A highly sensitive luminescent lectin sensor based on an α-d-mannose substituted Tb3+ antenna complex

Article abstract of DOI:10.1039/c3dt33023k

Lectin-carbohydrate interactions are the basis of many biological processes and essentially they constitute the language through which intercellular communications are codified. Thus they represent powerful tools in the examination and interpretation of changes that occur on cell surfaces during both physiological and, more importantly, pathological events. The development of optical techniques that exploit the unique properties of luminescent lanthanoid metal complexes in the investigation of lectin-carbohydrate recognition can foster research in the field of ratiometric biosensing and disease detection. Here we report the synthesis of a Tb³⁺-DO3A complex (Tb?1) bearing an α-d-mannose residue and the related study of binding affinity with concanavalin A (Con A) labeled with rhodamine-B- isothiocyanate (RITC-Con A). Luminescence spectroscopy and dynamic studies show changes in emission spectra that can be ascribed to a luminescence resonance energy transfer (LRET) from Tb?1 (donor) to RITC-Con A (acceptor). The binding constant value between the two species was found to be one order of magnitude larger than those previously reported for similar types of recognition. To the best of our knowledge this is the first example of the use of a pre-organized luminescent lanthanoid complex in the study of carbohydrate-protein interactions by LRET. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3dt33023k

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Transactions
An international journal of inorganic chemistry
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Volume 42 | Number 26 | 14 July 2013 | Pages 9391–9786
ISSN 1477-9226
COVER ARTICLE
Capobianco, Quici et al.
A highly sensitive luminescent lectin sensor based on an α-D-mannose
substituted Tb3+ antenna complex
1477-9226(2013)42:26;1-K

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PAPER
A highly sensitive luminescent lectin sensor based on
an α-D-mannose substituted Tb3+ antenna complex†
Cite this: Dalton Trans., 2013, 42, 9453
Emma Martín Rodríguez,a Nicoleta Bogdan,a John A. Capobianco,*a
Simonetta Orlandi,b,c Marco Cavazzini,b,c Chiara Scalerab,c and Silvio Quici*b,c
Lectin–carbohydrate interactions are the basis of many biological processes and essentially they consti-
tute the language through which intercellular communications are codified. Thus they represent power-
ful tools in the examination and interpretation of changes that occur on cell surfaces during both
physiological and, more importantly, pathological events. The development of optical techniques that
exploit the unique properties of luminescent lanthanoid metal complexes in the investigation of lectin–
carbohydrate recognition can foster research in the field of ratiometric biosensing and disease detection.
Here we report the synthesis of a Tb3+-DO3A complex (Tb⊂1) bearing an α-D-mannose residue and the
related study of binding affinity with concanavalin A (Con A) labeled with rhodamine-B-isothiocyanate
(RITC-Con A). Luminescence spectroscopy and dynamic studies show changes in emission spectra that can
be ascribed to a luminescence resonance energy transfer (LRET) from Tb⊂1 (donor) to RITC-Con A (accep-
tor). The binding constant value between the two species was found to be one order of magnitude
larger than those previously reported for similar types of recognition. To the best of our knowledge this
is the first example of the use of a pre-organized luminescent lanthanoid complex in the study of carbo-
hydrate–protein interactions by LRET.
Received 17th December 2012,
Accepted 14th February 2013
DOI: 10.1039/c3dt33023k
www.rsc.org/dalton
as probes for studying carbohydrates on cell membranes. The
nature and the concentration of saccharides or polysacchar-
Introduction
The ability to probe the activity and reactivity of biomolecules ides on the different organs and cells can significantly change
as they occur within living cells is fundamental to furthering depending on the cell type and more importantly on whether
biomedical science. Thus, the detection and simultaneous the cell is healthy or cancerous.2 Therefore the interaction
monitoring of chemical interactions between biological targets between lectins and carbohydrates is of paramount impor-
have become indispensable in medical diagnosis, targeted tance to quantitatively analyze carbohydrates of interest since
therapeutics and molecular biology. In this sense, the interest it can be used as a diagnostic marker for different types of
in lectins, which are proteins containing specific domains for diseases.3
the recognition of sugars, has intensified during the last few
The lectin–carbohydrate binding, which generally relies on
years. In fact lectins proved to be extremely valuable tools for the multivalent interactions of the protein with responsive
the analysis of cell surface sugars, for the assessment of the molecules containing several monosaccharide units (glyco-
role of the latter in cell growth and differentiation in both dendrimers), has been studied among others by micro-
physiological and pathological processes.1 Thanks to their gravimetric,4 electrochemical5 and optical methods.6 In
specific binding with sugars, lectins have been generally used comparison to other methods of investigation, the use of
luminescent glycodendrimers as biosensors for the recog-
nition of lectins presents some advantages: luminescence
aDepartment of Chemistry and Biochemistry and Centre for Research in NanoScience
measurements are very sensitive (even single molecule detec-
of Concordia University, 7141 Sherbrooke Street West, Montreal, QC H4B 1R6,
tion is possible), versatile and can be easily performed.7 More-
Canada. E-mail: capo@vax2.concordia.ca; Fax: (+) 1-515-848-2868;
over low detection limits would allow the monitoring
Tel: (+) 1-514-848-2424
bIstituto di Scienze e Tecnologie Molecolari (ISTM) of Consiglio Nazionale delle
of biological events as they occur, facilitating the understand-
ing of the origin and growth of several diseases, including
cancer.
Glycodendrimers have been synthesized on fluorescent
oligothiophenes,8 Ru(II), Re(II) and Ir(III) metal complexes,9–11
quantum dots,12 gold13 and NaGdF4:Er3+,Yb3+ up-converting
Ricerche (CNR), Via Golgi 19, I-20133 Milano, Italy
cPolo Scientifico Tecnologico (PST) – CNR, Via Fantoli 16/15, I-20138 Milano, Italy.
