U.S. patent application number 10/337004 was filed with the patent office on 2003-08-14 for purification of oligonucleotides.
Invention is credited to Krotz, Achim H., Ravikumar, Vasulinga T..
Application Number | 20030153742 10/337004 |
Document ID | / |
Family ID | 23968503 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030153742 |
Kind Code |
A1 |
Krotz, Achim H. ; et
al. |
August 14, 2003 |
Purification of oligonucleotides
Abstract
Methods and compounds useful for the purification of
oligonucleotides and their analogs are provided wherein the
oligonucleotides are contaminated with at least one oligonucleotide
having at least one abasic site by the formation of imines at the
aldehyde moiety of the abasic site and subsequent separation based
on extractions, precipitations or chromatography.
Inventors: |
Krotz, Achim H.; (San Diego,
CA) ; Ravikumar, Vasulinga T.; (Carlsbad,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE - 46TH FLOOR
PHILADELPHIA
PA
19103
US
|
Family ID: |
23968503 |
Appl. No.: |
10/337004 |
Filed: |
January 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10337004 |
Jan 2, 2003 |
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09495398 |
Jan 31, 2000 |
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Current U.S.
Class: |
536/25.4 |
Current CPC
Class: |
C07H 19/04 20130101;
C07H 21/00 20130101 |
Class at
Publication: |
536/25.4 |
International
Class: |
C07H 021/04 |
Claims
What is claimed is:
1. A method of purifying an oligonucleotide from a mixture, said
mixture including said oligonucleotide and at least one contaminant
comprising: treating said mixture with an amino reagent reactive
with said contaminant for a time and under conditions effective to
form an imine linkage with each said contaminant; and separating
said oligonucleotide from said imine linked contaminants.
2. The method of claim 1 wherein said amino reagent is an amine,
hydrazine, hydroxylamine, semicarbazide or thiosemicarbazide.
3. The method of claim 2 wherein said separation is based upon
differences in solubility of said oligonucleotide and said imine
linked contaminants in a selected solvent.
4. The method of claim 3 wherein said imine linked contaminants are
more soluble in a selected solvent than said oligonucleotide.
5. The method of claim 3 wherein said imine linked contaminants are
less soluble in a selected solvent than said oligonucleotide.
6. The method of claim 1 wherein said oligonucleotide is more
soluble in a first solvent than said imine linked contaminant and
said imine linked contaminant is more soluble in a second solvent
than said oligonucleotide and said first and said second solvents
are immiscible.
7. The method of claim 6 wherein said first solvent is water or an
aqueous solution and said second solvent is an organic solvent.
8. The method of claim 7 wherein said organic solvent is benzene,
diethyl ether, ethyl acetate, hexanes, pentane, chloroform,
dichloromethane or carbon tetrachloride.
9. The method of claim 1 wherein said oligonucleotide is more
soluble in a first solvent than said imine linked contaminant and
said imine linked contaminant is more soluble in a second solvent
than said oligonucleotide and said first and said second solvents
are miscible.
10. The method of claim 9 wherein said first solvent is water or an
aqueous solution.
11. The method of claim 9 wherein said second solvent is an organic
solvent.
12. The method of claim 11 wherein said organic solvent is acetone,
methanol, ethanol or isopropanol.
13. The method of claim 1 wherein said separating is effected by
selectively precipitating the oligonucleotide or the imine linked
contaminant.
14. The method of claim 13 wherein said separating is effected by
selectively precipitating the oligonucleotide.
15. The method of claim 1 wherein said separating is effected by
chromatography.
16. The method of claim 1 wherein said separating is effected by
liquid-liquid extraction.
17. The method of claim 1 wherein said amino reagent further
includes a surfactant.
18. The method of claim 17 wherein said surfactant is a non-ionic
surfactant.
19. The method of claim 17 wherein said amino reagent further
including said surfactant has the formula: 15wherein: x is from 0
to 20, y is from 0 to 5, n is from 0 to 150; and Z is --NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2 or
--NHC(S)NHNH.sub.2.
20. The method of claim 19 wherein x is 8, y is 1 and n is 12.
21. The method of claim 1 wherein said amino reagent further
includes an attachment to a polymeric support.
22. The method of claim 21 wherein said polymeric support is a
solid phase polymeric support.
23. The method of claim 21 wherein said polymeric support is a
hydroxylamine resin.
24. The method of claim 21 wherein said separating is effected by
rinsing or washing said oligonucleotide from said imine linked
contaminants bound to said polymeric support.
25. The method of claim 21 wherein said polymeric support is a
liquid phase polymeric support.
26. The method of claim 25 wherein said liquid phase polymeric
support is hydrophilic.
27. The method of claim 25 wherein said polymeric support is a
polyvinyl alcohol, a polyethylene glycol (PEG), a cellulose, or a
polyvinyl alcohol-poly(1-vinyl-2-pyrrolidinone).
28. The method of claim 25 wherein said amino reagent including
said polymeric support is a polyethylene glycol (PEG) amine,
polyethylene glycol (PEG) hydrazine, polyethylene glycol (PEG)
hydroxylamine, polyethylene glycol (PEG) semicarbazide,
polyethylene glycol (PEG) thiosemicarbazide.
29. A method of purifying an oligonucleotide from a mixture, said
mixture including said oligonucleotide and at least one
contaminant, comprising: treating said mixture with a compound of
formula I: 16I wherein: x is from 0 to about 20, y is from 0 to
about 5, n is from 0 to about 150, Z is a reactive nitrogenous
moiety capable of reacting with an aldehyde to form an imine; for a
time and under conditions effective to form an imine linkage with
each contaminant; and separating said oligonucleotide from said
imine linked contaminants.
30. The method of claim 29 comprising treating said mixture with
plurality of compounds of formula I wherein said compounds differ
with respect to the value of n.
31. The method of claim 29 wherein n is from about 8 to about
16.
32. The method of claim 29 wherein n is 12.
33. The method of claim 29 wherein Z is --NH.sub.2, --NHNH.sub.2,
--ONH.sub.2, --NHC(O)NHNH.sub.2 or --NHC(S)NHNH.sub.2.
34. The method of claim 29 wherein Z is --ONH.sub.2.
35. The method of claim 29 wherein Z is --ONH.sub.2, x is 8, y is 1
and [CH.sub.3(CH.sub.2).sub.x].sub.y-- is para to said
--(OCH.sub.2CH.sub.2).sub.n-Z.
36. The method of claim 35 wherein n is from about 8 to 16.
37. The method of claim 35 wherein n is 12.
38. The method of claim 29 wherein said separating is effected by
liquid-liquid extraction.
39. The method of claim 29 wherein said separating is effected by
selectively precipitating the oligonucleotide or the imine linked
contaminant.
40. The method of claim 39 wherein said separating is effected by
selectively precipitating the oligonucleotide.
41. The method of claim 29 wherein said separating is effected by
chromatography.
42. A method of modifying the solubility of an oligonucleotide
comprising: selecting an oligonucleotide having at least one abasic
site; treating said oligonucleotide with an amino reagent reactive
with said oligonucleotide for a time and under conditions effective
to form an imine linkage between said oligonucleotide and said
amino reagent at said abasic site.
43. The method of claim 42 wherein said amino reagent is an amine,
hydrazine, hydroxylamine, semicarbazide or thiosemicarbazide.
44. The method of claim 42 wherein said amino reagent further
includes a surfactant.
45. The method of claim 44 wherein said surfactant is a non-ionic
surfactant.
46. The method of claim 44 wherein said amino reagent further
including said surfactant has the formula: 17wherein: x is from 0
to 20, y is from 0 to 5, n is from 0 to 150; and Z is --NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2 or
--NHC(S)NHNH.sub.2.
47. The method of claim 46 wherein x is 8, y is 1 and n is 12
said.
48. The method of claim 42 wherein said amino reagent further
includes an attachment to a polymeric support.
49. The method of claim 48 wherein said polymeric support is a
solid phase polymeric support.
50. The method of claim 48 wherein said polymeric support is an
hydroxylamine resin.
51. The method of claim 48 wherein said polymeric support is a
liquid phase polymeric support.
52. The method of claim 51 wherein said liquid phase polymeric
support is hydrophilic.
52. The method of claim 51 wherein said polymeric support is a
polyvinyl alcohol, a polyethylene glycol (PEG), a cellulose, or a
polyvinyl alcohol-poly(1-vinyl-2-pyrrolidinone).
53. The method of claim 51 wherein said amino reagent further
included to a polymeric support is a polyethylene glycol (PEG)
amine, polyethylene glycol (PEG) hydrazine, polyethylene glycol
(PEG) hydroxylamine, polyethylene glycol (PEG) semicarbazide,
polyethylene glycol (PEG) thiosemicarbazide.
54. A compound of Formula I, 18x is from 0 to 20, y is from 0 to 5,
n is from 0 to 150; and Z is --NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC(O)NHNH.sub.2 or --NHC(S)NHNH.sub.2.
55. A composition comprising a plurality of compounds according to
claim 54 wherein said compounds differ with respect to the value of
n; wherein said value of n is from 0 to about 150.
56. The composition of claim 54 wherein said value of n is from
about 8 to about 16.
57. The compound of claim 54 wherein n is 12.
58. The compound of claim 54 wherein Z is --ONH.sub.2.
59. The compound of claim 54 wherein Z is --ONH.sub.2, x is 8, y is
1, [CH.sub.3(CH.sub.2).sub.x].sub.y is para to said
(OCH.sub.2CH.sub.2).sub.- n-Z, and n is from about 0 to about
150.
60. The compound of claim 59 wherein n is from about 8 to 16.
61. The compound of claim 59 wherein n is 12.
Description
FIELD OF THE INVENTION
[0001] The present invention provides methods and compounds useful
for modifying the solubilities of oligonucleotides and their
analogs. The present invention further provides methods for
purifying oligonucleotides from mixtures containing the
oligonucleotides and at least one contaminant wherein the
contaminant is an oligonucleotide having at least one abasic
site.
BACKGROUND OF THE INVENTION
[0002] Modern therapeutic efforts are generally focused on the
functions of proteins which contribute to many diseases in animals
and man. There have been numerous attempts to modulate the
production of such proteins by interfering with the function of
biomolecules, such as intracellular RNA, that are involved in the
synthesis of these proteins. It is anticipated that protein
production will thus be inhibited or abolished, resulting in a
beneficial therapeutic effect. The general object of such
therapeutic approaches is to interfere with or modulate gene
expression events that lead to the formation of undesired
proteins.
[0003] One such method for the inhibition of specific gene
expression is the use of oligonucleotides and oligonucleotide
analogs as antisense drugs. These oligonucleotide or
oligonucleotide analogs are designed to be complementary to a
specific target messenger RNA (mRNA) or DNA, that encodes for the
undesired protein. The oligonucleotide or oligonucleotide analog is
expected to hybridize with good affinity and selectivity to its
target nucleic acid, such that the normal essential functions of
the target nucleic acid are disrupted. Antisense therapeutics hold
great promise as evidenced by the large number of oligonucleotides
and oligonucleotide analogs that have been evaluated clinically in
recent times. Further, oligonucleotides and oligonucleotide analogs
have shown significant promise in the diagnosis of disease and have
also been used extensively as probes in diagnostic kits and as
research reagents.
[0004] There is, therefore, a great need for the large scale
production of oligonucleotides and oligonucleotide analogs for
commercial application. The predominant synthetic regime currently
in use for oligonucleotide synthesis is the phosphoramidite method
as developed by Caruthers (Caruthers, M. H. Gene Synthesis
Machines: DNA Chemistry and Its Uses. Science, 1985, 230, 281-85).
The phosphoramidite method transformed oligonucleotide synthesis
from a manual or semi-manual procedure carried out by a few
specialists into a commercialized process performed by a machine.
The oligonucleotides are synthesized on a solid-support via
sequential reactions in a predetermined order, typically controlled
by a computerized pumping system. The crux of this chemistry is a
highly efficient coupling reaction (>98%) between a 5'-hydroxyl
group of a support-bound deoxynucleoside and an alkyl
5'-O-DMTr-3'-O-(N,N-diisopropy- lamino-O-cyanoethyl)phosphoramidte
deoxynucleoside. For example, oligonucleotide synthesis typically
begins with a nucleoside linked to a solid-support, typically via a
linker molecule attached to the 3'-oxygen of the first nucleosidic
synthon. Deprotection (or "cleavage") of the 5'-hydroxyl group is
effected by treatment with an acid (3% dichloroacetic acid (DCA) in
dichloromethane or toluene) which removes the
5'-O-(4,4'-dimethoxytriphenylmethyl) hydroxyl protecting group
(DMTr) to provide an oligonucleotide having a free 5'-OH group.
Such protecting groups are routinely used in oligonucleotide
synthesis to allow selective reaction between two functional groups
while protecting all other functionalities present in the reacting
molecules.
[0005] The next step consists of premixing a nucleoside
phosphoramidite with an activator such as 1-H tetrazole. The very
reactive P(III) tetrazolide intermediate reacts almost immediately
with the 5'-OH group of the support bound nucleoside to generate a
dinucleoside phosphite with a phosphite triester internucleosidic
linkage. The unstable P(III) species is oxidized to a more stable
P(V) internucleosidic linkage with iodine to the phosphotriester
before proceeding with chain extension. A capping reaction with an
acylating reagent is performed to prevent the unreacted 5'-OH
groups from further extension. These steps are then repeated
iteratively until the desired oligonucleotide is obtained. A more
detailed treatment of oligonucleotide synthesis, and further
representative synthetic procedures can be found in
Oligonucleotides And Analogues A Practical Approach, Eckstein, F.,
Ed., IRL Press, N.Y, 1991.
[0006] One challenge facing commercialization of oligonucleotide
based therapeutic and diagnostic products is the ability to
manufacture and market these products at a reasonable cost and with
a high level of oligonucleotide purity.
[0007] The trityl group has been used for the temporary protection
of primary hydroxyl groups due to the generally good crystallizing
properties imparted by the trityl ether and its easy removal
through mild acid treatment or by hydrogenolysis (Agarawal, K. I.;
Yamazaki, A.; Cashion, P. L.; Khorana, H. G., Angew Chem. Int Ed.
Engl. 1972, 451 and Stanek, J. Top. Curr. Chem. 1990, 54, 234).
However, the literature indicates that detritylation is a
problematic operation. Low yields, formation of by-products, acyl
migration and glycosidic bond cleavage or depurination often arise
from protic acid-catalyzed detritylation reactions (e.g., 80%
acetic acid acid at reflux (Micheel, F., Ber. 1932, 65, 262), 80%
formic acid in ethyl acetate at room temperature (Soudheimer, S.
