Bicyclo[3.2.1]amide-DNA: A chiral, nonchiroselective base-pairing system

Article abstract of DOI:10.1002/1521-3765(20021202)8:23<5312::AID-CHEM5312>3.0.CO;2-M

The design, synthesis, and base-pairing properties of bicyclo[3.2.1]amide-DNA (bca-DNA), a novel phosphodiester-based DNA analogue, are reported. This analogue consists of a conformationally constrained backbone entity, which emulates a B-DNA geometry, to

Full text of DOI:10.1002/1521-3765(20021202)8:23<5312::AID-CHEM5312>3.0.CO;2-M

FULL PAPER
Bicyclo[3.2.1]amide-DNA: A Chiral, Nonchiroselective Base-Pairing System
Dae-Ro Ahn,[a] Anita Egger,[a] Christian Lehmann,[b] Stefan Pitsch,[c] and
Christian J. Leumann*[a]
Abstract: The design, synthesis, and
base-pairing properties of bicy-
clo[3.2.1]amide-DNA (bca-DNA),
novel phosphodiester-based DNA ana-
logue, are reported. This analogue con-
sists of a conformationally constrained
tigations revealed the duplexes with d-
or l-complements to be of similar sta-
bility and enantiomorphic in structure.
Bca oligonucleotides that contain all
four bases form strictly antiparallel,
left-handed complementary duplexes
with themselves and with complemen-
tary DNA, but not with RNA. Base-
mismatch discrimination is comparable
to that of DNA, while the overall
thermal stabilities of bca-oligonucleo-
tide duplexes are inferior to those of
DNA or RNA. A detailed molecular
modeling study of left- and right-handed
bca-DNA-containing duplexes showed
only minor changes in the backbone
a
backbone entity, which emulates
a
structure and revealed a structural
B-DNA geometry, to which the nucleo-
bases were attached through an extend-
ed, acyclic amide linker. Homobasic
adenine-containing bca decamers form
duplexes with complementary oligonu-
cleotides containing bca, DNA, RNA,
and, surprisingly, also l-RNA back-
bones. UV and CD spectroscopic inves-
switch around the base-linker unit to
be responsible for the generation of
enantiomorphic duplex structures. The
obtained data are discussed with respect
to the structural and energetic role of
the ribofuranose entities in DNA and
RNA association.
Keywords: chirality ¥ conformation
analysis
¥
DNA recognition
¥
oligonucleotides ¥ RNA
DNA[11])have been evaluated. Besides this, analogues of
increased (seven bonds)[7] or decreased (five bonds)[12] length
in the repetitive backbone unit have been evaluated.
Introduction
Oligonucleotide analogues are of interest in antisense therapy
and as tools in the area of functional genomics. In order to
map the structure/stability landscape of oligonucleotides in
complementary duplex formation, a number of DNA ana-
logues have been synthesized and their base-pairing and
antisense properties evaluated over recent years.[1] Major
changes in the association behavior of oligonucleotides arise,
as expected, from variation of the ribofuranose substructure
in DNA and RNA. In this context, six-membered ring
versions (e.g, homo-DNA,[2] HNA,[3] CeNA,[4] CNA,[5]
p-RNA[6])and four-membered ring versions (e.g, carbocyclic
oxetanocine-DNA[7] and cyclobutane-DNA[8]), as well as ring-
deficient analogues (such as seco-DNA[9, 10] and glycerol-
In an effort to investigate various aspects of conformational
restriction on the hybridization properties of DNA ana-
logues,[13] we recently prepared and analyzed bicyclo[3.2.1]-
16]
DNA (Figure 1).[14
This analogue is composed of a rigid
[a] Prof. C. J. Leumann, Dipl. Chem. D.-R. Ahn, Dipl. Chem. A. Egger
Department of Chemistry and Biochemistry
University of Bern
Figure 1. Chemical formulae of DNA, bicyclo[3.2.1]-DNA, and bicy-
clo[3.2.1]amide-DNA. The repetitive backbone bonds are highlighted in
red, those of the base-linker elements in blue.
Freiestrasse 3, 3012 Bern (Switzerland)
Fax : (‡41)31-631-3422
E-mail: leumann@ioc.unibe.ch
[b] Dr. C. Lehmann
backbone unit of B-DNA geometry, to which the bases are
flexibly attached through a methylenoxy linker unit. The main
goal was to determine the impact on the duplexation proper-
ties of the loss of ring structure between base and backbone.
We were able to show that bicyclo[3.2.1]-DNA is still a
competent, but less effective, duplex partner for DNA, RNA,
Institut de Chimie Organique
¬
Universite de Lausanne
Dorigny (Switzerland)
[c] Prof. S. Pitsch
¬
Institut de Chimie Moleculaire et Biologique (ICMB)
EPFL, 1015 Lausanne (Switzerland)
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Chem. Eur. J. 2002, 8, No. 23

5312 5322
and for itself. Furthermore, duplex formation is still orienta-
tion-specific (no parallel duplexes are formed), and base
mismatches are discriminated equally well as in DNA. Thus, a
number of necessary features for selective information trans-
fer are still fulfilled in bicyclo[3.2.1]-DNA.
subsequently tritylated to 4a d by treatment with dimethox-
ytrityl triflate (DMTOTf)in pyridine at slightly elevated
temperatures. Desilylation with Bu4NF in THF afforded 5a
d, which could be phosphitylated with 2-cyanoethoxy diiso-
propylamino chlorophosphine (CEP)to give the building
blocks for oligonucleotide synthesis 6a d, again in good
yields. With these building blocks in hand we approached the
automated oligonucleotide synthesis.
The basic framework of this bicyclo[3.2.1] scaffold offers a
number of possible ways to change the molecular attachment
of the nucleobases to the backbone. Thus, in analogy to the
above-mentioned systems with elongated or shortened repet-
itive backbone units, we designed the bicyclo[3.2.1]amide-
DNA (bca-DNA)as a DNA analogue in which the distance
between the base and the backbone is elongated by one bond
relative to DNA or bicyclo[3.2.1]-DNA, while the number of
bonds in the repetitive backbone unit (six)remains un-
changed. We report here on the synthesis, the base-pairing
properties, and on structural investigations (by CD spectros-
copy and molecular modeling)of bicyclo[3.2.1]amide-DNA in
complexation with itself as well as with complementary DNA
and RNA.
Synthesis of oligonucleotides: Bicyclo[3.2.1]amide (bca)oli-
gonucleotides 7 11 (Table 1)were prepared according to the
standard protocols for automated DNA synthesis on the
1.3 mmol scale. Because of the reversed orientation of the
Table 1. Yield after HPLC purification (1.3 mmol scale), as well as
sequence and MS data, of bca-oligonucleotides used in this study.
Sequence (5' ! 3')
[M À H]À
ESI-MS
(calcd)(found[%) ]
Yield OD260
7
8
9
10
11
bca(GAGACCCGT)3471.5
bca(ACGGGTCTC)3462.5
bca(CTCTGGGCA)3462.5
3471.2
3462.0
3462.0
12.6 (10)
16.6 (13)
24.4 (20)
4203.1[b]
4118.2[b]
bca(XA10)[a]
bca(XT10)[a]
4205.2
4115.1
42.3 (41)
37.4 (41)
Results
[a] X ˆ dT, arising from solid support. [b] MALDI-ToF MS.
Synthesis of monomers: We started the synthesis of the
monomeric building blocks 5a d with the bicyclic backbone
unit 1, for which we had already developed a convenient
access (Scheme 1).[17] Compound 1 was converted into com-
pounds 3a d by amide bond formation with the nucleobase-
bearing acetic acid derivatives 2a d, used previously for the
dimethoxytrityl (O(3')) and phosphityl (O(5')) groups in the
phosphoramidites 6a d relative to the deoxyribonucleoside
phosphoramidites, synthesis of the bca-oligonucleotides pro-
ceeded in the 5' ! 3' direction. As solid support, commercial
thymidine-derived CPG was used for the synthesis of 10 and
11, and a universal CPG solid support for oligonucleotides 7
9. While the use of the former results in the attachment of a
natural DNA unit at the 5'-end of the bca oligonucleotides,
through a 5' 5' junction, the advantage of the universal solid
support lies in its traceless removal after synthesis and
deprotection.
synthesis of the polyamide nucleic acids (PNAs), and the
20]
uronium-based coupling reagent TOTU.[18
Despite the
considerable steric hindrance of the amino group, acceptable
to good yields were obtained. Compounds 3a d were
The standard DNA synthesis cycle needed some adjust-
ments. For complete removal of the dimethoxytrityl groups,
10% trichloroacetic acid in dichloroethane had to be used.
Furthermore, the coupling time was extended to 6 10 min,
and the standard activator tetrazole was replaced by the more
active (S-benzylthio)-1H-tetrazole. With these changes, cou-
pling yields of 94 99% were obtained, according to trityl
assay. Crude oligonucleotides were deprotected and detached
from the solid support in ammonia. Standard conditions
(conc. NH3, 558C, 16 h)were sufficient to release and
deprotect the bca oligonucleotides prepared on dT solid
support. No products arising from cleavage of the amide bond
between base and backbone units were detected. Removal of
the universal solid support, however, needed harsher con-
ditions (NH3, 708C, 48 h)and resulted in partial cleavage of
(statistically)one base-linker unit (ESI-MS). These ™abasic∫
oligonucleotides could, however, easily be removed by HPLC.
All bca oligonucleotides were characterized by ESI mass
spectrometry. We examined the structural and thermodynam-
ic properties of duplex formation of bca oligonucleotides 7
11 with themselves and with the backbone systems of DNA,
RNA, and l-RNA by CD spectroscopy and UV melting curve
analysis.
Scheme 1. Synthesis of the monomer building blocks 6. a) N,N,N',N'-
tetramethyluronium tetrafluoroborate (TOTU), iPr2NEt, DMF, room
temperature; b)DMTOTf, pyr, 60 8C; c)Bu 4NF, THF, room temperature;
d) iPr2NEt, [(iPr2N)(NCCH2CH2O)]PCl, THF, room temperature.
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C. J. Leumann et al.
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Pairing properties within the homobackbone series: Table 2
summarizes the thermal melting temperatures (Tm)as well as
the thermodynamic data for duplex to single-strand transi-
tions as determined by the concentration variation method.[21]
Representative melting curves of the nonamer duplexes are
depicted in Figure 2.
over duplex formation imposed by the structurally preorgan-
ized backbone. The thermodynamic data are in agreement
with no additional strain in the complex, compared to DNA,
but with reduced affinity due to the increased flexibility of the
base-carrying unit.
