which display interesting recognition properties such as
size dependent homotropic allostery, chiral recognition,and control over guest folding.11 In this paper we continue
200228 ⼭梨 ⽂献セミナー Q&A
this line of inquiry by comparing and contrasting theproperties of acyclic glycoluril oligomers 4 !6に⽐べて⾼いのはなぜか？
Q. cucurbit[7]uril の⽔溶性が他の cucurbit[n]uril recognition(Figure 1) with those of their macrocyclic counterparts
A. ⽔和時のエネルギー変化と固体の分⼦間相互作⽤の 2 点による説明が可能です。
CB[6] and CB[7]. We delineate some key factors governing
現在単離されている CB[n](n = 5, 6, 7, 8, 10)を⽐べると、CB[5]とCB[7]が数⼗〜数mM と⾼い⽔溶性を
the recognition propertiesof CB[n]-type
receptors (e.g.,preorganizationand macrocyclic effect).
⽰すのに対し、残りの CB[6]、CB[8]、CB[10]は 0.05
mM 以下と低い⽔溶性を⽰すことが分かっています。

p
e
su
b
w
a
w
a

分⼦動⼒学法によって解析したところ、CB[n]の⽔和時のギブス⾃由エネルギー変化は、n が⼤きくなるにつれて⼤きくなり、n が⼤きいほど溶解が熱⼒学的に不利になることが分かりました。これは、n が⼤S

きいほど⽔分⼦のネットワークを多く破壊し、溶解がエントロピー的に不利になるからだと考察されています。また、分⼦内の CH…O の相互作⽤の違いに起因して、固体の CB[n]はアモルファス(n = 5, 7)か結晶(n = 6, 8)のどちらかを取ることが知られていて、結晶はアモルファスよりも強い分⼦間相互作⽤を形成します。したがって、CB[5]と CB[7]の⽔溶性が⾼く、その中でも n が⼩さい CB[7]の⽔溶性が最も⾼くなると解釈することができます。
(Malaspina, T., et al., J. Phys. Chem. B 2016, 120, 7511.)
Q. cucurbit[6]uril と 6C とで diammonium ion 11 とのアフィニティに⼤きな差が出るのはなぜか？
A. diammonium ion 11 は CB[6]の cavity に対しては⼤きすぎることが原因です。
6C(下図 host 6)は CB[6]に⽐べて、より⼤きいゲ
スト分⼦に対するアフィニティが⼤きいことが分かっています。これは、⾮環状の 6C が環状の CB[6]よりも⼤きな cavity を持つことに起因すると考えられています。

Table 1. Binding Constants (Ka, M!1) Measured for Host•Guest Complexes between Hosts 3!6 , CB[6], and CB[7] and Guests 7 !15

Figurehost
1. Hosts
and guests CB[6]used in this study.
6

host 4ahost 57
8
9!
!
(5.6 ( 0.4) " 103(4.7 ( 0.5) " 10(1.4 ( 0.1) " 105(1.0 ( 0.2) " 106

10
11
12
13
14
15

!
(1.5 ( 0.1) " 104!
!
!
!

(1.0 ( 0.1) " 104(1.2 ( 0.1) " 106(2.7 ( 0.4) " 104nbe(1.1 ( 0.2) " 106(6.1 ( 0.9) " 105

guest

a

4

CB[7]

(5.0 ( 0.3) " 10(2.0 ( 0.2) " 10
!
6
c
(1.6
0.3) " 10(1.5reported( 0.1) " 108the!
We( have
previously
synthesisof methylene(2.2 ( 0.4) " 106(4.5 ( 0.8) " 108 b(9.0 ( 1.4) " 107 b
bridged glycoluril pentamer
5 and hexamer 6 and their(2.9 ( 0.2) " 108 c
purification
ion-exchange(4.9 ( 0.6) " 104by time-consuming(1.9 ( 0.1) " 103 b DOWEX(2.1 ( 0.3)
" 106 b7b10d(2.2 ( 0.4) " 10550
30(1.8 ( 0.3)
109 bchromatography.
In(orderto streamlinethe" synthesis(6.8 ( 1.4) " 105
1.4 " 106 d(2.3 ( 0.4) " 107 b
andof 5 nb
we considered the use
bis-ns-CB[10](2.6purification( 0.3) " 104(8.9of( 1.4)" 108 b
7
b
(1.8
(
0.4)
"
10
!
(4.2
(
1.0)
"
1012
as a readily available
starting material (Figure 1).11a
Bis-ns64b(6.0 ( 1.3) " 10!
(2.5 ( 0.4) " 10
4

