Host-guest chemistry based bioconjugation:
progress toward in cell application

Literature seminar #1
B4 Yuki Yamanashi
2020/02/28(Fri)Contents- Introduction- Bioorthogonal molecular conjugation in cells/ in vivo
- Current options- Host-guest association- Characteristics of cucurbit[n]uril- Applications of cucurbit[n]uril- Cucurbit[n]uril for in cell bioimaging- Characteristics of cucurbit[n]uril based bioimaging- Further applications- Future directions and challanges- Summary2

Contents- Introduction- Bioorthogonal molecular conjugation in cells/ in vivo
- Current options- Host-guest association- Characteristics of cucurbit[n]uril- Applications of cucurbit[n]uril- Cucurbit[n]uril for in cell bioimaging- Characteristics of cucurbit[n]uril based bioimaging- Further applications- Future directions and challanges- Summary3Bioorthogonal molecular conjugation in cells/ in vivo

4stry & Biology

Review
McKay, C. S.; Finn, M. G. Chem. Biol.
2014, 21 (9), 1075–1101.

-

visual tag (e.g., fluorescent molecular) →detection of target molecular

Figure 1. Strategies for Introducing and Conjugating Functionality to Biomolecule Targets(A) The two-step bioorthogonal labeling strategy: in the first step exogenous functionality is introduced genetically, metabolically or chemically, and the second
step involves highly specific bioorthogonal reaction.
(B) Site-specific bioconjugation based on native functionalities present in proteins.

-immobilization agent (e.g., magnetic bead) →isolation of target molecular-therapeutic agent (e.g., radioactive molecular) →pre-targeting therapeutics

condensation processes that produce water as the only byprod- amino acid residues of peptides or proteins with high site selec
uct. Most applications
involve a two-step
approach that requires tivity
has become
important. The addressable functional groups- catalyst→selective-modificationof target
molecular

Current options: Host-guest chemistry has many advantages.

5- “Click” reactions (azide-alkyne cycloaddition, Diels-Alder reaction, …)
Wide variety of reactions
Slow reaction rate (kon ~ 101-104 M-1s-1)- Protein tagging (Halo-tag, CLIP-tag, Snap-tag, …)High selectivity in mild conditions
Large size (~ 20-30 kDa) and instability of protein- Biological host-guest pairs (biotin-avidin, eDHFR-TMP, aptamers, …)
Strong and rapid association (kon ~ 109-1010 M-1s-1)
Large size (~ 10-50 kDa) and instability of protein/oligonucleotides- Synthetic host-guest pairs (β-cyclodextrin, cucurbit[n]uril, …)
Strong and rapid association (kon ~ 109-1010 M-1s-1)
Small(~1 kDa) and stable molecular

Schreiber, C. L.; Smith, B. D. Nat. Rev. Chem. 2019, 3 (6), 393–400.
Sasmal, R.; Das Saha, N.; et al. Anal. Chem. 2018, 90 (19), 11305–11314.

Synthetic host-guest pairs: Cucurbit[n]uril(CB[n]) family is suitable for bioconjugations.

6* (Strept)avidin-biotin: Kd ~ 10-15- β-Cyclodextrin – hydrophobic molecule
low affinity (Kd ~ 10-6-10-3)- Pillar[n]ene – hydrophobic molecule
low affinity (Kd ~ 10-6-10-4)- Tetralactam – squaraine
high affinity (Kd ~ 10-11-10-7)- Calixarene – hydrophobic cation
low affinity (Kd ~ 10-6-10-4)- Cucurbit[n]uril(CB[n]) – hydrophobic cation
very high affinity (Kd ~ 10-15-10-9)
wide variety of guests
Schreiber, C. L.; Smith, B. D. Nat. Rev. Chem. 2019, 3 (6), 393–400.

Contents- Introduction- Bioorthogonal molecular conjugation in cells/ in vivo
- Current options- Host-guest association- Characteristics of cucurbit[n]uril- Structure- Host-guest chemistry- Synthesis- Applications of cucurbit[n]uril- Cucurbit[n]uril for in cell bioimaging- Characteristics of cucurbit[n]uril based bioimaging- Further applications- Future directions and challanges- Summary7Structure of CB[n]

8

n
5
6
7
8
10

MW
830.7
996.8
1162.91329.1
1661.4Assaf, K. I.; Nau, W. M. Chem. Soc. Rev. 2015, 44 (2), 394–418.

Host-guest
chemistryof CB[n]Chart
1. Guests
Used in This Paper

9

Review Article

Open Access Article. Published on 15 October 2014. Downloaded on 1/29/2020 4:15:45 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

: H(CH2)mNH3+
: +H3N(CH2)mNH3+

View Article Online

Fig. 15of 1-(pReview Article

High-

CBn b
high ainclustanes,
cene98(Tableappro
covale
The feKim98
ferrocthe retions i
ustration of the release of high-energy water molecules from the CB7 cavity upon binding of a hydrophobic guest.
perpenthis toFig. 14 (a) Relationship between the binding constant (log Ka) versus chain
13
+
+
+
quadr
length m for H(CH2)mNH3 (K) and H3N(CH2)mNH3 (m). (b) Dimensionalhe 242 Å3 large CB7 cavity does not allow thecomparison between CB6 and various a,o-alkane diammonium ions.
on ferr
14
lecules to arrange in a structurally stableby intrAd
group being highest, that of an alkoxy group being lowest, andnergy of each individual internal water molecomp
128that of an alkylthio group being in between.
It should besmall CBn, such as CB5 and CB6, but thenoted that the driving force due to desolvation of convex guests and co
15,16
17,18
easing all water from the inner cavity of CB7
is mainly related to their surface area. It produces a ‘‘classical’’ measu
he larger number of water molecules (moreAssaf, K. I.; hydrophobicNau, W. M.
Chem.
Rev.entropic
2015,component,
44 (2), 394–418.
eﬀectwith aSoc.
favorable
which water,

