Metal-mediated base pairing within the simplified nucleic acid GNA

Article abstract of DOI:10.1039/b816142a

Hydroxypyridone and pyridopurine homo- and hetero-base pairs have been investigated in the context of duplex GNA (glycol nucleic acid). Phosphoramidites for automated GNA solid phase synthesis were synthesized economically in a few steps starting from com

Full text of DOI:10.1039/b816142a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Metal-mediated base pairing within the simplified nucleic acid GNA†
Mark K. Schlegel, Lilu Zhang, Nicholas Pagano and Eric Meggers*
Received 17th September 2008, Accepted 24th October 2008
First published as an Advance Article on the web 1st December 2008
DOI: 10.1039/b816142a
Hydroxypyridone and pyridopurine homo- and hetero-base pairs have been investigated in the context
of duplex GNA (glycol nucleic acid). Phosphoramidites for automated GNA solid phase synthesis were
synthesized economically in a few steps starting from commercially available enantiopure glycidol.
Similar to their behavior in DNA, the hydroxypyridone and pyridopurine homo-base pairs display a
metal-dependent base pairing, with the hydroxypyridone base pair exhibiting a preference for copper(II)
ions and the pyridopurine a preference for nickel(II) ions. However, these metallo-base pairs show
modulated properties in GNA with respect to metal-dependent pairing stabilities and metal selectivities.
Most interestingly, the hydroxypyridone homo-base pair and hydroxypyridone-pyridopurine
hetero-base pair are particularly well accommodated in the GNA duplex and form
copper(II)-dependent base pairs that are more stable compared to a Watson-Crick A:T base pair at the
same position by nearly 20 ◦C and 24 ◦C, respectively. The structure of the copper(II)-hydroxypyridone
homo-base pair is discussed based on a recent metallo-GNA duplex crystal structure.
Introduction
Due to its programmable self assembly, DNA is a promising
biomaterial for the construction of nanoscale architectures (struc-
tural DNA nanotechnology) and has already been used for the
assembly of a variety of periodic arrays in two dimensions and
the construction of materials with predictable three-dimensional
structures such as polyhedra and catenanes.1 In this respect, an
important current goal is the functionalization of DNA in order
to create nanoscale devices with novel properties.2 One recently
developed strategy is the incorporation of metal-mediated base
pairs into DNA, which enables one to place metal ions within
˚
the nucleobase p-stacking thus providing an A-scale control of
the patterning of metal ions along a helix axis.3–6 It can easily
be envisioned that a one dimensional array of redox-active metal
ions, positioned along a DNA double helix by metal-mediated
base pairing, may lead to nucleic acids with interesting magnetic
and electronic properties. However, despite such exciting prospects
for metal-mediated base pairing, the multistep synthesis of such
Fig. 1 Constitution of the (S)-GNA backbone and the metallo-base pairs
investigated in this study.
artificial nucleotides, including the chromatographical separation
concentrate on introducing additional functionality into GNA,
of anomeric mixtures, is often painstaking and thus makes their
such as redox-active metal ions. As a first step into this direction,
future large scale utilization in nanoscale devices questionable.
we recently reported the X-ray crystal structure of a GNA duplex
Aware of this general problem, we recently developed the
containing two copper(II)-mediated base pairs.8 Here we present
simplified nucleic acid GNA (glycol nucleic acid) which contains
the synthesis, incorporation, and pairing properties of three
an acyclic backbone with propylene glycol nucleosides that are
different metal-mediated base pairs in the context of GNA and
connected by phosphodiester bonds (Fig. 1).7–10 Due to its
discuss the structure of a GNA duplex containing two copper(II)-
hydroxypyridone homo-base pairs.
unique combination of high duplex stability, high base pairing
fidelity, and easy synthetic access of its nucleotide building
blocks, GNA comprises an interesting scaffold for future nucleic
acid nanotechnology. Current efforts in our laboratory therefore
Results and discussion
Synthesis of chelating nucleotides and incorporation into GNA
Fachbereich Chemie, Philipps-Universita¨t Marburg, Hans-Meerwein-
Strasse, 35043 Marburg, Germany. E-mail: meggers@chemie.uni-marburg.
For our initial studies on metal-mediated base pairing in GNA,
we chose a copper(II)-mediated hydroxypyridone homo-base pair
(H–Cu–H, Fig. 1) and a nickel(II)-mediated pyridopurine homo-
base pair (P–Ni–P, Fig. 1) scheme, both previously developed
de; Fax: (+49) 6421 2821534; Tel: (+49) 6421 2822189
† Electronic supplementary information (ESI) available: 1H NMR spectra
of compounds; GNA oligonucleotide synthesis and purification details.
See DOI: 10.1039/b816142a
476 | Org. Biomol. Chem., 2009, 7, 476–482
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Scheme 1 Synthesis of phosphoramidites 1 and 2 for solid phase GNA oligonucleotide synthesis. DPC = diphenylcarbamoyl, DMTr = dimethoxytrityl.
for metal-mediated base pairing in DNA by Shionoya et al.
and Switzer et al., respectively.4,5 Accordingly, we developed
synthetic routes to the phosphoramidites 1 and 2 (Scheme 1).
