Thiosemicarbazone Dynamic Combinatorial Chemistry: Equilibrator-Induced Dynamic State, Formation of Complex Libraries, and a Supramolecular On/Off Switch

Article abstract of DOI:10.1021/acs.joc.7b01161

Dynamic combinatorial libraries that equilibrate under thermodynamic control and can be trapped kinetically when desired are key to creating complex systems that can mimic dynamic biological systems, such as the biochemical system of life. A much-sought-after feature is the ability to turn off the dynamic exchange of the system, in order to investigate a transient state away from thermodynamic equilibrium, and then turn on the dynamic exchange again. We describe here the first use of thiosemicarbazone exchange to form dynamic combinatorial libraries. The libraries were found to require a nucleophilic catalyst, or equilibrator, in order to reach thermodynamic equilibrium. This equilibrator approach adds a supramolecular level of control over the dynamic system and allows the dynamic exchange to be turned off by addition of 18-crown-6, which binds the equilibrator in a nonnucleophilic complex. The dynamic exchange can be restarted by addition of potassium ions that competitively bind 18-crown-6, thus liberating the equilibrator. The highly complex thiosemicarbazone-based macrocyclic libraries contain both [2]catenanes and sequence isomers, which can be distinguished by HPLC-MS/MS.

Full text of DOI:10.1021/acs.joc.7b01161

Article
pubs.acs.org/joc
Thiosemicarbazone Dynamic Combinatorial Chemistry: Equilibrator-
Induced Dynamic State, Formation of Complex Libraries, and a
Supramolecular On/Off Switch
†
‡
§
Dennis Larsen, Anne Jeppesen, Claudia Kleinlein, and Michael Pittelkow*
Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark
*
S Supporting Information
ABSTRACT: Dynamic combinatorial libraries that equilibrate
under thermodynamic control and can be trapped kinetically
when desired are key to creating complex systems that can
mimic dynamic biological systems, such as the biochemical
system of life. A much-sought-after feature is the ability to turn
off the dynamic exchange of the system, in order to investigate
a transient state away from thermodynamic equilibrium, and
then turn on the dynamic exchange again. We describe here
the first use of thiosemicarbazone exchange to form dynamic
combinatorial libraries. The libraries were found to require a
nucleophilic catalyst, or equilibrator, in order to reach
thermodynamic equilibrium. This equilibrator approach adds a supramolecular level of control over the dynamic system and
allows the dynamic exchange to be turned off by addition of 18-crown-6, which binds the equilibrator in a nonnucleophilic
complex. The dynamic exchange can be restarted by addition of potassium ions that competitively bind 18-crown-6, thus
liberating the equilibrator. The highly complex thiosemicarbazone-based macrocyclic libraries contain both [2]catenanes and
sequence isomers, which can be distinguished by HPLC-MS/MS.
1
. INTRODUCTION
off the equilibrating reaction, and that the molecules produced
in the DCL experiment are stable enough to be isolated and
studied under the conditions of the equilibrating mixture. For
example, the use of acylhydrazones as the reversible reaction in
1
2
Abiogenesis, one theory on the origin of life, describes how
life on Earth could have developed from inanimate matter
through a wealth of interconnected equilibria by building up
more and more complex dynamic systems, which eventually
4a,7
DCLs has received significant attention.
The reaction
3
between an aldehyde and a hydrazide produces acylhydrazones
mediated by acidic conditions, typically the addition of 2−5%
trifluoroacetic acid (TFA). This generally gives DCLs that
equilibrate under thermodynamic control: the template-
amplified species from the DCL can be isolated, and the
interactions of the DCLs with the template can be studied.
However, the amplification process occurs in the presence of
TFA, and the molecular recognition event cannot be studied
resulted in the formation of living cells. In recent decades, the
fields of dynamic combinatorial chemistry and systems
chemistry have investigated how interconnected equilibria can
lead to highly complex dynamic systems from mixtures of
simple molecular building blocks, thus providing a bottom-up
approach toward understanding how complex systems, such as
life itself, could have evolved from relatively simple small
4
,5
6
molecules.
(
e.g., by NMR spectroscopy) under the same conditions as the
Dynamic combinatorial chemistry provides an appealing
5
DCL, as it would cause the DCL to start equilibrating again.
Here we establish thiosemicarbazone exchange by the
reaction between an aldehyde and a thiosemicarbazide as a
new reversible reaction for dynamic combinatorial chemistry
entry point to study systems chemistry. In dynamic
combinatorial chemistry, reversible chemical reactions mediate
the formation of dynamic combinatorial libraries (DCLs) that
equilibrate under thermodynamic control. The addition of
templates to a DCL enables the selection of strong receptors
for added templates from the DCLs.
Ideal” reversible chemical reactions must live up to a
number of requirements to create functioning DCLs; these
requirements may differ depending on the individual
application of the DCL. Universally, it is essential that the
reversible chemical reaction proceeds to the thermodynamic
equilibrium, and it is necessary to confirm this each time a new
DCL is studied. Other requirements, which are often more
difficult to achieve experimentally, are the ability to turn on and
4a
(
Figure 1). We also demonstrate the use of a nucleophilic
4b
hydrazide catalyst, termed an equilibrator, to make the system
reach a thermodynamic equilibrium. By exploiting the require-
ment for an equilibrator, we show how the dynamic system can
be turned on and off via a supramolecular capture/release
mechanism. It was found that the thermodynamic equilibration
can be turned off by capturing the equilibrator as its ammonium
“
Received: June 2, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
scaffold in which one amino group was converted into a
thiosemicarbazide, while an amide coupling strategy was used
to append an aromatic aldehyde-containing moiety onto the
other amino group (Chart 1). Use of the C -symmetric 1R,2R-
2
diaminocyclohexane as a central scaffold ensures that only one
stereoisomer can result from this type of modular synthesis.
Chart 1. Catalyst Structures
Building block C is based on a simple benzene scaffold with a
thiosemicarbazone and a protected aldehyde in a 1,3-
substitution pattern. While conventional aldehyde protection
schemes proved sufficiently stable for building blocks A and C
Figure 1. Principle of thiosemicarbazone-based dynamic systems
presented in this study. Use of a nucleophilic catalyst, or equilibrator,
allows for turning on or off the dynamic state of the system. (Inset)
Structure of thiosemicarbazone motif, with thiourea and imine
moieties highlighted.
(
acetals of methanol and ethylene glycol, respectively),
successful isolation of building block B was achieved only
after forming the kinetically stabilized acetal of 2,2-dimethyl-
propane-1,3-diol in a practical example of applying the
ion complex with 18-crown-6, and by exploiting 18-crown-6’s
stronger affinity for potassium over ammonium ions, it was also
possible to revert the static mixture to its dynamic state. We
describe two different types of equilibrators: one that interferes
with the DCL (addition of excess thiosemicarbazide) and one
that mediates the equilibration but does not incorporate into
the DCL (addition of excess hydrazide).
11
Thorpe−Ingold effect.
2
.2. Reaching Thermodynamic Equilibrium: The Need
for an Equilibrator. With building block A in hand, we
initiated experiments to determine whether thiosemicarbazone
exchange can be used to form dynamic systems. Initially, we
dissolved A (1.0 mM) in chloroform with TFA (5 vol %),
thereby exposing the acetal-protected aldehyde function and
starting thiosemicarbazone formation (Figure 2). These initial
conditions were similar to those used to achieve equilibration of
acylhydrazone DCLs. By use of high-performance liquid
chromatography (HPLC) with UV detection coupled with
mass spectrometry (MS), we were able to identify several of the
expected macrocyclic oligomers, A , ranging from dimer A to
2
. RESULTS AND DISCUSSION
Thiosemicarbazones were traditionally used to precipitate
aldehydes from solution, and more recently they have been
8
applied in anion recognition. Furthermore, the last couple of
decades has seen a vastly expanded use of thiourea-based
9
receptors, for example, for organocatalysis, and the inherent
n
2
thiourea moiety of thiosemicarbazones is a desirable recog-
nition motif to incorporate into DCLs.
heptamer A in a distribution pattern systematically favoring
7
the smallest macrocyclic species (black trace in Figure 2).
Macrocycle formation was fast, as evidenced by the fact that
macrocycles were present in the library in high concentrations
immediately after preparation.
To verify whether this distribution was a true dynamic
system at thermodynamic equilibrium, we isolated the
In comparison to acylhydrazones, thiosemicarbazones are
expected to be more resilient to hydrolysis (and thus exchange)
because of attenuation of the electron-withdrawing effect from
the thiocarbamoyl in thiosemicarbazones in comparison to the
acyl in hydrazones. Therefore, use of an equilibrator is expected
to be required for efficient thiosemicarbazone exchange, as has
also been found necessary for some electron-deficient
macrocyclic dimer A by preparative chromatography and set
2
up a library under identical conditions to the library started
from monomer building block A. In a truly thermodynamic
system, the library should equilibrate to give the same
distribution, but as is evident from Figure 2, the library started
10
acylhydrazone-based DCLs. This makes thiosemicarbazone
exchange ideal for the formation of DCLs whose exchange
reaction can be stopped by supramolecular means.
