Garry Kiefer - Academia.edu (original) (raw)

Papers by Garry Kiefer

Research paper thumbnail of Alpha-Methyl Functionalization of Electron-Poor Heterocycles and Synthesis of Novel Dipyridine and Phenanthroline Metallocyclic Palladium Complexes

Research paper thumbnail of Unexpected Changes in the Population of Coordination Isomers for the Lanthanide Ion Complexes of DOTMA-Tetraglycinate

Inorganic chemistry, Jan 7, 2016

Lanthanide complexes with DOTA-tetraglycinate (DOTA-(gly)4) heavily favor the square antiprismati... more Lanthanide complexes with DOTA-tetraglycinate (DOTA-(gly)4) heavily favor the square antiprismatic (SAP) coordination isomer in aqueous solution, a structural feature that has made them useful as water-based paraCEST agents. In an effort to create amide-based paraCEST agents with rapid water exchange rates, we prepared the analogous tetraglycinate complexes with DOTMA, a ligand known to favor the twisted square antiprismatic (TSAP) coordination structures. Unexpectedly, NMR investigations show that the LnDOTMA-(gly)4 complexes, like the LnDOTA-(gly)4 complexes, also favor the SAP isomers in solution. This observation led to density functional theory (DFT) calculations in order to identify the energy terms that favor the SAP structures in lanthanide complexes formed with macrocyclic DOTA- and DOTMA-tetraamide ligands. The DFT calculations revealed that, regardless the nature of the ligand, the TSAP isomers present more negative hydration energies than the SAP counterparts. The extent...

Research paper thumbnail of Amplifying the Sensitivity of Zinc(II) Responsive MRI Contrast Agents by Altering Water Exchange Rates

Journal of the American Chemical Society, 2015

Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI con... more Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI contrast agent, GdDOTA-diBPEN, new structural targets were rationally designed to increase the rate of water exchange to improve MRI detection sensitivity. These new sensors exhibit fine-tuned water exchange properties and, depending on the individual structure, demonstrate significantly improved longitudinal relaxivities (r 1). Two sensors in particular demonstrate optimized parameters and, therefore, show exceptionally high longitudinal relaxivities of about 50 mM −1 s −1 upon binding to Zn(II) and human serum albumin (HSA). This value demonstrates a 3-fold increase in r 1 compared to that displayed by the original sensor, GdDOTA-diBPEN. In addition, this study provides important insights into the interplay between structural modifications, water exchange rate, and kinetic stability properties of the sensors. The new high relaxivity agents were used to successfully image Zn(II) release from the mouse pancreas in vivo during glucose stimulated insulin secretion.

Research paper thumbnail of A pH-Responsive MRI Agent that Can Be Activated Beyond the Tissue Magnetization Transfer Window

Angewandte Chemie, 2015

A terbium-based complex that displays a water exchange CEST resonance well-outside the normal mag... more A terbium-based complex that displays a water exchange CEST resonance well-outside the normal magnetization transfer (MT) frequency range of tissues provides a direct readout of pH by MRI. Deprotonation of the phenolic proton in this complex results in a frequency shift of 56 ppm in a bound water molecule exchange peak between pH 5 and 8. This allows direct imaging pH without prior knowledge of the agent concentration and with essentially no interference from the tissue MT signal.

Research paper thumbnail of Facile radiolabeling of monoclonal antibodies and other proteins with zirconium-89 or gallium-68 for PET imaging using p-isothiocyanatobenzyl-desferrioxamine

Research paper thumbnail of TmDOTA-Tetraglycinate Encapsulated Liposomes as pH-Sensitive LipoCEST Agents

Research paper thumbnail of A lanthanide complex with dual biosensing properties: CEST (chemical exchange saturation transfer) and BIRDS (biosensor imaging of redundant deviation in shifts) with europium DOTA-tetraglycinate

NMR in Biomedicine, 2011

Responsive contrast agents (RCAs) [R1.12] composed of lanthanide III ion (Ln 3+) complexes with a... more Responsive contrast agents (RCAs) [R1.12] composed of lanthanide III ion (Ln 3+) complexes with a variety of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA 4−) derivatives have shown great potential as molecular imaging agents for magnetic resonance (MR). A variety of LnDOTA-tetraamide complexes have been demonstrated as RCAs for molecular imaging with chemical exchange saturation transfer (CEST). The CEST method detects proton exchange between bulk water and any exchangeable sites on the ligand itself or an inner sphere of bound water that is shifted by a paramagnetic Ln 3+ bound in the core of the macrocycle. It has also been shown that molecular imaging is possible when the RCA itself is observed (i.e., not its affect on bulk water) using a method called biosensor imaging of redundant deviation in shifts (BIRDS). The BIRDS method utilizes redundant information stored in the non-exchangeable proton resonances emanating from the paramagnetic RCA for ambient factors like temperature and/or pH. Thus CEST and BIRDS rely on exchangeable and non-exchangeable protons, respectively, for biosensing. We posited that it is feasible to combine these two biosensing features into the same RCA (i.e., dual CEST and BIRDS properties). A complex between europium ion (Eu 3+) and DOTA-tetra-glycinate (DOTA-(gly) 4 4−) is used to demonstrate that its CEST characteristics are preserved while BIRDS properties are detected. In vitro temperature sensitivity of EuDOTA-(gly) 4 − is used to show that qualitative MR contrast with CEST can be calibrated using quantitative MR mapping with BIRDS, thereby enabling quantitative molecular imaging at high spatial resolution.

