Incorporation of biotinylated manganese-salen complexes into streptavidin: New artificial metalloenzymes for enantioselective sulfoxidation

Article abstract of DOI:10.1016/j.jorganchem.2008.11.023

Incorporation of achiral biotinylated manganese-salen complexes into streptavidin yields artificial metalloenzymes for aqueous sulfoxidation using hydrogen peroxide. Four biotinylated salen ligands were synthesized and their manganese complexes were teste

Full text of DOI:10.1016/j.jorganchem.2008.11.023

Journal of Organometallic Chemistry 694 (2009) 930–936
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Journal of Organometallic Chemistry
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Incorporation of biotinylated manganese-salen complexes into streptavidin:
New artificial metalloenzymes for enantioselective sulfoxidation
A. Pordea a, D. Mathis a, T.R. Ward a,b,
*
a Institute of Chemistry, University of Neuchâtel, Avenue Bellevaux 51, CP 158, CH-2009 Neuchâtel, Switzerland
b Department of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Incorporation of achiral biotinylated manganese-salen complexes into streptavidin yields artificial metal-
loenzymes for aqueous sulfoxidation using hydrogen peroxide. Four biotinylated salen ligands were syn-
thesized and their manganese complexes were tested in combination with several streptavidin mutants,
yielding moderate conversions (up to 56%) and low enantioselectivities (maximum of 13% ee) for the sul-
foxidation of thioanisole.
Received 17 September 2008
Received in revised form 10 November 2008
Accepted 11 November 2008
Available online 19 November 2008
Ó 2008 Elsevier B.V. All rights reserved.
This work is dedicated to Prof. G. Jaouen on
the occasion of his 65th birthday.
Keywords:
Enantioselective catalysis
Artificial metalloenzyme
Biotin–avidin technology
Oxidation
Schiff base
1. Introduction
lyze different enantioselective transformations including: ester
hydrolysis [2], reductions (hydrogenation, transfer hydrogena-
The steric and electronic properties of organometallic com-
plexes synthesized by chemists enable them to catalyze many
chemical transformations. Their application in asymmetric cataly-
sis is one of the most efficient routes to enantiopure compounds. In
nature, it is estimated that half of all enzymes contain a metal ion
[1]. The complex structures and functions of such metalloenzymes
have inspired chemists and biochemists in designing highly effi-
cient and enantioselective catalytic systems. However, the highly
organized protein framework around the metal center provides
very precise second coordination sphere interactions, which are
difficult to mimic in traditional organometallic chemistry. The de-
sign of artificial metalloenzymes for asymmetric catalysis is based
on combining the high activity of an organometallic complex with
the enantioselectivity provided by the protein environment.
Several strategies can be envisaged for the incorporation of a
catalytically active metal center inside a protein. Supramolecular,
dative or covalent anchoring have been successfully imple-
mented for the creation of artificial metalloenzymes that cata-
tion) [3–14], oxidations (dihydroxylation, epoxidation, sulfoxida-
tion) [15–23] or C–C bond formations (Diels–Alder, Michael
additions and allylic alkylation) [24,25]. In this context, we have
demonstrated that the biotin-(strept)avidin technology, first re-
ported by Whitesides et al. [3], offers a great potential for the
design and optimization of hydrogenation [7], transfer hydroge-
nation [13,14], alcohol oxidation [26] and allylic alkylation reac-
tions [25]. With the aim of expanding the reaction range of this
type of hybrid catalysts, we focused our attention on developing
enantioselective oxidations and we selected the sulfoxidation
reaction as a starting point.
Among the metal catalysts for asymmetric oxidations, salen-
based complexes have received considerable attention, due to their
synthetic accessibility, versatility and high catalytic activity [27–
29]. Highly enantioselective aqueous oxidation systems based on
salen, salan or salalen complexes of Ti, Fe and Al have been
reported recently [30–33]. Although their application in homoge-
neous asymmetric sulfoxidation is limited [34–36], manganese-
and chromium-salen derivatives are water compatible and have
already been used in the context of artificial metalloenzymes as
mimics of heme, the native co-factor of myoglobin [20,21]. Herein,
we describe the creation of streptavidin-based artificial metalloen-
zymes for sulfoxidation reactions using achiral biotinylated
Mn-salen complexes. The proposed mechanism of the oxidation
* Corresponding author. Address: Department of Chemistry, University of Basel,
Spitalstrasse 51, CH-4056 Basel, Switzerland. Tel.: +41 61 267 1004; fax: +41 61 267
1005.
E-mail address: thomas.ward@unibas.ch (T.R. Ward).
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2008.11.023

A. Pordea et al. / Journal of Organometallic Chemistry 694 (2009) 930–936
931
reactions catalyzed by Mn- or Cr-salens, which does not require
binding of the substrate to the metal [37,38], suggests that the
chiral environment provided by the protein could have a determi-
nant influence on the enantiodiscrimination step. Cationic achiral
Mn-salen complexes have previously been used as chiral catalysts
in the presence of an optically active donor axial ligand, which fa-
vors one particular conformation of the five-membered chelate
ring formed between the manganese ion and the ethylenediimine
part [39]. In this context, we reasoned that the host protein could
influence either delivery of the incoming prochiral substrate, or the
conformation of the organometallic moiety, to provide enantiose-
lectivity (Scheme 1).
tion of the benzyl group by hydrogenation in presence of Pd/C.
Condensation with 3,5-di-tert-butyl-2-hydroxybenzaldehyde and
biotinylation of the corresponding diiminopyrrolidine yielded
the biotinylated salen ligand Sal-1 (see the Section 4 for the
description of the synthetic procedures and the characterization
of the intermediates). The manganese-salen complex, Mn-Sal-1,
was prepared by treatment with manganese acetate,
Mn(OAc)2 ꢀ 4H2O, followed by ligand exchange with NaCl
(replacement of acetate by the chloride ion) [41] and purification
on silica gel.
