Labeling lactam derivatives

Balfour, using tritium-labeled CR to investigate uptake and metabolism, found that both intact cornea of the guinea pig and corneal homogenates take up CR readily and metabolize It to a lactam derivative. The metabolic process appeared to take place in the corneal cells. Homogenization of the corneas supported this idea after homogenization, the metabolic activity was found in supernatant fluid. The author speculated that the lactam derivative of CR Is not involved in the process of irritation, but that methyl derivatives are involved. 

Figure 44.8 Structures of TcfCOlj-labeled lactam bridge-cyclized a-MSH derivatives obtained using different pyrazolyl-diamine BFCAs.

The amount of label bound to the MIP in the absence of the analyte is known as B0 and B is the amount of label bound to the MIP in the presence of each concentration of analyte. The plot of the ratio B/B0 as a function of the log[analyte], or the log [interferent], is a sigmoid curve such as the one shown in Fig. 7 for the penicillin G assay described above. As the concentration of penicillin G increases in the sample, the amount of bound PAAP decreases as does the B/B0 ratio. Another p-lactam antibiotic not derived from penicillin, such as cephapirin, did not show any cross-reactivity (Fig. 7). 

It must be underlined that, for the development of a successful FILA based on the use of non-related tracers, the latter should also show sufficient affinity for the specific binding sites of the imprinted polymer otherwise the assay will not be selective. For instance, in order to facilitate the competition between the labeled derivative and the analyte, Moreno-Bondi et al. have developed a FILA for the analysis of penicillins [34, 36] using novel fluorescently labeled [5-lactam antibiotics with a close resemblance to the analyte (Fig. 12) [95]. 

The manufacture of the large variety of polyamides (commonly referred to as nylons) occurs through polycondensation of amino carboxylic acids (or functional derivatives of them, e.g. lactams) and from diamines and dicarboxylic acids. Labeling the amino groups with A and the carboxyl groups with B allows differentiation of the different chemical structures between the two types AB (from amino carboxylic acids) and AA-BB (from diamines and dicarboxylic acids). The number of C atoms in the monomers acts as a code number for the identification of the polyamides. The polycaprolactam manufactured from caprolactam (type AB) is then called polyamide 6 (PA 6). The number of carbon atoms in the diamine is given first for type AA-BB followed by the number of atoms in the dicarboxylic acid, e.g. PA 66 for polyhexamethylenedia-dipic amide from hexamethylenediamine and adipic acid. For copolymers the components are separated by a slash, e.g. PA 66/6 (90 10) is a copolymer composed of 90 parts PA 66 and 10 parts PA 6. 

Related 1,2-additions to imines in the -lactam area employing organocuprates have also met with considerable success. Net substitution reactions, proceeding via intermediate imino derivatives, have been achieved with lithium diallyl- and dialkyl-cuprates on educts such as (105) and (106). When a stereochemical label is present as in (107) and (108), the major products reflect trans addition to imines (109) and (110), respectively (Scheme 18). 

A stereoselective synthesis of aminoalkyl-substituted P-lactams (41) has been developed, the key step of which is [2+2] cycloaddition of imines to ketenes generated photochemically from diazoketones (42), which are derived from protected a-amino acids. A number of steroidal diazoketones related to progesterone have been synthesized as potential photoaffinity labelling reagents for the mineralocorticoid receptor. ... 

C-labeled porphobilinogen, requir for biosynthetic studies, has been prepared via reductive C-methylation using C-formaldehyde (derived from C-methanol). Further syntheses of 2-aminomethyl pyrroles and related lactams have been described, utilizing the azaindole route, and this method has also been adapted to the preparation of porphobilinogen labeled with C in the propionic acid side chain. °... 

The biosynthesis of camptothecin (102) follows that of terpenoid indole alkaloids through strictosidine (79) The lactam (101) derived from (79), and not the C-3 epimer, is the next intermediate.Several unknown steps follow, included in which is dehydrogenation of ring d [see (101)]. In this process a proton is lost from C-14. Tritium label at this site in (101) is retained by a primary isotope effect, the result of non-stereospecific deprotonation.This indicates that removal of the proton is not enzyme-controlled (c/. papaverine biosynthesis ref. 8, p. 19). 

Penicillins and cephalosporins contain a P-lactam ring, which is fused with a thiazolidine ring. These types of compounds are synthesized by both prokaryotic and eukaryotic microorganisms, including Streptomyces, Penicillium, Aspergillus, and Cephalosporium species (Luckner, 1990). Important precursors are L-2-aminoadipic acid, L-cysteine, and L-valine. An important intermediate is 6-(L-aminoadipyl)-L-cysteinyl-D-valine (Luckner, 1990). The valine unit with a d-configuration found in one portion of the molecule is labeled when radioactively labeled L-valine is added to the culture. The most important antibiotic of this group, penicillin G (3) from Penicillium spp., is derived from similar precursors. 

In order to determine the origin of the P-lactam 3-methoxyl group the authors fed L-[methyl- C]-methionine. The resulting C-labelled (252) was purified by paper electrophoresis then heated with hydroiodic acid to liberate the methoxyl methyl group as methyl iodide which was trapped and counted. It was found that 87% of the C-label of (252) was recovered in the methyl iodide showing that the methoxyl methyl group was specifically derived from methionine as is the case in cephamycin biosynthesis. 

Hydrolysis of the labeled compound with 2 N sulfuric acid furnished 3-amino-4-methyl-2-hexanone which, on treatment with iodine in the presence of sodium hydroxide, furnished isoleucine and iodoform. The iodoform was oxidized to carbon dioxide, while treatment of isoleucine with ninhydrin furnished 2-methylbutanal, isolated as its 2,4-dinitrophenylhydrazone. Kuhn-Roth oxidation of the aldehyde gave acetic acid which was degraded by the Schmidt procedure to carbon dioxide and methylamine. The 2-methylbutanal was also degraded stepwise by the method of Strassman. Determination of the radioactivity of the various degradation products showed that 94% of the activity was equally shared between the lactam C(2) and side-chain C(10) carbonyl atoms. The remaining activity was shared between C(4) of the lactam ring and the a> carbon atom of the ec-butyl side chain. The results are consistent with derivation of tenuazonic acid from isoleucine and two molecules of acetic acid. However, the direct incorporation of isoleucine into tenuazonic acid was not investigated. 

The biosynthesis of erythroskyrin was investigated by Shibata et al. (1966), who established the distribution of in the molecule which was obtained by cultivation of the mold on Czapek-Dox media containing various "Relabeled substrates. The degradation reactions which were carried out on the labeled erythroskyrin are summarized in Fig. 1 (10-12). Erythroskyrin containing the label from added DL-[l-R"RC]valine was decomposed into A-methylvaline (10) by ozonolysis. The product was converted into its N-(2,4-dinitrophenyl) derivative, which was decomposed photochemically into isobutanal (11). The 2,4-dinitrophenylhydrazone derivative of the aldehyde contained no radioactivity, while the A-(2,4-dinitrophenyl)-A-methyl-valine contained 79% of the activity of the erythroskyrin. The result showed that [l-R"RC]valine was incorporated (essentially) intact into the lactam portion of erythroskyrin, with the radioactivity located mainly at C(4). 

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