Robert Bruce Merrifield

• Robert Bruce Merrifield:1921-2006

• American Chemist
• Born: Fort Worth, Tex., Ph.D. Univ.
of California at Los Angeles, 1949.
• As a researcher at the Rockefeller
Institute for Medical Research
(later Rockefeller Univ.), with which
he was associated until his death,
Merrifield developed a
comparatively simple method for
synthesizing polypeptides.
• The innovation, which won
Merrifield the 1984 Nobel Prize in
Chemistry, greatly facilitated the
manufacture of insulin, interferon,
and other drugs.
 Combinatorial Chemistry
• Combinatorial chemistry is a sophisticated set of
techniques used to synthesize, purify, analyze, and
screen large numbers of chemical compounds, far faster
and cheaper than was previously possible.
• The direct precursor of combinatorial chemistry was
the solid-phase synthesis of polypeptides developed by
American biochemist Robert Bruce Merrifield in the
1960s, followed by the advances in laboratory
automation since then.
• Initial development of the field has been led by the
pharmaceutical industry in the search for new drugs,
but its applications are spreading into other fields of
chemistry.
• Other terms associated with this field are parallel array
synthesis and high-throughput chemistry.
 Combinatorial Chemistry
• Whereas classical synthetic chemistry involves the
stepwise synthesis and purification of a single
compound at a time, combinatorial chemistry makes it
possible to synthesize thousands of different molecules
in a relatively short amount of time, usually without the
intermediate separation of compounds involved in the
synthetic pathway, and with a high degree of
automation.
• Such procedures result in the production of new
compounds faster and in greater numbers than is
possible with standard synthetic methods.
• The first and still the most common type of
combinatorial synthesis involves attaching a molecular
species onto a macroscopic substrate such as a plastic
bead and performing one or several well-characterized
chemical reactions on the species.
 Combinatorial Chemistry
• After each reaction, the product mixture can be
split among several reaction containers and
then recombined after the reaction (a
procedure called mix and split), or else carried
out in parallel containers.
• The resulting mixture of compounds is referred
to as a molecular library and can contain many
thousands of individual compounds. The
analysis, or screening, of these libraries to
identify the compounds of interest, along with
their subsequent isolation and identification,
can be completed by a variety of methods.
 Combinatorial Chemistry
• One example is iterative deconvolution; it involves the
successive identification of each of the units backward
along the chain of synthesized units.
• Another, called positional scanning, requires the
multiple synthesis of a library, each time varying the
location of a known unit along the chain and
comparing the activities of the resulting libraries.
• More recent advances in library screening involve the
"tagging" of a substrate with tiny radio frequency
transmitters or unique two-dimensional barcodes.
• Another important recent advance by researchers
allows combinatorial syntheses to be carried out in
solution, which further extends the scope and utility of
this field.
 Combinatorial Chemistry
• It is a collection of methods that allow the
simultaneous chemical synthesis of large numbers of
compounds using a variety of starting materials.
• The resulting compound library can contain all of
the possible chemical structures that can be
produced in this manner.
• Multiple parallel synthesis is a related group of
methodologies used to prepare a selected smaller
subset of the molecules that could in theory have
been prepared.
• The content of libraries prepared by multiple
parallel synthesis is more focused and less diverse
than those constructed with combinatorial
technology.
 Combinatorial Chemistry
• In Fig. 1.1, there is a hypothetical combinatorial
compound library of condensation products
produced by reacting every possible combination
of five starting materials.
• This results in a library containing 25 (5 ×5)
products. The library could be constructed by 25
individual reactions, with each product separate
from all of the others.
Figure 1.1. A combinatorial library constructed from
five reacting components.
Figure 1.2. A multiple parallel synthesis library constructed
from six participants.
 Combinatorial Chemistry
• Combinatorial + chemistry
• One entry found for combinatorial chemistry. Main
Entry:
• com·bi·na·to·ri·al chemistry
Pronunciation: käm-b -n - t r- - l-, k m- b -n -, - t r-
Function:
• A branch of applied chemistry concerned with the
rapid synthesis and screening of large numbers of
different but related chemical compounds generated
from a mixture of known building blocks in order to
recover new substances optimally suited for a
specific function using combinatorial chemistry to
develop new pharmaceuticals and catalysts.
