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Synthesis of oligonucleotides on cellulose by a phosphotriester method

Synthesis of oligonucleotides on cellulose by a phosphotriester method Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 volume 8 Number io 1980 Nucleic Acid s Research Roberto Crea and Thomas Horn Department of Organic Chemistry, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080, USA Received 22 January 1980 ABSTRACT The synthesis of oligothymidilic aoid3, (dT) (where m = U, 7, 10, 13i 16, 19, 22, and 25), was carried out using a solid phase approach In combi- nation with the modified phosphotriester methodology developed in solution. Cellulose was used as the solid support after it3 functionalization with a specially featured dlnucleoside diphosphate, 5'-0-p-chlorophenylphospho- 2'(3')_o-acetyluridilyl-[2'(3')-3']-5'-0-dimethoxytritylthymldine p-chloro- phenylester. The fully protected trideoxynucleoside triphosphate containing only thymidine wa3 repeatedly used to elongate the oligonucleotide chain in the 3'-5' direction. Individual coupling yields ranged from 45* to 75J- The total time needed to prepare (dTJjj was four days. Similarly, the tridecanucleotide d(AGAAGGTACTTTT) was synthesized in good yield. The results show that this approach can be used for a fast and economic way to synthesize oligodeoxynucleotides. IHTRODUCTION The synthesis in solution of oligodeoxynucleotides of defined sequence by a modified phosphotriester method ha3 been shown to be very success- ful and its effectiveness demonstrated in the chemical synthesis of genes for human insulin . The use of protected trideoxynucleotides as 2 3 building block3 ' and the application of high performance liquid chroma- tography (h.p.l.c.) as a purification tool have dramatically increased the speed by which the synthesis of DNA fragments, 12-15 bases long, can be carried out. However, the synthesis of the trinucleotide building blocks on a large scale, required to maintain a complete "codon" library, is a time consuming and costly process. Therefore a method which allows us to reduce the amount of trimer used in each elongation step is highly desirable. A way to reduce the losses of nucleotidic material and avoid the time consuming purification of intermediates, would be the synthesis of the oligonucleotides on a solid polymer. Several approaches to the synthesis of DNA fragments on a solid support have been published (for review see t,5). Although a wide range of polymers have been used, © IRL Press Limited, 1 Falconborg Court, London W1 V 5FG, U.K. 2331 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research chemical synthesis of oligodeoxynucleotide3 has been accomplished mainly Attempts to synthesize oligodeoxy- with the phO3phodiester method ' nucleotides by the phosphotrie3ter method on 3Olid-phase have been and recently a method to synthesize d'T^g wa* publishedd , the synthesis of homo- and hetero- reported. We describe here oligodeoxynucleotides on cellulose using the modified phosphotriester methodology and trideoxynucleotides as building blocks. RESULTS AND DISCUSSION Cellulose was chosen as the 3olid support because of its inherent polarity, its well known swelling properties in pola^ solvents (i.e. pyri- dlne) and its mechanical stability. The polymer was functionalized by reacting the cellulose with the dimer 51-0-p-chlorophenylphospho-2'(3')-0- acetyluridilyl-[2'(3')-3' ]-5'-O-dlmethoxytritylthymidine p-chlorophenyl- ester (Tb). . ,., ° 3 - CNCHiCHA /> o „ u CH cicrH i HO u o/oVa a ~ a-ZoVo' so ^ r vy ?^± k°y ———- k^> HO OH 0 0 1 r THF.BSA ^ r CH.CN, 1-Me-lmictazoJe Ni T \ X I 80% a CNCH^O 0 c.-®-o'V - TPSTa.Py 2 . EtjN: Py : H,o • R = Cf.CH,CH, SCHEME I Th» synthesis of (Jh_) is outlined in Scheme 1 and it3 features provide the following advantages: A) It can be synthesized from easily available starting materials. 3) Coupling of the ph03phodiester function to the cellulose with TPSTe provide the attachment of (7b) to the polymer (see experimental part). C) Deblocking of dimethoxytrityl group (DMT) from the deoxy- moiety in 2332 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research acidic medium affords the corresponding 5'-hydroxyl derivative for the subsequent coupling step; therefore chain extension of the oligomer on the cellulose occurs in the more favorable 3'—5" direction. D) The release of the synthesized oligonucleotide from the polymer is simple, fast, and essentially complete under the conditions used for the removal of the ba3e-labile protecting groups of the oligonucleo- tidic chain. The aqueous concentrated ammonia hydrolyses the acetyl group of the uridine used as anchoring group . Subsequent nucleo- philic attaok of the free 2'(31)-hydroxyl group on the phosphate moiety triggers the release of the oligonucleotidlc chain with a free 3' end (scheme 2). 7b U- llrocyl T" Thytnine R" p-chloroph«ny1 TPSTe, Cellulose TPSTe • Triitopropylbwzern- tulfonylutrozoli OMT • 4,4 '-dimtthoiytfityl CE • 2-cyanoithyl OMTO I 2% BenzenesuKonic Acid T T T I 9 I 9 I 9 2. DMTO TPSTe D hOPO LOPO, kOPO" , OR N OR n x N OB^ nnN OR 3. Ac O/Py DMT i i ?ii + °*A ° i — HO-kkOPOlkOH ^ P OPOH Cellulose V^ 6-/T -o- xo-l/ -^ SCHEME 2 2333 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Uridine was protected at it3 2', 3'-diol group by treatment with tri- methyl orthoacetate in THF in the presence of benzenesulfonic acid (BSA) as cataly3t. The 2', 3'-cyclic derivative (2) was phosphorylated with the monofunctional phosphorylating agent 2-cyanoethyl-p-chlorophenyl- 2 14 pho3phorochloridate ' (^3_, 2 equivalents) in acetonitrile and 1-methyl- imidazole (1 equivalents). The resulting compound (H) wa3 treated with 80S aqueous acetic acid (10 minutes) and the 2'(3')-0-acetyl derivatives (5_) isolated by silica gel column chromatography as a mixture of isomers. Finally, this material (5.) was reacted with (6), 5'-0-dimethoxytrityl-3'-0- p-chlorophenylthymidilyl phosphate, (1.1 equivalents) in pyridine with TPSTe (2.8 equivalents) a3 coupling agent. The dimer (2a) so obtained was purified on 3ilica gel and precipitated from petroleum ether (32J yield). The efficiency of the release mechanism was tested by treatment of (2a_, 10 rag) with concentrated ammonia. After 30 minutes at 50 °C, the starting material (7a_) had completely degraded into a base-line material and 5'-dimethoxytritylthymidine (TLC analysis) . Coupling of the dinucleotide to cellulose was accomplished in the fol- lowing way: the 2-cyanoethyl group was selectively removed from the fully protected dlmer (Ja, 0.2S mraole) by treatment with a solution of trlethyl- amine-pyridine-H 0 (1:3:1 v/v) for 25 minutes at room temperature. Under these conditions the acetyl group was completely 3table. The phosphodiester derivative (7b) so obtained was dried with pyridine and reacted with cellulose (Whatman CC31, 0.7 g) in anhydrous pyridine in the P"es«mce of TPST? (O.o mmole). The attachment of (7_o_) to the polymer was followed by aomtorir.? the release of DMT "esidue J at 194 nn after acidic t-eatment of a sraall sample of polymer (33° experimental). An average incorpo-at'.on of ca. 100 umole dime"/? cellulose was obtained. Elongation of the nucleotidic chain was carried out with the trideoxv- nucleotide (Q_) ; the synthesis of this compound has previously been de- scribed . The functionalized cellulose (J3) (100 mg, 9.7 umole of dimer) was first treated with 21 benzenesulfonic acid (BSA) in CH OH/CHC1 at 0° C in order to make the 5'-hydroxyl group of the thymidine available fo- the chain extension. After several coevaporations with pyridine, the dried polvraer was reacted with (0) (50 |jmoles) in pyridine containing TPSTe (220 ptaole). TPSTe is known to be a very powerful condensing agent ' ; therefore the coupling reaction was stopped after two hours. The solid support was collected by centrlfugatlon and the supernatant saved for the recovery of unreacted trimer (£) (see below). The cellulose was washed 2334 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research with pyridine and then partially acetylated in order to block the un- reacted 5'-hydroxyl groups. The resulting cellulose was washed (MeOH), treated with 2% BSA, and then coupled again with the same quantity of trlmer {$). Eight such cycles were repeated under the same conditions in four days. Table 1 lists the operations per coupling cycle. After each cycle a simple (ca. 10 mg) of the polymer was treated with cone. aq. NH followed by 80% aq. AcOH to remove all protecting groups. The unblocked oligonucleotidic material was then analyzed to calculate the yields per coupling. For this purpose we used two different methods: a) the release of DMT residue in acidic solution (494 nm) , which gave a semiquantitative estimate of the amount of trimer (£) reacted, and b) h.p.l.c. analysis , which allowed the accurate calculation of the yields per coupling derived from the peak areas (254 nm) (figs. 1 and 2) . Table 2 shows the yields based on h.p.l.c. analysis. H.p.l.c. analysis of the products showed that the major co-products were due to incomplete chain elongation. Small quantities of side product? that could not be accounted for are probably due to direct reaction of the trimer (£) with the hydroxyl groups of the cellulose or chain degradation TABLE 1 STEP OPERATION CHEMICAL USED VOL.