Influence of the methylene group between azadithiolate nitrogen atom and phenyl moiety on the protophilic properties of [FeFe]-hydrogenase model complexes

Article abstract of DOI:10.1007/s11696-017-0187-7

An [FeFe]-hydrogenase mimic 4 with functional benzyl moiety covalently linked to the azadithiolate ligand was synthesized. The structure, protonation, and electrochemical properties of 4 and a phenyl substituted analogue (coded as 3) were simultaneously s

Full text of DOI:10.1007/s11696-017-0187-7

Chem. Pap.
DOI 10.1007/s11696-017-0187-7
ORIGINAL PAPER
Influence of the methylene group between azadithiolate nitrogen
atom and phenyl moiety on the protophilic properties
of [FeFe]-hydrogenase model complexes
Shang Gao1 Ting-Ting Yang1 Jian-Xun Zhao1 Qian Duan1 Qing-Cheng Liang1
Da-Yong Jiang1
•
•
•
•
•
Received: 28 December 2016 / Accepted: 2 May 2017
Ó Institute of Chemistry, Slovak Academy of Sciences 2017
Abstract An [FeFe]-hydrogenase mimic 4 with functional
benzyl moiety covalently linked to the azadithiolate ligand
was synthesized. The structure, protonation, and electro-
chemical properties of 4 and a phenyl substituted analogue
(coded as 3) were simultaneously studied to explore the
influence of the methylene group between the bridgehead
nitrogen atom and functional phenyl moiety on the pro-
tophilic properties of the model complexes. X-ray single
crystal diffraction analysis revealed that the nitrogen atoms
of 3 and 4 possessed sp2 and sp3-hybridization, respec-
tively. Although the light-driven electron transfer was
prevented in the molecule of 4, the sp3-hybridized nitrogen
atom of 4 could be protonated in the presence of the proton
acid to give the [4(NH)]? cation. The generated positive
charge could be reduced at ca. -1.2 V versus Fc/Fc? with
a distinctly electrocatalytic proton reduction activity,
whereas the proton reduction catalysed by 3 occurred at ca.
-1.45 V. The catalytic proton reductions of 3 and 4 fol-
lowed ECCE and CECE mechanisms, respectively. It was
noteworthy that the potential of 4 was remarkably anodic
shifted and closer to that of the proton reduction catalysed
by natural enzymes.
Introduction
Hydrogenases are a series of metalloenzymes that catalyse
the reversible interconversion of protons and molecular
hydrogen (Lubitz et al. 2014). According to the metal ion
composition of their active sites, hydrogenases can be
classified as [Fe]-, [NiFe]-, or [FeFe]-hydrogenase. All of
these enzymes are of high interest in biotechnology while
[FeFe]-hydrogenases are particularly active in both
hydrogen production and oxidation (Birrell et al. 2016;
Sommer et al. 2017). Recently, spectroscopic and crystal-
lographic studies have revealed the organometallic nature
of [FeFe]-hydrogenase active site (so-called H-cluster,
Scheme 1), which consists of a butterfly [2Fe] subunit and
a [4Fe4S] cluster connected via a thiolate of a cysteine
residue (Peters et al. 1998; Nicolet et al. 1999). Both the
[2Fe] and the [4Fe4S] sub-clusters are redox active, and the
two electrons involved in the redox reaction of
2H? ? 2e- $ H2 are accommodated by the changing
oxidation states of the two sub-clusters (Sommer et al.
2017). Possible redox states of the H-cluster such as the
active oxidized state Hox, the active reduced state Hred and
the super-reduced state Hsred have been well characterised
by combined EPR, FTIR and electrochemical studies
(Lubitz et al. 2007; Adamska et al. 2012). The redox and
protonation events that occurred on the [2Fe] subunit and
the [4Fe4S] cluster in the catalytic cycle are also resolved
in detail, demonstrating the essential necessity of the two
sub-clusters (Adams 1990; Pereira et al. 2001; Adamska
et al. 2012; Adamska-Venkatesh et al. 2014; Mulder et al.
2014). Inspired by the structural and functional details of
H-cluster, artificial photocatalytic systems have been con-
structed in view of solar energy conversion (Eckenhoff and
Eisenberg 2012; Wang et al. 2012; Wu et al. 2014; Wang
et al. 2015). From a photochemical point of view, electron
Keywords Hydrogenase Á Enzyme mimic Á Azadithiolate Á
Electrochemistry Á Proton reduction
Electronic supplementary material The online version of this
article (doi:10.1007/s11696-017-0187-7) contains supplementary
material, which is available to authorized users.
& Shang Gao
custgaoshang@126.com
1
School of Materials Science and Engineering, Changchun
University of Science and Technology, 7989 Weixing Road,
Changchun 130022, People’s Republic of China
123

Chem. Pap.
Scheme 1 Proposed
intermediate in enzymatic
reduction of protons and
oxidation of H2 (middle) and the
synthetic model complexes 3
(left) and 4 (right)
transfer should be triggered by a preceding absorption of a
photon by a photosensitizer. We have focused on the
construction of inexpensive [FeFe]-hydrogenase mimics
with photocatalytic activity (Gao et al. 2014, 2017). One
and cyclic voltammetry are explored to give more insights
into the influence of the methylene group between the
azadithiolate nitrogen atom and functional phenyl moiety
on the protophilic properties of [FeFe]-hydrogenase model
complex. Herein, the synthesis, structures, N-protonation
and electrochemical properties of 4 and the phenyl sub-
stituted analogue 3 were discussed in detail.
successful model was complex
3 (as depicted in
Scheme 1), consisting of a noble-metal-free organic chro-
mophore, a [2Fe2S] proton reduction catalytic centre to
accomplish the photo-induced H2 evolution. The light-
driven intramolecular electron transfer has been eviden-
tially demonstrated, and a remarkably photocatalytic effi-
ciency was achieved. However, the other significant source
of reducing equivalents for converting protons to molecular
hydrogen, that is, electrochemistry, has not been thought-
fully investigated.
Experiment
Reagents and instruments
All organometallic reactions and operations were carried
out under a dry, oxygen-free argon atmosphere with stan-
dard Schlenk techniques. All solvents were dried and dis-
tilled prior to use by standard methods. Starting compound
[(l-S)2{Fe(CO)3}2] was prepared according to literature
procedure (Bogan et al. 1983). Other materials were
commercially available and used without further
purification.
