Nucleosides and Nucleotides. 174. Synthesis of Oligodeoxynucleotides Containing 4′-C-[2-[[N-(2-Aminoethyl)carbamoyl]oxy]ethyl]thymidine and Their Thermal Stability and Nuclease-Resistance Properties

Article abstract of DOI:10.1021/jo9720492

The synthesis and properties of oligodeoxynucleotides (ODNs) containing 4′-C-[2-[[N-(2-arninoethyl)-carbamoyl]oxy]ethyl]thymidine (3) are described. 4′α-(2-Hydroxyethyl)thymidine (4), which is a precursor for phosphoramidite 5, was synthesized using a newly developed intramolecular radical cyclization reaction at the 4′-position of thymidine derivative 7. The radical reaction of 4′β-(phenylseleno)-3′-O-(dimethylvinylsilyl)thymidine derivative 7, which was prepared from thymidine in several steps, with Bu₃SnH and AIBN, followed by Tamao oxidation, gave either 4′α-(2-hydroxyethyl) derivative 6 or 4′α-(1-hydroxy ethyl) derivative 13, respectively. With a low Bu₃-SnH concentration, the reaction gave 6, via 6-endo-radical-cyclized product 11, as a sole product in 87% yield. The reaction of 7 in the presence of excess Bu₃SnH gave 13 in 75% yield, via 5-exo-cyclized product 12, as a diastereomeric mixture. The 4′α-(2-hydroxyethyl) derivative 6 was then converted into a 4′-C-[2-[[N-(2-aminoethyl)carbamoyl]oxy]ethyl]thymidine derivative 14, which was phosphitylated to give phosphoramidite 5 in 72% yield. In this study, 3 was incorporated into a nonadecamer, d[CTGGCTCAGCTCGTCTCAT]-3′, and a heptadecamer, d[CTCGTACCATTCCGCTC]3′, instead of T at various positions. ODNs containing 3 were more resistant to nucleolytic hydrolysis by both snake venom phosphodiesterase (a 3′-exonuclease) and DNase I (an endonuclease) than unmodified parent ODNs, although ODNs containing 3 only slightly destabilized duplex formation with both complementary DNA and RNA strands. Furthermore, the duplex formed by an ODN containing 3 and its complementary RNA was a good substrate for Escherichia coli RNase H.

Full text of DOI:10.1021/jo9720492

1660
J . Org. Chem. 1998, 63, 1660-1667
Nu cleosid es a n d Nu cleotid es. 174. Syn th esis of
Oligod eoxyn u cleotid es Con ta in in g
4′-C-[2-[[N-(2-Am in oeth yl)ca r ba m oyl]oxy]eth yl]th ym id in e a n d
Th eir Th er m a l Sta bility a n d Nu clea se-Resista n ce P r op er ties1
Yoshihito Ueno, Yuki Nagasawa, Isamu Sugimoto, Naoshi Kojima, Makiko Kanazaki,
Satoshi Shuto, and Akira Matsuda*
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, J apan
Received November 6, 1997
The synthesis and properties of oligodeoxynucleotides (ODNs) containing 4′-C-[2-[[N-(2-aminoethyl)-
carbamoyl]oxy]ethyl]thymidine (3) are described. 4′R-(2-Hydroxyethyl)thymidine (4), which is a
precursor for phosphoramidite 5, was synthesized using a newly developed intramolecular radical
cyclization reaction at the 4′-position of thymidine derivative 7. The radical reaction of 4′â-
(phenylseleno)-3′-O-(dimethylvinylsilyl)thymidine derivative 7, which was prepared from thymidine
in several steps, with Bu3SnH and AIBN, followed by Tamao oxidation, gave either 4′R-(2-
hydroxyethyl) derivative 6 or 4′R-(1-hydroxyethyl) derivative 13, respectively. With a low Bu3-
SnH concentration, the reaction gave 6, via 6-endo-radical-cyclized product 11, as a sole product in
87% yield. The reaction of 7 in the presence of excess Bu3SnH gave 13 in 75% yield, via 5-exo-
cyclized product 12, as a diastereomeric mixture. The 4′R-(2-hydroxyethyl) derivative 6 was then
converted into a 4′-C-[2-[[N-(2-aminoethyl)carbamoyl]oxy]ethyl]thymidine derivative 14, which was
phosphitylated to give phosphoramidite 5 in 72% yield. In this study, 3 was incorporated into a
nonadecamer, d[CTGGCTCAGCTCGTCTCAT]-3′, and a heptadecamer, d[CTCGTACCATTCCGCTC]-
3′, instead of T at various positions. ODNs containing 3 were more resistant to nucleolytic hydrolysis
by both snake venom phosphodiesterase (a 3′-exonuclease) and DNase I (an endonuclease) than
unmodified parent ODNs, although ODNs containing 3 only slightly destabilized duplex formation
with both complementary DNA and RNA strands. Furthermore, the duplex formed by an ODN
containing 3 and its complementary RNA was a good substrate for Escherichia coli RNase H.
In tr od u ction
enzyme when methylphosphonate ODN is a complemen-
tary strand.5 Furthermore, phosphorothioate ODNs have
been reported to exhibit nonsequence-specific activity.6
On the other hand, several studies have demonstrated
that ODNs that have been modified at the 3′-end are
more resistant to 3′-exonucleases than unmodified ODNs.7
These 3′-modified ODNs form stable duplexes with
complementary DNA and RNA strands, and these du-
plexes are substrates for RNase H. Therefore, 3′-modi-
fied ODNs have the properties of antisense molecules,
except that they can be degraded by endonucleases inside
cells.
We recently reported the synthesis of ODNs with
natural phosphodiester linkages containing a 2′-deoxy-
uridine analogue 1 that carries an aminoalkyl linker at
the 1′-position (Figure 1).8 We found that ODNs contain-
ing 1 slightly destabilized duplex formation with a
complementary RNA, while these ODNs were more
resistant to nucleolytic hydrolysis by both snake venom
Antisense oligodeoxynucleotides (ODNs) have been
applied extensively to the regulation of cellular and viral
gene expression.2 They hybridize to mRNA targets by
Watson-Crick base-pairing and inhibit translation of
mRNA in a sequence-specific manner. One of the major
problems encountered when using naturally occurring
phosphodiester ODNs as antisense molecules is their
rapid degradation by nucleases found in cell culture
media and inside cells.2 Therefore, several types of
backbone-modified ODNs such as methylphosphonates,
phosphoramidates, and phosphorothioates have been
synthesized and used for antisense studies.2,3
However, the benefits of such enzymatic stabilizations
are sometimes counteracted by the loss of other proper-
ties that are important for antisense activity. Phospho-
rothioate ODNs tend to have lower binding affinity for
their complementary RNA targets than unmodified phos-
phodiester ODNs, possibly because they occur in diaster-
eomeric mixtures.4 Although the role of RNase H cleav-
age in the antisense strategy is not clearly understood,
it has been reported that RNA is not a substrate for the
(4) (a) Cosstick, R.; Eckstein, E. Biochemistry 1985, 24, 3630-3638.
