Enantiomeric selection properties of β-homoDNA: Enhanced pairing for heterochiral complexes

Article abstract of DOI:10.1002/anie.201301659

De gustibus: β-homoDNA has the singular property of being able to pair with homochiral complements of opposite chirality, with a greater stability than that observed in the corresponding isochiral complexes. Relevant to etiological investigations on nucleic acid structure, these results suggest the existence of a relationship between carbohydrate structure and stereoselectivity of the hybridization processes of the corresponding nucleic acids. Copyright

Full text of DOI:10.1002/anie.201301659

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DOI: 10.1002/anie.201301659
Nucleic Acids
Enantiomeric Selection Properties of b-homoDNA: Enhanced Pairing
for Heterochiral Complexes**
Daniele DꢀAlonzo,* Jussara Amato, Guy Schepers, Matheus Froeyen, Arthur Van Aerschot,
Piet Herdewijn,* and Annalisa Guaragna
The analysis of the physicochemical properties of sugar-
modified nucleic acids is currently at the core of intense
multidisciplinary investigations including chemistry, biology,
biotechnology, and medicine.[1] On one side, synthetic poly-
mers acting as RNA/DNA mimics have extensively been
devised for applications in therapy, diagnostics, and synthetic
biology.[2,3] On the other side, the construction of alternative
pairing systems has been explored either to consider their use
as orthogonal nucleic acid candidates[4] or with the aim to
potentially yield insights into the chemical evolution criteria
ultimately leading to the current genetic system.[5] In all cases,
structural changes of natural (deoxy)ribose cores have been
established to determine profound consequences in the
pairing potential of the resulting artificial nucleic acids.[6,7]
In some noteworthy examples, oligonucleotide systems
endowed with six-membered sugars in the backbone have
been observed[8–10] to hold the singular property (unique of its
kind) of pairing with homochiral complements having oppo-
Figure 1. Sugar-modified nucleic acids displaying pairing aptitude for
homochiral complements of opposite chirality.
site sense of chirality. Relevant to etiology-oriented inves-
tigations on nucleic acid structure,[5] these findings could
suggest the existence of a relationship between nature of the
sugar backbone and chiral-selection properties of nucleic
acids, thereby providing insights to enrich our understanding
of the structural prerequisites for base pairing. From a com-
parative analysis of the pairing behavior of six-membered
nucleic acids[2,5–7] we perceived that, despite the large
structural differences, oligonucleotide systems capable of
iso- and heterochiral hybridization (Figure 1) shared preor-
ganized carbohydrate conformations with equatorially-ori-
ented nucleobases.[11] This observation took us to wonder if
such an arrangement of the aglycon moiety, especially
whereas inducing strong backbone-base inclination[6] or
even enabling formation of quasilinear oligomeric struc-
tures,[5–8] could lead sugar chirality not to be crucial in
hybridization processes. In view of systematic investigations
aimed at addressing this question, we herein considered the
chiral selection properties of the well-known[5,12] pairing
system composed of (6’!4’)-linked b-erythro-hexopyranosyl
nucleotides (b-homoDNA; Figure 1). Based on above
assumptions and early experimental data,[8] we reasoned
that the strongly inclined[5,12] complexes provided by the “all-
equatorial” pyranose backbone of b-homoDNA could make
the latter an interesting candidate displaying potential for
heterochiral hybridization.
[*] Dr. D. D’Alonzo, Dr. A. Guaragna
Dipartimento di Scienze Chimiche
Universitꢀ degli Studi di Napoli Federico II
Via Cintia 21, 80126 Napoli (Italy)
E-mail: dandalonzo@unina.it
An investigation into the enantioselectivity of the hybrid-
ization processes of b-homoDNA required access to oligo-
meric sequences in both enantiomeric forms (b-d- and b-l-
homoDNA). From a synthetic standpoint, while access to
d-hexopyranosyl nucleosides was easily obtained by a carbo-
hydrate-based route,[13] the synthesis of the corresponding l-
enantiomers under the same reaction conditions was ham-
pered by the limited commercial availability of almost all l-
hexoses. In an alternative path, our long studied de novo
approach to l-monosaccharides[14] and other structurally-
related compounds[9] was recently exploited[15] for the prep-
aration of the l-nucleosides 2a,b (T and ABz acting as model
nucleobases) from the homologating agent 1 (Scheme 1).
