U.S. patent number RE34,069 [Application Number 07/481,572] was granted by the patent office on 1992-09-15 for process for the preparation of oligonucleotides.
This patent grant is currently assigned to Biosyntech GmbH. Invention is credited to Hubert Koster, Nanda D. Sinha.
United States Patent |
RE34,069 |
Koster , et al. |
September 15, 1992 |
**Please see images for:
( Certificate of Correction ) ( Reexamination Certificate
) ** |
Process for the preparation of oligonucleotides
Abstract
The invention relates to a process for the preparation of
oligonucleotides by the following steps: reaction of a nucleoside
with a phosphine derivative, reaction of the nucleotide derivative
thus obtained with a nucleoside bonded to a polymeric carrier,
oxidation of the carrier-bound nucleoside-nucleotide thus obtained
with formation of phosphotriester groups, blocking of free primary
5'--OH groups, elimination of a protective group from the terminal
5'--OH group, where appropriate single or multiple repetition of
the abovementioned steps to introduce further nucleoside phosphate
or oligonucleoside phosphate units, and cleavage of the
nucleoside-carrier bond and, where appropriate, elimination of all
protective groups present in the oligonucleoside phosphates. The
phosphine derivative used is a compound of the general formula III
##STR1## in which X and L can react with OH groups of the sugar
units in the oligonucleotides, and R.sup.3 is a protective group
which can be liberated by .beta.-elimination.
Inventors: |
Koster; Hubert (Concord,
MA), Sinha; Nanda D. (San Rafael, CA) |
Assignee: |
Biosyntech GmbH (Hamburg,
DE)
|
Family
ID: |
27191213 |
Appl.
No.: |
07/481,572 |
Filed: |
February 16, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
752178 |
Aug 10, 1984 |
04725677 |
Feb 16, 1988 |
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Current U.S.
Class: |
536/25.34;
536/25.3; 536/26.5; 987/189; 536/26.71 |
Current CPC
Class: |
C07H
21/00 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C07H 015/12 (); C07H
017/00 () |
Field of
Search: |
;536/27,28,29 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0040099 |
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Nov 1981 |
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EP |
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81302110.2 |
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Nov 1981 |
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EP |
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0061746 |
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Oct 1982 |
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EP |
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0064796 |
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Nov 1982 |
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EP |
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82200564.1 |
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Nov 1982 |
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EP |
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0090789 |
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Oct 1983 |
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EP |
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83870031.8 |
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Oct 1983 |
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EP |
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0131993 |
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Jan 1985 |
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EP |
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8420951.6 |
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Jan 1985 |
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EP |
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Other References
Clesen et al., "Tetrahedron Letters", vol. 25, No. 12, pp.
1307-1310, 1984. .
Lelsinger et al., "Jour. of the Amer. Chem. Soc." vol. 98, No. 12,
Jun. 1976, pp. 3655-3661. .
Narang, "Tetrahedron", vol. 39, No. 1, pp. 3-22, 1983. .
Marugg et al., "Recl. Trav. Chim. Pays-Bas" vol. 103, pp. 97-98,
1984. .
Ogilvie, K. K. et al., Can J. Chem 58:2686 (1980). .
V. Amarnath and A. D. Broom, Chemical Reviews 77(2)183 (1977).
.
H. Koster et al., Nucleic Acids Research Symposium Series No. 7
(1980) pp. 39-61. .
Caruthers, M. H., Science 230:281 (1985). .
Zon, G. et al., Nucleic Acids Research 13(22):8181 (1985). .
Urdea, M. S. et al. Nucleic Acids Research Symposium Ser. 16 (1985)
pp. 257-260. .
Gao, X. et al. Nucleic Acids Research 13(2):573 (1985). .
Beaucage and Caruthers (1981) Tet. Lett. 22:1859-1862..
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Primary Examiner: Griffin; Ronald W.
