Process for the capture and reuse of the 4,4′-dimethoxytriphenylmethyl group during manufacturing of oligonucleotides

Article abstract of DOI:10.1021/op980048l

In a standard amidite coupling-based automated oligonucleotide synthesis campaign, the 4,4′-dimethoxytriphenylmethyl (DMT) protecting group is discarded as waste along with large amounts of dichloromethane. Herein, we report a straightforward and simple process for complete capture and recovery of the DMT group and dichloromethane, rendering the oligonucleotide manufacturing process safer and more economical.

Full text of DOI:10.1021/op980048l

Organic Process Research & Development 1998, 2, 415−417
Process for the Capture and Reuse of the 4,4′-Dimethoxytriphenylmethyl Group
during Manufacturing of Oligonucleotides
Zhiqiang Guo, Henrik M. Pfundheller, and Yogesh S. Sanghvi*
DeVelopment Chemistry Department, Isis Pharmaceuticals, 2292 Faraday AVenue, Carlsbad, California 92008
Abstract:
In a standard amidite coupling-based automated oligonucleotide
synthesis campaign, the 4,4′-dimethoxytriphenylmethyl (DMT)
protecting group is discarded as waste along with large amounts
of dichloromethane. Herein, we report a straightforward and
simple process for complete capture and recovery of the DMT
group and dichloromethane, rendering the oligonucleotide
manufacturing process safer and more economical.
Introduction
Figure 1. Reaction, reagents, and abbreviations: i, 3% Cl
CHCO H in CH Cl ; ii, CH CN wash; iii, 0.2 M solution of 3
0.45 M 1H-tetrazole in CH CN; iv, oxidation step (see ref 6
2
-
2
2
2
3
The potential therapeutic use of oligonucleotides repre-
sents a new paradigm for modern drug discovery. Over the
+
3
for details); v, capping reagents (DMT, 4,4′-dimethoxytriph-
enylmethyl chloride; SS, linker solid support; Am., standard
CE amidite; Tet., activated amidite; P(III), phosphite triester
linkage; P(V) Nu., phosphorothioate linkage attached to a
1
2
past decade, oligonucleotide-based antisense, triplex, ri-
3
4
bozyme, and aptamer techniques have emerged as powerful
tools in the discovery of more specific and effective
oligonucleotide drugs. Among these technologies, the an-
tisense approach leads the way, with a dozen or more
oligonucleotides currently undergoing human clinical trials
for the treatment of viral infections, cancers, and inflamma-
Bz
iBu
nucleoside residue via SS; B: T, C , or G ).
with a nucleoside linked to a solid support, such as 1.
Treatment with 3% dichloroacetic acid (DCA) in dichlo-
romethane removes the 5′-O-(4,4′-dimethoxytriphenylmethyl)
group (DMT), to provide 2 with a free 5′-OH group. In
this step, the 5′-O-DMT group is released as a cation with a
characteristic bright red-orange color. The colorful cation
generated in this reaction not only allows monitoring
coupling efficiency but also allows the spectrophotometric
flow monitor and computer system to discontinue the flow
of 3% DCA deblock solution.
5
tory disorders. Recent advances in automated synthesis on
solid support and commercialization of synthetic nucleic acid
building blocks now allow the generation and screening of
an unprecedented number of synthetic oligonucleotides.
Manufacturing processes that allow synthesis of oligonucle-
otides at a reasonable cost not only are expediting the
discovery of these drugs but also will enable meeting the
market demand for such molecules.
It is noteworthy that a majority of oligonucleotides are
made via a common procedure, popularly known as the
phosphoramidite method, summarized in Figure 1. Briefly,
Detritylation under acidic conditions is reported to be a
7
reversible reaction. Therefore, removal of all DMT cation
from the solid support is crucial to the success of the
deblocking step. Next, in step ii, the support is washed with
dry acetonitrile (ACN) to remove traces of acid solution and
any trapped DMT cation. Step iii consists of premixing
amidite 3 with 1H-tetrazole to produce a very reactive P(III)
6
oligonucleotides are synthesized on a solid support via
sequential coupling and oxidation reactions (i-v, Figure 1)
in a predetermined order, all controlled by a computerized
liquid delivery system. For example, synthesis can begin
8
tetrazolide intermediate (4) that reacts almost immediately
with the 5′-OH group of 2, generating 5 with a phosphite
triester internucleosidic linkage. Unreacted excess 4 is then
washed from the support with dry ACN. Step iv is designed
to oxidize the unstable P(III) species of 5 to a more stable
P(V) internucleosidic linkage to furnish a dimer such as 7.
Capping (step v) is the final step, wherein unreacted 5′-OH
groups are acylated, generating 6 to prevent further elonga-
tion of oligonucleotide attached to the support. These five
(
(
(
(
1) Crooke, S. T. Biotechnology and Genetic Engineering ReViews; Intercept:
Newcastle upon Tyne, UK, 1998; Vol. 15, pp 121-157.
