U.S. patent number 6,762,298 [Application Number 09/792,799] was granted by the patent office on 2004-07-13 for thermolabile phosphorus protecting groups, associated intermediates and methods of use.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Serge L. Beaucage, Andrzej Grajkowski, Andrzej Wilk.
United States Patent |
6,762,298 |
Beaucage , et al. |
July 13, 2004 |
Thermolabile phosphorus protecting groups, associated intermediates
and methods of use
Abstract
The invention provides a method of thermally deprotecting the
internucleosidic phosphorus linkage of an oligonucleotide, which
method comprises heating a protected oligonucleotide in a fluid
medium at a substantially neutral pH, so as to deprotect the
oligonucleotide. The present invention further provides a method of
synthesizing an oligonucleotide using the thermal deprotection
method described above, and novel oligonucleotides and
intermediates that incorporate the thermolabile protecting group
used in accordance with the present invention.
Inventors: |
Beaucage; Serge L. (Silver
Spring, MD), Wilk; Andrzej (Bethesda, MD), Grajkowski;
Andrzej (Bethesda, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
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Family
ID: |
22421815 |
Appl.
No.: |
09/792,799 |
Filed: |
February 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCTUS0004032 |
Feb 16, 2000 |
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Current U.S.
Class: |
536/25.31;
536/25.3; 536/25.33; 536/25.34 |
Current CPC
Class: |
C07H
19/10 (20130101); C07H 19/20 (20130101); C07H
21/00 (20130101); Y02P 20/55 (20151101) |
Current International
Class: |
C07H
21/00 (20060101); C07H 19/10 (20060101); C07H
19/00 (20060101); C07H 021/00 (); C07H 021/02 ();
C07H 021/04 () |
Field of
Search: |
;536/25.33,26.7,26.8,27.62,27.81,28.51,28.53,25.34,26.74,27.8,25.3,25.31 |
References Cited
[Referenced By]
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WO 00/56749 |
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Sep 2000 |
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WO |
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Primary Examiner: Wilson; James O.
Assistant Examiner: Lewis; Patrick
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This patent application is a continuation-in-part of copending
international patent application No. PCT/US00/04032, filed Feb. 16,
2000, pending, which claims priority to U.S. provisional patent
application No. 60/125,867, filed Mar. 24, 1999.
Claims
What is claimed is:
1. A method of deprotecting an oligonucleotide, which method
comprises heating an oligonucleotide of the formula: ##STR40##
in a fluid medium, at a substantially neutral pH, at a temperature
up to about 100.degree. C. to produce an oligonucleotide of the
formula: ##STR41##
wherein: R is a thermolabile protecting group of the formula:
##STR42## R.sup.1 is H, R.sup.1a, OR.sup.1a, SR.sup.1a or NR.sup.1a
R.sup.1a', wherein R.sup.1a and R.sup.1a' are the same or different
and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an
aryl, or an aralkyl; or, when R.sup.1 is NR.sup.1a R.sup.1a',
R.sup.1a and R.sup.1a', together with the nitrogen atom to which
they are bonded, comprise a heterocycle containing from 3 to about
7 atoms in the ring skeleton thereof; X.sup.1 is O, S or Se; X is O
or S; Z is O, S, NR.sup.2a, CR.sup.2a R.sup.2a' or CR.sup.2a
R.sup.2a CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b
and R.sup.2b' are the same or different and each is H, an alkyl, an
alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; or
R.sup.1a or R.sup.1a', in combination with any of R.sup.2a,
R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X of the
protecting group to which they are bonded, comprise a ring
containing from 3 to about 7 atoms in the skeleton thereof;
provided that R.sup.1 is not R.sup.1a when Z is S, Z is not
CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b' when
R.sup.1 is SR.sup.1a, and Z is not O or S when R.sup.1 is H;
R.sup.2, R.sup.2', R.sup.3 and are the same or different and each
is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or
an aralkyl, or R.sup.2 or R.sup.2', in combination wit R.sup.3 or
R.sup.3', together with the carbon atoms to which they are bonded,
comprise a cyclic substituent of the formula: ##STR43## wherein p
is an integer from 0-6 and a-d are the same or different and each
is selected from the group consisting of H, an alkyl, a nitro, a
dialkylamino, an alkoxy, an alkylthio, a cyano and a halogen,
provided that the aromatic ring, which bears substituents a-d, is
one carbon removed from the phosphate oxygen of formula (IIIa),
wherein R.sup.1, R.sup.2a, R.sup.2a', R.sup.2b, R.sup.2b', R.sup.2,
R.sup.2', R.sup.3 or R.sup.3' is unsubstituted or substituted with
one or more substituents, which are the same or different, selected
from the group consisting of OR.sup.8, CN, NO.sub.2, N.sub.3, and a
halogen, wherein R.sup.8 is H or an alkyl; R.sup.4 and R.sup.15 are
the same or different and each is H, a hydroxyl protecting group,
or a solid support; Q.sup.1 is a nucleoside, an oligonucleotide or
an oligomer comprising an oligonucleotide; n is an integer from 1
to about 300; and Q is a nucleoside, an oligonucleotide or an
oligomer comprising an oligonucleotide and, when n is greater than
1, each Q is independently selected, provided that the deprotection
is not by an enzyme.
2. The method of claim 1, wherein Q or Q.sup.1 comprises a
nucleoside of the formula: ##STR44##
wherein: B is a labeling group, an alkyl, an alkenyl, an alkynyl, a
cycloalkyl, an aryl, a heteroaryl, a heterocycloalkyl, an aralkyl,
an amino, an alkylamino, a dialkylamino, a purine, a pyrimidine,
adenine, guanine, cytosine, uracil, or thymine, wherein B is
unsubstituted or substituted with one or more substituants, which
are the same or different, selected from the group consisting of a
nucleobase protecting group, R.sup.11, OR.sup.11, NHR.sup.11,
NR.sup.11 R.sup.12, N.dbd.C--NR.sup.11' R.sup.12', CN, NO.sub.2,
N.sub.3, and a halogen, wherein R.sup.11 and R.sup.12 are the same
or different and each is H, an alkyl or an acyl, and R.sup.11 and
R.sup.12' are the same or different and each is an alkyl or
R.sup.11' and R.sup.12', together with the nitrogen atom to which
they are bonded, form a heterocycle containing 3 to about 7 atoms
in the ring skeleton thereof; and E is H, a halogen, OR.sup.13,
NHR.sup.13, or NR.sup.13 R.sup.14, wherein R.sup.13 and R.sup.14
are the same or different and each is H, a protecting group, an
alkyl, or an acyl.
3. The method of claim 1, wherein R.sup.1 is H, an alkyl or
NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a', together with
the nitrogen atom to which they are bonded, comprise a heterocycle
containing from 3 to about 7 atoms in the ring skeleton
thereof.
4. The method of claim 1, wherein X.sup.1 is S.
5. The method of claim 1, wherein Z is CR.sup.2a R.sup.2a' or
CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b' and R.sup.2a, R.sup.2a',
R.sup.2b and R.sup.2b' are the same or different and each is H or
an alkyl.
6. The method of claim 1, wherein R.sup.2 or R.sup.2' is H or an
alkyl.
7. The method of claim 1, wherein R.sup.3 or R.sup.3' is H, an
alkyl or an
8. The method of claim 1, wherein R is a protecting group of the
formula: ##STR45##
9. The method of claim 1, wherein the temperature is from about
50.degree. C. to about 90.degree. C.
10. The method of claim 1, wherein the deprotection is carried out
at about pH 7.
11. The method of claim 1, wherein the fluid medium contains
water.
12. A method of producing an oligonucleotide, which method
comprises (a) reacting a nucleophile of the formula:
with an electrophile of the formula: ##STR46##
wherein W is a dialkylamino group that is displaced by the
nucleophile, under conditions to displace W and produce an adduct
comprising a tricoordinated phosphorus atom; (b) reacting the
product obtained in step (a) with a reagent selected from the group
consisting of oxidizing agents, sulfurizing agents, and selenizing
agents to produce a protected oligonucleotide of the formula:
##STR47## (c) cleaving R.sup.15 from the protected oligonucleotide
from step (b) to produce a nucleophile; (d) optionally repeating
steps (a)-(c) until an oligomer of a specified length is obtained;
and (e) heating the product from step (c) or (d) in a fluid medium,
at a substantially neutral pH, at a temperature up to about
100.degree. C. to produce a deprotected oligonucleotide of the
formula: ##STR48## wherein R is a thermolabile protecting group of
the formula: ##STR49## R.sup.1 is H, R.sup.1a, OR.sup.1a, SR.sup.1a
or NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a' are the same
or different and each is H, an alkyl, an alkenyl, an alkynyl, a
cycloalkyl, an aryl, or an aralkyl; or, when R.sup.1 is NR.sup.1a
R.sup.1a', R.sup.1a and R.sup.1a', together with the nitrogen atom
to which they are bonded, comprise a heterocycle containing from 3
to about 7 atoms in the ring skeleton thereof; X.sup.1 is O, S or
Se; X is O or S; Z is O, NR.sup.2a, CR.sup.2a B.sup.2a' or
CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b', wherein R.sup.2a,
R.sup.2a', R.sup.2b and R.sup.2b' are the same or different and
each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl,
or an aralkyl; or R.sup.1a or R.sup.1a', in combination with any of
R.sup.2a, R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X
of the protecting group to which they are bonded, comprise a ring
containing from 3 to about 7 atoms in the skeleton thereof;
provided that R.sup.1 is not R.sup.1a when Z is S, Z is not
CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b CR.sup.2b'
when R.sup.1 is SR.sup.1a, and Z is not O or S when R.sup.1 is H;
R.sup.2, R.sup.2', R.sup.3 and R.sup.3' are the same or different
and each is H, an alkyl, an alkenyl, alkynyl, a cycloalkyl, an
aryl, or an aralkyl, or R.sup.2 or R.sup.2', in combination R.sup.3
or R.sup.3', together with the carbon atoms to which they are
bonded, comprise a cyclic substituent of the formula: ##STR50##
wherein p is an integer from 0-6 and a-d are the same or different
and each is selected from the group consisting of H, an alkyl, a
nitro, an amino, a hydroxyl, a thio, a cyano and a halogen,
provided that the aromatic ring, which bears the substituents a-d,
is one carbon removed from the phosphate oxygen of formula (IIIa),
wherein R.sup.1, R.sup.2a, R.sup.2a', R.sup.2b, R.sup.2b' R.sup.2,
R.sup.2', R.sup.3 or R.sup.3' is unsubstituted or substituted with
one or more substituents, which are the same or different, selected
from the group consisting of OR.sup.8, CN, NO.sub.2, N.sub.3, and a
halogen, wherein R.sup.8 is H or an alkyl; R.sup.4 is H, a hydroxyl
protecting group, or a solid support; R.sup.15 is a hydroxyl
protecting group or a solid support; Q.sup.1 is a nucleoside, an
oligonucleotide or an oligomer comprising an oligonucleotide; n is
an integer from 1 to about 300; and Q is a nucleoside, an
oligonueleotide or an oligomer comprising an oligonucleotide and,
when a is greater than 1, each Q is independently selected,
provided that the deprotection is not by an enzyme.
Description
FIELD OF THE INVENTION
This invention pertains to thermolabile phosphate protecting
groups, intermediates therefor and methods of using them in
oligonucleotide synthesis.
BACKGROUND OF THE INVENTION
There are significant potential therapeutic applications for
oligonucleotides. The therapeutic application of oligonucleotides
is based on the selective formation of hybrids between antisense
oligonucleotides and complementary nucleic acids, such as messenger
RNAs (mRNAs). Such hybrids inhibit gene expression by preventing
protein translation. Nuclease-resistant oligonucleotides are highly
desirable in this regard. Nucleosides bearing phosphorothioate
internucleotide linkages are well-known for such nuclease
resistance and, thus, are undergoing rapid development.
In view of their significant potential therapeutic application,
there is a high demand for improved methods of preparing
oligonucleotides and analogues thereof. A number of methods for
synthesizing oligonucleotides have been developed. The most
commonly used synthetic method for the synthesis of thioated
oligonucleotides is the phosphoramidite method with stepwise
sulfurization (see, e.g., U.S. Pat. Nos. 4,415,732, 4,668,777,
4,973,679, 4,845,205, and 5,525,719). Essentially, a phosphate
precursor is sulfurized such that a sulfur atom is substituted for
one of the non-bridging oxygen atoms normally present in
phosphodiesters. This method uses tricoordinated phosphorus
precursors that normally produce products containing a mixture of
different thioated oligonucleotide stereoisomers, primarily due to
the use of non-stereoselective and non-stereospecific
acid-catalyzed nucleophilic substitution reactions.
Protecting groups for internucleosidic phosphorus linkages and
associated deprotection methods are well-known in the art, and have
been described, for example, in U.S. Pat. Nos. 4,417,046,
5,705,621, 5,571,902 and 5,959,099. However, the methods presently
used for removing internucleosidic phosphorus protecting groups are
disadvantageous in that they employ harsh reagents, such as bases
(e.g., ammonium hydroxide) and acids (e.g., trichloroacetic acid).
Under these deprotection conditions, there is a greater risk of
problems, such as by-product formation and degradation of the
desired oligonucleotide, which make oligonucleotide purification
more difficult and increase the overall cost, particularly in
large-scale production processes. Moreover, the range of structural
analogs that one can prepare is limited to those that are stable
under the acidic and/or basic deprotection conditions that are
commonly employed in the art.
Accordingly, there is a need for internucleosidic phosphorus
protecting groups that can be removed under milder conditions and
methods of making and using such protecting groups. Removal of such
protecting groups should be fast and should be carried out under
conditions that minimize the possibility for degradation of the
desired oligonucleotide. In addition, the intermediates that
introduce such protecting groups should be easy to synthesize
inexpensively on a large scale. It is, therefore, of prime
importance to develop low-cost, protected intermediates for
oligonucleotide synthesis which are easy to synthesize, couple
efficiently during stepwise synthesis, and are deprotected quickly
in high yield under mild conditions.
The invention provides such protecting groups and methods. These
and other objects and advantages of the invention, as well as
additional inventive features, will be apparent from the
description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The invention provides a method of thermally deprotecting an
oligonucleotide. The method comprises heating an oligonucleotide of
the formula: ##STR1##
in a fluid medium, at a substantially neutral pH, at a temperature
up to about 100.degree. C. to produce an oligonucleotide of the
formula: ##STR2##
wherein R is a thermolabile protecting group of the formula:
##STR3##
R.sup.1 is H, R.sup.1a, OR.sup.1a, SR.sup.1a or NR.sup.1a
R.sup.1a', wherein R.sup.1a and R.sup.1a' are the same or different
and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an
aryl, or an aralkyl. Alternatively, when R.sup.1 is NR.sup.1a
R.sup.1a', R.sup.1a and R.sup.1a', together with the nitrogen atom
to which they are bonded, comprise a heterocycle. Substituent
X.sup.1 is O, S or Se and substituent X is O or S. Substituent Z is
O, S, NR.sup.2a, CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a'
CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b and
R.sup.2b' are the same or different and each is H, an alkyl, an
alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl.
