Volume 9 Number 12 1931
`
`Nucleic Acids Research
`
`Synthesis of cligodeoxyfibonucleotides on silica gel support
`
`
`Flora Chcwl', Tomas Kempel' and Gunnar Palm
`
`
`KabiGen AB, Department of Chemistry, 112 87 Stockholm. Sweden
`
`
`Received 16 March 1981
`
`ABSTRACT
`
`A rapid solid phase method of oligonucleotide synthesis
`based on monomeric protected nucleosides has been developed.
`
`INTRODUCTION
`
`The solid phase synthesis of oligonucleotides on silica gel
`described by Caruthers et a1.1 using monomeric baseprotected
`5'-0-dimethoxytrityl-nucleoside-3'-O-methylphosphochloridites
`has been further studied. A new method for the functionalization
`
`of silica, deprotection of the 5'-0-dimethoxytrityl group with
`
`zinc bromide/nitromethane/water, and the use of acetic anhydride
`
`in N,N-dimethylaminopyridine as a capping reagent have been intro-
`duced.2
`These improved methods allow a dodecamer to be assem-
`bled in12 hours where each nucleoside addition cycle including
`
`all transformations and washes takes about
`
`1 hour. At present,
`
`several oligonucleotides (6-15 long} have been synthesized by
`
`this procedure. In the present paper are described the syntheses
`and isolations of a hexamer, a nonamer and two dodecamers (see
`
`table). The oligonucleotides are first isolated as 5‘-0-dimethoxy—
`
`tritylated compounds which are fully deprotected at the triester
`bond and at the purine/pyrimidine rings. They are purified on
`
`in a gradient sys-
`HPLC using reversed phase chromatography {C18}
`tem of 20-30% acetonitrile in 0.1 M triethylammonium acetate.
`
`The remaining 5'-0-dimethoxytrityl group is removed by 80% acetic
`
`acid in water and the fully deprotected oligomer is subsequently
`
`isolated in the same chromatographic system using a gradient of
`10-15% acetonitrile.
`
`
`
`@131. Pm- Umitud.1 Falconbcrg Court. London w1v 5FG. U.K.
`
`1’3“?
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`RESULTS AND DISCUSSION
`
`The solid phase support consists of 5'-0-dimethoxytrityl-
`N-benzoyl-Z'-deoxynucleoside-3'-succinic acids3 coupled to amin—
`ated silica. The silica. Porasil C, has been aminated with
`1'4 or dichlm‘odirnet:hy].silane‘1
`followed by aminoethanol. This silica was then treated with
`5'—O-dimethoxytrityl-N-benzoyl-Z'-deoxynucleoside—3'—0—succinic
`
`either triethoxysilylpropylamine
`
`acid in pyridine using dicyclohexylcarbodiimide (DCCI) as con-
`densing reagent {figure 1}. The degree of functionalization of
`
`T-silica was examined by 1} detritylation using ZnBr2 in nitro-
`methane and 2} ammonolysis.
`to give 5'-O-dimethoxytrityl-thymi-
`dine. The products were analyzed on HPLC and compared with re-
`
`ference samples. The dimethoxytrityl alcohol and 5'-0-dimethoxy-
`trityl-thymidine content were the same for both types of silica
`
`(0.01 mmole/100 mg]. The A. GIr and C-silica were also analyzed
`and showed the same degree of functionalization as T—silica. The
`
`at,
`
`-5.«3—5.:—o-fcx}-m—é’-{cqg-é-o
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`2803
`
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`Nucleic Acids Research
`
`extent of functionalization is comparable to a reported value5
`for Porasil C using dichlorodimethylsilane and it, therefore.can
`
`be assumed that the DCCI condensation is essentially a quantita-
`tive reaction. Such a high degree of functionalization allows
`
`us to make oligomers 9-15 nucleotides long using 150-200 mg of
`silica. The synthetic scheme is outlined in figure 1. Full
`
`cycles of operations and reaction times are given below. The
`
`"manual" machine used for the syntheses is shown in figure 2.
