Zn2+ complexes of 3,5-Bis[(1,5,9-triazacyclododecan-3-yloxy) methyl]phenyl conjugates of oligonucleotides as artificial RNases: The effect of oligonucleotide conjugation on uridine selectivity of the cleaving agent

Article abstract of DOI:10.1002/hlca.201200153

2-(3,5-Bis{[1,5,9-tris(trifluoroacetyl)-1,5,9-triazacyclododecan-3-yloxy] methyl}phenoxy)ethanol was synthesized and converted to a O-(2-cyanoethyl)-N,N- diisopropylphosphoramidite building block, 12. 2′-O-Methyl oligoribonucleotides incorporating a 2-[(2S,4S,5R)-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)ethyl 4-oxopentanoate or a 2-{2-[2-({[(2R,4S,5R)-4-hydroxy- 5-(hydroxymethyl)tetrahydrofuran-2-yl]acetyl}amino)ethoxy]ethoxy}ethyl 4-oxopentanoate non-nucleosidic unit close to the 3′-terminus were assembled on a solid support, the 4-oxopentanoyl protecting groups were removed by treatment with hydrazinium acetate on-support, and 12 was coupled to the exposed OH function. The deprotected conjugates were purified by HPLC, and their ability to cleave a complementary RNA containing either uridine or some other nucleoside at the potential cleaving site was compared. Somewhat unexpectedly, conjugation to an oligonucleotide did not enhance the catalytic activity of the Zn²⁺-bis(azacrown) complex and virtually abolished its selectivity towards the uridine sites. Copyright

Full text of DOI:10.1002/hlca.201200153

Helvetica Chimica Acta – Vol. 96 (2013)
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Zn2þ Complexes of 3,5-Bis[(1,5,9-triazacyclododecan-3-yloxy)methyl]phenyl
Conjugates of Oligonucleotides as Artificial RNases: The Effect of
Oligonucleotide Conjugation on Uridine Selectivity of the Cleaving Agent
by Teija Niittymꢀki, Ekaterina Burakova1), Evelina Laitinen, Anna Leisvuori, Pasi Virta*, and
Harri Lçnnberg*
Department of Chemistry, University of Turku, FIN-20014 Turku
(phone: þ 358-2-3336770; fax: þ 358-2-3336700; e-mail: harlon@utu.fi)
2-(3,5-Bis{[1,5,9-tris(trifluoroacetyl)-1,5,9-triazacyclododecan-3-yloxy]methyl}phenoxy)ethanol was
synthesized and converted to a O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite building block, 12. 2’-
O-Methyl oligoribonucleotides incorporating a 2-[(2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)ethyl 4-oxopentanoate or a 2-{2-[2-({[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl]acetyl}amino)ethoxy]ethoxy}ethyl 4-oxopentanoate non-nucleosidic unit close to the 3’-
terminus were assembled on a solid support, the 4-oxopentanoyl protecting groups were removed by
treatment with hydrazinium acetate on-support, and 12 was coupled to the exposed OH function. The
deprotected conjugates were purified by HPLC, and their ability to cleave a complementary RNA
containing either uridine or some other nucleoside at the potential cleaving site was compared.
Somewhat unexpectedly, conjugation to an oligonucleotide did not enhance the catalytic activity of the
Zn2þꢀbis(azacrown) complex and virtually abolished its selectivity towards the uridine sites.
Introduction. – Metal ion chelates conjugated to either an oligonucleotide or a
peptide nucleic acid (PNA) probe have received considerable interest as man-made
restriction enzymes, with which large RNA molecules could be tailored in a sequence-
selective manner [1]. Upon hybridization of the probe with its complementary target
sequence, the concentration of the cleaving agent in the vicinity of one particular
phosphodiester linkage is increased, resulting in accelerated chain cleavage. Since
metal ion chelates are able to cleave phosphodiester bonds only within single-stranded
regions [2], the cleaving agent is usually incorporated either into a terminal position of
the probe, or the base sequence of the probe is designed to form upon hybridization a
bulge opposite to an intrachain-cleaving agent. Owing to the proximity effect, the
cleaving activity of the conjugated chelate typically is 100-fold compared to the chelate
monomer [3], although, in special cases, more marked accelerations have been
reported. For example, a 2’-O-methyl oligoribonucleotide bearing two 3-(3-hydroxy-
propyl)-1,5,9-triazacyclododecane ligands on a 2-hydroxyethyl 3’-O-(2-hydroxyethyl)-
b-d-ribofuranoside branching unit (1; Fig. 1) exhibits, as a Zn2þ complex, 1000-fold
cleaving activity compared to monomeric Zn2þ chelate of 1,5,9-triazacyclododecane
[4], and a Cu2þ complex of 2,9-dimethyl-1,10-phenanthroline (2) experiences an even
greater increase in catalytic activity when tethered into an intrachain position within a
PNA probe creating a tetranucleotide bulge [5].
Interestingly, a base-moiety-selective cleavage may also be achieved by dinucleat-
ing ligands. (1,5,9-Triazacyclododecan-3-yl)oxy groups, when attached to an aromatic
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Helvetica Chimica Acta – Vol. 96 (2013)
Fig. 1. Structures of metal ion-based cleaving agents of RNA phosphodiester linkages
scaffold, 3, cleave RNA at the 5’-side, and, to a somewhat lesser extent, at the 3’-side, of
uridines [6]. Evidently, one of the Zn2þꢀazacrown moieties anchors the cleaving agent
to the uracil base, while the other azacrown moiety serves as a catalyst for the
phosphodiester transesterification. Owing to this proximity effect, the cleavage is up to
two orders of magnitude faster at a uridine site than at an adenosine or a cytidine site.
While this difference in reaction rate is sufficient for uridine-selective cleavage of short
oligonucleotides, random background cleavage still is too fast to allow controlled
manipulation of sequences longer than 20 – 30 nucleotides [6c]. Bearing in mind that
conjugation of a metal ion chelate to an oligonucleotide increases, owing to the
proximity effect, its cleaving activity, one might expect this to be the case also with 3,
leading to increasingly selective cleavage at a single uridine site. The present study is
aimed at elucidating whether exploitation of the uracil-anchoring ability of 3 in synergy
with sequence recognition by a 2’-O-methyl oligoribonucleotide probe really enhances
the catalytic efficiency and, hence, ensures cleavage precisely at a single phosphodiester
bond. Accordingly, oligonucleotide conjugates depicted in Fig. 2 have been prepared,
and their ability to cleave various oligoribonucleotide targets has been studied.