2
.1. Building Block Design. Three building blocks,
from A showed only minor signs of formation of larger
2
comprising a central scaffold onto which was placed a
thiosemicarbazide and a protected aldehyde, were synthesized
see Supporting Information). Building blocks A and B were
both designed around a central 1R,2R-diaminocyclohexane
macrocycles after 28 days (compare black and red traces in
Figure 2). Thus, the thiosemicarbazone exchange reaction is
significantly more resistant to equilibration than acylhydrazone
exchange.
(
B
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
amine. Therefore, we turned our attention to hydrazide and
thiosemicarbazide-based equilibrators 3 and 4.
Both aniline 1 and hydrazide 3 have been employed in
previous studies to afford faster exchange in dynamic
10,12
systems.
Whereas use of hydrazide 3 in acylhydrazone-
based dynamic combinatorial libraries results in formation of
linear acylhydrazone species along with the macrocyclic library
members, we were delighted to see that this was not the case
for our thiosemicarbazone-based system. Thus, use of hydrazide
3
(10 mM) was found to afford efficient exchange without any
detectable signs of linear acylhydrazones in the mixture.
Depending on conditions and what building blocks were
used, dynamic combinatorial libraries set up by use of
equilibrator 3 reached equilibrium within 5−15 days, as
illustrated in Figure 3, which shows the composition of two
Figure 2. (Top) Schematic representation of acid-catalyzed hydrolysis
of the acetal protecting group and subsequent macrocyclization.
(
Bottom) HPLC-UV chromatograms (at 325 nm) of libraries started
from either building block A or from isolated macrocycle A after 28
2
days of equilibration in CHCl with 5 vol % TFA.
3
To overcome this apparent barrier for equilibration, we
tested a range of nucleophilic catalysts for their ability to afford
efficient exchange between library members and thereby allow
the DCL to reach a thermodynamic equilibrium. The catalysts
are envisioned to perform a nucleophilic attack on the C−N
double bond of the thiosemicarbazones. This leads to a linear
thiosemicarbazide, which can then intermolecularly open
another macrocyclic library member or perform an intra-
molecular ring closure, resulting in faster scrambling of the
1
0,12
library.
Because of their specific purpose in this study, these
nucleophilic catalysts were termed equilibrators. Several
possible equilibrators were screened, ranging in nucleophilicity
from aniline 1 up to aromatic thiosemicarbazide 4 (Chart 2).
Figure 3. Applying equilibrator 3 afforded DCLs at thermodynamic
equilibrium, as evidenced by the fact that mixed A + B libraries (1:1,
1
.0 mM total building block concentration in CHCl with 15 vol %
3
Chart 2. Equilibrator Structures
methanol, 10 vol % acetonitrile, and 1 vol % TFA) reached the same
composition whether they were started by combining the building
blocks A and B or by mixing already formed libraries of A and B.
different libraries made from A and B in equimolar amounts
(
1.0 mM total building block concentration in CHCl with 15
3
vol % methanol, 10 vol % acetonitrile, and 1 vol % TFA). One
library was set up simply by mixing the two building blocks.
The other library was made by mixing a preformed library of A
with a preformed library of B. A few hours after the two
preformed libraries were mixed, macrocycles that contained
both A and B monomer units were detected, and after 15 days,
the two libraries had reached the same composition. This
observation proves that thiosemicarbazone exchange can be
used to form dynamic systems at true thermodynamic
equilibrium by use of an equilibrator such as hydrazide 3.
Use of thiosemicarbazide-based equilibrator 4 (10 mM) also
resulted in efficient exchange, giving rise to a dynamic system
that reached thermodynamic equilibrium within a matter of 4−
6 days. Not surprisingly, libraries with equilibrator 4 contained
Libraries starting from either A or A were set up in the
2
presence of equilibrator, and the composition of the resulting
libraries was followed by HPLC-UV daily. Use of aniline 1 (100
mM) did speed up the exchange, though the exchange was still
too slow to be practically useful. After 28 days in the presence
of 1, the two libraries started from A and A had not yet
2
reached identical compositions (Figure S1).
Though benzylamine 2 is orders of magnitude more
1
3
nucleophilic, use of 2 (100 mM) did not result in faster
exchange in comparison to aniline 1, presumably because of the
concomitant higher degree of protonation of the nucleophilic
C
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
linear thiosemicarbazone species along with the macrocyclic
library members (Figure S2). This illustrates that thiosemi-
carbazone exchange can be applied to dynamic systems
containing both macrocyclic and linear library members.
Furthermore, along with the notion that these DCLs formed
These aromatic cyclization reactions are, for all practical
applications, completely irreversible, and in light of the
aforementioned formation of linear library members when
equilibrator 4 was used, this side reaction results in trapping the
system in an undesired static state.
[
2]catenanes (as will be discussed), we propose that
A range of carboxylic acids were screened to test for this
thiosemicarbazone-based dynamic systems in a future study
can be the basis for further exploration of the recently proposed
expansion of the Jacobson−Stockmayer cyclization theory to
undesired condensation, and the extent of condensation was
found to rely on both the pK of the acid and the apparent
a
steric bulk of the carboxylic acid (Table S1). It was also found
that addition of as little as 0.5 vol % water efficiently prevented
the condensation with TFA, and therefore we developed
conditions in which up to 1 vol % water was used. This was
made possible by adding methanol (15 vol %) and/or
14
include reversible formation of [2]catenanes.
Use of an equilibrator adds another level of control over the
dynamic system, as we will exploit further, but it also adds to
the overall complexity of the system, and sometimes
unexpected side reactions start to occur. We found that using
either hydrazide 3 or thiosemicarbazide 4 as equilibrator
resulted in formation of unexpected condensation products 5
acetonitrile (10 vol %) to the otherwise CHCl -based solvent.
3
Further examination revealed that sulfonic acids such as
trifluoromethanesulfonic acid and p-toluenesulfonic acid could
also be used to furnish dynamic systems, and inorganic acids
such as aqueous HBr or HCl were also applicable when care
was taken not to use too high acid concentrations in methanol-
containing (15 vol %) solvent mixtures (Tables S2 and S3).
Due to their high acidities, use of 1 vol % concentrated aqueous
HBr or HCl was found to afford methylation of the library
members via protonation of methanol and subsequent
nucleophilic attack by a lone pair, presumably from a nitrogen
or sulfur atom, of the library member (Scheme 3). This
mechanism of methylation was confirmed by setting up a
and 6 with TFA when applied in CHCl (Scheme 1).
3
Scheme 1. Condensation Products 5 and 6
library with 15 vol % CD OD in place of nonisotopically
3
labeled methanol (Figures S3 and S4).
Scheme 3. Methylation of Library Members by Protonated
Methanol
If this condensation reaction is truly reversible, in itself it
does not pose a problem for the dynamic system. It was found,
however, that when hydrazide 3 was used, the effective
concentration of nucleophilic equilibrator became so low that
the exchange reaction slowly halted, and for thiosemicarbazide
4
it was found, as evidenced by LC−MS, that condensation
product 6 underwent further cyclization reactions to form
either of two isomers, triazole 7 or thiadiazole 8 (Scheme 2).
In summary, we identified suitable conditions under which
thiosemicarbazones can exchange to reach a true thermody-
namic equilibrium in a matter of days, mediated by use of a
nucleophilic hydrazide or thiosemicarbazide equilibrator under
acidic conditions in primarily organic solvents. We found that
the condensation products that follow from using a hydrazide
or a thiosemicarbazide equilibrator in conjunction with a
carboxylic acid can be avoided by addition of small amounts of
water (0.5−1 vol %) to the reaction mixture. Use of methanol
as a cosolvent can lead to unwanted methylation reactions over
time, but this occurs only in the presence of high excess of
strong mineral acids (HBr or HCl). Alternatively, thiosemi-
carbazone DCLs can be set up by use of sulfonic acids, such as
p-toluenesulfonic acid and trifluoromethanesulfonic acid.
a
Scheme 2. Cyclization Products 7 and 8
2
.3. Distinction of Macrocyclic Sequence Isomers by
HPLC−MS/MS. When building blocks A and C were used
1:1, 1.0 mM total building block concentration in CHCl with
(
1
3
5 vol % methanol, 10 vol % acetonitrile, and 1 vol % TFA) to
form a mixed A + C library, a complex mixture of both
homomeric (A , A , C , A , and others) and heteromeric
2
3
3
4
(
A C , A C , A C , A C , and others) macrocyclic oligomers
1 2 1 3 2 1 2 2
was formed. A chromatographic method capable of separating
most of the peaks in the diverse library was developed, and it
was found that two distinct tetrameric species with retention
times (RT) of 6.1 and 7.9 min, respectively, both consisted of
two A units and two C units (Figure 4a). Since the two
a
Reaction was performed in CHCl3 with TFA (10 vol %).
Chromatogram at 325 nm after 4 h showed formation of condensation
product 6, while the chromatogram taken after 4 days showed
predominant presence of cyclization products 7 and 8.