7c

h
w
fo
so
2
c
(S
v
a
sp
u
in
ex
C
sl
o
sh

CB[10] is a macrocycle composed of 10 glycoluril rings ande
Ka values taken from ref 13a. bKa values taken from ref 3b. c Ka 18
valuestakenfrom ref 3d. dKa values
taken from ref 12b.
nb = notwo
binding.
CH
connectivity
featuresglycoluril
2-bridges whose(Lucas, D. and Isaacs, L. Org. Lett. 2011, 13, 4112.)(9) (a)Uzunova,
V. D.;
Cullinane,
C.; Brix,
K.; Nau,ofW.
M.; Day,
Figure3b shows
a Jobplot preparedfor mixtures
5 and
A. I. Org.
Chem.
2010,
8, 2037–2042.
10 ([5Biomol.
] þ [10] =0.5 mM)which
confirms the(b)1:1McInnes,
nature of F. J.;
1 J. Org. Biomol. Chem.
Anthony,G.;
Kennedy,A. we
R.;performed
Wheate, N.
H NMR competithe N.
5 •10complex.
Next,
2010, 8, 765–773. (c) Hettiarachchi, G.; Nguyen, D.; Wu, J.; Lucas, D.;tionexperiments
between5•10
andguests8 , and 12
Ma, D.; Isaacs, L.; Briken, V. PLoS ONE 2010, 5,7 ,e10514.
(d)andKim, E.;
between
6 •13
andguestsS.;7 Selvapalam,and 10 by N.;
monitoring
Kim, D.;
Jung, H.;Lee,
J.; Paul,Yang, Y.;theLim, S.;chemicalshift
guestsChem.,
10 (for5 •10)
and 13
6 •13)
Park, C.
G.; Kim,
K. ofAngew.
Int.
Ed. 2010,
49,(for4405–4408.
(e)
which
undergo N.
fastexchangeon theTrabolsi,
chemicalA.;shift
timeH. A.;
Angelos,
S.; Khashab,M.;
Yang, Y.-W.;
Khatib,
Stoddart,
J. F.;J. I.
J. Am.
Soc.competitive
2009 , 131, 12912–12914.
scale
andZink,
fit thedata
to aChem.
standard
binding(10) model(a) Chakraborty,
Wu, A.; Witt,
D.; Lagona,
Fettinger,(SupportingA.;
Information)
to determine
Ka J.;
valuesJ. C.; Isaacs,
L.
J.
Am.
Chem.
Soc.
2002,
124,
8297–8306.
(b)
Lagona,J.;(Table 1). To determine the remaining values of Ka we
Fettinger, J. C.; Isaacs,
L. J. Org. Chem. 2005 , 70, 10381–10392. (c) Liu,
performed 1H NMR competition experiments between
S.; Kim, K.; Isaacs, L. J. Org. Chem. 2007 , 72, 6840–6847. (d) Huang,•11 (or P.
6 •11)and guests
for
W.-H.; 5Zavalij,Y.; Isaacs,
L. J. byAm.monitoring
Chem. Soc.the2008integrals, 130, 8446–8454.
the
free
and
binding
guest
11
which
exhibits
slow
exchange(e) Ma, D.; Gargulakova, Z.; Zavalij, P. Y.; Sindelar, V.; Isaacs, L. J.
on thechemical
shift time scale (Table 1). Table 1 also
Org. Chem.
2010,
75, 2934–2941.
theW.-H.;
Ka values
measuredforL.hosts
4 ,Chem.
(11) presents(a) Huang,
Liu, S.;
Zavalij,previously
P. Y.; Isaacs,
J. Am.