(b) LaPlanche, L. A.; J ames, T. L.; Powell, C.; Wilson, W. D.; Uznanski,
B.; Stec, W. J .; Summers, M. F.; Zon, G. Nucleic Acids Res. 1986, 14,
9081-9093. (c) Latimer, L. J . P.; Hampel, K.; Lee, J . S. Nucleic Acids
Res. 1989, 17, 1549-1561. (d) Hacia, J . G.; Wold, B. J .; Dervan, P. B.
Biochemistry 1994, 33, 5367-5369.
(5) (a) Tidd, S. M.; Hawley, P.; Warenius, H. M.; Gibson, I. Anti-
Cancer Drug Des. 1988, 3, 117-127. (b) Walder, R. Y.; Walder, J . A.
Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5011-5015.
* To whom reprint requests should be addressed. Phone: +81-11-
706-3228. Fax: +81-11-706-4980. E-mail: matuda@pharm.hokudai.ac.jp
(1) Part 173: Shuto, S.; Shirato, M.; Sumita, Y.; Ueno, Y.; Matsuda,
A. J . Org. Chem., in press.
(2) (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 544-584. (b)
Milligan, J . F.; Matteucci, M. D.; Martin, J . C. J . Med. Chem. 1993,
36, 1923-1937. (c) Crooke, S. T., Lebleu, B., Eds. Antisense research
and applications; CRC Press: Boca Raton, FL, 1993. (d) Agrawal, S.
Ed. Antisense therapeutics; Humana Press: Totowa, NJ , 1996.
(3) (a) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925-1963.
(b) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123-6194.
(6) (a) Stein, C. A. Antisense Res. Dev. 1995, 5, 241 and references
therein. (b) Agrawal, S. Trends Biotechnol. 1996, 14, 376-387.
(7) (a) Shaw, J .-P.; Kent, K.; Bird, J .; Fishback, J .; Froehler, B.
Nucleic Acids Res. 1991, 19, 747-750. (b) Gamper, H. B.; Reed, M.
W.; Cox, T.; Virosco, J . S.; Adams, A. D.; Gall, A. A.; Scholler, J . K.;
Meyer, R. B., J r. Nucleic Acids Res. 1993, 21, 145-150 and references
therein.
S0022-3263(97)02049-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/14/1998

Synthesis of Oligodeoxynucleotides
J . Org. Chem., Vol. 63, No. 5, 1998 1661
Sch em e 1
F igu r e 1. Structures of the modified nucleoside analogues.
phosphodiesterase (a 3′-exonuclease) and nuclease P1 (an
endonuclease) than unmodified ODNs. More recently,
we also synthesized ODNs containing a 2′-deoxyuridine
analogue 2, which carries an aminohexyl linker at the
5-position of uracil.9 These ODNs greatly stabilized
duplex formation with both complementary DNA and
RNA strands. Furthermore, these ODNs were more
resistant to nucleolytic hydrolysis by both snake venom
phosphodiesterase and nuclease S1 (an endonuclease)
than unmodified ODNs and were stable in medium
containing 10% fetal calf serum. These results indicate
that ODNs that have been suitably modified by ami-
noalkyl linkers are resistant not only to exonucleases but
also to endonucleases, even when they have natural
phosphodiester linkages.
The 4′R-position of nucleosides is much closer to the
internucleotide phosphodiester linkages in ODNs than
the 1′- and 5-positions. Therefore, we envisioned that
ODNs containing a nucleoside analogue with an ami-
noalkyl-linker at the 4′R-position would be more resistant
to endonucleases than ODNs containing a nucleoside
analogue with an aminoalkyl linker at the 1′- or 5-posi-
tion.
developed.10 Although only a few examples of 4′-
branched nucleoside analogues have been reported,10 the
necessary 2′-deoxy-4′R-(2-hydroxyethyl)nucleoside 4 (Fig-
ure 1), which can be a precursor for the synthesis of 5, is
unknown.
Recently, we developed a regio- and stereoselective
method for introducing a 1-hydroxyethyl or 2-hydroxy-
ethyl group at the â-position of a hydroxyl of halohydrins
and selenoalkanols with a dimethyl or diphenylvinylsilyl
group as a radical-acceptor tether via intramolecular
radical cyclization reaction and subsequent Tamao oxida-
tion reaction.11 Therefore, we planned to synthesize the
desired 4′R-branched nucleoside using this intramolecu-
lar radical cyclization reaction at the 4′-position of a
thymidine derivative with a dimethylvinylsilyl group as
a radical acceptor tether. Our synthetic strategy is
outlined in Scheme 1. A substrate 7 for the radical
reaction can be prepared from a known 4′-(phenylsele-
no)thymidine derivative 8.12 Radical reaction of 7 with
low concentrations of Bu3SnH and subsequent Tamao
oxidation13 will give the desired 4′R-branched nucleoside
6, which can be further derivatized to the corresponding
phosphoramidite unit 5.
In this paper, we report the synthesis of ODNs
containing 4′-C-[2-[[N-(2-aminoethyl)carbamoyl]oxy]eth-
yl]thymidine (3). The thermal stability of duplexes
consisting of these ODNs and their complementary DNA
or RNA strands, the resistance of these ODNs to nucle-
olytic hydrolysis by snake venom phosphodiesterase and
DNase I (an endonuclease), and whether the duplex of
an ODN containing 3 with a complementary RNA strand
can elicit RNase H activity were also studied.
The 4′-(phenylseleno)thymidine derivative 8 was pre-
pared starting from thymidine in six steps via a 5′-formyl
derivative of thymidine according to a method recently
(10) Although several methods for introducing carbon substituents
at the 4′-position of nucleosides have been reported, these methods
are not stereoselective and the carbon substituents that can be
introduced are limited: (a) Secrist, J . A., III; Winter, W. J ., J r. J . Am.