Phosphoramidite 1, leading to hydroxypyridone homo-base pairs,
was synthesized by the reaction of (S)-dimethoxytritylglycidol
3 with nucleobase 4 in the presence of catalytic amounts of
NaH (0.22 equiv.) to afford 5 in a regioselective and stereospe-
cific fashion in 65% yield. The benzyl protection group was
subsequently removed by catalytic hydrogenation (5→6, 98%),
followed by reprotection with a diphenylcarbamoyl (DPC) group
to provide compound 7 in 64%. Finally, the GNA nucleoside
7 was converted into the phosphoramidite 1 by reacting with
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (8) in the
presence of Hu¨nig’s base (86%). Overall, phosphoramidite 1 was
synthesized from commercially available (R)-(+)-glycidol in 5 steps
in an overall yield of 34%. This is a significant improvement over
the corresponding 2¢-deoxynucleotide for which a 9 step synthesis
(including one additional step for the separation of an anomeric
mixture) with an overall yield of 8.0% was reported starting from
2-methyl-3-benzyloxypyridone (4).4,11
Phosphoramidite 2 was synthesized in an analogous fashion
by reacting (S)-dimethoxytritylglycidol 3 with 6-chloropurine (9)
in the presence of K2CO3 (0.14 equiv.) to afford nucleoside 10
in 38% yield. After protection of the 2¢-OH group with a tert-
butyldimethylsilyl (TBS) group (10→11, 86%), the heterocycle of
11 was converted to the pyridopurine 13 by a Negishi coupling
with 2-pyridylzincbromide 12 and PdCl2(PPh3)3 (81%). Finally,
after TBAF deprotection to 14 (81%), the phosphoramidite 2
was formed in 65% by the reaction with 2-cyanoethyl-N,N-
diisopropylchlorophosphoramidite (8) in the presence of Hu¨nig’s
base. Overall, this phosphoramidite 2 was synthesized starting
from (R)-(+)-glycidol in 5 steps with an overall yield of 17%
compared to the analogous 2¢-deoxynucleotide for which starting
with 2¢-deoxy-D-ribose a 6 step synthesis with an overall yield of
26% was reported.5,12,13
With the phosphoramidites 1 and 2 in hand, the chelating
hydroxpyridone (H) and pyridopurine (P) phosphoramidites were
incorporated into GNA oligonucleotides by automated solid
phase oligonucleotide synthesis using standard protocols for 2-
cyanoethyl phosphoramidites. GNA oligonucleotides were synthe-
sized in trityl-on mode, cleaved from the resin with concentrated
ammonia at 55 ◦C overnight, purified by C18 reverse phase HPLC,
detritylated with 80% acetic acid, and subsequently purified once
again using a Waters XTerra column.
Metal-dependent duplex formation
In order to investigate the influence of metal ions on the thermal
stabilities of the homo-base pairs H:H and P:P in GNA, the chelat-
ing nucleotides were introduced into the middle of a 15mer duplex
(Table 1) and the melting temperatures (TM) were determined
by UV-monitored thermal melting. After removal of transition
metal traces in the oligonucleotide solutions by treatment with the
chelating resin Chelex 100 (Sigma), the pyridopurine base pair P:P
leads to a marked destabilization of the GNA duplex by 15.5 ◦C
compared to an A:T base pair at the same position (Table 1,
entries 1 and 5). However, the addition of two equivalents of NiCl2
strongly stabilizes the duplex, with an increase in the TM value of
17.9 ◦C (Fig. 2 and Table 1, entry 6). Thus, with a TM of 52.9 ◦C, the
nickel(II)-containing P:P base pair is 2.4 ◦C more stable compared
to an A:T Watson-Crick base pair at the same position (Table 1,
entries 1 and 6). In contrast, metal salts such as Pd(NO3)2, AuCl3,
ZnCl2, and CdSO4 have no notable stabilization effect (Table 1,
entries 10–13), whereas Co(NO3)2 (+7.0 ◦C), CuCl2 (+ 7.3 ◦C),
and AgNO3 (+12.5 ◦C) stabilize the P:P base pair significantly
(Table 1, entries 7–9).
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Table 1 Thermal stabilities of GNA duplexes in the presence of metal
ionsa
3¢-AATATTAXTATTTTA-2¢
2¢-TTATAATYATAAAAT-3¢
Entries
X:Y
Metal ions
TM (◦C)
DTM (◦C)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
A:T
A:T
A:T
A:T
P:P
P:P
P:P
P:P
P:P
P:P
P:P
none
NiCl2
CuSO4
ZnCl2
none
NiCl2
AgNO3
CuCl2
Co(NO3)2
CdSO4
ZnCl2
AuCl3
Pd(NO3)2
none
CuSO4
ZnCl2
Co(NO3)2
Ni(NO3)2
Cd(NO3)2
AuCl3
AgNO3
Pd(NO3)2
none
50.5
51.0
50.8
50.8
35.0
52.9
47.5
42.3
42.0
36.5
36.0
34.6
32.8
37.0
70.2
52.9
42.1
38.5
37.9
37.1
37.0
36.6
37.3
74.4
50.9
50.1
46.2
38.3
37.4
37.0
36.4
+0.5
+0.3
+0.3
+17.9
+12.5
+7.3
+7.0
+1.5
+1.0
-0.4
P:P
P:P
-2.2
H:H
H:H
H:H
H:H
H:H
H:H
H:H
H:H
H:H
H:P
H:P
H:P
H:P
H:P
H:P
H:P
H:P
H:P
+33.2
+15.9
+5.1
+1.5
+0.9
+0.1
0
-0.4
CuSO4
ZnCl2
+37.1
+13.6
+12.8
+8.9
+1.0
+0.1
-0.3
Ni(NO3)2
Co(NO3)2
CdSO4
AuCl3
AgNO3
Pd(NO3)2
-0.9
a Measurements performed in 10 mM sodium phosphate buffer, 100 mM
NaNO3, pH = 7.0. Each sample contained 2 mM of each strand and 4 mM
of the specified metal salt. Data were measured at least in duplicate and
the average taken. b Effect of the transition metal ion additives on the TM
values.