F(amechanism (Figure 6b), considering that the rate-determining
step is the dethreading of the supramolecular complex[10](Table 2). The analysis of the data for the dethreading process
pound
K[a] [m"1]
k + [b] [m"1 s"1]k"[c] [s"1]is much more simple, as the dissociation reaction is not affected by the presence of ions. The values of k" determined from$ CB7]2 +1.6 ! 107
6.9 ! 106
0.43
Q. 3.8PAINT$ 2 CB7]2 +! 104 の仕組みについて
3.5 ! 106
92,[d] 0.48[e]the fitting are introduced as a fixed parameter in the fitting
1$ CB7]2 +6.6 ! 106
2.5 ! 106
0.38
model for the threading experiments (see the Supporting InA. 説明不⾜な点があったため、詳細に説明します。
quilibrium constant determined from the ratio of the threading andformation for details). The values of k + and k" are gathered in
通常の光学顕微鏡では、回折限界以下の距離にある
2 点を識別することができません。そこで、様々
reading rate constants.
[b] Threading rate constant. [c] Dethreading
Table 2. The rate constant k + 1Z is almost one third of k + 1E,
constant. [d] k" for the dethreading of the first CB7 from [EEな⼯夫を施すことにより、回折限界を超えた解像度を持
which suggests a small but measurable kinetic effect of theCB7]2 + . [e] k" for the dethreading of the second CB7 from [EE2+
isomerization state of the azobenzene units (Table 2).[23] The
CB7] .
つ超解像顕微鏡が開発されています。今回紹介した
Thekprincipleofvalues of k + and" could be used for a more accurate estimaPAINT では、付いたり離れたりする蛍光分⼦を⽤いてい single-molecule
microscopytion of the thermodynamic conます。この蛍光分⼦はターゲット分⼦との結合に関係な
stants for the Z and E complexes(Table 2). It is worth noting that
く蛍光を放出しますが、ターゲット分⼦に付いている時
02/chem.201402821
Full Paper
the first dethreading process of
間と露光時間が同程度であれば、付いている分⼦のみが[EE-1$ 2CB7]2 + is faster than thesecond one, which, on its turn,
強く検出されます。⼀部のターゲット分⼦をまばらに観
takes place in the same time測することになるため、ターゲット分⼦全体を同時に検
scale of the dethreading from
出した際には重なる点であっても、区別することができboth [EE-1$ CB7]2 + and [ZZ1$ CB7]2 + . These results suggest
ます。これを繰り返し、得られた像を合計することで、that 1) as expected, after remov回折限界を超えた解像度を持つ顕微鏡像が得られます。
al of one CB7 from the 2:1 come 6. Stopped-flow kinetic traces recorded at 293 K for the absorbance change at 260 nm obtained upon
(https://www.nobelprize.org/prizes/chemistry/2014/press-release/)
plex, the other one shuttles back2+
"6g equal volumes of a) a solution of EE-1 and a solution of CB7 (concentration after mixing 9 ! 10 m and
"5
2+
[a]
[a]
[a,
c]
to the bipyridinium unit andb) a solutionCredi,of [EE-1$ 2 CB7]and a solution of AD (concentration after mixing
0 m, respectively)tina
Carboni,andAlberto
Elia Previtera,"6m and 1.1 ! 10"4 m, respectively)
(H
O,
5
mm
NaCl).
crepresents
thefittingoftheexperimental
data2) the rate of slippage of the2and Serena Silvi*[a]ext for details). Q. cucurbit[n]uril の細胞内での応⽤には他にどのようなものがあるか？ macrocycle over the azobenzene
A. ドラッグデリバリーへの応⽤などが挙げられます。
unit when it dethreads from the
1:1 complexes is not affected by
CB[n]とゲスト分⼦の結合を細胞内で形成させる例は、
ferentcomplexeswiththismolecule:a[2]pseudorotaxane,sforthe isomerization state. It is likely, therefore, that the Z axle
nformation for details). This model would imply the simulin which
the macrocycle shuttles fast along the length ofaded
調べた限りでは今回紹介したイメージングしかありませcannot arrange two CB7 rings for electronic reasons, rather
ous determinationof six rate constants, but some of these
the axle, and a [3]pseudorotaxane, in which two CB7 s aremodel
than forthe steric hindrance exerted by the Z isomer. It is acs can be setんでした。
as fixed parameters.
First
of
