Ligand−Protein Affinity Studies Using Long-Lived States of Fluorine-19 NucleiRoberto Buratto,*,† Daniele Mammoli,† Estel Canet,†,‡,§,∥ and Geoffrey Bodenhausen†,‡,§,∥

†Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland‡Departement de Chimie, Ecole Normale Superieure−PSL Research University, 24 Rue Lhomond, 75231 Paris Cedex 05, France§Sorbonne Universite, UPMC Univ Paris 06, 4 place Jussieu, 75005 Paris, France∥CNRS, UMR 7203 LBM, 75005 Paris, France

ABSTRACT: The lifetimes TLLS of long-lived states or TLLC oflong-lived coherences can be used for the accurate determination ofdissociation constants of weak protein−ligand complexes. Theremarkable contrast between signals derived from LLS or LLC infree and bound ligands can be exploited to search for weak binderswith large dissociation constants KD > 1 mM that are important forfragment-based drug discovery but may escape detection by otherscreening techniques. Alternatively, the high sensitivity of theproposed method can be exploited to work with large ligand-to-protein ratios, with an evident advantage of reduced consumptionof precious proteins. The detection of 19F−19F long-lived states insuitably designed fluorinated spy molecules allows one to performcompetition binding experiments with high sensitivity whileavoiding signal overlap that tends to hamper the interpretation ofproton spectra of mixtures.

■ INTRODUCTION

Fragment-based drug discovery (FBDD) has emerged as afruitful approach to develop new drugs.1 Initially, one mustidentify small molecular fragments that bind weakly to amacromolecular target with dissociation constants KD on theorder of 10 μM to 10 mM or greater.2 These ligands can besubsequently developed through medicinal chemistry in orderto optimize features such as absorption, distribution, metabo-lism, excretion, and toxicological (ADMET) properties.Nuclear magnetic resonance (NMR) seems particularly suitableto reveal such weak interactions. A plethora of NMRexperiments has been developed for screening fragmentlibraries, such as WaterLOGSY,3 saturation transfer difference(STD),4 and relaxation-edited experiments. The latter canexploit the contrast of longitudinal or transverse relaxationupon binding.5 Experiments that monitor the relaxation oflong-lived states (LLS)6,7 have been demonstrated to beparticularly sensitive to binding phenomena.8,9 The immunityof LLS to dipolar relaxation between the two participatingnuclei explains the dramatic contrast

=−

CR R

R100[%]LLS

LLSobs

LLSfree

LLSobs

(1)

that is obtained if the observed relaxation rate RLLSobs is compared

with the rate RLLSfree of the ligand in its free state. The contrast

CLLS is often larger for LLS than for other relaxation rates suchas T1selective, T2, and T1ρ. The more favorable the contrast, the

easier it is to screen weakly binding fragments and to determinetheir dissociation constants.10

Experiments based on the direct observation of ligands sufferfrom a number of limitations: nonspecific binders may givesimilar effects as specific ones, ligands are difficult to detect iftheir solubility is low, and strong ligands in slow exchange areeasily mistaken for nonbinders. To overcome these drawbacks,Dalvit and co-workers11 introduced so-called competitionexperiments for ligand screening. In this approach, a weak-affinity ligand is used as a spy molecule; a stronger binderdisplaces the spy molecule, and the latter’s expulsion affects therelaxation rates of nuclei on the spy. The concentration of acompetitor that is required to displace a spy molecule isinversely proportional to the former’s affinity for the macro-molecular target: the higher the affinity, the lower theconcentration needed. The study of weakly binding fragmentsturns out to be challenging since high concentrations andtherefore high solubility are required. Moreover, if mixtures ofpotential competitors are tested, the risk of signal overlap mustbe sidestepped by a careful choice of the cocktail of molecules.This work demonstrates that the excitation of LLS involving

pairs of 19F nuclei in spy ligands that have been designed tofeature a favorable contrast CLLS between free and bound formsprovides a very effective tool to study weak protein−ligandinteractions. Such experiments benefit from a good sensitivity