Alternatively, R.sup.1a or R.sup.1a', in combination with any of
R.sup.2a, R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X
of the protecting group to which they are bonded, comprise a ring
containing from 3 to about 7 atoms in the skeleton thereof. R.sup.1
is not R.sup.1a when Z is S, Z is not CR.sup.2a R.sup.2a' or
CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b' when R.sup.1 is SR.sup.1a,
and Z is not O or S when R.sup.1 is H.
Substituents R.sup.2, R.sup.2', R.sup.3 and R.sup.3' are the same
or different and each is H, an alkyl, an alkenyl, an alkynyl, a
cycloalkyl, an aryl, or an aralkyl. Alternatively, R.sup.2 or
R.sup.2', in combination with R.sup.3 or R.sup.3', together with
the carbon atoms to which they are bonded, comprise a cyclic
substituent of the formulae: ##STR4## wherein p is an integer from
0-6 and a-d are the same or different and each is selected from the
group consisting of H, an alkyl, a nitro, a dialkylamino, an
alkoxy, an alkylthio, a cyano and a halogen, provided that the
aromatic ring, which bears substituents a-d, is one carbon removed
from the phosphate oxygen of formula (IIIa).
Substituents R.sup.1, R.sup.2a, R.sup.2b, R.sup.2b', R.sup.2,
R.sup.2', R.sup.3 or R.sup.3' can be unsubstituted substituted, as
further described herein. Substituents R.sup.4 and R.sup.15 are the
same or different and each is H, a hydroxyl protecting group, or a
solid support.
Q and Q.sup.1 are the same or different and each is a nucleoside,
an oligonucleotide or an oligomer comprising an oligonucleotide.
Variable n represents an integer from 1 to about 300. When n is
greater than 1, each Q is independently selected.
The present invention further provides a novel compound selected
from the group consisting of compounds of the formulae:
##STR5##
wherein R is a thermolabile protecting group as defined herein,
R.sup.4, R.sup.15 and X.sup.1 are as defined herein, and W is a
dialkylamino group.
The present invention further provides method of producing an
oligonucleotide. The method comprises: (a) reacting a nucleophile
of the formula:
with an electrophile of the formula: ##STR6##
wherein R, R.sup.4, Q, Q.sup.1 and W are as defined herein, and
R.sup.15 is a protecting group, under conditions to displace W and
produce an adduct comprising a tricoordinated phosphorus atom; (b)
reacting the product obtained in step (a) with a reagent selected
from the group consisting of oxidizing agents, sulfurizing agents,
and selenizing agents to produce a protected oligonucleotide of the
formula: ##STR7##
wherein n=1; (c) cleaving R.sup.15 from the protected
oligonucleotide from step (b) to produce a nucleophile; (d)
optionally repeating steps (a)-(c) until an oligomer of a specified
length is obtained; and (e) thermally deprotecting the thermolabile
protecting group R in accordance with the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A generally illustrates the thermal deprotection of a
tetracoordinated phosphorus internucleosidic linkage.
FIG. 1B illustrates the thermal deprotection of a
phosphate/thiophosphate internucleosidic linkage.
FIG. 1C illustrates the thermal deprotection of the
phosphate/thiophosphate internucleosidic linkage of an
oligonucleotide prepared from a phosphoramidite precursor.
FIG. 1D illustrates the thermal deprotection of various
thermolabile phosphate/thiophosphate protecting groups.
FIG. 2A illustrates the synthesis of various phosphoramidite
precursors.
FIG. 2B illustrates the structures of various phosphoramidite
precursors.
FIG. 3 illustrates the synthesis of various N-acylphosphoramidite
of various.
FIG. 4 illustrates the solid phase synthesis of an oligonucleotide
using an N-acylphosphoramidite precursor.
FIG. 5 illustrates the solid phase stereocontrolled synthesis of
phosphorothioate oligonucleotides using an N-acylphosphoramidite
precursor.
FIG. 6 illustrates the synthesis of various
N-acylphosphoramidites.
FIG. 7 illustrates the preparation of acyclic
N-acylphosphoramidites and their application in solid phase
synthesis.
FIG. 8 illustrates the preparation of cyclic and acyclic
N-acylphosphoramidites.
FIG. 9 illustrates the preparation of an oligonucleotide using
either cyclic or acyclic N-acylphosphoramidite precursors.
FIG. 10A illustrates the structure of a P-chiral (S.sub.P)
N-acylphosphoramidite.
FIG. 10B illustrates the structure of a P-chiral (R.sub.P)
N-acylphosphoramidite.
FIG. 11 illustrates the structure of a P-diastereomeric (R.sub.P,
S.sub.P) N-acylphosphoramidite.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated, at least in part, on the
surprising and unexpected discovery of a method for thermally
deprotecting the internucleosidic phosphorus linkage of an
oligonucleotide, new thermolabile protecting groups that can be
removed under such conditions and intermediates that incorporate
them. The methods and protecting groups of the present invention
simplify, and improve the efficiency and cost-effectiveness
effectiveness of, oligonucleotide synthesis by avoiding the use of
harsh reagents, such as alkaline or acidic reagents. In one
embodiment, the present invention provides a method of deprotecting
an oligonucleotide, which method comprises heating an
oligonucleotide of the formula: ##STR8##
in a fluid medium, at a substantially neutral pH, at a temperature
up to about 100.degree. C. to produce an oligonucleotide of the
formula: ##STR9##
wherein:
R is a thermolabile protecting group of the formula: ##STR10##
The deprotection method of the present invention can be performed
in any suitable fluid medium. Suitable fluid media include, for
example, liquid media and gaseous media. A preferred fluid medium
comprises or contains water. Liquid media include, for example,
solvents, preferably solvents that are liquid at room temperature.
Suitable solvents include organic solvents and inorganic
solvents.
Organic solvents preferably include those that are easily removed
by evaporation. Preferably, the organic solvent is a polar organic
solvent. Preferred polar organic solvents include, for example,
acetonitrile; cyclic ethers such as, for example, dioxane and
tetrahydrofuran; alcohols such as, for example, methanol, ethanol
and isopropanol; mixtures thereof; and the like. Non-polar organic
solvents such as, for example, hydrocarbons, e.g., hexane,
cyclohexane and heptane; aromatic hydrocarbons, e.g., toluene and
benzene; mixtures thereof, and the like can be included in the
fluid medium, for example, as co-solvents.
Inorganic solvents include, for example, water. In a particularly
preferred embodiment, the solvent is water or a mixture of one or
more organic solvents and water.
The liquid medium can be a homogeneous solution or heterogeneous
mixture, but is preferably a homogeneous solution. Most preferably,
the liquid medium is a homogeneous solution that contains water as
a co-solvent.
Suitable solvents include, for example, acetonitrile/water mixtures
ranging from about 10:1 (v/v) to about 1:10 (v/v)
acetonitrile/water. Suitable acetonitrile/water mixtures include,
for example, about 9:1 (v/v) acetonitrile/water, about 5:1 (v/v)
acetonitrile/water, about 2:1 (v/v) acetonitrile/water, about 1:1
(v/v) acetonitrile/water, about 1:2 (v/v) acetonitrile/water, about
1:5 (v/v) acetonitrile/water, and about 1:9 (v/v)
acetonitrile/water.
Suitable solvents also include, for example, dioxane/water mixtures
ranging from about 10:1 (v/v) to about 1:10 (v/v) dioxane/water.
Suitable dioxane/water mixtures include, for example, about 9:1
(v/v) dioxane/water, about 5:1 (v/v) dioxane/water, about 2:1 (v/v)
dioxane/water, about 1:1 (v/v) dioxane/water, about 1:2 (v/v)
dioxane/water, about 1:5 (v/v) dioxane/water, and about 1:9 (v/v)
dioxane/water.
Suitable solvents also include other organic solvent/water
mixtures, e.g., using the ratios described herein. Other suitable
solvents include organic solvents, such as, for example,
acetonitrile, dioxane, mixtures thereof, and the like, that contain
a trace amount of water (e.g., from about 0.05-2 wt. %, from about
0.1-2 wt. %, from about 0.5-2 wt. %, from about 1-2 wt. %, and the
like).
The method of the present invention can be performed in a gaseous
medium, most preferably a gaseous medium that contains water in a
gaseous or fluid state (e.g., steam, hot water mist or vapor, or
the like). The gaseous medium also can include the gaseous phases
of any of the organic solvents or solvent mixtures described
herein. In a particularly preferred embodiment, the method of the
present invention includes contacting the oligonucleotide of
formula (IIIa) (e.g., bound to a solid support) with steam.
As indicated above, the method of the present invention is carried
at a substantially neutral pH. As utilized herein, the term
"substantially neutral pH" means a pH in the range from about
5.5-7.5, preferably from about 6-7.5, most preferably about 7
(e.g., about 7.0-7.4). Optionally, a buffer can be added to the
solvent system to maintain a substantially neutral pH throughout
the course of the deprotection reaction. Suitable buffers include,
for example, phosphate buffers, trialkylammonium acetate buffers
(e.g., 0.1 M triethylammonium acetate), and the like.
The deprotection method of the present invention is preferably
performed at a temperature that is sufficient to remove the
protecting group at a rate that is practical for commercial scale
production (e.g., about 3 hours or less), but should be low enough
to avoid thermal degradation of the desired oligonucleotide.
Typically, the deprotection is performed at a temperature up to
about 100.degree. C. (at about 100.degree. C. or less), e.g., from
above about ambient temperature (e.g., above about 20-25.degree.
C.) to about 100.degree. C. Preferably, the deprotection is
performed at a temperature from about 50-100.degree. C., more
preferably from about 60-100.degree. C., still more preferably from
about 70-100.degree. C., most preferably from about 80-100.degree.
C. About 90.degree. C. or about 100.degree. C. is especially
preferred. When a liquid solvent medium is used, the deprotection
is preferably performed from about 50-90.degree. C., more
preferably from about 60-90.degree. C., still more preferably from
about 70-90.degree. C., even still more preferably from about
80-90.degree. C., and most preferably at about 90.degree. C.
However, in some circumstances, it may be desirable to carry out
the deprotection at somewhat higher temperatures (e.g., up to about
110.degree. C., e.g., from about 100-105.degree. C.).
The structure of the thermolabile protecting group (substituent R
of formula IIIa) can vary considerably in terms of different
combinations of R.sup.1, R.sup.2, R.sup.2', R.sup.3, R.sup.3', Z
and X, while maintaining thermal lability. In other words, the bond
linking protecting group R to the non-bridging phosphate,
phosphorothioate or phosphoroselenoate oxygen can be thermally
cleaved using different combinations of R.sup.1, R.sup.2 ,
R.sup.2', R.sup.3, R.sup.3', Z and X.
While R.sup.1 can be any suitable substituent, R.sup.1 preferably
is H, R.sup.1a, OR.sup.1a, SR.sup.1a or NR.sup.1a R.sup.1a',
wherein R.sup.1a and R.sup.1a' are the same or different and each
is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or
an aralkyl. Alternatively, when R.sup.1 is NR.sup.1a R.sup.1a',
R.sup.1 and R.sup.1a', together with the nitrogen atom to which
they are bonded, comprise a heterocycle containing from 3 to about
7 atoms in the ring skeleton thereof.
Preferably, X.sup.1 is O, S or Se; X is O or S; and Z is O, S,
NR.sup.2a, CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b
R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b and R.sup.2b' are
the same or different and each is H, an alkyl, an alkenyl, an
alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively,
R.sup.1a or R.sup.1a', in combination with any of R.sup.2a,
R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X of the
protecting group to which they are bonded, comprise a ring
containing from 3 to about 7 atoms in the skeleton thereof.
It is preferred that thioesters are not utilized in the methods or
the protecting groups of the present invention as they are believed
to have a tendency to hydrolyze rather easily in the presence of
water. Thus, when Z is S, it is preferred that R.sup.1 is not
R.sup.1a. Similarly, when R.sup.1 is SR.sup.1a, Z is not CR.sup.2a
R.sup.2a' or CR.sup.2a R.sup.2a' C.sup.R2b R.sup.2b'. Further, it
is preferred that formate esters or formate thioesters are not
utilized in the methods or the protecting groups of the present
invention as they also are believed to have a tendency to hydrolyze
rather easily in the presence of water. Thus, when R.sup.1 is H, it
is preferred that Z is not O or S.
While R.sup.2, R.sup.2', R.sup.3 and R.sup.3' can be any suitable
substituent, R.sup.2, R.sup.2', R.sup.3 and R.sup.3' preferably are
the same or different and each is H, an alkyl, an alkenyl, an
alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively,
R.sup.2 or R.sup.2', in combination with R.sup.3 or R.sup.3',
together with the carbon atoms to which they are bonded, can
comprise a cyclic substituent of the formula: ##STR11##
wherein p is an integer from 0-6 and a-d are the same or different
and each is selected from the group consisting of H, an alkyl, a
nitro, a dialkylamino, an alkoxy, an alkylthio, a cyano and a
halogen, provided that the aromatic ring, which bears substituents
a-d, is one carbon removed from (i.e., is benzylic relative to) the
phosphate oxygen of formula (IIIa).
The foregoing substituents can be unsubstituted or substituted.
Preferably, R.sup.1, R.sup.2a, R.sup.2a', R.sup.2b, R.sup.2b',
R.sup.2, R.sup.2', R.sup.3 or R.sup.3' is unsubstituted or
substituted. Preferably, R.sup.1, substituents, which are the same
or different, selected from the group consisting of OR.sup.8, CN,
NO.sub.2, N.sub.3, and a halogen, wherein R.sup.8 is H or an
alkyl.
Substituents R.sup.4 and R.sup.15 are the same or different and
each is preferably H, a hydroxyl protecting group, or a solid
support. Substituent Q.sup.1 represents a nucleoside, an
oligonucleotide or an oligomer comprising an oligonucleotide. The
variable n is an integer from 1 to about 300, preferably from about
3 to about 200, more preferably from about 10 to about 40, and most
preferably from about 15 to about 25. Substituent Q represents a
nucleoside, an oligonucleotide or an oligomer comprising an
oligonucleotide. When n is an integer greater than 1, each Q is
independently selected, i.e., each Q in each monomeric unit can be
the same or different.