Research paper thumbnail of Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine

Research paper thumbnail of Multifunctional Polymeric Scaffolds for Enhancement of PARACEST Contrast Sensitivity and Performance: Effects of Random Copolymer Variations

Macromolecules, 2010

A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) tetraamide ligand having a s... more A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) tetraamide ligand having a single acrylamide side-chain (M1) was copolymerized with either 2-methylacrylic acid (MAA), 2-(acryloylamino)-2-methyl-1-propanesulfonic acid (AMPS) or N-isopropylacrylamide (NIPAM) to create a series of linear random copolymers using classical free radical chain polymerization chemistry. The metal ion binding properties of hydrolyzed M1 were investigated by pH potentiometry and the europium (III) complexes of the resulting heteropolymers were evaluated as PARACEST imaging agents. All polymeric agents were found to possess similar intermediate-toslow water exchange and CEST characteristics as the parent EuDOTA-tetraamide monomer. Consistent with basic multiplexing principles, the highest molecular weight polymer, Eu-DMAA 3.1, also showed the highest CEST sensitivity with a detection limit of 20 ± 2 μM. The second arylamide component gave polymers with widely different chemical characteristics and CEST properties. In particular, the Eu-DNIPAM 4.0 and Eu-DMAA 4.1 polymers displayed different solubility characteristics as a function of pH or temperature which, in turn, affected the water exchange and CEST properties of the corresponding agents. It was concluded that introduction of hydrophobic groups into the polymer backbone reduces solvent accessibility to the Eu 3+ component, effectively slowing water exchange between the inner-sphere water coordination position at each Eu 3+ center with bulk water. The CEST properties of the heteropolymers when dissolved in plasma suggest that the more hydrophobic characteristics of these polymers could be advantageous for in vivo applications.

Research paper thumbnail of A Responsive Europium(III) Chelate That Provides a Direct Readout of pH by MRI

Journal of the American Chemical Society, 2010

A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric CEST... more A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric CEST principles. Deprotonation of a single phenolic proton between pH 6 and 7.6 results in an ~5 ppm shift in the water exchange CEST peak that is easily detected by MRI. Collection of two CEST images at two slightly different activation frequencies provides a direct readout of solution pH without the need of a concentration marker.

Research paper thumbnail of Synthesis, Relaxometric and Photophysical Properties of a New pH-Responsive MRI Contrast Agent:  The Effect of Other Ligating Groups on Dissociation of a p-Nitrophenolic Pendant Arm

Journal of the American Chemical Society, 2004

Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdeca... more Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triAcetate) and GdNP-DO3AM (1-methlyene(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide), containing a single nitrophenolic pendant arm plus either three acetate or three amide pendant arms were synthesized and characterized. The properties of the gadolinium, terbium, and dysprosium complexes of these ligands were examined as a function of pH. The extent and mechanism of the changes in water relaxivity with pH of each gadolinium complex was found to differ substantially for the two complexes. The water relaxivity of Gd(NP-DO3A) increases from 4.1 mM −1 s −1 at pH 9 to 7.0 mM −1 s −1 at pH 5 as a result of acid-catalyzed dissociation of the nitrophenol from the lanthanide. The nitrophenol group in Gd(NP-DO3AM) does not dissociate from the metal center even at pH 5; therefore, the very modest increase in relaxivity in this complex must be ascribed to an increase in prototropic exchange rate of the bound water and/or phenolic protons.