J.; Eby, R.; Schuerch, C., Carbohydr. Res. 1978, 60, 187 and
Bessodes, M.; Komiotis, D.; Antonakis, K., Tetrahedron Lett. 1986,
27, 579), hydrogen chloride in methanol (Verkade, P. E.; Vander
Lee, J.; Meerburg, W. Rec. Trav. Chim., 1935, 54, 716) or other
solvent (Choy, Y. M.; Unrau, A. M., Carbohydr. Res. 1971, 17, 439),
and hydrogen bromide in acetic acid (Roy, N.; Timell, T. E.,
Carbohydr. Res. 1968, 7, 82 and Barker, G. R., Methods Carbohydr.
Chem. 1963, 2, 68), among others (Helferich, B., Adv. Carbohydr.
Chem. 1948, 3, 79).
[0008] The deprotection of a trityl group is usually performed
under acidic conditions using a protic or a Lewis acid in an
organic solvent. Alternate deprotection protocols have been
attempted such that acidic conditions are avoided in an attempt to
ameliorate the problems of depurination in purine rich
oligonucleotides. These techniques, however, have not been reported
to have been successfully applied to the large scale manufacture of
oligonucleotides, as is required for research or commercial
purposes.
[0009] The importance of a good separation technique for synthetic
oligonucleotides is often neglected. The impurities from a large
number of reactions are stored upon the support and must all be
resolved, preferably in a single step. Powerful separation methods
have been developed to purify oligonucleotides. For example,
polyacrylamide gel electrophoresis separates oligonucleotides by
virtue of their charge differences. Other techniques include high
performance liquid chromatography (HPLC), including ion exchange
chromatography which resolves by charge differences and reverse
phase chromatography which separates according to hydrophobicty.
These chromatographic techniques, however, have their limitations.
These limitations are either that the purification is limited to
milligram quantities of materials, or there is an insufficient
resolution between the desired oligonucleotide and the contaminant
impurity.
[0010] For the foregoing reasons, there exists a need for new
methods that address the shortcomings of the large scale production
and purification of oligonucleotides as discussed above.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to methods and compounds
useful for purifying oligonucleotides and oligonucleotide analogs
from mixtures having contaminants comprising oligonucleotides or
oligonucleotide analogs that have undesired abasic sites. Mixtures
are treated with amino reagents reactive with and capable of
forming imine linkages with the contaminants. Chemical modification
of undesired contaminants using the present method enhances
separation of the desired oligonucleotides from undesired
contaminants by methods that are ineffective for the parent
mixtures prior to such modification.
[0012] In preferred embodiments, the imine-linked contaminants are
separated from the oligonucleotide to be purified using
chromatography.
[0013] In other preferred embodiments, the imine-linked
contaminants are separated from the oligonucleotide to be purified
by selectively precipitating the imine-linked contaminants with
respect to the oligonucleotide to be purified.
[0014] In other preferred embodiments, the imine-linked
contaminants are separated from the oligonucleotide to be purified
by selectively precipitating the oligonucleotide to be purified
with respect to the imine-linked contaminant.
[0015] In other preferred embodiments, the imine-linked
oligonucleotides are separated from the oligonucleotide to be
purified by a liquid-liquid extraction.
[0016] In some preferred embodiments, the imine-linked contaminants
are separated from the oligonucleotide to be purified using
chromatography with a single solvent, or with two or more miscible
solvents.
[0017] In other preferred embodiments of the invention, the
imine-linked contaminants oligonucleotides are separated from the
oligonucleotide to be purified by precipitation using two or more
immiscible solvents, or two or more miscible solvents.
[0018] In other preferred embodiments, the imine-linked
contaminants are separated from the oligonucleotide to be purified
by liquid-liquid extraction using two or more immiscible
solvents.
[0019] In a preferred embodiment of the invention, the imine-linked
contaminants are separated from the oligonucleotide to be purified
based upon differences in solubility of the oligonucleotide and the
imine-linked contaminant in a selected solvent.
[0020] In one preferred embodiment, the imine-linked contaminants
are more soluble in a selected solvent than the oligonucleotide to
be purified. In another preferred embodiments, the imine-linked
contaminants are less soluble in a selected solvent than the
oligonucleotide to be purified.
[0021] In one preferred embodiment of the invention, the difference
in solubility is a difference wherein the oligonucleotide to be
purified is more soluble in a first solvent, preferably water or an
aqueous solvent, than the imine-linked contaminants and the
imine-linked contaminants are more soluble in a second solvent,
preferably an organic solvent, than the oligonucleotide to be
purified and the first and second solvents are immiscible. In
further preferred embodiments, the organic solvent includes
benzene, diethyl ether, ethyl acetate, hexane, pentane, chloroform,
dichloromethane, carbon tetrachloride, and the like. In other
preferred embodiments the first and second solvents are miscible
and the second solvent is preferably an organic solvent that is
miscible with water such as, acetone, methanol, isopropanol,
ethanol and the like.
[0022] In preferred embodiments, the amino reagents include amines,
hydrazines, hydroxylamines, semicarbazides, and
thiosemicarbazides.
[0023] In one preferred embodiment of the present invention, the
amino reagent is linked to a polymeric support thereby forming a
linked amino reagent. The amino reagents may be linked to
solid-phase polymeric supports, to form, for example, a
hydroxylamine resin, or may be linked to liquid-phase support,
preferably hydrophilic supports. In preferred embodiments, the
liquid-phase polymeric support is a polyvinyl alcohol, a
polyethylene glycol (PEG), a cellulose, or a polyvinyl
alcohol-poly(1-vinyl-2-pyrrolidinone). Preferred liquid-support
linked amino reagents are polyethylene glycol (PEG) amine,
polyethylene glycol (PEG) hydrazine, polyethylene glycol
hydroxylamine, polyethylene glycol semicarbazide, and polyetheylene
glycol thiosemicarbazide.
[0024] In further embodiments the amino reagent may include a
surfactant, such as a non-ionic surfactant. A preferred amino
reagent including such a surfactant has the formula: 1
[0025] wherein:
[0026] x is from 0 to 20,
[0027] y is from 0 to 5,
[0028] n is from 0 to 150; and
[0029] Z is --NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC(O)NHNH.sub.2 or --NHC(S)NHNH.sub.2.
[0030] In a preferred embodiment x is 8, y is 1 and n is 12.
[0031] In some preferred embodiments of the present invention,
methods for purifying an oligonucleotide from a mixture wherein the
mixture includes the oligonucleotide and at least one contaminant,
wherein the contaminant comprises at least one aldehyde moiety,
comprise the steps of:
[0032] treating the mixture with a compound of formula I: 2
[0033] wherein:
[0034] x is from 0 to about 20, preferably 8;
[0035] y is from 0 to about 5, preferably 1;
[0036] n is from 0 to about 150, preferably 12; and
[0037] Z is a reactive nitrogenous moiety, such as --NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, or --NHC(S)NH.sub.2,
that is capable of reacting with an aldehyde to form an imine;
[0038] for a time and under conditions effective to form imine
linkages with each contaminant; and
[0039] separating said oligonucleotide from said imine linked
contaminants.
[0040] In some preferred embodiments of the present invention,
methods for purifying an oligonucleotide from a mixture wherein the
mixture includes the oligonucleotide and at least one contaminant,
wherein the contaminant comprises at least one aldehyde moiety,
comprise the steps of:
[0041] treating the mixture with a plurality of compounds of
formula I: 3
[0042] wherein the compounds differ with respect to the value of n,
and wherein all other variables are as described above.
[0043] In another aspect of the invention, a method is provided for
modifying the solubility of an oligonucleotide comprising:
[0044] selecting an oligonucleotide having at least one abasic
site;
[0045] treating the oligonucleotide with an amino reagent reactive
with the oligonucleotide for a time and under conditions effective
to form an imine linkage between the abasic site of the
oligonucleotide and the amino reagent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] These and other features, aspects, and advantages of the
present invention will become better understood with reference to
the following description, appended claims and accompanying
drawings where:
[0047] FIG. 1 shows the S.sub.N1 mechanism pathway which has been
suggested for the hydrolysis of nucleosides to generate an abasic
site.
[0048] FIG. 2 shows an abasic site is a mixture of four chemical
species in a tautomeric equilibrium.
[0049] FIG. 3 shows the deoxyribose fragment in its open chain
aldehydic form can undergo a base catalyzed .beta.-elimination
reaction leading to scission of the phosphodiester backbone at the
3'-end of the abasic site.
[0050] FIG. 4 shows the amino reagents reacting with the carbonyl
moiety an oligonucleotide abasic site to form the corresponding
imine.
[0051] FIG. 5 is a drawing of the mass spectra of phosphorothioate
oligodeoxyribonucleotide, PS-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 1)
that is contaminated with an oligonucleotide having an abasic
site.
[0052] FIG. 6 is a drawing of the mass spectra of phosphorothioate
oligodeoxyribonucleotide, PS-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 1)
where the abasic sites are either totally absent or are greatly
diminished after treatment of the oligonucleotide mixture with
IGEPAL.TM. CO-720 hydroxylamine.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0053] The methods and compounds of the present invention are
useful for the purification of oligonucleotides. Purification of
oligonucleotides is especially required in the areas of both
antisense and PCR technology.
[0054] Virtually all diseases are associated with inadequate or
inappropriate production or performance of proteins. Antisense
technology involves the use of synthetic segments of DNA or RNA
called oligonucleotides to stop the production of such disease
related proteins. These oligonucleotides block the transmission of
genetic information between the nucleus and the protein production
sites within a cell. For a protein to be synthesized, the gene that
specifies its composition must be copied from double stranded DNA
into molecules of single-stranded RNA, called messenger RNA, which
carry the genetic information necessary for protein synthesis from
the nucleus of the cell to the cytoplasm where synthesis occurs.
Oligonucleotides designed precisely on the basis of the genetic
code bind specifically with the messenger RNA and effectively jam
its genetic signal, thereby preventing the production of disease
associated proteins.
[0055] PCR is an acronym which stands for polymerase chain
reaction. The PCR technique is basically a primer extension
reaction for amplifying specific nucleic acids in vitro. The use of
a thermostable polymerase allows the dissociation of newly formed
complimentary DNA and subsequent annealing or hybridization of
primers to the target sequence with minimal loss of enzymatic
activity. PCR will allow a short stretch of DNA (usually fewer than
3000 bp) to be amplified to about a million fold so that one can
determine its size and nucleotide sequence. The particular stretch
of DNA to be amplified, called the target sequence, is identified
by a specific pair of DNA primers called oligonucleotides, which
are usually about 20 nucleotides in length. A primer is a an
oligonucleotide which is complementary to a section of the DNA
which is to be amplified in the PCR reaction. Primers are annealed
to the denatured DNA template to provide an initiation site for the
elongation of the new DNA molecule. Primers can either be specific
to a particular DNA nucleotide sequence or they can be universal.
Universal primers are complementary to nucleotide sequences which
are very common in a particular set of DNA molecules. Thus, they
are able to bind to a wide variety of DNA templates.
[0056] The present invention is directed to methods and compounds
that are useful for the purification of oligonucleotides from
milligram to multi-kilogram quantities. These methods include
purifying an oligonucleotide from a mixture of the oligonucleotide
and at least one contaminant oligonucleotide (hereinafter referred
to as a "contaminant") wherein the contaminant comprises an
oligonucleotide having at least one abasic site. For example, an
abasic site can be an apurinic or apyrimidinic site located on an
oligonucleotide wherein an aldehyde moiety is present as shown in
FIG. 4.
[0057] In preferred embodiments of the invention, methods are
provided for purifying an oligonucleotide from a mixture of the
oligonucleotide and at least one contaminant. In some preferred
embodiments the method includes treating a mixture comprised of
oligonucleotides and at least one contaminant with an amino reagent
that is reactive with the aldehyde moiety located at the abasic
site of the contaminant, thereby forming an imine linkage with the
aldehyde moiety, and subsequently separating the imine-containing
or imine-linked oligonucleotides from the oligonucleotide to be
purified.
[0058] Oligonucleotides, including those having abasic sites, are
polar molecules that are soluble in polar solvents such as water,
aqueous solutions, and aqueous buffers. While not wishing to be
bound by any specific theory, it is believed that imine formation
at the abasic site modifies the solubility of the contaminant
oligonucleotide. Separation is facilitated based on the differences
in solubility between the imine-linked contaminants and the
oligonucleotide to be purified.
[0059] Imine formation at the abasic site is also believed to
modify the partitioning behavior between the imine-linked
contaminant and the oligonucleotide to be purified, thereby
facilitating chromatographic separation. Chromatography is a
separations method that relies on differences in partitioning
behavior between a flowing mobile phase and a stationary phase to
separate the components in a mixture. Thus, in some preferred
embodiments of the invention, the separation of the oligonucleotide
to be purified from the imine-linked contaminants is based on the
differing partitioning behavior between the two.
[0060] Oligonucleotide
[0061] As used herein, the term "oligonucleotide" refers to a
polynucleotide formed from a plurality of joined nucleotide units,
including linear sequences of nucleotides, in which the 5' linked
phosphate or other internucleotide linkage on one sugar group is
covalently linked to either the 2'-, 3'-, or 4'-position on the
adjacent sugars. Also included within the definition of
"oligonucleotide" are double stranded oligonucleotides including
DNA, RNA and plasmids, vectors and the like. Thus, the term
"oligonucleotide" includes linear sequences having 2 or more
nucleotides, and any variety of natural and non-natural
constituents as described below.
[0062] The term nucleoside has its accustomed meaning as a
pentofuranosyl sugar (ribose or deoxyribose) which is linked
glycosidically to a nucleosidic base (i.e., an amino heterocyclic
base or nucleobase), including but not limited to a purine or
pyrimidine base, but lacking the phosphate residues that would make
it a nucleotide. The term nucleotide refers to a phosphoric ester
of a nucleoside; the basic structural unit of nucleic acids (DNA or
RNA).
[0063] It will be appreciated that the methods of the present
invention can be applied to the purification of oligonucleotides
synthesized by a number of different chemical approaches such as
phosphodiester, phosphotriester, phosphite triester or
phosphoramidite, and H-phosphonate chemistries and by solution or
solid phase reactions, as has been widely reported in the
literature.
[0064] The nucleotide building blocks and therefore the
oligonucleotides synthesized and purified using the methods of the
invention may have both naturally occurring and non-naturally
occurring constituent sugars, internucleoside linkages and/or
nucleobases. Non-naturally occurring sugars, internucleoside
linkages and nucleobases are typically structurally distinguishable
from, yet functionally interchangeable with, naturally occurring
sugars (e.g. ribose and deoxyribose), internucleoside linkages
(i.e. phosphodiester linkages), and nucleosidic bases (e.g.,
adenine, guanine, cytosine, thymine). Thus, non-naturally occurring
moieties include all such structures which mimic the structure
and/or function of naturally occurring moieties, and which aid in
the binding of the oligonucleotide analog to a target, or otherwise
advantageously contribute to the properties of the synthesized
oligonucleotide.
