U.S. patent number 6,858,720 [Application Number 09/999,623] was granted by the patent office on 2005-02-22 for method of synthesizing polynucleotides using ionic liquids.
This patent grant is currently assigned to Agilent Technologies, Inc.. Invention is credited to Douglas J. Dellinger, Geraldine F Dellinger, Joel Myerson, Michel G. M. Perobost.
United States Patent |
6,858,720 |
Myerson , et al. |
February 22, 2005 |
Method of synthesizing polynucleotides using ionic liquids
Abstract
A method of synthesizing polynucleotides is disclosed. The
method involves contacting a first nucleotide with a selected
reactive group in the presence of an ionic liquid. The selected
reactive group may be on a second nucleotide, a polynucleotide, or
on a moiety on an insoluble substrate, for example in an
oligonucleotide synthesizer.
Inventors: |
Myerson; Joel (Berkeley,
CA), Perobost; Michel G. M. (Bethany, CT), Dellinger;
Douglas J. (Boulder, CO), Dellinger; Geraldine F
(Boulder, CO) |
Assignee: |
Agilent Technologies, Inc.
(Palo Alto, CA)
|
Family
ID: |
25546541 |
Appl.
No.: |
09/999,623 |
Filed: |
October 31, 2001 |
Current U.S.
Class: |
536/25.3;
536/22.1; 536/23.1; 536/25.33; 536/25.34; 536/25.4; 536/25.41 |
Current CPC
Class: |
C07D
213/20 (20130101); C07D 231/12 (20130101); C07H
21/00 (20130101); C07D 233/56 (20130101); C07D
249/08 (20130101); C07D 233/54 (20130101) |
Current International
Class: |
C07D
213/00 (20060101); C07D 213/20 (20060101); C07D
233/00 (20060101); C07D 233/54 (20060101); C07H
21/00 (20060101); C07D 521/00 (20060101); C07H
021/00 (); C07H 021/02 (); C07H 021/04 () |
Field of
Search: |
;536/25.3,25.33,25.34,22.1,23.1,25.4,25.41 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0742287 |
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Sep 1996 |
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EP |
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WO96/28457 |
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Sep 1996 |
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WO |
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WO98/39348 |
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Mar 1998 |
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WO |
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WO99/54509 |
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Apr 1998 |
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WO |
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WO00/18778 |
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Sep 1998 |
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WO |
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WO00/61594 |
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Apr 2000 |
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WO |
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Other References
Earle et al., Paradigm Confirmed: The First Use of Ionic Liquids to
Dramatically Influence the Outcome of Chemical Reactions Organic
Letters, vol. 6, No. 5, pp. 707-710, 2004.* .
Earle et al., "Ionic Liquids, Green solvents for the future", Pure
Applied Chemistry, vol. 72, No. 7, pp. 1391-1398, 2000.* .
Kenneth R. Seddon, Room Temperature Ionic Liquids: Neoteric
Solvents for Clean for Clean Catalysis, Ionic Liquids Review, pp.
1-8;www.ch.qub.ac.uk/krs/krs.htmll. .
Serge L. Beaucage and Radhakrishnan P. Iyer, "Advances in the
Synthesis of Oligonucleotides by the Phosphoramidite Approach";
Tetrahedron Report No. 309, vol. 48, No. 12, pp. 2223-2313, 1992.
.
Ahmad Hasan, Hong Li, Jeno Toamsz and Barbara Ramsay Shaw;
"Base-boronated Dinucleotides: synthesis and effect of
N7-cyanoborane substitution on he base protons"; Nucleic Acids
Research, 1996 vol. 24, No. 11; pp. 2150-2157. .
Frank Bergman, Erich Kueng, Patrick Iaiza, and Willi Bannwarth; "
Allyl as Internucleotide Protecting Group in DNA Synthesis to be
cleaved off by Ammonia"; Tetrahedron, vol. 51, No. 25, 99.
6971-6976, 1995. .
Frank Bergman and Wolfgang Pfleiderer; "Nucleotides"; The
2-Dansylethoxycarbonyl
(=2-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl} ethoxycarbonyl;
Dnseoc) Group for Protection of the 5'-Hydroxy Function in
Ologodeoxyribonucleotide Synthesis; Helvetica Chimica Acta--vol.
77(1994); pp. 203-213. .
Michael C. Pirrung and Lora Fallon; Glenn McGall "Proofing of
Photolithographic DNA Synthesis with 3',
5'--Dimethoxybenzoinyloxycarbonyl-Protected Deoxynucleoside
Phosphoramidites"; 1998 American Chemical Society, pp. 241-246.
.
Harald Sigmund, Thomas Maier and Wolfgang Pfeiderer; "A New Type of
Fluorescence Labeling of Nucleosides Nucleotides and
Oligonucleotides"; Nucleosides & Nucleotides, 16(5&6), pp.
685-696 (1997). .
Glenn H. McGall, Anthony D. Barone, Martin Diggelman, Stephen P. A.
Foder, Erik Gentalen, and Nam Ngo; "The Efficiency of
Light-Directed Synthesis of DNA Arrays of Glass Substrates";
Journal of the American Chemical Society, vol. 119, No. 22, Jun. 4,
1997, pp. 5081-5090. .
Michael C. Pirrung and Jean Claude-Bradley; "Comparison of Methods
for Photochemical Phosphoramidite-Based DNA Synthesis", J. Org.
Chem. 1995, vol. 60, pp. 6270-6276. .
Hayes Dougan, John B. Hobbs, Jeffrey I. Weitz and Donald M. Lyster;
"Sunthesis and Raioiodination of a Stannyl
Oligofeoxyribonucleotide"; Nucleic Acids Research, 1997, vol. 25,
No. 14, pp. 2897-2901. .