The nickel(II)-induced stabilization of the pyridopurine base
pair in GNA is comparable with the stabilization of this base pair
in the context of a DNA duplex (DTM = +17.6 and 18.1 ◦C after the
addition of two equivalents of NiCl2 and Ni(NO3)2, respectively)
reported by Switzer et al.5 Similarly, in DNA CoCl2 leads to a
stabilization of the P:P base pair by +10.3 ◦C, whereas AgNO3
and CuCl2, in contrast to what is seen in GNA, do not provide
significant stabilizations (DTM = +2.0 and +2.9 ◦C, respectively).
Thus, the pyridopurine base pair displays a modulated metal ion
selectivity within the GNA duplex with a preference order of
Fig. 2 Metal-dependent stabilities of duplexes containing hydroxy-
pyridone (H) and pyridopurine (P) homo- and hetero-base pairs.
A) Cu2+-dependent UV-melting curves with the H:H base pair in GNA.
B) Ni2+-dependent UV-melting curves with the P:P base pair in GNA.
C) Cu2+-dependent UV-melting curves with the H:P base pair in GNA. All
measurements were performed in 10 mM sodium phosphate buffer,
100 mM NaNO3, pH = 7.0. Each sample contained 2 mM of each 15mer
GNA strand (see Table 1 for sequences). CuSO4 or NiCl2 were added to a
concentration of 4 mM.
Ni2+ > Ag+ > Cu2+ ª Co2+, compared to Ni2+ > Co2+ >> Cu2+
ª
Ag+ in DNA.5
Whereas the pyridopurine base pair displays a preference for
nickel(II) ions, the hydroxypyridone base pair is stabilized the
strongest with copper(II) ions. In the absence of transition metal
ions, the hydroxypyridone base pair H:H is destabilized in the
context of the GNA duplex by 13.5 ◦C compared to an A:T
base pair at the same position (Table 1, entries 1 and 14). The
addition of two equivalents of CuSO4 results in a strong increase
◦
in the TM value by 33.2 C (Fig. 2 and Table 1, entry 15). With
at the same position (Table 1, entries 1 and 15). No significant
stabilization can be observed with Pd(NO3)2, AgNO3, AuCl3,
Cd(NO3)2 or Ni(NO3)2 (Table 1, entries 18–22); but Co(NO3)2
a TM of 70.2 ◦C, the copper(II)-containing H:H base pair is
19.7 ◦C more stable compared to an A:T Watson-Crick base pair
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and ZnCl2 stabilize the H:H base pair by 5.1 ◦C and 15.9 ◦C,
respectively (Table 1, entries 16 and 17).
Within the metallo-base pair, a copper ion is coordinated by two
hydroxypyridone nucleobases in an almost perfectly square planar
fashion (Fig. 3D) with only a slight propeller twist of around 15◦
between the two chelating ligands. Coordinative bonds between
The copper(II)-induced stabilization of the hydroxypyridone
base pair in GNA is surprisingly strong and significantly exceeds
the reported stabilization for the analogous 2¢-deoxynucleotide
hydroxypyridone base pair in DNA for which for a stabilization
˚
the copper and oxygen ligands vary between 1.8 and 2.0 A.
The high TM values of duplexes containing the copper(II)-
hydroxypyridone base pair and the crystal structure demonstrate
that this metallo-base pair fits very well into the overall GNA
duplex structure. This is surprising since the C1¢–C1¢ distance in this
◦
of only 13.1 C was reported in a 15mer duplex.4 This indicates
that the Cu2+-mediated H:H base pair is particularly well accom-
modated in the GNA duplex compared to DNA.
˚
Finally, we investigated the metal-mediated crosspairing of the
hydroxypyridone and pyridopurine chelates (Table 1, entries 23–
31). Overall, this hetero-base pair H:P behaves similarly to the
hydroxypyridone homo-base pair H:H in its metal selectivities.
In fact, the stabilization of H:P by Cu2+ exceeds the copper(II)-
induced stabilization of H:H, affording a duplex with a TM value of
74.4 ◦C. Thus, H:P is 23.9 ◦C more stable than an A:T base pair at
the same position in the presence of Cu+2 ions. This higher stability
can be rationalized by the higher hydrophobicity of P compared
to H, whereas the steric problems within metal-coordinated P:P
are absent in H:P.
H:H base pair is 12.7 A and thus exceeds the analogous C1¢–C1¢
˚
distance of the natural A:T and G:C base pairs by around 2.0 A
(Fig. 3E). However, a closer look at the structure reveals that
the hydroxypyridone nucleotide backbone compensates for this
by rotating around the C2¢–C3¢ bond. In contrast to the natural
nucleotides, in which the vicinal C–O bonds assume a gauche
orientation, the glycolic C–O bonds of the hydroxypyridone
nucleotides are oriented anti (Fig. 3C), which reduces the distances
between the oxygens of paired nucleotides.
