Chemical synthesis of a novel coenzyme A-based affinity probe

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Chemical synthesis of a novel coenzyme A-based affinity probe
TCNP Protocol – Cole Lab
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A Selective Chemical Probe for Coenzyme-A Requiring Enzymes
Coenzyme A-requiring enzyme
A novel coenzyme A-based affinity probe is developed using a sulfoxycarbamate
functionality. This probe can selectively identify several acetyltransferases
relative to other enzymes and proteins and leaves behind a biotin tag which can
be used for western blotting and mass spectrometric characterization.
A selective chemical probe for coenzyme A-requiring enzymes.
Hwang Y, Thompson PR, Wang L, Jiang L, Kelleher NL, Cole PA.
Angew Chem Int Ed Engl. 2007;46(40):7621-7624.
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1. Synthesis of Chemical Probe
b, c, d
e, f
-O P O
-O P O
Figure 1. Synthesis of CoA-desthiobiotin-thiocarbamate sulfoxide, 2.a) Ethylchlorothiol formate, 0 C, aqueous
NaOH/diethylether; b) 6 N HCl, MeOH, RT; c) TSTU, desthiobiotin, RT, dioxane/H2O/DMF; d) Oxone, RT, MeOH/H2O; e)
CoASH, LiOH, MeOH/H2O; f) Oxone, 0 C, MeOH/H2O then reverse-phase clolumn chromatography. CoA=coenzyme A,
TSTU= N,N,N’,N’-tetramethyl-O-(N-succinimidyl) uranium tetrafluoroborate
[3-(Ethylsulfanylcarbonyl-methyl-amino)-propyl]-carbamic acid tert-butyl ester
A solution of ethyl chlorothiolformate (1.0 g, 8.0 mmol) in diethyl ether (4 ml) was added
dropwise to (3-methylamino-propyl)-carbamic acid tert-butyl ester12 (3 , 1.51 g, 8.0
mmol) in a mixture of diethyl ether (20 ml) and aqueous NaOH (8 ml, 1 M). After 30
min, addition of water (20 ml), followed by phase separation and washing the organic
phase with ddH2O, dilute hydrochloric acid (0.1 M), and ddH2O sequentially, drying over
anhydrous Na2SO4 and concentration lead to a clear liquid (2.18 g, 99% yield).
ES-TOF HRMS (M+Na+) calcd for C12H24N2O3NaS 299.1405, found m/z 299.1401.
H NMR (400MHz, CDCl3) _ 5.30 (s, 1H), 3.48 (m, 2H), 3.09 (m, 2H), 2.96 (s, 3H), 2.92
(q, J=7.6Hz, 2H), 1.69 (m, 2H), 1.44 (s, 9H), 1.29 (t, J=7.6Hz, 3H); 13C NMR (100MHz,
CDCl3) _ 169.17, 156.08, 79.00, 46.16, 37.12, 34.63, 28.43, 27.46, 24.79, 15.41.
Desthiobiotin thiocarbamate, 4
Desthiobiotin (214 mg, 1.0 mmol) was dissolved in a 1:1:1 mixture of
DMF/dioxane/ddH2O (6 ml). To the solution, diisopropylethylamine (523 µl, 3.0 mmol)
and N,N,N’,N’-tetramethyl-O-(N-succinimidyl) uranium tetrafluoroborate (TSTU, 380
mg, 1.26 mmol) were added.13 The mixture was stirred for 3.5 h at rt. Separately, the
thiocarbamate above was subjected to Boc hydrolysis in aqueous methanolic HCl (6 N
aqueous HCl : methanol=1/5 (v/v)) for 3 h at rt. After removing the solvent under
reduced pressure, the concentrate was dissolved in DMF (1.5 ml) and diisopropylamine
(300 µl) and added to the activated desthiobiotin mixture and stirred for additional 2 h at
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rt. The reaction mixture was concentrated, directly loaded onto a silica gel column and
eluted with methanol and methylene chloride (1:20 v/v) to give a foamy material (250
mg, 67% yield)
ES-TOF HRMS (M+H+) calcd for C17H33N4O3S 373.2273, found m/z 373.2267.
H NMR (400MHz, CDCl3) _ 6.82 (s, 1H), 6.04 (s, 1H), 5.49 (s, 1H), 3.84 (m, 1H), 3.69
(m, 1H), 3.48 (m, 2H), 3.20 (m, 2H), 2.67 (s, 3H), 2.89 (q, J=7.6Hz, 2H), 2.21 (t, 7.6Hz,
2H), 1.66 (m, 4H), 1.32-1.50 (m, 6H), 1.29 (t, J=7.6Hz, 3H), 1.12 (d, J=6.8Hz, 3H); 13C
NMR (CDCl3, 100MHz) _ 173.31, 172.97, 164.22, 56.05, 51.42, 46.17, 36.27, 35.67,
34.67, 29.52, 28.81, 26.87, 25.91, 25.38, 24.81, 15.68, 15.39.
