Research Collection
Doctoral Thesis
Photochemical transformations in continuous flow devices
Author(s): Laurino, Paola
Publication Date: 2011
Permanent Link: https://doi.org/10.3929/ethz-a-006682166
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ETH Library DISS. ETH NO. 19754
PHOTOCHEMICAL TRANSFORMATIONS IN CONTINUOUS FLOW DEVICES
A dissertation submitted to ETH ZURICH
for the degree of Doctor of Sciences
presented by PAOLA LAURINO
Dipl. Pharm. Chem., Universitá degli Studi di Milano
Date of birth 1.4.1981
citizen of Italy
accepted on the recommendation of
Professor Dr. Hansjörg Grützmacher Professor Dr. Peter H. Seeberger Professor Dr. Dieter A. Schlüter
2011
A Maria Emilia, Silvia e Antonio
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“The fun of chemistry is in its unexpectedness. There are times when you come to face-to- face with an unexpected phenomenon while carrying out experiments. You simply have to be sufficiently aware and open to accept the seemingly unbelievable. There are still many more valuable ideas remaining to be discovered. The question is how to find them and how to develop them into new possibilities.” (Mukaiyama, Angew. Chem. Int. Ed. 2004, 43, 5590)
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Acknowledgements
I would first like to thank Professor Peter H. Seeberger for his support and encouragement during my entire stay in his group. The enthusiasm and scientific freedom that he provided were strong driving forces for me to develop new ideas, and his belief in my abilities helped me to be more confident and successfully carry on my research.
I am grateful to Professor Hansjörg Grützmacher, his discussions which helped me to better understand the problems encountered in the polymerization project and improved the quality of my work and our collaboration.
Thanks to Professor Dieter A. Schlüter who accepted to be my co-examiner.
I thank Dr. Klaus Tauer, who patiently explained and taught me a lot about polymerization, helped me evaluate my data and supported me to develop the polymerization project.
I especially want to thank Dr. Kikkeri Raghavendra to help me develop the QD projects. His creativity and imagination taught me a lot about a different way of pursuing science.
Thanks to Dr Nahid Azzouz to carry most of the bioassays of my projects.
I am grateful to Dr. Riccardo Castelli who was always available to help me with chemical mechanisms and reactions.
Thanks to Dr. Hugo Hernandez who helped me understand the polymerization mechanism and scientifically supported me in the first period in Berlin.
Thanks to Dr. Bastien Castagner who initially contributed to develop the flow reactor.
I would like to thank the microreactor team, the former members who introduced me in the micro world and also the present members.
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Thanks to Dr. Farhan Bou Hamdan and Dr. Alexander O´Brien to proof read the first versions of the thesis. I am grateful to Dr. Xiao Yin Mak, Dr. Mattan Hurevich, Dr. Hugo Hernandez and Dr. Klaus Tauer to correct and help me organize different parts of this thesis. Thanks to Mr. Bopanna Monnanda Ponnapa to proof read my thesis. Finally a special acknowledgement to Dr. Claney Lebev Pereira for going carefully through my entire thesis and helping me improve it with critical suggestions and corrections.
I would like to thank Herr Mario Kessinger and Frau Dorothee Böhme their help in the administrative issues was fundamental.
Thanks to
My parents for supporting me consistently during these years.
Silvia my sister who taught me a lot about life, without her advices I would have often felt lost.
My Italian friends who were always there for me, and the friends that I met in Leiden, Zürich and Berlin, even if spread around the world, I bring part of them always with me.
Professor Ad IJzerman and Dr. Laura Fumagalli without them I would have never been able to start my PhD in ETH.
My boyfriend who endured me during this last period, his support was endless.
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TABLE OF CONTENTS
Abstract 1 Riassunto 4
1. Microreactors for Continuous Flow Organic Synthesis 7 1.1 Concepts and Definition 8 1.2 Continuous Flow Microreactor Design 10 1.3 Particle Synthesis in Continuous Flow 16 1.4 Continuous Flow Photochemistry 22 1.5 Advantages and Limitations 31 1.6 Conclusions 33 1.7 References 34
2. Synthesis and Functionalization of Quantum Dots 41 2.1 Introduction 42 2.2 QDs Batch Synthesis 48 2.3 Continuous Flow QD Synthesis and Functionalization 53 2.4 Photophysical Properties of QDs Produced in Continuous Flow 58 2.5 Biological Studies Using Carbohydrate-Capped QDs 63 2.6 Conclusions 67 2.7 Experimental Part 68 2.8 References 83
3. Continuous Flow Microreactors and Phosphine Oxide Initiators: an Unique Combination 86 3.1 Introduction 87 3.2 Continuous Flow Microreactor Design 95 3.3 Emulsification 100 3.4 Extraordinarily High Molecular Weight Polymer in Small Particles 104 3.5 Ultrafast Polymerization of Styrene 110 3.6 Investigations on BAPO-AA 113 3.7 Studies on Different Phosphine Oxide Initiators 121
VII
3.8 Molecular Weight Distribution and Average Size of Polymer Particles 123 3.9 Snowballing Radical Generation Mechanism 125 3.10 Snowballing Radical Generation Simulation 133 3.11 Seed Polymerization of Polymer Particles 138 3.12 Applications of SRG Mechanism 140 3.13 Conclusions 145 3.14 Experimental Part 146 3.15 References 151
4. Final Conclusions and Outlook 155
Appendix 159 5. Glyco-dendronized Polylysine for Bacteria Detection 161 5.1 Indroduction 162 5.2 Synthesis of Glyco-dendrons 165 5.3 Photofunctionalization of Polylysine 166 5.4 Bacteria Detection with Glyco-dendronized Polylysine 168 5.5 Conclusions 170 5.6 Experimental Part 171 5.7 References 183
List of Abbreviations 185
Curriculum Vitae 187
VIII
IX
Abstract
Microreactor enables precise and automated manipulation of reaction mixture in a defined volume of space. In recent times there has been an increasing interest in the miniaturization of chemical and biochemical reactors. The microreactor is an efficient and versatile alternative to the traditional batch reactor techniques and has facilitated the development of new methods for organic and polymer chemists. Microreactors, when used in continuous flow permit the precise control and screening of reaction parameters. This thesis emphasizes the benefits of using microreactor technology for the synthesis of nanoparticles and the study of photochemical reactions. Herein two successful applications were studied and described: 1) The use of continuous flow device for the synthesis and functionalization of quantum dots (QDs). 2) Discovery of a new mechanism for the reaction of phosphine oxide initiators during the emulsion polymerization in a continuous flow microreactor.
Preliminary results regarding the synthesis of QDs in a round bottomed flask resulted in major limitations in the control of particle nucleation and growth, which were primarily due to the non-homogeneous mixing and heating of the reaction mixture. To overcome these limitations, a versatile new strategy was developed and applied for producing carbohydrate- or dihydrolipoic acid-capped CdSe/ZnS and CdTe/ZnS quantum
1
dots (QDs) for biological applications. This method involved a three-step flow synthesis in single-phase by using a continuous flow microreactor.
The first step involved screening different reaction time and temperature conditions to produce QDs of different size with precise photophysical properties. The second and third step involved the shell formation (ZnS) to obtain QDs and functionalization of the same with thiol linked carbohydrate or dihydrolipic acid. The method described has the following advantages: 1) Production of QDs in the range of 2–4 nm with narrow particle size distribution. 2) Precise control of the nucleation and growth of QD cores in a single-phase flow and lower temperature than previous syntheses. 3) Easy screening of reaction parameters. 4) The procedure can be scaled up to produce large amounts of QDs. In the second part of this thesis a photo-initiated phosphine oxide mediated polymerization reaction accelerated by snowballing radical generation (SRG) in a continuous flow microreactor was described.
This method afforded narrow size distributed particle in a scalable and reproducible manner. Surprisingly, high molecular weight polymer chains were detected in small particles generated at short residence times (36.5 s). The use of microreactor permitted an in-depth investigation of the reaction mechanism, screening parameters and conditions not obtainable in a batch reactor. The classical emulsion polymerization could not justify the
2
observed screening results. A new mechanism for the reaction was proposed whereby upon irradiation phosphine oxide was able to generate more radicals for particles than expected. The discovery of this new mechanism of reaction (Snowballing Radicals Generation) permitted to produce materials with defined morphologies that were difficult to synthesize previously.
3
Riassunto
Il microreattore permette di manipolare in modo preciso e automatizzato miscele di reazione in un volume di spazio definito. Recentemente l´interesse per la miniaturizzazione di reattori chimici e biochimici è aumentato. L´uso del microreattore si presenta come un´alternativa efficiente e versatile ai tradizionali reattori e ha facilitato lo sviluppo di nuovi metodi in chimica organica e polimerica. I microreattori quando usati in flusso continuo, permettono il controllo preciso e lo screening di parametri di reazione.
Questa tesi enfatizza i vantaggi nell´uso della tecnologia del microreattore per la sintesi di nanoparticelle e lo studio di reazioni di fotochimica. Due esempi sono stati studiati con successo e saranno riportati:
1) L´uso di un microreattore in flusso continuo per la sintesi e funzionalizzazione di quantum dots (QDs). 2) La scoperta del meccanismo di reazione degli ossidi di fosfina, usati come iniziatori della polimerizzazione in emulsione in un microreattore in flusso continuo.
I risultati preliminari della sintesi di QDs in un reattore tradizionale hanno mostrato delle limitazioni nel controllo della crescita e nucleazione delle particelle, le quali erano principalmente dovute a una mancanza di riscaldamento e miscelazione omogenei della
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miscela di reazione. Per superare queste limitazioni, una nuova e versatile strategia è stata sviluppata e poi utilizzata per produrre QDs (CdSe/ZnS e CdTe/ZnS) funzionalizzati con diversi carboidrati o con l´acido diidrossilipoico per studi biologici. Questo metodo ha coinvolto tre reazioni in fase omogenea usando un microreattore in flusso continuo.
La prima reazione è stata ottimizzata utilizzando diverse temperature e tempi di reazione per produrre QDs con diverse dimensioni e precise proprietà fotofisiche. Il secondo e terzo passaggio hanno generato lo strato esterno (ZnS) dei QDs e la loro funzionalizzazione con carboidrati o con l´acido diidrossilipoico. Il metodo descritto presenta i seguenti vantaggi: 1) La produzione di QDs monodispersi con una dimensione di 2–4 nm. 2) Il controllo preciso della nucleazione e crescita del nucleo dei QDs in fase omogenea e a temperature piú basse di quelle riportate precedentemente. 3) Uno screening facile dei parametri di reazione. 4) La procedura può essere facilmente riprodotta su vasta scala. Nella seconda parte di questa tesi una reazione di polimerizzazione inizializzata dagli ossidi di fosfina e accelerata da un´inaspettata generazione di radicali (SRG) è stata studiata usando un microreattore in flusso continuo.
Questo metodo ha permesso di sintetizzare particelle con grandezza uniforme in un modo riproducibile anche in vasta scala. Il peso molecolare delle catene dei polimeri
5
che costituivano le particelle si è rivelato sorprendentemente elevato per la grandezza delle particelle e per la velocità con cui erano sintetizzate (36.5 s.). L´uso del microreattore ha permesso un´investigazione dettagliata del meccanismo di reazione, uno screening di parametri e di condizioni di reazione difficilmente ottenibili in un reattore tradizionale. Un nuovo meccanismo di reazione è stato proposto sulla base del quale se irradiati gli ossidi di fosfina sono in grado di generare in una particella di polimero più radicali di quelli previsti. La scoperta di questo nuovo meccanismo di reazione (Snowballing Radical Generation) ha permesso di produrre materiali con morfologia definita che prima erano difficili da sintetizzare.
6 1. Continuous Flow Microreactor for Organic Synthesis
1. Microreactor for Continuous Flow Organic
Synthesis
7 1. Continuous Flow Microreactor for Organic Synthesis
1.1 Concepts and Definitions
A microreactor or a microstructured reactor is a device where a chemical reaction takes place within a confined space.1 The devices consist generally, but not exclusively,2 of a microtubular system that permits chemical reactions to take place in channels of µm 1 diameter size. In contrast to the round-bottomed flask, microreactors can be operated in continuous flow by constantly feeding in reagents and harvesting the products. Continuous flow reactors can be classified based on the reaction volume and thus the size. Continuous flow microreactors and microfluidic devices are miniaturized systems where reactions occur in a volume scale of µL to few mL. Continuous flow reactors of 5 mL scale and up are called mesoscale reactors (Figure 1.1).1 In particular, continuous flow microfluidic device controls and manipulates continuous liquid flow in microchannels.
Figure 1.1 Examples of different volume continuous flow reactors: (a) 6 µL borosilicate glass reactor; 3 (b) 2.4 mL borosilicate glass reactor;3 (c) 20 mL stainless steel coil reactor.4 Pictures not to scale.
Continuous flow reactors or microreactors can be classified according to the flow generated into the channels either in single-phase or segmented-phase (Figure 1.2). Homogeneous-liquid phase reactions are often carried out in single-phase flow mode where reagent streams are brought into contact. Heterogeneous phase reactions (liquid- liquid or gas-liquid) often exploit the two phase segmented flow approach characterized by the formation of reaction aliquots containing different phases.5
8 1. Continuous Flow Microreactor for Organic Synthesis
Figure 1.2 Schematic representations of single phase and segmented gas–liquid flow microreactor.
The flow process depends on three important parameters: flow rate, residence time, and reaction throughput. The flow rate is the volume of the reaction mixture that passes through the device per unit time and is measured in [mL min-1]. The residence time is the average amount of time that a reaction mixture spends in a continuous flow microreactor measured in [min]. The residence time is calculated based on the reactor volume and the flow rate using (eq. 1).
(1) τ = V/q
τ (residence time); V (reactor volume); q (flow rate).
