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11
Chapter • Combinatorics: Chemistry with Big Numbers
ids). After testing these 400 substances, the biologically
most active mixture is the starting point for the second
round of synthesis. Another 400 libraries are generated,
this time with the form XA(Aa1)(Aa2)BX. Aa1 and Aa2
are the amino acids from the most active mixture from
the rst testing. These are also subjected to the test. This
identies the “best” amino acids for positions2 and5.
The strategy is followed step by step until the most active
sequence is identied.
In asimpler procedure, the amino acids are varied
in one position at atime. By starting with 20libraries
AXXXXX the most active amino acid (Aa1) is determined in the rst position. The starting point for the
next synthetic cycle is the most active mixture (Aa1)
XXXXX. By varying the adjacent position, the second
amino acid (Aa2) is ascertained. This is repeated and
6 × 20 = 120 hexapeptide libraries are prepared in the
form of AXXXXX, (Aa1)AXXXX,… (Aa1)(Aa2)(Aa3)
(Aa4)(Aa5)A until the “best” amino acids in all positions
are determined.
Another method allows the targeted construction of
alibrary in afew working steps. The conceptual design
of the synthesis ensures that adened compound is produced on each polymeric support bead. This is achieved
by using the so-called “split-and-combine” technique
(. Fig.11.4). For example, it is possible to synthesize
all 8000 possible tripeptides from the 20proteinogenic
amino acids in only 60reaction steps. They are produced
as 20mixtures of 400 substances each. In the end, one
denite compound is located on each polymer bead. The
individual beads are available as abatch that is easily
separated mechanically and individually tested.
. Fig. 11.4 The construction of a compound library according to
the split-and-combine technique starts with acertain amount of resin beads. These are evenly distributed among nreaction vessels. Only
three are considered here for the sake of simplicity. In the rst ask reagentA (e.g., amino acidA) is coupled to the resin. ReagentsB andC
are analogously added to ask2 and3. In the next step, adipeptide is
constructed. To solve the problem of different reaction rates between
the different amino acidsA, B, andC, only one soluble reaction partner is added in excess to the mixture of solid-phase-bound starting
materials. After the rst reaction step, the resin, which is now loaded
with an amino acid, is combined and mixed. It is again distributed between three (or more) reaction asks. The next reaction is carried out.
In the case of apeptide synthesis, amino acidA is added to ask1,
B to ask2, andC to ask3. The resin is combined and mixed thoroughly. In the meantime, all nine possible dipeptides are on the beads.
After separating the beads again, the third step follows. In case the
peptide chain is to be extended by another amino acid, amino acidA
is added to ask1, B to ask2, andC to ask3. Now all 27imaginable sequential tripeptides are on the resin after three parallel reaction
steps. Aclearly identiable compound is found on each resin bead.
The library can be tested directly on the polymer or it can be tested in
solution after cleavage from the support
11.7 Which Compound in the Solid
Support Combinatorial Library
Is Biologically Active?
The libraries generated on the solid support are biologically tested. This can be done directly on the polymer-immobilized compounds. As with the testing of
bacteriophage libraries, there is arisk that the support
material will interfere with the test, for example, by steric
hindrance or unspecic interactions. Furthermore, it is
important that the protein to be tested is in asoluble
form. Membrane-bound receptors will, therefore, be
excluded from the assay. Alternatively, the compound
library can be cleaved from the resin. To do this, the resin
must be coupled to the library component by asuitable
“linker” that allows the library to be selectively released.
This linker can be cleaved at low pH or photochemically
with UV light. However, it must not interfere with the
synthetic assembly of the library and must not be cleaved
during the course of synthesis. Final cleavage from the
resin must not destroy the products. Testing the cleaved
products certainly correlates better with physiological
. • Combinatorial Libraries with Large Diversity: AChallenge for Synthetic Chemistry
conditions. Spreading the cleaved compounds over
alarge area or embedding them in agel provides spatial
separation so that compounds interacting with the test
protein occur in local high concentrations. In this way,
binding to insoluble proteins (e.g., membrane-bound
receptors) can be tested. However, the advantage of
mechanical manipulation of a polymer-bound library
is lost upon release.
