2'-Deoxy-8-(propyn-1-yl)adenosine-containing oligonucleotides: Effects on stability of duplex and quadruplex structures

Article abstract of DOI:10.1016/S0960-894X(00)00381-4

2'-Deoxy-8-(propyn-1-yl)adenosine has been incorporated in synthetic oligodeoxyribonucleotides and its influence on thermal stability of duplex and quadruplex structures investigated by UV, CD and ¹H NMR. The obtained results seem to indicate t

Full text of DOI:10.1016/S0960-894X(00)00381-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2005±2009
20-Deoxy-8-(propyn-1-yl)adenosine-Containing Oligonucleotides:
E€ects on Stability of Duplex and Quadruplex Structures
Bruno Catalanotti,a Aldo Galeone,a Luigi Gomez-Paloma,b Luciano Mayola,*
and Antonietta Pepec
aDip. di Chimica delle Sostanze Naturali, Univ. di Napoli ``Federico II'', Via D. Montesano, 49 I-80131 Napoli, Italy
bDip. di Scienze Farmaceutiche, Univ. di Salerno, Via Ponte don Melillo I-84084 Fisciano (SA), Italy
cDip. di Chimica, Univ. della Basilicata, Via N. Sauro, 85 I-85100 Potenza, Italy
Received 28 April 2000; revised 19 June 2000; accepted 30 June 2000
AbstractÐ-20-Deoxy-8-(propyn-1-yl)adenosine has been incorporated in synthetic oligodeoxyribonucleotides and its in¯uence on
thermal stability of duplex and quadruplex structures investigated by UV, CD and 1H NMR. The obtained results seem to indicate
that the presence of the modi®ed base negatively a€ects the stability of double stranded DNA whereas remarkably increases the
stability of parallel quadruplex structures. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
types of quadruplex structures with G-tetrads have been
reported: parallel strands, alternating antiparallel
strands etc.8 Each of these structures is characterized by
particular conformational requirements for G-tetrads;
the four parallel-stranded quadruplex demands all dG
in an anti conformation, whereas in antiparallel quad-
ruplex structures a syn conformation for deoxyguan-
osine in given positions is required.8 For example, 15-mer
50-dGGTTGGTGTGGTTGG-30 (a thrombin activity
inhibitor) adopts a chairlike structure, with alternating
antiparallel strands and a syn conformation for dG1,
dG5, dG10 and dG14.9 11 As a matter of fact, it has
already been shown that the introduction of several
substituents in 8 position of dG1, dG5, dG10 and dG14
determines an increased activity, probably due to the
stabilizing e€ect on syn conformation of dG.12
There is a growing interest in synthetic modi®ed oligo-
nucleotides containing nucleoside-analogues. The
incorporation of modi®ed bases into oligonucleotides
may indeed produce useful changes in physical and
biological properties of the resulting DNA fragments,
which can be used as e€ective tools for investigation of
nucleic acid structures as well as protein±nucleic acid
interaction. As far as the former research theme is con-
cerned, non-canonical DNA structures (hairpins, three-
and four-way junctions, triplexes, etc.)1,2 are currently
the subject of extensive investigations because of their
important biological implications. Particularly, quad-
ruplex structures are an attractive topic of a large number
of researches ranging from chemistry to molecular biology
and pharmacology, since they can be present in the single-
stranded G-rich telomere sequence3,4 and it has been
demonstrated that G-quadruplex formation inhibits
initiation of telomerase activity in Oxytricha.5 Further-
more, a number of quadruplex forming oligonucleotides
have resulted to be potent inhibitors of thrombin6 as
well as HIV-1 integrase,7 the enzyme responsible for the
insertion of viral DNA into the host genome.
Recently, several non-G-tetrads13,14 have been described
as well. Particularly the presence of an A-tetrad14 (with
all dA in a syn conformation)(Fig. 1) in parallel stranded
quadruplex 50-d(AGGGT)4-30 (I) have been evidenced.
However, authors were not able to observe A-tetrads in
the parallel stranded quadruplex 50-d(TAGGGT)4-30
(II), probably due to the lack of H bond between 50-OH
and N-3 of A2 promoting syn conformation.
It is well known that G-rich oligonucleotides may adopt
a quadruplex structure at physiological concentration of
monovalent ions forming stacked G-tetrads. Several
In this frame we have synthesized 20-deoxy-8-(propyn-1-yl)-
adenosine-containing oligonucleotides (dApr-ODNs)
and investigated their behavior in formation and stabil-
ity of both duplex and quadruplex structures by UV
1
*Corresponding author. Fax:+39-81-678552; e-mail: mayoll@unina.it
melting, CD and H NMR spectra measurements.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00381-4