[0065] Modifications on the furanosyl portion of the nucleotide
subunits may also be effected, as long as the essential tenets of
this invention are adhered to. Representative examples of
non-naturally occurring sugars include sugars having any of a
variety of substituents attached to any one or more of the
positions on the sugar. Examples of such modifications are
2'-O-alkyl, -halogen, O-aminoalkyl, O-alkyloxyalkyl, N-protected
O-aminoalkyl, O alkylaminoalkyl, O-dialkylaminoalkyl,
O-imidazolylalkyl, dialkylamino-oxyalkyl, O-alkylaminooxyalkyl, and
polyethers of the formula (O-alkyl).sub.m, where m is 1 to about 10
substitued nucleotides. Preferred among these polyethers are linear
and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups
such as crown ethers and those which are disclosed by Ouchi et al.,
Drug Design and Discovery, 1992, 9, 93, Ravasio et al., J. Org.
Chem., 1991, 56, 4329, and Delgardo et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1992, 9, 249, herein incorporated
by reference. Further sugar modifications are disclosed in Cook,
Anti-Cancer Drug Design, 1991, 6, 585. Cook, Medicinal Chemistry
Strategies for Antisense Research; in Antisense Research and
Applications, Crooke et al., CRC Press Inc., Boca Raton, Fla.,
1993, all of which are herein incorporated by reference. 2'-Fluoro,
O-alkyl, O-aminoalkyl, O-imidazolylalkyl, O-alkylaminoalkyl, and
O-aminoalkyl substitutions are described in U.S. patent application
Ser. No. 08/398,901, filed Mar. 6, 1995, entitled Oligomeric
Compounds having Pyrimidine Nucleotide(s) with 2' and
5'-Substitutions, the disclosure of which is hereby incorporated by
reference.
[0066] Some specific examples of such modifications at the 2'
position of sugar moieties which are useful in the present
invention are OH, SH, SCH.sub.3, F, OCN, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3 where n is from 1 to about 10; C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl;
Cl, Br, CN, CF.sub.3, OCF.sub.3, O--, S--, or -alkyl; O--, S--, or
N-alkenyl; SOCH.sub.3, SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2;
N.sub.3; NH.sub.2; heterocycloalkyl; heterocycloalkaryl;
aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving
group; a conjugate; a reporter group; an intercalator; a group for
improving the pharmacokinetic properties of an oligonucleotide; or
a group for improving the pharmacodynamic properties of an
oligonucleotide and other substituents having similar
properties.
[0067] Oligonucleotides bearing sugars having O-substitutions on
the ribosyl ring are also amenable to the present invention.
Representative substitutions for ring O include S, CH.sub.2, CHF,
and CF.sub.2 (Sanghvi and Cook in Carbohydrate Modifications in
Antisense Research, ACS Symposium Series 580, ACS Publication,
Washington, D.C., 1994).
[0068] Sugar mimetics may also be used in place of the
pentofuranosyl group. Exemplary modifications are disclosed in U.S.
patent applications: Ser. No. 463,358, filed Jan. 11, 1990,
entitled Compositions And Methods For Detecting And Modulating RNA
Activity; Ser. No. 566,977, filed Aug. 13, 1990, entitled Sugar
Modified Oligonucleotides That Detect And Modulate Gene Expression;
Ser. No. 558,663, filed Jul. 27, 1990, entitled Novel Polyamine
Conjugated Oligonucleotides; Ser. No. 558,806, filed Jul. 27, 1991,
entitled Nuclease Resistant Pyrimidine Modified Oligonucleotides
That Detect And Modulate Gene Expression; and Serial Number
PCT/US91/00243, filed Jan. 11, 1991, entitled Compositions and
Methods For Detecting And Modulating RNA Activity; Ser. No.
777,670, filed Oct. 15, 1991, entitled Oligonucleotides Having
Chiral Phosphorus Linkages; Ser. No. 814,961, filed Dec. 24, 1991,
entitled Gapped 2' Modified Phosphorothioate Oligonucleotides; Ser.
No. 808,201, filed Dec. 13, 1991, entitled Cyclobutyl
Oligonucleotide Analogs; and Ser. No. 782,374, filed 782,374,
entitled Derivatized Oligonucleotides Having Improved Uptake &
Other Properties, all assigned to the assignee of this invention.
The disclosures of all of the above noted patent applications are
incorporated herein by reference.
[0069] Other sugar mimetics such as a morpholino may also be used
in place of the pentofuranosyl group. Summerton, J. E. and Weller,
D. D., U.S. 5,034,506 issued Jul. 23, 1991 entitled Uncharged
Morpholino-Based Polymers having Achiral Intersubunit Linkages. The
disclosure of which are incorporated herein by reference.
[0070] Representative internucleotide linkages that may be present
in the oligonucleotides include, but are not limited to,
phosphodiester, phosphorothioate, phosphoroselenoate,
phosphorodithioate, H-phosphonate, methyl phosphonate, alkyl
phosphonate and various alkyl amino groups including but not
limited to: CH.sub.2--NH--O--CH.sub.2,
CH.sub.2--N(CH.sub.3)--O--CH.sub.2,
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 and
O--N(CH.sub.3)--CH.sub.2- --CH.sub.2. These linkages may be between
the 5'-O nucleotide unit and any one of the 2'-, 3'-, or
4'-positions of another nucleotide unit. See generally, Sanghvi in
DNA with Altered Backbones in Antisense Applications in
Comprehensive Organic Natural Product Chemistry, Vol. 7, Elservier
Science Ltd., Oxford, 1998, which is hereby incorporated herein by
reference in its entirety.
[0071] Exemplary among these are the phosphorothioate and other
sulfur-containing species which are known for use in the art. In
accordance with some preferred embodiments, at least some of the
phosphodiester bonds of the oligonucleotide have been substituted
with a structure which functions to enhance the stability of the
oligonucleotide or the ability of the oligonucleotide to penetrate
into the region of cells where the viral RNA is located.
[0072] In accordance with other preferred embodiments, the
phosphodiester bonds are substituted with other structures which
are, at once, substantially non-ionic and non-chiral, or with
structures which are chiral and enantiomerically specific. Still
other linkages include the those disclosed in U.S. patent
applications Ser. No. 566,836, filed Aug. 13, 1990, entitled Novel
Nucleoside Analogs; Ser. No. 703,619, filed May 21, 1991, entitled
Backbone Modified Oligonucleotide Analogs; Ser. No. 903,160, filed
Jun. 24, 1992, entitled Heteroatomic Oligonucleoside Linkages;
Serial Number PCT/US92/04294, filed May 21, 1992, entitled Backbone
Modified Oligonucleotides; and Serial Number PCT/US92/04305, all
assigned to the assignee of this invention. Persons of ordinary
skill in the art will be able to select other linkages for use in
practice of the invention.
[0073] Oligonucleotides may also include species which include at
least some modified base forms. Thus, purines and pyrimidines other
than those normally found in nature may be so employed. For
example, deaza or aza purines and pyrimidines may be used in place
of naturally purine or pyrimidine bases and pyrimidine bases having
substitutent groups at the 5- or 6-positions; purine bases having
altered or replacement substituent groups at the 2-, 6- or
8-positions are also provided in some aspects of the present
invention.
[0074] Representative nucleobases that may be present in the
oligonucleotides used in the methods of the invention include,
adenine, guanine, cytosine, uridine, and thymine, as well as other
non-naturally occurring and natural nucleobases such as xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil,
8-halo, oxa, amino, thio, thioalkyl, hydroxyl and other
8-substituted adenines and guanines, 5-trifluoromethyl and other
5-substituted uracils and cytosines, and 7-methylguanine. Further
naturally and non-naturally occurring nucleobases include those
disclosed by Metrigan et al. in U.S. Pat. No., 3,687,808, by
Sanghvi, in Chapter 15, Antisense Research and Applications, Ed. S.
T. Crooke and B. Lebleu, CRC Press, Boca Raton, Fla., 1993, by
Englisch et al., Angewandte Chemie, Int. Ed., 1991, 30, 613, in The
Concise Encyclopedia of Polymer Science and Engineering, Ed. J. I.
Kroschwitz, John Wiley and Sons, 1990, pp. 858-859, and by Cook,
Anti-Cancer Drug Design, 1991, 6, 585. The disclosures of each of
the foregoing is incorporated by reference in their entirety. The
terms nucleosidic base and nucleobase are further intended to
include heterocyclic compounds that can serve as nucleosidic bases,
including certain universal bases that are not nucleosidic bases in
the most classical sense, but function similarly to nucleosidic
bases. One representative example of such a universal base is
3-nitropyrrole. For useful protecting groups for nucleobases see
Greene, T. W.; Wuts, P. G. M., Protective Groups in Organic
Synthesis, 2d ed., John Wiley & Sons, New York, 1991, which is
incorporated herein by reference in its entirety.
[0075] Oligonucleotides may also comprise other modifications
consistent with the spirit of this invention. Such oligonucleotides
are best described as being functionally interchangeable with yet
structurally distinct from natural oligonucleotides. All such
oligonucleotides are comprehended by this invention so long as they
effectively function as subunits in the oligonucleotide. Thus,
purine containing oligonucleotides are oligonucleotides comprising
at least one purine base or analog thereof. In other embodiments of
the present invention, compounds of the present invention may be
subunits of a species comprising two or more compounds of the
present invention which together form a single oligonucleotide.
[0076] Abasic Sites
[0077] In the context of the present invention an abasic site
refers to a nucleotide unit in an oligonucleotide in which the
purine or pyrimidine (nucleobase) group has been removed or
replaced by a hydroxyl group. One or more abasic sites may become
incorporated into one or more nucleotide bases of an
oligonucleotide. N-glycosidic bonds between a purine base and its
deoxyribose moiety are most susceptible to hydrolysis. Depurination
can occur spontaneously with a relatively high frequency under
physiological conditions (Lindahl, T., Prog. Nucleic Acid Res. Mol.
Biol., 1979, 22, 135 and Lindahl, T., Nature, 1993, 362, 709), and
is especially accelerated at low pH and high temperatures (Roger,
M.; Hotchkiss, R., Proc. Natl. Acad. Sci. USA, 1961, 47, 653).
[0078] Abasic sites are common lesions in DNA and are considered to
be important intermediates in mutagenesis and carcinogenesis (Loeb,
L. A.; Preston, B. D. 1986, Annu. Rev. Genet. 20, 201). In view of
the biological significance of abasic sites, a number of methods
have been developed to detect and quantitate abasic sites in DNA.
These include: assays utilizing alkali elution (Brent, T. P.;
Teebor, G. W.; Duker, N. J. in DNA Repair Mechanisms (Hanawalt, P.
C., Friedberg, E. C., Fox, C. F., Eds. 1978, Academic Press, New
York), DNA unwinding (Kohn, K. W.; Ewing, R. G.; Erikson, L. C.;
Zwelling, L. A. in DNA Repair: A Manual of Research Procedures
(Freidberg, E. C.; Hanawalt, P., Eds.) Vol. 1, Part B, 379, Marcel
Dekker, New York, Birnboim, H. C.; Jevcak, J. J. Cancer Research
1981, 41, 1889), .sup.32P-post-labeling (Weinfeld, M.; Liuzzi, M.;
Paterson, M. C. Biochemistry 1990, 29 1737), and, modification of
abasic sites by [.sup.14C]-methoxyamine (Talpaert-Borle, M.;
Liuzzi, M. Biochim. Biophys. Acta 1983, 740, 410, Liuzzi, M.;
Talpaert-Borle, M. Int. J. Radiat. Biol. 1988, 54, 709), or
O-4-(nitrobenzyl) hydroxylamine (NBHA) (Kow, Y. W. Biochemistry,
1989, 28, 3280, Chen, B. X.; Kubo, K.; Ide, H.; Erlanger, B. F.;
Wallace, S. S.; Kow, Y. W. Mutat. Res. 1992, 273, 253), or with
9-aminoellipticine (9-AE) (Bertrand, J. R.; Vasseur, J. J.; Rayner,
B.; Imbach, J. L.; Paoletti, J., Nucleic Acids Research, 1989, 17,
10307).
[0079] Kubo has reported an assay for the detection and
quantitation of abasic sites wherein the abasic site is modified by
a probe bearing a biotin residue, called the Aldehyde Reactive
Probe (ARP) and then the tagged biotin is quantified by an
ELISA-like assay (Kubo, K.; Ide, H.; Wallace, S. S.; Kow, Y. W.
Biochemistry, 1992, 31, 3703, Kubo, K.; Ide, H.; Akamatsu, K.;
Kimura, Y.; Michiue, K.; Makino, K.; Asaeda, a.; Takamori, Y.
Biochemistry, 1993, 32, 8276, Nakamura, J.; Walker, V. E.; Upton,
P. B.; Chiang, S. Y.; Kow, Y. W.; Swenberg, J. A. Cancer Research,
1998, 58, 222).
[0080] Imbach's group has studied the mechanism of conjugation of
several amines, such as 3-amino carbazole, 9-aminoellipticine, and
4'-aminomethyl-4,5',8-trimethyl-psoralen, to oligonucleotides via
chemically generated abasic sites (Vasseur, J. J.; Peoch, D.;
Rayner, B., Imbach, J. L., Nucleosides and Nucleotides, 1991, 10,
107).
[0081] These procedures, however, have been used to either detect
and quantify, or to study the mechanism for formation of abasic
sites in DNA or an oligonucleotide at the femtomole level. The
above procedures have not been shown to be amenable to purifying
DNA or oligonucleotides and their analogs, especially on a
multi-gram or multi-kilogram scale suitable for commercial
applications.
[0082] Two alternative pathways (S.sub.N1 or S.sub.N2 mechanism)
have been suggested for the hydrolysis of nucleosides. Many
experimental evidences support the S.sub.N1 mechanism as shown in
FIG. 1. Those include to lack of anomerization (Olivanen, M.;
Lonnberg, H., Tetrahedron, 1987, 43, 1133), linear pH-profiles and
entropies of activation near zero or positive (Zoltewicz, J. A.;
Clark, D. F.; Sharpless, T. W.; Grahe, G., J. Am. Chem. Soc., 1970,
92, 1741, Hevesi, L.; Wolfson-Daavidson, E.; Nagy, J. B.; Nagy, O.
B.; Bruylants, A., J. Am. Chem. Soc., 1972, 94, 4715 and Garrett,
E. R.; Mehta, P. J., J. Am. Chem. Soc., 1972, 94, 8532). In the
S.sub.N1 mechanism, the first step of the reaction is the
protonation of the heterocyclic base. The second step of the
S.sub.N1 mechanism involves the cleavage of the N-glycosidic bond
which is a slow reaction and likely to be the rate determining step
(Oivanen, M.; Lonnberg, H.; Zhou, X. X.; Chattopadhyaya, J.,
Tetrahedron, 1987, 43, 1133). The last step of the S.sub.N1
mechanism, is a fast reaction of the addition of water.
