Biomedical Engineering Reference
In-Depth Information
(Scheme 8.4) [35]. The strategy hinges on the differentiation between the outer
layer and the inner layer of TentaGel resin beads as reported by Lam and co-workers
(e.g.,
22
) [36]. First, resin beads were functionalized with a
-Ala-
-Ala-Arg-Met
handle using standard Fmoc peptide synthesis. The two
-Ala residues serve as
a spacer, the Arg residue functions as a constant ionization handle for subsequent
MALDI-MS analysis, and the Met residue is critical to effect CNBr cleavage in order
to analyze bead contents during synthesis and after screening. Subsequently, resin
beads were soaked in water and reacted with Fmoc-Glu(OSu)-OAll. This simple step,
which exploits the resin's physicochemical properties, partially shrinks the bead and
leaves only the outer-layer amino groups available for reaction in water with the
succinimidyl side chain of Fmoc-Glu(OSu)-OAll [36].
Subsequent Fmoc peptide chemistry in organic solvents allowed the introduction
of Fmoc-Glu(O
t
-Bu)-OH on the inner layer only. The outer layer was subsequently
functionalized into the macrocycle, and the inner layer (
23
to
26
) served to introduce a
linear tag sequence identical to that of the macrocycle for MALDI-MS sequence deter-
mination after CNBr cleavage and partial Edman degradation [37]. Subsequently, the
authors elaborated the library using a split-and-pool technique. After introduction
of additional residues to provide the diversity elements, the allyl ester was cleaved
and the octapeptide macrocyclized using PyBOP reagent on the outer-layer-anchored
sequence, whereas the inner-layer sequence, which does not contain a free carboxylic
acid, could not macrocyclize. Finally, all side-chain protections were cleaved with
TFA. This approach allowed the refinement of the one bead-one compound approach
to new ligands [37,38].
The screening of the above and similar libraries on several targets led to the
identification of ligands of the prolactin receptor (
K
d
=
2.0
M) [35], inhibitors of
the calcineurin/NFAT interaction (
K
d
=
0.7
M) [39], inhibitors of the Grb2/SH2
interaction (
K
d
=
45 to 900 nM) [40], as well as tyrocidine A analogs with antibacterial
properties [41]. The approach exemplifies how a judicious exploitation of the resin's
physical properties allows the development of a robust tagging methodology, which
facilitates subsequent deconvolution steps.
8.3.2 Synthesis of Small-to-Medium-Sized Macrocycles
Using Amphoteric Reagents
Yudin and co-workers reported an innovative three-component coupling reaction for
the synthesis of medium-sized peptidic macrocycles based on the amphoteric aziri-
dine carboxaldehyde reagent
11
(Scheme 8.2) [22,23]. This class of macrocycles is
very difficult to synthesize, owing to ring strain, and as a result, exploitation of its
chemical space has been limited by synthetic accessibility. Macrocyclization in this
series was performed at room temperature and at 0.2 M concentration, as opposed
to the usual millimolar or lower range. This remarkably high concentration was
attributed to the conservation of an ionic pair throughout every intermediate step of
the three-component coupling, which favored the folded precursor (
9
, Scheme 8.1)
and contributed to overcoming the entropic penalty associated with ring closure. This
