U.S. patent application number 12/568229 was filed with the patent office on 2010-04-08 for purification of oligonucleotides.
This patent application is currently assigned to Girindus AG. Invention is credited to Olaf Grossel, Andreas Hohlfeld, Meinolf Lange, Fritz Link, Andreas Schonberger.
Application Number | 20100087635 12/568229 |
Document ID | / |
Family ID | 34933485 |
Filed Date | 2010-04-08 |
United States Patent
Application |
20100087635 |
Kind Code |
A1 |
Grossel; Olaf ; et
al. |
April 8, 2010 |
PURIFICATION OF OLIGONUCLEOTIDES
Abstract
A method for purifying a protected oligonucleotide comprising
the steps of: a) providing a solution of the protected
oligonucleotide in at least one solvent A having a boiling point
below the boiling point of a solvent B, heating the solution at a
temperature of at least 30.degree. C. and below the boiling point
of the at least solvent A, adding solvent B until precipitation of
a material is visible in the solution, said solvent B being an
alcohol having 1 to 6 C-atoms or a diol having 2 to 6 C-atoms,
allowing the solution to cool down under stirring until formation
of a supernatant and a residue, removing the supernatant or b)
providing solvent B, said solvent B being an alcohol having 1 to 6
C-atoms or a diol having 2 to 6 C-atoms, heating solvent B at a
temperature above 30.degree. C. and below the boiling point of
solvent B, adding a solution of a protected oligonucleotide in at
least one solvent A until precipitation of a material is visible in
the solution, allowing the solution to cool down under stirring
until formation of a supernatant and a residue, removing the
supernatant.
Inventors: |
Grossel; Olaf;
(Halle/Westfalen, DE) ; Hohlfeld; Andreas; (Porta
Westlalica, DE) ; Lange; Meinolf; (Bielefeld, DE)
; Link; Fritz; (Bensberg, DE) ; Schonberger;
Andreas; (Muden/Aller, DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
Girindus AG
Bergisch-Gladbach
DE
|
Family ID: |
34933485 |
Appl. No.: |
12/568229 |
Filed: |
September 28, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12402128 |
Mar 11, 2009 |
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12568229 |
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12196605 |
Aug 22, 2008 |
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12402128 |
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11814429 |
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PCT/EP2006/050503 |
Jan 30, 2006 |
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12196605 |
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60647457 |
Jan 28, 2005 |
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Current U.S.
Class: |
536/25.4 |
Current CPC
Class: |
C07H 21/00 20130101 |
Class at
Publication: |
536/25.4 |
International
Class: |
C07H 1/06 20060101
C07H001/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 2005 |
EP |
05001768.0 |
Claims
1-18. (canceled)
19. A method for purifying a protected oligonucleotide comprising:
a1) providing a solution of said protected oligonucleotide in at
least one solvent (A) having a boiling point below the boiling
point of a solvent (B); a2) heating said solution of said protected
oligonucleotide in at least one solvent (A) at a temperature of at
least 30.degree. C. and below the boiling point of said at least
one solvent (A); a3) adding solvent (B) to said solution until
precipitation of a material is visible, said solvent (B) being an
alcohol having 1 to 6 carbon atoms or a diol having 2 to 6 carbon
atoms; a4) allowing said solution to cool down under stirring until
formation of a supernatant and a residue; and a5) removing said
supernatant; or b1) providing solvent (B), said solvent (B) being
an alcohol having 1 to 6 carbon atoms or a diol having 2 to 6
carbon atoms; b2) heating said solvent (B) at a temperature above
30.degree. C. and below the boiling point of said solvent (B); b3)
adding a solution of a protected oligonucleotide in at least one
solvent (A) to said solvent (B) until precipitation of a material
is visible in the solution; b4) allowing said solution to cool down
under stirring until formation of a supernatant and a residue; and
b5) removing said supernatant.
20. The method of claim 19, wherein said protected oligonucleotide
comprises from 2 to 30 nucleotides.
21. The method of claim 19, wherein said protected oligonucleotide
comprises a 3' and 5' protection group, a 5' protection group and
no 3' protection group, or a 3' protection group and no 5'
protection group.
22. The method of claim 21, wherein said 5'protection group is
DMTr, MMTr, tert-butyl dimethylsilyl, levulinyl, benzoyl,
fluorenemethoxycarbonyl, or 9-phenylthioxanthen-9-yl.