If biological activity is found in the assay, the next
step is to determine which compound from the library is
responsible. If the library is well dened by the synthesis
protocol, then it is known which compounds have been
tested. The active compounds are narrowed down by
deconvolution and resynthesis of sublibraries. The one-
bead one-compound technique produces only one dened
compound on each resin bead. However, it is not known
which compound it is. The characterization of the compound is attempted only after the activity is identied.
There are many ways to do this: they can be tested on
the resin by separating the relevant resin beads and analyzing the compounds. If the library consists of peptides
or oligonucleotides, peptide sequencing is performed by
Edman degradation (works even at 0.1picomolar!), or
polymerase chain reaction (Sect.12.1) allows amplication and enrichment of oligonucleotides.
More sophisticated techniques are also used. During
synthesis, the library is allowed to “grow” on several different linkers. The individual library compounds can be
released from these linkers under different conditions
(e.g., different pH values or photochemically at different
wavelengths). First, the compound is cleaved from the
rst linker to perform the assay. Cleavage from the second linker is performed after mechanical separation of
the desired resin beads. This method effectively “labels”
the resin beads. The technique is, therefore, an elegant
variation on library testing in the detached state. The
different linker-bound compounds on the resin bead
need not be identical. Thus, atest library of peptides
can be linked to the resin bead by oligonucleotides used
as labels. Halogenated aromatics have also been proposed
as labels because they are easily identied by mass spectrometry, even in minute amounts. The labels can even
be encoded with abinary code based on their sequence
or the number of monomer building blocks.
In recent years, the new technology of DNA-encoded
chemical libraries (DECL) has emerged. It starts with an
oligonucleotide that is equipped with achemical linker
group. At this linker, molecules for biological testing are
assembled in successive steps by chemical synthesis. Each
individual synthesis step is documented by attaching
anucleotide to the original starting nucleotide. Comparable to abarcode, aDNA-encoded label is created. Many
DNA-compatible reactions have been developed in order
to link new synthesis building blocks to the growing test
compounds. The technique does not necessarily rely on
syntheses on aresin. It is also possible to create libraries
purely in solution. This greatly simplies the subsequent
testing of the library in abiochemical activity assay with
the target protein. Thus, the target protein can be immobilized on asolid support. The library then ows past
the target protein on acolumn. Detected hits captured
from the library are released from the protein again, e.g.,
by washing or with aknown displacement ligand, and
they are subsequently isolated. They are then analyzed
by decoding the genetic information of the DNA label.
PCR amplication (Sect.12.1) is used for this purpose,
followed by high-throughput DNA sequencing. In the
meantime, however, techniques have been developed that
do not require immobilization of the target proteins at
all. Even testing in living cells has been reported. Extensive libraries have meanwhile been created. New clinical
candidates have already been identied using this screening approach.
11.8 Combinatorial Libraries
with Large Diversity: AChallenge
for Synthetic Chemistry
Another aspect speaks for the last above-mentioned concept. In the meantime, alarge number of organic reactions have been transferred to solid-phase synthesis. For
each solid-phase synthesis, aspecial strategy, a specic
linker, and asuitable cleavage method must be developed.
Each single synthetic step must be compatible with the
protecting groups, the polymer support, and the linker.
However, a whole new dimension of chemical diversity
is made available than is possible with peptides and nucleotides.
Careful design of the target molecules to be synthesized is indispensable for combinatorial chemistry.
Limitations arise from the accessibility, that is, the development of an appropriate synthetic scheme, and
furthermore from the desired structural diversity of the
resulting library. Computer methods help to nd a“reasonable” selection of synthetic components. How is the
optimal composition obtained? This highly depends on
what the constructed library should be tested for. Alibrary can be developed for general-purpose screening.
It should then be “optimally diverse.” Their composition
is based on general criteria such as molecular weight,
overall lipophilicity, abalanced distribution of hydrogen-bond donor and acceptor groups, and the size of
the hydrophobic surface. These criteria are important for
the similarity or diversity of drug molecules (Chap.17).