CD spectroscopic investigation of the DNA and bca-DNA
duplexes corresponding to entries 1 and 4 (Table 2)led to a
surprise (Figure 3). The CD traces of the two duplexes are
virtually enantiomorphic, indicating a left-handed Watson
Crick base-paired structure for the bca-NA duplex. Thus, the
preference for left-handed duplex formation, as already
observed in the A/T-homobasic bca-DNA series,[22] seems
not to be restricted to the homo-A/T sequence context.
Table 2. Tm and thermodynamic data of duplex formation in the pure bca
series.[a]
[b]
Entry
Tm DH
DS
DG298K
[kJ molÀ1] [JKÀ1 molÀ1] [kJ molÀ1
]
1
2
3
4
5
bca(GAGACCCGT)31.7
(CTCTGGGCA)bca
bca(GAGACCCGT)nd
bca(CTCTGGGCA)
bca(XA10)21.6
À 281.1
À 809.7
À 39.7
À 300.6
À 280.1
À 291.6
À 911.5
À 756.0
À 849.3
À 28.9
À 54.6
À 38.5
bca(XT10)
d(GAGACCCGT)48.5
(CTCTGGGCA)d
d(A10)32.4
d(T10)
[a] Buffer conditions: 10mm Na cacodylate, 1m NaCl, pH ˆ 7.0, total
oligonucleotide concentration ˆ 5mm. [b] nd ˆ not detected.
Figure 3. CD spectra (58C)of bca(GAGACCCGT)¥ bca(ACGGGTCTC)
(solid line)and d(GAGACCCGT)¥ d(ACGGGTCTC)(dotted line.) Ex-
perimental conditions as indicated in Table 2.
Pairing properties with complementary DNA and RNA in the
mixed-base sequence series: Heterobackbone duplex forma-
tion between the mixed-sequence bca-oligonucleotides 7
9
and complementary DNA and RNA was analyzed in a similar
way by UV melting curve analysis. The corresponding
thermodynamic and Tm data are reproduced in Table 3. Bca
oligonucleotides of mixed sequence do base-pair to antipar-
allel complementary DNA, with thermal affinity reduced by
approximately 2.8 K per base pair (entries 1, 2). No duplex
formation occurs with parallel oriented DNA complements
(entry 3). In contrast to the homobackbone series (Table 2),
Figure 2. UV melting curves of the duplexes bca(GAGACCCGT)¥
bca(ACGGGTCTC)(squares)and
d(GAGACC¥CGT)
d(ACGGGTCTC)(circles.) Experimental conditions as indicated in
Table 2.
As can be seen, bicyclo[3.2.1]amide-DNA forms duplexes
not only in the homobasic A/T series, but also in a mixed-base
context. With the nonsymmetric nonamer sequences 7 9 we
could clearly show that antiparallel strand alignment (Table 2,
entry 1)leads to stable duplexes, while parallel strand align-
ment within the same sequence context failed to produce a
duplex (Table 2, entry 2). The thermal stability of bca-
duplexes is diminished by 1.0 1.8 K per base-pair, relative
to DNA. The reduced thermodynamic stability of the
duplexes is not of enthalpic, but of entropic origin, as can be
seen from the vant×Hoff transition enthalpies (DH).
From these data we conclude that elongation of the
distance between base and backbone by one atom is still
compatible with Watson Crick duplex formation in short
oligomers (ꢀ10 base-pairs). As in the case of DNA, duplex
formation is restricted to antiparallel strand alignment,
although bca-DNA shows enhanced structural flexibility in
the attachment of the bases to the backbone units. This
selectivity indicates a high degree of stereochemical control
Table 3. Tm and thermodynamic data of duplex formation for mixed bca/
DNA and bca/RNA duplexes.[a]
[b]
Entry
Tm DH
DS
[JKÀ1 molÀ1
DG298K
[kJ molÀ1
]
[kJ molÀ1
]
]
1
2
3
4
5
6
bca(GAGACCCGT)23.7
(CTCTGGGCA)d
bca(ACGGGTCTC)23.5
(TGCCCAGAG)d
bca(CTCTGGGCA)nd
d(GAGACCCGT)
bca(ACGGGTCTC)nd
(UGCCCAGAG)rD
bca(CTCTGGGCA)nd
rD(GAGACCCGU)
d(GAGACCCGT)51.1
(CTCTGGGCA)rD
À 201.3
À 198.8
À 566.6
À 557.2
À 32.4
À 32.7
À 296.1
À 799.3
À 57.8
[a] Buffer conditions: 10mm Na cacodylate, 1m NaCl, pH ˆ 7.0, total
oligonucleotide concentration ˆ 5mm [b] nd ˆ not detected.
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Bicyclo[3.2.1]amide-DNA
5312 5322
the destabilization in the bca-DNA/DNA duplexes is mostly
enthalpic in origin, most likely expressing some structural
strain within the hydrogen-bonded complex. Interestingly, no
duplex formation was observed either with parallel or with
antiparallel oriented RNA. Thus, bca-DNA shows a remark-
able selectivity towards DNA in the mixed sequence context.
We investigated the duplex structure of 8 with its DNA
complement (Table 3, entry 2)and found its CD trace again to
be enantiomorphic to that of the DNA/DNA and the DNA/
RNA duplex (Figure 4).
Figure 4. CD spectra (58C)of the duplexes bca(ACGGGTCTC)¥ d(GA-
GACCCGT)(solid line,) d(GAGACCCGT)¥ d(ACGGGTCTC)(dotted
line), and d(GAGACCCGT) ¥ rD(ACGGGUCUC)(dash-dotted line).
This behavior is opposite to that in the homobasic A/T
[22]
series, in which the CD spectra of bca(XA10)¥ d(T 10
)
and
bca(XA10) ¥ rD(XT10)(Figure 5)do not show inversion of sign
in their CD spectra although both contain a DNA or RNA
complement. It thus emerges that, in a mixed-base context,
bca-DNA can drive a complementary DNA strand into an
enantiomorphic duplex conformation.
Pairing properties of homobasic A/T bca-DNA with d- and l-
RNA: The picture becomes different in the homo-A/T
sequences. As we noted earlier, no complementary base-
pairing is observed for the bca-pyrimidine oligonucleotide 11
with DNA and RNA. Oligonucleotide 10, on the other hand,
was found to form duplexes both with complementary DNA
and with complementary RNA (poly-U).[22] The particular
observation that duplexes containing bca-DNA can adopt two
enantiomorphic forms was intriguing enough to explore
whether bca-DNA has the potential to form duplexes with
both l- and d-configured RNA. Because no stable bca-DNA/
RNA duplex occurred in the mixed-base sequence series, we
investigated these features in the homobasic A/T-sequence
series (Table 4).
Inspection of the data in Table 4 reveals that, indeed,
oligonucleotide 10 forms duplexes with both the d- and the l-
RNA complement. The difference in thermal stability is
rather small (1 K per base pair)and underlines the chiral
degeneracy of 10 in complementary strand recognition. A CD
spectroscopic investigation on the structure of the homo- and
heterochiral duplexes showed a typical right-handed Wat-
son Crick helix in the case of rD(T10)X, and a left-handed
helix in the case of rL(T10)X as the complement (Figure 5).
Figure 5. Top: UV melting curves of bca(XA10) ¥ rD(T10)X (squares) and
bca(XA10) ¥ rL(T10)X (circles); middle: CD spectra of bca(XA10) ¥ rD(T10)X;
bottom: CD spectra of bca(XA10) ¥ rL(T10)X; buffer conditions: 10 mm Na
cacodylate, 1m NaCl, pH 7.0, total oligonucleotide concentration ˆ 5 mm.
Table 4. Tm and thermodynamic data of duplex formation for bca/rD and
bca/rL and the corresponding DNA/rD and DNA/rL duplexes.[a]
[b]
Entry
Tm
DH
[kJ molÀ1
DS
[JKÀ1 molÀ1
DG298 K
[kJ molÀ1
]
]
]
D
1
2
3
4
bca(XA10) / r (T10)X 30.7 À 228.2
À 637.0
À 966.0
na[c]
À 38.3
À 28.5
na[c]
L
bca(XA10) / r(T10)X 20.1 À 316.5
D
d(A10) / r (T10)X
37.0 na[c]
X nd
L
d(A10) / r(T10)
[a] Buffer conditions: 10mm Na cacodylate, 1m NaCl, pH 7.0, total
oligonucleotide concentration ˆ 5mm. [b] nd ˆ not detected. [c] na ˆ not
analyzed.
Thus, in contrast to the mixed sequences investigated
before, bca oligonucleotide 10 can be accommodated in both a
left- and a right-handed double helix. A plot of the relative
stoichiometry of strands against absorption at constant
oligonucleotide concentration (Job plot)in the homo- and
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C. J. Leumann et al.
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À
heterochiral duplexes clearly indicated duplex formation and
ruled out triplex formation under the experimental conditions
applied (Figure 6).
O C(5')bonds. In the base linker unit, conformational
À
diversity occurs around the C N bond and (to a lesser extent)
ˆ À À
around the O C C N(base)bond. From the three possible
staggered conformations around the C N bond in a bca-
À
nucleoside monomer, it appears that the endo[3.2] conformer
(in which the carbonyl oxygen of the base-linker unit lies in-
between the three- and the two-center bridges of the bicyclo
scaffold)and the endo[3.1] conformer are slightly more stable
than the endo[2.1] conformer, by about 1 1.5 kcalmolÀ1
(Figure 7).
An extension of the endo[3.2] and the endo[3.1] conformers
to the dimer level opens the possibility of inter-residue
hydrogen-bond formation between the base-linker amide
functions of two consecutive nucleotide residues. By con-
straining the phosphodiester backbone in a B-DNA geometry,
the endo[3.1] conformer is intrinsically preorganized for right-
handed helix formation and the endo[3.2] conformer for left-
handed helix formation (Figure 8).
With this as the background we modeled the three different
antiparallel, duplexes bca(A10)¥ bca(T 10), bca(A10) ¥ rL(T10),
and bca(A10) ¥ rD(T10)(Figure 9; see Experimental Section.)
Indeed, the two left- and the right-handed duplex structures
also show uninterrupted base-pairing after unconstrained
energy minimization. All structures show a spine of hydrogen
bonds along the base-linker units in the bca-DNA strands. In
the right-handed bca(A10) ¥ rD(T10)duplex (Figure 9)the r DT
strand completely conserves its native backbone geometry,
À
while the bca-DNA strand shows the base-linker C N bonds
in the endo[3.1] conformation. The left-handed bca(A10) ¥
rL(T10)duplex (Figure 9)shows the r LT strand in an almost
perfect mirror image conformation with respect to the rDT
backbone. The bca(A10)strand almost exclusively uses the
described amide switch from the endo[3.1] to the endo[3.2]
conformation in order to accommodate itself in both helical
forms.
