Physicochemical characterization of the dimeric lanthanide complexes [en{Ln(DO3A)(H2O)}(2)] and [pi{Ln(DTTA)(H2O)}(2)](2-): a variable-temperature O-17 NMR study

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1315

Physicochemical characterization of the dimeric

lanthanide complexes [enfLn(DO3A)(H

2

O)g

2

] and

[pifLn(DTTA)(H

2

O)g

2

]

2

−

: a variable-temperature

17

_{O NMR study}

Tzu-Ming Lee,

Tsan-Hwang Cheng,

Ming-Hung Ou,

C. Allen Chang,

Gin-Chung Liu

and

Yun-Ming Wang

1_{School of Medicinal and Applied Chemistry, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan} 2_{Department and Institute of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan} 3_{Department of Radiology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan}

Received 16 June 2003; Revised 18 September 2003; Accepted 23 September 2003

The Gd(III) complexes of the two dimeric ligands [en(DO3A)2] fN,N

-bis[1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-10-yl-methylcarbonyl]-N,N-ethylenediamineg and [pi(DTTA)2]8−

[bisdiethylene-triaminepentaacetic acid (trans-1,2-cyclohexanediamine)] were synthesized and characterized. The 17_O

NMR chemical shift of H2O induced by [enfDy(DO3A)g2] and [pifDy(DTTA)g2]2− at pH 6.80 proved

the presence of 2.1 and 2.2 inner-sphere water molecules, respectively. Water proton spin–lattice relaxation rates for [enfGd(DO3A)(H2O)g2] and [pifGd(DTTA)(H2O)g2]2− at 37.0± 0.1◦C and 20 MHz

are 3.60± 0.05 and 5.25 ± 0.05 mM−1s−1 per Gd, respectively. The EPR transverse electronic relax-ation rate and 17_{O NMR transverse relaxation time for the exchange lifetime of the coordinated H}

2O

molecule and the 2_{H NMR longitudinal relaxation rate of the deuterated diamagnetic lanthanum}

complex for the rotational correlation time were thoroughly investigated, and the results were com-pared with those reported previously for other lanthanide(III) complexes. The exchange lifetimes for [enfGd(DO3A)(H2O)g2] (769± 10 ns) and [pifGd(DTTA)(H2O)g2]2− (910± 10 ns) are significantly higher

than those of [Gd(DOTA)(H2O)]− (243 ns) and [Gd(DTPA)(H2O)]2− (303 ns) complexes. The rotational

correlation times for [enfGd(DO3A)(H2O)g2] (150± 11 ps) and [pifGd(DTTA)(H2O)g2]2−(130± 12 ps) are

slightly greater than those of [Gd(DOTA)(H2O)]−(77 ps) and [Gd(DTPA)(H2O)]2−(58 ps) complexes. The

marked increase in relaxivity (r1) of [enfGd(DO3A)(H2O)g2] and [pifGd(DTTA)(H2O)g2]2−result mainly

from their longer rotational correlation time and higher molecular weight. Copyright 2004 John Wiley & Sons, Ltd.

KEYWORDS:NMR; EPR;1_{H NMR;}2_{H NMR;}17_{O NMR; Gd(III) complexes; paramagnetic complexes; proton relaxation}

INTRODUCTION

Several lanthanide [e.g. Gd(III)] and transition metal [e.g. Mn(II) and Fe(III)] complexes of polyaminocarboxylates are either commercially available or in clinical trials for use as magnetic resonance imaging (MRI) contrast agents. The MR signal of body fluids can be altered by the presence of paramagnetic water relaxation agents to result in enhanced image contrast. The general design criteria for safe and efficacious MRI contrast agents have been reviewed by a number of investigators.1,2 _{However, they remain areas for} further research and development of MRI contrast agents, Ł_{Correspondence to: Yun-Ming Wang, School of Medicinal and} Applied Chemistry, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. E-mail: ymwang@kmu.edu.tw

Contract/grant sponsor: National Science Council of the Republic of China; Contract/grant number: NSC 91-2623-7009-002-NU.

particularly those with enhanced relaxivity and improved tissue targeting functions. The main barrier to this endeavor is the low sensitivity of MRI contrast agents when they are coupled with the low flux of most biochemical processes. This problem can be addressed by developing contrast agents that possess higher spin–lattice relaxivity
r1. The dimeric MRI contrast agents possess two gadolinium(III) ions and will increase relaxivity.

