The Zn2+ complex of 1,4,7,10-tetraazacyclododecane as an artificial nucleobase

Article abstract of DOI:10.1080/15257770.2011.583809

{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4, 4'-dimethoxytrityl)-D-erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2, 2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1-yl]ethyl}acetamide (1) was prepared and incorporated into a 2'-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2'-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn2+. Introduction of Zn2+ moderately stabilized the duplex with U-bulged targets. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.583809

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The Zn2+ Complex of 1,4,7,10-
Tetraazacyclododecane as an Artificial
Nucleobase
Ms Maarit Laine a , Ms Maria Aromaa a , Dr Pasi Virta a , Tuomas
Lönnberg a , Dr Päivi Poijärvi-Virta a & Dr Harri Lönnberg a
a Department of Chemistry , University of Turku , Turku , Finland
Published online: 02 Sep 2011.
To cite this article: Ms Maarit Laine , Ms Maria Aromaa , Dr Pasi Virta , Tuomas Lönnberg , Dr Päivi
Poijärvi-Virta & Dr Harri Lönnberg (2011) The Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane
as an Artificial Nucleobase, Nucleosides, Nucleotides and Nucleic Acids, 30:7-8, 609-618, DOI:
10.1080/15257770.2011.583809
To link to this article: http://dx.doi.org/10.1080/15257770.2011.583809
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Nucleosides, Nucleotides and Nucleic Acids, 30:609–618, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.583809
THE Zn2+ COMPLEX OF 1,4,7,10-TETRAAZACYCLODODECANE
AS AN ARTIFICIAL NUCLEOBASE
Maarit Laine, Maria Aromaa, Pasi Virta, Tuomas Lo¨ nnberg,
Pa¨ivi Poija¨rvi-Virta, and Harri Lo¨ nnberg
Department of Chemistry, University of Turku, Turku, Finland
{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4ꢁ -dimethoxytrityl)-α-D-
2
erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1-
yl]ethyl}acetamide (1) was prepared and incorporated into a 2ꢁ-O-methyl oligoribonucleotide.
The hybridization of this oligonucleotide with complementary 2ꢁ-O-methyl oligoribonucleotides
incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence
and presence of Zn2+. Introduction of Zn2+ moderately stabilized the duplex with U-bulged targets.
Keywords Oligonucleotide; azacrown; Zn2+ complex; conjugate
INTRODUCTION
Zn2+ complexes of azacrown-conjugated oligonucleotides have been
shown to serve as artificial ribonucleases that cleave oligoribonucleotides
in a sequence-selective manner.[1] In addition, the Zn2+ azacrown chelates
have another interesting property; they recognize uracil and thymine
bases.[2] This binding is so tight that a trinuclear Zn2+ complex of 1,3-
tris[(1,3,5-triazacyclododecan-3-yloxy)methyl]benzene, for example, cleaves
at submillimolar concentrations of oligoribonucleotides selectively at UpU
sites.[3] Two of the Zn2+ azacrown moieties anchor the cleaving agent to
the uracil bases and the third one cleaves the intervening phosphodiester
linkage. For the same reason, oligonucleotide conjugates bearing a single
Zn2+ azacrown moiety are unable to cleave a U5-bulge opposite to the
cleaving agent, although an A5-bulge is cleaved; the azacrown chelate is
engaged in uracil binding and this prevents its catalytic action.[1b] The
latter finding raises the question of the applicability of Zn2+ chelates as
adenine base surrogates that recognize uracil and thymine bases. For this
purpose, {2-deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-
Received 2 February 2010; accepted 19 April 2010.
Address correspondence to Harri Lo¨nnberg, Department of Chemistry, University of Turku,
FIN-20014, Turku, Finland. E-mail: harlon@utu.fi
609

610
M. Laine et al.
FIGURE 1 Structure of {2-deoxy-3-O-[2-cyanoethoxy (diisopropylamino)phosphino]-5-O-(4,4ꢁ-dimeth-
oxytrityl)-α-D-erythro-pentofuranosyl}-N -{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-
tetraazacyclododecan-1-yl]ethyl}acetamide (1).
