The effect of an amino-acid bridge on binding affinity and cleavage efficiency of pyrenyl-macrocyclic polyamine conjugates toward DNA

Article abstract of DOI:10.1002/cbdv.200800196

A series of pyrenyl-macrocyclic polyamines 5a-5c have been prepared and characterized. Their DNA-cleavage properties were examined under physiological conditions. Without the presence of other additives, the DNA cleavage ability of 5a -5c showed the order

Full text of DOI:10.1002/cbdv.200800196

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The Effect of an Amino-Acid Bridge on Binding Affinity and Cleavage
Efficiency of Pyrenyl-Macrocyclic Polyamine Conjugates toward DNA
by Qiao-Sen Lua), Yu Huanga), Jing Lia), Zhong-Wei Zhangb), Hong-Hui Lin*b), and Xiao-Qi Yu*a)
a) Department of Chemistry, Key Laboratory of Green Chemistry and Technology (Ministry of
Education), Sichuan University, Chengdu, 610064, P. R. China
b) Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life
Sciences, Sichuan University, Chengdu, Sichuan 610064, P. R. China
(fax: þ862885415886; e-mail: xqyu@tfol.com)
A series of pyrenyl-macrocyclic polyamines 5a–5c have been prepared and characterized. Their
DNA-cleavage properties were examined under physiological conditions. Without the presence of other
additives, the DNA cleavage ability of 5a–5c showed the order of 5c>5a>5b. Absorption and
fluorescence experiments showed the binding affinity of 5a–5c to DNA. The interactions of 5a–5c with
CT-DNA indicated that the DNA binding ability followed an order according to their cleavage efficiency.
All the results indicated that the structures of amino-acid bridge in the ligands may affect the DNA
binding and cleavage ability. The cleavage-mechanism studies indicated that singlet oxygen and
superoxide free radicals were involved in the catalytic DNA cleavage process.
1. Introduction. – In recent years, artificial nucleases have attracted increasing
interest due to their potential applications in the fields of molecular-biological
technology and drug development [1–3]. Chemical nucleases show some advantages
over conventional enzymatic nucleases for their smaller size and the ability to avoid
more sterically hindered regions of a macromolecule. Among these synthetic
compounds, a series of polycyclic aromatic hydrocarbons (PAHs) have been studied
extensively [4][5]. PAHs can form stable and depurination adducts with DNA [6], or
enter futile redox cycles to generate reactive oxygen species that can lead to the
formation of oxidatively damaged nucleobases [7]. In the large family of PAHs, pyrene
and their derivatives were widely used as biomarkers to study human PAH exposure
[8–11]. It was reported that they can form DNA covalent adducts or cause DNA strand
breakages upon light irradiation [12][13]. Pyrenyl peptides were also investigated, and
their potential to induce protein strand scission was demonstrated [14][15]. The
research on the photocleavage ability also proved that the aromatic residues
remarkably affected the binding affinity and photocleavage efficiency of pyrenyl
peptide [16]. However, along with the occurrence of the PAHs and hundreds of their
derivatives in the literature, only a few PAH-including receptors with an acid-bridged
cyclen-pyrene structure have been reported as nuclease mimics for the cleavage of
phosphodiester bonds [17].
As a typical macrocyclic polyamine, 1,4,7,10-tetraazacyclododecane (cyclen) was
widely studied for its good solubility under physiological conditions and strong
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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coordination ability toward metal ions [18–21]. Recently, our group has reported
several cyclen derivatives, which were used to mimic natural nucleases and to catalyze
the cleavge of DNA [22–24]. Comparing to metal–cyclen complexes, free 1,7-
dimethylcyclen (DMC) also displayed good DNA cleavage ability [25]. We report
here, for the first time, the design and synthesis of a series of novel amino acid-bridged
cyclen-pyrene compounds. Unlike pyrene [26], these compounds showed excellent
water solubility. The applications of these compounds on catalytic DNA cleavage were
studied by agarose gel electrophoresis, absorption, and fluorescence experiments.
