Delivery of floxuridine derivatives to cancer cells by water-soluble organometallic cages

Article abstract of DOI:10.1021/bc200472n

The self-assembly of 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine (tpt) triangular panels with p-cymene (pPrⁱC₆H₄Me) ruthenium building blocks and 2,5-dioxydo-1,4-benzoquinonato (dobq) or 5,8-dioxydo-1,4-naphthoquinonato (donq) bridges, in the presence of a pyrenyl-nucleoside derivatives (pyreneR), affords the triangular prismatic host-guest compounds [(pyrene-R)?Ru₆(pPrⁱC ₆H₄Me)₆(tpt)₂(dobq) ₃]⁶⁺ ([(pyrene-R)?1]⁶⁺) and [(pyrene-R)?Ru₆(pPrⁱC₆H₄Me) ₆(tpt)₂(donq)₃]⁶⁺ ([(pyrene-R)?2]⁶⁺), respectively. The inclusion of six monosubstitutedpyrenyl-nucleosides (pyrene-R1 = 5′-(1-pyrenyl butanoate)-2′-deoxyuridine, pyrene-R2 = 5-fluoro-5′-(1-pyrenyl butanoate)-2′-deoxyuridine, pyrene-R3 = 5′-{N-[1-oxo-4-(1-pyrenyl) butyl]-glycyl}-2′-deoxyuridine, pyrene-R4 = 5-fluoro-5′-{N-[1-oxo-4- (1-pyrenyl)butyl]-glycyl}-2′-deoxyuridine, pyrene-R5 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl} -2′-deoxyvuridine, pyrene-R6 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl) butyl]-phenylalanyl}-2′-deoxyuridine) has been accomplished. The carceplex nature of [(pyrene-R)?1]⁶⁺ with the pyrenyl moiety firmly encapsulated in the hydrophobic cavity of the cage with the nucleoside groups pointing outward was confirmed by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS), while the host-guest nature of [(pyrene-R)?2] ⁶⁺ was studied in solution by NMR techniques. In contrast to the floxuridine compounds used in the clinic, the host-guest complexes are highly water-soluble. Consequently, the cytotoxicities of these water-soluble compounds have been established using human ovarian A2780 and A2780cisR cancer cells. All the host-guest systems are more cytotoxic than the empty cages alone [1][CF₃SO₃]₆ (IC₅₀ = 23 μM) and [2][CF₃SO₃]₆ (IC₅₀ = 10 μM), the most active compound [pyrene-R4?1][CF₃SO₃] ₆being 2 orders of magnitude more cytotoxic (IC₅₀ = 0.3 μM) on these human ovarian cancer cell lines (A2780 and A2780cisR).

Full text of DOI:10.1021/bc200472n

Article
pubs.acs.org/bc
Delivery of Floxuridine Derivatives to Cancer Cells by Water-Soluble
Organometallic Cages
Jeong Wu Yi,† Nicolas P. E. Barry,‡ Mona A. Furrer,‡ Olivier Zava,§ Paul J. Dyson,§ Bruno Therrien,*,‡
and Byeang Hyean Kim*,†
†Department of Chemistry, Division of IBB, Pohang University of Science and Technology, Pohang 790-784, Korea
‡Institut de Chimie, Universite
§Institut des Sciences et Ingen
́
de Neuchat
̂
el, 51 Ave de Bellevaux, 2000 Neuchat
̂
el, Switzerland
́
ierie Chimique, Ecole Polytechnique Fed
́ ́
erale de Lausanne (EPFL), 1015 Lausanne, Switzerland
ABSTRACT: The self-assembly of 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine (tpt) trian-
gular panels with p-cymene (pPriC6H4Me) ruthenium building blocks and 2,5-
dioxydo-1,4-benzoquinonato (dobq) or 5,8-dioxydo-1,4-naphthoquinonato (donq)
bridges, in the presence of a pyrenyl-nucleoside derivatives (pyreneR), affords the
triangular prismatic host−guest compounds [(pyrene-R)⊂Ru6(pPriC6H4Me)6-
(tpt)2(dobq)3]6+ ([(pyrene-R)⊂1]6+) and [(pyrene-R)⊂Ru6(pPriC6H4Me)6-
(tpt)2(donq)3]6+ ([(pyrene-R)⊂2]6+), respectively. The inclusion of six monosubstituted
pyrenyl-nucleosides (pyrene-R1 = 5′-(1-pyrenyl butanoate)-2′-deoxyuridine, pyrene-R2 =
5-fluoro-5′-(1-pyrenyl butanoate)-2′-deoxyuridine, pyrene-R3 = 5′-{N-[1-oxo-4-(1-pyrenyl)-
butyl]-glycyl}-2′-deoxyuridine, pyrene-R4 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-
2′-deoxyuridine, pyrene-R5 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′-deoxy-
vuridine, pyrene-R6 = 5-fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′-deoxyuridine)
has been accomplished. The carceplex nature of [(pyrene-R)⊂1]6+ with the pyrenyl
moiety firmly encapsulated in the hydrophobic cavity of the cage with the nucleoside groups pointing outward was confirmed by
NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS), while the host−guest nature of [(pyrene-R)⊂2]6+
was studied in solution by NMR techniques. In contrast to the floxuridine compounds used in the clinic, the host−guest
complexes are highly water-soluble. Consequently, the cytotoxicities of these water-soluble compounds have been established
using human ovarian A2780 and A2780cisR cancer cells. All the host−guest systems are more cytotoxic than the empty cages
alone [1][CF3SO3]6 (IC50 = 23 μM) and [2][CF3SO3]6 (IC50 = 10 μM), the most active compound [pyrene-R4⊂1][CF3SO3]6
being 2 orders of magnitude more cytotoxic (IC50 = 0.3 μM) on these human ovarian cancer cell lines (A2780 and
A2780cisR).
Chart 1
INTRODUCTION
■
Synthetic nucleoside derivatives are commonly employed as anti-
viral and anticancer drugs. They include floxuridine, 5-fluorouracil,
fludarabine, cladribine, zidovudine, as well as others (Chart 1).1
5-Fluorouracil is a widely used antimetabolite for the treatment
of human solid tumors including colorectal and breast cancers.2
Similarly, 5-fluoro-2′-deoxyuridine (FdUrd, Floxuridine) is a
5-fluorouracil derivative known for its high antitumor activity
against metastases of cancer.3,4 Floxuridine has been utilized to
treat human solid tumors; however, its therapeutic effect is
limited by the efficiency of cellular uptake and bioavailability of
the drug. In order to overcome these limitations, various syn-
thetic approaches have been envisaged to enhance the cell per-
meability and cytotoxicity of floxuridine derivatives as potent
anticancer drugs.5−7
Pyrenyl-functionalized nucleic acids are popular conjugate
molecules in biological chemistry.8 Indeed, the pyrenyl moiety
fits perfectly between base pairs of DNA,9 possesses interesting
fluorescence properties,10 and functionalization of pyrene is
synthetically easy.11 Therefore, several derivatives of pyrenyl-
functionalized nucleic acids have been prepared in recent
years.12−17 However, despite such popularity and interest, low
Received: September 13, 2011
Revised: December 19, 2011
Published: January 22, 2012
© 2012 American Chemical Society
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Figure 1. Metalla-prisms [{Pt(acac)2}⊂Ru6(pPriC6H4Me)6(tpt)2(dobq)3]6+ ([Pt(acac)2⊂1]6+)15 and [guest⊂Ru6(pPriC6H4Me)6(tpt)2(donq)3]6+
([guest⊂2]6+).36
cell internalization remains one of the limitations of pyrenyl-
functionalized nucleic acids for biological applications.18
of the nucleoside on the biological activity together with the
ability of the metalla-prisms to deliver these hydrophobic mol-
ecules are discussed.
