DNA-Encoded Libraries: Hydrazide as a Pluripotent Precursor for On-DNA Synthesis of Various Azole Derivatives

Article abstract of DOI:10.1002/chem.202100850

DNA-encoded combinatorial chemical library (DEL) technology, an approach that combines the power of genetics and chemistry, has emerged as an invaluable tool in drug discovery. Skeletal diversity plays a fundamental importance in DEL applications, and relies heavily on novel DNA-compatible chemical reactions. We report herein a phylogenic chemical transformation strategy using DNA-conjugated benzoyl hydrazine as a common versatile precursor in azole chemical expansion of DELs. DNA-compatible reactions deriving from the common benzoyl hydrazine precursor showed excellent functional group tolerance with exceptional efficiency in the synthesis of various azoles, including oxadiazoles, thiadiazoles, and triazoles, under mild reaction conditions. The phylogenic chemical transformation strategy provides DELs a facile way to expand into various unique chemical spaces with privileged scaffolds and pharmacophores.

Full text of DOI:10.1002/chem.202100850

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doi.org/10.1002/chem.202100850
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DNA-Encoded Libraries: Hydrazide as a Pluripotent
Precursor for On-DNA Synthesis of Various Azole
Derivatives
Fei Ma,[a] Jie Li,[a] Shuning Zhang,[a] Yuang Gu,[a] Tingting Tan,[a] Wanting Chen,[a]
Shuyue Wang,[a] Peixiang Ma,[a] Hongtao Xu,[a] Guang Yang,*[a] and Richard A. Lerner*[b]
Abstract: DNA-encoded combinatorial chemical library (DEL)
technology, an approach that combines the power of
genetics and chemistry, has emerged as an invaluable tool in
reactions deriving from the common benzoyl hydrazine
precursor showed excellent functional group tolerance with
exceptional efficiency in the synthesis of various azoles,
including oxadiazoles, thiadiazoles, and triazoles, under mild
reaction conditions. The phylogenic chemical transformation
strategy provides DELs a facile way to expand into various
unique chemical spaces with privileged scaffolds and pharma-
cophores.
drug discovery. Skeletal diversity plays
a fundamental
importance in DEL applications, and relies heavily on novel
DNA-compatible chemical reactions. We report herein a
phylogenic chemical transformation strategy using DNA-
conjugated benzoyl hydrazine as a common versatile pre-
cursor in azole chemical expansion of DELs. DNA-compatible
Introduction
protein 1 (RIP1) inhibitor GSK3145095 for pancreatic cancer,[5]
RIP1 kinase inhibitor GSK2982772 for autoimmune diseases,[6]
soluble epoxide hydrolase (sEH) inhibitor GSK2256294 for
diabetes,[7] and selective sphingosine-1-phosphate (S1P) recep-
tor agonist GSK18427099 for multiple sclerosis, etc.[8]
In 1992, an elegant approach using DNA barcoding technology,
a.k.a DNA-encoded combinatorial chemical libraries (DELs), was
first described by Lerner and Brenner,[1] in which genetics and
chemistry are linked to track and playback the complete
transformation processes of chemical molecules. The unique
feature of DELs has gained wide attention in both the
pharmaceutical industry and academia.[2] DELs make use of the
combination of molecular engineering, combinatorial synthesis,
deep-sequencing and advanced informatics, including machine
learning, to enable the creation and screening of enormously
diverse new chemical entities at an unprecedented level in a
short period of time. The numerous number of structurally
diverse molecules in DELs endows the technology with great
potential for hit identification of drug targets with exquisite
specificity through library interrogation.[3] DELs consisting of
large numbers (millions to billions) can be readily constructed
using a strategy called “split and pool”,[4] and screened against
a broad spectrum of validated protein targets via affinity-based
selection. Serval drug candidates have since been discovered
and entered into clinical studies, including receptor interacting
As an on-going effort to improve the diversity and quality
of DELs, a number of labs, including ours, have been working to
develop novel DNA-compatible reactions that generate spatial
and regioselective diversities efficiently.[9] However, most tradi-
tional reaction conditions in conventional organic chemistry are
not compatible with the presence of DNA.[10] Chemical trans-
formations in the presence of DNA require aqueous media and
extremely low concentrations in comparison with conventional
chemical syntheses (e.g. 0.1–1 mM vs. 0.1—1 M, respectively).
