A conformationally locked aminomethyl C-glycoside and studies on its N-pyren-1-ylcarbonyl derivative inserted into oligodeoxynucleotides

Article abstract of DOI:10.1002/ejoc.200500977

A new conformationally locked aminomethyl C-glycoside has been synthesized and incorporated into oligonucleotides (ONs). The applicability of the monomer in posi-ON synthesis (i.e., conjugation by organic synthesis performed on ONs attached to resin) has

Full text of DOI:10.1002/ejoc.200500977

FULL PAPER
DOI: 10.1002/ejoc.200500977
A Conformationally Locked Aminomethyl C-Glycoside and Studies on Its
N-Pyren-1-ylcarbonyl Derivative Inserted into Oligodeoxynucleotides
Carlo Verhagen,[a] Torsten Bryld,[a] Michael Raunkjær,[a] Stefan Vogel,[a]
Katerina Buchalová,[a] and Jesper Wengel*[a]
Keywords: C-Glycosides / Pyrenes / Oligodeoxynucleotides / LNA / Locked nucleic acid
A new conformationally locked aminomethyl C-glycoside
has been synthesized and incorporated into oligonucleotides
(ONs). The applicability of the monomer in post-ON synthe-
sis (i.e., conjugation by organic synthesis performed on ONs
attached to resin) has been successfully demonstrated by
condensation with pyren-1-ylcarboxylic acid. The abilities of
the pyrenyl-derivatized ONs to recognize abasic sites in com-
plementary strands were investigated by thermal denatur-
ation studies, which showed the obtained pyrenyl-derivat-
ized ONs to be promising candidates for this purpose. The
synthetic route also allowed synthesis of a conformationally
locked dipeptide mimetic suitable for use in peptide and
carbohydrate chemistry.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
monomer Z in relation to monomer W. The higher flexibil-
ity would presumably allow the pyrene moiety to orientate
for optimal base stacking. ONs with incorporated mono-
mer Z were subjected to thermal denaturation studies in
order to evaluate the effect of the change in design relative
to monomer W.
Introduction
A variety of modified oligonucleotides (ONs) have been
synthesized in attempts to obtain antisense molecules with
significantly improved pharmaceutical properties. The in-
troduction of Locked Nucleic Acid (LNA)[1–3] has contrib-
uted to this development through its unprecedented ability
to recognize complementary RNA targets. The constitution
of LNA (i.e., the presence of the conformationally locked
bicyclic sugar unit in place of the flexible ribose unit of
the natural nucleotide-based backbone) is the factor that
mediates this behavior. We now present further exploitation
of the LNA scaffold through the introduction of the C-gly-
coside precursor X for use in conjugation chemistry on a
post-ON synthesis level (Figure 1).[4] The advantage of such
a methodology is the potential to synthesize a vast number
of conjugates from only one precursor by means of peptide
coupling chemistry. We have previously published the syn-
thesis and hybridization properties of a monomer contain-
ing a pyrene moiety directly attached to a bicyclic locked
skeleton (monomer W, Figure 1),[5] but putative modeling
indicated an advantage in having the pyrene moiety located
further away from the ribose into the core of the
DNA:DNA duplex, resulting in enhancement of base stack-
ing. Monomer Z (Figure 1) was designed to investigate this
hypothesis, as the pyrene moiety here should have a higher
degree of positional freedom due to the longer linker in
Figure 1. Chemical structures of monomers.
Here we further demonstrate the applicability of mono-
mer Z to recognition of abasic sites (Φ, Figure 1). This
capability has previously been demonstrated for ONs incor-
porating monomer K,[6–8] having a pyrene moiety directly
substituting for a nucleobase. Monomer K[8] and monomer
W have shown interesting universal base properties, and
similar behavior is reported here for monomer Z.
[a] Nucleic Acid Center, Department of Chemistry, University of
Southern Denmark,
5230 Odense M, Denmark
Fax: +45-66158780
E-mail: jwe@chem.sdu.dk
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Conformationally Locked Aminomethyl C-Glycoside
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Scheme 1. i) TMSCN, BF3·Et2O, CH3CN, 68%. ii) a) BH3·S(Me)2, THF, b) HCl, THF, c) NaOH, EtOH, d) Boc2O, CH2Cl2, 56%.
iii) a) 2,2-dimethoxypropane, TsOH, THF (R1 = R1 = CMe2 indicates isopropylidene group), b) BzCl, TEA, CH2Cl2, 84%. iv) a) TsOH,
MeOH, b) MsCl, TEA, 73%. v) NaOH, THF, 84%. vi) a) NaOBz, DMF, b) NaOH, EtOH, 73%. vii) a) H2, 10% Pd/C, HCl, EtOH,
b) FmocCl, MgO, 77%. viii) DMTCl, TEA, CH2Cl2, CH3CN, 71%. ix) (iPr)2NP(Cl)OCH2CH2CN, DIPEA, DCM, 57%. x) a) TFA,
b) FmocCl, MgO, THF, 86%. xi) a) Dess–Martin periodinane, CH2Cl2, b) NaH2PO4, NaClO2, tBuOH, 2-methylbut-2-ene, 76%.
Furthermore, only a few changes in the synthetic strategy from a cyano-derivatized carbohydrate. Our choice of strat-
enabled the synthesis of the novel, rigid LNA-based linker egy involved Schmidt coupling chemistry[23–25] on the
12 (Scheme 1). The six-atom linker is suitable for use in so- known carbohydrate 1,[26] providing a suitable precursor for
lid-phase peptide synthesis, whilst the structural design of further synthesis. It was anticipated that the anchimeric as-
the building block contributes to the still expanding family sistance offered by the 3-O-acetyl group should induce ster-
of sugar amino acids (SAAs)[9–11] resembling conformation- eoselective formation of the desired β-anomer. Treatment of
ally restrained dipeptide isosters.[12–17] Members of this 1 with TMSCN and BF3·Et2O afforded allononitrile 2 in
class of SAAs have found application in foldamers,[18,19] 68% yield without detectable formation of the correspond-
host-guest studies,[20] and as turn inducers.[21] We suggest ing α-anomer. The cyano moiety was reduced with
SAA 12 as a promising candidate for such applications, due BH3·Me2S, followed by base-mediated cleavage of the ester
to the rigid nature of the molecule.
bonds. The primary amino group was subsequently allowed
to react with Boc2O to afford C-glycoside 3 in a yield of
56%. Use of NaBH4, LiAlH4, or NaBH3TFA as reducing
agents in order to achieve fully reduced products was also
investigated, but those approaches proved unsuccessful due
to workup difficulties.
