Organic Letters
Letter
ASSOCIATED CONTENT
Supporting Information
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Calibration curves, detailed procedures, additional experi-
ments, and characterization of Ser-His (PDF)
AUTHOR INFORMATION
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Figure 4. Extent of amide substrate reaction, as judged by
development of fluorescence after incubation with 10 mM Ser-His
for 48 h, 50 °C, 1.8 μg/mL substrates 7−10, 40 mM Britton−
Robinson buffer (40 mM phosphoric acid, 40 mM acetic acid and 40
mM boric acid) titrated to pH 6 with NaOH. Control was the same
reaction in the absence of the dipeptide. Reactions were monitored in
a M1000 PRO Microplate reader, via fluorescence intensity measure-
ments with λex = 485 nm and λem = 530 nm. Percent conversion was
calculated based on the assumption that both amide bonds involving
fluorophore amines were hydrolyzed to generate rhodamine 110.
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This work was supported by DARPA Cooperative Agreement
N66001-15-2-4023 (S.H.G.) and the Howard Hughes Medical
Institute (L.D.L.). We thank Prof. J. Burstyn (University of
WisconsinMadison) for access to the fluorimeter in her
laboratory and Profs. T. Kodadek (The Scripps Research
Institute) and B. Paegel (The Scripps Research Institute) for
helpful comments.
REFERENCES
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with BSA. For each substrate, a control solution lacking Ser-
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His was prepared and analyzed alongside the solution
containing Ser-His. Figure 4B shows the fluorescence increase
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as percent conversion, which was calculated based on the
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release rhodamine 110. We observed only small increases in
fluorescence intensity after 48 h (<1% in each case). There was
no significant difference between samples containing Ser-His
relative to analogous samples lacking Ser-His. Experiments
His or 10 mM imidazole (Figure S3). Background hydrolysis
was more substantial at this elevated pH after 48 h, but neither
Ser-His nor imidazole caused an increase in the extent of this
hydrolysis. We therefore conclude that Ser-His does not
function as a general catalyst of amide hydrolysis.
1
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Developing enzyme-mimetic catalysts that significantly
enhance the rates of challenging reactions near neutral pH
and room temperature remains a profound challenge in
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de novo enzyme design for a few reactions, but chemical
transformations as demanding as amide hydrolysis have yet to
be addressed in this arena. It is noteworthy that DNA-based
(
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catalysis of aromatic amide hydrolysis has been achieved. In
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1
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be interpreted as evidence that Ser-His or any other short His-
containing peptide is competent to catalyze amide hydrolysis.
Auweraer, M.; Blank, K.; Hofkens, J. Bioconjugate Chem. 2011, 22,
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Org. Lett. XXXX, XXX, XXX−XXX