Supplementary Information
Supplementary Experimental Procedures
Mutagenesis analysis
Cysteines were mutated to appropriated amino acids (C71S, C223T, C240L and
C421S)
according
to
Soft
Intolerant
to
Tolerant
(SIFT)
prediction
(http://sift.bii.a-star.edu.sg/) and PolyPhen-2 sequence and structural features to retain
PDK1 activity maximally for following enzymatic assays. Point mutation was
generated using Site-directed Gene Mutagenesis Kit (SBS Genetech, Beijing, China)
following manufacture's instruction to generate PDK1 C71S, C223T, C240L and
C421S mutants. These mutants were expressed and purified the same as wide type
PDK1 as mentioned above.
siRNA transfection
PDK1
siRNAs
(PDK1-#1:AGUCGCAUUUCAAUUAGAA,
siPDK1-#2:
CUACAUGAGUCGCAUUUCA) were purchased from Ribobio (China). Cells were
transfected with PDK siRNAs or scramble siRNA as control using Lipofectamine™
RNAimax (Invitrogen). The knockdown efficacy was measured 72 h after the
transfection using immunoblotting.
Plasmid transfection
Cells were transfected with PDK1 WT, C240L mutant or empty vector as control
using Lipofectamine™ 2000 (Invitrogen). When transfected to PDK1 depleted cells,
the PDK1 plasmid was made synonymous mutations to resist the interference. The
expression efficacy was measured 48 h after the transfection using immunoblotting.
Mass spectrometry analysis
To map the modification site of JX06, PDK1 protein was incubated with the
JX06 for 2 h at 4 ℃. Then, it was further separated with the SDS-PAGE and trypsin
digested in-gel. Resulted tryptic peptides were then subjected to the Nano-LC/MS/MS.
Briefly, the lyophilized tryptic peptides were dissolved in 4µL of loading buffer (0.1%
formic acid and 2% acetonitrile) and injected into a trap column (75µm inner diameter
x2cm, packed with, 5 µm particle size, 100 Å pore size, Dikma Technologies Inc.,
Lake Forest, CA) through the auto-sampler at a maximum pressure of 250 bar in
100% buffer A (0.1% formic acid in HPLC grade water). After loading and washing,
the peptides were transferred to an analytical column (10 cm length with 75 μm ID,
packed with C18 resin, 3 μm particle size, 90 Å pore size, Dikma Technologies Inc.,
Lake Forest, CA) and separated using a 70-min gradient from 2 to 35% of buffer B
(0.1% formic acid in acetonitrile) at 300 nl/min flow rate on the EASY-nLC 1000
HPLC system (Thermo Fisher Scientific Inc., Waltham, MA). The eluted peptides
were ionized and introduced into an Q-Exactive mass spectrometer (Thermo Fisher
Scientific Inc., Waltham, MA), which was operated in data-dependent mode,
automatically switching between MS and MS2. Survey full-scan MS spectra (from
m/z 300 to 2,000) were acquired in the Orbitrap with resolution R = 24,000 at m/z
400. Internal calibration was performed using the ion signal (Si(CH3)2O)6H+ at
m/z445.120025 present in ambient laboratory air. The ten most intense ions were
sequentially isolated by the quadrapole and subjected to higher-energy collisionally
induced dissociation (HCD) with a normalized energy of 28%. The exclusion duration
for the data-dependent scan was 30 sec, the repeat count was 2, and the exclusion
window was set at +1 Da and −1 Da.
All the acquired MS data were analyzed by Mascot (v2.3, Matrix Science Ltd.,
London, UK). Peak lists were generated by Proteome Discoverer software (version
1.4) from Thermo Fisher. Search parameters were as follows: variable modification of
M oxidation, trypsin as proteolytic enzyme with up to two miss cleavages, precursor
ion mass tolerance of 10 ppm, fragment ion mass tolerance of 0.01Da. In addition, we
specified the cysteine plus 118.986341 or 160.996965 as variable modifications to
identify the modification sites of the compounds.