E-mail: silvio.quici@istm.cnr.it; Fax: (+) 0039 02 503 14159;
Tel: (+) 0039 02 503 14164
†Electronic supplementary information (ESI) available: 1H NMR and 13C NMR
spectra of all new compounds are given. See DOI: 10.1039/c3dt33023k
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nanoparticles,14 and applied to monitoring carbohydrate–
protein recognition events.
An important issue is the development of new sensors for
continuous in vivo detection of glucose in diabetic patients. To
this aim several fluorescent receptors have been investigated
and in many cases lectin concanavalin A (Con A) has been
used as the glucose-binding unit. The technique generally
used consists of measuring changes in fluorescence (or
Förster) resonance energy transfer (FRET), which involves the
non-radiative energy transfer from a fluorescent donor mole-
cule to an acceptor molecule in close proximity. This results in
a decrease of fluorescence intensity and the lifetime of the
donor.15 On the other hand the use of FRET on biological
systems has some limitations that are strictly related to the
photophysical properties of fluorescent probes (short lifetime
of excited state, poor signal to background limit, small
amount of energy transfer above 80 Å).16
For these reasons luminescent lanthanoid complexes, such Fig. 1 Schematic representation of luminescence resonance energy transfer
as Eu3+ or Tb3+ chelates, have been widely used as donors in a
(LRET) involving the Tb⊂1 complex and RITC-Con A.
simple modification to the standard FRET generally referred to
as luminescence (or lanthanoid-based) resonance energy trans-
fer (LRET).16 The LRET system relies on the same fundamental
the concentration of Con A increases. Since in this system
both the intensity of the emission and lifetimes are related to
mechanism, however the unique optical properties of lantha-
the concentration of Con A, the studied complex is behaving
noid emission (narrow and intense bands, long emission life-
as a sensor with respect to Con A.
time, absence of luminescence self-quenching processes)
offer several potential advantages that can overcome FRET
drawbacks. Hamachi and coauthors showed that ratiometric
sensing of complicated saccharide derivatives is possible using
Results and discussion
Design and synthesis of ligands and the complex
LRET between an engineered lectin biosensor complexed with
Tb3+.17 To the best of our knowledge, however, no examples of
the use of pre-organized lanthanoid antenna complexes in the The structure of ligand 1 was designed to perform efficiently
study of carbohydrate–protein interactions have been reported the two functions of lectin recognition and sensitization of the
in the literature. In our opinion, the high values of emission metal emission. 1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic
quantum yields of the complexed lanthanoid ion and the acid (DO3A) was selected as the Ln-chelating subunit because
excellent spectral overlap with acceptor counterparts could of the high thermodynamic and kinetic stabilities which are
have a strong effect in the resonance energy transfer process, well assessed for its lanthanoid complexes.19 An acetophenone
increasing the precision of energy transfer measurements over was introduced at the 10 position of the macrocycle. The
long distances based on donor lifetime. In addition the choice of such a chromophore was dictated by its efficiency in
remarkable chemical, thermal, kinetic and photophysical the sensitization process that overcomes the low absorption
stability of lanthanoid metal complexes together with cross section of Tb3+ (ε < 1 M−1 cm−1).20 In addition the func-
their very low toxicity18 make them suitable candidates for the tionalization at the para position of the phenyl ring with a
development of in vivo biosensing technologies.
short alkyl chain allows the easy introduction of a primary
In this paper we describe (i) the synthesis of the highly amino group that can be used to bond a tailored lectin
luminescent Tb3+-DO3A complex (Tb⊂1) bearing an α-D- binding residue, i.e. α-D-mannose.
mannose residue on the side chain; (ii) the complete photo-
physical characterization of Tb⊂1 and of rhodamine-B-isothio- group on the lateral chain is shown in Scheme 1.
cyanate-labeled Con A (RITC-Con A) in phosphate buffer Reaction of commercially available 4-hydroxyacetophenone
The synthesis of the protected ligand 8 bearing the amino
solution; and (iii) the study of complex Tb⊂1 emission in the 2 with 3-bromopropylphthalimide 3 was carried out in CH3CN
presence of increasing amounts of RITC-Con A. The results and refluxed for 3 days, in the presence of solid Na2CO3 as a
obtained indicate a decrease of the Tb3+ ion emission intensity base, and afforded 3-(4-acetylphenoxy)-propylphthalimide 4 in
and sensitization of RITC emission due to the binding 97% yield. Treatment of 4 with N-bromosuccinimide (NBS)
between Con A and the mannose residue of the Tb3+ complex, and p-toluenesulphonic acid (p-TsOH) in CH3CN at reflux for
thus promoting the LRET process in which the terbium 2 h produced 3-[4-(2-bromoacetyl)phenoxy]-propylphthalimide
complex behaves as the donor and RITC as the acceptor 521 with a 68% yield. Alkylation of DO3A tris(tert-butyl) ester
(Fig. 1). The lifetime of the luminescence originating from the hydrochloride 622,23 with 5 carried out in CH3CN at reflux
7
5D4 excited state to the F6 level of the Tb3+ ion decreases as in the presence of solid Na2CO3 as a base afforded 7 in 91%
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Scheme 1 Synthesis of ligand 8 bearing the amino group on the lateral chain.
yield, after purification of the crude product by column
chromatography. Selective deprotection of the phthalimido
group of 7 was obtained with hydrazine hydrate in EtOH at
reflux for 3 h to afford 8 in 84% yield as a yellow solid foam.
The preparation of related complex Tb⊂1 has been carried
out following the synthesis reported in Scheme 2.
Scheme 2 Synthesis of lanthanoid complex Tb⊂1.
inorganic salts, and then with 90 : 10 H2O–MeCN (v/v) afforded
the Tb⊂1 in 83% yield as a white crystalline solid.
