Extremely high purity oligonucleotides and methods of synthesizing them using dimer blocks

The present invention comprises an improved method of synthesizing oligonucleotides. The method comprises employing dinucleotides (or "dimer blocks") as the basic synthetic unit building block. The method results in extremely high purity oligonucleotides in which the N-1 content is very low, generally less than 1-2% of the full length, N, oligonucleotide. We have found that synthesis using dinucleotide phosphorothioates results in oligonucleotides having very little phosphodiester content. Furthermore, we have found that the amount of dimer required in each coupling step can be less than about 6 and is preferably about 2 equivalents. Synthesis of oligonucleotides according to the dimer block approach described herein can also be conducted without the capping step that has heretofore been deemed necessary after each coupling.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to the field of chemical synthesis of 
oligonucleotides. More particularly, the invention relates to the 
synthesis of extremely high purity oligonucleotides. 
2. Summary of the Related Art 
Since the discovery by Zamecnik and Stephenson (Proc. Natl. Acad. Sci. 75, 
280 (1978)) that synthetic oligonucleotides can inhibit Rous sarcoma virus 
replication, there has been great interest in the use of oligonucleotides 
and oligonucleotide analogs having modified internucleotide linkages to 
control gene regulation and to treat pathological conditions. There have 
been many reports of successful use of antisense oligonucleotides to 
inhibit gene expression both in vitro and in vivo, either directly by 
binding to double stranded DNA, or, primarily, indirectly by inhibiting 
translation of mRNA. 
Many reports of successful antisense inhibition of nucleic acid expression 
in vitro have been reported. For example, Rapaport and Zamecnik (U.S. Pat. 
No. 5,616,564) disclosed successful antisense inhibition of malaria in 
parisitized erythrocytes. See also Barker et al. (Proc. Natl. Acad. Sci. 
USA 93, 514 (1996)). Oligodeoxyribonucleotide phosphorothioates have been 
found to inhibit immunodeficiency virus (Agrawal et al., Proc. Natl. Acad. 
Sci. USA 85, 7079 (1988); Agrawal et al., Proc. Natl. Acad. Sci. USA 86, 
7790 (1989); Agrawal et al., in Advanced Drug Delivery Reviews 6, 251 (R. 
Juliano, Ed., Elsevier, Amsterdam, 1991); Agrawal et al. in Prospects for 
Antisense Nucleic Acid Therapy of Cancer and AIDS, 143 (E. Wickstrom, Ed., 
Wiley/Liss, New York, 1991); and Zamecnik and Agrawal in Annual Review of 
AIDS Research, 301 (Koff et al., Eds., Dekker, New York, 1991)), and 
influenza virus (Leiter et al., Proc. Natl. Acad. Sci. USA 87, 3420-3434 
(1990)) in tissue culture. In addition, oligodeoxyribonucleotide 
phosphorothioates have been the focus of a wide variety of basic research 
(e.g., Agrawal et al., Proc. Natl. Acad. Sci. USA 87, 1401 (1990) and 
Eckstein and Gish, Trends Biochem. Sci. 14, 97 (1989)), enzyme inhibition 
studies (Mujumdar et al., Biochemistry 28, 1340 (1989)), regulation of 
oncogene expression (Reed et al., Cancer Res. 50, 6565 (1990)) and IL-1 
expression (Manson et al., Lymphokine Res. 9, 35 (1990)) in tissue 
culture. A number of review articles report the many published studies of 
successful antisense inhibition in vitro. E.g., Uhlmann and Peyman, Chem. 
Rev. 90, 543 (1990). 
A number of published reports disclose the successful antisense inhibition 
of nucleic acid expression in vivo. For example, Offensperger et al. (EMBO 
J. 12, 1257 (1993)) demonstrated in vivo inhibition of duck hepatitis B 
virus. Nesterova and Cho-Chung (Nat. Med. 1, 528 (1995)) demonstrated 
inhibition of tumor growth by a single subcutaneous injection of antisense 
phosphorothioate oligonucleotide targeted to the RI, subunit of protein 
kinase A in nude mice. Several general reviews of in vivo antisense 
inhibition have appeared that discuss these and other studies 
demonstrating successful in vivo antisense inhibition of nucleic acid 
expression as well as applications for therapeutic use. See, e.g., 
Agrawal, TIBTECH 14, 376 (1996); Field and Goodchild, J. Exp. Opin. 
Invest. Drugs 4, 799 (1995). 
These and other studies have proven sufficiently successful to justify 
extension to humans. A number of human clinical trials are currently 
ongoing, testing antisense oligonucleotides against a variety of disease 
causing targets, including HIV, CMV retinitis, ICAM, PKC, c-myb, and 
c-raf. 
A necessary precursor to using antisense oligonucleotides to inhibit 
nucleic acid expression is the synthesis of the oligonucleotides. Various 
methods have been developed for the synthesis of oligonucleotides for such 
purposes. Early synthetic approaches included phosphodiester and 
phosphotriester chemistries. Khorana et al. (J. Molec. Biol. 72, 209 
(1972)) discloses phosphodiester chemistry for oligonucleotide synthesis. 
Reese (Tetrahedron Lett. 34, 3143 (1978)) discloses phosphotriester 
chemistry for synthesis of oligonucleotides and polynucleotides. These 
early approaches have largely given way to the more efficient 
phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and 
Caruthers (Tetrahedron Lett. 22, 1859 (1981)) discloses the use of 
deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and 
Zamecnik (U.S. Pat. No. 5,149,798) discloses optimized synthesis of 
oligonucleotides by the H-phosphonate approach. 
Both of these modem approaches have been used to synthesize 
oligonucleotides having a variety of modified internucleotide linkages. 
Agrawal and Goodchild (Tetrahedron Lett. 28, 3539 (1987)) teaches 
synthesis of oligonucleotide methylphosphonates using phosphoramidite 
chemistry. Connolly et al. (Biochemistry 23, 3443 (1984)) discloses 
synthesis of oligonucleotide phosphorothioates using phosphoramidite 
chemistry. Jager et al. (Biochemistry 27, 7237 (1988)) discloses synthesis 
of oligonucleotide phosphoramidates using phosphoramidite chemistry. 
Agrawal et al. (Proc. Natl. Acad. Sci. USA 85, 7079 (1988)) discloses 
synthesis of oligonucleotide phosphoramidates and phosphorothioates using 
H-phosphonate chemistry. 
A number of treatises and review articles have appeared that discuss the 
various synthetic approaches. E.g., Methods in Molecular Biology, Vol. 20, 
Protocols for Oligonucleotides and Analogs, p. 63-80 (S. Agrawal, Ed., 
Humana Press 1993); Methods in Molecular Biology, Vol. 26: Protocols for 
Oligonucleotide Conjugates (Agrawal, Ed., Humana Press, Totowa, NJ 1994); 
Oligonucleotides and Analogues: A Practical Approach pp. 155-183 
(Eckstein, Ed., IRL Press, Oxford 1991); Antisense Res. and Applns. pp. 
375 (Crooke and Lebleu, Eds., CRC Press, Boca Raton, FL 1993); Gene 
Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds., 
Raven Press, New York, 1992). 
Both phosphoramidite and H-phosphonate chemical syntheses are carried out 
on a solid support that is stored in a reaction vessel. The required 
reaction steps for coupling each nucleotide are detritylation, coupling, 
capping, and oxidation. For small scale (up to 1 .mu.mole) synthesis, the 
total time for the addition of one nucleotide is about 6 minutes. An 
oligonucleotide, 30-mer in length, can be assembled in 180 minutes. Under 
these conditions, synthesized oligonucleotides are chemically pure and 
biologically active. However, when oligonucleotides are synthesized on a 
larger scale (up to 1 mmole), the time for addition of each nucleotide 
onto CPG is in the range of 30 to 60 minutes, requiring approximately 
12-25 hours for assembling a 25-mer oligonucleotide. The increase in time 
is due to the volume of the solid support being used in synthesis. This 
increase in cycle time exposes the already assembled oligonucleotide 
sequence to all reaction steps (including dichloroacetic acid 
detritylation step, coupling step, oxidation step and capping step) for a 
longer time. This increase in total assembly time affects the yield as 
well as chemical and biological properties of the compound. The chemical 
and biological properties are mainly affected by depurination, base 
modifications, and the like. 
To reduce the effects of these problems, it is possible to synthesize 
oligonucleotides using dimeric or multimeric synthons, thereby reducing 
the number of cycles, and thus the time required for synthesizing 
oligonucleotides. To this end, several investigators have worked toward 
developing acceptable dimeric or multimeric synthon approaches. Khorana 
(Science 203, 614 (1979)) introduced the concept of multimeric synthons, 
using a phosphodiester approach. Crea and Itakura (Proc. Natl. Acad. Sci. 
USA 75, 5765 (1978)), Reese (Tetrahedron Lett. 34, 3143 (1978)), and 
Ohtsuka et al. (Nucleic Acids Res. 10, 6553 (1982)) all disclose use of 
dimeric or multimeric synthons in a phosphotriester approach. Kumar and 
Poonian (J. Org. Chem. 49, 4905 (1984)) and Wolter et al. (Nucleosides and 
Nucleotides 5, 65 (1986)) disclose synthesis of oligonucleotide 
phosphodiesters using dimeric phosphoramidite synthons. Marugg et al. 
(Nucleic Acids Res. 12, 9095 (1984)) teaches use of a dinucleotide 
thiophosphotriester to produce oligonucleotides containing one 
phosphorothioate linkage. Connolly et al. (Biochemistry 23, 3443 (1984)) 
and Cosstick and Eckstein (Biochemistry 24, 3630 (1985)) disclose addition 
of one dinucleotide phosphorothioate to a growing oligonucleotide chain 
using a phosphoramidite approach. Brill and Caruthers (Tetrahedron Lett. 
28, 3205 (1987)) discloses synthesis of thymidine dinucleotide 
methylphosphonothioates. Roelen et al. (Nucleic Acids Res. 16, 7633 
(1988)) discloses a solution phase approach, using a reagent obtained in 
situ by treating methylphosphonothioic dichloride with 
1-hydroxy-6-trifluoromethyl benzotriazole to introduce a 
methylphosphonothioate internucleotide linkage into a dinucleotide in 
60-70% yield, and produces a hexamer containing the linkage by two 
consecutive condensations of dimers. Roelen et al. (Tetrahedron Lett. 33, 
2357 (1992)), discloses reagents for alkylphosphonate and 
alkylphosphonothioate chemistry. It discloses the solution phase synthesis 
of TG methyl, n-butyl, and n-octyl phosphonate and phosphonothioate 
dimers. 
Leba dev et al. (Tetrahedron. Lett. 31, 855 (1990)) discloses a solution 
phase approach to produce dinucleotides containing a stereospecific 
methylphosphonothioate internucleotide linkage in 50-60% yield. Katti and 
Agarwal (Tetrahedron Lett. 27, 5327 (1986)) discloses 3' 
1-methoxycarbonate methylphosphonate dimers. 
