A new approach to oligonucleotide N3′-→P5′ phosphoramidate building blocks

Article abstract of DOI:10.1081/NCN-200060068

A new synthetic approach to 5′-phosphoramidites of 3′-aminonucleosides was developed. The methodology relies upon the use of 3′-amino-2′,3′-dideoxy nucleosides as the key starting materials. The final phosphoramidite products were obtained with high yields via 2-3-step efficient chemical transformations using selective introduction of orthogonal protective groups to the 3′-aminonucleoside sugar and base moieties. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200060068

This article was downloaded by: [Northeastern University]
On: 09 October 2014, At: 17:39
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
A NEW APPROACH TO OLIGONUCLEOTIDE N3′⇉P5′
PHOSPHORAMIDATE BUILDING BLOCKS
Daria Zielinska a , Krisztina Pongracz a & Sergei Gryaznov a
a Geron Corporation , Menlo Park, California, USA
Published online: 15 Nov 2011.
To cite this article: Daria Zielinska , Krisztina Pongracz & Sergei Gryaznov (2005) A NEW APPROACH TO OLIGONUCLEOTIDE
N3′⇉P5′ PHOSPHORAMIDATE BUILDING BLOCKS, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 1063-1067, DOI:
10.1081/NCN-200060068
To link to this article: http://dx.doi.org/10.1081/NCN-200060068
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):1063–1067, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060068
A NEW APPROACH TO OLIGONUCLEOTIDE N3’!P5’
PHOSPHORAMIDATE BUILDING BLOCKS
5
Daria Zielinska, Krisztina Pongracz, and Sergei Gryaznov
Geron Corporation,
Menlo Park, California, USA
5
A new synthetic approach to 5’-phosphoramidites of 3’-aminonucleosides was developed. The
methodology relies upon the use of 3’-amino-2’,3’-dideoxy nucleosides as the key starting materials. The
final phosphoramidite products were obtained with high yields via 2---3-step efficient chemical
transformations using selective introduction of orthogonal protective groups to the 3’-aminonucleoside
sugar and base moieties.
Keywords 3’-Aminonucleosides, Oligonucleotide Phosphoramidates
RESULTS AND DISCUSSION
Oligonucleotide N3’!P5’ thio-phosphoramidates (NPS) are currently under
development as telomerase-addressed potential anti-cancer agents.[1] At the same
time oligonucleotide N3’!P5’ phosphoramidates (NP) have been successfully used
as FISH probes.[2] These oligonucleotide analogues are currently prepared using an
amidite transfer method of choice that utilizes key 3’-aminonucleoside-5’-
phosphoramidite building blocks (I) (Figure 1). Hence, the availability and
accessibility of these monomer compounds may play an important role in
successful development of the oligonucleotide phosphoramidates as therapeutic
and diagnostic agents.
The earlier described synthesis of these monomers, unfortunately, is a highly
complex, labor-intensive, low-yielding multi-step process, especially for purine
nucleosides (10 steps for purines and 6–7 steps for pyrimidines).[3] The total yield of
the phosphoramidite products (on multi-gram to ꢀkg scale) reaches only ꢀ5% for
purines and ꢀ15–20% for pyrimidines. The natural 2’-deoxynucleosides with 3’-
hydroxyl group are used as starting materials for these procedures.
Address correspondence to Sergei Gryaznov, Geron Corporation, 230 Constitution Drive, Menlo Park, CA
94025, USA.
1063
Order reprints of this article at www.copyright.rightslink.com

1064
D. Zielinska, K. Pongracz, and S. Gryaznov
FIGURE 1 General structure of 3’-aminonucleoside-5’-phosphoramidites.
Here we present a new synthetic approach to the purine (Adebz and Guaibu
)
and thymidine (Thy) phosphoramidites (1a,g,t, respectively). The developed
methodology relies upon the application of 3’-amino-2’,3’-dideoxynucleosides as the
key starting materials. The final phosphoramidite products were obtained, with high
yields, via 2–4-step efficient chemical transformations using a selective introduction
of orthogonal protective groups to the 3’-aminonucleoside sugar and base moieties.
The 3’-amino purine nucleosides, 3’-amino-2’, 3’-dideoxyadenosine, and 3’-amino-
2’, 3’-dideoxyguanosine, were acquired from Metkinen Oy Company (Finland). These
compounds were prepared by an enzymatic trans-glycosylation process starting
from 3’-amino-3’-deoxythymidine, which was obtained via reduction of readily
available 3’-azido-3’-deoxythymidine (AZT). The performed chemical reactions are
outlined in Schemes 1–3.
Thus, preparation of the thymidine monomer 1t involves only two chemical
steps: 3’-NH-selective (3’-amino- vs. 5’-hydroxyl group) tritylation in pyridine of the
nucleoside 3’-amino group (yield ꢀ95%). Importantly, in general, the regioselective
3’-NH-tritylation is a key step enabling this new approach to phosphoramidites
1a,g,t. The tritylation reaction was subsequently followed by a standard 5’-O-
phosphitylation of the 5’-OH-3’-NH-Tr thymidine precursor, resulting in 1t. The
total yield of the phosphoramidite product 1t was ꢀ70% based on the starting 3’-
amino-3’-deoxythymidine (Scheme 1).
The adenosine and guanosine-based monomers 1a,g were prepared in a
similar manner (Schemes 2 and 3). However, presence of the second reactive
heterocyclic amino group at purine bases (N6 or N2), as well as the low solubility of
these nucleosides in anhydrous organic solvents (particularly 3’-aminoguanosine),
required the use of different tritylation conditions.
Hence, adenosine phosphoramidite 1a was prepared as follows: 3’-amino-
adenosine was 3’-NH-tritylated in a mixture of DMF and pyridine in the presence of
triethylamine. The 3’-NH-tritylated intermediate was isolated via aqueous wash and
extraction procedure with >90% yield, and it was taken to the next N6-benzoylation
step without any further purification (Scheme 2). Formation of only small amounts
(<5%) of the bis-5’-O-, 3’-NH-trityl byproduct was detected. The subsequent N6
benzoylation (using either a per-benzoylation process or TMS-Cl based transient
protection protocols) resulted in 5’-OH-3’-NH-Tr-adenosine precursor (Scheme 2).
This compound was purified by silica gel column chromatography (isolated yield

