Intrinsic anisotropy parameters of a series of lanthanoid complexes deliver new insights into the structure-magnetism relationship

Abstract

•Lanthanoids display ligand-field-like effects at room temperature•Anisotropic paramagnetic susceptibility parameters are reported for all lanthanoids•Site-specific isotope labels yield assignments of up to 1,400 ppm wide 1H-NMR spectra Lanthanoid tri-cations are considered to be chemically indistinguishable. A general dogma on the 4 f-electrons states that they are shielded by the outer electrons such that they do not show ligand-field effects, nor contribute to directional bonding, but merely play a role as isotropic Coulomb clouds. Recently, research in the field of single-molecule magnetism and others has questioned this and demonstrated ligand-field effects for lanthanoids at low temperatures. Here, we present a complete series of isostructural lanthanoid complexes with a markedly non-symmetrical chelating ligand, designed for pseudocontact shift NMR spectroscopy. Unexpectedly, the orientation of the principal magnetic axis revealed a distinct correlation with the ground state f-electron distribution. We thus show, for the first time, a crucial interaction of the ligand field with the prolate or oblate f-electron distribution at room temperature, disproving the aforementioned dogma. Lanthanoid chelating tags (LCTs) are widely used for advanced paramagnetic NMR of biomacromolecules. The magnitude of their induced pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) depends critically on the anisotropy of the magnetic susceptibility tensor, which is usually determined by the resonances of the conjugated protein, inevitably reducing the effect by motional averaging. Here, for the first time, we present the intrinsic anisotropy parameters for the full lanthanoid series determined experimentally from resonances on the ligand itself. The strongly shifted proton spectra could only be assigned by extensive, site-specific isotope labeling. The extremely large anisotropies obtained deliver an upper limit for future PCS applications. To our great surprise, at least at room temperature, we observed an unprecedented correlation between the oblate or prolate f-electron distribution of the lanthanoid and the orientation of the main magnetic axis as well as the size of the magnetic anisotropy. Lanthanoid chelating tags (LCTs) are widely used for advanced paramagnetic NMR of biomacromolecules. The magnitude of their induced pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) depends critically on the anisotropy of the magnetic susceptibility tensor, which is usually determined by the resonances of the conjugated protein, inevitably reducing the effect by motional averaging. Here, for the first time, we present the intrinsic anisotropy parameters for the full lanthanoid series determined experimentally from resonances on the ligand itself. The strongly shifted proton spectra could only be assigned by extensive, site-specific isotope labeling. The extremely large anisotropies obtained deliver an upper limit for future PCS applications. To our great surprise, at least at room temperature, we observed an unprecedented correlation between the oblate or prolate f-electron distribution of the lanthanoid and the orientation of the main magnetic axis as well as the size of the magnetic anisotropy. IntroductionLanthanoids and their rich structural chemistry have found wide-spread utilization in the last few decades.1Levason W. Chemistry and applications of the lanthanides.Coord. Chem. Rev. 2017; 340: 1-298Crossref Scopus (5) Google Scholar The peculiar electronic properties of lanthanoids resulted in applications ranging from superconductivity2Sun W. Kuang X. Keen H.D.J. Lu C. Hermann A. Second group of high-pressure high-temperature lanthanide polyhydride superconductors.Phys. Rev. 