To achieve longer residence times, reagents can be purged more slowly or a larger volume reactor used. Production rates can vary from nL min-1 to L min-1. The throughput of a reaction in a continuous flow microreactor defines the productivity. The throughput measured in [mol min-1] is directly proportional to the flow rate and concentration of the desired product. In the past decades, continuous flow microreactors have emerged as an alternative to the traditional round bottom flask in chemical laboratories.6 Particle synthesis and photochemical transformations in continuous flow have been explored and will be the focus of this thesis.
9 1. Continuous Flow Microreactor for Organic Synthesis
1.2 Continuous Flow Microreactor Design
Microreactors are often custom-designed for the desired chemical process, and the materials chosen for fabrication may depend on the reaction conditions. Commonly used materials, that each offer advantages and disadvantages include polymers, glass, silicon, ceramics and metals (Figure 1.3).
Figure 1.3 Examples of microreactors fabricated from: (a) glass;7 (b) ceramic;8 (c) metal9 and (d) silicon.10
Polymers are generally the cheapest solution, but can swell during the fabrication process.11 Recently, perfluorinated polymers have been used, most commonly in the form of tubing and despite temperature limitations, are inert to most reagents and solvents.12 Glass is difficult to handle during fabrication but is the most chemically inert material available.13 Ceramics are another chemically inert option and stable at high temperature, but difficult to handle, during the fabrication process.14 Other choices include steel and silicon, both of which are resistant to high temperature, pressure and have good thermal conductivity. Silicon is not compatible with bases and the manufacturing costs are very high.15 The reactor is usually integrated into a system composed of many components like an injection system, mixer, purification apparatus and analytical system that are described in detail below (Figure 1.4).
10 1. Continuous Flow Microreactor for Organic Synthesis
Figure 1.4 Components of continuous flow microreactor.
1.2.1 Injection System
Continuous flow microreactors are typically driven by an injection system, that can be operated by a mechanical or a non-mechanical pump. Mechanical pumps depend on moving parts such as valves or pistons to pump liquid with a precise flow rate. Three examples of common mechanical pumps are syringe pumps, peristaltic pumps and high- performance-liquid chromatography (HPLC) pumps (Figure 1.5).
Figure 1.5 Schematic representation of mechanical pumps: (a) syringe pump; (b) peristaltic pump; (c) HPLC pump.
Syringe pumps are small infusion pumps where the liquid is stored in syringes. In a peristaltic pump the rotation of a rotor compresses a flexible tube, which forces the liquid to move through the tubing.16 HPLC pumps use the action of small pistons to displace the volume of liquid through the tubing. A non-mechanical pump, on the other hand, utilizes an activation force such as electro-hydrodynamic, electro-osmotic or ultrasonic flow
11 1. Continuous Flow Microreactor for Organic Synthesis generation to move the fluids. 17 Electro-hydrodynamic pumps use an electric field generated between two electrodes to create a flow. Operating at a defined voltage, the electrodes can accelerate charged molecules thereby generating a fluidic stream. The absence of moving elements in non-mechanical pumps renders them easier to integrate in the continuous flow microreactor. The connections between the microreactor and other devices in the system are created by polymer, silicon, glass or metal tubing. The tubing as well as the connections must often withstand high pressures of up to 10 MPa. Connection can be established via compression sealing with rubber O-rings, that allows for the connection to be easily interchanged.18 Another option is a permanent connection with epoxy-glue, glass brazing or metal soldering while these connections are more robust, they require elaborate fabrication techniques. 15b, 19
1.2.2 Mixing Systems
Reagents used in continuous flow microreactors can be premixed or simultaneously injected by passing through a mixing system (for example, via T or V connections, micromixers, etc) before reaching the microreactor. 20 The mixing system can be designed whereby the fluid flow is constrained by a laminar regime, as a result of small-scale tubing.1c Flow is defined as laminar when the Reynolds number that represents the ratio of convective to viscous forces, is below 2100 for cylindrical channels (eq. 2).
(2) Re = Ud/ ρ
Re (Reynolds number); U (average velocity); d (hydraulic diameter of the channel); ρ (viscosity).
12 1. Continuous Flow Microreactor for Organic Synthesis
In a laminar regime, the mixing is mainly based on the diffusion between fluids. The Peclét number,1c which represents mass transfer as the ratio of convection and diffusion describes this situation (eq. 3).
(3) Pe = Ud/D
Pe (Peclét number); U (average velocity); d (hydraulic diameter of the channel); D (diffusion coefficient).
A flow microreactor can have an integrated mixer or an external mixer, depending on whether the microdevice has multiple inlet channels that combine into the mixer or whether it is made of just a single inlet. The mixer can be either passive or active. In the first case, mixing is due to the passive diffusion of the fluids.21 The laminar effect between the two fluids increases the surface of contact of fluids and consequently improves the diffusion of reactants between the phases. With multi-laminar flow the above effect can be further enhanced (Figure 1.6).
Figure 1.6 Schematic representation of (a) laminar flow, or (b) multi-laminar flow generated by Y connection.
In the case of active mixing, forces such as ultrasonic waves, thermal gradient, electric or magnetic field fluctuations are applied to enhance the diffusion process.22 Active mixing requires more elaborate fabrication skills and the chemicals used during the reaction must be compatible with the forces applied.23 Active mixing and the geometry of the channel networks of the device can give rise to turbulent flow generated by fluctuations of flow velocity and pressure, and characterized by a chaotic and stochastic regime (Figure 1.7).24
13 1. Continuous Flow Microreactor for Organic Synthesis
Figure 1.7 (a) Laminar versus (b) turbulent flow.
1.2.3 Purification Apparatus and Analytical Systems
The ability to incorporate purification and analytical devices makes continuous flow microreactors a very versatile system. According to the needs and nature of the microreactor, other tools can be integrated downstream of the device such as an microextractor,25 a distillatory apparatus,26 a column containing a scavenger27 or even other reactors for multistep reactions.28 Liquid-liquid extraction in continuous flow microreactors can be achieved by using a thin porous fluoropolymer membrane that selectively wets organic solvent. To retain the reaction byproduct, a scavenger supported on a solid support and packed into a column can be used in continuous flow as a purification apparatus (Figure 1.8).
Figure 1.8 Schematic representation of the principle for (a) liquid-liquid microextractor and (b) scavenger column. The continuous flow reactor can be used as a platform for rapid reaction screening when combined with an analytical system. An automated system to measure changes in temperature and pressure can be integrated in-line with the flow device. For example, an
14 1. Continuous Flow Microreactor for Organic Synthesis internal thermocouple allows for the real time monitoring of reaction temperatures.29 Incorporation of analytical systems such as HPLC, FTIR, IR etc to facilitate the control and screening of parameters is also possible.30
15 1. Continuous Flow Microreactor for Organic Synthesis
1.3 Particle Synthesis in Continuous Flow
Continuous flow microreactor can be exploited for the efficient synthesis of nano- and micro-particles.31 The major drawback of synthesizing these kinds of particles in round-bottomed flasks is the general lack of reproducibility and difficulty in scaling up, due to non-uniform mixing and heating of the reaction mixing. These limitations significantly affect particle nucleation and growth, both of which are important features in obtaining high quality material. The main advantage of continuous flow microreactor compared to batch processes is the easy scale up by simply prolonging the run time while keeping the other parameters and conditions consistent throughout the process. Excellent composition and morphology of the final material can result from the high performance intrinsic property of the microreactor system (better heating and pressure control, mixing, etc).32 Furthermore, the synthesis of complex heterogeneous particles, multi-shell particles, novel materials and structures can be achieved using the microreactor device due to the ease and safety with which high temperatures and pressures can be attained in the device.18a, 33 However, one major challenge encountered in the synthesis of particles by continuous flow microreactor is to maintain the controlled growth of these particles so as to avoid clogging of the flow system. To highlight the contributions of microreactors to the synthesis of particles, some examples are discussed below.
1.3.1 Organic Particles
The polymerization of monomer droplets in a continuous flow microreactor can result in the formation of particles of different shapes and sizes.34 The polymerization can also occur at the interface of the polymer droplets to generate capsules.35 Furthermore, the
16 1. Continuous Flow Microreactor for Organic Synthesis formation of different sized particles can be achieved by using immiscible solvents, taking advantage of droplet formation in a heterogeneous system.36 Kumacheva et al. have reported the effects of changing the channel dimensions to achieve the required polymer particle characteristics (discoid or spherical shapes). 37 A solution of monomer (dimethylacrylate oxypropyldimethylsiloxane, DMOS) containing a photoinitiator (1-hydroxycyclohexyl phenyl ketone, HCPK), was dispersed in water and stabilized by a surfactant (sodium dodecyl sulfate, SDS) (Figure 1.9).
Figure 1.9 (a) A schematic bidimensional increase of the reaction zone where the particles are generated and irradiated (UV lamp with an output of 400W at a wavelength of 330-380 nm). The monomer phase is injected as core flow and the water phase as sheath flow. Depending to the height of the channel, disk or spherical particles can be generated (b) Image of the solidified disk (scale bar = 200 nm).38
Different particle shapes were generated using this method by forcing the monomer droplets at different heights of the channel and by controlling the flow rate into the reactor and varying the viscosity of the solution according to the capillary number (eq. 4).
(4) Ca = µυ/γ
Ca (capillary number); µ (dynamic viscosity of the aqueous phase); υ (velocity of the aqueous phase); γ (interfacial tension between the oil and aqueous phase).
17 1. Continuous Flow Microreactor for Organic Synthesis
After generating circular or discoid shapes, a lattice structure was trapped by UV irradiation for 30–180 s (Figure 1.7). Depending on whether the droplets relax faster or slower after entering the inlet and on their viscosity before the irradiation, the shape of the disk (spherical or hexagonal or pentagonal) was controlled. The resulting particles had controlled size distribution and shape. 38
18 1. Continuous Flow Microreactor for Organic Synthesis
1.3.2 Inorganic Particles
The synthesis of inorganic particles using microreactors has been widely investigated and syntheses of silver,39 gold,40 palladium41 or copper42 nanoparticles have been reported. Köhler et al. have described the synthesis of gold nanoparticles in a continuous flow glass–silicon microreactor.40 Gold nanoparticles with mean diameters of
5–50 nm were obtained directly from the gold salt (HAuCl4) and a reducing agent (ascorbic acid). Optimization and rapid screening of flow rate, pH, amount of reducing agent and concentration of poly(vynilpyrrolidone) (PVP) resulted in gold nanoparticles that were twice as narrow in size distribution than those synthesized using a round-bottomed flask (Figure 1.10).
Figure 1.10 Picture of microreactor used for the synthesis of gold nanoparticles [PVP = poly(vynilpyrrolidone)].40
The synthesis of inorganic particles often requires the addition of a ligand or surfactant in order to avoid clogging of microreactor channels, and can be carried out under homo- or heterogeneous conditions. The main advantage of using the continuous flow reactor for homogenous synthesis of particles is the possibility to add reagents consistently at different time points to the reaction stream. Furthermore, the segmented flow of a heterophasic system can be a valid option to control nucleation and particles growth.43 The use of metal oxides to generate particles containing iron,44 silica45 or titanium dioxide has also been reported.46 Tadaki et al. prepared titanium dioxide nanoparticles on the insoluble interface of a continuous flow ceramic microreactor (Figure 1.11).46
19 1. Continuous Flow Microreactor for Organic Synthesis
Figure 1.11 Ceramic microreactor used for the synthesis of TiO2 nanoparticles. Two inlets and two outlets are shown.46
The nanoparticles with a diameter below 10 nm were prepared by hydrolysis of titanium alkoxide. The selection of proper solvents and optimum flow rate allowed for a stable interface between two insoluble solvents and this interface was exploited as the reaction chamber where particle nucleation and growth occurred. Coalescence of droplets containing reagents is another approach for the synthesis of metal oxide nanoparticles. In the continuous flow microreactor two electrodes can be placed to generate droplet currents. The coalescence of these droplets can be fast enough that the mixing of the reagents is optimal for the preparation of narrow size distribution particles.47 Other examples reported in the literature use small seed particles as nucleus- precursors for the growing particles.48
1.3.3 Miscellaneous Particles
Other types of particles that combine two or more organic and/or inorganic materials, such as shell particles, can also be synthesized in continuous flow reactors. One example are the particles made of different semiconducting materials that we have synthesized and will be discussed in the chapter 2 of this thesis.49. These semiconducting particles can be covered by a shell of polyethylene glycol, where the optical properties of
20 1. Continuous Flow Microreactor for Organic Synthesis the core particles combined with the biocompatible characteristics of PEG yielded particles suitable for in vitro or in vivo imaging studies.50
21 1. Continuous Flow Microreactor for Organic Synthesis
1.4 Continuous Flow Photochemistry
Since the beginning of the 20th century, the importance of photochemical reactions has been recognized, and much effort has been made to improve the efficiency of these reactions.51 The advantages of photochemical transformations are well documented. 52 Reactions can be carried out in mild and environmental friendly conditions. Light is used to activate the starting materials, minimizing or avoiding the use of other reagents which can often result in the generation of side products.53 Photochemical reactions are key synthetic organic chemistry.54 The disadvantages associated with photochemical reactions are usually related to the light source and reactor design. The light source is usually a lamp with a specific life time (e. g. xenon lamp, halogen lamp, medium, low or high pressure mercury lamps). The emission of the lamp typically changes over time, and usually a cooling system is required to dissipate the heat that is generated. Drawbacks related to the traditional batch photochemical reactions include a lack of reproducibility, due to the difficulty in maintaining the ratio between the irradiated area and the reaction volume consistent especially during reaction scaling up. Continuous flow reactors permit excellent control of the irradiation time as it is directly related to the flow rate. According to the Beer-Lambert law, the penetration of light decreases exponentially with the distance from the light source (eq. 5).
