Near quantitative synthesis of urea macrocycles enabled by bulky N-substituent

Article abstract of DOI:10.1038/s41467-021-21678-3

Macrocycles are unique molecular structures extensively used in the design of catalysts, therapeutics and supramolecular assemblies. Among all reactions reported to date, systems that can produce macrocycles in high yield under high reaction concentration

Full text of DOI:10.1038/s41467-021-21678-3

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https://doi.org/10.1038/s41467-021-21678-3
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1 Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 2 Department of Nuclear, Plasma, and
Radiological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 3 Beckman Institute for Advanced Science and Technology,
University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 4 Department of Chemistry, Rice University, Houston, TX 77005, USA. 5 George L. Clark
X-Ray Facility & 3M Materials Laboratory, School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 6 Pritzker School
of Molecular Engineering, University of Chicago, Chicago, IL 60637, USA. 7 Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL
61801, USA. 8 Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 9 Materials Research Laboratory, University
10
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of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. These authors contributed equally: Yingfeng Yang, Hanze Ying. email: jianjunc@illinois.edu
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1

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acrocycles are unique structural units of very broad Results
interest. Owing to their relatively rigid and defined Rationale for the efficient construction of HUMs. In an amide
M
scaffold, macrocycles are promising in biological sen- with chemical structure of R1C(O)NHR2, the R1 and R2 moieties
sing1–3, ion transporting4, drug discovery5–7 and molecular should be ‘trans’ to each other (Fig. 1a) because of the coplanar
sieving8. Building on their propensity for self-assembly, macro- resonance structure of the amide bond (C(O)–NH) as the other-
cycles can also serve as precursors to mechanically interlocked wise ‘cis’ conformation of these two moieties would result in sig-
structures9,10 and molecular machinery11, and play an important nificant steric hinderance and is therefore thermodynamically
role in catalysis12 by providing nano-confinement13. The selective disfavored. Same ‘trans’ bond structure should also hold true in
binding and associated host–guest interaction further dictate their the case of an urea with chemical structure of R1N1HC(O)N2HR2,
usage in environmental pollutant removal14 or drug delivery15. the close analog of amide, with R1N1H moiety ‘trans’ to the R2
Despite all these widespread applications, synthesis of macro- moiety in the coplanar resonance structure of the urea bond
cycles remains challenging, in particular under high concentra- (N1C(O)–N2H) (Fig. 1b). These have been the basis forming the
tion in large scale.
zigzag molecular structures of the polyamide or polyurea back-
Traditionally, macrocycles are synthesized through kinetic bones. However, such R1/R2‘trans’ bond structures of the amide
control in very dilute concentrations, which suffers from low R1C(O)NHR2 may no longer hold true when the hydrogen of the
yields and tedious purification processes16. With the advent of N–H was replaced by a substituent much bulkier than R2. It has
dynamic covalent chemistry (DCC), syntheses of macrocycles been reported that when the N–H was changed to N-t-Bu in
with improved yields through thermodynamic control have been arylopeptoids or α-peptoids, the amide bonds adopted the R1/R2
reported17. Aided by the ‘error checking’ and ‘self-correction’ cis conformation exclusively (Fig. 1c)32. We previously reported
features enabled by reversible reactions, DCCs like boronic HUB as a new DCC tool33, which can be synthesized from iso-
ester18, imine19, alkene20 and alkyne21 metathesis have been cyanates and hindered amines, and can dissociate back to the
demonstrated for macrocycle construction with moderate yields. starting compounds (Fig. 1e). As the t-Bu has been predominately
Since the linkage between the building blocks can greatly impact used in our design of HUB, we postulate that replacing the
the physical and chemical properties of the material, new, viable hydrogen of N2H-R2 in the urea by t-Bu (bulkier than R2) would
DCCs are desired for the construction of macrocycles, in parti- result in R1-N1H ‘cis’ to R2 (i.e., bulky t-Bu N-substitute ‘cis’ to
cular those of intrinsic hydrogen bonding capabilities which are C(O)) (Fig. 1d). Throughout this paper, we use ‘cis-urea’ to define
of huge implications in molecular recognitions, self-assembly, such bond structure with bulky N-substitute cis to C(O), as shown
molecular machinery and catalysis.