The use of such synthons in the synthesis of oligonucleotides has also been 
disclosed. Kumar and Poonian, supra, demonstrated the use of 3' 
phosphoramidite methyl phosphotriester dimers in the solid phase manual 
synthesis of a 29-mer with an overall yield of 93.4%. Wolter, supra, 
demonstrated the automated, solid phase synthesis of a 101-mer using 
.beta.-cyanoethyl-protected phosphoramidite dimers. Miura et al. (Chem. 
Pharm. Bull. 35, 833 (1987)) discloses automated solid-phase synthesis of 
pentadecathymidilate with phosphoramidite dimers. And Bannwarth (Helv. 
Chim. Acta 68, 1907 (1985) disclosed the use of phosphoramidite 
dinucleotides in the synthesis of oligonucleotides of modest length 
(N=8-11). 
Krotz et al. recently reported the synthesis of phosphorothioate dimers 
having low phosphodiester dimer content. They used these dimers to 
synthesize phosphorothioate oligo(T) and oligo(TC) nucleotides, wherein 
they observed that the N/N-1 ratio was on the order of 99:1 as measured by 
capillary gel electrophoresis (CGE). The phosphodiester content of the 
oligomers was on the order of 1% as determined by .sup.31 P NMR for 
oligomers synthesized with phosphorothioate dimers wherein the 
phosphorothioate linkage is protected by a .beta.-cyanoethyl group on the 
non-bridging oxygen. A similar reduction of the phosphodiester content was 
not observed for dimers wherein the phosphorothioate linkage was protected 
by a .beta.-cyanoethyl group on the (non-linking) sulfur. 
Once synthesized, the desired oligonucleotide (being "N" nucleotides in 
length) must be isolated from failure sequences (i.e., sequences with 
fewer than "N" nucleotides, such as N-1, N-2, etc.) and other impurities. 
While automated synthesizers have proven an invaluable tool for obtaining 
oligonucleotides, 1-3% of the reactions fail during each cycle in which a 
nucleotide monomer is to be added. Consequently, the resulting products 
are generally a heterogenous mixture of oligonucleotides of varying 
length. For example, in a typical 20mer synthesis, the 20mer product 
represents only 50-80% of the recovered oligonucleotide product. 
Furthermore, preparation of oligodeoxynucleotides on a solid phase support 
requires that the oligodeoxynucleotide be cleaved from the support. 
Cleavage of the oligonucleotide from the support is typically accomplished 
by treating the solid phase with concentrated ammonium hydroxide. The 
ammonium hydroxide is conventionally removed under reduced pressure using, 
for example, a rotary evaporator. This method for removing the ammonium 
hydroxide, however, is not ideal for use in large scale isolation of 
oligodeoxynucleotides. 
For most purposes (e.g., therapeutic or diagnostic) the purity of the 
compounds is extremely important. Consequently, there has been an interest 
in developing chromatographic techniques for purifying oligonucleotides. 
Because of their therapeutic potential, much of the focus has been on 
purifying oligonucleotide phosphorothioates. 
Conventional methods for purifying oligodeoxynucleotides employ 
reverse-phase liquid chromatography. Manufacturing facilities using such 
methods require explosion-proof equipment because acetonitrile is 
typically used in the elution buffer. 
Methods of oligodeoxynucleotide phosphorothioate purification have been 
published. Metelev and Agrawal (Anal. Biochem. 200, 342 (1992)) reported 
the ion-exchange HPLC analysis of oligodeoxyribonucleotide 
phosphorothioates on a weak anion-exchange column (Partisphere WAX) in 
which the weak anion exchanger utilizes a dimethylaminopropyl functional 
group bonded to Partisphere silica. This medium, with an ion-exchange 
capacity of 0.18 meq/g, exhibits an interaction with anions weaker than 
those observed with strong anion-exchange media. The authors of this study 
found that separation was length dependent for oligonucleotide 
phosphorothioates up to 25 nucleotides in length. Furthermore, N-1 peaks 
were separated from the parent peak. They also found that 30-mer and 
35-mer oligonucleotide phosphorothioates were separable with the same 
gradient, although better separation could be obtained with a shallower 
gradient. 
Metelev et al. (Ann. N.Y Acad. Sci. 660, 321-323 (1992)) reported the 
analysis of oligoribonucleotides and chimeric 
oligoribo-oligodeoxyribonucleotides using ion-exchange HPLC. They found 
that the retention time of the oligonucleotides studied depended on the 
number of ribonucleotide moieties in the oligonucleotide. In addition, the 
retention time of oligoribonucleotides was found to be length dependent. 
The authors noted that oligoribonucleotides of length up to 25 nucleotides 
could be purified and analyzed. 
Bigelow et al. (J. Chromatography 533, 131 (1990)) reported the use of 
ion-pair HPLC to analyze oligonucleotide phosphorothioates. Stec. et al. 
(J. Chromatography 326, 263 (1985)) and Agrawal and Zamecnik (Nucleic 
Acids Res. 19, 5419 (1990)), reported HPLC analysis of 
oligodeoxyribonucleotides containing one or two phosphorothioate 
internucleotide linkages using a reversed-phase column. 
Tang et al. (WO 95/27718) disclosed a purification techniques suitable for 
large scale separation of oligonucleotide phosphorothioates. The method 
uses DMAE Fractogel EMD column with an organic solvent-free, low salt, 
elution buffer. The method does not require elevated temperatures, making 
it more amenable for large scale chromatography. 
Puma et al. (WO 96/01268) disclosed a purification method not requiring the 
removal of ammonium hydroxide or the use of conventional C-18 silica gel 
reverse-phase liquid chromatography. The disclosed methods use hydrophobic 
interaction chromatography and DEAE-5PW anion ion-exchange chromatography. 
As antisense oligonucleotides proceed through human clinical trials, there 
is an ever-increasing demand for extremely pure oligonucleotides in large 
quantities. Regulatory agencies around the world are addressing the 
requisite standards for antisense oligonucleotides as drug compounds. 
E.g., Kambhampati et al, Antisense Res. Dev. 3, 405 (1993). Consequently, 
there remains a need for new methods of producing large quantities of 
highly pure oligonucleotides. 
SUMMARY OF THE INVENTION 
The invention provides new methods for producing dimeric nucleotide 
synthons, hereafter called "dimer blocks," having modified internucleotide 
linkages, e.g,. alkylphosphonate, phosphoramidate, phosphorothioate, or 
alkylphosphonothioate. Phosphorothioates are the preferred modified 
internucleotide linkage. According to this aspect of the invention, 
synthesis of dimer blocks proceeds in a single pot solution phase 
reaction, regardless of the type of internucleotide linkage in the dimer 
block. For example, to synthesize dimer block alkylphosphonates, 
condensation of a nucleoside 3'-alkylphosphonamidite with a 3'-protected 
nucleoside is carried out. For synthesis of dimer block phosphoramidates, 
alkylamine is added after H-phosphonate condensation of nucleotides. For 
synthesis of dimer block phosphorothioates, sulfurization using an 
appropriate sulfur reagent follows solution phase coupling of the 
protected monomeric nucleotides to yield a dimer. For preparing dimer 
block alkylphosphonothioates, an alkylphosphonamidite is used in the same 
one pot reaction as described for dimer block phosphorothioates. This 
simple chemistry allows for the synthesis of all possible dimer block 
methylphosphonothioates and promotes preparation of dimer blocks having 
3'-condensing groups. 
Thus, in a second aspect the invention provides novel dimer blocks 
comprising the nucleotides GG, GA, GT, GC, AG, AA, AT, AC, TG, TA TC, TT, 
CG, CA, CT or CC linked together by alkylphosphonate, phosphoramidate, 
phosphorothioate or alkylphosphonothioate linkages, and having various 
combinations of protective groups and condensing groups. These dimer 
blocks also give rise to a method of using such dimer blocks to assemble 
oligonucleotides containing alkylphosphonate, phosphoramidate, 
phosphorothioate, or alkylphosphonothioate linkages. Moreover, the dimer 
blocks allow assembly of oligonucleotides having exclusively 
alkylphosphonate, phosphoramidate, phosphorothioate or 
alkylphosphonothioate internucleotide linkages, or mixtures thereof. 
Thus, in a third aspect, the invention provides methods of using dimer 
blocks to assemble oligonucleotides having alkylphosphonate, 
phosphorothioate, phosphoramidate or alkylphosphonothioate linkages or 
having combinations of two or more of these. It is an object of the 
invention to provide efficient methods that reduce total assembly time of 
oligonucleotides. It is a further object of the invention to provide 
efficient methods that reduce total solvent consumption required for 
oligonucleotide assembly. It is also an object of the invention to provide 
efficient methods that ease purification of oligonucleotides by increasing 
the yield of full length oligonucleotides. It is an additional object of 
the invention to provide efficient methods that reduce side reactions by 
reducing the exposure of partially assembled oligonucleotides to 
chemicals. It is also an object of this aspect of the invention to provide 
highly pure dimers that facilitate synthesis and purification of extremely 
highly pure oligonucleotides. Finally, it is an object of the invention to 
reduce overall cost of oligonucleotide assembly by allowing the use of 
inexpensive solution phase chemistry to achieve half of the total 
synthesis. 
The present invention also provides oligonucleotides and methods for 
synthesizing them with a heretofore unobtainable purity. In one aspect, 
the invention provides a population of oligonucleotides having a purity of 
greater than 98%. In one embodiment of this aspect of the invention, 
oligonucleotides are provided that have an N-1 content of less than 2%, 
preferably less than 1%, and more preferably less than 0.5% of the content 
of the desired oligonucleotide (the N oligonucleotide). 
In one embodiment, the present invention comprises a population of 
oligonucleotides having all phosphorothioate internucleoside linkages, 
wherein the amount of phosphodiester impurity at each of the 
phosphorothioate linkages is less than 1%, preferably less than 0. 1%, and 
most preferably undetectable, by .sup.31 P NMR. 
The extremely high purity of oligonucleotides according to the invention 
sets a new industry standard. 
In another aspect, the present invention comprises a method for large scale 
(.gtoreq.ca. 100 .mu.mol) synthesis of oligonucleotides of extremely high 
purity. The method comprises synthesizing oligonucleotides using 
nucleotide dimers (herein called "dimer blocks") in place of 
mononucleotides as the basic synthetic unit. In the method of the present 
invention, oligonucleotide populations with extremely high purity are 
obtained. 
In another embodiment of this aspect of the invention, synthesis is 
conducted using standard techniques except that dimer blocks are used and 
the normal capping step is eliminated. We have found that excellent 
results (i.e., high yields of high purity oligonucleotide) can be obtained 
without the usual capping step. 
In another embodiment of this aspect of the invention, synthesis by either 
of the two previous embodiments is conducted using about six or less 
equivalents of dimer per coupling. Preferably, four equivalents and most 
preferably two equivalents of dimer are used per coupling step. 
