Synthesis and Characterization of 4,4'-Di(p-aminophenylethynyl)-6,6'- bis(N,N-bis(t-butoxycarbonylmethyl)amino methyl)-2,2'-bipyridine

Article abstract of DOI:10.14233/ajchem.2014.15686

To obtain a new solid phase time-resolved fluorescence immunoassay bifunctional chelate, a new intermediate compound, 4'-di(paminophenylethynyl)- 6,6'-bis[N,N-bis(t-butoxycarbonylmethyl)amino methyl]-2,2'-bipyridine was synthesized using 4,4'-dinitro-2,2'- bipyridine-6,6'-dimethyl bis(trifluoroacetate) as the initial material by four steps viz., hydrolyzation, bromination, esterification and substitution. The structure of the compound was charactered by IR, ¹H NMR and HRMS.

Full text of DOI:10.14233/ajchem.2014.15686

Asian Journal of Chemistry; Vol. 26, No. 8 (2014), 2256-2258
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.15686
Synthesis and Characterization of 4,4'-Di(p-aminophenylethynyl)-6,6'-
bis(N,N-bis(t-butoxycarbonylmethyl)amino methyl)-2,2'-bipyridine
1
1
1
2
3
2
2
XIU-YUN YANG , DAN-DAN DANG , WEI ZHU , WEN-BING XIE , WEN-BO CUI , YU CHANG , YUN-HUI LI1,* and LI-HUA PAN
1School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022, P.R. China
2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P.R. China
3Jilin Province Design and Research Institute for Petrochemical Engineering, Changchun 130021, P.R. China
*Corresponding author: Fax: +86 431 84727678; Tel: +86 431 85582361; E-mail: liyh@cust.edu.cn; 982618911@qq.com
Received: 25 April 2013;
Accepted: 6 September 2013;
Published online: 15 April 2014;
AJC-15003
To obtain a new solid phase time-resolved fluorescence immunoassay bifunctional chelate, a new intermediate compound, 4'-di(p-
aminophenylethynyl)-6,6'-bis[N,N-bis(t-butoxycarbonylmethyl)amino methyl]-2,2'-bipyridine was synthesized using 4,4'-dinitro-2,2'-
bipyridine-6,6'-dimethyl bis(trifluoroacetate) as the initial material by four steps viz., hydrolyzation, bromination, esterification and
substitution. The structure of the compound was charactered by IR, 1H NMR and HRMS.
Keywords: Solid-phase time-resolved fluorescence immunoassay, Bifunctional chelate, Intermediate.
4,4'-Dinitro-2,2'-bipyridine-6,6'-dimethylene bis trifluoroacetate
(compound 1) was prepared in accordance with reference9.
4,4'-Di(p-aminophenylethynyl)-6,6'-bis(N,N-bis(t-butoxy-
carbonylmethyl)amino methyl)-2,2'-bipyridine (compound 5)
was prepared in accordance with reference8.
INTRODUCTION
Time-resolved fluorescence immunoassay (TRFIA)
analysis method1 is a ultramicro analytical techniques in vitro,
which based on lanthanide chelates as labels. There are two
different systems in the field of TRFIA:DELFIA (dissociation
enhanced lanthanide fluoroimmunoassay, DELFIA) and DSLFIA
(direct solid lanthanide fluoroimmunoassay, DSLFIA)2. DELFIA
system, although highly sensitivie, detection range have some
limitatoins.
Elemental analysis were performed with a Vario EL
analyzer. 1H NMR spectra were obtained from Unity-400 M
NMR spectrometer with CDCl3 as a solvent. Fourier transform
infrared spectrometer:Vertex 70; fluorescence spectra: F-7000
analyzer; Fourier transform ion cyclotron resonance mass
spectrometer: 7.0T superconducting magnet type.
Direct solid lanthanide fluoroimmunoassay can be
measured directly, from which we can get more biological
information. For example, DSLFIA can be used in such as in
hybrization, immuno histochemsity, homogeous assays and
fluoresence imaging3. However, its sensitivity is not high due
to the lack of chelating agent which fluorescence intensity is
not ideal and other factors, The synthesis of an ideal chelating
agent has become a challenging research in the field of bio-
chemistry and immunology analysis.
Caution! Although we did not encounter any problems,
there are potentially explosive in organic synthesis process.
They should be used in small quantities and handled with extreme
care. Synthetic routes of 4,4'-di(p-aminophenylethynyl)-6,6'-
bis(N,N-bis(t-butoxycarbonylmethyl)amino methyl)-2,2'-
bipyridine is shown in Fig. 1.
Preparation of compound 2: Compound 1 (2 g) was
dissolved in 40.8 mL THF and 40.8 mL deionized water in a
flask. The reaction mixture was stirred for 5 min at room
temperature, then added NaHCO3 (1.14 g, 0.136 mmol) and
reacted for 20 h.After completion of the reaction, the solution
was filtrated. The filtrate was precipitated with water and
filtrated again, the precipitate got through the twice filtration
mentioned above was dried to obtain a pale yellow solid of
compounds 2.Yield: 1.19 g, (97 %). m.p. 241.58 ºC. Elemental
analysis (%) calcd for compound 2: C, 47.06, H, 3.268, N,
18.30; found: C, 46.47, H, 2.966, N, 17.55. IR (KBr, νmax, cm-1):
According to the chelating agents on the needs of certain
functional groups, a newintermediate compound 4,4'-di(p-
aminophenylethynyl)-6,6'-bis(N,N-bis(t-butoxycarbonyl-
methyl)amino methyl)-2,2'-bipyridine on the basis of the
chelating agent having been synthesized4,5 and a variety of
other intermediates6-8.
EXPERIMENTAL
All the reagents and solvents employed were commer-
cially available and used as received without further purification.