Chem. Soc. 1978, 100, 2554-2556. (b) Youssefyeh, R. D.; Verheyden,
P. H.; Moffat, J . G. J . Org. Chem. 1979, 44, 1301-1309. (c) Haraguchi,
K.; Tanak, H.; Miyasaka, T. Tetrahedron Lett. 1990, 31, 227-230. (d)
Haraguchi, K.; Tanak, H.; Itoh, Y.; Saito, S.; Miyasaka, T. Tetrahedron
Lett. 1992, 33, 2841-2844. (e) J ohnson, C. R.; Esker, J . L.; Van Zandt,
M. C. J . Org. Chem. 1994, 59, 5854-5855. (f) Haraguchi, K.; Tanaka,
H.; Itoh, Y.; Yamaguchi, K.; Miyasaka, T. J . Org. Chem. 1996, 61, 851-
858. (g) Marx, A.; Erdmann, P.; Senn, M.; Korner, S.; J ungo, T.;
Petretta, M.; Imwinkelried, P.; Dussy, A.; Kulicke, K. J .; Macko, L.;
Zehnder, M.; Giese, B. Helv. Chim. Acta 1996, 79, 1980-1994. (h)
Waga, T.; Ohrui, H.; Meguro, H. Nucleosides Nucleotides 1996, 15,
287-304. (i) O.-Yang, C.; Wu, H. Y.; Frase-Smith, W. B.; Walder, K.
A. M. Tetrahedron Lett. 1992, 33, 37-40. (j) Bousquie, I.; Madiot, M.;
Florent, J .-C.; Monneret, C. Bioorg. Med. Chem. Lett. 1996, 6, 1815-
1818.
Resu lts a n d Discu ssion
Syn th esis. To synthesize ODNs containing 3, a 4′R-
branched nucleoside phosphoramidite unit 5 is needed.
However, efficient general synthetic methods for prepar-
ing 4′-branched nucleoside analogues have not been
(8) (a) Dan, A.; Yoshimura, Y.; Ono, A.; Matsuda, A. Bioorg. Med.
Chem. Lett. 1993, 3, 615-618. (b) Ono, A.; Dan, A.; Matsuda, A.
Bioconjugate Chem. 1993, 4, 499-508.
(9) (a) Ono, A.; Haginoya, N.; Kiyokawa, M.; Minakawa, N.; Mat-
suda, A. Bioorg. Med. Chem. Lett. 1994, 4, 361-366. (b) Haginoya, N.;
Ono, A.; Nomura, Y.; Ueno, Y.; Matsuda, A. Bioconjugate Chem. 1997,
8, 271-280. (c) Nomura, Y.; Ueno, Y.; Matsuda, A. Nucleic Acids Res.
1997, 25, 2784-2791. (d) Ueno, Y.; Kumagai, I.; Haginoya, N.;
Matsuda, A. Nucleic Acids Res. 1997, 25, 3777-3782.
(11) Shuto, S.; Kanazaki, M.; Ichikawa, S.; Matsuda, A. J . Org.
Chem. 1997, 62, 5676-5677.
(12) Giese, B.; Erdmann, P.; Schafer, T.; Schwitter, U. Synthesis
1994, 1310-1312.
(13) Tamao, K.; Ishida, N.; Kumada, M. J . Org. Chem. 1983, 48,
2122-2124.

1662 J . Org. Chem., Vol. 63, No. 5, 1998
Ueno et al.
Sch em e 2
Sch em e 3
developed by Giese and co-workers.12 Introduction of a
dimethylvinylsilyl group as a radical acceptor tether was
performed by treating 10 with dimethylvinylchlorosilane
and Et3N in the presence of DMAP in toluene14 to afford
a vinylsilyl derivative 7, a substrate for the radical
reaction, in 95% yield (Scheme 2).
The radical reactions were performed with Bu3SnH
using AIBN as a radical initiator, and the products were
isolated after Tamao oxidation. When a solution of 1.8
equiv of Bu3SnH and AIBN (0.5 equiv) in benzene was
added slowly over 4 h to a solution of 7 in benzene (0.01
M) at 80 °C, the desired 4′R-(2-hydroxyethyl)thymidine
derivative 6, which was derived from a 6-endo-cyclized
product 11, was isolated in 87% yield.15 The regioselec-
tivity could be completely reversed depending on the
reaction conditions used. When 7 was treated with 3.0
equiv of Bu3SnH at 60 °C in benzene in the presence of
AIBN, a diastereomeric mixture of 4′R-(1-hydroxyethyl)
derivatives 13, which was derived from a 5-exo-cyclized
product 12, was obtained in 75% yield.16 Therefore, the
radical reaction with the vinylsilyl tether may be useful
for the regio- and stereoselective introduction of a 1- or
2-hydroxyethyl group to various systems.
To synthesize the phosphoramidite unit 5, 6 was then
converted into carbonylimidazolide, which was reacted
with 1,2-diaminoethane. Without purifying the product,
the amino group was protected with a trifluoroacetyl
group to give 14, which was then phosphitylated by a
standard procedure17 to give the corresponding nucleoside
3′-phosphoramidite 5 (Scheme 3).
and 2. The average coupling yield of 5 was 96% using a
1.2 M solution of the amidite in CH3CN and a coupling
time of 360 s.17 The fully protected ODNs (1 µmol) linked
to the solid supports were treated with concentrated NH4-
OH at 55 °C for 16 h, followed by C-18 column chroma-
tography; detritylation gave ODNs (16-20 and 22-24)
in 32-95 OD260 units. Each ODN analogue obtained
showed a single peak on reversed-phase HPLC. Fur-
thermore, ODNs 16 and 22 were analyzed by electrospray
ionization (ESI) mass spectrometry, and the observed
molecular weights supported their structures (see Ex-
perimental Section).
Th er m a l Sta bility. The stability of the duplexes
formed by these ODNs and a complementary DNA, 5′-
d[ATGAGACGAGCTGAGCCAG]-3′ (25), or RNA, 5′-
r[GAGCGGAAUGGUACGAG]-3′ (26), was studied by
thermal denaturation. To confirm the effect of the
terminal ammonium ion of the aminoalkyl linker of 3 on
the thermal stability of the duplexes, thermal denatur-
ation was performed under both low ionic strength (0.05
M NaCl) and high ionic strength (0.5 M NaCl) in a buffer
of 0.01 M sodium phosphate (pH 7.0). One transition was
observed in the melting profile of each duplex. Melting
temperatures (Tm’s) are listed in Tables 1 and 2.
