Nucleosides and nucleotides. 121. Synthesis of oligonucleotides

Tetsuya Kodama, Satoshi Shuto, Satoshi Ichikawa, and Akira Matsuda .... Noriyasu Haginoya, Akira Ono, Yukari Nomura, Yoshihito Ueno, and Akira Matsuda...
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Bioconjugate Chem. 1003, 4, 499-508

Nucleosides and Nucleotides. 121. Synthesis of Oligonucleotides Carrying Linker Groups at the 1’-Position of Sugar Residues? Akira Ono,? Akihito Dan, and Akira Matsuda’ Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060, Japan. Received June 8, 1993’

Novel 2‘-deoxyuridine analogues carrying aminoalkyl linkers at the 1’-position of the sugar residues were synthesized and incorporated into oligonucleotides, then intercalating groups such as an anthraquinone derivative and a pyrene derivative were attached to the amino groups. Duplexes consisting of the oligonucleotides carrying the linker groups and a complementary ribonucleotide were more stable than an unmodified parent duplex, but the duplexes consisting of the oligonucleotides and a complementary deoxyribonucleotide were less stable. The oligonucleotides carrying the linker groups were more resistant to nuclease P1 and venom phosphodiesterase than an unmodified oligonucleotide. Furthermore, a duplex formed by the oligonucleotide analogue and the complementary ribonucleotide was a substrate for ribonuclease H.

INTRODUCTION Oligonucleotide analogues containing modified backbones or carrying various functional groups have been synthesized and used for biological and biophysical studies such as antisense studies ( 1 , 2 ) . From these studies, it is apparent that the potency of any given antisense oligonucleotide is highly dependent on its resistance to nucleolytic digestion. Therefore, oligonucleotidescontaining nuclease-resistant phosphodiester analogues, such as oligonucleotides with phosphorothioate and methylphosphonate linkages, have been synthesized (I, 2). However, no complete oligonucleotide analogue for antisense study has been established. For instance, methylphosphonate and a-anomeric oligonucleotides with target RNAs form duplexes that are not substrates for RNase H. Thus, the oligonucleotide analogues need relatively high concentration for antisense activity since RNase H-mediated degradation is a major mechanism by which antisense oligonucleotides inactivate mRNA ( 1 , 3 ) . Phosphorothioate oligonucleotides are taken up into cells more slowly than normal oligonucleotidesand have affinity for certain proteins (4-6). Also, the oligonucleotides with methylphosphonate and phosphrothioate linkages have been practically synthesized as mixtures of stereoisomers because of asymmetry about the phosphorus, so that only a small part of the isomers can form stable duplexes with target mRNAs (7).

* Author to whom all correspondence and reprint requests should be addressed. + Part 120: Kakefuda,A., Yoshimura, Y., Sasaki,T., Matsuda, A. (1993) Tetrahedron. In press. t Present address: Department of Chemistry, Faculty of Science,TokyoMetropolitanUniversity,Minamiosawa, Hachioji, Tokyo 192-03,Japan. Abstract published in Advance ACS Abstracts, October 1, @

1993. 1 Abbreviationsused AIBN, 2,2’-azobis(isobutyronitrile); Bur SnH,tributyltin hydride; BQNF, tetrabutylammoniumfluoride; DMTr, 4,4’-dimethoxytrityl;DMTrC1,4,4’-dimethoxytrityl chloride; Fmoc, 9-fluorenylmethoxycarbonyl;FmocC1, 9-fluorenylmethyl chloroformate; HEPES, N-(2-hydroxyethyl)piperazineN’-2-(ethanesulfonicacid);HPLC, high-pressure liquid chromatography; RNase H, ribonuclease H; TIPDS; 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl;TIPDSC12, 1,3-Dichloro-l,l,3,3-tetraisopropyldisiloxane;TEAA, triethylammonium acetate.

Several studies have demonstrated that oligonucleotides modified at the 3’ end are more resistant to nucleases than unmodified ones (8,9).It suggested that oligonucleotides are largely degraded by 3’-exonucleases and that a single modification of the oligonucleotides at the 3‘-end is sufficient to inhibit these enzymes. With target mRNAs, the 3‘-modified oligonucleotidesform more stable duplexes than the oligonucleotides modified at every phosphate, such as phosphorothioates and methylphosphonates, and the duplexes are substrates for RNase H. Therefore, the 3’-modified oligonucleotides have almost complete properties, except that the 3‘-modified oligonucleotides could be degraded by endonucleases in cells. In this report, we describe a new method for introducing functionalgroups into oligonucleotidesto provide nuclease resistance, even if the oligonucleotides contain normal phosphodiester linkages. Many methods to attach the functional groups at several sites on the oligonucleotides, such as the 5’- and 3’-ends, the 5-position of uracil residues, the N-4 of cytosine residues, phosphate residues, and the 2-position of adenine residues, have been reported ( 1 , 2 , I O ) . On the other hand, few methods to attach the functional groups at the sugar moieties have been reported ( 1 1 , 12). However, it can be supposed that the bulky functional group attached at sugar moieties may inhibit hydrolysis by nucleases since the functional groups are close to phosphodiester linkages. Therefore, we introduced nucleosides carrying amino linkers at the 1’-position, 1 and 2 (Figure 1) (13), into oligonucleotides, then functional groups such as an anthraquinone derivative and a pyrene derivative were attached to the amino group to give oligonucleotides containing 3-6. On the other hand, the functional groups may not destabilize duplex formation as follows. The functional groups should be accommodated in the minor groove when the oligonucleotide analogues form duplexes with complementary strands. Since the 1’-position of the sugar moiety in a duplex is in the center of the minor groove, the linker groups may not sterically disorder the duplex formation, especially in the minor groove of RNA-DNA heteroduplexes since the minor grooves of the heteroduplexes are wider than those of DNA-DNA duplexes (14). RNase H recognizes sugar puckering of duplexes distinctly so that duplexes formed by RNAs and oligonu-

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500 Bioconjqte Chem., Vol. 4, No. 6, 1993

Table I. Synthesized Oligonucleotides I-NHz I-AC I-An II-NHz 11-AC 11-An 11-Py 111-NH2

l,n=Z,R=H

Py =

Figure 1. Structureand numberingof the nucleoside analogues.

cleotides containing 2‘-modified sugar moieties are not substrates for the enzyme (15, 16). However, it is also known that “chimeric” oligonucleotides composed of regions containing modified sugar derivatives linked to an unmodified region can form duplexes with target RNAs and the duplexes are substrates for RNase H (17). Therefore, duplexes formed by RNAs and the oligonucleotides containing new nucleoside analogues, 1-6, could be substrates for the enzyme. RESULTS AND DISCUSSION The oligonucleotide analogues used in this study were synthesized by the phosphoramidite method on a commercially available DNA synthesizer. A scheme for a synthesis of 3’-phosphoramidite derivatives of the 2‘deoxyuridine analogues is shown in Figure 2. 1“,3‘,5‘Tri-O-benzoyl-l’-(hydroxymethyl)-2’-deoxyuridinewas synthesized by the reported method (18) with several modifications (Figure 2). The 0,2‘-anhydro linkage of 7 was hydrolyzed under acidic conditions to give 8, then 8 was converted into (thiocarbony1)imidazolide9, which was treated with BusSnH (19) to give 2’-deoxy derivative 10