Among the variety of oxidants used in combination with Mn-
salen complexes for catalytic oxidations, we selected hydrogen
peroxide for initial sulfoxidation experiments, because of its water
compatibility and due to a low extent of background reaction
(uncatalyzed oxidation leading to racemic sulfoxide, Table 1, entry
1). In combination with H2O2, Mn-Sal-1 displayed a very modest
activity for the sulfoxidation of thioanisole 1 (Table 1, entry 2).
As the complex was only poorly soluble in water, the experiment
was repeated in a H2O/EtOH 1/1 mixture (v/v), thus affording a rea-
sonable conversion (Table 1, entry 3). Reasoning that incorporation
into streptavidin would solve the solubility problem, we proceeded
to test the catalyst in the presence of the host protein. Upon adding
WT Sav (hereafter, Sav refers to streptavidin; wild-type streptavi-
din is abbreviated WT Sav) to the reaction mixture, a slight in-
crease in conversion was observed, but the reaction displayed
little selectivity (Table 1, entry 4).
Two approaches can be envisaged for the optimization of such
artificial metalloenzymes: genetic modification of the host pro-
tein or chemical modifications of the first coordination sphere
of the metal. During this study, in the absence of precise informa-
tion on the localization of the metal center inside streptavidin,
the L7,8 loop region (residues 112–121) was selected for the ge-
netic optimization, as it lied close to the carboxylate of biotin’s
side chain and therefore could perturb the environment around
the organometallic fragment. In a first attempt to optimize the
activities and selectivities of Mn-Sal-1 ꢁ Sav (ꢁ refers to the
incorporation of the biotinylated complex into Sav), we tested
the influence of point mutations at the 112 position, expected
to lie in the proximity of the organometallic moiety. A small in-
crease in activity was observed upon using the S112G Sav mutant
(Table 1, entry 5). Interestingly, S112D and S112G mutations
afforded a reversed selectivity compared to WT Sav, suggesting
that the host protein has an influence on the enantioselection
(Table 1, entries 5–6).
2. Results and discussion
Metal-salen complexes have already been used by the groups
of Watanabe and Lu for the creation of artificial metalloenzymes
for asymmetric sulfoxidation, by incorporation into apo-myoglo-
bin. In one case, the binding affinity of apo-myoglobin for man-
ganese- and chromium-salen complexes was ensured by
hydrophobic interactions, as well as by a dative bond between
the metal and a hystidine residue. The rational design based
on structural information allowed chemical and genetic fine-tun-
ing of the metal position inside the protein and thus, optimiza-
tion of the binding and of the enantioselectivity (up to 33% (S)
for the sulfoxidation of thioanisole) [19,20]. On the other hand,
Lu and coworkers have used a dual covalent anchoring of a man-
ganese-salen into apo-myoglobin, thus restraining the flexibility
of the metal and improving enantioselectivity (up to 51% (S)
for the sulfoxidation of thioanisole) [21]. In this context, and in-
spired by Jacobsen’s catalyst [40], one of the most successful me-
tal-salen complexes for the epoxidation of alkenes, we designed
and synthesized the achiral biotinylated salen ligand Sal-1, in
which the diimine bridge was derived from a sterically con-
strained cis-3,4-diaminopyrrolidine (Scheme 2). The diamine pre-
cursor was synthesized in five steps starting from N-
benzylmaleimide by ruthenium-catalyzed dihydroxylation,
reduction of the dihydroxyimide and ditosylation of the corre-
sponding diol, followed by conversion to the diazidopyrrolidine,
which was reduced to the diamine with simultaneous deprotec-
Previous results obtained for hydrogenation [7], transfer hydro-
genation [13] and allylic alkylation reactions [25] demonstrated
that chemical optimization brings more diversity than genetic
modifications. Therefore, in a second approach, three additional
biotinylated Schiff base tetradentate ligands were synthesized
and their Mn complexes, formed in situ, were tested in the sulfox-
idation of thioanisole.
To maximize the possible interactions upon incorporation into
streptavidin, the biotinylated ligands were designed to offer a large
structural diversity, by varying the ligand type (aromatic or ali-
phatic imines), the biotin anchor position (on the diimine back-
bone or on the 2-hydroxybenzaldehyde moiety), and the spacer
between the biotin and the coordinating moiety. The three achiral
salen ligands Sal-2, Sal-3 and Sal-4 (Scheme 3) were synthesized
by condensation of the appropriate diamine with the correspond-
ing 2-hydroxybenzaldehydes. 3,5-Di-tert-butyl-2-hydroxybenzal-
dehyde was used to form the symmetric diimines Sal-2 and Sal-
4, and the biotin anchor was attached to the diamine component
before (Sal-2) or after Schiff base formation (Sal-4). The non-sym-
metric diimine Sal-3 was obtained upon mixing ethylenediamine,
3,5-di-tert-butyl-2-hydroxybenzaldehyde and the aldehyde bear-
ing the biotin anchor (see Section 4).
Scheme 1. Schematic representation of possible interactions during the sulfoxida-
tion of thioanisole catalyzed by biotinylated manganese-salen complexes incorpo-
rated into streptavidin.

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A. Pordea et al. / Journal of Organometallic Chemistry 694 (2009) 930–936
Scheme 2. Synthesis of the biotinylated manganese complex Mn-Sal-1.
Table 1
T115A and E116A Sav mutants afforded a considerably increased
Sulfoxidation of thioanisole catalyzed by Mn-Sal-1.a
activity (Table 2, entries 8–9). Finally, the addition of coordinating
axial ligands such as N-methylimidazole or pyridine-N-oxide,
known to be beneficial for the Mn-catalyzed reactions with
H2O2 [42,43], did not improve the activity or the selectivity of
the catalysts (Table 2, entries 10–11).