all,
the
kinetic
結合は細胞外で形成させているものの、のちにconfined at the extremities of the axle. Upon trans to cis isoworktually recognized that the Z isomer is more polar with respect
tants
formerization
the equilibria
of Equations (5) and (6) can beof the azobenzene moieties, the [3]pseudorotaxrt on細胞へ応⽤している例は複数存在します。
特に多くの応⽤[26]+to the E one, and this could be the reason for the slightly
toreasonablevalues,knowingthatthebindingofNatoane is destabilized, and only one CB7 resides on the axle,
n be[18b]が報告されている例として、CB[n]と複合体を形成させる(see
the
Supporting
Informationfordetails).
is
fast
lower
affinity of the CB7 for the ZZ-12 + axle.
surrounding the bipyridinium unit. The system was successcular2+
over,dethreading
can be anddeterminedand [EEinserted
intoratethe constantscore of liposomes,
preliminary
in- The photochemical properties of [EE-1$ CB7]azo- thefully
ことで薬剤分⼦を細胞内へ輸送し、細胞内で放出するとい
dependent
experiments,
by means
a competitive
guest.ability.
1$ 2CB7]2 + have also been investigated: irradiation of [EEvestigations
confirmedthat itofmaintains
its switchingo difうドラッグデリバリーシステムへの応⽤が挙げられます。
1$ CB7]2 + and [EE-1$ 2CB7]2 + with 365 nm light affords in
kþ1E
þ
2þ
2+
ð1Þ
þ CB7 !!!
(Das,
D., etal. Front.
Chem. 2019
7, 1.)!! ½EE-1 $ CB7%
both cases [ZZ-1$ CB7]
withquantum
efficiencies
of ,0.10
k "1E
and 0.08, respectively (Table 1). Therefore the encapsulation of
kþ2E
ð2Þ$ CB7%2þ þ CB7 !!!
$ 2 CB7%2þ
!! ½EE-1[2, 9]axle into the wheel does not influence the photochemical
theof their cavity,
CBs
show
a
good
affinityforcationic
guests,k"2E
アゾベンゼンなどを利⽤した光スイッチへの応⽤例はあるか？
Q.
kþ1Zof the azobenzene units. It is worth noting thatgivinghost–guest complexes the stability of which can be propertiescon2þ
ð3Þ [11]
þ CB7 !!!
!! ½ZZ-1 $ CB7%2þ
[6, 10]trolled
by
means
of
pH
changes,
de ofelectrochemistry,
comwhen the axle is in the Z configuration it cannot host two
k "1Z A. 複数の報告例があります。
[2, 7]
kþNa1
petitive reactions,haveþ !!!
and light.[6, 12]þCB7s;
therefore upon irradiation of the [3]pseudorotaxane, theð4Þþ Na !! CB7 ( シクロデキストリンと同様に、cucurbit[n]uril
Naにも、アゾベンゼンなどを利⽤した光スイッチへの応⽤
o re- k"Na1 The large association constants of CBs with various guests
inreleaseof one CB7 is achieved (Scheme 3). Indeed the shape
kþNa2
sNa
hyaqueoussolution
and
the
of
þ
þ !!!
þ
þ possibility to control the stability つのアゾベンゼン部位を持つ例が複数報告されています。下図の例では、2と cucurbit[7]uriloftheabsorption spectra of an bipyridinium
irradiated solutionof [EEð5Þþ Na !! Na ( CB7 ( Naonylicthe adducts
by means of different inputs make these macrocy2+
2+k"Na2
1$ 2CB7] and of a solution of [ZZ-1$ CB7] (obtained eitherとのホストゲスト相互作⽤を利⽤し、光によって構成成分の⽐を切り替えることができる擬ロタキサン
es ofcles good candidates for the construction of molecular maby titration of ZZ-12 + or by irradiation of a solution of [EEs known[2] that adamantane derivatives like 1-adamantylaを形成しています。
(AD) are suitable guests for CB7, with association cons as large as 1012 m"1. Therefore on addition of AD to a son of E or Z pseudorotaxanes, fast and complete dissociais achieved,[10] as confirmed by titration experiments of$ 2CB7]2 + and [ZZ-1$ CB7]2 + with AD. Stopped-flow exments have been performed under simplified conditions,ely
by mixing
a solution of each pseudorotaxane with a soe examinedcompounds.
Scheme3. SchematicM.,
representationof the-light-controlled
release(Baroncini,
et al. Chem.
A Eur. J. 2014
, 20and, 10737.)n of AD in excess. The data were fitted with a first-order
uptake of CB7.

