Synthesis of an unlocked nucleic acid terpyridine monomer and binding of divalent metal ion in nucleic acid duplexes

Article abstract of DOI:10.1021/jo9019978

(Chemical Equation Presented) Herein we present the synthesis and thermal stability studies of modified oligonucleotides containing an unlocked nucleic acid (UNA) terpyridine monomer. Incorporation of this monomer into both strands of a DNA duplex allowed

Full text of DOI:10.1021/jo9019978

pubs.acs.org/joc
nucleotides with the capability of reversibly modulating
Synthesis of an Unlocked Nucleic Acid Terpyridine
Monomer and Binding of Divalent Metal Ion
in Nucleic Acid Duplexes
DNA duplex thermal stability in the presence or absence of
divalent transition metal ions.9
Several examples of highly defined, self-assembled struc-
tures based on the interactions between transition metals and
specific organic compounds are known.10 In addition, inter-
actions between ONs and transition metals have been
extensively explored. Research has been published by nu-
merous groups during the past decade dealing with DNA
base pairing mediated by transition metals.11-16 Even
though metal-mediated base pairing does affect duplex
stability, the technologies described were primarily aimed
at constructing synthetic nanowires rather than modulating
duplex stability. Furthermore, a number of modified nucleo-
tide monomers have been prepared containing metal chela-
tors directed toward the major or minor groove in a duplex,
mostly aiming at artificial RNA cleavage.17,18
Kasper K. Karlsen, Troels B. Jensen, and Jesper Wengel*
Nucleic Acid Center, Department of Physics and Chemistry,
University of Southern Denmark, Odense, Denmark
jwe@ifk.sdu.dk
Received September 16, 2009
Modulation of duplex stability depending on the presence
or absence of transition metals involving modified mono-
mers containing metal chelators situated outside the DNA
helix core has previously been reported.9,19,20 Duplex stabi-
lization has been achieved with modified nucleotide mono-
mers placed in the central part of the ON9,19 as well as with
non-nucleotide chelators situated at the ON termini.20 Kalek
et al. used modified nucleosides based on N20-substituted
20-amino LNA21 (locked nucleic acid) or 20-amino-20-deoxy-
uridine monomers functionalized with a terpyridine unit as
metal chelator.9 It was shown that metal chelators incorpo-
rated into both strands of a duplex in what was termed a “þ1
zipper” arrangement,22 in the presence of 1 equiv of a metal
ion, generated dramatic effects on duplex thermal stability.
Interestingly, reduced thermal stability was induced upon
Herein we present the synthesis and thermal stability
studies of modified oligonucleotides containing an un-
locked nucleic acid (UNA) terpyridine monomer. Incor-
poration of this monomer into both strands of a DNA
duplex allowed reversible thermal stability modulation
upon addition or withdrawal of divalent metal ions. A
likely explanation of this phenomenon is interstrand
complexation between two terpyridine units and a metal
ion. This system could be useful in the development of
nanoscale devices based on DNA hybridization.
(9) Kalek, M.; Madsen, A. S.; Wengel, J. J. Am. Chem. Soc. 2007, 129,
9392–9400.
(10) Leininger, S.; Olenyuk, B.; Stang, P. J. Chem. Rev. 2000, 100, 853–
907.
(11) Miyake, Y.; Togashi, H.; Tashiro, M.; Yamaguchi, H.; Oda, S.;
Kudo, M.; Tanaka, Y.; Kondo, Y.; Sawa, R.; Fujimoto, T.; Machinami, T.;
Ono, A. J. Am. Chem. Soc. 2006, 128, 2172–2173.
(12) Clever, G. H.; Polborn, K.; Carell, T. Angew. Chem., Int. Ed. 2005,
44, 7204–7208.
(13) Brotschi, C.; Leumann, C. J. Nucleosides Nucleotides Nucleic Acids
2003, 22, 1195–1197.
(14) Switzer, C.; Sinha, S.; Kim, P. H.; Heuberger, B. D. Angew. Chem.,
Int. Ed. 2005, 44, 1529–1532.