Shegenori Iwai and Eiko Ohtsuka; "5'-Levulinyl and
2-tetrahydrofuranyl protection for the synthesis of
oligoribonucleotides by the Phosphoramidite approach"; Nucleic
Acids Research, vol. 16, No. 20, 1988..
|
Primary Examiner: Wilson; James O.
Assistant Examiner: McIntosh, III; Traviss C.
Attorney, Agent or Firm: Beck; Michael J.
Parent Case Text
RELATED APPLICATION
This application relates to a U.S. Patent application Ser. No.
10/001,044 entitled "Use of Ionic Liquids for Fabrication of
Polynucleotide Arrays", filed on the same day as this application
in the names of Myerson et al.
Claims
What is claimed is:
1. A method of forming an internucleotide bond comprising
contacting a free hydroxyl of a growing polynucleotide with a
solution comprising a nucleotide monomer and at least 25% by weight
of an ionic liquid.
2. The method of claim 1 wherein the ionic liquid is an organic
salt comprising a substituted heterocyclic organic cation.
3. The method of claim 2 wherein the organic salt further comprises
an anion selected from chloride (Cl.sup.-), bromide (Br.sup.-),
tetrafluoroborate ([BF.sub.4 ].sup.-), hexafluorophosphate
([PF.sub.6 ].sup.-), [SbF.sub.6 ].sup.-, [CuCl.sub.2 ].sup.-,
[AlCl.sub.4 ].sup.-, [Al.sub.2 Cl.sub.7 ].sup.-, [Al.sub.3
Cl.sub.10 ].sup.-, methylsulfate (CH.sub.3 SO.sub.4.sup.-),
trifluoroacetate (CF.sub.3 CO.sub.2.sup.-), heptafluorobutanoate
(CF.sub.3 (CF.sub.2).sub.2 CO.sub.2.sup.-), triflate (CF.sub.3
SO.sub.2.sup.-), nonaflate (C.sub.2 F.sub.5 SO.sub.2.sup.-),
bis(trifluoromethylsulfonyl)imide ((CF.sub.3 SO.sub.2).sub.2
N.sup.-), bis(perfluoroethylsulfonyl)imide ((C.sub.2 F.sub.5
SO.sub.2).sub.2 N.sup.-), and tris(trifluoromethylsulfonyl)methide
((CF.sub.3 SO.sub.2).sub.3 C.sup.-).
4. The method of claim 2 wherein the organic salt is characterized
as being a liquid when being >98% pure and at standard
temperature and pressure.
5. The method of claim 2 wherein the cation is an N-substituted
pyridine having the ##STR6## wherein R is alkyl and each R' is
independently selected from hyrido, alkyl, or halogen group.
6. The method of claim 2 wherein the cation has the formula
##STR7## wherein each R is independently selected from alkyl, each
R' is independently selected from hydrido, alkyl, or halogen, and
R" is selected from hydrido or methyl.
7. The method of claim 1 wherein the ionic liquid is an organic
salt comprising a cation selected from an N-substituted pyridine
and a 1,3-disubstituted imidazole.
8. A method of forming an internucleotide bond between a first
nucleoside moiety and a second nucleoside moiety, the method
comprising (a) obtaining a solution comprising the first nucleoside
moiety and at least 25 percent by weight of an ionic liquid, and
(b) contacting the second nucleoside moiety with the solution of
(a) to form the internucleotide bond.
9. The method of claim 8, further comprising (c) contacting the
internucleotide bond with an oxidizing reagent to oxidize the
internucleotide bond.
10. The method of claim 8 wherein the second nucleoside moiety is
immobilized on a solid support.
11. The method of claim 8 wherein the ionic liquid is an organic
salt comprising a substituted heterocyclic organic cation.
12. The method of claim 11 wherein the organic salt further
comprises an anion selected from chloride (Cl.sup.-), bromide
(Br.sup.-), tetrafluoroborate ([BF.sub.4 ].sup.-),
hexafluorophosphate ([PF.sub.6].sup.-), [SbF.sub.6 ].sup.-,
[CuCl.sub.2 ].sup.-, [AlCl.sub.4 ].sup.-, [Al.sub.2 Cl.sub.7
].sup.-, [Al.sub.3 Cl.sub.10 ].sup.-, methylsulfate (CH.sub.3
SO.sub.4.sup.-), trifluoroacetate (CF.sub.3 CO.sub.2.sup.-),
heptafluorobutanoate (CF.sub.3 (CF.sub.2).sub.2 CO.sub.2.sup.-),
triflate (CF.sub.3 SO.sub.2.sup.-), nonaflate (C.sub.2 F.sub.5
SO.sub.2.sup.-), bis(trifluoromethylsulfonyl)imide ((CF.sub.3
SO.sub.2).sub.2 N.sup.-), bis(perfluoroethylsulfonyl)imide
((C.sub.2 F.sub.5 SO.sub.2).sub.2 N.sup.-), and
tris(trifluoromethylsulfonyl)methide ((CF.sub.3 SO.sub.2).sub.3
C.sup.-).
13. The method of claim 11 wherein the organic salt is
characterized as being a liquid when being >98% pure and at
standard temperature and pressure.
14. The method of claim 11 wherein the cation is an N-substituted
pyridine having the formula ##STR8## wherein R is alkyl and each R'
is independently selected from hyrido, alkyl, or halogen group.
15. The method of claim 11 wherein the cation has the formula
##STR9## wherein each R is independently selected from alkyl, each
R' is independently selected from hydrido, alkyl, or halogen, and
R" is selected from hydrido or methyl.
16. The method of claim 8 wherein the ionic liquid is an organic
salt comprising a cation selected from an N-substituted pyridine
and a 1,3-disubstituted imidazole.
Description
FIELD OF THE INVENTION
The invention relates generally to methods of polynucleotide
synthesis. The invention more specifically relates to forming
internucleotide bonds in a solution containing ionic liquid.