Results. – Preparation of 2-{3,5-Bis[(1,5,9-triazacyclododecan-3-yloxy)methyl]-
phenoxy}ethanol and Its Conversion to a Phosphoramidite Building Block. Commer-
cially available dimethyl 5-hydroxyisophthalate was converted to its allyl ether 4, and
the COOMe groups were reduced to CH2OH functions with LiAlH4 in Et2O
(Scheme 1). The dimethanol 5 obtained was then tosylated in dioxane to 6, and the
Ts groups were replaced by 1,5,9-tris[(tert-butoxy)carbonyl]-1,5,9-triazacyclododecan-
3-ol (7), prepared as described in [6a]. The allyloxy group of the bis(azacrown)
conjugate 8 was converted to 2-oxoethoxy group by OsO4 hydroxylation and
subsequent NaIO4 oxidation, and the resulted aldehyde 9 was reduced with NaBH4
to 10. Finally, the Boc protections were replaced with CF3CO (Tfa) groups (!11), and

Helvetica Chimica Acta – Vol. 96 (2013)
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Fig. 2. The underlying principle of action of the artificial RNases prepared
the OH function was phosphitylated to 2-cyanoethyl N,N-diisopropylphosphoramidite
12.
Preparation of Non-Nucleosidic Phosphoramidite Building Blocks. Non-nucleosidic
building blocks allowing attachment of the bis(azacrown) conjugate as a phosphor-
amidite reagent, 12, to the oligonucleotide chain were prepared as follows. 2-
[(2S,4S,5R)-5-{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxytetrahydro-
furan-2-yl]ethyl 4-oxopentanoate (13) was obtained as described in [7]. Its analog 17,
with a longer side chain at C(2), was synthesized by subjecting the earlier prepared
[7] ethyl [(2R,4S,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-{[(tert-bu-
tyl)(dimethyl)silyl]oxy}tetrahydrofuran-2-yl]acetate (14) to aminolysis with 2-[2-(2-
aminoethoxy)ethoxy]ethanol to obtain 15, acylating the OH function with (Lev)2O
(!16), and removing the TBDMS group with Bu4NF in THF (!17; Scheme 2).
Compounds 13 and 17 were then converted to phosphoramidite building blocks 18 and
19, respectively, by conventional methods.
Synthesis of Oligonucleotide Bis(azacrown) Conjugates 20 and 21. Conjugates of 2’-
O-methyl oligoribonucleotides bearing the bis(azacrown) cleaving agent at the
penultimate site from the 3’-terminus were assembled by the conventional phosphor-
amidite chemistry on a commercially available support with N4-benzoyl-5’-O-[bis(4-
methoxyphenyl)(phenyl)methyl]-2’-O-methylcytidine as the 3’-terminal nucleoside

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Scheme 1. Synthesis of 2-[3,5-Bis({[1,5,9-tris(trifluoroacetyl)-1,5,9-triazacyclododecan-3-yl]oxy}meth-
yl)phenoxy]ethyl 2-Cyanoethyl N,N-Diisopropylphosphoramidite (12)
i) Allyl bromide, EtNiPr2, DMF. ii) LiAlH4, Et2O. iii) TosCl, NaOH, H2O, dioxane. iv) 7, NaH, DMF. v)
OsO4, NaIO4, H2O, dioxane. vi) NaBH4, EtOH. vii) 1. TfaOH, CH2Cl2; 2. TfaOMe, Et3N, MeOH; 3.
(Tfa)2O, pyridine, CH2Cl2; 4. Et3N, MeOH. viii) 2-Cyanoethyl N,N-diisopropylphosphonamidic chloride,
Et3N, CH2Cl2.
(Scheme 3). The 5’-O-protection was removed, and the non-nucleosidic building block,
either 18 or 19, was coupled manually using a prolonged coupling time. The Lev group
was removed from the non-nucleosidic block by H2NNH3OAc treatment in pyridine,
and the bis(azacrown) block 12 was coupled manually, using again a prolonged
coupling time. The support was then loaded on the synthesizer, and the 2’-O-methyl
oligoribonucleotide sequence 5’-ACA CAG ACA CGC CZC-3’ (Z stands for the non-
nucleosidic block) was assembled, and the support was subjected to ammonolysis to
obtain the fully deprotected conjugate 20 or 21.

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Helvetica Chimica Acta – Vol. 96 (2013)
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Hybridization of Oligonucleotide Bis(azacrown) Conjugate 21 with Chimeric
Oligoribonucleotide/2’-O-Methyl Oligoribonucleotide Targets. Table 1 records the
melting temperatures for duplexes of conjugate 21 with several chimeric oligoribonu-
cleotide/2’-O-methyl oligoribonucleotide targets, 22 – 27. All the targets are fully
complementary to 21 over the 3’-terminal sequence, 3’-UGU GUC UGU GCG C-5’,
but contain different 5’-terminal ribonucleotide overhangs. For comparative purposes,
the melting temperatures of the duplexes of unconjugated 2’-O-methyl oligoribonu-
cleotide 5’-ACA CAG ACA CGC C-3’ (28) with the same targets are also included.
Table 1. Melting Temperatures (Tm [8]) of the Duplexes of Oligonucleotide Conjugate 21 and Reference
Oligonucleotide 28 with Chimeric Targets 22 – 27 in the Absence and Presence of Zn2þ Ion at pH 7.0. The
data refers to [oligomer] ¼ 2 mm and [Zn2þ] ¼ 10 mm at the ionic strength of 0.1m, adjusted with NaCl. pH
was adjusted with 10 mm phosphate buffer. Bold letters refer to ribonucleotides, normal letters to 2’-O-
methyl ribonucleotides.
Target
21
28
with Zn2þ without Zn2þ with Zn2þ without Zn2þ
3’-UGU GUC UGU GCG GAC AAC AA-5’ (22) 76.1
3’-UGU GUC UGU GCG GUA AAC AA-5’ (23) 76.7
3’-UGU GUC UGU GCG GAU AAC AA-5’ (24) 76.7
3’-UGU GUC UGU GCG GAA UAC AA-5’ (25) 77.5
3’-UGU GUC UGU GCG GAA AUC AA-5’ (26) 78.2
3’-UGU GUC UGU GCG GAA AAU AA-5’ (27) 75.9
78.3
82.0
79.7
79.8
78.5
77.9
73.8
73.3
73.5
75.0
74.5
75.5
74.9
75.0
76.1
74.6
74.4
74.4
The data in Table 1 clearly show that conjugate 21 hybridizes with all the targets
even slightly better than its unconjugated counterpart 28. The duplexes formed are
stable at 358, the temperature at which the kinetic measurements were carried out.