-εlc (5) I/I0 = e
I (Intensity transmitted light); I0 (Intensity incident light); ɛ (extinction coefficient); l (path length that the light has to pass through); c (concentration of the substance in the solution).
In a continuous flow device, the penetration of light is maximized and consistent throughout the system (Figure 1.12).
22 1. Continuous Flow Microreactor for Organic Synthesis
Figure 1.12 Schematic representation of a vertical section of batch photoreactor (a) and continuous flow photoreactor (b). (l is the path length that light has to go through in the reaction solution)
Reactions can be scaled up without changing the parameters. Scaling down allows for screening various reaction parameters. Finally the compactness of the system facilitates efficient cooling of the system. 17c, 55 For smaller flow systems, a good alternative light source that was recently exploited, are light emitting diodes (LEDs). LEDs are small, efficient and do not require powerful cooling system because they do not warm up excessively. LEDs emit light at a specific wavelength and avoid side reactions that can occur at different wavelengths thereby simplifying product purification.56
23 1. Continuous Flow Microreactor for Organic Synthesis
1.4.1 Miniaturized Systems for Photochemical Transformations
Different miniaturized systems have been used for photochemical reactions, ranging from commercially available systems to in-house designs. One of the systems initially introduced for photochemical reaction is the FFMR-cyl, which is a thin falling- film reactor, where the channels are parallel and engraved around a metal cylinder (Figure 1.13).57
Figure 1.13 Cylindrical falling film micro reactor (FFMR-cyl) for scale-up and its standard version FFMR.
The type of material used for the fabrication of the microreactor is of critical importance. Jensen and coworkers have fabricated two reactor prototypes, one made of glass for reactions in the wavelength of 365 nm and the other made of quartz for shorter wavelength reactions. In both cases, the microreactors are serpentine shaped in order to fit the longest channel in the smallest space possible, with one inlet and one outlet (Figure 1.14). The mask has been designed according to the channel shape and then covered by a Pyrex or Quartz wafer.58
24 1. Continuous Flow Microreactor for Organic Synthesis
Figure 1.14 Serpentine shaped microreactor fabricated by Jensen et al.59
De Mello et al. reported the fabrication of a glass microreactor that is serpentine shaped and has two inlets, one for a gaseous reagent and other for liquids.59 Fukuyama et al. have used a pre-fabricated reactor Mikroglas Dwell Device, FOTURAN®60 which is made of glass and integrated with a cooling/heating system (Figure 1.15).6c
Figure 1.15 A glass microreactor with integrated cooling system (1000 µm width and 500 µm depth). View of microreactor (a) from the top; (b) from lateral. (c) Picture of the system.
Another approach is a tubular reactor, coiled around the lamp with a cooling system placed in-between the lamp and the tubing reactor. In this case, the channel is made of flexible Teflon tubing and not of glass or quartz (Figure 1.16) 2, 61
25 1. Continuous Flow Microreactor for Organic Synthesis
Figure 1.16 FEP (fluorinated ethylene propylene) flow reactor (a) and picture of the system (b).61a
The most commonly used types of Teflon tubing are polytetrafluoroethylene (PTFE) and fluorinated ethylene propylene (FEP) because of high light transmittance, high pressure resistance and inertness to most organic compounds.62
26 1. Continuous Flow Microreactor for Organic Synthesis
1.4.2 Photochemical Reactions in Continuous Flow
Photochemical cycloaddition reactions have been investigated with different substrates from different groups. Booker-Milburn et al. initially reported the cycloaddition of malemide with 1-hexyne to yield substituted cyclobutene (Scheme 1.1).61a The reaction was carried out in a FEP tubing coiled around a medium pressure Hg lamp (Figure 1.16). This system produced more than 500 g of 3 in a continuous 24 h processing time.
Scheme 1.1 [2 + 2] cycloaddition between maleimide 1 and 1-hexyne 2.
Fukuyama et al. reported the [2 + 2] addition of vinyl acetates with various cyclohexenone derivatives using a microflow system (Scheme 1.2).51c, 63
Scheme 1.2 [2 + 2] Cycloaddition of cyclohexenone 4 and vinyl acetate 5.
Yields were improved and reaction times significantly reduced, as compared to the same reaction run in batch using a pyrex flask. Two serially-connected micro-photoreactors used in tandem at twice the flow-rate used for a single reactor provided a similar result (Table 1.2).
27 1. Continuous Flow Microreactor for Organic Synthesis
Table 1.2 Comparison between microreactor and flask conditions.
Device Residence time (h) Yield (%) Microreactor 2 88 Two serially connected 2 85 microreactors Pyrex flask 2 8 Pyrex flask 4 22
In addition to their studies of [2 + 2] cycloadditions, Fukuyama et al. investigated the use of a Barton reaction to construct steroid 8, an important intermediate in the synthesis of an endothelial receptor antagonist (Scheme 1.3). Initial optimization studies of this reaction involved the comparison of various glass covers (quartz, pyrex, and soda lime glass) for the stainless steel continuous flow microreactor, as well as examination of the light source (300 W high-pressure Hg lamp, or 15 W black light lamp) and its distance from the reactor. A scale up synthesis was achieved (up to 20 h of run) under optimal conditions using two serially-connected reactors (total volume of 8 mL, a residence time of 32 min and irradiation by eight 20 W black light lamps, 60 % yield).55c
Scheme 1.3 Barton nitrite photolysis of steroid 8.
Booker-Milburn et al. used the same flow system for an intramolecular [5 + 2] cycloaddition to form the key bicyclic azepine intermediate for the synthesis of the Stemona alkaloid (±)-neostenine (Scheme 1.4). The flow reaction was performed continuously for 24 h, demonstrating that scale-up was possible. The yields were improved to 63% in microreactor compared to batch reactor results (20% in flask) with shorter residence time.64
28 1. Continuous Flow Microreactor for Organic Synthesis
Scheme 1.4 Key step in the synthesis in (±)-neostenine
Another relevant example was reported by Aggarwal et al. The thermal [4 + 2] cycloaddition involved a thioaldehyde 13 generated by photolysis of a corresponding thioester 12, and reaction with cyclopentadiene yielded intermediate 14 for the synthesis of a chiral sulphide, a synthetically useful reagent for organo-catalytic reactions (Scheme 1.5). After optimizing the reaction conditions in a continuous flow reactor they reported a large scale synthesis of the intermediate (38 g substrate, 75% yield). The superior performance of a continuous flow photoreactor compared to a batch photoreactor was demonstrated clearly.
Scheme 1.5 Photolysis of thioester 12.
Photochemical reactions have been also used in polymer synthesis. Kumacheva et al. investigated a multistep polymerization in continuous flow microreactor combining a polyaddition of acrylate monomer [tri(propylene glycol) diacrylate, TPGDA] photoinitiated by 2-diethoxyacetophenone (DEAP) and a thermal polycondensation of urethane oligomer (Scheme 1.6). The first step was a free radical photoinitiated polymerization reaction that was able to generate the necessary heat to activate a second
29 1. Continuous Flow Microreactor for Organic Synthesis polycondensation. The result is the formation of an interpenetrating polymer network structure. The resulting cross-linked polymer particles were of narrow size distribution and controlled morphology. Furthermore, the microfluidic device provided for an easy screening of monomers.65
Scheme 1.6 Step 1, Photopolyaddition of tri(propylene glycol) diacrylate, TPGDA initiated by 2- diethoxyacetophenone (DEAP) under UV irradiation (Dr Hönle UVA Print 40C, F-lamp, 400 W, λ = 330–380 nm); step 2, thermal polycondensation of urethane oligomer catalyzed by dibutyltin dilaurate (DBTDL).
30 1. Continuous Flow Microreactor for Organic Synthesis
1.5 Advantages and Limitations
The benefits of continuous flow microreactor technology has been widely reviewed and several examples have demonstrated the utility of microreactor and their influence on chemistry.1f, 15b, 66 Drug discovery and process development can be improved by the use of a continuous flow system by shortening the screening process and optimizing reaction conditions and reagents.27, 67 Compared to the traditional round bottom flask, the geometry of the channels of the continuous flow microdevice permits a larger surface-to-volume ratio thereby enhancing the mass-heat transfer that influences the time,68 yield and selectivity of the reactions.69 The continuous flow microreactors can be used for the synthesis of hazardous compounds due to the highly controlled nature of the system and isolation of the reaction from air and moisture. In principle, reactions in a continuous flow microdevice can be performed easily under inert conditions.70 The minimal use of solvent and energy helps to conserve resources and helps practice environmentally friendly chemistry. Scaling up can be done by extending the run time, or numbering up the number of reactor (Figure 1.17).51c
Figure 1.17 Continuous flow approaches to large scale production numbering up system.
The continuous flow device also permits to optimize many reactions in a microscale by reducing the cost and limiting the output of hazardous byproducts. The above factors allow for process optimization and later scaling up for production.71
31 1. Continuous Flow Microreactor for Organic Synthesis
Continuous flow microreactor nevertheless have some limitations arising from the limited solubility of the reactants and products, incompatibility of solid reactants and the necessity of using of a unique solvent or miscible solvent for multistep reaction when an extractor cannot be integrated into the system. The combination of different operation units is not always efficient due to mixing problems, the handling of multi-step reactions or different phase reactions and finally the separation of these phases.72 The identification of products in a very small scale reaction is another issue, which however if feasible can be overcome by the incorporation of an on-line analytical system.73 In addition, the cost of construction and technical complexity of such systems has to be considered.74 The decision of running a reaction whether in continuous flow or in a batch reactor must be evaluated on a case-by-case basis considering the purpose of the study (rapid screening or optimization of conditions, scale up, high throughput etc.) and important factors such as mixing, heat transfer, or reaction time.75
32 1. Continuous Flow Microreactor for Organic Synthesis
1.6 Conclusions
Continuous flow techniques have been used as alternative to batch reactions. Applications have been shown in diverse fields of research ranging from biology, biotechnology, polymer chemistry to traditional organic chemistry. Typical uses of the continuous flow microreactors require that these systems be inexpensive and simple to operate. Eventually these devices can become powerful tools for chemical reactions, analysis, cells sorting, high-throughput screening and particle production. Despite successful examples in many research areas on how the continuous flow microreactors can facilitate and improve various transformations, their potential has not been realized entirely. One main limitation of these technologies is related to the fact that even if modular systems are available, there are no flexible systems that allow for a wide set of chemical transformation. Still each system remains quite specific for one particular chemical reaction. Another main disadvantage that chemists have to face is its incompatibility with solids and the clogging of the system due to precipitation of components of the reaction mixture. The aim of this dissertation is two-fold: first, to investigate and improve upon the synthesis of particles, benefiting from the highly controlled parameters and reaction conditions of the microreactor. The second part of the thesis focuses on photo-initiated emulsion polymerization in continuous flow microreactor and reports on the reaction mechanism of phosphine oxide initiators.
33 1. Continuous Flow Microreactor for Organic Synthesis
1.7 References
1 For reviews and books on microreactor technology, see: (a) Ehrfeld, W.; Hessel, V.; Löwe, H. Microreactors: New Technology for Modern Chemistry, Wiley-VCH, Weinheim, 2000. (b) Fletcher, P. D. I.; Haswell, S. J.; Pombo-Villar, E.; Warrington, B. H.; Watts, P.; Wong, S. Y. F.; Zhang, X. L. Tetrahedron 2002, 58, 4735–4757. (c) Hessel, V.; Hardt, S.; Löwe, H. Chemical Micro Process Engineering, Wiley-VCH, Weinheim, 2004. (d) Thayer, A. M. Chem. Eng. News 2005, 83, 43–87. (e) Geyer, K.; Codée, J. D. C.; Seeberger, P. H. Chem. Eur. J. 2006, 12, 8434–8442. (f) Brivio, M.; Verboom, W.; Reinhoudt, D. N. Lab Chip 2006, 6, 329–344. (g) Mason, B. P.; Price, K. E.; Steinbacher, J. L.; Bogdan, A. R.; McQuade, D. T. Chem. Rev. 2007, 107, 2300–2318. (h) Wiles, C.; Watts, P. Eur. J. Org. Chem. 2008, 10, 1655–1671. (i) Microreactors in Organic Synthesis and Catalysis ed. T., Wirth; Wiley–VCH, Weinheim, 2008; (f) Luis, S. V.; Garcia- Verdugo, E. Chemical Reactions and Processes under Flow Conditions RSC Green Chemistry Series 2010. 2 Anzenbacher, P.; Palacios, M. A. Nat. Chem. 2009, 1, 80–86. 3 www.micronit.com 4 www.uniqsis.com 5 Köhler, J.M.; Henkel, Th.; Grodrian, A.; Kirner, Th.; Roth, M.; Martin; Metze, K. J.
Chem. Eng. J. 2004, 201, 201–216. 6 Hook, B. D. A.; Dohle, W.; Hirst, P. R.; Pickworth, M.; Berry, M. B.; Booker-Milburn,
K. I. J. Org. Chem. 2005, 70, 7558–7564. 7 www.pumpen-ventile.de 8 www.emeraldinsight.com 9 www.nanobits.com 10 Snyder, D. A.; Noti, C.; Seeberger, P. H.; Schael, F.; Bieber, T.; Rimmel, G.; Ehrfeld, W. Helv. Chim. Acta 2005, 88, 1-9. 11 Holger, B. H.; Gärtner, C. Rev. Mol. Biotech. 2001, 82, 89–99.
