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A series of di- and tetraamide derivatives of DOTA were synthesized, and their lanthanide(III) complexes were examined by multinuclear 1H-, 13C-, and 17O-NMR spectroscopy, and compared with literature data of similar, known complexes (Table). All ligands formed structures similar to the parent [LnIII(DOTA)]− complexes, with four N-atoms and four O-atoms from DOTA and one O-atom from the inner-sphere water molecules. Interestingly, the lifetimes τM of the inner-sphere, metal-bound water molecules vary widely, ranging from nano- to milliseconds, depending on the identity of the pendent amide side chains. In general, positively charged [LnIII(DOTA-tetraamide)]3+ complexes display the longest residence times (high τM values), while complexes with additional charged functional groups on the extended amides display much smaller τM values, even when the side groups are not directly coordinated to the central Ln3+ ions. The design of novel [LnIII(DOTA-tetraamide)]3+ complexes with a wide, tunable range of τM values is of prime importance for the application of fast-responding, paramagnetic chemical-exchange-saturation-transfer (PARACEST) imaging agents used for the study of physiological and metabolic processes.

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3′-5-triiodo-l-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats ( fa/fa) after a 24-h fast. 2H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by 2H NMR analysis. In T3-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. 13C NMR analysis also revealed increased phospho enolpyruvate carboxykinase flux (4×), increased pyruvate cycling flux (4×), and increased TCA flux (5×) in T3-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated 2H and 13C NMR analysis of plasma glucose.

To investigate the role of Angptl4, an inhibitor of lipoprotein lipase that is induced by >3-fold in the heart after rosiglitazone treatment, we generated transgenic mice that overexpress Angptl4 in the heart (MHC-Angptl4). We show that MHC-Angptl4 mice exhibit cardiac-restricted expression of the transgene and inhibition of cardiac lipoprotein lipase (LPL) activity. However, LPL activities in other tissues or that released into plasma by heparin are not affected. In addition, MHC-Angptl4 mice also exhibit hypertriglyceridemia after 6 h of fasting. We use echocardiography to show that MHC-Angptl4 mice develop left-ventricular dysfunction. Comparison of the metabolic profiles of isolated working hearts demonstrates that cardiac impairment in MHC-Angptl4 mice is positively associated with decreased triglyceride (TG) utilization. When bred to transgenic mice that overexpress acyl-CoA synthetase in the heart, a strain that exhibits elevated cardiac TG accumulation, cardiac TG content in double transgenic mice is reversed to that of wild-type mice. Taken together, our data support the hypothesis that induction of Angptl4 in the heart inhibits lipoprotein-derived fatty acid delivery. This mouse model will be useful to elucidate the role of reduced fatty acid supply in the pathogenesis of heart failure and related disorders.

A recently introduced tracer, [3,4-(13)C(2)]glucose, was compared to the widely used tracer, [6,6-(2)H(2)]glucose, for measurement of whole-body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4-(13)C(2)]glucose, [6,6-(2)H(2)]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by (13)C NMR (for [3,4-(13)C(2)]glucose) or (1)H and (2)H NMR (for [6,6-(2)H(2)]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 +/- 4 micromol/kg/min. Since (2)H and (13)C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4-(13)C(2)]glucose does not interfere with measurements of (13)C or (2)H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously.

The chemistry of Gd(3+)-based MRI agents has advanced considerably during the past decade toward agents with higher relaxivity and agents that respond to physiology and/or metabolism. This review describes various approaches that have been taken toward the development of responsive contrast agents and discusses the importance of fast water exchange for advancement of targeted Gd(3+)-based agents with higher sensitivity. The recent discovery of Eu(3+) complexes having extraordinarily slow water exchange has opened a new avenue in contrast agent design based upon the chemical exchange saturation transfer (CEST) mechanism. These new paramagnetic complexes called PARACEST agents offer new possibilities of imaging biological functions such as tissue pH and metabolite levels. The lower detection limits that may apply to each class of contrast agent (Gd(3+)-based versus PARACEST) are discussed and the extent to which they may be applied to the imaging of β-cells is considered.

Addition of a benzyl substituent to the macrocyclic ring of DOTA has a substantial impact on the conformational ring flipping motion of the macrocycle in the resulting LnDOTA complexes. The p-NO2-benzyl substituent in the Ln(p-NO2-Bn-DOTA)- complexes lies in an equatorial position and effectively "locks" the conformation of the ring into the deltadeltadeltadelta configuration. The presence of the p-NO2-benzyl group also increases the population of the square antiprismatic (SAP) coordination isomer for all Ln(p-NO2-Bn-DOTA)- complexes relative to that seen for the respective LnDOTA- complexes. Despite this increase in SAP isomer population, the rate of water exchange in these complexes remains comparatively fast. The kinetic and thermodynamic stabilities of the Ln(p-NO2-Bn-DOTA)- complexes are also slightly lower than the corresponding LnDOTA- complexes but appear to be sufficiently high for in vivo use.

Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using 13 C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.5 mM (U- 13 C)glucose plus 50 mU/mL insulin until newly synthesized glycogen recovered to new steady-state levels ( � 60% of pre-depleted values). After a short wash-out period, the perfusate glucose was then switched to (1- 13 C)glucose, and glycolysis and glycogenolysis were stimulated by addition of glucagon (1mg/ml). A 13 C NMR multiplet analysis of the methyl resonance of lactate provided an estimate of pyruvate derived from glucose vs glycogen while a multiplet analysis of the C4 resonance of glutamate provided an estimate of acetyl-CoA derived from glycolytic pyruvate vs glycogenolytic pyruvate. These two indices were not equivalent and their difference was further magnified in the presence of insulin during the stimulation phase. These combined observations are consistent with functional compartmentation of glycolytic and glycogenolytic enzymes that allows pyruvate generated by these two processes to be distinguished at the level of lactate and acetyl-CoA. Copyright # 2004 John Wiley & Sons, Ltd.

