Combinatorial Chemistry and

Synthesis in Parallel

Fernando Cardozo-Pelaez, MS PhD

Professor of Neurotoxicology ,
Email: Fernando.Cardozo@utp.edu.co
The “omics”:Genomics
3 billion DNA base pairs
20,000 to 25,000 genes in the human
genome codes for an average of
three proteins
Sequencing is determining the exact
order of the bases in a strand of DNA
DNA sequencing to search for
genetic variations and/or mutations
that may play a role in the
development or progression of a
disease
The “omics”:Transcriptomics
Gene readouts are called transcripts,
and a transcriptome is a collection of
all the gene readouts present in a
cell
Analyzing the entire collection of
RNA sequences in a cell (the
transcriptome) researchers can
determine when and where each
gene is turned on or off in the cells
and tissues of an organism
The function of most genes is not yet
known
The “omics”:Proteomics
The large-scale study of a set of proteins
produced in an organism, system, or biological
context
When and where proteins are expressed
Rates of protein production, degradation, and
steady-state abundance;
How proteins are modified (for example, post-
translational modifications (PTMs) such as
phosphorylation);
Movement of proteins
between subcellular compartments;
Involvement of proteins in metabolic pathways;
how proteins interact with one another
interactions with the genome and the
environment
The “omics”:Lipidomics
Large-scale study of lipids using
the principles of analytical
chemistry
Lipids represent unique reporters
of sub-cellular, cellular and organ
health and disease in mammals
The applications of lipidomics for
studying lipid metabolism
broadens our understanding of
the molecular mechanisms that
underpin metabolic disease states
The “omics”:Metabolomics
The large-scale study of small
molecules, commonly known as
metabolites, within cells,
biofluids, tissues or organisms
which are influenced by both
genetic and environmental factors
In humans, there are thought to
be around 3,000 endogenous or
common metabolites

thesis in medicinal chemistry
ects Where do you need Synthetic Chemistry?
past, medicinal chemistry involved the identifica-
a lead compound having a useful activity which
en modified to develop a clinically useful drug.
cation of the molecular target for the drug, and
chanism by which it worked, often took many
o establish. Today, most medicinal chemistry pro-
art with an identifiable target, and the emphasis is
covering a lead compound that will interact with
Find a Find a Isolate active
target lead structure
Compound
synthesis
designed to produce mixtures of different compound
within each reaction vessel, whereas those used in pa
allel synthesis produce a single product in each vesse
In general, parallel synthesis is favoured because it
easier to identify the structures that are synthesize
However, there is still scope for combinatorial chemi
try in finding lead compounds, especially as this proc
dure can generate significantly more structures in a s
period of time, thus increasing the chances of findin
a lead compound. Both methods generally involve th
use of solid phase techniques, which are discussed
the next section.
Identify Optimize
SAR
structure lead
Compound
synthesis
Combinatorial chemistry
Objetive: a defined reaction route to produce a large number of
compounds in a short period of time
Compound library
Finding lead compounds
Combinatorial synthesis are designed to produce mixtures of different
compounds

https://www.youtube.com/watch?v=MVgsX7PM4F4
 ease with which excess reagent can be removed; • a means of cleaving the product or the intermediates
• intermediates in a reaction sequence are bound to the from the linker;
bead and do not need to be purified; • protecting groups for functional groups not involved
• the polymeric support can be regenerated and reused in the synthetic route.

Solid phase techniques

if appropriate cleavage conditions and suitable anchor/
linker groups are chosen (see later);
• automation is possible;
• if a combinatorial synthesis is being carried out, a
range of different starting materials can be bound to
separate beads. The beads can be mixed together such
16.2.1 The solid support
The first successful example of solid phase synthesis was
the Merrifield resin peptide synthesis. The resin involved
consisted of polystyrene beads where the styrene is

Used to carry out reactions where

that all the starting materials are treated with another
reagent in a single experiment. The starting materials
and products are still physically distinct, as they are
bound to separate beads. In most cases, mixing all the
Cl
+
HO2C
partially cross-linked with 1% divinylbenzene. The beads
are derivatized with a chloromethyl group (the anchor/
linker) to which amino acids can be coupled via an ester
group (Fig. 16.2). This ester group is stable to the reaction
the starting material is linked to a