On the other hand, the dithiol ligand has been
unequivocally confirmed as an azadithiolate (Silakov et al.
2009; Erdem et al. 2011; Berggren et al. 2013). The
bridging amine group plays an important role for the
heterocyclic cleavage or formation of hydrogen in enzy-
matic process. The amine moiety close to the Fed (‘‘distal’’
to [4Fe4S] cluster) atom with open coordination site would
accept or donate a proton from a transient hydride in
hydrogen production (Berggren et al. 2013; Esselborn et al.
2013; Lubitz et al. 2014), acting as a proton shuttle
between the protein and the H-cluster (Sommer et al.
2017). Studies on the protonation of the amino headgroup
in diiron azadithiolates might contribute to the under-
standing of the mechanism of enzymatic hydrogen pro-
duction and/or uptake. Likewise, after protonation, the
nitrogen-bridged diiron complex exhibited milder reduc-
tion potential (Ott et al. 2004; Wang et al. 2007; Jiang et al.
2007; Capon et al. 2008), which was closer to that (ca.
-1.0 V versus Fc/Fc?) of the proton reduction catalysed
by natural enzymes (Holm et al. 1996; Butt et al. 1997).
Relative to the phenyl species as a substituent on the
azadithiolate nitrogen atom, the benzyl group seemed to
have an influence on the architectural, protonated and
electrochemical properties of the diiron dithiolate complex.
We, therefore, set out to synthesize a new [FeFe]-hydro-
genase active site mimic which contains a functional
benzyl group covalently embedded to the azadithiolate
ligand (coded as 4, Scheme 1). The structure, acid titration
IR spectra were recorded on JASCO FT/IR 430 spec-
trophotometer. 1H and 13C spectra were collected on a
Bruker AVANCE II/400 NMR spectrometer. HR-MS
determinations were made on a GCT-MS instrument (Mi-
cromass, England). UV–Vis spectra were measured on a
PerkinElmer Lambda 35 spectrophotometer. Steady-state
emission spectra were determined on JASCO FP-6500
spectrophotometer.
Synthesis and characterization
2-[4-(bromomethyl)phenyl]benzothiazole (coded as 1a,
Scheme 2) was synthesized from 4-methylbenzoic acid and
2-aminothiophenol according to literature procedures
(Palmer et al. 1971; Yoshino et al. 1986). The solution of
1a (4.5 g, 15 mmol) in anhydrous CHCl3 (50 mL) was
added dropwise to a solution (40 mL anhydrous CHCl3) of
hexamethylenetetramine (2.1 g, 15 mmol). The mixture
was refluxed for 5 h with vigorous stirring. The resulting
precipitate was washed several times with deionized water
and added to a mixture of ethanol and concentrated HCl
123

Chem. Pap.
Scheme 2 Synthetic procedure of the model complex 4
(4:1, 100 mL). The resulting solution was kept stirring at
70 °C for 12 h and then allowed to stand at room tem-
perature overnight. An HCl salt was obtained by filtration,
washed with 10% KHCO3 (50 mL) and extracted into
CHCl3 (50 mL). The organic layer was dried over anhy-
drous MgSO4. The solvent was removed in vacuo to give
the primary amine 1b as a yellow solid (3.06 g, 85%). A
mixture of 1b (1.8 g, 7.5 mmol) and paraformaldehyde
(0.6 g, 19.5 mmol) in 20 mL CH2Cl2 was stirred for 5 h
and treated dropwise with 2.25 mL (31 mmol) of SOCl2.
After 1.5 h, the solvent and unreacted SOCl2 were removed
under vacuum to give the product 1c. The generated solid
was added to the THF solution of [(l-LiS)2Fe2(CO)6],
freshly prepared by the reaction of super hydride LiEt3BH
(1 mol L-1 solution in THF, 8 mL) and [(l-S)2-
{Fe(CO)3}2] (2) (1.38 g, 4 mmol in 30 mL THF). The
mixture was stirred for 2 h at -78 °C and then 1 h at room
temperature. The solvent was removed on a rotary evapo-
rator. The crude product was purified by column chro-
matography (silica, 10% dichloromethane in hexane as
eluent) to give complex 4 (1.77 g, 72%) as a red solid.
1H NMR (400 MHz, CDCl3): d = 7.93 (d, 2H, C6H4),
7.52 (s, 2H, NC6H4S), 7.42 (s, 2H, NC6H4S), 7.34 (s, 2H,
C6H4), 3.78 (s, 2H, PhCH2), 3.37 (s, 4H, SCH2) ppm; 13C
NMR (100 MHz, CDCl3): d = 207.9, 167.6, 154.3, 139.2,
135.2, 133.4, 129.4, 128.0, 126.6, 125.5, 123.5, 121.8,
61.7, 52.6 ppm; IR (CH2Cl2): m~/cm-1 2073, 2030, 1995
(CO); HR-MS (EI): m/z calc. for [M?]: 609.8713; found:
609.8708.
program (Sheldrick 1996). The structures were solved by
direct methods and refined by full-matrix least-squares
techniques on F2O using the SHELXTL 97 crystallographic
software package (Sheldrick 1997). All non-hydrogen
atoms were refined anisotropically. All hydrogen atoms
were located using the geometric method, and their posi-
tions and thermal parameters were fixed during the struc-
ture refinement. Details of crystal data, data collections and
structure refinements were summarized in Table 1.
Electrochemistry
Electrochemical measurements were made with a BAS
100B electrochemical workstation at a scan rate of
100 mV s-1. All voltammograms were obtained in a con-
ventional three-electrode cell under argon and at ambient
temperature. The working electrode was a glassy carbon
disc (diameter 3 mm) that was successively polished with
3- and 1 lm diamond pastes and sonicated for 15 min prior
to use. The reference electrode was a non-aqueous Ag/Ag?
electrode (0.01 mol L-1 AgNO3 and 0.1 mol L-1 n-Bu4-
NPF6 in CH3CN) and the counter electrode was a platinum
wire. The potentials were reported versus ferrocene
(Fc)/ferrocenium (Fc?) couple.
