Synthesis of cyclopentane amide DNA (cpa-DNA) and its pairing properties

Article abstract of DOI:10.1021/jo034143q

We recently reported on the synthesis and pairing properties of the DNA analogue bicyclo[3.2.1]-amide DNA (bca-DNA). In this analogue the nucleobases are attached via a linear, 4-bond amide-linker to a structurally preorganized sugar-phosphate backbone un

Full text of DOI:10.1021/jo034143q

Syn th esis of Cyclop en ta n e Am id e DNA (cp a -DNA) a n d Its P a ir in g
P r op er ties
Dae-Ro Ahn, Markus Mosimann, and Christian J . Leumann*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3,
CH-3012 Bern, Switzerland
leumann@ioc.unibe.ch
Received February 1, 2003
We recently reported on the synthesis and pairing properties of the DNA analogue bicyclo[3.2.1]-
amide DNA (bca-DNA). In this analogue the nucleobases are attached via a linear, 4-bond amide-
linker to a structurally preorganized sugar-phosphate backbone unit. To define the importance of
the degree of structural rigidity of the bca-backbone unit on the pairing properties, we designed
the structurally simpler cyclopentane amide DNA (cpa-DNA), in which the bicyclo[3.2.1]-scaffold
was reduced to a cyclopentane unit while the base-linker was left unchanged. Here we present a
synthetic route to the enantiomerically pure cpa-DNA monomers and the corresponding phos-
phoramidites containing the bases A and T, starting from a known, achiral precursor in 9 and 12
steps, respectively. Fully modified oligodeoxynucleotides were synthesized by standard solid-phase
oligonucleotide chemistry, and their base-pairing properties with complementary oligonucleotides
of the DNA-, RNA-, bca-DNA-, and cpa-DNA-backbones were assessed by UV melting curves and
CD-spectroscopic methods. We found that cpa-oligoadenylates form duplexes with complementary
DNA that are less stable by -2.7 °C/mod. compared to DNA. The corresponding cpa-oligothymidy-
lates do not participate in complementary base-pairing with any of the investigated backbone
systems except with its own (homo-duplex). As its congener bca-DNA, cpa-DNA seems to prefer
left-handed helical duplex structures with DNA or with itself as indicated by the CD spectra.
In tr od u ction
Oligonucleotide analogues are of considerable interest
in antisense therapy to specifically inhibit the expression
of disease-related genes1 and in genomics to specify the
hitherto unknown function of new proteins.2 Carbohydrate-
modified DNA analogues are of particular interest, as the
replacement of deoxyribose units by synthetic constructs
opens many degrees of freedom to tune the pairing
properties of corresponding oligonucleotides.3 In this
context we recently focused on the concept of conforma-
tional restriction as a designer tool to enhance the
functional performance of DNA analogues.4
One of these analogues is bicyclo[3.2.1]amide-DNA
(bca-DNA, Figure 1), containing a backbone unit that is
conformationally locked in a B-type geometry, to which
the nucleobases are attached via a linear amide linker
with extended distance between the base and the back-
bone units relative to DNA (4 instead of 3 bonds).5 The
main characteristics of this system as determined so far
F IGURE 1. Representation of the chemical structures of
DNA, bca-DNA, and cpa-DNA, highlighting the relative dif-
ferences in the distance between base and backbone.
are a strong discrimination of RNA relative to DNA as
the complementary pairing partner in random-base
sequences, accompanied by a generally lower affinity
relative to DNA. Strand association is constrained to
antiparallel duplex formation, and Watson-Crick base-
pairing is mismatch sensitive as in the case of DNA. An
unexpected and interesting feature is the accommodation
of bca-homo-adenine sequences in right- as well as left-
handed helical structures, and their chirodegeneracy in
complementary recognition of D- and L-RNA (both are
complex partners in this sequence context).
To investigate the structure/property relationship of
bca-DNA in more detail, we wished to evaluate the
structurally simpler cyclopentane amide DNA (cpa-DNA,
Figure 1), which as the only structural change lacks the
pyranose ring. Here we report on the synthesis of the
corresponding nucleoside building blocks containing the
bases A and T, the synthesis of corresponding oligonucle-
otide analogues, as well as their pairing properties.
* Address correspondence to this author. Phone: +41-31-631 4355.
Fax: +41-31-631 3422.
(1) Uhlmann, E. Curr. Opin. Drug Discovery Dev. 2000, 3, 203-213.
(2) Dean, N. M. Curr. Opin. Biotechnol. 2001, 12, 622-625.
(3) For recent reviews on sugar backbone modified nucleotides see:
(a) Herdewijn, P. Liebigs Ann. 1996, 1337-1348. (b) Kool, E. T. Chem.
Rev. 1997, 97, 1473-1487. (c) Braasch, D. A.; Corey, D. R. Chem. Biol.
2001, 8, 1-7.
(4) Leumann, C. J . Bioorg. Med. Chem. 2002, 10, 841-854.
(5) (a) Egger, A.; Hunziker, J .; Rihs, G.; Leumann, C. Helv. Chim.
Acta 1998, 82, 734-743. (b) Egger, A.; Leumann, C. Synlett 1999, 913-
916. (c) Ahn, D.-R.; Egger, A.; Pitsch, S.; Lehmann, C.; Leumann, C.
Chem. Eur. J . 2002, 8, 5275-5322.
10.1021/jo034143q CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/19/2003
J . Org. Chem. 2003, 68, 7693-7699
7693

Ahn et al.
SCHEME 1a
SCHEME 2a
a
a
Reagents and conditions: (a) (i) m-CPBA, CH2Cl2, rt, 42%; (ii)
Reagents and conditions: (a) EDC, HOOC-CH2-base, DMF,
rt, 65% for 9a , 66% for 9b. (b) (i) 9-Chloro(9-phenyl)xanthene,
pyridine/DMSO, rt; (ii) TBDMS-Cl, imidazole, DMF, rt; 33% for
10a , 31% for 10b. (c) Bu4NF, THF, rt, 92% for 11a , 98% for 11b.
(d) [(iPr2N)(NCCH2CH2O)]PCl, iPr2NEt, THF, rt, 59% for 12a , 63%
for 12b.
Bu4NF, THF, rt, 93%. (b) BnNCO, toluene, 90 °C, 79%. (c) NaH,
DMF, rt, 76%. (d) Ac2O, DMAP, pyridine, rt, 97%. (e) PLE,
phosphate buffer, pH 7, 30 °C, 31%. (f) 8 N KOH, 80 °C EtOH,
82%. (g) 10% Pd/C, H2, MeOH, rt, 98%.
Resu lt a n d Discu ssion
(chiral GLC) at 31-37% conversion was observed. Car-
bamate (+)4 was subsequently hydrolyzed to the phenyl-
amine 6, which was directly deprotected by hydrogenoly-
sis to give the dihydroxyamine 7 in 80% yield over the
two steps. Although it is known that PLE preferentially
hydrolyzes R configured esters,11 we preferred to rigor-
ously confirm the absolute configuration of (+)4. This was
done by converting (+)4 into the corresponding (-)-
camphanic acid ester 8, and subjecting crystals of it to
X-ray analysis. The X-ray data are in full agreement with
the absolute configuration of (+)4 given in Scheme 1 (see
Supporting Information).
Syn th esis of cp a -Mon om er s. 2-Amino-1,3-cyclopen-
tanediol 7 constitutes the basic substructure that serves
for the synthesis of the corresponding nucleoside building
blocks. This unit, already known in racemic form,6 was
retrosynthetically related to epoxide 2 (Scheme 1).