Attorney, Agent or Firm: Hamilton, Brook, Smith &
Reynolds
Claims
We claim:
1. A process for the preparation of oligonucleotides of the
.[.general.]. formula I ##STR13## in which B denotes a nucleoside
base, R.sup.1 denotes hydrogen, hydroxyl or hydroxyl which is
protected by .Iadd., where appropriate, .Iaddend.a removable
protective group and n denotes an integer from 1 to 200, comprising
the steps of
(a) reacting a nucleoside of the .[.general.]. formula II ##STR14##
in which R.sup.1 as defined as above, and R.sup.2 denotes a
removable protective group and B' denotes the nucleotide base B
protected by the protective groups which can be eliminated, with a
phosphine derivative of the .[.general.]. formula III ##STR15## in
which R.sup.3 is a protective group which can be eliminated, and X
and L are groups which react with hydroxyl groups in the sugar
moieties of the nucleotides or nucleosides, in the presence of a
base to thereby form a nucleotide phosphite
(b) reacting the nucleotide phosphite obtained in step (a) and
represented by the formula IV: ##STR16## in which B', R.sup.1,
R.sup.2, R.sup.3 and L are as defined above, with a nucleoside, of
the .[.general.]. formula V, bound to a polymeric carrier ##STR17##
in which B' and R.sup.1 are as defined above and C denotes the
polymeric carrier;
(c) oxidizing the carrier-bound nucleoside-nucleotides obtained in
step (b) and represented by the formula: ##STR18## in which B',
R.sup.1, R.sup.2, R.sup.3 and C are as defined above, with
formation of phosphotriester groups,
(d) blocking free primary 5'--OH groups, which have not been
reacted in the reaction according to step (b), with permanent
protective groups;
(e) eliminating the protective group R.sup.2 ;
(f) optionally repeating steps (a) to (e) to introduce further
nucleoside phosphate or oligonucleoside phosphate units; and
(g) cleaving the nucleoside carrier bond and optionally eliminating
the protective groups present in the oligonucleoside
phosphates,
which process comprises using in step (a) as the phosphine
derivative of the .[.general.]. formula III a compound in which
R.sup.3 denotes a group of the formula VII ##STR19## in which the
groups Y, which can be identical or different, represent hydrogen,
methyl, and/or ethyl and Z represents an electron-attracting group,
where, in the phosphine derivative of the formula III, X is
chlorine, bromine, CN or SCN and L is CN or SCN, a secondary amino
radical of the formula (VIII)
where the groups R.sup.4 are primary, secondary, or tertiary alkyl
radicals having .Badd.1-10 carbon atoms, or together form a
cycloalkyl radical having 5-7 carbon atoms, which can contain one
or two nitrogen, oxygen, or sulfur atoms as hereoatoms, or are
imidazole, triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole,
pyrrole, benzotriazole, benzohydroxytriazole, imidazole substituted
in the phenyl moiety, triazole substituted in the phenyl moiety,
tetrazole substituted in the phenyl moiety, 3-nitro-1,2,4-triazole
substituted in the phenyl moiety, thiazole substituted in the
phenyl moiety, pyrrole substituted in the phenyl moiety,
benzotriazole substituted in the phenyl moiety, or
benzohydroxytriazole substituted in the phenyl moiety.
2. The process as claimed in claim 1, in which is used a phosphine
derivative of the formula III in which X is chlorine or bromine,
and L is a secondary amino radical of the formula (VIII)
where the groups R.sup.4 are primary, secondary or tertiary alkyl
radicals having 1-10 carbon atoms, or together form a cycloalkyl
radical having 5-7 carbon atoms, which can contain one or two
nitrogen, oxygen or sulfur atoms as heteroatoms, or are imidazole,
triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole, pyrrole,
benzotriazole, benzohydroxytriazole, imidazole substituted in the
phenyl moiety, triazole substituted in the phenyl moiety, tetrazole
substituted in the phenyl moiety, 3-nitro-1,2,4-triazole
substituted in the phenyl moiety, thiazole substituted in the
phenyl moiety, pyrrole substituted in the phenyl moiety,
benzotriazole substituted in the phenyl moiety, or
benzohydroxytrizole substituted in the phenyl moiety.
3. The process as claimed in claim 1 or 2, in which is used a
phosphine derivative of the formula (III) in which X is chlorine, L
is an N,N-dimethylamino, diethylamino or -diisopropylamino group or
N-morpholino group, and R.sup.3 is a .beta.-cyanoethyl group.
