(η5-Cyclopentadienyl)Fe(CO)2-complexes of sulfonamide anions. Acylation of the amino group in the sulfanilamide complex and its relevance to the protein labelling

Article abstract of DOI:10.1016/S0022-328X(98)00756-6

Illumination with visible light of [CpFe(CO)₂I] (Cp=η⁵-C₅H₅) with NH₂SO₂C₆H₄-p-R (R=Me, NO₂, NH₂) in benzene in the presence of diisopropylamine gives [CpFe(CO)₂NHSO₂C₆H₄-p-R] in 62-91% yield. The primary amino group in [CpFe(CO)₂NHSO₂C₆H₄-p-NH ₂] was acylated by treatment of this complex with neat carboxylic acid anhydrides (acetic, propionic and isobutyric) or with carboxylic acid (acetic, iodoacetic, β-maleimidopropionic) and 1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide (EDC) in aqueous solutions at pH 5-6 at room temperature. The latter reaction shows that [CpFe(CO)₂NHSO₂C₆H₄-p-NH ₂] can be used as an IR-detectable metallo-carbonyl reagent labelling the COOH groups in proteins and as a parent compound for a new family of reagents labelling the thiol function.

Full text of DOI:10.1016/S0022-328X(98)00756-6

Journal of Organometallic Chemistry 568 (1998) 171–175
(p5-Cyclopentadienyl)Fe(CO)2-complexes of sulfonamide anions.
Acylation of the amino group in the sulfanilamide complex and its
relevance to the protein labelling
Janusz Zakrzewski *, Maria Pawlak
*
*
Department of Organic Chemistry, Uni6ersity of Lo´dz´, 90-136 Lo´dz´, Narutowicza 68, Poland
Received 9 April 1998
Abstract
Illumination with visible light of [CpFe(CO)2I] (Cp=p5-C5H5) with NH2SO2C6H4-p-R (R=Me, NO2, NH2) in benzene in the
presence of diisopropylamine gives [CpFe(CO)2NHSO2C6H4-p-R] in 62–91% yield. The primary amino group in
[CpFe(CO)2NHSO2C6H4-p-NH2] was acylated by treatment of this complex with neat carboxylic acid anhydrides (acetic,
propionic and isobutyric) or with carboxylic acid (acetic, iodoacetic, i-maleimidopropionic) and 1-[3-(dimethylamino)propyl]-3-
ethyl-carbodiimide (EDC) in aqueous solutions at pH 5–6 at room temperature. The latter reaction shows that
[CpFe(CO)2NHSO2C6H4-p-NH2] can be used as an IR-detectable metallo-carbonyl reagent labelling the COOH groups in
proteins and as a parent compound for a new family of reagents labelling the thiol function. © 1998 Elsevier Science S.A. All
rights reserved.
Keywords: Iron carbonyls; Cyclopentadienyl; Sulfonamide anions; N-acylation; Carbodiimide
1. Introduction
introduction of IR-detectable labels into these (and
other) biopolymers. This is the case of [CpFe(CO)2(p1-
N-maleimidato)] and [CpFe(CO)2(p1-N(1)-isothio-
cyanatophthalimides)], which were succesfully used for
labeling of model aminoacids and bovine serum albu-
mine ([1]c, f).
Sulfonamides constitute a class of N–H acidic com-
pounds which display very interesting pharmacological
properties, especially p-aminobenzenesulfonamide (sul-
fanilamide) and its derivatives [2]. However, very little
is known about transition-metal complexes of sulfon-
amides or their anions [3], and, to our knowledge, there
is no example of a complex in which such a ligand is
co-ordinated to a metallo-carbonyl moiety.
We thought that it would be of interest to look if
sulfonamides undergo reaction shown in Eq. (1) and
afford CpFe(CO)2-complexes of their anions. The
derivative of sulfanilamide would be of special interest
as containing NH2 group which is of interest as a
reactive function for coupling with the COOH func-
We are currently investigating the synthetic potential
of the photochemical substitution of iodide in
[CpFe(CO)2I] (Cp=p5-C5H5) by anions of N–H acids
(Eq. (1); H–N(X)(Y)=N–H acid, B=diisopropyl-
amine).