(4,4ꢁ-dimethoxytrityl)-α-D-erythro-pentofuranosyl}-N -{2-[4,7,10-tris(2,2,2-tri-
fluoroacetyl)-1,4,7,10-tetraazacyclododecan-1-yl]ethyl}acetamide (1) has
been prepared and incorporated into a 2ꢁ-O-methyl oligoribonucleotide
(Figure 1). The hybridization of this oligonucleotide with complementary
2ꢁ-O-methyl oligoribonucleotides incorporating one to five uracil bases
opposite to the azacrown moiety has been studied in the absence and
presence of Zn2+.
RESULTS AND DISCUSSION
Preparation of {2-deoxy-3-O-[2-cyanoethoxy(diisopropylamino)
phosphino]-5-O-(4,4ꢀ-dimethoxytrityl)-α-D-erythro-
pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-
tetraazacyclododecan-1-yl]ethyl}acetamide (1)
Commercially available 1,4,7,10-tetraazacyclododecane (cyclen) was
first converted to its N -(2-aminoethyl) derivative (6) to allow tethering to
previously[1a] prepared 5-O-(4,4ꢁ-dimethoxytrityl)-1C-(2-ethoxy-2-oxoethyl)-
1,2-dideoxy-α-D-erythro-pentofuranose (7). For this purpose, 2-ethanolamine
was converted to N -(4-nitrophenylsulfonyl)aziridine[4] (2), with which cyclen
was alkylated in MeCN to afford N -[2-(1,4,7,10-tetraazacyclododecan-1-
yl)ethyl]-4-nitrobenzenesufonamide (3) (Scheme 1). To allow purification
by silica gel chromatography, all amino functions were protected with tert-
butoxycarbonyl (BOC) groups, giving N -{2-[4,7,10-tris(tert-butoxycarbonyl)-
1,4,7,10-tetraazacyclododecan-1-yl]ethyl}-N -(tert-butoxycarbonyl)-4-nitrobe-
nzenesulfonamide (4). Removal of the 4-nitrosulfonyl group yielded
BOC-protected 1-(2-aminoethyl)-1,4,7,10-tetraazacyclododecane (5), which
by acidolytic deprotection was converted to 6.
The preparation of ethyl [2-deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-α-D-erythro-
pentofuranosyl]acetate (7) has been described earlier.[1a] The ester function
was hydrolyzed in aqueous alkali and the resulting potassium carboxylate
was converted to pyridinium salt (8) and coupled to N -(2-aminoethyl)cyclen

Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane
611
SCHEME 1 Synthesis of N -(2-aminoethyl)cyclen. Reagents: i: DCM-Py (2:1, v/v); ii: KOH, DCM, H2O;
iii: Cyclen, MeCN; iv: Boc2O, Et3N, Py; v: HSCH2COOH, DBU, DMF; vi: CF3COOH, DCM.
(6) by O-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium haxafluoropho-
sphate/N ,N -diisopropylethylamine (HBTU/DIEA) activation in 1,4-
dioxan to obtain amide 9 (Scheme 2). The amino groups of the
cyclen moiety were protected with trifluoroacetyl groups giving 10,
and finally, the 3-OH group of the sugar moiety was phosphitylated
with 2-cyanoethyl-N ,N -diisopropylchlorophosphoramidite to obtain [2-
deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4ꢁ-dimeth-
oxytrityl)-α-D-erythro-pentofuranosyl]-N -{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-
1,4,7,10-tetraazacyclododecan-1-yl]ethyl}acetamide (1).
SCHEME 2 Synthesis of the cyclen-derived building block. Reagents: i: KOH, H2O, 1,4-dioxan; ii: Dowex
(50WX8-200) resin in HPy+ form; iii: 5, DIEA, HBTU, 1,4-dioxan; iv: CF3COOMe, Et3N, MeOH; v:
NCCH2CH2OP(Cl)N(iPr)2, Et3N, DCM.