Conclusions
We here presented a full account of the incorporation of three
metal-mediated base pairs into the simplified nucleic acid GNA
by characterizing the pairing properties of pyridopurine and
hydroxypyridone homo- and hetero-base pairs. Interestingly, it
turns out that the properties of these metallo-base pairs are
modulated in GNA compared to DNA with respect to base
pairing stabilities and metal-selectivities. Most interestingly, the
copper(II)-dependent stability of the GNA hydroxypyridone
homo-base pair significantly exceeds the stability of this base pair
in the context of duplex DNA and renders it, together with the
hydroxypyridone-pyridopurine hetero-base pair, currently by far
the most stable base pair in GNA. The application of such metallo-
GNAs for the generation of duplexes with interesting electronic
and magnetic properties is underway.
Structure of the copper-mediated hydroxypyridone base pair in a
GNA duplex
˚
We recently determined the 1.3 A crystal structure of an (S)-
GNA duplex formed from the self-complementary strand 3¢-
CGHATHCG-2¢ in which two copper(II)-hydroxypyridone base
pairs served as handles to site-selectively introduce two heavy
atoms per duplex for phasing the crystallographic data (Fig. 3).8
Within the crystal, individual duplexes are coaxially stacked,
thereby forming a continuous helix with a periodic pattern of
˚
copper(II) ions along the helix axis with Cu-Cu distances of 14.5 A
˚
within a duplex and 23.7 A between adjacent duplexes (Fig. 3A,B).
Experimental section
General procedures and reagents
NMR spectra were recorded on a Bruker DRX-500 (500 MHz),
DRX-400 (400 MHz), DMX-360 (360 MHz), or DMX-300
(300 MHz) spectrometer. High-resolution mass spectra were
obtained with a Micromass AutoSpec or Thermo LTQ-FT instru-
ment using ES ionization. Infrared spectra were recorded either
on a Perkin Elmer 1600 or Nicolet 510 series FTIR spectrometer.
Solvents and reagents were used as supplied from Aldrich or
Acros. Reactions were performed under an atmosphere of argon
unless otherwise specified. Compound 3 was prepared as described
previously.7
Fig. 3 Crystal structure of an 8mer GNA duplex with the self-comple-
mentary strand 3¢-CGHATHCG-2¢ in the presence of copper(II) (ref. 8).
The metallo-base pairs are shown in green. A) Coaxially stacked 8mer
duplexes form a continuous helix in the crystal with defined pattern
of copper(II)-ions. B) An individual 8mer duplex. C) Superimposed
hydroxypyridone and thymine nucleoside from the crystal structure
indicating the differences in the conformation along C2¢–C3¢. D), E)
Copper(II) coordination to hydroxypyridone nucleobases of opposite
strands. Indicated are the coordinative bond length and the C1¢–C1¢ distance
Compound 5
Compound 4 (1.3 g, 6.0 mmol) and NaH (60% in mineral oil,
50 mg, 1.3 mmol) were combined in DMF (12 mL) and stirred
at room temperature under argon for one hour. A solution
of 3 (2.1 g, 5.7 mmol) in DMF (12 mL) was added and the
resulting mixture was heated to 110 ◦C overnight. The DMF
was then evaporated, the residue taken up in ethyl acetate and
concentrated to dryness. The crude product was purified by
˚
(in A).
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column chromatography starting with EtOAc:Et3N (100:1), then
eluted with EtOAc:MeOH:Et3N (40:3:0.01) to afford compound 5
as a light yellow foam (2.2 g, 65%). 1H-NMR (500 MHz, CDCl3)
d (ppm) 7.42 (d, J = 8.1 Hz, 2H), 7.37–7.20 (m, 13H), 6.83 (d,
J = 8.2 Hz, 4H), 6.11 (d, J = 7.3 Hz, 1H), 5.08 (d, J = 11.3 Hz,
1H), 4.90 (d, J = 11.3 Hz, 1H), 4.14 (m, 2H), 3.79 (s, 6H), 3.52
(dd, J = 14.6, 9.7 Hz, 1H), 3.35 (dd, J = 9.2, 4.3 Hz, 1H), 3.10
(t, J = 8.4 Hz, 1H), 2.16 (s, 3H). 13C-NMR (125 MHz, CDCl3) d
(ppm) 173.0, 158.8, 145.9, 144.9, 142.2, 140.4, 137.8, 136.2, 136.0,
130.3, 129.1, 128.5, 128.4, 128.2, 128.1, 127.1, 116.5, 113.4, 86.5,
73.4, 68.9, 65.5, 58.6, 55.4, 12.9. IR (film) v (cm-1) = 3197, 3063,
2933, 2836, 1626, 1608, 1561, 1508, 1462, 1398, 1301, 1251, 1220,
1176, 1073, 1033, 980, 910, 827, 790, 753, 728, 701. HRMS calcd
for C37H38NO6 (M + H)+ 592.2699, found (M + H)+ 592.2727.
Compound 1
To an argon purged solution of compound 7 (355 mg,
0.51 mmol) and diisopropylethylamine (510 mL, 2.9 mmol)
in dichloromethane (8.5 mL) was added 2-cyanoethyl N,N¢-
diisopropylchlorophosphoramidite 8 (235 mL, 1.1 mmol) dropwise
and the solution stirred for two hours at room temperature
under argon. The solution was washed one time with saturated
aqueous NaHCO3, extracted into dichloromethane, dried over
Na2SO4, and finally concentrated by rotary evaporation. The
crude product was purified by column chromatography eluting
with hexanes:acetone:Et3N (1:1:0.01) to afford compound 1 as a
white foam (395 mg, 86%). 31P NMR (121 MHz, CDCl3) d (ppm)
150.6, 150.0. HRMS calcd for C52H58N4O8P (M + H)+ 897.3987,
found (M + H)+ 897.3973.