H
NHBoc

R1 O
O
solid support
NHBoc
Resin bead
H
R

De-protection with TFA

1
Several reactions can then be
HO2C

H
NHBoc

R2
carried out in sequence on the
attached molecule
O O
O O Coupling O
NH NHBoc NH2
H H
2 1
R H R
R1

O HF
HO2C aa1aa2aa3 aan NH2
O OH
aa1aa2aa3 aan NH2

FIGURE 16.2 Peptide synthesis on a solid support (Boc = tert-butyloxycarbonyl (t-BuO-CO); TFA = trifluoroacetic acid).

Patrick97397.indb 314 11/28/2012 9:16:20 PM

Solid phase advantages
Excess reagents or unbound by- The polymeric support can be
products from each reaction can be regenerated and reused if
easily removed by washing the resin appropriate cleavage conditions and
Large excesses of reagents can be suitable anchor/ linker groups are
used to drive the reactions to chosen
completion (greater than 99%)
because of the ease with which
excess reagent can be removed
intermediates in a reaction sequence
are bound to the bead and do not
need to be purified
 Solid phase requirements
A cross-linked insoluble polymeric Protecting groups for functional
support which is inert to the groups not involved in the synthetic
synthetic condition route
An anchor or linker covalently linked
to the resin—the anchor has a
reactive functional group that can be
used to attach a substrate
A bond linking the substrate to the
linker, which will be stable to the
reaction conditions used in the
synthesis
A means of cleaving the product or
the intermediates from the linker

range of different starting materials can be bound to
separate beads. The beads can be mixed together such
ing material;
address this, more polar solid phases were developed,
the Merrifield resin peptide synthesis. The resin involved such as Sheppard's polyamide resin. Other resins have • the functional group which is desired on the final
consisted of polystyrene beads where the styrene been is developed to be more suitable for the synthesis of
product once it is released.

Merrifield resin peptide synthesis

that all the starting materials are treated with another
reagent in a single experiment. The starting materials
and products are still physically distinct, as they are
bound to separate beads. In most cases, mixing all the
Cl HO2C NHBoc
partially cross-linked with 1% divinylbenzene. The beads
are derivatized with a chloromethyl group (the anchor/
linker) to which amino acids can be coupled via an ester
group (Fig. 16.2). This ester group is stable to the reaction
non-peptides. For example, Tentagel resin is 80% poly-
ethylene glycol grafted to cross-linked polystyrene, and
provides an environment similar to ether or tetrahydro-
furan. Regardless of the polymer that is used, the bead
should be capable of swelling in solvent while remaining
stable. Swelling is important because most of the reac-
Polystyrene beads partially cross-linked with 1%
tions involved in solid phase synthesis take place in the
divinylbenzene
interior of the bead rather than on the surface. It is wrong
to think of resin beads as being like miniature marbles
with an impenetrable surface. Each bead is a polymer
Resins having different linkers are given different
names (Fig. 16.5). For example, the Wang resin has a
linker which is suitable for the attachment and release of
carboxylic acids. It can be used in peptide synthesis by
linking an N-protected amino acid to the resin by means
of an ester link. This ester link remains stable to coupling
and de-protection steps in the peptide synthesis, and
can then be cleaved using trifluoroacetic acid (TFA) to
release the final peptide from the bead (Fig. 16.6). One
problem with the Wang resin is that the first amino acid

+
O Derivatized with a chloromethyl group (the anchor/
and swelling involves unfolding of the polymer chains
such that solvent and reagents can move between the
H R 1 O linker)
chains into the heart of the polymer (Fig. 16.3).
NHBoc Although beads are the common shape for the solid
H Amino acids can be coupled via an ester group
support, a range of other shapes, such as pins, have been
Resin bead R1 designed to maximize the surface area available for reaction
linked to the resin is prone to racemization. The Barlos
resin contains a trityl linker and was designed to avoid
this problem. The final product can be cleaved under
very mild conditions (e.g. HOAc/TFE/CH2Cl2 or TFA/
CH2Cl2) owing to the high stability of the trityl cations
that are formed. Molecules can also be linked to the resin