34 1. Continuous Flow Microreactor for Organic Synthesis
12 (a) Willis, P. A.; Hunt, B. D.; White, V. E.; Lee, M. C.; Ikeda, M.; Bae, S.; Pelletier, M. J.; Grunthaner, F. J. Lab Chip 2007, 7, 1467–1474; (b) Grover, W. H.; von Muhlen, M. G.; Manalis, S. R. Lab Chip 2008, 8, 913–918; (c) Yoon, T. H.; Park, S: H.; Min, K. I.; Zhang,
X. L.; Hanswell, S. J.; Kim, D. P. Lab Chip 2008, 8, 1454–1459. 13 Kikutami, Y.; Hibara, A.; Uchiyama, K.; Hisamoto, H.; Tokeshi, M.; Kitamori, T. Lab
Chip 2002, 2, 193–196. 14 Knitter, R.; Gohring, D.; Risthaus, P.; Hausselt, J. Microsyst. Technol. 2001, 7, 85–90. 15 (a) Jahnisch, K.; Hessel, V.; Lowe, H.; Bearns, M. Angew. Chem. Int. Ed. 2004, 43, 406– 446. (b) Murphy, E. R.; Inoue, T.; Sahoo. H. R.; Zaborenko, N.; Jensen, K. F. Lab Chip,
2007, 7,1309–1314. 16 Strike, D. J.; Thiébaud, P.; van der Sluis, A. C.; Koudelka-Hep, M.; de Rooij, N. F.; Microsys. Tech. 1994, 1, 48–50. 17 (a) Watts, P.; Haswell, S. J. Chem. Soc. Rev. 2005, 34, 235–246. (b) Okamoto, H.; Ushijima, T.; Kitoh, O. Chem. Eng. J. 2004, 101, 57–63. (c) Hasegawa, T.; Koyama, D.; Nakamura, K.; Ueha S. J. Electroceram. 2008, 20, 145–151. 18 (a) Marre, S.; Park, J.; Rempel, J.; Guan, J.; Bawendi, M. G.; Jensen, K. J. Adv. Mater. 2008, 20, 4830–4834. (b) Marre, S.; Adamo, A.; Basak, S.; Jensen, K. Lab Chip 2010, DOI: 10.1039/c000156b. (c) Muller, A.; Cominos, V.; Hessel, V.; Horn, B.; Schurer, J.; Ziogas, A.; Jahnisch, K.; Hillmann, V.; Grosser, V.; Jam, K. A.; Bazzanella, A.; Rinke, G.;
Kraute, A.; Chem. Eng. J. 2005, 107, 205–214. 19 (a) Yen, B. K. H.; Gunther, A.; Schmidt, M. A.; Jensen, K. F.; Bawendi, M. G. Angew. Chem. Int. Ed. 2005, 44, 5447–5451. (b) Trachsel, F.; Hutter, C.; von Rohr, P. R. Chem.
Ing. J. 2008, 135, S309–S316. 20 Jähnisch, K.; Hessel, V.; Löwe, H.; Baerns, M. Angew. Chem., Int. Ed. 2004, 43, 406. 21 (a) Hessel, H.; Lowe, H.; Schonfeld, F. AIChE J. 2003, 49, 599–577. (b) Nguyen, N. T.;
Wu, Z. G. J Micromech. Microeng. 2005, 15, R1–R16. 22 (a) Hessel, H.; Lowe, H.; Schonfeld, F.; Chem. Eng. Sci. 2005, 60, 2479–2501. (b) Liu, R. H.; Yang, J. N.; Pindera, M. Z.; Athavale, M.; Grodzinski, P. Lab Chip 2002, 2,151–
157. 23 El Moctar, A. O.; Aubry N.; Batton J. Lab Chip 2003, 3, 273–280.
35 1. Continuous Flow Microreactor for Organic Synthesis
24 Strrock, A. D.; Dertinger, A. A.; Mezic, I.; Stone, H. A.; Whitesides G. M. Science 2002,
295, 647–651. 25 (a) Kralij, J. G.; Sahoo, H. R.; Jensen, K. F. Lab Chip 2007, 7, 256–263. (b) Aota, A.;
Nonaka, M.; Hibara, A.; Kitamori, T. Angew. Chem. Int. Ed., 2007, 46, 878–880. 26 Hartman, R. L.; Sahoo, H. R.; Yen, Be. C.; Jensen, K. F. Lab Chip 2009, 9, 1843–1849. 27 (a) Baxendale, I. R.; Lee, A.-L.; Ley, S. V. J. Chem. Soc., Perkin Trans. 1 2002, 1850– 1857. (b) Baxendale, I. R.; Ley, S. V.; Smith, C. D.; Tranmer, G. K. Org. Lett. 2006, 8, 5231–5234. (c) Iglesias-Sigüenza, F. J.; Smith, C. J.; Baxendale, I. R; Ley, S. V. Org. Biomol. Chem. 2007, 5, 2758–2761. (d) Ley, S. V.; Baxendale, I. R. Chimia 2008, 62, 162–168. 28 (a) Sahoo, H. R.; Kralj, J. G.; Jensen, K. F. Angew. Chem. Int. Ed. 2007, 46, 566–577. (b) Baxendale, I. R.; Griffiths-Jones, C. M.; Ley, S. V.; Tranmer, G. K. Synlett 2006, 427–
430. 29 Losey, M. W.; Schmidt, M. A.; Jensen, K. F. Ind. Eng. Chem. Res. 2001, 40, 2555–2562. 30(a) Löbbecke, S.; Ferstl, W.; Pani, S.; Türcke, T. Chem. Eng. Tech. 2005, 28, 484–493. (b) Zhang, X. L.; Wiles, C.; Painter, S. L.; Watts, P.; Haswell, S. J. Chim. Oggi-Chem. Today 2006, 24, 43–45. (b) Hartman, R. L.; Naber, J. R.; Buchwald, S. L.; Jensen, K.F.; Angew. Chem. Int. Ed. 2010, 49, 899–903. 31 (a) Leng, J.; Salmon, J. B. Lab Chip 2009, 9, 24–34. (b) Engl, W.; Backov, R.; Panizza, P. Curr Opin. Colloid Interface Sci. 2008, 13, 206–216. (c) Dendukuri, D.; Doyle, P. S. Adv. Mater. 2009, 21, 4071–4086. (d) Marre, S.; Jense, K. F. Chem Soc Rev. 2010, 39, 1183–1202. 32 (a) Tumarkin, E.; Kumacheva, E. Chem. Soc. Rev. 2009, 38, 2161–2168. (b) LoPresti, C.; Lomas, H.; Massignani, M.; Smart, T.; Battaglia, G. J. Mater. Chem. 2009, 19, 3567–
3590. 33 (a) Sue, K.; Kimura, K.; Arai, K. Mater. Lett. 2004, 58, 3229–3231. (b) Sue, K.; Hattori, H.; Satp, T.; Komoriya, T.; Kawai-Nakamura, A.; Tanaka, S.; Hiaki, T.; Kawasaki, S.;
Takebayashi, Y.; Yoda, S.; Furuya, T. Chem Lett. 2009, 38, 792–793. 34 Shah, R. K.; Kim, J. W.; Agresti, J. J.; Weitz, D. A.; Chu, L. Y. Soft Matter 2008, 4,
2303–2309.
36 1. Continuous Flow Microreactor for Organic Synthesis
35 Zhang, H.; Ju, X. J.; Xie, R.; Cheng, C. J.; Ren, P. W.; Chu, L. Y. J. Colloid Interface
Sci. 2009, 336, 235–243. 36 Xu, S.; Nie, Z.; Seo, M.; Lewis, P.; Kumacheva, E.; Stone, H. A.; Garstecki, P.; Weibel, D. B.; Gitlin, I.; Whitesides G. M. Angew. Chem. Int. Ed. 2005, 44, 724–728. 37 Seo, M.; Nie, Z.; Xu, S.; Lewis, P. C.; Kumacheva, E. Langmuir 2005, 21, 4773–4775. 38 (a) Hayward, R. C.; Utada, A. S.; Dan, N.; Weitz, D.A. Langmuir 2006, 22, 4457–4461. (b) Kim, J. W.; Utada, A. S.; Fernandez-Nieves, A.; Hu, Z. B.; Weitz, D. A. Angew. Chem. Int. Ed. 2007, 46, 1819–1822. (c) Karnik, R.; Gu, F.; Basto, P.; Cannizarro, C.; Dean, L.;
Kyei, M. W.; Langer, R.; Farokhzad, O. C. Nano Lett. 2008, 8, 2906–2912. 39 Lin, X. Z.; Terepka, A. D.; Yang, H. Nano Lett. 2004, 4, 2227–2232. 40 Köhler, J. M.; Wagner, J. Nano Lett. 2005, 5, 685–691. 41 Song, Y.; Cumar, C. S.; Hormes, J. Nanosci. Nanotechnol. 2004, 7, 788–793. 42 Hung, L. H.; Lee, A. P. J. Med. Biol. Eng. 2007, 27, 1–6. 43 (a) Kumar, D. V. R.; Kasture, M.; Prabhune, A. A.; Ramana, C. V.; Prasad, B. L. V.; Kulkarni, A. A. Green Chem. 2010, 12, 609–615. (b) Hartlieb, K. J.; Saunders, M.; Jachuck, R. J. J.; Raston, C. L. Green Chem. 2010, 12, 1012–1017. (c) Iyer, K. S.; Raston, C. L.; Saunders, M. Lab Chip 2007, 7, 1800–1805. (d) Nishikawa, H.; Morita, T.;
Sugiyama, J.; Kimura, S. J. Colloid Interface Sci. 2004, 280, 506–510. 44 Salazar-Alvarez, G.; Muhammed, M.; Zagorodni, A. A. Chem. Eng. Sci. 2006, 61,
4625–4633. 45 (a) He, P.; Greenway, G.; Haswell, S. J. Chem. Eng. J. 2011, 167, 694–699. (b) Corradi, A. B.; Bondioli, F.; Ferrari, A. M.; Focher, B.; Leonelli, C. Pow. Tech. 2006, 167, 45–48. 46 Yonemoto, T.; Kubo, M.; Doi, T.; Tadaki, T. Chem. Eng. Res. Des. 1997, 75, 413–419. 47 (a) Abou-Hassan, A.; Bazzi, R.; Cabuil, V. Angew. Chem. Int. Ed. 2009, 48, 7180–7183. (b) Wang, H.; Nakamura, H.; Uehara, M.; Miyazaki, M.; Maeda, H. Chem. Comm. 2002,
1462–1463. 48 Wagner, J.; Köhler, J. M. Nano Lett. 2005, 5, 685–691. 49 Kikkeri, R.; Laurino, P.; Odedra, A.; Seeberger, P. H. Angew. Chem. Int. Ed. 2010, 49,
2054–2057. 50 (a) Marre, S.; Park, J.; Rempel, J.; Guan, J.; Bawendi, M. G.; Jensen, K. F. Adv. Mater. 2008, 20, 4830–4834. (b) Sounart, T. L.; Safier, P. A.; Voigt, J. A.; Hoyt, J.; Tallant, D. R.;
37 1. Continuous Flow Microreactor for Organic Synthesis
Matzke, C. M.; Michalske, T. A. Lab Chip 2007, 7, 908–915. (c) Yen, B. K. H.; Günther, A.; Schmidt, M. A.; Jensen, K. F.; Bawendi, M. G. Angew. Chem. Int. Ed. 2005, 44, 5447– 5451. (d) Shestopalov, I.; Tice, J. D.; Ismagilov, R. F. Lab Chip 2004, 4, 316–321. (e) Edel, J. B.; Fortt, R.; deMello, J. C.; deMello, A. J. Chem. Commun. 2002, 1136–1137. (f)
Dideko, Y. T.; Suslick, K. S. J. Am Chem Soc. 2005, 127, 12196–12197. 51 (a) Paternó, E. Gazz. Chim. It. 1909, 39, 237–250. (b) Ciamacian, G. Science 1912, 36,
385–394. 52 (a) Protti, S.; Dindi, D.; Fagnoni, M.; Albini, A. Green Chem. 2009, 11, 239–249. (b) Protti, S.; Fagnoni, M. Photochem. Photobiol. Sci. 2009, 8, 1499–1516. (c) Coyle, E. E.;
Oelgemöller, M. Photochem. Photobiol. Sci. 2008, 7, 1313–1322. 53 Cook, T. R.; Dogutan, D. K.; Reece, S. Y.; Surendranath, Y.; Teets, T. S.; Nocera, D. G.