Animal studies
Male BALB/c-nude mice were inoculated subcutaneously with 5×106 A549 or
5×106 HT-29 cells in right forearm and treatment was started when tumor volume
reached 100 mm3. Mice grouped into 6 mice per group were intraperitoneally treated
with saline or JX06 at a dose of 80 or 40 mg/kg daily. Tumor growth was monitored
by measurement of tumor size slide caliper every other day. Mice were sacrificed and
analyzed after last dosing. All procedures were performed in accordance with the
National Institutes of Health Guidelines for the Care and Use of Laboratory Animals
and European Union directives and guidelines and were approved by the local ethics
committees.
Molecular dynamic simulation
Molecular dynamic simulation was performed on the open conformation structure of
PDK1 (PDB entry: 2Q8F). Missing residues were complemented by Discovery Studio
2.5 (Accelrys Software Inc., San Diego, CA). Gromacs software package version
4.5.3 and the AMBER03 force field with SPC/E water were used1, 2, 3. The water box
was 10 Å from the protein in all six directions. The system was neutralized by adding
Na+ and Cl-. Hydrogen bonds were constrained using the linear constraint solver
(LINCS) algorithm11 and the particle-mesh Ewald method was employed to
accommodate long-range electrostatic interactions
4, 5
. To avoid edge effects, the
periodic boundary conditions were applied. The system was subjected to energy
minimization using the steepest-descents algorithm and then heated to 300 K
6, 7
. The
water, ions and protein are separately coupled to temperature reservoirs of 300K using
Berendsen thermostat method16 with a coupling time of 1ps 8. Firstly, a 100ps
simulation was performed to heat the solvent molecules and ions. Secondly, 50ps and
20ps simulations were carried out to heat all the atoms with the restriction of main
chain and protein Cα atoms, respectively. Finally, the conventional 200ns MD
simulation was performed. The coordinates were saved every 10 ps.
Chemical synthesis
The reagents (chemicals) were purchased from Alfa Aesar, Acros, and Titan Chemical
Co. and used without further purification. Analytical thin-layer chromatography (TLC)
was HSGF 254 (150-200 µm thickness, Yantai Huiyou Company, China). Yields were
not optimized. Melting points were measured in capillary tube on a SGW X-4 melting
point apparatus without correction. Nuclear magnetic resonance (NMR) spectroscopy
was performed on a Bruker AMX-400 NMR (IS as TMS). Chemical shifts were
reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton
coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). Low- and high-resolution mass spectra (LRMS and
HRMS) were given with electric and electrospray ionization (EI and ESI) produced
by a Finnigan MAT-95 and LCQ-DECA spectrometer. Generally, LC-MS was
recorded on Agilent 1200 HPLC/6110 SQ system by the following conditions:
Method A:
Mobile Phase: A: Water (10 mM NH4HCO3), B: ACN. Gradient: 5% B increase
to 95% B within 1.4 min, 95% B for 1.6 min, back to 5% B within 0.01min. Flow rate:
1.8 mL/min. Column: XBridge C18, 4.6 x 50 mm, 3.5 μm. Column temperature:
50 °C.
Method B:
Mobile Phase: A: Water (0.01% TFA), B: ACN (0.01% TFA). Gradient: 5% B
increase to 95% B within 1.2 min, 95% B for 1.3 min, back to 5% B within 0.01min.
Flow rate: 2.0 mL/min. Column: XBridge C18, 4.6 x 50 mm, 3.5 μm. Column
temperature: 50 °C.
Synthesis and Spectroscopic Data of JX01-JX12 and JX86
General Synthesis of Bis(dialkylaminethiocarbonyl)disulfide derivatives (Scheme 1)[1]
Scheme 1
S
R
S
KOH
NH + CS2
R
Water
N
S
K+
NaNO2/c. HCl
Water
R
R
N
S
N
S
R
R
S
JX01, JX05-JX08, JX86
A mixture of secondary amine (50 mmol), carbon disulfide (51 mmol), potassium
hydroxide (110 mmol), and water (250 mL) was heated (50 °C) under stirring for 12
hours. The mixture was cooled to 25 °C, to this solution was added 3 g of sodium
nitrite followed by 3 mL of methanol, and under cooling (0 °C) and stirring
concentrated HCl (10 mL) was added dropwise. After stirred for 5mins, the desired
compound was extracted into the organic phase using EA (2 x 100 mL). The
combined organic layer was dried over Na2SO4, filtered, concentrated in vacuo. Then
the residue was purified by flash chromatography (EA/PE) on silica to obtain the
products.