4. A method of preparing oligonucleotides of the .[.general.].
formula: ##STR20## wherein B is a nucleoside base, R.sup.1 is
hydrogen, hydroxyl or hydroxyl which is protected by removable
nucleoside protective groups, and n denotes an integer from 1 to
200, comprising the steps of:
(a) reacting a nucleotide phosphite represented by the formula:
##STR21## wherein B' is a nucleoside base B protected by .Iadd.,
where appropriate, .Iaddend.a base protective group which can be
eliminated, R.sup.1 is as defined above, R.sup.2 is 4,4'
dimethoxytrityl or 4,4',4" trimethoxytrityl; and L is
N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino, or
N-morpholino, with a nucleoside bound to a polymeric carrier, of
the .[.general.]. formula: ##STR22## wherein B' and R.sup.1 are as
defined above and C represents the polymeric carrier, to produce a
carrier bound nucleoside-nucleotide of the formula: ##STR23##
wherein B', R.sup.1, R.sup.2, R.sup.3 and C are as defined above;
(b) oxidizing the carrier bound nucleoside-nucleotide;
(c) blocking free primary 5'--OH groups, which have not been
reacted in the reaction of step (a), with permanent protective
groups;
(d) eliminating the protecting group R.sup.2 ;
(f) repeating steps (a) to (d) to introduce further nucleoside
phosphate units; and
(g) cleaving the nucleoside-carrier bond and optionally eliminating
protective groups present in the oligonucleoside phosphates.
5. A method of synthesizing oligonucleotides, comprising the steps
of:
(a) coupling a nucleoside .beta.-cyanoethyl-protected
phosphoramidite to a carrier-bound nucleoside to produce a carrier
bound nucleoside-nucleotide having a phosphite triester
linkage;
(b) oxidizing the phosphite triester to form a phosphate
triester;
(c) optionally coupling additional nucleoside
.beta.-cyanoethyl-protected phosphoramidites to the carrier bound
nucleoside-nucleotide and, after each coupling step, oxidizing the
resulting phosphite triester to form a phosphate triester, to form
a carrier bound polynucleotide;
(d) removing the .beta.-cyanoethyl protecting groups; and
(e) removing the polynucleotide from the carrier.
6. A method of claim 5, wherein the nucleoside .beta.-cyanoethyl
phosphoramidite is a nucleoside .beta.-cyanoethyl
N,N-dimethylphosphoramidite, N,N-diethylphosphoramidite,
.[.N,N-dipropylphosphoramidite.].
.Iadd.N,N-diisopropylphosphoramidite .Iaddend.or N,N-morpholino
phosphoramidite.
7. A method of claim 6, wherein the carrier is controlled port
glass.
8. A method of claim 7, wherein the .beta.-cyanoethyl protecting
group is removed with simultaneous removal of the polynucleotide
from the carrier, by concentrated aqueous ammonia.
9. In a method of polynucleotide synthesis, comprising sequentially
coupling nucleotide phosphoramidites to produce a polynucleotide,
wherein the phosphorus atoms of the nucleotide phosphoramidites are
protected by methyl groups, the improvement wherein the phosphorus
protecting group are cyanoethyl groups.
10. A method of synthesizing oligonucleotides, comprising the steps
of:
a. coupling a nucleoside .beta.-cyanoethyl-protected
phosphoramidite to a nucleoside, the nucleoside being bound to a
polymeric carrier via an ester bond to produce a carrier-bound
nucleoside-nucleotide having a phosphite triester linkage;
b. oxidizing the phosphite triester to form a phosphate triester
linkage;
c. sequentially coupling additional nucleoside .beta.-cyanoethyl
protected phosphoramidite to the carrier-bound
nucleoside-nucleotide, and after each coupling step, oxidizing the
resulting phosphite triester linkage to a phosphate triester to
produce a carrier-bound polynucleotide;
d. treating the carrier bound polynucleotide with concentrated
ammonia to remove the .beta.-cyanoethyl phosphate protecting group
and hydrolyzing the ester bond to the carrier to remove the
polynucleotide from the carrier.
11. A method of claim 10, wherein the nucleoside .beta.-cyanoethyl
phosphoramidite is a nucleoside .beta.-cyanoethyl
N,N-dimethylphosphoramidite, N,N-diethylphosphoramidite;
.[.N,N-dispropylphosphoramidite.].
.Iadd.N,N-diispropylphosphoramidite .Iaddend.or N,N-morpholino
phosphoramidite.
12. A method of claim 10, wherein the carrier is controlled pore
glass.