20, n = 4 21,n=6

111-An IV- (NHda IV-(An)z IV-(PY)Z V-(NHz)s V-(An)s V-(PY)a

(T)76(T)6 (T)d(TM(T)a (T)s~(T)~~(T)s (T)aG(T)rG(T)a (T)d(T)sl(T)sl(T)~ (T)s4(T)s4(T)s4(T)r O“)sG(T)s6(T)sG(T)r

in good yield. After 3’- and 5’-hydroxyl groups were protected by a TIPDS group (20),diaminoalkanes were conjugated with the unprotected 1”-hydroxyl group of 11 to give deoxyuridine analoguescarrying aminoalkyl linkers at the 1’-position, 12 and 13, in good yields. After the amine function was protected with an Fmoc group, the TIPDS group was deprotected, then the nucleosides were converted into corresponding nucleoside 3’-phosphoramidites, 20 and 21. In this study, the nucleoside analogues were introduced into oligonucleotides with a model sequence, (TIn. Oligonucleotides were synthesized on a DNA synthesizer by the phosphoramidite method (21). An average coupling yield of the nucleoside 3’-phosphoramidites 20 and 21 was about 85 % . The yield was lower than that of the thymidine 3’-phosphoramidite probably due to a steric effect of the linkers at the 1’-position, and not due to a purity problem of the amidite samples since the purity of the nucleoside 3’-phosphoramiditesmeasured by 31P-NMRwas more than 99 % (data not shown). Fully protected oligonucleotides were deprotected and purified by the same procedure as for the purification of natural oligonucleotides. Then, the oligonucleotides were treated with acetic anhydride, N- [(anthraquinone-2-carbony1)oxyl succinimide(221,or Nsuccinimidylpyrenebutanolate(22) to give the oligonucleotides containing 3-6. The oligonucleotidessynthesized in the study are listed in Table I. Purity and nucleoside composition of the oligonucleotides in this preparation were analyzed by HPLC. The oligonucleotides had single peaks by HPLC analysis with the (2-18 silica gel column. An example is shown in Figure 3a. Also, each oligonucleotide phosphorylated with 32Pat the 5’-end had a single spot by polyacrylamide gel

0

O P C N

1(T)is 3(T)16 4(T)lS (Th1(T)s (T)73(T)s (T)74(T)s (T)76(T)s (T)72(T)8

I

16, n = 4, R = H 17, n = 6 , R = H 18, n = 4, R = DMTr 119, n = 6, R = DMTr

’K I

I

Figure 2. Synthesis of nucleoside 3’-phosphoramidites. ( a ) a mixture of 2 N HCl and DMF (2:5, v/v), rt, 95% yield; ( b ) N,”thiocarbonyldiimidazole,DMF; ( c ) BusSnH, AIBN, toluene, 92% yield from 8; (d) (1)NaOMe, MeOH, (2) TIPDSC12,57%yield; (e) N,”-carbonyldiimidaole, diaminoalkane, dioxane, 95% yield; U, 9-fluorenylmethyl chloroformate, pyridine, 86 % yield; (g) BQNF, THF,98% yield;(h)DMTrC1,pyridine, 83 % yield; (i) 2-cyanoethylNJV-diisopropylphosphoramid~hlorite,N,N-diisopropylethy~ine,

CHzC12, 80% yield.

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Oligonucleotide with Modified Sugar

Table 11. T,s of Duplexes'

T, ("C) complementarystrands (T)l6

I-NHz I-AC I-An 11-NHz 11-AC 11-An 11-Py 111-NH2 111-An 1'0

20

30

31 34 32 38 30 30 35 33 31 34

33 33 33 35 25 24 29 29 25 21

a Experimental conditions are described in ExperimentalProcedures.

Table 111. T,s of Dudexesa

T, PC) of comdementarv strands 10

20

30

Figure 3. Examples of HPLC profiles of the oligonucleotides: (a) 11-NEz,a linear gradient of CH&N from 10 to 20% (20min) in 0.1 M TEAA buffer pH 6.8; (b) 11-Ac, 10 to 20%; (c) 11-An, 20 to 33%; (d) the nucleoside mixture obtained by complete hydrolysis of 11-Ac, a linear gradient of MeOH from 8 to 20 % (20min) in water; (e)the nucleosidemixture obtainedby complete hydrolysis of 11-An, 20 to 45% (10 min) to 80% (5 min) to 80% (15 min). The peaks were observed at 254 nm. electrophoresis under denaturing conditions (23)(data not shown). Each oligonucleotide was completely hydrolyzed by venom phosphodiesterase and alkaline phosphatase to the corresponding nucleosides, then the nucleoside composition was confirmed by HPLC with the C-18 silica gel column; however, no sharp peak corresponding to 1 was observed (data not shown). Therefore, to certify the existence of the amino group, oligonucleotide 11-NHzwas treated with acetic anhydride to give acetylated oligonucleotide 11-Ac, the retention time of which was slightly longer than that of 11-NHz (Figure 3b). 11-Ac was completely hydrolyzed and the nucleoside composition was analyzed by HPLC (Figure 3d). A peak corresponding to 3 was observed clearly and the nucleoside composition calculated from areas of the peaks (Figure 3d) was approximately T:3 = 15:l. Also, the oligonucleotide carrying the anthraquinone derivative 11-An,the retention time of which was longer than those of 11-NH2and 11-Ac (Figure 3c), was hydrolyzed and the existence of the nucleoside analogue 4 was confirmed (Figure 3e). 4 had a longer retention time than that of 3, owing to a hydrophobicity of the An group. The peaks for 3 and 4 were confirmed by coelution with authentic samples. The applicability of antisense oligonucleotides as inhibitors of gene expression is limited by various factors. For instance, solubility in water, ability to hybridize to complementary targets, and resistance to nucleolitic hydrolysis are required for the antisense oligonucleotides. Furthermore, target RNAs forming duplexes with the antisense oligonucleotides must be substrates for RNase H in order to degrade the target RNAs. The first key factor examined was the stability of duplexes consisting of the oligonucleotides carrying the functional groups and complementary strands. Tms were measured by thermal denaturation. One-transition curves that were similar each other were observed for all melting profiles (data not shown). Tmsare shown in Tables I1and 111. The stability of the duplexes was examined by changingthe kinds of the functional groups, their positions, and complementary strands (Table I). The stability of the duplexes depended upon the position of the functional groups and the complementary strands. With (dA)16 as

33 25 18 7 29 22 12 29 26 17 4 Experimental conditions are described in Experimental Procedures.

the complementary strand, the Tms of the duplexes carrying the linker groups in the center of them, 11-NHz(dA)16, II-Ac-(dA)16, II-An-(dA)16, and II-py-(dA)16, were lower than the T m of the control duplex (T)ls-(dA)le, but the Tmsof the duplexes carrying the linker groups at the 5'-end, I-NHz-(dA)u, I-Ac-(dA)ls, and I-An-(dA)ls, were similar to that of the control. On the other hand, when poly(rA) was used as the complementary strand, Tmsof the duplexes carrying the linker groups at their centers were similar to that of the control. Furthermore, Tmsof the duplexes carrying the linker groups at the 5'ends were higher than that of the control. The functional groups should be accommodated in the minor groove when the oligonucleotide analogues form duplexes with the complementary strands since the 1'position of the sugar moiety in a duplex is in the minor groove. Since the minor groove of the B-type duplex (DNA-DNA duplexes) was narrower than that of the A-type duplex (DNA-RNA and RNA-RNA duplexes) (141, attachment of the bulky group at the 1'-position of the sugar in the center of the B-type duplexes may disturb the duplex conformation, but attachment at the 5'-end may not. In contrast, modification in the minor groove of the A-type duplexes did not destabilize the duplex formation. The phenomenon was supported by the CD spectroscopic study of the duplexes. The study indicated that the spectra of the duplexes containing poly(rA) as the complementary strand were almost identical to that of the control duplex (T)16-p01y(rA),but the spectra of the duplexes containing (*)I6 were different in intensity from the spectra of (T)16(*)I6 even though their splitting patterns are similar (data not shown). Besides the minor-groove substitutions, another factor which might be considerred to estimate the stability of the duplex formation, is the lack of the methyl group at 5-position of the nucleoside analogues. Since it is wellknown that the 5-methyl group of thymidine stabilizes the duplex formation (24), the stability of the duplexes