Main driving forces:congeners. The molecular recognition and self-assembly- ion(dipole)-dipole interaction
properties of CB[6] sthe oldest and most widely studiedhydrophobic
effect been delineated by themember of- theCB[n] familyshave→largelyon theCB[6]cavitysize
pioneering workof Mockdepends
and Kim.
undergoeshigh affinity, highly selective, constrictive binding interactions

to control
independeapplicatio
collectiVeof comple

CB[7] has wide range of affinity to various types of guests.

RTICLES

10

Affinity in 50 mM NaO2CCD3-buffered D2O (pD=4.74)

Liu e

Kd ~ 10-5-10-4

art 1. Guests Used in This Paper

Cucurbit[7]uril(n = 2)-6-11-10-10
Kd ~ 1310-7-10molecular
Kd ~ 10-10-12and CB
ngeners.
The
recognition andKd
self-assembly
toKd
control
those
processes,
which suggests
that-13CB[7]~ 10-9-10-8~ 10
operties of CB[6]14sthe oldest and most widely studied
independently, will prove as useful as CB[6] forMe
nanotechnolo
6,7
J. Am.
Soc. 2005,
127, 15959–15967.
(modiﬁed)
ember
ofChem.
the CB[n]familyshave
been delineatedby theapplications. In this paper, we argue that the +CB[n] fam
Me components for NtheMe
15,16
17,18
oneering work of MockcollectiVely, constitute prime
preparatand Kim.
CB[6] undergoes·2IMe N+Meof complex self-sorting systems not only because of the w
gh affinity, highly selective, constrictive binding interactions
15recognition properties
Me (e.g. affinity and selectivity)
definedth cationic species, especially ammonium ions, driven by a4deach member of the family but also because Kdof thehighmbination of ion-dipole interactions, hydrogen bonds, and-15lev~ 10of selectivity exhibited by different members of the familye hydrophobic effect. Aside from well-defined thermodynamic
31
a commonects,Cao,CB[6]also excels
theP.viewpoint
of-Majerski,
kinetic control
L.; Šekutor,
M.;from
Zavalij,Y.; Mlinaric̈
K.; Glaser, R.;
Isaacs, L. guest.
Angew. Chemie - Int. Ed. 2014, 53 (4), 988–993.
19 For example, CB[6] accelerates therecognition
processes.
Liu, S.; Ruspic,
C.; Mukhopadhyay,P.; Chakrabarti, S.; Zavalij, P. Y.; Isaacs, L. J. Am. Chem. Soc. 2005, 127 (45), 15959–15967.

- K value ranges from 10 to 10 .

20

Results

Monofunctionalized CB[n] derivatives are accessible.

11

Methylene-bridged glycoluril hexamer (6C)
Scheme 2 (a) Synthesis of Me2CB7, CycH2CB7, and MePhCB7. (b) Synthesis of a monofunctionalized CB7 derivative; dots mark connection points to thesecond identical half of the symmetric structures. Vinciguerra, B.; Cao, L.; et al. J. Am. Chem. Soc. 2012, 134 (31), 13133–13140.

400 | Chem. Soc. Rev., 2015, 44, 394--418

This journal is © The Royal Society of Chemistry 2015

Cucurbit[n]uril (n = 5, 6, 7, 8)
Ayhan, M. M.; Karoui, H.; et al. J. Am. Chem. Soc. 2015, 137 (32), 10238–10245.

ammonium ions (e.g., p-xylylenediammonium ion 11)

Synthesis of 6C and CB[n] (1): Mechanism of CB[n] formation
Scheme 1. Mechanism of CB[n] Formation

a

12

a

Color code: reversible equilibria, green arrows; irreversible reactions, red arrows.

dx.doi.org/10.1021/ja208229d |J. Am. Chem. Soc. 2011, 133, 17966–- CB[n] (n = 5, 6, 7, 8, 10) is synthesized17967
by the condensation
reaction of
glycoluril 1 and formaldehyde under strongly acidic conditions.
- Each CB[n] was purified by fractional crystallization and dissolution.

Lucas, D.; Minami, T.; Iannuzzi, G.; Cao, L.; Wittenberg, J. B.; Anzenbacher, P.;
Isaacs, L. J. Am. Chem. Soc. 2011, 133 (44), 17966–17976.