13. A protected nucleotide having the formula: ##STR24## where, B'
is a nucleoside base protected .Iadd., where appropriate,
.Iaddend.by a base protective group which can be eliminated;
R.sup.1 is H, OH, or a hydroxyl group which is protected by a
removable nucleoside protective group;
R.sup.2 is a removable protective group;
R.sup.3 is ##STR25## L is CN, SCN, or NR.sub.2.sup.4 ; R.sup.4 is a
primary, secondary or tertiary alkyl radical having 1-10 carbon
atoms, or R.sub.2.sup.4 is a cycloalkyl radical having 5-7 carbon
atoms or a cycloalkyl radical having 5-7 atoms comprising atoms and
one or two nitrogen, oxygen or sulfur atoms as heteroatoms;
Y is H, CH.sub.3, or CH.sub.2 CH.sub.3 ; and Z is a .[.halgen.].
.Iadd.halogen .Iaddend.CN, NO.sub.2, phenyl substituted in the o,
o' or p positions with a halogen, CN or NO.sub.2 radical,
phenylthio, phenylsulfoxy, or phenylsulfonyl, where the phenyl
radicals, may be substituted in the o, o' or p positions with a
halgen, CN or NO.sub.2 radical, or where the group ##STR26## may be
replaced by CF.sub.3, CCl.sub.3, or CBr.sub.3.
14. A protected nucleotide as in claim 13, wherein Z is CN.
15. A protected nucleotide as in claim 14, wherein R.sub.3 l is
CH.sub.2 --CH.sub.2 --CN.
16. A protected nucleotide as in claim 13, wherein R.sub.2 is
4,4'-dimethoxytrityl or 4,4"-trimethoxytrityl.
17. A protected nucleotide having the formula: ##STR27## where,
R.sup.1 is H, OH, or a hydroxyl group which is protected .Iadd.,
where appropriate, .Iaddend.by a removable nucleoside protective
group;
R.sup.2 is 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl;
B' is a nucleoside base protected by a base protective group which
can be eliminated
R.sup.3 is CH.sub.2 --CH.sub.2 --CN; and
L is N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino or
N-morpholino.
18. A protected nucleotide having the formula: ##STR28## where,
R.sup.1 is H, OH, or a hydroxyl group which is protected by a
protective group selected from the group consisting of trityl
groups, acyl groups and silyl ether groups;
R.sup.2 is 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl;
B' is a nucleoside base selected from the group consisting of
adenine, guanine, cytosine, thymine, uracil and analogs thereof
which are protected by acyl groups or Schiff bases;
R.sup.3 is CH.sub.2 --CH.sub.2 --CN; and
L is N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino, or
N-morpholino.
19. A protected nucleotide of claim 18, wherein .[.R.sub.1 .].
.Iadd.R.sup.1 .Iaddend. is H and B' is adenine, guanine, cytosine,
thymine or uracil wherein the adenine or .[.guanine.].
.Iadd.cytosine .Iaddend.is protected by a benzoyl group and the
guanine is protected by an .[.isobutyl.]. .Iadd.isobutyryl
.Iaddend.group.
Description
DESCRIPTION
The invention relates to a process for the preparation of
oligonucleotides of the general formula I indicated in claim 1. The
oligonucleotides prepared according to the invention have defined
sequences and can be used as specific primers and probes and are of
great importance for the synthesis of complete genes
(Arzneimittelforschung 30, 3a, 548, (1980)).
According to the most recent state of the art, oligonucleotides are
prepared by either the phosphate or phosphite triester method using
polymeric carriers (Nachr. Chem. Tech. Lab. 29, 230 (1981)). In
order to be able to construct defined sequences, it is necessary
for the individual units (nucleoside or nucleotides) to be provided
with suitable protective groups. In this context, base-labile acyl
groups are generally used for the protection of the exocyclic amino
groups on the heterocyclic nucleobases, and a base-labile ester
bond is used to attach the oligonucleotide chain to the polymeric
carrier in a customary manner, and acid-labile trityl ether groups
are used to protect the primary 5'--OH group. The phosphate
protective group used in the phosphate triester method is
customarily either the 2-chlorophenyl or the 4-chlorophenyl group,
with an ester-type bond, which can only be removed by attack of a
base or a nucleophile on the phosphorus atom. This type of step is
inherently undesirable since it involves the risk of cleavage of
the internucleotide phosphate ester bond. This risk has been
greatly reduced by the use of oximate anions (Tetrahedron Lett. 19,
2727 (1978)), although these also attack the phosphorus atom in an
undesired manner in the crucial step and, moreover, have the
disadvantage that a relatively small amount of desired
oligonucleotide is contaminated with every large amounts of
involatile salts which are difficult to extract. This not only
makes the working up and subsequent purification of the synthesized
oligonucleotide difficult but also leads to considerable material
losses.