ODNs containing 3 were synthesized on a DNA syn-
thesizer by the phosphoramidite method.18 In this study,
3 was incorporated into a nonadecamer, d[CTGGCT-
CAGCTCGTCTCAT]-3′, and a heptadecamer, d[CTCG-
TACCATTCCGCTC]-3′, instead of T. The sequences of
the ODN analogues synthesized are shown in Tables 1
With DNA (25) as a complementary strand, the Tm’s
for duplexes containing 3 were the same as or only
slightly smaller than that of the control duplex [64.5 °C
(0.05 M NaCl) and 76.5 °C (0.5 M NaCl), respectively]
(Table 1). The modified nucleoside 3 did not greatly
destabilize duplex formation. Even when four molecules
of 3 were incorporated into the parent ODN instead of
(14) Sieburth, S. M.; Fensterbank, L. J . Org. Chem. 1992, 57, 5279-
5281.
(15) The ratio of 11:12 was 14:1 from the 1H NMR spectra of the
radical reaction product.
(16) The ratio of 11:12 was 1:23 from the 1H NMR spectra of the
radical reaction product.
(17) Gait, M. J ., Ed. Oligonucleotides synthesis: a practical ap-
proach; IRL Press: Oxford, U.K., 1984.
(18) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22,
1859-1862.

Synthesis of Oligodeoxynucleotides
J . Org. Chem., Vol. 63, No. 5, 1998 1663
Ta ble 1. Syn th esized ODNs a n d ODN-DNA Hybr id iza tion Da ta a
ODNsb
0.05 M NaCl Tm (°C)
0.5 M NaCl Tm (°C)
∆Tm (°C)
15
16
17
18
19
20
5′-CTG GCT CAG CTC GTC TCA T-3′
5′-CTG GCT CAG CTC GTC 3CA T-3′
5′-C3G GCT CAG CTC GTC TCA T-3′
5′-C3G GCT CAG CTC GTC 3CA T-3′
5′-C3G GCT CAG C3C GTC 3CA T-3′
5′-C3G GC3 CAG C3C GTC 3CA T-3′
64.5
64.0
63.5
64.5
64.0
64.0
76.5
76.5
76.5
76.0
76.0
75.5
12.0
12.5
13.0
11.5
12.0
11.5
a
b
Experimental conditions are described in the Experimental Section. 3, see Figure 1.
Ta ble 2. Syn th esized ODNs a n d ODN-RNA Hybr id iza tion Da ta a
ODNsb
0.05 M NaCl Tm (°C)
0.5 M NaCl Tm (°C)
∆Tm (°C)
21
22
23
24
5′-CTC GTA CCA TTC CGC TC-3′
5′-CTC GTA CCA 3TC CGC TC-3′
5′-CTC G3A CCA T3C CGC TC-3′
5′-C3C G3A CCA 3TC CGC 3C-3′
61.0
60.0
60.5
60.0
72.5
71.5
71.5
70.5
11.5
11.5
11.0
10.5
a
b
Experimental conditions are described in the Experimental Section. 3, see Figure 1.
T, Tm values were 64.0 °C (0.05 M NaCl) and 75.5 °C
(0.5 M NaCl), respectively. Furthermore, ∆Tm’s [Tm’s (0.5
M NaCl) - Tm’s (0.05 M NaCl)] for duplexes containing
3 were similar to that of the control duplex (12.0 °C) and
were almost independent of the number of 3. Therefore,
the terminal ammonium ion of the amino linker of 3 did
not seem to affect the thermal stability of the ODN-DNA
duplex.
When RNA (26) was used as a complementary strand,
the Tm’s for ODNs containing 3 followed a trend similar
to those for ODN-DNA duplexes, although the Tm values
were smaller than those of ODN-DNA duplexes (Table
2). Although ∆Tm’s [Tm (0.5 M NaCl) - Tm (0.05 M NaCl)]
for duplexes containing 3 became slightly smaller than
that of the control duplex (11.5 °C) as the number of 3
increased, the differences were small. Therefore, the
terminal ammonium ion of the amino linker of 3 seemed
to hardly affect the thermal stability of the ODN-RNA
duplex.
In previous papers,8,9 we described the thermal stabil-
ity of duplexes constructed from ODNs containing 1′-[[[N-
(4-aminobutyl)carbamoyl]oxy]methyl]-2′-deoxyuridine (1)
or 5-[N-(6-aminohexyl)carbamoyl]-2′-deoxyuridine (2).
ODNs containing 2 stabilized duplex formation with a
complementary DNA, while those containing 1 greatly
destabilized duplex formation with a complementary
DNA. When an ODN analogue containing 2 forms a
duplex with a complementary DNA, the aminohexyl
linker of 2 should be accommodated in the major groove,
since the 5-position of the deoxyuridine is in the major
groove. On the other hand, when an ODN analogue
containing 1 forms a duplex with a complementary DNA,
the aminobutyl-linker of 1 should be accommodated in
the minor groove, since the 1′-position of the sugar moiety
is located in the minor groove. The minor groove of a
DNA-DNA duplex is narrower and deeper than its major
groove.19 Therefore, a bulky amino linker may not fit into
the narrow minor groove as compared with the wide
major groove. The 4′-position of the nucleoside is located
in a minor groove in a duplex. However, ODNs contain-
ing 3 did not greatly destabilize duplex formation with a
complementary DNA. The 4′-position of the nucleoside
is closer to the phosphate backbone than the 1′-position
in a duplex and is located at the edge of the minor groove.
Therefore, the modified nucleoside 3 might not greatly
destabilize ODN-DNA duplex formation.
F igu r e 2. CD spectra of ODN-DNA and ODN-RNA du-
plexes at 15 °C in 0.01 M sodium phosphate (pH 7.0) contain-
ing 0.05 M NaCl: (s) 15-25; (---) 20-25; (s s) 21-26; (s‚s)
24-26.