A general synthetic route to two DOTA-linked N-Fmoc amino acids (DOTA-F and DOTA-K) is described that allows insertion of DOTA at any endo-position within a peptide sequence. Three model pentapeptides were prepared to test the general utility of these derivatives in solid-phase peptide synthesis. Both DOTA derivatives reacted smoothly by means of standard HBTU activation chemistry to the point of insertion of the DOTA amino acid, but extension of the peptide chain beyond the DOTA-amino acid insertion required the use of pre-activated C-pentafluorophenyl ester N-alpha-Fmoc amino acids. Three Gal-80 binding peptides (12-mers) were then prepared by using this methodology with DOTA positioned either at the N terminus or at one of two different internal positions;the binding of the resulting GdDOTA-12-mers to Gal-80 were compared. The methodology described here allows versatile, controlled introduction of DOTA into any location within a peptide sequence. This provides a potential method for the screening of libraries of DOTA-linked peptides for optimal targeting properties.

Type 1 and type 2 diabetes are both diseases of insulin insufficiency, although they develop by distinct pathways. The recent surge in the incidence of type 2 diabetes and the chronic ailments confronted by patients with either form of the disease highlight the need for better understanding of beta-cell biology. In this review, we present recent work focused on this goal. Our hope is that basic research being conducted in this and other laboratories will ultimately contribute to the development of methods for enhancing beta-cell function and survival in the context of both major forms of diabetes. Our strategy for understanding the beta-cell involves a multidisciplinary approach in which tools from the traditional fields of biochemistry, enzymology, and physiology are teamed with newer technologies from the fields of molecular biology, gene discovery, cell and developmental biology, and biophysical chemistry. We have focused on two important aspects of beta-cell biology in our studies: beta-cell function, specifically the metabolic regulatory mechanisms involved in glucose-stimulated insulin secretion, and beta-cell resistance to immune attack, with emphasis on resistance to inflammatory cytokines and reactive oxygen species.

Patients with HIV taking protease inhibitors were selected for the presence (five subjects) or absence (five subjects) of lipoatrophy. Following an overnight fast, subjects were given oral (2)H(2)O in divided doses (5 mL/kg body water), [U-(13)C(3)] propionate (10 mg/kg), and acetaminophen (1000 mg). Glucose (from plasma) or acetaminophen glucuronide (from urine) were converted to monoacetone glucose for (2)H NMR and (13)C NMR analysis. The fraction of plasma glucose derived from gluconeogenesis was not significantly different between groups. However, flux from glycerol into gluconeogenesis relative to glucose production was increased from 0.20 +/- 0.13 among subjects without lipoatrophy to 0.42 +/- 0.12 (P < 0.05) among subjects with lipoatrophy, and the TCA cycle contribution was reduced. Lipoatrophy was associated with an abnormal profile of glucose production as assessed by (13)C and (2)H NMR of plasma and urine.

A critical analysis of the lanthanide induced paramagnetic shift (LIS) data for several series of Ln3+ complexes of C3 symmetry in terms of structural changes, crystal-field effects and/or variation of hyperfine constants along the lanthanide series was undertaken using a combination of the two-nuclei and three-nuclei techniques together with the classical one-nucleus technique. The crystal-field independent two-nuclei technique to study the isostructurality of a series of lanthanide complexes, is usefully complemented by the three nuclei shift ratio method, which is based exclusively on the experimental shift data, requiring no knowledge of B02, 〈Sz〉 or Cj values. However, this later method cannot provide quantitative values for Fi and Gi. The combined use of the three methods was found to be a powerful analytical tool of the solution structure of lanthanide complexes. Isostructurality of whole series of complexes, either with no change of the Fi, Gi and B02 parameters (L5 and L6), or with changes of the Fi and B02 parameters (L7 and L8), is clearly defined by the combination of the two first methods. In these cases, the three-nuclei method sometimes fully supports such an isostructurality (L6, L8), but in other cases, due to the high structural sensitivity of its α and β parameters, it is able to detect small, unnoticed, structural changes in the complexes of L5 and L7. Clear structural changes, involving the Fi, Gi and B02 parameters, are observed for the series of complexes of (L9), where the three methods agree, involving hydration and carboxylate coordination changes. More subtle structural changes, involving the internal dynamics of the bound ligands, are proposed in other cases (L1–L4). These could also result from a magnification, by the present graphical analysis, of the breaks expected from the gradual structural changes along the series due to the lanthanide contraction.

The lanthanide(III) complexes of three tetraamide DOTA bearing pyridyl, phenolic and hydroxypyridyl substituents have been studied by NMR, luminescence and cyclic voltammetry. The relaxivity profiles of the gadolinium complexes of the pyridyl and phenolic ligands were flat and essentially the same between pH 2 and 8. The hydroxypyridyl ligand, however, exhibited two regions of enhanced relaxivity. The small relaxivity enhancement (25 %) at lower pH (pH 2-4) has been attributed to an increase in the prototropic exchange of the coordinated water molecule while the slightly larger enhancement (84 %) at higher pH (pH 6-9) reflects deprotonation of the ligand amide protons. Deprotonation of the amides results in the formation of an intramolecular acid-base pair interaction with the phenolic protons and this, in turn, causes a highly organized second hydration sphere to come into effect, thereby increasing the relaxivity. The water relaxivity of the Gd(3+)-hydroxypyridyl complex is further enhanced upon binding to serum albumin.

