All organic precursors and solvents were obtained from commercial sources and used as received unless otherwise stated. Human serum albumin (HSA, 66.7 kDa) was purchased from Fluka. The monoclonal antibody (3G4) was generously provided by Paragrine Pharmaceuticals. 1
H and 13
C NMR spectra were acquired on a JEOL Eclipse 270 spectrometer operating at 270.17 and 67.93 MHz, respectively. Chemical shifts (δ) are reported in parts per million. CEST spectra and T1
values were measured using either a JEOL Eclipse 270 or a Varian INOVA 500 spectrometer (operating at 499.99 MHz). Prior to data acquisition, samples were warmed to room temperature and equilibrated in the probe at 25 °C for at least 10 min before measurement. T1
was measured by using an inversion-recovery sequence with 15 different delay times and 8 averages. CEST spectra were recorded by application of a long presaturation pulse at selected frequencies across the spectrum followed by a single observe pulse to measure the residual water signal. Infrared spectra were recorded on a Nicolet Avatar 360 FT-IR spectrometer as either KBr pellets or as neat liquids. UV-vis absorption spectra were recorded using a Shimadsu UV-1601 double beam diode array spectrophotometer. Hydrogenations were performed using a Parr hydrogenation apparatus. [Eu3+
] was determined analytically using an Elan 6100 DRC (PE Scien) ICP Mass Spectrometer. Electrospray ionization mass spectrometry (ESI-MS) was performed by HT Laboratories, San Diego, California. Fast atom bombardment mass spectrometry (FAB-MS) was performed by the Mass Spectrometry Facility at University of Alabama at Tuscaloosa. Elemental analyses were obtained from Galbraith Labs, Knoxville, Tennessee. Gel electrophoresis was performed on Pharmacia LKB (Phast System) using phast gel (Gradient 4–15 Amersham Bioscience). 1-Benzyloxycarbonyl-1,4,7,10-tetraazacyclododecane trihydrochloride salt (referred to as mono-protected CBz-cyclen)33
, and 1,4,7,10–tetra(methylcarbonylamide)-1,4,7,10-tetraaza cyclododecane (DTMA)28
were prepared using published methods.
(55.3 g, 400 mmol) and methylamine hydrochloride (13.5 g, 200 mmol) were added to dichloromethane (700 mL) and the suspension was cooled to 0 °C. Bromoacetyl bromide (39.75 g, 200 mmol) was then added dropwise with vigorous stirring, and the reaction mixture was stirred at 0 °C for an additional 1 h followed by 2 h at room temperature. Water (70 mL) was then added and the organic layer that separated was dried overnight over Na2
. The solvent were removed under reduced pressure and the residue was crystallized from diethylether to afford a colorless compound (23.4 g, 77% yield). 1
H NMR (270 MHz, CDCl3
): δ = 2.8 (3H, d, CH3
), 3.9 (2H, s, CH2
), 6.8 (1H, s br, NH); 13
C NMR (67.5 MHz, CDCl3
): δ = 27.0 (CH3
), 29.2 (CH2
), 166.1 (CONH). NMR data is consistent with published data 28
2-p-Nitrobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10 -tetra(methylcarbonylamide) (5)
K2CO3 (1.25 g, 9 mmol) was added to a solution of p-NO2 benzyl cyclen 4 (0.6 g, 1.95 mmol) in acetonitrile (10 mL) and the suspension was heated at 70 °C for 30 min. 2-bromo-N-methylacetamide (1.25 g, 8.21 mmol) was then added in one portion. The reaction mixture was heated for 7 days at 65 – 70 °C, allowed to cool and filtered. The solvents were then removed under reduced pressure. The resulting residue was purified by column chromatography over silica gel. The column was first eluted with dichloromethane to remove impurities followed by elution of the title compound with methanol. The residue obtained from the column was dissolved in chloroform, filtered and solvent was removed by vacuum to afford the title compound as a yellow solid (0.68 g, 71 % yield). Rf = 0.4 (SiO2, MeOH). 1H NMR (270 MHz, D2O, pD 14): δ = 2.5 (4H, m br, ring CH2), 2.75–2.8 (12H, m, CH3), 3.01–3.12 (4H, m br, ring CH2), 3.2–3.4 (7H, m, ring CH2), 3.7–4.14 (10H, m, CH2Ar and CH2CO), 7.5 (2H, d, Ar), 8.2 (2H, d, Ar). 13C NMR (67.5 MHz, D2O pD 14): δ = 25.7–26.1 (CH3), 32.0 (CH2Ar), 51.01–52.5 (CH2 ring br), 56.1–56.4 (CH2CO) 124.1 (Ar), 130.5 (Ar), 146.4 (Ar), 147.8 (Ar), 171.0–173.7(CONH, br). FTIR (KBr): 1181, 1203, 1348, 1382, 1518, 1561, 1673, 2850, 2971, 3083, 3250. m/z (ESI+) 614 ([M+Na]+), 630 ([M+K]+). Anal. Found C = 51.7%, H = 7.8%, N =19.8% C27H45N9O6.2H2O requires C = 51.7%, H = 7.9%, N = 20.1%.