Research paper thumbnail of Polymeric PARACEST Agents for Enhancing MRI Contrast Sensitivity

Journal of the American Chemical Society, 2008

Linear polymers of PARACEST agents were prepared by using classical free radical chain polymeriza... more Linear polymers of PARACEST agents were prepared by using classical free radical chain polymerization conditions. The Eu 3+-polymers exhibited similar intermediate-to-slow water exchange and CEST characteristics as the Eu 3+-monomers. This provided an avenue to lower the detection limit of these imaging agents substantially and makes them potentially useful as MRI sensors for molecular imaging. Magnetic resonance imaging (MRI) is an important clinical tool for anatomical imaging and monitoring certain tissue characteristics, such as perfusion and diffusion. Although MRI contrast agents are often used to improve diagnostic specificity, MRI is limited in molecular imaging applications because of its inherently low sensitivity when compared to nuclear medicine or fluorescence imaging. 1 Consequently, the search for new agents that can be detected by MRI at much lower concentrations continues to be an active area of research. The most widely used contrast agents are low molecular weight Gd 3+-based complexes that shorten

Research paper thumbnail of A Bridge to Coordination Isomer Selection in Lanthanide(III) DOTA-tetraamide Complexes

Inorganic Chemistry

Interest in macrocyclic lanthanide complexes such as DOTA is driven largely through interest in t... more Interest in macrocyclic lanthanide complexes such as DOTA is driven largely through interest in their use as contrast agents for MRI. The lanthanide tetraamide derivatives of DOTA have shown considerable promise as PARACEST agents, taking advantage of the slow water exchange kinetics of this class of complex. We postulated that water exchange in these tetraamide complexes could be slowed even further by introducing a group to sterically encumber the space above the water coordination site, thereby hindering the departure and approach of water molecules to the complex. The ligand 8O 2-bridged-DOTAM was synthesized in a 34% yield from cyclen. It was found that the lanthanide complexes of this ligand did not possess a water molecule in the inner coordination sphere of the bound lanthanide. The crystal structure of the ytterbium complex revealed that distortions to the coordination sphere were induced by the steric constraints imposed on the complex by the bridging unit. The extent of the distortion was found to increase with increasing ionic radius of the lanthanide ion, eventually resulting in a complete loss of symmetry in the complex. Because this ligand system is bicyclic, the conformation of each ring in the system is constrained by that of the other, in consequence inclusion of the bridging unit in the complexes means only a twisted square antiprismatic coordination geometry is observed for complexes of 8O 2-bridged-DOTAM.

Research paper thumbnail of p-Isothiocyanatobenzyl-desferrioxamine: a new bifunctional chelate for facile radiolabeling of monoclonal antibodies with zirconium-89 for immuno-PET imaging

European Journal of Nuclear Medicine and Molecular Imaging, 2009

Purpose Immuno-PET is an emerging imaging tool for the selection of high potential antibodies (mA... more Purpose Immuno-PET is an emerging imaging tool for the selection of high potential antibodies (mAbs) for imaging and therapy. The positron emitter zirconium-89 (89 Zr) has attractive characteristics for immuno-PET with intact mAbs. Previously, we have described a multi-step procedure for stable coupling of 89 Zr to mAbs via the bifunctional chelate (BFC) tetrafluorophenol-N-succinyldesferal (TFP-N-sucDf). To enable widespread use of 89 Zr-immuno-PET, we now introduce the novel BFC p-isothiocyanatobenzyl-desferriox amine B (Df-Bz-NCS) and compare its performance in 89 Zrimmuno-PET with the reference BFC TFP-N-sucDf. Methods Three mAbs were premodified with Df-Bz-NCS and labeled with 89 Zr at different pHs to assess the reaction kinetics and robustness of the radiolabeling. Stability of both 89 Zr-Df-Bz-NCS-and 89 Zr-N-sucDf-conjugates was evaluated in different buffers and human serum. Comparative biodistribution and PET studies in tumor-bearing mice were undertaken. Results The selected conjugation conditions resulted in a chelate:mAb substitution ratio of about 1.5:1. Under optimal radiolabeling conditions (pH between 6.8-7.2), the radiochemical yield was >85% after 60 min incubation at room temperature, resulting in radioimmunoconjugates with preserved integrity and immunoreactivity. The new radioimmunoconjugate was very stable in serum for up to 7 days at 37°C, with <5% 89 Zr release, and was equally stable compared to the reference conjugate when stored in the appropriate buffer at 4°C. In biodistribution and imaging experiments, the novel and the reference radioimmunoconjugates showed high and similar accumulation in tumors in nude mice. Conclusions The novel Df-Bz-NCS BFC allows efficient and easy preparation of optimally performing 89 Zr-labeled mAbs, facilitating further exploration of 89 Zr-immuno-PET as an imaging tool.

