Compounds dehydroalanine

The Michael addidon of nitto compounds is a useful method for the preparadon of various naniral products The Michael kldidon of nittoalkanes to dehydroalanines gives ynitto-ct-amino acids, which provides a convenient synthesis of side-chain modified ct-amino acids fEq 4 114 " Transformadons of Y-nitto-ct-amino acid derivadves into ct-amino adds occur by... 

TV-Benzylidene dehydroalanine, however, could not be isolated by this method because the compound decomposed during work up at room temperature.[ 181 CDI was inert toward a series of dipeptides containing a serine residue in the TV-terminal position... 

FIGURE 3.10 Deprotection of functional groups by beta-elimination.17 (A) Removal of a labile proton beta to a good leaving group leads to release of the protector as the didehydro compound. (B) Recently developed protectors (Samukov et al., 1988) also designated untra-ditionally as 4-nitrophenyl- (C) Transformation of an O-protected serine residue into a dehydroalanine residue by hefa-elimination. 

Addition to the multiple bonds in ethyl A-acetyI-a,)8-dehydroalaninate by nitrones—for example, by phenylnitrone—afforded compound 29 (83TL2193). 

Due to the widespread use of structurally diverse amino acid derivatives in practically all areas of the physical and life sciences, the synthesis and applications of these compounds are of fundamental importance. Heterocyclic f)-substituted-a-alanincs are non-proteinogenic amino acids that are widely found in nature [138-147]. Naturally occurring (l-amino acids are also compounds with interesting pharmacological aspects. N,N-Bis(tert-butyloxycarbonyl)-dehydroalanine methyl ester (186) was reacted at room... 

Addition Reaction. The double bond of dehydroalanine and e-methyl dehydroalanine formed by the e-elimination reaction (Equation 6) is very reactive with nucleophiles in the solution. These may be added nucleophiles such as sulfite (44). sulfide (42), cysteine and other sulfhydryl compounds (20,47), amines such as a-N-acetyl lysine (47 ) or ammonia (48). Or the nucleophiles may be contributed by the side chains of amino acid residues, such as lysine, cysteine, histidine or tryptophan, in the protein undergoing reaction in alkaline solution. Some of these reactions are shown in Figure 1. Friedman (38) has postulated a number of additional compounds, including stereo-isomers for those shown in Figure 1, as well as those compounds formed from the reaction of B-methyldehydroalanine (from 6 elimination of threonine). He has also suggested a systematic nomenclature for these new amino acid derivatives (38). As pointed out by Friedman the stereochemistry can be complicated because of the number of asymmetric carbon atoms (two to three depending on derivative) possible. 

Addition to the double bond of dehydroalanine (or B-methyl-dehydroalanine) involves nucleophilic attack by compounds containing S, 0 or NH as shown by Figure 1. The overall reaction may be written as shown in Equation 13... 

The rate of nucleophile addition to the double bond is dependent on the nature of the nucleophile as would be expected. Finley et al. (47) have measured the relative effectiveness of the sulfhydryl group of L-cysteine and the e-amino group of a-N-acetyl-L-lysine in adding to the double bond of N-acetyl dehydroalanine. At equal concentrations of the reactive species, cysteine adds some 31 times more rapidly to the double bond than does a-N-acetyl-L-lysine (47). However, when one compares these two compounds at the same pH the relative rates in favor of cysteine (pK of sulfhydryl group = 8.15) versus a-N-acetyl-L-lysine (pK of e-amino group = 10.53) are most impressive at lower pH s (Table VI). Therefore, it has been recommended that cys-... 

The reactions of proteins in alkaline solution are very important from a number of standpoints. We have already discussed several uses of alkali treatment in food processing in the introduction. When contact between the food and alkali is kept to a minimum at the lowest temperature possible with adequate control of mixing, etc. there is presently no apparent reason to discontinue its use. Low levels of lysinoalanine occur in food which has been processed in the absence of added alkali, even at pH 6 and in the dry state (20). For example, the egg white of an egg boiled three minutes contained 140 ppm of lysinoalanine while dried egg white powder contained from 160 to 1820 ppm of lysinoalanine depending on the manufacturer (20). No lysinoalanine was found in fresh egg white, 3 Elimination and addition of lysine to the double bond of dehydroalanine reduce the level of the essential amino acid lysine. This can be prevented by adding other nucleophiles such as cysteine to the reaction. Whether lysinoalanine (and other compounds formed by addition reactions) is toxic at low levels in humans is not known. 

