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Homocysteine-Thiolactone

3.1 3.1.1

Chemical Synthesis Demethylation of Methionine

Hcy-thiolactone was discovered by serendipity in 1934 as a by-product of an early assay for the quantification of methionine in proteins [187]. The assay involves boiling with hydriodic acid (128
C, 3 h). This causes demethylation of methionine with the formation of methyl iodide, whose quantitative recovery (99.5  2.0 %) by absorption in alcoholic solution of silver nitrate was the basis of the methionine assay. The residue from methionine that remains in the acid has been identified by elemental analysis as Hcy-thiolactone. The recovery of Hcy-thiolactone from methionine digestion is also quantitative (97.0  2.1 %) [187]. Recent studies have found that during hydriodic acid digestion, L-methionine is racemized to D,L-Hcy-thiolactone (Reaction 3.1 and Fig. 3.1). Although no longer used as a methionine assay, the hydriodic acid digestion procedure became a convenient method for the preparation of D,L-[35S]Hcy-thiolactone for biological studies [188]. Enzymatic digestion with bleomycin hydrolase (which exhibits essentially absolute stereoselectivity for L-Hcy-thiolactone) [85] resolves racemic D,L-[35S]Hcy-thiolactone to L-[35S]Hcy and D-[35S]Hcy-thiolactone (Fig. 3.1) [188]. In contrast to hydriodic acid, the digestion with hydrochloric acid is ineffective in demethylation of methionine [3]. For example, during incubation with 6 N HCl at 135 or 120
C, the rate of conversion of methionine to Hcy-thiolactone is 1,000- and 10,000-fold slower than that observed with hydriodic acid [79]. Concentrated sulfuric acid (18 N) also causes demethylation of methionine at 125–135
C. However, due to oxidative properties of sulfuric acid, homocystine (in 42.5 % yield) is obtained in this case [3].

H. Jakubowski, Homocysteine in Protein Structure/Function and Human Disease, DOI 10.1007/978-3-7091-1410-0_3, © Springer-Verlag Wien 2013

19

20

3 Homocysteine-Thiolactone

Reaction 3.1 The hydriodic acid-dependent conversion of methionine to Hcythiolactone (Reprinted from [68])

NH2

NH2.HI O o

HI, 120 C OH

O

-CH3I

S

S CH3 NH2

1.0 O

0.8 S

Abs

Fig. 3.1 Absorption spectra of L-Hcy-thiolactoneHCl (0.2 mM in water, 25
C). D,L-Hcy (0.2 mM), shown for comparison, does not appreciably absorb UV light above 220 nm (Reproduced from [68])

0.6 NH3+

0.4 0.2

COO-

HS

0.0 220

240

260

280

300

Wavelength (nm)

3.1.2

Acid-Dependent Cyclization of Homocysteine

In the presence of hydrochloric acid, L-Hcy undergoes intramolecular condensation to L-Hcy-thiolactone (Reaction 3.2) [4]. The rate of ring closure depends on the acid concentration and temperature. For example, in 0.1 N, 0.6 N, or 6 N hydrochloric acid at 100
C, 50 % of ring closure condensation occurs in 3 h, 15 min, or 220 nm

~30 h ~1 h 6.67a

~2 h ~2 h 9.04, 9.71b 9.02, 9.69 (thiol group)b  Condenses to form Hcy-thiolactone  Reacts with aldehydes to afford tetrahydrothiazinesa  Oxidized to disulfides  Reacts with nitric oxide to afford S-nitroso-Hcyd

 Acylates amino g