Large-scale synthesis of 5′-O-pixyl protected 2′- deoxynucleosides useful for oligonucleotide synthesis

Article abstract of DOI:10.1081/NCN-200065966

Synthesis of 5′-O-pixylated 2′-deoxynucleosides 4 has been accomplished and the products are commercially available. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200065966

This article was downloaded by: [Australian National University]
On: 26 January 2015, At: 01:32
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
LARGE-SCALE SYNTHESIS OF 5′-O-PIXYL PROTECTED 2′-
DEOXYNUCLEOSIDES USEFUL FOR OLIGONUCLEOTIDE
SYNTHESIS
Robert T. Day a , Dean Williams a , Priscilla Soriano a & Yogesh S. Sanghvi b
a RI Chemical, Inc. , Orange, California, USA
b Rasayan Inc. , Encinitas, California, USA
Published online: 15 Nov 2011.
To cite this article: Robert T. Day , Dean Williams , Priscilla Soriano & Yogesh S. Sanghvi (2005) LARGE-SCALE SYNTHESIS OF
5′-O-PIXYL PROTECTED 2′-DEOXYNUCLEOSIDES USEFUL FOR OLIGONUCLEOTIDE SYNTHESIS, Nucleosides, Nucleotides and
Nucleic Acids, 24:5-7, 1135-1138, DOI: 10.1081/NCN-200065966
To link to this article: http://dx.doi.org/10.1081/NCN-200065966
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):1135–1138, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200065966
LARGE-SCALE SYNTHESIS OF 5’-O-PIXYL PROTECTED
2’-DEOXYNUCLEOSIDES USEFUL FOR
OLIGONUCLEOTIDE SYNTHESIS
5
Robert T. Day, Dean Williams, and Priscilla Soriano
RI Chemical, Inc., Orange,
California, USA
5
Yogesh S. Sanghvi
Rasayan Inc., Encinitas, California, USA
5
Synthesis of 5’-O-pixylated 2’-deoxynucleosides 4 has been accomplished and the products are
commercially available.
INTRODUCTION
In polyfunctional molecules such as nucleosides, selective protection becomes
an issue that has been addressed by the development of a number of new reagents.
Among these, the dimethoxytrityl (DMTr) ethers have proven to be one of the most
commonly used protecting groups (PG) for oligonucleotide synthesis, first
introduced by Khorana.[1] Chattopadhyaya and Reese developed the 9-phenyl-
xanthen-9-yl 2 (Px) as an alternative for trityl group useful for oligonucleotide
synthesis.[2,3] The Px group is removed by acid treatment at approximately the same
rate as the DMTr group. The key attributes of the Px group are summarized below.
.
The nucleosides protected with the 5’-O-Px group tend to crystallize easily;
therefore products can be purified without expensive column chromatography.
The Px group possesses a highly rigid and planar backbone that results in an
.
increased stability of the Px carbocation over other analogous group currently in
use such as DMTr.
The Px group has been shown to undergo photochemically induced heterolytic
.
carbon-oxygen bond cleavage to generate the Px cation in neutral aqueous
solution.[4]
.
The Px cation has a UV extinction coefficients that is ꢀ100 times greater than for
the DMTr group.
Address correspondence to Robert T. Day, RI Chemical, Inc., 632 W. Angus Ave., Orange, CA 92868, USA.
1135
Order reprints of this article at www.copyright.rightslink.com