Results and discussion
In the present report, commercially available materials
were employed to generate the target product. Bromination
of 2-p-tolylbenzothiazole with N-Bromosuccinimide
(NBS) gave the bromomethyl compound 1a for subsequent
reactions. Then the aminomethyl compound 1b was pre-
pared via a short and high-yielding procedure (Gunn-
laugsson et al. 2004) instead of the classical Gabriel amine
synthesis (Fyles and Suresh 1994). The transformation of
1a with the inexpensive hexamethylenetetramine in dry
chloroform was easy to yield an insoluble amine com-
pound, which could be dissolved in a mixture of ethanol
Crystal structure determination
The X-ray diffractions of all single crystals were made on a
SMART APEX II diffractometer. Data were collected at
273 K using graphite monochromatic Mo-Ka radiation
˚
(k = 0.71073 A) in the x-2h scan mode. Data processing
was accomplished with the SAINT processing program
(Siemens Energy and Automation Inc., 1996). Intensity
data were corrected for absorption by the SADABS
123

Chem. Pap.
Table 1 Crystallographic data
and processing parameters for
complexes 3 and 4
Complex
3
4
Formula
C21H12Fe2N2O6S3
596.21
C22H14Fe2N2O6S3
610.23
Mw
Crystal system
Space group
Monoclinic
P2(1)/c
Orthorhombic
P2(1)2(1)2(1)
6.651(3)
14.208(7)
25.854(12)
2443.2(19)
4
˚
a (A)
13.8606(12)
7.6900(7)
21.5052(19)
2290.1(4)
4
˚
b (A)
˚
c (A)
3
˚
V (A )
Z
D
calcd./g cm-3
1.729
1.659
l (mm-1
)
1.582
1.485
F(000)
1200
1232
Crystal size (mm)
Reflns collected
Independent reflections
Rint
0.45 9 0.22 9 0.10
12,350
0.95 9 0.08 9 0.07
11,654
4509
3991
0.0204
0.0301
Parameters refined
GOF on F2
307
316
1.035
0.982
R1 [I [ 2r(I)]
wR2 [I [ 2r(I)]
Residual electron density (e A
0.0231
0.0270
0.0620
0.0495
-3
)
˚
0.281/-0.232
0.223/-0.162
and concentrated HCl. After the filtration of the HCl salt,
compound 1b was isolated as a product in ca. 85% yield.
The resulting primary amine could react readily with
paraformaldehyde and SOCl2 to generate bis(chlor-
omethyl)amine 1c (Lawrence et al. 2001a, b). The treat-
ment of 1c with [(l-S)2{Fe(CO)3}2]2- dianion, freshly
derived from [(l-S)2{Fe(CO)3}2] in THF, gave the iron
thiolate carbonyl dimer 4 in a reasonable yield (Scheme 2).
summarized in Table 2. Both complexes consisted of a
butterfly architectonic [2Fe] core with distorted square-
pyramidal geometry around each iron atom (Tard and
Pickett 2009; Simmons et al. 2014). The Fe–Fe bond
˚
lengths [3: 2.5036(4) A, 4: 2.5013(12) A] were in good
˚
agreement with those of previously reported diiron aza-
˚
dithiolates (2.48–2.52 A) (Georgakaki et al. 2003; Liu et al.
2005; Tard et al. 2005; Gloaguen and Rauchfuss 2009),
whereas slightly shorter than those in the structures of
enzymes Clostridium pasterianum and Desulfovibrio
1
Complex 4 was fully characterized by H NMR, 13C
NMR, IR and HR-MS spectroscopies. The molecular
structure was further confirmed by X-ray crystallography.
ORTEP plots of 4 and complex 3 were shown in Fig. 1,
while the selected bond lengths and angles were
˚
desulfuricans (ca. 2.6 A) (Peters et al. 1998; Nicolet et al.
1999, 2001). Indeed, the models 3 and 4 involved two
fused six-membered rings in which the N-substituted aza-
dithiolate ligands were g2:g2-coordinated to the Fe(CO)3
moieties. One ring (Fe1–S1–C7–N1–C8–S2) adopted a
chair conformation, while the other ring (Fe2–S1–C7–N1–
C8–S2) had a boat conformation. Although the structures
of the Fe2S2(CO)6 subunits of 3 and 4 appeared quite
similar, the tertiary amine moieties featured apparently
distinct conformations. The substituted phenyl group of 3
lay in the axial position relative to above-mentioned met-
alloheterocycle, while the benzyl moiety resided in an
equatorial position in 4. In the light of the C–N–C angles,
the sum of ca. 357° for 3 and 332° for 4 suggested that the
bridgehead azadithiolate nitrogen atoms were sp2 and sp3-
hybridized, respectively. It was noteworthy that N1 atom in
3 possessed a pseudo-triangular conformation, leading to
slightly weakened p-p conjugation between the p-orbital of
Fig. 1 Molecular structures of complexes 3 (a) and 4 (b) with 30%
probability level ellipsoids (the hydrogen atoms have been omitted for
clarity)
123

Chem. Pap.
˚
Table 2 Selected bond lengths (A) and angles (deg) for complexes 3
and 4
Complex
3
4
Bond lengths
Fe(1)–Fe(2)
2.5036(4)
2.2663(5)
2.2647(5)
2.2717(5)
2.2594(5)
1.423(2)
2.5013(12)
2.2485(11)
2.2487(11)
2.2466(11)
2.2534(11)
1.448(4)
Fe(1)–S(1)
Fe(1)–S(2)
Fe(2)–S(1)
Fe(2)–S(2)
N(1)–C(7)
N(1)–C(8)
1.432(2)
1.445(3)
N(1)–C(9)
1.404(2)
1.472(3)
N(1)ÁÁÁC(6)
S(1)ÁÁÁS(2)
3.4891(23)
3.0609(6)
3.1781(41)
3.0226(17)
Bond angles
Fe(1)–S(1)–Fe(2)
Fe(1)–S(2)–Fe(2)
S(1)–Fe(1)–Fe(2)
S(1)–Fe(2)–Fe(1)
S(2)–Fe(1)–Fe(2)
S(2)–Fe(2)–Fe(1)
C(1)–Fe(1)–Fe(2)
C(6)–Fe(2)–Fe(1)
C(7)–N(1)–C(8)
C(7)–N(1)–C(9)
C(8)–N(1)–C(9)
Dihedrala
66.969(15)
67.201(15)
56.619(14)
56.412(13)
56.300(13)
56.499(14)
67.62(3)
67.50(4)
56.15(3)
56.23(3)
56.34(3)
56.16(3)
147.69(6)
154.19(6)
113.03(15)
121.62(15)
122.20(15)
71.7
147.16(11)
148.57(11)
111.8(2)
109.4(2)
110.7(2)
78.5
Fig. 2 FT-IR spectra of (a) 4 in CH3CN, (b) 4 ? 5 equiv. of HOTf in
CH3CN, (c) 4(NH) ? 0.5 equiv. of TEOA in CH3CN, (d) 4(NH) ? 1
equiv. of TEOA in CH3CN
processes of 4 and 4(NH) were reversible. With the addi-
tion of 0.5 equiv. of TEOA to the CH3CN solution of
a
Defined by the intersection of the two SFe2 planes
~
~
4(NH), the m(CO) bands of 4 recovered while the m(CO)
bands of 4(NH) still existed (Fig. 2c), indicative of the
equilibrium between 4 and 4(NH). Further addition of
TEOA up to 1 equiv. resulted in quantitative recovery of 4.
