A practical guide to the construction of radiometallated bioconjugates for positron emission tomography

Positron emission tomography (PET) has become a vital imaging modality in the diagnosis and treatment of disease, most notably cancer. A wide array of small molecule PET radiotracers have been developed that employ the short half-life radionuclides (11)C, (13)N, (15)O, and (18)F. However, PET radiopharmaceuticals based on biomolecular targeting vectors have been the subject of dramatically increased research in both the laboratory and the clinic. Typically based on antibodies, oligopeptides, or oligonucleotides, these tracers have longer biological half-lives than their small molecule counterparts and thus require labeling with radionuclides with longer, complementary radioactive half-lives, such as the metallic isotopes (64)Cu, (68)Ga, (86)Y, and (89)Zr. Each bioconjugate radiopharmaceutical has four component parts: biomolecular vector, radiometal, chelator, and covalent link between chelator and biomolecule. With the exception of the radiometal, a tremendous variety of choices exists for each of these pieces, and a plethora of different chelation, conjugation, and radiometallation strategies have been utilized to create agents ranging from (68)Ga-labeled pentapeptides to (89)Zr-labeled monoclonal antibodies. Herein, the authors present a practical guide to the construction of radiometal-based PET bioconjugates, in which the design choices and synthetic details of a wide range of biomolecular tracers from the literature are collected in a single reference. In assembling this information, the authors hope both to illuminate the diverse methods employed in the synthesis of these agents and also to create a useful reference for molecular imaging researchers both experienced and new to the field.     

Targeting abasic sites and single base bulges in DNA with metalloinsertors

The site-specific recognition of abasic sites and single base bulges in duplex DNA by sterically expansive rhodium metalloinsertors has been investigated. Through DNA photocleavage experiments, Rh(bpy)2(chrysi)3+ is shown to bind both abasic sites and single base bulges site-specifically and, upon irradiation, to cleave the backbone of the defect-containing DNA. Photocleavage titrations reveal that the metal complex binds DNA containing an abasic site with high affinity (2.6(5) x 106 M-1), comparably to the metalloinsertor and a CC mismatch. The complex binds single base bulge sites with lower affinity (approximately 105 M-1). Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)2(chrysi)3+ binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites, a binding mode in which the mismatched or unpaired bases are extruded from the helix and replaced in the base stack by the sterically expansive ligand of the metalloinsertor.     

Binding of Ru(bpy)2(eilatin)2+ to matched and mismatched DNA

The DNA-binding properties of Ru(bpy)2(eilatin)(2+) have been investigated to determine if the sterically expansive eilatin ligand confers specificity for destabilized single-base mismatches in DNA. Competitive DNA photocleavage experiments employing a sequence-neutral metallointercalator, Rh(bpy)2(phi)(3+) (phi = 9,10-phenanthrenequinonediimine), and a mismatch-specific metalloinsertor, Rh(bpy)2(chrysi)(3+) (chrysi = chrysene-5,6-quinonediimine), reveal that the eilatin complex binds to a CC mismatched site with an apparent binding constant of 2.2(2) x 10(6) M(-1). Nonetheless, the selectivity in binding mismatched DNA is not high: competitive titrations with Rh(bpy)2(phi)(3+) show that the complex binds also to well-matched B-form sites. Thus, Ru(bpy)2(eilatin)(2+), despite containing the extremely expansive eilatin ligand, displays lower selectivity for the mismatch than does Rh(bpy)2(chrysi)(3+), a metalloinsertor containing the smaller, though still bulky, chrysene-5,6-quinonediimine ligand. In summary, the size and shape of the eilatin ligand allow stacking with both well-matched and mismatched DNA.     

The discretized flow on domains of attraction: a structural stability result

It is well known that, for stepsize sufficiently small, compactattractors of ordinary differential equations persist underdiscretization. The present paper describes the structure ofthe discrete-time dynamical system obtained via discretizationon A(M_h)\M_h where M_h is the approximate attractor and A(M_h)is its domain of attraction. The existence of a smooth embeddinginto a continuous-time parallelizable flow is proved. The constructioncan be used to define sections for discretizations and can beinterpreted as a justification of the method of modified equations.     

Unexpected oxidative C-C cleavage in the metallation of 2-substituted imidazolium salts to give N-heterocyclic carbene complexes

Imidazolium salts blocked at C2 with methyl or benzyl groups unexpectedly react with silver oxide to give N-heterocyclic carbene complexes of silver via an oxidative carbon-carbon bond cleavage.     

A mismatch-selective bifunctional rhodium-Oregon Green conjugate: a fluorescent probe for mismatched DNA

A fluorescent metallointercalator conjugate that selectively targets DNA base mismatches has been synthesized by coupling an organic fluorophore to a bulky Rh intercalator containing the chrysenequinone diimine ligand. Ion pairing between the cationic Rh and anionic fluorophore moieties dramatically quenches the fluorescence of the conjugate in solution and in the presence of matched DNA. However, in the presence of mismatched DNA, the fluorescence of the conjugate is increased >300%. This increase in fluorescence is attributed to the loss in intramolecular quenching associated with DNA binding; intercalation of the Rh moiety into the mismatched site can lead to electrostatic repulsion of the anionic fluorophore away from the DNA phosphate backbone and Rh. Denaturing PAGE experiments with 32P-labeled oligonucleotides indicate that the conjugate selectively binds the mismatched DNA with a binding affinity of 6 x 105 M-1 and, upon irradiation, cleaves the DNA backbone neighboring the mismatched site.     

Metallo-intercalators and metallo-insertors

Since the elucidation of the structure of double helical DNA, the construction of small molecules that recognize and react at specific DNA sites has been an area of considerable interest. In particular, the study of transition metal complexes that bind DNA with specificity has been a burgeoning field. This growth has been due in large part to the useful properties of metal complexes, which possess a wide array of photophysical attributes and allow for the modular assembly of an ensemble of recognition elements. Here we review recent experiments in our laboratory aimed at the design and study of octahedral metal complexes that bind DNA non-covalently and target reactions to specific sites. Emphasis is placed both on the variety of methods employed to confer site-specificity and upon the many applications for these complexes. Particular attention is given to the family of complexes recently designed that target single base mismatches in duplex DNA through metallo-insertion.     

Building Blocks for the Construction of Bioorthogonally Reactive Peptides via Solid-Phase Peptide Synthesis

The need for post-synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents. In this regard, the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far-reaching implications in both the laboratory and the clinic. Herein, we report the synthesis and development of a series of modular and versatile building blocks for inverse electron-demand Diels–Alder copper-free click chemistry: tetrazine-functionalized artificial amino acids. Following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines, we successfully demonstrated its effectiveness and applicability. This versatile methodology has the potential to have a transformational impact, opening the door for the rapid, facile, and modular synthesis of bioorthogonally reactive peptide probes.