Spy vs. spy: selecting the best reporter for 19F NMR competition experiments

Characterization of a series of fluorinated compounds for competitive 19F NMR reveals the principles that can guide developing highly sensitive assays.


SUPPORTING INFORMATION
6 For describing the synthesis and characterisation of spy molecules and intermediates, compounds were numbered as shown in the two schemes below. The respective compound numbering in the main text is indicated where applicable (e.g. compound S3a corresponds to spy molecule 1 in the main text, and so on). Compounds S1a, S1c and S8a were purchased from commercial sources and used without further purification. The spectroscopic characterization and yields for intermediates S7b, S8b and S9b can be found elsewhere, as these were previously prepared by our group, [2] while all the remaining compounds were synthesized and characterized as described below. For the NMR characterization of compounds S3a-d and S9a-b, either a chloride or a formate salt was obtained depending on the purification strategy used.

General procedure i. Coupling of aryl bromides with 4-methylthiazole -Synthesis of S1b, S1d and S7b
The aryl bromide (1 equiv.) was dissolved in dimethylacetamide (3 mL per mmol of bromide), followed by the sequential addition of 4-methythiazole (2 equiv.), potassium acetate (2 equiv.) and palladium (II) acetate (0.02 equiv.). The reaction was stirred for approximately two hours at 150 °C under nitrogen atmosphere. The mixture was extracted with brine and dichloromethane. Combined organic phases were concentrated and DMA was removed in the vacuum pump. The desired product was then purified by flash column chromatography with an increasing elution of ethyl acetate (0-100%) in heptane.

General procedure ii. Reduction of nitriles to amines -Synthesis of S2a-d and S8b
To a stirring solution of the nitrile (1 equiv.) in THF under nitrogen atmosphere, was added a solution of LiAlH4 (1 equiv. from a 1M solution in THF). After an overnight period, the mixture was cooled down in an ice bath and diluted with diethyl ether. Water was then slowly added (1 μL per mg of LiAlH4 added), followed by the addition of 15% NaOH (1 μL per mg of LiAlH4 added) and once more water (3 μL per mg of LiAlH4 added). The ice bath was removed and the mixture. MgSO4 was added and after 15 additional minutes stirring the mixture was filtered, then extracted with HCl solution (pH ≈ 2). The organic phase was discarded and the pH of the aqueous phase was raised to 12 by adding NaOH 1 M. This solution was extracted three times with DCM and the organic phase was concentrated and purified (purification strategy specified for each compound).

(4-bromo-3-(trifluoromethyl)phenyl)methanaminium formate (S2a):
Prepared from 0.264 g (1.06 mmol) of the respective nitrile (S1a), resulting in 0.110 g (0.35 mmol, 35%) of the desired product as a white solid. Compound obtained as a formate salt after purification in reverse phase HPLC using a 5 to 95% gradient of formic acid 0.1% and acetonitrile. 1   Prepared from 0.221 g (0.82 mmol) of the respective nitrile (S1b), resulting in 0.092 g (0.29 mmol, 36%) of the desired product as a yellow solid. Compound obtained as a formate salt after purification in reverse phase HPLC using a 5 to 95% gradient of formic acid 0.1% and acetonitrile. 1   Prepared from 0.523 g (2.09 mmol) of the respective nitrile (S1c), resulting in 0.195 g (0.65 mmol, 31%) of the desired product as a white solid. Compound obtained as a formate salt after purification in reverse phase HPLC using a 5 to 95% gradient of formic acid 0.1% and acetonitrile. 1   Prepared from 0.398 g (1.49 mmol) of the respective nitrile (S1d), resulting in 0.157 g (0.49 mmol, 33%) of the desired product as a yellow solid. Compound obtained as a free amine after purification in reverse phase HPLC using a 5 to 95% gradient of ammonia 0.1% and acetonitrile. 1