23. The method of claim 19, wherein said solvent A is
CH.sub.2Cl.sub.2, CHCl.sub.3, tetrahydrofuran, acetonitrile,
methanol, ethanol, dichloroethane, tetrachloroethane, dioxane, or
acetone.
24. The method of claim 19, wherein said solution of a protected
oligonucleotide in at least one solvent (A) comprises a mixture of
solvents selected from the group consisting of CH.sub.2Cl.sub.2,
CHCl.sub.3, tetrahydrofuran, acetonitrile, methanol, ethanol,
dichloroethane, tetrachloroethane, dioxane, and acetone.
25. The method of claim 19, wherein said solution of a protected
oligonucleotide in at least one solvent (A) in a2) and said solvent
B in b2) is heated at a temperature between 40 and 70.degree.
C.
26. The method of claim 19, wherein said solvent (B) is added drop
wise in a3).
27. The method of claim 19, wherein the ratio between the volume of
said solution of a protected oligonucleotide in at least one
solvent (A) and the volume of said solvent (B) is from 1:1 to
1:100.
28. The method of claim 27, wherein said ratio is from 1:1 to
1:10.
29. The method of claim 19, wherein said residue is a gel, an oil,
or crystalline.
30. The method of claim 19, further comprising adding an ether to
said residue and then removing said ether together with
impurities.
31. The method of claim 19, further comprising dissolving said
residue to form a solution comprising at least one solvent (A) and
repeating a1) through a5) or b1) through b5) at least once.
32. The method of claim 19, wherein a) said solvent (B) is added in
a3) until precipitation of a material is visible in the solution
even upon further heating; or b) said solution of a protected
oligonucleotide in at least one solvent (A) is added in b3) until
precipitation of a material is visible in the solution even upon
further heating.
33. The method of claim 19, wherein said solvent (B) is selected
from the group consisting of methanol; ethanol;
1,2-dihydroxyethane; 1-propanol; 2-propanol; butanol; and
pentanol.
34. The method of claim 19 wherein said solvent (A) is
CH.sub.2Cl.sub.2 and said solvent (B) is 2-propanol.
35. A method for purifying a protected, non-ionic oligonucleotide
comprising a) providing a solution of said protected, non-ionic
oligonucleotide in at least one solvent (A); b) combining said
solution with a solvent (B) to form a solvent (A+B), said solvent B
being an alcohol having 1 to 6 carbon atoms or a diol having 2 to 6
carbon atoms, wherein said protected, non-ionic oligonucleotide is
more soluble (weight/volume) at 25.degree. C. in said solvent (A)
than in said solvent (B), and wherein the amounts of said solvent
(A) and said solvent (B) are selected to have a saturated solution
of said protected, non-ionic oligonucleotide in said solvent (A+B);
c) waiting until formation of a supernatant and a residue; and d)
removing said supernatant.
36. The method of claim 35, wherein said solvent (A) is
CH.sub.2CH.sub.2, CHCl.sub.3, tetrahydrofuran, or dioxane.
37. The method of claim 35, wherein said solvent (B) is
isopropanol, 1-propanol, or ethanol.
38. The method of claim 35, wherein said solution (A+B) is cooled
down at least 10.degree. C. to enhance formation of said
supernatant and residue.
Description
[0001] The present invention relates to a method for purifying
oligonucleotides.
[0002] The synthesis of oligonucleotides has been the subject of
intensive research for a long period of time. Automated synthesis
procedures have been developed and apparatus for the automated
synthesis are commercially available. Most of these procedures have
been developed for rather small quantities of oligonucleotides (in
the range of mg). These amounts are sufficient for most research
purposes.
[0003] Especially with the development of antisense therapeutics,
large scale synthesis became a matter of considerable importance.
Although relative large scale synthesis procedures have been
developed, one major drawback is the purification of the
intermediates and products. For small scale synthesis
reversed-phase HPLC is an appropriate method allowing the
purification of mg quantities in short time with good purification
results. For large scale production reversed-phase HPLC becomes a
difficult procedure requiring large amounts of solvents, expensive
apparatus and the like.
[0004] There is an ongoing need for improved methods for purifying
the products of oligonucleotide synthesis.
