LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides

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Abstract

LNA guanine and 2,6-diaminopurine (D) phosphoramidites have been synthesized as building blocks for antisense oligonucleotides (ON). The effects of incorporating LNA D into ON were investigated. As expected, LNA D containing ON showed increased affinity towards complementary DNATm +1.6 to +3.0 °C) and RNATm +2.6 to +4.6 °C) ON. To evaluate if LNA D containing ON have an enhanced mismatch sensitivity compared to their complementary LNA A containing ON thermal denaturation experiments towards singly mismatched DNA and RNA ON were undertaken. Replacing one LNA A residue with LNA D, in fully LNA modified ON, resulted in higher mismatch sensitivity towards DNA ON (ΔΔTm −4 to >−17 °C). The same trend was observed towards singly mismatched RNA ON (ΔΔTm D–a = −8.7 °C and D–g = −4.5 °C) however, the effect was less clearcut and LNA A showed a better mismatch sensitivity than LNA D towards cytosineTm +5.5 °C).

Introduction

The promising aspects of using analogues of natural nucleic acids in antisense oligonucleotides (ON) has stimulated much interest in this field during recent years. Locked nucleic acid (LNA)2 was introduced in 1998[1], [2], [3] as a novel class of conformationally restricted ON analogues. It is well established that LNA is the single nucleic acid modification that contributes the highest increase in affinity (Tm) ever obtained in Watson–Crick hydrogen bonding.4 The 2,4-linked bicyclic structure provide high affinity and high specificity to both fully and partially LNA modified ON. LNA monomers can be mixed with DNA and RNA monomers and in principle all other known nucleic acid analogues based on the phosphoramidite oligomerization approach, for example, phosphorothioates and 2-O-alkyl modified RNA.5 These unique properties positions LNA as a versatile probe in nucleic acid chemistry.

In order to design and synthesize LNA therapeutics it is essential to have efficient and robust syntheses of the necessary LNA phosphoramidites. The LNA thymine, 5-methylcytosine and adenine monomers are now routinely synthesized on a 100+g scale, however the essential LNA guanine monomer has until recently caused problems due to several troublesome synthetic steps. In particular the Vorbrüggen coupling[6], [7] of the guanine nucleobase (or derivatives thereof) to the carbohydrate moiety has been problematic, inevitably leading to mixtures of the N7 and N9 regioisomers.8 Many different strategies have been devised to enhance the selectivity in favour of the N9 isomer.[9], [10], [11], [12] We found a method employing 2-amino-6-chloropurine in the Vorbrüggen coupling particularly appealing for several reasons.[9], [13], [14], [15] Besides potentially being able to furnish a regiospecific coupling a number of other advantages could be envisaged: (1) guanine and guanine nucleosides are very polar compounds, complicating synthetic work and the use of the less polar 2-amino-6-chloropurine was likely to be an advantage; (2) 2-amino-6-chloropurine is cheap and commercially available in large quantities; (3) 2-amino-6-chloropurine can be transformed into a number of different nucleobase analogues such as 6-arylpurines,16 2-aminopurine,[17], [18] 6-thiopurine[19], [20] and 2,6-diaminopurine[21], [22] essentially making it a convergent synthesis of a whole range of LNA purine monomers. The potential of 6-substituted purines as nucleoside drugs23 and the ability to modulate the properties of ON by introducing modified nucleobases24 are areas of considerable interest and has recently also turned our attention towards modified nucleobases in a LNA context.

Here we present the first regio- and stereospecific synthesis of the LNA G phosphoramidite employing 2-amino-6-chloropurine, unambiguously showing that the N9 isomer is the only product of the Vorbrüggen coupling. In addition, the synthesis of the LNA 2,6-diaminopurine (LNA D) phosphoramidite and ON containing this novel LNA monomer has been accomplished. To evaluate LNA D containing ON potential as probes for antisense ON, mismatch discrimination experiments of LNA D containing ON towards singly mismatched DNA and RNA sequences have been performed, and compared to LNA A containing ON. Moreover, the hybridization properties of LNA D containing ON, towards complementary RNA and DNA sequences, have been studied in an effort to gain a better understanding of what effects govern 2,6-diaminopurines ability to stabilize certain duplexes.

Section snippets

Results and discussion

The convergent synthesis of LNA monomers relies on the coupling of different nucleobases to the same coupling sugar 1 (Scheme 1) followed by ring closure to give the bicyclic nucleosides.8 The coupling of 1 with 2-N-isobutyrylguanine has been reported previously.8 Although conditions favouring the formation of the LNA guanine N9 isomer are employed, significant amounts (approximately 10–15%) of the N7 isomer were always detected in our hands and the N9 and N7 isomers were very difficult to

Conclusion

An efficient synthesis of the LNA G phosphoramidite has been developed taking advantage of the regiospecific Vorbrüggen coupling of 2-amino-6-chloropurine to the carbohydrate moiety, eliminating the issue of traces of the N7 regioisomer in the final product. The guanine nucleobase was protected with the dimethylformamidine protection group allowing for the use of fast ON deprotection protocols when this is required. Moreover, the NMR spectra of both the N7 and N9 regioisomers after the

Experimental

For reactions conducted under anhydrous conditions glassware was dried overnight in an oven at 150 °C and was allowed to cool in a dessicator over anhydrous KOH. Anhydrous reactions were carried out under an atmosphere of argon using anhydrous solvents. Solvents were HPLC grade, of which DMF, pyridine, acetonitrile and dichloromethane were dried over molecular sieves (4 Å from Grace Davison) and THF was freshly distilled from Na · benzophenone to a water content below 20 ppm. TLC was run on Merck

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    Present address: Cambridge University, University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, UK.

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