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1 2 3 4 5 6 7 8

b)

1 2

3 4

P(T) 2 PT

PT

oligonucleotides

Figure 5. Polyacrylamidegel electrophoresisof oligonucleotides hydrolyzedby nucleases: (a) (T)l6(lanes 1-4) and 11-NH2(lanes 5-8) were incubated with venom phosphodiesterase for 0 min (lanes 1and 5), 10 min (lanes 2 and 6), 20 min (lanes 3 and 7), and 30 min (lanes 4 and 8); (b) (T)16(lanes 1and 2) and 11-NH2 (lanes 3 and 4) were incubated with nuclease P1 for 0 min (lanes 1and 3 ) and 10 min (lanes 2 and 4). Experimental conditionsare described in Experimental Procedures.

oligonucleotides

Figure 4. (a) Tmsof the duplexes consisting of the oligonucleotides and poly(rA). (b) T,s of the duplexes consisting of the oligonucleotidesand (dA)16.

could be improved by alkyl substitution at the 5-position of the nucleoside analogues 1-6. Depending upon the kinds of the functional groups in the minor groove, the following orders of Tms were observed: I-An-poly(rA) > I-NH2-poly(rA) 1I-Ac-poly(rA) (in the duplexes modified at the 5’-end) and 11-Anpoly(rA) = 11-Py-poly(rA) > II-NH2-poly(rA) 1 II-Acpoly(rA) (in the duplexes modified a t the center). The same order was observed for duplexes containing (dA)16 as the complementary strand. The results are described as An and Py-conjugated duplexes > free aminobutyl duplex 2 acetylated duplex, and the phenomenon can be interpreted as follows. A positive charge on the terminal amino group of the linker was expected to stabilize the duplex formation electrostatically. However, the acetylation (i.e. the erasure of the positive charge) did not destabilize the duplex formation much, probably because the effect of the positive charge was not crucial under the conditions used for the thermal denaturation study. In contrast to the acetylation, the An and P y derivatization efficiently stabilized the duplex formation even though these modifications,like the acetylation,erased the positive charge on the free amino group. Intercalation of An and P y into the base pairs or hydrophobic interaction between the functional groups and the surface of the minor groove could stabilize the duplex formation effectively. There were no significant difference between Tms of the duplexes carrying the diaminobutane linker (11-NH2

and its derivatives) and those of duplexes carrying the diaminohexane linker (111-NH2and 111-An). Therefore, we used the diaminobutane linker for further studies. Then the stability of the duplexes depending upon the number of the functional groups attached to the oligonucleotides was examined. T,s are summarized in Table I11 and Figure 4. As expected from above studies, the duplexes formed by (dA)16 and the oligonucleotide analogues became less stable as the numbers of the functional groups attached to them increased (Figure 4B). In contrast, the duplexes formed by poly(rA) and the oligonucleotideanalogues carrying two or three functional groups, IV-(An)2-poly(rA), V-(An)3-poly(rA), and VI(Py)2-poly(rA), were more stable than the control duplex (T)lcpoly(rA), except that the duplex carrying three P y groups, V-(Py)3-poly(rA), was slightly less stable than the control (Figure 4A). Consequently,the attachment of one functional group stabilized the duplex formation with the complementary polyribonucleotide greatly and the increases of the number of the functional groups did not largely destabilize the duplex formation. The second key factor examined was the stability of the oligonucleotidesto nucleolyticdegradation. Two kinds of nucleases, snake venom phosphodiesterase (a 3’-exonuclease) and nuclease P1 (an endonuclease), were used in this study. The oligonucleotideslabeled at the 5’-end with 32P(23)were incubated with an appropriate nuclease and the reaction was analyzed by polyacrylamide gel electrophoresis under denaturing conditions (23) (Figure 5). Although the control (T)16was hydrolyzed randomly by venom phosphodiesterase after 30 min (Figure 5a, lanes 2-4), 11-NH2 was hydrolyzed only at the 3’-side from the nucleoside analogue (lanes 6-8). The phosphodiester linkage at the 3’-side of the nucleoside carrying the linker group was resistant to the nuclease. However, the linkage was not perfectly resistant to the nuclease and was hydrolyzed under the conditions for complete digestion (See Experimental Procedures; data not shown). Also, the phosphodiester linkages at the 3‘- and 5’-sides of the nucleoside analogue were more resistant to nuclease P1 than the phosphodiester linkages beside thymidines (Figure 5b).

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1 2 3 4 5 6 7 8 9

A13

PT

Figure6. Polyacrylamidegel electrophoresisof oligonucleotides hydrolyzed by nucleaseP1. (lanes 1-3), V-(NHz)s (lanes443, and V-(An)s (lanes 7-9) were incubated with nuclease P1 for 0 h (lanes 1, 4, 7), 12 h (lanes 2, 5, 8), and 24 h (lanes 3, 6, 9). Experimental conditions are described in Experimental Procedures.

To investigate the resistance of the phosphodiester linkages between the nucleoside analogues to the nuclease, the oligonucleotidescarryingtwo or three functionalgroups were incubated with P1 (Figure 6). Under conditions in which the (T)16Was hydrolyzed completely after 12h (lane 2), the oligonucleotidecarrying three aminobutane linkers, V-(NH2)3, was also hydrolyzed almost completely (lane 5). In contrast, a spot for intact V-(An)3, the oligonucleotide carrying three An groups,was still observed (lane 8)under the same conditions. A bulky group, An, attached to the sugar moiety could increase resistance to the endonuclease. Interestingly, in lane 8, just three spots, corresponding to Pi (phosphoric acid), pT, and intact V-(An)3, and a very faint spot for a fragment of the oligonucleotide hydrolyzed at the middle of it were observed. This lot of nuclease P1 seemed to contain a certain phosphatase activity that might hydrolyze the phosphomonoester at the 5’-end to release Pi. Therefore, it can be interpreted that the spot for intact V-(An)3 could be decreased by releasing 32Pifrom the 5’-end by a phosphatase activity, hydrolyzing the phosphodiester linkage at the 5’-end by the phosphodiesterase activity, but phosphodiester linkages inside the oligonucleotides were not hydrolyzed seriously. Severalstudies investigated how the oligonucleotides modified at the 3’-ends were resistant to 3’-exonucleases (8, 9). Consequently, moderately and properly modified oligonucleotidescontaining the nucleoside analogues not only inside the oligonucleotide but also at the 3‘- and 5’-ends could be highly resistant to the endonuclease. The last key factor examined was degradation of the target RNA by RNase H. The duplexes consisting of V-(An)3 and (rA)16 labeled with 32Pa t the 5’-end was incubated with RNase H, and the products were analyzed by polyacrylamide gel electrophoresis (Figure 7). (rA)16 in the V-(An)3-(rA)ls molecules duplex (lanes 4 and 5) and the (T)16-(rA)16 duplex (lanes 6 and 7) were similarly hydrolyzed by RNase H. It has been known that RNase H recognize sugar puckering distinctly so that duplexes formed by target RNAs and sugar-modified oligonucleotides were not substrates for RNase H (15). However, it has also been known that “chimeric” oligonucleotides composed of regions containingmodified sugar derivatives linked to an unmodified region can form duplexes with target RNAs and the duplexes are substrates for RNase

Figure7. Polyacrylamidegelelectrophoresisof (rA)l~ hydrolyzed by RNase H in the presence of complementary strands: Lane 1,

(rA)16;lane 2, (rA)16+ enzyme; lane 3, (rA)l6+ (T)l6;lanes 4 and + enzyme; lanes 6 and 7, (rA)16+ V-(An)3 + enzyme. Same experiments were repeated in lanes 4 and 5 or lanes 6 and 7. Experimental conditions are described in Experimental Procedures. 5, (rA)16 + (T)16