in Fig. 1d. If such ‘cis-urea preference’ holds true in a HUB con-
Cyclic peptides have been important hydrogen bonding- taining bulky t-Bu group, we envision that the [1:1] adduct of a
capable moiety in self-assembly22 and are key structures of diisocyanate and a hindered diamine would result in a hindered
numerous natural pharmacophores23. Synthesis of cyclic peptides polyurea with ‘bulky urea-turn’ (‘cis’ structure of R1NH and R2,
in high yields, however, is quite challenging. Synthesis of mac- Fig. 1f) in their backbone instead of forming a zigzag urea back-
rocyclic amides, which share the identical backbone functional bone. For a typically [1:1] adduct of a diisocyanate and a diamine
group as cyclic peptides, have been extensively reported with with perfect zigzag urea chain without bulky groups, since the
structurally rigid aromatic precursors with pre-designed bond backbone units all prefer to stay trans to each other, macro-
angles or by employing intramolecular hydrogen bonding to pre- cyclization would be difficult in the linear extended backbone
organize the building blocks24. However, there have been no structure and can only occur in highly dilute solution. While for
viable DCC reported so far for efficient macrocyclic amide hindered urea chains with ‘bulky urea-turn’ in each HUB, such
synthesis. As a structural surrogate, urea shares desirable features hindered polyurea should easily undergo macrocyclization with
with the amide group, such as rigidity, planarity, polarity and several bond rotations (Fig. 1g). This resembles to some extent the
hydrogen bonding capacity. There have been numerous reports so-called ‘gem-dimethyl effect’ where large substituents favor ring-
on binding, recognition, catalysis or assembly of acyclic oligour- closure and intramolecular reactions34,35. Thus, we envision that
eas25–27. Synthesis and applications of urea macrocycles have also the properly selected diisocyanate and hindered diamine adducts
been explored. For instance, Shimizu reported bisurea macro- adopting significant ‘bulky urea-turn’ conformation would enable
cycles as nanocontainer to facilitate photodimerization, selective efficient construction of urea macrocycles.
cycloaddition and to stabilize radicals28–30. Gong pioneered an
efficient approach for one-pot formation of aromatic tetraurea
macrocycles via specific oligomeric structures preorganized by Quantitative synthesis of the hindered urea macrocycle
their intrinsic intramolecular hydrogen bonds31. Gale reported a (HUM1). In a preliminary trial, two commercially available
tetraurea macrocycle that can bind chloride in aqueous solution3. building blocks, methylene diphenyl diisocyanate (N1) and N, N′-
However, these reports typically involve synthesis of urea mac- Di-tert-butylethylenediamine (A1), were mixed (Fig. 2a) in 1:1
rocycles in mediocre yields or with specific building blocks, and molar ratio and maintained at 60 °C. The system initially gave
some even require stringent reaction condition and reagents. mixtures of multiple species. Interestingly, after incubating for 2 h,
Here, we report a simple, robust method for constructing urea the originally messy and multimodal peaks transformed to one set
macrocycles with hindered urea bond (HUB) associated dynamic of sharp resonances in 1H NMR (Fig. 2b), implying the formation
chemistry. A variety of hindered urea macrocycles (HUMs) are of possibility a low molecular weight residue with well-defined
synthesized in near quantitative yields under high concentrations, structure in very high purity. The residue was characterized by
with inexpensive and widely available diisocyanates and bulky multiple methods without any purification (Supplementary Fig. 2).
diamines. The unusual high efficiency is attributed to the steric Evidences pointed to the anticipated formation of a dimeric
conformation lock and weak association interactions mediated by HUM1 in quantitative yield with the unnecessity of any purifica-
the bulky group as well as the ‘self-correction’ property enabled tion, by simply mixing equal molar N1 and A1. The structure of
by the dynamic bond. The bulky tert-butyl (t-Bu) group can be HUM1 was further confirmed by the single-crystal X-ray diffrac-
readily removed by acid to stabilize the macrocycle structure, tion (XRD) analysis (Fig. 2c).
facilitating their applications in self-assembly, binding and
molecular recognition and therapeutics.