[0005] US 2003/0055241 discloses a method for preparing purified
oligonucleotides comprising treating a solution of oligonucleotide
with an aggregating agent and a precipitation enhancer. The method
is used for the purification of unprotected oligonucleotides or 5'
protected oligonucleotides. An ionic form is needed for the
precipitation enhancer to interact with the molecule.
[0006] EP 0 512 768 A1 discloses a method for purifying DNA. The
DNA is prepared from natural sources.
[0007] Both methods described above can only be used for
unprotected or nearly completely unprotected oligonucleotides.
During synthesis, oligonucleotides are protected, therefore, the
methods of US 2003/0055241 and EP 0 512 768 A1 are not
applicable.
[0008] It is an object of the present invention to overcome the
drawbacks of prior art and provide an efficient purification method
for oligonucleotides and intermediates, especially for large scale
synthesis.
[0009] In one embodiment, the present invention provides a method
for purifying a protected oligonucleotide comprising the step of
[0010] providing a solution of the protected oligonucleotide in at
least one solvent A having a boiling point below the boiling point
of a solvent B, [0011] heating the solution at a temperature of at
least 30.degree. C. and below the boiling point of the at least
solvent A, [0012] adding solvent B until the solution becomes
cloudy, said solvent B being an alcohol having 1 to 6 C-atoms or a
diol having 2 to 6 C-atoms, [0013] allowing the solution to cool
down under stirring until formation of a supernatant and a residue,
[0014] removing the supernatant.
[0015] In a further embodiment, the invention provides a method for
purifying a protected oligonucleotide comprising the steps of
[0016] providing solvent B, said solvent B being an alcohol having
1 to 6 C-atoms or a diol having 2 to 6 C-atoms, [0017] heating
solvent B at a temperature above 30.degree. C. and below the
boiling point of solvent B, [0018] adding a solution of a protected
oligonucleotide in at least one solvent A until the solution
becomes cloudy, [0019] allowing the solution to cool down under
stirring until formation of a supernatant and a residue, [0020]
removing the supernatant.
[0021] "Becomes cloudy" is an observation describing that the
solution is saturated, e.g. unable to dissolve the protected
oligonucleotide. A solution is cloudy if precipitation of a
material is visible.
[0022] "Cooled down" means to reduce the temperature of the
solution. Typically cooling down is effected to reach room
temperature (about 25.degree. C.). In some cases, it is preferred
to cool further, i.e. to reach at least 20.degree. C. or at least
10.degree. C. or 0.degree. C.
[0023] A "protected oligonucleotide" is an oligonucleotide
comprising one or more protective groups for example at the 5', 3',
2' position of the sugar moiety and/or at the heterocyclic bases
and/or at the P-linkage (e.g. phosphate or thiophosphates).
[0024] The term "protective groups" includes groups which are not
removed prior to use of the oligonucleotides, e.g. modifications
used to increase stability of the oligonucleotide.
[0025] A suitable protection for the 2'-hydroxyl-group include but
are not limited to tert-butyl dimethylsilyl (TBDMS),
triisopropylsilyloxymethyl (TOM), fluoro-phenylmetoxypiperidinyl
(FPMP), and CH.sub.2-O-Et, and non-cleavable modifications like 2'F
or 2'MeO
[0026] Suitable protecting groups for the 3'-hydroxyl-group include
but are not limited to tert-butyl dimethylsilyl (TBDMS), levulinyl,
benzoyl.
[0027] Suitable modifications include LNA (2'O-4'C-methylene
bridge).
[0028] Suitable protected nucleobases are known to persons skilled
in the art for example N-4-benzoylcytosine, N-6-benzoyl adenine,
N-2-isobutyryl guanine, N-4-acetyl or isobutyryl cytosine,
N-6-phenoxyacetyl adenine, N-2-tert-butyl phenoxyacetyl guanine.
Suitable non-base residues include also hydrogen (H) leading to the
1',2'-dideoxyribose (dSpacer from Glen Research) which can be used
as linker or to mimic a basic sites in an oligonucleotide
(Takeshita et al., J. Biol. Chem., 1987, 262, 10171).
[0029] Suitable 5'-protection group include, but are not limited to
trityl groups, preferably a dimethoxytrityl group (DMTr) or a
monomethoxytrityl group (MMTr). These protection groups are used in
conventional prior art solid phase oligonucleotides synthesis.
