Synthesis of 2′-amino-LNA purine nucleosides

Article abstract of DOI:10.1080/15257770600793729

The first reported synthesis of 2′-amino-LNA purine nucleosides via a transnucleosidation is accomplished enabling the preparation of oligonucleotides incorporating 2′-amino-LNA with all four natural bases. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770600793729

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Synthesis of 2′-Amino-LNA Purine
Nucleosides
Jacob Ravn a , Christoph Rosenbohm a , Signe M. Christensen a b
Troels Koch a
&
a Santaris Pharma A/S , H⊘rsholm, Denmark
b Novo Nordisk A/S , DK-2760, Mål⊘v, Denmark
Published online: 16 Feb 2007.
To cite this article: Jacob Ravn , Christoph Rosenbohm , Signe M. Christensen & Troels Koch (2006)
Synthesis of 2′-Amino-LNA Purine Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 25:8,
843-847, DOI: 10.1080/15257770600793729
To link to this article: http://dx.doi.org/10.1080/15257770600793729
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Nucleosides, Nucleotides, and Nucleic Acids, 25:843–847, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600793729
SYNTHESIS OF 2ꢀ-AMINO-LNA PURINE NUCLEOSIDES
Jacob Ravn, Christoph Rosenbohm, Signe M. Christensen, and Troels Koch
Santaris Pharma A/S, Hørsholm, Denmark
2
The first reported synthesis of 2ꢁ-amino-LNA purine nucleosides via a transnucleosidation is
2
accomplished enabling the preparation of oligonucleotides incorporating 2ꢁ-amino-LNA with all
four natural bases.
Keywords Amino-LNA; LNA; Transnucleosidation
INTRODUCTION
The first synthesis of 2ꢁ-amino-LNA was achieved by Wengel and
coworkers[1] in 1998 for the thymine nucleoside 1 (Figure 1), and five years
later the synthesis of 5-methylcytosine nucleoside 2 was reported.[2] The
nitrogen atom in the bicyclic carbohydrate structure of 2ꢁ-amino-LNA is
located in the minor groove of a nucleic acid heteroduplex. This positioning
makes the amino group well suited for conjugation purposes. An illustrative
example of this is the conjugation of photochemical reporter groups,
such as pyrene, that may serve as useful tools for structure elucidation
and in diagnostics.[3,4] Recently, the diastereomeric 2ꢁ-amino-α-l-LNA-T
nucleoside 3 has also been synthesized, giving access to the location of the
reporter groups into the major groove of a nucleic acid duplex.[5,6] In vitro
and in vivo data[7] on 2ꢁ-amino-LNA oligonucleotides are very limited due
to the restriction that only the pyrimidines thymidine and 5-methylcytosine
of amino-LNA have been available. In this article, we present for the first
time the synthesis of the 2ꢁ-amino-LNA adenine and guanine nucleosides 4
and 5. A key intermediate in the synthesis is derived from the synthesis of
the 2ꢁ-amino-LNA-T 1.[2]
RESULTS AND DISCUSSION
In our previously reported synthesis of 2ꢁ-amino-LNA thymine
analogs,[2] an azide is incorporated at the 2ꢁ-position by nucleophilic
Received 7 February 2006; accepted 3 April 2006.
Current address for S. M. Christensen: Novo Nordisk A/S, DK-2760 Ma˚løv, Denmark.
Address correspondence to Jacob Ravn, Santaris Pharma A/S, 3 Bøge Alle, DK-2970 Hørsholm,
Denmark. E-mail: jra@santaris.com
843

844
J. Ravn et al.
FIGURE 1 2ꢁ-amino-LNA nucleoside derivatives.
substitution of a d-threo--configurated OTf. The preceding inversion of
configuration at C-2ꢁ relies on the formation of a 2,2ꢁ-anhydro intermediate
limited to the pyrimidine nucleobases. Inspired by this strategy, different
approaches for the synthesis of D-threo-configurated purine nucleosides
were explored (Scheme 1). However, all attempts at nucleophilic substitu-
tion with O-nucleophiles of 2ꢁ-OTf 6 also gave significant amount of the
elimination product 7 like the elimantion products observed by Sharma
and Nair.[8] The best conditions were reacting with NaOBz in DMSO,
yielding a 1:1 mixture of the desired d-threo-configurated purine nucleoside
8 and the elimination product. Substitution with different halogens also
resulted in elimination reactions, but refluxing 6 with LiI in CH3CN yielded
the 2ꢁ-I 9 in 90% yield. However, all attempts to displace the 2ꢁ-I with
N-nucleophiles such as NaN3, TMSN3, BnNH2, and phtalimide salts in
different solvents either failed to react at all or gave the elimination product
10 like the elimination products obtained by Haraguchi et al.[9] Under
SCHEME 1 Reagents and conditions: i) NaOBz, DMSO; ii) LiI, CH3CN, 90% iii) N-nucleophiles.