Ruthenium-based anticancer agents have attracted much
interest in recent years,19,20 and organometallic Ru(arene) com-
pounds are the latest trend.21−23 By modification of the ligand
sphere and linking more than one metal center, the properties
of the complexes can be altered to obtain desired biological
characteristics.24−26 Recently, new hybrid drug delivery systems
built from arene ruthenium complexes have been developed.27−30
These water-soluble organometallic metalla-cages are able to
encapsulate guest molecules. For example, the hexacationic prism
[Ru6(pPriC6H4Me)6(tpt)2(dobq)3]6+ ([1]6+) (tpt =2,4,6-tris-
(pyridin-4-yl)-1,3,5-triazine; dobq = 2,5-dioxydo-1,4-benzoqui-
nonato) was synthesized,31 which was capable of encapsulating
planar Pt and Pd acetylacetonate complexes (Figure 1) as
well as planar aromatic compounds of various sizes.32,33
Similarly, larger metalla-cages were synthesized such as
[Ru6(pPriC6H4Me)6(tpt)2(donq)3]6+ ([2]6+) (tpt = 2,4,6-tris-
(pyridin-4-yl)-1,3,5-triazine; donq = 5,8-dioxydo-1,4-naphthoquino-
nato) in which the guest molecule was free to escape from the
cavity of the host in solution, thus providing host−guest systems
(Figure 1).34,35 In these complexes the encapsulated guest is stable,
with the physical properties of the host being retained following
encapsulation. The biological activity of these systems has
been evaluated, and encouraging results were obtained. The
metalla-prisms exhibit some activity which increases with the
encapsulation of a guest, suggesting transport and leaching of
the guest once inside the cell. Indeed, fluorescence experi-
ments have been used to monitor the uptake of the fluorescent-
labeled pyrenyl derivative 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene
(pyrene-X) to cancer cells demonstrating an uptake of the
carceplex [pyrene-X⊂Ru6(pPriC6H4Me)6(tpt)2(dobq)3]6+ being 1
order of magnitude greater than that of pyrene-X alone.36
With a view to combine the drug delivery ability of the arene
ruthenium metalla-prisms with the biological properties of pyrenyl-
functionalized nucleic acids, a series of pyrenyl-nucleosides
(pyrene-R) including derivatives analogous to floxuridine
have been synthesized and encapsulated in the metalla-prisms
[1]6+ and [2]6+. The in vitro anticancer activity of these
water-soluble systems, [pyrene-R⊂1]6+ and [pyrene-R⊂2]6+,
was evaluated against human ovarian A2780 and A2780cisR
(acquired resistance to cisplatin) cancer cells. The implications
RESULTS AND DISCUSSION
■
The synthesis of the pyrenyl-nucleoside derivatives starts with
conjugation of the butyryl pyrene derivatives, N-[1-oxo-4-
(1-pyrenyl)butyl]-glycine ethyl ester and N-[1-oxo-4-
(1-pyrenyl)butyl]-phenylalanine ethyl ester, with O-protected
nucleosides. The butyryl pyrene derivatives conjugated with
glycine and phenylalanine are obtained using typical peptidic
coupling from 1-pyrene butyric acid. 3′-O-TBDMS-2′-deoxyur-
idine and 5-fluoro-3′-O-TBDMS-2′-deoxyuridine are prepared
according to a published procedure37 from the corresponding
unprotected nucleoside in two steps (Scheme 1).
The pyrenyl modified nucleosides are synthesized through an
esterification reaction between the butyl pyrenyl amino acids
and deprotected silyl group via in situ reaction using tetra-
butylammonium fluoride in THF (Scheme 2). These couplings
give six pyrenyl-nucleoside derivatives which are characterized by
1H and 13C{1H} NMR spectroscopy and mass spectrometry.
The encapsulation of the pyrenyl-nucleosides (pyrene-R1−6) in
the metalla-prism [1]6+ is performed in a two-step process, in
which the p-cymene ruthenium dobq (2,5-dioxydo-1,
4-benzoquinonato) dinuclear complex is first reacted with
silver triflate to produce a reactive intermediate, which is then
combined with a 2:1 mixture of tpt (2,4,6-tris(pyridin-4-yl)-
1,3,5-triazine) and the functionalized pyrenyl derivative
(Scheme 3), affording the corresponding carceplex systems
[(pyrene-R1−6)⊂1][CF3SO3]6. These metalla-prisms are isolated
in 69−73% yield and are characterized by 1H and 13C{1H} NMR
spectroscopy and electrospray ionization mass spectrometry.
The encapsulation of the pyrenyl-nucleosides (pyrene-R1−6) in
the larger metalla-prism [2]6+ is achieved following the same
strategy as for [(pyrene-R1−6)⊂1][CF3SO3]6 using the
p-cymene ruthenium donq (5,8-dioxydo-1,4-naphthoquinonato)
dinuclear complex (Scheme 4). The host−guest systems are
isolated as their triflate salts [(pyrene-R1−6)⊂1][CF3SO3]6 in
good yield.
Under the electrospray ionization mass spectrometry (ESI-MS)
conditions employed (see Experimental Section), all inclusion
complexes are remarkably stable. The ESI mass spectra of all
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Scheme 1. Synthesis of Butyryl Pyrene Conjugated Amino Acids
Scheme 2. Synthesis of Modified Pyrenyl-Nucleoside Derivatives (Pyrene-R1-6)
Scheme 3. Synthesis of the Carceplex Systems [(Pyrene-R1-6)⊂1]6+
carceplex systems [pyrene-R⊂1][CF3SO3]6 show peaks correspond-
ing to [pyrene-R + 1 + (CF3SO3)3]3+ at m/z 1132.1 (R1), 1137.8
(R2), 1151.5 (R3), 1157.5 (R4), 1181.5 (R5), and 1186.8 (R6),
respectively. Similarly, the ESI mass spectra of the host−guest
systems [pyrene-R⊂2][CF3SO3]6 show peaks corresponding to
[pyrene-R + 2 + (CF3SO3)3]3+ at m/z 1182.8 (R1), 1188.1 (R2),
1201.5 (R3), 1207.5 R4), 1231.5 (R5), and 1236.5 (R6), respec-
tively. These peaks are assigned unambiguously on the basis of
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Scheme 4. Synthesis of the Host-Guest Systems [(Pyrene-R1-6)⊂2]6+
Figure 2. ESI mass of the peak envelopes corresponding to [pyrene-R + 1 + (CF3SO3)3]3+ and [pyrene-R + 2 + (CF3SO3)3]3+.
their characteristic Ru6 isotope pattern (Figure 2). In addition
to these parent peaks [pyrene-R + 1 + (CF3SO3)3]3+ and [pyrene-
R + 2 + (CF3SO3)3]3+, a series of characteristic fragmentation
peaks corresponding to the cages are observed. A typical spectrum
is given in Figure 3, identifying the fragmentation peaks observed
for [pyrene-R5⊂1][CF3SO3]6. This fragmentation pattern has
been previously observed for related pyrene-R⊂cage systems.32,33
These systems are not only stable under ESI mass conditions,
they are stable in solution at room and elevated temperature
(45 °C) in D2O, as well as in biological media (aqueous
solution containing RPMI 1640 medium with 10% fetal calf
serum (FCS) and antibiotics), showing no leaching of the guest
molecules or breaking up of the cages. Consequently, the anti-
proliferative activity of the empty metalla-cages [1]6+, [2]6+
and all the inclusion systems [pyrene-R1−6⊂1]6+ and [pyrene-
R1−6⊂2]6+ was evaluated against the human ovarian A2780
and A2780cisR (acquired resistance to cisplatin) cancer cell
lines using the MTT assay, which measures mitochondrial
dehydrogenase activity as an indication of cell viability. The
IC50 values (IC50 is the drug concentration necessary for 50%
inhibition of cell viability) are listed in Table 1 and are reported
together with that of cisplatin for comparison purposes. It was
not possible to obtain IC50 values for the pyrene-R compounds,
i.e., in the absence of the metalla-cage, due to solubility problems
in biological media.
All pyrene-R⊂cage complexes show higher cytotoxicity than
their corresponding empty metalla-cages [1]6+ and [2]6+ on
both A2780 and A2780cisR cancer cells. Moreover, they show
activity comparable or superior to cisplatin with essentially the
same level of activity on both nonresistant and cisplatin
acquired resistance cell lines. In addition, as control experi-
ments, the antiproliferative activity of the two butyryl pyrene
derivatives N-[1-oxo-4-(1-pyrenyl)butyl]-glycine ethyl ester and
N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanine ethyl ester was eval-
uated, and both show IC50 values >200 μM when tested alone,
while the corresponding pyrenyl⊂cage systems possess IC50 values
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Figure 3. ESI mass spectrum of the [pyrene-R5⊂1][CF3SO3]6 system (clip = {Ru2(pPriC6H4Me)2(dobq)}2+).
Table 1. IC50 Values for [1]6+, [2]6+, [(Pyrene-R1-6)⊂1]6+,
EXPERIMENTAL SECTION
■
and [(Pyrene-R1-6)⊂2]6+ Determined on Human Ovarian
General. [Ru2(pPriC6H4Me)2(dobq)Cl2],31 [Ru2-
(pPriC6H4Me)2(donq)Cl2],34 and 2,4,6-tris(4-pyridyl)-1,3,5-
triazine38,39 (tpt) were prepared according to published
methods and all other reagents were commercially available
(Sigma-Aldrich, S&T Pharma, TCI Europe) and used as received.
The 1H, 13C{1H} NMR spectra were recorded either on a Bruker
AvanceII 400 MHz (University of Neuchatel) or a FT-300 MHz
Bruker Apect 3000 (POSTECH, Korea) spectrometers by using
the residual protonated solvent as internal standard. Infrared
spectra were recorded as KBr pellets on a Bruker FT-IR PS55+
spectrometer. Mass spectra (FAB) were obtained using a Jeol
JMS700 high-resolution mass spectrometer at the Korea Basic
Science Center, Daegu, Korea. The melting range of the solids
was determined with the melting point Gallenkamp apparatus.
