synthesis of mono-functionalized cucurbit[n]urils and

Synthesis of Mono-Functionalized

Cucurbit[n]urils and Exploration of their

Applications

by

Shuai Zhang

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Chemistry

Approved Dissertation Committee

Prof. Dr. Werner M. Nau, Jacobs University Bremen

Prof. Dr. Mathias Winterhalter, Jacobs University Bremen

Prof. Dr. Uwe Pischel, University of Huelva

Dr. Andreas Hennig, Jacobs University Bremen

Date of Defense: 26th June, 2019 9

Department of Life Sciences and Chemistry

To my dear Grandma

i

Table of Contents Abstract .................................................................................................................................... iii

Acknowledgements ................................................................................................................... v

List of Publications ................................................................................................................. vii

Participation in Scientific Conferences ................................................................................. ix

Chapter 1. Introduction ........................................................................................................... 1

1.1 Supramolecular Chemistry ............................................................................................... 3

1.2 Supramolecular Chemistry of Cucurbit[n]urils ................................................................ 4

1.2.1 Basic Properties of Cucurbit[n]urils .......................................................................... 5

1.2.2 Specific Host–Guest Binding of Cucurbit[n]urils ...................................................... 8

1.3 Functionalization of CBn and Their Applications .......................................................... 10

1.4 Cu (I)-Catalyzed Azide-Alkyne Cycloaddition .............................................................. 12

1.5 Förster Resonance Energy Transfer (FRET) .................................................................. 14

1.6 References ...................................................................................................................... 16

Chapter 2. Synthesis of Mono-Functionalized CB6 and CB7 ............................................ 25

2.1 Introduction .................................................................................................................... 27

2.2 Synthesis and Separation of CBn ................................................................................... 28

2.3 Synthesis of CB6-OH and CB7-OH ............................................................................... 29

2.4 Synthesis of CB6-OPr and CB7-OPr .............................................................................. 31

2.5 Synthesis of CB6-Carboxyfluorescein (CB6-CF) .......................................................... 34

2.6 Designed FRET Pair Based on CB6-CF and R-S ........................................................... 35

2.6.1 Spectral Characterization of the Designed FRET pair ............................................. 37

2.6.2 Spectral Properties of the Designed FRET Pair ....................................................... 37

2.6.3 Quenching Mechanism ............................................................................................ 39

2.7 Further Synthesis of Mono-Functionalized CB7 ............................................................ 40

2.8 References ...................................................................................................................... 45

Chapter 3. Ratiometric DNA Sensing with a Host-Guest FRET Pair ............................... 47

3.1 Introduction .................................................................................................................... 49

3.2 Synthesis of CB7-CF ...................................................................................................... 50

3.3 Optical Characterizations of FRET Pair ......................................................................... 51

3.4 DNA Sensing by FRET Pair ........................................................................................... 56

3.5 Conclusions .................................................................................................................... 58

3.6 References ...................................................................................................................... 58

Chapter 4. Precise Supramolecular Control of Surface Coverage Densities on Polymer

Micro- and Nanoparticles ...................................................................................................... 63

ii

4.1 Introduction .................................................................................................................... 65

4.2 Particle Synthesis ............................................................................................................ 67

4.3 Quantification of Surface-Bound CB7 ........................................................................... 68

4.4 Supramolecular Surface Functionalization ..................................................................... 72

4.5 Supramolecular Control of Surface Coverage Densities ................................................ 76

4.6 Conclusions .................................................................................................................... 80

4.7 References ...................................................................................................................... 80

Summary and Outlook ........................................................................................................... 87

Appendices .............................................................................................................................. 89

Supporting Information for Chapter 3 .................................................................................. 91

Supporting Information for Chapter 4 .................................................................................. 99

Curriculum Vitae ................................................................................................................. 121

iii

Abstract

The present doctoral thesis describes the synthetic procedure of clickable mono-functionalized

cucurbit[n]urils (n = 6, 7) and mainly on the exploration of new applications based on the mono-

functionalized CBn. In principle, with the functionalized clickable group, various

functionalized CBn derivatives can be achieved. One explored application is based on the

functionalization of CB7 on the surface of nano-/macro-particles, thus can be applied to

quantify the surface coverage densities of particles. The other one is based on a chromophore

attached to CB7 which makes the host molecule fluorescent and enables it to form a host-guest

FRET pair with a corresponding fluorescent guest, which can be applied to DNA sensing.

Besides these, the binding constants between CBn and inorganic cations were systematically

studied.

The first part of the thesis focuses on the synthesis and characterizations of mono-

functionalized CB6 and CB7, including mono-hydroxylated CB6 or CB7, propargyl attached

CB6 or CB7, and fluorophore attached CB6 or CB7, and related compounds.

The second part of the thesis reports a host-guest FRET pair based on the macrocyclic

host CB7 labelled with carboxyfluorescein as acceptor and the nucleic stain DAPI as donor and

guest. This supramolecular FRET pair is to be used for quantitative sensing of DNA with an

excellent linear dependence of the ratiometric fluorescence intensities. Such approach can be

applied to quantify DNA accurately and potentially be used in real-time PCR.

The third part of the thesis demonstrates a strategic supramolecular application to

precisely control the coverage densities on the surface of nano-/macro-particles. The key is to

functionalize CB7 on the surface of particles. After that, incubation of CB7-functionalized

particles with two high-affinity guests, resulted in a simple linear relationship between surface

coverage densities of one fluorescent guest and the mole fraction of this guest in the incubation

mixture. This suggests a highly modular supramolecular strategy for the stable immobilization

of application-relevant molecules on particle surfaces and a precise control of their surface

coverage densities.

In the last part, I summarize the main projects during my PhD and give the outlook about

exploring more applications based on mono-functionalized CB7.

v

Acknowledgements

First of all, I would like to express my great appreciation to my supervisors, Prof. Dr. Werner

Nau and Dr. Andreas Hennig, for their patient guidance, thoughtful discussions, and

enthusiastic encouragement. I am thankful to Prof. Dr. Werner Nau, who gave me a chance to

study in supramolecular chemistry, provided me with scientific insights throughout my doctoral

studies, and encouraged me not to give up when I felt very frustrated in my second year. His

positive attitude motivates me not only in research work but also in my life outside the lab. And

I am thankful to Dr. Andreas Hennig, who led me into my beloved research topic-material

science. Without his continuous support and supervision, I would not have been able to finish

the most interesting project during my PhD.

I would also like to thank my former and present colleagues I worked with during my

four-year study in the Nau group for providing a very harmonious and knowledgeable

environment. In particular, I am thankful to Ms. Yan-cen Liu for helpful discussions about

fundamental knowledge of supramolecular chemistry and all the fun during our many trips in

Germany. I thank Dr. Khaleel I. Assaf for answering my questions all the time with patience

and a smile, for his guidance on NMR analysis and his encouragement from time to time. I

thank Dr. Chusen Huang for his guidance with cell culture experiments and fruitful suggestions

in one of my research projects. I thank Dr. Maik Jacob for his help to calculate the Förster

distance. I thank Dr. Andrea Barba-Bon for the help in running the first column in my life and

the beautiful memory of our Berlin trip. I thank Dr. Suhang He, Mr. Mohammad Ata Alnajjar,

Mr. Nilam Mohamed, and Ms. Yao Chen for sharing with me a pleasant time in the lab. Also,

I thank Dipl.-Chem. Thomas Schwarzlose for his encouragement and help. I thank Dr. Frank

Biedermann, Dr. Haibo Zhang, and Dr. Xiaojuan Wang for the shared projects.

I thank Prof. Mathias Winterhalter and Prof. Dr. Uwe Pischel for being kind enough to

act as committee members of my doctoral defence.

I am thankful to the China Scholarship Council (CSC) for the financial support of my

4-year doctoral study. Also, I am thankful to the Deutsche Forschungsgemeinschaft (DFG) for

the financial support for the attendance of the 5th ICCB conference in 2017 and the SupraChem

conference in 2019.

My special thanks are extended to my grandmother, my parents, and my brother. I thank

them for always being there for me, both financially and emotionally, which gives me courage

and confidence to live abroad for such a long time.

vi

Last but not the least, I would like to thank my dearest husband, Xiaofei, for his

everlasting love, perpetual understanding, constant encouragement, and endless support.

vii

List of Publications

1. Shuai Zhang, Khaleel I. Assaf, Chusen Huang, Andreas Hennig, and Werner M. Nau.

“Ratiometric DNA sensing with a host–guest FRET pair.” Chemical Communications, 2019,

55, 671-674.

2. Huang, Haihong, Baosheng Ge, Shuai Zhang, Jiqiang Li, Chenghao Sun, Tongtao Yue, and

Fang Huang. “Using Fluorescence Quenching Titration to Determine the Orientation of a

Model Transmembrane Protein in Mimic Membranes.” Materials, 2019, 12, 349.

3. Huang, Haihong, Baosheng Ge, Chenghao Sun, Shuai Zhang, and Fang Huang. “Membrane

Curvature affects the Stability and Folding Kinetics of Bacteriorhodopsin.” Process

Biochemistry, 2019, 76, 111-117.

4. Shuai Zhang, Zoe Domínguez, Khaleel I. Assaf, Mohamed Nilam, Thomas Thiele, Uwe

Pischel, Uwe Schedler, Werner M. Nau, and Andreas Hennig. “Precise supramolecular control

of surface coverage densities on polymer micro-and nanoparticles.” Chemical Science, 2018,

9, 8575-8581.

5. Wang, Wenjing, Xiaoqiang Wang, Jin Cao, Jun Liu, Bin Qi, Xiaohai Zhou, Shuai Zhang,

Detlef Gabel, Werner M. Nau, Khaleel I. Assaf, and Haibo Zhang. “The Chaotropic Effect as

an Orthogonal Assembly Motif for Multi-responsive Dodecaborate-cucurbituril

Supramolecular Networks.” Chemical Communications, 2018, 54, 2098-2101.

6. Qi, Bin, Chenchen Wu, Ling Xu, Wenjing Wang, Jin Cao, Jun Liu, Shuai Zhang, Detlef

Gabel, Haibo Zhang, and Xiaohai Zhou. “From Boron Clusters to Gold Clusters: New Label-

free Colorimetric Sensors.” Chemical Communications, 2017, 53, 11790-11793.

7. Wang, Xiaojuan, Yanan Wang, Hua He, Xiqi Ma, Qi Chen, Shuai Zhang, Baosheng Ge,

Shengjie Wang, Werner M. Nau, and Fang Huang. “Deep-red Fluorescent Gold Nanoclusters

for Nucleoli Staining: Real-time Monitoring of the Nucleolar Dynamics in Reverse

Transformation of Malignant cells.” ACS Applied Materials & Interfaces, 2017, 9, 17799-

17806.

8. Yue, Tongtao, Mingbin Sun, Shuai Zhang, Hao Ren, Baosheng Ge, and Fang Huang. “How

transmembrane peptides insert and orientate in biomembranes: a combined experimental and

simulation study.” Physical Chemistry Chemical Physics, 2016, 18, 17483-17494.

viii

9. Norouzy, Amir, Khaleel I. Assaf, Shuai Zhang, Maik H. Jacob, and Werner M. Nau.

“Coulomb Repulsion in Short Polypeptides.” The Journal of Physical Chemistry B, 2014, 119,

33-43.

10. Ge, Baosheng, Yan Li, Haixiang Sun, Shuai Zhang, Peijie Hu, Song Qin, and Fang Huang.

“Combinational Biosynthesis of Phycocyanobilin using Genetically-engineered Escherichia

Coli.” Biotechnology Letters, 2013, 35, 689-693.

Manuscript in Preparation

Shuai Zhang, Laura Grimm, Frank Biedermann and Werner M. Nau. “Systematic Investigation

on Binding Affinities of Cucurbit[n]uril with Common Cations.” Manuscript in preparation.

ix

Participation in Scientific Conferences

1. 02/2019 SupraChem 2019, Würzburg, Germany. Poster entitled: “Ratiometric DNA

sensing with a host–guest FRET pair”.

2. 06/2016 5th International Conference on Cucurbiturils (ICCB), Brno, Czech Republic.

Poster entitled “Cucurbit[7]uril-Functionalized Polymer Microparticles”.

Chapter 1 Introduction

1

Chapter 1. Introduction


2


3

1.1 Supramolecular Chemistry

Supramolecular chemistry, which is one of the today’s most interested research fields in

chemistry, was introduced by Jean-Marie Lehn in 1978 for the definition, consolidation and

generalization of the domain of crown ether chemistry, the chemistry of molecular recognition,

and host-guest chemistry. In 1967, Charles Pederson had investigated the selective binding of

crown ethers to specific metals and cation transfer in biphasic solvent.1 This accidental

discovery paved the way for Donald J. Cram and Jean-Marie Lehn to express their creativity in

unique ways and wholly develop the field of supramolecular chemistry.2-4 They were jointly

awarded the 1987 Nobel Prize in chemistry for a recognition of crown ether, host-guest and

molecular recognition chemistries.

Supramolecular chemistry has been described as “chemistry beyond the molecule”.

Over the past 50 years, this field has grown into a major branch of chemistry and has promoted

innumerable developments: from fundamental knowledge to practical applications, from

noncovalent interactions to drug delivery, and from polymer materials to solid-state

engineering. As a result, it has led to the appearance and establishment of supramolecular

science and technology, as a wide range of multidisciplinary and interdisciplinary field that

provides highly fertile soil for scientists’ creativity in all disciplines.

The importance of supramolecular chemistry was again established by the 2016 Nobel

Prize for Chemistry, which was awarded to Jean-Pierre Sauvage, Sir J. Fraser Stoddart, and

Bernard L. Feringa in recognition of their work in molecular machines, in which

supramolecular chemistry was basic in showing the noncovalent bond into visibility.

Particularly, they used supramolecular chemistry as a kit to create mechanical power, promoting

molecules that can operate as machines, including molecular knots,5, 6 molecular elevator,7, 8

molecular motor9, 10 and nanocar.11

Until now, numerous families of supramolecular host molecules (natural and synthetic)

have been reported and investigated. Cucurbit[n]urils (CBn) as one of most young macrocycles

is very much related to my research during my 4-year PhD study. Therefore, in Chapter 1 I will

focus on the CBn properties, functionalizations and applications, as well as the mechanisms of

Cu(I)–catalyzed azide-alkyne cycloaddition related to CBn functionalizations and the principle

of förster resonance energy transfer associated with CBn applications.


4

1.2 Supramolecular Chemistry of Cucurbit[n]urils

Cucurbit[n]urils (CBn, n = 5-8, 10, 13-15) are cyclic oligomers composed of different glycoluril

units connected by methylene groups (Figure 1.1), which represent as a remarkable water

soluble molecular hosts. CB6 was the first one to be synthesized in 1905 by Behrend,12 but its

structure was not shown until 1981.13 Shortly after 2000 CBn family was expanded to have

more species, such as CB5, CB7, CB8 and CB10, by Kim,14, 15 Day16, 17 and Isaacs.18-20

Recently, CB13, CB14 and CB15 which show twisted crystal structures (Figure 1.2) have been

discovered by Tao.21, 22 In the recent two decades, although applications of large CBn are very

limited, CBn (n = 5-8) have been well explored in fundamental and applied science, including

formation of supramolecular hydrogels,23-25 supramolecular tandem enzyme assays,26-29

catalysis,30-32 biological molecules recognition,33-36 and materials science.37-41

Figure 1.1 Chemical structures of CB5, CB6, CB7 and CB8.

Figure 1.2 X-ray crystal structure of twisted CB14, hydrogen atoms are hidden for clarity.22


5

1.2.1 Basic Properties of Cucurbit[n]urils

CBn are barrel-shaped macrocycles with highly symmetric structure, negatively charged

carbonyl portal and hydrophobic cavity. Like the cyclodextrins, the CBn homologues have the

same depth (9.1 Å), but their portal diameter, inner cavity diameter and cavity volume vary

systematically with the number of united glycoluril units (Figure 1.3 and 1.4). The CBn

structures are rather rigid, so the suitable guests for CBn to form host-guest complex are size

selective. As shown in Table 1.1, the thermal stability of CBn is high enough for CBn to keep

complete structure under 370 °C. The solubility of CBn varies on sizes, CB5 and CB7 show

modest solubility in water, but CB6 and CB8 are slightly soluble in water. Therefore, the poor

solubility of CB6 and CB8 is one of the limitations of the CBn family to be explored for some

applications in aqueous solution.

Figure 1.3 Model of the strict CBn macrocycles.

Table 1.1 Physical properties of CBn (n = 5-8).

CBn p (Å) d (Å) h (Å) V(Å3) s (mM) Stability (°C)

CB5 2.4 4.4 9.1 68 20-30 420

CB6 3.9 5.8 9.1 142 0.01842 42543

CB7 5.4 7.3 9.1 242 20-30 370

CB8 6.9 8.8 9.1 367 0.01 420

p: portal diameter; d: inner cavity diameter; h: the height; V: cavity volume;

s: water solubility; Values are taken from ref.15, 42, 43.

Not only the size of portal decides what kind of guest fits with CBn, but also the packing

coefficient (PC). PC representing the ratio of the guest size and the host cavity volume, as one

of testing standards to estimate the fit goodness of host-guest complex, was introduced by

Rebek and Mecozzi in 1998.44 A value of ca. 0.55 was reported to get the best binding between

host and guest molecules, while larger or smaller PC value accompanied by lower affinities.

PC value has been proven useful for a couple of macrocyclic hosts, as well as CBn, which was

reported by Nau group to be applied for predicting the stability of CBn complex successfully.45


6

Figure 1.4 (a) Top and side views of different cavity definitions. (b) Schematic illustration of

an ideal cavity filling of 55% by encapsulation of a spherical guest. Reprinted by permission

from ref.46.

The CBn host-guest chemistry is obviously and intuitively related with dimensions of

CBn structures, also with other driving forces, such as electrostatic effect and hydrophobic

effect. Electrostatic effect plays an important role in biochemical molecular recognitions47, as

well as in supramolecular chemistry.48 Figuer 1.5 shows the calculated electrostatic potential of

CBn (n = 5-8), which spotlights the preference of the positive charge associated and the

reluctance of the anions with the portals of CBn.19, 49

Indeed, the electrostatic potential has a significant influence for the recognition behavior

of CBn. The first reports of CBn complex was formed with metal cations being as a lid bound

to the portals of CBn.50 The common cations (inclusive of alkali and alkaline earth metal

cations,50-54 transition metal cations,55, 56 lanthanides and actinides,57-59 as well as ammonium

ions60) have been proven to bind with CBn with varying affinities and increase the CBn

solubility significantly.


7

Figure 1.5 Calculated electrostatic potential (EP) at the B3LYP/6-31G* level of theory for (a)

CB5, (b) CB6, (c) CB7, and (d) CB8 in the h plane (left) and in the v plane (right). Reprinted

by permission from ref. 61.

Figure 1.6 Schematic illustration of the release of high-energy water molecules from the CB7 cavity

upon binding of a hydrophobic guest. Reprinted by permission from ref. 46.

The advantage of applying CBn into molecular recognition is that the formation of CBn

based complex can be conducted in aqueous solution with high binding affinity. Generally,

compared with organic solvents the presence of water decreases the host-guest binding affinity


8

as water molecules compete strongly to form hydrogen bond. However, this is not the case for

CBn, which are able to solve this problem by encapsulating high-energy water (Figure 1.6) in

the cavity.62 And the release of high-energy water accounts for a large proportion of the overall

hydrophobic effect for the complex formation. The number of high-energy water molecules

accommodated in the cavity is depending on CBn cavity volume. The molecular dynamics

simulations conducted by Biedermann shows that high host-guest binding affinity can be gained

by the removal of all water molecules. CB7 was found to have the highest energy gain compared

with other homologues, as it encapsulates more high energy water molecules than CB5 and

CB6 (more than twice), and has more suitable cavity size to host higher energy water molecules

than CB8.63

1.2.2 Specific Host–Guest Binding of Cucurbit[n]urils

CB5 is considered to be the smallest macrocycle in CBn family until now. In respect to its small

cavity volume (68 Å3), CB5 has been limited to use in the formation of numerous guest

molecules compared to the larger family members. Even with such small cavity and portal sizes,

CB5 and its derivative have found to be available for the formation of host-guest complex with

alkali and alkaline earth cations64 and ammonium cations64, as well as divalent transition metal

cations.55 Instead of these small cations, CB5 and derivatives are reported to encapsulate small

gas molecules, such as O2, N2, Ar, CO, N2O, CO2, He, H2, Ne, Kr, Ar, Xe, Rn and CH4.65, 66 In

particular the very recent study by the Nau group on CB5 binding affinities with noble gases

by replacement of ethane or methane as NMR probes in water is attracting attention.66 In this

study, the contributions to host–guest binding was identified and a conclusion was drawn that

the binding process is driven by differential cavitation energies rather than dispersion

interactions. Their discovery show that the cavitation energy drives the noble gas into CB5

cavity, which supplies have an impact on the improvement of gas storage materials and the

understanding of biological receptors.