[CpFe(CO)2I]+H–N(X)(Y)+B
hw
ꢀꢀꢀꢀꢀꢀꢀꢀꢁ [CpFe(CO)2N(X)(Y)] (1)
+
−
I
−BH
This reaction leads to a variety of stable CpFe(CO)2-
complexes of anions of pyrroles, indoles, cyclic imides
and uracils [1]. Some of these complexes contain func-
tional groups able to react with functional groups
present in lateral chains of proteins and can be used for
* Corresponding author. Tel.: +48 42 355755; fax: +48 42
786583; e-mail janzak@krysia.uni.lodz.pl
0022-328X/98/$19.00 © 1998 Elsevier Science S.A. All rights reserved.
PII S0022-328X(98)00756-6

172
J. Zakrzewski, M. Pawlak / Journal of Organometallic Chemistry 568 (1998) 171–175
tions in biomolecules. This could be easily tested in the
reactions with some model carboxylic acids. Herein we
report results of this study.
2. Results and discussion
Irradiation of [CpFe(CO)2I] with sulfonamides 1a–c
and diisopropylamine in benzene brings about forma-
tion of orange, air stable complexes 2a–c in 64–91%
yield (Scheme 1).
The reaction proceeds in the same manner with 1c as
with 1a–b, suggesting that amino group in the benzene
ring is not involved, and does not hamper the reaction.
We reported earlier a similar phenomenon in the case
of reaction with aminophthalimides ([1]f).
The IR spectra of 2a–c in the region 1400–1100
cm−1 are very similar to the spectra of 1a–c and
display characteristic, strong bands assignable to the
symmetric and antisymmetric stretching vibrations of
the –SO2– moiety (shifted around 50 cm−1 towards
lower wavelengths in comparison to the same bands in
1a–c). This is in full accord with the structure in which
CpFe(CO)2-moiety is bonded to deprotonated sulfon-
amide nitrogen causing repulsion between iron d-elec-
trons and the lone pair at nitrogen. In the region of the
N–H stretching vibrations 2a–b show one band at
3290 cm−1 (wNH), whereas 2c shows three bands: 3370,
3310 (wNH2) and 3280 cm−1 (w NH), accordingly to the
presented structure.
The derivative of sulfanilamide 2c is sparingly water-
soluble (ca. 1 mg ml−1) and its aqueous solutions are
stable providing they are stored in the dark.
Attempted reactions of 2c with acetyl chloride and
triethylamine in dichloromethane failed. However, we
have found that 2c react neat liquid anhydrides (acetic,
propionic and isobutyric anhydride) at r.t. to afford
corresponding amides 3a–c, which precipitate directly
Scheme 2.
from the solutions in analytically pure form (Scheme 2).
IR spectra of these products display in the 1700–
1500 cm−1 region bands characteristic for compounds
of the type PhNHCOR: 1680–1685 cm−1 (Amide I)
and 1540 cm−1 (Amide II, characteristic for N-substi-
tuted amides). This is consistent with the structures
3a–c, acetylation taking place at the NH2 nitrogen.
1H-NMR spectra of 3a–c in DMSO-d6 show at low
field two one-proton singlets: the first at 10–10.5 ppm,
which can be assigned to the amide proton, and the
second at 7.70–7.75 ppm, which by comparison with
the value of 7.71 ppm in 2c can be assigned to the
NHSO2 proton. Another evidence supporting the struc-
ture of 3a–c is provided by their IR spectra, showing
two absorption bands in the region of NH stretching
vibrations. The band appearing at 3280–3290 cm−1
can be assigned to the NH stretching vibrations in the
sulfonamide moiety, whereas the band at 3310–3320
cm–1 to the same vibrations in the amide moiety.
Finally, we have found that illumination with visible
light of an aerated solution of 3a in methanol leads to
decomposition of the complex and liberated 4-acetami-
dobenzenesulfonamide 4 was isolated and identified by
comparison with an authentic sample.