To better understand the formation process of HUM1, the
reaction was conducted at room temperature in high concentration
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and monitored by gel permeation chromatography. A gradual time (entries 1 and 8–22, Table 1, Supplementary Figs. 9–25).
transition from higher molecular weight multimodal peaks to one Among the 20 combinations of NxAy, 13 of them gave quanti-
sharp peak with the calculated molecular weight consistent with tative yield of macrocycles. While most combinations gave
HUM1 was observed (Fig. 2d), which suggests that the final [NxAy]2 dimeric macrocycles as shown in Table 1, [NxAy]1 type
thermodynamic equilibration has been reached. Furthermore, of monomeric macrocycles were formed in some systems invol-
HUM1 can be quantitatively obtained within a wide range of ving N2, presumably because the relatively flexible benzyl linker
concentration, from 1 to 500 mM (entries 1–4, Table 1) at any of N2 can effectively release the ring strain for the monomeric
temperature ranging from 20 to 75 °C (entries 5–7, Table 1). macrocycle species. The combinations of N2 with A2, A3 and A5
Reactions of equal molar N1 and A1 in different aprotic solvents, even reached exclusive macrocycle product at room temperature
such as chloroform, THF, ethyl acetate, dimethyl formamide and within 3 min after mixing (entries 9, 10 and 12), much faster than
dimethyl sulfoxide, all result in the formation of pure HUM1 in the dynamic exchange of the reversible bond33,36. It is thus evi-
quantitative yields, with only differences in their time of reaching dent that the HUB is a remarkable macrocycle enabling structural
the equilibrium to form HUM1.
motif, given the unusually high yield of macrocycles from the off-
the-shelf, readily available building blocks.
The macrocycle formation was also tested in a three-
HUB as macrocycle enabling structural motif for the synthesis component system where two different diisocyanates N3 and
of other HUMs. The exceptionally high efficiency of HUM1 N4 were mixed with one diamine A2 (entry 23, Table 1,
synthesis prompts us to explore if the reaction is universal Supplementary Fig. 28).
A self-sorting phenomenon was
to other substrates. Four commercially available diisocyanates Nx observed, in which HUMs [N3A2]2 and [N4A2]2 formed,
(x = 1–4) were chosen, as well as five hindered diamines Ay (y = respectively, with no hybrid products detected. The self-sorting
1–5) which are either commercially available or can be facilely presumably results from the dynamicity of the system which
synthesized from t-butylamine and the corresponding halides allows it to collapse to its thermodynamically most stable state, as
(Table 1). The macrocycle formation efficiencies were monitored otherwise mismatch of the building blocks will possibly incur ring
by 1H NMR and the final products were further confirmed by 13
C
strain and thus increase total energy of the system. Postmodifica-
NMR and matrix-assisted laser desorption/ionization-time of tion of macrocyclic scaffold offers a higher level of manipulation.
flight (MALDI-TOF). Most of the combinations in the library We demonstrated that the presence of the pyridyl and the
gave HUMs as the predominant species within short period of pendant alkynyl groups in monomers A4 and A5 did not
N
A
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3

A
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c
a
b
a
N1
A1
c
d
P1
HUM1
> 99%
b
5 min
36 h
72 h
d
1 min
2 h
a
b
c
d
8
7
6
5
4
3
2
1
10
15
Elution time (min)
20
ꢀ (ppm)
Fig. 2 Quantitative synthesis of the hindered urea macrocycle HUM1. a
S
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interfere with the macrocycle formation process, implying the between cis/trans conformations of the t-Bu substituted model
potential for further functionalization and structural diversity compound MC2, which is much larger than its i-Pr substituted
extension in future designs. What’s more interesting, when the counterpart MC2′ (~2 kT) (Fig. 3d). Similar NOE and DFT cal-
t-Bu group in A2 was replaced with even bulkier ones (A6 and culation results were obtained for different sets of model com-
A7), similar macrocycles formation behaviors were shown pounds representing aromatic/aliphatic ureas (Supplementary
(entries 24–25, Supplementary Figs. 26 and 27), further Figs. 35–38, Supplementary Table 1, Supplementary Informa-
substantiating the versatility of this strategy.