(15) Switzer, C.; Shin, D. Chem. Commun. 2005, 1342–1344.
(16) Weizman, H.; Tor, Y. J. Am. Chem. Soc. 2001, 123, 3375–3376.
€
(17) Niittymaki, T.; Lonnberg, H. Org. Biomol. Chem. 2006, 4, 15–25.
(18) Trawick, B. N.; Daniher, A. T.; Bashkin, J. K. Chem. Rev. 1998, 98,
939–960.
(19) Babu, B. R.; Hrdlicka, P. J.; McKenzie, C. J.; Wengel, J. Chem.
Commun. 2005, 1705–1707.
(20) Freville, F.; Pierre, N.; Moreau, S. Can. J. Chem. 2006, 84, 854–858.
(21) Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 6078–
6079.
(22) The “zipper” nomenclature for naming different arrangements of
modified monomers in the two strands of duplexes will be used in this article.
An “n zipper” describes the arrangement of two modified nucleosides of
interest, positioned in opposite strands of a DNA duplex. The number n
indicates the distance in base pairs between the two nucleosides. If n is
positive, the two modified monomers are positioned relatively toward the 50-
end of the two strands, and if n is negative, the two modified monomers are
positioned relatively toward the 30-ends of the two strands. The two X
monomers in the duplex 50-d(GTG AXA TGC):30-d(CAC TAX ACG) are as
an example positioned in a “þ1 zipper” constitution.
The unique properties of DNA regarding specific recogni-
tion and self-assembly offers researchers a significant tool for
the construction of nanoscale devices.1-5 Often, these de-
vices assemble into structures incapable of further transi-
tions. In order to expand the repertoire within DNA-based
nanotechnology, it is important to investigate novel adjus-
table systems. This issue has previously been addressed, e.g.,
using photoreactive caged oligonucleotides (ONs) which are
capable of duplex formation only after photoirradiation,6
macromolecular folding constraints based on DNA duplex
formation,7,8 or terpyridinyl 20-amino-LNA-modified oligo-
(1) Liu, D. S.; Balasubramanian, S. Angew. Chem., Int. Ed. 2003, 42,
5734–5736.
(2) Gothelf, K. V.; Labean, T. H. Org. Biomol. Chem. 2005, 3, 4023–4037.
(3) Liao, S. P.; Seeman, N. C. Science 2004, 306, 2072–2074.
(4) Sherman, W. B.; Seeman, N. C. Nano Lett. 2004, 4, 1203–1207.
(5) Wengel, J. Org. Biomol. Chem. 2004, 2, 277–280.
(6) Hobartner, C.; Silverman, S. K. Angew. Chem., Int. Ed. 2005, 44,
7305–7309.
(7) Miduturu, C. V.; Silverman, S. K. Angew. Chem., Int. Ed. 2006, 45,
1918–1921.
(8) Zelin, E.; Silverman, S. K. Chem. Commun. 2009, 767–769.
8838 J. Org. Chem. 2009, 74, 8838–8841
Published on Web 10/29/2009
DOI: 10.1021/jo9019978
r
2009 American Chemical Society

Karlsen et al.
JOCNote
U-UNA monomers all follow the same procedure, under-
lining that UNA monomers in general are easier to obtain
and less expensive to use for functionalizing ONs than
20-amino-LNA monomers. If the reduction of duplex stabi-
lity caused by insertion of UNA monomers is found to be
problematic, we envision that LNA monomers can be added
to increase duplex stability.