[0083] An abasic site is a mixture of four chemical species in a
tautomeric equilibrium as shown in FIG. 2 (Manoharan, M.; Ransom,
S. C.; Mazumder, A.; Gerlt, J. A., J. Am. Chem. Soc., 1988, 110,
1620). An abasic site can exist as: (A) an open chain aldehyde, (B)
a .beta.-hemiacetal, (C) an .alpha.-hemiacetal, or (D) an
open-chain hydrate. The open chain aldehyde structure constitutes
approximately 1% of the population of the abasic site (Manoharan,
M.; Ransom, S. C.; Mazumder, A.; Gerlt, J. A., Nucleosides and
Nucleotides, 1989, 8, 879). The ring opened aldehyde form of
deoxyribose is responsible for the reactivity of the abasic sites
even if the cyclic deoxyribose form is predominant (Takeshita, M.,
Chang, C. N.; Johnson, F.; Will, S.; Grollman, A. P., J. Biol.
Chem., 1987, 262, 10171, Vasseur, J. J.; Rayner, B.; Imbach, J. L.,
Biochem. Biophys. Res. Commun., 1986, 134, 1204). Thus, an "abasic
site" exists in an equilibrium which includes an open chain
aldehyde. As used herein, the term "aldehyde moiety" as applied to
the term "abasic site" is intended to denote the aldehyde group of
such open chain aldehyde.
[0084] It is well established that the deoxyribose fragment in its
open chain aldehyde form can undergo a base catalyzed
.beta.-elimination reaction leading to scission of the
phosphodiester backbone at the 3'-end of the abasic site as shown
in FIG. 3. The mechanism for cleavage of the 3'-phosphodiester bond
is thought to occur by abstraction of the acidic 2'-hydrogen
followed by the .beta.-elimination. An alternative postulated
mechanism of this breakage involves formation of an imine, which
facilitates abstraction of the 2'-deoxyribose proton leading to the
phosphate bond scission to give a 2',3'-ethylenic imine (Vasseur,
J. J.; Rayner, B.; Imbach, J. L.; Bertrand, J. R.; Malvy, C.;
Paoletti, C., Nucleosides & Nucleotides, 1989, 8, 863, Organic
Chemistry of Nucleic Acids, Eds. Kochetkov, N. K.; Budovskii, E.
I., Plenum Press, London-New York, 1972, Part B, Chapt. 10 (III),
Jones, A. S.; Mian, A. M.; Walker, R. T., J. Chem. Soc. (C), 1968,
2042).
[0085] Imine
[0086] Amino reagents, such as primary, secondary, and tertiary
amines can add to aldehydes and ketones to give different kinds of
products. (For a review of reactions of aldehydecompounds leading
to the formation of C.dbd.N bonds, see Dayagi; Degani, in Patai The
Chemistry of the Carbon-Nitrogen Double Bond; Ref. 40, pp.64-68;
Reeves, in Patai, Ref. 2, pp. 600-614). Primary amines react with
aldehydes to give imines. In contrast to imines in which the
nitrogen is attached to a hydrogen, (C.dbd.NH--H), substituted
imines are stable enough for isolation. The reaction of an aldehyde
or ketone with a primary amine (R--NH.sub.2) as shown below is the
best way to prepare them: 4
[0087] The above reaction is straightforward and proceeds in high
yields. The initial N-substituted hemiaminals lose water to give
the stable imine. These imines are usually called a Schiff base if
R is an aromatic group.
[0088] In accordance with the present invention, imines include any
moiety that contains a carbon-nitrogen double bond (C.dbd.N--),
such as imines (C.dbd.N--), oximes (C.dbd.N--O--), hydrazones
(C.dbd.N--NH--), semicarbazones (C.dbd.N--NH--C(O)NH--),
thiosemicarbazones (C.dbd.N--NH--C(S)NH--), azines
(C.dbd.N--N.dbd.C) and osazones [(C.dbd.N--).sub.2].
[0089] Oximes can be prepared by the addition of hydroxylamine to
aldehydes or ketones: 5
[0090] O-substituted oximes may be prepared in an analogous
reaction wherein the hydroxylamine is substituted, having the
formula NH.sub.2OR.
[0091] The product of condensation of a hydrazine (NH.sub.2NHR) and
an aldehyde or ketone is called a hydrazone: 6
[0092] Another hydrazine derivative frequently used to prepare the
corresponding hydrazone of an aldehyde or ketone is semicarbazide
(NH.sub.2NHC(O)NH.sub.2), or thiosemicarbazide
(NH.sub.2NHC(S)NH.sub.2) in which case the hydrazone is called a
semicarbazone or a thiosemicarbazone, respectively: 7
[0093] Hydrazine (NH.sub.2NH.sub.2) itself gives hydrazones only
with aryl ketones. With other aldehydes and ketones, either no
useful product can be isolated, or the remaining NH.sub.2 group
condenses with a second mole of aldehydecompound to give an azine:
8
[0094] .alpha.-Hydroxy aldehydes and ketones and
a-dialdehydecompounds give osazones, in which two adjacent carbons
have carbon-nitrogen double bonds: 9
[0095] Amine reagents in accordance with the present invention
include but are not limited to primary amines, hydroxylamines,
hydrazines, semicarbazides, and thiosemicarbazides.
[0096] The forgoing amino reagents can also react with the aldehyde
of an oligonucleotide abasic site to form the corresponding imine
as shown in FIG. 4. It should be noted that the above described
imines can be further modified, for example, by a reduction
sequence to give the corresponding saturated systems (--C.dbd.N--
to --CH--NH--) including but not limited to treatment of the imine
with hydrogen and a hydrogenation catalyst (For reviews, see
Rylander, Hydrogenation Methods; Academic Press: New York, 1985,
82), or a reductive amination sequence with sodium cyanoborohydride
(Tetrahedron Letters, 1994, 35, 2775), or sodium borohydride
(Schellenberg, J. Org. Chem., 1963, 28, 3259), or sodium
triacetoxyborohydride (Abdel-Magid; Maryanoff; Carson, Tetrahedron
Letters, 1990, 28, 3259), or zinc and hydrochloric acid (Borch;
Bernstein; Durst, J. Am. Chem. Soc., 1971, 93, 2897).
[0097] Purification
[0098] The combined aldehydic impurities from the abasic sites, the
.beta.-elimination reaction products and their degradation products
are major contaminants in the large scale production of
oligonucleotides. The feasibility of the large scale manufacture of
oligonucleotides requires the availability of suitable separation
and purification techniques. Some of the purification and
separation techniques that are well known in the art include
chromatography, extractions and precipitations.
[0099] Partitioning of a solute between two phases is the basis for
chromatographic separations and extractions. A partitioning is
based on the differing solubilities of components in a mixture. A
chromatography is a separations method that relies on differences
in partitioning behavior between a flowing mobile phase and a
stationary phase to separate the components in a mixture. A column
holds the stationary phase and the mobile phase carries the sample
through it. Sample components that partition strongly into the
stationary phase spend a greater amount of time in the column and
are separated from components that stay predominantly in the mobile
phase and pass through the column faster. As the components elute
from the column they can be quantified by a detector and/or
collected for further analysis. An analytical instrument can be
combined with a separation method for on-line analysis.. Examples
of such "hyphenated techniques" include gas and liquid
chromatography with mass spectrometry (GC-MS and LC-MS),
Fourier-transform infrared spectroscopy (GC-FTIR), and diode-array
UV-VIS absorption spectroscopy (HPLC-UV-VIS). Examples of
chromatographic techniques include: gas chromatography,
high-performance liquid chromatography (HPLC), liquid
chromatography (LC), size-exclusion chromatography (SEC) also
called gel-permeation chromatography (GPC), gel electrophoresis,
polyacrylamide gel electrophoresis, and thin-layer chromatography
(TLC).
[0100] An extraction is a technique that has been useful for
separations and purifications. An extraction uses two immiscible
phases to separate a solute from one phase into the other. The
distribution of a solute between two phases is an equilibrium
condition described by partition theory. A liquid-liquid extraction
is accomplished based on the different solubilities of components
in a mixture. Solvents that are immiscible with water are generally
less polar than water and tend to dissolve relatively non-polar
solutes to a greater extent. Conversely, relatively polar solutes
are more readily dissolved in water. The differences in
solubilities between non-polar and polar solutes allows for their
separation by a liquid-liquid extraction technique.
[0101] Precipitation techniques such as crystallization,
re-crystallization, and a titration, are used extensively in
organic chemistry to separate and purify compounds from a mixture.
These techniques allow for the selective precipitation of a
selected component compound from a mixture of compounds while in
solution. By taking advantage of the differing solubilities of a
compound in different solvents, a component compound can be
selected out of solution as a pure compound, as a solid
precipitate. Subsequent removal of the solvents gives the desired
purified solid compound.
[0102] The chromatographic, liquid-liquid extraction and
precipitation techniques, however, are not amenable to the
separation or purification of mixtures of oligonucleotides and
contaminant oligonucleotides having at least one abasic site due to
their similar polarities and thus, their similar solubilities.
[0103] In accordance with the preferred embodiments of the
invention, the attachment of a suitable moiety including but not
limited to non-ionic surfactants, solid phase polymeric supports,
and liquid-phase polymeric supports, to any of the previously
described amino groups (primary amines, hydroxylamines, hydrazines,
semicarbazides, and thiosemicarbazides), however, gives a useful
amino reagent for the purification of an oligonucleotide. These
non-ionic surfactant amino reagents and polymeric support amino
reagents are useful for purifying an oligonucleotide from a mixture
of the oligonucleotide and a contaminant oligonucleotide having at
least one abasic site.
[0104] The formation of imines between any of the amino reagents
and contaminant oligonucleotides having at least one abasic site
gives modified contaminant oligonucleotides, also defined
throughout the specification as imine-linked contaminants. The
modified contaminant oligonucleotides have a modified or different
solubility than the oligonucleotide to be purified. This difference
in solubility allows for the separation and purification of the
oligonucleotide from a mixture of the oligonucleotide and the
modified contaminant oligonucleotides.
[0105] According to one aspect of the present invention, the
modification of the solubility of the contaminant oligonucleotide
involves a change of state from a more soluble form to a less
soluble form in selected solvents such as water, aqueous solutions,
and aqueous buffers. Alternatively, the modification of the
solubility of the contaminant oligonucleotide involves a change of
state from a less soluble form to a more soluble form in selected
solvents such as methanol, ethanol, and isopropanol.
[0106] The difference in solubilities of the oligonucleotide to be
purified and the modified contaminant oligonucleotide in different
solvents gives a convenient and easy way to facilitate their
separation through chromatography or liquid-liquid extraction. A
chromatographic separation of the oligonucleotide to be purified
and the imine-linked contaminant relies on the differing
solubilities or partitioning behavior of these molecules. Some
representative chromatographic methods useful in the present
invention include gas chromatography, high-performance liquid
chromatography (HPLC), liquid chromatography (LC), flash column
silica gel chromatography, gel electrophoresis, polyacrylamide gel
electrophoresis, size-exclusion chromatography (SEC), also called
gel-permeation chromatography (GPC), thin-layer chromatography
(TLC), and others as described above. These chromatographic
techniques are referred to as a "chromatography" in the context of
the present invention.
[0107] In some preferred embodiments of the present invention, the
separation step comprises a liquid-liquid extraction wherein the
oligonucleotide to be purified has a greater solubility in a first
solvent than the imine-linked contaminants, and the imine-linked
contaminants have a greater solubility in a second solvent than the
oligonucleotide to be purified. Preferably, the first solvent
includes water and aqueous solutions, such as aqueous buffers.
Preferably, the second solvent is an organic solvent. More
preferably, the second solvent is benzene, diethyl ether, ethyl
acetate, hexanes, pentane, chloroform, dichloromethane or carbon
tetrachloride. Preferably, the first and second solvents are
immiscible.
[0108] In other preferred embodiments of the present invention, the
difference in solubilities of the oligonucleotide to be purified
and the modified contaminant oligonucleotide in different solvents
gives a convenient way to facilitate their separation by a
precipitation. A precipitation is useful when the oligonucleotide
to be purified has a greater solubility in a first solvent than the
imine-linked contaminants, and the imine-linked contaminants have a
greater solubility in a second solvent than the oligonucleotide to
be purified, wherein the first and second solvents are miscible.
Preferably, the first solvent is water or an aqueous solutions,
such as an aqueous buffer and the second solvent is an organic
solvent, such as acetone, methanol, ethanol or isopropanol.
[0109] It should be noted that the difference in solubilities
between the oligonucleotide to be purified and the imine-linked
contaminants may be one of degree. That is, the oligonucleotide to
be purified may be somewhat more or less soluble in a selected
solvent than the modified contaminant oligonucleotide, and the
modified contaminant oligonucleotide may be more or less soluble in
a selected solvent than the oligonucleotide.
[0110] In some preferred embodiments, the amino reagents used to
form the imines with the contaminant oligonucleotides include
surfactants. Non-ionic surfactants are preferred. Especially
preferred are amino reagents including surfactants of the following
formula: 10
[0111] wherein:
[0112] x is from 0 to 20,
[0113] y is from 0 to 5,
[0114] n is from 0 to 150; and
[0115] Z is a reactive nitrogenous moiety capable of reacting with
an aldehyde to form an amine.
[0116] In preferred embodiments, the separation of the contaminants
is accomplished by either a liquid-liquid extraction with a
suitably selected solvent such as those described herein or by the
selective precipitation of the oligonucleotide to be purified by
the addition of a suitably selected solvent, such as those
described herein.
[0117] In other preferred embodiments, the amino reagents used to
form the imines with the contaminant oligonucleotides comprise
solid phase polymeric supports. Thus, upon imine formation, the
modified contaminants are bound to these supports. The separation
is preferably accomplished by a wash or a rinse of the insoluble
solid phase support with a suitably selected solvent, such as those
described herein. These separation techniques are well known to
those of skill in the art.
[0118] In still other preferred embodiments, the amino reagents
used to form the imines with the contaminant oligonucleotides
comprise liquid-phase polymeric supports. Thus, upon imine
formation, the modified contaminants are bound to these supports.
In one aspect of the invention, the separation is preferably
accomplished by either a liquid-liquid extraction with a suitably
selected solvent such as those described herein, or by a selective
precipitation of the oligonucleotide by the addition of a suitably
selected solvent such as those described herein. Both of these
methods are well known in the art.
[0119] Surfactants
[0120] Molecules or ions which are amphiphilic, that is which
contain both a hydrophobic (non-polar) and a hydrophilic (polar)
part, in aqueous solution frequently assemble at interfaces. This
property has given them the name surface-active agents or
"surfactants". A surfactant is capable of reducing the surface
tension of a liquid in which it is dissolved.
[0121] Generally, a surfactant is composed of a hydrophobic
non-phobic tail group and a hydrophilic polar head group. The
hydrophobic tail can be aliphatic, alicyclic, aryl or aromatic, or
a mixture thereof. The hydrophobic tail is preferably a hydrocarbyl
group as described below.