However, the desired diversity of a library can also be
considered in terms of its biological properties at agiven
receptor (target-oriented). Criteria that make molecules
“similar” or “diverse” for one receptor are not necessarily identical for another receptor (Sect.17.7). Thus, with
respect to the wide range of proteins on which combinatorial libraries will be tested, there is no absolute measure
Chapter • Combinatorics: Chemistry with Big Numbers
11
of diversity. On the other hand, combinatorial chemistry
plays an important role in establishing initial structure–
activity relationships for a target protein. This requires
very rapid chemical variations in different regions of
alead structure that has been found to be suitable. The
design and synthesis of targeted compound libraries provides arapid solution to this problem.
11.9 Nanomolar Ligands for G-Protein-
Coupled Receptors
The following two sections present examples of how
combinatorial libraries can be designed and successfully
synthesized. Chemists at the company Chiron synthesized
alibrary of trimeric N-substituted oligoglycines (peptoids)
by using the split-and-combine method (. Fig.11.5). In
their design of the nitrogen substituents, the scientists had
G-protein-coupled receptors in mind. These receptors are
the targets of many neurotransmitters and hormones. In
the construction of their peptoids, they combined at least
one aromatic group and a side chain with an H-bond
donor in the form of a hydroxyl group (. Fig. 11.5,
groupsA andO). Furthermore, abasic nitrogen atom is
present in the molecules with X=H. These groups match
those also found in neurotransmitters and hormones. For
the remaining third substituents (groupD), the substitu-
ent composition was chosen to be as diverse as possible.
From these groups, apeptoid library of approximately
5000 di- and tripeptoids was prepared.
Different mixtures were tested on the adrenergic receptors. The H–ODA–NH2 partial library was identied
as the most active one. It served as astarting point for
the stepwise deconvolution of the library. Partial libraries were resynthesized, rst by keeping the hydroxy side
chain from groupO constant, then the members of the
diverse groupD, and nally the aromatic substituent
from subsetA. In the end, 11.7 remained as ananomolar
ligand (. Fig.11.6).
The same peptoid library was tested on another
GPCR, the opiate receptor. In this case the most active
partial library H–ADO–NH2 was found in the rst step.
The relevant deconvolution through resynthesis delivered
11.8 as ananomolar ligand. The molecule has ap-hy-
droxyphenelethyl moiety and adiphenylmethane group
on both ends of the tripeptoid. It is known from detailed
studies on Met-enkephalin 11.9 that the amino acids tyrosine and phenylalanine are essential for the activity.
There are analogous groups for both moieties on the
tripeptoid (. Fig.11.6).
. Fig. 11.5 Peptoids are oligoglycines that have substitu-
tions at the nitrogen. Alibrary of di- and tripeptoids was
constructed according to the split-and-combine technique.
Three Xgroups were added to the N-terminus. Three
groupsO with ahydroxy function, four groupsA with
an aromatic ring, and 17groupsD with diverse groups
were used as nitrogen side chains. Eighteen mixtures (six
permutations ofA, O, andD with three end groups) gave
ca.5000 di- and tripeptides. The H–ODA–NH2 library
showed activity on the α-adrenergic receptor. First, the
hydroxy groupsO were deconvoluted. The compounds with
p-hydroxyphenethyl groups were the most active ones. In
the next synthesis round, 17partial libraries were composed
with this Ogroup held constant, and dened groups were
used from the diverse Dgroup. Compounds with adiphenyl
or diphenyl ether group were particularly active. With these
groups in the Dposition, the work was continued. Deconvolution of the aromatic side chainsA in the last position
resulted in eight individual compounds
. • Parallel or Combinatorial, in Solution or on aSolid Support?
. Fig. 11.6 The derivative 11.7 is the most
potent compound from the H–ODA–NH2 library
with aKi = 5 nM on the α-adrenergic receptor.