BACKGROUND OF THE INVENTION
Much interest has been focused on reactions for coupling
nucleotides to form polynucleotide chains, and various chemical
schemes have been described for the synthesis of polynucleotides.
Typically these methods use a nucleoside reagent of the formula:
##STR1##
in which: A represents H or an optionally protected hydroxyl group;
B is a purine or pyrimidine base whose exocyclic amine functional
group is optionally protected; one of M or Q is a conventional
protective group for the 3' or 5'--OH functional group while the
other is: ##STR2## where x may be 0 or 1, provided that: a) when
x=1: R' represents H and R" represents a negatively charged oxygen
atom; or R' is an oxygen atom and R" represents either an oxygen
atom or an oxygen atom carrying a protecting group; and b) when
x=0, R' is an oxygen atom carrying a protecting group and R" is
either a hydrogen or a di-substituted amine group.
When x is equal to 1, R' is an oxygen atom and R" is an oxygen
atom, the method is in this case the so-called phosphodiester
method; when R" is an oxygen atom carrying a protecting group, the
method is in this case the so-called phosphotriester method.
When x is equal to 1, R' is a hydrogen atom and R" is a negatively
charged oxygen atom, the method is known as the H-phosphonate
method.
When x is equal to 0, R' is an oxygen atom carrying a protecting
group and R" is a halogen, the method is known as the phosphite
method, and when R" is a leaving group of the disubstituted amine
type, the method is known as the phosphoramidite method.
The conventional sequence used to prepare an oligonucleotide using
reagents of the type of formula (I), basically follows four
separate steps: (a) coupling a selected nucleoside which also has a
protected hydroxy group, through a phosphite linkage to a
functionalized support in the first iteration, or a nucleoside
bound to the substrate (i.e. the nucleoside-modified substrate) in
subsequent iterations; (b) optionally, but preferably, blocking
unreacted hydroxyl groups on the substrate bound nucleoside; (c)
oxidizing the phosphite linkage of step (a) to form a phosphate
linkage; and (d) removing the protecting group ("deprotection")
from the now substrate bound nucleoside coupled in step (a), to
generate a reactive site for the next cycle of these steps. The
functionalized support (in the first cycle) or deprotected coupled
nucleoside (in subsequent cycles) provides a substrate bound moiety
with a linking group for forming the phosphite linkage with a next
nucleoside to be coupled in step (a). Final deprotection of
nucleoside bases can be accomplished using alkaline conditions such
as ammonium hydroxide, in a known manner.
The foregoing methods of preparing polynucleotides are well known
and described in detail, for example, in Caruthers, Science 230:
281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356;
Hunkapillar et al., Nature 310: 105-110, 1984; and in "Synthesis of
Oligonucleotide Derivatives in Design and Targeted Reaction of
Oligonucleotide Derivatives, CRC Press, Boca Raton, Fla., pages 100
et seq., U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S.
Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No.
5,869,643, EP 0294196, and elsewhere. The phosphoramidite and
phosphite triester approaches are most broadly used, but other
approaches include the phosphodiester approach, the phosphotriester
approach and the H-phosphonate approach. Such approaches are
described in Beaucage et al., Tetrahedron (1992) 12:2223-2311. A
more recent approach for synthesis of polynucleotides is described
in U.S. Pat. No. 6,222,030 B1 to Dellinger et al, Issued Apr. 24,
2001.
In the typical phosphoramidite method of solid phase
oligonucleotide synthesis, the synthesis typically proceeds in the
3' to 5' direction (referring to the sugar component of the added
nucleoside), although the synthesis may easily be conducted in the
reverse direction. The added nucleoside generally has a
dimethoxytrityl protecting group on its 5' hydroxyl and a
phosphoramidite functionality on its 3' hydroxyl position. Beaucage
et al. (1981) Tetrahedron Lett. 22:1859. See FIG. 1 for a schematic
representation of this technology. In FIG. 1 "B" represents a
purine or pyrimidine base, "DMT" represents dimethoxytrityl
protecting group and "iPr" represents isopropyl. In the first step
of the synthesis cycle, the "coupling" step, the 5' end of the
growing chain is coupled with the 3' phosphoramidite of the
incoming monomer to form a phosphite triester intermediate (the 5'
hydroxyl protecting group prevents more than one monomer per
synthesis cycle from attaching to the growing chain). Matteucci et
al. (1981) J. Am. Chem. Soc. 103:3185. Next, the optional "capping
reaction" is used to stop the synthesis on any chains having an
unreacted 5' hydroxyl, which would be one nucleotide short at the
end of synthesis. The phosphite triester intermediate is subjected
to oxidation (the "oxidation" step) after each coupling reaction to
yield a more stable phosphotriester intermediate. Without
oxidation, the unstable phosphite triester linkage would cleave
under the acidic conditions of subsequent synthesis steps.
Letsinger et al. (1976) J. Am. Chem. Soc. 98:3655. Removal of the
5' protecting group of the newly added monomer (the "deprotection"
step) is typically accomplished by reaction with acidic solution to
yield a free 5' hydroxyl group, which can be coupled to the next
protected nucleoside phosphoramidite. This process is repeated for
each monomer added until the desired sequence is synthesized.
According to some protocols, the synthesis cycle of couple, cap,
oxidize, and deprotect is shortened by omitting the capping step or
by taking the oxidation step `outside` of the cycle and performing
a single oxidation reaction on the completed chain. For example,
oligonucleotide synthesis according to H-phosphonate protocols will
permit a single oxidation step at the conclusion of the synthesis
cycles. However, coupling yields are less efficient than those for
phosphoramidite chemistry and oxidation requires longer times and
harsher reagents than amidite chemistry.