Results and Discussion
The synthetic approach for construction of the bicyclic
scaffold was based on the reported LNA synthesis method-
Conversion of the primary hydroxy groups into good
ology,[22] though the introduction of a novel aminomethyl- leaving groups by regioselective sulfonylation was attempted
ene functionality implied a change of strategy. We expected in order to facilitate intramolecular attack from the O3
that the amino functionality should be readily accessible atom. We have previously taken advantage of regioselective
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FULL PAPER
sulfonylation and acylation procedures in our work.[27,28] tempt to oxidize the aldehyde with aqueous NaClO2, how-
Here, however, we obtained mixtures of partially sulfo- ever, gratifyingly provided LSAA 12 in 76% yield (from 11)
nylated products and our efforts to control regioselectivity after workup.
turned out to be unsuccessful. This prompted the design of
The obtained phosphoramidite 10 was used for incorpo-
a new synthetic route involving protection of the primary ration of monomer X into oligonucleotides (see Exp. Sec.
hydroxy groups through the formation of a five-membered for details) The oligonucleotide synthesis was carried out
cyclic acetal. The reaction was carried out on C-glycoside without cleavage of the O5Ј-DMT group from the last
3 by treatment with 2,2-dimethoxypropane in the presence monomer and the oligonucleotides were subjected to on
of TsOH. The crude acetal was O3-benzoylated by treat- resin conjugation reactions by a previously published
ment with benzoyl chloride, affording compound 4 in 84% method.[4,33] The primary amine was thus selectively depro-
yield. The primary hydroxy groups were liberated by acidic tected with 20% piperidine in DMF and subsequently
hydrolysis and subsequently mesylated with mesyl chloride treated with pyrene-1-carboxylic acid in the presence of
to give dimesylate 5 in 73% yield. Treatment of glycoside 5 HOBt, HBTU, and DIPEA, producing ONs with monomer
with aqueous base ensured ester hydrolysis, which was fol- Z incorporated. The ONs were released from the solid sup-
lowed by in situ ring closure to give the locked bicyclic C- port, deprotected, and purified by standard procedures to
glycoside 6 in 84% yield. Although a tedious protection- yield ON2, ON9, and ON14–ON17 (see the Experimental
deprotection procedure had been necessary, glycoside 6 was Section for details).
obtained in a satisfactory overall yield of 51% from diol 3.
The remaining mesyl group was removed by treatment of 6
with NaOBz in DMF, followed by base-mediated hydrolysis
of the intermediate benzoyl ester to afford glycoside 7 in
Hybridization Studies
73% yield.
Modified monomers that bind equally well to each of the
natural bases constitute a research area of great interest.
Universal bases have been used as primers for PCR reac-
tions and as universal hybridization probes in diagnostic
systems.[34] Previously published universal bases, based on
a 2-deoxy-β--ribofuranosyl moiety, include monomer K
(Figure 1)[6] and 3-nitropyrrole,[35–37] 5-nitroindole,[38] iso-
carbostyril,[39] and 8-aza-7-deazaadenine analogues.[40]
These monomers showed the desired properties of dis-
playing only small variations in the thermal stabilities (Tm
values) of the duplexes formed with complements contain-
ing any of the four natural bases opposite the universal base
monomer, but also induced decreases in thermal stability
relative to the matched unmodified DNA duplexes (∆Tm
values of –4 to –10 °C). Replacement of the nucleobase with
a pyrene moiety directly attached to an LNA skeleton (W,
Figure 1)[5] had previously been shown to produce good
universal base properties, but a significant decrease in over-
all thermal stability relative to the unmodified matched
DNA:DNA duplex containing an A:T basepair as reference
(Table 1) was also observed in this case.[5]
The need for a protecting group applicable in a post-ON
synthetic approach resulted in the replacement of the acid-
labile Boc protecting group by the base-labile Fmoc protect-
ing group. Conversion of glycoside 7 into a suitable phos-
phoramidite building block furthermore implied O4-deben-
zylation. Acidic hydrogenolysis of glycoside 7 resulted in a
fully deprotected C-glycoside and the amino functionality
was subsequently Fmoc-protected under chemoselective
conditions[29] to afford diol 8 in 77% yield. The primary
hydroxy group was regioselectively DMT-protected by
treatment with DMTCl and DIPEA in a 1:1 mixture of
CH3CN and DCM. The use of this solvent mixture ensured
a fast reaction without cleavage of the Fmoc group, and
the DMT-protected glycoside 9 was obtained in 71% yield.
Treatment of glycoside 9 with 2-cyanoethyl N,N-diisopro-
pylphosporamidochloridite provided phosphoramidite 10
in 57% yield.
A different strategy was chosen in order to obtain a suit-
able SAA building block. The Boc group was replaced by a
Fmoc group in order to satisfy solid-phase peptide coupling
protocols, though for reasons of solubility it was considered
an advantage to maintain the 4-O-benzyl protecting group.
The Boc protecting group was removed by treatment of gly-
coside 7 with TFA and the crude amine was subsequently
Fmoc-protected under chemoselective conditions to provide
C-glycoside 11 in 86% yield. Oxidation of the primary hy-
droxy functionality with TEMPO and BAIB as co-oxidant
was attempted.[30,31] To our surprise, the direct oxidation to
the carboxylic acid turned out to be difficult to perform
and we were not able to obtain a pure product. The oxi-
dation was then carried out by conversion of the hydroxy
group into an aldehyde with Dess–Martin periodinane and
the aldehyde was subsequently oxidized by use of
AgNO3.[32] In this way, a 15% yield of the desired SAA 12
Table 1. Thermal denaturation temperatures measured as the max-
ima of the first derivatives of the melting curves (A260 vs. tempera-
ture; 5 °C to 80 °C with an increase of 1 °Cmin–1) recorded in me-
dium salt buffer [“110 m Na+”]. For buffer conditions, see Gene-
ral Procedure. For structures of modified monomers, see Figure 1.
Thermal denaturation temperature (Tm values) in °C
5Ј-d(GCATHTCAC)
H = A
H = T
H = G
H = C
ON1 5Ј-d(GTGATATGC) 28
ON2 5Ј-d(GTGAZATGC) 21
ON3 5Ј-d(GTGAWATGC) 18[8]
19
12
11
21
19
20
19[8]
18[8]
17[8]
The hybridization properties of the modified ON2 (with
was obtained. Oxidation of the aldehyde with NaIO4 and monomer Z incorporated into a mixed 9-mer sequence) dis-
RuCl3 resulted in a disappointing 14% yield. A final at- played a reduction in duplex stability (–7 °C) in relation to
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Conformationally Locked Aminomethyl C-Glycoside
FULL PAPER
the reference duplex containing an A:T basepair. However,
ON2 showed only minor variations in Tm value when hy-
bridized with any of the four natural nucleobases opposite
monomer Z. This indicates that monomer Z is a good can-
didate as a universal base, and increases in thermal stabilit-
ies – relative to monomer W – were observed (Table 1).