As utilized herein, the term "alkyl" means a straight-chain or
branched-chain alkyl radical which, unless otherwise specified,
contains from about 1 to about 20 carbon atoms, preferably from
about 1 to about 10 carbon atoms, more preferably from about 1 to
about 8 carbon atoms, and most preferably from about 1 to about 6
carbon atoms. Examples of such alkyl radicals include methyl,
ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,
pentyl, isoamyl, hexyl, octyl, dodecanyl, and the like.
The term "alkenyl" means a straight-chain or branched-chain alkenyl
radical, which has one or more double bonds and, unless otherwise
specified, contains from about 2 to about 20 carbon atoms,
preferably from about 2 to about 10 carbon atoms, more preferably
from about 2 to about 8 carbon atoms, and most preferably from
about 2 to about 6 carbon atoms. Examples of alkenyl radicals
include vinyl, allyl, 1,4-butadienyl, isopropenyl, and the
like.
The term "alkynyl" means a straight-chain or branched-chain alkynyl
radical, which has one or more triple bonds and contains from about
2 to about 20 carbon atoms, preferably from about 2 to about 10
carbon atoms, more preferably from about 2 to about 8 carbon atoms,
and most preferably from about 2 to about 6 carbon atoms. Examples
of alkynyl radicals include ethynyl, propynyl (propargyl), butynyl,
and the like.
The terms "alkylamino" and "dialkylamino" mean an alkyl or a
dialkyl amine radical, wherein the term "alkyl" is defined as
above. Examples of alkylamino radicals include methylamino
(NHCH.sub.3), ethylamino (NHCH.sub.2 CH.sub.3), n-propylamino,
isopropylamino, n-butylamino, isobutylamino, sec-butylamino,
tert-butylamino, n-hexylamino, and the like. Examples of
dialkylamino radicals include dimethylamino (N(CH.sub.3).sub.2),
diethylamino (N(CH.sub.2 CH.sub.3).sub.2), di-n-propylamino,
diisopropylamino, di-n-butylamino, diisobutylamino,
di-sec-butylamino, di-tert-butylamino, di-n-hexylamino, and the
like.
The term "cycloalkyl" means a monocyclic alkyl radical, or a
polycyclic alkyl which comprises one or more alkyl carbocyclic
rings, which can be the same or different when the polycyclic
radical has 3 to about 10 carbon atoms in the carbocyclic skeleton
of each ring. Preferably, the cycloalkyl has from about 4 to about
7 carbon atoms, more preferably from about 5 to about 6 carbons
atoms. Examples of monocyclic cycloalkyl radicals include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclodecyl, and the like. Examples of polycyclic cycloalkyl
radicals include decahydronaphthyl, bicyclo[5.4.0]undecyl,
adamantyl, and the like.
The term "aryl" refers to an aromatic carbocyclic radical, as
commonly understood in the art, and includes monocyclic and
polycyclic aromatics such as, for example, phenyl and naphthyl
radicals, which radicals are, unless indicated otherwise,
unsubstituted or substituted with one or more substituents selected
from the group consisting of a halogen, an alkyl, an alkoxy, an
amino, a cyano, a nitro, and the like. Preferably, the aryl has one
or more six-membered carbocyclic rings including, for example,
phenyl, naphthyl, and biphenyl, and are unsubstituted or
substituted as set forth herein.
The term "aralkyl" means alkyl as defined herein, wherein an alkyl
hydrogen atom is replaced by an aryl as defined herein. Examples of
aralkyl radicals include benzyl, phenethyl, 1-phenylpropyl,
2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl,
3-naphthylpropyl, 3-naphthylbutyl, and the like.
The terms heterocycle and heterocyclic refer to both
heterocycloalkyls and heteroaryls. The term "heterocycloalkyl"
means a cycloalkyl radical as defined herein (including
polycyclics), wherein at least one carbon of a carbocyclic ring is
substituted with a heteroatom such as, for example, O, N, or S. The
heterocycloalkyl optionally has one or more double bonds within a
ring, and may be aromatic, but is not necessarily aromatic. The
heterocycloalkyl preferably has 3 to about 10 atoms (members) in
the skeleton of each ring, more preferably from about 3 to about 7
atoms, more preferably from about 5 to about 6 atoms. Examples of
heterocycloalkyl radicals include epoxy, aziridyl, oxetanyl,
tetrahydrofuranyl, ribose, dihydrofuranyl, piperidinyl,
piperazinyl, pyranyl, morpholinyl, and the like.
The term "heteroaryl" means a radical defined by an aromatic
heterocyclic ring as commonly understood in the art, including
monocyclic radicals such as, for example, imidazole, thiazole,
pyrazole, pyrrole, furane, pyrazoline, thiophene, oxazole,
isoxazole, pyridine, pyridone, pyrimidine, cytosine,
5-methylcytosine, thymine, pyrazine, and triazine radicals, and
polycyclics such as, for example, quinoline, isoquinoline, indole,
purine, adenine, guanine, N6-methyladenine, and benzothiazole
radicals, which heteroaryl radicals are unsubstituted or
substituted with one or more substituents, which are the same or
different, selected from the group consisting of a halogen, an
alkyl, an alkoxy, an amino, a cyano, a nitro, and the like. The
heteroaryl preferably has 3 to about 10 atoms (members) in the ring
skeleton of each ring, more preferably from about 3 to about 7
atoms, more preferably from about 5 to about 6 atoms.
It will be appreciated that the heterocycloalkyl and the heteroaryl
substituents can be coupled to the compounds of the present
invention via a heteroatom, such as nitrogen (e.g., 1-imidazolyl),
or via a carbon atom (e.g., 4-thiazolyl). It will also be
appreciated that heteroaryls, as defined herein, are not
necessarily "aromatic" in the same context as phenyl is aromatic,
although heteroaryls nonetheless demonstrate physical and chemical
properties associated with aromaticity, as the term is understood
in the art.
The term "carboxyl" means any functional group with a carbonyl
backbone, and includes functional groups such as, for example, a
carboxylic acid, an esters (e.g., ethoxycarbonyl), and amides
(e.g., benzamido).
The term "nucleoside" includes all modified and naturally occurring
nucleosides, including all forms of furanosides found in nucleic
acids. Naturally occurring nucleosides include, for example,
adenosine, guanosine, cytidine, thymidine, and uridine.
Nucleoside "derivatives" or "analogs" include synthetic nucleosides
as described herein. Nucleoside derivatives also include
nucleosides having modified base moieties, with or without
protecting groups. Such analogs include, for example, deoxyinosine,
2,6-diaminopurine-2'-deoxyriboside, 5-methyl-2'-deoxycytidine, and
the like. The base rings most commonly found in naturally occurring
nucleosides are purine and pyrimidine rings. Naturally occurring
purine rings include, for example, adenine, guanine, and N.sup.6
-methyladenine. Naturally occurring purine rings include, for
example, cytosine, thymine, and 5-methylcytosine. The compounds and
methods of the present invention include such base rings and
synthetic analogs thereof, as well as unnatural
heterocycle-substituted base sugars, and even acyclic substituted
base sugars. Moreover, nucleoside derivatives include other purine
and pyrimidine derivatives, for example, halogen-substituted
purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines,
N.sup.6 -ethyladenine, N.sup.6 -(alkyl)-cytosines, 5-ethylcytosine,
and the like.
The term "oligonucleotide" as used herein includes linear oligomers
of natural or modified nucleosides, and modified ologonucleotides,
as described herein. Oligonucleotides include deoxyribonucleosides,
ribonucleosides and anomeric forms thereof, and the like.
Oligonucleotides are typically linked by phoshodiester bonds, or
the equivalent thereof, ranging in size from a few monomeric units
(e.g., 2-4) to several hundred monomeric units. Preferably, the
oligonucleotides of the present invention are oligomers of
naturally-occurring nucleosides ranging in length from about 12 to
about 60 monomeric units, and more preferably, from about 15 to
about 30 monomeric units. Whenever an oligonucleotide is
represented by a sequence of letters, such as "AGTC" it will be
appreciated that the nucleotides are in the 5'-3' orientation from
left to right.
In accordance with the present invention, Q and/or Q.sup.1 can be a
natural nucleoside or a modified/unnatural nucleoside. Q and/or
Q.sup.1 also can be an oligomer comprising one or more natural or
modified/unnatural nucleosides. Modified nucleosides can be
obtained, for example, by any suitable synthetic method known in
the art for preparing nucleosides, derivatives, or analogs thereof.
Modified nucleosides include, but are not limited to, chemically
modified nucleosides used as building blocks for "labeled"
oligonucleotides, or suitable precursors or analogs used in the
preparation of such modified nucleosides. Various chemically
modified nucleosides are described, for example, in Smith et al.,
Nucleosides & Nucleotides, 15(10), 1581-1594 (1996) ("Smith et
al."). Smith et al. describes the synthesis of nucleosides (and
oligomers which include such nucleosides) in which the base ring is
replaced by a carboxylic acid to which is appended various
"labeling" groups (e.g., biotin, cholesterol,
fluorenylmethoxycarbonyl (Fmoc), and trifluoroacetyl) via a
modified amide linker. Modified nucleosides also include other
chemically modified nucleosides, for example, nucleosides described
in Smith et al. in which the base ring is replaced by a
hydroxyethyl, a cyano, or a carboxylic acid (including esters and
amides thereof). Modified nucleosides further include nucleosides
in which the base ring is replaced by a cyclic substituent, for
example, an aryl, a cycloalkyl, a heterocycloalkyl, or a heteroaryl
(other than a base naturally occurring in nucleosides).
Q and/or Q.sup.1 also include oligonucleotides, which can be
natural or modified. Modified oligonucleotides include, for
example, oligonucleotides containing a modified nucleoside (as
described herein), oligonucleotides containing a modified
internucleotide linkage, or oligonucleotides having any combination
of modified nucleosides and internucleotide linkages (even if a
natural nucleoside is present in the oligomer chain).
Oligonucleotides whose nucleosides are connected via modified
internucleotide linkages can be found, for example, in Waldner et
al., Bioorg. Med. Chem. Letters, 6, 19, 2363-2366 (1996) ("Waldner
et al."), which describes the synthesis of oligonucleotides
containing various amide internucleotide linkages.
The term "oligomer comprising a nucleoside" as utilized herein
means an oligomer in which at least one of the monomeric units
comprises nucleoside, and at least one of the other monomeric units
is not a nucleoside. For example, one of the monomeric units in the
oligomer can be an amino acid, an organic spacer (e.g., an
aliphatic or aromatic spacer, an alkylene glycol, or the like), or
a carbohydrate (e.g., a sugar). Moreover, one of the non-nucleoside
units of the oligomer can itself be oligomeric, for example, a
peptide, an oligosaccharide, a polyalkylene glycol, or the
like.
It will be appreciated that protecting groups (sometimes referred
to as a blocking groups) other than the thermolabile protecting
groups described herein can be utilized in accordance with the
present invention. Generally, the term "protecting group," as used
herein, means a substituent, functional group, salt, ligand, or the
like, which is bonded (e.g., via covalent bond, ionic bond, or
complex) to a potentially reactive functional group and prevents
the potentially reactive functional group from reacting under
certain reaction conditions. Potentially reactive functional groups
include, for example, amines, carboxylic acids, alcohols, double
bonds, and the like. Preferably, the protecting group is stable
under the reaction conditions for which the protecting group is
employed, and also can be removed under reasonably mild
deprotection conditions. It will be appreciated that any additional
protecting groups to be used in accordance with the present
invention should be chosen based on the type of substituent that is
being protected. Thus, in general, it is not uncommon to use a
different protecting group for each of a phosphite oxygen, a
phosphate oxygen, an amine, a thiol, a hydroxyl, and the like. It
will also be appreciated that the choice of protecting groups will
depend on other factors such as, for example, the reaction
conditions employed in a particular synthetic step, the pH, the
temperature, and the relative reactivities of the reactants and/or
products.
Protecting groups for hydroxyls include, for example, silyl ethers
(e.g., trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl,
tert-butyldiphenylsilyl, dimethylphenylsilyl, and
diphenylmethylsilyl), benzyl carbonates, trityl, monomethoxytrityl,
dimethoxytrityl, esters (e.g., acetate, benzoate, and the like),
pixyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc),
a tetrahydropyranyl group, and the like. When the hydroxyl is a
sugar hydroxyl, preferred protecting groups include, for example,
pixyl, acetyl, 9-fluorenylmethyloxycarbonyl (Fmoc),
t-butyldimethylsilyl (TBDMS), trityl, monomethoxytrityl ("MMT" or
"MMTr"), dimethoxytrityl ("DMT" or "DMTr"), and the like.
Protecting groups for nitrogen include, for example, amides (e.g.,
trifluoroacetyl, benzoyl, and isobutyryl), carbamates (e.g.,
tert-butyloxycarbonyl and N-benzyloxycarbonyl), trityl, and the
like.
When an amine to be protected is part of a nucleoside base ring,
suitable protecting groups can include amides, for example,
benzoyl, isobutyryl, and the like. Other protecting groups are
defined in the literature. See, e.g., Iyer, Current Protocols in
Nucleic Acid Chemistry, Vol.1 (Beaucage S. L., Bergstrom, D. E.,
Glick, G. D. Jones R. A. eds); John Wiley and Sons: New York, 2000,
pp. 2.1.1-2.1.17; Beaucage, et al., Tetrahedron, 48, 2223-2311
(1992); and McBride et al., J. Am. Chem. Soc., 108, 2040-2048
(1986).
Suitable protecting groups also include, for example,
2-[N,N-(dialkylamino)oxy]ethyl (Prakash et al., Org. Lett., 2,
2995-3998 (2000)), a (2-methoxy)ethoxy (Martin, Helv. Chim. Acta.,
78, 486-504 (1995)), triisopropylsilyloxymethyl and those groups
defined by Wincott, Current Protocols in Nucleic Acid Chemistry,
Vol.1 (Beaucage S. L., Bergstrom, D. E., Glick, G. D. Jones R. A.
eds); and John Wiley and Sons: New York, 2000, pp.
3.5.1-3.5.12.
Any suitable solid support can be used in accordance with the
present invention. Solid supports are commonly known in the art and
include, for example, organic solid supports (e.g., crosslinked
polystyrene) and inorganic solid supports. Preferably, the solid
support is inorganic, and is more preferably a silica support. It
will be appreciated that the solid support includes all linkers,
spacers, arms, and other moieties (organic or inorganic) known in
the art for manipulating attachment to a solid support. It will
also be appreciated that the solid support can be bonded to the
molecule directly, without using any of the aforesaid linkers,
spacers, arms, or other connecting moieties. Some aspects of the
invention are common with known approaches to solid phase synthesis
of oligonucleotides, for example, selection of suitable protecting
groups, selection of suitable solid phase supports, and the like.
