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Article https://doi.org/10.1038/s41557-025-01750-x

Supramolecular coordination cages as

crystalline sponges through a symmetry
mismatch strategy

Received: 29 May 2024 Wei He 1, Yikuan Yu1, Kenta Iizuka , Hiroki Takezawa
1 1
&
Makoto Fujita 2,3
Accepted: 21 January 2025

Published online: xx xx xxxx

The crystalline sponge method enables single-crystal X-ray diffraction
Check for updates analysis of guests absorbed within single-crystalline porous materials.
However, its application with large or highly polar guests remains
challenging. In this study, we addressed some of these limitations using
palladium-based octahedron-shaped M6L4 (Td) coordination cages as
crystalline sponges. The key to facilitate the crystallization of the cage is the
addition of large aromatic polysulfonates (‘sticker’ anions); the symmetry
mismatch between the cage and the sticker (D2h) results in a low-symmetry
̄ preventing guest disorder and leading to the formation of
space group (P 1),
guest-accessible channels in the crystal. Guests can be encapsulated either
before or after cage crystallization. The size and host–guest properties of
the cavity enable analysis of a broad range of compounds, including
water-soluble molecules, large amphiphilic molecules (molecular weight of
~1,200) and molecular aggregates. We have demonstrated the versatility of
the cage–sticker strategy through its application to a triaugmented
triangular-prism-shaped M9L6 cage, extending the guest scope to
medium-sized pharmaceutical molecules.

Among the structural analysis methods currently available, single- We are particularly interested in the remarkable guest-binding
crystal diffraction is useful to determine the three-dimensional struc- properties of octahedron-shaped M6L4 discrete cage 1 in solution29
ture of a given molecule. In 2013, our group introduced an X-ray struc- (Fig. 1a) and have frequently studied the rich host–guest chemistry of
tural analysis method termed the crystalline sponge (CS) method to its host–guest complexes by crystallographic analysis30–39. The results
eliminate the need for analyte crystallization1. In this method, a small prompted us to explore the possibility of transforming this cage unit
crystal of a porous complex (CS) is immersed in a solution containing into a crystalline porous material with CS properties. However,
the target compound, which is then absorbed and aligned within the attempts to directly crystallize cage 1 from aqueous solution resulted
CS pores. The post-oriented (or ‘post-crystallized’) target compound in close-packed crystals devoid of the pores or channels that would
can then be observed alongside the host CS framework through crystal- allow guests to infiltrate the large cavity of 1 (Fig. 1b). Previously, we
lographic analysis2–12. Despite its innovation, the CS method has certain synthesized a networked M6L4 cage complex featuring a Co2+ ion at
limitations related to, for example, the molecular size and polarity of each corner of the octahedral cage unit and shared with adjacent
analytes. Therefore, there is eager anticipation for a CS variant that units40. This networked complex exhibits a large void space in its crys-
can overcome these limitations13–28. talline state and can absorb analytes from solution, functioning as a

1
Department of Applied Chemistry, School of Engineering, The University of Tokyo, Mitsui Link Lab Kashiwanoha 1, FS CREATION, Kashiwa, Japan.
2
Tokyo College, UT Institutes for Advanced Study (UTIAS), The University of Tokyo, Mitsui Link Lab Kashiwanoha 1, FS CREATION, Kashiwa, Japan.
3
Division of Advanced Molecular Science, Institute for Molecular Science (IMS), Okazaki, Japan. e-mail: [email protected];
[email protected]

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a M
12+
N
N
12 NO3–
–
O3S SO3–
N N
N N
N
N
N N
M N N
N M –
O3S SO3–
N N
M N
N N
N
N 2
N

N
N N
M M Pd
N
1

b 1

Crystallization Crystallization

1 alone With sticker 2

• Hard to crystallize • Easy to crystallize

• High-symmetry space group (I41/a) • Low-symmetry space group (P 1)
• No guest-accessible channels • Guest-accessible channels
Potent crystalline sponge

Fig. 1 | Sticker-induced crystallization of a coordination cage into a CS. the help of sticker 2. In contrast to the direct crystallization of cage 1, sticker-
a, Chemical structures of coordination cage 112+ and sticker anion 24−. The induced crystallization of cage 1 produced effective CS sc-1. The large channels
coordination cage 1 used in this study is self-assembled from six metal corners of sc-1 enable efficient guest inclusion, while the low-symmetry space group
(cis-capped Pd(II) complexes) and four planar ligands (2,4,6-tris(4-pyridyl)-1,3,5- prevents the disorder of guests in X-ray crystallographic analysis.
triazine). b, A schematic outline of the crystallization of cage 1 without and with