Scientific interest in optimizing the properties of gadolinium (III) complexes as MRI contrast agents has led to many new insights into lanthanide ion coordination chemistry in the last two decades. Among these was the surprising observation that water exchange in lanthanide (III) derivatives of DOTA can be modulated dramatically by judicious choice of ligand side chain and Ln(3+) ionic radii. This resulted in the discovery of paramagnetic CEST agents for altering MRI image contrast based upon the chemical exchange saturation transfer mechanism. The goal of this article is to review the factors that govern water molecule and water proton exchange in these complexes and to compare the potential sensitivity of PARACEST agents versus Gd(3+)-based T(1) relaxation agents for altering tissue contrast.

A new and effective synthetic pathway to 6,7:13,14-dibenzo-1,8,4,11-dioxadiazacyclotetradecane (1) has been developed. Beginning from salicylic acid, this synthetic pathway involves five high yielding steps to afford the macrocycle 6,7:13,14-dibenzo-1,8,4,11-dioxadiazacyclotetradecane-3,10-dione. Reduction of this macrocycle afforded the dioxadiaza crown 1 with an overall yield of 60%.

Liver-specific PEPCK knockout mice, which are viable despite markedly abnormal lipid metabolism, exhibit mild hyperglycemia in response to fasting. We used isotopic tracer methods, biochemical measurements, and nuclear magnetic resonance spectroscopy to show that in mice lacking hepatic PEPCK, 1) whole-body glucose turnover is only slightly decreased; 2) whole-body gluconeogenesis from phosphoenolpyruvate, but not from glycerol, is moderately decreased; 3) tricarboxylic acid cycle activity is globally increased, even though pyruvate cycling and anaplerosis are decreased; 4) the liver is unable to synthesize glucose from lactate/pyruvate and produces only a minimal amount of glucose; and 5) glycogen synthesis in both the liver and muscle is impaired. Thus, although mice without hepatic PEPCK have markedly impaired hepatic gluconeogenesis, they are able to maintain a near-normal blood glucose concentration while fasting by increasing extrahepatic gluconeogenesis coupled with diminishing whole-body glucose utilization.

DOTA-tetraamide ligands having extended phenol or pyridine substituents have been synthesized. The (5)D(0) --(7)F(J) deltaJ = 1 and 2 emission bands in the corresponding europium(III) complexes differ in their sensitivity to solution pH. This offers the potential for developing pH responsive probes for in vivo imaging that are independent of probe concentration.

Rapid water exchange is normally considered a prerequisite for efficient Gd3+-based MRI contrast agents. Yet recent measures of exchange rates in some Gd3+ complexes have shown that water exchange can become limiting when such complexes are attached to larger macromolecular structures. A new class of lanthanide complexes that display unusually slow water exchange (bound water lifetimes (τM298) > 10 μs) has recently been reported. This apparent disadvantage may be taken advantage of by switching the metal ion from gadolinium(III) to a lanthanide that shifts the bound water resonance substantially away from bulk water. Given appropriate water exchange kinetics, one can then alter the intensity of the bulk water signal by selective presaturation of this highly shifted, Ln3+-bound water resonance. This provides the basis of a new method to alter MR image contrast in tissue. We have synthesized a variety of DOTA-tetra(amide) ligands to evaluate as potential magnetization transfer (MT) contrast agents and found that the bound water lifetimes in these complexes are sensitive to both ligand structure (a series of Eu3+ complexes have τM298 values that range from 1 to 1300 μs) and the identity of the paramagnetic Ln3+ cation (from 3 to 800 μs for a single ligand). This demonstrates that it may be possible either to fine-tune the ligand structure or to select proper lanthanide cation to create an optimal MT agent for any clinical imaging field.

Perturbations of renal and systemic pH accompany diseases of the kidney, such as renal tubular acidosis, and the ability to image tissue pH would be helpful to assess the extent and severity of such conditions. A dual-contrast-agent strategy using two gadolinium agents, the pH-insensitive GdDOTP5− and the pH-sensitive GdDOTA-4AmP5−, has been developed to generate pH maps by MRI. The renal pharmacokinetics of the structurally dissimilar pH-insensitive contrast agents GdDTPA2− and GdDOTP5− were found to be similar. On that basis, and on the basis of similarity of structure and charge, the renal pharmacokinetics of GdDOTP5− and GdDOTA-4AmP5− were assumed to be identical. Dynamic T1-weighted images of mice were acquired for 1 hr each following boluses of GdDOTP5− and GdDOTA-4AmP5−. The time-varying apparent concentration of GdDOTP5− and the time-varying enhancement in longitudinal relaxation rate following GdDOTA-4AmP5− were calculated for each pixel and used to compute pH images of the kidneys and surrounding tissues. MRI pH maps of control mice show acidic regions corresponding to the renal papilla, calyx, and ureter. Pretreatment of mice with the carbonic anhydrase inhibitor acetazolamide resulted in systemic metabolic acidosis and accompanying urine alkalinization that was readily detected by this dual-contrast-agent approach. Magn Reson Med 49:249–257, 2003. © 2003 Wiley-Liss, Inc.

Copper-64 ( T(1/2)=12.7 h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. In this paper, we evaluated three tetraaza macrocyclic ligands with two, three, and four pendant methanephosphonate functional groups. DO2P [1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid)], DO3P [1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonic acid)], and DOTP [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)] were all radiolabeled with (64)Cu in high radiochemical yields. Copper-64-labeled DO2P and DOTP were highly stable in rat serum out to 24 h, while (64)Cu-DO3P remained 73% intact, with the remainder possibly forming a (64)Cu(.)2DO3P dimer by 24 h. The biodistribution experiments were performed in normal Sprague-Dawley rats. Of the three complexes, (64)Cu-DO2P demonstrated the most optimal clearance through the blood and liver. Copper-64-DO3P and (64)Cu-DOTP exhibited higher liver uptake and longer retention of liver activity, possibly because of the large negative charge of the complexes under physiological conditions. All three (64)Cu-labeled complexes showed high accumulation in bone, likely due to the binding of the methanephosphonate groups to hydroxyapatite. These results suggest that this series of methanephosphonate macrocyclic ligands may be useful as potential bone-imaging agents. The thermodynamic stability constants of the Cu(II) complexes with these three ligands were determined, and were found to be significantly higher than those of their acetate analogues. The Cu(II)-DO2P complex exhibited the highest stability constant among divalent transition metal ion DO2P complexes. Metabolism studies of (64)Cu-DO2P in rat liver suggest that the DO2P ligand may be used as a bifunctional chelator for copper radionuclides in radiodiagnostic or radiotherapeutic studies.