Compound 6
Compound 10
Compound 5 (1.5 g, 2.5 mmol) and Pd/C (10% on carbon,
750 mg) were combined in ethyl acetate (70 mL) and the resulting
suspension was purged with argon, then H2, and allowed to stir
under a H2 atmosphere for two hours. Filtration through Celite
using EtOAc:Et3N (100:1) afforded compound 6 as a tan foam
6-Chloropurine (9, 2.0 g, 12.9 mmol) and K2CO3 (230 mg,
1.7 mmol) were combined in DMF (20 mL). A solution of
epoxide 3 (4.7 g, 12.5 mmol) in DMF (20 mL) was added
and the resulting mixture was heated to 90 ◦C overnight. The
DMF was then evaporated, the residue taken up in ethyl acetate
and concentrated to dryness. The crude product was purified
by column chromatography starting with hexanes:EtOAc:Et3N
(1:1:0.01), then eluting with hexanes:EtOAc:Et3N (1:2:0.01) to
afford compound 10 as a white foam (2.5 g, 38%). 1H-NMR
(360 MHz, CDCl3) d (ppm) 8.69 (s, 1H), 8.16 (s, 1H), 7.40 (m,
2H), 7.34–7.20 (m, 7H), 6.83 (m, 4H), 4.51 (dd, J= 14.3, 3.0 Hz,
1H), 4.35 (dd, J= 14.3, 7.1 Hz, 1H), 4.22 (m, 1H), 3.81 (s, 6H),
3.48 (b, 1H), 3.20 (d, J= 5.6 Hz, 2H). 13C-NMR (90 MHz, CDCl3)
d (ppm) 159.0, 152.2, 152.0, 151.3, 146.8, 144.6, 135.73, 135.71,
131.7, 130.2, 128.31, 128.25, 127.4, 87.0, 69.5, 64.8, 55.6, 48.0.
IR (film) v (cm-1) = 3317, 2933, 2835, 1666, 1602, 1592, 1562,
1508, 1464, 1445, 1405, 1336, 1302, 1253, 1179, 1076, 1032, 948,
909, 830, 727, 702, 643. HRMS calcd for C29H27N4O4Cl (M + H)+
531.1799, found (M + H)+ 531.1805.
1
(1.25 g, 98%) which was used without further purification. H-
NMR (500 MHz, CDCl3) d (ppm) 7.45 (d, J = 7.8 Hz, 2H),
7.37–7.15 (m, 8H), 6.86 (d, J = 8.5 Hz, 4H), 5.98 (br m, 1H), 5.42
(br s, 2H), 4.25 (d, J = 13.7 Hz, 1H), 4.03 (br s, 1H), 3.81 (s, 6H),
3.65 (m, 1H), 3.41 (m, 1H), 3.18 (m, 1H), 2.39 (s, 3H). 13C-NMR
(90 MHz, CDCl3) d (ppm) 168.7, 158.9, 145.7, 144.8, 138.5, 136.0,
135.8, 130.2, 129.4, 128.24, 128.18, 127.2, 113.5, 113.4, 110.8, 86.7,
69.2, 65.2, 58.0, 55.5, 12.4. IR (film) v (cm-1) = 3199, 3071, 2933,
2825, 1626, 1607, 1577, 1508, 1464, 1444, 1366, 1302, 1248, 1174,
1076, 1032, 909, 825, 791, 727, 702, 643, 584. HRMS calcd for
C30H32NO6 (M + H)+ 502.2229, found (M + H)+ 502.2248.
Compound 7
To an argon purged solution of compound 6 (415 mg, 0.83 mmol)
and diphenylcarbamoyl chloride (230 mg, 0.99 mmol) in anhy-
drous pyridine (6.6 mL) was added diisopropylethylamine (175 mL,
0.99 mmol) and the solution stirred for one hour at room
temperature under argon. The solution was poured into saturated
aqueous NaHCO3, extracted two times into dichloromethane,
dried over Na2SO4, and finally concentrated by rotary evaporation.
After coevaporation with toluene, the crude product was purified
by column chromatography starting with EtOAc:Et3N (100:1),
then EtOAc:MeOH:Et3N (50:1:0.01), and finally eluting with
EtOAc:MeOH:Et3N (40:3:0.01) to afford compound 7 as a light
yellow foam (370 mg, 64%). 1H-NMR (500 MHz, 373K, DMSO-
d6) d (ppm) 7.50–7.20 (m, 20H), 6.89 (d, J = 8.8 Hz, 4H), 6.08
(d, J = 7.6 Hz, 1H), 4.13 (d, J = 11.4 Hz, 1H), 3.84 (m, 2H),
3.76 (s, 6H), 3.14 (dd, J = 9.3, 3.2 Hz, 1H), 3.00 (dd, J = 9.3,
6.1 Hz, 1H), 2.25 (s, 3H). 13C-NMR (125 MHz, 373K, DMSO-
d6) d (ppm) 169.2, 157.9, 150.9, 144.2, 142.3, 140.6, 140.4, 139.5,
135.3, 129.2, 128.3, 127.4, 127.2, 126.4, 126.1, 125.6, 114.5, 112.9,
85.5, 68.6, 64.9, 62.5, 55.2, 54.7, 12.0. IR (film) v (cm-1) = 3400
(br), 3059, 2928, 2833, 1742, 1644, 1608, 1598, 1512, 1494, 1352,
1306, 1250, 1218, 1200, 1176, 1151, 1053, 1025, 1002, 819, 757, 693.
