MASAD JOSE DAMHA, NASSIM USMAN et KELVIN KENNETH OGILVIE. Can. J. Chem ..... under vacuum to obtain derivative 6a as a white powder. The product.
Solution and solid phase chemical synthesis of arabinonucleotides MASADJ O S DAMHA,' ~ NASSIMUS MAN,^
AND
KELVINKENNETH OGILVIE~
Department of Chemistry, McGill University, Montreal, P. Q., Canndn H3A 2 K6
Received August 29, 1988
MASAD J O SDAMHA, ~ NASSIMUSMAN, and KELVIN KENNETH OGILVIE. Can. J . Chem. 67, 831 (1989). A fast and convenient procedure for the chemical synthesis of arabinonucleotides, which eliminates the multistep protection of the arabinonucleoside building blocks, is described. The results of these studies were successfully applied to the automated chemical synthesis of the hexanucleotide 5'-aUpaApaUpaApaUpaA-3'. Both solution and solid phase phosphite triester procedures are described. Key rvords: arabinonucleotides, arabinophosphoramidites, automated chemical synthesis, protected arabinonucleosides.
MASAD JOSEDAMHA, NASSIM USMAN et KELVIN KENNETH OGILVIE. Can. J. Chem. 67, 831 (1989). Une mCthode gCnCrale et rapide a CtC developpCe pour la synthkse chimique d'arabinonuclCotides en solution et sur support solide. La mCthode a CtC appliquee a la synthkse automatiske de l'hexanucleotide 5'-aUpaApaUpaApaUpaA-3'. Mots cle's : arabinonuclCotides, arabinophosphoramidites, synthese chimique automatisie, arabinonuclCosides protCgCs.
Introduction Arabinonucleosides, analogues of naturally occurring nucleosides, have been shown to possess a wide range of biological activities including antiviral and antitumor properties (1, 2). Unfortunately, the cytotoxicity, enzymic modification, and relative insolubility of these analogues (e.g., 9-P-D-arabinofuranosyladenine, aA) limit their utility for systemic administration. Some of these problems have been bypassed by chemically modifying the sugar or base moieties of these molecules (3). A different approach, which might be envisaged to improve the biological action and solubility of arabinonucleosides, is their incorporation into a nucleotide chain (4, 5). In addition to possible resistance to enzymic modifications and cleavage of the phosphodiester linkage, arabinonucleotides are expected to possess an inherent stability comparable to that of deoxyribonucleic acids (DNA). The unfavourable (trans) orientation of the 2'-hydroxyl function in arabinonucleotides prevents 2'-hydroxyl assisted cleavage of the vicinal 3'-5' phosphodiester linkage (4). The chemical synthesis of arabinonucleic acids (ANA, I), like that of ribonucleic acids (RNA), is difficult due to the presence of the 2'-hydroxyl function. The selective protection of the 2'-hydroxyl group is generally difficult due to its orientation towards the hindered P face of the arabinofuranosyl sugar. In the original work by Wechter (4), the 2'- and 3'-hydroxyls were left unprotected during nucleotide coupling steps, resulting in a mixture of 2'-5'- and 3'-5'-linked dinucleoside monophosphates. Srnrt and Sorm (6) reported the incorporation of arabinouridine (aU) and arabinocytidine (aC) as the terminal 3'-end nucleoside of two trinucleotide diphosphates. Syntheses of aU-containing nucleotides were presented by Nagyvary and Provenzale (7), Ogilvie and Iwacha (8), and Schramm (9) but these procedures are limited to nucleosides that possess a C-2 keto function (pyrimidines) as they require formation of an 02,2'-anhydro intermediate. Chattopadhyaya and co-workers (10) described the regiospecific, stepwise 'Present address: J. Tuzo Wilson Research Laboratories, Department of Chemistry, University of Toronto, Erindale College, ~ i s s i s s a u ~ a , Ont., Canada L5L 1C6. 2~resentaddress: Department of Biology, 16-739, Massachusetts Institute of Technology, Cambridge, MA 02 139, U .S .A. 3 ~ u t h o rto whom correspondence may be addressed. Present address: Vice-President (Academic), Acadia University, Wolfville, N.S., Canada BOP 1x0.
synthesis of the 3'-5'-linked nucleotides aA,aA and aA,aA,aA but protection of the nucleoside monomers required numerous steps. Our laboratory has previously described the use of 2'-0-silyl-protected ribonucleosides as building blocks for the synthesis of ribonucleotides (11, 12). The successful use of the tert-butyldimethylsilyl group (TBDMS) in providing a rapid method for protecting the 2'-hydroxyl in ribonucleosides suggested that the same approach should be investigated in the arabinonucleoside area. Indeed, 5'-0-monomethoxytrityl 2'0-TBDMS arabinonucleosides have been successfully prepared (13) and incorporated into nucleotide chains (5). In this report we describe a more practical and efficient procedure for the preparation of arabinonucleotide fragments. This method minimizes the protection of the arabinonucleoside units and affords 3'-5'-linked arabinonucleotide oligomers in good yields. A preliminary report of some of these results has appeared (14).