CS, as demonstrated in our original report1,40. Unfortunately, the high well-suited to the absorption of guest molecules (Fig. 1b). These prop-
symmetry of the host environment ( Fm3m ̄ cubic space group) makes erties allow the accommodation of hydrophobic or hydrophilic por-
modelling guests with multiple orientations very difficult owing to the tions of guests, eliminating the need for complete inclusion within the
dominance of the host’s symmetry in the system (see Supplementary cavity of the M6L4 cage unit. Consequently, a broad range of guests,
Methods for more details), unless four guest molecules per cavity align including large amphiphilic molecules (molecular weight (MW) of
along the tetrahedral symmetry axes of the cage unit41–43. This is likely ~1,200), highly polar molecules, synthetic intermediates and
because the rigid and large symmetrical cavity hampers anisotropic non-covalent molecular aggregates, can now be analysed. We have
distortion of the crystalline lattice through host–guest or guest– demonstrated the versatility of the cage–sticker strategy through its
guest interactions. application to a triaugmented triangular-prism-shaped M9L6 expanded
In this study, we successfully crystallized cage 1 in a low-symmetry cage (D3h symmetry)44, extending the scope of the CS method to
̄ facilitated by large aromatic polysulfonates (‘sticker’
space group (P 1), medium-sized pharmaceutical molecules. This development repre-
anions). Symmetry mismatch between the cage (Td) and the sticker sents a substantial advance in the practical utility of the CS method.
(D2h) is key to the low-symmetry crystallization and formation of
guest-accessible void channels in the crystal, resulting in a potent CS Results and discussion
(single-crystalline 1, hereafter referred to as sc-1, Fig. 1b) that over- Sticker-induced crystallization of sc-1 and its use as a CS
comes several limitations of the conventional method. Unlike crystals Large aromatic anions are often used to facilitate the crystallization
formed from cage 1 alone, sc-1 features a large void space with channels of non-crystalline or hard-to-crystallize cationic compounds45–48.

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a Analyte
(liquid or solution)

CS Guest-absorbed CS Diffraction study

Fig. 2 | Crystallized cage sc-1 as a CS for X-ray crystallographic observation sc-1 (left) and guest-absorbed sc-1 (right); the red-boxed area is enlarged in c.
of guest molecules. a, Schematic outline of guest inclusion in a CS and the c, Enlarged view of guest-absorbed sc-1. Two molecules of guest 3a are closely
subsequent diffraction study. b, X-ray crystal structures of as-synthesized CS packed in each cage cavity, as shown by the space-filling models.

When tetrasodium pyrene-1,3,6,8-tetrasulfonate (sticker 24−) was added When a trace amount of guest (~20 μg) was brought into contact
to an aqueous solution of cage 1 (or 112+·(NO3−)12), we observed the fac- with a crystal of sc-1, rapid inclusion of the guest occurred (Fig. 2a).
ile and rapid growth of single crystals of sc-1 (see Methods for details). After 12 h, the crystal was subjected to X-ray diffraction analysis. As
X-ray crystallographic analysis of sc-1 revealed the anion-exchanged expected, the accommodation of 3a in the M6L4 cage cavity of sc-1 was
cage formulated as [(112+)·(24−)1.5·(NO3−)6]. Anion 24− interacts with the observed (Fig. 2b). Two guest molecules were detected in the cage
ligands on the octahedral faces of 112+, forming infinite donor–acceptor cavity with ~100% guest occupancy (Fig. 2c). Hydrophobic interac-
stacking (Extended Data Fig. 1). The sc-1 crystal is expected to be a tions, a major driving force for guest binding with discrete cage 1 in
potent CS as it crystallizes in a low-symmetry space group (P 1), ̄ leaving aqueous solution, also seems to operate in the cage cavity of sc-1. The
a large void space (channels) that is accessible to guests. In the absence inclusion reduces the space group symmetry of sc-1 to P1, preventing
of sticker anion 2, the crystallization of cage 1 produces close-packed disorder of the guests because of the absence of symmetry elements
crystals of high symmetry (I41/a) with no guest-accessible channels in the unit cell. The absolute stereochemistry of 3a was confirmed
(Extended Data Fig. 2), making it unsuitable for use as a CS. Symmetry by the low Flack parameter value of 0.251(12), which was attributed
mismatch in the salt formation between the cation (112+, Td) and to induced anomalous scattering from the heavy atoms (Pd and S) in
anion (24−, D2h) seems to be a key factor for the crystallization in the the achiral host and sticker. The chiral environment around the heavy
low-symmetry space group. atoms is a result of efficient guest-to-host chirality transfer49. Thus,
The ability of sc-1 to act as a CS was examined with a steroidal the CS method with sc-1 enables the absolute configuration of chiral
guest, methylandrostenediol (3a), on a one-crystal scale (Fig. 2). guests to be determined.