Entry
Protein
Catalyst
Solvent
Conv. (%)
ee (%)
1
2
3
4
5
6
–
–
H2O
10
10
50
21
34
25
–
–
–
–
–
Mn-Sal-1
Mn-Sal-1
Mn-Sal-1
Mn-Sal-1
Mn-Sal-1
H2O
H2O/EtOH 1/1
H2O
H2O
H2O
WT Sav
S112G Sav
S112D Sav
6 (R)
11 (S)
5 (S)
3. Conclusion
a
Reaction conditions: 1.5% DMF in H2O; Sav 60
lM (tetrameric); Mn-Sal-1
In summary, we showed that incorporation of achiral biotinyl-
ated manganese-salen complexes into streptavidin yields active
artificial metalloenzymes for the sulfoxidation of thioanisole.
Although the enantioselectivities obtained are low compared to
other strategies based on the association of metal-salen complexes
with proteins [19–21], they suggest that the protein participates in
the enantioselection mechanism. Moreover, the tight binding being
ensured by the biotin anchor, a high diversity might be introduced
by chemical derivatization without noticeable loss in affinity. The
structures of the organometallic salen-type catalysts can be readily
tuned [27] to provide a variety of biotinylated ligands, which can
be combined with genetic variations of the host protein, in order
to reduce the flexibility of the metallic moiety and thus increase
enantioselectivity. In contrast to methods relying on the incorpora-
tion of an isolated metal ion into a protein [15–18,23], this method
overcomes the problem of affinity between the two counterparts
and offers the advantage of a well-defined binding site for the cat-
alytic center. Ongoing research is directed towards computer mod-
200
l
M; thioanisole 1 10 mM; H2O2 1 equiv.; room temperature; 4 h.
The new biotinylated Mn-salen complexes catalyzed the
aqueous sulfoxidation of thioanisole with modest activities in
the presence of H2O2 as stoichiometric oxidant (Table 2, entries
1–3). A slight decrease in conversion was obtained with all the
catalysts in the presence of S112D Sav (Table 2, entries 4–6)
and the enantioselectivities remained low (maximum 13% ee (R)
for Mn-Sal-3 ꢁ S112D Sav, Table 2, entry 5). It is worthy to note
that replacing the Sal-1 ligand with the non-symmetric diimine
Sal-3 lead to an inversion of the enantioselectivity in the presence
of the S112D Sav mutant (from 5% ee (S) – Table 1, entry 6, to 13%
ee (R) – Table 2, entry 5). Several streptavidin isoforms bearing
glycine or alanine mutations in the L7,8 loop region were then
tested for enantioselective sulfoxidation after incorporation of
the Mn-Sal-3 catalyst (Table 2, entries 7–9). Interestingly, the

A. Pordea et al. / Journal of Organometallic Chemistry 694 (2009) 930–936
933
Scheme 3. Biotinylated salen ligands Sal-2, Sal-3 and Sal-4, tested for the sulfoxidation of thioanisole 1.
Table 2
IT (Finnigan) spectrometer. The relative intensities are given in
parenthesis (%). Streptavidin (wild-type and mutants) was pre-
pared according to published results [46].
Sulfoxidation of thioanisole catalyzed by Mn-Sal-2–Mn-Sal-4.a
Entry
Protein
Catalyst
Additive
Conv. (%)
ee (%)
1
2
3
4
5
6
7
8
–
–
–
Mn-Sal-2
Mn-Sal-3
Mn-Sal-4
Mn-Sal-2
Mn-Sal-3
Mn-Sal-4
Mn-Sal-3
Mn-Sal-3
Mn-Sal-3
Mn-Sal-3
Mn-Sal-3
–
–
–
–
–
–
–
–
44
38
19
30
32
3
32
56
56
36
35
–
–
–
4.2. Synthesis of (meso)-N-biotinyl-3,4-(N0,N00-bis(3,5-di-tert-
butylsalicylidene))-pyrrolidine, Sal-1
S112D Sav
S112D Sav
S112D Sav
T114G Sav
T115A Sav
E116A Sav
S112D Sav
S112D Sav
1 (S)
13 (R)
0
8 (R)
6 (R)
7 (R)
13 (R)
11 (R)
4.2.1. (meso)-N-Benzyl-3,4-dihydroxy-2,5-pyrrolidinedione [48]
To a solution of N-benzylmaleimide (3 g, 16.0 mmol) in AcOEt/
CH3CN 1/1 (total volume 195 mL) cooled in an ice bath at 0 °C was
added a solution of RuCl3 (233 mg, 1.13 mmol) and NaIO4 in water
(32 mL). The biphasic mixture was vigourously stirred for 3 min at
0 °C, following which the reaction was quenched with 100 mL
Na2S2O3 saturated solution. The organic phase was separated and
the aqueous layer was extracted three times with AcOEt. The com-
bined organic layers were dried over MgSO4 and evaporated in va-
cuo to afford the crude product, which was purified by flash
chromatography on silicagel using CH2Cl2/MeOH (98/2) to afford
a white solid (2.5 g, 70% yield). 1H NMR (200 MHz, CD3OD,
298 K): dH (ppm) 4.48 (s, 2H, N-CH2-Ph), 4.67 (s, 2H, 2 ꢂ CH-OH),
7.28–7.34 (m, 5H, HCaromatic).
9
10
11
–
Pyridine-N-oxide
N-Methylimidazole
a
Reaction conditions: 1% DMF in H2O; Sav 33 lM (tetrameric); Mn-Sal-X 100 lM;
thioanisole 1 5 mM; H2O2 1 equiv.; room temperature; 5 h.
eling and structural characterization, which would allow gaining
more information on the localization of the active site and thus
optimization of the activity and of the selectivity of these
metalloenzymes.