Among various combinations with exclusive macrocycle preference’ which is induced by the bulky t-Bu substituent.
formations, two types of processes were observed: the mixings We next elucidated how the ‘cis-urea preference’ can facilitate
tion 3.3.2, 3.3.3). These results clearly demonstrated the ‘cis-urea
of diisocyanate and diamine either gave high-purity macrocycle macrocycle formation by using the linear analog MC4 (the [1:1]
immediately (entries 9, 10 and 12, Table 1) as kinetically favored adduct of N2 and A2 with reactive chain ends, upper left inset,
products or gave mixture which later turned to macrocycles Fig. 3f, Supplementary Fig. 39). Twelve different conformations
exclusively as thermodynamically favored products. These two were generated by rotating around the C(O)–N(tBu) bond of the
types were further investigated to understand the underlying adduct, which converged into five different metastable conforma-
mechanisms.
tions after local energy minimization. The DFT calculations on
the five different conformers of MC4 revealed the general trend
that with increased degree of folding (i.e., smaller θ1 or shorter
chain-end distance d), the free energy decreases, substantiating
that the conformer with the shortest reactive chain-end distance
predominates. It is also observed that the most stable conformers
adopt ‘cis-urea’ conformations (lower right inset, Fig. 3f),
elucidating the correlations between ‘cis-urea preference’ and
proximity of reactive chain ends, which contributes to the
efficient macrocycle formation.
While only HUM2 was discussed here, kinetic effects should also
play a role in dimeric macrocycle systems. This argument is
supported by the cis-urea conformation of several macrocycles and
linear analogs shown by the single-crystal structures (Supplemen-
tary Figs. 54–57, Supplementary Tables 4–7), and the fact that most
high-yielding HUMs systems underwent a burst formation with
more than 40% yields of the target macrocycles in less than 3 min,
which was much higher than the statistic yields at such
concentration (Supplementary Fig. 12, Supplementary Informa-
tion 3.2.4). Thus, the kinetic aspect of the ‘cis-urea preference’
facilitated efficient HUM formation can be rationalized from two
aspects: conformational flexibility and effective molarity. On one
Validation of t-Bu induced ‘cis-urea preference’. The observa-
tion of kinetically controlled macrocycle formation process is
consistent with our initial hypothesis (Fig. 1g). To validate the
relationships between the ‘cis-urea’ conformation and size of
substituents, the kinetically favored macrocycle HUM2 ([N2A2]1)
was selected as well as a series of acyclic model compounds (MC1
and MC1′, which differ only in one substituent) (Fig. 3). Their
conformations were studied by Nuclear Overhauser Effect Spec-
troscopy (NOESY). While substituted urea usually adopts various
conformations due to the relatively low-rotational barriers of urea
C-N bond37,38, both HUM2 (Fig. 3a) and the t-Bu substituted
MC1 (Fig. 3b) adopted a relatively fixed cis conformation, with the
N–H hydrogen (labeled as ‘a’) in close proximity only to the less
bulky benzyl/methyl hydrogens (labeled as ‘b’) but not to the t-Bu
hydrogens (labeled as ‘c’). On the contrary, if the t-Bu group was
changed to an ethyl group as in MC1′, the NH became spatially
close to both the methyl and ethyl group (Fig. 3c), showing the C
(O)‒N(Me)Et amide bond can rotate more freely with both con-
formations coexisting. Density functional theory (DFT) calcula-
tions revealed more than 4.2 kcal/mol (~7 kT) energy differences
4
N
A
T
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R
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C
O
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|
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1
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4
6
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2
1
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2
1
6
7
8
-
3
|
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.
c
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a
t
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n
s

N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
h
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:
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1
0
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1
0
3
8
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6
7
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2
1
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2
1
6
7
8
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3
A
R
T
I
C
L
E
Table 1 Substrate universality of HUB based macrocycles.
