Inherently Chiral Bambus[4]urils

Article abstract of DOI:10.1021/acs.joc.0c01174

A new class of bambus[4]urils (BU[4]s) composed of asymmetric N,N′-disubstituted glycoluril subunits with different alkyl groups were designed, synthesized, and fully characterized by NMR techniques and X-ray crystallography. Structural studies showed that four macrocyclic diastereoisomers are possible: two Sn symmetric achiral macrocycles and two macrocycles that are inherently chiral. The relative head-to-tail arrangement of the N-substituents in Bn4Me4BU[4], 5a, clearly observed by X-ray spectroscopy analysis, determines the overall symmetry of the bambusuril structure. Chiral Pr4Me4BU[4], 4b, was resolved by chiral high-performance liquid chromatography (HPLC) into its enantiomers, and all four inherently chiral bambusuril pairs (two Pr4Me4BU[4] and two Bn4Me4BU[4] stereoisomers, 4b, 4d, 5b, and 5d) were clearly observed by 1H NMR spectroscopy with the aid of (R)-BINOL as a chiral solvating agent. This latter methodology provides a rapid and powerful approach for investigating the enantiopurity of inherently chiral cavitands, which complements and augments the conventional chromatographic approaches.

Full text of DOI:10.1021/acs.joc.0c01174

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Article
Inherently Chiral Bambus[4]urils
Amar Ramchandra Mohite, and Ofer Reany
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01174 • Publication Date (Web): 10 Jun 2020
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Inherently Chiral Bambus[4]urils
†
†,
Amar Ramchandra Mohite and Ofer Reany
†
Department of Natural Sciences, The Open University of Israel, Ra’anana 4353701, Israel.
E-mail: oferre@openu.ac.il
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Abstract
A new class of bambus[4]urils (BU[4]s) composed of asymmetric N,N’-disubstituted glycoluril
subunits with different alkyl groups were designed, synthesized and fully characterized by NMR
techniques and X-ray crystallography. Structural studies showed that four macrocyclic
diastereoisomers are possible: two S symmetric achiral macrocycles and two macrocycles that
n
are “inherently” chiral. The relative “head-to-tail” arrangement of the N-substituents in
Bn Me BU[4], 5a, clearly observed by X-ray spectroscopy analysis, determine the overall
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symmetry of the bambusuril structure. Chiral Pr Me BU[4], 4b, was resolved by chiral HPLC
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into its enantiomers and all four inherently chiral bambusuril pairs (two Pr Me BU[4] and two
Bn Me BU[4] stereoisomers, 4b, 4d, 5b and 5d) were clearly observed by H NMR
spectroscopy with the aid of (R)-BINOL as a chiral solvating agent. This latter methodology
provides a rapid and powerful approach for investigating the enantiopurity of inherently chiral
cavitands, which complements and augments the conventional chromatographic approaches.
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TOC
Introduction
Chirality in macrocycles with large cavities has attracted great attention due to their extensive
use in chiral recognition, especially in their role for resolution of racemates and in selectively
transporting chiral guests through bulk liquid membrane technologies. In addition, chiral host
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systems are often regarded as enzyme mimics, acting as catalysts in asymmetric reactions to
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afford high yields and enantiomeric excess (ee) of chiral products. Therefore, development of
novel chiral receptors or supramolecular frameworks, provide tools for the stereochemical
understanding of complex biological systems. For example, chiral cyclodextrins (CDs) have
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been used as artificial hosts mimicking various enzyme activities, and chiral corands were
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utilized as transacylase mimics. Similarly, chiral porphyrins have been designed to model
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oxygen uptake and oxygen transport processes, and chiral crown-ethers were employed to
mimic natural ion channels.6
Glycoluril-based cavitands such as cucurbiturils (CBs) and bambusurils (BUs) are rigid achiral
macrocycles, exhibiting D and S point group of symmetry, respectively. A general criterion for
nh
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chirality is the absence of any improper symmetry elements, i.e. S , including mirror image,,
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or a center of inversion, i. As such, these cavitands can be made chiral by either appending an
intrinsically chiral functionality to the macrocyclic backbone or by using a non-symmetric subunit
to produce stereogenic cavitands (Scheme 1). To date, several chiral macrocycles have been
reported, including chiral hemicucurbiturils, biotinurils and chiral BU[6], all of which are
constructed from symmetrical cavity with stereogenic centers at their backbone or include
tethered linkers with “external” stereogenic groups.
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Chirality based on non-symmetric functionalization encompasses many examples of chiral
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calixarenes and resorcinarenes. Hence, substituents strategically positioned may give rise to
a macrocycle whose mirror image is not superimposable onto itself. In calixarenes, the
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expression inherently chiral was first coined by Bohmer et al. and later extended by Mandolini,
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Schiaffino and co-workers to include non-symmetrically substituted molecules that exhibit a
curvature structure. In the last decade the concept of inherent chirality was demonstrated in the
synthesis and applications of molecules that present curvature.13,14 Interestingly, only rare
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examples of inherently chiral cucurbiturils are known in the literature and, to the best of our
knowledge, there are no reports of such chirality in bambusurils. ()-bis-nor-seco-CB[6], was
the first reported cavitand where enantio- and diastereoselective recognition was demonstrated
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by cavity selection, without the need for a chiral auxiliary. Similarly, the nor-seco-CB[10] and
the Eu+3 complex of twisted CB[14] exhibit asymmetric cavities but no applications in chiral
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recognition were demonstrated.
Recently, we introduced a new family of cavitands, dubbed hetero-bambus[n]urils (BU[n]s).19
These macrocycles are rigid molecules due to the high steric demands of their subunits, which
are locked in an alternate arrangement. Moreover, we demonstrated that sulfur analogs of
BU[4], which are too small to accommodate any type of guest, have shown much potential for
the formation of metal-organic chain polymers20,21 and in anchoring self-assembled monolayers
(
SAMs) on gold surfaces.22 Therefore, designing inherently chiral hetero-BUs would be
appealing for developing applications in the field of chiral sensing technologies.
