CA2200552A1 - Method for loading solid supports for nucleic acid synthesis - Google Patents
Method for loading solid supports for nucleic acid synthesisInfo
- Publication number
- CA2200552A1 CA2200552A1 CA002200552A CA2200552A CA2200552A1 CA 2200552 A1 CA2200552 A1 CA 2200552A1 CA 002200552 A CA002200552 A CA 002200552A CA 2200552 A CA2200552 A CA 2200552A CA 2200552 A1 CA2200552 A1 CA 2200552A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleoside
- loading
- cpg
- diisopropylcarbodiimide
- solid support
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Abstract
New methods of loading solid supports for oligonucleotide synthesis are presented. The new methods comprise coupling a succinylated nucleoside to a solid support using diisopropylcarbodiimide (DIC) as an activator and N-hydroxybenzotriazole (HOBT) as an acid catalyst. DIC is substantially cheaper than the currently used activating agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC). Furthermore, in the preferred embodiment, when it is used in combination with HOBT, coupling is more efficient, requiring less nucleoside to achieve the same loading densities as in the prior art. The loading process is faster than prior art methods and the overall cost savings of about 43 % are realized.
Description
' 2 ~ ~ ~ 5 ~ 2 METHOD FOR LOADING SOLID SUPPORTS FOR
NIJCLEIC ACID SYNTHESIS
BACKGROUND OF THE INVENTION
Field of the Invention ~ This invention relates to the field of oligonucleotide synthP!ci~, and, more particularly, to methods of loading mononucleosides on a solid support.
S Description of the Related Art Since 7~mernik and SL~l-he~ on, Proc. Natl. Acad. Sci. USA 75, 280-284 (1978) first d~m~ l.dLe;d virus replic~tion inhibition by synthetic oli~ol llcleotides, great interest has been ge~ dlcd in oligonucleotides as thc.d~GuLic agents. In recent years the development of oligonucleotides as the.dl)cuLic agents and as agents of gene GA~ sion modulation has gained great mo.. G.. I.~.~. The ~alG~L
development has been in the use of so-called ~nti~n~e oligonucleotides, which form Watson-Crick d~ple~s with target mRNAs. Agrawal, Trends in Biotechnology 10, 152-158 (1992), e~lc-l~ivcly reviews the development of ~nti~n~e oligonucleotides as antiviral agents. See also Uhlm~nn and Peymann, Chem. Rev. 90, 543 (1990).
Various methods have been developed for the synthesis of oli~o~ llcleotides for such ~oses. See generally, Methods in Molecular Biolof~, Vol. 20:
Protocols for Oligonucleotides and Analogs (S. Agrawal, Ed., ~llm~n~ Press, i993), Oligonucleotides and Analogues: A Pracfical Approach ~. Eckstein, Ed., . 20 1991); Uhlm~nn and Peyman, supra Early synthetic approaches included phosFhndiester and phosphotriester ~h~mi~tries. Khorana et al., J. Molec. Biol. 72, 209 (1972) discloses phosphodiester ~hPmi~Ty for oligonucleotide synth~
WO 96/09314 . ~ PCT/l~S9S/12196 Reese, Tetrahedron Lett. 34, 3143-3179 (1978), discloses phosphotriester ~h~mi~try for synthesis of oligonucleotides and polynucleotides. These early a~.oaches have largely given way to the more efficient phosIhoramidite and H-phosrhcl~te approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859- 1862 (1981), discloses the use of deoxynucleoside phosphor~mi-lites in polynucleotidesynlhesis. Agrawal and 7.~m~rnik, U.S. Patent No. 5,149,798 (1992), discloses oplhlli~d synthesis of oligomlcleotides by the H-ph- sphnn~te al~ploacll.
Both of these modern approaches have been used to Syl~ ii7J~
oligonucleotides having a variety of mor1ified inttormlcleotide link~ges. Agrawal and Goodchild, Tetrahedron Lett. 28, 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23, 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using rhosphoramidite çhPmi~y. Jager et al., Biochemistry 27, 7237 (1988), discloses synthesis of oligonucleotide phosrh-r~mi-l~tes using phosphoramidite ch~mi~try. Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988), discloses syllllle~is of oligonucleotide phosphor~mi~l~tes and phosphorothio~tes using H-phosphon~te eh~mi~try.
Solid phase synthesis of oligonucleotides by the r~ l~gOi~lg methods involves the same generalized protocol. Briefly, this approach compn~es anchoring the 3'-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion.Desired int~rmlcleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the S'-most nucleoside of the n~cçnt~ support-bound oligonucleotide.
NIJCLEIC ACID SYNTHESIS
BACKGROUND OF THE INVENTION
Field of the Invention ~ This invention relates to the field of oligonucleotide synthP!ci~, and, more particularly, to methods of loading mononucleosides on a solid support.
S Description of the Related Art Since 7~mernik and SL~l-he~ on, Proc. Natl. Acad. Sci. USA 75, 280-284 (1978) first d~m~ l.dLe;d virus replic~tion inhibition by synthetic oli~ol llcleotides, great interest has been ge~ dlcd in oligonucleotides as thc.d~GuLic agents. In recent years the development of oligonucleotides as the.dl)cuLic agents and as agents of gene GA~ sion modulation has gained great mo.. G.. I.~.~. The ~alG~L
development has been in the use of so-called ~nti~n~e oligonucleotides, which form Watson-Crick d~ple~s with target mRNAs. Agrawal, Trends in Biotechnology 10, 152-158 (1992), e~lc-l~ivcly reviews the development of ~nti~n~e oligonucleotides as antiviral agents. See also Uhlm~nn and Peymann, Chem. Rev. 90, 543 (1990).
Various methods have been developed for the synthesis of oli~o~ llcleotides for such ~oses. See generally, Methods in Molecular Biolof~, Vol. 20:
Protocols for Oligonucleotides and Analogs (S. Agrawal, Ed., ~llm~n~ Press, i993), Oligonucleotides and Analogues: A Pracfical Approach ~. Eckstein, Ed., . 20 1991); Uhlm~nn and Peyman, supra Early synthetic approaches included phosFhndiester and phosphotriester ~h~mi~tries. Khorana et al., J. Molec. Biol. 72, 209 (1972) discloses phosphodiester ~hPmi~Ty for oligonucleotide synth~
WO 96/09314 . ~ PCT/l~S9S/12196 Reese, Tetrahedron Lett. 34, 3143-3179 (1978), discloses phosphotriester ~h~mi~try for synthesis of oligonucleotides and polynucleotides. These early a~.oaches have largely given way to the more efficient phosIhoramidite and H-phosrhcl~te approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859- 1862 (1981), discloses the use of deoxynucleoside phosphor~mi-lites in polynucleotidesynlhesis. Agrawal and 7.~m~rnik, U.S. Patent No. 5,149,798 (1992), discloses oplhlli~d synthesis of oligomlcleotides by the H-ph- sphnn~te al~ploacll.
Both of these modern approaches have been used to Syl~ ii7J~
oligonucleotides having a variety of mor1ified inttormlcleotide link~ges. Agrawal and Goodchild, Tetrahedron Lett. 28, 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23, 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using rhosphoramidite çhPmi~y. Jager et al., Biochemistry 27, 7237 (1988), discloses synthesis of oligonucleotide phosrh-r~mi-l~tes using phosphoramidite ch~mi~try. Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988), discloses syllllle~is of oligonucleotide phosphor~mi~l~tes and phosphorothio~tes using H-phosphon~te eh~mi~try.
