Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1(1H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak “spy ligands” can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput

Bodenhausen, Geoffrey

Bornet, Aurélien

Buratto, Roberto

Gerber-Lemaire, Sandrine

Jannin, Sami

Laguerre, Aurélien

Mammoli, Daniele

Milani, Jonas

Passemard, Solène

Salvi, Nicola

Singh, Maninder

Vuichoud, Basile

ChemMedChem

PubMed

Harvard University - DASH 

Drug Screening Boosted by Hyperpolarized Long-Lived States in NMR

Transverse and longitudinal relaxation times (T11 and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1(1H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak “spy ligands” can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput.LRMBISIC-GELSP

Bornet, Aurelien

Laguerre, Aurelien

Passemard, Solene

Infoscience - École polytechnique fédérale de Lausanne

Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2014Drug Screening Boosted by Hyperpolarized Long-LivedStates in NMRRoberto Buratto,[a] Aurlien Bornet,*[a] Jonas Milani,[a] Daniele Mammoli,[a] Basile Vuichoud,[a]Nicola Salvi,[a, b] Maninder Singh,[a, c] Aurlien Laguerre,[a, d] Solne Passemard,[a]Sandrine Gerber-Lemaire,[a] Sami Jannin,[a, e] and Geoffrey Bodenhausen*[a, f, g, h]cmdc_201402214_sm_miscellaneous_information.pdf S1Synthesis of a spin-pair-labeled tripeptide  Abbreviations: ACN   Acetonitrile Anal. Calc.  Analysis calculated Atm   Atmosphere DCM   Dichloromethane DIPEA   N, N'-Diisopropylethylamine DMF   N, N-Dimethylformamide DMSO   Dimethyl sulfoxide Equiv   Equivalent ESI   Electrospray ionization Fmoc   Fluorenylmethyloxycarbonyl chloride HOBt   Hydroxybenzotriazole HPLC   High-performance liquid chromatography hr   Hour HRMS   High resolution mass spectrometry IR   Infrared LC-MS   Liquid chromatography - mass spectrometry MeOH   Methanol min   Minute NMR   Nuclear magnetic resonance Pbf   2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl rpm   Revolutions per minute  rt   Room temperature SPPS   Solid phase peptide synthesis SN1   Nucleophilic substitution 1 TBTU   O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TFA   Trifluoroacetic acid TIS   Triisopropylsilane tR   Retention time  I- Synthesis  The synthesis of the spin-pair-labeled tripeptide 3-bromothiophene-2-carboxamido-Gly-Gly-Arg (henceforth ‘BT-GGR’) was performed by solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and Fmoc protected amino acids (Scheme S1). The first step of this synthesis is a SN1 substitution of Fmoc-Arg(Pbf)-OH on the resin. All remaining reactive 2-chlorotrityl groups were then capped with MeOH. The tripeptide was elaborated by successive couplings of Fmoc protected amino acids in the presence of HOBt and TBTU followed by deprotection of the N-terminus under basic conditions. Finally, 3-bromothiophene-2-carboxylic acid was conjugated at the N-terminus of the tripeptide. Cleavage from the resin, followed by deprotection of the arginine side chain, afforded the spin-pair-labeled tripeptide 6, which was purified by HPLC. Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S2 Scheme S1: Synthesis of spin-pair-labeled linear tripeptide BT-GGR 6.  II- Synthetic steps N-Fmoc-Arg(Pbf)-O-resin (2): After swelling with dry DCM (80 mL) for 30 min, the 2-chlorotrityl chloride resin (0.83 mmol.g-1, 1 equiv, 2.49 mmol, 3 g) was treated with a solution of Fmoc-Arg(Pbf)-OH (1) (2 equiv, 4.98 mmol, 3.23 g) in dry DCM/dry DMF (80 mL/10 mL) and DIPEA (8 equiv, 19.92 mmol, 2.58 g, 3.52 mL) at rt for 1.5 hr agitated at 125 rpm. The reaction was performed in a 400 mL filtration tube with a polyethylene frit. The resin was washed with DMF (3 × 100 mL) and DCM (3 × 100 mL) and dried for 12 hr in vacuo to give the N-Fmoc-Gly-O-resin (2). MeOH (30 mL) was added to cap the free sites, and the reaction mixture was shaken for 15 min at 125 rpm. The resin was washed with DMF (3 × 100 mL) and DCM (3 × 100 mL).  