Here, we demonstrate a synthetic strategy for the preparation of a new family of bambus[4]urils
(BU[4]s) composed of asymmetric N,N’-disubstituted glycoluril subunits with different alkyl
groups (Scheme 1). Accordingly, the synthesis of Pr Me BU[4] and Bn Me BU[4] afforded
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mixtures of four possible diastereoisomers: two of which are symmetric achiral (S and S
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symmetry), and two “inherently” chiral macrocycles (C and C symmetry). Each one of the
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diastereoisomers was isolated and fully characterized by spectroscopic techniques including
NMR and X-ray spectroscopy. The solid state structures of the achiral stereoisomers of
Pr Me BU[4] and Bn Me BU[4] reveal two possible orientation modes, head-to-tail and head-
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to-head, derived from the different spatial orientation of the substituents around the bambusuril
cavity. We also succeeded to separate between the enantiomers of each of the two inherently
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chiral bambusurils and determined their purity by H NMR spectroscopy, using 1,1’-bi-2-
naphthol (BINOL) derivatives as chiral solvating agents. We also show that the enantiomeric
purity results obtained by this technique are closely matched to values determined by chiral
HPLC analysis.
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Scheme 1. Different categories of stereogenicity in BU[4]s depending on the alkyl group identity
in N,N’-disubstituted glycoluril subunit: A. achiral BU[4], B. BU[4] equipped with stereogenic
centers and C. BU[4] exhibiting four possible diastereoisomers: two of which are symmetric
achiral (S and S symmetry), and two “inherently” chiral macrocycles (C and C symmetry).
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Results and Discussion
Synthesis and characterization
For the synthesis of BU[4] with different N-substituents, asymmetric N,N’-disubstituted glycoluril
bearing different alkyl substituents were initially prepared. Commercially available propyl or
benzyl isocyanates were treated with methylamine to produce the corresponding asymmetric
dialkyl ureas, followed by reaction with trans-4,5-dihydroxyimidazolidin-2-one (DHI) to afford
a mixture of enantiomeric glycoluril products, 3a and 3b (Scheme 2).
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Scheme 2. Synthesis of asymmetric glycoluril (racemic mixture) from asymmetric dialkylurea.
Conditions: a) MeNH (1.5 equiv.), 0 C for 2 h, then, RT, 6 h in dry THF; b) DHI (1.1 equiv.),
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con. HCl (0.1 mL), reflux in water, 1 h.
Condensation of the racemic mixtures of 3a and 3b with paraformaldehyde in acidic (PTSA)
chloroform solution followed by microwave irradiation for 2 h, respectively afforded all possible
Pr Me BU[4] stereoisomers, 4a-d, and Bn Me BU[4] stereoisomers, 5a-d in good overall
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yields(Figure 1). All stereoisomers could be separated by preparative TLC and fully
characterized by NMR and HRMS (vide infra and in experimental section). Interestingly,
significant amounts of every diastereomer could be found in the mixture and the ratio between
the four stereoisomers was found to be 1:1:1:1 and 0.8:1:1:0.6, for 4 and 5 respectively.
Figure 1. Top: drawings of all BU[4] diastereoisomers, 4a-d and 5a-d in 1,3-alternate structure
(color code: red, a square plane representing the equator part of the bambusuril; black, dialkyl
imidazolidinone rings above the square plane; grey, dialkyl imidazolidinone rings below the
square plane). Bottom: schematic presentation of the larger substituents in all possible
orientations. Arrowheads represent the larger group above or below the square plane of BU[4]
in either head-to-tail or head-to-head arrangement.
The 1,3-alternate structure of BU[4] equipped with eight identical alkyl groups exhibits a high
degree of symmetry (S ), which is reflected in a limited set of nonequivalent hydrogen atom
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signals in the H NMR spectrum. For example, the number of observable nonequivalent
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hydrogen atoms of R BU[4] in the H NMR spectrum consists of just three signals, assigned to
the N-CH R groups, methylene groups (NCH N) and the methine groups. Unfortunately,
Me BU[4] has not been prepared; however, the number of limited nonequivalent hydrogen
atoms in R BU[4] with various alkyl groups (R = Pr, Bn, allyl)
Me BU[4] confirm the high degree of symmetry and the H NMR spectra show identical pattern
of three signals.
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as well as in semithio-
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If one of the methyl groups in glycoluril is substituted by a different group (Scheme 2), then a
mixture of two enantiomers is obtained. The cyclotetramerization of the glycoluril racemate with
paraformaldehyde generates four possible stereoisomers of R Me BU[4] (R  Me) (Fig. 1). The
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H NMR spectra of these stereoisomers are expected to differ both in the chemical shift and in
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the number of nonequivalent protons (number of signals). Indeed, we observed that the H NMR
spectra of stereoisomers 4a-d and 5a-d are remarkably distinct and display variation in the
number of nonequivalent hydrogen NMR signal as a result of their singular symmetric
environment (vide infra Figures 2 & 3). For example, the structure of stereoisomer 5a exhibits
S symmetry. In this arrangement, each of the methyl groups is surrounded by an identical
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chemical environment, resulting in a single peak at 2.87 ppm (Figure 2, 5a). Replacement of
one of the glycoluril units by its enantiomer (Scheme 2) affords stereoisomer 5b with C1
symmetry, which is manifested by the appearance of four different singlets at 2.85, 2.87, 3.06
and 3.07 ppm assigned for each of the four diastereotopic methyl groups (Figure 2, 5b).
Similarly, replacing two neighboring glycoluril units by their enantiomers affords stereoisomer
5c with S symmetry (inversion) and, consequently, two singlets at 2.85 and 3.08 ppm are
observed in the H NMR spectrum (Figure 2, 5c). In contrast, if the two glycouril enantiomers
are positioned opposite to each other, then 5d with C symmetry is obtained, where all four
chemically equivalent methyl groups appear at the same chemical shift, in this case at 3.04 ppm
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(Figure 2, 5d).