Solid phase synthesis of oligonucleotides by the r~ l~gOi~lg methods involves the same generalized protocol. Briefly, this approach compn~es anchoring the 3'-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion.Desired int~rmlcleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the S'-most nucleoside of the n~cçnt~ support-bound oligonucleotide.
WO 96/09314 PCT/US9~i/12196 Oligonucleotide synthesis generally begins with coupling, or "loading," of the 3'-most nucleoside of the desired oligonucleotide to a filnrtion~li7p~l solid phase support. A variety of solid ~u~oll~ and methods for their plcl)alaLion areknown in the art. E.g., Pon, "Solid-Phase SU~O1L ~ for Oligonucleotide Synth~ci~,"
in Methods in Molec. Biol., Vol. 20,: Protocols for Oligonucleotides and Analogs, p. 465 (Agrawal, Ed., Humana Press, 1993). Generally, the functionalized support has a plurality of long chain alkyl amines (LCAA) on the surface that serve as sites for nucleoside coupling. Controlled pore glass (CPG)is the most widely used support. It consists of appro~ tely 100-200 ~m beads with pores rsm~ing from a few hundred to a few thousand angstroms.
CPG su~olL~ are generally loaded by ~tt~hin~ a nucleoside-3'-succinate to the support through the succinyl group via an amide bond. Early efforts used dicyclohexylcarbo--1iimi~1e (DCC) to activate the nucleoside-3'-sl.rcin~te Activation was accomplished by converting the nucleoside-3'-succinate into the symmetrical anhydride (Sproat and Gait, in Oligonucleotide Synthesis: a Practical Approach p. 83-115 (Gait, Ed., IRL Press, 1984)), or e~ ;ryillg with p-nitrophenol (Atkinson and Smith, id. at 35-81; Koster et al., Tetrahedron 40, 103-112 (1984)) or pent~hlorophenol (Gough et al, Tetrahedron Lett. 22, 4177-4180 (1981); Kl~r7~l~ Biochem. 25, 7840-7846 (1986)).
~(--~L~ L et al., Nucleosides & Nucleotides 12, 967 (1993) reported that DCC and l-hydoxybenzotriazole (HOBT) (used in equimolar amounts in a 14:1 dichloromethane/dim~Lllylrol...~ e solvent) resulted ir higher loading ~1~n~iti~os than when DCC was used alone.
WO 96/09314 ~ ~ ~ ~ 5 5 2 PCTiUS95/12196 The use of DCC suffers from a number of disadvantages, however. First, DCC is highly toxic. Second, loading was tedious and gave only moderate yields (50-75%). Third, the coupling reactions were lengthy, requiring 3-4 d to make the activated ~uccill~Lles and an additional 4-7 d to couple them to the CPG. And S finally, loading values were quite variable, and c~lilll~ll loading of 30-40 ,umol/g was not always obtained.
Pon et al., Biotechniques 6, 768-775 (1988), improved on this method by employing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC). Using this reagent along with a catalytic amount of dimethylaminopyridine (DMAP) in triethylarnine/pyridine, Pon et al. observed direct coupling of the nucleoside-3'-succinate to the support. DEC, a smaller, less rigid carbo~1iimicle (colllpa,~,d to DCC) was found to give much better results -- loadings of up to 50 - 60 ~lmol/g could be obtained in 24 hours.
In an ~ . "s.l; ~e procedure, Damha et al., Nucl. Acids Res. 18, 3813-3821 (1990), showed that loading could be accolll~lished by succillylating the LCAA of the solid support, Ihe~eby providing a carboxylic acid functional group, followed by direct ~ nt of a nucleoside by esterification with DEC and DMAP in pyridine.
Tong et al., J. Org. Chem 58, 2223 (1993), followed the approach of Dalnha et al., supra, and colllpaled the efficiency of DCC, DEC, and DIC in loading 3' ullprol~cl~d cytidine onto a succinylated CPG support under basic conditions. The reaction was con~luctecl in the presence of DMAP in dry pyridine.
Los3-1in~s of 22 (DCC), 18 (DEC), and 30 (DIC) ~Lmol/g were obtained.
wo 96109314 PCT/US951121g6 A major drawback of the current methods for loading solid support is that the main solvent is pyridine. Pyridine is toxic, has an obnoxious odor, and, - thcl~ r~ , is a work place and enviro~ nt~l hazard. In addition, DEC is costly, particularly when used in a large, production scale synthçses. Consequently, improved methods of column loading for oligonucleotide ~yl~lhesis are desirable.
SUMMARY OF THE INVENTION
The present invention cc~ es new and irnproved methods of loading mlrleQsirles onto a solid support for solid phase oligonucleotide synthesis. Them~tho~l~ of the present invention provide several advantages over prior art methods. First, they are more cost effici~nt Cheaper, more efficient catalysts and a~;liv,llo,~ used in the present invention result in cost savings as lesser amounts of both catalyst and mononucleoside rca~;L~ are required. Savings of approxim~tçly 43% have been obs~ t;d for loading ~l~on~itiçs of about 70-80 ,umol/g. Second, we are able to çl;.~ e pyridine as a solvent, which not only effects cost savings, but ilnl)l'Ov~S the safety of the process, both to the worker and to the c llvilvllllLc~ll.
Conco...;~ y, fewer hazardous wastes are produced.
In a first aspect of the present invention, diis~-u~ylcarbo-liimide (DIC) is used as an activator in the acid catalyzed loading of succi,lylated monomlr.1eoside.
It has been un~l e~itedly found that the use of DIC in acid cataly~d loading is more erre.i~ive than standard techniques using DEC, DIC is also more effective than DEC in acid catalyzed loading. DIC offers the further advantage of being cheaper per unit mass. DEC ~lcse,lLly costs about $308/100 g while DIC costs about $97/100 g. In ~ ihon, DIC can be used in an amount that is about 20%
that of DEC.
Wo 96/09314 ~ 5 5 ~ PCT/US95112196 In a second aspect ofthe present invention, 1-hydo~ybc;l.~ull;~ole (HOBT) is used in comhin~tion with DIC to catalyze linkage of a mononucleoside to an activated solid support. HOBT acts more efficiently and economically than other compounds such as N-hydro~y~lcci~ (NHS), paratoluenesulfonic acid S (pTSA), and trifluoroacteic acid (TFA). Loading d~n~ities appro~hing 120 ~moVg on controlled pore glass (CPG) solid support are readily obtained. Higher loading ~en~itie~ are also observed on other solid :iU~JOll:i such as the "TE,NTAGEL" (Rapp Polymere) and "HLP" (ABI). Nitrobel.~uLIiazole (NBT) acts as ~fflci~ntly as HOBT, but is somewhat more ~x~c.lsi~re.
In a the third aspect of the present invention, a method is p lese,lled for lo~ling a non-linker-attached nucleoside (i.e., a nucleoside having a free 3' hydroxyl group) onto a solid support bearing a linker groug, e.g., a succinyl moiety. In this aspect of the invention, a solid support to which a linker has been ~tt~hP~l is loaded by cont~ting it with a nucleoside having a free 3' hydroxyl group in the presence of DIC and HOBT.