In the phosphite triester method, the methyl group with an
ester-type bond is customarily used as the phosphate protective
group which can be removed by attack of a nucleophile on the methyl
C atom (J. Amer. Chem. Soc. 99, 3526 (I1977)). Since attack on the
P atom is avoided, there is likewise avoidance of the risk of
cleavage of the internucleotide bond. The nucleophile customarily
used is thiophenol/triethylamine, which are unpleasant to
manipulate and, moreover, lead to involatile compounds which are
difficult to extract and which, as mentioned above, both make
work-up difficult and lead to considerable material losses.
Although the actual synthesis of oligonucleotides by the solid
phase/phosphite or phosphate triester method takes place very
efficiently and rapidly, the preparation of oligonucleotides of
defined sequence remains very time-consuming. This is primarily due
to the problems of the subsequent work-up and purification which
take up a multiple of the actual synthesis time. The process of the
invention operates at this point and provides in this connection a
crucial technical improvement.
In order to obtain compounds of the formula I indicated in claim 1,
##STR2## in which B denotes a nucleobase, for example adenine (A),
guanine (G), cytosine (C), thymine (T) or uracil (U) or their
analogs, and R.sup.1 denotes hydrogen, hydroxyl or hydroxyl which
is protected by the protective groups customary in nucleotide
chemistry, and n denotes an integer from 1 to 200, according to the
invention a variety of defined reaction steps are carried out, as
follows:
(a) Reaction of a nucleoside of the general formula II.
##STR3##
R.sup.1 of the general formula II can be hydrogen; in this case the
compounds of the formula I are oligodeoxynucleotides. The group
R.sup.1 can also be hydroxyl or hydroxyl which is, where
appropriate, protected by the protective groups customary in
nucleotide chemistry. Examples of protective groups of this type
are trityl, monomethoxytrityl and dimethoxytrityl, acyl, for
example acetyl, benzoyl; tetrahydropyranyl,
methoxytetrahydropyranyl, o-nitrobenzyl and silyl ethers, such as,
for example, t-butyldiphenylsilyl ethers. A general review of the
protective groups customary in nucleotide chemistry is to be found
in, for example, Tetrahedron 1981, pages 363-369, Liebigs Ann.
Chem. 1978, 839-850, and Nucleic Acids Research, Symposium Series
No. 7, 1980, 39-59.
R.sup.2 is likewise a protective group customary in nucleotide
chemistry according to the above mentioned publications, preferably
the acid-labile 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl
group. B' can likewise have a protective group customary in
nucleotide chemistry according to the above mentioned prior
publications.
The nucleoside of the formula II is reacted according to the
invention with a phosphine derivative of the general formula III
according to claim 1. ##STR4##
In the general formula, X denotes chlorine, bromine, CN or SCN; L
denotes chlorine, bromine, CN, SCN or an amino radical of the
formula--NR.sub.2.sup.4 (formula VIII), where the groups R.sup.4
denote primary, or secondary or tertiary alkyl radicals having 1-10
carbon atoms, or together denote a cycloalkyl radical having 5-7
carbon atoms, optionally with alkyl branches, and/or can contain
one or two nitrogen, oxygen and/or sulfur atoms as heteroatoms. The
group L can also form a reactive heterocyclic radical, the
imidazolyl, triazolyl, tetrazolyl, 3-nitro-1,2,4-triazolyl,
thiazolyl, pyrrolyl, benzotriazolyl (optionally with substituents
in the phenyl moiety) or benzohydroxytriazolyl (optionally with
substituents in the phenyl ring) and the like.
R.sup.3 is the phosphine derivative of the general formula (III)
is, according to the invention, a group of the general formula VII,
##STR5## which can be removed with the aid of bases by
.beta.-elimination and in which Y denotes hydrogen, methyl or
ethyl. Z represents an electron-attracting group, for example,
halogen, such as fluorine, chlorine or bromine, CN or NO.sub.2. Z
can also denote phenyl, phenylthio, phenylsulfoxy or
phenylsulfonyl, it being possible for the phenyl radicals to be
substituted in the o,o'-position and/or p-position with halogen, CN
or NO.sub.2. It is also possible for one of the groups CF.sub.3,
CCl.sub.3 or CBr.sub.3 to replace the group ##STR6##
The reaction according to step a takes place in the presence of an
organic base.
