Design and synthesis of DNA-encoded libraries based on a benzodiazepine and a pyrazolopyrimidine scaffold

Article abstract of DOI:10.1039/c6md00243a

Selection-based screening of large DNA-encoded libraries of drug-like small molecules is a validated method to identify bioactive compounds. Among the chemical space of bioactive compounds certain scaffold structures are well represented. These are commonly called "privileged scaffolds". We have synthesized DNA-encoded libraries based on two representatives of these scaffolds, a benzodiazepine and a pyrazolopyrimidine, and additionally a third library based on propargyl glycine. All three core structures possess a carboxylic acid to couple them to aminolinker-modified DNA. For subsequent library synthesis they contained an amino function to which a set of carboxylic acid building blocks were coupled, and a terminal alkyne that was reacted with a set of azides to furnish triazoles. The two sets of building blocks, 114 carboxylic acids and 104 azides, were selected with the help of chemoinformatic methods in order to control the physicochemical properties of the final libraries, remove unwanted substructures, and maximize diversity. The set of building blocks contained desthiobiotin allowing for validation of library synthesis. The DNA-encoded libraries were synthesized by split-and-pool combinatorial chemistry yielding three libraries that contain 28.254 compounds together. For DNA barcoding, 5′-phosphorylated double-stranded coding DNA sequences with four base overhangs were ligated with T4 ligase. The resulting DNA-encoded libraries were compared to bioactivity databases and, though being based on core structures well-established in medicinal chemistry, showed novelty with respect to the known bioactive chemical space.

Full text of DOI:10.1039/c6md00243a

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Design and synthesis of DNA-encoded libraries based on a
benzodiazepine and a pyrazolopyrimidine scaffold‡
M. Klika Škopić,a† O. Bugain,a† K. Jung, a S. Onstein,a S. Brandherm,a T. Kalliokoski,b and A.
Brunschweigera*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Selection-based screening of large DNA-encoded libraries of drug-like small molecules is a validated method to identify
bioactive compounds. Among the chemical space of bioactive compounds certain scaffold structures are well represented.
These are commonly called “privileged scaffolds”. We have synthesized DNA-encoded libraries based on two
representatives of these scaffolds, a benzodiazepine and a pyrazolopyrimidine, and additionally a third library based on
ethynyl glycine. All three core structures possess a carboxylic acid to couple them to aminolinker-modified DNA. For
subsequent library synthesis they contained an amino function to which a set of carboxylic acid building blocks were
coupled, and a terminal alkyne that was reacted with a set of azides to furnish triazoles. The two sets of building blocks,
114 carboxylic acids and 104 azides, were selected with the help of chemoinformatic methods in order to control the
physicochemical properties of the final libraries, remove unwanted substructures, and maximize diversity. The set of
building blocks contained desthiobiotin allowing for validation of library synthesis. The DNA-encoded libraries were
synthesized by split-and-pool combinatorial chemistry yielding three libraries that contain 28.254 compounds together.
For DNA barcoding, 5’-phosphorylated double-stranded coding DNA sequences with four base overhangs were ligated with
T4 ligase. The resulting DNA-encoded libraries were compared to bioactivity databases and, though being based on core
structures well-established in medicinal chemistry, showed novelty with respect to the known bioactive chemical space.
www.rsc.org/
connected. One of the archetypal privileged scaffolds is the
which can be found in
5
benzodiazepine core
5
(
figure 1
)
Introduction
hundreds of bioactive compounds, among them natural
products such as asperlicin C, clinical candidates, and approved
drugs. An exemplary structure of this compound class is the
clinically evaluated farnesyltransferase inhibitor BMS214662
The selection of large, pooled DNA-encoded libraries (DELs) of
drug-like small molecules is a validated method for the target-
based identification of bioactive compounds.1 It is an
operationally simple, economic screening technology that
(2,
figure 1A).6
allows to interrogate large chemical space in
a single
experiment. The method holds promise to make a contribution
to the validation of the plethora of putative drug targets
emerging from the “–omics” technologies.2 Indeed, several
valuable chemical biology probes that aid in gaining a deeper
understanding of pathophysiology trace their ancestry to the
screen of a DEL.3 Analysis of the chemical space of bioactive
compounds has revealed that certain scaffold structures are
highly represented.4 In the literature these are often referred
to as “privileged scaffolds”. A scaffold is here defined as the
core element of a molecule to which the substituents are
Place figure 1 here
Figure 1: (A) The approved drug ibrutinib 1 and the clinical
candidate BMS214662
the core structures
encoded library synthesis
based on the structures
2
; (
C
B
) reduction of these compounds to
) core structures functionalized for
; ( ) DNA-encoded libraries 11
3-
5; (
6
-
8
D
9-
6
-
8.