Research paper thumbnail of Amphiphilic EuDOTA‐Tetraamide Complexes Form Micelles with Enhanced CEST Sensitivity

European Journal of Inorganic Chemistry, 2012

The synthesis and characterization of four new DOTA‐tetraamide ligands having variable alkyl chai... more The synthesis and characterization of four new DOTA‐tetraamide ligands having variable alkyl chain lengths (C1, C12, C14, and C16) and their respective europium(III) complexes are reported. The three EuL complexes having long alkyl chains spontaneously form micelles of variable size. The critical micelle concentration differed for each complex (lower for the C16 complex than the C12 complex) while micelle size increased with increasing alkyl chain length. Chemical exchange saturation transfer (CEST) experiments showed that all four EuIII complexes displayed slow‐to‐intermediate water exchange kinetics. As expected, the CEST signals in these complexes decreased with increasing temperatures due to faster water exchange but, interestingly, the CEST signals for the C14 and C16 complexes approached a maximum near 25 °C consistent with exchange limited CEST at or near room temperature. The water residence lifetimes obtained by fitting the CEST spectra to the Bloch equations increased in p...

Research paper thumbnail of Investigations into whole water, prototropic and amide proton exchange in lanthanide(iii) DOTA-tetraamide chelates

Dalton Transactions, 2011

Research paper thumbnail of A europium(iii)-based PARACEST agent for sensing singlet oxygen by MRI

Dalton Transactions, 2013

A europium (III) DOTA-tetraamide complex was designed as a MRI sensor of singlet oxygen (1 O 2). ... more A europium (III) DOTA-tetraamide complex was designed as a MRI sensor of singlet oxygen (1 O 2). The water soluble, thermodynamically stable complex reacts rapidly with 1 O 2 to form an endoperoxide derivative that results in an ∼3 ppm shift in the position of the Eu(III)-bound water chemical exchange saturation transfer (CEST) peak. The potential of using this probe to detect accumulation of the endoperoxide derivative in biological media by ratiometric CEST imaging was demonstrated.

Research paper thumbnail of The pH sensitivity of –NH exchange in LnDOTA–tetraamide complexes varies with amide substituent

Contrast Media & Molecular Imaging, 2011

The amide proton exchange rates in various lanthanide(III) DOTA–tetraamide complexes were investi... more The amide proton exchange rates in various lanthanide(III) DOTA–tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA–(gly)4− (Yb‐1), YbDOTA–(NHCH2PO3)45− (Yb‐2) and YbDOTA–(NHCH2PO3Et2)43+ (Yb‐3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb‐1, Yb‐2 and Yb‐3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the –NH protons in Yb‐1 and Yb‐2 were similar (−17 ppm) while the –NH proton in Yb‐3 was at −13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb‐3 is substantially weaker than that in the other two complexes. In ...

Research paper thumbnail of Abstract 5275: Conjugation and radiolabeling of monoclonal antibodies and other proteins with 89Zr or 68Ga for PET imaging using p-isothiocyanatobenzyl-desferrioxamine

Cancer Research, 2011

Presently, hundreds of intact monoclonal antibodies (mAbs) and mAb fragments are under clinical d... more Presently, hundreds of intact monoclonal antibodies (mAbs) and mAb fragments are under clinical development because of their excellent potential treatment of cancer and other pathological conditions. Positron emission tomography (PET) offers an exciting imaging option to confirm and quantify selective tumor uptake of such targeting molecules. To enable PET imaging of mAbs (immuno-PET), an appropriate positron emitter, with a half-life (t1/2) that is compatible with the time needed to achieve optimal tumor-to-non tumor ratios (typically 2-4 days for intact mAbs and Since the aforementioned multi-step labeling approach is relatively complicated and time consuming the new BFC p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS; Macrocyclics, TX) was developed. In this study, we present an efficient and more rapid preparation of 89Zr-mAbs and 68Ga-Nanobodies, whereby conjugates are now prepared in 2 instead of 6 steps. In vitro stability measurements were performed, followed by biodistribution and PET studies in nude mice bearing tumor xenografts. Labeling of mAbs with 89Zr using the Df-Bz-NCS BFC resulted in radiochemical yields >85% after 60 min at room temperature (RT). Labeling of Nanobodies with 68Ga via the same BFC resulted in radiochemical yields of 55-70% (not corrected for decay) after 5 min at RT. All labeled compounds showed preserved integrity and immunoreactivity while the stability in human serum at 37°C was good: The novel Df-Bz-NCS BFC allows efficient, rapid and easy preparation of radiolabeled mAbs or mAb-fragments with high tumor uptake in nude mice bearing human tumor xenografts. These achievements facilitate further exploration of immuno-PET as imaging tool in antibody development, target characterization and patient selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5275. doi:10.1158/1538-7445.AM2011-5275

Research paper thumbnail of Strategies for labeling proteins with PARACEST agents

Bioorganic & Medicinal Chemistry, 2011

Reactive surface lysine groups on the chimeric monoclonal antibody (3G4) and on human serum album... more Reactive surface lysine groups on the chimeric monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4-10.1 chelates were added per 3G4 molecule and between 5.6-5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53 μs for the non-conjugated chelate to 65-77 μs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73-75 μs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein.

