Volume 9 Number 12 1981
`
`Nucleic Acids Research
`
`Synthesis of oligodeoxyribonucleotides onsilica gel support
`
`
`Flora Chowt, Tomas Kempet 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 al.! using monomeric baseprotected
`5'-O-dimethoxytrityl-nucleoside-3'-O-methylphosphochloridites
`has been further studied. A new method for the functionalization
`
`of silica, deprotection of the 5'-O-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.
`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'-O-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'-O-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.
`
`
`
`© IRLPress Limited, 1 Falconberg Court, London W1V 5FG, U.K.
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`RESULTS AND DISCUSSION
`
`The solid phase support consists of 5'-O-dimethoxytrityl-
`N-benzoy1-2'-deoxynucleoside-3'-succinic acids? coupled to amin-
`ated silica. The silica, Porasil C, has been aminated with
`r4
`either triethoxysilylpropylamine|
`or dichlorodimethylsilane
`followed by aminoethanol. This silica was then treated with
`5'-O-dimethoxytrityl-N-benzoyl-2'-deoxynucleoside-3'-O-succinic
`
`4
`
`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 ZnBr 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'-O-dimethoxy-
`trityl-thymidine content were the same for both types of silica
`
`(0.01 mmole/100 mg). The A, G, and C-silica were also analyzed
`and showed the same degree of functionalization as T-silica. The
`
`2Z6r,
`
`OOnTr
`
`o
`
` 1
`
`Fig. 7
`o
`:
`9%
`“Si-0-$3-O-FCH}WH-Cthe0
`e a
`Silica I
`
`;
`one
`o
`o
`~$i-0-Si 4KWH-E-4CH)s E-0-4
`et
`
`8,
`
`,onnre
`
`Silica v/a
`
`8,
`
`o-P of
`- @)-0
`_CMy
`on
`(P)-o
`Zuo140fie» Orolob-len Cycle
`
`B
`
`8
`
`*
`
`DMT
`
`is
`
`OCH,
`
`New
`
`8,
`
`a
`
`vPro
`cH,o
`
`ODNTr
`
`z,
`346):0/pyanp
`
`9
`bux
`
`8= protected base, OMAP = 4-dimethylominepyridine
`OAT = 4,4.—dimethexyérityt } @ = polymer support =
`silica Tor I
`
`
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`extent of functionalization is comparable to a reported value”
`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.
`
`is pumped through the
`
`1. A wash of dry tetrahydrofuran (THF)
`system with a flow of 1 ml/minute.
`2. The 5'-O-dimethoxytrityl group is removed by a 1% water-
`nitromethane v/v mixture which has been saturated with ZnBr..
`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-nucleoside-3'-O-methyl-
`phosphochloridite in THF/sym-collidine
`is injected into the
`loop. 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 I,/H,0/THF solution (5 minutes).
`
`Fig.2
`
`23 outlet from loop
`
`inlet
`/
`injection valve system
`2
`injection leep
`2.1
`2.2 inlet to loep with syringe
`
`pump
`3
`column
`4
`recycling valve
`5
`6
`ovtlet
`
`
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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).
`
`a reaction is unclear. In early experiments ZnBr
`
`The use of 2nBr. as a detritylating agent has only been mention-
`ed briefly!'®,
`It is reported to remove the dimethoxytrityl pro-
`tecting group fast and selectively though the mechanism for such
`2 was found to
`be an inconsistent reagent; i.e., the detritylation was some-
`times very efficient and at other times slow. Presumably this
`was due to different concentrations of the reagent in nitro-
`methane. However, when 1% water was added, that mixture of ZnBr.
`(excess) and nitromethane gave a very powerful detritylating
`
`agent. For example, 200 mg of functionalized silica (in the
`column) could be detritylated with 12 ml of this anBr5 solution.
`There was no indication of side reactions such as depurination
`from the use of this "wet" ZnBr./CH,NO, 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'-0O-
`
`dimethoxytrity1-N-benzoy1-2'-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-dimethylaminopyridine’ 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 oligomers prepared by the solid
`phase method is given in the following table. Compounds 1,2 and 4
`
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`Table of oligonucleotides made by solid phase (FP) and
`solution chemistry (5)
`
`Acetonitrile &,
`Method of
`Preparation in 0.1 M Et,NH OAc
`
`yield & full
`. Retention yield 4 yield 4
`time min.