Furthermore, different structures of the amino-acid moiety exhibited dissimilar effects
on the binding and cleavage ability toward DNA. The studies of the cleavage
mechanism showed that singlet oxygen and superoxide free radicals are involved in the
cleavage process. We also assume that pyrenyl-macrocyclic polyamines form DNA
covalent adducts and cause DNA single-strand cleavage [27].
2. Results and Discussion. – 2.1. Preparation of Pyrenyl-Cyclen Compounds. The
synthetic route is depicted in the Scheme. Compounds 2a–2c could be prepared
through the coupling reaction between N-Cbz-protected amino acids and Boc3-cyclen
in the presence of DCC and HOBt with high yields. The Cbz group of 2a–2c was then
removed with Pd/C by catalytic hydrogenation under H2 atmosphere. Subsequent
coupling reactions between deprotected products 3a–3c and pyrene-1-carboxylic acid 6
was performed by using DCC in CH2Cl2. The isolated yields of products 4a–4c ranged
from 55 to 60% depending on the substituted group R. All of the Boc groups were
finally removed by adding trifluoroacetic acid in dichloromethane solution dropwise.
All the structures of new compounds were confirmed by IR, NMR, ESI-MS and
HR-MS.
2.2. DNA Binding and Cleavage Studies. 2.2.1. Absorption Spectrum Investigation.
First, absorption spectra were ultilized to study the binding ability of compounds 5a–5c
to DNA. The absorption titration data were fitted to Eqn. 1:
[DNA]/Deap ¼[DNA]/Deþ1/(KDNADe)
(1)
where [DNA] is the concentration of DNA in base pairs, Deap ¼ea ꢀeF, and De¼eb ꢀeF.
The apparent extinction coefficient, ea, was obtained by calculating the value of A[obs]
/
[5]. eb and eF correspond to the extinction coefficients of the bound form and the free
form of compounds 5a–5c, respectively.
As shown in Fig. 1, the addition of CT-DNA to a solution of 5c resulted in a gradual
decrease in the absorbance at 341 nm, which corresponds to the pyrene chromophore.
Furthermore, a strong hypochromic effect along with a red shift of 13 nm in the
absorption spectra of 5c was also observed. This could be attributed to the pꢀp
stacking between the pyrene chromophore and the nucleobases of DNA [28][29]. The
maximum hypochromism (ca. 47%) was observed at high DNA concentrations
(83 mm); meanwhile, a new band formed at 354 nm. The half-reciprocal analysis
[30][31] of the absorption titration data according to Eqn. 1 gave a linear plot (as
shown in the Inset of Fig. 1) and resulted in an intrinsic binding constant KDNA of 1.6ꢁ
104 mꢀ1 for 5c. Similar changes in absorption were observed in the cases of the other
conjugates, 5a and 5b, with intrinsic binding constants of 1.4ꢁ104 and 0.8ꢁ104 mꢀ1
,

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Scheme. Synthetic Route to Pyrenyl-Macrocyclic Polyamine
respectively. Different structures of the spacing group might affect the DNA binding
constants, and the order of binding abilities of the compounds to CT-DNA is 5c>5a>
5b. The hydrophilic OH group (as in 5b) might be disadvantageous to the interaction
between 5a–5c and DNA. On the contrary, some aliphatic side chain (as in 5c) might
favor the interaction.
2.2.2. Fluorescence Spectroscopy. Ethidium bromide (EB), a well-known molecular
probe, emits intense fluorescence in the presence of DNA upon its strong intercalation
between the adjacent DNA base pairs. It was previously reported that this enhanced
fluorescence could be quenched, at least partly by the addition of a second molecule
[32][33]. This feature could be used to examine the binding strength between DNA
and the test compound. The fluorescence quench results were quite consistent with the
classical Stern–Volmer equation [32][34].
F0/F¼1þKSV [Q]
where F0 and F are the fluorescence intensities in the absence and presence of 5a–5c,
respectively. KSV is the Stern–Volmer quenching constant, and [Q] is the concentration
of 5a–5c. The fluorescence quenching curves of EB bound to CT-DNA by 5a–5c were
shown in Fig. 2. The KSV values for 5a–5c were estimated as 1.2ꢁ104, 0.99ꢁ104, and

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. Absorption spectra of 5c (3.3ꢁ10ꢀ5 m) in the presence of CT-DNA in Tris·HCl buffer (10 mm,
pH 7.0). [DNA]: 8.3–83 mm. Inset: corresponding half-reciprocal plot of 5c.