On the other hand, we previously reported that ODNs
containing 1 only slightly destabilized ODN-RNA duplex
formation.8 The result obtained for ODNs containing 3
is similar to that for ODNs containing 1. Since the minor
groove of A-type duplexes (DNA-RNA and RNA-RNA
duplexes) is shallower and flatter than that of DNA-
DNA duplexes, the amino linker of 1 or 3 could be well
accommodated in its minor groove.
Cir cu la r Dich r oism . To study the global conforma-
tion of duplexes, CD spectra of duplexes composed of
ODNs containing 3 and either the complementary DNA
(25) or RNA (26) were measured in a buffer of 0.01 M
sodium phosphate (pH 7.0) containing 0.05 M NaCl at
15 °C. With DNA (25) as a complementary strand, the
spectra of the duplexes (15-25 and 20-25) showed a
positive CD band at around 275 nm and a negative CD
band at around 245 nm that were attributable to B-like
DNA conformation (Figure 2). The shapes of the spectra
were quite similar to each other, although the intensity
of the positive band in the spectrum of the duplex (20-
25) containing 3 was clearly less than that for the
unmodified duplex (15-25).
On the other hand, when RNA (26) was used as a
complementary strand, the spectra of duplexes (21-26
(19) Saenger, W. Principle of nucleic acid structure; Springer-
Verlag: New York, 1984.

1664 J . Org. Chem., Vol. 63, No. 5, 1998
Ueno et al.
and 24-26) showed a positive CD band at around 260
nm and a large negative CD band at around 210 nm
(Figure 2). Although the shapes of the spectra were
similar to each other, the positive band in the spectrum
of the duplex (24-26) containing 3 was shifted to a
shorter wavelength by about 3 nm relative to that in the
spectrum of the unmodified duplex (21-26), and the
intensity of the negative band at around 210 nm was
slightly reduced compared with that for the unmodified
duplex (21-26). These results suggest that the modified
nucleoside 3 slightly affects the global conformation of
both ODN-DNA and ODN-RNA duplexes. The Tm
values for duplexes containing 3 may reflect these
conformational changes of the duplexes.
P a r tia l Digestion of ODNs w ith Sn a k e Ven om
P h osp h od iester a se a n d DNa se I. Since resistance to
nucleolytic hydrolysis by nucleases is an important factor
in antisense studies, the stability of ODNs containing 3
to nucleolytic digestion was examined. Two kinds of
nucleases, snake venom phosphodiesterase (a 3′-exonu-
clease) and DNase I (an endonuclease), were used in this
study. ODNs (16 and 20) containing one and four
molecules of 3 were labeled at the 5′-end with 32P and
incubated with an appropriate nuclease, and the reac-
tions were then analyzed by polyacrylamide gel electro-
phoresis under denaturing conditions.20
When snake venom phosphodiesterase was used in the
reactions, the control ODN 15 was hydrolyzed almost
completely by the enzyme after 120 min of incubation
(Figure 3a). In contrast, the phosphodiester linkage at
the 5′-side of 3 was highly resistant to the nuclease, and
the phosphodiester linkage at the 3′-side of 3 was also
slightly resistant to the enzyme (Figure 3b).
F igu r e 3. Polyacrylamide gel electrophoresis of 5′-32P-labeled
ODNs hydrolyzed by snake venom phosphodiesterase: (a) 15;
(b) 16. ODNs were incubated with snake venom phosphodi-
esterase for 0 min (lane 1), 10 min (lane 2), 20 min (lane 3),
30 min (lane 4), 60 min (lane 5), and 120 min (lane 6).
Experimental conditions are described in the Experimental
Section.
On the other hand, when DNase I was used in the
reactions, the phosphodiester linkages around 3 were
more resistant to enzymatic hydrolysis than those beside
thymidine. The half-lives of ODN 15 and ODN 20 were
9 and 140 min, respectively. The modified ODN 20 was
15 times more stable than the unmodified ODN (Figure
4). In a previous paper,9d we reported that the hepta-
decanucleotide containing four molecules of 2 was four
times more stable to hydrolysis by nuclease S1 (an
endonuclease) than an unmodified ODN. Also, the ODN
containing 2 was as resistant to DNase I as to nuclease
S1.21 Therefore, the ODN containing 3 seemed to be
more resistant to nucleolytic hydrolysis by endonucleases
than the ODN containing 2.
hydrolyzed by the enzyme. Therefore, the ODN-RNA
duplex containing 3 is a substrate for E. coli RNase H.
Con clu sion s
In this paper, we reported the synthesis of ODNs
containing 4′-C-[2-[[N-(2-aminoethyl)carbamoyl]oxy]eth-
yl]thymidine (3). 4′R-(2-Hydroxyethyl)thymidine (4) was
synthesized using a newly developed intramolecular
radical cyclization reaction at the 4′-position of thymidine
derivative 7 with a dimethylvinylsilyl group as a radical
acceptor tether. ODNs (16-20 and 22-24) containing
3 were synthesized on a DNA synthesizer using 5. These
ODNs containing 3 were more resistant to nucleolytic
hydrolysis by both snake venom phosphodiesterase and
DNase I than unmodified parent ODNs. ODN 20 was
15 times more stable to nucleolytic hydrolysis by DNase
I than an unmodified ODN. The stability of duplexes
consisting of ODNs (15-24) and either their complemen-
tary DNA or RNA strands were studied by thermal
denaturation. ODNs containing 3 did not greatly desta-
bilize duplex formation with either the complementary
DNA or RNA strand. Furthermore, the duplex formed
by an ODN containing 3 and the complementary RNA
was a substrate for E. coli RNase H. These results
suggest that an ODN containing 3 is a good candidate
as a novel antisense molecule.
Degr a d a tion of th e Ta r get RNA by RNa se H. It
has been postulated that the antisense activity of anti-
sense ODNs is due, at least in part, to cleavage of the
RNA strand of a DNA-RNA duplex by RNase H. We
next examined whether the DNA-RNA heteroduplex of
ODN containing 3 and its complementary RNA can elicit
RNase H activity. The duplex consisting of an ODN (21
or 24) and RNA (26) labeled with 32P at the 5′-end was
incubated with Escherichia coli RNase H, and the
products were analyzed by polyacrylamide gel electro-
phoresis. As shown in Figure 5, RNA in the ODN-RNA
duplex (24-26) containing the modified nucleoside and
the control ODN-RNA duplex (21-26) were similarly
(20) Maniatis, T.; Fritsch, E. F.; Sambrook, J . Molecular cloning: a
laboratory manual; Cold Spring Harbor University Press: Cold Spring
Harbor, NY. 1982.
