Oligodeoxynucleotides containing diastereomeric O2′,C3′-linked bicyclic nucleotide units for functionalization of the major groove of nucleic acid duplexes: A summary and novel derivatives

Article abstract of DOI:10.1081/CAR-200067044

The synthesis and evaluation of a range of piperazino-derivatized diastereomeric O2′,C3′-linked bicyclic nucleotides are described. A new and optimized protocol is presented for the synthesis of the bicyclic scaffold on which the piperazino moiety is appe

Full text of DOI:10.1081/CAR-200067044

This article was downloaded by: [University of Colorado at Boulder Libraries]
On: 27 December 2014, At: 07:48
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lcar20
Oligodeoxynucleotides Containing
Diastereomeric O2′,C3′‐linked Bicyclic
Nucleotide Units for Functionalization
of the Major Groove of Nucleic Acid
Duplexes: A Summary and Novel
Derivatives
Michael Raunkjær a , Kim F. Haselmann b & Jesper Wengel a
a Nucleic Acid Center, Department of Chemistry , University of
Southern Denmark , Odense M, Denmark
b Mass Spectrometry Laboratory, Department of Chemistry ,
University of Southern Denmark , Odense M, Denmark
Published online: 16 Aug 2006.
To cite this article: Michael Raunkjær , Kim F. Haselmann & Jesper Wengel (2005)
Oligodeoxynucleotides Containing Diastereomeric O2′,C3′‐linked Bicyclic Nucleotide Units for
Functionalization of the Major Groove of Nucleic Acid Duplexes: A Summary and Novel Derivatives ,
Journal of Carbohydrate Chemistry, 24:4-6, 475-502
To link to this article: http://dx.doi.org/10.1081/CAR-200067044
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Carbohydrate Chemistry, 24:475–502, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200067044
Oligodeoxynucleotides
Containing Diastereomeric
O20,C30-linked Bicyclic
Nucleotide Units for
Functionalization of the
Major Groove of Nucleic
Acid Duplexes: A Summary
and Novel Derivatives
Michael Raunkjær
Nucleic Acid Center, Department of Chemistry, University of Southern Denmark,
Odense M, Denmark
Kim F. Haselmann
Mass Spectrometry Laboratory, Department of Chemistry, University of Southern
Denmark, Odense M, Denmark
Jesper Wengel
Nucleic Acid Center, Department of Chemistry, University of Southern Denmark,
Odense M, Denmark
The synthesis and evaluation of a range of piperazino-derivatized diastereomeric
O20,C30-linked bicyclic nucleotides are described. A new and optimized protocol is
presented for the synthesis of the bicyclic scaffold on which the piperazino moiety is
Received March 29, 2005; accepted April 25, 2005.
Dedicated to the memory of professor Jacques H. van Boom.
Address correspondence to Jesper Wengel, Nucleic Acid Center, Department of
Chemistry, University of Southern Denmark, Campusvej 55, DK-5230, Odense M,
Denmark. E-mail: jwe@chem.sdu.dk
475

M. Raunkjær, K. F. Haselmann, and J. Wengel
476
appended. At low salt concentration, the C200-S-configured piperazino-modified oligonu-
cleotides display significantly enhanced hybridization affinity toward complementary
DNA and RNA targets relative to the unmodified oligonucleotide control, whereas no
melting transition is observed for hybrids formed with the C200-R-configured pipera-
zino-modified oligonucleotides. Upon derivatization of the piperazino moiety with a
1-pyrenebutanoyl group, all modified oligonucleotides display strong DNA binding
and profound DNA hybridization selectivity.
Keywords Oligonucleotide, Bicyclic nucleotide, Fluorescence, Pyrene, Low salt
hybridization, DNA selectivity, RNA selectivity
INTRODUCTION
During the past two decades, chemically modified oligonucleotides (ONs) have
been intensively investigated toward the development of novel therapeutic and
diagnostic agents. The desire to create ONs with enhanced nuclease stability,
cell penetration ability, or increased binding affinity toward complementary
nucleic acids has motivated the discovery of, for example, radically modified
analogs as peptide nucleic acid (PNA, Fig. 1),[1] bicyclic ONs as locked
nucleic acid (LNA, Fig. 1),[2,3] or six-membered analoges like hexitol nucleic
acid (HNA, Fig. 1).[4] The hybridization properties of modified ONs have also
Figure 1: C ¼ C40-(pyren-1-yl)carbonylpiperazinomethyl-DNA and D ¼ N20-(pyren-1-yl)
methyl-20-amino-LNA.

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
477
been modulated by introduction of analogs with cationic groups embarking on a
gain in duplex stability by overall net charge reduction. This has been studied
by modifying the base,[5–11] phosphate,[12–14] or sugar[15–21] moieties of
nucleotides.
The scope of ON applications can be broadened considerably by conjugation
of an ON with additional moieties.[22] For synthetic convenience, the position
most frequently used for conjugation is the 50-end of an ON. Nevertheless, it
is desirable if conjugation can also be performed internally in the sequence.
Recently, we have demonstrated the utility of 20-amino-LNA nucleotides[23]
and piperazino-derivatized nucleotides[24–26] for this purpose, thereby extend-
ing the scope of piperazino-derivatized ONs previously investigated by our
group.[27] Thus, a pyrene-functionalized 40-C-piperazinomethyl nucleotide
(C, Fig. 1), when incorporated into an ON, induces DNA-selective hybridization
with the pyrene moiety facing the minor groove of the duplex.[25] Likewise,
pyrene-derivatives of 20-amino-LNA nucleotides D (Fig. 1) induced enhanced
thermal stabilities (Tm values) when placed in one strand, or in both strands
in a zipperlike constitution, in all cases with the pyrene moiety facing the
minor groove.[23]
In 1997, our group reported the synthesis of the O20,C30-linked bicyclic
nucleotide P (Fig. 2) in the search for novel bicyclic nucleotide analogs.[28]
Incorporation of monomer P in 14-mer thymidylate sequences resulted in a
decrease in affinity toward the complementary DNA targets when compared
to the unmodified reference duplex. Overall, the affinity was observed to
decrease significantly when several monomers P were incorporated into the
ON. Furthermore, it was not possible to detect any melting transition for
50-P13T: DNA. In general, the modified sequences displayed a decreased
affinity toward complementary RNA targets, except for a slight increase of
18C in Tm value observed when investigating 50-T5P4T5 : RNA. Interestingly,
a large increase in thermal stability (DTm ¼ 148C) was obtained for
50-P13T: RNA. This result suggests the helical structure of 50-P13T: RNA to
be different from 50-T14 : RNA, since sequences having a single or several inter-
dispersed monomers P display destabilizing behavior when targeting comp-
lementary RNA.
With the observed RNA selectivity in mind, we decided to study further the
applicability of the bicyclic monomer P. Appending a conjugation point to the
bicyclic scaffold would allow attachment of different moieties such as alkyla-
mines, oligopeptides, oligonucleotides, or pyrene groups to the modified ON.
The position of attachment(s) in the ON could be decided simply by choosing
the order of incorporation of unmodified and modified monomers. Also, the
position of conjugation to the bicyclic frame was of importance, and we
decided to install the conjugation moiety on C200. In this way, a conjugated
group was enforced to face the major groove of the helical structure obtained
when hybridizing toward a complementary strand.

M. Raunkjær, K. F. Haselmann, and J. Wengel
478
Figure 2: Structures of the O20, O30-linked bicyclic nucleotide monomers.
The initial work of constructing a bicyclic scaffold with a conjugation point
attached was carried out as shown in Scheme 1.[29] Starting from nucleoside
1,[28] a dihydroxylation reaction was performed using OsO4 and as co-oxidant
NMO. In this way, triol 2 was provided in 88% yield as an inseparable
Scheme 1: Reagents and conditions : i) OsO4, NMO, THF, H2O, rt, 15 hr, 88%; ii) TBDMSCl,
pyridine, rt, 15 hr, 3S : 53%, 3R : 31%, mixture : 13%. T ¼ thymin-1-yl.

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
479
mixture of diastereomers (ratio 1 : 2, R : S). Regioselective TBDMS protection of
the primary hydroxyl group allowed separation of the diastereomers providing
nucleoside 3R (31% yield) and 3S (53% yield) as well as a mixture of the two
diastereomers (14%).[29]
Regioselective tosylation followed by a base-mediated intramolecular ring
closure afforded the O20,C30-linked bicyclic nucleoside 5R (from 3S) and 5S
(from 3R) in yields of 45% and 52%, respectively (Sch. 2). For convenience of
ON synthetic protocols, the TBDMS group was exchanged with an acetyl
group providing nucleoside 7R in 76% yield from 5R. Hydrogenolysis using
H2 and Pd(OH)2 on carbon as catalyst afforded the debenzylated nucleoside
8R in 94% yield. The monomer building block for ON synthesis was obtained
by regioselective O50-DMT protection and O30 phosphitylation affording phos-
phoramidite 10R in 58% yield from 8R. Overall, phosphoramidite 10R was
provided in 19% yield from 3S. The same synthetic transformations were
carried out for the C200-S-configured nucleosides, thereby providing a synthetic
route yielding phosphoramidite 10S in an overall yield of 20% from 3R (the use
of “ꢀ” indicates that reactions are independently carried out on each
diastereomer).[29]
Scheme 2: Reagents and conditions : i) TsCl, pyridine, rt, 72 hr; ii) K2CO3, 18-crown-6, DMF,
708C, 24 hr, 5R : 45% (from 3S) and 5S : 52% (from 3R); iii) TBAF, THF, 3 hr, rt, 6R : 80% and 6S : 94%;
iv) Ac2O, pyridine, rt, 15 hr; 7R : 96% and 7S : 92%; v) H2, Pd(OH)2/C, EtOH, rt, 96 hr, 8R : 94% and
8S : 95%; vi) DMTCl, pyridine, rt, 15 hr, 9R : 92% and 9S : 96%; vii) 2-cyanoethyl N,N-
diisopropylphosphoramidochloridite, DIPEA, DCM, rt, 18 hr, 10R : 64% and 10S : 50%; viii) DNA-
synthesizer. T ¼ thymin-1- yl. The use of “ꢀ” indicates that reactions are independently carried
out on each diastereomer.

