Chemical optimization of artificial metalloenzymes based on the biotin-avidin technology: (S)-selective and solvent-tolerant hydrogenation catalysts via the introduction of chiral amino acid spacers

Article abstract of DOI:10.1039/b509015f

Incorporation of biotinylated-[rhodium(diphosphine)]⁺ complexes, with enantiopure amino acid spacers, in streptavidin affords solvent-tolerant and selective artificial metalloenzymes: up to 91% ee (S) in the hydrogenation of N-protected dehydroamino acids. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b509015f

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Chemical optimization of artificial metalloenzymes based on the
biotin-avidin technology: (S)-selective and solvent-tolerant
hydrogenation catalysts via the introduction of chiral amino acid
spacers{
Myriem Skander, Christophe Malan, Anita Ivanova and Thomas R. Ward*
Received (in Cambridge, UK) 27th June 2005, Accepted 5th August 2005
First published as an Advance Article on the web 1st September 2005
DOI: 10.1039/b509015f
Incorporation of biotinylated-[rhodium(diphosphine)]+ com-
commercially available N-Boc-protected amino acids, the two
pairs of epimeric ligands can be prepared in three steps in 34–58%
overall yield.
plexes, with enantiopure amino acid spacers, in streptavidin
affords solvent-tolerant and selective artificial metalloenzymes:
up to 91% ee (S) in the hydrogenation of N-protected
dehydroamino acids.
Traditionally, enantioselective catalysis has been subdivided
into three areas: homogeneous-, heterogeneous- and enzymatic-
catalysis.1,2 In recent years, two additional areas have witnessed a
‘‘rebirth’’: organocatalysis3–8 and artificial metalloenzymes.9–12
In this later area, an achiral catalyst precursor is incorporated
into a protein to produce an enantioselective hybrid catalyst with
properties reminiscent both of enzymes and of homogeneous
catalysts. Both covalent and supramolecular anchoring strategies
have been pursued successfully to yield sulfoxidation, ester
hydrolysis and hydrogenation artificial metalloenzymes.9,13–16
Inspired by Whitesides’ early report, we have recently exploited
the biotin-avidin technology to produce artificial hydroge-
nases.10,12,17–20 Relying on both chemical- and genetic-optimiza-
tion strategies (e.g. chemogenetic), we produced both (R)- and
(S)-selective catalysts for the hydrogenation of N-protected
dehydroamino acids.21 Herein, we report on our efforts to improve
both on the stability and on the selectivity of artificial
metalloenzymes based on the biotin-avidin technology.
Having demonstrated that the introduction of a short achiral
amino acid spacer between the biotin anchor and the rhodium-
diphosphine moiety has, in some cases, a positive influence on
both the activity and the selectivity of hybrid catalysts, we
proceeded to incorporate enantiopure amino acid spacers.
For initial studies, we focused on phenylalanine ((R)- or (S)-Phe)
and proline ((R)- or (S)-Pro) as spacers. As the biotin-binding
pocket in streptavidin is lined with four tryptophane residues, we
speculated that an aromatic side chain on the spacer may lock, via
p–p interactions, the catalyst in a privileged position. In the same
spirit, proline was selected for its restricted degrees of freedom
imposed by the pyrrolidine ring.
The synthesis of Biot-(R)-Phe-1, Biot-(S)-Phe-1, Biot-(S)-Pro-1
and Biot-(R)-Pro-1 is outlined in Scheme 1. Starting from the
Institute of Chemistry, University of Neuchaˆtel, Av. de Bellevaux 51,
CP2, CH-2007, Neuchaˆtel, Switzerland. E-mail: thomas.ward@unine.ch;
Fax: +41 32 718 25 11; Tel: +41 32 718 25 16
{ Electronic supplementary information (ESI) available: Full experimental
details of the synthesis of ligands and hydrogenation procedures. See
http://dx.doi.org/10.1039/b509015f
Scheme 1 Synthesis of biotinylated ligands bearing an enantiopure
amino acid spacer. Reagents: (i) chlorodimethoxytriazine, NMO, CH3CN,
RT, 48 h; (ii) trifluoroacetic acid, anisole, CH2Cl2, RT, 3 h; (iii) (+)-biotin-
OC6F5, N(i-Pr)2Et, DMF, RT, 48 h.
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Table 1 Hydrogenation experimentsa
N-AcAla
N-AcPhe
Entry
Ligand
ee (%)
Conversion (%)
ee (%)
Conversion (%)
1
2
3
4
5
6
7
8
9
a
Biot-(R)-Phe-1
Biot-(S)-Phe-1
Biot-(S)-Pro-1
Biot-(R)-Pro-1
Biot-(R)-Pro-1/45% dmso
Biot-(R)-Pro-1/biphasic EtOAc
Biot-1
Biot-1/45% dmso
Biot-1/biphasic EtOAc