Synthesis. 50-O-4,40-Dimethoxytrityluridine was subjected
to oxidative cleavage of the vicinal diol function using NaIO4
in 1,4-dioxane and water followed by NaBH4 reduction27
affording 20,30-seconucleoside derivative 1 as described ear-
lier.24,27,28 The next conversion involved three consecutive
reactions. First benzoylation of the 20-hydroxy group carried
out at -78 °C using benzoyl chloride in a mixture of DCM
and pyridine, then O30-silylation using TBDMSCl in pyr-
idine, and finally hydrolysis under basic condition of the
O20-benzoyl group to give derivative 2 in 62% yield. To
install the piperazino group, the 20-hydroxy group was
mesylated using mesyl chloride in pyridine, whereupon this
intermediate was treated with a large excess of piperazine
affording compound 3 in 70% yield. Compound 3 thus
obtained was treated with 6-(2,20:60,200-terpyridin-40-yloxy)
hexanoic acid and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU) as cou-
pling reagent to give compound 4 in 83% yield. Desilylation
using TBAF in THF (to give compound 5, 88% yield)
followed by O30-phophitylation by reaction with 2-cya-
noethyl-N,N-diisopropylphosphoramidochlorodite afforded
phosphoramidite 6 (94% yield) suitable for incorporation
of monomer X into ONs on an automated DNA synthesizer
(ON1-ON6; Table 1). This was accomplished using 1H-
tetrazole as activator and 15 min coupling time. The stepwise
coupling yield for the phosphoramidite 6 was ∼80% and
∼99% for unmodified DNA amidites (2 min coupling time).
ON1-ON4 were purified using RP-HPLC while ON5 and
ON6 were purified by precipitation only. The composition
of all ONs were confirmed by MALDI-MS analysis
(Supporting Information) and their purity confirmed as
>80% by ion-exchange HPLC.
Thermal Denaturation Studies. Initially, 9-mers ON1-
ON3 were synthesized to evaluate the effect on thermal
stability of monomers X in “þ1 and -1 zipper”22 arrange-
ments with and without metal ions added. As we discovered
(vide infra) that the “-1 zipper” arrangement with 1 equiv of
metal ion displayed a positive effect on thermal stability, we
decided to also synthesize ON4-ON6 to evaluate if a similar
effect would be observed also for “-2 and -3 zipper”
arrangements. Thermal denaturation studies were carried
out for all duplexes with and without metal ions added
(Table 1). Incorporation of monomer X in one strand only,
with or without metal ions added, significantly reduces
thermal duplex stability relative to the stability of the un-
modified DNA control duplex (Tm = 28 °C). This decrease
was expected from previous reports on ONs modified with
UNA monomers.24,27 When comparing the results for du-
plexes containing one X monomer with or without metal ions
itis clearthat addition of metalions in some casesincreases the
melting temperature (ON3:DNA, ON4:DNA, ON6:DNA),
FIGURE 1. Structure of DNA and UNA (unlocked nucleic acid)
monomers.
SCHEME 1. Synthetic Route to Monomer Xa
aKey: (i) (a) BzCl, anhyd DCM, -78 °C, 1 h, (b) TBDMSCl, anhyd pyri-
dine, rt, 19 h, (c) NaOH, MeOH, rt, 2.5 h (62%, three steps); (ii) (a) MsCl,
anhyd pyridine, rt, 2 h, (b) piperazine, anhyd pyridine, 100 °C, 2 h (70%,
two steps); (iii) 6-(2,20:60,200-terpyridin-40-yloxy)hexanoic acid, HATU,
DMF, DIPEA, rt, 1.5 h (83%); (iv) TBAF, THF, rt, 24 h (88%); (v) NC-
(CH2)2OP(Cl)N(i-Pr)2, DCM, DIPEA, rt, 4.5 h (94%); (vi) DNA syn-
thesizer.
addition of ethylenediamine tetraacetic acid (EDTA), which
binds strongly to divalent metal ions rendering them un-
available for interstrand complexation.9
Herein we explore a nucleic acid modification based on
unlocked nucleic acid (UNA, Figure 1)23,24 for metal ion
binding, namely 20-deoxy-20-piperazino-20,30-secouridine
conjugated with a terpyridine unit (monomer X, Scheme 1).
Our focus is on interstrand complexation involving two X
monomers, one in each strand. Due to the high flexibility of
the UNA monomer, interstrand complexation is expected to
be less position dependent than with the previously studied
system based on 20-amino-LNA monomers having a locked
furanose ring.9 Furthermore, the synthesis of the UNA
terpyridine monomer is reported herein and involves signifi-
cantly fewer steps than synthesis of the 20-amino-LNA
terpyridine monomer.9,21,25,26 Furthermore, in contrast to
20-amino-LNA derivatives, the synthesis of A-, C-, G-, and
(23) Jensen, T. B.; Langkjær, N.; Wengel, J. Nucleic Acids Symp. 2008, 52,
133–134.
