Metallo-carbonyl conjugates of biotin and biocytin

Article abstract of DOI:10.1016/S0022-328X(02)02145-9

Coupling of biotin with CpFe(CO)₂(η¹- N(1)-4-aminosulfonamido) (1a) and CpFe(CO) (P(OMe)₃) (η¹-N(1)-4-aminosulfonamide) (1b) afforded corresponding metallo-carbonyl biotin amides 2a-b. Reaction of N_E-

Full text of DOI:10.1016/S0022-328X(02)02145-9

Journal of Organometallic Chemistry 668 (2003) 95Á100
/
www.elsevier.com/locate/jorganchem
Metallo-carbonyl conjugates of biotin and biocytin
Bogna Rudolf a, Maria Makowska a, Anna Domagala a, Agnieszka Rybarczyk-Pirek b,
Janusz Zakrzewski a,*
a Department of Organic Chemistry, University of Lodz, 90-136 Lodz, Narutowicza 68, Poland
b Department of Crystallography, University of Lodz, 90-236 Lodz, Pomorska 149/153, Poland
Received 2 October 2002; accepted 26 November 2002
Abstract
Coupling of biotin with CpFe(CO)2(h1-N(1)-4-aminosulfonamido) (1a) and CpFe(CO)(P(OMe)3) (h1-N(1)-4-aminosulfonamide)
(1b) afforded corresponding metallo-carbonyl biotin amides 2aÁ
/
b. Reaction of No-biotinyl-
-lysine (biocytin) with CpFe(CO)2(h1-
L
N-3-isothiocyanatophtalimidato) (4a), CpFe(CO)2(h1-N-4-isothiocyanatophtalimidato) (4b), CpFe(CO)2(h1-N(1)-4-isothiocyana-
tosulfonamido) (5, this compound was prepared from 1a and thiophosgene and its structure confirmed by spectral data and by the
X-ray diffraction method) gave bioconjugates 6aÁc. The HABA-avidin test revealed high affinities of all synthesised bioconjugates
/
to avidin.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Biotin; Biocytin; Labelling; Cyclopentadienyl iron carbonyl; Isothiocyanate
1. Introduction
ganese tricarbonyl and cyclopentadienyl iron dicarbonyl
(h1-N-succiminato) [8]. Multiple labelling was achieved
by attachment of both biotin and metal carbonyl
fragments to a protein or a dendrimer core. Herein,
we report the synthesis of metallo-carbonyl conjugates
The extraordinarily strong affinity of biotin to
(strept)avidin (Kmꢀ
spread applications in biochemical research, medical
diagnosis and radioimmunotherapy [1Á3]. Bioanalytical
applications of the biotin-(strepta)avidin system require
biotin conjugates containing various reporter groups
used as markers in competitive assays. IR-detectable
metallo-carbonyl markers constitute an new alternative
/
1014Á1015 Mꢁ1) has found wide-
/
of biotin, and its derivative, biocytin (No-biotinyl-L-
/
lysine), based on the CpFe(CO)2(h1-N-sulfonamide)
and CpFe(CO)2(h1-N-phtalimidato) systems described
earlier [9,10], that are of interest for developing a IR-
detectable biotinylated probe for biochemical applica-
tions.
to frequently used fluorescent ones [4Á7]. Their sensitive
/
detection is based on the presence of very strong
absorption bands due to the stretching vibrations of
co-ordinated CO ligands in the region 1850Á ,
2100 cmꢁ1
/
2. Results and discussion
which is virtually free from any absorption originating
from biomolecules or biological matrices. Metallo-
carbonyl markers have been successfully applied in a
study of hormone-receptor interactions and immunoas-
says (carbonylmetalloimmunoassay). We have recently
reported labelling of biotin with metal-carbonyl moieties
such as dicobalt hexacarbonyl, cyclopentadienyl man-
2.1. Metallo-carbonyl conjugates of biotin
We have recently described the synthesis of the
complex, (h5-C5H5)Fe(CO)2[h1-N-(4-aminobenzenosul-
fonamido)], (1a), and acylation of its primary amino
function with carboxylic acids in the presence of 1-ethyl-
3-[3-(dimethylamino)]carbodiimide
hydrochloride
(EDC) [9]. We applied this reaction for coupling of 1a
with biotin (Scheme 1). Since 1a is sparingly soluble in
water the reaction was carried out in this solvent. The
* Corresponding author. Tel.: ꢂ
6583.
