Synthesis and base pairing properties of 1′,5′-Anhydro-l- hexitol nucleic acids (L-HNA)

Article abstract of DOI:10.1002/chem.200901847

Oligonucleotides composed of 1′,5′-anhydro-arabino-hexitol nucleosides belonging to the L series (L-HNA) were prepared and preliminarily studied as a novel potential base-pairing system. Synthesis of enantiopure L-hexitol nucleotide monomers equipped with

Full text of DOI:10.1002/chem.200901847

FULL PAPER
DOI: 10.1002/chem.200901847
Synthesis and Base Pairing Properties of 1’,5’-Anhydro-l-Hexitol
Nucleic Acids (l-HNA)
Daniele DꢀAlonzo,[b] Arthur Van Aerschot,[a] Annalisa Guaragna,[b] Giovanni Palumbo,[b]
Guy Schepers,[a] Stefania Capone,[b] Jef Rozenski,[a] and Piet Herdewijn*[a]
Abstract: Oligonucleotides composed
of 1’,5’-anhydro-arabino-hexitol nucleo-
sides belonging to the l series (l-
HNA) were prepared and preliminarily
studied as a novel potential base-pair-
ing system. Synthesis of enantiopure l-
hexitol nucleotide monomers equipped
with a 2’-(N6-benzoyladenin-9-yl) or a
2’-(thymin-1-yl) moiety was carried out
by a de novo approach based on a
domino reaction as key step. The l oli-
gonucleotide analogues were evaluated
in duplex formation with natural com-
plements as well as with unnatural
sugar-modified oligonucleotides. In
many cases stable homo- and hetero-
chiral associations were found. Besides
Tm measurements, detection of hetero-
chiral complexes was unambiguously
confirmed by LC-MS studies. Interest-
ingly, circular dichroism measurements
of the most stable duplexes suggested
that l-HNA form left-handed helices
with both d and l oligonucleotides.
Keywords:
domino reactions
nucleic acids · oligonucleotides
DNA analogues
·
·
·
HNA
Introduction
the most prominent examples of this class of conformation-
ally constrained oligonucleotide analogues is represented by
the HNA system (1’,5’-anhydro-d-arabino-hexitol nucleic
acids,[6] Figure 1), which has been found to hold great se-
quence-dependent RNA binding properties.[7] The great
thermal stability exhibited by d-HNA-based duplexes has
prompted research towards other base-pairing systems
based on a six-membered sugar backbone, such as d-CNA,[8]
d-ANA[9] and d-CeNA.[10]
From the dawn of antisense research, the challenge of syn-
thesising artificial nucleic acids has engaged chemists in the
construction of a great number of oligonucleotide analogues
that have more and more daring structural changes from the
original model, but are capable of keeping the potential for
selective cross communication.[1] One of the major efforts
over the years has concerned the preparation of oligonu-
cleotide architectures equipped with a sugar moiety deviat-
ing from the natural (deoxy)ribose backbone.[2] In some of
these modifications, replacement of the five-membered fura-
nose moiety with a six-membered ring, such as that present
in pyranosyl nucleosides (homo-DNA,[3] Figure 1) has been
designed to obtain highly preorganised structures towards
duplex formation and resulted in potentially excellent candi-
dates for cross pairing with natural complements.[4,5] One of
[a] Prof. Dr. A. Van Aerschot, G. Schepers, Prof. Dr. J. Rozenski,
Prof. Dr. P. Herdewijn
Rega Institute for Medical Research, Katholieke Universiteit Leuven
Minderbroederstraat 10, 3000 Leuven (Belgium)
Fax : (+32)16-337340
E-mail: Piet.Herdewijn@rega.kuleuven.be
[b] Dr. D. DꢀAlonzo, Dr. A. Guaragna, Prof. G. Palumbo, Dr. S. Capone
Dipartimento di Chimica Organica e Biochimica
Universitꢁ Federico II, Napoli, via Cinthia 4, 80126 Napoli (Italy)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901847.
Figure 1. Sugar modified d series oligonucleotides.
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In this area, great opportunities were also provided by
structures having the sugar moiety belonging to l-series
(Figure 2). Some of them, such as l-LNA,[11] effectively dis-
played unprecedented hybridisation with both DNA and
RNA complements. Studies involving modified l oligonu-
cleotides (particularly l-CNA) were also carried out to ex-
amine the role of conformational diversity in the chiral se-
lection of nucleic acid antipodes.[12] Recently, important
findings arose from the use of l-DNA as oligonucleotide
spiegelmers (from the German “Spiegel”, for mirror) with
many successful applications in the aptamer field[13] as well
as in intracellular nucleic acid detection.[14] On the basis of
these reports, our ongoing interest in sugar-modified oligo-
nucleotides prompted us to examine the potential of l-hexi-
tol nucleic acids (l-HNA, Figure 2) as a novel base-pairing
system. In this paper, attention was primarily given to hy-
bridisation properties of l-HNA with their natural comple-
ments; studies were also carried out to examine the hybridi-
sation capacity of l-HNA with l-DNA[15] as well as with
other d- and l-unnatural oligonucleotides.
primary alcohol, acetonide removal and acetalation by
double intramolecular cyclisation (89%). Subsequently, di-
thioethylene bridge removal from 2, followed by reductive
cleavage of 1,6-anhydrosugar function of 3 afforded pseudo-
glucal 4 (55% yield over two steps). With the aim to create
a C-2 electrophilic site for nucleobase insertion, 4 was first
deacetylated, then the resulting allylic alcohol was stereose-
lectively epoxidized to provide oxirane 5 (89% overall
yield). Finally, isopropylidene protection[20] of 5 afforded
key intermediate 6 (80% yield).
With the protected epoxide 6 in our hand, nucleobase in-
sertion into the required C-2’ axial position[21] was studied.
Under mild basic conditions (DBU/DMF) both thymine and
adenine l-altritol nucleosides 7 and 8 were obtained in good
yields (89 and 74%, respectively; Scheme 1). Then, 3’-OH
group removal in 7 and 8 under slightly modified Barton–
McCombie conditions[17] promptly yielded the corresponding
deoxynucleosides 10 (80% overall yield) and 12 (91% over-
all yield). Acetonide deprotection of the latter gave the
pure 1’,5’-anhydro-2’-(thymin-1-yl)-2’,3’-dideoxy-l-arabino-
hexitol (13) and 1’,5’-anhydro-2’-(adenin-9-yl)-2’,3’-dideoxy-
l-arabino-hexitol (14) in quantitative yields (Scheme 1).
The analytical data of free nucleosides 13 and 14 obtained
1
by H and 13C NMR spectroscopy, ESI-MS and CHNX anal-
ysis were identical to those of the corresponding d anti-
podes.[22] In addition, the absence of enantiomeric impuri-
ties[23] was confirmed by polarimetric measurements, and the
optical rotations of nucleosides 13 and 14 were compared
with those of the previously synthesised d antipodes
(Scheme 1).
Incidentally, it must be noted that since some key en-
zymes involved in the development of viral infections are
endowed with poor enantioselectivity,[24] nucleosides 13 and
14, which have structures resembling l-nucleosides, were
also considered with the aim to evaluate their antiviral po-
tential.[25]
In order to examine the properties of l-hexitol nucleic
acids, conversion of nucleosides 13 and 14 into fully protect-
ed phosphoramidite nucleotides was carried out (Scheme 2).
The primary hydroxyl group of thymine nucleoside 13 was
selectively protected (DMTCl/Py) to afford tritylated 15
(75%). Then, conversion into its phosphoramidite derivative
16 (DIPEA/2-cyanoethyl-N,N-diisopropyl chlorophosphor-
amidite) was easily achieved (74%). As far as adenine nu-
cleoside 14 is concerned, transient protection[26] was used to
selectively introduce the N6-benzoyl group (99% yield),
hence standard dimethoxytritylation of 17 and subsequent
phosphoramidite installation in 18 afforded the fully protect-
ed nucleotide building block 19 (57% yield over two steps).
Figure 2. Sugar modified l series oligonucleotides.