Conventional synthesis protocols of oligonucleotides are not
without disadvantages. For example, cleavage of the DMT protecting
group under acidic conditions gives rise to the
resonance-stabilized and long-lived bis(p-anisyl)phenylmethyl
carbocation. Gilham et al. (1959) J. Am. Chem. Soc. 81:4647.
Protection and deprotection of hydroxyl groups with DMT are thus
readily reversible reactions, resulting in side reactions during
oligonucleotide synthesis and a lower yield than might otherwise be
obtained. To circumvent such problems, large excesses of acid are
used with DMT to achieve quantitative deprotection. As bed volume
of the polymer is increased in larger scale synthesis, increasingly
greater quantities of acid are required. The acid-catalyzed
depurination which occurs during the synthesis of
oligodeoxyribonucleotides is thus increased by the scale of
synthesis. Caruthers et al., in Genetic Engineering: Principles and
Methods, J. K. Setlow et al., Eds. (New York: Plenum Press, 1982).
Solvent use in larger scale synthesis becomes increasingly
prohibitive as well, as more washing is required. In particular,
the reagents used in the coupling step typically are highly
susceptible to hydrolysis, which requires dry solvents, further
increasing the cost of solvents.
Salts that are fluid at room temperature have been investigated as
environmentally friendly solvents. These salts have been termed
`room temperature ionic liquids` (herein simply referred to as
`ionic liquids`) and are generally composed of a heterocyclic
cation, e.g. a substituted imidazole or pyridine, and an anion such
as tetrafluoroborate or hexafluorophosphate, although certain
organic anions such as methylsulfate (CH.sub.3 SO.sub.4.sup.-),
among others, have been discovered to be effective as the anion in
certain organic liquids. Ionic liquids are known to dissolve a wide
range of substances, both organic and inorganic. Ionic liquids
typically are non-corrosive, have little or no vapor pressure under
standard conditions, and exhibit low viscosity. More information
regarding ionic liquids may be gleaned from two review articles by
Hussey (Hussey, C. L., Adv. Molten Salt Chem. (1983) 5:185; and
Hussey, C. L., Pure Appl. Chem. (1988) 60:1763).
SUMMARY OF THE INVENTION
The invention is thus addressed to the aforementioned deficiencies
in the art, and provides a novel method for synthesizing
oligonucleotides, wherein the method has numerous advantages
relative to prior methods such as those discussed above. The method
involves forming an internucleotide bond between a first nucleoside
moeity and a second nucleoside moiety in an environment that
includes an ionic liquid.
In a preferred embodiment of the invention, the second nucleoside
moiety is immobilized to an insoluble substrate. The second
nucleoside moiety on the insoluble substrate is contacted with a
solution having the first nucleoside moiety in a solution
containing ionic liquid. An internucleoside bond is thus formed
between the first and second nucleoside moieties. The product of
the reaction is a polynucleotide wherein the first and second
nucleoside moieties have been bonded together.
In some embodiments, the first nucleoside moiety corresponds to a
nucleoside phosphoramidite monomer, as in conventional
polynucleotide synthesis as described above. The invention also
encompasses the formation of an internucleoside bond between two
polynucleotides or oligonucleotides, or between a polynucleotide
and an oligonucleotide. In such case, the first nucleoside moiety
corresponds to the one of the polynucleotides or oligonucleotides,
and the second polynucleotide moiety corresponds to the
polynucleotide or oligonucleotide to be joined to the first
nucleoside moiety.
In particular embodiments, the reaction is geared to producing
"native" polynucleotides, i.e. substantially identical to those
that might be isolated from nature. In other embodiments, the
reaction is used to synthesize polynucleotide analogues, which may
have `modified` (not occurring in nature) phosphodiester backbones
or modified bases attached to the sugar groups in the
phosphodiester backbones.
In another embodiment, after the internucleotide bond has been
formed, it is modified, e.g. by oxidation, to form the ultimate
polynucleotide product. The present invention in its broadest sense
encompasses materials and methods for use in forming
polynucleotides, polynucleotide intermediates, and polynucleotide
analogues. The invention also encompasses reagents and methods for
synthesis of oligonucleotides allowing the synthesis to be
conducted under a wide range of conditions and allowing for the use
of a variety of protecting groups. This wide range includes the use
of co-solvents along with the ionic liquid in the coupling
reaction.
Additional objects, advantages, and novel features of this
invention shall be set forth in part in the descriptions and
examples that follow and in part will become apparent to those
skilled in the art upon examination of the following specifications
or may be learned by the practice of the invention. The objects and
advantages of the invention may be realized and attained by means
of the instruments, combinations, compositions and methods
particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will be understood from
the description of representative embodiments of the method herein
and the disclosure of illustrative apparatus for carrying out the
method, taken together with the Figures, wherein
FIG. 1 schematically illustrates a prior art oligonucleotide
synthesis method using phosphoramidite monomers. The known prior
art methods, including the one illustrated, do not describe the use
of ionic liquids in the coupling step where the internucleotide
bond is formed.
DETAILED DESCRIPTION
Before the invention is described in detail, it is to be understood
that unless otherwise indicated this invention is not limited to
particular materials, reagents, reaction materials, manufacturing
processes, or the like, as such may vary. It is also to be
understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting. It is also possible in the present invention that steps
may be executed in different sequence where this is logically
possible. However, the sequence described below is preferred.
It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "an insoluble support" includes a
plurality of insoluble supports. In this specification and in the
claims that follow, reference will be made to a number of terms
that shall be defined to have the following meanings unless a
contrary intention is apparent:
As used herein, polynucleotides include single or multiple stranded
configurations, where one or more of the strands may or may not be
completely aligned with another. The terms "polynucleotide" and
"oligonucleotide" shall be generic to polydeoxynucleotides
(containing 2-deoxy-D-ribose), to polyribonucleotides (containing
D-ribose), to any other type of polynucleotide which is an
N-glycoside of a purine or pyrimidine base, and to other polymers
in which the conventional backbone has been replaced with a
non-naturally occurring or synthetic backbone, e.g with a modified
sugar group, or in which one or more of the conventional bases has
been replaced with a non-naturally occurring or synthetic base.