(b) Reaction of the nucleoside-.[.phosphorous.]. .Iadd.phosphorus
.Iaddend.acid derivative, of the formula IV, obtained in step a.
##STR7##
The reaction of the compound according to formula IV is carried out
with a nucleoside of the general formula V according to claim 1,
which is bound to a polymeric carrier. ##STR8## It is possible to
use soluble or insoluble, that is to say crosslinked, polymeric
carriers, for example modified silica gel, glass, especially
"controlled pore glass", polyester, polyamide, polyvinyl alcohol,
polysiloxane, polystyrene or the like. Ester bonds are suitable and
preferred for the attachment between the carrier and the
nucleoside, including those derived from the levulinyl or
.beta.-benzoylpropionyl radical; the latter ester bonds can be
cleaved with hydrazine under neutral conditions. The acid-labile
trityl ether bond, optionally with substituents in the phenyl
rings, is also suitable as a method of attachment, compare Liebigs
Ann. Chem. 1974, 959.
(c) Oxidation of the carrier-bond nucleotide-nucleoside, of the
general formula VI, obtained in step b. ##STR9##
Oxidation leads to a phosphate group; this can be carried out with,
for example, iodine/H.sub.2 O, H.sub.2 O.sub.2 or organic peracids
or, in general, by oxidation by introduction of O, S or Se.
(d) Blocking of free primary 5'--OH groups which have not been
reacted in the reaction according to step b (in the product of the
formula V).
These free hydroxyl groups are blocked with a permanent protective
group, for example by reaction with acetic anhydride.
(e) Elimination of the protective groups(s) R.sup.2.
The elimination is carried out using, for example, a protonic acid
or Lewis acid, such as ZnBr.sub.2 or dialkylaltuminum chloride,
when R.sup.2 represents a trityl group or a methoxy derivative
thereof.
(f) Introduction of further nucleoside phosphate or oligonucleoside
phosphate units.
Steps a-e can be repeated to introduce at least one nucleoside
phosphate moiety. Of course, when oligonucleoside phosphate units
are employed, the chains are lengthened by more than one nucleoside
phosphate unit.
(g) Elimination of all protective groups.
This elimination can be carried out in such a manner that, using
aqueous ammonia, in one step the N-acyl groups on the heterocyclic
bases, the ester bond between the oligonucleotide and the carrier
(the latter can, where appropriate, also be cleaved with hydrazine
under neutral conditions) an the phosphate protective group are
eliminated by .beta.-elimination in accordance with the general
scheme 1 at the end of the description. An oligonucleotide having
only a 5'-terminal trityl protective group is then obtained, and
this can be purified directly in a manner known per se, after
removal of the volatile base (ammonia), by high-pressure liquid
chromatography (HPLC) on reverse phase material.
The intermediates of the general formula IV according to claim 1
are new compounds. They are in the form of very stable compounds
which can be prepared in the pure form and are easy to manipulate
but nevertheless are very reactive in the sense of forming
internucleotide bonds. The use of R.sup.3 as a protective group
which can be removed by bases via .beta.-elimination makes is
possible for the first time to eliminate all the protective groups,
apart from the 5'-trityl group, in one step where, in an
advantageous manner, by the use of volatile bases the desired
oligonucleotide is contaminated with foreign materials to only a
very small extent and thus directly afterwards can be purified by
reverse phase HPLC due to the hydrophobic 5'-trityl group which is
still present.
A further advantage of the process of the invention results from
the fact that, due to the removal of the protective group by
.beta.-elimination, no attack on the P-atom takes place and thus
none of the newly formed internucleotide bonds can be cleaved
during the deprotection. Thus, the process of the invention takes
very much less time and leads to overall purer products than do the
processes hitherto available.
The invention is illustrated in detail below by means of examples,
the phosphine derivatives used being those in which R.sup.3 is a
.beta.-cyanoethyl group. Details of the reaction and physical
characteristics of the compounds prepared can be seen in schemes 2
and 3, Table 1, and FIGS. 1-7 at the end of the description.
EXAMPLE 1
Preparation of phosphine derivatives of the general formula
III:
.beta.-Cyanoethyl phosphoramidochloridite:
A general summary of the reaction can be seen in scheme 2.