D. Zielinska, K. Pongracz, and S. Gryaznov
1065
SCHEME 1 Synthesis of thymidine phosphoramidite 1t.
of ꢀ70%), and then 5’-O-phosphitylated, resulting in the desirable phosphoramidite
1a with total yield of ꢀ60% based on the starting amino nucleoside.
Alternatively, using a one-pot synthetic approach, 3’-amino-2,’3’-deoxyadeno-
sine was 3’-NH-tritylated in a solution of pyridine in the presence of triethylamine,
and the reaction mixture was N6-benzoylated further (without isolation of the 5’-
tritylated product) using TMS-Cl-based transient protection methodology. The total
yield of the 5’-OH-3’-NH-Tr-adenosine intermediate was ꢀ70% after silica gel col-
umn chromatography. This synthetic route resulted in generation of a by-product
(<10% by weight), which was readily separated from the desirable compound during
silica gel chromatography. The exact chemical structure of the byproduct is yet
unknown (ESI MH + 1382.8, 691.38, possible M.W. 1381 D, main fragment 690 D)
1
and H NMR spectrum of this compound is markedly different from the main
product. However, it appears that its formation is caused by the presence of
triethylamine during the N6-benzoylation step.
Synthesis of guanosine phosphoramidite 1g was also performed in three
steps as follows, (Scheme 3). First, 3’-amino-2’,3’-dideoxyguanosine was selectively
SCHEME 2 Synthesis of adenosine phosphoramidite 1a.

1066
D. Zielinska, K. Pongracz, and S. Gryaznov
SCHEME 3 Synthesis of guanosine phosphoramidite 1g.
3’-NH-tritylated in the mixture of DMF and pyridine at 50°C. Formation of
relatively small amounts of a single impurity bis-3’-NH,-N2-trityl-guanosine (<5%) was
detected. The desirable product, 5’-hydroxy-3’-NH-trityl guanosine, was isolated by
precipitation from dichloromethane with yield of ꢀ95%. Further, this compound
was reacted with iso-butyryl chloride (iBu-Cl) to form the phosphoramidite pre-
cursor N2-iBu-protected 3’-NH-Tr-guanosine. Either N,O per-acylation with iBu-Cl,
or transient protection with TMS-Cl followed by iBu-Cl acylation was used for the
N2-iso-butyrylation step, resulting in product yields of 60 and 53%, respectively, after
crystallization from acetonitrile. Finally, the phosphoramidite 1g was formed using
common phosphitylation procedure with bis-diisopropyl phosphoramidite, result-
ing in the final product 1g (yield 80%).
Interestingly, if isobutyric anhydride (rather than iBu-Cl) was used for N2-amino
group protection, then formation of primarily 5’-O-iBu-3’-NH-Tr guanosine with un-
acylated N2 group was observed. This compound was isolated with ꢀ90% yield via
crystallization from dichloromethane/acetonitrile. Moreover, the quality of iBu-Cl
plays an important role in achieving good yield of the product. Apparently, the
presence of products of hydrolysis of iBu-Cl in the reaction mixture led to formation
of significant amounts (up to ꢀ50%) of a major byproduct: 3’-NH-iBu-N2-Tr
guanosine (the molecular weight is the same as for the desirable product) during the
N2 protection reaction.
In conclusion, we developed an efficient and relatively simple method for prepa-
ration of 3’-aminonucleoside-5’-phosphoramidites, key building blocks used for as-
sembly of oligonucleotide N3’!P5’ phosphoramidates and thio-phosphoramidates.
REFERENCES
1. Herbert, B.-S.; Pongracz, K.; Shay, J.W.; Gryaznov, S.M. Oligonucleotide N3’!P5’ phosphoramidates as
efficient telomerase inhibitors. Oncogene 2002, 21, 638–642.

D. Zielinska, K. Pongracz, and S. Gryaznov
1067
2. Ford, L.P.; Zou, Y.; Pongracz, K.; Gryaznov, S.M.; Shay, J.W.; Wright, W.E. Telomerase can inhibit the
recombination based pathway of telomere maintenance in human cells. J. Biol. Chem. 2001, 276, 32198–
32203.
3. Nelson, J.S.; Fearon, K.; Nguyen, M.Q.; McCurdy, S.N.; Frediani, J.F.; Foy, M.F.; Hirschbein, B.L. N3’!P5’
oligodeoxyribonucleotide phosphoramidates: a new method of synthesis based on a phosphoramidite amine-
exchange reaction. J. Org. Chem. 1997, 62, 7278–7287.