Or
Nx
Ay
[NxAy]1
[NxAy]2
Nx
Ay
N1
A1
N2
A2
N3
A3
N4
A4
A5
A6
A7
Entry
Isocyanates
Amines
HUMs
Conc. (mM)
T (°C)
t (h)
Yields (%)
1
N1
N1
N1
N1
N1
N1
N1
A1
A1
A1
A1
A1
A1
A1
A1
A2
A3
A4
A5
A1
A2
A3
A4
A5
A1
A2
A3
A4
A5
A2
A6
A7
[N1A1]2
[N1A1]2
[N1A1]2
[N1A1]2
[N1A1]2
[N1A1]2
[N1A1]2
[N2A1]2
[N2A2]1
[N2A3]1
[N2A4]1
[N2A5]1
[N3A1]2
[N3A2]2
[N3A3]2
[N3A4]2
[N3A5]2
[N4A1]2
[N4A2]2
[N4A3]2
[N4A4]2
[N4A5]2
5
1
0
6
0
0
0
0
~
~
~
~
~
~
~
~
<
<
<
<
~
~
~
~
~
~
~
~
~
~
1
0
1
2
4
1 2
0 .5
2 0
3
3
3
3
3
2
>
>
>
>
>
>
>
6
>
>
>
9
8
>
>
>
>
6
>
>
>
>
>
>
>
9
9
9
9
9
9
9
1
9
9
9
1
5
5
5
5
5
5
5
2
3
4
5
6
7
8
9
6
6
6
.5
5
2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
2
5
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.
a
2 0
3 7
7 5
8
N2
N2
N2
N2
N2
N3
N3
N3
N3
N3
N4
N4
N4
N4
N4
N3 + N4
N3
N3
6
0
2
2
2
2
0
0
0
0
m
m
m
m
i
i
i
i
n
n
n
n
5
5
5
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
6 0
6 0
6 0
6 0
6 0
6 0
6 0
6 0
6 0
6 0
6 0
6 0
6 0
1
9
9
9
9
8
9
9
9
9
9
9
9
5
5
5
5
1
3
2
2
2
1
.
5
5
5
5
5
5
5
5
2
2
2
2
2
2
0
1
2
3
4
5
3
2
[N3A2]2; [N4A2]2
[N3A6]2
[N3A7]2
5
;
2
5
;
5
0
/
/
/
0
0
U
p
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[
a
N
x
s
A
;
y
b
]
o
r
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o
[
N
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m
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:
y
Y
]
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o
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1
2
c
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i
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u
(
x
=
1
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,
y
=
1
–
.
O
n
g
e
i
i
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:
h
i
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d
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d
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1
Y
P
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d
w
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d
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t
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a
b
t
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N
M
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.
a
o
u
c
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c
i
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u
s
w
h
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c
r
y
s
t
a
l
s
b
e
c
a
u
s
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o
f
l
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d
s
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a
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c
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c
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e
.
hand, the ‘cis-urea preference’ would reduce the conformational structurally more rigid, dimeric macrocycle structures that can
flexibility of the urea chain and thus, the minimum of the energy accommodate four ‘bulky urea turns’ (Fig. 1f) are favored and can
landscape can be reached faster39. On the other hand, the folded be formed with several bond rotations. Furthermore, in a situation
structure will position the reactive chain ends close to each other, where one urea bond of the macrocycle was opened up, the
thus increasing the effective molarity for the ring-closing step. In preorganized intermediate would provide a high effective molarity
cases where the building blocks are suitably angled with chain ends driving rapid ring-closure before another urea bond opens up,
in proximity, monomeric macrocycles can be quantitatively formed which in turn contributes to the higher macrocycle stability against
upon mixing, such as HUM2. In cases where building blocks are exchange reactions. To prove this, HUM2 and its linear model
N
A
T
U
R
E
C
O
M
M
U
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5

A
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a
b
c
a
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a
c
c
c
a
c
c
a
b
a
b
MC1
MC1’
b
b
c
HUM2
b
b
4.1
4.0
ꢀ (ppm)
3.9
6.4
6.2
ꢀ (ppm)
6.0
6.5
6.0
ꢀ (ppm)
d
e
0
-0.5
-1
ꢁGtrans-cis (kcal/mol)
Compounds
PBE(6-31G(d))
4.24
B3LYP(6-31G(d))
MP2
5.11
1.90
k
ex= 0.25 h-1
MC2
4.62
1.35
-1.5
HUM2
MC3
MC2’
1.26
0
2
4
Time (h)
6
f
15.54 Å
6.3260
5.9279
15.05 Å
v2
5.0157
10.42Å
v1
d
θ1
v4
θ2
v3
16
12
8
cis-urea
8.62 Å
2.1750
4.46 Å
trans-urea
4 6
4
0
0
2
Relative energy (kcal/mol)
θ1
compounds MC3 were exchanged with butylisocyanate (Fig. 3e). phenomenon of equilibrium was beyond the explanation. In the
The linear analog underwent fast exchange reactions while HUM2 first example of HUM1 we discussed, a mixture of species with
remained unchanged even after prolonged incubation of over 50 h. various sizes were formed first, which was later transformed to an
exclusive macrocyclic compound during a relatively long period
of time compared to kinetically favored macrocycles. It was a
Role of t-Bu group in thermodynamic stabilization. While ‘cis- typical thermodynamic equilibrium process, which indicated that
urea preference’ well explained the fast HUMs formation, the HUM1 has reached a locally minimum energy state. In one of the
6
N
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Fig. 3 Validation of the t-Bu induced ‘cis-urea preference’.