Received: October 9, 2015Published: January 22, 2016

Article

pubs.acs.org/jmc

© 2016 American Chemical Society 1960 DOI: 10.1021/acs.jmedchem.5b01583J. Med. Chem. 2016, 59, 1960−1966

since 19F has a high gyromagnetic ratio and 100% naturalabundance, and since 19F spectra do not suffer from signaloverlap.Long-lived states6,12−14 decay with a time constant TLLS that

can be much longer than the longitudinal relaxation timeconstant T1 under suitable conditions. Likewise, long-livedcoherences (LLC)15 relax with a time constant TLLC that can belonger than the transverse relaxation time constant T2. Insystems with two coupled spins-1/2, singlet states |S0⟩correspond to antisymmetric linear combinations of the twoproduct states |αβ⟩ and |βα⟩. If a “triplet−singlet populationimbalance”, or TSI,16 is created, it can only be dissipated in thepresence of chemical shift anisotropy (CSA), dipolarinteractions with external spins, or dipolar interactions toparamagnetic species.To perform an LLS experiment, it is convenient to start with

a weakly coupled two-spin AX system, temporarily suppress thechemical shift difference by radio frequency (RF) irradiation toobtain an A2-like spin system, and eventually revert to the AXsystem by interrupting the RF irradiation. In the experimentalprocedure that we commonly use,7 (i) one converts theBoltzmann equilibrium populations of an AX system into anLLS, which amounts to setting up a triplet−singlet imbalance(Figure 1 a-b);6,7 (ii) one sustains the LLS in an interval τm by

applying a resonant RF field with the carrier (νRF) placedhalfway between the two chemical shifts6,14,17,18 (Figure 1b,c);(iii) finally, after turning off the RF field, one applies a suitablepulse sequence to convert the remaining triplet−singletimbalance TSI (which is equivalent to an LLS) back intoobservable magnetization (Figure 1c,d). The lifetime TLLS of along-lived state can be determined by fitting the signal intensityas a function of the locking interval τm (Figure 1b,c).LLC15 are closely related to LLS. Their lifetimes TLLC can be

longer than transverse relaxation times T2 and are also stronglyaffected by interactions with proteins. Therefore, TLLC can alsobe a useful observable for protein−ligand binding studies. LLCoscillate in the interval τm with the scalar coupling constantJ(19FI, 19FS). The Fourier transform of this oscillatory decayyields J-spectra with narrow line widths (ΔνLLC = 1/(πTLLC)).In the singlet−triplet basis set, an LLC can be defined as |S0⟩⟨T0| + |T0⟩⟨S0|. This is equivalent to a linear combination of Ix− Sx and 2IySz − 2IzSy in the Cartesian product operator basis.An LLC experiment comprises the same three intervals forexcitation, locking, and detection. First, the Boltzmannequilibrium population of a magnetically inequivalent AX spinsystem is excited to yield a spin density operator correspondingto Ix − Sx (Figure 2a). Then, the chemical shift differencebetween the two spins is temporarily suppressed by a RF field,

with the carrier νRF halfway between the chemical shifts of thetwo spins to obtain two magnetically equivalent spins A2 in theinterval τm (Figure 2b,c). Finally, after interrupting the RFirradiation, the system reverts to a magnetically inequivalent AXconfiguration, and the signal is acquired after point c in Figure2. At this point, the density operator can be expressed as afunction of the duration τm of the RF field:15

σ τ π τ

π ττ

= − + −

−⎛⎝⎜

⎞⎠⎟

I S J I S I S

JT

( ) [( ) cos(2 ) (2 2 )

sin(2 )] exp

x x IS y z z y

ISLLC

m m

mm

(2)

Thus, the terms Ix − Sx give oscillatory signals that slowlydecay as a function of the interval τm.The J-modulation can be suppressed by inserting a π/2 pulse

at the midpoint of a double spin echo in the interval τm, so thatthe LLC decay can be fitted to a simple exponential function.19