H (I7).Furthermore, it was obvious from our results that RNase H can cleave the duplex carrying the bulky group at every fourth nucleoside. In above study, we demonstrated that the duplexes formed by the oligonucleotide analogues and the ribonucleotide were substrates for RNase H since the oligonucleotides were substituted moderately so that the enzyme can recognize regions containing unmodified backbone as substrates. Furthermore, the moderate substitution could be enough for providing resistance to exo and endonucleases, even if the oligonucleotides contain unmodified nucleosides and phosphodiester linkages. Consequently, the results may indicate that the properly modified oligonucleotides containing the unmodified phosphodiester linkages can be applied for antisense study. CONCLUSION It has been shown in this study that the oligonucleotide analogueswith normal phosphodiester linkagescan be used as potential antisense agents. The duplexes formed by the oligonucleotides carrying the functional groups at the 1’-position of the sugar moiety and the complementary polyribonucleotidewere more stable than the unmodified control duplex. Also, the oligonucleotide analogues were resistant to degradation by exo and endonuclease. Moreover, the duplexesformed by the oligonucleotideanalogues and the ribonucleotideswere substrates for RNase H. The results indicated that the moderately substituted oligonucleotides could be applicable to antisense studies. EXPERIMENTAL PROCEDURES General Experimental. Melting points were measured on a Yanagimoto MP-3 micromelting point apparatus (Yanagimoto, Japan) and are uncorrected. Mass spectra (MS) were measured on a JEOL JMX-DX303 spectrometer (JEOL, Japan) a t an ionizing voltage of 70 eV. Field desorption mass spectra (FD-MS) and fast atom bombardment mass spectra (FAB-MS) were measured on a JEOL JMS-HX110 a t an ionizing voltage of 70 eV. The lH NMR spectra were recorded on a JEOL JNM-FX lOOFT (100-MHz)spectrometer or a JEOL JNM-GX 270 (270-MHz) spectrometer or a JEOL JNM-EX 400 (400MHz) spectrometer with tetramethylsilane as an internal standard. Chemicalshifts are reported in parts per million (6), and signals are expressed as s (singlet), d (doublet),

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t (triplet),m (multiplet), or br (broad). All exchangeable protons were detected by disappearance on the addition of DzO. TLC was done on Merck Kieselgel F254 precoated plates (Merck). The silica gel, the neutralized silica gel, or the silanized silica gel used for column chromatography were YMC gel 60A (70-230 mesh) (YMC Co., Ltd.), or ICN silica 60A (ICN Biochemicals), or preparative C18 125A (Waters),respectively. UV absorption spectra were recorded with a Shimadzu UV-240 spectrophotometer (Shimadzu Corp.). 1-( 1,4,6-Tri-0-benzoyl-P-D-fructofuranosyl) uracil (8). 2,3’-0-Anhydro-1-(1,4,6-tri-0-benzoyl-P-D-fructofuranosyl)uracil(7)(18) (12.0 g, 21.1 mmol) was dissolved in a mixture of DMF (500 mL) and 2 N HCl(200 mL), and the mixture was stirred at room temperature for 2 days. The reaction mixture was neutralized by adding 4 N NaOH (100 mL), and precipitates were filtered off. The liquid layer was concentrated to dryness in uucuo,and the residue was dissolved in ethyl acetate. The solution was successively washed with water (three times) and then brine, dried over Na2S04, and concentrated to about one-fourth. The resulting precipitates were collected to give 1 (11.2 g, 91 % 1. The filtrate was chromatographed over a silica gel column (2.7 X 7 cm) with 30-70 % ethyl acetate in n-hexane as eluents. Fractions were concentrated to give a further amount of 1 (0.6 g, 5%1. The physical data were identical with those of an authentic sample previously reported (25). 1-(1,4,6-TriO-benzoyl-3-deoxy-~-~-psicof uranosy1)uracil (10). A mixture of 8 (14.8 g, 24.8 mmol) and N,”thiocarbonyldiimidazole (29) (14.1 g, 74.4 mmol) in DMF (130 mL) was stirred at room temperature for 3 days under an Ar atmosphere. The reaction mixture was diluted with ethyl acetate (500 mL) and the solution was successively washed with water (50 mL, 5 times) and then brine, dried over Na2S04, and concentrated to give thiocarbonylimidazolide 9. After being dehydrated by coevaporation with toluene twice, 9 was dissolved in toluene (200 mL) containing AIBN (200 mg) and BusSnH (19) (19 mL, 74.4 mmol). The mixture was heated under reflux for 40 min, then the solvent was removed by evaporation. The residue was directly crystallized from a mixture of MeOH and CHC13 to give 10 (12.2 g, 87%). The mother liquid was concentrated and chromatographed over a silica gel column (6 X 15 cm) with 10-5076 ethyl acetate in n-hexane as eluents. Fractions were concentrated to give a further amount of 10 (0.7 g, 5%): IH NMR (270 MHz, CDCl3) 8.21 (1H, br s, 3-NH), 8.11-7.37 (16 H, m, Ph and H-6), 5.69 (1H, m, H-4’),5.54 (1H, d, H-5, J5,6 = 8.8 Hz), 4.924.78 (4 H, m, H-l’a, H-5’, and H-6’),4.50 (1H, d, H-l’b, Jljgem = 9.3 Hz), 3.12 (2 H, m, H-B’a,b). 1-[3-Deoxy-4,6-0-( 1,1,3,3-tetraisopropyldisiloxane1,3-diyl)-~-~-psicofuranosyl]uracil(ll). (20)NaOMe (1N, 15 mL) was added to a suspension of 10 (9.5 g, 16.7 mmol) in MeOH (140 mL) and the mixture was stirred at room temperature for 13 h. The reaction mixture was neutralized by addition of Dowex 50 (H+ form) and the resin was filtered and washed with hot MeOH. The combined filtrate and washings were concentrated to dryness in uacuo. After it was successively coevaporated with benzene (three times) and then DMF (once), the deprotected compound was dissolved in DMF (100 mL). Imidazole (2.5 g, 36.8 mmol) was added to the solution and the mixture was stirred at -30 OC under an Ar atmosphere. TIPDSClz (4.9 mL, 15.5 mmol) was added dropwise over 20 min to the solution and the mixture was stirred for 2 h at -30 OC, then overnight at room temperature. MeOH (5 mL) was added to the reaction mixture, and the solvents were concentrated. The residue