Chem Rev. 2010, 110, 6474–6502. 54 (a) Photochemical Key Steps in Organic Synthesis, ed. Mattay J., Griesbeck A., VCH- Wiley, Weinheim, 1994. (b) Synthetic Organic Chemistry, ed. Griesbeck A., Mattay J., Dekker M., New York, 2005. (c) CRC Handbook of Organic Photochemistry and Photobiology, Horspool W., Lenci F., 2nd Ed, CRC, Orlando, 2004. (d) Ciana, C. L.; Bochet, C. Chimia 2007, 61, 650–654. (f) Hoffmann, N. Chem. Rev. 2008, 108, 1052– 1103. (g) Esser, P.; Pohlmann, B.; Scharf, H.–D. Angew. Chem. Int. Ed. 1994, 33, 2009– 2023. (h) Oelgemoller, M.; Jung, C.; Mattay, J. Pure Appl. Chem. 2007, 79, 1939–1948. (i) Pohlmann, B.; Scharf, H. D.; Jarolimek, U.; Mauermann, P. Solar Energy 1997, 61, 159– 168. (l) Oelgemoller, M.; Schiel, C.; Mattay, J.; Frohlich, R. Eur. J. Org. Chem. 2002, 2465–2474. (m) Schiel, C.; Oelgemoller, M.; Ortner, J.; Mattay, J.; Zimmermann, E. Green Chem. 2005, 7, 35–38. (n) Schiel, C.; Oelgemoller, M.; Ortner, J.; Mattay, J. Green Chem. 2001, 3, 224–228. (o) Oelgemoller, M.; Healy, N.; de Oliveira, L.; Jung, C.; Mattay, J. Green Chem. 2006, 8, 831–834. (p) Wilkins, F. W.; Blake, D. M., Chem. Eng. Progr. 1994, 90, 41–49. 55 (a) Chin, P.; Barney, W. S.; Pindzola, B. A. Curr. Opin. Drug Discov. Dev. 2009, 12, 848–861. (b) Hartman, R. L.; Jensen, K. F. Lab Chip 2009, 9, 2495–2507. (c) Fukuyama,
T.; Rahman, M. T.; Sato, M.; Ryu, I. Synlett 2008, 151–163. 56 (a) Lapkin, A. A.; Boddu, V. M.; Aliev, G.N.; Goller, B.; Polisski, S.; Kovalev, D. Chem Eng. J. 2008, 136, 331–336. (b) Chen, H. W.; Ku, Y.; Irawan, A. Chemosphere 2007, 69,
38 1. Continuous Flow Microreactor for Organic Synthesis
184–190. (c) Chen, D. H.; Ye, X. J.; Li, K. Y. Chem Eng Technol. 2005, 28, 95–97. (d) Kriesel, G.; Meyer, S.; Tietze, D.; Fidler, T.; Gorges, R.; Kirsch, A.; Schäfer, B.; Rau, S.; Chem. Ing. Tech. 2007, 79, 153–159. (e) Landgraf, S. Spectrochim. Acta, Part A, 2001, 57, 2029–2048. (f) Sugimoto, A.; Sumino, Y.; Takagi, M.; Fukuya, T.; Ryu, I. Tetrahedron Lett. 2006, 47, 6197–6200. 57 (a) Hessel, V.; Löwe, H.; Stange, T. Lab Chip 2002, 2, 14N–21N. (b) He, Z.; Li, Y.;
Zhang, Q.; Wanga, H. App. Cat. B: Env. 2010, 93, 376–382. 58 Lu, H.; Schimdt, M. A.; Jensen, K. F. Lab. Chip 2001, 1, 22–28. 59 Wootton, R. C. R.; Fortt, R.; de Mello, A. J. Org. Process Res. Dev. 2002, 6, 187–189. 60 www.mikroglas.com 61 (a) Hook, B. D. A.; Dohle, W; Hirst P., L.; Pickworth, M.; Berry, M. B.; Booker- Milburn, K.M J. Org. Chem. 2005, 70, 7558–7564. (b) Laurino, P.; Kikkeri, R.; Azzouz
N., Seeberger P. H. Nano Lett 2011, 11, 73–78. 62 Funayama, H.; Sugarawa, T. Bull Chem Soc. Jpn 1987, 70, 2245–2249. 63 Tsutsumi, K.; Terao, K.; Yamaguchi, H.; Yoshimura, S.; Morimoto, T.; Kakiuchi, K.;
Fukuyama, T.; Ryu I. Chem. Lett. 2010, 39, 828–829. 64 Booker-Milburn, K.; Anson, C. E.; Clissold, C.; Costin, N. J.; Dainty, R. F.; Murray, M.; Patel, D.; Sharpe, A. Eur. J. Org. Chem. 2001, 1473–1482. 65 Li, W.; Pham, H. H.; Nie, Z.; MacDonald, B.; Güenther, A.; Kumacheva, E. J. Am.
Chem. Soc. 2008, 130, 9935–9941. 66 (a) Geyer, K.; Gustafsson, T.; Seeberger, P. H. Synlett 2009, 15, 2382–2391. (b) Watts,
P.; Haswell, S. J. Chem. Soc. Rev. 2005, 34, 235–346. 67 (a) Bogdan, A. R. Poe, S. L. Kubis, D. C. Broadwater, S. J. McQuade, D. T. Angew. Chem. Int. Ed. 2009, 48, 8547–8550. (b) Kawaguchi, T. Miyata, H. Ataka, K. Mae, K. Yoshida, J. Angew. Chem. Int. Ed. 2005, 44, 2413–2416. (c) Suga, S. Nagaki, A. Yoshida, J.-I. Chem. Commun. 2003, 354–355. 68 Fukuyama, T., Chino, Y.; Katama, N.; Ryu, I. Chem. Lett. 2004, 33, 1430. 69 (a) Schwalbe, T.; Autze, V.; Hohmann, M.; Stirner, W. Org. Process. Res. Dev., 2004, 8, 440–454. (b) Roberge, D. M.; Ducry, L.; Bieler, N.; Cretton, P.; Zimmermann, B. Chem.
Eng. Technol. 2005, 28, 318–323.
39 1. Continuous Flow Microreactor for Organic Synthesis
70 (a) Gustafsson, T.; Gilmour, R.; Seeberger, P. H. Chem. Comm. 2008, 3022–3024. (b)
O'Brien, A. G.; Lévesque, F.; Seeberger, P. H. Chem. Comm. 2011, 2688–2690. 71 Schwalbe, T.; Kadzimirsz, D.; Jas, G. QSAR Comb. Sci. 2005, 24, 758–768. 72 Watts, P.; Wiles, C. Org. Biomol. Chem. 2007, 5, 727–732. 73 Martha, C. T.; Heemskerk, A.; Hoogendoorn, J.-C.; Elders, N.; Niessen, W. M. A.; Orru R., V. A.; Irth H. Chem. Eur. J. 2009, 15, 7368–7375. 74 Brandt, J. C.; Wirth, T.; Beilstein J. Org. Chem. 2007, 5, No.30. 75 Valera, F. E.; Quaranta, M.; Moran, A.; Blacker, J.; Armstrong, A.; Cabral, J. T.; Blackmond, D. G. Angew. Chem. Int. Ed. 2010, 49, 2478–2485.
40 2. Synthesis and Functionalization of Quantum Dots
2. Synthesis and Functionalization of Quantum Dots
This part of the thesis was performed with Dr. R. Kikkeri (collaboration on project design and data evaluation), Dr. A. Odedra. (attempts for the first QDs synthesis in continuous flow microreactor) and Dr. B. Lepenies (studies on in vitro and in vivo imaging).
Parts of this chapter have been published in: • “In Vitro Imaging and in Vivo Liver Targeting with Carbohydrate Capped Quantum Dots” Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger, P. H. J. Am. Chem. Soc., 2009, 131, 2110–2112. • “Synthesis of Carbohydrate-Functionalized Quantum Dots in Microreactors” Kikkeri, R.; Laurino, P.; Odedra, A.; Seeberger, P. H: Angew. Chem. Int. Ed. 2010, 49, 2054–2057. • “Continuous Flow Reactor Based Synthesis of Carbohydrate and Hydrolipoic Acid Capped Quantum Dots” Laurino, P.; Kikkeri, R.; Seeberger, P. H. Nat. Protoc. 2011, in press.
41 2. Synthesis and Functionalization of Quantum Dots
2.1 Introduction
Nanomaterials have been applied broadly fields raging from electronics to life sciences.1 Semiconducting nanoparticles also called “quantum dots” (QDs) are of significant importance owing to their tunable emission wavelength and long luminescence lifetimes (compared to organic dyes).2 QDs are semiconducting crystalline solids composed of an inorganic core stabilized by an organic shell of surfactant (ligand). In solid state physics semiconducting materials are characterized by a defined energy bandgap where no electron states can exist, between the top of the valence band and the bottom of the conduction band. The electrons can absorb photon (light) or phonon (heat) and gain enough energy to overcome this bandgap. In QDs, the bandgap and size inversely proportional, smaller the size of the particle bigger will be the bandgap.3
Figure 2.1 Relationship between bandgap energy and size of QDs.4 Due to the strong relationship between the size and bandgap energy, QDs with a high controlled size and very precise photo-physical properties can be produced.5 The tuneability of QDs in the visible and near-infrared (NIR) spectral region confers a broad
42 2. Synthesis and Functionalization of Quantum Dots absorption and a narrow emission wavelength for these particles (Figure 2.2).6 These optical properties of QDs find very important applications in various fields.
Figure 2.2 Absorbance and fluorescence spectra of CdSe/ZnS, CdTe and CdTe/CdSe quantum dots of various sizes.
The synthesis of QDs can be accomplished by the pyrolysis of organometallic precursors of Cd/S or Se or Te.7 QDs are commonly prepared by the reaction of dimethyl 8 cadmium (CdMe2), with trioctylphosphine selenide (TOPSe) in a mixture of coordinating solvents such as trioctylphosphine (TOP) and trioctylphosphine oxide (TOPO) at higher temperatures (300 °C). Improvements of the above process included the addition of non- volatile Cd precursors (cadmium chloride, cadmium oxide, cadmium acetate or cadmium carbonate) to a mixture of alkylphosphonic acid or alkyl amines at higher temperatures. These changes allowed for better control of particle nucleation and growth, and consequentially yielded QDs of different size.9, 1c The oxidation of QDs or leaching of the core metal into the solution necessitates the core protection with a suitable semiconductor such as ZnS, CdS, CdSe, ZnSe or ZnTe.10 The shell can also have more specific functions depending on the bandgaps and the relative position of the electronic energy state of the involved semiconductors. The core/shell particles can be divided into three categories depending on the differences between the bandgap energies of the core and shell (Figure 2.3).
43 2. Synthesis and Functionalization of Quantum Dots
Figure 2.3 Schematic representation of the energy-level alignment in type I and type II core/shell QDs.11
The core material of type I QDs are characterized by a smaller bandgap energy than the shell material (e.g. CdSe/ZnS).12 The emission peak of type I QDs depends on the size of the core, and the shell has the function of improving the optical properties of the core and stabilizing it from photodegradation. In type II QDs the energy level of the shell is upper or lower compared to the bandgap of the core (e.g. CdTe/CdSe or CdSe/CdTe) and in these particles the emission wavelength can be changed by increasing or decreasing the thickness of the shell (700–1000 nm). The quantum yield of the type II QDs particles is lower than to type I QDs.13 The third class, reverse type I QDs has a bandgap larger for the core than that of the shell. The emission spectra of these particles can be varied in the range of 450–700 nm by varying the thickness of the shell without substantial decrease in the quantum yield of the particles (e.g. CdS/HgS, CdS/CdSe or ZnSe/CdSe).14 To further improve the stability and biocompatibility of QDs, the shell can be capped with polymers, bio-organic compounds or silica. The properties of the shell can improve the solubility of the nanoparticles and can act as a handle for bioconjugation while retaining the optical properties of the QDs.15 The shell can be functionalized by exchanging the ligand on the surface of the particle with organic componds. In the case of polymers or dendrimers having hydrocarbon chains, the functional group can be bound to the external shell through hydrophobic or electrostatic interactions.16 Bio-organic compounds such as proteins, peptides, sugars,17 can also be functionalized by a thiol group that forms a dative bond with the external surface of the shell.2a In case of capped silica QDs a base catalyzed hydrolysis of tetraethoxysilane (TES) is carried out on the surface of QDs to encapsulate the QDs shell.18
44 2. Synthesis and Functionalization of Quantum Dots
2.1.1 Previous work on the Continuous Flow Synthesis of QDs
The increasing use of QDs for different applications necessitates the development of a scalable procedure for their preparation.19 The scale up of conventional batch QDs syntheses is limited, due to the lack of precise control of nucleation and QD growth. This limitation arises in batch syntheses due to the poor homogenous heating and mixing of the reactants. To overcome these difficulties, some research groups have reported that the high surface-to-volume ratio of the microreactor channels enable precise temperature control as well as efficient mixing, resulting in the preparation of QDs with high control of particle nucleation and growth.20 In addition, injecting large volumes of precursor in the system facilitates the scale up in a straightforward manner in the continuous flow microreactor providing enough material for applications. The preparation of high quality CdSe QDs in a continuous flow microreactor requires a solution of selenium and cadmium precursors containing surfactant stabilizers. The selenium precursor is generally tri-n-octylphosphine selenide (TOPSe) formed from elemental Se dissolved in trioctylphosphine (TOP). To afford cadmium precursor, cadmium sources (CdO, CdMe2, and Cd(Ac)2) and a surfactant (stearic acid or oleic acid) are typically dissolved in an high-boiling solvent like oleylamine, squalene, or toluene. The reactants are introduced into the system by two syringes that are filled with selenium and cadmium precursor respectively and then simultaneously injected into the microreactor or pre-mixing the precursors and then injecting using one single syringe. The synthesis of QDs occurs in the heated section of the microreactor followed by the quenching of the reaction at room temperature in the later part of the microreactor. The approach mainly used for the synthesis of QDs in continuous flow is the two phase flow (gas-liquid phase). In the gas-liquid phase a premixed solution of the precursors is first injected followed by addition of an inert gas (e.g. Argon)21 and in the liquid phase the two precursor solutions are delivered into the microreactor in two separated flows.22
45 2. Synthesis and Functionalization of Quantum Dots
Figure 2.4 Schematic representation of QDs synthesis in a continuous flow reactor using gas-liquid or liquid phases.
The first two-phase synthesis of QDs in continuous flow was reported by Bawendi et al.21b who used a gas-liquid segmented flow microreactor for the synthesis of QDs at high temperature. The advantage of using a two phase system is that every droplet undergoes a re-circulatory motion which results in better mixing of the reactants (Figure 2.5).
Figure 2.5 Schematic representation of recirculatory motion in a two phase reaction (segmented flow).