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a OH
OH HO O
HO O
S
H 3a 3b 3c
O N
HO H
H H
HO HO NH

b c

e +

Sticker 2

Sticker-induced
crystallization

1 sc-1
Method a
Guest inclusion Guest absorption
in solution in crystalline state
G G
Method b

Sticker 2

Sticker-induced
crystallization

1·G (sc-1)·G

Fig. 3 | Crystal structures of guest molecules determined by sc-1. a, Chemical and a nitrate anion involved in the network are shown. e, Schematic outlining
structures and stick models of guest molecules 3a–3c. b,c, Crystal structures of the preparation of guest-absorbed crystals (sc-1)·G by two methods. Method
3a (b) and 3c (c) (with the cage framework) determined by guest absorption in the a: guest inclusion in crystalline sc-1, as illustrated in Fig. 2a. Method b: sticker-
crystallized cage of sc-1 (left, method a) and by sticker-induced crystallization of induced crystallization of host–guest complex 1·G. Non-hydrogen cage atoms
guest-included cage 1 (right, method b). d, Crystal structure of 3b determined by are represented as green wires (with green spheres for Pd(II) corners), while guest
method a (left) and image highlighting the hydrogen-bonding network around molecules are shown as space-filling models.
the hydrophilic part of the guest (right). Solvent water molecules, a sticker anion

In a similar manner, CS crystallography with sc-1 success- aqueous solution (Supplementary Fig. 22). For the more hydrophilic
fully revealed the structures of amphiphilic molecule 3b and guest 3c, crystallographic analysis showed that its hydrophilic por-
water-soluble molecule 3c (Fig. 3a). It is worth mentioning that tion is fixed to the cage portal through hydrogen bonding, while its
crystalline host sc-1 exhibits ‘amphiphilic’ molecular recognition in hydrophobic and electron-rich indole plane is stacked on the planar
the inclusion of 3b, with the hydrophobic and hydrophilic portions ligand outside the cage structure (Fig. 3c, left). Therefore a great
of the guest recognized by the cavity and portal of the cage, respec- advantage of sc-1 is that the portals and guest-accessible channels
tively (Fig. 3d). This amphiphilic recognition was also suggested in sc-1 also provide binding opportunities for both hydrophobic
by 1H NMR analysis of the complexation of discrete cage 1 and 3b in and hydrophilic guests.