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How the ligand field in lanthanide coordination complexes determines magnetic susceptibility anisotropy, paramagnetic NMR shift, and relaxation behavior.Acc. Chem. Res. 2020; 53: 1520-1534Crossref PubMed Scopus (61) Google Scholar Recently, lanthanoid single-molecule magnets were developed that show blocking temperatures up to liquid nitrogen temperature.7Randall McClain K.R. Gould C.A. Chakarawet K. Teat S.J. Groshens T.J. Long J.R. Harvey B.G. High-temperature magnetic blocking and magneto-structural correlations in a series of dysprosium(III) metallocenium single-molecule magnets.Chem. Sci. 2018; 9: 8492-8503Crossref PubMed Google Scholar,8Guo F.S. Day B.M. Chen Y.C. Tong M.L. Mansikkamäki A. Layfield R.A. Magnetic hysteresis up to 80 kelvin in a dysprosium metallocene single-molecule magnet.Science. 2018; 362: 1400-1403Crossref PubMed Scopus (942) Google Scholar Especially, useful lanthanoid applications have been developed for nuclear magnetic resonance spectroscopy (NMR), starting with the classic “lanthanoid shift reagents” 9Geraldes C.F.G.C. Lanthanide shift reagents.Methods Enzymol. 1993; 227: 43-78Crossref PubMed Scopus (14) Google Scholar up to the more recent field of paramagnetic biomolecular NMR spectroscopy. Trivalent lanthanoid cations can be tethered site-specifically to proteins using lanthanoid chelating tags (LCTs).10Nitsche C. Otting G. Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags.Prog. Nucl. Magn. Reson. Spectrosc. 2017; 98–99: 20-49Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar,11Joss D. Häussinger D. Design and applications of lanthanide chelating tags for pseudocontact shift NMR spectroscopy with biomacromolecules.Prog. Nucl. Magn. Reson. Spectrosc. 2019; 114-115: 284-312Crossref PubMed Scopus (34) Google Scholar LCTs induce pseudo contactshifts (PCSs) for NMR investigations of protein structures, interactions, and dynamics.12Bertini I. Luchinat C. Parigi G. Paramagnetic constraints: an aid for quick solution structure determination of paramagnetic metalloproteins.Concepts Magn. Reson. 2002; 14: 259-286Crossref Scopus (107) Google Scholar,13Brewer K.D. Bacaj T. Cavalli A. Camilloni C. Swarbrick J.D. Liu J. Zhou A. Zhou P. Barlow N. Xu J. et al.Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution.Nat. Struct. Mol. Biol. 2015; 22: 555-564Crossref PubMed Scopus (104) Google Scholar The size of the PCS is proportional to the anisotropy parameters Δχax and Δχrh of the magnetic susceptibility tensor of the lanthanoid ion (cf. Equation 1).14Bertini I. Luchinat C. Parigi G. Magnetic susceptibility in paramagnetic NMR.Prog. Nucl. Magn. Reson. Spectrosc. 2002; 40: 249-273Abstract Full Text Full Text PDF Scopus (397) Google Scholar Not only do these anisotropy parameters play a crucial role for PCS NMR, they are also discussed in the context of single-molecule magnets5Woodruff D.N. Winpenny R.E.P. Layfield R.A. Lanthanide single-molecule magnets.Chem. Rev. 2013; 113: 5110-5148Crossref PubMed Scopus (2088) Google Scholar and low-temperature EPR spectra.15Pineda E.M. Chilton N.F. Marx R. Dörfel M. Sells D.O. Neugebauer P. Jiang S.-D. Collison D. van Slageren J. McInnes E.J.L. Winpenny R.E.P. Direct measurement of dysprosium(III) ···dysprosium(III) interactions in a single-molecule magnet.Nat. Commun. 2014; 5: 1-7Google Scholar,16Razzaghi S. Brooks E.K. Bordignon E. Hubbell W.L. Yulikov M. Jeschke G. EPR relaxation-enhancement-based distance measurements on orthogonally spin-labeled T4-lysozyme.Chembiochem. 2013; 14: 1883-1890Crossref PubMed Scopus (18) Google Scholar In general, the anisotropy parameters for different LCTs, and hence their performance, are determined from PCSs that are observed on the protein conjugated to the tag. The severe drawback of this method is that any flexibility in the linkage between the LCT and the protein will inevitably lead to an averaging of the anisotropy and, as a consequence, smaller Δχax and Δχrh. Despite the continuous development of new LCTs17Keizers P.H.J. Saragliadis A. Hiruma Y. Overhand M. Ubbink M. Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment.J. Am. Chem. Soc. 2008; 130: 14802-14812Crossref PubMed Scopus (145) Google Scholar, 18Yang F. Wang X. Pan B.B. Su X.C. Single-armed phenylsulfonated pyridine derivative of DOTA is rigid and stable paramagnetic tag in protein analysis.Chem. Commun. (Camb). 