The 3-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosylthio)propa-
noic acid 10 was obtained in 69% yield according to a
procedure reported for an analogous galactose-derivative,24 by
reaction of pentaacetyl α-D-mannose 9 with 3-mercaptopropio-
nic acid in anhydrous CH2Cl2 and in the presence of BF3·Et2O
at 0 °C for 9 h. Reaction of 10 with the DO3A amino derivative
8 was carried out in anhydrous CH2Cl2 in the presence of
1-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide
(DCC).25 The temperature was raised from 0 °C to RT overnight
and the reactants were continuously stirred. This procedure
afforded 11 as a solid yellow foam in 65% yield after purifi-
cation by column chromatography. Deprotection of 11 was
achieved in two steps: it was first treated with CF3COOH in
anhydrous CH2Cl2 for 48 h at room temperature to remove the
three tert-butyl ester protecting groups and then it was reacted
with sodium methoxide in CH3OH for 24 h at RT to remove
the four acetate groups on the mannose residue, affording the
crude ligand 1. This product was purified by a short Amberlite
XAD 1600T column (from 100% H2O to 80 : 20 H2O–acetone)
to give 61% yield (over two steps) of the title compound as a
white crystalline solid.
Photophysical characterization and LRET
In order to verify the effectiveness of Tb⊂1 as a sensor for the
detection of lectins by means of luminescent energy transfer
(LRET), we focused our attention on model plant lectin con-
canavalin A (Con A) labeled with rhodamine-B-isothiocyanate
(RITC-Con A).
Upon excitation at 355 nm, Fig. 2 shows the spectral behav-
iour of Tb⊂1 (donor), which emits strong fluorescence at 480,
550, 580 and 625 nm from the 5D4→7F6, 5D4→7F5, 5D4→7F4
and 5D4→7F3 transitions of the Tb3+ ion, respectively. Less
intense peaks are observed in the 650 to 700 nm region of the
spectrum and are attributed to the 5D4→7F2,1,0 transitions of
the Tb3+ ion.
The emission spectra of mixed solutions of the lanthanoid
complex Tb⊂1 (1.9 mM) and increasing concentrations of
RITC-Con A (from 0.5 to 8.3 μM) in PBS at pH 7.4 were
recorded and the results are reported in Fig. 3a as a function
of acceptor concentration. The LRET process was evidenced by
the decrease in the emission intensities of the peaks at 550
5
5
and 480 nm corresponding to the D4→7F5 and D4→7F6 tran-
sitions of Tb3+ respectively. In addition the occurrence of
lanthanoid-based energy transfer phenomena was also sup-
ported visually by the color change under UV irradiation of
the solutions, from green (typical of Tb3+ emission) to reddish
(Fig. 3b).
The Tb⊂1 complex was prepared by addition of a solution
of TbCl3·6H2O in H2O into a water solution of ligand 1 under
magnetic stirring and adjusting the pH at 6.5–7.0 with 2%
aqueous NaOH. The reaction mixture was then heated at reflux
for 4 h. Purification of the residue by a short Amberlite XAD
1600T column eluting first with 100% H2O, to remove all
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Fig. 2 Absorption and emission spectra (λex = 557 nm) of RITC-Con A and the
emission spectrum (λex = 355 nm) of the Tb⊂1 complex.
Fig. 4 Lifetime of the 5D4 state of Tb3+ measured at 550 nm for different con-
centrations of RITC-Con A. The inset shows the decay curves used to calculate
the lifetime values.
Table 1 Lifetimes, energy transfer rates and efficiencies for the different solu-
tions used in this article
[RITC-Con A]/ [RITC-Con A]/
μM
E from
τ/ms kET/ms−1 E from τ Itotal
[Tb⊂1] 10−3
0
1.75
1.47
1.25
1.03
0.73
0.46
0.52
1.04
2.08
4.17
8.33
0.268
0.536
1.07
2.15
4.30
0.11
0.23
0.40
0.80
1.60
0.16
0.29
0.41
0.58
0.74
0.16
0.23
0.32
0.46
0.59
and Table 1, where the lifetime data at 550 nm are collected,
the donor-only signal is a single exponential with a lifetime of
1.75 ms. By adding increasing concentrations of the acceptor
the lifetime of the donor decreases to a value of 0.46 ms
(Table 1). The decrease in the excited state lifetime of the
donor is due to the fact that the acceptor provides an
additional relaxation pathway of the donor excited state.
In order to exclude the intervention of non-selective LRET
processes in the observed phenomena, we compared the be-
havior of Tb⊂1 with that of a similar complex, namely
CH3O-Tb3+·DO3A26 that does not have mannose residues
able to give recognition of the lectin (Fig. 5). The lifetime
decay curves of two solutions at the same concentration
(1.5 × 10−5 M) of Tb⊂1 and CH3O-Tb3+·DO3A, respectively,
were measured in the presence and in the absence of a fixed
concentration of RITC-Con A (0.5 μM). Due to the overlap of
emission spectra of the 5D4→7F4 transition of the Tb3+ ion and
RITC-Con A, at this wavelength more noticeable differences are
evidenced.
Fig. 3 (a) Emission spectra of solutions containing a fixed concentration of
Tb⊂1 (1.9 mM) and increasing concentration of RITC-Con A (from 0.5 to
8.3 μM); (b) image of the same solutions containing a fixed concentration of
Tb⊂1 (1.9 mM) and increasing concentration of RITC-Con A (from 0.5 to 8.3 μM,
from left to right) illuminated with a UV-lamp (325 nm).
Whereas the decay curve of CH3O-Tb3+·DO3A maintains the
same exponential shape both in the presence and in the
absence of the acceptor counterpart, by adding RITC-Con A to
Tb⊂1 the temporal behaviour of the intensity shows a short
initial rise time, associated with the population of the
The LRET process was confirmed by measuring the lumi-
5
nescence lifetime of the D4→7F5 transition of the Tb3+ ion as
a function of the acceptor concentration. As shown in Fig. 4
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transfer rate may be calculated from eqn (1) and (2) and may
be expressed as
1
1
kET
¼
ꢀ
ð3Þ
τDA τD
Table 1 shows the calculated energy transfer rates (kET),
which increase with increasing concentration of the acceptor.