Epoxidation of the racemic starting material (()17 with
m-CPBA led to the corresponding epoxides in a ratio of
3:1 (anti:syn) in 70% yield. Chromatographic separation
of the anti isomer, followed by desilylation with Bu4NF
yielded (()2 in 93% yield, which was subsequently
converted into the carbamate (()3 (79%) with benzyl
isocyanate in toluene. The nitrogen in (()3 thus serves
as the source of the amino function in 7.
The intramolecular cyclization of (()3 was effected in
analogy to a known procedure8 by treatment with NaH
in DMF and gave the cyclic carbamate (()4, in 76% yield.
To split (()4 into pure enantiomers we followed two
strategies based on enzymatic kinetic resolution. First
we tried a lipase-catalyzed esterification.9 This enzyme
is known to preferentially catalyze the esterification of
R configured alcohols. However, with pseudomonas fluo-
rescens lipase (PFL) no sufficient level of enantiomeric
resolution could be obtained. The second approach was
based on an esterase-catalyzed kinetic resolution10 of (()-
5 that was easily obtained from (()4 by standard acety-
lation. Both yield and enantioselectivity in the hydrolysis
by pig liver esterase (PLE) proved to be superior. An
enantiomeric excess of (+)4 on the order of 93.0-99.5%
The direct and selective introduction of the base units
into 7 with use of typical peptide coupling reagents
(Scheme 2) needed some optimization. The use of
N,N,N′,N′-tetramethyluronium tetrafluorborate (TOTU)
i
and Pr2NH in DMF led to a mixture of amides and
esters. The same was observed when DCC in DMF was
used in the presence of DMAP. The best results were
obtained with EDC in DMF. Under these conditions, only
the amino group in 7 reacted with the base units to give
the desired amides in acceptable yields (66% of 9a , and
65% of 9b).
The next problem to be solved was the selective
addressing of one of the two secondary hydroxyl groups
in 9. Direct pixylation of 9a gave two regioisomers in a
∼4:1 ratio (1H NMR) in 55% yield. The two regioisomers
were not separable by column chromatography. However,
after TBDMS protection of the remaining OH function,
the isomers could be separated by column chromatogra-
phy to yield pure 10a as the major isomer in 33% yield
over the two steps. After deprotection of the TBDMS
(6) Tai, V. W-F.; Imperiali, B. Tetrahedron Lett. 1998, 39, 7215-
7218.
(7) Livinghouse, T.; Stevens, R. V. J . Am. Chem. Soc. 1978, 100,
6479-6482.
(8) Roush, W. R.; Adam, M. A. J . Org. Chem. 1985, 50, 3752-3757.
(9) Faber, K. In Biotransformations in Organic Chemistry; Springer-
Verlag: Berlin, Germany, 1997; pp 88-115.
(10) (a) Laumen, K.; Schneider, M. Tetrahedron Lett. 1984, 25,
5875-5878. (b) Wang, Y.-F.; Sih, C. J . Tetrahedron Lett. 1984, 25,
4999-5002.
(11) Sabbioni, G. J . J . Org. Chem. 1987, 52, 4565-4570.
7694 J . Org. Chem., Vol. 68, No. 20, 2003

Synthesis of Cyclopentane Amide DNA
TABLE 1. Yield a fter HP LC P u r ifica tion (1.3 µm ol
sca le), Sequ en ce, a n d MS Da ta of cp a -Oligon u cleotid es
14 a n d 15 Syn th esized a n d Used in th e P r esen t Stu d y
[M - 1]-
ESI-MS
(found)
yield
sequence
13 d(tcpa
(calcd)
OD260 (%)
)
3389.5
3125.4
3390.1
3125.7
2.7 (2.5)
2.3 (1.6)
10
14 d(acpa
)
9
TABLE 2. Tm Da ta (°C, 260 n m ) of Du p lexesa
13
15
19
d(T)9
18
d(T)10
d(tcpa
)
dT(tbca
)
10
10
14 d(acpa
)
15.5
<1b
∼4
∼4
9
17 d(A)10
22 r(A)10
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
21.6
28.3
20.1
19.1
32.1
24.5
23.6
16 dT(abca
)
10
a
Total concentration of oligonucleotides: 5 mM in 10 mM Na-
cacodylate, 1 M NaCl; pH 7 Concentration) 4 µM. c n.d. ) Tm
not detectable.
b
F IGURE 2. Representative UV melting curves of the duplexes
13/14, 17/19, and 14/19. Total concentration of oligonucleo-
tides: 5 µM, in 10 mM Na-cacodylate, 1 M NaCl; pH 7.0.
group, the resulting compound 11a (92%) was trans-
formed into the phosphoramidite 12a under standard
conditions in 59% yield. The corresponding adenine-
containing intermediate 10b was obtained in the same
way and was subsequently transformed into the phos-
phoramidite building block 12b (63%). The position of the
pixyl group at O-C(5) and the TBDMS group at O-C(2)
in 10 was rigorously proven in the case of 10b by NMR
duplex melting are summarized in Table 2. Figure 2
contains representative melting curves of selected du-
plexes.
1
methods (see the Supporting Information). From H-13C
COSY spectra, H-C(5) could be correlated with the
The Tm data show that the oligoadenylate 14 still forms
a weak duplex with complementary DNA while the
oligothymidylate 13 was unable to base-pair with any
oligoadenylate sequence, except the one of the same
backbone type. Given the Tm of 24 °C for the reference
quaternary sp3 carbon of the pixyl group. Alternatively,
a
1H-NOE between H-C(2) and the Si-CH3 of the
TBDMS group etablished the TBDMS group to be at-
tached at O-C(2). Thus the remaining task was to verify
the configuration of the centers C(2) and C(5) relative to
C(1). This was unambiguously done by NOE spectros-
copy. A strong NOE between the amide NH and H-C(5)
established the trans arrangement of substituents at C(1)
and C(5). Alternatively, strong mutual NOEs between
H-C(1) and H-C(2), carrying the TBDMS group, estab-
lished the cis substitution pattern of C(1) and C(2).
Syn th esis of cp a -Oligon u cleotid es. cpa-oligonucle-
otides 13 and 14 were prepared according to the standard
protocols for automated solid-phase DNA synthesis on
the 1.3-µmol scale (Table 1). As a solid support, com-
mercially available universal solid support was used. The
standard DNA synthesis cycle needed some adjustments.
The coupling time was extended to 6 min and the
standard activator tetrazole was replaced by the more
active ethylthio-1H-tetrazole. With these conditions,
coupling yields of 92-99% for cpa-T and 73-99% for
cpa-A were obtained according to the trityl assay. Depro-
tection and detachment from solid support of crude
oligomers was effected in concentrated NH3:40% methyl-
amine (1:1), 55 °C, 16 h. Under these conditions, byprod-
ucts arising from incomplete removal of the 3′-linker were
observed by HPLC in nonnegligeable amounts. The
oligonucleotides were purified by HPLC and character-
ized by ESI-MS (Table 1).
12
duplex d(A)9/d(T)9 we find a stronger duplex destabili-
zation for the oligoadenylate with the cpa-backbone (-2.2
°C/mod.) as compared to the bca-backbone (-1.0 °C/mod.).
In general, the pairing preference of the cpa-oligonucleo-
tide correlates with that of the corresponding bca-
backbone although on a more reduced level of affinity.