** TIME (min.) Removal of DMT 2X BSA in CHCl /MeOH* 10 10 Washing MeOH, Pyridine 50,20 5 3 Drying Pyridine 10 10 A Coupling Trimer, TPSTe, Py. 1 120 Washing Pyridine 2 5 Acetylation Ac.O/Pyridine 1 30 Washing Pyridine 8 10 Methanol 30 10 * At 0 C (ice-wate r bath). ** Milliliter s per 100 mg polymer. 2335 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research TIM E (mtnuccs) Fig.l. H.p.l.c. analysis of the oligothymidilic acids obtained after two (n=2), three (n=3), and four (n=i) coupling cycles. during deblocking. A method to block reactive hydroxyl groups after functionalization (i.e. coupling of (Jb_) to the cellulose) without changing the mechanical properties of the polyrasr support is currently under investigation. Purification of the products was performed by h.p.l.c. on a strong anionlc exchange resin (Pennaphase AAX) by collecting the material corresponding to the last eluted peak. The pooled solution (ca. 1.0 O.D. unit at 25t nm) was concentrated and the nucleotidlc material desalted on 2336 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research 16 18 16 Time (minutes) Fig.2. H.p.l.c. analysis of dT obtained after five (n=5), six (n=6), and eight (n=8) coupling cycles. Bio-gel P-2. The size of each compound wa3 confirmed by gel electro- phoretic analysis (fig. 3). Two-dimensional sequence analysis of (dT) showed the expected pattern for this compound (results not shown). The synthesis in solution of oligonucleotides of defined sequence using the trinucleotide block approach required the use of mo3t of the 61 pos- sible trlnucleotide triphosphates. In order to investigate the reactivity of trimers with different base composition, we synthesized the following TABLE 2 Cycle Compound Yield No. % 2 (dT) <dT>,o 4 (dT) 62 ( 3 < dT \ 6 <dT> 57 |9 WT) a (dT) ^ 48 2337 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research oligonueleotide: d(AGAAGGTACTTTT) (J_6) . This oligoraer, 13-base3 long, wa3 selected because its synthesis requires the use of four trimer3, each hav- ing a different ba9e at their 3'-phosphate end (3ee scheme 3)- Starting from the polymer (1.0) (n=l, 120 mg), the synthesis of the fully protected oligonueleotide derivative was accomplished by sequential addition of the th-ee trlmera (50 pinoles each): DMT-Tp ABp CBp (12), o o o — SCHEME 3 10 ( n=l ) 2% BSA I HO-Tp Tp Tp TpAcUp -Cellulose 1) DMT-Tp ABp CBp (12) o o o 2) Ac O/Py ( 40% ) 3) 2X BSA Bz Bz Ac,, HO-Tp A p C p Tp Tp Tp Tp Up -Cellulose o o o r o o o o o Bz iBu iBu DMT-A Ac 0 / 2) Ac 0/Py 2 ( 421 ) 3) 2% BS A Bz iBu IBu Bz Bz Ac, HO-A p G p G p Tp A p C p Tp Tp Tp Tp Up -Cellulose *o o Ko o Ko Ko po o o ^o Ko Bz iBu Bz DMT-A p G p A p (1_4) ( 451 ) Bt iBu Bz Bz IBu iBu Bz Bz Ac., DMT-A pGpApApGpGpTpApCpTpTpTpT p Up -Cellulos e o o *o o o o o o o Ko o ro o ro (15 ) 1 ) cone. NH, 2 ) 80% AcOH HO-ApGpApApGpGpTpApCpTpTpTpT-OH (16 ) 2338 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Fig. 3. Gel electrophoretic analysis on a 201 polyacrila- tnide slab, after incubation with [ T - 32 P ] ATP and poly- nucleotide ktnase. dT dT dT 19 22 2 5 DMT-ABp C^G 18 " V±) and DMT-ABp CiB>UABp (U), where p p-chlorophenyl- phosphate. Tie trimers (_12), (1_3_), and (Vi) were prepared from the corresponding fully protected derivatives by removal of the cyanoethyl group from the 3'-terminal phosphotriesters with Et N-Py-H 0 (1:3:1 v/v) . The three cycles were carried out under the same conditions as de- scribed in the synthesis of oligothymldilic acids. Scheme 3 outlines the synthesi3 of (.16) and the yields per coupling calculated from h.p.l.c. analysis (see fig. 5) are shown in parentheses. The final polymer (1^, 85 mg) wa3 treated with base (cone. aq. NH ) and acid (80* aq. AcOH), the cellulose residue was pelleted off and the supernatant evaporated to dryness. The residue, dissolved in t> aq. NH (? ml), wa3 washed with ether and used for the isolation of (JJS). This was accomplished by repeated runs on h.p.l.c; the material corresponding to the last eluted peak wa3 collected and pooled. After desalting on Bio-Gel ?2, the product (1^6) was analyzed as described for the oUgothymidilic acid3. From 0.5 ml of cnjde solution ca. 10 A__., units of pure product (V5) was obtained. Gel electrophoretic analysis showed that (16) 2339 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Fig. h. Two-dimen- sional analysis of a partial venom phosphodiesterase digesc of d(AGAAGGTACTTTT).Dimension 1, electrophoresis on cellulose acetate strip at pH 3.5, dimen- sion 2, homochroraatography on DEAE cellulose thin layer plate. has the same mobility as (dT) (see fig. 5) and two-dimensional sequence ll ft ft analysis of the partial vanom phosphodiesterase digest confirmed that (16) had the expected sequence (fig. H). The recovery of the excess trimers was accomplished by one of the following procedures: a) purification of the trimer 3'-phosphodiester by gel exclusion column chromatog-aphy on Sephadex LH-60 , or b) reuse of the filtrate solution for further condensation either on polymer support or in solution. From the data obtained 30 far we calculated that 35-701 of the excess trime^ could be recovered. CONCLUSIONS This study demonstrates that a solid phase approach combined with the trinucleoti'le phosphotrlester methodology ha3 several advantages over the synthesis of oligonucleotides in solution . Only one kind of 2340 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Fig.5. (a) Synthesis of d(AGAAGGTACTTTT) monitored by h.p.l.c. after the second, third, and fourth coupling cycle. (b) Gel electrophoretic analysis of the product (P) purified by h.p.l.c. References are and a dodecamer (Ref). 10 12 M 16 IB 20 Time tminultt) trinucleotide building blocks (3'-phosphate) 19 necessary for the chain elongation. The synthesis is faster, because washing procedures substitute column chromatography on silica gel. This also reduces the loss of oligonucleotidic material. Because of the recovery of the unreacted trinucleotide at the end of each coupling step, and the reduced scale of synthesis on the cellulose, the amount of trimer used per coupling is significantly reduced. Functionalization of cellulose with a ribo- deoxvribo dinucleo3ide diphosphate containing uridine and one of the fou- 2341 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research deoxyribonucleosides provides the starting polymer necessary for the solid-phase synthesis of DNA fragments with different bases at the 3'-end. In the synthesis of an oligonucleotlde 13 bases long with trinucleo- tides of different sequence, the yields per coupling were lower than the ones in the synthesis of homopolymer3 (dT) . However, no base speci- ficity has so far been observed. MATERIALS AND GENERAL PROCEDURES Analytical grade reagent3 were U3ed throughout thi3 synthetic study. Pyridine was dried over KOH, distilled, and stored over molecular sieves (type HA). Thin layer chromatography (TLC) was performed on silica gel plates (Merck, Silica Gel 60 F254) with methanol/chloroform (1:9 v/v) as solvent system. The presence of the DMT group wa3 monitored by 3praying the TLC plate with 10J aq. ^SO ^ and heating the plate to 60° C. Puri- fication by column chromatography was performed on 3ilica gel (Merck, Silica Gel 60H). H.p.l.c. was performed with a Spectra-Physics SP 8000 Liquid Chroma- tograph on Permaphase AAX (du Pont) (50 cm x 0.1) cm I.D.) using a linear gradient of water to 1M KC1, pH M.5, at a rate of 3* per minute. The elu- tion was performed at constant flow (3 ml/min.) at 60°C. Cellulose (Whatman CC 3 D was washed with pyridine, rin3ed extensively with methanol, and dried in vacuo over p °5" A^1 reactions at the polymer level were carried out under magnetic stirring. The fully protected mono- and trinucleotides were synthesized by following the procedures described previously . 