Vol. 26, No. 8 (2014) Synthesis of 4,4'-Di(p-aminophenylethynyl)-6,6'-bis(N,N-bis(t-butoxycarbonylmethyl)amino methyl)-2,2'-bipyridine 2257
NO2
NO2
NO2
NO2
Br
Br
NaHCO3
H2O
HBr/HAc
H2C
O
N
N
F3C
CH2
O
H2C
OH
N
N
CH2
OH
N
N
C
O
CF3
C
Br
Br
O
1
2
3
NH2
NH2
Br
N
Br
HC
NH2
C
C
C
C
NH(CH2COOt–Bu)2
Na2CO3
N
[(C6H5)3P]2PdCl3
N
N
COOt–Bu
COOt–Bu
COOt–Bu
COOt–Bu
N
N
N
N
4
COOt–Bu
COOt–Bu
COOt–Bu
COOt–Bu
5
Fig. 1. Synthetic route of target compound
3183 (OH), 1533 and 1355 (NO2), 3069(C-H). 1H NMR
(CDCl3): 4.85 (d, 4H); 5.95 (t, 2H); 8.23 (d, 2H); 8.82 (d, 2H).
Preparation of compound 3: Compound 3 was synthe-
sized by the condensation of the compound 2 in solution of
hydrobromic acid and CH3CH2OH in a 33 % (w/w) molar ratio
under stirring at 101 ºC for 5 h, then the mixture was poured
on crushed ice and the precipitated solid complex was
separated from the solution by filtration, purified by washing
several times with deionized water until pH = 7, then dried to
obtain 1.39 g solid powder. The solid powder was washed with
ethyl ether, then purified through column chromatography
(dichloromethane: n-hexane = 5:4, silica gel particle size 50-
75 µm) and tracked the received liquid by TLC, after collecting
and drying, 1.14 g solid was obtained. Yield: 1.14 g, (47 %).
m.p. 220.5 ºC. Elemental analysis (%) calcd. for compound 3:
C, 28.84, H, 1.163, N, 5.16, Br, 63.95; found: C, 28.70, H,
2.087, N, 5.57, Br, 63.48. IR (KBr, νmax, cm-1):1556 (C=N).
1H NMR (CDCl3): 4.56 (d, 4H); 8.54(d, 2H); 7.66 (d, 2H).
Preparation of compound 4: 1.5 g compound 3, 1.6 g of
anhydrous sodium carbonate and 1.6 g iminodiacetic acid di-
tert-butyl ester were successively added to a three-necked flask,
then 150 mL anhydrous acetonitrile was added under stirring.
The mixture was refluxed for 24 h at 80 ºC. After completion
of the reaction, filtrated the solution and concentrated the
filtrate to get a pale yellow solid. Then it was recrystallizated
with n-hexane, obtain 1.86 g white solid, yield 75 %, m.p.
132.67 ºC, elemental analysis (%) calcd. for compound 4: C,
52.17, H, 6.28, N, 6.76, O, 15.46, Br, 19.32. Found: C, 52.15,
H, 6.14, N, 6.82. O, 15.54, Br, 19.34. IR (KBr, νmax, cm-1):
Preparation of compound 5: 1.5 g compound 4, 0.050 g
bis(triphenylphosphine) dichloride palladium(II) and 0.027 g
cuprous iodide were successively added to a flask, then tetra-
hydrofuran (45 mL), triethylamine (45 mL) and p-aminophenyl
acetylene (0.47 g) were added under nitrogen atmosphere. The
mixture was stirred for 24 h in the temperature range of 50-
52 ºC.After completion of the reaction, the solution was cooled,
filtered. Then the filtrate which was concentrated was dissolved
in chloroform (86 mL) and dried with anhydrous sodium
sulfate after washing. 0.90 g compound 5 was obtained through
column chromatography (petroleum ether: ethyl acetate = 1:1,
silica gel particle size 50-75 µm), yield 55 %, decomposition
temperature: 221.62 ºC, elemental analysis (%) calcd. for
compound 5: C, 69.33, H, 7.11, N, 9.33; found: C, 69.31, H,
7.14, N, 9.28. IR (KBr, νmax, cm-1): 3374 (N-H), 1729 (C=O),
2200 (Ca≡C). 1H NMR (CDCl3): 8.36(s, 2H); 7.69(s, 2H); 7.36
(d, 2H); 6.65 (d, 2H); 4.13 (s, 4H); 3.88 (s, 4H); 3.54 (s, 8H);
1.48 (s, 36H); EI-MS: 901.54837 (M + H), 923.53365 (M +
Na), 939.50632 (M + K).
RESULTS AND DISCUSSION
Design of intermediate molecule of the bifunctional
chelator: Bipyridine and some derivatives of the macrocyclic
compounds in combination with some rare earth ions can form
a stable chelate cryptand10, where the rare earth ions can not
be easily substituted, also with high fluorescence stability11
and the low influence of solvent. Therefore, 4,4'-di(p-amino-
phenylethynyl)-6,6'-bis(N,N-bis(t-butoxycarbonyl-methyl)-
amino methyl)-2,2'-bipyridine was synthesized as the interme-
diate. Isothiocyano groups as the activated moieties for coup-
ling to protein could be introduced through isothiocyanation
of the amino- phenylethynyl.
1
1550 (C=N), 1750 (C=O), 1146 (CH3). H NMR (CDCl3):
1.47(s, 36H); 3.50(s, 8H); 4.17 (s, 4H); 7.88 (d, 2H);8.46 (d,
2H).