The inventive method provides extremely high purity oligonucleotides with 
both a savings in cost of production as well as time. The oligonucleotides 
produced by the inventive method are ideally suited for in vivo 
therapeutic methods of treatment. 
The foregoing merely summarizes certain aspects of the present invention 
and is not intended, nor should it be construed as limiting the invention 
in any way. All patents and other publications recited herein are hereby 
incorporated by reference in their entirety. 
DETAILED DESCRIPTION OF THE INVENTION 
The invention relates to reagents and methods for assembling 
oligonucleotides. More particularly, the invention relates to the assembly 
of oligonucleotides having modified internucleotide linkages. 
Synthesis of Dimer Blocks 
In a first aspect, the invention provides new processes for making dimer 
blocks. Dimer blocks are dimeric nucleotides having modified 
internucleotide linkages and blocking groups at the 5'-hydroxyl. Preferred 
blocking groups include tert-butyldimethylsilyl, dimethoxytrityl, 
levulinyl, monomethoxytrityl and trityl groups. The 3' position of the 
dimer block may have a blocking group, a free hydroxyl, or a 
.beta.-cyanoethylphosphoramidite group. Preferred modified internucleotide 
linkages include phosphorothioate, alkylphosphonate and 
alkylphosphonothioate linkages. The modified linkage of the dimer block 
has an alkoxy or alkyl group. 
In a very general sense, the method of synthesizing dimer blocks according 
to the invention can be considered to be a method of synthesizing a dimer 
block having an alkylphosphonate, phosphoramidate, phosphorothioate or 
alkylphosphonothioate internucleotide linkage, the method comprising the 
steps of: 
(a) condensing together a first nucleoside derivative having a protective 
group at a 5' end and a condensing group at a 3' end with a second 
nucleoside derivative having a protective group at a 3' end and a hydroxyl 
group at a 5' end to form a dinucleotide derivative having a reduced 
internucleotide linkage, and 
(b) oxidizing the internucleotide linkage with an appropriate oxidizing 
agent to yield a dimer block alkylphosphonate, phosphoramidate, 
phosphorothioate or alkylphosphonothioate. 
The precise dimer block obtained, of course will depend upon the nature of 
the first nucleoside derivative and the oxidizing agent, as shown in Table 
1: 
TABLE 1 
______________________________________ 
First Nucleoside 
Dimer Block Type 
Derivative Oxidizing Agent 
______________________________________ 
alkylphosphonate 
nucleoside-3'-alkyl N,N di- 
iodine 
isopropyl phosphonamidite 
phosphoramidate 
nucleoside-3'-H phosphonate 
alkyl- or arylamine 
phosphorothioate 
nucleoside-3'-O-alkyl N,N, 
sulfurizing reagent 
diisopropyl phosphoramidite 
alkylphosphono- 
nucleoside-3'-alkyl N,N 
sulfurizing reagent 
thioates diisopropyl phosphonamidite 
______________________________________ 
In a first embodiment of this aspect of the invention, the method produces 
a dimer block having 5' and 3' blocking groups and a phosphorothioate 
internucleotide linkage. In this embodiment, the method comprises the 
steps of (a) joining together, by phosphoramidite chemistry, a nucleoside 
having a 5' blocking group and a nucleoside having a 3' blocking group, 
and (b) adding an appropriate sulfurizing agent, such as the Beaucage 
reagent. The Beaucage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide) is 
taught in U.S. Pat. No. 5,003,097. 
Examples of the preferred method of synthesizing phosphorothioate dimers 
are given below. We have found that synthesis of phosphorothioate dimers 
by this method results in a population of phosphorothioate dimers having 
very low phosphodiester dimer impurity. The use of these phosphorothioate 
dimer blocks in the synthesis of oligonucleotides in turn results in 
phosphorothioate oligonucleotides with less phosphodiester impurity. 
In a second embodiment of this aspect of the invention, the method produces 
a dimer block having a 5' blocking group, a 3' free hydroxyl group, and a 
phosphorothioate internucleotide linkage. In this embodiment, the method 
comprises steps (a) and (b) of the first embodiment above, and further 
comprises the step of (c) deprotecting the 3'-hydroxyl group. This is 
achieved by the use of conditions selective for removal of the 3' 
protective group only. For example, if the 5' protective group is 
dimethoxytrityl, monomethoxytrityl or trityl, and the 3' group is 
tert-butyldimethylsilyl, then selective removal of the 3' group is 
obtained by treatment with tetrabutylammonium fluoride. Alternatively, if 
the 5' group is dimethoxytrityl, monomethoxytrityl or trityl and the 3' 
group is levulinyl, selective removal of the 3' group is obtained by 
treatment with hydrazine monohydrate in pyridine/acetic acid. 
In a fourth embodiment of this aspect of the invention, the method produces 
a dimer block having a 5' blocking group, a 3' .beta.-cyanoethyl 
phosphoramidite group and a phosphorothioate internucleotide linkage. In 
this embodiment the method comprises steps (a), (b) and (c) of the first 
two embodiments described above, and further comprises the step of (d) 
converting the free 3' hydroxyl group to a 13-cyanoethyl phosphoramidite 
group. 
Those skilled in the art will recognize that as an alternative to the third 
and fourth embodiments, dimer blocks having phosphotriester 3' groups can 
be prepared according to well known procedures. 
In additional embodiments of this aspect of the invention the method 
produces dimer blocks having a 5' blocking group, an 
alkylphosphonothioate, alkylphosphonate or phosphoramidate internucleotide 
linkage, and a 3' group that may be a blocking group, a free hydroxyl, a 
.beta.-cyanoethyl phosphoramidite group, or a phosphotriester group. In 
these embodiments, the method is carried out exactly as described for the 
four embodiments above to produce dimer block alkyl-phosphonothioates, 
except that the starting material is a nucleoside alkylphosphonamidite. 
Analogous dimer block alkylphosphonates are prepared in identical fashion 
to the dimer block alkylphosphonothioates, except that an iodine solution 
is used in place of the sulfurizing agent. Analogous dimer block 
phosphoramidates are prepared by H-phosphonate condensation followed by 
oxidation of the linkage with an alkyl- or arylamine in carbon 
tetrachloride. 
This first aspect of the invention offers a method of producing dimer block 
products that are useful as intermediates for assembling oligonucleotides 
having modified internucleotide linkages. The ability to produce these 
dimer blocks in a one pot reactions greatly simplifies their production. 
Dimer Blocks 
In a second aspect, the invention provides novel dimer block products 
having a 5' blocking group, a modified internucleotide linkage and a 3' 
group that may be a blocking group, a free hydroxyl, an H-phosphonate 
group, or in some cases a .beta.-cyanoethyl phosphoramidite. The method 
for producing these dimer blocks is independent of the sequence of the 
nucleotides in the dimer block, thus allowing production of all possible 
dimer sequences containing alkylphosphonate, phosphoramidate, 
phosphorothioate or alkylphosphonothioate linkages, i.e., GG, GA, GT, GC, 
AG, AA, AT, AC, TG, TA, TT, TC, CG, CA, CT, and CC. Such dimers are 
illustrated below: 
##STR1## 
Where B.sub.1 and B.sub.2 are the same or different nucleotide base (e.g., 
G, A, T, C, or modifications thereof, and R.sub.1 is a protective group 
such as dimethoxytrityl, monomethoxytrityl or trityl, L is an 
alkylphosphonate, phosphoramidate, thiophosphotriester, or 
alkylphosphonothioate, and R.sub.2 is an H, a 
.beta.-cyanoethylphosphoramidite, or a protective group such as levulinyl 
or t-butyldimethylsilyl. Although the foregoing illustrates DNA/DNA 
dimers, RNA/RNA, RNA/DNA, and DNA/RNA dimers are also encompassed within 
the scope of the invention disclosed herein. 
Synthesis of Ultra-pure Oligonucleotides Using Dimer Blocks 
In a third aspect, the invention provides a method of using dimer blocks to 
assemble oligonucleotides having modified internucleotide linkages. In 
this aspect, dimer blocks having modified internuclcotide linkages (e.g., 
phosphorothioate, alkylphosphonate, phosphoramidate, or 
alkylphosphonothioate), are used to assemble oligonucleotides having such 
modified internucleotide linkages (dimer blocks having 5' blocking groups 
and 3' .beta.-cyanoethyl phosphoramidite are used). Synthesis is then 
conducted according to the phosphoramidite approach by condensing the 
dimer block with the nascent oligonucleotide. (As used herein, the term 
"nascent oligonucleotide" means the less-than-full-length, solid 
support-bound synthetic nucleic acid that upon elongation results in the 
desired synthetic oligonucleotide.) Support of oligonucleotide synthesis 
with dimer blocks can be, for example, soluble polymers as well as 
insoluble CPG and polymer beads. 
In order to ensure obtaining oligonucleotides of extremely high purity as 
disclosed herein, it will be appreciated that measures should be taken at 
each step in the synthetic/purification process to maximize the purity. 
According to the present invention, this begins with using dimers as the 
elemental oligonucleotide building block. In the synthetic methods 
disclosed herein, dimer phosphoramidites are used. Preferably the dimer 
phosphoramidite is at least 90% pure (as determined by HPLC). More 
preferably it is at least 96% pure. Most preferably it is at least 98% 
pure. 
In the phosphoramidite approach, .beta.-cyanoethyl 
tetraisopropylphosphorodiamidite and tetrazole are used to activate a 
5'-DMT, .beta.-cyanoethyl protected dimer to yield the phosphoramidite 
dimer, such as depicted in the following in which a phosphorothioate dimer 
is illustrated: 
##STR2## 
Alternatively, 2-cyanoethyl diisopropylchlorophosphoramidite can be used 
instead of the diamidite displayed in the scheme above. 
Three principal types of impurities generally arise in this reaction. The 
first results from hydrolysis of the dimer amidite to yield an 
H-phosphonate dimer. The second impurity is the starting material itself, 
the 3'-hydroxy dimer. Both of these impurities are "inert" in the sense 
that they will not react with the nascent oligonucleotide and contribute 
to the N-x or N+x content of the final oligonucleotide product (where N is 
the number of nucleotides in the final, full-length oligonucleotide and x 
is an integer.gtoreq.1). 
The third principal type of impurity is the tetrazole-activated 
phosphorodiamidite. This impurity is not inert and measures should be 
taken to minimize its presence. Preferably, the tetrazole and dimer are 
first combined and then added together to the diamidite under conditions 
in which the tetrazole is not in great excess of the diamidite. This 
results in a low probability that two molecules of tetrazole will react 
with the diamidite. Preferably, the tetrazole and diamidite are used in 
.about.1:1 ratio and each in slight excess of the dimer. A suitable amount 
of each is 1.3 cq diamidite and 1.2 eq tetrazole per eq of dimer. After 
the reaction is complete, the product is preferably washed several times 
as described in the synthesis of dimer 24 in Example 4, below. Following 
the foregoing protocol minimizes the amount of non-inert impurity. 
