1. Synthesis of Activity-Based Probes for Acyl Protein Thioesterases
Yiming Chen, Michael A. Zompa, Dr. Christopher T. Seto
Department of Chemistry, Brown University, Providence RI 02912
Introduction Synthesis Conclusion
An activity-based probe (ABP) can employed to The synthetic scheme for the ABP is outlined in Scheme 2. The synthesis of the ABP has been finished, and
investigate acyl protein thioesterase enzymes (APTs), important synthetic intermediates have been
which catalyze the depalmitoylation of proteins in the characterized. Successful construction of the final ABP
O O O
cell. An ABP designed to react specifically with active SOCl2, MeOH benzaldehyde target compound has been confirmed by mass
HO OH HO O HO O
form of APTs has been synthesized. This ABP will be NH2 16 hr, 0 oC, 97% NH2 MeOH, rt, 1hr N Ph spectrometry.
used to study the catalytic activities of thioesterase and O
O
esterase enzymes. NaBH3CN, MeOH
HO O paraformaldehyde
HO O
Pd(OH)2
18 hr, rt HN
Bn 5 hr, rt, NaBH3CN, 47% N 55 psi H2, 24 hr, 78%
Bn
Synthesis of the ABP has been accomplished via a 21- O
step synthetic pathway. The reactivity of the ABP will be HO
NH
O
va l i d a te d th r o u g h ki n e ti c a ssa ys a n d m a ss
spectrometry. Then, the ABP will be used to study the OH +
p-CH3C6H4SO3H n-BuLi, n-PrBr, -78 oC
activities of APTs in cell lysates. O
0 oC, 4 hr, 97%
O O HMPA, THF, 12 hr, 76% O O
Mechanism
p-CH3C6H4SO3H Li, TMEDA, 70 oC CrO3, H2SO4
OH
MeOH, rt, 2 hr, 90% t-BuOK, 10 hr, 78% OH 0 oC, 1 hr, 85%
O NH2
The activity-based probe consists of four major O
+ O
DCC, CH2Cl2, rt
NH2 DMAP, 18 hr, 65%
HN O
components. The substrate component (red) binds to OH H2N O O
the active site of the target enzyme, and will be H
OH
hydrolysed. Then the reactive group (green) will react EDC, THF, HOBt, rt, 18hr
HN
O
O
N Fmoc-Cl, DIPEA
CH2Cl2, rt, 8hr
with a nucleophile on the enzyme to give covalent 4-hydroxymandelic acid, 87% O
O
OH
modification. Finally, the alkyne handle (blue) will be H
OH OSiEt3 Figure 1. ESI-MS of ABP
H
reacted with a reporter tag (pink) to detect the “trapped” HN
O
O
N TES-Cl, DIPEA
CH2Cl2, rt 4 hr, 52% HN
O
O
N
enzyme.. The mechanism of our ABP is shown in
Future Plans
O O
O OFmoc O OFmoc
Scheme 1. H
OSiEt3
O (CH2)14CH3 APT
NEt3, Pyridine HN
O
O
N COCl2, CH2Cl2 • Scale up the synthesis to generate enough ABP for
O
O
H
N
F
O
rt, 2 hr, 85% O
O
OH
DIPEA, 0 oC, 1 hr use in assays
N
H
O
O
O
OSiEt3 O
• Demonstrate reaction of APTs with our ABP through
enzymatic
hydrolysis
O N CO2Me
HN
O
O
H
N
O
H
N
O DIPEA, CH2Cl2
kinetic assays and mass spectrometry
O F O
O
O Cl
+
HO rt, 0.5 hr, 59% • React the ABP in cell lysates to investigate how APT
H
N
O
O
N
O
O
OSiEt3
activity differs between normal vs. abnormal cells.
H H
O O N OH
O N CO2Me HN O O CH2Cl2, CH3(CH2)14COCl
cyclization O O
References
elimination O O N DIPEA, rt, 2 hr
O
O F O (CH2)14CH3
H OSiEt3
O N H
O
• Greaves, J.; Chamberlain, L. H., J. Cell Biol. 2007,
N O + O N O
H CO2Me HN O O
O TBAF, THF, rt, 1 hr
O O N O O
O O N
O
176, 249–254.
1,6-elimination
Nu APT • Valeur, E.; Bradley, M., Chem. Soc. Rev. 2009, 38,
O
O
H
N H
OH
O (CH2)14CH3
606–631.
N
H
O
O
HN
O
O
N
O
O DAST, CH2Cl2 • Lenger, J.; Schröder, M.; Ennemann, E. C.; Müller,
O
B.; Wong, C.-H.; Noll, T.; Dierks, T.; Hanson, S. R.;
O O -78 oC, 1 hr
O O N
nucleophilic
O
addition
Nu APT
O (CH2)14CH3 Sewald, N., Bioorg. Med. Chem. 2012, 20, 622–627.
O F
H H
O N O N O
N O HN O O
H
O O O
O O O N
click O
X-N3
Acknowledgements
chemistry
Nu APT
N N O
H
X N O N
N O
H
O
Scheme 2. Synthesis of ABP for APTs • Rachel H. Bisiewicz
O X=fluorescent marker or biotin
• Dr. Russell Hopson
• Koki Nishimura • Dr. Tun-Li Shen
• Stephanie M. London
Scheme 1. Mechanism of reaction of ABP