Apart from some improvements, dichloro-.beta.-cyanoethoxyphosphine
(1) is prepared as in Can. J. Chem. 58, 2686 (1980):
300 ml of ether and 79.0 g (1 mol) of pyridine are added through a
dropping funnel to 137.5 g (1.0 mol) of PCl.sub.3 in a three-neck
flask; the mixture is cooled to -78.degree. C. under argon. Then a
solution of 71.0 g (1 mol) of .beta.-cyanoethanol in 150 ml of dry
ether is added dropwise over the course of 1 to 1.5 hours. The
cooling bath is removed; stirring is continued at room temperature
for a further 3 hours (where necessary, another 300 ml of ether are
added in order to ensure better stirrability). The stirrer and
dropping funnel are removed under argon; the mixture is stored at
0.degree. C. overnight. The solid salts are removed under argon;
the precipitate is washed twice with 75 ml of ether each time. The
combined organic phases are concentrated in vacuo; the residue is
finally distilled in vacuo; boiling point 70.degree.-75.degree.
C./0.4 mm Hg.
.beta.-Cyanoethyl phosphoramidochloridite (3):
A solution of 17.2 g (0.1 mol) of .beta.-cyanoethyl
phosphorodichloridite (1) in 60 ml of ether is added dropwise, over
the course of 1 to 1.5 hours, to a solution of the
N-trimethylsilylated secondary amine (0.1 mol) or secondary amine
(0.2 mol) in 30 ml of ether at -20.degree. C. under argon. After
stirring at room temperature for 20 hours, the amine hydrochloride
is removed; the remaining solution is concentrated. The residue is
finally distilled in vacuo in a short-path distillation
apparatus.
The physical properties of the compounds thus obtained are
summarized in Table 1.
FIGS. 1a, 1b and 1c show .sup.31 P NMR spectra of three different
.beta.-cyanoethyl phosphoramidochloridites.
The N-morpholine derivative is too unstable to heat for
distillation to be possible. Nevertheless, the preparation is so
pure that the residue can be used directly for the preparation of
the activated nucleoside derivatives. The purity is usually greater
than 95% according to the .sup.31 P NMR spectra.
Nucleoside .beta.-cyanoethyl phosphoramidites:
The preparation of the appropriately protected nucleoside
.beta.-cyanoethyl phosphoramidites can be seen in scheme 3.
The synthesis is analogy to Tetrahedron Lett. 22, 1859 (1981), with
some improvements, provides good yields.
3.0 mmol of the N-protected 5'-dimethoxytritylated deoxynucleoside
are dried azeotropically using THF/toluene, dissolved in 15 ml of
dry THF, and 12.0 mmol of N,N,N-diisopropylethylamine are added.
6.0 mmol of the .beta.-cyanoethyl phosphoramidochloridite are added
dropwise to the solution under argon, with vigorous stirring, over
the course of 2 minutes. After a short time (2 to 5 minutes), the
amine hydrochloride precipitates out. The suspension is stirred for
a further 30 to 40 minutes. The amine hydrochloride is filtered off
under argon and thoroughly washed with dry THF (10 to 15 ml). The
entire organic phase is concentrated and dissolved in
argon-saturated ethyl acetate (100 ml). The organic phase is
extracted twice with 50 ml each time of argon-saturated 10% aqueous
sodium carbonate solution. The organic phases are dried with sodium
sulfate and evaporated under reduced pressure to give a foam. The
foam is dissolved in a little ethyl acetate or toluene and
precipitated in n-hexane at -78.degree. C. The activated
nucleosides are stable for several months when stored at
-20.degree. C. under argon.
FIG. 2 shows the .sup.31 P NMR spectrum of one of the activated
deoxynucleosides.
Synthesis of d(CGGTACCG)
100 mg of "controlled pore glass" (CPG) loaded with a total of 8
umol of N-isobutyryldeoxyguanine (compare Tetrahedron Lett. 24, 747
(1983)) are consecutively condensed with the 5'-dimethoxytritylated
N-acylated .beta.-cyanoethyl N,N-diisopropylphosphoramidites of the
deoxynucleosides C, C, A, T, G, G and C, in each case 20 to 25
equivalents of the phosphoramidite in acetonitrile being activated
with 75-80 equivalents of sublimed tetrazole. The condensations are
complete within 30 minutes at the most; the coupling yield is
greater than 94%. After each condensation, oxidation with I.sub.2
/H.sub.2 O and blocking of unreacted 5'--OH groups with acetic
anhydride are carried out. Then the dimethoxytrityl group is
eliminated either with 3% trichloroacetic acid in nitromethane/1%
methanol or ZnBr.sub.2 /nitromethane/1% H.sub.2 O.
