Reaction discovery by using a sandwich immunoassay

Article abstract of DOI:10.1002/anie.201201451

Mmm, a reaction sandwich Using an immunoassay-based technique able to monitor any kind of cross-coupling reaction, a systematic and rapid evaluation of a large panel of random reactions was carried out. This approach led to the discovery of two new copper-promoted reactions: a desulfurization reaction of thioureas leading to isoureas and a cyclization reaction leading to thiazole derivatives from alkynes and N-hydroxy thioureas. Copyright

Full text of DOI:10.1002/anie.201201451

Angewandte
Chemie
DOI: 10.1002/anie.201201451
High-Throughput Screening
Reaction Discovery by Using a Sandwich Immunoassay**
Julia Quinton, Sergii Kolodych, Manon Chaumonet, Valentina Bevilacqua, Marie-Claire Nevers,
Hervꢀ Volland, Sandra Gabillet, Pierre Thuꢀry, Christophe Crꢀminon, and Frꢀdꢀric Taran*
Despite the bredth of the synthetic chemistꢀs arsenal, there is
still a need to develop new organic reactions, to allow for
unprecedented connection of reactive functions. A recent and
still underdeveloped approach to reaction discovery involves
the use of efficient screening techniques that allow test
reactions to be systematized in a format that maximizes the
opportunity for discovering new reactions. This “forced
serendipity” approach is based on the assumption that the
probability of serendipitous findings increases when a large
number of chemical reactions are performed. Robust and
general high-throughput screening techniques allowing the
quick identification of new reactions are the critical point of
such an approach.
Improvements in the automation of mass spectroscopy
(MS) coupled to gas (GC) or liquid (LC) chromatography
were the initial driving force of this serendipity-based
strategy. Weber et al. reported in 1999 the discovery of
a new Ugi-type reaction after screening thousands of reaction
mixtures.[1] Since this pioneering work, a few examples of new
reactions discovered by MS-based screening have been
reported. Porco, Jr. and co-workers developed a so-called
multidimensional screening, wherein reactions are run in an
array format and analyzed by LC-MS techniques.[2] Several
new interesting reactions were successfully identified, but
because of moderate throughput of the screening, the
approach was rather focused on one particular type of
designed substrate or reaction process, which may leave
many areas of chemical reactivity unexplored. Recently,
MacMillan and co-workers discovered a useful photoredox-
An alternative strategy for discovery of new reactions,
initially used to facilitate MS analysis,[5] relies on the use of
tagged reactants. Liu and co-workers developed a powerful
method based on DNA-tagged reactants allowing the quick
identification of new chemical transformations.[6] However,
DNA tags require particular chemistry and might interact
with the catalysts.
Herein, we discuss an approach to discover chemical
reactions using an immunoassay screening technique that uses
tags that are easier to synthesize and more compatible with
the chemical reactions studied. As we reported,[7] sandwich
immunoassays can be adapted to monitor cross-coupling
reactions by connecting small-molecule tags to chemically
reactive groups. Products of coupling reactions can then be
specifically detected by two anti-tag monoclonal antibodies:
one antibody capturing the double-tagged coupling product
onto a solid phase and a second acting as a detector. The
required tags are respectively a tert-butoxycarbonyl (Boc)-
protected imidazole and a tert-butyldimethylsilyl (TBDMS)-
protected guaiacol derivative, both are linked to functional
groups A and B by standard peptide coupling (Figure 1). We
recently showed that any kind of cross-coupling products can
be detected by this technique if the tags are separated by at
least eight atoms.[8]
Typically our sandwich immunoassay is run in microtiter
plates and allows the measurement of at least 1000 reaction
yields in a single day (for one person, without robotization).
We anticipated that this throughput should be high enough to
apply the technique to reaction discovery projects.
Our strategy (Figure 1) is therefore based on a three-step
procedure: 1) parallel reactions of combinations of tagged
functional groups A and B and catalysts are run in 96-well
plates, 2) quenching, dilution, and transfer of crude reaction
mixtures to determine cross-coupling yields by sandwich
immunoassay, and 3) validation and evaluation of hits by
reproducing the active combination with nontagged func-
tional groups.
