Apolar carbohydrates as DNA capping agents

Article abstract of DOI:10.1039/c2cc17093k

Mono- and disaccharides have been shown to stack on top of DNA duplexes stabilizing sequences with terminal C-G base pairs. Here we present an apolar version of glucose and cellobiose as new capping agents that stack on DNA increasing considerably its stability with respect to their natural polyhydroxylated mono- and disaccharide DNA conjugates.

Full text of DOI:10.1039/c2cc17093k

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Apolar carbohydrates as DNA capping agentswz
´ ´
Ricardo Lucas, Empar Vengut-Climent, Irene Gomez-Pinto, Anna Avino, Ramon Eritja,
a
a
b
c
c
˜
´
b
a
´
Carlos Gonzalez and Juan C. Morales*
Received 15th November 2011, Accepted 27th January 2012
DOI: 10.1039/c2cc17093k
Mono- and disaccharides have been shown to stack on top of DNA
duplexes stabilizing sequences with terminal C–G base pairs. Here
we present an apolar version of glucose and cellobiose as new
capping agents that stack on DNA increasing considerably its
stability with respect to their natural polyhydroxylated mono- and
disaccharide DNA conjugates.
DNA double helix is only observed with C–G or G–C terminal
base pairs.
Herein, we report the synthesis of oligonucleotides with
permethylated mono- and disaccharides covalently linked to
their 50-end. These apolar carbohydrates act as new capping
molecules capable of stacking on double-stranded DNA
(Fig. 1). Permethylated glucose and cellobiose were found to
stabilize DNA duplexes much more than natural glucose and
cellobiose.
Non-covalent forces direct molecular interactions between
biomolecules and their combination and interplay in biology
rules life. DNA being the central molecule of life also gives the
chance to study molecular interactions in aqueous media.
Aromatic p–p stacking interactions have extensively been studied
using DNA as a model. Both natural1 and non-natural2–4
aromatic bases attached to the 30-end or 50-end of double
stranded DNA have shown enhanced stabilization of DNA
duplexes, acting as capping agents. These molecular ‘‘caps’’ are
usually planar aromatic rings of different size and shape that take
advantage of p–p stacking interactions.5–8 The only non-planar
compounds described to stack on DNA are steroids such as
cholic acid which showed a high increase in DNA stability via
CH–p interactions.9 Recently, binaphthyl and phenylcyclohexyl
nucleosides10,11 with nonplanar aromatic bases have been included
inside DNA but no data as capping entities were reported.
Our group has studied carbohydrate–aromatic stacking
interactions using carbohydrate oligonucleotide conjugates
(COCs) with dangling-ends as a model. First, we evaluated
monosaccharide–phenyl interactions as a double dangling
motif at the edge of a duplex of DNA.12 We found that
stabilization varies from ꢀ0.15 to ꢀ0.40 kcal molꢀ1 and
depends on the number of hydroxy groups and stereochemistry.
Recently, we have shown that highly polar carbohydrates can
act as DNA capping molecules. Sugar stacking is observed for
mono- and disaccharides on top of C–G or T–A base pairs as
the edge of the DNA duplex.13 Nevertheless, stabilization of the
Synthesis of the permethylated carbohydrate oligonucleotide
conjugates started with the preparation of the corresponding
permethylated glucose and cellobiose phosphoramidite derivatives
(5 and 10, respectively) (Scheme 1). Glycosylation of the O-benzyl
protected ethylene glycol spacer followed by deprotection of the
acetyl groups yielded intermediate 2. Methylation under standard
conditions produced compound 3 in good overall yield (70%,
3 steps). Further hydrogenation and standard phosphoramidite
preparation proceeded uneventfully to yield permethylated
glucose phosphoramidite 5 (76%, 2 steps). A similar synthetic
strategy was followed to prepare permethylated cellobiose
phosphoramidite 10 (48% yield, 5 steps).
Preparation of the apolar saccharide oligonucleotide conjugates
was carried out by standard solid phase oligonucleotide synthesis
using compounds 5 or 10 at the last coupling step. Both apolar
carbohydrates were attached to self-complementary sequences
CGCGCG, GGCGCC, AGCGCT and TGCGCA. Solutions
of the COCs were subjected to UV melting analysis and
thermodynamic parameters were calculated (Table 1).
Conjugates containing permethylated glucose and cellobiose on
sequences terminated on a C–G base pair (conjugates 15 and 19)
increased considerably their melting points (7.8 1C and 8.3 1C,
respectively) over those of the natural control sequence 11.
a
´
Instituto de Investigaciones Quımicas, CSIC - Universidad de Sevilla,
Americo Vespucio, 49, 41092 Sevilla, Spain.
E-mail: jcmorales@iiq.csic.es
b
c
´
´
Instituto de Quımica Fısica ‘Rocasolano’, CSIC, C/. Serrano 119,
28006 Madrid, Spain
´
´
Instituto de Investigacion Biomedica de Barcelona, IQAC, CSIC,
CIBER - BBN Networking Centre on Bioengineering, Biomaterials
and Nanomedicine, Baldiri Reixac 10, E-08028 Barcelona, Spain
w Dedicated to Professor Soledad Penades on her 70th birthday.
´
Fig. 1 Schematic drawing of COCs with dangling-ends and details of
z Electronic supplementary information (ESI) available: Detailed
experimental procedures. See DOI: 10.1039/c2cc17093k
one of them (permethylated glucose stacking on top of a C–G base pair).
c
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Chem. Commun., 2012, 48, 2991–2993 2991

