Chemistry Reference
In-Depth Information
and proteins [1, 3a]. Imperfections in this process (i.e., aberrant forms of glycosy-
lation) are associated with a variety of diseases, including rheumatoid arthritis, an
assortment of congenital disorders, certain muscular dystrophies, and cancer [5]. Gly-
can changes can mark malignancy, tumor cell invasiveness and metastasis potential
[5d, 6]. Glycopeptides are therefore attractive targets for the development of therapeu-
tics, but unfortunately progress has been limited by the complexity and heterogeneity
of the oligosaccharides [7].
Almost all glycoproteins exhibit microheterogeneity, that is, a polymorphism asso-
ciated with their glycan [8]. These various glycoforms—where the peptide backbone
is identical but the site and/or nature of the glycosylation can differ—can have a range
of different physical, biochemical, and biological properties [9]. While initially they
were thought to be of a random nature resulting from a lack of fidelity during their
synthesis, today it is apparent that the populations of glycoforms are highly regulated,
with the glycans being used by the organism as a means to fine-tune the biological
activity of the protein [8, 9]. Unfortunately, these variable patterns of glycosylation
present a range of problems to the researcher as the exact structure-activity relation-
ship is difficult to determine, as well as causing regulatory difficulties [9b].
Clearly, a need exists for readily obtainable sources of fully characterized, homo-
geneous glycoforms. Regrettably, oligosaccharides—unlike other biopolymers—are
produced by a template-independent pathway, and so direct genetic methods for
manipulating glycosylation patterns and generating a single, homogenous glycoform
do not exist [7]. Recombinant expression techniques may not be suitable, as glycosy-
lation is organism specific; artificial cultivation conditions may also lead to aberrant
glycosylation patterns [1]. Furthermore, the heterogeneity of the glycoprotein mix-
ture makes isolation of pure glycoforms from natural sources very difficult, requiring
abundant source material and many rounds of repetitive chromatography [9b].
One solution to this problem is chemical synthesis which can provide access to
homogenous glycopeptides, enabling a quantitative structure activity assessment [1].
Such homogeneous glycopeptides can be used along with synthetic peptide segments
in the chemical construction of glycoprotein molecules of defined structure. De
novo chemical synthesis of proteins offers many advantages: it provides exquisite
atom-level control, also allowing incorporation of unnatural amino acids, labels and
markers which may help in unravelling the mechanism of function [10].
While many methods and techniques exist for the rapid and efficient chemical
synthesis of peptides (such as Nobel-Prize winning solid-phase peptide synthesis
(SPPS) [11]) and proteins (native chemical ligation (NCL) [12]), the limiting factor
for the synthesis of glycopeptides is often the construction and attachment of the
oligosaccharide. Two main synthetic strategies are usually pursued (Scheme 10.1)
[2, 13]. One is to construct the putative glycan-protein link early, in the form of a
building block which is then incorporated into SPPS (linear synthesis). Alternatively,
the glycan-protein link can be formed late in the synthesis, when the peptide scaffold
is complete (convergent synthesis).
Unfortunately, both routes are not without their drawbacks. Construction of the
glycosylated amino acid building block is often complex and time consuming, and
requires the protection of the carbohydrate hydroxyl groups. Subsequent removal of
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