Desthiobiotin sulfone, 5
A solution of Oxone® (369 mg) in ddH2O (3 ml) was added to a cooled solution of sulfide
(110 mg, 0.3 mmol) in methanol (3 ml). The resulting cloudy slurry was stirred for 4 h at
rt. The reaction mixture was diluted with ddH2O (40 ml) and extracted 3 times with
chloroform (20 ml). The combined extracts were washed with ddH2O and brine, dried
over anhydrous Na2SO4, and concentrated under reduced pressure to give a clear oil (64.3
mg, 83% yield).
ES-TOF HRMS (M+H+) calcd for C17H33N4O5S 405.2172, found m/z 405.2160.
H NMR (400MHz, CDCl3) _ 6.75 (t, J=5.6Hz, 1H), 6.65 (t, J=6.0Hz, 1H), 6.08 (s, 1H),
5.94 (s, 1H), 5.34 (s, 1H), 5.32 (s, 1H), 3.84 (m, 2H), 3.77 (t, J=7.2Hz, 2H), 3.69 (m, 2H),
3.46 (t, J=7.2Hz, 2H), 3.38 (q, J= 7.2Hz, 4H), 3.35 (s, 3H), 3.26 (m, 4H), 3.02 (s, 3H),
2.19 (m, 4H), 1.91 (m, 2H), 1.82 (m, 2H), 1.65 (m, 4H), 1.43 (t, J=7.2Hz, 6H), 1.26-1.51
(m, 12H), 1.12 (d, J=6.8Hz, 6H); 13C NMR (100MHz, CDCl3) _ 173.53, 173.39, 164.16,
164.10, 161.17, 160.75, 56.01(2), 51.39 (2), 47.91, 46.36, 46.11, 46.08, 36.09, 36.04,
35.99, 35.72, 34.74, 34.27, 29.51, 29.47, 28.70, 28.67, 27.43, 26.32, 25.86, 25.81, 25.28,
25.22, 15.71 (2), 6.81, 6.77.
CoASH (10 mM 700 µl in methanol), desthiobiotin sulfone 4 (200 mM, 350 µl in
methanol) and LiOH (100 mM, 140 µl in ddH2O) were mixed at rt. After 2 h, the mixture
was precipitated by adding diethyl ether and pelleted by centrifugation. The precipitate
was dissolved in methanol and then precipitated again with diethyl ether, and this
procedure was repeated three times to remove any residual biotin-sulfone. Finally the
precipitate was dissolved in ddH2O and checked for its concentration and purity by
analytical reversed C-18 HPLC (buffer A: KH2PO4 50 mM pH 4.5, buffer B:
acetonitrile/water=4/1, a gradient elution from 0% B to 60% B for 30min with a flow rate
of 1ml/min monitoring UV absorbance at 260 nm, retention time 19.0 min, 96% purity
based on peak area)
MALDI HRMS (M+H+) calcd for C36H63N11O19P3S 1078.3230, found m/z 1078.3172.
H NMR (400MHz, D2O) _ 8.37 (s, 1H), 8.06 (s, 1H), 5.97 (d, J=7.2Hz, 1H), 4.65 (m,
1H), 4.60 (m, 1H), 4.39 (m, 1H), 4.04 (m, 2H), 3.83 (s, 1H), 3.66 (m, 2H), 3.54 (m, 1H),
3.35 (dd, J=4.8, 9.6Hz, 1H), 3.26 (t, J=7.6Hz,), 3.17-3.21 (m, 2H), 3.16 (t, J=6.4Hz), 2.97
(m, 2H), 2.75 (m, 5H), 2.25 (t, J=6.4Hz, 2H), 2.03 (t, J=7.2Hz, 2H), 1.61 (m, 1H), 1.53
(m, 1H), 1.39 (m, 2H), 1.26 (m, 2H), 1.11 (m, 4H), 0.88 (d, J=6.4Hz, 3H), 0.70 (s, 3H),
0.55 (s, 3H); 31P NMR (160 MHz) _ 1.65 (s), -9.71 (d, J=21.0Hz), -10.27 (d, J=21.0Hz).
