pH dependent thermal stabilization of DNA by Glcβ(1→3) GlcNAcβ1-→STol

Article abstract of DOI:10.1002/jccs.200400098

A disaccharide, Glcβ(1→3)GlcNAcβ1→STol (GGS, 1), was synthesized and demonstrated to stabilize ct-DNA during the denaturing process. GGS at 50 μM shifted T_m of ct-DNA by 23 °C and the behavior was pH dependent. Poly(dA-dT)₂ was found

Full text of DOI:10.1002/jccs.200400098

Journal of the Chinese Chemical Society, 2004, 51, 655-658
655
pH Dependent Thermal Stabilization of DNA by Glcb(1®3)GlcNAcb1®STol
Chun-Cheng Lina,* (
Kuo-Cheng Lua (
), Sue-Mei Hungb (
),
) and Chien-Chung Chengb,* (
)
aInstitute of Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan, R.O.C.
bDepartment of Chemistry, Tamkang University, Tamsui 251, Taiwan, R.O.C.
A disaccharide, Glcb(1®3)GlcNAcb1®STol (GGS, 1), was synthesized and demonstrated to stabilize
ct-DNA during the denaturing process. GGS at 50 mM shifted Tm of ct-DNA by 23 °C and the behavior was pH
dependent. Poly(dA-dT)2 was found to be the preferable type of DNA for GGS stabilization by circular
dichroism spectroscopy study.
Keywords: DNA-carbohydrate interaction; Hydrogen-bond; pH dependent; Thermal stabilization.
Interactions of molecules with specific sequences of
DNA or RNA to retrieve information stored in them are es-
sential at various stages of a cell’s life cycle. Molecules
which bind with DNA/RNA and disrupt the normal functions
of cancer cells and virus/bacteria can be used as antitumor or
antiinfective agents. Many carbohydrate-containing antibiot-
ics such as calicheamicin and erythromycin1 have been proved
to be useful therapeutic agents and their carbohydrate moi-
eties were demonstrated to serve as the recognition domains
to provide specific binding toward DNA2 or RNA.3 Recently,
one of the elegant designs by using a monosaccharide to
recognize a specific DNA sequence has been reported by
Toshima and co-workers.4 Neocarzinostatin, a carbohydrate-
containing antibiotic, was simplified by replacing its agly-
cone moiety with a photoactive intercalator, and the resulting
mono-amino sugar derivative was still found to direct the
specific DNA sequence cleavage. Therefore, study of carbo-
hydrate-DNA/RNA interactions may provide a new approach
for the design of a new generation of antibiotics.
nucleic acids. However, saccharides should be good candi-
dates in association with DNA or RNA because a large num-
ber of hydrogen-bonds (H-bonds), potentially, can be gener-
ated from interactions of their hydroxyl groups with the phos-
phate anions of DNA/RNA backbones, and/or amino and car-
bonyl groups of nucleobases. Indeed, a single-stranded RNA
has been demonstrated to bind with schizophyllan, a neutral
b(1®3)-glucan with b(1®6) branch polysaccharide, through
H-bond interactions.6 Also, DNA ligands that bind tightly
and selectively to neutral cellobiose were discovered7 by
SELEX8 (systemic evolution of ligands by exponential en-
richment) selection. These results demonstrate that neutral
and common carbohydrates9 can interact specifically with
DNA or RNA through H-bond interactions and prompt us to
study the capability of GGS 1, Glcb(1®3)GlcNAcb1®STol,10
to interact and stabilize a specific type of DNA in the denatur-
ing process.
The synthesis of GGS11 is illustrated in Scheme I. Se-
lectively protecting the primary hydroxyl group of 4 using
TBDPSCl followed by per-benzoylation gave 5. Removal of
thiocresol from 5 with NBS was followed by treating with
trichloroacetonitile to yield imidate 6. Glycosylation of do-
nor 6 with acceptor 7 gave disaccharide 8. After manipulation
of deprotection and protection steps, precursor 9 was ob-
tained. The ester protecting groups of 9 were further hydro-
lyzed to afford GGS. Sulfation12 and oxidation13 of the pri-
mary hydroxyl group of 9 followed by basic hydrolysis
yielded sulfated compound 2 and acid compound 3, respec-
tively.
The carbohydrate domains of antibiotics usually are
presented as deoxy and/or amino sugars to increase hydro-
phobic and electrostatic interactions with DNA/RNA.5 Thus,
common uncharged carbohydrates are generally considered
to have obscurity in binding with highly negatively-charged
HO
HO
HO
O
O
HO
S
O
HO
AcHN
HO
CH3
1
Dedicated to Professor Sunney I. Chan on the occasion of his 67th birthday.
* Corresponding author. E-mail: cclin@chem.sinica.edu.tw; cccheng@chem.sinica.edu.tw