Addition of Zn2þ still enhanced the hybridization of 21, the influence being most
prominent with target 23. In this case, the melting temperature, Tm, is increased by 5.38,
while with the unconjugated reference oligonucleotide 28 the increment is 1.78, and
with target 22, containing no uridine within the overhang, addition of Zn2þ increases the
Tm value of the duplex with conjugate 21 by 2.28. Evidently, the cleaving agent really
recognizes the uracil base within the overhang of 23.
Cleaving Activity of the Zn2þ Complexes of Oligonucleotide Bis(azacrown)
Conjugates 20 and 21. It has been shown previously [6c] that the dinuclear Zn2þ
complex of bis(azacrown) monomer 3 cleaves oligoribonucleotide 5’-CAAUAC-3’ 32
times faster than oligoribonucleotide 5’-CAACAC-3’, the cleavage taking place
predominantly on both sides of the uridine residue [6c]. The rate constants referring
to 100 mm concentration of 3(Zn2þ)2 at pH 7.5 and 358 (I ¼ 0.1m) have been reported to
be (7.70 ꢁ 0.05) · 10ꢀ6 sꢀ1 and (0.24 ꢁ 0.03) · 10ꢀ6 sꢀ1, respectively. As discussed in more
detail in [6], the marked rate acceleration accompanying the replacement of the
intrachain cytidine with uridine may in all likelihood be attributed to anchoring of
3(Zn2þ)2 through one of the azacrown chelates to the uracil base, the other chelate
serving as the cleaving agent. Since oligonucleotide conjugate 21 in the presence of
Zn2þ clearly recognizes the uracil base within the overhang of target 23, one might
expect this anchoring to lead to enhanced, highly specific cleavage of the overhang at
the site of anchoring. However, this is not the case. Table 2 contains the rate constants

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for the cleavage of targets 22 – 27 by conjugates 20 and 21 in the presence of Zn2þ. On
the contrary, target 22 containing no uridine in the overhang was cleaved even more
efficiently than the uridine-containing targets. In other words, tethering of 3 to a
sequence recognizing oligonucleotide does not enhance but rather retards its uridine-
selective cleaving ability. Tentatively, one might speculate that the tethering of 3 results
in constrain that prevents 3 to interact simultaneously with the uracil base and the
adjacent phosphodiester linkage. This assumption is supported by the finding that even
slightly higher cleavage rates were observed when, instead of conjugate 20 of 21, a
mixture of unconjugated complementary oligonucleotide 28 and monomeric 3 was used
at [Zn2þ] of 90 mm to promote the reaction (Table 2). Accordingly, the situation
appears to be similar to that reported in [3c] for mono(azacrown) conjugates of
oligonucleotides: uracil base in the target sequence retards the cleavage by binding to
the cleaving agent. Consistent with this, the slower cleavage of target 23 compared to
target 24 probably results from the presence of guanosine next to uridine. It is known
[6b] that 3(Zn2þ)2 tends to interact with both bases in 3’-GpU-5’, although a monomeric
Zn2þ chelate binds to guanine base less firmly than to uracil base. Hence, both azacrown
groups on 20 or 21 may be largely involved in anchoring to 23.
Table 2. Rate Constants (k [10ꢀ6 sꢀ1]) for the Cleavage of Chimeric Oligonucleotides 22 – 27 by
Complementary Oligonucleotide Conjugates 20 and 21 at pH 7.3 and 358 (I ¼ 0.1m). The data refers to
[oligomer] ¼ [3] ¼ 18 mm and [Zn2þ] ¼ 90 mm at the ionic strength of 0.1m. pH was adjusted with a HEPES
buffer. Bold letters refer to ribonucleotides, the rest to 2’-O-methyl ribonucleotides.
Target
20
21
28 þ 3
3’-UGU GUC UGU GCG GAC AAC AA-5’ (22)
3’-UGU GUC UGU GCG GUA AAC AA-5’ (23)
3’-UGU GUC UGU GCG GAU AAC AA-5’ (24)
3’-UGU GUC UGU GCG GAA UAC AA-5’ (25)
3’-UGU GUC UGU GCG GAA AUC AA-5’ (26)
3’-UGU GUC UGU GCG GAA AAU AA-5’ (27)
2.44 ꢁ 0.07
1.09 ꢁ 0.04
2.70 ꢁ 0.10
0.39 ꢁ 0.04
1.66 ꢁ 0.08
0.19 ꢁ 0.03
0.44 ꢁ 0.03
0.25 ꢁ 0.02
0.82 ꢁ 0.05
0.79 ꢁ 0.08
0.8 ꢁ 0.1
0.9 ꢁ 0.1
4 ꢁ 1
0.5 ꢁ 0.1
0.8 ꢁ 0.1
1.5 ꢁ 0.2
Experimental Part
General. DMF and THF were dried over 3-ꢂ, and CH2Cl2, MeCN, and pyridine over 4-ꢂ molecular
sieves. Et3N was dried by distillation and storage over CaH2. NMR Spectra: Bruker Avance at 500 MHz.
The chemical shifts, d, in ppm from internal Me4Si and the coupling constants J in Hz; appropriate 2D-
NMR methods (COSY, HSQC, and HMBC) used for peak assignment. ESI-MS: in m/z.
Dimethyl 5-(Allyloxy)isophthalate (¼ Dimethyl 5-(Prop-2-en-1-yloxy)benzene-1,3-dicarboxylate; 4).
Dimethyl 5-hydroxyisophthalate (¼dimethyl 5-hydroxybenzene-1,3-dicarboxylate; 4.9 g, 23 mmol),
EtNiPr2 (8.2 ml, 46 mmol), and allyl bromide (5.6 g, 46 mmol) were dissolved in DMF (5.0 ml), and the
mixture was stirred at r.t. for 48 h. H2O (50 ml) was added, and the product was extracted with Et2O (3 ꢂ
30 ml). The combined org. layers were washed with sat. NaHCO3, dried (Na2SO4), and evaporated to
dryness. The crude product was purified by silica-gel column chromatography (CC) to yield 4 (5.0 g,
86%). Colorless solid flakes. 1H-NMR (500 MHz, CDCl3): 8.29 (t, J ¼ 1.2, HꢀC(2)); 7.77 (d, J ¼ 1.3,
HꢀC(4), HꢀC(6)); 6.06 (m, CH2CH¼CH2); 5.46 (m, 1 H, CH2CH¼CH2); 5.34 (m, 1 H, CH2CH¼CH2);
4.64 (m, CH2CH¼CH2); 3.95 (s, 2 MeO). 13C-NMR (125 MHz, CDCl3): 166.1 (COOMe); 158.6 (C(5));
132.4 (CH2CH¼CH2); 131.7 (C(1), C(3)); 123.1 (C(2)); 120.1 (C(4), C(6)); 118.2 (CH2CH ¼ CH2); 69.2
(CH2CH¼CH2); 52.4 (MeO).