CB6 is the most abundant homologue from the synthesis of CBn, with a moderate

volume of 142 Å3, which is well known to complex with aliphatic amines. Alkylammonium

and alkyldiammonium ions were firstly studied to bind with CB6 by Mock in aqueous formic

acid, with the determination of their binding constants.67 It can be well explained by the strong

interactions between the negative carbonyl rim and the positively charged protonated amines.

With further investigations Mock and coworkers found that aliphatic amines bind with CB6 has

structure selectivity.68, 69 Consequently, CB6 shows a chain length dependent selectivity with


9

strongest binding affinities towards pentano- and hexano-bridged diammonium ions. Moreover,

the binding affinity of CB6 with imidazolium-based ions have also been studied broadly,70-72

and applied to make monofunctionalized-CB673 as well as to separate the CBn homologues.74

In addition, the small cavity of CB6 limits to include fluorescent moiety into the cavity, while

CB6 is successfully used for fluorescence sensor designs with alkyldiammonium-dye as smartly

synthesized guest to explore applications for indicator displacement assays.75-77

Compared with CB6, CB7 with the advantages of relative large cavity volume and better

water solubility, has reported to be the most popular host in CBn family.49 Compared with other

host, CB7 can recognize and host with a wider scope of guests with high binding affinities. On

account of the special properties of CB7, a couple of fluorescent dyes such as acridine orange,78

berberine chloride79 and DNA stain dye DAPI,80 are shown to form stable complex with CB7

with moderate binding constants of ca. 106, which makes CB7 favoured in many indicator

displacement strategies. What is more, CB7 is reported to bind with diamantane diammonium

with a binding constant up to 1017 M1,81 which goes over the biotin-(strept)avidin pair as the

strongest non-covalent interaction found in nature. More three dimensional compounds like

other adamantane derivatives and ferrocene also form very stable complex with CB7 (1012 -

1015 M1). The ultra-high binding of CB7-guest pair implicates that this system can be used in

some applications where biotin-(strept)avidin pair plays a role, which makes the transition from

fundamental research into practical applications.82

CB8 has a cavity volume of 367 Å3 which is 1.5 times larger than that of CB7, while

CB8 has the similar binding properties with smaller CBn homologues. CB8 also shows

relatively strong binding with adamantane and ferrocene derivatives.83 With a large cavity

volume, CB8 can also bind with large guests, for example cyclen and cyclam, to form “a

macrocycle inside macrocycle” complex.84 As well as fullerene which can bind with CB8

portals, with each portal sitting one fullerene molecule.85 Very interestingly, alkylammonium

ions armed with long aliphatic chains are reported to form U-shape within CB8 cavity.86-88

Moreover, nitroxide derivatives have also been proven to form complex with CB8.89, 90 One of

the unique properties of CB8 is the ability to encapsulate two guests, and the two guests can be

the same91 or two different molecules.92-94 This unique trait makes CB8 applicable in many

fields such as sensing,95, 96 catalysis,40, 97, 98 and supramolecular polymeric materials.99-102


10

1.3 Functionalization of CBn and Their Applications

Acting as cyclodextrin macrocycles, the functionalization expanded their applications in

numerous varying areas, the development and prosperity of CBs family also demand

functionalization. The initial motivation to modify CBs may be ascribed to the poor solubility

of CB6 and CB8 in common solvents, which limited this host family to explore more practical

applications. The first synthesized functionalized CBs was Me10CB5 derived from

dimethylglycouril by Stoddart and co-workers in 1992.103 Since then a wide range of fully or

partially alkyl-modified CBs derivatives are reported, and the derivatives are coming from

various sources, including dimethylglycoluril,104, 105 cyclohexanoglycoluril,106, 107

diphenylglycoluril,108 and cyclopentanoglycoluril.109, 110 In 2011, Isaacs and coworkers

reported the p-xylylenediammonium ion as a template to synthesize methylene-bridged

glycoluril hexamer.111 This hexamer is available to further react with substituted

phthalaldehydes to achieve monofunctionalized CB6 derivatives (Figure 1.7). In addition, they

specially designed a “clickable” group onto the skeleton of CB6 derivative which provides a

convenient way for introducing further functionality.112 Furthermore, modified CB6 with a

fluorophore (naphthalene group) were applied by Isaacs to detect cancer-associated

nitrosamines and basic amino acids as supramolecular sensors.113, 114 This principle can also be

used to obtain CB7 derivatives by reacting the hexamer with modified glycoluril. With the

advantage of the special properties of CB7, Isaacs and co-workers used functionalized CB7 for

targeting delivery drug molecule to cancer cells,115 forming vesicle-type assemblies,116

endowing biopharmaceuticals with PEGylation,117 releasing drug molecules,118 as well as

sensing and imaging the cells.119


11

Figure 1.7 Functionalized CB6 and CB7 derivatives from Isaacs’s group.

Generally the alkyl-modified CBs derivatives can improve the solubility of host molecules in

common solvents, but they are synthesized indirectly and difficult to be functionalized further.

Therefore, direct functionalization of CBs would be appealing. Kim and co-workers achieved

a breakthrough in this field to obtain fully-hydroxylated CBn (n = 5-8) by direct oxidation of

CBs in the presence of K2S2O8 in 2003.120 Through this method CB5 and CB6 derivatives can

be reacted very efficiently with yields of 42% and 45%, however, for CB7 and CB8, the yields

are lower than 5%. This functionalization creates a platform for further modifications and

countless potential applications, including CBs-based supramolecular polymers,121-123

fluorescent capsules,124, 125 drug delivery,126-128 enrichment of proteins,37, 129 and biocompatible

supramolecular hydrogels.130 In 2012, Scherman and co-workers separated monohydroxylated

CB6 through the oxidation of CB6 in the presence of bisimidazolium salts, and further

functionalization with reactive alkenyl and alkynyl was also reported. In 2013, Kim and co-

workers improved their previous method and separated monohydroxylated CB7 and they

successfully applied monohydroxylated CB7 to form supramolecular velcro for reversible

underwater adhesion. Two years later, Kim group conjugated a fluorophore Cy3 with CB7

(CB7-Cy3) from monohydroxylated CB7.131 The conjugation was proven to form fluorescence

resonance energy transfer (FRET) pair with adamantylamine-Cy5, and this high affinity host-

guest FRET pair was used for single vesicle content mixing assay. The ultra-high affinity

between CB7-Cy3 and adamantane or ferrocene derivatives makes this dye conjugated CB7

applicable for protein imaging in cell culture.132 Very recently, CB7 functionalized with


12

alkenyl133 and alkynyl134 were reported by following Kim’s method, applying to form

supramolecular assembly and for drug encapsulation, respectively.

Figure 1.8 Synthesis of monohydroxylated-CBn (n = 5-8).

In 2015, Bardelang and Ouari described a photochemical method to functionalize CBn

(n = 5-8) with single alcohol in high conversions by using H2O2 and UV light (Figure 1.8).135

This method was considered to be the milestone of functionalized CBn with respect to easy

operation and excellent yields. Following this procedure, Nau and Hennig synthesized one CB7

derivative with propargyl conjugated which is clickable for further reactions. They

functionalized CB7 on the surface of macro- and nanoparticles to achieve precise control of

surface coverage densities on polymers.41 They also attached a fluorescent dye with CB7 (CB7-

CF) and a FRET pair was made between CB7-CF and DNA stain dye DAPI. The special

properties of host-guest interactions motivated them to apply this system into sensing DNA and

explore potential application in real time PCR.36

1.4 Cu (I)-Catalyzed Azide-Alkyne Cycloaddition

The Cu (I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) reaction, often referred to as

click chemistry, was firstly reported by Tornøe and Meldal for the synthesis of solid state

peptide in 2001.136 Subsequently, the CuAAC effect was described by the groups of Meldal and

Sharpless with two independent publications in 2002.137, 138 Since then this reaction has received

considerable attention among researchers from different disciplines in the fact that the reaction

is insensitive to harsh conditions and quantitative to couple azido and ethynyl molecules

through the formation of chemical stable 1,2,3-triazole. Additionally, the triazole formed is

chemically inert to usual reactive conditions, including reduction, oxidation and hydrolysis.139

As a result, CuAAC reaction is proved to be one of the most straightforward ways to connect


13

two molecules and has been applied in numerous research areas, including biochemistry,

materials science and medical science.140

Figure 1.9 Examples of efficient N-donor ligands for CuAAC reations. Donor atoms for copper

bonding are in red.

In principle, a CuAAC reaction only needs three components, a terminal azide, a

terminal alkyne and a Cu catalyst, and it is worth noting that any source of copper can be

accepted as a precatalyst for versatile CuAAC reaction. All other reagents (such as ligands,

solvents, and base) and reactions conditions (temperature, N2 atmosphere) are elective. Among

these, ligands (Figure 1.9) which can coordinated with Cu have been proven to accelerate the

reaction rate and protect the Cu catalyst from oxidation.140, 141 Polydentate N-donor chelators

are popular ligands used in CuAAC reactions, since the auxiliary nitrogen donors has strong

electron donating effect which make it easier for Cu to coordinate thus to be more reactive than

those with oxygen or sulfur donors.


14

Figure 1.10 a) the mononuclear mechanism of CuAAC reaction. b) The dinuclear mechanism

of CuAAC, X is a bridging ligand.

Since CuAAC reaction is used currently in numerous fields, to understand the intrinsic

mechanism of this reaction becomes more and more urgent. Tedious work has been performed

to demonstrate the mechanism of CuAAC reaction, not only experimentally142-146 but also

theoretically.147-151 Sharpless and co-workers firstly proposed a mononuclear mechanism which

started with ethynyl copper followed by the subsequent formation of three intermediates (Figure

1.10 a). Following experiments found that the mononuclear mechanism questionable and more

than one copper ion involved in this reaction, which was further proved by density functional

theory (DFT) investigations. One remarkable experiment was conducted by Fokin and co-

workers applying isotopic labelling in CuAAC reaction, through this method bis-copper-

intermediates (binuclear) were observed by electrospray ionization mass spectrometric

technique.145 Plus the outstanding work from other groups, the dinuclear mechanism of CuAAC

reaction is increasingly accepted.143, 146, 152 In the dinuclear mechanism, the second copper was

involved to form the metallacycle structure which could alleviate the ring strain and lower the

activation barrier (Figure 1.10 b).

1.5 Förster Resonance Energy Transfer (FRET)

Förster resonance energy transfer, also known as fluorescence resonance energy transfer or

resonance energy transfer, is a mechanism proposed by Theodor Förster in 1948 to describe the

energy transfer between two chromophores (referred to as a donor and an acceptor).153 FRET

is an electrodynamic happening that non-radiative energy can be transferred through long range

dipole-dipole interactions between the two chromophores in the absence of photon, therefore

this energy transfer is distance dependent. This process occurs between a donor chromophore


15

in excited state and an acceptor in ground state. The prerequisite of FRET to occur is the

fluorescence spectrum of donor molecules overlaps with the absorbance spectrum of acceptor

molecules (Figure 1.11).

500 600

0.0

0.5

1.0

0.0

0.5

1.0R-S

Flu

ore

sce

nceA

bsorb

an

ce

Wavelength (nm)

CB6-CF

Figure 1.11 Spectral overlap between the absorbance spectrum of CB6-carboxyfluorescein

(CB6-CF, donor) and the fluorescence spectrum of rhodamine-spermine (R-S, acceptor).

The theory of FRET is relatively complicated, while it is well explained by taking into account

one donor and one acceptor separated by a distance.154 With this assumption, the rate of FRET

is given by equation 1.1. QD represents the quantum yield of the donor in the absence of

acceptor. The term 2 represents the relative orientation of the donor and acceptor transition

dipoles, which is usually assumed to be 2/3. N represents Avogadro’s number. D is the donor

lifetime, n is the refractive index of the solution (typically assumed to be 1.4 for biomolecules

in aqueous solution), and r is the distance between donor and acceptor. In equation 1.1 the

transfer rate kT(r) is described as a function of r to highlight its distance dependence.

𝑘T(𝑟) = 𝑄𝐷2

D𝑟6 (9000(ln10)

1285𝑁𝑛4 ) ∫ 𝐹𝐷()𝐴()4d∞

0 (1.1)

Generally, the rate of FRET from a donor to an acceptor is given by equation 1.2. R0 is known

as Förster distance, a distance between the donor and acceptor at which the FRET efficiency is

50%. Typically, R0 is in the range of 20 to 90 Å, which are comparable to the size of biological

molecules. r is the distance between the donor and acceptor. It is obvious that the rate of FRET

is proportional to r6 with a significant dependence on the distance.

𝑘T(𝑟) =1

D(

𝑅0

𝑟)

6 (1.2)


16

J() is the spectral overlap integral, which represents the degree of spectral overlap between

the emission spectrum of donor and the absorbance spectrum of acceptor. Where FD() is the

donor emission spectrum normalized to an area of 1, A() represents the molar extinction

coefficient of the acceptor which can be obtained from acceptor absorption spectrum. As the

name suggests, is the wavelength.

𝐽() = ∫ 𝐹𝐷()𝐴()4d∞

0 (1.3)

From equation 1.1 and 1.2, equation 1.4 can be deduced as followed. This equation expresses

that the Förster distance can be calculated from donor emission spectrum and acceptor

absorption spectrum plus the quantum yield of the donor.

𝑅06 =

9000(ln10)𝑄𝐷2

1285𝑁𝑛4 ∫ 𝐹𝐷()𝐴()4d∞

0 (1.4)

Once the value of Förster distance (R0) is known, equation 1.1 can be easily transferred to

equation 1.4. The FRET efficiency (E) is given by equation 1.5. With the substitution of

equation 1.2 into 1.5, equation 1.6 can be easily derived. From this equation, one can predict

the FRET efficiency with an estimated or calculated r value.

𝐸 =𝑘T(𝑟)

D−1+ 𝑘T(𝑟)

(1.5)

𝐸 =𝑅0

6

𝑅06+𝑟6 (1.6)

The FRET efficiency can be measured by both steady state fluorescence spectroscopy and time-

resolved fluorescence spectroscopy. The calculation is given by equation 1.7, where FDA and

FD are the fluorescence intensity of the donor in the presence and absence of acceptor, DA and

D are the fluorescence lifetime of the donor in the presence and absence of acceptor.

𝐸 = 1 −𝐹DA

𝐹D= 1 −

DA

D (1.7)

With the experimental proof of FRET theory in 1967, this theory has been well explored

in countless applications, particularly in biochemistry, such as protein folding,155 medical

diagnostics, optical imaging, membrane fusion, DNA analysis and so on.

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23

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231.

Chapter 2 Synthesis of Mono-Functionalized CB6 and CB7

25

Chapter 2. Synthesis of Mono-Functionalized

CB6 and CB7


26


27

In the past decade, the supramolecular chemistry of the CBn family of molecular containers has

rapidly developed due to the exceptionally high affinity and selectivity in aqueous solution.1-7

Accordingly, unfunctionalized CBn have been used to create functional supramolecular

systems including molecular machines, materials for capture and release of volatile compounds,

supramolecular polymers, solubilizing agents for insoluble drugs, supramolecular catalysts, and

chemical sensing ensembles. In order to further extend the supramolecular chemistry of CBn it

is necessary to develop efficient synthetic methods to prepare functionalized CBn derivatives.

2.1 Introduction

A big step in this direction is accomplished by Kim group who performed the direct

perhydroxylation of cucurbituril using K2S2O8 as an oxidant to yield a series of hydroxy-

substituted (OH)2nCBn,8 which has served as a trigger for the synthesis of many other

functionalized cucurbituril.9-14 These reactions represent the first direct covalent derivatizations

of CBn. These functionalized products show various potential applications, including the

synthesis of chromatographic materials,15 formation of ion channels, construction of sensors as

well as their utility in ion separation and capture applications.16 They also show promise in

molecular devices,17 as antibacterial agents that are stable against enzymatic degradation,18 as

functional CBn-based polymers,19-23 and for the formation of CBn-based supramolecular

polymers.24 Further areas include application in drug delivery,25, 26 as fluorescent capsules,27

and as supramolecular biocompatible hydrogels.28

Scherman and coworkers isolated monohydroxylated CB6 by harnessing the host-guest

chemistry of bisimidazolium salts with CB6 with a much high solubility.9 Afterwards, Kim

group improved their method and obtained monohydroxylated CB7 in one step with a weak

point of low quantitative reaction conversion.29 Recently, Bardelang and Ouari developed a

convenient photochemical method to synthesize monohydroxylated CBn directly with

relatively high yield by using hydrogen peroxide and UV light,30 and what we are following is

this direct synthesis method.


28

2.2 Synthesis and Separation of CBn

Figure 2.1 The reaction between glycoluril and formaldehyde.

60 mL 9 M sulfuric acid is mixed with 20 mL formaldehyde (37% aqueous solution) at room

temperature and then cooled down the temperature to 2-5 C using an ice bath. Then glycoluril

is then added in small portions with vigorous stirring. The temperature is increased to 95 C in

an oil bath and the mixture is refluxed for 72 h.

200 mL distilled water and 800 mL acetone are added into the reaction solution with

vigorous stirring, in order to participate all CBn homologues. The suspension is settled down,

filtered and washed with 250 mL mixed solution of acetone and water (v/v: 4:1). The filtrate is

collected and decanted subsequently with small portion of acetone/water solution in order to

remove the concentrated acid. The precipitate is dissolved in 400 mL distilled water. CB5 and

CB7 would dissolve but not CB6 and CB8 whose solubilities in water are very low. By this

step, the mixture of CB6 and CB8 is the solid in the filter and mixture of CB5 and CB7 is in

the filtrate.

To get CB6, 10 g of the mixture of CB6 and CB8 (dry solid) is dissolved in 100 mL 3 M

HCl. Then the solution is filtrated, CB6 stays in the solution and little CB6 and CB8 are left in

the filter. The solution is evaporated to get the solid, 200 mL methanol is added to disperse the

solid again, and then the solution is filtrated following by 3 times water (3 50 mL) washing

and 3 times methanol (3 30 mL) washing.

To get CB7, a volume of 300 mL acetone is added to the filtrate, which would precipitate

mainly the CB7. Then the solid is washed a couple of times with acetone and dried under

vacuum, relatively pure CB7 can be obtained.


29

2.3 Synthesis of CB6-OH and CB7-OH

Figure 2.2 Synthesis of CB6-OH and CB7-OH.

Take CB7 as an example, CB6 follows the same procedure. 1 g (0.86 mmol) CB7 was dissolved

in 125 mL of a mixture of Millipore water and 12 M HCl (3:2 v/v) and introduced in a 250 mL

quartz glass round bottom flask under nitrogen. 65 µL (0.62 mmol) 30% hydrogen peroxide in

H2O was added and the solution was vigorously stirred during irradiation of UV light (254 nm)

for 48 h. The reaction was monitored by 1H NMR by taking aliquots of the reaction mixture.

The solvent was then evaporated under reduced pressure affording a white solid. The

crude product containing a mixture of CB7-(OH)n (with n = 0, 1, 2, 3)30 was separated by

column chromatography. Therefore, the mixture was dissolved in 950 µL H2O/HCOOH 1:1

and loaded onto silica gel 60 (0.04-0.063 mm) and the column was eluted with

H2O/AcOH/HCOOH 10:10:1.5. The eluent was collected in fractions of 2 mL (>250 fractions)

and the fractions containing pure CB7-OH were combined. Evaporation of the solvent gave 150

mg CB7-OH as a white solid. The 1H NMR was in accordance with the reported spectrum30 and

the identity and purity of the obtained material was additionally confirmed by mass

spectrometry (Figure 2.3 and 2.4). The ESI-MS spectra were obtained in the presence of

cystamine, as cystamine can dramatically increase the water solubility of CBn.


30

Figure 2.3 Mass spectrum of CB7-OH with 1 mM cystamine in Millipore water. Traces of CB6

were presumably enriched during column chromatography.

Figure 2.4 Mass spectrum of CB6-OH with 1 mM cystamine in Millipore water.


31

2.4 Synthesis of CB6-OPr and CB7-OPr

Figure 2.5 Synthesis of CB6-OPr and CB7-OPr.

Take CB7-OPr for example. 20 mg (17 µmol) CB7-OH was dissolved in 1.5 mL anhydrous

DMSO. 10 mg (0.4 mmol) NaH (95% purity as solid) was added, and the mixture was stirred

at room temperature for 3 h. Subsequently, the mixture was cooled to 0 °C, 0.5 mL (4.4 mmol)

propargyl bromide was added, and the reaction mixture was stirred at room temperature for 12

h. 50 mL diethyl ether was added, and the resulting precipitate was three times triturated with

25 mL MeOH. Drying under high vacuum afforded a pale yellow solid, which was subjected a

second time to the same reaction conditions. This gave the desired CB7-OPr quantitatively as

confirmed by mass spectrometry (Figure 2.7), 1H NMR (Figure 2.8), MS (ESI, +ve): 685.3,

[CB7-OPr+Cys+2H]2+.