Results and discussion Preparation of arabinonucleoside phosphoramidites The key reagents used for the synthesis of arabinonucleotides were the nucleoside 3'-phosphoramidite derivatives 3 a and 3b, which were easily prepared, in good yields, in a manner analogous to the preparation of ribonucleoside 3'-phosphoramidites (12b). Thus, N,N-diisopropyl-P-cyanoethylphosphonamidic chloride (1.1 equiv.) selectively reacted with the 5'-tritylated arabinonucleosides 2a and 26 (1.0 equiv.) at O°C (2 h) to afford the corresponding 3'-phosphoramidites 3 a and 3b in 90 and 60% yields, respectively (Scheme 1). The 2',3'-0bisphosphoramidites 5a and 5b were also isolated in ca. 5% yield and were easily separated from the desired 3'-0-phosphoramidites by silica gel chromatography. The arabinouridine 2'-0-phosphoramidite 4 a was formed in ca. 3-5% yield ( 3 ' NMR) ~ and was separated from the 3'-0-phosphoramidite isomer 3a by repeated purifications on a silica gel column. The arabinoadenosine 2'-0-phosphoramidite 4b could not be detected. These results clearly indicate the marked difference in the steric environments of the 2'- and 3'-hydroxyl functions of arabinonucleosides. Solution phase synthesis of arabinonucleotides To evaluate the arabinonucleoside 3'-0-phosphoramidites as building blocks for arabinonucleotide synthesis, 3 a (1.0 equiv.) was activated with tetrazole (4 equiv.) in THF in the presence of 2',3 '-0-diacetylarabinouridine (6a, 1.3 equiv.). After 30 min at 20°C, the resulting 3 ' 3 ' dinucleoside phosphite
CAN. J . CHEM. VOL. 67, 1989
DNA
RNA
triester intermediate 7a was reacted in situ with a mixture of acetic anhydridel4-dimethylaminopyridine/collidine (5.01 0.517.0 equiv.) for 30 min to block the 2'-hydroxyl position. Upon completion of the capping reaction, an excess of an aqueous iodine solution was added to oxidize the fully protected phosphite intermediate (Scheme 2). Thus, activation of the nucleoside 3'-phosphoramidite with tetrazole, acetylation of the 2'-hydroxyl, and iodine oxidation were all carried out sequentially without intermediate product isolation. The workup of the condensation reaction involved removal of excess iodine using an aqueous sodium bisulfite solution followed by purification of the product by silica gel chromatography. This procedure resulted in the isolation of phosphotriester aUpaU (8a) in 78% yield based on the 3'-phosphoramidite 3a. The reaction of the adenosine 3'-phosphoramidite 3b, nucleoside 66, and tetrazole by this condensation procedure gave the dinucleoside phosphotriester aA,aA (86) in 60% yield. It is worth noting that, unlike ribonucleoside phosphite
ANA, 1
triesters (15), the phosphite triester linkage of aU,aU (7a) and aApaA (7b) does not undergo 2'-hydroxyl assisted cleavage, because of the unfavourable (trans) orientation of the 2'hydroxyl group. During the coupling step one may envisage the possibility of the formation of branches at the unprotected 2'-hydroxyl of phosphite intermediates aUpaU (7a) and aApaA (7b). However, branched trinucleotides did not form under the condensation conditions. It appears that the ratio of nucleoside amidite 3 to nucleoside 6 employed in these couplings (1:1 .3), as well as the steric hindrance of the 2'-hydroxyl group, disfavour this side reaction. Chain branching does occur in solid phase syntheses where the ratio of phosphoramidites to solidsupport-bound arabinonucleoside reaches 20: 1 (vide infra). Solid phase synthesis of arabinonucleotides The use of a solid phase approach eliminates the need for purification of the protected nucleotides, allows the rapid
DAMHA ET AL.
MMTOvB MMTO
Ac20/DMAP
r
0
I
P-OCH
I
AcO
?CH2CN
I
AcO 8 R=MMT 9 R=H
synthesis of long sequences, and reduces the amount of (expensive) nucleoside 3'-phosphoramidite needed. Because our methodology employs the same synthetic strategy currently used for the solid phase synthesis of DNA and RNA chains, the preparation of ANA oligomers was attempted on a DNA/RNA synthesizer (12b). Preparation of solid supports To carry out the solid phase syntheses it was first necessary to prepare a suitable solid support, which avoided the preparation of a derivative of the arabinonucleosides. Thus, a support bearing a carboxylic acid would be most suitable as the carboxyl moiety could simply be esterified with one of the hydroxyl groups of compound 2. Four different supports were evaluated: aminopropyl controlled pore glass (CPG), long chain alkylarnine (LCAA) CPG, carboxyl CPG, and aminopropylsuccinylated Vydac TP silica gel. In the case of the two amino derivatized CPG supports a succinyl linker was attached according to the literature procedure (16), which resulted in supports bearing a terminal carboxylic acid. All four supports were then treated with compound 2 and DCC followed by p-nitrophenol (to esterify any remaining carboxyl groups). Finally, piperidine was added to convert the p-nitrophenyl esters to amides. None of the three CPG supports gave
satisfactory results. The aminopropylsuccinylated Vydac TP silica gel gave loadings of 20-25 pmol g-'. Therefore all solid phase syntheses were performed on the silica gel support (Fig. 1). Solid phase synthesis The first syntheses performed were those of the trimer of aU using 60- and 300-s recycle times in the cycle shown in Table 1. At the end of each coupling cycle any unreacted 5'- and all of the 2'-hydroxyls of the newly added nucleoside were acetylated using an AczO solution. The average coupling yields obtained, determined by trityl cation analysis, were 94 and 115%, respectively. The coupling yield higher than 100% obtained in the latter synthesis indicated that a side reaction (i.e., chain branching) was occumng at the unprotected 2'-hydroxyl group. The formation of side products was confirmed by polyacrylamide gel electrophoresis (Fig. 2) and HPLC analyses (data not shown) of the crude products after complete deprotection. Consistent with the formation of branches during the coupling step is the observation that the amount of side product increases with increasing coupling time (Fig. 2). To eliminate chain branching two approaches were taken. The first was to decrease the coupling time, to favour only the reaction of the amidite at the less hindered 5'-OH, and the
CAN. J . CHEM. VOL. 67. 1989
834
coupling yields, 39 units for method 1 and 57 units for method 2. Gel electrophoresis analysis indicated some small amount of branching in the case of method 1 (data not shown). The branching reaction appeared to occur early in the synthesis (at the dimer or trimer level).