Electrospray ionization mass spectra were obtained in positive-ion
mode on a Bruker FTMS 4.7T BioAPEX II mass spectrometer.
Synthesis of Butyryl Pyrene Conjugated Amino Acids.
1-Pyrene butyric acid (1.0 mmol), EDC·HCl (1.5 mmol),
HOBT (1.5 mmol), and DMAP (1.5 mmol) in anhydrous
DMF 10 mL were stirred at room temperature for 1 h. Amino
acid ethyl ester (1.2 equiv) was added to the reaction mixture
and stirred for an additional 2 h. The reaction mixture was
concentrated in vacuo. The residue was diluted with dichloro-
methane (100 mL) and extracted with saturated NaHCO3
aqueous solution, water, and brine sequentially. The organic
layer was dried over anhydrous MgSO4 and filtered. The filtrate
was evaporated in vacuo, and the residue was recrystallized with
EtOH to give pale-yellow solid.
A2780 and A2780cisR Cancer Cell Lines
IC50/μM
compound
A2780
A2780cisR
resistance factor (RF)
cisplatin
1.6 0.3
23 2.1
8.6 1.2
25 2.5
5.4
1.1
0.9
1.0
0.9
3.3
1.0
1.1
1.1
1.0
2.0
1.0
0.8
0.8
1.2
[1]6+
[2]6+
6.0 1.2
2.1 0.14
1.3 0.05
0.4 0.03
0.4 0.10
5.6 0.9
1.3 0.3
0.3 0.03
0.2 0.02
5.9 0.3
1.7 0.06
1.2 0.03
1.4 0.18
5.2 0.9
2.2 0.35
1.2 0.04
1.3 0.13
0.4 0.11
6.1 1.5
1.4 0.3
0.3 0.03
0.4 0.05
5.7 1.0
1.4 0.06
1.0 0.04
1.7 0.21
[pyrene-R1⊂1]6+
[pyrene-R1⊂2]6+
[pyrene-R2⊂1]6+
[pyrene-R2⊂2]6+
[pyrene-R3⊂1]6+
[pyrene-R3⊂2]6+
[pyrene-R4⊂1]6+
[pyrene-R4⊂2]6+
[pyrene-R5⊂1]6+
[pyrene-R5⊂2]6+
[pyrene-R6⊂1]6+
[pyrene-R6⊂2]6+
ranging from 2−5 μM. These observations are in agreement
with results previously reported with other pyrenyl derivatives
encapsulated in water-soluble arene ruthenium metalla-
cages.29−33,35,36 On closer inspection of the IC50 data, it is
apparent that the fluorinated compounds (pyrene-R2, pyrene-
R4, pyrene-R6) are more cytotoxic than their nonfluorinated
counterparts (pyrene-R1, pyrene-R3, pyrene-R5). The high
level of cytotoxicity of the pyrene-R4 derivatives of floxuridine,
especially that involving the less cytotoxic metalla-cage, viz.
[1]6+, are good candidates to improve the therapeutic efficiency
of this clinically used compound.
Synthesis of N-[1-oxo-4-(1-pyrenyl)butyl]-glycine ethyl
ester was prepared according to the general procedure as men-
tioned above from glycine ethyl ester hydrochloride (1.14 g,
8.17 mmol). Yield 2.24 g (73%); M.p.: 146−147 °C; 1H NMR
(300 MHz, CDCl3): δ 8.25 (d, J = 9.0 Hz, 1H), 8.14−
8.06 (m, 4H), 7.99−7.93 (m, 3H), 7.81 (d, J = 9.0 Hz, 1H),
5.94 (s, 1H, NH) 4.19 (q, J = 9.1 Hz, 2H, OCH2CH3), 4.00
(d, J = 6.0 Hz, 2H), 3.35 (t, J = 7.5 Hz, 2H, PyCH2CH2CH2−),
2.33 (t, J = 7.5 Hz, 2H, PyCH2CH2CH2−) 2.19 (m, 2H,
PyCH2CH2CH2−), 1.25 (t, J = 9.1 Hz, 3H, OCH2CH3);
13C{1H} NMR (75 MHz, CDCl3): δ 172.1, 159.1, 156.8, 156.6,
144.0, 143.9, 141.5, 138.6, 132.7, 132.5, 128.0, 127.3, 125.3,
172.8, 170.1, 135.8, 131.4, 130.9, 130.0, 128.8, 127.5, 127.4,
CONCLUSION
■
In this study, water-soluble arene ruthenium metalla-cages were
used to deliver pyrenyl-nucleosides to cancer cells. The differ-
ent floxuridine-metalla-cage combinations exhibit excellent anti-
proliferative effects on both A2780 ovarian cancer cells and
their cisplatin resistant strains. Hence these molecules could be
a good therapeutic alternative to floxuridine which suffers from
poor cellular uptake and bioavailability.
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126.7, 125.8, 125.1 125.0, 124.9, 124.8, 124.8, 123.4, 61.5, 41.4,
35.6, 32.7, 27.2, 14.1; IR (neat): 3310, 3037, 2937, 2867, 1739,
1651, 1537, 1198 cm−1; HRMS-FAB (m/z): calcd for
(m, 1H, C5′H), 2.54 (br s, 1H, exch D2O, OH), 2.31−2.27
(m, 2H, C2′H), 0.89 (s, 9H, C(CH3)3), 0.09 (s, 6H, Si(CH3)2);
13C{1H} NMR (75 MHz, CDCl3): δ 163.2, 150.2, 141.1, 102.5,
87.6, 86.7, 71.4, 61.8, 40.9, 25.7, 18.0, −4.7, −4.9; IR (neat):
3447, 2963, 2930, 2857, 1699, 1559, 1472, 1274, 1082 cm−1;
HRMS-FAB (m/z): calcd for C15H27N2O5Si+ [M + H]+, 343.1689;
found, 343.1687.
C24H24NO3 [M + H]+, 374.1756; found, 374.1751.
+
Synthesis of N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanine
ethyl ester was prepared according to the general procedure
as mentioned above from phenylalanine ethyl ester hydro-
chloride (1.27 g, 5.53 mmol). Yield 2.08 g (81%); M.p.: 157−
5-Fluoro-3′-O-(tert-butyldimethylsilyl)-2′-deoxyuridine was
prepared according to the general procedure as mentioned
above from 5-fluoro-2′-deoxyuridine (1.11 g, 4.51 mmol). Yield
1
158 °C; H NMR (300 MHz, CDCl3): δ 8.24 (d, J = 9.0 Hz,
1H), 8.13−8.03 (m, 4H), 7.98−7.94 (m, 3H), 7.78 (d, J =
9.0 Hz, 1H), 7.22−7.17 (m, 3H), 7.10 (m, 2H), 5.95 (d, J =
6.1 Hz, 1H), 4.90 (q, J = 6.1 Hz, 1H), 4.12 (q, J = 6.0 Hz, 2H),
3.29 (t, J = 9.1 Hz, 2H), 3.17−3.03 (m, 2H), 2.28−2.23 (m, 2H),
2.19−2.11 (m, 2H), 1.20 (t, J = 6.0 Hz, 3H); 13C{1H} NMR
(75 MHz, CDCl3): δ 172.2, 171.8, 136.0, 135.8, 131.4, 130.9,
130.0, 129.3, 128.8, 128.6, 127.5, 127.4, 127.3, 127.1, 126.7,
125.9, 125.1, 125.0, 124.9, 124.8, 123.42, 61.6, 53.1, 38.0, 35.8,
32.7, 27.2, 14.2; IR (neat): 3308, 3289, 3098, 2947, 2865, 1709,
1660, 1557, 1451, 1198 cm−1; HRMS-FAB (m/z): calcd for
C31H30NO3+ [M + H]+, 464.2226; found, 464.2224.
1
1.24 g (76%); M.p.: 140.6−142 °C; H NMR (300 MHz,
CDCl3): δ 8.84 (br s, 1H, exch D2O, NH), 7.97 (d, 1H, J =
6.4 Hz, C6H), 6.23 (t, 1H, C1′H), 4.51 (m, 1H, C3′H), 4.00−
3.95 (m, 2H, C4′H and C5′H), 3.83−3.78 (m, 1H, C5′H), 2.35−
2.17 (2 m, 2H, C2′H), 0.90 (s, 9H, C(CH3)3), 0.09 (s, 6H,
Si(CH3)2); 13C{1H} NMR (75 MHz, CDCl3): δ 156.9, 148.7,
142.0, 138.9, 125.2, 124.8, 87.6, 86.3, 71.4, 61.8, 41.2, 25.7, 18.0,
4.7, −4.9; IR (neat): 3446, 3193, 2955, 2929, 2857, 1716, 1699,
1559, 1404, 1260, 1097 cm−1; HRMS-FAB (m/z): calcd for
C15H26FN2O5Si+ [M + H]+, 361.1595; found, 361.1597.