Whether monomer Z was able to recognize abasic sites
Φ when positioned opposite to these in a duplex was next
examined. Pyrene recognition of abasic sites had previously
been demonstrated by monomer K, mediated by the forma-
tion of non-hydrogen-bonded base pairs.[6,41] ONs contain-
ing an abasic site Φ were recognized by ONs containing
monomer Z positioned in the complementary position to
the abasic site, with a large increase in duplex stability rela-
Figure 2. Model structure of ON2:ON10 based on geometry opti-
tive to native DNA (Table 2) This can be explained by the mization (Spartan02). Only modified monomers and surrounding
pyrene moiety’s ability to intercalate with the nucleobases,
followed by enhanced base stacking. In order to achieve
phosphates were subjected to optimization. The unmodified nucle-
otides were frozen during optimization. The pyrene moiety is
shown in yellow with reduced van der Waals radii and the LNA
increased base stacking the pyrene moieties have to fit
skeleton is shown in green.
within the core of the duplex, which has been shown to be
the case for monomer K.[6,8] However, monomer Z has a
more rigid furanose structure with the pyrene moiety at- complementary RNA demonstrating DNA selective bind-
tached through an amide bond, resulting in a longer dis- ing for these ONs.
tance between the pyrene moiety and the furanose ring and
Fluorescence spectroscopy could potentially provide in-
possibly in a more favorable orientation of the pyrene moi- direct evidence of intercalation by the pyrene moiety.[43,44]
ety than for monomer K. Accordingly, putative molecular The fluorescence emission bands (Figure 3) of the duplexes
modeling showed the pyrene moiety to fit “like a hand in obtained with ON9 all displayed higher intensities than
an glove” opposite an abasic site in a DNA:DNA duplex those of ON9 in the single-stranded form. The fluorescence
(Figure 2).[42]
of the unhybridized ON9 gave a fairly weak, unstructured
The mixed 9-mer ONs modified with a single monomer monomer emission band, which could be the result of inter-
were hybridized against each other and against complemen- actions with the neighboring nucleobases giving some
tary DNA and RNA, with an A:T(U) basepair as reference quenching of the fluorescence.[43,44] When ON9 was hy-
(Table 2). It was clearly seen that ONs containing an abasic bridized against the ON with an abasic site (ON9:ON5) the
site formed stable duplexes only when hybridized against intensity of the fluorescence increased approximately two-
ONs modified with monomer Z as the opposite “base”, fold and the emission became more structured, with clear
giving Tm values of 22 °C (ON5:ON9) and 25 °C I1 and I3 bands indicating smaller interactions with the sur-
(ON2:ON10). No hybridization was observed when ONs rounding nucleobases. Hybridization of ON9 against DNA
with abasic sites were mixed with complementary RNA or (ON1) resulted in a strong, unstructured monomer emission
DNA strands. These results support intercalation of the py- band, probably due to a dynamic equilibrium between dif-
rene unit and provision of stability to the duplex through ferent orientations of the pyrene moiety, one being the in-
additional base stacking. When ONs containing incorpo- tercalated form, probably with the opposing adenine base
rated monomer Z were hybridized against DNA, stable du- flipped out of the duplex, and another being one in which
plexes were obtained, although with decreases in stability the pyrene moiety is flipped out of the duplex, explaining
relative to DNA:DNA (∆Tm values = –5 °C and –8 °C). No the increased fluorescence. With two pyrene moieties oppo-
duplex formation above 10 °C was observed when ONs site each other (ON9:ON2), the intensity is almost the same
(ON2 and ON9) containing monomer Z were mixed with as or slightly lower than the intensity seen with one pyrene
Table 2. Thermal denaturation temperatures measured as the maxima of the first derivatives of the melting curves (A260 vs. temperature;
5 °C to 80 °C with an increase of 1 °Cmin–1) recorded in medium salt buffer [“110 m Na+”]. For structures of modified monomers, see
Figure 1. For buffer conditions, see General Procedure.
Thermal denaturation temperatures (Tm values) measured in °C
ON7
ON8
ON9
ON10
ON11
d(GCATATCAC) r(GCAUAUCAC) d(GCATZTCAC) d(GCATΦTCAC) d(GCATXTCAC)
ON1 d(GTGATATGC)
ON4 r(GUGAUAUGC)
ON2 d(GTGAZATGC)
ON5 d(GTGAΦATGC)
ON6 d(GTGAXATGC)
29
28
24
Ͻ10
Ͻ10
27
37
Ͻ10
Ͻ10
Ͻ10
21
Ͻ10
21
22
20
Ͻ10
Ͻ10
25
Ͻ10
Ͻ10
Ͻ10
Ͻ10
25
Ͻ10
Ͻ10
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Figure 3. Steady-state fluorescence spectra of ON9 with 0.15 µ of each strand recorded in air-saturated medium salt buffer [“110 m
Na+”] at 19 °C.
Figure 4. The “2+2” and “4+0” systems.
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Conformationally Locked Aminomethyl C-Glycoside
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moiety hybridized against DNA (ON9:ON1), and dis- Table 3. For conditions, see Table 1.
playing an unstructured band. This observation can only be
Thermal denaturation temperatures (Tm values) mea-
sured in °C
explained in terms of dynamic equilibrium of the two py-
rene moieties interchanging as intercalating units. The two
pyrene moieties would not be anticipated both to interca-
late simultaneously since this would probably result in the ON15
formation of an excimer band in the fluorescence emission
ON12
ON14
ON16
ON13
43
28
ON17
22
spectrum, which is not observed.
The relative stable duplexes formed by ONs modified
with a single monomer Z (ON2 and ON9) positioned oppo-
site to abasic sites Φ prompted us to expand into more com-
plex systems (Figure 4 and Table 3). We therefore designed
two systems containing four abasic sites with monomer Z
placed opposite these. In the first system, four Z monomers
were placed consecutively in the same strand (the “4+0”
system). Alternatively, two Z monomers alternating with
two abasic sites Φ were positioned in each of the comple-
mentary strands (the “2+2” system). As described earlier,
post-ON synthesis conveniently provided us with ON14–
ON17. The stabilities of the corresponding duplexes were
investigated by thermal denaturation studies (Table 3), and
the orientations of the pyrene moieties were investigated by
fluorescence spectroscopy.
ever, the notable point is the fact that the ONs modified
with monomer Z are able to form stable duplexes targeting
abasic sites, which is not the case for the corresponding
DNA strand containing a DNA thymine monomer instead
of the Z monomer. The best way to obtain stable duplexes
is also shown to be the zipper model, rather than by placing
all the pyrene moieties in one strand. A very similar system
using monomer K has been published independently of us,
but resulted in the formation of a substantially less stable
complex.[45]
We investigated whether the pyrene moieties were posi-
tioned in a sandwich-like fashion by fluorescence emission
spectroscopy, as induction of excimer band emission would
be expected (see Figure 5).[46] In the system with four con-
secutively placed pyrenes (“4+0”) the fluorescence spectra
in the hybridized form and in the unhybridized form are
virtually identical displaying similar excimer band inten-
sities, with the only difference being a stronger monomer
fluorescence in the hybridized form. This shows that the
pyrenes are located in close proximity to each other in both
The presence of pyrenes and abasic monomers placed in the hybridized and unhybridized forms, but does not pro-
a “2+2 zipper”-like manner (ON14:ON15) gave a decrease vide any information relating to the orientations of the py-
in the Tm value of 15 °C relative to the duplex with native rene moieties within the duplex. In the spectra of the sys-
A:T basepairs. A larger decrease of 21 °C was observed tems containing four pyrene moieties placed in a “2+2 zip-
when all four pyrene moieties were placed in one strand per”, the intensity of the monomer fluorescence of the du-
opposite four consecutive abasic sites (ON16:ON17). How- plex is identical to the monomer fluorescence intensity of
Figure 5. Steady-state fluorescence spectra with 0.15 µ of each strand recorded in air-saturated medium salt buffer [“110 m Na+”] at
19 °C.
