Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 24

3.2.4 Reactivity Toward Amino Groups

Well before biological relevance of Hcy-thiolactone has been established, it was
recognized that “the S-C bond of homocysteine thiolactone is that of a thioester, and
its reactivity, particularly in the peptide bond formation, deserves investigation”
[201]. Early studies have shown that at alkaline pH values, Hcy-thiolactone in
concentrated solutions (0.1-1 M) reacts with itself to form N-homocysteinyl-Hcy-
thiolactone, which then converts to diketopiperazine of homocysteine, which in turn
oxidizes to insoluble polymeric adducts [202]. (These reactions do not occur in
diluted Hcy-thiolactone solutions.) To prevent these reactions, Hcy-thiolactone was
acetylated with acetic acid anhydride, and the resulting N-acetyl-Hcy-thiolactone
was used in studies of its reactivity toward amines. In 1956 it was found that N-acetyl-
Hcy-thiolactone can react with amines and amino acids in alkaline solutions
(pH

9) with the formation of a peptide bond [203]. In 1960s, a few studies
examined the reactivity of N-acetyl-Hcy-thiolactone with a goal of introducing
sulfhydryl groups into proteins [204]. However, because N-acetyl-Hcy-thiolactone
is much less reactive than Hcy-thiolactone, these studies required long reaction times,
relatively high pH [205], or the presence of silver ions as a catalyst [205, 206].
Nevertheless, N-acetyl-Hcy-thiolactone has been used to introduce a thiol function-
ality into small molecules and macromolecules, including aminoglycosides, amino
lipids, muramyl dipeptide, cell-surface carbohydrates, human serum albumin,
lactoglobulin, enzymes, and oligonucleotides for a variety of applications [207].
Another Hcy-thiolactone congener,

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-thiobutyrolactone, is used in the synthesis of
pH-responsive polymeric hydrogels for biomedical applications [208].
For example, a report published in 1991 describes the modification with
N-acetyl-Hcy-thiolactone as a facile method to introduce a thiol group at the
5 0 -end of an oligonucleotide, which, in contrast to other methods, does not require
the deprotection step [209]. An oligonucleotide is synthesized using the
phosphoramidite method, and an amino group is introduced using the amino
modifier II. The oligo is purified and reacted with 5 mg of N-acetyl-Hcy-thiolactone
in 0.5 mL pH 8 phosphate buffer for 3 h, after which the reaction is

γ

95 %
complete. The thiol-substituted oligonucleotide is then labeled with a thiol-specific
reagent, such as N-(4-dimethylaminoazobenzene-4 0 )-iodoacetamide or fluorescent
N-(3-prenyl)-maleimide. In this way, oligonucleotides containing a desired dye are
prepared. The presence of the dye attached to an oligonucleotide is confirmed
by UV/vis and fluorescence spectral analyses [209].
Strong incentives to examine the reactions of Hcy-thiolactone with proteins
under physiological conditions appeared in 1990s after Hcy-thiolactone has been
discovered in living cells and the mechanism of its biological formation elucidated
[63, 138, 197, 210] and when elevated Hcy has been recognized as a risk factor for
cardiovascular and neurodegenerative diseases [211]. Subsequent studies demon-
strate that the thioester bond of Hcy-thiolactone is highly susceptible to reactions
with nucleophiles under physiological conditions, particularly with the side chain
amino group of protein lysine residues (Table 3.3 ). For example, at pH 7.4 and

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