Labelling of macromolecules

The naturally occurring aromatic amino acids phenylalanine, tryptophane and tyrosine (Fig. 1) have been labelled with fluorine-18 through similar electrophilic substitution methods [7]. Aromatic residues contained in peptides have been labelled with CH3C02[ F]F [105,106], an example of direct labelling of macromolecules. However, direct labelling of macromolecules is usually not the method of choice nowadays (see Section 6). 

F. Dolle, C F]Fluoropyridines From conventional radiotracers to the labelling of macromolecules such as proteins and oligonucleotides, in Ernst Sobering Research Foundation (Ed.), PETChemistry Vol. 62, Springer-Verlag, Berlin - Heidelberg - New York, 2007, pp. 111-143. 

Casanova-Schmitz M, Starr TB, Heck Hd A. 1984a. Differentiation between metabolic incorporation and covalent binding in the labeling of macromolecules in the rat nasal mucosa and bone marrow by inhaled [ " C]- and [ H] formaldehyde. Toxicol Appl Pharmacol 76 26-44. 

FIGURE 3 The stable nitroxide radical used for labelling of macromolecules containing aminogroups (1) and spin label attached to protein macromolecule (2). 

C] Cyanogen bromide can also be used for unspecific labeling of macromolecules. The reactions can be performed at near physiological conditions, with respect to temperature and pH. 

Kuhnast B, Hinnen F, Tavitian B, DoUe F (2008) [ F]FPyKYNE, a fluoropyridine-based alkyne reagent designed for the fluorine-18 labeling of macromolecules using click chemistry. J Label Compd Radiopharm 51(9) 336-342... 

Under current treatment of statistical method a set of the states of the Markovian stochastic process describing the ensemble of macromolecules with labeled units can be not only discrete but also continuous. So, for instance, when the description of the products of living anionic copolymerization is performed within the framework of a terminal model the role of the label characterizing the state of a monomeric unit is played by the moment when this unit forms in the course of a macroradical growth [25]. 

An exhaustive statistical description of living copolymers is provided in the literature [25]. There, proceeding from kinetic equations of the ideal model, the type of stochastic process which describes the probability measure on the set of macromolecules has been rigorously established. To the state Sa(x) of this process monomeric unit Ma corresponds formed at the instant r by addition of monomer Ma to the macroradical. To the statistical ensemble of macromolecules marked by the label x there corresponds a Markovian stochastic process with discrete time but with the set of transient states Sa(x) constituting continuum. Here the fundamental distinction from the Markov chain (where the number of states is discrete) is quite evident. The role of the probability transition matrix in characterizing this chain is now played by the integral operator kernel ... 

Knowing the functions (26) and(27) it is possible by means of the formalism of the theory of Markovian processes [53] to find any statistical characteristic in an ensemble of macromolecules with labeled units. A subsequent label erasing procedure is carried out by integration of the obtained expressions over time of the formation of monomeric units. Examples of the application of this algorithm are reported elsewhere [25]. 

The following generalized protocol relates to the labeling of IgG with NHS-rhodamine. Optimization of the level of rhodamine incorporation may have to be done with other proteins or other macromolecules. 

The following protocol is a general guide for labeling biological macromolecules with Lissamine rhodamine B sulfonyl chloride. Optimization of the fluorophore incorporation level (F/P ratio) may have to be done for specific labeling experiments. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

The particular case of macromolecule labelling with fluorine-18 45... 

From a chemical point of view, the half-life of fluorine-18 allows multi-step synthetic approaches that can be extended over hours. Fluorine-18 has therefore, in spite of its somewhat limited chemical repertoire, been effectively used for the labelling of numerous both relatively simple and complex bioactive chemical structures [3,5-9], including high-molecular-weight macromolecules such as peptides, proteins [10-13] and oligonucleotides [14-18]. General considerations on radiochemistry involving short-lived positron emitters will be discussed in Section 2.3. 

THE PARTICULAR CASE OF MACROMOLECULE LABELLING WITH FLUORINE-18... 

Noteworthy is the labelling of so-called peptide nucleic acids (PNAs). These constitute a class of synthetic macromolecules where the deoxyribose phosphate backbone of DNA is replaced by the pseudo-peptide A/-(2-aminoethyl)glycyl backbone, while retaining the nucleobases of DNA [270,271]. PNAs have been labelled at a terminal cysteine-site using A/-(4-[ F]fluorobenzyl)-2-bromoaceta-mide [272-274], a reagent belonging to another class of thiol-selective reagents. 

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