Consequently, considerable guidance in making such selections in
the context of the present invention can be found in literature,
e.g. Beaucage et al., Tetrahedron, 49, 6123-6194 (1993). Desirably,
R.sup.4 and R.sup.15 are not both solid supports.
Preferably, Q or Q.sup.1 comprises a nucleoside of the formula:
##STR12##
wherein B is a labeling group, an alkyl, an alkenyl, an alkynyl, a
cycloalkyl, an aryl, a heteroaryl, a heterocycloalkyl, an aralkyl,
an amino, an alkylamino, a dialkylamino, a purine, a pyrimidine,
adenine, guanine, cytosine, uracil, or thymine, wherein B is
unsubstituted or substituted with one or more substituents, which
are the same or different, selected from the group consisting of a
nucleobase protecting group, R.sup.11, OR.sup.11, NHR.sup.11,
NR.sup.11 R.sup.12, an amidine (e.g., N.dbd.CH--NR.sup.11'
R.sup.12' or N.dbd.C(alkyl)--NR.sup.11' R.sup.12'), CN, NO.sub.2,
N.sub.3, and a halogen, wherein R.sup.11 and R.sup.12 are the same
or different and each is H, an alkyl or an acyl, and R.sup.11' and
R.sup.12' are the same or different and each is an alkyl.
Alternatively, R.sup.11' and R.sup.12', together with the nitrogen
atom to which they are bonded, form a heterocycle containing 3 to
about 7 atoms in the ring skeleton thereof. Substituent E is
preferably H, a halogen, OR.sup.13, NHR.sup.13, or NR.sup.13
R.sup.14, wherein R.sup.13 and R.sup.14 are the same or different
and each is H, a protecting group, an alkyl, or an acyl. In a
preferred embodiment, Q and/or Q.sup.1 is a nucleoside substituent
of the formula: ##STR13##
wherein B and E are as defined herein.
It will be appreciated that certain combinations of R.sup.1,
R.sup.2, R.sup.2, R.sup.3, R.sup.3', Z and X, can be chosen to
promote thermal cleavage of the bond linking the protecting group
to the non-bridging phosphate, phosphorothioate or
phosphoroselenoate oxygen. For example, R.sup.3 can be chosen from
among substituents that may increase the lability of the bond
linking the organic moiety to the non-bridging phosphate,
phosphorothioate or phosphoroselenoate oxygen, e.g., an
electron-withdrawing group or a cation-stabilizing group, e.g., an
aryl, preferably a phenyl. Alternatively, R.sup.3 and/or R.sup.3'
can be a substituent that makes the carbon to which it is attached
less hindered (e.g., R.sup.3 and R.sup.3' are H) and, possibly,
more susceptible to a thermally-mediated deprotection mechanism,
e.g., internal displacement by the C.dbd.X residue.
In one embodiment, R.sup.1 is H, an alkyl or a heterocycle defined
by NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a', together
with the nitrogen atom to which they are bonded, comprise a
heterocycle containing from 3 to about 7 atoms in the ring skeleton
thereof. In another embodiment X.sup.1 is S. In yet another
embodiment, Z is CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a'
CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b and
R.sup.2b' are same or different and each is H or an alkyl. In still
another embodiment, R.sup.2 or R.sup.2' is H or an alkyl. In yet
another embodiment, R.sup.3 or R.sup.3' is H, an alkyl or an aryl.
It will be appreciated that other combinations of substituents not
specifically described herein also can be used in connection with
the method of the present invention. Examples of specific
protecting groups (R) used in accordance with the present invention
include protecting groups of the formulae: ##STR14##
The thermal deprotection method of the present invention is
generally illustrated in FIG. 1A. The thermal cleavage of the bond
that links the protecting group to the non-bridging phosphate,
phosphorothioate or phosphoroselenoate oxygen is indicated by the
dotted lines shown in FIG. 1A. See also FIG. 1B. As indicated
above, thermal cleavage can be advantageous in that the use of
harsh chemicals, such as ammonium hydroxide, is avoided. As such,
thermal cleavage provides a mild alternative that can be used in
the production of monomeric, oligomeric, or polymeric compounds,
particularly those that incorporate nucleoside monomers, which are
substituted with substituents that are chemically labile under
standard acidic or basic deprotection conditions.
The thermal cleavage of various protecting groups is shown in FIGS.
1B-1D. FIG. 1B illustrates the thermal cleavage of an acetamide
protecting group. The thermal cleavage illustrated in FIG. 1B
(i.e., wherein X.sup.1 is O or S), for example, can be carried out
to completion in about 80 minutes at about 80.degree. C. FIG. 1C
illustrates the thermal deprotection of a formamide protecting
group. FIG. 1D illustrates the thermal deprotection of various
protecting groups with various combinations of substituents Z and
R.sup.1.
The thermolabile protecting groups of the present invention can be
employed in oligonucleotide synthesis methods that are well-known
in the art. For example, oligonucleotides that incorporate
thermolabile protecting groups can be obtained from phosphoramidite
precursors such as, for example, compound 101 (FIG. 1C). The
phosphoramidite precursors can be prepared using well-known
synthetic methods, e.g., as illustrated in FIG. 2A.
The synthesis shown in FIG. 2A can be carried out, for example, by
adding anhydrous N,N-diisopropylamine to a solution of phosphorus
trichloride in dry benzene to produce
bis(N,N-diisopropylamino)chlorophosphine, and reacting it in situ
with 2-(N-formyl-N-methyl) aminoethan-1-ol to produce
phosphordiamidite 120 in about 73% yield. Phosphordiamidite 120 can
then be reacted with a suitably protected nucleophile, such as
5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine (Barone et al., Nucl.
Acids Res., 12, 4051-4061 (1984)) to produce deoxyribonucleoside
phosphoramidite 101 (FIG. 2A).
Phosphoramidite precursors incorporating structurally diverse
thermolabile protecting groups can be prepared in a manner similar
to that shown in FIG. 2A, using structurally diverse alcohol
derivatives. Such alcohol derivatives include, for example,
N-acetylethanolamine (commercially available from Aldrich Chemical
Co., Milwaukee, Wis.), 2-(N-acetyl-N-methyl)aminoethanol (Saegusa
et al., Makromol. Chem., 177, 2271-2283 (1976)),
2-(N-formyl-N-methyl)aminoethanol (Shibanuma et al., Chem Pharm.
Bull., 28, 2609-2613 (1980)), 1-(2-hydroxyethyl)-2-pyrrolidinone
(commercially available from Aldrich Chemical Co., Milwaukee,
Wis.), N-methyl-4-hydroxybutyramide (Wilk et al., J. Org. Chem.,
64, 7515-7522 (1999)), N-tert-butyl-4-hydroxybutyramide (Bigg et
al., Synthesis, 277-278 (1992)),
N,N-dimethyl-1-hydroxyethylcarbamate (Probst et al., Makromol.
Chem., 177, 2681-2695 (1976)), 3-acetyl-1-propanol (commercially
available from Aldrich Chemical Co., Milwaukee, Wis.), and the
like. Alternatively, phosphoramidite precursors can be prepared by
reacting a suitably protected nucleophile, such as
5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine, with
hexaethylphosphorus triamide and diethylammonium tetrazolide in dry
acetonitrile for 30 min at 25.degree. C. to produce the
corresponding deoxyribonucleoside 3'-O-phosphordiamidite, which can
be reacted in situ with an equimolar amount of any of the alcohol
derivatives described above (Wilk et al., J. Org. Chem., 62,
6712-6713 (1997)).
Exemplary phosphoramidite precursors of the present invention are
shown in FIG. 2B. Dinucleoside phosphotriesters 110-117 (FIG. 1D)
can be made from the corresponding phosphoramidite precursors shown
in FIG. 2B, for example, by activating with 1H-tetrazole, and
manually coupling to a suitably protected nucleophile, such as,
e.g., 5'-unprotected thymidine covalently attached to long-chain
alkylamine-controlled pore glass (LCAA-CPG). Standard aqueous
iodine oxidation or sulfurization, e.g., by
3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage et al., Ann. New
York Acad. Sci., 616, 483-485 (1990); Iyer et al., J. Org. Chem.,
55, 4693-4699 (1990); and Regan et al., Org. Prep. Proc. Int., 24,
488-492 (1992)), followed by release from LCAA-CPG by treatment
with pressurized methylamine gas for 3 min at 25.degree. C. (Boal
et al., Nucl. Acids Res., 24, 3115-3117 (1996)), produces
dinucleoside phosphotriesters 110-117 (FIG. 1D). Removal of the
phosphate protecting groups from purified dinucleoside
phosphotriesters 110-112 and 115-117 (X.dbd.O) in aqueous solvents
(e.g., water or an eluent from chromatographic purification, e.g.,
a water/acetonitrile mixture), at about pH 7 (without the aid of
concentrated ammonium hydroxide), occurs in less than 3 h at
-90.degree. C. affording the corresponding dithymidylyl
monophosphate 118 in essentially quantitative yields. Removal of
the phosphate protecting groups from phosphotriesters 113 and 114
under these conditions typically occurs in about 14 h and 4 h,
respectively. Removal of the thiophosphate protecting groups from
thiophosphate triesters 110, 112, 114, 116 and 117 (X.dbd.S) in
aqueous solvents, at about pH 7, occurs in less than 3 h at
-90.degree. C. affording the corresponding dithymidylyl
monothiophosphate 119 in essentially quantitative yields. Removal
of the thiophosphate protecting groups from thiophosphate triesters
111, 113 and 115 also is accomplished under these conditions,
although a desulfurization side reaction has been observed in some
cases.
Oligonucleoside phosphotriesters bearing protecting group R can be
readily prepared from phosphoramidite precursors of the present
invention using standard methods that are well-known in the art,
e.g., solid-phase synthesis. Thermal deprotection of
oligonucleoside phosphotriesters bearing protecting group R is
accomplished using mild conditions, for example, by heating at
about 90.degree. C. or less, for 3 h or less, in an aqueous solvent
such as, for example, water or 2:3 (v/v) acetonitrile/water, with
or without a buffer (e.g., 0.1M triethylammonium acetate), at pH
7.0, to afford the corresponding oligonucleotide in high yield.
Using this procedure, oligonucleotides such as dT.sub.18 and
d(AG).sub.10 have been prepared in high yield and high purity.
The present invention further provides a method of producing an
oligonucleotide, which method comprises:
(a) reacting a nucleophile of the formula:
with an electrophile of the formula: ##STR15##
wherein R.sup.15 is a protecting group as defined herein and W is a
dialkylamino group that is displaced by the nucleophile, under
conditions to displace W and produce an adduct comprising a
tricoordinated phosphorus atom;
(b) reacting the product obtained in step (a) with a reagent
selected from the group consisting of oxidizing agents, sulfurizing
agents, and selenizing agents to produce a protected
oligonucleotide of the formula: ##STR16##
wherein n=1;
(c) cleaving R.sup.15 from the protected oligonucleotide from step
(b) to produce a nucleophile;
(d) optionally repeating steps (a)-(c) until an oligomer of a
specified length is obtained; and
(e) heating the product from step (c) or (d) in a fluid medium, at
a substantially neutral pH, at a temperature up to about
100.degree. C. to produce a deprotected oligonucleotide of the
formula: ##STR17##
wherein R is a thermolabile protecting group of the formula:
##STR18##
wherein W, R.sup.1, X.sup.1, X, Z, R.sup.2, R.sup.2', R.sup.3,
R.sup.3', R.sup.4, Q.sup.1, Q and n are as defined above
The present invention further provides novel thermolabile
internucleosidic phosphorus protecting groups and novel
intermediates that incorporate such protecting groups. Preferably,
the present invention provides a compound selected from the group
consisting of compounds of the formula: ##STR19##
wherein R is a thermolabile protecting group of the formula:
##STR20##
wherein W, R.sup.1, X.sup.1, X, Z, R.sup.2, R.sup.2', R.sup.3,
R.sup.3', R.sup.4, R.sup.15, Q.sup.1, Q and n are as provided,
however, that when R.sup.1 is not H, Z is not NR.sup.2a, wherein
R.sup.2a is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an
aryl, or an aralkyl.
Preferably, Q or Q.sup.1 in the compound of the present invention
comprises a nucleoside of the formula: ##STR21##
wherein B and E are as defined above. In a preferred embodiment, Q
and/or Q.sup.1 is a nucleoside substituent of the formula:
##STR22##
wherein B and E are as defined above.
As indicated above, certain combinations of R.sup.1, R.sup.2,
R.sup.2', R.sup.3, R.sup.3', Z and X, can be chosen to promote
thermal cleavage of the bond linking the protecting group to the
non-bridging phosphate, phosphorothioate or phosphoroselenoate
oxygen. In one embodiment, R.sup.1 is H, an alkyl or a heterocycle
defined by NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a',
together with the nitrogen atom to which they are bonded, comprise
a heterocycle containing from 3 to about 7 atoms in the ring
skeleton thereof. In another embodiment X.sup.1 is S. In yet
another embodiment, Z is CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a'
CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2 and
R.sup.2b' are same or different and each is H or an alkyl. In still
another embodiment, R.sup.2 or R.sup.2' is H or an alkyl. In yet
another embodiment, R.sup.3 or R.sup.3' is H, an alkyl or an aryl.
It will be appreciated that other combinations of substituents not
specifically described herein also can be used in accordance with
the present invention. Examples of novel protecting groups used in
accordance with the present invention include protecting groups of
the formulae: ##STR23##
The phosphoramidite coupling approach in oligonucleotide synthesis
is well-known in the art and typically involves displacement of an
amino functionality on phosphorus. Acidic conditions are required
for the displacement of the amino functionality. The
phosphorus-nitrogen bond in a standard phosphoramidite is labile
under acidic conditions (even when a mild acid such as tetrazole is
used), invariably resulting in epimerization of the phosphorus atom
in the resulting coupled adduct. Although attempts have been made
to control the extent of epimerization in coupling reactions using
phosphoramidites, there is inevitably some epimerization, which
promotes the formation of diastereomers. Even if the formation of
undesired diastereomers occurs in minute quantities, the overall
yield of the target product decreases exponentially.
This problem can be overcome by utilizing N-acylphosphoramidites as
alternative coupling vehicles, for example, to couple
nucleoside-containing fragments. See WO 00/56749.
N-acylphosphoramidites are advantageous in that the coupling
reactions can be performed without any epimerization at phosphorus.