Bis(N,N-dimethyl thiocarbamoyl)-disulfide (JX01, Thiram)
S
N
S
S
N
S
Light yellow powder, mp: 156–167 °C, yield: 45%. 1H NMR (400 MHz CDCl3) δ
3.64 (s, 6H), 3.61(s, 6H); HRMS (EI) m/z calcd for C6H12N2S4 (M+) 239.9883, found
239.9886, 88.0213 (100%); Purity: 100% (214 nm, RT=1.74 min, Method A).
Bis(N,N-diethyl thiocarbamoyl)-disulfide (JX05)
S
N
S
S
N
S
Light yellow powder, mp: 69–70 °C, yield: 55%. 1H NMR (400 MHz CDCl3) δ 4.03
(q, J = 6.8 Hz, 8H), 1.48 (t, J = 6.8 Hz, 6H), 1.31 (t, J = 6.8 Hz, 6H); HRMS (EI) m/z
calcd for C10H20N2S4 (M+), 296.0509, found 296.0513, 149.0316 (100%); Purity:
100% (214 nm, RT=2.13 min, Method A).
Bis(1-pyrrolidinyl thiocarbonyl)-disulfide (JX07)
S
N
S
S
N
S
White powder, mp: 125 °C, yield: 60%. 1H NMR (400 MHz CDCl3) δ 4.00-3.95 (m,
8H), 2.18-2.15 (m, 4H), 2.06-2.01 (m, 4H); HRMS (EI) m/z calcd for C10H16N2S4
(M+), 292.0196, found 292.0201, 114.0370 (100%); Purity: 91% (214 nm, RT=1.90
min, Method A).
Bis(1-piperidinyl thiocarbonyl)-disulfide (JX08)
S
N
S
S
N
S
Light yellow powder, mp: 121-222 °C, yield: 60%. 1H NMR (400 MHz CDCl3) δ 4.24
(br s, 8H), 1.77 (s, 12H); HRMS (EI) m/z calcd for C12H20N2S4 (M+), 320.0509, found
320.0524, 128.0522 (100%); Purity: 96% (214 nm, RT=2.16 min, Method A).
Bis(4-morpholinyl thiocarbonyl)-disulfide (JX06)
S
N
O
S
O
S
N
S
Light yellow powder, mp: 143-145 °C, yield: 75%. 1H NMR (400 MHz CDCl3) δ 4.31
(br s, 8H), 3.85 (t, J = 5.0 Hz, 8H); HRMS (EI) m/z calcd for C10H16N2O2S4 (M+),
324.0095, found 324.0099, 130.0319 (100%); Purity: 100% (214 nm, RT=1.73 min,
Method A).
Bis(2-((p-tolyloxy)methyl)morpholineyl thiocarbonyl)-disulfide (JX86)
Yellow oil, yield: 65%. 1H NMR (400 MHz CDCl3) δ 7.10-7.08 (d, J = 8.0 Hz, 4H),
6.84-6.82 (d, J = 8.0 Hz, 4H), 5.64-4.62 (m, 4H), 4.09-3.88 (m, 8H), 3.88-3.75 (m,
2H), 3.69-3.35 (m, 4H), 2.29(s, 6H); HRMS (ESI) m/z calcd for C10H16N2S4Na
[M+Na]+, 587.1134, found 587.1147. Purity: 97% (254 nm, RT=7.13 min). The purity
of compound JX86 was determined by Agilent 1100, and checked with a UV/vis
detector setting λ = 254 nm. Compound was separated by using a reverse phase C18
analytical column (Zorbax Eclipse Plus, 4.6×150 mm, 5 μm particle size).
Sodium morpholine-4-carbodithioate (JX12)
S
N
O
S Na+
To a stirred solution of morpholine (1.00 g, 11.5 mmol) in acetonitrile (50 mL),
carbon disulfide (0.874 g, 11.5 mmol) and sodium hydroxide (50% aqueous solution,
11.5 mmol) was added below 5 °C. After stirring for 6 hours, the solution was left to
evaporate at room temperature. The crude product was wash with acetonitrile to give
JX12 as white solid (0.8 g, 38%).