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Guest inclusion before crystallization as an alternative The crystallographic analysis of (sc-1)·4d revealed a P1 space
protocol group, with two sets of host–guest complexes observed in each unit
As sc-1 is prepared from molecular cage 1, the guest can be accommo- cell (Supplementary Fig. 46). It also confirmed our expectation that
dated in the cage either before or after the sticker-induced crystalliza- only the hydrophobic steroidal head of 4d enters the cage cavity, while
tion of 1 (Fig. 3e). In addition to the standard protocol discussed above the hydrophilic polysaccharide tail protrudes from the cage, remaining
(method a), we developed an alternative protocol (method b) in which in the aqueous environment. Despite the large unit cell, we successfully
host–guest complexation in solution is followed by sticker-induced assigned all of the non-hydrogen atoms without any restraints on 1,2-
crystallization. To examine the scope of method b, the same guests and 1,3-distances, except for some parts of the relatively disordered
3a–3c (~1 mg of each) were analysed using this method. For guest 3a, terminal sugar unit. One of the guest molecules shows near-perfect
almost the same host–guest structure (sc-1)·3a2 was observed (Fig. 3b, coordinates with atomic precision, as supported by an unambiguous
right). Guest 3c was observed inside the cage (while outside the cage electron density map (Supplementary Fig. 44). The high accuracy is
in method a), most likely due to host–guest complexation within the probably due to the fixation of both the hydrophobic and hydrophilic
cage before crystallization (Fig. 3c, right). Hydrophilic guest 3b seems portions of guest 4d. Thus, the absolute configuration of guest 4d was
to easily escape from the cage during crystallization, leading to low unambiguously determined, as confirmed by the low Flack parameter
occupancy of the guest and an ambiguous electron density map for of 0.094(6).
the analyte (Supplementary Fig. 19). These observations suggest that Digitonin (4e, MW = 1,229.32), with molecular formula C56H92O29,
method b reproduces the host–guest chemistry in the solution state, is one of the largest members of the saponin family. The molecular
while method a facilitates guest recognition using both the inside and size, the presence of a spiro atom in the steroid moiety and the high
outside (or surface) of the cage in the crystalline state. flexibility of the polysaccharide moiety presents great challenges in the
Either method a or b can be used for most compounds. When structure determination of 4e. 1H NMR analysis of the complex formed
only a minute amount of sample (submicro- to microgram quantity) between 4e and cage 1 indicated that only the steroidal moiety of 4e is
is available, method a is recommended because guest-absorbed encapsulated in cage 1 (Supplementary Fig. 57).
sc-1 can be prepared with only one tiny crystal of sc-1, similar to the The crystallization of host–guest complex 1·4e with sticker 2
conventional CS method. Method b is applicable when a sufficient resulted in high-quality crystals (see Supplementary Information,
amount of sample (micro- to milligram quantity) is available as the section 6.6 for details)50. Despite the large hydrophilic portion with
guest inclusion in solution in method b sometimes requires an excess five sugar residues, we successfully assigned the whole skeleton of 4e
amount of the guest. From a practical point of view, method b is through crystallographic analysis, except for one of the terminal sugar
more recommended as the inclusion of the analyte into the cage residues located in the middle of a large channel in sc-1. Owing to the
under solution conditions proceeds readily without the need for lack of effective interactions with the host framework, the configura-
the trial-and-error optimization of guest-soaking conditions often tions of the stereogenic carbon atoms in this residue remain somewhat
required in method a. For practical reasons, method b was applied ambiguous. However, efficient amphiphilic guest recognition was again
in the following experiments. observed, with the hydrophobic aglycone of 4e fully encapsulated in
the cage cavity and the five sugar residues protruding out (Fig. 4e,f
Analysis of large amphiphilic molecules and Extended Data Fig. 4). The absolute structure was confirmed by
We expected that the CS method with sc-1 would overcome the limi- the low Flack parameter of 0.072(9). It is worth mentioning that the
tations of the conventional method in terms of substrate scope. The stereochemistry of the spiro carbon in the steroid moiety of 4e is sup-
size limitation of analyte molecules is the most notable issue in the ported by an unambiguous electron density map, in which the carbon
conventional CS method that needs to be addressed. Therefore, and oxygen atoms attached to the spiro carbon atom are clearly dis-
we examined several large steroidal molecules of pharmaceutical tinguished (Supplementary Fig. 52).
interest, 4a–4e, with MWs ranging from 400 to 1,200. Surprisingly, The successful structural determination of these compounds indi-
we succeeded in analysing all of these analytes using the CS method cates that the scope of guests can be considerably extended with sc-1
with sc-1. to potentially include large amphiphilic molecules such as amphiphilic
The 3D structures of mifepristone (4a, MW = 429.60), glyco- polypeptides, glycolipids and polynucleotides.
cholic acid (4b, MW = 465.63) and beclomethasone dipropionate
(4c, MW = 521.04), including their absolute stereochemistries, were CS crystallography of highly polar pharmaceuticals
confirmed crystallographically (Fig. 4a–c). While the relatively com- We also explored the scope of highly polar, even water-soluble mol-
pact 4a is almost fully accommodated in the cage cavity, 4b and 4c ecules of pharmaceutical interest, such as monensin (5a, MW = 692.86,
show amphiphilic binding, as observed for 3b, with the hydrophilic as the sodium salt), a representative polyether antibiotic, as guests
tails of 4b and 4c protruding from the cage and fixed at the cage portal with sc-1. CS crystallography successfully showed the structure of 5a
through hydrogen bonding. trapped in the crystalline host sc-1 using 200 μg of the guest (Fig. 4g).
The packing structure of amphiphilic molecule 4b shows that The flexible skeleton of 5a adopts a relatively extended conformation
the guest molecule occupies only a small portion of the void space in in the cavity of sc-1, which is notably different from the typical cyclic
the CS (Extended Data Fig. 3), which suggests that even larger amphi- conformation of 5a (ref. 51), presumably due to the geometric con-
philic molecules could be accommodated in the cavity. Consequently, straints of the cage cavity (Extended Data Fig. 5). The Flack parameter
we also examined two further steroidal polysaccharides, digoxin value of 0.193(17) and an unambiguous electron density map support
(4d, MW = 780.95) and digitonin (4e, MW= 1,229.32). the absolute stereochemistry of 5a, including the spiro chiral centre
1
H NMR analysis of the complex formed between 4d and cage 1 in in the skeleton.
D2O indicated that only the steroidal moiety of 4d is encapsulated in The structure of levosulpiride (5b, MW = 341.43), a sulfonamide,
cage 1 (Supplementary Fig. 49). Specifically, signals from the steroidal was also determined using 80 μg of the guest (Fig. 4h). In the crystal
portion were markedly shifted upfield, while those from the sugar structure, almost the entire skeleton of 5b is captured in the hydropho-
moiety remained unchanged. The addition of sticker 2 to an aqueous bic cavity of the cage through efficient π–π interactions between the
solution of 1·4d led to the formation of single crystals. X-ray analysis electron-rich benzene moiety of 5b and an electron-deficient ligand
of these crystals unambiguously revealed the 3D structure of 4d as in an octahedral face of the cage. The sulfonamide moiety is located
well as the amphiphilic recognition (that is, partial encapsulation) of at the portal of the cage, exposed to the hydrophilic environment
the guest (Fig. 4d). (Supplementary Fig. 65).

