Imine-based reactions have found wide application in the conjugation of biomolecules as a result of their high chemoselectivity. [1][2][3][4][5][6][7][8] Aldehydes and ketones can be readily introduced into biomolecules and are virtually inert towards reaction with other functional groups in these molecules. Under acidic conditions the carbonyl group reacts with primary amines to form a reversible imine (Scheme 1 a), and the equilibrium favors the free carbonyl. However, when a-effect nitrogens [9] such as aminooxy groups and hydrazides are used, the equilibrium favors the imine. [1][2][3][4][5][6][7][8] Oxime ligations are often called upon to link complex and precious macromolecules. [7,8] The oxime bond is stable under physiological conditions, whereas more dynamic imines, such as hydrazones, are often reduced to obtain a stable linkage. Oxime ligations proceed with modest reaction rates in acidic solution but are poorly reactive at pH 7. which limits their use in many biological applications. To improve the reaction rate, oxime ligations typically require millimolar concentrations of Scheme 1. a) Imine formation and b) transimination under acidic conditions.

We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. Keywordsglycoprotein; sialic acid; oxime ligation; aniline; periodate oxidation; metabolic labeling; live cell labelingThe expanding interest in glycoproteomics and the biological roles of glycoconjugates has increased efforts to develop efficient tools to label cell surface glycoproteins. Several elegant approaches have exploited metabolic labeling of cells, and even whole model organisms, using analogs of glycan precursors that carry bio-orthogonal groups (e.g. azide, alkyne, ketone or aldehyde), allowing the chemical ligation of reporter groups onto cell surface glycoconjugates 1, 2.The chemistries used for conjugation with these functional groups each have both advantages and disadvantages for use with living cells. The conjugation of azides with substituted triphenylphosphines using the Staudinger-Bertozzi ligation can be performed on living cells, but suffers from slow reaction kinetics 3 . Conjugation of azides with substituted-alkynes (or vice versa) with the Huisgen cycloaddition, or 'click chemistry', has rapid reaction kinetics, but requires a copper catalyst that is toxic to living cells4 , 5. The newly described ligation of azides with ring-strained alkynes is compatible with living cells and has rapid reaction kinetics, but requires reagents that are not currently commercially available 2 . Finally, imine (oxime or A limitation of all these methods is the need for culturing cells with a glycan precursor containing a bio-orthogonal group prior to labeling. As an alternative, aldehydes can be readily introduced into cell surface glycans by mild periodate oxidation, known for nearly 40 years to selectively oxidize the polyhydroxy side chain of sialic acids 7,8 . The recent demonstration that oxime ligation on complex biomolecules is dramatically accelerated using aniline as a nucleophilic catalyst 9-12 inspired us to explore the efficiency of this reaction with aldehydes on living cells introduced by metabolic labeling or periodate oxidation. While aniline can be efficiently used as a nucleophilic catalyst for labeling biomolecules in solution by oxime and hydrazone ligations, we chose to employ the oxime ligation, which gives a more stable product than the hydrazone ligation 13 . NIH Public AccessThe two approaches used for the introduction of aldehydes onto cell surface sialic acids for subsequent ligation with aminooxy-biotin are illustrated in Fig. 1a. Cells were subjected to mild periodate oxidation (1 mM NaIO 4 at 4 °C for 30 min; Supplementary Fig. 1) to selectively introduce an aldehyde at C-7 of sialic acid...

A high yielding and rapid chemoselective ligation approach is presented that uses aniline catalysis to activate aromatic aldehydes towards two amine nucleophiles, namely 6-hydrazinopyridyl and aminooxyacetyl groups. The rates of these ligations are resolved for model reactions with unprotected peptides. The resulting hydrazone and oxime conjugates are attained under ambient conditions with rate constants of 10 1 -10 3 M -1 s -1 . These rate constants exceed those of current chemoselective ligation chemistries and enable efficient labeling of peptides and proteins at low μM concentrations, at neutral pH, without using a large excess of one of the components. The utility of the approach is demonstrated by the p-fluorobenzylation of Human Serum Albumin and by the fluorescent labeling of an unprotected peptide with Alexa Fluor 488.

Aniline accelerates hydrazone formation and transimination through nucleophilic catalysis. To demonstrate the method, unprotected peptides are reacted and then scrambled using a hydrazone reaction under conditions relevant for biological applications. The strong enhancement in the rate of hydrazone equilibration broadens the scope of this stable imine in the field of dynamic covalent chemistry.

Anilin agiert in Form des orange gezeichneten Intermediats als nucleophiler Katalysator in der Oxim‐Ligation in wässriger Lösung. Die Oxim‐Ligation zweier ungeschützter Peptidfragmente wurde bei pH 4.5 und pH 7 beschleunigt, was die Anwendungsbreite dieser Reaktion auf Bedingungen ausdehnt, die für die Biokonjugation relevant sind (siehe Schema).

The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumorbearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and decreased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature. [Cancer Res 2008;68(18):7676-83]

A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.

The multivalent character of dendrimers has positioned these well-defined, highly branched macromolecules at the forefront in the development of new contrast agents for biomedical magnetic resonance imaging (MRI). By modifying the periphery of the dendrimer with gadolinium(III) chelates, the relaxivity of the resulting MRI contrast agent is increased considerably, compared to low molecular weight Gd(III) chelates. The monodisperse character of dendrimers creates a unique opportunity to introduce dendritic MRI contrast agents into clinics. In addition, a prolonged vascular retention time is obtained due to the larger size of the dendritic molecules. By using dendrimers as multivalent scaffolds carrying multiple ligands, the interaction between ligand and marker can be enhanced through multivalent interactions. Current research focuses on the combination of multivalent targeting and enhanced relaxivity. This paper describes the application of dendrimers in biomedical MRI.