While the benzodiazepine core targets proteins from diverse
families thus meeting the initial definition of the term
“privileged”, i.e. a structure capable of binding multiple
targets,5 the pyrazolopyrimidine
4, exemplified by the
, falls into a different
approved kinase inhibitor ibrutinib 1 7
class of privileged scaffolds. This structure resembles the
nucleobase adenine and is thus biased towards adenine (-
nucleotide) binding sites, e.g. in kinases.8,9
Several strategies have been developed for the synthesis of
DNA-encoded libraries.1,3 These libraries can be furnished in a
templated manner,10 exemplified by synthesis of encoded
libraries of macrocycles and the yoctoreactor approach.
However, templated synthesis requires coupling of all building
blocks to oligonucleotides prior library synthesis, and building
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blocks used for library synthesis need to be bifunctional. Also, evaluation of these building blocks and the synthesis of three
oligonucleotides can be used to encode and to template encoded libraries 11 figure 1D) that each consist of a central
9
-
(
building blocks for fragment screening, e.g. in the ESAC scaffold structure serving as vector to project two sets of
(Encoded Self-Assembling Chemical libraries) format.11 The building blocks.
most common format for DNA-encoded library synthesis is the
combinatorial split-and-pool approach with iterative synthesis
and encoding steps.12 The synthesis steps are recorded by
chemical or enzymatic DNA-ligation techniques; Klenow fill-in
Results and discussion
Synthesis of trifunctionalized tetrahydrobenzodiazepine and
is an efficient method to encode libraries composed of two
pyrazolopyrimidine scaffolds
building blocks.12,13 The present libraries were planned to
The functionalized pyrazolopyrimidine
7 was synthesized in
consist of three building blocks. Each contains a central
scaffold serving as vector for two sets of building blocks, thus
enzymatic ligation of double-stranded, 5’-phosphorylated DNA
sequences using T4 DNA ligase which efficiently connects was
chosen for encoding. There is only a limited repertoire of
organic synthesis reactions available that are amenable for DEL
synthesis: mostly, carbonyl reactions such as amide bond or
(thio)urea formation and reductive amination, C-C cross
coupling reactions, e.g. the Suzuki reaction, nucleophilic
(aromatic) substitution of reactive halides, and the Cu(I)-
catalyzed azide-alkyne cycloaddition were used for library
synthesis.1,3 These reactions allow for appendage of building
blocks to properly functionalized structures. Synthesis
strategies to substituted (hetero)cyclic structures from simple
starting materials are less established for DNA-encoded library
synthesis, and encompass for instance the Diels-Alder reaction,
condensation reactions leading e.g. to benzimidazoles and
straightforward manner from 6-aminopyrazolopyrimidine 12 (figure
2A). A Mitsunobu reaction with protected serine introduced the
required carboxylic acid and amine functionalities to the core
heterocycle, and iodination of the heterocycle and Sonogashira
coupling with TMS-protected acetylene introduced the terminal
alkyne moiety, yielding compound 16 in a few steps. Protective
group chemistry then gave the properly substituted and protected
scaffold 7.9 However, in the basic deprotection step to compound
16a (figure 2A, and SI part) that removed both the TMS-group and
the tert-butyl ester, we noticed racemization of the amino
substituent. The tetrahydrobenzodiazepine was accessed through a
somewhat lengthier synthesis route that started with the
bromination of isatoic anhydride 17 (figure 2B), the condensation of
this product (18) with glycine methylester to the core heterocycle
19, and the reduction of the carbonyl groups of 19 yielding the
tetrahydrobenzodiazepine structure 20.6,10 This was N-protected
with a boc-group and reacted with acrylic acid tert-butylester by
Heck reaction to introduce the carboxylic acid function. Protection
of the amino function was needed to prevent N-alkylation by the
acrylic acid ester. The Heck reaction was followed by hydrogenation
of the double bond to remove the α,β-unsaturated Michael
acceptor function (23). Alkylation of the aryl amine with propargyl
bromide introduced the terminal alkyne. Finally, protective group
chemistry furnished the target compound 8.
imidazolidinones, and lately also
spirocyclic structures.14-16
a cascade reaction to
For the synthesis of the present libraries, compounds
were reduced to their core pyrazolopyrimidine structure
benzodiazepine figure 1B), respectively. They, and the
amino acid , which served initially to develop the library
1
and
2
4
and
5
(
6
synthesis strategy, display functionalities for encoded library
synthesis with DNA-compatible preparative organic synthesis
methods (7, 8, figure 1C): a carboxylic acid to couple the
scaffolds to 5’-aminolinker modified DNA, an Fmoc-protected
amine for appendage of carboxylic acid building blocks by
amide coupling, and a terminal alkyne for appendage of azide
building blocks by Cu(I) catalyzed azide-alkyne cycloaddition
(CuAAC), respectively. Two orthogonal reactions were chosen
for library synthesis as this obviated the requirement of
additional protective group chemistry. Amide synthesis is a
workhorse reaction in the synthesis of DELs for which
thousands of carboxylic acid building blocks are available.18-20
The CuAAC was less employed in reported DEL synthesis,
although this reaction is compatible with DNA,21 has been
extensively used in the synthesis of bioactive compounds,22-25
has broad functional group tolerance, does not demand
protective group chemistry, and is a high-yielding reaction,
which is important for library quality, i.e. an even distribution
of the individual library members.26 Moreover, although only
few azides are available,20 they are readily accessible from
abundantly available precursors such as aryl amines and
halides.27 In situ synthesis of reactants was described in only a
few reports on DNA encoded library synthesis.14,26,28 To the set
of building blocks elected for library synthesis we added the
streptavidin binder desthiobiotin to validate library
synthesis.12b
Place figure 2 here
Figure
combinatorial DEL-synthesis. (
. Reagents and conditions: a) N-iodosuccinimide, DMF, 80 °C, 14 h; b)
2
:
Synthesis of privileged scaffolds functionalized for
A
) Synthesis of aminopyrazolopyrimidine
7
PPh3, DIAD, THF, rt, 16 h; c) Pd(PPh3)4, CuI, Et3N, TMS-acetylene, DMF,
rt, 18 h; d) K2CO3, MeOH, rt, 16 h; e) TFA, DCM, rt, 16 h; f)
NaHCO3,Fmoc-OSu, H2O, 1,4-dioxane, rt, 16 h.
(B) Synthesis of
tetrahydrobenzodiazepine 8. Reagents and conditions: a) bromine in
H2O at 50 °C for 1 h; b) glycine in H2O, Et3N, rt, 4 h; c) BH3 in dry THF,
reflux, 18 h; d) “diboc” in dry MeOH, rt, 18 h; e) Pd(OAc)2, P(O-tol)3,
tert-butyl acrylate, Et3N, dry CH3CN, 100 °C, 18 h; f) Pd/C, dry MeOH,
rt, 18 h; g) Cs2CO3, propargyl bromide, dry DMF, 60 °C, 18 h; h) TFA in
dry CH2Cl2, rt, 19 h; i) Fmoc-Osu, NaHCO3, H2O, dry 1,4-dioxane.
Chemoinformatics-supported selection of carboxylic acid and
azide building blocks for library synthesis
The scaffolds 6-8 allow for combinatorial substitution with building
blocks by amide synthesis and by Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC). Both reactions are high yielding and have a
Here, we show the synthesis of compounds 7 and 8, the
chemoinformatics-supported selection of building blocks, the
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broad scope, two properties that are important for sampling by the three scaffolds and the selected building blocks and then
chemical space, and also for library quality.1,3,18,19,26
calculating the nearest-neighbour (NN) similarity of these structures
using Morgan- and Feat Morgan-fingerprints to the bioactivity
database ChEMBL 21 (~2 million compounds), the patent database
SureChEMBL (April 2016 edition, ~16 million compounds) and the
bioactivity database PubChem (March 2016 edition, ~89 million
compounds). The NN-Tanimoto similarities from these large
databases to the generated library are shown in table 1. For
instance, the nearest neighbour to library 10, which is based on the
aminopyrazolopyrimidine, an adenine-mimicking scaffold often
found in kinase inhibitors, is in the ChEMBL database compound
CHEMBL2023774. This compound displays the same 1,3-
disubstituted aminopyrazolopyrimidine core scaffold, and inhibited
the Src kinase.35 However, the maximum Morgan/Feat Morgan-
Tanimoto-similarity of the chemical space described in the
bioactivity databases to the libraries 9-11 is generally rather low.
Place figure 3 here
Figure 3: Chemoinformatic filtering cascade to select from a large pool of
commercially available building blocks a diverse set of carboxylic acids and
halides endowing the projected DELs with defined properties.
However, as only a limited number of azides are commercially
available,20 we chose to use aliphatic and benzylic halides, and to
convert these in situ into azides.27 There are thousands of
aliphatic/benzylic halides and carboxylic acid building blocks
available for library synthesis,20 so in order to facilitate the building
block selection, we applied a chemoinformatic filtering cascade to a
database of commercially available chemicals. For the calculation of
the physicochemical properties the free carboxylic acid of
compounds 6-8 was substituted with an ethyl amide. In the first
step, we removed those building blocks that would yield library
members with physicochemical properties outside pre-defined
values: the calculated logP of all library members was to fall in to
the range of -2 – 5, and the molecular weight was not to exceed 650
Da including the linker structure connecting the core structure to
the DNA barcode. In the second step, we removed building blocks
that may show unwanted reactivity, for instance redox reactions or
covalent reactions with the DNA barcode or the target (PAINS).29
Then, from the remaining two sets of building blocks, we made a
selection of each 150 chemicals with RDKit diversity picker using
Morgan fingerprints in order to maximize the diversity of the
screening library.30,31 Finally, the collections of carboxylic acids and
halides were manually curated by removing overly expensive
compounds, and those that would likely not react or give rise to
side products, for instance acids with an unprotected aliphatic
amino group, dicarboxylic acids, or dihalides. We finally arrived at a
set of 114 carboxylic acids (compounds A-DF, table S1, supporting
information) and 104 halides (compounds 1-104, table S1,
supporting information) for the synthesis of the DNA-encoded
libraries 9-11 (figure 1). Within both sets of building blocks,
aliphatic, cyclic aliphatic, aromatic and heteroaromatic structures,
Surprisingly, it is much higher to library
9 than to the
pyrazolopyrimidine- and benzodiazepine-based libraries 10 and 11.
This can be judged either beneficial, as these libraries cover novel
chemical space which is desirable for a screening collection, or
detrimental, as the DNA-compatible chemistry used to substitute
the scaffolds 4 and 5 might bias the libraries towards chemical
space that was recently described as “dark chemical matter”,
compound classes in screening library collections that rarely show
biological activity.36
Table 1: Analysis of the calculated physicochemical properties1 of DNA-
encoded libraries 9-11 and nearest neighbours (NN)2 to these libraries in
public databases.
Descriptor
Library 9
CHEMBL2023-
774 (0.688)
SCHEMBL1262
7717 (0.750)
98041443
(0.780)
Library 10
CHEMBL1683-
305 (0.597)
SCHEMBL1316
0649 (0.605)
44235677
(0.685)
Library 11
NN-ChEMBL
CHEMBL1922-
546 (0.569)
NN-Sure-ChEMBL
SCHEMBL1328
8590 (0.600)
NN-Pub-Chem
42499651
(0.645)
Mean MW
Mean ClogP
Mean Fsp3
433 Da
566 Da
578 Da
3.6
1.3
1.2
0.483
120 Å2
0.387
190 Å2
0.454
115 Å2
most of them substituted with heteroatoms, some of them Mean TPSA
Mean NROT
Mean HBA
Mean HBD
10
7
3
11
12
4
11
8
2
displaying functional groups, are presented. Inspection of these
fragment-sized building blocks (tables S1 and S2) revealed a
number of structures that can be found in bioactive compounds:
For instance, the benzopyrazole AI (table S1) is a fragment binding
to the kinase CDK2;32 dihydrouracil BK was identified as binding
motif for members of the family of PARP enzymes from a screen of
a DNA-encoded library,33 and the uracil BN is a feature of a
compound binding to the activated complement factor C3d.34
Other fragments as for instance the indole R, the benzofuran AC
and the benzimidazole 75 (table S2) are found in numerous
bioactive compounds. Although the projected DNA-encoded
libraries 9-11 are based on scaffolds that constitute core structures
of several bioactive compounds, and include several fragments that
are well presented in bioactive molecules, they sample chemical
space that is hitherto not covered. The novelty of the compounds
was assessed by first enumerating all of the compounds produced
1mean molecular weight (MW), calculated logP (ClogP), fraction of the sp3-
carbons (Fsp3), topical polar surface area (TPSA), number of rotatable bonds
(NROT), number of hydrogen bond acceptors (HBA) and number of
hydrogen bond donors (HBD). All of these values were calculated using the
combination of RDKit and ChemFP
2 maximum Morgan/Feat Morgan-Tanimoto-similarity in parenthesis.
The properties of the three libraries were analyzed by typical
descriptors that were found statistically associated with peroral
bioavailability (table 1):37,38 molecular weight, calculated logP to
assess the mean lipophilicity of the libraries, the topological surface
area, the number of rotatable bonds, the number of hydrogen bond
donors and acceptors (mean values given). The fraction of sp3-
hybridized carbon atoms has been suggested as a metric associated
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with clinical success.39 While the mean values of all parameters of
Journal Name
the 1 µmol-scale (see supporting information). Prior removal
of the protective group, unreacted amines were capped with
acetic acid anhydride. We found both MMt-amino-PEG(8)-
linker and Fmoc-protected amino-PEG(4)-linker suitable (table
S1). However, prolonged deprotection of the Fmoc-group with
piperidine/DMF led to formation of a lipophilic side product.
This side product was likely due to a transamidation reaction
and has also been noticed by others.43 Reducing the
deprotection time to 5 minutes suppressed formation of this
side product effectively. In the next step, compounds 6-8 were
coupled to the DNA-PEG-linker conjugates 25 (from the MMt-
protected PEG-linker) and 26 (from the Fmoc-protected PEG-
linker) on the 1 µmol-scale, unreacted amines were again
capped, and the Fmoc-group of the scaffolds was removed
with piperidine/DMF. Library synthesis continued with the
library fall into ranges statistically associated with peroral
bioavailability, libraries 10 and 11 that are based on larger scaffold
structures show higher mean molecular weight and the
9
a
aminopyrazolopyrimidine library 10 has a higher mean topical polar
surface area.
Large diversity in the shapes of the molecules is also a desired
feature in a screening library.40,41 Here, the shape diversity of the
libraries was investigated by first generating single low-energy
conformation for each of the library compounds using Schrödinger
Suite 2016.1 and then calculating two 3D-diversity metrics for the
molecules: the normalized principal moments of inertia ratios
(NPRs) and Plane of Best Fit score (PBF score). Briefly, NPRs are
numeric values that describe the overall three-dimensional shapes
of the library molecules. When these values are plotted against
each other, they form a triangle where the corners represent rods,
spheres and disks (for further information, see ref. 40). PBF score
describes how different conformation of a molecule is from its 2D
representation. It is a value of usually between zero and two for
drug-like molecules, the higher value indicating higher 3D character
(additional information is available in ref 41) The NPR-plots show
clearly that all libraries 9-11 cover a wide range of shape diversity as
libraries 9-11 all focus on different regions of NPR-space (figure
S17). In addition, most of the compounds have PBF-score above 1,
which indicates a high 3D-character for the DEL library (figure S18).
parallel coupling of the carboxylic acid building blocks
A-DJ
(
table S1). It was most convenient to couple sets of 20
carboxylic acids to the three DNA conjugates 27-29, i.e.