G. Schepers, Prof. Dr. M. Froeyen, Prof. Dr. A. Van Aerschot,
Prof. Dr. P. Herdewijn
Laboratory for Medicinal Chemistry, Rega Institute for Medical
Research, KU Leuven
Minderbroedersstraat 10, 3000 Leuven (Belgium)
E-mail: Piet.Herdewijn@rega.kuleuven.be
Dr. J. Amato
Dipartimento di Farmacia
Universitꢀ degli Studi di Napoli Federico II
Via D. Montesano 49, 80131 Napoli (Italy)
[**] Financial support from KU Leuven (GOA/10/13) and FWO (Flemish
Scientific Research grant G.0784.11N) is gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201301659.
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self-association. Pairing priority[16] (Ah:Ah > Ah:Th) was dem-
onstrated by comparing the Tm value (558C) of the self-
complementary strand (entry 2) with the higher Tm value
(588C) exhibited by the shorter, self-complementary
sequence comprising both Ah:Ah and Ah:Th base pairs.
The pairing properties of b-l-homoDNA were then
examined in annealing studies with some (un)natural d-
complements (Table 1, entries 4–12). Sugar-modified oligo-
nucleotide systems adopt quasilinear structures by virtue of
the equatorial nucleobase arrangement.[5–8] The stability of
the resulting complexes was compared with those containing
its d-enantiomer (b-d-homoDNA). Formation of isochiral/
heterochiral hybrids was either directly determined or
indirectly deduced by studies in the “mirror-image world”.[17]
Neither in homopurine nor homopyrimidine form did b-l-
homoDNA show any significant pairing aptitude toward
natural complements (Table 1, entries 4–7). Analogous
results have already been reported for b-d-homoDNA.[5,18]
In contrast, b-l-homoDNA exhibited from good to excellent
hybridization properties when annealed with preorganized d-
oligonucleotide partners (entries 8–12). Notably, the stability
of the complexes obtained between strands of opposite
chirality was generally higher than that of the corresponding
isochiral associations. For example, b-l-homoDNA formed
stronger complexes with d-CNA (Tm up to 608C) than those
formed between b-d-homoDNA and d-CNA (Tm up to 228C;
entries 8–9). Conversely, when annealed with d-HNA, b-l-
homoDNA gave hybrids of comparable stability (entry 10). In
line with previous observations,[7] the stability of heterochiral
complexes increased with the preorganization of nucleic acid
complements (entries 8–10). Along this line, annealing
experiments between homochiral b-homoDNA complements
having the same or opposite sense of chirality (b-homoDNA
acting as the most preorganized pairing system) were
eventually performed (entries 11 and 12). Compared with
the weak transition likely related to the isochiral duplex Ah:Th
(Tm = 358C), the melting curve observed from mixing equi-
molar amounts of d-(Ah)13 and l-(Th)13 was referred to
formation of a complex having far greater stability (Tm =
858C). Unexpectedly, when examining the UV melting
behavior of the heterochiral mixture, no trace of the exceed-
ingly stable isochiral Ah:Ah association (Tm > 908C) was
detected. Conversely, such an association largely occurred
when d-(Ah)13 and d-(Th)13 were mixed[19] (entry 11). We were
also surprised to find some discrepancies between UV- and
CD-melting measurements of the same mixtures (entry 12).
In the latter case, the heterochiral Ah:Th association (Tm =
878C) resulted thermodynamically more stable than both the
isochiral Ah:Th (Tm not detected)[20] and Ah:Ah complexes
(Tm = 838C).[21]
Scheme 1. The b-l-erythro-hexopyranosyl nucleotides 5a,b as building
blocks for b-l-homoDNA synthesis. DIPEA=diisopropylethylamine,
MMTCl=p-monomethoxytrityl chloride, PMB=para-methoxybenzyl,
Py=pyridine.