De-protection with TFA

Peptide cleaved at the end of the synthesis using
and, hence, maximize the amount of compound linked to
the solid support. Functionalized glass surfaces have also
by means of an alcohol group.
Starting materials with a carboxylic acid (RCO2H)
HO2C NHBoc vigorous acidic conditions (hydrofluoric acid)
been used and are suitable for oligonucleotide synthesis. can be linked to the Rink resin via an amide link. Once
the reaction sequence is complete, treatment with TFA
releases the product with a primary amide group, rather
H R2 Sheppard's
16.2.2 polyamide
The anchor/linker resin than the original carboxylic acid (R′CONH2; Fig. 16.7).
O O The anchor/linker is a molecular unit covalently attached Primary and secondary alcohols (ROH) can be linked
O O Coupling O Tentagel resin (80% poly- ethylene glycol grafted to
to the polymer chain making up the solid support. It con- to a dihydropyran-functionalized resin. Linking the
tains a reactive functional group with which the starting alcohol is done in the presence of pyridinium 4-toluene-
NH NHBoc NH2 cross-linked polystyrene) similar environment to
material in the proposed synthesis can react and hence sulfonate (PPts) in dichloromethane. Once the reaction
H H
R2 H R1 R1 ether or THF
become attached to the resin. The resulting link must be
stable to the reaction conditions used throughout the
sequence has been completed, cleavage can be carried
out using TFA (Fig. 16.8).

Swelling of the bead

O HF
HO2C aa1aa2aa3 aan NH2 Starting material,
Swelling
O OH Resin bead reagents
and solvent
aa1aa2aa3 aan NH2 Linkers

FIGURE 16.2 Peptide synthesis on a solid support (Boc = tert-butyloxycarbonyl (t-BuO-CO); TFA = trifluoroacetic acid). FIGURE 16.3 Swelling of a resin bead allowing access of reagents and solvent.
The anchor/linker
It is a molecular unit covalently attached The functional group which will be
to the polymer chain making up the present on the starting material
solid support The functional group which is desired
It contains a reactive functional group on the final product once it is released
with which the starting material in the Wang resin
proposed synthesis can react and hence
become attached to the resin Barlos resin
It must be stable to the reaction Rink resin
conditions used throughout the Dihydropyran derivatized resin
synthesis 316 Chapter 16 Combinatorial and parallel synthesis

It must be easily cleaved to release the

final compound once the synthesis is Starting
material Synthesis
Y

complete Bead
X
Y
Z Z
Cleavage

Linker X
Building
blocks
316 Chapter 16 Combinatorial and parallel synthesis

The linkers
Bead
Starting
material
Y
Synthesis
Cleavage
Y

X Z Z
Linker X
Building
blocks

FIGURE 16.4 The principles of an anchor/linker. X, Y, Z are functional groups.

Wang resin has a linker which is
suitable for the attachment and
OH NH2
release of carboxylic acids
The Barlos resin contains a trityl
O O OMe

Wang resin Rink resin OMe linker and was designed to avoid
O
O
Cl racemization
Rink resin for starting materials
Cl

with a carboxylic acid linked via an

Dihydropyran
derivatized resin Barlos resin

FIGURE 16.5 Types of resin with the linkage point circled.

amide link
O
Dihydropyran-functionalized resin
Bead Linker
OH + HO2C NH (Fmoc) O C NH (Fmoc)
primary and secondary alcohols
can be linked
H R H R

Peptide
 FIGURE 16.5 Types of resin with the linkage point circled.