10% palladium (0.4 g) on carbon was added to a solution of 5 (0.41 g, 0.69 mmol) in ethanol (10 mL). The reaction mixture was placed in a hydrogenation vessel for 24 h with the H2 pressure set to 45 psi. The sample was mixed twice during this 24 h period for 10 – 15 min each time. The reaction mixture was filtered through Celite®545 (Aldrich) and the filtrate concentrated under reduced pressure to afford the title compund as a pale solid (0.39 g, 99 % yield). 1H NMR (270 MHz, D2O pD 14): δ = 2.60–3.67 (37H, m), 6.78 (2H, d, Ar), 7.02 (2H, d, Ar). 13C NMR (67.5 MHz, D2O pD 14): δ = 26.8-26.02 (CH3), 30.9 (CH2Ar), 51.3–52.6 (CH2 ring, br), 57.0–57.4 (CH2CO, br), 116.9 (Ar), 130.3 (Ar), 145.5 (Ar), 146.4 (Ar), 173.7–174.6 (CONH, br). FTIR (KBr): 1156, 1247, 1357, 1410, 1464, 1557, 1676, 2827, 2968, 3076, 3450 cm−1. m/z (ESI+) 562 ([M+H]+), 584 ([M+Na]+).
A solution of thiophosgene (0.051 g, 446 μmol) in chloroform (2 mL) was added to a solution of 6 (0.065 g, 116 μmol) in water (1 mL) and the mixture was stirred at room temperature for 2 hours. The water fraction was separated and the organic layer extracted with water (2 × 1 mL). The water fractions were combined and freeze dried to give the title compound as a pale solid (0.07 g, 99% yield). 1H NMR (270 MHz, D2O pD = 2) δ = 2.62–2.77 (12H, m, CH3), 3.05–4.11 (25H, m, CH2Ar, CH2CO and ring CH2), 7.31 (4H, m, Ar). FTIR (KBr): 1158, 1236, 1362, 1410, 1471, 1571, 1689, 2116 (N=C=S), 2947, 2975, 3099, 3267. m/z (ESI+) 604 ([M+H]+), 626 ([M+Na]+).
K2CO3 (3.37 g, 24.4 mmol) was added to a solution of mono-protected CBz-cyclen 7 (1.1 g, 2.44 mmol) in acetonitrile (20 mL), the suspension was heated warmed to 70 °C, and 2-bromo-N-methylacetamide (1.15 g, 24.9 mmol) was added in one portion. The reaction mixture was stirred at 65 – 70 °C for 12 hours, filtered, then concentrated under vacuum. The resulting residue was dissolved in water, extracted with dichloromethane (3 × 50 mL), and the combined organic fractions dried over Na2SO4 overnight. After filtering, the solvent was removed under reduced pressure and the residue purified by silica gel column chromatography, eluting with 10% methanol in chloroform to afford the title compound as a colorless gum (1.08 g, 85 % yield). Rf = 0.3 (SiO2, MeOH/CHCl3, 1:9). 1H NMR (270 MHz, CD3CN): δ = 2.82 (8H, s br, ring CH2), 2.89 (6H, s, CH3), 2.93 (3H, s, CH3), 3.14 (4H, s br, ring CH2), 3.30 (4H, s br, ring CH2), 3.53 (2H, s, CH2CO), 3.74 (4H, s, CH2CO), 5.3 (2H, s, OCH2Ph), 7.52 (2H, m br, NH), 7.58 (5H, m, Ph), 7.7 (1H, m br, NH). 13C NMR (67.5 MHz, CD3CN) δ = 25.4 (CH3), 25.6 (CH3), 47.0 (CH2 ring), 47.4 (CH2 ring), 53.4 (CH2 ring br), 58.3 (NCH2CO), 67.0 (OCH2Ph), 128.0 (Ph), 128.3 (Ph), 129.9 (Ph), 137.6 (Ph), 156.7 (CONH), 171.7 (CONH), 171.9.9 (COO). FTIR (NaCl pallet, CHCl3): 1049, 1119, 1157, 1219, 1417, 1533, 1673, 2827, 3013, 3056, 3352 cm−1. m/z (ESI+) 558 (100% [M+K]+).