The highest transfer rate of 1.60 ms−1 is achieved at an
acceptor/donor ratio of 4.30 × 10−3 : 1. The LRET efficiencies,
E, can be obtained from the measured lifetime values or the
luminescence intensities using eqn (4) or (5) respectively:
τDA
E ¼ 1 ꢀ
E ¼ 1 ꢀ
ð4Þ
ð5Þ
τD
IDA
ID
where τDA and τD represent the luminescence lifetimes and IDA
and ID represent the luminescence intensities in the presence
and absence of the acceptor respectively. As shown in Table 1,
the LRET efficiency E is enhanced with increasing acceptor
concentration and the efficiencies calculated using the
emission intensities are in good agreement with the ones
calculated with the lifetime. The high energy transfer
efficiency is attributed to the excellent spectral overlap of the
donor emission and the acceptor absorption.
Fig. 5 Comparison of the decay curves of the 5D4 state of Tb3+ measured at
588 nm for the CH3O-Tb3+·DO3A (top) and Tb⊂1 (bottom) in the presence (red)
and absence (black) of RITC-Con A.
From the emission spectra in Fig. 3a, the binding constant
between the Tb⊂1 complex and RITC-Con A was calculated
using eqn (6):27
RITC-Con A excited level via LRET from the Tb3+, followed by
an analogous exponential decrease as obtained at 550 nm
wavelength. This different behaviour in the lifetime curves
between the CH3O-Tb3+·DO3A (which does not present a rise
time or a decrease in the lifetime) and the Tb⊂1 (which pre-
sents the rise time and the decrease in the lifetime) demon-
strates that efficient LRET is possible only after recognition
of RITC-Con A protein by Tb⊂1 via the mannose molecule
which ensures close proximity between donor and acceptor
molecules.
½RITC-ConAꢁF0 ½RITC-ConAꢁF0
F0
ΔFmaxKa
¼
þ
ð6Þ
ΔF
ΔFmax
where ΔF = F0 − F, ΔFmax = F0 − Fmax. F0 and F represent the
intensity of the Tb⊂1 complex before and after the addition of
RITC-Con A, respectively. Fmax is the maximum intensity at
585 nm obtained in the presence of the lectin while Ka is the
binding constant (in M−1). Fig. 6 shows the product of the
The extent of energy transfer may be evaluated from the
change in the excited state lifetime of the donor. It is well
established that the measured lifetime (τD) in the absence of
the acceptor can be expressed as
1
k
1
τD
¼
¼
ð1Þ
kr þ knr
where kr and knr are the radiative and non-radiative rates
respectively. In the presence of the acceptor the lifetime of the
donor may be expressed as
1
1
τDA
¼
¼
ð2Þ
kDA kr þ knr þ kET
where kET represents the energy transfer rate. Since the radia-
tive and non-radiative rates may be considered constant, the
decrease in the lifetime of the donor in the presence of the
Fig. 6 Product of the concentration of RITC-Con A and F0/ΔF as a function of
acceptor is due to energy transfer. Therefore, the energy the concentration of RITC-Con A calculated from the spectra.
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concentration of RITC-Con A and F0/ΔF as a function of the the Tb⊂1 complex was obtained with an electrospray ion-trap
concentration of RITC-Con A, which yields a straight line (R2 = mass spectrometer ICR-FTMS APEX II (Bruker Daltonics)
0.9852). From the slope and the intercept the binding constant by the Centro Interdipartimentale Grandi Apparecchiature
Ka was calculated to be (4.3 0.7) × 105 M−1. This value is one (C.I.G.A.) of the University of Milano. Elemental analyses
order of magnitude larger than values previously reported,14,28 were carried out by the Departmental Service of Microanalysis
which clearly indicates that the Tb⊂1 is a very efficient donor (University of Milano).
for energy transfer assays to study the binding-interactions
3-(4-Acetylphenoxy)-propylphthalimide (4). A mixture of
between α-D-mannose and the lectin Con A.
4-hydroxyacetophenone (1.36 g, 10 mmol), 3-bromo-
2
propylphthalimide 3 (3.0 g, 11 mmol) and Na2CO3 (3.18 g,
30 mmol) in MeCN (30 mL) was refluxed for 72 h under mag-
netic stirring. After cooling, the mixture was diluted with
CH2Cl2, filtered through a glass frit and the solvent was
Conclusions
We have synthesized a new luminescent lanthanoid complex, removed at reduced pressure. The solid residue was purified by
namely Tb⊂1, bearing a mannose function on a lateral chain. crystallization from MeCN (20 mL) to give the title compound
The binding affinity of such a compound towards concanavalin (2.3 g) as a white solid. The mother liquor was concentrated at
A labeled with rhodamine-B-isothiocyanate (RITC-Con A) was reduced pressure and the residue purified by chromatography
demonstrated by optical methods. In fact the spectroscopic (silica gel, CH2Cl2–MeOH 98.5 : 1.5) to give a second portion of
investigation of mixed solutions of lanthanoid complex Tb⊂1 the title compound (846 mg) as a white solid (overall yield:
and RITC-Con A showed that an effective luminescence reson- 97%). 1H NMR (400 MHz, CDCl3): δ 2.22 (quin, 3J(H,H) =
ance energy transfer took place from the excited state of the 6.0 Hz, 2H, NCH2CH2CH2O), 2.54 (s, 3H, –COCH3), 3.93 (t,
3
Tb3+ ion to rhodamine, resulting in a decrease of fluorescence 3J(H,H) = 6.0 Hz, 2H, NCH2CH2CH2O), 4.11 (t, J(H,H) = 6.0 Hz,
3
intensity and lifetime of the donor. To the best of our knowl- 2H, NCH2CH2CH2O), 6.82 (d, J(H,H) = 8.9 Hz, 2H, Ar-H), 7.73
edge the one presented here is the first example of the use of a (m, 2H, Phthal-H), 7.84 (m, 2H, Phthal-H), 7.88 (d, 3J(H,H) =
pre-organized complex of the lanthanoid ion in the study of 8.9 Hz, 2H, Ar-H); 13C NMR (100.6 MHz, CDCl3): δ 26.3
carbohydrate–protein interactions by LRET. The binding con- (COCH3), 28.2 (NCH2CH2CH2O), 35.4 (NCH2CH2CH2O), 65.9
stant Ka between Tb⊂1 and RITC-Con A was calculated and its (NCH2CH2CH2O), 114.1 (Ar-CH), 123.3 (Phthal-CH), 130.4,
value was found to be one order of magnitude larger than 130.5 (Ar-CH), 132.1 (Phthal-C), 134.0 (Phthal-CH), 162.6,
those previously reported for similar compounds. However, 168.4 (Phthal-CO), 196.7 (COCH3); ESI-MS: m/z = 346.2 ([M +
since lectin–carbohydrate adhesion generally involves multi- Na+]), calcd for C19H17NaNO4 = 346.1; elemental analysis calcd
valent interactions, we intend to investigate in the future glyco- (%) for C19H17NO4: C 70.58, H 5.30; found: C 70.66, H 5.31.