[3]
À
catalyzed C H arylation reaction using robotic GC-MS.
A
copper-catalyzed alkyne hydroamination and two nickel-
catalyzed hydroarylation reactions were also recently discov-
ered by Robbins and Hartwig using a simpler MS instru-
ment.[4]
[*] Dr. J. Quinton, S. Kolodych, Dr. M. Chaumonet, V. Bevilacqua,
S. Gabillet, Dr. F. Taran
CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage
91191 Gif sur Yvette (France)
E-mail: frederic.taran@cea.fr
A particularly critical design element of this strategy relies
on the choice of reactive functional groups that, in theory,
should not be based on preconceived ideas of what will react.
Herein, we conducted the core experiment with 21 tagged A
functional groups and 16 tagged B functional groups (see
Supporting Information for synthesis), most of them contain-
ing common functional groups (such as alcohol, nitro, amine,
alkene, alkyne, azide, aldehyde, and nitrile). Assuming that
the probability of identifying new reactions should be higher
with functionalities whose chemistry has been less explored,
we also selected some less common functional groups (such as
skipped alkyne, N-hydroxy thiourea, alkynyl oxime, and
amidoxime). Alkane groups were also added to the library as
M.-C. Nevers, Dr. H. Volland, Dr. C. Crꢀminon
CEA, iBiTecS, Service de Pharmacologie et d’Immunoanalyse
91191 Gif sur Yvette (France)
Dr. P. Thuꢀry
CEA, IRAMIS, Service Interdisciplinaire sur les Systꢁmes
Molꢀculaires et les Matꢀriaux
91191 Gif sur Yvette (France)
[**] This work was supported by the European Union, BioChemLig
project and by ANR, ClickScreen project.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201201451.
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hits were identified. Among them we focused our interest on
the 22 hits which generated more than 5% of the cross-
coupled products. Of these hits, 13 combinations, confirmed
by LC/MS analysis, corresponded to well-known reactions:
Henry, Knoevenagel, Michael reactions (11H, 13H, 11L, and
19L), Cu-catalyzed alkyne–azide cycloaddition (2D, 5D, and
6D), alkyne–alkyne dimerization (2E, 5E, and 6E), and Pd-
catalyzed Sonogashira and Heck reactions (2G and 3G). The
remaining nine combinations were thus carefully investigated
by reproducing the reactions on the mmole scale using non-
tagged reactants. These experiments highlighted only two
interesting reactions resulting from copper-mediated coupling
of phenols and thioureas (combination 19F) and coupling of
alkynes with N-hydroxy-thioureas (combination 2O). The
other seven combinations gave complex mixtures containing
several coupling products and by-products and were therefore
considered less promising.
Examination of the combination 19F was first carried out
using para-cresol 19 and 1-pentyl-3-phenylthiourea F under
the reaction conditions used in the screening. Two products
were isolated: the isourea 19F-1 and the byproduct FF
(Scheme 1).
Scheme 1. Target reaction 1 (yields of isolated product).
Figure 1. Outline of the immunoassay-based reaction discovery pro-
cess. PG: Tag protecting groups.