`The solid phase silica (150-200 mg}, functionalized with either
`nucleoside T, C, A, or G.
`is loaded into the column.
`
`1. A wash of dry tetrahydrofuran [THE]
`
`is pumped through the
`
`system with a flow of 1 ml/minute.
`
`2. The 5'-0-dimethoxytrity1 group is removed by a 1% water-
`nitromethane Viv mixture which has been saturated with ZnBrz.
`This reagent continues to be fed in as long as the orange
`color of the trityl cation is observed. In general,
`the de-
`tritylation is complete within 15 minutes.
`
`3. Excess reagent is removed by a wash cycle of THF {5 minutes).
`
`4. The incoming 5'—O—dimethoxytrityl-nuc1eoside—3'—0—methy1-
`
`is injected into the
`phosphochloridite in THF/gym—collidine
`100p. A 10 times excess is used and recycled in the system
`for 10-15 minutes.
`
`5. Excess reagent is then removed by a THF wash {5 minutes).
`
`6. The phosphite triester is oxidized to the phOSphate triester
`
`with a 12/HZO/THF solution [5 minutes}.
`
`Fr'92
`
`I
`
`inlet
`
`2
`injection valve system
`2.:
`injection loop
`2.2 51-13:! £0 loop wiéh garb-19¢
`
`‘a'
`
`column
`
`23 001342! {rum 600,0 3 PM?
`
`recycling valve
`5'
`cutlef
`6
`
`
`2309
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`Nucleic Acids Research
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`7. The oxidation reagent is then removed by a THF wash {10 min-
`utes).
`
`8. The cycle is completed by a capping of unreacted 5'-hydroxy
`
`groups using acetic anhydride/N.N-dimethylaminopyridine/THF.
`The reagent is in about 50 times excess and the reaction is
`
`complete within 5 minutes.
`
`9. Finally the capping reagent is washed out with THF (5 min-
`utes).
`
`The use of ZnBr2 as a detritylating agent has only been mention-
`ed briefly”6
`. It is reported to remove the dimethoxytrityl pro-
`tecting group fast and selectively though the mechanism for such
`
`a reaction is unclear. In early experiments ZnBr
`
`2 was found to
`be an inconsistent reagent; i.e., the detritylation was some-
`
`firesumably this
`times very efficient and at other times slow.
`was due to different concentrations of the reagent in nitro-
`
`methane. However, when 1% water was added, that mixture of ZnBr2
`{excess} and nitromethane gave a very powerful detritylating
`
`agent. For example.r 200 mg of functionalized silica {in the
`
`column} could be detritylated with 12 m1 of this ZnBr2 solution.
`There was no indication of side reactions such as depurination
`
`from the use of this "wet" ZnBr2/CH3N02 reagent. Since phosphites
`as coupling reagents do not give quantitative yields it became
`necessary to find a reagent to inactivate the unreacted 5'-
`
`hydroxyl groups. This concept of a capping reagent is particul—
`
`arly important when purines are coupled to the support; 5'—0*
`
`dimethoxytrityl-N-benzoyl-Z'—deoxyadenosine-3'—O-methylphospho-
`
`chloridite and the corresponding deoxyguanosine derivative give
`70-90% coupling, whereas the pyrimidine couplings are near
`
`quantitative. These yields have been determined from dimethoxy-
`
`trityl alcohol content and analyzed on C18 using 70% methanol-
`
`water v/v. The most useful capping reagent is a mixture of
`acetic anhydride/N,N-dimethylaminopyridine7 in THF. Silica
`{200 mg, 0.02 mmole of free hydroxy groups} is fully acetylated
`within 10 minutes with a 50 times excess of reagent. Should such
`
`a powerful reagent react with the nucleoside bases it could be
`
`easily removed in the subsequent ammonolysis of the oligonucleo-
`
`tide. A summary of the synthetic cligomers prepared by the solid
`
`phase method is given in the following table. Compounds 1,2 and 4
`
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`Table of oliqonucleotidss Bade by solid phase It} and
`solution chemists:
`IS:
`
`Acetonitvile I.