Considering the position of the benzyl group by using the arrowheads drawing presentation (see
schematic representation in Figure 1, bottom), two types of linking modes between neighboring
glycoluril subunits in the 1,3-alternate geometry are defined: “head-to-tail” and “head-to-head”
modes. Accordingly, the benzyl groups in 5a are all arranged in head-to-tail mode and in 5d the
benzyl groups are all arranged in head-to-head mode, whereas stereoisomers 5b and 5c exhibit
mixed modes. Apparently, these configurations have a profound effect on the chemical shift of
the corresponding protons (Figure 2). For example, the neighboring methyl groups in head-to-
tail mode undergo a large shielding effect (upfield shift), probably due to an anisotropic effect
exerted by the phenyl ring. Thus, the mode by which the substituents are arranged can be
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unequivocally determined from the chemical shifts in the H NMR spectrum.
Further detailed analysis can be inferred from the NMR spectra of 5, particularly, by identifying
all other signals. For instance, the diastereotopic methine protons in 5a give rise to two-
separated AB spin system at 5.59 and 5.52 ppm, each corresponding to 4 protons with a 3J
coupling of 8.4 Hz, which is typical when the vicinal protons are trans-coplanar (i.e., the vicinal
dihedral angle  = 0°). In contrast, the chemical shift difference between the coupled methine
protons in 5d is almost indistinguishable. This trend is consistent with other coupled protons of
both H NMR spectra. Thus, the benzyl protons in 5a give rise to two doublets at 4.76 and 4.33
ppm, each corresponding to 4 protons with a J coupling of 16 Hz, whereas the observed
chemical shift difference between the coupled protons in the H NMR spectrum of 5d is less
than 0.1 ppm. Similarly, the bridging methylene protons appear in the spectrum of 5a at 4.04
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and 3.98 ppm as two doublets ( J = 15.3 Hz), whereas in the spectrum of 5d these protons
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appear as two singlets at 4.64 and 3.76 ppm. These spectroscopic features allow us to identify
and discriminate between the linking modes of neighboring glycoluril subunits. Thus, if both
benzyl groups are in close proximity to the bridging methylene groups (e.g. in 5d), then the
chemical shift differences between the methylene hydrogens will be smaller than the difference
when only one of the benzyl groups is in close proximity to the bridging methylene hydrogens
(e.g. 5a). Indeed, the two linking modes, head-to-tail and head-to-head, are clearly manifested
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in the H NMR spectra of stereoisomers 5b and 5c (marked accordingly in Figure 2).
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Figure 2. Aliphatic region of the H NMR spectra (400 MHz, CDCl ) of stereoisomers of 5,
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highlighting the influence of different binding modes on the chemical shift of the corresponding
signals.
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The chemically nonequivalent methine groups are also evident in the C NMR spectrum of 5a
displaying two peaks at 71.9 and 71.6 ppm. Likewise, two separated peaks at 72.1 and 71.2
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ppm are observed in the C NMR spectrum of 5d. On the other hand, four signals are observed
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in the C NMR spectra of 5b and 5c, confirming the presence of both linking glycoluril modes
in the structures of these stereoisomers (see SI Figures S7-S10). 2D-NMR experiments (COSY,
HMQC and 2D-NOESY) were recorded and support the structure of each stereoisomer of 5
(Figures S11-S18).
Similar NMR spectroscopy analyses were performed to characterize each stereoisomer of
Pr Me BU[4], 4 (Figure 3 and in SI). Interestingly, the N-methyl groups appear as a single singlet
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at 3.0 ppm corresponding to all 12 methyl protons for all the stereoisomers, except for 4b.
Moreover, replacing all benzyl substituents by propyl groups is obviously manifested by
diminished anisotropic effects in the chemical shifts of methine, bridging methylene and N-
methyl groups. Consequently, the distinction between the linking modes between two
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neighboring glycoluril subunits in 4 stereoisomers is less evident from their H NMR spectra.
Nevertheless, the chemical nonequivalence is fully retained for each given type of protons. For
example, the chemical shifts of the non-equivalent methine protons are separated by 0.13-0.19
ppm and that of the non-equivalent methylene protons in the propyl groups by 0.35 ppm.
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Figure 3. H NMR spectra (400 MHz, CDCl ) of stereoisomers of 4. Hashtag represents the
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water signal in chloroform and asterisks show solvent impurities.
X-ray crystallography.
The solid-state structures of bambus[4]uril with different substituents was then investigated.
Single crystal (Figure 4 and Table 1) of 5a, suitable for X-ray crystallography, was obtained by
slow evaporation of a mixture of CHCl and o-dichlorobenzene solutions, and the data was
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collected at 110 K (more crystallographic details in Table S1). Although the phenyl groups are
highly disordered and several disordered solvent molecules were observed, we were able to
solve the structure by using heavy restrains during refinements (see also Table S1). Expectedly,
the rigid structure of 5a exhibits the 1,3-alternate arrangement of glycoluril subunits, consistent
with previously reported solid-state structures of hetero-BU[4]s.24-26 Accordingly, the crystal
structure of 5a is monoclinic, containing four molecules in the unit cell with space group Cc and
cell constants of a = 23.264(5), b = 18.390(4), c = 17.423(4) Å; and  = 127.66(3)˚, respectively.
A top view of the crystal structures reveals the relative orientation between substituents (Figure
4A). Hence, in the crystal structure of 5a all four benzyl groups are oriented anticlockwise in a
head-to-tail relationship confirming the anticipated S symmetry.
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Recently, four intramolecular distances, d -d and a torsional angle,  (vide infra), were defined
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to characterize the conformations of the various heteroisomers of BU[4]. Accordingly, d is the
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distance between the peripheral carbonyl heteroatoms, d is the distance between the C
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symmetry related methine carbon atoms, d is the distance between the two equatorial carbonyl
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oxygen atoms (on the same side), and d is the distance between the planes that include all four
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methine groups of each portal (Figure 4B). In addition, each glycoluril can be viewed as two
rigid flaps fused together at an angle of 113˚: one flap participates in the molecular portal
whereas the other flap constitutes the molecular equator. Table 1 highlights the similarity of the
measured intramolecular distances in 5a to the values measured in other achiral BU[4]
analogues, and that indeed it adopts a double-cup, jigger-like conformation, which is persistently
preserved regardless of the nature of their substituents. An analogous trend was also observed
for , which is defined as the torsion angle between two adjacent flaps that are connected by a
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methylene group (see also in Figure 1 colored in red). Hence, in a typical jigger conformation,
where   150˚, the portal flaps are effectively perpendicular to one another.
