Note: Descriptions are shown in the official language in which they were submitted.
CA 02439407 2003-08-26
IS
25
WO 02/068437 PCT/US02/05601
PROTECTED DEOXYADENOSINES AND DEOXYGUANOSINES
Field of the Invention
The invention relates to the synthesis and purification of protected
nucleosides, and more
particularly to methods for the synthesis and purification of protected
nucleosides without the
use of pyridine as a solvent.
Nucleosides are compounds of importance in physiological and medical research,
obtained
during partial decomposition, i.e., hydrolysis, of nucleic acids, and
containing a purine or
pyrimidine base linked to either D-ribose (forming ribonucleosides) or D-
deoxyribose (forming
deoxyribonucIeosides). They are nucleotides minus the phosphate group. Well-
known
nucleosides include adenosine (A), cytidine (C), uridine (U), and guanosine
(G), as well as the
deoxynucleosides deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG),
and
deoxythymidine (dT). It should be noted that thymidine is actually a
deoxynucIeoside, and may
be referred to in the literature as either thymidine (T) or deoxythymidine
(dT).
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The "deoxy" site in earn of the four deoxynucleosides dA~~~'tlC;
t1G=a~t~,~"~i~ti:::~i~~hpo~:Y~~iy3~:~t~,t.~~~~"
position of the furan ring. The active hydroxy sites are at the 3' and 5'
positions. In each of dA,
dC and dG there is an exocyclic NI-IZ group which is protected, preferably by
acylation, as
discussed below.
Nucleosides are mufti-functional compounds, having both amino and hydroxy
functional groups.
They may be used in automatic synthesizers to produce oligonucleotides as well
as synthetic
genes. Oligonucleotides and genes are formed by stringing together nucleosides
in a
predetermined sequence through phosphate ester linkages between the 3'-
hydroxyl group of one
nucleoside and the S'-hydroxyl group of the next.
l5
1n order to conduct syntheses selectively and efficiently, it is necessary to
block or "protect"
specific functional groups in order to achieve reaction at the desired sites.
The "protecting"
groups are designed to be removed under specific carefully controlled
conditions, usually under
relatively mild and typically acidic conditions. To be useful as precursors in
the synthesis of
high value pharmaceuticals, it is necessary that protected nucleosides be of
very high purity (i.e.,
greater than about 99%, preferably greater than about 99.5%) Unless otherwise
indicated, purity
percentages herein axe expressed as percent area as measured by HPLC, but may
be expressed as
percent by weight, where indicated.
Reported Developments
Typically, the protection of nucleosides involves the derivatization of both
amino and hydroxy
functional groups, except for thymidine, which requires only the protection of
hydroxyl groups.
Various schemes are employed to achieve these protected nucleosides, but
usually the N-
protected derivatives (most often N-acylated) are isolated and purified before
protecting the
hydroxyl groups. For example, the 2-amino group of dC, and the 6-amino group
of dA are
protected by benzoyl groups, while the 2-amino group of dG is protected by an
isobutyryl group.
The hydroxyl group, typically a 5'-hydroxyl group in all of the nucleosides,
is generally
protected by a 4,4'-dimethoxytrityl (DMT) group.
2
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S When phosphorylated!~ protected nucleoside will react a~vliE~~-3~~-
h~yd~.~~yr :group. A
phosphorylated protected nucleoside will then react with a second nucleoside
at the 5' position
after the 5' position has been deblocked by removal of the DMT group. The
phosphorylation
thus occurs between the 3'-hydroxyl group of the first nucleoside and the 5'-
hydroxyl group of
the second nucleoside to form a dinucleotide. By repeating the phosphorylation
procedure, the
synthesizer can produce oligonucleotides containing a predetermined sequence
of nucleosides.
Discussions of the synthesis and protection of nucleosides by derivatization
may be found in
many references, including the following, all of which are incorporated herein
by reference. One
method of protecting nucleosides is described in Ti, et al., "Transient
Protection: Efficient One-
flask Syntheses of Protected Deoxynucleosides", J. Am. Chem. Soc., Vol. 104,
1316-1319
(1982), which is discussed in more detail below in regard to the examples.
Other methods of
synthesizing protected nucleosides are set forth in Charubala, et al.,
"Nucleotides XXIII:
Synthesis of Protected 2'-Deoxyribonucleoside-3'-phosphotriesters Containing
the
p-Nitrophenylethyl Phosphate Blocking Group", S~rnthesis, 965 (1984). Still
other methods for
synthesizing such protected nucleosides are set forth in Kierzek, "The
Synthesis of 5'-O-
dimethoxytrityl-N-acyl-2'-deoxynucleosides, Improved 'Transient Protection'
Approach",
Nucleosides & Nucleotides, 4(5), 641-649 (1985). In all of these references,
protection by N-
acylation is effected with benzoyl chloride on adenosine and cytidine
derivatives, and with
isobutyric anhydride on guanosine derivatives, as is well known in the art.
The compounds are
then further protected by the introduction of methoxytrityl or dimethoxytrityl
groups, also as is
well known in the art. An earlier article on the protection of such
nucleosides may be found in
Schaller, et al., J. Amer. Chem. Soc., Vol. 85, 3821-3827 (1963). Another
article on protected
nucleosides is McGee, et al., "A Simple High Yield Synthesis of N2-(2-
Methylpropanoyl)-2'-
deoxyguanosine", Synthesis, 540 (1983). In all of the reported syntheses, the
protected
nucleosides must be subjected to purification prior to their use in
pharmaceutical syntheses.
A detailed description of the preparation of protected deoxynucleosides is
provided in Jones,
"Preparation of Protected Deoxyribonucleosides", Oli~onucleotide Synthesis: A
Practical
Approach, IRL Press, 23-34 (1984), incorporated herein by reference. This
reference describes
various processes for protecting such nucleosides, protecting the 5'-hydroxyl
group as a trityl
3
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ether and the 3'-hydr~l group with benzoyl chloride
ori~'~f~v~i!iW~~'t~~ctft~i~: h'b~'~
ribonucleosides, protection of the 2'-hydroxyl group is also needed, most
commonly provided by
tert-butyldimethylsilyl (TBDMS) group. The exocyclic amino groups are
protected by acylation
with a benzoyl moiety (Bz) or an isobutyryl group (iB), as discussed above.