Analysis of the LIS data for several series of Ln3+ complexes of C4 symmetry in terms of structural changes, crystal-field effects and/or variation of hyperfine constants along the lanthanide series was undertaken using a combination of the two-nuclei and three-nuclei techniques together with the classical onenucleus technique. Isostructurality of whole series of complexes, with changes of the Fi, and B02 parameters, was clearly defined for the complexes of L by the combination of the two first methods. Small changes, involving the three Fi, Gi and B02 parameters, are observed for the series of complexes of L-L4, using the three data plotting methods. Some of the plots according to the two- and three-nuclei methods are accidentally linear, without necessarily implying isostructurality of the complexes, as they involve parameters, which may be insensitive to any small structural changes occurring in these systems. These parameter variations could result from a magnification, by the present graphical analysis, of the breaks expected from the gradual structural changes along the series due to the lanthanide contraction. The α and β parameters of the three-nuclei method are not diagnostic of the type of structures the complexes have in solution, due to their very indirect dependence on the geometric factors.

PARACEST redox sensors containing the NAD(+)/NADH mimic N-methylquinolinium moiety as a redox-active functional group have been designed and synthesized. The Eu(3+) complex with two quinolinium moieties was nearly completely CEST-silent in the oxidized form but was "turned on" upon reduction with β-NADH. The CEST effect of the Eu(3+) complex containing only one quinolinium group was much less redox-responsive but showed an unexpected sensitivity to pH in the physiologically relevant pH range.

Hyperpolarized [1-13C]-pyruvate has shown tremendous promise as an agent for imaging tumor metabolism with unprecedented sensitivity and specificity. Imaging hyperpolarized substrates by magnetic resonance is unlike traditional MRI because signals are highly transient and their spatial distribution varies continuously over their observable lifetime. Therefore, new imaging approaches are needed to ensure optimal measurement under these circumstances. Constrained reconstruction algorithms can integrate prior information, including biophysical models of the substrate/target interaction, to reduce the amount of data that is required for image analysis and reconstruction. In this study, we show that metabolic MRI with hyperpolarized pyruvate is biased by tumor perfusion and present a new pharmacokinetic model for hyperpolarized substrates that accounts for these effects. The suitability of this model is confirmed by statistical comparison with alternates using data from 55 dynamic spectroscopic measurements in normal animals and murine models of anaplastic thyroid cancer, glioblastoma, and triple-negative breast cancer. The kinetic model was then integrated into a constrained reconstruction algorithm and feasibility was tested using significantly undersampled imaging data from tumor-bearing animals. Compared with naïve image reconstruction, this approach requires far fewer signal-depleting excitations and focuses analysis and reconstruction on new information that is uniquely available from hyperpolarized pyruvate and its metabolites, thus improving the reproducibility and accuracy of metabolic imaging measurements. Cancer Res; 75(22); 4708–17. ©2015 AACR.

There is a growing appreciation for the complexity of the pathways involved in glucose-stimulated insulin secretion (GSIS) from pancreatic islet β-cells. In our laboratory, this has stimulated the development of an interdisciplinary approach to the problem. In this study, we review recent studies combining the tools of recombinant adenovirus for gene delivery, the development of novel cell lines that exhibit either robust or weak GSIS, and nuclear magnetic resonance imaging for metabolic fingerprinting of glucose-stimulated cells. Using these tools, we demonstrate a potentially important role for pyruvate carboxylase−mediated pyruvate cycling pathways in the control of GSIS, and discuss potential coupling factors produced by such pathways.

The linear graphical analysis of the LIS NMR data available for the axially symmetric complexes [Ln(DOTA)]- (M and m isomers), [Ln(DOTP)]5- (pH 10.0, 7.0 and 3) and [Ln(DOTEA)]3+ using the classical crystal field dependent method and a crystal field independent method were compared. As the second method provides ratios of geometric structural terms G rather than G values, the effect of lanthanide contraction was reduced. Thus, the large breaks in plots observed for all nuclei of those systems using the classical method are still present in the plots of the second method, only in a few of the nuclei and much reduced. This shows that the large breaks at the middle of the lanthanide series present in plots of the classical method as well as the anomalies often present for those plots for the Tm and Yb ions are mostly due to changes of the crystal field coefficient A2Â 0 along the lanthanide series, while both the hyperfine coupling constants and the ratios of geometric terms also change as a result of the lanthanide contraction, leading to small breaks at the middle of the lanthanide series. Analysis of the proton shifts of [Ln(DOTP)] complexes at pH 10, 7 and 3 indicates that protonation of the complexes results in a decrease on the crystal field coefficient. The dipolar shift ratios and absolute shifts obtained were also interpreted in terms of the structural models for those complexes in solution and their available X-ray crystal structures. http://www.sciencedirect.com/science/article/B6TG5-45S49G7-1/1/7fee3ca7ba8b89327bec4ab48419d290

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.

The physical, magnetic and nuclear properties of the lanthanide ions have rendered them ideal for use in both diagnostic and therapeutic pharmaceuticals. Recent innovations in biotechnology have opened new possibilities for the design of such pharmaceuticals. By conjugating a suitable targeting vector to the metal chelate it may be possible to target tissue type, cell type, individual proteins and even specific nucleic acid sequences. This review describes recent advances in the targeting of these complexes and discusses their potential in the development of new inorganic pharmaceuticals.