A "nucleotide" refers to a sub-unit of a nucleic acid (whether DNA
or RNA or analogue thereof) which includes a phosphate group, a
sugar group and a nitrogen containing base, as well as analogs of
such sub-units. A "nucleoside" references a nucleic acid subunit
including a sugar group and a nitrogen containing base. A
"nucleoside moiety" refers to a molecule having a sugar group and a
nitrogen containing base (as in a nucleoside) as a portion of a
larger molecule, such as in a polynucleotide, oligonucleotide, or
nucleoside phosphoramidite. A "nucleotide monomer" refers to a
molecule which is not incorporated in a larger oligo- or
poly-nucleotide chain and which corresponds to a single nucleotide
sub-unit; nucleotide monomers may also have activating or
protecting groups, if such groups are necessary for the intended
use of the nucleotide monomer. A "polynucleotide intermediate"
references a molecule occurring between steps in chemical synthesis
of a polynucleotide, where the polynucleotide intermediate is
subjected to further reactions to get the intended final product,
e.g. a phosphite intermediate which isoxidized to a phosphate in a
later step in the synthesis, or a protected polynucleotide which is
then deprotected. An "oligonucleotide" generally refers to a
nucleotide multimer of about 2 to 100 nucleotides in length, while
a "polynucleotide" includes a nucleotide multimer having any number
of nucleotides. It will be appreciated that, as used herein, the
terms "nucleoside" and "nucleotide" will include those moieties
which contain not only the naturally occurring purine and
pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C),
guanine (G), or uracil (U), but also modified purine and pyrimidine
bases and other heterocyclic bases which have been modified (these
moieties are sometimes referred to herein, collectively, as "purine
and pyrimidine bases and analogs thereof"). Such modifications
include, e.g., methylated purines or pyrimidines, acylated purines
or pyrimidines, and the like, or the addition of a protecting group
such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl,
benzoyl, or the like. The purine or pyrimidine base may also be an
analog of the foregoing; suitable analogs will be known to those
skilled in the art and are described in the pertinent texts and
literature. Common analogs include, but are not limited to,
1-methyladenine, 2-methyladenine, N6-methyladenine,
N6-isopentyladenine, 2-methylthio-N-6-isopentyladenine,
N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine,
3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,
4-acetylcytosine, 1-methylguanine, 2-methylguanine,
7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,
8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
5-ethyluracil, 5-propyluracil, 5-methoxyuracil,
5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,
5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,
2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,
uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,
pseudouracil, 1-methylpseudouracil, queosine, inosine,
1-methylinosine, hypoxanthine, xanthine, 2-aminopurine,
6-hydroxyaminopurine, 6-thiopurine and 2,6-diaminopurine.
An "internucleotide bond" refers to a chemical linkage between two
nucleoside moieties, such as a phosphodiester linkage in nucleic
acids found in nature, or such as linkages well known from the art
of synthesis of nucleic acids and nucleic acid analogues. An
internucleotide bond may comprise a phospho or phosphite group, and
may include linkages where one or more oxygen atoms of the phospho
or phosphite group are either modified with a substituent or
replaced with another atom, e.g. a sulfur atom, or the nitrogen
atom of a mono- or di-alkyl amino group.
A "group" includes both substituted and unsubstituted forms.
Typical substituents include one or more lower alkyl, any halogen,
hydroxy, or aryl, or optionally substituted on one or more
available carbon atoms with a nonhydrocarbyl substituent such as
cyano, nitro, halogen, hydroxyl, or the like. Any substituents are
typically chosen so as not to substantially adversely affect
reaction yield (for example, not lower it by more than 20% (or 10%,
or 5% or 1%) of the yield otherwise obtained without a particular
substituent or substituent combination). An "acetic acid" includes
substituted acetic acids such as dichloroacetic acid (DCA) or
tri-chloroacetic acid (TCA).
A "phospho" group includes a phosphodiester, phosphotriester, and
H-phosphonate groups. In the case of either a phospho or phosphite
group, a chemical moiety other than a substituted 5-membered furyl
ring may be attached to 0 of the phospho or phosphite group which
links between the furyl ring and the P atom.
A "protecting group" is used in the conventional chemical sense to
reference a group which reversibly renders unreactive a functional
group under specified conditions of a desired reaction. After the
desired reaction, protecting groups may be removed to deprotect the
protected functional group. All protecting groups should be
removable (and hence, labile) under conditions which do not degrade
a substantial proportion of the molecules being synthesized. In
contrast to a protecting group, a "capping group" permanently binds
to a segment of a molecule to prevent any further chemical
transformation of that segment. A "hydroxyl protecting group"
refers to a protecting group where the protected group is a
hydroxyl. A "reactive-site hydroxyl" is the terminal 5'-hydroxyl
during 3'-5' polynucleotide synthesis and is the 3'-hydroxyl during
5'-3' polynucleotide synthesis. An "acid labile protected hydroxyl"
is a hydroxyl group protected by a protecting group that can be
removed by acidic conditions. Similarly, an "acid labile protecting
group" is a protecting group that can be removed by acidic
conditions. Preferred protecting groups that are capable of removal
under acidic conditions ("acid-labile protecting groups") include
those such as tetrahydropyranyl groups, e.g. tetrahydropyran-2-yl
and 4-methoxytetrahydropyran-2-yl; an arylmethyl group with n aryl
groups (where n=1 to 3) and 3-n alkyl groups such as an optionally
substituted trityl group, for example a monomethoxytrityl for
oligoribonucleotide synthesis and a dimethoxytrityl for
oligodeoxyribonucleotide synthesis, pixyl; isobutyloxycarbonyl;
t-butyl; and dimethylsilyl. A trityl group is a triphenylmethyl
group. Suitable protecting groups are described in "Protective
Groups in Organic Synthesis" by T. W. Green, Wiley
Interscience.