E-mail address: janzak@krysia.uni.lodz.pl (J. Zakrzewski).
/
48-42-678-4731; fax: ꢂ48-42-678-
/
0022-328X/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0022-328X(02)02145-9

96
B. Rudolf et al. / Journal of Organometallic Chemistry 668 (2003) 95Á100
/
2.2. Metallo-carbonyl conjugates of biocytin
Biocytin (No-biotinyl- -lysine) 3 is a commercially
L
available biotin derivative containing both carboxylic
and primary amino function. We though that it would
be of interest to attach the metallo-carbonyl moiety to
the amino function of 3 because the presence in such
conjugates of the carboxylic group will allow their
attachment to biomolecules. We have decided to use
for labelling of 3 organometallic isothiocyanates 4aÁ
/
b
[10].
Scheme 1.
product 2a precipitates directly from the reaction
mixture in 57% yield.
The IR spectrum of 2a exhibits two strong absorption
bands characteristic of the (h5-C5H5) Fe(CO)2 moiety at
2050 and 2005 cmꢁ1. As the positions and number of
n(CO) bands is critical for the eventual use of this
complex in quantitative bioassays, we attempted to
transform 2a into a monocarbonyl complex, 2b, for
We have also synthesised a new metallo-carbonyl
isothiocyanate 5 via reaction of 1a with thiophosgene in
the presence of triethylamine in DMF (Eq. (1)).
which only one n(CO) band was expected at Â1980
/
cmꢁ1. We attempted to obtain 2b by refluxing 2a with
trimethylphosphite in ethanol or by photolysing this
complex in DMF in the presence of trimethylphosphite,
but all our attempts failed. We have, therefore decided
to try to exchange one CO ligand in 1a by trimethylpho-
sphite to obtain 1b and then couple this complex with
biotin.
ð1Þ
The isothiocyanate 5 was isolated in 76% yield and its
structure confirmed by IR, 1H-NMR and elemental
analysis. The molecular structure of 5 was also con-
firmed by the X-ray diffraction method. The crystal data
are gathered in Table 1, selected bonds lengths and
angles in Table 2. The view of the molecule of 5 is shown
in Fig. 1.
We have found that 1a can be transformed in modest
yield (34%) into 1b, by photolysis in DMF containing a
fourfold excess of trimethylphosphite. 1b was character-
ized by IR, 1H-NMR and elemental analysis. The
coupling of 1b with biotin was carried out in DMF
using EDC as the carboxyl group activator. The product
The reaction of 3 with isothiocyanates 4aÁ
carried out in DMFÁwater in the presence of an excess
of triethylamine (Scheme 2).
The conjugates 6aÁc were isolated as orange air-
/
b and 5 was
/
2b was isolated in Â
1b). Conjugates 2aÁ
assuming molecular formulae 2a×
Their FAB mass spectra (nitrobenzyl alcohol matrix)
displayed peaks corresponding to Mꢂ
Hꢂ and MꢁHꢁ
/
30% yield (59% based on reacted
b gave correct CHNS analysis
H2O and 2b×4H2O.
/
/
/
/
stable microcrystalline solids in 82Á
/
94% yield and their
1
structures were confirmed by IR, H-NMR elemental
/
/
analyses and FAB MS. They are soluble in aqueous
Na2CO3 and precipitate from such solutions on acid-
ification with diluted HCl.
ions in the positive and negative ions mode, respectively.
Interestingly, in the spectrum of 2b the peak correspond-
ing to m/e 742 (Mꢂ4H2O) was observed in both modes
/
confirming suggested composition and revealing excep-
tionally tight water binding. As expected, IR spectrum
of 2b (KBr) displayed the very strong absorption band
at 1965 cmꢁ1 due to the stretching vibrations of the CO
ligand.