The pure cpa-DNA duplex shows also slightly reduced
thermal stability compared to that of the pure bca-DNA
duplex (-0.4 °C/mod.) and to the pure natural duplex
(-0.9 °C/mod.). Interestingly, the two structurally related
cpa- and bca-backbone types hardly communicate with
each other within the oligoadenine/oligothymine sequence
motif. The Tm data of both mixed-backbone, cpa/bca
duplexes 13/16 and 14/15 are <1 °C, thus amounting to
a loss in thermal stability of >2.4 °C/mod.
CD Sp ectr a of cp a -DNA Sin gle Str a n d s a n d Du -
p lexes. CD spectra were measured under the same
conditions as for Tm analysis. The oligonucleotide cpa-
(A9) (14) seems to be preorganized in a left-handed helix
as a single strand, as deduced from the enantiomorphic
shape of its CD trace relative to that of d(A10) (17) (Figure
3a). However, there are differences with respect to the
CD trace of oligonucleotide 16, carrying the bca-backbone.
For this system, the single-strand structure and helicity
is not clear. The CD curves of duplexes of 14 with
complementary cpa-oligonucleotide 13, and especially
with complementary DNA 19, are also reminiscent of left-
handed duplex structures. This was concluded based on
Du p lex F or m a tion P r op er ties of cp a -DNA w ith
Com p lem en ta r y DNA, bca -DNA, RNA, a n d L-RNA.
To compare the pairing preferences of cpa-DNA with
those of bca-DNA the following oligonucleotides 15-22,
prepared and investigated earlier, were included in the
UV melting curve assays. The corresponding Tm data for
(12) Steffens, R.; Leumann, C. J . J . Am. Chem. Soc. 1999, 121,
3249-3255.
J . Org. Chem, Vol. 68, No. 20, 2003 7695

Ahn et al.
F IGURE 4. CD spectra (experimental conditions as indicated
in Table 2, T ) 5 °C) of cpa-DNA/RNA duplexes.
duplex by ca. 1.3 °C/base-pair. This is opposite to bca-
DNA, which in the same sequence context prefers D-RNA
over L-RNA by ca. 1.1 °C/base-pair. This may again be
the consequence of the intrinsically left-handed, helical
nature of the single stranded cpa-oligonucleotide.
While in the case of bca-oligonucleotide 16 the CD
spectra of the duplexes with D- and L-RNA virtually
reflected enantiomorphic structures, the situation is less
clear in the case of the duplexes of cpa-oligonucleotide
14 with D- and L-RNA (Figure 4). While the CD trace of
the duplex with D-RNA (14/20) largely indicates a right-
handed helix, that of the duplex with the L-RNA comple-
ment (14/21) is not enantiomorphic and may well reflect
also a non-Watson-Crick base-paired structure. Thus the
two related bca- and cpa-backbone systems deviate
structurally from each other in this context. One expla-
nation for this fact lies in the difference of the cyclopen-
tane ring conformation in both systems.
F IGURE 3. CD spectra of selected single strands (a) and
duplexes (b). Experimental conditions as indicated in Table 2
(T ) 5 °C).
TABLE 3. Tm Da ta of Selected Du p lexesa
duplex
14/20
14/21
16/20
16/21
17/20
Although the cpa-DNA backbone mimicks the D- (and
not L-) ribose structure, the CD of their single strands
indicate left-handed helical conformations. Recently,
Urata et al. have reported the formation of a right-
handed double helix by an L-oligodeoxyribonucleotide
analogue in which the bases were fixed in the low anti-
glycosyl conformation.13 This is in contrast to the corre-
sponding single strands, which according to CD spec-
troscopy occur in a left-handed helical arrangement. Thus
the chirality of the deoxyribose unit does not imperatively
determine the sense of helicity of a corresponding double
helix. These observations are in full agreement with our
results on the bca-DNA and the cpa-DNA analogue. In
these cases it is not the glycosylic angle but the orienta-
tion of the base-linker unit that participates in defining
the chiral sense of the double helix.
Structurally related but conformationally more flexible
nucleoside analogues based on a serinol backbone unit
to which the base thymine was connected in the same
way as in cpa-DNA were incorporated into oligonucleo-
tides in the past.14 All oligonucleotides containing these
modifications were strongly destabilizing when intro-
duced in the center of their sequence. Thus a similar
Tm
7.0
19.1
30.7
20.1
37.0
a
Experimental conditions as indicated in Table 2.
the inverted signs of maxima and minima of the bands
at ca. 250 and 265 nm, relative to that of the duplex 18/
22 (Figure 3b). Thus again, removal of the six-membered
ring in bca-DNA, leading to cpa-DNA, does not cause
substantial structural differences on the duplex level. It
seems though that the cpa-DNA single strand 14 has a
higher propensity for left-handed helix formation, com-
pared to bca-DNA.
P a ir in g P r op er ties of 15 w ith Com p lem en ta r y D-
a n d L-RNA. As we have already reported, the bca-
homoadenine oligonucleotide 16 displays chirodegenerate
recognition properties in that it forms duplexes with both
enantiomeric forms of complementary RNA (D-RNA 20
and L-RNA 21). We therefore investigated whether the
cpa-DNA series shares these properties too. Initially we
studied the stability of duplexes by means of UV melting
experiments (Table 3).
As shown in Table 3, the cpa-DNA oligonucleotide 14
pairs to both enantiomeric forms of complementary RNA,
as is the case for bca-DNA. Interestingly, the cpa-DNA/
L-RNA duplex is more stable than the cpa-DNA/D-RNA
(13) Urata, H.; Miyagoshi, H.; Kumanshiro, T.; Mori, K.; Shoji, K.;
Akagi M. J . Am. Chem. Soc. 2001, 123, 4845-4846.
7696 J . Org. Chem., Vol. 68, No. 20, 2003

Synthesis of Cyclopentane Amide DNA
1H), 1.93-1.86, 1.76-1.65, 1.58-1.51 (3m, 4H). 13C NMR (75
MHz, CDCl3) δ 155.7 (s), 138.2 (s), 128.7 (d), 128.5 (d), 127.5
(d), 127.4 (d), 127.2 (d), 74.5 (d), 56.7 (d), 56.4 (d), 45.1 (t), 26.7
(t), 25.2 (t). HRMS (ESI+) m/z calcd for C13H15NO3 234.1130
[M + H]+, found 234.1188 [M + H]+.
situation as for the cpa-DNA modification is encountered.
Unfortunately there are no indications on the structural
and pairing properties of fully modified oligonucleotide
analogues of that backbone type. Thus, no conclusions
on the effect of the five-membered-ring structure in the
backbone unit of cpa-DNA can be drawn.
In summary, we presented the synthesis of the cpa-
DNA monomers containing the bases adenine and thy-
mine from the known compound 1 in 12 steps. Fully
modified oligomers of this new DNA analogue were
synthesized by standard oligonucleotide chemistry. Fully
modified cpa-oligoadenylates form duplexes with comple-
mentary DNA that are less stable by -2.7 °C/mod.
compared to DNA. The corresponding cpa-oligothymidy-
lates do not participate in complementary base-pairing
with any of the investigated backbone systems except
with its own (homo-duplex). As its congener bca-DNA,
cpa-DNA seems to prefer left-handed helical duplex
structures with DNA or with itself as indicated by the
CD spectra.
