Drug Screening Boosted by Hyperpolarized Long-Lived States in NMR

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.


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 SN 1 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.

Protein-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 |S 0 〉⟨T 0 | -|T 0 〉⟨S 0 | and |S 0 〉⟨T 0 | + |T 0 〉⟨S 0 |. These terms are equivalent to I x -S x and 2I y S z -2I z S y 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 I z + S z of an IS pair of chemically inequivalent spins is transformed into a density operator I x -S x ( Figure S2), or alternatively into 2I y S z -2I z S y . Then, during a variable interval τ m , the two spins are rendered magnetically equivalent by applying a resonant rf field with a carrier ν rf 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 singlequantum coherences are best described in the product basis as a function of τ m : The observation of I x -S x as function of τ m gives an oscillatory decay that can be Fourier transformed to obtain a so-called J-spectrum with very narrow line widths Δν LLC = 1/(πT LLC ). A LLC has a lifetime T LLC that is often much longer than the conventional transverse relaxation time T 2 of the corresponding protons. Just like the lifetimes T LLS of Long-Lived States, the lifetimes T LLC can be dramatically reduced if the ligand that carries the LLC binds to a protein. Typically, τ 1 = 1/2Δν IS to achieve an efficient conversion of I y + S y into I x -S x and τ aq = 500 μs, ∆τ m = 50 ms to have a bandwidth of the J-spectrum of 20 Hz, τ aq = 500 μs, τ LLC + 2τ aq + τ π = ∆τ m = 50 ms.
This oscillatory decay can be sampled by incrementing τ m 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 τ m in the vicinity of the maxima that appear at multiples of 1/J IS (Fig.S 3). We have acquired signals for three τ m 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 S7 the sinusoidal signal (Fig.S 4). The value of T LLC 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 N 1 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 τ aq, refocused by a π 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),   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 Δν LLC free = 0.20 Hz (T LLC free = 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 (T LLC free = 1.5 s) is ideal for ligand screening.
With a ligand/protein ratio [L] 0 /[P] 0 = 40, a contrast C LLC 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 T LLC obs = 0.81 s and LLC spectra with fairly broad lines Δν LLC obs = 1/(πT LLC obs ) = 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, T LLC obs becomes longer, the peak narrower, and its intensity higher (see Figure 5c and 5d of the main text).