2016; 52: 11535-11538Crossref PubMed Google Scholar, 19Joss D. Bertrams M. Häussinger D. A sterically overcrowded, isopropyl-substituted, lanthanide-chelating tag for protein pseudocontact shift NMR spectroscopy: synthesis of its macrocyclic scaffold and benchmarking on ubiquitin S57 C and hCA II S166 C.Chem. Eur. J. 2019; 25: 11910-11917Crossref PubMed Scopus (11) Google Scholar, 20Müntener T. Kottelat J. Huber A. Häussinger D. New lanthanide chelating tags for PCS NMR spectroscopy with reduction stable, rigid linkers for fast and irreversible conjugation to proteins.Bioconjug. Chem. 2018; 29: 3344-3351Crossref PubMed Scopus (26) Google Scholar, 21Loh C.T. Ozawa K. Tuck K.L. Barlow N. Huber T. Otting G. Graham B. Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins.Bioconjug. Chem. 2013; 24: 260-268Crossref PubMed Scopus (70) Google Scholar and their applications22Gao J. Liang E. Ma R. Li F. Liu Y. Liu J. Jiang L. Li C. Dai H. Wu J. et al.Fluorine pseudocontact shifts used for characterizing the protein–ligand interaction mode in the limit of NMR intermediate exchange.Angew. Chem. Int. Ed. Engl. 2017; 56: 12982-12986Crossref PubMed Scopus (18) Google Scholar,23Zimmermann K. Joss D. Müntener T. Nogueira E.S. Schäfer M. Knörr L. Monnard F.W. Häussinger D. Localization of ligands within human carbonic anhydrase II using 19F pseudocontact shift analysis.Chem. Sci. 2019; 10: 5064-5072Crossref PubMed Google Scholar in the last 2 decades, the precise anisotropy parameters of a LCT itself, i.e., without motional averaging due to the flexible attachment to the protein, have never been characterized so far. We would like to coin the term “intrinsic anisotropy parameters” for the anisotropy tensor that is determined on the tag itself, but of course, this “intrinsic” property is a function of temperature and the nature of the lanthanoid, among others.PCS NMR spectroscopy conveniently provides the anisotropy parameters Δχax and Δχrh, as well as the three Euler angles, α, β, and γ, if a 3D structural model of the lanthanoid complex and PCSs for at least five nuclei are available14Bertini I. Luchinat C. Parigi G. Magnetic susceptibility in paramagnetic NMR.Prog. Nucl. Magn. Reson. Spectrosc. 2002; 40: 249-273Abstract Full Text Full Text PDF Scopus (397) Google Scholar (cf. Equation 1). The crucial, but especially in cases of strongly paramagnetic LCTs challenging task, is the assignment of the paramagnetically shifted set of resonances, as strong paramagnetic relaxation effects (PREs) prevent classical assignment strategies via two- or higher-dimensional NMR experiments. While axiallyδpcs=112πr3[Δχax2z2−x2−y2r2+32Δχrhx2−y2r2]whereΔχax=χzz−χxx+χyy2andΔχrh=χxx−χyy(Equation 1) symmetric tensors often allow the assignment of the paramagnetic spectra if several different lanthanoid metal data sets show similar trends,24Di Pietro S. Piano S.L. Di Bari L. Pseudocontact shifts in lanthanide complexes with variable crystal field parameters.Coord. Chem. Rev. 2011; 255: 2810-2820Crossref Scopus (38) Google Scholar,25Hiller M. Maier M. Wadepohl H. Enders M. Paramagnetic NMR analysis of substituted Biscyclooctatetraene lanthanide complexes.Organometallics. 2016; 35: 1916-1922Crossref Scopus (15) Google Scholar rhombic tensors yield more complex spectra. An option for rhombic cases could be to choose the assignment based on the correlation coefficient obtained from iteratively fitting the anisotropy parameters for all possible combinations of assignments. In practice, however, the combinatorial space has to be restricted,26Suturina E.A. Mason K. Geraldes C.F.G.C. Kuprov I. Parker D. Beyond Bleaney’s theory: experimental and theoretical analysis of periodic trends in lanthanide-induced chemical shift.Angew. Chem. Int. Ed. Engl. 2017; 56: 12215-12218Crossref PubMed Scopus (39) Google Scholar as it scales with the factorial of the number of signals.Here, we present the first complete characterization of the intrinsic anisotropy parameters exhibited for the