In the fourth aspect of the present invention pyridine is eli....~ as a solvent. Pyridine has generally been used in the loading process as a solvent and to dlissolve and wash a~vay all catalyst, unreacted starting materials, and reaction by-products. Because of pyridine's toxicity, its elimin~tion from the lo~q-lin~
process increases the safety of the process. In addition, fewer environm~nt~lly hazardous wastes are produced. We have found that ~r~lol.;l.ile can be used in place of pyridine without affecting loading effici~nciçs Cost savings are also thereby rç~li7~1 ~ wo 96/09314 2 ~ O 0 5 5 2 PCT/US95112196 Another benefit provided by the methods of the present invention is the ability to adjust loading den~iti~s to any desired level, up to the m~x;.~.l..., possible Pmpiric~l value obtainable under optimal con-litio~. This is useful because eql-irm~nt limit~tin~.c (and/or other factors) may restrict the degree of loading to S values ~-lb~ 11y less than the emririç~l m~x;.. ---.-.
The present methods can be used to load any nucleoside onto any functionalized solid ~u~olL. The mlcleo~ may comprise any unmodified base (e.g., A, T, C, G, or U) or m~ -lifi~ocl base, and a modified or unmodified ribose moiety. Any solid support suitable for use in oligonucleotide ~yllLlle;,is can be used with the present methods.
The foregoing merely sllmm~ri7~s certain aspects of the present invention and is not in~t.n~e~l, nor should it be co~ d, to limit the invention in any manner.
All patents and publications cited in this specification are hereby incorporated by l~;r~rellce in their elllil~ly.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention colnpri~es new mt~tho-le for loading mon- n~lcleosides on a solid ~u~olL. As used herein "loading" refers to the chPmic~l linkage of a nucleoside (which will be the 3'-most nl-cleosi~le of the oligonucleotide to be syn~h~si7P~l) to a fimctio~l group on a solid support. The degree of lo~rling ise A~i~s~ed in ~mol monomer/g solid support. A "f mrtic)n~l group" is a chrmir~l moiety, such as an amino or hY~ AY1 moiety, capable of being joined to a mlcleosil1e either dire~;Lly or via a linker. A "functionalized support" is a su~oll having such functional groups.
WO 96/09314 2 ~2 ~ 0 5 5 ~ PCT/US9S/12196 Current methods of loading solid supports are slow, often t~lcing up to about one week to load a controlled pore glass (CPG) support, and require about 90-100 g (or about 0.15 mol) of DMT-protected succinylated monomer to load 500 g o~F CPG. Typical ~ 1X;I~ ll loading ~lPn~ities are about 75 ~lmol/g. While loadling in a 24 hour period has been reported, the loading densities are typically low. The methods plesenlt;d herein s lhst~nti~lly improve on these values. We have found that we are able to obtain loading ~lPn~itiçs in the range of 70 to 80 ~Lmol/g using one third of the amount of DMT-plo~;led succinylated monomer and that by increasing the amount of DMT-plole~ d succinylated mon-mPr, a iliv~lol, and catalyst, loading ~l~n~ities of about 120 llmol/g are easily obtained on CPG. We have observed higher loading ~l~n~ities on other supports having a greater conce,lll~lion of ~ln-,tion~l groups. In ~ litic)n to achieving higher loading densities, the new methods are faster as well, allowing complete loading of the column (with high loading density) in just two days. Thus, use of the methods disclosed herein will sllbst~nti~lly reduce costs and time.
The new mPtho~ lose~l herein are also ~i~ifil ~ntly less hazardous for the worker and safer for the e,l~L..i~.n~ Pyridine is ~;ullc;lllly used as a reaction solvent and, alone or in comhin~tion with other solvents, to wash away unreacted fe~ catalyst, and u~lw~l~d reaction by-products. We have found that the amount of pyridine can be reduced to about 5% in ac~lvl il.ile or el;,.. i~
completely. Because pyridine is toxic and has an obnoxious odor, the present methods are less hazardous than the prior art methods.
The present methods also require lesser amounts of re~gPnt~ rçslllting in lesser costs and amounts of waste. We have found that column loading densities wo 96/09314 ~2 2 0 0 5 5 2 PCT/US95/12196 in the range of about 70-80 ,umol/g can be ~tt~inP,l using less than about 3 x 10-3 mol of nucleoside per 25 g of CPG. Thus, the current methods provide additional cost savings by these means as well. Overall, savings of about 43% are realized with the new methods for loading (1en~itiPS in the range of about 70-80 ,umol/g.S The present invention provides meth~ of loading a filnrtion~li7P-l solid su~o,lforoligonucleosidesynthesiscomrri~in~cont~r~tin~thefllnrti~n~li7e~l solid support with a solvent, diis~,u~ylcarbo~1iimide, and a nucleoside having a 3' linker group ~tt~rhed at a pH of less than 7Ø
In the first embodiment of the invention, dii~o~,u~lcarbo-liinniAP (DIC) is used as an a;Liv~lol for the acid catalyzed loading of nucleoside-3'-succinates. The a~;Liv~lul forms an intermefli~t~ with the tPrmin~l carboxylic acid moiety of the nucleoside-3~ ccil .A~ rPn-1erin~ it ~ tible to nucleophilic attack by a support-bound amino fimrtio~l group. It has been ullc~e~ dly found that DIC in the ~lcsence of l-hy~llo~ybel~uli~ole (HOBT) is more crreclivc than DEC at activating the succinyl carboxylic acid moiety. Under ideal conditions, wh~ei the coupling of the nucleoside-3'-succin~te to the solid support is 100% complete, 1 equivalent of DIC is l~ ed to couple 1 equivalent of nucleoside-3'-~uccil~al~
to the support. DIC is moisture sensilive, however, and the.erore, in general, more than 1 equivalent is ~luil.,d. This will not detract appreciably from the cost benefit of using DIC since it is s~lbst~nti~lly cheaper than DEC. We have found that about 2.5 to 3.0 eq of DIC per 1 eq of mo~oml~leosicle s~lccin~ts results in llent lo~ling~ luulinely yielding loading cl~.n~ities of about 75 to about 85 ~mol/g.
In the second embodiment of the invention, HOBT is used to catalyze DIC
activated lo~-ling. Expt~rim~nt~ using pyridine as a solvent (pLeselll~d infia) Wo 96/09314 ~ 2 0 6~ 5 5 2 PCTiUS95/12196 0 .l~mcn.etrate that HOBT is a more effective than other compounds such as N-hydloxysuccinimi~le (NHS), paratoluenesulfonic acid (pTSA), and trifluoroacetic acidl (TFA). Our ~ e~ ents demon~ le that the use of HOBT results in loading d~n~itiP~e r~ngin~ from 30 to 100% greater than those ~ inecl using NHS, pTSA, and TFA. The amount of HOBT is not critical; it should be sufficient to catalyzethe reaction. We have found that about 0.08 to about 0.16 g HOBT per ml DIC
work ç~cee~lin~ly well, although lesser or greater amounts are likely to work just as well. Most preferably, a lesser a-m--ount of HOBT is used, generally about 0.08 g-In ~-olll~. aspect of this embo-lim~nt, nitro-HOBT is used as a catalyst.
Our e~.,lln,ents show that catalysis with nitro-HOBT results in e~esçnti~lly equ;valent loading deneities as col~ d to when HOBT is used. Nitro-HOBT is used in the amount as described above for HOBT.
The m~tho-lc of the present invention can also be used to load a non-linker-sltt?rh.ofl nucleoside (i.e., a nucleoside having a free 3' hydroxyl group) onto a column bearing a linker groug, e.g., a ~uccillyl moiety. Accordingly, in a thirdembodiment of the present invention, a solid support to which a linker, most preferably ~lcçinic acid, has been ~tt~chP-l is loaded by c- nt~ctin~ it with a nucleoside having a free 3' hy~yl group in the ~e3ence of DIC and HOBT.