Other suitable 5'-protection groups include, but are not limited to
tert-butyl dimethylsilyl (TBDMS), levulinyl, benzoyl,
fluorenemethoxycarbonyl (FMOC), 9-phenylthioxanthen-9-yl
(S-pixyl).
[0030] In a preferred embodiment, the protected oligonucleotide is
a non-ionic compound.
[0031] An "oligonucleotide" is an oligomer of monomeric units
comprising sugar units connected to heterocyclic bases, said
monomeric units being connected via linkages. Typical linkages are
derivatives of phosphor, for example phosphates, thiophosphates or
derivatives thereof. The term also covers oligonucleosides,
oligonucleotide analogs, modified oligonucleotides, nucleotide
mimetics and the like in the form of RNA and DNA. In general, these
compounds comprise a backbone of linked monomeric subunits where
each linked monomeric subunit is directly or indirectly attached to
a heterocyclic base moiety. The linkages joining the monomeric
sub-units, the monomeric subunits and the heterocyclic base
moieties can be variable in structure giving rise to a plurality of
motives for the resulting compounds.
[0032] According to preferred embodiments of the invention, the
protected oligonucleotide has a length of 2 to 30 nucleotides,
preferably 2 to 9, preferably 2 to 6.
[0033] Modifications known in the art are the modification of the
heterocyclic bases, the sugar or the linkages joining the monomeric
subunits. Variations of inter-nucleotide linkages are for example
described in WO 2004/011474, starting at the bottom of page 11,
incorporated by reference.
[0034] Typical derivatives are phosphorthioates,
phosphorodithioates, methyl and alkyl phosphonates and
phosphonoaceto derivatives.
[0035] Further typical modifications are at the sugar moiety.
Either the ribose is substituted by a different sugar or one or
more of the positions are substituted with other groups such as F,
O-alkyl, S-alkyl, N-alkyl. Preferred embodiments are 2'-methyl and
2'-methoxyethoxy. All these modifications are known in the art.
[0036] Concerning the heterocyclic base moiety, there are a number
of other synthetic bases which are used in the art, for example
5-methyl-cytosine, 5-hydroxy-methyl-cytosine, xanthin, hypoxanthin,
2-aminoadenine, 6- or 2-alkyl derivatives of adenine and guanine,
2-thiouracyl. Such modifications are also disclosed in WO
2004/011474 starting from page 21.
[0037] The protected oligonucleotide may have a 5' and 3'
protection group or a 5' protection group and no 3' protection
group or a 3' protection group and no 5' protection group. It
may--depending on the structure of the oligonucleotide--have a 2'
protection group.
[0038] Is preferred, that the P-linkage and the heterocyclic bases
will be suitably protected.
[0039] In one embodiment, the protected oligonucleotide is
dissolved in a solvent or a mixture of solvents wherein one of the
solvents is designated as solvent A. Suitable solvents A are for
example CH.sub.2Cl.sub.2, CHCl.sub.3, tetrahydrofuran, acetonitril,
methanol, ethanol, dichlorethane, tetrachlorethane, dioxane,
acetone.
[0040] While one solvent might be sufficient for some protected
oligonucleotides in another embodiment it is preferred to use a
mixture of solvents for example selected from CH.sub.2Cl.sub.2,
CHCl.sub.3, tetrahydrofuran, acetonitrile, methanol, ethanol,
dichlorethane, tetrachlorethane, dioxane, acetone.
[0041] According to the invention, the solution of the protected
oligonucleotide is heated at a temperature of at least 30.degree.
C. but below the boiling point of the at least one solvent A. If a
mixture of solvents is used for the solution of the protected
oligonucleotide it is preferred that the solution is not heated
above the boiling point of any ingredient of the mixture.
[0042] Thereafter, a solvent B, an alcohol or diol is added to the
solution.
[0043] It is preferred that solvent A has a boiling point below the
boiling point of solvent B but good results can also be achieved
with solvents A and B having a similar boiling point or even
situations wherein the boiling point of solvent A is higher than
the boiling point of solvent B.
[0044] A very preferred solvent A comprises CH.sub.2Cl.sub.2
(preferably at least 90% by weight) and a very preferred solvent B
comprises isopropanol (preferably at least 90% by weight). A
combination of CH.sub.2Cl.sub.2 and isopropanol is especially
useful and, therefore, preferred.