Bawendi et al.21b achieved the synthesis of monodisperse CdSe QDs exploiting the two phase system at 280 °C. The inert gas used in the study was argon and the liquid phase consisted in a mixture of cadmium 2,4-pentanedionate in oleic acid, squalane, oleylamine and SeTOP. A systematic study for screening residence times and different ratio between gas and liquid phase was facilitated by the use of a continuous flow microreactor. This investigation illustrated how a microreactor synthesis facilitated the systematic study of the
46 2. Synthesis and Functionalization of Quantum Dots mechanism and control of QD formation. Later studies used this two phase continuous flow microreactor system for the synthesis of QDs.21a, b, f Compared to the synthesis of QDs under homogenous condition the two phase approach requires complex experimental setup and involves longer residence time as major disadvantages. To achieve narrow residence time distribution for two-phase systems higher temperature are involved. Therefore, the synthesis and functionalization of QDs under homogeneous condition is very challenging. In this chapter, we present the synthesis of type-I core/shell crystals (CdSe/ZnS and CdTe/ZnS) and their functionalization in batch and an improved synthesis in continuous flow in homogenous phase at lower temperature. The obtained QDs were later functionalized and used for binding assays and imaging studies.
47 2. Synthesis and Functionalization of Quantum Dots
2.2 Quantum Dots Batch Synthesis
Our initial studies were directed toward the synthesis of QDs using a batch process. CdSe/ZnS quantum dots with an emission maximum of 635 nm were chosen for the synthesis.6 The QDs were prepared by heating a mixture of cadmium oxide, trioctylphosphine oxide (TOPO), lauric acid, and hexadecylamine under nitrogen at 310 °C. A solution of tri-n-octylphosphine selenide (TOPSe) was rapidly injected into the reaction mixture at 310 °C which was immediately cooled down to 270 °C (Scheme 2.1).
Scheme 2.1 QDs synthesis in batch reactor. (a) 310 °C to 270 °C 5 min; cooled to 60 °C; (b) 160 °C, 1h (TOP: tri-n-octylphosphine; TOPO: tri-n-octylphosphine oxide).
The reaction was stirred for five minutes at 270 °C and then cooled to 60 °C to stop particle growth (Figure 2.6).2b After purification of the CdSe QDs, the ZnS precursor was added to a solution of CdSe at 160 °C to afford CdSe/ZnS QDs. 2b The resulting CdSe/ZnS QDs were stabilized by a layer of surfactant (TOPO/TOP). The carbohydrate–PEGylated linker and PEGylated linker (polyethylenglycol) that were required for the functionalization of CdSe/ZnS QDs, were then synthesized. The synthesis of the PEG derivatives begin from PEG2000 1, which was coupled to thioctic acid to give ester 2 (Scheme 2.2).
48 2. Synthesis and Functionalization of Quantum Dots
Scheme 2.2 Synthesis of DHLA-PEG2000 (3) and DHLA-PEG2000-NH2 (6). Reagents and conditions: (a) DL-Thioctic acid, DCC, DMAP, rt, 12 h (12%); (b) NaBH4, MeOH/H2O, 0°C, 3h
(36%); (c) MsCl, TEA, NaN3, rt, 12 h (84%); Ph3P, H2O, rt, 12 h (63%); (d) Thioctic acid, DIC,
NHS, rt, 12 h (48%); (e) NaBH4, MeOH/H2O, rt, 3h (47%).
Subsequent reductive opening of 2 afforded 3, which was used as control during imaging studies. On the other hand reaction of 1 with methanesulfonyl chloride, sodium azide and triphenylphosphine afforded diamino-PEG2000 4. Monoacylation of 4 by treatment with thioctic acid N-hydroxysuccinimide (NHS) ester afforded 5, which underwent reductive ring opening to give 6. The DHLA-PEG2000-NH2 6 was used for functionalization of QDs and further modification. The thiol linked monosaccharide mannose 10 and galactose 14 that were required as sugar moiety for the functionalization of QDs, were prepared. The corresponding commercial available monosaccharides 7 and 11 were acetylated and then glycosylated using 2-[2-(2-chloroethoxy)ethoxy]ethanol and BF3·Et2O (Scheme 2.3).
49 2. Synthesis and Functionalization of Quantum Dots
Scheme 2.3 Synthesis of mannose-SH (10) and galactose-SH (14) derivatives. Reagents and conditions: (a) Ac2O/Py, rt, 1 h (quant.); (b) 2-[2-(2-chloroethoxy)ethoxy]ethanol, BF3·Et2O, rt, overnight (64%); (c) KSAc, DMF, rt, 3 h (84%); (d) NaOMe, MeOH Amberlite 120 (H+), rt, 1 h
(87%); (e) Ac2O/Py, rt, 1 h (quant.); (f) 2-[2-(2-chloroethoxy)ethoxy]ethanol, BF3·Et2O, rt, overnight (68%); (g) KSAc, DMF, rt, 3 h (82%); (h) NaOMe, MeOH Amberlite 120 (H+), rt, 1 h (89%)
Substitution of the chloride group with the thio-acetyl residue followed by basic deacetylation under standard conditions afforded compounds 9 and 13 respectively. The synthesis of thiol linked galactosamine 19 was achieved by treatment of peracetylated galactal 15 with trimethylsilyl azide, bis(acetoxy)iodobenzene and diphenyl diselenide (Scheme 2.4).
Scheme 2.4 Synthesis of galactosamine-SH (19). Reagents and conditions: (a) TMSN3, PhI(AcO)2, diphenyl diselenide, -30 °C, 12 h (97%); (b) 2-[2-(2-chloroethoxy)ethoxy]ethanol, N- Iodosuccinimide, TfOH, rt, overnight (61%); (c) KSAc, DMF, rt, 12 h (78%); (d) NaOMe, MeOH, + Amberlite 120 (H ), rt, 2 h (80%); (e) PMe3, H2O, rt, 12 h (81%).
50 2. Synthesis and Functionalization of Quantum Dots
Glycosylation with 2-[2-(2-chloroethoxy)ethanol followed by substitution gave the thiol derivative 18, before deprotection and reduction of which provided access to 19. The prepared CdSe/ZnS nanoparticles were functionalized by the exchange of the ligand (TOP/TOPO) with 6 to give 22 (Scheme 2.5).
Scheme 2.5 QD functionalization. Reagents and conditions: (a) Chloroform/ethanol, 60 °C, 12 h CdSe/ZnS; (b) borate buffer (pH = 8.5), overnight; (c) borate buffer (pH = 7.3), 3 h, rt, monosaccaride linked thiols.
The PEGylated particles 22 were coupled with 4-maleimidopropanoic acid NHS ester 21 to give 23, which upon treatment with thiol linked monosaccharide gave functionalized QDs 24.
51 2. Synthesis and Functionalization of Quantum Dots
Figure 2.6 Carbohydrate PEGylated QDs. Mannose-PEG2000-QD 24a, Galactosamine-PEG2000- QD 24b, Galactose-PEG2000-QD 24c.
CdSe QDs exhibited absorption at 400 nm and emission at 635 nm, which remained constant for the CdSe/ZnS and the coated particles 24. The quantum yield (Φ), calculated according to equation 4 using a standard sample (Fluorescein) was 0.33 for core particles and coated particles.
(4)
Φref = 0.93 (quantum yield of the reference in this case fluorescence is used as a reference sample); S = area under the emission peak.
The particle size was calculated from the fluorescence spectra by extrapolating from the full width at half maximum (FWHM) values, which showed the formation of particles with 15–20 nm diameters. The main problem during the synthesis in batch was limited control over mixing and the temperature of the reaction mixture that did not allow for a precise control of the particle size. The difficulties encountered in cooling down the batch system allowed only for the synthesis of particles with long emission wavelengths (635 nm).
52 2. Synthesis and Functionalization of Quantum Dots
2.3 Continuous Flow QD Synthesis and Functionalization
The problems associated with the synthesis of QDs in a batch process mainly due to temperature control, homogenous mixing and reproducibility were overcome by synthesizing of QDs in continuous flow microreactor. In this section the three steps synthesis in homogenous phase of CdSe or CdTe/ZnS quantum dots capped with monosaccharides or dihydrolipoic acid has been reported. In the first approach every intermediate of the synthesis step was purified, isolated and analyzed and in the second approach only the final product was isolated and analyzed (Figure 2.7).23 Furthermore, different residence time and temperature for the core formation were screened and resulted in the formation of narrow distributed QDs with different sizes and optical properties of the core.24
Figure 2.7 Continuous microreactor setup for the synthesis of functionalized QDs (OA: oleic acid; TOP: tri-n-octylphosphine).
53 2. Synthesis and Functionalization of Quantum Dots
2.3.1 System Set-up
A commercially available 1 mL glass microreactor (Syrris FRX volcano) was used for QD synthesis.25 The glass microreactor is composed of a micromixer, which allows controlled and homogeneous mixing of different starting materials and a tubular reactor for that enables to reach laminar flow and homogenous heating of the reaction mixture (Figure 2.8).
Figure 2.8 Schematic overview of continuous flow system used for QD synthesis. (1) Syringe pump; (2) Gas-tight syringes; (3) and (3’) PTFE tubing; (4) and (4’) Inlets; (5) Hot plate; (6) Thermocouple; (7) Mixing system; (8) Microreactor ; (9) Outlet; (10) PTFE tubing with back pressure regulator; (11) Collection vessels.
The injection system consists of a syringe pump that controls the flow rate and injection volume according to the residence time required. Using two gas-tight syringes the starting materials were simultaneously injected. The microreactor was washed with the reaction solvent to equilibrate the system and warmed to the required temperature prior to performing the reaction. It is very important to reach a stable temperature before starting the run as any oscillations of the reaction temperature can produce broader and irreproducible distribution particle sizes. In addition,
54 2. Synthesis and Functionalization of Quantum Dots the first 0.1 mL and the last 0.1 mL of each run were discarded to avoid dilution effects that can reduce the quality and the consistency of the QDs.
2.3.2 QD Synthesis
Initially, CdSe and CdTe QDs with different emission maxima were prepared by injection of a 1:1 ratio of cadmium precursor and selenium or tellerium precursor (Figure 2.9).
Figure 2.9 First step of the QDs synthesis: the core particle (OA: oleic Acid; TOP: tri-n- octylphosphine).
The cadmium precursor was prepared by the addition of oleic acid (OA) and dedecanoic acid to a solution of cadmium oxide dissolved in lauric acid at 150 °C. The selenium and tellerium precursors were prepared by dissolving elemental Se or Te powder in tri-n-octylphosphine (TOP). Reaction times varied from three to thirty minutes (3, 10, 20 and 30 min). In case of CdTe, three residence times (3, 10 and 20 min) were investigated, while for CdSe four residence times (3, 10, 20 and 30 min) and four different temperatures at 3 min residence time (160, 180, 200 and 220 °C) were investigated. The produced CdSe or CdTe core QDs were used for further modification via of two approches. In the first approach, the core was directly injected into the second reactor, whereas in the second approach the core was precipitated using a mixture of methanol/chlorofom/hexane, characterized and then injected into the second reactor.
55 2. Synthesis and Functionalization of Quantum Dots
A freshly prepared mixture of hexamethyl disilathiane, TOP, diethylzinc in toluene and zinc sulfide was injected simultaneously with the QDs core precursor dissolved in toluene and TOP into the microreactor. The temperature was maintained at 90 °C with a residence time of 30 min (Figure 2.10).
Figure 2.10 Second step of the QD synthesis: the core/shell particle (OA: oleic Acid; TOP: tri-n- octylphosphine).
The resulting ZnS coated CdTe or CdSe QDs were purified by precipitation using methanol/chloroform or used directly as it was in the second approach of synthesis process.
2.3.3 QD Functionalization
The ZnS capped CdTe or CdSe QDs were functionalized by ligand exchange with pyridine in continuous flow. Freshly prepared oleic acid capped CdTe or CdSe /ZnS QDs were dissolved in pyridine and injected into the microreactor at 60 °C for 30 min. The resulting pyridine capped QDs served to install carbohydrates on the QD nanoparticles. A mixture of freshly prepared dihydrolipoic acid, mercapto-PEG α-mannose 10 or mercapto- PEG β-galactose 14 or dihydrolipoic acid in an ethanol/ethylenedichloride mixture (1:1) and a solution of pyridine-coated QDs in dichloroethane were simultaneously injected into the microreactor at 60 °C with 30 min residence time.
56 2. Synthesis and Functionalization of Quantum Dots
Figure 2.11 Third step of the QD synthesis: capping of core shell particles (EDC: Ethylenedichloride).
The resulting QDs were purified by precipitation with hexane/chloroform/methanol and redispersed in water for further characterization. All mannose, galactose and dihydrolipoic acid-modified QDs had a narrow size distribution, a spherical shape and emission spectra as the CdSe/ZnS QDs.
57 2. Synthesis and Functionalization of Quantum Dots
2.4 Photophysical Properties of QDs Produced in Continuous Flow
In order to understand how different parameters could influence the synthesis and subsequently the properties of the final product, the optical properties of the QDs generated using the microreactor were determinated. The CdSe or CdTe core of QDs particles were isolated and characterized by UV-Vis spectroscopy. The UV-Vis spectra of the CdSe and CdTe core particles prepared at different residence time at 160 °C showed a shift of the curves at higher wavelength and the absorbance peaks (λmax) increased proportionally with the residence times which were directly related to the size, shape and 26 composition of the QDs (Figure 2.12).
Figure 2.12 UV-visible absorption spectra of (a) CdSe QDs and (b) CdTe QDs in chloroform.
The optical properties of QDs showed a time dependent bathochromic shift in the band- edge emission and enhanced intensity. The fluorescence emission peaks of CdTe QDs with maximum emission at 521, 575, and 598 nm were sharp, thus indicating a very narrow size distribution. (Figure 2.13). More importantly, an increased quantum yield was observed with residence times increasing from 3 to 20 min (Table 2.1).
58 2. Synthesis and Functionalization of Quantum Dots
Figure 2.13 Fluorescence spectra of CdTe QDs in chloroform prepared at different residence times: 3, 10 and 20 min.
Table 2.1 Photophysical properties of CdTe QDs.