dendrimer derivatives of the luminescent lanthanoid complex
3-[4-(2-Bromoacetyl)phenoxy]-propylphthalimide (5). N-Bromo-
as optical biosensors in the detection of many biological succinimide (1.25 g, 7 mmol) was added in one portion to
events.
a solution of 4 (2.26 g, 7 mmol) and p-toluenesulfonic acid
monohydrate (2.0 g, 10.5 mmol) in MeCN (100 mL). The reac-
tion mixture was refluxed under magnetic stirring for 2 h.
After cooling, the solvent was removed at reduced pressure and
the residue dissolved in CH2Cl2 (150 mL). The organic phase
was washed with water (100 mL), dried over MgSO4 and after
Experimental section
Materials and methods
All available chemicals and solvents were purchased from com- filtration, the solvent was removed at reduced pressure. The
mercial sources and were used without any further purifi- solid residue was purified by chromatography (silica gel,
cation. Thin layer chromatography (TLC) was conducted on CH2Cl2–MeOH 98.5 : 1.5) to give the title compound as a white
1
plates precoated with silica gel Si 60-F254 (Merck, Darmstadt, solid (1.91 g, 68%). H NMR (400 MHz, CDCl3): δ 2.22 (quin,
3
Germany). Column chromatography was conducted by using 3J(H,H) = 6.0 Hz, 2H, NCH2CH2CH2O), 3.92 (t, J(H,H) = 6.0 Hz,
silica gel Si 60, 230–400 mesh, 0.040–0.063 mm (Merck, 2H, NCH2CH2CH2O), 4.11 (t, 3J(H,H)
= 6.0 Hz, 2H,
1
3
Darmstadt, Germany). H and 13C NMR spectra were recorded NCH2CH2CH2O), 4.38 (s, 2H, CH2Br), 6.84 (d, J(H,H) = 7.0 Hz,
on a Bruker Avance 400 (400 and 100.6 MHz, respectively); 2H, Ar-H), 7.72 (m, 2H, Phthal-H), 7.84 (m, 2H, Phthal-H), 7.91
3
chemical shifts are indicated in parts per million downfield (d, J(H,H) = 7.0 Hz, 2H, Ar-H); 13C NMR (100.6 MHz, CDCl3):
from SiMe4, using the residual proton (CHCl3 = 7.26 ppm, δ 28.2 (NCH2CH2CH2O), 30.7 (CH2Br), 35.3 (NCH2CH2CH2O),
(CH3)2SO = 2.50 ppm, HOD = 4.80 ppm) and carbon (CDCl3 = 66.1 (NCH2CH2CH2O), 114.4 (Ar-CH), 123.3 (Phthal-CH), 127.0,
77.0 ppm, (CD3)2SO = 40.45 ppm) solvent resonances as the 131.3 (Ar-CH), 132.1 (Phthal-C), 134.0 (Phthal-CH), 163.3,
internal reference. Protons and carbon assignments were 168.4 (Phthal-CO), 189.9 (COCH2Br); ESI-MS m/z = 402.1 ([M +
achieved by 13C-APT, 1H–1H COSY, and 1H–13C heteronuclear H+]), calcd for C19H17BrNO4 = 402.0; elemental analysis calcd
correlation experiments. Coupling constant values J are given (%) for C19H16BrNO4: C 56.73, H 4.01, N 3.48; found: C 56.68,
in Hz. Low resolution mass spectra were recorded on a H 4.02, N 3.47.