In either case, the LLC contrast is

=−

CR R

R100[%]LLC

LLCobs

LLCfree

LLCobs

(3)

■ RESULTS AND DISCUSSIONTo the best of our knowledge, long-lived states have so far onlybeen observed in systems comprising pairs of 1H, 13C or 15Nnuclei.20 Here we explore the possibility of observing LLS andLLC using pairs of 19F nuclei. We have synthesized a moleculethat contains a pair of diastereotopic aliphatic fluorine atoms:1,1-difluoro-1-phenylacetyl-Gly-Arg, abbreviated as DFPA-GR(Figure 3). The presence of an arginine residue gives rise to a

weak affinity for the active site of trypsin, thus allowing one toexplore the behavior of LLS and LLC of pairs of 19F nucleiupon binding. Aliphatic fluorine atoms have been preferred toaromatic ones, in order to minimize the relaxation of LLS dueto chemical shift anisotropy (CSA.) The CSAs of 19F nuclei in

Figure 1. Pulse sequence for excitation, locking, and observation oflong-lived states (LLS) involving a pair of 19F nuclei. Note that it issufficient to apply proton decoupling during signal observation inorder to remove J(1H, 19F) splittings if desired.

Figure 2. Pulse sequence for excitation, locking, and observation oflong-lived coherences (LLC) involving a pair of 19F nuclei. As inFigure 1, proton decoupling can be used during signal observation toremove J(1H, 19F) splittings if desired.

Figure 3. Structure of the custom-synthesized spy ligand 1,1-difluoro-1-phenylacetyl-Gly-Arg (DFPA-GR) containing a pair of aliphaticdiastereotopic fluorine atoms.

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aliphatic positions in amino acids are smaller than those of 19Fnuclei in aromatic rings.21,22

No less than seven bonds separate the fluorine nuclei fromthe closest chiral center (Cα of the arginine residue, indicatedby a star in Figure 3). The chemical shift difference ΔυI,Sbetween the two diastereotopic fluorine nuclei is only 0.8 ppm,or 300 Hz, at B0 = 9.4 T (400 and 376 MHz for 1H and 19F,respectively), while the scalar coupling constant is 2JI,S = 252Hz. Since an RF field amplitude υ1 = 5ΔυI,S is generallysufficient to sustain LLS,8 a weak field υ1 = 1.5 kHz allows oneto achieve TLLS = 2.63 s in DFPA-GR at B0 = 9.40 T.In general, ΔυIS can be reduced by increasing the distance

between the fluorine nuclei and the chiral center. Conversely,when the glycine residue in DFPA-GR is deleted, one observesan increase of the chemical shift difference between the twodiastereotopic fluorine nuclei to ΔυIS = 2.05 ppm, thusrequiring an increased RF field amplitude of about υ1 = 3.5kHz to lock the LLS. The resulting TLLS = 2.06 s is slightlyshorter than the one in DFPA-GR. On the other hand, theinsertion of an additional Gly residue would lead to a furtherreduction of the chemical shift difference ΔυIS.The excitation of LLS for the pair of fluorine nuclei of DFPA-

GR was achieved with the pulse sequence shown in Figure 1. Inthe absence of protein, the LLS relaxation rate of the ligand wasfound to be RLLS = 0.38 s−1, much smaller than the longitudinalrelaxation rate R1 = 1.64 s−1, leading to a favorable ratio R1/RLLS> 4. This shows that it is possible to obtain LLS withsufficiently long lifetimes for pairs of fluorine nuclei. Attemptsto use pairs of aromatic 19F nuclei lead to smaller ratios R1/RLLSthat are less favorable for drug screening.Of all parameters that can be exploited in 19F NMR, the

transverse relaxation rate R2(19F) = 1/T2(

19F) is widelyconsidered to be one of the most sensitive to bindingphenomena because R2(

