Carboxy groups specificity

Also deoxycholic acid (6) crystallizes in an inclusion lattice with channel-shaped cavities 13). Figure 3 shows that they are formed by facing molecules of deoxycholic acid, 4). This characteristic structural unit is a double layer of head-to-tail linked deoxycholic acid molecules at which specific H-bridges between hydroxy and carboxy groups are the decisive fact. The channels as such (e.g. in case of the orthorhombic crystal, see Fig. 3) are lined with lipophilic groups. Thus only van der Waals contacts are kept between the included guest molecules (also for polar molecules like acetone, Fig. 3) and the molecules of the channel wall. 

Mammalian esterases have been classified into three groups according to specificity for substates and inhibitors (110). In terms of overall hydrolytic activity in mammals, the most important class of esterases is that of the B-esterases, which are principally active with aliphatic esters and amides. A-Esterases are important for aromatic esters and organophosphorus esters, and C-esterases are active with acetyl esters. In general, the specificity of mammalian esterases is determined by the nature of substituent groups (acetyl, alkyl, or aryl) rather than the heteroatom (O, N, or S) that is adjacent to the carboxy group. That is, the same esterase would likely catalyze hydrolysis of an ester, amide, or thioester as long as the substituents were identical except for the heteroatom (110). 

In order to ligate at the C-terminus without the risk of unwanted conjugation through side-chain carboxy groups, it is usually best to direct reactivity to this site, and to this site only, using the specificity for the a-carboxy shown by many proteases. This is best done by using reverse proteolysis to substitute the a-carboxy with a chemically activated group. 

Shihunine.—Preliminary results139 indicated that the orchid alkaloid shihunine (153) was derived from (152), an important intermediate in naphthoquinone biosynthesis.140 Further details are now available in a full paper.141 The intact incorporation of (152) is affirmed by the observation that (152), labelled with 13C at C-l, was an efficient and specific precursor for (153). [l-14C]Acetate was examined as a shihunine precursor and was found only to label C-5. This is consistent with the expected formation of (152) from shikimic acid (144) and a-ketoglutarate (151),140 the latter gaining acetate label in its carboxy-groups through the tricarboxylic acid cycle. 

Transfer and termination reactions may involve a number of different compounds present in the polymerization system these are either added intentionally or present as impurities. The intentionally added compounds, usually chain transfer agents, are used simply either to decrease the molecular weight or to obtain polymers with controlled molecular weights and specific end groups (e.g. hydroxy and carboxy groups) which can then be utilized for further reactions. 

In addition to suitable protection of other coreactive functionalities, activation of the carboxy group prior to reaction with the amino group is required for a controlled formation of the amide bond between two a-amino acid or peptide components (Scheme 1). For this purpose activated species can be generated separately, isolated, and stored for subsequent use. Alternatively, the activation can be carried out in situ with specific coupling reagents. Independently of the mode of activation, an electron-withdrawing group X must be incorporated at the acyl carbon in a transient mode in the in situ activation or in the formation of reactive intermediates. 

Af -2,2-Bis(ethoxycarbonyl)vinyl-protected amino acids are prepared by reaction of commercially available diethyl 2-(ethoxymethylene)malonate (127) with the respective amino acid in methanolic KOH. This rapid reaction is complete within 5 minutes and leads to the potassium salts. Subsequent acidification with 1M HCl yields the amino acid derivative in 75-90% yield.f This intermediate enamine-type N-protection is of particular interest in chemistry to be performed on the carboxy groups of the amino acids such as esterification with alkyl bromides in the presence of a base. Since cleavage of the enamine entity is achieved by treatment with bromine in chloroform at room temperature, it cannot be used for amino acids sensitive to halogenation such as tyrosine, tryptophan, and methionine (Scheme 61). Based on the experience gained with the enamine-type protection the Al-2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) and N-2-(4,4-dimethyl-2,6-dioxocyclohex-ylidene)isovaleryl derivatives were developed as specific side-chain protecting groups (see Section 2.1.2.2.5.2). 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

Thermitase, a thermostable extracellular serine protease from the thermophrhc microorganism Thermoactinomyces vulgaris, with an esterase/protease activity ratio >1000 1 shows a broad amino acid side-chain tolerance and cleaves methyl, ethyl, benzyl, ethox-ybenzyl, and ferf-butyl esters from a variety of Nps-, Boc-, Bpoc-, and Z-protected di- and oligopeptides in high yields at pH 8 and 33-55 °C (Scheme 15 ).[28,29,60-62] jjj addition, it is specific for the a-carboxy groups of Asp and Glu. 

Enzyme-mediated carbonyl reductions are not restricted to aldehydes and ketones. With some organisms, carboxy groups can be reduced to primary alcohols. The example in Scheme 53 proceeds with enantiomeric specificity, ... 

Azetidine-2-carboxytic Acid.—DL-[l- C]methionine has been found to be a specific precursor for azetidine-2-carboxylic acid (158) in Nicotiana tabacum as it is in other species. The label was located on the carboxy-group, the expected site. 

The twenty L-amino acids (actually, nineteen a-amino acids and one a-imino acid (Table 1.1)) which, in preparation for their role in protein synthesis, are joined in vivo through their carboxy group to tRNA to form a-aminoacyl-tRNAs, are organised by ribosomal action into specific sequences in accordance with the genetic code (Chapter 8). 

Sometimes the coupling reaction of an activated carboxy group and a deprotected amino group is difficult to accomplish. These difficult couplings are usually sequence-dependent and not residue-specific. In these cases, a double coupling is required (i.e., repeat step 5-6 before step 7). 

Further evidence implicating betalamic acid (176) in betalain biosynthesis comes from a study of the biosynthesis of the acid itself in Portulaca grandifiora Both [2- CJ- and [l- C]-DL-dopa were incorporated into betalamic acid. Degradation of the betalamic acid (176) derived from [l- C]dopa showed that the incorporation was specific, almost all the activity being confined to the carboxy-groups (and presumably only C-19 is labelled). This pattern of dopa incorporation is the same as for the betalains, a requirement if (176) is to be an intermediate in betalain biosynthesis. 

Structures analogous to the tyrosine-derived portion of anthramycin are to be seen in the propyl- and ethyl-proline moieties of lincomycin (167) and (168), which are produced by another Streptomyces species. These substituted proline residues had been shownearlier to arise from tyrosine too C-1 of tyrosine was incorporated efficiently and specifically into the carboxy-groups of (169) and (170), and a study showed that the nitrogen atom of the proline ring came from... 

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