Acyl protection is
also discussed in Foster, et al., "N-Acyl Protecting Groups for
Deoxynucleosides", Tetrahedron,
37(2), 363-369 (1981). TBDMS is discussed for protection of any hydroxyl
group, using 4-
(dimethylamino)pyridine (DMAP) as a catalyst, in Chaudhary, et al., "4-
Dimethylaminopyridine:
An Efficient and Selective Catalyst for the Silylation of Alcohols",
Tetrahedron Letters, No. 2,
99-102 (1979).
All of these reported preparation procedures use pyridine as a solvent in the
synthesis processes.
Pyridine has been found to be chemically compatible with the nucleosides, the
protected
nucleosides, and with the various reactants used to form such products.
However, pyridine is
highly toxic and its ability to dissolve nucleosides is limited. Large amounts
of pyridine have to
be used in the reaction to maintain the nucleosides in solution. These large
amounts of pyridine
are difficult to remove from the protected nucleoside products. Thus, the
pyridine solvent
processes have very low productivity, and the processing costs are high. It
would, therefore, be
desirable to have a process for preparing protected nucleosides that does not
require the use of
pyridine as a solvent.
As discussed in Chaudhary, et aL, cited above, 4-(dimethylamino)pyridine
(DMAP) is often used
as a catalyst in processes in which pyridine is the solvent. In fact, DMAP has
become a standard
catalyst for use in such processes. However, DMAP is also considered highly
toxic, is very
expensive, and is difficult to separate from the desired product. It is,
therefore, also desirable to
provide a process which does not require the use of DMAP as a catalyst.
Another reason for the use of pyridine is that it is a mild base, and can
neutralize acids formed in
the reaction mixture. In this capacity it acts as an "acid scavenger". Many of
the chemical
reactions in the protection process produce acid byproducts, such as HCI. For
example, benzoyl
chloride or isobutyryl chloride are used to acylate the amino group. In the
acylation reaction,
these acid chloride compounds react with the amino group to produce HCl as a
byproduct.
4
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S Similarly, in the trityT"~ion reaction, dimethoxytrityl chlor~ide~(I~MT=~Cl)-
"c~r~c~th~r l~lt~t'~151'd'i-t~les
react with hydroxy groups to generate NCI as a byproduct of this reaction. It
is necessary to
neutralize the acid produced by these reactions to prevent the acid from
undesirably reacting with
the nucleosides, and therefore an acid scavenger is needed in such processes.
I O The automatic synthesis of oligonucleotides as well as synthetic genes has
previously been
carried out on a milligram scale for research purposes. More recently,
oligonucleotides are being
used in larger quantities in the formation of commercial products such as anti-
sense drugs that
prevent the synthesis of disease-causing proteins in the human body. The very
sensitive nature
of the protecting groups together with the variety of polar and non-polar
impurities generated
I 5 during the syntheses of these derivatives makes their purifications
complicated, expensive, and
difficult to scale-up to industrial-scale production. Therefore, procedures
are now needed to
manufacture nucleosides in relatively large industrial quantities.
Column chromatography, especially flash silica gel chromatography, has been
used extensively
20 to purify protected nucleosides on a small to medium scale. This method
requires the use of
large volumes of high purity solvents in proportion to the amount of material
purified. The
method is also labor-intensive, requiring precise monitoring to make the
fraction cuts at the
appropriate times to maximize yield of desired product. For these reasons,
large-scale use of this
method of purification can be very costly.
The equipment required to conduct flash silica gel chromatography on a mufti-
kilogram scale is
expensive to purchase and operate. For example, one commercially available
production scale
chromatography unit is capable of separating up to about 4 kg of material per
run. Run times can
vary from 18 to 36 minutes, at an elution rate of 7 liters per minute. The
basic unit investment is
very expensive, coupled with the cost (and subsequent disposal cost) of 125 to
250 liters of
expensive high purity solvent per run. In addition, some products need to be
purified multiple
times by chromatography to reach the desired purity, each time at a
significant yield loss. The
high costs associated with chromatographic purification make it unattractive
to industrial-scale
operations.
5
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For the above reasons ~s desnable to provide a process
~'car,pr~t~.ct~z~!g=",~t~~~~;b~~ct~~~~~;t~s~.;q;~:~s
not required the use of pyridine as a solvent, which does not require the use
of DMAP as a
catalyst, and which does not require the use of separative chromatography to
purify the product.
The present invention meets these needs and offers additional advantages as
discussed in the
following detailed description.
The process of acylation for protection of exocyclic amino groups is well
known in the art and is
described, far example, in Jones, "Preparation of Protected
Deoxyribonucleosides," op. cit. As
discussed in that reference, a key problem in the preparation of protected
deoxyribonucleosides
is chemically differentiating between hydroxyl and amino groups. Only in the
case of dC has it
I S been possible to selectively acylate the amino group (N-acylation) without
acylating the
hydroxyl group. The dA and dG amino groups have been found to be too weakly
basic for such
a selective reaction. However, it is possible to selectively de-acylate, that
is, to differentiate by
making use of the more rapid hydrolysis, at pH greater than 10, of the esters
versus the amides.
Thus the chemical procedure which has been used is to per-acylate the
nucleoside, acylating both
the hydroxyl groups and the amino group, and then to selectively hydrolyze the
esters to leave
the desired N-acylated nucleoside. Such a per-acylation process, however, has
been found to be
difficult to use, because it requires isolation of the per-acylated
intermediate. An alternative
procedure is discussed in the Jones reference for selective N-acylation based
on temporary
protection of the hydroxyl groups as trimethylsilyl ethers, and is referred to
as "TMS-transient
protection." Unlike ester groups, the trimethylsilyl ethers can be hydrolyzed
in solution without
the need for isolation. For these reasons, the TMS-transient protection method
of N-acylation is
the preferred method for use with dA and dG. However, the acylation procedures
described in
the Jones reference require the use of pyridine as a solvent.
In PCT Published application WO 0075154, the applicants disclosed a process
for selectively
protecting the exocyclic amino group of a starting deoxyribonucleoside which
has an exocyclic
amino group which is to be protected by acylation and at least one hydroxyl
group which is to be
left unprotected, the process comprising:
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a) dispers~g the starting deoxyribonucleosid~"iii:va j5ol'a><~=~0-
7~'~~iSfWliic~>7 ice' '
substantially free of pyridine and which is a solvent for the protected
product formed in step b);
and
b) selectively acyIating said exocyclic amino group of said starting
deoxyribonucleoside to form a protected product in which said hydroxyl groups)
are
unprotected.