Research paper thumbnail of Alpha-Methyl Functionalization of Electron-Poor Heterocycles and Synthesis of Novel Dipyridine and Phenanthroline Metallocyclic Palladium Complexes

Research paper thumbnail of Unexpected Changes in the Population of Coordination Isomers for the Lanthanide Ion Complexes of DOTMA-Tetraglycinate

Inorganic chemistry, Jan 7, 2016

Lanthanide complexes with DOTA-tetraglycinate (DOTA-(gly)4) heavily favor the square antiprismati... more Lanthanide complexes with DOTA-tetraglycinate (DOTA-(gly)4) heavily favor the square antiprismatic (SAP) coordination isomer in aqueous solution, a structural feature that has made them useful as water-based paraCEST agents. In an effort to create amide-based paraCEST agents with rapid water exchange rates, we prepared the analogous tetraglycinate complexes with DOTMA, a ligand known to favor the twisted square antiprismatic (TSAP) coordination structures. Unexpectedly, NMR investigations show that the LnDOTMA-(gly)4 complexes, like the LnDOTA-(gly)4 complexes, also favor the SAP isomers in solution. This observation led to density functional theory (DFT) calculations in order to identify the energy terms that favor the SAP structures in lanthanide complexes formed with macrocyclic DOTA- and DOTMA-tetraamide ligands. The DFT calculations revealed that, regardless the nature of the ligand, the TSAP isomers present more negative hydration energies than the SAP counterparts. The extent...

Research paper thumbnail of Amplifying the Sensitivity of Zinc(II) Responsive MRI Contrast Agents by Altering Water Exchange Rates

Journal of the American Chemical Society, 2015

Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI con... more Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI contrast agent, GdDOTA-diBPEN, new structural targets were rationally designed to increase the rate of water exchange to improve MRI detection sensitivity. These new sensors exhibit fine-tuned water exchange properties and, depending on the individual structure, demonstrate significantly improved longitudinal relaxivities (r 1). Two sensors in particular demonstrate optimized parameters and, therefore, show exceptionally high longitudinal relaxivities of about 50 mM −1 s −1 upon binding to Zn(II) and human serum albumin (HSA). This value demonstrates a 3-fold increase in r 1 compared to that displayed by the original sensor, GdDOTA-diBPEN. In addition, this study provides important insights into the interplay between structural modifications, water exchange rate, and kinetic stability properties of the sensors. The new high relaxivity agents were used to successfully image Zn(II) release from the mouse pancreas in vivo during glucose stimulated insulin secretion.

Research paper thumbnail of A pH-Responsive MRI Agent that Can Be Activated Beyond the Tissue Magnetization Transfer Window

Angewandte Chemie, 2015

A terbium-based complex that displays a water exchange CEST resonance well-outside the normal mag... more A terbium-based complex that displays a water exchange CEST resonance well-outside the normal magnetization transfer (MT) frequency range of tissues provides a direct readout of pH by MRI. Deprotonation of the phenolic proton in this complex results in a frequency shift of 56 ppm in a bound water molecule exchange peak between pH 5 and 8. This allows direct imaging pH without prior knowledge of the agent concentration and with essentially no interference from the tissue MT signal.

Research paper thumbnail of Facile radiolabeling of monoclonal antibodies and other proteins with zirconium-89 or gallium-68 for PET imaging using p-isothiocyanatobenzyl-desferrioxamine

Research paper thumbnail of TmDOTA-Tetraglycinate Encapsulated Liposomes as pH-Sensitive LipoCEST Agents

Research paper thumbnail of A lanthanide complex with dual biosensing properties: CEST (chemical exchange saturation transfer) and BIRDS (biosensor imaging of redundant deviation in shifts) with europium DOTA-tetraglycinate

NMR in Biomedicine, 2011

Responsive contrast agents (RCAs) [R1.12] composed of lanthanide III ion (Ln 3+) complexes with a... more Responsive contrast agents (RCAs) [R1.12] composed of lanthanide III ion (Ln 3+) complexes with a variety of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA 4−) derivatives have shown great potential as molecular imaging agents for magnetic resonance (MR). A variety of LnDOTA-tetraamide complexes have been demonstrated as RCAs for molecular imaging with chemical exchange saturation transfer (CEST). The CEST method detects proton exchange between bulk water and any exchangeable sites on the ligand itself or an inner sphere of bound water that is shifted by a paramagnetic Ln 3+ bound in the core of the macrocycle. It has also been shown that molecular imaging is possible when the RCA itself is observed (i.e., not its affect on bulk water) using a method called biosensor imaging of redundant deviation in shifts (BIRDS). The BIRDS method utilizes redundant information stored in the non-exchangeable proton resonances emanating from the paramagnetic RCA for ambient factors like temperature and/or pH. Thus CEST and BIRDS rely on exchangeable and non-exchangeable protons, respectively, for biosensing. We posited that it is feasible to combine these two biosensing features into the same RCA (i.e., dual CEST and BIRDS properties). A complex between europium ion (Eu 3+) and DOTA-tetra-glycinate (DOTA-(gly) 4 4−) is used to demonstrate that its CEST characteristics are preserved while BIRDS properties are detected. In vitro temperature sensitivity of EuDOTA-(gly) 4 − is used to show that qualitative MR contrast with CEST can be calibrated using quantitative MR mapping with BIRDS, thereby enabling quantitative molecular imaging at high spatial resolution.