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was chromatographed over a silica gel column (5 X 12 cm) with 0-20% EtOH in CHCl3 as eluents. Fractions were concentrated to give 11 (4.8 g, 57% as a white foam): EIMS mlz 469 (M+-CH20H), 457 (M+-isopropyl),389 (M+base); IH NMR (270 MHz, CDCl3) 8.26 (1H, br s, 3-NH), 7.95 (1H, d, H-6, J5,6 = 8.1 Hz), 5.67 (1H, dd, H-5, J ~ , N H = 2.4 Hz),4.34 (1H, ddd, H-4’, J4!,5’ = 8.1,J3fa,4?= 7.0, J3!b,4’ = 10.6 Hz), 4.13-3.95 (4 H, m, H-6’a,b and H-l’a,b, J6’gem = 13.2, J5j,6? = 2.6, Jl’gem = 12.0 Hz), 3.84 (1H, dt, H-5’, J5/,ty = 2.6, J4#,5f = 8.1 Hz), 2.93 (1H, dd, H-3’a, J3#gem = 13.6,Jya,4t= 7.0 Hz), 2.43 (1H, dd, H-3’b, J3’b,4’ = 10.6 Hz), 1.05 (28 H, m, isopropyl). 1’-[[[N-(4-Aminobutyl)carbamoyl]oxy]methyl]-3’,5’0(1,1,3,3-tetraisopropyldisiloxane1,3-diyl)-2’deoxyuridine (12). After it was dehydrated by coevaporation three times with toluene, 11 (300 mg, 0.6 mmol) was dissolved in dioxane (5 mL). N,”-Carbonyldiimidazole (146 mg, 0.9 mmol) was added to the solution and the mixture was stirred for 2 h at room temperature, then a further amount of N,N‘-carbonyldiimidazole(49 mg) was added to the mixture, which was stirred for a further 2 h. The reaction mixture was diluted with H2O (30 mL) and the solution was extracted with ethyl acetate (40 mL, three times). The combined organic layers (about 100 mL) were added slowly to a solution of lP-diaminobutane (0.3 mL, 3.0 mmol) in ethyl acetate (30 mL). The reaction proceeded immediately. The solution was successively washed with water (five times) and then brine, dried over NazSO4, and concentrated to give 12 (360 mg, 99% as a foam),which was used for the next reaction without further purification: EI-MS mlz 614 (M+), 571 (M+-isopropyl), 503 (M+-base);1H NMR (270 MHz, CDCl3) 10.70 (1H, br s, 3-NH), 7.93 (1 H, d, H-6, J5,6 = 8.3 Hz), 5.99 (1 H, br t, OCONH), 5.73 (1H, d, H-5),4.81 (1H, d, H-l”a, Jl”gem = 11.5 Hz), 4.38-4.25 (2 H, m, H-l”b and H-3’), 4.02 (2 H, m, H-5’a,b), 3.86 (1H, m, H-4’),3.05 (5 H, m, H-2’a, H-2”, and H-5’9, 2.26 (1H, dd, H-2’b), 1.75, 1.55 (each 2 H, m, H-3”a,b, or H-4”a,b), 1.00 (28 H, m, isopropyl). 1’-[ [[N-[ 4-[(9-Fluorenylmethoxycarbonyl)amino]-

butyl]carbamoyl]oxy]met hyl]-3’,5’-0-( 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)%’-deoxyuridine( 14). Pyridine (65 wL, 0.8 mmol) and 9-fluorenylmethyl chloroformate (FmocC1, 207 mg, 0.8 mmol) were added to a solution of 12 (326 mg, 0.53 mmol) in CHzClz (5 mL). The reaction mixture was stirred for 2 h at room temperature. Ethyl acetate (50 mL) was added to the reaction mixture and the solution was successively washed with water (three times) and then brine, dried over NazS04, and concentrated. The residue was chromatographed over a silica gel column (1.7 X 8 cm) with 0-4% EtOH in CHCl3 as eluents. Fractions were concentrated to give 13 (391 mg, 88% as a white foam): FD-MS mlz 836 (M+); IH NMR (400MHz, CDC13) 8.18 (1H, br s, 3-NH), 7.90 (1H, d, H-6, J5,6 = 8.3Hz), 7.76-7.30 (8H, m, Fmoc), 5.66 (1H, d, H-5), 4.75 (1H, d, H-l”a, J1”gem = 11.3 Hz), 4.41 (2 H, d, FmocCH2, J A =~ 7.3 Hz), 4.36 (1 H, d, H-l”b), 4.35 (1 H, m, H-3’), 4.22 (1H, t, H-9 of Fmoc, J A =~ 7.3 Hz), 4.20 (2 H, dd, H-5’a,b, J5tgem = 13.0, J4t.5’ = 3.0 Hz), 3.85 (1 H, dt, H-4’, J3t,4‘ = 8.3 Hz), 3.19 (4 H, m, H-2” and H-5”), 3.05 (1H, dd, H-2’a, J2’gem = 13.7, Jya,3’ = 6.6 Hz), 2.34 (1H, dd, H-2’, Jyb,3? = 9.9 Hz), 1.50 (4 H, m, H-3” and 4”),1.00 (28 H, m, isopropyl).

1’-[[ [N-[ 4-[(9-Fluorenylmethoxycarbonyl)amino]hexyl]carbamoyl]oxy]methyl]-3’,5’-0-( 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-deoxyuridine (15). Compound 11 (860 mg, 1.7 mmol) was treated with N,Ncarbonyldiimidazole (837 mg, 5.1 mmol) and 1,g-diaminohexane (3.6 g, 31 mmol) to give a nucleoside carrying a