Synthesis of 6C and CB[n] (2): Template synthesis of 6C

13ming reactions conScheme 2. Templated Synthesis of 6C and Bis-nor-secoess than 2 equiv of
CB[10]
e deficient reaction] is slowed down27
enhanced.
ion of the different
valent bond formingnderson and Sanders
e.33 Template molc product by either NH3+
odynamic templates-8 M
K(6C·11)
=
4.5
×
10
+
d
odynamic
control by 11
H 3Nothers. Kinetic tem-3 M
K(CB[6]·11)
=
1.8
×
10
d
duct by changing the
e the rate of productclassified as positive
p or slow down the- Diammmonium ion 11 acts as template.
respectively. Finally,- 11 slows down transformation of 6C and paraformaldehyde into CB[6] bytransition state enforming stable 6C·11 complex.
es or products, they
particularly efficacious to use 11 as template and simultaneouslnces on the reaction.
D.; Minami, T.; Iannuzzi, G.; Cao, L.; Wittenberg, J. B.; Anzenbacher, P.;
as an in Lucas,
situ
probe
for
analysisof133thecontentof CB[n] reaction
Isaacs,
L.
J.
Am.
Chem.
Soc.
2011,
(44),
17966–17976.
and Bis-nor-seco-Applications of CB[n]14

Cucurbit[n]uril

Wide range of affinity
Monofunctionalized derivative
High water solubility
High stability
Low toxicity
Bio-orthogonal binding

molecular
machinecatalysis

bio-sensing-

proteinimmobilization

pre-targetingtherapyproteinmodification

imaging

material
fabricationgas
purification
drugdeliveryContents- Introduction- Bioorthogonal molecular conjugation in cells/ in vivo
- Current options- Host-guest association- Characteristics of cucurbit[n]uril- Applications of cucurbit[n]uril- Cucurbit[n]uril for in cell bioimaging- Characteristics of cucurbit[n]uril based bioimaging- Further applications- Future directions and challanges- Summary

15

(Strept)avidin-biotin based bioimaging

16

Streptavidin: 53 kDa(tetramer)high affinity (Kd ~ 10-15 M)

effect of endogenous bioting. 2 (left)of biotin/streptavidin
with loop3–4 clo
rapidPictureassociation
low complex
cell permeability
e a lid, (right) important hydrogen bonds within the biotin/streptav
signal amplification
enzymatic degradation
11
nding pocket.
many applications

low resolution

irreversible binding
high costLiu, W.; Samanta, S. K.; Smith, B. D.; Isaacs, L. Chem. Soc. Rev. 2017, 46 (9), 2391–2403.
Kim, K. L.; Sung, G.; Sim, J.; Murray, J.; Li, M.; Lee, A.; Shrinidhi, A.; Park, K. M.; Kim, K. Nat. Commun. 2018, 9 (1).

CB[n]-guest based bioimaging

CB[n]

17

ligandligandligandligandligandtargetligandguest

1. Characteristics of CB[n] based bioimaging
a. Specificity
b. Cell permeability: live cell imaging
c. Stability
2. Further applications
a. Combination with gold nanoparticle
b. Cell surface specific labelingc. Super-resolution imaging

CB[n]-guest based bioimaging

CB[n]

18

ligandligandligandligandligandtargetligandguest

1. Characteristics of CB[n] based bioimaging
a. Specificity
b. Cell permeability: live cell imaging
c. Stability
2. Further applications
a. Combination with gold nanoparticle
b. Cell surface specific labelingc. Super-resolution imaging

fixed cell

1-a. Specificity: vs. Streptavidin-biotin+H N
2target: Golgi

CB[7]-ADA

19

+H N
2

+H N
2

CB[7]

nd

Bright fielde

AdA-2 antibody
Cy3-CB[7]AF647-2nd antibody

f

g

Merge

h

100

80

60

64
7SA

i

O
V1
/N
AF

d

C
y3
-C

c

N

b

O
V2
/N

a

Merge
N

AF555-2nd antibody

Bright field

Colocalization efficiency (%)

BT-2nd antibody
AF647-SAB[
7]

MUNICATIONS | DOI: 10.1038/s41467-018-04161-4

40

20

0

Kim, K. L.; Sung, G.; Sim, J.; Murray, J.; Li, M.; Lee, A.; Shrinidhi, A.; Park, K. M.; Kim, K. Nat. Commun. 2018, 9 (1).

living cell

1-b. Cell permeability: live cell imaging

ARTICLECB[7]-ADA

20

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04161

AdA

H2 –
Cl
N
+

OH =

OH

O
O

O

N
H

Cy3-CB[7]Alexa 647

BF

Merge

Mitochondria
Nucleus

AdA

OH

H2O2
CB[7]

Cy3

1. Anti-V5
primary antibody
2. Alexa 647-conjugatedsecondary antibody

V5 tag

AdA

OH

V5 tag
APEX2

AdA

O*

Nucleusg. 6 Visualization of a subcellular organelle (mitochondria). Schematic illustration for proximity-dependent adamantylation to mitochondria proteinsing mito-V5-APEX2 and immunoﬂuorescence labeling of mito-V5-APEX2, and confocal laser scanning microscopy images of living COS7 cells whicve mito-V5-APEX2 expressed in mitochondria. Scale bar = 20 μm

Kim, K. L.; Sung, G.; Sim, J.; Murray, J.; Li, M.; Lee, A.; Shrinidhi, A.; Park, K. M.; Kim, K. Nat. Commun. 2018, 9 (1).

