Abstract
amino acids with bulky or reactive side chains.
Sixty years ago Merrifield first introduced the idea of
solid phase peptide synthesis, and in 1984 was
awarded the Nobel Prize in Chemistry for the same.
Since then Solid-phase peptide synthesis has been the
cornerstone of combinatorial chemistry, allowing for
the rapid generation of diverse compound libraries for
drug discovery and other applications. However, some
limitations in this method of combinatorial synthesis
led to the development of solution-phase synthesis as
an alternative approach, presenting certain
advantages over the latter. Despite these advantages,
the development of solution-phase combinatorial
chemistry has been slower compared to solid-phase
techniques, primarily due to challenges in product
separation and removal of impurities at each step.
Also, the limited library size is one of the major
constraints.
While some compounds are best synthesized in
solution phase due to their chemical properties, the
difficulty in purification and the limited library size
have hindered the widespread adoption of solution-
phase methods. Consequently, two main approaches,
encoding, and deconvolution methods, have been
utilized to decipher the synthesised sequences. This
paper focuses exclusively on encoding-based
strategies, providing a comprehensive review of major
methods introduced in existing literature and research
papers. Understanding the working and development
of encoding-based strategies can provide insights into
overcoming limitations and expanding the scope of
solution-phase combinatorial chemistry for future
research and innovation.
Introduction to solution-phase synthesis
For a long time now, solid-phase synthesis has been
the major method employed for the development of
combinatorial libraries. However, this method has a
few limitations, described below
1. Not all amino acid residues are compatible
with synthesis on an immobilised solid
support (or resin). Some amino acid residues
may undergo side reactions or racemization
under the harsh conditions used in SPPS,
leading to reduced yield and purity of the
desired peptide product. Additionally, the rigid
environment of the solid support may limit
the diffusion of reagents and hinder the
progress of chemical reactions, particularly for
2. The chemical, physical and thermal stability of
the resin is a concern.
3. Large amounts of product is difficult to
synthesise, except for a few approaches, like
the tea-bag method.
Most of these limitations can be overcome by
solution-phase synthesis, which allows for chemical
compatibility for synthesis of a larger pool of peptides,
eliminates the need to worry about the resin’s
stability, and is much more scalable for synthesis of
large amounts of product. However, the main
limitation of this method is regarding product
purification and removal of excess reagents. In this
method there is no phase separation between the
reactant and the product, so product extraction is
extremely difficult. Also, the use of excess reagents
leads to increased impurities in the mixture. Also,
when a large number of reagents are taken together in
a solution, it can result in several side reactions and
may lead to polymerization giving a tarry mass.
However, several methods have been developed to
serve as a solution to these problems like the use of
polymer-supported reactants, use of scavenger resins,
the use of fluorous solvents like CF72, and doing the
synthesis on dendrimers, which become soluble under
a certain temperature range.
Encoding Approaches
However, in all of these approaches, we need a way to
decipher the sequence of building blocks after the
combinatorial library has been synthesised and the
target compound has been isolated. This can be done
either using deconvolutional-based methods or using
encoding methods. In most of the encoding methods
(in most, not all) some kind of identifier molecules
called tags are used. Thus at each step in peptide
synthesis, alongside the addition of amino acid to the
peptide, a tag molecule (or a sequence of tags) is
added simultaneously to the coding strand. Later this
coding sequence is cleaved off the peptide and read,
and we get to know about the exact sequence of the
peptide chain.
There is a very limited amount of literature published
concerning encoding in solution phase. Here we will
focus mainly on the method introduced by Rudresha
Kottani, Roman A. Valiulin, and Andrei G. Kutateladze.
Kottani’s approach for encoding and
screening of solution phase libraries based
on the conditional photorelease of
externally sensitized photolabile tags
This tagging methodology is based on dithiane
adducts of carbonyl compounds, which are capable of
efficient photoinduced fragmentation in the presence
of an external electron transfer (ET) sensitizer.
A single unit consists of 3 elements: a ligand, tether,
and tag. Here, the ligands used are biotin,
glucosamine, and aminoundecanoic acid. Each kind of
ligand can be encoded with 3 different tags, which are
dithiane adducts of carbonyl compounds and differ in
the carbonyl chain. Upon addition of a detergent like
dodecyl phosphocholine (DPC), a micelle-like structure
is formed around the tag and linker, in which the tag
remains buried and the ligand is free on the outside.
Figure 1 : Thio ortho esters-encoded biotin.
Besides the fact that amphiphiles displaying
phosphocholine are common in biological settings,
utilization of DPC micelles offers several additional
benefits.