`Method of
`Preparation in 0.1 it Stalin on:
`
`yield l full
`I
`_ Retention yield I yield.
`can Mn. mrvon nflTr-calpound. daprotactad.
`
`1a 6 [Imrlmnmm]
`‘Ib dlml’l‘cml
`
`.
`
`.
`
`30 ninutes
`3O minute:
`
`4a d[lm:11tms]
`
`2a d [ (amrirmmmnccl
`2h dlrormmnccl
`
`P, s
`P, S
`
`in minutes
`30 minutes
`
`30 minutes
`
`30 ninutcs
`
`were made by independent routes using standard triester synthes-
`158_ The compounds were identical as the partially deprotected
`tritylated compound and as the fully deprotected oligomers.
`Yields for this method of synthesis have been determined in two
`
`different ways. The amount of tritylated compound attached to
`
`the support was measured as the amount of trityl alcohol obtain-
`
`ed upon treatment with zinc bromide. They show coupling efficien—
`
`cy to be 70—100%. However, a drop in yield is encountered upon
`
`deprotection and cleavage of the compound from the support. The
`
`isolated yields are about 25% of the expected overall yields.
`This discrepancy has been observed by other workers, and was
`explained as a loss on 018 columns during isolation9 . The amount
`isolated, hOWever, was sufficient
`(in each case, greater than
`
`10 0.D.). When a larger quantity was desired, a larger capacity
`
`column was used. Figure 3 shows the separation profiles for
`
`compounds 1-3 at the two stages of HPLC purification. Gel elec—
`
`shows that the solid phase oligo—
`trophoresis {20% acrylamide)
`nucleotides are of a higher purity than those from solution
`
`chemistry, all of which have been purified to a single peak by
`HPLC. Structural determination was performed according to the
`standard methods of sequencing by homochromatography.1D
`___—._.—,——_
`
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`
`la
`
`Fig.
`
`3—
`
`33
`
`
`23 &
`
`HPLC profiles of dimethoxytritylated oligomers 1—3a with a flow
`
`of 1 m1/minute and a gradient of 20-30% acetonitrile in 0.1 M
`triethvlammonium acetate over 30 minutes.
`
`3b
`
`lb
`
`2b
`
`HPLC profiles of fully deprotected oligomers 1-3h with a flow
`
`It'll/minute and a gradient of 10-15% acetonitrile in 0.1 M
`of 1
`triethylammonium acetate over 30 minutes.
`
`2812
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`EXPERIHENTAL SECTION
`
`The following chemicals were purchased from commercial
`
`sources: deoxycytidine,
`
`thymidine. deoxyadenosine and deoxyguan-
`
`osine (Pharma-Waldhof}, 4.4Ldimethoxytrity1 chloride (Aldrich),
`triethoxysilylproPylamine, dimethyldichlorosilane. aminoethanol.
`
`nitromethane, zinc bromide, succinic anhydride. acetic anhydride,
`
`N,N-dimethylaminoPyridine,
`collidine,
`triethylamine.
`
`tetrahydrofuran. benzoylcyanide. gym-
`thiophenol, n-butylamine {Fluka}. Poracil
`
`C(37-75p] silica (Waters). For the reversed-phase C18 column
`chromatography. analytical columns from Altex and HPLC, aceto-
`
`nitriletFluka, HPLC-grade].
