Scalable synthetic oligodeoxynucleotide purification with use of a catching by polymerization, washing, and releasing approach

Article abstract of DOI:10.1021/ol101316g

Synthetic oligodeoxynucleotides are purified with use of a catching by polymerization, washing, and releasing approach. The method does not require any chromatography, and purification is achieved by simple operations such as shaking, washing, and extraction. It is therefore useful for large-scale purification of synthetic oligonucleotide drugs. In addition to purification of oligonucleotides, this catching by polymerization concept is expected to be equally useful for purification of other synthetic oligomers such as peptides and oligosaccharides.

Full text of DOI:10.1021/ol101316g

ORGANIC
LETTERS
2
010
Vol. 12, No. 16
720-3723
Scalable Synthetic Oligodeoxynucleotide
Purification with Use of a Catching by
Polymerization, Washing, and Releasing
Approach
3
Shiyue Fang* and Suntara Fueangfung
Department of Chemistry, Michigan Technological UniVersity, 1400 Townsend DriVe,
Houghton, Michigan 49931
shifang@mtu.edu
Received July 13, 2010
ABSTRACT
Synthetic oligodeoxynucleotides are purified with use of a catching by polymerization, washing, and releasing approach. The method does
not require any chromatography, and purification is achieved by simple operations such as shaking, washing, and extraction. It is therefore
useful for large-scale purification of synthetic oligonucleotide drugs. In addition to purification of oligonucleotides, this catching by polymerization
concept is expected to be equally useful for purification of other synthetic oligomers such as peptides and oligosaccharides.
There is a high expectation on using synthetic oligonucle-
otides (ONs) as therapeutics to cure human diseases.
Currently, one such agent is used clinically and more are on
various stages of clinical trials. In addition, numerous projects
with the goal of developing ON drugs through selective gene
silencing are in progress. As a result, large quantities
crude ON contains various impurities, which must be
removed for most applications. Current purification technolo-
gies include trityl-on reverse phase (RP) HPLC, anion-
exchange HPLC, polyacrylamide gel electrophoresis (PAGE),
1
3
and various types of affinity chromatography. These meth-
ods are either unsuitable or highly expensive for large-scale
purification, and ON purification has been considered as a
(
kilograms to metric tons) of pure synthetic ONs are needed
2
for preclinical research, clinical trials, and patient use. Due
to the advancement of solid phase synthesis technology,
large-scale ON synthesis has become possible. However, the
2
bottleneck in antisense drug development. In this com-
munication, we report the use of an unprecedented concept;
catching by polymerization;for the development of a simple
(
1) Recent reviews: (a) Caruso, G.; Caffo, M.; Raudino, G.; Alafaci,
C.; Salpietro, F. M.; Tomasello, F. Recent Pat. CNS Drug DiscoVery. 2010,
, 53. (b) Hau, P.; Jachimczak, P.; Bogdahn, U. Expert ReV. Anticancer
5
(3) Examples: (a) In Current Protocols in Nucleic Acid Chemistry;
Beaucage, S. L., Bergstrom, D. E., Glick, G. D. , Jones, R. A., Eds.; John
Wiley & Sons: New York. (b) Fang, S.; Bergstrom, D. E. Tetrahedron
Lett. 2004, 45, 7987. (c) Fang, S.; Bergstrom, D. E. Nucleic Acids Res.
2003, 31, 708. (d) Fang, S.; Bergstrom, D. E. Bioconjugate Chem. 2003,
14, 80. (e) Sproat, B. S.; Rupp, T.; Menhardt, N.; Keane, D.; Beijer, B.
Nucleic Acids Res. 1999, 27, 1950. (f) Pearson, W. H.; Berry, D. A.; Stoy,
P.; Jung, K. Y.; Sercel, A. D. J. Org. Chem. 2005, 70, 7114. (g) Beller, C.;
Bannwarth, W. HelV. Chim. Acta 2005, 88, 171.
Ther. 2009, 9, 1663. (c) Chi, K. N.; Gleave, M. E. Drug Future 2009, 34,
5
1
25. (d) Richardt-Pargmann, D.; Vollmer, J. Oligonucleotide Ther. 2009,
175, 40. (e) Murao, S.; Fujii, M. Curr. Org. Chem. 2009, 13, 1366. (f)
Seguin, R. M.; Ferrari, N. Expert Opin. InVest. Drugs 2009, 18, 1505. (g)
Akhtar, S. J. Drug Targeting 2009, 17, 491. (h) Zhao, X. B.; Pan, F.; Holt,
C. M.; Lewis, A. L.; Lu, J. R. Expert Opin. Drug DeliVery 2009, 6, 673.
(2) Schulte, M.; Luhring, N.; Keil, A.; Sanghvi, Y. S. Org. Process Res.
DeV. 2005, 9, 212.
10.1021/ol101316g  2010 American Chemical Society
Published on Web 07/21/2010