Most interestingly, the backbone torsion angles a z in the
bca-DNA strands are very similar in both the left- and the
right-handed duplex. The switch from the right- to the left-
handed duplex (Figure 9)is operated through sign changes of
all of the backbone torsional angles for the oligoribothymi-
Figure 6. Job plots of bca(XA10)with r D(T10)X (top), and with rL(T10)X
(bottom)in 10 m m Na cacodylate, 1m NaCl, pH 7.0 at 58C. c(tot.) ˆ 5 mm.
Interestingly, the thermodynamic data of duplex formation
show a more favorable enthalpy term in the case of the
heterochiral duplex than in that of the homochiral duplex.
Thus it appears that bca-DNA shows enthalpic advantages in
duplex formation whenever its complement intrinsically
prefers left-handedness (l-RNA, bca-DNA)and a relative
enthalpic disadvantage when the complement prefers right-
handedness (DNA, d-RNA). As expected, the free enthalpies
of duplex formation (DG)re-
flect the thermal stabilities in-
ferred from the Tm values in any
of the cases investigated (Ta-
ble 4).
Molecular modeling: The unex-
pected, free and easy, structural
accommodation of the homo-
chiral bca-DNA backbone in
both a left- and a right-handed
double-helix in the homo-A/T
series is unique, and prompted
us to search for relevant struc-
tural parameters. It is evident
that the conformational flexi-
bility of the backbone resides in
À
the two bridging P O bonds,
Figure 7. Conformational analysis of the bridgehead amide orientation in an isolated bicyclo[3.2.1]-NA
backbone unit. The bases are omitted for clarity.
À
and in the adjacent O C(3'),
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Bicyclo[3.2.1]amide-DNA
5312 5322
er the latter system may cross-
pair with enantio-RNA, as bca
oligonucleotides do, has not
been investigated so far.
Thus, it appears that elonga-
tion of the linker between the
base and a preorganized back-
bone facilitates the formation
of left-handed duplex struc-
tures and, hence, promotes the
loss of chiroselectivity. An in-
spection of available data
shows that promiscuous hetero-
chiral strand recognition oc-
curs in a number of oligonu-
cleotidic systems, but is typical-
ly much weaker than homochi-
ral strand recognition. For ex-
ample, it has been shown that
Figure 8. Representation of a bca-dinucleotide unit in both helical conformations, as determined by molecular
modeling. The bases are held in place by inter-residue hydrogen bonds.
enantiomeric homodeoxyadenylates can bind to poly(U).[24]
This, however, seems to be an exception as, in a mixed base
context, recognition of DNA and RNA by l-DNA does not
occur.[25] It has also been shown that oligo-homothymidylates
of the RNA analogue LNA can bind to complementary
l-RNA, forming enantiomorphic duplex structures.[26] The
cyclohexanyl DNA analogue CNA (the carba analogue of the
hexitol nucleic acid analogue HNA)forms heterochiral
duplexes in its own series (homo-A/homo-T strands)but not
with DNA and RNA.[5] The same feature is known for the
RNA isomer pRNA, which can form heterochiral duplexes
within its backbone type in a non-natural base-context,[27] but
typically has a strong preference for homochiral duplex
formation.[28]
dine strand, but only very subtle (Æ258)changes of the angles
a and z for the bicyclo[3.2.1]amide backbone (Table 5).
Remarkably, the geometries are very consistent for the
modified backbone strands when proceeding from the hetero-
to the homoduplex.
Bca strands use the intermediate amide function as a relay
to switch between a right- and a left-handed conformation.
This relay leads to almost superimposable sugar units but
enantiomorphically arranged bases, as can be seen from
Figure 10, which displays a bca-A unit out of a left- and a
right-handed double helix.
Inspection of the backbone torsion angles of bca strands in
left- and right-handed duplexes, as obtained from molecular
modeling, highlights the relative ease with which the helicity
of a duplex can be inverted by minor structural changes in the
phosphodiester backbone. While torsion angles g and d are
invariant for obvious reasons, changes of torsion angles a and
z of less than 258 can induce inversion of handedness of the
backbone. The attachment of the bases to the backbone
through a cyclic structural element (such as the ribofuranose
unit of DNA and RNA)seems to be a measure to increase
enantiodifferentiation in duplex formation, as it constrains the
attainable conformational space of the nucleobases and
reduces the possibility of enantiomorphic arrangements of
the bases in a manner such as displayed in Figure 10.
There is a pronounced difference in the thermal stabilities
of duplexes of homobasic against mixed-base bicyclo[3.2.1]-
amide-DNA. In the mixed-base context, bca/bca duplexes are
more stable than bca/DNA duplexes, while bca/RNA duplex
formation has not been observed. In the homobasic (A/T)
context, only the purine bca oligonucleotides form duplexes
with complementary DNA and RNA with thermal stabilities
relatively similar to those of the duplex consisting of two bca
strands. Whatever the molecular basis of this differential
behavior might be, triple-helix formation in the homopurine/
homopyrimidine system can be excluded as a potential cause,
from the corresponding Job plots (Figure 6).
Discussion
Besides bicyclo[3.2.1]-DNA (Figure 1),[14 16] there were only a
few other known DNA analogues in which the bases are
linked to a phosphodiester backbone through a non-cyclic
unit. These are glycerol-DNA,[11] the seco-DNAs,[9, 10] and
inverse DNA.[23] While fully modified single strands of the
former two systems are known to be incompetent in duplex
formation, no data on fully modified strands are available in
the case of inverse DNA. With respect to bicyclo[3.2.1]-DNA,
in which the number of bonds (three)between the base unit
and the backbone is identical to that in DNA, the following
common properties were found for bicyclo[3.2.1]amide-DNA,
in which the base is more distant by one bond (four in total)
from the backbone. Both systems base-pair to themselves and
to complementary DNA with reduced affinity relative to
DNA. Both systems show strong preferences for antiparallel
strand alignment in duplexes and show strong discrimination
of base-mismatch formation. Two evident differences are the
complementary base-pairing with RNA and the structural
differences as observed by CD spectroscopy. In a mixed-base
sequence context, bca oligonucleotides show highly reduced
affinity for complementary RNA, which is not the case for the
bicyclo[3.2.1]-DNA. In contrast to bca oligonucleotides, there
were no signs of left-handed duplex formation by bicy-
clo[3.2.1]-oligonucleotides with either DNA or RNA. Wheth-
Chem. Eur. J. 2002, 8, No. 23
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5317

C. J. Leumann et al.
FULL PAPER
Figure 9. Stereoview of duplexes perpendicular and parallel to the helix axis. Top: right-handed bca(A10) ¥ rD(T10); middle: left-handed bca(A10) ¥ rL(T10);
bottom: left-handed bca(A10)¥ bca(T 10).
Table 5. Backbone torsional angles[a] [8] for the three duplexes indicated,
obtained from molecular modeling.
Bicyclo[3.2.1]amide-DNA has some structural similarity
31]
with the polyamide nucleic acid analogue PNA,[29
as it
Torsion angle bca(A10)¥ bca(T 10)bca(A 10) ¥ rD(T10)bca(A 10) ¥ rL(T10)
shares the same base linker unit with PNA. While it is evident
that PNA, due to its achiral nature, can be accommodated in
right- and left-handed double helices, it appears that PNA is
somewhat less selective, in that antiparallel and parallel
duplex formation with complementary DNA and RNA can be
observed, this in spite of the fact that there is no change in the
backbone-to-base distance (three bonds)relative to DNA. In
terms of antisense applications, however, bicyclo[3.2.1]amide-
DNA is not competitive relative to PNA as it discriminates
RNA binding and shows overall reduced affinity to DNA.
The linker amide functions in bca oligonucleotides offer the
possibility of inter-residue hydrogen-bond formation. During
molecular modeling (Figure 8, Figure 9)such hydrogen bonds
A strand
T strand
a
b
g
d
e
À 37 (Àsc)
‡ 160 (‡ap)
‡ 68 (‡sc)
À 10 (Àsc)
‡ 162 (‡ap)
‡ 68 (‡sc)
À 35 (Àsc)
‡ 159 (‡ap)
‡ 68 (‡sc)
‡ 157 (‡ap)
‡ 126 (‡ac)
À 145 (Àac)
‡ 157 (‡ap)
‡ 127 (‡ac)
À 134 (Àac)
‡ 157 (‡ap)
‡ 124 (‡ac)
À 146 (Àac)
z
a
b
g
d
e
À 36 (Àsc)
‡ 159 (‡ap)
‡ 68 (‡sc)
À 61 (Àsc)
‡ 158 (‡ap)
‡ 60 (‡sc)
‡ 86 (‡sc)
À 159 (Àap)
À 79 (Àsc)
‡ 63 (‡sc)
À 159 (Àap)
À 61 (Àsc)
À 85 (Àsc)
‡ 160 (‡ap)
‡ 76 (‡sc)
‡ 157 (‡ap)
‡ 127 (‡ac)
À 146 (Àac)
z
[a] sc ˆ synclinal, ac ˆ anticlinal, ap ˆ antiperiplanar.
5318
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Chem. Eur. J. 2002, 8, No. 23

Bicyclo[3.2.1]amide-DNA
5312 5322
SIL-G-25 UV254 (Machery Nagel). Visualization by UV and/or by
dipping into
a solution of Ce(SO4)2 (10.5 g), phosphomolybdic acid
(21 g), H2SO4 (60 mL), and H2O (900 mL). Flash chromatography: silica
gel 60 (230 400 mesh).
Compound 3a: O-[(Ethoxycarbonyl)-cyanomethylene-amino]-N,N,N',N'-
tetramethyluronium tetrafluoroborate (TOTU)(36 mg, 0.11 mmol)was
added at room temperature to a solution of 1 (30 mg, 0.11 mmol), (iPr)2NEt
(0.1 mL), and 2a (36 mg, 0.11 mmol)in DMF (5 mL.) After stirring
overnight the mixture was concentrated (HV, 408C)and extracted (AcOEt/
sat. NaHCO3). Purification by flash chromatography (AcOEt) gave 3a
(56 mg, 88%)as a white solid. Rf ˆ 0.61 (EtOAc); 1H NMR (300 MHz,
[D]chloroform, 258C): d ˆ 0.07, 0.11 (2s, 6H; (CH3)2Si), 0.88 (s, 9H;
(CH3)3C), 1.36 (s, 9H; (CH3)3CPh), 1.64 (m, 1H; C(7)H), 1.84 (m, 1H;
C(4)H), 2.42 (dd, 3J(H,H) ˆ 12.5, 3.7 Hz, 1H; C(4)H), 2.59 (ddd,
3
3J(H,H) ˆ 15.8, 10.7, 5.9 Hz, 1H; C(7)H), 3.65 (dd, J(H,H) ˆ 11.8, 6.6 Hz,
1H; C(3)H), 3.83 (s, 1H; C(8)H), 4.04 4.16 (m, 1H; C(1)H, 1H; C(3)H),
4.36, 4.62 (2d, 3J(H,H) ˆ 14.3 Hz, 2H; CH2), 4.45 4.48 (m, 1H; C(6)H),
4.64 (s, 1H; C(6)OH), 6.91 (s, 1H; NH), 7.53 (d, 3J(H,H) ˆ 8.5 Hz, 2H;
3
3
ArH), 7.65 (d, J(H,H) ˆ 7.4 Hz, 1H; ArH), 7.73 (d, J(H,H) ˆ 7.4 Hz, 1H;
ArH), 7.81 (d, 3J(H,H) ˆ 8.5Hz, 2H; ArH), 8.64 ppm (s, 1H; NH);
13C NMR (75 MHz, [D]chloroform, 258C): d ˆ À5.05, À4.48 (2q), 17.80
(s), 25.58, 31.02 (2d), 31.58, 34.76 (2t), 35.16, 38.57 (2s), 53.50, 59.29 (2t),
66.53 (s), 75.41, 78.56, 80.86, 97.36 (4d), 126.06, 127.50 (2q), 129.73 (s),
149.19 (d), 157.33, 163.01, 168.01 ppm (3s); HRMS, ESI-MS calcd for
C30H44N4O6Si: 583.2958; found: 583.2971 [MÀ À H].