2,H,6-T^llsopropyl- benzenesulfonyltetrazole (TPSTe) wa3 synthesized according to the proce- dure reported in the literature and recrystallized from benzene- petroleum ether. 2'(3')-0-Acetylurldlne-5'-0-2-eyanoethyl-p-chlorophenylphosphat e (5) . Uridine , Q), (61 mmole) was suspended in a solution of anhydrous THF (200 ml) containing trimethvl orthoacetate (125 mmole) and anhydrous ben- zenesulfonl c acid (BSA) (0.6 g) . After stirring at 25° C for ten min- ute s under exclusion of moi3ture, the solution had become clear. TLC showed complete conversion of uridine into a new compound with higher R . The reaccion was quenched by addition of methanol saturated with NH (5 ml) and ths solvents removed under reduced pressure. The oily residue was dissolved In CHC1 (300 ml) and extracted with saturated aq. NaHCO 2342 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research solution (150 ml) and water (150 ml). The organic layer wa3 dried (MgSO^) and evaporated to drynes3. Without further purification, 21 ,3'-0-methoxyethylideneuridine (.2) (ca. 60 mmole) was dissolved In anhydrous acetonitrile (200 ml) containing 1-N-methylimidazole (200 mmole). The solution was chilled (ice-water) 2 14 and freshly prepared 2-cyanoethyl-p-chlorophenylphosphorochloridate ' (90 mmole) in CH CN (25 ml) was added dropwise over a 15 minute period. The solution was then left to warm up to room temperature. TLC analysis showed virtually complete phosphorylatlon of (2)- The solvent was removed under reduced pressure, and the residual oil partitioned between CHC1, (200 ml) and aq. NaHCO (150 ml). The CHC1 layer was washed with water (150 ml) and then concentrated to a small volume (50 ml). This solu- tion was triturated twice with pentane (500 ml) to remove most of the 1-N-methylimidazole. The product obtained (^) was treated with 80t aqueous acetic acid (200 ml) at room temperature for 10 minutes. Finally, the ace- tic acid was removed under reduced pressure by coevaporation with absolute ethanol. TLC of the glass residue showed only one product. Purification of (5_) was performed on a silica gel column (150 g) . After washing with CHC1, the pure product was eluted with a solution of MeOH in CHC1, (1 to 4J v/v). The fractions containing the product were pooled and con- centrated to a small volume. Precipitation of (5) from petroleum ether (35°- 60° C) gave a white solid (14.5 g; 50% yield based on uridine). Ft =0.31; UV (95* EtOH): X 258 nm ( E 9,600) , X . 226 nra ( E 1,800). i max mln Anal: C^H^N O^PCl (529-5); calcd: C 45.32, H 3-96, Cl 6.70, N 5-90, P 7.90; found: C 45.2, H 4.09, Cl 6.91, N 5.88, P 7.98. 5'-0-2-Cyanoethyl-p-chloropho;phophenyl-2'(3')-0-acetyluridllyl- C2'(3' )-3' ]-5'-0-dlmethoxytrltylthynildlne p-chloroptienyle3ter(7a). 5'-0-Dimethoxytritylthymidine-3'-0-2-cyanoethyl-p-chlorophenylphosphate (7.0 mmole) was treated with a solution of triethylamlne/pyridine/water (1:3:1 v/v) (50 ml) at room temperature for 25 minutes. The phosphodiester derivative (j>) so obtained was dried by coevaporation with anhydrous pyri- dlne (3 x 25 ml). 2'(31 )-0-Acetylu'-idine-5'-0-2-cyanoethyl-p-chlorophenyl- phosphate (5_) (6.6 mmole) was added to derivative (^) and the mixture d-ied again with pyrldine (30 ml). TPSTe U0.5 mmole) in anhydrous pyidine (50 ml) was added to this solution. The reaction mixture was kept in vacuo and protected from the light for 16 hours. The reaction was stopped by addition of water, and the solvents removed under reduced pressure. The 2343 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research residual material in CHC1 (250 ml) was extracted with aqueous NaHCO (100 ml), and then washed with water (100 ml). The combined CHC1 extracts were _e_vaporated to dryness. Chromatography of the product on silica gel (75 g) was performed using MeOH/CHCl (1:99 v/v) as solvent system. The fractions containing the product were evaporated to a glass. Precipitation of this material from petroleum ether (35° - 60° C) gave a colorless solid (2.6 g, 32% yield). Its homogeneity was confirmed by TLC analysis (R =0.65). Anal: C H^N 0 P (1247); calcd: C 54.90, H 4.44, Cl 5.68, N 5.61, P 4.96; found C 54.61, H 4.44, Cl 5.92, N 5-91, P 5.31. To confirm the release mechanism, this compound (Ja, 10 mg) was treated with cone, aqueous aramonia/pyridine (9:1 v/v; 2 ml) for 30 minutes at 60° C TLC analysis showed a complete degradation of (Ja.) into a baseline material and a compound containing the DMT group. This latter compound was identi- fied as 5'-DMT-thyraidine (TLC). Removal of DMT with 2% BSA gave a compound identical to thymidine. FuncUonalizatlon of cellulose with (7b) The fully protected dimer (7.3.) was treated with a solution of pyridine- triethvlamine-water (3:1:1 v/v) (20 nil) at room temperature. After 25 min- utes, TLC showed complete conversion of (J_a) into its phosphodiester deri- vative (base-line material (7b)). The latter material was dried by coevap- oratlon with pyridine (3 x 15 ml) and then reacted with dry cellulose (Whatman CC 3D (0.7 g) in anhydrous pyridlne (3 ml) in the presence of TPSTe (0.23 g). The mixture was stirred overnight (16 hours) at room temperature. The polymer was then filtered (sintered glass filter M) and washed with dry pyridine. The filtrate was stored for the recovery of un- reacted dime" (Jb). The cellulose was washed extensively with MeOH (60 ml) and dried in a dessicator over KOH. The extent of coupling of (£b) to the polymer was determined by treat- ing a sample of cellulose (8, 10 mg) with 2% BSA in CHC1 -HeOH (7:3 v/v) (2 ml) at 0°C for 10 minutes. After centrifugation of the 3olid particles, the ^bsorbancy of the released DMT group was measured at 494 nm. A stan- dard curve was obtained by using different amounts of 5'-O-dimethoxyt^ityl thymidine In 2% BSA 3tock solution. From the data obtained, we calculated an average incorporation of ca 100 pmole3 dimer {Jb) per g cellulose. Another sample (.&_, 10 mg) was treated with cone, aqueous ammonia for 8 hours at 60°C. The polymer was centrifuged off and the supernatant concen- 2344 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research trated to dryness. The residual material was dissolved in CHC1 (1 ml) and analyzed by TLC. The only product detected wa3 5'-O-dimethoxytrityl thymidine. Treatment of the chloroform solution with U% BSA in CH 0H- CHC1 (1 ml) gave thymidine and DMT residue. From the total amount of DMT (194 run) we calculated that the ammonia treatment had released ca 95$ of the 5'-0- DMT-thymidine from the polymer. Synthesis of ollqothymidilic acids, (dT)m (m=4,7,10,13,16,19,22, and 25) . The functionalized polymer ((3, 100 mg; 9.7 pmole of dimer) was fir3t treated with 2% BSA in CHC1 /MeOH (7:3 v/v, 10 ml) at 0°C for 10 minutes a3 described above. After washing the polymer on the filter with methanol (50 ml) the cellulose was dried by flushing with anhydrous pyridine (20 ml) The trimer (£) (50 u moles) was mixed with the polymer in a 10 ml round- bottom flask. The mixture was dried by coevaporation with pyridine (2x 5 ml) and finally resuspended in anhydrous pyridine (1.0 ml) . TPSTe (75 mg) was added quickly and the reaction mixture kept in vacuo. After two hours of continuous stirring, pyridine (2 ml) wa3 added to the reaction vessel and the polymer recovered by centrifugation. The supernatant was collected separately for the recovery of excess trimer (5.). The polymer so obtained was resuspended in a solution of pyridine-acetic anhydride (10:1 v/v, i ml) and stirred fo^ 30 minutes. After washing the cellulose on a filter with pyidine (8 ml) and methanol (30 ml), the polymer was ready for the next coupling step (see Table 1) . The following cycle: a) deblocking of DHT group from the polymer with BSA, b) coupling of the polymer so obtained with trimer (9_) in pyridine containing TPSTe, and c) acetylation of the resulting polymer with pyri.dine/Ac 