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given in Hz. Assignments of NMR spectra are based on 2D spectra
and follow the standard carbohydrate and von Baeyer nomencla-
ture. Matrix-Assisted Laser Desorption Ionization Mass Spectrom-
etry (MALDI-MS) was performed on a 4.7 Tesla Ultima (IonSpec,
Irvine, CA) Fourier transform ion cyclotron resonance (FTICR)
mass spectrometer.
unhybridized ON15 and only marginally stronger than the
monomer fluorescence intensity of unhybridized ON14.
The excimer band originating from the duplex is of the
same intensity as the combined excimer bands of the two
strands in their single-stranded forms. No enhanced inten-
sity of the excimer band of the fluorescence of the duplex
relative to the excimer fluorescence band of the combined
ssONs’ (ON14 + ON15) fluorescence is observed. The firm
conclusion that can be drawn from these experiments is that
the pyrene units remain within a few Ångstrøms distance
in both the “2+2” and the “4+0” duplex systems, as test-
ified by the significant excimer bands. This indicates that
the pyrene moieties are positioned in a sandwich-like man-
ner within the core of the duplexes.
3-O-Acetyl-2,5-anhydro-6-O-benzoyl-5-C-[(benzoyloxy)methyl]-4-O-
benzyl-D-allononitrile (2): TMSCN (475 µL, 3.56 mmol) and
BF3·Et2O (250 µL, 1.96 mmol) were added to a stirred solution of
1[26] (990 mg, 1.75 mmol) in anhydrous acetonitrile (9.0 mL). The
reaction mixture was stirred for 30 min at room temp. before ad-
dition of a sat. aq. solution of NaHCO3 (10 mL). The mixture was
stirred for 15 min at room temp. and extracted with ethyl acetate
(50 mL). The organic phase was washed successively with sat. aq.
solutions of NaHCO3 (3×25 mL, 10%) and brine (25 mL), dried
(MgSO4), filtered, and concentrated to dryness under reduced pres-
sure. The residue was purified by silica gel column chromatography
with ethyl acetate in petroleum ether [0% Ǟ 25% (v/v)] as eluent,
Conclusion
1
yielding the allononitrile 2 (635 mg, 68%). H NMR (CDCl3): δ =
8.04–8.00 (m, 4 H, Harom), 7.58–7.55 (m, 2 H, Harom), 7.46–7.40
(m, 4 H, Harom), 7.26–7.20 (m, 5 H, Harom), 5.68 (dd, J = 5.2 Hz,
J = 2.7 Hz, 1 H, 3-H), 4.88 (d, J = 12.4 Hz, 1 H, CHaHb), 4.87 (d,
J = 2.4 Hz, 1 H, 4-H), 4.66 (d, J = 12.2 Hz, 1 H, CHaHbЈ), 4.64
(d, J = 11.3 Hz, 1 H, CHaHbЈЈ), 4.59 (d, J = 5.2 Hz, 1 H, 2-H),
4.50 (m, 1 H, CHaHbЈЈ), 4.48 (d, J = 11.4 Hz, 1 H, CHaHbЈ), 4.44
(d, J = 12.6 Hz, 1 H, CHaHb), 2.18 (s, 3 H, CH3) ppm. 13C NMR
(CDCl3): δ = 169.4, 166.1, 166.0 (CO), 136.2, 133.4, 133.3, 129.8,
129.7, 128.6, 128.5, 128.1 (Ar), 116.0 (CN), 84.7 (C-2), 78.3, 74.2,
74.0, 69.0, 64.0, 63.4 (C-3, C-4, C-5, C-6, C-6Ј, CH2-Bn), 20.7
(CH3) ppm. HRMS: calcd. for C30H27NO8Na 552.1629; found
m/z 552.1630.
The synthesis of the conformationally locked ami-
nomethyl C-glycoside monomer X and its incorporation
into oligonucleotides have been achieved. The potential of
this monomer as a very useful conjugation site for the intro-
duction of functional groups within the core of
a
DNA:DNA duplex was demonstrated by the unprecedented
introduction of four identical groups by a post-ON syn-
thetic approach. The pyrene-functionalized aminomethyl
C-glycoside monomer Z demonstrated versatility by func-
tioning as a universal base with a satisfactory affinity
towards DNA. ONs incorporating monomer Z show no hy-
bridization with RNA, but are able to recognize DNA
strands containing abasic sites Φ positioned opposite to
monomer Z. This ability was utilized for the creation of
fairly complex systems that, by virtue of the bicyclic core
structure in combination with the short aminomethyl link-
age, are stabilized relative to similar systems using a mono-
cyclic DNA scaffold. This is evidence for the utility of bicy-
clic LNA-like monomers for the construction of compli-
cated and unnatural nucleic acid systems, which may find
applicability in nanotechnology.
2,5-Anhydro-4-O-benzyl-1-[(tert-butoxycarbonyl)amino]-1-deoxy-5-
C-(hydroxymethyl)-D-allitol (3): BH3·S(Me)2 in THF (2 , 3.4 mL,
6.8 mmol) was slowly added to a stirred and cooled (0 °C) solution
of 2 (362 mg, 0.684 mmol) in freshly distilled THF (3.5 mL). The
reaction mixture was stirred for 16 h at room temp. The reaction
mixture was cooled (0 °C), followed by dropwise addition of aq.
HCl (6 , 7 mL, 42 mmol). The mixture was stirred for 30 min at
room temp. and the solvents were evaporated to dryness under re-
duced pressure. The residue was dissolved in a mixture of EtOH
and 2  aq. NaOH (1:1, v/v, 10 mL, 10 mmol) and was stirred for
2 h at room temp. DCM (10 mL) and Boc2O (200 µL, 0.871 mmol)
were added to the reaction mixture and stirring was continued for
another 16 h at room temp. The mixture was extracted with ethyl
acetate (3×15 mL) and the combined organic phase was washed
successively with a sat. aq. solution of NaHCO3 (3×15 mL) and
brine (15 mL), dried (MgSO4), filtered, and concentrated to dry-
ness under reduced pressure. The residue was purified by silica gel
column chromatography with MeOH in EtOAc [0% Ǟ 10% (v/v)]
Experimental Section
All reagents were obtained from commercial suppliers and were
used without further purification. Reactions were carried out under
nitrogen or argon when anhydrous solvents were used. Column
chromatography was performed with Silica gel 60 (particle size
0.040–0.063 µm, Merck). Dichloromethane (DCM) used for col-
umn chromatography was distilled prior to use. TLC analysis was
conducted with Fluka silica gel 60 F254 alumina sheets. UV absorb-
ing compounds were detected at 254 nm and charred either with
20% H2SO4 in ethanol or with (NH4)6Mo7O24·4H2O (25 g),
(NH4)4Ce(SO4)4·2H2O (10 g), and H2SO4 (100 mL) in H2O
(900 mL). Unsaturated hydrocarbons and aldehydes were sprayed
with KMnO4 (2%)/K2CO3 (1%) in H2O. Amines were charred with
ninhydrin (0.3 g) and acetic acid (3 mL) in 1-butanol (100 mL).
NMR spectra were recorded with a Varian Unity 300 spectrometer.