M. Raunkjær, K. F. Haselmann, and J. Wengel
480
Phosphoramidites 10R and 10S were used as building blocks for incorpor-
ation of monomers R and S, respectively (Sch. 2). Thermal denaturation
studies revealed monomer S to behave like parent monomer P when incorpor-
ated into a 14-mer homothymidylate sequence (Table 1). An increase in
thermal stability (DTm of þ148C) was observed for 50-S13T: RNA, whereas no
thermal transition was detectable for 50-S13T: DNA. Hence, monomer S
seems a promising candidate for attachment of different moieties to duplexes
formed with RNA complements since the properties of monomer P are pre-
served. Interestingly, monomer R behaves differently from monomer S since
Tm values of 618C were observed for both 50-R13T: RNA, 50-R13T: DNA (ON3
in Table 1) and for 50-R13T alone. Accordingly, we rationalized that monomer
R induces a strong self-complexation in the almost fully modified sequence,
suggesting 50-R13T to be organized in a complex based on T: T basepairing.[29]
CD experiments showed neither an A-type nor a B-type helical structure of the
homo-complex. The introduction of a C200 hydroxymethyl group on the bicyclic
scaffold was clearly influencing the properties of the corresponding ONs. In the
case of the R monomer, one could easily imagine the C200 hydroxymethyl group
to interact with the O2 carbonyl group in the thyminyl moiety via an intramo-
lecular hydrogen bond. This suggested the thymine moiety to be preorganized
in a syn-conformation allowing the formation of a homocomplex.[29]
We have recently introduced monomer M (Fig. 2), a methylated derivative
of monomer R.[26] This monomer was synthesized as a variant of monomer R
that lacks the hydroxyl group needed to form the intramolecular hydrogen
Table 1: Thermal denaturation studies of known ON derivatives at 110 mM [Naþ]
and pH 7.
DNA target
Tm
RNA target
Tm
ON
Sequence
Entry
Ref A
DTm
DTm
50-T14
31
29
ON1
ON2
ON3
50-T7RT6
50- T5R4T5
50- R13T
27a
10a
60a
24
25a
25a
61a
24
221
24
b
b
ON4
ON5
ON6
50-T7MT6
50- T5M4T5
50- M13T
27
10
<10
24
221
24
25
38
25
24
þ9
Thermal denaturation experiments were performed with 1.5 mM of the two complementary
strands in a medium salt buffer: 0.1 mM EDTA, 100 mM sodium chloride, 10 mM sodium phos-
phate, pH 7.0. The melting temperatures [Tm (8C)] were determined as the local maximum of
the first derivative of the melting curve (A260 vs. temperature). DTm values are calculated
relative to the relevant unmodified reference. aTm values are found in ref. 29. bA value of 618C
was obtained in an analogous thermal denaturation experiment without the addition of
complementary strand.

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
481
bond with the base suggested in the case of monomer R. Accordingly, no self-
complexation was observed for 50-M13T (ON6, Table 1). In fact, ON6 displays
similar hybridization properties to those of 50-P13T and 50-S13T with increased
affinity toward complementary RNA (DTm ¼ þ98C) and RNA selective hybrid-
ization. One or four consecutive modifications of M induce a moderate decrease
in thermal stability toward RNA (ON4 and ON5, Table 1), also in analogy to
the results obtained for ONs containing modification R (ON1 and ON2,
Table 1) and corresponding well with the behavior of ONs modified with the
parent compound P. The thermal stability studies of ONs modified with
monomer M showed the RNA binding capability to be preserved when the
C200 hydroxymethyl moiety was chemically functionalized. This result reveals
the structural design of monomer R to be a promising candidate for further con-
jugation that apparently is compatible with both stereochemical configurations
of C200.
These promising results prompted us to design monomer Q (Fig. 2) for
studying the hybridization properties at various pH values and salt concen-
trations of modified ONs carrying the basic piperazino functionality.[26] Hybrid-
ization experiments were carried out on partly modified mixed 9-mer
sequences and showed increased affinity toward RNA targets, relative to the
unmodified duplex, at pH 5 in a 40 mM Naþ buffer. At pH 7, there are no
increases in duplex stability. The thermal stability studies demonstrate the
applicability of the piperazino-moiety for enhancement of duplex stability
under selected conditions. This motivated further use of the distal nitrogen
atom of the piperazino group as a conjugation site enabling further molecular
designs, and we herein report our studies in this direction.
RESULTS AND DISCUSSION
In addition to involving many synthetic steps, the original synthesis of
monomers R and S (Sch. 2) had another drawback : the regioselective tosyla-
tion of the mixture 3R/3S and the base mediated ring closure of the mixture
4R/4S were both low yielding. This prompted us to search for a new synthetic
route toward the ring-closed key nucleosides 6R and 6S. The initial silyl protec-
tion of the primary hydroxyl group of nucleosides 2R/2S (Sch. 1) was omitted.
Instead, the primary hydroxyl group was regioselectively tosylated using tosyl
chloride in anhydrous pyridine (Sch. 3). The crude tosylated nucleoside
mixture 11R/11S was, upon treatment with saturated methanolic ammonia,
converted into an inseparable mixture of the desired ring-closed nucleosides
6S and 6R (in a 1 : 2 ratio). The overall yield calculated from starting nucleo-
sides 2R/2S was 81%. We argue the reaction to take place via an intramolecu-
lar nucleophilic attack of the 20-OH on the C200 of the in situ formed epoxide
12R/12S, thereby forming the kinetically favored ring-closed products
6R/6S. The overall isolated yield was somewhat lower (55%) when the

M. Raunkjær, K. F. Haselmann, and J. Wengel
482
Scheme 3: Reagents and conditions : i) TsCl, pyridine, rt, 12 hr; ii) sat. NH3 in MeOH, MeOH,
08C, 1 hr, (82%) from 2R/2S. T ¼ thymin-1- yl.
ring-closing step was performed with NaH as base and DMF as solvent.
Although the products were obtained as inseparable mixtures, the new syn-
thetic protocol offers fewer synthetic steps and is highly rewarding with
respect to yield and ease of the work-up procedures.
Alternative ways of synthesizing nucleoside mixture 12R/12S were inves-
tigated in an attempt to influence the diastereochemical outcome of the
reaction. Treatment of alkene 1[28] (Sch. 1) with m-CPBA resulted in an 80%
yield of diastereomeric product mixture 12R/12S in a 1 : 1 ratio (100 mg
reaction scale). Unfortunately, the yield was significantly reduced (40–61%
isolated of epoxide mixture 12R/12S) upon scaling up the reaction. Employing
1,1,1-trifluoroacetone and Oxone for the in situ formation of methyl(trifluoro-
methyl)dioxirane[30] as well as use of acetonitrile or benzonitrile in the
presence of H2O2 (the Payne epoxidation)[31,32] failed to give the desired
epoxide. However, with easy access to nucleoside mixture 6R/6S, it was now
possible to investigate several ways of introducing the piperazino moiety.
Attachment via amide bond formation, as in monomer Q,[26] was abandoned
due to instability (slow amide bond cleavage) observed during deprotection
after ON synthesis. Oxidation of the primary alcohol to an aldehyde, a sub-
strate for reductive amination,[33] proved troublesome. Instead, we attempted
to install the piperazino group by a nucleophilic attack of piperazine on a tri-
flated nucleoside target. The triflation of mixture 6R/6S in DCM or anhydrous
pyridine was inefficient as byproducts were formed during the reaction even at
low temperature (–208C). Analytical data suggested these byproducts to be
formed via an elimination reaction of the triflated product. Alternatively, the
use of TsCl in anhydrous pyridine afforded tosylated nucleoside mixture
13R/13S (Sch. 4) as an inseparable mixture of diastereomers without the

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
483
Scheme 4: i) TsCl, pyridine, rt, 12 hr, 85%; ii) piperazine, DMF, 708C, 4 hr, 74%; iii) H2, Pd(OH)2/C,
MeOH, rt, 144 hr, 90%; iv) FmocCl, MgO, THF, H2O, 08C, 30 min, 16R : 65%, 16S : 32%, 17R : 49%,
and 17S : 20%. T ¼ thymin-1-yl.
formation of byproducts. The crude mixture 13R/13S was treated with piper-
azine (10 eq.) in DMF at elevated temperature affording amine mixture 14R/
14S in 74% yield from the mixture 6R/6S. Hydrogenolysis of the benzyl groups
using H2 and Pd(OH)2 on carbon in MeOH as solvent provided the deprotected
nucleoside mixture 15R/15S in 90% yield. Importantly, the 1 : 2 ratio of the
inseparable diastereomeric products was maintained during the high-
yielding (68%) sequence including tosylation, introduction of the piperazino
group, and debenzylation. In this way, the diastereomeric assignment of the
relative ratio between nucleosides 15R and 15S was determined according to
the known diasteromeric ratio known of diol mixture 2R/2S.[34]
The secondary amine in nucleoside mixture 15R/15S was Fmoc protected
by application of regioselective conditions (Sch. 4) : FmocCl was added to a
solution of nucleosides 15R/15S and MgO in a mixture of H2O and THF at
08C, thereby avoiding additional reaction with the hydroxyl groups.[35] The
two diastereomeric products were separated by use of column chromatography,
affording nucleosides 16R and 16S in yields of 65% and 32%, respectively. Fur-
thermore, nucleoside mixture 15R/15S was treated with 1-pyrenebutyric acid
in an EDC-mediated coupling reaction in MeOH as solvent providing nucleo-
sides 17R and 17S in yields of 49% and 20%, respectively,[36] after separation
by column chromatography. The four diastereomerically pure nucleosides

M. Raunkjær, K. F. Haselmann, and J. Wengel
484
thus obtained were each treated with DMTCl and DIPEA in a mixture of DCM
and CH3CN as solvent (1 : 1 ratio) affording nucleosides 18R and 18S (Fmoc
protected) and nucleosides 19R and 19S (pyrene-derivatized) in yields of
85%, 88%, 77%, and 75%, respectively (Sch. 5). Finally, the four O50-DMT pro-
tected nucleosides were each reacted with 2-cyanoethyl N,N-diisopropylpho-
sphoramidochloridite and DIPEA in anhydrous DCM, yielding the target
amidites 20R, 20S, 21R, and 21S in yields of 60%, 68%, 61%, and 24%,
respectively.
Amidites 20R and 20S were used as building blocks for incorporation of
monomers X and W in mixed 9-mer ONs (Table 2) by use of standard
coupling procedures (see the Experimental section for details). Furthermore,
one W monomer was built into a 14-mer oligothymidylate (ON7, Table 2).
The Fmoc moieties were removed during base-mediated deprotection of the
phosphate and nucleobase protecting groups. Caution had to be exercized as
intramolecular strand cleavage was observed when using prolonged
Scheme 5: Reagents and conditions : i) DMTCl, DIPEA, DCM, CH3CN, rt, 30 min, 18R : 84%,
18S : 88%, 19R : 77%, and 19S : 75%; ii) 2-cyanoethyl N,N diisopropylphosphoramidochloridite,
DIPEA, anhydrous DCM, rt, 1 hr, 20R : 60%, 20S : 68%, 21R : 61%, and 21S : 24%; iii) DNA-
synthesizer. T ¼ thymin-1-yl. The use of “ꢀ” indicates that reactions are independently carried
out on each diastereomer.