An advantage of the present method is that oligonucleotide synthesis can be 
conducted on commercially available synthesizers using standard cycles, 
substituting dimers for the usual monomers. This is demonstrated, for 
example, in Example 6-9, below. 
Following synthesis, the oligonucleotide product is cleaved from the solid 
support and subject to purification by Ion-Exchange Chromatography (IEX). 
Optionally, the oligonucleotide can first be purified by Reverse Phase 
Chromatography (RPC) before IEX. As described by Puma et al. (WO 
96/01268), Hydrophobic Interaction Chromatography (HIC) can be useful as 
the initial chromatographic step. For the purposes of the present 
invention, however, RPC is preferred. Advantageously, standard protocols 
of purifying oligonucleotides by these techniques can be employed. 
Although High Pressure Liquid Chromatography (HPLC) can also be used, the 
present method offers the additional advantage of using Medium Pressure 
Liquid Chromatography (MPLC), which is preferable because it is cheaper 
and more easily adaptable to large scale synthesis. 
To obtain the high purity levels as disclosed herein, careful screening and 
pooling of chromatography fractions is preferable. Screening of fractions 
and/or trial pools of fractions is preferably conducted by analytical 
Capillary Electrophoresis (CE) to determine the degree of purity and yield 
of each fraction. Then, fractions having an acceptable degree of purity 
and yield are pooled in a pre-determined manner to yield a final 
population of oligonucleotides having a sufficiently high degree of purity 
and yield. Depending on the application, one may be willing to sacrifice 
some yield to obtain a higher degree of purity, and vice versa. Such 
pooling methods are well known to those skilled in the art. 
Following this general protocol results in oligonucleotides of heretofore 
unrealized purity, as demonstrated in the Examples below. The degree of 
purity obtainable according to the methods of this aspect of the invention 
is described in detail below. 
In addition, we have surprisingly found that synthesis of ultra-pure 
oligonucleotides according to this aspect of the invention can be 
conducted without compromising purity or yield using an amount of dimer 
block that is much less than has previously been thought possible. Prior 
art methods, such as those disclosed by Wolter et al., supra, have used a 
large excess of dimer (often in the range of tens of equivalents) in order 
to drive the reaction to completion and ensure high yield. We have 
surprisingly found, however, that fewer than about 6 equivalents can be 
used and still obtain high yields of highly pure oligonucleotide. 
Accordingly, in a preferred embodiment of this aspect of the invention, 
.ltoreq.6 equivalents of dimer are used in each coupling step. More 
preferably .about.2 equivalents of dimer are used per coupling step. 
Traditional methods of synthesis, be it using monomers or dimers, employ a 
capping step during each synthetic cycle to block unreacted reactive 
sites, thereby minimizing subsequent addition of nucleotides to these 
sites rather than to the nascent oligonucleotide. Capping was thought to 
be a necessary step to achieve high yields of pure oligonucleotides. We 
have surprisingly found, however, that synthesis of oligonucleotides using 
dimer blocks as disclosed herein can be conducted without the capping 
step. The elimination of the capping step also results in a tremendous 
savings in time and money without sacrificing yield. 
Accordingly, in another preferred embodiment of this aspect of the 
invention, dimer block synthesis of oligonucleotides is conducted without 
the capping step. 
Purification of oligonucleotides prepared by dimer block synthesis may be 
accomplished using two approaches. When the purity of the crude 
oligonucleotide is relatively low, the crude oligonucleotide is preferably 
purified by RPC followed by IEX. Oligonucleotides having a N:N-1 ration of 
100:0 (i.e., non-detectable amounts of N-1 oligonucleotide) can be 
obtained. 
Alternatively, for crude products of higher purity, RPC can be omitted and 
the crude oligonucleotide purified by IEX to give an oligonucleotide with 
an N:N-1 ratio of 100:0 (i.e., no detectable N-1 impurity). 
As evidenced in Table 5, crude products having a range of purities have 
been obtained. Depending on the purity of the crude product, a two-step 
chromatographic purification (RPC followed by IEX) or a single-step 
chromatographic purification (IEX only) may be employed. As evidenced in 
Table 4, crude products of lower purity (e.g., 15 and 300 .mu.mol scale, 
synthesized with capping) are purified by RPC to yield an intermediate 
material whose purity is approximately equivalent to that of a crude 
product having high purity as originally synthesized (e.g., 300 .mu.mol 
scale, synthesized without capping). In the current comparison, relative 
purities of crude products are clearly differentiated on the basis of IEX 
and CE analyses. Relative purities of feedstocks taken for purification by 
IEX are well defined by IEX analysis of the detritylation mixtures. 
As evidenced in Table 4, the single-step chromatographic purification 
provides higher overall recoveries. Taken together, the two approaches 
(i.e., the one step and two step approaches) provide robust and versatile 
tools for purification of oligonucleotide prepared by dimer synthesis. 
Crude products having a range of purities can be accommodated. 
A strongly preferred technique in achieving required purity is rigorous 
screening of trial pools by CE analysis. During initial work at 15 .mu.mol 
scale (infra), this technique was not employed. In that work, product 
purity was 97% by CE and (N+x) content was 2.4%. In subsequent work, 
rigorous use of CE analysis was incorporated and product 
purities.gtoreq.98% by CE were achieved, and (N+x) content was reduced to 
0.3% or less. 
The data indicate that dimer synthesis, performed with or without capping, 
provides a crude product which can be brought to.gtoreq.98% final purity. 
Achievement of 98+% product purity can be seen as arising from the 
following factors: 
(1) Use of dimer synthesis. This approach has the inherent potential to 
entirely eliminate (N-1) impurity. Such impurity presents the greatest 
challenge to chromatographic purification. 
(2) Use of high-resolution preparative chromatography. 
(3) Rigorous use of capillary electrophoresis to analyze trial pools 
prepared from chromatographic fractions. 
The synthetic methods of the invention can be conducted at both small 
(e.g., 1 .mu.mol) scale as well as large (e.g., .gtoreq.100 .mu.mol) 
scale, resulting in oligonucleotide product with similar purity and yield. 
In any of the embodiments according to this aspect of the invention, both 
monomers of any dimer block used in the synthetic method can be 
deoxyribonucleotides, or one can be a deoxyribonucleotide and the other 
can be a ribonucleotide. 
In a preferred embodiment of the present method, dimer phosphorothioates 
are employed to yield an oligonucleotide comprising entirely 
phosphorothioate internucleoside linkages. Oligonucleotide 
phosphorothioates with a PO impurity level of less than 0.5%, preferably 
0.3%, more preferably .ltoreq.0.4%, and most preferably non-detectable by 
.sup.31 P NMR can be obtained. 
In a preferred embodiment, the purity of the crude oligonucleotide 
phosphorothioate (i.e., before purification by chromatographic means) 
produced by the method according to the invention is .gtoreq.75% as 
determined by CE. Preferably in this embodiment: 
a) the N-1 content is non-detectable; 
b) the N-1 content is non-detectable, the N-2 content is &lt;6%, the N-x 
content for x&gt;2 is .ltoreq.15% and the PO content is .ltoreq.0.6%; 
c) the N-1 content is non-detectable and the N-2 content is &lt;1%; 
d) the N-1 content is &lt;2%; 
e) the N-1 content is less than or equal to 0.5%; or 
f) the N+x content is &lt;8%. 
Another preferred dimer block is one having a phosphotriester 
internucleotide linkage. 
Those skilled in the art will recognize that this approach also allows the 
convenient synthesis of mixed phosphate backbone oligonucleotides, e.g., 
oligonucleotides having any combination of one or more phosphorothioate, 
alkyl-/arylphosphonothioate, phosphodiester, and/or alkyl-/arylphosphonate 
linkages. 
The method according to this aspect of the invention provides several 
advantages over monomeric synthesis of oligonucleotides. First, since half 
as many assembly cycles are required, the total assembly time is reduced 
by half, which, for large scale synthesis, can be a saving of 12 hours or 
more for a single oligonucleotide. This reduction in time also results in 
fewer side reactions, since partially assembled oligonucleotides are 
exposed to chemicals for a shorter time. The method also facilitates 
purification of oligonucleotides by increasing the proportion of full 
length oligonucleotides, since that proportion varies inversely with the 
number of cycles performed. Finally, the method reduces cost of synthesis 
by cutting solvent consumption by half and by allowing one half of the 
total synthesis to be carried out using inexpensive solution phase 
chemistry. The present method extends these advantages to oligonucleotides 
having exclusively phosphorothioate, alkylphosphonate, phosphoramidate, or 
alkylphosphonothioate linkages as well as to oligonucleotides having any 
combination thereof. 
Ultra-Pure Oligonucleotides 
The methods of the third aspect of the invention produce oligonucleotides 
of heretofore unobtainable purity. Accordingly, in a fourth aspect, the 
invention provides oligonucleotides produced by the methods of the third 
aspect of the invention and having a purity described below. 
Oligonucleotide phosphorothioates are a preferred oligonucleotide 
according to this aspect of the invention. As used herein, the term 
oligonucleotide phosphorothioate is an oligonucleotide having all 
phosphorothioate internucleotide linkages. 
Oligonucleotide product of length N contains two major types of impurities, 
size impurity (type "A") and composition impurity (type "B") such that the 
"total purity" of a population of oligonucleotides can be defined by: 
EQU total purity=100% -(% impurity A+% impurity B) 
where "% impurity A" is the total percentage of oligonucleotides of length 
other than N (i.e., % (N+x)+% (N-x), where x is an integer other than 0 
and N+x represents all oligonucleotides of length greater than N 
nucleotides and N-x represents all oligonucleotides of length less than N 
nucleotides). Type A impurities are detectable and quantitatable by 
capillary electrophoresis. 
"% impurity B" relates to oligonucleotides having all modified (i.e., 
non-phosphodiester) linkages and is the total percentage of 
oligonucleotides of length N having one or more phosphodiester 
internucleotide linkages in place of the modified linkage. In the 
preferred oligonucleotide phosphorothioates, the type B impurity is an 
oligonucleotide having at least one phosphorothioate linkage replaced by a 
phosphodiester linkage. Type B impurities (commonly called "PO" 
impurities) are detectable along with N-x' impurity by ion exchange 
chromatographic (IEX) analysis of oligonucleotide (DMT-off form). The peak 
in the IEX chromatogram corresponding to the PO impurity plus the N-x' 
impurity appears just before the peak of the desired N-mer 
oligonucleotide. With reference to this impurity peak, the term N-x' 
refers to the sum of N-2 plus N-3 and/or N-4. (The terms N-2, N-3, and N-4 
as employed in this calculation are defined further below.) As defined, 
the type B (or PO) impurity is estimated for oligonucleotides by using the 
following formula: 
EQU %PO=[.alpha..sub.PO/N-x'].sub.IEX [.alpha..sub.N-2 
+.alpha..sub.N-3,4)].sub.CE 
where 
##EQU1## 
and A.sub.i is the area under peak "i" of the chromatogram, the sum in the 
denominator of the definition of ".alpha." is over all peaks in the 
chromatogram, and the subscripts "IEX" and "CE" indicate the 
chromatographic technique from which the data were obtained. 