This method for the N-acylation of deoxyribonucleosides does not use pyridine
as a solvent or
DMAP as a catalyst and eliminates the need for the difficult step of
separating product from
pyridine solvent and/or DMAP catalyst, as generally required in prior
processes. This process
provides also protected nucleosides that may be purified by liquid-liquid
extraction and/or
selective adsorption, and then solidified by precipitation or crystallization
by adding a solvent in
which the product is insoluble. The PCT publication discloses that the
pyridine-free synthetic
method varies for each of the three deoxyribonucleosides that require
acylation, dA, dC and dG.
The method requires that dA and dG be acylated indirectly via TMS-transient
protection while
dC can be acylated directly by selective acylation.
Applicants have improved upon the pyridine-free synthetic method as it relates
to the dA and dG
thereby simplifying and improving the overall yield of the protection method
for the synthesis of
protected dA and dG.
Summary of the Invention
The present invention relates to a process for the preparation of an N-acyl
deoxynucleoside,
which is either an N-acyl deoxyadenosine or an N-acyl deoxyguanosine,
comprising acylating
the hydroxyl groups and the exocyclic amino group on said deoxynucleoside with
anhydride to
form a 3'-, 5'-O-acyl, N-acyl deoxynucleoside and selectively removing the
acyl groups from the
hydroxyl groups to form an N-acyl deoxyadenosine or N-acyl deoxyguanosine.
7
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S Another aspect of the~resent invention relates to a process f'or the
prepar~timn~~of~~~n-~1'~l'wac~l
derivative of a deoxynucleoside containing 3'- and 5'-hydroxyl groups and an
exocyclic amino
group, comprising the steps of:
(1) reacting said deoxynucleoside with a first anhydride under conditions
effective in
selectively acylating said hydroxyl groups;
(2) reacting the 3'-, 5'-O-acylated product of step (1) with a second
anhydride under
conditions effective in acylating said primary amino group to form an N-
acylated, 3'-, 5'-
O-acylated deoxynucleoside; and
(3) subjecting said N-acylated, O-acylated deoxynucleoside to conditions
effective to
selectively remove said O-acyl groups to form N-acyl deoxynucleoside,
wherein said deoxynucleoside is either deoxyadenosine or deoxyguanosine.
The present invention is further an improvement in a process for the
protection of the exocyclic
amino group and 5'-hydroxyl group contained in either deoxyadenosine or
deoxyguanosine,
comprising the transient protection of the 3'- and 5'-hydroxyl groups, the
acylation of said amino
group, the selective removal of said hydroxyl group protection and a non-
transient protection of
said 5'-hydroxyl group, wherein the improvement comprises the selective
transient acylation of
said deoxynucleoside hydroxyl groups, the acylation of the exocyclic amino
group, and the
selective removal of the O-acyl protecting groups.
The practice of the present invention is an unexpected improvement in the
prior art pyridine-free
nucleoside synthetic method providing for simplified processing and higher
yields than available
using the methods described in the prior art.
Brief Description of the Drawings
FIGURE 1 presents a schematic representation of the preparation of protected
N6-benzoyl-5'-O-
(4,4-dimethoxytrityl)-2'-deoxyadenosine from deoxyadenosine free-base.
FIGURE 2 presents a schematic representation of the preparation of protected
5'-0-
dimethoxytrityl N2-isobutyryl-2'-deoxyguanosine from deoxyguanosine free-base.
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Detailed Description
The process of the present invention is applicable to the preparation of
protected
deoxyribonucleosides, and is particularly pertinent to improving the methods
used in the prior
pyridine-free synthetic process of protected deoxynucleosides discussed above.
In that process,
the deoxynucleosides dA, dC and d,G, each of which have an exocyclic amino (NI-
3Z), are first
protected by acylation of that amino group. This step is necessary, because
the highly reactive
amino groups would otherwise react with the compounds being used to protect
the hydroxyl
group. As discussed above, the amino groups of dA and dC are protected
preferably by benzoyl
groups, while the amino group of dG preferably is protected by an isobutyryl
group. These
acylated compounds will be referred to herein as Bz-dA, Bz-dC and iB-dG,
respectively.
The preferred acylation procedures for protecting exocyclic amino groups vary
for each of the
three deoxyribonucleosides that require acylation, dA, dC and dG. The prior
art disclosed that
dC could be acylated directly by selective N-acylation, while dA and dG could
not be so acylated
and therefore preferably are acylated indirectly via TMS-transient protection.
Applicants have
discovered a process modification that enables the direct N-acylation of dA
and dG in a pyridine
free solvent system and that avoids the need for transient TMS protection.
The solvent chosen for the protective acylation reactions should be one in
which the N-acylated
deoxynucleoside produced is soluble., The starting deoxynucIeoside need not be
fully soluble in
the solvent provided it can be dispersed to carry on the acylation process.
The preferred polar
solvent to use depends on the particular deoxynucleoside being acylated, as
discussed further
below.
The process according to the present invention achieves the N - and O-
acylation using organic
anhydride reagents. The use of anhydrides in the present method prevents the
generation of
strong acids during the acylation reaction, and as applicants have discovered,
permits the choice
of conditions for selective O-acylation of dG and dA, as well as simultaneous
O- and N-
acylation, if desired.
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S
Preferred anhydride reagents comprise the alkyl anhydrides and the aryl
anhydrides. More
preferred anhydrides include the lower alkyl anhydrides and the phenyl
anhydrides. Lower alkyl
groups as described herein mean straight chain or branched alkyl groups
containing one to about
6 carbon atoms. Exemplary lower alkyl groups include methyl, ethyl, n-propyl,
isopropyl, n-
butyl, isobutyl, n-pentyl, and n-hexyl. Preferred anhydrides include acetic
anhydride, isobutyryl
anhydride and benzoic anhydride.
The present process selectively removes the O-acyl protecting groups under
conditions that leave
the N-acyl group intact. Selective removal may be achieved by the use of a
nucleophilic reagent
in a non-aqueous polar erotic solvent. A preferred nucleophilic reagent is a
salt of a lower alkyl
alkoxide, such as methoxide, ethoxide or n-propoxide. Metal salt alkoxides are
preferred. The
erotic polar solvent typically corresponds to the choice of metal alkoxide,
and is preferably
methanol, ethanol or propanol, respectively. The de-protection reaction is
typically conducted at
low temperature to provide for selective hydroxyl de-protection. A preferred
temperature range
is from about -35°C to about 0°C, more preferably from about -25
°C to about -10 °C, and most
preferably at about -15 °C.