Scheme 1.

J. Zakrzewski, M. Pawlak / Journal of Organometallic Chemistry 568 (1998) 171–175
173
The above results indicate that 2c can be of interest
for modification of proteins (and other biopolymers) by
coupling of their COOH groups (present in the C-ter-
minal aminoacid and in aspartate and glutamate
residues) with the NH2-group in 2c. However, to be
applicable for biological samples such reactions should
work in aqueous (or polar organic solvent-water) media
in the presence of reagents activating the carboxylic
function (carbodiimides, Woodward,’s reagent) [4].
Protein modification with 2c is of interest since
modified biomolecules are expected to be detectable,
even at very low concentrations, by means of FTIR
spectroscopy in the region of 1900–2100 cm−1 (region
of stretching vibrations of co-ordinated CO ligands)
where biomolecules and biological matrices are practi-
cally transparent.. The idea of using transition-metal
complexes at IR-detectable labels of biomolecules was
put forward by Jaouen [5,6] and was successfully ap-
plied for the study of hormone–receptor interactions [7]
and in immunoassays (carbonylmetallo-immunoassays,
CMIA) [8]. The labelling procedures applied up to now
for the introduction of the organometallic label were
mainly based on the reaction of organometallic active
N-succinimidyl (NS) esters with the amino groups
(lysine residues in proteins) [9]. These groups were also
labeled with the oganometallic isothiocyanates ([1]f),
imidoester [10] and pyrylium salts [11]. Thiol groups
present in cysteine residues can be labelled by the use of
[CpFe(CO)2(p1-N-maleimidato)] ([1]c). However, de-
spite the potential importance [4,12] there was no exam-
ple of a metallo-carbonyl label reacting with the COOH
function.
Taking advantage of solubility of 2c in water we
decided to study the reactions of 2c with carboxylic
acids in the presence of a water-soluble carbodiimide,
1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide hy-
drochloride (EDC). We chose some simple water-solu-
ble acids to mimic conditions used in protein labeling.
The reaction was carried out at r.t. in weakly acidic
solutions (pH 5–6). This fact is very important for an
eventual application for labelling of proteins since in
basic media (pH\8) polycondensation or cyclization
of these molecules due to reaction of activated car-
boxylic function with the nonprotonated, nucleophilic
amino functions [4]. Obviously, the amino function in
the labeling reagent must not be completely protonated
under coupling conditions, i.e. it should be only weakly
basic (pKaB5–6). We think that this condition is fulfi-
lled for 2c. Although we did not measure the pKa value
for this complex, we expect that will not differ very
much from the value measured for 1c (2.1 in H2O) [13].
No detectable protonation of 2c is therefore expected at
pH 5–6. We confirmed this from 1H-NMR spectra
which show no pH-dependence of the chemical shifts of
the signals of this complex in the pH range 5–7. We
have found that 2c can be readily acylated with car-
boxylic acids in aqueous solutions containing an excess
of EDC. When equimolar amounts of carboxylic acid
and 2c were used the best yields were obtained in the
presence of a 4-fold excess of EDC. The products 3a,d,e
crystallize directly from the reacting solution in 54–
81% yield in analytically pure form. NMR spectra of
mother solutions indicate that they contain some
amount of the product but its separation from EDC
and the product of its hydrolysis is troublesome. In
reaction with acetic acid the product obtained was
1
identical (IR and H-NMR) with that obtained in the
reaction of 2c with acetic anhydride. The entries (d) and
(e) deserve some comments. Iodoacetamides and
maleimides are considered as reagents reacting specifi-
cally with the cysteine thiol groups and are frequently
used in protein labeling [4]. Consequently, 3d,e are
potential metallocarbonyl markers for thiol-containing
biomolecules. Complex 2c can be, therefore, a starting
compound for creation of a family of metallocarbonyl
markers for various functional groups.