Mp: 350 °C . 1H NMR (400 MHz DMSO-d6) δ 430 (t, J = 4.8 Hz, 4H), 3.48 (t, J = 4.8
Hz, 4H); HRMS (ESI) m/z calcd for C5H8NS2ONa2 [M+Na]+, 207.9837, found
207.9850.
Scheme 2 depicts the synthetic route for the preparation of compounds JX02–JX04
and JX09.
Scheme 2
S
I
N
ACN
S
S
CS2
NH
TEA
N
Cl
S
S
I
S
S
N
JX02
N
S
NH
N
ACN
S
N
H2O
N
S
N
S
S
JX04-a
S
JX09
N
S
S
O
N
ACN
Br2
JX03
O
Cl
O
S
N
P4S10/Al2O3
N
N
S
S
JX04
Methylene bis(dimethylcarbamodithioate) (JX02)
S
N
S
S
S
N
A mixture of dimethylamine (0.1mol in methanol), carbon disulfide (9.2 g, 0.12 mol)
and triethylamine (30.3 g, 0.3 mol) in 100 mL acetonitrile was stirred at room
temperature for 16 h, the reaction mixture was concentrated in vacuo. The residue was
washed thoroughly with diethyl ether and dried under reduced pressure to furnish the
dithiocarbamic acid salt (15 g, 68%) as white solid, which was used in next step
without further purification.
To the dithiocarbamic acid salt (488 mg, 2.2 mmol) in acetonitrile (20 mL) was added
diiodomethane (267 mg, 1 mmol), and the mixture was stirred at room temperature
overnight. The solvent was evaporated under reduced pressure, and the residue was
diluted with ethyl acetate (150 mL) and washed with water, brine, and dried over
Na2SO4, filtered, concentrated in vacuo. Then the residue was purified by flash
chromatography on silica with ethyl acetate-hexane (1/4, v/v) to give JX02 (0.42 g).
Light yellow powder, mp: 150-151 °C, yield: 75%. 1H NMR (400 MHz CDCl3) δ 5.37
(s, 2H), 3.56 (s, 6H), 3.35 (s, 6H); HRMS (EI) m/z calcd for C7H14N2S4 (M+),
254.0040, found 254.0039, 165.9810 (100%); Purity: 96% (214 nm, RT=1.92 min,
Method A).
Bis(dimethylamino thiocarbonyl)-monosulfide (JX03)
S
N
S
S
N
To the dithiocarbamic acid salt (488 mg, 2.2 mmol) in acetonitrile (20 mL) was added
dimethylcarbamothioic chloride (272 mg, 2.2 mmol) and the mixture was stirred at
room temperature overnight. The solvent was evaporated under reduced pressure, and
the residue was diluted with ethyl acetate (150 mL) and washed with water, brine, and
dried over Na2SO4, filtered, concentrated in vacuo. Then the residue was purified by
flash chromatography on silica with ethyl acetate-hexane (1/3, v/v) to give JX03 (0.36
g).
Light yellow powder, mp: 106-107 °C, yield: 80%. 1H NMR (400 MHz CDCl3) δ 3.53
(s, 6H), 3.44 (s, 6H); HRMS (EI) m/z calcd for C6H12N2S3 (M+), 208.0163, found
208.0164, 88.0217 (100%); Purity: 100% (214 nm, RT=1.66 min, Method A).
Dimethylthiocarbamoyl dimethylcarbamoyl disulfide (JX09)
O
N
S
S
N
S
To a solution of JX03 (2 g , 9.6 mmol) in 100 mL of toluene was added bromine (3.84
g, 24 mmol) in carbon tetrachloride (10 mL) at 25 °C over 5 mins. After 4 hours
stirring, the reaction mixture was filtered to give orange powder which was suspended
in ice-water. The suspension was stirred at 0 °C until all the orange solid had reacted
and a lemon-yellow solution formed with colorless precipitation. The solid was
collected and wash with EA to afford JX09 (0.36 g).
White powder, mp: 95 °C, yield: 17%. 1H NMR (400 MHz CDCl3) δ 3.60 (s, 3H),
3.57 (s, 3H), 3.23 (s, 3H), 3.07 (s, 3H); HRMS (EI) m/z calcd for C6H12N2OS3 (M+),
224.0112, found 224.0114, 88.0217 (100%); Purity: 96% (214 nm, RT=1.57 min,
Method A).