Testing on the opiate receptor gave compound
11.8 as the candidate with highest afnity
(Ki = 6 nM) from the H–ADO–NH2 library after
deconvolution. Met-enkephalin 11.9 is apotent
opiate receptor ligand. The relationship between
the p-hydroxyphenyl group in 11.8 and the tyro-
sine side chain in 11.9, and aphenyl portion in the
diphenylmethane groups of 11.8 and the benzyl
groups of phenylalanine in 11.9 is obvious. Tyr
and Phe are essential for the activity of Met-en-
kephalin
11.10 More Potent than Captopril: AHit
from aCombinatorial Library of
Substituted Pyrrolidines
The Affymax company prepared alibrary of ca.500 dif-
ferently substituted pyrrolidines by 1,3-dipolar cycload-
dition. In the rst step, the resin was loaded with pro-
tected amino acids (Gly, Ala, Leu, and Phe; . Fig.11.7).
Then the transformation to an imine was made with four
different aromatic aldehydes. Cycloaddition with ve dif-
ferent alkenes led to ve-membered-ring heterocycles. In
the last step, the pyrrolidines were N-substituted with
three different thiols.
This last step was done in view of testing these ligands
on the angiotensin-converting enzyme (ACE, Sect.25.4).
Inhibitors of this enzyme contain afunctionalized proline residue at their C-terminus. The iterative deconvolution of the library afforded 11.10 as apotent ACE inhibitor (. Fig.11.7; Ki = 160 pM). It is distinctly astronger
binder than the marketed product captopril and belongs
to the most potent thiol-containing ACE inhibitors.
11.11 Parallel or Combinatorial, in
Solution or on aSolid Support?
While solid-supported combinatorial chemistry has
enabled the automated synthesis of alarge number of
molecules, it has also posed problems. The difculties
of testing on resins or deconvolution and resynthesis of
libraries have already been mentioned. Labeling is an elegant but laborious alternative. Another way to avoid
deconvolution of alibrary but still take advantage of
combinatorial chemistry is parallel synthesis in spatially
separated reaction vessels. Throughout the entire reaction sequence, it is clear which reactant and product are
in each vessel. There is no need for laborious deconvolution. At rst glance, this strategy seems impractical.
How can athousand reaction components be reasonably transformed into athousand reaction vessels? The
reaction vessels should not be considered in the classical
sense of organic chemistry. Instead, miniaturized reaction “automats” are developed, in which all reaction
steps are carried out in parallel. Alternatively, methods
have been developed in which the resin beads are lled
into many small reaction capsules. These are open to the
solution phase for compound transport, but the beads
are mechanically enclosed. Each capsule has alabel that
can be read by aradio transmitter. All the capsules are
then placed in aconventional round-bottomed ask and
the usual chemistry is carried out. The capsules can be
mechanically separated and brought into contact with
different reagents. Which reaction sequence is performed
on which capsule is tracked by the registration system
with the radio transmitter. In this way, one molecule per
reaction capsule can be produced by combinatorial principles, virtually as in parallel synthesis. The individual
compounds are then available for testing.
Synthesis on asolid support has disadvantages compared to solution phase chemistry. In general, the transformations are slower and the analysis to follow the reactions is much more complicated. Coupling to the solid
support requires asuitable linker. Such alinker should be
removed from the library prior to testing. Most importantly, removal of the linker (“traceless linker”) should not
leave any functional groups in the library that could unintentionally be part of the pharmacophore. The chemistry
used to attach and remove the linker must be compatible
with all other reactions in the synthesis of the solid-sup-
Chapter • Combinatorics: Chemistry with Big Numbers
ported library. This can limit the chemistry that can be
applied. In preparative chemistry, molecules are preferably
constructed using aconvergent synthesis strategy. For this,
asynthesis strategy is developed in which the components
of the nal product are prepared in separate steps, each in
parallel. In the subsequent reaction steps, the previously
prepared components are brought together and coupled
to form the nal product. Such astrategy is more efcient
and leads to higher yields than alinear synthetic route.
However, a convergent strategy cannot be implemented by sequentially building on aresin. Therefore, for
some syntheses, the tables have been turned. The prepared
libraries are not bound to the solid support, but to the
reagents with which they are treated. The advantage of
carrying out reactions on the solid support remains. Good
mechanical separation of reaction components, ease of
working with large excess of reagents, and automated reactions are part of this technique. An advantage is that
convergent syntheses are now possible. Even toxic reagents
can be used, as their separation is ensured by their rm
adhesion to asolid support. The usual analytical methods
typically applied to the solution phase can also be used.