Results and Discussion
Synthesis of l-hexitol-based nucleotide building blocks: The
well-known poor commercial availability of almost all l-
hexoses along with the small number of practical synthetic
approaches[16] towards such compounds has long hampered
the preparation of l-hexitol-based oligonucleotide ana-
logues. Herein, synthesis of enantiopure l-hexitol nucleoside
monomers 13 and 14 (Scheme 1) as building blocks for the
preparation of l-HNA was carried out on the basis of a re-
cently reported procedure[17] already devoted to l-hexose
synthesis.[18] According to this strategy, construction of the
1,6-anhydrosugar backbone 2 was achieved by a domino re-
action[19] by treating acetate 1 with DDQ (Scheme 1). Such
a reaction involves five formal synthetic transformations in
one step: MPM group removal, oxidation of the resulting
Oligonucleotide synthesis and hybridisation properties of l-
HNAs: Assembly of A*- and T*-containing modified oligo-
nucleotides (dACTHUNTRGENN(UG Al*) and dCAHTUNGTREN(NUGN Tl*)) was accomplished by using
the common phosphoramidite method on a solid support.[27]
Fully modified sequences were assembled on a solid support
containing a propane-1,3-diol linker. The 3’ tail generated
did not interfere with physicochemical studies.[28] Several
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Synthesis and Base Pairing Properties of l-HNA
FULL PAPER
Scheme 1. Synthesis of l-arabino-hexitol nucleosides 13 and 14. i) ref. [19], 89%; ii) Ra-Ni, acetone, room temperature, 70%; iii) TMSOTf, Et3SiH,
CH2Cl2, 08C to room temperature, 78%; iv) a) MeONa, MeOH, room temperature, b) MCPBA, CH2Cl2, 08C to room temperature, 89% overall yield;
v) DMP, PPTS, acetone, room temperature, 80%; vi) T or A, DBU, DMF, 908C, 6–8 h, 89 and 74%, respectively; vii) NaOHaq, CS2, BrCH2CH3, DMF,
08C, 93%; viii) Bu3SnH, AIBN, toluene, reflux, 15 min, 98%; ix) 80% AcOHaq, 4 h, 608C, quant.; x) NaOHaq, CS2, BrCH2CH2CN, DMSO, 08C, 82%; xi)
Bu3SnH, AIBN, toluene, reflux, 15 min, 98%; xii) 80% AcOHaq, 4 h, 608C, quant.
nine/thymine oligonucleotides. Deconvoluted electrospray
ionisation mass spectrometric analysis showed all oligonu-
cleotides to be of correct molecular weight (Table 1).
The pairing properties of l-HNA containing oligonucleo-
tides were examined by hybridising oligomers with their
complementary strands and determining the melting points
(Tm) of the hybrids by temperature-dependent UV spectros-
copy. To determine whether the presence of l-hexitol nu-
cleosides into a short natural oligodeoxyribonucleotide se-
quence can perturb the structure of the corresponding DNA
duplex, incorporation of a single thymine or adenine nucleo-
side 13 or 14 at the X position of the 5’-d(CACCGXTGC-
TACC)-3’ sequence was examined. The stability of the re-
sulting duplexes was compared to those of the fully natural
corresponding double stranded (ds) DNA. The strength of
hybridisation was studied by thermal denaturation experi-
ments, which were determined at 260 nm in NaCl (0.1m)
buffer with KH2PO4 (20 mm, pH 7.5) and EDTA (0.1 mm) at
Scheme 2. Preparation of nucleotides 16 and 19 as building blocks for oli-
gonucleotide synthesis. i) DMTCl, Py, 75%; ii) (iPr)2NP(Cl)(OCH2-
a concentration of 4 mm for each strand. Compared to the
corresponding fully natural DNA strands, incorporation of a
single nucleoside 13 or 14 into the matched sequence led to
greatly reduced thermal stability of the resulting duplex
(Table 2, entries 1 and 5). In particular, introduction of thy-
CH2CN), DIPEA, CH2Cl2, 74%; iii) TMSCl, BzCl, Py, 99%; iv) DMTCl,
Py, 68%; v) (iPr)2NP(Cl)(OCH2CH2CN), DIPEA, CH2Cl2, 84%.
10–13-mer modified oligonucleotides were synthesised, in-
cluding oligoadenylates, oligothymidylates and mixed ade-
mine nucleoside 13 (dACTHNUTRGNEUN(G Tl*)) led to a Tm of 46.18C (versus
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P. Herdewijn et al.
Table 1. ESI-MS data of oligonucleotides containing l-hexitol nucleosides.
responding d-hexitol oligonu-
cleotides (d-HNA). Complexes
between fully modified oligo-
nucleotides and their natural
complements were also consid-
ered.
l-HNA formed very stable
homochiral self-complementa-
ry duplexes (ds-l-HNA) in
agreement with those obtained
with ds-d-HNA.[6] The complex
Entry
Sequence
MS calcd
MS found
1
2
3
4
5
6
7
8
5’-d(CAC CGTl* TGC TAC C)
5’-d(CAC CGAl* TGC TAC C)
5’-d(AGT ATT GTl*C CTA)
5’-d(AGT ATT GAl*C CTA)
6’-d(Tl*Tl*Tl* Tl*Tl*Tl* Tl*Tl*Tl* Tl*Tl*Tl* Tl*)
6’-d(Al*Al*Al* Al*Al*Al* Al*Al*Al* Al*Al*Al* Al*)
6’-d(Al*Tl*Tl* Tl*Al*Tl* Al*Tl*Tl* Al*Tl*Tl* Al*)
6’-d(Al*Tl*Al* Al*Al*Tl* Tl*Tl*Al* Tl*)
3882.7
3891.7
3647.7
3656.7
4210.9
4328.0
4255.9
3301.7
3882.7
3891.7
3647.7
3656.7
4211.1
4328.3
4255.8
3301.9
dACHTUNGTRENNUNG(Al*): l-hexitol adenine nucleoside; dACHTUGNTERN(NGUN Tl*): l-hexitol thymine nucleoside.
between d
gave the same melting profile (Tm 778C) as that of between
(Td*)13 and d(Ad*)13 (Tm 788C). Both duplexes were signifi-
ACHTUNGTNERNG(U Tl*)13 and dACHTUNGTRENNUNG(Al*)13
57.18C of natural dsDNA). Similarly, incorporation of the
corresponding adenine nucleoside moiety 14 (d(Al*)) led to
N
dN
ACHTUNGTRENNUNG
a Tm of 45.48C (versus 57.08C of natural dsDNA). Single
Tl*–T, Tl*–C and Tl*–G mismatches in this sequence were
also introduced (Table 2, entries 2–4); this resulted in a de-
crease of DTm values, ranging from 2.5 to 8.28C. Likewise,
one Al*–A, Al*–C and Al*–G mismatch led to a slight DTm
from À2.3 to À4.38C (entries 6–8).
cantly more stable than the corresponding natural com-
plexes (dsDNA and DNA–RNA). The stereochemical rela-
tionship between d- and l-HNA was confirmed by circular
dichroism (CD) experiments, in which the single CD spectra
of the self complementary 6’-dACTHGNURTEN(NNGU Al*Tl*AHCNUTTREGG(NUNN Al*)3CAHTUNGTRENN(NGU Tl*)3Al*Tl*)
sequence and of its d enantiomer exhibited a perfect mirror-
image behaviour (Figure 3).[34]
Table 2. Hybridisation studies between the mixed DNA sequence 5’-
d(CACCGXTGCTACC)-3’
and
its
natural
complement
3’-
d(GTGGCYACGATGG)-5’ after a single d
N
ACHTUNGTRENNUNG
tion, including mismatches.
3’-d(CCA TCG TXG CCA C)-5’
5’-d(GGT AGC AYC GGT G)-3’
X=Tl*
Y
X=T
Entry
Tm [8C]
DTm [8C]
Tm [8C]
DTm [8C]
1
2
3
4
A
T
C
G
46.1
39.9
37.9
43.6
–
57.1
46.7
43.2
50.9
–
À6.2
À8.2
À2.5
À10.4
À13.9
À6.2
Figure 3. CD spectra of single strands related to 6’-d
(Tl*)3Al*Tl*) (g) and its d enantiomer (c).