612
M. Laine et al.
TABLE 1 HPLC retention times and ESI-MS for the 2ꢁ-O-methyl oligoribonucleotides prepared
Sequence
tR/minutea
m/z obsd.
m/z calcd.
11
12
13
14
15
16
17
5ꢁ-CGC GCX GGC CC-3ꢁb
5ꢁ-CGC GCA GGC CC-3ꢁ
5ꢁ-GGG CCU GCG CG-3ꢁ
5ꢁ-AAG CCU GCG AA-3ꢁ
5ꢁ-GCC UGC GC-3ꢁ
30.5
23.8
25.6
26.6
23.4
22.0
23.2
3723.9
3632.7
3689.7
3665.8
2611.5
4329.9
4971.0
3723.9
3631.7
3688.7
3664.7
2611.5
4328.8
4968.9
5ꢁ-GGG CCU UUG CGC G-3ꢁ
5ꢁ-GGG CCU UUU UGC GCG-3ꢁ
aFor the chromatographic conditions, see the Experimental section. bX refers to (2-deoxy-α-D-erythro-
pentofuranosyl)-N -[2-(1,4,7,10-tetraazacyclododecan-1yl)ethyl]acetamide (cf. compound 9).
Synthesis of Oligonucleotides
The 11-mer 2ꢁ-O-methyl oligoribonucleotide 11 incorporating the
azacrown-functionalized non-nucleosidic unit and the unmodified 2ꢁ-O-
methyl oligoribonucleotides 12–17 were assembled by the standard phospho-
ramidite protocol of solid-phase synthesis. The coupling yield for the non-
nucleosidic block 1 phosphoramidite was 94%. The crude oligonucleotides
were purified by reversed-phase high-performance liquid chromatogra-
phy (RP-HPLC) and their identity was verified by electrospray ionization
mass spectrometry (ESI-MS) analysis. Table 1 records the oligonucleotides
prepared.
Melting Temperature Measurements
Melting temperatures (Tm) for the duplexes of the azacrown contain-
ing oligonucleotide 11 with unmodified targets 13–17 were measured at
various concentrations of ZnCl2 in a 20-mmol L−1 cacodylate buffer at pH
7.4, the ionic strength being adjusted to 1.0 or 0.1 mol L−1 with NaCl. The
concentration of the oligonucleotides was 2 µmol L−1. For comparison, the
hybridization efficiency of oligonucleotide 12, an analog of 11 containing
2ꢁ-O-methyladenosine instead of the azacrown block, was quantified. Table 2
summarizes the results.
Belousoff et al.[5] have previously attempted to increase the affinity of
peptide nucleic acid (PNA) to the HIV-1 trans-activation response (TAR)
mRNA by conjugating cyclen to the amino terminus of the peptide chain.
The base sequence of the conjugate was designed in such a manner that the
azacrown moiety could interact with the uracil base within the hairpin hex-
aloop. Introduction of the azacrown actually slightly stabilized the duplex
formation, but contrary to expectations, addition of Zn2+ did not result in
extra stabilization. The results of the present study are rather different. Re-
placement of 2ꢁ-O-methyladenosine within a 2ꢁ-O-methyl oligoribonucleotide
with the cyclen-derived 2ꢁ-deoxyriboside (10) dramatically destabilizes the

Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane
613
TABLE 2 Melting points for the duplexes of the azacrown functionalized oligonucleotide 11 and its
adenine analog 12 with 2ꢁ-O-methyl oligoribonucleotide targets in 20 mmol L−1 cacodylate buffer (pH
