Biomedical Engineering Reference
In-Depth Information
priming or extension at one end and the use of an enzyme to perform a single
transformation on an almost complete synthetic molecule at the other.
There are a series of factors to take into consideration when deciding where to
introduce one's desired substrate analogue along this continuum. In the extreme of
precursor simplicity, unnatural primer or extender units, the utility of precursor-
directed biosynthesis is the most obvious. The unnatural molecules are often
extremely simple and frequently are commercially available, completely removing
the need for any synthetic effort. If synthesis is required, it is frequently only well-
established coupling chemistry for the activation of these molecules as thioesters of
coenzyme A or some surrogate thereof. While this type of application is the most
obvious case of precursor-directed biosynthesis, it does have several limitations.
Unnatural priming of biosynthetic systems allows only variation at a limited
number of positions on the natural product—those corresponding to the primer unit.
In the biosynthesis of many molecules, such as polyketides, the extender units are
identical in each chain elongation cycle, making the selective insertion of a single
unnatural extender molecule virtually impossible in the absence of some clever
engineering of the proteins responsible for the recruitment of these extenders. Both
these approaches aremade less useful by the fact that they rely on loading an unnatural
substrate onto what have frequently evolved to be the most discriminating enzymatic
components of the biosynthetic pathway.
Inmanycases, the limiting factor on the success of aprecursor-directedapproach
in a given system is the promiscuity of the enzymes responsible for accepting the
unnatural substrate. Short of experimentation, there is noway to rationally predict this
problem. However, it can often be circumvented by choosing a later entry point along
the biosynthetic pathway. The reasoning goes as follows: as the biosynthetic inter-
mediate increases in complexity, there is lower evolutionary pressure on the corre-
sponding enzymes to have high specificity because the likelihood of occurrence of an
endogenous molecule that resembles the natural intermediate is remote.
At the opposite end of the complexity continuum is the use of only a single
biosynthetic enzyme to perform a single transformation on an almost entirely
synthetic molecule. During the discussion of polyketide and nonribosomal peptide
biosynthesis, it was noted that these systems possess a terminal thioesterase. These
enzymes frequently catalyze an entropically disfavored reaction in the cyclization of
more than 14-membered chains. This makes them extremely useful in synthetic
efforts as getting these macrocyclizations to occur at a reasonable rate and in good
yield often proves difficult.
Apart from thioesterases, many of the enzymes responsible for polyketide and
nonribosomal peptide tailoring are used in this type of capacity. In the remainder of
this chapter, we will discuss examples using oxidases on a synthetic scaffold to
introduce late-stage oxidations in a stereospecific manner, wild-type glycosylation
of unnatural scaffolds, and unnatural glycosylation of wild-type scaffolds. Despite
the utility of these type of transformations, they do require the use of much more
complex precursors and as such fail to exploit one of the real strengths of precursor-
directed biosynthesis.
In summary, examples exist in which unnatural precursor molecules have
been introduced at all stages of assembly line biosynthesis of polyketides and
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