`DMTr-OH DMTr-compound deprotected
`
`dad [ tore)ren<ac ]
`
`lad [ cowrey acarereac ]
`Tb d(ACATCTGAG)
`
`'
`
`i
`
`20-308,
`10-15%,
`
`30 minutes
`30 minutes
`
`aad [ towne)roraaarcracc] P, &
`2b d(TGTAAATGTACC)
`Pp, &
`
`20-30%, 30 minutes
`10-15%, 30 minutes
`
`jad [ torecrencaaarcac ]
`3b d(CTTTGAAATGAC)
`
`P
`PB
`
`20-30%, 30 minutes
`10-15%, 30 minutes
`
`were made by independent routes using standard triester synthes-
`is®. 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 C18 columns during isolation? . The amount
`isolated, however, was sufficient
`(in each case, greater than
`10 O.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-
`trophoresis (20% acrylamide)
`shows that the solid phase oligo-
`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. !°
`—ieeSse
`
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`
`Fig. 3
`aeEela
`
`
`3aL
`
`HPLC profiles of dimethoxytritylated oligomers 1-3a with a flow
`
`of 1 ml/minute and a gradient of 20-30% acetonitrile in 0.1M
`triethylammonium acetate over 30 minutes.
`
`3b
`
`1b
`
`eb
`
`HPLC profiles of fully deprotected oligomers 1-3b with a flow
`
`of 1 ml/minute and a gradient of 10-15% acetonitrile in 0.1 M
`triethylammonium acetate over 30 minutes.
`
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`EXPERIMENTAL SECTION
`
`The following chemicals were purchased from commercial
`
`thymidine, deoxyadenosine and deoxyguan-
`sources: deoxycytidine,
`osine (Pharma-Waldhof), 4,4-dimethoxytrityl chloride (Aldrich),
`triethoxysilylpropylamine, dimethyldichlorosilane, aminoethanol,
`nitromethane, zinc bromide, succinic anhydride, acetic anhydride,
`N,N-dimethylaminopyridine,
`tetrahydrofuran, benzoylcyanide, sym-
`collidine,
`triethylamine,
`thiophenol, n-butylamine (Fluka), Poracil
`C(37-75y) silica (Waters). For the reversed-phase C18 column
`chromatography, analytical columns from Altex and HPLC, aceto-
`nitrile (Fluka, 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 ml), and dimethyldichlorosilane (0.1 mole, 13 g)
`was added. The mixture was shaken at room temperature for 30
`minutes after which the pyridine solution was decanted off. The
`vessel was cooled to 0°c, and an excess of aminoethanol
`(0.3
`mole, 18 g) was added. The mixture was then stirred for 24 hours
`
`at room temperature. The silica was filtered, washed with
`
`pyridine (200 ml) and ethyl ether (100 ml), and dried ina
`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 ml), and dried.
`
`Silica II. Functionalization of Silica Gel
`Triethoxysilylpropylamine
`To Porasil C (10 g) was added triethoxysilylpropylamine
`(15 ml) and toluene (50 ml), and the mixture was refluxed for
`7 hours. The mixture was cooled. The silica was filtered off and
`
`(Porasil C) with
`
`washed with pyridine (2 x 100 ml). 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 ml)
`and ethyl ether (3 x 50 ml), and dried in a desiccator.