(24) Langkjær, N.; Pasternak, A.; Wengel, J. Bioorg. Med. Chem. 2009,
17, 5420–5425.
(25) Ravn, J.; Rosenbohm, C.; Christensen, S. M.; Koch, T. Nucleosides
Nucleotides Nucleic Acids 2006, 25, 843–847.
(26) Rosenbohm, C.; Christensen, S. M.; Sorensen, M. D.; Pedersen,
D. S.; Larsen, L. E.; Wengel, J.; Koch, T. Org. Biomol. Chem. 2003, 1, 655–663.
(27) Nielsen, P.; Dreioe, L. H.; Wengel, J. Bioorg. Med. Chem. 1995, 3,
19–28.
(28) Pandolfi, D.; Rauzi, F.; Capobianco, M. L. Nucleosides Nucleotides
1999, 18, 2051–2069.
J. Org. Chem. Vol. 74, No. 22, 2009 8839

Karlsen et al.
JOCNote
TABLE 1. Thermal Denaturation Temperatures (Tm Values) of Duplexes Containing Monomer X Measured at Different Concentrations of Divalent
Metal Ionsa
þ Ni2þ
þ Cu2þ
þ Zn2þ
code
sequence
þ EDTAb
16.5
(-11.5)
1 equivc
5 equivc
1 equivc
5 equivc
1 equivc
5 equivc
ON1
DNA
50-d(GTG AXA TGC)
30-d(CAC TAT ACG)
12.5
(-15.5)
13.5
(-14.5)
11.0
(-17.0)
9.5
(-18.5)
11.5
(-16.5)
11.5
(-16.5)
DNA
ON2
50-d(GTG ATA TGC)
16.5
(-11.5)
13.0
(-15.0)
9.5
(-18.5)
9.0
(-19.0)
12.5
(-15.5)
13.0
(-15.0)
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
30-d(CAC TAX ACG)
DNA
ON3
50-d(GTG ATA TGC)
30-d(CAC XAT ACG)
11.0
(-17.0)
11.5
(-16.5)
12.5
(-15.5)
12.0
(-16.0)
n.t.
n.t.
ON4
DNA
50-d(GTG ATA XGC)
30-d(CAC TAT ACG)
13.0
(-15.0)
15.0
(-13.0)
14.0
(-14.0)
14.0
(-14.0)
14.0
(-14.0)
15.0
(-13.0)
ON5
DNA
50-d(GTG AAXTGC)
30-d(CAC TTA ACG)
13.0
(-15.0)
10.5
(-17.5)
10.0
(-18.0)
10.0
(-18.0)
10.0
(-18.0)
10.0
(-18.0)
DNA
ON6
50-d(GTG AAT TGC)
30-d(CAC XTA ACG)
11.0
(-17.0)
14.0
(-14.0)
12.5
(-15.5)
12.0
(-16.0)
14.0
(-14.0)
13.5
(-14.5)
ON1
ON2
50-d(GTG AXA TGC)
30-d(CAC TAX ACG)
14.0
(-14.0)
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
ON1
ON3
50-d(GTG AXA TGC)
30-d(CAC XAT ACG)
14.0
(-14.0)
38.0
(þ10.0)
34.5
(þ6.5)
34.5
(þ6.5)
18.0
(-10.0)
ON5
ON6
50-d(GTG AAX TGC)
30-d(CAC XTA ACG)
n.t.
n.t.
n.t.
n.t.
n.t.