D
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 4. (a) HPLC-UV chromatogram (325 nm) of a library of
building blocks A and C. (b) Tandem mass spectrometry (MS/MS) of
the molecular ion (m/z 959.3, shown in blue) of the A C species
2
2
eluting at ca. 6.1 min. (c) MS/MS of the molecular ion (m/z 959.3,
shown in blue) of the A C species eluting at ca. 7.9 min. Fragment
2
2
ions corresponding to loss of one or two monomer units are shown in
red.
isomeric A C oligomers are cyclic (i.e., not linear), only two
2
2
structural isomers are possible: an alternating isomer (-A-C-A-
C-) and a nonalternating isomer (-A-A-C-C-). All possible
[
[
2]catenane structures ([A ]·[A C ], [A ]·[C ], [A C ]·
1 1 2 2 2 1 1
A C ], and [A C ]·[C ]) were ruled out on the basis of the
1
1
2
1
1
Figure 5. (a) HPLC-UV chromatogram (325 nm) of a library of
building block A with peaks assigned on the basis of MS. (b) MS/MS
at a collision cell energy of 70 eV of hexameric species at RT = 3.9 min
experimental fact that neither A , C , A C , nor C was
detected experimentally, nor do they seem likely on the basis of
simple molecular mechanics.
HPLC-MS/MS study of the two species revealed remarkably
different fragmentation patterns, underlining that the two
isomers are indeed distinct molecular entities and not two
quasi-stable conformations of otherwise identical molecules.
The species eluting at 6.1 min had only one large dimeric
1
2
1
1
1
(left) and RT = 4.1 min (right). (c) Distribution of intensities of
hexameric, pentameric, tetrameric, trimeric, and dimeric ions in MS/
MS analysis of both hexameric species as a function of applied collision
cell energy. (d) Structural representations of distinct hexameric species
corresponding to the peak at RT = 3.9 min (left) and 4.1 min (right).
+
fragment ion corresponding to [A C + H] , as well as trimeric
fragments corresponding to [A C + H] and [A C + H]
filter and fragmented in a hexapole collision cell, and finally the
intensity of daughter ions (and surviving molecular ion) was
measured by passing the ions through a time-of-flight mass
spectrometer. The two hexamers showed clearly divergent ion
breakdown patterns. The species in the early peak (RT 3.9
min) produced several identifiable daughter ions (at a collision
cell energy of 70 eV) corresponding to loss of exactly one, two,
or three monomer units, with each daughter ion at lower
intensity than the molecular ion (Figure 5b). On the other
hand, at the same collision cell energy, the later-eluting hexamer
(RT 4.1 min) produced only one daughter ion corresponding
to loss of exactly three monomer units, and the intensity of this
daughter ion was twice that of the molecular ion.
1
1
+
+
1
2
2 1
(
Figure 4b). The MS/MS spectrum of the peak eluting at 7.9
min also contained all of these fragment ions, albeit at lower
intensities, but a dimeric fragment ion corresponding to [A +
H] was also evident (Figure 4c). If the unlikely scenario of
ion−ion crossover is ruled out, the only isomer that can
produce [A + H] fragment ions is the nonalternating isomer
2
+
15
+
2
(
-A-A-C-C-).
Similar MS/MS analyses were also used to distinguish two
isomers of A B . Thus, a nonalternating version (-A-A-B-B-)
was found to elute after 4.2 min, while the alternating isomer
(
2
2
-A-B-A-B-) eluted after 4.8 min (peaks marked A B in Figure
2 2
3
; further details in Figure S5).
Upon sweeping the collision cell energy from 40 to 85 eV in
a series of consecutive runs, a clear pattern emerged: the
hexamer at 3.9 min produced pentameric, tetrameric, and
trimeric daughter ions, while the hexamer at 4.1 min produced
only trimeric daughter ions (Figure 5c). In both cases, dimeric
daughter ions were also seen at higher collision cell energies.
This evidence strongly suggests that the species eluting at 3.9
2.4. Identification of a [2]Catenane from Dynamic
System of Macrocyclic Thiosemicarbazones. It was found
that dynamic libraries of building block A contained two
distinct hexameric species (Figure 5a). To identify the
structures of these two hexamers, we performed a HPLC-
MS/MS study. The molecular ion corresponding to a hexamer
+
of A (m/z 1814.9, [M + H] ) was selected by a hexapole mass
min is a macrocyclic hexamer (i.e., a cyclic A species), while
6
E
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 6. A dynamic mixture of macrocyclic oligomers of A is formed by aid of equilibrator 3. The dynamic exchange of this mixture is then turned
off by addition of 18-crown-6, which binds the equilibrator to form nonnucleophilic complex 3·18C6. Turning the dynamic state of the system back
+
on can be achieved by addition of a potassium salt, since the K ion will competitively bind to 18-crown-6 and thereby liberate the equilibrator.
the species eluting at 4.1 min consists of two mechanically
interlocked macrocyclic trimers (i.e., two A species forming a
3
[
2]catenane, [A ]·[A ] (Figure 5d). The HPLC-UV−MS/MS
3
3
data do not allow for the distinction between the two
diastereotopic [A ]·[A ] [2]catenanes, nor is it possible to
3
3
verify whether the library produces both diastereomers or only
one. The formation of this [2]catenane, which is the first
thiosemicarbazone-based catenane reported, illustrates the
propensity for dynamic systems to build up molecular
complexity.
2
.5. From Dynamic to Static and Back: Supra-
molecular On/Off Switch. The ability to reach a thermody-
namic equilibrium is a defining trait of a true dynamic
combinatorial library, but the ability to stop the equilibration
process can be of equal importance in dynamic systems. For
instance, in order to study a specific transient composition or
isolate a short-lived library member, it is useful, and sometimes
required, to be able to turn off the dynamic exchange reaction
at any given time. Ideally, the method to stop the dynamic
exchange should be simple, work instantaneously, and be
reversible, in the sense that it is possible to turn back on the
dynamic exchange, that is, reinitiate the exchange reaction to re-
establish a dynamic state.
The fact that the thiosemicarbazone dynamic system
developed herein requires a nucleophilic catalyst equilibrator
was exploited to implement such an on/off protocol. Addition
of 18-crown-6 (1.6-fold excess compared to equilibrator) to a
library of building block A resulted in trapping of the
equilibrator, as it forms an ammonium ion−crown ether
complex, 3·18C6 (Figure 6). This effectively stopped the
dynamic exchange reaction as evidenced by the lack of change
in composition (Figure 7). In essence, 18-crown-6 turns off the
dynamic state of the system by preventing the dynamic
exchange reaction from taking place.
Figure 7. Composition of dynamic library of A (chromatograms at 325
nM) as a function of time (days). Addition of 18-crown-6 completely
stops library evolution by effectively removing equilibrator 3 from the
mixture. Library evolution is started again immediately upon addition
+
of KBr to release 3 by competitive binding of 18-crown-6 by K ions.
3
. CONCLUSIONS
We have developed the first example of a thiosemicarbazone-
based dynamic combinatorial library. With the set of building
blocks produced so far, we showcase that a nucleophilic
catalyst, dubbed an equilibrator, is necessary to facilitate the
exchange reaction, allowing us to obtain a dynamic system of
covalently bound oligomers at thermodynamic equilibrium.
We have shown that the dynamic system produces complex
mixtures of macrocyclic oligomers, including formation of a
[2]catenane, which was identified by HPLC−MS/MS. Finally,
we have demonstrated how the dynamic state of the system can
be turned off by addition of 18-crown-6, which binds the
equilibrator in an ammonium ion−crown ether complex. The
equilibration between oligomers can be reinitiated by addition
of potassium ions, which preferentially bind to 18-crown-6 and
therefore liberate the equilibrator.
Finally, by exploiting 18-crown-6’s high affinity for potassium
ions (log K = 6.10, compared to log K = 4.27 for ammonium
4
. EXPERIMENTAL SECTION
Unless stated otherwise, all starting materials and solvents were
purchased from commercial suppliers and used as received. HPLC-
grade solvents were used for synthesis and library setup, and when
needed they were dried prior to synthesis by standing over molecular
sieves. Water content of dried solvents and reagents was measured on
a Metrohm 737 KF coulometer. Dry column vacuum chromatography
16
ions in methanol), it was possible to restart the exchange
reaction by shaking the DCL with KBr (ca. 3-fold excess
compared to equilibrator 3 was added, though not all of the
KBr dissolved). Thus the dynamic system, which was
deliberately frozen long before it had reached its thermody-
namic equilibrium, started adjusting composition immediately
after equilibrator 3 was released from its 18-crown-6 trap
(
DCVC) was performed with 60 Å, 400−800 mesh silica and solvents
of technical or HPLC grade. Melting points are uncorrected.