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
Table 2: Thermal denaturation studies of piperazino-modified ONs
485
[Naþ] ¼ 110 mMa
[Naþ] ¼ 10 mMb
DNA
target
RNA
target
DNA
target
RNA
target
Entry
Sequence
Tm
DTm Tm DTm
Tm
DTm
Tm
,10
DTm
Ref A 50-T14
ON7
31
32
28
16
17
50-T6WT7
þ1
28
0
þ1 ,10
,10
.þ7
Ref B 50-GTG-ATA-TGC
29
29
27
28
28
26
19
,10
17
21 .þ11
ON8
ON9
50-GTG-AWA-TGC
0
22
22
þ1
þ1
16
.þ6
50-GWG-AWA-WGC 27
21 .þ11
ON10 50-GTG-AXA-TGC
ON11 50-GXG-AXA-XGC
27
22 ,10
28 ,10
,10
,10
17 212
See caption below Table 1. a0.1 mM EDTA, 100 mM NaCl, 10 mM sodium phosphate, pH 7.0.
b0.1 mM EDTA, 10 mM sodium phosphate, pH 7.0.
deprotection time. MS analysis suggested a hydrolysis of the O30 phosphate
bond of the modified monomers to occur (data not shown).
The hybridization affinity of ON7 toward complementary DNA and RNA
targets did not decrease in a medium salt buffer (110 mM [Naþ], Table 2),
relative to the unmodified reference Ref A. Similarly, comparable hybridiz-
ation affinity relative to Ref A was observed toward complementary DNA in
the low salt buffer (10 mM [Naþ], Table 2). However, no melting transition
was detected with RNA as complementary target. Relative to Ref B, the
mixed 9-mer ON with one W monomer incorporated showed comparable
affinity toward DNA and RNA targets in the medium salt buffer (ON8,
Table 2). The same tendency was observed for ON9, an ON with three
W monomers incorporated. Apparently, the hybridization affinity toward comp-
lementary targets is not influenced when the piperazino moiety is pointing
away from the nucleobase, that is, with S-configuration of C200. Furthermore,
complexes formed between ON8 or ON9 and complementary strands displayed
a significant increase in Tm value, relative to the reference duplexes, when the
salt concentration was lowered to 10 mM [Naþ]. This enhancement in hybrid-
ization affinity is probably explained by a net charge reduction of the duplex,
because the amino functionality of the piperazino moiety of a monomer W,
anticipated to be at least partially protonated at pH 7,[37] compensates for
the loss of positively charged counterions at reduced salt concentration.
Interestingly, ONs with monomer X incorporated displayed different
hybridization properties than ONs with monomer W incorporated. Thus,
ON10 showed a small decrease in duplex stability toward complementary
DNA and RNA targets, and the binding affinity of ON11 was significantly
reduced (Table 2). Hence, in the low salt buffer (10 mM [Naþ]), neither ON10

M. Raunkjær, K. F. Haselmann, and J. Wengel
486
nor ON11 showed any thermal transition. This indicates that the protonated
piperazino moiety of monomer X is disturbing Watson-Crick base-pairing.
Furthermore, with these experiments we have gained information on how to
covalently attach the piperazino moiety to the C200-R-configured nucleoside.
The choice of an amide bond linkage, as seen for monomer Q, resulted in a
small decrease in Tm toward DNA and RNA targets in a medium salt buffer
at pH 7 (one monomer incorporated). Like ON11, a large decrease in Tm
value was observed in a low salt buffer with three Q monomers incorporated.
Based on these results it seems of less importance whether the piperazino
group is appended to the nucleotide via an amide bond or via an alkylamino-
linkage. The similar hybridization properties observed indicate that it is the
distal amino group in the piperazino moieties that is at least partially proto-
nated at pH 7.
The pyrenyl-derivatized monomers Y and Z were incorporated in mixed
9-mer ONs, and their influence on hybridization properties was investigated
by thermal denaturation studies (Table 3). ONs with one monomer incorpor-
ated, Y in ON12 and Z in ON13, showed a significant increase in hybridization
affinity toward complementary DNA targets with a DTm of þ11 and þ98C
relative to Ref B, respectively. Duplexes of ON12 and ON13 with complemen-
tary RNA also displayed increased Tm values, although not to the same extent
as seen with the DNA target. The same tendency was observed at low salt
concentration.
An ON with two incorporations of either monomer Y or monomer Z (ON14
and ON15, Table 3) was furthermore studied. ON14 displayed the same
tendency as ON12, that is, an enhanced Tm value toward both the DNA
(DTm ¼ þ108C) and the RNA (DTm ¼ þ28C) target in a medium salt buffer.
Table 3: Thermal denaturation studies of pyrenebutanoyl piperazino-modified ONs.
[Naþ] ¼ 110 mMa
[Naþ] ¼ 10 mMb
DNA
target
RNA
target
DNA
target
RNA
target
Entry
Ref B
Sequence
Tm
DTm
Tm
DTm
Tm
DTm
Tm
,10
DTm
50-GTG-ATA-TGC
29
27
,10
ON12 50-GTG-AYA-TGC
ON13 50-GTG-AZA-TGC
40
38
þ11
þ9
33
30
þ6
þ3
25
25
.þ15
.þ15
19 .þ9
17 .þ7
Ref C 50-GCA-ATA-CAC
ON14 50-GCA-YAY-CAC
ON15 50-GCA-ZAZ-CAC
29
39
38
28
30
29
,10
,10
þ10
þ9
þ2
þ2
23 . þ 13 ,10
25
.þ15 ,10
See caption below Table 1. a0.1 mM EDTA, 100 mM NaCl, 10 mM sodium phosphate, pH 7.0.
b0.1 mM EDTA, 10 mM sodium phosphate, pH 7.0.

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
487
ON15 displayed the same characteristics as ON14 in a medium salt buffer. The
DNA selectivity of ON14 and ON15 was even more pronounced in the low salt
buffer. Thus, increases in DTm values of .þ138C and .þ158C relative to Ref
C, respectively, were observed against complementary DNA, whereas no
melting transitions were observed with RNA as complementary target. Thus,
while monomers X or W induce preferential hybridization toward RNA,
monomers Y or Z clearly induce preferential hybridization toward DNA. No
difference in hybridization properties for the pyrenyl-derivatized ONs was
observed for the two different C200 configurations. This observation suggests
that the pyrene moieties are interacting with the duplex, for example, by inter-
calation or by groove binding, and that this effect is dominating the diastereo-
selective effect of the piperazino moiety. Alternatively, the conversion of the
distal amine functionality of the piperazino derivatives into the neutral
amide functionality of the pyrenyl derivatives may suggest that a positive
charge is playing a role for the observed diastereoselective hybridization
characteristics of the piperazino derivatives.
Thermal denaturation studies of duplexes with modified building blocks
incorporated in both strands were also carried out (Table 4). In general, high
Tm values were obtained in these experiments, with the combination
ON13 : ON15 forming the most thermally stable complex both in medium
and low salt buffer. This confirms the duplex stabilizing behavior of ONs
with monomer Y or monomer Z incorporated, and convincingly demonstrates
the applicability of the piperazino-functionalized ONs for display of additional
functionalities in the major groove.
In light of the enhanced hybridization properties induced by incorporation
of monomer Y or Z into ONs, their base-discriminating properties were
evaluated (Table 5). Satisfactory Watson-Crick selectivity was observed for
both ON12 and ON13 as the enhanced Tm values, for the A : T matched
base pair significantly differed from the Tm values measured with the corre-
sponding mismatch base pairs. The ability of a DNA nucleotide placed
between two monomers of Y (or Z) to discriminate mismatched complements
was investigated as well. As depicted in Table 5, incorporation of the pyrene-
derivatized monomers into ON14 and ON15 resulted in very satisfactory
Table 4: Thermal denaturation studies of complexes formed between
two modified strands.
110 mM
[Naþ]
10 mM
[Naþ]
ON14
ON15
ON14
ON15
ON12
ON13
42
43
44
46
ON12
ON13
26
31
31
35
See caption below Table 1. Measurements performed in pH 7.0 buffers.

M. Raunkjær, K. F. Haselmann, and J. Wengel
Table 5: Base pairing selectivity studies, [Naþ] ¼ 110 mM, pH ¼ 7.0.
Target 50-GCA-TBT-CAC
488
B ¼ A
B ¼ C
B ¼ T
B ¼ G
Entry
Sequence
(Tm)
(Tm)
(Tm)
(Tm)
Ref B
ON12
ON13
50-GTG-ATA-TGC
50-GTG-AYA-TGC
50-GTG-AZA-TGC
29
40
38
13
,10
21
18
27
23
18
28
30
Target 50-GTG-ABA-TGC
Ref C
ON14
ON15
50-GCA-TAT-CAC
50-GCA-YAY-CAC
50-GCA-ZAZ-CAC
,10
22
20
12
23
26
29
39
38
20
26
23
See caption below Table 1.
base-discriminating behavior of the central DNA nucleotide, notably with
improved discrimination between the T (matched) and G (mismatched) nucleo-
tides relative to the unmodified reference Ref C.
The fluorescence properties of the pyrene-modified ONs were investigated
using steady-state fluorescence emission spectroscopy. The experiments were
conducted for the single-stranded ONs and for the duplexes formed with comp-
lementary DNA or RNA. Low monomer fluorescence emission bands at
l ¼ 378 nm and l ¼ 398 nm were observed for the singly modified ON12 and
ON13 upon excitation at lmax ¼ 340 nm. The low level of excimer fluorescence
can be explained by a low concentration of ONs, for example, the unlikely event
of having two pyrene units from two different ONs in proximity. Furthermore,
the nucleobases can function as monomer fluorescence quenchers, and the
monomer emission is expected to be reduced by the presence of nearby nucleo-
bases in the flexible ON.[38–42] The fluorescence experiments showed the fluor-
escence monomer emission of the modified ONs to decrease upon duplex
formation with a complementary sequence. This can be explained if the
pyrene units align in the major groove in proximal distance to the nucleobases.
Also, intercalation of the complex by the pyrene unit(s) is a possibility.
Okamoto et al. have investigated the temperature dependence of lmax of an
intercalating pyrene moiety.[43] Upon heating, the pyrenyl-modified ON and
the complementary target dissociated, leading to a red shift of lmax by 6 nm.
Employment of the same strategy on ON12 furnished a lmax red shift of
3 nm, neither ruling out nor confirming intercalation as the mechanism of
action.
The reduced fluorescence emission could also be described by the fluor-
escence quantum yield f. The fluorescence quantum yields were calculated
for the modified ONs alone and for the complexes formed with complementary
targets (Table 6).[44] Clearly, the value of f is reduced when the modified ON is