ON4
ON3
50-d(GTG ATA XGC)
30-d(CAC XAT ACG)
n.t.
n.t.
n.t.
n.t.
n.t.
aThermal denaturation temperatures (Tm values, °C). ΔTm values (°C) are shown in parentheses, i.e., the change in Tm value relative to the Tm values
of the unmodified DNA:DNA duplex of the same sequence (28.0 °C). Tm values were measured as the maximum of the first derivatives of the melting
curve (A260 vs temperature) recorded in medium salt buffer (100 mM NaCl, adjusted to pH 7.0 with 10 mM NaH2PO4/5 mM Na2HPO4), using 1 μM
concentration of the two complementary strands; Tm values are averages of at least two measurements. The structure of monomer X is shown in
Scheme 1. All samples were denatured for 15 min at 80 °C before measurement. b[EDTA] = 0.1 mM. cM2þ equivalents are relative to duplex. n.t. = no
transition.
while addition of metal ions in other cases lowers the melting
temperatures (ON1:DNA, ON2:DNA, ON5:DNA). These
data indicate a sequence dependence which might be of steric
origin, i.e., caused by different possibilities of structurally
encompassing the bulky terpyridine unit complexed to a metal
ion. The duplexes with two modifications, one in each strand,
generally show either no melting transition or strongly in-
creased melting temperatures. This depends on the concentra-
tion of the metal ions, on the nature of the metal ion (Zn2þ
,
Cu2þ, or Ni2þ) and, most prominently, on the positioning of
the monomers in the two strands. The monomers are posi-
tioned either in a “þ1 zipper” (ON1:ON2), a “-1 zipper”
(ON1:ON3), a “-2 zipper” (ON5:ON6), or a “-3 zipper”
(ON4:ON3) constitution. Upon addition of 1 equiv of metal
ion, a large thermal stabilization was induced exclusively in
the “-1 zipper” arrangement. On the basis of previous9 and
all current results, the most plausible explanation of the large
stabilization induced by addition of 1 equiv of metal ion is the
joint coordination from both terpyridine moieties of ON1
and ON3 to the metal ion. No thermal stabilization was
observed upon addition of 5 equiv of metal ions. We explain
this by the likely scenario that both terpyridine units coordi-
nate a metal ion which disables interstrand complexation.
These data thus add support to the theory that the large
thermal stability of the “-1 zipper” arrangement with 1 equiv
of metal ion is caused by interstrand complexation. Despite
the flexible structure of monomer X, our data indicate that
interstrand coordination is impossible for the “þ1, -2, or -3
zipper” arrangements. Additionally, the large thermal stabi-
FIGURE 2. CD spectra of the ON1:ON3 duplex in the presence
(1 equiv) and absence of Ni2þ and the DNA:DNA reference duplex.
The spectra were recorded at 10 °C using the same conditions as
described for the thermal denaturation studies (Table 1).
lity of “-1 zipper” (ON1:ON3) with 1 equiv of metal ion was
reversed upon addition of 5 equiv of EDTA and denaturation
for 15 min. The structure of the “-1 zipper” (ON1:ON3)
duplex was studied using circular dichroism (CD) spectros-
copy. CD spectra were recorded for the “-1 zipper” duplex in
the presence (1 equiv) and absence of Ni2þ and compared with
the spectrum obtained for the unmodified reference duplex
(Figure 2). All three curves display similar overall character-
istics which indicate that the structure of the “-1 zipper”
duplex, even when interstrand complexed, is of a standard B
8840 J. Org. Chem. Vol. 74, No. 22, 2009

Karlsen et al.
JOCNote
TABLE 2. MALDI-MS Analysis of ON1-ON6
containing pure oligonucleotides were collected and evaporated
on a speed-vac, followed by detritylation (80% aq AcOH, 20
min), precipitation (abs EtOH, -18 °C, 12 h) and washing with
abs EtOH. The composition of oligonucleotides was verified by
MALDI-MS analysis (Table 2), whereas the purity (>80%) was
verified by ion-exchange HPLC (100 mm ꢀ 4.6 mm column
size). The following eluent system was used: eluent A, 25 mM
Tris-Cl, 1 mM EDTA, pH 8.0; eluent B, 1 M NaCl, gradient,
0-5 min isocratic hold of 95% eluent A followed by a linear
gradient to 70% eluent B over 41 min at a flow rate of
0.75 mL/min.