4
method for ON purification. This method is easy to perform
acrylated with methacryloyl chloride to give 3, which was
coupled with thymidine by diisopropylsilyl bis(trifluo-
romethane sulfonate) to give 4. Phosphinylation of 4 with
Beaucage’s method provided phosphoramidite 1.
To prove the purification concept, the 20-mer ODN 5 (see
Scheme 2) was synthesized on a 1 µmol scale with standard
and uses inexpensive materials, and therefore is suitable for
large-scale purification.
3
a
The major impurities in crude ON are the failure sequences
generated in the coupling step in each synthetic cycle. For a
typical 20-mer oligodeoxynucleotide (ODN) synthesis, the
yield of full-length ODN is 40-70% depending on the scale
of the synthesis, and 30-60% of ODNs are failure
3
f
sequences. During automated synthesis, the failure se-
quences are capped with acetic anhydride in each synthetic
cycle to prevent them from coupling with phosphoramidite
monomers in the next synthetic cycles. We reasoned that if
a phosphoramidite that contains a reversibly linked poly-
merizable function is used in the last synthetic cycle, it will
only be coupled to the 5′-end of the full-length ODN. As a
result, after synthesis, cleavage, and deprotection, all full-
length ODN will contain a polymerizable group while the
failure sequences will not. The full-length sequence can then
be incorporated into a polymer through a simple copoly-
merization process, and failure sequences and other impuri-
ties can be removed by simple washing. Pure ODN can then
be obtained by cleaving from the polymer. If successful, this
purification method does not need any chromatography and
expensive solid phase affinity extraction materials, and
purification can be achieved by simple operations such as
shaking, washing, and extraction.
Scheme 2. Purification of ODN with Use of the Catching by
Polymerization, Washing, and Releasing Approach
The polymerization reaction we chose for this process was
the radical acrylamide polymerization reaction. This reaction
uses inexpensive materials, and is widely used by biologists
and biochemists for PAGE. The required phosphoramidite
1
that contains the polymerizable function (the methacryla-
mide) and the cleavable linker (the diisopropyl silyl acetal)
was synthesized using the route shown in Scheme 1. The
Scheme 1. Synthesis of Phosphoramidite 1
phosphoramidite chemistry on CPG on a synthesizer. To
perform the cleavage and deprotection with ammonium
hydroxide at room temperature, the UltraMild DNA synthesis
6
conditions were used (see the Supporting Information). In
the last synthetic cycle, phosphoramidite 1 was used to
incorporate the last nucleotide and the polymerizable meth-
acrylamide group.
The ODN synthesis result was analyzed with RP HPLC
(Figure 1, trace a). The full-length sequence 5 appeared at
5
7 min due to the hydrophobic group at its 5′-end. Most
impurities have a retention time between 15 and 20 min.
For polymerization, the crude ODN (∼50 nmol) was
dissolved in water. The polymerization monomer N,N-
dimethylacrylamide and a small amount of cross-linking
agent N,N′-methylenebis(acrylamide) were added. The po-
lymerization was initiated by ammonium persulfate and
TMEDA (Scheme 2). The reaction is not very sensitive to
amino tertiary alcohol 2 was prepared by using a known
procedure we reported previously. This compound was
5
(6) The diisopropylsilyl acetal linker can also survive normal ODN
cleavage and deprotection conditions such as NH OH (29%)/H NMe (40%)
4 2
(
1:1), 65 °C, 30 min. When these conditions are used, the 2-cyanoethyl
(
(
4) Fang, S. U.S. Patent pending US20080081902.
5) Tang, W.; Fang, S. Tetrahedron Lett. 2008, 49, 6003.
phosphoramidites benzoyl-dA, isobuyryl-dG, acetyl-dC, and dT can be used
for the synthesis. See ref 3c.
Org. Lett., Vol. 12, No. 16, 2010
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3
Excess HF was quenched with Me SiOMe (Scheme 2).
The supernatant was removed, and the gel was washed
with water. The supernatant and water were combined.
3 2 2
Volatiles, which included Me SiF, (i-Pr) SiF , MeOH,
pyridine, DMF, and water, were evaporated, and the ODN
was analyzed with RP HPLC. As shown in trace c, failure
sequences (6) and other impurities were completely
removed, and ODN 8 was pure. Due to its lower
lipophilicity than 5, 8 has a retention time of 19 min. The
recovery yield for the purification process was estimated
to be 72% by comparing the area of the peak in trace c at
1
9 min with that in trace a at 57 min. This yield is higher
than those typically obtained with trityl-on and fluorous
phase purification methods. We anticipated that the yield
of this new technology could be further increased when
the process is used for larger scale ODN purification. By
comparing traces a, b, and c, we can also conclude that
the diisopropyl silyl acetal linker is stable during ODN
synthesis, cleave, deprotection, acrylamide gel formation,
and extraction of failure sequences.
The identity of ODN 8 was established by comparing with
an authentic sample synthesized under standard conditions
and purified with RP HPLC by a company (Figure 1). As
shown in trace d, a coinjection of 8 purified by using our
technology with an authentic sample gave a single peak. The
HPLC profile for the authentic sample is shown in trace e.
MALDI-TOF analysis of 8 also gave correct molecular
weight. Because damaged ODNs resulting from nucleobase
modifications under radical polymerization conditions may
not be able to be detected by HPLC and MALDI-TOF, the
four nucleosides, adenosine, thymidine, guanosine, and
cytidine, were subjected to the polymerization conditions,
and then recovered from the polyacrylamide gel by extrac-
tion. HPLC analysis showed that these compounds are
completely stable under the polymerization conditions (see
the Supporting Information).
This new ON purification technology has significant
advantages over known ones. The most widely used method
for ON purification is trityl-on RP HPLC. Compared with
this method and other methods that use more hydrophobic
Figure 1
full-length sequence 5, failure sequences 6, and other impurities;
b) failure sequences 6 and other impurities that were removed from
the gel after catching the full-length sequence 5 by polymerization;
c) ODN 8 purified by catching by polymerization, washing, and
. RP HPLC traces of ODNs: (a) crude ODN that contains
3
e
tags than DMTr, our technique does not need any capital
expenses including those for the highly expensive and
consumable preparative columns. In addition, our technique
only needs a minimum amount of organic solvents. The
polymerization, removing failure sequences, and extraction
of full-length ON are all carried out with water as the solvent.
Furthermore, our method is expected to give better results
for purification of ONs that are prone to adopt secondary
structures and phosphorothioates that contain different di-
astereoisomers. These materials usually gave broad peaks
(
(
releasing; (d) ODN 8 and authentic sample; and (e) authentic
sample.
air but it was performed under a nitrogen atmosphere. The
polyacrylamide gel 7 was formed within 30 min at room
temperature. To ensure completion of the reaction, the gel
was allowed to stand for another 30 min. At this stage,
the full-length ODN 5 was incorporated into the polymer
7
in chromatography. In the literature, ONs have also been
3f,g
purified with fluorous affinity chromatography
biotin-avidin enabled affinity extraction.
and
7
while impurities including failure sequences 6 remained
3b-d
Compared with
in solution. Water was added to the gel to extract the
impurities. The extract was analyzed with RP HPLC. As
shown in trace b, the full-length ODN 5 was completely
incorporated into the gel. After drying the gel under
vacuum thoroughly, the full-length unmodified ODN 8 was
cleaved with HF-pyridine in DMF at room temperature.
these methods, our technique does not need any expensive
affinity materials such as fluorous affinity column and avidin-
coated beads.
(
7) Sanghvi, Y. S.; Schulte, M. Curr. Opin. Drug DiscoVery DeV. 2004,
7, 765.
3722
Org. Lett., Vol. 12, No. 16, 2010