`
`Silica I. Functionalization of Silica Gel (Porasil C) with
`
`Dichlorodimethylsilane and Aminoethanol
`
`The silica {Porasil C. 10 g} was dried at 200°C for 24
`hours. After cooling in a desiccator,
`the silica was mixed with
`
`dry pyridine [75 m1)Ir and dimethyldichlorosilane {0.1 moleIr 13 g)
`
`was added. The mixture was shaken at room temperature for 30
`minutes after which the pyridine solution was decanted off. The
`
`[0.3
`vessel was cooled to 0°CIr and an excess of aminoethanol
`mole. 18 g} was added. The mixture was then stirred for 24 hours
`
`at room temperature. The silica was filtered. washed with
`
`pyridine (200 m1) and ethyl ether (100 m1), and dried in a
`
`desiccator at room temperature. The silica was mixed with pyri—
`dine (75 ml), and trimethylsilylchloride (0.1 mole) was added.
`
`The mixture was shaken for 5 hours at room temperature. The
`
`silica was then filtered, washed with pyridine (100 ml) and
`ethyl ether (100 m1); and dried.
`
`Silica II. Functionalization of Silica Gel
`
`(Porasil C) with
`
`Triethoxysilylpropylamine
`
`To Porasil C {10 g} was added triethoxysilylpropylamine
`{15 ml) and toluene [50 m1}, and the mixture was refluxed for
`7 hours. The mixture was cooled. The silica was filtered off and
`
`washed with pyridine (2 x 100 m1). Trimethylsilylchloride (11 ml)
`
`was added and the mixture was shaken for 4 hours at room temper-
`ature. The silica was filtered, washed with pyridine (3 x 50 m1)
`and ethyl ether {3 x 50 ml), and dried in a desiccator.
`
`
`
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`General Procedure for the Preparation of 5'—O—Dimethoxytrityl-
`
`2'-Deoxynucleoside-3'-0-Succinic Acids
`[dTIr dABz,
`The 5'-O-dimethoxytrity1-2'-deoxynucleoside
`or 6032.
`2 mmole} and N,N-dimethylaminopyridine (2 mmole)
`were dissolved in pyridine (50 m1). To the Clear solution was
`
`11,12
`
`dGBz
`
`added in portions succinic anhydride (2.1 mmole] at room temper-
`ature. The mixture was stirred for 48 hours. Water
`[3 ml} was
`
`then added to consume unreacted succinic anhydride. The mixture
`
`was concentrated at reduced pressure and the pyridine removed by
`
`coevaporation with toluene. The residue was taken up in dichloro-
`methane {200 ml). and the organic phase was washed with an aque-
`ous solution [100 m1} of citric acid (4 mmole). The free 3'-0-
`
`succinic acid was mainly partitioned to the organic phase. The
`
`solution was dried over Nazso4 and concentrated at reduced
`pressure. The solid residue was dissolved in dichloromethane
`
`{50 ml} and precipitated from n-hexane [300 ml). The nucleoside-
`3'—0—succinic acids were isolated in 70-80% yield. TLC analysis
`on silica {10% methanol-dichloromethaneJ showed the reaction
`
`product as a spot below the 5'-0-dimethoxy-2‘-deoxynuc1eoside
`
`starting material [trace amount). The precipitated succinyl
`
`nucleosides were used without further purifications.
`
`General Procedure for the Reaction of 5'-0-Dimethoxytrityl—2'—
`
`Deoxynucleoside-B'-0-Succinic Acid with Silica-I and Silica-II
`Silica-I or Silica-II (6 g}
`in pyridine [10 ml) was treated
`
`with triethylamine (1 g)
`
`to obtain the silica free amine.
`
`Excess triethylamine was removed by coevaporation with pyridine.
