Lysine dansylation

Although stack injection has been employed previously [316,582], the benefit of stacking for sample pre-concentration was only studied in detail later [346]. With the sample buffer (0.5 mM) at a 10-fold lower conductivity than the separation buffer (5 mM), simple EK stacking using the gated injection was observed. This was applied to the separation of dansylated amino acids (dansyl-lysine, didansyl-lysine, dansyl-isoleucine, and didansyl-isoleucine) [346]. 

A dansyl-containing lysine analogue, monodansylcadaverine (MDC), was used in initial TGase linking, because the dansyl UV absorption peak allowed quantification by reverse-phase HPLC using the absorbance at 280 nm. The reacted Qll was dissolved in TFA along with a known dansyl standard. Peak areas of the standard were then compared with product to establish the amormt of MDC present into Qll. Six Qll peaks were measured and ascribed to Qll with zero to five MDCs attached (Fig. 33). 

Repeated addition of MDC to Q11 did occur, but the dominant product was Q11 with a single MDC. The fraction of Qll with higher numbers of attached MDC decreased for increasing MDC number. Separately, a lysine peptide that contained the bioactive RGD [73] sequence ( -dansyl-GLKGGRGDS-Am) was successfully TGase crosslinked with self-assembled Qll five distinct Qll-dansyl RGD were detected by mass spectrometry. 

Suitably protected amino acids (112) (cysteine, serine, and lysine) have been added via the side-chain heteroatom (S, O, and N, respectively) to conjugated alkynones, alkynoic ester and alkynoic amide (113). The expected heterosubstituted vinyl product (114) was formed in each case, mainly as the ii-isomer. In an accompanying paper, this type of addition was applied to the derivatives of fluorescein, 7-hydroxycoumarin, Sudan 1, and dansyl chloride with linker arms containing a conjugated terminal alkyne. 

These techniques have been used successfully in the micro-Zdman degradation of the enzyme mouse sarcoma dihydrofolate reductase to obtain the amino acid sequence of the first 25 amino acids 455). Similarly, RPC has been used in coqjunction with the automated Edman technique for sequencing 32 residues of myoglobin 456). Methionine and its oxidation products, methionine sulfoxide and methionine sulfone, in methionine fortified foods have been analyzed as their dansyl derivatives 457). Lysine has been determined as its dansyl derivative in a study in which the stability of lysine in fortified wheat flour was evaluated (458). 

Another useful reaction of amino side chains is that with dansyl chloride (Eq. 3-29). Many lysine derivatives can be determined quantitatively by amino acid analysis.280... 

Comparison with in vivo procedures Although the FDNB procedure proved to be a suitable reference method, there is no doubt that all methods should be ultimately compared to in vivo procedures. For this reason selected samples were also analyzed by plasma amino acid and digestibility methods. Preliminary results ( Table II ) show that plasma lysine results correlated very well with results for lysine digestibility and FDNB lysine ( r =0.95 ), reasonably well with those for dansyl chloride lysine, succinic anhydride reactive lysine and dye binding lysine, but poorly with total lysine. Although the absolute values were in many cases very different, it is apparent that all methods except total lysine can be used to at least indicate the extent of lysine damage. 

Fig. 4.12 Dansyl-decorated oligo-lysine dendrimer (2nd generation) as dendritic antenna with sensor properties towards metal ions (according to Balzani, Vogtle et al)...

As described previously for dansylleucine-modified CDs, the hydrophobic side chain of leucine affected the guest binding of the fluorescent CDs. To examine the presence of hydrophobic units near the dansyl moiety, monensin-incorpo-rated dansyl-L-lysine modified (3-CD (41) was prepared as an environment-re-... 

The compound l-fluoro-2,4-dinitrobenzene (FDNB) reacts with free amino, imidazole, and phenolic groups at neutral to alkaline pH to yield the corresponding, colored dinitrophenyl (DNP) compounds. Thus, FDNB will react with the free, unprotonated a-amino groups on amino acids, as well as with the side chains of lysine, histidine, and tyrosine (Fig. 6-1). Dansyl chloride is another compound that is known to react with the unprotonated, N-terminal amino groups of peptides. De-rivatization of peptides with this compound yields fluorescent products that provide a very sensitive method of detection of the amino acid derivatives (Fig. 6-2). 