HRMS calcd for C43H41N2O7 (M + H)+ 697.2908, found (M + H)+
697.2897.
Compound 11
Compound 10 (2.6 g, 4.9 mmol) was dissolved in dichloromethane
(22 mL) under argon and TBSCl (1.7 g, 11.3 mmol), imidazole
(3.0 g, 44.1 mmol), and catalytic DMAP added to the solution.
This was allowed to stir overnight at room temperature and
then concentrated by rotary evaporation the next morning.
The crude product was purified by column chromatography
starting with hexanes:EtOAc:Et3N (6:1:0.01), then eluting with
hexanes:EtOAc:Et3N (4:1:0.01) to afford compound 11 as a white
foam (2.7 g, 86%). 1H-NMR (360 MHz, CDCl3) d (ppm) 8.73 (s,
1H), 8.12 (s, 1H), 7.43 (d, J = 7.3 Hz, 2H), 7.35–7.19 (m, 7H),
6.82 (m, 4H), 4.56 (dd, J = 14.1, 3.9 Hz, 1H), 4.44 (dd, J = 14.0,
6.6 Hz, 1H), 4.15 (m, 1H), 3.80 (s, 6H), 3.14 (dd, J = 9.8, 4.2 Hz,
1H), 3.00 (dd, J = 9.6, 7.1 Hz, 1H), 0.79 (s, 9H), -0.16 (s, 3H),
-0.33 (s, 3H). 13C-NMR (75 MHz, CDCl3) d (ppm) 158.76, 158.74,
152.3, 151.9, 150.9, 146.5, 144.6, 135.8, 135.7, 131.4, 130.0, 128.2,
128.0, 127.1, 113.3, 86.6, 69.8, 64.7, 55.3, 48.0, 25.8, 17.9, -4.8,
-5.4. IR (film) v (cm-1) = 2954, 2927, 2858, 1608, 1590, 1560, 1508,
1464, 1442, 1403, 1333, 1302, 1246, 1176, 1080, 1036, 997, 940,
831, 779, 726, 700. HRMS calcd for C35H41N4O4SiCl (M + H)+
645.2664, found (M + H)+ 645.2683.
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Compound 13
The crude product was purified by column chromatography over
basic alumina starting with hexanes:EtOAc:Et3N (1:1:0.01), then
using hexanes:EtOAc:Et3N (1:2:0.01), and finally eluting with
EtOAc:Et3N (100:1) to afford compound 2 as a light yellow foam
(620 mg, 65%). 31P-NMR (121 MHz, CDCl3) d (ppm) 150.5,
150.0. HRMS calcd for C43H48N7O5P (M + H)+ 774.3533, found
(M + H)+ 774.3527.
To an argon purged solution of compound 11 (2.7 g, 4.2 mmol) and
PdCl2(PPh3)2 (265 mg, 0.4 mmol) in anhydrous THF (85 mL) was
added 2-pyridylzinc bromide (12, 0.5 M in THF, 13.5 mL) and the
solution heated to 65 ◦C for two hours. After cooling to room
temperature, the solution was washed with saturated aqueous
NaHCO3, extractedintodichloromethane, driedover Na2SO4, and
concentrated by rotary evaporation. The crude product was puri-
fied by column chromatography over basic alumina starting with
hexanes:EtOAc:Et3N (3:1:0.01), then using hexanes:EtOAc:Et3N
(1:1:0.01), and finally eluting with EtOAc:Et3N (100:1) to afford
GNA oligonucleotide synthesis and purification
GNA oligonucleotides were prepared on an ABI 394 DNA/RNA
synthesizer on a one micromole scale. GNA phosphoramidites
(A, T, G, C, 1, 2) were used at a concentration of 100 mM with a
standard protocol for 2-cyanoethyl phosphoramidites, except that
the coupling was extended to 3 minutes (A, T, G, C), or 8 minutes
(1, 2). After the trityl-on synthesis, the resin was incubated with
concentrated aqueous ammonia at 55–60 ◦C for 12 hours and then
evaporated. The tritylated oligonucleotides were purified by C18
reversed-phase HPLC (Varian Dynamax 250 ¥ 10 mm, Microsorb
300–10, C18) with aqueous triethylammonium acetate (50 mM
TEAA) and acetonitrile as the eluent. The oligonucleotides
were then detritylated with 80% acetic acid for 20 min and
precipitated with iPrOH after addition of 3 M sodium acetate. All
oligonucleotides were finally purified at 55–60 ◦C using a Waters
XTerra column (MS C18, 4.6 ¥ 50 mm, 2.5 mm) with aqueous
TEAA (50 mM) and acetonitrile as the eluent. All identities
were confirmed by MALDI-TOF MS and purities confirmed
by HPLC and MALDI-TOF analysis. Extinction coefficients for
GNA (strands shown in 3¢→2¢ direction) were calculated from
deoxynucleotide increments and yielded the following values for
e260 (M-1cm-1): AAT ATT ATT ATT TTA (153630), TAA AAT
AAT AAT ATT (171720), AAT ATT AHT ATT TTA (150606),
TAA AAT AHT AAT ATT (162666), AAT ATT APT ATT TTA
(159660), TAA AAT APT AAT ATT (171720), TAA AAT ATT
AAT ATT (165690).