The term "alkyl" as used herein, unless otherwise specified, refers
to a saturated straight chain, branched or cyclic hydrocarbon group
of 1 to 24, typically 1-12, carbon atoms, such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,
cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,
3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term
"lower alkyl" intends an alkyl group of one to eight carbon atoms,
and includes, for example, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,
neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,
2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term "cycloalkyl"
refers to cyclic alkyl groups such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
The term "alkenyl" as used herein, unless otherwise specified,
refers to a branched, unbranched or cyclic (in the case of C5 and
C6) hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms
containing at least one double bond, such as ethenyl, vinyl, allyl,
octenyl, decenyl, and the like. The term "lower alkenyl" intends an
alkenyl group of two to eight carbon atoms, and specifically
includes vinyl and allyl. The term "cycloalkenyl" refers to cyclic
alkenyl groups.
The term "alkynyl" as used herein, unless otherwise specified,
refers to a branched or unbranched hydrocarbon group of 2 to 24,
typically 2 to 12, carbon atoms containing at least one triple
bond, such as acetylenyl, ethynyl, n-propynyl, isopropynyl,
n-butynyl, isobutynyl, t-butynyl, octynyl, decynyl and the like.
The term "lower alkynyl" intends an alkynyl group of two to eight
carbon atoms, and includes, for example, acetylenyl and propynyl,
and the term "cycloalkynyl" refers to cyclic alkynyl groups.
The term "aryl" as used herein refers to an aromatic species
containing 1 to 5 aromatic rings, either fused or linked, and
either unsubstituted or substituted with 1 or more substituents
typically selected from the group consisting of amino, halogen and
lower alkyl. Preferred aryl substituents contain 1 to 3 fused
aromatic rings, and particularly preferred aryl substituents
contain 1 aromatic ring or 2 fused aromatic rings. Aromatic groups
herein may or may not be heterocyclic. The term "aralkyl" intends a
moiety containing both alkyl and aryl species, typically containing
less than about 24 carbon atoms, and more typically less than about
12 carbon atoms in the alkyl segment of the moiety, and typically
containing 1 to 5 aromatic rings. The term "aralkyl" will usually
be used to refer to aryl-substituted alkyl groups. The term
"aralkylene" will be used in a similar manner to refer to moieties
containing both alkylene and aryl species, typically containing
less than about 24 carbon atoms in the alkylene portion and 1 to 5
aromatic rings in the aryl portion, and typically aryl-substituted
alkylene. Exemplary aralkyl groups have the structure --(CH2)j--Ar
wherein j is an integer in the range of 1 to 24, more typically 1
to 6, and Ar is a monocyclic aryl moiety.
EXAMPLES
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of synthetic organic chemistry,
biochemistry, molecular biology, and the like, which are within the
skill of the art. Such techniques are explained fully in the
literature.
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to prepare and use the compounds disclosed and
claimed herein. Efforts have been made to ensure accuracy with
respect to numbers (e.g., amounts, temperature, etc.) but some
errors and deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, temperature is in .degree. C.
and pressure is at or near atmospheric. Standard temperature and
pressure are defined as 20.degree. C. and 1 atmosphere.
The synthesis of polynucleotides has been well-studied, and methods
incorporate both aqueous and organic solvents. It is well known
that changing the solvent in a reaction system frequently affects
the performance of the reaction, sometimes profoundly. The ionic
nature of ionic liquids fundamentally differs from molecular nature
of aqueous or organic solvents used in various steps of the
polynucleotide synthesis cycle. Potential problems include changes
of chemical mechanism, possibly favoring different products due to
the ionic nature of the solvent. Stabilization of charged reaction
intermediates due to interaction with the ionic liquid, or chemical
reaction with components of the ionic liquid itself might be
expected. Will the short-lived reaction intermediates found in
conventional solvents be long-lived stable intermediates in an
ionic liquid? Will changes in the relative stabilities of reaction
intermediates change the available reaction pathways? Will the
expected changes in reaction kinetics shift the balance between
thermodynamic and kinetic control, and hence produce different
products?
We determined to study the effect of the coupling reaction in ionic
liquid solvent as an alternative to molecular solvents (aqueous and
organic solvents). We have discovered that, despite the previously
mentioned potential problems, we were able to achieve coupling of
nucleoside moieties via formation of an internucleotide bond in
ionic liquids. We have now found that various advantages exist in
performing the coupling reaction in ionic liquids. One advantage we
found was that the hydrophobicity of ionic liquid led to reduced
problems in dealing with hydrolysis of the reactants due to water
in the reaction environment. Less solvent may be used to wash in
between coupling steps, and ionic liquid solvents may be recovered
more easily, when compared to prior art methods. This may be
particularly useful in large-scale synthesis, where lots of washing
and solvents are required.
Particularly useful phosphoramidites, their preparation, and their
use are described in detail in U.S. Pat. No. 5,902,878; U.S. Pat.
No. 5,700,919; U.S. Pat. No. 4,668,777; U.S. Pat. No. 4,415,732;
PCT publication WO 98/41531 and the references cited therein, among
others.
The chemical synthesis of thymidine-thymidylate dimers in ionic
liquid were preformed by the following protocol:
3 .ANG. molecular sieves were activated by drying in a vacuum oven
at 200.degree. C. overnight. A small number of sieves were placed
in a 5 ml, round bottom flask with a 14/20 ground glass joint that
was then sealed with a rubber septum. 3 ml of
1-ethyl-3-methyl-1H-imidazolium trifluoromethanesulfonate (Aldrich
Chemical Company, Milwaukee, Wis. USA) was added to the flask and
the liquid allowed to dry overnight.