`
`
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`Bz
`
`General Procedure for the Preparation of 5'-O-Dimethoxytrityl-
`2'-Deoxynucleoside-3'-O-Succinic Acids
`(dT, aaBZ,
`The 5'-O-dimethoxytrityl-2'-deoxynucleoside
`or ac®,
`2 mmole) and N,N-dimethylaminopyridine (2 mmole)
`dG”
`were dissolved in pyridine (50 ml). To the clear solution was
`added in portions succinic anhydride (2.1 mmole) at room temper-
`ature. The mixture was stirred for 48 hours. Water
`(3 ml) was
`
`11,12
`
`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 ml) of citric acid (4 mmole). The free 3'-0O-
`
`succinic acid was mainly partitioned to the organic phase. The
`solution was dried over Na SO, and concentrated at reduced
`pressure. The solid residue was dissolved in dichloromethane
`(50 ml) and precipitated from n-hexane (300 ml). The nucleoside-
`3'-O-succinic acids were isolated in 70-80% yield. TLC analysis
`on silica (10% methanol-dichloromethane)
`showed the reaction
`
`product as a spot below the 5'-O-dimethoxy-2'-deoxynucleoside
`starting material (trace amount). The precipitated succinyl
`
`nucleosides were used without further purifications.
`
`General Procedure for the Reaction of 5'-O-Dimethoxytrityl-2'-
`Deoxynucleoside-3'-O-Succinic Acid with Silica-I and Silica-II
`Silica-I or Silica-II (6 g)
`in pyridine (10 ml) was treated
`
`to obtain the silica free amine.
`with triethylamine (1 g)
`Excess triethylamine was removed by coevaporation with pyridine.
`
`To the silica was added the deoxynucleoside-3'-O-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 mole, 13 g)
`in 50 ml of
`pyridine for 3 hours. The silica was again filtered, washed with
`pyridine (200 ml) 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
`ZnBr.
`in nitromethane to detritylate and 2) ammonolysis, to
`cleave the 5'-O-dimethoxytritylnucleoside. Analyses were perform-
`ed on HPLC using Lichrosorb silica. Detritylation of 100 mg of
`5550560606065
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`(1.5% methanol-
`T-silica yielded 0.012 mmole of trityl alcohol
`dichloromethane,
`2 ml/minute, R(t)=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 ml/minutes, R(t)=3.5 minutes). The PF-, cre. and
`ce2_silica were similarly analyzed and found also to be func-
`tionalized 0.01 mmole/100 mg silica.
`
`General Procedure for the Preparation of 5'-O-Dimethoxytrityl-
`2'=-Deoxynucleoside-3'-O-Methyl Phosphochloridites
`The baseprotected 5 '-O-dimethoxytrityl-nucleoside!!’!? (5.5
`mmole)
`in dry THF (20 ml) was added over 30 minutes to a stirred
`and cooled solution (-78°) of methyldichlorophosphite!?’'4 (5.0
`mmole, 0.48 ml) and sym-collidine (25 mmole, 3.3 ml)
`in THF (20
`ml) under argon. After an additional 30 minutes,
`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 ZnBr5
`To nitromethane (500 ml) 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'-O-Dimethoxytrityl1-N-benzoyl1-deoxyguanosine-3'-p-chlorophenyl-
`(4-cyanoethyl phosphate and b) 5'-O-dimethoxytrityl-N-benzoyl-
`deoxyadenosine-3'-p-chlorophenyl-4-cyanoethyl phosphate were
`both treated with this ZnBr. 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 mmole, 2.4 g)
`
`in THF (40 ml) was made. Acetic anhydride (10 mmole,
`
`1 g) and
`
`sym-collidine (10 mmole, 1.2 g) were added to 10 ml 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 Support
`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 temperature!’ !° » 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 ao and ge. 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 48 hours at room temperature the mixture was again
`concentrated. The residue was taken up in 1 ml of 0.1 M triethyl-
`ammonium bicarbonate (TEAB) and extracted with ethyl acetate and
`
`ethyl ether.
`
`Isolation of the Partially Deprotected 5'-O-Dimethoxytrityl-
`nucleotides and the Fully Deprotected Oligonucleotides
`The reaction mixture in 1 ml of 0.1 M TEAB was analyzed on
`HPLC using C18 columns !®,
`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 ml/minute (see table).
`
`
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`a A
`
`CKNOWLEDGEMENTS
`
`The authors would like to thank KabiGen for supporting this
`
`research project and Marianne Magnusson for typing this
`
`manuscript.
`
`t
`
`Present address: Molecular Genetics Incorporated, 10300 Bren
`Road East, Minnetonka, MN
`55343
`USA
`
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`
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`
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`
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`
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`
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