[0122] Hydrocarbyl groups of the invention include aliphatic,
alicyclic and aryl groups. These groups can be combined together or
substituted to give various combinations of aliphatic, alicyclic
and aryl groups.
[0123] Aliphatic and alicyclic groups suitable for use in the
invention include saturated and unsaturated, straight and branch
chain and alicyclic, alkyl, alkenyl and alkynyl groups including
methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl and other
higher carbon straight-chain alkyl groups; 2-methylpropyl,
2-methyl-4-ethylbutyl, 2,4-diethylpropyl, 3-propylbutyl,
2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6-butyloctyl,
2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2-ethylhexyl and
other branched-chain groups; vinyl, allyl, crotyl, propargyl,
2-pentenyl and other unsaturated groups; and cyclohexane,
cyclopentane, adamantane as well as other alicyclic groups.
[0124] Aryl groups suitable for use in the invention include
phenyl, tolyl, benzyl, naphthyl, anthracyl, phenanthryl, xylyl and
other aromatic groups.
[0125] The four major classifications of surfactants are anionic,
cationic, zwitterionic, and non-ionic. Surfactants are grouped into
one of these categories depending on the nature of the hydrophillic
head group. Anionic surfactants are water soluble and have a
negative charge in aqueous solution, as in sodium dodecyl sulfate
(CH.sub.3(CH.sub.2).sub- .11 SO.sub.4.sup.-Na.sup.+). Cationic
surfactants have a positive charge in aqueous solution, as in
dodecylamine hydrochloride (CH.sub.3(CH.sub.2).sub.11.sup.+NH.sub.3
Cl.sup.-). Zwitterionic surfactants have two ionogenic groups
producing a cation and an anion. Zwitterionic surfactants can be
ampholytic and can behave as either a cationic, anionic, or
non-ionic species depending on the pH of the solution, as in
N-dodecyl-N,N-dimethyl betaine, (C.sub.12H.sub.25N.sup.+(-
CH.sub.3).sub.2CH.sub.2COO.sup.-). Non-ionic surfactants have at
least one uncharged head group which is polar in nature. In some
preferred embodiments they are prepared by attaching repeating
units of ethylene oxide to a water insoluble molecule to form a
polyoxyethylene chain. The resulting polyethylene glycol (PEG)
units attached to the terminal primary alcohol are polar and
hydrophillic (water soluble) while the hydrocarbon derived
non-polar groups are hydrophobic (water insoluble), as in
polyethyleneglycol mono[4-(1,1,3,3-tetra-methylbutyl)phenyl]ether
(CH.sub.3C(CH.sub.3).sub.2CH.sub.2C(CH.sub.3).sub.2C.sub.6H.sub.4OCH.sub.-
2CH.sub.2OH). This non-ionic surfactant is also available
commercially under the name TritonX-100.TM. (Union Carbide). In
these non-ionic surfactants, the head group is usually larger than
the hydrocarbon tail. Non-ionic surfactants with small head groups
also exist, such as dodecyl sulfinyl ethanol
(C.sub.12H.sub.25SOCH.sub.2CH.sub.2OH)
[0126] In each case, the hydrophilic head group of a surfactant is
strongly attracted to the water molecules due to hydrogen bonding,
whereas the force of attraction between the hydrophobic tail group
and water is only slight. This is due to the strong interactions
between the water molecules, arising from dispersion forces, and
hydrogen bonding acting cooperatively to squeeze the hydrocarbon
tail out of the water. As a result, the surfactant molecules align
themselves at the surface and internally so that the hydrophilic
end is toward the water and the hydrophobic end is squeezed away
from the water.
[0127] Besides assembly at interfaces, surfactants can undergo a
process of self-assembly which is more commonly known as
micellization. The formation of micelles is another way by which
surfactants can sequester their non-polar part from contact with
the aqueous phase and thus reduce the free energy of surfactant
systems.
[0128] The polar head groups of a non-ionic surfactant are based on
repeating polyethylene glycol units (PEG) attached to a primary
alcohol. The alcohol is a polar group which forms hydrogen bonds
with water. Other groups that are uncharged, are polar in nature,
and can hydrogen bond with water can be substituted for the
alcohol. These groups, which include the previously described amino
groups (primary amines, hydroxylamines, hydrazines, semicarbazides
and thiosemicarbazides), when substituted for the alcohol give a
non-ionic surfactant amino reagent.
[0129] Due to their polar nature, the oligonucleotide and the
contaminant oligonucleotide are usually soluble in water, including
aqueous solutions and aqueous buffers. The addition of a non-ionic
surfactant amino reagent to an aqueous solution or aqueous buffer
which contains an oligonucleotide and a contaminant gives a
modified contaminant having an imine linkage. The contaminant is
modified so that it has a different solubility than the
oligonucleotide in a selected solvent. The non-polar hydrocarbyl
tail of the non-ionic surfactant now incorporated into the
contaminant as its imine, modifies the solubility of the
contaminant oligonucleotide. The presence of the non-ionic
surfactant bound imine or the modified contaminant allows for the
separation of the oligonucleotide from the modified contaminent
based on their differing solubilities. This separation is
accomplished by either a liquid-liquid extraction of the modified
contaminant oligonucleotide by the addition of a selected solvent,
or by the selective precipitation of the oligonucleotide by the
addition of a selected solvent.
[0130] In some preferred embodiments, a liquid-liquid extraction is
used to separate and purify the desired oligonucleotide from the
modified contaminant oligonucleotide. The separation is
accomplished by the addition of a selected solvent to an aqueous
solution or aqueous buffer solution which contains the
oligonucleotide and the modified contaminant oligonucleotide bound
to the amino non-ionic surfactant reagent. In preferred embodiments
the selected solvents are immiscible with water, and include
benzene, diethyl ether, ethyl acetate, hexane, pentane, petroleum
ether, toluene, choroform, dichloromethane, and carbon
tetrachloride. The liquid-liquid extraction thus gives an aqueous
layer which contains mostly the oligonucleotide to be purified and
an organic layer which contains mostly the modified contaminant
oligonucleotide and any excess amino non-ionic surfactant
reagent.
[0131] An oligonucleotide also can be purified from the non-ionic
surfactant bound modified contaminant oligonucleotide by a
precipitation. In some preferred embodiments, the oligonucleotide,
the modified contaminant oligonucleotide, and the surfactant amino
reagent are soluble in aqueous solutions and aqueous buffers. In
addition, the modified contaminant oligonucleotide is soluble in
water miscible solvents including acetone, methanol, ethanol, and
isopropanol. Oligonucleotides, however, are mostly insoluble in
these solvents.
[0132] For example, in some embodiments, treatment of an aqueous
solution or aqueous buffer which contains the oligonucleotide to be
purified and the imine-linked contaminants, wherein the imines are
bound to a non-ionic surfactant, with a miscible solvent, for
example, ethanol, and cooling to a temperature of from about
-40.degree. C. to 10.degree. C., preferably to a temperature of
about -20.degree. C. to 0.degree. C., and most preferably to about
-20.degree. C. causes the oligonucleotide to be purified to
precipitate from the solution. The imine-linked contaminants stay
soluble in the ethanol-water solution. Centrifugation or filtration
of the solution, removal of the solvent which contains the modified
contaminant oligonucleotides and a wash or rinse of the solids with
ethanol, gives a purified solid oligonucleotide.
[0133] In preferred embodiments of the present invention, non-ionic
surfactants available under the trademark IGEPAL.TM. are employed.
IGEPAL.TM. is a non-ionic surfactant that is inexpensive and
commercially available from Aldrich Chemical Company (Milwaukee,
Wis.). IGEPALS.TM. have the general formula III: 11
[0134] wherein:
[0135] x is 7 to 8;
[0136] y is 1 to 2; and
[0137] n is 0 to 150.
[0138] In some preferred embodiments, a plurality of these
compounds are employed, wherein the value of n varies from about 0
to about 150, and is preferably from about 0 to about 16, even more
preferably from about 8 to about 16. In some embodiments, n is 12.
The surfactants are reacted with nitrogenous reagents to form
surfactant-linked amino reagents, useful for the methods of the
present invention.
[0139] Other non-ionic surfactants may have other hydrophobic tails
which are selected from hydrocarbyl groups such as aliphatic,
alicyclic and aryl groups, as were previously described. For
example, a preferred embodiment of the invention is directed to
branched side chain hydrophobic tails.
[0140] It should be noted that polymers exist as a distribution of
molecular weights, however, the polydispersity of commercial PEG's
is quite narrow (Harris, J. M., In Poly(Ethylene Glycol) Chemistry:
Biotechnical and Biomedical Applications; Harris, J. M., Ed.;
Plenum Press: New York, 1992, p2). In the context of the present
invention, n is a whole number which represents that polymers exist
as a distribution of molecular weights as is defined above.
[0141] A list of commercially available (Stepan) IGEPALS.TM.
include:
1 IGEPAL .TM. CA-210 OCTYL PHENOL 1.5 MOLE ETHOXYLATE IGEPAL .TM.
CA-520 OCTYL PHENOL 5 MOLE ETHOXYLATE IGEPAL .TM. CA-620 OCTYL
PHENOL 7 MOLE ETHOXYLATE IGEPAL .TM. CA-630 OCTYL PHENOL 9 MOLE
ETHOXYLATE IGEPAL .TM. CA-720 OCTYL PHENOL 12 MOLE ETHOXYLATE
IGEPAL .TM. CA-880 OCTYL PHENOL 12 MOLE ETHOXYLATE IGEPAL .TM.
CA-887 OCTYL PHENOL 30 MOLE ETHOXYLATE IGEPAL .TM. CA-890 OCTYL
PHENOL 40 MOLE ETHOXYLATE IGEPAL .TM. CA-897 OCTYL PHENOL 40 MOLE
ETHOXYLATE IGEPAL .TM. CO-210 NONYL PHENOL 1.5 MOLE ETHOXYLATE
IGEPAL .TM. CO-430 NONYL PHENOL 4 MOLE ETHOXYLATE IGEPAL .TM.
CO-530 NONYL PHENOL 6 MOLE ETHOXYLATE IGEPAL .TM. CO-580 NONYL
PHENOL 7 MOLE ETHOXYLATE IGEPAL .TM. CO-610 NONYL PHENOL 8.5 MOLE
ETHOXYLATE IGEPAL .TM. CO-620 NONYL PHENOL 8 MOLE ETHOXYLATE IGEPAL
.TM. CO-630 NONYL PHENOL 9 MOLE ETHOXYLATE IGEPAL .TM. CO-660 NONYL
PHENOL 10 MOLE ETHOXYLATE IGEPAL .TM. CO-670 NONYL PHENOL 10.2 MOLE
ETHOXYLATE IGEPAL .TM. CO-680 NONYL PHENOL 10 MOLE ETHOXYLATE
IGEPAL .TM. CO-710 NONYL PHENOL 10.5 MOLE ETHOXYLATE IGEPAL .TM.
CO-720 NONYL PHENOL 12 MOLE ETHOXYLATE IGEPAL .TM. CO-730 NONYL
PHENOL 15 MOLE ETHOXYLATE IGEPAL .TM. CO-738 NONYL PHENOL 15 MOLE
ETHOXYLATE IGEPAL .TM. CO-850 NONYL PHENOL 20 MOLE ETHOXYLATE
IGEPAL .TM. CO-858 NONYL PHENOL 20 MOLE ETHOXYLATE IGEPAL .TM.
CO-880 NONYL PHENOL 30 MOLE ETHOXYLATE IGEPAL .TM. CO-887 NONYL
PHENOL 30 MOLE ETHOXYLATE IGEPAL .TM. CO-890 NONYL PHENOL 40 MOLE
ETHOXYLATE IGEPAL .TM. CO-897 NONYL PHENOL 40 MOLE ETHOXYLATE
IGEPAL .TM. CO-897 NONYL PHENOL 40 MOLE ETHOXYLATE IGEPAL .TM.
CO-970 NONYL PHENOL 50 MOLE ETHOXYLATE IGEPAL .TM. CO-977 NONYL
PHENOL 50 MOLE ETHOXYLATE IGEPAL .TM. CO-990 NONYL PHENOL 100 MOLE
ETHOXYLATE IGEPAL .TM. CO-997 NONYL PHENOL 100 MOLE ETHOXYLATE
IGEPAL .TM. NP-100 NONYL PHENOL.
[0142] Other non-ionic surfactants that include a non-polar
(hydrophobic) hydrocarbyl tail and a polar (hydrophilic) PEG head
group are within the scope of the invention and are useful in other
embodiments of the invention.
[0143] The alcohol of a non-ionic surfactant can be converted into
any of the previously listed amino groups (e.g., primary amines,
hydroxylamines, hydrazines, semicarbazides and thiosemicarbazides),
with procedures that are well known in the art. For example,
[0144] 1. The conversion of an alcohol to an amine is accomplished
by treatment of the alcohol with hydrazoic acid (HN.sub.3),
diisopropyl azodicarboxylate (i-Pr-OOCN.dbd.NCOO-i-Pr), and excess
triphenylphosphine (Ph.sub.3P) in tetrahydrofuran, followed by the
addition of water or aqueous acid (Fabiano; Golding; Sadeghi,
Synthesis, 1987, 190).
[0145] 2. The conversion of an alcohol to a hydroxylamine is
accomplished by treatment of the alcohol with triphenylphosphine
(PPh.sub.3), N-hydroxphthalimide, diethyl azodicarboxylate (DEAD)
(Et-OOCN.dbd.NCOO-Et) (3 eq each) in tetrahydrofuran, followed by
the addition of hydrazine in tetrahydrofuran-ethanol (Floyd, C. D.;
Lewis, C. N.; Patel, S. R.; Whittaker, M., Tetrahedron Letters,
1996, 37, 8045).
[0146] 3. The conversion of an alcohol to a hydrazine is
accomplished by treatment of the alcohol with mesyl chloride and
triethylamine in dichloromethane.sub.2, followed by the addition of
hydrazine hydrate (4 eq) in ethanol at 0.degree. C. (Yaun, C.; Li,
C., Synthesis, 1995, 4, 507).
[0147] 4. The conversion of an alcohol to a semicarbazide is
accomplished by converting the alcohol into the amine as described
above (1) and treating the amine with ethyl chloroformate and
pyridine, followed by the addition of hydrazine in ethanol (Indian
J. Chem., 1985, Sect. B, 24B (11), 1115).
[0148] 5. The conversion of an alcohol to a thiosemicarbazide is
accomplished by treatment of the above described semicarbazide with
2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide
(Lawesson's reagent) (Cava; Levinson, Tetrahedron, 1985, 41,
5061-5087).
[0149] Preferred embodiments of the invention are directed to
IGEPALS.TM. that can be transformed into the corresponding
O-IGEPAL.TM. amine, O-IGEPAL.TM. hydroxylamine, O-IGEPAL.TM.
hydrazine, O-IGEPAL.TM. semicarbazide, or O-IGEPAL.TM.
thiosemicarbazide derivatives by procedures as described above.
