De novo synthesis of a new diethylenetriaminepentaacetic acid (DTPA) bifunctional chelating agent

Diethylene triamine pentaacetic acid (DTPA) has been in extensive use as a metal chelator in the development of radiopharmaceuticals and contrast agents. The former application uses DTPA mostly as a bifunctional chelating agent (BCA) conjugated to tumor-targeting vehicles (TTVs) such as monoclonal antibodies (MAbs) and receptor-directed peptides. A new bifunctional DTPA derivative was synthesized by a fully organic scheme. This compound, N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-[pentakis(carboxymethyl)]-N(4)-(carboxymethyl)-2,6-diamino-4-azahexanoic hydrazide (20) was prepared by a convergent synthesis strategy using N(alpha)-benzyloxycarbonyl-2,3-diaminopropionic acid as the starting compound. This commercially available material was used to build a functionalized triamine which served as the molecular core template for assembling the target molecule. To evaluate the conjugation and radiolabeling capabilities of this new molecule, it was covalently attached to the anti-TAG-72 MAb, Delta CH2HuCC49, and the conjugate was radiolabeled in near-quantitative yields with yttrium-90 ((90)Y) and lutetium-177 ((177)Lu). Biodistribution of the (177)Lu-labeled DTPA-Delta CH2HuCC49 in tumor-bearing nude mice demonstrated preservation of the immunoreactivity of the MAb as indicated by high tumor uptake. In addition to the introduction of a new bifunctional DTPA, this work reports on a novel synthetic approach for preparation of this useful metal chelator and introduces a new conjugation protocol.     

Methods for MAG3 conjugation and 99mTc radiolabeling of biomolecules

The chelator mercaptoacetyltriglycine (MAG3) forms a single stable chelate with technetium-99m (99mTc) oxotechnetate. The bifunctional N-hydroxysuccinimidyl ester of mercaptoacetyltriglycine with S-acetyl protection of the sulfhydryl group may be used to conjugate MAG3 to primary amine functionalized biomolecules for the purpose of radiolabeling with 99mTc for gamma detection or single photon emission computed tomography imaging (SPECT). We report here an improved MAG3 conjugation and 99mTc radiolabeling method capable of generating high radiochemical yield and high specific radioactivity. Post-labeling purification will not be needed if the protocol is followed as presented. Apart from the preparation of reagents, the conjugation and purification requires about 4 h, while the labeling with 99mTc requires about an additional 30 min.     

Fast and easy access to efficient bifunctional chelators for MRI applications

Novel bifunctional agents based on the AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) structure with hydroxyl, carboxyl and chloro functional groups were prepared. The Gd^III complexes show optimal magnetic properties making them very good candidates for conjugation to biomolecules for molecular imaging applications. An example of conjugation to a bile acid derivative is also reported.     

An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with (99m)Tc at room temperature

The mercaptoacetyltriglycine (MAG3) chelator has been shown to stably complex technetium-99m (99mTc) for nuclear imaging and radiorhenium (186/188Re) for tumor radiation therapy studies. The bifunctional N-hydroxysuccinimidyl ester of MAG3 with S-acetyl protection (N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycinate (NHS-MAG3)) has been successfully used to covalently conjugate a MAG3 chelator to primary amine functionalized biomolecules. We describe herein a simplified synthesis of NHS-MAG3 that begins with the preparation of the N-hydroxysuccinimidyl ester of S-acetylmercaptoacetic acid (N-succinimidyl S-acetylmercaptoacetate (SATA)) from mercaptoacetic acid and is followed by the synthesis of S-acetylmercaptoacetyltriglycine from SATA, together requiring about 14 days. Finally, the synthesis of NHS-MAG3 from S-acetylmercaptoacetyltriglycine requires a further 5 days. We had earlier described a method for the preparation of MAG3-conjugated and 99mTc-radiolabeled biomolecules that required elevated temperatures during postconjugation purification. We now report a modified method for the preparation that is accomplished at room temperature and therefore applicable to temperature-sensitive biomolecules. The conjugation and radiolabeling of bovine serum albumin is used as an example. The conjugation and purification requires about 2-3 h and the radiolabeling and postlabeling purification requires about an additional 2 h.     

An N2S2 bifunctional chelator for technetium-99m and rhenium: complexation, conjugation, and epimerization to a single isomer.