5'-Dimethoxytrityl-2'-deoxyThymidine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 0.38 grams,
0.5 mmol, was added to the flask and the solution shaken until the
reagent had dissolved. A small amount of the solution was removed
from the flask and placed in an NMR tube for analysis by .sup.31 P
NMR using an external lock. The resulting NMR spectrum showed the
presence of the starting material nucleoside phosphoramidite at
.delta.147.18 ppm relative to phosphoric acid.
3'-Acetyl Thymidine (ChemGenes Corp., Waltham Mass. USA) 0.14
grams, 0.5 mmol was added to the mixture along with tetrazole 0.18
grams, 2.5 mmol. The solution was shaken on a wrist action shaker
until the reagents were completely dissolved. An aliquot of the
reaction mixture was removed from the flask and placed in an NMR
tube for analysis by .sup.31 P NMR using an external lock. The
resulting NMR spectrum showed complete conversion of the starting
material nucleoside phosphoramidite at .delta. 147.18 ppm to the
phosphite triester at .delta. 139.16 ppm.
In another example, 3 .ANG. molecular sieves were activated by
drying in a vacuum oven at 200.degree. C. overnight. A small number
of sieves were placed in a 5 ml, round bottom flask with a 14/20
ground glass joint that was then sealed with a rubber septum. In
this example, 3 ml of 1-butyl-3-methyl-imidazolium
tetrafluoroborate (Solvent Innovation GmbH, 50679 Koln, Germany)
was added to the flask and the liquid allowed to dry overnight.
5'-Dimethoxytrityl-2'-deoxyThymidine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 0.38 grams,
0.5 mmol, was added to the flask and the solution shaken until the
reagent had dissolved. A small amount of the solution was removed
from the flask and placed in an NMR tube for analysis by .sup.31 P
NMR using an external lock. The resulting NMR spectrum showed the
presence of the starting material nucleoside phosphoramidite at
.delta. 146.94 ppm relative to phosphoric acid.
3'-Acetyl Thymidine (ChemGenes Corp., Waltham Mass.) 0.14 grams,
0.5 mmol was added to the mixture along with tetrazole 0.18 grams,
2.5 mmol. The solution was shaken on a wrist action shaker until
the reagents were completely dissolved. An aliquot of the reaction
mixture was removed from the flask and placed in an NMR tube for
analysis by .sup.31 P NMR using an external lock. The resulting NMR
spectrum showed complete conversion of the starting material
nucleoside phosphoramidite at .delta. 146.94 ppm to the phosphite
triester diastereomers centered at .delta. 138.98 ppm.
In general, the product of the coupling reaction, when performed in
a solid phase system, may be represented by the following
structural formula: ##STR3##
Wherein:
.largecircle. represents the solid support or a support-bound
oligonucleotide chain;
A represents H or an optionally protected hydroxyl group;
B is a purine or pyrimidine base whose exocyclic amine functional
group is optionally protected; and
R is a suitable protecting group,
"Y" is hydrido or hydrocarbyl, typically alkyl, alkenyl, aryl,
aralkyl, or cycloalkyl. Preferably, Y represents: lower alkyl;
electron-withdrawing .beta.-substituted aliphatic, particularly
electron-withdrawing .beta.-substituted ethyl such as
.beta.-trihalomethyl ethyl, .beta.-cyanoethyl, .beta.-sulfoethyl,
.beta.-nitro-substituted ethyl, and the like; electron-withdrawing
substituted phenyl, particularly halo-, sulfo-, cyano- or
nitro-substituted phenyl; or electron-withdrawing substituted
phenylethyl. Most preferably, Y represents methyl,
.beta.-cyanoethyl, or 4-nitrophenylethyl.
In this formula, the sugar and the base to the 5' side of the
phosphorus atom (P) corresponds to one nucleoside moiety, and the
sugar and the base to the 3' side of the phosphorus atom (P)
correspond to the other nucleoside moiety.
Ionic liquids that may be used include organic salts that are fluid
below about 80.degree. C. at around normal atmospheric pressure
(about 1 atmosphere at sea level). The organic salts generally have
an organic cation and either an inorganic or organic counterion.
The organic cation is preferably an N-substituted pyridine having
the following structure: ##STR4##
wherein R is alkyl and each R' is independently selected from
hyrido, alkyl, or halogen;
or a 1,3 di-substituted imidazole having the following structure:
##STR5##
wherein each R is independently selected from alkyl, each R' is
independently selected from hydrido, alky, or halogen, and R" is
selected from hydrido or methyl.
Preferred organic cations include 1,3-dimethyl-imidazolium,
1-ethyl-3-methyl-imidazolium, 1-butyl-3-methyl-imidazolium,
1-hexyl-3-methyl-imidazolium, 1-decyl-3-methyl-imidazolium,
1-dodecyl-3-methyl-imidazolium, 1-methyl-3-octyl-imidazolium,
1-methyl-3-tetradecyl-imidazolium,
1,2-dimethyl-3-propyl-imidazolium,
1-ethyl-2,3-dimethyl-imidazolium, 1-butyl-2,3-dimethyl-imidazolium,
N-ethylpyridinium, N-butylpyridinium, N-hexylpyridinium,
4-methyl-N-butyl-pyridinium, 3-methyl-N-butyl-pyridinium,
4-methyl-N-hexyl-pyridinium, 3-methyl-N-hexyl-pyridinium,
4-methyl-N-octyl-pyridinium, 3-methyl-N-octyl-pyridinium,
3,4-dimethyl-N-butyl-pyridinium, and
3,5-dimethyl-N-butyl-pyridinium.