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A.
B.
Figure 4. A. Top view of the X-ray single structure of 5a represented in stick model revealing
S symmetry and disordered substituents. Thermal ellipsoids are shown at the 50% probability
4
level. Solvent molecules were omitted for clarity. Color code: nitrogen, blue; oxygen, red;
carbon, grey; hydrogen: white. B. Side view structure of 5a as a model used to define distances
d −d and torsional angle θ.
1
4
Table 1. Structural parameters of various BU[4].
Entry
BU[4]
d1 (Å)
10.6-10.7 5.1-5.5
8.5-8.9 4.3-4.9
10.4-10.5 5.0-5.6
d2 (Å)
d3 (Å)
5.4
6.1-6.2
5.3-5.5
d4 (Å)
 (˚)
24
1
2
3
allyl BU[4]
3.91
3.93
3.91
150.6
131.0
148.3
8
25
Bn BU[4]
8
5a (Bn Me BU[4])
4
4
Enantiodifferentiation of chiral bambusurils
Since 4b and 4d, as well as 5b and 5d, are inherently chiral cavitands, each one of them
comprises a pair of enantiomers. Hence, we sought after an efficient technique to resolve them.
Initially, attempts were focused on the enantiomeric separation of 5b by chiral high-performance
liquid chromatography (HPLC) techniques. Enantiomer separation of 5b with various chiral
HPLC columns was unsuccessful. In contrast, a satisfactory distinction between enantiomers of

4
b was achieved on a Chiralcel OD-RH column using water-acetonitrile mixture (65:35, v/v) as
eluent (Figure S19). Notably, the separation was better at isocratic conditions. Even though the
peak’s width increased with retention time, the resulting selectivity factor ( = 1.13) was
sufficient to collect each one in high purity (>95 %) by fractional separation.
To circumvent the tedious, expensive and slow chromatographic analytical procedure, and to
validate the HPLC separation results, we turned our attention towards alternative
enantiodifferentiation methods that could be more straightforward and efficient. Among the
commonly used NMR methodologies to investigate enantiomeric separation conditions,28 we
assumed that lanthanide-based chiral shift reagents (LSRs) would be the preferred choice
because lanthanides are hard Lewis acids that primarily form complexes with oxygen- and
nitrogen-containing donors. However, all attempts to differentiate between the enantiomers of
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4b in the presence of Europium(III) tris[3-(hepta-fluoropropylhydroxymethylene)-d-camphorate]
(
Eu(hfc) ) or Eu(OTf) failed.
3 3
Another commonly used methodology to differentiate between enantiomers comprises chiral
solvating agents (CSAs), which form diastereoisomeric solvation complexes with solute
enantiomers via rapidly reversible equilibria in competition with the bulk solvent. We investigated
a series of chiral 1,1’-bi-2-naphthol (BINOL) derivatives (Figure 5, (R)-BINOL I-V), which are
broadly applicable CSAs, in solutions containing mixtures of all stereoisomers of 4.
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Figure 5. Various chiral BINOL compounds employed as chiral solvating agents in this study.
These atropisomeric compounds can form complexes using the hydroxy groups as hydrogen
bond donors and can lead to different chemical shifts for the otherwise undistinguishable
enantiomer protons.29 Indeed, an in-situ mixture of all stereoisomers of 4 was treated with
1
BINOL I (10 equiv.) giving rise to dramatic changes in the chemical shift values of all H NMR
signals (Figure 6).
A.
B.
C.
#
*
*
*
1
Figure 6. A. Fragments of stacked H NMR spectra (400 MHz, CDCl ) of a mixture of 4 (3 mg,
3
1
equiv) diastereoisomers exhibiting  variation of the methyl singlets in the presence of
H
1
different (R)-BINOLs (10 equiv). B. H NMR spectra (400 MHz) of all 4 (3 mg, 1 equiv)
diastereoisomers in different solvents presenting variation in the  of the methyl signals before
H
and C. after addition of (R)-BINOL I (20 equiv). Asterisks refer to residual trace solvent (acetone
and water) and hashtag refer to benzylic protons of (R)-BINOL III.
The analysis of the signals corresponding to the methyl groups of 4 is simpler to monitor
because their hydrogen resonances originally appear as two singlets at ca. 3.01 ppm and then
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split to multiple singlets. To our satisfaction, the recorded spectrum showed almost complete
separation of all diastereoisomers, with an average shift of  = 0.175 ppm between the
H
stereoisomeric mixture of 4 (rac = 3.01 ppm) and their complexes with BINOL I (average = 2.825
ppm) (Figure 6A). A similar tendency was observed with (R)-BINOLs II-V, albeit the differences
in the chemical shift values for the methyl groups were smaller ( = 0.025, 0.060 and 0.013
H
ppm, respectively) and the signals were less resolved.
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Interestingly, a strong solvent effect could also be observed in the NMR mixture. Nonpolar
solvents such as chloroform and toluene were shown to maximize the observed anisochrony
between the diastereoisomeric complexes after the addition of (R)-BINOL I, while more polar
solvents such as methanol and acetone preferentially solvate BU[4] and the corresponding H
values were smaller (Figure 6B and 6C). Noticeably, there were no observable changes in the
1H NMR spectrum of 4 in DMSO before and after addition of (R)-BINOL I. Apparently, DMSO
disrupts the BINOL binding to the BU[4].30.
To elucidate the association between BINOL I and stereoisomers of 4, nuclear Overhauser
effect (NOE) spectroscopy was employed. Hence, correlation peaks between several (R)-
BINOL I naphthyl signals and the signals of propyl protons of 4 mixture were clearly detected
by 2D ROESY (rotating frame Overhauser spectroscopY),31 indicating the close proximity of
naphthyl rings to the propyl groups (Figure 7).