Fig. 2. Stern–Volmer quenching curves of 5a–5c. lex ¼497 nm. [EB]¼26.7 mg/ml, [DNA]¼3.3 mg/ml,
[5]¼0–8.0 mm.
1.7 ꢁ104 mꢀ1, respectively. This result was consistent with the UV-titration results (5c>
5a>5b).
2.2.3. Agarose Gel Electrophoresis. The interaction of 5a–5c with pUC19 was
studied under physiological conditions (378 and pH 7.4, in 100 mm Tris·HCl buffer
without additional reagent). The cleavage of supercoiled pUC19 plasmid DNA was
studied by monitoring the conversion of circular supercoiled DNA (Form I) to nicked
(Form II) and linear (Form III) DNA. The amount of strand scission was assessed by
agarose gel electrophoresis. Fig. 3 shows the DNA-cleavage results obtained at pH 7.4
(100 mm Tris·HCl buffer, at 378) after 16 h. All three compounds 5a–5c could cleave
supercoiled DNA (Form I) to nicked (Form II) or linear DNA (Form III). In the

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presence of 5a, supercoiled DNA could be efficiently cleaved at the concentration of
0.143 mm (85% conversion; Fig. 3,a, Lane 4). When the concentration of 5a increased
over 0.250 mm (Lanes 8 and 9), Form II and Form III DNA degraded completely into
small pieces. In comparison, 5c showed a slightly higher DNA-cleavage efficiency than
5a, and a much higher compared to 5b (Fig. 3,b). When the concentration of 5b
increased to 0.714 mm, the supercoiled DNA could be completely cleaved. The
quantified data for different forms of DNA produced were listed in Table 1. Under the
same concentration of 5a–5c (0.143 mm), 5c showed better cleavage selectivity than 5a
(no linear DNA formed), and a better cleavage efficiency than 5b (higher content of
Form II).
Fig. 3. Agarose gel-electrophoresis patterns for the cleavage of pUC19 plasmid DNA (7 mg/ml) by 5a–5c
for 16 h in Tris·HCl buffer (100 mM, pH 7.4) at 378. a) Lane 1: DNA control; Lanes 2–9: DNAþ(0.036,
0.071, 0.143, 0.179, 0.214, 0.250, 0.286, 0.429 mm) of 5a; b) Lane 1: DNA control; Lanes 2–9: DNAþ
(0.714, 0.286, 0.214, 0.143, 0.071, 0.036, 0.018, 0.009 mm) of 5b; c) Lane 1: DNA control; Lanes 2–9:
DNAþ(0.036, 0.071, 0.143, 0.179, 0.214, 0.250, 0.286, 0.429 mm) of 5c.
Fig. 4 shows the effect of the reaction time on the cleavage reaction catalyzed by 5c.
Increasing the reaction time resulted in increase of catalytic ability. When extending the
reaction time to 16 h (Fig. 4, Lane 8), supercoiled DNAwas almost converted to nicked
(Form II) DNA completely.
The experiments carried out with H2O2 could enhance the cleavage activity
dramatically (data are not shown); this result confirmed that one of the reactive oxygen
species is involved in causing DNA cleavage. Therefore, the cleavage mechanism was
studied by using a series of scavengers that could inhibit the reactive oxygen species
(ROS). For example, NaN3 was used as singlet oxygen scavenger, superoxide
dismutase (SOD) was used as superoxide scavenger, DMSO and t-BuOH were used
as scavenger of hydroxyl radicals. Plasmid pUC19 DNA was incubated with 5a in the
presence of NaN3, SOD, DMSO, and t-BuOH, respectively, and the results were listed
in Table 2. It is obvious that SOD and NaN3 obviously inhibited the cleavage process.
However, DMSO and t-BuOH did not show any inhibition of against DNA cleavage.