Finnigan LCQ Advantage Thermo-spectrometer equipped with
10-[4-(3-Phthalimidopropyloxy)benzoylmethyl]-1,4,7,10-tetra-
an electrospray ion trap. The high resolution mass spectrum of azacyclododecane-1,4,7-triacetic acid tris(1,1-dimethylethyl)
9458 | Dalton Trans., 2013, 42, 9453–9461
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ester (7). A mixture of 1,4,7,10-tetraazacyclododecane-1,4,7- 7.69 mmol) in dry dichloromethane (40 mL) was added under
triacetic acid tris(1,1-dimethylethyl) ester hydrochloride 6 an inert atmosphere 3-mercaptopropionic acid (2.7 mL,
(0.551 g, 1 mmol), 5 (0.480 g, 1 mmol) and Na2CO3 (1.06 g, 31 mmol) and the solution was cooled to 0 °C. After the slow
10 mmol) in MeCN (20 mL) was refluxed for 30 h under mag- addition of BF3·Et2O (3.0 mL, 23.7 mmol), the mixture was
netic stirring. After cooling, the mixture was filtered through a stirred at 0 °C for 9 h and then diluted with dichloromethane
glass frit and the solvent was removed at reduced pressure. (20 mL). The mixture was washed with water (3 × 20 mL) and
The residue was dissolved in CH2Cl2 (150 mL) and the organic brine (20 mL) and dried over MgSO4. After filtration, the
phase was washed with water (100 mL) and dried over MgSO4. solvent was removed at reduced pressure and the crude was
After filtration, the solvent was removed at reduced pressure purified by chromatography (silica gel, n-hexane–AcOEt 50 : 50,
and the residue was purified by chromatography (silica gel, 1% AcOH) to give the title compound (2.3 g, 69%) as a trans-
CH2Cl2–MeOH 95 : 5) to give the title compound as a white parent oil. 1H NMR (400 MHz, DMSO-d6): δ 1.92 (s, 3H,
1
solid (778 mg, 93%). H NMR (400 MHz, CDCl3): δ 1.25–1.51 OCOCH3), 2.00 (s, 3H, OCOCH3), 2.01 (s, 3H, OCOCH3), 2.09 (s,
(m, 27H, C(CH3)3), 2.0–3.5 (m, 24H), 3.92 (m, 4H), 4.08 (t, 3J(H, 3H, OCOCH3), 2.57 (t, 3J(H,H) = 7.1 Hz, 2H, SCH2CH2CO2H),
H) = 6.0 Hz, 2H, NCH2CH2CH2O), 6.83 (d, 3J(H,H) = 8.8 Hz, 2.79 (t, 3J(H,H) = 7.1 Hz, 2H, SCH2CH2CO2H), 4.01 (dd, 2J(H,H)
2H, Ar-H), 7.73 (m, 2H, Phthal-H), 7.85–7.81 (m, 4H, Phthal-H = 12.0 Hz, 3J(H,H) = 2.3 Hz, 1H, 6a-H), 4.16 (dd, 2J(H,H) =
3
and Ar-H); 13C NMR (100.6 MHz, CDCl3): δ 27.8 (C(CH3)3), 27.9 12.0 Hz, J(H,H) = 5.8 Hz, 1H, 6b-H), 4.23–4.27 (m, 1H, 5-H),
(C(CH3)3), 28.2 (HNCH2CH2CH2O), 35.3 (HNCH2CH2CH2O), 5.00 (dd, 3J(H,H) = 10.0 Hz, 3J(H,H) = 3.4 Hz, 1H, 3-H), 5.09
3
55.6 (N–C(2)H, N–C(3)H, N–C(5)H, N–C(6)H), 55.9 (N–C(8)H, (t, J(H,H) = 10.0 Hz, 1H, 4-H), 5.16–5.18 (m, 1H, 2-H), 5.5 (s,
N–C(9)H, N–C(11)H, N–C(12)H), 59.8 (N(1)CH2CO2tBu, N(4)- 1H, 1-H); 13C NMR (100.6 MHz, DMSO-d6); δ 20.0 (OCOCH3),
CH2CO2tBu, N(7)CH2CO2tBu), 65.8 (HNCH2CH2CH2O), 81.8 (C- 26.4 (SCH2CH2CO2H), 34.7 (SCH2CH2CO2H), 62.4 (C-6), 66.1
(CH3)3), 81.9 (C(CH3)3), 114.2 (Ar-CH), 123.3 (Ar-CH), 128.9, (C-4), 69.0 (C-5), 69.4 (C-3), 70.4 (C-2), 82.1 (C-1), 169.9
129.8 (Phthal-CH), 132.1 (Phthal-C), 134.1 (Phthal-CH), 162.9, (OCOCH3), 170.0 (OCOCH3), 170.1 (OCOCH3), 170.5
168.4 Phthal-CO), 172.8 (CO2tBu), 197.8 (Ar-CO); ESI-MS m/z = (OCOCH3), 173.3 (COOH); ESI-MS m/z = 459.1 ([M + Na+]),
858.5 ([M + Na+]), calcd for C45H65NaN5O10 = 858.5; elemental calcd for C17H24NaO11S = 459.1; 434.9 ([M − H]−, calcd for
analysis calcd (%) for C45H65N5O10: C 64.65, H 7.84, N 8.38; C17H23O11S
= 435.1; elemental analysis calcd (%) for
found: C 64.55, H 7.86, N 3.46.
C17H24O11S: C 46.78, H 5.54; found: C 46.84, H 5.55.
10-[4-(3-Aminopropyloxy)benzoylmethyl]-1,4,7,10-tetraaza-
10-{4-[3-(3-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosylthio)-
cyclododecane-1,4,7-triacetic acid tris(1,1-dimethylethyl) ester propanamido)propyloxy]-benzoylmethyl}-1,4,7,10-tetraazacyclo-
(8). To a solution of 7 (1.55 g, 1.85 mmol) in EtOH (20 mL) dodecane-1,4,7-triacetic acid tris(1,1-dimethylethyl) ester
was added hydrazine monohydrate (0.9 mL) and the solution (11). To a solution of 8 (705 mg, 1 mmol) and 10 (452 mg,
refluxed under magnetic stirring for 3 h. After cooling, the 1.04 mmol) in dry dichloromethane (30 mL) was added under
solvent was removed at reduced pressure and the residue was an inert atmosphere 1-hydroxybenzotriazole (HOBt, 147 mg,
dissolved in CH2Cl2 (20 mL). The suspension was filtered 1.09 mmol) and the mixture cooled to 0 °C. After the dropwise
through a glass frit to remove the phthalhydrazide and the addition of a solution of dicyclohexylcarbodiimide (DCC,
solution was concentrated at reduced pressure. This treatment 225 mg, 1.09 mmol) in dry dichloromethane (10 mL), the
was repeated three times. After evaporation of the solvent, the mixture was allowed to reach room temperature and further
title compound was obtained as a yellow foam (1.12 g, 84%). stirred overnight. After filtration of precipitated dicyclohexy-
This product was used without further purification. 1H NMR lurea, the mixture was washed with saturated NaHCO3
(400 MHz, MeOD, 60 °C): the signals from the cyclen moiety, (2 × 20 mL) and brine (20 mL) and dried over MgSO4. After fil-
NCH2CH2N and NCH2CO, appear in the range δ 2.0–3.3 as a tration, the solvent was removed at reduced pressure and the
set of broad signals overlapped with signals from the aliphatic crude product was purified by chromatography (silica gel,
chain. Selected signals: δ 1.2–1.5 (m, 27H, C(CH3)3), 3.98 (br s, CH2Cl2–MeOH 95 : 5) to give the title compound (732 mg,
1
2H, N(10)CH2CO), 4.16 (t, 3J(H,H)
=
6.1 Hz, 2H, 65%) as a yellow foam. H NMR (400 MHz, CDCl3): the signals
3
NCH2CH2CH2O), 6.99 (d, J(H,H) = 8.8 Hz, 2H, Ar-H), 7.94 (d, from the cyclen moiety, NCH2CH2N and NCH2CO, appear in
3J(H,H) = 8.8 Hz, 2H, Ar-H); 13C NMR (100.6 MHz, CDCl3): the range δ 2.0–3.5 as a set of broad signals overlapped with
δ 26.9 (C(CH3)3), 27.0 (C(CH3)3), 30.6 (HNCH2CH2CH2O), 37.9 signals from the acetate groups and the aliphatic chains.