N-Fmoc-Gly-Arg(Pbf)-O-resin (3): The Fmoc-protected resin (2) was suspended in a solution of 20% piperidine in DMF (30 mL) for 30 min and agitated at 125 rpm to give the N-deprotected resin. The resin was washed with DMF (3 × 100 mL), DCM (3 × 100 mL), and DMF (1 × 100 mL).  Fmoc-Gly-OH (1.5 equiv, 3.73 mmol, 1.11 g), HOBt (3 equiv, 7.47 mmol, 1.01 g), TBTU (3 equiv, 7.47 mmol, 2.40 g) Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S3and DIPEA (6 equiv, 14.94 mmol, 1.94 g, 2.63 mL) were dissolved in dry DMF (30 mL). The solution was added to the N-deprotected resin. The reaction mixture was shaken at rt for 1 hr at 125 rpm to give Fmoc-Gly-Arg(Pbf)-O-resin (3) which was washed with DMF (3 × 100 mL), DCM (3 × 100 mL), DMF (1 × 100 mL).   N-Fmoc-Gly-Gly-Arg(Pbf)-O-resin (4): The Fmoc-protected resin (3) was suspended in a solution of 20% piperidine in DMF (30 mL) for 30 min and agitated at 125 rpm to give the N-deprotected resin. The resin was washed with DMF (3 × 100 mL), DCM (3 × 100 mL), and DMF (1 × 100 mL). Fmoc-Gly-OH (1.5 equiv, 3.73 mmol, 1.11 g), HOBt (3 equiv, 7.47 mmol, 1.01 g), TBTU (3 equiv, 7.47 mmol, 2.40 g), and DIPEA (6 equiv, 14.94 mmol, 1.94 g, 2.63 mL) were dissolved in dry DMF (30 mL). The solution was added to the N-deprotected resin. The reaction mixture was shaken at rt for 1 hr at 125 rpm to give Fmoc-Gly-Gly-Arg(Pbf)-O-resin (4) which was washed with DMF (3 × 100 mL), DCM (3 × 100 mL), and DMF (1 × 100 mL).   3-bromothiophene-2-carboxamido-Gly-Gly-Arg(Pbf)-O-resin (5): The Fmoc-protected resin (4) was suspended in a solution of 20% piperidine in DMF (30 mL) for 30 min and agitated at 125 rpm to give the N-deprotected resin. The resin was washed with DMF (3 × 100 mL), DCM (3 × 100 mL), and DMF (1 × 100 mL). 3-bromothiophene-2-carboxylic acid (1.5 equiv, 3.73 mmol, 0.77 g), HOBt (3 equiv, 7.47 mmol, 1.01 g), TBTU (3 equiv, 7.47 mmol, 2.40 g), and DIPEA (6 equiv, 14.94 mmol, 1.94 g, 2.63 mL) were dissolved in dry DMF (30 mL). The solution was added to the N-deprotected resin. The reaction mixture was shaken at rt for 1 hr at 125 rpm to give (5) which was washed with DMF (3 × 100 mL), DCM (3 × 100 mL), and DMF (1 × 100 mL).   3-bromothiophene-2-carboxamido-Gly-Gly-Arg (6): After swelling with dry DCM (80 mL) for 30 min and washing with DMF (80 mL), (5) was treated with a solution of 5 % TFA in DCM (80 mL) for 30 min at rt to give the protected tripeptide. The reaction mixture was collected and the resin was washed with DCM (3 × 100 mL). The solution was concentrated and dried for 12 hr in vacuo. Finally, deprotection of the side chain group was carried out with 50 mL TFA:H2O:TIS (95:2.5:2.5) for 2 hr. TFA was then removed by evaporation and the final product (6) was obtained by purification with HPLC (Figure S1) (tR = 7.91 min).    (6)  1H NMR (400 MHz, DMSO-d6): δ 8.30 (td, J = 5.5, 2.6 Hz, 2H, NH), 8.08 (d, J = 7.8 Hz, 1H, CH(5)), 7.85 (d, J = 5.2 Hz, 1H, NH), 7.61 (d, J = 7.0 Hz, 1H, CH(4)), 7.22 (d, J = 5.2 Hz, 4H, NH), 4.19 (td, J = 8.2, 5.0 Hz, 1H, CH(9')), 3.94 (d, J = 5.5 Hz, 2H, CH2(6')), 3.87 – 3.70 (m, 2H, CH2(3')), 3.08 (q, J = 6.7 Hz, 2H, CH2(3'')), 1.74-1.59 (m, 2H, CH2(1'')), 1.49 (qt, J = 7.4 Hz, 2H, CH2(2'')). 13C-NMR (100 MHz, CDCl3): δ 173.4, 168.6, 160.2, 156.7, 132.9, 132.0, 130.7, 124.0, 118.9, 110.1, 51.6, 42.7, 41.8, 28.3, 25.1. IR (neat): 3330, 3190, 3100, 2970, 2930, 1630, 1530, 1415, 1245, 1200, 1135, 1085, 1050, 880, 760, 720, 685. HRMS-ESI: calculated for C15H22BrN6O5S+: 477.0478; found for C15H22BrN6O5S+: 477.0556.  Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S4a  b         Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00%31#1,1:4 1#1,1:5120803AE 1: Scan ES+ TIC1.31e97.917.525.064.782.884.153.596.718.479.3814.2713.579.95 12.4411.46m/z200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150%0100120803AE 225 (7.909) Cm (219:229) 1: Scan ES+ 4.60e7479.10477.10480.10481.04955.18Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S5c  d  Figure S1 a): HPLC spectrum: 2 to 60 % of solvent B (ACN:HCOOH 99.9:0.1) in solvent A (H2O:HCOOH 99.9:0.1) in 10 min. b) LC-MS spectrum after HPLC purification. c) 1H NMR of 6 in DMSO-d6 at 400 MHz and 25o C. d) 13C NMR of 6 in CDCl3 at 100 MHz and 25o C.    Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S6Protein-Ligand Screening using Long-Lived Coherences (LLC) boosted by Dynamic Nuclear Polarization (DNP) In the singlet-triplet basis set, Long-Lived Coherences (LLC) (1-3) can be defined as a linear combination of |S0〉⟨T0| - |T0〉⟨S0| and |S0〉⟨T0| + |T0〉⟨S0|. These terms are equivalent to Ix – Sx and 2IySz – 2IzSy in the product basis. An LLC experiment comprises similar steps as required for the observation of Long-Lived States (LLS): excitation, sustaining and detection. First, the Boltzmann equilibrium population Iz + Sz of an IS pair of chemically inequivalent spins is transformed into a density operator Ix – Sx (Figure S2), or alternatively into 2IySz – 2IzSy. Then, during a variable interval τm, the two spins are rendered magnetically equivalent by applying a resonant rf field with a carrier νrf positioned at the average of the chemical shifts of the two spins that are involved in the LLC. After interrupting the rf irradiation, the free induction signal is acquired as usual. The detectable single-quantum coherences are best described in the product basis as a function of τm:    = 	  −	 cos2 +	2 −	2 sin2 exp "− #$%&&'(		       (S1)  The observation of Ix – Sx as function of τm gives an oscillatory decay that can be Fourier transformed to obtain a so-called J-spectrum with very narrow line widths ΔνLLC = 1/(πTLLC). A LLC has a lifetime TLLC that is often much longer than the conventional transverse relaxation time T2 of the corresponding protons. Just like the lifetimes TLLS of Long-Lived States, the lifetimes TLLC can be dramatically reduced if the ligand that carries the LLC binds to a protein.    Figure S2: Pulse sequences designed to excite, sustain and detect LLC. (a) in the manner of two-dimensional spectroscopy,  with N1 steps τm = n1∆τm, n1 = 0, 1, 2, ..., (N1-1), and (b) using a one-dimensional ‘on-the-fly’ fast acquisition method. Typically, τ1 = 1/2ΔνIS to achieve an efficient conversion of Iy + Sy into Ix – Sx and τaq = 500 μs, ∆τm = 50 ms to have a bandwidth of the J-spectrum of 20 Hz, τaq = 500 μs, τLLC + 2τaq + τπ = ∆τm = 50 ms.   This oscillatory decay can be sampled by incrementing τm in the manner of two-dimensional spectroscopy (Fig. S2a). To reduce the experimental time, it is possible to sample the exponential decay of the sinusoidal signal by choosing delays τm in the vicinity of the maxima that appear at multiples of 1/JIS (Fig.S 3). We have acquired signals for three τm delays that are close to each of these maxima. A local fit of these three signal amplitudes permits one to determine one of the maxima of Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S7the sinusoidal signal (Fig.S 4). The value of TLLC is obtained by a mono-exponential fit of the envelope of consecutive maxima (Fig S5).  By alternating intervals for signal observation and for sustaining the LLC, one can also observe N1 points of an ‘on-the-fly’ LLC signal in a single shot (1) in the manner of one-dimensional spectroscopy (Fig. S2b).  The rf irradiation is briefly interrupted so that part of the oscillating LLC is temporarily transformed into observable single-quantum coherence in a window τaq, refocused by a pi pulse, before being transformed back into LLC by switching the rf irradiation on again. One of the advantages of this fast acquisition scheme is to make the LLC method fully compatible with dissolution DNP (4),   Figure S3: Sampling the oscillating LLC decay. Sampling around the maxima of the sinusoidally modulated decay of the LLC signals (green dots) allows one to determine the time constant TLLC by fitting to an exponential curve (red).    