This means that singlet oxygen and superoxide were the most possible ROSs, and the
.
possibility of a HO radical in the mechanism could be ruled out. This result was

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Table 1. Cleavage of pUC19 Plasmid DNA by 5a–5c at Different Concentrations
Compound
Concentration [mm]
Supercoiled [%]
Nicked [%]
Linear [%]
None
5a
5a
5a
5a
5a
5b
5b
5b
5b
5b
5b
5b
5c
0
92
60
44
15
12
10
80
70
64
58
34
27
19
38
25
12
8
8
35
50
75
77
75
20
30
36
42
66
69
76
62
75
88
90
90
0
5
6
10
11
15
0
0
0
0
0
4
5
0
0
0
2
5
0.036
0.071
0.143
0.179
0.214
0.009
0.018
0.036
0.071
0.143
0.214
0.286
0.036
0.071
0.143
0.179
0.214
5c
5c
5c
5c
5
Fig. 4. Agarose gel-electrophoresis patterns for the cleavage of pUC19 plasmid DNA (7 mg/ml) by 5c
(0.179 mm) in a Tris·HCl buffer (100 mm, pH 7.4) at 378. a) Lane 1: DNA control; Lanes 2–8: 5c, 1, 2, 4,
6, 8, 12, and 16 h. b) Quantization of % plasmid relaxation Form II relative to Form I per lane.
.
different from many other DNA cleavage reactions, in which HO radicals acted as
ROSs, and singlet oxygen scavenger could not inhibit the cleavage process.
Although it is still not clear how singlet oxygen causes DNA cleavage, we proposed
.
that compounds 5a–5c produce the radical cation Py þ , which was subsequently
.
trapped by O2 to form the superoxide anion O2ꢀ. A DNA or DNA adduct radical also
might be formed that caused DNA cleavage; the interpretation is also in agreement
with previous observations [18][19][27][35][36].

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Table 2. Effect of Scavengers on DNA Cleavage by 5aa)
Scavenger
ROS quenched
Effect on DNA cleavage [%]
DMSO
t-BuOH
NaN3
OH
OH
1O2
No effect
No effect
ꢀ31
.
SOD
O2ꢀ
ꢀ43
a) pUC19 Plasmid DNA and 5a (0.179 mm) in the presence of standard radical scavengers in a Tris·HCl
buffer (100 mm, pH 7.4) at 378 for 16 h.
3. Conclusions. – The DNA binding and cleavage efficiency of three pyrenyl-cyclen
compounds 5a–5c containing different amino-acid bridges were investigated. Their
DNA binding abilities were studied by UV-titration and fluorescence spectroscopy. The
results indicated that the hydrophilic OH group (as in 5b) might be disadvantageous to
the interaction between 5a–5c and DNA, while a lipophilic side chain (as in 5c) might
favor the interaction. The order of binding abilities of the compounds is 5c>5a>5b,
and the same order was found for their cleavage efficiencies [37]. The interaction
between 5a–5c and pUC19 indicated that the DNA cleavage abilities of 5a–5c
depended on the concentration of 5a–5c as well as on the incubation time. Mechanistic
studies showed that singlet oxygen and superoxide free radicals were involved in DNA
cleavage. We also assume that pyrenyl-macrocyclic polyamines form DNA covalent
adducts, leading to DNA single-strand cleavage.
Experimental Part
1. General. All chemicals and reagents were obtained commercially and used without further
purification. Cyclen and 1,4,7-tris[(tert-butoxy)carbonyl]-1,4,7,10-tetraazacyclododecan (Boc3-cyclen)
were prepared as reported in [38][39]. Compound 6 was synthesized according to the literature [40].
Electrophoresis grade agarose and plasmid DNA (pUC19) were purchased from Takara Biotechnology
Company. Anh. MeCN, CHCl3, and CH2Cl2 were dried and purified under N2 according to standard
methods and were distilled immediately before use. All aq. solns. were prepared from deionized or
distilled H2O. CT-DNA was directly dissolved in H2O at a concentration of 1 or 5 mg/ml and stored at 48
in the dark. IR Spectra: Shimadzu FTIR-4200 spectrometer as KBr pellets or thin films on KBr plates.