Chemical shift values are given relative to tetramethylsilane as in-
ternal standard (1H and 13C NMR) and relative to 85% H3PO4 as
external reference (31P NMR). Coupling constants (J values) are
1
as eluent, yielding the allitol 3 (145 g, 56%). H NMR (CDCl3): δ
= 7.35–7.31 (m, 5 H, Harom), 4.73 (d, J = 11.2 Hz, 1 H, CHaHb),
4.56 (d, J = 11.3 Hz, 1 H, CHaHb), 4.38 (m, 1 H, 4-H), 4.03 (d, J
= 8.6 Hz, 1 H, 2-H), 3.97 (m, 2 H, 3-H, CHaHbЈ), 3.77 (d, J
11.4 Hz, 1 H, CHaHbЈЈ), 3.57–3.29 (m, 3 H, CHaHbЈ, CHaHbЈЈ,
CHaHbЈЈЈ), 3.09–3.04 (m, 1 H, CHaHbЈЈЈ), 1.44 (s, 9 H, C(CH3)
3) ppm. 13C NMR (CDCl3): δ = 157.2 (CO), 137.5, 128.6, 128.1,
127.8 (Ar), 87.0 (C-5), 85.3 (C-4), 80.0 (Cq, Boc), 78.1 (C-2), 73.1
(C-3), 71.9 (CH2Bn), 63.3, 62.5 (C-6, C-6Ј), 43.5 (C-1), 28.3
(C(CH3)3) ppm. HRMS: calcd. for C19H29NO7Na 406.1836; found
m/z 406.1821.
2,5-Anhydro-3-O-benzoyl-4-O-benzyl-1-[(tert-butoxycarbonyl)-
amino]-1-deoxy-5-C-hydroxymethyl-6,5-(C-hydroxymethyl)-di-O-iso-
2544
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 2538–2548

Conformationally Locked Aminomethyl C-Glycoside
propylidene- -allitol (4): A catalytic amount of p-toluenesulfonic
FULL PAPER
4-H and CHaHbЈЈ), 4.32 (d, J = 5.6 Hz, 1 H, 2-H), 4.27 (d, J =
11.6 Hz, 1 H, CHaHb), 4.18 (d, J = 11.0 Hz, 1 H, CHaHbЈЈ), 3.42
(m, 2 H, CHaHbЈЈЈ and CHaHbЈЈЈ), 3.03 (s, 3 H, Ms), 3.01 (s, 3 H,
D
acid (50 mg, 0.29 mmol) and 2,2-dimethoxypropane (29.0 mL,
181 mmol) were added to a stirred solution of 3 (8.86 g, 23.1 mmol)
in anhydrous THF (400 mL). The reaction mixture was stirred for Ms) 1.45 (s, 9 H, C(CH3)3) ppm. 13C NMR (CDCl3): δ = 165.5,
2 h at room temp. before addition of a sat. aq. solution of NaHCO3
(25 mL) and DCM (300 mL). The resulting mixture was stirred for
5 min at room temp., after which the phases were separated and
the organic phase was washed successively with a sat. aq. solution
of NaHCO3 (3×150 mL) and brine (150 mL), dried (MgSO4), fil-
tered, and concentrated to dryness under reduced pressure. The
residue was dissolved in anhydrous DCM (400 mL) and the solu-
tion was cooled to 0 °C, followed by addition of benzoyl chloride
(8.7 mL, 75 mmol) and TEA (14 mL, 100 mmol). The reaction mix-
ture was stirred for 15 min at 0 °C. The mixture was allowed to
reach room temperature and then stirred for another 16 h at room
temp. before addition of a sat. aq. solution of NaHCO3 (25 mL).
The organic phase was separated and washed successively with a
sat aq. solution of NaHCO3 (3 × 150 mL) and brine (100 mL),
dried (MgSO4), filtered, and concentrated to dryness under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy with ethyl acetate and petroleum ether [0% Ǟ 30% (v/v)] as
156.0 (CO), 136.6, 133.6, 129.8, 129.1, 128.6, 128.5, 128.3 (Ar),
82.3, 81.5, 78.4, 74.2, 72.7 (C-5, C-4, C-3, C-2, C(CH3)3, CH2Bn)
68.1, 67.6 (C-6, C-6Ј), 42.3 (C-1), 37.9 (Ms), 28.3 (C(CH3)3) ppm.
MALDI-MS: m/z 666.2 [M + Na]+ . HRMS: calcd. for
C28H37NO12S2Na 666.1649; found m/z 666.1648.
(1R,3S,4S,7S)-7-Benzyloxy-3-[(tert-butoxycarbonyl)amino-
methyl]-1-[(methylsulfonyloxy)methyl]-2,5-dioxabicyclo[2.2.1]-
heptane (6): Aq. NaOH (4 , 150 mL, 0.6 mol) was added to a
stirred solution of 5 (9.27 g, 14.8 mmol) in THF (150 mL). The
reaction mixture was stirred for 16 h at room temp., after which
the mixture was extracted with ethyl acetate (3×150 mL). The com-
bined organic phase was collected, washed with brine (50 mL),
dried (MgSO4), filtered, and concentrated to dryness under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy with ethyl acetate in petroleum ether [0% Ǟ 50% (v/v)] as
1
eluent, yielding 6 (5.36 g, 84%). H NMR (CDCl3): δ = 7.38–7.35
(m, 5 H, Harom), 4.80 (m, 1 H, NH), 4.70 (d, J = 11.6 Hz, 1 H,
CHaHb), 4.57 (d, J = 11.6 Hz, 1 H, CHaHb), 4.49 (d, J = 10.3 Hz,
1 H, CHaHbЈ), 4.40 (d, J = 11.7 Hz, 1 H, CHaHbЈ), 4.29 (s, 1 H,
7-H), 4.09–4.02 (m, 3 H, CHaHbЈЈ, 3-H, 4-H), 3.90 (d, J = 7.8 Hz,
1 H, CHaHbЈЈ), 3.14–3.03 (m, 5 H, CHaHbЈЈЈ, CHaHbЈЈЈ and CH3),
1.44 (s, 9 H, C(CH3)3) ppm. 13C NMR (CDCl3): δ = 156.1 (CO)
137.3, 128.5, 128.4, 127.8 (Ar), 84.0 (C-1), 83.1 (C-3), 78.5 (CH2),
77.5 (C-7), 72.5, 72.1 (CH2, C-4) 66.2 (CH2), 42.3 (CH2), 37.7
(CH3), 28.3 (C(CH3)3, Boc) ppm. MALDI-MS: m/z 466.1 [M +
Na]+. HRMS: calcd. for C19H29NO7Na 466.1506; found m/z
466.1486.