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50 mM
10 mM
5 mM
b
a
b’
a’
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c
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d
1 mM
1.52
HUM1
MC5
1.50
1.48
ꢀꢂ(ppm)
1.46
c
a
a’
b’
b
c
c’
d d’
8
7
6
5
4
3
2
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ꢀꢂ(ppm)
Fig. 4 Role of t-Bu group in thermodynamic stabilization. a
U
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n HUM1, MC5
. c
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a
k
s
.
b
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a
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.
C
o
l
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r
c
o
d
:
g
r
a
,
C
l
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r
e
O
.
explorative studies, we performed a similar reaction with N1 and mixture was characterized by NOESY (Fig. 4a). When the proton
a new diamine with only the t-Bu group changed to i-Pr (A1′). In on t-Bu of HUM1 (peak d) was irradiated, the signals from t-Bu
this case, a mixture of oligomeric molecules was obtained even protons of MC5 showed up (peak d’) as well as ones from the
with prolonged incubation (Supplementary Fig. 42). The mixture aromatic region of MC5. This demonstrated that the t-Bu groups
was proven to reach chemical equilibrium after 15 days without of HUM1 and MC5 are in close proximity in space, supporting
any trend towards an exclusive macrocyclic product. It implied the argument of existence of intermolecular interaction involving
some unique driving forces t-Bu group may provide to the for- t-Bu group.
mation of macrocycle that less bulky groups do not have. Then, a
The single-crystal XRD showed how t-Bu group may interact
concentration-dependent NMR study was performed for HUM1 with the macrocycle in the solution state. As shown in Fig. 4c, two
solutions in CDCl3 from 1 to 50 mM. Interestingly, the proton adjacent macrocycles both have one of their t-Bu groups pointing
signal from the t-Bu group showed a clear trend of upfield shift towards the C-shaped cavity of each other, forming a ‘host-guest’
with the increase of concentration (Fig. 4b, Supplementary pair. The corresponding t-Bu—cavity centroid distance is ~0.272
Fig. 45), based on which the dimeric association constant of nm, suggesting that the macrocycle HUM1 may be stabilized by
HUM1 in CDCl3 was determined to be around 5.5 M−1 (Sup- the presence of energetically favorable t-Bu−macrocycle interac-
plementary Fig. 46). On the contrary, the urea proton showed no tions. As a further support, atomistic level molecular simulation
clear change to the concentration, indicating no association was performed in [N1 + A1] system to calculate the average
through hydrogen bonds. As a control, no such phenomenon was monomeric non-bonding energy in macrocycles with various
observed for its acyclic analog MC9 (Supplementary Fig. 47). It sizes n (denoted as nmer), either with or without inter-
implies the existence of an intermolecular interaction involving macrocycle interactions considered (Supplementary Fig. 48a).
