Chemistry Reference
In-Depth Information
On-bead activity assays may be subject to artifacts presumably caused by the very
high concentration of compound on the solid support [16]. Activities visible on the
bead thus often disappear once the compound is cleaved from the support and tested in
solution. To allow direct screening of our OBOC libraries in solution, we have
established a so-called “off-bead” screening protocol in which the peptide dendrimers
are cleaved from the solid support by photolysis of a photolabile linker. The cleaved
dendrimer then diffuses in the underlying silica gel plate that is impregnated with the
assay reagent, for example, a fluorogenic substrate [17]. In this manner, beads
carrying active peptide dendrimers are identified by the halo formed on the matrix
surrounding the beads, as shown for the identification of esterase peptide dendrimers
(Figure 15.2b).
The results of OBOC library screening are analyzed by determining the structure of
the compounds present on either the “positive” hits, the “negative” hits, or a random
selection of beads. From such analysis, a structure-activity relationship may emerge
leading to the identification of active compounds. Importantly, results from library
screening must be confirmed by resynthesis, purification, and testing of pure sub-
stances. For example,
the esterase dendrimer
RG3
(AcTyrThr)
8
(DapTrpGly)
4
(DapArgSer)
2
DapHisSerNH
2
(AcIlePro)
8
(DapIleThr)
4
(DapHisAla)
2
DapHisLeuNH
2
were initially identified by testing a focused “core-
active site” combinatorial library featuring catalytic residues at the core positions only
using an on-bead activity assay with the fluorogenic 8-butyryloxypyrene-1,3,6-
trisulfonate
and its
analog
HG3
[18]. A subsequent investigation of the same combinatorial library
using an off-bead activity assay confirmed the esterase activity of
1
with
acyloxypyrene trisulfonates.
17
The off-bead assay also allowed to analyze the negative
hits, which revealed that activity of the catalytic histidine residues at the dendrimer
core is controlled by the nature of the residues in the dendrimer outer branches. Thus,
histidine-containing peptide dendrimers were also found in the nonhits, but these
contained anionic residues in the outer dendrimer branches. Resynthesis confirmed
the absence of activity in these histidine-containing anionic dendrimers, for example,
D16
RG3
and
HG3
(AcGluThr)
8
(DapGluPhe)
4
(DapHisLeu)
2
DapHisAspNH
2
, which simply lack
any binding to the trianionic substrate, while partly anionic dendrimers such as
D14
(AcIleThr)
8
(DapIlePro)
4
(DapHisAsp)
2
DapArgLeuNH
2
are active but have weak
substrate binding (high K
M
).
15.2.3 TAGSFREE Decoding of Combinatorial Libraries
One of the bottlenecks in testingOBOC libraries is the identification of the structure
of the compounds on the beads. This can be realized by either reading an encoding
tag that has been separately introduced on the bead or analyzing the bead directly by
mass spectrometry or, in the case of linear peptidewith a freeN-terminus, byEdman
microsequencing [19]. These methods are complex, expensive, and mostly not
applicable to peptide dendrimers. To circumvent these limitations, we have
developed a library design algorithm for OBOC libraries of peptides and peptide
dendrimers called TAGSFREE, which allows direct sequence determination from
the total amino acid analysis (AAA) of the bead [20]. AAA is a one-step