1
eluent, yielding the allitol 4 (10.25 g, 84%). H NMR (CDCl3): δ
= 8.12–8.09 (m, 1 H, Harom), 8.03–8.00 (m, 1 H, Harom), 7.61–7.41
(m, 4 H, Harom), 7.22 (m, 4 H, Harom), 5.29–5.26 (m, 1 H, 3-H),
4.62 (d, J = 11.5 Hz, 1 H, CHaHb), 4.57 (d, J = 11.4 Hz, 1 H,
CHaHb), 4.29 (dd, J = 4.9 Hz and J = 10.3 Hz, 1 H, 2-H), 4.24 (d,
J = 12.3 Hz, 1 H, CHaHbЈ), 4.03 (d, J = 5.0 Hz, 1 H, 4-H), 3.91
(d, J = 12.2 Hz, 1 H, CHaHbЈ), 3.76–3.68 (m, 2 H, CHaHbЈЈ and
CHaHbЈЈ), 3.45–3.20 (m, 2 H, CHaHbЈЈЈ and CHaHbЈЈЈ), 1.43 (m,
15 H, C(CH3)3, 2×CH3, isoprop) ppm. 13C NMR (CDCl3): δ =
165.7 (CO), 137.4, 133.4, 129.8, 129.4, 128.5, 128.4, 128.3, 128.1
(Ar), 98.2 (C(CH3)2), 79.5, 79.4, 78.4, 74.1, 73.1 (C-5, C-4, C-3,
C-2, C(CH3)3, CH2Bn), 66.4, 62.7 (C-6, C-6Ј), 42.5 (C-1), 28.3
(C(CH3)3), 23.9, 23.2 (2 × C(CH3)2) ppm. HRMS: calcd. for
C29H37NO8Na 550.2411; found m/z 550.2420.
(1S,3S,4S,7S)-7-Benzyloxy-3-[(tert-butoxycarbonyl)aminomethyl]-1-
(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (7): NaOBz (2.67 g,
18.5 mmol) was added to a stirred solution of 6 (5.20 g, 11.7 mmol)
in anhydrous DMF (200 mL). The reaction mixture was stirred for
14 h at 100 °C, was allowed to reach room temp., and was sub-
sequently filtered. EtOAc (250 mL) was added to the filtrate and
the resulting mixture was washed successively with H2O
(3×200 mL) and brine (2×150 mL). The organic phase was dried
(MgSO4), filtered, and concentrated to dryness under reduced pres-
sure. The residue was dissolved in a mixture of ethanol (125 mL)
and aq. NaOH (4 , 125 mL, 0.5 mol) and stirred for 2 h at room
temp. Ethyl acetate (200 mL) was added and the organic phase was
separated, washed successively with H2O (2×100 mL) and brine
(50 mL), dried (MgSO4), filtered, and concentrated to dryness un-
der reduced pressure. The residue was purified by flash silica gel
column chromatography with ethyl acetate in petroleum ether [0%
Ǟ 20 % (v/v)] as eluent, yielding 7 (3.12 g, 73 %). 1H NMR
(CDCl3): δ = 7.36 (m, 5 H, Harom, Bn), 4.91 (br.s, 1 H, NH), 4.67
(d, J = 10.2 Hz, 1 H, CHaHb), 4.62 (d, J = 9.9 Hz, 1 H, CHaHb),
4.20 (s, 1 H, 7-H), 4.08–4.01 (m, 2 H, CHaHbЈ, 3-H), 3.85–3.79 (m,
2 H, CHaHbЈ, 4-H), 3.37 (m, 2 H, CHaHbЈЈ and CHaHbЈЈ), 3.19–
3.13 (m, 1 H, CHaHbЈЈЈ), 3.06–2.97 (m, 1 H, CHaHbЈЈЈ), 1.47 (s, 9
H, C(CH3)3) ppm. 13C NMR (CDCl3): δ = 156.3 (CO), 137.5,
128.4, 127.8, 127.6 (Ar), 86.6 (C-1), 82.6 (C-3), 79.8 (C(CH3)3),
78.2 (CH2), 77.6 (C-7), 72.6 (C-6, rotamer), 72.0 (C-4), 69.2 (C-6,
rotamer), 58.6 (CH2), 42.5 (CH2), 28.3 (C(CH3)3) ppm. HRMS:
calcd. for C19H27NO6Na 388.1731; found m/z 388.1726.
2,5-Anhydro-3-O-benzoyl-4-O-benzyl-1-[(tert-butoxycarbonyl)-
amino]-1-deoxy-6-O-(methylsulfonyl)-5-C-[(methylsulfonyloxy)-
methyl]-D-allitol (5): p-Toluenesulfonic acid (760 mg, 4.80 mmol)
was added to a stirred solution of 4 (10.25 g, 19.43 mmol) in
MeOH (200 mL). After 45 min, a sat. aq. solution of NaHCO3
(25 mL) was added and stirring was continued for 15 min. The mix-
ture was concentrated to dryness by evaporation under reduced
pressure and ethyl acetate (300 mL) was added to the resulting resi-
due. The separated organic phase was washed successively with a
sat. aq. solution of NaHCO3 (3× 100 mL) and brine (100 mL),
dried (MgSO4), filtered, and concentrated to dryness under reduced
pressure. The residue was dissolved in anhydrous DCM (250 mL)
and cooled to 0 °C. MsCl (7.7 mL, 100 mmol) and TEA (16.7 mL,
120 mmol) were added to the resulting mixture. After 3.5 h of stir-
ring, a sat. aq. solution of NaHCO3 (25 mL) was added to the
reaction mixture. The mixture was stirred for 15 min and was then
concentrated to dryness under reduced pressure. The residue was
dissolved in ethyl acetate (300 mL) and the resulting mixture
washed successively with a sat. aq. solution of NaHCO3
(3×100 mL) and brine (100 mL), dried (MgSO4), filtered, and con-
centrated to dryness under reduced pressure. The residue was puri-
fied by silica gel column chromatography with ethyl acetate in pe-
troleum ether [0% Ǟ 50% (v/v)] as eluent, yielding allitol 5 (9.20 g,
1
73%). H NMR (CDCl3): δ = 8.07–8.04 (m, 2 H, Harom), 7.61 (t,
J = 7.4 Hz, 1 H, Harom), 7.47 (t, J = 7.7 Hz, 2 H, Harom), 7.29–7.20 (1S,3S,4S,7S)-3-{[(Fluorenylmethoxy)carbonyl]aminomethyl}-7-
(m, 5 H, Harom), 5.48 (t, J = 4.2 Hz, 1 H, 3-H), 5.06 (m, 1 H, NH),
4.77 (d, J = 11.7 Hz, 1 H, CHaHb), 4.64 (d, J = 11.1 Hz, 1 H,
hydroxy-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (8): HCl
[37% (wt.-%), five drops] and a catalytic amount of Pd on charcoal
CHaHbЈ), 4.46 (d, J = 11.2 Hz, 1 H, CHaHbЈ), 4.40–4.35 (m, 2 H, (10 wt.-%, 20 mg) were added to a solution of 7 (1.50 g, 4.11 mmol)
Eur. J. Org. Chem. 2006, 2538–2548
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2545

C. Verhagen, T. Bryld, M. Raunkjær, S. Vogel, K. Buchalová, J. Wengel
FULL PAPER
in ethanol (40 mL). N2 was bubbled through the solution for 5 min,
after which H2 was flushed through the system. The reaction mix-
ture was stirred for 14 h at room temp. under an atmosphere of H2.