2.3. Affinity of 2aÁ
/
b and 6aÁc to avidin
/
To assess the affinity of 2aÁ
have used the HABA-Avidin reagent (Sigma). The
/
b and 6aÁc to avidin we
/

B. Rudolf et al. / Journal of Organometallic Chemistry 668 (2003) 95Á
/100
97
Table 1
Crystallographic data
Table 2
˚
Selected bond lengths (A) and angles (8) in 5
Molecular formula
Molecular weight
Crystal description and size
(mm)
C
390.21
14H10FeN2O4S2
Bond lengths
Fe1 Á
Fe1 Á
Fe1 Á
Fe1 Á
Fe1 Á
Fe1 Á
Fe1 Á
Fe1 Á
/
N2
1.965(4)
2.071(6)
2.076(6)
2.052(8)
2.045(8)
2.033(6)
1.750(6)
1.770(6)
1.565(4)
1.430(3)
1.451(3)
Orange cubic block 0.25ꢃ
/
0.25ꢃ
/
0.3
/
C11
C12
C13
C14
C15
C30
C40
/
Crystal system
Space group
Triclinic
¯
/
P/
1/
/
˚
a (A)
8.544(1)
12.953(1)
7.617(1)
94.51(1)
93.80(1)
101.70(1)
820.0(1)
2
/
˚
b (A)
/
˚
c (A)
/
a (8)
b (8)
g (8)
S2 Á
S2 Á
S2 Á
/
N2
/O21
/O22
˚
V (A3)
Z
Bond angles
N2 Á
N2 Á
N2 Á
/
S2 Á
S2 Á
S2 Á
S2 Á
S2 Á
S2 Á
N2 Á
Fe1 Á
Fe1 Á
Fe1 Á
/
O21
O22
C21
110.2(2)
109.7(2)
109.7(2)
105.8(2)
104.0(2)
117.0(2)
129.8(2)
96.4(5)
dx (g cmꢁ3
F(000)
)
1.580
396
/
/
/
/
m (cmꢁ1
T (K)
)
99.43
293(2)
72.69
C21
C21
O21
Á
Á
Á
/
/O21
/
/O22
Max u (8)
hkl ranges
/
/
O22
S2
ꢁ
/
10.9; ꢁ
/16.15; ꢁ9.9
/
Fe1 Á
/
/
No. of reflns measd
No. of indep reflns
Rint
3345
3114
0.030
F2
C30
C40
Á
Á
/
/N2
/
/N2
91.2(2)
94.2(3)
C30 Á
/
/N2
Refinement type
Hydrogen atoms
No. of params refined
Reflns/params
Mixed
205
15
a
R(F)
wR(F2)
0.0983
0.1193
b
c
a
R(F)
0.0437 for 1663 reflections with Iꢀ
/
2s(I)
b
wR(F2)
0.0839 for 1663 reflections with I ꢀ
/
c
2s(I)
0.938
GOF
˚
Difference peak/hole (e A
ꢁ3
)
0.269/ꢁ
/
0.452
a
R(F)ꢀ
/
a( j Foꢁ
/
Fc j )/a j Fo j .
b
c
2 1/2
] .
wR(F2)ꢀ
/
[aw( j Foꢁ
/
Fc j )2/a j Fo
j
wꢀ
/
(exp1.3(sinu/l)2]/[s2(Fo2)ꢂ
/
(0.0338P)2] where Pꢀ
/
[(F2o)ꢂ
/
2(F2c)]/3.
Fig. 1. The view of the molecule of 5 with atom-numbering scheme.
Displacement ellipsoids are shown at 30% probability level.
biotinÁ/avidin system at least at micromolar concentra-
formation the complex (avidinÁbiotin conjugate) was
/
tions.
monitored by disappearance of the absorbance at 500
nm, characteristic for the HABA-avidin complex ac-
cording to the Eq. (2).
3. Experimental
(HABA-avidin)ꢂbiotin conjugate
0 HABAꢂ(avidin-biotin conjugate)
(2)
All reactions were carried out under argon. Chroma-
tographic separations were carried out using silica gel 60
1
400 mesh ASTM) H-NMR spectra were
We have found that changes caused by addition 2aÁ
/
b
(Merck, 230Á
/
or 6aÁc are practically the same as those caused by
addition of the equivalent amount of biotin. For
example, Fig. 2 shows plots of the changes of absor-
/
recorded on a Varian Gemini 200BB (200 MHz)
spectrometer. IR spectra were recorded on a Biorad
spectrometer. Mass spectra were recorded on a Finnigan
bance at 500 nm against concentration of 6aÁ
/
b (or
MAT 95 spectrometer. Compounds 4aÁb and 5 were
/
biotin).