ACHTUGTNRENNUG(Al*Tl*ACHTUNGTRENNUNG(Al*)3-
Y
X=Al*
X=A
Entry
AHCTUNGTRENNUNG
Tm [8C]
DTm [8C]
Tm [8C]
DTm [8C]
5
6
7
8
T
45.4
43.0
41.1
43.1
–
57.0
46.9
45.0
53.2
-
A
C
G
À2.4
À4.3
À2.3
À10.1
À12.0
À3.8
It is noteworthy that, besides A–T associations, T–T and
A–A interactions were formed. In particular, a d(Tl*)13–d-
(Tl*)13 association was found, and stability of the association
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
was heavily dependent on salt buffer concentration (Tm
values range from 33 to 518C with increasing NaCl concen-
tration from 0.1 to 1m; Table 3). This result fits with that
In a second set of experiments, the pairing properties of
fully modified l-HNA sequences were examined by hybrid-
ising the oligomers with both their natural complements and
other unnatural oligonucleotides,[29] namely d- and l-cyclo-
hexanyl-NA[8] (CNA), a- and b-d-hexopyranosyl-NA[30] (d-
homo-DNA), d-HNA,[6] l-DNA[31] (Figures 1 and 2) and
peptide-NA[32] (PNA). Synthesis of such unnatural sequen-
ces has already been reported elsewhere.[6,8,29–32] For most
experiments, hybridisation was examined within the simplest
recognition mode (oligoA vs. oligoT).[33] On the basis of our
data and literature information, it is reasonable to assume
the formation of double helices, even though the presence
of triplexes or higher-order complexes cannot be fully ex-
cluded.
found for the d
(Al*)13–d(Al*)13 association was also detected and its Tm
(Td*)13 hybrid.[6] A somewhat weak
ACHUTNGTREN(NUG Td*)13–dACHUTNGTRENNGUN
dN
ACHTUNGTRENNUNG
linearly increased with salt concentration (Table 3). This is
indicative for an intermolecular complex formation between
dACHTUNRTGNEUNG(Al*)13 strands, even though the broad melting curve does
not suggest a single stable association.[35]
Much more unstable associations were found between l-
HNA and the natural DNA and RNA. In particular, given
the fact that dACHTNUTGRENNUG(Al*)13–dT13 and dAHCTUNTGREN(NUGN Al*)13–dT2rU13dT2 hybrids
had the same melting profiles as well as similar Tm values
(28–318C), it can be conjectured that such associations
might be better related to a d
N
ACHTNUGTNER(UNGN Al*)13 interaction.
The stability of complexes formed by self complementary
In the case of d(Tl*)13–dA13 and d
N
ACHTUNGTRENNUNG
base pairing between d
ied first. Melting curves were compared to those of the cor-
(Tl*)13 and d
E
sigmoidal but broad melting curves did not suggest one
clear transition.
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Synthesis and Base Pairing Properties of l-HNA
FULL PAPER
Table 3. Thermal stability studies of duplexes containing l-HNA, d-HNA, d-DNA and d-RNA oligonucleo-
tides. Melting points were determined at 260 nm (unless otherwise specified) in 0.1m NaCl, 20 mm KH2PO4
(pH 7.5), 0.1 mm EDTA.
and PNA-A10 (738C, Table 4,
entry 9). Finally, it is worth
mentioning that no stable asso-
ciation was found for the l-
Complement (Tm [8C])
Self hybridisation
ACHTUNGTERN(NUNG Tm [8C])
Entry
Oligomer (sequence)
d-DNA
d-RNA
l-HNA
d-HNA
HNA–l-DNA
duplex[38]
1
2
3
4
5
6
7
d-DNA-d
d-RNA-r(T13)
d-RNA-d(T2rU13dT2)
l-HNA-d(Tl*)13
d-HNA-d(Td*)13
l-HNA-d(Al*)13
d-HNA-d(Ad*)13
G
34[a]
34[a]
14
32
31[b]
21[a]
–
–
–
(Table 4, entry 10). This result
matches the aforementioned
investigations[6] regarding the
selectivity of d-HNA for d-
RNA rather than d-DNA.
The high Tm values reported
in Table 4 and Figure 4 for the
interaction between modified
oligonucleotides with the same
G
ND
26[c]
21[d]
48[c]
28[b,h]
33[c]
ND
ND
28[b]
33[c]
G
29[d]
58[a]
31[b]
21[a]
77
37,[e] 53[f]
78[a]
33,[e] 51[f]
33,[e] 46[a,f]
31,[e] 41[f]
31,[e] 41[f]
40,[e] 55[f]
77
E
40,[e] 55[f]
78[a]
37,[e] 53[f]
ND: not determined; [a] taken from ref. [6]; [b] Tm related to dACHTNUGTRENN(UG Al*)13–dACHTUNTGREN(NUGN Al*)13 interaction; [c] taken from
ref. [33]; [d] very broad transition; [e] determined in 0.1m NaCl; [f] determined in 1m NaCl; [g] determined at
270 nm; [h] d(T2rU13dT2) used as complement.
The formation of hetero-oligomers between d- and l-
Table 4. Tm values of l-HNA with unnatural oligonucleotides. Data were
compared to those related to associations with d-HNA.
HNA was then investigated. Equimolar amounts of d
ACHTNUGRTEN(NGNU Td*)13
and d(Al*)13 at 0.1 and 1m NaCl were mixed and the sharp
melting curves for the resulting mixture had higher Tm
values (40 and 558C, respectively) than those related to ds-
ACHTUNGTRENNUNG
Entry
Oligonucleotide (sequence)
Complement (Tm [8C])
l-HNA
d-HNA
1
2
3
4
5
6
7
8
9
l-CNA-d(Al)13
l-CNA-d(Tl)13
d-CNA-d(Ad)13
d-CNA-d(Td)13
a-d-homo-DNA-d(Ad)13
a-d-homo-DNA-d(Td)13
b-d-homo-DNA-d(Ad)13
b-d-homo-DNA-d(Td)13
PNA-A10
72
61
–
ND
ND
72[a]
62[a]
69[a]
64[a]
ND
62[a]
ND
ND
dACHTUNGTRENNUNG(Td*)13 (33 and 468C) or ds-dCAHTUNGTRENN(GUN Al*)13 (31 and 418C). The
same results were provided by the mirror-image mixture
(Table 3). This might suggest the possibility of heterochiral
complex formation. To ascertain this conjecture, we tested
31.5[b,c]
56
30
78
the mixed 6’-d
quence[36] after hybridisation with complementary d-HNA
sequences 6’-d(Td*(Ad*)2Td*(Ad*)2Td*Ad*Td*(Ad*)3Td*)
and 6’-d(Td*(Ad*)3Td*Ad*Td*(Ad*)2Td*(Ad*)2Td*), which
ACHTUNGTRENNUNG(Al*ACHTUNGTRENNUNG(Tl*)3Al*Tl*Al*AHCNUTEGTRG(NNUN Tl*)2Al*CAHTNUGTREN(NUNG Tl*)2Al*) se-
35
73
A
E
N
ACHTUNGTRENNUNG
10
l-DNA-d(Tl)10
31[b,c]
A
E
N
ACHTUNGTRENNUNG
ND: not determined; [a] data from ref. [33]; [b] broad melting curve;
[c] Tm is probably related to a d(Al*)13–d(Al*)13 interaction.
could also allow the determination of the orientation of
complementary strands in the duplex. However, the melting
curves of the mixtures had temperatures and shapes that
were similar to those of the single strands. CD experiments
of such d- and l-mixed A/T sequences were also carried
out, but the spectrum of each d-HNA/l-HNA mixture ap-
peared in fact as the sum of those of the separate strands,
with Cotton effects that elide each other. Owing to these
last results, regular Watson–Crick base pairing can be ex-
cluded, even though the possible formation of a complex be-
N
ACHTUNGTRENNUNG
tween dACHTUNGTRENNUNG(Al*)13 and dCAHUTNGTREN(NUGN Td*)13, as well as the mirror-image
mixture, needed further examination with different experi-
ments (see below for LC-MS studies).
l-HNA strands in either homopurine or homopyrimidine
form were finally hybridised with the aforementioned un-
natural complements, and showed, in some cases, from good
to excellent hybridisation capacity (Table 4, Figure 4). In
particular, high Tm values were found for the l-HNA–l-
CNA duplexes (72 and 618C, Table 4, entries 1 and 2) in
analogy with their d-HNA–d-CNA counterparts. On the
other hand, no stable duplexes were detected after l-HNA–
d-CNA hybridisation[37] (Table 4, entries 3 and 4). Interest-
ingly, even though an obvious preferable aptitude for associ-
ation with l oligomers occurred, a remarkable Tm value was
found for the l-HNA-T13–a-d-homo-DNA-A13 duplex
(568C, Table 4, entry 5). The Tm values obtained after asso-
ciation between l-HNA-T13 and b-d-homo-DNA-A13 was
even higher (788C, Table 4, entry 7). A very stable duplex
was also detected after hybridisation between l-HNA-T13
Figure 4. Melting curves for l-HNA based homo- and heterochiral oligo-
mers: l-HNA-T13 mixed with a-d-homo-DNA-A13 (*); l-HNA-A13
mixed with l-CNA-T13 (ꢃ); l-HNA-T13 mixed with PNA-A10 (+); l-
HNA-T13 mixed with l-CNA-A13 (~); l-HNA-T13 mixed with l-HNA-
A13 (&); l-HNA-T13 mixed with b-d-homo-DNA-A13 (&).