This has been confirmed for H6BO
DO3A2,3 in which the Gd(III) complex incorporates several desirable features. As the ligand is a DOTA [DOTA D 1,4,7,10-tetraaza-1,4,7,10-tetrakis(carboxymethyl)cyclododecane] derivative, we can expect high kinetic inertness and thermodynamic stability, and as a neutral complex it is preferable from the point of view of application (a less painful injection because of lower osmolality). The fact that two Gd3C ions are bound to one molecule allows for smaller injection volumes for the same total amount in mmol Gd kg
1body weight. Finally,

HOOC HOOC HOOC HOOC HOOC HOOC N H N H COOH COOH N HN N N N H N H N N N N COOH COOH COOH N N N N NH NH O O N H N H 3 COOCH₃ + SOCl2 MeOH H3COOC H3COOC MeOH K2CO3 2 EDDA DO3A 1

Scheme 1. Synthesis of en(DO3A)2.

the increased molecular weight and volume may result in a longer rotational correlation time, and thus in a higher proton relaxivity.

To extend these observations, [en
DO3A2]6
and [pi
DTTA2]8
, the derivatives of DO3A (1,4,7,10-tetraaza-cyclododecane-1,4,7-triaacetic acid) and DTPA [1,1,4,7,7-pentakis(carboxymethyl)-1,4,7-triazaheptane] were synthe-sized. The number of inner-sphere water molecules was determined from the17_{O NMR chemical shift of the water} as a function of Dy(III) concentration. The water proton spin–lattice relaxivity r1 of the [enfGd
DO3A
H2Og2] and [pifGd
DTTA
H2Og2]2
complexes at various temperatures and pH values are described. The EPR and17_{O NMR} trans-verse relaxation rate data were analyzed together in a simultaneous multiple-parameter least-squares fitting pro-cedure to determine the water residence lifetime.2_{H NMR} spectroscopy was used to determine the rotational correla-tion time.

EXPERIMENTAL

Materials

The acid forms of the free ligand, DO3A, were synthesized and characterized in accordance with the literature.4

Ethylenediaminediacetic acid dimethyl ester (1).

To a solution of EDDA (ethylenediaminediacetic acid, 28.41 mmol) in MeOH (150 ml) warmed to 40–50°C was added SOCl2(8.24 ml, 113.64 mmol) dropwise slowly. After

20 h, solvent was removed from the fraction containing the product by rotary evaporation. The residue was dried under vacuum and a white powder was obtained (4.76 g, 80.21%).1_{H NMR (D}

2O), υ (ppm): 3.48
NCH2CH2N, 3.75
NCH2COOCH3, 4.03
NCH2COOCH3.

en(DO3A)

(2) (Scheme 1).

DO3A (1.27 g, 3.66 mmol) and anhydrous methanol (120 ml) were mixed in a single-necked flask and the pH of the solution was adjusted to 11.59 with NH4OH. A water-bath was used to keep the temperature at 40–50°C. Compound 1(0.49 g, 2.4 mmol) was dissolved in anhydrous methanol (240 ml) and slowly added to a single-necked flask which contained DO3A and K2CO3 (10 g). After 48 h, the reaction solution was concentrated under reduced pressure to a pale yellow oil. The residue was dissolved in 50 ml of distilled water and made alkaline to pH 11.0 with ammonia solution. The solution was then applied to an AG1-X8 anion-exchange resin column (200–400 mesh, HCO2
form, 60 ml of resin and 3.0 cm column diameter). After passing through an anion-exchange resin column, the product eluted in the 0.035Mformic acid fraction. Solvent was removed from the

fraction containing the product by rotary evaporation and co-evaporated five times with 200 ml of water to remove the formic acid. The residue was dried in vacuum and a pale yellow hygroscopic powder was obtained (1.374 g, 88%) 13_{C NMR (50 MHz, D}