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
Table 6: Fluorescence quantum yields.
489
Quantum
yield f
Target
DNA
Target
RNA
Without
target
ON14
ON15
ON12
ON13
ON14
ON15
0.014
0.012
0.018
0.019
0.012
0.032
0.022
0.024
0.022
0.034
0.024
0.030
0.026
0.047
0.037
0.045
Fluorescence quantum yields (f) determined in a 110 mM [Naþ] buffer at pH 7.0.
targeting complementary DNA or RNA. In general, the most significant
decrease in f was observed for the complexes formed with monomers incorpor-
ated in each strand.
CONCLUSION
The synthetic route toward diastereomeric O20,C30-linked bicyclic nucleotides
with a hydroxymethyl handle has been successfully optimized. In each of the
two diastereomeric structures, the conjugation handle was derivatized with a
piperazino moiety, and the corresponding phosphoramidites were used for
incorporation of piperazino-modified nucleotides into modified ONs. The C200-
S-configured piperazino-modified ONs displayed significantly enhanced
hybridization affinity toward complementary DNA and RNA targets in a low
salt buffer. No thermal transition was detected when applying the same
conditions to C200-R-configured piperazino-modified ONs. This observation
suggests the protonated piperazino moiety of this monomer to disturb
Watson-Crick base-pairing. We have demonstrated the applicability of both
diastereomeric piperazino-derivatized structures to function as attachment
points by introducing a 1-pyrenebutyric acid moiety by amide bond formation.
A preference for DNA hybridization was revealed for all ONs modified with
pyrene-derivatized nucleotides due to a significant increase in DNA binding
relative to that obtained with the reference ON. Notably, the DNA preference
and excellent base-pairing selectivity were preserved after incorporation of
more than one pyrene-functionalized monomer in an ON. The results
obtained testify to the utility of ONs containing diastereomeric O20,C30-
linked bicylic nucleotide units for functionalization of the major groove of
nucleic acid duplexes.
EXPERIMENTAL
The atom numbering of compounds follows the conventional nucleoside style,
for example, furanose atoms are numbered with a prime and atoms originating

M. Raunkjær, K. F. Haselmann, and J. Wengel
490
from the allyl-moiety (structure 1[28]) are numbered with a double prime. All
compounds are named according to the von Baeyer nomenclature.
General
All reagents were purchased from commercial suppliers and used without
further purification. THF was distilled from sodium and benzophenone ketyl.
Reactions were carried out under an atmosphere of nitrogen when anhydrous
solvents were used. Organic phases were dried using Na2SO4 followed by fil-
tration and evaporation under reduced pressure to achieve crude product
residues. Column chromatography was performed using Silica gel 60 (0.040–
0.063 mm), and fractions containing the product were pooled and evaporated
to dryness. Dichloromethane (DCM) used for column chromatography was dis-
tilled prior to use. TLC analysis was conducted with Merck silica gel plates
(60 F254) with UV absorption (254 nm); with spraying (ammonium molybdate
(25 g/L), ceric ammonium sulfate (10 g/L), and charring at 1508C; or with
H2SO4 (5 vol%) in EtOH and charring at 1508C as methods applied for detec-
1
tion. H, 13C, and 31P NMR spectra were recorded with a Bruker WM-300
(300/75.1 MHz) spectrometer; chemical shifts are given in ppm (d) relative to
tetramethylsilane as internal standard and relative to 85% H3PO4 as
1
external reference (31P NMR). When given, assignments of H and 13C peaks
are based on 1H-1H COSY and 1H-13C HMQC spectra. HRMS (high resolution
mass spectrometry) was performed by matrix-assisted laser-desorption ioniz-
ation-mass spectrometry (MALDI-MS) on a 4.7 Tesla Ultima (IonSpec, Irvine,
CA) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.
Nano-electrospray mass spectrometry was performed on the same instrument.
Fluorescence measurements were performed on a Perkin-Elmer LS 55 lumines-
cence spectrometer equipped with a Peltier temperature controller.
(1S,3R,5R,6R,8R)-5-Benzyloxy-6-benzyloxymethyl-3-hydroxymethyl-8-
(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane (6R)[29] and (1S,3S,5R,-
6R,8R)-5-Benzyloxy-6-benzyloxymethyl-3-hydroxymethyl-8-(thymin-1-
yl)-2,7-dioxabicyclo[3.3.0]octane (6S).[29] To a solution of compounds 2R/
2S (4.01 g, 7.83 mmol) in anhydrous pyridine (50 mL) was added TsCl (2.23 g,
11.72 mmol). The reaction mixture was stirred at rt for 12 hr before addition
of a sat. aq. solution of NaHCO3 (10 mL). The resulting mixture was evaporated
to dryness under reduced pressure and the residue was dissolved in a mixture
of EtOAc (100 mL) and H2O (50 mL). The organic phase was separated and
washed successively with a sat. aq. solution of NaHCO3 (3 ꢁ 50 mL) and H2O
(50 mL), and subsequently dried and evaporated to dryness under reduced
pressure. The residue was dissolved in a precooled mixture of MeOH and sat.
methanolic NH3 (50 mL, 1 : 1 v/v) and stirred at 08C for 1 hr. The reaction
mixture was evaporated to dryness under reduced pressure and the residue

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
491
was purified by silica gel column chromatography with MeOH in DCM [0–4%
(v/v)] as eluent. An inseparable mixture of diastereomers 6R/6S (ratio 1 : 2;
S : R) was obtained in yield of 3.17 g (82%). Analytical data were consistent
with the data reported in literature.
(1S,3R,5R,6R,8R)-5-Benzyloxy-6-benzyloxymethyl-3-piperazinometyl-
8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane (14R) and (1S,3S,5R,-
6R,8R)-5-Benzyloxy-6-benzyloxymethyl-3-piperazinomethyl-8-(thymin-
1-yl)-2,7-dioxabicyclo[3.3.0]octane (14S). TsCl (2.24 g, 11.74 mmol) was
added to a solution of compound 6R/6S (2.90 g, 5.87 mmol) in anhydrous
pyridine. The reaction mixture was stirred at rt for 12 hr before addition of a
sat. aq. solution of NaHCO3 (10 mL). The resulting mixture was evaporated
to dryness under reduced pressure and the residue was dissolved in a
mixture of EtOAc (100 mL) and H2O (50 mL). The organic phase was washed
successively with a sat. aq. solution of NaHCO3 (3 ꢁ 50 mL) and H2O
(50 mL), and was dried and evaporated to dryness under reduced pressure.
The residue was dissolved in anhydrous DMF, and piperazine (5.22 g,
60.7 mmol) was added. The reaction mixture was stirred at 708C for 4 hr and
then evaporated to dryness under reduced pressure. The product was purified
by silica gel column chromatography with MeOH in DCM [2–6% (v/v)] as
eluent. The products were obtained as an inseparable mixture of diastereomers
14R/14S (ratio 1 : 2; S : R) in a yield of 2.4 g (74%). 13C NMR dC (CDCl3) (major
diastereomer) 163.8, 150.7, 137.6, 137.5, 137.4, 128.5, 128.4, 127.9, 127.8 127.1,
108.9, 93.2, 85.2, 82.6, 79.9, 73.5, 68.7, 67.2, 62.7, 54.8, 46.6, 45.6, 45.3, 37.3, and
12.4; 13C NMR dC (CDCl3) (minor diastereoisomer) 163.7, 150.1, 137.6, 137.5,
137.4, 128.5, 128.4, 127.9, 127.8, 127.1, 109.2, 92.6, 84.3, 81.8, 79.7, 73.7, 68.0,
67.6, 63.3, 54.6, 46.7, 45.6, 45.3, 41.1, and 12.7; HRMS (14R/14S) [M þ Na]þ
m/z 585.2661 (calcd. 585.2611).
(1S,3R,5R,6R,8R)-5-Hydroxy-6-hydroxymethyl-3-piperazinomethyl-8-
(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane (15R) and (1S,3S,5R,6R,-
8R)-5-hydroxy-6-hydroxymethyl-3-piperazinomethyl-8-(thymin-1-yl)-
2,7-dioxabicyclo[3.3.0]octane (15S). 20% Pd(OH)2/C (850 mg) was added
to a solution of nucleosides 14R/14S (2.10 g, 3.70 mmol) in MeOH (15 mL)
and the reaction mixture was degassed with hydrogen gas and stirred in an
atmosphere of hydrogen at rt for 168 hr. The reaction mixture was filtered
and the filtrate was evaporated to dryness affording a mixture of nucleosides
15R/15S (diastereomeric ratio of 2 : 1) as a white foam (563 mg, 90%).
13C NMR dC (DMSO-d6) (major diastereomer) 166.4, 151.9, 139.8, 109.5, 90.0,
88.1, 84.7, 84.2, 81.6, 62.4, 61.2, 51.4, 44.7, 40.8 and 12.8; 13C NMR dC
(DMSO-d6) (minor diastereomer) 166.4, 151.9, 139.1, 109.5, 86.5, 85.8, 84.6,
81.7, 81.5, 62.6, 62.4, 51.4, 44.7, 40.4, and 12.4. HRMS [M þ H]þ m/z 383.1925
(calcd. 383.1931).