3-Ben zyl-4-h yd r oxy-h exa h yd r o-cyclop en t a oxa zol-2-
on e ((()4). To a suspension of 55% NaH (300 mg, 7.2 mmol)
in DMF (45 mL) was added (()3 (300 mg, 1.29 mmol). This
suspension was stirred at room temperature overnight, then
cooled to 0 °C and slowly quenched with sat. NH4Cl (50 mL).
The aqueous phase was extracted with CH2Cl2 (4×), and the
organic phase was dried over Na2SO4 and evaporated to
remove as much DMF as possible. Purification by CC (hexane/
ethyl acetate 1:1 f pure ethyl acetate) gave (()4 (228 mg, 76%)
as a yellowish solid. TLC (hexane/ethyl acetate 1:1) Rf 0.22.
IR (CHCl3, film) 3684 m, 3620 m, 3448 br, 3020-2896 s, 2400
s, 1740 s, 1672 m, 1522 s, 1422 s, 1228 s, 1046 s, 929 s, 740 s
cm-1. NMR assignments from 1H,1H- and from 1H,13C-COSY:
1H NMR (300 MHz, CDCl3) δ 7.35-7.27 (m, 5H, Ar-H), 4.94
(t, J ) 6.3 Hz, 1H, C(6a)-H), 4.65, 4.19 (2d, J ) 15.1 Hz, 2H,
CH2Bn), 4.14 (d, J ) 3.3 Hz, 1H, C(4)-H), 3.80 (d, J ) 7.4 Hz,
1H, C(3a)-H), 2.19-2.00 (m, 2H, C(6)-H), 1.96 (br, 1H, OH),
1.91-1.78 (m, 1H, C(5)-H), 1.75-1.68 (m, 1H, C(5)-H). 13C
NMR (75 MHz, CDCl3) δ 158.3 (s, C(2)), 135.9 (s, C(ar)), 128.8
(d, C(ar)), 128.1 (d, C(ar)), 128.0 (d, C(ar)), 79.0 (d, C(6a)), 73.8
(d, C(4)), 66.9 (d, C(3a)), 47.3 (t, C(benzyl)), 30.9 (t, C(5)), 30.5
(t, C(5)). HRMS (ESI+) m/z calcd for C13H15NO3 234.1130 [M
+ H]+, found 234.1133 [M + H]+.
Thus the main effect of the removal of the six-
membered ring in bca-DNA (f cpa-DNA) is a reduced
pairing affinity to complementary DNA, RNA, and bca-
DNA, and a higher propensity of the single strand for
left-handed helical structure formation.
Acet ic Acid 3-Ben zyl-2-oxo-h exa h yd r o-cyclop en t a -
oxa zol-4-yl Ester ((()5). To a solution of (()4 (500 mg, 2.15
mmol) in pyridine (20 mL) containing DMAP (39.3 mg, 320
µmol) was added acetic anhydride (310 µL, 3.23 mmol). The
solution was stirred at room temperature for 6 h, then
quenched with 140 mL of sat. NaHCO3 (aq) and extracted with
3 × 100 mL of CH2Cl2, and then the organic phase was
evaporated. The residual brown oil was purified by CC
(hexane/ethyl acetate 3:1 f 2:1 f 1:1) to give (()5 (575 mg,
97%) as a colorless viscous oil that crystallized upon drying
at HV. TLC (hexane/ethyl acetate 1:1) Rf 0.5. Mp 49-50 °C.
Exp er im en ta l Section
2,3-Ep oxycyclop en ta n ol ((()2). To a solution of m-CPBA
(54 g, 70% purity, 0.22 mol) in CH2Cl2 (150 mL) in an ice bath
was added slowly a solution of (()1 (36.4 g, 0.18 mol) in CH2-
Cl2 (150 mL). The mixture was stirred overnight at room
temperature. The resulting white precipitate was removed by
filtration and the filtrate was extracted with 10% Na2S2O3,
followed by 5% NaOH, water, and brine. The organic phase
was dried with Na2SO4 and evaporated and the residue was
purified by CC (hexane/ethyl acetate 50:1, Rf 0.25 (anti isomer),
0.15 (syn isomer)). The desired anti isomer (16.1 g, 42%) was
subsequently dissolved in THF (60 mL) and treated with Bu4-
NF‚3H2O (36.8 g, 0.12 mol), and the mixture was stirred at
room temperature overnight. After evaporation of THF, the
residue was purified by column chromatography (hexane/ethyl
acetate 1:2) to give (()2 (7.06 g, 93%) as a colorless oil. TLC
(hexane/ethyl acetate 1:1) Rf 0.26. IR (CHCl3, film) 3676 m,
3614 m, 3444 m, 3032-2860 m, 1436 w, 1394 w, 1316 w, 1232
s, 998 s, 918 w, 844 s, 800 m cm-1. 1H NMR (300 MHz, CDCl3)
δ 4.36 (d, J ) 5.1 Hz, 1H), 3.55 (br s, 1H), 3.42 (d, J ) 2.2 Hz,
1H), 2.03-1.96, 1.90-1.79, 1.72 - 1.53 (3m, 5H). 13C NMR
(75 MHz, CDCl3) δ 71.3 (d), 58.5 (d), 56.8 (d), 29.2 (t), 24.8 (t).
MS (EI+) m/z 100 [M+].
1
IR (KBr) 2940 m, 1731 s, 1455 m, 759 s, 704 s. H NMR (300
MHz, CDCl3) δ 7.40-7.30 (m, 5H), 5.03 (d, J ) 4.1 Hz, 1H),
4.90 (dd, J ) 7.4, 7.4 Hz, 1H), 4.77, 4.25 (2d, J ) 15.1 Hz,
2H), 3.83 (d, J ) 7.0 Hz, 1H), 2.01 (s, 3H), 2.18-2.07, 2.00-
1.86 (2m, 4H). 13C NMR (75 MHz, benzene-d6) 170.08 (s),
157.64 (s), 136.00 (s), 128.56 (d), 128.37 (d), 127.80 (d), 78.26
(d), 76.18 (d), 64.91 (d), 46.84 (t), 31.05 (t), 27.79 (t), 20.81 (q).
HRMS (ESI+) m/z calcd for C15H17NO4 276.1235 [M + H]+,
found 276.1229 [M + H]+.
3-Ben zyl-4-h yd r oxy-h exa h yd r o-cyclop en t a oxa zol-2-
on e ((+)4). Ester (()5 (750 mg, 2.73 mmol) was suspended
in 0.1 M phosphate buffer (35 mL, pH 7.0) and treated with
2.2 mg (210 U/mg) of pig liver esterase. The pH was kept
constant at 7.0 by continuous addition of 1 N NaOH. The
mixture was extracted with ethyl acetate (3 × 30 mL) and the
organic phase was dried over Na2SO4. The solvent was
evaporated and the residue purified by CC (hexane/ethyl
acetate 1:1) to give (+)4 (95 mg, 31%) as a white solid. ee >99%
1-((N-Ben zylca r b a m oyl)oxy)-2,3-ep oxycyclop en t a n e
((()3). To a solution of (()2 (4.6 g, 46 mmol) in toluene (40
mL) was added benzylisocyanate (8.4 mL, 69 mmol) at room
temperature. The mixture was heated to 95 °C and stirred
overnight. After evaporation of solvent, the residue was
dissolved in ethyl acetate (50 mL) and washed with sat.