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Scheme 1 Synthesis of permethylated glucose and cellobiose phosphoramidites 5 and 10. Reaction conditions: (a) BnOCH2CH2OH, BF3ꢁOEt2,
CH2Cl2; (b) Na2CO3, MeOH; (c) MeI, NaH, DMF; (d) H2, Pd(OH)2, THF–MeOH; (e) 2-cyanoethyl-N,N0-diisopropylamino-chlorophos-
phoramidite, DIEA, CH2Cl2; (f) H2, Pd(OH)2, AcOEt–MeOH.
apolar carbohydrates were attached to the TGCGCA
Table 1 Thermodynamic parameters for COCs
sequence (see Table S3, ESIz). This decrease of COC stabilization
X-DNA sequencea,b,c,d Tme/1C ꢀDH1 ꢀDS1 ꢀDG3o7 DDGo37
on sequences with A–T or T–A base pairs at the edge of the
X = nonef
duplex with respect to the sequences with C–G or G–C base pairs
was also observed for COCs with the natural mono- and
disaccharides. This effect may be due to the larger entropy cost
of reducing the fraying in the more flexible terminal A–T base
pair that counteracts the stabilization obtained with the stacking
of the apolar sugar.13
CGCGCG 11
AGCGCT 12
X = glucose–C2f
CGCGCG 13
AGCGCT 14
X = glc(Me)–C2
CGCGCG 15
AGCGCT 16
X = cellobiose–C2f
CGCGCG 17
AGCGCT 18
X = cellob(Me)–C2
CGCGCG 19
AGCGCT 20
40.9
33.5
46.5
40.3
123
107
8.2
7.1
—
—
44.0
33.6
52.1
37.3
140
98
8.7
7.0
ꢀ0.5
0.1
48.7
34.5
55.0
44.8
147
121
9.4
7.2
ꢀ1.2
ꢀ0.2
The structures of the conjugates containing the permethylated
glucose unit 15 and 16 were studied by NMR spectroscopy.
Proton assignment was carried out following standard procedures.
The DNA duplex structures are barely distorted by the
presence of the apolar sugars as can be inferred by comparison
of the DNA chemical shifts of the conjugates and the control
sequences (see ESIz, Fig. S2). Chemical shift changes are
mostly observed in the neighboring residues of the permethylated
glucose (C1 in the CGCGCG sequence and A1 in the AGCGCT
sequence), indicating that the carbohydrate is interacting mainly
with the terminal residues. This capping interaction is also
supported by a significant number of NOEs (see Fig. 2 and
Table S2, in ESIz). The number and intensities of these NOE
contacts are comparable with those observed in the disaccharide
conjugates studied in our previous work.13 Strong and medium
NOEs are observed between several protons of the terminal
base-pairs with H3 and H5 of the apolar glucose unit, suggesting
that the permethylated glucose interacts with the terminal base-pair
of the duplex predominantly through its a face. In the case of
conjugate 16 some low intensity NOEs are also observed with
H4 proton. These NOEs may arise from spin-diffusion or from
minor species with different carbohydrate conformations, and
were not used in the structural calculations. Interestingly,
many of the DNA-permethylated glucose NOEs involve
exchangeable protons of the terminal base-pair. In both conjugates,
these protons exhibit narrow signals, indicating that they are
protected from water exchange. As in the case of the natural
disaccharide–DNA conjugates studied previously, the capping
carbohydrate reduces strongly the internal dynamics of the
terminal base-pairs. This effect is especially pronounced in
conjugate 16, where the terminal base-pair is AT.
45.9
34.4
49.2
39.1
130
103
8.9
7.1
ꢀ0.7
0.0
49.2
37.7
55.0
43.9
146
117
9.7
7.7
ꢀ1.5
ꢀ0.6
a
b
–C2– states for –CH2–CH2–OPO2ꢀ–. Buffer: 10 mM Na
c
phosphate, 1 M NaCl, pH 7.0. Estimated errors are: Tm ꢂ 0.8 1C
d
and ꢂ6% in DG1. Units for DH1 and DG1 are kcal molꢀ1 and for
DS1 are cal Kꢀ1 molꢀ1
.
Average value of three experiments measured
e
f
at 5 mM conc. From ref. 13.
When conjugates with apolar glucose 15 and apolar cellobiose
19 are compared with their corresponding natural hydroxy-
lated versions glucose–DNA conjugate 13 and cellobiose–
DNA conjugate 17, Tm’s are increased by 4.7 1C and 3.3 1C,
respectively. A similar trend is observed when DG values are
compared; conjugates 15 and 19 stabilize CGCGCG duplexes
by ꢀ1.2 and ꢀ1.5 kcal molꢀ1, respectively, with respect to
unmodified CGCGCG. This stabilization is similar to that
found for a benzene nucleoside in the same context.2 As a
result, the duplex stabilizations of conjugates with the apolar
version of glucose 15 and cellobiose 19 are 2.4 and 2.1 times