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a N
b OH
O c O
O O O O
OH N OH
H HO
H
H O H
H H
H F H
HO OH
O 4a 4b O 4c

d O O
e O
H

OH HO H
HO
OH OH OH OH H O
H
HO HO O HO O O HO H
H OH O H H OH
O O O O
O O O O HO O H
H
HO HO OH
O
4d 4e
O
HO OH OH
O

HO
OH OH

g OH HO
HO
h O
NH2 i
O S
O O
O O
O H H
H O O O
NH
O O H
N O H
O O
HO
H
H 5a 5b 6

Fig. 4 | Crystal structures of large amphiphilic compounds of pharmaceutical model of 4e. Non-hydrogen cage atoms are represented as green wires
interest with MWs up to 1,200 determined using the CS method. a–e, Chemical (with green spheres for Pd(II) corners), while the guest molecule is shown as
structures (top) and crystal structures (bottom) of steroidal molecules of a space-filling model. g–i, Chemical structures (upper) and crystal structures
pharmaceutical interest: mifepristone (4a, MW = 429.60) (a), glycocholic acid (lower) of highly polar pharmaceuticals and a rare synthetic intermediate:
(4b, MW = 465.63) (b), beclomethasone dipropionate (4c, MW = 521.04) (c), monensin (5a, MW = 670.87) (g), levosulpiride (5b, MW = 341.43) (h) and
digoxin (4d, MW = 780.95) (d) and digitonin (4e, MW = 1,229.32) (e). Non- synthetic intermediate 6 (MW = 404.59) (i). The crystal structures of the analytes
hydrogen cage atoms are represented as green wires (with green spheres for are shown as stick models. For synthetic intermediate 6, 20 μg of the analyte was
Pd(II) corners), while guest molecules are shown as stick models. f, Space-filling used in the CS method.

Application in total synthesis has been established by NMR analysis, but crystallographic study could
In natural product total synthesis, the stereochemical confirmation of offer more convincing assignments, particularly with respect to the
synthetic intermediates at key points of the total synthesis is extremely chiralities of two of the quaternary carbon atoms and the spiro carbon
important to prevent the synthesis of the wrong compounds. Com- atom as there are no reliable empirical or spectroscopic methods to
pound 6 is a synthetic intermediate in the total synthesis of natural determine the configurations of these chiral centres. We received a
products developed by Porco and co-workers52,53. Its stereochemistry small amount of compound 6 from the Porco group and, using only

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a b c d
O O
N N
8O 8O
HO 10 10

7 9 9 11

e f
O O
N N
O O O
N
O
O O
N N
O O
8 12

Fig. 5 | Visualization of host–guest guest chemistry using the CS method. aggregate of dienophile 8 and pyrene (9). This aggregate, originally proposed in
Chemical structures and crystal structures of molecular aggregates in sc-1. 2007, undergoes a [2 + 2] addition upon photoirradiation59. c,d, (sc-1)·(9·10) (c)
a, (sc-1)·(7·8) incorporates a reactive aggregate of anthracene 7 and dienophile and (sc-1)·(10·11) (d) incorporate aggregates of all-trans-cyclododecatriene (10)
8 within the cage. This aggregate, originally proposed in 2006, is notable for with aromatic compounds 9 or 11, respectively, within the cage. e,f, (sc-1)·(8)2 (e)
undergoing an unusual Diels–Alder reaction upon heating58. The proximity of and (sc-1)·(12)3 (f) incorporate aggregates formed by two molecules of 8 or three
dienophile 8 to the terminal ring of anthracene 7, as observed, is the most likely molecules of 12, respectively, within the cage. Guest molecules are shown as stick
reason for the unusual Diels–Alder reaction occurring at the 1,4-positions of 7 models with superimposed space-filling models. The host cage is represented as
rather than at the most reactive 9,10-positions. b, (sc-1)·(8·9) includes a reactive a wire-frame model in a and omitted in b–f for clarity.