10% palladium on carbon (0.2 g) was added to a solution of 8 (0.93 g, 1.5 mmol) in ethanol (30 mL). The reaction mixture was placed in a hydrogenation vessel for 72 h with the H2 pressure set to 50 psi. The sample was mixed for 10 – 15 min twice per day during this 72 h period. The catalyst was filtered through Celite®545 (Aldrich), the solvents were removed under reduced pressure to afford the title compound colorless oil (0.57 g, 99 % yield). 1H NMR (270 MHz, D2O pD 9): δ = 2.54 (4H, s br, ring CH2), 2.63 (4H, s br, ring CH2), 2.69–2.70 (9H, m, NHCH3), 2.77 (4H, s br, ring CH2), 3.06–3.10 (6H, m, CH2CONH), 3.23–3.24 (4H, s br, ring CH2). 13C NMR (67.5 MHz, D2O pD 9) δ = 25.8 (CH3), 26.9 (CH3), 44.9 (CH2 ring), 49.5 (CH2 ring), 51.1 (CH2 ring), 53.4 (CH2 ring), 56.3 (CH2CONH), 58.3 (CH2CONH), 174.1 (CONH), 174.5 (CONH). FTIR (NaCl pallet, CHCl3): 1111, 1212, 1305, 1417, 1546, 1647, 2842, 2924, 3091, 3317 cm−1. m/z (ESI+) 386 ([M+H]+), 408 ([M+Na]+). Anal. Found C = 45.3%, H = 9.0%, N = 21.5% C17H35N7O3 × 3.5H2O requires C = 45.5%, H = 9.4%, N = 21.8%.
N-(2-Bromoacetyl) glycine benzyl ester (10)
K2CO3 (13.83 g, 10 mmol) was added to a solution of glycine benzyl ester, p-toluenesulfonate salt (1.68 g, 5 mmol) in dichloromethane (50 mL). The suspension was cooled to 0 °C and, with vigorous stirring, bromoacetyl bromide (0.99 g, 5 mmol) was added drop wise over ~5 min. After the addition was complete, the reaction mixture was stirred at 0 °C for 1 h and at room temperature for an additional 2 h. Water (10 mL) was then added and the organic layer was separated and dried over Na2SO4 overnight. The drying agent was then filtered and the solvent removed under reduced pressure. The resulting residue was crystallized from hexane to afford the title compound as a colorless solid (1.04 g, 73% yield). 1H NMR (270 MHz, CDCl3): δ = 3.9 (2H, s, CH2CO), 4.11 (2H, d, CH2CO2), 5.2 (2H, s, CH2Ph), 6.97 (1H, s br, NH), 7.36 (5H, m, Ph). 13C NMR (67.5 MHz, CDCl3): δ = 28.7 (CH2CONH), 42.0 (CH2CO2), 67.6 (CH2Ph), 128.6 (Ph), 128.8 (Ph), 134.9 (Ph), 165.8 (CONH), 169.1 (CO2). FTIR (KBr): 940, 1041, 1188, 1262, 1382, 1444, 1538, 1662, 1763, 2877, 2966, 3064, 3282 cm−1. m/z (ESI−) 284 (100% [M-H]−).