.alpha..sub.PO/N-x refers to the peak immediately preceding the peak 
corresponding to the "N" oligomer in IEX and has contributions from both 
N-x oligonucleotides and N oligonucleotides having a PO internucleoside 
linkage. [.alpha..sub.N- 2].sub.CE corresponds to oligonucleotides of 
length N-2, and [.alpha..sub.N-3,4 ].sub.CE corresponds to the peak 
immediately preceding the N-2 peak in CE chromatograms and is believed to 
arise primarily from oligonucleotides of length N-3 and/or N-4. The above 
formula for calculating estimated values for %PO was applied only to 
product purified by IEX (DMT-off). Results appear in Table 4. A second 
approach was applied to calculating estimated values for %PO in crude 
oligonucleotides (DMT-on): 
##EQU2## 
Wherein [A.sub.PO/N-x' ].sub.IEX refers to the area of the peak 
immediately preceding that corresponding to the DMT-on form of the N 
oligonucleotide. This calculation tends to over-estimate the PO content as 
it does not subtract the contribution from N-x' impurities. Thus, this 
approach provides a conservative estimate of purity with respect to PO 
content. Values based on this calculation appear in Table 5. For both pure 
and crude oligonucleotide, %PO can also be determined by .sup.31 P. 
Other calculations employed herein are based on the PO/N-x' peak. %DMT-on 
(by IEX) is calculated as follows: 
EQU %DMT-on=[.alpha..sub.N +.alpha..sub.PO/N-x' ].sub.IEX-DMT-n 
where .alpha..sub.PO/N-x' refers to the peak immediately preceding that 
for the DMT-on form of the N oligonucleotide. The calculation provides a 
useful estimate of combined phosphorothioate and phosphodiester forms of 
DMT-on oligonucleotide, N-oligomer. No correction is made for N-x' content 
in the .alpha..sub.PO/N-x' peak. Results based on this calculation appear 
in Tables 4 and 5. 
In a preferred embodiment, oligonucleotide phosphorothioates according to 
this aspect of the invention have a total purity of 98% or more. More 
preferably, the total purity is greater than 99%. Preferably in these 
embodiments 
a) the N-1 content is non-detectable; 
b) the N-1 content is non-detectable, the N-2 content is less than 1%, and 
the N-x content for x&gt;2 is less than 2%; 
c) the N-1 content is non-detectable and the N-2 content is less than 1%; 
d) the N-1 content is non-detectable and the N-x content for x&gt;1 is less 
than 2%; 
c) the N-1 content is &lt;2% 
f) the N-1 content is less than or equal to 0.5%; 
g) the N+x content is &lt;2%; 
h) the N+x content is &lt;1%; or 
i) the N+x content is &lt;0.5%. 
Unless expressly indicated otherwise, all percentages of oligonucleotides 
of a particular length mean percentages as measured by capillary gel 
electrophoresis. As used herein, "non-detectable" mean non-detectable by 
capillary gel electrophoresis, which can detect a single oligonucleotide 
size impurity (e.g., N-x and N+x) down to 0.15%. Typically in capillary 
electrophoresis, the noise level is .about.20-30 mV. The minimum 
detectable peak has a S/N of .about.3:1, or about 60 mV. 
Oligonucleotides according to this aspect of the invention can be of 
essentially any conventionally synthesizable length and can be made 
according to the third aspect of the invention. Preferably, 
oligonucleotides according to this aspect of the invention are 50 or fewer 
nucleotides in length, more preferably, 30 or fewer, and most preferably 
of length of from about 15 to about 30 nucleotides. 
In yet another embodiment of this aspect of the invention, the 
oligonucleotide phosphorothioates have less than 0.5% phosphodiester 
content (i.e., the number of phosphodiester linkages comprises less than 
0.5% of the number of phosphorothioate linkages as measured by .sup.31 P 
NMR). Preferably, the PO content is less than or equal to 0.3%. More 
preferably, the phosphodiester content is less than or equal to 
0.03%-0.04%, the lower limit of detection of .sup.31 P NMR. Even more 
preferably, the PO content is non-detectable by .sup.31 P NMR. 
The following examples are intended to further illustrate certain preferred 
embodiments of the methods according to the invention, and arc not 
intended to be limiting in nature.

EXAMPLES 
Example 1 
Solution Phase Synthesis Of 5'-O-dimethoxytrityl-thymidine-3'-O-methyl 
phosphorothioate-5'-O-N.sup.4 -benzoyl-2'-deoxycytidine 
The synthesis steps for this protected dimer block for synthesis of 
phosphorothioate containing oligonucleotides are shown below: 
##STR3## 
wherein (a) is anhydrous acetonitrile and tetrazole, (b) is Beaucage 
reagent, (c) is tetrahydrofuran and tetrabutyl ammonium fluoride, and DMT 
is dimethoxytrityl and TBDMS is tert-butyldimethylsilyl. 
A mixture of 5'-O-dimethoxytrityl-thymidine-3'-O-methyl N,N-diisopropyl 
phosphoramidite, 1, (1.4 g, 2 mmol) and N.sup.4 
-benzoyl-3'-O(tert-butyldimethylsilyl)-2'-deoxycytidine, 2, (0.88g, 2 
mmol) was dissolved in anhydrous acetonitrilc (25 ml) and a solution of 
tetrazole (0.45 M, 10 ml) was added. The reaction mixture was stirred at 
room temperature for 15 min. Beaucage reagent (0.6 g in anhydrous 
acetonitrile 15 ml) was added and the mixture was further stirred for 15 
min. The reaction mixture was evaporated to remove most of the 
acetonitrile under reduced pressure. The crude reaction product was 
extracted with dichloromethane and washed with brine. The organic layer 
was dried over Na.sub.2 SO.sub.4 and evaporated to dryness to obtain 3. 
Product 3 was re-dissolved in tetrahydrofuran (16 ml) and treated with a 1 
M solution of tetrabutylammonium fluoride (3 ml, THF) for 15 min. The 
reaction mixture was evaporated to almost dryness and partitioned between 
dichloromethane and water. The organic layer was dried over Na.sub.2 
SO.sub.4 and evaporated to a small volume. The product was purified by 
column chromatography using silica gel (2.5.times.20 cm). The dimer block 
product, 4, was eluted with 0-7% methanol in dichloromethane (0.5% 
pyridine); obtain 1.3 g (70% yield); not optimized. 
.sup.31 P NMR=70.06. 
Example 2 
Solution Phase Synthesis Of 5'-O-dimethoxytrityl-N.sup.4 
-benzoyl-2'-deoxycylidine-3'-O-methyl phosphorothioate-5'-O-thymidine 
The synthesis steps for this dimer block for synthesis of 
phosphorothioate-containing oligonucleotides are shown below: 
##STR4## 
wherein (a) is acetonitrile and tetrazole, (b) is Beaucage reagent, and 
(c) is pyridine acetic acid and hydrazine hydrate. DMT is dimethoxytrityl 
and Lev is levulinyl. 
A mixture of N4-benzoyl 5'-O-dimethoxytrityl-2'-deoxycytidine-3'-O-methyl 
N,N-diisopropyl phosphoramidite 6 (1.6 g, 2 mmol) and 
3'-O-levulinyl-thymidine, 7, (0.68 g, 2 mmol) was dissolved in anhydrous 
acetonitrile (25 ml) and a solution of tetrazole (0.45 M, 10 ml) was 
added. The reaction mixture was stirred for 15 minutes at room 
temperature. Beaucage reagent (0.6 g in anhydrous acetonitrile 15 ml) was 
added and the reaction mixture was further stirred for 15 minutes. The 
reaction mixture was then evaporated to remove most of the solvent. The 
crude reaction product was extracted with dichloromethane and washed with 
brine to obtain 8. Dichloromethane was evaporated, the solid residue was 
re-dissolved in 20 ml pyridine and mixed with 20 ml of 1 M hydrazine 
hydrate solution in pyridine/acetic acid (3/2). The reaction mixture was 
stirred for 5 min. The reaction mixture was then cooled on an ice-bath and 
4 ml acetyl acetone was added to quench the excess amount of hydrazine 
hydrate. The mixture was evaporated to a small volume and then directly 
applied to silica gel column chromatography (2.5.times.25 cm). The dimer 
block product, 9, was eluted by using 0-7% methanol in dichloromethane 
(0.5% pyridine) to obtain 1.25 g (67% yield). .sup.31 P NMR=69.86. 
Example 3 
Solution Phase Synthesis Of 5'-O-dimethoxytrityl-N.sup.4 
-benzoyl-2'-deoxycytidine-3'-O-methylphosphorothioate 5'-O-N.sup.6 
-benzoyl-2 ' deoxyadenosine 
The synthetic steps for this dimer block for synthesis of 
phosphorothioate-containing oligonucleotides are shown below: 
##STR5## 
wherein (a)-(c) are the same as in Example 2. 
A mixture of N.sup.4 
-benzoyl-5'-O-dimethoxytrityl-2'-deoxycytidine-3'-O-methyl N,N-diisopropyl 
phosphoramidite, 15, (4 g, 5 mmol) and 3'-O-levulinyl-N.sup.6 
-benzoyl-2'-deoxyadenosine, 16, (2 g, 4.4 mmole) was dissolved in 
anhydrous acetonitrile (18 ml) and a solution of tetrazole (0.45 M, 22 ml) 
was added. The reaction mixture was stirred for 30 minutes at room 
temperature, then treated with Beaucage reagent (1.4 g in anhydrous 
acetonitrile 25 ml) for 15 minutes. The reaction mixture was then 
evaporated to remove most of the solvent. The crude reaction product was 
extracted with dichloromethane and washed with brine. Dichloromethane was 
evaporated to obtain a solid product, 17, which was then redissolved in 40 
ml pyridine, and 40 ml of 1 M hydrazine hydrate solution in 
pyridine/acetic acid (3/2) was added. After 7 minutes, the reaction was 
quenched with ice and the product was extracted with dichloromethane, then 
washed with water. The organic layer was dried over sodium sulfate and 
then co-evaporated with toluene to dryness. The mixture was re-dissolved 
in a small volume of dichloromethane and applied to silica gel column 
chromatography (5.times.12 cm). The dimer block product, 18, was eluted by 
using 0-7% methanol in dichoromethane (0.5% pyridine) to obtain 3.8 g (79% 
yield). .sup.31 P NMR=69.84, 69.89. 