In addition, DNA-compatible reactions generally must be carried
out under mild reaction conditions to ensure the stability and
integrity of DNA tags. The incorporation of privileged scaffolds
and pharmacophores into DELs would be greatly facilitated by
the ability to carry out the required synthetic reactions in the
presence of DNA. We describe here a phylogenic approach, in
which a family of on-DNA chemical transformations for a
diverse range of azole compounds can be carried out using a
common precursor molecule.
Substituted azole derivatives with five- or six-membered
heterocyclic rings have been frequently used as the fundamen-
tal pharmacophores in new drug design due to their highly
desirable biochemical properties.[11] Among all the possible
azole derivatives, 1,3,4-oxadiazole, 1,3,4-thiadiazole-2-amine
and 1,2,4-triazole derivatives have been widely exploited for
various applications.[12] Many drugs containing these azole
moieties are approved for clinical use, including raltegravir and
maraviroc, two anti-HIV agents;[13] nesapidil, an antihypertensive
agent;[14] and acetazolamide, an anti-glaucoma and mild cardiac
[a] Dr. F. Ma, Dr. J. Li, S. Zhang, Y. Gu, T. Tan, W. Chen, S. Wang, Dr. P. Ma,
Dr. H. Xu, Prof. G. Yang
Shanghai Institute for Advanced Immunochemical Studies
ShanghaiTech University, 201210 Shanghai (P. R. China)
E-mail: yangguang@shanghaitech.edu.cn
[b] Prof. R. A. Lerner
Department of Chemistry, The Scripps Research Institute
La Jolla CA 92037 (USA)
E-mail: rlerner@scripps.edu
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/chem.202100850
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edema agent.[15] The important pharmaceutical potential of
azole derivatives has led to continuing efforts to development
methods for on-DNA synthesis of azole heterocyclic com-
pounds. For example, DNA-compatible synthesis of thiazole,[16]
oxazole,[17] tetrazole,[18] imidazole,[19] 1,3,4-oxadiazole[19] and
1,2,4-oxadiazole[20] have been reported in recent years. Among
the synthetic transformations of azoles in conventional organic
chemistry, benzoyl hydrazine has been used as a versatile
functionality with a wide range of substrates to form the
corresponding intermediates which can subsequently be trans-
formed into azole derivatives under mild reaction conditions.
Compared to conventional organic reactions, DEL chemical
reactions usually occur at much lower, e.g. micromolar,
substrate concentrations. Moreover, the DNA tag makes the
separation of end-products from substrates easy and fast. These
properties allow a DEL reaction to be driven kinetically,[21] thus
similar organic reactions can be readily clustered around a
common precursor. We chose DNA-conjugated benzoyl
hydrazine as a common precursor to synthesize various on-DNA
azole compounds. Here, as shown in Figure 1, we report a
phylogenic approach through condensation cyclization of DNA-
conjugated benzoyl hydrazine with different cyclization agents
to construct azole-focused DELs. Using five-membered azole
ring derivatives including 1,3,4-oxadiazole, 1,3,4-thiadiazole-2-
amine, and 1,2,4-triazole as examples, we demonstrated that
highly efficient on-DNA syntheses from the same precursor can
be achieved.
NH2 (Figure S1), with 4a–4j (Scheme S2). To remove the Boc-
protection group of HP-ArHBs, different buffer systems were
screened with the Na2HPO4À NaH2PO4 buffer (pH=6.0, 200 mM)
showing the highest conversion yield (Table S1). Under the
optimized reaction conditions, the de-Boc product DNA-con-
jugated benzoyl hydrazine HP-ArHDs was obtained in excellent
°
yields at 60 C in 12 hours (Scheme S3). As expected, all the
DNA-conjugated (hetero)aryl hydrazide derivatives, HP-ArHD-1–
HP-ArHD-10, could be obtained in high yields following the
same synthetic route (Scheme S3).