NMR spectra were recorded on a Bruker Ultrashield Plus 500
1
13
19
(Figure 7).
spectrometer, operating at 500 MHz ( H) and 126 MHz ( C). All F
F
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
spectra, however, were recorded on an Oxford NMR 300
spectrometer, operating at 282 MHz. Unless specified otherwise, all
spectra were obtained at 298 K and are referenced to the internal
solution was cooled in an ice bath, a 60% aqueous solution of
hexafluorophosphoric acid (125 mL, 0.85 mol) were added at such a
rate that the solution temperature did not exceed 5 °C (approximately
10 min). After addition of water (0.5 L), the organic phase was
separated, dried over Na SO , filtered, and concentrated in vacuo. The
1
13
19
solvent residue for H and C and to TFA in a sealed tube for F.
CDCl was dried by standing over molecular sieves (4 Å) and treated
3
2
4
with oven-dried basic aluminum oxide when the presence of water
and/or acid sensitivity was an issue. Chemical shifts (δ) are quoted in
parts per million (ppm), and coupling constants (J) are listed in hertz.
The following abbreviations are used for convenience in reporting the
multiplicities of NMR resonances: s, singlet; d, doublet; t, triplet; q,
quartet; p, pentet; m, multiplet; br s, broad singlet. The NMR data
were processed by use of MestReNova v8.0.0 from MestreLab
remaining crude tetramethylchloroformadinium hexafluorophosphate
salt was dissolved in acetonitrile (200 mL), and KF (50 g, 0.86 mol;
predried at 125 °C overnight) was added. The solution was stirred at
room temperature for approximately 20 h before the precipitate was
filtered off. The mother liqueur was concentrated in vacuo, and the
resulting precipitate was recrystallized by redissolving it in acetonitrile
(50 mL), adding diethyl ether (100 mL), and stirring the resulting
two-phase system vigorously at ÷42 °C (dry ice/acetonitrile bath).
Overall yield: 111 g (50%) of white crystals.
1
13
Research S.L. Assignment of H and C resonances was achieved by
standard one- and two-dimensional NMR techniques: correlation
spectroscopy (COSY), total correlation spectroscopy (TOCSY), 13
C
Melting point 98−101 °C (lit. 108−110 °C). 1H NMR (500
17
13
attached proton test (APT), heteronuclear single quantum coherence
MHz, CD CN) δ 3.14 (s). C NMR (126 MHz, CD CN) δ 157.6 (d,
3
3
1
13
(
(
HSQC; H and C), and heteronuclear multiple-bond correlation
J = 270.4), 40.4.
1
13
HMBC; H and C).
4.2. 4-Dimethoxymethylbenzoic Acid. To a solution of 4-
formylbenzoic acid (20.2 g, 0.13 mol) in methanol (380 mL, 0.35 M)
was added ammonium chloride (30.0 g, 0.56 mol) in one portion. The
solution, which contained some undissolved ammonium chloride, was
heated to reflux for 24 h before the methanol was evaporated in vacuo.
The remaining solid was dissolved in diethyl ether (250 mL) and
filtered to remove undissolved ammonium chloride. The ethereal
phase was washed with water (100 mL), dried over Na SO , and
Mass spectrometry, including high-resolution mass spectrometry,
was performed on the LC−MS apparatus described below. Isotopic
patterns were calculated and visualized by use of IsotopePattern, a part
of the LC−MS software package DataAnalysis developed by Bruker
Daltonics GmbH. This software package was also used to visualize
HPLC chromatograms.
HPLC analysis was performed on a Dionex UltiMate 3000 system,
which incorporates an UltiMate 3000 diode-array UV−vis detector
capable of measuring absorbance of light in the 190−800 nm range.
LC−MS was carried out by connecting the HPLC apparatus to a
Bruker MicrOTOF-QII system equipped with an electrospray
ionization (ESI) source with nebulizer gas at 1.2 bar, dry gas at 8
L/min, dry temperature at 200 °C, capillary at 4500 V, and end plate
offset at −500 V. For m/z values in the 100−1000 range, ion transfer
was conducted with funnel 1 and funnel 2 voltage at 200.0 peak-to-
peak voltage (Vpp) and hexapole voltage at 100.0 Vpp while the
quadrupole ion energy was set at 5.0 eV with a low mass cutoff at
2
4
filtered before the solvent was removed in vacuo to give 28.75 g (83%)
of a white powder.
18
Melting point 119−120 °C (lit. 118−119 °C). 1H NMR (500
MHz, CDCl ) δ 11.30 (br s, 1H), 8.12 (d, J = 8.3 Hz, 2H), 7.58 (d, J =
3
13
8.2 Hz, 2H), 5.46 (s, 1H), 3.34 (s, 6H). C NMR (126 MHz, CDCl3)
δ 170.8, 144.0, 130.3, 129.3, 127.1, 102.4, 52.9. Anal. Calcd for
C H O : C, 61.22; H, 6.16. Found: C, 61.41; H, 6.03. HRMS (ESI-
1
0
12
4
+
+
TOF) m/z [M + Na] calcd for C H O Na 219.0628, found
1
0
12
4
−
−
219.0632; [M − H] calcd for C H O 195.0663, found 195.0666.
4.3. N-[(1R,2R)-2-Aminocyclohexyl]-4-(dimethoxymethyl)-
benzamide. 4-Dimethoxymethylbenzoic acid (1.34 g, 6.83 mmol)
10
11
4
1
00.00 m/z. In the collision cell, collision energy was set at 8.0 eV and
collision voltage at 100.0 Vpp, and a transfer time of 80.0 μs and
prepulse storage of 1.0 μs were used. For detection of m/z values in
the 500−2500 range, the following parameters were changed: transfer
was conducted with funnel 1 voltage at 300.0 Vpp, funnel 2 voltage at
and TFFH (1.81 g, 6.85 mmol) were dissolved in CH Cl (200 mL,
2 2
0.03 M) at 0 °C, and triethylamine (4.8 mL, 35 mmol) was added.
After 5 min, (1R,2R)-1,2-diaminocyclohexane (0.82 g, 7.2 mmol) was
added and the reaction mixture was stirred for 3 days at room
temperature. The reaction mixture was washed with water (200 mL),
4
00.0 Vpp, and hexapole voltage at 400.0 Vpp, while the low mass
cutoff in the quadrupole was changed to 300.00 m/z. The collision cell
energy was raised to 10.0 eV and the collision voltage to 630.0 Vpp.
Generally, both detection ranges were used when analyzing libraries to
ensure identification of all peaks, with low-range settings used during
the first minutes and high-range settings used for the remainder of the
analysis.
Solvents and additives of LC−MS grade were purchased from
commercial suppliers and used as received. Water was purified on a
Millipore Milli-Q Integral 5 system.
and the resulting aqueous phase was washed with CH
mL) before the combined organic phases were dried (Na
2
Cl
2
2
(3 × 60
4
SO ) and
the product was purified by DCVC. A column of 5 cm diameter
packed with 6 cm of silica was used, and all fractions contained 0.1%
triethylamine and had a volume of 50 mL. Gradient: pure heptane to
pure EtOAc in 25% increments and then to 30% methanol in EtOAc
in 5% increments. Product coeluted with an unidentified impurity (as
observed by thin-layer chromatography) in fractions containing 5−
25% methanol. Therefore, these fractions were combined and
subjected to further purification by DCVC, this time first eluting the
byproduct by running four fractions of EtOAc on a column of the
same dimensions and finally eluting the pure product by addition of
methanol to the EtOAc fractions in 10% increments until all the
product had eluted. Evaporation of solvent yielded 1.12 g (60%) of
Separations were achieved by use of one of four columns I−IV, all
maintained at 20 °C unless stated otherwise: (I) Dionex Acclaim rapid
separation liquid chromatography (RSLC) 120 C18, 2.2 μm, 120 Å,
2
.1 × 50 mm column; (II) same as I except a C8 stationary phase and
dimensions of 2.1 × 150 mm; (III) Dionex Acclaim RSLC
PolarAdvantage II (PA2) C18, 2.2 μm, 120 Å, 2.1 × 50 mm column;
pale yellow crystals.
Melting point 168−169 °C (decomp). H (500 MHz, CDCl
1
(
(
IV) Waters Acquity ultraperformance liquid chromatography
UPLC) high-strength silica (HSS) C18, 1.8 μm, 100 Å, 2.1 × 50
3
) δ
7.78 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 6.16−6.00 (m, 1H),
5.44 (s, 1H), 3.78−3.67 (m, 1H), 3.32 (s, 6H), 2.55−2.44 (m, 1H),
2.21−2.11 (m, 1H), 2.06−1.97 (m, 1H), 1.81−1.72 (m, 2H), 1.47−
mm column. The mobile-phase solutions prepared were (A) 0.1%
formic acid in water and (B) 0.1% formic acid in acetonitrile. Injection
volumes were generally between 0.10 and 0.30 μL. The column was
conditioned to the starting eluent with at least five column volumes
prior to each injection.
1.17 (m, 6H). 13C NMR (126 MHz, CDCl
) δ 167.4, 141.4, 134.7,
3
126.9, 126.7, 102.2, 56.6, 55.7, 52.5, 35.7, 32.5, 25.1, 25.0. HRMS
+
(ESI-TOF) m/z [M + H] calcd for C16H N O 293.1860, found
25 2 3
4
.1. Tetramethylfluoroformadinium Hexafluorophosphate
293.1866.