Synthesis of 3′-O-Protected Nucleoside Derivatives.
3′-O-(tert-Butyldimethylsilyl)-2′-deoxynucleosides were pre-
pared according to the published procedures.37 A solution of
2′-deoxynucleoside (1.0 mmol), 4,4′-dimethoxytrityl chloride
(DMTrCl) (1.15 mmol), and anhydrous pyridine (5.0 mmol)
in anhydrous DMF (10 mL) was stirred at room temperature
for 5 h. To the reaction mixture was then added methanol
(1 mL) for quenching, and the mixture was stirred for 30 min.
The reaction mixture was partitioned between dichloromethane
(250 mL) and saturated NaHCO3 aqueous solution (250 mL).
The organic layer was washed with water two times and brine,
dried over anhydrous Na2SO4, and filtered. The filtrate was
concentrated and coevaporated with pyridine two times. The
residue was dissolved in 10 mL of anhydrous DMF. To the
solution was added imidazole (5.0 mmol) and the solution was
stirred at room temperature for 30 min. After adding tert-
butyldimethylsilyl chloride (1.5 mmol) the resulting reaction
mixture was stirred overnight at room temperature. Then 100
mL of saturated NaHCO3 aqueous solution was added, and the
mixture was extracted with dichloromethane two times. The
combined organic layer was washed with water three times then
brine, dried with Na2SO4, and filtered. The filtrate was con-
centrated in vacuo and coevaporated with toluene three times.
The residue was dried over 6 h in vacuo and used to the next
step without further purification.
The concentrated residue was dissolved in 10 mL of an-
hydrous dichloromethane and stirred at 0−5 °C for 30 min.
Then 10 mL of 5% trichloroacetic acid in dichloromethane was
slowly added to the reaction mixture and the resulting reaction
mixture was stirred at room temperature for 3 h. To the
reaction mixture was then added 1 mL of methanol and 50 mL
of saturated NaHCO3 aqueous solution was added to the
mixture. The organic layer was collected, dried over anhydrous
Na2SO4, and filtered. The filtrate was evaporated in vacuo, and
the residue was purified by column chromatography to give a
white solid.
Synthesis of Modified Nucleosides Conjugated Pyrenyl
Derivatives. Butyryl pyrene conjugated amino acids (1.0 mmol)
and lithium hydroxide monohydrate (3.0 mmol) was stirred in
THF/H2O (4/1) at room temperature for 2−3 h. The resulting
reaction mixture was neutralized with Dowex 50WX8−200 ion-
exchange resin, filtered, and concentrated in vacuo. The residue
was coevaporated with pyridine three times and dried in vacuo.
Without further purification, the residue was dissolved with 20 mL
of anhydrous DMF. To the solution were added EDC·HCl
(1.5 mmol), HOBT (1.5 mmol), and DMAP (1.5 mmol) and
stirred at room temperature for 1 h. 3′-O-TBDMS nucleosides
(1.0 mmol) was added to the reaction mixture. The resulting
reaction mixture was stirred at ambient temperature for 6 h.
The reaction mixture was concentrated in vacuo. The residue
was extracted between dichloromethane (150 mL) and 1 N
HCl aqueous solution (10 mL). The organic layer was washed
with water three times, dried over anhydrous MgSO4, and
filtered. The filtrate was evaporated in vacuo. The residue was
dissolved with anhydrous THF and cooled at 0−5 °C. 1 M
TBAF in THF (1.5 mL) was added slowly to the reaction mixture.
The resulting reaction mixture was warmed up at room
temperature and stirred for 6 h. Afterward, ethyl acetate was
added to the mixture and the solution was washed with satu-
rated NaHCO3 aqueous solution, water, and brine sequentially.
The organic layer was dried over anhydrous MgSO4 and filtered.
The filtrate was evaporated in vacuo, and the residue was purified
by column chromatography to give a pale-yellow solid.
5′-(1-Pyrenyl butanoate)-2′-deoxyuridine (pyrene-R1) was
prepared according to the general procedure as mentioned
above from 3′-O-(tert-butyldimethylsilyl)-2′-deoxyuridine (212
mg, 0.62 mmol). Yield: 280 mg (91%); M.p.: 223−224 °C;
1H NMR (300 MHz, acetone-d6): δ 10.2 (s, 1H), 8.41(d, J =
9.6 Hz, 1H), 8.24−8.18 (m, 6H), 8.01 (m, 1H), 7.92 (m, 1H),
7.73 (d, J = 8.1 Hz, 1H, C6H), 6.34 (t, J = 6.6 Hz, 1H,
C1′H), 5.67 (d, J = 8.1 Hz, 1H, C5H), 4.48 (m, 1H, C4′H),
4.42−4.18 (m, 2H, C5′H), 4.17−4.15 (m, 1H, C3′H), 3.40 (t,
J = 7.5 Hz, 2H, PyCH2CH2CH2), 2.56 (t, J = 7.2 Hz, 2H,
PyCH2CH2CH2), 2.41−2.32 (m, 2H, PyCH2CH2CH2), 2.30−
2.08 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, acetone-d6): δ
172.3, 162.5, 150.0, 139.6, 135.8, 131.1, 130.6, 129.6, 128.3,
127.1, 126.9, 126.2, 125.6, 124.6, 124.5, 124.4, 123.1, 101.6,
70.8, 63.5, 39.4, 32.9, 31.9, 26.5; IR (neat): 3421, 3044, 2947,
1698, 1653, 1460, 1383, 1272, 1248, 1089 cm−1; HRMS-FAB
3′-O-(tert-Butyldimethylsilyl)-2′-deoxyuridine was prepared
according to the general procedure as mentioned above from
2′-deoxyuridine (1.04 g, 4.56 mmol). Yield 1.23 g (79%); M.p.:
1
206−207 °C; H NMR (300 MHz, CDCl3): δ 9.10 (br s, 1H,
exch D2O, NH), 7.67 (d, 1H, J = 8.1 Hz, C6H), 6.18
(t, 1H, J = 6.6 Hz, C1′H), 5.75 (d, 1H, J = 8.1 Hz, C5H), 4.51−
4.47 (m, 1H, C3′H), 3.95−3.91 (m, 2H, C4′H and C5′H), 3.78
466
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Bioconjugate Chemistry
Article
(m/z): calcd for C29H27N2O6 [M + H]+, 499.1869; found,
499.1866.
(m, 1H, C4′H), 4.48−4.43 (m, 1H, CαH), 4.24−4.05 (m, 3H,
C3′H and C5′H), 3.35−3.18 (m, 2H, PyCH2CH2CH2), 3.11−3.09
(m, 2H, phenyl), 2.40−2.29 (m, 4H, PyCH2CH2CH2), 2.14−
2.12 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, acetone-d6):
δ 172.7, 171.9, 162.8, 150.4, 140.3, 137.2, 136.7, 131.0, 129.9,
129.2, 128.7, 128.4, 127.6, 127.5, 127.3, 126.8, 126.5, 126.0,
124.9, 124.8, 123.7, 102.6, 84.9, 84.4, 71.5, 64.7, 54.5, 40.0,
37.3, 34.9, 32.4, 27.5; IR (neat): 3327, 3043, 2946, 1704, 1698,
1559, 1457, 1248, 1184, 1086 cm−1; HRMS-FAB (m/z): calcd
+
5-Fluoro-5′-(1-pyrenyl butanoate)-2′-deoxyuridine (pyrene-R2)
was prepared according to the general procedure as mentioned
above from 5-fluoro-3′-O-(tert-butyldimethylsilyl)-2′-deoxyuri-
dine (209 mg, 0.58 mmol). Yield: 267 mg (89%); M.p.: 216−
1
218 °C; H NMR (300 MHz, CDCl3): δ 9.54 (s, 1H), 8.18
(d, 1H, J = 9.0 Hz), 8.08−7.95 (m, 7H), 7.75 (d, 1H), 7.51 (d, 1H,
J = 5.7 Hz), 6.05 (t, 1H, C1′H), 4.30−4.26 (m, 1H, C4′H), 4.13−
4.02 (m, 3H, C5′H and C3′H), 3.31 (m, 2H, PyCH2CH2CH2), 2.43
(t, 2H, PyCH2CH2CH2), 2.28−2.16 (m, 2H, PyCH2CH2CH2),
1.88−1.83 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, CDCl3): δ
173.1, 156.9, 148.6, 142.0, 138.9, 135.1, 131.3, 130.1, 130.0, 128.7,
127.5, 127.3, 126.8, 125.9, 125.0, 124.9, 124.8, 123.9, 123.4, 123.1,
85.5, 84.3, 80.7, 63.3, 40.3, 33.5, 32.4, 26.4; IR (neat): 3447, 3198,
3043, 2949, 1734, 1717, 1473, 1418, 1264, 1086 cm−1; HRMS-FAB
(m/z): calcd for C29H26FN2O6+ [M + H]+, 517.1775; found,
517.1778.