If the N-protecting group is a lower alkyl-protecting group, then the N-
acylation step may be
achieved in a non-selective first acylation, followed by a selective de-
protection of the acylated
hydroxyl groups. In this case, only one anhydride need be used in the practice
of the present
invention. This is the preferred method of preparing 5'-O-dimethoxytrityl Na-
isobutyryl-2'-
deoxyguanosine described in more detail below.
The direct acylation of dG will be discussed first. In this acylation
procedure, a starting material
of dG monohydrate preferably is used as the starting deoxynucleoside. The dG
is dispersed in a
polar aprotic solvent that is free of pyridine. The solvent is preferably as
anhydrous as possible,
because any water present must be removed or otherwise tied up prior to
acylation. Suitable
aprotic solvents include tetrahydrofuran (THF), acetonitrile, amides, such as
dimethylformamide
(DMF), dimethylacetamide and N-methylpyrrolidone, as well as dimethylsulfoxide
(DMSO),
dimethylsulfone and hexamethylphosphate (HMPA). Preferred polar aprotic
solvents for use in
CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
the direct acylation of'"dG are aprotic solvents such as
dirri'etl'i'elf'bi~nav'i~it'd~'''(I:~'IVIr')':""Ai~"~'cylat~ng
reagent, preferably an anhydride is added to the dG solution. Isobutyric
anhydride is a suitable
and preferred reagent. A preferred molar ratio of anhydride to dG is about 4
to 5. The reaction
temperature should be maintained at about 70 to about 90 degrees C, and most
preferably
between about 75 to about 80 degrees C. The reaction is slower at lower
temperatures, which
may result in an incomplete reaction that may contaminate the product with
unreacted starting
materials. The reaction mixture may be monitored to ensure complete reaction
of the starting
material and conversion of all partially acylated intermediates to the anal
tri-acylated product. A
number of monitoring means may be used including NMR, UV, IR and various forms
of
chromatography. The preferred monitoring means is high-pressure liquid
chromatography
(NPLC).
The anhydride acylating agent added to the starting deoxyribonucleoside
solution produces
organic acid byproducts upon reacting with the nucleosides. In such cases, the
acid byproduct
must be neutralized, or it may cause decomposition of the nucleoside.
Therefore, an acid
scavenger preferably is included in the reaction mixture. A preferred acid
scavenger is a tertiary
amine, which forms a salt with the acid, with a particularly preferred
tertiary amine being
triethylamine (TEA). Other suitable tertiary amines include any tri-alkyl
amine of the general
formula N(R)3, wherein R represents the same or different CI to C6 alkyl
groups. In addition to
TEA, such tri-alkyl amines include trimethylamine, tripropylamine and di-
isopropyl-monoethyl
amine (DIPEA). Other suitable tertiary amines include triethanolamine.
The acylation of dA is achieved by selective O-acylation using a lower alkyl
anhydride, followed
by N-benzoylation, and then selective deprotection of the hydroxyl sites to
leave just the amino
site benzoylated, thus forcing benzoylated dA (Bz-dA). The solvent in which
the starting dA is
dispersed is preferably an aprotic solvent, preferably as anhydrous as
possible. In a preferred
embodiment of this process, dA monohydrate (dA~H20) is dissolved in toluene
with about x
equivalents of triethylamine (TEA). Of the x eq of TEA, 7 eq is to neutralize
acetic acid
produced from the chemical reactions and 3 eq to buffer the reaction mixture
to ensure a slightly
basic environment. About 2-4 equivalents of acetic anhydride is added slowly
while maintaining
the reaction at a relatively low temperature of about I 5 to 30 degrees C,
preferably about 20 to
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25 degrees C. The ac~f~c anhydride selectively reacts at ~~t'ie'~a~ct~iv~e
~?~'W°~i;-d vyclr~~j~~rSt~e'~:~' =1'~i~e
reaction should be monitored, as by TLC and/or HPLC, until all of the dA is
consumed. The
reaction is quenched with an aqueous mild base such as sodium bicarbonate to
consume all
unreacted anhydride.
I O The next step uses the organic reaction mixture containing the O-acyIated
compound without
isolation to acylate the unreacted exocyclic amino group. The typical
protecting group for dA is
benzoyl, and is the preferred N-acyl protecting group in the present method,
although other
anhydride may also be used in the N-acyl reaction. The N-acylation conditions
are harsher than
the earlier O-acylation reaction in that the reaction temperature should be
maintained at about 75
to about 95 degrees C, preferably between about 80 to about 90 degrees C, and
most preferably
between about 83 to about 88 degrees C. The reaction is slower at lower
temperatures, which
may result in an incomplete reaction that may contaminate the product with
unreacted starting
materials. To ensure completion of the reaction, the reaction mixture may be
monitored to
ensure complete reaction of the starting material and conversion of all
partially acylated
intermediates to the final N-benzoylated product. A number of monitoring means
may be used
including NMR, UV, IR and various forms of chromatography. The preferred
monitoring means
is high-pressure liquid chromatography (HPLC).
As in the first anhydride reaction, the anhydride produces organic acid
byproducts, such as
benzoic acid, upon reacting with the dA. In such cases, the acid byproduct
must be neutralized,
or it may cause decomposition of the nucleoside. Therefore, an acid scavenger
preferably is
included in the reaction mixture. A preferred acid scavenger is a tertiary
amine, which forms a
salt with the acid, with a particularly preferred tertiary amine being
triethylamine (TEA). Other
suitable tertiary amines include any tri-alkyl amine of the general formula
N(R)3, wherein R
represents the same or different C~ to C6 alkyl groups. In addition to TEA,
such tri-alkyl amines
include trimethylamine, tripropylamine and di-isopropyl-monoethyl amine
(DIPEA). Other
suitable tertiary amines include triethanolamine.
The acetylated hydroxyl groups are then deprotected and restored to their
original hydroxyl form
by hydrolysis. The hydrolysis reaction selectively deprotects the hydroxyl
sites, while leaving
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CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
the amino sites benzoyatea. H preferred hydrolysis metlio~'~-
i"s'a'chie~'ed"h'y'first ti=ea'fing tie°""
protected dA with an alcoholic alkoxide, such as methanolic sodium methoxide
to remove the
acetyl groups and form the sodium salts of the N-benzoyl dA. Subsequent
treatment with an
aqueous buffered salt solution, preferably an aqueous solution of a tertiary
amine acid addition
salt, such as triethyl amine hydrochloride, converts the sodium hydroxyl salt
of dG to the original
hydroxyl groups. The deprotecied N-benzoyl dA is separated in the organic
phase of the reaction
mixture, which phase is separated and dried for subsequent use to form the 5'-
O-protected N-
benzoyl dA.