e 2. Thermodynamic and kinetic constants for the threading andreading processes.

The distance between each protein > 0.2 µm

Microscope

High-resolution
image

Singlefluorescentproteiny and Motion in Pseudorotaxanes
Wheel and an Azobenzene-[8]

chines.
are simple prototypes of10741
artificial
Eur. J. 2014,
20, 10737Pseudorotaxanes– 10744
www.chemeurj.org
molecular machines, wherein an axle-like molecule is surrounded by (at least) one macrocyle. These supramolecular systems
can perform threading-dethreading motions, and are convenient precursors for the synthesis of more complex interlocked[13]

Illustration: © Johan Jarnestad/The Royal Swedish Academy of Sciences" 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Q. cucurbit[n]uril の⽣体内での応⽤例としてはどのようなものがあるか？
A. ⽣体内イメージングへの応⽤などが報告されています。
CB[7]を繋いだ抗体と、放射/蛍光標識された adamantane 誘導体を⽤いることで、⽣体内イメージングを可能にした例が報告されています。この⽅法により、素早い結合形成が可能であるという⾮共有結合的な戦略のメリットを残しつつ、biotin-streptavidin の系で発⽣する内在性ビオチンによる偽陽性や
4

Molecular Imaging

streptavidin の免疫原性の問題を解消することが期待されます。

nterfaces

Research Articleof BDP630/650-AdA in live C.

supports not only conjugation of CB[7] units on Erbitux again
but also accessibility of the cavity of CB[7] units on Erbitux to
AdA using the ultrastable and bio-orthogonal host−guest
ﬀect of supramolecular latching ofchemistry. In in vivo assessments with cancer-bearing mice,g the localization of Cy3-CB[7]only the mouse pretreated with Erbitux-CB[7] showed
RET, as described above without
selective accumulation of the Cy5-AdA on the cancer site
istent with the results shown in11
injection
ofCB7.
Cy5-AdA(Figure7d).molecules
It clearly
Figure 3. Biodistributionafterof 2 tointravenous
4 in the absence andpresence ofThree different
C-labeledshow distinct biodistribution in
still forms an in situ host−guest
2018, 17, 1.)
M. G.,
et al.ofMol.
mice when administered individually. The left image column represents sagittal views (Strebl,
of a 3-dimensional
rendering
dynamicImaging
PET data averaged
ere was a strong FRET
signalatfrom 30 to 60 minutes postradiotracer injection overlaid on a CT of the same animal. 1 mg/mL of CB7 was used for precomplexed experiments.
The transverse
the epithelial system (Figure5i).view to the right shows the same animal from a different perspective for clarity. In the right column of images, the distribution of
the same 11C-labeled molecules is shown when precomplexed with CB7. CB7 indicates cucurbit[7]uril; PET, positron emission tomography; CT,
A was localized in intestinal
cells
computed tomography.
ith this alone (Figure 5b), the
AdA near the epithelial systemin
The experiments
were carried out with low effective specific design of 18F-radiotracers generally, since they are practically
activity
reporters.
It
is conceivable that the mass of injected accessible and stable, yet to our knowledge not utilized. Whilefurther demonstrates noncovareporter is a variable that needs to be optimized for distinct formal H to F substitutions may lead to purification issues,r latching, of BDP630/650-AdA
imaging protocols, and the molecules employed can all be alternative aliphatic fluorination methods are available that
lized Cy3-CB[7] via speciﬁcandin high specific activity simply by adding addi- employ starting materials more easily separable from fluorides
synthesizedthan the unsubstituted compounds (eg, carboxylates).36
tional purification steps.
nition occurring in a complicated