(
TFFH). Tetramethylurea (100 mL, 0.85 mol) and phosphorus
4.4. Building Block A. N-[(1R,2R)-2-Aminocyclohexyl]-4-
(dimethoxymethyl)benzamide (1.17 g, 4.00 mmol) was dissolved in
anhydrous methanol (35 mL, 30 ppm). Carbon disulfide (2.42 mL, 40
mmol) and triethylamine (0.73 mL, 4.0 mmol) were added and the
resulting solution was stirred for 20 min before the reaction mixture
was cooled in an ice bath. Di-tert-butyl dicarbonate (840 mg, 3.85
oxychloride (90 mL, 0.96 mol) were stirred at 100 °C under N2
atmosphere for 1 h before the solution was allowed to reach room
temperature. The solution was transferred to a 5 L three-necked
round-bottomed flask equipped with a mechanical stirrer, an addition
funnel, and a thermometer, and CH Cl (2 L) was added. While the
2
2
G
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
mmol) dissolved in methanol (5 mL) and 1,4-diazabicyclo[2.2.2]-
octane (DABCO; 4.4 mg, 1 mol %) were added, and after the mixture
was stirred for 5 min, the ice bath was removed. The solution was
stirred for an additional 15 min before the solvent was removed in
4.7. Methyl 2-(Benzyloxy)-5-formylbenzoate. Methyl 5-
formyl-2-hydroxybenzoate (2.31 g, 12.8 mmol) was dissolved in
CH Cl /methanol (1:1, 90 mL), and benzyl bromide (7 mL, 0.06
2
2
mol) and K
heated to boiling and kept at refluxing temperature for 2 days before it
was allowed to reach room temperature. Excess K CO was removed
CO (9.56 g, 69 mmol) were added. The mixture was
2
3
vacuo. The remaining raw isothiocyanate was redissolved in CH Cl2
2
(
1
40 mL) and cooled in an ice bath before hydrazine hydrate (60%,
.94 mL, 40 mmol) was added dropwise over 5 min. After addition,
the ice bath was removed and the solution was stirred for an additional
5 min before the reaction mixture was quenched with water (30 mL).
2
3
by filtration, and the solvent was removed by rotary evaporation. The
crude product was purified by DCVC with a 5% gradient of ethyl
acetate in heptane (column diameter 6 cm packed with ca. 6 cm silica,
fraction volume 100 mL). Fractions 5−8 were combined and
concentrated and the resulting residue was purified once more by
DCVC with the same parameters as before. Fractions 7 and 8 were
combined and concentrated to reveal the title compound as a clear
1
The organic phase was collected, and the aqueous phase was extracted
with CH Cl (3 × 20 mL). The combined organic phases were dried
2
2
over Na SO , filtered, and concentrated on Celite in vacuo for
2
4
purification by DCVC. A column of 4 cm diameter packed with 6 cm
of silica was used, and all fractions were 50 mL and contained 0.5%
triethylamine. A gradient of 20% ethyl acetate in heptane, followed by
a gradient of 2% methanol in ethyl acetate, was used. Fractions 6−9
were collected and concentrated in vacuo to yield 1.17 g (80%) of a
light yellow oil. Yield 2.20 g (64%).
1
H NMR (500 MHz, CDCl ) δ 9.91 (s, 1H), 8.36 (d, J = 2.2 Hz,
3
1
H), 7.98 (dd, J = 8.7, 2.2 Hz, 1H), 7.49 (d, J = 7.5 Hz, 2H), 7.41 (t, J
=
(
7.5 Hz, 2H), 7.34 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H), 5.29
s, 2H), 3.94 (s, 3H). 13C NMR (126 MHz, CDCl ) δ 190.0, 165.5,
slightly yellow powder.
Melting point 176−178 °C. H NMR (500 MHz, CDCl ) δ 7.91 (d,
J = 8.3 Hz, 2H), 7.53 (s, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.46 (s, 1H),
3
1
1
7
2
62.6, 135.7, 134.6, 134.3, 129.3, 128.7, 128.2, 126.8, 121.1, 113.7,
3
+
+
0.8, 52.3. HRMS (ESI-TOF) m/z [M + H] calcd for C H O
16 15
4
71.0965, found 271.0967.
6
3
1
.87 (s, 1H), 5.41 (s, 1H), 4.56−4.45 (m, 1H), 3.98−3.89 (m, 1H),
4.8. Methyl 2-(Benzyloxy)-5-(5,5-dimethyl-1,3-dioxan-2-yl)-
.67 (s, 2H), 3.32 (s, 6H), 2.32−2.26 (m, 1H), 2.17−2.11 (m, 1H),
13
benzoate. Methyl 2-(benzyloxy)-5-formylbenzoate (2.20 g, 8.15
mmol) was dissolved in toluene (125 mL), and 2,2-dimethyl-1,3-
propanediol (4.97 g, 48 mmol) and p-toluenesulfonic acid (0.15 g,
.86−1.77 (m, 2H), 1.48−1.32 (m, 4H). C NMR (126 MHz,
CDCl ) δ 182.6, 167.2, 141.4, 134.5, 127.4, 127.0, 102.6, 56.6, 56.0,
3
5
7
2.8, 32.6, 32.6, 25.1, 24.7. Anal. Calcd for C H N O S: C, 55.71; H,
17 26 4 3
0
.87 mmol) were added. The mixture was heated to boiling, and water
.15; N, 15.29. Found: C, 55.90; H, 7.14; N, 15.08. HRMS (ESI-TOF)
+
+
was removed by azeotropic distillation with a Dean−Stark trap. After 5
h, no more water distilled over, and the reaction mixture was allowed
to reach room temperature before the solvent was removed by rotary
evaporation. The product was purified by DCVC with a 1% gradient of
ethyl acetate in toluene with 1% triethylamine (column diameter 6 cm,
packed with ca. 6 cm silica, fraction volume 100 mL). Fractions 3−6
were combined and concentrated to reveal the desired product as light
m/z [M + H] calcd for C H N O S 367.1798, found 367.1801; [M
+
1
7
27
4
3
+
+
Na] calcd for C H N NaO S 389.1618, found 389.1618.
17 26 4 3
4
.5. Dimer A . Building block A (100.0 mg) was dissolved in
2
CH Cl (2 L), and TFA (2.5 mL) was added. After the mixture was
2
2
stirred for 20 h at room temperature, the bulk of the solvent was
removed by rotary evaporation before the crude product was
thoroughly dried in high vacuum. The crude product was redissolved
in CH Cl and evaporated onto Celite for purification by DCVC. A
yellow crystals. Yield 1.39 g (48%).
2
2
1
Melting point 114−115 °C. H NMR (500 MHz, CDCl ) δ 7.97 (d,
3
column of 3 cm diameter packed with 6 cm of silica was used. All
fractions were 30 mL and contained 4% triethylamine. A gradient of
J = 2.3 Hz, 1H), 7.59 (dd, J = 8.7, 2.3 Hz, 1H), 7.49−7.47 (m, 2H),
7
(
2
1
1
.38−7.35 (m, 2H), 7.31−7.29 (m, 1H), 7.00 (d, J = 8.6 Hz, 1H), 5.36
1
0% ethyl acetate in heptane was used, and 18.6 mg of the pure dimer
s, 1H), 5.20 (s, 2H), 3.90 (s, 3H), 3.77−3.74 (m, 2H), 3.65−3.62 (m,
was obtained as a slightly yellow powder from fractions 9 and 10.
Fractions 11 and 12 were combined and another column (same
parameters) was run on the crude product from these fractions to yield
another 21.3 mg of the pure dimer. Yield 40.3 mg (49%).
H), 1.28 (s, 3H), 0.80 (s, 3H). 13C NMR (126 MHz, CDCl ) δ
3
66.5, 158.6, 136.8, 131.3, 131.2, 130.1, 128.7, 127.9, 126.9, 120.5,
13.9, 101.0, 77.8, 70.8, 52.1, 30.3, 23.2, 22.0. MS (EI) 356.2. Anal.
Calcd for C H O : C, 70.77; H, 6.79. Found: C, 70..86; H, 6.66.