With HP-ArHDs in hand, we investigated synthetic reactions
of 1,3,4-oxadiazole derivatives in the presence of DNA. Conven-
tional syntheses for 1,3,4-oxadiazole derivatives were reported
using various oxidizing agents such as chloramine T,[22] ceric
ammonium
nitrate
(CAN),[23]
bis-(trifluoroacetoxy)
iodobenzene,[24] trichloroisocyanuric acid (TCCA),[25] N-
chlorosuccinimide,[26] Cu(OTf)2,[27] and oxidized iodine.[28] Using
HP-ArHD-1 and benzaldehyde as model substrates, oxidizing
agents suitable for on-DNA synthesis of 1,3,4-oxadiazole
derivatives were extensively screened and validated under
various reaction conditions (Table S2). For all the examined
oxidizing agents including iodine, chloramine T, CAN, TCCA,
Dess–Martin, and IBX, only iodine showed a low but encourag-
°
ing 23% yield in DMSO at 25 C (Table S2). Subsequently, iodine
was chosen as the oxidizing agent for further optimization
including buffer, solvent, temperature, and stoichiometry, etc.
(Table S2). It was found that under a condition, in which 80
equiv. of benzaldehyde (in 10 μL DMSO) were mixed with
80 equiv. of I2 (in 10 μL DMSO), 1 equiv. of HP-ArHD-1 (in 10 μL
°
Results and Discussion
ddH2O), and 5 μL 250 mM borate buffer (pH=9.4) at 60 C for
3 h, the reaction proceeded smoothly and reached a nearly
complete conversion (96% yield). In order to gauge the scope
of substrates, various substituted aldehyde derivatives were
next tested. As shown in Figure 2a, the aromatic derivatives of
benzaldehyde with functional groups both electron-rich (RA2-
RA4) and electron-deficient (RA5-RA7) at para-substituted
positions of the aromatic ring afforded the corresponding
products in medium to excellent yields (69%-98%). Notably,
the benzaldehyde derivatives with fluorine- (RA8), chlorine-
(RA9), bromine- (RA10), and iodine-substitution (RA11) afforded
the desired products with excellent yields of 96%, 92%, 94%,
and 92%, respectively. Gratifyingly, reactive functional groups
such as ester (RA12), nitrile (RA13) and acid (RA14) were all
well-tolerated in the reaction (65%-94%). Moreover, the meta-
substituted (RA15-RA17) and sterically hindered ortho-substi-
tuted benzaldehyde derivatives (RA18-RA20) with electron-rich
groups (À CH3, À OCH3) and electron-deficient (À Cl) afforded the
corresponding products in excellent yields (89%–95%). The
dual-substituted benzaldehyde derivatives (RA21-RA22) also
showed excellent conversions of 89% and 94%, respectively. In
addition, oxygen-, nitrogen- and sulfur-containing heteroaryl
aldehyde derivatives (RA23-RA30) were shown to react effi-
ciently with HP-ArHD-1 in good to excellent yields (67%–97%).
It was noted the naphthalene analog, 1-naphthaldehyde,
reacted with HP-ArHD-1 in an excellent yield (82%). To our
delight, the aliphatic aldehyde derivatives (RA32-RA34) also
gave the desired products with excellent yields (80%–92%).
In order to synthesize the starting materials HP-ArHDs (Fig-
ure 1), the synthetic route was designed as shown in Scheme S1
(see Support Information). First, using (hetero)aryl acid deriva-
tives 1a–1j, the corresponding Boc-protected (hetero)aryl
hydrazide derivatives 4a–4j were synthesized using a conven-
tional three-steps reaction (Scheme S1). The DNA-conjugated
benzoyl hydrazine with Boc-protection, HP-ArHBs, was next
prepared by acylation of the DNA headpiece compound, HP-
Figure 1. Synthesis of on-DNA azole derivatives.