performing in total 60 reactions in parallel. Each solid phase
(400 nmol, ca. 16 mg) containing the conjugates 27-29 was
split in 20 nmol aliquots into a 96well plate by suspending in
the bulk solid phase in DMF and splitting the suspension (see
supporting information). Then, 20 carboxylic acid building
blocks (table S1) were coupled in parallel to each DNA scaffold
conjugate using HATU as coupling reagent. The solid support-
bound DNA conjugates were transferred to a 96well filter
plate, thoroughly washed, and deprotected and cleaved from
the CPG with aq. ammonia/methylamine on the filter plate
which was connected to a receiver plate and sealed for this
purpose. The conjugates were then purified by ion pair reverse
phase HPLC. Evaluating this process after coupling two sets of
carboxylic acid building blocks, i.e. 40 carboxylic acids, we
Synthesis of the DNA-encoded libraries 9-11
The synthesis of the DNA-encoded libraries 9-11 was
performed as outlined in figure 4: Scaffold structures
6-8
(figure 1) were coupled to a fully protected, solid phase-bound
5’-aminolinker modified single-strand DNA sequence, and the
Fmoc-group was removed (27-29).19,33 Then, 114 carboxylic
noted that ten (H, W, AC, AD, AE, CW, CX, CY, CZ, DA, table S1)
of these building blocks did not yield the target amide
products. As efficient Fmoc-peptide chemistry has been
reported for the synthesis of peptide conjugates of shorter
solid support-bound DNA oligonucleotides,43 we changed the
initial sequence from a 23mer DNA to a shorter 14mer. This
shorter DNA-sequence allowed us to obtain amide coupling
acid building blocks
A-DJ (table S1) were reacted with the
three DNA-conjugates 27-29. All conjugates 27A-DJ – 29A-DJ
were cleaved from the solid phase and each DNA-conjugate
was purified to a single peak by ion pair reversed phase HPLC
in order to synthesize the DNA-encoded library from a uniform
set of DNA-conjugates which we deemed beneficial for library
quality.26 These purified conjugates were then encoded with
double-stranded DNA sequences by two successive DNA
ligation reactions with T4 ligase. The library synthesis was
concluded with appendage of a second set of azide building
products from five of the ten carboxylic acid building blocks (
H,
W
,
AC, AD, AE). However, lack of detection of an amide
coupling product could either be due to low reactivity of the
carboxylic acid or to susceptibility of the amide bond to
hydrolytic cleavage in the deprotection step.
blocks 1-104 (table S2) by copper(I)-catalyzed alkyne-azide
cycloaddition on DEAE sepharose.27,42
Having coupled the whole set of 114 carboxylic acid building
blocks
A-DJ (table S1) to the three DNA-conjugates 27-29 we
obtained 99 products for the DNA-amino acid conjugate 27, 84
Place figure 4 here
Figure 4: Synthesis of the DEL by a two stage synthesis strategy. a) products for the DNA-pyrazolopyrimidine conjugate 28, and 94
products for DNA-benzodiazepine conjugate 29. For a statistic
see figure S1. The coupling efficiencies were very variable,
while the amino acid 27 and the secondary amine of the
benzodiazepine 29 yielded more than 80 of the target amides
with good conversion rates, the pyrazolopyrimidine gave only
52 amides in acceptable to good yields. In hindsight, the
varying coupling efficiency of the carboxylic acids justified the
effort to purify and isolate every conjugate. HPLC analysis
indicated a purity of more than 95 % of the ion pair
HATU; b) piperidine in DMF; c) aq. NH3/aq. MeNH2; d) encoding by
T4 DNA ligation; e) NaN3 in DMF; f) DEAE sepharose, Cu(I), TBTA,
Na-ascorbate.
Library synthesis was initiated with coupling of a protected
amino-PEG-carboxylic acid using HATU as coupling reagent to a
solid phase-coupled 5’-C6-aminolinker modified 23mer DNA
containing the primer and scaffold code by amide synthesis on
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chromatography-purified conjugates 27A-CV – 29A-CV and A chemically synthesized 69mer dsDNA VI
MALDI MS analysis confirmed the identity of the products served as surrogate of the DNA-encoded library was used to
table S1, exemplary HPLC traces of amide coupling products: evaluate the primer efficiency. Even a high concentration of
figures S19 S28). Interestingly, while most building blocks gave 100 pM template DNA VI VI´ required more than 10 cycles of
a shift to longer retention times, a number of building blocks amplification with its primer pair VIII IX table S8) to detect
caused a shift to shorter retention times. These were polar initiation of amplification with SYBR green, and 1 pM template
structures such as the primary amide and the dihydrouracil required 17 cycles to detect initiation of amplification, while
BL. Thus, through HPLC-purification and isolation of the DNA- with the primer pair VIII IX table S8) alone we detected
/VI´ (table S8) that
(
-
/
/
(
H
/
(
conjugates 27A-CV – 29A-CV we obtained a uniform set of 277 initiation of amplification already at 21 cycles (figure S9a) due
DNA-small molecule conjugates for encoding and to formation of primer dimers. We therefore tested a number
combinatorial library synthesis.