t-Bu group in macrocycles, and it was further supported by the The results suggest that the interactions between macrocycles
NOESY study. As it is difficult to tell apart the ‘through bond’ or stabilize the 2mers and drive it to be the much more favored
‘through space’ coupling in the same molecule, the acyclic model species. Dimerization potential of mean force (PMF) calculations
compound MC5 was added to the HUM1 solution and the showed that 2mer had a higher stabilization free energy (4.0 kT)
N
A
T
U
R
E
C
O
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M
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C
A
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2
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7

A
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6
7
8
-
3
a
c
HUM1
UM1
HUM1+amine
UM1+amine
reversible
irreversible
a’
b’
c’
d’
c
c’
b
b
b’
a
a’
acid
d’
HUM1
UM1
a
b
c
9
8
7
6
ꢀ (ppm)
Amine exchange
No exchange
e
d
De-[N2A4]-C12
MIC (mg/L)
USA100
USA200
USA300
De-[N2A4]-C12
Vancomycin
16
4
16
2
8
2
Fig. 5 Stabilization of the HUMs through de-tert-butylation and the potential applications. a
S
n
c
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e UM1
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than other-sized macrocycles (Supplementary Fig. 48c). Further- urea macrocycles can also be used on molecular recognition. For
more, a ‘pocket effect’ was clearly observed in the 2mer system, instance, UM1 was found to be a potent receptor for several
with t-Bu group from one macrocycle sitting in the cavity of different anions (Supplementary Fig. 51). The binding constant
another one (Supplementary Fig. 48e), which was in consistence between UM1 and phosphates, for example, can reach 104 M−1
with the structure in the solid state.
even in competitive solvents like DMSO (Fig. S34).
Inspired by the structural similarity between urea macrocycles
and cyclic peptides and the notion that cyclic structures usually
have improved membrane activity compared with their linear
analogs, we further explored the biological activities of urea
macrocycles as antimicrobial agents. [N2A4]1 was selected
because of its rigid structure and the ease of postmodification
via its pyridinyl moiety. Through alkylation, the charged urea
macrocycle with C12 alkyl tail showed high antimicrobial
activities against three different strains of methicillin-resistant
staphylococcus aureus (MRSA) (Fig. 5e), with the minimum
inhibitory concentration comparable to Vancomycin, a clinically
widely used antibiotics for MRSA treatment. Following these
preliminary studies, explorations of other applications of urea
macrocycles are under way.
Stabilization of the macrocycles through de-tert-butylation
reaction. Macrocycles constructed from DCCs often suffer from
stability issue because the reversible property of dynamic bonds
still exist in ring molecules. We attempted to transform dynamic
HUB structures to a regular urea to remove the dynamicity and
stabilize the macrocycle. Given the structural similarity of HUB to
t-Bu ester which can be readily cleaved by acid40, we attempted to
use acid to remove the t-Bu group from HUB (Fig. 5a). HUM1
was treated with trifluoracetic acid for 5 min. As expected, the
stable urea macrocycle UM1 was obtained in nearly quantitative
yield from the de-tert-butylation of HUM1 (Fig. 5c). To assess its
stability, we treated UM1 with excessive amount of N-tert-
butylmethylamine (Fig. 5b). UM1 stayed unchanged with amine
exchange reaction, substantiating the complete elimination of the
dynamicity. In comparison, HUM1 was quickly degraded via Discussion
rapid amine exchange reaction with N-tert-butylmethylamine In this research, urea macrocycles are synthesized efficiently
(Fig. 5c).
through DCC under ambient conditions from readily available
Apart from the increased stability, de-tert-butylated urea starting materials. The strategy shows high versatility to different
macrocycles show much stronger tendency to self-assemble. When substrates, reaction conditions and bulky substituents, and toler-
UM1 was dissolved in DMSO and set aside for 2 days, it self- ance to functional groups. We also demonstrated that the efficient
assembled into fibers with 200~300 nm in width and greater than HUM formation is uniquely driven by both kinetic and thermo-
200 µm in length, presumably assisted by the intermolecular dynamic effects, which, more interestingly, share a common
interactions, such as π–π interaction, and the availability of NH for contributor: the steric N-substituent. Bulky groups are playing
hydrogen bonding after de-tert-butylation (Fig. 5d). As expected, multiple roles to achieve high-efficient macrocycle synthesis here:
8
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first of all, it activates the covalent urea bond to provide reversible Data availability
The data supporting this study are provided in the Supplementary Information and are
also available from the authors upon reasonable request. All single-crystal data of HUM1,
HUM3, [N4A2]2 and MC5 have been deposited in the Cambridge Crystallographic Data
Centre (CCDC) and can be downloaded free of charge from http://www.ccdc.cam.ac.uk/
data_request/cif. The accession numbers are CCDC: 1959553, 1959555, 1959556 and
feature and enable systems to find their minimum energy state;
secondly, it promotes the formation speed of the macrocycles by
conformation lock, which increases the effective molarity of local
reactants and the probability of the ring-closing steps; thirdly, it
reduces the energy and promote the dominance of the macrocycle 1959562, respectively.