Wang resin for peptide synthesis

O
OH + HO2C NH (Fmoc) O C NH (Fmoc)
Bead Linker
H R H R

Peptide
O synthesis O
O C NH2 O C
aa1aa2aa3 aan NH2
Piperidine
H R
TFA
cleavage

Fmoc = OH HO2C aa1aa2aa3 aan NH2

Peptide
O

O
Rink resin synthesis
Solid phase techniques 31

O
C R
NH2 + HO2C R NH
Further modifications
Bead Linker or additions to R

O O
C R! C R!
NH TFA H2N

FIGURE 16.7 Solid phase synthesis with a Rink resin (R contains functional groups
which allows further modifications of the molecule to give R'). The structure of the
linker is shown in Fig. 16.5.
 Bead Linker or additions to R

O O

Dihydropyran-functionalized
C R!
NH TFA HN
C R! resin synthesis
2

FIGURE 16.7 Solid phase synthesis with a Rink resin (R contains functional groups
which allows further modifications of the molecule to give R'). The structure of the
linker is shown in Fig. 16.5.

ROH
R!
O O OR O O
PPts Further modifications
Bead Linker
or additions to R
TFA

HO R!

FIGURE 16.8 Solid phase synthesis with a dihydropyran-functionalized resin (R contains functional
 14.8.2), and so a large amount of research was carried
out to extend solid phase synthetic methods to the syn-
thesis of small non-peptide molecules. The first move
away from natural peptides was to use the same peptide
coupling procedures, but with non-natural amino acids.
phase synthesis began when it became possible to pr
duce heterocyclic structures. Heterocycles are less su
ceptible to metabolism, and have better pharmacokinet
properties. They are more rigid, and diversity is possib
by varying the substituents around the heterocyclic ‘cor

Evolution of solid phase synthesis
Peptides could also be modified once they were built by
reactions such as N-methylation. N-Substituted glycine
units were used to produce structures known as peptoids
where the side chain is attached to the nitrogen rather
1,4-Benzodiazepines have been synthesised by lin
ing a selection of amino acids to resin beads through th
carboxylic acid group (Fig. 16.9). Reaction with a varie
of imines gave the adducts shown. Treatment with TF

Peptides pose particular problems as drugs in terms of their O

pharmacokinetic properties R4
NH NH
R 1
O
R4
O
O R4 N
R3
To extend solid phase synthetic methods to the synthesis of + R1 ∆ NH N
small non-peptide molecules R2 O
Cl(C H2)2Cl 2 TFA
R1
R N
NH2
The use of non-natural amino acids (N-methylation- R3
peptoids) R3 R2
Imines Amino acid Adducts
Heterocyclic structures
More rigid FIGURE 16.9 Benzodiazepine synthesis involving a cyclo-release strategy.

Increased diversity
Aldol condensations 318 Chapter 16 Combinatorial and parallel synthesis
DIBAL reductions
R2
Wittig reactions Patrick97397.indb 317 O O
N
11/28/
H O
R2 N O + H2N R2 HN O
LDA reductions C
O
N
O TFA
NH
Heck couplings R1 O R1
R1
Stille couplings Hydantoins
Mitsunobu reaction
FIGURE 16.10 Synthesis of hydantoins.
 reactions are all possible. Automated or semi-automated sis are planned in advance to ensure that they contain dif-
synthesizers can cope with 6, 12, 42, 96, or 144 reac- ferent functional groups on their arms, placed at different

Generating a compound library: the use of

tion vials depending on the instrument and the size of distances from the central scaffold.
the reaction tubes used. The addition of solvent, starting
materials, and reagents can be carried out automatically
16.3.2 Designing ‘drug-like’ molecules

scafolds
using syringes. Automated work-up procedures, such as
the removal of solvent, washing and liquid–liquid sepa- The ‘spider-like’ approach increases the chances of find-
rations are also possible. Reactions can be stirred and ing a lead compound which will interact with a target
carried out under inert atmospheres, and the reactions
can be heated or cooled as required.
Structural diversity to increase the
Centroid
or scaffold

chances of success 16.3 Planning and designing a

compound library
Synthesis of ‘spider-like’
molecules (spiderThto eproduce
scaffolds)
techniques of solid phase synthesis have been used
large quantities of compounds from a par-
ticular reaction sequence. These can be stored as com-
The arms containpounddifferent
libraries and then accessed to search for new lead
compounds capable of interacting with novel or existing
functional groupsdrug which
targets. It is are used
Functional groups
important that the molecules in these Substituent
libraries are structurally diverse to increase the chances ‘arms’