Some reactions, especially ring closure reactions or
condensations, compete with intermolecular transfor-
mations. To avoid this, highly dilute solution conditions
are used. When asolid supported reactant is used, the
local concentration of the reactant is reduced because
it is xed to the solid support and spatially separated.
Reactions that occur over atrapped reaction product
can be simplied if the trapping reagent is coupled to
asolid support. Mechanical ltration is sufcient to separate the trapped components. Similarly, products can
be separated and puried by trapping them on asolid
support. Acids and bases can be separated for purication by treatment with an immobilized amine or sulfonic
acid. Metal complex formation or hydrophobic adhesion
groups are now being used for the purication of combinatorially produced compound libraries.
How will combinatorial chemistry further evolve?
The miniaturization of reaction vessels and synthetic
automats seems to be apromising perspective. The lab-
on-a-chip concept is already widely used for bioanalytical
methods. Small reaction volumes, integrated separation
columns, miniaturized valves and pumps controlled by
piezo elements are integrated on small chip cards. It remains to be seen whether such serial reaction automats
are the laboratories of the future.
11
. Fig. 11.7 The amino acids AA=Gly, Ala, Leu,
or Phe are coupled to the support resin(a). Next,
they are transformed to imines with four different
aromatic aldehydes (Ar–CHO);(b), which react with
alkenes under 1,3-dipolar cycloaddition conditions
to give pyrrolidines(c). In the last step, the free NH
proton on the heterocycle is treated with different
thiol compounds (Thio-COCl);(d). With the help of
the split-and-combine technique the library is cleaved
from the polymer with release of an acid function.
Its ability to inhibit the angiotensin-converting
enzyme was tested. By resynthesis and renewed
testing, the library was deconvoluted to the active
compound. In doing so, compound 11.10 was identied as ahigh-afnity inhibitor
. • The Protein Finds Its Own Optimal Ligand: Click Chemistry and Dynamic Combinatorial Chemistry
11.12 The Protein Finds Its Own
Optimal Ligand: Click Chemistry
and Dynamic Combinatorial
Chemistry
Could aprotein simply produce its own best inhibitor?
This would have to be synthesized with an ideal geometry
and optimal interactions directly in the binding pocket
of the target enzyme or bound there immediately after
synthesis. Which chemical reactions are suitable for such
aconcept? They must be reactions that can be performed
in an aqueous medium. They should be orthogonal to the
chemistry used by the biological systems themselves. In
this way, unwanted cross reactions can be avoided. They
must proceed with high delity, enthalpically driven, fast,
and with nearly complete turnover. Such reactions have
been developed under the term “click chemistry” in the
groups of Barry Sharpless in La Jolla, California, USA,
and Morten Meldal at the University of Copenhagen,
Denmark. Carolyn Bertozzi, now at Stanford University
in California, USA, was able to use these reactions in
living organisms. For this work, the three scientists were
awarded the Nobel Prize in Chemistry in 2022.
Cycloadditions of unsaturated compounds (1,3-dipolar cycloadditions, Diels–Alder reactions); nucleophilic
substitutions, especially ring-opening reactions; nonaldol-like carbonyl reactions; and additions to C–C multiple bonds fulll these requirements. These can be applied
using combinatorial principles. The 1,3-dipolar cycloaddition (Huisgen reaction) is particularly well suited for the
construction of ve-membered triazole and tetrazole heterocycles (. Fig.11.8). 1,4-Disubstituted 1,2,3-triazoles
can be regiospecically prepared by the reaction of an
azide and an alkyne in the presence of Cu(I) salts at room
temperature. 1,5-Disubstituted triazoles are formed when
copper ions are excluded or other ions such as ruthenium
are added. The reaction proceeds over awide pH range
between4 and12. The reaction type can be extended to
tetrazoles. This requires nitriles as dipolarophile reactants in the presence of zinc ions.