Oligonucleotide with Modified Sugar

Biocon/ugate Chem., Vol. 4, No. 6, 1993 505

J 5 , 6 = 8.2 Hz), 7.76-7.20 (8 H, m, Fmoc), 7.33-6.78 (13 H, hexylamine group (13),which was treated with FmocCl m, DMTr), 5.49 (1H, dd, H-51, 5.02 and 4.95 (each 1H, (480 mg, 1.9 mmol) as described above. The mixture was br t, OCONH), 4.65 (1 H, d, H-l”a, Jl-gem = 11.5 Hz), chromatographed over a silica gel column (2.2 X 12 cm) 4.42-4.20 (7 H, m, H-3’, 4’, 5’a,b, H-9 of Fmoc, and Fmocwith 0-2% EtOH in CHCl3 as eluents. Fractions were CH3, 3.77 (6 H, s, OMe), 3.17 (4 H, m, H-2” and H-5”), concentrated to give 15 (1.0 g, 68% from 11, as a white 2.84 (1H, dd, H-2’a, = 15.3, J2’a,3’ = 5.8 Hz), 2.66 (1 foam): FAB-MS mlz 865 (M+ + 1); IH NMR (270 MHz, H, br d, H-2’b), 2.47 (1H, br s,3’-OH), 1.48 (4 H, m, H-3” CDCl3) 8.21 (1H, br s, 3-NH), 7.89 (1H, d, H-6, J5,6 = 8.2 and H-4”). Hz), 7.78-7.28 (8 H, m, Fmoc), 5.65 (1H, d, H-5),4.88 and 4.74 (each 1H, br t, OCONH), 4.72 (1H, d, H-l”a, J1”gem 1’-[[[N-[ 4 4(9-Fluorenylmethoxycarbonyl)amino]= 11.5 Hz), 4.45-4.30 (4 H, m, H-3’, H-l”b, and Fmochexyl]carbamoyl]oxy ]methyll-5’-0-(dimethoxytrity1)CH3, 4.22 (1H, t, H-9 of Fmoc), 4.08 (2 H, m, H-5’a,b), 2’-deoxyuridine (19). After it was dehydrated by co3.84 (1H, dt, H-4’, J3’,41= 8.2 Hz), 3.18 (4 H, m, H-2” and evaporation with pyridine (three times), 17 (365 mg, 0.59 H-7”), 3.04 (1H, dd, H-2’a, J2”gem 13.7, J2f8,3/ = 7.1 Hz), mmol) was dissolved in pyridine (3 mL), then DMTrCl 2.33 (1H, dd, H-2’b, J2q,,3< = 10.0 Hz), 1.50 (4 H, m, H-3” (208 mg, 0.62 mmol) was added to the solution. The and H-6’9, 1.30 (4 H, m, H-4” and H-5’9, 1.00 (28 H, m, mixture was stirred for 2.5 h at room temperature, then isopropyl). Anal. Calcd for C4H64N4010Si2: C, 61.08; H, MeOH (I mL) was added to the reaction and the solvents 7.46; N, 6.48. Found: C, 61.09; H, 7.36; N, 6.16. were removed. The residue was dissolved in ethyl acetate and the solution was washed with water, dried over Naz1’-[[[N-[ 4-[ (9-Fluorenylmethoxycarbonyl)amino]SO4,and concentrated. The residue was chromatographed butyl]carbamoyl]oxy]methyl]-2’-deoxyuridine (16). over a neutralized silica gel column (2.7 X 6 cm) with 35BQNF (1M, 1.2mL,THFsolution)wasaddedtoasolution 100% ethyl acetate in n-hexane as eluents. Fractions were containing 14 (391 mg, 0.47 mmol) and acetic acid (150 concentrated to give 19 (445 mg, 82% as a white foam). pL) in THF (10 mL), and the whole was stirred overnight FAB-MS m/z 926 (M+ 1); ‘H NMR (270 MHz, CDCl3) at room temperature. MeOH (1 mL) was added to the 8.35 (1 H, br s, 3-NH), 7.77 (1H, d, H-6, J5,6 = 8.2 Hz), mixture and the solution was concentrated to dryness. 7.77-7.20 (8 H, m, Fmoc), 7.29-6.81 (13 H, m, phenyl), The residue was chromatographed over a silica gel column 5.47 (1H, dd, H-51, 4.90 (2 H, br s, OCONH), 4.62 (1H, (1.7 X 6 cm) with &lo% EtOH in CHCl3 as eluents. d, H-l”a, J1”gem = 11.7 Hz), 4.42-4.18 (7 H, m, Fmoc-CHz, Fractions were concentrated to give 16 (273 mg, 98% as a white foam): FAB exact mass calcd for C ~ O H ~ ~ O Q H-9 N ~ of Fmoc, H-l”b, H-3’, H-4’, and H-5’a,b), 3.77 (6 H, s,OMe),3.18 (4H,m,H-2”andH-7”),2.82 (1H,m,H-2’a), 595.2403, found 595.2375; lH NMR (270 MHz, DMSO-de) 2.64 (1H, m, H-2’b), 2.62 (1H, br s, 3’-OH), 1.62 (8 H, m, 7.88 (1H, d, H-6, J 5 , 6 = 7.7 Hz), 7.90-7.30 (8 H, m, Fmoc), H-3”, H-4”, H-5”, and H-6”). 7.29 and 7.12 (each 1H, br t, OCONH), 5.46 (1H, d, H-5), 5.21 (1H, d, 3’-OH, J = 5.3 Hz), 4.60 (1H, d, H-l”a,J1ftgem /3-Cyanoethyl[ 1’-[[ [N-[4-[(9-Fluorenylmethoxy= 11.4 Hz), 4.31-3.99 (6 H, m, H-l”b, H-3’, H-4’, H-9 of carbonyl)amino]butyl]carbamoyl]oxy ]methyl]-5'4Fmoc,and Fmoc-CH2),3.45 (2 H,m,H-5’a,b),2.95 (4 H, (dimethoxytrityl)-2’-deoxyuridin-3’0-ylI-NJV-diisom, H-2” and H-5”), 2.65 (1 H, dd, H-2’a, J2”gem = 14.5, propylphosponamidite (20). After it was dehydrated J2’a,3’ = 6.5 Hz), 2.27 (1H, dd, H-2’b, Jyb,3’ = 3.5 Hz), 1.34 by coevaporation with pyridine twice, 18 (172 mg, 0.19 (4 H, m, H-3” and H-4”). mmol) was dissolved in CH2Clz (5 mL) containing N,Ndiisopropylethylamine (50 pL, 0.29 mmol). 2-Cyanoethyl 1’-[[ [N-[ 4-[ (9-Fluorenylmethoxycarbonyl)amino]N,N-diisopropylphosphoramidochloridite(26)(50pL, 0.23 hexyl]carbamoyl]oxy]met hyl]-2’-deoxyuridine( 17). mmol) was added to the solution and the reaction mixture Compound 15 (980 mg, 1.1mmol) was treated with Budwas stirred for 0.5 h at room temperature. CHC13 and NF as described above. The product was purified by silica aqueous saturated NaHC03 were added to the mixture, gel chromatography (2.8 X 10 cm) with 0-12% EtOH in and the separated organic layer was washed with aqueous CHC13 as eluents. Fractions were concentrated to give 17 saturated NaHC03 (three times), dried over Na~S04,and (660 mg, quant. as a white foam): FAB exact mass calcd concentrated. The residue was chromatographed over a for C ~ ~ H ~ Q623.2717, N ~ O Q found 623.2719; ‘H NMR (270 silica gel column (1.8 X 5.5 cm) with ethyl acetate as an MHz, CDC13) 9.41 (1H, br s, 3-NH), 7.98 (1H, d, H-6, J5,6 eluent. Fractions were concentrated to give 19 (167 mg, = 8.2 Hz), 7.74-7.25 (8 H, m, Fmoc), 5.60 (1H, d, H-5), 80% as a foam): FAB exact mass calcd for C&70N&P 5.42 (1H, br s, 3’-OH), 5.19 (1H, br s, 5’-OH), 4.67 (1H, 1097.4789, found 1097.4860; 31PNMR (270 MHz, CDCl3) d, H-l”a, J l f r g e m = 12.1 Hz), 4.45-4.16 (6 H, m, Fmoc-CHz, 149.32, 149.12 (diastereomers). H-9 of Fmoc, H-l”b, H-3’, and H4’), 3.28 (2 H, m, H-S’a,b), 3.09 (4 H, m, H-2” and H-7”), 2.87 (1 H, m, H-2’a), 2.50 /3-Cyanoethyl[ 1’-[[ [N-[4-[ (9-Fluorenylmethoxy(1H, m, H-2’b), 1.75-1.60 (8 H, m, H-3”, H-4”, H-5”, and carbonyl)amino]hexyl]carbamoyl]oxy]methyl]-5’-0(dimethoxytrityl)-2’-deoxyuridin-3’0-yl]-NJV-diisoH-6”). propylphosphonamidite (21). Compound 19 (388 mg, 1’-[ [[N-[4-[ (9-Fluorenylmethoxycarbonyl)amino]0.42 mmol) was phosphitylated as described above to give butyl]carbamoyl]oxy]methyl]-5’-0-(dimethoxytrity1)21 (472 mg, 98% as a yellow foam). 2’-deoxyuridine (18). After it was dehydrated by co1‘-[ [ [N-(4-Aminobutyl)carbamoyl]oxy]methyl]-2’evaporation with pyridine (three times), 16 (279 mg, 0.47 deoxyuridine(1). Compound 16 (75mg, 0.13 mmol) was mmol) was dissolved in pyridine (3 mL). DMTrCl (167 dissolved in concentrated NH40H (28%, 4 mL) and the mg, 0.49 mmol) was added to the solution and the mixture solution was kept overnight at room temperature. The was stirred a t room temperature. After 3 h, 8 mg of mixture was concentrated to dryness and the residue was DMTrCl was added and the reaction was stirred overnight. chromatographed over a silanized silica gel column (0.7 X MeOH (1 mL) was added to the reaction mixture, the 1.5 cm) with water as an eluent. Fractions were concensolvents were removed, and the residue was chromatotrated and the residue was successively coevaporated with graphed over a silica gel column (1.7 X 7 cm) with 0-4% EtOH and then toluene to give 1 (44 mg, 95% as a solid): EtOH in CHCL as eluents. Fractions were concentrated FAB exact mass calcd for C15H25N407 373.1723, found to give 18 (350 mg, 83 % as a white foam): FAB exact mass 373.1731; ‘H NMR (270 MHz, DMSO-&) 7.86 (1 H, d, calcd for C51H53011N4897.3711,found 897.3738; ‘H NMR H-6, J5.6 = 8.3 Hz), 7.13 (1 H, br t, OCONH), 5.45 (1H, (270 MHz, CDC13)8.25 (1H, br s, 3-NH), 7.78 (1H, d, H-6,