Fluorescence spectra: at r.t. in air with a Hitachi FL-4500 fluorescence spectrometer, and corrected for
the system response. Absorption spectra: in aq. soln. on a TU-1901 spectrophotometer. 1H-NMR
Spectra: Bruker AV II-400 MHz and the d scale in ppm, referenced to residual solvent peaks or internal
TMS. ESI-MS and HR-MS: Finnigan LCQDECA and a Bruker Daltonics Bio TOF mass spectrometer,
resp.
2. Synthesis of Pyrenyl-Macrocyclic Polyamines. 2.1. Preparation of 2a–2c. To a soln. of Boc3-cyclen
(3 mol), 1 (3.5 mmol), and 1-hydroxybenzotriazole (HOBt; 3.3 mmol) in CH2Cl2 (50 ml) was added
N,N’-dicyclohexylcarbodiimide (DCC; 3.5 mmol) in an ice bath. The mixture was stirred at 08 for 10 min,
then at r.t. overnight. The white precipitate was filtered, and the filtrate was concentrated in vacuo. The
crude material was purified by flash chromatography (FC; SiO2; AcOEt/hexane 1:2) to give white
amorphous solid 2.
Tris(tert-butyl) 10-{N-[(Benzyloxy)carbonyl]glycyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarbox-
ylate (2a). White amorphous solid. Yield 86%. IR (KBr): 3421, 2974, 2931, 1696, 1647, 1466, 1411, 1366,
1250, 1164, 1043, 966, 864, 775. 1H-NMR (400 MHz, CDCl3, TMS): 1.44–1.48 (m, 3 t-Bu); 3.42–3.46 (m,

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8 CH2); 4.01–4.02 (d, J¼4.8, CH2); 5.11 (s, PhCH2); 5.75 (s, NH); 7.34–7.38 (m, 5 arom. H). HR-MS-
ESI: 686.3735 ([MþNa]þ , C33H53N5O9þ ; calc. 686.3729).
Tri(tert-butyl) 10-{N-[(Benzyloxy)carbonyl]seryl}-1,4,7,10-tetraazacyclodecane-1,4,7-tricarboxylate
(2b). White amorphous solid. Yield 82%. IR (KBr): 3437, 2975, 2931, 1687, 1639, 1466, 1413, 1366,
1250, 1164, 1019, 966, 858, 777. 1H-NMR (400 MHz, CDCl3, TMS): 1.44–1.48 (m, 3 t-Bu); 2.65 (s, CH2O);
3.39–3.80 (m, 8 CH2); 4.69–4.74 (m, OH); 5.12 (s, PhCH2); 5.73 (d, J¼7.6, NH); 7.26–7.37 (m, 5 arom.
H). HR-MS-ESI: 716.3841 ([MþNa]þ , C34H55N5O1þ0 ; calc. 716.3868).
Tri(tert-butyl) 10-[N-[(Benzyloxy)carbonyl]leucyl}-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxy-
late (2c). White amorphous solid. Yield 80%. IR (KBr): 3421, 2974, 2931, 1696, 1647, 1466, 1411, 1366,
1250, 1164, 1043, 966, 864, 775. 1H-NMR (400 MHz, CDCl3, TMS): 0.93 (d, J¼6.8, Me); 1.01 (d, J¼6.4,
Me); 1.45–1.48 (m, 3 t-Bu); 1.74–1.76 (m, CH2); 3.49 (m, 8 CH2); 4.63 (t, J¼8.4, CH); 5.07 (s, PhCH2);
5.44 (d, J¼8.8, NH); 7.33 (m, 5 arom. H). HR-MS-ESI: 742.4361 ([MþNa]þ , C37H61N5O9þ ; calc.
742.4349).
2.2. Preparation of 3a–3c. Pd/C (10 mol-%, 70 mg) was added to 2 (1.0 mmol) dissolved in MeOH
(50 ml), and the resulting mixture was stirred under H2 (1 atm) for 16 h at r.t. The mixture was filtered
through a Celite pad, and the filtrate was evaporated off. The crude material was purified by FC (SiO2;
CHCl3/MeOH 9 :1) to give white amorphous solid 3.
