Tyrosine sulfate ester

With TFA at a lower temperature, the results clearly indicate that the hydrolysis of the tyrosine 0-sulfate ester is sufficiently low to be acceptable for preparative purposes. 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

Difficulties encountered in the postsynthetic chemical sulfation of peptides and the correspondingly low yields have led to the proposal of an alternative approach. This approach makes use of appropriate tyrosine 0-sulfate derivatives for the chain elongation steps in solution and on solid supports by applying protection strategies compatible with the acid sensitivity of the sulfate ester. Moreover, the analytical characterization of the peptides synthesized with tyrosine 0-sulfate derivatives is greatly facilitated since contaminations deriving from the preparation of the intermediates are easily detected by chromatographic (HPLC and CE) and spectroscopic methods (see Table 2). 

Factor IXa causes a rapid activation of factor X only if Ca2+, phospholipid,553 554 and the accessory factor Villa555 are present. The IXa Villa complex acts on X about 2 x 105 times faster than does IXa alone. This complex cleaves the same bonds in X as does the VIIa Va complex formed in the tissue factor pathway.514 The 2332-residue factor VIII and factor V have similar structures that include three repeats of a domain homologous to the blue copper-containing plasma protein ceruloplasmin (Chapter 16).556-559 Tyrosine 1680 of VIII apparently must be converted to a sulfate ester for full activity.560... 

While esters of sulfuric acid do not play as central a role in metabolism as do phosphate esters, they occur widely. Both oxygen esters (R-0-S03 , often referred to as O-sulfates) and derivatives of sulfamic acid (R-NH-SOg, N-suIfates) are found, the latter occurring in mucopolysaccharides such as heparin. Sulfate esters of mucopolysaccharides and of steroids are ubiquitous and sulfation is the most abundant known modification of tyrosine side chains. Choline sulfate and ascorbic acid 2-sulfate are also found in cells. Sulfate esters of phenols and many other organic sulfates are present in urine. 

Spectral alterations of the tyrosyl spectrum resulting from the formation of an ether linkage with the phenolic oxygen may be quite small, as with 0-methyltyrosine (Wetlaufer et al. 1958), or substantial, as with the aromatic ether thyronine (Gemmill, 1955). The ester linkage is also of some interest in this connection, since tyrosine-O-sulfate has been shown by Bettelheim (1954) to occur in bovine fibrinogen. The spectrum of tyrosine sulfate .. . differs markedly from that of tyrosine, showing a much weaker absorption with a maximum near 2630 A. ... 

The opioid peptides stem from a large precursor molecule in which several copies of the enkephalins are present, the ratio between Met-enkephalin and Leu-enkephalin being 6 1 [39]. The same precursor, pro-opiocortin, also contains a modified form of the enkephalin sequence in which the N-terminal tyrosine is present as the sulfate ester [40]. This kind of post-translational change has been discussed in connection with gastrin, cholecystokinin and caerulein (p. 165). The last mentioned peptide prompts us to recall dermorphin (p. 186), an opioid peptide found in the skin of an amphibian. 

Less reveahng are the ir spectra of larger peptide molecules. Unless sophisticated instruments, such as high resolution Fourier-transform spectrophotometers are applied, the large amide absorption overshadows other carbonyl frequencies. In some special cases, however, ir spectra can be uniquely informative. For example esterification of the phenolic hydroxyl in tyrosine with sulfuric acid is best revealed through the characteristic ir spectrum of sulfate esters, clearly distinguishable from the spectrum of the sulfonic acid derivatives which are the potential by-products of the sulfation process. 

Peptides and amino acids may be sulfated the hydroxyl groups of serine and threonine reacted with chlorosulfonic acid in trifluoroacetic acid to give the O-sulfate esters. The free amino groups did not react since they were completely protonated in the strongly acidic media and the rates of sulfonation were determined. Peptides containing the above amino acids were also sulfated. The aromatic amino acids tyrosine and tryptophan, by treatment with chlorosulfonic acid-trifluoroacetic acid, afforded the arylsulfonic acid derivatives. In the solid phase synthesis of cholecystokinin-33, tyrosine has been sulfated to the O-sulfate with chlorosulfonic acid and this was incorporated into the peptide sequence. ... 

Tyrosine (9-sulfate is stable under alkaline conditions, thus allowing for its quantification by amino acid analysis upon alkaline hydrolysis [0.2 M Ba(OH)2, 110 °C, 24 h] of sulfated tyrosine peptides and proteins.[6 331 Conversely, more than 95% of the ester is hydrolyzed after five minutes in 1M hydrogen chloride at 100 °C. Despite this pronounced acid lability, sulfated tyrosine peptides are sufficiently stable to short exposures of TFA134 35 or aqueous TFA[36 as required in peptide synthesis for removal of add-labile protecting groups. 

Conversely, exposure to hydrogen fluoride at 0 °C over short periods of time leads to almost quantitative hydrolysis.[37-39] In aprotic organic solvents, decomposition takes place unless the sulfate hemi-ester is neutralized with strong counterions.[37,381 The results of a detailed study are summarized in Figures 1 and 2t4°l which show the effect of various acids used in peptide chemistry in deprotection and in cleavage from the resin as well as the effect of temperature on the hydrolysis of tyrosine 0-sulfate as a sodium salt. 

A further approach using UV-visible spectrometry involves colorigenic substrates. These molecules change color when hydrolyzed in the enzymatic reaction under study. A good example is provided by -nitrophenol esters, which are colorless, but are hydrolzyed by appropriate enzymes to yellow /r-nitrophenol, which can be determined at ca. 405 nm. Many substrates of this type are readily available, e.g., jp-nitrophenyl phosphate as a phosphata.se substrate, the corresponding sulfate as a substrate for aryl sulfatases, Af-carboben-zoxy-L-tyrosine-/7-nitrophenyl ester as a substrate for proteolytic enzymes such as chymotrypsin, etc. 

The occurrence of organic esters of sulfuric acid in biological material suggested that these substances are formed under physiological conditions. The excretion of arylsulfuric acids by animals fed with phenolic substances has been known since Baumann (4S), and recently Tallan et al. isolated tyrosine-O-sulfate from the urine of normal animals (43). Important information on the enzymic mechanism of sulfuric add ester formation was obtained by De Meio et al. (44) and by Bernstein and McGilvery (43). The mechanism was clarified by Lipmann and his associates (46, 47) and by Wilson and Bandurski (48). The enz3unic reactions leadii to arylsulfuric acid S3mthesis are as follows ... 

For the covalent approach, standard protocols include bioconjugation techniques that have been routinely used in protein derivatization (Ma et al., 2012). The most widely used reactions include the reactive side chains of lysine (Lys), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), and tyrosine (lyr) residues, which are available to form biocompatible covalent bonds (Fig. 15.5A). Lysine residues and N-termini provide reactive moieties in primary amines form (R-NH ) that have been mainly targeted with A-hydroxysuccinimidyl-esters (NHS-esters) or NHS-ester sulfate derivatives (Smith et al., 2013). 

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