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CoA-desthiobiotin-thiocarbamate-sulfoxide, 2
To an ice-bath cooled aqueous solution of the above biotin-CoA-thiocarbamate (700 µl,
10 mM) was added Oxone® (466 µl, 50 mM aqueous solution). The reaction mixture was
incubated at rt for 20 min and after dilution with ddH2O (2 ml) was injected into
preparative-reversed phase HPLC column (buffer A: KH2PO4 50 mM, pH 4.5, buffer B:
acetonitrile/ddH2O=4/1, a gradient elution profile: 0% buffer B at 0 min, 0% buffer B at 5
min, 60% buffer B at 35 min, flow rate 10 ml/min, UV absorbance monitored at 260 nm).
The fractions eluted at about 24 min were collected, lyophilized and dissolved in ddH2O.
The KH2PO4 salt was precipitated by adding the same volume of acetone and removed by
centrifugation. After the acetone was removed under reduced pressure, the aqueous
solution of the product was lyophilized and dissolved in ddH2O to determine its
concentration by UV absorbance at 260 nm and its purity by analytical HPLC (the same
method used for the starting material; total 6 mL of 865 µM in 92% purity based on
HPLC peak area).
MALDI HRMS (M-H+2K+) calcd for C36H61N11O20P3SK2 1170.2316, found m/z
H NMR (400MHz, D2O) _ 8.38 (s, 1H), 8.15 (s, 1H), 5.95 (d, J=5.6Hz, 1H), 4.62 (m,
1H), 4.62 (m, 1H), 4.55 (m, 1H), 3.99 (m, 2H), 3.74 (s, 1H), 3.58 (m, 2H), 3.47 (m, 1H),
3.18-3.35 (m, 7H), 3.06 (m, 2H), 2.94 (m, 2H), 2.74 (s, 3H), 2.23 (t, J=7.4Hz, 2H), 1.96
(t, J=7.4Hz, 2H), 1.57 (m, 2H), 1.32 (m, 2H), 1.20 (m, 2H), 1.05 (m, 4H), 0.081 (d,
J=6.4Hz, 3H), 0.64 (s, 3H), 0.52 (s, 3H); 31P NMR(160MHz) _ 1.06 (s), -9.76 (d, J=21.1
Hz), -10.33 (d, J=21.1Hz).
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2. Protein Labeling
Figure 2. A) In vitro labeling experiments on non-CoA requiring proteins with 2 (25 µM). B) In vitro labeling experiments on
CoA-requiring proteins with 2 (25 µM) and competition by actCoA (3 mM). C) In vitro labeling of cell lysates with 2 (50
µM). The lysates prepared from HeLa nucleus were either spiked with recombinant p300-HAT (approximately 1/50) or not.
D) Labeled cell lysates were probed by Western blotting with anti-Hat1 antibody after streptavidin-agarose purification
Preparation of HeLa nuclear extract.14
HeLa (human cervical adenocarcinoma) cells were grown to 80% confluency in DMEM
supplemented with fetal bovine serum (10%), penicillin (100 µg/µl) and streptomycin
(100 µg/µl). Cells were harvested, Dounce-homogenized in lysis buffer (10 mM KCl, 10
mM HEPES pH 7.9, 0.5 mM DTT, 1.5 mM MgCl2) and centrifuged 5 min at 10,000 x g,
to remove the cytoplasmic fraction. The crude nuclei pellet was resuspended in extract
buffer (25% glycerol, 0.5 mM PMSF, 0.5 mM DTT, 1.5 mM MgCl2, 20 mM HEPES, 0.2
mM EDTA) and shaken gently for 30 min. After centrifugation at 20,000 x g for 5 min,
the supernatant was transferred and dialyzed against dialysis buffer (0.5 mM PMSF, 0.1
M KCl, 0.2 mM EDTA, 0.5 mM EDTA, 20% glycerol, 20 mM HEPES pH 7.9).
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Procedure for labeling recombinant proteins with CoA-desthiobiotin-thiocarbamatesulfoxide, 2
Purified proteins (10 µl of 0.5µg/µl) were treated with 25 µM 2 (865 µM stock in ddH2O)
either with or without 3 mM actCoA in assay buffer (50 mM HEPES pH 8.0, 100 mM
NaCl, DTT 0.5 mM) at 30 oC for 1 h. The assay was then quenched with a standard
5XSDS-PAGE loading buffer (reducing). Proteins were separated by SDS-PAGE and
transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% BSA
in TBS with 0.1 % Tween 20 (TBST) at rt, followed by 1 h incubation with HRPstreptavidin in TBST. After four washes with changes every 15 min in TBST, the
biotinylated proteins were visualized by enhanced chemiluminescence.