Noteworthy, the functionalization of propargyl group with CB6 and CB7 need to be

reacted twice to achieve a high yield. This process can be well followed by MS spectra. As can

be seen from Figure 2.6 and 2.7, after one time reaction free mono-hydroxylated CB6 and CB7

is still left in a large amount. However, with one more time reaction under the same conditions,

nearly 100% yield is obtained finally. In addition, this functionalization lowers the solubility of

CB6-OH and CB7-OH. Consequently, for both CB6-OPr and CB7-OPr, NMR spectra can only

be shown in the presence of guest molecules, such as p-xylylenediamine with CB7-OPr.


32

Figure 2.6 Mass spectra of product started with CB6-OH with 1 mM cystamine in Millipore

water.


33

Figure 2.7 Mass spectrum of product started with CB6-OH with 1 mM cystamine in

Millipore water.


34

Figure 2.8 1H NMR spectra of CB7-OPr in 1% DCl in D2O in absence (top) and presence of

substoichiometric amounts (middle) or excess (bottom) of the cavity binder p-xylylenediamine.

2.5 Synthesis of CB6-Carboxyfluorescein (CB6-CF)

Figure 2.9 Synthesis of CB6-CF.


35

21 mg (20 μmol) CB6-OPr was dissolved in DMSO/H2O (3.4 mL/0.9 mL, 4.3 mL in total).

Subsequently, 6-Caboxyfluorescein-Azide (40 μmol), CuSO4 (40 μmol), sodium ascorbate

(40 μmol) and TBTA (20 μmol) were added. The solution is stirred at room temperature for

24 h. 50 mL diethyl ether was added, and the resulting precipitate was washed three times with

25 mL MeOH. Drying under high vacuum afforded a dark solid. The crude product containing

unreacted CB6-OPr, 6-FAM-Azide, and CB6-CF, was purified by column chromatography. In

detail, the mixture was dissolved in 600 μL H2O/HCOOH 1:1 and loaded onto silica gel 60

(0.04-0.063 mm) and the column was eluted with H2O/AcOH/HCOOH 10:10:1.5. The eluent

was collected in fractions of 2 mL (ca. 50 fractions) and the fractions containing pure CB6-CF

were combined. Evaporation of the solvent gave 8 mg (7.6 μmol, 38% yield) CB6-CF as a

brown solid. The product identity was confirmed by mass spectrometry (Figure 2.10), [CB6-

CF+Cys+2H+]2+ is calculated at 832 and found at 832.

Figure 2.10 The mass spectrum of CB6-CF.

2.6 Designed FRET Pair Based on CB6-CF and R-S

Our initial aim is to design a FRET pair based on host-guest interactions and try to explore its

potential applications. Therefore, after the successful synthesis of CB6-CF, we designed a guest

molecule rhodamine-spermine (R-S) which was afforded by Prof. Pischel (structures are shown

in Figure 2.11 a, b). Figure 2.11 c illustrates a FRET pair based on supramolecular host-guest

interaction between CB6-CF and R-S. In this FRET pair, CB6-CF and R-S are considered as

the donor and the acceptor respectively. As a result of the simulated calculation based on the

complex of CB6-CF and R-S (as shown in Figure 2.12), the maximum distance between

acceptor and donor depends on the molecules orientation, which is calculated to be 0.9 nm and

1.4 nm in two possible structures. This calculation demonstrates the designed FRET system

possible to occur theoretically.


36

Figure 2.11 Illustration of host-guest FRET pair based on CB6-CF and R-S.

Figure 2.12 DFT calculation of the structure in different possible orientations for the CB6-

CF/R-S complex in gas phase.


37

2.6.1 Spectral Characterization of the Designed FRET pair

300 400 500 600 700 800

0.0

0.5

1.0

0.0

0.5

1.0R-S

Flu

ore

sce

nceA

bsorb

an

ce

Wavelength (nm)

CB6-CF

Figure 2.13 Normalized absorption (dashed) and emission (solid) spectra of CB6-CF (black)

and R-S (green) in 10 mM (NH4)2HPO4, pH 7.5. exc,CB6-CF = 490 nm and exc,R-S = 560 nm.

Figure 2.13 shows the spectroscopic properties of CB6-CF and R-S. The spectral overlap

between the CB6-CF emission spectrum (black solid line in Figure 2.13) and the R-S absorption

spectrum (green dashed line) demonstrates this FRET pair works in principle. The results from

control experiments show that CB6-CF could be selectively excited around 450 nm, where the

absorbance of R-S is insignificant.

2.6.2 Spectral Properties of the Designed FRET Pair

Steady state fluorescence spectroscopy is used to characterize the spectral properties of this

FRET pair. Titrations of 50 nM CB6-CF with increasing amounts of R-S were carried out to

investigate the performance of the FRET system (Figure 2.14 a, b). Overall, the spectral changes

during the titrations can be grouped into three different concentration regions of R-S. At low

concentrations of R-S (0-0.12 µM), the fluorescence of CB6-CF steeply decreases by a factor

of two and R-S is not fluorescent at all (Figure 2.14 a). We presume that this is due to formation

of the host-guest complex leading to fluorescence quenching of the fluorescein dye due to the

close spatial proximity of the rhodamine dye. In fact, analysis of the fitting curve of this

concentration region suggested a nearly quantitative binding affording a lower limit of the

binding constant of Ka > 6 × 108 M1. This is accordance with the very strong binding reported

for spermine to CB6 (Ka ca. 1012 M1). With increasing concentrations of R-S (0.36-0.76 µM),

the fluorescence intensity of CB6-CF starts to slightly increase. We believe that this is due to a

residual fluorescence emission from excess R-S. The solution now contains ca. 10 times more


38

R-S than CB6-CF. This is supported by Figure 2.14 d (only R-S), where a slight increase in

fluorescence around 521 nm becomes visible in this concentration range.

500 550 600 650 700

0

2

4

6

Flu

ore

sce

nce

Wavelength (nm)

a) CB6-CF and R-S (0-0.12 µM)

CR-S

0

0,0049

0,0149

0,0348

0,0744

0,1234

500 550 600 650 700

0

2

4

6

Flu

ore

sce

nce

Wavelength (nm)

b) CB6-CF and R-S (0.12-4.36 µM)

CR-S

0,1234

0,2200

0,3614

0,5437

0,7621

1,0112

1,2854

1,5789

1,8864

2,2027

2,5233

2,8443

3,2249

3,4747

3,7791

4,0740

4,3582

500 550 600 650 700

0

2

4

6

8

Flu

ore

sce

nce

Wavelength (nm)

c)CR-S

0

0,0049

0,0149

0,0348

0,0744

0,1234

0,2200

0,3614

0,5437

0,7621

1,0112

1,2854

1,5789

1,8864

2,2027

2,5233

2,8443

3,2249

3,4747

3,7791

4,0740

4,3582

6-FAM-Azide and R-S (0-4.36 µM)

500 550 600 650 700

0.0

2.5

5.0F

luore

sce

nce

Wavelength (nm)

d)CR-S

0

0,0049

0,0149

0,0348

0,0744

0,1234

0,2200

0,3614

0,5437

0,7621

1,0112

1,2854

1,5789

1,8864

2,2027

2,5233

2,8443

3,2249

3,4747

3,7791

4,0740

4,3582

only R-S (0-4.36 µM)

Figure 2.14 Fluorescence titrations (exc = 450 nm, slits: 10/10) and control experiments with

increasing amounts of R-S (0 to 4.36 µM) in presence of a) 50 nM CB6-CF and 0-0.12 µM R-

S, b) 50 nM CB6-CF and 0.12-4.36 µM R-S, c) 50 nM 6-FAM azide, and d) in absence of

fluorescein. Experiments were performed in 10 mM (NH4)2HPO4, pH 7.5.

Addition of even larger amounts of R-S (>0.76 µM, >15fold excess) then leads to a

continuous decrease in the fluorescence intensity of fluorescein. At these high concentrations,

R-S shows significant absorbance in the spectral region of fluorescein emission ( =

110000 M1cm1, A > 0.08). This may lead to reabsorption of the emitted photons and thus a

decrease in fluorescence. This is supported by a similar decrease in fluorescence upon addition

of R-S to a solution containing 6-FAM-azide lacking the CB6 receptor (Figures 2.14 c).

In summary, it becomes apparent that a host-guest complex between CB6-CF and R-S

is formed. The spectral changes of the titration and the control experiments are in clear

accordance with this interpretation. However, a fluorescence increase in the R-S emission at

low concentrations of R-S (Figure 2.14 a) was not observed, which currently suggests that

FRET is not the quenching mechanism.


39

2.6.3 Quenching Mechanism

As noted above, the absence of a fluorescence increase in the region of rhodamine emission

during the titration of CB6-CF with R-S suggests that FRET is not the (only) operative

fluorescence quenching mechanism. We thus considered that static quenching, either of

fluorescein by rhodamine or of rhodamine by fluorescein after FRET, may cause the lack of

fluorescence increase.

600 650 700

0

5000

10000

a)

Inte

nsity

Wavelength (nm)

0

0,0015

0,0047

0,0111

0,0426

0,0886

0,1333

0,2601

0,4137

0,5845

0,7637

0,9361

1,0856

1,2167

1,3324

CCB6-CF110 nM R-S

0.0 0.4 0.8 1.2

8500

9000

9500

10000b)

Inte

nsity

[CB6-CF] (M)

Figure 2.15 a) Titration of R-S (110 nM) with CB6-CF (From 0 to 1.33 µM) in 10 mM

(NH4)2HPO4, pH 7.5 using direct excitation of R-S (λex= 550 nm). b) CB6-CF concentration

dependence of the fluorescence intensity of R-S at 580 nm.

600 650 700

0

1500

3000

4500

a)

Inte

nsity

Wavelength (nm)

0 10 20 30

4250

4500

4750

5000b)

Inte

nsity

[CB6] ()

Figure 2.16 a) Titration of 0.5 µM R-S with CB6 (0 to 30 µM) in 10 mM (NH4)2HPO4, pH 7.5.

(λexc = 560 nm). b) CB6 concentration dependence of the fluorescence intensity of R-S at 580

nm.

To test whether there is static quenching of rhodamine by fluorescein (after FRET) in

this system or not, a control titration experiment was conducted. We used a constant

concentration of R-S and added increasing amounts of CB6-CF (Figure 2.15). The excitation

wavelength was 550 nm, such that rhodamine is directly excited without exciting CB6-CF.


40

Under these conditions, any change in rhodamine fluorescence must occur because of the

formed complex. In fact, we observed a slight decrease of R-S fluorescence intensity, which

was, however, not strong enough to fully explain the absence of rhodamine emission upon

excitation of CB6-CF.

In another control experiment, parent unmodified CB6 was added to a solution

containing 0.5 µM R-S (shown in Figure 2.16). At a lower concentration of CB6, from 0 to 0.3

µM, the intensity somehow decreased. When the concentration was higher than 0.6 µM, the

intensity started to increase. Also here, the fluorescence changes are small and do not explain

the absence of the expected increase in rhodamine fluorescence after FRET.

Based on the proposed analysis of the spectral properties, the designed FRET pair based

on CB6-CF and R-S is not suitable for further study. We need to select another FRET pair with

proper spectral properties. A new FRET pair based on CB7 host-guest interaction is later

achieved and this content will be described in detail in Chapter 3.

2.7 Further Synthesis of Mono-Functionalized CB7

Figure 2.17 Examples of mono-functionalized CB7 derivatives.


41

Figure 2.18 MALDI-TOF spectra of CB7-TAMRA and CB7-Cy3.


42

Besides CB6-CF, we further synthesized four mono-functionalized CB7 derivatives, one will

be explained in the next Chapter and the other three includes two fluorescent dyes in different

emission range and one functional molecule with –COOH at the end (Figure 2.17 and 2.18).

CB7-TAMRA and CB7-Cy3 are reacted by CB7-OPr with commercial 5-TAMRA azide and

Cyanine 3 azide. CB7-O2O is reacted with synthesized azido molecule with functional groups

from Prof. Dr. Leif Schroeder. The synthesis procedures to get CB7-TAMRA and CB7-Cy3

are the same with CB6-CF procedure. The procedure to get CB7-O2O is quite different which

takes tedious work to optimize the reaction conditions.

Figure 2.19 The synthesis route of CB7-NIe and CB7-O2O.

From our collaborator Prof. Dr. Leif Schroeder, we received two chemicals (1 and 2,

Figure 2.19) designed to react with CB7-OPr. The first try was following the proved successful

procedure of CB6-CF, unfortunately no product was acquired by characterizations with TLC

and MS. However, click reaction is well known as efficient reaction and it should work in

principle between azide and alkyne. Therefore, twelve different reaction conditions were tried

for the reaction by varying the Cu source, ligand and solution composition (Table 2.1). The

stock solutions of chemicals used in table 2.1: CB7-OPr: 30 mM in DMSO; NaHCO3: 1 M in

H2O; NaAsc: 1 M in H2O; TBTA: 376 mM in DMSO; BPDS: 388 mM in H2O; CuSO4: 1M in

H2O; CuI: 0.5 M in DMSO; CuBr: 0.5 M in DMSO; DIPEA: 0.742 g/mL in H2O. For each of

the reaction it combines 3 steps. Firstly it was reacted for 24 h in room temperature. Without

new spot observed on TLC temperature was increased to 50 °C for another 24 h reaction time.


43

If still no new spot shown on TLC, the temperature then increased to 90°C. The first and second

step were characterized by TCL, as well as the third step, no new spot appeared by all of them.

Finally we chose MALDI-TOF to detect products after the third step reaction due to the very

low solubility of mono-functionalized CB7 derivatives.

Table 2.1 Different conditions for the reactions between CB7-OPr and 1 or 2.

No. Azide CB7-

OPr

Cu

source Ligand Azide NaAsc/Base Solvent Temperature

1 1

15 µL

1 µmol

10 mM

CuSO4

6 µL

12 µmol

120 mM

BPDS

8 µL

6 µmol

60 mM

10 µL

2 µmol

20 mM

NaAsc

6 µL = Cu2+

12 µmol

120 mM

50 µl

DMSO/H2

O

1:1

r.t./24 h

no product 50 °C/24 h


2 1

15 µL

1 µmol

10 mM

CuSO4

1.5 µL

3 µmol

30 mM

BPDS

8 µL

6 µmol

60 mM

10 µL

2 µmol

20 mM

NaAsc

1.5 µL = Cu2+

3 µmol

30 mM

50 µl

DMSO/

100 mM

NaHCO3

1:1

r.t./24 h



3 1

15 µL

1 µmol

10 mM

CuSO4

1.5 µL

3 µmol

30 mM

none

10 µL

2 µmol

20 mM

NaAsc

3 µL = 2 x Cu2+

6 µmol

60 mM

50 µl

DMSO/H2

O

1:1

r.t./24 h



4 1

15 µL

1 µmol

10 mM

CuSO4

2 µL

4 µmol

40 mM

TBTA

5 µL

4 µmol

40 mM

10 µL

2 µmol

20 mM

NaAsc

2 µL

4 µmol

40 mM

50 µl

DMSO/

100 mM

NaHCO3

1:1

r.t./24 h



5 1

15 µL

1 µmol

10 mM

CuSO4

1.5 µL

3 µmol

30 mM

none

10 µL

2 µmol

20 mM

NaAsc

3 µL = 2 x Cu2+

6 µmol

60 mM

50 µl

DMSO/H2

O/t-BuOH

45:45:10

r.t./24 h



6 2

15 µL

1 µmol

10 mM

CuSO4

6 µL

12 µmol

120 mM

BPDS

8 µL

6 µmol

60 mM

10 µL

2 µmol

20 mM

NaAsc

6 µL = Cu2+

12 µmol

120 mM

50 µl

DMSO/H2

O

1:1

r.t./24 h



7 2

15 µL

1 µmol

10 mM

CuSO4

1.5 µL

3 µmol

30 mM

BPDS

8 µL

6 µmol

60 mM

10 µL

2 µmol

20 mM

NaAsc

1.5 µL = Cu2+

3 µmol

30 mM

50 µl

DMSO/

100 mM

NaHCO3

1:1

r.t./24 h



8 2

15 µL

1 µmol

10 mM

CuSO4

1.5 µL

3 µmol

30 mM

none

10 µL

2 µmol

20 mM

NaAsc

3 µL = 2 x Cu2+

6 µmol

60 mM

50 µl

DMSO/H2

O

1:1

r.t./24 h



9 2

15 µL

1 µmol

10 mM

CuSO4

2 µL

4 µmol

40 mM

TBTA

5 µL

4 µmol

40 mM

10 µL

2 µmol

20 mM

NaAsc

2 µL

4 µmol

40 mM

50 µl

DMSO/

100 mM

NaHCO3

1:1

r.t./24 h



10 2

30 µL

2 µmol

20 mM

CuI

2 µL

2 µmol

20 mM

none

10 µL

2 µmol

20 mM

DIPEA

0.7 µL

4 µmol

40 mM

50 µL

DMSO

only

r.t./24 h



11 2

30 µL

2 µmol

20 mM

CuI

2 µL

2 µmol

20 mM

TBTA

5 µL

4 µmol

40 mM

10 µL

4 µmol

40 mM

DIPEA

0.7 µL

4 µmol

40 mM

50 µL

DMSO

only

r.t./24 h



12 2

30 µL

2 µmol

20 mM

CuBr

2 µL

2 µmol

20 mM

none

10 µL

2 µmol

20 mM

DIPEA

0.7 µL

4 µmol

40 mM

50 µL

DMSO

only

r.t./24 h




44

Figure 2.20 MALDI-TOF spectra of reactants from condition 11 and 12.

As shown in Figure 2.20, MS of Samples from condition 11 and 12 show the peak 1574

m/z, which comes from the complex of CB7-O2O with Na+, demonstrating that CB7-O2O can


45

be successfully synthesized by using condition 11 and 12. The peak is very tinny because of the

low solubility of the aimed product. The explanation of no new spot shown on TLC plate is still

missing. In conclusion, the difficulty of the click reactions based on CB7-OPr is depend on the

azido molecules. To successfully synthesize the target product, optimization of reaction

conditions plays a crucial role.

2.8 References

(1) W. L. Mock and N. Y. Shih, J. Org. Chem., 1986, 51, 4440-4446.


Chem. Soc., 2005, 127, 15959-15967.

(3) L. Cao, M. Sekutor, P. Y. Zavalij, K. Mlinaric-Majerski, R. Glaser and L. Isaacs, Angew.

Chem. Int. Ed., 2014, 53, 988-993.

(4) S. Moghaddam, C. Yang, M. Rekharsky, Y. H. Ko, K. Kim, Y. Inoue and M. K. Gilson, J.

Am. Chem. Soc., 2011, 133, 3570-3581.

(5) S. Kasera, F. Biedermann, J. J. Baumberg, O. A. Scherman and S. Mahajan, Nano Lett.,

2012, 12, 5924-5928.

(6) W. M. Nau, M. Florea and K. I. Assaf, Isr. J. Chem., 2011, 51, 559-577.

(7) K. I. Assaf and W. M. Nau, Chem Soc Rev., 2015, 44, 394-418.

(8) S. Y. Jon, N. Selvapalam, D. H. Oh, J. K. Kang, S. Y. Kim, Y. J. Jeon, J. W. Lee and K.

Kim, J. Am. Chem. Soc., 2003, 125, 10186-10187.

(9) N. Zhao, G. O. Lloyd and O. A. Scherman, Chem. Commun., 2012, 48, 3070-3072.

(10) W. H. Huang, P. Y. Zavalij and L. Isaacs, Org. Lett., 2008, 10, 2577-2580.

(11) D. Lucas, T. Minami, G. Iannuzzi, L. Cao, J. B. Wittenberg, P. Anzenbacher, Jr. and L.

Isaacs, J. Am. Chem. Soc., 2011, 133, 17966-17976.

(12) B. Vinciguerra, L. Cao, J. R. Cannon, P. Y. Zavalij, C. Fenselau and L. Isaacs, J. Am.

Chem. Soc., 2012, 134, 13133-13140.

(13) L. Cao and L. Isaacs, Org. Lett., 2012, 14, 3072-3075.

(14) L. Gilberg, M. S. Khan, M. Enderesova and V. Sindelar, Org. Lett., 2014, 16, 2446-2449.

(15) S. M. Liu, L. Xu, C. T. Wu and Y. Q. Feng, Talanta, 2004, 64, 929-934.

(16) S. Kushwaha and P. P. Sudhakar, Anal., 2012, 137, 3242-3245.

(17) Y. J. Jeon, H. Kim, S. Jon, N. Selvapalam, D. H. Oh, I. Seo, C. S. Park, S. R. Jung, D. S.

Koh and K. Kim, J. Am. Chem. Soc., 2004, 126, 15944-15945.

(18) J. Kim, Y. Ahn, K. M. Park, Y. Kim, Y. H. Ko, D. H. Oh and K. Kim, Angew. Chem. Int.

Ed. , 2007, 46, 7393-7395.


46

(19) E. R. Nagarajan, D. H. Oh, N. Selvapalam, Y. H. Ko, K. M. Park and K. Kim, Tetrahedron

Lett., 2006, 47, 2073-2075.