[0045] A further embodiment of the invention is a method comprising
[0046] providing a solution of a protected, non-ionic
oligonucleotide in at least one solvent A, [0047] combining said
solution with solvent B to form a solvent A+B wherein the amounts
of solvent A and solvent B are selected to have a saturated
solution of the non-ionic, protected oligonucleotide in solvent
A+B, [0048] and waiting until formation of a supernatant and a
residue.
[0049] This solvent B is an alcohol having 1 to 6 C-atoms or a diol
having 2 to 6 C-atoms.
[0050] Unless the necessary amount is exactly known, it is
preferred to add solvent B drop wise. Upon addition of solvent B
the solution becomes cloudy but in most cases, the solution becomes
clear again upon further stirring and/or heating. As the boiling
point of solvent B is higher than the boiling of solvent A, the
temperature of the solution increases upon further heating. The
preferred amount of solvent B is the amount which has to be added
to have the solution stay cloudy or a little bit less of solvent
B.
[0051] In one embodiment, the solution is heated at a temperature
in the range of 5 to 10.degree. C. below the boiling point. Once
the solution is cloudy, it is heated at the boiling point for one
minute and then allowed to cool down.
[0052] Heating of the solution is especially preferred if the
impurities are large, i.e. the purity of the oligonucleotide is
below 85%. In this case, the heating increases the purification
efficiency. If purity is higher (i.e. about at least 90%) or a
reduced purification efficiency is acceptable, the method of the
present invention can also be effected at a temperature of about 25
to 30.degree. C. or even at a temperature between 15 and 25.degree.
C. Purification is less efficient at lower temperatures.
[0053] In some embodiments, it is useful to repeat the method of
the present invention. In these cases the first purification step
could be effected at a temperature of at least 30.degree. C. and
subsequent purification steps at a lower temperature, i.e.
20.degree. C. or 25.degree. C. Thereafter, the solution is allowed
to cool down under stirring. Cooling can be effected to room
temperature or even to lower temperatures for example with a
refrigerator.
[0054] It is preferred to stir these solutions during cooling. In a
preferred embodiment stirring is continued for at least three
hours, preferably six hours and most preferred overnight.
[0055] According to the method of the invention, a supernatant and
a residue are formed. The supernatant comprises impurities and is
removed and discarded. The residue can be of different forms, it
can be a gel, an oil or it can be in a crystalline form. In some
embodiments, it is helpful to add an ether to the residue and
remove the ether again together with further impurities. The
protected oligonucleotide is not soluble in the ether.
[0056] In some cases, this treatment with ether increases the
tendency to form crystalline or powder forms of the residue.
[0057] In a preferred embodiment, the purification treatment is
repeated at least once. A solvent or solvent mixture of the solvent
type A is added and the oligonucleotide is redissolved. Thereafter,
the purification method of the present invention is repeated.
[0058] The method of the present invention is especially suitable
for larger scale synthesis, i.e. starting from about 100 mg
quantities to quantities of kg. The equipment needed for the method
of the present invention are cheap compared to a large scale
HPLC.
[0059] The method of the present invention is especially useful to
remove catalysts such as tetrazole, DCI or BMT and sulfurizing
agents such as PADS.
[0060] The invention is further explained with reference to the
following non-limiting examples:
[0061] 1) 5'-O-DMTr-C.sup.Bz-P(S,OCNE)-C.sup.Bz-3'-O-Lev (10.8
mmol)
were dissolved in 25 ml dichloromethane and heated to 40.degree. C.
Addition of 120 ml 2-propanol (1:4.8) while the solution was heated
until 55-60.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 77.2 to 88.0% after one purification. By using
5'-O-TBDMS-C.sup.Bz-P(S,OCNE)-C.sup.Bz-3'-O-Lev the purity
increased from 78.1% to 94.8% after 2 purifications.
[0062] 2) 5'-O-DMTr-A.sup.Bz-P(S,OCNE)-G.sup.iBu-3'-O-Lev (15
mmol)
were dissolved in 50 ml dichloromethane and heated to 40.degree. C.
Addition of 450 ml 2-propanol (1:9) while the solution was heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 50.8 to 79.5% after 2 purifications.
[0063] 3) 5'-O-DMTr-A.sup.Bz-P(S,OCNE)-G.sup.iBu-3'-OH (1.63
mmol)
were dissolved in 20 ml dichloromethane and heated to 40.degree. C.