`
`To the silica was added the deoxynucleoside-3'—0—succinic acid
`
`(5 mmole} in pyridine [100 ml) and dicyclohexylcarbodiimide
`(44 mmole , 9 g). The mixture was shaken at room temperature for
`
`2 days. The silica was filtered, washed with pyridine [300 ml]
`and reacted with benzoylcyanide (0.1 moleIr 13 g)
`in 50 m1 of
`
`pyridine for 3 hours. The silica was again filtered, washed with
`
`pyridine (200 m1} and ethyl ether (200 ml}, and dried. The degree
`
`of 5'-O-dimethoxytrityl-2'-deoxynucleoside incorporated into
`
`silica was determined by the following methods:
`
`1}
`
`treatment with
`
`ZnBr2 in nitromethane to detritylate and 2} ammonolysis, to
`cleave the 5'-0-dimethoxytritylnucleoside. Analyses were perform-
`
`ed on HPLC using Lichrosorb silica. Detritylation of 100 mg of
`____———-—-—————
`
`2814
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`[1.5% methanol-
`T-silica yielded 0.012 mmole of trityl alcohol
`dichloromethane,
`2 mllminute, Rtt}=4.0 minutes}. Treatment of a
`
`second 100 mg of silica with concentrated ammonia gave 0.011
`mmole of 5'-O-dimethoxytrityl-thymidine (5% methanol-dichloro-
`
`methane, 2 mI/minutes, thl=3.5 minutes}. The ABz—, CBz-, and
`GBz-silica were similarly analyzed and found also to be func-
`tionalized 0.01 uncle/100 mg silica.
`
`General Procedure for the Preparation of S'-O-Dimethoxxtrityl-
`
`2'-Deoxynucleoside-3'-O-Methyl Phosphochloridites
`The baseprotected 5'-O-dimethoxytrityl-nucleoside‘1'12 [5.5
`mmole)
`in dry THF [20 ml) was added over 30 minutes to a stirred
`and cooled solution {-780} of methyldichlorophosphite13'14 {5.0
`mmole, 0.48 ml} and sym-collidine (25 mmole, 3.3 ml} in THF [20
`m1) under argon. After an additional 30 minutes.r the reaction
`
`mixture was centrifuged. The clear supernatant was removed from
`
`the collidine hydrochloride and transferred to a round bottom
`
`flask. The solution was concentrated to a gum. and was then
`diluted to a convenient concentration (0.2 mmole/ml] with five
`
`equiv. of sym—collidine in THF.
`
`Detritylating Reagent
`
`(5 ml) was added ZnBr2
`To nitromethane {500 m1) and water
`(70 g], and the mixture was stirred at room temperature for
`24 hours. A test on the facility of trityl removal was done.
`
`5'-0-Dimethoxytrityl-N-benzoy1-deoxyguanosine-3'—p-chlorophenyl-
`
`fi-cyanoethyl phosphate and b} 5'—O—dimethoxytrityl-N-benzoyl-
`
`deoxyadenosine-B'-p-chloropheny1-/3-cyanoethy1 phosphate were
`
`both treated with this ZnBr2 solution at room temperature. The
`reaction was followed by TLC (silica,
`10% methanol-dichloro-
`
`methane v/v}. The detritylation was complete within 5 minutes.
`
`The mixture was checked periodically for two days; no evidence
`of depurination could be detected.
`
`Capping Reagent
`A solution of N,N-dimethylaminopyridine (20 mmoleIr 2.4 g)
`
`in THF (40 ml} was made. Acetic anhydride (10 mmole,
`
`1 g) and
`
`gym-collidine {10 mmoleIr 1.2 g) were added to 10 m1 of the stock
`solution. The capping solution darkened during the course of a
`
`day and was consequently remade every day. The capping activity
`
`
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`and selectivity, however, appeared unchanged after 24 hours
`storage at room temperature.
`
`Removal of the Oligonucleotide from the Solid Suppgrt
`The silica containing the synthesized oligonucleotides was
`
`washed with 1.4-dioxane after the last cycle and transferred to
`
`a vial. The methyl protecting group of the internucleotide
`
`phosphotriester was removed by treatment with thiophenol-
`triethylamine in dioxane at room temperature1'15. The reaction
`was fast and selective.