1-c. Stability

tical Chemistry

fixed cellCB[7]-ADA

21

A

e 1. Host−guest interaction mediated bioorthogonal labeling in cells. (A) Structure of CB[7] and ADA. (B) Schematic showing the strancovalent labeling. Microtubules were tagged with CB[7] using CB[7]−antibody. Fluorophore conjugated ADA ﬁnds its high aﬃnity
h molecular recognition, resulting in speciﬁc ﬂuorescentlabelingof microtubules.
(C)
SIMChem.
image2018,of microtubulesfrom CB[7]−Sasmal,R.; DasSaha, N.; et al.
Anal.
90 (19), 11305–11314.
2. In situ binding kinetics of CB[7]−ADA based labeling platform. (A−G) Structured illumination imaging of microtubule after incfixed cell

1-c. Stability: vs. TCO-Tz ligationorCB[7]-ADA

22

or
or

Sasmal, R.; Das Saha, N.; et al. Anal. Chem. 2018, 90 (19), 11305–11314.

fixed cell

1-c. Stability: vs. TCO-Tz ligation

CB[7]-ADA

23

or
or
or

separately kept at 37 ℃
in culture media + 10% FBS
for 0 or 16 h

Figure S30. Stability study of CB[7]−ADA interaction based labeling platform. Labeling efficiency
before (a) and after (b) incubation of the reactive reagents in physiologically relevant condition. 25
nM ADA−Cy5 and CB[7] labeled microtubules in HeLa cells were kept separately at 37°C in phenol
red free culture media containing 10% FBS for 16 h. Fluorescence images were acquired after 15 min

CB[7]-ADA

TCO-Tzincubation of ADA−Cy5 with CB[7] labeled microtubules. Minimal change in labeling efficiency wasobserved from this experiment. Scale bars: 5 µm (a and b).

Figure S30. Stability study of CB[7]−ADA interaction based labeling platform. Labeling efficiency

Figure S31. Stability study of TCO−Tz interaction based labeling platform. Labeling efficiency

before (a) and after (b) incubation of the reactive reagents in physiologically relevant condition. 25

before (a) and after (b) incubation of the reactive reagents in physiologically relevant condition. 25

0h

16 h

°

0h

16 h

nM Tz−Cy5 and TCO labeled microtubules in HeLa cells were kept separately at 37°C in phenol red
Sasmal, R.; Das Saha,N.; et al. Anal. Chem. 2018, 90 (19), 11305–11314.
free culture media containing 10% FBS for 16 h. Fluorescence images were acquired after 15 min

nM ADA−Cy5 and CB[7] labeled microtubules in HeLa cells were kept separately at 37 C in phenol
red free culture media containing 10% FBS for 16 h. Fluorescence images were acquired after 15 minCB[n]-guest based bioimaging

CB[n]

24

ligandligandligandligandligandtargetligandguest

1. Characteristics of CB[n] based bioimaging
a. Specificity
b. Cell permeability: live cell imaging
c. Stability
2. Further applications
a. Combination with gold nanoparticle
b. Cell surface specific labelingc. Super-resolution imaging

2-a. Combination with gold-nanoparticle

living cellCB[7]-ADA

25

Figure 5. Multiplexed labeling. (A) Schematic showing the use of CB[7]−ADA and TCO−Tz orthogonal labeling platlabeling and multiplexed imaging. (B) Multiplexed SIM imaging of microtubules and mitochondria. Microtubules and mit
using CB[7] and TCO conjugated antibodies, respectively. Multiplexed images were captured after addition of ADA−Rh(C) Multiplexed imaging of actin and microtubule in thoracic muscle tissue of Drosophila using orthogonal labeling pairs. A
phalloidin−CB[7] and microtubule was targeted using antibody−TCO conjugate. Scale bars: 5 μm (parts B and C).

Kd ~ 10-7 M

Kd ~ 10-13 M

Figure S40. Gold nanoparticle (AuNP) assisted delivery strategy for CB[7] conjugated Phalloidin
derivatives. (A) HMD guest functionalized AuNP (d~2 nm) was used to deliver CB[7] conjugated
Phalloidin derivative inside live HeLa cell. (B) HMD guest binds with CB[7] with an affinity of ~10–7

M-1 (Kd) whereas ADA posses an affinity of ~10–13 M-1 (Kd) towards CB[7]. Therefore, the initial

complex between HMD-CB[7] can be disassembled by presentation of the orthogonal guest molecule1-adamantylamine (ADA), which Figure
has relatively
high affinity for CB[7]. This guest exchange process
6. Live cell imaging using CB[7]−ADA interaction. (A) RFP (pseudo-colored green) transfected SVEC cells. (BSasmal,R.;CB[7]−ADA
Das Saha,interaction.
N.; et al.Cells
Anal. Chem.
90 (19),VCAM
in SVEC cell complex,
surfaceusing
imaged2018,
after exciting
with11305–11314.
a 642 nm laser line
leads to the formation of more stable
CB[7]-ADA
leading to efficient
targetwerelabeling.

living cell2-b. Cell surface specific labelingCB[7]-FcA

26
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04161-

ARTICLEDAPI

Cy3-CB[7]Overlay

Cy3-CB[7]Overlayb

FcA-COOH
EDC, Sulfo-NHS
4 °C

Cy3-CB[7]

4 °C

4 °C

37 °C

DAPI

a

Cy3-CB[7]Overlay

DAPI

CIH–
H
N

O

OH

O

Fe

O

c

FcA-COOH
N

Fe

2CI

–

BAFc

N

K (CB[7]-FcA-COOH) ~ 10
K (CB[7]-BAFc)
~ 10

M
M

ig. 3 Selective visualization of intracellularly translocated proteins. Schematic illustration of the ferrocenylation -10of plasma membrane proteins anddisualization of those proteins using Cy3-CB[7] and confocal laser scanning microscope images. a COS7 cells treated with FcA-COOH, EDC, and sulfo-13
NHS at 4 °C for 30 min, b the cells further incubated with BAFc d
at 4 °C for 20 min after a, and c the cells treated with BAFc at 4 °C for 20 min after
dditional incubation for 1 h at 37 °C of the cells in a. Scale bar = 10 μm

hat plasma membrane proteins can be selectively ferrocenylated translocation of proteins, as a part of the normal metabolism owith FcA-COOH through EDC coupling at 4 °C at which intra- living cells. After treatment with BAFc at 4 °C, the ﬂuorescenc
25.M.;Kim,
K. L.; Sung,was
G.;signiﬁcantly
Sim, J.; Murray,
J.; Li,
Shrinidhi,
A.; Park, K.wasM.;only
Kim, visible
K. Nat. inside
Commun.
9 (1).3c
ellular uptake of
FcA-COOH
reducedTo Lee,test A.;signalof Cy3-CB[7]the 2018,
cells (Fig.