1136
R. T. Day et al.
.
The pyrrole (pKa—0.27) has been identified as an excellent irreversible scavenger
for Px cation during oligonucleotide synthesis.
Interestingly, these traits of Px group has culminated into a recent publication
by Reese recommending that Px group is indeed a better choice over DMTr for the
protection of the 5’-OH group during oligonucleotide synthesis.[5] For these reasons,
we believe that it is important to synthesize 5’-O-Px protected nucleosides on large-
scale and make them commercially available.
CHEMISTRY AND DISCUSSION
In order to synthesize 5’-O-pixylated nucleosides 4, we needed a good source of
Px-Cl.[2,3] Aldrich and Advanced Asymmetrics are the only two commercial
suppliers of Px-Cl in the market place. Surprisingly, the cost of 1g of Px-Cl is $23,
compared to $9 for 1 g of DMTr. The exorbitant price of the commercial Px-Cl
from the current sources was a big roadblock in our ability to make 5’-O-pixylated
nucleosides. As a result, we considered buying Px-OH[1] that was significantly
cheaper ($6/g). The commercial 1 was chlorinated with excess of (COCl)2 in DCM/
toluene at RT in 2 hours to furnish 2 in quantitative yield.[6] Due to the hygroscopic
nature of 2, the crude product was used as such for pixylation studies. Recently, we
*
have identified an Asian supplier of good quality Px-Cl at lower cost. As a result,
the chlorination step has been avoided.
The classical method for the introduction of the Px group involved the reaction
of a nucleosidic primary alcoholic group with Px-Cl in pyridine. Although Px group
has been extensively used for the protection of the 5’-OH of nucleosides; it has been
also reported for the protection of a secondary hydroxyl group.[7] Therefore, it is
not uncommon to observe the formation of 3’-O- and 5’-O-bis pixilated nucleosides
5 as a byproduct. The formation of 5 was minimized by portion-wise slow addition
of Px-Cl to a solution of nucleoside in pyridine at room temperature.
The reaction of Px-Cl with nucleosides in pyridine is generally slow and
may take 6–12 hours for completion. The reaction rate could be accelerated by
addition of DMAP or DBU. However, this may lead to an increased amount of
bis-pixylated products.
First, we repeated the synthesis of 5’-O-pixylated nucleosides 4 reported by
Reese.[2,3] Following the literature protocol reaction of base protected nucleosides 3
with 2 furnished 4 with trace amounts of bis-pixylated products 5 (Scheme 1).
Thus, the 5’-O-pixylated derivatives of 1) 2’-deoxyuridine, 2) thymidine, 3) N4-Bz-2’-
deoxycytidine, 4) N6-Bz-2’-deoxyadenosine and 5) N2-Ibu-2’-deoxyguanosine were
isolated in 65, 60, 45, 57, 50% yields respectively, after crystallization from
appropriate solvents. All pixylated nucleosides 4 were purified by silica gel column
chromatography to obtain analytically pure samples. The structure pixylated 4
1
were confirmed by H NMR and ES MS. Interestingly, pyridine was employed in
*
Px-Cl is now available from Innovassynth, India (www.innovasynth.com).

R. T. Day et al.
1137
SCHEME 1
this protocol that is a toxic and hazardous solvent. Although this is a common
practice in academia, it is not a first choice for scale-up in the industry. Selecting a
less hazardous solvent during scale-up preparation not only decreases risks to
operators and community but also reduces the inconvenience and additional costs.
Therefore, we selected acetonitrile as an alternative solvent of choice that is safer
and cheaper than pyridine. Preliminary results indicate that the pixylation of
thymidine was complete in 6–8 hours and furnished 80% isolated yield of the
desired product 4 (B = T). Presently, we are optimizing the scale-up protocols for
the synthesis of 5’-O-pixylated nucleosides.
SUMMARY
Synthesis of 5’-O-pixylated nucleosides has been accomplished using the
literature protocols and further scale-up is in progress. A low-cost commercial
source of Px-Cl has been identified in Asia.[6] With easy access to Px-Cl, we plan to
make several 5’-O-pixylated nucleosides and make them commercially available for
R&D purpose.y
yContact RI Chemical Inc. for any 5’-O-pixylated nucleosides.

1138
REFERENCES
R. T. Day et al.
1. Schaller, H.; Weimann, G.; Lerch, B.; Khorana, H.G. Studies on polynucleotides. XXIV. The stepwise
synthesis of specific deoxyribopolynucleotides (4). Protected derivatives of deoxyribonucleosides and new
syntheses of deoxyribonucleoside-3’-phosphates. J. Am. Chem. Soc. 1963, 85, 3821–3827.
2. Chattopadhyaya, J.B.; Reese, C.B. The 9-phenylxanthen-9-yl protecting group. J. Chem. Soc., Chem.
Commun. 1978, 639–640.
3. Balgobin, N.; Josephson, S.; Chattopadhyaya, J.B. A general approach to the chemical synthesis of
oligodeoxyribonucleotides. Acta Chem. Scand., B 1981, 35, 201–212.
4. Misetic, A.; Byod, M.K. The pixyl (Px) group: a novel photocleavable protecting group for primary alcohols.
Tetrahedron Lett. 1998, 39, 1653–1656.
5. Reese, C.B.; Yan, H. Alternatives to the 4,4’dimetoxytrityl (DMTr) protecting group. Tetrahedron Lett. 2004,
45, 2567–2570.
6. Oka, N.; Sanghvi, Y.S.; Theodorakis, E.T. 3,3’-Oxybis(dimethyoxytrityl chloride) (O-DMTCl): synthesis and
applications of a novel bifunctional protecting group. Bioorg. Med. Chem. Lett. 2004, 14, 3241–3244.
7. Pompizi, I.; Haberli, A.; Leumann, C.J. Oligodeoxynucleotides containing conformationally constrained basic
sites: a UV and fluorescence spectroscopic investigation on duplex stability and structure. Nucleic Acids Res.
2000, 28, 2702–2708.