20 μg of 6, successfully analysed its molecular structure by reducing the structure of (sc-1)·(7·8) revealed the close proximity of the ethylene
amount of solution in a small sample tube (Fig. 4i and Supplementary moiety of 8 to the terminal anthracene ring of 7 at a distance of
Information, section 8.2 for details). 3.2 Å (Fig. 5a). Thus, this X-ray visualization converted a specula-
After preparing 6-absorbed sc-1, the crystal structure was solved tive explanation for the origin of the unusual site selectivity into
in space group Pc and all of the non-hydrogen atoms of 6 were assigned a reliable conclusion.
on the basis of an unambiguous electron density map. The analysis Upon photoirradiation, dienophile 8 also undergoes [2 + 2] pho-
revealed that the polycyclic skeleton of 6 is perfectly encapsulated toaddition with other stable aromatic compounds in cage 1 (ref. 59).
within the hydrophobic cavity of the sc-1 cage, while the acetal moi- The preorganization of 8 with pyrene (9) was also visualized by the CS
ety protrudes through the cage portal to contact the sticker anion 2 method (Fig. 5b). The crystal structure of (sc-1)·(8·9) shows that the
(Extended Data Fig. 6). The complete stereochemistry of the analyte closest distance between the reaction sites of 8 and 9 in the cage is
was determined to be (4S,5R,9R,10R,13R,14S,23R)-6, fully consistent 5.5 Å. Although the two substrates are placed in much closer proxim-
with the NMR relative structure. We carefully examined the configu- ity than in solution reactions, this distance still exceeds the Schmidt
ration at the spiro atom, where the oxygen atom attached to the spiro rule requirement of 3.5 Å in solid-phase reactions60. Cage confinement
carbon is clearly distinguished from the carbon atoms by its higher restricts the guest orientation, but presumably allows tumbling motion
electron density (Supplementary Fig. 69). This demonstrates that our to violate the Schmidt rule.
method is of great help in total synthesis studies as the construction All-trans-cyclododecatriene (10), a triterpene analogue and an
of the proper frameworks and stereochemistries can be confirmed at olefin ligand for transition metals, attracts attention owing to the
key steps in the total synthesis. unique structures and reactivities of its complexes, ascribed to the
propeller-like arrangement of its double bonds after metal coordina-
Visualization of the host–guest chemistry tion61. The pairwise inclusion of 10 with aromatic compounds 9 or 11
M6L4 cage 1 has a cavity that is large enough to accommodate two was also visualized by the CS method using sc-1 (Fig. 5c,d). In both
or more small molecules. Owing to their close contact, aggregated cases, triene 10 adopts a propeller-like conformation to form pairs of
guest molecules in the cavity often exhibit unique properties and racemates in which the propeller can have a left-hand or right-hand
reactivities54–57. We previously reported an unusual Diels–Alder screw. This indicates that the confinement effect of the cage cavity is
reaction between anthracene 7 and dienophile 8, where the reac- comparable to metal coordination for the conformational fixation of
tion took place at the 1,4-positions of the anthracene, rather than flexible molecules.
at the commonly most reactive 9,10-positions58. This unusual site Binding multiple guests in a cavity has been recognized as an
selectivity can be explained by the preorganization of the two important mode of host–guest complexation as unique properties of
substrates, where the reaction sites are fixed in close proximity. molecules can be created through the precise confinement of multiple
Although molecular mechanics calculations supported the preor- molecules62. Interestingly, guests 8 and 12, with similar structures,
ganization of the reaction sites, there was no experimental evidence resulted in (sc-1)·(8)2 (Fig. 5e) and (sc-1)·(12)3 (Fig. 5f), respectively,
to support this preorganization. Therefore, the sticker-induced with different host/guest ratios. This indicates that, in multiple guest
crystallization of cage 1 was applied to visualize the preorganiza- binding, the host/guest ratio and guest orientation are sensitive to
tion of the two substrates in the cavity. As expected, the crystal subtle changes in guest structure. Consequently, the complexation

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a b

HO

O O
OH O
F B F
O O O
NH

O
O
O OH
O 15

Fig. 6 | An M9L6 cage as a CS. a, Crystal structure of the inclusion complex Hydrogen atoms of the cage and of the guest (shown with ellipsoids), as well
(sc-13)·(15)2 formed from rifampicin derivative 15 (MW = 772.58) and M9L6. as solvent and anion molecules, have been omitted for clarity. b, Chemical
One of the molecules of 15 is shown with 50% anisotropic displacement structure of rifampicin derivative 15. c, Electron density map (Fo, observed
parameters and the other as a space-filling model. Non-hydrogen cage structure factor amplitude, blue isomesh, 1.8 e Å−3, σ = 1.5) superimposed on the
atoms are represented as green wires, with green spheres for Pt(II) corners. refined structure of 15.