Synthesis was based on a key stereoselective N-glycosidation
involving in situ anomerization of a/b nucleosides (b/a up to
20:1). Conversion of 2a,b into the phosphoramidite nucleo-
tides 5a,b was then carried out under common reaction
conditions (Scheme 1). Fully modified sequences containing
b-l-erythro-hexopyranosyl nucleotides were synthesized
using the phosphoramidite method on solid support.[13a]
In early annealing experiments we assessed that b-l-
homoDNA [l-(Bh)n, B = A/T] formed isochiral self-comple-
mentary duplexes (ds-b-l-homoDNA) with the same melting
profiles as those reported for ds-b-d-homoDNA[16] (Table 1,
entries 1–3). Besides common l-Ah:l-Th pairing, formation of
l-Ah:l-Ah complexes was indicated by the UV melting curve
of l-(Ah)6 (entry 1), and strongly suggested intermolecular
Table 1: Thermal stability studies of complexes containing b-(d- and/or
l-) homoDNA. Melting points were determined in 0.1m NaCl, 20 mm
KH2PO4 (pH 7.5), 0.1 mm Na2EDTA (4 mm concentration of each strand
unless otherwise specified).
Entry Oligonucleotide Sequence
Complement (Tm [8C])
d-homoDNA l-homoDNA
1
2
3
4
5
6
7
8
l-homoDNA
l-homoDNA
l-homoDNA
d-DNA
d-DNA
d-RNA
d-RNA
d-CNA
d-CNA
d-HNA
l-(Ah)6
n.d.
46[a,b]
55[a]
l-(Ah)6(Th)6 n.d.
l-(Ah)6(Th)4 n.d.
58[a]
[c]
[c]
d-(dT)13
d-(dA)13
d-(rU)13
d-(rA)13
d-(Tc)13
d-(Ac)13
d-(Th)13
d-(Ah)13
–
–
–
–
–
–
–
–
[c]
[c]
[c]
[c]
[c]
[c]
>90[b]
29,[d,e] >90[b]
9
22[d,f]
60
78
85
87
Because of their singular behavior, b-homoDNA-based
complexes were subjected to further comparative studies. CD
analysis of iso- and heterochiral mixtures (Figure 2) con-
firmed hybrid formation between b-homoDNA complements
with opposite sugar chirality. Likewise, large conformational
differences among these complexes were suggested, as
a result of the presence of oligomeric strands providing
matching/mismatching chiroptical contributions. For exam-
ple, although all complexes displayed almost superimposable
10
11
12
86
d-homoDNA
d-homoDNA
35, >90[b]
83[b]
d-(Ah)13
[g]
[a] Used 8 mm of the self-complementary sequence. [b] Referred to an
Ah:Ah association. [c] No clear cooperative transition detected.
[d] Determined by evaluation in the mirror-image world (Ref. [17]).
[e] UV, CD, and PAGE data suggested formation of heterochiral duplexes
and triplexes. [f]Ttaken from Ref. [18]. [g] Melting points determined by
CD analysis. n.d.=not determined.
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Figure 4. Close view of the simulated d-(Ah)13:l-(Th)13 duplex. Green:
carbon atoms in the d-(Ah)13 strand. Yellow: carbon atoms in the
l-(Th)13 strand.
Figure 2. Normalized CD spectra of: ds-d-(Ah)13 (g); d-(Ah)13 + d-
(Th)13 (1:1 mixture; a); d-(Ah)13 + l-(Th)13 (1:1 mixture; c); and
d-(Th)13 (l). All measurements were taken at 208C in 0.01m tris-
HCl, 0.15m NaCl buffer (pH 7.0).
1
homoDNA and a C4 form for b-l-homoDNA). In line with
positive bands around l = 275 nm, only isochiral duplexes
showed a negative band around l = 250 nm. At about the
same wavelength, the heterochiral complex exhibited a very
weak absorption. Isochiral and heterochiral Ah- and Th-
containing mixtures also roughly displayed mirrored Cotton
effects around l = 215 nm (Figure 2).
A clear view of the pairing behavior of b-homoDNA was
provided by PAGE analysis under nondenaturating condi-
tions (Figure 3). While pairing of isochiral strands led to co-
experimental data, the duplex model was found to be very
stable, as the temperature increase to 360 K did not affect its
structural integrity.