configuration can be explained by taking into account the
high degree of preorganisation that six-membered oligonu-
cleotides hold as a consequence of the relatively rigid chair
structure of the sugar backbone. Also, the stable duplex
formed between l-HNA and PNA confirms that PNA can
adopt both left-handed and right-handed structures, depend-
ing on the configuration of its hybridisation partner.[12,32] On
Chem. Eur. J. 2009, 15, 10121 – 10131
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10125

P. Herdewijn et al.
the other hand, formation of double helices by oligonucleo-
tides with opposite configuration was a much less predicta-
ble result, which has seldom been observed before[12] and
needed to be more accurately confirmed. With this aim, CD
experiments were carried out involving l-HNA–l-CNA, ds-
l-HNA, l-HNA–b-d-homo-DNA and l-HNA–a-d-homo-
DNA duplexes, which gave the most thermally stable com-
plexes (Figures 5 and 6).
ochiral complexes as indicated by the Tm studies. Moreover,
it is noteworthy that spectra of all the examined mixtures
had negative Cotton effects. In the case of ds-l-HNA and l-
HNA–l-CNA duplexes (Figure 5) this result easily leads to
the conclusion that these complexes form left-handed
double helices.[39]
On the other hand, in case of l-HNA–a-d-homo-DNA
and of l-HNA–b-d-homo-DNA complexes (Figure 6) it can
be only conjectured that, since l-CNA–b-d-homo-DNA
duplex is in a left-handed form[12] and given the structural
similarity between the CNA and HNA constructs, the nega-
tive Cotton effects should be related to duplexes existing in
left-handed forms.
All these homo- and heterochiral mixtures gave CD spec-
tra that were different from those of the corresponding
single strands; this strongly suggests the formation of heter-
Detection of heterochiral complexes by LC-MS experi-
ments: Even though Tm and CD studies already provided
striking indications on the formation of heterochiral com-
plexes, LC-MS experiments gave the final evidence that con-
firmed such interactions. Figure 7 shows the LC-MS behav-
iour of the complex formed between l-HNA-T13 and b-d-
homo-DNA-A13. LC profiles of the single oligothymidylate
and oligoadenylate strands and of the corresponding mix-
ture already showed different retention times; this clearly
suggests an interaction. Interestingly, LC profile of b-d-
homo-DNA-A13 displayed two peaks, MS analysis of which
proved that they are related to the single strand ([A]4À, m/z
1081.3; [A]3À, m/z 1441.7) and to the duplex based on A–A
interactions ([A–A]5À, m/z 1730.4), respectively (Figure 7a).
MS analysis of l-HNA-T13 also showed the faint signal of a
T–T duplex[40] ([T–T]5À, m/z 1683.7), in addition to the sig-
nals of the single strand ([T]4À, m/z 1051.7; [T]3À, m/z
1402.6; Figure 7b). Then, MS analysis of the l-HNA–b-d-
homo-DNA complex revealed a major peak related to the
A–T duplex ([A–T]5À, m/z 1707.1), while the peak associat-
ed to A–A interaction had disappeared (Figure 7c).
Figure 5. Normalised CD spectra of: l-HNA-A13 (&); l-HNA-T13 (&);
l-HNA-A13–l-HNA-T13 (*); l-CNA-A13 (~); l-HNA-T13–l-CNA-A13
(ꢃ). Measures were taken at 208C in 0.1m NaCl, 20 mm KH2PO4
(pH 7.5), 0.1 mm EDTA.
LC-MS studies of the complex formed between l-HNA-
T13 and a-d-homo-DNA-A13 demonstrated that the a-d-
homo-DNA-A13 as single strand on its own also had a A–A
interaction ([A–A]5À, m/z 1730.4). Analysis of the l-HNA-
T13–a-d-homo-DNA-A13 complex mainly highlighted the
peaks related to the single strands, even though three more
weak signals associated with duplexes (ds-l-
a-d-(homo-A)13 and l-(HNA-T)13–a-d-(homo-A)13) could
be detected (see the Supporting Information).
ACHTUNGRTNEN(UNG HNA-T)13, ds-
AHCTUNGTRENNUNG
Finally, in order to consider the interaction between l-
HNA-A13 and d-HNA-T13, which was suggested by Tm stud-
ies, LC-MS runs were carried out. LC-MS profile of l-
HNA-A13 (Figure 8a) confirmed the existence of a duplex
based on a A–A interaction ([A–A]5À, m/z 1731.1) while the
spectrum of d-HNA-T13 was identical to its mirror image
(Figure 7b) and was not reported. Again, the LC profile of
the mixture clearly demonstrated the occurrence of the in-
teraction, owing to the different retention time compared to
the single strands. The existence of the complex was then
further confirmed by ESI-MS ([A–T]5À, m/z 1707.6; Fig-
ure 8b).
Figure 6. Normalised CD spectra of: l-HNA-T13 (*); a-d-homo-DNA-
A13 (~); b-d-homo-DNA-A13 (&); l-HNA-T13–a-d-homo-DNA-A13 (ꢃ);
l-HNA-T13–b-d-homo-DNA-A13 (&). Measures were taken at 208C in
0.1m NaCl, 20 mm KH2PO4 (pH 7.5), 0.1 mm EDTA.
10126
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Chem. Eur. J. 2009, 15, 10121 – 10131

Synthesis and Base Pairing Properties of l-HNA
FULL PAPER
Conclusions
1’,5’-Anhydro-l-arabino-hexitol
nucleic acids (l-HNA) with
either thymine or adenine as
the base moieties were pre-
pared and studied. Synthesis of
the corresponding l-hexitol nu-
cleotide building blocks was
carried out by a convenient
and scalable de novo approach.
As in the case of d-HNA, l-
HNA oligonucleotides showed
self association; the homochi-
ral duplexes were significantly
more stable than the corre-
sponding natural associations.
Moreover, even though the l-
HNA system was found not to
be able to hybridise with natu-
ral DNA and RNA (or at least
not able to give a single stable
interaction), strong associa-
tions were found after hybridi-
sation with homochiral oligo-
nucleotides with
a six-mem-
bered sugar backbone. Re-
markably, Pu–Py pairing was
observed between l-HNA and
hexose oligonucleotides with
the d configuration. In addi-
tion, CD experiments seem to
suggest that l-HNA always
form left-handed helices, either
with d or l oligonucleotides.
Further in-depth examination
of this topic is in progress; in
particular, efforts are currently
focusing on structure determi-
nation of l-HNA based du-
plexes, in order to provide new
insight into the actual confor-
mation of the hexitol backbone
following double helix forma-
tion.
Experimental Section
Chemical synthesis of l-arabino-hexi-
tol-based nucleotide building blocks:
All moisture-sensitive reactions were
performed under nitrogen atmos-
phere by using oven-dried glassware.
Solvents were dried over standard
drying agents and freshly distilled
prior to use. Reactions were moni-
tored by TLC (precoated silica gel
Figure 7. LC-MS profiles of b-d-homo-DNA-A13, l-HNA-T13 and l-HNA-T13–b-d-homo-DNA-A13.