1) It ensures that the screening is always
compatible with aqueous media, regardless of
the aqueous solubility of the tested libraries.
2) It allows us to center the design of the tagging
system around readily available hydrophobic
alkyl dithianes, which can be selectively
extracted after irradiation with hexane or
other nonpolar solvents for unobstructed
GCMS analysis.
3) It spatially segregates the photofragmentation
chemistry from molecular recognition,
eliminating potential interference between
them.
4) It restricts the photochemistry to the micelle
interior, improving the quantum efficiency of
fragmentation, as it is known to increase in
the nonpolar environment.
5) The micelle-assisted design offers an option of
solubilizing certain target proteins that are not
water-soluble.
Figure 2: Ligand-receptor binding and photo-induced cleavage of
tag
The receptor, ImmunoPure avidin (Pierce, Rockford,
IL), was equipped with xanthone as an ET-sensitizer.
Upon ligand binding to the receptor, the sensitizer
approaches the tether closely. Upon irradiation, the
photosensitive sensitizer becomes activated, initiating
photochemically induced electron transfer. This
process cleaves off the tag from the tether and linker.
All tags encoding individual molecules of the bound
ligand are collectively released into the solution,
unveiling the identity of the lead compound. The
cleaved tags can be detected using electron capture
gas chromatography.
The following considerations have been kept in mind
while designing tags :
1) It should be amenable for detection at very
low concentrations by ubiquitous analytical
techniques.
2) It should not possess any functional groups
implicated in biological interactions.
3) It should not interfere with synthetic steps.
4) It should be easy to separate from the
aqueous screening environment, for example,
due to significant hydrophobicity.
5) It should be accessible via a simple and
straightforward synthetic approach.
This methodology can be further extended for
synthesis and screening of peptides. The peptides can
be synthesized using regular BOC/FMOC protection.
Here for every different amino acid, for every different
position a unique kind of tag is used. Say if we want to
make a 4-member peptide out of 5 amino acids, we
need 4*5 = 20 unique tags. Upon ligand-receptor
binding and exposure to photo radiation, sequential Other methods include Synthesis of DECLs by
electron transfer takes place between the tag sequence encoded routing (as suggested by Halpin
molecules, which are then separated from the peptide amd Harboury), DECLs formed by using a yoctoliter-
and recognised using electron capture gas scale reactor (by Hansen et al.), Dual pharmacophore
chromatography, thus deciphering the electron DECLs (by Melkko et al.39 and Scheuermann and
sequence Neri), and Single pharmacophore DECLs (by Mannocci
et al. and Clarck et al). In the paper by Olivier B. C.
Monty, Nicholas Simmons, Srinivas Chamakuri, Martin
Soluble DNA-encoded libraries M. Matzuk, and Damian W. Young methods for Fmoc-
Based Peptide Synthesis for DECLs have been
Despite of the difficulties associated with solution explored.
phase synthesis, researches are going on to develop
effective methods. In their paper from 2000, Harbury
and Halpin omitted the solid support in the synthesis Conclusion
of DNA-encoded libraries and replaced it with the
Encoding in solution phase is not a popularly used
encoding DNA oligomers and ended up with soluble
method for synthesis and screening in combinatorial
libraries. Here the amount of peptide encoded is no
chemistry, as evident by the lack of published
longer limited by the number of beads. Franzini et al.
literature in this field. Though encoding is the most
developed a purification method for filtering out
popular mode of synthesis in solid phase, its
excess reagents and byproducts in this encoding
applicability in solution phase still remains limited,
technique. The excess of DNA is biotinylated and
one of the reasons being existence of alternate
captured on streptavidin-coated sepharose. Since
deconvolution approaches. Kotti’s approach is a
then, various methods for DNA-encoded soluble small
significant development. Work is going on to devise
molecule organic libraries have been published. For
methods to synthesise DEFCL in solution phase, some
example in the DTS approach, developed by Prof. Liu
of which have been successful. This is a highly
from Harvard University, , two libraries are used. One
developing field and we can expect some significant
of them is the combinatorial template library.
developments in the near future.
Members of the libraries are DNA strands with
attached BB (red circle) at one end (Figure 3). The DNA
strand has annealing regions for attachment of the
second, third, and so forth BBs. The second library
contains a BB attached by a cleavable bond to the DNA
coding region. When the two libraries are mixed, the
coding region of the second BBs anneals with the
annealing region on the strand on the template
library. After annealing, the second BB is in proximity
to the first one and can make a covalent bond with it.
After cleaving the bond between the BB and its coding
region, DECL is formed.

Figure 3 : The DTS approach

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