Compound 3b: This compound was prepared as described for 3a, from 1
(192 mg, 0.71 mmol), 2b (238 mg, 0.44 mmol), and TOTU (258 mg,
0.78 mmol)in DMF (5 mL). Compound 3b (390 mg, 81%)was obtained
after flash chromatography (AcOEt ! AcOEt/MeOH 9:1)as a white solid.
This material was used without characterization for the next step.
Figure 10. Representation of a bca-A unit in the right- (top)and left-
handed (bottom)helical conformation. While the backbone bonds of the
two units are almost superimposable, the bases adopt enantiomorphic
positions.
were spontaneously formed in both the right- and the left-
handed duplex structures. In principle, such a hydrogen-bond
network between consecutive base elements could ideally add
to the preorganization of single strands for duplex formation.
An NMR structural investigation of a bca-(AT)dimer,
however, has not so far provided compelling evidence that
such a hydrogen-bonding network exists in solution (data not
shown). A definitive answer to this question requires a
detailed structural analysis of a stable duplex. As a note of
caution we add that intra- and inter-residue hydrogen-bond
formation in PNA, involving the base-linker amide function,
has been proposed on the basis of molecular modeling,[32, 33]
but has never been observed in high-resolution structures of
PNA duplexes and triplexes.[34, 35]
Previous studies on DNA analogues have shown that the
number of bonds in the repetitive DNA backbone can be
varied to five[12, 36] or seven[7] without breakdown of comple-
mentary recognition of RNA and DNA. Bicyclo[3.2.1]amide-
DNA is the first analogue to demonstrate that the distance
between the base and the backbone can also be extended from
three to four bonds without breakdown of complementary
Watson Crick base-pairing.
Compound 3c: This compound was prepared as described for 3a, from 1
(195 mg, 0.71 mmol), 2c (270 mg, 0.99 mmol), and TOTU (270 mg,
0.82 mmol)in DMF (10 mL). Compound 3c (227 mg, 60%)was obtained
after flash chromatography (MeOH/CH2Cl2 1:9)as a white solid. Rf ˆ 0.34
1
(MeOH/CH2Cl2 ˆ 1:9); H NMR (300 MHz, [D6]DMSO, 258C): d ˆ À0.11,
À0.03 (2s, 6H; (CH3)2Si), 0.82 (s, 9H; (CH3)3Si), 1.10 (d, 3J(H,H) ˆ 6.6 Hz,
6H; 2CH3,), 1.49 (dd, 3J(H,H) ˆ 16.1, 1.8 Hz, 1H; C(7)H), 1.63 (dd,
3J(H,H) ˆ 12.5, 3.3 Hz, 1H; C(4)H), 2.28 (m, 1H; C(4)H, 1H; C(7)H), 2.76
(sept, 3J(H,H) ˆ 7.0 Hz, 1H; CH), 3.51 (dd, 3J(H,H) ˆ 11.0, 6.6 Hz, 1H;
3
(3)H), 3.78 (d, J(H,H) ˆ 5.5 Hz, 1H; C(1)H), 4.18 (s, 1H; C(8)H), 4.20
4.24 (m, 1H; C(6)H), 4.73 (s, 2H; CH2CO), 4.98 (d, 3J(H,H) ˆ 4.1, 1H;
C(6)OH), 7.75 (s, 1H; NH), 8.16 (s, 1H; CH), 11.62, 12.05 ppm (2s, 2H;
NH); 13C NMR (75 MHz, [D6]DMSO, 258C): d ˆ À4.90, À4.88 (2q), 17.69
(s), 18.95, 19.20, 25.75 (3q), 30.21, 34.54 (2t), 34.86 (d), 45.50, 58.79 (2t),
65.14 (s), 73.86, 78.03, 78.40 (3d), 119.69 (s), 140.79 (d), 148.03, 149.18,
155.06, 165.97, 180.28 ppm (5s); HRMS, LSI-MS calcd for C24H39N6O6Si:
‡
535.2697; found: 535.2700 [M ‡H].
Compound 3d: This compound was prepared as described for 3a, from 1
(100 mg, 0.36 mmol), 2d (84 mg, 0.46 mmol), and TOTU (132 mg,
0.40 mmol)in DMF (5 mL). Compound 3d (128 mg, 76%)was obtained
after flash chromatography (AcOEt ! AcOEt/MeOH 9:1), as a white
solid. Rf 0.18 (AcOEt/CHCl3 9:1); [a]2D5 ˆ À16.40 (c ˆ 0.5 in CHCl3);
1H NMR (300 MHz, [D6]DMSO, 258C): d ˆ À0.03, À0.01 (2s, 6H;
(CH3)2Si), 0.81 (s, 9H; (CH3)3C), 1.49 (dd, 3J(H,H) ˆ 15.1, 1.8 Hz, 1H;
C(7)H), 1.66 (dd, 3J(H,H) ˆ 12.5, 3.5 Hz, 1H; C(4)H), 1.73 (d, 4J(H,H) ˆ
3
1.1 Hz, 3H; CH3), 2.21 (dt, J(H,H) ˆ 12.1, 7.0 Hz, 1H; C(4)H), 2.31 (ddd,
3
3J(H,H) ˆ 16.2, 10.3, 5.9 Hz, 1H; C(7)H), 3.51 (dd, J(H,H) ˆ 11.4, 7.0 Hz,
1H; C(3)H), 3.78 (dt, 3J(H,H) ˆ 12.1, 3.7 Hz, 1H; C(3)H), 3.86 (dd,
3J(H,H) ˆ 5.5, 1.1 Hz, 1H; C(1)H), 4.13 (s, 1H; C(8)H), 4.19 4.24 (m, 1H;
C(6)H), 4.24, 4.30 (2d, 3J(H,H) ˆ 16.2 Hz, 2H; CH2CO), 4.93 (d,
3J(H,H) ˆ 3.3 Hz, 1H; C(6)OH), 7.30 (s, 1H; NH), 8.06 (s, 1H; CH),
11.23 ppm (s, 1H; NH); 13C NMR (75 MHz, [D]chloroform, 258C): À5.08,
À4.48, 12.42 (3q), 17.80 (s), 25.53 (q), 31.96, 34.78, 51.29, 59.20 (4t), 66.67
(s), 75.13, 78.41, 81.05 (3d), 111.4 (s), 140.79 (d), 151.07, 164.34, 168.32 ppm
Experimental Section
General: Reactions were carried out under Ar in distilled, anhydrous
solvents. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite was from
Sigma and 4,4'-(dimethoxytriphenyl)methyl triflate (DMT-OTf) was pre-
pared as described.[37] All other reagents were from Fluka (highest quality
available). All NMR spectra were measured at room temperature. 1H NMR
spectra: d in ppm relative to solvent ([D]chloroform ˆ 7.24, [D6]DMSO ˆ
2.49, or [D6]benzene ˆ 7.20), J in Hz. 13C NMR spectra: d in ppm relative to
solvent ([D]chloroform ˆ 77.00, [D6]DMSO ˆ 39.70, or [D6]benzene ˆ
128.00), multiplicities from DEPT spectra. 31P NMR spectra: d in ppm
relative to 85% H3PO4 as external standard). ESI-MS: VG platform Fisons
instruments. LSI-MS: Micromass Autospec Q VG. TLC: pre-coated plates
(3s); HRMS, LSI-MS calcd for C20H34N3O6Si: 440.2217; found: 440.2227
‡
[M ‡H]; elemental analysis calcd (%)for
C 20H33O6SiN3 ¥ 1.25H2O
(462.10): C 51.98, H 7.43, N 9.09; found C 51.89, H 7.19, N 8.80.
Compound 4a: DMT-OTf (154 mg, 0.34 mmol)was added at room
temperature to
a solution of 3a (100 mg, 0.17 mmol)in pyridine
(0.9 mL). The reaction mixture was stirred at 608C. An additional 1 equiv
Chem. Eur. J. 2002, 8, No. 23
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5319

C. J. Leumann et al.
FULL PAPER
(76 mg, 0.17 mmol)of DMT-OTf was added in two portions over 3 h. After
5 h the mixture was extracted (AcOEt/sat. NaHCO3), the organic phase
was evaporated, and the residue was purified by flash chromatography
(EtOAc/hexane 1:2 ! 1:1)to give 4a (131 mg, 87%)as a pale yellow solid.
Rf ˆ 0.30 (AcOEt/hexane 1:2, silica gel pre-deactivated by 1% TEA in
(2d, 3J(H,H) ˆ 15.8 Hz, 2H; CH2), 3.90 (dd, 3J(H,H) ˆ 11.4, 6.6 Hz, 1H;
C(3)H), 4.10 (d, 3J(H,H) ˆ 4.0 Hz, 1H; C(1)H), 4.36 (dd, 3J(H,H) ˆ 9.9,
3
3.7 Hz, 1H; C(6)H), 4.41 (dt, J(H,H) ˆ 11.8, 3.7 Hz, 1H; C(3)H), 4.78 (s,
1H; C(8)H), 5.30 (s, 1H; NH), 6.42 (s, 1H; CH), 6.88 6.98 (m, 4H; ArH),
7.16 7.31 (m, 3H; ArH) 7.54 7.59 (m, 4H; ArH), 7.68 7.71 (m, 2H; ArH),
10.61 ppm (s, 1H; NH); 13C NMR (75 MHz, [D6]benzene, 258C): d ˆ
À4.94, 12.28 (2q), 17.90 (s), 25.78 (q), 31.23, 34.15, 49.98 (3t), 54.91, 54.99
(2q), 59.87 (t), 66.50 (s), 77.29, 78.25, 78.93 (3d), 87.40, 110.24 (2s), 113.70,
113.89, 127.35, 128.75, 131.07, 131.30 (6d), 136.58, 137.15 (2s), 140.64 (d),
146.66, 151.39, 159.48, 159.49, 164.22, 165.68 ppm (6s); FAB-MS: 780.20
1
hexane); H NMR (300 MHz, [D6]benzene, 258C): d ˆ À0.10, 0.02 (2s, 6H;
(CH3)2Si), 0.81 (s, 9H; (CH3)3C), 1.19 (s, 9H; (CH3)3CPh), 1.58 (d,
3J(H,H) ˆ 15.1 Hz, 1H; C(7)H), 1.81 (ddd, 3J(H,H) ˆ 15.1, 10.0, 5.9 Hz,
1H; C(7)H), 2.30 (m, 1H; C(4)H), 3.04 (dt, 3J(H,H) ˆ 12.5, 7.3 Hz, 1H;
C(4)H), 3.48, 3.44 (2s, 6H; 2CH3), 3.90 (dd, 3J(H,H) ˆ 11.0, 7.0 Hz, 1H;
3
‡
C(3)H), 4.01 (d, J(H,H) ˆ 5.1 Hz, 1H; C(1)H), 4.13, 4.37 (2d, 3J(H,H) ˆ
[M ‡K].