2-c. Super-resolution imaging: mechanism of PAINT

27

Communication

PAINT: Points Accumulation for Imaging in Nanoscale Topography

Fig. 1 Concept of host–guest mediated super-resolution imaging. (a) The
Sasmal, R.; Das Saha, conjugatedN.; Schueder, F.; Joshi,guest
D.; Sheeba,molecule
V.; Jungmann, R.; interacts
Agas<, S. S. Chem.transiently
Commun. 2019, 55with(96), 14430–14433.
fluorophoreCB[7]in vitro

2-c. Super-resolution imaging: optimization of guest

=

CB[7]
guest

CB[7]-HMD

+H N
3

28

(propylamine)

+H N
3

N
H2

(HMD)

+

Glass slide

Glass slide

N
H2

(ADA)

+

guest

Kd
[M]

kon
[M-1s-1]

kon·Kd= koff [s-1]τb = koff-1 [ms](residence time)

prolylamine

10-4-10-3

108

105-106

0.01-0.001

HMD

10-7-10-6

108

101-10210-100ADA

10-13-10-12

108

10-5-10-6

107-108

Sasmal, R.; Das Saha, N.; Schueder, F.; Joshi, D.; Sheeba, V.; Jungmann, R.; Agasti, S. S. Chem. Commun. 2019, 55 (96), 14430–14433.

in vitro

2-c. Super-resolution imaging: optimization of guest

=

CB[7]
guest

CB[7]-HMD

+H N
3

29

(propylamine)

+H N
3

N
H2

(HMD)

+

Glass slide

Glass slide

N
H2

(ADA)

+

HMD (interval 1 min)Figure S35| Single-molecule imaging of BSA-CB[7] immobilized surface using Alexa Flour 647 conjugated
HMD guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart). Scalebar: 5 µm (a-f).

guest

Kd
[M]

kon
[M-1s-1]

kon·Kd= koff [s-1]τb = koff-1 [ms](residence time)

resultprolylamine

10-4-10-3

108

105-106

0.01-0.001

no fluorescence-7 immobilized-6
8 Alexa Flour 6471 conjugated2
Figure S35| Single-molecule imaging of BSA-CB[7]surface using

HMD

10 -10

10

10 -1010-100ON/OFF switching

ADA

10-13-10-12

108

10-5-10-6

107-108

bleachHMD guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart). Scalebar: 5 µm (a-f).

Sasmal, R.; Das Saha, N.; Schueder, F.; Joshi, D.; Sheeba, V.; Jungmann, R.; Agasti, S. S. Chem. Commun. 2019, 55 (96), 14430–14433.

fixed cell

2-c. Super-resolution imaging

CB[7]-HMD

30

+H N
2

+H N
2

+H N
2

CB[7]
target: F-Actin

Phalloidin

NH3+

Phalloidin

NH3+

Phalloidin

NH3+

Sasmal, R.; Das Saha, N.; Schueder, F.; Joshi, D.; Sheeba, V.; Jungmann, R.; Agasti, S. S. Chem. Commun. 2019, 55 (96), 14430–14433.

living cell

2-c. Super-resolution imaging

CB[7]-HMD

31

+H N
2

AuNP

NH3+

+H N
2

AuNP-assisted
Cellular uptake+H N
2Communication Phalloidin

Phalloidin
CB[7]

AM.

+H N
2

+H N
2

NH3+

Phalloidin

NH3+

NH3+

NH3+

Sasmal, R.; Das Saha, N.; Schueder, F.; Joshi, D.; Sheeba, V.; Jungmann, R.; Agasti, S. S. Chem. Commun. 2019, 55 (96), 14430–14433.

Contents- Introduction- Bioorthogonal molecular conjugation in cells/ in vivo
- Current options- Host-guest association- Characteristics of cucurbit[n]uril- Applications of cucurbit[n]uril- Cucurbit[n]uril for in cell bioimaging- Characteristics of cucurbit[n]uril based bioimaging- Further applications- Future directions and challenges- Summary32