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized 13 C-enriched metabolites offers considerable potential as a rapid, non-invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ-[1-13 C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPPox ) in isolated perfused mouse livers. The appearance of hyperpolarized (HP) H13 CO3- within seconds after exposure of livers to HP-δ-[1-13 C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPPox pathway to produce HP-H13 CO3- after three enzyme catalyzed steps (6P-gluconolactonase, 6-phosphogluconate dehydrogenase, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H13 CO3- after exposure to low levels of H2 O2 , while livers perfused with glucose and insulin showed a twofold increase in H13 CO3- after exposure to peroxide. This indicates that flux through the PPPox is stimulated by H2 O2 in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non-polarized [1,2-13 C]glucose followed by 1 H NMR analysis of lactate in the perfusate verified that flux through the PPPox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized δ-[1-13 C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.

Keywords: dynamic nuclear polarization ; hyperpolarization ; NMR spectroscopy ; radicals ; relaxation ; Signal Reference EPFL-ARTICLE-169192doi:10.1002/chem.201102037View record in Web of Science Record created on 2011-10-03, modified on 2017-05-12

Inversion transfer (IT) is a well-established technique with multiple attractive features for analysis of kinetics. However, its application in measurement of ATP synthesis rate in vivo has lagged behind the more common saturation transfer (ST) techniques. One well-recognized issue with IT is the complexity of data analysis in comparison with much simpler analysis by ST. This complexity arises, in part, because the γ-ATP spin is involved in multiple chemical reactions and magnetization exchanges, whereas Pi is involved in a single reaction, Pi → γ-ATP. By considering the reactions involving γ-ATP only as a lumped constant, the rate constant for the reaction of physiological interest, kPi→γATP , can be determined. Here, we present a new IT data analysis method to evaluate kPi→γATP using data collected from resting human skeletal muscle at 7 T. The method is based on the basic Bloch-McConnell equation, which relates kPi→γATP to m˙Pi, the rate of Pi magnetization change. The kPi→γATP value is accessed from m˙Pi data by more familiar linear correlation approaches. For a group of human subjects (n = 15), the kPi→γATP value derived for resting calf muscle was 0.066 ± 0.017 s(-1) , in agreement with literature-reported values. In this study we also explored possible time-saving strategies to speed up data acquisition for kPi→γATP evaluation using simulations. The analysis indicates that it is feasible to carry out a (31) P IT experiment in about 10 min or less at 7 T with reasonable outcome in kPi→γATP variance for measurement of ATP synthesis in resting human skeletal muscle. We believe that this new IT data analysis approach will facilitate the wide acceptance of IT to evaluate ATP synthesis rate in vivo. Copyright © 2015 John WileySons, Ltd.

Flux through pyruvate dehydrogenase (PDH) in the heart may be reduced by various forms of injury to the myocardium, or by oxidation of alternative substrates in normal heart tissue. It is important to distinguish these two mechanisms because imaging of flux through PDH based on the appearance of hyperpolarized (HP) [13C]bicarbonate derived from HP [1-13C]pyruvate has been proposed as a method for identifying viable myocardium. The efficacy of propionate for increasing PDH flux in the setting of PDH inhibition by an alternative substrate was studied using isotopomer analysis paired with exams using HP [1-13C]pyruvate. Hearts from C57/bl6 mice were supplied with acetate (2 mM) and glucose (8.25 mM).13C NMR spectra were acquired in a cryogenically cooled probe at 14.1 Tesla. After addition of hyperpolarized [1-13C]pyruvate,13C NMR signals from lactate, alanine, malate, and aspartate were easily detected, in addition to small signals from bicarbonate and CO2. The addition of propionate (2 mM) increased appearance of HP [13C]bicarbonate >30-fold without change in O2consumption. Isotopomer analysis of extracts from the freeze-clamped hearts indicated that acetate was the preferred substrate for energy production, glucose contribution to energy production was minimal, and anaplerosis was stimulated in the presence of propionate. Under conditions where production of acetyl-CoA is dominated by the availability of an alternative substrate, acetate, propionate markedly stimulated PDH flux as detected by the appearance of hyperpolarized [13C]bicarbonate from metabolism of hyperpolarized [1-13C]pyruvate.

The diseased myocardium lacks metabolic flexibility and responds to stimuli differently compared with healthy hearts. Here, we report the use of hyperpolarized 13C NMR spectroscopy to detect sudden changes in cardiac metabolism in isolated, perfused rat hearts in response to adrenergic stimulation.Metabolism of hyperpolarized [1-(13)C]pyruvate was investigated in perfused rat hearts. The hearts were stimulated in situ by isoproterenol shortly after the administration of hyperpolarized [1-(13)C]pyruvate. The hyperpolarized 13C NMR results were corroborated with 1H NMR spectroscopy of tissue extracts.Addition of isoproterenol to hearts after equilibration of hyperpolarized [1-(13)C]pyruvate into the existing lactate pool resulted in a sudden, rapid increase in hyperpolarized [1-(13)C]lactate signal within seconds after exposure to drug. The hyperpolarized H(13)CO3 (-) and hyperpolarized [1-(13)C]alanine signals were not affected by the isoproterenol-induced elevated cardiac workload. Separate experiments confirmed that the new hyperpolarized [1-(13)C]lactate signal that arises after stimulation by isoproterenol reflects a sudden increase in total tissue lactate derived from glycogen.These results suggest that hyperpolarized pyruvate and 13C MRS may be useful for detecting abnormal glycogen metabolism in intact tissues.