1- N-Acetyl benzyl glycinate-1,4,7,10-tetraazacyclododecane-4,7,10-tri(methylcarbonyl amide) (11)
K2CO3 (0.21 g, 1.5 mmol) was added to a solution of 9 (0.5 g, 1.3 mmol) in acetonitrile (10 mL), the suspension was heated for 20 min at 70 °C, and N-(2-bromoacetyl) glycine benzyl ester 10 (0.43 g, 1.5 mmol) was added in one portion. The reaction mixture was stirred at 65 – 70 °C overnight, then filtered and solvent removed under reduced pressure. The resulting residue was purified by silica gel column chromatography. The column was first eluted with CHCl3/MeOH (9.5:0.5) to remove impurities, and the title compound was then eluted with CHCl3/MeOH/NH4OH (3:2:1) as the slowest moving fraction. The fractions were concentrated, dissolved in 2% methanol in chloroform, and filtered and the solvents were removed under vacuum to afford title compound (0.61 g, 79.5 % yield). Rf = 0.65 (SiO2, CHCl3/MeOH/NH4OH, 3:2:1). 1H NMR (270 MHz, CD3CN ): δ = 2.56–3.19 (33H, ring CH2, CH3, CH2CO), 3.95 (2H, d, NHCH2CO2), 5.13 (2H, s, OCH2Ph), 6.73 (4H, m br, CONH), 7.38 (5H, m, Ph). 13C NMR (67.5 MHz, CD3CN) δ = 25.3 (CH3), 25.5 (CH3), 41.0 (CH2COO), 50.1–50.9 (CH2 ring br), 56.9 (NCH2CO), 57.1 (NCH2CO), 66.5 (CH2Ph), 128.2 (Ph), 128.3 (Ph), 129.7 (Ph), 136.1 (Ph), 169.7 (CONH), 172.0 (CONH), 172.5 (CO2). FTIR (NaCl pallet, CHCl3): 672, 761, 1216, 1642, 1988, 3080, 3394 cm−1. m/z (ESI+) 591 ([M+H]+, 613 ([M+Na]+).
10% palladium on carbon (0.1 g) was added to a solution of 11 (0.45 g, 0.76 mmol) in ethanol (10 mL). The suspension was placed on a hydrogenation apparatus operating at 45 psi and room temperature for 96 hours. The catalyst was filtered through Celite®545 (Aldrich) and the solvents removed under reduced pressure to afford the title compound as a colorless amorphous solid (0.37 g, 97 % yield). 1H NMR (270 MHz, D2O pD = 8): δ = 2.72–2.73 (9H, 2 s, CH3), 2.9 (4H, s br, ring CH2), 3.1–3.4 (12H, m br, ring CH2), 3.5 (4H, s, CH2CONH), 4.73–4.75 (6H, m, CH2CO). 13C NMR (67.5 MHz, D2O) δ = 25.9 (CH3), 26.0 (CH3), 43.5 (CH2CO2), 49.81 (CH2 ring, br), 50.2 (CH2 ring, br), 51.3 (CH2 ring, br), 51.9 (CH2CONH), 54.6 (CH2CONH), 56.1 (CH2CO2), 166.2 (CONH), 169.5 (CONH), 172.0 (CONH), 172.9 (CONH), 172.9 (CONH), 176.0 (CO2). FTIR (NaCl pallet, CHCl3): 769, 1212, 1642, 1992, 3060, 3414 cm−1. m/z (ESI−) 499 (100 % [M-H]−).
General procedure for preparation of lanthanide chelates
An aqueous solution of the appropriate ligand was adjusted to a pH of ~5.5 using 1 M NaOH/1 M HCl as necessary. A stoichometric quantity of standardized lanthanide chloride aqueous solution was added and the reaction mixture was stirred at room temperature with constant adjustment of pH to 5.0 – 5.5 using 1 M NaOH. The extent of the chelation reaction was monitored by following changes in the reaction pH, and were typically observed to be complete ~30 min. The absence of free lanthanide ions was established with xylenol orange (0.15 M acetate buffer, pH 5.5). The pH was then adjusted to 7 and the samples were filtered and freeze-dried to give the chelates. Eu-1: FTIR (KBr): 2116 (N=C=S) cm−1; m/z (FAB+) 754 and 756 [Eu3+1 – 2H]+; Eu-2: m/z (FAB+) 649 and 651 [Eu3+2− - H]+; Eu-6: m/z (FAB+) 710 and 712 [Eu3+6− - H]+ with an appropriate isotope pattern was observed.