1
21 24
5
Melting point 248−255 °C (decomp). H NMR (500 MHz,
4
.9. 2-(Benzyloxy)-5-(5,5-dimethyl-1,3-dioxan-2-yl)benzoic
Acid. Methyl 2-(benzyloxy)-5-(5,5-dimethyl-1,3-dioxan-2-yl)benzoate
1.11 g, 3.11 mmol) was dissolved in tetrahydrofuran (50 mL), and
CD CN) δ 9.38 (s, 2H, NH), 7.98 (d, J = 4.6 Hz, 2H, NH), 7.82 (d, J
3
=
8.3 Hz, 4H), 7.71 (s, 2H, imine-CH), 7.52 (d, J = 8.3 Hz, 4H), 7.24
(
(
d, J = 8.4 Hz, 2H, NH), 4.38 (qd, J = 11.8, 3.7 Hz, 2H), 3.77 (tt, J =
aqueous LiOH (1 M, 4 mL, 4 mmol) was added. The mixture was
stirred at ambient temperature for ca. 60 h, after which an additional
amount of aqueous LiOH (1 M, 4 mL, 4 mmol) was added. After
stirring for 5 h, aqueous NaOH (2 M, 3 mL, 6 mmol) was added, and
the mixture was stirred at room temperature for 22 h before it was
heated to boiling and kept at refluxing temperature for ca. 4 h. After
the mixture was cooled to room temperature, aqueous HCl (2 M, 35
mL) was added and the mixture was extracted with CH Cl (3 × 100
1
1
4
0.9, 4.3 Hz, 2H), 2.99−2.93 (m, 2H), 2.01 (d, J = 12.0 Hz, 2H),
.88−1.75 (m, 4H), 1.63 (qd, J = 12.5, 3.6 Hz, 2H), 1.56−1.36 (m,
13
H), 1.35−1.23 (m, 2H). C NMR (126 MHz, CD CN) δ 180.0
3
(
6
CS), 167.9 (CO), 141.8 (imine-C), 137.8, 134.9, 128.9, 127.7,
+
1.4, 53.0, 32.9, 32.9, 26.3, 25.0. HRMS (ESI-TOF) m/z [M + Na]
+
calcd for C H N NaO S 627.2295, found 627.2301.
30
36
8
2 2
4.6. Methyl 5-Formyl-2-hydroxybenzoate. 5-Formyl-2-hydrox-
2
2
ybenzoic acid (2.22 g, 0.134 mol) was suspended in methanol, and
concentrated sulfuric acid (1 mL) was added dropwise over ca. 5 min.
The mixture was heated to boiling and kept at refluxing temperature
for 24 h. After cooling to room temperature, the mixture was
concentrated by rotary evaporation. The crude product was dissolved
in ethyl acetate and filtered through a plug of silica. Ethyl acetate was
removed by rotary evaporation. The crude product was dissolved in
mL). The organic extracts were dried (Na SO ) and concentrated to
2
4
reveal the desired product as a light yellow oil. Yield 1.03 g (97%).
1
H NMR (500 MHz, CDCl ) δ 8.31 (d, J = 2.2 Hz, 1H), 7.72 (dd, J
3
=
8.6, 2.2 Hz, 1H), 7.43−7.38 (m, 5H), 7.11 (d, J = 8.6 Hz, 1H), 5.38
(s, 1H), 5.29 (s, 2H), 3.77−3.73 (m, 2H), 3.65−3.62 (m, 2H), 1.27 (s,
13
3
H), 0.79 (s, 3H). C NMR (126 MHz, CDCl ) δ 165.5, 157.7,
3
1
34.4, 133.2, 132.9, 132.2, 129.2, 127.9, 117.9, 113.3, 100.6, 77.7, 72.4,
+
methanol (35 mL), and CH Cl was added. The solution was washed
2
2
30.3, 23.2, 22.0. HRMS (ESI-TOF) m/z [M + H] calcd for
C H O 343.1540, found 343.1543.
+
with water and extracted with CH Cl (3 × 100 mL). The combined
2
2
20
23
5
organic phases were dried (Na SO ) and concentrated to reveal the
2
4
4.10. N-(1R,2R)-2-Aminocyclohexyl-2-(benzyloxy)-5-(5,5-di-
desired product. Yield 2.31 g (96%).
methyl-1,3-dioxan-2-yl)benzamide. 2-(Benzyloxy)-5-(5,5-dimeth-
yl-1,3-dioxan-2-yl)benzoic acid (1.03 g, 3.0 mmol) was dissolved in
CH Cl and cooled to 0 °C (ice bath). Tetramethylfluoroformadinium
1
Melting point 79−80 °C. H NMR (500 MHz, CDCl ) δ 11.36 (s,
3
1
1
H), 9.89 (s, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.01 (dd, J = 8.6, 2.0 Hz,
2
2
H), 7.11 (d, J = 8.6 Hz, 1H), 4.01 (s, 3H). 13C NMR (126 MHz,
hexafluorophosphate (0.88 g, 7.4 mmol) and triethylamine (2.0 mL,
14 mmol) were added, and the mixture was stirred for 10 min before
(1R,2R)-1,2-diaminocyclohexane (0.38 g, 3.3 mmol) was added. The
mixture was stirred for ca. 20 h at room temperature. CH Cl (50 mL)
CDCl ) δ 190.0, 170.1, 166.5, 135.8, 134.0, 128.8, 118.9, 112.8, 53.0.
3
MS: (EI) 180.1. Anal. Calcd for C H O : C, 60.00; H, 4.48. Found: C,
9
8
4
6
0.08; H, 4.53.
2
2
H
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
and water (100 mL) was added, and the mixture was extracted with
4.13. 2-(3-Nitrophenyl)-1,3-dioxolane. 3-Nitrobenzaldehyde
(15 mmol, 2.27 g, 1 equiv), p-toluenesulfonic acid (1.5 mmol, 285.3
mg, 0.1 equiv), and ethylene glycol (37.5 mmol, 2.1 mL, 2.5 equiv)
were mixed in toluene (0.5 M, 30 mL), and the mixture was heated to
reflux overnight with a Dean−Stark apparatus fitted to the setup. The
CH Cl (3 × 100 mL). The combined organic phases were dried
2
2
(
Na SO ) and concentrated. The product was purified by DCVC in
2 4
three consecutive runs with a 20−25% gradient of ethyl acetate in
heptane, followed by a 1−5% gradient of methanol in ethyl acetate.
The final compound was a light yellow solid. Yield 0.27 g (21%).
solution was cooled to room temperature, and saturated NaHCO
3
1
H NMR (500 MHz, CDCl ) δ 8.28 (d, J = 2.2 Hz, 1H), 7.70 (d, J
solution (40 mL) and EtOAc (40 mL) were added. The phases were
separated, and the aqueous phase was extracted with EtOAc (2 × 20
mL). The combined organic extracts were filtered through a plug of
silica and evaporated to dryness to yield a yellow oil. This was further
purified by dry column chromatography. A column of 2 cm diameter
was packed with 5 cm of silica. A gradient of 20% CH Cl in heptane,
3
=
=
8.5 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.44−7.39 (m, 5H), 7.05 (d, J
8.5 Hz, 1H), 5.38 (s, 1H), 5.13 (s, 2H), 3.75−3.71 (m, 2H), 3.68−
3.60 (m, 3H), 2.10−2.00 (m, 1H), 1.89−1.84 (m, 2H), 1.68−1.57 (m,
4H), 1.27 (s, 3H), 1.15−1.08 (m, 2H), 0.78 (s, 3H). C NMR (126
13
MHz, CDCl ) δ 165.0, 156.9, 135.4, 132.2, 130.8, 130.2, 128.9, 128.4,
3
2
2
1
2
4
21.8, 112.5, 101.1, 77.6, 71.6, 56.1, 55.8, 34.6, 32.2, 30.2, 25.0, 24.9,
followed by a gradient of 1% MeOH in CH Cl , was used to yield the
2 2
+
+
3.1, 21.9. HRMS (ESI-TOF) m/z [M + H] calcd for C H N O
target compound as a yellow solid (2.9 g, 99%).
26
35
2
4
1
39.2591, found 439.2610.
.11. N-(1R,2R)-2-Aminocyclohexyl-5-(5,5-dimethyl-1,3-di-
oxan-2-yl)-2-hydoxybenzamide. N-(1R,2R)-2-Aminocyclohexyl 2-
benzyloxy)-5-(5,5-dimethyl-1,3-dioxan-2-yl)benzamide (0.27 g, 0.62
H NMR (500 MHz, CDCl ) δ 8.35 (s, 1H), 8.22 (d, J = 8.2, 1H),
3
4
7.801 (d, J = 7.6, 1H), 7.56 (t, J = 7.9, 1H), 5.89 (s, 1H), 4.18−4.11
1
3
(m, 2H), 4.11−4.04 (m, 2H). C NMR (126 MHz, CDCl ) δ 148.4,
3
(
140.5, 132.8, 129.5, 124.1, 121.8, 102.4, 65.6. HRMS (ESI-TOF) m/z
+
+
mmol) was dissolved in methanol (30 mL), and Pd/C (10%, 27 mg)
suspended in methanol (10 mL) was added. By use of a three-way
valve equipped with a H2 balloon, three vacuum/H2 cycles were
performed to exchange the air in the flask for H , and the reaction
mixture was stirred for ca. 18 h. The mixture was filtered through a
plug of Celite, and the solvent was removed to reveal the desired
product as a white solid. Yield 0.18 g (86%).
[M + H] calcd for C H NO 196.0604, found 196.0600.