+
for C38H36N3O7 [M + H]+, 646.2553; found, 646.2550.
5-Fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′-
deoxyuridine (pyrene-R6) was prepared according to the general
procedure as mentioned above from 5-fluoro-3′-O-(tert-butyldi-
methylsilyl)-2′-deoxyuridine (224 mg, 0.62 mmol). Yield: 342 mg
1
(83%); M.p.: 234−236 °C; H NMR (300 MHz, acetone-d6):
δ 10.5 (s, 1H), 8.39 (d, 1H, J = 8.1 Hz), 8.24−8.13 (m, 6H),
8.04−7.99 (m, 3H), 7.67 (d, 1H, J = 7.5 Hz), 7.28−7.17 (m,
4H), 6.32 (t, 1H, J = 5.7 Hz, C1′H), 4.84−4.79 (m, 1H, C4′H),
4.55−4.51 (m, 1H, CαH), 4.37−4.13 (m, 3H, C3′H and C5′H),
3.33−3.22 (m, 2H, PyCH2CH2CH2), 3.20−3.16 (m, 2H, phenyl),
2.38−2.11 (m, 4H, PyCH2CH2CH2), 2.09−2.06 (m, 2H, C2′H);
13C{1H} NMR (75 MHz, acetone-d6): δ 172.8, 171.7, 156.9,
149.0, 142.2, 139.1, 137.1, 136.6, 131.4, 130.0, 129.1, 128.7, 128.4,
127.5, 127.2, 126.7, 126.5, 126.0, 124.9, 124.8, 124.6, 124.5, 124.1,
123.7, 85.3, 84.7, 71.0, 64.5, 54.2, 39.6, 37.2, 34.9, 32.4, 27.5; IR
(neat): 3402, 2964, 2929, 1717, 1700, 1653, 1540, 1473, 1361,
1264, 1079 cm−1; HRMS-FAB (m/z): calcd for C38H35FN3O7+
[M + H]+, 664.2459; found, 664.2463.
5′-{N-[1-Oxo-4-(1-pyrenyl)butyl]-glycyl}-2′-deoxyuridine
(pyrene-R3) was prepared according to the general procedure
as mentioned above from 3′-O-(tert-butyldimethylsilyl)-2′-
deoxyuridine (224 mg, 0.65 mmol). Yield: 316 mg (87%);
M.p. M.p.: 231−233 °C; 1H NMR (300 MHz, acetone-d6): δ 10.1
(s, 1H), 8.45(d, 1H, J = 9.0 Hz), 8.24−8.15 (m, 5H), 8.08−
7.93 (m, 3H), 7.75 (d, 1H, J = 8.1 Hz, C6H), 6.32 (t, 1H, J =
6.90 Hz, C1′H), 5.76 (d, 1H, J = 8.1 Hz, C5H), 4.72 (s, 1H),
4.50−4.48 (m, 1H, C4′H), 4.39−4.37 (m, 3H, C3′H and C5′H),
4.08 (d, 2H, J = 5.7 Hz, CαH), 3.42 (t, 2H, J = 7.5 Hz,
PyCH2CH2CH2), 2.45 (t, 2H, 7.2 Hz, PyCH2CH2CH2), 2.34−
2.28 (m, 2H, PyCH2CH2CH2), 2.20−2.08 (m, 2H, C2′H);
13C{1H} NMR (75 MHz, acetone-d6): δ 173.1, 169.9, 162.9,
150.4, 140.2, 136.7, 131.4, 131.0, 129.9, 128.7, 127.6, 127.3,
126.5, 125.9, 124.9, 124.8, 124.7, 123.7, 102.2, 84.9, 84.5, 71.1,
64.2, 41.1, 39.7, 34.9, 32.5, 27.6; IR (neat): 3333, 3046, 2940,
1753, 1696, 1540, 1460, 1271, 1191, 1088 cm−1; HRMS-FAB
(m/z): calcd for C31H30N3O7+ [M + H]+, 556.2084; found,
556.2086.
Encapsulation of Modified Nucleosides Conjugated
Pyrenyl Derivatives (Pyrene-R1−6) in Metalla-Cage [1]6+.
A mixture of [Ru2(pPriC6H4Me)2(dobq)Cl2] (0.15 mmol),
AgCF3SO3 (0.30 mmol), tpt (0.10 mmol), and pyrenyl
derivatives (pyrene-R) (0.05 mmol) in MeOH (30 mL) was
stirred at 60 °C for 15 h. The reaction mixture was kept in the
dark. The resulting reaction mixture was filtered and con-
centrated in vacuo. The residue was dissolved in CH2Cl2
(2 mL), and diethyl ether (50 mL) was slowly added to precipi-
tate the product, which was kept in the refrigerator for 2 h. The
product was filtered and dried in vacuo.
5-Fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-2′-deoxy-
uridine (pyrene-R4) was prepared according to the general
procedure as mentioned above from 5-fluoro-3′-O-(tert-
butyldimethylsilyl)-2′-deoxyuridine (204 mg, 0.57 mmol).
[Pyrene-R1⊂1][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
1
Yield: 276 mg (85%); M.p.: 227−229 °C; H NMR (300
pyrene-R1 (27.1 mg, 0.05 mmol). Yield 136 mg (71%). H
MHz, DMSO-d6): δ 8.39 (s, 1H), 8.36 (d, 1H, J = 9.3 Hz),
8.24−8.14 (m, 4H), 8.08 (s, 2H), 7.99 (t, 1H), 7.89 (t, 2H),
6.13 (t, 1H, J = 6.3 Hz, C1′H), 5.60 (br, 1H, NH), 4.30−4.25
(m, 3H, C4′H and C5′H), 3.96−3.89 (m, 3H, C3′H and CαH),
3.31 (t, 2H, J = 7.5 Hz, PyCH2CH2CH2), 2.32 (t, 2H, J = 7.2
Hz, PyCH2CH2CH2), 2.22−2.14 (m, 2H, PyCH2CH2CH2),
2.04−1.99 (m, 2H, C2′H); 13C{1H} NMR (75 MHz, acetone-d6):
δ 173.6, 170.4, 157.8, 157.4, 149.5, 142.1, 139.0, 136.9, 131.3,
130.8, 129.7, 128.6, 128.0, 127.9, 127.7, 126.9, 126.6, 125.4, 125.3,
125.2, 125.0, 124.7, 124.6, 123.9, 85.1, 84.3, 79.4, 70.4, 64., 41.2,
3.1, 32.5, 27.9; IR (neat): 3429, 3027, 2941, 1696, 1664, 1559,
1194, 1027 cm−1; HRMS-FAB (m/z): calcd for C31H29FN3O7+
[M + H]+, 574.1990; found, 574.1986.
NMR (300 MHz, acetone-d6): δ 9.88 (s, 1H, N3H), 8.58
(s, 12H, Hα), 8.04 (br, 12H, Hβ), 7.87 (d, 1H, J = 8.1 Hz, C6H),
7.05 (d, 1H, J = 9.0 Hz, HPy), 6.89 (d, 1H, J = 15.6 Hz, HPy), 6.84
(d, 1H, J = 15.6 Hz, HPy), 6.57 (d, 1H, J = 9.3 Hz, HPy), 6.42
(t, 1H, J = 6.3, C1′H), 6.38 (d, 1H, J = 3.9 Hz, HPy), 6.24
(d, 12H, J = 6.0 Hz, Har), 6.19 (br, 8H, Hq and HPy), 6.02 (d,
12H, J = 6.0 Hz, Har), 5.99 (m, 1H, HPy) 5.85 (d, 1H, J =
7.5 Hz, HPy), 5.60 (d, 1H, J = 8.1 Hz, C5H), 4.68−4.58 (m, 3H,
C3′H and C5′H), 4.44−4.43 (m, 1H, C4′H), 2.99 (sept,
6H, J = 6.9 Hz, CH), 2.57−2.55 (m, 6H, PyCH2CH2CH2
and C2′H), 2.23 (s, 18H, CH3), 1.72−1.69 (m, 2H,
PyCH2CH2CH2), 1.42 (d, J = 6.90 Hz, 36H, CH3); 13C{1H}
NMR (75 MHz, acetone-d6): δ 184.2, 172.8, 167.6, 162.7, 154.0,
150.5 143.6, 140.8, 135.5, 129.7, 129.1, 128.2, 127.5, 127.2, 126.9,
125.8, 124.9, 124.6, 124.4, 123.6, 122.8, 122.7, 122.2, 119.3, 104.1,
101.9, 99.2, 84.6, 83.8, 82.3, 71.1, 64.3, 38.7, 32.9, 31.8, 31.2, 26.4,
21.6, 17.2; IR (neat): 3446, 2964, 2927, 1717, 1521, 1376, 1258,
1030, 811 cm−1; ESI-MS (m/z): 1132.14 [pyrene-R1 + 1 +
(CF3SO3)3]3+.