19F) is strongly affected by exchangebroadening.23 The rates R2(

19F) of our spy ligand DFPA-GRhave been determined in the presence or absence of trypsin.The resulting R2(

19F) contrast lies in the range 32 < C2 < 40%for the two diastereotopic 19F nuclei for 370 μM DFPA-GRwith 2 μM trypsin (i.e., for a 185-fold excess). However, if weswitch our attention to LLS, the contrast CLLS increases to 87%under the same conditions (Table 1). This confirms that RLLS isextremely sensitive to binding.LLC can also be readily excited and sustained in DFPA-GR.

Using the pulse sequence introduced by Singh and Kurur,19 theLLC decay could be fitted to a monoexponential function. The

LLC relaxation rate is RLLC = 2.30 s−1, while R2 = 2.61 and 2.65s−1, respectively, for the two diastereotopic fluorine nuclei, sothat the ratios are R2/RLLC = 1.13 and 1.15 for these two nuclei,i.e., less favorable than the ratio R1/RLLS > 4. Furthermore, therates RLLC are sensitive to binding. If the experiment isperformed in a solution of 370 μM DFPA-GR and 2 μMtrypsin (185-fold excess), RLLC increases to 4.55 s−1, leading toa respectable contrast CLLC = 49%, which is however far belowthe remarkable contrast CLLS = 87% under the same conditions.In order to determine the dissociation constant KD of the

DFPA-GR/trypsin complex, a titration experiment wasperformed to monitor TLLS = 1/RLLS, while stepwise increasingthe ligand concentration [Ltot] with an initial concentration of 2μM trypsin. The addition of small aliquots of a concentratedsolution of DFPA-GR allows one to increase [Ltot], while theprotein concentration is hardly affected. The titration curve wasfitted to the following equation:

= − +RPLL

R R R[ ][ ]

( )LLSobs

totLLSbound

LLSfree

LLSfree

(4)

where RLLSbound and RLLS

free are the LLS relaxation rates of the ligandin its fully bound and free forms, respectively. The molarfraction [PL]/[L]tot of the bound form can be calculated from

=+ + − + + −PL

LP L K P L K P L

L[ ][ ]

[ ] [ ] ([ ] [ ] ) 4[ ][ ]

2[ ]tot

tot tot D tot tot D tot tot

tot

2

(5)

The dissociation constant of the DFPA-GR/trypsin complexthat can be derived from the titration curve (Figure 4) is KD =

106 ± 26 μM. The thermodynamic constant KD must be equalto the ratio of the kinetic dissociation and association rateconstants, KD = kof f/kon. Since the latter is usually assumed to belimited by diffusion (107 < kon< 109 M−1 s−1),24 kof f must be inthe range 103−105 s−1. Thus, the system easily fulfills the fastexchange condition, i.e., kex = (kon[P] + kof f) ≈ kof f ≫ Δω,where [P] is the concentration of the free protein and Δω =2πΔν is the difference in 19F chemical shifts between the boundand free forms of the ligand. Even if the assumption that kon is

Table 1. Transverse Relaxation Rates R2(19F) of the Two

Diastereotopic 19F Nuclei, Relaxation Rate RLLC(19F) of the

Long-Lived Coherences and Relaxation Rate RLLS(19F) of the

Long-Lived States of the Spy Molecule DFPA-GR of Figure3, and Contrast Observed in the Presence or Absence ofTrypsina

370 μMDFPA-GR

370 μM DFPA-GR+ 2 μM trypsin contrast

R2(s−1)

19Fpro‑R 2.61 3.85 C2 = 32%19Fpro‑S 2.65 4.39 C2 = 40%

RLLC (s−1) 2.30 4.55 CLLC = 49%RLLS (s

−1) 0.38 2.85 CLLS = 87%

aThe selective longitudinal relaxation rates R1,s (19F) could not be

determined because of the difficulty of selectively inverting only one ofthe two spins.