The (13) C-labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of (13) C-enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α-ketoglutarate into glutamate in neurons, and incorporation of α-ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single (13) C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate-to-glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2-(13) C]acetate and [1,6-(13) C]glucose, and proton decoupled (13) C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that (13) C-labeled acetate and glucose contributed approximately equally to acetyl-CoA (0.96) in astrocytes. As this method relies on a single (13) C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions. Differences in (13) C labeling of brain glutamate and glutamine have been attributed to metabolic compartmentation. The acetate:glucose ratio, introduced for description of a (13) C NMR (nuclear magnetic resonance) spectrum, is an index of glucose and acetate oxidation in brain tissue. A simple mathematical method is presented to quantify the ratio of acetate-to-glucose oxidation in astrocytes from a single NMR spectrum. As kinetic analysis is not required, the method is readily applicable to analysis of tissue extracts. α-KG = alpha-ketoglutarate; CAC = citric acid cycle; GLN = glutamine; GLU = glutamate.

The development of responsive or "smart" magnetic resonance imaging (MRI) contrast agents that can report specific biomarker or biological events has been the focus of MRI contrast agent research over the past 20 years. Among various biological hallmarks of interest, tissue redox and hypoxia are particularly important owing to their roles in disease states and metabolic consequences. Herein we review the development of redox-/hypoxia-sensitive T1 shortening and paramagnetic chemical exchange saturation transfer (PARACEST) MRI contrast agents. Traditionally, the relaxivity of redox-sensitive Gd(3+) -based complexes is modulated through changes in the ligand structure or molecular rotation, while PARACEST sensors exploit the sensitivity of the metal-bound water exchange rate to electronic effects of the ligand-pendant arms and alterations in the coordination geometry. Newer designs involve complexes of redox-active metal ions in which the oxidation states have different magnetic properties. The challenges of translating redox- and hypoxia-sensitive agents in vivo are also addressed.

Slow water exchange in Gd3+ complexes is generally considered detrimental to their use as MR contrast agents. The objective of this work was to demonstrate how this feature may serve as a useful template for the design of responsive MR agents.Lanthanide (Ln) complexes of two 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA)-tetraamide phosphonate (1) and phosphonate ester (2) ligands were studied by multinuclear (1H, 13C, 31P, and 17O) nuclear MR spectroscopy.The inner-sphere water lifetime in the Ln(2) complexes was much longer (tauM298 = 0.8-1.3 ms) than in the corresponding Ln(1) complexes. This allowed direct detection of the bound-water molecule in europium(2) in water at 40 degrees C by 1H nuclear MR. The water relaxivity of gadolinium(2) was independent of pH between 8.5 and 6.0, whereas the relaxivity of gadolinium(1) increased more than twofold in this pH range.T1-weighted images of phantoms containing gadolinium(1) at different pH values demonstrate the efficacy of this complex as a pH-sensitive MR contrast agent.

The synthesis of a polylysine dendron containing eight GdDOTA units conjugated to a peptoid dimer known to have a high affinity for the vascular endothelial growth factor receptor 2 (VEGFR2) is described. This simple low molecular weight system with a molecular r(1) relaxivity of ∼48 mM(-1) s(-1) is shown to enhance MR images of tumors grown in mice in vivo.

A kinetic model of the citric acid cycle for calculating oxygen consumption from13C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MV˙o2) was compared with MV˙o2predicted by the model with13C NMR data obtained from rat hearts perfused with glucose and either [2-13C]acetate or [3-13C]pyruvate. The accuracy of MV˙o2measured from three subsets of NMR data was compared: glutamate C-4 and C-3 resonance areas; the doublet C4D34 (expressed as a fraction of C-4 area); and C-4 and C-3 areas plus several multiplets of C-2, C-3, and C-4. MV˙o2determined by set 2(C4D34 only) gave the same degree of accuracy as set 3(complete data); both were superior to set 1(C-4 and C-3 areas). Analysis of the latter suffers from the correlation between citric acid cycle flux and exchange between α-ketoglutarate and glutamate, resulting in greater error in estimating MV˙o2. Analysis of C4D34 is less influenced by correlation between parameters, and this single measurement provides the best opportunity for a noninvasive measurement of oxygen consumption.

Cyclic pentamine 1 was synthesized in good yields from fragments containing two and three nitrogen atoms.

C isotopomers, thereby permitting a direct evaluation ofanaplerosis.MATERIALS AND METHODSHeart PerfusionsHearts from anesthetized, adult rats were rapidly excisedand arrested in ice-cold Krebs-Hensleit buffer. Hearts ingroups I–IV were perfused in the Langendorff mode withone of four different mixtures of propionate (2 mM) andacetate (5 mM): I, [1,2,3

Isolated, perfused rat livers were examined by single-quantum (SQ) and double-quantum-filtered (DQ-filtered) 23Na spectroscopy during prolonged global ischemia and during perfusion with ouabain, low-buffer potassium, or lithium-enriched buffer. Baseline separation of the intracellular (Na(i)+) and extracellular (Na(e)+) sodium resonances using TmDOTP5- allowed a direct comparison of temporal changes in SQ versus DQ-filtered Na(i)+. The SQ Na(i)+ signal increased approximately 150% during the first 15 min of global ischemia and then remained relatively constant over the next 45 min, while the DQ-filtered signal steadily increased approximately 400% over the same 60 min period. In similar experiments in which all perfusate sodium was replaced by lithium, the DQ-filtered Na(i)+ signal increased approximately 180% over a similar period of ischemia. Exposure of livers to ouabain also resulted in larger increases in DQ-filtered versus SQ signal of Na(i)+. The approximately 290% increase in DQ-filtered sodium observed during perfusion of livers with a hypokalemic buffer (1.2 mM K+) could be completely reversed by continued perfusion with a buffer containing normal levels of K+ (4.7 mM). These data suggest that the DQ-filtered Na(i)+ signal of liver does not simply report an increase in [Na(i)+], but may be exquisitely sensitive to other intracellular events initiated by altered physiology.