7.4) at various concentrations of Zn2+
Duplex
[Zn2+]/µmol L−1a
I/mol L−1b
Tm/◦C
5ꢁ-CGC GCA GGC CC-3ꢁ (12)
3ꢁ-CG CGU CCG-5ꢁ (15)
0
20
50
100
1.0
1.0
1.0
1.0
81.8
81.9
81.0
81.2
5ꢁ-CGC GCX GGC CC-3ꢁ (11)c
3ꢁ-CG CGU CCG-5ꢁ (15)
0
20
50
100
1.0
1.0
1.0
1.0
51.6
51.6
50.7
51.6
5ꢁ-CGC GCX GGC CC-3ꢁ (11)
3ꢁ-GCG CGU CCG GG-5ꢁ (13)
0
20
50
100
1.0
1.0
1.0
1.0
75.6
75.7
75.2
75.7
5ꢁ-CGC GCX GGC CC-3ꢁ (11)
3ꢁ-GCG CGU3CCG GG-5ꢁ (16)
0
20
50
100
1.0
1.0
1.0
1.0
75.3
76.5
76.2
77.6
5ꢁ-CGC GCX GGC CC-3ꢁ (11)
3ꢁ-GCG CGU5CCG GG-5ꢁ (17)
0
20
50
100
1.0
1.0
1.0
1.0
72.9
74.8
74.3
76.0
5ꢁ-CGC GCA GGC CC-3ꢁ (12)
3ꢁ-GCG CGU CCG GG-5ꢁ (13)
0
50
0.1
0.1
83.7
84.6
5ꢁ-CGC GCX GGC CC-3ꢁ (11)
3ꢁ-GCG CGU CCG GG-5ꢁ (13)
0
50
0.1
0.1
65.2
67.9
5ꢁ-CGC GCX GGC CC-3ꢁ (11)
3ꢁ-AAG CGU CCG AA-5ꢁ (14)
0
50
0.1
0.1
30.6
33.7
5ꢁ-CGC GCX GGC CC-3ꢁ (11)
3ꢁ-CG CGU CCG-5ꢁ (15)
0
50
0.1
0.1
45.8
47.6
aIntroduced as ZnCl2. bAdjusted with NaCl. c X refers to (2-deoxy-α-D-erythro-pentofuranosyl)-N -[2-
(1,4,7,10-tetraazacyclododecan-1yl)ethyl]acetamide (cf. compound 9).
hybridization to 2ꢁ-O-methyl oligoribonucleotide targets containing uridine
opposite to the non-nucleosidic block. The melting temperature for a duplex
with an 8-mer (15) and 11-mer (13) sequence dropped by 30◦C (I = 1 mol
L−1) and 20◦C (I = 0.1 mol L−1), respectively. Addition of Zn2+ up to 100
µmol L−1 did not change the situation at high ionic strength: the difference
between the Tm values of duplex 11/15 and 12/15 remained unchanged. At

614
M. Laine et al.
low ionic strength, the addition of Zn2+ slightly decreased the melting tem-
perature difference between 12/13 and 11/13. Interestingly, hybridization
of 11 to targets that formed a U3- or U5-bulge opposite to the cyclen-derived
unit was modestly enhanced upon increasing concentration of Zn2+ from 0
to 100 µmol L−1: the Tm was increased by 2◦C–3◦C. Evidently, formation of
a ternary complex between uracil base, cyclen, and Zn2+ ion is not possible
without disruption of the normal double helical structure, and hence, even
a modest stabilization is observed only when interaction with a bulged uracil
base is possible.
EXPERIMENTAL
General Methods
1
The H, 13C, and 31P NMR spectra were recorded by a Bruker 400 or
500 NMR spectrometer and the chemical shifts are given in ppm. The mass
spectra were recorded on a Bruker micrOTOF-Q ESI-MS system. Oligonu-
cleotides were prepared by using ABI-392 DNA/RNA synthesizer. Oligonu-
cleotides were purified by RP-HPLC on a Hypersil ODS column C18 (250 ×
4.6 mm or 250 × 4 mm, 5 µm; flow rate 1 mL minute−1; buffer A = 0.1 mol
L
−1 TEAA and B = 0.1 mol L−1 TEAA containing 50% MeCN; a linear gra-
dient from 0% to 50% buffer B in buffer A in 25 minutes, followed by 50%
buffer B in buffer A for 5 minutes).