A bifunctional chelator 6 was prepared bearing an N2S2 core for binding rhenium or technetium and a carboxylic acid group for conjugation to amino groups of biomolecules. Complexation of 6 with rhenium(V) resulted in two kinetic isomers, anti-7 and syn-7, being formed in approximately equal amounts. Epimerization with 0.5 M NaOH yields a single isomer anti-7, as determined by NMR spectroscopy and single-crystal X-ray analysis. The 99mTc complex was prepared at the tracer level by reaction of the ligand with 99mTcO4-, tin(II) chloride and sodium gluconate giving a mixture of two isomers, but showing a preference for the anti isomer. Chelation in the presence of 1 M NaOH results in anti-8 being formed as the sole product. The bifunctional ability of the ligand was explored by amide formation with (S)-alpha-phenethylamine, either by direct DCC coupling or through the N-hydroxy succinimidyl ester 9 intermediate. The deprotected bioconjugate 11 was complexed with rhenium, yielding similar amounts of two isomeric rhenium complexes, anti-12 and syn-12, which were isolated and characterized by NMR spectroscopy. Treatment of the kinetic mixture of anti-12 and syn-12 with 1 M NaOH resulted in quantitative conversion to a single rhenium complex anti-12. With technetium-99m in 0.1 M sodium acetate, bioconjugate 11 yielded both technetium-99m complexes anti-13 and syn-13, in a 2:1 ratio, respectively. In contrast, complexation in the presence of 1 M NaOH gave only one technetium-99m complex, assigned the structure anti-13.     

Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, (64)Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of (64)Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with (64)Cu.     

Gallium-68 complex of a macrobicyclic cage amine chelator tethered to two integrin-targeting peptides for diagnostic tumor imaging

Tumor-targeting peptides radiolabeled with positron-emitting (68)Ga are promising candidates as new noninvasive diagnostic agents for positron emission tomography (PET). The targeting peptides are tethered to a chelator that forms a stable coordination complex with Ga(3+) that is inert to dissociation of Ga(3+)in vivo. Metal complexes of macrobicyclic hexaamine "sarcophagine" (sar = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) ligands exhibit remarkable stability as a result of the encapsulating nature of the cage amine ligand. A Ga(3+) sarcophagine complex, [Ga-(1-NH(3)-8-NH(2)-sar)](4+), has been characterized using X-ray crystallography, demonstrating that Ga(3+) is coordinated to six nitrogen atoms in a distorted octahedral complex. A bifunctional derivative of (NH(2))(2)sar, possessing two aliphatic linkers with carboxylic acid functional groups has been attached to two cyclic-RGD peptides that target the α(v)β(3) integrin receptor that is overexpressed in some types of tumor tissue. This dimeric species can be radiolabeled with (68)Ga(3+) in >98% radiochemical yield and (68)Ga(3+) does not dissociate from the ligand in the presence of transferrin, an endogenous protein with high affinity for Ga(3+). Biodistribution and micro-PET imaging studies in tumor-bearing mice indicate that the tracer accumulates specifically in tumors with high integrin expression. The high tumor uptake is coupled with low nonspecific uptake and clearance predominantly through the kidneys resulting in high-quality PET images in animal models.     

N-terminal α-amino group modification of antibodies using a site-selective click chemistry method

Site-specific conjugation of small molecules to antibody molecules is a promising strategy for generation of antibody-drug conjugates. In this report, we describe the successful synthesis of a novel bifunctional molecule, 6-(azidomethyl)-2-pyridinecarboxyaldehyde (6-AM-2-PCA), which was used for conjugation of small molecules to peptides and antibodies. We demonstrated that 6-AM-2-PCA selectively reacted with N-terminal amino groups of peptides and antibodies. In addition, the azide group of 6-AM-2-PCA enabled copper-free click chemistry coupling with dibenzocyclooctyne-containing reagents. Bifunctional 6-AM-2-PCA mediated site-specific conjugation without requiring genetic engineering of peptides or antibodies. A key advantage of 6-AM-2-PCA as a conjugation reagent is its ability to modify proteins in a single step under physiological conditions that are sufficiently moderate to retain protein function. Therefore, this new click chemistry-based method could be a useful complement to other conjugation methods.     