full stable lanthanoid series (La to Lu, but without Pm) of isostructural 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based complexes with the rigid, high-affinity LCT DOTA-M7FPy.20Müntener T. Kottelat J. Huber A. Häussinger D. New lanthanide chelating tags for PCS NMR spectroscopy with reduction stable, rigid linkers for fast and irreversible conjugation to proteins.Bioconjug. Chem. 2018; 29: 3344-3351Crossref PubMed Scopus (26) Google Scholar The combinatorial space was restricted by using different selective 2H and 13C isotope labeling schemes of the LCT. The resulting intrinsic anisotropy parameters of Ln-DOTA-M7FPy are compared with Δχax and Δχrh of a ubiquitin S57C construct conjugated to Ln-DOTA-M7FPy, and to an axial symmetric lanthanoid complex, Ln-DOTA-M8. This in-depth study allowed us to experimentally assign the 1H-NMR spectra of a full series of lanthanoid complexes and provide an upper boundary for Δχax and Δχrh of ideally rigid and non-flexible LCT conjugated to proteins.Besides a new insight into the dynamic properties of the LCTs, we unexpectedly found a strong dependence of the orientation of the main magnetic axis on the ground state electron distribution of the lanthanoid27Sievers J. Asphericity of 4f-Shells in Their Hund’s Rule Ground States.Z. Physik B - Condensed Matter. 1982; 45: 289-296Crossref Scopus (141) Google Scholar and the symmetry of the LCT (cf. Figure 1). Similar observations have been reported for lanthanoid complexes in solid state at 2 K.28Boulon M.E. Cucinotta G. Luzon J. Degl’Innocenti C. Perfetti M. Bernot K. Calvez G. Caneschi A. Sessoli R. Magnetic anisotropy and spin-parity effect along the series of lanthanide complexes with DOTA.Angew. Chem. Int. Ed. Engl. 2013; 52: 350-354Crossref PubMed Scopus (253) Google Scholar Here, for the first time, we demonstrate that sufficiently non-symmetric ligands can deliver such ligand-field-like effects in solution and at room temperature as well.Figure 2Proton spectrum of Dy-DOTA-M7FPyShow full caption1H-NMR spectrum of Dy-DOTA-M7FPy (20 mM) with assignment (for the central region of the spectrum, see Figure S28). The spectrum was recorded at 298 K in phosphate buffer (50 mM, pH = 6.0) at 600.13 MHz. The full spectrum was pieced together from 10 single spectra, each with a sweep width of 154 ppm. Abbreviations: a, axial; e, equatorial; n, near; f, far.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Results and discussionStructure of Ln-DOTA-M7FPyDOTA-based lanthanoid complexes provide the highest anisotropy parameters and, hence, the largest PCSs for structural biomolecular NMR applications. We have chosen the LCT DOTA-M7FPy for this attempt to unravel the intrinsic anisotropy parameters of the susceptibility tensors of all its lanthanoid complexes because it is a very rigid, high-affinity chelator that yields excellent PCSs. Ln-DOTA-M7FPy complexes display eleven stereogenic centers with defined stereochemistry (1R, 2S, 4S, 5S, 7S, 8S, 10S, 11S, 2′R, 2″R, 2″′R) and can adopt in principle four different conformers, however, due to the steric bulk introduced by the ligand design, solely the Λ(δδδδ) or square antiprism (SAP) conformer is formed. This can be safely assumed for both early and late lanthanoids, based on the NOE patterns observed for La-, Sm-, and Lu-DOTA-M7FPy (Figures S34, S51, and S83) and data in the literature.20Müntener T. Kottelat J. Huber A. Häussinger D. New lanthanide chelating tags for PCS NMR spectroscopy with reduction stable, rigid linkers for fast and irreversible conjugation to proteins.Bioconjug. Chem. 2018; 29: 3344-3351Crossref PubMed Scopus (26) Google Scholar,29Opina A.C.L. Strickland M. Lee Y.S. Tjandra N. Swenson R.E. Vasalatiy O. Comparison of solution properties of polymethylated DOTA-like lanthanide complexes with opposite chirality of the pendant arms.Inorg. Chem. 2019; 58: 15788-15800Crossref PubMed Scopus (7) Google Scholar,30Joss D. Walliser R.M. Zimmermann K. Häussinger D. Conformationally locked lanthanide chelating tags for convenient pseudocontact shift protein nuclear magnetic resonance