Figure 4. Lifetimes TLLS of the long-lived state LLS associated with thetwo 19F nuclei of the spy ligand DFPA-GR in the presence of 2 μMtrypsin as a function of the concentration [DFPA-GR] at 298 K and9.4 T. The curve shows a fit of the experimental data to eq 4.

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determined by diffusion were violated, additional experimentalevidence demonstrates that the DFPA-GR/trypsin system is infast exchange. For example, the relaxation rates RLLS in Figure 4show a trend typical of a weighted average of the rates of freeand bound states. In the presence of a system in slow exchange,the observed relaxation rate would not change during a titrationsince only the signals of the ligand in the free state would beobservable (the small fraction of ligand in the bound statewould not contribute to the average rate).In practice, it may be challenging to identify a spy ligand that

contains a pair of 19F nuclei and fulfills these conditions. Inmost cases, one should begin by selecting a spy molecule withsuitable exchange rates, and then insert a 19F spin pair bysynthesis, as we have demonstrated for spin-pair labelscontaining two protons.9

Once a suitable spy molecule such as DFPA-GR that fulfillsthe conditions of fast exchange has been identified, libraries ofpotential binders can be screened by competition bindingexperiments,11 by observing changes in the LLS or LLC decay

rates of the spy molecule. The competing ligand leads to apartial expulsion of the spy molecule and hence to a change inthe molar fraction Xspy

bound. The larger the contrast CLLS, thesmaller the changes of Xspy

bound that can be detected and thehigher the sensitivity to competitive binding.Figure 5 shows the LLS spectrum of 500 μM DFPA-GR in

the absence of protein (top), in the presence of 2 μM trypsin(center) and with 2 μM trypsin plus 485 μM morin, a knowntrypsin inhibitor25 (bottom). In the absence of protein thesignal is intense because the LLS relaxation rate RLLS is small,while in the presence of protein RLLS increases so that thesignals are attenuated. In the presence of both the protein and acompeting molecule, the competitor hinders the interactionbetween the spy molecule and the protein, thus leading to adecrease of RLLS. Nevertheless, since the inhibitor nevercompletely displaces the spy molecule from the active site ofthe protein, a small fraction of the spy molecule remains inexchange with the protein, so that the signal is not completelyrestored. Note that the relative intensities of the four signals are

Figure 5. (Top) Signals derived from 19F−19F LLS of 500 μM DPFA-GR without protein after τm = 0.7 s. (Center) The same in the presence of 2μM trypsin. (Bottom) The same in the presence of 2 μM trypsin and 485 μM morin as competitor, which partly displaces DPFA-GR from thebinding site of the protein, leading to a partial restoration of its signals. A total of 128 scans were recorded for each spectrum, with acquisition andrepetition times of 0.7 and 3 s.

Figure 6. (Left, black dots) LLS decays of 495 μM DFPA-GR in the absence of protein and competitor; (blue dashed line) in the presence of 2 μMtrypsin and 500 μM morin; (green continuous line) in the presence of 2 μM trypsin and 500 μM BT-GGR; and (red dashed-dotted line) in thepresence of 2 μM trypsin only. (Right) 19F spectra derived from 19F−19F LLS of the spy DFPA-GR in the four solutions described above, afterlocking for τm = 0.7 s.

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different in the presence or absence of the protein. This effect islikely due to cross-correlated relaxation interference amplifiedby the slow motion regime of the ligand in the bound state.Once a competitor has been identified, its dissociation

constant needs to be determined. To do so, the spy ligandDFPA-GR can be titrated in the presence of a fixedconcentration of competitor or vice versa.11 The curve of TLLS= (1/RLLS) vs [spy], respectively, vs [competitor] can be fittedusing eq 4 to determine an apparent dissociation constantKD, app

spy . The true dissociation constant KDcompetitor can be

calculated from the relationship:

=−

KL K

K K

[ ]Dcompetitor

competitorD truespy

D appspy

D truespy

,

, , (6)

where KD, truespy = 106 μM is the true dissociation constant of the

spy ligand DFPA-GR and [Lcompetitor] is the total concentrationof the competitor. A quick estimate of KD

competitor can be obtainedfrom a single titration point: knowing RLLS

obs , the mole fraction ofthe spy molecule in its bound form can be estimated using eq 4.At this point, KD, app

spy can be calculated by rearranging eq 5.Following this approach, we have estimated KD

competitor = 28 μMfor morin and KD

competitor = 250 μM for BT-GGR, in reasonableagreement with values reported in the literature (30 and 200μM).9,25 This shows that estimates of the affinities ofcompetitors can be obtained quickly. A more accuratedetermination of KD

competitor requires a full titration experiment.The results demonstrate the applicability of the method over

a broad range of competitor affinities. Morin can be consideredas a typical medium-affinity binder, while BT-GGR can beconsidered as a representative of binding fragments, with aweak affinity for trypsin and a low molecular weight(MWBT−GGR = 385 Da) (Figure 6).Often, screening is performed by testing mixtures (also

known as “cocktails”) of 3−10 putative competitors in order toreduce experimental time and minimize protein consumption.

If one would observe proton signals, such experiments wouldneed a careful choice of the mixtures in order to avoid overlapof signals of the ligands with those of the spy molecule. Forexample, Figure 7 shows a comparison between the 1H spectraof the spy molecule DFPA-GR alone and mixed with BT-GGR,a competitor with a similar molecular structure. The overlapsbetween the proton signals of the spy molecule and those of thecompetitor are too severe to allow one to perform reliablecompetition experiments.The problem can become even more severe when looking for

weak binders. Indeed, the larger the dissociation constants, thehigher the required concentrations of the competitors. In thiscontext, 19F NMR has no rivals. It allows one to performexperiments with high sensitivity while avoiding problems ofoverlap with protonated buffers and signals of mixtures. Thecombination of the high sensitivity to binding phenomenaoffered by the LLS method and the lack of overlap in 19F NMRcan be put to good use for fragment-based drug discovery.Since competition experiments are based on indirect detectionof competing ligands through changes in the properties of thespy molecule, a deconvolution step may be needed, wheremixtures containing fewer or single compounds are testedseparately.

■ CONCLUSIONS

The lifetimes TLLS of long-lived states and, to a lesser extent,the lifetimes TLLC of long-lived coherences can be used veryeffectively to characterize protein−ligand interactions.8 In thecontext of fragment-based drug discovery, the exquisitesensitivity to ligand−protein binding, as evidenced by afavorable contrast CLLS or CLLC, can be exploited to searchfor weak binders with large dissociation constants KD

competitor > 1mM, which may escape detection by other screeningtechniques.10 Alternatively, the high sensitivity of the proposedmethod can be exploited to work with large ligand-to-proteinratios, with an evident advantage in terms of protein saving. By

Figure 7. (Top) Proton spectrum of 495 μM DFPA-GR (spy). (Bottom) Spectrum of 495 μM DFPA-GR mixed with 500 μM BT-GGR. Therectangle shows the range where the signals of the α-protons of the glycine residues appear.

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combining LLS experiments with dissolution DNP so that theconcentrations of both ligands and proteins can be drasticallyreduced, one can alleviate solubility problems that may arisewhen high concentrations of competitors are required todisplace a spy molecule from its binding site.9

We have shown for the first time that long-lived states andcoherences can be observed using pairs of 19F nuclei.Fluorinated spy molecules allow one to avoid signal overlapwith resonances of buffers and competitors, so that one canincrease the number of putative ligands that can be testedsimultaneously, thus speeding up the entire screening process.Competition experiments allow one to detect the binding ofarbitrary molecules that do not contain any fluorine nuclei. Theaffinity of competing ligands can be estimated quickly withoutperforming a full titration. The new method should facilitatethe identification of lead compounds that can be optimized inlater stages of drug design.