2
(HNCH2CH2CH2O), 50.5 (N(10)CH2CO), 55.4 (N–C(2)H, N–C(3)- Selected signals: δ 1.10–1.80 (m, 27H, C(CH3)3), 4.09 (dd, J(H,
H, N–C(5)H, N–C(6)H), 55.6 (N–C(8)H, N–C(9)H, N–C(11)H, H) = 12.0 Hz, 3J(H,H) = 2.3 Hz, 1H, (Man)6a-H), 4.18 (t, 3J(H,H)
N–C(12)H), 59.6 (N(1)CH2CO2tBu, N(4)CH2CO2tBu, N(7)- = 6.6 Hz, 2H, HNCH2CH2CH2O), 4.33 (dd, 3J(H,H) = 12.0 Hz,
CH2CO2tBu), 66.0 (HNCH2CH2CH2O), 81.7 (C(CH3)3), 114.1 3J(H,H) = 5.3 Hz, 1H, (Man)6b-H), 4.39–4.41 (m, 1H, (Man)5-H),
(Ar-CH), 129.1, 129.7 (Ar-CH), 163.4, 173.2 (CO2tBu), 198.3 (Ar- 5.20–5.32 (m, 2H, (Man)3-H, (Man)4-H), 5.33–5.34 (m, 2H,
3
CO); ESI-MS m/z = 728.4 [M + Na+], calcd for C37H63NaN5O8 = (Man)1-H and (Man)2-H), 6.98 (d, J(H,H) = 8.9 Hz, 2H, Ar-H),
3
3
728.4; elemental analysis calcd (%) C37H63N5O8: C 62.95, H 7.71 (t, J(H,H) = 5.7 Hz, 1H, HNCH2CH2CH2O), 7.82 (d, J(H,
8.99, N 9.92; found: C 62.89, H 9.01, N 9.90.
H) = 8.9 Hz, 2H, Ar-H); 13C NMR (100.6 MHz, CDCl3): δ 20.6
3-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosylthio)propanoic (OCOCH3), 20.7 (OCOCH3), 20.8 (OCOCH3), 20.9 (OCOCH3),
acid (10). To a solution of pentaacetyl α-D-mannose 9 (3.0 g, 27.1 (SCH2CH2CONH), 27.8 (C(CH3)3), 27.9 (C(CH3)3), 28.9
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(HNCH2CH2CH2O), 36.1 (SCH2CH2CONH), 36.3 (HNCH2-
10-{4-[3-(3-(α-D-Mannopyranosylthio)propanoyl)aminopropyl-
CH2CH2O), 55.6 (N–C(2)H, N–C(3)H, N–C(5)H, N–C(6)H), 55.8 oxy]benzoylmethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triace-
(N–C(8)H, N–C(9)H, N–C(11)H and N–C(12)H), 59.7 (N(1)- tic acid terbium(III) complex (Tb⊂1). A solution of TbCl3·6H2O
CH2CO2tBu, N(4)CH2CO2tBu, N(7)CH2CO2tBu), 62.5 ((Man) (53.4 mg, 0.14 mmol) in water (5 mL) was slowly added to a
C-6), 66.5 ((Man)C-4), 66.8 (HNCH2CH2CH2O), 68.8 ((Man)C-5), solution of 1 (100 mg, 0.13 mmol) in water (15 mL) keeping
69.6 ((Man)C-3), 70.9 ((Man)C-2), 82.2 (C(CH3)3), 82.4 (C- the pH of the solution at 6.5–7.0 by the addition of 2%
(CH3)3), 82.5 ((Man)C-1), 114.7 (CH-Ar), 128.2, 129.9 (CH-Ar), aqueous sodium hydroxide. The reaction mixture was refluxed
163.7, 169.7 (OCOCH3), 169.8 (OCOCH3), 169.9 (OCOCH3), for 4 h, cooled to room temperature and concentrated at
170.8 (OCOCH3), 171.3 (CONH), 172.6 (CO2tBu), 197.5 (ArCO); reduced pressure. The residue was purified by a short Amber-
ESI-MS m/z = 1146.7 ([M + Na]+), calcd for C54H85NaN5O18S = lite XAD 1600T column eluting first with 100% H2O to remove
1146.5; elemental analysis calcd (%) for C54H85N5O18S: all inorganic salts and then with 90 : 10 H2O–MeCN to give the
C 57.69, H 7.62, N 6.23. Found: C 57.61, H 7.62, N 6.22.