Using N-acylphosphoramidites, oligonucleotides bearing the
thermolabile protecting group R can be readily prepared. Exemplary
oligomers that can be prepared using the N-acylphosphoramidites
described herein include, e.g., oligonucleoside phosphotriesters of
the formula: ##STR24##
wherein R.sup.1, X.sup.1, X, R.sup.2, R.sup.2', R.sup.3, R.sup.3',
R.sup.4, R.sup.15, Q.sup.1, Q and n are as defined above.
Oligonucleotides bearing other thermolabile protecting groups also
can be prepared from other N-acylphosphoramidites (e.g., acyclic
N-acylphosphoramidites). Moreover, when N-acylphosphoramidites are
used, post-coupling reactions and transformations, for example,
oxidation, sulfurization, and deprotection, occur without
epimerization at the phosphorus atom. Thus, utilizing
N-acylphosphoramidites provides for the facile production of
P-chiral oligomeric or polymeric products, with complete control of
stereochemistry with respect to the phosphorus atom. Moreover,
stereochemistry can be controlled for tricoordinated and
tetracoordinated phosphorus atoms.
In view of the above, the N-acylphosphoramidites that can be
utilized in accordance with the present invention are preferably
selected from the group consisting of compounds of the formulae:
##STR25##
wherein R, R.sup.1, X, R.sup.2, R.sup.2', R.sup.3, R.sup.3',
R.sup.4, R.sup.15, Q.sup.1, Q and n are as defined above group
consisting of OR.sup.7, CN, NO.sub.2, N.sub.3, and a halogen,
wherein R.sup.7 is an alkyl, an aryl, or an aralkyl and wherein
R.sup.7 is unsubstituted or is substituted with one or more halogen
atoms.
The N-acylphosphoramidites provide for the stereospecific
substitution of tricoordinated phosphorus compounds under basic
conditions. In this regard, the monomeric compounds of formulae (I)
and (II), and the oligomeric compounds of formula (III), are useful
in the synthesis of polymers, particularly oligonucleotide
polymers, bearing thermolabile protecting groups on the
internucleosidic phosphorus linkage.
Preferably, the N-acylphosphoramidites are hydroxyl-protected
monomer-O-(O-protected)-(N-acyl)phosphoramidites, or hydroxyl
protected
oligomer/polymer-O-(O-protected)-(N-acyl)phosphoramidites,
exemplified by formulae (I)-(III). In a preferred embodiment, the
compound is a hydroxyl-protected
monomer-O-(N-acyl)-1,3,2-substituted oxazaphospholane (formula
(I)), which can be isolated as the Rp or Sp chiral form, to be used
in the synthesis of polymers containing stereogenic phosphorus
centers of predetermined configuration in a site-specific
manner.
With respect to the N-acylphosphoramidites, any suitable N-acyl
moiety can be used. Suitable acyl moieties include R.sup.1
(C.dbd.X)N--groups which render the phosphorus-(N-acyl) bond
sufficiently reactive to allow displacement of the N-acyl group by
a nucleophile, preferably under basic conditions. The C.dbd.X bond
of the N-acylphosphoramidites includes carbonyl and carbonyl
equivalents. Thus, the N-acyl group includes carbonyl (wherein X is
O) and thiocarbonyl (wherein X is S). Typically, the N-acyl group
is a carbonyl, wherein X is O.
The Q in the N-acylphosphoramidites of formulae (I) and (II), and
the Q and Q.sup.1 in the intermediates obtained therefrom (formula
(III)), include nucleosides (natural and modified) and oligomers
which include one or more of such nucleosides, as described herein.
Any suitable monomer-monomer, monomer-oligomer, oligomer-monomer,
or oligomer-oligomer coupling reaction can be accomplished,
stereospecifically, using the compounds and methods of the present
invention. For example, the N-acylphosphoramidite of formula (I) or
(II) can be used to stereospecifically couple a suitably protected
nucleoside (or even a suitably protected oligonucleotide) to an
oligonucleotide. Thus, the N-acylphosphoramidites described herein
can be attached to an oligomer such as, for example, an
oligonucleotide (i.e., wherein Q is an oligonucleotide), as well as
a monomer (i.e., wherein Q is a nucleoside). The nucleophile which
is coupled to the N-acylphosphoramidite also can be monomeric or
oligomeric. Accordingly, Q.sup.1 also includes oligomers that
contain, as a component thereof, a nucleoside substituent as
described herein.
In a preferred embodiment, Q and/or Q.sup.1 is a nucleoside
substituent of the formula: ##STR26##
In this embodiment, R.sup.4 is advantageously a solid support or a
protecting group. The protecting group is most preferably a
4,4'-dimethoxytrityl protecting group.
Examples of monomeric N-acylphosphoramidites that can be used in
accordance with the present invention include compounds of the
formulae: ##STR27## ##STR28##
wherein B is as defined above.
Stereospecific coupling reactions can be carried out successively
"n" times, for example, starting with a nucleophile R.sup.4
--O--Q.sup.1 --OH (wherein R.sup.4 and Q.sup.1 are as defined
above), and continuing thereafter, to provide an intermediate of
formula (III), wherein n is an integer from 1 to about 300. It will
be appreciated that, when a compound of formula (I) is reacted with
a nucleophile R.sup.4 --O--Q.sup.1 --OH, "R.sup.4 " of formula (I)
is represented by "R.sup.15 " of formula (III). When the protecting
group R.sup.15 is removed, then R.sup.15 becomes a hydrogen.
R.sup.4 and R.sup.15 desirably are not both solid supports in
formula (III). When R.sup.15 is hydrogen, then another coupling
reaction can be carried out, and the process repeated successively,
until a polymer of desired length or structure is obtained. In each
successive reaction, the Q substituent of formula (I) can be can be
independently selected, as desired to obtain a variety of different
combinations. As such, Q can be the same or different in each of
the units defined by n, when n is greater than 1. In other words, Q
is independently selected when n is greater than one. Preferably, n
is in the range of from about 3 to about 200; more preferably, n is
in the range from about 10 to about 40; and most preferably n is in
the range from about 15 to about 25.
Typically, the monomeric units in the polymers prepared in
accordance with the present invention are connected via phosphorus
diester linkages, for example, phosphate or chiral phosphate
(P-chiral) linkages, as desired. However, the compounds and methods
of the present invention are not limited to the synthesis of
polymers having only phosphorus-linked monomeric units. For
example, the compounds of the present invention also can be used to
introduce one or more phosphorus-linked units into a polymer having
another type of linkage in the structure thereof, for example, a
carbonate, a urea, an ester, an ether, or any suitable combination
thereof.
Preferred N-acylphosphoramidites include N-acylphosphoramidites of
the formula: ##STR29##
wherein R.sup.1 -R.sup.4, B, and E are as defined above.
As indicated above, particular substituents for R.sup.1 -R.sup.3
can be selected which facilitate thermal cleavage of the protecting
group on the non-bridging phosphate or phosphorothioate oxygen
after coupling has been carried out.
Generally, oligonucleotide synthesis using an N-acylphosphoramidite
can be carried out by the steps of:
(a') reacting a nucleophile that can displace the N-acyl group of
an N-acylphosphoramidite of formula (I) or (II), wherein R.sup.4 is
a protecting group with an N-acylphosphoramidite of formula (I) or
(II), preferably in the presence of a base, to produce an adduct of
the N-acylphosphoramidite and the nucleophile, the adduct
comprising a tricoordinated phosphorus atom;
(b') reacting the adduct with a reagent selected from the group
consisting of oxidizing agents, sulfurizing agents, and selenizing
agents to produce a product, wherein the tricoordinated phosphorus
atom is converted into a phosphorus atom with a valence of greater
than three (e.g., a tetracoordinated phosphorus atom);
(c') removing the protecting group R.sup.4 from the product;
(d') optionally repeating steps (a') through (c'), one or more
times as necessary, until a polymer of specified length is
obtained; and
(e') thermally cleaving the bond linking the resulting protecting
group bonded to the non-bridging phosphate, phosphorothioate or
phosphoroselenoate oxygen atom after step (a'), (b'), (c') or (d').
While the thermal deprotection can be carried out at any stage
after any of steps (a')-(d'), it is preferably carried out after
step (c') or (d').
Preferably, the N-acylphosphoramidite is a P-chiral
N-acylphosphoramidite. When a P-chiral N-acylphosphoramidite is
used, the resulting adduct also is P-chiral, since the coupling
reaction (step (a')) occurs with stereo specificity. Moreover,
reaction of the resulting adduct of step (a') with an oxidizing, a
sulfurizing, or a selenizing agent (step (b')) occurs
stereospecifically, that is, without any epimerization at
phosphorus. For example, sulfurization of the P-diastereomerically
pure adduct of step (a'), obtained by using a P-diastereomerically
pure N-acylphosphoramidite, results in a P-diastereomerically pure
adduct. Although sulfurization reactions are applied to adducts
prepared from standard phosphoramidite coupling chemistry, the
phosphorothioate products obtained thereby contain a mixture of
phosphorus stereoisomers (i.e., they are not stereopure) because
the phosphorus adducts prepared via standard phosphoramidite
chemistry contain a mixture of stereoisomers. As indicated above,
standard phosphoramidite coupling reactions are not stereospecific.
Thus, P-chiral coupling adducts can be stereospecifically produced
using the N-acylphosphoramidites described herein and, thus, can
provide access to oligonucleotides which are stereochemically pure
at phosphorus (e.g., oligonucleotide phosphorothioates).
Any suitable base can be used in coupling step (a') including, for
example, inorganic and organic bases. Preferably, the base used in
step (a') is a relatively non-nucleophilic base, which is more
preferably a relatively non-nucleophilic amine base such as, for
example, tetramethylguanidine (TMG). Advantageously, and
preferably, the coupling is carried out under basic conditions. As
a result, the use of an acid in the coupling reaction is avoided,
and the P-diastereomerically pure adduct formed in step (a') does
not epimerize. Since the coupling reaction of step (a') occurs with
complete stereospecificity, the stereochemical purity with respect
to phosphorus can be governed by the stereochemical purity of the
N-acylphosphoramidite used therein.
Desirably, the N-acylphosphoramidite approach described herein
further includes the step of capping the unreacted nucleophilic
group after step (b') or (c'). Capping is usually done as a
prophylactic measure to prevent the unreacted nucleophilic groups,
left over from prior condensation reactions, from reacting in
subsequent condensation cycles. Capping promotes synthetic
advantages such as, for example, preventing the formation of
undesirable side products. When the nucleophile (or oligomeric
adduct, if steps (a')-(c') are repeated at least once) is a sugar
hydroxyl, capping typically involves acylation of the unreacted
sugar hydroxyls.
Typically, the reaction in step (a') leads to formation of a
tricoordinated P-chiral product, thereby enabling, in step (b'),
the formation of a P-chiral product. Deprotection of the preferred
tetracoordinated P-chiral products can provide a P-chiral polymer
of predetermined chirality and length. Preferably, the nucleophile
is a nucleoside, an oligonucleotide, or a derivative thereof, step
(a') utilizes a P-chiral N-acylphosphoramidite, and step (b')
comprises sulfurization. Repeating the steps (a')-(c') can be
continued as many times as desired, until a polymer of a particular
length and chirality is obtained.
As discussed above, formation of a tricoordinated P-chiral product
in step (a') can be achieved by using any suitable P-chiral
N-acylphosphoramidite, most preferably a P-chiral analog of
compound (I) or (II). In accordance with the present invention,
P-chiral N-acylphosphoramidites can be obtained by any suitable
method such as, for example, chiral synthesis, chromatographic
resolution, or any suitable combination thereof. Chromatographic
separation of a mixture of P-chiral isomers can be facilitated, for
example, if the monomeric subunit of the N-acylphosphoramidite is a
chiral molecule, as illustrated, for example, in Scheme 2.
##STR30##
Using this technique, P-chiral products having any desired
phosphorus stereochemistries can be stereospecifically prepared
simply by selecting the appropriate P-chiral N-acylphosphoramidite
and using it in accordance with the method of the present
invention. When P-chiral phosphate analogue linkages are desired,
the N-acylphosphoramidite approach described herein makes it
possible to prepare polymers having a predetermined sequence of
P-chirality along the polymer backbone. P-chiral oligonucleotides
derived from N-acylphosphoramidites can be employed as
hybridization probes, therapeutic agents, e.g., selective protein
expression inhibitors, and the like.
There are other advantages to using N-acylphosphoramidite
precursors, such as, for example, moisture stability. In
particular, the N-acylphosphoramidites described herein are far
more stable to moisture under the coupling conditions of step (a')
than are the conventional phosphoramidite synthons for which mild
acid conditions are required. Moisture instability is one
disadvantage inherent in oligonucleotide synthesis using standard
phosphoramidite chemistry. In particular, standard phosphoramidite
precursors can hydrolytically degrade upon contact with moisture
under standard (acidic) conditions which are required to accomplish
a coupling reaction. As such, acid-promoted phosphoramidite
nucleoside couplings typically are carried out in a moisture-free
environment, particularly if the target polymer comprises a large
number of monomeric units. Since the N-acylphosphoramidites
described herein undergo hydrolytic degradation sluggishly, or not
at all, under the coupling conditions of step (a'), the problem of
competitive hydrolytic cleavage has essentially been eliminated. As
such, the utilization of N-acylphosphoramidites as described herein
does not require a scrupulously water-free environment.
In a preferred embodiment, the nucleophile coupled to the
N-acylphosphoramidite is attached to a solid support. When the
nucleophile is attached to a solid support, the nucleophile is
preferably a compound of the formula:
wherein Q is a nucleoside, an oligonucleotide comprising a
nucleoside, or an oligomer comprising a nucleoside, wherein the
nucleoside is of the formula: ##STR31##
wherein B and E are as defined herein, or an oligomer which
includes one of these nucleosides as a component thereof, and
R.sup.4 is the solid support.
Desirably, the nucleophile is a monomer. In a preferred embodiment,
the nucleophile is a monomer and is attached to a solid phase
support through a linking group that will resist cleavage in the
presence of a base, for example, a base used in step (a'), thereby
allowing the resulting oligomer/polymer to remain attached to the
solid support throughout each successive coupling step. When a
solid support is used in connection with a nucleophile (e.g., a
nucleophilic monomer), Q is preferably a nucleoside of the formula:
##STR32##
wherein B and E are as defined above. In one preferred embodiment,
Q is a nucleoside substituent having a defined stereochemistry, and
is represented by the formula: ##STR33##
wherein B and E are as defined above.