3. Experimental
3.1. General remarks
Photochemical syntheses of CpFe(CO)2-complexes of
sulfonamide anions were carried out under an atmo-
sphere of argon. Chromatographic separations were
carried out on silica gel 60 (230–400 mesh ASTM).
Solvents were dried by using standard procedures. The
1H-NMR spectra were obtained by using a Varian
Gemini 200BB spectrometer at 200 MHz on solutions
in DMSO-d6 and were referenced toward internal
Me4Si. IR spectra were run on a Specord 75 IR spec-
trometer. FAB MS was obtained on a Finnigan MAT
95 spectrometer. The elemental analyses were deter-
mined by the Analytical Services of the Center of
Molecular and Macromolecular Studies of the Polish
*
Academy of Sciences, Lo´dz´. All reagents are available
commercially.
3.2. Synthesis of CpFe(CO)2-complexes of sulfonamide
anions
A magnetically stirred suspension of [CpFe(CO)2I]
(460 mg, 1.5 mmol), sulfonamide (1.0 mmol) in benzene
(15 ml) and diisopropylamine (1 ml) was illuminated
with visible light (4×150 W domestic tungsten lamps)
at 0–5°C (external cooling in a water-ice bath). The
colour gradually changes from black to orange. After
2.5 h (3.5 h in the case of sulfanilamide) the illumina-
tion was stopped and the reaction mixture evaporated
to dryness to give an orange solid which was treated
with 5% aq. K2CO3 (10 ml) and extracted with chloro-

174
J. Zakrzewski, M. Pawlak / Journal of Organometallic Chemistry 568 (1998) 171–175
form (3×30 ml). After drying (MgSO4) and concentra-
tion to a small volume, the reaction mixture was intro-
duced on a short silicagel column. Chloroform eluted a
black band of unreacted FpI, and then the product was
eluted as orange crystals with CHCl3–methanol (95/5).
Analytical samples were crystallized from CH2Cl2–
ether.
Anal. Calc. for C16H16N2O5SFe: C 47.54, H 3.99, N
6.93, S 7.93%. Found: C 47.63, H 4.35, N 6.99, S
7.89%.
[CpFe(CO)2NHSO2C6H4-p-NHCOCH(CH3)2],
3c:
Yield 94%. IR (KBr, cm−1): 3310 (NH, amide); 3280
(NH, sulfonamide); 2040, 1990 (FeCO) 1680 (amide I)
1
1540 (amide II) 1320, 1140 (n SO2). H-NMR (DMSO-
[CpFe(CO)2NHSO2C6H4-p-CH3], 2a: Yield 62%. IR
d6, l ppm): 10.03, s, 1H (NH, amide); 7.74, s, 1H (NH,
sulfonamide); 7.67, d, J=8.6 Hz, 2H and 7.56, d,
J=8.6 Hz, 2H (aromatic H’s); 5.15, s, 5H (Cp); ca. 2.6,
m, partly obscured by the solvent signal, (CH); 1.10, d,
J=7.5 Hz, 6H (CH3). Anal. Calc. for C17H18N2O5SFe:
C 48.80, H 4.34, N 6.70, S 7.65%. Found: C 48.69, H
4.44, N 6.70, S 7.80%.
(KBr, cm−1): 3290 (NH); 2045, 2000 (FeCO), 1290,
1
1130 (SO2). H-NMR (CDCl3, l ppm): 7.71, d, J=7.9
Hz, 2H and 7.67, d, J=7.9 Hz, 2H (aromatic protons);
5.29, s, 5H (Cp); 2.65, s, 3H (CH3). Anal.: Calc. for
C14H13NO4SFe: C 48.44, H 3.77, N 4.03, S 9.23.
Found: C 48.66, H, 3.91, N 3.88, S 9.42.
[CpFe(CO)2NHSO2C6H4-p-NO2], 2b: Yield 91%.
IR(KBr, cm−1): 3290 (NH); 2055, 2000 (FeCO); 1530,
1345 (NO2), 1305, 1140 (SO2). 1H-NMR (CDCl3, l
ppm): 8.37, d, J=8.0 Hz, 2H and 7.91, d, J=8.0 Hz,
2H (aromatic protons); 5.20, s, 5H (Cp). Anal.: Calc.
for C13H10N2O6Fe: C 41.29, H 2.67, N 7.41, S 8.48.