The pyridine-free deoxyribonucleosides which have had their exocyclic amino
groups protected
by acylation in accordance with the present invention may now be used to form
5'-protected
deoxyribonucleoside products as described in the prior pyridine-free process.
In that process, the
5'-hydroxyl group of the deoxyribonucleoside is protected, and the protected
product is purified
and separated out of solution as a solid. Purification and solidification
comprise liquid-liquid
extraction steps that take advantage of the fact that the protected nucleoside
products are
insoluble in water, while soluble in selected polar and non-polar solvents.
There is therefore provided an improved process for preparing an essentially
pure 5'-protected
deoxyribonucleoside comprising:
a) dissolving a starting deoxyribonucleoside prepared in accordance with the
present
invention, in a polar, aprotic solvent which is inert to the starting
deoxyribonucleoside, to the 5'-
protected deoxyribonucleoside, and to the other reactants, wherein any
exocyclic amino groups
of the starting deoxyribonucleoside are protected, preferably by acyI
protection;
b) reacting the starting deoxyribonucleoside in said solution with a
protecting
reagent to form a 5'-protected deoxyribonucleoside product;
c) removing polar impurities by one or more liquid-liquid extractions using
immiscible polar and non-polar solvent systems in which the product
preferentially partitions
into the non-polar phase, and the impurities preferentially partition into the
polar phase; and
d) removing non-polar impurities by solidifying the product out of solution
while
leaving the non-polar impurities in solution.
13
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In step (a), the starting deoxyribonucleoside may be one
t'ftatA~l~'e~dy'°°h'~~'1't~"e'~bcy~t5'c -aii~i't~t~"'
group protected. Protection of the exocyclic amino group may be made by the
above-described
acylation protection process of the present invention. The nucleoside to be
protected is dissolved
in a polar, aprotic organic solvent. Examples of polar, aprotic solvents
suitable for use in this
step include amides, such as dimethylformamide (DMF), dimethylacetamide and N-
methyl-
I O pyrrolidone, as well as dimethylsulfoxide (DMSO), dimethylsulfone and
hexamethylphosphate
(HMPA). DMF is particularly preferred. For the reasons discussed above,
pyridine is not used
as a solvent in the processes of the present invention.
In step (b), a protecting reagent is added to the starting deoxyribonucleoside
solution that
I S selectively protects the 5'-hydroxyl group of the deoxyribonucleoside. The
protecting reagent
must react with the 5' hydroxyl group preferentially over the 3' hydroxyl
group to form a
removable protecting group at the 5' position. A preferred method of
protecting this hydroxyl
group is to form a trityl derivative compound, a process referred to as
"tritylation". Preferred
protecting groups are trityl, methoxytrityl and dimethoxytrityl groups.
Preferred protecting
20 reagents are trityl chloride and substituted trityl chlorides. For example,
dimethoxytrityl chloride
(DMT-Cl) is used in the examples set forth below. During the tritylation
reaction, the
3'-hydroxyl group should be left unreacted. This is necessary to permit
selective bonding of the
5' and 3' positions during subsequent oligonucleotide synthesis. The
tritylation reaction is
preferably conducted at a temperature ranging from about -10°C to about
40°C, more preferably
25 from about 10°C to about 25°C.
Protecting reagents such as trityl chloride or substituted trityl chlorides
produce an acid
byproduct upon reacting with nucleosides. In such cases, the acid byproduct
must be
neutralized, or it may cause decomposition of the nucleoside. Therefore, an
acid scavenger is
30 preferably included in the reaction mixture. Preferably the acid scavenger
is present in a mole
ratio to the tritylation agent of about 1:1 to about 3:1. A preferred acid
scavenger is a tertiary
amine, which forms a salt with the acid. A particularly preferred tertiary
amine is triethylamine
(TEA). Other suitable tertiary amines include any tri-alkyl amine of the
general formula N(R)3,
wherein R represents the same or different C, to C6 alkyl groups. In addition
to TEA, such tri-
35 alkyl amines include trimethylamine, tripropylamine and di-isopropyl-
monoethyl amine
I4
CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
(D1PEA). Other suitable terfiary amines include triethanolainirie:
ATfhoug7i~pyridipe is~not used
as a solvent in the process of the present invention, a small amount of
pyridine can be used as an
acid scavenger. When used in small amounts, the pyridine can be purified out
of the product
with the other impurities.
The polar solvents and acid scavengers must be chemically compatible with the
starting
nucleosides, the reactants, and the nucleoside products to which they are
exposed. They must
also not interfere adversely with the reaction of the reactants with the
nucleosides.
Because the DMT-Cl used in tritylation is highly reactive towards water, all
materials involved
in the tritylation must be anhydrous. In the preferred acylation processes of
the present
invention, Bz-dA may pick up water from the TMS-transient protection acylation
reactions, and
therefore need to be dehydrated. It was found that ordinary thermal and vacuum
drying
techniques were unsatisfactory for removing the water contained in this
material, because the
water may exist in hydrate form. In accordance with another aspect of the
present invention, an
azeotropic dehydration process has been developed which is effective to remove
water in the
Bz-dA.
The azeotropic dehydration process of the present invention is applied to the
solution of the
starting deoxyribonucleoside dissolved in the polar aprotic solvent prior to
the addition of the
protecting reagent. The dehydration process comprises adding a dehydrating
solvent to the
solution of step (a) that forms an azeotrope, with water and the polar aprotic
solvent, and
distilling the azeotrope from the mixture. For a starting solution of
deoxyribonucleoside in the
polar aprotic solvent dimethylformamide (DMF) suitable dehydrating solvents
include one or
more Cs to C~o hydrocarbons, which may be linear, branched or cyclic, and may
be substituted or
unsubstituted. Preferred dehydrating solvents include pentane, hexane, toluene
and heptane,
particularly hexane. It was also found desirable to include a small amount of
a tertiary amine,
such as TEA, to stabilize the acylated nucleosides during the azeotropic
dehydration step.
The protecting reagent should be added to the solution of the starting
deoxyribonucleoside under
controlled conditions of temperature arid addition rate. The progress of the
reaction should be
CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
monitored, as by HPLC: analysis. The objective of the momrormg ls-ro control
ano opumtze the
conversion without generating too much of over-tritylated impurities. The
reaction is quenched
by the addition of water when the optimal point is reached.