66 (R)
73 (S)
23 (R)
86 (S)
87 (S)
83 (S)
94 (R)
16 (R)
30 (R)
100
100
100
100
100
90
100
71
56
64 (R)
64 (S)
23 (R)
91 (S)
86 (S)
87 (S)
93 (R)
24 (R)
31 (R)
100
88
100
100
94
85
85
9
5
Both substrates (260 ml, 50 eq. each/Rh cat., 24 mM) were dissolved in 0.38 M MES, added to a solution containing [Rh(COD)(L)]BF4
(100 ml, 1 eq., 0.62 mM in dmso) and streptavidin (tetrameric, 100 ml, 0.33 eq./Rh cat., 0.21 mM in H2O) in a 3 mL Pyrex tube. The final
volume was adjusted with solvent (640 ml, water, dmso or EtOAc) to 1100 mL and the mixture hydrogenated at 5 bar H2 for 15 h.
The new ligands were tested in the rhodium catalyzed hydroge-
nation of both a-acetamidoacrylic and a-acetamidocinnamic acids
in the presence of streptavidin to yield N-acetamidoalanine
(N-AcAla) and N-acetamidophenylalanine (N-AcPhe) respectively
(Scheme 2). For comparison purposes, catalytic runs were
performed with the biotinylated ligand devoid of spacer Biot-1
(Scheme 3).18 The results are summarized in Table 1.{
Introduction of a phenylalanine spacer, yields the reduction
products in up to 73% ee (Table 1, entries 1, 2). Interestingly,
depending on the absolute configuration of the Phe-spacer, both R
and S products are obtained with nearly identical but opposite
enantioselectivity.
In order to test the stability and robustness of the (S)-selective
artificial metalloenzyme, we tested [Rh(COD)(Biot-(R)-Pro-
1)]+,streptavidin in the presence of increasing amounts of
dimethyl sulfoxide (dmso). Increasing the organic solvent content
from 9% (used so far for all screening experiments) to 45%, only a
very modest erosion in activity and selectivity is observed (Table 1,
entry 5). Very similar results are obtained under biphasic reaction
conditions using ethylacetate as organic phase (Table 1, entry 6).
In strong contrast, the (R)-selective catalyst [Rh(COD)(Biot-
1)]+,streptavidin, performs poorly in the presence of increasing
amounts of organic (water miscible or non-miscible) solvents
(Table 1, entries 7–9).
Introduction of an (S)-proline spacer, produces (R)-N-AcAla and
(R)-N-AcPhe with very modest enantioselectivity (entry 3). In the
presence of the (R)-proline spacer however, good enantioselectivities
and conversions are obtained for both (S)-N-AcAla (86% ee) and
(S)-N-AcPhe (91% ee). In contrast to the phenylalanine spacer
where both combinations are equally effective in terms of activity
and selectivity, the ligand with a proline spacer yields clear matched-
and mismatched-combinations.
These experiments demonstrate that incorporation of (R)-Pro as
a spacer yields an (S)-selective hydrogenation catalyst with
enhanced stability towards organic solvents (both miscible and
non-miscible). Due to the poor solubility of olefin substrates in
water, coupled to the straightforward incorporation of enantiopure
amino acid spacers between biotin and the ligand, these findings
thus significantly broaden the scope of artificial metalloenzymes
based on the biotin-avidin technology.
These results suggest that the chiral environment, and possibly
the position of the catalyst within the streptavidin binding site,
changes dramatically upon inverting the configuration of the
spacer. This emphasizes the importance of second coordination
sphere interactions between the spacer and the host protein in
positioning the rhodium moiety within the biotin binding pocket.
We thank Prof. C. R. Cantor for the streptavidin gene as well as
Prof. P. Schu¨rmann and J.-M. Neuhaus for their help in setting up
the protein production. This work was funded by the Swiss
National Science Foundation (Grants FN 620–57866.99, and FN
200021–105192/1 as well as NRP 47 ‘‘Supramolecular Functional
Materials’’), CERC3 (Grant FN20C321–101071), the Roche
Foundation, the Canton of Neuchaˆtel as well as FP6 Marie
Curie Research Training Network (IBAAC network, MRTN-CT-
2003–505020). Umicore Precious Metals Chemistry is acknowl-
edged for a generous loan of rhodium.
Notes and references
Scheme 2 Hydrogenation of both a-acetamidoacrylic (R 5 H) and
a-acetamidocinnamic acids (R 5 Ph).
{ In the absence of streptavidin but otherwise identical reaction conditions,
all catalyst precursors afford quantitatively the reduction products in very
low ee (ee , 10% in all cases).
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Scheme 3 Structure of Biot-1.
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