With hydroxylamine, al -trans, ll-c/5-, and 13-cw-retinal gave a mixture of the syn- and anti-oximes, whereas 11,13-di-cw-retinal gave only the syn-oxime/ The synthesis of the dansyl-lysyl-lysine-N-retinylidene Schiff base has been described/ The products of a colour reaction of retinoic acid (121) in 74% H2SO4 have been identified as (122) and (123)/ The oxidation and isomerization of retinoic acid by I2 and light have been used to prepare the a -trans- and 13-CI5-isomers of 4-oxoretinoic acid (124) which were separated by h.p.l.c/ The photoisomerization of the retinoid (125) has been studied. The many isomers produced were separated by h.p.l.c. and characterized by H and n.m.r. ... 

Hill, R.D. and Laing, R.R., Specific reaction of dansyl chloride with one lysine residue in rennin, Biochim. Biophys. Acta 132, 188-190, 1967 Chen, R.F., Huorescent protein-dye conjugates. I. Heterogeneity of sites on serum albumin labeled by dansyl chloride. Arch. Biochem. Biophys. 128, 163-175, 1968 Chen, R.F., Dansyl-labeled protein modified with dansyl chloride activity effects and fluorescence properties. Anal Biochem. 25, 412M16, 1968 Brown, C.S. and Cunningham, L.W., Reaction of reactive sulfydryl groups of creatine kinase with dansyl chloride. Biochemistry 9, 3878-3885, 1970 Hsieh, W.T. and Matthews, K.S., Lactose repressor protein modified with dansyl chloride activity effects and fluorescence properties. 

A brilliant emitter. Certain naphthalene derivatives exhibit a weak yellow fluorescence when they are in a highly polar environment (such as water) and an intense blue fluorescence when they are in a markedly nonpolar environment (such as hexane). The binding of e-dansyl-lysine to specific antibody is accompanied by a marked increase in its fluorescence intensity and a shift in color from yellow to blue. What does this finding reveal about the hapten-antibody complex ... 

The synthesis of dansyl amino penicillanic acid (DNS-APA, I) was carried out according to the procedure outlined in Ref. (8). The e-dansyl monocyclic p-lactam of lysine (II) prepared essentially as described for another monocyclic p-lactam (9). 

Figure 7. Electropherogram of dansyl amino acids. A = -labelled lysine B = dilabelled lysine C = isoleucine D methionine ...

Experiments were conducted to determine the free amino groups available for reaction with dansyl chloride. Only one faint fluorescent area in the region of dansyl-lysine was observed. No dansyl-ethanolamine was observed. However, if the pol3nner was first treated with 6 N HCl to hydrolyze the amino acids, dansyl amino acids were obtained. Quantitative analysis for ethanolamine from the amino acid analyzer gave 1.3 ethanolamine residues for 2 proline residues. The low solubility of the polymer in aqueous solution may have been the primary factor leading to lack of derivatization of the intact glycopeptide. 

Dansyl chloride (DNS-Cl) (l-dimethylaminonaphthalene-5-sulphonyl-chloride). This reagent was originally introduced into protein chemistry for end-group analysis over twenty years ago (Gray and Hartley, 1963) and has been widely used because of the simplicity of the reaction and its ability to react with both primary and secondary amines, unlike OPA and fluram. Furthermore, in contrast to other fluorescent reagents, the dansyl derivatives are stable to acid hydrolysis, and can therefore be used in N-group labelling before hydrolysis. HPLC separations of dansyl derivatives have recently been published (Tapuhi et al., 1981). Sensitivity of detection is at the low picomole level. The sensitivity is limited because of the side-reactions which can occur with lysine and, to a lesser extent, histidine and tyrosine. 

Uenc, A. Ikeda, A. Ikeda. H. Ikeda, T. Toda, F. Fluorescent cyclodextrins responsive to molecules and metal ions. Fluorescence properties and inclusion phenomena of A7 -dansyl-L-lysine-) -cyclodextrin and mon-ensin-incorporated A7 -dansyl-L-lysine- 8-cyclodextrin. J. Org. Chem. 1999. 64 (2). 382 - 387. 

The Meg X-chain dimer appears to have at least three distinct binding sites. One is located on the rim of the funnel-shaped cleft, a second is at the constriction between funnel and cavity, and a third is at the bottom of the cavity. These sites bind a whole range of compounds including -dansyl lysine, colchicine, 1,10-phenanthroline, methadone, morphine, meperidine, 5-acetyluracil, caffeine, theophylline, menadione, triacetin, and other compounds (Schiffer et al, 1973). 

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