656 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004
Lin et al.
Scheme I Synthesis of GGS (1) and compounds 2 and 3
OH
O
R
O
HO
HO
HO
S
O
AcHN
HO
CH3
1 R = CH2OH (GGS)
TBDPSO
TolO
3 R = CO2Na
2 R = CH2OSO3Na
OH
O
TBDPSO
O
b, c
a
O
HO
STol
TolO
TolO
TolO
STol
HO
O
CCl3
NH
TolO
HO
TolO
5
6
4
O
O
TBDPSO
O
O
STol
HO
O
O
O
7
TolO
TolO
e, f, g, h
S
TrocHN
d
O
TrocHN
TolO
CH3
8
HO
AcO
i
O
1
2
3
O HO
TolO
S
O
TolO
j, k
l, k
AcHN
TolO
CH3
9
Reagents and conditions: a) i. TBDPSCl, imidazole, DMF, rt, 3 h; ii. toluoyl chloride, pyr., DMAP (78% for two
steps); b) NBS, acetone/H2O (9/1), rt, 30 min, 73%; c) trichloroacetonitrile, CH2Cl2, Cs2CO3 (cat.), 0oC to rt,
1.5 h, 81%; d) TMSOTf (cat.), CH2Cl2, 0oC to rt, 1 h, 63%; e) TFA, CH2Cl2, H2O, rt, 30 min, 74%; f) Ac2O, pyr.,
DMAP (cat.), CH2Cl2, rt, 3 h, 96 %; g) i. Zn. HOAc, overnight; ii. Ac2O, pyr. DMAP (cat), (96% in two steps); h)
HF,/pyr, HOAc, THF, 85%; i) NaOMe (cat), MeOH, 83%; j) (CH3)3N.SO3, DMF, 50oC, overnight, then Dowe50,
MeOH, 1 h, 82%; k) 2M NaOH, THF, 0oC 6 h, 90%; l) PDC, DMF, 68%.
Interactions of these carbohydrates with double-
stranded DNA were evaluated by measuring the changes of
the DNA melting temperature (Tm).14 Sonicated calf thymus
DNA (ct-DNA) was used as the norm DNA in this study, and
its Tm at 100 mM is 44 °C at pH 4.9 in 10 mM sodium phos-
phate buffer solution. After adding GGS (10 mM)15 to the
DNA solution, the Tm shifted to 51 °C. When the concentra-
tion of GGS was increased to 50 mM,15 the Tm reached 67 °C
(as shown in Fig. 1). The 23-degree shift of ct-DNA Tm sug-
gested that GGS was capable of interacting with and stabiliz-
ing the double-stranded DNA during the denaturing process.
Furthermore, the shift in Tm was found to be pH dependent
under the same GGS concentration (50 mM).15 When the pH
value was changed from 4.9 to higher values (6.0, 7.1 and
8.2), the Tm shifts were reduced substantially. The pH de-
pendent behaviors suggested that DNA was protonated at the
nucleobases under acidic conditions, thus enhancing the
H-bond interaction with GGS. Because the experiment was
performed in phosphate buffer, the possibilities of the H-
bond interactions being generated by the hydroxyl groups of
disaccharide and DNA phosphate6 moieties can be excluded.
When the hydroxyl group at position 6¢ of GGS was
shifted by only 4-5 °C under similar conditions. These results
suggested that the hydroxyl group at position 6¢ may play an
important role in the disaccharide-DNA interactions.
Fig. 1. DNA melting temperatures in the presence of
various disaccharides. The absorption intensity
is the value at the wavelength at 260 nm. DNA
solution contained 100 mM of ct-DNA and 10
mM sodium phosphate (pH 4.86) with 100 uM
of compounds 11-14, respectively, or 50 mM of
GGS.
modified to anionic sulfate (2) or carboxylate (3), the Tm