Research paper thumbnail of Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine

Research paper thumbnail of Multifunctional Polymeric Scaffolds for Enhancement of PARACEST Contrast Sensitivity and Performance: Effects of Random Copolymer Variations

Macromolecules, 2010

A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) tetraamide ligand having a s... more A DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) tetraamide ligand having a single acrylamide side-chain (M1) was copolymerized with either 2-methylacrylic acid (MAA), 2-(acryloylamino)-2-methyl-1-propanesulfonic acid (AMPS) or N-isopropylacrylamide (NIPAM) to create a series of linear random copolymers using classical free radical chain polymerization chemistry. The metal ion binding properties of hydrolyzed M1 were investigated by pH potentiometry and the europium (III) complexes of the resulting heteropolymers were evaluated as PARACEST imaging agents. All polymeric agents were found to possess similar intermediate-toslow water exchange and CEST characteristics as the parent EuDOTA-tetraamide monomer. Consistent with basic multiplexing principles, the highest molecular weight polymer, Eu-DMAA 3.1, also showed the highest CEST sensitivity with a detection limit of 20 ± 2 μM. The second arylamide component gave polymers with widely different chemical characteristics and CEST properties. In particular, the Eu-DNIPAM 4.0 and Eu-DMAA 4.1 polymers displayed different solubility characteristics as a function of pH or temperature which, in turn, affected the water exchange and CEST properties of the corresponding agents. It was concluded that introduction of hydrophobic groups into the polymer backbone reduces solvent accessibility to the Eu 3+ component, effectively slowing water exchange between the inner-sphere water coordination position at each Eu 3+ center with bulk water. The CEST properties of the heteropolymers when dissolved in plasma suggest that the more hydrophobic characteristics of these polymers could be advantageous for in vivo applications.

Research paper thumbnail of A Responsive Europium(III) Chelate That Provides a Direct Readout of pH by MRI

Journal of the American Chemical Society, 2010

A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric CEST... more A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric CEST principles. Deprotonation of a single phenolic proton between pH 6 and 7.6 results in an ~5 ppm shift in the water exchange CEST peak that is easily detected by MRI. Collection of two CEST images at two slightly different activation frequencies provides a direct readout of solution pH without the need of a concentration marker.

Research paper thumbnail of Synthesis, Relaxometric and Photophysical Properties of a New pH-Responsive MRI Contrast Agent:  The Effect of Other Ligating Groups on Dissociation of a p-Nitrophenolic Pendant Arm

Journal of the American Chemical Society, 2004

Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdeca... more Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triAcetate) and GdNP-DO3AM (1-methlyene(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide), containing a single nitrophenolic pendant arm plus either three acetate or three amide pendant arms were synthesized and characterized. The properties of the gadolinium, terbium, and dysprosium complexes of these ligands were examined as a function of pH. The extent and mechanism of the changes in water relaxivity with pH of each gadolinium complex was found to differ substantially for the two complexes. The water relaxivity of Gd(NP-DO3A) increases from 4.1 mM −1 s −1 at pH 9 to 7.0 mM −1 s −1 at pH 5 as a result of acid-catalyzed dissociation of the nitrophenol from the lanthanide. The nitrophenol group in Gd(NP-DO3AM) does not dissociate from the metal center even at pH 5; therefore, the very modest increase in relaxivity in this complex must be ascribed to an increase in prototropic exchange rate of the bound water and/or phenolic protons.