2) Soyfer, V. N., Potaman, V. N., Eds. Triple Helical Nucleic Acids; Springer-
Verlag: New York, 1996.
3) Eckstein, F., Lilley, D. M., Eds. Catalytic RNA; Springer-Verlag: New
York, 1996.
4) Bischofberger, N.; Shea, R. G. In Nucleic Acid Targeted Drug Design;
Propst, C. L., Perun, T. J., Eds.; Marcel Dekker: New York, 1992; Chapter
1
3, pp 579-612.
(
5) Sanghvi, Y. S. In Vol. 7: DNA and Aspects of Molecular Biology; Kool,
E., Ed.; In ComprehensiVe Natural Product Chemistry; Barton, D. H. R.,
Nakanishi, K., Eds. in chief; Elsevier Science: Oxford, UK, 1998 (in press).
6) Beaucage, S. L.; Caruthers, M. In Bioorganic Chemistry Nucleic Acids;
Hecht, S. M., Ed.; Oxford University Press: New York, 1996; Chapter 2,
pp 36-74.
(7) Ravikumar, V. T.; Krotz, A. H.; Cole, D. L. Tetrahedron Lett. 1995, 36,
6587.
(8) Dahl, B. H.; Nielsen, J.; Dahl, O. Nucleic Acids Res. 1987, 15, 1729. Berner,
S.; M u¨ hlegger, K.; Seliger, H. Nucleic Acids Res. 1989, 17, 853.
(
1
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Vol. 2, No. 6, 1998 / Organic Process Research & Development
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Published on Web 09/04/1998

Scheme 1a
steps are then repeated until an oligonucleotide of desired
length and sequence is completed.
It is important to note that the amidite coupling process
described creates two high-molecular-weight waste products.
First, the DMT group used for 5′-OH protection amounts to
approximately 35% of the weight of incoming monomeric
phosphoramidite units 3. This mass is released as the DMT
cation in waste dichloromethane. Second, a 0.5 mol excess
of building block 4 is also discarded in waste ACN along
with 1H-tetrazole. Gratifyingly, methods to capture 4 for
a
Reagents and conditions: a, concentration under reduced pressure to an
oil; b, add MeOH and 3 N NaOH solution; c, extraction of DMT-OH in organic
solvent; d, a f AcCl in toluene, reflux 3 h, f a f crystallization from
cyclohexane.
During a standard 60 mmol synthesis of oligonucleotide
1
8
on an OligoProcess reactor, one complete column volume
of deblock solution was collected manually upon completion
of a detritylation step (i in Figure 1). This solution is acidic
9
its recovery by conversion to 3 are already in place.
However, methods to capture the valuable DMT group and
for its reconversion to useful DMT-Cl have never been
published. In synthesizing a high-molecular-weight (∼6000)
drug, such as an oligonucleotide, it becomes very important
to maintain a high level of atom efficiency and atom
economy. In this note, we describe, for the first time, a
convenient process for the capture of currently wasted DMT
group and its conversion to useful DMT-Cl.
(
pH 0.6) and bright red-orange in color. The color is due to
DMT cation present in the solution. The concentration of
this cation is approximately 4.8 mmol/L, measured by its
characteristic UV absorption at 504 nm. The next step in
the recovery (a in Scheme 1) involves concentration of the
red-orange solution under vacuum. During this step, most
of the dichloromethane is removed from the solution and
collected. The dichloromethane recovery is about 90% in
this step, with a purity of >95%. We believe that
dichloromethane recovered by this process is pure enough20
for reuse as a solvent for organic synthesis.
The residual red oil is then dissolved in a minimum
volume of methanol and the solution quenched with 3 N
NaOH solution (step b, Scheme 1). The rationale for use of
NaOH as a quenching base is three-fold. First, it is
inexpensive and environmentally safe; second, it neutralizes
DCA in a very effective manner; and last, it provides
hydroxide ion for generation of DMT-OH. At this point,
the bright red color of the solution fades away, indicating
Results and Discussion
1
9
There are two main reasons why the DMT group has been
widely used for the protection of hydroxyl groups in
1
0
carbohydrate, nucleoside, and oligonucleotide chemistry.
First, a triphenylmethyl group imparts crystallinity to many
compounds that are low-melting solids, and second, the
triphenylmethyl group is easily removed under a variety of
1
1
conditions. Use of the DMT group in oligonucleotide
1
2
synthesis was first described by Nobel laureate Khorana
in 1962. After all these years, DMT is still the preferred
hydroxyl protecting group for oligonucleotide synthesis, both
1
3
14
in solid-supported and in solution-phase syntheses.