14.7 Hz, 2H; CH2), 4.32 (m, 1H; C(6)H), 4.43 (m, 1H; C(3)H), 4.70 (s, 1H;
C(8)H), 5.99 (s, 1H; NH), 6.91 (m, 4H; ArH), 7.14 (d, 3J(H,H) ˆ 7.3 Hz,
2H; ArH), 7.31 (m, 4H; ArH), 7.42 (d, 3J(H,H) ˆ 7.2 Hz, 2H; ArH), 7.56
(m, 5H; ArH), 7.72 (d, 3J(H,H) ˆ 7.7Hz, 2H; ArH), 8.06 (d, 3J(H,H) ˆ
7.7 Hz, 2H; ArH), 9.68 ppm (s, 1H; NH); 13C NMR (75 MHz, [D6]benzene,
258C): d ˆ À4.94, À4.83 (2q), 17.90 (s), 25.87, 31.02 (2q), 31.19, 34.02 (2t),
34.90 (s), 52.86 (t), 54.88, 54.95 (2q), 59.98 (t), 66.58 (s), 77.23, 78.38, 78.97
(3d), 87.31 (s), 113.77, 113.94, 125.87, 127.29, 128.34, 128.81, 131.04, 131.25
(8d), 136.75, 137.12, 146.75 (3s), 150.05 (d), 156.18, 159.40, 159.42, 163.54,
166.21 ppm (5s); HRMS, LSI-MS calcd for C51H62N4O8Si: 887.4422; found:
Compound 5a: Bu4NF (24 mg, 0.076 mmol)was added at room temper-
ature to a solution of 4a (34 mg, 0.038 mmol)in THF (1 mL,) and the
mixture was stirred overnight. After evaporation of the solvent the crude
product was purified by flash chromatography (CH2Cl2/MeOH ˆ 10:1)to
give 5a (23 mg, 81%)as a pale yellow solid. Rf ˆ 0.24 (AcOEt, silica gel
1
pre-deactivated by 1% TEA in hexane); H NMR (300 MHz, [D6]benzene,
258C): d ˆ 1.18 (s, 9H; (CH3)3CPh), 1.39 (m, 1H; C(7)H), 2.08 (m, 1H;
C(7)H), 2.41 (s, 1H; C(8)OH), 2.44 (m, 1H; C(4)H), 2.84 (m, 1H; C(4)H),
3.42, 3.45 (2s, 6H; 2CH3), 3.89 (m, 1H; C(3)H), 4.21 (d, 1H; 3J(H,H) ˆ
4.8 Hz, C(1)H), 4.30 4.42 (m, 1H; one of CH2CO, 1H; C(6)H, 1H;
C(3)H), 4.60 (s, 1H; C(8)H), 4.77 (d, 3J(H,H) ˆ 7.7 Hz, 1H; one of
CH2CO), 5.78 (s, 1H; NH), 6.60 (br, 1H; ArH), 6.85 (m, 4H; ArH), 7.14 (m,
1H; ArH), 7.30 (m, 5H; ArH), 7.55 (m, 4H; ArH), 7.71 (d, 3J(H,H) ˆ
‡
887.4415 [M ‡H].
Compound 4b: This compound was prepared as described for 4a, from 3b
(390 mg, 0.71 mmol)and DMT-OTf (930 mg, 2.02 mmol)in pyridine
(4 mL)overnight. Compound 4b (415 mg, 69%)was obtained as a white
solid after flash chromatography (AcOEt/CH2Cl2 9:1). Rf ˆ 0.34 (AcOEt/
CH2Cl2 9:1); [a]2D5 ˆ ‡10.43 (c ˆ 0.58 in CHCl3); 1H NMR (300 MHz,
[D]chloroform, 258C): d ˆ À0.26, À0.12 (2s, 6H; (CH3)2Si), 0.67 (s, 9H;
(CH3)3C), 1.48 (d, 3J(H,H) ˆ 15.4 Hz, 1H; C(7)H), 1.75 (ddd, 3J(H,H) ˆ
15.8, 9.1, 5.1 Hz, 1H; C(7)H), 2.04 (dd, 3J(H,H) ˆ 12.9, 3.7 Hz, 1H; C(4)H),
2.64 (dt, 3J(H,H) ˆ 12.9, 7.4 Hz, 1H; C(4)H), 3.81 (brs, 6H; 2CH3, 1H;
C(3)H), 4.13 4.22 (m, 1H; C(3)H), 4.3 (s, 1H; C(8)H), 4.37, 4.54 (2d,
3J(H,H) ˆ 16.2 Hz, 2H; CH2), 5.10 (s, 1H; NH), 6.88 6.93 (m, 4H; ArH),
7.26 7.61 (m, 12H; ArH), 7.98 8.01 (m, 2H; ArH), 8.07, 8.69 (2s, 2H;
2CH), 9.06 ppm (brs, 1H; NH); 13C NMR (75 MHz, [D]chloroform, 258C):
d ˆ À5.31, À4.94 (2q), 17.56 (s), 25.42 (q) 30.45, 33.69, 45.83 (3t), 55.22,
55.26 (2q), 59.60 (t), 66.29 (s), 76.39, 77.49, 78.50 (3d), 86.82 (2s), 113.35,
113.41 (2d), 122.15 (s), 127.21, 127.7, 128.02, 128.33, 128.75, 130.54, 130.65,
132.62 (8d), 133.71, 136.10, 136.51 (3s), 143.62(d), 145.54, 149.37, 151.86,
152.49, 158.84, 158.87, 164.03 ppm (7s); HRMS, LSI-MS calcd for
3
7.4 Hz, 2H; ArH), 8.12 (d, J(H,H) ˆ 7.0 Hz, 2H; ArH), 9.89 ppm (s, 1H;
NH); 13C NMR (75 MHz, [D6]benzene, 258C): d ˆ 31.03 (q), 32.11 (t), 34.91
(s), 35.07, 53.05 (2t), 54.90, 55.00 (2q), 60.00 (t), 68.09 (s), 77.06, 77.75, 78.45
(3d), 87.09 (s), 113.75, 113.84, 125.81, 127.20, 128.29, 128.92, 131.16, 131.26
(8d), 137.08, 137.38, 146.75, 146.93 (4s), 150.10 (d), 156.23, 159.32, 159.34,
163.69, 167.50 ppm (5s); HRMS, LSI-MS calcd for C45H48N4O8: 773.3535;
‡
found: 773.3536 [M ‡H].
Compound 5b: This compound was prepared as described for 5a, from 4b
(415 mg, 0.49 mmol)and Bu 4NF (296 mg, 0.95 mmol)in THF (3 mL.)
Compound 5b (314 mg, 87%)was obtained after flash chromatography
(Et2O/MeOH 9:1 ! 8:2)as a white solid. Rf 0.5 (Et2O/MeOH 8:2); [a]D25
ˆ
‡1.32 (c ˆ 0.76 in CHCl3); 1H NMR (300 MHz, [D6]DMSO, 258C): d ˆ
0.57 (d, 3J(H,H) ˆ 16.6 Hz, 1H; C(7)H), 1.37 (ddd, 3J(H,H) ˆ 15.3, 8.8,
6.4 Hz, 1H; C(7)H), 2.18 2.30 (m, 1H; C(4)H), 2.44 2.49 (m, 1H;
C(4)H), 3.61 3.63 (m, 1H; C(1)H, 1H; C(8)H), 3.69 3.75 (m, 6H; CH3,
1H; C(3)H), 3.88 (dt, 3J(H,H) ˆ 12.0, 3.6 Hz, 1H; C(3)H), 4.26 (dd,
‡
3
3J(H,H) ˆ 9.4, 2.8 Hz, 1H; C(6)H), 5.04, 5.11 (2d, J(H,H) ˆ 17.1 Hz, 2H;
C48H55O7N6Si: 855.3902; found: 855.3908 [M ‡H].
3
CH2), 5.59 (d, J(H,H) ˆ 2.2 Hz, 1H; C(8)OH), 6.93 7.00 (m, 4H; ArH),
Compound 4c: This compound was prepared as described for 4a, from 3c
(235 mg, 0.44 mmol)and DMT-OTf (600 mg, 1.32 mmol)in pyridine
(2.3 mL)overnight. Compound 4c (202 mg, 55%)was obtained after flash
chromatography (MeOH/EtOAc 1:9)as a pale yellow solid. Rf ˆ 0.70
(MeOH/CH2Cl2 ˆ 1:9, silica gel pre-deactivated by 1% TEA in hexane);
1H NMR (300 MHz, [D6]benzene, 258C): d ˆ À0.10, À0.01 (2s, 6H;
(CH3)2Si), 0.82 (s, 9H; (CH3)3Si), 1.19 (d, 3J(H,H) ˆ 6.6 Hz, 6H; 2CH3),
1.62 (m, 1H; C(7)H), 1.86 (m, 1H; C(7)H), 2.42 (m, 1H; C(4)H), 2.91 (sept,
3J(H,H) ˆ 6.9 Hz, 1H; 2CH3), 3.00 (m, 1H; (4)H) 3.54, 3.61 (2s, 6H;
2CH3), 3.93 (dd, 3J(H,H) ˆ 7.3, 7.0 Hz, 1H; C(3)H), 4.01 (d, 3J(H,H) ˆ
5.2 Hz, 1H; C(1)H), 4.40 4.52 (m, 1H; C(6)H, 1H; C(3)H), 4.60 (s, 1H;
C(8)H), 4.76 4.90 (2d, 3J(H,H) ˆ 16.5 Hz, 2H; CH2CO), 5.83 (s, 1H; NH),
6.97 (m, 4H; ArH), 7.24 (d, 3J(H,H) ˆ 7.3 Hz, 1H; ArH), 7.36 (t, 3J(H,H) ˆ
7.22 7.67 (m, 12H; ArH), 7.85 (s, 1H; NH), 8.04 8.06 (m, 2H; ArH), 8.34,
8.72 (2s, 2H; 2CH), 11.17 ppm (s, 1H; NH); 13C NMR (75 MHz,
[D]chloroform, 258C): d ˆ 31.58, 34.34, 45.92 (3t), 55.21 55.23 (2q), 59.54
(t), 67.44 (s), 76.46, 76.98, 77.80 (3d), 86.63 (s), 113.34, 113.38 (2d), 121.94
(s), 127.08, 127.88, 127.99, 128.30, 128.59, 130.54, 132.64 (7d), 133.32, 136.38,
136.61 (3s), 144.04 (d), 145.68, 149.16, 151.70 (3s), 152.23 (d), 158.71, 158.77,
164.93, 165.21 ppm (4s); HRMS, LSI-MS calcd for C42H41O7N6: 741.3036;
‡
found: 741.3037 [M ‡H].