M. Raunkjær, K. F. Haselmann, and J. Wengel
492
(1S,3R,5R,6R,8R)-5-Hydroxy-6-hydroxymethyl-3-(N-[9-fluorenylmet-
hoxycarbonyl]piperazino)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]-
octane (16R) and (1S,3S,5R,6R,8R)-5-Hydroxy-6-hydroxymethyl-3-(N-
[9-fluorenylmethoxycarbonyl]piperazino)methyl-8-(thymin-1-yl)-2,7-
dioxabicyclo[3.3.0]octane (16S). To a stirred solution of nucleosides 15R/
15S (418 mg, 1.09 mmol) in a mixture of THF (15 mL) and H2O (5 mL) was
added MgO (306 mg, 7.59 mmol), and the mixture was cooled to 08C. A
solution of FmocCl (312 mg, 1.21 mmol) in THF (5 mL) was added dropwise
to the reaction mixture and stirring was continued at 08C for 30 min before
addition of EtOAc (20 mL). The resulting mixture was filtered and the
residue was washed with EtAOc (2 ꢁ 10 mL). The combined filtrate was evap-
orated to dryness under reduced pressure and coevaporated with abs. EtOH
(2 ꢁ 20 mL). The residue was purified by silica gel column chromatography
with MeOH in DCM [5 : 95 (v/v)] as eluent to afford as white foams the separ-
ated diastereomers 16R and 16S in yields of 394 mg (65%) and 195 mg (32%),
respectively. Analytical data for 16R : 1H NMR dH (CDCl3) 7.75 (d, J ¼ 7.3 Hz,
2H, ArFmoc), 7.54 (d, J ¼ 7.3 Hz, 2H, ArFmoc), 7.39 (t, J ¼ 7.3 Hz, 2H, ArFmoc),
7.30 (t, J ¼ 7.3 Hz, 2H, ArFmoc), 7.20 (s, 1H, H-6), 6.05 (br s, 1H, H-10), 4.53–
4.19 (m, 5H, H-20, H-200, CHCH2Fmoc and CHCHF2moc), 3.86 (m, 3H, H-40, Ha-50
and Hb-50), 3.43 (m, 4H, 2 ꢁ CH2pip), 2.77–2.65 (m, 3H, Ha-300 and CHp2ip),
2.63–2.45 (m, 4H, Ha-100, Hb-300 and CH2pip), 1.89 (s, 3H, CH3), 1.72 (m, 1H,
Hb-100); 13C NMR dC (CDCl3) 163.9, 155.1 and 150.8 (CO), 143.9, 143.8,
141.2, 127.7, 127.5, 127.0, 124.9 and 120.0 (ArFmoc), 137.8 (C6), 109.6 (C5),
87.5 (C30), 88.1, 83.2, 82.0, 78.5, 77.2 and 47.2 (CH), 67.3, 62.0, 60.7, 53.4,
43.4, 39.7 and 29.6 (CH2) and 12.5 (CH3); MALDI-MS [M þ Na]þ m/z 627.2
(calcd. 627.3). Analytical data for 16S : 1H NMR dH (CDCl3) 8.72 (1H, s,
NH), 7.76 (d, J ¼ 7.7 Hz, 2H, ArFmoc), 7.55 (d, J ¼ 7.4 Hz, 2H, ArFmoc),
7.41 (t, J ¼ 7.0 Hz, 2H, ArFmoc), 7.32 (t, J ¼ 7.6 Hz, 2H, ArFmoc), 7.16
(d, J ¼ 0.9 Hz, 1H, H-6), 5.97 (d, J ¼ 5.0 Hz, 1H, H-10), 4.45 (m, 4H, H-20,
H-200 and CHCH2Fmoc), 4.23 (t, J ¼ 6.6 Hz, CHCHF2moc), 4.08–3.47 (m, 3H,
H-40, Ha-50 and Hb-50), 3.47 (m, 4H, 2 ꢁ CH2pip), 2.77–2.65 (m, 3H, Ha-300
and CHp2ip), 2.48–2.34 (m, 4H, Ha-100, Hb-300 and CHp2ip), 1.92 (d, J ¼ 0.8 Hz,
3H, CH3), 1.87 (m, 1H, Hb-100); 13C NMR dC (CDCl3) 164.0, 155.0, 150.4
(CO), 143.8, 141.3, 127.7, 127.0, 124.9 and 120.0 (ArFmoc), 136.8 (C6), 109.7
(C5), 86.7 (C30), 88.1, 83.8, 82.3, 81.1, 77.2, and 47.2 (CH), 67.3, 62.2, 60.8,
54.2, 43.3, 39.2, and 29.6 (CH2) and 12.6 (CH3); MALDI-MS [M þ Na]þ m/z
627.2 (calcd. 627.3).
(1S,3R,5R,6R,8R)-5-Hydroxy-6-hydroxymethyl-3-(N-[pyren-1-ylbutanoyl]
piperazino)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane
(17R)
and (1S,3S,5R,6R,8R)-5-Hydroxy-6-hydroxymethyl-3-(N-[pyren-1-ylbuta-
noyl]piperazino)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]-octane
(17S). EDC hydrochloride (230 mg, 1.2 mmol) and DIPEA (210 mL, 1.2 mmol)

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
493
were added to a stirred solution of nucleosides 15R/15S (399 mg, 1.04 mmol)
and 1-pyrenebutyric acid (346 mg, 1.2 mmol) in MeOH (15 mL). The reaction
mixture was stirred at rt for 12 hr and then evaporated to dryness under
reduced pressure. The residue was dissolved in a mixture of EtOAc (20 mL)
and H2O (5 mL), and the separated organic phase was washed successively
with brine (2 ꢁ 10 mL) and H2O (10 mL) and was dried and evaporated to
dryness under reduced pressure. The residue was purified by silica gel
column chromatography using MeOH in DCM [5 : 95 (v/v)] as eluent to give
as white foams the separated diastereomers 17R and 17S in yields of 332 mg
(49%) and 136 mg (20%), respectively. Analytical data for 17R : 1H NMR dH
(CDCl3) 8.30 (d, J ¼ 9.4 Hz, 1H, Arpyr), 8.15–7.94 (m, 7H, Arpyr), 7.83
(d, J ¼ 7.7 Hz, 1H, Arpyr), 7.12 (d, J ¼ 1.3 Hz, 1H, H-6), 5.97 (d, J 4.3 Hz, 1H,
H-10), 4.45 (m, 1H, H-200), 4.29 (d, J ¼ 4.5 Hz, 1H, H-20), 3.85–3.70 (m, 3H,
H-40, Ha-50 and Hb-50), 3.41–3.21 (m, 5H, CHaCHb(pip) and 2 ꢁ CHp2ip), 2.71
(m, 1H, CHaCHb(pip)), 2.42–2.13 (m, 2H, CH2pip), 2.63–2.13 (m, 10H, Ha-300,
Hb-300, CHp2ip and 3 ꢁ CH2butanoyl), 1.83 (m, 3H, Ha-100 and CH3), 1.63 (m, 1H,
Hb-100); 13C NMR dC (CDCl3) 171.2, 163.7, and 150.6 (CO), 137.8, 131.3,
130.8, 129.8, 128.7, 127.4, 127.3, 127.2, 127.1, 126.6, 125.8, 124.9, 124.8,
124.7 (2 peaks) and 123.3 (Arpyr), 135.9 (C6), 109.4 (C5), 88.0, 87.4, 83.1, 81.8,
81.7, 61.8, 60.5, 53.9, 53.4, 44.8, 41.1, 39.6, 32.7, 32.3, 26.8, and 12.4; HRMS
[M þ Na]þ m/z 675.2761 (calcd. 675.2789). Analytical data for 17S : 1H NMR
dH (CDCl3) 8.31 (d, J ¼ 9.1 Hz, 1H, Arpyr), 8.17–7.95 (m, 7H, Arpyr), 7.84
(d, J ¼ 7.8 Hz, 1H, Arpyr), 7.14 (d, J ¼ 1.4 Hz, 1H, H-6), 5.93 (d, J ¼ 4.7 Hz, 1H,
H-10), 4.39 (d, J ¼ 5.0 Hz, 1H, H-20), 4.35 (m, 1H, H-200), 4.04–3.89 (m, 3H,
H-40, Ha-50 and Hb-50), 3.74 (m, 1H, CHaCHb(pip)), 3.53 (m, 1H, CHaCHb(pip)),
3.43–3.31 (m, 2H, CHp2ip), 2.63–2.13 (m, 13H, Ha-100,Ha-300, Hb-300, 2 ꢁ CHp2ip
and 3 ꢁ CH2butanoyl), 1.92 (d, J ¼ 1.3 Hz, 3H, CH3), 1.79 (dd, J ¼ 2.0 and
13.6 Hz, 1H, Hb-100); 13C NMR dC (CDCl3) 171.1, 164.0, 150.3 (CO), 136.9,
131.3, 130.8, 129.8, 128.7, 127.4, 127.3, 127.1, 126.7, 126.6, 125.8, 124.9, 124.8,
124.7, 123.4, and 123.3 (Arpyr), 135.9 (C6), 109.4 (C5), 87.9, 86.7, 83.8, 82.5,
80.7, 61.9, 60.7, 54.2, 54.0, 44.8, 41.1, 39.2, 32.7, 32.2, 26.8, and 12.5; HRMS
[M þ Na]þ m/z 675.2782 (calcd. 675.2789).
(1S,3R,5R,6R,8R)-6-((4,40-Dimethoxytrityl)oxymethyl)-5-hydroxy-3-(N-
[9-fluorenylmethoxycarbonyl]piperazino)methyl-8-(thymin-1-yl)-2,-
7-dioxabicyclo[3.3.0]octane (18R). DMTCl (769 mg, 2.28 mmol) and DIPEA
(660 mL, 3.79 mmol) were added to a stirred solution of nucleoside 16R (458 mg,
0.758 mmol) in a mixture of anhydrous DCM (10 mL) and anhydrous CH3CN
(10 mL). The reaction mixture was stirred for 30 min at rt whereupon DCM
(20 mL) and H2O (5 mL) were added. The organic phase was washed succes-
sively with a sat. aq. solution of NaHCO3 (3 ꢁ 5 mL) and H2O (10 mL) and sub-
sequently dried and evaporated to dryness under reduced pressure. The
residue was purified by column chromatography using sat. methanolic