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Figure 2. Examples of building blocks used in phylogenic chemical transformation strategy for the synthesis of on- DNA azole derivatives derived from HP-
ArHD-1. (a) Reaction conditions: 1 equiv. of HP-ArHD-1 (10 μL, 1 mM in water), 80 equiv. of RA1 (10 μL, 100 mM in DMSO), 80 equiv. of I2 (10 μL, 80 mM in
°
DMSO), 5 μL borate buffer (pH=9.4, 250 mM), 60 C, 3 h; (b) Reaction conditions: 1 equiv. of HP-ArHD-1 (10 μL, 1 mM in water), 200 equiv. of SA (10 μL,
°
200 mM in EtOH), 800 equiv. of K2CO3 (10 μL, 800 mM in water), 60 C, 3 h; (c) Reaction conditions: 1 equiv. of HP-ArHD-1 (10 μL, 1 mM in water), 200 equiv. of
°
NA (10 μL, 200 mM in EtOH), 1000 equiv. of K2CO3 (10 μL, 1000 mM in water), 60 C, 3 h. Conversion of HP-ArHD-1 was determined by LC-MS.
Under the same reaction conditions, the remaining DNA-
conjugated (hetero) aryl hydrazide derivatives HP-ArHD-2–HP-
ArHD-10 (Scheme S3) were investigated in the syntheses of on-
DNA 1,3,4-oxadiazoles derivatives (Scheme S4). As shown in
Scheme S4, compared to HP-ArHD-1, all the alternative aryl-
hydrazides showed reduced reactivities (19%–76%), of which
the indole derivative (HP-ArHD-10) failed to react with
benzaldehyde to afford the desired product.
1,3,4-thiadiazol-2-amine derivatives are another important
family of azoles that have demonstrated versatile pharmaco-
logical activities. Various synthetic routes for 1,3,4-thiadiazol-2-
amine derivatives were reported including reactions between
hydrazide and isothiocynate,[29] acid hydrazides and
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dithiocarbamates,[30] hydrazination,[11c] and using Vilsmeier and
Lawesson’s reagents,[31] etc. Taking into account the tolerability,
integrity and water-solubility of the DNA barcode in the
reaction, the reactant pair between HP-ArHD-1 and isothiocya-
natobenzene (SA1) was chosen for the on-DNA synthesis of
1,3,4-thiadiazol-2-amine derivatives. The desired product HP-
ArHD-SA1 was obtained in 60% yield using EtOH (volume ratio:
33%) as the co-solvent (Table S3) suggesting that introduction
of isocyanates was a feasible route to synthesize on-DNA 1,3,4-
thiadiazol-2-amine derivatives. To improve the conversion yield,
reaction conditions were examined and optimized. The reaction
with 200 equiv. of isothiocyanatobenzene (SA1), 800 equiv. of
thetic organic approaches for 1, 2, 4-triazole derivatives, which
includes microwave irradiation of acid hydrazide and S-methyl
isothioamide hydroiodide on the surface of silica gel;[32]
condensation of nitrile and hydrazide under microwave irradi-
[33]
°
ation at 150 C; reacting aldehydes with hydrazonoyl hydro-
chlorides via 1,3-dipolar cycloaddition;[34] and dimerization of
amidine hydrochloride in the presence of Cu(OAc)2.[35] These
conventional methods required high reaction temperature and
strong base, which are likely not to be tolerated with DNA
oligonucleotides. In order to synthesize on-DNA 1,2,4-triazole
derivatives, a new synthetic route taking advantage of the
findings that the acylamidrazone obtained by condensation of
imino ether and acylhydrazine could serve as an intermediate in
triazole cyclization[36] was designed and exploited. HP-ArHD-1
and benzimidate hydrochloride (NA1) were chosen as model
substrates in the reaction. In the presence of 1000 equiv. of
°
K2CO3, and EtOH (volume ratio: 33%) at 60 C for 3 h proceeded
smoothly with an excellent conversion (84%) (Table S3). In
order to explore the substrate scope for the synthesis of on-
DNA 1,3,4-thiadiazol-2-amine derivatives, multiple aryl isothio-
cyanates with various substituents were next tested. As shown
in Figure 2b, the derivatives bearing either electron-donating
group (SA2-SA5, SA21) or electron-withdrawing group (SA6-
SA7) reacted readily with HP-ArHD-1 in good to excellent yields
(74%–89%). In addition, fluorine-, chlorine-, bromine-, or
iodine-substituted isothiocyanatobenzene derivatives at para-
positions (SA8-SA11) afforded the desired products in the yields
of 86%, 79%, 86%, and 87%, respectively. However, methyl 4-
isothiocyanatobenzoate (SA13) and 1-isothiocyanato-4-nitro-
benzene (SA14) failed to react with HP-ArHD-1 suggesting that
a reactive group like ester (SA13) or the strong electron-
withdrawing group like nitro (SA14) in the para-position of the
benzene ring was not tolerated under the reaction conditions.