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B. Catalanotti et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2005±2009
Chemistry
the exocyclic amino-group by benzoyl chloride in dry
pyridine; iii) removal of the 30- and 50-OH protecting
groups by aqueous ammonia. The so-obtained N-benzoyl-
20-deoxy-8-(propyn-1-yl)adenosine (4) was treated with
5,50-dimethoxytritylchloride in dry pyridine18 for the
®nal protection of the 50-OH group and the derived com-
pound (5) was, in turn, transformed into the corresponding
phosphoramidite monomer (6) by 2-cyano-ethyl-N,N-
diisopropyl chlorophosphoramidite.19 Compound 6 was
used for the preparation of dApr-ODNs following the
usual protocols. During the automated syntheses, 6
exhibited similar coupling eciencies as the common,
commercially available phosphoramidite derivatives.
The key step in the synthesis of dApr-ODNs is the pre-
paration of the suitably protected dApr phosphoramidite
monomer to allow its site-speci®c incorporation into
DNA oligomers. The synthesis of the fully protected
dApr monomer was carried out through the reactions
outlined in Scheme 1.
20-Deoxyadenosine (1) was brominated by bromine in
CH3COOH/CH3COONa aqueous bu€er according to
Eason et al.15 a€ording 8-bromo-20-deoxyadenosine (2).
Compound 2 was coupled with propyne following the
De Clercq procedure16 with minor modi®cations [in par-
ticular Pd0(PPh3)4 was used instead of (PPh3)2PdCl2], to
give 20-deoxy-8-(propyn-1-yl)adenosine (3). The following
step consists in the protection of the exocyclic amino-
group. We adopted the one-pot procedure known as
transient protection, developed by Gait,17 that provides:
i) temporary protection of 30- and 50-hydroxyl groups by
trimethylchlorosilane in dry pyridine; ii) protection of
Results and Discussion
Duplex stability of 50-d(CGCGAAprTTCGCG)2-30 (III)
The e€ect of the insertion of dApr was checked by
comparison of the well-studied Dickerson dodecamer
Figure 1. Scheme of an A-tetrad and syn and anti conformations. The H-bonds are indicated by dotted lines.
Scheme 1. Synthesis of fosforoamidite derivative 6. (a) CH3COOH/CH3COONa pH 4.0; Br2: 12 h; room temp; (b) triethylamine, CuI, PD0(PPh3)4],
propyne gas in dry deoxygenated DMF at 50 ꢀC, 6 h; (c) (CH3)3SiCl in dry Py, 0 ꢀC 30 min; (d) BzCl, 30 ꢀC, 2 h; (e) NH4OH, 30 min; (f) 4,40-dim-
etoxytrytil chloride, triethylamine, 4-(dimethylamino)pyrridine in dry Py, 6 h room temp; (g) N,N-diisopropylethylamine, 2-cyanoethyl-N,N-diiso-
propyl chlorophosphoramidite in dry CH2Cl2, room temp. 80 min.

B. Catalanotti et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2005±2009
2007
50-d(CGCGAATTCGCG)2-3020 versus the modi®ed
counterpart 50-d(CGCGAAprTTCGCG)2-30 (III). Table
1 lists the NMR chemical shifts of non-exchangeable
protons of III obtained by standard methods.21 NMR
properties of III suggested that this oligonucleotide
adopts B-DNA-like global folding, although minor
local deviation may exist, especially in proximity of the
modi®ed nucleotide. The CD spectrum of III con®rms
the B-DNA-like structure (data not shown).
310 K a reduction of the intensity of signals at
13.45 ppm (T7N1H of Apr6:T7 base pair) and at
13.30 ppm (G2N1H of G2:C11 base pair) is evident. At
315 K the resonance at 13.45 ppm is broader and just
visible showing an increased exchange rate with the solvent
due to the Apr:T base pair opening. At 320 K the signal
at 13.20 ppm also disappears indicating a further
advance of the melting process.
In Fig. 2 (left) the same experiment for the Dickerson
dodecamer is shown. A di€erent pattern of melting is
evident. At 300 K ®ve resonances of the internal base
pairs are present. At 310 K the signal at 13.50 ppm
(G2N1H of G2:C11 base pair) shows a lower intensity
and completely disappears at 320 K. At 325 K the residue
signals are broadened and reduced in intensity. Finally,
at 330 K the melting process is complete.
In Fig. 2 (right) the immino-proton resonances of III,
taken at several temperatures are shown (assignments
were obtained by standard methods21). At 300 K ®ve
resonances occur between 12.5 and 14.0 ppm, suggesting
that all the base residues are involved in hydrogen
bonding with exception of the frying end bases. At
Table 1. 1H NMR chemical shift values (ppm) of 50-d(CGCGA
AprTTCGCG)2-30 (III)
Thermal NMR experiments clearly point out to a minor
stability of the Apr:T base pair when compared to a
canonical A:T base pair. This could be tentatively
explained by the steric hindrance between the bulky C8-
substituent and the sugar moiety which forces, as
observed in similar derivatives,15 the nucleoside to
adopt a syn glycosidic conformation in contrast with the
anti conformation of normal nucleoside residues in B-
DNA duplex.
H8/H6
H2/H5/CH3
5.89
H10
H20
H200
C1
G2
C3
G4
A5
Apr
T7
T8
C9
7.62
7.93
7.26
7.84
8.05
Ð
7.13
7.33
7.49
7.89
7.31
7.92
5.73
5.85
5.67
5.28
6.22
5.92
5.86
6.08
5.55
5.83
5.72
6.12
1.94
2.61
1.82
2.61
2.62
2.87
1.94
2.09
2.12
2.39
1.85
2.33
2.38
2.66
2.23
2.69
Ð
2.88
1.94
2.09
2.12
2.39
2.29
2.59
5.34
6
/2.22
1.36
1.46
5.63
By the way, the reduced stability of Apr:T base pair
might also result in a local variation of DNA ¯exibility
thus pointing to dApr-ODNs as tools for studying
DNA/DNA-bending protein interactions. In fact, the
target selection by DNA-bending proteins depends on
local, sequence dependent variation in DNA ¯exure, as
indicated by recent studies on 5-hydroxymethyl-20-
deoxy-uridine containing DNA fragments.22 24
G10
C11
G12
5.40
Quadruplex stability of 50-d(AprGGGT)4-30 (IV) and 50-
d(TAprGGGT)4-30 (V)
A preliminary study of the e€ect of dApr in quadruplex
structures was carried out by UV melting pro®les. The
melting temperature of quadruplex structures can be
estimated following the thermal denaturation at the l
Figure 2. Comparison of imino region of 1H NMR spectra at di€erent
temperatures of III (right) and Dickerson dodecamer (left).
Figure 3. Melting UV pro®les at pH 7.0 for the ODNs II at 295 nm
(blue) and V at 315 nm (red).