Chem. Eur. J. 2009, 15, 10121 – 10131
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www.chemeurj.org
10127

P. Herdewijn et al.
4-O-Acetyl-1,5-anhydro-2,3-dideoxy-
l-erythro-hex-2-enitol (4): TfOTMS
(0.34 mL, 1.89 mmol) was added
dropwise to a stirring solution of 1,6-
anhydrosugar derivative
3 (1.64 g,
9.64 mmol) and triethylsilane
(2.11 mL, 13.20 mmol) in CH2Cl2
(60 mL) at 08C. After 30 min, the re-
action was quenched with solid
NaHCO3, heated to room tempera-
ture, diluted with CH2Cl2 (300 mL)
and washed with brine (300 mL). The
organic layer was dried (Na2SO4) and
the solvent evaporated under reduced
pressure. Chromatography of the
crude residue over silica gel (CH2Cl2)
gave the pure 4 (1.47 g; 78%) as a
colourless oil. 1H NMR (500 MHz,
CDCl3, 258C): d=2.12 (s, 3H;
CH3CO), 3.49–3.54 (m, 1H; H-5),
3.58–3.63 (m, 1H; H-1a), 3.72–3.78
(m, 1H; H-1b), 4.18–4.25 (m, 2H; 2ꢃ
H-6), 5.29 (brs, 1H, H-4), 5.76 (brd,
J
3,2 =9.9 Hz, 1H; H-3), 5.93 ppm
(brd, 2,3 =9.9 Hz, 1H; H-2);
J
13C NMR (100 MHz, CDCl3, 258C):
d=20.4 (CH3), 63.5 (CH2), 64.5
(CH2), 65.6 (CH), 78.7 (CH), 127.8
(CH), 128.9 (CH), 181.3 ppm (C); el-
emental analysis calcd (%) for
C8H12O4: C 55.81, H 7.02; found: C
55.68, H 7.19.
1,5:2,3-Dianhydro-l-allo-hexitol (5):
Zemplꢄn deacetylation was accom-
plished by treatment of
4 (1.20 g,
6.97 mmol) in MeOH (100 mL) with
MeONa (0.75 g, 13.94 mmol) for 4 h
at room temperature. Then the mix-
ture was neutralised with acetic acid
and the solvents were evaporated
Figure 8. LC-MS profiles of l-HNA-A13 and d-HNA-T13–l-HNA-A13.
under reduced pressure. The crude
plate F254, Merck). Column chromatography: Merck Kieselgel 60 (70–
230 mesh); flash chromatography: Merck Kieselgel 60 (230–400 mesh).
Optical rotations were measured at (25Æ2)8C in the stated solvent. 1H
and 13C NMR spectra were recorded on NMR spectrometers operating at
200, 300, 400 or 500 MHz and 50, 75, 100 or 125 MHz, respectively. Com-
bustion analyses were performed by using CHNS analyzer. Exact mass
measurements were performed by using a quadrupole time-of-flight mass
spectrometer (Q-Tof-2, Micromass, Manchester, UK) equipped with a
standard electrospray ionisation (ESI) interface.
residue was dissolved in CHCl3 and filtered through a short pad of silica
gel; the resulting filtrate was concentrated to dryness. The residue was
dissolved in anhydrous CH2Cl2 (30 mL), then mCPBA (1.44 g,
8.36 mmol) was added at 08C. The resulting reaction mixture was stirred
at room temperature, overnight; then the insoluble materials were fil-
tered off, washed and the filtrate was evaporated. Chromatography of
the crude residue over silica gel (CH2Cl2/MeOH 9:1) gave pure 5 (0.98 g;
89% overall yield) as a colourless oil. [a]2D5 =À35.8 (c=1.0, CHCl3);
1H NMR (500 MHz, CD3OD, 258C): d=3.23 (ddd, J5,4 =2.0, J5,6a =5.9,
J
5,6b =8.8 Hz, 1H; H-5), 3.40–3.43 (m, 1H; H-3), 3.51 (t, J=4.2 Hz, 1H;
4-O-Acetyl-1,6-anhydro-2,3-dideoxy-b-l-erythro-hex-2-enopyranose (3):
A solution of 2 (4.0 g, 15.36 mmol; prepared according to ref. [19]) in
acetone (200 mL) was added in one portion to a stirred suspension of
Raney-Ni (W2; 40 g, wet; previously washed with H2O) at 08C and
under nitrogen atmosphere. The suspension was stirred for 4 h, then the
solid was filtered off and washed with acetone. The filtrate was evaporat-
ed under reduced pressure to afford a crude residue, which after chroma-
tography over silica gel (CH2Cl2) gave pure 3 (1.96 g, 70%) as a colour-
less oil. [a]2D5 =À185.1 (c=2.0 in CHCl3); 1H NMR (400 MHz, CDCl3,
258C): d=2.12 (s, 3H; CH3CO), 3.54 (dd, J6a,5 =2.1, J6a,6b =8.0 Hz, 1H;
H-6a), 3.97 (dd, J6b,5 =6.7, J6b,6a =8.0 Hz, 1H; H-6b), 4.71 (bdd, J5,6a =2.1,
H-2), 3.55 (dd, J6a,5 =5.9, J6a,6b =11.7 Hz, 1H; H-6a), 3.77–3.82 (m, 3H;
H-4, H-6b, H-1a), 4.08 ppm (dd, J1b,2 =4.2, J1b,1a =13.2 Hz, 1H; H-1b);
13C NMR (125 MHz, CD3OD, 258C): d=55.4 (CH), 56.5 (CH), 63.1
(CH2), 65.6 (CH2), 66.8 (CH), 76.8 ppm (CH); elemental analysis calcd
(%) for C6H10O4: C 49.31, H 6.90; found: C 49.15, H 6.93.
4,6-O-Isopropylidene-1,5:2,3-dianhydro-l-allo-hexitol (6): 2,2-Dimethox-
ypropane (DMP, 1.05 mL, 10.09 mmol) and pyridinium p-toluenesulfo-
nate (PPTS, 1.68 g, 6.69 mmol) were added to a solution of 5 (0.97 g,
6.69 mmol) in anhydrous acetone (50 mL) and the resulting reaction mix-
ture was stirred for 12 h at room temperature. Then the solvent was
evaporated, the crude residue dissolved in EtOAc and washed with satu-
rated NaHCO3 (2ꢃ300 mL) and brine (2ꢃ200 mL). The organic layer
was dried (Na2SO4) and the solvent evaporated under reduced pressure.
Chromatography of the crude residue over silica gel (CH2Cl2) gave pure
J
5,6b =6.7 Hz, 1H; H-5), 4.80 (d, J4,3 =4.4 Hz, 1H; H-4), 5.60 (d, J1,2
3.5 Hz, 1H; H-1), 5.81 (ddd, J3,5 =1.7, J3,4 =4.4, J3,2 =9.6 Hz, 1H; H-3),
6.20 ppm (dd, 2,1 =3.5,
2,3 =9.6 Hz, 1H; H-2); 13C NMR (100 MHz,
=
1
6 (1.01 g; 80%) as a colourless oil. [a]2D5 =+1.2 (c=0.7, CHCl3); H NMR
J
J
CDCl3, 258C): d=21.1 (CH3), 63.2 (CH2), 68.0 (CH), 74.7 (CH), 95.5
(CH), 122.5 (CH), 132.6 (CH), 170.6 ppm (CO); elemental analysis calcd
(%) for C8H10O4: C 56.47, H 5.92; found: C 56.28, H 5.94.
(400 MHz, C6D6, 258C): d=1.29 (s, 3H; CH3), 1.50 (s, 3H; CH3), 2.68
(appt, J2,1 =3.5, J2,3 =4.4 Hz, 1H; H-2), 3.09 (brd, J2,3 =4.4 Hz, 1H; H-3),
3.55 (dd,
J1a,2 =3.5, J1a,1b =13.4 Hz, 1H; H-1a), 3.58 (t, J6a,6b =J6a,5 =
10128
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Chem. Eur. J. 2009, 15, 10121 – 10131

Synthesis and Base Pairing Properties of l-HNA
FULL PAPER
10.3 Hz, 1H; H-6a), 3.65 (d, J1b,1a =13.4 Hz, 1H; H-1b), 3.71–3.78 (m,
1H; H-5), 3.79–3.85 ppm (m, 2H; H-6b, H-4); 13C NMR (125 MHz, C6D6,
258C): d=18.8 (CH3), 29.3 (CH3), 51.4 (CH), 52.6 (CH), 62.6 (CH2), 64.2
(CH2), 65.8 (CH), 71.2 (CH), 99.8 ppm (C); elemental analysis calcd (%)
for C9H14O4: C 58.05, H 7.58; found: C 58.24, H 7.54.