Recently, Fensholdt22 and Wang23 reported that ODNs
containing 4′-C-(hydroxymethyl)thymidine, 4′-C-(meth-
(21) Ueno, Y.; Kumagai, I.; Matsuda, A. Unpublished work.

Synthesis of Oligodeoxynucleotides
J . Org. Chem., Vol. 63, No. 5, 1998 1665
F igu r e 4. Polyacrylamide gel electrophoresis of 5′-32P-labeled
ODNs hydrolyzed by DNase I: (a) 15; (b) 20. ODNs were
incubated with DNase I for 0 min (lane 1), 10 min (lane 2), 20
min (lane 3), 30 min (lane 4), 60 min (lane 5), and 120 min
(lane 6). Experimental conditions are described in the Experi-
mental Section.
F igu r e 5. Polyacrylamide gel electrophoresis of 5′-32P-labeled
RNA 26 hydrolyzed by E. coli RNase H in the presence of
complementary strands: lane 1, 26; lane 2, 26 + 21; lane 3,
26 + 24; lane 4, 26 + enzyme; lane 5, 26 + 21 + enzyme; lane
6, 26 + 24 + enzyme. Experimental conditions are described
in the Experimental Section.
oxymethyl)thymidine, or 4′-C-(aminomethyl)thymidine
exhibited hybridization to both complementary DNA and
RNA strands that were similar to or better than those of
unmodified ODNs and were more resistant to nucleolytic
hydrolysis by snake venom phosphodiesterase than un-
modified ODNs, although endonuclease-resistance was
not examined. Various 4′R-branched nucleoside ana-
logues with aminoalkyl-linkers of various lengths can be
synthesized using the radical cyclization reaction with a
silicon tether described here. The synthesis, thermal
stability and nuclease-resistance properties of ODNs
containing these 4′R-branched nucleoside analogues are
currently under investigation in our laboratory. These
results will be reported shortly.
(Na2SO4), and evaporated under reduced pressure. The resi-
due was dissolved in THF (80 mL), TBAF (1 M in THF, 14
mL, 14 mmol) was added, and the resulting mixture was
purified by column chromatography (SiO2, 70-100% AcOEt
in hexane) to give 10 (4.04 g, 63%) as a foam: 1H NMR (270
MHz, CDCl3) δ 8.12 (br s, 1H), 7.78 (s, 1H), 7.54-6.78 (m, 18H),
6.73 (dd, 1H, J ) 8.0, 7.1), 4.65 (m, 1H), 3.80 (s, 6H), 3.64 (d,
1H, J ) 10.3), 3.47 (d, 1H, J ) 10.3), 3.03 (d, 1H, J ) 3.0),
2.52-2.47 (m, 2H), 1.88 (s, 3H); FABMS m/z 574 (M+ - base).
Anal. Calcd for C37H36N2O7Se: C, 63.52; H, 5.19; N, 4.00.
Found: C, 63.58; H, 5.39; N, 4.09.
1-[2-Deoxy-5-O-(d im eth oxytr ityl)-3-O-(d im eth ylvin yl-
silyl)-4-C-(p h e n ylse le n o)-r-L -t h r eo-p e n t o-1,4-fu r a n o-
syl]th ym in e (7). A mixture of 10 (1.00 g, 1.43 mmol), DMAP
(17 mg, 0.14 mmol), Et3N (560 µL, 4.0 mmol), and dimethylvi-
nylchlorosilane (500 µL, 4.4 mmol) in toluene (20 mL) was
stirred at room temperature for 2 h under argon. Insoluble
materials were filtered off, and the filtrate was evaporated
under reduced pressure. The residue was partitioned between
AcOEt and H2O. The organic layer was washed with brine,
dried (Na2SO4), and evaporated under reduced pressure. The
residue was purified by column chromatography (SiO2, hex-
ane-AcOEt ) 3:2) to give 7 (1.23 g, 95%) as a foam: 1H NMR
(270 MHz, CDCl3) δ 8.12 (br s, 1H), 7.54-6.72 (m, 29H), 6.61
(dd, 1H, J ) 7.0, 7.0), 6.15-5.57 (m, 3H), 4.58 (dd, 1H, J )
5.5, 3.7), 3.84 (d, 1H, J ) 10.6), 3.78 (s, 6H), 3.14 (d, 1H, J )
10.6), 2.45 (ddd, 1H, J ) 13.3, 7.0, 3.7), 2.33 (ddd, 1H, J )
13.3, 7.0, 5.5), 1.91 (s, 3H): FABMS m/z 751(M+ - PhSe). Anal.
Calcd for C51H48N2O7SeSi: C, 67.46; H, 5.33; N, 3.09. Found:
C, 67.28; H, 5.35; N, 3.39.
Exp er im en ta l Section
NMR spectra were recorded at 270 or 500 MHz (1H) and at
202 MHz (31P) and are reported in ppm downfield from TMS
or 85% H3PO4. J values are given in hertz. Mass spectra were
obtained by fast atom bombardment (FAB). Thin-layer chro-
matography was done on Merck coated plates 60F254
. The
silica gel or the neutralized silica gel used for column chro-
matography were Merck silica gel 5715 or ICN silica 60A,
respectively.
1-[2-Deoxy-5-O-(d im eth oxytr ityl)-4-C-(p h en ylselen o)-
r-L-th r eo-p en to-1,4-fu r a n osyl]th ym in e (10). A mixture of
812 (4.69 g, 9.18 mmol) and DMTrCl (6.22 g, 18.4 mmol) in
pyridine (90 mL) was stirred at room temperature. After 3 h,
DMTrCl (1.56 g, 4.60 mmol) was added further, and the
mixture was stirred at room temperature for 2 h. Ice-water
and AcOEt were added, the resulting mixture was partitioned,
and the organic layer was washed with H2O and brine, dried
5′-O-(Dim e t h oxyt r it yl)-4′-C-(2-h yd r oxye t h yl)t h ym i-
d in e (6). A solution of Bu3SnH (1.9 mL, 7.1 mmol) and AIBN
(340 mg, 2.07 mmol) in benzene (70 mL) was added slowly over
4 h to a solution of 7 (3.13 g, 4.00 mmol) in benzene (250 mL)
at 80 °C. The solvent was evaporated under reduced pressure,
(22) Fensholdt, J .; Thrane, H.; Wengel, J . Tetrahedron Lett. 1995,
36, 2535-2538.
