Synthesis of a water-soluble ytterbium porphyrin-bovine serum albumin conjugate

Article abstract of DOI:10.1023/B:RUBI.0000015779.37503.8d

The ytterbium complex of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin was synthesized as an IR-fluorescent label and covalently bound to bovine serum albumin. The resulting conjugate fluoresces at 985 nm and is of interest for use in IR-fluorescent tumor diagnostic, immunoassay, and energy transfer studies.

Full text of DOI:10.1023/B:RUBI.0000015779.37503.8d

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 1, 2004, pp. 89–93. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 1, 2004, pp. 99–104.
Original Russian Text Copyright © 2004 by Chudinov, Rumyantseva, Lobanov, Chudinova, Stomakhin, Mironov.
Synthesis of a Water-Soluble Ytterbium Porphyrin–Bovine
Serum Albumin Conjugate
1
A. V. Chudinov*, V. D. Rumyantseva**, A. V. Lobanov***, G. K. Chudinova***,
A. A. Stomakhin*, and A. F. Mironov**
*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, GSP Moscow, 117984 Russia
**Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 119571 Russia
***Research Center for Photochemistry, Russian Academy of Sciences, Moscow, Russia
Received July 30, 2002; in final form, September 18, 2002
Abstract—The ytterbium complex of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin was synthesized as an
IR-fluorescent label and covalently bound to bovine serum albumin. The resulting conjugate fluoresces at
985 nm and is of interest for use in IR-fluorescent tumor diagnostic, immunoassay, and energy transfer studies.
Key words: conjugates, fluorescence, near IR region, serum albumin, ytterbium porphyrins
1
INTRODUCTION
bility of the development of an IR-fluorescent immun-
osensor on a model system.
Previously, we have obtained fluorescent probes on
the basis of palladium metalloporphyrins with isocyan-
ate group [1]. The probes on the basis of metallopor-
The metalloporphyrin–albumin conjugate is of
interest for studying the process of energy transfer in
lipid membranes (including lipid LB films), and its use
may partially clear the problem of the role of protein–
lipid environment in the migration of exciting energy
[10]. Therefore, the synthesis of ytterbium metallopor-
phyrins and the obtaining of their conjugates with pro-
teins appear to be important problems of bioorganic
chemistry.
2
phyrins with some lanthanides that fluoresce in the near
IR region of spectrum (900–1050 nm) are especially
interesting [2]. Any irradiance of biogenic substances is
practically absent, and, subsequently, the sensitivity of
the probe detection is higher in this range [3]. Among
the lanthanide ions whose metalloporphyrin derivatives
exhibit an IR-fluorescence, the complexes with Yb3+,
which has the least ionic radius (0.642 Å), are the most
stable.
RESULTS AND DISCUSSION
The ability of porphyrins to accumulate in malig-
nant tumors underlies the photodynamic therapy and
We prepared TCMPP (I) as the starting compound
the fluorescent diagnostics of cancer diseases [4]. In for the synthesis of YbTCPP (IV) by the method [11]
this case, the main problem is the enhancement of and hydrolyzed it by warm caustic potash in aqueous
selectivity of the porphyrin accumulation in tumor pyridine. The reaction mixture was evaporated and
cells. The selectivity of porphyrin delivery to cancer acidified by hydrochloric acid. The precipitated green
cells increases when metalloporphyrin–albumin conju- solid was TCPP dihydrochloride (II).
gates are used [5]. The possibility of using the IR fluo-
YbTCPP (IV) is usually synthesized according to
rescence of YbTCPP for the diagnostics of tumors and
the procedure of obtaining tetrasulfophenyllantanides
an extremely low phototoxicity of YbTCPP were dem-
described in [12]. This procedure results in the water-
onstrated in [6, 7].
soluble form of YbTCPP, its tetrasodium salt
(Ybíëêê4– · 4Na+) (III). The acid YbTCPP, soluble in
The use of highly ordered oriented systems, such as
LB films, make realizable the creation of immunosen-
sor systems with the IR-fluorescent detection principle
[8]. We have previously demonstrated [9] a basic possi-
anhydrous organic solvents, is necessary for activation
of carboxyl groups.