Novel chemically modified analogues of neuropeptide Y for tumor targeting

The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y 1/Y 2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the N(epsilon) side chain of Lys(4) of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y 1-receptor selective [Lys(DOTA)(4), Phe(7), Pro(34)]NPY was labeled with (111)In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In vitro and in vivo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.     

Labeling of proteins using [105Rh]Rh-4-(p-aminobenzyl)-diethylenetriamine

A bifunctional ligand 4-(p-aminobenzyl)-diethylenetriamine has been synthesized. ¹⁰⁵Rh complexes with this ligand were prepared with an overall yield of 79% at pH 9.0 in bicarbonate buffer. The preformed complex was converted to the isothiocyanate derivative using thiophosgene. Conjugation yields of 75% with IgG and 85% with HSA could be obtained for a 4 h conjugation reaction. Affinity chromatography of human-IgG coupled to the rhodium complex in an anti-human IgG agarose gel indicated no denaturation of the labeled protein. The procedure reported here can be adapted for the preparation of ¹⁰⁵Rh-labeled antibodies for therapeutic applications.     

Conjugation of chelating agents to proteins and radiolabeling with trivalent metallic isotopes

Peptides and proteins may be tagged with metallic elements in order to use them as imaging reporters or for other applications. The polypeptide of interest is first conjugated to a suitable chelating agent that forms stable complexes with the element of interest. This conjugation step is undertaken either in aqueous or in non-aqueous conditions depending on the solubility of the substrate. For polypeptides of greater than approximately 10 kDa in size, this is normally done in aqueous medium. Most commonly the chelators are reacted with lysine amino groups. The protein is first desalted into a suitable buffer at pH 8-9 and a molar excess of a bifunctional chelating agent is added. After a suitable period of incubation, excess, unreacted or hydrolyzed chelator is removed and the protein conjugate is desalted into an acidic buffer. The conjugate can then be tagged by addition of a suitable metal salt followed, if necessary, by removal of unchelated metal. As described in the protocol that follows, the entire conjugation, purification and labeling procedure takes about 2 d.     

The production of 99mTc-labeled conjugated antibodies using a cyclam-based bifunctional chelating agent

A bifunctional chelating agent (BFCA) based on a macrocyclic amine, cyclam, was used to form specifically ^99mTc labeled rabbit-anti-HSA-antibodies. This study demonstrates the feasibility of forming a highly stable cyclam-based ^99mTc-BFCA with its subsequent conjugation to antibodies.     

Site-specific conjugation on serine right-arrow cysteine variant monoclonal antibodies

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (C(H)3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.     

A novel bifunctional maleimido CHX-A' chelator for conjugation to thiol-containing biomolecules

A novel bifunctional maleimido CHX-A' DTPA chelator 5 was developed and conjugated to the monoclonal antibody trastuzumab (Herceptin) and subsequently radiolabeled with (111)In. The resulting (111)In labeled immunoconjugate 2 was demonstrated to bind to SKOV-3 ovarian cancer cells comparably to an isothiocyanato CHX-A' DTPA modified native trastuzumab, 1. Through efficient thiol-maleimide chemistry, antibodies, peptides or other targeting vectors can now be modified with an established radioactive metal chelating agent CHX-A' DTPA for imaging and/or therapies of cancer.     

In vivo behavior of copper-64-labeled methanephosphonate tetraaza macrocyclic ligands

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.     

Synthesis of a cross-bridged cyclam derivative for peptide conjugation and 64Cu radiolabeling

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.     