 Combinatorial Chemistry
• Combinatorial chemistry is a technology
for creating molecules en masse and
testing them rapidly for desirable
properties - continues to branch out
rapidly.
• Compared with conventional one-
molecule-at-a-time discovery strategies,
many researchers see combinatorial
chemistry as a better way to discover new
drugs, catalysts, and materials.
 Combinatorial Chemistry
• Combinatorial chemistry (or CombiChem) is an
innovative method of synthesizing many different
substances quickly and at the same time.
• Combinatorial chemistry contrasts with the time-
consuming and labor intensive methods of
traditional chemistry where compounds are
synthesized individually, one at a time.
• While combinatorial chemistry is primarily used
by organic chemists who are seeking new drugs,
chemists are also now applying combinatorial
chemistry to other fields such as semiconductors,
superconductors, catalysts and polymers.
 Combinatorial Chemistry
• CombiChem is used to synthesize large number of
chemical compounds by combining sets of building blocks.
• Each newly synthesized compound's composition is slightly
different from the previous one.
• A traditional chemist can synthesize 100-200 compounds
per year. A combinatorial robotic system can produce in a
year thousands or millions compounds which can be tested
for potential drug candidates in a high-throughput
screening process.
• Over the last few years, the combinatorial chemistry has
emerged as an exciting new paradigm for the drug
discovery. In a very short time the topic has become the
focus of considerable scientific interest and research
efforts.
Dynamic combinatorial Chemistry
• Dynamic combinatorial chemistry is a new approach to
synthetic molecules that are good at molecular
recognition.
• It is a powerful strategy to discover new ligand for
biomolecules and to identify and prepare synthetic
receptors for specific guest molecules.
• Dynamic combinatorial libraries are made by linking
simple building blocks together using a reversible
reaction, allowing a constant interchange of building
blocks resulting in a mixture of library members that is
in equilibrium.
• The amount of each of the library members is directly
related to their thermodynamic stability.
 The Combinatorial Approach
• Many chemicals are pieced together through combinations of
smaller building blocks. For example, benzene is a chemical
consisting of six carbon atoms connected in an aromatic ring
structure, with a hydrogen atom bound to each carbon.
Substituting one of the hydrogen with a hydroxyl (-OH) group
forms the chemical phenol.
• Substituting a methyl (-CH3) group instead forms toluene, and
substituting an amino (-NH2) group forms aniline. Because of
their different "functional groups" or side groups, all of these
compounds have very different physical and chemical
properties.
• More variations can be synthesized by substituting additional
side groups with more than one of the hydrogens.
• By substituting one of just these three groups (or by not adding
any groups) for any of the six hydrogens in a benezene ring,
there are 46, or 4,096, possible combinations (the number of
different compounds is much smaller, because benzene is
symmetrical, and many of the combinations represent
equivalent structures).
 The Combinatorial Approach
• Side groups can also be placed onto other side
groups. For example, a single chlorine atom
can substitute for one of the hydrogens of the
methyl group in toluene to form benzyl
chloride.

• By using a moderately sized collection of side

groups, placing them onto a “scaffold"
molecule that is more complex than benzene
(such as cholesterol, which has three six-carbon
rings and a five-carbon ring), and by using
additional levels of side groups, combinatorial
chemists can synthesize vast numbers of
distinct but related compounds.
 The Combinatorial Approach
• Although the utility of combinatorial chemistry
was not fully appreciated by scientists until the
1980s, nature uses this strategy over and over.
Genes, after all, are composed of different
combinations of only four different nucleotides,
and just twenty different amino acids form the
building blocks of all proteins.
• In the immune systems of mammals, B
lymphocytes use an elaborate scheme for
mutating and combining different segments of
antibody genes to generate a diverse pool of
antibody molecules that can recognize and bind
a wide array of alien molecules that enter the
body with a pathogen infection.