Future directions and challenges

33

e complex (11a). Related changes arecomplexation of the hydrophobic
Supporting Information) indicating
nclusion and NH3+ portal binding is
ry. Changes are also observed in thehost 1 in the 1•H-Phe-NH2 complex.
rotons in C2v-symmetric 1 are symdisplay a sharp singlet (Figure 3(f))
d and split upon formation of the C1mmetry) 1•H-Phe-NH2 complex. Thisxplained by the fact that the chiral
Figure4. (Colour online) Schematic illustrations of the geome- Expanding
thediastereotolimited scopeof guestsnders
all four Hf atoms
try of (a) 1•H-Phe-NH and (b) 1•H-Lys-NH2 complexes.
kinetics- ofDevelopment
exchange, typiﬁedof
beautiCB[n] derivatives will 2expand the guest
scope.
-NH2 -complex,
twoCB[n](Calabadion
doublets are
Acyclic
1) had high affinity toward a wider range
pporting ofInformation,
FigureS86). acids than normal CB[n].
N-terminal
amino
upon complexation that removes edge-to-face C-H•••π
are observed with host 2. None of- Further applications
interactions that occur in the uncomplexed host that result
en 1 or 2 and the amino acid amides
Application
of
CB[n]
has
been limited to relatively simple
in upﬁeld shifting (2c). In contrast, for the aromatic aminos of exchange on the 1H NMR timeapplications.
acid amides (e.g. H-Trp-NH2, H-Tyr-NH2, H-Phe-NH2) only
complexes
between 1 (2) and the- Furtherapplications
wide
ofinfields
areshift
expected.
very
smallrangechangeschemicalare observed for Hf
matic aminoacid amides,
we typically in awhichbelievereﬂectsa Supramol.
balance Chem.
betweenexpectedﬁeld shift for host aromatic sidewall
Zebazewe
Ndendjio,
S. A.;
Isaacs, L.
2019,the31 (7),
432–441.

Summary: Host guest chemistry for bioconjugation in cells/ in vivo

- Development of methodologies for bioorthogonal molecular
conjugation is desired.
- Cucurbit[n]uril based host guest chemistry has many
desirable characteristics for in cell/ in vivo applications.
- Cucurbit[n]uril based bioimaging has several advantages
over protein based bioimaging.
- Some unique applications are realized by utilizing thecharacteristics of cucurbit[n]uril-guest interaction.
- Expanding the limited scope of guests by developing new
host molecules and further applications in a wide range offields are expected.

34

Appendix

Contents(appendix)- Introduction- Affinity of CB[8]- Synthesis of monohydroxy-CB[n]- Toxicity of CB[n]- Cellular uptake of CB[n]- Cucurbit[n]uril for in cell bioimaging- 2-c. Super-resolution imaging- Other applications- Future directions and challenges- Recognition of N-terminal Phe of insulin by CB[7]36Affinity of CB[8]37- Kd value ranges from 104 to 1015.
R T I C -L E Cucurbit[7]urilSand cucurbit[8]uril has whole different guest preference.

Liu e

art 1. Guests Used in This Paper

Kd ~ 10-5-10-4
Cucurbit[8]uril(n = 3)-11-10

Kdwhich~ 10 suggests-10 that CB[7] and CB~ 10
ngeners.13 The molecular recognitionno
andbinding
self-assembly
to Kd
control
those-10
processes,
operties of CB[6]14sthe oldest and most widely studied
independently, will prove as useful as CB[6] for nanotechnol
J. Am.
Soc. 2005,
127, 15959–15967.
(modified)emberofChem.
the CB[n]familyshave
been delineatedby theapplications.6,7 In this paper, weMeargue that the CB[n] fam
N+ Meoneering work of Mock15,16 and Kim.17,18 CB[6] undergoes
collectiVely,
for the preparat
Meconstitute prime components·2IMe self-sorting
Me only because of the wof complex
systems not
gh affinity, highly selective, constrictive binding interactionsN+defined recognition
properties (e.g. affinity and selectivity
th cationic species, especially ammonium ions, driven by aMe
Kd ~ 10-12
each member of the family but also because of the high lev
mbination of ion-dipole interactions, hydrogen bonds, andCao,L.;Šekutor,M.;
Zavalij,P.
Y.;
Mlinaric̈
Majerski,
K.;
Glaser,R.;
L. Angew.
ChemieInt. Ed. 2014,
53 (4),
ofIsaacs,
selectivity
exhibitedby -different
membersof 988–993.
the familye hydrophobic effect. Aside from well-defined thermodynamic
31
Liu,CB[6]S.; Ruspic,
C.; Mukhopadhyay,P.; Chakrabarti,
Zavalij, P. Y.;aIsaacs,
L. J.guest.
Am. Chem.
Soc. 2005, 127 (45), 15959–15967.
commonfects,
also excels
from the viewpointof kineticS.;
control-9-8Synthesis of monohydroxy-CB[n]38Ayhan, M. M.; Karoui, H.; et al. J. Am. Chem. Soc. 2015, 137 (32), 10238–10245.

lution of this poorly water-soluble macrocycle would present

Toxicity of CB[n]accessible concentration range was limited (Fig. 3). At the high39

ww.nature.com/scientificreports/

Figure 1. Structureof cucurbit[7]uril(CB[7]).
Relative cell viability of CHO-K1
in dependenceon Brix,
CB[7]concentrationmM)
incubation
time determinedusing
the MTT as
Uzunova,cells
V. D.;
Cullinane,C.;
K.;
Nau, W. M.;(0–1Day,
A. I.and
Org.
Biomol. Chem.
2010, 8 (9),2037–2042.
onitoring formazan absorbance at 570 nm. Mean values and standard deviations were obtained from five independent experimental determinatio| Org. Biomol. Chem., 2010, 8, 2037–2042

This journal is © The Royal Society of Chemistry 20

Zhang, X.; Xu, X.; Li, S.; Wang, L. H.; Zhang, J.;
Wang, R. Sci. Rep. 2018, 8 (1), 1–7.