2258 Yang et al.
Asian J. Chem.
Comparison of synthetic routes and methods: The yield
Conclusion
of complex 4 was increased to 75 % by the reaction of compound
3 and iminodiacetic acid di-t-butyl ester instead of iminodi-
acetic acid diethyl ester. The yield of compound 5 was increased
to 55 %. The complex 4 was purified through the processing
step of recrystallization instead of column chromatography,
which not only simplified the process of purification but also
avoided the use of methanol which has higher toxicity.
Structure and features of 4,4'-di(p-aminophenylethynyl)-
6,6'-bis(N,N-bis(t-butoxycarbonylmethyl)amino methyl)-
2,2'-bipyridine: Due to the super conjugation effect of tertiary
butyl carbon cations, the hydrolysis reaction rate and yield of
complex 5 are all higher than 4,4'-di(p-aminophenylethynyl)-
6,6'-bis(N,N-bis(ethoxycarbonylmethyl)amino methyl)-2,2'-
bipyridine7.
Synthesis of the present compound will be conducive to
the improvement of the sensitivity of DSLFIA. So this experi-
ment provides an important intermediate compound reference
for the final synthesis of solid-phase time-resolved fluore-
scence immunoassay chelate.
ACKNOWLEDGEMENTS
The authors thank the project supported by the National
Natural Science Foundation of China (Grant No. 30471606)
and Changchun science and technology project (12SF26).
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0
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550
600
650
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Wavelength (nm)
Fig. 2. Fluorescence spectrum of 4,4'-di(p-aminophenylethynyl)-6,6'-
bis(N,N-bis(carboxymethyl)amino methyl)-2,2'-bipyridine-Eu3+