The short half-life of 11C presents an intrinsic practical limitation to the proposed application. 18F is easily available Summary
upramolecular latchingtool tothroughout the continental United States, as it is the most comto investigate its potential
monly forusedinpositron emitter in the clinical setting, and fluori- We establish herein the preliminary compatibility of the adamantracers
e localization of CB[7]natedunits
on aare currently the most useful in a logistic capacity tane/CB7 host–guest complex interaction with the concept of preallow for more stable syntheses due to the longer half-life targeted immuno-PET. We show for a variety of radiolabeled
he monocarboxylic andacid-HEG
compared to 11C. More recent advances in 18F fluorination reporters that association complexes with CB7, albeit without antiderivative was conjugatedto the direct labeling of adamantyl residues with body conjugation, are stable and form rapidly in vitro and in vivo
chemistry allow
18 35
me of cetuximab, a recombinant
F. In order to probe whether associated structural changes and that biodistribution of radiolabeled adamantane derivatives
changed by addition of CB7 for both moledisrupt
the complexation, experiments analogous to Figure 3 can be fundamentally
ody that targets thehuman18cules labeled with 11C and 18F. In addition to our preliminary
F-labeledfluoroadamantylamineinwere conducted with Figure( Li,M., et ofal.cancer-bearing
ACS Appl. Mater.
Interfaces 2019, 11, 43920.)
In vivo ﬂuorescenceimaging
mice (a)eptor with high aﬃnity
for the rats. As seen 7.
Sprague-Dawley
in Figure 4B and C, not only findings, we report a potential caveat in that the extremely rapid
45
untreated,(b)
Cy5-AdA
treated,(c)Erbitux
andCy5-AdA
treatedcomplexwill
leavecomplexformationin the blood which could be
Matrixk, and lung cancers.was
the retention time of the probe on a high-performance
We alsonote thesequenpossibility of the rate of clearance
sequentially,and significantly(d) Erbitux-CB[7]and hurdle.
Cy5-AdA
treatedliquid chromatography(HPLC) shifted
when a major
onization time of ﬂight
mass
tially.
CB7
was added but also
the ratio of kidney-to-liver uptake at of the molecules to be too rapid for tumor targeting. We are hopeMS) revealed successful
conjugalater time points was increased over 4-fold consistent with the ful that this concept will be widely applicable to pretargetedn Erbitux (Erbitux-CB[7];Figure changes observed earlier. The change in distri- immuno-PET as well as pretargeted radiotherapy. Applications
biodistribution
demonstrates that Cy5-AdA can be noncovalently anchored onments, in vitro cell experiments
bution was reproducible by injecting CB7 separately 5 minutes of CB7 in pharmaceutical formulation can profit from information
Erbitux-CB[7] that is prelocalized in a cancer site in a live
before injection of radiotracer. This alludes to the potential that about its in vivo distribution disclosed herein as well.

targeting ability of Erbitux after
mouse.
This
resultwas also
cross-checked by detecting
kinetics will
be sufficientfor future
experimentsofdentical ﬂuorescence association
signals forthis system with antibody-conjugated
CB7.strong
It is further
worth Materials
distinguishably
ﬂuorescencesignals andof Cy5-AdA
Methods fromgated to Alexa 555 between
bothnoting thatno bone uptake
fluoride wasobserved,
indicating
the ofextracted
cancertissuesof theRadiosynthesis
mouse treated with Erbituxmetabolicstability of the C–F bond on a PET timescale, whichwith Erbitux-CB[7] andErbitux
CB[7] (Figure S12d). Taken together, this noncovalent in situ
is atypicalfor aliphatic fluorides. These results suggest that Compound 1 (Martinostat) was prepared as previouslyb,c) and ﬂow cytometry(Figure37
anchoring,supramolecularoccurseﬃciently
under
Molecular
weight:354.49a g/mol.
fluoroadamantyl moieties
should receive
more attention inlatching,
the described.
ns its cancer cell targeting ability
dynamic
bloodstream
condition,
resulting
in
in
vivo
cancermolecular latching of Cy5-AdA31
imaging of a mammalian model.
ancer cells analyzed by CLSM
metry (Figure 6k) additionally
CONCLUSIONS
We have demonstrated a new supramolecular tool fornoncovalent in situ anchoring of small synthetic molecules inlive C. elegans using a fully synthetic high-aﬃnity binding pairconsisting of Cy3-CB[7] and BDP630/650-AdA. Emergenceof FRET signals upon formation of an ultrastable host−guest
complex between them demonstrated speciﬁc host−guest■