Drug Targets:
• Combinatorial chemistry is most often
used to synthesize "small molecules“, in
contrast to macromolecules such as DNA,
RNA, proteins, and polysaccharides,
which are polymers containing long
chains of monomer subunits.
• Because of their enormous size,
macromolecules cannot easily enter cells,
which is an important requirement for
compounds intended for use as drugs.
 Drug Targets:
• In many cases the combinatorial chemist is looking for
a compound that will bind tightly and specifically to a
cellular molecule, such as the catalytic, or "active“, site
on the inside of an enzyme. Small molecules can fit into
the holes and crevasses leading to the active site.
• By binding the enzyme, the synthetic compound may
prevent it from binding to its natural substrate or from
carrying out its catalytic reaction. Defective enzymes
that resist normal cellular restraints on their activities
are responsible for many diseases, including certain
cancers.
• Chemical inhibitors of such rogue enzymes hold
promise as powerful drugs. Alternatively, binding of a
small molecule to an enzyme could enhance the
enzyme's normal activity. Such molecules have
potential as drugs for diseases caused by insufficient
activity of a crucial enzyme.
Drug Targets:
• For two molecules to bind to one another, they
must have a proper fit, like a key in a lock. The
fit depends on the shapes of the two molecules
as well as on the chemical interactions between
them.
• For example, two positively charged side
groups will repel each other, but negative and
positive groups can attract.
• Not surprisingly, a synthetic compound that
binds a particular molecule often has chemical
properties and a shape mimicking the natural
ligand for the molecule. Such compounds are
termed analogues.
Drug Targets:
• When the drug target is known, its structure
can be used as a template to create analogues
with complementary shapes.
• Alternatively, if an analogue is already in hand
that binds the target but has undesirable
properties (such as weak binding, poor
solubility, or serious side effects), this structure
can be used as a starting point.
• Even without such clues, the speed and
automation of the combinatorial approach
makes it feasible to randomly synthesize and
test millions of compounds.
 High-Throughput Screening
• A library of a billion or more different
molecules is only useful if the molecules
can be quickly and economically screened
for the desired function.
• "High-throughput" techniques have been
developed that automate most of the steps
required to combine the molecules with
their targets and evaluate the extent of any
reaction.
 High-Throughput Screening
• Typically, the molecules are arrayed on a solid
surface and the target is added. Unbound target is
washed away.
• Fluorescent tags are often added to the target, to
allow easy (and automated) visualization of the
results.
• Robotic systems controlled by computers can react
and evaluate billions of separate compounds in the
time it would take a human to screen a dozen.
• One such approach is used in DNA microarrays, in
which thousands of genes from a DNA library are
attached to a solid base.
• These are reacted with messenger RNAs from a
cell, and the results are visualized fluorescently.
 Selex
• In addition to its use in drug development, combinatorial
chemistry can be applied to other areas of biomedical research,
such as the design of molecules for diagnosing medical conditions.
• Compounds for these applications can be larger than
pharmaceutical compounds, and do not have to be designed to
enter the body.
• Using a novel combinatorial chemistry method called in vitro
selection or SELEX (systematic evolution of ligands by
exponential enrichment), a short DNA or RNA molecule (termed
an oligonucleotide) with a desired property, such as the ability to
specifically recognize and bind a molecule associated with a
particular disease, can be selected in a single experiment from a
library containing approximately 1015 different compounds.
• First, a library of oligonucleotides is created in a machine called
an oligonucleotide synthesizer. This apparatus can make
oligonucleotides with either a defined or random sequence.
 Selex
• Oligonucleotides for SELEX are designed to have a
central region containing random sequence and outer,
flanking regions with defined sequences. These defined
sequences will be used as primer-binding sites for the
polymerase chain reaction (PCR).
• The oligonucleotide library is prepared as a mixture,
usually containing about 1014 to 1015 different
sequences. These specialized oligonucleotides, termed
aptamers, are then exposed to target ligand molecules,
which are typically attached to a solid support, such as
a filter membrane.