1
compound 13 as a tan foam (2.3 g, 81%). H-NMR (400 MHz,
CDCl3) d (ppm) 9.12 (s, 1H), 8.97 (m, 1H), 8.86 (d, J = 8.0 Hz,
1H), 8.23 (s, 1H), 7.93 (td, J = 7.7, 1.6 Hz, 1H), 7.45 (m, 3H), 7.31
(m, 6H), 7.21 (t, J = 7.2 Hz, 1H), 6.82 (dd, J = 8.9, 2.5 Hz, 4H),
4.60 (dd, J = 14.1, 3.7 Hz, 1H), 4.50 (dd, J = 14.1, 6.7 Hz, 1H),
4.21 (m, 1H), 3.77 (s, 6H), 3.16 (dd, J = 9.8, 4.2 Hz, 1H), 3.07 (dd,
J = 9.8, 7.0 Hz, 1H), 0.79 (s, 9H), -0.16 (s, 3H), -0.34 (s, 3H). 13C-
NMR (125 MHz, CDCl3) d (ppm) 158.73, 158.71, 154.0, 153.7,
153.3, 152.6, 150.6, 146.8, 144.7, 136.8, 136.0, 135.9, 130.1, 128.2,
128.0, 127.0, 126.0, 124.9, 113.3, 86.6, 69.9, 64.9, 55.3, 47.7, 25.8,
17.9, -4.8, -5.3. IR (film) v (cm-1) = 3499, 2956, 2929, 2853, 1612,
1581, 1510, 1463, 1441, 1384, 1325, 1301, 1251, 1174, 1116, 1068,
1030, 988, 929, 825, 773, 720, 692, 635, 570, 531. HRMS calcd for
C40H45N5O4Si (M + H)+ 688.3319, found (M + H)+ 688.3314.
Compound 14
To an argon purged solution of compound 13 (1.25 g, 1.8 mmol)
in anhydrous THF (38 mL) was added TBAF (1 M in THF,
4 mL) and the solution stirred for 30 minutes at room temperature.
The solution was washed with water, extracted into ethyl acetate,
dried over Na2SO4, and concentrated by rotary evaporation. The
crude product was purified by column chromatography over basic
alumina starting with EtOAc:Et3N (100:1), then eluting with
EtOAc:MeOH:Et3N (40:3:0.01) to afford compound 14 as a tan
foam (840 mg, 81%). 1H-NMR (400 MHz, CDCl3) d (ppm) 9.04
(s, 1H), 8.95 (m, 1H), 8.82 (m, 1H), 8.22 (s, 1H), 7.93 (td, J = 7.8,
1.8 Hz, 1H), 7.46–7.38 (m, 3H), 7.31–7.25 (m, 6H), 7.21 (m, 1H),
6.81 (m, 4H), 4.54 (dd, J = 14.4, 2.8 Hz, 1H), 4.39 (dd, J = 14.3,
7.0 Hz, 1H), 4.22 (m, 1H), 3.76 (s, 6H), 3.24 (dd, J = 9.8, 5.9 Hz,
1H), 3.17 (dd, J = 9.7, 5.6 Hz, 1H). 13C-NMR (100 MHz, CDCl3) d
(ppm) 158.8, 153.8, 153.5, 153.4, 152.3, 150.5, 146.9, 144.6, 136.9,
135.7, 135.6, 130.0, 128.08, 128.07, 127.1, 125.9, 125.0, 113.4, 86.7,
69.6, 64.7, 55.3, 47.9, 25.8. IR (film) v (cm-1) = 3369, 3060, 2953,
2931, 2833, 1612, 1582, 1509, 1462, 1444, 1328, 1302, 1252, 1209,
1175, 1152, 1069, 1031, 905, 828, 726, 697, 637, 577. HRMS calcd
for C34H31N5O4 (M + H)+ 574.2454, found (M + H)+ 574.2449.
Metal dependent thermal stability of (S)-GNA duplexes monitored
by UV spectroscopy
The melting studies were carried out in 1 cm path length quartz
cells (200 mL sample solution covered by mineral oil) on a Beckman
DU800 spectrophotometer equipped with a thermoprogrammer.
Stock solutions of GNA strands (10 mM) were prepared in a
10 mM sodium phosphate buffer, pH 7.0, containing 100 mM
NaNO3 and subsequently treated with Chelex resin overnight.
The stock oligos were then combined with the respective metals
(2 equivalents) and diluted to a final duplex concentration of
2 mM. Melting temperatures were calculated using a nonlinear
fit to the heating curves. Experiments were performed at least in
duplicate and the averages taken.
Compound 2
Acknowledgements
To an argon purged solution of compound 14 (705 mg,
1.2 mmol) and diisopropylethylamine (1.0 mL, 5.9 mmol)
in dichloromethane (20 mL) was added 2-cyanoethyl N,N¢-
diisopropylchlorophosphoramidite (560 mL, 2.5 mmol) dropwise
and the solution stirred for two hours at room temperature
under argon. After the addition of dichloromethane, the solution
was washed one time with saturated aqueous NaHCO3, dried
over Na2SO4, and finally concentrated by rotary evaporation.
We thank the Philipps-University Marburg for financial support.
References
1 For DNA nanotechnology, see: N. C. Seeman, Chem. Biol., 2003, 10,
1151–1159; N. C. Seeman, Mol. Biotechnol., 2007, 37, 246–257.