A.
B.
Figure 7: A. Portion of the 2D ROESY spectrum (400 MHz, CDCl at 298 K) of (R)-BINOL I and
3
the mixture of 4 stereoisomers in 5:1 ratio. B. Suggested structure of the (R)-BINOL I complex
with Pr Me BU[4] mixture of all four stereoisomers. Arrows represent the observed
4
4
intermolecular NOEs between hydrogens of the glycoluril units (H , green; H , blue; H red; and
1
2
3
H yellow) and the corresponding aromatic hydrogens in BINOL molecule (H ).
4
a-f
Furthermore, by performing a Job plot analysis the binding stoichiometry of the formed
32
complexes was elucidated (for details see SI). Accordingly, the Job plot displayed a sigmoidal
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curvature with a maxima BINOL mol fraction (BINOL) at 0.5-0.55. This behavior is clearly
indicative of 1:1 complex formation.
Following the above results, the applicability of CSA-mediated NMR analysis in
enantiodifferentiation between inherently chiral cavitands was probed on the enantiomers of 4b
with (R)-BINOL I. Recently, CSA-mediated NMR was successfully applied to challenging
pharmaceutical development problems, which were not amenable to chromatographic
solutions, and provided a rapid and powerful solution for investigating enantiomeric purity that
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33
complements and augments conventional chromatographic approaches. Hence, the 4b
1
enantiomers were separated by chiral HPLC and their H NMR spectra were recorded in CDCl
3
(Figure 8). Expectedly, both spectra were identical and overlapped the spectrum of the racemic
mixture (Figure 8B). However, upon addition of (R)-BINOL I (5 equiv) the formation of
diastereomeric complexes (exhibiting C symmetry) resulted in two sets of four separable
1
singlets assigned for each enantiomer (Figure 8C). The results obtained from the integration of
1H NMR signals for each enantiomer allowed us to estimate the purity of each enantiomer (ca.
95 ee-%).
A.
B.
C.
Figure 8: A. HPLC chromatogram displaying separation between enantiomers of rac-4b on a

1
Chiralcel OD-RH column (more details in SI); B. Fragments of H NMR spectra (400 MHz,
CDCl .) of rac-4b and its separated enantiomers and C. following addition of (R)-BINOL I (5
3
equiv).
To validate the generality of this technique in the context of inherently chiral bambusurils, NMR
titration of both BINOL I enantiomers to a solution containing racemic mixture of 5b was
performed. Upon stepwise addition of (R)-BINOL I (0.5-20 equiv.), all four-nonequivalent methyl
1
groups in the H NMR spectrum of the racemic mixture of 5b displayed an upfield shift and split
into eight discrete singlet signals; four singlets for each enantiomer (Figure 9). Interestingly,
1
matching H NMR spectra were recorded upon stepwise addition of (S)-BINOL I (0.5-20 equiv.),
indicating that the structures of the formed complexes are symmetrically identical and that both
BINOL I enantiomers produce identical chemical shift anisochrony. Hence, both enantiomeric
BINOL-based solvating agents can be used to discriminate between inherently chiral
stereoisomers of BU[4].
In view of the fact that BINOL compounds form weak intermolecular interactions with
1
bambusurils, we also observed changes in the chemical shift pattern of H NMR spectra of
achiral bambusurils such as 5a (Figure 10A). Compound 5a exhibit S symmetry and therefore
4
all four methyl groups display a single singlet signal at  = 2.835 ppm. Upon stepwise addition
H
of (R)-BINOL I to the solution, the methyl protons peak in 5a split into two discrete singlets
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following complexation with (R)-BINOL I. Hence, the observed upfield shift of the methyl group
signals at complex = 2.82 and 2.765 ppm is explained by the loss of one or more symmetry
elements in the structure of 5a. Consequently, the chemically non-equivalent protons are
exposed by their chemical shift differences, which are also concentration-dependent with
respect to the added amount of chiral BINOL (as expected for an equilibrium mixture). A similar
observation was obtained with (S)-BINOL I (Figure 10B).
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1
Figure 9. Fragments of stacked spectra from H NMR titration (400 MHz, CDCl ) of (R)- and
3
(S)-BINOL-I with rac-5b (5 mM).
A.
B.
1
Figure 10. Fragments of stacked spectra from H NMR titration (400 MHz, CDCl ) of 5a (5 mM)
3
with (R)-BINOL I (0-60 equiv.) (A) and with either enantiomer of BINOL I (0-20 equiv.) (B).
Summary
A series of novel chiral bambus[4]urils containing asymmetric glycoluril subunits with different
alkyl substituents have been prepared and all four possible stereoisomers of the X Me BU[4]
4
4
family (X = Pr or Bn) have been isolated. These stereoisomers were fully characterized by NMR
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techniques and the solid-state structure of 5a, was analyzed by X-ray crystallography. The
crystal structure confirm the induced “head-to-tail” arrangement of the N-substituents, following
the different spatial orientation of the substituents around the bambusuril cavity. Moreover, it
was shown that the different arrangements have a significant effect on the NMR chemical shifts
of the corresponding atoms.
All stereoisomers were found to form complexes with chiral BINOLs, probably based on
intermolecular hydrogen-bond interactions between the diol moiety of the BINOL molecule and
the carbonyl groups at both portals of the bambusuril. These interactions were also evident from
the NOE correlations observed between the substituent’s protons in the bambusuril molecule
and the arene group protons of the BINOL compound.
1
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Finally, the role of BINOL I as a chiral solvating agent (CSA) to differentiate between bambusuril
1
stereoisomers was manifested in the H NMR spectra of two inherently chiral stereoisomers.
The chemical shift differences were found to be markedly dependent on: (1) the nature of
solvent, (2) the nature of substituents in the BINOL structure and (3) the extent of CSA
necessary for maximal nonequivalence chemical shifts of the enantiopure bambusuril. This
methodology provides a rapid and powerful approach for investigating enantiopurity of
inherently chiral cavitands, which complements and augments conventional chromatographic
approaches.