It was noted that the meta-substituted (SA15-SA17) and ortho-
substituted aryl isocyanates (SA18-SA20), despite possible steric
hindrance, afforded final products in good to excellent yields
(76%-97%). HP-ArHD-1 was also shown to react readily with
the heterocyclic 3-isothiocyanatopyridine (SA22) in 82% yield,
and the naphthalene analog, 1-isothiocyanatonaphthalene
(SA23), 88% yield. Furthermore, the aliphatic 1-isothiocyanato-
propane (SA24) could also afford the desired product with a
moderate 65% yield.
°
K2CO3 at 60 C for 2 h, to our satisfaction, HP-ArHD-1 reacted
with benzimidate hydrochloride (NA1) affording the desired
product HP-ArHD-NA1 at a 65% yield (Table S4). Subsequently,
reaction conditions including base, solvent, temperature, time,
and stoichiometry were examined and optimized (Table S4).
Results showed that in the presence of 1000 equiv. of K2CO3
with EtOH (%volume: 33%) as a co-solvent, 200 equiv. of
benzimidate hydrochloride could react readily with HP-ArHD-1
°
at 60 C and afforded the final product with an excellent 94%
yield in 4 h. In order to investigate the substrate scope of this
reaction, dozens of imidate hydrochloride derivatives were
synthesized from nitrile using the facile and efficient Pinner
reaction (Scheme S6).[37] As shown in Figure 2c, these substi-
tuted imidate hydrochloride derivatives were examined with
HP-ArHD-1 under the optimized reaction conditions. Electron-
donating substituents (NA2-NA4, NA16-NA17) appeared to
afford higher yields (43%–94%) compared to electron-with-
drawing substituents (NA5-NA6), which had low to moderate
yields (22%–75%). The fluorine-, bromine-, chlorine- and
iodine-substituted benzimidate hydrochloride derivatives (NA7-
NA10) afforded the desired products in yields of 96%, 75%,
78%, and 44%, respectively. No product formation was
observed with an ester derivative (NA15) that contains a
sensitive functional group and ethyl isonicotinimidate
hydrochloride (NA18) that contains a heterocyclic pyridine.
Nevertheless, heterocycles bearing thiophene (NA19), furan
(NA20), pyrrole (NA21), or benzothiophene (NA22) afforded the
corresponding products with moderate yields (35%–62%).
Significantly, the aliphatic imidate hydrochloride (NA24)
could also react with HP-ArHD-1 in an excellent 84% yield.
We also tested the above established reaction conditions
with all the remaining DNA-conjugated (hetero) aryl-hydrazide
derivatives (HP-ArHD-2–HP-ArHD-10), and showed moderate to
excellent conversion (41%–94% yield) (Scheme S7).