An example of the use of click chemistry is the search
for an inhibitor of the target protein acetylcholinesterase
(AChE, Sect.23.7). The enzyme was added to amixture
of potential azide and alkyne reactants for the Huisgen
reaction. Of the many possible reaction products, the enzyme captures two femtomolar inhibitors (11.11, 11.12)!
Provided with aphenylphenanthridine moiety at one end
and atacrine head group at the shallow entrance, azide
and alkyne react in the center of the hose-like binding
pocket to form atriazole (. Fig.11.9). Only afew products are formed. They are determined by the possible
placement of the starting materials. Crystal structures
have been determined for two potent products. The newly
generated triazole ring forms awater-mediated H-bond
to the catalytically active Ser 203 of the protein. It appears that the triazole ring is not formed exclusively as an
. Fig. 11.8 The 1,3-dipolar cycloaddition (Huisgen reaction) is
atypical click chemistry reaction and leads to ve-membered triazole
and tetrazole heterocycles. In the presence of Cu(I) salts, azides and
alkynes react regiospecically at room temperature to form 1,4-disubstituted 1,2,3-triazoles, in the absence of copper but with ruthenium
ions, 1,5-disubstituted products are formed. If anitrile is used instead
of the alkyne component, and the reaction is catalyzed by zinc ions,
1,5-disubstituted tetrazoles will be obtained as products
entropically favored linkage product. Presumably, the polar interaction with Ser 203 exerts adirectional inuence.
Jean-Marie Lehn’s research group in Strasbourg,
France, took adifferent approach. They developed “dy-
namic combinatorial chemistry” through the spontaneous
construction of molecules from suitable starting materials via reversible chemical reactions (. Fig.11.10). All
conceivable combinatorial products are formed from
amixture of different building blocks. Adynamic exchange equilibrium is established between them. The
target receptor (e.g., aprotein) is added to such an equilibrium system. In this way, the mixture components with
the best protein-binding properties have an advantage,
as the protein captures the best binders and shifts the
equilibrium. This leads to aself-perpetuating selection
of the ligands that t best into the binding pocket. In
this way, the added protein is effectively seeking its own
best inhibitor.
A suitable reversible equilibrium reaction is the
formation of acylhydrazones from ahydrazide and an
aldehyde under acidic conditions (. Fig. 11.11). Inspired by an earlier fragment screening on the aspartic
protease endothiapepsin (Sect.7.9), Milon Mondal and
Anna Hirsch at the University of Gronningen in the
Netherlands added this enzyme to amixture of several
acylhydrazides and aldehydes. STD NMR spectroscopy
(Sect.7.8) followed the formation of the products selected by the enzyme. Two micromolar inhibitors, 11.13
and 11.14, were crystallographically characterized as inhibitors of the catalytic center of the protease. Based on
these structures, asecond round of design and dynamic
Chapter • Combinatorics: Chemistry with Big Numbers
. Fig. 11.9 The library produced from alkynes
bearing an acetylcholinesterase (AChE)-suitable
tacrine side chain and AChE custom-made
phenylphenanthridine-substituted azides. In the
presence of AChE the products 11.11 (green)
and 11.12 (gray) are formed, which proved to
be potent enzyme inhibitors. They differ in the
topology on the ve-membered ring. Crystal
structure determinations were accomplished
with both inhibitors. The surface around the
protein is shown with the bound ligand11.12.
Both ligands occupy the hose-like binding pocket of AChE. Compound 11.11 binds via awater
molecule (red sphere) to the hydroxy function of
Ser 203. (7 https://sn.pub/vekrXl)
11
Synopsis
. Fig. 11.10 A mixture of different library components that interact
under equilibrium conditions in dynamic combinatorial chemistry is
furnished. Numerous products can form in the equilibria. They represent potential “keys” that can t in the “lock” of the target protein.
The added receptor protein binds to the best-tting ligands from the
combinatorial synthesis followed. It was now performed
with adialdehyde. A54 nM inhibitor (11.15) was isolated
from the reaction mixture.
Click chemistry and dynamic combinatorial chemistry in the presence of the target protein offer the perspective of very rapidly obtaining new potent compounds.