In a particularly preferred embodiment, a cyclic
N-acylphosphoramidite of formula (I) is used to effect the desired
coupling, and is represented by the formula: ##STR34##
wherein R.sup.1 -R.sup.4, B, and E are as defined above.
Preferably, B is a purine, a pyrimidine, adenine, guanine,
cytosine, uracil, or thymine, wherein B is unsubstituted or
substituted with one or more substituents, which are the same or
different, selected from the group consisting of a protecting
group, R.sup.11, OR.sup.11, NHR.sup.11, NR.sup.11 R.sup.12, CN,
NO.sub.2, N.sub.3, and a halogen, wherein R.sup.11 and R.sup.12 are
as defined herein.
In one embodiment, R.sup.1 is an alkyl, which is unsubstituted or
substituted with one or more substituents selected from the group
consisting of fluorine, OR.sup.7 and SR.sup.7, wherein R.sup.7 is
an alkyl or an aryl. For example, R.sup.1 can be a C.sub.1 -C.sub.6
alkyl, which is unsubstituted or substituted with one or more
fluorine atoms, e.g., a methyl, which is unsubstituted or
substituted with one or more fluorine atoms (e.g., fluoromethyl).
In another embodiment, R.sup.2, R.sup.2', R.sup.3, or R.sup.3' is a
vinyl group, a phenyl or a benzyl. A preferred protecting group for
R.sup.4 is the 4,4'-dimethoxytrityl group.
Oxidizing agents that can be used in accordance with the present
invention include any suitable reagent that can oxidize a
tricoordinated phosphorus atom, particularly a phosphite, to
provide a phosphorus atom having a valence of higher than three,
preferably a tetracoordinated phosphorus atom such as, for example,
a phosphate, or an equivalent thereof. Suitable oxidizing agents
include, for example, I.sub.2 /H.sub.2 O, peroxides, such as
tert-butylhydroperoxide, and the like.
Sulfurizing agents that can be used in accordance with the present
invention include any suitable reagent that can sulfurize a
tricoordinated phosphorus atom, particularly a phosphite, to
provide a phosphorus atom with a valence of greater than three,
preferably a tetracoordinated phosphorus atom such as, for example,
a phosphorothioate, or an equivalent thereof. Suitable sulfurizing
agents include, for example, 3H-1,2-benzodithiol-3-one 1,1-dioxide
("Beaucage Reagent"), phenylacetyl disulfide,
bis(O,O-diisopropoxyphosphinothioyl) disulfide, and the like.
Selenizing agents that can be used in accordance with the present
invention include any suitable reagent that can selenize a
tricoordinated phosphorus atom, particularly a phosphite, to
provide a phosphorus atom having a valence of greater than three,
preferably a tetracoordinated phosphorus atom such as a
phosphoroselenoate, or an equivalent thereof. Suitable selenizing
agents include, for example, potassium selenocyanate (KSeCN) or
elemental selenium.
N-acylphosphoramidites also can be applied toward the synthesis of
unmodified oligonucleotides and to the non-stereospecific synthesis
of oligonucleotide analogues, for example, by performing the steps
of:
(i) providing a nucleophile;
(ii) reacting the nucleophile, in the presence of a mild acid, with
a synthon of the formula: ##STR35##
wherein X and R.sup.1 -R.sup.3' are as defined herein, and W is a
leaving group amenable to nucleophilic displacement (e.g., a
dialkylamino), to produce an adduct of the nucleophile and the
synthon, which is an N-acylphosphoramidite having a tricoordinated
phosphorus atom;
(iii) reacting, in the presence of a base, the resulting adduct
with a nucleoside, having at least one nucleophilic group and at
least one suitably protected nucleophilic group, to produce a
product;
(iv) oxidatively transforming the tricoordinated phosphorus atom
into a tetracoordinated one;
(v) deprotecting the protected nucleophilic group of the resulting
product; and
(vi) repeating the steps (ii)-(v) until an oligomer or polymer of
predetermined length is obtained.
Preferably, this method further comprises the step of capping
unreacted nucleophilic groups after step (iii) or (iv), as
discussed herein. It is further preferred to attach the first
monomer (i.e., the nucleophile in the first coupling reaction of a
synthesis) to a solid phase support through a linking group that
will resist cleavage, when in the presence of the base used in step
(iii).
Preferably, W is displaced by a monomer of the formula R.sup.4
--O--Q--OH or R.sup.4 --O--Q.sup.1 --OH, wherein R.sup.4, Q, and
Q.sup.1 are as defined herein. In a preferred embodiment, W is a
dialkylamino having from 2 to about 8 carbon atoms (e.g.,
dimethylamino, diethylamino, N-methyl-N-isopropylamino, and the
like), or a cyclic amine substituent having from 2 to about 6
carbon atoms (e.g., pyrrolidinyl, piperidinyl, morpholinyl,
aziridinyl, and the like), wherein one or more carbon atoms of the
dialkylamino and cyclic amine substituents are unsubstituted or
substituted with one or more heteroatoms, which are the same or
different. More preferably W is a dialkylamino, or a cyclic amino.
Most preferably, W is a di(C.sub.1 -C.sub.6 alkyl)amino (e.g., a
diethylamino or a diisopropylamino).
The reactions in step (iii) enable the formation of the
tricoordinated P-chiral product and, preferably, step (vi) causes
formation of the tetracoordinated P-chiral product in a
stereospecific manner. Moreover, thermal deprotection preferably
gives either a P-achiral or a P-chiral polymer of predetermined
length. In step (iii), suitably protected nucleosides comprise
unmodified and/or modified nucleosides. Step (iv) preferably
comprises oxidation and/or sulfurization.
When an N-acylphosphoramidite is used, it is preferred that an
N-acylphosphoramidite of formula (I) is used. Thus, in a preferred
embodiment, the resulting product of steps (a')-(c'), (a')-(d'),
(iii), or (iii)-(v) described herein is a compound of formula
(III). Compounds of formula (III) are dimeric, when one coupling
step is performed (n=1). However, any desired number of subsequent
coupling steps can be performed, typically requiring deprotection
(step (c) or step (v)) prior to subsequent coupling reactions,
wherein each monomeric unit defined by "n" is the same or
different, and the substituents R.sup.1 -R.sup.4, R.sup.15, X,
Q.sup.1, and Q are as defined herein. Compounds of formula (III)
are useful in the synthesis of polymers, particularly
phosphodiester-linked polymers, more particularly P-chiral
phosphodiester-linked polymers, which can be obtained from (III)
via thermal cleavage of the 2-amidoethoxy protecting group bonded
to the non-bridging phosphate, phosphorothioate or
phosphoroselenoate oxygen atom, as described herein.
Oligomers and polymers synthesized in accordance with the present
invention are typically represented by the formula: ##STR36##
wherein: Q, X.sup.1, and n are as defined above, and Y is any
suitable heteroatom or organic substituent, preferably hydroxyl (or
a suitable salt thereof). Preferably n is in the range from about 3
to about 200; more preferably, n is in the range from about 10 to
about 40; and most preferably in the range from about 15 to about
25. In the polymers synthesized using the methods and compounds of
the present invention, Q, X.sup.1, and Y, or any combination
thereof, can be the same or different when n is 1, and can be the
same or different in each of the units defined by n when n is
greater than 1.
Typically, R.sup.4 is a hydrogen or a hydroxyl protecting group
such as, for example, a 4,4'-dimethoxytriphenylmethyl (DMTr),
4-methoxy-triphenylmethyl (MMTr), pixyl, acetyl,
9-fluorenylmethyloxycarbonyl (Fmoc), t-butyldimethylsilyl (TBDMS),
and the like. Alternatively, R.sup.4 is a reporter group such as,
for example, an amine, a mercapto, a phosphate, a phosphorothioate,
and the like. Reporter groups preferably contain an active moiety
for further reaction with radioactive label such as, for example,
.sup.32 P-phosphate, .sup.125 I-iodinated Bolton-Hunter reagent,
and the like, or a non-radioactive label such as, for example,
fluorescein isothiocyanate (FITC), dansyl chloride, and the like,
or any other biologically active group such as, for example,
biotin, digoxigenin, and the like. Reporter groups can be
introduced by means known to those skilled in the art including,
for example, introduction of appropriate linkers, spacers, arms, or
other reagents used for manipulating the distance between the
reporter group and the polymer.
X.sup.1 in formula (IIIA) is preferably S, O, or Se. If desired,
X.sup.1 also can be a substituted imino of the
formula.dbd.NR.sup.16, wherein R.sup.16 is an alkyl, an aryl, or an
alkenyl-substituted aryl substituent. Preferably, Y is an OH (or
suitable salt thereof).
In a preferred embodiment, P-chiral polymers that are prepared in
accordance with the present invention are of formula (IIIA) above,
wherein X.sup.1 and Y, or any combination thereof, can be the same
or different in any of the units being defined by n. More
preferably, P-chiral oligonucleotides prepared in accordance with
the present invention are of the formula: ##STR37##
wherein X.sup.1, Y, B, E and R.sup.4 are as defined herein, and
E.sup.1 includes the same groups defined herein with respect to E,
and E and E.sup.1 can be the same or different. B is preferably a
natural or a synthetically modified nucleic base, or B is a
synthetic analog or reporter group, preferably a reporter group
comprising a carboxyl, an alkyl, or an alkylamine. E.sup.1 is
preferably a 3'-hydroxyl (optionally protected), and E is
preferably a hydrogen, a halogen, a hydroxyl, or an appropriately
protected hydroxyl, an amine, or an appropriately protected amine,
or the like.
A polymer of any suitable length can be prepared in accordance with
the method of the present invention. Preferably, n is in the range
from about 3 to about 200, but is more preferably in the range from
about 12 to about 60. It is understood that the P-chiral
oligonucleotides of the invention can include linkages, for
example, 5'-3', 5'-2', 5'-5', 3'-3', 2'-2', and 3'14 2' linkages,
between nucleosides by the appropriate selection of Q and Q.sup.1,
as defined herein.
The compounds represented by formulae (I) and (II) are typically
prepared from a synthon of the formula: ##STR38##
wherein R, R.sup.1 -R.sup.3', and R.sup.5 are as defined herein,
and W is a leaving group amenable to nucleophilic attack by a free
group of the monomer, preferably a monomer of the formula R.sup.4
--O--Q--OH or R.sup.4 --O--Q.sup.1 --OH, wherein R.sup.4, Q, and
Q.sup.1 are as defined herein. Preferably, W is halogen, a
dialkylamino having from 2 to about 8 carbon atoms, or a cyclic
amine substituent having from 2 to about 6 carbon atoms, wherein at
least one carbon of the alkyl groups in the dialkylamino and cyclic
amine substituents is optionally substituted with one or more
heteroatoms, which are the same or different. More preferably W is
a dialkylamino, or a cyclic amino. Most preferably, W is a
dialkylamino (e.g., a diethylamino or a diisopropylamino).
EXAMPLES
The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its
scope.
Example 1
This example demonstrates the preparation of
2-(N-formyl-N-methyl)aminoethan-1-ol (FIG. 2A).
2-(Methylamino)ethanol (51.0 g, 0.68 mol) was placed in a 250 mL
round-bottom flask equipped with a reflux condenser, and cooled to
5.degree. C. by immersion in an ice bath. Ethyl formate (75.0 g,
1.01 mol) was then added, in portions through the condenser to the
stirred amino alcohol over a period of 5 min at 5.degree. C. The
solution is removed from the ice bath and brought to reflux for 1
h. The solution was then distilled at atmospheric pressure to
remove excess ethyl formate, and then carefully distilled under
high-vacuum to afford 2-(N-formyl-N-methyl)aminoethan-1-ol as a
clear colorless liquid (63.1 g, 0.61 mol, 90%) boiling at
120-122.degree. C. @ 0.15 mm Hg. .sup.1 H-NMR (300 MHz,
DMSO-d.sub.6): .delta.[2.75 (s) and 2.94 (s, 30%) (3H)], 3.27 (m,
2H), 3.47 (m, 3H), [7.94 (s) and 7.99 (s, 30%) (1H)]. .sup.13 C-NMR
(75 MHz, DMSO-d.sub.6): .delta.29.2, 34.9, 46.2, 51.2, 57.8, 57.9,
58.1, 58.2, 162.7, 163.0. EI-MS: calcd for C.sub.4 H.sub.9 NO.sub.2
(M.sup..multidot.+) 103, found 103.
Example 2
This example demonstrates the preparation of
N,N,N',N'-tetraisopropyl-O-[2-[(N-formyl-N-methyl)amino]ethyl]phosphordiam
idite (compound 120, FIG. 2A).
To an oven-dried 100 mL round-bottom flask containing 50 mL of dry
benzene under a dry argon atmosphere, 876 .mu.L of freshly
distilled phosphorus trichloride (10 mmol) were added by syringe
through a rubber septum. The stirred solution was cooled to
5.degree. C. by immersion in an ice bath and then, 7.7 mL of
anhydrous N,N-diisopropylamine (55 mmol) were added by syringe
under argon over a period of 30 min. The reaction mixture was
removed from the ice bath and allowed to warm to 25.degree. C.
under a positive pressure of argon until the formation of
bis(N,N-diisopropylamino)chlorophosphine is complete. The rate of
the reaction was monitored by .sup.31 P NMR spectroscopy; after -48
h, the expected chlorophosphine was observed as the major (>96%)
reaction product (132.0 ppm downfield relative to a phosphoric acid
external standard). 2-(N-Formyl-N-methyl)aminoethan-1-ol (1.03 mL,
10 mmol) was then added to the suspension. The resulting mixture
was stirred for 2 h at 25.degree. C. under a positive pressure of
argon. .sup.31 P NMR analysis of the reaction mixture indicates
that the generation of compound 120 is essentially quantitative
(-96%) and reveals two singlets at 118.0 and 118.7 ppm in C.sub.6
D.sub.6. The suspension was filtered through a sintered glass
funnel (coarse porosity, 60 mL) and washed with 20 mL of dry
benzene. The filtrates were evaporated under reduced pressure to an
oil and dissolved in a minimum amount (-3 mL) of benzene and
triethylamine (95:5 v/v). The viscous solution was then applied
uniformly to the top of a chromatography column (3.times.20 cm)
packed with a Silica Gel 60 .ANG. (Merck 230-400 mesh, 30 g) slurry
in a solution of benzene:triethylamine (95:5 v/v). The column was
eluted isocratically with benzene:triethylamine (95:5 v/v) and
fractions of 8 mL each were collected. Fractions containing the
phosphordiamidite 120 were evaporated to an oil. Residual
triethylamine was removed from the product by co-evaporation with
toluene (4.times.10 mL). The phosphordiamidite was then left under
high vacuum for at least 3 h. Yield: 2.43 g (7.3 mmol, 73%). .sup.1
H-NMR (300 MHz, C.sub.6 D.sub.6): .delta.[1.14 (d, J=6.9 Hz), 1.16
(d, J=6.7 Hz) 1.18 (d, J=6.7 Hz) (24H)], [2.40 (s, 34%) and 2.64
(s, 66%) (3H)], 2.80 (t, J=5.4 Hz, 2H), 3.43 (m, 4H), [3.29 (dt,
J=5.4 Hz, J.sub.CP =8.5 ), Hz) and 3.60 (dt, J=5.4 Hz, J.sub.HP
=6.6 Hz)(2H)], [7.82 (s, 34%) and 7.98 (s, 66%) (1H)]. .sup.13
C-NMR (75 MHz, C.sub.6 D.sub.6): .delta.24.1, 24.2, 24.6, 24.7,
44.7, 44.9, 45.8 (d, .sup.2 J.sub.CP =8.5 Hz), 50.4 (d, .sup.2
J.sub.CP =8.5 Hz), 61.3, 61.5, 61.9, 62.2, 161.9, 162.3. .sup.31
P-NMR (121 MHz, C.sub.6 D.sub.6): .delta.118.0, 118.7. EI-HRMS:
calcd for C.sub.16 H.sub.36 N.sub.3 O.sub.2 P (M.sup.19 +)
333.2545, found 333.2528.
