A higher-yield route to FP–PEG–biotin ABPP probe
Reworking Cravatt’s FP–PEG–biotin synthesis cuts chromatography and raises overall yield from 1% to 28.5%.
Source
An improved synthesis of a fluorophosphonate-polyethylene glycol-biotin probe and its use against competitive substrates
Inspect source passages
A higher-yield route to FP–PEG–biotin ABPP probe
These passages occur verbatim in the stored paper text. They support the points listed below; they do not verify every statement in the explanation.
The new route gives 28.5% overall yield of 1 over nine steps.
Starting from common precursor 2 , our synthesis requires only four chromatographic purifications over nine steps and provides a 28.5% overall yield of 1 .
The Cravatt sequence had an overall yield of 1%.
In contrast, the Cravatt sequence requires six chromatographic purifications over eight steps with an overall yield of 1%.
What they did
Authors redesigned the route from tetraethylene glycol via tosylate/iodide, Arbuzov phosphonate, hydrogenolysis, succinimidyl carbonate, biotin coupling, and DAST fluorination, then tested the probe on serine hydrolases and reversible-substrate competition.
What they found
Nine steps, four chromatographies, 28.5% overall yield versus six chromatographies/eight steps and 1% in the original sequence. Key steps: 90% to iodide 4b, 92% Arbuzov, 96% debenzylation, 87% carbonate, 76% biotin coupling, 80% DAST. The probe labels serine hydrolases in proteomes.
The limits
What it doesn't show
Competition with reversible substrates is preliminary; this is not a new enzyme-inhibitor campaign with full Ki tables.
Key terms
- Fluorophosphonate (FP) probe
- Covalent serine-hydrolase warhead derived from DFP, here biotin-tagged.
- ABPP
- Activity-based protein profiling using covalent probes and enrichment.
- Arbuzov reaction
- Alkyl iodide plus triethyl phosphite to install a phosphonate.
- DAST
- Diethylaminosulfur trifluoride, used to convert phosphonate to fluorophosphonate.
- PEG linker
- Polyethylene glycol spacer between warhead and biotin handle.
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New overall yield of FP–PEG–biotin:
Common questions
What overall yield was achieved?
28.5% over nine steps vs 1% previously.
How many chromatographies?
Four, versus six in the Cravatt sequence.
Last step?
DAST fluorination at −42 °C, 80% yield.
What enzymes does it target?
Serine hydrolases in proteome samples.
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