Recently Befroy and colleagues measured TCA cycle flux and anaplerosis in human liver by analyzing 13C enrichment in carbon 1 and carbon 5 of glutamate during [1-13C]acetate infusion1. The entry of [1-13C]acetyl-CoA into the cycle yields [5-13C]glutamate on each turn of the cycle but [1-13C]glutamate only on subsequent turns. Anaplerosis, for instance via pyruvate carboxylation, results in the influx of unlabeled oxaloacetate. The subsequent loss of 13C-oxaloacetate from the cycle to make phosphoenolpyruvate (PEP) decreases the enrichment in glutamate-C1 but not glutamate-C5. Hence, the steady-state ratio of [1-13C] to [5-13C]glutamate should reflect anaplerosis relative to TCA cycle activity (i.e. citrate synthase flux = 1). PEP formed from anaplerosis has at least two fates, either in gluconeogenesis (GNG) or to regenerate pyruvate via pyruvate kinase (PK). PK, and other pathways (e.g. malic enzyme), contribute to a futile pyruvate cycle whereby oxaloacetate → pyruvate → oxaloacetate. The relationship among these variables is simple: GNG = anaplerosis – PK flux. This pathway has been included in virtually all models of hepatic GNG2-9. In humans PK flux, relative to citrate synthase (CS), was 1.5 to 5.8 when determined using a [3-14C]lactate tracer with radioisotope detection,8 [3-13C]lactate tracer with mass isotope detection3 or [U-13C]propionate tracer with NMR detection6,9. In fact, pyruvate cycling accounted for the majority of anaplerosis in these human studies3,6,8,9, yet was excluded from anaplerosis by Befroy et al.1 This exclusion complicates the analysis considerably because pyruvate cycling in the presence of H13CO3-, as observed in the in vivo 13C NMR spectra1, regenerates [1-13C]glutamate. Hence, the steady-state ratio of [1-13C] to [5-13C]glutamate no longer reflects total anaplerosis. Given the implications, it is worth examining the effect of pyruvate cycling on 13C in glutamate C1 and C5 using the conditions reported by Befroy et al. Carbon-13 labeling in glutamate is a complex function of pyruvate cycling, anaplerosis and enrichment in acetyl-CoA and bicarbonate 4,10. Consequently, simulations must be used to explore these interactions10. Using published relationships10 and the conditions reported by Befroy et al., glutamate 13C1/13C5 is sensitive to total anaplerosis if 13CO2 = 0 and pyruvate cycling = 0 but much less sensitive when 13CO2 = 6% 1 and pyruvate cycling is active2-9 (Figure 1). The glutamate 13C1/13C5 ratio reported by Befroy et al. is indeed consistent with substantial pyruvate cycling (Figure 1), as previously described in human liver3,6,8,9. However, the presence of pyruvate cycling also means that a 5-fold range in anaplerosis is essentially undetectable by the glutamate 13C1/13C5 ratio. Thus, this method cannot be used to support the conclusion by Befroy et al., that other methods have overestimated anaplerosis1, since the glutamate 13C1/13C5 ratio cannot detect total anaplerosis under these conditions (Figure 1). Figure 1 The effect of pyruvate cycling on the steady-state 13C1/13C5 of glutamate. Data was simulated using program tcaSIM (see source data). All simulations were conducted using an [1-13C]acetyl-CoA fractional enrichment of 0.22 and 13CO2 fractional enrichment ... Futile cycles serve a role in the regulation of metabolism. Earlier studies, including those in humans, found that the futile cycle interconverting pyruvate and oxaloacetate is unquestionably active in liver regardless of whether lactate or propionate tracers are used2-9. Thus, it is best to assume that pyruvate cycling is always a feature of liver metabolism and to incorporate this possibility in data analysis.

The hyperfine shift reagent, TmDOTP5-, was used to resolve the 39K NMR resonances of intra- (Ki+) and extracellular (Ke+) potassium in isolated, perfused guinea pig hearts. [Ki+] as measured by 39K NMR was 25.9 +/- 10.3 mM, compared with 114.4 +/- 10.8 mM as measured by atomic absorption spectroscopy (AAS) using TmDOTP5- as a marker of extracellular space. Thus, only approximately 23% of intracellular potassium was detected by 39K NMR using our experimental conditions. The area of the Ki+ signal increased during early ischemia then returned to baseline levels during reperfusion. In an effort to learn more about the Ki+ not detected by 39K NMR, hearts were perfused with a Rb+-enriched, K+-depleted buffer for an extended period. This resulted in loss of the entire 39K NMR signal, and Ki+, as measured by AAS, decreased from approximately 60 to approximately 6 to 7 micromol/g wet weight. When K+-depleted hearts were subjected to global ischemia, a small 39K NMR signal reappeared, suggesting that at least a portion of the nonexchangeable Ki+ becomes detectable by NMR during ischemia. This newly visible K+ signal subsequently dissipated during reperfusion of ischemic hearts. We conclude that ischemia induces changes in the NMR visibility of 39K in perfused guinea pig hearts.

The use of copper radioisotopes in imaging and therapy applications has created a greater need for bifunctional chelates (BFCs) for complexing copper radioisotopes to biomolecules. It has been demonstrated that the charge and lipophilicity of the Cu-BFC complex has a significant effect on the in vivo behavior of the radiolabeled Cu-BFC-biomolecule conjugate. To evaluate the effects of charge, stability, and macrocyclic backbone size on the biological behavior of 64Cu complexes, a series of macrocyclic 64Cu complexes have been prepared, and the biodistributions of these agents were evaluated in normal Sprague-Dawley rats. Two macrocyclic backbones, dodecane and tetradecane, were evaluated; cyclen, DOTA, and DO2A were dodecane backbone derivatives, and cyclam, TETA, and et-cyclam were tetradecane backbone derivatives. The biodistributions of the 64Cu-labeled complexes correlated with differences in the size of the macrocycle backbone and the formal charge of the complex. All compounds showed uptake and clearance through the liver and kidneys; however, the positively charged 64Cu complexes showed significantly higher uptake in both of these organs than did the negatively charged or neutral complexes. 64Cu-TETA, a negatively charged complex with the tetradecane backbone, had the most efficient clearance by 24 hours' postinjection. These data suggest that negatively charged complexes may have more favorable clearance properties when used as BFCs.

