg, is performed simultaneously with cleavage of the PNA oligomer from a solid support.

5 . The method of claim 1 , wherein LG is a group of formula II:

wherein the bracket “}” is used to indicate the points of attachment of the leaving group of formula II to the remainder of the reagent of formula I and wherein each R 2 is independently, hydrogen, fluorine, chlorine, bromine or iodine or an alkyl, an alkenyl, an alkynyl, a heteroalkyl, a heteroalkenyl, a heteroalkynyl, a heterocycloalkyl, an aryl, a heteroaryl, an arylalkyl, a heteroarylalkyl, a nitro (—NO 2 ), a cyano (—CN) or a sulfo (—SO 2 ) group.

6 . The method of claim 1 , wherein the reagent of formula I is generated in situ by combining reagents of formulas III and IV;

wherein each Pg is the same or a different amine protecting group.

7 . The method of claim 1 , wherein each Pg is independently, Aloc, Bhoc, Cbz, Cyoc, DDe, Fmoc or t-boc.

8 . The method of claim 1 , wherein the reagent of formula I is N,N′-bis-(t-boc)-guanylpyrazole (V) or N,N′-bis-Cbz-guanylpyrazole (VI)

9 . The method of claim 1 , wherein the one or more amine groups is each independently a terminal amine group or a side chain amine group.

10 . The method of claim 1 , wherein at least one of the amine groups is linked to a side chain of a subunit of the backbone of the PNA oligomer wherein the backbone subunit has the formula VII:

wherein n is an integer from 1 to 6 and Nb is a nucleobase.

11 . The method of claim 10 , wherein the at least one amine group is linked to the side chain of a subunit of the backbone of the PNA oligomer wherein the backbone subunit has the formula VIII:

wherein Nb is a nucleobase.

12 . The method of claim 11 , wherein deprotection of both protecting groups Pg of the protected guanidinium group, after reaction of the PNA oligomer with the reagent of formula I according to claim 1 , produces a backbone subunit of formula VIII′ or a protonated version thereof represented by formula VIII″:

wherein, Nb is a nucleobase.

13 . The method of claim 1 , wherein at least one amine group is linked to a side chain of a subunit of the backbone of the PNA oligomer wherein the backbone subunit has the formula IX:

wherein n is an integer from 1 to 6.

14 . (canceled)

15 . The method of claim 1 , wherein at least one of the amine groups is an N-terminal amine group of the PNA oligomer.

16 . (canceled)

17 . The method of claim 1 , wherein the PNA oligomer is reacted with the reagent of formula I when it is support bound.

18 . The method of claim 1 , wherein the PNA oligomer is reacted with the reagent of formula I when it is in solution.

19 . The method of claim 1 , wherein the PNA oligomer comprises at least one covalently linked fluorophore and/or at least one covalently linked quencher.

20 . The method of claim 1 , wherein the PNA oligomer comprises alternating subunits wherein every other PNA subunit comprises an amine group linked to a side chain of the PNA backbone wherein each said side chain amine group is capable of being guanylated by reaction with the reagent of formula I under basic conditions.

21 . The method of claim 20 , wherein the PNA oligomer comprises four PNA subunits of formula XI′:

or the protonated form thereof represented by formula XI″:

wherein the structure within each [ ] represents a PNA backbone subunit, each indicates a point of attachment to the remainder of the PNA oligomer and each Nb is a nucleobase, wherein each nucleobase can be the same of different as compared with the other nucleobases of the PNA oligomer.

22 . A PNA oligomer comprising at least one guanidinium group and at least one covalently linked fluorophore and/or covalently linked quencher.

23 . The PNA oligomer of claim 22 , wherein the PNA oligomer comprises at least one PNA subunit of formula VIII′:

or a protonated version thereof represented by formula VIII″:

24 . The PNA oligomer of claim 22 , comprising at least one covalently linked dabcyl quencher and at least one PNA subunit of formula VIII′:

or a protonated version thereof represented by formula VIII″:

wherein Nb is a nucleobase.

25 . (canceled)

26 . A method comprising:

a) performing an amplification reaction using a PNA oligomer probe capable of sequence specifically hybridizing to a target sequence in a nucleic acid of interest, wherein said nucleic acid of interest can be amplified, if present, and wherein the PNA oligomer probe has the general formula XV, or the protonated form thereof represented by formula XV′:

wherein each DL is independently of the other, a covalently linked fluorophore or a covalently linked quencher provided that only one of the two DLs can be a quencher.

27 . The method of claim 26 , wherein the PNA oligomer comprises at least one PNA subunit of formula VIII′:

or a protonated version thereof represented by formula VIII″:

wherein Nb is a nucleobase.

28 . (canceled)

29 . The method of claim 26 wherein the amplification reaction is a Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Q-beta replicase amplification (Q-beta) or Rolling Circle Amplification (RCA)

30 - 32 . (canceled)