The conditions for this reaction are the same as described herein for the loading of nucleosides bearing a 3' linker onto functi~ n~li7~ ;U~)O~
In a fourth embodiment of the present invention, methods of loading a solid support are ~esc.-led in which the amount of pyridine used as the reaction solvent and wash solvent is sllhst~nti~lly reduced or el;~ e~l We have found that ~ wo 96l09314 2! 2 ~ 0 5 5 2 PCT/lUSg5/12196 pyridine need not be the main solvent in the loading reaction. Any solvent that dissolves the re~rt~nt~ but does not react itself can be used. We have found that both ~ct;lo~ ;le and dichloromethane are suitable solvents. Ac~lo~ ile is the most ~,~r~l..,d solvent. In one aspect of this embo-1imPnt a ~ lu~ of pyridine S with ~c~ ;le and/or dichloromPth~nP is used as the ~hllaly solvent for loading nucleosides. A small amount of pyridine (e.g., about 5%) may be used to ensure a minim~l amount of detritylation, but is not required. Accordingly, in another aspect of this embo-lim~nt no pyridine is used. Because of the reduced costs, in the most prer~l~,d embodiment the solvent is ~cc;lo~ ;le with very little (e.g., 5%
or less) or no pyridine.
The present invention can be used with any function~li7P~cl solid support.
A number of such supports are known in the art. E.g, Pon in Methods in Molec.
Biol., supra. We ~l~m~ l-dl~ below that both "TENTAGEL S" (Rapp Polyrnere, Tiibingen, Ge.~ll~ly)(a support in which polyethyleneglycol spacers are grafted on a gel-type support) and HLP (ABI, Foster City, CA) (a PEG-Poly~Lylelle ~u~oll) can be loaded with the current m~thn-1~ to extremely high tll~n~ities. CPG is the most plc;r~c;d support for DNA synthesis.
While the results ples_l.led below were obtained using succinylated thymidine mrmom~r, those skilled in the art will appreciate that any suitably plvl~cled nucleoside monomer (n~ r~lly occurring or modified) can be used with the present methods. Dimer blocks and other mnltin1lr.leoside synthons can also be loaded according to the methods of the present invention. In addition, although succinic acid is the plere..~,d linker, any suitable linker can be used. Such a linker will preferably have a free carboxyl group that a support-bound amino group can wo 96/09314 2 ~ Q ~ 5 5 2 PCT/US95112196 ~0 attack to form an amide bond, thereby binding the linker and its :Itt~ch~l nucleoside to the support. F~mrles of ~lt~ tive linkers are disclosed by Pon in Met.hods in Molec. Biol., supra.
The following F.~mplec are int~n-lçd for illustrative purposes and are not S intelltle-1, nor should they be consl.ued, as limiting the invention in any way.
EXAMPLES
Example 1 Standard Method of Loading of DMT-dT-Succinic Alcid on Controlled Pore Glass 500 g of CPG (Schott, Ho7h~im, Germany) (particle size- 100-130 ,um;
pore si_e: D50: 41.6nm), 6.1 g of dimethylamino pyridine (Aldrich, Milwaukee, WI), 50 g of triethylamine (Aldrich), and 100 g of ethyl-3-(3-dimethylamino propyl) carbo-liimi-le (DEC, mol. wt. 191.7) (Sigma, St. Louis, MO) were placed in a 5 1 Schott bottle and hand shaken for 20 ~ s. 60 g of DMT-dT-succinic acid (Monomer Sciences, Huntsville, AL) was added and the bottle capped and shaken in an orbital shaker at 160 rpm for 18 hours.
A small analytical sample of the resin w~ withdrawn from the Schott bottle, sllcce~;vely washed with pyridine (3 x 5 ml) (Baxter, Muskegon, MI), m~lh~n~l (3 x 5 ml) (Baxter), and methylene chloride (3 x 5 ml ) (EM Science, Cincinnati, OH) sllcce~;vc;ly, and dried in vacuo.
A~lo~;"~tely 20 mg of dry resin was weighed, 200 ~l perchloric acid/ethanol (6:4) was added, and the reslllting solution diluted to 100 ml withmelhylene çhl~ le The absoll,~ce was measured at 498 nm. The same procedure was repeated on a second analytical sample and the average loading ~ WO 96/09314 2 2 0 0 5 5 2 PCT~US95/l2l96 value calculated using Beer's law with a molar absorption coefficient of 70 l/(mol cm) for DMT. A loading value of 66.7 ~Lmol/g was obtained.
- An ~d~1ition~l 20.0 g of DMT-dT-succinic acid was added to the Schott bottle and the llfi~ shaken for 18 hours at anbient le~ .dlulc;. Another analytical sample was removed and worked-up as described above. The absolb~lce of the sample was measured and a loading value of 66.5 ~lmol/g obtained.
Another 20.0 g of DMT-dT-succinic acid was added to the Schott bottle and the ~ shaken for 18 hours at ambient ten~ dlu,e. A third analytical sample was removed and worked-up as rle~sçribed above. The absoll,~lce of the sample was measured and a loading value of 69.6 ,umol/g obtained.
The ""~ ,e re...~ g in the Schott bottle was filtered and the resin washed with pyridine (3 x 1 1). The dry solid was ll~rell~d to a Schott bottle and Cap A (1.0 1 of acetic anhydride in tetrahydrofuran) (Crll~ch~m, Lillv;~ o~
United Kingdom) and Cap B (1.5 1 of N-methylimidzole, pyridine in tetrahy&oru,~l) (Millipore, Bedford, MA) were added. The llli~ , was shaken for 18 hours at ambient te~ . The solid (CPG-T) was filtered and s~ ce~i~;vely washed with methanol (3 x 1 1) and methylene chloride (3 x 1 1) and dried in vacuo to yield 502.5 g. The resin was subjected to the same procedure described above for each of the small ~mpl~s A loading value of 71.4 ~lmol/g was obtained.
Example 2 New Method of Loading DMT-d~-Succinic Acid on Controlled Pore Glass WO 96109314 2 ~ O 0 5 5 2 PCT/US95/12196 0 250.0 g of CPG (Schott), 0.8 g of hydroxybel~.,LL;azole (mol. wt. 135.13) (Aldrich), and 10 ml of 1,3-diisopr~ylcarbodiimide (DIC, mol. wt. 126.20, - density = 0.806 g/ml) (Aldrich) were mixed with 50 ml of pyridine (Baxter) and 1 1 of acetorlitrile (Baxter) in a 2 1 Schott bottle and shaken for 20 mimltec 15 g of DMT-T-succinic acid (mol. wt. 644) (Monomer Sciences) were added, the bottle stoppered and shaken in an orbital shaker at a rate of 180 rpm for 16 hours.
A small analytical sample of the resin was withdrawn from the Schott bottle and washed with 5% pyridine in acetonitrile (3 x 5 ml), methanol (3 x 5 ml)l, and methylene chloride (3 x 5 ml) s~lccç~ively and dried under a stream of0 in vacuo.
A~r~ lately 20 mg of the dry resin were weighed and 200 ml perchloric acid/ ethanol (6:4) added. The solution was diluted to 70 ml with methylene chloride and the absorbance measured at 498 nm. The entire procedure was repeated and the average value of the absorbencies used to calculate the loading.
The loading w~ calculated as above. An average loading value of 78.0 ~lmol/g was obtained.