Helvetica Chimica Acta – Vol. 96 (2013)
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(5-Allyloxy-1,3-phenylene)dimethanol (¼ [5-(Prop-2-en-1-yloxy)benzene-1,3-diyl]dimethanol; 5).
LiAlH4 (0.93 g, 25 mmol) was slowly added to a mixture of 4 (2.1g, 8.3 mmol) in Et2O (25 ml) at 08.
The mixture was allowed to warm up and stirred for 3 h at r.t. The reaction was quenched by addition of
H2O (100 ml), and the product was extracted with AcOEt (3 ꢂ 50 ml). The combined org. layers were
washed with sat. aq. NaCl, dried (Na2SO4), and evaporated to dryness. The residue was purified by CC
1
(5% MeOH in CH2Cl2) to afford 5 (1.4 g, 88%). Colorless oil. H-NMR (500 MHz, CDCl3): 6.87 (s,
HꢀC(2)); 6.78 (s, HꢀC(4), HꢀC(6)); 6.05 (m, CH2CH¼CH2); 5.41 (m, 1 H, CH2CH¼CH2); 5.29 (m, 1 H,
CH2CH¼CH2); 4.55 (d, J ¼ 5.0, 2 CH2OH); 4.51 (m, CH2CH¼CH2); 3.08 (t, J ¼ 5.0, CH2OH). 13C-NMR
(125 MHz, CDCl3): 158.9 (C(5)); 142.8 (C(1), C(3)); 133.2 (CH2CH¼CH2); 117.74 (CH2CH¼CH2);
117.71 (C(2)); 112.3 (C(4), C(6)); 68.8 (CH2CH¼CH2); 64.8 (CH2OH).
[(5-Allyloxy-1,3-phenylene)bis(methylene)]bis(4-methylbenzenesulfonate) (¼ [5-(Prop-2-en-1-yloxy)-
benzene-1,3-diyl]dimethanediyl Bis(4-methylbenzenesulfonate); 6). TosCl (6.2 g, 33 mmol) in Et2O
(6.0 ml) was slowly added under stirring to a mixture of 5 (2.1 g, 11 mmol), NaOH (1.7 g, 43 mmol), H2O
(9.0 ml), and THF (5.0 ml) at 08. Stirring was continued for 4 h at 08, sat. aq. NaHCO3 was added, and
then the crude product was extracted with AcOEt (3 ꢂ 50 ml). The combined org. layers were dried
(Na2SO4) and evaporated to dryness. CC (3% MeOH in CH2Cl2) of the residue yielded 3.4 g (62%) of 6
as colorless oil and 0.76 g of incompletely tosylated diol (i.e., monotosylated 5 as colorless oil).
Data for 6. 1H-NMR (500 MHz, CDCl3): 7.79 (m, 4 arom. H, Tos); 7.34 (m, 4 arom. H, Tos); 6.74 (m,
HꢀC(4), HꢀC(6)); 6.69 (m, HꢀC(2)); 5.99 (m, CH2CH¼CH2); 5.39 (m, 1 H, CH2CH¼CH2); 5.30 (m,
1 H, CH2CH¼CH2); 4.96 (s, 2 CH2OH); 4.44 (m, CH2CH¼CH2); 2.46 (s, 2 Me). 13C-NMR (125 MHz,
CDCl3): 158.9 (C(5)); 145.1 (C(1), C(3)); 135.3 (Tos)); 133.1 (Tos)); 132.6 (CH2CH¼CH2); 129.9 (Tos));
128.0 (Tos)); 120.4 (CH2CH¼CH2); 118.0 (C(2)); 115.1 (C(4), C(6)); 71.2 (CH2CH¼CH2); 68.9
(CH2OH); 21.7 (Me).
Hexa(tert-butyl) 3,3’-{{[5-(2-Oxoethoxy)-1,3-phenylene]bis(methylene)}bis(oxy)}bis(1,5,9-triazacy-
clododecane-1,5,9-tricarboxylate) (¼ Hexa(tert-butyl) 3,3’-{[5-(2-Oxoethoxy)benzene-1,3-diyl]bis(me-
thanediyloxy)}bis(1,5,9-triazacyclododecane-1,5,9-tricarboxylate); 9). NaH (39 mg of 60% dispersion
in mineral oil, 0.97 mmol) was added to a mixture of 6 (0.15 g, 0.30 mmol), tri(tert-butyl) 3-hydroxy-
1,5,9-triazacyclododecane-1,5,9-tricarboxylate (7; 0.31 g, 0.64 mmol) and DMF. The mixture was stirred
for 1 h at r.t., the reaction was quenched by slow addition of MeOH (0.5 ml) and H2O (20 ml), and the
product was extracted with Et2O (6 ꢂ 15 ml). The org. fractions were combined, dried (Na2SO4), and
evaporated to dryness. The residue was purified by CC (40% AcOEt in CH2Cl2) to yield 0.28 g (82%) of
hexa(tert-butyl) 3,3’-{{[5-(allyl)oxy-1,3-phenylene]bis(methylene)}bis(oxy)}bis(1,5,9-triazacyclodode-
cane-1,5,9-tricarboxylate) (¼ hexa(tert-butyl) 3,3’-{[5-(prop-2-en-1-yloxy)benzene-1,3-diyl]bis(methane-
diyloxy)}bis(1,5,9-triazacyclododecane-1,5,9-tricarboxylate); 8) as colorless oil.
