Synthesis of phosphoramidites of isoGNA, an isomer of glycerol nucleic acid

Article abstract of DOI:10.3762/bjoc.10.220

IsoGNA, an isomer of glycerol nucleic acid GNA, is a flexible (acyclic) nucleic acid with bases directly attached to its linear backbone. IsoGNA exhibits (limited) base-pairing properties which are unique compared to other known flexible nucleic acids. He

Full text of DOI:10.3762/bjoc.10.220

Synthesis of phosphoramidites of isoGNA, an isomer of
glycerol nucleic acid
Keunsoo Kim, Venkateshwarlu Punna, Phaneendrasai Karri
and Ramanarayanan Krishnamurthy*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 2131–2138.
Department of Chemistry, The Scripps Research Institute, 10550
North Torrey Pines Rd, La Jolla, CA 92037, USA
doi:10.3762/bjoc.10.220
Received: 01 May 2014
Email:
Accepted: 15 August 2014
Published: 08 September 2014
Ramanarayanan Krishnamurthy* - rkrishna@scripps.edu
* Corresponding author
This article is part of the Thematic Series "Nucleic acid chemistry".
Guest Editor: H.-A. Wagenknecht
Keywords:
acyclic nucleic acids; glycerol nucleic acids; isoGNA; oligonucleotides;
phosphoramidites
© 2014 Kim et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
IsoGNA, an isomer of glycerol nucleic acid GNA, is a flexible (acyclic) nucleic acid with bases directly attached to its linear back-
bone. IsoGNA exhibits (limited) base-pairing properties which are unique compared to other known flexible nucleic acids. Herein,
we report on the details of the preparation of isoGNA phosphoramidites and an alternative route for the synthesis of the adenine
derivative. The synthetic improvements described here enable an easy access to isoGNA and allows for the further exploration of
this structural unit in oligonucleotide chemistry thereby spurring investigations of its usefulness and applicability.
Introduction
Acyclic nucleic acids have garnered a lot of attention and are of the backbone exerts a strong influence on the disposition of
becoming an important component in nucleic acid chemistry the nucleobases, and there seems to be no straightforward corre-
[1-3]. Lately, there has been an explosion in the number of lation between the nature of the backbone and base-pairing
investigated candidates [4-7]. We have recently reported on the properties [9].
base-pairing properties of isoGNA, an isomer of glycerol
derived nucleic acid (Figure 1) [8]. Our motivation was driven We are continuing our investigation of isoGNA oligonu-
by the question “how structurally simple and minimal can an cleotides within the context of chimeric sequences and collabo-
oligonucleotide be and still exhibit base-pairing?” When we rative intercalation studies and thus have a continuing need for
studied isoGNA we were surprised to find that the base-pairing the synthesis of isoGNA building blocks. While iso-glycerol
properties were unpredictably different from other closely nucleosides are known [10-15], the published synthetic routes
related acyclic nucleic acids [8]. This indicated that the nature and efficiencies of these vary widely and primarily depend on
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Beilstein J. Org. Chem. 2014, 10, 2131–2138.
Figure 1: A constitutional and conformational (idealized) representation of isoGNA, an isomer of glycerol nucleic acid.
the nature of the nucleobase. Herein, we detail our synthetic detected. Therefore, in the adenine case, we tried a direct SN2
efforts and the current improvements for the synthesis of all reaction of N6-benzoyladenine with tosyl derivative 6, which
four isoGNA nucleosides and their phosphoramidites.
was only moderately successful (30% yield of 7). Unexpect-
edly, in both the thymine and adenine cases, the subsequent
debenzylation reaction failed under a variety of conditions (H2,
10% Pd/C, MeOH or EtOAc; Pd/C, DMF, cyclohexadiene; 10%
Results and Discussion
Initial synthetic routes
We started with commercially available (S)-solketal (1), which Pd/C, NH4HCOO, acetone, reflux; 10% Pd-black, DMF, cyclo-
was protected as the benzyl ether 2. Ketal deprotection fol- hexadiene; (CH3)3SiI, CHCl3, 25 °C), for reasons that were not
lowed by the silylation of the primary hydroxy group with tert- obvious to us. Consequently, we abandoned this approach and
butyldiphenylsilyl chloride provided the substrate 4, which is pursued a different protecting group approach.
suitable for the introduction of the canonical nucleobases by a
SN2 displacement reaction. At this point, we chose the We silylated 1 to afford 8 whose ketal was deprotected fol-
Mitsunobu reaction (Scheme 1), which has been widely lowed by tritylation to give the derivative 10, which is expected
employed [16]. The reaction with the N3-benzoyl protected to be suited for the introduction of nucleobases by an SN2 reac-
thymine delivered 5 in good yield. However, the reaction with tion (Scheme 2). Once again, the Mitsunobu reaction with
N6-benzoyladenine proved problematic and no product was thymine proceeded smoothly. However, the reaction with
Scheme 1: Initial routes towards the synthesis of iso-glycerol nucleosides.