These are represented by the following formulas: 12
[0150] A preferred embodiment of the invention is directed to
IGEPAL.TM. that is transformed into the corresponding O-IGEPAL.TM.
hydroxylamine derivative as described above and in Example 1
(Floyd, C. D.; Lewis, C. N.; Patel, S. R.; Whittaker, M.,
Tetrahedron Letters, 1996, 37, 8045, herein incorporated by
reference). This procedure gives an O-IGEPAL.TM. hydroxylamine of
the formula: 13
[0151] wherein:
[0152] x is 7 to 8;
[0153] y is 1 to 2; and
[0154] n is 0 to 150.
[0155] A preferred embodiment of the invention is the O-IGEPAL.TM.
CO-720 hydroxylamine of the formula: 14
[0156] (X)
[0157] wherein:
[0158] x is 8;
[0159] y is 1;
[0160] n is from about 0 to about 150.
[0161] In especially preferred embodiments of the invention, x is
8, y is 1, n is from about 8 to 16, and
[CH.sub.3(CH.sub.2).sub.x].sub.y is para to
(OCH.sub.2CH.sub.2).sub.n--ONH.sub.2. In more preferred
embodiments, n is about 12. The compound of the formula X is
utilized to purify an oligonucleotide as is described in Example
8.
[0162] The purification of an oligonucleotide that is contaminated
with an oligonucleotide having at least one abasic site with
IGEPAL.TM. CO-720 hydroxylamine is described in Examples 8 through
15.
[0163] The mass spectra of phosphorothioate
oligodeoxyribonucleotide, PS-d(GCCCAAGCTGGCATCCGTCA) that is
contaminated with an oligonucleotide having an abasic site is shown
in FIG. 5. The oligonucleotide has an m/z value of 2121.4
(m=6,364.2 and z=3). Two abasic contaminants are present in the
mixture as is shown from the spectral peaks at m/z values of 2082.4
(loss of adenine) and 2071.4 (loss of guanine). Several bis abasic
contaminants are also present in the mixture as is shown from the
spectral peaks at m/z values of 2043.6 (loss of two adenines),
2038.2 (loss of one adenine and one guanine), and 2032.2 (loss of
two guanines).
[0164] As is described in Example 8, the mixture of the
oligonucleotide and the contaminant oligonucleotide that has the
above described abasic sites, are treated with IGEPAL.TM. CO-720
hydroxylamine in a sodium phosphate buffer for 12 to 24 hours. The
oligonucleotide to be purified is precipitated from the solution by
the addition of ethanol and cooling to about -20.degree. C. The
precipitated solid oligonucleotide is isolated by centrifugation
(or filtration) and removal of the solvents. The solid
oligonucleotide is dissolved in sodium acetate buffer and is
subjected to mass spectral analysis.
[0165] As is shown in FIG. 6, the two abasic sites at the m/z
values of 2082.4 and 2017.4, and the three bis abasic sites for the
m/z values of 2043.6, 2038.2, and 2032.2 are either totally absent
or are greatly diminished after treatment of the oligonucleotide
mixture with IGEPAL.TM. CO-720 hydroxylamine.
[0166] Polymeric Supports
[0167] The use of polymeric supports or resins, have advanced the
areas of both peptide synthesis and more recently combinatorial
synthesis. Polymeric supports can be divided into two classes of
insoluble solid phase polymeric supports, and soluble liquid-phase
polymeric supports.
[0168] Solid Phase Supports
[0169] Since the introduction of the Merrifield method for peptide
synthesis (Merrifield, R. B., J. Am. Chem. Soc., 1963, 85, 2149),
solid phase supports have been incorporated into numerous synthetic
methodologies to facilitate synthesis and product purification
(Gallop, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.;
Gordon, E. M., J. Med. Chem., 1994, 37, 1233, Gold, L.; Polisky,
B.; Uhlenbeck, O.; Yarus, M., Annu. Rev. Biochem., 1995, 64, 763,
Thompson, L. A.; Ellman, J. A., Chem. Rev., 1996, 96, 555). The
majority of peptide and combinatorial libraries to date have been
synthesized on solid phase supports. An advantage of solid phase
support synthesis is the ease of purification. The isolation of the
support bound reaction products is accomplished simply by washing
or rinsing away any excess reagents from the support bound
material. Subsequent cleavage of the support bound materials gives
compounds that are of high purity.
[0170] Polystyrene is the most common core resin used in solid
phase supports, but other core matrices include polyacrylate,
polyethylene glycol, and polyacrylamide. The two most important
factors in solid phase organic synthesis is the swelling factor and
the bead size of the resin. The swelling characteristics are
affected by factors such as the degree of crosslinking,
hydrophobicity of the substrate, and the nature of the core matrix
itself. The swelling factor is important in that the reaction
kinetics in solid phase organic synthesis are diffusion controlled.
The resin that swells more will have a higher diffusion rate of
substrate into the core of the matrix resulting in shorter reaction
times, and more complete chemical conversions.
[0171] Polystyrene is very hydrophobic and swells in relatively
non-polar solvents such as tetrahydrofuran, toluene, and
dichloromethane, and shrinks in polar solvents such as methanol and
water (aqueous solutions and aqueous buffers). Polystyrene that is
crosslinked with 1% to 2% divinylbenzene provides a material that
retains it shape (generally in the form of a bead,.a disk or a thin
film), has good mechanical resistance to breakage, and has a
mechanically stable gel-like consistency when in the presence of
solvents. The swelling factor of 1% crosslinked polystyrene in
various solvents (mL/g of resin) is given in Table 1.
(NOVABIOCHEM.TM., The Combinatorial Chemistry Catalog & Solid
Phase Organic Synthesis Handbook, 1999, S8, Christensen, J. W.,
Advanced Chemtech Handbook of Combinatorial & Solid Phase
Organic Chemistry, A Guide to Principles, Products & Protocols,
1998, 99). It should be kept in mind, however, that a resin which
is substituted with a particular substrate may depart from this
first order estimation depending on the nature of solvent, degree
of substitution and chemical nature of the substrate.
2 TABLE 1 (mL/g of resin) Solvent Polystyrene TG PEGA
Tetrahydrofuran (THF) 8 6 13 Toluene 7 5 12 Dichloromethane (DCM) 7
5 13 Dimethylformamide (DMF) 3 5 11 Methanol (MeOH) 2 4 13 Water 1
4 16
[0172] Another important factor to consider in solid phase
chemistry is that of bead size. Polystyrene beads are available in
diameter sizes ranging from less than a micron to 750 microns. Bead
size is commonly reported in Tyler Mesh size which is inversely
proportional to the nominal diameter. The two most commonly used
resin sizes are 100-200 and 200-400 mesh (75-100 micron and 35-75
micron respectively). Reaction kinetics are generally faster using
smaller beads due to the higher surface area to volume ratio.
However, to small a bead can lead to extended filtration times. The
range of 100-200 mesh offers the best balance of reaction kinetics
versus reliability.
[0173] Scavenger resins have become useful for the workup of
solution phase reactions to remove excess reagents, substrates or
byproducts. A simple wash, rinse or a filtration of the resin
allows for a simple purification of the reaction products. For
example, nucleophilic scavenger resins such as aminomethyl
polystyrene resin, N-(2-aminoethyl)aminomethyl polystyrene resin,
and tris(2-aminoethyl)amine polystyrene resin
(bis(2-aminoethyl)-2-aminoethyl- benzyl amine polystyrene resin)
have been used to remove excess reagents such as isocyanates, acid
chlorides, alkyl chloroformates and sulfonyl chlorides from
solution phase chemical libraries. (Flynn, D. L.; Crich, J. Z.;
Devraj, R. V.; Hockerman, S. L.; Parlow, J. J.; South, M. S.;
Woodard, S., J. Am. Chem. Soc., 1997, 119, 4874, Booth, R. J.;
Hodges, J. C., J. Am. Chem. Soc., 1997, 119, 4882). In addition,
the triethylamino analog of Merrifield's resin (polyamine resin)
has been used to sequester any excess unreacted aldehyde by
removing it as a resin bound imine adduct (Flynn, D. L.; Crich, J.
Z.; Devraj, R. V.; Hockerman, S. L.; Parlow, J. J.; South, M. S.;
Woodard, S., J. Am. Chem. Soc., 1997, 119, 4874).
[0174] These solid phase scavenger resins have a hydrophobic
polystyrene core that is crosslinked with 1% divinylbenzene. These
resins swell in non-polar solvents like tetrahydrofuran, toluene
and dichloromethane, but shrink in polar solvents such as methanol
and water, aqueous solutions and aqueous buffer solutions. Due to
their polar nature, the oligonucleotides to be purified and the
contaminant oligonucleotides having at least one abasic site are
hydrophilic and are soluble in water, aqueous solutions, and
aqueous buffer solutions. They are far less soluble to insoluble in
non-polar solvents like tetrahydrofuran, toluene and
dichloromethane. Thus, due to the inherent differences in their
solubilities, the above described scavenger resins are not amenable
to the purification of mixtures of oligonucleotides and a
contaminant oligonucleotides having at least one abasic site.
[0175] Hydrophobic polystyrene supports do not swell well in polar
solvents such as methanol and water. Furthermore, the hydrophobic
environment of the polymer matrix repels charged ionic species,
which is a particular problem associated with an oligonucleotide
synthesis. A variety of commercially available resins have been
developed to overcome the problems associated with polystyrene
resins. These include TentaGel.TM. (TG) resins, (available from
Advanced ChemTech, Louisville, Ky.) and PEGA resins (Table 1).
These resins utilize either a polyethylene glycol (PEG) chain (TG
resin) or a polyacrylamide chain or a combination thereof (PEGA
resin), that is grafted onto the polystyrene core. These resins
include PEG polystyrene based resins such as NovaSyn.TM. TG resins
(Calbiochem-Novabiochem Corp, San Diego, Calif.) and TentaGel.TM.
resins (Advanced ChemTech) as well as several PEG polyacrylamide
based resins such as NovaSyn.TM. P500 resins, NovaSyn.TM. K125
resins, PEGA resins, and NovaGel.TM. resins (all
Calbiochem-Novabiochem Corp), and the polyacrylamide SPAR.TM.-50
resins (Advanced ChemTech).
[0176] The above mentioned PEG polystyrene and polyacrylamide
resins are soluble in polar solvents such as methanol and water,
due to the hydrophilic character of the PEG chain and the amide
functionalities that have been grafted onto the polystyrene core.
The resulting resins have a hydrophobic as well as a hydrophilic
character and swell well in polar solvents such as methanol and
water (Bayer, E., Angew. Chem. Int. Ed. Engl., 1991, 30, 113,
Hutchins, S. M.; Chapman, K. T., Tetrahedron Letters, 1994, 35,
4055, Adams, J., et al, J. Org. Chem., 1998, 63, 3706, Meldal, M.,
Tetrahedron Letters, 1992, 33, 3077).
[0177] The nucleophilic amine forms of the above mentioned resins
include NovaSyn.TM. TG amino resin (Bayer, E., Angew. Chem. Int.
Ed. Engl., 1991, 30, 113), Aminomethyl NovaGel.TM. (Adams, J. H.,
et al, J. Org. Chem., 1998, 63, 3706), NovaSyn.TM. TGR resin, Rink
amide NovGel.TM. ((Rink, H., Tetrahedron Letters, 1987, 28, 3782,
Bernatowicz, M. S.; et al, Tetrahedron Letters, 1989, 30, 4645,
Story, S. C., et al, Int. J. Peptide Protein Res., 1992, 39, 87,
Albericio, F.; et al, J. Org. Chem., 1990, 55, 3730), NovaSyn.TM.
TG Sieber resin (Sieber, P., Tetrahedron Letters, 1987, 28,
2107).
[0178] Other nucleophilic amine forms of the above mentioned resins
include TentaGel.TM. S NH.sub.2 resin (Svensson, A.; Fex, T.;
Kihlberg, J., Tetrahedron Letters, 1996, 37, 7649, Sucholeiki, I.,
Tetrahedron Letters, 1994, 35, 73207, Johnson, C. R.; Zhang, B.,
Tetrahedron Letters, 1995, 36, 9253), TentaGel.TM. S RAM Fmoc resin
(Larhed, M.; Lindeberg, G.; Hallberg, A., Tetrahedron Letters,
1996, 37, 8219, Virgilio, A. A.; Ellman, J., J. Am. Chem. Soc.,
1994, 116, 11580), and TentaGel.TM. S AM.
[0179] SPAR.TM.-50 resin is a polyacrylamide resin that swells in
polar protic solvents such as methanol and water (Sparrow, J. T.,
et al, Peptide Research, 1996, 9, 297, Kanda, P., et al, Intl. J.
Peptide Prot. Res., 1991, 38, 385, and is described in one or more
of the following U.S. Pat. Nos. 4,973,638, 5,028,675, 5,084,509,
5,126,399, 5,296,572, 5,512,648, the disclosures of which are
herein incorporated by reference). Other commercially available
analogs of SPAR-50 include the benzylic alcohol HMBA SPAR.TM.-50
resin and the Fmoc protected Rink-SPAR-50 resin,
Fmoc-Phe-HMBA-SPAR.TM.-50 resin, and the Fmoc-Gly-HMBA-SPAR.TM.-50
resin.
[0180] The nucleophilic hydroxyl forms of the above mentioned
resins include NovaSyn.TM. TG hydroxy resin (Bayer, E., Angew.
Chem. Int. Ed. Engl., 1991, 30, 113), NovaSyn.TM. TG HMP resin,
NovaSyn.TM. TGA resin, HMPA NovaGel.TM. and HMPA-PEGA resin
(Sieber, P, Tetrahedron Letters, 1987, 28, 6147), and NovaSyn.TM.
TG HMBA resin and HMBA-PEGA resin (Atherton, E.; Sheppard, R. C.,
Solid Phase Peptide Synthesis, A Practical Approach, IRL Press,
Oxford, 1989, 512). Other nucleophilic hydroxyl resins include
TentaGel.TM. S OH resin (Kocis, P., et al., Tetrahedron Letters,
1995, 36, 6623, Hauske, J. R., et al., Tetrahedron Letters, 1995,
36, 1589), TentaGel.TM. PAP resin, TentaGel.TM. S PHB resin,
TentaGel.TM. S AC resin (Ngu, K.; Patel, D. V., Tetrahedron
Letters, 1997, 38, 973), TentaGel.TM., and TentaGel.TM. S HMB resin
(Cheng, Y.; Chapman, K. T., Tetrahedron Letters, 1997, 38,
1497).