An additional advantage of tritylating the starting deoxyribonucleoside in the
polar aprotic
solvent rather than in pyridine is that the reaction does not require the use
of a catalyst. As
discussed above, tritylation in pyridine generally requires the use of a
catalyst, such as 4-
(dimethylamino)pyridine (DMAP) which is a toxic material that is very
expensive and difficult
to separate from the final product.
I 5 Step (c) is a purification step that removes polar impurities from the
product. The solution of the
protected nucleoside inevitably contains polar and non-polar impurities that
must be removed
from the final nucleoside product. As discussed above, the final product
should be at least about
98% pure, preferably at least about 99% pure, and more preferably at least
about 99.5% pure, by
weight. Among the polar impurities, which need to be removed, are the amino-
protected
nucleosides which either did not react with the hydroxyl protecting agent, or
which may have
two acyl protecting groups (identified as bis-acylated products). Among the
non-polar impurities,
which need to be removed, are the bis-tritylated materials, which contain two
trityl protecting
groups, and impurities derived from DMT-Cl. These tend to be the most
difficult impurities to
remove because their polarity is relatively close to that of the desired
protected nucleosides.
Polar impurities are removed from the solution of protected nucleoside product
by a liquid-liquid
extraction process, which takes advantage of the difference in solubility
between protected
nucleosides and polar impurities in polar solvent system. The protected
nucleosides are
generally insoluble in water and basic aqueous salt solutions, while many of
the polar impurities
are soluble in water or such solutions. Therefore water, or preferably an
aqueous solution of
water and a basic salt such as a soluble carbonate or bicarbonate salt, can be
used to extract polar
impurities from the protected nucleoside product. By adding water or a basic
aqueous solution to
the initial product solution in polar, aprotic solvent, the protected
nucleoside is maintained to the
non-polar phase, while the majority of polar impurities are left in the polar
phase. Preferred
basic salts include sodium and potassium carbonate and bicarbonate,
particularly sodium
16
CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
bicarbonate. Preferably, the basic aqueous salt solution ctipt~a~rn~
~bii'~1t"'I"°~o-'t'o~'about"I"~~~'o,
preferably about 2% to about 5%, by weight salt. Other halogenated solvents,
such as
chloroform and 1,2-dichloroethane may also be employed as the non-polar
solvent. The product
solution in the non-polar solvent can be repeatedly extracted in this manner
with water or a basic
aqueous solution to achieve higher purity level. It has been found that an
aqueous solution
comprising about 1 to about 10% by weight Nal-iC03, preferably about 2% to
about 5%, and
about 0 to about 40% DMF, is suitable for the removal of polar impurities.
After the polar
solvent system has been mixed with the non-polar phase, the mixture is allowed
to settle, and is
separated. The desired protected product remains in the non-polar phase, while
the undesired
polar impurities are carried away in the polar phase. The process may be
repeated as many times
as necessary to remove the polar impurities and obtain the desired product
purity.
This polar solvent system selectively extracts out all or most of the polar
impurities. To remove
additional polar impurities, the resulting product solution optionally may be
treated further with a
suitable adsorbent, such as activated carbon. Other suitable adsorbents, such
as silica, aIumina,
and molecular sieves, are well known to those skilled in the art. When polar
impurities are below
desirable levels, the adsorbent with adsorbed impurities may be readily
removed, as by filtration.
This has been found to be a desirable step in the purification of the
protected dA products, but
unnecessary in the purification of dG.
In step (d), the product is solidified out of solution by a process, which
further purifies the
product. The product may be solidified out of solution by crystallization, in
the case of a
crystalline product, by precipitation in the case of an amorphous product, or
by a combination of
crystallization and precipitation in the case of products, which form both
crystalline and
amorphous solids. Thus the term solidification is intended to encompass the
processes of
crystallization, precipitation and combinations thereof by which a solid
product comes out of
solution.
Solidification may be effected by the use of either non-polar solvents or
polar solvents. In non-
polar solvent solidification, a non-polar phase containing the dissolved
product is combined with
a miscible solvent in which the product is insoluble in an amount effective to
crystallize a
17
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WO 02/068437 PCT/US02/05601
crystalline product, or to precipitate an amo~hous product froiri the-riori-
poiax~phase: f-or
crystallization, preferably, the miscible solvent in which the product is
insoluble is added slowly
to the product solution. For precipitation, preferably, the product solution
is added slowly to the
miscible solvent in which the product is insoluble.
When the solidification is taking place in a polar solvent, the product is
maintained, transferred
or re-dissolved into a polar phase, which comprises a polar solvent that is
miscible with water.
Because the present protected nucleosides are all insoluble in water, water
can be used to solidify
the product from the polar phase. To solidify the product from the polar
phase, the polar phase
containing the dissolved product may be combined with a suitable amount of
water effective to
solidify the product out of solution. The water may be added to the polar
phase, or the polar
phase may be added to the water.
Many polar solvents are suitable for use in this solidification process. In
addition to being
miscible with water and a solvent for the product, the polar solvent must also
be inert to the
product. Preferably, the polar solvent also has a boiling point of less than
100 degrees C, the
boiling point of water, to facilitate subsequent removal by evaporation.
Suitable solvents include
acetonitrile, acetone, and lower (Ci to C3) alcohols, particularly methanol.
. When the product is solidified in non-polar solvents, crystallization
effectively removes most of
the non-polar impurities. The non-polar impurities remain dissolved in the non-
polar solvents as
the product crystallizes out of solution. Such a crystallization process is
preferably used with
Bz-DMT-dA. However, when the product is solidified by precipitation to an
amorphous form, it
was found that additional purification might be needed to remove non-polar
impurities. In such
a case, an additional step of liquid-liquid extraction is used to remove non-
polar impurities.
Such a process is preferably used with protected nucleosides which form
amorphous solids, as
was found to be the case with iB-DMT-dG. In liquid-liquid extraction to remove
non-polar
impurities, the crude product is isolated in a polar phase in which it is
soluble. A mixture of
DMF and water is preferred for use in the liquid-liquid extraction
purification of these products.
The impurities are then extracted with a non-polar solvent, or a non-polar
solvent system. The
non-polar solvent should be a solvent for the non-polar impurities, but not
for the nucleoside
18
CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
product. In addition, the polar phase and non-polar solvent system must be
~mm~sc~ble mth each
other. This will cause the non-polar impurities to partition into the non-
polar solvent phase,
while keeping the majority of the product in the polar solvent phase. The non-
polar impurities
can then be removed by phase separation. Mixtures of aromatic hydrocarbons and
aliphatic
hydrocarbons are suitable non-polar solvent systems, particularly ones
containing about 6 to 12
I 0 carbon atoms, wilh or without heteroaloms. A mixture of cumene and hexane
is suitable
although a mixture of methyl t-butyl ether (MTBE) and toluene is a preferred
composition.