0 was repeated seven more times under the same conditions. Release of the ollaonucleotldes rrom the polymer and removal of protecting The polymer (H), n=l-8, 10 mg) suspended in pyridine (0.5 ml) was treated with concentrated aqueous ammonia (4 ml) at 60° C for 8 hours. The solid polymer was removed by centrifugation and discarded. The supernatant was evaporated to dryness, and the residue treated with 80? aq. acetic acid at room temperature for 15 minutes. The acetic acid was removed by evapor- ation under reduced pressure and the solid residue redissolved in t% aque- ous ammonia (2 ml). After washing with ether (3x 1 ml) the aqueous solu- tion was analyzed on h.p.l.c. For each coupling step the chromatogram 2345 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research showed the formation of a new product with higher retention time (3ee figs. 1 and 2) . The yields per coupling were calculated from the peak areas and are based on the next shorter oligomer. For each elongation step the final product, after complete deblocking, was isolated by preparative h.p.l.c on Permaphase AAX. The fractions corresponding to the last peak were pooled (0.5 - 1.0 O.D. units at 25*4 nm), the solution concentrated and desalted on Bio-gel P-2. Gel electrophoretic analysis on a 20$ acrylamide slab of the oligomer3 (dT) (m=13,lb,19, 22, and 25) after their incubation with [y - P]-ATP and T. polynucleotide kinase, showed that each fragment released from the polymer had the expected 3ize (fig. 3). Furthermore, [ P] (dT) wa3 analyzed by two dimensional sequence analysis of its partial venom phosphodiesterase digestion products . The analysis con- firmed the structure of this compound. Synthesis of d(AGAAGGTACTTTT) (16). The functionalized polymer {B_, 125 mg) was treated with 2$ BSA as des- c-ibed above and used for the synthesis of the 13-base long oligonucleotide of defined sequence (1_6). This was accomplished by using/four dffferent trimers: DMT-Tp Tp Tp (9), DMT-Tp ABp CBp (U), DMT-A^G^Vp^ (_13), o o o o o o and DMT-A p G p A p (_14), where p = p-chlorophenylphosphate. The coupling of these triraers (50 umoles each) to the polymer was carried out in the sequential order shown in scheme 3, under the same conditions as reported above for the 3ynthe3is of oligothymidilates. The yields obtained for each coupling are shown in scheme 3 and were calculated by h.p.l.c. analysis based on the next shorter fragment. The fully protected cligomer obtained after the fourth cycle was re- leased from the polymer and deblocked with ammonia and acetic acid as de- scribed above. The isolated product (_16) (h.p.l.c.) had the same mobility as (dT) on 20$ polyacrylamide slab (see fig. 5). Furthermore, two-dimensional sequence analysis of the partial venom phosphodiesterase digest of (lj>) confirmed the expected sequence (fig. 4). Recovery of the exoes3 trlnucleotldes A. Chromatography on Sephadex LH-60. The filtrate pyridine solution (3 ml) containing the excess trinucleotid e (DMT-Tp Ap Cp , ca <10 umole) and TPSTe was used for the recovery of thi3 compound (\2) by gel exclusion chromatography. Water (0.5 2346 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research ml) wa3 first added and the solution evaporated to dryness. The residue wa3 dissolved in THF/MeOH (95:5 v/v, 1 ml) and applied to a column of Sephadex LH-fiO (0.7 x 100 cm) preequilibrated in the same solvent. Isocratic elution of the product was obtained with THF/MeOH (95:5 v/v). Fractions of 1 ml were collected. The fractions containing product (fraction 38 to 50) (detected as DMT-posltive baseline material) were pooled, concentrated and the trimer precipitated from petroleum ether (25 mg, 35S recovery yield). B. Reuse of the excess trimer for synthesis in solution . The pyrldlne solution containing the excess DMT-Ap Ap Gp (ca MO o Q o ymole) and TPSTe was dried by coevaporation with pyridine (2x2 ml). 3'_0-Ani9oyl- 2-N-isobutrylguanosine (It mg, 29 umole) was added and the mixture dried by coevaporation with pyridine (2x2 ml). Additional TPSTe (50 mg) was added, and the reactants dissolved in pyridine (1 ml). The reaction mixture wa3 kept iri vacuo for 6 hours. TLC analysis showed complete conversion of the nucleoside into the product tetramer. This latter compound was purified on a silica gel column and obtained as a homogeneous solid after precipitation from petroleum ether (23 mg) . C. Reuse for solid-phase synthesis. The pyidine solution (3 ml) recovered from a coupling step containing the t>-iraer DMT-Tp Tp Tp (°_, ca M0 uraole), was reused to elongate the oligonucleotidic chain on cellulose. The trimer was added to the polymer (10) (n=l, 100 mg) and the suspension dried by coevaporation with pyridine. An additional portion of TPSTe (75 mg) was added and the reaction mixture suspended in pyridine (1 ml). The fully protected ollgomer was released from the polymer and deblocked with aq. ammonia and aq. acetic acid as described above. H.p.l.c. analysis showed a coupling yield of 43> for d(T) based on dtT^ . ACKNOWLEDGEMENTS The authors are grateful to Dr. D.G. Kleid, Dr. P.M.J. Burgers and Dr. J.H. van Boom for their valuable comments and critical reading of the manuscript. REFEREHCES Abbreviations: DMT = M,1'-dimethoxytrityl, BSA = benzenesulfonic acid, TPSTe = 2,1,6,-Triisopropylbenzenesulfonyltetrazole, CE = 2-cyanoethyl, Bz = benzoyl, iBu = isobutryl, Ac = acetyl. 2347 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research 1 . Crea, R., Hiro3e T. and Itakura, K. (1979) Tetrahedron Letters, 5, 395-398. 2. Crea, R., Kraszewski, A., Hirose, T. and Itakura, K. (1978) Proc. Natl. Acad. Sci. (U.S.A.), 75, 5765-5759. 3. Hiro3e, T., Crea, R. and Itakura, K. (1978) Tetrahedron Letters, 28, 2449-2452. U. Ko33el, H. and Seliger, H. (1975) Fortsuch. Chem. Org. Natur3toffe 32, 5. Gait, H.J. (1979) Polymer-Supported Reagents, Catalysts and Protecting Groups (P. Hodge and D.C. Sherrington, Eds), Wiley, London, in press. 6. Belagaje, R-, Brown, E.L., Gait, H.J., Khorana, H.G. and Norris, K.E. (1979) J. 3iol . Chem. 254, 5754 and subsequent papers. 7. Narang, C.K., Brunfeldt, K. and Norris, K.E. (1977) Tetrahedron Letters 21 , 1819-1822. r.ait , M.J. and Sheppard, R.C. (1977) Nucleic Acids Res. 4, 1135-1158. Potapov, V.K., Veiko, V.P., Koroleva, O.N. and Shabarova, Z.A. (1979) Nucleic Acids Res. 6, 2041-2056. 8. Pless, R.C. and Letsinger, R.L. (1975) Nucleic Acid3 Research 2, 773- Seliger , H. (1975) Die Hacromoleculare Chemie 176, 1611-1627. 9- Miyosht, K. and Itakura, K. (1979) Tetrahedron Letters 38, 3635-3638. 10. Stawinski, J. , Hozumi, T., Narang, S.A., Bahl, C.P. and Wu, R. (1977) Nucleic Acid3 Re3. 4, 353-371. 11 . Koster, H. and Heyns, K. (1972) Tetrahedron Letters, 16, 1531-1534. 12. The three "ribo-deoxyribo" dinucleoside diphosphates made up by uridine and 4-N-benzoylcytidlne, 6-N-benzoyladenosine, and 2-N-isobutryl- guanoslne, rsspectively, were synthesized in a similar manner and isolate d as homogeneous solid3 in comparable yield. The relevant data wil l be published elsewhere. 13- Griffin , B.E., Jarraan, H., Ree3e, C.B., Sulstow, J.E. (1970) Tetrahedron 26, 1023. 14. Crea, R., et al , manuscript in preparation. 15. Seliger, H. , Holupirek, M. and Gortz, H.H. (1973) Tetrahedron Letters, 24, 2115-2118. 16. Jay, E., Bambara, R., Padmanabhan, P. and Wu, R. C1974) Nucleic Acids Res. 1, 331-353- 17. -le Rooij, J.F.M., Arentzsn, R., den Hartog, .I.A.J., van der Marel, G. and van Boom, J.H. (1979) J. Chromatography 171, 453-U59- 18. After thi3 paper was submitted for publication methods for the synthesis of ollgodeoxynucleotides on solid supports were reported using a phosphite-triester1" 8 and a phosphotriester1"*5 approach, respectively : a. Matteucci, M.D. and Caruthera, M.H. (1980) Tetrahedron Letters, 21 , 719-722. b. Gait, M.J.,Singh, H. .Sheppard, R.C,Edge, M.D.,Green, A.R., Heathcliffe , G.R., Atkinson, T.C., Newton, C.R., and Markham, A.F. (1980) Nucleic Acids Research, 8, 1081-1096. 2348 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Synthesis of oligonucleotides on cellulose by a phosphotriester method

Crea, Roberto; Horn, Thomas
Nucleic Acids Research , Volume 8 (10) – May 24, 1980
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Oxford University Press
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Copyright © 2022 Oxford University Press