The new inherently chiral BU[4]s offer interesting opportunities and future applications in the
area of supramolecular chemistry. Currently, we are exploring these opportunities with hetero-
bambusurils in our laboratory.
Experimental Section
General Information. All chemicals, including 1a and 1b, and solvents were commercially
available and used without further purification. 4,5-dihydroxyimidazolidin-2-one (DHI) was
prepared according to ref. 23. Analytical TLC was performed using Merck silica gel F254 (230-
400 mesh) plates and analyzed by UV light or by staining upon heating with vanilline solution
Melting points (mp) were determined in open capillaries using a Stuart SMP11 melting point
1
13
apparatus. The H and C NMR spectra were recorded in CDCl or DMSO-d , on Bruker
3
6
AVIII400 or Bruker DPX 400 spectrometers. The chemical shifts are reported in parts per million
(
ppm) relative to residual solvent signals: CDCl :  = 7.26 ppm,  = 77.23 ppm; DMSO-d : 
3
H
C
6
H
= 2.50 ppm,  = 39.51 ppm.
C
Single crystals of compound 5a were obtained by standing a period in o-dichlorobenzene-
chloroform (1:1) mixture. The single-crystals of each compound were immersed in Paratone–N
oil and mounted on an APEX 2 (Bruker AXS) diffractometer at a temperature of 110 K. Data
collection was performed with a monochromatic Mo K radiation ( = 0.71073 Å) and using φ
a
34
and ω scans to cover the Ewald sphere. Using Olex2 program, the structure was solved with
35
the olex2.solve; a structure solution program using Charge Flipping method, and refined with
36
the ShelXL refinement package using Least Squares minimization. Non-hydrogen atoms were
refined with anisotropic displacement parameters. The hydrogen atoms were refined
isotropically on calculated positions using a riding model with their Uiso values constrained to
3
1
.5 times the U of their pivot atoms for terminal sp carbon atoms and 1.2 times for all other
eq
carbon atoms. Software used for molecular graphics: Mercury 4.0.0.37
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The chromatographic separation of enantiomers was performed on an YL9100 HPLC System,
consisting of a YL9101 Vacuum Degasser, a YL9110 Quaternary Pump and a YL9160 PDA
Detector. Chromatographic resolution of enantiomers was carried out on a reverse-phase
carbamate-based cellulose (Daicel chemical industries Ltd. Tokyo, Japan) under isocratic and
isothermal (at 30 C) conditions using water/acetonitrile mixture as the appropriate mobile
phase giving in the main text. Chiral column was analytical type, dimension 150 mm4.6mm.
Sample preparation: rac-4b (10 mg) was dissolved in a 65:35 (v/v) mixture of water/acetonitrile
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(10 mL) with 10 µL of sample solution then injected for HPLC analysis. Detection wavelength:
214 nm.
General procedure for the synthesis of N,N’-disubstituted ureas: To a stirred solution
of 1a or 1b (2 g) in dry THF (10 mL) was added dropwise a solution of methyl amine in THF
o
o
(
2M, 1.5 equiv) at 0 C. The reaction mixture was stirred at 0 C for 2 h and then at room
temperature for additional 6 h. After removal of the solvent under reduced pressure, the residual
precipitate was washed with diethyl ether (DEE, 50 mL) filtered and dried under high vacuum.
1
1-methyl-3-propylurea (2a).
White solid (1.9 g, 70% yield).mp 65-67 °C. H NMR (400
MHz, CDCl )  5.21 (bs, 2H), 3.11 (q, J = 7 Hz, 2H), 2.73 (d, J = 4.4 Hz, 3H), 1.47 (sextet, J =
3
1
3
1
7 Hz, 2H), 0.89 (t, J = 7 Hz, 3H). C{ H} NMR (100 MHz, CDCl )  160.1, 42.5, 27.2, 23.8,
3
-
1
1.7. HRMS (ESI) m/z: [M - H] Calcd for C H N O 163.0871; Found 163.0870.
9
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2
1
1-benzyl-3-methylurea (2b).
White solid (2.12 g, 86% yield). mp 95-96 °C. H NMR
(
400 MHz, DMSO-d )  7.32–7.19 (m, 5H), 6.37 (bt, J = 5.6 Hz, 1H), 5.81 (bq, J = 4.4 Hz, 1H),
6
13
1
4
1
.21 (d, J = 6.4 Hz, 2H), 2.58 (d, J = 4.4 Hz, 3H); C{ H} NMR (100 MHz, DMSO-d )  159.6,
6
-
42.0, 129.1, 127.9, 127.4, 43.9, 27.4; HRMS (ESI) m/z: [M - H] Calcd for C H N O 163.0871;
9
11
2
Found 163.0870.
General procedure for the synthesis of N,N’-disubstituted glycolurils: A suspension
solution containing 4,5-dihydroxyimidazolidin-2-one (1 g, 8.5 mmol) and urea 2a or 2b (1 equiv)
in water (3 mL) were heated up to 60 °C under vigorous stirring and conc. HCl (35%, 0.15 mL)
was added. Then the heating temperature was raised and kept at 105 °C for additional 75 min.
Upon cooling, the gummy precipitate solidified and then filtered, washed with water and dried
under high vacuum to afford 3a or 3b respectively.
1
-methyl-3-propyl-tetrahydroimidazo-[4,5-d]imidazole-2,5(1H,3H)-dione (3a). White solid
1
(
1.2 g, 71% yield). m.p. 265-266 °C; H NMR (400 MHz, DMSO-d )  7.52 (s, 1H), 7.49 (s, 1H),
6
5.23 (dd, J = 8 Hz and 1 Hz, 1H), 5.13 (dd, J = 8 Hz and 1 Hz, 1H), 3.16–3.09 (m, 1H), 2.97–
13
1
2.91 (m, 1H), 2.63 (s, 3H), 1.58–1.37 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H); C{ H} NMR (100 MHz,
-
DMSO-d )  162, 158.6, 68.3, 66.5, 43.5, 28.6, 21.5, 12.1; HRMS (ESI) m/z: [M - H] Calcd for
6
C H N O 197.1039; Found 197.1037.