+

508 Bioconjugate Chem., Vol. 4, No. 6, 1993

d, H-5), 4.61 (1H, d, H-l”a, J1”gem = 11.0 Hz), 4.15-3.98 (3 H, m, H-l”b, H-3’, and H-4’1, 3.50 (2 H, m, H-5’a,b), 2.90 (4 H, m, H-2” and H-5’9, 2.65 (1H, dd, H-2’a), 2.27 (1H, dd, H-2’b), 1.30 (4 H, m, H-3” and H-4”). 1’-[[[N-( 4-Acetamidobutyl)carbamoyl]oxy]methyl]%’-deoxyuridine(3). Triethylamine (42 pL, 0.3 mmol) and acetic anhydride (23 pL, 0.24 mmol) were added to a solution of 12 (0.2 mmol) in CH2C12 (3 mL), and the reaction mixture was stirred for 20 min at room temperature. MeOH (1mL) was added to the reaction, and the solvents were evaporated. The residue was chromatographed over a silica gel column (1.8 X 6 cm) with 0-496 EtOH in CHCl3 as eluents. Fractions were concentrated and white crystals were collected to give the TIPDS derivative of 3 (111mg, 85 % ). An analytical sample was recrystallized from a mixture of ethyl acetate and ethyl ether: mp 129.5-130.5 “C; FAB-MS mlz 657 (M++ 1);lH NMR (270 MHz, CDCl3) 8.38 (1 H, br s, 3-NH), 7.90 (1 H, d, H-6, J 5 , 6 = 8.2 Hz), 5.88 (1H, br s, OCONH), 5.66 (1 H, dd, H-5, J N H = ,2.3 ~ Hz), 4.87 (1 H, br t, NH of CHsCONH), 4.83 (1H, dd, H-l”a, J1”gem = 11.5 Hz), 4.35 (1 H, ddd, H-3’, Jya,3. = 6.6, Jyb,3) = 9.9, J3!,4! = 8.2 Hz), 4.30 (1H, d, H-l”b), 4.03 (2 H, dd, H-5’a,b, J5’gem = 6.6, J41,51 = 2.8 Hz), 3.86 (1H, dt, H-4’),3.23 (4 H, m, H-2” and H-5”), 3.08 (1 H, dd, H-2’a, Jztgem = 13.7 Hz), 2.35 (1H, dd, H-2’b), 2.03 (3 H, s, Ac), 1.48 (4 H, m, H-3” and H-4”), 1.00 (28 H, m, isopropyl). Anal. Calcd for C2gH52N409Si2: C, 53.02; H, 7.98; N, 8.53. Found: C, 53.03; H, 8.00; N, 8.62. A 1M BmNF solution (THF, 0.3 mL) was added to the solution of the above nucleoside (92 mg, 0.14 mmol) in THF (2 mL). The whole was stirred for 10 min at room temperature, then MeOH (1mL) was added to the reaction, and the solvents were evaporated. The residue was chromatographed over a silica gel column (1.8 X 6.5 cm) with 0-20% EtOH in CHC13 as eluents. Fractions were concentrated to give 3 (49 mg, 84% as a solid): FAB-MS mlz 415 (M+ 1); lH NMR (270 MHz, D M s 0 - d ~ 11.12 ) (1H, br s, 3-NH), 7.86 (1H, d, H-6, J5,6 = 8.2 Hz), 7.76 and 7.14 (each 1 H, br t, CHsCONH), 5.46 (1 H, d, H-5), 5.22 (1H, d, 3’-OH, J = 3.8 Hz), 4.88 (1H, t, 5’-OH, J = 5.5 Hz), 4.61 (1H, d, H-l”a, J l ” g e m = 11.6 Hz), 4.12 (2 H, m, H-l”b and H-3’), 3.99 (1 H, m, H-4’), 3.42 (2 H, m, H-5’a,b), 2.91 (4 H, m, H-2” and H-5’9, 2.64 (1 H, dd, H-2’a, J2’gem = 14.8, J2’a,3‘ = 6.1 Hz), 2.27 (1H, dd, H-2’b, J2’b,3’ = 3.0 Hz), 1.77 (3 H, s, CH&O), 1.30 (4 H, m, H-3” and H-4”). 1’-[[ [ N - [4-[(2-Anthraquinonylcarbonyl)amino]butyl]carbamoyl]oxy]methyl]-2’-deoxyuridine (4). Thionyl chloride (0.5 mL) was added to a solution of anthraquinone-2-carboxylicacid (980 mg, 0.32 mmol) in DMF (1mL) and the mixture was concentrated, then the residue was dissolved in pyridine (5 mL). The solution was added dropwise toa solution of 14 (105 mg, 0.17 mmol) in pyridine (5 mL) at 0 “C.The whole was stirred for 0.5 h a t 0 “C, then the solvent was evaporated and the residue was dissolved in ethyl acetate. The solution was successively washed with water (three times) and then brine, dried over Na2S04, and concentrated. The residue was chromatographed over a silica gel column (2.8 X 7 cm) with 0-3% EtOH in CHCl3 as eluents. Fractions were concentrated to give TIPDS derivative of 4 (127 mg, 87 5% as a solid): mp 167.5-169 “C; FAB-MS mlz 849 (M+). Anal. Calcd for C42H~N4011Si2:C, 59.41; H, 6.65; N,6.60. Found: C, 59.27; H, 6.61; N, 6.61. Bu4NF (1 M, THF, 0.23 mL) was added to a solution of the above nucleoside (65 mg, 77 pmol) in T H F (2 mL). After 15min, the mixture was concentrated and the residue

+

Ono et al.

was chromatographed over a silica gel column (1.8 X 7 cm) with 0-12% EtOH in CHCl3 as eluents. Fractions were concentrated to give 4 (37 mg, 79% as a gum): UV (MeOH) Am, (nm) 257,330; lH NMR (270 MHz, DMSOd6) 11.10 (1H, br s, 3-NH), 8.91 and 7.15 (each 1H, br t, OCONH), 8.63-7.91 (7 H, m, An), 7.85 (1H, d, H-6, J5,6 = 8.2 Hz), 5.45 (1H, d, H-5), 5.20 (1H, d, 3’-OH, J = 3.3 Hz), 4.85 (1H, t, 5’-OH, J = 5.2 Hz), 4.59 (1H, d, H-l”a, Jltjgem = 11.0 Hz), 4.11 (2 H, m, H-l”b and H-3’), 3.98 (1 H, m, H-4’1, 3.40 (2 H, m, H-5’a,b), 3.15 and 2.92 (each 2 H, m, H-2” and H-5”), 2.65 (1H, dd, H-2’a, J 2 ~ g e m= 14.7, J2’a,3’ = 5.9 Hz), 2.20 (1H, m, H-2’b), 1.50 (4 H, m, H-3” and H-4”). N-[(Anthraquinone-2-carbonyl)oxy]succinimide (22). l-Ethyl-3-[(3-(dimethylamino)propyllcarbodiimide hydrochloride (976 mg, 0.4 mmol) and N-hydroxysuccinimide (45 mg, 0.4 mmol) were added to a solution of anthraquinone-2-carboxylic acid (51 mg, 0.2 mmol) in a mixture of THF and DMF (lO:l, 5.5 mL), and the whole was stirred for 2 h at room temperature. The solvents were evaporated, the residue was dissolved in CH2C12, and the solution was successively washed with water, aqueous saturated NaHC03, and then brine, dried over Na2S04, and concentrated. A mixture of ethyl acetate and acetone was added to the residue and the resulting precipitate was collected to give 22 (57 mg, 82%): EI-MS mlz 349 (M+); lH NMR (100 MHz, CDC13) 9.08-7.24 (7 H, m, anthraquinone), 2.96 (4 H, m, -CHzCH2-). Anal. Calcd for C19HllN06: C, 65.33; H, 3.17; N, 4.01. Found: C, 65.04; H, 3.05; N, 3.97. Synthesis of Oligonucleotides. Oligonucleotides were synthesized on a DNA synthesizer (Applied Biosystem Model 381A) by the phosphoramidite method (21). Synthesis was monitored by spectrophotometricmeasurement of released dimethoxytrityl cation at 500 nm on each addition. Then, fully protected oligonucleotides were deprotected and purified by the same procedure as for the purification of natural oligonucleotides. That is, each oligonucleotide linked on the resin was treated with concentrated NH4OH for 5 h at 55 “C, then the released oligonucleotide was chromatographed over a C-18 silica gel column (1X10 cm, Waters) with a linear gradient of acetonitrile in 0.1 M TEAA buffer (pH 7.0). Fractions were concentrated and the residue was treated with 80% acetic acid for 20 min a t room temperature. The solution was concentrated and the residue was coevaporated with water. The residue was dissolved in water and the solution was washed with ethyl ether, then the water layer was concentrated to give a deprotected oligonucleotide. The sample was purified by HPLC with the (2-18 silica gel column (Inertsil ODS-2, GL Science Inc.) when necessary. Acetylation of Oligonucleotides. A solution containing each oligonucleotide (1 OD unit at 254 nm) and acetic anhydride (2 pL) in 0.2 M HEPES buffer (500 pL, pH 7.8) was kept for 50 min at room temperature. The oligonucleotide was passed through a short C-18 column (Dispo SPE (3-18, YMC) and was used for next studies without further purification. Reaction of Oligonucleotides with N-[(Anthraquinone-2-carbony1)oxyIsuccinimide. A solution of 22 (0.5 mg) in DMF (70 pL) was added to a solution of each oligonucleotide (1.5 OD units at 254 nm) in 50 mM NaHC03-Na2C03 (70 pL, pH 10.3) and the mixture was kept overnight at room temperature. After the pH of the mixture was adjusted to 3 by adding 0.1 N HC1, the solution was washed with ethyl acetate (four times), then saturated NaHC03 was added to the water layer to neutralize it.