to probe a binding site

of success, andfor binding
so some thought has to be put into plan-
ning and designing a compound library.
Planning and designing a compound library
FIGURE 16.11 ‘Spider-like’ molecule.
31

regions Binding
regions

the ‘arms’ should be evenly

spread around the scaffold
Screen compound library
Receptor
Patrick97397.indb 318 binding 11/28/2012 9:16:23 PM
site
 capacity to form hydrogen bonds with target binding sites.
They are easy to synthesize and a large variety of different
substituents are possible by using the amino acid build-
ing blocks. Further substitution is possible on the terminal
amino and carboxylic acid functions. The substituents are
widely distributed along the peptide chain allowing a good
exploration of conformational space. If we consider Lipinski's
rule of five, the peptide scaffold should, ideally, be restricted
to di- and tripeptides in order to keep the molecular weight
below 500. It is interesting to note that the orally active
antihypertensive agents captopril and enalapril are dipeptide-
like, whereas larger peptides, such as the enkephalins, are
and de-protection strategies. Nevertheless, the potential of
sugar-based drugs is so great that a lot of progress has been
made in developing solid phase syntheses based on sugar
scaffolds.
Steroids might appear attractive as scaffolds. However, the
molecular weight of the steroid skeleton itself (314) limits
the size and number of the substituents which can be added
if we wish to keep the overall molecular weight below 500.
The indole scaffold shown suffers a disadvantage in hav-
ing its variable substituents located in the same region of
the molecule, preventing a full exploration of conformational
space (i.e. it is a tadpole-like scaffold).

Me
R4
Privileged scaffolds
O R5

benzodiazepine, hydantoin,
OR5 O
H Me N
R1 C N R4
O C N R4O O R1 O

tetrahydroisoquinoline, biaryls
R3 O OR1 N
R2 O R3 R2
OR2 R1 R2 R3
Dipeptide Glucose Steroid Hydantoin

R2
O Me O O
R1 R2 R3 R3
N R1 R4
HO2C
R3

Remember Lipinski
R3 H2N
R2
N N
X R1 N R2 O R5 N
Ar R1
1,4-Benzodiazepine Pyridine β-Lactam
R2
Indole
di-tri-peptide
R1 R2 N R2
R1 R1 R3
N

Biphenyl Pyrimidine Diphenylmethane

Examples of scaffolds.

that ought to be present, and their relative positions.

16.3.6 Computer-designed libraries
For example, if the target is a zinc-containing protease
(e.g. angiotensin-converting enzyme), a library of com- It has been claimed that half of all known drugs involve
pounds containing a carboxylic acid or thiol group only 32 scaffolds. Furthermore, it has been stated that a
 Research Article

pubs.acs.org/acscombsci

Solid-Phase Synthesis of 4,7,8-Trisubstituted 1,2,3,4-Tetrahydro-

benzo[e][1,4]diazepin-5-ones
Barbora Lemrová and Miroslav Soural*
Department of Organic Chemistry, Institute of Molecular and Translational Medicine, Faculty of Science, Palacky University,
771 46 Olomouc, Czech Republic
*
S Supporting Information

ABSTRACT: Solid-phase synthesis of 1,2,3,4-tetrahydro-

benzo[e][1,4]diazepin-5-ones with use of polystyrene resin
is described. The starting material was polymer supported
1,2-diaminoethane and as a key synthon, 4-chloro-2-fluoro-
5-nitrobenzoic acid was used. The synthetic approach allows
the preparation of derivatives with variable substitution at positions 4 and 8. Additionally, a skeletal diversity was increased when the
nitro group was reduced and some benzene fused heterocycles were prepared. An expansion of a diazepinone to a benzodiazocinone
scaffold was also successful although some limitations in a diversity of target derivatives were observed.
KEYWORDS: benzodiazepinones, benzodiazocinones, thiazines, thiazepines, bisheterocycles, solid-phase synthesis

