Synthesis of an amino-functionalized model of the Fe-only hydrogenase active site

Article abstract of DOI:10.1002/chem.200390058

A dinuclear 2Fe2S mimic 6 of the active site of the Fe-only hydrogenases has been synthesized. Complex 6 contains a free amino group which enables linkage to a protein backbone or to a redox active species for the study of electron transfer processes in p

Full text of DOI:10.1002/chem.200390058

FULL PAPER
Synthesis of an Amino-Functionalized Model of the Fe-Only Hydrogenase
Active Site
Szabolcs Salyi,[a] Mikael Kritikos,[b] Bjˆrn äkermark,*[a] and Licheng Sun*[a]
Abstract: A dinuclear 2Fe2S mimic 6 of the active site of the Fe-only hydrogenases
has been synthesized. Complex 6 contains a free amino group which enables linkage
to a protein backbone or to a redox active species for the study of electron transfer
processes in proteins or in supramolecular systems. The structures of the complex 6
and its Boc-protected precursor 5 could be verified by X-ray crystallography.
Keywords: bioinorganic chemistry ¥
biomimetic synthesis ¥ iron carbonyl
sulfides ¥ iron-only hydrogenase
Because of our interest in photochemical hydrogen pro-
duction,[17] and a general interest in attaching a 2Fe2S unit to a
protein structure, we decided to try to develop synthetic
procedures for preparing such units which contain a suitable
functional group. The protected and unprotected amine
substituted Fe dimer carbonyl sulfide complexes 5 and 6 have
therefore been synthesized and characterized.
Introduction
The iron-only hydrogenases form a wide-spread class of
enzymes in microorganisms, which mediate both the metab-
olism of hydrogen and the reverse reaction, formation of
7]
molecular hydrogen from protons.[1
The X-ray crystal structures of two types of Fe-only
hydrogenases, CpI (Clostridium pasteurianum)[8] and DdH
(Desulfovibrio desulfuricans)[9] were reported recently. The
active sites in both systems contain an uncommon six-Fe
cluster(H-cluster), which consists of a 4Fe-4S cubane struc-
ture, bridged by cysteine-S to a novel 2Fe2S subunit. The 4Fe-
4S unit probably mediates electron transfer while the 2Fe-2S
subunit is responsible for the formation and activation of
hydrogen. In this subunit, the two Fe ions are linked by two
non-cysteine thiolate groups and have CO and CNÀ diatomic
terminal ligands.[10] Very recent crystallographic studies
suggest that the bridging dithiolate ligand has the structure
of SCH2NHCH2SÀ, or the N-protonated equivalent.[11]
À
Since the elucidation of the X-ray crystal structures of Fe-
only hydrogenases, some synthetic 2Fe-2S complexes have
15]
been prepared as structural mimics of the active site.[12
Results and Discussion
Rauchfuss et al. have reported a model complex with the
structure of {Fe2[m-S2(CH2)3](CN)(CO)4(PMe3)}À.[16] Using
electrochemistry, they could demonstrate catalytical hydro-
gen production with this complex in homogeneous solution in
the presence of strong acid.
In the 1980s, Winter, Zsolnai, and Huttner[18] prepared
Fe2(SR)2(CO)6 derivatives through the reaction of propane-
1,3-dithiol and [Fe3(CO)12]. However, very few diiron hex-
acarbonyl complexes, functionalized at 2-position in the
dithiolate ring, have been described, probably because of
the limited availability of functionalized 1,3-dithiols. We
would therefore like to present a general synthetic route for
such 1,3-dithiols, whereby sulfur bridged diiron complexes can
be synthesized in a relatively simple procedure (Scheme 1).
The synthesis of the 2-functionalized 1,3-dithiolpropane
subunit 4 from 1,3-dibromopropan-2-ol turned out to be a
challenging problem, because the dibromo functionality is
both heat and base sensitive. The acid-catalyzed esterification
[a] Prof. Dr. B. äkermark, S. Salyi, Dr. L. Sun
Department of Organic Chemistry, Arrhenius Laboratory
Stockholm University, Stockholm (Sweden)
Fax : (‡46)-8-154908
E-mail: bjorn.akermark@organ.su.se
licheng.sun@organ.su.se
[b] Dr. M. Kritikos
Department of Structural Chemistry, Arrhenius Laboratory
Stockholm University, Stockholm (Sweden)
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557

B. äkermark, L. Sun et al.
FULL PAPER
Scheme 1. The synthetic procedure of complexes 5 and 6.