2O) (ppm): 179.2, 178.0, 173.0, 166.7, 58.6, 57.4, 54.2, 53.1, 51.8, 50.8, 47.1, 46.8, 45.0, 42.6. Anal.

HOOC HOOC HOOC HOOC COOH COOH COOH N HOOC N N O N N N O COOH COOH _COOH COOH O O O O H2N K2CO3 NH2 N N N N N N COOH COOH O O N H N H 4 DTPA 3 Pyridine Acetic Anhydride Acetonitrile

Scheme 2. Synthesis of pi(DTTA)2.

Calculated for C35H64N10O15: C, 48.60; H, 7.46; N, 16.19. Found: C, 48.55; H, 7.40; N, 16.10%.

Diethylenetriamine-N

_{-acetic acid-N,N}

_{-dianhydride (3).}

Pyridine (50 ml) and acetonitrile (50 ml) warmed to 50°C were mixed with a solution of DTPA (50 mmol, 19.7 g) in acetic anhydride (318 mmol, 32.4 g). After 24 h, solvent was removed from the fraction and the residue was washed with acetic anhydride and diethyl ether. The solid was then dried under vacuum and a white powder was obtained (16.6 g, 92%).1_{H NMR (200 MHz, DMSO-d}

6), υ (ppm): 3.71 (s, 8H, terminal NCH2CO2), 3.30 (s, 2H, central NCH2CO2), 2.72 (t, 4H, NCH2CH2N), 2.59 (t, 4H, NCH2CH2N).

[pi(DTTA)

] (4) (Scheme 2).

Compound 3 (2.3 g, 6.88 mmol), K2CO3 (10 g) and DMSO (100 ml) were mixed in a single-necked flask and trans-1,2-cyclohexanediamine (0.33 ml, 2.75 mmol) was slowly added. After 12 h, the solution was fractioned and filtered by ultrafiltration using ultrafiltration membranes YM3 (diameter 25 mm, MW cut-off D 3000) and YM1 (diameter 25 mm, MW cut-off D 1000). Solvent was removed by rotary evaporation. The residue was dried under vacuum and a pale-yellow oil was obtained (1.37 g, 43%).13_{C NMR} (100 MHz, D2O), υ (ppm): 172.91, 170.80, 166.81, 166.72, 56.64, 56.22, 54.40, 52.93, 52.44, 52.34, 53.03, 50.67, 50.38, 49.89, 31.28,

24.04. Anal. Calculated for C34H56N8O18: C, 47.22; H, 6.53; N, 12.96. Found: C, 47.16; H, 6.50; N, 12.64%.

General

GdCl3Ð6H2O (99.9%), DyCl₃Ð6H2O (99.9%) and LaCl3Ð7H2O (99.9%) were obtained from Aldrich and used without further purification. The concentrations of Gd3C, Dy3C and La3C were determined by chelatometric titration with EDTA using xylenol orange as indicator. All other reagents used for the synthesis of the ligand were purchased from commercial sources unless noted otherwise. 1_{H and} 13_{C NMR spectra} and elemental analyses were used to confirm the composition of the products.17_{O-enriched water (20.1%) was purchased} from Isotec.