modes should be carefully analysed for all guests, even when similar absolute structure. Despite the unanticipated reaction during crys-
guests have already been analysed. tallization, the high-quality data allowed for precise identification
It is noteworthy that the successful crystallographic analysis of the structure of 15. The incorporation of ‘BF2’ from BF4− into the
of multiple host–guest (G) binding structures, such as (sc-1)·(G)2, 1,8-dihydroxynaphthalene unit and hydrolysis of the hydrazone moiety
(sc-1)·(G)3 and (sc-1)·(G1)·(G2), is due to the crystallization of sc-1 in a are visualized in the crystal structure and further validated by electro-
low-symmetry space group. This is in stark contrast to direct crystal- spray ionization mass spectrometry and 19F NMR spectroscopy (Sup-
lization from solutions of 1·(G)n complexes (without 2), where high plementary Figs. 96–98). Notably, the combined molecular weight of
symmetry (typically I41/a) often causes the disorder of accommodated the two accommodated guest molecules exceeds 1,500, highlighting
guests (Supplementary Figs. 93 and 94). the potential of this CS method for applications involving pharmaceuti-
cal macrocyclic molecules, for which reliable methods for determining
An M9L6 cage as a CS their stereochemistry are currently lacking.
We also demonstrated the versatility of the sticker approach with our
recently synthesized M9L6 cage 13 with D3h symmetry, the cavity of which Conclusion
is expanded fourfold compared with the M6L4 cage, allowing for molecu- In summary, we have established a general approach to obtain atomi-
lar recognition of medium-sized molecules with a MW exceeding 1,000 cally precise structural information on analytes included in a coordi-
(ref. 44). As with the M6L4 cage, salt formation of 13 with sticker 2 (D2h) nation cage through sticker-induced crystallization via single-crystal
resulted in the crystallization of the salt (hereafter sc-13) with a lower X-ray diffraction. The crystal structure provides an unsymmetrical
symmetry space group (P212121). Therefore, we examined the use of environment to secure the position of the analyte, meeting the require-
sc-13 as a CS to determine the structure of a medium-sized molecule. ments of the CS method. This second-generation CS method expands
Using method b, rifampicin (14), an ansamycin antibiotic with a MW of the range of suitable analytes to medium-sized molecules with highly
~800, was added to an aqueous solution of cage 13 and the host–guest polar moieties or even flexible sugar chains, as well as preorganized
complex formed was crystallized with sticker 2 and NaBF4 as additive molecular aggregates. The sticker method also allows capture of snap-
(see Supplementary Information, section 10.2 for details). X-ray crystal- shots of the host–guest structure with various guests, unveiling the
lographic analysis of a dark-orange crystal revealed the 3D structure of origins of unusual chemical phenomena.
the guest molecule, rifampicin (14), as its difluoroborate derivative 15 Understanding the structure of molecules is a most crucial and
(MW = 772.58; Fig. 6). This unexpected borate 15 seems to be formed by indispensable fundamental process in scientific research across vari-
the action of solvent water and BF4− during crystallization. The asym- ous disciplines, ranging from life sciences to materials science. As
metric unit of the crystal contains a complete framework of the M9L6 history has proven, the emergence of new techniques for molecular
cage with no symmetry elements passing through it. The symmetry structure analysis has always led to breakthroughs in natural science
mismatch in the salt formation was essential for low-symmetry crystal research. We believe that the CS method, which has paved the way to
packing as more symmetric packing (P63/mmc) was observed in the access atomic-resolution structures of non-crystalline substances and
crystallization of 13 alone (see Supplementary Information, section trace amounts of materials, represents a substantial breakthrough
10.4 for details). and innovation in molecular science. Traditionally, new methods
Two crystallographically independent molecules of 15 are of molecular structure analysis have been brought about through
observed in the cavity, and the configurations of all nine asymmet- instrument development. The importance of the CS method also
ric carbon atoms have been determined unambiguously. The Flack lies in the fact that a molecular analysis method, rivalling or even
parameter of 0.028(3) confirms the reliable determination of the surpassing traditional approaches, has emerged from foundational,

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curiosity-driven chemical research such as host–guest chemistry and 17. Zhang, S., Fairen‐Jimenez, D. & Zaworotko, M. J. Structural
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Methods 2357187 ((sc-1)·3a2 with method a), 2357188 ((sc-1)·3a2 with method b),
X-ray structural analysis 2357189 ((sc-1)·3b with method b), 2357190 ((sc-1)·3c with method b),
Single-crystal X-ray diffraction data were collected on Synergy-S and 2357191 ((sc-1)·4a), 2357192 ((sc-1)·4b), 2357193 ((sc-1)·4c), 2357196
Synergy Custom (Rigaku Oxford Diffraction) diffractometers. The ((sc-1)·4e), 2357197 ((sc-1)·5a), 2357198 ((sc-1)·5b), 2357199 ((sc-1)·6),
diffraction data were reduced and corrected using the CrysAlisPro pro- 2357200 ((sc-1)·(8·9)), 2357201 ((sc-1)·(9·10)), 2357202 ((sc-1)·(10·11))
gram. The crystal structures were solved by direct methods and refined and 2386991 ((sc-13)·(15)2). Copies of the data can be obtained free of
using the SHELXL-2018/3 program suite63,64. Hydrogen atoms were charge via https://www.ccdc.cam.ac.uk/structures/.
placed at the calculated positions and refined using a riding model.
The occupancies of the guest molecules were refined on the basis of References
the electron densities using the free variables of the SHELXL program. 63. Sheldrick, G. M. Crystal structure refinement with SHELXL.
Acta Crystallogr. C 71, 3–8 (2015).
Crystallization of sc-1 64. Hübschle, C. B., Sheldrick, G. M. & Dittrich, B. ShelXle: a Qt
To an aqueous solution of 1 (0.1 mmol, 20 mM, 5 ml), an aqueous solu- graphical user interface for SHELXL. J. Appl. Crystallogr. 44,
tion of 2 (0.2 equiv. with respect to cage 1, 20 μmol, 20.0 mM, 1 ml) was 1281–1284 (2011).
added and the mixture was allowed to stand at 20 °C for 12 h to obtain
single-crystalline sc-1 as yellow block-shaped crystals. Acknowledgements
This research was supported by AMED AIMGAIN (JP24zf0227103, to
General procedure for the preparation of guest-absorbed sc-1 M.F.), Grants-in-Aid for Scientific Research(S) (JP24H00054, to M.F.),
(method a) Early-Career Scientists (JP21K14641, to H.T.) and Scientific Research(B)
A piece of as-synthesized sc-1 crystal was placed in a microvial with (JP23H01783, to H.T.). This work was also supported by JST SPRING
20 μl of the mother liquor. Then the guest (20–50 μg) was added to the (JPMJSP2108, to W.H. and K.I.). We thank J. A. Porco Jr and F. Yang for
supernatant and the microvial was capped and stored at 20 °C for 12 h providing us with valuable analytical samples.
to give guest-absorbed sc-1 crystal, which was picked and mounted
in a laboratory X-ray diffractometer and crystallographic data were Author contributions
collected. Details of the individual guests are described in the Sup- W.H., Y.Y. and K.I. performed the described experiments. W.H., Y.Y.,
plementary Information. K.I. and H.T. analysed the data. W.H., H.T. and M.F. co-wrote the paper.
H.T. and M.F. supervised the research project.
General procedure for CS crystallography with sc-1 (method b)
The host–guest complex 1·G was prepared by suspending guest pow- Competing interests
der (~1 mg) in an aqueous solution of cage 1 (10 μmol, 20 mM, 500 μl) The authors declare no competing interests.
and stirring the mixture at 60 °C for 15 min. The resulting mixture was
passed through a membrane filter unit to remove insoluble solids com- Additional information
pletely and transferred into a microtube. Then an aqueous solution of Extended data is available for this paper at
sticker 2 (0.2 equiv. with respect to cage 1) was added and the resulting https://doi.org/10.1038/s41557-025-01750-x.
mixture was stored at 20 °C for 12 h to afford crystals of guest-absorbed
sc-1. A piece of crystal was mounted in a laboratory X-ray diffractometer Supplementary information The online version
and diffraction data were collected. Details of individual guests are contains supplementary material available at
described in the Supplementary Information. https://doi.org/10.1038/s41557-025-01750-x.