(20) D. Kim, E. Kim, J. Kim, K. M. Park, K. Baek, M. Jung, Y. H. Ko, W. Sung, H. S. Kim, J.

H. Suh, C. G. Park, O. S. Na, D.-k. Lee, K. E. Lee, S. S. Han and K. Kim, Angew. Chem. Int.

Ed., 2007, 119, 3541-3544.

(21) D. W. Lee, K. M. Park, M. Banerjee, S. H. Ha, T. Lee, K. Suh, S. Paul, H. Jung, J. Kim,

N. Selvapalam, S. H. Ryu and K. Kim, Nat. Chem., 2011, 3, 154-159.

(22) D. Kim, E. Kim, J. Lee, S. Hong, W. Sung, N. Lim, C. G. Park and K. Kim, J. Am. Chem.

Soc., 2010, 132, 9908-9919.

(23) K. Baek, G. Yun, Y. Kim, D. Kim, R. Hota, I. Hwang, D. Xu, Y. H. Ko, G. H. Gu, J. H.

Suh, C. G. Park, B. J. Sung and K. Kim, J. Am. Chem. Soc., 2013, 135, 6523-6528.

(24) M. Munteanu, S. Choi and H. Ritter, Macromolecules, 2009, 42, 3887-3891.

(25) H. K. Lee, K. M. Park, Y. J. Jeon, D. Kim, D. H. Oh, H. S. Kim, C. K. Park and K. Kim,

J. Am. Chem. Soc., 2005, 127, 5006-5007.

(26) K. M. Park, K. Suh, H. Jung, D. W. Lee, Y. Ahn, J. Kim, K. Baek and K. Kim, Chem.

Commun., 2009, 0, 71-73.

(27) E. Kim, J. Lee, D. Kim, K. E. Lee, S. S. Han, N. Lim, J. Kang, C. G. Park and K. Kim,

Chem. Commun., 2009, 0, 1472-1474.

(28) K. M. Park, J. A. Yang, H. Jung, J. Yeom, J. S. Park, K. H. Park, A. S. Hoffman, S. K.

Hahn and K. Kim, ACS Nano, 2012, 6, 2960-2968.

(29) Y. Ahn, Y. Jang, N. Selvapalam, G. Yun and K. Kim, Angew. Chem. Int. Ed., 2013, 52,

3140-3144.



Chapter 3 Ratiometric DNA Sensing with a Host-Guest FRET Pair

47

Chapter 3. Ratiometric DNA Sensing with a

Host-Guest FRET Pair


48


49

This chapter is derived from the content of the following publication:

Shuai Zhang, Khaleel I. Assaf, Chusen Huang, Andreas Hennig* and Werner M. Nau*,

Ratiometric DNA Sensing with a Host-Guest FRET Pair, Chem. Comm., 2019, 55, 671-674.

In this chapter, a supramolecular host-guest FRET pair based on a carboxyfluorescein-labelled

cucurbit[7]uril (CB7-CF, as acceptor) and the fluorescent dye 4',6-diamidino-2-phenylindole

(DAPI, as donor) is developed for sensing of DNA. In comparison to the commercial DNA

staining dye SYBR Green I, the new chemosensing ensemble offers dual-emission signals,

which allows a linear ratiometric response over a wide concentration range.

3.1 Introduction

Fluorescent chemosensors are receiving increasing attention, especially for biological

analytes.1-2 For example, numerous commercially available fluorescent dyes, such as SYBR-

Green I, bind to the minor groove of double-stranded DNA (dsDNA), which can be exploited

for sensing in medical diagnostics, e.g., for real-time PCR.3-6 Most of these dye-intercalation

techniques are based on single-wavelength monitoring, whereas dual emitting probes, which

allow ratiometric sensing for more precise quantification, are mostly based on the recognization

of DNA hybridization. This has been achieved by using pyrene-functionalized molecular

beacons (with either oligonucleotide7 or peptide8 backbones), locked nucleic acids,9 and

perylene-based probes,10-12 but these probes only respond to complementary DNA sequences.

An alternative sensing strategy to direct binding, such as intercalation, is indicator

displacement. Macrocycles such as cyclodextrins,13-15 calixarenes,16-18 crown ethers,19-21 and

cucurbit[n]urils22-23 (CBn) have been abundantly used in combination with fluorescent dyes to

quantify analytes by the latter strategy, including the cited examples of the most desirable

ratiometric sensing. With the aim to monitor analyte formation or depletion and to measure the

kinetics of enzymatic reactions, macrocycle/dye reporter pairs have also been implemented in

supramolecular tandem assays,24-31 including the enzymatic conversion of nucleotides,22, 32-41

but they have not yet been employed for DNA sensing.

CBn macrocycles, and in particular the medium-sized homologue CB7, stand out as

recognition motifs due to their high analyte affinities and selectivities.42-44 The known assays

rely on a direct affinity between analyte and CBn, which results in a competitive displacement

of the fluorescent indicator dye. However, although indirect interactions between DNA or

nucleotides and CBn, mediated by other guests, have been described,45-52 applications to DNA

sensing are elusive. Herein, we establish a nonconventional approach to DNA sensing, which

relies neither on a simple intercalation of a fluorescent dye with the analyte (DNA) nor on a

direct binding of DNA to a recognition site, but rather on a competitive binding of a fluorescent


50

dye to an acceptor-labelled CB7 and DNA; this allows a ratiometric DNA sensor based on

fluorescence resonance energy transfer (FRET) between the two chromophores to be set up

according to Figure 3.1. The new application complements recent work on fluorescent dye-

conjugated CBn derivatives with cyanine-3 and tetramethylrhodamine for the detection of

vesicle fusion53-55 and cellular imaging.56

Figure 3.1 (a) Molecular structures of CB7-CF and DAPI. (b) Schematic illustration of a DNA

chemosensing ensemble based on FRET between DAPI (donor) and CB7-CF (acceptor).

3.2 Synthesis of CB7-CF

The synthesis of CB7-carboxyfluorescein (CB7-CF) involved labelling through click

reaction of the azide derivative of carboxyfluorescein (CF, using commercial 6-FAM-

azide), with monofunctionalised propargyl-CB7 (CB7-OPr)57-58 (see Figure 3.2). Since

the CF chromophore is equatorially attached to the outer wall of CB7, its hydrophobic

cavity remains accessible for guest binding. Intramolecular or intermolecular complexation of

CF in the cavity, which presents a common obstacle when using monofunctionalized CBn

derivatives,59 does not interfere, because of the anionic nature of CF, which is incompatible

with the cation-receptor propensity of CBn homologues.60


51

Figure 3.2 Synthesis of CB7-CF.

3.3 Optical Characterizations of FRET Pair

300 400 500 600

0.0

0.5

1.0

0.0

0.5

1.0

Wavelength (nm)

DAPI

CB7-CF

(a)

400 500 600

0

250

500

Wavelength (nm)

(b)

Figure 3.3 (a) Normalized absorption (solid) and emission (dashed) spectra of DAPI

(blue) and CB7-CF (green). (b) Emission spectra (ex = 360 nm) of 2 µM DAPI (blue),

1 µM CB7-CF (green), and a mixture of 2 µM DAPI and 1 µM CB7-CF (orange).

The encapsulation of the fluorescent DNA stain 4',6-diamidino-2-phenylindole (DAPI, as

chloride salt), by CB7-CF allows efficient FRET to occur. The spectral characterization of the

FRET pair was performed by UV-Vis absorption and fluorescence spectroscopy (Figure 3.3).

The emission spectrum of DAPI has a significant overlap with the absorption spectrum of CB7-

CF, which fulfills the principal requirement of FRET. The distance between DAPI and CB7-

CF was modelled to be between 5.9 Å (minimum) and 10.8 Å (maximum), which falls


52

sufficiently far below the calculated critical Förster radius of the system (R0 = 33.3 Å, Figure

3.4) to allow quantitative FRET (>99%).

Figure 3.4 DFT-optimized structures (B3LYP/3-21G*) of different possible co-conformations

for the CB7-CF/DAPI complex (in gas phase). The distance (d) between the center of mass of

the CF and DAPI is given in Å. The structure shown in (a) was found to be more stable than

that in (b) by 4.9 kcal/mol.

Optical titration experiments were limited by the reduced water solubility of CB7-CF

(ca. 5 M in pure water and ca. 30 M in the presence of 0.5% DMSO), but the fluorescence

intensities were sufficiently high to allow an analysis also in this solubility range. The

fluorescence titration of the donor (DAPI, ex = 360 nm) upon addition of acceptor (CB7-CF)

was performed first in the presence of 0.5% DMSO (Figure 3.5 a). As expected, there was a

strong rise in fluorescence of CB7-CF, which afforded, after correction for direct excitation of

CF at the excitation wavelength of DAPI, a binding constant of (1.4 0.1) × 106 M–1.

Noteworthy, the fluorescence of the donor did not decrease, as expected for quantitative FRET,

but showed a slight increase. This phenomenon is well known in FRET experiments with

commercial labelling agents (6-FAM-azide) and can be traced back to a nonquantitative

labelling with acceptor.61 The comparison of the fluorescence intensity of DAPI alone and that

extrapolated to high concentrations of CB7-CF, along with the expected fluorescence

enhancement factor in an unlabelled CB7 cavity (ca. 12, see Figure 3.6) allowed us to estimate

the labelling degree with CF as 90%.


53

400 500 600 7000

100

200

300

400

I

I

(b)

[DAPI]

CB7-CF fluorescence

DAPI fluorescence

/nm

0 1 2 3 4 5 6 7 80

200

400

[DAPI]/M

400 500 600 7000

150

300

450

I/I 0

I(a)

/nm

[CB7-CF]0 3 6 9 12 15

0

10

20

30

[CB7-CF]/M

Figure 3.5 (a) Fluorescence spectral changes (ex = 360 nm) with increasing amounts of

CB7-CF (0 to 15 µM) in the presence of 1 µM DAPI; the inset shows the fluorescence

titration (em = 520 nm) with increasing concentration of CB7-CF (R2 = 0.998).

Corrected for direct excitation of CF. (b) Fluorescence spectral changes (ex = 360 nm)

with increasing amounts of DAPI (0 to 7.4 µM) in the presence of 1 µM CB7-CF; the

inset shows the fluorescence titration (em = 450 nm) with increasing concentration of

DAPI in the presence of only DAPI (black curve, R2 = 0.998), 1 µM CB7-CF (red curve,

R2 = 0.999), and 1 µM CB7 (blue curve, R2 = 0.998). Fitting curves were obtained by a

1:1 host-guest binding isotherm.

400 450 500 550 600 650 700

0

100

200

300

400

I

I

/nm

0 2 4 6 8 100

150

300

450

[CB7]/M

Ka = (2.8 0.2)

Figure 3.6 Variation of the fluorescence spectrum (ex = 360 nm) of 1 μM DAPI in 10 mM

(NH4)2HPO4, pH 7.2, upon addition of CB7. Inset: CB7 concentration dependence of the

fluorescence intensity at 470 nm, R2 = 0.998. The curve was fitted by 1:1 binding stoichiometry.

The reverse titration allowed us to demonstrate operation of the FRET system in neat

water (10 mM ammonium phosphate buffer, pH 7.2, Figure 3.5 b), where the solubility of CB7-


54

CF was limited to ca. 5 M. Upon addition of increasing amounts of donor (DAPI) to a CB7-

CF solution (1 µM) its fluorescence (ex = 360 nm, obs = 450 nm) increased. However, the

fluorescence increase (red line) fell far below the 12-fold increase observed upon addition to a

corresponding CB7 solution (1 µM, blue line). This is due to the fact that the DAPI fluorescence

in the CB7-CF cavity is effectively quenched by FRET (also reflected in an increased CF

fluorescence) and not enhanced as in unlabelled CB7, where a microenvironmental effect

enhances the fluorescence.62 Comparison of the fluorescence intensity of DAPI in the presence

of CB7-CF with that in the absence of any host further indicates a slight increase (compare red

and black lines), which can be attributed to the nonquantitative acceptor labelling already

exposed in the titration with acceptor (see above, Figure 3.5 a). Fitting of the fluorescence

titrations by taking into account these boundary conditions afforded a binding constant between

DAPI and CB7-CF of (5.2 0.5) × 106 M1, comparable to that between DAPI and CB7 in the

same ammonium phosphate buffer ((2.8 0.2) × 106 M–1), but below that in neat water (11

× 106 M–1),62 due to the competition of ammonium cation binding to CB7 in the buffer.63

400 500 600 700

0

150

300

450

I

(a)

1 M CB7-CF+20 M AMADA+[DAPI] [DAPI] 0 M

[DAPI] 0.04 M

[DAPI] 0.15 M

[DAPI] 0.38 M

[DAPI] 0.82 M

[DAPI] 1.55 M

[DAPI] 2.50 M

[DAPI] 3.76 M

[DAPI] 5.17 M

[DAPI] 6.36 M

[DAPI] 7.37 M

/nm

400 500 600 700

0

150

300

450

/nm

I

32 M AMADA

1 M CB7-CF 7.4 M [DAPI]

(b)

Figure 3.7 (a) Fluorescence titrations (ex = 360 nm) with increasing amounts of DAPI

(0 to 7.4 µM) in the presence of 1 µM CB7-CF as well as 20 µM AMADA. (b) Fluorescence

spectra (ex = 360 nm) of 1 μM CB7-CF and 7.4 μM DAPI without (black line) and with (red

line) 32 μM AMADA in 10 mM (NH4)2HPO4, pH 7.2. The addition of AMADA leads to a

strong fluorescence decrease of CB7-CF.

Note that the value in the aqueous buffer lies slightly above that determined in the

presence of 0.5% DMSO (1.4 × 106 M–1, see above), a co-solvent which is expected to slightly

reduce binding as a consequence of a reduced hydrophobic driving force.64 The reversibility of

the FRET process and the analyte responsiveness of the system was established through control

experiments with 1-aminomethyladamantane (AMADA), a known competitive binder of CB7

(see Figure 3.7).


55

0 10 20 30 40 500

1

2

3

4

DAPI+CB7-CF

DAPI+CB7

DAPI

IRF

Lo

g (

Co

un

ts)

t /ns

Figure 3.8 Time-resolved fluorescence decay traces of 5 µM DAPI alone (blue) and in

the presence of 10 µM CB7-CF (black) and CB7 (red); instrument response function is

shown in magenta. exc = 373 nm, obs = 450 nm.

Table 3.1 Fitting results of lifetime decays of DAPI with and without CB7 or CB7-CF

in 10 mM (NH4)2HPO4, pH 7.2 with 0.5% DMSO.

Sample τ/ns B Rel% χ2

DAPIa 1 = 0.29 0.056 81.1

2.24

2 = 2.77 0.001 18.9

DAPI 1 = 0.31 0.054 79.2

2.66

2 = 2.74 0.002 20.8

DAPI+CB7 1 = 1.59 0.026 100 2.40

DAPI+CB7-CF 1 = 0.65 0.021 30.2

1.94

2 = 3.05 0.010 69.8

a Without DMSO.

The FRET pair was also characterized by nanosecond time-resolved fluorescence spectroscopy

(Figure 3.8 and Table 3.1). DAPI has a long fluorescence lifetime component (2.75 ns).65 Upon

complexation by CB7, the lifetime is reduced to ca. 1.6 ns, which presents an unusual but

previously documented response;66 generally, an increase in fluorescence intensity upon CB7

complexation is accompanied by an increased fluorescence lifetime.60 This unusual behavior

points to an increase of the radiative lifetime () of DAPI upon CB7 complexation, which is

likely related to a conformational realignment of the chromophore. From the Förster radius of

the FRET pair, a quantitative (>99%) fluorescence quenching of DAPI was expected in the

DAPI-CB7-CF complex. Unfortunately, due to the interference of nonquantitative (90%)

acceptor labelling, a residual long-lived component attributable to DAPI remained even in the


56

presence of an excess of CB7-CF. Noteworthy, the fluorescence lifetime of the CF

chromophore in CB7-CF (3.6 ns) remained virtually the same as that in free CF,67 which

demonstrates that the CF chromophore in CB7-CF and also in the DAPI-CB7-CF complex is

not quenched.

3.4 DNA Sensing by FRET Pair

20 30 40 50 60 70 80 90

150

300

450

20 °C to 90 °C Em = 450 nm

20 °C to 90 °C Em = 520 nm

90 °C to 20 °C Em = 450 nm

90 °C to 20 °C Em = 520 nm

I

T/°C

30 60 90

0.8

1.2

1.6

2.0

I 45

0/I

52

0

T/°C

(b)

400 500 600 700

0

100

200

300

400

I

/nm

(a)

0 5 10 15 200.0

0.3

0.6

0.9y = 43x + 0.08

R2 = 0.995

I 45

0/I

52

0

[DNA]/(gmL1)

Figure 3.9 (a) Fluorescence spectra (ex = 360 nm) of 0.5 M DAPI and 1 M CB7-CF

with increasing concentration of salmon sperm DNA (0 to 20 g/mL). The inset shows

the linear relationship between the fluorescence intensity ratio (I450/I520) and the

concentration of salmon sperm DNA (0 to 20 g/mL). (b) Temperature-dependent

fluorescence intensity at 450 nm and 520 nm (ex = 360 nm) from 20 to 90 °C. The inset

shows the reversible changes in I450/I520 ratios between 20 and 90 °C.

DAPI is well known to intercalate strongly into the minor groove of DNA (binding affinity of

107 M–1 per nucleotide, or 1010 M–1 for a typical DNA with ca. 1000 base pairs),68-69 but binds

with micromolar affinity to CB7,62 which should allow for an efficient relocation of the dye

from CB7 upon addition or formation of DNA if the concentrations are favourably selected.70

Accordingly, we conducted a fluorescence titration of the pre-assembled FRET pair (0.5 µM

DAPI and 1 µM CB7-CF) with Type III salmon sperm DNA (Figure 3.9 a). With increasing

concentration of DNA, DAPI is relocated from CB7-CF, where it no longer serves as FRET

donor and, thereby, reduced the CF fluorescence at 520 nm, into DNA, where its own

fluorescence at 450 nm becomes enhanced. The ratio of the fluorescence intensity values

(I450/I520) increased linearly in the picomolar range (up to 20 µg/mL), which sets up an excellent

DNA chemosensing ensemble with an LOD value (limit of detection) of ca. 60 ng/mL (inset in

Figure 3.9 a). A plateau value was reached at DNA concentrations above 0.1 mg/mL (Figure

3.10 a), indicating that DAPI relocation from CB7 to DNA is quantitative.


57

400 500 600 700

0

100

200

300

400

I

/nm

I450

/I520

0.0 0.1 0.2 0.3 0.4 0.5

0.0

0.5

1.0

1.5

[DNA]/(mgmL1)

(a)

400 500 600 700

0

100

200

300

400

500

I

/nm

(b)

0.0 0.1 0.2 0.3 0.4 0.50

250

500I

[DNA]/(mgmL1

)

550 600 650 700

0

250

500

750

1000

I

/nm

0.0 0.1 0.2 0.3

0

500

1000I

[DNA]/(mgmL1

)

(c)

0.00 0.01 0.02 0.03 0.04

0.0

0.5

1.0

0.5 M DAPI

0.5 M SYBR Green

0.5 M DAPI + 1 M CB7-CF

[DNA]/(mgmL1

)

I/I

(d)

Figure 3.10 (a) Fluorescence spectra (ex = 360 nm) of 0.5 M DAPI and 1 M CB7-CF with

increasing concentration of salmon sperm DNA (0 to 0.5 mg/mL). The inset shows the

corresponding change in the fluorescence intensity ratio at 450 nm and 520 nm. (b) (c) Variation

of the fluorescence spectrum of 0.5 μM DAPI (ex = 360 nm) and of 0.5 μM SYBR (ex =

497 nm) upon addition of salmon sperm DNA. The inset shows the dependence of the

fluorescence intensity at 450 nm on DNA concentration and at 520 nm on DNA concentration.

(d) Normalized fluorescence intensity of DAPI, SYBR-Green, and DAPI/CB7-CF with dame

concentration of fluorescence dye in dependence on DNA concentration. Note that for

DAPI/CB7-CF the ratiometric response is shown.

The performance of the dual-wavelength monitoring system was directly contrasted

(Figure 3 b-d) to DAPI alone and also to commercial SYBR-Green I, a gold standard for the

detection of DNA in PCR technologies.71 Although the conventional intercalators excelled with

respect to the absolute fluorescence enhancement factor upon DNA addition, the DAPI/CB7-

CF system showed a much larger linear-response range, which is advantageous for absolute

quantification. With identical instrumental settings, SYBR-Green I showed an approximately

linear response up to 3 g/mL, while the range of the FRET system extends up to 20 g/mL

(Figure 3.9 a). Consequently, we propose that the DAPI/CB7-CF FRET pair can be used as a


58

complementary and non-proprietary indicator dye for nucleotide sensing technologies,

including real-time PCR. As a proof of principle, we carried out fluorescence-based DNA

melting experiments, which demonstrated that the FRET system responds reversibly to the

melting of DNA in dependence on temperature (Figure 3.9 b); interestingly, the heating and

cooling curves displayed a large hysteresis, presumably as a consequence of the more complex

competitive equilibria involved when compared to a simple dye-intercalation approach.