of primer pairs, arriving at the sequences
X/XI which amplified
The second set of building blocks was introduced by Cu(I)- its template DNA VII
/
VII´ much more efficiently (figure S9b
)
catalyzed azide-alkyne cycloaddition (CuAAC). As the analysis and showed no formation of primer dimers over 30 cycles of
of complex mixtures of hundreds of DNA conjugates is not a amplification. Thus, an optimized ligation protocol and
trivial task, we evaluated the reactivity of the in situ efficient primer pairs were established. These were confirmed
synthesized azides with a DNA-alkyne conjugate 30
(
table S2
)
by comparison of the amplification plots (figure S12) of equal
prior library synthesis.12a,33 Each 400 pmol of DNA-alkyne amounts of the chemically synthesized reference template
conjugate 30 was immobilized on DEAE sepharose in 96well DNA VII/VII´ (table S8) and the DNA duplex 29CP-XVI/XVII
plates,40 while the azides were prepared by substitution of the (sequence: see table S10) containing the same primer and
halides with NaN3/tetrabutylammonium iodide.27 The coding sequence that was accessed from the desthiobiotin-
cycloaddition reaction was then performed according to a substituted benzodiazepine DNA-conjugate 29CP (table S1)
previously established procedure with a 2500fold excess of the through the optimized ligation protocol (figures S9, and S10,
azide at 45°C for 16 h.27 Prior to elution of the triazole sequences see table S10, for the ligation scheme see figures
products, the resin was extensively washed to remove the S13 and S14).
excess of reactants and reagents. A 1N buffered aqueous With the optimized conditions for T4 DNA ligation, a validated,
solution of EDTA was used to remove Cu-ion contaminants as efficient pair of primers, a purified set of DNA-small molecule
these might compromise DNA stability during storage. The conjugates 27A
conjugates were analyzed by MALDI MS (table S2). From the building blocks in hand we synthesized the three DNA-encoded
104 azides tested in this experiment, 102 yielded the triazole libraries 11 figure 4). In the first encoding step, an amount
products, for a statistical analysis, see figure S1. For 82 azides of 40 pmol of each purified and characterized DNA conjugate
complete conversion was detected by MALDI MS, while in case 27A-CV 29A-CV was ligated in one pot with 5’-
of azide building blocks 10 16 38 44 51 53 62 68 75 76 phosphorylated dsDNA that contained the optimized primer
77 101 102 and 104 we noticed incomplete conversion, yet sequence and the code of the scaffold, and 5’-phosphorylated
-CV - 29A-CV, and a validated set of azide
9-
(
–
a
,
,
,
,
,
,
,
,
,
,
,
,
the yield for these building blocks exceeded 50% as estimated short 12mer dsDNA sequences encoding the building blocks A-
by MALDI MS analysis which we judged sufficient for library CV with T4 DNA ligase (scheme for encoding: figures S13 and
synthesis.12a These building blocks are mostly heterocyclic S14). The ligation reactions from each scaffold 27
-
29 were
and 87 did not yield the pooled and the DNA was precipitated with 70 % aqueous
target triazoles at all. We then tested also a set of azide ethanol for overnight. The pellets were re-dissolved, an aliquot
building blocks with a DNA conjugate of the amino acid 31 of each pooled library was taken and analyzed to confirm
see supporting information, figure S29 and detected successful ligation (figure S15) and again precipitated for two
quantitative conversion to the target triazoles by CuAAC. hours with 70 % aqueous ethanol. The pellet was dissolved in
The target DNA-encoded libraries 11 were encoded by T4 water, and split into 102 wells of two 96well plates, and, after
structures. However, only azides
6
6
(
,
)
9
-
ligation of 5’-phosphorylated dsDNA containing overhangs.12a,c 5’-phosphorylation with polynucleotide kinase, ligated with a
The conditions for enzymatic ligation were optimized with set of 102 dsDNA sequences containing the code for the azide
dsDNA sequences (figures S2 and S7, tables S5 and S6) that building blocks 1-102 and the reverse PCR primer. These 102
contained tetramer overhangs. Several parameters were dsDNA sequences were added in 2fold excess to drive the
tested in combinatorial manner: ligation time, temperature, ligation to completion. The encoded DNA-conjugates were
different buffer systems, ratio of 5’-phosphorylated to non- directly transferred to DEAE sepharose and reacted with the
phosphorylated counter strand, and concentration of the T4 azides
1-102 as described above. After the reaction, the library
ligase (table S4, figures S3-S6 and S8). Suitable conditions for was eluted from the resin, pooled, and twice precipitated. The
ligation of dsDNA sequences were found for encoding of the pellet was dissolved, and an aliquot was analyzed by gel
DNA-encoded libraries: equal amounts of 5’-phosphorylated to analysis (figure S16) and by qPCR (table S9). The qPCR analysis
non-phosphorylated counter strand, use of
a
10fold indicated a loss of one ct-value, as compared the chemically
VII’, i.e. a loss of 50% of
concentrated buffer that allowed for higher concentrations of synthesized reference DNA VII
/
the enzyme and the dsDNA, and a ligation temperature of amplifiable DNA. This loss might be due to some degradation
25°C. The dsDNAs were ligated either for 4 hours or overnight of the DNA because of Cu(I)-mediated oxidation and
with equal efficiency.