Conjugation of Eu-1 to HAS
HSA (40 mg, 0.6 μmol) was dissolved in 1.5 mL of HEPES/HCl buffer (5 mM, 140 mM NaCl, pH 8.6). Eu-1 (11.3 mg, 12 μmol) was dissolved in 0.15 mL of water and added to the protein solution. The reaction mixture was incubated at room temperature for 3 hours, then purified by dialysis against HEPES/HCl buffer (5 mM, 140 mM NaCl, pH 7.4) for 48 h (4 × 2L) at room temperature and followed by gel permeation chromatography over Sephadex (G-25) equilibrated with HEPES/HCl buffer (5 mM, 140 mM NaCl, pH 7.4). The purified conjugate (~2.2 mL) was concentrated to ~0.2 mL using an Ultrafree-MC Microcentrifuge filter (NMWL 5000 Da). After the first concentration step, 0.3 mL of fresh buffer was added and the volume was reduced again to ~0.4 mL.
Conjugation of Eu-1 to 3G4
3G4 (23 mg/mL, 77 nmol) was dialyzed in a Tube-O-Dialyzer™ (2 mL, GBioscences) against HEPES/HCl buffer (5 mM, 140 mM NaCl, pH 8.6) for 24 h (2 × 2L) at 4 °C. The final concentration was obtained by measuring the absorbance at 280 nm (ε = 1.4 mL/(cm × mg)). Eu-1 (3.6 mg, 3.85 μmol) was dissolved in 0.1 mL of water and added to the 3G4 solution (22 mg/mL). The mixture was incubated at room temperature for 3 hours, followed by dialysis against HEPES/HCl buffer (5mM, 140 mM NaCl, pH 7.4) for 48 h (4×2L) at 4°C. The purified conjugate was concentrated using absorbent (Spectra/Gel Absorbent, Spectrum®, Spectrum Lab) for 6 h at 4 °C.
Europium 1-N-Hydroxysuccinimidylacetatoamido glycinate-1,4,7,10-tetraazacyclododecane- 4,7,10-tri(methylcarbonylamide) chloride was obtained by the method of Lewis et al. (29). Eu-2 (0.11 g, 0.12 mmol) was dissolved in water (0.86 mL, 4 °C), and the pH adjusted to 5.5, followed by addition of N-hydroxysuccinimide (13.7 mg, 0.12 mmol) in 0.1 mL of water. The reaction mixture was stirred for 5 min and 0.02 mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (12.2 mg, 60 μmol) aqueous solution was added. The reaction mixture was stirred for 30 minutes at 0 °C followed by pH adjustment to 7.5 with 0.25 M Na2HPO4. The europium activated ester chelate 12 was used for conjugation without isolation.
Conjugation of activated chelate 12 to HAS
HSA (40.0 mg, 0.6 μmol) was dissolved in 0.78 mL of HEPES/HCl buffer (5 mM, 140 mM NaCl, pH 7.4) followed by addition of the europium activated ester chelate 12 (theoretical amount: 60 μmol). The reaction mixture was then incubated at room temperature for 2 hours. The reaction mixture was purified by dialysis against HEPES/HCl buffer (pH 7.4) for 48 h (4 × 2L) at room temperature followed by gel permeation chromatography over Sephadex (G-25) equilibrated with HEPES/HCl buffer (pH 7.4). The purified conjugate (~2.2 mL) was concentrated to ~0.2 mL using Ultrafree-MC Microcentrifuge filters (NMWL 5000 Da). After the first concentration step, 0.3 mL of fresh buffer was added and the once again the volume was reduced to ~0.4 mL.
Determination of Eu-Antibody/Protein Ratio
The number of europium chelates per antibody or HSA was measured by Inductively Coupled Plasma Mass Spectrometry (ICP MS). The uncertainty in the measurement reflects the deviation of three parallel measurements (n = 3). The peak intensities were fitted to a four point calibration curve to obtain a mean value. The antibody/protein concentrations were determined by standard Lowry assay. Three measurements were performed per each conjugate preparation.