Compound 8 (0.40 g, 0.35 mmol) was dissolved in a mixture of 1,4-dioxane (4.0 ml) and H2O
(1.0 ml), and a soln. of OsO4 in tBuOH (1:39 (w/w); 56 ml, 4.4 mmol) was added. The mixture was stirred
for 30 min at r.t., cooled to 08, a soln. of NaIO4 (0.17 g, 0.80 mmol) in H2O (1.0 ml) was added, and stirring
was continued for 2 h. Sat. aq. NaHCO3 (10 ml) was added to the mixture, and the product was extracted
with AcOEt (3 ꢂ 5 ml). The combined org. layers were evaporated to dryness, and the residue was
1
subjected to a CC (3% MeOH in CH2Cl2) to yield 9 (0.30 g, 74%). White foam. H-NMR (500 MHz,
CDCl3): 9.85 (s, CH2CH¼O); 6.85 (m, HꢀC(2), HꢀC(4), HꢀC(6)); 4.62 (s, CH2CH¼O); 4.58 (s, 2
CH2OH); 4.20 – 2.90 (m, 26 H, azacrown); 2.02 (m, 4 H, azacrown); 1.77 (m, 4 H, azacrown); 1.55 (s,
18 Me). 13C-NMR (125 MHz, CDCl3): 199.1 (CH2CH¼O); 158.1 (C(5)); 156.3 (Boc); 156.0 (Boc); 140.5
(C(1), C(3)); 119.7 (C(2)); 112.8 (C(4), C(6)); 80.0 (CH2CH¼O); 79.7 (Boc); 75.6 (azacrown); 72.7
(CH2O); 71.3, 53.4, 49.3, 47.0, 44.9, 29.5 (azacrown); 28.4 (Boc).
3,3’-{{[5-(2-Hydroxyethoxy)-1,3-phenylene]bis(methylene)}bis(oxy)}bis(1,5,9-triazacyclododecane-
1,5,9-triyl)tris(2,2,2-trifluoroethanone) (¼1,1’,1’’,1’’’,1’’’’,1’’’’’-{[5-(2-Hydroxyethoxy)benzene-1,3-diyl]-
bis(methanediyloxy-1,5,9-triazacyclododecane-3,1,5,9-tetrayl)}hexakis(trifluoroethanone); 11). Aldehyde
9 0.30 g (0.26 mmol) was dissolved in a mixture of CH2Cl2 (3.0 ml) and EtOH (4.0 ml), and NaBH4
(27 mg, 0.71 mmol) was added to the mixture. The mixture was stirred for 4 h at r.t., the reaction was
quenched by addition of sat. aq. NH4Cl (10 ml), and the mixture was extracted with CH2Cl2 (4 ꢂ 10 ml).
The combined org. fractions were dried (Na2SO4), evaporated to dryness, and the residue was subjected
to CC (5% MeOH in CH2Cl2) to yield 0.25 g (85%) of hexa(tert-butyl) 3,3’-{{[5-(2-hydroxyethoxy)-1,3-

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Helvetica Chimica Acta – Vol. 96 (2013)
phenylene]bis(methylene)}bis(oxy)}bis(1,5,9-triazacyclododecane-1,5,9-tricarboxylate) (¼ hexa(tert-bu-
tyl) 3,3’-{[5-(2-hydroxyethoxy)benzene-1,3-diyl]bis(methanediyloxy)}bis(1,5,9-triazacyclododecane-
1,5,9-tricarboxylate); 10). HR-ESI-MS: 1137.7235 ([M þ H]þ, C58H101N6O1þ6 ; calc. 1137.7274).
TfaOH (1.0 ml) was added to 10 (0.25 g) in CH2Cl2 (1.0 ml), and the mixture was stirred overnight at
r.t. and evaporated to dryness. The residue was co-evaporated with H2O, MeOH, and pyridine, dissolved
in MeOH (1.0 ml), and Et3N (0.33 ml, 2.4 mmol) and TfaOMe (0.24 ml, 2.4 mmol) were added. The
mixture was stirred overnight at r.t. and evaporated to dryness. Sat. aq. NaHCO3 (10 ml) was added, and
the incompletely N-trifluoroacetylated products were extracted with AcOEt (3 ꢂ 5 ml). The combined
org. layers were dried (Na2SO4), evaporated to dryness, and the residue was dissolved in a mixture of
CH2Cl2 (1.0 ml) and pyridine (0.1 ml). (Tfa)2O (0.17 ml, 1.2 mmol) was added, and the mixture was
stirred overnight at r.t. and evaporated to dryness. To expose the trifluoroacetylated OH group, the
residue was dissolved in MeOH (1.0 ml) containing Et3N (50 ml), mixed for 5 h, and evaporated to
dryness. NaHCO3 (10 ml) was added to the residue, and the product was extracted with AcOEt (3 ꢂ
5 ml). The org. layers were combined, dried (Na2SO4), and evaporated to dryness. The crude product
was purified by CC (40% AcOEt in CH2Cl2) to yield 11 (0.16 g, 54%). White foam. 1H-NMR (500 MHz,
CDCl): 6.67 – 6.70 (m, HꢀC(2), HꢀC(4), HꢀC(6)); 4.49 – 4.54 (m, 2 Ar-CH2O); 3.86 – 4.16 (m,
CH2CH2OH, 2 HꢀC(3’)); 2.45 – 3.86 (m, 24 H, azacrown); 2.04 – 2.44 (m, 4 H, azacrown); 1.54 – 2.01
(m, 4 H, azacrown). HR-ESI-MS: 1135.2849 ([M þ Na]þ, C40H46F18N6NaOþ10 ; calc. 1135.2886.
3,3’-{{{5-(2-[(2-Cyanoethoxy)(diisopropylamino)phosphinooxy]ethoxy}-1,3-phenylene}bis(methyl-
ene)}bis(oxy)}bis(1,5,9-triazacyclododecane-1,5,9-triyl)tris(2,2,2-trifluoroethanone) (¼2-[3,5-Bis({[1,5,9-
tris(trifluoroacetyl)-1,5,9-triazacyclododecan-3-yl]oxy}methyl)phenoxy]ethyl 2-Cyanoethyl Dipropan-2-
ylphosphoramidoite; 12). 2-Cyanoethyl N,N-diisopropylaminophosphoro chloridite (¼ 3-[chloro-
(diisopropylamino)phosphinooxy]propanenitrile ¼ 2-cyanoethyl dipropan-2-ylphosphoramidochlori-
doite; 21 ml, 94 mmol) was added to a mixture of 11 (80 mg, 72 mmol), Et3N (50 ml, 0.36 mmol), and
CH2Cl2 (1.0 ml) under N2. The mixture was stirred for 1 h at r.t. and then directly subjected to CC.