4. Experimental
4.2.2. (meso)-N-Benzyl-3,4-dihydroxypyrrolidine
This synthesis was adapted from a published procedure [48]. All
the manipulations were carried out using standard Schlenk tech-
niques. To a suspension of LiAlH4 (4.3 g, 113 mmol) in THF
(130 mL) under nitrogen was added a solution of (meso)-N-ben-
zyl-3,4-dihydroxy-2,5-pyrrolidinedione (5.0 g, 22.6 mmol) and
the reaction mixture was refluxed during 62 h. The reaction was
cooled with an ice bath and AcOEt (120 mL) was carefully added
with vigourous stirring, followed by water (18 mL) and 3 M NaOH
solution (4.25 mL). After 1 h stirring at room temperature, the alu-
minium salts filtered on celite and washed with AcOEt. The filtrate
was evaporated to yield the crude product, which was purified by
flash chromatography on silicagel using AcOEt/MeOH/Et3N (95/5/
1) to afford a yellow oil (1.13 g, 26% yield). 1H NMR (200 MHz,
CDCl3, 298 K): dH (ppm) 2.59 (br, 2H, OH), 2.68–2.70 (m, 4H,
2 ꢂ CH2-N), 3.6 (s, 2H, N-CH2-Ph), 4.16–4.21 (m, 2H, 2 ꢂ CH-OH),
7.28–7.37 (m, 5H, HCaromatic).
4.1. General considerations
Solvents were of analytical grade and were purchased from
Aldrich, Fluka or Acros and used without further purification.
Water was purified to milliQ degree of purity. D-Biotin was pur-
chased from Chanzou Huaren (purity P 99%). Biotin pentafluor-
ophenol ester (BPFP) and biotinyl-N-hydroxysuccinimide ester
(BNHS) were prepared according to reported procedures [44,45].
1H and 13C were acquired on a 200 MHz (Varian) or on a
400 MHz (Bruker) spectrometer using deuterated chloroform
(99.8% D), dimethyl sulfoxide (99.5% D) or methanol (99.8% D) from
Cambridge Isotope Laboratories. Chemical shifts are reported in
ppm and signals are quoted as s (singulet), d (doublet), t (triplet),
br (broad) or m (multiplet). Electron Spray Ionization and Atmo-
spheric Pressure Chemical mass spectra were recorded on a LCQ-

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A. Pordea et al. / Journal of Organometallic Chemistry 694 (2009) 930–936
4.2.3. (meso)-N-Benzyl-3,4-dihydroxypyrrolidine di-p-
4.2.6. (meso)-N-Biotinyl-3,4-(N0,N00-bis(3,5-di-tert-
toluenesulfonate
butylsalicylidene))-pyrrolidine, Sal-1
This synthesis was adapted from a published procedure [49]. To
a solution of (meso)-N-benzyl-3,4-dihydroxypyrrolidine (1.13 g,
5.85 mmol) in pyridine (32 mL) was added p-toluenesulfonyl chlo-
ride (3.4 g, 17.8 mmol) in one portion. The resulting solution was
stored at 4 °C for 4 days. The mixture was then added dropwise
to a cooled solution of citric acid (160 g in 1 L water) and the
resulting aqueous solution was extracted four times with Et2O.
The combined organic layers were dried over Na2SO4 and evapo-
rated in vacuo. The crude product was purified by flash chromatog-
raphy on silicagel using CH2Cl2/Et3N (100/1) to afford a white solid
(2.36 g, 81% yield). 1H NMR (200 MHz, CDCl3, 298 K): dH (ppm)
2.47 (s, 6H, 2 ꢂ CH3), 2.69–2.76 (m, 2H, CH2-N), 3.02–3.11 (m,
2H, CH2-N), 3.6 (s, 2H, N-CH2-Ph), 4.71–4.83 (m, 2H, 2 ꢂ CH-OH),
7.19–7.36 (m, 9H, HCaromatic), 7.74 (s, 2H, HCaromatic), 7.78 (s, 2H,
HCaromatic).
To a solution of (meso)-3,4-(N,N0-bis(3,5-di-tert-butylsalicylid-
ene))-pyrrolidine (36 mg, 0.07 mmol) and BNHS (25 mg,
0.07 mmol) in DMF (1 mL) was added Et3N (14 lL, 0.1 mmol) and
the reaction mixture was stirred at room temperature during
24 h. The solvent was evaporated and the crude product was puri-
fied by flash chromatography on silicagel using CH2Cl2/MeOH (90/
10) to afford Sal-1 as a yellow solid (45 mg, 86% yield).
1H NMR (200 MHz, CDCl3, 298 K): dH (ppm) 1.27–1.37 (m, 36H,
4 ꢂ C(CH3)3), 1.40–1.90 (m, 6H, CH2CH2CH2-CH2CO), 2.41 (m, 2H,
CH2-CO), 2.6–3.0 (m, 2H, CH2-S), 3.11–3.25 (m, 1H, CH-S), 3.8–
3.97 (m, 4H, 2 ꢂ CH2-N), 4.0–4.25 (m, 2H, 2 ꢂ CH-N@CH), 4.30–
4.40 (m, 1H, CH-N), 4.45–4.50 (m, 1H, CH-N), 5.30 (d, J = 5.6 Hz,
1H, CH-NH), 5.97 (d, J = 9.6 Hz 2H, CH-NH), 7.05 (dd, J = 2.4 Hz,
J = 4.4 Hz, 2H, 2 ꢂ HCaromatic), 7.36 (d, J = 2.4 Hz, 2H, 2 ꢂ HCaromatic),
8.35 (s, 1H, CH@N), 8.42 (s, 1H, CH@N), 13.0 (s, 1H, OH), 13.07 (s,
1H, OH).