• The unbound aptamers are then washed away, leaving
only the rare aptamers that can bind the ligand
adhering to the filter. These aptamers can then be
recovered from the filter by washing it with a solution
that disrupts the binding.
 Selex
• These binding candidate aptamers represent a minuscule
fraction of the original library. Some may bind the target
ligand tightly, but others may bind weakly.
• Since all the aptamers have defined primer-binding sites on
the ends, this much-reduced population can now be
amplified exponentially by PCR.
• After amplification, the aptamers can be subjected to
another round of ligand binding, now using more strigenent
washing conditions, in which only the tightest-binding
molecules will stay bound.
• These high-affinity binders can be recovered again
subjected to still more cycles of PCR amplification, binding,
washing, and recovery, until the population of aptamers
consists exclusively of very tightly binding molecules.
• For some applications, these molecules are useful directly.
They can also be studied to design non-DNA molecules that
have similar shapes but that will have more potential as
drugs.
Dynamic combinatorial Chemistry
• This characteristic makes such libraries ideal to
identify new compounds that are good at molecular
recognition: exposing a dynamic combinatorial library
of potential receptors to a guest molecule will lead to an
increase in the stability of those receptors that bind the
guest.
• As a result the equilibrium will shift in favor of the best
binders at the expense of the poor receptors:
amplification of the fittest!
• The increase in concentration of the best binders makes
these easy to detect and identify. After switching off the
exchange reaction (usually by changing the pH or
removing the catalyst that is necessary for the process)
the selected products can be isolated and used.
Principle of Combinatorial Chemistry
• Combinatorial chemistry is a technique by which
large numbers of structurally distinct molecules
may be synthesized in a time and submitted for
pharmacological assay.
• The key of combinatorial Chemistry is that a
large range of analogues is synthesized using the
same reaction conditions, the same reaction
vessels. In this way, the chemist can synthesize
many hundreds or thousands of compounds in
one time instead of preparing only a few by
simple methodology.
Principle of Combinatorial Chemistry
• In the past, chemists have traditionally
made one compound at a time.
• For example compound A would have been
reacted with compound B to give product
AB, which would have been isolated after
reaction work up and purification through
crystallization, distillation, or
chromatography.
Principle of Combinatorial Chemistry
• In contrast to this approach, combinatorial
chemistry offers the potential to make every
combination of compound A1 to An with
compound B1 to Bn.
Principle of Combinatorial Chemistry
• The range of combinatorial techniques is
highly diverse, and these products could be
made individually in a parallel or in
mixtures, using either solution or solid
phase techniques. Whatever the technique
used the common denominator is that
productivity has been amplified beyond the
levels that have been routine for the last
hundred years.
 Combinatorial Chemistry
• Finding of novel drug is a complex process. Historically, the
main source of biologically active compounds used in drug
discovery programs has been natural products, isolated from
plant, animal or fermentation sources.
• Combinatorial chemistry is one of the important new
methodologies developed by researchers in the pharmaceutical
industry to reduce the time and costs associated with
producing effective and competitive new drugs.
• By accelerating the process of chemical synthesis, this method
is having a profound effect on all branches of chemistry, but
especially on drug discovery.
• Through the rapidly evolving technology of combi-chemistry,
it is now possible to produce compound libraries to screen for
novel bioactivities. This powerful new technology has begun to
help pharmaceutical companies to find new drug candidates
quickly, save significant money in preclinical development
costs and ultimately change their fundamental approach to
drug discovery.
Conclusion:
• Combinatorial chemistry is a new field of
chemistry that employs novel concepts and
techniques to rapidly synthesize and screen
large numbers of compounds.
• Researchers in this field rely predominantly on
journals rather than books. Fortunately for
library collections, except for a few specialized
journals, nearly all the literature in this field
appears in the well-known journals that most
chemistry libraries already own.
• Two world wide web portals provide
convenient access to the abundant web based
information sources in combinatorial
chemistry.