Tri(tert-butyl) 10-Glycyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (3a). White amorphous
solid. Yield 80%. IR (KBr): 3433, 2974, 2931, 1695, 1647, 1467, 1411, 1366, 1249, 1164, 966, 787. 1H-NMR
(400 MHz, CDCl3, TMS): 1.46–1.48 (m, 3 t-Bu); 3.37–3.46 (m, 8 CH2, CH2CO). HR-MS-ESI: 530.3548
([MþH]þ , C25H48N5Oþ7 ; calc. 530.3558).
Tri(tert-butyl) 10-Seryl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (3b). White amorphous
solid. Yield 80%. IR (KBr): 3420, 2975, 2930, 1696, 1467, 1412, 1367, 1249, 1162, 1108, 1055, 978, 847, 787.
1H-NMR (400 MHz, CDCl3, TMS): 1.45–1.46 (m, 3 t-Bu); 3.37–3.71 (m, 8 CH2, CH2OH); 3.79–3.83 (m,
OH); 3.97 (t, J ¼ 7.2, CH). HR-MS-ESI: 560.3654 ([MþH]þ , C26H49N5O8þ ; calc. 560.3650).
Tri(tert-butyl) 10-Leucyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (3c). White amorphous
solid. Yield 79%. IR (KBr): 3433, 2974, 2931, 1695, 1647, 1467, 1411, 1366, 1249, 1164, 966, 787. 1H-NMR
(400 MHz, CDCl3, TMS): 0.94–0.97 (m, 2 Me); 1.47–1.49 (m, 3 t-Bu); 1.78–1.86 (m, CH2CH); 3.17–
3.75 (m, CH, 8 CH2). HR-MS-ESI: 586.4174 ([MþH]þ , C29H55N5O7þ ; calc. 586.4189).
2.3. Preparation of 4a–4c. To a soln. of 3 (1.0 mmol) and 6 (1.1 mmol) in CH2Cl2 was added DCC
(1.1 mmol) at 08, and the mixture was stirred for 2 h at the same temp., and then stirred at r.t. overnight
under N2. The mixture was filtered, and the filtrate was dried (Na2SO4). The solvent was removed under
reduced pressure, then the residue was purified by CC (SiO2; for 4a and 4c, AcOEt/hexane 2 :1, for 4b,
AcOEt/hexane 4 :1).
Tri(tert-butyl) 10-[N-(Pyren-1-ylcarbonyl)glycyl]-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxy-
late (4a). Pale yellow amorphous solid. Yield 56%. IR (KBr): 3403, 2974, 2929, 1694, 1648, 1470, 1410,
1366, 1248, 1163, 851, 777, 622. 1H-NMR (400 MHz, CDCl3, TMS): 1.43–1.49 (m, 3 t-Bu); 3.44–3.49 (m,
8 CH2); 4.46–4.47 (d, J¼4, CH2CO); 8.03–8.05 (d, J¼7.6, 1 pyrene H); 8.07–8.09 (d, J¼9.2, 1 pyrene
H); 8.13–8.24 (m, 6 pyrene H); 8.65–8.67 (d, J¼7.6, 1 pyrene H). ESI-MS: 780.4 ([MþNa]þ ).
Tri(tert-butyl) 10-[N-(Pyren-1-ylcarbonyl)seryl]-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate
(4b). Pale yellow amorphous solid. Yield 55%. IR (KBr): 3427, 2975, 2929, 1695, 1470, 1411, 1367, 1248,
1
1163, 852, 777, 619. H-NMR (400 MHz, CDCl3, TMS): 1.44–1.56 (m, 3 t-Bu); 3.41–3.87 (m, 8 CH2);
4.04–4.05 (d, J¼4.4, CH2OH); 5.35–5.37 (t, J¼6.8, CHCO); 8.04–8.23 (m, 8 pyrene H); 8.64–8.66 (d,
J¼9.2, 1 pyrene H). ESI-MS: 810.4 ([MþNa]þ ).
