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. 2014 Jan 24;15(2):223-7.
doi: 10.1002/cbic.201300661. Epub 2013 Dec 20.

A general method for artificial metalloenzyme formation through strain-promoted azide-alkyne cycloaddition

Hao Yang  1 , Poonam Srivastava, Chen Zhang, Jared C Lewis
Affiliations

A general method for artificial metalloenzyme formation through strain-promoted azide-alkyne cycloaddition

Hao Yang et al. Chembiochem. .

Abstract

Strain-promoted azide-alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p-azido-L-phenylalanine (Az) residue and catalytically active bicyclononyne-substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor-based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both Si-H and olefin insertion reactions involving these carbene precursors.

Keywords: artificial metalloenzymes; biocatalysis; click chemistry; cofactors; dirhodium.

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Figures

Figure 1
Figure 1
A/B) Structure of wt-tHisF (PDB number 1THF[21a]); colored residues are positions 199 (blue), 50 (orange), and 176 (red). C) HR-ESI-MS of wt-tHisF, tHisF-Az50, and tHisF-Az50-RhBCN. D) Fluorescence spectra (290 nm) of wt-tHisF, tHisF-Az50 (in buffer, 60% ACN, 6M GdmCl), and tHisF-Az50-RhBCN (ACN = acetonitrile; GdmCl = guanidinium chloride).
Figure 2
Figure 2
A) Cartoon schematic of tHisF. B) “Top” (A50/C174) and C) “bottom” (A50/C343) dual-labeled constructs for FRET analysis
Scheme 1
Scheme 1
ArM preparation via SPAAC
Scheme 2
Scheme 2
Syntheses of cofactors 3, 6, and 7; structure of probe 8.
Scheme 4
Scheme 4
tHisF-RhBCN catalyzed A) cyclopropanation and B) Si-H insertion
See this image and copyright information in PMC

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