Carbohydrate DNA Interaction
J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 657
A variety of sugars, as listed in Fig. 2, were also exam-
ined to demonstrate the unique properties of GGS in reaction
with DNA. Disaccharide 10 was used to compare the influ-
ence of different disarccharide conformations (b1®3 vs
b1®4 O-linkage). Disaccharides 11-13 were applied to study
the orientation effects of the hydroxyl groups at the axial and
equatorial positions. Sugars 14-20 were examined to see the
binding affinity differences between mono- and di-saccha-
rides, and to ascertain the effects of the functional groups
such as methylthiophenyl and amino groups. The results
showed that most of the saccharides did not have any influ-
ence in shifting the Tm of ct-DNA, except for 17, 18 and 20
which possess free amino groups and slightly increased the
Tm by only 2-3 °C in the DNA denaturing process. These re-
sults also demonstrated that the interactions of GGS with
DNA were not through intercalator (methylthiophenyl) with
nucleobases because both compounds 10 and 15 contained
the same intercalator but could not shift Tm.
The preferable type of DNA for GGS was determined
by measuring the shift of the absorption wavelength16 of
DNA in circular dichroism (CD) spectroscopy. Disaccharide
GGS itself did not show any change in CD spectra even with
increasing its concentration up to 200 mM, suggesting that the
aggregation effect of carbohydrate can be ignored. When
poly(dA-dT)2 was used to react with GGS (10 - 80 mM) as
shown in Fig. 3, the absorption wavelength of DNA CD spec-
tra was shifted from 272 to 262 nm, while those of disaccha-
rides 2-6 remained unchanged. Furthermore, the use of
poly(dG)-poly(dC), poly(dA)-poly(dT) or poly(dG-dC)-
poly(dG-dC) to react with GGS only slightly increased the
DNA absorption intensities without shifting wavelength un-
der similar conditions. The results indicated that disaccharide
GGS preferred to bind with poly(dA-dT)2.
HO
HO
OH
O
HO
OH
O
OH
O
OH
O
O
HO
HO
OH
S
CH3
O
HO
HO
AcHN
HO
AcHN
11
10
HO
O
HO
HO
OH
O
OH
O
HO
HO
OH
O
OH
O
HO
O
OH
OH
HO
AcHN
HO
AcHN
CH3
HO
12
13
HO
OH
In brief, we have demonstrated that GGS, 1, can stabi-
lize poly(dA-dT)2 double helix DNA in a thermal denature
process. This novel phenomenon is pH dependent and may be
attributed by the H-bond interactions between the hydroxyl
groups of disaccharide and the protonated amino bases of
DNA. The disaccharide interacted and changed the local en-
vironment of double-stranded DNA resulting in Tm shift.
More detailed information at the molecular level is needed to
investigate this pH dependent thermal stabilization interac-
tion. Study of interactions between well-defined oligonucleic
acid and GGS is in progress.
HO
OH
OH
HO
CO2Me
S
H3C
O
O
OH
HO
HO
HO
H2N
OH
O
OH
O
O
AcHN
OH
14
15
AcHN
16
HO
HO
HO
HO
HO
HO
HO
HO
NH2
O
OH
O
O
O
OH
OH
HO
OH
OH
HO
HO
H2N
HO
H2N
18
17
20
19
Fig. 2. List of mono- and di-saccharides used for DNA
binding studies.
ACKNOWLEDGEMENTS
This work was supported by Academia Sinica (AS92IC6)
and the National Science Council of Taiwan (NSC91-2113-
M-001-013).
Received August 7, 2003.
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Fig. 3. CD absorption spectra of Poly(dA-dT)2 titrated
with compound 1. [Poly(dA-dT)2] = 100 mM,
[compound 1] = 0, 10, 20, 30, 40 and 50 mM, re-
spectively, at 10 mM sodium phosphate buffer
(pH 4.86), at 25 °C.
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