Chemistry Reference
In-Depth Information
Synthetic or
natural ligands
X-S
X
X
Binding site and
ligand discovery
X
SH
S-X
Allosteric site and
ligand discovery
(a) Covalent capture using native cysteines
Activation or
inhibition
NH
2
NH
2
NH
2
NH
2
N
N
O
O
NaCNBH
3
(b) Covalent capture using imine chemistry
Figure 10.4
Examples of covalent capture methods. (A) Covalent modification of native
cysteines has been shown to modulate ion-channel activity and in the case of enzymes lead to
allosteric inhibition. These findings can be important for structural-functional characterization
or as starting points for drug discovery. (B) Reversible covalent capture using imine chemistry.
and specificity for proteins and refining the sequence specificity through traditional methods
proved intractable. Kohda and colleagues were able to apply covalent capture techniques
to solve the problem. They used a single cysteine residue in the cytosolic domain of Tom20
to capture 19-residue peptides containing a C-terminal cysteine residue.
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They made the
peptidemore amenable to noncovalent interactions by inserting a glycine residue as a spacer
between the cysteine and the recognition elements of the peptide.
They screened seven libraries, each containing 19 peptides of different masses, and
found the sequences that bound most tightly to Tom20. Matrix-assisted laser desorp-
tion/ionization time-of-flight mass spectrometry (MALDI-TOF MS) served as a readout
that allowed the ionized peptides to be observed directly. Using this approach, Kohda
and colleagues discovered that the recognition site spans six residues, not the previously
established five, and were able to refine sequence preferences further.
10.3.8 Covalent Capture Using Imines
Linkers other than disulfides can also be used for reversible site-directed ligand discovery
(Figure 10.4B). Imine bonds, which form reversibly inwater, have been used for performing
dynamic combinatorial chemistry on an enzyme.
31
-
33
In particular, Abell and colleagues
took advantage of the fact that the protein aspartate decarboxylase (ADC) contains an
N-terminal pyruvoyl group at the active site; the ketone of this reactive functionality can
form imines with amine-containing molecules.
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]
They reacted 55 primary amines with
ADC along with the reductant sodium cyanoborohydride. This trapped the metastable imine
as the stable secondary amine and the resulting complexes could be analyzed usingMALDI-
TOF MS. Nine of the 55 amines formed adducts with ADC at 2 mM. Two of these were