■ EXPERIMENTAL SECTIONAll solutions for screening and titration experiments were preparedwith 20 mM TRIS buffer, 150 mM NaCl, and 5 mM MgCl2. Stocksolutions were prepared in d6-DMSO containing 200 mM BT-GGR,9

200 mM morin (Sigma-Aldrich), and 200 mM DFPA-R or 200 mMDFPA-GR. All experiments were performed using type IX-S trypsinfrom porcine pancreas (Sigma-Aldrich). During the titrations, 2 μLaliquots of 150 mM DFPA-GR were added to 500 μL of the startingsolution. The ligand concentrations were measured by the PULCONtechnique.26 The rates RLLS and RLLC were obtained by fitting thedecays with monoexponentially decaying functions, using 10 delays τmranging from 10 ms to 5 times the expected relaxation time TLLS. TheR2 measurements were recorded using a CPMG sequence with a π/2pulse at the top of an echo to suppress J-modulations in the manner ofPROJECT,27 combined with continuous-wave decoupling of theprotons during acquisition, but not during the echo sequence, to avoidinterference. The spin−echo de- and refocusing delays were τ = 20 ms.Titration curves were fitted to eq 2. In the presence of a competitor,the apparent dissociation constant of the spy molecule was used asinput to eq 4 to calculate the true dissociation constant of thecompetitor. All NMR measurements were performed at 25 °C and B0= 9.4 T where 19F and 1H nuclei resonate near 376.38 and 400 MHz,respectively.The synthesis of 1,1-difluoro-1-phenylacetyl-Gly-Arg (DFPA-GR)

was performed by solid-phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and Fmoc-protected amino acids. The firststep is a SN1 substitution of Fmoc-Arg(Pbf)-OH on the resin. Allremaining reactive 2-chlorotrityl groups were then capped withMeOH. The product was obtained by coupling of Fmoc-protectedGly in the presence of HOBt and TBTU, followed by deprotection ofthe N-terminus of the dipeptide. Finally, difluoro-phenyl-acetic acidwas conjugated at the N-terminus of the dipeptide. Cleavage from theresin, followed by deprotection of the arginine side chain, affordedDFPA-GR.N-Fmoc-Arg(Pbf)-O-resin (2). After swelling with dry DCM (80

mL) for 5 min, the 2-chlorotrityl chloride resin (0.83 mmol·g−1, 1equiv, 1 mmol, 1.2 g) was treated with a solution of Fmoc-Arg(Pbf)-OH (1) (1.2 equiv, 1.2 mmol, 0.78 g) in dry DCM (10 mL) andDIPEA (2.5 equiv, 6.23 mmol, 0.81 g) shaken at 125 rpm at rt for 2 h.The reaction was performed in 4 × 10 mL filtration tubes with apolyethylene frit. MeOH (10 mL) was added to cap the free sites, andthe reaction mixture was shaken for 1 h at 125 rpm. The resin waswashed with DCM (3 × 12 mL), DCM/MeOH 1:1 (3 × 12 mL),MeOH (3 × 12 mL), and diethyl ether (3 × 12 mL) and dried for 12 hin vacuo to give the N-Fmoc-Arg(Pbf)-O-resin (2).N-Fmoc-Gly-Arg(Pbf)-O-resin (3). N-Fmoc-Arg(Pbf)-O-resin (2)

was suspended in a solution of 20% piperidine in DMF (10 mL) for 30min and shaken at 125 rpm to give the N-deprotected resin. The resinwas washed with DMF (4 × 10 mL) and DCM (4 × 10 mL). Fmoc-Gly-OH (4 equiv, 4 mmol, 1.19 g), HOBt (4 equiv, 4 mmol, 0.54 g),

TBTU (4 equiv, 4 mmol, 1.29 g), and DIPEA (4 equiv, 4 mmol, 0.52g) were dissolved in DMF (10 mL). The solution was added to the N-deprotected resin. The reaction mixture was shaken at rt for 3 h at 125rpm to give Fmoc-Gly-Arg(Pbf)-O-resin (3), which was washed withDMF (4 × 10 mL).