We partially modified the procedure of the ytter-
bium metalloporphyrin synthesis. A mixture of TCPP
(II) and ytterbium acetylacetonate was fused in imida-
zole for 40 min. The reaction mixture was dissolved in
a small quantity of methanol and treated with diethyl
ether. The unreacted TCPP and resultingYbTCPP were
precipitated, whereasYb acetylacetonate and imidazole
remained in solution. The TCPP andYbTCPP (IV) pre-
1
Corresponding author; phone: +7 (095) 135-9950; e-mail:
chud@genome.eimb.relarn.ru
2
Abbreviations: BSA, bovine serum albumin; CTAB, cetyltrimeth-
ylammonium bromide; Im, imidazole; LB, Langmuir–Blodgett
technology; NSI, N-hydroxysuccinimide; PBS, phosphate-buff-
ered saline; TCMPP, 5,10,15,20-tetrakis(4-carbomethoxyphe-
nyl)porphyrin; TCPP, 5,10,15,20-tetrakis(4-carboxyphenyl)por-
phyrin; and YbTCPP, TCPP ytterbium complex.
1068-1620/04/3001-0089 © 2004 MAIK “Nauka /Interperiodica”

90
CHUDINOV et al.
COOCH3
COOH
NH
N
N
NH
N
N
11––22)
H3COOC
COOCH3
HOOC
COOH
HN
HN
COOCH3
COONa
COOH
(I)
(II)
3–4
COOH
Im
N
Im
Yb
Im
N
N
N
N
N
5)
5
NaOOC
Yb
COONa
HOOC
COOH
N
N
Im
NH
Im =
N
COONa
COOH
(III)
(IV)
Scheme. Reagents and reaction conditions: (1) 2 M KOH, pyridine, 1.5 h; (2) 0.1 M HCl; (3)Yb(acac) , imidazole, 240°C, 40 min;
3
(4) 0.5 M Na CO ; (5) 0.1 N HCl
2
3
MALDI mass spectrometry and 1H NMR spectros-
copy allowed us to find that two imidazole molecules
are coordinated with ytterbium ion (Fig. 1). A similar
situation was previously found for an ytterbium sul-
foporphyrin [12].
cipitate was dissolved in aqueous solution of sodium
carbonate, applied onto a chromatographic column
packed with Trisacryl, and eluted with water. The first
colored zone contained TCPP sodium salt and the sec-
ond, YbTCPP sodium salt. The aqueous solution of
(Ybíëêê4– · 4Na+ (III)) was then slowly acidified with
0.1 N HCl, which led to the crystallization of YbTCPP
acid (IV). This procedure should be carried out care-
fully, because an excessive acidification was observed
to cause destruction of the metallocomplex. The precip-
itated YbTCPP acid is soluble in a number of organic
solvents.
The homogeneity of the resulting porphyrins was
confirmed by TLC. The benzene–methanol–triethyl-
amine systems were efficiently used for the separation
of mixtures of porphyrins containing no metal ions.
Addition of various quantities of methanol allowed the
regulation of the eluent polarity.
Ytterbium complex (IV) demonstrates the zero
mobility when being analyzed in the systems suitable
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

SYNTHESIS OF A WATER-SOLUBLE YTTERBIUM
91
A
NH
NH
1
N
N
COOH
Yb
2
N
N
HOOC
COOH
3
N
N
4
250
300
350
400
450
500
nm
HOOC
Fig. 2. Electronic spectra of (1) covalent conjugate BSA–
YbTCPP, (2) YbTCPP, (3) noncovalent conjugate of BSA
with YbTCPP, and (4) BSA in PBS, pH 7.4
Fig. 1. Spatial structure of YbTCPP
(280)
BSA
(417)
YbTCPP
for the porphyrins devoid of metal ions. This is proba- the wavelength 280 or 417 nm; ε
and ε
are the
molar absorption coefficients of BSA (36 000 M–1 cm–1
at 280 nm) andYbTCPP (450 000 M–1 cm–1 at 417 nm)
(see table). The α value reached 5.4–6.6 when the ini-
tial quantity of YbTCPP was equal or more than
100 mol/mol of BSA. At such a conjugate composition,
no substantial disturbances in the structure or functions
of protein are usually observed and no concentrational
quenching of the porphyrin fluorescence occurs.
bly connected with the presence of the third valence of
ytterbium, which is partially free. An addition of 1%
acetylacetone to the methanol–triethylamine system
(60 : 1) increases the chromatographic mobility of the
YbTCPP acid complex to Rf 0.19; TCPP has Rf 0.27 in
this system.