nitrogen atom and the phenyl ring. On the other hand, the
sp3-hybridized N1 atom in 4 holds a distorted tetrahedral
conformation with the nitrogen lone-pair pointing towards
Fe2 nucleus. The nonbonding CÁÁÁN distance between the
azadithiolate nitrogen atom and the nearest carbonyl car-
˚
bon atom was significantly shortened in 4 (ca. 3.18 A) than
˚
in 3 (ca. 3.49 A).
~
The m(CO) absorptions shifted to the frequencies essen-
tially equal to those of 4 (Fig. 2d). Meanwhile, addition of
strong acid to solutions of the related phenyl-substituted
analogue 3 did not result in the changed IR spectra, sug-
gesting that the bridgehead nitrogen atom of 3 could not be
protonated under present conditions.
The UV/Vis spectrum of complex 4 was dominated by
an intense absorption band in the UV region around
315 nm, which could be attributed to the p–p* excitation
within the phenylbenzothiazole chromophore (Fig. 3a). A
low-energy shoulder was visible in the near UV region and
featured r–r* and d–r* character involved with the [2Fe]
unit (Goy et al. 2013). The metal-based band determined
the photophysical and photochemical properties of 4.
Likewise, a broad and featureless absorption reaching up to
550 nm was similar to the characteristic bands of previ-
ously reported [FeFe]-hydrogenase mimics (Eilers et al.
2007) and was responsible for the dark red colour of 4
solution. Excitation of the p–p* absorption of 4 resulted in
a fluorescence emission band around 370 nm, with a much
Protonation of 4 occurred in the CH3CN solution upon
addition of a strong organic acid such as triflic acid (HOTf,
pKa * 2.6 in CH3CN) (Eilers et al. 2007). Complex 4 in
~
CH3CN solution exhibited three characteristic m(CO) bands
at 2073, 2030 and 1995 cm-1 in IR spectrum (Fig. 2a).
Addition of an excess of HOTf to the CH3CN solution of 4
~
resulted in a shift of m(CO) bands to higher frequencies
with an average value of ca. 18 cm-1 (Fig. 2b), which
represented a protonation of the azadithiolate nitrogen
(Lawrence et al. 2001a, b; Schwartz et al. 2006) and a
generation of N-protonated product 4(NH). Subsequent
titration of an organic base such as triethanolamine
(TEOA) revealed that the protonation and deprotonation
123

Chem. Pap.
event might involve the transfer of two electrons occurred
at closely separated potentials (Borg et al. 2004; Capon
et al. 2007, 2009; Felton et al. 2007, 2009a) or the two-
electron processes coupled with chemical reaction (Zeng
et al. 2010; Xiao et al. 2011; Zhao et al. 2012; Qian et al.
2015). In either case, the redox process of [FeIFeI] to
[FeIFe0] undoubtedly occurred around -1.53 V for 3 and
-1.60 V for 4. Likewise, electrochemically irreversible
oxidation events had been established at 0.53 V for 3 and
0.41 V for 4. The ratios of the oxidation peak current of 3
and 4 over the primary reduction peak current were both
ca. 1.5. Hereby, we assumed the oxidations of 3 and 4 to be
combined results of two overlapping processes of [FeIFeI]
to [FeIFeII] and the oxidation of phenylbenzothiazole
moiety. Moreover, the redox potential could be usually
considered as an indicator to detect the electron density
around the iron nuclei in [FeFe]-hydrogenase mimics. The
more negative reduction potentials and less positive oxi-
dation potential of 4 relative to those of 3 indicated that 4
was easier to be oxidized and carried a decreased reduction
capability. The azadithiolate nitrogen atom of 3 was part of
an aniline. The N lone pair could overlap with the two anti-
bonding r*C–S orbitals to form an orbital mixing that
represented the hyperconjugation. The hyperconjugation
effect led to a relatively strong communication between the
azadithiolate nitrogen atom and the [2Fe] site, which was
responsible for the more positive reduction potential of 3.
The electrocatalytic proton reduction of complexes 3
and 4 were studied by CV in the presence of p-toluene-
sulfonic acid (HOTs, pKa *8.0 in CH3CN) (Fujinaga and
Sakamoto 1977) with the concentration of 2–8 mM. Upon
addition of 2 equiv. of HOTs to the CH3CN solution of 3, a
new reduction event was observed at ca. -1.45 V while the
peak of the initial [FeIFeI]/[FeIFe0] reduction process at ca.
-1.53 V still existed. The current intensity of the former
reduction exhibited a well linear increase with sequential
increments of the acid concentration, while the potential
slightly shifted towards a more negative value (Fig. 5).
These electrochemical behaviours featured a catalytic
proton reduction process (Bhugun et al. 1996; Gloaguen
et al. 2001) and demonstrated the electrocatalytic activity
of 3. Moreover, the CVs did not display any largely anodic
shifted peak in the whole process, implying that the pro-
tonation of azadithiolate nitrogen atom of 3 had not taken
place in the catalytic proton reduction (vide infra).
Fig. 3 UV-Vis absorption (left) and emission (right) spectra of
complexes 3, 4, and compound 2-p-tolylbenzothiazole, recorded in
CH3CN
stronger emission intensity relative to that of 3 (Fig. 3b).