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0305-1048
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10.1093/nar/8.10.2331
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Abstract

Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 volume 8 Number io 1980 Nucleic Acid s Research Roberto Crea and Thomas Horn Department of Organic Chemistry, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080, USA Received 22 January 1980 ABSTRACT The synthesis of oligothymidilic aoid3, (dT) (where m = U, 7, 10, 13i 16, 19, 22, and 25), was carried out using a solid phase approach In combi- nation with the modified phosphotriester methodology developed in solution. Cellulose was used as the solid support after it3 functionalization with a specially featured dlnucleoside diphosphate, 5'-0-p-chlorophenylphospho- 2'(3')_o-acetyluridilyl-[2'(3')-3']-5'-0-dimethoxytritylthymldine p-chloro- phenylester. The fully protected trideoxynucleoside triphosphate containing only thymidine wa3 repeatedly used to elongate the oligonucleotide chain in the 3'-5' direction. Individual coupling yields ranged from 45* to 75J- The total time needed to prepare (dTJjj was four days. Similarly, the tridecanucleotide d(AGAAGGTACTTTT) was synthesized in good yield. The results show that this approach can be used for a fast and economic way to synthesize oligodeoxynucleotides. IHTRODUCTION The synthesis in solution of oligodeoxynucleotides of defined sequence by a modified phosphotriester method ha3 been shown to be very success- ful and its effectiveness demonstrated in the chemical synthesis of genes for human insulin . The use of protected trideoxynucleotides as 2 3 building block3 ' and the application of high performance liquid chroma- tography (h.p.l.c.) as a purification tool have dramatically increased the speed by which the synthesis of DNA fragments, 12-15 bases long, can be carried out. However, the synthesis of the trinucleotide building blocks on a large scale, required to maintain a complete "codon" library, is a time consuming and costly process. Therefore a method which allows us to reduce the amount of trimer used in each elongation step is highly desirable. A way to reduce the losses of nucleotidic material and avoid the time consuming purification of intermediates, would be the synthesis of the oligonucleotides on a solid polymer. Several approaches to the synthesis of DNA fragments on a solid support have been published (for review see t,5). Although a wide range of polymers have been used, © IRL Press Limited, 1 Falconborg Court, London W1 V 5FG, U.K. 2331 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research chemical synthesis of oligodeoxynucleotide3 has been accomplished mainly Attempts to synthesize oligodeoxy- with the phO3phodiester method ' nucleotides by the phosphotrie3ter method on 3Olid-phase have been and recently a method to synthesize d'T^g wa* publishedd , the synthesis of homo- and hetero- reported. We describe here oligodeoxynucleotides on cellulose using the modified phosphotriester methodology and trideoxynucleotides as building blocks. RESULTS AND DISCUSSION Cellulose was chosen as the 3olid support because of its inherent polarity, its well known swelling properties in pola^ solvents (i.e. pyri- dlne) and its mechanical stability. The polymer was functionalized by reacting the cellulose with the dimer 51-0-p-chlorophenylphospho-2'(3')-0- acetyluridilyl-[2'(3')-3' ]-5'-O-dlmethoxytritylthymidine p-chlorophenyl- ester (Tb). . ,., ° 3 - CNCHiCHA /> o „ u CH cicrH i HO u o/oVa a ~ a-ZoVo' so ^ r vy ?^± k°y ———- k^> HO OH 0 0 1 r THF.BSA ^ r CH.CN, 1-Me-lmictazoJe Ni T \ X I 80% a CNCH^O 0 c.-®-o'V - TPSTa.Py 2 . EtjN: Py : H,o • R = Cf.CH,CH, SCHEME I Th» synthesis of (Jh_) is outlined in Scheme 1 and it3 features provide the following advantages: A) It can be synthesized from easily available starting materials. 3) Coupling of the ph03phodiester function to the cellulose with TPSTe provide the attachment of (7b) to the polymer (see experimental part). C) Deblocking of dimethoxytrityl group (DMT) from the deoxy- moiety in 2332 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research acidic medium affords the corresponding 5'-hydroxyl derivative for the subsequent coupling step; therefore chain extension of the oligomer on the cellulose occurs in the more favorable 3'—5" direction. D) The release of the synthesized oligonucleotide from the polymer is simple, fast, and essentially complete under the conditions used for the removal of the ba3e-labile protecting groups of the oligonucleo- tidic chain. The aqueous concentrated ammonia hydrolyses the acetyl group of the uridine used as anchoring group . Subsequent nucleo- philic attaok of the free 2'(31)-hydroxyl group on the phosphate moiety triggers the release of the oligonucleotidlc chain with a free 3' end (scheme 2). 7b U- llrocyl T" Thytnine R" p-chloroph«ny1 TPSTe, Cellulose TPSTe • Triitopropylbwzern- tulfonylutrozoli OMT • 4,4 '-dimtthoiytfityl CE • 2-cyanoithyl OMTO I 2% BenzenesuKonic Acid T T T I 9 I 9 I 9 2. DMTO TPSTe D hOPO LOPO, kOPO" , OR N OR n x N OB^ nnN OR 3. Ac O/Py DMT i i ?ii + °*A ° i — HO-kkOPOlkOH ^ P OPOH Cellulose V^ 6-/T -o- xo-l/ -^ SCHEME 2 2333 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Uridine was protected at it3 2', 3'-diol group by treatment with tri- methyl orthoacetate in THF in the presence of benzenesulfonic acid (BSA) as cataly3t. The 2', 3'-cyclic derivative (2) was phosphorylated with the monofunctional phosphorylating agent 2-cyanoethyl-p-chlorophenyl- 2 14 pho3phorochloridate ' (^3_, 2 equivalents) in acetonitrile and 1-methyl- imidazole (1 equivalents). The resulting compound (H) wa3 treated with 80S aqueous acetic acid (10 minutes) and the 2'(3')-0-acetyl derivatives (5_) isolated by silica gel column chromatography as a mixture of isomers. Finally, this material (5.) was reacted with (6), 5'-0-dimethoxytrityl-3'-0- p-chlorophenylthymidilyl phosphate, (1.1 equivalents) in pyridine with TPSTe (2.8 equivalents) a3 coupling agent. The dimer (2a) so obtained was purified on 3ilica gel and precipitated from petroleum ether (32J yield). The efficiency of the release mechanism was tested by treatment of (2a_, 10 rag) with concentrated ammonia. After 30 minutes at 50 °C, the starting material (7a_) had completely degraded into a base-line material and 5'-dimethoxytritylthymidine (TLC analysis) . Coupling of the dinucleotide to cellulose was accomplished in the fol- lowing way: the 2-cyanoethyl group was selectively removed from the fully protected dlmer (Ja, 0.2S mraole) by treatment with a solution of trlethyl- amine-pyridine-H 0 (1:3:1 v/v) for 25 minutes at room temperature. Under these conditions the acetyl group was completely 3table. The phosphodiester derivative (7b) so obtained was dried with pyridine and reacted with cellulose (Whatman CC31, 0.7 g) in anhydrous pyridine in the P"es«mce of TPST? (O.o mmole). The attachment of (7_o_) to the polymer was followed by aomtorir.? the release of DMT "esidue J at 194 nn after acidic t-eatment of a sraall sample of polymer (33° experimental). An average incorpo-at'.on of ca. 100 umole dime"/? cellulose was obtained. Elongation of the nucleotidic chain was carried out with the trideoxv- nucleotide (Q_) ; the synthesis of this compound has previously been de- scribed . The functionalized cellulose (J3) (100 mg, 9.7 umole of dimer) was first treated with 21 benzenesulfonic acid (BSA) in CH OH/CHC1 at 0° C in order to make the 5'-hydroxyl group of the thymidine available fo- the chain extension. After several coevaporations with pyridine, the dried polvraer was reacted with (0) (50 |jmoles) in pyridine containing TPSTe (220 ptaole). TPSTe is known to be a very powerful condensing agent ' ; therefore the coupling reaction was stopped after two hours. The solid support was collected by centrlfugatlon and the supernatant saved for the recovery of unreacted trimer (£) (see below). The cellulose was washed 2334 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research with pyridine and then partially acetylated in order to block the un- reacted 5'-hydroxyl groups. The resulting cellulose was washed (MeOH), treated with 2% BSA, and then coupled again with the same quantity of trlmer {$). Eight such cycles were repeated under the same conditions in four days. Table 1 lists the operations per coupling cycle. After each cycle a simple (ca. 10 mg) of the polymer was treated with cone. aq. NH followed by 80% aq. AcOH to remove all protecting groups. The unblocked oligonucleotidic material was then analyzed to calculate the yields per coupling. For this purpose we used two different methods: a) the release of DMT residue in acidic solution (494 nm) , which gave a semiquantitative estimate of the amount of trimer (£) reacted, and b) h.p.l.c. analysis , which allowed the accurate calculation of the yields per coupling derived from the peak areas (254 nm) (figs. 1 and 2) . Table 2 shows the yields based on h.p.l.c. analysis. H.p.l.c. analysis of the products showed that the major co-products were due to incomplete chain elongation. Small quantities of side product? that could not be accounted for are probably due to direct reaction of the trimer (£) with the hydroxyl groups of the cellulose or chain degradation TABLE 1 STEP OPERATION CHEMICAL USED VOL.