Cellular uptake of CB[7]-dye

Li, M.; Lee, A.; Kim, S.; Shrinidhi, A.; Park, K. M.; Kim, K. Org. Biomol. Chem. 2019, 17 (25), 6215–6220.

40

in vitro

2-c. Super-resolution imaging: optimization of guest

CB[7]-HMD

41

propylamine
Figure S37| Single-molecule imaging of BSA-CB[7] immobilized surface using Alexa Flour 647 conjugatedpropylamine guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart).
Scale bar: 5 µm (a-f).

HMD
Figure S37| Single-molecule imaging of BSA-CB[7] immobilized surface using Alexa Flour 647 conjugatedFigure S35| Single-molecule imaging of BSA-CB[7] immobilized surface using Alexa Flour 647 conjugatedpropylamine guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart).
HMD guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart). Scale
Scale bar: 5 µm (a-f).
bar: 5 µm (a-f).

ADA
Figure S35| Single-molecule imaging of BSA-CB[7] immobilized surface using Alexa Flour 647 conjugatedFigure S39| Single-molecule imaging of BSA-CB[7] immobilized surface using Alexa Flour 647 conjugated
HMD guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart).
ADAScaleguest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart). Scaleguestµm (a-f).
Kbar:
d 5[M]

kon [M-1s-1]

kon·Kd= koff [s-1]prolylamine

10-4-10-3

108

105-106τb = koff-1 [ms]resultFigureS38| Single-molecule imaging
of unmodified BSA immobilized surface using Alexa Flour 647(residencetime)bar: 5 µm (a-f).

conjugated propylamine guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200

0.01-0.001

frames apart). Scale bar: 5 µm (a-f).

-7 S39| -6
8of BSA-CB[7] immobilized surface 1using Alexa2Flour 647 conjugatedFigureSingle-molecule imaging

HMD

10 -10

10

10 -10ADA

5 µm (a-f). -12
10bar:-13-10108

10-5-10-6

no fluorescence34

10-100ON/OFF switching107-108

bleachADA guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames apart). Scale

Figure S36| Single-molecule imaging of unmodified BSA immobilized surface using Alexa Flour 647

Figure S38| Single-molecule imaging of unmodified BSA immobilized surface using Alexa Flour 647

conjugated HMD guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200 frames

conjugated propylamine guest (1 nM). Six frames (a-f) correspond to the images at 1 min interval (i.e. 1200
Sasmal, R.; Das Saha, N.;
Schueder, F.; Joshi, D.; Sheeba, V.; Jungmann, R.; Agas<,
S. S. Chem. Commun. 2019, 55 (96), 14430–14433.
apart). Scale bar: 5 µm (a-f).
frames apart). Scale bar: 5 µm (a-f).

fixed cell

2-c. Super-resolution imaging: in fixed cellCB[7]-HMD

42

+H N
2

+H N
2

CB[7]
NH3+

NH3+

+H N
2

target: microtubule

ChemComm

NH3+

Fig

Sasmal, R.; Das Saha, N.; Schueder, F.; Joshi, D.; Sheeba, V.; Jungmann, R.; Agasti, S. S. Chem. Commun. 2019, 55 (96), 14430–14433.

cence signal. This indicated that not every cell is also used cells that were expressing eGFP without a FLAG
43
cted.
Pleasingly, when
the cellsimaging:were treatedwith applications
Cy3-CB observed negligible fixed
ﬂuorescencefrom Cy3-CB[7cell signals
CB[7]-AdA
Cucurbituril
for in-cellOtherly the FLAG-eGFP expressed cells showed good overlap of plementary Figure 7), which indicated that no Cy3-CB
orescence signals from eGFP with that from Cy3-CB[7]. present due to the absence of AdA in the cells. Thi
on-transfected cells rarely showed any signal from Cy3-CB supports that the speciﬁc adamantylation in Fig. 4 was fa
ce FLAG-eGFP was not present. This result suggested that by the FLAG-tag-based immunospeciﬁcally anchored
HO

H2O2

HO

OH
AdA

AdA

AdA

OH

AdA

AdA

AdA

AdA

HRP

AdA

O Proximity-dependent
adamantylation

Cy3-CB[7]

Flag
eGFP

AdA

In cytoplasm
FLAG-tag

BF

eGFP

Anti-FLAG
primary antibody

HRP-conjugatedsecondary antibody

H2 CI–
N
+

O
O

DAPI

Cy3-CB[7]AdA-phenol

OH

O

OH

N
H

Merge

Visualization of intracellular proteins. Schematic illustration for adamantylation on cytoplasmic proteins using immunospeciﬁcally introducednd ﬂuorescence microscopy images of the ﬁxed COS7 cells which have FLAG-eGFP expressed in cytoplasm. Scale bar = 20 μm

AdA

OH

Cy3-CB[7]Kim, K. L.; Sung, G.; Sim, J.; Murray, J.; Li, M.; Lee, A.; Shrinidhi, A.; Park, K. M.; Kim, K. Nat. Commun. 2018, 9 (1).

Cucurbituril for in-cell imaging: Other applications
emistry

fixed cellCB[7]-AdA

44

exed labeling. (A) Schematic showing the use Sasmal,of CB[7]−ADA
andorthogonallabeling
platforms for
R.; Das Saha,
N.;TCO−Tz
et al. Anal. Chem.
2018, 90(19), 11305–11314.