Research paper thumbnail of Polymeric PARACEST Agents for Enhancing MRI Contrast Sensitivity

Journal of the American Chemical Society, 2008

Linear polymers of PARACEST agents were prepared by using classical free radical chain polymeriza... more Linear polymers of PARACEST agents were prepared by using classical free radical chain polymerization conditions. The Eu 3+-polymers exhibited similar intermediate-to-slow water exchange and CEST characteristics as the Eu 3+-monomers. This provided an avenue to lower the detection limit of these imaging agents substantially and makes them potentially useful as MRI sensors for molecular imaging. Magnetic resonance imaging (MRI) is an important clinical tool for anatomical imaging and monitoring certain tissue characteristics, such as perfusion and diffusion. Although MRI contrast agents are often used to improve diagnostic specificity, MRI is limited in molecular imaging applications because of its inherently low sensitivity when compared to nuclear medicine or fluorescence imaging. 1 Consequently, the search for new agents that can be detected by MRI at much lower concentrations continues to be an active area of research. The most widely used contrast agents are low molecular weight Gd 3+-based complexes that shorten

Research paper thumbnail of A Bridge to Coordination Isomer Selection in Lanthanide(III) DOTA-tetraamide Complexes

Inorganic Chemistry

Interest in macrocyclic lanthanide complexes such as DOTA is driven largely through interest in t... more Interest in macrocyclic lanthanide complexes such as DOTA is driven largely through interest in their use as contrast agents for MRI. The lanthanide tetraamide derivatives of DOTA have shown considerable promise as PARACEST agents, taking advantage of the slow water exchange kinetics of this class of complex. We postulated that water exchange in these tetraamide complexes could be slowed even further by introducing a group to sterically encumber the space above the water coordination site, thereby hindering the departure and approach of water molecules to the complex. The ligand 8O 2-bridged-DOTAM was synthesized in a 34% yield from cyclen. It was found that the lanthanide complexes of this ligand did not possess a water molecule in the inner coordination sphere of the bound lanthanide. The crystal structure of the ytterbium complex revealed that distortions to the coordination sphere were induced by the steric constraints imposed on the complex by the bridging unit. The extent of the distortion was found to increase with increasing ionic radius of the lanthanide ion, eventually resulting in a complete loss of symmetry in the complex. Because this ligand system is bicyclic, the conformation of each ring in the system is constrained by that of the other, in consequence inclusion of the bridging unit in the complexes means only a twisted square antiprismatic coordination geometry is observed for complexes of 8O 2-bridged-DOTAM.

Research paper thumbnail of p-Isothiocyanatobenzyl-desferrioxamine: a new bifunctional chelate for facile radiolabeling of monoclonal antibodies with zirconium-89 for immuno-PET imaging

European Journal of Nuclear Medicine and Molecular Imaging, 2009

Purpose Immuno-PET is an emerging imaging tool for the selection of high potential antibodies (mA... more Purpose Immuno-PET is an emerging imaging tool for the selection of high potential antibodies (mAbs) for imaging and therapy. The positron emitter zirconium-89 (89 Zr) has attractive characteristics for immuno-PET with intact mAbs. Previously, we have described a multi-step procedure for stable coupling of 89 Zr to mAbs via the bifunctional chelate (BFC) tetrafluorophenol-N-succinyldesferal (TFP-N-sucDf). To enable widespread use of 89 Zr-immuno-PET, we now introduce the novel BFC p-isothiocyanatobenzyl-desferriox amine B (Df-Bz-NCS) and compare its performance in 89 Zrimmuno-PET with the reference BFC TFP-N-sucDf. Methods Three mAbs were premodified with Df-Bz-NCS and labeled with 89 Zr at different pHs to assess the reaction kinetics and robustness of the radiolabeling. Stability of both 89 Zr-Df-Bz-NCS-and 89 Zr-N-sucDf-conjugates was evaluated in different buffers and human serum. Comparative biodistribution and PET studies in tumor-bearing mice were undertaken. Results The selected conjugation conditions resulted in a chelate:mAb substitution ratio of about 1.5:1. Under optimal radiolabeling conditions (pH between 6.8-7.2), the radiochemical yield was >85% after 60 min incubation at room temperature, resulting in radioimmunoconjugates with preserved integrity and immunoreactivity. The new radioimmunoconjugate was very stable in serum for up to 7 days at 37°C, with <5% 89 Zr release, and was equally stable compared to the reference conjugate when stored in the appropriate buffer at 4°C. In biodistribution and imaging experiments, the novel and the reference radioimmunoconjugates showed high and similar accumulation in tumors in nude mice. Conclusions The novel Df-Bz-NCS BFC allows efficient and easy preparation of optimally performing 89 Zr-labeled mAbs, facilitating further exploration of 89 Zr-immuno-PET as an imaging tool.