NaHCO3. The organic phase was dried over Na2SO4 and
evaporated, and the residue was purified by CC (hexane/ethyl
acetate 4:1) to give (()3 (8.51 g, 79%) as a white solid. TLC
(CH2Cl2) Rf ) 0.3. IR (CHCl3, film) 3684 w, 3620 w, 3448 m,
3016-2873 s, 2400 m, 2254 w, 1802 m, 1724 s, 1514 s, 1228 s,
(chiral GLC). Mp 86-87 °C. [R]22 +37.5 (c 0.0032, MeOH).
D
N-Ben zyl-2-a m in ocyclop en ta n -1,3-d iol (6). Alcohol (+)-
4 (1.35 g, 5.79 mmol) was dissolved in ethanol (20 mL), and 8
N KOH (15 mL) was added to this mixture. The mixture was
stirred overnight under reflux. After cooling to room temper-
ature, the mixture was extracted with ethyl acetate (3 × 50
mL). The organic phase was dried over Na2SO4, the solvent
was evaporated, and the residue was purified by CC (CH2Cl2/
MeOH/25% NH4OH 9:1:0.1) to give 6 (979 mg, 82%) as a white
solid. TLC (CH2Cl2/MeOH/25% NH4OH 9:1:0.1) Rf 0.68. Mp
1047 s, 907 s, 846 m, 754 s cm-1. H NMR (300 MHz, DMSO-
d6) δ 7.80 (t, J ) 6.1 Hz, 1H), 7.33-7.20 (m, 5H), 5.00 (br s,
1H), 4.17 (d, J ) 6.2 Hz, 2H), 3.60 (s, 1H), 3.49 (d, J ) 2.2 Hz,
1
90-92 °C. [R]22 -50.5 (c 0.0019, MeOH). IR (KBr) 3386 s,
(14) (a) Rana, V. S.; Kumar, V. A.; Ganesh, K. N. Tetrahedron 2001,
57, 1311-1321. (b) Ramasamy, K. S.; Seifert, W. Bioorg. Med. Chem.
Lett. 1996, 6, 1799-1804. (c) Rana, V. S.; Kumar, V. A.; Ganesh, K.
N. Bioorg. Med. Chem. Lett. 1997, 7, 2837-2842. (d) Benhida, R.;
Devys, M.; Fourrey, J .-L.; Lecubin, F.; Sun, J .-S. Tetrahedron Lett.
1998, 39, 6167-6170.
D
1
2907 s, 1584 w, 1044 m, 757 s. H NMR (300 MHz, MeOD-d3)
δ 7.36-7.26 (m, 5H), 4.19 (dt, J ) 8.07, 3.30 Hz, 1H), 4.06 (q,
J ) 6.86 Hz, 1H), 3.87 (2d, J ) 12.8 Hz, 2H), 2.79 (t, J ) 2.58
Hz, 1H), 2.25-1.97, 1.73-1.44 (2m, 4H). 13C NMR (75 MHz,
J . Org. Chem, Vol. 68, No. 20, 2003 7697

Ahn et al.
MeOD-d3) δ 141.37 (s), 129.77 (d), 129.74 (d), 128.44 (d), 77.08
(d), 71.37 (d), 70.44 (d), 53.37 (t), 31.38 (t), 31.30 (t). HRMS
(ESI+) m/z calcd for C12H18NO2 208.1337 [M + H]+, found
208.1321 [M + H]+.
and extracted with sat. NaHCO3 (aq) and brine. The organic
phase was dried over Na2SO4 and evaporated. The residue was
purified by CC (hexane/ethyl acetate 2:1 f 3:2) to give the
major isomer 10a (40 mg, 33% over 2 steps) as a white solid.
TLC (hexane/ethly acetate 1:1) Rf 0.44 (major isomer), 0.32
2-Am in ocyclop en ta n -1,3-d iol (7). To a solution of 6 (910
mg, 4.39 mmol) in ethanol (10 mL) in an autoclave was added
10% Pd/C (1.3 g). The autoclave was then charged with H2
(10 atm) and the mixture was stirred overnight (∼20 h) at
room temperature. The reaction mixture was filtered over
Celite, and the filtrate was evaporated to give pure 7 (503 mg,
98%) as a yellowish oil. TLC (CH2Cl2/MeOH/25% NH4OH 9:1:
0.1) Rf 0.09. [R]22D -51.2 (c 0.0025, MeOH). 1H NMR (300 MHz,
MeOD-d3) δ 4.09 (dt, J ) 5.52, 2.94 Hz, 1H), 3.93 (q, J ) 7.11
Hz, 1H), 2.80 (dd, J ) 7.35, 5.16 Hz, 1H), 2.22-2.03, 1.69-
1.43 (2m, 4H). 13C NMR (75 MHz, MeOD-d3) δ 78.50 (d), 73.31
(d), 64.03 (d), 31.09 (t), 30.90 (t). HRMS (ESI+) m/z calcd for
C5H12NO2 118.0868 [M + H]+, found 118.0873 [M + H]+.
N-[(2,5-Dih yd r oxycyclop en t yl)-1-(t h ym in -1-yl)]-a cet -
a m id e (9a ). To a solution of 7 (20 mg, 0.17 mmol) in DMF (2
mL) were added thymin-1-yl acetic acid (38 mg, 0.20 mmol)
and EDC (49 mg, 0.25 mmol). The mixture was stirred at room
temperature overnight. After evaporation of the solvent, the
residue was purified by CC (CH2Cl2/MeOH 95:5 f 85:15) to
give 9a (31 mg, 66%) as a white solid. TLC (CH2Cl2/MeOH
85:15) Rf 0.44. Mp >210 °C dec. [R]22D -55.7 (c 0.0023, DMSO).
IR (KBr) 3405 s, 3342 s, 1673 s, 1242 m, 1030 m. 1H NMR
(300 MHz, DMSO-d6) δ 11.24 (s, 1H), 7.96 (d, J ) 8.07 Hz),
7.37 (d, J ) 1.08 Hz, 1H), 4.68, 4.62 (2d, J ) 5.13, 3.69 Hz,
2H), 4.33 (s, 2H), 3.94 (m, 2H), 3.64 (dt, J ) 7.32, 5.13 Hz,
1H), 2.01-1.85, 1.52-1.30 (2m, 4H), 1.74 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) 167.21 (s), 164.57 (s), 151.20 (s), 142.63
(d), 107.95 (s), 74.06 (d), 69.85 (d), 65.18 (d), 49.23 (t), 30.25
(t), 29.93 (t), 12.06 (q). HRMS (ESI+) m/z calcd for C12H17N3O5
284.1246 [M + H]+, found 284.1241 [M + H]+.
(minor isomer). Data for 11a : [R]22 +12.5 (c 0.002, MeOH).
D
IR (KBr) 3423 m, 2950 w, 1697 s, 1448 m, 1242 m, 1066 m,
757 m. 1H NMR (300 MHz, benzene-d6) δ 10.24 (br s, 1H), 7.69
(d, J ) 7.71 Hz, 2H), 7.41 (d, J ) 5.88 Hz, 1H), 7.26-6.96 (m,
10H), 6.50 (s, 1H), 5.51 (d, J ) 8.1 Hz, 1H), 4.39 (dt, J ) 8.82,
4.41 Hz, 1H), 3.89 (s, 2H), 4.35-3.82 (m, 2H), 1.72 (s, 3H),
1.51-1.33, 1.27 (3m, 4H), 0.86 (s, 9H), -0.005, -0.144 (2s, 6H).
13C NMR (75 MHz, benzene-d6) δ 166.11 (s), 164.37 (s), 152.17
(s), 151.59 (s), 151.30 (s), 148.92 (s), 140.45 (d), 132.03 (d),
131.49 (d), 129.71 (d), 129.48 (d), 128.08 (d), 127.91 (d), 126.94
(d), 124.87 (s), 124.17 (s), 123.78 (d), 123.32 (d), 116.50 (d),
116.34 (d), 10.57 (s), 76.99 (d), 76.45 (s), 71.02 (d), 59.63 (d),
50.32 (t), 30.62 (t), 27.40 (t), 25.86 (q), 18.02 (s), 12.29 (q), -4.81
(q), -5.09 (q). HRMS (ESI-) m/z calcd for C37H42N3O6Si
652.8498 [M - H]-, found 652.2836 [M - H]-.
N-[2-(ter t-Bu tyld im eth ylsilyloxy)-5-(9-ph en yl-xa n th en -
9-yloxy)-cyclop en tyl-1-(N6-p-m eth oxyben zoyl)a d en in -1-
yl]-a ceta m id e (10b). To a solution of 9b (80 mg 0.187 mmol)
in DMSO (0.15 mL) were added pyridine (0.7 mL) and
9-phenyl-xanthenyl chloride (27 mg, 0.09 mmol) at room
temperature. The mixture was stirred for 5 h. Then another
portion of 9-phenyl-xanthenyl chloride (27 mg, 0.09 mmol) was
added. After an additional 15 h, the reaction mixture was
quenched with sat. NaHCO3(aq) and extracted with ethyl
acetate (3 × 20 mL). The combined organic layers were washed
with water, dried over Na2SO4, and evaporated. CC of the
residue (CH2Cl2/MeOH/TEA 96.5:2.5:1 f 94.5:5:1) gave the
mixture of monopixylated isomers (67 mg, ∼3:1) that was
subsequently dissolved in DMF (1 mL) and treated with
TBDMSCl (30 mg, 0.196 mmol) and imidazole (26 mg, 0.392
mmol) at room temperature. This mixture was stirred at room
temperature overnight. After removal of the solvent the
residue was redissolved in ethyl acetate (20 mL) and washed
with sat. NaHCO3 (aq) and brine. The organic phase was dried
over Na2SO4 and evaporated. The residue was purified by CC
(hexane/ethyl acetate (+1% TEA) 1:1) to give the major isomer
10b (46 mg, 31% over 2 steps) as a white solid. TLC (ethyl
acetate) Rf 0.59 (major isomer), 0.42 (minor isomer). Data for
N-[(2,5-Dih yd r oxycyclop en t yl)-1-(N6-(p -m et h oxyb en -
zoyl)a d en in -1-yl)]-a ceta m id e (9b). To a solution of 7 (11 mg,
0.094 mmol) in DMF (0.5 mL) were added N6-(p-methoxyben-
zoyl)adenin-1-yl acetic acid (32 mg, 0.103 mmol) and EDC (22
mg, 0.115 mmol). The mixture was stirred at room tempera-
ture overnight. After evaporation of the solvent, the residue
was purified by CC (CH2Cl2/MeOH 95:5 f 88:12) to give 9b
(26 mg, 65%) as a white solid. TLC (CH2Cl2:MeOH 85:15) Rf
0.33. Mp >230 °C dec. [R]22 -28.8 (c 0.0017, DMSO-d6). IR
D
10b: [R]22 +8.2 (c 0.0022, MeOH). IR (KBr) 3417 s, 2927 m,
D
(KBr) 3282 s, 2938 m, 1702 s, 1246 m, 1177 m, 1093 m, 1033
m, 759 m.1H NMR (300 MHz, DMSO-d6) δ 10.99 (s, NH), 8.68
(s, 1H), 8.37 (s, 1H), 8.29 (d, J ) 8.1 Hz, 1H), 8.03 (d, J ) 8.5
Hz, 2H), 7.06 (d, J ) 8.8 Hz, 2H), 5.03 (s, 2H), 4.78, 4.76 (2d,
J ) 3.7, 5.5 Hz, 2H), 3.98 (m, 2H), 3.84 (s, 3H), 3.68 (dd, J )
12.5, 7.7 Hz, 1H), 2.02-1.90, 1.52-1.50, 1.36-1.32 (3m, 4H).
13C NMR (75 MHz, DMSO-d6) δ 166.38 (s), 165.05 (s), 162.69
(s), 152.77 (s), 151.50 (d), 150.38 (s), 145.67 (d), 130.71 (d),
125.77 (s), 125.03 (s), 113.86 (d), 74.05 (d), 69.83 (d), 61.66 (d),
55.66 (q), 45.34 (t), 30.22 (t), 29.92 (t). HRMS (ESI+) m/z calcd
for C20H23N6O5 427.1729 [M + H]+, found 427.1824 [M + H]+.
1
1604 s, 1449 s, 1247 s, 1173 m, 759 m, 666 m. H NMR (300
MHz, CDCl3) δ 9.06 (s, 1H), 8.69 (s, 1H), 8.13 (s, 1H), 8.00 (d,
J ) 8.8 Hz, 2H), 7.43-6.97 (m, 15H), 5.20 (d, J ) 8.1 Hz, 1H,
N-H), 4.73, 4.57 (2d, J ) 16.1 Hz, 2H), 4.06 (m, 1H, C(1)-H),
3.95 (t, J ) 4.0, 1H, C(2)-H), 3.88 (s, 3H), 3.60 (q, J ) 8.1 Hz,
1H, C(5)-H), 1.79, 1.26-1.11 (2m, 4H), 0.72 (s, 9H), -0.13,
-0.20 (2s, 6H). 13C NMR (75 MHz, benzene-d6) δ 165.23 (s),
164.39 (s), 163.12 (s), 152.77 (s), 152.39 (s), 152.10 (d), 151.62
(s), 150.77 (s), 148.79 (s), 143.87 (d), 132.00 (d), 131.56 (d),
130.51 (d), 129.75 (d), 129.50 (d), 128.08 (d), 127.87 (d), 126.99
(d), 126.84 (s), 124.63 (s), 124.31 (s), 123.80 (d), 123.33 (d),
116.51 (d), 116.38 (d), 114.00 (d), 77.08 (d), 76.52 (s), 71.13
(d), 59.89 (d), 54.86 (q), 46.20 (t), 30.75 (t), 27.65 (t), 25.84 (q),
18.00 (s), -4.88 (q), -5.13 (q). HRMS (ESI-) m/z calcd for
N-[2-(ter t-Bu tyld im eth ylsilyloxy)-5-(9-p h en yl-xa n th en -
9-yloxy)-cyclop en tyl-1-(th ym in -1-yl)]-a ceta m id e (10a ). To
a solution of 9a (50 mg. 0.18 mmol) in DMSO (0.2 mL) were
added pyridine (0.8 mL) and 9-phenyl-xanthenyl chloride (25
mg, 0.09 mmol) at room temperature. The mixture was stirred
for 5 h, and another portion of 9-phenyl-xanthen chloride (25
mg, 0.09 mmol) was added. After another 15 h, the reaction
mixture was diluted with sat. NaHCO3 and extracted with
ethyl acetate (3 × 20 mL). The combined organic layers were
washed with water, dried over Na2SO4, and evaporated. CC
(CH2Cl2/MeOH/TEA 94:5:1) of the residue gave the mixture
of the monopixylated isomers (52 mg, ∼4:1) as well as the
dipixylated product (19 mg). The mixture of monopixylated
isomers was subsequently dissolved in DMF (1 mL), and to
this solution were added TBDMS-Cl (20 mg, 0.145 mmol) and
imidazole (18 mg, 0.289 mmol) at room temperature. The
mixture was stirred overnight followed by removal of the
solvent. The residue was redissolved in ethyl acetate (20 mL)
C
45H47N6O6Si 795.3326 [M - H]-, found 795.4184 [M - H]-.
N-[2-Hyd r oxy-5-(9-p h en yl-xa n th en -9-yloxy)-cyclop en -
tyl-1-(th ym in -1-yl)]-a ceta m id e (11a ). To a solution of 10a
(46 mg, 0.07 mmol) in THF (1 mL) was added Bu4NF‚3H2O
(45 mg, 0.14 mmol) at room temperature. The mixture was
stirred overnight and the solvent removed. The residue was
purified by CC (CH2Cl2/MeOH 19:1) to give 11a (35 mg, 92%)
as a white solid. TLC (CH2Cl2/MeOH 95:5) Rf 0.27. [R]22D +11.5
(c 0.0013, MeOH). IR (KBr) 3443 s, 1681 s, 1449 m, 1242 m.
1H NMR (300 MHz, CDCl3) δ 9.73 (s, 1H), 7.41-6.99 (m, 14H),
5.90 (d, J ) 8.10 Hz, 1H), 4.19, 3.82 (2d, J ) 15.8 Hz, 2H),
4.07 (dt, J ) 14.3, 9.24 Hz, 1H), 3.97 (br, 1H), 3.58 (q, J )
7.84 Hz, 1H), 3.46 (br s, 1H), 1.86 (s, 3H), 1.33-1.11 (m, 4H).
13C NMR (75 MHz,CDCl3) δ 166.33 (s), 164.30 (s), 151.41 (s),
7698 J . Org. Chem., Vol. 68, No. 20, 2003

Synthesis of Cyclopentane Amide DNA
151.38 (s), 150.85 (s), 148.62 (s), 140.97 (d), 131.38 (d), 130.93
(d), 129.33 (d), 129.08 (d), 127.64 (d), 127.13 (d), 126.55 (d),
123.98 (s), 123.54 (s), 123.09 (d), 122.72 (d), 116.39 (d), 116.26
(d), 111.00 (s), 76.86 (d), 75.73 (s), 69.58 (d), 60.09 (d), 50.55
(t), 30.07 (t), 29.66 (t), 12.24 (q). HRMS (ESI-) m/z calcd for
100:1 f 2:0.5, 1% Et3N) to give a yellowish viscous oil. The oil
was redissolved in ethyl acetate (1 mL) and this solution was
added dropwise to stirred hexane (40 mL). The resulting white
precipitate was filtered off and washed with cold hexane. The
precipitate was dried to give 12b (130 mg, 63%) as a white
solid. TLC (CH2Cl2/MeOH ) 40:1): Rf 0.32. 1H NMR (300 MHz,
benzene-d6): δ 10.03, 8.76 (2br s, 1H), 8.17, 8.08 (2s, 1H), 8.13-
6.72 (m, 14H), 6.16, 6.10 (2d, J ) 8.43, 8.82 Hz, 1H), 4.75-
4.53 (m, 3H), 4.19-4.05, 3.91 (2m, 2H), 3.58-3.53, 3.20-3.07
(2m, 4H), 3.32 (s, 3H), 2.29-2.11 (m, 2H), 1.87-1.79, 1.67,
1.32-1.20 (3m, 4H), 1.12-0.86 (m, 12H). 13C NMR (75 MHz,
benzene-d6) 165.84 (s), 165.69 (s), 165.03 (s), 164.94 (s), 163.24
(s), 152.57 (d), 151.93 (s), 151.85 (s), 151.57 (s), 151.55 (s),
150.70 (s), 149.10 (s), 148.99 (s), 144.57 (d), 144.34 (d), 132.10
(d), 132.05 (d), 131.74 (d), 131.63 (d), 130.79 (d), 130.71 (d),
129.94 (d), 129.80 (d), 129.53 (d), 129.42 (d), 128.08 (d), 127.79
(d), 126.98 (d), 124.55 (s), 124.17 (s), 124.09 (d), 123.65 (s),
123.50 (s), 123.36 (d), 123.22 (d), 118.83 (s), 118.30 (s), 116.47
(d), 116.44 (d), 116.41 (d), 116.36 (d), 114.04 (d), 77.30 (d), 76.51
(s), 76.49 (s), 73.55 (dd, J C-P ) 16.5 Hz), 72.58 (J C-P ) 9.75
Hz), 59.72 (dd, J C-P ) 3.75 Hz), 59.48 (dd, J C-P ) 4.50 Hz),
58.48 (t), 58.24 (t), 58.16 (t), 58.09 (t), 54.98 (q), 54.96 (q), 46.18
(t), 43.34 (d), 43.21 (d), 43.18 (d), 43.06 (d), 27.99 (t), 27.86 (t),
24.68 (q), 24.60 (q), 24.52 (q), 24.41 (q), 24.31 (q), 20.47 (t),
20.39 (t), 20.20 (t), 20.12 (t). 31P NMR (161.9 MHz, benzene-
d6) δ 148.86, 147.56. HRMS (ESI-) m/z calcd for C48H50N8O7P
881.3540 [M - H]-, found 881.4448 [M - H]-.
C
31H28N3O6 538.1978 [M - H]-, found 538.1960 [M - H]-.
N-[2-Hyd r oxy-5-(9-p h en yl-xa n th en -9-yloxy)-cyclop en -
tyl-1-(N6-p-m eth oxyben zoyl)aden in -1-yl]-acetam ide (11b).
To a solution of 10b (38 mg, 0.0476 mmol) in THF (1 mL) was
added Bu4NF‚3H2O (30 mg, 0.0952 mmol) at room tempera-
ture. The mixture was stirred overnight and the solvent
removed. The residue was purified by CC (CH2Cl2/MeOH/TEA
94:5:1) to give 11b (32 mg, 98%) as a white solid. TLC (CH2-
Cl2/MeOH 95:5) Rf 0.45. [R]22 +16.3 (c 0.0019, MeOH). IR
D
(KBr) 3390 s, 1674 m, 1602 s, 1456 s, 1246 s, 1174 m, 759 m.
1H NMR (300 MHz, MeOD-d3) δ 8.58 (s, 1H), 8.41 (s, 1H), 8.10
(d, J ) 8.8 Hz, 2H), 7.37-7.07 (m, 15H), 4.99, 4.90 (2d, J )
16.9 Hz, 2H), 4.22 (dd, J ) 8.1, 5.2 Hz, 1H), 3.99 (m, 1H), 3.92
(s, 3H), 1.97, 1.43-0.89 (2m, 4H). 13C NMR (75 MHz, MeOD-
d3) δ 168.16 (s), 167.74 (s), 165.26 (s), 154.08 (s), 153.44 (d),
153.02 (s), 152.79 (s), 151.41 (s), 150.78 (s), 146.90 (d), 132.85
(d), 132.65 (d), 131.85 (d), 130.83 (d), 130.79 (d), 128.86 (d),
128.48 (d), 127.82 (d), 127.22 (s), 125.69 (s), 125.14 (s), 124.96
(d), 124.48 (s), 124.34 (d), 117.56 (d), 117.53 (d), 115.26 (d),
78.84 (d), 77.49 (s), 71.16 (d), 61.67 (d), 56.38 (q), 47.04 (t),
31.65 (t), 29.55 (t). HRMS (ESI-) m/z calcd for C39H33N6O6
681.2461 [M - H]-, found 681.2459 [M - H]-.