Entry Time (min) Temperature (°C) QDs diameter (nm) Quantum Yield (Φ)
1 3 160 1.67 ± 0.27 14 ± 1
2 10 160 3.01 ± 0.36 21 ± 1
3 20 160 3.24 ± 0.49 23 ± 1
The photoluminescence peaks of CdSe QDs were sharp indicating the narrow size distribution of the QDs, as evident from the observed 40 to 50 nm FWHM (full width at half maximum) of the band-edge luminescence (Figure 2.14).
59 2. Synthesis and Functionalization of Quantum Dots
Figure 2.14 Fluorescence spectra of CdSe QDs prepared at different residence times at 160 °C with different residence times: 3, 10, 20 and 30 min.
An attempt to increase the reaction time to 30 min resulted in an increase in the FWHM (90 nm) and a decrease in quantum yield. It became obvious that longer reaction times allowed for saturated nucleation to occur after 20–30 min at 160 °C leading to a alterated dispersity and subsequent decrease in quantum yield (Table 2.2).
60 2. Synthesis and Functionalization of Quantum Dots
Table 2.2 Photophysical properties and yields of CdSe QDs prepared at different temperature and residence times. FWHM values calculated according to the fluorescent spectra. Quantum yield was compared to fluorescein at 470 nm (0.93).
Time Temperature Particle FWHM Quantum Yield Entry (min) (°C) diameter (nm) (nm) Yield (Φ) (%) 1 3 160 1.45 ± 0.22 40–50 8 ± 1 72 2 10 160 1.83 ± 0.26 40–50 11 ± 1 71 3 20 160 2.64 ± 0.43 40–60 19 ± 1 67 4 30 160 3.36 ± 0.34 40–90 15 ± 1 66 5 3 180 1.87 ± 0.39 40–45 12 ± 1 72 6 3 200 2.61 ± 0.24 35–40 16 ± 1 79 7 3 220 3.06 ± 0.41 30–35 21 ± 1 88
Constant shift of the absorption and emission curves of the CdSe or CdTe particles according to the residence time was a clear indication for the formation of bigger sized QDs with increased reaction times. It was also evident that higher temperature resulted in the formation of QDs with narrower size distribution, as shown by the fluorescence spectra (Figure 2.15). The FWHM values decreased with an increase in temperature and the diameter increased thus indicating a complete saturation of the particles nucleation (Table 2.2)
61 2. Synthesis and Functionalization of Quantum Dots
Figure 2.15 Fluorescence spectra of CdSe QDs prepared at different temperatures (--- 160 °C, --- 180 °C, ---200 °C and ---220 °C) with 3 min residence time.
The yield of the reaction was calculated according to equation 5 using a known concentration of the starting materials, in this case the concentration of Cd precursor was used as a reference sample (eq. 5)
(5)
The efficiency of the QDs preparation process, determined by the yield of QDs, were satisfying in most of the cases (ca. 70%). A direct correlation with temperature was observed wherein higher yields were obtained at higher temperatures (Table 2.2). The UV- Vis spectra also demonstrated that the core/shell QDs exhibited similar trends to the core QDs. More importantly, the quantum yield increased from 23% to 31% due to the stabilization of the core by the ZnS shell. TEM analysis of the CdTe, CdTe/ZnS and CdTe/ZnS/mannose QDs showed that the QDs did not change in shape and geometry even if covered by a layer of ZnS or ZnS and mannose linker. The QDs maintained their monodispersity and spherical shape (Figure 2.16).
Figure 2.16 TEM images of CdTe core QDs (a); CdTe/ZnS QDs (b); CdTe/ZnS/mannose QDs (c); (scale bar 50 nm).
62 2. Synthesis and Functionalization of Quantum Dots
2.5 Biological Studies Using Carbohydrate-Capped QDs
The development and use of quantum dots for biological and biomedical applications is a fast emerging field of nanotechnology. 27 The unique optical properties of these nanometer-sized semiconducting crystals make them an interesting fluorescent tool for in vivo and in vitro imaging as well as for sensor applications.28 We used sugar-capped PEGylated QDs for targeting hepatic carcinoma cells and as fluorescent biosensor for the detection of carbohydrate-lectin interactions. The asialoglycoprotein (ASGP-R) that is only expressed in hepatic cells,29 is a receptor that specifically binds to galactosyl-terminal proteins. We utilized the galactose capped QDs to target ASGP-R mediated endocytosis using the carcinoma cell line HepG2.2f, 30 The CdSe/ZnS QDs prepared in batch and functionalized with galactosamine (GalN)-PEG2000 24b and PEG2000 linker 24d, were exploited in this study (Figure 2.17).
Figure 2.17 PEGylated QDs involved in the biological studies: Galactosamine-PEG2000-QD 24b, PEG2000-QD 24d.
HepG2 cells were incubated overnight with 20 nmol of PEG2000 QDs 24d or 20 nmol of GalN-PEG2000 QDs 24b. As negative control, PBS was added to the cells. After washing, cells were collected and uptake of QDs was measured by flow cytometry. Compared to the negative control (gray, Figure 2.18), PEG2000 QDs 24d were taken up only to a minor extent (dashed line, Figure 2.18). In contrast, GalN-capped QDs 24b were taken up to a
63 2. Synthesis and Functionalization of Quantum Dots higher extent by HepG2 cells (solid line, Figure 2.18). Further studies demonstrate the specific up–take of QDs by liver cells in vivo.2f
Figure 2.18 Specific uptake of D-GalN capped QDs by HepG2 cells.
As a proof-of-principle, QDs prepared and functionalized in continuous flow were used as fluorescent biosensors for specific carbohydrate-protein interaction. The lectin concanavalin A (ConA), a protein well known to specifically bind mannose, was incubated with α-mannose-functionalized QDs 25 and with β-galactose QDs 26.
64 2. Synthesis and Functionalization of Quantum Dots
Figure 2.19 Mannose-QDs 25 and Galactose-QDs 26.
Immediately after incubation, the binding between CoA and α-mannose-QDs resulted in turbidity whereas β-galactose-QDs did not (Figure 2.20). The disappearance of the turbidity upon the addition of a concentrated solution of mannose (0.1 M), a competitive inhibitor, demonstrated that the turbidity was caused by specific carbohydrate-protein interactions.
Figure 2.20 Kinetics of turbidity by α-mannose (black line) and β-galactose (red line) CdTe/ZnS capped QDs.
In addition, the turbidity caused by the specific interaction between α-mannose capped-QDs and ConA resulted in a decreased intensity in the fluorescence spectra (Figure 2.21). The specific agglutination of α-mannose coupled QDs clearly shows the bioavailibity for protein binding of the monosaccharide on the surface of QDs.
65 2. Synthesis and Functionalization of Quantum Dots
Figure 2.21 Fluorescence spectra of CdTe/ZnS α-mannose capped QD before (black line) and after addition of ConA lectin (pink line).
66 2. Synthesis and Functionalization of Quantum Dots
2.6 Conclusions
The continuous flow three step syntheses of CdTe and CdSe/ ZnS/ monosaccaride or dihydrolipoic acid capped QDs was successfully performed in a continuous flow microreactor to overcome the limitations of QD syntheses in batch. The microreactor further allowed the syntheses of different sized QDs in efficient, reproducible and scalable fashion, and in an inert atmosphere and safe working conditions. The first step syntheses was carried out under homogeneous conditions and at lower temperature compared to the previously reported syntheses (160–220 °C vs 300 °C). To the best of our knowledge we believe that the lower temperatures suppressed the continuous particle nucleation through the high control of the precursor consume. After screening different residence times and temperatures the CdSe or CdTe QDs were capped with a shell of ZnS at 60 °C in continuous flow microreactor. The QDs were then conjugated with thiol linked monosaccharides, and used as sensors to detect the specific interactions with carbohydrates binding proteins. These studies showed that QDs are very potent tools for biological studies because of their extraordinary optical properties and minimal photodegradation. However the major drawback of QDs is the toxicity, and many efforts have been made to synthesize good alternative QD candidates (e.g. InP QDs31) but these particles are not comparable to the optical properties of CdSe and CdTe.
67 2. Synthesis and Functionalization of Quantum Dots
2.7 Experimental Part
2.7.1 General Information
All the reagents were purchased from Aldrich and used as received. Dichloromethane
(CH2Cl2) was purified by a Cycle-Tainer Solvent Delivery System. Triethylamine was distilled over CaH2 prior to use. Analytical thin layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm). Compounds were visualized by UV irradiation or dipping the plate in CAN solution followed by heating. Flash column chromatography was carried out on Fluka Kieselgel 60(230-400 mesh). Absorption spectra were recorded using a Varian CARY 50 spectrophotometer fitted with Hellma optical fibers (Hellma, 041.002-UV) and an immersion probe made of quartz suprazil (Hellma, 661.500-QX). Fluorescence emission spectra were recorded on a Perkin-Elmer LS-50B spectrofluorometer in Chloroform. IR spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer. Optical rotation measurements were conducted using a Perkin-Elmer 241 polarimeter. TEM images were performed with EFTEM Omega 912, Zeiss AG apparatus. 1H and 13C NMR spectra were recorded on a Varian VXR-300 (300 MHz) orBruker DRX500 (500 MHz) spectrometer. High-resolution mass spectra (HR MALDI MS) were recorded using mass Spectrometry service at the Laboratory for Organic Chemistry (ETH Zurich).
68 2. Synthesis and Functionalization of Quantum Dots
2.7.2 Description of the Experimental Set Up
The flow reactor setup consists of commercially available parts. A syringe pump injection system (Harvard PHD 2000, cat. # 702012) is used for reagent delivery and a glass microreactor (Syrris FRX reactor, cat. # 2100146) as the reaction device, which is held on a metal support (Syrris FRX Volcano, cat. # 2101346) mounted on a hotplate (Syrris FRX hotplate, cat. # 2101310). The dimensions of the glass device are 2.50 x 8.5 cm (width x length), with a glass rectangular micromixing zone of 161 x 1240 x 536 x103 µm (width x depth x length) and inner channel dimensions of microreactor are 391 x 1240 x 1844 x 103 µm (width x depth x length). The total volume of the continuous flow device is 1 mL. The reactor is connected with two inlets to the gastight syringes (Hamilton syringes 0.5 mL) via PolyTetraFluoroEthylene (PTFE) tubing (1/16″ OD, 0.030 ID) and the outlet is connected to the collecting flask through PTFE tubing (1/16″ OD, 0.030 ID) as well. Injection of reagents is controlled by adjusting flow rate of in the syringe pump. The syringes are refilled manually and the scale up is due to the use of bigger syringes. For every 1-mL run the first 0.1 mL and the last 0.1 mL were discavered and only the middle fraction (0.8 mL) was collected.
2.7.3 Batch Synthesis of CdSe/ZnS QDs
CdSe/ZnS quantum dots with an emission maximum centered at 635 nm were synthesized via pyrolysis of the organometallic precursors.2b Cadmium oxide (0.05 g, 4.0 mmol), trioctylphosphine oxide (TOPO) (2 g, 5.2 mmol), lauric acid (0.22 g, 1.1 mmol), and hexadecylamine (2 g, 8.3 mmol) were mixed and heated to 310 °C, under nitrogen flow, for 30 min. A selenium stock solution, prepared from selenium (0.03 g, 4.0 mmol) in trioctylphosphine (TOP) (2 mL, 5.4 mmol), was rapidly injected into the reaction mixture at 310 °C. The reaction mixture was then immediately cooled to 270 °C, the stirring was stopped after 5 min and the reaction vessel cooled to 60 °C. CdSe-QD cores were purified
69 2. Synthesis and Functionalization of Quantum Dots by precipitation in methanol/CHCl3. The CdSe particles were subsequently coated with ZnS layers by dropwise addition of a mixture of TOP (3 mL), diethylzinc in hexane (2.0 mL), and hexamethyldisilathiane (0.2 mL) over 1 h at 160 °C. Following cooling to room temperature, the 3 concentration of the resulting CdSe/ZnS QDs solution was then estimated.32
2.7.4 Synthesis of the PEG-Linker
TA-PEG2000-OH (2).
DL-thioctic acid (0.13 g, 0.63 mmol) and PEG2000 1 (12.0 g, 6.0 mmol) were added to a solution of DCC (0.17 g, 0.83 mmol) and N,N dimethylaminopyridine (0.10 g, 0.80 mmol) in dichloromethane at 0 °C, stirred at room temperature for 12 h and concentrated in vacuo. The crude residue was purified over silica gel using EtOAc/MeOH (2-5%) to afford ( TA- 1 PEG2000-OH 2 1.5 g, 12%). H NMR (300 MHz, CD3OD): δ 1.42-1.51 (m, 2H); 1.61- 1.70 (m, 4H); 1.87-1.95 (m, 2H); 2.35 (t, J = 7.5 Hz, 2H); 2.42–2.48 (m, 1H); 2.58–2.62 (t, J = 6.0 Hz, 1H); 3.12–3.18 (m, 2H); 3.41 (t, J = 6.0 Hz, 1H); 3.55–3.72 (m, 164H); 3.87 (t, 13 J = 6.0 Hz, 1H); 4.24 (t, J = 5.4 Hz, 2H); C-NMR (75 MHz, CD3OD); δ 22.3, 24.6, 26.6, 34.1, 88.7, 39.4, 42.7, 53.4, 61.7, 69.3, 70.5, 71.2, 71.3, 72.2, 72.7; HRMS-MALDI (m/z): + [M+Na] Calculated for C80H158O38S2Na + (C2H4O)n 1813.9770 + (44.026)n; Found: 1813.972 + (44.032)n.