Next, we conjugated YbTCPP with BSA. Free
amino groups, mainly ε-amino groups of Lys residues,
are located on the surface of BSA molecule. The BSA–
YbTCPP was synthesized in two stages. At the first
stage, the YbTCPP succinimide ester was prepared
under anhydrous conditions.An interaction of acid (IV)
with isobutyl chloroformate in pyridine resulted in the
active mixed anhydride, which was then transformed
into the less active N-succinimide ester. A TLC moni-
toring showed that, under the found reagent ratio (see
the Experimental section), theYbTCPP with one active
carboxyl group is preferentially formed. At the second
stage, the succinimide ester was conjugated with the
protein in water–organic medium at pH 9–9.3.
We carried out a control bare experiment to estimate
the degree of sorption (noncovalent) binding of BSA
withYbTCPP (IV). BSA was conjugated withYbTCPP
acid bearing nonactivated carboxyl groups. The value
of α ≤ 0.1 was found for such noncovalent conjugates
(Fig. 2), and, therefore, the proportion of nonspecifi-
cally bound porphyrin in the BSA–YbTCPP complex is
small.
A study of the IR-fluorescent properties ofYbTCPP
and BSA–YbTCPP complex showed that the IR fluo-
rescence of ytterbium porphyrins is due to the energy
transition of inner 4f electrons of Yb3+ ions from the
The composition of the resulting synthetic conju-
gate was determined after its gel-chromatographic puri-
fication. The number of porphyrin molecules per pro-
tein molecule, α, was determined by using spectropho-
tometric method [13]. In UV region, the conjugate
absorption is summed up from the absorptions of BSA
and (IV), whereas only YbTCPP absorbs visible light
(Fig. 2).
2
2F5/2 to the F7/2 state [2]. The excitation energy is
absorbed by the π-electron system of the ligand por-
phyrin and then migrates from its triplet level to the
ytterbium 2F5/2 resonance level. Therefore, the fluores-
cence in the near IR range is the result of the intramo-
lecular energy transfer, and, hence, it should substan-
tially depend on the complex environment.
A weak IR fluorescence is characteristic of aqueous
solutions of YbTCPP: the calculated value of its quan-
tum yield is 0.006. At the same time, increased fluores-
cence intensity with the maximum at 986 nm is
observed in water–micellar solutions. The fluorescence
intensity of the BSA–YbTCPP conjugate with 985-nm
maximum is even greater (Fig. 3). An increase in quan-
tum yields nf (see table) observed forYbTCPP upon the
Therefore, α was calculated according to the equa-
tion:
α =
(417)
BSA–YbTCPP
(280)
BSA
(280)
BSA–YbTCPP
(280)
YbTCPP
(417)
,
YbTCPP
= A
× ε
/A
– A
× ε
(417)
YbTCPP
where A
, AB(4S1A7–)YbTCPP , and A(280)
are the
BSA–YbTCPP
absorptions of YbTCPP, BSA–YbTCPP, and BSA at transitions buffer
water–micellar system
fixa-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

92
CHUDINOV et al.
Spectral properties of YbTCPP and BSA–YbTCPP
abs
max
Conditions
Compound
ε, M–1 cm–1
nf
λ
, nm
0.1 å Na2CO3, 0.9 M NaHCO3, pH 9.2
0.05% Triton ï-100
YbTëêê
417
4.5–105
3.9–105
4.0–105
1.00
2.00
3.03
YbTëêê
419
425
8.6 mM K2HPO4, 1.4 mM KH2PO4, 0.15 M NaCl, pH 7.35
BSA–YbTëêê
tion on protein may be explained by a certain decrease ethanol, pyridine, benzene, triethylamine, diethyl ether,
in the heat molecular mobility of complex (IV) and its sodium chloride, sodium bicarbonate, sodium carbon-
preferential occurrence in monomeric form. The contri- ate, hydrochloric acid, and potassium hydroxide of ana-
bution of some amino acid residues of BSA into the lytical or reagent purity grades were of domestic pro-
energy of YbTCPP molecules cannot be also excluded. duction (Reakhim, Russia). Pyridine was stored over
The table also lists the values of molar extinction coef- molecular sieves (4 Å), and BSA (Sigma, United
ficients of YbTCPP and BSA–YbTCPP at the wave- States), in lyophilized state at +4°ë.