Figure 1. ORTEP (ellipsoids at 30% probability level) view of C21H22Fe2-
NO10S2 (5).
of 1,3-dibromopropanol[19] gave only low yields (<5%). More
recently, Boyes and Hewson have reported[20] the esterifica-
tion of similar compounds by using N,N-dicyclohexylcarbo-
diimide (DCC) and 4-dimethylaminopyridine (DMAP). After
several trials, we managed to find reaction conditions, which
gave an ester derivative 2 of 1,3-dibromopropan-2-ol in
reasonable yields.
The most critical step in our synthesis seemed to be the
replacement of the bromides by thiol groups. The direct
preparation of thiols from alkyl halides and metal sulfides
should be facile, but in our hands direct methods gave only a
moderate yield of di-thiol.[21] Indirect methods involving
thiourea[22] and xanthate derivatives[23] are commonly utilised
for the synthesis of thiols, but these intermediates have to be
transformed to thiols by hydrolysis with base or by reduction
with lithium aluminium hydride, which will react also with the
ester group in compound 2.[24] However, treatment of
thioacetates with amines under mild conditions has recently
been shown to give thiols in good yields.[25, 26] In accordance,
we were able to prepare the dithioacetate 3 by treatment of 2
with potassium thioacetate.[27] Unfortunately, the reaction of 3
with amines failed, but we were able to obtain the ester-dithiol
4 in excellent yields by treating 3 with hydrazine hydrate in
acetic acid solution.[28]
Reaction of the dithiol ligand 4 with [Fe3(CO)12] in T HF
gave the product 5 in a rather good yield (87%) after
purification by column chromatography on silica gel. Treat-
ment of 5 with trifluoroacetic acid (TFA) in dichloromethane
at room temperature[29] gave complex 6 which has a free
amino functional group. The synthetic steps for preparing
complexes 5 and 6 could be repeated; furthermore it was no
problem to scale-up the synthesis. It is worth to mention that
the Fe dimer core structures in 5 and 6 were stable even in
fairly strongly acidic conditions such as in the presence of
TFA. These complexes were also stable during purification by
column chromatography on silica gel.
Figure 2. ORTEP (ellipsoids at 30% probability level) view of C16H11Fe2-
NO8S2 (6).
similar and to a large extent analogous to those found in other
Fe2(m-SR)2(CO)6 structures.[13, 30] In the central Fe2(m-
SR)2(CO)6 unit the two Fe atoms and the two S atoms form
a butterfly conformation in which the metal atoms are
À
connected to each other through a Fe Fe single bond with
2.509(1) and 2.499(1) ä for 5 and 6, respectively. The first S2C3
coordination shell surrounding each Fe atom is best described
as a square-pyramidal coordination geometry with one of the
carbonyl C atoms in the apical position. The Fe atom is
displaced from the basal plane in the direction to the apical C
atom. Intermolecular N-H ¥¥¥ O hydrogen bonds are present in
both 5, N1 ¥¥¥ O8 2.988(2) ä, and 6, N1 ¥¥¥ O8 3.212(8) ä. A
potentially important structural feature of these complexes is
that the amino group is well separated from the dimeric Fe
core. (Fe1 ¥¥¥ N1 11.163(2), Fe2 ¥¥¥ N1 11.351(2) ä in 5 and
Fe1 ¥¥¥ N1 11.186(7), Fe2 ¥¥¥ N1 11.240(6) ä in 6). Selected
bond lengths of compounds 5 and 6 are listed in Table 1.
All new compounds synthesized herein were characterized
by 1H NMR spectroscopy and elemental analysis.