We had demonstrated that the luminescence quenching of
complex 3 was caused by the intramolecular oxidative
process with electron transfer from the excited chro-
mophore to [2Fe] unit in 3 (Gao et al. 2014). Such an
intramolecular electron transfer was recognized as one of
the important steps required for light-driven reduction of
protons to hydrogen performed by [FeFe]-hydrogenase
mimic. The differences in emissions between 3 and 4
suggested that the desirable electron transfer might be
prevented in latter molecule. It could be concluded that the
short rigid architecture of 3 was more favourable for the
light-driven intramolecular electron transfer.
The electrochemical behaviours of 3 and 4 were inves-
tigated by cyclic voltammetry (CV) in CH3CN with n-
Bu4NPF6 (0.05 mol L-1) as supporting electrolyte under
an argon atmosphere (Fig. 4). All potentials were in volts
versus Fc/Fc?. In dry solution, 3 and 4 exhibited similar
reduction processes, with one quasi-reversible (-1.53 V
for 3, -1.60 V for 4) and an irreversible reduction peak
(-1.96 V for 3, -2.03 V for 4). The primary reduction
Based on aforementioned electrochemical observations
and other similar cases (Capon et al. 2005, 2009; Felton
et al. 2009b), an ECCE (electrochemical–chemical–chem-
ical–electrochemical) process could be proposed to account
for the proton reduction catalysed by 3 in the presence of
HOTs. Initially, the azadithiolate [FeIFeI] complex 3 was
reduced at ca. -1.53 V to give the [Fe0FeI]- monoanion
(1st E step). In the presence of proton acid, the [Fe0FeI]-
Fig. 4 CVs of complexes 3 and 4 in CH3CN solution (0.05 mol L-1
n-Bu4NPF6). The scan rate is 100 mV s-1
123

Chem. Pap.
et al. 2007; Capon et al. 2008) and implied that the pro-
tonation occurred at the azadithiolate nitrogen atom rather
than at the Fe–Fe bond, which generally resulted in larger
potential shifts (Zhao et al. 2002; Gloaguen et al. 2002;
Eilers et al. 2007). The current of the peak at -1.2 V
increased linearly with the amount of acid added (2–8
equiv.), indicative of the catalytic reduction of protons at
this potential. Likewise, an acid-dependent reduction
around -1.54 V had also been evidenced. The slopes of
the plots of current versus acid concentration showed that
the process observed around -1.54 V exhibited a slightly
greater catalytic activity.
Obviously, the catalytic proton reduction of 4 followed a
distinguishing mechanism relative to that of 3. The pres-
ence of the moderately strong acid (HOTs) resulted in the
protonation of azadithiolate nitrogen atom of 4 (1st C step).
The generated [4(NH)]? was electrochemically reduced to
4(NH) at ca. -1.2 V (1st E step). The metal-centred
reduction likely increased the electron density of the diiron
site significantly so that HOTs was able to protonate 4(NH)
at the electron-rich Fe–Fe bond, producing a cationic
complex with a bridging hydride ligand (2nd C step). The
l-hydride diiron complex underwent a second reduction at
a potential close to that of [4(NH)]? (2nd E step), and then
the catalyst was liberated and made available for another
cycle. The catalytic cycle was proposed to be a CECE
process for the reduction behaviours of 4 at ca. -1.2 V.
However, the mechanism of the proton reduction path
observed around -1.54 V was somewhat complicated, and
the related investigations are now underway.
Fig. 5 Left CVs of complex 3 in CH3CN/n-Bu4NPF6 solution in the
absence and presence of 2, 4, 6, 8 equiv. of HOTs. The scan rate is
100 mV s-1. Right Dependence of current intensity of the electro-
catalytic reductions on the concentration of HOTs
species accepted a proton (1st C step) and underwent a
subsequent protonation to form the g2-H2 species (2nd C
step), which could be reduced at the potential (-1.45 V)
slightly more positive than -1.53 V (2nd E step) to release
molecular hydrogen and accomplish the electrocatalytic
proton reduction cycle.
In contrast to the electrochemical behaviours of complex
3, addition of HOTs to the solution of 4 in CH3CN resulted
in new reduction peak at ca. -1.2 V being observed
(Fig. 6). The peak was shifted by around 0.4 V towards
more positive potential relative to the primary reduction of
4 and could be ascribed to a one-electron reduction process
of the introduced [4(NH)]? cation. The difference between
the reduction potentials of [4(NH)]? and 4 was consistent
with those found for previously reported azadithiolate
diiron analogues (Ott et al. 2004; Wang et al. 2007; Jiang
Conclusions
In summary, a functional benzyl-containing diiron aza-
dithiolate complex (coded as 4) was synthesized as bio-
mimetic model of the active site of [FeFe]-hydrogenase.
The structure of synthetic mimic was fully characterized by
NMR, IR, HRMS spectra and X-ray single crystal
diffraction analysis. Complex 4 exhibited distinct pro-
tophilic property relative to the phenyl substituted analogue
(coded as 3), due to the existence of the methylene group
near the azadithiolate nitrogen atom. IR spectra evidenced
that the sp3-hybridized nitrogen atom of 4 underwent a
protonation in the presence of organic acid, which did not
occur for the case of 3 under same conditions. The redox
behaviours of 3 and 4 in the absence of proton acid were
quite similar. The most remarkable feature of 4 as a cata-
lyst for electrochemical proton reduction was the relatively
mild potential (ca. -1.2 V versus Fc/Fc?). The potential
shifted towards more positive value by ca. 250 mV related
to that of 3, which could be rationalized by considering the
altered catalytic mechanism. The catalytic cycle proposed
Fig. 6 Left CVs of complex 4 in CH3CN/n-Bu4NPF6 solution in the
absence and presence of 2, 4, 6, 8 equiv. of HOTs. The scan rate is
100 mV s-1. Right Dependence of current intensity of the electro-
catalytic reductions on the concentration of HOTs
123

Chem. Pap.
of proton reduction by [Fe2(CO)6{l-SCH2N(R)CH2S}] com-
plexes related to the [2Fe]H subsite of [FeFe]hydrogenase. Chem
Eur J 14(6):1954–1964. doi:10.1002/chem.200701454
for 4 commenced with the protonation of the azadithiolate
nitrogen atom, operated at moderately negative potential
and resembled the natural enzymes more closely.