9 10 4
4.14. 2-(1,3-Dioxolan-2-yl)aniline. 2-(3-Nitrophenyl)-1,3-dioxo-
lane (14.3 mmol, 2.8 g, 1 equiv) was dissolved in MeOH (0.2 M, 70
mL), and 10% Pd/C (10 wt %, 280 mg) suspended in methanol (5
mL) was added. Hydrazine hydrate (71.5 mmol, 4.5 mL, 5 equiv) was
2
added, and the reaction mixture was stirred under N overnight. The
2
reaction mixture was filtered through a plug of Celite and concentrated
for analysis. NMR confirmed conversion to the desired product. The
crude material was dissolved in 1:1 CH Cl /hexane (90 mL) and
1
Melting point 134−135 °C. H NMR (500 MHz, CDCl ) δ 7.56 (s,
3
1
5
=
H), 7.49 (d, J = 8.5 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.65 (s, 1H),
.33 (s, 1H), 3.76 (d, J = 11.1 Hz, 2H), 3.73−3.69 (m, 1H), 3.64 (d, J
2
2
filtered through a short plug of silica. The product was eluted with
11.1 Hz, 2H), 2.56−2.49 (m, 1H), 2.11−2.06 (m, 1H), 2.02−1.99
additional 1:1 CH Cl /hexane (3 × 90 mL) and concentrated to give a
2
2
(
(
m, 1H), 1.78−1.73 (m, 2H), 1.40−1.32 (m, 2H), 1.27 (s, 3H), 1.25
colorless oil (1.32 g), which was used without further purification.
s, 2H), 0.80 (s, 3H). 13C NMR (126 MHz, CDCl ) δ 170.0, 162.2,
1
3
H NMR (500 MHz, CDCl ) δ 7.11 (t, J = 7.8 Hz, 1H), 6.81 (d, J =
3
1
3
32.0, 128.8, 124.1, 118.7, 114.8, 101.2, 77.7, 56.1, 55.2, 35.6, 32.5,
0.3, 25.1, 23.3, 22.0. HRMS (ESI-TOF) m/z [M + H] calcd for
7.6 Hz, 1H), 6.77 (s, 1H), 6.65 (d, J = 8.0 Hz, 1H), 5.68 (s, 1H),
+
13
4.10−4.02 (m, 2H), 4.01−3.93 (m, 2H), 3.78 (br s, 2H, NH ).
C
2
+
C H N O 349.2122, found 349.2122.
NMR (126 MHz, CDCl ) δ 145.5, 138.9, 129.4, 116.8, 116.2, 113.1,
1
9
29
2
4
3
4.12. Building Block B as Its Acetone Condensation Product.
103.8, 65.3.
N-(1R,2R)-2-Aminocyclohexyl 5-(5,5-dimethyl-1,3-dioxan-2-yl)-2-hy-
doxybenzamide (185 mg, 0.53 mmol) was dissolved in dry methanol
4.15. Building Block C. 2-(1,3-Dioxolan-2-yl)aniline (11.9 mmol,
1.96 g, 1 equiv) was dissolved in CH Cl (0.2m, 60 mL) and placed
2
2
(
20 mL), and CS (0.35 mL) and triethylamine (0.08 mL) were added.
under N atmosphere. Et N (77.1 mmol, 10.7 mL, 6.5 equiv) and CS
2
2
3
2
After stirring for ca. 20 min at room temperature, the reaction mixture
was cooled in an ice bath before di-tert-butyl dicarbonate (120 mg,
(77.1 mmol, 4.6 mL, 6.5 equiv) were added via syringe, and the
reaction mixture was stirred overnight at room temperature. TFFH
(23.7 mmol, 6.3 g, 2 equiv) was added in one portion while a stream of
0
0
.55 mmol) in methanol (1 mL) and 4-dimethylaminepyridine (3 mg,
.025 mmol) were added. After stirring for 5 min, the reaction mixture
N was kept over the reaction mixture. The mixture was stirred at
2
was removed from the ice bath and allowed to reach room
temperature before it was concentrated by rotary evaporation. The
remainder was dissolved in CH Cl (20 mL), and the mixture was
cooled in an ice bath. Aqueous hydrazine (50−60%, 0.033 mL, 0.53
mmol) was added, and the mixture was stirred for 5 min before it was
removed from the ice bath and allowed to reach room temperature.
The mixture was stirred for ca. 1 h at room temperature. Water (25
room temperature for 4 h. Then the reaction mixture was cooled to 0
°C, hydrazine hydrate (0.28 mol, 17.4 mL, 24 equiv) was added via
syringe, and the reaction mixture was stirred for a further 4 h at room
temperature. Water (100 mL) was added, and the mixture was
extracted with CH Cl (3 × 200 mL). The solution was concentrated.
2
2
2
2
The residue was dissolved in CH Cl /heptane (3:1, 80 mL) and
2
2
directly subjected to dry column chromatography. A column of 3 cm
diameter was packed with 6 cm of silica. A gradient of 0.5% MeOH in
CH Cl was used, and all fractions were 50 mL. Fractions 5−8 were
mL) was added, and the resulting mixture was extracted with CH Cl2
2
(
3 × 30 mL). The combined organic phases were dried (Na SO ) and
2 4
2
2
concentrated by rotary evaporation. The compound was purified by
collected, concentrated, and stirred with a mixture of CH Cl /Et O
2 2 2
DCVC with a 20% gradient of ethyl acetate in heptane, followed by a
(1:2, 200 mL) to obtain 1.56 g (55%) of the product C as a white
1
% gradient of methanol in ethyl acetate (column diameter 4 cm,
solid.
1
packed with ca. 4 cm silica; fraction volume 50 mL, all fractions
contained 1% triethylamine). Fractions 7 and 8 were combined and
concentrated to reveal a light yellow oil. To this was added acetone
H NMR (500 MHz, DMSO-d ) δ 9.67 (br s, 1H, NH), 9.13 (br s,
1H, NH), 7.72 (br s, 1H), 7.65 (br s, 1H), 7.29 (t, J = 7.8 Hz, 1H),
6
7.14 (d, J = 7.4 Hz, 1H), 5.69 (s, 1H), 4.05−3.99 (m, 2H), 4.76 (br s,
13
(
10 mL), and the resulting solution was stirred for ca. 10 min before it
was concentrated to reveal the desired product as a white solid. Yield
3.9 mg (30%).
Melting point 159−160 °C. H NMR (500 MHz, CDCl ) δ 12.67
2H, NH ), 3.96−3.90 (m, 2H). C NMR (126 MHz, DMSO-d ) δ
2
6
179.4, 139.3, 138.2, 127.9, 124.3, 122.3, 121.7, 102.7, 65.8. HRMS
+
+
7
(ESI-TOF) m/z [M + H] calcd for C H N O S 240.0801, found
240.0803.
10 14 3 2
1
3
(
s, 1H), 8.21 (s, 1H), 8.05 (d, J = 6.8 Hz, 1H), 7.85 (d, J = 1.9 Hz,
4.16. 4-Methylbenzohydrazide (3). Ethyl 4-methylbenzoate
(3.28 g, 3.21 mL, 20.0 mmol) was dissolved in ethanol (50 mL, 0.4
M), and hydrazine hydrate (25%, 12 mL, 60 mmol, 3 equiv) was
added. The mixture was refluxed under stirring for 20 h before the
solvent was removed in vacuo. The remnant was redissolved in
CH Cl (100 mL) and washed with water (80 mL). The organic phase
1
H), 7.54−7.50 (m, 2H), 6.92 (d, J = 8.5 Hz, 1H), 5.33 (s, 1H), 4.59−
4
.52 (m, 1H), 3.92−3.85 (m, 1H), 3.76 (d, J = 11.1 Hz, 2H), 3.65 (d, J
=
11.1 Hz, 2H), 2.34−2.28 (m, 1H), 2.17 (s, 1H), 1.99 (s, 3H), 1.87−
13
1
.79 (m, 5H), 1.51−1.38 (m, 4H), 1.32 (s, 3H), 0.79 (s, 3H).
C
NMR (126 MHz, CDCl ) δ 177.8, 170.2, 162.3, 150.2, 132.0, 129.3,
3
2
2
1
2
4
25.3, 118.3, 113.9, 101.5, 77.8, 56.5, 56.1, 32.5, 32.4, 25.3, 25.1, 24.5,
was separated, dried (Na SO ), and concentrated to give a white solid,
which was recrystallized from absolute ethanol to yield 1.00 g (34%) of
white crystals.
2 4
+
+
3.4, 22.0. HRMS (ESI-TOF) m/z [M + H] calcd for C H N O S
23
35
4
4
63.2374, found 463.2370.
I
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
19
Melting point 114−115 °C (lit. 116−118 °C). 1H NMR (500
REFERENCES
■
MHz, CDCl ) δ 7.64 (d, J = 8.2 Hz, 2H), 7.32 (br s, 1H), 7.25 (d, J =
8
3
(
1) (a) Miller, S. L. Science 1953, 117, 528. (b) Urey, H. C. Proc. Natl.
13
.2 Hz, 2H), 4.08 (br s, 2H), 2.40 (s, 3H). C NMR (126 MHz,
Acad. Sci. U. S. A. 1952, 38, 351. (c) Oparin, A. I. The Origin of Life;
MacMillan: New York, 1938. (d) Sutherland, J. D. Angew. Chem., Int.