Single crystal X-ray diffraction shows that the two Fe2-
(m-S)2(CO)6 units of 5 (Figure 1) and 6 (Figure 2) are very
558
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Fe-Only Hydrogenases
557 560
Table 1. Selected bond lengths [ä] for 5 and 6.
for C15H19Br2NO4: C 41.21, H 4.38, Br 36.56, N 3.20; found: C 41.71, H 4.58,
Br 35.67, N 3.21.
5
6
1,3-Dithioacetyl-2-(4-butoxycarbonylaminobenzoyl)propanol (3): The di-
bromo derivative 2 (0.219 g, 0.5 mmol) was dissolved in acetone (15 mL),
and then potassium thioacetate (4.5 equiv; 0.257 g, 2.25 mmol) was added.
The reaction was stirred under N2 at ambient temperature and followed by
TLC. When starting material disappeared (ca. 4 h) the precipitated KBr
was filtered off, and the filtrate was evaporated in vacuo. The residue was
purified by flash column chromatography with toluene/EtOAc (6:1) as
À
Fe1 S1
2.2699(7)
2.2603(7)
2.5093(5)
2.2528(7)
2.2466(7)
1.797(3)
1.798(3)
1.807(3)
1.807(3)
1.797(3)
1.799(3)
1.833(2)
1.824(2)
2.278(2)
2.257(2)
2.499(1)
2.256(2)
2.261(2)
1.794(7)
1.787(7)
1.808(7)
1.804(7)
1.789(7)
1.815(7)
1.824(6)
1.827(6)
À
Fe1 S2
À
Fe1 Fe2
À
Fe2 S1
À
Fe2 S2
À
Fe1 C1
À
Fe1 C2
1
eluent, giving 3 (177 mg, 82.5%) as an oil. H NMR (CDCl3): d ˆ 1.52 (s,
À
Fe1 C3
9H), 2.32 (s, 6H), 3.26 (dd, J ˆ 10.5, 4.8 Hz, 1H), 3.29 (dd, J ˆ 10.5, 3.9 Hz,
1H), 5.22 5.28 (m, 1H), 6.71 (s, 1H), 7.42 7.44 (m, 2H), 7.91 7.93 (m,
2H); elemental analysis calcd (%) for C19H25Br2NO6S2: C 53.38, H 5.89, N
3.28, S 15.00; found: C 53.20, H 5.97, N 3.17, S 15.14.
À
Fe2 C4
À
Fe2 C5
À
Fe2 C6
À
S1 C8
1,3-Dithiolo-2-(4-butoxycarbonylaminobenzoyl)propanol (4): The thioace-
tate derivative 3 (124 mg, 0.29 mmol) was dissolved in DMF (5 mL). To
solution hydrazine hydrate (3.5 equiv; 50 mL, 1.027 mmol) was added
dropwise under N2 at ambient temperature, and then the solution was
stirred for 10 min. Afterwards, acetic acid (3.5 equiv; 60 mL, 1.027 mmol)
was added dropwise under N2 at ambient temperature. The reaction was
followed by TLC, and when the starting material disappeared (ca. 30 min)
the mixture was diluted with water (10 mL) and ethyl acetate (10 mL).
After phase separation the organic layer was washed with 2 Â 10 mL of
water, and then the water phase was extracted with ethyl acetate. The
combined organic phase was washed with water (10 mL) and brine
(10 mL), dried with Na2SO4, filtered, and concentrated in vacuo. The
crude product was purified by flash column chromatography with toluene/
À
S2 C7
Conclusion
An amino-functionalized model 6 for the active site of the Fe-
only hydrogenases has been prepared by covalently linking
4-aminobenzoic acid to a 2Fe2S structure. The Fe dimers of 6
and its precursor 5 are fully stable during the reaction
conditions invetsigated. The crystal structures of 5 and 6 show
that the functional group and the Fe dimer are separated from
each other by about 11 ä. This suggests that these complexes
will be useful for protein modification and the build up of
supramolecular systems, where a 2Fe2S unit is linked to one or
more redox components for the study of electron transfer
processes.