M. Raunkjær, K. F. Haselmann, and J. Wengel
494
ammonia and MeOH [0.5% sat. methanolic ammonia, 0–3% MeOH/(v/v/v)] in
DCM as eluent to give nucleoside 18R (580 mg, 84%) as a white foam. 1H NMR
dH (DMSO-d6) 11.4 (s, 1 H, NH) 7.87 (d, J ¼ 7.4 Hz, 2H, ArFmoc), 7.56
(d, J ¼ 7.2 Hz, 2H, ArFmoc), 7.43–7.20 (m, 14H, ArDMT, ArFmoc and H-6), 6.88
(d, J ¼ 7.8 Hz, 4H, ArDMT), 5.91 (d, J ¼ 4.5 Hz, 1H, H-10), 5.75 and 5.70
(s, 2H, NH and 30-OH), 4.35 (d, J ¼ 6.6 Hz, 2H, CHCHF2moc), 4.23
(t, J ¼ 6.4 Hz, 1H, CHCH2Fmoc), 4.15 (m, 2H, H-20 and H-200), 4.00 (m, 1H,
H-40), 3.74 (s, 6H, 2 ꢁ OCH3), 3.32 (m, 6H, Ha-300, Hb-300, Ha-50, Hb-5 and
CHp2ip), 2.46 (m, 2H, CH2pip), 2.24–2.07 (m, 5H, Ha-100 and 2 ꢁ CH2pip), 1.81
(s, 3H, CH3), 1.61 (m, 1H. Hb-100); 13C NMR dC (DMSO-d6) 163.7, 154.2 and
150.0 (CO), 158.1, 154.2, 144.8, 143.8, 140.7, 137.5, 135.5, 135.4, 129.7,
127.8, 127.7, 127.6, 127.1, 126.7, 125.0, 120.0, 113.2 and 107.4 (C5, C6 and
Ar), 87.6, 86.6, 85.7, 82.3, 81.0, 79.8, 66.4, 62.2, 60.7, 55.0, 54.9, 52.7, 46.8,
43.3 and 12.1; HRMS [M þ Na]þ m/z 929.3755 (calcd. 929.3732).
(1S,3S,5R,6R,8R)-6-((4,40-Dimethoxytrityl)oxymethyl)-5-hydroxy-3-(N-
[9-fluorenylmethoxycarbonyl]piperazino)methyl-8-(thymin-1-yl)-2,7-
dioxabicyclo[3.3.0]octane (18S). The procedure for preparation of
compound 18R was used. Compound 16S (282 mg, 0.47 mmol) was reacted
with DMTCl (473 mg, 1.40 mmol) in a mixture of anhydrous DCM (10 mL)
and CH3CN (10 mL) in the presence of DIPEA (325 mL, 0.742 mmol). Nucleo-
1
side 18S (373 mg, 88%) was obtained as a white foam. H NMR dH (DMSO-
d6) 11.5 (s, 1 H, NH) 7.87 (d, J ¼ 7.8 Hz, 2H, ArFmoc), 7.60 (d, J ¼ 7.1 Hz, 2H,
ArFmoc), 7.44–7.21 (m, 14H, ArDMT, ArFmoc and H-6), 6.89 (d, J ¼ 9.0 Hz, 4H,
ArDMT), 5.87 (d, J ¼ 3.6 Hz, 1H, H-10), 5.80 (s, 2H, NH and 30-OH), 4.36
(d, J ¼ 6.3 Hz, 2H, CHCH2Fmoc), 4.25 (t, J ¼ 6.1 Hz, 1H, CHCHF2moc), 4.18
(d, 1H, H-20), 4.06 (m, 1H, H-40), 3.92 (m, 1H, H-200), 3.74 (s, 6H, 2 ꢁ OCH3),
3.40–3.32 (m, 6H, Ha-300, Hb-300, Ha-50, Hb-5 and CH2pip), 2.48 (m, 2H, CHp2ip),
2.27–2.09 (m, 4H, 2 ꢁ CHp2ip), 1.96 (dd, J ¼ 5.4 and 12.8, 1H, Ha-100), 1.80
(s, 3H, CH3), 1.60 (dd, 1H, J ¼ 9.4 and 12.4, 1H Hb-100); 13C NMR dC (DMSO-
d6) 163.7, 158.1, 158.0, 149.9, 144.7, 142.5, 139.4, 137.4, 136.5, 135.4, 135.3,
129.8, 128.9, 127.8, 127.7, 127.2, 126.7, 121.4, 120.0, 113.2 and 109.7 (CO,
C5, C6 and Ar), 86.5, 86.4, 86.1, 85.8, 84.2, 79.1, 61.8, 55.0, 54.9, 54.5, 45.4,
43.3 and 12.1; HRMS [M þ Na]þ m/z 929.3755 (calcd. 929.3732).
(1S,3R,5R,6R,8R)-6-((4,40-Dimethoxytrityl)oxymethyl)-5-hydroxy-3-(N-
[pyren-1-ylbutanoyl]piperazino)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo
[3.3.0]octane (19R). The procedure for preparation of compound 18R was used.
Compound 17R (120 mg, 0.18 mmol) was reacted with DMTCl (186 mg,
0.55 mmol) in a mixture of anhydrous DCM (3 mL) and CH3CN (3 mL) in the
presence of DIPEA (160mL, 0.92 mmol). Nucleoside 19R (135 mg, 77%) was
1
obtained as a white foam. H NMR dH (CDCl3) 8.30 (d, J ¼ 9.2 Hz, 1H, Arpyr),
8.16–7.96 (m, 7H, Arpyr), 7.89 (d, J ¼ 7.8 Hz, 1H, Arpyr), 7.55–7.21 (m, 9H, H-6

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
495
and ArDMT), 6.83 (d, J ¼ 8.5 Hz, 4H, ArDMT), 6.05 (br s, 1H, H-10), 4.35
(d, J ¼ 4.0 Hz, 1H, H-20), 4.00 (m, 1H, H-200), 4.80–3.05 (m, 15H-40, Ha-50, Hb-50,
2 ꢁ OCH3, Ha-300, Hb-300 and 2 ꢁ CH2pip), 2.60–2.10 (m, 5H, Ha-100 and
2 ꢁ CHp2ip), 2.05–1.95 (m, 7H, 3 ꢁ CHb2utanoyl), 1.90 (d, J ¼ 0.9 Hz, 3H, CH3),
1.61 (m, 1H, Hb-100); 13C NMR dC (CDCl3) 170.9, 163.1, 158.7, 150.8, 144.7,
137.8, 136.3, 135.7, 135.7, 131.4, 130.9, 130.0, 129.9, 129.7, 128.8, 128.1, 127.9,
127.8, 127.4, 127.3, 127.1, 126.7, 126.6, 125.7, 125.0, 124.9, 124.8, 124.7, 123.6,
113.3 and 109.4 (CO, C6, C5 and Ar), 88.6, 88.5, 86.6, 82.7, 82.5, 77.1, 62.4,
62.0, 55.2, 53.9, 53.7, 53.6, 44.8, 41.1, 40.3, 40.2, 32.7, 32.2, 26.8 and 12.5;
HRMS [M þ Na]þ m/z 977.4104 (calcd. 977.4096).
(1S,3S,5R,6R,8R)-6-((4,40-Dimethoxytrityl)oxymethyl)-5-hydroxy-3-(N-
[pyren-1-ylbutanoyl]piperazino)methyl-8-(thymin-1-yl)-2,7-dioxabi-
cyclo[3.3.0]octane (19S). The procedure for preparation of compound 18R
was used. Compound 17S (100mg, 0.15 mmol) was reacted with DMTCl
(155 mg, 0.46 mmol) in a mixture of anhydrous DCM (2mL) and CH3CN
(2 mL) in the presence of DIPEA (133 mL, 0.74 mmol). Nucleoside 19S (110 mg,
1
75%) was obtained as a white foam. H NMR dH (CDCl3) 8.31 (d, J ¼ 9.3 Hz,
1H, Arpyr), 8.17–7.95 (m, 7H, Arpyr), 7.85 (d, J ¼ 7.7 Hz, 1H, Arpyr), 7.51–7.20
(m, 9H, H-6 and ArDMT), 6.83 (d, J ¼ 8.4 Hz, 4H, ArDMT), 5.98 (d, J ¼ 4.7 Hz,
1H, H-10), 4.41 (d, J ¼ 5.0 Hz, 1H, H-20), 4.27 (m, 1H, H-200), 4.20 (dd, J ¼ 3.8
and 6.5 Hz, 1H, H-40), 3.79 (m, 7H, Ha-50, Hb-50 and 2 ꢁ OCH3), 3.47–3.40
(m, 6H, Ha-300, Hb-300 and 2 ꢁ CH2pip), 2.58–2.45 (m, 4H, 2 ꢁ CHp2ip), 2.40–2.17
(m, 7H, Ha-100 and 3 ꢁ CHb2utanoyl), 1.90 (d, J ¼ 0.9 Hz, 3H, CH3), 1.60 (m, 1H,
Hb-100); 13C NMR dC (CDCl3) 171.0, 163.0, 158.5, 150.3, 144.6, 136.9, 135.9,
135.7, 135.6, 131.3, 130.1, 130.0, 129.9, 128.8, 128.1, 127.9, 127.4, 127.3, 127.1,
126.7, 126.6, 125.0, 124.9, 124.8, 124.7, 123.4, 113.2 and 109.5 (CO, C6, C5
and Ar), 88.8, 86.6, 86.5, 84.0, 81.8, 80.1, 62.2, 62.0, 55.2, 55.1, 55.0, 54.9, 44.8,
41.1, 39.2, 32.7, 32.2, 26.8 and 12.5; HRMS [M þ Na]þ m/z 977.4085 (calcd.
977.4096).
(1S,3R,5R,6R,8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-6-
((4,40-dimethoxytrityl)oxymethyl)-3-(N-[9-fluorenylmethoxycarbonyl]
piperazino)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane (20R).
2-Cyanoethyl N,N-diisopropylphosphoramidochloridite (188mL, 0.86 mmol)
was added dropwise to a stirred solution of nucleoside 18R (494mg,
0.55 mmol) dissolved in a mixture of anhydrous DCM (6mL) and DIPEA
(1.2mL). The reaction mixture was stirred for 1 hr at rt and subsequently
evaporated to dryness under reduced pressure. The residue was purified by
silica gel column chromatography using EtOAc in petroleum ether [1: 1 (v/v)]
as eluent to give amidite 20R (363 mg, 60%) as a white foam. 31P NMR
dP (DMSO-d6) 142.7 and 142.6; HRMS [M þ Na]þ m/z 1129.4815 (calcd.
1129.4811).