This reveals high affinity of synthesised conjugates to
avidin and their applicability for assays based on the
prepared according to earlier published procedures
[9,10]. Other reagents were commercially available and
used without prior purification.

98
B. Rudolf et al. / Journal of Organometallic Chemistry 668 (2003) 95Á100
/
HÁ
2CO]ꢂ. Anal. Found: C, 46.61; H, 4.79; N, 9.44; S,
10.53. Calc. For C23H28FeN4O7S2 (monohydrate): C,
46.63; H, 4.76; N, 9.46; S, 10.82.
/
3.2. Synthesis of 1b
A solution of 1a (160 mg, 0.46 mmol) and trimethyl-
phosphite (0.217 ml, 1.84 mmol) in DMF (5 ml) was
irradiated with visible light (a setup of 4ꢃ150 W
/
tungsten lamps) at 0 8C for 2 h. After evaporation to
dryness the residue was chromatographed collecting the
red fraction eluted with chloroformÁ
Crystallization (dichloromethaneÁether) afforded pure
2b. Yield: 77 mg (38%). H-NMR (dmso-d6, d ppm):
7.39 (d, Jꢀ8.0 Hz, 2H, Har), 6.58 (d, Jꢀ8.0 Hz, 2H,
Har), 5.50 (s, 2H, NH2), 4.44 (s, 5H, Cp), 3.65 (d, Jꢀ
11.1 Hz, 9H, OMe). IR (KBr, cmꢁ1): 3428, 3342, 3251
(NHꢂNH2), 1957 (FeÁCO), 1284, 1122 (SO2) 1020, 748
/
methanol (9:1).
/
1
/
/
/
/
/
Scheme 2.
(PO). Anal. Found: C, 40.30; H, 4.74; N, 6.61. Calc. For
C15H21FeN2O6PS2: C, 40.56; H, 4.76; N, 6.31.
3.1. Synthesis of 2a
3.3. Synthesis of 2b
A solution of 1a (150 mg, 0.43 mmol), biotin (70 mg,
0.29 mmol) and EDC (220 mg, 1.15 mmol) in water (30
ml) was stirred 3 h at r.t. The orange precipitate was
filtered, washed with water, ether and dried. Yield: 94
mg (57%).
A solution of 1b (80 mg, 0.18 mmol), biotin (44 mg,
0.18 mmol) and EDC (138 mg, 0.72 mmol) in DMF (3
ml) was stirred overnight at r.t. The solvent was
evaporated and the residue chromatographed
1H-NMR (dmso-d6, d ppm): 10.09 (s, 1H, NH
(chloroformÁ
were collected, the first being unreacted 1b and the
second 2b. Crystallization from dichloromethaneÁether
/methanol 9:1 as eluent). Two red bands
amide);7.75 (s, 1H, sulfonamide); 7.76 (d, Jꢀ
/8.0 Hz,
2H) and 7.56 (d, Jꢀ8.0 Hz, 2H) aromatic H’s; 6.46 (s,
/
/
1H, NH biotin); 6.38 (s, 1H, NH biotin); 5.15 (s, 5H,
Cp); 4.30 (m, 1H) and 4.15 (m, 1H), CH (biotin); 2.34
afforded analytically pure sample of 2b. Yield: 36 mg
(30%);
1H-NMR (dmso-d6, d ppm): 10.13 (s, 1H, NH
amide), 7.70 (m, 4H, Har), 6.45 (s, 1H, NH biotin),
6.38 (s, 1H, NH biotin), 4.43 (s, 5H, Cp), 4.31 (m, 1H,
(m, 2H, CH2CO); 1.6Á
cmꢁ1): 3324(NH); 2036, 1994 (FeÁ
amideꢂbiotin); 1531 (NH amide); 1307 and 1130
(SO2). FAB MS (nitrobenzyl alcohol matrix, m/e):
positive ions mode: 575 [Mꢂ HÁ
H]ꢂ, 519 [Mꢂ
2CO]ꢂ; negative ions mode: 573 [MꢂH]ꢂ, 517 [Mꢂ
/
1.4 (m, 6H, other CH2). IR (KBr,
/CO); 1699 (CO
/
CH biotin), 4.16 (m, 1H, CH biotin); 2.34 3.65 (d, Jꢀ
10.0 Hz, 9H, MeO). 2.35 (m, 2H, CH2CO); 1.6Á1.4 (m,
6H, other CH2). IR (KBr, cmꢁ1): 3295, 3195, 3090
/
/
/
/
/
/
/
Fig. 2. Plot of the changes of absorbance at 500 nm of the HABA-Avidin complex against concentration of biotin or biotin conjugates (4aÁb).