more stable, respectively, than their corresponding conjugates
with natural glucose 13 and cellobiose 17. The smaller increase
in cellobiose may be due to the fact that the increased surface
of the apolar version of cellobiose could be too large to fully
stack on top of the C–G base pair. Similar results were found
when the apolar sugars were attached to the GGCGCC
sequence (see ESIz, Table S3).
In the case of the AGCGCT sequence, both conjugates with
permethylated glucose 16 and cellobiose 20 show an increase
in Tm (1 1C and 4.2 1C, respectively) and in free energy (ꢀ0.1
and ꢀ0.6 kcal molꢀ1, respectively) with respect to the natural
sequence 12. Once again, similar results were found when the
Restrained molecular dynamics calculations were carried out
with the AMBER program. Resulting structures are shown in
Fig. 3. In both conjugates 15 and 16, the carbohydrate and the
linker adopt a similar and well-defined structure. Permethylated
c
2992 Chem. Commun., 2012, 48, 2991–2993
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Fig. 3 Structures of conjugate 15 (A), and conjugate 16 (B). Top:
details of the stacking. Bottom: superposition of ten calculated
structures.
We thank the MICINN of Spain (grants CTQ2006-01123,
CTQ2007-68014-C02-02, CTQ2009-13705, BFU2007-63287),
Generalitat de Catalunya (2009/SGR/208), and Instituto de
Salud Carlos III (CIBER-BNN, CB06_01_0019) for financial
support. RL thanks CSIC for a JAE contract.
Fig. 2 (a) Schematic drawing of conjugate 16 with arrows indicating
important observed NOEs; (b) selected region of NOESY spectra for
conjugate 16 (carbohydrate–DNA contacts are shown in cyan).
Notes and references
1 S. Bommarito, N. Peyret and J. SantaLucia, Jr., Nucleic Acids Res.,
2000, 28, 1929–1934.
2 K. M. Guckian, B. A. Schweitzer, R. X. F. Ren, C. J. Sheils,
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1996, 118, 8182–8183.
glucoses stack on top of the terminal base-pair, with their a sides
oriented towards the nucleobases. Carbohydrate conformation is
the usual 4C1 chair. Permethylation increases the carbohydrate size
and allows for an enhanced stacking interaction in which a single
monosaccharide covers most of the terminal base-pair surface
(Fig. 3). Although the main features of both conjugates are quite
similar, minor differences are observed (see Fig. 3, top). These
differences are probably due to the different adjacent nucleobase,
purine in the case of conjugate 15 and pyrimidine for 16.
3 E. T. Kool, J. C. Morales and K. M. Guckian, Angew. Chem., Int.
Ed., 2000, 39, 990–1009.
4 A. Zahn and C. J. Leumann, Chem.–Eur. J., 2008, 14, 1087–1094.
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7 J. Tuma, W. H. Connors, D. H. Stitelman and C. Richert, J. Am.
Chem. Soc., 2002, 124, 4236–4246.
These results are noteworthy since hydrophobic mono- and
disaccharides attached to DNA show a relevant increase in
stabilization of DNA duplexes especially with terminal C–G
or G–C base pairs. In this context, the stability of DNA with
apolar sugars 50-caps is approaching to that found with the
traditional aromatic caps. Further improvement may be obtained
modulating the hydrophobicity of the carbohydrate. NMR studies
confirmed that permethylated sugars stack on top of duplex DNA
similarly to other aromatic moieties. Finally, Our results have
implications in molecular recognition and may be useful in drug
design and in the assembly of supramolecular structures.
8 S. Egetenmeyer and C. Richert, Chem.–Eur. J., 2011, 17,
11813–11827.
9 C. F. Bleczinski and C. Richert, J. Am. Chem. Soc., 1999, 121,
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11 M. Kaufmann, M. Gisler and C. J. Leumann, Angew. Chem., Int.
Ed., 2009, 48, 3810–3813.
12 J. C. Morales, J. J. Reina, I. Dı
R. Eritja, Chem.–Eur. J., 2008, 14, 7828–7835.
13 R. Lucas, I. Gomez-Pinto, A. Avino, J. J. Reina, R. Eritja,
C. Gonzalez and J. C. Morales, J. Am. Chem. Soc., 2011, 133,
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az, A. Avino, P. M. Nieto and
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This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 2991–2993 2993