abs
The reagent Yb(acac)3 was obtained from YbCl3,
lengths of their absorption maxima, λ
.
max
and TCMPP was synthesized according to [14].
Thus, we demonstrated a possibility of synthesis of
proteins with an IR-fluorescent label by the example of
a BSA conjugate with a water-soluble Yb-porphyrin
(YbTCPP). This conjugate exhibits fluorescence at
985 nm, the quantum yield of which substantially
depends on its environment. An inclusion of the conju-
gate into biosensor films or into biological or artificial
membranes would probably enhance intensity of its IR
fluorescence. An appropriate choice of optical equip-
ment would allow a prospective use of this conjugate.
Trisacryl GF-05 (LKB, Sweden) was used for the
column chromatography of YbTCPP. The column size
was 10 × 300 mm, and the flow rate 1–1.5 ml/min. Pre-
coated Silufol UV-254 plates (20 × 70 mm, Kavalier,
Czechia) were used for TLC in systems: (A) 15 : 15 : 1
benzene–methanol–triethylamine and (B) 60 : 1 meth-
anol–triethylamine containing 1% acetylacetone. A
gel-chromatographic purification of BSA–YbTCPP
was carried out on a column (10 × 180 mm) packed
with Sephadex G-25 (Pharmacia, Sweden). The eluate
from the column was detected at 280 nm using an Uvi-
cord (LKB, Sweden) instrument.
EXPERIMENTAL
1H NMR spectra were registered on a Bruker
AMX400 spectrometer (Germany) at 295 K. The val-
ues of chemical shifts (δ, ppm) were calculated relative
to the residual signals of HOD (δ 4.63) for solutions in
D2O, CHCl3 (δ 7.26) for solutions in CDCl3, and
(CH3)2SO (δ 3.57) for solutions in DMSO-d6 [14]. The
spin–spin-interacting signals were revealed by means
of double resonance. Mass spectra were measured on a
Kompact MALDI 4 (KRATOS Analytical, United
States) instrument. Electronic spectra were obtained on
a Shimadzu UV-VIS 3100 spectrophotometer (Japan).
Melting points were determined on a Boetius hot plate
(Germany). Fluorescence spectra were measured on a
spectrofluorimeter Shimadzu RF 5000 (Japan). Quan-
tum yields were calculated by the procedure in [15]
using a solution of 5,10,15,20-tetraphenylporphyrin in
toluene as a standard (Q = 0.12). CTAB and concen-
trated solutions of HCl and NaOH were used for the
determination of molar extinction coefficients.
Isobutyl chloroformate and imidazole were from
Fluka (Switzerland); N-hydroxysuccinimide from
Merck (Germany); acetylacetone and potassium mono-
phosphate and diphosphate from Sigma (United
States); Triton X-100 and cetyltrimethylammonium
bromide (CTAB) from Ferak (Germany); methanol,
Intensity, arbitrary units
100
80
2
60
1
40
20
0
5,10,15,20-Tetrakis(4-carboxyphenyl)porphyrin
(II). Five ml of 2 M KOH were added to a solution of
TCMPP (50 mg, 0.046 mmol) in pyridine (5 ml), and
the mixture was refluxed for 1.5 h at intensive stirring
and evaporated on a rotary evaporator at 80°ë and
15 mmHg. The residue was diluted with 10 ml of dis-
tilled water and acidified with 0.1 N HCl to pH 2 at stir-
ring. The color of the solution was changed from red to
900
950
1000
1050
1100
nm
Fig. 3. Fluorescence spectra of (1)YbTCPP in 0.05% aque-
ous solution of Triton X-100 and (2) BSA–YbTCPP in tri-
distilled water at room temperature.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004

SYNTHESIS OF A WATER-SOLUBLE YTTERBIUM
93
green, and a green solid (II) was precipitated. The pre- Sephadex G-25 column (10 × 180 mm) preliminarily
cipitate was filtered, washed with water (100 ml), and equilibrated with PBS buffer (8.6 mM K2HPO4,
dried over phosphorus pentoxide; yield 33.5 mg (92%);
Rf 0.23 (A); mp >300°ë; electronic spectrum [5 : 1 ben-
zene–methanol, λmax, nm (intensity ratio)]: 420, 513,
1.4 mM KH2PO4, 0.15 M NaCl, pH 7.35), and the col-
umn was eluted with the same buffer at a flow rate of
0.2 ml/min. The fraction with retention time 25–35 min
was used for further studies. The fractions with lower
retention times (protein aggregates) were discarded.