Preferred anions of the ionic liquid are chloride (Cl.sup.-),
bromide (Br.sup.-), tetrafluoroborate ([BF.sub.4 ].sup.-),
hexafluorophosphate ([PF.sub.6 ].sup.-), [SbF.sub.6 ].sup.-,
[CuCl.sub.2 ].sup.-, [AlCl.sub.4 ].sup.-, [Al.sub.2 Cl.sub.7
].sup.-, [Al.sub.3 Cl.sub.10 ].sup.-, methylsulfate (CH.sub.3
SO.sub.4.sup.-), trifluoroacetate (CF.sub.3 CO.sub.2.sup.-),
heptafluorobutanoate (CF.sub.3 (CF.sub.2).sub.2 CO.sub.2.sup.-),
triflate (CF.sub.3 SO.sub.2.sup.-), nonaflate (C.sub.2 F.sub.5
SO.sub.2.sup.-), bis(trifluoromethylsulfonyl)imide ((CF.sub.3
SO.sub.2).sub.2 N.sup.-), bis(perfluoroethylsulfonyl)imide
((C.sub.2 F.sub.5 SO.sub.2).sub.2 N.sup.-), and
tris(trifluoromethylsulfonyl)methide ((CF.sub.3 SO.sub.2).sub.3
C.sup.-). Ionic liquids are available from Covalent Associates
(Woburn, Mass.), Solvent Innovation (Koln, Germany), Aldrich
Chemical Company (Milwaukee, Wis.), and Acros Organics (Geel,
Belgium).
In one embodiment, to perform the coupling reaction, at least one
of the chemical species having a nucleoside moiety is dissolved in
a solution having at least 98 percent by weight of ionic liquid,
whereupon the other chemical specie having a nucleoside moiety (the
second nucleoside moiety) is contacted with the solution containing
ionic liquid and the first nucleoside moiety. In other embodiments,
the solution has at least about 90% ionic liquid, or at least about
75% ionic liquid, or at least about 50% ionic liquid, or at least
about 25% ionic liquid, or at least about 10% ionic liquid.
Co-solvents that may be mixed into the ionic liquid include but are
not limited to acetonitrile, tetrahydrofuran, dimethylformamide,
methylene chloride, propylene carbonate, adiponitrile, toluene,
dioxane, dimethylsulfoxide, and N-methylpyrrolidone. An activator
compound is typically included in a concentration of about 0.05
molar up to about 0.5 molar. The activator is generally tetrazole,
S-ethyl-thiotetrazole, 4-nitrotriazole, or dicyanoimidazole,
although other acidic azoles may be used. One potential advantage
of using an ionic liquid is that the ionic liquid may serve as the
activator.
In the conventional synthesis method depicted schematically in FIG.
1, it is typical to use an aqueous solution of iodine for the
oxidation step. However, phosphoramidite reagents that have been
activated for coupling are highly reactive with water. The
invention may be extended to include using ionic liquids as
solvents elsewhere in the synthesis cycle to reduce or
substantially eliminate the presence of water during oxidation and
deprotection.
In one embodiment of the invention, the first nucleoside moiety is
a monomer nucleoside phosphoramidite, which is coupled to a free
hydroxyl of a second nucleoside moiety, analogous to conventional
polynucleotide synthesis. The invention also encompasses the
formation of an internucleoside bond between two polynucleotides or
oligonucleotides, or between a polynucleotide and an
oligonucleotide. In such case, the first nucleoside moiety
corresponds to the one of the polynucleotides or oligonucleotides,
and the second polynucleotide moiety corresponds to the
polynucleotide or oligonucleotide to be joined to the first
nucleoside moiety. The skilled practitioner in the art will realize
that one of the nucleoside moieties must be activated, as in a
phosphoramidite. Such modification is well known in the art.
As explained earlier herein, the method of the invention also lends
itself to synthesis of polynucleotides in the 5'-to-3' direction.
In such a case, the initial step of the synthetic process involves
attachment of an initial nucleoside to a solid support at the 5'
position, leaving the 3' position available for covalent binding of
a subsequent monomer. The coupling reaction in which the nucleoside
monomer becomes covalently attached to the 3' hydroxyl moiety of
the support bound nucleoside is conducted under reaction conditions
identical to those described for the 3'-to-5' synthesis. The
synthesis cycle is then continued with the (optional) capping step,
the oxidation of the internucleotide bond, and the deprotection of
the active site hydroxyl in preparation for the next synthesis
cycle, which is repeated until a polynucleotide having the desired
sequence and length is obtained. Following synthesis, the
polynucleotide may, if desired, be cleaved from the solid support.
The details of the synthesis in the 5'-to-3' direction will be
readily apparent to the skilled practitioner based on the prior art
and the disclosure contained herein.
The coupling reaction as described herein may easily be adapted to
be performed in a conventional automated oligonucleotide
synthesizer utilizing an insoluble substrate to immobilize the
polynucleotides during synthesis. Such methodology will be apparent
to those skilled in the art and is described in the pertinent texts
and literature, e.g., in D. M. Matteuci et al. (1980) Tet. Lett.
521:719 and U.S. Pat. No. 4,500,707. Examples of suitable support
materials include, but are not limited to, polysaccharides such as
agarose (e.g., that available commercially as Sepharose.RTM., from
Pharmacia) and dextran (e.g., those available commercially under
the tradenames Sephadex.RTM. and Sephacyl.RTM., also from
Pharmacia)
While the foregoing embodiments of the invention have been set
forth in considerable detail for the purpose of making a complete
disclosure of the invention, it will be apparent to those of skill
in the art that numerous changes may be made in such details
without departing from the spirit and the principles of the
invention. Accordingly, the invention should be limited only by the
following claims.
All patents, patent applications, and publications mentioned herein
are hereby incorporated by reference in their entireties.
* * * * *