(Dimethylthiocarbamoyl)methylene dimethylcarbamodithioate (JX04)
S
N
N
S
S
To the dithiocarbamic acid salt (488 mg, 2.2 mmol) in acetonitrile (20 mL) was added
2-chloro-N,N-dimethylacetamide (303 mg, 2.5 mmol) and the mixture was stirred at
room temperature overnight. The solvent was evaporated under reduced pressure, and
the residue was diluted with ethyl acetate (150 mL) and washed with water, brine, and
dried over Na2SO4, filtered, concentrated in vacuo. Then the residue was purified by
flash chromatography on silica with ethyl acetate-hexane (1/4, v/v) to give
(Dimethylcarbamoyl)methylene Dimethylcarbamodithioate (JX04-a, 0.27 g).
Light yellow powder, mp: 58 °C, yield: 60%. 1H NMR (400 MHz, CDCl3) δ 4.28 (s,
2H), 3.55 (s, 3H), 3.43 (s, 3H), 3.17 (s, 3H), 3.00 (s, 3H); HRMS (EI) m/z calcd for
C7H14N2OS2 (M+), 206.0548, found 206.0546, 88.0214 (100%); Purity: 96% (214 nm,
RT=1.28 min, Method B).
1 g of P4S10/Al2O3 (6 g of P4S10, 10 g of Al2O3) was suspended in a solution of the
JX1008 (515 mg, 2.5mmol) in 15 mL of 1,4-dioxane. The reaction was stirred under
reflux for 1 hour and filtered. The filtrate was poured onto ice (150 g), and the
resulting mixture was stirred for 30 mins, and then separated between EA and water,
the organic layer was washed with brine then dried over Na2SO4, filtered,
concentrated in vacuo. Then the residue was purified by flash chromatography on
silica with ethyl acetate-hexane (1/3, v/v) to give JX04 (0.28 g).
Light yellow powder, mp: 115-116 °C, yield: 50%. 1H NMR (400 MHz, CDCl3) δ
4.73 (s, 2H), 3.55 (s, 3H), 3.50 (s, 3H), 3.43 (s, 3H), 3.42 (s, 3H); HRMS (EI) m/z
calcd for C7H14N2S3(M+), 222.0319, found 222.0322, 189.0513 (100%); Purity: 100%
(214 nm, RT=1.71 min, Method A).
Bis(dimethyl carbamoyl) disulfide (JX10)
Compound JX10 were synthesized through the approach outlined in Scheme 3.
Scheme 3
O
S
N
S
S
N
S
NaClO
ACN/water
N
JX01
S
S
N
O
JX10
To a solution of JX01 (1 g, 4.2mmol) in acetonitrile(100 mL) was added 30 mL of
NaClO (5% in water), and the mixture was stirred at room temperature over night.
The solvent was evaporated under reduced pressure, and the residue was diluted with
ethyl acetate (250 mL) and washed with water, brine, and dried over Na2SO4, filtered,
concentrated in vacuo. Then the residue was purified by flash chromatography on
silica with ethyl acetate-hexane (1/6, v/v) to give JX10 (0.30 g).
Light yellow powder, mp: 70 °C, yield: 35%. 1H NMR (400 MHz, CDCl3) δ 3.15 (s,
3H), 3.06 (s, 3H); HRMS (EI) m/z calcd for C6H12N2O2S2 (M+), 208.0340, found
208.0339, 72.0439 (100%); Purity: 100% (214 nm, RT=1.71 min, Method B).
Isobutyric dithioperoxyanhydride (JX11)
Compound JX11 were synthesized through the approach outlined in Scheme 4.
Scheme 4
O
S, NaOH
Cl
toluene/PEG400
O
S
S
O
JX11
A three-neck flask was charged with sulfur (480 mg, 15 mmol), Sodium hydroxide
(400 mg, 10 mmol), PEG400 (5 mL), and toluene (100 mL), this suspension was
heated to reflux for 2 hours then cooled to 25 °C. After isobutyryl chloride (150 mg,
1.5 mmol) was added dropwised, the reaction was heated at 95 °C overnight. The
mixture was filtered and separated between EA and water, the organic layer was
washed with brine, dried over Na2SO4, filtered, concentrated in vacuo. Then the
residue was purified by flash chromatography on silica with ethyl acetate-hexane
(1/10, v/v) to give JX11 (0.1 g).
