FREE-RADICAL THIOL-ENE AND THIOL-YNE COUPLINGS AS CLICK PROCESSES FOR GLYCOCONJUGATION - Click Chemistry in Glycoscience: New Developments and Strategies - page 69

On closing this section, it may be concluded that two methodologies, one based
on TEC and the other on TYC, were established for the modification of BSA 72
at three different sites. Thus, multiple glycosylation and fluorolabeling of 72 have
been carried out. Noteworthy, both methodologies employ the unmodified protein
in its native form. Therefore, it can be deduced that a route has been paved toward
the dual conjugation of proteins bearing one or more cysteine residues. Although
free cysteine is relatively rare in protein sequences, in principle, modern molecular
biology methods allow the introduction of this amino acid anywhere in a protein
[42]. Alternatively, sulfhydryl groups can be introduced in natural proteins by a
totally chemical method. This consists of the nucleophilic ring opening of the readily
available N -acetylhomocysteine thiolactone by the

ε -NH 2 groups of abundant lysine

residues [43].

3.7 CONCLUSION

The above results taken from research carried out in our laboratory illustrate the
efficiency of TEC and TYC in the manipulation of complex and delicate substrates
such as carbohydrates, peptides, and proteins. The click status of TEC has been
amply validated by the successful applications in a number of different fields. The
reaction represents a metal-free click process that allows the assembly of complex
substrates to be carried in an efficient and simple manner. In most of the cases
examined the products were formed with nearly quantitative yields, while their iso-
lation required chromatographic techniques. Unfortunately, this fails to meet one
of the “click” criteria. Significantly, also the less exploited TYC is emerging as a
valuable metal-free click ligation tool. For some aspects TYC is complementary to
TEC due to its potential for the introduction of two different fragments on the same
substrate. Moreover, given the numerous methods for terminal alkyne synthesis that
have been made available for the execution of CuAAC, TYC can be considered a
complementary click method for which a wide range of applications can be foreseen
(Scheme 3.26).

R 2

N

R 2 -N 3

N
N

Cu I

R 1

H

R 1

SR 3

R 3 -SH

SR 3

R 1

h ν

SCHEME 3.26 Click ligation tools with alkynes: CuAAC and TYC.