(23) Wang, G.; Seifert, W. F. Tetrahedron Lett. 1996, 37, 6515-6518.

1666 J . Org. Chem., Vol. 63, No. 5, 1998
Ueno et al.
and the residue was partitioned between MeCN and hexane.
The MeCN layer was evaporated under reduced pressure, and
the residue was dissolved in MeOH/THF (1:1, 40 mL). Aque-
ous H2O2 (30%, 1.3 mL, 38 mmol), KF (1.05 g, 18.1 mmol),
and KHCO3 (400 mg, 4.00 mmol) were added to the above
solution, and the resulting mixture was stirred at room
temperature for 16 h. Aqueous Na2S2O3 solution (1 M, 60 mL)
was added, and the resulting insoluble materials were filtered
off. The filtrate was evaporated under reduced pressure, and
the residue was purified by column chromatography (SiO2,
1-3% MeOH in CHCl3) to give 6 (2.05 g, 87%) as a white
foam: 1H NMR (500 MHz, CDCl3) δ 8.97 (br s, 1H), 7.62 (s,
1H), 7.40-6.83 (m, 13H), 6.48 (t, 1H, J ) 7.2), 4.53 (m, 1H),
3.85 (m, 1H), 3.79 (s, 6H), 3.58 (ddd, 1H, J ) 10.3, 6.7, 3.5),
3.31 (d, 1H, J ) 9.9), 3.24 (d, 1H, J ) 9.9), 2.46-2.43 (m, 2H),
2.05 (ddd, 1H, J ) 15.0, 8.2, 3.5), 1.81 (ddd, 1H, J ) 15.0, 6.7,
3.3), 1.38 (s, 3H); FABMS m/z 589 [M+ + 1]; HRFABMS calcd
for C33H37N2O8 589.2549, found 589.2522.
NaHCO3 and brine, dried (Na2SO4), and concentrated. The
residue was purified by column chromatography (a neutralized
SiO2, 50-100% AcOEt in hexane) to give 5 (385 mg, 72%) as
a foam: 31P NMR (202 MHz, CDCl3) δ 150.28, 149.88; FABMS
m/z 971 [M+ + 1]; HRFABMS calcd for C47H59F3N6O11
P
971.3931, found 971.3894.
Syn th esis of ODNs. ODNs were synthesized on a DNA
synthesizer (Applied Biosystem model 381A) by the phos-
phoramidite method. The fully protected ODNs were then
deblocked and purified by the same procedure as for the
purification of normal ODNs. That is, each ODN linked to
the resin was treated with concentrated NH4OH at 55 °C for
16 h, and the released ODN protected by a DMTr group at
the 5′-end was chromatographed on a C-18 silica gel column
(1 × 10 cm, Waters) with a linear gradient of MeCN from 0 to
30% in 0.1 M TEAA buffer (pH 7.0). The fractions were
concentrated, the residue was treated with aqueous 80% AcOH
at room temperature for 20 min and then the solution was
concentrated and the residue was coevaporated with H2O. The
residue was dissolved in H2O and the solution was washed
with Et2O, and then the H2O layer was concentrated to give
deprotected ODN 16 (95), 17 (65), 18 (48), 19 (38), 20 (64), 22
(32), 23 (61), and 24 (33). The yields are indicated in
parentheses as OD units at 260 nm on a 1 µmol scale.
Electr osp r a y Ion iza tion Ma ss Sp ectr om etr y. Spectra
were obtained on a Quattro II (Micromass, Manchester, U.K.)
triple quadrupole mass spectrometer equipped with an ESI
source in the negative-ion mode. The HPLC-purified ODN
samples were dissolved in aqueous 50% 2-propanol containing
1% triethylamine (10 pmol ODN/µL) and introduced into the
ion source through a loop injector with a carrier solvent, 33%
aqueous MeOH, flowing at 10 mL/min flow rate. About 15
scans were acquired over approximately 1 min and combined
to obtain smoothed spectra. All molecular masses of the ODNs
were calculated from the multiple-charge negative-ion spectra.
The observed average molecular masses of 16 and 22 were
5860.9 and 5187.5, respectively, and fit the calculated molec-
ular weights (theoretical average molecular masses) for these
compounds, i.e., 5860.9 (for 16, C188H245N65O118P18) and 5187.5
(for 22, C167H220N56O105P16) within a commonly accepted error
range of ESI MS, 0.01%.
5′-O-(Dim e t h oxyt r it yl)-4′-C-(1-h yd r oxye t h yl)t h ym i-
d in e (13). A solution of 7 (313 mg, 0.400 mmol), Bu3SnH (320
µL, 1.19 mmol), and AIBN (20 mg, 0.12 mmol) in benzene (4
mL) was stirred at 60 °C for 4 h. The solvent was evaporated
under reduced pressure, and the residue was partitioned
between MeCN and hexane. The MeCN layer was evaporated
under reduced pressure. The residue was treated under
Tamao oxidation conditions as described above for 6 to give
13 (177 mg, 75%) as a white foam: 1H NMR (270 MHz, CDCl3)
δ 8.30 (br s, 1/3H), 8.23 (br s, 2/3H), 7.41-6.85 (m, 14H), 6.51
(t, 1/3H, J ) 7.1), 6.44 (dd, 2/3H, J ) 5.9, 8.7), 4.84 (m, 1/3H),
4.74 (m, 2/3H), 4.18-4.09 (m, 1H), 3.81 (s, 6H), 3.58 (d, 2/3H),
3.50 (d, 2/3H, J ) 10.0), 3.29 (d, 2/3H, J ) 10.0), 3.21 (s, 2/3H),
2.90 (d, 2/3H), 2.71 (d, 1/3H), 2.49-2.36 (m, 2H), 1.54 (s, 2H),
1.46 (s, 1H), 1.15-1.11 (m, 3H); FABMS m/z 588 [M+];
HRFABMS calcd for C33H36N2O8 588.2469, found 588.2488.
5′-O-(Dim e t h oxyt r it yl)-4′-C-[2-[[N -[2-[N -(t r iflu or o-
a cetyl)a m in o]eth yl]ca r ba m oyl]oxy]eth yl]th ym id in e (14).