Interestingly, striking structural differences arose from
comparison of the heteroduplex with a previous[6] MD
simulation of the isochiral ds-d-homoDNA. Contrary to the
quasilinear shape of the former, the latter is known[6,12] to
adopt a helical structure. In addition, the high backbone-base
inclination value of the isochiral duplex model (hB 378) was
lower than that calculated for the heteroduplex (hB 468). In
view of the correlation between backbone-base inclination
and interstrand stacking in nucleic acid duplexes,[22] it is
reasonable to hypothesize a greater interstrand stacking
contribution in the heterochiral complex, which could
explain, in comparison with the isochiral duplex, its higher
thermodynamic stability.
In summary, the preliminary analysis of the pairing
properties of b-l-homoDNA has revealed that, despite
sugar stereochemistry, it is able to strongly pair with
homochiral d-complements, in many cases with a greater
stability than that observed in the corresponding isochiral
complexes. The heteroduplex composed of enantiomeric b-
homoDNA complements has especially deserved attention
owing to a notable thermodynamic stability, it can be
currently considered as the strongest association between
homochiral oligomers of opposite sense of chirality. The
selectivity observed during formation (preferred to the
competitive, isochiral self-complementary pairing process)
also represents, to the best of our knowledge, an unprece-
dented event among either natural or artificial nucleic acids.
From an etiological standpoint, the reversed enantioselectiv-
ity in the pairing properties of b-homoDNA underlines the
“unsuitability” of hexose nucleic acids (b-homoDNA acting
as a model system) as “potentially natural” RNA alternatives.
Most generally, our results strengthen the hypothesis of a role
played by the sugar unit of six-membered nucleic acids in the
alteration of the stereoselectivity of the hybridization pro-
cesses. Previous and current experimental clues highlight the
importance of sugar conformation (involving equatorial
Figure 3. 20% PAGE under nondenaturing conditions of: d-(Th)13
(lane 1); d-(Ah)13 + d-(Th)13 (1:1 mixture; lane 2); d-(Ah)13 (lane 3); d-
(Ah)13 + l-(Th)13 (1:1 mixture; lane 4); d-(Ah)13 + l-(Th)13 (2:1 mixture;
lane 5); l-(Th)13 (lane 6). Measurements were taken in 0.01m Tris-HCl,
0.15m NaCl, pH 7.0 (2 mm concentration of each oligonucleotide
strand).
occurrence of Ah:Th and Ah:Ah duplexes (lane 2), the latter
totally disappeared owing to formation of a complex of
slightly faster mobility, thus resulting from pairing of hetero-
chiral Ah and Th strands (lane 4). The supposed formation of
triplexes such as d-Ah:d-Ah:l-Th (which could explain
disappearance of the Ah:Ah duplex) was also realistically
ruled out (lane 5).
The heteroduplex shape and stability were eventually
explored using MD simulations (Figure 4). Although slightly
right-handed, the antiparallel d-(Ah)13:l-(Th)13 duplex model
displayed almost no helicity, with an average helical twist of
6.08, as well as a strong backbone-base inclination with
a predominance for interstrand over intrastrand base stack-
ing. Sugar substituents adopted a classic all-equatorial
4
arrangement (corresponding to a C1 conformation for b-d-
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[11] In case of CNA, conformational flip of the sugar moiety to the
nucleobase arrangement), although participation of other
factors (above all, sugar rigidity) has also been herein
suggested. In-depth studies aimed at shedding light on this
topic are currently ongoing and will be published in due
course.
chair form bearing equatorially-oriented nucleobases has been
identified as a stringent prerequisite for heterochiral hybrid-
ization (Ref. [8]). Although this has not yet been verified for
HNA, we assume that the similar pairing properties of the two
oligonucleotide systems reflect an analogous conformational
behavior of the corresponding sugar units.
Received: February 26, 2013
Published online: May 13, 2013
[12] M. Egli, P. Lubini, P. S. Pallan, Chem. Soc. Rev. 2007, 36, 31.
[13] a) A. Augustyns, F. Vandendriessche, A. Van Aerschot, R.