Deuteration

The lanthanum complexes were synthesized by reaction of La2O3 with ligands in water and precipitated by addition of acetone. Deuteration of lanthanum complexes at the ˛-position with respect to carboxylate groups was performed using the procedure described by Wheeler and Legg.5 Deuteration was confirmed by1_{H NMR spectroscopy.}

Complexation

The Dy(III) and Gd(III) complexes were prepared by mixing solutions of hydrated LnCl3(10 mM) and ligand (10 mM) in a

Complex formation was instantaneous at room temperature. The solution was then evaporated under reduced pressure and the residue dried overnight at 60°C.

Proton T

measurements

The samples were prepared by dissolving a measured amount of the Gd(III) chelates in water at pH 6.80 using the buffer solution (0.10M) PIPES (PIPES D piperazine-N,N0

-bis-2-ethanesulfonic acid)–NaOH. The buffer solution was used to maintain a constant ionic strength (i.e. 0.10M). The

0.10Mbuffer was sufficient to maintain the solution pH at

6.80. The buffered Gd(III) chelate solutions were all allowed to equilibrate for at least 2 h. The pH of these solutions was determined immediately before relaxation time
T1 measurements.

Relaxation times of aqueous solutions of gadolinium(III) complexes with [en
DO3A2]6
and [pi
DTTA2]8
were measured to determine the relaxivity r1. All measurements were made at 20 MHz as a function of temperature on a Bruker Minispec NMS-120 NMR spectrometer. The samples were contained in 5 mm glass tubes. The spectrometer was tuned and calibrated before each measurement. The values of T1 were measured from eight data points generated by an inversion–recovery pulse sequence. The slope of plots of 1/T1versus the concentration of Gd(III) complex gives r1in mM
1s
1.

EPR measurements

EPR spectra were recorded at the X-band (0.34 T) using a Bruker ER 200D-SRC spectrometer operated in the continuous-wave mode. The samples were contained in the 1 mm glass tubes. The cavity temperature was stabilized using electronic temperature control of the gas flowing through the cavity. Temperature was verified by substituting a thermometer for the sample tube. Measurements were made from 273 to 363 K. The peak-to-peak linewidth was measured from the recorded spectra using the instrument’s software.

17

_{O NMR}

The hydration numbers of [enfDy
DO3Ag2] and [pifDy
DTTAg2]2
were determined using the method described by Alpoim et al.6 _The 17_{O NMR spectra were} recorded on a Varian Gemini-400 spectrometer at 25°C. Induced17_{O shift (d.i.s: dysprosium(III) induced}17_{O NMR} water chemical shift) measurements were made using D2O as an external standard. Dy(III) chelate solutions were prepared by combining solutions of Dy(III) and ligand in a 2 : 1 ratio, and a stoichiometric amount of standardized NaOH was added so that the complex was fully formed. Six solutions of various dysprosium(III) concentrations were prepared by serial dilution of the stock solution.

Measurement of the17_{O transverse relaxation rate was} carried out with a Varian Gemini-300 (7.05 T, 40.65 MHz) spectrometer, equipped with a 5 mm probe, by using an external D2O lock. Experimental settings were spectral width 10 000 Hz, pulse width 7µs, acquisition time 10 ms and no sample spinning. A Varian VT-J103 temperature control

– 12 – 10 – 8 – 6 – 4 – 2 0 10 20 30 Conc (mM) Chemical shift (ppm) [pi{Dy(DTTA)(H2O)}2]2– [en{Dy(DO3A)(H2O)}2] DyCl3

Figure 1. Dy(III)-induced water17_{O NMR shift versus Dy(III)}

chelate concentration in D2O at 25.0 š 0.1°C.

unit was used to stabilize the temperature. The value of the transverse relaxation rate was obtained by evaluating the linewidth at half-height
1/2of the water17O signal
R2 D1/2. Solutions containing 2.6% of the17O isotope were used.