Compound 5c: This compound was prepared as described for 5a, from 4c
(200 mg, 0.24 mmol)and Bu 4NF (150 mg, 0.48 mmol)in THF (1 mL.)
Compound 5c (124 mg, 72%)was obtained after flash chromatography
(MeOH/CH2Cl2 ˆ 1:19 ! 1:9)as a white solid. Rf ˆ 0.50 (MeOH/CH2Cl2 ˆ
3
1
7.3 Hz, 2H; ArH), 7.60 (t, J(H,H) ˆ 8.8 Hz, 4H; ArH), 8.00 (s, 1H; CH),
1:9, silica gel pre-deactivated by 1% TEA in hexane); H NMR (300 MHz,
10.54, 12.56 ppm (2s, 2H; NH); 13C NMR (75 MHz, [D6]benzene, 258C):
d ˆ À4.98, À4.86 (2q), 17.87 (s), 19.02, 19.20, 25.77 (3q), 31.90, 34.20 (2t),
36.08 (d), 46.49 (t), 55.06, 55.21 (2q), 60.03 (t), 66.79 (s), 77.68, 77.90, 78.87
(3d), 87.32 (s), 113.91, 113.79 (2d), 120.10 (s), 127.41, 128.33, 128.80, 131.02,
131.28 (5d), 136.81, 137.33 (2s), 141.03 (d), 146.75, 148.61, 149.75, 156.30,
159.37, 159.40, 165.62, 180.29 ppm (8s); HRMS, ESI-MS calcd for
C45H55N6O8Si: 835.3856; found 835.3838 [MÀ À H].
[D]chloroform, 258C): d ˆ 1.16, 1.19 (2d, 3J(H,H) ˆ 5.49 Hz, 6H; 2CH3),
1.61 (m, 1H; C(7)H), 2.20 (m, 1H; C(7)H), 2.37 (m, 1H; C(4)H), 2.65 (t,
1H; 3J(H,H) ˆ 6.2 Hz, C(4)H), 3.10 (m, 1H; CH), 3.75 (s, 6H; 2CH3),
3.78 4.09 (m, 2H; C(3)H, 1H; C(6)H, 1H; C(1)H), 4.57 (d, 3J(H,H) ˆ
7.7 Hz, 1H; C(8)H), 4.76 (2d, 3J(H,H) ˆ 22.5 Hz, 2H; CH2CO), 6.83 (m,
4H; ArH), 7.15 7.56 (m, 1H; NH, 9H; ArH), 7.63 (s, 1H; CH), 10.19,
12.12 ppm (2s, 2H; NH); 13C NMR (75 MHz, [D]chloroform, 258C): d ˆ
18.78, 19.00 (2q), 32.07, 34.15 (2t), 36.17 (d), 45.99 (t), 55.27, 55.33 (2q),
59.54 (t), 68.01 (s), 74.54, 77.53, 78.75 (3d), 86.34 (s), 113.29, 113.39 (2d),
119.48 (s), 126.90, 127.84, 128.38, 130.39, 130.74 (5d), 136.37, 136.99 (2s),
140.53 (d), 145.83, 147.94, 149.01, 155.77, 158.47, 158.62, 167.38, 179.58 ppm
(8s); HRMS, ESI-MS calcd for C39H41N6O8: 721.2991; found: 721.3002
[MÀ À H].
Compound 4d: This compound was prepared as described for 4a, from 3d
(84 mg, 0.19 mmol)and DMT-OTf (240 mg, 0.53 mmol)in pyridine
(0.9 mL)overnight. Compound 4d (110 mg, 78%)was obtained after flash
chromatography (Et2O/MeOH 9:1)as a white solid. Rf ˆ 0.19 (CHCl3);
[a]2D5 ˆ ‡15.47 (c ˆ 1.06 in CHCl3); 1H NMR (300 MHz, [D6]benzene,
258C): d ˆ À0.10, 0.01 (2s, 3H; CH3Si), 0.81 (s, 9H; (CH3)3C), 1.73 (s, 3H;
CH3), 1.73 (d, 3J(H,H) ˆ 9.9 Hz, 1H; C(7)H), 1.96 (ddd, 3J(H,H) ˆ 15.5, 9.9,
5.5 Hz, 1H; C(7)H), 2.22 (dd, 3J(H,H) ˆ 12.5, 3.7 Hz, 1H; C(4)H), 3.04 (dt,
3J(H,H) ˆ 12.9, 7.0 Hz, 1H; C(4)H), 3.47, 3.52 (2s, 6H; 2CH3), 3.46, 3.85
Compound 5d: This compound was prepared as described for 5a, from 4d
(110 mg, 0.14 mmol)and Bu 4NF (100 mg, 0.32 mmol)in THF (1 mL.)
Compound 5d (87 mg, 99%)was obtained after flash chromatography
5320
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0823-5320 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 23

Bicyclo[3.2.1]amide-DNA
5312 5322
(AcOEt)as a white solid. Rf ˆ 0.22 (Et2O/MeOH 9.5:0.5); 1H NMR
(400 MHz, [D6]DMSO, 258C): d ˆ 0.53 (d, 3J(H,H) ˆ 16.2 Hz, 1H; C(7)H),
1.36 (ddd, 3J(H,H) ˆ 15.7, 9.8, 5.9 Hz, 1H; C(7)H), 1.75 (d, 4J(H,H) ˆ
1.2 Hz, 3H; CH3), 2.13 (dt, 3J(H,H) ˆ 12.6, 7.0 Hz, 1H; C(4)H), 2.42 (dd,
[D6]benzene, 258C): d ˆ 0.85 1.17 (m, 12H; 4CH3, 6H; 2CH3), 1.23 1.34
(m, 1H; C(7)H), 1.80 2.00 (m, 2H; CH2CN), 2.32 (t, 3J(H,H) ˆ 7.0 Hz,
1H; C(7)H), 2.49 2.55 (m, 1H; C(4)H), 2.60 2.83 (m, 1H; C(4)H), 3.08
3.38 (m, 2H; CH2, 2H; 2CH), 3.50, 3.52, 3.57, 3.59 (4s, 6H; 2CH3), 3.68 (m,
1H; C(6)H), 3.96 (m, 1H; C(3)H), 4.19 (d, 3J(H,H) ˆ 5.21 Hz, 1H; C(4)H),
4.40 4.50 (m, 1H; C(3)H), 4.60 4.75 (m, 2H; CH2CO), 4.91, 5.02 (2s, 1H;
C(8)H), 6.33, 6.62 (2s, 1H; NH), 6.96 (m, 4H; ArH), 7.61 7.79 (m, 7H;
ArH), 7.97, 8.03 (2s, 1H; CH), 10.14, 12.34 ppm (br, 2H; NH); 13C NMR
(75 MHz, [D]chloroform, 258C): d ˆ 18.81, 18.89, 18.94, 19.37 (4q), 20.30,
20.38, 20.45, 20.53 (4t), 24.18, 24.28, 24.38, 24.44, 24.48, 24.55 (6q), 26.86,
29.00, 29.63, 30.79, 31.52, 33.59, 33.93, 34.61 (8t), 36.02, 36.17, 36.22, 41.29,
42.88, 42.98, 43.05, 43.14 (8d), 46.03, 46.25 (2t), 55.30, 55.36 (2q), 58.29,
58.56, 59.67, 59.82 (4t), 66.01, 66.08 (2s), 77.59, 77.73, 77.99, 78.18 (4d), 86.72
(s), 113.38, 113.46 (2d), 118.41, 120.37 (2s), 127.12, 127.21, 127.98, 128.22,
128.29, 130.37, 130.58, 130.76 (8d), 135.99, 136.12, 136.77, 136.86 (4s),
139.39, 139.62 (2d), 145.61, 145.69, 147.66, 148.47, 148.58, 155.48, 155.53,
158.68, 158.80, 158.83, 164.88, 165.28, 178.64, 178.72 ppm (14s); 31P NMR
(161.9 MHz, [D6]benzene, 258C): d ˆ 148.09, 149.70 ppm; HRMS, MS-ESI
calcd for C48H58N8O9P: 921.4069; found 921.4048 [MÀ À H].
3
3J(H,H) ˆ 12.4, 3.1 Hz, 1H; C(4)H), 3.58 (d, J(H,H) < 1 Hz, 1H; C(8)H),
3.61 (d, 3J(H,H) ˆ 5.7 Hz, 1H; C(1)H), 3.71 (dd, 3J(H,H) ˆ 11.2, 6.7 Hz,
1H; C(3)H), 3.74 (s, 6H; 2CH3), 3.84 (dt, 3J(H,H) ˆ 11.7, 3.4 Hz, 1H;
C(3)H), 4.23 (dd, 3J(H,H) ˆ 9.6, 3.0 Hz, 1H; C(6)H), 4.30, 4.48 (2d,
3J(H,H) ˆ 16.7 Hz, 2H; CH2), 5.65 (d, 3J(H,H) ˆ 2.2 Hz, 1H; C(8)OH),
6.91 6.96 (m, 4H; ArH), 7.20 7.45 (m, 9H; ArH, CH), 7.62 (s, 1H; NH),
11.31 ppm (s, 1H; NH); 13C NMR (75 MHz, [D6]DMSO, 258C): d ˆ 12.37
(q), 32.15, 33.87, 49.74 (3t), 55.54 (q), 59.30 (t), 67.63 (s), 75.79, 77.20, 77.72
(3d), 79.64, 86.14, 108.41 (3s), 113.76, 113.84, 127.20, 128.39, 130.58, 130.70
(6d), 136.71, 136.87 (2s), 142.66 (d), 146.44, 151.50, 158.72, 158.76, 164.79,
168.17 ppm (6s); HRMS (ESI-TOF-MS) calcd for C 35H36N3O8: 626.2502;
found: 626.2474 [MÀ À H].