Many serine proteinase inhibitors are active due to their ability to form a covalent link to Ser-195 of the enzyme. However, several proteinaceous compounds which are very potent serine proteinase inhibitors (K < 10" M) bind equa. y well to the parent enzyme and to the corresponding anhydroenzyme, in which the active-site serine has been replaced with dehydroalanine, proving that, with enough contact points, a covalent link to Ser-195 is not required for good activity [see section on proteinaceous inhibitors]. 

Dehydroalanine 116 desmosine 48, 49 diazo compounds 157 aryl diazonium salts, reactive properties 157 synthesis 160 diazoacetates, analysis of products 165 reactive properties 162 synthesis 164 diazoketones, analysis of products 162 conversion to haloketones 139 reactive properties 165 synthesis 140 diazomethane preparation 141 reactive properties 162 diazonium salts 89 diazonium-IH-tetrazole 90, 95 3,4-dihydroxyproline 52, 53 diimidoesters 69 diisopropylfluorophosphate 130 2,3-dimethylmaleic anhydride 83 dinitrophenylation 79 disulfide bond reduction 103... 

At a given temperature the overall rate of reaction depends on the rate of 13-elimination and on the conformation of the protein. This is because the accessibility of the dehydroalanine residue for the nucleophilic attack depends on the spatial arrangement of the reacting groups. The reaction can be inhibited by acylating the nucleophilic groups in proteins or by adding thiol compounds, which compete with a.a. residues for the dehydroalanine double bond ... 

We have shown that treatment of Se-(4-methoxybenzyl)-selenocysteine or related peptides with DBU in dimethylformamide produces in quantitative manner the related dehydroalanine-compounds as shown in Fig. 8 (101). As this method can be applied to the Fmoc/tBu strategy of solid phase peptide synthesis (102), if Fmoc removal is carried out with due precautions, it should represent a very efficient synthetic approach to microcystins. 

The kinetics of the Michael addition reaction with acetonitrile on reduced protein studied by Cavins and Friedman (19) served as an excellent model for later studies with N-acetyldehydroalanine methyl ester (20) where the formation of a variety of dehydroalanine adducts of amino acids were reported. It is necessary to use the dehydroalanine methyl ester in these studies because the reaction kinetics are much faster than with the free N - acetyl dehydroalanine (21). The free amino compounds decomposes to ammonia and pyruvic acid when synthesis is attempted. 

Figure 2 summarizes some of the compounds that can be formed through the addition of dehydroalanine to a variety of amino acids in proteins. Although there are no reports in the literature of such reactions, one is tempted to raise the question as to whether dehydroalanine reacts with nucleic acids containing reactive amino groups. More complete discussion of the dehydroalanine addition reactions have been made earlier by Friedman (25), Asquith et al. (26) and Feairheller et al. (27). 

The reaction of lysine with dehydroalanine was studied by Snow et al. (21) using the N-a-acetyl-dehydroalanine methyl ester as a model. The studies with the model compound are difficult to compare with reactions that occur in proteins because proteins are such complex molecules and analysis is difficult. The comparison of the variety of proteins which form LAL suggests that although dehydroalanine may be formed by a variety of pathways, the Michael addition between lysine and dehydroalanine is rapid, particularly at higher pHs. 

Studies in several laboratories have shown that strongly hepatotoxic cyclic heptapeptides of a common type occur in different strains of Microcystis [59]. Microcystis aeruginosa, the species most frequently investigated contains the peptide formulated in Fig. 25. The two letter suffix LA designates the two L-amino acids in positions 2 and 4 (X, Y = Leu, Ala) which have been found variable in all of the toxins examined to date. X can also be arginine (R) or methionine (M). The 10-carbon chain jS-amino acid is unique for this type of hepatotoxic compound. Another not quite common structural element is the unsaturated side chain of dehydroalanine (No. 7), the formation of which can readily be imagined by elimination of H2O from serine or H2S from cysteine. 

It is obvious that AOA can be considered the aminooxy analogue of glycine, while AOPP is the aminooxy analogue of phenylalanine. Both of these compounds can be expected to interfer with amino acid metabolizing enzymes carrying a carbonyl group(e.g. that of pyridoxal phosphate in the case of transaminases or dehydroalanine in... 

Figure 12. Transformation of dehydroalanyl to lysinoalanine (LAL), S-B-(2-pyridylethyl)-L-cysteine (2-PEC), and lanthionine (LAN) residues in a protein. Hydroxide ions induce elimination reactions in cysteine and serine to form dehydroalanine. The double bond of dehydroalanine then interacts with the e-NH2 group of lysine to form LAL, with the SH group of cysteine to form LAN, and with the SH group of added 2-mercaptoethylpyridine to form 2-PEC. The latter is identical to the compound obtained from cysteine and 2-vinylpyridine.