3.5 Conclusions

In conclusion, we have introduced a host-guest FRET pair based on the macrocyclic host CB7

labelled with carboxyfluorescein as the acceptor and the nucleic stain DAPI as donor and guest.

We demonstrated that the resulting host-guest FRET ensemble can be used for quantitative

sensing of DNA with an excellent linear dependence of the ratiometric fluorescence intensities

(I450/I520). The approach opens a new avenue to highly accurate DNA quantification such as

real-time PCR, for which very few alternatives exist to the presently employed proprietary dyes.

Moreover, this approach provides potential strategy for new genetic method to convert single-

wavelength sensor into ratiometric probes.

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62

Chapter 4 Precise Supramolecular Control of Surface Coverage Densities on Polymer Micro-

and Nanoparticles

63

Chapter 4. Precise Supramolecular Control of

Surface Coverage Densities on Polymer Micro-

and Nanoparticles


and Nanoparticles

64


and Nanoparticles

65

This chapter is derived from the content of the following publication:

Shuai Zhang, Zoe Domínguez, Khaleel I. Assaf, Mohamed Nilam, Thomas Thiele, Uwe

Pischel, Uwe Schedler, Werner M. Nau, and Andreas Hennig, Precise Supramolecular Control

of Surface Coverage Densities on Polymer Micro- and Nanoparticles, Chem. Sci., 2018, 9,

8575-8581.

In this chapter we report the controlled surface functionalization of micro- and nanoparticles by

supramolecular host-guest interac-tions. Our idea is to exploit the competition of two high

affinity guests for binding to the surface-bound supramolecular host cucurbit[7]uril (CB7). To

establish our strategy, surface azide groups were introduced to hard-sphere

(poly)methylmethacrylate particles with a grafted layer of poly(acrylic acid), and a propargyl

derivative of CB7 was coupled to the surface by click chemistry. The amount of surface-bound

CB7 was quantified with the high-affinity guest aminomethyladamantane (AMADA), which

revealed CB7 surface coverage densities around 0.3 nmol/cm2 indicative of a 3D layer of CB7

binding sites on the surface. The potential for surface functionalization was demonstrated with

an aminoadamantane-labeled rhodamine (Ada-Rho) as a second high-affinity guest.

Simultaneous incubation of CB7-functionalized particles with both high-affinity guests,

AMADA and Ada-Rho, revealed a simple linear relationship between the resulting surface

coverage densities of the model fluorescent dye and the mole fraction of Ada-Rho in the

incubation mixture. This suggests a highly modular supramolecular strategy for the stable

immobilization of application-relevant molecules on particle surfaces and a precise control of

their surface coverage densities.

4.1 Introduction

The possibility to precisely control the attachment of application-relevant molecules to

the surfaces of micro- and nanoparticles creates a powerful platform technology with a

large number of conceivable applications. A vast number of different methods have

therefore been explored, but the performance of these materials is still limited by the

shortcomings of existing surface functionalization methods.1-3 For example, the highly

specific and strong (Ka 1015 M1) binding interaction between the small-molecule

ligand biotin with the proteins avidin or streptavidin is popular for surface attachment in

initial proof-of-principle studies with nanoparticles,1 but it has also been noted that the

tetrameric structure of the proteins and their large size (i) may induce crosslinking, (ii)

prevent a precise control of conjugate stoichiometry, and (iii) limit the maximum


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66

achievable surface coverage densities.4-6 Alternative and refined strategies to reliably

functionalize particle surfaces with molecular components are thus highly desired to

achieve the transition from proof-of-principle to real applications of nanobioconjugate

materials.1-3

Numerous supramolecular host-guest systems have been previously investigated

with the goal to equip nanoparticles with molecular recognition capabilities, and this has

enabled a large variety of potential applications.7-22 The typically µM to mM affinities

of most synthetic host-guest systems ensure reversible binding, which is highly

desirable, e.g., in nanoparticle polymer composites, to control nanoparticle assembly, or

for drug delivery. However, for surface functionalization aiming towards bioanalytical

applications, host-guest complexes are required, which are sufficiently stable at much

lower concentrations. To account for the low binding affinity, multivalent systems have

been explored, but this strategy sacrifices – similar to the (strept)avidin/biotin system –

control over the binding stoichiometry and induces particle crosslinking.11, 14, 23, 24

Cucurbit[n]urils (CBn, n = 5–8, 10, and 14) composed of n glycoluril units

comprise a class of biocompatible macrocycles, which stand out from all other

supramolecular host molecules by remarkably high binding affinities (Ka > 1017 M1)25-

30 towards certain guest molecules. This exceeds the affinity of biotin with (strept)avidin

and clearly suggests CBn hosts as a complementary tool in bioconjugation.4, 31

Moreover, CBs are highly biocompatible as shown in various applications, e.g., in

enzyme and membrane transport assays,32-36 for immobilization of proteins and cells on

planar surfaces,37-39 for enrichment and isolation of proteins by affinity-beads,40-42 for

supramolecular PEGylation of biopharmaceuticals,43 for multi-stimuli-responsive

release,44 and for protein imaging.45 CBn hosts were also adsorptively attached to metal

surfaces such as planar46, 47 and spherical48-50 gold surfaces, and to iron oxide

nanoparticles through multidentate binding of their carbonyl-fringed portals.51, 52

However, this adsorptive surface functionalization affects the host-guest recognition

properties of the cavity since one of the portals is involved in surface binding. Moreover,

the presence of two carbonyl-fringed portals may lead to particle aggregation.46, 51-53

Herein, we present for the first time the covalent surface modification of small, hard-

sphere core-shell particles with cucurbit[7]uril (CB7). To demonstrate this, we use polymer

microparticles, also known as beads or microspheres, as well as nanoparticles. These particles

play important roles, e.g., in optical tweezers,54 drug delivery,55 medical imaging,56, 57 and in

diagnostic, multiplexing bead-based assays,58-60 as well as lateral flow immunoassays.61, 62 Our


and Nanoparticles

67

approach affords, similar to the previously established planar surfaces37-39 and porous resins of

large (>40 µm) sepharose beads,40-42 a suitable supramolecular strategy to subsequently

immobilize application-relevant molecules. Moreover, we demonstrate herein, that host-guest

chemistry allows an unprecedented control of surface functionalization of particles in an easily

quantifiable manner.

4.2 Particle Synthesis

Figure 4.1 a) Synthesis of propargyl-CB7 (CB7-OPr). b) Synthesis of CB7-

functionalized PMMA microparticles. c) Optical microscopy image (at 40-fold

magnification) of 5 mg/ml CB7-functionalized PMMA particles in 10 mM (NH4)2HPO4,

pH 7.2.

For the synthesis of CB7-functionalized particles, we decided to use copper-catalyzed

azide-alkyne click chemistry (Figure 4.1). To obtain the required propargyl-CB7 (CB7-

OPr), a different procedure than the recently reported method by Zhang and coworkers

was established.63 First, monohydroxylated-CB7 was synthesized according to the


and Nanoparticles

68

method of Bardelang and coworkers.64 Quantitative conversion into CB7-OPr was then

achieved by repeated cycles of crude product resubmission to propargyl bromide and

sodium hydride in DMSO, and the identity and purity of CB7-OPr was confirmed by

comparison with the reported data (see Electronic Supplementary Information, ESI†).63

As particles, we used microparticles (mean diameter of 2.55 µm) composed of a

compact, hard-sphere poly(methylmethacrylate) (PMMA) core and a grafted layer (111

µmol/g) of poly(acrylic acid) (PAA). These particles were chosen for their ease of

handling, optical transparency, and the possibility for microscopic observation, and they

were previously characterized by us in detail.6, 65-69 In addition, commercially available

nanoparticles (mean diameter of 110 nm) with a hard-sphere polystyrene core and

surface carboxylic acids were also tested.

Azide groups were introduced by reaction of surface COOH groups with 11-azido-3,6,9-

trioxaundecan-1-amine (ATA) using standard amide coupling protocols,6, 65 and CB7

was finally covalently bound to the surface by Cu-catalyzed click chemistry (Figure 4.1

a, b). The resulting particles were washed into 10 mM (NH4)2HPO4, pH 7.2, which was

also used for all subsequent experiments. Inspection by optical microscopy indicated no

increased tendency for aggregation compared to the PAA- and ATA-functionalized

particles, and by IR spectroscopy, which was in accordance with surface-immobilized

CB7.

4.3 Quantification of Surface-Bound CB7

The most compelling evidence for surface functionalization with CB7 was obtained by

successful extraction of aminomethyladamantane (AMADA) from 10 mM (NH4)2HPO4,

pH 7.2 using CB7-functionalized particles (Figure 4.2). The ultra-strong affinity of

AMADA to CB7 (Ka ca. 1015 M1)27 was previously exploited by us to evaluate various

surface quantification methods.6, 65-69 Therein, AMADA-putrescine was used as a

chemical labelling agent for surface COOH groups by amide formation and the number

of surface-bound AMADA was determined by extraction of CB7 and subsequent

quantification of remaining CB7 by the fluorescent dye acridine orange (AO).6, 64

Similarly, remaining AMADA is now quantified by addition of a known concentration

of CB7 and AO to the supernatant.


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Figure 4.2 Quantification of CB7 on particle surfaces. a) Incubation of CB7-

functionalized particles with AMADA and subsequent centrifugation gives a

supernatant, which can be analyzed to afford the concentration of remaining AMADA

by addition of the fluores-cent dye acridine orange (AO) and CB7. b) Dependence of

fluorescence spectral changes (exc = 450 nm, obs = 510 nm) of the supernatant on the

volume of added CB7-functionalized particles stock solution (10 mg/mL) during

incubation with 25 µM AMADA. And the bottom figure compares CB7-functionalized

(filled circles) and ATA-functionalized particles as control (open circles) in 10 mM

(NH4)2HPO4, pH 7.2.

In accordance with our expectations, an increase of the fluorescence with

increasing volume of the particle stock solution immediately indicated the particle-

dependent extraction of AMADA and thus successful immobilization of CB7 on the

particle surface (Figure 4.2b). The clearly linear dependence with no indications of the

typical curvature of a reversible binding isotherm is consistent with quantitative binding

between surface-bound CB7 and AMADA, as well as with quantitative binding in the

supernatant analysis (Figure 4.3a). As a consequence, we can determine the average

(bulk) CB7 surface coverage densities from the intersection between the linear increase

and the plateau region in the inset of Figure 4.2, which indicates the amount of particles

required to extract all AMADA from solution.65, 66


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500 550 600 650 7000

50

100

150

200

250

0 1 20.2

0.6

1.0F

luo

rescen

ce

Wavelength (nm)

AMADA

a)

F/F

0

[AMADA] (µM)

500 600 700

0

100

200

300

0 30 60

1.0

1.2

1.4

1.6

Flu

ore

scen

ce

Wavelength (nm)

amount

of particles

during incubation

b)

F/F

0

Volume (µL)

Figure 4.3 a) Competitive fluorescence titration (λexc = 450 nm) of 2 µM AO and 1.1 µM CB7

in 10 mM (NH4)2HPO4, pH 7.2. The inset shows the corresponding fluorescence titration (λem

=510 nm) plot normalized to the initial fluorescence intensity and demonstrates quantitative 1:1

binding between AMADA and CB7 in solution. b) Dependence of fluorescence spectral

changes (exc = 450 nm, obs = 510 nm) of the supernatant on the volume of added CB7-

functionalized polymer particles stock solution (10 mg/mL) during incubation with

(dimethylaminomethyl)ferrocene (surface coverage density = 5.5 µmol/g). The inset compares

CB7-functionalized (filled circles) and ATA-functionalized particles as control (open circles).

This simple method to determine the resulting CB7 loading capacities and surface

coverage densities was highly reproducible (Table 4.1, coefficient of variation of ca. 2% for n

= 7) and enabled us to evaluate various reaction conditions and their reproducibility during

surface functionalization in a straightforward manner (Table 4.2). For example, we could show

that washing the particles after click reaction with buffer containing EDTA or not had no

influence on the resulting surface coverage, which suggests that no copper ions remained on

the particle surface (cf. entry #1 and #2 in Table 4.2). Furthermore, we could establish that the

click reaction is considerably reproducible. The resulting loading capacities of three different

reaction batches varied by less than 1% (Table 4.2, entry #1).


and Nanoparticles

71

Table 4.1 Reproducibility measurements for the quantification of surface-bound CB7.

Experiment loading capacity

(µmol/g)

coupling yield

(%)

CB7 surface density

(nmol/cm2)

1 5.66 5.10 0.286

2 5.81 5.23 0.294

3 5.52 4.97 0.279

4 5.88 5.30 0.297

5 5.63 5.07 0.285

6 5.74 5.17 0.290

7 5.80 5.23 0.293

average 5.72 5.15 0.289

standard deviation 0.11 0.10 0.006

coefficient of

variation ca. 2%

Table 4.2 Reaction results for CB7 surface functionalization.a

entry # reaction conditionsb

loading

capacity

(µmol/g)

coupling yield

(%)c

CB7 surface

density

(nmol/cm2)

1 1) 3 h at pH 5.0, 2) 24 h (with

EDTA)d 5.69 ± 0.04 5.13 ± 0.03 0.288 ± 0.002

2 1) 3 h at pH 5.0, 2) 24 h

(without EDTA) 5.67 5.11 0.287

3 1) 3 h at pH 5.0, 2) 48 h 6.2 5.6 0.32

4 1) 3 h at pH 7.2, 2) 24 h 4.4 4.0 0.22

5 1) 6 h at pH 7.2, 2) 24 h 4.8 4.3 0.24

a Values were determined with the AMADA assay (Section 5.1). b Functionalization of COOH

surface groups with 1) azide groups was performed in pH 5.0 or pH 7.2 buffer for 3 h or 6 h

and then with 2) CB7-OPr in a click reaction for 24 h or 48 h (see Section 4.1 and 4.2 for

details). c With respect to 111 µmol/g surface COOH. d From three replicates.


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The resulting CB7 surface coverage densities (ca. 0.3 nmol/cm2, i.e. 1.8 CB7

molecules/nm2 or a loading capacity of ca. 5.7 µmol/g) were significantly higher than

the value for a CB7 monolayer on planar gold surfaces (ca. 0.08 nmol/cm2),38 which is

in accordance with a grafted 3D layer of PAA on the particle surface and thus a 3D layer

of surface-bound CB7. Interestingly, the overall coupling yields in our two-step

functionalization protocol were in very good agreement with typical coupling yields for

amide formation only (ca. 5% with respect to COOH groups of surface PAA),6, 66 which

suggests that the second step, the click reaction to attach CB7 onto the surface, is nearly

quantitative. As controls, azide-functionalized particles lacking CB7 gave no change in

fluorescence intensity (inset of Figure 4.2b), which excluded unspecific binding of

AMADA to the particle surface. As additional controls, identical values for the CB7

surface coverage density were determined by extraction of

(dimethylaminomethyl)ferrocene (Figure 4.3b), which further excludes any unspecific

binding. Moreover, poly(styrene) nanoparticles with surface carboxylic acid groups

could be similarly surface-functionalized and analyzed, which afforded CB7 surface

coverage densities of ca. 0.1 nmol/cm2 (Figure 4.4).

500 600 700

0

200

400

600

Flu

ore

sce

nce

Wavelength (nm)

a)

increasing amount

of nanoparticles

0 50 100 150

150

300

450

600

Flu

ore

scen

ce

Volume (µL)

b)

Figure 4.4 a) Dependence of fluorescence spectral changes (exc = 450 nm, obs = 510 nm) of

the supernatant on the volume of added CB7-functionalized (poly)styrene nanoparticles stock

solution (10 mg/mL) during incubation with 4.56 µM AMADA. b) Respective titration plot.

4.4 Supramolecular Surface Functionalization

The possibility to reliably immobilize application-relevant molecules on the surface of

CB7-functionalized particles was demonstrated with an aminoadamantyl-labeled

rhodamine (Ada-Rho, from Prof. Uwe Pischel) as a model fluorescent dye (Figure 4.5).

Immobilization was simply achieved by addition of CB7-functionalized particles to a


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buffered aqueous solution containing Ada-Rho and washing, which gave a bright red

fluorescence from surface-bound Ada-Rho in fluorescence microscopy, whereas the size

of the particles remained same as judged by comparison of the bright-field images of

particles with and without Ada-Rho. The absorbance as well as the fluorescence of the

supernatant decreased linearly with increasing amounts of CB7-functionalized particles

indicating quantitative binding (Figure 4.5). As controls, when the CB7 cavity was

blocked by pre-incubation with the stronger binder AMADA,70 the particles were unable

to extract Ada-Rho. Similarly, no Ada-Rho extraction was observed with ATA-

functionalized particles (Figure 4.6).

Figure 4.5 a) Surface functionalization of CB7 particles with Ada-Rho. b) Fluorescence

microscopy images (exc = 546 nm) of CB7-functionalized particles (5 mg/mL) with surface-

bound Ada-Rho.


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550 600 650 700 7500

400

800

0 20 40 60

300

400

500F

luo

resce

nce

Wavelength (nm)

a)

Particles

Flu

ore

sce

nce

Volume (µL)

400 450 500 550 600

0.00

0.04

0.08

0 20 40 60

0.03

0.04

0.05

0.06

Abso

rban

ce

Wavelength (nm)

b)Particles

Abso

rban

ce

Volume (µL)

Figure 4.6 Variation of a) fluorescence intensity (λexc = 520 nm, λobs = 580 nm) and b)

absorbance (λobs = 570 nm) of the supernatant (150 µL) of a mixture of 10 µM Ada-Rho and

varying amounts of CB7 particles (10 mg/mL) after centrifugation and dilution to 2000 µL in

10 mM (NH4)2HPO4, pH 7.2.

550 600 650 700 7500

200

400

0 20 40 60

0.8

1.0

1.2

a)

Flu

ore

sce

nce

Wavelength (nm)

F0/F

Volume (µL)

400 500 600

0.00

0.01

0.02

0 20 40 60

0.8

1.0

1.2

b)

Abso

rban

ce

Wavelength (nm)

A0/A

Volume (µL)

Figure 4.7 Variation of a) fluorescence intensity (λexc = 520 nm, λobs = 580 nm) and b)

absorbance (λobs = 570 nm) of the supernatant (220 µL) of a mixture containing 1.7 µM Ada-

Rho and varying amounts of ATA-functionalized particles (10 mg/mL) after centrifugation and

dilution to 2000 µL in 10 mM (NH4)2HPO4, pH 7.2.

As in the case of AMADA (see above), the loading capacity of Ada-Rho could

be determined from the intersection between the linear decrease and the plateau region

of the titration plots, which revealed that the amount of Ada-Rho on the surface was

significantly lower (3.0 µmol/g) than the amount of CB7 (5.7 µmol/g). Such a

dependence of the surface coverage density on the size of the immobilized molecule is

common, and may be due to steric repulsion between molecules at adjacent binding sites,

a size-dependent diffusion through the grafted 3D PAA network, or a conformational


and Nanoparticles

75

rearrangement of the PAA chains in response to the presence of the hydrophobic dye,

which may block otherwise available binding sites.

500 600 700

0

100

200

300

400

500

amount of

particles

during incubation

Flu

ore

scen

ce

Wavelength (nm)

a)

0 20 40 60 80

250

300

350

400

450

V = 52.9 µL

loading capacity = 2.84 µmol/g

Flu

ore

scen

ce

Volume (µL)

b)

Figure 4.8 Quantification of remaining CB7 binding sites on Ada-Rho-functionalized particle

surfaces. a) Fluorescence spectra of different samples. b) Dependence of fluorescence spectral

changes (exc = 450 nm, obs = 510 nm) of the supernatant on the volume of added functionalized

polymer particles stock solution (10 mg/mL) during incubation with 25 µM AMADA in 10 mM

(NH4)2HPO4, pH 7.2.

0 20 40 60

0.5

0.6

0.7

0.8

0.9

1.0 Ada-Rho only

Ada-Rho + 0.5 eq AMADA

A0/A

Volume (µL)

Figure 4.9 Extraction with CB7-functionalized beads using solutions of 0.44 µM Ada-Rho

(solid circles) or a mixture of 0.44 µM Ada-Rho and 0.22 µM AMADA (open circles). The

intersections refer to final Ada-Rho surface loadings of 3.0 µmol/g and 1.8 µmol/g,

respectively.

The latter explanation can be ruled out, because particles, in which all binding

sites were saturated with Ada-Rho could still extract additional 2.8 µmol/g AMADA

from solution (Figure 4.8) and the sum of Ada-Rho (3.0 µmol/g) and AMADA (2.8

µmol/g) was in excellent agreement with the CB7 surface coverage density determined

with AMADA only (5.7 µmol/g). In other words, approximately 50% of all available


and Nanoparticles

76

CB7 binding sites were occupied when the particles were incubated with Ada-Rho first,

and then, the remaining binding sites could be occupied in a second incubation step with

AMADA.70 A different result was, however, obtained, when the CB7-functionalized

particles were incubated with a mixture of both, Ada-Rho and AMADA. This reduced

the amount of Ada-Rho that can be extracted with a specific amount of particles (Figure

4.9), which is consistent with a competitive occupation of the CB7 binding sites by

AMADA, and suggests an elegant method to control the surface coverage density of

CB7-functionalized particles.