Found: C 41.56, H 2.66, N 7.15, S 8.72.
[CpFe(CO)2NHSO2C6H4-p-NH2], 2c: Yield 87%. IR
(KBr, cm−1): 3370, 3310 (NH2), 3280 (NH), 2045, 2000
(FeCO), 1290, 1125 (SO2). 1H-NMR (DMSO-d6, l
ppm): 7.71, bs, 1H (NH, sulfonamide), 7.31, d, J=8.5
Hz, 1H, and 6.53, d, J=8.5 Hz, 1H (aromatic H’s);
5.52, bs, 2H (NH2); 5.12,s, 5H (Cp). Anal: Calc. for
C13H12N2O4SFe: C 44.85, H 3.47, N 8.05, S 9.21%.
Found: C 44.48, H 3.65, N 8.01, S 9.35%.
3.4. Decomplexation of 4-acetamidobenzenesulfonamide,
4, from 3a
An aerated solution of 3a (77 mg, 0.2 mmol) in
methanol (5 ml) was irradiated (setup as described
above) at 0–5°C for 1 h. The pale yellow solution
obtained was evaporated to dryness and triturated with
methanol (5 ml). The insoluble yellow amorphic mate-
rial was filtered of and the filtrate after evaporation of
the solvent afforded 4 as a gray solid. Crystallization
from water gave 4 as colorless microcrystals. M.p.
216–218°C. Yield 32 mg (75%). IR and 1H-NMR
spectra of this product were identical to those of an
authentic sample.
3.3. Acylation of [CpFe(CO)2NHSO2C6H4-p-NH2] (2c)
with acid anhydrides
3.5. Coupling of [CpFe(CO)2NHSO2C6H4-p-NH2] (2c)
with carboxylic acids/EDC
[CpFe(CO)2NHSO2C6H4-p-NH2], 2c,(100 mg, 0.29
mmol) was dissolved in the anhydride (1.5 ml) and
stand overnight at r.t. The solid formed was filtered off,
washed with diethyl ether and dried in vacuo (50°C/0.2
Tr).
To
a
magnetically
stirred
solution
of
[CpFe(CO)2NHSO2C6H4-p-NH2] (2c) (100 mg, 0.29
mmole) in water (5 ml) were added carboxylic acid
(0.30 mmol) and EDC (1.38 mmol). The pH of the
resulting mixture was 5–6. After 3 h the solid formed
was filtered off, washed with water (2 ml) and dried in
vacuo (40–50°C/0.2 Tr).
[CpFe(CO)2NHSO2C6H4-p-NHCOCH3], 3a: Yield
54%.
[CpFe(CO)2NHSO2C6H4-p-NHCOCH2I], 3d: Yield
81%. IR (KBr, cm−1): 3310 (NH, amide); 3280 (NH,
sulfonamide); 2045, 1995 (FeCO); 1690 (amide I) 1555
[CpFe(CO)2NHSO2C6H4-p-NHCOCH3], 3a: Yield
86%. IR (KBr, cm−1): 3320 (NH, amide); 3280 (NH,
sulfonamide); 2040, 1990 (FeCO) 1680 (amide I) 1540
1
(amide II) 1320, 1140 (SO2). H-NMR (DMSO-d6, l
ppm): 10.15, s, 1H (NH, amide); 7.75, s, 1H (NH,
sulfonamide); 7.61, m, 4H (aromatic H’s); 5.16, s, 5H
(Cp); 2.08, s, 3H (Me). FAB MS(NBA matrix, positive
ions): 391 (M+H); 335 (M+H–2 CO). Anal. Calc.
for C15H14N2O5SFe: C 46.16, H 3.62, N 7.18, S 8.22%.
Found: C 45.85, H 3.66, N 7.03, S 7.99%.