/

B. Rudolf et al. / Journal of Organometallic Chemistry 668 (2003) 95Á
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99
1
6c. Yield: 62%. H-NMR (dmso-d6, d ppm): 10.04 (s,
(NH), 1965 (FeÁ
(NH amide); 1311, 1131 (SO2), 1021, 751 (PO). FAB MS
(nba matrix m/e): positive ions mode: 742 [MꢂHꢂ
4H2O]ꢂ, 671 [MꢂH]ꢂ; negative ions mode: 742 [Mꢂ
Hꢂ
4H2O]ꢁ, 669 [MÁH]ꢁ, 517. Anal. Found: C, 40.56;
/
CO); 1694 (CO amideꢂ
/
biotin); 1534
2H, NH amideꢂCOOH), 8.3Á7.4 (m, 7H, HarꢂNH),
/
/
/
/
/
7.70 (m, 4H, Har), 6.4 (b, 2H, NH biotin), 5.17 (s, 5H,
Cp), 4.31 (m, 1H, CH biotin), 4.13 (m, 1H, CH biotin);
2.1 (b, 4H), 1.4 (m, 12H). IR (KBr, cmꢁ1): 3295, 3090
/
/
/
/
H, 5.74; N, 7.65. Calc. For C25H43FeN4O12PS2 (tetra-
hydrate): C, 40.44; H, 5.74; N, 7.56.
(NH), 2046, 1997 (FeÁ
positive ions mode: negative ions mode: 761 [MÁ
733 [MÁHÁ HÁ
CO]ꢁ, 705 [MÁ 2CO]ꢁ. Anal. Found: C,
/
CO). FAB MS (nba matrix m/e):
/
H]ꢁ,
/
/
/
/
44.08; H, 5.20; N, 9.90; S, 11.87. Calc. For C30H42Fe-
N6O11S3 (trihydrate): C, 44,11; H, 5.43; N, 10.29; S,
11.75.
3.4. Synthesis of 5
To a solution of 1a (522 mg, 1.5 mmol) in DMF (5 ml)
containing triethylamine (0.3 ml) thiophosgen (0.15 ml,
3.6. X-ray structure determination
1.96 mmol) was added at 0Á5 8C and the mixture was
/
stirred at temperature 0.5 h. The precipitated triethyla-
mine hydrochloride was filtered off and the solvent
evaporated. The residue was dissolved in chloroform (5
ml) and washed twice with water. After evaporation and
column chromatography (eluent chloroform) and crys-
Data were collected on a Rigaku AFC5S diffract-
˚
1.54178 A) X-ray source and a
ometer using Cu Ka (lꢀ
/
graphite monochromator. Analytical absorption correc-
tions was applied. Experimental details are given in the
Table 1. The crystal structure was solved by direct
methods using SHELXS86 [11] and refined by full-matrix
least square method using SHELXL97 [12]. All non-
hydrogen atoms were refined with anisotropic displace-
ment parameters. Hydrogen atoms except for H2 atom
were introduced in the calculated positions and refined
using the rigid body model. The position of the H2
atom, taking part in an intermolecular hydrogen bond-
ing with O22 of the other molecule, was found on the
Fourier-difference map and refined freely. The molecu-
lar geometry was calculated by PARST [13] and PLATON
[14].
tallization (dichloromethaneÁ
obtained. Yield: 444 mg (76%).