1
548, 592, 645, (1.0 : 0.05 : 0.028 : 0.019 : 0.013); H
NMR (δ, ppm): 8.82 (8 H, s, β-H), 8.34–8.39 (16 H, s,
PhH), 2.50 (4 H, s, COOH); MALDI MS: found å
790.84; calc. for C48H30N4O8 å 790.77.
ACKNOWLEDGMENTS
Ytterbium complex of 5,10,15,20-tetrakis(4-car-
boxyphenyl)porphyrin (IV).A mixture of (II) (50 mg,
10.5 µmol), ytterbium acetylacetonate (100 mg,
0.21 mmol), and imidazole (700 mg) was carefully trit-
urated on a watch crystal, transferred to a bulb equipped
with a reflux condenser, and heated on an air bath at
240°C in a nitrogen flow for 40 min. Then the mixture
was cooled, and imidazole excess was removed on a
rotary evaporator (100°ë, 15 mm Hg). The residue was
triturated with methanol to get a thick suspension,
treated with diethyl ether (20 ml), and kept for 30 min
to permit the flaky precipitate to settle. The supernatant
was decanted and discarded, and the procedure was
repeated thrice. The residue was air-dried, dissolved in
0.5 M Na2CO3, and applied onto a column packed with
Trisacryl GF-05, which was eluted with distilled water.
Complex (III) was eluted in the second colored zone.
The solution of (III) was filtered and treated dropwise
with 0.1 M HCl until the formation of fine crystals of
(IV) that worked to bottom. The mixture was centri-
fuged, supernatant was decanted, and the residue was
twice washed with 0.001 M HCl and distilled water and
air-dried to a constant mass. The yield of porphyrin
(IV) was 45.0 mg (64.7%); Rf 0.19 (B); electronic spec-
We thank A.P Mozoleva (Engelhardt Institute of
Molecular Biology, Russian Academy of Sciences,
Moscow) for registration of 1H NMR spectra.
This work was supported by the Russian Foundation
for Basic Research, project no. 02-04-48952.
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Conjugate of ytterbium complex of 5,10,15,20-
tetrakis(4-carboxyphenyl)porphyrin with bovine
serum albumin. A solution of isobutyl chloroformate
(5 µl, 35 µmol) in chloroform (40 µl) was added at
intensive stirring and –10°C to a solution of (IV)
(15.8 mg, 14.3 µmol) in pyridine (2 ml). The reaction
mixture was additionally stirred for 1 h at –10°C and
for 2 h at 0°C, then treated with N-hydroxysuccinimide
(7 mg, 60.8 µmol), stirred overnight at room tempera-
ture, and centrifuged at 14 000 rpm for 10 min. The
supernatant that contained a solution of N-succinimide
ester of YbTCPP in pyridine (200 µl) was added to a
stirred solution of BSA (2 mg, 0.03 µmol) in 0.1 M car-
bonate–bicarbonate buffer (1 ml, pH 9.1) at room tem-
perature. The mixture was stirred at room temperature
overnight, centrifuged at 14000 rpm for 10 min, and
supernatant that contained the BSA–YbTCPP conju-
gate was separated (~1 ml). It was applied onto a
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 1 2004