Example 4 
Synthesis of 
N-benzoyl-5'-O-dimethoxytrityl-P-cyanoethylthiophosphoryl-2'-deoxycytidyly 
l-3'-O-[(N,N-diisopropylamino)cyanoethoxyphosphino](3'.fwdarw.5') thymidine 
(24) 
The title phosphoramidite dimer was synthesized as depicted below: 
##STR6## 
Synthesis of dimer 22 
60 ml of 0.45 M solution of tetrazole in acetonitrile were added to a 
solution of phosphoramidite 20 (10.0 g, 12.0 mmol) and nucleoside 21 (4.0 
g, 12.0 mmol) in 150 ml acetonitrile. The reaction was stirred for 30 min 
at room temp. TLC analysis indicated that complete conversion of the 
starting materials to the intermediate phosphite, which was further 
oxidized by adding 88 ml acetonitrile solution of Beaucage Reagent (3.52 
g, 18.0 mmol). The reaction was stirred for 30 min and then was evaporated 
to dryness in vacuo to give dimer 22 as a yellow gum in quantitative yield 
(13.0 g, .sup.31 P NMR, .delta. 67.0). 
Synthesis of dimer 23 
Hydrazine monohydrate (2.1 g, 42.0 mmol) was added slowly to an ice-cold 
solution of crude dimer 22 (6.63 g, 6.0 mmol) in 65 ml pyridine/acetic 
acid mixture (3:2). After 20 min, excess hydrazine monohydrate was 
quenched with the slow addition of acetyl acetone (11 ml). The reaction 
was added to crushed ice and was extracted with methylene chloride 
(3.times.50 ml). Combined organic layer was dried and evaporated to give a 
yellow oil, which was chromatographed on flash silica gel. Elution with 
methylene chloride/methanol/pyridine (93:5:2) gave dimer 23 as a colorless 
foam (5.0 g, 82.7% .sup.31 P NMR, .delta., 67.0). 
Synthesis of dimer 24 
A solution of dimer 23 (31.5 g, 31.3 mmol) and tetrazole (2.75 g, 39.3 
mmol) in 150 ml acetonitrile was added to a solution of 
tetraisopropylphosphorodiamidite (13.0 g, 43.1 mmole) in 60 ml of 
acetonitrile. The reaction was stirred at room temperature for 45 minutes. 
It was then cooled in ice and the supernatant solution was decanted into 
another flask. The precipitate was washed with cold acetonitrile three 
times. The combined acetonitrile solution was evaporated to dryness. The 
residue was dissolved in dichloromethane and hexane was added. Supernatant 
solution was removed and the residual oil was washed with hexane. The 
dichloromethane/hexane treatment was repeated one more time. The resulting 
yellow foam was dissolved in ethyl acetate containing I% pyridine and was 
passed quickly through a short pad of silica gel. Evaporation of the 
pooled fractions afforded the dimer phosphoramidite 24 as colorless foam 
(31.7 g, 83.9%, .sup.31 P, .delta., 67.0 (P-V), .about.149.0 (P-III). 
Example 5 
Determining the purity of phosphorothioate dimers 
Authentic samples of CpoT dimer phosphoramidites were made by employing the 
same chemistry as that used to synthesize CpsT dimer phosphoramidites, 
except that t-butyl hydroperoxide was used instead of Beaucage reagent to 
oxidize the phosphite intermediate to give the phosphotriester dimer. 
Phosphotriester and phosphorothioate dimers can be distinguished by .sup.31 
P NMR. The phosphoramidite phosphorous signal in both cases appears at 
.about..delta. 149. There is a significant difference in the chemical 
shifts of the phosphorothioate and phosphotriester functions, however. 
Phosphorothioate triester signal is observed at .about..delta. 67.0, 
whereas the phosphotriester peak appears at .about..delta. -2.0. A trace 
amount of H-phosphonate byproduct is detected in the phosphorothioate 
dimer as a doublet centered at .delta. 14.5. Thus, .sup.31 P NMR can serve 
as an effective tool in determining the impurity of phosphotriester in a 
phosphorothioate triester compound. 
Two samples of phosphorothioate dimer phosphoramidite were spiked with a 
known amount of phosphotriester dimer phosphoramidite. A sample containing 
10% phosphotriester dimer and 90% of phosphorothioate dimer and a second 
sample constituting 5% phosphotriester dimer and 95% phosphorothioate 
dimer were prepared and the .sup.31 P NMR of the samples recorded. .sup.31 
P NMR spectra of both samples exhibited distinct, well-separated signals 
at .about..delta. 67.0 and .about..delta. -2.0, as expected. A 750 times 
enlargement of pure phosphorothioate dimer spectrum manifested no 
detectable peak at .about..delta. 2.0. 
Example 6 
0.2 .mu.mol scale synthesis of oligonucleotide phosphorothioates 
A fluidized bed technique was used for the synthesis of (CT).sub.10 T 
oligomer on a Perseptive Biosystem Expedite Synthesizer. The design of the 
synthesizer is such that the reagents like activator, amidite, and Cap A, 
Cap B are mixed before entering the column containing resin. There were 
two capping steps in this method, one before oxidation and another after 
oxidation. 37.6 equivalents of dimer were used in each coupling step. 
In this and all subsequent oligonucleotide syntheses described herein, the 
dimer phosphoramidite was made as described in Example 4. All other 
reagents were purchased from commercial sources and used as received. 
Fresh solutions of the reagents were made prior to the start of the 
oligonucleotide synthesis. At the end of the synthesis, resin was dried 
under vacuum. The oligonucleotide was cleaved from the support and 
deprotected by treating it with ammonium hydroxide at 55.degree. C. 
overnight. Crude product was analyzed by reverse phase chromatography, ion 
exchange chromatography and capillary gel electrophoresis. 
Detritylation 
3% trichloroacetic acid in dichloromethane were used for detritylation in 
three steps: 
a) 0.3 ml was delivered for 8 sec.; 
b) 0.3 ml was delivered for 8 sec.; and 
c) 0.45 ml was delivered for 30 sec. 
Wash 
Washing was conducted with acetonitrile. 0.9 ml was delivered for 30 sec. 
Coupling 
The activator used for coupling was 0.45 M 1-H tetrazole in acetonitrile; 
the amidite was 0.1 M solution of dimer phosphoramidite in acetonitrile. 
Coupling was conducted as follows: 
a) pre-couple wash with acetonitrile--0.075 ml for 1 see; 
b) 0.075 ml activator was introduced for 1 sec; 
c) 0.075 ml activator was delivered for 1 sec; 
d) 0.075 ml amidite was delivered for 1 sec; 
e) 0.075 ml activator was introduced for 1 sec; 
f) 0.090 ml acetonitrile wash for 2.4 sec; 
g) coupling was allowed to continue for 900 sec. 
Wash 
The resin was washed with 0.27 ml for 900 sec. 
Capping 
Cap A consisted of 10% acetic anhydride in tetrahydrofuran. Cap B was 10% 
N-methylimidazole, 20% pyridine, and 70% tetrahydrofuran. 0.12 ml of Cap A 
was delivered for 3 sec followed by delivery of 0.12 ml of Cap B for 3 
sec. 
Wash 
The reaction mixture was then washed with 0.375 ml of acetonitrile for 17 
sec. 
Sulfurization 
0.45 ml of 2% Beaucage reagent in acetonitrile was delivered for 7 sec. 
Wash 
The reaction mixture was then washed with 0.3 ml of acetonitrile delivered 
for 120 sec. 
Capping 
0.105 ml of each of Cap A and Cap B was delivered for 2 sec. 
Wash 
The reaction mixture was then washed with 0.9 ml of acetonitrile delivered 
for 15 sec. 
Example 7 
15 .mu.mole scale synthesis of oligonucleotide phosphorothioates 
The same solutions were used as described previously in this Example to 
synthesize the (CT).sub.10 T oligomer on a 15 .mu.mol scale. 5.02 
equivalents of dimer were used in each coupling step. 
Detritylation 
7.5 ml detritylation solution was delivered for 200 sec. 
Wash 
Washing was conducted in two steps: 
a) 0.75 ml of acetonitrile was delivered for 12.5 sec; and 
b) 6.0 ml of acetonitrile was delivered for 100 sec. 
Coupling 
a) 0.6ml acetonitrile as a pre-couple was added for 16 sec; 
b) 0.525 ml activator was delivered for 14 sec; 
c) 0.375 ml amidite was delivered for 10 sec; 
d) 0.375 activator was delivered for 10 sec; 
e) pause for 60 sec; 
f) 0.3 ml activator was delivered for 30 sec; 
g) 0.6 ml acetonitrile wash for 10 sec; 
h) 0.375 ml amidite was delivered for 10 sec; 
i) 0.375 ml activator was delivered for 10 sec; 
j) pause for 60 sec; 
k) 0.3 ml activator was delivered for 30 sec; 
l) coupling was allowed to continue for an additional 900 sec. 
Wash 
Washing was conducted in two steps: 
a) 1.5 ml of acetonitrile was delivered for 40 sec; and 
b) 1.5 ml of acetonitrile was delivered for 25 sec. 
Capping 
a) 1.125 ml Cap A was delivered for 30 sec; 
b) 1.125 ml Cap B was delivered for 30 sec. 
Wash 
a) 0.225 ml acetonitrile wash for 40 sec, followed by 
b) 1.5 ml acetonitrile wash for 25 sec. 
Sulfurization 
a) 1.875 ml of Beaucage solution was added over 50 sec and allowed to react 
for 60 sec. 
Wash 
a) 1.5 ml of acetonitrile wash was added for 25 sec. 
Capping 
a) 0.750 ml Cap A was delivered for 20 sec. 
b) 0.750 ml Cap B was delivered for 20 sec. 
Wash 
a) 5.1 ml acetonitrile was added for 85 sec. 
Example 8 
300 .mu.mole scale synthesis of oligonucleotide phosphorothioates 
Syntheses of (CT).sub.10 T oligomer was conducted on a 300 .mu.mol scale on 
a Pharmacia OligoPilot II Synthesizer. CPG-T was purchased from Glen 
Research (Sterling, Va.). 
Flow-through type column reactor was used for the synthesizer. CPG-T 
support was packed in the column. The amount and the rate at which the 
reagent was delivered to the reactor column depended on the scale of the 
synthesis and the size of the column. 
In one synthesis, 10.5 g CPG-T (29.0 .mu.mol/g) was used in a 46 ml size 
column. Two equivalents of dimer phosphoramidate were used in each 
coupling step for all 300 .mu.mol scale syntheses. The synthesis cycle 
consisted of the following steps: 
Detritylation 
3% dichloroacetic acid in dichloroethane was passed through the solid 
support for 3 minutes at a rate of 75 ml/min. 
Wash 
Washing was conducted in two steps: 
a) Acetonitrile was passed through the column for 6 min at a rate of 75 
ml/min. 
b) Acetonitrile was passed through the column for 1.92 min at a rate of 75 
ml/min. 
Coupling 
The activator solution and the amidite solution were injected in alternate 
fashion. The activator solution was introduced to the reactor for 1 min at 
a rate of 36 ml/min. This process was repeated eight times. The amidite 
solution was introduced for 0.2 min at a rate of 3.8 ml/min. This process 
was also repeated eight times. Again the activator solution was pumped for 
0.1 min at a rate of 36 ml/min. The line to the reactor was washed with 
acetonitrile for 0.1 min at a rate of 4 ml/min. This activity was repeated 
8 times. Combined solution of the activator and the amidite was then 
circulated in the reactor loop for 6 min at a rate of 25 ml/min. 