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B. Catalanotti et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2005±2009
Figure 4. CD experiments at several temperatures of the modi®ed 6-mer V (A) and the 6-mer II (B).
value corresponding to the maximum hyperchromism
e€ect upon G-quartet formation. This can be deter-
mined from the di€erence spectrum of each oligonu-
cleotide at 4 ꢀC and 90 ꢀC.25,26 As previously noted,
natural ODNs show an isosbestic point around 280 nm,
and the l value of the maximum hyperchromism e€ect
upon G-quartet formation is found to be 295 nm.
Modi®ed ODNs behave in a di€erent fashion: the 6-mer
V is characterized by a bathochromic shift of the iso-
sbestic point to 305 nm, while the UV pro®les of the
modi®ed 5-mer IV didn't show any isosbestic point.
Consequently, the melting pro®le of V (Fig. 3) was
recorded at 315 nm, giving a well-shaped sigmoid curve,
whereas no suitable melting curve could be obtained for
the 5-mer IV at any l value.
The results are summarized in Table 2. Analysis of
melting temperature (Tm), obtained by UV and CD
measurement, proves that in the 5-mer, as well as in the
6-mer the introduction of 20-deoxy-8-(propyn-1-yl)ade-
nosine in quadruplex forming oligonucleotides, strongly
enhances the stability of the structure in both cases. A
more detailed structural investigation is in order to clarify
the molecular bases of the observed increase in stability
and particularly to establish if, in the case of the 6-mer,
this is due to the presence of a non-50-end A-tetrad.
Conclusions
The above data indicate that the modi®ed base Apr if,
on the one hand, destabilizes B-DNA duplexes, on the
other hand may increase the stability of particular
quadruplexes, most likely assuming a prevalent glycosidic
syn conformation. This observation indicates that the
substitution of natural bases with modi®ed ones might
improve the performance of synthetic ODNs as potential
drugs since biological activity seems to be directly cor-
related to stability in quadruplex aptamers.12,29
Further information on the structure was obtained
recording a series of CD experiments at variable tem-
perature (Fig. 4). At room temperature, IV and V
showed a positive Cotton e€ect at 263 nm and a less
strong negative e€ect at 246 nm. These spectra were
similar to those described for parallel-stranded guanine
quadruplexes, such as the CD spectra of the sequences
dT4G4, dT4G4T etc.27
The molar ellipticity at 264 nm for the di€erent samples
has been indicated as a measure of quadruplex forma-
tion28 and could be used for monitoring transition of an
ODN from a quadruplex to a single strand. Single
strand CD spectra generally present a null point at this
wavelength and a positive band at 250 nm ca. This
means that the contribution to the band at 264 nm is
only due to a quadruplex structure.
Acknowledgements
This work is supported by Italian M.U.R.S.T. and
C.N.R. The authors are grateful to ``Centro Ricerche
Interdipartimentale di Analisi Strumentale'', C.R.I.A.S.,
for supplying NMR facilities.
References and Notes
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ODN
Tm (K)a
50d(AGGGT)4-30 (I)
328.4
333.5
332.3*
339.6
50d(TAGGGT)4-30 (II)
50d(AprGGGT)4-30 (IV)
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aObtained by UV melting experiments, except (*) obtained by CD
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