4.5 Supramolecular Control of Surface Coverage

Densities

400 500 600

0.00

0.03

0.06

0 50 100

0.03

0.04

0.05

0.06

Ab

so

rba

nce

Wavelength (nm)

Ada-Rho/AMADA

100 : 0

a)

Ab

so

rba

nce

Volume (µL)

2.95 µmol/g

Ada-Rho

400 500 600

0.00

0.03

0.06

0 50 100

0.020

0.025

0.030

0.035

Ada-Rho/AMADA

7.5 µM : 2.5 µM

Abso

rban

ce

Wavelength (nm)

b)

Abso

rban

ce

Volume (µL)

2.34 µmol/g

Ada-Rho

400 500 600

0.00

0.03

0.06

0 50 100

0.015

0.020

0.025

0.030

Abso

rban

ce

Wavelength (nm)

c)1.41 µmol/g

Ada-Rho

Ada-Rho/AMADA

5 µM : 5 µM

Abso

rban

ce

Volume (µL)

400 500 600

0.00

0.03

0.06

0 50 100

0.006

0.007

0.008

0.009

Ab

so

rba

nce

Wavelength (nm)

d)0.74 µmol/g

Ada-Rho

Ada-Rho/AMADA

25 : 75

Ab

so

rba

nce

Volume (µL)

Figure 4.10 Variation of absorbance (λobs = 570 nm) of the supernatant (370 µL) containing a

mixture of 2 nmol AMADA and Ada-Rho (molar fraction of Ada-Rho: a) 1.0, b) 0.75, c) 0.5,

and d) 0.25) incubated varying amounts of CB7 functionalized particles after centrifugation and

dilution to 2000 µL in 10 mM (NH4)2HPO4, pH 7.2. The insets show the dependence of

absorbance at λobs = 570 nm on the volume of added particle stock solution (7 mg/mL).


and Nanoparticles

77

In order to investigate in detail how the presence of AMADA during incubation with

Ada-Rho influences the resulting surface coverage densities, varying amounts of CB7

particles were incubated with mixtures containing different mole fractions of AMADA

and Ada-Rho. This indicated that the amount of particles, which are required to extract

Ada-Rho from solution, remained approximately the same despite varying total Ada-

Rho concentrations in the incubation solution (Figure 4.10). Consequently, the surface

coverage densities of Ada-Rho as determined by our extraction-based surface

quantification method de-pended linearly on the molar fraction of AMADA and Ada-

Rho over its entire range (Figure 4.11).

0.00 0.25 0.50 0.75 1.00

0

1

2

3

Surf

ace

Ada

-Rho

(µ

mo

l/g

)

[AMADA]/([AMADA]+[Ada-Rho])

Figure 4.11 Dependence of resulting Ada-Rho surface coverage densities on the mole

fraction of the competitor AMADA in mixtures of AMADA and Ada-Rho (6 µM total

concentration).

It is noteworthy that such a simple linear relationship came as a surprise, because

first, the number of available binding sites is different for AMADA and Ada-Rho (see

above), and second, the resulting surface concentrations of two competitors should, in a

thermodynamically equilibrated mixture, also depend on their binding affinities.30, 70 We

conclude that the occupation of the CB7 binding cavities is diffusion-limited, which

leads to a kinetically controlled competitive occupation of the CB7 binding sites with

AMADA and Ada-Rho. In addition to the mechanistic insights, our results clearly

demonstrate that the surface coverage densities of application-relevant functional

molecules on CB7-functionalized particles can be precisely adjusted by using two

competitive cavity binders. This allowed us to prepare a series of particles with exactly

known Ada-Rho surface coverage densities in a straightforward manner, which could

then be analyzed by fluorescence spectroscopy and microscopy (Figure 4.12 and 4.13).


and Nanoparticles

78

Therein, a linear increase in the fluorescence intensity was observed at low surface

coverage densities (up to ca. 1 µmol/g), whereas higher surface fluorophore coverage

densities did not lead to a further increase in fluorescence intensity.

550 600 650 700 7500

50

100

0 1 2 3

0.0

0.5

1.0

Flu

ore

scen

ce

Wavelength (nm)

surface

Ada-Rho

(II 0

)/(I

ma

xI 0

)

Surface Ada-Rho (µmol/g)

a)

0.0 0.5 1.0 1.5 2.0

40

60

80

100

Mea

n F

luore

sce

nce

Surface Ada-Rho µmol/g

b)

Figure 4.12 a) Steady-state fluorescence spectra (exc = 520 nm) of Ada-Rho-labeled

particles (0.2 mg/mL) in 10 mM (NH4)2HPO4, pH 7.2 with increasing surface coverage

densities of Ada-Rho (adjusted by using mixtures of Ada-Rho and AMADA, see Figure

4.10). The inset shows the dependence of the normalized fluorescence intensity (em =

585 nm) on the surface coverage densities. b) Dependence of the mean fluorescence

intensity within the regions of interest (ROIs) in fluorescence microscopy images (see

Figure 4.12). Error bars represent the standard deviation (n = 3).

This result is consistent with our previous observations with covalently bound

surface fluorophores and originates most likely from self-quenching due to an increased

probability of non-fluorescent aggregate formation at high surface coverage densities.6,

69 It is important to note that uncertainties arising from the covalent surface modification

protocol needed to be previously eliminated by control measurements with absolute

fluorometry involving an integrating sphere set-up,6 whereas in this report, we exploit

supramolecular host-guest chemistry to unambiguously determine and control the

fluorophore surface coverage densities. Another interesting consideration is that our

competition-based surface functionalization protocol applies an excess of two

competitors for a limited number of accessible binding cavities, which could lead to a

more homogeneous distribution of surface coverage densities within a particle

population than methods relying on substoichiometric amounts of reagent. The latter

require a very efficient mixing to prevent a local depletion during reagent addition to a

reaction mixture.


and Nanoparticles

79

Figure 4.13 Fluorescence microscopy images (546 nm bandpass filter) of Ada-Rho-labeled

particles (5 mg/mL) with increasing surface coverage densities of Ada-Rho (expressed as

loading capacities, LC). The white circles represent the automatically assigned ROIs by the

software ImageJ.

0 1 2

0

150

300100 mM AMADA

Flu

ore

sce

nce

Time (h)

no AMADA

50 mM AMADA

Figure 4.14 Time-dependent dissociation of Ada-Rho from CB7-functionalized particles in

presence of the competitor AMADA. Ada-Rho functionalized particles (2 mg/mL) were

incubated with 50 mM (open circles) or 100 mM (filled circles) AMADA in 10 mM

(NH4)2HPO4, pH 7.2 and the fluorescence of the supernatant (lexc = 520 nm, lobs = 585 nm) was

measured after certain time intervals. As control, the fluorescence of the supernatant did not

show any release of surface-bound Ada-Rho in absence of the competitor AMADA (filled

squares).

In contrast, when the particles were first surface-functionalized with Ada-Rho

and subsequently incubated with high concentrations of AMADA for a longer period, a


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80

slow dissociation of Ada-Rho from the surface was observed (Figure 4.14). This clearly

demonstrates the principal reversibility of the host-guest interaction despite the strong

affinity. Furthermore, it enables the determination of exchange kinetics on particles

surfaces and presents a complementary conceptual framework for kinetic studies of host-

guest systems.71, 72

4.6 Conclusions

In conclusion, we have introduced polymer particles surface-functionalized with the

supramolecular host molecule CB7. Therefore, we synthesized monofunctionalized CB7

bearing a propargyl group, which can be covalently bound to azide-functionalized

surfaces of polymer particles by click chemistry. The successful reaction and the

resulting number of CB7 molecules on the particle surface was reliably quantified and

the ease of subsequently introducing other molecular components was demonstrated

with the fluorescent dye Ada-Rho. Compared with covalent conjugation strategies,

simple mixing of the two components in water suffices and other additives such as

coupling reagents are not required. Overall, this provides a reliable host-guest-based

surface functionalization method in water with wide-ranging perspectives. We have

shown that it allows to precisely control surface coverage densities, which unfolds

numerous perspectives in varying areas. For example, it allows the investigation of

fluorescence quenching mechanisms on surfaces and validation of absolute

fluorometry,6, 69 the systematic testing of hitherto unverified, theoretical quantification

models for spherical substrates in x-ray photoelectron spectroscopy (XPS),68 as well as

straightforward construction of multimodal probes for medical imaging,73 potentially for

clinical applications, which require regulatory clearance and thus metrological

traceability.67 Varying surface coverage densities can have a significant impact on the

efficiency of nanoparticle-based diagnostics and therapy.1-3, 74-77 We also demonstrated

the controllable release of surface-bound Ada-Rho by a stronger cavity binder. This

suggests the use of CB7-functionalized particles for the construction of stimuli-

responsive release systems.

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(74) M. Colombo, L. Fiandra, G. Alessio, S. Mazzucchelli, M. Nebuloni, C. De Palma, K.

Kantner, B. Pelaz, R. Rotem, F. Corsi, W. J. Parak and D. Prosperi, Nat. Commun., 2016, 7,

13818.

(75) H. S. Choi, W. Liu, F. Liu, K. Nasr, P. Misra, M. G. Bawendi and J. V. Frangioni, Nat.

Nanotechnol., 2010, 5, 42-47.

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Wittrup, M. G. Bawendi and A. Y. Ting, Nat. Meth., 2008, 5, 397-399.

(77) M. Howarth and A. Y. Ting, Nat. Protoc., 2008, 3, 534-545.


and Nanoparticles

86

Summary and Outlook

87

Summary and Outlook

The thesis describes the synthetic procedure of clickable mono-functionalized cucurbit[n]urils

(n = 6, 7) and mainly on the exploration of new applications based on the mono-functionalized

CBn. we explore two applications based on mono-functionalized CB7.

One is to attach fluorescein on the outer rim of CB7, thus a host-guest FRET pair based

on the macrocyclic host CB7 labelled with carboxyfluorescein as acceptor and the nucleic stain

DAPI as donor and guest was designed. This supramolecular FRET pair is used for quantitative

sensing of DNA with an excellent linear dependence of the ratiometric fluorescence intensities.

Compared with commercial DNA staining dyes, the FRET pair with dual-wavelength can offer

more precise results. Such approach can be applied to quantify DNA accurately and potentially

be used in real-time PCR.

For the second application, mono-functionalized CB7 is introduced on the surface of

nano-/macro-particles. After that, incubation of CB7-functionalized particles with two high-

affinity guests, resulted in a simple linear relationship between surface coverage densities of

one fluorescent guest and the mole fraction of this guest in the incubation mixture. This suggests

a highly modular supramolecular strategy for the stable immobilization of application-relevant

molecules on particle surfaces and a precise control of their surface coverage densities.

For the future plan based on mono-functionalized CB7, I would like to suggest new

members in our group to synthesize CB7 derivatives attathed with organelle recognition

functional groups and apply this into cell culture. The other promising project is to connect two

CB7 toghther to dimerize guests and biomolecules, for example to form proteins dimer through

host-guest interactions.

Summary and Outlook

88

89

Appendices

Supporting Information for Chapter 3

91


Materials and Methods

CB7 and CB7-OPr were synthesized according to literature procedures.1 DAPI (4′,6-diamidine-

2′-phenylindole dihydrochloride) and Type III salmon sperm DNA (ca. 2000 base pair)2 were

commercial samples (Sigma Aldrich) and used as received without further purification. 6-

FAM-azide was purchased from Baseclick and used as received. Reagents for synthesis were

from Fluka, Carl Roth, and Sigma-Aldrich. TLC was performed on SIL G/UV254 (Macherey-

Nagel). Buffers and salts were of the highest purity available from Fluka and Sigma-Aldrich

and used as received.

UV-Vis absorption measurements were performed with a Varian Cary 4000

spectrophotometer and the fluorescence spectra were recorded on a Varian Cary Eclipse

spectrofluorometer. All measurements were performed at ambient temperature, except for the

fluorescence-based DNA melting experiment, which was recorded in a temperature range from

20 to 90 °C with rectangular quartz cuvettes with 1-cm optical path length. The fluorescence

lifetimes were measured by time-correlated single-photon counting (FLS920, Edinburgh

Instruments Ltd.). For the lifetime measurements, DAPI and CB7-CF were excited at 373 nm

by using a diode laser (PicoQuant, LDH-P-C 375, fwhm ca. 50 ps) and the fluorescence was

followed at 470 nm. 1H NMR spectra were recorded on a Jeol ECS400 MHz and chemical shifts

(δ) are reported in ppm relative to TMS (δ = 0 ppm). Mass spectra were recorded on a Bruker

MALDI TOF spectrometer; HCCA (α-cyano-4-hydroxycinnamic acid) was used as a matrix.

Synthesis and Characterization of CB7-CF

8.7 mg (7.2 μmol) CB7-OPr was dissolved in 0.7 mL anhydrous DMSO and 6 mg (13 μmol)

6-FAM-azide was added. Then, 10 mg (50 μmol) sodium L-ascorbate was added into 2.8 mL

55% DMSO aqueous solution containing 4.47 mg (28 μmol) CuSO4 and 14.86 mg (28 μmol)

tris(benzyltriazolylmethyl)amine (TBTA). These two solutions were mixed and stirred at room

temperature for 24 h. 50 mL diethyl ether was added, and the resulting precipitate was washed

three times with 25 mL MeOH. Drying under high vacuum afforded a dark solid. The crude

product containing unreacted CB7-OPr, 6-FAM-Azide, and CB7-CF, was purified by column

chromatography. In detail, the mixture was dissolved in 600 μL H2O/HCOOH 1:1 and loaded

onto silica gel 60 (0.04-0.063 mm) and the column was eluted with H2O/AcOH/HCOOH

10:10:1.5. The eluent was collected in fractions of 2 mL (50 fractions) and the fractions

containing pure CB7-CF were combined. Evaporation of the solvent gave 4 mg (2.4 μmol, 33%


92

yield) CB7-CF as a brown solid. The product identity was confirmed by mass spectrometry

(Figure S2) and NMR (Figure S3). 1H NMR (400 MHz, D2O with excess p-xylene diamine3),

(ppm) = 8.55 (H1), 8.44 (H2), 7.96 (H7), 7.75 (H6), 7.48 (H4), 7.46 (H3), 7.37 (H5, H10,

Hbfree), 6.58 (Hbbound), 5.73 (Hd), 5.52 (He), 4.79 (H11, HOD), 4.24 (H14, Hc), 4.00 (H13),

3.92 (Hafree), 3.49 (Habound), 1.90 (H12). MALDI-TOF MS calculated for [CB7-CF] 1675.43,

found 1675.67; calculated for [CB7-CFH++Na+] 1697.41, found 1697.63.

Fig. S1 Synthesis of CB7-CF.

Fig. S2 MALDI-TOF mass spectra of CB7-CF (HCCA matrix, positive mode).


93

Fig. S3 1H NMR spectra of CB7-CF in D2O with excess p-xylene diamine (see ref. 2) to aid in

solubilization (magenta peak assignments).

Fig. S4 DFT-optimized structures (B3LYP/3-21G*) of different possible co-conformations for

the CB7-CF/DAPI complex (in gas phase). The distance (d) between the center of mass of the

CF and DAPI is given in Å. The structure shown in (a) was found to be more stable than that

in (b) by 4.9 kcal/mol.


94

400 500 600 700

0

100

200

300

400

500

I

(a)

[DAPI] 0 M

[DAPI] 0.04 M

[DAPI] 0.15 M

[DAPI] 0.38 M

[DAPI] 0.82 M

[DAPI] 1.55 M

[DAPI] 2.50 M

[DAPI] 3.76 M

[DAPI] 5.17 M

[DAPI] 6.36 M

[DAPI] 7.37 M

1 M CB7+ [DAPI]

/nm

400 450 500 550 600 650 700

0

100

200

300

400

500

I

(b)

1 M CB7-CF+20 M AMADA+[DAPI] [DAPI] 0 M

[DAPI] 0.04 M

[DAPI] 0.15 M

[DAPI] 0.38 M

[DAPI] 0.82 M

[DAPI] 1.55 M

[DAPI] 2.50 M

[DAPI] 3.76 M

[DAPI] 5.17 M

[DAPI] 6.36 M

[DAPI] 7.37 M

/nm

400 500 600 700

0

100

200

300

400

500

I

[DAPI] 0 M

[DAPI] 0.04 M

[DAPI] 0.15 M

[DAPI] 0.38 M

[DAPI] 0.82 M

[DAPI] 1.55 M

[DAPI] 2.50 M

[DAPI] 3.76 M

[DAPI] 5.17 M

[DAPI] 6.36 M

[DAPI] 7.37 M

/nm

Only DAPI(c)

Fig. S5 Fluorescence titrations (ex = 360 nm) with increasing amounts of DAPI (0 to 7.4 µM)

in the presence of (a) 1 µM CB7, (b) 1 µM CB7-CF as well as 20 µM AMADA, and (c) only

DAPI. All experiments were performed in 10 mM (NH4)2HPO4, pH 7.2.

300 350 400 450 500 550 600

0

100

200

300

400

500

I

(a)

/nm

450 500 550 600 650

0

20

40

60

80

100

I

(b)

/nm

ex

/nm

340

345

352

355

360

365

370

380

Fig. S6 (a) Excitation spectrum (em = 520 nm) of 1 M CB7-CF in

10 mM (NH4)2HPO4, pH 7.2. (b) Emission spectra of 1 M CB7-CF at varying excitation

wavelengths.

Data Analysis

Binding constants were calculated from the fluorescence titrations (Fig. 3b, inset) by assuming

a 1:1 complex stoichiometry and performing a nonlinear fitting according to eq. S5. [G] and

[HG] are the concentrations of the uncomplexed guest and the host-guest complex, and [G]0


95

and [H]0 are the total concentrations of guest and host. Ka is the association constant of the guest

with the host. a and b are constants depending on instrumental settings of the fluorometer.

At a suitable low concentration, the fluorescence intensity of a fluorophore has a linear

relationship with fluorophore concentration (eq. S1).

𝐼 = 𝑎[G] + 𝑏[HG] (S1)

Conservation of mass requires that:

[G]0 = [G] + [HG] (S2)

From eq. 1 and eq. S2 one obtains:

𝐼 = (𝑎 − 𝑏) ∙ [G] + 𝑏 ∙ [G]0 (S3)

According to the law of mass action, the concentration of host-guest complex under equilibrium

conditions is:

[G] =[G]0−[H]0−

1

𝐾𝑎

2+ √([H]0+[G]0+

1

𝐾𝑎)

2

4− [H]0[G]0 (S4)

Substitution of eq. S4 into eq. S3 affords eq. S5, which can be implemented into fitting

programs and which was used in the fitting in Figure 3b, inset, in the main text.

𝐼 = (a − b) ∙[G]0−[H]0−

1

𝐾𝑎

2+ √([H]0+[G]0+

1

𝐾𝑎)

2

4− [H]0[G]0 + b ∙ [G]0 (S5)

400 450 500 550 600 650 700

0

100

200

300

400

I

I

/nm

0 2 4 6 8 100

150

300

450

[CB7]/M

Ka = (2.8 0.2)

Fig. S7 Variation of the fluorescence spectrum (ex = 360 nm) of 1 μM DAPI in 10 mM

(NH4)2HPO4, pH 7.2, upon addition of CB7. Inset: CB7 concentration dependence of the

fluorescence intensity at 470 nm, R2 = 0.998. The curve was fitted by 1:1 binding stoichiometry.


96

400 500 600 700

0

100

200

300

400

500

I

/nm

Fig. S8 Fluorescence spectra (ex = 360 nm) of 1 μM CB7-CF and 7.4 μM DAPI without (black

line) and with (red line) 32 μM AMADA in 10 mM (NH4)2HPO4, pH 7.2. The addition of

AMADA leads to a strong fluorescence decrease of CB7-CF.

0 10 20 30 40 500

1

2

3

4

DAPI+CB7-CF

DAPI+CB7

DAPI

IRF

Lo

g (

Co

un

ts)

t /ns

Fig. S9 Time-resolved fluorescence decay traces of 5 µM DAPI alone (blue) and in the

presence of 10 µM CB7-CF (black) and CB7 (red); instrument response function is

shown in magenta. exc = 373 nm, obs = 450 nm.

Table S1. Fitting results of lifetime decays of DAPI with and without CB7 or CB7-CF

in 10 mM (NH4)2HPO4, pH 7.2 with 0.5% DMSO.

Sample τ/ns B Rel% χ2

DAPIa 1 = 0.29 0.056 81.1

2.24

2 = 2.77 0.001 18.9

DAPI 1 = 0.31 0.054 79.2

2.66

2 = 2.74 0.002 20.8

DAPI+CB7 1 = 1.59 0.026 100 2.40

DAPI+CB7-CF 1 = 0.65 0.021 30.2

1.94

2 = 3.05 0.010 69.8

a Without DMSO.