Tri(tert-butyl) 10-[N-(Pyren-1-ylcarbonyl)leucyl]-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxy-
late (4c). Pale yellow amorphous solid. Yield 60%. IR (KBr): 3412, 2971, 2931, 1696, 1643, 1467, 1410,
1366, 1249, 1164, 851, 778, 620. 1H-NMR (400 MHz, CDCl3, TMS): 1.02–1.04 (d, J¼6.4, Me); 1.18–1.19
(d, J¼6.4, Me); 1.45–1.59 (m, 3 t-Bu); 1.72–1.99 (m, CH2); 2.05 (m, Me2CH); 3.28–3.76 (m, 8 CH2);
5.32–5.36 (m, CHCO); 6.81–6.83 (d, J¼8.4, NH); 8.03–8.24 (m, 8 pyrene H); 8.60–8.62 (d, J¼9.6, 1
pyrene H). ESI-MS: 836.5 ([MþNa]þ ).
2.4. Preparation of 5a–5c. To a soln. of 4 (0.5 mmol) in CH2Cl2 (30 ml) was added CF3COOH (TFA;
2.5 mmol). After stirring at r.t. for 4 h, the solvent was removed under reduced pressure to give an oil. To
the oil were added 50 ml of Et2O, and a white solid was formed by triturating. The solid was washed three

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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times with Et2O and dried under vacuum to give the product salt. The salt was dissolved in H2O, and
10 ml of 2n aq. NaOH soln. were added, and the mixture was then extracted with CH2Cl2 (3ꢁ30 ml). The
org. phase was washed with brine and then dried (Na2SO4). After removing the solvent under reduced
pressure, the product was obtained as a pale yellow solid.
N-[2-Oxo-2-(1,4,7,10-tetraazacyclododecan-1-yl)ethyl]pyrene-1-carboxamide (5a). Pale yellow solid.
Yield 84%. IR (KBr): 3402, 2925, 2846, 1641, 1524, 1457, 1291, 1118, 852, 717, 619. 1H-NMR (400 MHz,
CDCl3, TMS): 2.69–2.91 (m, 6 CH2); 3.61–3.65 (m, 2 CH2); 4.56–4.57 (d, J¼4, CH2CO); 8.05–8.25 (m,
8 pyrene H); 8.68–8.71 (d, J¼9.2, 1 pyrene H). HR-MS-ESI: 458.2551 ([MþH]þ , C27H31N5O2þ ; calc.
458.2556).
N-[1-(Hydroxymethyl)-2-oxo-2-(1,4,7,10-tetraazacyclododecan-1-yl)ethyl]pyrene-1-carboxamide
(5b). Pale yellow solid. Yield 84%. IR (KBr): 3424, 2926, 2851, 1636, 1523, 1456, 1117, 850, 714, 619.
1H-NMR (400 MHz, CDCl3, TMS): 2.56–3.49 (m, 8 CH2); 4.09–4.10 (d, J¼4.4, CH2OH); 4.35–4.38 (t,
J¼13.6, CHCO); 8.02–8.24 (m, 8 pyrene H); 8.65–8.67 (d, J¼9.2, 1 pyrene H). HR-MS-ESI: 488.2656
([MþH]þ , C28H33N5Oþ2 ; calc. 488.2660).
N-[3-Methyl-1-(1,4,7,10-tetraazacyclododecan-1-ylcarbonyl)butyl]pyrene-1-carboxamide (5c). Pale
yellow solid. Yield 85%. IR (KBr): 3427, 2925, 2865, 1633, 1523, 1456, 1117, 850, 714, 619. 1H-NMR
(400 MHz, CDCl3, TMS): 1.01–1.03 (d, J¼6.4, Me); 1.10–1.12 (d, J¼6.4, Me); 1.58–1.64 (m, Me2CH);
1.77–1.89 (m, CH2); 2.51–3.88 (m, 8 CH2); 5.34 (m, CHCO); 7.06 (d, J¼8.4, NH); 8.03–8.24 (m, 8
pyrene H); 8.57–8.59 (d, J¼9.2, 1 pyrene H). HR-MS-ESI: 514.3177 ([MþH]þ , C31H39N5O2þ ; calc.
514.3181).
This work was financially supported by the National Science Foundation of China (Nos. 20725206,
20732004, and 20572075), Specialized Research Fund for the Doctoral Program of Higher Education, and
Scientific Fund of Sichuan Province for Outstanding Young Scientist.
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Received June 3, 2008