Liquid, yield: 32%. 1H NMR (400 MHz, CDCl3) δ 3.02-2.92 (m, 2H), 1.29 (d, J = 6.8
Hz, 12H); HRMS (EI) m/z calcd for C8H14O2S2 (M+), 206.0435, found 206.0436,
71.0487 (100%); Purity: 100% (214 nm, RT=1.85 min, Method B).
Synthesis and Spectroscopic Data of Biotin-JX06 and Its Intermediates
Scheme 1 depicts the sequence of reactions that led to the preparation of biotin-JX06
using 1,2-bis(2-aminoethoxy)ethane as the starting material.
Scheme 1
O
H2N
O
O
Boc2O
NH2
TEA/THF
H2N
O
O
N
H
HN
H
Boc D-biotin/EDCI/HOBt
DIPEA/DMF
NH
H
H
N
S
O
biotin-JX06-a
TFA
HN
H
HO
NH
N
H
N
S
O
O
O
.TFA
NH2
HN
H
Boc
/EDCI/HOBt
O
H
DIPEA/DMF
H
H
N
O
O
O
N
Boc
NH
H
H
N
O
O
O
O
O
N
H
N
NH
O
H
N
S
O
O
O
N
H
O
i) CS2/KOH
ii)NaNO2/HCl
S
N
H
N
O
S
biotin-JX06-e
S
S
S
H
HN
O
N
H
biotin-JX06-d
O
S
H
Boc
biotin-JX06-b
O
biotin-JX06-c
O
TFA
N
H
NH
S
HN
H
HN
H
O
O
O
O
O
H
NH
N
H
O
O
H
N
O
O
biotin-JX06
O
Tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (biotin-JX06-a)
H2N
O
O
N
H
Boc
Boc anhydride (4.37 g, 20 mmol) in THF (20 mL) was added dropwise to a solution
of 1,2-bis(2-aminoethoxy)ethane (18.1 g, 122 mmol) and TEA(4.0 g 40 mmol) in 100
mL of THF within 2 hours. After additional stirring for 5 hours, the reaction mixture
was concentrated in vacuo. The resulting yellow oil was separated between EA and
water, the organic layer was washed with brine, dried over Na2SO4, filtered,
concentrated in vacuo. Then the residue was purified by flash chromatography on
silica with ethyl acetate-hexane (1/1, v/v) to give biotin-JX06-a (15.1 g, 50%) as
colorless oil.
1
H NMR (400 MHz, CDCl3) δ 5.15 (br s, 1H), 3.61 (s, 4H), 3.54 (br s, 4H), 3.46 (s,
2H), 3.31(s, 2H), 2.88(s, 2H), 1.44(s, 9H).
Tert-butyl
(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentana
mido)ethoxy)ethoxy)ethyl)carbamate (biotin-JX06-b)
O
HN
H
NH
H
H
N
S
O
O
O
N
H
Boc
A three-neck flask was charged with biotin-JX06-a (2.48 g, 10 mmol), D-biotin (2.44
g 10 mmol), HOBt (2 g, 15 mmol), EDCI (2.86 g, 15 mmol), DIPEA (3.87 g, 30mmol)
and DMF (30 mL), this mixture was stirred at 25 °C over night. The reaction mixture
was diluted with 200 mL DCM and washed by water, brine, then dried over Na2SO4,
filtered, concentrated in vacuo. The residue was purified by flash chromatography on
silica with MeOH-DCM (1/20, v/v) to give biotin-JX06-b (3.5 g, 73%) as light
yellow solid.
1
H NMR (400 MHz, CDCl3) δ 6.54 (br s, 1H), 6.25 (br s, 1H), 4.54 (s, 1H), 4.35 (br s,
1H), 3.62 (s, 4H), 3.56 (s, 4H), 3.45 (s, 2H), 3.31 (s, 2H), 2.93-2.91 (m, 1H),
2.78-2.75 (m, 1H), 2.25 (s, 2H), 1.68 (br s, 4H), 1.44 (s, 11H).