Eu-1 – 3G4 samples were diluted with 2% fetus bovine serum (FBS) in PBS buffer to a concentration of 2 mg/mL antibody. As a control, unmodified 3G4 was used at the same concentration. Phosphatidylserine (50 μL, 10 μg/mL) in chloroform was added to each well in the 96-well plate and chloroform was evaporated at room temperature. 0.2 mL of the blocking buffer (10% FBS in PBS) was added to each well and this was incubated at 37 °C for 1 hour. The blocking buffer was removed and the plate was washed three times with PBS buffer. Samples were prepared in duplicate using a two fold dilution of 2 mg/mL 3G4 with blocking buffer. After incubating the plate at 37°C for 2 hour, it was washed three times with PBS to remove unbound 3G4. 0.1 mL of 0.8 μg/mL HRP (Horseradish peroxidase) conjugated goat anti-human IgG was then added to each well and the samples were incubated at 37°C for 1 h. The plate was then washed three times with PBS buffer followed by addition 100 μL of a developing reagent (10 mL of 0.2 M Na2PO4, 10 mL of 0.1 M citric acid, 10 mg of 1,2 -diaminobenzene, 10 μL of H2O2) to detect the second antibody. The samples were incubated for 10 min, followed by addition of 0.1 mL of 0.18 M H2SO4 to stop the reaction. The optical density at 490 nm was read using a 96 well plate reader (7520-Microplate reader, Cambridge Technology, Inc).
Fitting of CEST spectra (Z-spectra) to a three pool exchange model
The water exchange kinetics of each chelate-conjugate were determined by fitting the acquired Z-spectra to the Bloch equations modified for exchange using a process described previously34
. In general EuDOTA-tetraamide chelates can only be fitted by using a 3-pool exchange model that includes bulk water, the Eu3+
-bound water molecule and the ligand amide protons (typically four per chelate). The amide protons in these Eu3+
chelates experience rather small paramagnetic shifts and are often not fully resolved from the bulk water peak itself. Nevertheless, inclusion of these known exchange sites in the fitting procedure is important in correctly fitting the shape of the bulk water peak and in obtaining the correct exchange rate constants for the more highly shifted Eu3+
-bound water exchange peak.
For Eu3+ chelates conjugated to a protein, the CEST fitting procedure is further complicated by a large number of exchangeable protons and/or water molecules closely associated with the protein. In order to fit these spectra it is important to be mindful of the way in which various parameters affect the fitting routine. Each saturation peak must be fitted to an appropriate intensity and width (shape). The intensity of the peak is predominantly determined by two factors the concentration of the exchanging pool and the T1 of protons within that pool. In general it is preferable to experimentally determine both these parameters. However, the exchangeable protons associated with the protein represent a diverse range of protons and environments consisting of: amide, hydroxyl and amine protons of the protein as well as water molecule hydrogen bound to the protein structure. For this reason determining a precise value of concentration or T1 is not possible. And yet, it is possible to take advantage of the nature of the fitting routine’s reliance on these two parameters for determining peak intensity to produce a valid fitting without knowing either value exactly. The T1 value of this pool was allowed to float substantially and even take unrealistic values. In so doing the fitting routine itself effectively self corrects for any error in the chosen concentration value – if the concentration value is too high then the T1 value used in fitting will be shorter to compensate.
Equally this third pool is inhomogeneous in chemical shift and will necessarily be broad. But line-width (shape) in the model is predominantly determined by the exchange rate of each pool with the bulk (note, there is no exchange in this model between the bound water pool and the third exchanging pool) and T2. One may take advantage of this to overcome the shift problem by allowing the center of the peak to shift over a reasonable diamagnetic range (1–6 ppm) and allowing T2 to adopt unrealistically short values. In this way the peak is allowed to become wide enough to encompass spins of all chemical shifts. Since there is little or no shift difference between the bulk water pool and this third pool, exchange between these pools will have little effect upon the shape of either peak and the shape of the peak of the third pool will be predominantly determined by T2.
Since it is exchange between bound and bulk water pools that is being probed the values concerning the bulk water and Eu3+
-bound water pools were then tightly constrained: T1
values were experimentally determined for the bulk and values previous determined for the bound pool in a related system employed.33
values were constrained to be shorter than T1
and fall within the range of values previously determined for similar systems. In this way the MATLAB™
fitting routine was constrained to fit the shape of the bound pool predominantly by altering water proton exchange alone, and was thus unaffected by the apparent increase in the width of the direct saturation peak at 0 ppm. This approach to fitting accommodates the signal arising from the protein without affecting the line-widths and shapes of the two exchange pools of interest. However, the fitting does as a consequence become inherently more dependent upon the line-shape of the bound pool; as a result, as B1
is increased and the spectrum broadens the values of τMH
obtained in the fitting shorten slightly because the shape of this peak is broader but less well defined and therefore provides a somewhat less reliable fit.