The le...~i"i"g sollltioî in the Schott bottle was filtered and washed with 5% pyridine in act;l~l~iLIile (3 x 500 ml). Dry solid was transferred to a Schott bottle and Cap A (500 rnl) and Cap B (750 ml) (Cap A and Cap B were as described above) were added and the l~ L~e shaken for 16 hours at ambient tem~.dlule. The solid (CPG-T) was filtered, washed sllccç~ively with methanol (3 x 500 ml) and then methylene chloride (3 x 500 ml), and dried in vacuo to yield 250 g of CPG-T. The loading value, 78 ,umol/g, was calculated as describedabove.
This entire procedure was repeated, ~ub~ varying amounts of the re~c~t~nt~ and using several catalysts and supports. The results are present in Table - 1.
From the foregoing it will be appreciated that although specific embo~l;,.lel.l~i of the present invention have been described herein for the purposes of illustration, various morlifir~tion may be made without deviating from the spirit or scope of the invention.
WO 96/09314 ~ 5 5 ~ PCT/US95/12196 0 0 ~ 0 3 ~ t ~ ~ , , , , , o ~ _ . . . . . . . .
~ ~ . . . . . .
~.~~.,.~~~~~~~~.~..
~i~ ' ' ~ ' ' ' --~ ' ' ~0 ~ O ~ 0 80 0 0 s ~ ~ s ~ ~ ~ r ~~~ ~ ~ ~ _ _ 0 ~1 C~
O ~ O "~ o Z_ ~ In, , g g ~ g 8 8 ~g" ~
in Methods in Molec. Biol., Vol. 20,: Protocols for Oligonucleotides and Analogs, p. 465 (Agrawal, Ed., Humana Press, 1993). Generally, the functionalized support has a plurality of long chain alkyl amines (LCAA) on the surface that serve as sites for nucleoside coupling. Controlled pore glass (CPG)is the most widely used support. It consists of appro~ tely 100-200 ~m beads with pores rsm~ing from a few hundred to a few thousand angstroms.
CPG su~olL~ are generally loaded by ~tt~hin~ a nucleoside-3'-succinate to the support through the succinyl group via an amide bond. Early efforts used dicyclohexylcarbo--1iimi~1e (DCC) to activate the nucleoside-3'-sl.rcin~te Activation was accomplished by converting the nucleoside-3'-succinate into the symmetrical anhydride (Sproat and Gait, in Oligonucleotide Synthesis: a Practical Approach p. 83-115 (Gait, Ed., IRL Press, 1984)), or e~ ;ryillg with p-nitrophenol (Atkinson and Smith, id. at 35-81; Koster et al., Tetrahedron 40, 103-112 (1984)) or pent~hlorophenol (Gough et al, Tetrahedron Lett. 22, 4177-4180 (1981); Kl~r7~l~ Biochem. 25, 7840-7846 (1986)).
~(--~L~ L et al., Nucleosides & Nucleotides 12, 967 (1993) reported that DCC and l-hydoxybenzotriazole (HOBT) (used in equimolar amounts in a 14:1 dichloromethane/dim~Lllylrol...~ e solvent) resulted ir higher loading ~1~n~iti~os than when DCC was used alone.
WO 96/09314 ~ ~ ~ ~ 5 5 2 PCTiUS95/12196 The use of DCC suffers from a number of disadvantages, however. First, DCC is highly toxic. Second, loading was tedious and gave only moderate yields (50-75%). Third, the coupling reactions were lengthy, requiring 3-4 d to make the activated ~uccill~Lles and an additional 4-7 d to couple them to the CPG. And S finally, loading values were quite variable, and c~lilll~ll loading of 30-40 ,umol/g was not always obtained.
Pon et al., Biotechniques 6, 768-775 (1988), improved on this method by employing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC). Using this reagent along with a catalytic amount of dimethylaminopyridine (DMAP) in triethylarnine/pyridine, Pon et al. observed direct coupling of the nucleoside-3'-succinate to the support. DEC, a smaller, less rigid carbo~1iimicle (colllpa,~,d to DCC) was found to give much better results -- loadings of up to 50 - 60 ~lmol/g could be obtained in 24 hours.
In an ~ . "s.l; ~e procedure, Damha et al., Nucl. Acids Res. 18, 3813-3821 (1990), showed that loading could be accolll~lished by succillylating the LCAA of the solid support, Ihe~eby providing a carboxylic acid functional group, followed by direct ~ nt of a nucleoside by esterification with DEC and DMAP in pyridine.
Tong et al., J. Org. Chem 58, 2223 (1993), followed the approach of Dalnha et al., supra, and colllpaled the efficiency of DCC, DEC, and DIC in loading 3' ullprol~cl~d cytidine onto a succinylated CPG support under basic conditions. The reaction was con~luctecl in the presence of DMAP in dry pyridine.
Los3-1in~s of 22 (DCC), 18 (DEC), and 30 (DIC) ~Lmol/g were obtained.
wo 96109314 PCT/US951121g6 A major drawback of the current methods for loading solid support is that the main solvent is pyridine. Pyridine is toxic, has an obnoxious odor, and, - thcl~ r~ , is a work place and enviro~ nt~l hazard. In addition, DEC is costly, particularly when used in a large, production scale synthçses. Consequently, improved methods of column loading for oligonucleotide ~yl~lhesis are desirable.
SUMMARY OF THE INVENTION
The present invention cc~ es new and irnproved methods of loading mlrleQsirles onto a solid support for solid phase oligonucleotide synthesis. Them~tho~l~ of the present invention provide several advantages over prior art methods. First, they are more cost effici~nt Cheaper, more efficient catalysts and a~;liv,llo,~ used in the present invention result in cost savings as lesser amounts of both catalyst and mononucleoside rca~;L~ are required. Savings of approxim~tçly 43% have been obs~ t;d for loading ~l~on~itiçs of about 70-80 ,umol/g. Second, we are able to çl;.~ e pyridine as a solvent, which not only effects cost savings, but ilnl)l'Ov~S the safety of the process, both to the worker and to the c llvilvllllLc~ll.
Conco...;~ y, fewer hazardous wastes are produced.
In a first aspect of the present invention, diis~-u~ylcarbo-liimide (DIC) is used as an activator in the acid catalyzed loading of succi,lylated monomlr.1eoside.
It has been un~l e~itedly found that the use of DIC in acid cataly~d loading is more erre.i~ive than standard techniques using DEC, DIC is also more effective than DEC in acid catalyzed loading. DIC offers the further advantage of being cheaper per unit mass. DEC ~lcse,lLly costs about $308/100 g while DIC costs about $97/100 g. In ~ ihon, DIC can be used in an amount that is about 20%
that of DEC.
Wo 96/09314 ~ 5 5 ~ PCT/US95112196 In a second aspect ofthe present invention, 1-hydo~ybc;l.~ull;~ole (HOBT) is used in comhin~tion with DIC to catalyze linkage of a mononucleoside to an activated solid support. HOBT acts more efficiently and economically than other compounds such as N-hydro~y~lcci~ (NHS), paratoluenesulfonic acid S (pTSA), and trifluoroacteic acid (TFA). Loading d~n~ities appro~hing 120 ~moVg on controlled pore glass (CPG) solid support are readily obtained. Higher loading ~en~itie~ are also observed on other solid :iU~JOll:i such as the "TE,NTAGEL" (Rapp Polymere) and "HLP" (ABI). Nitrobel.~uLIiazole (NBT) acts as ~fflci~ntly as HOBT, but is somewhat more ~x~c.lsi~re.