code
sequence
found m/z [M]
calcd m/z [M]
ON1
ON2
ON3
ON4
ON5
ON6
50-d(GTG AXA TGC)
50-d(GCA XAT CAC)
50-d(GCA TAX CAC)
50-d(GTG ATA XGC)
50-d(GTG AAX TGC)
50-d(GCA ATX CAC)
3169.7
3100.7
3098.5
3171.7
3170.7
3099.0
3169.9
3098.9
3098.9
3169.9
3169.9
3098.9
type. In conclusion, the UNA monomer introduced in this
paper is easy to synthesize and possess the ability to reversibly
change the thermal stability of a DNA duplex upon addi-
tion or withdrawal of divalent metal ions. Furthermore,
two complementary ONs modified with monomer X in a
“-1 zipper” arrangement can exist either as single strands
(Tm = 14 °C, with EDTA) or as a duplex (Tm = 38 °C, with
1 equiv of Ni2þ) at room temperature. Therefore, the techno-
logycanbe appliedtoswitchbetweenorderedand less ordered
structures at room temperature.
General Procedure for Thermal Denaturation Studies. Con-
centrations of oligonucleotides were calculated using the follow-
ing extinction coefficients (OD260/μmol): G, 10.5; A, 13.9; T/U,
7.9; C, 6.6; terpyridine, 8.0.29 Oligonucleotides (1.0 μM of each
strand) were thoroughly mixed in Tm buffer (100 mM NaCl, pH
7.0 adjusted with 10 mM NaH2PO4/5 mM Na2HPO4 and
0.1 mM EDTA (EDTA was included only in experiments with-
out divalent metal ions added)), denatured by heating at 85 °C
for 15 min, and subsequently cooled to the starting tempera-
ture of the experiment. In experiments with divalent metal
ions present, an appropriate amount of an aqueous solution
of CuCl2, ZnCl2, or NiSO4 (1 equiv; 10 μL, 5 equiv; 50 μL,
100 μM) was added after mixing the strands. Quartz optical cells
with a path length of 1.0 cm were used. Thermal denaturation
temperatures (Tm values/°C) were measured on a UV/vis spec-
trometer equipped with a Peltier temperature programmer and
determined as the maximum of the first derivative of the thermal
denaturation curve (A260 vs temperature). A temperature ramp
of 1.0 °C/min was used in all experiments. Reported thermal
denaturation temperatures are an average of two measurements
within (1.5 °C.
Experimental Section
General Procedure for Synthesis and Purification of Oligonu-
cleotides. Synthesis of oligonucleotides was performed in
0.2 μmol scale using an automated DNA synthesizer. Standard
cycle procedures were applied for unmodified phosphoramidites
using 0.45 M solution of 1H-tetrazole as activator. Stepwise
coupling yields, as determined by a spectrophotometric DMTþ
assay, were >99% for standard phosphoramidites and ∼80%
(15 min coupling time) for phosphoramidite 6. Removal of the
nucleobase protecting groups and cleavage from solid support
was effected using standard conditions (32% aqueous ammonia
for 12 h at 55 °C). Purification of oligonucleotides ON1-ON4
was performed by DMT-ON RP-HPLC using a C18-column
(10 μm, 300 mm ꢀ 7.8 mm). The following eluent system was
used: eluent A, 95% 0.1 M Et3NH3 5% CH3CN; eluent B, 25%
Acknowledgment. The Nucleic Acid Center is a research
center of excellence funded by the Danish National Research
Foundation for studies on nucleic acid chemical biology.
We gratefully acknowledge financial support from the
Danish National Research Foundation and the Villum Kann
Rasmussen Foundation.
0.1 M Et3NH HCO3, 75% CH3CN; gradient, 0-5 min isocratic
3
hold of 100% eluent A followed by a linear gradient to 55%
eluent B over 75 min at a flow rate of 1.0 mL/min. Fractions
Supporting Information Available: Detailed experimental
procedures and characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
(29) Cali, R.; Rizzarelli, E.; Sammartano, S.; Siracusa, G. Transition Met.
Chem. 1979, 4, 328–332.
J. Org. Chem. Vol. 74, No. 22, 2009 8841