4.2.4. (meso)-N-Benzyl-3,4-diazidopyrrolidine
ESIꢃ-MS: 758.7 (100, [MꢃH]ꢃ), 759.7 (50, M), 794.5 (80,
This synthesis was adapted from a published procedure [50]. All
the manipulations were carried out using standard Schlenk tech-
niques. To a solution of (meso)-N-benzyl-3,4-dihydroxypyrrolidine
di-p-toluenesulfonate (2.0 g, 4.0 mmol) in DMF (22 mL) under
nitrogen was added solid NaN3 (1.04 g, 16.0 mmol). The resulting
suspension was heated at 100 °C for 22 h and then cooled at room
temperature. Water (40 mL) was added and the mixture was ex-
tracted four times with CH2Cl2. The organic layers were dried over
Na2SO4 and evaporated in vacuo. The crude product was purified by
flash chromatography on silicagel using hexane/AcOEt (80/20) to
afford an incolor oil (880 mg, 90% yield). 1H NMR (400 MHz, CDCl3,
298 K): dH (ppm) 2.70 (dd, J = 10.2 Hz, J = 4.7 Hz, 2H, 2 ꢂ CH2-N),
3.01 (dd, J = 10.2 Hz, J = 6.5 Hz, 2H, 2 ꢂ CH2-N), 3.69 (s, 2H,
N-CH2-Ph), 4.04–4.07 (m, 2H, 2 ꢂ CH-N3), 7.32–7.38 (m, 5H,
HCaromatic). 13C NMR (100 MHz, CDCl3, 298 K): dC (ppm) 57.5 (2 ꢂ
CH2-N), 60.1 (N-CH2-Ph), 62.2 (2 ꢂ CH-N3), 127.8 (CaromaticH),
128.9 (2 ꢂ CaromaticH), 129.1 (2 ꢂ CaromaticH), 138.2 (Caromatic).
[MꢃH+2H2O]ꢃ), 795.4 (40, M+2H2O).
4.3. Synthesis of N,N0-bis-(3,5-di-tert-butylsalicylidene)-3,4-
diaminobenzoic acid, aminobiotinyl amide, Sal-2
4.3.1. 3,4-Diaminobenzoic acid, aminobiotinyl amide
Biotinamine was synthesized according to a reported procedure
[44]. 3,4-Diaminobenzoic acid (152 mg, 1 mmol), N-(3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide EDC (233 mg, 1.5 mmol), 1-
hydroxybenzotriazole HOBT (202 mg, 1.5 mmol), Et3N (268 lL,
2 mmol) and biotinamine (401 mg, 1.75 mmol) were mixed in
DMF (40 mL) and the resulting solution was stirred at 45 °C during
24 h. After this time, the DMF was evaporated and the crude prod-
uct was purified by flash chromatography on silicagel using
CH2Cl2/MeOH (initial mixture 9/1, final mixture 6/4) to afford the
product (203 mg, 0.56 mmol, 56% yield).
1H NMR (400 MHz, DMSO-d6, 298 K): dH (ppm) 1.21–1.61 (m,
8H, CH2CH2CH2CH2-CH2NH), 2.58 (d, J = 12.4 Hz, 1H, CH2-S), 2.82
(dd, J = 12.4 Hz, J = 5.1 Hz, 1H, CH2-S), 3.08–3.14 (m, 1H, CH-S),
3.38–3.52 (m, 2H, CH2-NH), 4.12–4.15 (m, 1H, CH-N), 4.29–4.33
(m, 1H, CH-N), 4.95 (br, 4H, NH2), 6.39 (s, 1H, CH-NH), 6.47 (s,
1H, CH-NH), 6.47 (d, J = 8.0 Hz, 1H, HCaromatic), 6.95 (dd, J = 8.0 Hz,
J = 1.9 Hz, 1H, HCaromatic), 7.04 (d, J = 1.9 Hz, 1H, HCaromatic), 7.88
(t, J = 5.6 Hz, 1H, CH2-HN).
4.2.5. (meso)-3,4-(N,N0-Bis(3,5-di-tert-butylsalicylidene))-pyrrolidine
This synthesis was adapted from a published procedure [51]. All
the manipulations were carried out using standard Schlenk tech-
niques. A suspension of palladium on charcoal (10% w/w, 47 mg,
0.02 mmol) in EtOH/CF3COOH (8/2 v/v) was added to a solution
of (meso)-N-benzyl-3,4-diazidopyrrolidine (100 mg, 0.41 mmol)
in EtOH/CF3COOH (8/2 v/v, total volume 2 mL). The suspension
was shaken under 5 bar H2 for 16 h, filtered through celite and con-
centrated under vacuum to afford a white solid. The crude residue
was dissolved in EtOH (3 mL) and neutralized with a solution of
NaOH 4.7 M (0.32 mL). Neat 3,5-di-tert-butyl-2-hydroxybenzalde-
hyde (197 mg, 0.84 mmol) was added at once and the reaction
was refluxed for 2 h. The reaction mixture was then poured into
3.3 mL saturated NaCl solution and extracted three times with
CH2Cl2. The organic layers were combined, washed with saturated
NaCl solution, dried over Na2SO4 and evaporated under vacuum.
The product was purified by flash chromatography on silicagel
using AcOEt/MeOH (initially pure AcOEt, then 95/5) to yield a foa-
my yellow glass (110 mg, 54% yield over the two steps). 1H NMR
(400 MHz, CDCl3, 298 K): dH (ppm) 1.28 (s, 18H, 2 ꢂ C(CH3)3),
1.29 (s, 18H, 2 ꢂ C(CH3)3), 3.39–3.41 (m, 4H, 2 ꢂ CH2-N), 4.05–
4.07 (m, 2H, CH-N@CH), 7.04 (d, J = 2.44 Hz, 2H, HCaromatic), 7.35
(d, J = 2.44 Hz, 2H, HCaromatic), 8.36 (s, 2H, 2 ꢂ CH@N), 13.34 (s,
2H, 2 ꢂ OH). 13C NMR (100 MHz, CDCl3, 298 K): dC (ppm) 29.8 (C-
(CH3)3), 31.9 (C-(CH3)3), 34.5 (2 ꢂ C-CH3), 35.4 (2 ꢂ C-CH3), 53.4
(2 ꢂ CH2-N), 73.6 (2 ꢂ CH-N), 118.0 (2 ꢂ Caromatic), 126.2 (2 ꢂ
CaromaticH), 127.5 (2 ꢂ CaromaticH), 137.1 (2 ꢂ Caromatic), 140.2 (2 ꢂ
Caromatic), 158.5 (Caromatic), 166.7 (2 ꢂ CH@N). APCI+-MS: 546.5
(30), 535.5 (50, [M+2H]2+), 534.5 (100, [M+H]+).