(4-(4-methylthiazol-5-yl)phenyl)methanamine (S8b):
Previously prepared and characterized. [2] General procedure iii. Amide coupling with Boc-L-hydroxyproline and deprotection -Synthesis of S3a-d and S9a-b To a solution of amine (1 equiv.) in DMF, Boc-L-hydroxyproline (1 equiv.) was added and the mixture was stirred at room temperature. DIPEA (2 equiv.) was added dropwise and the mixture was stirred for 5 minutes at room temperature. HATU (1.1 equiv.) was added and the mixture was stirred at room temperature for 30-90 minutes (LCMS monitoring). Water was added and the mixture was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over MgSO4 and evaporated under reduced pressure to give the corresponding crude, which was purified by flash column chromatography with an increasing gradient of DCM and 20% MeOH in DCM to yield the desired product. The Boc protected compound was dissolved in DCM, followed by the dropwise addition of a 4M HCl solution in dioxane (at least 3 equiv.). Often an insoluble precipitate starts to be formed. A few drops of MeOH were added to make the solution homogeneous, being kept stirring for approximately one hour. The DCM and the HCl were removed by flushing nitrogen into the solution and residual solvents were evaporated under reduced pressure. To remove traces of impurities, compounds S3a, S3b, S3d and S9a were furtherly purified by preparative HPLC, being obtained as a formate salt, while the remaining compounds were obtained as a chloride salt.

General procedure v. Amide coupling with 3,3-dimethylbutyric acid -Synthesis of S5a-d
To a solution of amine (1 equiv.) in DMF, 3,3-dimethylbutyric acid (1 equiv.) was added and the mixture was stirred at room temperature. DIPEA (2 equiv.) was added dropwise and the mixture was stirred for 5 minutes at room temperature. HATU (1.1 equiv.) was added and the mixture was stirred at room temperature for 60 minutes (LCMS monitoring). Water was added and the mixture was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over MgSO4 and evaporated under reduced pressure to give the corresponding crude, which was purified in the acidic Gilson preparative HPLC.

General procedure vi. Amide coupling with Boc-L-tert-leucine and deprotection -Synthesis of intermediates of compounds S6a-d and S12a-b
Same as "general procedure iii", just replacing the Boc-L-hydroxyproline with Boc-L-tert-leucine. All the crude intermediates prepared at this step were directly used in the next steps without further purification and characterization after deprotection of the Boc group.
General procedure vii. Amine trifluoroacetylation -Synthesis of S10a-b and S12a-b To a solution of the amine (1 equiv.) in dry MeOH (1 ml per mmol of amine) was added triethylamine (2 equiv.).
Ethyltrifluoroacetate (1.25 equiv.) was added and the reaction was stirred at room temperature for approximately 24 hours (LCMS monitoring). The solvent was evaporated under reduced pressure and the crude mixture was extracted with ethyl acetate and 1.0 M HCl solution. Combined organic phases were dried over MgSO4, concentrated and purified in the acidic Gilson preparative HPLC system, yielding the desired product.

General procedure viii. Amide coupling with 3,3,3-fluoropropanoic acid -Synthesis of S11a-b
To a solution of amine (1 equiv.) in DMF, 3,3,3-Trifluoropropanoic acid (1 equiv.) was added and the mixture was stirred at room temperature. DIPEA (2 equiv.) was added dropwise and the mixture was stirred for 5 minutes at room temperature. HATU (1.1 equiv.) was added and the mixture was stirred at room temperature for 30-90 minutes (TLC monitoring). Water was added and the mixture was extracted with ethyl acetate. The combined organic phases were washed with brine, dried over MgSO4 and evaporated under reduced pressure to give the corresponding crude, which was purified by flash column chromatography with an increasing gradient of DCM and 20% MeOH in DCM to yield the desired product. -1-(3,3,3-trifluoropropanoyl) 1-(3,3,3-trifluoropropanoyl)