N-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno
[3,4-d]imidazol-4-yl)pentanamide trifluoroacetate (biotin-JX06-c)
O
HN
H
NH
H
H
N
S
O
O
.TFA
NH2
O
In a round bottom flask, biotin-JX06-b (2.37 g, 5 mmol) was dissolved in 20 mL TFA,
stirred at 25 °C for 2 hours. The solvent was concentrated in vacuo to afford
quantitatively the crude product of biotin-JX06-c, which was used in next step
without further purification.
1
H NMR (400 MHz, CD3OD) δ 4.53-4.51 (m, 1H), 4.34-4.31 (m, 1H), 3.71 (t, J = 5.2
Hz, 2H), 3.67 (s, 6H), 3.57 (t, J = 5.8 Hz, 2H), 3.38 (t, J = 5.8 Hz, 2H), 3.24-3.19 (m,
1H), 3.13 (t, J = 5.0 Hz, 2H), 2.96-2.91 (m, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.23 (t, J =
7.4 Hz, 2H), 1.79-1.56 (m, 4H), 1.48-1.41 (m, 2 H).
Tert-butyl 2-((2-(2-(2-(5-((3aS,4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)
pentanamido)ethoxy)ethoxy)ethyl)carbamoyl)morpholine-4-carboxylate
(biotin-JX06-d)
O
HN
H
NH
H
H
N
S
O
O
O
O
N
H
O
N
Boc
A three-neck flask was charged with biotin-JX06-c (2.36 g, 5 mmol),
4-(tert-butoxycarbonyl)morpholine-2-carboxylic acid (1.16 g 5 mmol), HOBt (1.4 g,
10 mmol), EDCI (1.91 g, 10 mmol), DIPEA (3.87 g, 30mmol) and DMF (30 mL), this
mixture was stirred at 25 °C over night. The solution was diluted with 100 mL DCM
and washed by water, brine, then dried over Na2SO4, filtered, concentrated in vacuo.
The residue was purified by flash chromatography on silica with MeOH-DCM (1/10,
v/v) to give biotin-JX06-d (1.32 g, 45%) as light yellow oil.
1
H NMR (400 MHz, DMSO-d6) δ 7.84 (s, 1H), 7.76 (s, 1H), 6.43 (s, 1H), 6.37 (s, 1H),
4.30 (s, 1H), 4.13 (s, 1H), 4.01 (br s, 1H), 3.92-3.83 (m, 2H), 3.73-3.69 (m, 1H), 3.62
(br s, 2H), 3.49 (s, 4H), 3.45-3.38 (m, 4H), 3.28-3.07 (m, 6H), 2.92-2.79 (m, 2H),
2.59-2.56 (m, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.64-1.47 (m, 4H),1.41(s, 9H).
2-((2-(2-(2-(5-((3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)penta
namido)ethoxy)ethoxy)ethyl)carbamoyl)morpholine-4-carbothioicdithioperoxyanhy
dride (biotin-JX06)
O
HN
H
NH
H
H
N
S
O
O
O
O
O
N
H
N
S
S
S
S
N
O
S
H
HN
H
N
H
O
O
H
N
O
O
NH
O
In a round bottom flask, biotin-JX06-d (0.587 g, 1 mmol) was dissolved in 7 mL TFA,
stirred at 25 °C for 2 hours. The reaction solvent was was concentrated in vacuo to
afford the crude product of biotin-JX06-e (0.6 g), which was used in next step
without further purification.
A mixture of biotin-JX06-e (0.6 g, 1 mmol), carbon disulfide (0.23 g, 3 mmol),
potassium hydroxide (0.34 g, 6 mmol), and water (10 mL) was heated (50 °C) under
stirring over night. The mixture was cooled to 25 °C, to this solution was added 0.2 g
of sodium nitrite followed by 0.2 mL of methanol, and under cooling (0 °C) and
stirring concentrated HCl was added dropwise to adjust the pH value to 2. After
stirred for 5 mins, the desired compound was extracted into the organic phase using
DCM (3 x 50 mL). The combined organic layer was dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by flash chromatography on silica
with MeOH-DCM (1/10, v/v) to give biotin-JX06 (0.07 g, 12% over two steps) as
light yellow oil.