13C NMR (100 MHz, DMSO-d6, 298 K): dC (ppm) 27.4 (CH2), 29.1
(CH2), 29.2 (CH2), 30.1 (CH2), 39.7 (CH2-NH), 40.7 (CH2-S), 56.4 (CH-
S), 60.1 (CH-N), 61.9 (CH-N), 113.6 (CaromaticH), 114.7 (CaromaticH),
117.8 (CaromaticH), 124.1 (Caromatic-CO), 134.7 (Caromatic-NH2), 139.0
(Caromatic-NH2), 163.6 (HN-CO-NH), 167.6 (CO-NH).
ESI+-MS: 386.2 (10, [M+Na]+), 365.1 (20, [M+2H]2+), 364.1 (100,
[M+H]+).
4.3.2. N,N0-Bis-(3,5-di-tert-butylsalicylidene)-3,4-diaminobenzoic
acid, aminobiotinyl amide, Sal-2
To
(386 mg, 1.65 mmol) in MeOH (10 mL) was added a solution of
3,4-diaminobenzoic acid, aminobiotinyl amide (200 mg,
a solution of 3,5-di-tert-butyl-2-hydroxybenzaldehyde
0.55 mmol) in MeOH (10 mL) and the reaction mixture was re-
fluxed during 48 h. After this time, MeOH was evaporated and
the crude product was purified by flash chromatography on silica-
gel using CH2Cl2/MeOH (initially pure CH2Cl2, final mixture 95/5)
to afford Sal-2 as a yellow-orange solid (287 mg, 0.36 mmol, 65%
yield).
1H NMR (400 MHz, CDCl3, 298 K): dH (ppm) 1.33 (s, 18H,
2 ꢂ C(CH3)3), 1.45 (s, 18H, 2 ꢂ C(CH3)3), 1.33–1.67 (m, 8H,
CH2CH2CH2CH2-CH2NH), 2.62 (d, J = 12.8 Hz, 1H, CH2-S), 2.82 (dd,
J = 12.8 Hz, J = 4.7 Hz, 1H, CH2-S), 3.07–3.12 (m, 1H, CH-S), 3.36–

A. Pordea et al. / Journal of Organometallic Chemistry 694 (2009) 930–936
935
3.51 (m, 2H, CH2-NH), 4.19–4.22 (m, 1H, CH-N), 4.39–4.42 (m, 1H,
CH-N), 5.67 (s, 1H; CH-NH), 6.68 (s, 1H; CH-NH), 7.23–7.27 (m, 3H,
HCaromatic), 7.46 (d, J = 2.4 Hz, 1H, HCaromatic), 7.48 (d, J = 2.4 Hz, 1H,
HCaromatic), 7.78 (dd, J = 8.2 Hz, J = 1.8 Hz, 1H, HCaromatic), 7.84 (d,
J = 1.8 Hz, 1H, HCaromatic), 8.68 (s, 1H, CH@N), 8.79 (s, 1H, CH@N),
13.41 (s, 1H, OH), 13.45 (s, 1H, OH).
HCaromatic), 7.37 (d, J = 2.4 Hz, 1H, HCaromatic), 8.41 (s, 1H, CH@N),
8.42 (s, 1H, CH@N).
4.5. Synthesis of N-biotinyl-N0,N00-bis(3,5-di-tert-
butylsalicylidene)diethylenetriamine, Sal-4
13C NMR (100 MHz, CDCl3, 298 K): dC (ppm) 27.4 (CH2), 28.9
(CH2), 29.0 (CH2), 29.5 (CH2), 29.8 (3 ꢂ CH3), 31.9 (6 ꢂ CH3), 34.6
(3 ꢂ CH3), 35.5 (4 ꢂ C-CH3), 40.3 (CH2-NH), 40.9 (CH2-S), 56.3
(CH-S), 60.5 (CH-N), 62.3 (CH-N), 118.6 (Caromatic), 118.7 (Caromatic),
119.4 (Caromatic), 120.3 (Caromatic), 126.3 (Caromatic), 127.4 (Caromatic),
127.6 (Caromatic), 128.9 (Caromatic), 129.1 (Caromatic), 133.9 (Caromatic),
137.5 (Caromatic), 137.7 (Caromatic), 141.0 (2 ꢂ Caromatic), 143.0
(Caromatic), 145.6 (Caromatic), 159.0 (Caromatic), 159.1 (Caromatic), 164.6
(HN-CO-NH), 166.0 (2 ꢂ CH@N), 167.1 (CO-NH).
A solution of 3,5-di-tert-butyl-2-hydroxybenzaldehyde (150 mg,
0.64 mmol) and ethylenetriamine (34 lL, 0.32 mmol) in toluene
(5 mL) was stirred during 4 h at room temperature. The solvent
was evaporated and the crude product was purified by flash chro-
matography on silicagel using CH2Cl2/MeOH (96/4) to afford a yel-
low solid (171 mg, quantitative yield). As the product hydrolyzed
rapidly in solution, the purification was performed very quickly.
To a solution of N,N0-bis-(3,5-di-tert-butylsalicylidene)diethyl-
enetriamine (170 mg, 0.32 mmol) and BNHS (131 mg, 0.38 mmol)
ESI+-MS: 818.6 (10, [M+Na]+), 798.5 (20), 797.4 (50, [M+2H]2+),
796.5 (100, [M+H]+).
in DMF (5 mL) was added Et3N (134 lL, 1 mmol) and the reaction
mixture was heated at 50 °C during 24 h. The solvent was evapo-
rated and the crude product was purified by flash chromatography
on silicagel using CH2Cl2/MeOH/Et3N (95/5/2) to afford Sal-4 as a
yellow solid (168 mg, 69% yield).