** TIME (min.) Removal of DMT 2X BSA in CHCl /MeOH* 10 10 Washing MeOH, Pyridine 50,20 5 3 Drying Pyridine 10 10 A Coupling Trimer, TPSTe, Py. 1 120 Washing Pyridine 2 5 Acetylation Ac.O/Pyridine 1 30 Washing Pyridine 8 10 Methanol 30 10 * At 0 C (ice-wate r bath). ** Milliliter s per 100 mg polymer. 2335 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research TIM E (mtnuccs) Fig.l. H.p.l.c. analysis of the oligothymidilic acids obtained after two (n=2), three (n=3), and four (n=i) coupling cycles. during deblocking. A method to block reactive hydroxyl groups after functionalization (i.e. coupling of (Jb_) to the cellulose) without changing the mechanical properties of the polyrasr support is currently under investigation. Purification of the products was performed by h.p.l.c. on a strong anionlc exchange resin (Pennaphase AAX) by collecting the material corresponding to the last eluted peak. The pooled solution (ca. 1.0 O.D. unit at 25t nm) was concentrated and the nucleotidlc material desalted on 2336 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research 16 18 16 Time (minutes) Fig.2. H.p.l.c. analysis of dT obtained after five (n=5), six (n=6), and eight (n=8) coupling cycles. Bio-gel P-2. The size of each compound wa3 confirmed by gel electro- phoretic analysis (fig. 3). Two-dimensional sequence analysis of (dT) showed the expected pattern for this compound (results not shown). The synthesis in solution of oligonucleotides of defined sequence using the trinucleotide block approach required the use of mo3t of the 61 pos- sible trlnucleotide triphosphates. In order to investigate the reactivity of trimers with different base composition, we synthesized the following TABLE 2 Cycle Compound Yield No. % 2 (dT) <dT>,o 4 (dT) 62 ( 3 < dT \ 6 <dT> 57 |9 WT) a (dT) ^ 48 2337 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research oligonueleotide: d(AGAAGGTACTTTT) (J_6) . This oligoraer, 13-base3 long, wa3 selected because its synthesis requires the use of four trimer3, each hav- ing a different ba9e at their 3'-phosphate end (3ee scheme 3)- Starting from the polymer (1.0) (n=l, 120 mg), the synthesis of the fully protected oligonueleotide derivative was accomplished by sequential addition of the th-ee trlmera (50 pinoles each): DMT-Tp ABp CBp (12), o o o — SCHEME 3 10 ( n=l ) 2% BSA I HO-Tp Tp Tp TpAcUp -Cellulose 1) DMT-Tp ABp CBp (12) o o o 2) Ac O/Py ( 40% ) 3) 2X BSA Bz Bz Ac,, HO-Tp A p C p Tp Tp Tp Tp Up -Cellulose o o o r o o o o o Bz iBu iBu DMT-A Ac 0 / 2) Ac 0/Py 2 ( 421 ) 3) 2% BS A Bz iBu IBu Bz Bz Ac, HO-A p G p G p Tp A p C p Tp Tp Tp Tp Up -Cellulose *o o Ko o Ko Ko po o o ^o Ko Bz iBu Bz DMT-A p G p A p (1_4) ( 451 ) Bt iBu Bz Bz IBu iBu Bz Bz Ac., DMT-A pGpApApGpGpTpApCpTpTpTpT p Up -Cellulos e o o *o o o o o o o Ko o ro o ro (15 ) 1 ) cone. NH, 2 ) 80% AcOH HO-ApGpApApGpGpTpApCpTpTpTpT-OH (16 ) 2338 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Fig. 3. Gel electrophoretic analysis on a 201 polyacrila- tnide slab, after incubation with [ T - 32 P ] ATP and poly- nucleotide ktnase. dT dT dT 19 22 2 5 DMT-ABp C^G 18 " V±) and DMT-ABp CiB>UABp (U), where p p-chlorophenyl- phosphate. Tie trimers (_12), (1_3_), and (Vi) were prepared from the corresponding fully protected derivatives by removal of the cyanoethyl group from the 3'-terminal phosphotriesters with Et N-Py-H 0 (1:3:1 v/v) . The three cycles were carried out under the same conditions as de- scribed in the synthesis of oligothymldilic acids. Scheme 3 outlines the synthesi3 of (.16) and the yields per coupling calculated from h.p.l.c. analysis (see fig. 5) are shown in parentheses. The final polymer (1^, 85 mg) wa3 treated with base (cone. aq. NH ) and acid (80* aq. AcOH), the cellulose residue was pelleted off and the supernatant evaporated to dryness. The residue, dissolved in t> aq. NH (? ml), wa3 washed with ether and used for the isolation of (JJS). This was accomplished by repeated runs on h.p.l.c; the material corresponding to the last eluted peak wa3 collected and pooled. After desalting on Bio-Gel ?2, the product (1^6) was analyzed as described for the oUgothymidilic acid3. From 0.5 ml of cnjde solution ca. 10 A__., units of pure product (V5) was obtained. Gel electrophoretic analysis showed that (16) 2339 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Fig. h. Two-dimen- sional analysis of a partial venom phosphodiesterase digesc of d(AGAAGGTACTTTT).Dimension 1, electrophoresis on cellulose acetate strip at pH 3.5, dimen- sion 2, homochroraatography on DEAE cellulose thin layer plate. has the same mobility as (dT) (see fig. 5) and two-dimensional sequence ll ft ft analysis of the partial vanom phosphodiesterase digest confirmed that (16) had the expected sequence (fig. H). The recovery of the excess trimers was accomplished by one of the following procedures: a) purification of the trimer 3'-phosphodiester by gel exclusion column chromatog-aphy on Sephadex LH-60 , or b) reuse of the filtrate solution for further condensation either on polymer support or in solution. From the data obtained 30 far we calculated that 35-701 of the excess trime^ could be recovered. CONCLUSIONS This study demonstrates that a solid phase approach combined with the trinucleoti'le phosphotrlester methodology ha3 several advantages over the synthesis of oligonucleotides in solution . Only one kind of 2340 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research Fig.5. (a) Synthesis of d(AGAAGGTACTTTT) monitored by h.p.l.c. after the second, third, and fourth coupling cycle. (b) Gel electrophoretic analysis of the product (P) purified by h.p.l.c. References are and a dodecamer (Ref). 10 12 M 16 IB 20 Time tminultt) trinucleotide building blocks (3'-phosphate) 19 necessary for the chain elongation. The synthesis is faster, because washing procedures substitute column chromatography on silica gel. This also reduces the loss of oligonucleotidic material. Because of the recovery of the unreacted trinucleotide at the end of each coupling step, and the reduced scale of synthesis on the cellulose, the amount of trimer used per coupling is significantly reduced. Functionalization of cellulose with a ribo- deoxvribo dinucleo3ide diphosphate containing uridine and one of the fou- 2341 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research deoxyribonucleosides provides the starting polymer necessary for the solid-phase synthesis of DNA fragments with different bases at the 3'-end. In the synthesis of an oligonucleotlde 13 bases long with trinucleo- tides of different sequence, the yields per coupling were lower than the ones in the synthesis of homopolymer3 (dT) . However, no base speci- ficity has so far been observed. MATERIALS AND GENERAL PROCEDURES Analytical grade reagent3 were U3ed throughout thi3 synthetic study. Pyridine was dried over KOH, distilled, and stored over molecular sieves (type HA). Thin layer chromatography (TLC) was performed on silica gel plates (Merck, Silica Gel 60 F254) with methanol/chloroform (1:9 v/v) as solvent system. The presence of the DMT group wa3 monitored by 3praying the TLC plate with 10J aq. ^SO ^ and heating the plate to 60° C. Puri- fication by column chromatography was performed on 3ilica gel (Merck, Silica Gel 60H). H.p.l.c. was performed with a Spectra-Physics SP 8000 Liquid Chroma- tograph on Permaphase AAX (du Pont) (50 cm x 0.1) cm I.D.) using a linear gradient of water to 1M KC1, pH M.5, at a rate of 3* per minute. The elu- tion was performed at constant flow (3 ml/min.) at 60°C. Cellulose (Whatman CC 3 D was washed with pyridine, rin3ed extensively with methanol, and dried in vacuo over p °5" A^1 reactions at the polymer level were carried out under magnetic stirring. The fully protected mono- and trinucleotides were synthesized by following the procedures described previously . 