2

_{H NMR}

The rotational correction time values of [enfLa
DO3Ag2] and [pifLa
DTTAg2]2
were determined by2H NMR spec-troscopy. The samples were prepared by dissolving the La3C complexes in D2O at pH 6.80. The measurement was carried out in a 10 mm o.d. tube on a Varian Gemini-400 (9.4 T) spec-trometer equipped with a broadband probe and measured by a substitution technique as described elsewhere.5

RESULTS AND DISCUSSION

Dy(III)-induced water

_{O NMR shifts}

Figure 1 shows the Dy(III)-induced water 17_{O NMR shifts} versus Dy(III) chelate concentration for solutions of DyCl3, [enfDy
DO3Ag2] and [pifDy
DTTAg2]2
in D2O at 25°C. The slopes obtained for [enfDy
DO3Ag2] and [pifDy
DTTAg2]2
at pH 6.80 are
102.6 ppm mM
1
r2 D 0.9764 and
107.8 ppm mM
1
r2 _{D 0.9762. On the other} hand, the slope for DyCl₃is
382.8 ppm mM
1
r2 _D0.999, and eight hydration numbers have been proposed for the dysprosium(III) ion.7 – 9 _{Therefore, [enfDy
DO3Ag}

2] and [pifDy
DTTAg2]2
complexes contain 2.1 and 2.2 inner-sphere water molecules, respectively, at pH 6.80. The actual number of inner sphere water molecules coordinated to the metal center for [enfDy
DO3Ag2] and [pifDy
DTTAg2]2
is one per Dy(III) ion. This result is similar to those for [pipfGd
DO3A
H2Og2] and [bisoxafGd
DO3A
H2Og2].10

Relaxometric studies of the gadolinium(III)

complexes

The longitudinal relaxivity r1 values of [enfGd
DO3A
H2Og2] and [pifGd
DTTA
H2Og2]2
are 3.60 mM
1s
1

per Gd and 5.25 mM
1s
1 _{per Gd at pH 6.80,} 37.0 š 0.1°C and 20 MHz, respectively. The r1 value of [enfGd
DO3A
H2Og2] is significantly higher than those of [Gd
DOTA]
_{(3.38 m}

M
1s
1 per Gd,

37.0°C)11 _{and [BOfGd
DO3A
H}

2Og2] (4.61 mM
1s
1 per Gd, 37.0 š 0.1°C)3 _{but lower than those of} [pipfGd
DO3A
H2Og2] (5.8 mM
1s
1 per Gd, 40.0°C)12

and [bisoxafGd
DO3A
H2Og2] (4.9 mM
1s
1 per Gd,

40.0°C).12 _{Also, the longitudinal relaxivity r}

1 value of [pifGd
DTTA
H2Og2]2
is significantly higher than that of the monomer [Gd
DTPA]2
_.

The origin of paramagnetic relaxation enhancement is generally divided into two components, inner-sphere and outer-sphere:13

1/TipD
1/Tiinner
sphereC
1/Tiouter
sphere i D 1, 2
1

Inner-sphere relaxation refers to relaxation enhancement of a solvent molecule directly coordinated to the paramagnetic ion, and outer-sphere relaxation refers to relaxation enhance-ment of solvent molecules in the second coordination sphere and beyond (i.e. bulk solvent). The inner-sphere relaxation contribution is obtained with the equation14

ris1pDCq/[55.6
T1MCM]
2 where C is the molar concentration of the gadolinium(III) complex, q is the number of water molecules bound to metal ion, T1Mis the longitudinal relaxation time of the bound water protons and M298is the residence lifetime of the bound water. Because of the inverse temperature dependence of T1Mand M298, two cases can be considered: (1) fast water-exchange
T1M×M, ris1pincreases as temperature decreases; (2) slow water-exchange
T1M − M, ris1p decreases as temperature decreases. Figure 2 displays a monoexponential decrease of observed relaxivity with increasing temperature in the range 278–343 K. This is characteristic of fast chemical exchange behavior, occurring when the M298of the coordinated water molecule is much shorter than T1Mof the bound water proton. In fact, Eqn (3)15_{can express T}