Preferably, the mixture comprises about 10 to about 90 parts aromatic to about
90 to about 10
parts aliphatic, by volume, more preferably about 1 to about 3 parts aromatic
to 1 part aliphatic,
with a mixture of about 1 parts aromatic to 1 part aliphatic being
particularly preferred.
To solidify the product out of solution by precipitation or crystallization,
the polar aprotic phase
in which the product has been isolated, and from which the polar impurities
have been removed
is mixed with an immiscible non-polar solvent in which the product is
insoluble. A wide range
of solvents can be used for this purpose, so long as the non-polar phase and
the non-polar solvent
are miscible with each other. For crystalline products, the non-polar solvent
is preferably added
slowly to the non-polar phase comprising the product. This causes the product
to crystallize,
while keeping non-polar impurities in the mother liquor. For amorphous
products, the non-polar
phase comprising the product is preferably added slowly to the non-polar.
solvent. This causes
the product to precipitate, while keeping the non-polar impurities in
solution.
The solidification process is considered precipitation in the case of a
product, which is
amorphous in solid form, and crystallization in the case of a product, which
is crystalline in solid
form. By the use of this process, and repeating the process as needed,
essentially pure protected
nucleosides are obtained without the use of chromatography and without the use
of pyridine as a
solvent or DMAP as a catalyst. In addition, the process can be scaled up
readily to any desirable
scale. For purposes of this application, an essentially pure nucleoside is one
of greater than
about 98% purity, and preferably greater than about 99% purity, and most
preferably greater than
about 99.5% pure, all by weight.
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WO 02/068437 PCT/US02/05601
The process of the present W vention offers many advantages°~over prior
processes m wmcn
pyridine is used as the process solvent. Because the polar aprotic solvent
used in the present
process are better solvents for the nucleosides than pyridine, the reactions
can be run at much
higher nucleoside concentrations. Thus, higher volumetric efficiency is
obtained. As discussed
above, the present process eliminates, or at least greatly reduces the use of
pyridine and DMAP,
which are considered highly toxic substances. The purification process steps
of the present
invention, including extraction, adsorption, and precipitationlcrystallization
are common
operations, and are more efficient and less costly operations than
chromatography. Because of
the simplicity and other improvements of the present process, the overall
product yield is also
improved. Further, the combination of these improvements results in a
significant lowering of
the processing costs for protected nucleosides. Finally, the new processes are
scalable to any
desirable scale to meet industrial demand.
Figure I presents a schematic representation of a preferred method of
preparing protected N6-
Benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-deoxyadenosine from deoxyadenosine free-
base, with the
direct .acylation protection. The deoxyadenosine free-base is combined with
acetic anhydride to
form transiently O-protected dA, followed by N-benzoyIation. This material is
then hydrolyzed
to form the N-benzoyl compound which is tritylated by the addition of DMT-CI
to form the final
protected product N6-Benzoyl-5'-O-(4,4-dimethoxytrityl)-2'-deoxyadenosine as
shown in Figure
1.
Figure 2 presents a schematic representation of a preferred method for the
preparation of
protected 5'-O-dimethoxytrityl Nz-isobutyryl-2'-deoxyguanosine from
deoxyguanosine. In this
case dG is tri-acylated with isobutyric anhydride followed by selective O-
deprotection. This N-
butyryl dG material is then tritylated by the addition of DMT-CI to farm the
final protected
product S'-O-dimethoxytrityl Na-isobutyryl-2'-deoxyguanosine as shown in
Figure 2.
In the following examples, the solid protected form of dA is crystalline in
structure, and
therefore it is solidified out of solution by crystallization. On the other
hand, dG is amorphous,
and is therefore solidified by precipitation. Generally, the crystallization
process successfully
reduced the level of non-polar impurities to the desired level. Other
particular steps in the,
CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
S processing of the different nucleosides will be apparent fxbrri the tottowmg
detarted descriptions
of examples.
EXAMPLES
In the following examples protected forms of the nucleosides deoxyadenosine
(dA), and
l0 deoxyguanosine (dG) are made in accordance with the method of the present
invention.
Example 1 - Preparation of 5'-O-dimethoxytrityl NZ-isobutyryl-2'-
deaxyQuanosine
Step (la)' Nz- 3'-O 5'-O-tri-isobutyryl deoxy~uanosine
15 A mixture of 200 g (0.70 mol) of deoxyguanosine (dG} monohydrate, 480 g of
isobutyric
anhydride (3.03 mol, 4.33 eq based on dG), 353 g of TEA (3.5 mol, S.0 eq based
on dG), and 1.0
L of DMF is heated to about 75-80°C and stirred under nitrogen. The
reaction is monitored
using HPLC and samples taken every 30 minutes. After about 4 hr additional
amounts of
isobutyric anhydride is added to the reaction mixti.~re to lower the dG and iB-
dG level to below
20 3% (area%). When the combined dG and iB-dG level drops to less than 3%, the
reaction is
quenched with 1.5 I of S% NaHC03 solution. The quenched mixture is stirred for
about 30
minutes, cooled to 25°C and extracted with methylene chloride (3 x O.S
L). The methylene
chloride extracts are combined and washed with 5% NaHC03 solution (3 x 1 1),
and the organic
phase is separated and dehydrated by azeotropic distillation. The dehydrated
organic phase is
2S used as is in the next step.
Step (1b): N-isobut~l deoxy,~uanosine
A chilled 25 % methanolic solution of sodium methoxide (76 g, 0.35 mol, 0.5 eq
based
on dG) is added slowly to a cooled mixture (about -18 to about -20°C)
of the dehydrated organic
30 phase from step ( I a) above diluted in 2.01 of methanol. The temperature
of the mixture is
maintained at or below -15 °C during the addition, and during the
course of the de-protection
reaction. Samples are taken about every 30 min. Additional NaOCH3 is used to
keep the
reaction going and to lower the combined di- and tr-iacylated dG level to
below 3%. When the
reaction is completed, 100 g of triethylammonium chloride (0.7 mol, 1 eq based
on dG} is added
3S to the mixture, which is stirred for 30 min at -15 °C and warmed up
to 20°C and stirred for an
2I
CA 02439407 2003-08-26
WO 02/068437 PCT/US02/05601
additional hour (at 20 °C). 2.0 I of DMF is added to the mixture, which
is distilled at ambient
pressure at about 80°C to remove methanol, methylene chloride, methyl
isobutyrate,
triethylamine and other low-boiling components. When no more distillate is
collected, 2.0 1 of
hexane is added to the mixture, which is distilled again to azeotropically
remove any residual
methanol.