title compound (100 mg, 83% yield) as a white crystalline
10-{4-[3-(3-(α-D-Mannopyranosylthio)-propanamido)propyloxy]- solid. ESI-MS m/z = 966.22294 ([M + Na]+), calcd for C34H50-
benzoylmethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic NaN5O14STb = 966.22206; elemental analysis calcd (%) for
acid (1). To a solution of 11 (700 mg, 0.62 mmol) in dry C34H50N5O14STb: C 43.27, H 5.34, N 7.42. Found: C 43.30,
dichloromethane (12 mL) was added TFA (4 mL) and the H 5.35, N 7.41.
mixture was left stirring at room temperature. After 48 h the
Absorption, luminescence spectroscopy and dynamic studies
solvent was removed at reduced pressure, the residue was
redissolved in dichloromethane (10 mL) and the solvent
removed at reduced pressure. This procedure was repeated
three times. The residue was then triturated with diethyl ether
giving a white powder. After removal of the solvent, the solid
was dried under vacuum. 1H NMR analysis showed complete
removal of the tert-butyl groups. No further purification was
undertaken. The solid material was dissolved in dry methanol
(20 mL) and after the addition of NaOMe (260 mg, 4.8 mmol)
the mixture was left stirring at room temperature for 24 h. The
solution was adjusted to pH = 7 (pH paper) with Amberlite
IR-120, filtered through a glass frit and the solvent was
removed at reduced pressure. The solid residue was purified by
a short Amberlite XAD 1600T column (from 100% H2O to
80 : 20 H2O–acetone) to give the title compound (300 mg, 61%)
Concanavalin A-tetramethylrhodamine (RITC-Con A, Molecular
Probe®), which contains 4 moles of dye per mole of lectin, was
used. The absorption spectrum of RITC-Con A was recorded
using a UV-VIS Cary spectrophotometer (Varian 100 Bio).
Luminescence from the Tb⊂1 complex was obtained upon
excitation with the 3rd harmonic, 355 nm, of an Nd:YAG
Q-switched laser (Quanta Ray, Spectra Physics) with a pulse fre-
quency of 10 Hz and a pulse width of 6 ns. The luminescence
and lifetime measurements were performed on samples dis-
solved in a phosphate buffer saline (PBS) at pH 7.4 in a quartz
cuvette (1 cm path length). The emitted light was collected at
π/2 with respect to the incident beam and subsequently dis-
persed by a 1 m Jarrell–Ash Czerny–Turner double mono-
chromator with an optical resolution of ∼0.15 nm.
A
1
as a white crystalline solid. H NMR (400 MHz, D2O, 60 °C): δ
thermoelectrically cooled Hamamatsu R943-02 photomulti-
plier tube detected the visible emissions. A preamplifier,
model SR440 Standard Research Systems, processed the photo-
multiplier signals and a gated photon-counter model SR400
Standard Research Systems data acquisition system was used
as an interface between the computer and the spectroscopic
hardware. The signal was recorded under computer control
using the Standard Research Systems SR465 software data
acquisition/analyzer system. For the lifetime measurements
the emitted signal was sent directly from the photomultiplier
2.37 (quin, 2H, HNCH2CH2CH2O), 2.92 (t, 3J(H,H) = 6.7 Hz,
2H, SCH2CH2CONH), 3.17–3.30 (m, 2H, SCH2CH2CONH),
3.4–3.6 (br s, 8H, N–C(2)H, N–C(3)H, N–C(5)H and N–C(6)H),
3.72–3.9 (m, 10H, N–C(8)H, N–C(9)H, N–C(11)H, N–C(12)H,
HNCH2CH2CH2O), 3.95–4.30 (m, 12H, (Man)4-H, (Man)3-H
N(4)CH2CO2H, (Man)6b-H), N(1)CH2CO2H, N(7)CH2CO2H,
3
(Man)6a-H, (Man)5-H and (Man)2-H), 4.54 (t, J(H,H) = 6.2 Hz,
3
2H, HNCH2CH2CH2O), 5.62 (s, 1H, (Man)1-H), 7.43 (d, J(H,H)
= 8.9 Hz, 2H, Ar-H), 8.43 (d, 3J(H,H) = 8.9 Hz, 2H, Ar-H);
13C NMR (100.6 MHz, D2O, 60 °C): δ 27.4 (SCH2CH2CONH),
28.3 (HNCH2CH2CH2O), 36.2 (SCH2CH2CONH), 36.6
(HNCH2CH2CH2O), 48.7 (N–C(2)H or N–C(3)H), 48.9 (N–C(2)H
or N–C(3)H), 51.6 (N–C(8)H and N–C(9)H), 54.3 (N(4)-
CH2COOH), 56.9 (N(1)CH2COOH and N(7)CH2COOH), 57.6
(N(10)CH2COOH), 61.3 ((Man)C-6), 66.6 (HNCH2CH2CH2O),
tube to a Tektronix TDS 520A (500 MHz, 500 Ms s−1
oscilloscope.
)
Acknowledgements
67.6 ((Man)C-4 or (Man)C-3), 71.5 ((Man)C-3 or (Man)C-4), 72.1 JAC is a Concordia University Research Chair in Nanoscience
((Man)C-2), 73.7 ((Man)C-5), 85.4 ((Man)C-1), 115.2 (CH-Ar), and is grateful to Concordia University for financial support of
128.7, 130.9 (CH-Ar), 163.5, 170.7 (CONH), 174.5 (N(4)- his research. JAC is also grateful for financial support from the
CH2COOH), 174.8 (N(1)CH2COOH and N(7)CH2COOH), Natural Sciences and Engineering Research Council. EMR
197.9 (ArCO); ESI-MS m/z = 810.5 ([M + Na]+), calcd for acknowledges financial support from the Fundación Alfonso
C34H53NaN5O14S = 810; elemental analysis calcd (%) for Martín Escudero and from the European Commission under
C34H53N5O14S: C 51.83, H 6.78, N 8.89. Found: C 51.89, H 6.80, the Marie Curie Fellowship Program (FP7-PEOPLE-2010-
N 8.88.
IOF-274404 “LUNAMED”). SQ is grateful for financial support
9460 | Dalton Trans., 2013, 42, 9453–9461
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