Capon JF, Gloaguen F, Petillon FY, Schollhammer P, Talarmin J
(2009) Electron and proton transfers at diiron dithiolate sites
relevant to the catalysis of proton reduction by the [FeFe]-
hydrogenases. Coord Chem Rev 253(9–10):1476–1494. doi:10.
1016/j.ccr.2008.10.020
Acknowledgements We are grateful to the National Natural Science
Foundation of China (Nos. 21201022, 61106050), the Specialized
Research Fund for the Doctoral Program of Higher Education (New
Teachers, No. 20122216120001), and the Scientific and Technologi-
cal Development Project of Jilin Province (No. 20150311086YY) for
financial support.
Eckenhoff WT, Eisenberg R (2012) Molecular systems for light
Trans
driven
2012(41):13004–13021. doi:10.1039/c2dt30823a
hydrogen
production.
Dalton
Eilers G, Schwartz L, Stein M, Zampella G, De Gioia L, Ott S,
Lomoth R (2007) Ligand versus metal protonation of an iron
hydrogenase active site mimic. Chem Eur J 13(25):7075–7084.
doi:10.1002/chem.200700019
References
¨
Erdem OF, Schwartz L, Stein M, Silakov A, Kaur-Ghumaan S, Huang
Adams MWW (1990) The structure and mechanism of iron-hydro-
genases. Biochim Biophys Acta Bioenerg 1020(2):115–145.
doi:10.1016/0005-2728(90)90044-5
Adamska A, Silakov A, Lambertz C, Ru¨diger O, Happe T, Reijerse E,
Lubitz W (2012) Identification and characterization of the
‘‘Super-Reduced’’ state of the H-cluster in [FeFe] hydrogenase: a
new building block for the catalytic cycle? Angew Chem Int Ed
51(46):11458–11462. doi:10.1002/anie.201204800
Adamska-Venkatesh A, Krawietz D, Siebel J, Weber K, Happe T,
Reijerse E, Lubitz W (2014) New redox states observed in
[FeFe] hydrogenases reveal redox coupling within the H-Cluster.
J Am Chem Soc 136(32):11339–11346. doi:10.1021/ja503390c
Berggren G, Adamska A, Lambertz C, Simmons TR, Esselborn J,
Atta M, Gambarelli S, Mouesca JM, Reijerse E, Lubitz W,
Happe T, Artero V, Fontecave M (2013) Biomimetic assembly
and activation of [FeFe]-hydrogenases. Nature 499:66–69.
doi:10.1038/nature12239
P, Ott S, Reijerse EJ, Lubitz W (2011) A model of the [FeFe]
hydrogenase active site with a biologically relevant azadithiolate
bridge: a spectroscopic and theoretical investigation. Angew
Chem Int Ed 50(6):1439–1443. doi:10.1002/anie.201006244
Esselborn J, Lambertz C, Adamska-Venkatesh A, Simmons T,
Berggren G, Noth J, Siebel J, Hemschemeier A, Artero V,
Reijerse E, Fontecave M, Lubitz W, Happe T (2013) Sponta-
neous activation of [FeFe]-hydrogenases by an inorganic [2Fe]
active site mimic. Nat Chem Biol 9:607–609. doi:10.1038/
nchembio.1311
Felton GAN, Vannucci AK, Chen JZ, Lockett LT, Okumura N, Petro
BJ, Zakai UI, Evans DH, Glass RS, Lichtenberger DL (2007)
Hydrogen generation from weak acids: electrochemical and
computational studies of a diiron hydrogenase mimic. J Am
Chem Soc 129(41):12521–12530. doi:10.1021/ja073886g
Felton GAN, Petro BJ, Glass RS, Lichtenberger DL, Evans DH
(2009a) One- to two-electron reduction of an [FeFe]-hydroge-
nase active site mimic: the critical role of fluxionality of the
[2Fe2S] core. J Am Chem Soc 131(32):11290–11291. doi:10.
1021/ja904520x
´
Bhugun I, Lexa D, Saveant JM (1996) Homogeneous catalysis of
electrochemical hydrogen evolution by iron(0) porphyrins. J Am
Chem Soc 118(16):3982–3983. doi:10.1021/ja954326x
´
Birrell JA, Wrede K, Pawlak k, Rodriguez-Macia P, Ru¨diger O,
Felton GAN, Mebi CA, Petro BJ, Vannucci AK, Evans DH, Glass RS,
Lichtenberger DL (2009b) Review of electrochemical studies of
complexes containing the Fe2S2 core characteristic of [FeFe]-
hydrogenases including catalysis by these complexes of the
reduction of acids to form dihydrogen. J Organomet Chem
694(17):2681–2699. doi:10.1016/j.jorganchem.2009.03.017
Fujinaga T, Sakamoto I (1977) Electrochemical studies of sulfonates
in non-aqueous solvents: part III. Trifluoromethanesulfonic acid
as a strong acid in dipolar aprotic solvents and acetic acid.
Reijerse EJ, Lubitz W (2016) Artificial maturation of the highly
active heterodimeric [FeFe] hydrogenase from Desulfovibrio
desulfuricans ATCC 7757. Isr J Chem 56(9–10):852–863.
doi:10.1002/ijch.201600035
Bogan LE, Lesch DA, Rauchfuss TB (1983) Synthesis of
heterometallic cluster compounds from Fe3(l3-Te)2(CO)9 and
comparisons with analogous sulfide clusters. J Organomet Chem
250(1):429–438. doi:10.1016/0022-328X(83)85067-0
Borg SJ, Behrsing T, Best SP, Razavet M, Liu XM, Pickett CJ (2004)
Electron transfer at a dithiolate-bridged diiron assembly: elec-
J Electroanal Chem 85(1):185–201. doi:10.1016/S0022-
0728(77)80163-0
trocatalytic hydrogen evolution.
126(51):16988–16999. doi:10.1021/ja045281f
J
Am Chem Soc
Fyles TM, Suresh VV (1994) Photoionophores derived from crown
ether polycarboxylic acids: synthesis, ion binding, and spectro-
scopic characterization. Can J Chem 72(5):1254–1261. doi:10.
1139/v94-159
Gao S, Huang S, Duan Q, Hou JH, Jiang DY, Liang QC, Zhao JX
(2014) Iron–iron hydrogenase active subunit covalently linking
to organic chromophore for light-driven hydrogen evolution. Int
J Hydrog Energy 39(20):10434–10444. doi:10.1016/j.ijhydene.