Novel bimodal bifunctional ligands for radioimmunotherapy and targeted MRI

The structurally novel bifunctional ligands C-NETA and C-NE3TA, each possessing both acyclic and macrocyclic moieties, were prepared and evaluated as potential chelates for radioimmunotherapy (RIT) and targeted magnetic resonance imaging (MRI). Heptadentate C-NE3TA was fortuitously discovered during the preparation of C-NETA. An optimized synthetic method to C-NETA and C-NE3TA including purification of the polar and tailing reaction intermediates, tert-butyl C-NETA (2) and tert-butyl C-NE3TA (3) using semiprep HPLC was developed. The new Gd(III) complexes of C-NETA and C-NE3TA were prepared as contrast enhancement agents for use in targeted MRI. The T 1 relaxivity data indicate that Gd(C-NETA) and Gd(C-NE3TA) possess higher relaxivity than Gd(C-DOTA), a bifunctional version of a commercially available MRI contrast agent; Gd(DOTA). C-NETA and C-NE3TA were radiolabeled with (177)Lu, (90)Y, (203)Pb, (205/6)Bi, and (153)Gd; and in vitro stability of the radiolabeled corresponding complexes was assessed in human serum. The in vitro studies indicate that the evaluated radiolabeled complexes were stable in serum for 11 days with the exception being the (203)Pb complexes of C-NETA and C-NE3TA, which dissociated in serum. C-NETA and C-NE3TA radiolabeled (177)Lu, (90)Y, or (153)Gd complexes were further evaluated for in vivo stability in athymic mice and possess excellent or acceptable in vivo biodistribution profile. (205/6)Bi- C-NE3TA exhibited extremely rapid blood clearance and low radioactivity level at the normal organs, while (205/6)Bi- C-NETA displayed low radioactivity level in the blood and all of the organs except for the kidney where relatively high renal uptake of radioactivity is observed. C-NETA and C-NE3TA were further modified for conjugation to the monoclonal antibody Trastuzumab.     

Preactivated carboxyl linker for the rapid conjugation of alkylamines to oligonucleotides on solid support

A C10 linker phosphoramidite reagent terminated with a succinimidyl-activated carboxyl group was prepared and used to couple to the 5'-end of an oligonucleotide synthesized on a solid support. The succinimidyl-activated carboxyl functionality can be used for rapid conjugation of amines to oligonucleotides on solid support or it can be hydrolyzed to form a carboxylic acid functionality. The activated linker was successfully used for conjugation of several primary and secondary aliphatic amine derivatives (including biotin and fluorescein cadaverine) onto a solid support-bound 12-mer DNA oligonucleotide at scales ranging from 0.15 to 1.0 micromol. The overall yields of the conjugation products after AMA deprotection and cleavage from the solid support ranged from 43 to 75% of the total oligonucleotide product. This value is significant, as it includes oligonucleotide synthesis, coupling of the linker, and conjugation of the amine. In addition, the entire process of oligonucleotide synthesis, linker coupling, amine conjugation, deprotection, and cleavage of the oligonucleotide from solid support can be accomplished in 1 day.     

Conformational modulation of in vitro activity of cyclic RGD peptides via aziridine aldehyde-driven macrocyclization chemistry

Here, we demonstrate a conjugation strategy whereby cyclic RGD-containing macrocycles are prepared using aziridine aldehydes, isocyanides, and linear peptides, followed by conjugation to a cysteamine linker. Our method involves site-selective aziridine ring-opening with the nucleophilic sulfhydryl group of cysteamine. Fluorescein was then efficiently conjugated to the primary amine of cysteamine by NHS-chemistry. This strategy may be expanded to provide easy access to a wide variety of fluorescent dyes or radiometal chelators. Modeling studies showed that aziridine aldehyde cyclization chemistry stabilized the RGD motif into the required bioactive conformation and that this cyclization chemistry modulated the geometry of macrocycles of different residue lengths. In vitro studies showed that cPRGDA and cPRGDAA both selectively bound to α(V)β(3)-overexpressing U87 glioblastoma cells, and that cPRGDA had a better binding affinity compared to cPRGDAA. The improved binding affinity of cPRGDA was attributed to the fixed Pro-C(α)-Asp-C(α) distance surrounding the stabilized RGD motif in cPRGDA.     

New macrobicyclic chelator for the development of ultrastable 64Cu-radiolabeled bioconjugate

Ethylene cross-bridged cyclam with two acetate pendant arms, ECB-TE2A, is known to form the most kinetically stable (64)Cu complexes. However, its usefulness as a bifunctional chelator is limited because of its harsh radiolabeling conditions. Herein, we report new cross-bridged cyclam chelator for the development of ultrastable (64)Cu-radiolabeled bioconjugates. Propylene cross-bridged TE2A (PCB-TE2A) was successfully synthesized in an efficient way. The Cu(II) complex of PCB-TE2A exhibited much higher kinetic stability than ECB-TE2A in acid decomplexation studies, and also showed high resistance to reduction-mediated demetalation. Furthermore, the quantitative radiolabeling of PCB-TE2A with (64)Cu was achieved under milder conditions compared to ECB-TE2A. Biodistribution studies strongly indicate that the (64)Cu complexes of PCB-TE2A cleared out rapidly from the body with minimum decomplexation.