FIG. 1. Arabinonucleoside-derivatized silica gel. TABLE1. Synthesis cycle for the synthesis of arabinonucleotidesR Step
Reagent
Time (s)
1 2 3 4 5 6 7 8 9
Acetonitrile 5%TCA/DCE Acetonitrile 0.05 M Amidite 0.17 M tetrazole/acetonitrile Recycle 0.25 M Ac20/DMAP/collidine/THF 0.1 M I2 THF/pyridine H20: 71211 Acetonitrile 0.25 M AczO/DMAP/collidine/THF
30 90 60 15 15-885 60 30 50 30
+
"TCA = trichloroacetic acid; DCE aminopyridine.
=
dichloroethane; DMAP
= 4-dimethyl-
second was to use the amidites bearing 2'-OAc protection (10a and lob). In each case the sequences were deprotected (vide infra) and analyzed by gel electrophoresis and HPLC. In the first approach, coupling times of 30 and 60 s were used in the synthesis of dimers and trimers of both aA and aU. The use of the 30-s recycle resulted in almost the complete elimination of the branching reactions (Fig. 2); however, the stepwise coupling yields were low, only ca. 75%. The use of a 60-s recycle suppressed the branching somewhat compared to the 300-s recycle (Fig. 2); however, there was still an unacceptable amount of branching and the average coupling yields were ca. 90%. The use of a 900-s recycle time did not increase the amount of branching relative to the 300-s recycle time; therefore it appears there is a saturating level of branching occurring at approximately 300 s. In the second approach, compounds 10a and lob were used, in which no branching may occur due to the presence of the 2'-OAc protecting group. These derivatives were prepared in high yields by the reaction of their corresponding 2'-hydroxyl amidites 3 a and 3b with an Ac20/4-dimethylaminopyridine/ EtNiPr2/THF solution at room temperature. A trimer of aU was prepared with compound 10a using a 300-s recycle time, and an average coupling yield of 96% was obtained. The isolated material after deprotection appeared as one well-defined band and, as expected, the branching reaction was entirely eliminated (Fig. 2). From these experiments it appeared that the best approach was to use a 30-s recycle time with amidites 3 (method 1) or the 2'-OAc protected amidites 10 and a 300-s recycle time (method 2). As a test, the synthesis of 5'-aUpaApaUpaApaUpaA-3'was performed using the two methods. In the case of method 1 an average coupling yield of 86% was obtained and in the case of method 2, 98.5%. The overall yields following deprotection were consistent with what would be expected from the average
Deprotection and isolation of arabinonucleotides All nucleotides (5'-hydroxyl free) were fully deprotected in a single treatment with 15 M aqueous ammonia in ethanol at 20°C (sequences containing aU) or 50°C (sequences containing aA) for 16 h. The crude products aUpaU and aApaA (solution phase synthesis) were passed through a column of Dowex Na+ ion exchange resin and subsequently purified by size exclusion chromatography (Sephadex G-25) and cellulose TLC. The hexanucleotide 5'-aU paA paUpaApaUpaA-3' was purified by electrophoresis on a preparative polyacrylamide gel and desalted on a Sephadex G-25 column. Characterization of arabinonucleoside phosphoramidites and arabinonucleotides Arabinonucleoside phosphoramidites Because the phosphoramidite derivatives represent a new class of protected arabinonucleosides it was necessary to characterize the products in detail. The chromatographic and spectroscopic properties of the phosphorarnidite derivatives are presented in Table 2. The NMR data show that there is, as expected, only one pair of diastereomeric signals in the 3 1 NMR ~ spectra of 3 and 10, thereby establishing the isomeric purity of these compounds. The isomeric uridine 3'- and 2'-phosphoramidites 3 a and 4a were characterized by conversion to the acetylated derivatives 10a and 11, respectively. The same compounds 10a and 11 were prepared from the known 5'-MMT-2'- and 5'-MMT-3'TBDMS-arabinouridine (5, 13) as shown in Scheme 3. The position of the phosphoramidite moiety of the adenosine phosphoramidite 3b was established by 13cNMR. In the 13C spectrum of 3b (one diastereoisomer, Fig. 3) the C4', C3', and C2' resonances are coupled to 31Pwhile that of C1' is not. The presence of the phosphorarnidite group causes shielding and deshielding of the arabinose carbon resonances relative to the corresponding signals in 2b, the effect being more pronounced for the carbon directly attached to the phosphoramidite group (C3'). Protected nucleotides The chromatographic and spectral properties of the fully protected @a, 8b) and 5'-unprotected (9a, 9b) nucleotides are ~ spectra are in summarized in Table 2. The UV and 3 1 NMR complete agreement with the proposed structures. Free nucleotides The physical (UV, 'H, 13c, and 3 ' NMR, ~ and R fvalues) properties of aUpaU and aApaA (solution phase synthesis) are presented in Table 3. The 'H and 13cNMR base resonances clearly indicated the composite nature of the deprotected products. The 'H NMR spectrum of aApaA was identical to that reported by Doornbos et al. (17). As found by Wechter (4), the fully deprotected arabinonucleotides were stable to alkaline treatment (0.1 N KOH, 37"C, 16 h, HPLC analysis), conditions that degrade ribonucleotide chains. Furthermore, they were completely degraded by spleen phosphodiesterase (2'-5'-linked arabinonucleotides are not hydrolyzed by this enzyme) and snake venom phosphodiesterase to the corresponding monomer components (HPLC
DAMHA ET AL
I
1 1
I
FIG.2. Gel electrophoresis (24% polyacrylamide/7 M urea) of fully deprotected arab~nonucleotides.Lanes: 1 , aUpaUpaU prepared with the 2'-OAc amidite 10a using a 300-s recycle time; 2 , 3 , 4, aUpaUpaU prepared with the 2'-hydroxyl amidite 3a using, respectively, recycle times of 30, 60, and 300s; 5, crude 5'-aUpaApaUpaApaUpaA-3'(method I); 6, crude 5'-aUpaApaUpaApaUpaA-3'(method 2); 7, pure 5'-aUpaApaUpaApaU,aA-3' (method 1); 8, pure 5'-aUpaApaUpaApaUpaA-3'(method 2). All samples were loaded with xylene cyan01 (XC) and bromophenol blue (BPB).
TABLE2. Physical properties of arabinonucleoside phosphoramidites and protected arabinonucleotides
Compound
UV (EtOH) Xmax (Xmin)
"EtOH/EtOAc/CHCI3 5:50:45. bEther/C~ZCIZ 1: 1. 'MeOH/CHCl, 5:95. d ~ t ~ ~ ~ . 'THF. 'DMSO-d6.
3 1 NMR ~ (CDCl3, 121.5 MHz)
Rf
CAN. J. CHEM. VOL. 67, 1989
M
+
M S T~ O O I
~
u
(3) TBAF (2) i-Pr \N /i-Pr
I
(1) Ac20 (2) TBAF
0
I
1AcO
'P-OCH~CH'CN
1
N
FIG. 3. I3cNMR (DEFT, 75.4 MHz) spectra of arabinoadenosine phosphoramidite 3 6 (one diastereoisomer) and arabinonucleoside 2 6 in CDC13.
analysis). This established the integrity of the 3'-5'-phosphodiester linkages.
Conclusions The procedure described in this study permits the rapid and efficient synthesis of 3'-5'-linked arabinonucleotides with a minimum effort involved in the protection of the monomer units. This represents the first general procedure for direct conversion of arabinonucleosides to phosphoramidite derivatives, and these can be immediately incorporated into nucleotide chains. This feature, along with the facile removal of the protecting groups, makes this an attractive and efficient procedure.
Experimental All general procedures and techniques including chromatography, UV, NMR, enzyme assays, condensation and deprotection procedures, gel electrophoresis analyses, and general reagents are as previously described (15, 12b). Solid phase syntheses were performed according to ref. 12. High performance liquid chromatography (HPLC) analyses were carried out on a Spectra-Physics SP8000 HPLC equipped with a 254 nm UV detector and a chromatography data system under the following conditions: reverse phase 4.6 X 250 mm Whatman Partisil ODS-2 column (10-pm particles, Chromatographic Specialities); mobile phase, solvent A: 0.02M KH2P04 (pH = 5.5, adjusted with KOH), solvent B: methanol, gradient 0-50% solvent B in 30 min;
DAMHA ET AL
TABLE3. Physical properties of free aU,aU and aApaA 'H NMR (300 MHz)" Nucleotide
1'
8(6)
2(5)
3 ' NMR" ~ (121.5 MHz)
UV (EtOH) A,, (A,;,,)
Rr
I3C NMR (75.4 MHz)" Nucleotide
1'
2'
3'
4'
5'
2(5)
8(6)
"D20;p D = 7-8; concentration,5-10 m M . bn-Propanol:aq.NH3(15 M):H20, 55:10:35. '1sopropanol:aq. NH3(15 M):H20, 70:10:20.
flow rate 2 mL/min; temperature 30°C. About one unit (in 10 p L buffer) of crude nucleotide was injected in the HPLC column per run. An AZm unit is the amount of material that will produce an absorbance of 1.0 at 260 nm, when dissolved in 1 mL of solvent, in a 1-cm cell.
200 MHz) 6: 9.34 (b, 1, NH), 8.78 (s, 1, H8), 8.27 (s, 1, H2), 6.58 (d, JHI,-H2' = 4.6 HZ, 1 , Hl'), 2.12 (s, 3, COCH3), 1.71 (s, 3, COCH3). Physical properties of ~~-acet~l-~~-benzoyl-2',3'-di0-acetylarabinoadenosine:TLC (methanol/CHC13 5:95): R I 0.34; (ethanol/ether/CH2CI2 3:71:26): R f 0.19; (ethanol/ethyl acetate/ CHCI3 5:50:45): R f0.38; UV (95% EtOH/HIO): A,, 250, 274 nm, Amin 231, 263 nm; 'H NMR (CDC13, 200 MHz) 6: 8.71 (s, 1, H8), 8.28 (s, 1, H2), 6.55 (d, JHI,-H2* = 5.3 HZ, HI'), 2.60 (s, 3, COCH3), 2.13 (s, 3, COCH3), 1.59 (s, 3, COCH3).
Synthesis of arabinonucleosides Derivatives 2 a and 2 b The synthesis of compound 2b has been described (15). Compound 2a was prepared in 89% yield according to the procedure developed Synthesis of arabinonucleoside phosphoramidites by Ogilvie et al. (13). 5 ' - 0 -Monornethoxytritylarabinouridine -3'-0-N ,N-diisopropyl-P2 ',3'-0-Diace~larabinouridine(6a) cyanoethylphosphoramidite (3a) To a THF (15 mL) solution of 5'-0-monomethoxytritylarabinoTo a stirred THF (18 mL) of derivative 2a (6.0 mmol, 3.1 g) uridine (2a) (1.9 mmol, 1.0 g), 4-dimethylarninopyridine (0.8 mmol, and diisopropylethylamine (21.8 mmol, 3.8 mL) was added dropwise, 98 mg), and 2,4,6-collidine (8.0 mmol, 1.1 mL), was added acetic over 15 min, N,N-diisopropyl-P-cyanoethylphosphonamidic chloride anhydride (8.0 mmol, 750 pL) at room temperature. After stirring for (6.3 mmol, 1.34 mL) at 0°C. The reaction was then stirred for 2 h 1 h, the solution was diluted with CH2C12(75 mL) and washed with 5% at room temperature. Ethyl acetate (100 mL, prewashed with 5% NaHC0, solution (50 mL). The organic layer was dried (anhydrous NaHCO,) was added and the solution washed with saturated brine Na2S04) and evaporated under reduced pressure to give a gum. The solution (3 x 100 mL). The organic phase was dried (anhydrous monomethoxytrityl group was removed by treatment of the crude Na2S04) and the solvent removed under vacuum to yield the crude product with 80% acetic acid (40 mL) at 90°C. After 15 min, ethanol product as a white foam. The crude product was purified (twice) by was added (50 mL) and the solvents removed under vacuum. The silica gel chromatography by elution with CH2CI2/hexane/triethylresidue was diluted with CH2Cl2(5 rnL) and added to a stirred hexane amine, gradient: 50:45:5 to 50:40: 10. After evaporation of the pooled solution (100 mL). The resulting precipitate was filtered and dried fractions the product was coevaporated, first with 95% ethanol to under vacuum to obtain derivative 6 a as a white powder. The product remove traces of triethylamine, and then with ether to provide the crystallized (94% yield, 613 rng) from CH2C12 or CHIC12/hexanes product as a white foam. The purification was repeated until 3 a was (3:1), mp 164-165°C; TLC (methanol/CHC13, 5:95): R f 0.24; isomerically pure (3'P NMR). Derivative 3 a was obtained in 90% yield UV (95% EtOH/H20): A,, 262 nm, Amin 232 nm; 'H NMR (CDC13, (3.8 g). A faster moving product identified as 5 '-0-monomethoxy200 MHz) 6: 8.94 (b, 1, NH), 7.63 (d, 1, H6), 6.27 (d, JHI'-H2' tritylarabinouridine-2',3'-O-di-N,N-diisopropyl-~-cyanoethylphos4.8 Hz, 1, Hl'), 5.73 (d, 1, H5), 2.13 (s, 3, COCH3), 2.01 (s, 3, phorarnidite (5a) was also isolated in ca. 5% yield. The chromatoCOCH3). graphic and spectroscopic properties of 3 a , 4a, and 5 a are presented ~ ~ - B e n z o ~',3 l -'-0-diacerylarabinoadenosine 2 ( 6b) in Table 2. Anal. calcd. for C38H45N408P.1/2H20: C 62.88, H 6.39, Compound 66 was prepared from N '-benzoyl-5 '-0-monomethoxyN 7.72; found: C 62.33, H 6.37, N 7.92. tritylarabinoadenosine (2b) (2.0 mmol, 1.3 g) as described for the ~ ~ - B e n z o ~'-0-monomethoxytrifylarabinoadenosine-3 l-5 '-0-N,Npreparation of compound 6a. The product was isolated on silica gel diisopropyl-P-cyanoethylphosphoramidite( 3 b) column chromatography using ethanol/ethyl acetate/CHzC12 1:50:49 Compound 2b (3.0 mmol, 1.9 g) and N,N-diisopropyl-P-cyanoas solvent. The yield of 6b was 78% yield (708 mg). A faster moving ethylphosphonamidic chloride (3.2 mrnol, 672 p L ) were reacted product identified as ~~-acet~l-~~-benzoyl-2',3'-di-O-acetylarabinoaccording to the method described for the preparation of compound 3a. adenosine was also isolated in 13% yield (127 mg). 6b, TLC The products were separated on silica gel column chromatography (methanol/CHC13 5:95): R f 0.26; (ethanol/ether/CH2ClZ 3:71:26): using CH2C12/hexane/triethylamine 50:45:5 as solvent. The 3'-phosR f0.08; (ethanollethyl acetate/CHCI3 5:50:45): R f 0.11; UV (95% phorarnidite and 2',3'-bisphosphoramidite derivatives 3 b and 5b were EtOH/H20): A,, 236,282 nm, Amin 230,248 nm; 'H NMR (CDCI3,
838
CAN. J. CHEM. VOL. 67, 1989
obtained in 66% (1.65 g) and 1190 (335 mg) yields, respectively. The 1 h, ethyl acetate (prewashed with 590 NaHC0,) was added and the solution washed with saturated brine solution (5 X 100 mL). The physical properties of these derivatives are presented in Table 2. Anal. calcd. for 3b, C46H50N707P. H20: C 64.10, H 6.08, N 11.38; found: organic phase was dried (anhydrous Na2S04)and the solvent removed under vacuum to yield the crude product as a white foam. The C64.30,H6.15, N 11.45. crude product was purified by silica gel chromatography by elution 5 '-0-Monornetho.rytrity1arabinouridine-2 '-0-acetyl-3 '-0-N, Nwith CH,C12/hexane/triethylamine 4 0 5 5 5 . After evaporation of the diisopropyl-P-cyanoethylphosphoramidite(IOa) pooled fractions the product was coevaporated, first with 95% ethanol Frotn derivative 3a: To a THF (4 mL) solution of compound 3a to remove traces of triethylamine, and then with ether to provide the (0.5 mmol, 358 mg), 4-dimethylaminopyridine (0.2 mmol, 25 mg), product as a white foam. Derivative l o b was isolated in 75% yield and diisopropylethylamine (4.0 mmol, 0.7 mL) was added dropwise, (332mg). The physical and chromatographic properties of l o b are over 1 min, acetic anhydride (2.0 mmol, 187 pL) at room temperature. presented in Table 2. A faster moving product identified as ~ ~ - a c e t ~ l After stirring for 45 min, ethyl acetate (prewashed with 5% NaHC0,) ~ ~ - b e n z o ~'-0-monomethoxytritylarabinoadenosine-2 l-5 '-0-acetylwas added and the solution washed with saturated brine solution (5 x 3'-0-N,N-diisopropyl-P-cyanoethylphosphoramiditewas also isola100 mL). The organic phase was dried (anhydrous Na2S04)and the ted in 25% yield (1 16 mg); TLC (ether/CH2C12 1: I): R f0.83, 0.76; solvent removed under vacuum to yield derivative 10a as a white foam UV (95% Et0H/H20): A,, 231, 271 nm, Ami, 227, 261 nm; in 95% yield (360 mg). The purity of this material judged by TLC 'H NMR (CDCI,, 400 MHz) 6: 8.65 (s, 1, H8), 8.63 (s, 1, H8), 8.18 and ,'P NMR was greater than 9590. The physical properties of this (s, 1, H2), 8.16 (s, 1, H2), 6.60 (d, JHI,-H2' = 4.2Hz, 1, HI'), compound are presented in Table 2. = 4.2Hz, 1, HI'), 2.59 (s, 6, COCH3), 1.66 (s, 3, From S'-O-monomethoxytrity1-3'-O-tert-butyldimethylsilylarabino- 6.59 (d, JHI'-HZ' COCH,), 1.62 (s, 3, COCH,); I3P NMR (CDC13, 121.5 MHz) 6: uridine: (a) 5'-0-MMT-2'-0-acetylarabinouridine.To a THF (1.5 mL) solution of 5'-O-monomethoxytrityl-3'-O-tert-butyldimethy1si1y1ara- 152.0. 151.9. binouridine (13) (0.23 mmol, 145 mg), 4-dimethylaminopyridine (0.07 mmol, 8 mg), and 2,4,6-collidine (0.92 mmol, 130 pL) was Solution phase synthesis of arabinonucleotides added acetic anhydride (0.92 mmol, 86 pL) at room temperature. After Dinucleotides aUpaU (8a) and aUpaU (9a) stirring for 1 h, the crude product was diluted with CH2C12and the To a sealed vial containing 3a (0.50 mmol, 359 mg), 6a (0.67 mmol, solution washed with saturated brine solution (50 mL) followed by 219 mg), and tetrazole (2.0 mmol, 140 mg) was added THF (3.0 mL) water (50 mL). The organic phase was dried (anhydrous Na2S0,) and and the solution stirred at room temperature for 45 min. At this point the solvent removed under vacuum to yield an oil. Analysis of this a mixture of 4-dimethylaminopyridine(0.26 mmol, 33 mg) and 2,4,6product by TLC showed complete conversion of the starting material collidine (3.6 mmol, 0.5 mL) in THF (2.7 mL) was added and, after (TLC: methanol/CH2C12 5:95, R f 0.58) to a faster moving product 15 min, the phosphite triester intermediate aU,aU (7a) was oxidized (TLC: methanol/CH2C12 5:95, R f 0.68). The 3'-silyl group was to the phosphate with an aqueous iodine solution (0.1 M in THF/water removed by treatment of the crude product with 1 M TBAF/THF 3:1.5, excess, 5 min). The reaction mixture was diluted with CH2C12 (1.2 mmol, 1.2 mL) at room temperature. After 15 min, water was (75 mL) and washed with an aqueous sodium bisulfite solution added (1 mL) and the product extracted with CH2C12(50 mL). The (2-5 mL of 5% sodium bisulfite in 200 mL water). The organic phase organic phase was dried (anhydrous Na2S04)and the solvent removed was washed with saturated brine (2 X 100 mL), dried (anhydrous under vacuum to yield an oil. The crude product was diluted with Na2S04),and evaporated under reduced pressure to yield a foam. The CH2C12(5 mL) and added to a stirred hexane solution (100 mL). The crude product was purified by silica gel chromatography by elution resulting precipitate was filtered and dried under vacuum to obtain with ethanollethyl acetate/CHC13, gradient: 5:50:45 to 10:50:40. The 5'-MMT-2'-0-acetylarabinouridine in quantitative yield (155 mg); pure product 8 a was obtained in 7890 yield (392 mg) and its physical TLC (methanol/CH2C125:95): R f0.49; UV (95% Et0H/H20): A,, properties are presented in Table 2. 234,262 nm, Amin 227, 25 1 nm; 'H NMR (CDC13, 200 MHz) 6: 6.30 It should be noted that 5',3',2'-0-triacetylarabinouridine is obtained (d, JHI'~H2' = 5.7 HZ, 1, HI'), 1.98 (s, 3, COCH,). as by-product and is easily separated during the purification step; ( b ) 5'-0-MMT-2'-0-acetyl-3'-0-phosphoramidite IOU. To a TLC (ethanollethyl acetate/CHCI3 5:50:45): R f 0.63; UV (95% stirred THF (0.6 mL) solution of 5'-0-MMT-2'-0-acetylarabinouriEtOH/H20): A,, 231 nm, Anlin 262 nm. dine (0.20 mmol, 110 mg) and diisopropylethylamine (0.84 mmol, The monomethoxytrityl group was removed by treatment of 8 a 144 pL) was added N,N-diisopropyl-P-cyanoethylphosphonamidic (0.43 mmol, 430 mg) with 80% acetic acid (10 mL) at 90°C for 15 min. chloride (0.24 mmol, 51 pL) at room temperature. After stirring for At the end of this period, ethanol was added and the resulting solution 2 h, ethyl acetate (prewashed with 5% NaHC0,) was added and the was evaporated under reduced pressure to give an oil. This material mixture was worked up and purified as for 3a. Chromatographic was diluted with CH2C12(5 mL) and added to a stirred hexane solution solvent: CH2C12/hexane/triethylamine;gradient: 50:45:5. The pure (100 mL). The resulting precipitate was filtered and dried under product was obtained as a white foam in 50% yield (75 mg). The vacuum to obtain the 5'-deprotected dinucleotide 9a as a white powder chromatographic and spectral properties of this derivative are presented in 80% yield (249 mg). The physical properties of this derivative in Table 2. .. ., C 44.45, are presented in Table 2. Anal. calcd. for C27H32N5017P: H4.42, N 9.60; found: C 44.32, H4.48, N 9.13. 5'-0-Monomethoxytritylarabinouridine-3'-0-acetyl-2 '-0-N, Ndiisopropyl-P-cyanoethylphosphoramidite(11) Dinucleotides aApaA ( 8 b) and aApaA (9 b) Derivative 11 was prepared and purified as for I O U from 5'-0Compounds 3b (0.61 mmol, 5 18 mg) and 6b (0.67 mmol, 373 mg) MMT-3'-0-TBDMS-arabinouridine (13) (vide supra). Physical prowere coupled in the presence of tetrazole in dry THF as described for perties of the intermediate, 5 '-0-MMT-3 '-0-acetylarabinouridine: the preparation of 8a. The product 8b was purified as for 8a. The yield TLC (methanol/CHC13 5195): R f = 0.27; UV (95% EtOH/H20): of 8b was 60% (453 mg) and its physical properties are presented A,,, 234,264 nm, Anlin 230, 252 nm; 'H NMR (CDC13, 200 MHz) 6: in Table 2. It should be noted that N~-benzoyl-5',3',2'-0-triacetyl6.10(d, JHI'-H2' = 4.3Hz, 1, HI'), 2.08 (s, 3, COCH3). arabinoadenosine is obtained as by-product and is easily separated The chromatographic and spectral properties of 11are presented in during the purification step (TLC, ethanol/ethyl acetate/CHC13 Table 2. 5:50:45; R f 0.51). Anal. calcd. for 8b, C63H58N11016P.3/2H20: ~ ~ - ~ e n z o'-0-monomethoxytritylarabinoadenosine-2 ~l-5 '-0-aceC 58.97, H 4.79, N 12.00; found: C 59.00, H 4.92, N 11.30. tyl-3'-O-N,N-diisopropyl-~-cyanoethylphosphoramidite ( I 0 b) Compound 8b (0.25 mmol, 330 mg) was detritylated and purified To a THF (4 mL) solution of compound 3b (0.5 mmol, 422 mg), (silica gel column, methanol/CHCl,: gradient 3-10% methanol) in 4-dimethylaminopyridine (0.2 mmol, 25 mg), and diisopropylethyla similar way as described for the preparation of compound 9a to mine (4.0 mmol, 0.7 mL) was added dropwise, over 1 min, acetic give compound 9b (181 mg, 74%). The physical properties of this anhydride (2.0 mmol, 187 pL) at room temperature. After stirring for derivative are presented in Table 2.
839
DAMHA ET AL.
I
Solid phase synthesis of arabinonucleotides Preparation of Vydac TP silica gel supports In a 50-mL round bottomed flask under argon, 2a (129 mg, 0.25 mmol) or 26 (161 mg, 0.25 mmol) was shaken with aminopropylsuccinylated Vydac TP silica gel (1 .O g) (16), 4-dimethylaminopyridine (25 mg, 0.20 mmol), and dicyclohexylcarbodiimide (2.06 g, 10 mmol) in anhydrous pyridine (9 mL) for 5 days. To the slurry was added p-nitrophenol (70 mg, 0.50 mmol) and the reaction shaken for 16 h. The support was filtered on a sintered glass funnel (M) and washed with pyridine (2 x 5 mL) and THF (2 X 5 mL). The support was then transferred to a 50-mL round bottomed flask and treated with 0.25 M Ac20/2,4,6-collidine/4-dimethylaminopyridine in THF (20 mL) for 18 h. The support was filtered and washed with THF. The support was then treated in a 50-mL round bottomed flask with piperidine (20 mL) for 18 h. The silica support was filtered and successively washed with acetone, EtOH, MeOH, and Et20. The support was dried overnight in a MeOH drying pistol. Loadings of 20.6 pmol g-l and 25.3 pmol g-L for the aA and aU supports respectively were obtained. Loadings were determined by detritylation of 5.0-mg samples with 5% trichloroacetic acid/dichloroethane solution (extinction coefficient used for the MMT cation = 56 pmol-' cm- mL). Solid phase synthesis Approximately 100 mg of derivatized solid support was used in each synthesis (ref. 12b for details). In each synthesis either amidite 3 or 12 was used with the cycle shown in Table 1. The time of recycle was varied (30,60,300, and 900 s) to examine the branching reaction when amidites 3a and 36 were used. In the case of amidites 10a and l o b a 5-min recycle time was used. Deprotection and isolation of arabinonucleotides Deprotection of aUpaU (9a) This derivative (25 pmol, 18 mg) was fully deprotected in a single step by treatment with aqueous ammonia (15 M) in ethanol (3 mL: 1 mL) at room temperature for 16 h. At the end of this period, the ammonia was evaporated in a Speed-Vac concentrator to yield a white foam. This material was diluted with water (1 mL) and passed slowly through a column of Dowex Naf ion exchange resin and the eluant (40 mL) lyophilized. The residue was diluted with water (1 mL) and applied to a column packed with preswollen Sephadex G-25 size exclusion gel. The fractions containing the nucleotide were lyophilized to give 396 A 2 a units of aUpaU (87% yield, ca. 12 mg). 