1
Elution with Et3N/AcOEt/petroleum ether 5 :70 :25 yielded 12 (80 mg, 85%). White foam. H-NMR
(500 MHz, CD3CN:) 6.82 (m, HꢀC(2), HꢀC(4), HꢀC(6)); 4.53 – 4.61 (m, 2 ArꢀCH2O); 3.89 – 4.29 (m,
OCH2CH2O, OCH2CH2CN, 2 HꢀC(3’)); 2,97 – 3.89 (m, 26 H, azacrown, 2 NCHMe2); 2.67 – 2.78 (m,
OCH2CH2CN); 1.76 – 2.46 (m, 8 H, azacrown); 1.19 – 1.26 (m, 4 Me). 31P-NMR (200 MHz, CD3CN):
148.5. HR-ESI-MS: 1335.4007 ([M þ Na]þ, C49H63F18N8NaO11Pþ; calc. 1335.3964).
2-[(2R,4S,5R)-5-{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-{[(tert-butyl)(dimethyl)silyl]-
oxy}tetrahydrofuran-2-yl]-N-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}acetamide (15). Ethyl [(2R,4S,5R)-5-
{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-{[(tert-butyl)(dimethyl)silyl]oxy}tetrahydrofuran-
2-yl]acetate (14; 0.44 g, 0.71 mmol) [7] was dissolved in 1,4-dioxane (15 ml) and aq. KOH (0.20 g,
3.5 mmol in 5 ml of H2O) was added. The mixture was stirred overnight at r.t. Solvent was removed in
vacuo, the residue was dissolved in pyridine (15 ml), and the soln. was stirred with a Dowex-50 resin
(pyridinium form) for 5 h. The resin was filtered off, pyridine was evaporated, and, after co-evaporation
with dry pyridine, the residue was dissolved in dry DMF (2 ml). EtNiPr2 (0.15 ml, 0.85 mmol), HBTU
(0.32 g, 0.85 mmol), and 2-[2-(2-aminoethoxy)ethoxy]ethanol (0.15 g, 0.85 mmol) were added, and the
mixture was strirred for 2.5 h at r.t. DMF was removed by evaporation, the residue was dissolved in Et2O,
washed with sat. NaHCO3, dried (Na2SO4), and evaporated to dryness. Purification by CC (0.1% Et3N
and 3% MeOH in CH2Cl2) yielded 15 (0.33 g, 65%). Yellowish oil. 1H-NMR (500 MHz, CDCl3): 7.23 –
7.46 (m, 9 H, DMTr); 6.85 (d, J ¼ 8.8, 4 H, DMTr); 6.78 (t, J ¼ 5.5, NH); 4.46 – 4.49 (m, HꢀC(5)); 4.25 –
4.26 (m, HꢀC(2)); 3.95 – 3.97 (m, HꢀC(4)); 3.81 (s, 6 H, DMTr); 3.42 – 3.69 (m, NHCH2CH2(OCH2-
CH2)2OH); 3.14 (dd, J ¼ 9.9, 4.5, 1 H, C(5)ꢀCH2O); 3.09 (dd, J ¼ 9.9, 5.3, 1 H, C(5)ꢀCH2O); 2.53 (dd,
J ¼ 15.1, 3.8, 1 H, C(2)ꢀCH2C¼O); 2.46 (dd, J ¼ 15.1, 8.1, 1 H, C(2)ꢀCH2C¼O); 1.91 (ddd, J ¼ 12.8, 5.2,
t
1.3, 1 H, CH2(3)); 1.71 (m, 1 H, CH2(3)); 0.86 (s, Bu); 0.03 (s, MeSi); 0.01 (s, MeSi). 13C-NMR
(125 MHz, CDCl3): 171.0 (C¼O); 158.5, 144.8, 136.1, 130.1, 128.2, 127.8, 126.8, 113.1, 86.9 (DMTr); 86.1
(C(5)); 75.1 (C(4)); 74.1 (C(2)); 72.5, 70.3, 70.2, 69.9 (NHCH2CH2OCH2CH2OCH2CH2OH); 64.1
(C(5)ꢀCH2O); 61.7 (OCH2CH2OH); 55.2 (DMTr); 42.5 (C(2)); 41.1 (C(2)ꢀCH2C¼O); 39.1
(NCH2CH2O); 25.8 (TBDMS); 18.0 (TBDMS); ꢀ 4.7 (TBDMS); ꢀ 4.8 (TBDMS). HR-ESI-MS:
746.3642 ([M þ Na]þ, C40H57NNaO9Siþ; calc. 746.3695).

Helvetica Chimica Acta – Vol. 96 (2013)
41
2-{2-[2-({[(2R,4S,5R)-5-{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-{[(tert-butyl)(dimeth-
yl)silyl]oxy}tetrahydrofuran-2-yl]acetyl}amino)ethoxy]ethoxy}ethyl 4-Oxopentanoate (16). Levulinic
anhydride ((Lev)2O; 0.29 g, 1.4 mmol) was added to a soln. of 15 (0.33 g, 0.46 mmol) in pyridine. The
mixture was stirred overnight at r.t. and evaporated to dryness. The residue was dissolved in CH2Cl2 and
washed with sat. aq. NaHCO3. The org. phase was dried (Na2SO4) and evaporated to dryness. The crude
product was purified by CC (0.1% Et3N and 3% MeOH in CH2Cl2) to yield 16 (0.32 g, 85%). 1H-NMR
(500 MHz, CDCl3): 7.21 – 7.45 (m, 9 H, DMTr); 6.84 (d, J ¼ 8.9, 4 H, DMTr); 6.69 (t, J ¼ 5.5, NH); 4.43 –
4.50 (m, HꢀC(5)); 4.25 – 4.26 (m, HꢀC(2)); 4.21 (t, J ¼ 4.8, OCH2CH2OLev); 3.94 – 3.96 (m, HꢀC(4));
3.81 (s, 2 MeO); 3.43 – 3.64 (m, NHCH2CH2OCH2CH2OCH2CH2OLev); 3.12 (dd, J ¼ 9.9, 4.5, 1 H,
C(5)ꢀCH2O); 3.07 (dd, J ¼ 9.9, 5.3, 1 H, C(5)ꢀCH2O); 2.75 (t, J ¼ 6.5, 2 H, Lev); 2.60 (t, J ¼ 6.5, 2 H,
Lev); 2.52 (dd, J ¼ 15.1, 4.0, 1 H, C(2)ꢀCH2C¼O); 2.45 (dd, J ¼ 15.1, 8.1, 1 H, C(2)ꢀCH2C¼O); 2.20 (s,
3 H, Lev); 1.89 – 1.93 (m, 1 H, CH2(3)); 1.67 – 1.73 (m, 1 H, CH2(3)); 0.86 (s, tBu); 0.02 (s, MeSi); 0.01 (s,
MeSi). 13C-NMR (125 MHz, CDCl3): 206.6 (Lev); 172.7 (Lev); 170.9 (CH2C(O)NH); 158.5, 144.8, 136.0,
130.1, 128.2, 127.8, 126.8, 113.1, 86.9 (DMTr); 86.0 (C(5)); 75.1 (C(4)); 74.1 (C(2)); 70.5, 70.2, 69.9, 69.0
(NHCH2CH2OCH2CH2OCH2CH2OLev); 64.1 (OCH2CH2OLev); 63.7 (C(5)ꢀCH2O); 55.2 (DMTr);
42.6 (C(3)); 41.3 (C(2)ꢀCH2C¼O); 39.2 (NHCH2CH2O); 37.9 (Lev); 29.9 (Lev); 27.9 (Lev); 25.8
(TBDMS); 18.0 (TBDMS); ꢀ 4.6 (TBDMS); ꢀ 4.8 (TBDMS). HR-ESI-MS: 844.4068 ([M þ Na]þ,
C45H63NNaO11Siþ; calc. 844.4063).