Wash 
The column was washed with acetonitrile for 2 min at a rate of 25 ml/min. 
Sulfurization 
5% Beaucage reagent in acetonitrile was introduced to the column for 0.6 
min at a rate of 48 ml/min. The line was washed with acetonitrile for 0.1 
min at a rate of 15 ml/min. The Beaucage solution was circulated in the 
loop for 5 min at a rate of 50 ml/min. 
Wash 
The column was washed with acetonitrile for 1 min at a rate of 50 ml/min. 
Capping 
The two capping solutions used comprised: 
Cap A: 20% N-methylimidazole in acetonitrile; and 
Cap B: 20% acetic anhydride, 30% sym-collidine, 50% acetonitrile. 
Cap A and Cap B solutions were pumped into the reactor alternatively. Cap A 
solution was introduced for 0.1 min at a rate of 18 ml/min. This action 
was repeated eight times. Cap B solution was also injected for 0.1 min at 
a rate of 18 ml/min. This process was repeated eight times. 
Wash 
a) The first wash step was done for 4.17 min at a rate of 14.4 ml/min. 
b) This next wash step was performed for 1.28 min at a rate of 75 ml/min. 
Example 9 
300 .mu.mole scale synthesis of oligonucleotide phosphorothioates without 
capping This synthesis of the (CT).sub.10 T oligomer was conducted using 
8.0 g of CPG-T (38.0 .mu.mol/g) in a 24 ml size reactor. The reagents used 
for this synthesis were the same as those described in Example 8, but, as 
seen in the protocol below, a capping step was not employed. Two 
equivalents of dimer were used for each coupling step. The synthesis cycle 
is described below. 
Detritylation 
The detritylation solution was passed through the solid support for 3 min a 
rate of 50 ml/min. 
Wash 
a) The column was washed with acetonitrile. Acetonitrile was passed through 
the column for 6 min at a rate of 50 ml/min. 
b) This wash utilized acetonitrile for 1.44 min at a rate of 50 ml/min. 
Coupling 
The activator solution and the amidite solution were injected in alternate 
fashion. The activator solution was introduced to the reactor for 0.1 min 
at a rate of 24 ml/min. This process was repeated six times. The amidite 
solution was introduced for 0.2 min at a rate of 4.8 ml/min. This process 
was also repeated six times. Again the activator solution was pumped for 
0.1 min at a rate of 24 ml/min. The line to the reactor was washed with 
acetonitrile for 0.1 min at a rate of 4 ml/min. This activity was repeated 
eight times. Combined solution of the activator and the amidite was then 
circulated in the reactor loop for 6 min at a rate of 20 ml/min. 
Wash 
The column was washed with acetonitrile for 1 min at a rate of 20 ml/min. 
Sulfurization 
Beaucage solution was introduced to the column for 0.6 min at a rate of 24 
ml/min. The line was washed with acetonitrile for 0.2 min at a rate of 15 
ml/min. The Beaucage solution was circulated in the loop for 4.6 min at a 
rate of 28.8 ml/min. 
Wash 
The column was washed with acetonitrile for 1 min at a rate of 24 ml/min. 
Capping 
The program for capping was not changed. The Cap A and Cap B bottles were 
filled with acetonitrile instead of the respective reagents. Thus, the 
capping step for this protocol becomes the wash step. 
Wash 
a) This wash step was done for 1.25 min at a rate of 24 ml/min. 
b) This step was performed for 0.95 min at a rate of 50 ml/min. 
The results of three different syntheses conducted without capping and 
before chromatographic purification are presented as experiment numbers 
5-7 in Table 5, infra. 
Example 10 
Purification of (CT).sub.10 T prepared by dimer block synthesis 
Oligonucleotides prepared according to the foregoing examples were 
subjected to chromatography to purify them further. Products taken for 
purification are described in Table 5, lines 3, 4, and 6. These products 
correspond to the three entries for Crude Product in Table 4. As shown, 
products corresponding to the first two entries in Table 4 were purified 
by RPC followed by IEX. Product corresponding to the third entry in Table 
4 was purified using IEX as the sole chromatographic step. Following 
chromatography, desalted product was prepared by 
ultrafiltration/diafiltration and lyophilization, as required. Product of 
&gt;98% purity was obtained, as determined by both analytical IEX-HPLC and 
capillary electrophoresis (CE). .sup.31 P NMR was employed to determine PO 
content. 
The purification procedures described below were conducted using crude 
product prepared at the 15 .mu.mol and 300 .mu.mol scales. Experience 
obtained during work with the 15 .mu.mol scale was applied to the 
subsequent purification at the 300 .mu.mol scale resulting in enhanced 
purity. 
Reversed Phase Chromatography 
As noted, RPC was used for preliminary purification of crude 
oligonucleotide having lower purity, as synthesized. RPC was employed in 
purification of crude products described in the first two entries of Table 
4, but was omitted in purifying crude product described in the third entry 
of this table. Crude product produced at 15.mu.mole scale (with capping) 
is of higher purity than that produced at 300 .mu.mol scale (with 
capping). The higher purity of the product obtained at 15 .mu.mol scale 
probably arises from the greater excess of amidite (7-8 fold) employed 
during synthesis compared with that employed at 300 .mu.mol scale (2-fold 
excess). 
RPC was performed using Amberchrom CG-300sd (TosoHaas), a porous styrenic 
resin. A combination of gradient and isocratic elution was employed. 
Buffer A was 0.10 M aqueous ammonium acetate. Buffer B was 80/20 
v/v/acetonitrile/Buffer A. 
Chromatography at the 15 .mu.mol scale was performed using a 1.0 cm 
ID.times.14.1 cm column. Load and wash steps were conducted at 4.0 ml/min; 
isocratic and gradient elution were conducted at 2.0 ml/min. At the 300 
.mu.mol scale, a 2.5 cm ID.times.20.3 cm column was employed. Two runs 
were made. During the first run, load and wash steps were performed at 
24.5 ml/min; isocratic and gradient elution were conducted at 12.3 ml/min. 
During the second run, the flow rate was 12.3 ml/min throughout. 
Feedstock was prepared by addition of Picopure water to the crude product 
in ammonium hydroxide solution; ammonium acetate was added to provide a 
concentration of 0.2 M. At the 15 .mu.mol scale, feedstock had a 
concentration of approximately 20 A.sub.260 units/ml solution. The loading 
factor was approximately 175 A.sub.260 units/ml bed. At the 300 .mu.mol 
scale, corresponding values were 53 A.sub.260 unit/ml solution and 147 
A.sub.260 units/ml bed. 
Purification was accomplished using the non-optimized sequence of steps 
displayed in Table 2. Capping was employed the synthesis of each of the 
samples shown. 
TABLE 2 
______________________________________ 
# 15 .mu.mol scale 
300 .mu.mol scale (run 1) 
300 .mu.mol scale (run 2) 
______________________________________ 
1 Load Load Load 
2 Wash, 100% (A).sup.1, 
Wash, 100% (A), 
Wash, 100% (A), 
5.4 CV.sup.3 1.1 CV 1.2 CV 
3 Grad:.sup.3 0-21% 
Grad: 0-19% (B) @ 
Grad: 0-19% (B) @ 
(B).sup.2 @ 1%/min, 
1%/min, 1.1 CV 
1%/min, 1.1 CV 
1.1 CV 
4 Isocrat.sup.3 21% (B), 
Isocrat 19% (B), 
Isocrat 19% (B), 
3.4 CV 1.8 CV 1.8 CV 
5 Grad: 21-72% Grad: 19-32% (B) @ 
Grad: 19-31% (B) @ 
(B) @ 1%/min, 
1%/min, 1.5 CV 
1%/min, 1.5 CV 
9.2 CV 
6 Wash, 100% (B), 
Isocrat 32% (B) 
Isocrat 31% (B) @ 
7.9 CV 1.1 CV 1%/min, 1.2 CV 
7 -- Grad: 32-55% (B) @ 
Grad: 31-55% (B) @ 
1%/min, 2.5 CV 
1%/min, 2.5 CV 
8 -- Wash, 100% (B), 
Wash, 100% (B), 
Approx. 4 CV.sup.4 
Approx. 4 CV.sup.4 
______________________________________ 
.sup.1 "A" refers to Buffer A (0.1 M ammonium acetate) 
.sup.2 "B" refers to Buffer B (80/20 v/v acetonitrile/Buffer A) 
.sup.3 "CV" is column volume, "Grad" is gradient elution, and "isocrat" i 
isocratic elution. 
.sup.4 The initial 1.5 CV was collected as a chromatographic fraction, th 
remainder was diverted to waste. 
Pooled chromatographic fractions were drawn from the fourth and fifth steps 
in the 15 .mu.mol scale purification and from the sixth step in the 300 
.mu.mol scale purifications. 
Detritylation 
Crude product corresponding to the third entry in Table 4 was subjected to 
IEX purification without preliminary RPC purification. First, however, 
crude product in ammonium hydroxide solution was processed on a rotary 
evaporator to remove ammonia prior to detritylation. To the remaining 
solution, a quantity of pure water was added, as required, to yield a 
solution having A.sub.260 .ltoreq.50 OD/ml. Finally, a quantity of glacial 
acetic acid was added to provide a final concentration of 20% V/V glacial 
acetic acid. The resulting solution was stirred at room temperature for 
2.5 hr. 
Post-RPC pools were detritylated in a corresponding procedure, but 
excluding processing on a rotary evaporator. After addition of glacial 
acetic acid, the reaction period was 2.25 hr at the 15 .mu.mol scale and 
2.75 hr at the 300 .mu.mol scale. 
After these procedures, the oligonucleotide products were subject to IEX. 
Ion Exchange chromatography 
IEX was performed using TSK-GEL DEAE-5PW (TosoHaas), a DEAE-substituted 
methacrylic polymer. A combination of gradient and isocratic elution was 
employed. Buffer A was 25 mM TrisCl, pH 7.2. Buffer B was 25 mM TrisCl 
containing 2.0 M sodium chloride, pH 7.2. 
The detritylated oligonucleotides previously subjected to RPC were purified 
as follows: 
At the 15 .mu.mol scale, a column 0.66 cm ID.times.13. 3 cm was employed. 
Steps 1, 2, and 7 (Table 3), were conducted at 1.29 ml/min, while steps 
3-6 were conducted at 0.86 ml/min. The loading factor was 212 A.sub.260 
units/mi bed. 
At the 300 .mu.mol scale, a column 2.2 cm ID.times.19.0 cm was employed. 
Steps 1, 2, and 7 (Table 3) were conducted at 14.3 ml/min, while steps 3-6 
were conducted at 9.5 ml/min. The loading factor was 217 A.sub.260 
units/ml bed. 