Phosphoramidite 6a: Chloro-2-cyanoethyl-diisopropylaminophosphine
(0.29 mL, 1.3 mmol)was added at room temperature to a solution of 5a
(335 mg, 0.43 mmol)and iPr2NEt (0.40 mL, 2.3 mmol)in THF (5 mL.)
After stirring for 3 h under Ar at room temperature, the reaction mixture
was quenched with sat. NaHCO3 and extracted (EtOAc). Compound 6a
(378 mg, 88%)was obtained from the organic phase after flash chroma-
tography (AcOEt)as a white foam. Rf ˆ 0.70 (AcOEt, silica gel pre-
deactivated by 1% TEA in hexane); 1H NMR (300 MHz, [D6]benzene,
258C): d ˆ 1.15, 1.14 (2s, 9H; (CH3)3CPh), 1.13, 1.11, 1.07, 1.05, 1.03 (5s,
12H; 4CH3), 1.26 1.34 (m, 1H; C(7)H), 1.58 1.63 (m, 1H; C(7)H), 2.00
(m, 1H; C(4)H), 2.15(m, 2H; CH2CN), 2.33 (dd, 3J(H,H) ˆ 12.5, 3.3 Hz,
1H; C(4)H), 3.43, 3.48 (2s, 6H; 2CH3), 3.19 3.60 (m, 2H; CH2, 2H;
2CH), 3.76 (m, 1H; C(6)H), 3.89 (m, 1H; C(3)H), 4.20 (d, 3J(H,H) ˆ
5.5 Hz, 1H; C(1)), 4.36 4.51 (m, 1H; C(3)H, 2H; CH2CO), 4.70, 4.74
(2s, 1H; C(8)H), 6.17 (s, 1H; NH), 6.89 6.97 (m, 4H; ArH), 7.13 7.32 (m,
3H; ArH), 7.59 7.92 (m, 8H; ArH), 9.07 ppm (s, 1H; NH); 13C NMR
(75 MHz, [D6]benzene, 258C): d ˆ 20.35 (t), 24.54, 24.66, 31.99, 30.99 (4q),
34.59 (s), 34.89 (t), 43.25, 43.11, 42.94 (3d), 53.14 (t), 54.90, 54.90, 54.98
(3q), 59.12, 59.34, 60.02 (3t), 66.53, 66.58 (2s), 77.19, 77.35, 78.16 (3d), 87.31
(s), 113.82, 113.97 (2d), 119.02 (s), 125.89, 127.28, 128.35, 128.47, 131.07,
131.24 (6d), 146.80 (s), 149.98 (d), 156.23, 159.41, 159.45, 163.48,
166.43 ppm (5s); 31P NMR (161.9 MHz, [D6]benzene, 258C): d ˆ 146.23,
148.25 ppm; HRMS, ESI-MS calcd for C54H65N6O9P: 971.4477; found:
971.4462.
Phosphoramidite 6d: This compound was prepared as described for 6a,
from 5d (126 mg, 0.17 mmol), iPr2NEt (370 mL, 2 mmol), and chloro-2-
cyanoethyl-diisopropylaminophosphine (240 mL, 1 mmol)in THF (2 mL).
Compound 6d (256 mg, 86%)was obtained after flash chromatography
(Et2O/MeOH 9.5:0.5)as a white foam. Rf ˆ 0.59 (Et2O/MeOH 9.5:0.5);
1H NMR (300 MHz, [D]chloroform, 258C): d ˆ 1.01, 1.03, 1.06, 1.08, 1.10
(5s, 12H; 4CH3), 1.42 (d, 3J(H,H) ˆ 16.9 Hz, 1H; C(7)H), 1.75 1.86 (m,
1H; C(7)H), 1.88 (s, CH3), 2.07, 2.15 (2dd, 3J(H,H) ˆ 12.9, 2.6 Hz, 1H;
C(4)H), 2.52 2.61 (m, 2H; CH2CN), 2.70 2.80 (m, 1H; C(4)H), 3.56
3.48 (m, 2H; CH2), 3.63 3.71 (m, 2H; 2CH), 3.79 (s, 6H; 2CH3), 3.82
3
3.89 (m, 1H; C(3)H), 3.95 (d, J(H,H) ˆ 5.2 Hz, 1H; C(1)H), 3.99 (s, 1H;
C(8)H), 4.07 4.18 (m, 2H; CH2, 1H; C(3)H), 4.35, 4.41 (m, 1H; C(6)H),
4.98, 5.09 (2s, 1H; NH), 6.85 6.90 (m, 4H; ArH), 6.95, 7.04 (2d, 3J(H,H) <
0.1 Hz, 1H; CH), 7.23 7.48 (m, 9H; ArH), 8.29 ppm (brs, 1H; NH);
13C NMR (100 MHz, [D]chloroform, 258C): d ˆ 12.27 (q), 20.26, 20.32,
20.46, 20.51 (4d), 24.20, 24.27, 24.35, 24.44, 24.49, 24.54, 24.62, 24.70 (8q),
30.75, 30.88 (2t), 34.02, 34.21 (2t), 42.96, 43.08 (2d), 49.22, 49.38 (2t), 55.30
(q), 57.95, 58.17, 58.42, 58.61 (4t), 59.68, 59.81 (2t), 65.84, 65.95 (2s), 77.24,
77.41, 77.49, 77.55 (4d), 86.61, 110.23, 110.33 (3s), 113.39, 113.45 (2d), 118.32
(s), 127.12, 127.22, 128.05, 128.34, 130.57, 130.66 (6d), 136.13, 136.21, 136.48,
136.56 (4s), 140.84, 141.51 (2d), 145.58, 150.54, 150.66, 158.82, 158.86,
163.78, 165.48, 165.80 ppm (7s); 31P NMR (161.9 MHz, [D]chloroform,
Phosphoramidite 6b: This compound was prepared as described for 6a,
from 5b (340 mg, 0.46 mmol), iPr2NEt (470 mL, 2.54 mmol), and chloro-2-
cyanoethyl-diisopropylaminophosphine (300 mL, 1.25 mmol)in THF
(5 mL). Compound 6b (256 mg, 86%)was obtained after extraction
(AcOEt/sat. NaHCO3)and chromatography on Sephadex LH 20 as a white
foam. Rf ˆ 0.60 (AcOEt/MeOH 9:1); 1H NMR (300 MHz, [D]chloroform,
258C): d ˆ 0.95, 0.97, 1.00, 1.02, 1.02, 1.03, 1.05, 1.05 (8s, 12H; 4(CH3)), 1.41,
1.52 (2d, 3J(H,H) ˆ 14.5 Hz, 1H; C(7)H), 1.74 1.85 (m, 1H; C(7)H), 2.06,
2.17 (2dd, 3J(H,H) ˆ 13.2, 3.6 Hz, 1H; C(4)H), 2.49 2.54 (m, 2H;
CH2CN), 2.69 2.72 (m, 1H; C(4)H), 3.29 3.41 (m, 2H; CH2), 3.53 3.70
(m, 2H; 2CH), 3.80 (s, 6H; CH3), 3.81 3.86 (m, 1H; C(3)H), 3.95, 4.01
(2d, 3J(H,H) ˆ 5.2 Hz, 1H; C(1)H), 4.15 4.22 (m, 1H; C(3)H, 1H;
C(8)H), 4.35, 4.41 (2d, 3J(H,H) ˆ 9.9 Hz, 1H; C(6)H), 4.41, 4.55, 4.56,
4.64 (4d, 3J(H,H) ˆ 15.8 Hz, 2H; CH2), 5.12, 5.42 (2s, 1H; NH), 6.88 6.94
(m, 4H; ArH), 7.25 7.61 (m, 12H; ArH), 7.99 8.01 (m, 2H; ArH), 8.11,
8.16, 8.71, 8.73 (4s, 2H; 2CH), 9.05, 9.06 ppm (2bs, 1H; NH); 13C NMR
(75 MHz, [D]chloroform, 258C): d ˆ 20.15, 20.25, 20.35, 20.43 (4t), 24.19,
24.28, 24.32, 24.38, 24.40, 24.49, 24.51, 24.62 (8q), 30.66, 30.76, 34.05, 34.21
(4t), 42.88, 43.05 (d), 45.68, 46.04 (t), 55.26, 55.29 (q), 57.83, 58.12, 58.16,
58.43, 59.57, 59.76 (6t), 66.04, 66.10 (2s), 76.58, 77.42, 77.48, 77.57 (4d),
86.81, 86.85 (2s), 113.40, 113.46 (2d), 118.06 (s), 127.19, 127.26, 127.78,
128.04, 128.33, 128.79, 130.53, 130.62, 132.64 (9d), 136.10, 136.21, 136.51,
136.56, 143.71, 143.76 (6s), 144.02, 145.00, 145.50, 145.56, 152.46, 158.83,
158.84, 158.87, 158.89, 164.43, 164.82 ppm (11s); 31P NMR (161.9 MHz,
‡
258C): d ˆ 154.02, 154.55 ppm; LSI-MS: 828 [M ‡H].
Synthesis and purification of the oligonucleotides: All oligonucleotides and
analogues were synthesized on the 1.3 mmol scale on a Pharmacia Gene-
Assembler Special DNA-synthesizer. Oligodeoxyribonucleotides were
synthesized by phosphoramidite chemistry and were assembled, depro-
tected, and purified according to standard protocols. RNA synthesis was
performed with 2'O-TBDMS protected phosphoramidites (Glen Research)
according to standard protocols for RNA synthesis. Unnatural l-oligo-
ribonucleotides were synthesized with the corresponding 2'O-TOM
protected l-phosphoramidites as described.[38, 39]
Bicyclo[3.2.1]amide-oligonucleotides were assembled either on CPG-
support carrying a natural deoxyribonucleoside unit, or on universal
¬
CPG-support from CT-Gen, San Jose. In the former case, a natural
nucleoside remained attached to the 5'-end in a 5' 5' phosphate linkage,
while universal support could tracelessly be removed. Synthesis proceeded
with the following changes relative to a DNA synthesis cycle: 1)a 10%
trichloroacetic acid solution in dichloroethane was used for detritylation
(90 sec), 2)the coupling time was extended to 6 min, and 3)tetrazole
(0.45m in CH3CN)was replaced by the more active 5-(benzylthio-)1 H-
tetrazole (0.25m in CH3CN)as the activator. Coupling yields were typically
ꢁ95%. After removal of the last trityl group, oligonucleotides were
detached from the support and deprotected by treatment with conc. NH3
(558C, 17 h for nucleoside modified CPG, 658C, 72 h for universal solid
support). The crude oligonucleotides were purified by DEAE-HPLC
(Mono Q HR 10/10 column, Pharmacia Biotech). Oligonucleotides were
desalted over SEP-PAK C-18 cartridges (Waters). All modified oligonu-
cleotides were routinely characterized by ESIÀ-TOF mass spectrometry
(see Table 1).