The positive inotropic effect of cardiac glycosides has been attributed to inhibition of the Na-K-ATPase, accumulation of intracellular sodium and enhanced calcium availability due to Na-Ca exchange. However, few measurements of intracellular sodium in the functioning left ventricle following ouabain exposure at therapeutic doses are available. Our experimental objective was to quantitate the relationship between contractile state and intracellular sodium measured by 23Na nuclear magnetic resonance spectroscopy or atomic absorption in the intact heart. Isolated guinea-pig hearts, perfused in the Langendorff mode, were paced and then exposed to ouabain (3x10(-7)m) for 30 min. Left-ventricular pressure was monitored continuously. Intracellular sodium was measured either at 1-min intervals throughout the perfusion by shift reagent-aided 23Na nuclear magnetic resonance spectroscopy in the beating heart or following 30 minutes of perfusion by atomic absorption in myocardial tissue. While treatment with ouabain was associated with almost a two-fold rise in developed pressure, there was no significant increase in intracellular sodium measured by either technique. Thus, the positive inotropic effect of ouabain in this model is not associated with significant changes in bulk intracellular sodium. However, these results do not exclude the possibility of shifts between intracellular pools which would not be detected in bulk measurements, or changes in NMR-invisible intracellular pools which are not detectable by single quantum spectroscopy techniques.

The affinities of 1,4,7-tris(2-hydroxyalkyl)-1,4,7-triazacyclononane derivatives for MgII and CaII were found to differ greatly. Whereas 1,4,7-tris(2-hydroxyethyl)- (L1), 1,4,7-tris(2-hydroxy-2-methylpropyl)- (L2) and the unsymmetrical 1,4,7-tris(2-hydroxypropyl)-1,4,7-triazacyclononane derivative L3b barely discriminated between MgII and CaII, the symmetrical isomer L3a was more than 500 times more selective for MgII than for CaII. Similar selectivity differences were observed between the diastereomers of 1,4,7-tris(2-hydroxy-2-phenylethyl)- (L4) and 1,4,7-tris(2-hydroxydodecyl)-1,4,7-triazacyclononane (L5). These selectivities were related to the structure of the magnesium complexes as shown by molecular mechanics (MMX) calculations. Only those ligands favoring the formation of a magnesium complex with a large twist angle between the planes of co-ordinating oxygens and ring nitrogens resulting in a small, tight cavity showed a large preference for MgII over CaII. The MMX calculations predicted large twist angle structures for phosphinate derivatives, and for a phosphonate monoester derivative, and these ligands were found to have a high selectivity for MgII. Similarly, the calculated preference for the smaller twist angle correctly predicted the lack of MgII/CaII selectivity for acetate and amide derivatives and for a phosphonate diester derivative. Equilibration of the complexes of the ligands in the presence of both MgII and CaII was slow, as shown for example by kd,0 = 2.1 × 10–4 s–1 for CaL3a. These dissociation rates were a factor of 100 times larger at the boundary of a two-phase (water–chloroform) system.

The water molecule exchange rates in a series of DyDOTA-(amide)X chelates were fine-tuned to maximize the effects of T2-exchange line broadening and improve T2 contrast.Four DyDOTA-(amide)X chelates having a variable number of glycinate side-arms were prepared and characterized as T2-exchange agents. The nonexchanging DyTETA chelate was also used to measure the bulk water T2 reduction due solely to T2*. The total transverse relaxivity (r2tot) at 22, 37, and 52°C for each chelate was measured in vitro at 9.4 Tesla (400 MHz) by fitting plots of total T2 (-1) versus concentration. The water molecule exchange rates for each complex were measured by fitting (17)O line-width versus temperature data taken at 9.4 Tesla (54.3 MHz).The measured transverse relaxivities due to water molecule exchange (r2ex) and bound water lifetimes (τM) were in excellent agreement with Swift-Connick theory, with DyDOTA-(gly)3 giving the largest r2ex = 11.8 s(-1) mM(-1) at 37°C.By fine-tuning the water molecule exchange rate at 37°C, the transverse relaxivity has been increased by 2 to 30 times compared with previously studied Dy(3+)-based chelates. Polymerization or dendrimerization of the optimal chelate could yield a highly sensitive, molecule-sized T2 contrast agent for improved molecular imaging applications.

A new macrocyclic ligand 1,4,7,10,-tetraazacyclododecane-1,4,7,10-tetrakis(methanephosphonic acid mono(2‘,2‘,2‘-trifluoroethyl) ester) (F-DOTPME) has been prepared and some of its metal binding properties examined. The ligand protonation constants (log K1 = 10.5, log K2 = 6.7) and its stability constant with CaII (log KCaL = 8.4) were determined by pH potentiometry. The stability of the LaIII complex could not be determined by potentiometry so it was evaluated in an EDTA competition experiment using 31P NMR to monitor the reaction. The dissociation kinetics of La(F-DOTPME)- was slow, with a kd,obs of only 1.0 × 10-7 s-1. A series of Ln(F-DOTPME)- complexes (LnIII = La, Gd, Dy, Tm, and Yb) were examined by multinuclear NMR. The spectra show that these complexes exist in aqueous solution as a mixture of stereoisomers of nearly equal energy (energy differences of less than 1 kJ/mol). Several resolved 19F resonances in the NMR spectra of the Dy(F-DOTPME)-, Tm(F-DOTPME)-, and Yb(F-DOTPME)- complexes have been ...