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Scheme 2: Preparation of thymine and adenine containing phosphoramidites of isoGNA.
N6-benzoyladenine proved problematic and did not proceed Boc derivative 16, which was synthesized starting from
even with the tosyl derivative 14. The solubility issues with N6-benzoyladenine in two steps. N6-Benzoyladenine was
benzoylated adenine and the bulk of the O-TBDPS group may treated with 2.6 equiv (Boc)2O to afford N6-benzoyl-N6,N9-di-
be the underlying reasons behind this difficulty. Therefore, we Boc-adenine 15; sat. aq NaHCO3/MeOH (1:2) conditions
proposed to use a doubly N6-protected adenine, the N6-benzoyl- cleaved the N9-Boc group of 15 specifically [17,18] to yield 16.
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The use of 16 in the subsequent Mitsunobu reaction afforded With all the four phosphoramidites in hand we proceeded to the
68% of the desired product 17. With derivatives 11 and 17 in automated synthesis of the oligomers, which proceeded
hand we proceeded to prepare the corresponding phosphor- smoothly with ≥95% coupling efficiency as determined by trityl
amidite derivatives 13 and 19, respectively, under standard assays. It was at the stage of the removal of the protecting
conditions as outlined in Scheme 2.
groups to afford the free oligos that we encountered a severe
problem with the adenine derivative. While the aqueous
We proceeded to prepare the cytosine- and guanine-containing ammonia/methylamine base deprotection removed all base
phosphoramidite by using the tritylated intermediate 10 as the labile protecting groups (acetate, isobutyrate, diphenylcar-
starting point (Scheme 3). In the cytosine series we found that bamoyl groups), the N6-Boc protecting group on adenine was
the normal N4-benzoyl or N4-acetyl protected cytosine did not impervious. We tried different acidic conditions (trifluoroacetic
give satisfactory yields under the Mitsunobu conditions. The acid, acetic acid with heat, AlCl3 [23-25]), but all of these
best yields were achieved with N4-isobutyrylcytosine as the conditions started to degrade the oligomer without completely
nucleophile [19-21]. In the guanine series, the N2-isobutyryl, removing the tert-Boc group (as seen by MALDI–TOF mass
O6-diphenylcarbamoyl protected guanine worked well spectral data in combination with ion-exchange HPLC moni-
(Scheme 3) [22].
toring) [8].
Scheme 3: Preparation of guanine- and cytosine-containing phosphoramidites of isoGNA.
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Improved synthesis of adenine-isoGNA
building block
ered the option of removing these Boc groups after the
Mitsunobu reaction is performed, and to introduce the
We went back in our synthesis in order to address the adenine N6-benzoyl group before the oligo stage.
protecting group problem and to re-work the inefficient
Mitsunobu reaction of N6-benzoyladenine. We explored various The Schneller group has reported that N6-amino-di-Boc-
conditions (different solvents, temperature etc.) and found that protected adenine undergoes an efficient reaction under classic
the sonication [26] of the reaction mixture by using N6-benzoyl- Mitsunobu conditions as a result of its increased solubility [28].
adenine in anhydrous dioxane led to respectable yields The regioselective attack at the N9-position is a consequence of
(34–36%) of 26 accompanied by a (unidentified) byproduct the steric bulk of the di-Boc protection at the N6-position, which
(Scheme 4). Without sonication no product 26 was formed (by renders the N7-position inaccessible. As expected, a high yield
TLC).
was observed under mild conditions with substrates containing
a secondary alcohol [28].
However, the yield of the sonication reaction varied capri-
ciously and the isolation of the compound was complicated by Di-Boc protected adenine was readily prepared via protection
interference from the DEAD side-products during column and followed by selective deprotection according to a literature
chromatographic purification. We did manage to get enough procedure [17,18]. The Mitsunobu reaction between the di-Boc-
amounts of 26 to prepare the phosphoramidite 28, and adenine and 10 was investigated, and it was found that dioxane
proceeded with the oligomer synthesis for our studies. as a solvent almost doubled the yield (74%) compared to THF
However, this was far from satisfactory, and the continued (36%; Scheme 5). Interestingly, sonication afforded no tangible
demand for isoGNA oligomers within our group and our collab- advantage. Doubling the amount of DIAD or di-Boc-adenine or
orative work prompted us to look for an improvement of the PPh3 did not give increased yields. The desired product 29 was
preparation of the adenine building block. Considering the easily purified on silica gel due to its lower polarity (when
possible reasons for the inefficient reaction of the protected compared with the N6-benzoyladenine reaction).