[0181] The hydroxyl forms of the above mentioned resins can be
converted into any of the previously listed amino groups (primary
amines, hydroxylamines, hydrazines, semicarbazides and
thiosemicarbazides), with procedures that are known in the art of
organic chemistry, as was previously described. These procedures
give other preferred embodiments of the invention. These
embodiments include but any of these solid phase polymeric support
amino reagents. Other embodiments of the invention include but are
not limited to PEG polystyrene based resins such as NovaSyn.TM. TG
resins, TentaGel.TM. resins, and PEG polyacrylamide based resins
such as NovaSyn.TM. P500 resins, NovaSyn.TM. K125 resins, PEGA
resins, NovaGel.TM. resins, and the polyacrylamide SPAR.TM.-50
resins, and others that are functionalized with any of the
previously described amino groups (primary amine, hydrazine,
hydroxylamine, semicarbazide, thiosemicarbazide).
[0182] As is shown in Example 2, NovaSyn TG.TM. hydroxy resin is
converted into NovaSyn.TM. TG hydroxylamine resin by treatment of
the alcohol with triphenylphosphine (PPh.sub.3),
N-hydroxphthalimide, diethylazodicarboxylate (DEAD,
Et-OOCN.dbd.NCOO-Et) (3 eq each) in tetrahydrofuran, followed by
hydrazine in tetrahydrofuran-ethanol (Floyd, C. D.; Lewis, C. N.;
Patel, S. R.; Whittaker, M., Tetrahedron Letters, 1996, 37,
8045).
[0183] NovaSyn TG.TM. hydroxylamine resin swells in a polar solvent
such as water, aqueous solutions or aqueous buffer solutions, and
reacts with an abasic site of a contaminant oligonucleotide to form
a resin bound imine. The purification of a mixture of an
oligonucleotide that is contaminated with an oligonucleotide that
has at least one abasic site, with NovaSyn TG.TM. hydroxyamine
resin is described Examples 16 through 23. Imine formation, removal
of the solvents, and a wash or rinse of the resin with water and
ethanol gives a purified oligonucleotide.
[0184] Liquid Phase Supports
[0185] Liquid phase supports are polymers that dissolve in the
reaction solvent. The term liquid phase synthesis was first used to
contrast the differences between solid phase peptide synthesis and
a method of synthesis on soluble polyethylene glycol (Mutter, M.;
Hagenmaier, H.; Bayer, E., Angew. Chem., Int. Ed. Engl., 1971, 10,
811, Bayer, E.; Mutter, M., Nature (London), 1972, 237, 512). The
advantage of liquid phase synthesis is that the solubilities of the
resin allows for homogeneous reaction conditions. The homogeneous
reaction conditions of liquid phase synthesis are readily amenable
to the reactions and procedures of classical organic chemistry. In
contrast, solid phase synthesis affords heterogeneous reaction
conditions which have several shortcomings such as non-linear
kinetic behavior, unequal product distribution and/or access to the
chemical reaction, and salvation problems.
[0186] Liquid phase solid supports are polymers that are soluble in
a selected solvent. Soluble polymers that have been used in liquid
phase synthesis include homopolymers and copolymers. Homopolymers
include polystyrene (non-cross-linked), polyvinyl alcohol,
polyethylene imine, polyacrylic acid, polymethylene oxide,
polyethylene glycol (PEG), polypropylene oxide, cellulose, and
polyacrylamide. Copolymers include PEG with 3,5-diisocyanatobenzyl
chloride, PEG with 3-nitro-3-azapentane 1,5-diisocyanate, polyvinyl
alcohol-poly(1-vinyl-2-pyrrolidinone),
polystyrene-poly(vinyl-substituted monosaccharides), and
poly(N-isopropylacrylamide)-poly(acrylic acid derivatives)
(Gravert, D. J.; Janda, K. D., Chem. Rev., 1997, 97, 489).
[0187] Liquid phase hydrophilic polymeric supports have been found
to be more compatible for the preparation of oligonucleotides due
to their solubilities in polar solvents like water, aqueous
solutions and aqueous buffers. These hydrophilic polymers include
homopolymers such as polyvinyl alcohol, polyethylene glycol (PEG),
and cellulose as well as the copolymer polyvinyl
alcohol-poly(1-vinyl-2-pyrrolidinone).
[0188] Of the polymer supports listed above, polyethylene glycol
(PEG) has been most often used for liquid-phase synthesis. By
convention, PEG usually indicates the polyether of molecular weight
less than 20,000 g/mol. The term PEG is used herein to describe
polyethylene glycols of 2,000 to 20,000 molecular weight which have
been utilized as supports. These limits have been set by the
physical properties of the polymer. PEGs of molecular weight 2,000
to 20,000 are crystalline with loading capacities of 1 to 0.1
mmol/g; lower molecular weight PEGs exist as liquids at room
temperature, and higher molecular weight PEGs have low loading
capacities. Macromolecular size is reported herein using the
notation MeO-PEG 12,000 to represent polyethylene glycol methyl
ether with an average molecular weight ca. 12,000 g/mol. It is
emphasized again that polymers exist as a distribution of molecular
weights, however, the polydispersity of commercial PEG's is quite
narrow (Harris, J. M., In Poly(Ethylene Glycol) Chemistry:
Biotechnical and Biomedical Applications; Harris, J. M., Ed.;
Plenum Press: New York, 1992, p2).
[0189] Depending on polymerization conditions, PEG termini may
consist of hydroxyl groups or may be selectively functionalized.
Commercially available PEG is produced through anionic
polymerization of ethylene oxide to yield a polyether structure
possessing either hydroxyl groups at both ends, or a methoxy group
at one end and a hydroxyl group at the other. A PEG is used to
represent polyethylene glycol with hydroxyl functionalities at both
ends. Similarly, MeO-PEG 12,000 (polyethylene glycol monomethyl
ether) designates the polyether terminated by a methoxy group at
one end and a free hydroxyl at the other of a PEG polymer of an
average molecular weight of ca. 12,000 g/mol.
[0190] Many successful applications of the liquid-phase method have
resulted from the use of various polyethylene glycols as the
polymeric support. This linear homopolymer exhibits solubility in a
wide range of organic solvents such as methanol, ethanol and
acetone as well as other polar solvents like water, aqueous
solutions and aqueous buffers. PEG is insoluble in hexane, diethyl
ether, and tertiary butyl methyl ether, and these solvents have
been used to induce PEG precipitation. In the antisense field,
MeO-PEG 12,000 was used to synthesize a 20 mer oligonucleotide.
This choice was dictated by necessity to avoid the unfavorable
solubility properties of the growing oligonucleotide chain over
those of the polymeric support (Bonora, G. M.; Biancotto, M. M.;
Scremin, C. L., Nucleic Acids Research, 1993, 21, 1213).
[0191] The previously described hydrophilic polymeric resins all of
which contain a primary alcohol functionality, including
homopolymers such as polyvinyl alcohol, polyethylene glycol (PEG),
and cellulose as well as the copolymer polyvinyl
alcohol-poly(1-vinyl-2-pyrrolidinone), can be converted into any of
the previously listed amino reagents (primary amines,
hydroxylamines, hydrazines, semicarbazides and thiosemicarbazides),
with procedures that are known in the art of organic chemistry as
was previously described. These procedures give other preferred
embodiments of the invention. These embodiments include but are not
limited to any of these amino reagents. Other embodiments of the
invention include but are not limited to the previously described
liquid phase hydrophilic polymeric supports including (homopolymers
such as polyvinyl alcohol, polyethylene glycol (PEG), and cellulose
as well as the copolymer polyvinyl
alcohol-poly(1-vinyl-2-pyrrolidinone)), and others which are
functionalized with any one of the previously described amino
groups (primary amines, hydrazines, hydroxylamines, semicarbazides,
and thiosemicarbazides).
[0192] As is shown in Example 7, MeO-PEG 12,000 hydroxy resin is
converted into MeO-PEG 12,000 hydroxylamine resin by treatment of
the alcohol with triphenylphosphine (PPh.sub.3),
N-hydroxphthalimide, diethylazodicarboxylate (DEAD,
Et-OOCN.dbd.NCOO-Et) (3 eq each) in tetrahydrofuran, followed by
the addition of hydrazine in tetrahydrofuran-ethanol (Floyd, C. D.;
Lewis, C. N.; Patel, S. R.; Whittaker, M., Tetrahedron Letters,
1996, 37, 8045, herein incorporated by reference).
[0193] MeO-PEG 12,000 hydroxylamine resin swells in a polar solvent
such as water, aqueous solutions or aqueous buffer solutions, and
reacts with an abasic site of a contaminant oligonucleotide to form
a resin bound imine. The MeO-PEG bound contaminant oligonucleotide
is soluble in either acetone, methanol or ethanol whereas the
oligonucleotide is insoluble and precipitates. Separation of the
solid oligonucleotide from the solution containing the MeO-PEG
bound contaminant oligonucleotide can occur by either a
centrifugation and removal of the solvents or by a filtration and a
wash or a rinse of the solids which gives a purified
oligonucleotide.
[0194] The purification of a mixture of an oligonucleotide that is
contaminated with a contaminant having at least one abasic site,
with MeO-PEG 12,000 hydroxylamine resin is described in Example 24.
After treatment of the mixture with MeO-PEG 12,000 hydroxylamine,
the oligonucleotide is precipitated from the solution by the
addition of ethanol and cooling to about -20.degree. C. The
precipitated solid oligonucleotide is isolated by centrifugation or
a filtration. Removal of the solvents, and a wash or rinse of the
solids with ethanol, gives a purified solid oligonucleotide.
[0195] Additional advantages and novel features of this invention
will become apparent to those skilled in the art upon examination
of the examples thereof provided below, which should not be
construed as limiting the appended claims.
EXAMPLES
[0196] Reagents and solvents are purchased from Aldrich.TM., P.O.
Box 355, Milwaukee, Wis., 53201. Resins are purchased from
Aldrich.TM., Advanced ChemTech, Inc., 5609 Fern Valley Road,
Louisville, Ky., 40228-1075, Calbiochem-Novabiochem Corporation,
10394 Pacific Center Court, San Diego, 92121, and Union Carbide
Corporation, 39 Old Ridgebury Road, Danbury, Conn. Reactions are
performed under an argon atmosphere unless otherwise noted. Column
chromatography is carried out using normal phase silica gel.
Solvent ratios are given as volume/volume. Solvent gradients are
carried out step-wise. Evaporation of solvents are performed in
vacuo (50 torr) at 35.degree. C. unless otherwise specified. NMR
spectra are obtained with the following instruments: .sup.1H NMR:
Varian Gemini-200 (199.975 MHZ) or Varian Unity 400 (399.952 MHZ).
.sup.13C NMR: Varian Gemini-200 (50.289 MHZ). .sup.31P NMR: Varian
Gemini-200 (79.990 MHZ). NMR spectra are recorded using either
deuteriochloroform, dimethylsulfoxide-d.sub.6,
dimethylformamide-d.sub.7, or deuteriomethanol as solvent
(tetramethylsilane as internal standard). The following
abbreviations are used to designate the multiplicity of individual
signals: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,
dd=doublet of doublets, br s=broad singlet. Mass spectra analysis
are performed on an LCQ quadrupole ion trap mass spectrometer
equipped with an electrospray ionization source (Finnigan MATT).
HPLC analysis is performed on a WATERS HPLC System (600E System
Controller, 996 Photodiode Array Detector, 717 Autosampler), using
a VYDAC Protein C-4 column with a gradient of 2 to 99% acetonitrile
in Et.sub.3NHOAc (0.1M) in 30 minutes.
[0197] Additional advantages and novel features of this invention
will become apparent to those skilled in the art upon examination
of the examples provided which should not be construed as limiting
the appended claims.
Example 1
[0198] Preparation of O-IGEPAL.TM. CO-720 Hydroxylamine.
[0199] A solution of IGEPAL.TM. CO-720 (30.0 g, 40.0 mmol) in
anhydrous tetrahydrofuran (80 mL dried over 3 .ANG. molecular
sieves) is added to an ice cooled solution of N-hydroxyphthalimide
(8.2 g, 50.0 mmol) and triphenylphosphine (13.3 g, 50.0 mmol) in
anhydrous tetrahydrofuran (250 ml). A solution of
diethylazodicarboxylate (8.4 g, 48.0 mmol) in anhydrous
tetrahydrofuran (40 ml) is added dropwise over 15 minutes. The
cooling bath is removed and the mixture is stirred overnight. The
clear solution is concentrated in vacuo and the oily residue is
redissolved in dichloromethane (500 ml) and washed with water
(1.times.200 ml). The organic layer is dried over anhydrous
Na.sub.2SO.sub.4, concentrated under vacuum, and is purified by
flash silica gel chromatography using a ethyl acetate/hexanes and
then an ethyl acetate/methanol gradient to give the O-IGEPAL.TM.
CO-720 N-hydroxyphthalimide derivative. .sup.1H NMR (200 MHz)
7.9-6.7 (m, 8H), 4.4-3.4 (m, 51H), 1.8-0.4 (m, 19H).
[0200] The O-IGEPAL.TM. CO-720 N-hydroxyphthalimide derivative
(13.5 g) is dissolved in tetrahydrofuran (60 ml), cooled to
0.degree. C., and anhydrous hydrazine (1.0 g) is added dropwise
over 10 minutes. After 10 minutes, the solution is stirred at room
temperature for 1 hour. Diethyl ether is added and the mixture is
kept at -20.degree. C. overnight. The mixture is filtered and the
liquid phase is evaporated to give a residue. The residue is
purified by silica gel column chromatography using a 98:2 to 90:10
ethyl acetate and methanol gradient to give 9.6 g of the O-IGEPAL
hydroxyl amine as a colorless oil.
[0201] MS for
C.sub.9H.sub.19--(C.sub.6H.sub.4)--(OCH.sub.2CH.sub.2).sub.n-
--ONH.sub.3.sup.+
[0202] n=8, calc. 588.8, found 588.5,
[0203] n=9, calc. 632.8, found 632.4
[0204] n=10, calc. 676.9, found 676.5,
[0205] n=11, calc. 721.0, found 720.5,
[0206] n=12, calc. 765.0, found 764.5,
[0207] n=13, calc. 809.1, found 808.5,
[0208] n=14, calc. 853.1, found 852.7,
[0209] n=15, calc. 897.2, found 896.7,
[0210] n=16, calc. 941.2, found 940.7.
Example 2
[0211] Preparation of NovaSyn.TM. TG Hydroxylamine Resin.
[0212] NovaSyn TG hydroxy resin (2.00 g, loading capacity 0.27
mmol/g) is suspended in anhydrous tetrahydrofuran (8 ml).
N-hydroxy-phthalimide (282 mg, 1.73 mmol) and triphenylphosphine
(454 mg, 1.73 mmol) are added and the mixture is mildly agitated
for 30 minutes on an orbital shaker. Diethylazodicarboxylate (2.82
g, 1.62 mmol) is added and the mixture is agitated overnight. The
resin is filtered and washed sequentially with tetrahydrofuran (20
ml), dimethylformamide (20 ml), dichloromethane (20 ml), methanol
(20 ml) and dichloromethane (50 ml), and dried under vacuum. The
resin is suspended in DMF (10 ml) and hydrazine (0.8 ml) is added.