spectroscopy.J. Biomol. NMR. 2018; 72: 29-38Crossref PubMed Scopus (17) Google Scholar The resulting C1-symmetry turned out to be crucial for the magnetic properties of the lanthanoid complexes (see below). Even though the lanthanoid complexes display only C1-symmetry, they contain a C4-pseudosymmetric axis. The C4-pseudosymmetry axis is normal to the cyclen-N4-plane going through the lanthanoid. While a rough estimation of Δχax is possible by assigning only the strongest PCS, a reliable determination of Δχrh and of the orientation of the tensor also requires the careful assignment of the less strongly shifted resonances that vary tremendously for different lanthanoids. It was, therefore, of utmost importance to obtain trustworthy peak assignments for all Ln-DOTA-M7FPy complexes. The 3D structures of Ln-DOTA-M7FPy (Data S1) required to fit the anisotropy parameters were calculated using DFT geometry optimization (for details, see experimental procedures). As it is unclear whether a water molecule occupies the ninth coordination site, the structure was calculated for all four possible combinations of implicit and explicit water at the apical position. The best correlation coefficients were obtained for 8-fold coordination with implicit water.NMR analysis and synthesis of isotopically labeled Ln-DOTA-M7FPyLn-DOTA-M7FPy shows 29 resonances in the 1D proton spectra. From these signals, 26 can be used to fit the anisotropy parameters (Figure 2). The three resonances from the phenyl sulfone were excluded from the fitting process because the phenyl rotation induced large motional averaging of the PCS. We considered only the proton resonances as it was previously shown that they are not significantly affected by contact shifts.26Suturina E.A. Mason K. Geraldes C.F.G.C. Kuprov I. Parker D. Beyond Bleaney’s theory: experimental and theoretical analysis of periodic trends in lanthanide-induced chemical shift.Angew. Chem. Int. Ed. Engl. 2017; 56: 12215-12218Crossref PubMed Scopus (39) Google Scholar Without restrictions, 4∗1026 different assignments would be possible with 26 shifts. In order to restrict the combinatorial space to a manageable number, all 26 signals were classified using spectroscopic data. The proton NMR spectra of the diamagnetic (La, Lu) complexes, as well as the only weakly paramagnetic (Sm, Eu) compounds were assigned by classical 2D NMR (HSQC, HMBC, COSY, NOESY, or ROESY) spectroscopy. The stronger PREs in the cases of Ce, Pr, and Nd allowed only for the recording of COSY spectra. COSY correlations were still observable as the signal intensity profited from a paramagnetic contribution dampening the effect of the PRE.31Bertini I. Piccioli M. Tarchi D. Luchinat C. COSY spectra of paramagnetic macromolecules: observability, scalar effects, cross-correlation effects, relaxation-allowed coherence transfer.Concepts Magn. Reson. 1994; 6: 307-335Crossref Scopus (42) Google Scholar,32Bertini I. Luchinat C. Tarchi D. Are true scalar proton–proton connectivities ever measured in COSY spectra of paramagnetic macromolecules?.Chem. Phys. Lett. 1993; 203: 445-449Crossref Scopus (91) Google Scholar Based on COSY correlations, all proton signals of Ce-, Pr-, and Nd-DOTA-M7FPy were classified (supplemental information) (Tables S1–S3).For the late lanthanoids, no 2D NMR spectra were recorded as the magnetization lifetimes were extremely shortened by the much stronger PRE and did not allow for polarization transfer or dipolar interactions. Therefore, two different labeling schemes were employed that would in principle allow the classification of signals directly from the 1D spectra. The synthesis of the isotope labeled complexes was adapted from known synthetic strategies.20Müntener T. Kottelat J. Huber A. Häussinger D. New lanthanide chelating tags for PCS NMR spectroscopy with reduction stable, rigid linkers for fast and irreversible conjugation to proteins.Bioconjug. Chem. 2018; 29: 3344-3351Crossref PubMed Scopus (26) Google Scholar,33Müntener T. Thommen F. Joss D. Kottelat J. Prescimone A. Häussinger D. Synthesis of chiral nine and twelve-membered cyclic polyamines from natural building blocks.Chem. Commun. (Camb). 