then bromoethane (0.81 mL) were added at 08C to a stirring solution of
compound 8 (0.81 g, 2.52 mmol) in DMF (33 mL). After being stirred for
30 min, the solvent was evaporated under reduced pressure; the crude
residue was extracted with CH2Cl2 and washed with NH4Cl solution. The
organic layer was dried (Na2SO4) and the solvent evaporated to give
crude 11. To a refluxing solution of 11 dissolved in anhydrous toluene
(60 mL) and kept under a nitrogen flow, a solution of nBu3SnH (1.02 mL,
3.78 mmol) and AIBN (0.02 g, 0.1 mmol) in anhydrous toluene (60 mL)
was added dropwise over 1 h. After the addition, the brown solution was
stirred for an additional 30 min under reflux. The reaction mixture was
then concentrated under reduced pressure and the resulting residue was
purified by column chromatography on silica gel (CH2Cl2/MeOH 95:5) to
give pure 12 (0.72 g, 85% over two steps) as a white brilliant powder:
[a]2D5 =À19.7 (c=0.4, CHCl3); 1H NMR (300 MHz, CDCl3, 258C): d=
1.42 (s, 3H; CH3), 1.48 (s, 3H; CH3), 2.05 (ddd, J3’a,2’ =4.3, J3’a,4’ =11.8,
General procedure for nucleoside synthesis: Purine or pyrimidine nucleo-
base (6.21 mmol) and the epoxide 6 (2.68 mmol) were suspended in anhy-
drous DMF (7 mL) under nitrogen for 15 min at room temperature. Then
1,8-diazabicyloACHTUNGTRENNUNG[5.4.0]undec-7-ene (DBU, 6.21 mmol) was added and the
reaction mixture was heated at 908C for 6–8 h, after which the reaction
was cooled to room temperature, quenched with NH4Cl and concentrat-
ed. The residue was extracted with CH2Cl2 and washed with brine (3ꢃ
150 mL). The organic layer was dried (Na2SO4) and the solvent was
evaporated under reduced pressure. Chromatography of the crude resi-
due over silica gel (CH2Cl2/MeOH 98:2) gave the corresponding pure
purine or pyrimidine nucleoside (74–89%). Data for 1’,5’-anhydro-4’,6’-
J
3’a,3’b =13.5 Hz, 1H; H3’a), 2.40–2.52 (m, 1H; H3’b), 3.44 (ddd, J5’,6’b
5.2, J5’,4’ =9.6, J5’,6’a =10.4 Hz, 1H; H-5’), 3.73 (ddd, J4’,3’b =4.5, J4’,5’ =9.6,
4’,3’a =11.8 Hz, 1H; H-4’), 3.82 (t, J6’a,5’ =J6’a,6’b =10.4 Hz, 1H; H-6’a), 3.99
(dd, J6’b,5’ =5.2, J6’b,6’a =10.4 Hz, 1H; H-6’b), 4.12 (dd, J1’a,2’ =2.9, J1’a,1’b
=
O-isopropylidene-2’-deoxy-2’-(thymin-1-yl)-l-altro-hexitol
(7):
white
powder, [a]2D5 =À36.7 (c=0.6, CHCl3); 1H NMR (500 MHz, CDCl3,
258C): d=1.44 (s, 3H; CH3), 1.50 (s, 3H; CH3), 1.97 (s, 3H; CH3), 3.51
(d, JOH,3’ =5.0 Hz, 1H; OH), 3.67 (dd, J4’,3’ =2.7, J4’,5’ =9.7 Hz, 1H; H-4’),
3.76 (t, J6’a,5’ =J6’a,6’b =10.3 Hz, 1H; H-6’a), 3.88–3.94 (m, 1H; H-5’), 3.98
(dd, J6’b,5’ =5.3, J6’b,6’a =10.3 Hz, 1H; H-6’b), 4.03 (d, J1’a,1’b =13.7 Hz, 1H;
H-1’a), 4.07 (brs, 1H; H-3’), 4.34 (dd, J1’b,2’ =3.4, J1’b,1’a =13.7 Hz, 1H; H-
1’b), 4.49 (brt, J2’,1’b =J2’,3’ =3.4 Hz, 1H; H-2’), 7.84 (s, 1H; H-6), 8.43 ppm
(brs, 1H; NH); 13C NMR (75 MHz, CDCl3, 258C): d=12.7 (CH3), 19.2
(CH3), 28.9 (CH3), 57.2 (CH), 62.3 (CH2), 64.0 (CH2), 66.0 (CH), 67.2
(CH), 69.2 (CH), 100.0 (C), 111.4 (C), 137.9 (CH), 151.3 (C), 164.3 ppm
(C); elemental analysis calcd (%) for C14H20N2O6: C 53.84, H 6.45, N
8.97; found: C 53.85, H 6.49, N 8.99. Data for 1’,5’-anhydro-4’,6’-O-iso-
propylidene-2’-deoxy-2’-(adenin-9-yl)-l-altro-hexitol (8): white powder,
[a]2D5 =À9.9 (c=1.3, DMSO); 1H NMR (300 MHz, [D6]DMSO, 258C):
d=1.27 (s, 3H; CH3), 1.33 (s, 3H; CH3), 3.52–3.60 (m, 1H; H-4’), 3.72–
3.90 (m, 3H; H-5’, H-6’a, H-6’b), 4.00–4.10 (m, 1H; H-3’a), 4.18–4.29 (m,
2H; H-1’a, H-1’b), 4.52–4.56 (m, 1H; H-2’), 5.83 (d, J=4.3 Hz, 1H;
3’OH), 7.29 (brs, 2H; NH2), 8.17 (s, 1H, H-8), 8.29 ppm (s, 1H; H-2);
13C NMR (75 MHz, [D6]DMSO, 258C): d=19.5 (CH3), 29.4 (CH3), 56.3
(CH), 62.0 (CH2), 65.0 (CH2), 65.8 (CH), 67.6 (CH), 69.4 (CH), 99.5 (C),
118.6 (C), 139.6 (CH), 150.0 (C), 153.0 (CH), 156.5 ppm (C); elemental
analysis calcd (%) for C14H19N5O4: C 52.33, H 5.96, N 21.79; found: C
52.15, H 5.98, N 21.85.
J
=
13.0 Hz, 1H; H-1’a), 4.42 (brd, J1’b,1’a =13.0 Hz, 1H; H-1’b), 4.94–5.00 (m,
1H; H-2’), 5.60 (brs, 2H; NH2), 8.34 (s, 1H; H-8), 8.39 ppm (s, 1H; H-
2); 13C NMR (75 MHz, CDCl3, 258C): d=19.1 (CH3), 29.1 (CH3), 33.7
(CH2), 50.7 (CH), 62.4 (CH2), 66.3 (CH2), 69.8 (CH), 75.8 (CH), 99.8 (C),
117.7 (C), 139.7 (CH), 150.0 (C), 153.1 (CH), 155.4 ppm (C); elemental
analysis calcd (%) for C14H19N5O3: C 55.07, H 6.27, N 22.94; found: C
55.24, H 6.25, N 22.85.
1’,5’-Anhydro-2’-(thymin-1-yl)-2’,3’-dideoxy-l-arabino-hexitol (13): A so-
lution of nucleoside 10 (0.5 g, 1.69 mmol) in 80% AcOH (14 mL) was
heated at 658C for 2 h. The reaction mixture was then concentrated to
dryness under reduced pressure and the residue was coevaporated with a
mixture of toluene and EtOH (1:1 v/v, 5ꢃ50 mL). The residue was puri-
fied by column chromatography (CH2Cl2/MeOH 9:1) to yield thymine
nucleoside 13 (0.43 g, 99%) as white crystals: [a]2D5 =À53.9 (c=0.5,
DMSO); 1H and 13C NMR spectroscopy data coincide with those report-
ed in refs. [6,19]; elemental analysis calcd for C11H16N2O5: C 51.56, H
6.29, N 10.93; found: C 51.68, H 6.31, N 10.89.
1’,5’-Anhydro-2’-(adenin-9-yl)-2’,3’-dideoxy-l-arabino-hexitol (14): A so-
lution of nucleoside 12 (0.49 g, 1.62 mmol) in 80% AcOH (10 mL) was
heated at 658C for 2 h while being stirred. Then the reaction mixture was
concentrated to dryness and the residue was purified by column chroma-
tography (CH2Cl2/MeOH 9:1) to yield adenine nucleoside 14 (0.42 g,
99%) as white crystals: [a]2D5 =À11.8 (c=0.5, DMSO); 1H and 13C NMR
spectroscopy data coincide with those reported in refs. [6,19]; elemental
analysis calcd for C11H15N5O3: C 49.81, H 5.70, N 26.40; found: C 49.94,
H 5.72, N 26.31.