adenine derivatives in the Mitsunobu reaction, we focused on
the modification of adenine in order to improve its solubility. Initially, we tried to simultaneously deprotect both Boc groups
and the TBDPS group in the presence of TBAF in a one-pot
We considered three different derivatives of adenine based on
increased solubility: 6-chloroadenine has been utilized to over-
come the solubility and regioselectivity (N7 versus N9 nucleosi-
dation) issues. Yet, the conversion of the chloro to the NH2
group is not efficient enough to consider this option an attrac-
tive one [27]. The second option was the N6,N6-dibenzoylade-
nine derivative, but this compound was found to be highly
unstable for isolation and handling (one of the N6-benzoyl
group falls off very easily). The third option was the use of the
N6,N6-di-Boc-adenine derivative, even though this entails the
difficulty in removing these Boc groups, which are incompat-
Scheme 5: Mitsunobu reaction with the di-Boc-adenine.
ible with the oligonucleotide backbone. Therefore, we consid-
Scheme 4: Preparation of adenine containing phosphoramidite of isoGNA.
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Beilstein J. Org. Chem. 2014, 10, 2131–2138.
reaction [29]. However, only the TBDPS group was removed The trityl group was reintroduced on 33 under standard condi-
and a quantitative amount of mono-protected compound 30 was tions to give 34, and subsequent benzoylation afforded 26 in
produced (Scheme 6). The fluoride ion was not effective overall good yields (Scheme 7). The NMR spectra of 26 were in
enough to remove at least one of the Boc groups in 29. We good agreement with those data obtained previously [8] via
checked the deprotection conditions for the removal of the Boc Mitsunobu reaction with N6-benzoyladenine.
moiety by fluoride ions on the model substrate N6,N6-di-Boc-
adenine, and found that it was very challenging because free Overall, 26 was afforded in 43% yield from the Mitsunobu
adenine was not afforded even under reflux with 1 M TBAF for reaction of 10 with N6,N6-di-Boc-adenine. In addition to the
4 days.
slight improvement compared to the direct Mitsunobu reaction
of 10 with N6-benzoyladenine, this protocol allows for the syn-
Finally, we decided to remove the Boc groups on adenine with thesis of large amounts of 26 with an exact control of the
trifluoroacetic acid (TFA), even though we also expected the regioselectivity at the N9-position of adenine.
loss of the DMTr group on 29. When 29 was treated with 20%
of TFA in CH2Cl2, a mixture of 32 and 33 formed after 4 h. The Having solved the “adenine problem”, we focused on each of
TBDPS moiety was found to be stable under these conditions the remaining steps and optimized the synthesis of other
(Table 1, entry 1). 50% of TFA in CH2Cl2 completely removed isoGNA phosphoramidites. For example, the deprotection of 8
both Boc groups on adenine at either 4 °C or rt to afford the to 9 was initially achieved by the treatment with conc. HCl.
desired 33 in 87% yield.
However, in scaling up the reaction, the loss of the silyl group
Scheme 6: An attempt to remove both Boc and silyl groups simultaneously.
Table 1: Result of Boc deprotection of 29 with TFA.
Entry
Ratio of TFA (%)
Temp.a
Time (h)
Ratio (%)b
Yield
(31:32:33)
1
2
20
50
0 °C to rt
4
2
4
2
0:33:66
0:20:80
0:0:100
0:0:100
–
0 °C to 4 °C
–
–
3c
50
0 °C to rt
87%
aTFA (99%) was added under ice-bath (−2 to 2 °C) cooling. bThe ratio is roughly calculated by the intensity of visualized spots on TLC by UV lamp.
cThis reaction was performed on a large scale.
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Beilstein J. Org. Chem. 2014, 10, 2131–2138.
Scheme 7: The synthesis of 26 via tritylation and benzoylation.
was found to occur. Therefore, we sought an alternative route Grant CHE-1004570 and by NASA Astrobiology: Exobiology
and found that the deprotection mediated by vanadium trichlo- and Evolutionary Biology Program (Grant NNX09AM96G).
ride gave the desired product 9 cleanly in large-scale reactions
[30]. The desilylation of intermediates 11, 20 and 26 was References
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21 and 24 were phosphitylated under standard conditions to
obtain the phosphoramidites suitable for the automated solid
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This work was jointly supported by the NSF and NASA Astro-
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