Oilgonucieotlde with Modified Sugar

The reaction mixture was purified by HPLC to remove the reagents to give the oligonucleotide carrying An. Pyrene Derivatization of Oligonucleotides. A solution of N-succinimidyl pyrenebutanoate (22) (0.5 mg) in DMF (50 pL) was added to a solution of each oligonucleotide (1.5 OD units at 254 nm) in 50 mM NaHCOs-Na2C03 (50 pL, pH 10.3) and the mixture was kept for 2 h a t room temperature. Workup and purification procedure were done as described above to give the oligonucleotide carrying Py. Complete Hydrolysis of Oligonucleotides. Each oligonucleotide (0.1-0.5 OD units a t 254 nm) was incubated with venom phosphodiesterase (10 pg, Boehringer Mannheim) and alkaline phosphatase (0.4units,Takara Shuzo Co., Ltd.) in a buffer containing 0.1 M Tris-HC1 (pH 8.2) and 2 mM MgC12 (total 140 pL) for 24 h at 37 "C. The reaction mixture was heated for 5 min at 100"C and cooled at 0 "C, then cold EtOH (0 "C, 320 pL) was added to the solution. The whole was kept for 1 h at -20 "C, then the solution was centrifuged for 20 min at 0 "C (12 000 rpm). A supernatant was separated and concentrated. The residue was analyzed by HPLC. Thermal Denaturation. Each solution contained each oligonucleotide (84 pM/base) and poly(rA) (84 pM/base) in a buffer of 0.01 M sodium phosphate and 0.05 M NaCl (pH 7.0). The thermally induced transitionof eachmixture was monitored at 260 nm on a Gilford Response 11. Partial Hydrolysis of Oligonucleotide with Venom Phosphodiesterase. (27). Each oligonucleotide labeled with 32Pat the 5'-end (0.01 OD units) (23) was incubated with venom phosphodiesterase (0.4 pg) in the presence of Torula RNA (0.3OD units a t 260 nm) in a buffer containing 37.5 mM Tris-HC1 (pH 8.0) and 7.5 mM MgC12 (total 20 pL) at 37 "C. At appropriate periods, aliquots of the reaction mixture were separated and added to a solution of EDTA (5 mM, 10 pL), then the mixtures were heated for 3 min at 100 "C. The solutions were analyzed by electrophoresis on 20 % polyacrylamide gel containing 8 M urea (23). Partial Hydrolysis of Oligonucleotides with Nuclease P1. (28). Each oligonucleotide labeled with 32P at the 5'-end (0.01 OD units) was incubated with nuclease P1 (12 ng, Yamasa Shoyu Co., Ltd.) in the presence of Torula RNA (0.3 OD units) in a buffer containing 6.8 mM ammonium acetate (pH 5.3) and 0.1 mM ZnCl2 (total 22 pL) at 10 "C. At appropriate periods, aliquots of the reaction mixture were separated and added to a solution of EDTA (5 mM, 10 pL), then the solutions were heated for 5 min at 100 "C. The solutions were analyzed by gel electrophoresis as described above. Hydrolysis of Duplexes with RNase H. (rA)16 labeled with s2P at the 5'-end (50 pmol) was incubated with ribonuclease H (6 units) (Takara Shuzo Co., Ltd.) in the presence or in the absence of the complementary strand [(T)16or V-(An)sl (50 pmol) in a buffer containing 40 mM Tris-HC1 (pH 7.7), 4 mM MgC12, 1 mM DTT, 4% glycerol, and 0.003 5% bovine plasma albumin (total 10 pL) at 20 "C (17). After 20 min, reaction mixtures were heated for 1min at 100 "C, then the reactions were analyzed by gel electrophoresis as described above. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture, Japan.

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(20) Tatsuoka, T., Imao, K., and Suzuki, K. (1986)Phosphono nucleoside. 1. Synthesis of 2,3'-anhydro-l'-deoxy-l'-phosphonO-l-8-D-frUCtofuranOSylUraCil. Heterocycles 24,617-620. (21)Beaucage, S.L., Caruthers, M. H. (1981).Deoxynucleotide

phosphoramidite-A new class of key intermediates for deoxypolynucleotidesynthesis. Tetrahedron Lett. 22,18591862. (22) Telser, J., Cruickshank, K. A., Morrison, L. E., and Netzel, L. (1989)Synthesis and characterization of DNA oligomerand

duplexes containing covalently attached molecular labels; comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. SOC.111,6966-6976. (23) Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular cloning; Cold Spring Harbor Laboratory,New York. (24)Riley, M., Maling,B., and Chamberlii, M. J. (1966)Physical

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adenine-thymine and adenine-uracil homopolymercomplexes. J . Mol. Biol. 20,359-389. (25) Yoshimura,Y.,Otter, B., Ueda. T., and Matsuda, A. (1992) Synthesis and optical properties of syn-fixed carbon-bridged pyrimidine cyclonucleosides. Chem. Pharm. Bull. 40, 17611769. (26) Sinha,N. D.,Biernat, J.,andKoster,H. (1983)&Cyanoethyl N,N-dialkylaminolN-morpholinomonochlorophosphoamidites, new phosphitylating agents facilitating ease of deprotection and work-up of synthesized oligonucleotides. Tetrahedron Lett. 24, 5843-5846. (27) Jay, E.,Bambara, R., Padmanabhan, R., and Wu, R. (1974). DNA sequence analysis: a general, simple and rapid method for sequencing large oligonucleotide fragments for mapping. Nucleic Acids Res. 1, 331-353. (28)Silberklang,M.,Gillum,A.M.,andRajBhndary,U.L. (1977) Theuse of nucleaseP1 in sequenceanalysisof end grouplabeled RNA. Nucleic Acids Res. 4,4091-4108.