The reaction mixture was filtered, and the filtrate was evaporated
to dryness under reduced pressure. The residue was dissolved in a
mixture of THF and H2O (4:1 v/v, 50 mL) and MgO (0.82 g,
20.5 mmol) was added. The resulting mixture was cooled to 0 °C
and stirred for 5 min before addition of FmocCl (3.12 g,
12.3 mmol). The reaction mixture was stirred for 1 h at 0 °C fol-
lowed by filtration. The residue was washed with EtOAc (50 mL)
and the combined filtrate was evaporated to dryness under reduced
pressure. The residue was purified by flash silica gel column
chromatography with MeOH in DCM [0% Ǟ 10% (v/v)] as eluent,
yielding 8 (1.26 g, 77%). 1H NMR (CDCl3): δ = 7.73 (d, J = 7.6 Hz,
2 H, Harom), 7.54 (d, J = 7.4 Hz, 2 H, Harom), 7.38 (t, J = 7.6 Hz,
2 H, Harom), 7.27 (t, J = 7.4 Hz, 2 H, Harom), 5.54–5.50 (m, 1 H,
NH), 4.36 (d, J = 6.4 Hz, 2 H, CH2,Fmoc) 4.21 (s, 1 H, 4-H), 4.14
(t, J = 6.4 Hz, 1 H, CHFmoc) 4.09 (s, 1 H, 7-H), 3.98 (t, J = 6.3 Hz,
1 H, 3-H), 3.92 (d, J = 8.2 Hz, 1 H, CHaCHb), 3.26–3.19 (m, 3 H,
CH2, CHaHb), 3.26–3.19 (m, 1 H, CHaHbN), 3.10–3.01 (m, 1 H,
CHaHbN) ppm. 13C NMR (CDCl3): δ = 157.0 (CO), 143.7, 141.2,
127.7, 127.0, 124.9, 120.0 (Ar), 87.1 (C-1), 81.7 (C-3), 80.1 (C-7),
71.8 (C-6), 71.6 (C-4), 66.8 (CHFmoc), 58.0 (CH2), 47.0 (CHFmoc),
42.9 (CH2) ppm. HRMS: calcd. For C22H23NO6Na 420.1418;
found m/z 420.1418.
149.5 ppm. HRMS: calcd. for C52H58N3O9PNa: 922.3803; found
m/z 922.3808.
(1S,3S,4S,7S)-7-Benzyloxy-3-{[(fluorenylmethoxy)carbonylamino]-
methyl}-1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptane (11): A
solution of 7 (500 mg, 1.37 mmol) in TFA (5 mL) was stirred for
30 min. The reaction mixture was evaporated to dryness under re-
duced pressure and the residue was dissolved in a mixture of THF
and H2O (4:1 v/v, 20 mL) and was cooled to 0 °C. MgO (270 mg,
6.70 mmol) was added and the mixture was stirred for 5 min at
0 °C, followed by addition of FmocCl (390 mg, 1.51 mmol). The
resulting mixture was stirred for 1 h at 0 °C, and H2O (5 mL) and
ethyl acetate (20 mL) were added. The separated organic phase was
washed with sat. aq. NaHCO3 (3×10 mL) and brine (10 mL), dried
(MgSO4), filtered, and concentrated to dryness under reduced pres-
sure. The residue was purified by flash silica gel column chromatog-
raphy with MeOH and DCM [0% Ǟ 10% (v/v)] as eluent, yielding
11 (577 mg, 86%). 1H NMR (CDCl3): δ = 7.75–7.24 (m, 13 H,
H
arom), 5.14 (br. s, 1 H, NH), 4.65–4.53 (m, 2 H, CHaHb and
CHaHb), 4.46–4.33 (m, 2 H, CH2Fmoc), 4.19–4.15 (m, 2 H, 7-H,
CHFmoc), 4.05–3.99 (m, 3 H, CHaHbЈ, CHaHbЈ and 3-H), 3.84–3.78
(m, 3 H, CHaHbЈЈ, CHaHbЈЈ and 4-H), 3.25–3.18 (m, 1 H,
CHaHbЈЈЈ), 3.06–2.97 (m, 1 H, CHaHbЈЈЈ) ppm. 13C NMR (CDCl3):
δ = 156.9 (CO), 143.8, 141.4, 137.6, 128.5, 128.0, 127.8, 127.7,
127.1, 125.0, 125.0, 120.1 (Ar), 86.7 (C-1), 82.5 (C-3), 78.2 (CH2),
77.7 (C-7), 72.7, 72.1, 66.9 (CH2Fmoc), 58.7 (C-6), 47.2, (CHFmoc),
43.1 (CH2) ppm. HRMS: calcd. for C29H29NO6Na: 510.1887;
found m/z 510.1871.
(1R,3S,4S,7S)-1-[(4,4Ј-Dimethoxytrityl)oxymethyl]-3-{[(fluorenyl-
methoxy)carbonylamino]methyl}-7-hydroxy-2,5-dioxabicyclo[2.2.1]-
heptane (9): TEA (320 µL, 2.30 mmol) and DMTCl (560 mg,
1.65 mmol) were added to a solution of 8 (600 mg, 1.51 mmol) in
a mixture of anhydrous DCM and anhydrous acetonitrile (1:1 v/v,
30 mL). The reaction mixture was stirred for 1.5 h at room temp.,
after which H2O (25 mL) and ethyl acetate (30 mL) were added.
The organic phase was separated and washed successively with a
sat. aq. solution of NaHCO3 (3×25 mL) and brine (25 mL), dried
(MgSO4), filtered, and concentrated to dryness under reduced pres-
sure. The residue was purified by silica gel column chromatography
with TEA and ethyl acetate in petroleum ether [1%, 1% Ǟ 40%
(1S,3S,4S,7S)-7-Benzyloxy-3-{[(fluorenylmethoxy)carbonylamino]-
methyl}-2,5-dioxabicyclo[2.2.1]heptane-1-carboxylic
Acid
(12):
Dess–Martin periodinane (600 mg, 1.41 mmol) was added to a
solution of 11 (458 mg, 0.94 mmol) in anhydrous DCM (15 mL).
The reaction mixture was stirred for 3 h at room temp., after which
ethyl acetate (20 mL), a sat. aq. solution of NaHCO3, and 10%
(wt.-%) aq. Na2S2O3 (1:1 v/v, 20 mL) were added. The resulting
mixture was stirred at room temp. until both phases became clear.
The organic phase was separated, washed successively with sat. aq.