In a the third aspect of the present invention, a method is p lese,lled for lo~ling a non-linker-attached nucleoside (i.e., a nucleoside having a free 3' hydroxyl group) onto a solid support bearing a linker groug, e.g., a succinyl moiety. In this aspect of the invention, a solid support to which a linker has been ~tt~hP~l is loaded by cont~ting it with a nucleoside having a free 3' hydroxyl group in the presence of DIC and HOBT.
In the fourth aspect of the present invention pyridine is eli....~ as a solvent. Pyridine has generally been used in the loading process as a solvent and to dlissolve and wash a~vay all catalyst, unreacted starting materials, and reaction by-products. Because of pyridine's toxicity, its elimin~tion from the lo~q-lin~
process increases the safety of the process. In addition, fewer environm~nt~lly hazardous wastes are produced. We have found that ~r~lol.;l.ile can be used in place of pyridine without affecting loading effici~nciçs Cost savings are also thereby rç~li7~1 ~ wo 96/09314 2 ~ O 0 5 5 2 PCT/US95112196 Another benefit provided by the methods of the present invention is the ability to adjust loading den~iti~s to any desired level, up to the m~x;.~.l..., possible Pmpiric~l value obtainable under optimal con-litio~. This is useful because eql-irm~nt limit~tin~.c (and/or other factors) may restrict the degree of loading to S values ~-lb~ 11y less than the emririç~l m~x;.. ---.-.
The present methods can be used to load any nucleoside onto any functionalized solid ~u~olL. The mlcleo~ may comprise any unmodified base (e.g., A, T, C, G, or U) or m~ -lifi~ocl base, and a modified or unmodified ribose moiety. Any solid support suitable for use in oligonucleotide ~yllLlle;,is can be used with the present methods.
The foregoing merely sllmm~ri7~s certain aspects of the present invention and is not in~t.n~e~l, nor should it be co~ d, to limit the invention in any manner.
All patents and publications cited in this specification are hereby incorporated by l~;r~rellce in their elllil~ly.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention colnpri~es new mt~tho-le for loading mon- n~lcleosides on a solid ~u~olL. As used herein "loading" refers to the chPmic~l linkage of a nucleoside (which will be the 3'-most nl-cleosi~le of the oligonucleotide to be syn~h~si7P~l) to a fimctio~l group on a solid support. The degree of lo~rling ise A~i~s~ed in ~mol monomer/g solid support. A "f mrtic)n~l group" is a chrmir~l moiety, such as an amino or hY~ AY1 moiety, capable of being joined to a mlcleosil1e either dire~;Lly or via a linker. A "functionalized support" is a su~oll having such functional groups.
WO 96/09314 2 ~2 ~ 0 5 5 ~ PCT/US9S/12196 Current methods of loading solid supports are slow, often t~lcing up to about one week to load a controlled pore glass (CPG) support, and require about 90-100 g (or about 0.15 mol) of DMT-protected succinylated monomer to load 500 g o~F CPG. Typical ~ 1X;I~ ll loading ~lPn~ities are about 75 ~lmol/g. While loadling in a 24 hour period has been reported, the loading densities are typically low. The methods plesenlt;d herein s lhst~nti~lly improve on these values. We have found that we are able to obtain loading ~lPn~itiçs in the range of 70 to 80 ~Lmol/g using one third of the amount of DMT-plo~;led succinylated monomer and that by increasing the amount of DMT-plole~ d succinylated mon-mPr, a iliv~lol, and catalyst, loading ~l~n~ities of about 120 llmol/g are easily obtained on CPG. We have observed higher loading ~l~n~ities on other supports having a greater conce,lll~lion of ~ln-,tion~l groups. In ~ litic)n to achieving higher loading densities, the new methods are faster as well, allowing complete loading of the column (with high loading density) in just two days. Thus, use of the methods disclosed herein will sllbst~nti~lly reduce costs and time.
The new mPtho~ lose~l herein are also ~i~ifil ~ntly less hazardous for the worker and safer for the e,l~L..i~.n~ Pyridine is ~;ullc;lllly used as a reaction solvent and, alone or in comhin~tion with other solvents, to wash away unreacted fe~ catalyst, and u~lw~l~d reaction by-products. We have found that the amount of pyridine can be reduced to about 5% in ac~lvl il.ile or el;,.. i~
completely. Because pyridine is toxic and has an obnoxious odor, the present methods are less hazardous than the prior art methods.
The present methods also require lesser amounts of re~gPnt~ rçslllting in lesser costs and amounts of waste. We have found that column loading densities wo 96/09314 ~2 2 0 0 5 5 2 PCT/US95/12196 in the range of about 70-80 ,umol/g can be ~tt~inP,l using less than about 3 x 10-3 mol of nucleoside per 25 g of CPG. Thus, the current methods provide additional cost savings by these means as well. Overall, savings of about 43% are realized with the new methods for loading (1en~itiPS in the range of about 70-80 ,umol/g.S The present invention provides meth~ of loading a filnrtion~li7P-l solid su~o,lforoligonucleosidesynthesiscomrri~in~cont~r~tin~thefllnrti~n~li7e~l solid support with a solvent, diis~,u~ylcarbo~1iimide, and a nucleoside having a 3' linker group ~tt~rhed at a pH of less than 7Ø
In the first embodiment of the invention, dii~o~,u~lcarbo-liinniAP (DIC) is used as an a;Liv~lol for the acid catalyzed loading of nucleoside-3'-succinates. The a~;Liv~lul forms an intermefli~t~ with the tPrmin~l carboxylic acid moiety of the nucleoside-3~ ccil .A~ rPn-1erin~ it ~ tible to nucleophilic attack by a support-bound amino fimrtio~l group. It has been ullc~e~ dly found that DIC in the ~lcsence of l-hy~llo~ybel~uli~ole (HOBT) is more crreclivc than DEC at activating the succinyl carboxylic acid moiety. Under ideal conditions, wh~ei the coupling of the nucleoside-3'-succin~te to the solid support is 100% complete, 1 equivalent of DIC is l~ ed to couple 1 equivalent of nucleoside-3'-~uccil~al~
to the support. DIC is moisture sensilive, however, and the.erore, in general, more than 1 equivalent is ~luil.,d. This will not detract appreciably from the cost benefit of using DIC since it is s~lbst~nti~lly cheaper than DEC. We have found that about 2.5 to 3.0 eq of DIC per 1 eq of mo~oml~leosicle s~lccin~ts results in llent lo~ling~ luulinely yielding loading cl~.n~ities of about 75 to about 85 ~mol/g.
In the second embodiment of the invention, HOBT is used to catalyze DIC
activated lo~-ling. Expt~rim~nt~ using pyridine as a solvent (pLeselll~d infia) Wo 96/09314 ~ 2 0 6~ 5 5 2 PCTiUS95/12196 0 .l~mcn.etrate that HOBT is a more effective than other compounds such as N-hydloxysuccinimi~le (NHS), paratoluenesulfonic acid (pTSA), and trifluoroacetic acidl (TFA). Our ~ e~ ents demon~ le that the use of HOBT results in loading d~n~itiP~e r~ngin~ from 30 to 100% greater than those ~ inecl using NHS, pTSA, and TFA. The amount of HOBT is not critical; it should be sufficient to catalyzethe reaction. We have found that about 0.08 to about 0.16 g HOBT per ml DIC
work ç~cee~lin~ly well, although lesser or greater amounts are likely to work just as well. Most preferably, a lesser a-m--ount of HOBT is used, generally about 0.08 g-In ~-olll~. aspect of this embo-lim~nt, nitro-HOBT is used as a catalyst.