system through weak interactions with the ring pocket. The
contribution of ‘kinetic’ and ‘thermodynamic’ effect to the mac-
rocycle synthesis may vary, depending on variables in each specific
macrocycle structure, such as ring size, monomer length/angle,
etc. Even more uniquely, the bulky group can be instantly
removed with the catalysis of acid, leaving stable macrocycles
without further concerns of structural changes.
Apart from introducing an efficient way to obtain a class of
functional macrocyclic molecules, this research underscored the
power of harnessing bulky group to facilitate highly efficient
preparation of macrocycles both kinetically and thermo-
dynamically. We envision this strategy could also be applied to
the preparation of more complex architectures and to other DCC
systems.
Here we report a simple, high-yielding, and robust method for
constructing urea macrocycles with dynamic HUB chemistry and
acid-assisted de-tert-butylation. HUMs can be formed in nearly
quantitative yields under mild conditions with high concentra-
tions in a relatively short timescale. The unusual high efficiency
was attributed to the ‘self-correction’ property enabled by the
dynamic bond as well as the ‘cis-urea preference’ and weak sta-
bilization effect mediated by the bulky group. In addition, the
bulky group can be efficiently removed by acid to stabilize the
macrocycle structure, which in turn showed higher propensity for
self-assembly, strong binding affinity for anions and potential as
an antimicrobial peptide surrogate for killing of drug-resistant
microbials. We envision that this ‘bulky group effect’ may not be
limited to HUB but exist in other DCC systems as well. Given the
exceptional simplicity, high yield synthesis, easy functionaliza-
tion, flexibility in substrate selection and hydrogen bonding
capacity of the product, we expect this chemistry to become a
great platform for the study of macrocycle applications in areas,
such as self-assembly, selective binding, molecular recognition,
drug discovery, catalysis and rotaxane, etc.
Received: 12 October 2020; Accepted: 13 January 2021;
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Methods
Full experimental details and characterization of compounds can be found in
the Supplementary Information. Typical hindered diamine synthesis: The cor-
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K2CO3 (1 eq) were added to the solution. The suspension was stirred at room
temperature for about 24 h and the reaction was monitored by TLC. After com-
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DCM (30 mL × 3). The organic layer was combined, washed twice with brine and
then dried with anhydrous Na2SO4. Then solvent was removed and crude product
was purified by flash column chromatography.
Macrocycle library generation: The diisocyanate (0.1 mmol) and diamine (0.1
mmol) were mixed directly in CDCl3 (2 mL) and then incubated at 60 °C. The
reactions were monitored by 1H NMR until the thermodynamic equilibrium had
been reached (evidenced by the lack of change in 1H NMR). Final products were
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the integration from 1H NMR spectra.
Macrocycle formation kinetics: The diisocyanate (0.1 mmol in 1 mL CDCl3) and
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Supplementary information The online version contains supplementary material
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Correspondence and requests for materials should be addressed to J.C.
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and the other, anonymous, reviewer(s) for their contribution to the peer review of this
work. Peer reviewer reports are available.
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Acknowledgements
This work is supported by United States National Science Foundation (NSF CHE 17-
09820) and American Chemical Society Petroleum Research Fund (58671-ND7). We
thank R. Wang for the helpful discussion, M. Kang for the WAXS characterization and
J. Lai for the assistance with computation of the binding constants.
Author contributions
Y.Y. and H.Y. contributed equally to this work. Y.Y., H.Y. and J.C. conceived the idea of
the project. Y.Y. and H.Y. performed the experimental work. Z.L., J.W., A.F. and Y.Z.
© The Author(s) 2021
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