Example 3
This example demonstrates the general preparation of
5'-O-(4,4'-dimethoxytrityl)-3'-O-(N,N-diisopropylamino)[2-[(N-formyl-N-met
hyl)amino]ethoxy]phosphinyl-2'-deoxyribonucleosides.
A suitably protected deoxyribonucleoside (2 mmol) was dried under
high vacuum for 2 h in a 50 mL round-bottom flask. Anhydrous
acetonitrile (10 mL) was added to the dried nucleoside followed by
N,N,N',N'-tetraisopropyl-O-[2-[(N-formyl-N-methyl)amino]ethyl]phosphordiam
idite 20 (730 mg, 2.2 mmol). To this solution was added by syringe
4.4 mL of 0.45 M 1H-tetrazole in acetonitrile (2 mmol), dropwise,
over a period of 0.5 h. The rates of the reaction were monitored by
TLC using benzene:triethylamine (9:1 v/v) as an eluent.
Phosphinylation of suitably protected 2'-deoxynucleosides was
usually complete within 1 h at 25.degree. C. (for best results,
phosphinylation of properly protected 2'-deoxyguanosine is allowed
to proceed for 12 h). The reaction mixture was then concentrated
under reduced pressure, dissolved in benzene:triethylamine (9:1
v/v), and chromatographed on a silica gel column (4 cm.times.10 cm)
using the same solvent for equilibration and elution. Appropriate
fractions were pooled, concentrated, and each of the
deoxyribonucleoside phosphoramidites were isolated as a white
amorphous powder in yields exceeding 90%.
5'-O-(4,4'-dimethoxytrityl)-3'-O-(N,N-diisopropylamino)[2-[(N-formyl-N-met
hyl)amino]ethoxy]phosphinyl-deoxythymidine (compound 101, FIG. 2A).
.sup.31 P-NMR (121 MHz, C.sub.6 D.sub.6): .delta.145.3, 145.2,
145.0, 144.8. FAB-HRMS: calcd for C.sub.41 H.sub.53 N.sub.4 O.sub.9
P (M+Cs).sup.+ 909.2604, found 909.2544. N.sup.6
-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(N
N-diisopropylamino)[2-[(N-formyl-N-methyl)amino]ethoxy]phosphinyl-2'
deoxyadenosine. .sup.31 P-NMR (121 MHz, C.sub.6 D.sub.6):
.delta.145.7, 145.6, 144.9. FAB-HRMS: calcd for C.sub.48 H.sub.56
N.sub.7 O.sub.8 P (M+Na).sup.+ 912.3827, found 912.3843. N.sup.2
-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(N,N-diisopropylamino)[2-[(N-
formyl-N-methyl)amino]ethoxy]phosphinyl-2'deoxyguanosine. .sup.31
P-NMR (121 MHz, C.sub.6 D.sub.6): .delta.145.7, 140.9. FAB-HRMS:
calcd for C.sub.45 H.sub.58 N.sub.7 O.sub.9 P (M+Na).sup.+ is
894.3933, found 894.3978.
Example 4
This example describes generally the automated synthesis of
oligonucleotides using a phosphoramidite precursor.
The automated synthesis of DNA/RNA oligonucleotides is performed on
DNA/RNA synthesizers using the corresponding nucleoside
phosphoramidites (examples of which are shown in FIG. 1C (compound
101) and FIG. 2B (compounds 102-109)) according to the
manufacturers recommendation. A general description of the various
steps involved in, for example, solid-phase DNA synthesis is
described in Beaucage, Methods in Molecular Biology, Vol. 20:
Protocols for Oligonucleotides and Analogs, (S. Agrawal, ed.),
Humana Press: Totowa, N.J., pp. 33-61; Beaucage et al., Current
Protocols in Nucleic Acid Chemistry, Vol.1 (Beaucage S. L.,
Bergstrom, D. E., Glick, G. D. Jones R. A. eds), John Wiley and
Sons: New York, 2000, pp. 3.3.1-3.3.20; and Beaucage et al.,
Tetrahedron, 48, 2223-2311 (1992), and references therein.
Example 5
This example describes a general procedure for the thermolytic
cleavage of phosphate/thiophosphate protecting groups from
chemically synthesized oligonucleotides.
Upon completion of solid-phase oligonucleotide synthesis, the
solid-phase bound oligonucleotide is 5'-detritylated and then
N-deprotected by treatment with, for example, pressurized ammonia
gas (10 bar at 25.degree. C.) for at least 10 h. The partially
deprotected oligonucleotides is eluted from the column chamber with
an aqueous solution of acetonitrile (MeCN, 2 parts) in 0.1 M
triethylammonium acetate, pH 7.0 (TEAA, 3 parts). For a typical 0.2
.mu.mol synthesis column, 1 mL of the aqueous MeCN/TEAA solution is
sufficient for complete elution of the oligonucleotide. The
oligonucleotide solution is then heated at 90.degree. C. up to 3 h
in a sealed glass vial to effect the thermolytic cleavage of the
phosphate/thiophosphate protecting group. The time required for
such a deprotection depends on the nature of the
phosphate/thiophosphate protecting group that has been used.
Example 6
This example illustrates the general synthesis of an
N-acylphosphoramidite. The reaction schemes referenced in this
example are generally illustrated in FIG. 3.
Typically, the synthon precursor (FIG. 3) is synthesized by first
refluxing a mixture of acrolein (1), trimethylsilyl cyanide, and
catalytic amounts of zinc iodide according to the procedure
reported by Gardrat et al. (J. Heterocyclic Chem., 27, 811 (1990)).
Reduction of the resulting nitrile 2 with LiAlH.sub.4 in Et.sub.2 O
afforded amino-alcohol 3. Heating 3 with a slight excess (1.1 molar
equiv) of ethyl fluoroacetate at 120.degree. C. until all ethyl
alcohol has distilled off gave the hydroxylated amide 4 in 88%
yield (b.p. 83-84.degree. C./0.1 torr). An equimolar solution of
hexaethylphosphorus triamide and 4 was heated to 120.degree. C.
until all diethylamine has distilled off. Vacuum distillation
afforded the oxazaphospholane 5 in 69% yield.
Nucleoside cyclic acylphosphoramidite 7 was prepared by the
reaction of a suitably protected nucleoside 6 with equimolar
amounts of 5 and 1H-tetrazole in anhydrous dichloromethane for 4 h
at ambient temperature. Following evaporation of the reaction
mixture, the residue is purified using a short silica gel column
chromatography. The nucleosidic synthon 7 is rapidly eluted with a
solution of acetonitrile:chloroform (1:2 v/v). Removal of the
eluent under reduced pressure afforded 7 as a white foam. The
nucleoside cyclic acylphosphoramidite 9 is prepared in a similar
manner from nucleoside 8 and compound 5.
Example 7
This example illustrates a solid phase synthesis using an
N-acylphosphoramidite. The general reaction scheme is illustrated
in FIG. 4, in which nucleoside cyclic acylphosphoramidite 7 (FIG.
3) is specifically applied to the manual solid-phase synthesis of a
decanucleotide (dC.sub.10). A solid support is denoted in FIGS. 4
and 5 by a darkened sphere with "S" in the center.
Because of the sensitivity of standard succinyl linkers to strong
bases, the first nucleoside monomer was attached to long chain
alkylamine controlled pore glass (LCAA-CPG) to generate 10 has been
modified. The attachment of the leader nucleoside to LCAA-CPG is
accomplished via a sarcosine succinyl linkage according to the
method of Brown et al. (J. Chem. Soc. Chem. Commun., p. 891-893
(1989)). A column filled with 0.2 mmol of 10, wherein the 5'--OH
was protected with a DMTr group, was treated with 2.5 mL of 3%
trichloroacetic acid in dichloromethane for 1 min to ensure
complete cleavage of the 5'-O-dimethoxytrityl (DMTr) protecting
group. The column was then washed with 5 mL of acetonitrile (MeCN)
and treated with a solution of 7 (10 mg) in 200 mL of 7.5%
N,N,N',N'-tetramethylguanidine (TMG) in MeCN for 3 min. A solution
(1 mL) of Cap A and Cap B (1:1) was pushed through the column, left
for 1 min, and then washed with MeCN (5 mL), after which a solution
of 1 M tert-butylhydroperoxide in dichloromethane (1 mL) was pushed
through the column for 1 min, and washed with MeCN (5 mL). This
cycle was repeated 8 additional times.
Stepwise DMTr analysis indicated that each coupling yield proceeded
with high efficiency, typically 90% or greater. The content of the
column is then transferred into a glass vial, and deprotected. The
crude oligomer can be characterized by reversed phase (RP) HPLC and
polyacrylamide gel electrophoresis (PAGE).
To enable, for example, the synthesis of thioated oligonucleotides
stereogenically at phosphorus, the synthon 7 (FIG. 3) must first be
separated into its Rp and Sp diasteroisomers (see FIG. 5). This is
accomplished by chromatography on functionalized silica (C-1, C-2,
C-4, C-8, or C-18 reversed-phase silica).
Example 8
This example illustrates the application of the synthetic cycle
described in Example 7, in the stereospecific synthesis of
oligonucleotide phosphorothioates. The reaction scheme is
illustrated generally in FIG. 5.
A diastereomeric mixture of nucleosidic N-acylphosphoramidite 7 was
chromatographically separated into its Rp and Sp isomers, 7Rp and
7Sp, respectively. Each P-chiral isomer was coupled with
nucleophilic monomer 10 (FIG. 4), using the conditions of Example
7, to provide P-chiral adducts. The coupling reactions are
stereospecific. Sulfurization of the resulting adducts results in
the formation of the 11Sp and 11Rp isomers, as illustrated in FIG.
5. Deprotection of the solid support and the 2-amidoethoxy fragment
from the sulfurized products is therefore expected to provide
stereochemically pure Rp and Sp oligonucleotide products.
It should be noted that the oxidant in the oxidation step is
replaced by a sulfur-transfer reagent such as
3H-1,2-benzodithiol-3-one 1,1-dioxide, phenylacetyl disulfide,
bis(O,O-diisopropoxyphosphinothioyl) disulfide, and the like. In
order to ensure optimum sulfurization, a capping step should be
performed after the sulfur transfer step.
Example 9
This example illustrates the preparation of various nucleosidic
N-acylphosphoramidites, wherein the N-acyloxazaphospholane moiety
is introduced at different hydroxyls of a differentially protected
nucleoside core. The reaction schemes are illustrated generally in
FIG. 6.
Using the procedure of Example 6, nucleophilic monomers 12, 14, 16,
and 18 were coupled to synthon 5 using tetrazole, to provide
nucleosidic N-acylphosphoramidites 13, 15, 17, and 19,
respectively. The resulting nucleosidic N-acylphosphoramidites can
be used as a vehicle for one or more coupling reactions, to provide
oligomer or polymer products. Alternatively, the resulting
nucleosidic N-acylphosphoramidites can be separated into their Rp
and Sp isomers prior to their use as coupling reagents. The
phospholane moiety of nucleosidic N-acylphosphoramidites 13, 15,
17, and 19 are attached to either the 3'- or 5'-hydroxyl in the
case of 2'-deoxyribonucleosides or, additionally, to the
2'-hydroxyl in the case of ribonucleosides. These products also
represent various ribonucleoside monomers that can be used for
solid-phase synthesis (both stereospecific and non-stereospecific)
of oligoribonucleotides and their analogues as illustrated in FIG.
4 and FIG. 5.
Example 10
This example illustrates the preparation of acyclic
N-acylphosphoramidites. The nucleoside acylphosphoramidites can be
applied in a manner similar to that described in Examples 7 and 8,
and FIGS. 4-5. The reaction scheme is illustrated generally in FIG.
7. A solid support is denoted in FIG. 7 by a darkened sphere with
"S" in the center.
As illustrated in FIG. 7, the non-nucleosidic chlorophosphoramidite
derivative 20 is condensed with a suitable N-methylamide (21) to
generate the acylphosphoramidite 22. Reaction of 22 with suitably
protected nucleosides 6 (FIG. 3) in the presence of 1H-tetrazole
affords the corresponding nucleoside 3'-acylphosphoramidites 23 as
a mixture of P-diastereoisomers. These amidites are activated under
basic conditions and are expected to be useful in solid-phase
oligonucleotide synthesis in a manner similar to that shown in FIG.
4. Nucleoside 5'-acylphosphoramidites similar to 9 (FIG. 3) also
can be applied for the same purpose. Alternatively, separation of
the Rp- and Sp-diastereoisomers of 23 are expected to enable the
stereospecic synthesis of thioated oligonucleotides in a manner
similar to that illustrated in FIG. 5. In this context,
ribonucleoside acylphosphoramidites of formula ##STR39##
can be used in accordance with the present invention for
ribonucleotide syntheses, and are expected to work in the same
manner as the cyclic species, for example, 13, 15, 17, and 19 (FIG.
6).