fragmentation.
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selection on its target protein streptavidin.12b Currently, we are
using the synthesized DNA-encoded libraries to identify novel
binders for target proteins.
Place figure 5 here
Figure 5: qPCR-analysis of the selection of the synthetic
reference dsDNA VII/VII´ versus the encoded compound 29CP-
XVI/XVII on streptavidin beads.
Acknowledgements
This work was supported by the German Federal Ministry of
Education and Research (BMBF) Grant 131605. ChemAxon is
acknowledged for the academic license for some of the
software used in the chemoinformatics filtering cascade
(http://www.chemaxon.com). The authors declare no
competing interest.
Finally, the encoded desthiobiotin conjugate 29CP-XVI/XVII
and the chemically synthesized non-modified DNA VII/VII´
were used to establish the selection assay with streptavidin
beads as a model system.12b Both the protein binder 29CP-
XVI/XVII and the negative control dsDNA VII/VII´were
incubated at a 100 pM concentration with the beads. The
beads were washed eight times with washing buffer, and then
heat-denatured to release the oligonucleotides. These were
incubated with a fresh batch of streptavidin beads and again
washed eight times with washing buffer. The desthiobiotin
conjugate was recovered with only a minor loss of DNA (- 0.3
ct values) while the amount of the non-modified DNA was
reduced by 5 ct values, i.e. by ca. 97 %. In other words, the
binder was enriched by a factor of 23 (figure 5).
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(A) The approved drug ibrutinib 1 and the clinical candidate BMS214662 2; (B) reduction of these
compounds to the core structures 3-5; (C) core structures functionalized for encoded library synthesis 6-8;
(D) DNA-encoded libraries 9-11 based on the structures 6-8.
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Chemoinformatic filtering cascade to select from a large pool of commercially available building blocks a
diverse set of carboxylic acids and halides endowing the projected DELs with defined properties.
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Synthesis of privileged scaffolds functionalized for combinatorial DEL-synthesis. (A) Synthesis of
aminopyrazolopyrimidine 5. Reagents and conditions: a) N-iodosuccinimide, DMF, 80°C, 14 h; b) PPh3,
DIAD, THF, rt, 16 h; c) Pd(PPh3)4, CuI, Et3N, TMS-acetylene, DMF, rt, 18 h; d) K2CO3, MeOH, rt, 16 h; e)
TFA, DCM, rt, 16 h; f) NaHCO3,Fmoc-OSu, H2O, 1,4-dioxane, rt, 16 h. (B) Synthesis of
tetrahydrobenzodiazepine 6. Reagents and conditions: a) bromine in H2O at 50 °C for 1 h; b) glycine in
H2O, Et3N, rt, 4 h; c) BH3 in dry THF, reflux, 18 h; d) “diboc” in dry MeOH, rt, 18 h; e) Pd(OAc)2, P(O-
tol)3, tert-butyl acrylate, Et3N, dry CH3CN, 100°C, 18 h; f) Pd/C, dry MeOH, rt, 18 h; g) Cs2CO3, propargyl
bromide, dry DMF, 60°C, 18 h; h) TFA in dry CH2Cl2, rt, 19 h; i) Fmoc-Osu, NaHCO3, H2O, dry 1,4-dioxane.
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Synthesis of the DEL by a two stage synthesis strategy. a) HATU; b) piperidine in DMF; c) aq. NH3/aq.
MeNH2; d) encoding by T4 DNA ligation; e) NaN3 in DMF; f) DEAE sepharose, Cu(I), TBTA, Na-ascorbate.
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qPCR-analysis of the selection of the synthetic reference dsDNA VII/VII´ versus the encoded compound
29CP-XX/XXI on streptavidin beads.