Figure S4: Local fit near three points. The signal amplitudes at three points (blue dots) taken near a maximum of the sinusoidally modulated decay of an LLC signal allows one to determine the amplitude of the maximum (arrow) by local fitting (red curve).    Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S8 Figure S5: Experimental LLC decay. Fitting of 11 consecutive maxima of the sinusoidally modulated decay of an LLC signal allows one to determine TLLC.  Since Long-Lived Coherences (LLC) belong to the class of zero-quantum coherences, their precession and decay are not sensitive to the inhomogeneity of the static magnetic field. If one excites an LLC via zero-quantum coherences, the efficiency of its excitation is not sensitive to the homogeneity of the static magnetic field (5). When all shim coil currents are set to zero (so that normal proton spectra show line widths of about 50 Hz), LLC spectra of free BT-GGR can be obtained with line widths as narrow as ΔνLLCfree = 0.20 Hz (TLLCfree = 1.5 s), i.e., a resolution enhancement of a factor of 50 000 (5). As LLC spectra can be acquired in a single shot, the method can be combined with dissolution DNP (1, 4).  Although the lifetimes of Long-Lived Coherences (LLC) associated with the two diastereotopic protons of the central Glycine in GGR turned out to be rather disappointing, the LLC associated with the two aromatic protons of bromothiophene of BT-GGR (TLLCfree = 1.5 s) is ideal for ligand screening. With a ligand/protein ratio [L]0/[P]0 = 40, a contrast CLLC of 82 % was obtained. Figure 5c in the main text shows LLC spectra of 0.5 mM BT-GGR in the presence of 25 μM trypsin, either without competitor, or with 50 μM of the intermediate competitor apigenin, or with 50 μM of the stronger competitor myricetin. All LLC spectra were recorded “on the fly” with the sequence of Figure S2b. In the absence of competitor, the protein concentration available for binding with the “spy ligand” BT-GGR is [P]free = [P]0, so that one observes a fairly short TLLCobs = 0.81 s and LLC spectra with fairly broad lines ΔνLLCobs = 1/(πTLLCobs) = 0.39 Hz. In the presence of a binder that has a stronger affinity for the protein than the “spy ligand” BT-GGR, [P]free decreases, TLLCobs becomes longer, the peak narrower, and its intensity higher (see Figure 5c and 5d of the main text).  References 1. Bornet A, Jannin S, Konter JA, Hautle P, van den Brandt B, & Bodenhausen G (2011) Ultra High-Resolution NMR: Sustained Induction Decays of Long-Lived Coherences. J Am Chem Soc 133(39):15644-15649. Supplementary Information to:  Drug Screening boosted by Hyperpolarized Long-Lived States in NMR  S92. Sarkar R, Ahuja P, Vasos PR, & Bodenhausen G (2010) Long-Lived Coherences for Homogeneous Line Narrowing in Spectroscopy. Phys Rev Lett 104(5):053001. 3. Sarkar R, Ahuja P, Vasos PR, Bornet A, Wagnieres O, & Bodenhausen G (2011) Long-lived coherences for line-narrowing in high-field NMR. Prog Nucl Mag Res Sp 59(1):83-90. 4. Ardenkjaer-Larsen JH, Fridlund B, Gram A, Hansson G, Hansson L, Lerche MH, Servin R, Thaning M, & Golman K (2003) Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR. Proc. Natl Acad Sci USA 100(18):10158-10163. 5. Chinthalapalli S, Bornet A, Segawa TF, Sarkar R, Jannin S, & Bodenhausen G (2012) Ultrahigh-Resolution Magnetic Resonance in Inhomogeneous Magnetic Fields: Two-Dimensional Long-Lived-Coherence Correlation Spectroscopy. Phys Rev Lett 109(4):047602.   

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Drug Screening Boosted by Hyperpolarized Long-Lived States in NMR

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