Synthesis of 2 ꢁ-Amino-LNA Purine Nucleosides
845
these conditions, depurination was also detected. The same strategy was
attempted for other purine analogs with similar results. Oxidation of C-2ꢁ
followed by stereoselective reduction to invert the configuration[10] was also
tried, but that strategy turned also out to be unsuccessful.
We finally decided to explore an approach where the purine nucleobase
is incorporated after the 2ꢁ-azide, hence exchanging the thymine for a
purine in a transnucleosidation.[11,12] In the synthesis of 2ꢁ-amino-LNA
thymine analogs, the 2ꢁ-azide nucleoside 12 (Scheme 2) is a key inter-
mediate. Initial attempts to perform the transnucleosidation on 12 were
unsuccessful, which is in accordance with earlier observations.[12] Instead
it was anticipated that an acylated 2ꢁ-amine would be a suitable substrate
for a transnucleosidation reaction. 12 was reduced using trimethylphos-
phine under neutral condition, to avoid ring-closure, and the resulting
2ꢁ-amine was “trapped” by addition of trifluoroacetic anhydride directly
to the reaction mixture to give 13 in 79% yield. Subjecting 13 to the
SCHEME 2 Reagents and conditions: i) a, PMe3, THF; b, TFAA (79%); ii) N6-benzoyladenine, BSA,
TMSOTf, DCE (57%); iii) LiOH, THF/H2O (99%); iv) NaOBz, DMSO (97%); v) sat. NH3/MeOH
(86%); vi) HCO2NH4, Pd(OH)2/C, MeOH (76%); vii) 2-amino-6-chloropurine, BSA, TMSOTf, DCE
(58%); viii) NaOH, THF/H2O (93%); ix) BnOH, KOtBu, THF (62%); x) a, NaOBz, DMSO; b, LiOH,
THF/H2O (85%); xi) HCO2NH4, Pd(OH)2/C, EtOH/H2O (81%).

846
J. Ravn et al.
Vorbru¨ggen[13] conditions (BSA and TMSOTf) with an excess of either
N6-benzoyladenine or 2-animo-6-chloropurine resulted in the successful
formation of the corresponding β purine nucleosides 14 and 18 in 57% and
58% yield, respectively, after chromatography. It is likely that anchimeric
assistance from the trifluoroacetyl group is responsible for the preferential
formation of the β-isomer in the same way as seen for 2ꢁ-O-acetylated glycosyl
donors. Because of the nature of the reaction being a thermodynamic
competition between the thymine leaving group and the silylated purine
base, an excess of purine (3 eq.) was necessary to drive the reaction to
completion.
Hydrolysis of the trifluoroacetyl group in 14 led to a subsequent ring
closure to give the fully protected 2ꢁ-amino-LNA substituted adenosine
nucleoside 15 in 99% yield. During all the preceding transformations,
the 5ꢁ-OMs groups have proven extremely stable. In our experience it is
actually possible to hydrolyze these mesylates by prolonged exposure to
concentrated aqueous LiOH at elevated temperature, giving rise to very
impure products. Instead, we usually prefer substitution of the mesylate
with benzoyl using sodium benzoate to give, in this case, 16 followed by
deprotection of both benzoyl groups by methanolic ammonia resulting in
17. Finally, the fully deprotected 2ꢁ-amino-LNA adenosine 4 was obtained
after debenzylation with ammonium formate and a catalytic amount of
palladium hydroxide on charcoal. In a similar fashion, 18 was hydrolyzed
with subsequent ring closure to give 19 in 93% yield. Substitution of the
6-chloro group with benzylalcohol gave the protected guanosine derivative
20, which was further transformed at the 5ꢁ-position to give 21. Finally,
debenzylation of both 3ꢁ-O and 6-O resulted in the fully deprotected 2ꢁ-
amino-LNA guanosine 5. In all 1H-NMR spectra for the bicyclic compounds
(15–17, 4, 19–21, and 5) the signals for 1ꢁ-H, 2ꢁ-H, and 3ꢁ-H are all singlets,
confirming the nucleosides to be in a North-conformation.
In conclusion, we have developed a new practical method for the
preparation of 2ꢁ-amino-LNA purine nucleosides via a transnucleosidation.
The novel synthesis strategy is exemplified for the adenosine and guanosine
derivatives. We are currently utilizing this method in preparing the suitably
protected phosporamidate analogs to be used in an automated oligonu-
cleotide synthesis.
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Synthesis of 2 ꢁ-Amino-LNA Purine Nucleosides
847
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