2019; 55: 4715-4718Crossref PubMed Google Scholar, 34Häussinger D. Huang J.R. Grzesiek S. DOTA-M8: an extremely rigid, high-affinity lanthanide chelating tag for PCS NMR spectroscopy.J. Am. Chem. Soc. 2009; 131: 14761-14767Crossref PubMed Scopus (131) Google Scholar, 35Müntener T. Protein PCS NMR spectroscopy under physiological conditions: development of a new high yield cyclization method for rigidified DOTA-based tags with sulfhydryl-reactive activators forming a reductively stable linkage. Phd Thesis (University of Basel, Faculty of Science, 2019Google Scholar The isotope labels on the cyclen ring were either introduced by elongating the alanine based trimer 12 with an isotope enriched alanine derivative 7 (2H) (Figure 3B) or 27 (13C) (Figure S2) or by a reduction of macrocycle 47 with LiAlD4 (Figure S4). The alkylation of M4-cyclen (16, 34, or 49 (Figures 3B and S2–S4) or of the twice alkylated M4-cyclen (20 or 54 (Figures 3B, S2, and S4) leads to a scrambling of the isotope labels over four or two positions, respectively. If the isotopes were introduced through the labeled alanine 6 (2H) (Figure 3B) or 24 (13C) (Figure S2) an observed labeling of 50% or 25% at the respective positions was achieved (1–3 Figure 3A). The introduction of the isotope label at a later step in the synthesis by reduction of the macrocycle 44 with LiAlD4 (Figure S4) resulted in an observed labeling of 100% or 50% (4 and 5 Figure 3A), depending again on the order of the debenzylation and alkylation steps. Ln-DOTA-M7FPy (1) was synthesized for all lanthanoids (NMR spectra Figures S30–S85) apart from Pm; complexes 2 to 5 were only synthesized for the late lanthanoids Tb to Yb. Surprisingly, La-, Ce-, Pr-, and Nd-DOTA-M7FPy dissociated at pH <3, with the highest rate of dissociation found for La and the lowest for Nd, thus following the trend in the ionic radii.Figure 3Synthesis of labeled complexesShow full caption(A) All labeling schemes used throughout this study. Blue dots mark positions that were labeled with deuterium 2H. Red dots mark positions that were labeled with carbon 13C.(B) Exemplary synthetic route towards two (2 and 3) of the five labeled complexes 1–5: (i) (1) LiAlH4, THF, 0°–5°C; (2) Cbz-Cl, H2O, EtOAc, Na2CO3; (3) IBX, EtOAc, 77°C. (ii) (1) T3P, DIPEA, EtOAc; (2) NaBH4, MeOH, H2O, THF, 0°–5°C; (3) IBX, MeCN, 80°C. (iii) (1) NaBH(CH3COO)3, DCM; (2) Pd/C H2, MeOH. (iv) (1) NaBH(CH3COO)3, DCM; (2) BnBr, MeCN, 45°C. (v) (1) HBr, AcOH, 40°C; (2) HATU, MeCN. (vi) LiAlH4, DCM, −78°C. (vii) Pd/C H2, TFA, MeOH. (viii) OTf-Lac-O-t-Bu, K2CO3, DCM, MeCN. (ix) OTf-Lac-O-t-Bu, K2CO3, DCM, MeCN. (x) Pd(OH)2/C, NH4(HCOO), EtOH. (xi) OTf-Lac-O-t-Bu, K2CO3, DCM, MeCN. (xii) (1) K2CO3, MeCN; (2) HCl, MeCN, 80°C; (3) aq. NH4(CH3COO), Ln(III), 80°C. Below each labeled molecule, the observed labeling percentage is noted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Carbon 13C labeling in combination with selectively 1H decoupled 13C spectra (Figures S105–S109) could be used to classify the cyclen methyl signals. Attempts to extend selective decoupling to classify other proton signals by labeling different positions with 13C were not successful as the intensity gain from the decoupling was no longer observable due to the stronger PRE. Deuterium labeling (spectra Figures S113–S166) on the other hand, which is less sensitive to PRE, allowed the classification of all proton signals for the late lanthanoids (for more details, see supplemental information) (Tables S4–S7). Four different deuterium labeling schemes were synthesized in order to sufficiently restrict the combinatorial space. Due to the late stage scrambling of the isotope labels, the amount of labeled material and the synthetic effort were greatly reduced. Careful control of the pH (50 mM phosphate buffer, pH 6.0, Table S15) and of the residual trifluoro acetic acid (TFA) from the HPLC purification was necessary to obtain reproducible chemical shifts (Figure S86).Intrinsic anisotropy parametersClassification