1’,5’-Anhydro-2’-(N6-benzoyladenin-9-yl)-2’,3’-dideoxy-l-arabino-hexitol
(17): TMSCl (1.8 mL, 15.1 mmol) was added to a stirring solution of nu-
cleoside 14 (0.4 g, 1.51 mmol) in anhydrous pyridine (30 mL) and under
nitrogen atmosphere. After 30 min, BzCl (0.42 mL, 3.75 mmol) was
added at 08C. The resulting reaction mixture was warmed to room tem-
perature and further stirred for 16 h. Then H2O (3 mL) was added at 08C
and then NH4OH (3 mL) after 10 min. The reaction mixture was stirred
at the same temperature for 1 h; afterwards the solvent was removed
under reduced pressure. Chromatography of the crude residue (CH2Cl2/
MeOH 9:1) afforded the pure benzoylated nucleoside 17 (0.56 g, 99%)
as a white powder. 1H NMR (300 MHz, [D6]DMSO, 258C): d=1.96–2.15
(m, 1H; H-3’a), 2.49–2.62 (m, 1H; H-3’b), 3.59–3.73 (m, 1H; H-5’), 3.80
1’,5’-Anhydro-4’,6’-O-isopropylidene-2’-(thymin-1-yl)-2’,3’-dideoxy-l-ara-
bino-hexitol (10): Aqueous NaOH (5N; 2.55 mL), CS2 (2.55 mL) and
then b-bromopropionitrile (6.1 mL) were added at 08C to a stirring solu-
tion of compound 7 (0.75 g, 2.41 mmol) in DMSO (15 mL). After being
stirred for 30 min, the solvent was evaporated and the crude residue was
extracted with CH2Cl2 and washed with NH4Cl solution. The organic
layer was dried (Na2SO4) and the solvent removed under reduced pres-
sure to give crude 9. To a refluxing solution of 9 dissolved in anhydrous
toluene (25 mL), kept under nitrogen flow,
a solution of nBu3SnH
(0.48 mL, 1.78 mmol) and AIBN (0.01 g, 0.06 mol) in anhydrous toluene
(20 mL) was added dropwise over 1 h. After the addition, the brown so-
lution was stirred for an additional 30 min under reflux. The reaction
mixture was then concentrated under reduced pressure and the resulting
residue was purified by column chromatography on silica gel (CH2Cl2/
MeOH 95:5) to give the pure 10 (0.51 g, 1.70 mmol, 80% overall yield)
as a white powder: [a]2D5 =À51.5 (c=0.3, CHCl3); 1H NMR (300 MHz,
CDCl3, 258C): d=1.70 (s, 3H; CH3), 1.75 (s, 3H; CH3), 1.92 (ddd, J3’a,2’a
4.7, J3’a,4’ =12.6, J3’a,3’b =13.3 Hz, 1H; H-3’a), 1.99 (s, 3H; CH3), 2.28 (brd,
3’b,3’a =13.3 Hz, 1H; H-3’b), 3.35 (ddd, 5’,6’b =5.3, 5’,4’ =9.7, J5’,6’a =
=
J
J
J
(dd, J6’a,5’ =4.9, J6’a,6’b =12.0 Hz, 1H; H-6’a), 3.91 (dd, J6’b,5’ =2.2, J6’b,6’a
=
10.1 Hz, 1H; H-5’), 3.68–3.82 (m, 2H; H-4’, H-6’a), 3.92–4.02 (m, 2H;
H1’a, H6’b), 4.20 (brd, 1H, J1’b,1’a =14.0 Hz), 4.62 (brs, 1H; H-2’), 7.84 (s,
1H; H-6), 8.65 ppm (s, 1H, NH); 13C NMR (75 MHz, CDCl3, 258C): d=
12.8 (CH3), 19.1 (CH3), 29.1 (CH3), 33.4 (CH2), 51.5 (CH), 62.3 (CH2),
66.0 (CH2), 68.9 (CH), 75.5 (CH), 99.8 (C), 110.6 (C), 138.0 (CH), 150.7
(C), 163.4 ppm (C); elemental analysis calcd (%) for C14H20N2O5: C
56.75, H 6.80, N 9.45; found: C 56.91, H 6.78, N 9.41.
12.0 Hz, 1H; H-6’b), 4.08 (dd, J1’a,2’ =2.3, J1’a,1’b =12.9 Hz, 1H, H-1’a), 4.42
(d, J1’b,1’a =12.9 Hz, 1H; H-1’b), 4.61–4.74 (m, 1H; H-4’), 5.05 (brs, 1H,
H-2’), 7.57 (t, Jortho =7.5 Hz, 2H; H-arom), 7.66 (t, Jortho =7.5 Hz, 1H; H-
arom), 8.10 (d, Jortho =7.5 Hz, 2H; H-arom), 8.72 (s, 1H; H-8), 8.78 ppm
(s, 1H; H-2); 13C NMR (75 MHz, [D6]DMSO, 258C): d=35.8 (CH2), 51.6
(CH), 61.0 (CH2), 61.2 (CH2), 68.3 (CH), 83.2 (CH), 123.0 (C), 128.0
(CH), 128.4 (CH), 132.6 (CH), 133.6 (C), 143.6 (CH), 149.6 (C), 151.6
(CH), 152.3 (C), 166.8 ppm (C); exact mass calcd for C18H19N5O4 [M+
H]+: 370.1509; found: 370.1511.
1’,5’-Anhydro-4’,6’-O-isopropylidene-2’-(adenin-9-yl)-2’,3’-dideoxy-l-ara-
bino-hexitol (12): Aqueous NaOH (5N; 2.79 mL), CS2 (2.79 mL) and
Chem. Eur. J. 2009, 15, 10121 – 10131
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10129

P. Herdewijn et al.
General procedure for nucleoside dimethoxytritylation: Dimethoxytrityl
chloride (2.34 mmol) was added at room temperature to a solution of the
nucleoside (1.95 mmol) in anhydrous pyridine (10 mL) and under nitro-
gen atmosphere. After being stirred at room temperature for 3 h, saturat-
ed NaHCO3 solution (1 mL) was added, the reaction solvent was evapo-
rated and the resulting residue was diluted with CH2Cl2 (50 mL) and
washed with brine (3ꢃ50 mL). The organic layer was dried (Na2SO4),
evaporated under reduced pressure and the crude residue was purified
by flash chromatography (CH2Cl2/MeOH 98:2) to afford the correspond-
ing dimethoxytritylated nucleoside (70–75%) as a white foam. 1H and
13C NMR data for 1’,5’-anhydro-6’-O-dimethoxytrityl-2’-(thymin-1-yl)-
2’,3’-dideoxy-l-arabino-hexitol (15) and 1’,5’-anhydro-6’-O-dimethoxytri-
tyl-2’-(N6-benzoyladenin-9-yl)-2’,3’-dideoxy-l-arabino-hexitol (18) coin-
cide with those reported in ref. [6]. Exact mass calcd for thymine nucleo-
side 15: C32H34N2O7 [M+H]+: 559.2444, found 559.2433. Exact mass
calcd for adenine nucleoside 18: C39H37N5O6 [M+H]+: 672.2822, found
672.2788.
UV melting experiments: Oligomers were dissolved in a buffer solution
containing NaCl (0.1m), potassium phosphate (0.02m, pH 7.5) and EDTA
(0.1 mm). The concentration was determined by measuring the absorb-
ance in MilliQ water at 260 nm at 808C, and by assuming that hexitol nu-
cleosides have the same extinction coefficients per base moiety in the de-
natured state as the natural nucleosides (A*, e=15000; T*, e=8500).
The concentration for each strand was 4 mm in all experiments. Melting
curves were determined with a Varian Cary 300 BIO spectrophotometer.
Cuvettes were maintained at constant temperature by water circulation
through the cuvette holder. The temperature of the solution was mea-
sured with a thermistor that was directly immersed in the cuvette. Tem-
perature control and data acquisition were carried out automatically with
an IBM-compatible computer by using Cary WinUV thermal application
software. A quick heating and cooling cycle was carried out to allow
proper annealing of both strands. The samples were then heated from 10
to 808C at a rate of 0.28CminÀ1, and were cooled again at the same
speed. Melting temperatures were determined by plotting the first deriva-
tive of the absorbance as a function of temperature; data plotted were
the average of two runs. Up and down curves in general showed identical
Tm values.