Admittedly, the number of reactions suitable for this
method is still limited, but further chemical transformations are being searched for intensively. If it is possible
to elucidate the structures of the most potent products
with the target protein, these can be modied by targeted
design into structural analogs that also meet the criteria
we demand of typical drug molecules.
11.13 Synopsis
As aconsequence of the tremendous acceleration of
-
automated compound screening for biological activ-
ity, the number of compounds required for testing
has signicantly increased and stimulated the devel-
opment of automated parallel synthesis and combi-
natorial chemistry.
Nature produces an enormous chemical multiplicity
-
by combining either amino acids or nucleic acids to
reveal polymers that fold into 3D arrangements.
The chemical space of organic molecules with up to
-
25nonhydrogen atoms and that satisfy the require-
ments of drug-likeness has been estimated to host
about 1027 imaginable candidates. Adatabase system-
atically generated on the computer and containing up
compound mixture and shifts the equilibrium in the direction of increased formation of this product. It is then removed from the equilibrium by protein binding. (Redrawn after O.Ramström and J.-M.
Lehn, Nat. Rev. Drug Discov., 1, 27–36 (2002))
to 17C, N, O, S, and halogen atoms, comprises 166.4
billion molecules.
Chemical reactions on asolid support, usually or-
-
ganic polymer resins such as cross-linked polystyrene,
follow astepwise synthesis strategy to build up molecules sequentially on the solid phase. Complete conversions and easy purication can be achieved, and
product release from the solid phase is accomplished
as the nal step.
Sophisticated synthetic strategies have been devel-
-
oped to generate multiple products on the solid support from reagent mixtures in alimited number of
reaction steps. Elaborate protocols have been established to keep track of product formation that also
use elaborate chemical labeling techniques. In particular, DNA-encoded libraries are increasingly being
used as this technique does not necessarily require
syntheses on aresin. This greatly simplies the subsequent testing of the library in biochemical activity
assays.
The biological activity testing of compound libraries
-
generated by combinatorial chemistry on asolid support requires sophisticated protocols to detach and
deconvolute the library.
The design and selection of building blocks used for
-
library synthesis are purpose-oriented and consider
the properties of the target(s) at which the library is
subsequently screened.
Multiple protocols have been developed, either for
-
combinatorial chemistry or parallel synthesis that
immobilize either the library substrates on the solid
Chapter • Combinatorics: Chemistry with Big Numbers
11
. Fig. 11.11 Dynamic combinatorial chemistry was used to convert
amixture of acylhydrazides (red) and aldehydes (blue) to acylhydrazones in acidic conditions (1st round). From the mixture, 11.13 and
11.14 emerged as micromolar inhibitors of the aspartic protease endothiapepsin. 11.13 binds to the two aspartates of the enzyme with
its hydrazone NH function mediated by awater molecule. 11.14 uses
phase, or the reagents are immobilized and the library
is developed in the solution phase.
The target protein can be added to amixture of re-
-
agents in click chemistry and dynamic combinatorial
chemistry. From alarge variety of possible reaction
products, the protein binding pocket selects the best
binder as apotent inhibitor or antagonist of the tar-
get protein.
its free amino group for a direct interaction to the two residues. In
a2nd round, different acylhydrazides were again reacted but now with
an aromatic dialdehyde. This mixture yielded 11.15 as adouble-digit
nanomolar inhibitor. It uses one of its two amino groups to bind to
the aspartates
Bibliography and Further Reading
General Literature
G. Jung and A. G. Beck-Sickinger, Multiple peptide synthesis methods
and their applications. New synthetic methods. Angew. Chem. Int.
Ed. Engl., 31, 367–383 (1992)
W. H. Moos, G. D. Green and M. R. Pavia, Recent Advances in the
Generation of Molecular Diversity, Ann. Rep. Med. Chem., 28,
315–324 (1993)
L. Weber, The application of multi-component reactions in drug dis-
covery. Curr. Med. Chem., 9, 2085–2093 (2002)
R. M. Baum, Combinatorial Approaches Provide Fresh Leads for Me-
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