97

400 500 600 700

0

100

200

300

400

I

/nm

I450

/I520

0.0 0.1 0.2 0.3 0.4 0.5

0.0

0.5

1.0

1.5

[DNA]/(mgmL1)

Fig. S10 Fluorescence spectra (ex = 360 nm) of 0.5 M DAPI and 1 M CB7-CF with

increasing concentration of salmon sperm DNA (0 to 0.5 mg/mL). The inset shows the

corresponding change in the fluorescence intensity ratio at 450 nm and 520 nm.

400 500 600 700

0

100

200

300

400

500

I

/nm

0.0 0.1 0.2 0.3 0.4 0.50

250

500I

[DNA]/(mgmL1

)

Fig. S11 Variation of the fluorescence spectrum (ex = 360 nm) of 0.5 μM DAPI in 10 mM

(NH4)2HPO4, pH 7.2 upon addition of salmon sperm DNA. Inset: Dependence of the

fluorescence intensity at 450 nm on DNA concentration.

550 600 650 700

0

250

500

750

1000

I

/nm

0.0 0.1 0.2 0.3

0

500

1000I

[DNA]/(mgmL1

)

Fig. S12 Variation of the fluorescence spectrum (ex = 497 nm) of 0.5 μM SYBR Green in

10 mM (NH4)2HPO4, pH 7.2 upon addition of salmon sperm DNA. Inset: Dependence of the

fluorescence intensity at 520 nm on DNA concentration.


98

0.00 0.01 0.02 0.03 0.04

0.0

0.5

1.0

0.5 M DAPI

0.5 M SYBR Green

0.5 M DAPI + 1 M CB7-CF

[DNA]/(mgmL1

)

I/I

Fig. S13 Normalized fluorescence intensity of DAPI, SYBR-Green, and DAPI/CB7-CF in

dependence on DNA concentration. Note that for DAPI/CB7-CF the ratiometric response is

shown (compare Figures S10-S12).

CF Labelling Degree Calculation

As the FRET efficiency is 99%, the DAPI fluorescence in Fig. 3a should not increase

upon addition of CB7-CF. However, there is a slight fluorescence increase by ca. 30%

at 460 nm of DAPI, attributed to complexation by unlabelled CB7. For comparison,

complexation of DAPI by unlabelled (parent) CB7 affords a fluorescence increase by a

factor of 12 (Fig. S8, at 460 nm). The ratio of these two factors (1.3 divided by 12) is

11%, which represents the degree of unlabelled CB7 corresponding to ca. 90% labelling

efficiency.

Limit of Detection

The limit of detection4 (LOD) of the FRET probe was determined according to eq. S6 from the

slope of the calibration curve, b, and the standard deviation of the y-intercept (inset in Fig. 4a)

of the calibration curve, Sa (with n 7). The molecular weight of salmon sperm DNA was taken

from the literature as 1.3 106 g/mol.2

LOD = 3𝑆a/𝑏 (S6)

References



(2) K. Tanaka and Y. Okahata, J. Am. Chem. Soc., 1996, 118, 10679-10683.

(3) Y. Yang, P. He, Y. Wang, H. Bai, S. Wang, J. F. Xu and X. Zhang, Angew. Chem. Int. Ed.,

2017, 129, 16457-16460.

(4) A. Shrivastava and V. B. Gupta, Chron. Young Sci., 2011, 2, 21.


99


Materials and Methods

Reagents for synthesis were from Fluka, Sigma-Aldrich, or Acros Organics NV, Belgium.

Azobisisobutyronitrile (AIBN) was from Molekula GmbH, Germany. Analytical thin layer

chromatography (TLC) was performed on SIL G/UV254 (Macherey-Nagel). Buffers and salts

were of the highest purity available from Fluka, Sigma-Aldrich and used as received. Methyl

methacrylate was destabilized with aluminum oxide (neutral for chromatography 50-200 µm,

60A) prior to use. Poly(styrene) nanoparticles (average diameter 110 nm) with surface

carboxylic acid groups were from Kisker Biotech GmbH & Co. KG (Steinfurt, Germany).

Cucurbit[7]uril (CB7) was synthesized according to established literature methods.1

Functionalization of microspheres was carried out in standard Eppendorf plastic tubes.

Concentrations of fluorescent dye stock solutions were determined using an extinction

coefficient of 90800 M1cm1 in acetonitrile/phosphate buffer for Ada-Rho.2

The photoreactions were carried out in a Luzchem LZC-4V photoreactor with 14 G8T5

lamps from SANKYO DENKI (six lamps from top and four lamps from each side) in a 250-

mL quartz glass round bottom flask. IR spectra were recorded on a Bruker Equinox 55 equipped

with an IRScope and ATR unit and are reported as wavenumbers in cm1 with band intensities

indicated as s (strong), m (medium), w (weak), and br (broad). 1H spectra were recorded on a

Jeol ECS400 MHz and chemical shifts (δ) are reported in ppm relative to TMS (δ = 0 ppm).

ESI-MS was performed on a Bruker HCT ultra and mass spectra are reported as mass-per-

charge ratio m/z (intensity in %, [assignment]). Absorbance measurements were performed

with a Varian Cary 4000 spectrophotometer. Fluorescence was measured with a Varian Cary

Eclipse spectrofluorometer equipped with a temperature controller. All spectroscopic

measurements were performed in 3.5 mL polymethacrylate fluorimeter cuvettes (Sigma-

Aldrich) or 3.5-mL quartz glass cuvettes (Hellma Analytics, Müllheim, Germany).

Fluorescence microscopy images were captured by an Axiovert 200 (Zeiss) with a filter (BP

546/12, LP 590) through an Evolution QEi Media Cybernetics camera by using a 40× objective,

and processed with the software ImageJ 1.48 V (https://imagej.nih.gov/ij/index.html).


100

Abbreviations

AIBN: azobisisobutyronitrile; AMADA: 1-aminomethyladamantane; ATA: 11-azido-3,6,9-

trioxaundecan-1-amine; CB7: cucurbit[7]uril; EDC: 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride salt; DMSO: dimethyl sulfoxide; EDTA:

ethylenediaminetetraacetic acid; ESI-MS: electrospray ionization mass spectrometry; FT-IR:

Fourier transform infrared spectroscopy; HR-MS: high resolution mass spectrometry; MES: 2-

(N-morpholino)ethanesulfonic acid; m.p.: melting point; NMR: nuclear magnetic resonance

spectroscopy; IR: infrared; PAA: poly(acrylic acid); PMMA: poly(methyl methacrylate); rcf:

relative centrifugal force; r.t.: room temperature; SDS: sodium dodecyl sulfate; TBTA:

tris(benzyltriazolylmethyl)amine; TLC: thin layer chromatography.


101

Synthesis

Synthesis of CB-OH

1 g (0.86 mmol) CB7, synthesized as previously reported,1 was dissolved in 125 mL of a

mixture of Millipore water and 12 M HCl (3:2 v/v) and introduced in a 250-mL quartz glass

round bottom flask under nitrogen. 65 µL (0.62 mmol) 30% hydrogen peroxide in H2O was

added and the solution was vigorously stirred during irradiation of UV light (254 nm) for 48 h.

The reaction was monitored by 1H NMR by taking aliquots of the reaction mixture. The solvent

was then evaporated under reduced pressure affording a white solid. The crude product

containing a mixture of CB7-(OH)n (with n = 0, 1, 2, 3),3 was purified by column

chromatography. Therefore, the mixture was dissolved in 950 µL H2O/HCOOH 1:1 and loaded

onto silica gel 60 (0.04-0.063 mm) and the column was eluted with H2O/AcOH/HCOOH

10:10:1.5. The eluent was collected in fractions of 2 mL (>250 fractions) and the fractions

containing pure CB7-OH (as confirmed by TLC, see Fig. S2) were combined. Evaporation of

the solvent gave 150 mg CB7-OH as a white solid. The 1H NMR was in accordance with the

reported spectrum,4 and the identity and purity of the obtained material was additionally

confirmed by mass spectrometry (Fig. S1) and TLC (Fig. S2).


102

Fig. S1 Mass spectrum of CB7-OH with 1 mM cystamine in Millipore water. Traces of CB6 were presumably

enriched during column chromatography (cf. Fig. S2).

Fig. S2 TLC of CBn mixture and CB7 derivatives.


103

Synthesis of CB7-OPr

20 mg (17 µmol) CB7-OH was dissolved in 1.5 mL anhydrous DMSO. 10 mg (0.4 mmol) NaH

(95% purity as solid) was added, and the mixture was stirred at room temperature for 3 h.

Subsequently, the mixture was cooled to 0 °C, 0.5 mL (4.4 mmol) propargyl bromide was

added, and the reaction mixture was stirred at room temperature for 12 h. 50 mL diethyl ether

was added, and the resulting precipitate was three times triturated with 25 mL MeOH. Drying

under high vacuum afforded a pale yellow solid, which was subjected a second time to the same

reaction conditions. This gave the desired CB7-OPr quantitatively as confirmed by mass

spectrometry (Fig. S3), 1H NMR (Fig. S4), and IR spectroscopy: MS (ESI, +ve): 685.3 (100,

[CB7-OPr+Cys+2H]2+ ). IR (KBr) cm1 806 (s), 968 (s), 1234 (s), 1322 (s), 1376 (s), 1473 (s),

1733 (s), 2120 (w), 2933 (m), 2998 (w), 3432 (s).5

Fig. S3 Mass spectrum of CB7-OPr with 1 mM cystamine in Millipore water.


104

Fig. S4 1H NMR spectra of CB7-OPr in 1% DCl in D2O in absence (top) and presence of substoichiometric

amounts (middle) or excess (bottom) of the cavity binder p-xylene diamine (pXD).


105

Synthesis of Ada-Rho

A mixture of 1-bromoadamantane (0.5 g, 2.32 mmol) and 1,4-diaminobutane (1.89 g, 11.62

mmol) was heated in a sealed tube at 190 °C for 20 h. Then, 2 M HCl (60 mL) and diethylether

(60 mL) were added, and the aqueous layer was separated and made alkaline with 50% aq.

NaOH (60 mL). The product was extracted with diethylether, the organic layer was dried over

anhydrous MgSO4, and after removal of the solvent, a pale yellow semi-solid was obtained

(0.26 g, 1.17 mmol, 50% yield). The product was used without further purification in the next

step. 1H NMR (400 MHz, CDCl3) (ppm): 2.70 (t, J = 6.6 Hz, 2H), 2.58 (t, J = 6.6 Hz, 2H),

2.06 (s, 3H), 1.71-1.53 (m, 12H), 1.51-1.44 (m, 4H).

N-Adamantanyl-1,4-diaminobutane (15 mg, 0.067 mmol) and triethylamine (15 mg,

0.14 mmol) were dissolved in 1 mL dry tetrahydrofuran and the solution was stirred for

5 minutes. Afterwards, lissamine rhodamine B sulfonyl chloride (38.9 mg, 0.067 mmol),

dissolved in 5 mL dry tetrahydrofuran, was added and the mixture was heated at 70 ºC in a

sealed tube for 72 hours. After that time, the solvent was removed and the crude was subjected

to purification by silica gel column chromatography, using dichloromethane/methanol (9/1) as

eluent. This procedure yielded the final product Ada-Rho as purple solid (18 mg, 0.024 mmol,

35% yield). 1H NMR (400 MHz, (CD3)2SO) δ (ppm): 8.52 (m, 1H, NH-adamantyl), 8.43 (d, J

= 1.9 Hz, 1H, CH-phenylsulfonate), 8.05 (t, J = 6.0 Hz, 1H, SO2NH), 7.96 (dd, J = 8.0 and 1.9

Hz, 1H, CH-phenylsulfonate), 7.49 (d, J = 8.0 Hz, 1H, CH-phenylsulfonate), 7.11-6.92 (m, 6H,

6 × CH-xanthylium), 3.64 (q, J = 7.0 Hz, 8H, 4 × CH3CH2N), 2.92 (dt, J = 6.0 Hz, 2H,

SO2NHCH2CH2CH2CH2NH), 2.88-2.78 (m, 2H, SO2NHCH2CH2CH2CH2NH), 2.09 (s, 3H, 3


106

× CH-adamantyl), 1.84 (s, 6H, 3 × CH2-adamantyl), 1.71-1.48 (m, 10H, 3 × CH2-adamantyl,

SO2NHCH2CH2CH2CH2NH), 1.21 (t, J = 7.0 Hz, 12H, 4 × CH3CH2N) ppm; 13C NMR (101

MHz, (CD3)2SO) δ 157.3a, 157.1a, 155.0a, 147.9a, 141.6a, 133.0a, 132.6b, 130.7c, 126.6c, 125.7c,

113.7a, 113.4b, 95.4b, 56.1 (quart. C-adamantyl), 45.3 (4 × CH3CH2N), 42.0

(SO2NHCH2CH2CH2CH2NH), 38.5 (SO2NHCH2CH2CH2CH2NH), 37.5 (3 × CH2-adamantyl),

35.2 (3 × CH2-adamantyl), 28.4 (3 × CH-adamantyl), 26.3 (SO2NHCH2CH2CH2CH2NH), 23.5

(SO2NHCH2CH2CH2CH2NH), 12.5 (4 × CH3CH2N) ppm.

a quaternary C corresponding to xanthylium or phenylsulfonate skeleton; b CH corresponding

to xanthylium skeleton; c CH corresponding to phenylsulfonate skeleton

Position

1H NMR

(400 MHz,

DMSO-d6) δ

13C NMR

(101 MHz, DMSO-d6)

δ

Multiplicity,

integration

39 8.52 - (m, 1H)

17 8.43 126.6 (d, J = 1.9 Hz, 1H)

34 8.05 - (t, J = 6.0 Hz, 1H)

19 7.96 125.7 (dd, J = 8.0, 1.9 Hz,

1H)

20 7.49 130.7 (d, J = 8.0 Hz, 1H)

1, 3, 6, 11, 13, 14 7.11-6.92 95.4, 113.4, 132.6 (m, 6H) xanthylium

22, 23, 28, 29 3.64 45.3 (d, J = 7.1 Hz, 8H)

35 2.92 42.0 (dt, J = 6.0 Hz, 2H)

38 2.88-2.78 38.5 (m, 2H)

42, 44, 48 2.09 28.4 (s, 3H)

41, 45, 49 1.84 37.5 (s, 6H)

36, 37 1.71-1.48 26.3, 23.5 (m, 4H)

43, 46, 47 1.71-1.48 35.2 (m, 6H)

24, 25, 30, 31 1.21 12.5 (t, J = 7.1 Hz, 12H)


107

2, 4, 5, 8, 9, 10, 12,

15, 16, 18 -

113.7, 133.0, 141.6,

147.9, 155.0, 157.1,

157.3

quart. carbons

xanthylium

and phenylsulfonate

40 - 56.1 quart. C adamantyl

Fig. S5 1H NMR spectrum of Ada-Rho in (CD3)2SO.


108

Fig. S6 13C NMR spectrum of Ada-Rho in (CD3)2SO.

Preparation and Characterization of CB7-Functionalized

Particles

Synthesis of PMMA/PAA Microparticles

PMMA microparticles: Poly(methylmethacrylate) (PMMA) polymer microbeads were

prepared by dispersion polymerization.6,7 In order to obtain a narrow size distribution, a custom-

made modified reaction chamber was employed to control the polymerization temperature and

the stirring speed of the sealed polymerization flasks. The bead size can be controlled by

parameters like monomer, stabilizer, and radical initiator concentration as well as reaction

temperature. A typical procedure for bead preparation is given in the following: 7 g

poly(vinylpyrrolidine) K90 (average molecular weight 1.300.000) and 600 mg (1.35 mmol)

sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT) were dissolved in 170 ml methanol. The

mixture was transferred to the reaction flask containing 15 ml (14.1 g, 141 mmol), destabilized

methyl methacrylate, and 200 mg (1.2 mmol) azobisisobutyronitrile (AIBN). The sealed flask

was placed in the reaction chamber, where the polymerization was performed at a stirring speed

of 20 rpm at a temperature of 55 °C for 21 hours. After cooling down to room temperature, the

resulting bead suspension was poured into 600 ml water to precipitate the PMMA beads. After

decantation of the water, the beads were washed several times with water and removed from


109

the solution by centrifugation in 50 ml Falcon tubes. These washing-centrifugation cycles were

repeated until no more methyl methacrylate could be detected in the supernatant.

Bead functionalization:8 300 ml of a 0.17 % (w/v) suspension of 2.55 µm PMMA particles

were mixed with 9 g (125 mmol) acrylic acid, 150 mg (0.52 mmol) sodium dodecylsulfate

(SDS), and 1.50 ml of 0.15 M benzophenone as photoinitiator in methanol. After 5 min

equilibration time, the suspension was exposed for 8 min to UV light with 20 mW/cm2 intensity,

while the suspension was vigorously stirred. After irradiation, the suspension was centrifuged

and washed several times with distilled water to remove unreacted compounds, additives, and

homopolymer. Absence of PAA in solution was confirmed by conductometry (conductance <10

μS/cm).

Azide-Functionalized Particles

ATA-functionalized PMMA microparticles: 10 mg PMMA microparticles (111 µmol/g

COOH) were washed into 660 μL reaction buffer (0.1 M MES, pH 5.0 or 10 mM (NH4)2HPO4,

pH 7.2) by repeated centrifugation, supernatant removal and resuspension cycles.

Subsequently, 60 μL of 40 mM 11-azido-3,6,9-trioxaundecan-1-amine (ATA) in reaction buffer

were added. The reaction was started by adding 80 μL of 100 mg/mL (0.52 M) EDC

hydrochloride freshly dissolved in 4 °C cold water. Total reaction volume was 800 μL, final

conditions were 12.5 mg/mL microparticles (corresponding to 1.11 μmol COOH groups), 2.4

μmol ATA, and 42 μmol EDC. After 3 h or 6 h reaction time, the particles were washed into 1

mL 10 mM (NH4)2HPO4, pH 7.2 to afford a 10 mg/mL stock solution of ATA-functionalized

particles.

ATA-functionalized PS nanoparticles: 10 mg commercially available PS nanoparticles were

washed into 660 μL reaction buffer (0.1 M MES, pH 5.0) and 18 μL of 400 mM 11-azido-3,6,9-

trioxaundecan-1-amine (ATA) in reaction buffer were added. The reaction was started by

adding 74 μL of 1 g/mL (5.2 M) EDC hydrochloride freshly dissolved in 4 °C cold water. Total

reaction volume was 800 μL, final conditions were 12.5 mg/mL nanoparticles, 7.2 μmol ATA,

and 389 μmol EDC. After 3 h reaction time, the particles were washed into 1

mL 10 mM (NH4)2HPO4, pH 7.2 (by 10x centrifugation at 28600 rcf for 40 min) to afford a

10 mg/mL stock solution of ATA-functionalized particles.


110

Fig. S7 Optical microscopy images (at 40fold magnification) of a) PMMA-PAA and b) ATA-functionalized

microparticles.


111

CB7-Functionalized Particles

32 µL of a 3 mM CB7-OPr stock solution in DMSO was added to 400 µL ATA-functionalized

particles (10 mg/mL) in 10 mM (NH4)2HPO4, pH 7.2 buffer, and 10 µL of a freshly prepared

20 mM sodium ascorbate solution in 10 mM (NH4)2HPO4, pH 7.2 was mixed with 20 µL of a

10 mM Cu2+/tris(benzyltriazolylmethyl)amine (TBTA) in 55% DMSO stock solution. Both

solutions were combined and the resulting reaction mixture was shaken for 24 h. The solution

was then centrifuged (3.5 min, 16000 rcf) and the supernatant was discarded after

centrifugation. The particles were thoroughly washed (20 times) with 10 mM (NH4)2HPO4, pH

7.2 or 10 mM (NH4)2HPO4, 1 mM EDTA, pH 7.2 (which gave identical results), and the total

buffer volume was finally adjusted to afford a particle concentration of 10 mg/mL.

Nanoparticles were prepared in the same way except for centrifugation at 28600 rcf for 40 min.

4000 3000 2000 1000

0

20

40

60

80

C C

Tra

nsm

itta

nce (

%)

Wavenumbers (cm1

)

azide particles

CB7 particles

azide particles + CB7-OPr

(a)

2200 2100 2000

83.2

83.4

85.0

85.2

85.4(b)

Tra

nsm

itta

nce

(%

)

Wavenumbers (cm1

)

azide particles

CB7 particles

azide

Fig. S8 (a) IR spectra (KBr pellet) of ATA-functionalized particles before (black) and after (red) click reaction

with CB7-OPr, and a mixture of ATA-functionalized particles and CB7-OPr (blue). (b) IR spectra of ATA-

functionalized particles before (black) and after (red) click reaction with CB7-OPr in a narrow range.