1
EtOAc (6:1) as eluent, giving 4 (94.5 mg, 95.5%) as a thick oil. H NMR
(CDCl3): d ˆ 1.46 (t, J ˆ 8.8Hz, 2H), 1.53 (s, 9H), 2.97 (dd, J ˆ 5.7 Hz,1 Hz,
1H), 3.00 (dd, J ˆ 5.7 Hz, 1.5 Hz, 1H), 5.16 (app. pent., J ˆ 5.7 Hz, 1H),
6.66 (s, 1H), 7.43 7.46 (m, 2H), 7.96 7.99 (m, 2H); elemental analysis
calcd (%) for C19H25Br2NO6S2: C 52.45, H 6.16, N 4.08, S 18.67; found: C
52.45, H 6.21, N 4.03, S 18.58.
Synthesis of [m-(SCH2)2CHCO2C6H4NHCO2C(CH3)3]Fe2(CO)6 (5): T he
dithiol derivative 4 (90 mg, 0.207 mmol) was dissolved in dry THF (20 mL),
and then triiron dodecacarbonyl (105 mg, 0.208 mmol) was added. The
resulting solution was warmed up to 65 678C, stirred for 0.5 h under N2,
and then concentrated by evaporating the solvent in vacuo. The crude
product was purified by flash column chromatography on silica gel with
toluene/pentane (1:1) as eluent followed by pure toluene. The crystalline
product 5 was obtained by cooling the concentrated toluene solution at ca
À208C overnight (141.5 mg, 87%). 1H NMR (CDCl3): d ˆ 1.52 (s, 9H), 1.69
(dd, J ˆ 12.9, 12.0 Hz, 1H), 2.91 (dd, J ˆ 13.5, 4.5 Hz, 1H), 4.42 4.49 (m,
1H), 6.65 (s, 1H), 7.39 7.42 (m, 2H), 7.86 7.88 (m, 2H); elemental analysis
calcd (%) for C21H19Fe2NO10S2: C 40.6, H 3.08, N 2.25, S 10.32; found: C
43.76, H 3.46, N 2.11, S 9.37. The deviation is due to the presence of ca 20%
toluene solvent in the crystal structure.
Experimental Section
All reagents and solvents were purchased from Aldrich and used as
received. 1H NMR spectra were recorded on a Varian 400 spectrometer.
Elemental analysis was performed in Analytische Laboratorien, Lindlar
(Germany).
4-Butoxycarbonylaminobenzoic acid (1): 4-Aminobenzoic acid (2.74 g,
20 mmol) was suspended in water (40 mL) and then tert-butanol (30 mL)
was added. To this solution di-tert-butyl-dicarbonate (8.73 g, 40 mmol) and
solid NaOH (0.88 g, 22 mmol) were added and the temperature was
increased to 608C. After completion of reaction the mixture it was diluted
with water (100 mL), washed with CH2Cl2 (2 Â 50 mL) and the water phase
was cooled to 0 58C. Ethyl acetate was added (50 mL), and under
intensive stirring the pH was lowered to 2 by the addition of 2m HCl. The
organic phase was separated and the extraction was repeated with ethyl
acetate (2 Â 50 mL). Combined extracts were dried over Na2SO4, and
evaporated in vacuo giving 4-butoxycarbonylaminobenzoic acid (1; 4.62 g,
97.5%). The solid off-white powder was directly used for the next step.
1H NMR ([D6]DMSO): d ˆ 1.48 (s, 9H), 7.54 7.56 (m, 2H), 7,82 7.84 (m,
2H), 9.72 (s, 1H), 12.59 (s, 1H).