Example 11
This example demonstrates an alternate approach to the synthesis of
oligonucleotides via nucleoside cyclic acylphosphoramidites and
acylphosphoramidites, as illustrated in FIGS. 8 and 9. A solid
support is denoted in FIGS. 8 and 9 by a darkened sphere with "S"
in the center. The strategy was demonstrated by reacting
non-nucleosidic cyclic N-acylphosphoramidite 5 (FIG. 3) and
acylphosphoramidite 22 (FIG. 7) with the functionalized
solid-support-bound 10 (FIG. 4) in the presence of 1H-tetrazole to
generate 25 and 26, respectively, as shown in FIG. 8. The reaction
of suitably protected nucleoside 6 with 25, or 6 with 26, under
basic conditions, followed by oxidation, provided dinucleotides 11
and 27, respectively. Deprotection of 11 and 27 provides the same
dinucleotide, as shown in FIG. 9. The same strategy applies with
respect to the synthesis of ribonucleotide and the
non-stereospecific synthesis of thioated oligonucleotides. The
solid-phase synthesis of a decanucleotide (dC.sub.10) has been
achieved using a DNA synthesizer.
General Protocol for Examples 12-17
For the synthesis of oligonucleotides using
5'-O-dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,2-oxazaphosphola
nyl-2'-O-deoxyribonucleoside derivatives in examples 12-17, the
general protocol is as follows. The syntheses were performed in a
standard DNA synthesis column as available from many suppliers.
Standard LCAA-CPG from Applied Biosystems (Masterpiece) columns are
used.
The syntheses were carried out by way of the following general
steps. The steps were not necessarily done in numerical order
within a particular synthesis cycle. The particular sequence of
steps used is indicated separately in each example.
In step 1, the appropriate CPG-bound nucleoside is detritylated in
accordance with a standard procedure.
In step 2,
5'-O-Dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,2-oxazaphosphola
nyl-2'-O-deoxyribonucleoside derivatives (5 mg, ca. 5 .mu.mol) are
dissolved in acetonitrile (200 .mu.L). Tetramethylguanidine (TMG, 4
.mu.l, ca. 30 .mu.mol) is subsequently added and the mixture is
applied to the synthesis column.
In step 3, a standard oxidation or sulfurization reaction is
carried out after the reaction of step 2 is continued for 5
min.
Steps 2 and 3 are repeated to optimize the yield for a particular
synthesis cycle. Steps 2 and 3 need not be performed more than once
for a particular synthesis cycle. However, yields are typically
improved (e.g., resulting in nearly 100% overall yield) if steps 2
and 3 are repeated within a particular synthesis cycle. Optionally,
steps 2 and 3 can be repeated three or more times, as desired, to
optimize the yield for a particular synthesis cycle even
further.
In step 4, the synthesis cycle is concluded with a capping step.
Synthesis cycles can be repeated until the designed sequence length
is obtained.
In step 5, the synthetic oligonucleotide is subjected to
post-synthesis cleavage from the support, and deprotection.
Example 12
This example describes the synthesis of a dinucleotide using an
N-acylphosphoramidite, particularly T.sub.PO T. The following steps
were used in the present example.
Step 1: The bound nucleoside was treated with 3% trichloroacetic
acid/dichloromethane (Applied Biosystems DNA synthesis reagent (3
mL, 1 min)), followed by washing with acetonitrile (3 mL, 30
s).
Step 2:
5'-O-Dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,2-oxazaphosphola
nyl-2'-O-deoxythymidine (5 mg, ca. 5 .mu.mol) and
tetramethylguanidine (TMG, 4 .mu.l, ca. 30 .mu.mol) in acetonitrile
(200 .mu.l) were added to the column and reacted for 5 min.,
followed by washing with acetonitrile (3 mL, 30 s).
Step 3: The resulting product was treated with
iodine/water/pyridine/tetrahydrofuran (Applied Biosystems DNA
synthesis reagent), (500 .mu.l, 30 s), followed by washing with
acetonitrile (3 mL, 30 s).
Step 5: The dinucleotide can be cleaved from the support and
deprotected.
In the present example, a standard column DMT-T-LCAA-CPG (0.2
.mu.mol) can be used and subjected to the above steps in the
following sequence:
Example 13
This example describes the synthesis of P-diasteriomerically pure
phosphorothioate [Rp]-C.sub.PS C. The following steps were used in
the present example.
Step 1: The bound nucleoside was treated with 3% trichloroacetic
acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3
mL, 1 min), followed by washing with acetonitrile (3 mL, 30 s).
Step 2:
[Sp]-N4-Benzoyl-5'-O-dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,
2-oxazaphospholanyl-2'-O-deoxycytidine (5 mg, ca. 5 .mu.mol) and
tetramethylguanidine (TMG, 4 .mu.l, ca 30 .mu.mol) in acetonitrile
(200 .mu.l) were added to the column and reacted for 5 min,
followed by washing with acetonitrile (3 mL, 30 s).
Step 3: The resulting product was treated with
3H-1,2-benzodithiol-3-one 1,1-dioxide (1% Beaucage Reagent in
acetonitrile (w/v)), 3 min, followed by washing with acetonitirile
(3 mL, 30 s).
Step 5: The dinucleotide can be cleaved from the support and
deprotected.
In the present example, a standard column DMT-CBz-LCAA-CPG (0.2
.mu.mol) can be used and subjected to the above steps in the
following sequence:
Example 14
This example describes the synthesis of a P-diastereomerically pure
phosphorothioate-linked trinucleotide (trimer), [Rp, Rp]C.sub.PS
C.sub.PS C. The following steps were used in the present
example.
Step 1: The bound nucleoside was treated with 3% trichloroacetic
acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3
mL, 1 min), followed by washing with acetonitirile (3 mL, 30
s).
Step 2:
[Sp]-N4-Benzoyl-5'-O-dimethoxytrityl-3'-O-(5-phenyl-N-fluoroacetyl)-1,3,2-
oxazaphospholanyl-2'-O-deoxycytidine (FIG. 10A, 5 mg, ca. 5
.mu.mol) and tetramethylguanidine (TMG, 4 .mu.l,
oxazaphospholanyl-2'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,2-oxazaphospholanyl
-2'-O-deoxycytidine (5 mg, ca. 5 .mu.mol) and tetramethylguanidine
(TMG, 4 .mu.l, ca. 30 .mu.mol) in acetonitrile (200 .mu.l) were
added to the column and reacted for 5 min., followed by washing
with acetonitrile (3 mL, 30 s).
Step 3: The resulting product from step 2 was treated with
3H-1,2,-benzodithiol-3-one 1,1-dioxide (1% Beaucage Reagent in
acetonitrile (w/v)), 3 min, followed by washing with acetonitrile
(3 mL, 30 s).
Step 4: The resulting product from step 3 was capped with acetic
anhydride/lutidine/tetrahydrofuran (Applied Biosystems DNA
synthesis reagent), (1 mL), mixed with
1-methylimidazole/tetrahydrofuran (Applied Biosystems DNA synthesis
reagent), (1 mL), 2 min, followed by washing with acetonitrile (3
mL, 30 s).
Step 5: The trinucleotide can be cleaved from the support and
deprotected.
In the present example, a standard column DMT-CBz-LCAA-CPG (0.2
.mu.mol) can be used and subjected to the above steps in the
following sequence:
The product obtained in accordance with this example can be
analyzed by RP-HPLC. The P-diastereomeric purity can be confirmed
by co-injection of the trimer prepared in accordance with this
example and the corresponding P-diastereomeric mixture obtained by
the standard phosphoramidite method.
Example 15
This example describes the synthesis of a P-diastereomerically pure
phosphorothioate-linked trinucleotide (trimer), [S.sub.P, S.sub.P
]C.sub.PS C.sub.PS C. The following steps were used in the present
example.
Step 1: The bound nucleoside was treated with 3% trichloroacetic
acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3
mL, 1 min), followed by washing with acetonitrile (3 mL, 30 s).
Step 2:
[Rp]-N4-Benzoyl-5'-O-dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,
2-oxazaphospholanyl-2'-O-deoxycytidine (FIG. 10B, 5 mg, ca. 5
.mu.mol) and tetramethylguanidine (TMG, 4 .mu.l, ca. 30 .mu.mol) in
acetonitrile (200 .mu.l) were added to the column and reacted for 5
min, followed by washing with acetonitrile (3 mL, 30 s).
Step 3: The resulting product from step 2 was treated with
3H-1,2-benzodithiol-3-one 1,1-dioxide (1% Beaucage Reagent in
acetonitrile (w/v)), 3 min, followed by washing with acetonitrile
(3 mL, 30 s).
Step 4: The resulting product from step 3 was capped with acetic
anhydride/lutidine/tetrahydrofuran (Applied Biosystems DNA
synthesis reagent), (1 mL), 2 min., followed by washing with
acetonitrile (3 mL, 30 s).
Step 5: The trinucleotide can be cleaved from the support and
deprotected.
In the present example, a standard column DMT-CBz-LCAA-CPG (0.2
.mu.mol) can be used and subjected to the above steps in the
following sequence:
The product obtained in the present example can be analyzed by
RP-HPLC. P-diastereomeric purity can be confirmed by co-injection
of the trimer prepared in this example and the corresponding
P-diastereomeric mixture obtained by the standard phosphoramidite
method.
Example 16
This example describes the synthesis of a P-diastereomerically pure
phosphorothioate-linked tetramer [Rp, Sp, Rp]-C.sub.PS C.sub.PS
C.sub.PS C. The following steps were used in the present
example.
Step 1: The bound nucleoside was treated with 3% trichloroacetic
acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3
mL, 1 min), followed by washing with acetonitrile (3 mL, 30 s).
Step 2:
[Sp]-N4-benzoyl-5'-O-dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,
2,-oxazaphospholanyl-2'-O-deoxycytidine (5 mg, ca. 5 .mu.mol) and
tetramethylguanidine (TMG, 4 .mu.l, ca. 30 .mu.mol) in acetonitrile
(200 .mu.l) were added to the column and reacted for 5 min,
followed by washing with acetonitrile (3 mL, 30 s).
Step 2':
[Rp]-N4-Benzoyl-5'-O-dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,
2-oxazaphospholanyl-2'-O-deoxycytidine (FIG. 10B, 5 mg, ca. 5
.mu.mol) and tetramethylguanidine (TMG, 4 .mu.l, ca. 30 .mu.mol) in
acetonitrile (200 .mu.l)) were added to the column and reacted for
5 min, followed by washing with acetonitrile
(3 mL, 30 s). 3: The resulting product from step 2 or 2' was
treated with 3H-1,2-benzodithiol-3-one 1,1-dioxide (1% Beaucage
Reagent in acetonitrile (w/v)), 3 min, followed by washing with
acetonitrile (3 mL, 30 s).
Step 4: The resulting product from step 3 was capped with acetic
anhydride/lutidine/tetrahydrofuran (Applied Biosystems DNA
synthesis reagent), (1 mL), mixed with
1-methylimidazole/tetrahydrofuran (Applied Biosystems DNA synthesis
reagent), (1 mL), 2 min, followed by washing with acetonitrile (3
mL, 30 s).
Step 5: The trinucleotide can be cleaved from the support and
deprotected.
In the present example, a standard column DMT-CBz-LCAA-CPG (0.2
.mu.mol) can be used and subjected to the above steps in the
following sequence:
The product obtained in this example can be analyzed by RP-HPLC.
P-diastereomeric purity can be confirmed by co-injection of the
tetramer prepared in the accordance with this example and the
corresponding P-diastereomeric mixture obtained by the standard
phosphoramidite method.
Example 17
This example describes the synthesis of a P-diastereomerically pure
phosphorothioate-linked undecamer, [all Rp]-(Tps)11T (eleven
nucleoside units in the oligonucleotide chain). The following steps
were used in the present example.
Step 1: The bound nucleoside was treated with 3% trichloroacetic
acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3
mL, 1 min), followed by washing with acetonitrile (3 mL, 30 s).
Step 2:
[Sp]-5'-O-Dimethoxytrityl-3'-O-(5-phenyl-3-N-fluoroacetyl)-1,3,2-oxazaphos
pholanyl-2'-O-deoxythymidine (5 mg, ca. 5 .mu.mol) and
tetramethylguanidine (TMG, 4 .mu.l, ca. 30 .mu.mol) in acetonitrile
(200 .mu.l) were added to the column and reacted for 5 min.,
followed by washing with acetonitrile (3 mL, 30 s).
Step 3: The resulting product from step 2 was treated with
3H-1,2-benzodithiol-3-one 1,1-dioxide (1% Beaucage Reagent in
acetonitrile (w/v)), 3 min, followed by washing with acetonitrile
(3 mL, 30 s).
Step 4: The resulting product from step 3 was capped with acetic
anhydride/lutidine/tetrahydrofuran (Applied Biosystems DNA
synthesis reagent), (1 mL), 2 min, followed by washing with
acetonitrile (3 mL, 30 s).
Step 5: The trinucleotide can be cleaved from the support and
deprotected.
In the present example, a standard column DMT-T-LCAA-CPG (0.2
.mu.mol) can be used and subjected to the above steps in the
following sequence:
The product obtained in the present example is believed to be
P-diastereomerically pure.
Example 18
This example demonstrates the hydrolytic stability of an
N-acylphosphoramidite (FIG. 11), relative to the hydrolytic
stability of a corresponding phosphoramidite. The hydrolytic
stability for each type of reagent was determined under reaction
conditions normally employed for each type of coupling reagent.
Samples of the dinucleotide d(T.sub.PO G) were prepared by a
standard coupling method using a standard phosphoramidite that is
commonly used in the art. Samples of d(T.sub.PO G) also were
prepared by a coupling reaction using the N-acylphosphoramidite of
FIG. 11. Each coupling method was performed in the absence of
moisture and in the presence of moisture (0.1% water).
The products were analyzed by HPLC. The HPLC's confirmed that the
same product (d(T.sub.PO G)) was obtained by either method when the
reactions were carried out in a moisture-free environment. However,
when the same reactions were carried out in the presence of
moisture, the product obtained by the standard phosphoramidite
contained only a trace of the desired product, and was almost
entirely the uncoupled single nucleoside dG. Thus, the standard
phosphoramidite was hydrolytically unstable under coupling
conditions in which moisture was present. By contrast, the product
obtained using the N-acylphosphoramidite (FIG. 11) contained mostly
the desired product, and a relatively minor amount of the uncoupled
single nucleoside dG, even when the coupling reaction was performed
in the presence of significant moisture. These results demonstrate
the relative hydrolytic stability of the N-acylphosphoramidites
described herein.
All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the
same extent as if each reference were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the
context of the following claims) are to be construed to cover both
the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations of those preferred embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventors expect skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
as specifically described herein. Accordingly, this invention
includes all modifications and equivalents of the subject matter
recited in the claims appended hereto as permitted by applicable
law. Moreover, any combination of the above-described elements in
all possible variations thereof is encompassed by the invention
unless otherwise indicated herein or otherwise clearly contradicted
by context.
* * * * *