‡
[D]chloroform, 258C): d ˆ 154.28, 154.40 ppm; LSI-MS: 942 [M ‡H].
Phosphoramidite 6c: This compound was prepared as described for 6a,
from 5c (126 mg, 0.17 mmol), iPr2NEt (180 mL, 1.1 mmol), and chloro-2-
cyanoethyl-diisopropylaminophosphine (115 mL, 0.52 mmol)in THF
(1 mL). Compound 6c (78 mg, 50%)was obtained after flash chromatog-
raphy (MeOH/EtOAc ˆ 1:9)as a white foam. Rf ˆ 0.67 (MeOH/CH2Cl2
UV melting experiments and CD spectra: Oligonucleotides were mixed to
1:1 stoichiometry by using the UV extinction coefficients of natural
1
1:9, silica gel pre-deactivated by 1% TEA in hexane); H NMR (300 MHz,
Chem. Eur. J. 2002, 8, No. 23
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0823-5321 $ 20.00+.50/0
5321

C. J. Leumann et al.
FULL PAPER
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 interval of 0 908C were
applied, with a linear gradient of 0.58CminÀ1. Heating and cooling ramps
were superimposable in all cases, indicating equilibrium conditions. CD
spectra were measured on a JASCO J-715 spectrometer at the temper-
atures indicated. Thermodynamic data of duplex formation were deter-
[9] P. Nielsen, F. Kirpekar, J. Wengel, Nucleic Acids Res. 1994, 22, 703
710.
[10] L. Peng, H.-J. Roth, Helv. Chim. Acta 1997, 80, 1494 1512.
[11] K. C. Schneider, S. A. Benner, J. Am. Chem. Soc. 1990, 112, 453 455.
[12] K. Schoning, P. Scholz, S. Guntha, X. Wu, R. Krishnamurthy, A.
Eschenmoser, Science 2000, 290, 1347 1351.
[13] C. J. Leumann, Bioorg. Med. Chem. 2002, 10, 841 854.
[14] C. Epple, C. Leumann, Chem. Biol. 1998, 5, 209 216.
[15] C. D. Roberts, C. Epple, C. J. Leumann, Nucleosides Nucleotides 1999,
18, 1413 1415.
[16] B. M. Keller, C. J. Leumann, Angew. Chem. 2000, 112, 2367 2369;
Angew. Chem. Int. Ed. 2000, 39, 2278 2281.
À1
mined by the concentration variation method from a plot of Tm against
ln c, as described.[21]
Molecular modeling: The molecular models of the bicyclo[3.2.1]amide
nucleic acid duplexes were built with the molecular modeling program suite
Moloc.[40, 41] In particular, the highly resolved A-RNA homopurine/
homopyrimidine duplex (X-ray structure of rD(G11) ¥ rD(C11)determined
at 1.20 ä,[42] protein database code 1QCU)served as starting geometry. The
phosphate backbone was rendered neutral by uniform hydrogen saturation.
The structure was subsequently subjected to energy optimization within the
[17] A. Egger, J. Hunziker, G. Rihs, C. Leumann, Helv. Chim. Acta 1998,
81, 734 743.
¬
¬
¬
¬
[18] Z. Timar, L. Kovacs, G. Kovacs, Z. Schmel, J. Chem. Soc. Perkin 1
2000, 19 26.
force field MAB,[43] which generally leads to
a flattening out and
[19] D. W. Will, G. Breipohl, D. Langner, J. Knolle, E. Uhlmann,
Tetrahedron 1995, 51, 12069 12082.
regularization within the base-pair planes, whereafter the latter were
changed into adenine and thymine residues and reoptimized, first with
fixed backbone geometry, then by releasing the whole structure. This
™idealized∫ rD(A11)¥r D(T11)duplex was then transformed into r D(A10) ¥
rD(T10)and mirrored into r L(A10) ¥ rL(T10), and the energies of both were
reoptimized to form the prototypes for further transformations. In
particular, 10-mer homoadenine and homothymine strand constitutions
of bicyclo[3.2.1]amide nucleic acid were superimposed onto both the left-
handed and the right-handed duplex templates by use of a flexible
optimization match algorithm contained in the modeling suite. The
backbones were substituted and fused to the template base-pairs, which
were kept fixed in the first optimization cycles to establish standard bond
lengths at the attachment points. The backbone was then regularized with
weak hydrogen-bond constraints applied between intrastrand amide
groups: through this procedure the endo[3.2] amide group conformation
was identified to allow significant left-handed base-pair ™staircase∫ over-
lap, whereas the endo[3.1] amide conformer was found to fit with right-
handed base-pair overlap (Figure 8). The endo[2.1] amide rotamer does not
lead to any hydrogen-bond-like inter-residual b-turn, and was thus rejected
for inclusion in the right- and left-handed oligomer structures; moreover,
according to isolated energy terms, the endo[2.1] rotamer is intrinsically
disfavored by slightly more than 1 kcalmolÀ1 (Figure 7). After regulariza-
tion of the modified backbone geometries, all constraints were removed
and the duplexes were completely relaxed in the force field.
[20] K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen,
T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt, J.
Org. Chem. 1994, 59, 5767 5773.
[21] L. A. Marky, K. J. Breslauer, Biopolymers 1987, 26, 1601 1620.
[22] A. Egger, C. J. Leumann, Synlett 1999, S1 913 916.
[23] M. Marangoni, A. Van Aerschot, P. Augustyns, J. Rozenski, P.
Herdewijn, Nucleic Acids Res. 1997, 25, 3034 3041.
[24] S. Fujimori, K. Shudo, Y. Hashimoto, J. Am. Chem. Soc. 1990, 112,
7436 7438.
[25] A. Garbesi, M. L. Capobianco, F. P. Colonna, L. Tondelli, F. Arca-
mone, G. Manzini, C. W. Hilbers, J. M. E. Aelen, M. J. J. Blommers,
Nucleic Acids Res. 1993, 21, 4159 4165.
[26] V. K. Rajwanshi, A. E. Hakansson, M. D. Sorensen, S. Pitsch, S. K.
Singh, R. Kumar, P. Nielsen, J. Wengel, Angew. Chem. 2000, 112,
1722 1725; Angew. Chem. Int. Ed. 2000, 39, 1656 1659.
[27] R. Krishnamurthy, S. Pitsch, M. Minton, C. Miculka, N. Windhab, A.
Eschenmoser, Angew. Chem. 1996, 108, 1619 1623; Angew. Chem.
Int. Ed. Engl. 1996, 35, 1537 1541.
[28] S. Pitsch, R. Krishnamurthy, M. Bolli, S. Wendeborn, A. Holzner, M.
Minton, C. Lesueur, I. Schˆnvogt, B. Jaun, A. Eschenmoser, Helv.
Chim. Acta 1995, 78, 1621 1635.
[29] M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier,
D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, P. E. Nielsen, Nature
1993, 365, 566 568.
[30] E. Uhlmann, A. Peyman, G. Breipohl, D. W. Will, Angew. Chem. 1998,
110, 2954 2983; Angew. Chem. Int. Ed. 1998, 37, 2796 2823.
[31] B. Hyrup, P. E. Nielsen, Bioorg. Med. Chem. 1996, 4, 5 23.
[32] ÷. Almarsson, T. C. Bruice, Proc. Natl. Acad. Sci. USA 1993, 90,
9542 9546.
Acknowledgement
The authors thank the Swiss National Science Foundation (grant No. 20
63582.00)and Novartis AG, Basel, for continuing financial support.
[33] ÷. Almarsson, T. C. Bruice, J. Kerr, R. N. Zuckermann, Proc. Natl.
Acad. Sci. USA 1993, 90, 7518 7522.
[34] S. C. Brown, S. A. Thomson, J. M. Veal, D. G. Davis, Science 1994, 265,
777 780.
[35] L. Betts, J. A. Josey, J. M. Veal, S. R. Jordan, Science 1995, 270, 1838
1841.
[36] M. Koga, S. W. Schneller, Nucleic Acids Symp. Ser. 1993, 29, 63 65.
[37] M. Tarkˆy, M. Bolli, C. Leumann, Helv. Chim. Acta 1994, 77, 716 744.
[38] S. Pitsch, Helv. Chim. Acta 1997, 80, 2286 2314.
[39] S. Pitsch, P. A. Weiss, L. Jenny, A. Stutz, X. Wu, Helv. Chim. Acta 2001,
84, 3773 3795.
[40] P. R. Gerber, Moloc–A Molecular Design Software Suite (http://
www.moloc.ch).
[41] C. Lehmann, Chimia 2000, 54, 469.
[42] C. Klosterman, S. A. Shah, T. A. Steitz, Biochemistry 1999, 38,
14784 14792.
[43] P. R. Gerber, K. M¸ller, J. Comput.-Aided Mol. Des. 1995, 9, 251 268.
[1] S. M. Freier, K.-H. Altmann, Nucleic Acids Res. 1997, 25, 4429 4443.
[2] J. Hunziker, H.-J. Roth, M. Bˆhringer, A. Giger, U. Diederichsen, M.
Gˆbel, R. Krishnan, B. Jaun, C. Leumann, A. Eschenmoser, Helv.
Chim. Acta 1993, 76, 259 352.
[3] C. Hendrix, H. Rosemeyer, I. Verheggen, F. Seela, A. Van Aerschot, P.
Herdewijn, Chem. Eur. J. 1997, 3, 110 120.
[4] J. Wang, B. Verbeure, I. Luyten, E. Lescrinier, M. Froeyen, C.
Hendrix, H. Rosemeyer, F. Seela, A. Van Aerschot, P. Herdewijn, J.
Am. Chem. Soc. 2000, 122, 8595 8602.
[5] Y. Maurinsh, H. Rosemeyer, R. Esnouf, A. Medvedovici, J. Wang, G.
Ceulemans, E. Lescrinier, C. Hendrix, R. Busson, P. Sandra, F. Seela,
A. Van Aerschot, P. Herdewijn, Chem. Eur. J. 1999, 5, 2139 2150.
[6] S. Pitsch, S. Wendeborn, B. Jaun, A. Eschenmoser, Helv. Chim. Acta
1993, 76, 2161 2183.
[7] S. Honzawa, S. Ohwada, Y. Morishita, K. Sato, N. Katagiri, M.
Yamaguchi, Tetrahedron 2000, 56, 2615 2627.
[8] J.-M. Henlin, K. Jaeckel, P. Moser, H. Rink, E. Spieser, G. Baschang,
Angew. Chem. 1992, 104, 492 493; Angew. Chem. Int. Ed. Engl. 1992,
31, 482 483.
Received: June 6, 2002 [F4163]
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