Similarly, under the same reaction conditions of HP-ArHD-1,
the remaining DNA-conjugated (hetero)aryl hydrazide deriva-
tives (HP-ArHD-2 – HP-ArHD-10) were next examined in the
syntheses of on-DNA 1,3,4-thiadiazol-2-amine derivatives. As
shown in Scheme S5, both electron-donating (HP-ArHD-2, HP-
ArHD-4) and electron-withdrawing (HP-ArHD-3, HP-ArHD-5,
HP-ArHD-6) substitutions on the aromatic ring of HP-ArHDs
afforded satisfactory conversions (58%–83%). Aryl-hydrazide at
the meta-position on the aromatic ring (HP-ArHD-7) gave a
moderate 58% yield. Moreover, the heterocycle bearing
pyridine (HP-ArHD-8) and indole (HP-ArHD-10) afforded the
desired products in yields of 40% and 79%, respectively.
Notably, the naphthalene nucleus (HP-ArHD-9) gave an impres-
sive 72% yield.
To further validate the chemical transformation of on-DNA
1,3,4-oxadiazoles, 1,3,4-thiadiazol-2-amines, and 1,2,4-triazole
derivatives, the standard samples of 4-(5-phenyl-1,3,4-oxadia-
zol-2-yl)benzoic acid (RR1), 4-(5-(phenylamino)-1,3,4-thiadiazol-
2-yl)benzoic acid (SS1), and 4-(5-phenyl-4H-1,2,4-triazol-3-yl)
benzoic acid (NN1) were individually synthesized using conven-
tional organic synthetic method (Scheme S8) and confirmed by
Encouraged by the successful application of phylogenic
chemical transformation strategy for on-DNA syntheses of 1,3,4-
oxadiazole and 1,3,4-thiadiazol-2-amine derivatives, we next
exploited the on-DNA synthesis of 1, 2, 4-triazole derivatives.
Many studies reported previously demonstrated diverse syn-
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LC–MS and NMR (see Support Information). The authentic HP-
ArHD-RA1, HP-ArHD-SA1 and HP-ArHD-NA1 were then resyn-
thesized by reacting RR1, SS1 and NN1, respectively, with HP-
NH2 via simple amide coupling (Scheme S9). The resynthesized
authentic samples of HP-ArHD-RA1, HP-ArHD-SA1 and HP-
ArHD-NA1 were co-injected with those synthesized by on-DNA
reactions using UPLC-MS. As shown in Figure S2, S3 and S4, the
products from DNA-compatible reactions had identical reten-
tion times and molecule weights corresponding to the
resynthesized compounds.
To ensure the compatibility of this chemistry with DEL
synthesis, it was important to show that these reaction
conditions do not damage the DNA-based barcodes.[16b,38] Any
effect of these reactions on the integrity of oligo DNA barcodes
was next examined by carrying out the enzymatic ligation
reactions of a 50-mer oligo DNA primer with on-DNA 1,3,4-
oxadiazoles (HP-ArHD-RA1), 1,3,4-thiadiazol-2-amine (HP-ArHD-
SA1), and 1,2,4-triazole derivatives (HP-ArHD-NA1) individually
(Figure 3). Results were analyzed by agarose gel electrophoreses
zoles, 1,3,4-thiadiazol-2-amines, and 1,2,4-triazoles, could be
readily derived from a common DNA-conjugated benzoyl
hydrazine (HP-ArHD) and excessive amounts of aldehydes,
isocyanates, and imidate hydrochlorides, respectively. Moreover
the same pluripotent precursor HP-ArHD could be extended
into the on-DNA syntheses of [1,2,4]triazolo[4, 3-b]pyridazines,
4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diazepines, as well as six-
membered heterocyclic rings such as 1,2,4-triazines, etc.
Assuming a three-cycle DEL library construction, with the
current three azole families, an azole-focused DEL library
containing 106 unique azole structures (cycle1×cycle2×
cycle3=350×10×300 building blocks=1.05×106) could be
envisioned and assembled readily. It is foreseeable that the
described phylogenic chemical transformation strategy could
be applied to various different pharmacophore structures, and
expand the DELs approach to cover broader and deeper
chemical space.