2-{2-[2-({[(2R,4S,5R)-5-{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-hydroxytetrahydrofur-
an-2-yl]acetyl}amino)ethoxy]ethoxy}ethyl 4-Oxopentanoate (17). Bu4NF · H2O (0.30 g, 1.1 mmol) was
added to a soln. of 16 (0.31 g, 0.38 mmol) in THF (3 ml). The mixture was stirred for 2 h at r.t., and then
volatiles were removed. The residue was dissolved in CH2Cl2 and washed with H2O. The org. phase was
dried (Na2SO4) and evaporated to dryness. The crude product was purified by CC (0.1% Et3N and 3%
1
MeOH in CH2Cl2) to yield 17 (0.21 g, 78%). H-NMR (500 MHz, CDCl3): 7.23 – 7.46 (m, 9 H, DMTr);
6.84 (d, J ¼ 8.9, 4 H, DMTr); 6.68 (t, J ¼ 5.5, NH); 4.46 – 4.53 (m, HꢀC(5)); 4.33 – 4.34 (m, HꢀC(2)); 4.21
(t, J ¼ 4.8, OCH2CH2OLev); 3.98 – 4.00 (m, HꢀC(4)); 3.81 (s, 6 H, DMTr); 3.44 – 3.65 (m,
NHCH2CH2OCH2CH2OCH2CH2OLev); 3.24 (dd, J ¼ 9.7, 4.7, 1 H, C(5)ꢀCH2O); 3.11 (dd, J ¼ 9.7, 5.7,
1 H, C(5)ꢀCH2O); 2.75 (t, J ¼ 6.5, 2 H, Lev); 2.60 (t, J ¼ 6.5, 2 H, Lev); 2.41 – 2.54 (m, C(2)ꢀCH2C¼O);
2.19 (s, 3 H, Lev); 2.04 (ddd, J ¼ 15.1, 5.6, 2.0, 1 H, CH2(3)); 1.81 – 1.87 (m, 1 H, CH2(3)). 13C-NMR
(125 MHz, CDCl3): 206.8 (Lev); 172.8 (Lev); 170.7 (CH2C(O)NH); 158.5, 144.8, 136.0, 130.1, 128.2,
127.8, 126.8, 113.1, 86.2 (DMTr); 86.2 (C(5)); 75.0 (C(4)); 74.2 (C(2)); 70.5, 70.3, 69.8, 69.0
(NHCH2CH2OCH2CH2OCH2CH2OLev); 64.5 (OCH2CH2OLev); 63.7 (C(5)ꢀCH2O); 55.2 (DMTr);
42.6 (C(3)); 40.8 (C(2)ꢀCH2C¼O); 39.2 (NHCH2CH2O); 37.9 (Lev); 29.9 (Lev); 27.9 (Lev). HR-ESI-
MS: 730.3355 ([M þ Na]þ, C39H49NNaO1þ1 ; calc. 730.3198).
2-{2-[2-({[(2R,4S,5R)-5-{[Bis(4-methoxyphenyl)(phenyl)methoxy]methyl}-4-({(2-cyanoethoxy)[di-
(propan-2-yl)amino]phosphanyl}oxy)tetrahydrofuran-2-yl]acetyl}amino)ethoxy]ethoxy}ethyl 4-Oxopen-
tanoate (19). 2-Cyanoethyl N,N-diisopropylaminophosphorochloridite (30 ml, 0.14 mmol) was added to a
mixture of 17 (74 mg, 0.10 mmol) and Et3N (73 ml, 0.52 mmol) in CH2Cl2 (0.5 ml) under N2. The mixture
was stirred for 1 h at r.t. and then subjected directly to CC (5% Et3N in AcOEt) to give 19 (48 mg, 51%).
Colorless oil. 1H-NMR (500 MHz, CD3CN): 7.23 – 7.48 (m, 9 H, DMTr); 6.86 – 6.89 (m, 4 H, DMTr); 6.62
(t, J ¼ 4.4, NH); 4.37 – 4.41 (m, HꢀC(2), HꢀC(5)); 4.12 (t, J ¼ 4.7, OCH2CH2OLev); 3.95 – 4.02 (m,
HꢀC(4)); 3.77 (s, 6 H, DMTr); 3.28 – 3.76 (m, NHCH2CH2OCH2CH2OCH2CH2OLev, OCH2CH2CN);
3.10 – 3.16 (m, 1 H, C(5)ꢀCH2O); 3.02 – 3.05 (m, 1 H, C(5)ꢀCH2O); 2.70 (t, J ¼ 6.4, 2 H, Lev); 2.62 (m,
2 NCHMe2); 2.52 (t, J ¼ 6.4, 2 H, Lev); 2.48 (m, OCH2CH2CN); 2.42 – 2.40 (m, 1 H, C(2)ꢀCH2C¼O);
2.15 – 2.04 (m, 1 H, C(2)ꢀCH2C¼O); 2.10 (s, 3 H, Lev); 1.98 – 1.94 (m, 1 H, CH2(3)); 1.78 – 1.85 (m, 1 H,
CH2(3)); 1.05 – 1.25 (m, 2 NCHMe2). 13C-NMR (125 MHz, CD3CN) 206.8 (Lev); 172.6 (Lev); 170.1
(CH2C(O)NH); 158.7, 145.3, 136.1, 130.1, 128.1, 127.8, 126.8, 113.1, 85.9 (DMTr); 85.5 (C(5)); 75.6 (C(4));
75.1 (C(2)); 70.2, 69.9, 69.3, 68.7 (NHCH2CH2OCH2CH2OCH2CH2OLev); 64.2 (OCH2CH2OLev); 63.5
(C(5)ꢀCH2O); 60.0 (OCH2CH2CN); 54.9 (MeO); 43.0 (NCHMe2); 42.9 (C(3)); 41.9 (C(2)ꢀCH2C¼O);
39.6 (NHCH2CH2O); 38.8 (Lev); 29.0 (Lev); 27.7 (Lev); 23.9 (CHMe2); 20.1 (OCH2CH2CN). 31P-NMR
(200 MHz, CD3CN) 147.3, 147.2. HR-ESI-MS: 930.4545 ([M þ Na]þ, C48H66N3NaO12Pþ; calc. 930.4276).
