1. TIGP program in Chemical Biology (April, 1 2008)
Basic references for Chemical Biology:
Science, 235 (1987) Why Nature Chose Phosphates?
Nature Chemical Biology 3 (2007) 352. Rethinking relationships between natural products
Nature Chemical Biology 2 (2006) 3-6. Origins of chemical biology
Nature Chemical Biology 1 (2005) Chemical biology:
an educational challenge for chemistry departments
Chemical biology, broadly defined as the application of chemistry to the
study of molecular events in biological systems.
Activity-based Probes
1. J. Biol. Chem. 279, (2004)
2. Angew. Chem. Int. Ed. 44, (2005) 6888
3. Chem. Rev. 106, (2006) 3279.
4. ChemBioChem 5, (2004) 41.
5. ChemBioChem 8, (2007) 2187

2. Protein Purification and Characterization
Chromatographic
Methods
Proteins
Activity
Characterization

3. Differential-Display Proteomics
Cell state A
Cell state B
MALDI-MS
Harvest/sample preparation
Spot excision
2-DE pI
Image analysis
size

4. Application of 2-DE in Proteomics
Hundreds of proteins could be separated by a single gel.
Potential targets could be identified and located by comparing the differences between two gels
(State A)
(State B)
Size pI

5. Separating Proteins into Different Classes Would
Simplify the Analysis

6. Hydrolytic Activity Probes vs Suicide Substrates
Recognition
Reporter
Linker
group
Latent reactive
Activity Probes
Recognition
group
Latent reactive
Suicide Substrates

7. Construction of Hydrolytic Activity Probes
Recognition
Reporter
Linker
group
Latent reactive

8. Labeling and Selection of Biocatalysts
Activity Probe
Biocatalysts
Separation
Characterization

9. Important Hydrolases Involved in the Post-Translational Modifications of Proteins
Phosphatases
Glycosidases
Proteases

10. Prototype Activity Probes
Recognition
head
Reporter
Probe-2
J. Chin. Chem. Soc. 1999, 46, 715.
J. Proteome Res. 2002, 1, 35.

11. Synthesis of Activity Probe-1

12. Synthesis of Activity Probe-1

13. Labeling of PTP-1B with Probe-1

14. Hydrolysis of Probe-1 by PTP-1B: 19F NMR Analysis
Incubation of Probe-1 with PTP-1B in an NMR tube. F
Probe-1

15. Specific Labeling of PTP-1B by Probe-1
Coomassie
Streptavidin M - + - + - + - + - + - +
Probe-1
kDa
116
b-Galactosidase 66 45
PTP-1B 35 25
Trypsin 18 14
Lane 1 2 3 4 5 6 7 8 9 10 11 12 13

16. Probe-1 Does Not Cross-Label Other Proteins
Coomassie
Streptavidin + + + +
Probe-1
kDa
Phosphorylase b 97
Albumin 66
Ovalbumin 45
PTP-1B
Carbonic anhydrase 30
Trypsin inhibitor 20
Lane 1 2 3 4
J. Proteome Res. 2002, 1, 35.

17. Activity Probe for b-Glucosidasen i t h a d T r p v L k u

18. Synthesis of Activity Probe for b-Glucosidase

19. Synthesis of Activity Probe for b-Glucosidase

20. Labeling of b-Glucosidase with Probe-3

21. F
Probe-3

22. Labeling of b-Glucosidase with Probe-3
Coomassie
Streptavidin - + - +
Probe-3
kDa
150 -
100 -
75 -
b-Glucosidase
50 -
37 -
Lane 1 2 3 4
Org. Lett. 2002, 4, 3607.

23. Problem of Cross-Labeling

24. Possible Cause of Cross-Labeling Problem4

25. Suppression of Labeling by Cys

26. Tryptic Peptides for Labeled and Unlabeled PTP1B
Calculated
+ IAA
+ QM-4
Sequence
Fragment 1
HEASDFPCR
33-41
(C40) 2
SYILTQGPLPNTCQGFWEMVWEQ
88-111
(C100) 3
CAQYWPQK
(C129) 4
ESGSLSPEHGPVVVHCSAGIGR
(C223) 5
(2 QM-4), (1 IAA + 1 QM-4)
SGTFCLADTCLLLMDK
(C234, C239)
For each modification by QM-4 there would be an increase in molecular weight of 508 Da.

27. Tandem Mass Spectrum of the Peptide HEASDFPCR Labeled with Probe-1

28. Preparation of Probes for Glycosidase Families
b-Glc
b-Gal
b-Fuc
a-Man
a-Glc
a-Gal
a-Fuc

29. Activity Probes LCL-6X for b-Xylosidase

30. Specific Labeling of b-Xylosidase with LCL-6X

31. Selective Enrichment of a-L-Arabinofuranosidase

32. Conclusions
Activity probes for phosphatase and glycosidase were prepared.
The cassette-like design provides great flexibility for future alterations.
Cysteine residues are the preferred labeling site for the reactive quinone methide trapping device.
Many novel applications could be accomplished with the use activity probes.