of proton signals by either COSY spectra or labeling schemes reduced the possible assignments to a manageable number. The anisotropy parameters were therefore fitted for all possible remaining assignments (Figures S13–S25). Based on the spectroscopic data from Sm and Eu, we were able to show that the correct assignment is found among the ones that correlate best with the DFT structure. However, in two out of four simulations, it is not the one yielding the highest correlation coefficient. For the remaining lanthanoids, the anisotropy parameters could nonetheless be determined as they were highly conserved among the best correlated assignments. For Er, Tm, and Yb, the anisotropy parameters were determined unambiguously since only one set of anisotropy parameters was found, while for Ce, Pr, Nd, Tb, Dy, and Ho, two sets of anisotropy parameters were found, which had similar magnitude and rhombicity but show slightly differing orientation. For the further discussion, the set of anisotropy parameters that appears with overall higher correlation coefficients were chosen for the latter ones (for more details, see supplemental information) (Figure S7, Table S8). The final anisotropy parameters of Ln-DOTA-M7FPy determined on the free tag (final assignment Tables S9 and S10, full tensors Table S13) and on the protein are depicted in Figure 4. Uncertainties were determined using Monte Carlo simulations (for details, see experimental procedures). For most of the lanthanoids on ubiquitin, the uncertainty had to be estimated as the Monte Carlo simulation generated several sets of anisotropy parameters defining tensors of similar size but differing orientations. To avoid ambiguities, all anisotropy parameters are represented in the unique tensor representation (UTR).36Schmitz C. Stanton-Cook M.J. Su X.C. Otting G. Huber T. Numbat: an interactive software tool for fitting deltachi-tensors to molecular coordinates using pseudocontact shifts.J. Biomol. NMR. 2008; 41: 179-189Crossref PubMed Scopus (145) Google Scholar The most striking result is the size of Δχax and Δχrh—the intrinsic anisotropy parameters exceed by far the ones reported for typical LCT. Even the very recently published DOTA-M7-Nitro LCT,37Joss D. Winter F. Häussinger D. A novel, rationally designed lanthanoid chelating tag delivers large paramagnetic structural restraints for biomolecular NMR.Chem. Commun. (Camb). 2020; 56: 12861-12864Crossref PubMed Google Scholar which is unique in the size of its anisotropy parameters among LCT, has 5%–10% smaller values. A comparison between the anisotropy parameters on the free LCT and ubiquitin S57C shows that about 80% of the anisotropy is lost due to motional averaging of the protein tether.38Suturina E.A. Häussinger D. Zimmermann K. Garbuio L. Yulikov M. Jeschke G. Kuprov I. Model-free extraction of spin label position distributions from pseudocontact shift data.Chem. Sci. 2017; 8: 2751-2757Crossref PubMed Google Scholar,39Shishmarev D. Otting G. How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic metal tags ? A modelling study.J. Biomol. NMR. 2013; 56: 203-216Crossref PubMed Scopus (58) Google Scholar The loss shows a slightly increasing trend going from Ce to Yb. There are two likely reasons for this trend: (1) The small magnitude of the observed PCS of the early lanthanoids makes them more susceptible to experimental errors, which result in an overestimation of the anisotropy parameters, or (2) The higher probability of the early lanthanoids to coordinate an additional donor at the ninth coordination site slightly reduces the motional averaging. It is noteworthy that the residual dipolar coupling (RDC)-derived Δχax parameters are very similar to the values obtained independently by PCSs. The transfer of the full anisotropy potential to the protein by further rigidifying the attachment will result in the observation of significant and easy-to-detect PCSs in the range of 0.01 ppm for spins at a distance of up to 175 Å, thus opening a perspective for unique long-range restraints for structural work. In principle, the d