(Figure S5) and subjected to deep-sequencing after PCR Experimental Section
amplification (Figure S6). As shown in Figure S5 and S6, the
General procedure for the synthesis of DNA-conjugated (hetero)
ligation reactions proceeded successfully to afford the desired
58-bp products of HP-ArHD-RA-L, HP-ArHD-SA-L, and HP-
ArHD-NA-L, respectively. These reaction conditions did not
interfere with the DNA headpiece’s cohesive ends.
aryl Boc-protected hydrazide derivatives (HP-ArHBs): To a solution
of DNA headpiece (HP-NH2, 300 mol) in 600 μL borate buffer (pH=
9.4, 250 mM) was added a mixture of O-(7-Azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU, 60 μL,
200 mM in N,N-Dimethylaniline (DMA)), 4 (60 μL, 200 mM in DMA)
and N,N-Diisopropylethylamine (DIPEA, 60 μL, 200 mM in DMA).
°
The resultant mixture was vortexed and stood at 25 C for 12 h.
Aqueous NaCl (78 μL, 5.0 M) and cold EtOH (2.34 mL) were
Conclusion
°
sequentially added and the resultant mixture was stored at À 80 C
°
for 30 min. The mixture was centrifuged at 4 C for 30 min at
The scope of synthetic transformations that can be used for
construction of a DEL is constrained by the requirement that
the appended DNA barcode is not modified. Indeed, this
constraint has led to a set of studies where classical organic
reactions are modified to work under mild conditions in
water.[39] Over time, more and more classical organic chemistry
has been applied to reactions compatible with the presence of
DNA. The results reported herein are another example of this
trend. We developed a phylogenic chemical transformation
strategy to carry out on-DNA syntheses that cover broad
substrate scopes and diverse reaction conditions under conven-
tional organic synthesis. We demonstrated that three chemical
families consisting of important pharmacophores, 1,3,4-oxadia-
12000 rpm before the resultant supernatant was removed. The
pellet was dissolved in deionized water (300 μL), which was used in
the next reaction without further purification.
General procedure for the synthesis of DNA-conjugated (hetero)
aryl hydrazide derivatives (HP-ArHDs): To the above solution of
HP-ArHBs (300 μL, 300 nmol, 1 mM in water) was added 300 μL
Na2HPO4-NaH2PO4 buffer (pH=6.0, 200 mM), and the resultant
mixture was vortexed and stood at 60 C for 12 h. Aqueous NaCl
(60 μL, 5.0 M) and cold EtOH (1.8 mL) were sequentially added and
°
°
the resultant mixture was stored at À 80 C for 30 min. The mixture
°
was centrifuged at 4 C for 30 min at 12000 rpm before the
resultant supernatant was removed. The pellet was dissolved in
deionized water (300 μL), which was used in the next reaction
without further purification.
Figure 3. Enzymatic ligation of HP-ArHD-RA1, HP-ArHD-SA1 and HP-ArHD-NA1.
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General procedure for the synthesis of on-DNA 1,3,4-oxadiazole
derivatives: To the mixture of HP-ArHD-1 (10 μL, 10 nmol, 1 mM in
water) and 5μL borate buffer (pH=9.4, 250 mM) was added 80
equiv. of aldehydes (10 μL, 80 mM in DMSO). The mixture was
Acknowledgements
This work was supported by JPB Foundation 1097 for R.A.L., and
China Postdoctoral Science Foundation Grant (2019M651617).
The authors thank Dr. Wenzhang Chen and Jiakang Chen at
SIAIS analytical chemistry platform, ShanghaiTech University for
the help of LC-MS analysis.
°
vortexed and stood at 60 C for 1 h. After the reaction, added 80
equiv. of iodine (10 μL, 80 mM in DMSO) and heated the reaction
°
mixture at 60 C for another 2 h. Aqueous NaCl (10% by volume,
°
5.0 M) and cold EtOH (2.5 times by volume, EtOH stored at À 20 C)
were sequentially added and the resultant mixture was stored at
°
À 80 C for 30 min. The sample was centrifuged for around 30
°
minutes at 4 C in a microcentrifuge at 12000 rpm. The above
Conflict of Interest
supernatant was removed and the pellet (precipitate) was dissolved
in deionized water for LC-MS detection.