Synthesis of Oligonucleotide Conjugates 20 and 21. Oligonucleotide conjugates 20 and 21 were
assembled by the phosphoramidite chemistry on a commercially available support bearing DMTr-

42
Helvetica Chimica Acta – Vol. 96 (2013)
protected N4-benzoyl-2’-O-methylcytidine, 8 (1.0 mmol) as the 3’-terminal nucleoside. The DMTr group
was manually removed with 3% Cl2CHCOOH in CH2Cl2, and the deprotected support was washed with
CH2Cl2 and MeCN and dried. The non-nucleosidic building block, either 18 or 19 (10 mmol in 100 ml of
MeCN) was coupled manually using 5-(benzylthio)tetrazole (50 ml, 0.25m in MeCN) as activator and
0.5 h coupling time. Another portion of activator was added, and the reaction was allowed to proceed for
another 0.5 h. The support was washed with MeCN, CH2Cl2, and THF, and then subjected to the capping
and oxidation steps according to the standard RNA coupling protocol. The Lev protecting groups of the
support-bound non-nucleosidic block were removed with 0.5m H2NNH3OAc in pyridine (NH2NH2 · H2O/
pyridine/AcOH 0.124 :4 :1, 0.5 h). The support was washed with pyridine and MeCN, and dried. The
azacrown building block 12 (11 mg, 8.6 mmol in 33 ml of MeCN) was then coupled manually using 5-
(benzylthio)tetrazole (50 ml, 0.25m in MeCN) as activator. The mixture was kept 2 h at r.t., and then
another portion of activator was added. After 1 h, the support was washed with MeCN, CH2Cl2, and THF,
and subjected to the capping and oxidation steps according to the standard RNA coupling protocol. The
2’-O-methyl oligonucleotide sequence was then assembled on an Applied Biosystems 3400 DNA
synthesizer in 1.0-mmol scale applying the conventional phosphoramidite chemistry and the standard
RNA coupling protocol. The fully protected 2’-O-methyl oligoribonucleotide conjugates 20 and 21 were
released from the support and deprotected with conc. aq. NH3 (5 h at 558). The crude product mixture
was filtered, and the filtrate was evaporated to dryness, dissolved in H2O, and then subjected to RP-
HPLC purification. The authenticity of the conjugate was verified by ESI-MS spectroscopy. 20: ESI-HR-
MS: 1808.0987 ([M ꢀ 3 H]3ꢀ), giving 5426.31 ([M ꢀ H]ꢀ; calc. 5426.30). 21: ESI-HR-MS: 1857.2 ([M ꢀ
3 H]3ꢀ), 1392.6 ([M ꢀ 4 H]4ꢀ), 1113.9 ([M ꢀ 5 H]5ꢀ), giving 5573.5 ([M ꢀ H]ꢀ; calc. 5573.4).
Chimeric 2’-O-methyl oligoribonulceotides/oligoribonucleotides 22 – 27, used as targets for the
artificial ribonucleases 20 and 21, and 2’-O-methyl oligoribonucleotide 28, used as a reference material
for the melting-temperature (Tm) studies, were assembled from commercially available 2’-O-methyl and
2’-O-[(triisopropylsilyloxy)methyl]-protected 2-cyanoethyl N,N-diisopropylphosphoramidite building
blocks (Glen Research) by the conventional phosphoramidite strategy using a 1.0-mmol scale and
applying the standard RNA coupling protocol of Applied Biosystems 392 or 3400 DNA synthesizer. All
buffer solns. were prepared in sterilized H2O, and sterilized equipment was used for their handling.
Tm Studies. The melting curves (absorbance vs. temp.) were recorded at 260 nm on a Perkin-Elmer
Lambda 35 UV/VIS spectrometer equipped with a multiple cell holder and a Peltier temp. controller. The
temp. was changed at a rate of 0.58/min (from 15 to 908). The recordings were performed in 10 mm
potassium phosphate buffer (pH 7) containing 0.1m NaCl. The oligonucleotide conjugates 20 and 21,
reference oligonucleotide 28, and their targets 22 – 27 were used at a concentration of 2 mm. The Tm
values were determined as the maximum of the first derivative of the melting curve.
Kinetic Measurements. The reactions were carried out in Eppendorf tubes immersed in a water bath,
the temp. of which was kept at 35.0 ꢁ 0.18. The pH was adjusted to 7.3 with a HEPES buffer (0.1m), and
the Zn2þ ion was added as a nitrate salt to give the total metal ion concentration of 90 mm. The ionic
strength was adjusted to 0.1m with NaNO3. The concentrations of the azacrown-functionalized
oligonucleotides and their targets were 18 mm. 4-Nitrobenzenesulfonate ion was used as an internal
standard. The total volume of the reaction mixture was 200 ml in each kinetic run. Aliquots of 20 ml were
withdrawn at suitable intervals, and the reaction was quenched by adding aq. HCl (1.0 ml of 1.0m soln.).
The samples were analyzed immediately by cap. zone electrophoresis (Beckman Coulter P/ACE MDQ
CE System) using a fused silica capillary (inner diameter 50 mm, effective length 50 cm). The inverted
polarity, citrate buffer (0.2m, pH 3.1), and ꢀ 30 kV voltage were used. The temp. of the capillary was kept
at 258. The samples were injected using hydrodynamic injection with 2 psi for 8 s. The capillary was
flushed for 3 min with H2O, 10 mm aq. HCl, and the background electrolyte buffer between every anal.
run. The quantification of the target and product oligonucleotides was based on comparison of their UV
absorption at 254 nm to that of the internal standard. The peak area was first normalized by dividing it by
the migration time and then by the similarly normalized peak area of the internal standard. First-order
rate constants for the cleavage of the target oligonucleotides were calculated by applying the integrated
first-order rate law to the disappearance of the starting material.

Helvetica Chimica Acta – Vol. 96 (2013)
43
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Received March 26, 2012