Isoureas are versatile esterification and alkylation
reagents[9] usually prepared from reaction of alcohols or
phenols with carbodiimides.[10] To the best of our knowledge
intermolecular coupling between a thiourea and phenols has
only been described once using activated N,N’-bis-
(Boc)thiourea by way of the formation of the corresponding
carbodiimide upon addition of stoichiometric amounts of HgII
salts.[11] Anticipating that our reaction should give rise to an
easier and more general synthesis of isoureas, optimization of
the reaction conditions was investigated (Table 1). The
reaction was found to proceed efficiently at room temper-
negative controls. Reactants were combined in a parallel
manner and exposed to one set of reaction conditions with or
without transition-metals (Cu, Pd, Au, and Ru). To maximize
the chances of discovering new reactions, a stoichiometric
amount of metal was used. Reactions were run in dimethyl-
acetamide (DMA) at 508C in the presence or absence of
triethylamine (TEA) leading to a total of 3360 parallel
reactions performed in 96-well plates. It should be noted that,
because of the high sensitivity of the screening method,
reactions were carried out with only 20 mL total volume and
100 nmol of reactants. Following treatment by a solution of
tetrabutylammonium fluoride (TBAF) and HCl (quenching
and tag deprotection), the crude reaction mixtures were
transferred into microtiter plates coated with anti-Tag
1 monoclonal antibody (mAb). After washing, the enzyme-
labeled anti-Tag 2 mAb was added and the enzymatic activity
quantitated (Figure 1). Yields were calculated using a calibra-
tion curve obtained with a double-tagged model compound
(see Supporting Information). All combinations were
screened by sandwich immunoassay in 2 days to reveal 57
coupling reactions. Representative results obtained with Cu,
Pd, and Au catalysts in the presence of TEA are indicated in
Figure 2.
Table 1: Optimization of reaction 1 conditions.[a]
Entry
Copper
Base[b]
Conditions
F19[c]
FF[c]
1
2
3
4
5
6
7
8
CuCl (1 equiv)
CuCl (1 equiv)
CuCl (1 equiv)
CuCl (1 equiv)
CuCl (1 equiv)
CuCl (1 equiv)
CuCl (0.1 equiv)
CuI (0.1 equiv)
CuOTf (0.1 equiv)
Cu(OAc)2 (0.1 equiv)
CuCl2 (0.1 equiv)
CuCl2 (0.1 equiv)
-
N2, 508C
N2, 508C
N2, 508C
N2, 508C
N2, 508C
N2, 258C
N2, 258C
N2, 258C
N2, 258C
N2, 258C
N2, 258C
air, 258C
trace
30%
32%
38%
48%
47%
48%
39%
15%
46%
52%
78%
45%
13%
4%
19%
13%
20%
20%
11%
5%
TEA
tBuOK
NaH
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
9
10
11
12
14%
22%
16%
Yields were globally poor and did not exceed 30%,
probably because of the reaction conditions used, but clear
[a] Reactions were conducted with 0.1m substrates in DMF. [b] 3 equiv.
[c] Yields as determined by NMR spectroscopy.
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Figure 2. Screening results. Reactions were carried out with 5 mm reactants, 10 mm TEA, and with or without 5 mm metal in 40 mL of DMA.
ature using K2CO3 in the presence of catalytic amounts of CuII
salts under air.
To look at the scope of the reaction, a series of reactions
were conducted with the optimized reaction conditions. As
shown in Scheme 2, the reaction efficiency appeared to be
strongly influenced by the phenol nucleophilicity but seemed
less sensitive to the thiourea structure.
The reaction was then successfully extended to the
intramolecular version to form N-substituted 2-aminobenz-
oxazoles from 2-aminophenols. Upon treatment with alkyl- or
arylisothiocyanates in the presence of catalytic amounts of
CuCl2, aminophenols underwent rapid formation of the
corresponding thiourea intermediates followed by copper-
catalyzed cyclodesulfurization to form the desired amino-
benzoxazoles in good yields (Scheme 3). Cyclodesulfurization
methods were previously reported using stoichiometric
amounts of toxic heavy-metal oxides,[12] or strong oxidants.[13]
Therefore, this reaction represents an interesting milder
Scheme 2. Scope of target reaction 1 (yields of isolated product).
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Table 2: Optimization of reaction 2 conditions.[a]
Entry
Copper
Base[b]
Conditions
2O[c]
1
2
3
4
5
6
7
8
CuCl (1 equiv)
CuCl2 (1 equiv)
TEA
TEA
TEA
TEA
K2CO3
Cs2CO3
-
-
-
-
-
-
-
-
DMF, air, 508C
DMF, air, 508C
DMF, air, 508C
DMF, Ar, 508C
DMF, air, 508C
DMF, air, 508C
DMF, air, 508C
DMF, air, 1008C
H2O, air, 808C
CH3CN, air, 808C
DMSO, air, 808C
EtOH, air, 808C
EtOH, air, 808C
EtOH, air, 808C
7%
5%
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (1 equiv)
Cu(OAc)2 (3 equiv)
Cu(OAc)2 (3 equiv)[d]
12%
n.d.