Visualization of intracellular proteins. Schematic illustration for adamantylation on cytoplasmic proteins using immunospeciﬁcally introduced H
fixed cellCB[7]-AdA 45
Cucurbituril
for in-cell
imaging:
applications
andﬂuorescence microscopy
imagesof the ﬁxed Other
COS7 cells
which have FLAG-eGFP expressed in cytoplasm. Scale bar = 20 μm

AdA
H2 Cl–
N+

Anti-FLAG
HRP-conjugatedprimary antibody secondary antibody

IIO

Cy3-CB[7]

OH

O

O

OH

N
H

FLAG-tag

Nucleus

1

DAPI

2
FLAG-H3in the nucleus

H2O2

AdA

OH
OH

OH

H2O2
AdA

HO

OH

Protein
AdA

O

Merge
Proximity-dependant
Adamantylation
AdA

AdA

CB[7]
AdA

Cy3

Nucleus

AdA

AdA

Cy3-CB[7]AdA

Kim, K. L.; Sung, G.; Sim, J.; Murray, J.; Li, M.; Lee, A.; Shrinidhi, A.; Park, K. M.; Kim, K. Nat. Commun. 2018, 9 (1).

N

N

AdA

N

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04161-4
AdA

S

N

AdA

O

N
H2 Cl–

O

O

O

N

H2N

eGFP
O

H
N

Cy3-CB[7]

eGFP

BF

Merge

S

SH

Merge

Cy3-CB[7]

EGFR

N
N
H

AdA

N
H

BF

Cytoplasm

46

O

S

SNAP

CB[7]-AdA

N

N

AdA
Cy3-CB[7]ARTICLE

Selective
adamantylation on EGFR

S
H2N

N

living cell

Cy3-CB[7]eGFPCucurbituril for in-cell imaging:
Other applications
N
H

O

N

N
H

N
N
H

O

Cytoplasma target speciﬁc protein. Schematic
illustration of adamantylation on r-EGFR using SNAP-tag
method.
Confocal laser scanningN
AdA-BG
H N
NO
H in the plasma membrane. Scale bar = 20 μm
COS7 cells which have SNAP and eGFP-tagged recombinant EGFRO expressed
N
N
N

H2N

N

AdA

N
H

O

2

N

N
H2 Cl–

N
H

N
H

O

O

O

g to the immunospeciﬁclabeling of oftheV5- speciﬁcFig. 7 Visualization
a targetof adamantylation
on r-EGFR using SNAP-tag method. Confocal laser scanninga protein. Schematic illustrationO
H ClOe signal appeared asmicroscopy
a punctuatedN
OH
images structureof COS7 cells which have SNAP and eGFP-tagged
recombinant
EGFR
expressed in the plasma membrane. Scale bar = 20 μm
NO
HOeristic structural feature of mitochondria,
AdA-COOH-V5-APEX2 is well expressed and located insignalcorresponding
totheimmunospeciﬁclabelingofthe V5- M9 buffer
We also observed the signal corresponding to
EDC, Sulfo-NHS,
a
O
H Cl
O
epitope
(Fig.
6).
The
signalappearedasapunctuatedstructureNOH
nd that the signal was well-overlapped with
N
O
H
O
which
characteristic
rescence signal of V5(Fig.is6).a These
results structural feature of mitochondria,
AdA-COOH
suggesting
thatproteinsmito-V5-APEX2
well expressed and located in
C.iselegans
y restricted adamantylationof theinthe target organelle. We also observed the signal corresponding to
EDC,
CB[7]
Cy3Sulfo-NHS, M9 bufferr organelle in living cells can be performed
Cy3-CB[7] and found that the bsignal was well-overlapped with
X enzyme and the adamantylated proteinsthat of immunoﬂuorescence signal of V5 (Fig. 6). These results
isualized with Cy3-CB[7]ultrastable
C. elegans
showedvia
thatthespatially
restricted adamantylation of the proteins inCB[7]ons between CB[7] andAdA.
Inaddition,we
Cy3
a speciﬁc subcellular organellein living cells can be performedbs required at leastusing
25 μM
of
AdA-phenol
mito-V5-APEX enzyme and the adamantylated proteins
ient enzyme-assisted
can proximity-dependent
be speciﬁcally visualized with Cy3-CB[7] via the ultrastabletion to visualize thetarget interactions
proteins withhost–guest
between CB[7] and AdA. In addition, wer, 250 μM of AdA-phenol
seemedbefound that the cellsto required
at least 25 μM of AdA-phenol
nditions that we testedin
this
study.
treatment for efﬁcient enzyme-assisted proximity-dependent
protein adamantylation to visualize the target proteins with
Cy3-CB[7];however, the250 μM of AdA-phenol seemed to berget-speciﬁc proteins.
To understand
optimumfortheconditions
weVisualization
tested in thisteins involved in cellular functions, visuali- that
Fig. 8of astudy.
simple animal model (C. elegans). a Schematic
Kim,
Sung,
G.;inSim,J.; Murray,
J.; Li, M.;
Lee,
A.;andShrinidhi,
A.;microscopy
Park, K. M.; Kim, K. Nat. Commun. 2018, 9 (1).
oteins in living cells
is ofK.aL.;
greatinterest
illustrationof adamantylation
on C.
elegansb ﬂuorescence
2

–

2

–

Recognition of N-terminal Phe of insulin by CB[7]47Webber, M. J.; Appel, E. A.; Vinciguerra, B.; Cortinas, A. B.; Thapa, L. S.; Jhunjhunwala, S.; Isaacs,
L.; Langer, R.; Anderson, D. G. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (50), 14189–14194.