4.4. Synthesis of N-((5-O-Biotinyl)-2,5-dihydroxysalicylidene), N0-
(3,5-di-tert-butylsalicylidene)-ethylenediamine, Sal-3
1H NMR (400 MHz, CDCl3, 298 K): dH (ppm) 1.31 (s, 18H,
2 ꢂ C(CH3)3), 1.45 (s, 18H, 2 ꢂ C(CH3)3), 1.59–1.76 (m, 6H,
CH2CH2CH2-CH2CO), 2.41 (t, J = 7.5 Hz, 2H, CH2-CO), 2.73 (d,
J = 12.7 Hz, 1H, CH2-S), 2.90 (dd, J = 12.7 Hz, J = 4.9 Hz, 1H, CH2-S),
3.11–3.16 (m, 1H, CH-S), 3.68–3.69 (m, 4H, CH2-N@CH), 3.76 (t,
J = 5.9 Hz, 2H, CH2-N), 3.84 (t, J = 5.9 Hz, 2H, CH2-N), 4.26–4.29
(m, 1H, CH-N), 4.47–4.50 (m, 1H, CH-N), 5.24 (s, 1H, CH-NH),
5.68 (s, 1H, CH-NH), 7.07 (d, J = 2.4 Hz, 1H, HCaromatic), 7.09 (d,
J = 2.4 Hz, 1H, HCaromatic), 7.39–7.40 (m, 2H, HCaromatic), 8.34 (s,
1H, CH@N), 8.37 (s, 1H, CH@N), 13.35 (s, 1H, OH), 13.69 (s, 1H, OH).
13C NMR (100 MHz, CDCl3, 298 K): dC (ppm) 25.5 (CH2), 28.7
(CH2), 29.8 (CH3), 29.8 (CH3), 31.9 (CH3), 33.2 (CH2), 34.4 (CH2),
34.5 (C-CH3), 35.4 (C-CH3), 40.9 (CH2-S), 48.3 (CH2-N), 50.3 (CH2-
N), 55.7 (CH-S), 57.8 (CH2-N), 58.8 (CH2-N), 60.5 (CH-N), 62.1
(CH-N), 117.9 (Caromatic), 118.2 (Caromatic), 126.4 (CaromaticH), 126.6
(CaromaticH), 127.5 (CaromaticH), 127.9 (CaromaticH), 137 (Caromatic),
137.1 (Caromatic), 140.6 (Caromatic), 140.8 (Caromatic), 158.2 (Caromatic),
158.4 (Caromatic), 163.7 (HN-CO-NH), 168.1 (CH@N), 168.4 (CH@N),
173.9 (CO-NH).
4.4.1. 5-(O-Biotinyl)-2,5-dihydroxybenzaldehyde
2,5-Dihydroxybenzaldehyde (1 g, 7.24 mmol), BNHS (2.06 g,
6.03 mmol) and Et3N (1.21 mL, 9.05 mmol) were mixed in DMF
(30 mL) and the resulting solution was stirred at room temperature
for 48 h. After this time, the DMF was evaporated and the crude
product was purified by flash chromatography on silicagel using
CH2Cl2/MeOH (95/5) to afford the product (437 mg, 1.20 mmol,
20% yield).
1H NMR (400 MHz, DMSO-d6, 298 K): dH (ppm) 1.57–1.40 (m,
4H, CH2CH2-CH2CO), 1.63–1.70 (m, 2H, CH2-CHS), 2.58 (t,
J = 7.4 Hz, 2H, CH2CO), 2.60 (d, J = 12.7 Hz, 1H, CH2-S), 2.85 (dd,
J = 12.7 Hz, J = 5.1 Hz, 1H, CH2-S), 3.12–3.17 (m, 1H, CH-S), 4.14–
4.18 (m, 1H, CH-N), 4.31–4.34 (m, 1H, CH-N), 6.39 (s, 1H, CH-
NH), 6.48 (s, 1H, CH-NH), 7.03 (d, J = 12.7 Hz, 1H, HCaromatic), 7.29
(dd, J = 8.9 Hz, J = 3.0 Hz, 1H, HCaromatic), 7.35 (d, J = 3.0 Hz, 1H,
HCaromatic), 10.26 (s, 1 H, CHO).
13C NMR (100 MHz, DMSO-d6, 298 K): dC (ppm) 25.2 (CH2), 28.8
(CH2), 28.9 (CH2), 34.1 (CH2CO), 40.7 (CH2-S), 59.2 (CH-S), 60.1 (CH-
N), 61.9 (CH-N), 119.1 (CaromaticH), 121.4 (Caromatic), 123.3 (Caromatic),
130.8 (CaromaticH), 143.6 (Caromatic), 159.3 (Caromatic), 163.6 (HN-CO-
NH), 172.9 (CO-O), 191.1 (CH@O).
ESI+-MS: 776.4 (20), 763.5 (55, [M+2H]2+), 762.6 (100, [M+H]+).
4.6. Preparation of the manganese-salen complexes Mn-Sal-1–Mn-
Sal-4
ESI+-MS: 387.1 (100, [M+Na]+), 365.1 (10, [M+H]+).