Busson, C. Urbanke, P. Herdewijn, Nucleic Acids Res. 1992, 20,
4711; b) M. Bçhringer, H.-J. Roth, J. Hunziker, M. Gçbel, R.
Krishnan, A. Giger, B. Schweizer, J. Schreiber, C. Leumann, A.
Eschenmoser, Helv. Chim. Acta 1992, 75, 1416.
[14] A. Guaragna, D. DꢁAlonzo, C. Paolella, C. Napolitano, G.
Palumbo, J. Org. Chem. 2010, 75, 3558.
[15] D. DꢁAlonzo, A. Guaragna, A. Van Aerschot, P. Herdewijn, G.
Palumbo, J. Org. Chem. 2010, 75, 6402.
[16] J. Hunziker, H.-J. Roth, M. Bçhringer, A. Giger, U. Die-
derichsen, M. Gçbel, R. Krishnan, B. Jaun, C. Leumann, A.
Eschenmoser, Helv. Chim. Acta 1993, 76, 259.
[17] N. K. Christensen, T. Bryld, M. D. Sørensen, K. Arar, J. Wengel,
P. Nielsen, Chem. Commun. 2004, 282.
Keywords: carbohydrates · chirality · heterochiral hybridization ·
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homoDNA · nucleic acids
[1] Chemistry and Biology of Artificial Nucleic Acids (Eds.: M. Egli,
P. Herdewijn), Wiley-VCH, Zꢀrich, 2012.
[2] P. Herdewijn, Chem. Biodiversity 2010, 7, 1.
[3] V. B. Pinheiro, A. I. Taylor, C. Cozens, M. Abramov, M. Renders,
S. Zhang, J. C. Chaput, J. Wengel, S.-Y. Peak-Chew, S. H.
McLaughlin, P. Herdewijn, P. Holliger, Science 2012, 336, 341.
[4] M. Maiti, V. Siegmund, M. Abramov, E. Lescrinier, H. Rose-
meyer, M. Froeyen, A. Ramaswamy, A. Ceulemans, A. Marx, P.
Herdewijn, Chem. Eur. J. 2012, 18, 869.
[5] A. Eschenmoser, Angew. Chem. 2011, 123, 12618; Angew. Chem.
Int. Ed. 2011, 50, 12412.
[6] E. Lescrinier, M. Froeyen, P. Herdewijn, Nucleic Acids Res. 2003,
31, 2975.
[7] D. DꢁAlonzo, A. Guaragna, G. Palumbo, Chem. Biodiversity
2011, 8, 373.
[8] M. Froeyen, F. Morvan, J.-J. Vasseur, P. Nielsen, A. Van Aer-
schot, H. Rosemeyer, P. Herdewijn, Chem. Biodiversity 2007, 4,
803.
[9] D. DꢁAlonzo, A. Van Aerschot, A. Guaragna, G. Palumbo, G.
Schepers, S. Capone, J. Rozenski, P. Herdewijn, Chem. Eur. J.
2009, 15, 10121.
[10] R. Krishnamurthy, S. Pitsch, M. Minton, C. Miculka, N. Wind-
hab, A. Eschenmoser, Angew. Chem. 1996, 108, 1619; Angew.
Chem. Int. Ed. Engl. 1996, 35, 1537.
[18] L. Kerremans, G. Schepers, J. Rozenski, R. Busson, A. Van Aer-
schot, P. Herdewijn, Org. Lett. 2001, 3, 4129.
[19] Studies to understand this phenomenon are underway. An
uncommon kinetic control ruling the pairing process has been
preliminarily hypothesized to explain our experimental data.
[20] The isochiral Ah:Th duplex was not detected by CD-melting
analysis probabaly because of its limited occurrence with respect
to the coexisting Ah:Ah association (compare the UV-melting
spectrum of the same mixture).
[21] It must be stressed that UV- and CD-melting experiments
measure different properties of a duplex, therefore different
results are not contradictory. Efforts aimed at a full ration-
alization of this behavior are however in progress.
[22] P. S. Pallan, P. Lubini, M. Bolli, M. Egli, Nucleic Acids Res. 2007,
35, 6611.
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