112

Surface Quantification Methods

Quantification of Surface-Bound CB7 with AMADA

For quantification of surface-bound CB7, aliquots from the as prepared CB7-functionalized

particle stock solution (10 mg/mL) were diluted with 10 mM (NH4)2HPO4, pH 7.2 to afford a

final volume of 475 µL, to which 25 µL 60 µM AMADA was added (final AMADA

concentration 3 µM). The mixture was briefly vortexed, sonicated, and then shaken for 5 min.

After centrifugation for 10 min at 16000 rcf, 400 μL of the supernatant was transferred to a new

Eppendorf tube and centrifugation was repeated. 350 µL of the final supernatant were

transferred into a 3-mL poly(methylmethacrylate) cuvette containing 1290 µL

10 mM (NH4)2HPO4, pH 7.2, and 160 μL 10 μM CB7 and 200 μL 10 μM acridine orange were

added. The final volume was 2000 µL and final concentrations were 0.8 µM CB7, 1 µM AO,

and 0-0.525 µM AMADA (depending on the amount of AMADA extracted).

Then, a fluorescence spectrum was recorded (λexc = 450 nm) and the fluorescence intensities

at λem = 520 nm were plotted against the volume of particle stock solution and normalized to

the fluorescence intensity in absence of particles (V = 0 µL). Linear fitting of the initial linear

increase of the titration plot gave the slope of the fitted line, a, and the y-intercept, b (see, for

example, inset of Fig. 2b in the main manuscript).

Assuming quantitative binding between AMADA and CB7 on the particle surface, the

loading capacity of the particles, i.e. the amount of CB7 per particle mass, can be obtained from

the intersection of the fitted line and the final plateau value in the titration plot, y∞, indicating

that all extraction of the molecule by the CB7-functionalized particles is complete. The volume

of particle stock solution needed to completely extract the molecule, x, is thus:

𝑥 =𝑦∞−𝑏

𝑎 [EQ1]

The mass of particles needed to completely extract the molecule, m, is then obtained by the

mass concentration of particle stock solution, ρParticle:

𝑚 =𝜌Particle (𝑦∞−𝑏)

𝑎 [EQ2]

This gives the loading capacity of the particles as the mass of particles needed to extract a

specific amount of molecules, n = c·V, as

Loading capacity (in μmol

gof particles) =

𝑎∙𝑐∙𝑉

𝜌Particle∙(𝑦∞−𝑏) [EQ3]

where a is slope and b the y-intercept of the fitted line, y∞ is the final plateau value in the

titration plot, c·V is the amount of the molecule to be extracted (e.g., 25 µL of 60 µM AMADA

stock solution or 500 µL of 3 µM during incubation), and ρParticle is the mass concentration of


113

particle stock solution (here: 10 mg/mL). The reproducibility of the method was evaluated by

repeated measurements with a randomly selected batch of CB7-functionalized particles (Table

S1).

Quantification of Surface-Bound Ada-Rho

Varying volumes of the CB7-functionalized particles (10 mg/mL) were transferred into 1.5 mL

Eppendorf tubes and 10 mM (NH4)2HPO4, pH 7.2 was added to achieve a total volume of 80

µL. Then, 100 µL of 20 µM Ada-Rho was added and the mixture was incubated for 17 min.

After addition of 20 µL Triton X-100 to prevent unspecific absorption of Ada-Rho (sodium

dodecylsulfate performed equally well), the mixture was briefly vortexed and then centrifuged

for 27 min at 16000 rcf. Afterwards, 150 µL of the supernatant was diluted into 1850 µL 10

mM (NH4)2HPO4, pH 7.2 and absorption and fluorescence spectra were recorded.

The fluorescence intensities at λem = 580 nm and the absorbance values at λ = 570 nm were

plotted against the volume of particle stock solution (Fig. S10), and linear fitting of the initial

linear decrease of the titration plot gave the slope of the fitted line, a, and the y-intercept, b.

The loading capacity of the particles, i.e. the mass of particles needed to extract a specific

amount of Ada-Rho, was calculated similarly as above (see EQ3) by additionally considering

the amount of unspecifically absorbed Ada-Rho, nunspecific:

Loading capacity (in μmol

gof particles) =

𝑎∙(𝑐∙𝑉−𝑛unspecific)

𝜌Particle∙(𝑦∞−𝑏) [EQ4]

As controls, AMADA-blocked CB7-functionalized particles were prepared by incubation of

100 µL 10 mg/mL CB7-functionalized particles and 1 mL 10 µM AMADA in 10 mM

(NH4)2HPO4, pH 7.2 for 17 min and subsequent centrifugation. The pellet was resuspended in

10 mM (NH4)2HPO4, pH 7.2 and then subjected to the procedure above to test whether Ada-

Rho could still bind to the particles (Fig. S12).

Unoccupied binding sites on the Ada-Rho and CB7-functionalized were quantified by the

AMADA-based method (Fig. S13, see Section 5.1 for experimental procedure).


114

Supramolecular Control of Surface Coverage Densities

In order to prepare particles with different surface coverage densities of Ada-Rho, varying

volumes of CB7 functionalized particles (7 mg/mL) were transferred into 1.5 mL Eppendorf

tubes and 10 mM (NH4)2HPO4, pH 7.2 was added to achieve a total volume of 140 µL. Then,

200 µL of a solution containing Ada-Rho and AMADA (10 µM total concentration) was added

and the mixture was incubated for 17 min. After addition of 100 µL 1% SDS, the mixture was

briefly vortexed and then centrifuged for 27 min at 16000 rcf. Afterwards, 370 µL of the

supernatant was diluted into 1630 µL 10 mM (NH4)2HPO4, pH 7.2 and absorption spectra were

recorded.

The absorbance values at λ = 570 nm were plotted against the volume of particle stock

solution (Fig. S15), and linear fitting of the initial linear decrease of the titration plot gave the

slope of the fitted line, a, and the y-intercept, b. The surface coverage density with Ada-Rho

was then calculated using EQ4, in which c is the concentration of Ada-Rho only. The

dependence of the resulting surface coverage density on the molar fraction of AMADA is

shown in Fig. 4 in the main text.

Particles for fluorescence microscopy were then prepared by incubating 40 µL of 10 mg/mL

CB7 functionalized particles with 200 µL of a solution containing Ada-Rho and AMADA (10

µM total concentration) for 17 min, addition of 100 µL 1% SDS and centrifugation for 27 min.

The particles were finally washed with 10 mM (NH4)2HPO4, pH 7.2 and the particle

concentration was adjusted to 5 mg/mL. The fluorescence of the particle suspensions was

determined by fluorescence spectroscopy (Fig. 5a in the main text) and by fluorescence

microscopy (Fig. 5b and Fig. S16). For the latter, 5 µL of the particle suspension were deposited

in the center of a Thermo SCIENTIFIC 76 26 mm clear-white glass slide and a 22 22 mm

ROTH #1 cover glass was placed on the particle suspension to ensure that particles distribute

homogeneously between glass slide and cover glass. The images were captured with a Zeiss

Axiovert 200 and an Evolution QEi Media Cybernetics imaging camera through a 40× objective

using a BP 546/12, LP 590 filter set. The dependence of the fluorescence intensities within the

field of view on the surface coverage densities with Ada-Rho (Fig. 5b) was obtained by

automatic assignment of the regions of interest (ROIs) and averaging the intensity within all

ROIs with the software ImageJ 1.48 V (Fig. S16). The brightness and contrast of the image in

the main manuscript (Fig. 3b) was enhanced. The images in the SI (Fig. S16) are unedited.


115

Supporting Figures and Tables

Supporting Tables S1 and S2

Table S1. Reproducibility measurements for the quantification of surface-bound CB7.

Experiment loading capacity

(µmol/g)

coupling yield

(%)

CB7 surface density

(nmol/cm2)

1 5.66 5.10 0.286

2 5.81 5.23 0.294

3 5.52 4.97 0.279

4 5.88 5.30 0.297

5 5.63 5.07 0.285

6 5.74 5.17 0.290

7 5.80 5.23 0.293

average 5.72 5.15 0.289

standard deviation 0.11 0.10 0.006

coefficient of variation ca. 2%

Table S2. Reaction results for CB7 surface functionalization.a

entry # reaction conditionsb loading capacity

(µmol/g)

coupling yield

(%)c

CB7 surface density

(nmol/cm2)

1 1) 3 h at pH 5.0, 2) 24 h (with EDTA)d 5.69 ± 0.04 5.13 ± 0.03 0.288 ± 0.002

2 1) 3 h at pH 5.0, 2) 24 h (without EDTA) 5.67 5.11 0.287

3 1) 3 h at pH 5.0, 2) 48 h 6.2 5.6 0.32

4 1) 3 h at pH 7.2, 2) 24 h 4.4 4.0 0.22

5 1) 6 h at pH 7.2, 2) 24 h 4.8 4.3 0.24

a Values were determined with the AMADA assay (Section 5.1). b Functionalization of COOH surface groups with

1) azide groups was performed in pH 5.0 or pH 7.2 buffer for 3 h or 6 h and then with 2) CB7-OPr in a click

reaction for 24 h or 48 h (see Section 4.1 and 4.2 for details). c With respect to 111 µmol/g surface COOH. d From

three replicates.


116

Supporting Figures S9 to S19

500 550 600 650 7000

50

100

150

200

250

0 1 20.2

0.6

1.0

Flu

ore

sce

nce

Wavelength (nm)

AMADA

F/F

0

[AMADA] (µM)

Fig. S9 Competitive fluorescence titration (λexc = 450 nm) of 2 µM AO and 1.1 µM CB7 in 10 mM (NH4)2HPO4,

pH 7.2. The inset shows the corresponding fluorescence titration (λem =510 nm) plot normalized to the initial

fluorescence intensity and demonstrates quantitative 1:1 binding between AMADA and CB7 in solution.

500 600 700

0

100

200

300

0 30 60

1.0

1.2

1.4

1.6

Flu

ore

sce

nce

Wavelength (nm)

amount

of particles

during incubation

F/F

0

Volume (µL)

Fig. S10 Dependence of fluorescence spectral changes (exc = 450 nm, obs = 510 nm) of the supernatant on the

volume of added CB7-functionalized polymer particles stock solution (10 mg/mL) during incubation with

(dimethylaminomethyl)ferrocene (surface coverage density = 5.5 µmol/g). The inset compares CB7-functionalized

(filled circles) and ATA-functionalized particles as control (open circles).


117

500 600 700

0

200

400

600F

luore

sce

nce

Wavelength (nm)

a)

increasing amount

of nanoparticles

0 50 100 150

150

300

450

600

Flu

ore

sce

nce

Volume (µL)

b)

Fig. S11 a) Dependence of fluorescence spectral changes (exc = 450 nm, obs = 510 nm) of the supernatant on the

volume of added CB7-functionalized (poly)styrene nanoparticles stock solution (10 mg/mL) during incubation

with 4.56 µM AMADA. b) Respective titration plot.

550 600 650 700 7500

400

800

0 20 40 60

300

400

500

Flu

ore

sce

nce

Wavelength (nm)

a)

Particles

Flu

ore

sce

nce

Volume (µL)

400 450 500 550 600

0.00

0.04

0.08

0 20 40 60

0.03

0.04

0.05

0.06

Absorb

an

ce

Wavelength (nm)

b)

Particles

Ab

so

rba

nce

Volume (µL)

Fig. S12 Variation of a) fluorescence intensity (λexc = 520 nm, λobs = 580 nm) and b) absorbance (λobs = 570 nm)

of the supernatant (150 µL) of a mixture of 10 µM Ada-Rho and varying amounts of CB7 particles (10 mg/mL)

after centrifugation and dilution to 2000 µL in 10 mM (NH4)2HPO4, pH 7.2.

550 600 650 700 7500

200

400

0 20 40 60

0.8

1.0

1.2

a)

Flu

ore

sce

nce

Wavelength (nm)

F0/F

Volume (µL)

400 500 6000.00

0.01

0.02

0 20 40 60

0.8

1.0

1.2

b)

Absorb

an

ce

Wavelength (nm)

A0/A

Volume (µL)

Fig. S13 Variation of a) fluorescence intensity (λexc = 520 nm, λobs = 580 nm) and b) absorbance (λobs = 570 nm)

of the supernatant (220 µL) of a mixture containing 1.7 µM Ada-Rho and varying amounts of ATA-functionalized

particles (10 mg/mL) after centrifugation and dilution to 2000 µL in 10 mM (NH4)2HPO4, pH 7.2.


118

550 600 650 700 750

0

40

80

120

Flu

ore

sce

nce

Wavelength (nm)

a)

0 10 20 30 40 500.0

0.5

1.0

b)

F/F

0

Volume (µL)

Fig. S14 Incubation of Ada-Rho with AMADA-blocked CB7-functionalized particles. a) Fluorescence spectra of

different samples. b) Dependence of fluorescence spectral changes (exc = 520 nm, obs = 570 nm) of the

supernatant on the volume of added AMADA-blocked CB7 functionalized polymer particles stock solution (10

mg/mL) during incubation with 2.5 µM Ada-Rho in 10 mM (NH4)2HPO4, pH 7.2.

500 600 700

0

100

200

300

400

500

amount of

particles

during incubation

Flu

ore

sce

nce

Wavelength (nm)

a)

0 20 40 60 80

250

300

350

400

450

V = 52.9 µL

loading capacity = 2.84 µmol/g

F

luo

resce

nce

Volume (µL)

b)

Fig. S15 Quantification of remaining CB7 binding sites on Ada-Rho-functionalized particle surfaces. a)

Fluorescence spectra of different samples. b) Dependence of fluorescence spectral changes (exc = 450 nm, obs =

510 nm) of the supernatant on the volume of added functionalized polymer particles stock solution (10 mg/mL)

during incubation with 25 µM AMADA in 10 mM (NH4)2HPO4, pH 7.2.

0 20 40 60

0.5

0.6

0.7

0.8

0.9

1.0

Ada-Rho only

Ada-Rho + 0.5 eq AMADA

A0/A

Volume (µL)

Fig. S16 Extraction with CB7-functionalized beads using solutions of 0.44 µM Ada-Rho (solid circles) or a

mixture of 0.44 µM Ada-Rho and 0.22 µM AMADA (open circles). The intersections refer to final Ada-Rho

surface loadings of 3.0 µmol/g and 1.8 µmol/g, respectively.


119

400 500 600

0.00

0.03

0.06

0 50 100

0.03

0.04

0.05

0.06A

bsorb

an

ce

Wavelength (nm)

a)

Ada-Rho/AMADA

100 : 0

Ab

so

rba

nce

Volume (µL)

2.95 µmol/g

Ada-Rho

400 500 600

0.00

0.03

0.06

0 50 100

0.020

0.025

0.030

0.035

Ada-Rho/AMADA

75 : 25

Absorb

an

ce

Wavelength (nm)

b)

Ab

so

rba

nce

Volume (µL)

2.34 µmol/g

Ada-Rho

400 500 600

0.00

0.03

0.06

0 50 100

0.015

0.020

0.025

0.030

Absorb

an

ce

Wavelength (nm)

c)

1.41 µmol/g

Ada-Rho

Ada-Rho/AMADA

50 : 50

Ab

so

rba

nce

Volume (µL)

400 500 600

0.00

0.03

0.06

0 50 100

0.006

0.007

0.008

0.009

Absorb

an

ce

Wavelength (nm)

d)

0.74 µmol/g

Ada-Rho

Ada-Rho/AMADA

25 : 75

Ab

so

rba

nce

Volume (µL)

Fig. S17 Variation of absorbance (λobs = 570 nm) of the supernatant (370 µL) containing a mixture of 2 nmol

AMADA and Ada-Rho (molar fraction of Ada-Rho: a) 1.0, b) 0.75, c) 0.5, and d) 0.25) incubated varying amounts

of CB7 functionalized particles after centrifugation and dilution to 2000 µL in 10 mM (NH4)2HPO4, pH 7.2. The

insets show the dependence of absorbance at λobs = 570 nm on the volume of added particle stock solution (7

mg/mL).


120

Fig. S18 Fluorescence microscopy images (546 nm bandpass filter) of Ada-Rho-labeled particles (5 mg/mL) with

increasing surface coverage densities of Ada-Rho (expressed as loading capacities, LC). The white circles

represent the automatically assigned ROIs by the software ImageJ.

0 1 2

0

150

300100 mM AMADA

Flu

ore

sce

nce

Time (h)

no AMADA

50 mM AMADA

Fig. S19 Time-dependent dissociation of Ada-Rho from CB7-functionalized particles in presence of the competitor

AMADA. Ada-Rho functionalized particles (2 mg/mL) were incubated with 50 mM (open circles) or 100 mM

(filled circles) AMADA in 10 mM (NH4)2HPO4, pH 7.2 and the fluorescence of the supernatant (exc = 520 nm,

obs = 585 nm) was measured after certain time intervals. As control, the fluorescence of the supernatant did not

show any release of surface-bound Ada-Rho in absence of the competitor AMADA (filled squares).

References

(1) Márquez, C.; Fang, H.; Nau, W. M. IEEE Trans. NanoBiosci. 2004, 3, 39-45.

(2) Wang, T.; Riegger, A.; Lamla, M.; Wiese, S.; Oeckl, P.; Otto, M.; Wu, Y.; Fischer, S.;

Barth, H.; Kuan, S. L.; Weil, T. Chem. Sci. 2016, 7, 3234-3239.

(3) Ayhan, M. M.; Karoui, H.; Hardy, M.; Rockenbauer, A.; Charles, L.; Rosas, R.; Udachin,

K.; Tordo, P.; Bardelang, D.; Ouari, O. J. Am. Chem. Soc. 2016, 138, 2060-2060.

(4) Ayhan, M. M.; Karoui, H.; Hardy, M.; Rockenbauer, A.; Charles, L.; Rosas, R.; Udachin,

K.; Tordo, P.; Bardelang, D.; Ouari, O. J. Am. Chem. Soc. 2015, 137, 10238-10245.

(5) Chen, H.; Huang, Z.; Wu, H.; Xu, J. F.; Zhang, X. Angew. Chem, Int. Ed. 2017, 129,

16802-16805.

(6) Shen, S.; Sudol, E. D.; El-Aasser, M. S. J. Polym. Sci. A 1994, 32, 1087-1100.

(7) Kim, J.-W.; Cho, S.-A.; Kang, H.-H.; Han, S.-H.; Chang, I.-S.; Lee, O.-S.; Suh, K.-D.

Langmuir 2001, 17, 5435-5439.

(8) Hennig, A.; Borcherding, H.; Jaeger, C.; Hatami, S.; Würth, C.; Hoffmann, A.; Hoffmann,

K.; Thiele, T.; Schedler, U.; Resch-Genger, U. J. Am. Chem. Soc. 2012, 134, 8268-8276.

CV

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Curriculum Vitae

CV

122

CV

123

CURRICULUM VITAE

Shuai Zhang

Birthdate: 02/01/1990 (date/month/year)

Nationality: Chinese

Address: Campus Ring 1

28759, Bremen, Germany

Tel: (+49) 15905389536

E-mail: [email protected]

Education

Sept 2015-Jul. 2019 Doctoral study at Jacobs University Bremen (Germany)

Oct 2014-Jan. 2015 Visiting Master of Jacobs University Bremen (Germany)

Sept 2012-Jun. 2015 Master of Science at China University of Petroleum (East China)

Sept 2008-Jun. 2012 Bachelor of Science at China University of Petroleum (East China)

Visits and Conferences

Feb 2019 2019SupraChem, Würzburg, Germany

Jun 2016 5th ICCB, Brno, Czech Republic

Oct 2014 Visiting master (DAAD scholarship) in Jacobs University Bremen, Germany

July 2013 Presentation, CB&B Summer Workshop, Qingdao, China

Nov 2011 17th International Biophysics Congress, Beijing, China

Internship

July 2010 Internship in Sinopec Qilu Petrochemical Company Ltd

Aug 2008 Internship in Guangda Property Company in Dongguan

Scholarships and Honors

2015-2019 CSC (China Scholarship Council) Scholarship

Oct 2014 DAAD Short-Term Scholarship

2013-2015 The Second Prize Scholarship

Oct 2011 The Third Prize in the national competition 12th National College

Challenge Cup Contest (Group Leader)

2009-2012 Scholarship for Excellent Students

mailto:[email protected]

CV

124

Articles

Chem. Comm., 2019, 55, 671-674 First-author

Materials, 2019, 12, 349 Co-author

Chem. Sci., 2018, 9, 8575-8581 First-author

Process Biochem., 2018, 76, 111-117 Co-author

Chem. Commun., 2018, 54, 2098 -2101 Co-author

Chem. Commun., 2017, 53, 11790 -11793 Co-author

ACS Appl. Mater. Interfaces, 2017, 9, 17799-17806 Co-author

Phys. Chem. Chem. Phys., 2016, 18, 17483 -17494 Co-author

J. Phys.Chem. B, 2015, 119: 33-43 Co-author

Biotechnol. Lett., 2013, 35: 689-693 Co-author

Languages

Chinese: Mother language; English: fluent; German: A2 level

Hobbies

Photography, Mountaineering, Traveling, Badminton, Jogging

synthesis of mono-functionalized cucurbit[n]urils and

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