DFPA-Gly-Arg(Pbf)-O-resin (4). Fmoc-Gly-Arg(Pbf)-O-resin (3)was suspended in a solution of 20% piperidine in DMF (30 mL) for 30min and shaken at 125 rpm to give the N-deprotected resin. The resinwas washed with DMF (4 × 10 mL) and DCM (4 × 10 mL).Difluoro-phenyl-acetic acid (4 equiv, 4 mmol, 0.69 g), HOBt (4 equiv,4 mmol, 0.54 g), TBTU (4 equiv, 4 mmol, 1.29 g), and DIPEA (4equiv, 4 mmol, 0.52 g) were dissolved in DMF (30 mL). The solutionwas added to the N-deprotected resin. The reaction mixture wasshaken at rt for 3 h at 125 rpm to give DFPA-Gly-Arg(Pbf)-O-resin(4), which was washed with DMF (4 × 10 mL) and DCM (4 × 10mL).

DFPA-Gly-Arg (5). Cleavage from the resin and deprotection ofthe side chain group was carried out with 10 mL of TFA/H2O/TIS(95:2.5:2.5) for 3 h. TFA was then removed by evaporation, and thefinal product (5) was obtained after lyophilization.

■ AUTHOR INFORMATIONCorresponding Author*Tel: (+41) 21 69 39428. E-mail: burattoroberto@yahoo.it.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors are grateful to Dr. Ranganath Gopalakrishnan andProf. Christian Heinis for helping in the synthesis of 19F-labelled molecules, Dr. Shivdas Sangram for helpful discussions,and Dr. Pascal Mieville for valuable assistance. This work wassupported by the Swiss National Science Foundation (SNSF),the Swiss Commission for Technology and Innovation (CTI),the EPFL, the CNRS, and the European Research Council(ERC project “Dilute para-water”).

■ ABBREVIATIONS USEDADMET, absorption, distribution, metabolism, excretion, andtoxicological properties; CPMG, Car−Purcell−Meiboom−Gillspin echo sequence; CSA, chemical shift anisotropy; DFPA-GR,1,1-difluoro-1-phenylacetyl-Gly-Arg; FBDD, fragment-baseddrug discovery; LLC, long-lived coherences; LLS, long-livedstates; PULCON, pulse length based concentration determi-nation; STD, saturation transfer difference; TSI, triplet-singletimbalance; Water-LOGSY, water-ligand observed via gradientspectroscopy

■ REFERENCES(1) Baker, M. Fragment-based lead discovery grows up. Nat. Rev.Drug Discovery 2013, 12, 5−10.(2) Hubbard, R. E. Structure-Based Drug Discovery: An Overview;Royal Society of Chemistry: Cambridge, 2006.(3) Dalvit, C.; Pevarello, P.; Tato, M.; Veronesi, M.; Vulpetti, A.;Sundstrom, M. Identification of compounds with binding affinity toproteins via magnetization transfer from bulk water. J. Biomol. NMR2000, 18, 65−68.(4) Mayer, M.; Meyer, B. Characterization of ligand binding bysaturation transfer difference NMR spectroscopy. Angew. Chem., Int.Ed. 1999, 38, 1784−1788.(5) Hajduk, P. J.; Olejniczak, E. T.; Fesik, S. W. One-dimensionalrelaxation- and diffusion-edited NMR methods for screeningcompounds that bind to macromolecules. J. Am. Chem. Soc. 1997,119, 12257−12261.(6) Carravetta, M.; Levitt, M. H. Long-lived nuclear spin states inhigh-field solution NMR. J. Am. Chem. Soc. 2004, 126, 6228−6229.

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Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.5b01583J. Med. Chem. 2016, 59, 1960−1966

1966