Research paper thumbnail of Amphiphilic EuDOTA‐Tetraamide Complexes Form Micelles with Enhanced CEST Sensitivity

European Journal of Inorganic Chemistry, 2012

The synthesis and characterization of four new DOTA‐tetraamide ligands having variable alkyl chai... more The synthesis and characterization of four new DOTA‐tetraamide ligands having variable alkyl chain lengths (C1, C12, C14, and C16) and their respective europium(III) complexes are reported. The three EuL complexes having long alkyl chains spontaneously form micelles of variable size. The critical micelle concentration differed for each complex (lower for the C16 complex than the C12 complex) while micelle size increased with increasing alkyl chain length. Chemical exchange saturation transfer (CEST) experiments showed that all four EuIII complexes displayed slow‐to‐intermediate water exchange kinetics. As expected, the CEST signals in these complexes decreased with increasing temperatures due to faster water exchange but, interestingly, the CEST signals for the C14 and C16 complexes approached a maximum near 25 °C consistent with exchange limited CEST at or near room temperature. The water residence lifetimes obtained by fitting the CEST spectra to the Bloch equations increased in p...

Research paper thumbnail of Investigations into whole water, prototropic and amide proton exchange in lanthanide(iii) DOTA-tetraamide chelates

Dalton Transactions, 2011

Research paper thumbnail of A europium(iii)-based PARACEST agent for sensing singlet oxygen by MRI

Dalton Transactions, 2013

A europium (III) DOTA-tetraamide complex was designed as a MRI sensor of singlet oxygen (1 O 2). ... more A europium (III) DOTA-tetraamide complex was designed as a MRI sensor of singlet oxygen (1 O 2). The water soluble, thermodynamically stable complex reacts rapidly with 1 O 2 to form an endoperoxide derivative that results in an ∼3 ppm shift in the position of the Eu(III)-bound water chemical exchange saturation transfer (CEST) peak. The potential of using this probe to detect accumulation of the endoperoxide derivative in biological media by ratiometric CEST imaging was demonstrated.

Research paper thumbnail of The pH sensitivity of –NH exchange in LnDOTA–tetraamide complexes varies with amide substituent

Contrast Media & Molecular Imaging, 2011

The amide proton exchange rates in various lanthanide(III) DOTA–tetraamide complexes were investi... more The amide proton exchange rates in various lanthanide(III) DOTA–tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA–(gly)4− (Yb‐1), YbDOTA–(NHCH2PO3)45− (Yb‐2) and YbDOTA–(NHCH2PO3Et2)43+ (Yb‐3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb‐1, Yb‐2 and Yb‐3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the –NH protons in Yb‐1 and Yb‐2 were similar (−17 ppm) while the –NH proton in Yb‐3 was at −13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb‐3 is substantially weaker than that in the other two complexes. In ...

Research paper thumbnail of Abstract 5275: Conjugation and radiolabeling of monoclonal antibodies and other proteins with 89Zr or 68Ga for PET imaging using p-isothiocyanatobenzyl-desferrioxamine

Cancer Research, 2011

Presently, hundreds of intact monoclonal antibodies (mAbs) and mAb fragments are under clinical d... more Presently, hundreds of intact monoclonal antibodies (mAbs) and mAb fragments are under clinical development because of their excellent potential treatment of cancer and other pathological conditions. Positron emission tomography (PET) offers an exciting imaging option to confirm and quantify selective tumor uptake of such targeting molecules. To enable PET imaging of mAbs (immuno-PET), an appropriate positron emitter, with a half-life (t1/2) that is compatible with the time needed to achieve optimal tumor-to-non tumor ratios (typically 2-4 days for intact mAbs and Since the aforementioned multi-step labeling approach is relatively complicated and time consuming the new BFC p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS; Macrocyclics, TX) was developed. In this study, we present an efficient and more rapid preparation of 89Zr-mAbs and 68Ga-Nanobodies, whereby conjugates are now prepared in 2 instead of 6 steps. In vitro stability measurements were performed, followed by biodistribution and PET studies in nude mice bearing tumor xenografts. Labeling of mAbs with 89Zr using the Df-Bz-NCS BFC resulted in radiochemical yields >85% after 60 min at room temperature (RT). Labeling of Nanobodies with 68Ga via the same BFC resulted in radiochemical yields of 55-70% (not corrected for decay) after 5 min at RT. All labeled compounds showed preserved integrity and immunoreactivity while the stability in human serum at 37°C was good: The novel Df-Bz-NCS BFC allows efficient, rapid and easy preparation of radiolabeled mAbs or mAb-fragments with high tumor uptake in nude mice bearing human tumor xenografts. These achievements facilitate further exploration of immuno-PET as imaging tool in antibody development, target characterization and patient selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5275. doi:10.1158/1538-7445.AM2011-5275

Research paper thumbnail of Strategies for labeling proteins with PARACEST agents

Bioorganic & Medicinal Chemistry, 2011

Reactive surface lysine groups on the chimeric monoclonal antibody (3G4) and on human serum album... more Reactive surface lysine groups on the chimeric monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4-10.1 chelates were added per 3G4 molecule and between 5.6-5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53 μs for the non-conjugated chelate to 65-77 μs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73-75 μs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein.