Biomedical Engineering Reference
In-Depth Information
produced by each strain.On the basis ofreference substances a number ofspots
on the chromatograms can be classified to be:
i) constituent of the nutrient broth,
ii) frequently formed and thus widely distributed microbial metabolite,
iii) a strain specific compound which merits further attention.
As a consequence, chemical screening results in the visualization of a nearly
complete picture of a secondary metabolite pattern which can be characterized
to be a metabolic finger-print of an individual strain [158,159].
Applied to a number of culture extracts from microbes (e.g.streptomycetes)
these screening concepts resulted in various structurally new secondary meta-
bolites.In a following step the obtained pure compounds have to be assayed in
different biological test systems.The availability of both,sufficient quantities of
pure metabolites,and a broad range of biological tests are the critical points for
success.As presumed [142],a certain percentage ofthese secondary metabolites
out of chemical screening progams showed striking biological effects and gave
reason for further more detailed biological studies towards new lead structures.
Chemical screening resulted in compounds out of nearly every structural class
known so far [158-192].Some selected examples are summerized in Fig. 8.
Recently,in our laboratories the sample preparation procedure (adsorption
of the metabolites present in the culture filtrate) has been further developed
towards automation and additional separation steps using modified Zymark
RapidTrace modules.On the basis of new adsorber resins high quality sample
preparation is now possible allowing a more detailed TLC-analysis of the meta-
bolites produced.This leads to an improvement in the separation ofoverlapping
spots and therefore to a higher reliability of the database assisted analysis.On
the other hand,sample application onto the TLC-plates is possible via commer-
cially available automated spotting stations.In consequence,chemical screening
was adopted to the 96-well format, thus efficiently supplementing biological
screening attempts.
Furthermore,the analytical power (fast,cheap,parallel,easy-to-handle,use of
UV/VIS and staining reagents) allows one to add physico-chemical information
on “hit-lists” out of target-directed screening approaches with samples from
natural sources.Therefore,integration of TLC-analysis in the course of secon-
dary biological screening and hit-verification is expected to be a remarkable
tool in lead-structure finding strategies, e.g. for the localization of the active
principle, for fast and efficient dereplication, and for speed-up isolation and
purification procedures. More recently, coupling techniques of mass spectro-
metry, or even TLC-FID coupling [193] have been described which obviously
can be integrated in chemical screening concepts.
In comparison to a TLC-based screening both the quality and sensivity of
HPLC separations of a physico-chemical screening via HPLC is better. On the
other hand,TLC allows a parallel,quick,and cheap handling of samples,and is
superior in the mode of detection (UV/VIS and staining). As well as eluted
compounds out of HPLC separations,spots from TLC can easily be subjected to
subsequent physico-chemical analysis (MS, IR, NMR etc.) via scrape off and
elution from the silica gel materials.
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