M. Raunkjær, K. F. Haselmann, and J. Wengel
496
(1S,3S,5R,6R,8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-
6-((4,40-dimethoxytrityl)oxymethyl)-3-(N-[9-fluorenylmethoxycarbonyl]-
piperazino)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane (20S).
The procedure for preparation of amidite 20R was used. Compound 18S
(260 mg, 0.29 mmol) dissolved in a mixture of anhydrous DCM (2 mL) and
DIPEA (0.4 mL) was reacted with 2-cyanoethyl N,N-diisopropylphosphorami-
dochloridite (70 mL, 0.32 mmol). The residue was purified by silica gel column
chromatography using EtOAc in petroleum ether [1 : 1 (v/v)] as eluent to
give amidite 20S (217 mg, 68%) as a white foam. 31P NMR dP (DMSO-d6)
143.3 and 143.1; HRMS [M þ Na]þ m/z 1129.4837 (calcd. 1129.4811).
(1S,3R,5R,6R,8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-
6-((4,40-dimethoxytrityl)oxymethyl)-3-(N-[pyren-1-ylbutanoyl]pipera-
zino)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane (21R). The
procedure for preparation of amidite 20R was used. Compound 19R (110 mg,
0.12 mmol) dissolved in a mixture of anhydrous DCM (1 mL) and DIPEA
(0.2 mL) was reacted with 2-cyanoethyl N,N-diisopropylphosphoramidochlori-
dite (40 mL, 0.18 mmol). The residue was purified by silica gel column chrom-
atography using EtOAc in petroleum ether [1 : 1 (v/v)] as eluent to give
amidite 21R (82 mg, 61%) as a white foam. 31P NMR dP (DMSO-d6) 142.7
and 142.6; HRMS [M þ Na]þ m/z 1177.5159 (calcd. 1177.5175).
(1S,3S,5R,6R,8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-
6-((4,40-dimethoxytrityl)oxymethyl)-3-(N-[pyren-1-ylbutanoyl]piperazi-
no)methyl-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane (21S). The pro-
cedure for preparation of amidite 20R was used. Compound 19S (100 mg,
0.10 mmol) dissolved in a mixture of anhydrous DCM (1 mL) and DIPEA
(0.2 mL) was reacted with 2-cyanoethyl N,N-diisopropylphosphoramidochlori-
dite (36 mL, 0.16 mmol). The residue was purified by silica gel column chrom-
atography using EtOAc in petroleum ether [1 : 1 (v/v)] as eluent to give
amidite 21S (30 mg, 24%) as a white foam. 31P NMR dP (DMSO-d6) 143.4 and
143.2; HRMS [M þ Na]þ m/z 1177.5180 (calcd. 1177.5175).
Oligonucleotide Synthesis
All ONs were prepared on a Biosearch 8700 Synthesizer using the phos-
phoramidite approach. Phosphoramidites 20R, 20S, 21R, and 21S were used
as building blocks using 10 min coupling time with pyridinium hydrochloride
as activator and tert-butylhydroperoxide as oxidant (stepwise coupling yield
of 98%). The unmodified DNA nucleotides were incorporated using 2 min
coupling time (stepwise coupling yields of .99%). The O50-DMT-ON ONs
were deprotected and cleaved form the solid support by treatment with sat.
methanolic NH3 for 2 hr at 558C and were purified by RP-HPLC. Detritylation

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
497
was performed using 80% AcOH for 20 min, and the O50-DMT-OFF ONs were
repurified by RP-HPLC to yield ONs of a satisfactory purity (.80% as evalu-
ated by gel capillary electrophoresis). Prior to thermal denaturation studies,
the ONs were desalted using NAPTM columns (Amersham Pharmacia AB,
Uppsala, SE).
Mass Spectrometric Analysis of Oligonucleotides
ON7-ON15 were analyzed by nanoelectrospray mass spectrometry using
the following procedure : 5-mL aliquot of SPE purified sample (using micro
columns with NAPTM resin, Amersham Pharmacia AB, Uppsala, SE) was
loaded onto a pulled glass capillary (Proxeon, Odense, DK). The glass capillary
was placed in a nanospray micromanipulator and a potential of approximately
2800 V was applied for negative ion mode. The electrospray source was a
standard hexapole based for external accumulation and with heated metal
capillary introduction. Due to the nature of the samples, only external cali-
bration was done, but with careful control of the signal magnitude. ON7
1096.00 (calcd. 1095.45 for [C147H203N30O97P13K]42), ON8 721.19 (calcd.
722.14 for [C96H120N36O54P8]42), ON9 1056.68 (calcd. 1056.59 for
[C110H145N40O56P8]32), ON10 963.23 (calcd. 963.19 for [C96H121N36O54P8]32),
ON11 1056.62 (calcd. 1056.59 for [C110H145N40O56P8]32), ON12 394.85
(calcd. 394.33 for [C116H130N36O55P8]82), ON13 397.59 (calcd. 397.08 for [C116-
H129N36O55P8Na]82), ON14 874.51 (calcd. 874.47 for [C141H159N37O55P8]42
and ON15 351.89 (calcd. 351.38 for [C141H152N37O55P8Na]102).
)
Thermal Denaturation Studies
The melting temperature experiments were performed on a Perkin-Elmer
lambda 20 spectrometer using a 1.0-mL cuvette with 1.5 mM of the two comp-
lementary strands in either buffer 1 (100 mM sodium chloride, 10 mM
sodium phosphate, and 0.1 mM EDTA, pH 7.0) or buffer 2 (10 mM sodium
phosphate and 0.1 mM EDTA, pH 7.0). The following example illustrates a
typical procedure of sample preparation (in buffer 1) : the two complementary
strands (dissolved in distilled H2O) were added to 500 mL of buffer A (200 mM
sodium chloride, 20 mM sodium phosphate, and 0.2 mM EDTA). Distilled H2O
was added until a total volume of 1000 mL was reached. Buffer A was prepared
by mixing appropriate volumes of buffer B (200 mM sodium chloride, 20 mM
NaH2PO4, and 0.2 mM EDTA) and buffer C (200 mM sodium chloride,
10 mM Na2HPO4, and 0.2 mM EDTA) until a pH value of 7.0 was obtained
(using a pH-meter for determination of the pH value). The samples were
heated to 708C and cooled to 58C before initiating the experiment with a
ramp of 18C/min. The melting temperature Tm was determined as the local
maximum of the first derivative of the melting curve (A260 vs. temperature).45

M. Raunkjær, K. F. Haselmann, and J. Wengel
498
Figure 3: Liner relationship of fluorescence emission area vs. absorbance.
Fluorescence Experiments
Steady-state fluorescence emission spectra (360–600 nm) were obtained
using quartz cuvettes with a pathlength of 1.0 cm and a concentration of
0.15 mM of strands in Tm buffer. Spectra were recorded at 208C with an
average of five scans using an excitation wavelength of 340 nm, excitation
slit of 4.0 nm, emission slit of 2.5 nm, and scan speed of 120 nm/min. The
quantum yield fS was determined using 1-pyrenebutyric acid as a reference.
The quantum yield is calculated according to the equation:
fS ¼ fP ꢁ ½AreaðSÞ=A340ðSÞꢂ=½AreaðPÞ=A340ðPÞꢂ ꢁ ½n2ðSÞ=n2ðPÞꢂ
where the quantum yield fP of 1-pyrenebutyric acid is 0.063 (measured relative
to known fluorescence quantum yield for 9,10-diphenylanthracene in cyclo-
hexane, fF ¼ 0.95)46 in MeOH. The value used for [Area(P)/A340(P)] is
290838 (slope of the linear relationship of fluorescence emission area vs. absor-
bance). Area (S) is the area under the fluorescence curve (360–600 nm) and
A340 is the absorbance at 340 nm. The values of refractive index n(S) and
n(P) equal 1.3328 (H2O) and 1.3288 (MeOH), respectively. To confirm the appli-
cability of the method, the linear correlation of absorbance and concentration
was verified for monomer 17R (fluorescence emission area vs. absorbance) at
low concentration (Fig. 3).
ACKNOWLEDGMENTS
Ms. Britta M. Dahl (University of Copenhagen), Ms. Kirsten Østergaard,
and Ms. Suzy W. Lena are thanked for oligonucleotide synthesis and

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
499
oligonucleotide purification. The Danish National Research Foundation and
the Danish Research Agency are thanked for financial support.
REFERENCES AND NOTES
[1] Wittung, P.; Nielsen, P.; Buchardt, O.; Egholm, M.; Norden, B. DNA-like double
helix formed by peptide nucleic-acid. Nature 1994, 368, 561.
[2] Koshkin, A.A.; Singh, S.K.; Nielsen, P.; Rajwanshi, V.K.; Kumar, R.; Meldgaard, M.;
Olsen, C.E.; Wengel, J. LNA (Locked nucleic acids) : synthesis of the adenine,
cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside
monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahe-
dron 1998, 54, 3607.
[3] Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.-I.; Morio, K.-I.; Doi, T.; Imanishi, T.
Stability and structural features of the duplexes containing nucleoside analogues
with a fixed N-type conformation, 20-O,40-C-methyleneribonucleosides. Tetra-
hedron Lett. 1998, 39, 5401.
[4] Van Aerschot, A.; Verheggen, I.; Hendrix, C.; Herdewijn, P. 1,5-Anhydrohexitol
nucleic acids, a new promising antisense construct. Angew. Chem. Int. Ed. Engl.
1995, 34, 1338.
[5] Heystek, L.E.; Zhou, H.Q.; Dande, P.; Gold, B. Control over the localization of
positive charge in DNA : the effect on duplex DNA and RNA stability. J. Am.
Chem. Soc. 1998, 120, 12165.
[6] Prakash, T.P.; Barawkar, D.A.; Kumar, V.; Ganesh, K.N. Synthesis of site-specific
oligonucleotide-polyamine conjugates. Bioorg. Med. Chem. Lett. 1994, 4, 1733.
[7] Ramasamy, K.S.; Zounes, M.; Gonzales, C.; Freier, S.M.; Lesnik, E.A.;
Cummins, L.L.; Griffey, R.H.; Monia, B.P.; Cook, D.P. Remarkable enhancement
of binding-affinity of heterocycle-modified DNA to DNA and RNA — synthesis,
characterization and biophysical evaluation of N-2-imidazolylpropylguanine and
N-2-imidazolylpropyl-2-aminoadenine modified oligonucleotides. Tetrahedron
Lett. 1994, 35, 215.
[8] Maier, M.A.; Barber-Peoc’h, I.; Manoharan, M. Postsynthetic guanidinylation of
primary amino groups in the minor and major grooves of oligonucleotides. Tetrahe-
dron Lett. 2002, 43, 7613.
[9] Hashimoto, H.; Nelson, M.G.; Switser, C. Zwitterionic DNA. J. Am. Chem. Soc.
1993, 115, 7128.
[10] Seela, F.; Zulauf, M. Oligonucleotides containing 7-deazaadenines : the influence of
the 7-substituent chain length and charge on the duplex stability. Helv. Chim. Acta
1999, 82, 1878.
[11] Lin, K.-I.; Matteucci, M.D. A cytosine analogue capable of clamp-like binding to a
guanine in helical nucleic acids. J. Am. Chem. Soc. 1998, 120, 8531.
[12] Letsinger, R.L.; Singman, C.N.; Histand, G.; Salunkhe, M. Cationic oligonucleo-
tides. J. Am. Chem. Soc. 1988, 110, 4470.
[13] Fathi, R.; Huang, Q.; Coppola, G.; Delaney, W.; Teasdale, R.; Krieg, A.M.;
Cook, A.F. Oligonucleotides with novel, cationic backbone substituents—
aminoethylphosphonates. Nucleic Acids Res. 1994, 22, 5416.