■ INTRODUCTION
Use of polyfunctional building blocks (i.e., more than three
Eaton reagent9 and subsequent ring expansion by Schmidt
reaction.10 Solid-phase synthesis of some 1,2,3,4-tetrahydro-
benzo[e][1,4]diazepin-5-ones has been published once with use
Parallel synthesis
Parallel synthesis involves the small- Allows for miniaturization
scale synthesis of large numbers of
compounds at the same time using Solid phase extraction Parallel synthesis 323

specialist miniaturized equipment

(solution or solid phase )
It is now an effective method of
producing large numbers of analogues
for drug discovery, studies into
structure–activity relation- ships, and
drug optimization
Quality rather than quantity
Lead optimization

FIGURE 16.15 Laboratory stations for microwave-assisted organic reactions

 FIGURE 16.22 Reduction of an aldehyde with a solid-supported borohydride reagent.
Borohydride reducing agent
NMe3resin
linked to a solid support BH4

FIGURE 16.22 Reduction of an aldehyde with a solid-supported

O borohydride reagent.
Borohydride reducing agent
linked to a solid (CH 2)4 S resin
support

Microwave assisted organic chemistry

O CH 3
O
O with a solid-supported borohydride reagent.
FIGURE 16.22 Reduction of an aldehyde O
OH Solid supported oxidizing
(CH2)4 S agent
used for aOSwern oxidation CH 3
O O
O
FIGURE 16.23
OH Swern oxidation using agent
Solid supported oxidizing
a solid supported reagent.
(CH2)4 S
used for a Swern
O oxidation CH 3
O O
OH 16.23 Swern
FIGURE oxidation oxidizing
using a solid supported reagent.

Amide formation
Solid supported agent
used for a Swern oxidation

FIGURE 16.23 Swern oxidation using

250a!Csolid supported reagent.
H
+ NH2 N
CO2H No solvent
10 mins O
250 !C H
+ NH2 N
CO2H 16.24 Amide formationNo
FIGURE using microwave technology.
solvent
10 mins O
250 !C H
+ NH2 N
CO2H
R" FIGURE 16.24 Amide
HO formationR#
using microwave technology.
No solvent R" R#

Suzuki couplings
10 mins
Pd(OAc)2 O
Cl + B
HO H O
HOAmide formation
FIGURE 16.24 R# using microwave
R" technology.
2
R" 175 !C, 5 min R#
Pd(OAc)2
Cl + B
FIGURE 16.25 A Suzuki
HO coupling carried H
outOunder microwave conditions.
2
R" HO R# 175 !C, 5 min R" R#
Pd(OAc)2
Cl + B
FIGURE 16.25
Mo(CO) A SuzukiHO
coupling carried out H
under
O microwave conditions. NBoc
(a) NO2 6
NH2 (b) 2 NBoc
EtOH 175 !C, 5 minHN
3 Equiv. Cl N
DBU Pd catalyst
FIGURE 16.25 A Suzuki coupling carried out under microwave conditions.

Metal mediated reductions and

Mo(CO)6 NBoc NBoc
(a) NO2 Reduction
EtOH NH 2 (b)
Amination
N N HN
N
3 Equiv. Cl
DBU Pd catalyst