Crude detritylated oligonucleotide that had not been subjected to RPC was 
purified using a column 1.1 cm ID.times.8.3 cm. Steps 1 and 2 were 
conducted at 3.6 ml/min., while steps 3-10 were conducted at 2.4 ml/min 
(Table 3). The loading factor was 249 A.sub.260 units/ml bed. 
The non-optimized elution steps listed in Table 3 were employed: 
TABLE 3 
______________________________________ 
15 .mu.mol scale.sup.3 
300 .mu.mol scale.sup.3 
300 .mu.mol scale.sup.4 
(capping) (capping) (no capping) 
______________________________________ 
1 LOAD LOAD LOAD 
2 Wash, 100% (A).sup.1, 
Wash, 100% (A), 
Wash, 100% (A), 
7.7 CV.sup.5 6.6 CV 8.5 CV 
3 Grad..sup.5, 10-40% 
Grad., 20-30% (B) @ 
Isocrat..sup.5, 22% (B), 
(B).sup.2 @ 0.5%/min., 
0.5%/min., 4.0 CV 
3.2 CV 
11.2 CV 
4 Isocrat. 40% (B), 
Isocrat. 35% (B), 
Isocrat. 30% (B), 
3.7 CV 3.6 CV 10.5 CV 
5 Grad., 40-60% 
Grad., 35-52% (B) @ 
Isocrat., 36% (B), 
(B) @ 0.5%/min., 
0.5%/min., 4.5 CV 
5.1 CV 
7.4 CV 
6 Isocrat., 75% (B), 
Grad., 52-75% (B) @ 
Isocrat. 46% (B), 
2.2 CV 1%/min., 3.0 CV 
5.9 CV 
7 Isocrat., 100% (B), 
Isocrat., 75% (B), 
Isocrat., 52% (B), 
5.0 CV 6.1 CV 7.5 CV 
8 -- Isocrat., 100% (B), 
Isocrat., 58% (B), 
4.0 CV 6.6 CV 
9 -- -- Isocrat., 75% (B), 
3.2 CV 
10 -- -- Isocrat., 100% (B), 
5.1 CV 
______________________________________ 
.sup.1 "A" refers to Buffer A (0.3 NaOH) 
.sup.2 "B" refers to Buffer B (0.3 NaOH + 2.0 NaCl) 
.sup.3 detritylated, postRPC pool 
.sup.4 detritylated crude product 
.sup.5 CV = column volume, "Grad" is gradient elution, and "Isocrat" is 
isocratic elution 
Pooled fractions were drawn from steps 4-7 in the 15 .mu.mol scale 
synthesis, steps 6-7 (pool 1) and 5-7 (pool 2) in the 300 .mu.mol scale 
synthesis with capping, and steps 5-8 (pool 1) and 6-9 (pool 2) in the 300 
.mu.mol scale synthesis without capping. 
In order to achieve purities.gtoreq.98% as measured by both IEX-HPLC and 
CE, it was necessary to make rigorous use of CE analysis during pooling 
decisions. Trial pools were prepared from chromatographic fractions so as 
to provide 99% and/or 98% purity as determined by IEX-HPLC. Various 
subsets of these trial pools were then prepared using a more restricted 
range of chromatographic fractions. These subsets were then analyzed by CE 
to assure that they met 98% or 99% purity goals, as required. This 
approach was successfully employed during purification of crude products 
prepared at 300.mu.mole scale. Pools of 98% and 99% purity were isolated, 
as determined by both IEX-HPLC and CE, and (n+x) values were reduced to 
0.3% or less. This rigorous use of CE analysis was not employed during 
purification of product prepared at 15 .mu.mole scale. Here, CE purity of 
the product was 97%, and (n+x) content was 2.4%. 
Desalting and Lyophilization 
As required, desalting was performed by diafiltration using an Amicon 
stirred cell fitted with a 1000 MWCO membrane. Conductivity of the final 
diafiltrate was reduced to &lt;60 .mu.mho. Lyophilization of the desalted 
solution followed. 
The results presented in Table 4 and 5 below are based, in part, on 
definitions and formulae set forth in the "Detailed Description of the 
Invention" section, particularly the section "Ultrapure Oligonucleotides." 
Selected definitions and formulae are presented or restated in footnotes 
to these tables for clarity. Additional definitions, formulae, and 
calculation procedures are presented in the text immediately following 
Table 4. 
TABLE 4 
__________________________________________________________________________ 
% Recovery 
Purity After 
% Purity by CE Chromatog. 
Scale 
% DMT- 
% DMT- 
% Purity .SIGMA. N - x 
% Step (by 
(.mu.mol) 
on RP.sup.11 
on IEX.sup.3 
IEX.sup.4 
N N - 1 
N - 2 
(X &gt; 2) 
.SIGMA. N + x 
PO.sup.12 
IEX) 
__________________________________________________________________________ 
Crude Product 
15.sup.1 
82 80 65.sup.9 
74 ND 4.6 
18.8 
3.0 -- 
300.sup.1 
83 75 65.sup.9 
65 0.8 
2.4 
25.3 
7.0 -- 
300.sup.2 
83 88 81.sup.9 
80 0.7 
3.9 
12.0 
3.4 -- 
Purification by RPC (on Amberchrom CG-300sd) 
15.sup.1 
-- 93 73.sup.9 
-- -- -- -- -- -- 70.sup.7 
300.sup.1 : 
Run 1 
-- see pool 
see pool 
-- -- -- -- -- -- 59.sup.7 
Run 2 
-- see pool 
see pool 
-- -- -- -- -- -- 63.sup.7 
Pool 
-- 86 76.sup.9 
-- -- -- -- -- -- 61.sup.7 
Runs 
1 + 2 
300.sup.2 
N/A.sup.5 
N/A N/A N/A 
N/A 
N/A 
N/A N/A N/A 
N/A 
Detritylation (Analysis of reaction mixture) 
15.sup.1 
-- -- 71.sup.10 
-- -- -- -- -- -- 
300.sup.1 
-- -- 77.sup.10 
87 -- -- -- -- -- 
300.sup.2 
-- -- 73.sup.10 
-- -- -- -- -- -- 
Purification by IEX (on DEAE-5PW) 
15.sup.1 
-- -- 98.sup.10 
97 ND.sup.6 
0.8 
ND 2.4 1.0 
88.sup.8 
300.sup.1 : 
Pool 1 
-- -- 98.sup.10 
98 ND 0.5 
1.5 0.1 0.2 
76.sup.8 
Pool 2 
-- -- 99.sup.10 
99 ND 0.4 
0.5 0.2 0.4 
54.sup.8 
300.sup.2 : 
Pool 1 
-- -- 98.sup.10 
99 ND 1.2 
0.2 ND 0.6 
69.sup.8 
Pool 2 
-- -- 99.sup.10 
99 ND 0.7 
0.2 0.3 0.0 
47.sup.8 
__________________________________________________________________________ 
.sup.1 with capping 
.sup.2 without capping 
.sup.3 [.alpha..sub.N + .alpha..sub.PO/N-x' ].sub.IEX,DMTon 
.sup.4 [.alpha..sub.N ].sub.IEX 
.sup.5 N/A = not applicable 
.sup.6 ND = not detected 
.sup.7 % recovery of DMTon form. Calculated as described in text below. 
.sup.8 % recovery of product expressed as IEX units. Calculated as 
described in text below. 
.sup.9 Analysis of DMTon form 
.sup.10 Analysis of DMToff form 
.sup.11 [.alpha..sub.N ].sub.RP 
.sup.12 Estimated % PO 
Chromatographic recoveries presented in Table 5 are expressed in two forms. 
Recovery after RPC purification is expressed with respect to the DMT-on 
form as percent RPC units recovered. Recovery after IEX purification is 
expressed as percent IEX units of product (DMT-off) recovered. RPC units 
and IEX units present in a quantity of the stock (i.e., load) or in 
individual or pooled fractions may be calculated as follows: 
(1) calculate the number of A.sub.260 units in a given volume of solution: 
(A.sub.260 units)=(ml of solution) (A.sub.260 determined using 1 cm path 
cuvette) 
(2) determine the percent purity by analytical IEX or analytical RPC: 
(3) calculate RPC units in a given volume of solution: 
EQU (RPC units)=[(A 260 units) (% DMT-on purities by RPC)]/100 
(4) calculate IEX units in a given volume of solution: 
EQU (IEX units)=[(A.sub.260 units)(% purity by IEX)]/100 
Using the above values, chromatographic recoveries are calculated: 
(1) calculate percent recovery of DMT-on form: 
##EQU3## 
(2) calculate percent recovery of product as IEX units: 
##EQU4## 
The overall chromatographic yield for the two step purification (defined as 
"(% recovery DMT-on by RPC).times.(% recovery by IEX)) was 62% at the 15 
.mu.mol scale and 46% and 29% for pools 1 and 2, respectively, at the 300 
.mu.mol scale (with capping). The overall chromatographic yield for the 
single-step purification is identical to % recovery by IEX and was thus 
69% and 47% for pools 1 and 2, respectively, obtained after purification 
at the 300 .mu.mol scale (no capping). 
Values for estimated %PO in Table 4 were calculated as set forth in the 
"Detailed Description of the Invention" section, under the heading 
"Ultrapure Oligonucleotides." Purified product described in the final 
entry of Table 4 (Pool2, 300 .mu.mol scale, without capping) was also 
analyzed by .sup.31 P NMR to determine PO content. In this analysis no PO 
was detected. 
Additional 21-mer oligonucleotides were synthesized according to the 
foregoing protocols. The results are presented in Table 5. The 
oligonucleotide was obtained in the range of 65-81% as detected by 
capillary electrophoresis (see Table 5). Interestingly, in experiment 2, 
when 75% of the 21-mer was observed, the N-1 content was undetectably 
small. The average stepwise coupling yield in experiment 2 was 97.2%. 
Ion-exchange chromatography estimates of the phosphodiester content per 
linkage in the phosphorothioate 21-mer was generally below 1.0%. 
The average stepwise coupling yield for the 15.0 .mu.mol scale synthesis 
was 97.0%, whereas for the 300 .mu.mol scale synthesis it was 97.2%. 
TABLE 5 
__________________________________________________________________________ 
Scale % IEX 
% CE 
# (.mu.mol) 
ODs % RP 
DMT-on 
N N - 1 
N - 2 
N + x 
% PO (IEX) 
__________________________________________________________________________ 
1* 
0.2 24 
84 86 65 10.6 
3.11 
5.7 
0.7 
2 0.2 23 
87 81 75 ND 5.4 4.9 
1.2 
3 15.0 
1700 
82 80 74 ND 4.6 3.0 
0.9 
4 300 30000 
83 75 65 0.8 
2.4 7.0 
0.7 
5 289 30894 
79 72 75 1.1 
2.3 7.4 
0.4 
6 304 33360 
83 87 80 0.7 
3.9 3.4 
0.4 
7 304 33583 
87 87 81 0.6 
3.1 3.9 
0.4 
__________________________________________________________________________ 
*monomer synthesis 
ND = not detectable