8%
11%
15%
7%
9
5%
10
11
12
13
14
n.d.
n.d.
24%
33%
54%
Scheme 3. Extension of reaction 1 to aminobenzoxazoles synthesis
(yields of isolated product).
alternative to known procedures. Although no clear mecha-
nism of the reaction can be provided at this time, control
experiments conducted with thiourea F in the presence of
CuCl2 indicated that no carbodiimide was formed during the
reaction. The formation of compound 19F-14 from N-
disubstituted thiourea also supports the absence of carbodi-
imide intermediate formation during the reaction process. No
H2S or elemental sulfur was detected in this desulfurization
reaction, suggesting an oxidative mechanism from the Cu2+/
air system.
[a] Experiments were carried out with 1 equiv of N-hydroxy thiourea O.
[b] 3 equiv. [c] Yields as determined by NMR spectroscopy. [d] 3 equiv of
N-hydroxy thiourea O were used. n.d.=not detected
Examination of combination 2O was then performed
using 4-ethynylbiphenyl 2 and 1-hydroxy-1-methyl-3-pro-
pylthiourea O as model substrates under the conditions
used during the screening procedure. LC/MS analysis of the
crude material revealed a minor coupling product with
a molecular weight equal to the sum of the starting reactants
with loss of H2O. Purification of the crude afforded two
products: the 2-iminothiazolidine 2O-1 obtained in 7% yield
together with the alkyne dimer product (53% yield) resulting
from Glaser–Hay oxidative coupling (Scheme 4).[14]
Scheme 5. Scope of target reaction 2 (yields of isolated product).
troscopy analysis and further confirmed by X-ray diffraction
analysis of products 2O-3 and 2O-7 (see Supporting Infor-
mation). Although the product yields could still be improved,
an interesting feature of this reaction is its regioselectivity. In
all cases, only one regioisomer (surprisingly the most steri-
cally hindered one) was formed during the reaction. No other
heterocycles were isolated; the only byproducts were the
alkyne dimer and products from N-hydroxy thiourea degra-
dation.
Scheme 4. Target reaction 2 (yields of isolated product).
Convinced by the usefulness of this new reaction, we
performed a systematic optimization study by varying all the
parameters that might influence the reaction efficiency. Part
of this study is given in Table 2. Varying bases and copper salts
was unsuccessful (Table 2, entries 1–6). The use of high
temperatures or controlled atmosphere (to alleviate the
formation of the alkyne dimer) also did not improve the
reaction (Table 2, entries 4 and 8). Improvement of the
reaction was finally possible by using excess Cu(OAc)2 in
refluxing EtOH under air (Table 2, entries 13 and 14).
We then examined the scope of the reaction using 3
equivalents of Cu(OAc)2 in EtOH at 808C under an
atmosphere of air. The reaction was tolerant of electron-
rich and electron-poor alkynes affording the desired 2-
iminothiazolidines 2O in moderate yields (Scheme 5). The
structures of the products were determined by NMR spec-
Whereas a detailed mechanism remains to be determined,
À
thiolation of the C H bond of the terminal alkyne by way of
copper-oxidative coupling[15] followed by subsequent cycliza-
tion seems to be one possible mechanism. In support of this
hypothesis, heating Cu(OAc)2 with the N-phenyl-N’-hydroxy
À
thiourea O’ interestingly undergoes C S bond formation
through C-H activation at only 508C to yield benzothiazole
2O-8 (structure determined by X-ray analysis; Scheme 6).
In conclusion, this work shows that sandwich immuno-
assays are a suitable and effective screening tool for
“serendipitously” discovering new chemical transformations.
Using this method, thousands of reactions were run and
screened in a few days, revealing two new reactions: a copper-
catalyzed desulfurization reaction of thioureas leading to
isoureas and a copper-promoted cyclization reaction leading
to thiazole derivatives from alkynes and N-hydroxy thioureas.
4
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Scheme 6. Reaction of O’ with Cu2+ (the structure of the copper
complex is hypothetical).
The presented screening strategy has no particular limitations
and could be used to explore any kind of bond formation
reactions in a large panel of experimental conditions.
Although the need to connect tags to reactive functions
may be a drawback of the screening technique, because it
requires some time for tagged reactant synthesis, it is also
a strong advantage. The tags allow for easy isolation of cross-
coupling products, ensuring a high level of selectivity of the
screen. Control experiments showed that the method is able
to detect the coupling products in very complex reaction
mixtures, including biological media, allowing identification
of reactions with yields as poor as 0.01% if necessary. This
represents a significant advantage over described screening
methods that cannot determine reaction yields in a high
throughput manner. We envision in the near future using the
high selectivity, sensitivity, and throughput of this screening
approach to search for new chemoselective, bioorthogonal
reactions, which represents one of the fascinating next steps of
the “forced serendipity” approach.
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Experimental Section
General procedure for parallel reactions and screening by sandwich
immunoassay: Reactant 1–21 (1 equiv; 5 mL of a 20 mm DMA
solution), reactant A-P (1 equiv; 5 mL of a 20 mm DMA solution),
metal salts (1 equiv; 5 mL of a 20 mm DMA solution), and TEA
(2 equiv; 5 mL of a 40 mm DMA solution) were mixed in a 96-well
microtiter plate. No precautions were taken to exclude oxygen. The
reaction plates were incubated at 508C with stirring for 24 h and then
quenched by addition of 110 mL of a 0.1m HCl/TBAF aqueous
solution.
ˇ ´
[11] ꢄ. Goonan, A. Kahvedzic, F. Rodriguez, P. S. Nagle, T. McCabe,
The crude mixtures were then diluted 1:105 in EIA buffer
(containing 0.1m phosphate buffer and 1 mgmLÀ1 BSA, pH 7.4).
100 mL of these diluted solutions was transferred to the wells of
a microtiter plate previously coated with anti-Tag 1 mAb (directly
adsorbed to the polystyrene support). After 3 h of incubation at room
temperature, the plates were washed and 100 mL of anti-Tag 2 mAb-
acetylcholinesterase (AChE) conjugate was added (mAb-AChE
conjugate was prepared and stored as previously described).[16]
After 12 h of immunological incubation at 48C, the plates were
washed and Ellmanꢀs reagent was added. The absorbance related to
the solid phase-bound AChE activity was measured at 414 nm.
Immunological reagents (antibody for the capture step and enzyme
conjugate antibody) were used in excess compared with the concen-
tration of the samples. All measurements for standards or samples
were made in duplicate.
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À
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Received: February 21, 2012
Revised: April 10, 2012
Published online: && &&, &&&&
Keywords: copper · high-throughput screening ·
.
immunoassays · reaction discovery
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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Angewandte
Communications
Communications
High-Throughput Screening
Mmm, a reaction sandwich… Using an
immunoassay-based technique able to
monitor any kind of cross-coupling reac-
tion, a systematic and rapid evaluation of
a large panel of random reactions was
carried out. This approach led to the
discovery of two new copper-promoted
reactions: a desulfurization reaction of
thioureas leading to isoureas and a cycli-
zation reaction leading to thiazole deriv-
atives from alkynes and N-hydroxy thio-
ureas.
J. Quinton, S. Kolodych, M. Chaumonet,
V. Bevilacqua, M.-C. Nevers, H. Volland,
S. Gabillet, P. Thuꢁry, C. Crꢁminon,
F. Taran*
&&&& — &&&&
Reaction Discovery by Using a Sandwich
Immunoassay
6
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
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