1
H NMR (400 MHz, DMSO-d6) δ 7.96 (br s, 2H), 7.85 (s, 2H), 6.43 (s, 2H) 6.37 (s,
2H), 5.27 (br s, 1H), 4.81 (br s, 2H), 4.36-3.96 (m, 8H), 3.80-3.54 (m, 5H), 3.50 (s,
8H), 3.47-3.36 (m, 10H), 3.27 (br s, 4H), 3.18 (br s, 4H), 3.09 (br s, 2H), 2.84-2.78 (m,
2H), 2.59-2.56 (m, 2H), 2.06 (t, J = 6.2 Hz, 4H), 1.67-1.20 (m, 12H); 13C NMR (100
MHz CD3OD) δ 195.3, 173.0, 170.5, 166.1, 76.1, 71.3, 71.3, 70.6, 70.4, 63.4, 61.5,
57.0, 41.1, 40.3, 39.9, 36.8, 29.8, 29.5, 26.9; HRMS (ESI) m/z calcd for
C44H72N10O12S6Na [M+Na]+ 1147.3547, found 1147.3569; Purity: 90% (214 nm,
RT=1.43 min, Method A).
Supplementary References
1.
Van der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC.
GROMACS: Fast, flexible, and free. J Comput Chem 26, 1701-1718 (2005).
2.
Yong D, et al. A point-charge force field for molecular mechanics simulations
of proteins based on condensed-phase quantum mechanical calculations. J
Comput Chem 24, 1999-2012 (2003).
3.
Berendsen HJC, Grigera JR, Straatsma TP. The missing term in effective pair
potentials. J Phys Chem 91, 6269-6271 (1987).
4.
Hess B, Bekker H, Berendsen HJC, Fraaije J. LINCS: A linear constraint
solver for molecular simulations. J Comput Chem 18, 1463-1472 (1997).
5.
Darden T, York D, Pedersen L. Particle mesh ewald - an n.log(n) method for
ewald sums in large systems. J Chem Phys 98, 10089-10092 (1993).
6.
Felippa CA. Variational-methods for the solution of problems of equilibrium
and vibrations. International Journal for Numerical Methods in Engineering
37, 2159-& (1994).
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Goheen H. Principles of numerical analysis. Econometrica 22, 540-541
(1954).
8.
Berendsen HJC, Postma JPM, Vangunsteren WF, Dinola A, Haak JR.
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3684-3690 (1984).
Supplementary Figures Legends
Supplementary Fig. S1. Thiram inhibits PDK activity in A549 cells. A549 cells
were treated with indicated compounds at effective dose suggested by the enzymatic
assay. PDK activity was indicated by PDHA1 phosphorylation measured by high
content analysis. Mean ± SE (n = 3), **P < 0.01.
Supplementary Fig. S2. JX06 exhibits no inhibitory effect on FAK at the cellular
level. A549 cells were treated with JX06 at 10 μM for indicated durations. FAK
activity was indicated by FAK and Src phosphorylation detected by immunoblotting.
Supplementary Fig. S3. PDK inhibition by JX06 in different cancer cells. Cells
were treated with JX06 at indicated concentrations for 24 h and PDHA1
phosphorylation was detected using immunoblotting.
Supplementary Fig. S4. The variable abundance of PDK1 in different cell lines.
The expression of PDK1 was detected using immunoblotting.
Supplementary Fig. S5. The variable growth dependency of different cell lines on
PDK1. Cell survival was measured for 72 h after PDK1 was depleted using two
siRNAs. Mean ± SE (n = 3)
Supplementary Fig. S6. Chemical structure of biotin-conjugated JX06 and
activity in PDK1 inhibition.
Supplementary Fig. S7. Cysteine 240 is critical for the enzymatic activity of
PDK1 in A549 cells. The proliferative rate of A549 cells as well as the
phosphorylation level of PDHA-1 was measured in PDK1 depleted cells after the
transfection of WT or C240L mutant, using empty vector as the control. Mean ± SE
(n = 3), **P < 0.01
Supplementary Fig. S8. Comparison of MMP level in four indicated cell lines
after JX06 treatment.
Supplementary Movie S1. A 200 ns molecular dynamic simulation of the open
conformation PDK1, in which the sidechain of R286 swung towards different
sides.