Syn th esis a n d P u r ifica tion of Oligon u cleotid es. All
oligonucleotides and analogues were synthesized on the 1.3-
µmol scale on a Pharmacia Gene-Assembler Special DNA-
synthesizer. Oligodeoxyribonucleotides were synthesized by
phosphoramidite chemistry, using commercial building blocks
(Glen Research) and reagents, and were deprotected and
purified according to standard protocols. cpa-oligonucleotides
13 and 14 were assembled on universal CPG-support from CT-
Gen, San J ose´. The synthesis proceeded with the following
changes to the synthetic cycle: The coupling time was ex-
tended to 6 min. Tetrazole (0.45 M in CH3CN) was replaced
by the more active 5-(ethylthio)-1H-tetrazole (0.25 M in CH3-
CN) as the activator. Coupling yields of 12a ,b were typically
∼95%. After removal of the last trityl group, oligonucleotides
were detached from the support and deprotected by treatment
with 25% NH3/40%MeNH2 (1:1) at 55 °C for 16 h. The crude
oligonucleotides were purified by DEAE-HPLC (Nucleogen,
Macherey-Nagel). Oligonucleotides were desalted over SEP-
PAK C-18 cartridges (Waters). Purities of all modified oligo-
nucleotides were controlled by RP-FPLC (PepRPC HR 5/5) and
routinely characterized by ESI--TOF mass spectrometry (see
Table 1 and Table S1 in the Supporting Information).
UV Meltin g Exp er im en ts a n d CD Sp ectr a . Oligonucleo-
tides were mixed to 1:1 stoichiometry using the UV extinction
coefficients of natural oligodeoxynucleotides. UV melting
curves were recorded on a Cary 3E UV/vis spectrophotometer
(Varian) at 260 nm. Consecutive heating-cooling-heating
cycles in the temperature intervall of 0-90 °C with a linear
gradient of 0.2-0.5 °C/min were applied. Heating and cooling
ramps were always superimposable indicating equilibrium
conditions. CD spectra were measured on a J ASCO J -715
spectrometer at the temperatures indicated.
2′-(2-Cya n oeth yl)-[1′-(2-(th ym in -1-yl)-a cetyla m in o)-3′-
(9-p h en yl-xa n th en -9-yloxy)-cyclop en tyl]-N,N′-d iisop r o-
p ylp h osp h or a m id ite (12a ). To a solution of 11a (25 mg,
0.046 mmol) in THF (1 mL) were added N,N′-diisopropylethyl-
amine (48 µL, 0.276 mmol) and chloro-2-cyanoethoxydiisopro-
pylaminophosphine (31 µL, 0.138 mmol) at room temperature.
The reaction mixture was stirred at room temperature for 1
h, quenched with sat. NaHCO3(aq), and extracted with ethyl
acetate (3 × 20 mL). The combined organic phases were dried
over Na2SO4 and evaporated and the residue was purified by
CC (CH2Cl2/MeOH 50:1) to give 12a (20 mg, 59%) as a white
solid. TLC (ethyl acetate) Rf 0.66. 1H NMR (300 MHz, benzene-
d6) δ 10.35, 10.00 (2s, 1H), 7.69 (t, J ) 7.74 Hz, 2H), 7.49-
6.70 (m, 10H), 6.75 (t, J ) 8.10 Hz, 1H), 6.61, 6.48 (2d, J )
1.11 Hz, 1H), 5.87 (t, J ) 8.46 Hz, 1H), 4.55 (m, 1H), 4.36-
3.59 (m, 4H), 3.50-3.10 (m, 4H), 2.43-2.34, 2.21-2.12 (2m,
2H), 1.76, 1.74 (2s, 3H), 1.66-1.27 (m, 4H), 1.16-1.07 (m,
12H). 13C NMR (75 MHz, benzene-d6) δ 166.39 (s), 166.35 (s),
164.36 (s), 164.24 (s), 152.10 (s), 152.02 (s), 151.54 (s), 151.52
(s), 151.39 (s), 149.06 (s), 140.90 (d), 140.64 (d), 132.10 (d),
131.65 (d), 131.39 (d), 129.88 (d), 129.66 (d), 129.53 (d), 129.48
(d), 128.11 (d), 127.89 (d), 127.81 (d), 127.30 (s), 126.94 (d),
124.81 (s), 124.75 (s), 124.11 (d), 124.08 (s), 123.85 (d), 123.40
(d), 123.33 (d), 118.77 (s), 118.09 (s), 116.52 (d), 116.41 (d),
116.38 (d), 116.31 (d), 77.18 (d), 76.96 (d), 76.46 (s), 76.45 (s),
73.19 (dd, J C-P ) 15.7, Hz), 72.52 (dd, J C-P ) 9.10 Hz), 59.35
(dd, J C-P ) 5.46 Hz), 59.28 (dd, J C-P ) 3.64 Hz), 58.82 (t), 58.59
(t), 58.43 (t), 58.19 (t), 50.35 (t), 50.15 (t), 45.28 (s), 44.53 (s),
43.41 (d), 43.33 (t), 43.24 (t), 43.16 (t), 27.58 (t), 27.43(t), 24.70
(q), 24.67 (q), 24.60 (q), 24.57 (q), 24.51 (q), 24.38 (q), 24.67
(q), 20.43 (t), 20.34 (t), 20.16 (t), 20.09 (t), 12.35 (q). 31P NMR
(161.9 MHz, benzene-d6) δ 148.69, 147.47. HRMS (ESI-) m/z
calcd for C40H45N5O7P 738.3056 [M - H]-, found 738.3047 [M
- H]-.
Ack n ow led gm en t. The authors thank the Swiss
National Science Foundation (grant no. 20-63582.00) for
financial support of this project. The BENEFRI small
molecule X-ray crystal service directed by Prof. H.
Sto¨ckli-Evans is thanked for providing the structure of
compound 9.
Su p p or tin g In for m a tion Ava ila ble: Details on the syn-
thesis and characterization of compound 8, the X-ray structure
of 8, 1H NMR spectra of compounds 2-12, as well as 2D 1H,13C-
COSY, and difference 1H-NOE spectra of 10b. This material
is available free of charge via the Internet at http://pubs.acs.org.
2′-(2-Cyan oeth yl)-[1′-(2-(N6-(p-m eth oxyben zoyl)aden in -
1-yl)-a cet yla m in o)-3′-(9-p h en yl-xa n t h en -9-yloxy)-cyclo-
p en tyl]-N,N′-d iisop r op ylp h osp h or a m id ite (12b). To a so-
lution of 11b (159 mg, 0.23 mmol) in THF (5 mL) were added
N,N′-diisopropylethylamine (120 µL, 0.69 mmol) and chloro-
2-cyanoethoxy-diisopropylaminophosphine (77 µL, 0.345 mmol)
at room temperature. The reaction mixture was stirred for 1
h. The mixture was quenched with sat. NaHCO3 (aq) and
extracted with ethyl acetate (3 × 20 mL). The combined
organic layers were washed with brine, dried over Na2SO4, and
evaporated. The residue was purified by CC (CH2Cl2/MeOH
J O034143Q
J . Org. Chem, Vol. 68, No. 20, 2003 7699