aminations
NO216.26 Microwave-assisted
FIGURE
(a)
Mo(CO)6
NHtransition metal-mediated
(b) reactions. (a) Reductions
NBoc and (b) aminations.
NBoc
Reduction
EtOH 2 Amination
N N HN
3 Equiv. Cl N
DBU Pd catalyst
parallel syntheses
FIGURE 16.26onMicrowave-assisted
microchips (Fig. 16.27 ) usingmetal-mediated
transition a con- each reactions.
reaction extremely accurately.
(a) Reductions and (bAnother advantage of
) aminations.
Reduction
tinuous flow of reactants
N in microfluidic channels.
N Th e microreactors is the potential
Amination to handle a vast number of
channels are designed such that various reactants are parallel reactions on microchips. The channels through
parallel
mixedsyntheses
and
FIGURE on microchips
reacted
16.26 they flow(Fig.
as Microwave-assisted16.27transition
through )the
using a con-
microchip. each
eachreaction
metal-mediatedchip extremely
can
reactions. accurately.
be(afabricated to and
) Reductions Another
(ball
allow advantage
possible
) aminations. of
mixing
tinuous flow of reactants in microfl uidic channels. Th e microreactors is the potential to
Several reactions have already been carried out success- combinations of the various reactants, either on sepa- handle a vast number of
channels are microscale
fully at the designed suchand that
it is various
found that reactants are parallel
many reac- reactions on
rate microchips or microchips. The channelsmicrochip.
on a three-dimensional through
parallel
mixed syntheses onthey
microchips (Fig. 16.27 )microchip.
using a con- each
each reaction extremely accurately. Another advantage of
and reacted as fl ow through the
tion times are shortened from hours to minutes. Some The example in Fig. 16.27 is a simple illustrationmixing
chip can be fabricated to allow all possible of how
tinuous
Several flow ofhave
reactions reactants
alreadyin been
microfl uidic out
carried channels.
success-The combinations
microreactors ofisthe thevarious
potentialreactants,
to handleeither
a vaston
number
sepa- of
Parallel shynthesis in microchips Parallel synthesis 327
Parallel synthesis 327
(a) BD Products
(a) BD Products
BC
Reagents BC
Reagents
D

Reactants are mixed and reacted

D AD
D AD
B D
C B AC
C C AC
C B

as they flow through the

D B
D
B A
B C A
C

microchip
A
A Y-shaped A
Microchips
A Microchips
capillary Y-shaped
capillary
connections
Syringe pumps connections

Reaction times are shortened

Syringe pumps

from hours to minutes

(b) C D
(b) B C
A B
A

Control the temperature

Upper plate
Upper plate

BD
BC BD
AD BC

Vast number of parallel reactions

AC AD
Middle plate AC
Middle plate
C D
CC D

on microchips D
C
D

Bottom plate
Bottom plate B
A B
A
Parallel on-chip gene synthesis and application to
Parallel on-chip gene synthesis and
optimization of protein expression application to
LETTERS