Crystal data for 5: C21H19Fe2NO10S2: M ˆ 621.20, monoclinic space group,
P21/c (No. 14), a ˆ 11.1598(10), b ˆ 11.9372(12), c ˆ 22.735(3) ä, b ˆ
92.002(12)8, Vˆ 3026.8(6) ä3, Z ˆ 4, Tˆ 170 K, 1calcd ˆ 1.466 gcmÀ3
,
m(MoKa) ˆ 1.15mmÀ1, F(000) ˆ 1356, 22301 reflections measured, 5651
unique (Rint ˆ 0.048), 4956 observed (I > 2s(I)), 365 parameters refined,
absorption correction (numerical): Tmin/Tmax ˆ 0.788/0.916. R1 ˆ 0.0335, (I
> 2s(I)), wR(F2) ˆ 0.1050, S ˆ 1.11 (all data). Max./min. residual electron
density: 0.52/ À 0.46. Data collection: STOE-IPDS image plate diffrac-
tometer with a rotating-anode using MoKa radiation (l ˆ 0.71073 ä). The
intensities of the reflections were integrated with the STOE software, and
the numerical absorption correction was performed with the programs
X-RED and X-SHAPE.[31] The structure was solved by direct methods[32]
and refined by full matrix least-squares on F2.[33]
1,3-Dibromo-2-(4-butoxycarbonylaminobenzoyl)propanol (2): DMAP
(0.128 g, 1.05 mmol) and 1,3-dibromopropan-2-ol (0.26 mL, 2.54 mmol)
were added to a suspension of compound 1 (0.6 g, 2.53 mmol) in dry CH2Cl2
(50 mL). The resulting suspension was stirred at room temperature for
15 min, and then a CH2Cl2 (15 mL) solution of DCC (0.87 g, 4.21 mmol)
was added dropwise (ꢀ15 min) under N2 atm at ambient temperature. The
reaction was followed by TLC, and when the starting material disappeared
(ꢀ4 h) the solvent was evaporated in vacuo. The residue was purified by
column chromatography on silica with toluene/EtOAc (6:1) as eluent.
After evaporation of the solvent 2 was obtained as a white powder (0.905 g,
Synthesis of [m-(SCH2)2CHCO2C6H4NH2]Fe2(CO)6 (6): Trifluoroacetic
acid (0.51 mL, 3 mmol) was added to a solution of the diiron complex 5
(90 mg, 0.14 mmol) in dry dichloromethane (10 mL), and then stirred at
ambient temperature. The reaction was followed by TLC, and when the
starting material disappeared (3 4 h) the solvent and unreacted TFA were
evaporated in vacuo. The residue was purified by flash chromatography
with toluene/EtOAc (6:1) as eluent to give 6 (67 mg, 89%). A single
crystalline product was obtained by cooling the concentrated toluene/
1
86%). H NMR (CDCl3): d ˆ 1.53 (s, 9H), 3.72 (dd, J ˆ 11.0, 5.4 Hz, 1H),
1
3.77 (dd, J ˆ 11.0, 4.9 Hz, 1H), 5.35 (app. pent., J ˆ 5.4 Hz, 1H), 6.67 (s,
pentane (1:1) solution of 6 at about À208C overnight. H NMR (CDCl3):
1H), 7.44 7.47 (m, 2H), 7.98 8.01 (m, 2H); elemental analysis calcd (%)
d ˆ 1.67 (dd, J ˆ 13.0, 11.5 Hz, 1H), 2.90 (dd, J ˆ 13.0, 4.1 Hz, 1H), 4.09 (s,
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B. äkermark, L. Sun et al.
FULL PAPER
2H), 4.39 4.44 (m, 1H), 6.59 6.62 (m, 2H), 7.73 7.76 (m,2H); elemental
analysis calcd (%) for C16H11Fe2NO8S2: C 36.88, H 2.13, N 2.69, S 12.30;
found: C 37.04, H 2.31, N 2.63, S 12.19.
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(No. 2), a ˆ 8.4739(14), b ˆ 9.9920(16), c ˆ 13.296(2) ä, a ˆ 69.81(2)8, b ˆ
71.99(2)8, g ˆ 85.96(2)8, Vˆ 1004.0(3) ä3, Z ˆ 2, Tˆ 293 K, 1calcd
ˆ
1.724(1) gcmÀ3, m(MoKa) ˆ 1.70mmÀ1, F(000) ˆ 524, 7898 reflections meas-
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refined, absorption correction (numerical): Tmin/Tmax ˆ 0.723/0.822. R1 ˆ
0.0483 (I > 2s(I)), wR2 ˆ 0.1743, S ˆ 1.19 (all data). Max./min. residual
electron density: 0.558/ À 0.767. Data collection: STOE-IPDS image plate
diffractometer with a rotating-anode using MoKa radiation (l ˆ 0.71073 ä).
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Received: October 14, 2002 [F4498]
560
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Chem. Eur. J. 2003, 9, No. 2