2,H,6-T^llsopropyl- benzenesulfonyltetrazole (TPSTe) wa3 synthesized according to the proce- dure reported in the literature and recrystallized from benzene- petroleum ether. 2'(3')-0-Acetylurldlne-5'-0-2-eyanoethyl-p-chlorophenylphosphat e (5) . Uridine , Q), (61 mmole) was suspended in a solution of anhydrous THF (200 ml) containing trimethvl orthoacetate (125 mmole) and anhydrous ben- zenesulfonl c acid (BSA) (0.6 g) . After stirring at 25° C for ten min- ute s under exclusion of moi3ture, the solution had become clear. TLC showed complete conversion of uridine into a new compound with higher R . The reaccion was quenched by addition of methanol saturated with NH (5 ml) and ths solvents removed under reduced pressure. The oily residue was dissolved In CHC1 (300 ml) and extracted with saturated aq. NaHCO 2342 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research solution (150 ml) and water (150 ml). The organic layer wa3 dried (MgSO^) and evaporated to drynes3. Without further purification, 21 ,3'-0-methoxyethylideneuridine (.2) (ca. 60 mmole) was dissolved In anhydrous acetonitrile (200 ml) containing 1-N-methylimidazole (200 mmole). The solution was chilled (ice-water) 2 14 and freshly prepared 2-cyanoethyl-p-chlorophenylphosphorochloridate ' (90 mmole) in CH CN (25 ml) was added dropwise over a 15 minute period. The solution was then left to warm up to room temperature. TLC analysis showed virtually complete phosphorylatlon of (2)- The solvent was removed under reduced pressure, and the residual oil partitioned between CHC1, (200 ml) and aq. NaHCO (150 ml). The CHC1 layer was washed with water (150 ml) and then concentrated to a small volume (50 ml). This solu- tion was triturated twice with pentane (500 ml) to remove most of the 1-N-methylimidazole. The product obtained (^) was treated with 80t aqueous acetic acid (200 ml) at room temperature for 10 minutes. Finally, the ace- tic acid was removed under reduced pressure by coevaporation with absolute ethanol. TLC of the glass residue showed only one product. Purification of (5_) was performed on a silica gel column (150 g) . After washing with CHC1, the pure product was eluted with a solution of MeOH in CHC1, (1 to 4J v/v). The fractions containing the product were pooled and con- centrated to a small volume. Precipitation of (5) from petroleum ether (35°- 60° C) gave a white solid (14.5 g; 50% yield based on uridine). Ft =0.31; UV (95* EtOH): X 258 nm ( E 9,600) , X . 226 nra ( E 1,800). i max mln Anal: C^H^N O^PCl (529-5); calcd: C 45.32, H 3-96, Cl 6.70, N 5-90, P 7.90; found: C 45.2, H 4.09, Cl 6.91, N 5.88, P 7.98. 5'-0-2-Cyanoethyl-p-chloropho;phophenyl-2'(3')-0-acetyluridllyl- C2'(3' )-3' ]-5'-0-dlmethoxytrltylthynildlne p-chloroptienyle3ter(7a). 5'-0-Dimethoxytritylthymidine-3'-0-2-cyanoethyl-p-chlorophenylphosphate (7.0 mmole) was treated with a solution of triethylamlne/pyridine/water (1:3:1 v/v) (50 ml) at room temperature for 25 minutes. The phosphodiester derivative (j>) so obtained was dried by coevaporation with anhydrous pyri- dlne (3 x 25 ml). 2'(31 )-0-Acetylu'-idine-5'-0-2-cyanoethyl-p-chlorophenyl- phosphate (5_) (6.6 mmole) was added to derivative (^) and the mixture d-ied again with pyrldine (30 ml). TPSTe U0.5 mmole) in anhydrous pyidine (50 ml) was added to this solution. The reaction mixture was kept in vacuo and protected from the light for 16 hours. The reaction was stopped by addition of water, and the solvents removed under reduced pressure. The 2343 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research residual material in CHC1 (250 ml) was extracted with aqueous NaHCO (100 ml), and then washed with water (100 ml). The combined CHC1 extracts were _e_vaporated to dryness. Chromatography of the product on silica gel (75 g) was performed using MeOH/CHCl (1:99 v/v) as solvent system. The fractions containing the product were evaporated to a glass. Precipitation of this material from petroleum ether (35° - 60° C) gave a colorless solid (2.6 g, 32% yield). Its homogeneity was confirmed by TLC analysis (R =0.65). Anal: C H^N 0 P (1247); calcd: C 54.90, H 4.44, Cl 5.68, N 5.61, P 4.96; found C 54.61, H 4.44, Cl 5.92, N 5-91, P 5.31. To confirm the release mechanism, this compound (Ja, 10 mg) was treated with cone, aqueous aramonia/pyridine (9:1 v/v; 2 ml) for 30 minutes at 60° C TLC analysis showed a complete degradation of (Ja.) into a baseline material and a compound containing the DMT group. This latter compound was identi- fied as 5'-DMT-thyraidine (TLC). Removal of DMT with 2% BSA gave a compound identical to thymidine. FuncUonalizatlon of cellulose with (7b) The fully protected dimer (7.3.) was treated with a solution of pyridine- triethvlamine-water (3:1:1 v/v) (20 nil) at room temperature. After 25 min- utes, TLC showed complete conversion of (J_a) into its phosphodiester deri- vative (base-line material (7b)). The latter material was dried by coevap- oratlon with pyridine (3 x 15 ml) and then reacted with dry cellulose (Whatman CC 3D (0.7 g) in anhydrous pyridlne (3 ml) in the presence of TPSTe (0.23 g). The mixture was stirred overnight (16 hours) at room temperature. The polymer was then filtered (sintered glass filter M) and washed with dry pyridine. The filtrate was stored for the recovery of un- reacted dime" (Jb). The cellulose was washed extensively with MeOH (60 ml) and dried in a dessicator over KOH. The extent of coupling of (£b) to the polymer was determined by treat- ing a sample of cellulose (8, 10 mg) with 2% BSA in CHC1 -HeOH (7:3 v/v) (2 ml) at 0°C for 10 minutes. After centrifugation of the 3olid particles, the ^bsorbancy of the released DMT group was measured at 494 nm. A stan- dard curve was obtained by using different amounts of 5'-O-dimethoxyt^ityl thymidine In 2% BSA 3tock solution. From the data obtained, we calculated an average incorporation of ca 100 pmole3 dimer {Jb) per g cellulose. Another sample (.&_, 10 mg) was treated with cone, aqueous ammonia for 8 hours at 60°C. The polymer was centrifuged off and the supernatant concen- 2344 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research trated to dryness. The residual material was dissolved in CHC1 (1 ml) and analyzed by TLC. The only product detected wa3 5'-O-dimethoxytrityl thymidine. Treatment of the chloroform solution with U% BSA in CH 0H- CHC1 (1 ml) gave thymidine and DMT residue. From the total amount of DMT (194 run) we calculated that the ammonia treatment had released ca 95$ of the 5'-0- DMT-thymidine from the polymer. Synthesis of ollqothymidilic acids, (dT)m (m=4,7,10,13,16,19,22, and 25) . The functionalized polymer ((3, 100 mg; 9.7 pmole of dimer) was fir3t treated with 2% BSA in CHC1 /MeOH (7:3 v/v, 10 ml) at 0°C for 10 minutes a3 described above. After washing the polymer on the filter with methanol (50 ml) the cellulose was dried by flushing with anhydrous pyridine (20 ml) The trimer (£) (50 u moles) was mixed with the polymer in a 10 ml round- bottom flask. The mixture was dried by coevaporation with pyridine (2x 5 ml) and finally resuspended in anhydrous pyridine (1.0 ml) . TPSTe (75 mg) was added quickly and the reaction mixture kept in vacuo. After two hours of continuous stirring, pyridine (2 ml) wa3 added to the reaction vessel and the polymer recovered by centrifugation. The supernatant was collected separately for the recovery of excess trimer (5.). The polymer so obtained was resuspended in a solution of pyridine-acetic anhydride (10:1 v/v, i ml) and stirred fo^ 30 minutes. After washing the cellulose on a filter with pyidine (8 ml) and methanol (30 ml), the polymer was ready for the next coupling step (see Table 1) . The following cycle: a) deblocking of DHT group from the polymer with BSA, b) coupling of the polymer so obtained with trimer (9_) in pyridine containing TPSTe, and c) acetylation of the resulting polymer