5-Fluoro-5′-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2′-
deoxyuridine (pyrene-R5) was prepared according to the
general procedure as mentioned above from 3′-O-(tert-butyl-
dimethylsilyl)-2′-deoxyuridine (207 mg, 0.60 mmol). Yield: 316 mg
1
(81%); M.p.: 238−239 °C; H NMR (300 MHz, acetone-d6):
δ 10.2 (s, 1H), 8.42 (d, 1H), 8.26−8.00 (m, 7H), 7.90 (t, 1H),
7.71 (d, 1H, J = 8.1 Hz, C6H), 7.28−7.17 (m, 4H), 6.32 (t, 1H,
J = 6.4 Hz, C1′H), 5.69 (d, 1H, J = 8.1 Hz, C5H), 4.78−4.72
[Pyrene-R2⊂1][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
467
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Bioconjugate Chemistry
Article
1
1
pyrene-R2 (27.1 mg, 0.05 mmol). Yield 145 mg (72%). H
NMR (300 MHz, acetone-d6): δ 10.4 (s, 1H, N3H), 8.57
(s, 12H, Hα), 8.04 (br, 12H, Hβ), 8.02 (d, 1H, J = 6.9 Hz),
7.08 (d, 1H, J = 8.7 Hz, HPy), 6.93 (d, 1H, J = 7.5 Hz, HPy),
6.87(d, 1H, J = 8.4 Hz, HPy), 6.58 (d, 1H, J = 9.3 Hz, HPy),
6.40−6.34 (m, 2H, HPy and C1′H), 6.21 (m, 19H, Har, Hq, and
HPy), 6.01 (m, 13H, Har and HPy), 5.85 (d, 1H, J = 7.2 Hz,
HPy), 4.76−4.58 (m, 3H, C3′H and C5′H), 4.45−4.43 (m, 1H,
C4′H), 2.98 (sept, 1H, J = 6.9 Hz, CH), 2.57−2.52 (m, 6H,
PyCH2CH2CH2 and C2′H), 2.22 (s, 18H, CH3), 1.73−1.68 (m,
2H, PyCH2CH2CH2), 1.42 (d, 36H, J = 6.9 Hz, CH3); 13C{1H}
NMR (75 MHz, acetone-d6): δ 184.2, 172.8, 167.6, 153.8,
150.5 148.9, 143.6, 140.8, 135.4, 129.7, 129.1, 128.2, 127.9,
127.2, 127.0, 126.4, 125.8, 124.6, 124.2, 123.6, 122.9, 122.2,
119.3, 104.1, 101.9, 99.2, 85.2, 83.8, 82.3, 70.9, 66.7, 64.3, 38.7,
33.9, 31.2, 26.5, 21.6, 17.2; IR (neat): 3447, 2965, 2927, 1717,
1521, 1376, 1258, 1058, 1030, 811 cm−1; ESI-MS (m/z):
1137.78 [pyrene-R2 + 1 + (CF3SO3)3]3+.
pyrene-R5 (33.0 mg, 0.05 mmol). Yield 142 mg (70%). H
NMR (300 MHz, acetone-d6): δ 10.15 (s, 1H, N3H), 8.57
(s, 12H, Hα), 8.30−7.91 (br, d, 13H, Hβ and C6H), 7.53 (m, 5H,
Hph), 7.00 (d, 1H, J = 9.0 Hz, Hpy), 6.87 (t, 1H, J = 8.1 Hz,
Hpy), 6.78 (m, 1H, Hpy), 6.55 (m, 1H, Hpy), 6.37−6.22 (m, 8H,
C1′H, HPy and Hq), 6.20 (d, 12H, J = 6.3 Hz, Har), 6.08−
6.07 (m, 2H, HPy), 6.00 (m, 13H, Har and HPy), 5.94 (d,
1H, J = 8.1 Hz, Hpy), 5.62 (d, 1H, J = 8.1 Hz, C5H), 4.89−4.77
(m, 1H, C4′H), 4.58−4.51 (m, 1H, CαH), 4.47−4.12
(m, 3H, C3′H and C5′H), 3.42−3.33 (m, 2H, PyCH2CH2CH2),
3.00 (sept, 6H, J = 6.9 Hz, CH), 2.55−2.53 (m, 2H,
PyCH2CH2CH2), 2.41−2.38 (m, 2H, C2′H), 2.22 (s, 18H,
CH3), 1.62−1.60 (m, 2H, PyCH2CH2CH2), 1.41 (d, 36H, J =
6.9 Hz, CH3); 13C{1H} NMR (75 MHz, acetone-d6): δ 184.2,
173.3, 172.1, 167.6, 163.1, 162.8, 153.8, 150.4, 143.3, 140.7,
140.5, 137.6, 135.7, 129.7, 129.1, 128.8, 128.1, 127.8, 127.1,
126.9, 126.2, 125.8, 125.3, 124.7, 124.2, 123.5, 122.8, 122.3,
119.3, 115.0, 104.1, 102.0, 99.2, 84.8, 83.8, 82.3, 71.6, 65.1, 55.6,
39.8, 37.5, 35.0, 31.7, 31.2, 26.3, 21.6, 17.2; IR (neat): 3447,
2965, 2927, 1717, 1521, 1376, 1259, 1058, 1031,
811 cm−1; ESI-MS (m/z): 1181.47 [pyrene-R5 + 1 + (CF3SO3)3]3+.
[Pyrene-R6⊂1][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
[Pyrene-R3⊂1][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
pyrene-R3 (27.6 mg, 0.05 mmol). Yield 134 mg (69%). H
NMR (300 MHz, acetone-d6): δ 10.02 (s, 1H, N3H), 8.57
(s, 12H, Hα), 7.96 (br, 12H, Hβ), 7.81 (d, 1H, J = 8.1 Hz,
C6H), 7.04 (d, 1H, J = 9.0 Hz, HPy), 6.91 (d, 1H, J = 7.8 Hz,
HPy), 6.79 (d, 1H, J = 8.7 Hz, HPy), 6.56 (d, 1H, J = 9.0 Hz,
HPy), 6.42 (t, 1H, J = 7.2 Hz, C1′H), 6.22 (m, 20H, Har, Hq, and
HPy), 6.00 (d, 12H, J = 6.3 Hz, Har), 5.80 (m, 2H, HPy), 5.60 (d,
1H, J = 8.1 Hz, C5H), 4.60−4.52 (m, 3H, C3′H and C5′H),
4.26 (m, 3H, C4′H and CαH), 2.98 (sept, 6H, J = 6.9 Hz, CH),
2.56−2.55 (m, 2H, PyCH2CH2CH2), 2.42 (m, 4H,
PyCH2CH2CH2 and C2′H), 2.22 (s, 18H, CH3), 1.64 (m,
2H, PyCH2CH2CH2), 1.40 (d, 36H, J = 6.9 Hz, CH3); 13C{1H}
NMR (75 MHz, acetone-d6): δ 184.2, 173.0, 170.1, 162.9,
153.8, 150.4, 140.3, 136.7, 131.4, 131.0, 129.9, 128.7, 127.6,
127.3, 126.5, 125.9, 125.5, 124.9, 124.8, 124.7, 124.2, 123.7,
104.1, 102.0, 102.2, 99.3, 84.9, 84.5, 83.9, 82.2, 71.1, 64.2, 41.1,
39.7, 34.9, 32.5, 31.2, 27.6, 21.6, 17.2; IR (neat): 3447, 2965,
2927, 1684, 1540, 1375, 1259, 1058, 811 cm−1; ESI-MS (m/z):
1151.47 [pyrene-R3 + 1 + (CF3SO3)3]3+.
1
pyrene-R6 (33.1 mg, 0.05 mmol). Yield 142 mg (71%). H
NMR (300 MHz, acetone-d6): δ 10.46 (s, 1H, N3H), 8.58 (s,
12H, Hα), 8.21−7.92 (br, d, 13H, Hβ and C6H), 7.55−7.38 (m,
5H, HPh), 7.02 (m, 1H, HPy), 6.95 (t, 1H, J = 6.3 Hz, HPy),
6.80 (d, 1H, J = 8.4 Hz, HPy), 6.77 (m, 1H, HPy), 6.54−6.22 (m,
8H, C1′H, HPy and Hq), 6.22 (d, 12H, J = 6.3 Hz, Har), 6.08−
6.02 (m, 3H, HPy), 6.01 (d, 12H, J = 6.0 Hz, Har), 5.99 (m, 1H,
HPy), 4.88−4.68 (m, 1H, C4′H), 4.54−4.42 (m, 1H, CαH),
4.28−4.24 (m, 3H, C3′H and C5′H), 3.38−3.36 (m, 2H,
PyCH2CH2CH2), 3.00 (sept, 6H, J = 6.9 Hz, CH), 2.55−2.53
(m, 2H, PyCH2CH2CH2), 2.41−2.38 (m, 2H, C2′H), 2.24
(s, 18H, CH3), 1.62−1.60 (m, 2H, PyCH2CH2CH2), 1.41 (d,
36H, J = 6.9 Hz, CH3); 13C{1H} NMR (75 MHz, acetone-d6):
δ 184.2, 173.2, 172.1, 167.7, 153.9, 148.9, 143.3, 137.4, 129.7,
129.4, 129.1, 128.6, 128.1, 127.8, 127.1, 126.9, 126.7, 126.2,
125.8, 125.2, 124.7, 124.5, 124.2, 123.5, 122.8, 122.7, 122.3,
119.3, 115.0, 104.1, 102.0, 85.3, 84.6, 83.8, 82.3, 71.2, 64.8, 55.4
39.5, 37.2, 35.0, 31.6, 31.2, 26.3, 21.6, 17.2; IR (neat): 3447,
2966, 2926, 1716, 1521, 1377, 1258, 1057, 1030, 811 cm−1;
ESI-MS (m/z): 1186.82 [pyrene-R6 + 1 + (CF3SO3)3]3+.