N-{2-[4,7,10-Tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecan-1-
yl]ethyl}-N-(tert-butoxycarbonyl)-4-nitrobenzenesulfonamide (4)
4-Nitrobenzenesulfonyl chloride (24.01 g, 108 mmol) was dissolved in
a mixture of dichloromethane (DCM) and pyridine (2:1). Ethanolamine
(2.18 mL, 36 mmol) was slowly added on an ice bath. The ice bath was
removed and the reaction mixture was stirred for 3 hours at room tempera-
ture, diluted with DCM (300 mL), and washed with 2 mol L−1 aqueous HCl
(3 × 100 mL) and 2 mol L−1 aqueous KOH (5 × 200 mL). The organic
phase was dried with Na2SO4 and the solvent was removed under reduced
pressure affording N -(4-nitrophenylsulfonyl)aziridine (2). Compound 2 in
dry MeCN (82 mL) was added dropwise into the solution of cyclen (6.29 g,
36.5 mmol) in dry MeCN during 4 hours at 0◦C. The reaction mixture was
allowed to warm up to room temperature, stirred overnight, and evapo-
rated to dryness affording N -[2-(1,4,7,10-tetraazacyclododecan-1-yl)ethyl]-4-
nitrobenzenesulfonamide (3). Di-tert-butyl dicarbonate (42 mL, 183 mmol)
and Et3N (51 mL, 365 mmol) were added into the solution of compound 3
in dry pyridine and the mixture was stirred overnight at room temperature.
To ensure the completeness of the reaction, another portion of di-tert-butyl
dicarbonate (17 mL, 74 mmol) and Et3N (51 mL, 365 mmol) was added.
The mixture was stirred for another 72 hours and evaporated to dryness. The

Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane
615
residue was subjected to DCM/aqueous sodium bicarbonate workup and the
organic phase was dried with Na2SO4. After evaporation, the residue was pu-
rified by silica gel chromatography (3%–5% MeOH in CH2Cl2 and a mixture
1
of petroleum ether and EtOAc, 7:3, v/v). The yield was 4.09 g (14%). H
NMR (400 MHz, CDCl3) δppm 8.41–8.35 (m, 2H), 8.21 (br. s, 2H), 3.95 (dd,
2H, J = 7.6 Hz, 8.4 Hz), 3.54–3.35 (m, 12H), 2.95 (dd, 2H, J = 7.6 Hz,
8.4 Hz), 2.79 (br. s, 4H), 1.47–1.38 (m, 36H). 13C NMR (125 MHz, CDCl3)
δppm 171.1, 155.8, 150.3, 145.4, 129.6, 124.4, 123.9, 85.6, 80.5, 79.5, 77.0,
60.4, 54.7, 53.7, 50.5, 50.0, 48.0, 42.4, 28.5, 21.0, 14.18. HRMS(ESI): obsd.
801.4040 [M+H]+, calcd. 801.4069 [M+H]+.
N-(2-Aminoethyl)cyclen (6)
Compound
4
(1.23 g, 1.5 mmol) was dissolved in N ,N -
dimethylformamide (DMF) (5 mL). Thioglycolic acid (250 µL, 14.7 mmol)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.2 mL, 14.7 mmol) were
added and the reaction mixture was stirred for 2.5 hours at room tem-
perature. The mixture was diluted with EtOAc (30 mL) and washed with
saturated aqueous solution of NaHCO3 (3 × 15 mL). The organic phase
was dried with Na2SO4 and evaporated to dryness. The residue was purified
by silica gel chromatography (petroleum ether/EtOAc, 1:1, v/v) affording
compound 5. The yield was 0.44 g (46%). To a solution of compound 5 (1.22
g, 2.0 mmol) in DCM, trifluoroacetic acid (5 mL) was added and the mix-
ture was stirred overnight at room temperature. The volatiles were removed
under reduced pressure and the residue was coevaporated with H2O (5 mL)
and MeOH (5 mL). Yield 40%. HRMS(ESI): calcd. 216.2188 [M+H]+, obsd.
216.2011 [M+H]+.