8
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4
2
1
-benzyl-3-methyl-tetrahydroimidazo[4,5-d]imidazole-2,5(1H,3H)-dione (3b). White solid
1
(
1.55 g, 75% yield). mp 215-216 °C; H NMR (400 MHz, DMSO-d )  7.61 (s, 1H), 7.58 (s, 1H),
6
7
.35–7.32 (m, 2H), 7.28–7.24 (m, 3H), 5.16 (dd, J = 8 Hz and 1.6 Hz, 1H), 5.03 (dd, J = 8 Hz
13
1
and 1.6 Hz, 1H), 4.60 (d, J = 15.6 Hz, 1H), 4.01 (d, J = 15.6 Hz, 1H), 2.69 (s, 3H); C{ H} NMR
(
100 MHz, DMSO-d )  162, 158.4, 138.4, 129.4, 128.7, 128.1, 68.2, 65.9, 44.8, 28.7; HRMS
6
-
(ESI) m/z: [M - H] Calcd for C H N O 245.1039; Found 245.1030.
12
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4
2
General procedure for the synthesis of BU[4] derivatives.38 A mixture containing
compound 3a or 3b (0.246 g, 0.1 mol), paraformaldehyde (36 mg, 1.2 equiv) and p-toluene
sulfonic acid (0.172g, 1 equiv) in CHCl (10 mL) was subjected to microwave irradiation for 2 h
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under continuous stirring at 75 °C (Pmax = 200 watt, T = 75 °C). Then the reaction mixture was
cooled and diluted with CHCl (30 mL). The organic layer was washed with saturated NaHCO
3
3
(50 mL), water (50 mL) and brine (50 mL), and the organic layer was dried over anhydrous
Na SO . After filtration, the solution was concentrated. After addition of diethyl ether, the
2
3
bambusuril stereoisomer mixture was precipitated as white solid immediately thereafter. After
filtration and washing with diethyl ether, the white solid was dried under high vacuum. The
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mixture of stereoisomer products was re-dissolved in CHCl and run on preparative thin layer
3
chromatography (PTLC) with ethyl acetate as mobile phase to give each of the
diastereoiasomers of either 4a-d or 5a-d isolated in the following ratio: 4a:4b:4c:4d (1:1:1:1) or
5a:5b:5c:5d (0.8:1:1:0.6).
1
Pr Me BU[4] (4, as mixture of diastereoisomers): White solid (0.173 g, 69% yield). H NMR (400
4
4
MHz, CDCl )  5.93–5.85 (m, 16H), 5.77–5.69 (m, 16H), 4.77-4.63 (m, 32H), 3.53-3.42 (m,
3
16H), 3.18-3.10 (m, 16H), 3.00 (s, 24H), 2.99 (s, 24H), 1.74-1.71 (m, 16H), 1.62-1.52 (m, 16H),
13
1
0
7
3
.95-0.91 (m, 48H); C{ H} NMR (100 MHz, CDCl )  159.9, 159.8, 158.2, 158.2, 72.5, 72.4,
3
0.6, 70.52, 70.46, 70.38, 52.1, 51.96, 51.83, 51.69, 51.54, 51.42, 51.29, 44.57, 30.20, 30.15,
0.10, 30.05, 30.02, 21.45, 21.39, 11.60, 11.55.
1
Pr Me BU[4] (4a). White solid; H NMR (400 MHz, CDCl )  5.93 (d, J = 8 Hz, 4H), 5.74
4
4
3
(
d, J = 8 Hz, 4H), 4.61 (dd, J = 16 Hz and 20 Hz, 8H), 3.51–3.44 (m, 4H), 3.19–3.11 (m, 4H), 3
13
1
(s, 12H), 1.80 (sextet, J = 8 Hz, 4H), 1.62 (sextet, J = 8 Hz, 4H), 0.96 (t, J = 8 Hz, 12H); C{ H}
NMR (100 MHz, CDCl )  159.8, 158.2, 72.4, 70.6, 51.3, 44.6, 30.2, 30, 21.5, 11.6.
3
1
Pr Me BU[4] (4b). White solid; H NMR (400 MHz, CDCl )  5.92 (dd, J = 8 Hz, 4H), 5.77
4
4
3
(
6
dd, J = 8 Hz, 4H), 4.78–4.64 (m, 8H), 3.54–3.44 (m, 4H), 3.18–3.11 (m, 4H), 3.01 (s, 6H), 3 (s,
13
1
H), 1.74 (bs, 4H), 1.62 (bs, 4H), 0.96 (sextet, J = 4 Hz, 12H); C{ H} NMR (100 MHz, CDCl )
3

159.8, 158.2, 72.7, 72.6, 72.5, 72.4, 70.6, 70.5, 70.49, 70.4, 69.8, 52, 51.9, 51.6, 51.5, 44.6,
+
30.2, 30.1, 21.5, 21.4, 21.3, 11.6, 11.58; HRMS (ESI) m/z: [M + H] Calcd for C H N O
36
57 16
8
841.4545; Found 841.4555.
1
Pr Me BU[4] (4c). White solid; H NMR (400 MHz, CDCl )  5.91 (t, J = 8 Hz, 4H), 5.78 (t,
4
4
3
J = 8 Hz, 4H), 4.77–4.66 (m, 8H), 3.54–3.46 (m, 4H), 3.19–3.12 (m, 4H), 3 (s, 12H), 1.80 (sextet,
J = 8 Hz, 4H), 1.64 (sextet, J = 8 Hz, 4H), 0.97 (t, J = 8 Hz, 12H); 13C{ H} NMR (100 MHz,
1
CDCl )  160, 159.8, 158.2, 158.19, 72.6, 72.4, 70.5, 70.4, 51.8, 51.7, 51.6, 44.7, 44.6, 30.1,
3
+
30, 21.4, 11.6; HRMS (ESI) m/z: [M + H] Calcd for C H N O 841.4545; Found 841.4555.