4.4.2. N-((5-O-Biotinyl)-2,5-dihydroxysalicylidene), N0-(3,5-di-tert-
butylsalicylidene)-ethylenediamine, Sal-3
4.6.1. (meso)-N-Biotinyl-3,4-(N0,N00-bis(3,5-di-tert-
butylsalicylidene))-pyrrolidine, manganeseIII chloride complex,
Mn-Sal-1
To
a solution of 5-(O-biotinyl)-2,5-dihydroxybenzaldehyde
(300 mg, 0.82 mmol) and 3,5-di-tert-butyl-2-hydroxybenzalde-
hyde (192 mg, 0.82 mmol) in MeOH (30 mL) was added a solu-
tion of Et3N (53 lL, 0.8 mmol) in MeOH (10 mL) and the
This synthesis was adapted from a published procedure [41]. To
a solution of Mn(OAc)2 4H2O (44 mg, 0.18 mmol) in EtOH (1.5 mL)
was added a solution of Sal-4 in toluene (1.8 mL) and the resulting
mixture was refluxed for 1 h. Saturated aqueous NaCl (0.2 mL) was
then added and air was bubbled through the solution during
30 min. The reaction mixture was cooled to room temperature, tol-
uene (4 mL) and CH2Cl2 (10 mL) were added and the organic phase
was separated and washed two times with water. The aqueous
phase was extracted with CH2Cl2 until it was colourless and the
collected organic layers were dried over Na2SO4 and evaporated
under vacuum. The Mn complex was purified by flash chromatog-
raphy on silicagel using CH2Cl2/MeOH (75/15) to afford a brown
solid (28.5 mg, 56% yield). ESI+-MS: 812.5 (100, [MꢃCl]+), 813.5
(40, [MꢃCl + H]+), 814.3 (10, [MꢃCl + 2H]+).
resulting reaction mixture was stirred at room temperature for
10 min. The Schiff bases were precipitated by adding water
(50 mL) and the solid was collected after centrifugation of the
resulting suspension, dissolved in CH2Cl2 and dried over MgSO4.
After evaporation of the CH2Cl2, the three Schiff bases were sep-
arated by flash chromatography on silicagel using CH2Cl2/MeOH
(initially pure CH2Cl2, final mixture 97/3) to afford the non-sym-
metric Schiff base Sal-3 as a yellow solid (141 mg, 0.23 mmol,
28% yield).
1H NMR (400 MHz, CD3OD, 298 K): dH (ppm) 1.29 (s, 9H,
C(CH3)3), 1.41 (s, 9H, C(CH3)3), 1.42–1.82 (m, 6H, CH2CH2CH2-
CH2CO), 2.60 (d, J = 12.7 Hz, 1H, CH2-S), 2.73 (t, J = 7.4 Hz, 2H,
CH2-CO), 2.95 (dd, J = 12.7 Hz, J = 4.9 Hz, 1H, CH2-S), 3.18–3.27
(m, 1H, CH-S), 3.94–3.96 (m, 4H, NH-CH2CH2-NH), 4.30–4.36 (m,
1H, CH-N), 4.48–4.54 (m, 1H, CH-N), 6.87 (d, J = 7.8 Hz, 1H,
HCaromatic), 7.04 (m, 2H; 2 ꢂ HCaromatic), 7.12 (d, J = 2.4 Hz, 1H,
4.6.2. Mn-Sal-2–Mn-Sal-4
The three Mn-Sal-X complexes (X = 2, 3 or 4) were prepared by
mixing solutions of known concentrations of Mn(OAc)2 4H2O
(0.08 M) and Sal-X (0.04 M) in DMF (1/1 volumes of the two solu-

936
A. Pordea et al. / Journal of Organometallic Chemistry 694 (2009) 930–936
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tions) and heating the resulting mixtures in closed tubes at 65 °C.
Thus, the corresponding Mn-Sal-X complexes were available in
0.02 M solutions in DMF and were used without further isolation
and purification.
4.7. Typical procedure for the sulfoxidation of thioanisole
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4.7.1. Using Mn-Sal-1 as catalyst (Table 1)
A solution of streptavidin in water (400
concentration) was mixed in a test tube with the biotinylated me-
tal complex, Mn-Sal-1 (4 L of a 0.02 M stock solution in DMF,
200 M final concentration). After 5 min incubation time, thioani-
sole was added (4 L of a 1 M DMF stock solution). The reaction
lL, 60 lM tetrameric
l
l
l
was initiated by adding 1 equivalent of H2O2 and the test tube
was sealed. After 4–5 h stirring at room temperature, the reaction
mixture was extracted four times with Et2O. The organic phase was
dried over Na2SO4 and subjected to HPLC analysis using a chiral
OD-H column (Daicel Chemical Industries, Tokyo) with hexane:iso-
propanol 90:10 at 0.8 mL/min; tR = 13.1 min, tS = 17.1 min (UV
detection at 215 nm). The absolute configuration of the sulfoxides
was assigned according to published results [32].
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4.7.2. Using Mn-Sal-2–Mn-Sal-4 as catalysts (Table 2)
A solution of streptavidin in water (400
concentration) was mixed in a test tube with the biotinylated me-
tal complex, Mn-Sal-X (2 L of a 0.02 M stock solution in DMF,
100 M final concentration). After 5 min incubation time, thioani-
sole was added (2 L of a 1 M DMF stock solution). The reaction
lL, 33 lM tetrameric
l
l
l
was initiated by adding 1 equivalent of H2O2 and the test tube
was sealed. After 4–5 h stirring at room temperature, the reaction
mixture was extracted four times with Et2O. The organic phase was
dried over Na2SO4 and subjected to HPLC analysis using a chiral
OD-H column (Daicel Chemical Industries, Tokyo) with hexane:iso-
propanol 90:10 at 0.8 mL/min; tR = 13.1 min, tS = 17.1 min (UV
detection at 215 nm). The absolute configuration of the sulfoxides
was assigned according to published results [32].
Acknowledgements
This work was supported by the Canton of Neuchâtel, and the
Swiss National Science Foundation (Grants FN 200021-105192
and 200020-113348), we thank Prof. C.R. Cantor for providing us
with the streptavidin gene.
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Kegley, Bioconjugate Chem. 4 (2000) 569.
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Technology, Academic Press, San Diego, 1990.
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