[2-Deoxy-5-O-(4,4ꢀ-dimethoxytrityl)-α-D-erythro-pentofuranosyl]-N-{2-[4,7,10-
tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1-yl]ethyl}acetamide
(10)
Previously[1a] prepared ethyl [2-deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-α-D-
erythro-pentofuranosyl]acetate (7) (0.49 g, 0.9 mmol) was dissolved in 1,4-
dioxane (20 mL). Aqueous KOH (0.29 g in 5 mL water) was added dropwise,
and the reaction mixture was stirred overnight and evaporated to dryness
under reduced pressure. The residue was dissolved in pyridine (20 mL),
Dowex resin in HPy+ form was added, and the resulting mixture stirred
for 2 hours at room temperature. The resin was collected by filtration
and washed with pyridine (4 × 10 mL), and the filtrate was evaporated to
dryness to obtain pyridinium [2-deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-α-D-erythro-
pentofuranosyl]acetate 8 (0.66 g). N -(2-Aminoethyl)cyclen (6, 0.25 g, 1.15
mmol) was dissolved in 1,4-dioxan (4 mL) and DIEA (1.2 mL, 6.89 mmol) was
added. To a solution of compound 8 in 1,4-dioxan (2 mL), DIEA (0.34 mL,
1.95 mmol) and HBTU (0.55 g, 1.45 mmol) were added and the mixture was

616
M. Laine et al.
stirred for 15 minutes. The solution of compound 8 was added dropwise to
N -(2-aminoethyl)cyclen (6) in 1,4-dioxane during 1 hour. The reaction mix-
ture was stirred overnight at room temperature and evaporated to dryness
to give [2-deoxy-5-O-(4,4ꢁ-dimethoxytrityl)-α-D-erythro-pentofuranosyl]-N -[2-
(1,4,7,10-tetraazacyclododecan-1-yl)ethyl]acetamide 9. Trifluoroacetic acid
(20 mL) and methanol (10.5 mL) were stirred at room temperature and the
methyl ester formed was isolated by distillation. Et3N (2 mL, 14.42 mmol)
and CF3COOMe (1.17 mL, 11.63 mmol) were added into the solution of
compound 9 in MeOH (2 mL) and the reaction mixture was stirred at room
temperature. After 72 hours, another portion of Et3N (1 mL, 7.21 mmol) and
CF3COOMe (0.58 mL, 5.76 mmol) was added and the mixture was stirred
for 3 hours. Pyridine (5 mL) was added and the solvent was removed under
reduced pressure. The residue was dissolved in EtOAc (100 mL) and washed
with saturated aqueous solutions of NaHCO3 (100 mL) and NaCl (100 mL).
The organic phase was dried with Na2SO4 and evaporated to dryness. The
residue was purified by silica gel chromatography (MeOH/CH2Cl2, 5:95,
v/v containing 0.1% Et3N and MeOH/EtOAc, 95:5, v/v containing 0.1%
Et3N). The yield of 10 was 0.34 g (36%). 1H NMR (500 MHz, CDCl3) δppm
7.45–6.75 (m, 13H), 4.46–4.41 (m, 1H), 4.29 (s, 1H), 4.15–4.10 (m, 1H),
3.87–3.32 (m, 12H), 3.80 (s, 6H), 3.25–3.20 (m, 2H), 3.10–3.07 (m, 1H),
2.95–2.71 (m, 4H), 2.70–2.46 (m, 4H), 1.77–1.72 (m, 1H). 13C NMR (125
MHz, CDCl3) δppm 171.95, 158.56, 144.78, 135.85, 130.00, 128.05, 127.92,
126.94, 113.15, 86.20, 85.30, 77.38, 77.05, 76.79, 75.05, 74.19, 74.01, 64.00,
60.42, 56.80, 55.23, 53.45, 49.35, 49.08, 49.06, 49.04, 49.00, 48.98, 47.29,
46.89, 46.86, 46.83, 46.00, 45.96, 45.89, 45.86, 45.84, 42.77, 42.74, 42.54,
42.52, 40.30, 39.97, 38.62. HRMS(ESI): obsd. 986.3321 [M+Na]+, calcd.
986.3363 [M+Na]+.