'The chromatographic and spectral properties of aUpaU are summarized in Table 3. aUpaU was enzymatically degraded to aU by a mixture of snake venom and alkaline phosphatase. Degradation of aUpaU with spleen phosphodiesterase resulted in the formation of aU3'p and aU in a 1:l ratio. Deprotection of aA,aA (9b) Compound 96 (25 pmol, 25 mg) was deprotected as for aUpaU 9a. The following conditions were used: ( a ) 15 M ammonium hydroxide/ ethanol (3 mL: 1 mL), 55"C, 18 h; (b) ion exchange chromatography (Dowex Na', 90 mL water as eluant); (c) cellulose TLC (solvent F); (d) size exclusion (Sephadex G-25, water as eluant). A 32% yield (216 AZa units) of aApaA was obtained. The chromatographic and spectral properties of aApaA are presented in Table 3. aA,aA was enzymatically degraded to aA by a mixture of snake venom and alkaline phosphatase. Degradation of aApaA with spleen phosphodiesterase resulted in the formation of aA3'p and aA in a 1:1 ratio. Deprotection of 5'-aUpaApaUpaApaUpA-3' (methods 1 and 2) Silica gel (50 mg) bearing the protected sequences was placed in a sterile 5-mL polypropylene test tube to which was added aqueous ammonia (15 M) in ethanol (3:1, 3 mL) and the tube sealed with a septum (the septum was taped to the tube using commonly available electrical tape). The tube was placed in a thermostated oil bath at 55OC for 18 h to effect the concomitant decyanoethylation, removal of the sequence from the support, and the removal of the exocyclic amino benzoyl protecting groups. After cooling the tube in an EtOH/Dry Ice bath, the washings were removed with a sterile silanized pipette and
'
placed in a sterile 15-mL test tube. The support was further washed with aqueous ammonia (15 M) in ethanol (3:1, 4 x 2 mL). The combined washings were frozen in an EtOH/Dry Ice bath and evaporated in a Speed-Vac evaporator overnight. The residue was extracted with sterile H 2 0 (5 x 1 mL) to yield 57 and 39 AZmunits of 5'-aUpaApaUpaApaU,A-3', methods 1 and 2, respectively. Each sequence isolated from methods 1 and 2 (20 A2m units each) was loaded into 15 cm wide slots on a 1.5 mm x 20 cm x 20 cm 24% polyacrylamide gel for purification. The gels were visualized by UV shadowing and the desired bands cut out. The gel pieces were cut into small pieces and extracted by soaking in 0.05 M NH40Ac, pH 7.0, at room temperature (16 h). The resulting supernatants were applied to a Sephadex G-25 column packed and run in 0.05 M NH40Ac, pH 7.0 (126). A final desalting step was carried out on Waters C18-SepPak cartridges. The final yields were 3.6 and 4.7 ,4260 units for the hexamers prepared by methods 1 and 2, respectively; UV (H20): A, 262. All sequences were stored as 1 A 2 a units110 pL aqueous solutions. Analytical gels were run with 0.5 ,4260 units of the sequence.
Acknowledgements We gratefully acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada and the FCAR program of Quebec. M.J.D. and N.U. acknowledge, respectively, a Dalbir-Bindra fellowship from the Graduate Faculty of McGill University and the award of post-graduate scholarship funds by NSERC. D. W. HENRY, and L. M. BEACHAM 111(Editors). 1. J. L. RIDEOUT, Nucleosides, nucleotides, and their biological activity. Academic Press, New York. 1983, and references cited therein. 2. R. S. K. YOUNGand G. A. FISCHER.Biochem. Biophys. Res. Commun. 32, 23 (1968). 3. R. VINCE,S. DALUGE, H. LEE, W. M. SHANNON, G. ARNETT, T. W. SCHAFER, T. L. NAGABHUSHAN, P. REICHERT, and H. TSAI.Science, 221, 1405 (1983). J. Med. Chem. 10, 762 (1967). 4. W. J. WECHTER. 5. D. P. C. MCGEE.M.Sc. thesis, McGill University, Montreal, Canada (1981). 6. (a) J. SMRTand F. SORM.Biochim. Biophys. Acta, 138, 210 (1967); (b) Collect. Czech. Chem. Commun. 32, 3 169 (1967). and J. NAGYVARY. Biochemistry, 9,1744 7. (a) R. G. PROVENZALE (1970); (b) J. NAGYVARY and G. PROVENZALE. Biochemistry, 8, 4769 (1969). Can. J. Chem. Chem. 48, 862 8. K. K. OGILVIEand D. IWACHA. (1970). Angew. Chem. Int. Ed. Engl. 6, 460 (1967). 9. G. SCHRAMM. 10. C. GIOELI,J. B. CHATTOPADHYAYA, A. F. DRAKE,and B. OBERG.Chem. Scr. 19, 13 (1982). 11. (a) K. K. OGILVIE, A. L. SCHIFMAN, and C. L. PENNEY. Can. J. Chem. 57, 2230 (1979); (b) K. K. OGILVIE, S. L. BEAUCAGE, A. L. SCHIFMAN, N. Y. THERIAULT, and K. L. SADANA. Can. J. Chem. 56,2768 (1978). 12. (a) R. T. PONand K. K. OGILVIE. Nucleosides Nucleotides, 3, 485 (1984); (b) N. USMAN,K. K. OGILVIE,M.-Y. JIANG,and R. J. CEDERGREN. J. Am. Chem. Soc. 109,7845 (1987). 13. K. K. OGILVIE,D. P. C. MCGEE, S. M. BOISVERT,G. H. HAKIMELAHI, and Z. A. PROBA.Can. J. Chem. 61, 1204 (1983). N. USMAN,and K. K. OGILVIE. Tetrahedron Lett. 14. M. J. DAMHA, 28, 1633 (1987). ~ ~OGILVIE. K. J. Org. Chem. 53,3710 (1988). 15. M. J. D A M H A ~ K. J. Am. Chem. Soc. 101, 16. ( a ) P. TUNDOand P. VENTURELLO. 6607 (1979); (b) R. L. KAASand J. L. KARDOS.Polym. Eng. Sci. 11, 11 (1971); (c) R. E. MAJORSand M. J. HOPPER. J. Chromatogr. Sci. 12, 767 (1974); ( d ) R. SCHWARZENBACH. J. Chromatogr. 117, 206 (1976). 17. J. DOORNBOS, J. L. BARASCUT, H. LAZREK, J. L. IMBACH, J. VAN WESTRENEN, G. M. VISSER,J. H. VAN BOOM,and C. ALTONA. Nucleic Acids Res. 11, 4583 (1983).