Encapsulation of Modified Nucleosides Conjugated
Pyrenyl Derivatives (Pyrene-R1−6) in Metalla-Cage [2]6+.
A mixture of [Ru2(pPriC6H4Me)2(donq)Cl2] (0.17 mmol),
AgCF3SO3 (0.34 mmol), tpt (0.11 mmol), and pyrenyl
derivatives (pyrene-R) (0.055 mmol) in MeOH (30 mL) was
stirred at 60 °C for 15 h. The reaction mixture was kept in the
dark. The resulting reaction mixture was filtrated and concen-
trated in vacuo. The residue was dissolved in CH2Cl2 (2 mL),
and diethyl ether (50 mL) was slowly added to precipitate the
product, which was kept in the refrigerator for 2 h. The product
was filtered and dried in vacuo.
[Pyrene-R4⊂1][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
pyrene-R4 (28.7 mg, 0.05 mmol). Yield 143 mg (73%). H
NMR (300 MHz, acetone-d6): δ 10.46 (s, 1H, N3H), 8.57
(s, 12H, Hα), 8.21−7.93 (br, d, 13H, Hβ and C6H), 7.04
(d, 1H, J = 9.0 Hz, HPy), 6.91 (d, 1H, J = 7.5 Hz, HPy), 6.78
(d, 1H, J = 9.0 Hz, HPy), 6.56 (d, 1H, J = 9.0 Hz, HPy), 6.39
(t, 1H, J = 7.8 Hz, C1′H), 6.21 (d, m, 18H, J = 6.0 Hz, Har
and Hq), 6.12−5.81 (m, 3H, HPy), 6.00 (m, Har and HPy), 5.81
(d, 1H, J = 7.8 Hz, HPy) 4.65−4.48 (m, 3H, C3′H and C5′H),
4.28 (br, 3H, C4′H and CαH), 2.98 (sept, 6H, J = 6.9 Hz, CH),
2.58 (m, 2H, PyCH2CH2CH2), 2.43 (m, 4H, PyCH2CH2CH2
and C2′H), 2.22 (s, 18H, CH3), 1.62 (m, 2H, PyCH2CH2CH2),
1.40 (d, J = 6.90 Hz, 36H, CH3); 13C{1H} NMR (75 MHz,
acetone-d6): δ 184.2, 173.5, 170.3, 167.6, 157.0, 153.8, 148.9,
143.3, 135.8, 129.7, 129.1, 128.1, 127.5, 127.2, 126.7, 126.2,
125.8, 125.4, 124.8, 124.5, 124.2, 123.5, 122.9, 122.7,
122.4, 119.3, 115.0, 104.1, 102.0, 99.2, 85.3, 84.8, 83.9, 82.2,
70.9, 64.5, 61.7, 41.1, 39.4, 35.1, 31.7, 31.2, 26.5, 21.6,
17.2; IR (neat): 3446, 2966, 2929, 1734, 1521, 1375, 1259,
1058, 811 cm−1; ESI-MS (m/z): 1157.47 [pyrene-R4 + 1 +
(CF3SO3)3]3+.
[Pyrene-R1⊂2][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
pyrene-R1 (28 mg, 0.055 mmol). Yield: 164 mg (75%); H
NMR (300 MHz, acetone-d6): δ 9.89 (s, 1H, N3H), 8.64
(s, 12H, Hα), 7.80 (m, 13H, Hβ and C6H), 7.59 (s, 12H, Hq),
6.96 (m, 4H, HPy), 6.44 (m, 4H, HPy), 6.37 (m, 1H, C1′H), 6.01
(d, 12H, J = 5.7 Hz, Hcym), 5.78 (d, 12H, J = 5.7 Hz, Hcym), 5.50
(d, 1H, C5H), 5.12 (m, 1H, HPy), 4.65−4.40 (m, 4H, C3′H and
C5′H), 3.40 (t, 2H, PyCH2CH2CH2), 2.94 (sept, 6H, J = 6.9, Hz,
[Pyrene-R5⊂1][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
468
dx.doi.org/10.1021/bc200472n | Bioconjugate Chem. 2012, 23, 461−471

Bioconjugate Chemistry
Article
CH(CH3)2), 2.38 (m, 6H, PyCH2CH2CH2 and C2′H), 2.12 (s,
18H, CH3), 1.35 (d, 36H, J = 6.9 Hz, CH(CH3)2); 13C{1H}
NMR (75 MHz, acetone-d6): δ 172.8, 171.1, 167.8, 162.6,
152.7, 150.4, 143.1, 141.0, 137.9, 135.0, 129.8, 128.9, 128.4,
127.9, 127.2, 126.1, 125.3, 125.1, 124,2, 123,7, 123.6, 122.9,
121.7, 119.3, 103.7, 102.3, 100.2, 71.2, 64.5, 38.6, 33.6, 31.6,
30.6, 26.5, 21.6, 16.4; IR (neat): 3447, 3066, 2967, 1734, 1535,
1460, 1275, 853 cm−1; ESI-MS (m/z): 1182.80 [pyrene-R1 +
2 + (CF3SO3)3]3+.
[Pyrene-R5⊂2][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
pyrene-R5 (31.0 mg, 0.048 mmol). Yield: 145 mg (73%); H
NMR (300 MHz, acetone-d6): δ 10.04 (s, 1H, N3H), 8.62
(s, 13H, Hα and C6H) 8.25−7.73 (m, 14H, Hβ and HPh), 7.57
(s, 12H, Hq), 7.31 (m, 3H, HPh), 6.92 (br, 4H, HPy), 6.51 (m,
2H, HPy), 6.23 (m, 3H, HPy and C1′H), 5.99 (d, 12H, J = 6.0
Hz, Hcym), 5.79 (m, 1H, C5H), 5.76 (d, 12H, J = 6.0 Hz, Hcym),
5.57 (d, 1H, HPy), 4.90 (m, 1H, C4′H), 4.54 (m, 1H, CHα),
4.28−4.11 (m, 3H, C3′H and C5′H), 3.35 (m, 2H,
PyCH2CH2CH2), 2.95 (sept, 6H, J = 6.9 Hz, CH(CH3)2), 2.32
(m, 4H, PyCH2CH2CH2 and C2′H), 2.11 (s, 18H, CH3), 1.62
(m, 2H, PyCH2CH2CH2), 1.37 (d, 36H, J = 6.9 Hz,
CH(CH3)2); 13C{1H} NMR (75 MHz, acetone-d6): δ 173.4,
172.7, 171.9, 167.9, 162.8, 152.7, 150.4, 143.2, 134.2, 137.8,
129.5, 129.4, 128.8, 128.6, 127.8, 126.9, 126.0, 125.0, 124.1,
123.7, 123.6, 119.3, 115.0, 111.4, 103.7, 102.4, 100.1, 84.9, 83.1,
71.2, 64.5, 39.9, 37.3, 35.2, 31.8, 30.5, 26.6, 21.6, 16.4; IR
(neat): 3446, 2966, 2925, 1717, 1534, 1275, 1030, 852 cm−1;
ESI-MS (m/z): 1231.50 [pyrene-R5 + 2 + (CF3SO3)3]3+.