1M: 1 T1M D 2 15 _H2g2S
S C 1ˇ2 r6 H
3C1 1 C ω2 H2C1 C 7C2 1 C ω2 SC22 
3 1 Ci D 1 R C 1 M C 1 Si
4 where S is the electron spin quantum number (7/2 for Gd3C), H is the proton nuclear magnetogyric ratio, ˇ is the Bohr magneton, g is the Land´e factor for the free electron, rHis

the distance between the metal ion and the bound water protons, ωH and ωS are the respective proton and electron Larmor frequencies and Ci
i D 1, 2 is the correlation time of the modulation of the dipolar electron-proton coupling. The overall correlation time Cireceives contributions from M298, R298and S(the electronic relaxation time of the metal ion) [Eqn (4)]. To understand how M298and R298influence

0 2 4 6 8 10 12 14 16 18 273 293 313 T (K) 333 353 373 [pi{Gd(DTPA)(H2O)}2]2– [en{Gd(DO3A)(H2O)}2] r1 /mM –1 s –1

Figure 2. Temperature dependence of the relaxivity for [enfGd
DO3A
H2Og2] and [pifGd
DTTA
H2Og2]2
at pH

6.80 and 20 MHz.

r1of [enfGd
DO3A
H2Og2] and [pifGd
DTTA
H2Og2]2
, 17_{O and}2_{H NMR spectra were used to determine the values} of Mand R.

Water-exchange lifetime studies of Gd(III)

complexes

The measured peak-to-peak line widths, Hpp, of the derivative spectrum can be related to the overall transverse electronic relaxation rate, 1/T2e, vi`a Eqn (5), where gLis the isotropic Land´e g factor (gLD2.0 for Gd3C):16

1 T2e D gLB p 3 h Hpp
5

The temperature dependence of transverse electronic relaxation rates at the X-band (0.34 T) at pH 6.80 for 50 mM solution of [enfGd
DO3A
H2Og2] and [pifGd
DTTA
H2Og2]2
are shown in Figs 3 and 4. The data were fitted simultaneously with the following17_{O NMR} results. Analysis of the temperature dependence of the transverse relaxation rate for the 17_{O water nuclei is the} most accurate method for evaluating the exchange lifetime of the water molecules directly coordinated to the metal in a paramagnetic Gd3C chelate.17_{According to the Swift and} Connick theory,14_{the paramagnetic contribution
R}O

2pto the observed transverse relaxation rate is given by

RO 2pD Cq 55.6
 O M
1 RO 2M 2 C
O_M
1RO 2MCωOM 2  RO2MC
MO
1 2 CωO_M2
6

T (K) In (1/ T2e ) 19 273 293 313 333 20 21 22 23 24

Figure 3. Temperature dependence of transverse electronic relaxation rates at the X-band (0.34 T) and pH 6.80 for a 50 mM solution of [enfGd
DO3A
H2Og2].

ln (1/ T2e ) 20 21 22 23 24 25 T (K) 273 293 313 333

Figure 4. Temperature dependence of transverse electronic relaxation rates at the X-band (0.34 T) and pH 6.80 for a 50 mM solution of [pifGd
DTTA
H2Og2]2
.

where RO

2M represents the 17O transverse relaxation rate of the coordinated water molecule and ωO

Mthe chemical shift difference between the coordinated and bulk water17_{O NMR} resonances. R2Mis expressed by RO 2MD 1 3
A ¯h 2 S
S C 1
e1C e2 1 C ω2 s2e2 
7 and _ei
1DO_M
1CT
1ie
8

where S is the electronic spin quantum number [7/2 for Gd(III)], A/¯h is the Gd–17_{O scalar coupling constant and }

ei
i D 1, 2 represents the correlation time of the processes

modulating the scalar interaction. This modulation may occur through both the longitudinal and the transverse average electronic relaxation times (T1e and T2e) and the mean residence lifetime
O

M of the water molecule at the paramagnetic site.