36
57 16
8
1
Pr Me BU[4] (4d). White solid; H NMR (400 MHz, CDCl )  5.92 (d, J = 8 Hz, 4H), 5.74
4
4
3
(d, J = 8 Hz, 4H), 4.73 (d, J = 20 Hz, 8H), 3.52–3.45 (m, 4H), 3.18–3.10 (m, 4H), 3.01 (s, 12H),
13
1
1
.80 (sextet, J = 8 Hz, 4H), 1.64 (sextet, J = 8 Hz, 4H), 0.97 (t, J = 8 Hz, 12H); C{ H} NMR
(
100 MHz, CDCl )  159.8, 158.2, 72.5, 70.5, 51.8, 44.6, 30.2, 21.5, 11.6.
3
Bn Me BU[4] (5, as mixture of diastereoisomers): White solid (0.158 g, 63% yield).
4
4
1
Bn Me BU[4] (5a). White solid; H NMR (400 MHz, CDCl )  7.34–7.24 (m, 20H), 5.59 (d,
4
4
3
J = 8.4 Hz, 4H), 5.52 (d, J = 8.4 Hz, 4H), 4.76 (d, J = 16.0 Hz, 4H), 4.33 (d, J = 16.0 Hz, 4H),
13
1
4
.04 (d, J = 16 Hz, 4H), 3.98 (d, J = 16 Hz, 4H), 2.83 (s, 12H); C{ H} NMR (100 MHz, CDCl )
3

159.3, 158.6, 137.7, 129.2, 128, 127.9, 72.3, 71.9, 51, 47.8, 29.9; HRMS (ESI) m/z: [M + H]+
Calcd for C H N O 1033.4545; Found 1033.4539.
52
57 16
8
1
Bn Me BU[4] (5b). White solid; H NMR (400 MHz, CDCl )  7.35–7.23 (m, 16H), 7.16–
4
4
3
7.13 (m, 4H), 5.66–5.44 (m, 8H), 4.81–4.64 (m, 4H), 4.46–4.29 (m, 6H), 4.10–3.95 (m, 4H), 3.75
13
1
(
s, 2H), 3.03 (s, 3H), 3.015 (s, 3H), 2.83 (s, 3H), 2.81 (s, 3H); C{ H} NMR (100 MHz, CDCl ) 
3
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2
2
2
2
2
2
2
2
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3
3
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3
3
3
3
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3
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159.62, 159.34, 159.29, 158.97, 158.64, 158.59, 158.52, 158.44, 137.67, 137.62, 137.27,
1
7
3
1
37.18, 129.19, 129.18, 129.12, 128.11, 128.08, 128.06, 128.03, 127.88, 127.86, 127.83,
2.46, 72.42, 72.16, 71.78, 71.55, 71.27, 51.33, 51.23, 51.19, 50.84, 47.81, 47.76, 47.25, 30.29,
+
0.26, 29.95, 29.81; HRMS (ESI) m/z: [M + H] Calcd for C H N O 1033.4545; Found
52
57 16
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033.4540.
1
Bn Me BU[4] (5c). White solid; H NMR (400 MHz, CDCl )  7.33–7.27 (m, 16H), 7.15–
4
4
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
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0
7.14 (m, 4H), 5.67 (d, 8.4 Hz, 2H), 5.62 (d, 8.4 Hz, 2H) 5.47 (s, 4H), 4.80 (d, J = 16 Hz, 2H),
4.72 (s, 2H), 4.37 (d, J = 2 Hz, 4H), 4.33 (d, J = 16 Hz, 2H), 4.08 (dd, J = 20 Hz and 16 Hz, 4H),
13
1
3
.72 (s, 2H), 3.04 (s, 6H), 2.82 (s, 6H); C{ H} NMR (100 MHz, CDCl )  159.6, 159, 158.6,
3
1
58.5, 137.6, 137.3, 129.2, 129.1, 128.1, 128, 127.9, 127.9, 72.6, 72.3, 72, 71.3, 51.6, 51, 47.7,
+
47.3, 30.2, 29.9; HRMS (ESI) m/z: [M + H] Calcd for C H N O 1033.4545; Found
52 57 16
8
1033.4540.
1
Bn Me BU[4] (5d). White solid; H NMR (400 MHz, CDCl )  7.30-7.28 (m, 12H), 7.17 (m,
4
4
3
8H), 5.57 (dd, J = 8.4 Hz and 2.4 Hz, 8H), 4.64 (s, 4H), 4.45 (dd, J = 16 Hz and 4 Hz, 8H), 3.76
13
1
(
1
s, 4H), 3.00 (s, 12H); C{ H} NMR (100 MHz, CDCl )  159.4, 158.5, 137.2, 129.1, 128.1,
3
+
27.8, 72.4, 71.6, 51.2, 47.2, 30.3; HRMS (ESI) m/z: [M + H] Calcd for C H N O 1033.4545;
52
57 16
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Found 1033.4578.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publication website at DOI:
01021/acs.joc.XXXXXX. Details of the X-ray structural characterization and NMR studies of
1
both characterization, and CSA titrations:
SI file (PDF)
X-ray data for 5a (CIF)
AUTHOR INFORMATION
Corresponding Author
E-mail: oferre@openu.ac.il
ORCID
Amar Mohite: 0000-0001-6039-7028
Ofer Reany: 0000-0003-1048-1947
ACKNOWLEDGMENTS
Financial support for this work was provided by The Ministry of Science Technology and Space
(MOST grant No. 3-10855). We thank Dr. Natalia Fridman from the crystallographic lab at the
Schulich Faculty of Chemistry, Technion, for her valuable contribution to the X-ray
crystallographic efforts. We thank Prof. Doron Pappo from Department of Chemistry, Ben-
Gurion University of the Negev for providing optically pure (R)-BINOL II-V derivatives for this
study. We also thank Prof. N. Gabriel Lemcoff for his valuable comments on this manuscript.
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