[Pyrene-R6⊂2][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
[Pyrene-R2⊂2][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
pyrene-R2 (29.0 mg, 0.06 mmol). Yield: 166 mg (74%); H
NMR (300 MHz, acetone-d6): δ 9.89 (s, 1H, N3H), 8.64
(s, 12H, Hα), 7.99−7.74 (m, 13H, Hβ and C6H), 7.59 (s, 12H,
Hq), 6.98 (m, 4H, HPy), 6.38 (m, 4H, HPy), 6.29 (t, 1H, C1′H),
6.01 (d, 12H, J = 5.7 Hz, Hcym), 5.78 (d, 12H, J = 5.7 Hz, Hcym),
5.12 (m, 1H, HPy), 4.68−4.43 (m, 4H, C3′H, C4′H and C5′H),
3.64 (t, 2H, PyCH2CH2CH2), 2.96 (sept, 6H, J = 7.2 Hz,
CH(CH3)2), 2.38 (m, 4H, PyCH2CH2CH2 and C2′H), 2.11 (s,
18H, CH3), 1.37 (d, 36H, J = 7.2 Hz, CH(CH3)2); 13C{1H} NMR
(75 MHz, acetone-d6): δ 172.8, 171.1, 167.8, 162.6, 156.7, 152.6,
150.3, 142.9, 141.0, 137.7, 135.1, 129.7, 128.9, 128.4, 127.9, 127.2,
126.1, 125.3, 125.0, 124,2, 123,7, 123.6, 123.0, 121.7, 119.4, 104.0,
100.2, 71.2, 64.4, 38.6, 33.5, 31.7, 30.6, 26.4, 21.5, 16.4; IR (neat):
3448, 3076, 2968, 1735, 1539, 1457, 1275, 1148, 853 cm−1;
ESI-MS (m/z): 1188.13 [pyrene-R2 + 2 + (CF3SO3)3]3+.
1
pyrene-R6 (35.0 mg, 0.053 mmol). Yield: 162 mg (74%); H
NMR (300 MHz, acetone-d6): δ 10.40 (s, 1H, N3H), 8.64
(s, 13H, Hα and C6H) 8.16−7.75 (m, 14H, Hβ and HPh), 7.56
(s, 12H, Hq), 7.31 (m, 3H, HPh), 6.84 (br, 4H, HPy), 6.47 (m,
2H, HPy), 6.27 (m, 3H, HPy and C1′H), 6.00 (d, 12H, J =
6.0 Hz, Hcym), 5.77 (d, 12H, J = 6.3 Hz, Hcym), 5.34 (d, 1H,
HPy), 4.86−4.79 (m, 1H, C4′H), 4.56 (m, 1H, CHα), 4.30−
4.22 (m, 3H, C3′H and C5′H), 3.38 (m, 2H, PyCH2CH2CH2),
2.95 (sept, 6H, J = 6.9 Hz, CH(CH3)2), 2.32 (m, 4H,
PyCH2CH2CH2 and C2′H), 2.14 (s, 18H, CH3), 1.42 (m, 2H,
PyCH2CH2CH2), 1.39 (d, 36H, J = 6.9 HZ, CH(CH3)2);
13C{1H} NMR (75 MHz, acetone-d6): δ 173.1, 172.1, 171.0,
168.0, 156.9, 152.8, 148.8, 143.3, 137.9, 129.8, 129.5, 128.8,
128.7, 127.8, 127.1, 126.9, 126.1, 124.9, 124.1, 123.7, 123.6,
123.0, 121.6, 119.3, 115.1, 111.4, 103.7, 100.1, 85.3, 84.9, 83.1,
71.2, 64.8, 54.8, 39.4, 37.3, 35.1, 31.7, 26.6, 21.6, 16.4; IR
(neat): 3447, 2966, 2925, 1734, 1559, 1273, 1147, 1030, 852
cm−1; ESI-MS (m/z): 1236.48 [pyrene-R6 + 2 + (CF3SO3)3]3+.
Cell Culture and Inhibition of Cell Growth. Human
A2780 ovarian carcinoma cells were obtained from the
European Centre of Cell Cultures (ECACC, Salisbury, UK)
and maintained in culture as described by the provider. The
cells were routinely grown in RPMI 1640 medium containing
10% fetal calf serum (FCS) and antibiotics at 37 °C and 6%
CO2. For the evaluation of growth inhibition tests, the cells
were seeded in 96-well plates and grown for 24 h in complete
medium. Complexes were added to the required concentration
and added to the cell culture for 72 h incubation. Solutions of
the compounds were applied by diluting a freshly prepared
stock solution of the corresponding compound in DMSO, with
the final concentration of 0.05% in the medium. The MTT test
was performed in the last 2 h without changing the culture
medium. Following drug exposure, MTT (Sigma) was added to
the cells at the final concentration of 0.2 mg/mL and incubated
for 2 h, then the culture medium was aspirated and the violet
formazan precipitate dissolved in DMSO. The optical density
was quantified at 540 nm using a multiwell plate reader (iEMS
Reader MF, Labsystems, US), and the percentage of surviving
cells was calculated from the ratio of absorbance of treated to
untreated cells. The IC50 values for the inhibition of cell growth
were determined by fitting the plot of the percentage of surviving
[Pyrene-R3⊂2][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
pyrene-R3 (27.4 mg, 0.05 mmol). Yield: 141 mg (71%); H
NMR (300 MHz, acetone-d6): δ 8.64 (s, 13H, Hα and C6H),
8.16−7.75 (m, 12H, Hβ), 7.60 (s, 12H, Hq), 6.96 (m, 4H, HPy),
6.53 (m, 2H, HPy), 6.31 (m, 3H, HPy and C1′H), 6.00 (d, 12H,
J = 6.0 Hz, Hcym), 5.77 (m, 13H, Hcym and C5H), 5.22 (d,
1H, HPy), 4.61−4.54(m, 4H, C3′H, C4′H and C5′H), 4.26
(m, 4H, PyCH2CH2CH2 and CH2α), 2.96 (sept, 6H, J = 6.9 Hz,
CH(CH3)2), 2.35−2.20 (m, 6H, PyCH2CH2CH2 and C2′H),
2.12 (s, 18H, CH3), 1.38 (d, 36H, J = 6.9 Hz, CH(CH3)2);
13C{1H} NMR (75 MHz, acetone-d6): δ 173.5, 171.1, 170.3,
168.0, 162.9, 152.7, 150.4, 143.1, 140.5, 137.9, 135.0, 129.8,
128.8, 128.3, 127.9, 127.2, 126.7, 126.1, 124.9, 124.1, 123.7, 123.6,
123.0, 122.9, 121.5, 119.3, 115.1, 111.4, 103.7, 102.4, 100.2,
84.9, 83.0, 71.1, 64.6, 41.3, 39.5, 35.2, 31.6, 30.3, 26.6, 21.5,
16.4; IR (neat): 3447, 3067, 2967, 1734, 1536, 1457, 1275,
1030, 853 cm−1; ESI-MS (m/z): 1201.46 [pyrene-R3 +
2 + (CF3SO3)3]3+.
[Pyrene-R4⊂2][CF3SO3]6. This complex was synthesized
according to the procedure described above using compound
1
pyrene-R4 (27.3 mg, 0.05 mmol). Yield: 148 mg (76%); H
NMR (300 MHz, acetone-d6): δ 8.64 (d, 13H, Hα and C6H),
8.16−7.75 (m, 12H, Hβ), 7.60 (s, 12H, Hq), 6.95 (m, 4H, HPy),
6.55 (m, 2H, HPy), 6.33 (m, 3H, HPy and C1′H), 6.00 (d, 12H,
J = 6.3 Hz, Hcym), 5.77 (d, 12H, J = 6.0 Hz, Hcym), 5.24 (m, 1H,
HPy), 4.61−4.54(m, 4H, C3′H, C4′H, and C5′H), 4.08 (m, 4H,
PyCH2CH2CH2 and CH2α), 2.89 (sept, 6H, J = 6.9 Hz,
CH(CH3)2), 2.38−2.20 (m, 6H, PyCH2CH2CH2 and C2′H),
2.12 (s, 18H, CH3), 1.32 (d, 36H, J = 6.9 Hz, CH(CH3)2);
13C{1H} NMR (75 MHz, acetone-d6): δ 173.4, 171.1, 170.3,
168.0, 156.9, 156.3, 152.7, 148.9, 143.2, 142.1, 137.9, 129.8,
128.8, 127.8, 127.2, 126.7, 126.1, 124.8, 124.4, 123.7, 123.6,
123.0, 119.3, 115.0, 111.4, 103.7, 100.2, 85.2, 84.7, 83.0, 70.9,
64.5, 41.2, 39.3, 35.2, 31.7, 30.6, 26.6, 21.6, 16.4; IR (neat):
3447, 3066, 2967, 1735, 1539, 1457, 1275, 1057, 853 cm−1;
ESI-MS (m/z): 1207.50 [pyrene-R4 + 2 + (CF3SO3)3]3+.
469
dx.doi.org/10.1021/bc200472n | Bioconjugate Chem. 2012, 23, 461−471

Bioconjugate Chemistry
Article
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AUTHOR INFORMATION
■
Corresponding Author
*E-mails: bhkim@postech.ac.kr, bruno.therrien@unine.ch.
ACKNOWLEDGMENTS
■
Financial support of this work by the Strategic Korean-Swiss
Cooperative program and a generous loan of ruthenium(III)
chloride hydrate from Johnson Matthey Research Centre are
gratefully acknowledged as well as the KNRRC program for
modified nucleosides.
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