2 For modified nucleic acids, see: S. M. Freier and K.-H. Altmann,
Nucleic Acids Res., 1997, 25, 4429–4443; A. Eschenmoser, Science,
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 476–482 | 481
©

View Article Online
1999, 284, 2118–2124; E. T. Kool, Acc. Chem. Res., 2002, 35, 936–943;
C. J. Leumann, Bioorg. Med. Chem., 2002, 10, 841–854; A. A. Henry
and F. E. Romesberg, Curr. Opin. Chem. Biol., 2003, 7, 727–733; S. A.
Benner, Acc. Chem. Res., 2004, 37, 784–797.
5 C. Switzer, S. Sinha, P. H. Kim and B. D. Heuberger, Angew. Chem.,
Int. Ed., 2005, 44, 1529–1532.
6 For reviews on metallo-base pairing, see: H.-A. Wagenknecht, Angew.
Chem., Int. Ed., 2003, 42, 3204–3206; M. Shionoya and K. Tanaka,
Curr. Opin. Chem. Biol., 2004, 8, 592–597; G. H. Clever, C. Kaul and
T. Carell, Angew. Chem., Int. Ed., 2007, 46, 6226–6236.
7 L. Zhang, A. Peritz and E. Meggers, J. Am. Chem. Soc., 2005, 127,
4174–4175; L. Zhang and E. Meggers, Synthesis, 2006, 645–653; M. K.
Schlegel, A. E. Peritz, K. Kittigowittana, L. Zhang and E. Meggers,
ChemBioChem, 2007, 8, 927–932.
8 M. K. Schlegel, L.-O. Essen and E. Meggers, J. Am. Chem. Soc., 2008,
130, 8158–8159.
9 Y.-W. Yang, S. Zhang, E. O. McCullum and J. C. Chaput, J. Mol. Evol.,
2007, 65, 289–295; C.-H. Tsai, J. Y. Chen and J. W. Szostak, Proc.
Natl. Acad. Sci. U. S. A., 2007, 104, 14598–14603; A. T. Horhota, J. W.
Szostak and L. W. McLaughlin, Org. Lett., 2006, 8, 5345–5347; R. S.
Zhang, E. O. McCullum and J. C. Chaput, J. Am. Chem. Soc., 2008,
130, 5846–5847.
10 For metallo-base pairing in PNA, see: D.-L. Popescu, T. J. Parolin and
C. Achim, J. Am. Chem. Soc., 2003, 125, 6354–6355; R. M. Watson,
Y. A. Skorik, G. K. Patra and C. Achim, J. Am. Chem. Soc., 2005, 127,
14628–14639; B. P. Gilmartin, K. Ohr, R. L. McLaughlin, R. Koerner
and M. E. Williams, J. Am. Chem. Soc., 2005, 127, 9546–9555.
11 A. Gold and R. Sangaiah, Nucleosides Nucleotides, 1990, 9, 907–912.
12 M. Hoffer, Chem. Ber., 1960, 93, 2777–2781.
3 E. Meggers, P. L. Holland, W. B. Tolman, F. E. Romesberg and P. G.
Schultz, J. Am. Chem. Soc., 2000, 122, 10714–10715; S. Atwell, E.
Meggers, G. Spraggon and P. G. Schultz, J. Am. Chem. Soc., 2001, 123,
12364–12367; N. Zimmermann, E. Meggers and P. G. Schultz, J. Am.
Chem. Soc., 2002, 124, 13684–13685; N. Zimmermann, E. Meggers and
P. G. Schultz, Bioorg. Chem., 2004, 32, 13–25; J. Mueller, D. Boehme,
P. Lax, M. M. Cerda and M. Roitzsch, Chem. Eur. J., 2005, 11, 6246–
6253; G. H. Clever, K. Polborn and T. Carell, Ang. Chem., Int. Ed.,
2005, 44, 7204–7208; C. Switzer and D. Shin, Chem. Commun., 2005,
1342–1344; Y. Miyake, H. Togashi, M. Tashiro, H. Yamaguchi, S. Oda,
M. Kudo, Y. Tanaka, Y. Kondo, R. Sawa, T. Fujimoto, T. Machinami
and A. Ono, J. Am. Chem. Soc., 2006, 128, 2172–2173; G. H. Clever,
Y. Soeltl, H. Burks, W. Spahl and T. Carell, Chem. Eur. J., 2006, 12,
8708–8718; K. Tanaka, G. H. Clever, Y. Takezawa, Y. Yamada, C. Kaul,
M. Shionoya and T. Carell, Nat. Nanotechnol., 2006, 1, 190–194; G. H.
Clever and T. Carell, Angew. Chem., Int. Ed., 2007, 46, 250–253; D. Shin
and C. Switzer, Chem. Commun., 2007, 42, 4401–4403; D. Boehme, N.
Duepre, D. A. Megger and J. Mueller, Inorg. Chem., 2007, 46, 10114–
10119; F.-A. Polonius and J. Mueller, Angew. Chem., Int. Ed., 2007, 46,
5602–5604; B. D. Heuberger, D. Shin and C. Switzer, Org. Lett., 2008,
10, 1091–1094.
4 K. Tanaka, A. Tengeiji, T. Kato, N. Toyama, M. Shiro and M.
Shionoya, J. Am. Chem. Soc., 2002, 124, 12494–12498.
13 Z. Kazimierczuk, H. B. Cottam, G. R. Revankar and R. K. Robins,
J. Am. Chem. Soc., 1984, 106, 6379–6382.
482 | Org. Biomol. Chem., 2009, 7, 476–482
This journal is
The Royal Society of Chemistry 2009
©