optimization of protein expression

Jiayuan Quan , Ishtiaq Saaem , Nicholas Tang , Siying Ma , Nicolas Negre3, Hui Gong1,
1,2,4 1,2,4 1 1,2 and
JiayuanPQuan1,2,4
3 ,&Ishtiaq Saaem1,2,41,2
, Nicholas Tang1, Siying Ma1,2, Nicolas Negre3, Hui Gong1, selec
Kevin White Jingdong Tian
Kevin P White3 & Jingdong Tian1,2 erro
Surv
Low-cost, high-throughput gene synthesis and precise High-throughput gene synthesis technology has been driven by
Auto
Low-cost,
control ofhigh-throughput
protein expression gene are
synthesis importance to High-throughput
and precise
of critical recent advances geneinsynthesis technologythat
DNA microarrays hascanbeen driven pools
produce by of up to a
control of protein expression are of critical
synthetic biology and biotechnology
importance
1–3. Here we describeto recent advances in DNA microarrays that
million oligonucleotides for gene1,16–19
can produce pools of up to a
assembly1,16–19, albeit in minute quan- 84%
synthetic biology and biotechnology 1–3. Here we describe million oligonucleotides for gene assembly , albeit in minute
5–106 molecules per sequence). The presence of too many quan- nucl
the development of an on-chip gene synthesis technology, tities (~10
the development of an on-chip gene synthesis technology,
tities (~10
5–10 6 molecules per sequence). The presence of too many
which integratesonona single
which integrates a single microchip
microchip the the synthesis
synthesis of of oligo sequences
oligo sequences in a poolin a pool
makes makes ittodifficult
it difficult effectively touse
effectively
the entireuse the entire
the s
DNA oligonucleotides using inkjet printing,
DNA oligonucleotides using inkjet printing, isothermal isothermal oligo pool for gene assembly, as similar sequences
oligo pool for gene assembly, as similar sequences can cross-hybridize. can cross-hybridize. acco
oligonucleotide amplification Practical solutions include more assembly
efficientstrategies
assembly strategies 19,20,
oligonucleotide amplification andand parallel
parallel genegene assembly. Practical
assembly. solutions include more efficient 19,20 , stud
Use of aamismatch-specific
mismatch-specific endonuclease for error selective amplification ofor,
oligos 20 or,here,
as we do here, physical division In situ
Use of endonuclease for error selective amplification of oligos20 as we do physical division com
correction resultsininanan
correction results error
error raterate of ~0.19
of ~0.19 errors
errors of thepool.
per kb. of the oligo
per kb. oligo pool.
To
We applied
We appliedthis thisapproach
approach to to
synthesize
synthesize pools of thousands To effectively
of thousands
pools use all the
To effectively useoligos
all thesynthesized on aFigure
oligos synthesized microarray,
on
1 The weintegratedwe
a microarray, on-chip oligo array synthesis, amplification
of codon-usage variants of lacZ a and 74
of codon-usage variants of lacZa and 74 challengingchallenging divided the whole microarray into subarrays,
divided the whole microarray into subarrays, each containing only
eachassembly
containing only Small pools of oligos are synthesized in expr
and gene process.
Drosophila protein
Drosophila proteinantigens,
antigens,which
whichwere thenthen
were screened for forthe oligos
screened the that arethat
oligos neededare to assemble
needed a longer DNA
to assemble molecule
aseparate
longer DNA of molecule
chambers on a of
plastic DNA microchip using an inkjet DNA of a
expression in Escherichia coli. In one round of synthesis and about 0.5–1 kb in total length. Subarrays are physically isolated from tein,
expression in Escherichia coli. In one round of synthesis and about 0.5–1 kb in total length. Subarrays are physically
microarray isolated from
synthesizer. The chambers are then filled with a combined
screening, we obtained DNA sequences that were expressed the rest of the chip by being located in individual wells, eliminating
screening, we obtained DNA sequences that were expressed
at a wide range of levels, from zero to almost 60% of the
the rest of the chip by being located in individual amplification
the need for post-synthesis partitioning of the oligo pool. Oligos are
wells,and eliminating
assembly reaction mixture and sealed. In a nicking and stud
at a wide range of levels, from zero to almost
total cell protein mass. This technology may facilitate 60% of the the need for post-synthesis partitioning
synthesized on an embossed plastic microchip using a custom-made of the
strand oligo pool.
displacement Oligos are
amplification reaction, a DNA polymerase (Bst large the p
total cell protein
systematic mass.ofThis
investigation technology
the molecular may facilitate
mechanisms synthesized
inkjet DNA microchipon synthesizer
an embossed plastic
21. The microchip
printing area in using
fragment, each showna custom-made
sub- in yellow) extends and displaces the proceeding strand we d
systematic investigation of the molecular mechanisms inkjet DNA microchip synthesizer 21. Thewhile printing
a area
nicking in each sub-
endonuclease (Nt.BstNBI, shown in teal) separates the
of protein translation and the design, construction and array was patterned with 150- m spots of silica thin film to reduce table
of proteinoftranslation
evolution and the
macromolecular design,metabolic
machines, construction and
networks array, was
‘edge-effects’ which patterned
could leadwith 150-oligo
to poor m spots
synthesis construction
of silica
22 . Ourthin
design oligos
film to from
reduce the universal primer (in red) and generates new
allowed‘edge-effects’
a standard 1, which 3 -ends asfor 22. Our design
extension. After amplification, the free oligos in each chamber equa
evolution
and syntheticof macromolecular
cells. machines, metabolic networks × 3 chipcould lead to be
surface poor oligo
divided synthesis
into many
and synthetic cells. as 30 subarrays,
allowed aeach containing
standard 1 × 361 3 chipsilicasurface
spots for are assembled
tosynthesizing
be divided ainto intoasgene
many products by polymerase chain assembly. a lib
A number of regulatory elements, such as promoters4,5 and ribos- uniqueas DNA oligonucleotide sequence. With the setup used
30 subarrays, each containing 361 silica spots for synthesizing a in this E. co
6,7
Example of high-throughput synthesis and
testing
https://www.youtube.com/watch?v=vCO4Dd7kynY