Data availability Correspondence and requests for materials should be addressed to

All data supporting the findings of this study are available within this Hiroki Takezawa or Makoto Fujita.
article and its Supplementary Information. Crystallographic data
for the structures reported in this paper have been deposited at the Peer review information Nature Chemistry thanks the anonymous
Cambridge Crystallographic Data Centre (CCDC) under deposi- reviewer(s) for their contribution to the peer review of this work.
tion numbers 2278392 ((sc-1)·(7·8)), 2278393 ((sc-1)·(12)3), 2278394
((sc-1)·(8)2), 2278395 ((sc-1)·4d), 2278396 (sc-1), 2278397 ((sc-1)·3b with Reprints and permissions information is available at
method a), 2278398 ((sc-1)·3c with method a), 2357186 (non-porous-1), www.nature.com/reprints.

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Extended Data Fig. 1 | Crystal structure of the sc-1, the cage-sticker crystal. a, An enlarged view of one cage, to which three stickers are attached. b, The packing
structure of cages and stickers among 8 unit cells. In a and b, cages and stickers are represented in the stick model. All hydrogen atoms, solvent molecules, and nitrate
anions are omitted for clarity. Color code: cage, grey; sticker, yellow.

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Extended Data Fig. 2 | Crystal structure of nonporous-1, the cage crystal guest-accessible channel. Cages are represented in the stick model. All hydrogen
without the sticker anion. The packing structure of cages among 8 unit cells atoms, solvent molecules, and nitrate anions are omitted for clarity. Cages along
is represented. In this case, cages are densely packed without leaving any the a axis of the crystal are represented in the same color.

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Extended Data Fig. 3 | Crystal structure of host-guest complex 1·4b. are represented in the space-fill model. All hydrogen atoms (except those on the
The packing structure of cages, stickers, and guest molecules among 8 unit cells guest), solvent molecules, and anions (except the sticker) are omitted for clarity.
is represented. Cages and stickers are represented in the stick model, and guests Color code: cage, grey; sticker, yellow; guest, C: orange, O: red, N: blue, H: white.

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Extended Data Fig. 4 | Crystal structure of host-guest complex 1·4e. are represented in the space-fill model. All hydrogen atoms (except those on the
The packing structure of cages, stickers, and guest molecules among 8 unit cells guest), solvent molecules, and anions (except the sticker) are omitted for clarity.
is represented. Cages and stickers are represented in the stick model, and guests Color code: cage, grey; sticker, yellow; guest, C: orange, O: red, H: white.

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Extended Data Fig. 5 | Crystal structure of host-guest complex 1·5a. An enlarged view of one cage is represented. The cage and the guest are represented in the stick
model (palladium atoms of the cage are represented in the space-fill model). Hydrogen atoms of the cage, solvent molecules, and anions are omitted for clarity.
Color code: cage, grey; guest, C: orange, O: red, H: white.

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Extended Data Fig. 6 | Crystal structure of host-guest complex 1·6. An enlarged view of one cage is represented. The cage and the guest are represented in the stick
model (palladium atoms of the cage are represented in the space-fill model). Hydrogen atoms of the cage, solvent molecules, and anions are omitted for clarity.
Color code: cage, grey; guest, C: orange, O: red, H: white.

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