`(Reaction times of 10 minutes and 20
`
`hours gave the same yield of isolated oligonucleotide}. After
`30 minutes,
`the reagent was removed, and the silica was washed
`with dioxane and water. Concentrated ammonia was added and the
`
`mixture heated at 50°C for 4 hours to deprotect A32 and Caz. At
`the same time the ester bond which linked the oligonucleotide
`
`to the support was hydrolyzed. The silica was removed by filtra-
`
`tion and the 5'-O-dimethoxytrityl-oligonucleotide in ammonia
`
`was concentrated at reduced pressure. To the residue was added
`
`2 ml of 1:1 butylamine-methanol V/v to remove the benzamide of
`G. After 43 hours at room temperature the mixture was again
`
`concentrated. The residue was taken up in 1 ml of 0.1 H triethyl—
`ammonium bicarbonate [TEAB) and extracted with ethyl acetate and
`
`ethyl ether.
`
`Isolation of the Partially Deprotected 5'-O—Dimethoxytrityl-
`
`nucleotides and the Fully DeErotected Oligonucleotides
`The reaction mixture in 1 m1 of 0.1 M TEAB was analyzed on
`HPLC using C18 columns16.
`A gradient system of 20-30% aceto-
`nitrile in 0.1 M triethylammonium acetate over 30 minutes was
`
`used for the separation of hexamers to dodecamers. The tritylated
`oligonucleotide could easily be identified and was isolated on
`
`either analytical or semipreparative columns. The fraction
`
`containing the desired compound was concentrated and treated
`with 80% acetic acid—water to remove the trityl protecting grOup.
`
`the solution was
`After complete deprotection (10-30 minutes).
`concentrated and residual acetic acid coevaporated with water.
`
`The fully deprotected oligonucleotide was analyzed and isolated
`
`by HPLC, using a gradient of 10-15% acetonitrile over 30 minutes
`at 1 mllminute {see table).
`
`
`2816
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`_—_———————
`
`ACKNOWLEDGEMENTS
`
`The authors would like to thank KabiGen for supporting this
`
`research project and Marianne Magnuason for typing this
`
`manuscript.
`
`?
`
`Present address: Molecular Genetics Incorporated, 10300 Bren
`Road East, Minnetonka, MN
`55343
`USA
`
`REFERENCES
`
`(1930) Nucleic
`
`(1974) J. Chromatogr. Sci.
`
`(1913) Anal. Chem. 3;.
`
`1a. Matteucci, M.D. and Caruthers, M.H. {1980) Tetrahedron
`Letters,719-722.
`1b. Caruthers, M.H., Beaucage, S.L., Efcavitch, J.W., Fisher,
`E.F., Matteucci, M.D. and Stabinsky, Y.
`(1980) Nucleic
`Acids Res. Symposium Ser. 2, 215—223.
`1c. Matteucci, M.D. and Caruthers, M.H.
`(1980) Tetrahedron
`Letters, 3243-3246.
`2. Results previously summarized in a Technical Report
`(1980-11-24)
`to KabiGen AB.
`3. Gait, M.J., Singh, M. and Sheppard, R.C.
`Acids Res. 2, 1081-1096.
`4. Majors, R.E. and Hopper, M.J.
`12, 767—778.
`5. Gilpin, R.K. and Burke, M.F.
`1383—1339.
`6a. Koster, H.,HoPpe, N., Kohli, V., Krdpelin, M., Kaut, H.
`and Kulikowski, K.
`{1980) Nucleic Acids Res. Symposium
`Ser. 1, 39-60.
`6b. Kohli, V., Blocker, H. and Roster, H. {1980} Tetrahedron
`Letters, 2683-2686.
`7. Hfifle, G., Steglich, W. and Vorhrflggen, H.
`Chem. 22, 602—615.
`Narang, S.A., Brousseau, R., Hsiung, H.M. and Michniewicz,
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