Addition of 400 ml 2-propanol (1:20) while the solution was heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 79.5 to 93.2% after 2 purifications.
[0064] 4) 5'-O-DMTr-C.sup.Bz-P(S,OCNE)-G.sup.iBu-3'-O-Lev (7.58
mmol)
were dissolved in 12 ml dichloromethane and heated to 40.degree. C.
Addition of 130 ml 2-propanol (1:10.8) while the solution was
heated until 50-55.degree. C. After the addition of 2-propanol the
mixture was cooled to room temperature and stirred over night. The
purified product was isolated as a colourless residue. The purity
increased from 62.5 to 88.7% after 2 purifications.
[0065] 5) 5'-O-DMTr-C.sup.Bz-P(S,OCNE)-G.sup.iBu-3'-OH (2.0
mmol)
were dissolved in 10 ml dichloromethane and heated to 40.degree. C.
Addition of 70 ml 2-propanol (1:4.8) while the solution was heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue, The purity increased
from 88.2 to 95.5% after 2 purifications.
[0066] 6) 5'-O-DMTr-T-P(S,OCNE)-G.sup.iBu-3'-O-Lev (10.2 mmol)
were dissolved in 30 ml dichloromethane and heated to 40.degree. C.
Addition of 450 ml 2-propanol (1:15) while the solution was heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 68.0 to 96.2% after one purification.
[0067] 7)
5'-O-DMTr-T-P(S,OCNE)-C.sup.Bz-P(S,OCNE)-C.sup.Bz-P(S,OCNE)-C.su-
p.Bz-3'-O-Lev (1.41 mmol)
were dissolved in 15 ml dichloromethane and heated to 40.degree. C.
Addition of 50 ml 2-propanol (1:3.3) while the solution was heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 68.0 to 89.6% after 2 purifications.
[0068] 8)
5'-O-H-T-P(S,OCNE)-C.sup.Bz-P(S,OCNE)-C.sup.Bz-P(S,OCNE)-C.sup.B-
z-3'-O-Lev (0.33 mmol)
were dissolved in 5 ml dichloromethane and heated to 40.degree. C.
Addition of 11 ml 2-propanol (1:2.2) while the solution is heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 59.2 to 83.7% after two purifications.
[0069] 9)
5'-O-DMTr-T-P(S,OCNE)-C.sup.Bz-P(S,OCNE)-T-P(S,OCNE)-C.sup.Bz-P(-
S,OCNE)-C.sup.Bz-P(S,OCNE)-C.sup.Bz-3'-O-Lev (0.26 mmol)
were dissolved in 5 ml dichloromethane and heated to 40.degree. C.
Addition of 11 ml 2-propanol (1:2.2) while the solution is heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 57.6 to 94.3% after three purifications.
[0070] 10)
5'-O-DMTr-T-P(S,OCNE)-C.sup.Bz-P(S,OCNE)-T-P(S,OCNE)-C.sup.Bz-P-
(S,OCNE)-C.sup.Bz-P(S,OCNE)-C.sup.Bz-3'-OH (0.22 mmol)
were dissolved in 10 ml dichloromethane and heated to 40.degree. C.
Addition of 30 ml 2-propanol (1:3) while the solution is heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 90.1 to 95.2% after one purification.
[0071] 11)
5'-O-DMTr-C.sup.Bz-P(S,OCNE)-G.sup.iBu-P(S,OCNE)-T-P(S,OCNE)-G.-
sup.iBu-3'-O-Lev (0.74 mmol)
were dissolved in 10 ml dichloromethane and heated to 40.degree. C.
Addition of 60 ml 2-propanol (1:6) while the solution is heated
until 55-60.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 47.6 to 80.6% after two purifications.
[0072] 12)
5'-OH-C.sup.Bz-P(S,OCNE)-P(S,OCNE)-T-P(S,OCNE)-G.sup.iBu-3'-O-L- ev
(0.23 mmol)
were dissolved in 20 ml dichloromethane and heated to 40.degree. C.
Addition of 100 ml 2-propanol (1:5) while the solution is heated
until 55-60.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 70.2 to 85.2% after two purifications.