{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4ꢀ-
dimethoxy-trityl)-α-D-erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-
trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1-yl]ethyl}acetamide (1)
Compound 10 (0.34 g, 0.35 mmol) was dissolved in dry MeCN
(4 mL). Dry Et3N (250 µL, 1.77 mmol) and 2-cyanoethyl-N ,N -
diisopropylchlorophosphoramidite (102 µL, 0.46 mmol) were added under
argon. The reaction mixture was shaken for 1 hour and purified by silica gel
chromatography (Et3N/EtOAc, 5:95, v/v). The product was coevaporated
1
with MeCN. The yield was 0.31 g (75%). H NMR (500 MHz, CDCl3) δppm
7.49–6.88 (m, 13H), 4.52–4.49 (m, 1H), 4.46–4.40 (m, 1H), 4.11–4.06 (m,
1H), 3.93 (m, 1H), 3.78 (s, 6H), 3.76–3.47 (m, 13H), 3.46–3.38 (m, 1H),
3.38–3.29 (m, 2H), 3.17–3.04 (m, 2H), 2.95–2.77 (m, 4H), 2.64 (t, 1H, J =
6.0, 6.0 Hz), 2.56–2.53 (m, 1H), 2.52–2.50 (t, 1H, J = 6.0, 6.0 Hz), 2.48–2.38
(m, 2H), 2.01–1.96 (m, 1H), 1.85–1.75 (m, 1H), 1.18–1.13 (m, 12H),
1.09–1.05 (m, 2H). 13C NMR (125 MHz, CDCl3) δppm 170.60, 158.64, 145.26,

Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane
617
136.12, 130.05, 128.10–126.78, 118.50, 117.20, 113.06, 85.83, 83.79, 77.26,
75.50, 64.00, 58.48–57.82, 65.11, 54.90, 50.78, 49.20, 48.55–48.40, 47.00,
45.33, 44.06, 42.96–42.84, 42.67–42.62, 41.30, 39.89, 39.53–39.40, 37.88,
35.88, 33.41–33.11, 30.03, 28.69, 25.75, 23.93–23.84, 20.10–19.94, 14.95,
0.84–(−0.15). 31P NMR (200 MHz, CDCl3) δppm 147.7, 147.6. HRMS(ESI):
obsd. 1186.4451 [M+Na]+, calcd. 1186.4441 [M+Na]+.
Synthesis of Oligonucleotides
Oligonucleotides 11–17 were synthesized by conventional phospho-
ramidite strategy from 2ꢁ-O-methyl-protected phosphoramidite building
blocks (Glenn Research) on a 1-µmol L−1 scale, following the standard
RNA-coupling protocol of ABI-392 DNA/RNA synthesizer. The products
were released from support and protecting groups of base moiety were re-
moved by concentrated aqueous ammonia treatment (33% aq. NH3, 2 hours
at room temperature and 4–10 hours at 55◦C). The crude oligonucleotides
were purified and desalted by RP-HPLC. The identity of the products was
verified by ESI-MS analysis (Table 1).
Determination of the Duplex Melting Temperatures
The melting curves (absorbance versus temperature) were measured
using a Perkin Elmer Lambda 35 ultraviolet/visible (UV/VIS) spectrometer
equipped with a Peltier temperature programmer. The melting curves were
collected by using a 0.5◦C/minute temperature ramp from 10◦C to 90◦C (for
both forward and reverse temperature ramps) measuring the absorbance at
260 nm every half minute. The measurements were performed in 20 mmol
L−1 cacodylate buffer (pH 7.4) containing 1.0 mol L−1 (or I = 0.1 mol
L−1) NaCl. The oligonucleotide concentration was 2 µmol L−1 and ZnCl2
(dissolved in water from the anhydrous salt) was used at the concentrations
of 20, 50, and 100 µmol L−1. The measurements were also performed without
ZnCl2. All aqueous solutions were prepared in sterilized water and sterilized
equipment was used for their handling. At the beginning of each melting
temperature experiment, all samples were held at room temperature for 1
hour and after that at 10◦C for 5 minutes. The resulting absorbance versus
temperature profiles were analyzed by using a UV WinLab Data Processor &
Wiewer program. The Tm values were determined as the maximum of the
first derivate of the melting curve.
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