Protein expression, purification and biotinylation
The VHL E3 ligase is a multi-protein complex composed of five proteins: VHL protein (pVHL), elongin B (eloB), elongin C (eloC), Cullin-2 (Cul2) and Ring-box protein 1 (Rbx1). [3] Since the compounds developed in this work bind solely to the VHL protein, the VBC complex (equimolar complex of pVHL54-213, eloB1-104 and eloC17-112) was used in all experiments, as it can be readily expressed in E. coli with high yields, [4] while the full E3 ligase would require baculovirus-insect cells expression system. [5] The expression and purification of VBC was performed as described previously by our group [2] and employed directly in all NMR experiments.
For the surface plasmon resonance (SPR) experiments, a VBC complex containing an AviTag TM in the Nterminus of eloB (AviVBC) was purified using the same procedure described for VBC. The modified eloB/eloC expression plasmid was previously developed in-house by Dr. Michael Roy and kindly shared. The AviVBC complex was site-specifically biotinylated in the AviTag using the GST-BirA method previously described by Fairhead and Howarth. [6]

Surface plasmon resonance experiments
The SPR experiments were performed with a Biacore T200 instrument (GE Healthcare). All measurements were performed at 20 °C with buffer containing 10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP, 0.005%  to 1 and 9). From this first screen, the binding affinities were roughly estimated using the Biacore T200 evaluation software (GE Healthcare), then measurements were repeated using concentrations above and below the KD obtained in the first round to generate better curves for fitting the data accurately. Contact and dissociation times varied across the different compounds tested, but in general fast binding kinetics were observed for all compounds, fully reaching steady-state or being completely dissociated from the surface in less than sixty seconds.
Data analysis was performed using the steady state responses of the double-referenced sensorgrams (raw data subtracted from blank and reference surface injections) obtained for each concentration tested. These responses were plotted against the respective concentrations and the data fitted to a 1:1 binding model using the Biacore T200 evaluation software and the following equation: Where Req is the steady-state response at a given concentration C. Deviations in Req were corrected by adding an 'offset' term to the equation. KD is the dissociation constant to be determined and RMAX is the maximum response expected for a given compound according to the equation below:

= ×
Where n is the stoichiometry of the interaction (in this case, n = 1) and RProtein is the immobilization level of protein. MWCompound and MWProtein are the molecular weights of compound and protein, respectively. Sensorgrams and fitting parameters for all spy molecules can be found in section 7.

Measurement of the transverse relaxation rates by 19 F CPMG NMR
All the NMR experiments were performed in a 500 MHz Bruker AVANCE NMR spectrometer equipped with a CPQCI-F cryoprobe. To measure the transverse relaxation rates (R2), a solution of each spy molecule at 100 μM was prepared in 50 mM potassium phosphate monobasic (KH2PO4), pH 7.5, 100 mM NaCl, 1 mM TCEP,

Competition experiments by 19 F NMR
As full measurements of R2 would be very time consuming, the competition experiments were performed using a single 19 F CPMG experiment (decoupled) per sample with a fixed CPMG delay. To determine the best CPMG delay for a given spy molecule and assay condition, the procedure described in Figure S5 was developed based on the transverse relaxation rate equations. [7] By knowing the relaxation rates of the spy molecule free in solution and in presence of protein, the CPMG delay where the difference between the NMR peaks is maximum is hereon referred as dmax. The dmax for all the conditions tested for each spy molecule can be found in section 8, together with the respective values of R2 and C2.

Method for selecting the best CPMG delay for competition experiments.
With the transverse relaxation rates of the spy molecule free in solution (R2 F , resulting in the blue plot) and bound to protein (R2 B , resulting in the red plot), the difference between the two curves is described by I(t) D (cyan curve). To obtain dmax, the delay where the difference curve reaches its maximum, the first derivative of I(t) D was obtained and equalled to 0, subsequently isolating t (CPMG filter).
After the dmax for each condition was established, the competition experiments with spy molecules 6 ( Figure   S3), 11 ( Figure S4) and 19 ( Figure 5  Data fitting using the Biacore T200 evaluation software. As responses were lower than the theoretical RMAX, fitting was performed with a fixed RMAX.