21
the formation of DMT-OH, confirmed by MS analysis. The
In a standard automated synthesis15 of a 20-mer phos-
phorothioate oligonucleotide on 100 mmol scale, the 5′-O-
DMT group is removed 19 times using a total of approxi-
mately 380 L of 3% DCA in dichloromethane. Interestingly,
all of that material goes to waste and is typically incinerated
at a premium price.16 Our original manufacturing process
for oligonucleotides not only generated toxic acid waste but
22
pH of the solution is ∼9-10, which does not affect the
DMT-OH.
The basic solution is concentrated under vacuum to
remove most of the methanol, providing a thick orange-
yellow syrup. In the next step (c in Scheme 1), the syrup is
dissolved in toluene and the solution washed with water
several times. Water washing removes DCA as a soluble
sodium salt, an easily disposable waste stream. The toluene
solution is then concentrated to 1/10 of original volume,
which helps remove trace water as an azeotrope, leaving the
solution dry and ready for the chlorination step (d in Scheme
1
7
also threw away an expensive protecting group of high
molecular weight. For this reason, we embarked on a project
to allow (i) salvage of a protecting group which constitutes
35% of the weight of amidite 3, (ii) neutralization of the
acidic halogenated waste stream, and (iii) potential for
recovery and reuse of the toxic solvent dichloromethane. The
process described herein addresses all these issues and is
amenable to scale-up for ton-scale manufacture scenario.
2
3
1
). The literature method for chlorination of DMT-OH
calls for the use of neat acetyl chloride under refluxing
conditions. However, we thought it was unnecessary to use
such a large excess of acetyl chloride. After a few attempts,
we were able to perform the chlorination with only 1/10 of
(9) Scremin, C. L.; Zhou, L.; Srinivasachar, K.; Beaucage, S. L. J. Org. Chem.
1
994, 59, 1963.
(
(
(
(
10) Sonveaux, E. In Protocols for Oligonucleotide Conjugates; Agrawal, S.,
Ed.; Humana: Totowa, NJ, 1994; Chapter 1, pp 1-72.
11) Wang, Y.; McGuigan, C. Synth. Commun. 1997, 27, 3829. Ding, X.; Wang,
W.; Kong, F. Carbohydr. Res. 1997, 303, 445.
12) Smith, M.; Rammler, D. H.; Goldberg, I. H.; Khorana, H. G. J. Am. Chem.
Soc. 1962, 84, 430.
13) White, H. A. In Solid Supports and Catalysts in Organic Synthesis; Smith,
K., Ed.; Ellis Horwood: New York, 1992; Chapter 8, pp 228-249.
14) Reese, C. B.; Song, Q. Bioorg. Med. Chem Lett. 1997, 7, 2787.
15) Using phosphoramidite chemistry, see ref 6 for details.
16) The current cost of disposal by incineration of halogenated waste is $260
per 55-gallon drum.
(18) OligoProcess is the largest automated oligonucleotide synthesizer built to
assemble 10-180 mmol of 20-mer oligonucleotide in less than 12 h. For
more details, contact Amersham Pharmacia Biotech, Piscataway, NJ (phone
1-800-526-3593).
(19) Determined by GC.
(20) Preliminary deblocking and phosphitylation experiments with recovered
dichloromethane indicated no side reactions.
(21) Bleasdale, C.; Ellwood, S. B.; Golding, B. T. J. Chem. Soc., Perkin Trans.
1 1990, 803.
(22) The pKR+ value for DMT-OH is - 1.24, cited from: Deno, N. C.;
Jaruzelski, J. J.; Schriesheim, A. J. Am. Chem. Soc. 1955, 77, 3044.
(23) Rathore, M.; Rani, P.; Mathur, N. K.; Narang, C. K. Indian J. Chem. 1995,
343, 634.
(
(
(
(17) The Aldrich catalog price for DMT-Cl is $81 for 25 g.
416
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Vol. 2, No. 6, 1998 / Organic Process Research & Development

the acetyl chloride recommended in the literature report.
DMT-OH solution in toluene is refluxed with acetyl chloride
for 3 h, and the solution is concentrated. The residue is
dissolved in cyclohexane and cooled to provide pure crystal-
line DMT-Cl. The H and C NMR, UV spectra, elemental
analysis, and MS analysis of the regenerated DMT-Cl
indicated that it was identical with that reported in the
literature.23 Additionally, we took this DMT-Cl and prepared
in DCM containing triphenylmethyl cation) and concentrated
under vacuum to remove most of the dichloromethane. The
oily red residue was then dissolved in MeOH (1.8 L), and
to this was added an aqueous NaOH solution (3 N, 1.5 L)
over a period of 1 h at room temperature. The resulting
solution was further stirred for 16 h at room temperature.