Step (2): 5'-O-dimethox trityl NZ-isobutyryl-2'-deox~~uanosine
1.5 I of a methylene chloride solution of 98% DMT-Cl (285 g ;0.84 mol, 1.20 eq
based
on initial dG) is added over a 30 min period to the mixture of triethylamine
(141 g; I .4 mol, 2.0
eq based on dG) and the organic residue from step ( I b). The temperature of
the mixture is
controlled to about 25 °C during the addition after which the mixture
is stirred for 30 min at 25
°C. The mixture is analyzed by HPLC, and additional amounts of DMT-CI
(as solid) are added
to lower the residual iB-dG level to below 3%. The mixture is washed with 3%
NaHC03
solution (I x 5.01), a mixture of DMF and 3% NaHCO3 solution (1:2, 2 x 5.01),
and 3%
NaHC03 solution (2 x 5.0 1). The organic phase is analyzed by HPLC after each
extraction and
combined with a 50-50 mixture of MTBE and toluene (7 I) while vigorously
stirring for about 30
min, further stirred for 30 min and filtered. The filter cake is dried under
vacuum suction for l 5
min and re-dissolved in 21 of methylene chloride, re-precipitated with 7 L of
50-50
MTBE/toluene, and the filtering process repeated until the purity of the
material is greater than
99.3%. The wet cake is dried in a vacuum oven until it passed the Loss on
drying (LOD). The
typical yield is 75%.
Example 2:
The Preparation of N6-Benzoyl-5'-O-(4,4-dimethox~rit~l-2'-deoxyadenosine
Sten (1a1: 3'-O. 5'-O-diacetvl-2'-deoxyadenosine
A mixture of 2'-deoxyadenosine monohydrate (dA)(454 g), toluene (51),
anhydrous
triethylamine (TEA)(860g), and of acetic anhydride (686 g) is stirred under
nitrogen at 20 °C and
sampled every 30 min until the combined residual dA and mono-acetyIated dA
(two isomers) is
less than 2°J°. The mixture is cooled to 1 S°C. 3 I of S%
NaHC03 solution is added while
maintaining the temperature below 25 °C. The mixture is stirred at 25
°C for 30 min to
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WO 02/068437 PCT/US02/05601
S hydrolyze the excess acetic anhydride. The mixture is allowed to settle, and
the lower phase rs
removed. 3 1 of S% NaHC03 solution is added to the organic phases, the phases
are mixed, and
combined with 0.2 1 of TEA and 2 1 of hexane. The mixture is heated to 70
°C to strip off
hexane and residual water. The process is continued until the water content is
lower than 0.2%
by Karl-Fisher titration. The O-acetylated product is used as is in the next
step.
Step (1b): N6-Benzoyl-3'-O S'-O-diacetyl -2'-deoxyadenosine
The dried organic phase obtained in step (la) is combined with TEA (330 g) and
benzoic
anhydride (Bn20) (S70 g), stirred under nitrogen at 8S °C, sampled on
an hourly basis until
benzoylation is completed (<2% residual 2Ac-dA), and cooled to 4S °C. A
S% NaHC03
1 S solution (41) is added to the reaction mixture while keeping the
temperature below 4S °C. Upon
the completion of the addition, the mixture is stirred at 4S °C for 1
hr to hydrolyze the residual
Bn20, and allowed to phase separate. The organic phase is separated and washed
with 1%
NaHC03 solution (2 x S.0 L) to remove all water-soluble components.
Step (lc): N6-Benzoyl-2'-deoxyadenosine.
A methanolic NaOCH3 solution (2S%; 362 g) is added to a cooled mixture of the
washed
organic phase obtained in step (1b) and methanol (2 1) while maintaining the
temperature below
-13 °C. Upon completion of the addition, the mixture is stirred at -1 S
°C, and sampled every 30
minutes until the reaction is complete and is supplemented with additional
NaOCH3 solution as
2S necessary. The reaction is quenched with 4 I of 10% TEA-HCL solution, is
stirred at 0 °C for 20
min, and the temperature raised to 70 °C. When all solids dissolve, the
mixture is allowed to
settle, and the aqueous phase removed. The organic phase is washed with 3 1 of
1 % NaHC03
solution at 70 °C, the aqueous phase removed and the organic phase
distilled to remove 3 I of
toluene phase under reduced pressure at 70 °C. The residue is diluted
with TEA (4S0 g), DMF
(3 1), and cooled to 20 °C, and used as is in the next step.
Step (2)- N6-Benzoyl-S'-O-(4 4-dimethox~yl)-2'-deoxyadenosine
A methylene chloride solution of DMT-CI (683 g in 31 of CHaCLz) is added to
the
diluted organic phase obtained in step (1 c) while maintaining the mixture
temperature below
3S 2S°C. Upon the completion of the addition, the mixture is stirred at
2S°C and sampled every 30
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WO 02/068437 PCT/US02/05601
min until the residual Bz-dA is less than 5% (area). Suitable amounts of
additional DMT-Cl are
added to the mixture as necessary. When the desired conversion is achieved,
the mixture is
quenched with 4 I of S% NaHC03 solution. The organic phase is washed twice
with a mixture of
DMF and 5% NaHC03 solution (1:3, 4 1 each time), and then again with a 5%
NaHC03 solution
(4 1). The washed organic is combined gradually with 3 1 of MTBE-hexane
mixture (1:l) with
l 0 vigorous agitation. Upon the completion of the addition, the mixture is
cooled to 5°C and stirred
at mild agitation rate to allow nucleation to take place. When the mixture
turns into a slurry, 2 1
of hexane are added, and the resulting mixture stirred at 5°C for 2 hr.
The solid is filtered,
washed with 41 of MTBE-hexane mixture (1:1), and vacuum dried until the
residual solvent
level is lower than 15% (by GC). The solid is re-dissolved in 2 I of CHZCIz,
and the product re-
I 5 precipitated by slowly adding 71 of MTBE-hexane mixture. The mixture is
cooled to 5°C, and
the solid was filtered. The filtering step is repeated until the purity is
greater than 99.0%. The
material is dried in a vacuum oven at SO°C until it passes LOD. The
overall yield is 65%.
24