1
(amide II); 1325, 1130 (SO2). H-NMR (DMSO-d6, l
ppm): 10.52, s, 1H, (NH, amide); 7.70, s,(NH, sulfon-
amide) 7.61 s, 4H (aromatics); 5.16, s, 5H (Cp); 3.84, s,
2H, CH2. Anal.: Calc. for C15H13N2O5ISFe·0.5H2O: C
34.31, H 2.69, N 5.33, S 6.16, I 24.17. Found: C 34.42;
H 2.50; N 5.45; S 6.24, I 23.82.
[CpFe(CO)2NHSO2C6H4-p-NHCOCH2CH2NC4H2-
O2], 3e: Yield 62%. IR (KBr, cm−1): 3320 (NH, amide);
3280 (NH, sulfonamide); 2055, 2000 (FeCO); 1710,
[CpFe(CO)2NHSO2C6H4-p-NHCOCH2CH3],
3b:
Yield 80%. IR (KBr, cm-1): 3315 (NH, amide); 3280,
(NH, sulfonamide); 2050, 1990 (FeCO) 1685 (amide I)
1540 (amide II) 1310, 1140 (SO2). 1H-NMR (DMSO-d6,
l ppm): 10.07, s, 1H (NH, amide); 7.73, s, 1H (NH,
sulfonamide); 7.66, d J=8.5 Hz, 2H and 7.56, d,
J=8.5 Hz, 2H (aromatic H’s); 5.14,s, 5H (Cp), 2.34, q,
J=7.4 Hz, 2H (CH2); 1.08, t, J=7.4Hz, 3H, (CH3).

J. Zakrzewski, M. Pawlak / Journal of Organometallic Chemistry 568 (1998) 171–175
175
[2] P.G. Sammes, in: C. Hansch, P.G. Sammes, J.B. Taylor (Eds),
Comprehensive Medicinal Chemistry, vol. 2, Pergamon Press,
Oxford, 1990, Chapter 7.1.
[3] See, for example: F. Blasco, L. Perello´, J. Borra´s, S. Garcia-
Granda, J. Inorg. Biochem. 61 (1996) 143.
[4] M. Brinkley, Bioconjugate Chem. 3 (1992) 2.
[5] M. Salmain, A Vessie`res, G. Jaouen, I.S. Butler, Anal. Chem. 63
(1991) 2323.
[6] G. Jaouen, A. Vessie`res, I.S. Butler, Acc. Chem. Res. 23 (1993)
361.
1680 (sh) (imide CO+amide I) 1540 (amide II); 1315,
1140 (SO2). H-NMR (DMSO-d6, l ppm): 10.20, 1H,
1
(NH, amide); 7.71 s, 1H, (NH, sulfonamide); 7.58 s,
4H, aromatics; 7.03, s, 2H, (olefinic); 5.15, s, 5H, (Cp);
3.72, t, J=6.8 Hz, 2H, (CH2), (second CH2 signal
presumably obscured by the solvent signal). Anal. Calc.
for C20H17N3O7SFe: C 48.09, H 3.43, N 8.42, S 6.41.
Found: C 47.76; H 3.50; N 8.84; S 6.26.
[7] S. Top, H. El Hafa, A. Vessieres, J. Quivy, J. Vaissermann,
D.W. Hughes, M.J. McGlinchey, J.-P. Mornon, E. Thoreau,
G.J. Jaouen, J. Am. Chem. Soc. 117 (1995) 8372.
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Commun. Chem. Pathol. Pharmacol. 84 (1994) 81. (b) A.
Varenne, A. Vessie`res, M. Salmain, P. Brossier, G. Jaouen,
Immunoanal. Biol. Spec. 9 (1994) 205. (d) A. Varenne, A.
Vessie`res, M. Salmain, S. Durand, P. Brossier, G. Jaouen, Anal.
Biochem. 242 (1996) 172.
Acknowledgements
This work was financially supported by the Polish
State Committee for Scientific Research (KBN) (Grant
3 T09A 008 08).
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gate Chem. 3 (1992) 471. (b) M. Salmain, M. Gunn, A. Gorfti,
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