The temperature dependence of RO

2M is determined by the temperature effect on O

M, v (the correlation time for modulation of the zero field splitting interaction) and ωO M according to
j
1T D
j
1298.15T 298.15 exp  Hj R
1 298.15
1 T 
9 ω_MOD gLBS
S C 1B 3kBT A ¯h
10

where the subscript j refers to the different correlation times, Hjis the activation enthalpy for the corresponding dynamic

process, B is the applied magnetic field strength and kBis the Boltzmann constant.

The water-exchange rates for [enfGd
DO3A
H2Og2] and [pifGd
DTTA
H2Og2]2
were obtained by measuring the17_{O NMR transverse relaxation rate
R}O

2pas a function of temperature. The data and its best simulation according to Eqns (5)–(10)16,18 _{are shown in Figs 5 and 6. As there} are a large number of parameters to be determined in the quantitative analysis of the 17_{O NMR transverse} relaxation rate
RO

2p versus T profiles, it is convenient to fix some of them. On this basis, in addition to the values of q and A/¯h

3.8 ð 106_{rad s}
1_{, the value of H}

M is fixed at 30 kJ mol
1.10 _{The parameters which provide} the best fit of the data for [enfGd
DO3A
H2Og2] and [pifGd
DTTA
H2Og2]2
are listed in Table 1. By varying the temperatures over a wide range, RO

2p is dominated by 1/M in the slow kinetic region at low temperatures and is dominated by 1/ei in the fast kinetic region at high temperature.

As shown in Table 1, the water-exchange lifetime M298 of [enfGd
DO3A
H2Og2] (769 š 10 ns) is simi-lar to those of [pipfGd
DO3A
H2Og2] (666 ns)10 and [bisoxafGd
DO3A
H2Og2] (714 ns)12but higher than that of [Gd
DOTA
H2O]2
(243 ns).18The higher water-exchange lifetimes for [enfGd
DO3A
H2Og2], [pipfGd
DO3A
H2Og2] and [bisoxafGd
DO3A
H2Og2] is perhaps due to the decreased number of carboxylate moieties which bind to the Gd(III) ion, so that the ligand is pulled less tightly around the metal center and is therefore less crowded around the water binding site.10 _{The water-exchange lifetime }

M298 of

Table 1. Kinetic and NMR parameters obtained from the simultaneous fit of17_{O NMR and EPR data for Gd(III)} complexes Complex 2
_s
2_ð1020_{ } v298(ps) M298(ms) R298(ps) H
kJ mol
1 Gd3Ca 1.19 7.3 1.2 41 15.3 [Gd(DOTA)
H2O]
a 0.16 11 243 77 49.8 [Gd(DTPA)
H2O]2
b 0.46 25 303 58 51.6 [pipfGd(DO3A)
H2Og2]b 0.17 š 0.01 19 š 2 666 171 š 12 34.2 š 1.8 [bisoxafGd(DO3A)
H2Og2]b 0.21 š 0.02 15 š 1 714 106 š 14 38.5 š 1.8 [enfGd(DO3A)
H2Og2] 0.49 š 0.02 17 š 1 769 š 10 105 š 11 35.2 š 1.0 [pifGd(DTTA)
H2Og2]2
0.90 š 0.01 14 š 2 910 š 10 130 š 12 45.0 š 1.8 a_{Data from Ref. 11.}

b_{Data from Ref. 15.}

T (K) 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 280 290 300 310 320 330 340 350 (s –1 ) R o 2p

Figure 5. Temperature dependence of the transverse water 17_{O relaxation rate at 7.05 T and pH 6.80 for a 50 mMsolution}

of [enfGd (DO3A)
H2Og2].

than that of [Gd
DTPA
H2O]2
(303 ns).10As described in the literature,19_{a }

M298of 1000 ns creates a situation wherein the exchange rate of water is a significant limiting factor determining relaxivity
r1when r1increases due to the R298 increase in these multidentate chelates, making a major con-tribution even for dimers. Therefore, the difference in r1 is not only one between rigid and non-rigid linkages, but is also due to R298in the higher molecular weight molecule.18