ON purification methods involving capping failure se-
quences with reagents that contain reactive groups such as
a diene were also proposed. Using these methods, failure
With use of this method, purification was achieved by simple
operations such as shaking, washing, and extraction; and the
method does not require any expensive equipment and
materials. As a result, it is suitable for large-scale purification.
Application of this new concept for the purification of other
8
sequences could be removed by extraction with dienophile
functionalized microspheres through a Diels-Alder reaction.
Compared with these methods, our technology does not need
any reactive group functionalized microspheres, which could
be highly expensive. In addition, due to the intrinsically lower
reaction rate of heterogeneous reactions than homogeneous
reactions, and the fact that only groups on the surface of
solids can participate in reactions and the majority of groups
within the solids cannot, to efficiently remove failure
sequeces, the previously proposed methods require a large
excess of solid phase extraction materials, which will prevent
them from being used for large-scale ON purification. In
contrast, our technology does not rely on any heterogeneous
reactions for purification. In principle, the ratio of the
polymerization monomer over ON can be kept to a minimum
as long as the steric bulkness of ON does not significantly
slow down the polymerization.
3b
9
synthetic oligomers such as RNA, modified ON, peptides,
1
0
and oligosaccharides is in progress.
Acknowledgment. Financial support from the U.S. NSF
(CHE-0647129), Michigan Universities Commercialization
Initiative, MTU Chemistry Department, MTU Biotech
Research Center, and The Royal Thai Government Scholar-
ship (S.F.); the assistance from P. P. Rao (preliminary
synthetic studies of 1); and an NSF equipment grant (CHE-
9512445) are all gratefully acknowledged.
Supporting Information Available: Experimental details,
images of NMR spectra of all new compounds and MALDI-
TOF mass spectrum of ODN 8, and images of original HPLC
profiles. This material is available free of charge via the
Internet at http://pubs.acs.org.
In conclusion, we have developed a new method for the
purification of synthetic ON. This method is based on an
OL101316G
4
unprecedented concept, which is catching by polymerization.
(
9) Montanari, V.; Kumar, K. J. Am. Chem. Soc. 2004, 126, 9528.
(
8) Pieken, W.; Wolter, A.; Leuck, M. U.S. Patent US20030195351 A1,
(10) Palmacci, E. R.; Hewitt, M. C.; Seeberger, P. H. Angew. Chem.,
Int. Ed. 2001, 40, 4433.
2
003.
Org. Lett., Vol. 12, No. 16, 2010
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