[0073] 13)
5'-O-DMTr-A.sup.Bz-P(S,OCNE)-G.sup.iBu-P(S,OCNE)-C.sup.Bz-P(S,O-
CNE)-G.sup.iBu-P(S,OCNE)-T-P(S,OCNE)-G.sup.iBu-3'-O-Lev (0.2
mmol)
were dissolved in 10 ml dichloromethane and heated to 40.degree. C.
Addition of 45 ml 2-propanol (1:4.5) while the solution is heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 45.2 to 77.9% after two purifications.
[0074] 14)
5'-O-DMTr-A.sup.Bz-P(S,OCNE)-G.sup.iBu-P(S,OCNE)-C.sup.Bz-P(S,O-
CNE)-G.sup.iBu-P(S,OCNE)-T-P(S,OCNE)-G.sup.iBu-3'-OH (0.1 mmol)
were dissolved in 5 ml dichloromethane and heated to 40.degree. C.
Addition of 8 ml 2-propanol (1:1.6) while the solution is heated
until 50-55.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from to 72.2% 88.4 after three purifications.
[0075] 15)
5'-OH-C.sup.Bz-P(S,OCNE)-C.sup.Bz-P(S,OCNE)-A.sup.Bz-P(S,OCNE)--
T-3'-O-Lev (0.72 mmol)
were dissolved in 25 ml dichloromethane and heated to 40.degree. C.
Addition of 120 ml 2-propanol (1:4.8) while the solution is heated
until 55-60.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 75.3 to 92.1% after two purifications.
[0076] 16)
5'-O-H-C.sup.Bz-P(S,OCNE)-G.sup.iBu-P(S,OCNE)-C.sup.Bz-P(S,OCNE-
)-C.sup.Bz-P(S,OCNE)-A.sup.Bz-P(S,OCNE)-T-3'-O-Lev (0.69 mmol)
were dissolved in 100 ml dichloromethane and heated to 40.degree.
C. Addition of 300 ml 2-propanol (1:3) while the solution is heated
until 55-60.degree. C. After the addition of 2-propanol the mixture
was cooled to room temperature and stirred over night. The purified
product was isolated as a colourless residue. The purity increased
from 55.3 to 95.3% after three purifications.
[0077] 17)
5'-O-DMTr-C.sup.Bz-P(S,OCNE)-A.sup.Bz-P(S,OCNE)-T-3'-O-Lev (6.0
mmol)
were dissolved in 25 ml dichloromethane and 1 ml Methanol and
heated to 40.degree. C. Addition of 120 ml 2-propanol (1:4.8) while
the solution is heated until 50-55.degree. C. After the addition of
2-propanol the mixture was cooled to room temperature and stirred
over night. The purified product was isolated as a colourless
residue. The purity increased from 66.2 to 91.8% after two
purifications.
[0078] 18) 5'-O-DMTr-T-P(O,OCNE)-C.sup.Bz-3'-OH (15.1 mmol)
were dissolved in 50 ml dichloromethane at 20.degree.. Addition of
700 ml 2-propanol (1:14) while the solution was stirred at
30.degree.. After the addition of 2-propanol the mixture was
stirred and cooled to room temperature over night. The purified
product was isolated as a colorless residue. Purity increased from
79.2% to 91.3% after two purification.
[0079] 19)
5'-O-DMTr-G.sup.iBu-P(O,OCNE)-T-P(O,OCNE)-T-P(O,OCNE)-G.sup.iBu-
-3'-O-Lev (0.76 mmol)
were dissolved in 10 ml dichloromethane and 1 ml methanol at
20.degree.. Addition of 30 ml 2-propanol (1:3) while the solution
was stirred at 30.degree.. After the addition of 2-propanol the
mixture stirred and cooled to room temperature over night. The
purified product was isolated as a colorless residue. Purity
increased from 79.2% to 91.3% after one purification.
[0080] 20)
5'-O-DMTr-P(O,OCNE)-C.sup.Bz-P(O,OCNE)-G.sup.iBu-P(O,OCNE)-T-P(-
O,OCNE)-T-P(O,OCNE)-G.sup.iBu-3'-O-Lev (0.38 mmol)
were dissolved in 10 ml dichloromethane and 1 ml methanol at
20.degree.. Addition of 50 ml 2-propanol (1:5) while the solution
was stirred at 30.degree.. After the addition of 2-propanol the
mixture stirred and cooled to room temperature over night. The
purified product was isolated as a colorless residue. Purity
increased from 50.8% to 70.3% after one purification.
* * * * *