Ed. 2016, 55, 104.
2) Pereto, J. Int. Microbiol. 2005, 8, 23 ( http://revistes.iec.cat/
index.php/IM/article/view/9494/9490).
3) Pross, A. J. Syst. Chem. 2011, 2, 1.
4) (a) Corbett, P. T.; Leclaire, J.; Vial, L.; West, K. R.; Wietor, J. L.;
Sanders, J. K. M.; Otto, S. Chem. Rev. 2006, 106, 3652. (b) Rasmussen,
B.; Sørensen, A.; Beeren, S. R.; Pittelkow, M. Dynamic Combinatorial
Chemistry. In Organic Synthesis and Molecular Engineering; Nielsen, M.
B., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2013; Chapt. 14, pp
93−436; DOI: 10.1002/9781118736449.ch14. (c) Mondal, M.;
Hirsch, A. K. Chem. Soc. Rev. 2015, 44, 2455.
5) Ludlow, R. F.; Otto, S. Chem. Soc. Rev. 2008, 37, 101.
6) (a) Lehn, J. M. Angew. Chem., Int. Ed. 2013, 52, 2836. (b) Cady,
C. W.; Robinson, D. M.; Smith, P. F.; Swiegers, G. F. Conclusion and
Future Perspectives: Drawing Inspiration from the Complex System
that Is Nature. In Bioinspiration and Biomimicry in Chemistry: Reverse-
Engineering Nature; Swiegers, G. F., Ed.; John Wiley & Sons, Inc.:
012; Chapt. 15, pp 455−471; DOI: 10.1002/9781118310083.ch15.
(c) Ruiz-Mirazo, K.; Briones, C.; de la Escosura, A. Chem. Rev. 2014,
14, 285. (d) Mahon, C. S.; Fulton, D. A. Nat. Chem. 2014, 6, 665.
e) de la Escosura, A.; Briones, C.; Ruiz-Mirazo, K. J. Theor. Biol. 2015,
CDCl ) δ 168.7, 142.4, 129.8, 129.4, 126.8, 21.5. Anal. Calcd for
3
C H N O: C, 63.98; H, 6.71; N, 18.65. Found: C, 63.89; H, 6.80; N,
8
10
2
+
+
1
1
8.64. HRMS (ESI-TOF) m/z [M + H] calcd for C H N O
8 11 2
(
51.0866, found 151.0868.
.17. 4-(4-Butylphenyl)thiosemicarbazide (4). 4-Butylaniline
1.58 mL, 10 mmol) was dissolved in CH Cl (20 mL), and
4
(
(
(
2
2
triethylamine (2.77 mL, 20 mmol) and CS (1.22 mL, 20 mmol) were
2
added. The solution was stirred at room temperature for 5 h before it
was cooled in an ice bath, and TFFH (2.64 g, 10 mmol) was added.
After the addition, the ice bath was removed and the solution was
stirred for 1.5 h at room temperature. Once again the solution was
cooled in an ice bath, and hydrazine hydrate (5 mL, 0.1 mol) was
added carefully over 5 min. The ice bath was removed and the solution
was stirred for 2 days at room temperature before it was quenched
with water (50 mL). The aqueous phase was extracted with ethyl
acetate (3 × 50 mL), and the combined organic phases were
backwashed once with water (50 mL) before they were dried
3
(
(
(
Na SO ) and concentrated in vacuo. The crude product was
2 4
recrystallized from ethanol and air-dried to yield 1.62 g (73%) of
2
white crystals.
1
Melting point 113−115 °C. H NMR (500 MHz, CDCl ) δ = 9.15
3
1
(
(
3
(
1
br s, 1H), 7.94 (br s, 1H), 7.52−7.33 (m, 2H), 7.24−7.12 (m, 2H),
.97 (br s, 2H), 2.63−2.56 (m, 2H), 1.62−1.54 (m, 2H), 1.40−1.30
13
381, 11. (f) Zayed, J. M.; Nouvel, N.; Rauwald, U.; Scherman, O. A.
Chem. Soc. Rev. 2010, 39, 2806.
m, 2H), 0.95−0.90 (m, 3H). C NMR (126 MHz, CDCl ) δ 180.1,
3
29.6, 129.0, 125.5, 124.5, 35.3, 33.7, 22.5, 14.1. Anal. Calcd for
(
7) Cousins, G. R. L.; Poulsen, S.-A.; Sanders, J. K. M. Chem.
Commun. 1999, 1575.
8) (a) Raposo, M. M.; Garcia-Acosta, B.; Abalos, T.; Calero, P.;
Martinez-Manez, R.; Ros-Lis, J. V.; Soto, J. J. Org. Chem. 2010, 75,
922. (b) Bonizzoni, M.; Fabbrizzi, L.; Taglietti, A.; Tiengo, F. Eur. J.
Org. Chem. 2006, 2006, 3567. (c) Suganya, S.; Zo, H. J.; Park, J. S.;
Velmathi, S. Ind. Eng. Chem. Res. 2014, 53, 9561.
9) Jakab, G.; Schreiner, P. R. Brønsted Acids: Chiral (Thio)urea
Derivatives. In Comprehensive Enantioselective Organocatalysis: Cata-
lysts, Reactions, and Applications; Dalko, P. I., Ed.; Wiley−VCH: 2013;
Chapt. 12, pp 315−341; DOI: 10.1002/9783527658862.ch12.
(10) Beeren, S. R.; Pittelkow, M.; Sanders, J. K. M. Chem. Commun.
011, 47, 7359.
11) Beesley, R. M.; Ingold, C. K.; Thorpe, J. F. J. Chem. Soc., Trans.
915, 107, 1080.
12) (a) Dirksen, A.; Dirksen, S.; Hackeng, T. M.; Dawson, P. E. J.
Am. Chem. Soc. 2006, 128, 15602. (b) Dirksen, A.; Yegneswaran, S.;
Dawson, P. E. Angew. Chem., Int. Ed. 2010, 49, 2023. (c) Bhat, V. T.;
Caniard, A. M.; Luksch, T.; Brenk, R.; Campopiano, D. J.; Greaney, M.
F. Nat. Chem. 2010, 2, 490.
C H N S: C, 59.16; H, 7.67; N, 18.81. Found: C, 59.45; H, 7.54; N,
1
1
17
3
+
+
1
2
8.89. HRMS (ESI-TOF) m/z [M + H] calcd for C H N S
11 18 3
(
24.1216, found 224.1213.
2
ASSOCIATED CONTENT
■
* Supporting Information
S
(
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.7b01161.
Additional text, five figures, one scheme, and three tables
1
13
describing dynamic combinatorial libraries; H and
C
2
(
1
(
NMR spectra of synthesized compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
E-mail pittel@chem.ku.dk.
*
ORCID
(
3
13) (a) Brotzel, F.; Chu, Y. C.; Mayr, H. J. Org. Chem. 2007, 72,
679. (b) Kanzian, T.; Nigst, T. A.; Maier, A.; Pichl, S.; Mayr, H. Eur. J.
Michael Pittelkow: 0000-0002-3371-9500
Org. Chem. 2009, 2009, 6379.
(14) Di Stefano, S.; Ercolani, G. J. Phys. Chem. B 2017, 121, 649.
Present Addresses
†
(
D.L.) Department of Chemistry, Stanford University,
(15) (a) Schiltz, H.; Chung, M.-K.; Lee, S. J.; Gagne, M. R. Org.
Stanford, CA 94305-5017.
Biomol. Chem. 2008, 6, 3597. (b) Chung, M. K.; White, P. S.; Lee, S. J.;
Waters, M. L.; Gagne, M. R. J. Am. Chem. Soc. 2012, 134, 11415.
‡
(
A.J.) Chemistry Research Laboratory, Department of
Chemistry, University of Oxford, Oxford OX1 3TA, U.K.
(16) Izatt, R. M.; Bradshaw, J. S.; Nielsen, S. A.; Lamb, J. D.;
§
(C.K.) Department of Chemistry and Chemical Biology,
Christensen, J. J.; Sen, D. Chem. Rev. 1985, 85, 271.
Harvard University, 12 Oxford St., Cambridge, MA 02138.
(17) Boas, U.; Pedersen, B.; Christensen, J. B. Synth. Commun. 1998,
2
(
8, 1223−1231.
Notes
18) Clerici, A.; Pastori, N.; Porta, O. Tetrahedron 1998, 54, 15679.
The authors declare no competing financial interest.
(
08.
19) Li, W.; Wang, X.; Zhao, Z.; Han, T. J. Chem. Res. 2010, 34, 106−
1
ACKNOWLEDGMENTS
■
We acknowledge financial support from the Lundbeck
Foundation for a Young Group Leader Fellowship (M.P.),
the German National Foundation (Studienstiftung des
Deutschen Volkes e.V.) for a fellowship (C.K.), and the
Lundbeck Foundation and the Department of Chemistry,
University of Copenhagen, for a Ph.D. scholarship (D.L.).
J
DOI: 10.1021/acs.joc.7b01161
J. Org. Chem. XXXX, XXX, XXX−XXX