The authors declare no conflict of interest.
General procedure for the synthesis of on-DNA 1,3,4-thiadiazol-2-
amine derivatives: To the mixture of HP-ArHD-1 (10 μL, 10 nmol,
1 mM in water) and 200 equiv. of isocyanates (10 μL, 200 mM in
EtOH) was added 800 equiv. of K2CO3 (10 μL, 800 mM in water). The
Keywords: azole · DNA-compatible reactions · DNA-encoded
libraries · hydrazide
°
mixture was vortexed and stood at 60 C for 3 h. After the reaction,
aqueous NaCl (10% by volume, 5.0 M) and cold EtOH (2.5 times by
°
volume, EtOH stored at À 20 C) were sequentially added and the
°
resultant mixture was stored at À 80 C for 30 min. The sample was
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°
centrifuged for around 30 minutes at 4 C in a microcentrifuge at
12000 rpm. The above supernatant was removed and the pellet
(precipitate) was dissolved in deionized water for LC-MS detection.
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General procedure for the synthesis of on-DNA 1,2,4-triazole
derivatives: To the mixture of HP-ArHD-1 (10 μL, 10 nmol, 1 mM in
water) and 1000 equiv. of K2CO3 (10 μL, 1000 mM in water) was
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°
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°
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°
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°
The sample was centrifuged for around 30 minutes at 4 C in a
microcentrifuge at 12000 rpm. The above supernatant was removed
and the pellet (precipitate) was dissolved in deionized water for
LC–MS detection.
Co-injection experiment: Samples were dissolved in an appropriate
amount of distilled and deionized water (ddH2O) and injected or
co-injected into a reverse-phase chromatography column (Xbridge
Oligonucleotide BEH C18 column, 1.7 μm, 2.1×50 mm). The elution
was carried out as followings: 5–30% solvent B over 30 min,
0.4 mL/min, λ=260 nm; solvent A: 0.75% v/v hexafluoroisopropa-
nol/ 0.038% v/v triethylamine in methanol/water=5/95; solvent B:
0.75% v/v hexafluoroisopropanol/ 0.038% v/v triethylamine in
methanol/water=90/10. The effluents were analyzed by a Xevo G2-
XS Q-TOF with electrospray ionization source.
Enzymatic ligation experiment: HP-ArHD-RA1 or HP-ArHD-SA1 or
HP-ArHD-NA1 (5 nmol) was dissolved in 5 μL ddH2O. To the tube
was added the Primer (LGC Biosearch Technologies, 1 mM in
°
ddH2O, 6 μL, Primer-F and Primer-R were freshly annealed at 95 C
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analyzed via gel electrophoresis on 4% agarose gel. The amplified
DNA samples were subjected to deep-sequencing.
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Reporting summary: Further information is available in the support
information.
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doi.org/10.1002/chem.202100850
Chemistry—A European Journal
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Manuscript received: March 8, 2021
Accepted manuscript online: April 2, 2021
Version of record online: ■■■, ■■■■
Chem. Eur. J. 2021, 27, 1–8
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© 2021 Wiley-VCH GmbH
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These are not the final page numbers!

FULL PAPER
The phylogenic chemical transforma-
tion approach to synthesize on-DNA
1,3,4-oxadiazole, 1,3,4-thiadiazol-2-
amine and 1,2,4-triazole derivatives
using DNA-conjugated benzoyl
Dr. F. Ma, Dr. J. Li, S. Zhang, Y. Gu, T.
Tan, W. Chen, S. Wang, Dr. P. Ma, Dr. H.
Xu, Prof. G. Yang*, Prof. R. A. Lerner*
1 – 8
DNA-Encoded Libraries: Hydrazide as
a Pluripotent Precursor for On-DNA
Synthesis of Various Azole Deriva-
tives
hydrazine as pluripotent precursor are
reported here, which shows great
potential in facilitating rapid construc-
tion of azole-focused DELs.