2014.05.003
Gao S, Zhang WY, Duan Q, Liang QC, Jiang DY, Zhao JX, Hou JH
(2017) An artificial [FeFe]-hydrogenase mimic with organic
chromophore-linked thiolate bridges for the photochemical
production of hydrogen. Chem Pap 71(3):617–625. doi:10.
1007/s11696-016-0049-8
Georgakaki IP, Thomson LM, Lyon EJ, Hall MB, Darensbourg MY
(2003) Fundamental properties of small molecule models of Fe-
only hydrogenase: computations relative to the definition of an
entatic state in the active site. Coord Chem Rev
238–239:255–266. doi:10.1016/S0010-8545(02)00326-0
Butt JN, Filipiak M, Hagen WR (1997) Direct electrochemistry of
Megasphaera elsdenii iron hydrogenase: definition of the enzyme’s
catalytic operating potential and quantitation of the catalytic
behaviour over a continuous potential range. Eur J Biochem
245(1):116–122. doi:10.1111/j.1432-1033.1997.00116.x
Capon JF, Gloaguen F, Schollhammer P, Talarmin J (2005) Catalysis
of the electrochemical H2 evolution by di-iron sub-site models.
Coord Chem Rev 249(15–16):1664–1676. doi:10.1016/j.ccr.
2004.11.018
´
Capon JF, Ezzaher S, Gloaguen F, Petillon FY, Schollhammer P,
Talarmin J, Davin TJ, McGrady JE, Muir KW (2007) Electro-
chemical and theoretical investigations of the reduction of
[Fe2(CO)5L{l-SCH2XCH2S}] complexes related to [FeFe]
hydrogenase. New J Chem 31(12):2052–2064. doi:10.1039/
B709273C
´
Capon JF, Ezzaher S, Gloaguen F, Petillon FY, Schollhammer P,
Talarmin J (2008) Electrochemical insights into the mechanisms
123

Chem. Pap.
Gloaguen F, Rauchfuss TB (2009) Small molecule mimics of
hydrogenases: hydrides and redox. Chem Soc Rev
38(1):100–108. doi:10.1039/B801796B
Palmer PJ, Hall G, Trigg RB, Warrington JV (1971) Antimicrobials.
1. Benzothiazolylbenzylamines. J Med Chem 14(12):1223–1225.
doi:10.1021/jm00294a024
Gloaguen F, Lawrence JD, Rauchfuss TB (2001) Biomimetic
hydrogen evolution catalyzed by an iron carbonyl thiolate.
J Am Chem Soc 123(38):9476–9477. doi:10.1021/ja016516f
Pereira AS, Tavares P, Moura I, Moura JJG, Huynh BH (2001)
¨
Mossbauer characterization of the iron–sulfur clusters in Desul-
fovibrio vulgaris hydrogenase. Am Chem Soc
123(12):2771–2782. doi:10.1021/ja003176?
J
´
Gloaguen F, Lawrence JD, Rauchfuss TB, Benard M, Rohmer MM
(2002) Bimetallic carbonyl thiolates as functional models for Fe-
only hydrogenases. Inorg Chem 41(4):6573–6582. doi:10.1021/
ic025838x
Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC (1998) X-ray
crystal structure of the Fe-only hydrogenase (CpI) from
Clostridium pasteurianum to 1.8 Angstrom resolution. Science
282(5395):1853–1858. doi:10.1126/science.282.5395.1853
Qian GF, Wang HL, Zhong W, Liu XM (2015) Electrochemical
investigation into the electron transfer mechanism of a diiron
hexacarbonyl complex bearing a bridging naphthalene moiety.
Electrochim Acta 163:190–195. doi:10.1016/j.electacta.2015.02.163
Schwartz L, Eilers G, Eriksson L, Gogoll A, Lomoth R, Ott S (2006)
Iron hydrogenase active site mimic holding a proton and a
hydride. Chem Commun 2006(5):520–522. doi:10.1039/
B514280F
¨
Goy R, Apfel U, Elleouet C, Escudero D, Elstner M, Gorls H,
Talarmin J, Schollhammer P, Gonzalez L, Weigand W (2013) A
´
silicon-heteroaromatic system as photosensitizer for light-driven
hydrogen production by hydrogenase mimics. Eur J Inorg Chem
2013(25):4466–4472. doi:10.1002/ejic.201300537
Gunnlaugsson T, Davis AP, Hussey GM, Tierney J, Glynn M (2004)
Design, synthesis and photophysical studies of simple fluores-
cent anion PET sensors using charge neutral thiourea receptors.
Org Biomol Chem 2(13):1856–1863. doi:10.1039/B404706K
Holm RH, Kennepohl PJ, Solomon EI (1996) Structural and
functional sspects of metal sites in biology. Chem Rev
96(7):2239–2314. doi:10.1021/cr9500390
Sheldrick GM (1996) SADABS absorption correction program.
¨
¨
University of Goffingen, Goffingen
Sheldrick GM (1997) SHELXTL 97 program for the refinement of
˚
Jiang S, Liu J, Shi Y, Wang Z, Akermark B, Sun LC (2007) Fe–S
¨
¨
crystal structure. University of Goffingen, Goffingen
Siemens Energy & Automation Inc, Inc Automation (1996) Software
packages SMART and SAINT. Siemens Energy & Automation
Inc, Inc Automation, Madison
complexes containing five-membered heterocycles: novel mod-
els for the active site of hydrogenases with unusual low
reduction potential. Dalton Trans 2007(8):896–902. doi:10.
1039/B615037C
Silakov A, Wenk B, Reijerse E, Lubitz W (2009) 14N HYSCORE
investigation of the H-cluster of [FeFe] hydrogenase: evidence
for a nitrogen in the dithiol bridge. Phys Chem Chem Phys
11(31):6592–6599. doi:10.1039/b905841a
Simmons TR, Berggren G, Bacchi M, Fontecave M, Artero V (2014)
Mimicking hydrogenases: from biomimetics to artificial
enzymes. Coord Chem Rev 270–271:127–150. doi:10.1016/j.
ccr.2013.12.018
Lawrence JD, Li H, Rauchfuss TB (2001a) Beyond Fe-only
hydrogenases: N-functionalized 2-aza-1,3-dithiolates Fe2[(-
SCH2)2NR](CO)x
2006(16):1482–1483. doi:10.1039/B104195A
(x = 5,
6).