N,N′-Carbonyldiimidazole (80 mg, 0.5 mmol) and DMAP (40
mg, 0.3 mmol) were added to a solution of 6 (966 mg, 1.6 mmol)
in DMF (80 mL), and the mixture was stirred at room
temperature. After 2, 4, and 6 h, further amounts of N,N′-
carbonyldiimidazole (80 mg, 0.5 mmol) were added to the
mixture. After 15 h, 1,2-diaminoethane (0.55 mL, 8.2 mmol)
was added to the mixture, which was stirred at room temper-
ature. After 3 h, the solvent was removed by evaporation. The
residue was diluted with CHCl3. The organic layer was
washed with aqueous 0.1 M NaH2PO4, dried (Na2SO4), and
evaporated under reduced pressure. The residue was dissolved
in MeOH (80 mL). Ethyl trifluoroacetate (976 µL, 8.2 mmol)
and Et3N (1.1 mL, 8.2 mmol) were added to the solution with
stirring at room temperature. After 14 h, the solvent was
removed by evaporation. The residue was diluted with CHCl3.
The organic layer was washed with H2O and brine, dried (Na2-
SO4), and evaporated under reduced pressure. The residue
was purified by column chromatography (SiO2, 1-10% EtOH
in CHCl3) to give 14 (818 mg, 65%) as a white foam: 1H NMR
(270 MHz, CDCl3) δ 8.92 (br s, 1H), 7.80 (br t, 1H), 7.42-6.84
(m, 14H), 6.42 (t, 1H, J ) 6.9), 5.37 (br t, 1H), 4.49 (m, 1H),
4.20 (m, 1H), 4.07 (m, 1H), 3.81 (s, 6H), 3.45 (m, 2H), 3.35 (m,
2H), 3.29-3.28 (m, 2H), 2.98 (d, 1H, J ) 5.4), 2.42-2.37 (m,
2H), 2.14-1.92 (m, 2H), 1.57 (s, 3H); FABMS m/z 771 [M++1];
HRFABMS calcd for C38H42N4O10F3 771.2850, found 771.2841.
5′-O-(Dim e t h oxyt r it yl)-4′-C-[2-[[N -[2-[N -(t r iflu or o-
a cetyl)a m in o]eth yl]ca r ba m oyl]oxy]eth yl]th ym id in e 3′-
O-(2-Cya n oeth yl N,N-d iisop r op ylp h osp h or a m id ite) (5).
After successive coevaporation with pyridine, 14 (423 mg, 0.55
mmol) was dissolved in CH2Cl2 (10 mL) containing N,N-
diisopropylethylamine (0.192 mL, 1.1 mmol). Chloro(2-cya-
noethoxy)(N,N-diisopropylamino)phosphine (0.184 mL, 0.83
mmol) was added to the solution, and the reaction mixture
was stirred for 20 min at room temperature. After 30 min,
further amounts of N,N-diisopropylethylamine (48 µL) and
chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine (61 µL)
were added to the mixture. After 1.5 h, aqueous saturated
NaHCO3 and CHCl3 were added to the mixture, and the
separated organic layer was washed with aqueous saturated
Th er m a l Den a t u r a t ion a n d CD Sp ect r oscop y. Each
solution contains ODN (3 µM) and the complementary DNA
25 (3 µM) or RNA 26 (3 µM) in an appropriate buffer. The
solution containing each ODN was heated at 90 °C for 10 min
and then cooled gradually to an appropriate temperature and
used for the thermal denaturation studies. Thermal-induced
transitions of each mixture were monitored at 260 nm on a
Perkin-Elmer Lambda2S. Sample temperature was increased
0.5 °C/min. Samples for CD spectroscopy were prepared by
the same procedure used in the thermal denaturation study,
and spectra were measured on a J ASCO J 720 spectropola-
rimeter at 15 °C. The ellipticities of duplexes were recorded
from 200 to 320 nm in a cuvette with a path length 1 mm. CD
data were converted into mdeg‚mol of residues-1‚cm-1
.
P a r tia l Hyd r olysis of ODN w ith Sn a k e Ven om P h os-
p h od iester a se. Each ODN labeled with 32P at the 5′-end (10
pmol) was incubated with snake venom phosphodiesterase (0.4
µg) in the presence of Torula RNA (0.26 OD units at 260 nm)
in a buffer containing 37.5 mM Tris-HCl (pH 8.0) and 7.5 mM
MgCl2 (total 20 µL) at 37 °C. At appropriate periods, aliquots
of the reaction mixture were separated and added to a solution
of EDTA (5mM, 10 µL), and then the mixture were heated for
3 min at 90 °C. The solutions were analyzed by electrophoresis
on 20% polyacrylamide gel containing 7 M urea.
P a r tia l Hyd r olysis of ODN w ith DNa se I. Each ODN
labeled with 32P at the 5′-end (10 pmol) was incubated with
DNase I (1 unit) in the presence of Torula RNA (0.26 OD units
at 260 nm) in a buffer containing 100 mM sodium acetate (pH
6.0) and 5 mM MgCl2 (total 20 µL) at 37 °C. At appropriate
periods, aliquots of the reaction mixture were separated and
added to a solution of EDTA (5mM, 10 µL), and then the
mixtures were heated for 3 min at 90 °C. The solutions were
analyzed by gel electrophoresis as described above.

Synthesis of Oligodeoxynucleotides
J . Org. Chem., Vol. 63, No. 5, 1998 1667
Hyd r olysis of Du p lexes w ith RNa se H. RNA labeled
with 32P at the 5′-end (50 pmol) was incubated with E.coli
RNase H (6 units) in the presence or in the absence of the
complementary strand (50 pmol) in a buffer containing 40 mM
Tris-HCl (pH 7.7), 4 mM MgCl2, 1 mM DTT, 4% glycerol, and
0.003% bovine plasma albumin (total 10 µL) at 20 °C. After
20 min, reaction mixtures were heated for 1 min at 90 °C, and
then the reactions were analyzed by gel electrophoresis as
described above.
Ack n ow led gm en t . This investigation was sup-
ported in part by a Grant-in-Aid for Encouragement of
Young Scientists from the Ministry of Education, Sci-
ence, Sports, and Culture of J apan and a Grant from
“Research for the Future” Program of the J apan Society
for the Promotion of Science (J SPS-RFTF97I00301).
J O9720492