Our e~.,lln,ents show that catalysis with nitro-HOBT results in e~esçnti~lly equ;valent loading deneities as col~ d to when HOBT is used. Nitro-HOBT is used in the amount as described above for HOBT.
The m~tho-lc of the present invention can also be used to load a non-linker-sltt?rh.ofl nucleoside (i.e., a nucleoside having a free 3' hydroxyl group) onto a column bearing a linker groug, e.g., a ~uccillyl moiety. Accordingly, in a thirdembodiment of the present invention, a solid support to which a linker, most preferably ~lcçinic acid, has been ~tt~chP-l is loaded by c- nt~ctin~ it with a nucleoside having a free 3' hy~yl group in the ~e3ence of DIC and HOBT.
The conditions for this reaction are the same as described herein for the loading of nucleosides bearing a 3' linker onto functi~ n~li7~ ;U~)O~
In a fourth embodiment of the present invention, methods of loading a solid support are ~esc.-led in which the amount of pyridine used as the reaction solvent and wash solvent is sllhst~nti~lly reduced or el;~ e~l We have found that ~ wo 96l09314 2! 2 ~ 0 5 5 2 PCT/lUSg5/12196 pyridine need not be the main solvent in the loading reaction. Any solvent that dissolves the re~rt~nt~ but does not react itself can be used. We have found that both ~ct;lo~ ;le and dichloromethane are suitable solvents. Ac~lo~ ile is the most ~,~r~l..,d solvent. In one aspect of this embo-1imPnt a ~ lu~ of pyridine S with ~c~ ;le and/or dichloromPth~nP is used as the ~hllaly solvent for loading nucleosides. A small amount of pyridine (e.g., about 5%) may be used to ensure a minim~l amount of detritylation, but is not required. Accordingly, in another aspect of this embo-lim~nt no pyridine is used. Because of the reduced costs, in the most prer~l~,d embodiment the solvent is ~cc;lo~ ;le with very little (e.g., 5%
or less) or no pyridine.
The present invention can be used with any function~li7P~cl solid support.
A number of such supports are known in the art. E.g, Pon in Methods in Molec.
Biol., supra. We ~l~m~ l-dl~ below that both "TENTAGEL S" (Rapp Polyrnere, Tiibingen, Ge.~ll~ly)(a support in which polyethyleneglycol spacers are grafted on a gel-type support) and HLP (ABI, Foster City, CA) (a PEG-Poly~Lylelle ~u~oll) can be loaded with the current m~thn-1~ to extremely high tll~n~ities. CPG is the most plc;r~c;d support for DNA synthesis.
While the results ples_l.led below were obtained using succinylated thymidine mrmom~r, those skilled in the art will appreciate that any suitably plvl~cled nucleoside monomer (n~ r~lly occurring or modified) can be used with the present methods. Dimer blocks and other mnltin1lr.leoside synthons can also be loaded according to the methods of the present invention. In addition, although succinic acid is the plere..~,d linker, any suitable linker can be used. Such a linker will preferably have a free carboxyl group that a support-bound amino group can wo 96/09314 2 ~ Q ~ 5 5 2 PCT/US95112196 ~0 attack to form an amide bond, thereby binding the linker and its :Itt~ch~l nucleoside to the support. F~mrles of ~lt~ tive linkers are disclosed by Pon in Met.hods in Molec. Biol., supra.
The following F.~mplec are int~n-lçd for illustrative purposes and are not S intelltle-1, nor should they be consl.ued, as limiting the invention in any way.
EXAMPLES
Example 1 Standard Method of Loading of DMT-dT-Succinic Alcid on Controlled Pore Glass 500 g of CPG (Schott, Ho7h~im, Germany) (particle size- 100-130 ,um;
pore si_e: D50: 41.6nm), 6.1 g of dimethylamino pyridine (Aldrich, Milwaukee, WI), 50 g of triethylamine (Aldrich), and 100 g of ethyl-3-(3-dimethylamino propyl) carbo-liimi-le (DEC, mol. wt. 191.7) (Sigma, St. Louis, MO) were placed in a 5 1 Schott bottle and hand shaken for 20 ~ s. 60 g of DMT-dT-succinic acid (Monomer Sciences, Huntsville, AL) was added and the bottle capped and shaken in an orbital shaker at 160 rpm for 18 hours.
A small analytical sample of the resin w~ withdrawn from the Schott bottle, sllcce~;vely washed with pyridine (3 x 5 ml) (Baxter, Muskegon, MI), m~lh~n~l (3 x 5 ml) (Baxter), and methylene chloride (3 x 5 ml ) (EM Science, Cincinnati, OH) sllcce~;vc;ly, and dried in vacuo.
A~lo~;"~tely 20 mg of dry resin was weighed, 200 ~l perchloric acid/ethanol (6:4) was added, and the reslllting solution diluted to 100 ml withmelhylene çhl~ le The absoll,~ce was measured at 498 nm. The same procedure was repeated on a second analytical sample and the average loading ~ WO 96/09314 2 2 0 0 5 5 2 PCT~US95/l2l96 value calculated using Beer's law with a molar absorption coefficient of 70 l/(mol cm) for DMT. A loading value of 66.7 ~Lmol/g was obtained.
- An ~d~1ition~l 20.0 g of DMT-dT-succinic acid was added to the Schott bottle and the llfi~ shaken for 18 hours at anbient le~ .dlulc;. Another analytical sample was removed and worked-up as described above. The absolb~lce of the sample was measured and a loading value of 66.5 ~lmol/g obtained.
Another 20.0 g of DMT-dT-succinic acid was added to the Schott bottle and the ~ shaken for 18 hours at ambient ten~ dlu,e. A third analytical sample was removed and worked-up as rle~sçribed above. The absoll,~lce of the sample was measured and a loading value of 69.6 ,umol/g obtained.
The ""~ ,e re...~ g in the Schott bottle was filtered and the resin washed with pyridine (3 x 1 1). The dry solid was ll~rell~d to a Schott bottle and Cap A (1.0 1 of acetic anhydride in tetrahydrofuran) (Crll~ch~m, Lillv;~ o~
United Kingdom) and Cap B (1.5 1 of N-methylimidzole, pyridine in tetrahy&oru,~l) (Millipore, Bedford, MA) were added. The llli~ , was shaken for 18 hours at ambient te~ . The solid (CPG-T) was filtered and s~ ce~i~;vely washed with methanol (3 x 1 1) and methylene chloride (3 x 1 1) and dried in vacuo to yield 502.5 g. The resin was subjected to the same procedure described above for each of the small ~mpl~s A loading value of 71.4 ~lmol/g was obtained.
Example 2 New Method of Loading DMT-d~-Succinic Acid on Controlled Pore Glass WO 96109314 2 ~ O 0 5 5 2 PCT/US95/12196 0 250.0 g of CPG (Schott), 0.8 g of hydroxybel~.,LL;azole (mol. wt. 135.13) (Aldrich), and 10 ml of 1,3-diisopr~ylcarbodiimide (DIC, mol. wt. 126.20, - density = 0.806 g/ml) (Aldrich) were mixed with 50 ml of pyridine (Baxter) and 1 1 of acetorlitrile (Baxter) in a 2 1 Schott bottle and shaken for 20 mimltec 15 g of DMT-T-succinic acid (mol. wt. 644) (Monomer Sciences) were added, the bottle stoppered and shaken in an orbital shaker at a rate of 180 rpm for 16 hours.