The overall yield of the protected octanucleotide at the end of all
condensation steps is 55% based on carrier-bound
deoxyguanosine.
Complete deprotection and cleavage off from the carrier is achieved
in one step by reaction of the glass beads with concentrated
aqueous ammonia (3 ml) at 50.degree. C. for 16 hours. The glass
beads are then thoroughly washed with 50% aqueous methanol (3 times
with 3 ml each time). The liquid phase is removed by evaporation
(removal of the methanol) and freeze-drying. Then an aliquot is
filtered through a millipore filter and purified by HPLC or RP 18
as can be seen in FIG. 3.
The fractions which contain the 5'-dimethoxytritylated
oligonucleotide are collected; the volatile buffer is removed in a
rotary evaporator in vacuo, 1 ml of 80% strength acetic acid is
added to the residue. After 45 minutes at room temperature, the
acetic acid is removed by freeze-drying.
The material thus obtained is phosphorylated in the customary
manner (Liebigs Ann. Chem. 1978, 982) with T4-polynucleotide kinase
and .gamma.-.sup.32 P-ATP. The resulting product is characterized
by polyacrylamide gel electrophoresis comparing with a
homo-oligo-dT chain length standard (Nucleic Acids Res. 6, 2096
(1979), FIG. 4) and by sequencing according to FIG. 5 (Liebigs Ann.
Chem. 1978, 982).
FIGS. 6a to 6c show the results (HPLC, gel electrophoresis,
sequencing) of the synthesis of d(GGGATCCC) using the nucleoside
.beta.-cyanoethyl N,N-dimethylphosphoramidites, FIGS. 6a to 6c show
the results (HPLC, g, electrophoresis, sequencing) of the synthesis
of d(GGGATATCCC) using the nucleoside .beta.-cyanoethyl
N,N-morpholinophosphoramidites.
The results given in FIGS. 3, 6a and 7a were obtained by using a
gradient from 10 to 25 vol. % CH.sub.3 CN, 5 min, and 25 to 29 vol.
% CH.sub.3 CN, 30 min, in 0.1M triethylammonium acetate at pH 7.0.
##STR10##
TABLE 1
__________________________________________________________________________
Physical data of .beta.-cyanoethyl phosphoramidochloridites 3a 3b
3c Compound L = N,N-dimethylamino L = N,N-diisopropylamino L =
N-morpholino
__________________________________________________________________________
Boiling point 90-92.degree./0.6 nm 103-5.degree./0.06 nm --
Chemical shift.sup.(2) 175.97 ppm 179.82 ppm 168.22 ppm in .sup.31
P NMR in CH.sub.3 CN Chemical shift in 4.01, 4.17(2t, POCH.sub.2,
2H) 4.02, 4.2(2t, POCH.sub.2, 2H) 3.96, 4.1(2t, POCH.sub.2, 2H)
.sup.1 H NMR in ppm 2.71(t, CH.sub.2CN, 2H) 3.8(m, N(CH).sub.2, 2H)
3.67(t, O(CH.sub.2).sub.2, 4H) 2.7(d, N(CH.sub.3).sub.2, 6H)
2.77(t, CH.sub.2 CH, 2H) 3.17(m, PN(CH.sub.2).sub.2, 4H) 1.29(d,
NCH(CH.sub.3).sub.2, 12H) 2.74(t, CH.sub.2CN.sub.2, 2H) Mass
spectrum ##STR11## ##STR12## (Cl), 136(C.sub.2 H.sub.6 N), 110
(Cl), 166(C.sub.3 H.sub.4 NO), (Cl), 152(C.sub.3 H.sub.4 NO),
(C.sub.3 H.sub.4 NO) 136(C.sub.6 H.sub.14 N) 136(C.sub.4 H.sub.8 O)
__________________________________________________________________________
.sup.(1) The crude product after removal of amine hydrochloride and
compounds volatile under high vacuum at room temperature has a
purity of 93-95% according to the .sup.31 P NMR spectrum .sup.(2)
The chemical shifts are determined in acetoned.sub.6 with 80%
strength H.sub.3 PO.sub.4 as the external standard.
* * * * *