1H-NMR (dmso-d6, d ppm): 7.84 (s, 1H, NH), 7.68
(d, Jꢀ6.0 Hz, 2H, Har), 7.52 (d, Jꢀ6.0 Hz, 2H, Har),
5.15 (s, 5H, Cp). IR (KBr, cmꢁ1): 3254 (NH), 2109
(NCS) 2039, 1991 (FeÁCO).
/hexane) the red 5 was
/
/
/
3.5. Coupling of 4aÁb and 5 with biocytin
/
Biocytin (30 mg, 0.08 mmol) and the organometallic
isothiocyanate 4aÁb or 5 (0.1 mmol) were dissolved in
DMF (2.1 ml), water (0.5 ml) and triethylamine (0.5 ml)
/
and stirred 2 h at r.t. After evaporation to dryness the
residue was dissolved in water (Â5 ml). The products
precipitate on acidification to pH 3 with 0.6 M HCl.
1
6a. Yield: 94%. H-NMR (dmso-d6, d ppm): 9.97 (s,
/
4. Supplementary material
Crystallographic data for the structural analysis have
been deposited with the Cambridge Crystallographic
Data Centre, CCDC No. 194449 for compound 5.
Copies of this information may be obtained free of
charge from The Director, CCDC, 12 Union Road,
1H, NH), 9.35 (b, 1H, NH), 8.67 (d, Jꢀ
Har) 7.8 (b, 1H NH), 7.46 (t, Jꢀ7.7 Hz, Har), 7.21 (d,
Jꢀ7.7 Hz, 1H, Har), 6.43 (s, 1H, NH biotin), 6.38 (s,
/
7.7 Hz, 1H,
/
/
1H, NH biotin), 5.38 (s, 5H, Cp), 4.30 (m, 1H, CH
biotin), 4.12 (m, 1H, CH biotin); 2.1 (m, 4H), 1.4 (m,
12H). IR (KBr, cmꢁ1): 2047, 1998 (FeÁ
/
CO). FAB MS
(nba matrix m/e): negative ions mode: 751 [MÁ
H]ꢁ, 695
[MÁHÁ
2CO]ꢁ. Anal. Found: C, 51.39; H, 4.49; N,
Cambridge, CB2 1EZ, UK (Fax: ꢂ44-1223-336033;
/
e-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.
cam.ac.uk).
/
/
/
10.05. Calc. For C32H36FeN6O8S2: C, 51.07; H, 4.82; N,
11.17.
6b. Yield: 93%. 1H-NMR (dmso-d6, d ppm): 10.09 (s,
Acknowledgements
1H, NH), 8.20 (d, Jꢀ
7.80 (b, 1H, NH), 7.56 (d, Jꢀ
Jꢀ7.6 Hz, 1H, Har), 6.45 (s, 1H, NH biotin), 6.35 (s,
/
7.6 Hz, 1H, NH), 7.98 (s, 1H Har),
/7.6 Hz, 1H, Har), 7.45 (d,
This research was financially supported from the
Polish State Committee for Scientific Research (Grant
PBZ-KBN 15/09/T09/99/01).
/
1H, NH biotin), 5.34 (s, 5H, Cp), 4.30 (m, 1H, CH
biotin), 4.12 (m, 1H, CH biotin); 2.1 (m, 4H), 1.4 (m,
12H). IR (KBr, cmꢁ1): 2046, 1997 (FeÁ
/
CO). FAB MS
(nba matrix m/e): negative ions mode: 751 [MÁ
H]ꢁ, 695
[MÁHÁ
2CO]ꢁ. Anal. Found: C, 49.87; H, 4.85; N,
/
References
/
/
10.61; S, 8.13. Calc. For C32H38FeN6O9S2 (monohy-
drate): C, 49.84; H, 4.67; N, 10.90; S, 8.31.
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B. Rudolf et al. / Journal of Organometallic Chemistry 668 (2003) 95Á100
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[2] G.T. Hermanson, Bioconjugate Techniques (Chapter 13), Aca-
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[10] A. Kazimierczak, J. Zakrzewski, M. Salmain, G. Jaouen,
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tion, University of Gottingen, Gottingen, Germany, 1990.
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