A small analytical sample of the resin was withdrawn from the Schott bottle and washed with 5% pyridine in acetonitrile (3 x 5 ml), methanol (3 x 5 ml)l, and methylene chloride (3 x 5 ml) s~lccç~ively and dried under a stream of0 in vacuo.
A~r~ lately 20 mg of the dry resin were weighed and 200 ml perchloric acid/ ethanol (6:4) added. The solution was diluted to 70 ml with methylene chloride and the absorbance measured at 498 nm. The entire procedure was repeated and the average value of the absorbencies used to calculate the loading.
The loading w~ calculated as above. An average loading value of 78.0 ~lmol/g was obtained.
The le...~i"i"g sollltioî in the Schott bottle was filtered and washed with 5% pyridine in act;l~l~iLIile (3 x 500 ml). Dry solid was transferred to a Schott bottle and Cap A (500 rnl) and Cap B (750 ml) (Cap A and Cap B were as described above) were added and the l~ L~e shaken for 16 hours at ambient tem~.dlule. The solid (CPG-T) was filtered, washed sllccç~ively with methanol (3 x 500 ml) and then methylene chloride (3 x 500 ml), and dried in vacuo to yield 250 g of CPG-T. The loading value, 78 ,umol/g, was calculated as describedabove.
This entire procedure was repeated, ~ub~ varying amounts of the re~c~t~nt~ and using several catalysts and supports. The results are present in Table - 1.
From the foregoing it will be appreciated that although specific embo~l;,.lel.l~i of the present invention have been described herein for the purposes of illustration, various morlifir~tion may be made without deviating from the spirit or scope of the invention.
WO 96/09314 ~ 5 5 ~ PCT/US95/12196 0 0 ~ 0 3 ~ t ~ ~ , , , , , o ~ _ . . . . . . . .
~ ~ . . . . . .
~.~~.,.~~~~~~~~.~..
~i~ ' ' ~ ' ' ' --~ ' ' ~0 ~ O ~ 0 80 0 0 s ~ ~ s ~ ~ ~ r ~~~ ~ ~ ~ _ _ 0 ~1 C~
O ~ O "~ o Z_ ~ In, , g g ~ g 8 8 ~g" ~
Claims (27)
1. A method of loading a functionalized solid support for oligonucleotide synthesis comprising simultaneously contacting the functionalized solid support with diisopropylcarbodiimide, nucleoside having a 3' linker group attached, and an acid catalyst.
2. The method of claim 1 wherein the acid catalyst is N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or a combination thereof.
3. The method of claim 2 wherein the solid support is CPG and less than about 3 x 10-3 mol of nucleoside is used per 25 g of CPG.
4. The method of claim 3 wherein about 2 to about 3 moles of diisopropylcarbodiimide are used per mole of nucleoside.
5. The method of claim 4 wherein the amount of N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or combination thereof is about 0.1 times that of the diisopropylcarbodiimide.
6. The method of claim 2 wherein the solvent comprises about 5% or less pyridine.
7. The method of claim 6 wherein the solid support is CPG and less than about 3 x 10-3 mol of nucleoside is used per 25 g of CPG.
8. The method of claim 7 wherein about 2 to about 3 moles of diisopropylcarbodiimide are used per mole of nucleoside.
9. The method of claim 8 wherein the amount of N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or combination thereof is about 0.1 that of the diisopropylcarbodiimide.
10. The method of claim 2 wherein the solvent contains no pyridine.
- 16a
- 16a
11. The method of claim 10 wherein the solid support is CPG and less than about 3 x 10-3 mol of nucleoside is used per 25 g of CPG.
12. The method of claim 11 wherein about 2 to about 3 moles of diisopropylcarbodiimide are used per mole of nucleoside.
13. The method of claim 12 wherein the amount of N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or combination thereof is about 0.1 that of the diisopropylcarbodiimide.
14. A method of loading a functionalized solid support for oligonucleotide synthesis comprising simultaneously contacting a functionalized solid support having a linker moiety with diisopropylcarbodiimide, nucleoside having a free 3 ' hydoxy group, and an acid catalyst.
15. The method of claim 14 wherein the acid catalyst is N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or a combination thereof.
16. The method of claim 15 wherein the functionalized solid support is CPG.
17. The method of claim 16 wherein the solid support is CPG and less than about 3 x 10-3 mol of nucleoside is used per 25 g of CPG.
18. The method of claim 17 wherein about 2 to about 3 moles of diisopropylcarbodiimide are used per mole of nucleoside.
19. The method of claim 18 wherein the amount of N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or combination thereof is about 0.1 times that of the diisopropylcarbodiimide.
20. The method of claim 15 wherein the solvent comprises about 5% or less pyridine.
21. The method of claim 20 wherein the solid support is CPG and less than about 3 x 10-3 mol of nucleoside is used per 25 g of CPG.
22. The method of claim 21 wherein about 2 to about 3 moles of diisopropylcarbodiimide are used per mole of nucleoside.
23. The method of claim 22 wherein the amount of N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or combination thereof is about 0.1 that of the diisopropylcarbodiimide.
24. The method of claim 15 wherein the solvent contains no pyridine.
25. The method of claim 24 wherein the solid support is CPG and less than about 3 x 10-3 mol of nucleoside is used per 25 g of CPG.
26. The method of claim 25 wherein about 2 to about 3 moles of diisopropylcarbodiimide are used per mole of nucleoside.
27. The method of claim 26 wherein the amount of N-hydroxybenzotriazole or nitrohydroxybenzotriazole, or combination thereof is about 0.1 that of the diisopropylcarbodiimide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/311,156 | 1994-09-23 | ||
| US08/311,156 US5554744A (en) | 1994-09-23 | 1994-09-23 | Method for loading solid supports for nucleic acid synthesis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2200552A1 true CA2200552A1 (en) | 1996-03-28 |
Family
ID=23205659
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002200552A Abandoned CA2200552A1 (en) | 1994-09-23 | 1995-09-22 | Method for loading solid supports for nucleic acid synthesis |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US5554744A (en) |
| AU (1) | AU3724295A (en) |
| CA (1) | CA2200552A1 (en) |
| WO (1) | WO1996009314A2 (en) |
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| US5149798A (en) * | 1989-04-06 | 1992-09-22 | Worcester Foundation For Experimental Biology | Process for synthesizing oligonucleotides and their analogs adaptable to large scale syntheses |
| US5204455A (en) * | 1989-06-15 | 1993-04-20 | Froehler Brian C | Monomethoxytrityl protected oligonucleotides bound to a solid support |
| AU7579991A (en) * | 1990-02-20 | 1991-09-18 | Gilead Sciences, Inc. | Pseudonucleosides and pseudonucleotides and their polymers |
-
1994
- 1994-09-23 US US08/311,156 patent/US5554744A/en not_active Expired - Fee Related
-
1995
- 1995-09-22 AU AU37242/95A patent/AU3724295A/en not_active Abandoned
- 1995-09-22 WO PCT/US1995/012196 patent/WO1996009314A2/en active Application Filing
- 1995-09-22 CA CA002200552A patent/CA2200552A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU3724295A (en) | 1996-04-09 |
| US5554744A (en) | 1996-09-10 |
| WO1996009314A2 (en) | 1996-03-28 |
| WO1996009314A3 (en) | 1996-05-30 |
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