NaHCO3 (3×10 mL) and brine (10 mL), dried (MgSO4), filtered,
and concentrated to dryness under reduced pressure. The residue
was dissolved in a mixture of tBuOH (10 mL) and 2-methylbut-2-
ene (1 mL) followed by addition of aq. NaH2PO4 (5.05 , 10 mL,
50.5 mmol). To this solution was added NaClO2 (914 mg,
10.1 mmol, in small portions over 15 min). The reaction mixture
was stirred vigorously for 2 h and then cooled to 0 °C, aq. HCl
(1 , 20 mL, 20 mmol) was added, and extraction was performed
with CHCl3 (3×20 mL). The combined organic phase was dried
(MgSO4), filtered, and concentrated to dryness under reduced pres-
sure. The residue was purified by silica gel column chromatography
with acetic acid in ethyl acetate [1%, (v/v)] as eluent, yielding 12
(358 mg, 76%). 1H NMR (CD3OD): δ = 7.77–7.23 (m, 13 H,
Harom), 4.56 (br.s, 2 H, CHaHb and CHaHb), 4.42–3.95 (m, 8 H,
CHFmoc, CH2Fmoc, CHaHbЈ, CHaHbЈ, CHaHbЈЈ, CHaHbЈЈ, 3-H, 4-
H, 7-H), 3.17–3.07 (m, 2 H, CHaHbЈЈЈ and CHaHbЈЈЈ) ppm. 13C
NMR (CD3OD): δ = 170.6 (COOH), 158.8 (CONH), 145.2, 142.6,
138.9, 129.4, 128.8, 128.7, 128.1, 126.2, 126.1, 120.9 (Ar), 85.1
(C-1), 84.7, 82.4, 80.2, 73.9, 73.1, 67.8 (CH2Fmoc), 48.3 (CHFmoc),
43.3 (CH2) ppm. HRMS: calcd. for C29H27NO7Na: 524.1680;
found m/z 524.1660.
1
(v/v/v)] as eluent, yielding 9 (0.740 g, 71%) . H NMR (CDCl3): δ
= 7.76–7.74 (m, 2 H, Harom), 7.58–7.56 (m, 2 H, Harom), 7.44–7.20
(m, 13 H, Harom), 6.84–6.81 (m, 4 H, Harom), 5.00 (br.s, 1 H, NH),
4.42–4.40 (m, 2 H, CHaHb and CHaHb), 4.18–4.08 (m, 1 H,
CHFmoc), 4.06–3.99 (m, 6 H, CHaHbЈ, CHaHbЈ, CH2Fmoc, 3-H and
7-H), 3.77 (s, 6 H, OMe), 3.42–3.33 (m, 2 H, CHaHbЈЈ and 4-H),
3.08–3.04 (m, 1 H, CHaHbЈЈ) ppm. 13C NMR (CDCl3): δ = 158.5
(CO), 149.8, 144.5, 143.8, 141.3, 135.6, 135.5, 130.0, 128.1, 127.9,
127.7, 127.1, 126.9, 125.0, 120.0, 113.2, (Ar), 86.3, 85.9, (C-1, Cq
DMT), 81.4 (C-3), 80.2 (C-7), 73.7, 72.4 (CH2, C-4), 66.9
(CH2Fmoc), 60.8 (CHFmoc), 55.2 (OMe), 47.2 (C-6) 43.0 (CH2) ppm.
HRMS: calcd. For C43H41NO8Na: 722.2724; found m/z 722.2740.
(1R,3S,4S,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphanyl-
oxy]-1-[(4,4Ј-dimethoxytrityl)oxymethyl]-3-{[(fluorenylmethoxy)-
carbonylamino]methyl}-2,5-dioxabicyclo[2.2.1]heptane (10): DIPEA
(0.9 mL) and 2-cyanoethyl N,N-diisopropylphosporamidochlorid-
ite (200 µL, 0.965 mmol) were added to a solution of 9 (575 mg,
0.823 mmol) in anhydrous DCM (2.6 mL). The reaction mixture
was stirred for 1 h at room temp. before addition of ethyl acetate
(10 mL) and H2O (5 mL). The organic phase was separated and
washed successively with a sat. aq. solution of NaHCO3 (3×5 mL)
and brine (5 mL), dried (MgSO4), filtered, and concentrated to dry-
ness under reduced pressure. The residue was purified by silica gel
column chromatography with TEA and ethyl acetate in petroleum
ether [1%, 1% Ǟ 29% (v/v/v)] as eluent, yielding the phosphoram-
Oligonucleotide Synthesis: Oligonucleotides were synthesized under
standard phosphoramidite coupling conditions on a 0.2 µol scale
with polystyrene as the solid support and without use of the final
detritylation step in order to leave the 5Ј-end DMT-protected. The
idite 10 (423 mg, 57 %). 31P NMR: ([D6]DMSO), δ = 150.3, stepwise coupling efficiencies for phosphoramidite 10 were 95%
2546
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 2538–2548

Conformationally Locked Aminomethyl C-Glycoside
FULL PAPER
with 15 min coupling time and with 1H-tetrazole as catalyst. The
coupling yield for unmodified deoxynucleoside phosphoramidites
Fluorescence Experiments: Steady-state fluorescence emission spec-
tra (360–600 nm) were obtained on a Perkin–Elmer LS 55 lumines-
(with 2 min standard coupling time) was Ͼ99% with 1H-tetrazole cence spectrometer fitted with a Peltier temperature controller in
as activator. After completion of oligomerization the support was
transferred from the reaction column to an Eppendorf tube and
treated with piperidine in DMF (20%, 1 mL) for 20 min. The su-
pernatant was removed by syringe (small needle), and the polysty-
rene was washed with acetonitrile (4×1 mL). A mixture of
pyrene-1-carboxylic acid, O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-tetra-
methyluronium hexafluorophosphate (3.7 mg, 9.5 µmol), and diiso-
propylethylamine (10 µL, 5.7 µmol) in DMF (1 mL) was added to
the Eppendorf tube containing the polystyrene, which was then
vortexed gently for 45 min. The supernatant was removed by sy-
ringe (small needle) and the polystyrene was washed with DMF
(2×1 mL) and MeOH (2×1 mL). The oligonucleotide was released
from the solid support by treatment with saturated aqueous ammo-
nia for 14 h at 55 °C, which also cleaved off the protecting groups
on the nucleobases. The 5Ј-O-DMT-protected oligonucleotides
were subsequently purified (minimum 80% purity) on a Waters
Prep LC 4000 system fitted with a Xterra MS C18-column (10 µm,
300 mm×7.8 mm) with use of an isocratic hold of 100% A-buffer
for 5 min followed by a linear gradient to 55% B-buffer over 75 min
at a flow rate of 1.0 mLmin–1 (A-buffer: 95% 0.1  NH4HCO3,
5% CH3CN; B-buffer: 25% 0.1  NH4HCO3, 75% CH3CN). The
DMT group was cleaved off by treatment with AcOH (80%,
100 µL) for 20 min followed by addition of sodium acetate (3 ,
50 µL), water (100 µL), and ethanol (600 µL). The solution was
cooled to –18 °C for 1 hour, which caused the oligonucleotide to
precipitate. The oligonucleotide was isolated after centrifugation at
5 °C by decanting of the supernatant.
quartz cuvettes with a path length of 1.0 cm and a concentration
of strands in Tm buffer of 0.15 µ. Spectra were recorded at 20 °C
with an average of 5 scans with use of an excitation wavelength of
340 nm, excitation slit of 4.0 nm, emission slit of 2.5 nm, and scan
speed of 120 nmmin–1.
Supporting Information (for details see the footnote on the first
page of this article): Copies of 13C NMR spectra of compounds 2–
12.
Acknowledgments
We thank the Danish National Research Foundation for funding
the Nucleic Acid Center. This work was supported by Sixth Frame-
work Programme Marie Curie Host Fellowships for Early Stage
Research Training under contract number MEST-CT-2004-504018.
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Received: December 15, 2005
Published Online: March 30, 2006
2548
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