M. Raunkjær, K. F. Haselmann, and J. Wengel
500
[14] Michel, T.; Debart, F.; Vasseur, J.-J. Efficient guanidination of the phosphate
linkage towards cationic phosphoramidate oligonucleotides. Tetrahedron Lett.
2003, 44, 6579.
[15] Hickman, D.T.; Tan, T.H.S.; Morral, J.; King, P.M.; Cooper, M.A.; Micklefield, J.
Design, synthesis, conformational analysis and nucleic acid hybridisation proper-
ties of thymidyl pyrrolidine-amide oligonucleotide mimics (POM). Org. Biomol.
Chem. 2003, 1, 3277.
[16] Meier, R.; Gru¨schow, S.; Leumann, C. Nucleic-acid analogs with restricted conforma-
tional flexibility in the sugar-phosphate backbone (‘bicyclo-DNA’) - part 7—synthesis
and properties of oligodeoxynucleotides containing [(30S,50S,60R)-60-amino-20-deoxy-
30,50-ethano-b-D-ribofuranosyl]thymine (¼(60R)-60-amino-bicyclothymidine). Helv.
Chim. Acta 1999, 82, 1813.
[17] Pfundheller, H.M.; Bryld, T.; Olsen, C.E.; Wengel, J. Oligonucleotides containing novel
40-C- or 30-C-(aminoalkyl)-branched thymidines. Helv. Chim. Acta 2000, 83, 128.
[18] Aurup, H.; Tuschl, T.; Benseler, F.; Ludwig, J.; Eckstein, F. Oligonucleotide
duplexes containing 20-amino-20-deoxycytidines—thermal-stability and chemical-
reactivity. Nucleic Acids Res. 1994, 22, 20.
[19] Kanazaki, M.; Ueno, Y.; Shuto, S.; Matsuda, A. Highly nuclease-resistant
phosphodiester-type oligodeoxynucleotides containing 40-a-C-aminoalkylthymidines
form thermally stable duplexes with DNA and RNA. A candidate for potent anti-
sense molecules. J. Am. Chem. Soc. 2000, 122, 2422.
[20] Griffey, R.H.; Monia, B.P.; Cummins, L.L.; Freier, S.; Greig, M.J.; Guinosso, C.J.;
Lesnik, E.; Manalili, S.M.; Mohan, V.; Owens, S.; Ross, B.R.; Sasmor, H.;
Wancewicz, E.; Weiler, K.; Wheeler, P.D.; Cook, P.D. 20-O-aminopropyl ribonucleo-
tides : a zwitterionic modification that enhances the exonuclease resistance and
biological activity of antisense oligonucleotides. J. Med. Chem. 1996, 39, 5100.
[21] Wang, G.; Seifert, W.E. Synthesis and evaluation of oligodeoxynucleotides contain-
ing 40-C-substituted thymidines. Tetrahedron Lett. 1996, 37, 6515.
[22] Goodchild, J. Conjugates of oligonucleotides and modified oligonucleotides : a
review of their synthesis and properties. Bioconjugate Chem. 1990, 1, 165.
[23] Hrdlicka, P.J.; Babu, B.R.; Sørensen, M.D.; Wengel, J. Interstrand communication
between 20-N-(pyren-1-yl)methyl-20-amino-LNA monomers in nucleic acid duplex-
es : directional control and signalling of full complementarity. Chem. Commun.
2004, 1478.
[24] Raunkjær, M.; Bryld, T.; Wengel, J. N-Methylpiperazinocarbonyl-20,30-BcNA and
40-C-(N-methylpiperazino)methyl-DNA : introduction of basic functionalities
facing the major groove and the minor groove of a DNA : DNA duplex. Chem.
Commun. 2003, 1604.
[25] Bryld, T.; Højland, T.; Wengel, J. DNA-selective hybridization and dual strand
invasion of short double-stranded DNA using pyren-1-ylcarbonyl-functionalized
40-C-piperazinomethyl-DNA. Chem. Commun. 2004, 1064.
[26] Raunkjær, M.; Sørensen, M.D.; Wengel, J. Synthesis and thermal denaturation
studies of novel 20-O,30-C-linked bicyclic oligonucleotides with a methoxy or a piper-
azino group facing the major groove of nucleic acid duplexes. Org. Biomol. Chem.
2005, 3, 130.
[27] Petersen, G.V.; Wengel, J. Synthesis and characterization of short oligonucleotide
segments containing nonnatural internucleoside amine and amide linkages.
Nucleosides Nucleotides 1995, 14, 925.

O20,C30-Linked Bicyclic Nucleotide Units for Functionalization
501
[28] Nielsen, P.; Pfundheller, H.M.; Olsen, C.E.; Wengel, J. Synthesis of 20-O,30-C-linked
bicyclic nucleosides and bicyclic oligonucleotides. J. Chem. Soc. Perkin Trans. 1
1997, 3423.
[29] Raunkjær, M.; Olsen, C.E.; Wengel, J. Oligonucleotide analogues containing (200S)-
and (200R)-20-O,30-C-((20-C-hydroxymethyl)ethylene)-linked bicyclic nucleoside
monomers : synthesis, RNA-selective binding, and diastereoselective formation of
a very stable homocomplex based on T: T base pairing. J. Chem. Soc. Perkin.
Trans. 1 1999, 2543.
[30] Wang, D.; Wong, M.-K.; Yip, Y.-C. Epoxidation of olefins using methyl(trifluoro-
methyl)dioxirane generated in-situ. J. Org. Chem. 1995, 60, 3887.
[31] Payne, G.B. A simplified procedure for epoxidation by benzonitrile-hydrogen
peroxide — selective oxidation of 2-allylcyclohexanone. Tetrahedron 1962, 18, 763.
[32] Payne, G.B.; Deming, P.H.; Williams, P.H. Reactions of hydrogen peroxide. 7.
Alkali-catalyzed epoxidation and oxidation using a nitrile as co-reactant. J. Org.
Chem. 1961, 26, 659.
[33] Borch, R.F.; Bernstein, M.D.; Durst, H.D. Cyanohydridoborate anion as a selective
reducing agent. J. Am. Chem. Soc. 1971, 93, 2897.
[34] NOE experiments did not reveal a significant difference between the diastereo-
mers. It was not possible to obtain a crystal of the separated piperazino-
modified nucleosides for use in X-ray analysis. The stereochemical assignment
of structures was based on the known 1 : 2 ratio of diol 2. Phosphoramidites
derived from C200-R-configured compounds all displayed values below 143 ppm
(
31P NMR). Phosphoramidites derived from C200-S-configured compounds all dis-
played values above 143 ppm (see the Experimental section).
[35] Kim, D.-H.; Rho, H.-S.; You, J.W.; Lee, J.C. Chemoselective N-acylation of amino
alcohols promoted by magnesium oxide in aqueous organic solution. Tetrahedron
Lett. 2002, 43, 277.
[36] The highest yield was achieved by use of EDC in MeOH. Other coupling reagents
(DIC or DCC) with or without activators (HOBt or DMAP) and in different solvents
such as DMF or DCM were lower yielding.
[37] Hall, H.K., Jr. Field and inductive effects on the base strengths of amines. J. Am.
Chem. Soc. 1956, 78, 2570.
[38] Shafirovich, V.V.; Courtney, S.H.; Ya, N.Q.; Geacintov, N.E. Proton-coupled photo-
induced electron-transfer, deuterium-isotope effects, and fluorescence quenching
in noncovalent benzo[a]pyrenetetraol-nucleoside complexes in aqueous solutions.
J. Am. Chem. Soc. 1995, 117, 4920.
[39] Geacintov, N.E.; Zhao, R.; Kuzmin, V.A.; Kim, S.K.; Pecora, L. Mechanisms of
quenching of the fluorescence of a benzo[a]pyrenetetraol metabolite model-
compound by 20-deoxynucleosides. J. Photochem. Photobiol. 1993, 58, 185.
[40] Margulis, L.; Pluzhnikov, P.F.; Mao, B.; Kuzmin, V.A.; Chang, Y.J.; Scott, T.W.;
Geacintov, N.E. Photoinduced electron-transfer in a covalent benzo[a]pyrene diol
epoxide-20-deoxyguanosine complex. Chem. Phys. Lett. 1991, 187, 597.
[41] Zahavy, E.; Fox, M.A. Photophysical quenching mediated by guanine groups in
pyrenyl-N-alkylbutanoamide end-labeled oligo nucleotides. J. Phys. Chem. B
1999, 103, 9321.
[42] Steenken, S.; Jovanovic, S.V. How easily oxidizable is DNA? One-electron
reduction potentials of adenosine and guanosine radicals in aqueous solution.
J. Am. Chem. Soc. 1997, 119, 617.

M. Raunkjær, K. F. Haselmann, and J. Wengel
502
[43] Okamoto, A.; Ichiba, T.; Saito, I. Pyrene-labeled oligodeoxynucleotide probe for
detecting base insertion by excimer fluorescence emission. J. Am. Chem. Soc.
2004, 126, 8364.
[44] The quantum yield, or quantum efficiency is defined as the number of photo
physical events occuring (e.g., fluorescence emission) per photon absorbed. The
calculations are described in the Experimental section.
[45] Mergny, J.-L.; Lacroix, L. Analysis of thermal melting curves. Oligonucleotides
2003, 13, 515.
[46] Morris, J.V.; Mahaney, M.A.; Huber, J.R. Fluorescence quantum yield determi-
nations — 9,10-diphenylanthracene as a reference-standard in different solvents.
J. Phys. Chem. 1976, 80, 969.