General procedure for nucleoside phosphitylation: Freshly dried diiso-
propylethylamine (3.3 mmol) and 2-cyanoethyl-N,N-diisopropyl chloro-
phosphoramidite (1.6 mmol) were added to a solution of the dimethoxy-
tritylated nucleoside (1 mmol) in anhydrous CH2Cl2 (6 mL) at 08C and
under argon atmosphere. The reaction mixture was stirred at 08C for
90 min after the reaction was completed, as indicated by TLC. Saturated
NaHCO3 solution (2 mL) was added, the solution was stirred for another
10 min and partitioned between CH2Cl2 (50 mL) and aqueous NaHCO3
(30 mL). The organic layer was washed with brine (3ꢃ30 mL) and the
aqueous phases were extracted with CH2Cl2 (30 mL). After solvent evap-
oration, the resulting oil was purified by flash chromatography (hexane/
acetone/TEA 62:36:2). The yellow solid was then dissolved in CH2Cl2
(3 mL) and precipitated twice in cold hexane (160 mL, À608C) to afford
the desired corresponding phosphoramidite nucleoside (74% yield for
compound 16, 84% yield for compound 19) as a white powder. Each
product was dried under vacuum and stored, overnight, under nitrogen at
À208C. Data for compound 16: exact mass calcd for C41H51N4O8P [M+
H]+: 759.3522, found 759.3510; 31P NMR: d=148.41, 148.87 ppm. Data
for compound 19: exact mass calcd for C48H54N7O7P: [M+H]+: 872.3900,
found 872.3887; 31P NMR: d=148.40, 148.67 ppm.
Circular dichroism measurements: CD spectra were measured at 208C
with a Jasco J-715 spectropolarimeter equipped with a Peltier-type tem-
perature control system (model PTC-348WI) in thermostatically con-
trolled 0.1 cm cuvettes. The molar ellipticity [q] (degcm2 dmolÀ1) was cal-
culated from the equation: [q]=[q]obs ꢃ(mrw)/10ꢃlc, where [q]obs is the
ellipticity (deg), mrw is the sample molecular weight (gmolÀ1), c is the
sample concentration (gmLÀ1), l is the optical path length of the cell
(cm). The cells had a 0.1 cm path length and sample concentration was
about 0.16 mgmLÀ1. The oligomers were dissolved and analysed in buffer
containing NaCl (0.1m), potassium phosphate (0.2m, pH 7.5) and EDTA
(0.1 mm).
LC-MS experiments: Oligonucleotides were dissolved at a concentration
of 100 mm in H2O containing HCO2NH4 (1m). Samples (100 nL) were in-
jected on a reverse phase column (C18 PepMap 0.5ꢃ15 mm, Dionex)
and eluted with a N,N-dimethylaminobutane/1,1,3,3,3-hexafluoro-2-prop-
anol and acetonitrile system. As reported above, electrospray spectra
were acquired by using an orthogonal acceleration/time-of-flight mass
spectrometer (Q-Tof-2, Micromass, Manchester, UK) in negative ion
mode.
Oligonucleotide synthesis: Oligonucleotide assembly was performed with
an Expedite DNA synthesizer (Applied Biosystems) by using the phos-
phoramidite approach. The oligomers were deprotected and cleaved
from the solid support by treatment with methylamine (40% in water)
and concentrated aqueous ammonia (1:1, 308C). After gel filtration on a
NAP-10 column (Sephadex G25-DNA grade; Pharmacia) with water as
eluent, the crude mixture was analysed by using a Mono-Q HR 5/5 anion
Acknowledgements
exchange column, after which purification was achieved by using
a
The authors are grateful to K. U. Leuven (OT) for financial support.
D.D. is indebted to Paola Carullo and Luigi Martino for helpful discus-
sions on CD experiments. CD measurements as well as most 1H and
13C NMR spectroscopy experiments were performed at Centro Interdi-
partimentale di Metodologie Chimico-Fisiche (CIMCF), Universitꢁ di
Napoli Federico II. The Varian Inova 500 MHz instrument is the property
of Consorzio Interuniversitario Nazionale La Chimica per l’Ambiente
(INCA) and was used in the frame of a project by INCA and M.I.U.R.
(L. 488/92, Cluster 11 A).
Mono-Q HR 10/10 column (Pharmacia) with the following gradient
system: A=10 mm NaOH, pH 12.0, 0.1m NaCl; B=10 mm NaOH,
pH 12.0, 0.9m NaCl. The low-pressure liquid chromatography system con-
sisted of a Merck–Hitachi L-6200A intelligent pump, a Mono-Q HR 10/
10 column (Pharmacia), a Uvicord SII 2138 UV detector (Pharmacia-
LKB) and a recorder. The product-containing fraction was desalted on a
NAP-10 column and lyophilised. Oligonucleotides were purified by RP-
HPLC on a C-18 column prior to mass spectrometric analysis. A linear
gradient of A: ammonium bicarbonate (25 mm in H2O, pH 7.0), and B:
acetonitrile (80% in H2O) was applied.
Characterisation of oligonucleotides by mass spectrometry: The purity of
the oligonucleotides was checked by HPLC/MS on a capillary chromato-
graph (CapLC, Waters, Milford, MA, USA). Columns of 150 mmꢃ
0.3 mm length (LC Packings, San Francisco, CA, USA) were used. Oligo-
nucleotides were eluted with a triethylammonium/1,1,1,3,3,3-hexafluoro-
2-propanol/acetonitrile solvent system; flow rate was 5 mLminÀ1. Electro-
spray spectra were acquired by using an orthogonal acceleration/time-of-
flight mass spectrometer (Q-Tof-2, Micromass, Manchester, UK) in nega-
tive ion mode; scan time used was 2 s. The combined spectra from a
chromatographic peak were deconvoluted by using the MaxEnt algo-
rithm of the software (Masslynx 3.4, Micromass, Manchester, UK). Theo-
retical oligonucleotide masses were calculated by using the monoisotopic
element masses.
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ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 10121 – 10131

Synthesis and Base Pairing Properties of l-HNA
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feline corona and feline herpes viruses (CRFK cells), HSV-1, HSV-2
and vaccinia virus (Hel cells), respiratory syncytial virus (HeLa
cells), vesicular stomatitis virus (Hel and HeLa cells), coxsackie B4
virus (Vero and HeLa cells), influenza A H1N1 subtype, influenza A
H3N2 subtype and influenza B viruses (MDCK cells).
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[35] This result disagrees with previous experiments on the thermal be-
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(Ad*)13–d(Ad*)13 interaction was not found (even though it was not
completely excluded). As two enantiomers must have the same ther-
mal behaviour, d(Ad*)13–d(Ad*)13 hybridisation was again studied
and found to have the same complex d(Al*)13–d(Al*)13 (Table 3).
[36] The 6’-d(Al*(Tl*)3Al*Tl*Al*(Tl*)2Al*(Tl*)2Al*) strand on its own
ACHTNUGTRNEN(NUG Ad*)13 (ref. [6]), in which a d-
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
gave a Tm of 50.58C; considering that the latter is a noncomplemen-
tary system, an overhanged Watson–Crick base pairing (provided
with some mismatches) of two separate strands can be conjectured:
[37] The weak association reported in Table 4 (entry 4) most probably
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[25] Compounds 13 and 14 had already been evaluated as potential in-
hibitors of HIV (strain IIIB in MT4 cells), HCMV (strain AD169 in
MRC5 cells) and HSV-1 (Vero cells) infections. Unfortunately, they
were inactive in these assays, see: M. W. Andersen, S. M. Daluge, L.
Kerremans, P. Herdewijn, Tetrahedron Lett. 1997, 38, 8147–8150. In
corresponds to ds-l-HNA-(A*)13, which had very similar melting
curves.
[38] This weak association is again probably related to ds-l-HNA-(A*)13.
[39] This result is corroborated by the X-ray analysis of ds-d-HNA,
which has demonstrated that the latter forms a right-handed double
helix: R. Declercq, A. Van Aerschot, R. J. Read, P. Herdewijn, L.
Van Meervelt, J. Am. Chem. Soc. 2002, 124, 928–933.
[40] The accurate amount of double strand compared to that of the
single strands in each LC-MS experiment can obviously depend on
how stable the complex is, and therefore this experiment should not
be taken into account for a quantitative analysis.
Received: July 3, 2009
Published online: September 8, 2009
Chem. Eur. J. 2009, 15, 10121 – 10131
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10131