Rotational correlation time studies of La(III)

complexes

In diamagnetic molecules, the relaxation rate of the 2_H nucleus is predominantly determined by the quadrupolar mechanism,5_{which is strictly intramolecular and modulated} by the sole rotation of the molecule. For fast-tumbling systems, the relaxation rate is thus directly related to the rotational correlation time
R298:

R1D 1 T1 D 3 8
e2_qQ h 2 R
11

where the quadrupolar coupling constant
e2_{qQ/h depends} on the hybridization state of the C-atom carrying the 2_H

0 500 1000 1500 2000 2500 275 285 295 T (K) 305 315 325 (s –1 ) R o 2p

Figure 6. Temperature dependence of the transverse water 17_{O relaxation rate at 7.05 T and pH 6.80 for a 50 mMsolution}

of [pifGd
DTTA
H2Og2]2
. The line represents the

simultaneous least-squares fit to all data points as described in the text.

atom, its value being ¾170 kHz in the case of an sp3 C-atom. The measurement was performed on diamagnetic lanthanum(III) complexes deuterated in the ˛-position to the carboxylate groups. The values of R298for La(III) complexes with en(DO3A)26
, pip(DO3A)2

6

,12 _bisoxa(DO3A)

26
,11

[pi(DTTA)2]8
, DTPA10 and DOTA11 at 310 K are given in Table 1. The R298 values for Gd(III) dimers are significantly higher than those for Gd(III) monomers, [La

2_H

10DOTA]
(77 ps)11_{and [La}

2_H

10DTPA]2
(58 ps).11Thus, the change in the molecular weight in these complexes alters R sig-nificantly. On the other hand, the lower rotational cor-relation time
R298 values for [enfLa

2H10(DO3A)g2] and [bisoxafLa

2_H

10(DO3A)g2] compared with that of [pipfLa

2_H

10(DO3A)g2]18indicates that the more flexible linker between the two macrocyclic chelating moieties in en(DO3A)2

6

and bisoxa(DO3A)2 6

decreases the R value and causes the lower relaxivity
r1of the Gd(III) complexes. The relationship between r1 and the molecular weight of

0 1 2 3 4 5 6 7 0 200 400 600 800 Molecular Weight r1 /Gd (mM –1 s –1 ) 1000 1200 1400 1600

Figure 7. Correlation of molecular weight of gadolinium(III) complexes with HP-DO3A (
_{), PA-DO3A (}), B22F (

ž

°

Gd(III) monomer and dimer complexes19_{is shown in Fig. 7.} The results show that there is a strong correlation between

r1 and molecular weight, which means that the relaxivity value of the monomer generally increases with molecular weight. However, in order to maximize the relaxivity gain, the linking group and molecular weight must be taken into account for Gd(III) dimer complexes.

CONCLUSION

From analysis of the 17_{O NMR relaxometric properties,} the larger water-exchange lifetime M298 for [enfGd
DO3A
H2Og2] is perhaps due to a decrease in the number of carboxylate moieties, so that the ligand is pulled less tightly around the metal center and there is less crowding around the water binding site. We have demonstrated relaxivity
r1/Gd mM
1s
1 enhancement through the incorporation of rigidifying elements in the linkers or increasing the molecular weight of chelates. The water proton spin–lattice relaxivity of [pifGd
DTTA
H2Og2]2
is higher than that for the monomer [Gd(DTPA)]2
_{owing to its longer rotational} correlation time and greater molecular weight. Furthermore, approaches aimed at enhancing relaxivity by modulating the water-exchange lifetime, M298, will be important for the future development of molecular MRI contrast agents used in imaging biochemical processes.

Acknowledgement

We are grateful to the National Science Council of the Republic of China for financial support under Contract No. NSC 91-2623-7009-002-NU.

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