The reaction mixture was then concentrated under vacuum
to remove most of the MeOH. The remaining aqueous layer
was extracted with toluene (200 mL × 3), and the organic
1
13
5′-O-DMT protected nucleosides in yield and purity similar
to those obtained by the use of fresh DMT-Cl.
2 4
phases were combined and dried (Na SO ). A small sample
(
50 mL) of that toluene solution was evaporated to give 4,4′-
2
1
Conclusion
dimethoxytriphenylmethyl alcohol (DMT-OH in Scheme
1
1
6
5
1
): H NMR (200 MHz, CDCl
3
) δ 3.08 (s, 1Η), 3.81 (s,
In summary, a convenient and inexpensive process for
capture and regeneration of the DMT group has been
developed for the first time. Overall cost, operational safety,
and environmental considerations were taken into account
in process development. Though this process may not be
economical to practice on a 100 mmol level for a 20 kg/
year oligonucleotide pilot plant, it is certainly very attractive
for a manufacturing plant25 where antisense or any other
1
3
H), 6.84-7.50 (m, 13H); C NMR (50 MHz, CDCl ) δ
3
5.25, 86.45, 113.10, 126.70, 127.82, 128.30, 129.21, 130.15,
+
36.24, 144.99, 158.45; MS (FAB) m/z 343 (MNa ). The
toluene solution was then concentrated to a minimum volume
(80 mL). AcCl (8.5 mL, 9.42 g, 0.12 mol) was added to
the DMT-OH in toluene, and the solution was refluxed for
2 h under argon. The reaction mixture was then cooled to
room temperature, and the solution was concentrated under
vacuum. Cyclohexane (100 mL) was added to the residue,
and the mixture was allowed to stand in a refrigerator for
2
4
oligonucleotides will be made in ton quantities per year.
Furthermore, if 5′-O-DMT-protected nucleosides and related
amidites were to be manufactured at the same site, both
regenerated DMT-Cl and recaptured dichloromethane could
be utilized directly on site for the synthesis of various
synthons and for the preparation of deblock solution (i in
Figure 1), respectively. This method of harvesting and
recovery of the protecting group may also be applicable to
1
6 h. The crystallized material was then collected by
filtration and the product washed with cold cyclohexane (50
mL × 2) to furnish DMT-Cl (17.5 g, 89.7%): mp 122-124
2
2
1
°
3
C [lit. mp 119-123 °C]; H NMR (200 MHz, CDCl ) δ
3
.84 (s, 6H), 6.87 (d, 4H), 7.20-7.36 (m, 9H); 13C NMR
solution-phase26 and blockmer synthesis27 of oligonucle-
(50 MHz, CDCl ) δ 55.4, 82.1, 113.0, 127.8, 129.76, 131.1,
3
1
2
37.8, 145.8, 159.1. Anal. Calcd for C21H19ClO (338.83):
otides. We believe that our method will help in reducing
the ecological burden posed by the need for proper disposal
of the reagent and solvent alike. Also, this is an excellent
example of a multistep organic synthesis, where atom
efficiency and atom economy are now maintained effectively.
C, 74.44; H, 5.65. Found: C, 74.66; H, 5.82. The indentity
of recaptured DMT-Cl was further confirmed by TLC
cospotting with an authentic sample in three different solvents
2 2 f 3 f
(CH Cl , R 0.25; CH CN, R 0.9; 30% EtOAc in hexanes,
R
f
0.4). In addition, this DMT-Cl was also used for the
preparation of 5′-O-DMT-protected nucleosides, and the
products were found to be identical to the literature report.
Experimental Section
A complete deblocking cycle (Figure 1, step i) from a 60
mmol campaign was collected manually (12 L of 3% DCA
Acknowledgment
(
24) For every kilogram of purified 20-mer antisense oligonucleotide made using
an OligoProcess synthesizer, approximately 3.6 kg of DMT-Cl can be
prepared. However, at this scale, the labor in recovery of DMT-Cl will be
much higher than the cost of buying fresh DMT-Cl.
We thank Mr. Rick Carty and Max Moore for their help
in collection of DMT solutions from manufacturing cam-
paigns. We also thank Dr. Douglas L. Cole for his constant
encouragement and support for this project and careful
reading of the manuscript.
(
25) Sanghvi, Y. S.; Andrade, M.; Deshmukh, R. R.; Holmberg, L.; Scozzari,
A. N.; Cole, D. L. In Manuals of Antisense Methodology; Hartman, G.,
Endres, S., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands,
1
998 (in press).
26) Bonora, G. M.; Biancotto, G.; Maffini, M.; Scremin, L. Nucleic Acids Res.
993, 21, 1213.
27) Krotz, A. H.; Klopchin, P.; Cole, D. L.; Ravikumar, V. T. Nucleosides
Nucleotides 1997, 16, 1637.
(
(
1
Received for review June 3, 1998.
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