Chem
Commun
´
Lawrence JD, Li H, Rauchfuss TB, Benard M, Rohmer MM (2001b)
Diiron azadithiolates as models for the iron-only hydrogenase
active site: synthesis, structure, and stereoelectronics. Angew
Chem
Int
Ed
40(9):1768–1771.
doi:10.1002/1521-
Sommer C, Adamska-Venkatesh A, Pawlak K, Birrell JA, Ru¨diger O,
Reijerse EJ, Lubitz W (2017) Proton coupled electronic
3773(20010504)40:9\1768:AID-ANIE17680[3.0.CO;2-E
Liu XM, Ibrahim SK, Tard C, Pickett CJ (2005) Iron-only hydroge-
nase: synthetic, structural and reactivity studies of model
compounds. Coord Chem Rev 249(15–16):1641–1652. doi:10.
1016/j.ccr.2005.04.009
Lubitz W, Reijerse E, van Gastel M (2007) [NiFe] and [FeFe]
hydrogenases studied by advanced magnetic resonance tech-
niques. Chem Rev 107(10):4331–4365. doi:10.1021/cr050186q
Lubitz W, Ogata H, Ru¨diger O, Reijerse E (2014) Hydrogenases.
Chem Rev 114(8):4081–4148. doi:10.1021/cr4005814
Mulder DW, Ratzloff MW, Bruschi M, Greco C, Koonce E, Peters
JW, King PW (2014) Investigations on the role of proton-
coupled electron transfer in hydrogen activation by [FeFe]-
hydrogenase. J Am Chem Soc 136(43):15394–15402. doi:10.
1021/ja508629m
rearrangement within the H-cluster as an essential step in the
catalytic cycle of [FeFe] hydrogenases. J Am Chem Soc
139(4):1440–1443. doi:10.1021/jacs.6b12636
Tard C, Pickett CJ (2009) Structural and functional analogues of the
active sites of the [Fe]-, [NiFe]-, and [FeFe]-hydrogenases.
Chem Rev 109(6):2245–2274. doi:10.1021/cr800542q
Tard C, Liu XM, Ibrahim SK, Bruschi M, De Gioia L, Davies SC,
Yang X, Wang LS, Sawers G, Pickett CJ (2005) Synthesis of the
H-cluster framework of iron-only hydrogenase. Nature
433:610–613. doi:10.1038/nature03298
Wang F, Wang M, Liu X, Jin K, Dong W, Sun LC (2007) Protonation,
electrochemical properties and molecular structures of halogen-
functionalized diiron azadithiolate complexes related to the
active site of iron-only hydrogenases. Dalton Trans
2007(34):3812–3819. doi:10.1039/B706178A
Wang F, Wang WG, Wang HY, Si G, Tung CH, Wu LZ (2012)
Artificial photosynthetic systems based on [FeFe]-hydrogenase
mimics: the road to high efficiency for light-driven hydrogen
evolution. ACS Catal 2(3):407–416. doi:10.1021/cs200458b
Wang M, Han K, Zhang S, Sun LC (2015) Integration of
organometallic complexes with semiconductors and other nano-
materials for photocatalytic H2 production. Coord Chem Rev
287:1–14. doi:10.1016/j.ccr.2014.12.005
Nicolet Y, Piras C, Legrand P, Hatchikian EC, Fontecilla-Camps JC
(1999) Desulfovibrio desulfuricans iron hydrogenase: the
structure shows unusual coordination to an active site Fe
binuclear center. Structure 7(1):13–23. doi:10.1016/S0969-
2126(99)80005-7
`
Nicolet Y, Lacey AL, Vernede X, Fernandez VM, Hatchikian EC,
Fontecilla-Camps JC (2001) Crystallographic and FTIR spec-
troscopic evidence of changes in Fe coordination upon reduction
of the active site of the Fe-only hydrogenase from Desulfovibrio
desulfuricans. J Am Chem Soc 123(8):1596–1601. doi:10.1021/
ja0020963
Wu LZ, Chen B, Li ZJ, Tung CH (2014) Enhancement of the
efficiency of photocatalytic reduction of protons to hydrogen via
molecular assembly. Acc Chem Res 47(7):2177–2185. doi:10.
1021/ar500140r
Xiao ZY, Wei ZH, Long L, Wang YL, Evans DJ, Liu XM (2011)
Diiron carbonyl complexes possessing a Fe(II)Fe(II) core:
˚
Ott S, Kritikos M, Akermark B, Sun LC, Lomoth R (2004) A
biomimetic pathway for hydrogen evolution from a model of the
iron hydrogenase active site. Angew Chem Int Ed
43(8):1006–1009. doi:10.1002/anie.200353190
123

Chem. Pap.
synthesis, characterisation, and electrochemical investigation.
Dalton Trans 40(16):4291–4299. doi:10.1039/C0DT01465F
Yoshino K, Kohno T, Uno T, Morita T, Tsukamoto G (1986) Organic
phosphorus compounds. 1. 4-(Benzothiazol-2-yl)benzylphospho-
nate as potent calcium antagonistic vasodilator. J Med Chem
29(5):820–825. doi:10.1021/jm00155a037
Zeng XH, Li ZM, Xiao ZY, Wang YW, Liu XM (2010) Using
pendant ferrocenyl group(s) as an intramolecular standard to
probe the reduction of diiron hexacarbonyl model complexes for
the sub-unit of [FeFe]-hydrogenase. Electrochem Commun
12(3):342–345. doi:10.1016/j.elecom.2009.12.023
Zhao X, Hsiao YM, Reibenspies JH, Darensbourg MY (2002)
Oxidative addition of phosphine-tethered thiols to iron carbonyl:
binuclear
SCH2CH2PPh2)2Fe2(CO)4, and hydride derivatives. Inorg
phosphinothiolate
complexes,
(l-
Chem 41(4):699–708. doi:10.1021/ic010741g
Zhao J, Wei ZH, Zeng XH, Liu XM (2012) Three diiron complexes
bearing an aromatic ring as mimics of the diiron subunit of
[FeFe]-hydrogenase: synthesis, electron transfer and coupled
chemical reactions. Dalton Trans 41(36):11125–11133. doi:10.
1039/C2DT31083J
123