Procedures for labeling HeLa nuclear extracts with 2
Protein mixtures (70 µl of 1.60 µg/µl, either HeLa nuclear extract or HeLa nuclear extract
spiked with p300-HAT (1/50 by weight) were treated with 50 µM CoA-desthiabiotinthiocarbamate sulfoxide, 2 and the rest was done as described in the above section.
Procedures for probing the labeled lysates by Western blotting after streptavidin-agarose
affinity purification.
The labeled lysates (20 µl of 1.9 µg/µl HeLa nuclear extract was treated with 100 µM 2
either in the presence of 5 mM acetonyl CoA or not for 1 hr at 30 oC) were treated with
100% trichloroacetic acid (final concentration 10 %) , incubated on ice for 30 min then
centrifuged at 14K rpm for 15 min. After removing the supernatant, the pellet was
washed with cold acetone and dried at rt in the fume hood for 20 min. The dried pellet
was dissolved in TBS-T (0.2 % SDS) heating at 65 oC for 15 min and incubated with
stereptavidin-agarose for 1 hr at rt. The supernatant was removed by centrifugation at 7K
rpm for 5 min and the remaining beads were washed three times with TBS-T (0.1%
tween, 10 times volume). Finally, the biotinylated proteins on beads were eluted boiling
in 2xsample buffer for 5 min. The eluted proteins were separated on SDS-PAGE gel and
transferred to nitrocellulose and the membrane was blocked with 5% nonfat milk in
TBST for 1 hr at 5 oC and then incubated with anti-Hat1 antibody in blocking buffer for
1.5 hr After three washes for 20 min in TBST, the membrane was incubated with antigoat antibody for 1h at rt, and washed with TBST three more times. The captured Hat1
protein was visualized by chemiluminescence.
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3. Trypsin digestion, purification and detection of biotinylated peptides
Figure 3. A) MALDI-MS analysis of the streptavidin-enriched peptides from p300-HAT and yESA1 after labeling with 2
and tryptic digestion. B) Labeled peptides and confirmation of labeling site from p300-HAT by MS/MS analysis. Upward
and downward slashes represent the observed c and z’ ions, respectively. All the c or z ions in red are modified while all
the c or z ions in blue are not modified, which leads to the localization of biotinylation to residues colored in yellow
Trypsinization and streptavidin enrichment
The labeled proteins (1 µM of p300-HAT, yESAI, were treated with 25 µ M CoAdesthiobiotin sulfoxide 2 as described above) were dialyzed against TBS to remove 2.
The dialysate was digested overnight at 37 oC with trypsin, then incubated with
streptavidin-agarose beads for 1 h at rt. The beads were then washed 3 times with 10
volumes of low salt wash buffer TBS (50 mM Tris pH 7.4, 150 mM NaCl) and 3 times
with high salt wash buffer (50 mM Tris pH 7.4, 500 mM NaCl) and finally with 10
volumes of ddH2O (twice). After washing, the beads were eluted with 30% aqueous
acetonitrile with 0.5% trifluoroacetic acid (TFA). The eluted peptides were partially dried
by vacuum centrifugation and analyzed by either MALDI-TOF or LC-MS/MS.
Peptide samples were combined with the MALDI matrix (α-cyano-4 hydroxycinnamic
acid, 20% acetonitrile, 0.1% TFA in water). Spectra were acquired on a Perseptive
Biosystems Voyager-DE Pro in the reflector mode.
ESI-Q-FTMS/MS and Data Analysis
Peptide samples were fractionated in a linear gradient of 30-70% CH3CN in 0.1% TFA
over 1 h at 1 mL/min using a Jupiter C4 reverse-phase column (4.6 _ 150 mm). Fractions
were lyophilized and then resuspended in 50 _L of electrospray solvent (49% CH3CN,
50% water, 1% formic acid) for mass spectrometry analysis. The peptides were directly
introduced into a custom built quadrupole-FTMS instrument using an Advion Nanomate
100 for automated nanospray. The ions were externally accumulated for a total of 1 s in
an accumulation octapole and shuttled to the ICR cell through a quadrupole that can
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function either as a simple ion guide or as a selective m/z filter.15 The targeted species
were isolated by Stored Waveform Inverse Fourier Transform (SWIFT)16 and fragmented
by Electron Capture Dissociation (ECD).17 Collected data were analyzed using
THRASH18 producing sets of intact mass and fragment ion peak list, which were
uploaded onto the ProSight PTM web server for single protein mode search
(prosightptm.scs.uiuc.edu).19 The mass difference between the observed ion and the
theoretical ion indicates modifications on the observed ions.
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