with pyri.dine/Ac 0 was repeated seven more times under the same conditions. Release of the ollaonucleotldes rrom the polymer and removal of protecting The polymer (H), n=l-8, 10 mg) suspended in pyridine (0.5 ml) was treated with concentrated aqueous ammonia (4 ml) at 60° C for 8 hours. The solid polymer was removed by centrifugation and discarded. The supernatant was evaporated to dryness, and the residue treated with 80? aq. acetic acid at room temperature for 15 minutes. The acetic acid was removed by evapor- ation under reduced pressure and the solid residue redissolved in t% aque- ous ammonia (2 ml). After washing with ether (3x 1 ml) the aqueous solu- tion was analyzed on h.p.l.c. For each coupling step the chromatogram 2345 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research showed the formation of a new product with higher retention time (3ee figs. 1 and 2) . The yields per coupling were calculated from the peak areas and are based on the next shorter oligomer. For each elongation step the final product, after complete deblocking, was isolated by preparative h.p.l.c on Permaphase AAX. The fractions corresponding to the last peak were pooled (0.5 - 1.0 O.D. units at 25*4 nm), the solution concentrated and desalted on Bio-gel P-2. Gel electrophoretic analysis on a 20$ acrylamide slab of the oligomer3 (dT) (m=13,lb,19, 22, and 25) after their incubation with [y - P]-ATP and T. polynucleotide kinase, showed that each fragment released from the polymer had the expected 3ize (fig. 3). Furthermore, [ P] (dT) wa3 analyzed by two dimensional sequence analysis of its partial venom phosphodiesterase digestion products . The analysis con- firmed the structure of this compound. Synthesis of d(AGAAGGTACTTTT) (16). The functionalized polymer {B_, 125 mg) was treated with 2$ BSA as des- c-ibed above and used for the synthesis of the 13-base long oligonucleotide of defined sequence (1_6). This was accomplished by using/four dffferent trimers: DMT-Tp Tp Tp (9), DMT-Tp ABp CBp (U), DMT-A^G^Vp^ (_13), o o o o o o and DMT-A p G p A p (_14), where p = p-chlorophenylphosphate. The coupling of these triraers (50 umoles each) to the polymer was carried out in the sequential order shown in scheme 3, under the same conditions as reported above for the 3ynthe3is of oligothymidilates. The yields obtained for each coupling are shown in scheme 3 and were calculated by h.p.l.c. analysis based on the next shorter fragment. The fully protected cligomer obtained after the fourth cycle was re- leased from the polymer and deblocked with ammonia and acetic acid as de- scribed above. The isolated product (_16) (h.p.l.c.) had the same mobility as (dT) on 20$ polyacrylamide slab (see fig. 5). Furthermore, two-dimensional sequence analysis of the partial venom phosphodiesterase digest of (lj>) confirmed the expected sequence (fig. 4). Recovery of the exoes3 trlnucleotldes A. Chromatography on Sephadex LH-60. The filtrate pyridine solution (3 ml) containing the excess trinucleotid e (DMT-Tp Ap Cp , ca <10 umole) and TPSTe was used for the recovery of thi3 compound (\2) by gel exclusion chromatography. Water (0.5 2346 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research ml) wa3 first added and the solution evaporated to dryness. The residue wa3 dissolved in THF/MeOH (95:5 v/v, 1 ml) and applied to a column of Sephadex LH-fiO (0.7 x 100 cm) preequilibrated in the same solvent. Isocratic elution of the product was obtained with THF/MeOH (95:5 v/v). Fractions of 1 ml were collected. The fractions containing product (fraction 38 to 50) (detected as DMT-posltive baseline material) were pooled, concentrated and the trimer precipitated from petroleum ether (25 mg, 35S recovery yield). B. Reuse of the excess trimer for synthesis in solution . The pyrldlne solution containing the excess DMT-Ap Ap Gp (ca MO o Q o ymole) and TPSTe was dried by coevaporation with pyridine (2x2 ml). 3'_0-Ani9oyl- 2-N-isobutrylguanosine (It mg, 29 umole) was added and the mixture dried by coevaporation with pyridine (2x2 ml). Additional TPSTe (50 mg) was added, and the reactants dissolved in pyridine (1 ml). The reaction mixture wa3 kept iri vacuo for 6 hours. TLC analysis showed complete conversion of the nucleoside into the product tetramer. This latter compound was purified on a silica gel column and obtained as a homogeneous solid after precipitation from petroleum ether (23 mg) . C. Reuse for solid-phase synthesis. The pyidine solution (3 ml) recovered from a coupling step containing the t>-iraer DMT-Tp Tp Tp (°_, ca M0 uraole), was reused to elongate the oligonucleotidic chain on cellulose. The trimer was added to the polymer (10) (n=l, 100 mg) and the suspension dried by coevaporation with pyridine. An additional portion of TPSTe (75 mg) was added and the reaction mixture suspended in pyridine (1 ml). The fully protected ollgomer was released from the polymer and deblocked with aq. ammonia and aq. acetic acid as described above. H.p.l.c. analysis showed a coupling yield of 43> for d(T) based on dtT^ . ACKNOWLEDGEMENTS The authors are grateful to Dr. D.G. Kleid, Dr. P.M.J. Burgers and Dr. J.H. van Boom for their valuable comments and critical reading of the manuscript. REFEREHCES Abbreviations: DMT = M,1'-dimethoxytrityl, BSA = benzenesulfonic acid, TPSTe = 2,1,6,-Triisopropylbenzenesulfonyltetrazole, CE = 2-cyanoethyl, Bz = benzoyl, iBu = isobutryl, Ac = acetyl. 2347 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research 1 . Crea, R., Hiro3e T. and Itakura, K. (1979) Tetrahedron Letters, 5, 395-398. 2. Crea, R., Kraszewski, A., Hirose, T. and Itakura, K. (1978) Proc. Natl. Acad. Sci. (U.S.A.), 75, 5765-5759. 3. Hiro3e, T., Crea, R. and Itakura, K. (1978) Tetrahedron Letters, 28, 2449-2452. U. Ko33el, H. and Seliger, H. (1975) Fortsuch. Chem. Org. Natur3toffe 32, 5. Gait, H.J. (1979) Polymer-Supported Reagents, Catalysts and Protecting Groups (P. Hodge and D.C. Sherrington, Eds), Wiley, London, in press. 6. Belagaje, R-, Brown, E.L., Gait, H.J., Khorana, H.G. and Norris, K.E. (1979) J. 3iol . Chem. 254, 5754 and subsequent papers. 7. Narang, C.K., Brunfeldt, K. and Norris, K.E. (1977) Tetrahedron Letters 21 , 1819-1822. r.ait , M.J. and Sheppard, R.C. (1977) Nucleic Acids Res. 4, 1135-1158. Potapov, V.K., Veiko, V.P., Koroleva, O.N. and Shabarova, Z.A. (1979) Nucleic Acids Res. 6, 2041-2056. 8. Pless, R.C. and Letsinger, R.L. (1975) Nucleic Acid3 Research 2, 773- Seliger , H. (1975) Die Hacromoleculare Chemie 176, 1611-1627. 9- Miyosht, K. and Itakura, K. (1979) Tetrahedron Letters 38, 3635-3638. 10. Stawinski, J. , Hozumi, T., Narang, S.A., Bahl, C.P. and Wu, R. (1977) Nucleic Acid3 Re3. 4, 353-371. 11 . Koster, H. and Heyns, K. (1972) Tetrahedron Letters, 16, 1531-1534. 12. The three "ribo-deoxyribo" dinucleoside diphosphates made up by uridine and 4-N-benzoylcytidlne, 6-N-benzoyladenosine, and 2-N-isobutryl- guanoslne, rsspectively, were synthesized in a similar manner and isolate d as homogeneous solid3 in comparable yield. The relevant data wil l be published elsewhere. 13- Griffin , B.E., Jarraan, H., Ree3e, C.B., Sulstow, J.E. (1970) Tetrahedron 26, 1023. 14. Crea, R., et al , manuscript in preparation. 15. Seliger, H. , Holupirek, M. and Gortz, H.H. (1973) Tetrahedron Letters, 24, 2115-2118. 16. Jay, E., Bambara, R., Padmanabhan, P. and Wu, R. C1974) Nucleic Acids Res. 1, 331-353- 17. -le Rooij, J.F.M., Arentzsn, R., den Hartog, .I.A.J., van der Marel, G. and van Boom, J.H. (1979) J. Chromatography 171, 453-U59- 18. After thi3 paper was submitted for publication methods for the synthesis of ollgodeoxynucleotides on solid supports were reported using a phosphite-triester1" 8 and a phosphotriester1"*5 approach, respectively : a. Matteucci, M.D. and Caruthera, M.H. (1980) Tetrahedron Letters, 21 , 719-722. b. Gait, M.J.,Singh, H. .Sheppard, R.C,Edge, M.D.,Green, A.R., Heathcliffe , G.R., Atkinson, T.C., Newton, C.R., and Markham, A.F. (1980) Nucleic Acids Research, 8, 1081-1096. 2348 Downloaded from https://academic.oup.com/nar/article/8/10/2331/2359753 by DeepDyve user on 22 July 2022 Nucleic Acids Research

Journal

Nucleic Acids Research – Oxford University Press

Published: May 24, 1980

Keywords: oligonucleotides; thymidine; economics; solids; diphosphates; oligodeoxyribonucleotides

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