The mixture is warmed to 60.degree. C. for 1 hour and kept
overnight at room temperature. The resin is filtered and washed
sequentially with tetrahydrofuran (20 ml), dimethylformamide (20
ml), dichloromethane (20 ml), methanol (20 ml) and dichloromethane
(50 ml), and is dried under vacuum.
Example 3
[0213] Preparation of O-IGEPAL.TM. CO-720 Amine.
[0214] The conversion of O-IGEPAL.TM. CO-720 to O-IGEPAL.TM. CO-720
Amine is accomplished by treatment of O-IGEPAL.TM. CO-720 with
hydrazoic acid (HN.sub.3), diisopropyl azodicarboxylate
(i-Pr-OOCN.dbd.NCOO-i-Pr), and excess triphenylphosphine
(Ph.sub.3P) in tetrahydrofuran followed by water or aqueous acid
(According to the procedure of Fabiano et al., Synthesis, 1987,
190.)
Example 4
[0215] Preparation of O-IGEPAL.TM. CO-720 Hydrazine.
[0216] The conversion of O-IGEPAL.TM. CO-720 to O-IGEPAL.TM. CO-720
Hydrazine is accomplished by treatment of O-IGEPAL.TM. CO-720 with
mesyl chloride and triethylamine in dichloromethane, followed by
the addition of hydrazine hydrate (4 eq) in ethanol at 0.degree. C.
(Yaun, C.; Li, C., Synthesis, 1995, 4, 507).
Example 5
[0217] Preparation of O-IGEPAL.TM. CO-720 Semicarbazide.
[0218] The conversion of O-IGEPAL.TM. CO-720 to O-IGEPAL.TM. CO-720
Semicarbazide is accomplished by converting O-IGEPAL.TM. CO-720
into the O-IGEPAL.TM. CO-720 Amine as described in Example 2 and
treating O-IGEPAL.TM. CO-720 Amine with ethyl chloroformate and
pyridine, followed by the addition of hydrazine in ethanol (Indian
J. Chem., 1985, Sect. B, 24B (11), 1115).
Example 6
[0219] Preparation of O-IGEPAL.TM. CO-720 Thiosemicarbazide.
[0220] The conversion of O-IGEPAL.TM. CO-720 to O-IGEPAL.TM. CO-720
Thiosemicarbazide is accomplished by converting O-IGEPAL.TM. CO-720
into the O-IGEPAL.TM. CO-720 Semicarbazide as described in Example
4 and treating O-IGEPAL.TM. CO-720 Semicarbazide with
2,4-bis(4-methoxyphenyl)--
1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lawesson's reagent)
(Cava; Levinson, Tetrahedron, 1985, 41, 5061-5087).
Example 7
[0221] Preparation of MeO-PEG-12,000 Hydroxylamine.
[0222] MeO-PEG-12,000 hydroxylamine may be synthesized from
MeO-PEG-12,000 monomethylether which is available from Union
Carbide-USA (Bio-PEG). The conversion of an alcohol to a
hydroxylamine is accomplished by treatment of the alcohol with
triphenylphosphine (PPh.sub.3), N-hydroxphthalimide, diethyl
azodicarboxylate (DEAD) (Et-OOCN.dbd.NCOO-Et) (3 eq each) in
tetrahydrofuran, followed by the addition of hydrazine in
tetrahydrofuran-ethanol (Floyd, C. D.; Lewis, C. N.; Patel, S. R.;
Whittaker, M., Tetrahedron Letters, 1996, 37, 8045).
Example 8
[0223] Purification of Phosphorothioate oligodeoxyribonucleotide,
PS-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 1)
[0224] A solution of phosphorothioate oligodeoxyribonucleotide,
PS-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 1) (0.100 grams, 0.014 mmol)
in sodium phosphate buffer (2 mL, 0.1 M, pH 7.2) is added to a
solution of O-IGEPAL.TM. CO-720 hydroxylamine (0.020 grams, 0.026
mmol) in sodium phosphate buffer (2 mL, 0.1 M, pH 7.2). After 18
hours, the solution is cooled to -20.degree. C. and ethanol (40 mL)
is added to precipitate the oligonucleotide. The heterogenous
mixture is kept at -20.degree. C. for 15 minutes. The precipitate
is spun down by centrifugation and the ethanol is removed. The
oligonucleotide is dissolved in sodium acetate buffer (2 mL, 0.1 M,
pH 7.2) and is analyzed by reversed phase HPLC and by mass
spectrometry.
Example 9
[0225] Purification of Phosphodiester Oligodeoxyribonucleotide
PO-D(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 2).
[0226] Phosphodiester oligodeoxyribonucleotide
PO-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 2) is treated with a
solution of O-IGEPAL.TM. CO-720 hydroxylamine in sodium phosphate
buffer (pH 7.2) overnight. Ethanol is added, and the heterogenous
mixture is kept at -20.degree. C. for 15 min. The precipitate is
spun down by centrifugation and the ethanol phase is removed. The
oligonucleotide is dissolved in NaOAc buffer and subjected to
reversed phase HPLC purification. HPLC fractions containing the
oligonucleotide are concentrated and the oligonucleotide is
isolated by ethanol precipitation.
Example 10
[0227] Purification of Phosphorothioate Oligodeoxyribonucleotide
PS-d(TCCGTCATCGCTCCTCAGGG) (SEQ ID NO. 3).
[0228] Phosphorothioate oligodeoxyribonucleotide
PS-d(TCCGTCATCGCTCCTCAGGG- ) (SEQ ID NO. 3) is treated with a
solution of O-IGEPAL.TM. CO-720 hydroxylamine in sodium phosphate
buffer (pH 7.2) overnight. Ethanol is added, and the heterogenous
mixture is kept at -20.degree. C. for 15 min. The precipitate is
spun down by centrifugation and the ethanol phase is removed. The
oligonucleotide is dissolved in NaOAc buffer and subjected to
reversed phase HPLC purification. HPLC fractions containing the
oligonucleotide are concentrated and the oligonucleotide is
isolated by ethanol precipitation.
Example 11
[0229] Purification of Phosphodiester Oligodeoxyribonucleotide
PO-d(TCCGTCATCGCTCCTCAGGG) (SEQ ID NO. 4).
[0230] Phosphodiester oligodeoxyribonucleotide
PO-d(TCCGTCATCGCTCCTCAGGG) (SEQ ID NO. 4)is treated with a solution
of O-IGEPAL.TM. CO-720 hydroxylamine in sodium phosphate buffer (pH
7.2) overnight. Ethanol is added, and the heterogenous mixture is
kept at -20.degree. C. for 15 min. The precipitate is spun down by
centrifugation and the ethanol phase is removed. The
oligonucleotide is dissolved in NaOAc buffer and subjected to
reversed phase HPLC purification. HPLC fractions containing the
oligonucleotide are concentrated and the oligonucleotide is
isolated by ethanol precipitation.
Example 12
[0231] Purification of Phosphorothioate Oligodeoxyribonucleotide
PS-d(GTTCTCGCTGGTGAGTTTCA) (SEQ ID NO. 5).
[0232] Phosphorothioate oligodeoxyribonucleotide
PS-d(GTTCTCGCTGGTGAGTTTCA- ) (SEQ ID NO. 5) is treated with a
solution of O-IGEPAL.TM. CO-720 hydroxylamine in sodium phosphate
buffer (pH 7.2) overnight. Ethanol is added, and the heterogenous
mixture is kept at -20.degree. C. for 15 min. The precipitate is
spun down by centrifugation and the ethanol phase is removed. The
oligonucleotide is dissolved in NaOAc buffer and subjected to
reversed phase HPLC purification. HPLC fractions containing the
oligonucleotide are concentrated and the oligonucleotide is
isolated by ethanol precipitation.
Example 13
[0233] Purification of Phosphodiester Oligodeoxyribooligonucleotide
PO-d(GTTCTCGCTGGTGAGTTTCA) (SEQ ID NO. 6).
[0234] Phosphodiester oligodeoxyribooligonucleotide
PO-d(GTTCTCGCTGGTGAGTTTCA) (SEQ ID NO. 6) is treated with a
solution of O-IGEPAL.TM. CO-720 hydroxylamine in sodium phosphate
buffer (pH 7.2) overnight. Ethanol is added, and the heterogenous
mixture is kept at -20.degree. C. for 15 min. The precipitate is
spun down by centrifugation and the ethanol phase is removed. The
oligonucleotide is dissolved in NaOAc buffer and subjected to
reversed phase HPLC purification. HPLC fractions containing the
oligonucleotide are concentrated and the oligonucleotide is
isolated by ethanol precipitation.
Example 14
[0235] Purification of Phosphorothioate Oligodeoxyribonucleotide
PS-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 7).
[0236] Phosphorothioate oligodeoxyribonucleotide
PS-d(TCCCGCCTGTGACATGCATT- ) (SEQ ID NO. 7) is treated with a
solution of O-IGEPAL.TM. CO-720 hydroxylamine in sodium phosphate
buffer (pH 7.2) overnight. Ethanol is added, and the heterogenous
mixture is kept at -20.degree. C. for 15 min. The precipitate is
spun down by centrifugation and the ethanol phase is removed. The
oligonucleotide is dissolved in NaOAc buffer and subjected to
reversed phase HPLC purification. HPLC fractions containing the
oligonucleotide are concentrated and the oligonucleotide is
isolated by ethanol precipitation.
Example 15
[0237] Purification of Phosphodiester Oligodeoxyribooligonucleotide
PO-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 8).
[0238] Phosphodiester oligodeoxyribooligonucleotide
PO-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 8) is treated with a
solution of O-IGEPAL.TM. CO-720 hydroxylamine in sodium phosphate
buffer (pH 7.2) overnight. Ethanol is added, and the heterogenous
mixture is kept at -20.degree. C. for 15 min. The precipitate is
spun down by centrifugation and the ethanol phase is removed. The
oligonucleotide is dissolved in NaOAc buffer and subjected to
reversed phase HPLC purification. HPLC fractions containing the
oligonucleotide are concentrated and the oligonucleotide is
isolated by ethanol precipitation.
Example 16
[0239] Purification of Phosphorothioate Oligodeoxyribonucleotide
PS-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 1).
[0240] Phosphorothioate oligodeoxyribonucleotide
PS-d(GCCCAAGCTGGCATCCGTCA- ) (SEQ ID NO. 1) is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrifugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 17
[0241] Purification of Phosphodiester Oligodeoxyribonucleotide
PO-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 2).
[0242] Phosphodiester oligodeoxyribonucleotide
PO-d(GCCCAAGCTGGCATCCGTCA) (SEQ ID NO. 2) is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrifugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 18
[0243] Purification of Phosphorothioate Oligodeoxyribonucleotide
PS-d(TCCGTCATCGCTCCTCAGGG) (SEQ ID NO. 3).
[0244] Phosphorothioate oligodeoxyribonucleotide
PS-d(TCCGTCATCGCTCCTCAGGG- ) (SEQ ID NO. 3) is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrifugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 19
[0245] Purification of Phosphodiester Oligodeoxyribonucleotide
PO-d(TCCGTCATCGCTCCTCAGGG) (SEQ ID NO 4).
[0246] Phosphodiester oligodeoxyribonucleotide
PO-d(TCCGTCATCGCTCCTCAGGG) (9SEQ ID NO. 4) is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrifugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 20
[0247] Purification of Phosphorothioate Oligodeoxyribonucleotide
PS-d(GTTCTCGCTGGTGAGTTTCA) (SEQ ID NO. 5).
[0248] Phosphorothioate oligodeoxyribonucleotide
PS-d(GTTCTCGCTGGTGAGTTTCA- ) (SEQ ID NO. 5)is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrifugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 21
[0249] Purification of Phosphodiester Oligodeoxyribonucleotide
PO-d(GTTCTCGCTGGTGAGTTTCA) (SEQ ID NO. 6).
[0250] Phosphodiester oligodeoxyribonucleotide
PO-d(GTTCTCGCTGGTGAGTTTCA) (SEQ ID NO. 6) is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrifugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 22
[0251] Purification of Phosphorothioate Oligodeoxyribonucleotide
PS-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 7).
[0252] Phosphorothioate oligodeoxyribonucleotide
PS-d(TCCCGCCTGTGACATGCATT- ) (SEQ ID NO. 7) is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrigugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 23
[0253] Purification of Phosphodiester Oligodeoxyribonucleotide
PO-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 8).
[0254] Phosphodiester oligodeoxyribonucleotide
PO-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 8) is treated with a
suspension of NovaSyn TG hydroxylamine resin in sodium phosphate
buffer (pH 7.2) overnight. The liquid phase is isolated by
centrifugation and the oligonucleotide is isolated by ethanol
precipitation.
Example 24
[0255] Purification of Phosphodiester Oligodeoxyribonucleotide
PO-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 8).
[0256] Phosphodiester oligodeoxyribonucleotide
PO-d(TCCCGCCTGTGACATGCATT) (SEQ ID NO. 8) is treated with a
suspension of MeO-PEG 12,000 hydroxylamine resin in sodium
phosphate buffer (pH 7.2) overnight. After treatment of the mixture
with MeO-PEG 12,000 hydroxylamine, the oligonucleotide is
precipitated from the solution by the addition of ethanol and
cooling to about -20.degree. C. The precipitated solid
oligonucleotide is isolated by centrifugation or a filtration.
Removal of the solvents, and a wash or rinse of the solids with
ethanol, gives a purified solid oligonucleotide.
[0257] It is intended that each of the patents, applications,
printed publications, and other published documents mentioned or
referred to in this specification be herein incorporated by
reference in their entirety.
[0258] Those skilled in the art will appreciate that numerous
changes and modifications may be made to the preferred embodiments
of the present invention, and that such changes and modifications
may be made without departing from the spirit of the invention. It
is, therefore, intended that the appended claims cover all such
equivalent variations that fall within the true spirit and scope of
the invention.
Sequence CWU 1
1
8 1 20 DNA Artificial Sequence Synthetic construct 1 gcccaagctg
gcatccgtca 20 2 20 DNA Artificial Sequence Synthetic construct 2
gcccaagctg gcatccgtca 20 3 20 DNA Artificial Sequence Synthetic
construct 3 tccgtcatcg ctcctcaggg 20 4 20 DNA Artificial Sequence
Synthetic construct 4 tccgtcatcg ctcctcaggg 20 5 20 DNA Artificial
Sequence Synthetic construct 5 gttctcgctg gtgagtttca 20 6 20 DNA
Artificial Sequence Synthetic construct 6 gttctcgctg gtgagtttca 20
7 20 DNA Artificial Sequence Synthetic construct 7 tcccgcctgt
gacatgcatt 20 8 20 DNA Artificial Sequence Synthetic construct 8
tcccgcctgt gacatgcatt 20
* * * * *