Thus, depending on the pH of the reaction, sulfhydryl groups of cysteine react about 34 to 5000 times faster than the e-amino groups of lysine with vinyl compounds such as N-acetyl dehydroalanine methyl ester (Friedman and Wall, 1964 Friedman et al., 1965 Cavins and Friedman, 1967 Snow et al., 1976). Therefore, by adding thiols such as cysteine it may be possible to prevent the formation of dehydroalanine residues during alkali-treatment of proteins. 

The formation of these compounds is based on the following reactions 1,2-elimination in the case of hydroxy amino acids and thio amino acids results in 2-amino-acrylic acid (dehydroalanine) or 2-aminocrotonic acid (dehydro-aminobutyric acid) ... 

The side chains of proteins can undergo post-translational modification in the course of thermal processes. The reaction can also result in the formation of protein cross-links. A known reaction which mainly proceeds in the absence of carbohydrates, for example, is the formation of dehydroalanine from serine, cysteine or serine phosphate by the elimination of H2O, H2S or phosphate. The dehydroalanine can then lead to protein cross-links with the nucleophilic side chains of lysine or cysteine (cf. 1.4.4.11). In the presence of carbohydrates or their degradation products, especially the side chains of lysine and arginine are subject to modification, which is accompanied by a reduction in the nutritional value of the proteins. The structures of important lysine modifications are summarized in Formula 4.95. The best known compounds are the Amadori product -fructoselysine and furosine, which can be formed from the former compound via the intermediate 4-deoxyosone (Formula 4.96). To detect of the extent of heat treatment, e. g., in the case of heat treated milk products, furosine is released by acid hydrolysis of the proteins and quantitatively determined by amino acid analysis. In this process, all the intermediates which lead to furosine are degraded and an unknown portion of already existing furosine is destroyed. Therefore, the hydrolysis must occur under standardized conditions or preferably by using enzymes. Examples showing the concentrations of furosine in food are presented in Table 4.13. 

Fig. 2 Advanced ligation strategies, (a) NCL and subsequent desulfurization of the unprotected cysteine/sele-nocysteine. DHA (dehydroalanine) is thiol reactive and can be further modified with SH-bearing compounds, (b) Examples for ligation-desulfurization strategies leaving amino acids different from cysteine at the junction site. MFD metal-free desulfurization, Pen penicillamine, Afpf mercaptD-pyrrolidine-2-carboxylic acid...

Substantial progress has been made with the concept to convert the required selenocysteine/cysteine residue upon ligation into an alanine [7-9] or dehydroalanine [10] by a process called desulfurization. In turn, the generated dehydroalanine moiety can then further react with other thiol-fimctionalized compounds... 

Benzoylserine compounds have also found only occasional application in P-elimination reactions in the alkaline methanolysis of N-benzyloxycarbonyl-S-benzoylcysteinyl-O-benzoylserine ester, Zervas and Ferderigos observed the formation of cyclolanthionine derivatives, formed by inter- or intramolecular Michael addition of the SH-function to the dehydroalanine unit formed by simultaneous elimination of benzoic acid (448, 449). 

Patchornik and coworkers studied the elimination reactions of S-2,4-dinitrophenyl-cysteine compounds 390, 391). The method proved suitable for the conversion of cysteine peptides into dehydroalanine peptides, in which the double bond can then be exploited as the site of attack in the specific (hydrolytic or oxidative) cleavage of the peptide 298—300). 

Thermolysis of 3-alkylsulflnyl-a-amino acid derivatives is an alternative reaction which has been applied to the synthesis of dehydroamino acid compounds 325, 326). Esters and peptides with dehydroalanine, dehydroaminobutyric acid, dehydrovaline and dehydrophenylalanine units have in many cases been obtained in excellent yields. The method is especially suitable for peptides with base sensitive groups. The elimination appears to take place with particular facility in compounds in which the amino acid unit bearing the sulfoxide group is present as a tertiary amide 326). Dehydrophenylalanine and dehydroaminobutyric acid derivatives are formed as mixtures of Z- and E-isomers on sulfoxide elimination. 

Thermal cleavage of a sulfone has been exploited in a synthesis of racemic versimide 18), while Gross 159) mentions a base catalyzed elimination from an S-methylcysteinesulfone derivative to yield the dehydroalanine compound. 

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