70 2. Synthesis and Functionalization of Quantum Dots
DHLA-PEG2000-OH (3). TA-PEG2000-OH 2 (1.0 g, 0.5 mmol) was dissolved in an ethanol/water (1:1) mixture and cooled to 0 °C. Sodium borohydride (20 mg, 0.54 mmol) was added and stirred at 0 °C for 3 h. The solution was neutralized with 1 N HCl solution, extracted with chloroform, dried over sodium sulfate, concentrated and purified over silica gel using CH2Cl2/CH3OH (2- 1 5%) to afford 0.45 g (46%) of DHLA-PEG2000-OH 3. H NMR (300 MHz, CD3OD): δ 1.28–137 (m, 2H); 1.48-1.75 (m, 4H); 1.82–1.92 (m, 1H); 2.34 (t, J = 7.5 Hz, 2H); 2.42– 2.48 (m, 2H); 2.68–2.672 (m, 2H); 2.84–2.92 (m, 1H); 3.55-3.72 (m, 164H); 4.21 (t, J = 13 6.0 Hz, 1H); C NMR (75 MHz, CD3OD); δ 21.8, 24.1, 26.1, 33.5, 38.3, 38.9, 42.4, 61.2, 63.1, 68.7, 69.9, 70.2, 71.3, 71.8, 72.2, 72.5; HRMS-MALDI (m/z): [M+Na]+ Calculated for C80H160O38S2Na + (C2H4O)n: 1815.9770 + (44.026)n; Found: 1815.975 + (44.031)n.
Diazido-PEG2000 (4a). PEG2000 1 (10 g, 5.0 mmol) was coevaporated with 50 mL toluene to remove water, and then 50 mL of THF and methane sulfonyl chloride (1.22 mL. 15.0 mmol) were added. The solution was stirred at 0 °C, and a solution of triethylamine (2.29 mL, 16.5 mmol) in THF (20 mL) was added dropwise over 30 min. After 1 h the ice bath was removed and the mixture was stirred overnight. The formed solid was dissolved by the addition of water (50 mL), forming two layers, that were cooled on an ice bath. Then, 1 N solution of sodium bicarbonate (5 mL) was added followed by sodium azide (1.3 g, 20.0 mmol). THF was removed and the water layer was refluxed for 24 h. The aqueous layer was extracted with dichloromethane (4 x 20 mL). Each dichloromethane layer was washed with an additional
100 mL saturated NaCl solution. The combined organic extracts, were dried (Na2SO4) and concentrated to afford (8.4 g, 84%) diazido-PEG2000 4a as white solid flakes. 1H NMR 13 (300 MHz, CDCl3): δ 3.37 (t, J = 5.1 Hz, 4H); 3.63–3.68 (m, 168H). C NMR (75 MHz,
CDCl3); δ 50.7, 67.8, 68.1, 68.9, 69.6, 70.5, 70.6, 71.6, 72.6, 3.0, 73.2.
71 2. Synthesis and Functionalization of Quantum Dots
Diamino-PEG2000 (4). Diazido-PEG2000 4a (8 g, 4.0 mmol) was dissolved in 50 mL THF and triphenylphosphine (3.14 g, 12.0 mmol) was added. The solution was stirred at room temperature for 10 h, water (3 mL) was added and stirred overnight. THF was removed in vacuo, and additional 100 mL of water was added. The triphenyl phosphine oxide precipitate was removed by filtration and the water was removed in vacuo to afford 1 (diamino- PEG2000 4 5.1 g, 63%) as white solid. H NMR (300 MHz, CD3OD): δ 2.77 (t, J = 5.4 Hz, 4H); 3.52 (t, J = 5.4 Hz, 4H); 3.63–3.68 (m, 164H). 13C NMR (75 MHz,
CD3OD); δ 39.7, 68.1, 68.6, 68.8, 69.6, 70.7.
TA-PEG2000-NH2 (5). DL-thioctic acid (0.3 g, 1.45 mmol) and N-hydroxysuccinimide (0.16 g, 1.6 mmol) were added to diisopropyl carbodiimide (0.27 g, 1.74 mmol) in CH2Cl2 and stirred at 0 °C for 2 h. This mixture was added dropwise to diamino-PEG2000 4 (4.0 g, 2.0 mmol) in 30 mL of THF. The reaction mixture was stirred at room temperature for 12 h and concentrated in vacuo. The crude residue was purified over silica gel using CH2Cl2/CH3OH (10-15%) as 1 eluent to afford 2.25 g (48%) of TA-PEG2000-NH2 5. H NMR (300 MHz, CDCl3): δ 1.42–1.51 (m, 2H); 1.62–1.70 (m, 4H); 1.89–1.93 (m, 2H); 2.17–2.2 (br.s, 2H); 2.42–2.48 (m, 1H); 2.58–2.62 (m, 1H); 3.12–3.18 (m, 2H); 3.41–3.46 (m 4H); 3.55–3.72 (m, 164H); 13 C NMR (75 MHz, CDCl3); δ 24.5, 24.9, 28.6, 33.8, 34.5, 38.4, 40.1, 61.56, 63.4, 68.5, 68.6, 69.1, 69.7, 70.5, 71.2, 71.3, 72.5, 73.3; HRMS-MALDI (m/z): [M+Na]+ Calculated for C82H164O37N2S2Na +(C2H4O)n 1856.03466 + (44.026)n; Found: 1856.0346 + (44.032)n.
DHLA-PEG2000-NH2 (6).
TA-PEG2000-NH2 5 (2.0 g, 0.5 mmol) was dissolved in an ethanol/water (1:1) mixture, cooled to 0 °C and sodium borohydride (20 mg, 0.54 mmol) was added and stirred at 0 ºC for 3 h and overnight stirring at room temperature. The solution was extracted with chloroform, dried over sodium sulfate and concentrated to afford (0.94 g, 47%) of DHLA- 1 PEG2000-NH2 6. H NMR (300 MHz, CD3OD): δ 1.28-137 (m, 2H); 1.48–1.75 (m, 4H); 1.82-1.92 (m, 1H); 2.34 (t, J = 7.5 Hz, 2H); 2.42-2.48 (m, 2H); 2.68-2.672 (m, 2H); 2.84– 13 2.925 (m, 1H); 3.55–3.72 (m, 164H); C-NMR (75 MHz, CD3OD); δ 21.6, 24.2, 26.1, 33.5, 38.2, 38.9, 42.5, 61.2, 63.1, 68.9, 69.9, 70.2, 71.2, 71.8, 72.1, 72.5, HRMS-MALDI
72 2. Synthesis and Functionalization of Quantum Dots
+ (m/z): [M+Na] Calculated for C82H166O37N2S2Na +(C2H4O)n: 1858.03466 + (44.026)n Found: 1858.0342 + (44.022)n.
2.7.5 Synthesis of Thiol Linked Monosaccharide
General procedure A. The acetylated sugar (1 equiv) and bis(2-chloroethoxy)ethanol (3 equiv) were dissolved in 10 mL CH2Cl2 and cooled to 0 ºC. BF3.Et2O (4 equiv) was added and stirred at room temperature for 48 h. The reaction was quenched with triethylamine and extracted with dichloromethane/water mixture. The organic layer was concentrated and purified over silica gel to afford sugar-PEG-Cl.
General procedure B. Sugar-PEG-Cl (1 equiv) and potassium thioacetate (3 equiv) were dissolved in 10 mL of anhydrous DMF and stirred at room temperature for 12 h. Ethyl acetate was added and the organic layer was washed five times with water. Purification by flash silica column chromatography afforded sugar-PEG-SAc.
General Procedure C. Sugar-PEG-Sac (1 equiv) and sodium methoxide (10 mol%) were dissolved in methanol (10 mL) and stirred at room temperature for 15 min. The reaction mixture was neutralized with Amberlite-H+ and solvent was evaporated to afford the free thiol.
2-(2-(2-Chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (9a). General procedure A with with 1,2,3,4,6-penta-O-acetyl-D-mannopyranoside 8 (1.5 g, 3.8 mmol), 2-[2-(2-Chloroethoxy)ethoxy]ethanol (1.92 mL, 11.4 mmol) and BF3 Et2O (2.43 mL, 15.2 mmol), followed by purification over silica gel using hexane/ethyl acetate (40:60) afforded 1.23 g (64%) of 2-(2-(2-Chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O- 24 1 acetyl-α-D-mannopyranoside 9a [α]D = +0.17 (c = 1.0, CHCl3); H NMR (300 MHz,
CDCl3): δ 1.91 (s, 3H); 1.96 (s, 3H); 2.01 (s, 3H); 2.07 (s 3H); 3.53–3.72 (m, 11H); 4.02-
73 2. Synthesis and Functionalization of Quantum Dots
4.04 (m, 2H); 4.18 (dd, J =5.4, 4.2 Hz, 1H); 4.79 (d, J = 2.1 Hz, 1H); 5.17–5.26 (m, 4H); 13 CNMR (75 MHz, CDCl3): δ 20.5, 42.7, 61.6, 66.4, 66.2, 67.3, 68.3, 68.5, 69.1, 69.4, 69.6, 70.3, 71.3, 72.5, 97.6, 97.7, 169.6, 169.8, 169.9, 170.5; HRMS-MALDI (m/z): [M]+
Calculated for C20H31O12Cl: 498.1504; Found: 498.1501.
2-(2-(2-Thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (9). General procedure B with 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-α-D galactopyranoside 9a (0.5 g, 1.0 mmol) and potassium thioacetate (0.29 g, 3.0 mmol), followed by purification over silica gel using hexane/ethylacetate (40:60) afforded 0.51 g (84%) of 2-(2-(2-Thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-α-D- 24 1 mannopyranoside 9 [α]D = +0.19 (c = 1.0, CHCl3); H NMR (300 MHz, CDCl3): δ 1.98 (s, 3H); 2.03 (s, 3H); 2.10 (s, 3H); 2.15 (s 3H); 2.33 (s, 3H); 3.11 (t, J = 6.6 Hz, 2H); 3.58– 3.68 (m, 9H); 4.08–4.15 (m, 2H); 4.31 (dd, J = 5.4, 4.2 Hz, 1H); 4.87 (d, J = 2.1 Hz, 1H); 13 5.25–5.38 (m, 4H); C NMR (75 MHz, CDCl3): δ 20.7, 21.1, 28.8, 30.5, 30.7, 60, 62.2, 62.4, 66.2, 67.4, 68.3, 68.5, 68.9, 69.5, 69.7, 87.0, 70.3, 70.6, 97.7, 169.5, 169.7, 169.8, + 170.5. HRMS-MALDI (m/z): [M] Calculated for C22H34O13S: 538.1723; Found: 538.1721.
2-(2-(2-Thioethoxy)ethoxy)ethoxy-α-D-mannopyranoside (10). General procedure C with 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-
α-D galactopyranoside 9 (0.2 g, 0.33 mmol) and sodium methoxide (20 mg, 10%) afforded r.t 0.11 g (87%) of 2-(2-(2-Thioethoxy)ethoxy)ethoxy-α-D-mannopyranoside 10 [α]D = 1 +7.82 (c = 1.0, MeOH); H NMR (300 MHz, CD3OD): δ 2.67 (t, J = 6.6 Hz, 2H); 3.58–- 13 3.71 (m, 12H); 3.81–3.85 (m, 4H), 4.92 (s, 1H); C NMR (75 MHz, CD3OD); δ 21.9, 60.2, 65.1, 65.8, 68.5, 68.6, 68.8, 69.3, 69.4, 69.8, 71.4, 71.8, 98.9; HRMS-MALDI (m/z): + [M] Calculated for C12H24O8S 328.1192; Found: 328.1192.
74 2. Synthesis and Functionalization of Quantum Dots
2-(2-(2-Chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (13a). General procedure A with 1,2,3,4,6-penta-O-acetyl-D-galactopyranoside 12 (1g, 2.5 mmol), 2-[2-(2-Chloroethoxy)ethoxy]ethanol (1.28 mL, 7.6 mmol) and BF3 .Et2O (1.6 mL, 10.1 mmol), followed by purification over silica gel using hexane/ethyl acetate (40:60) afforded 0.88 g (68%) of 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-β-D- r.t 1 galactopyranoside 13a. [α]D = +6.8 (c = 1.0, CHCl3); H NMR (300 MHz, CDCl3): δ 1.96 (s, 9H); 2.02 (s, 9H); 2.04 (s, 9H); 2.14 (s, 9H); 3.58–3.84 (m, 25H); 4.08 -4,12 (m, 6H); 4.49 (d, J = 8.0 Hz, 1H); 4.99 (dd, J = 10.9, 3.4 Hz, 1H); 5.37 (d, J = 3.5 Hz, 1H); 5.29- 13 5.43 (m, 4H); C NMR (75 MHz, CDCl3): δ 20.6, 28.3, 36.4, 37.0, 39.1, 41.7, 45.6, 59.7, 61.2, 66.9, 67.0, 67.2, 68.7, 69.1, 70.6, 101.1, 169.5, 169.6, 169.7, 169.9, 170.1, 170.2;
+ HRMS-MALDI (m/z): [M] Calculated for C20H31O12Cl: 498.1504; Found: 498.1501.
2-(2-(2-Thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-β-D galactopyranoside (13). General procedure B with 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-α-D- galactopyranoside 13a (0.5 g, 1.0 mmol) and potassium thioacetate (0.29 g, 3.0 mmol), followed by purification over silica gel using hexane/ethylacetate (40:60) afforded 0.51 g (84%) of 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl-β-D-
r.t 1 galactopyranoside 13. [α]D = +11.5 (c = 1.0, CHCl3); H NMR (300 MHz, CDCl3): δ 1.96 (s, 9H); 2.02 (s, 9H); 2.04 (s, 9H); 2.14 (s, 9H); 3.58–3.84 (m, 25H); 4.08–4.12 (m, 6H); 4.49 (d, J = 8.0 Hz, 1H); 4.99 (dd, J = 10.9, 3.4 Hz, 1H); 5.37 (d, J = 3.5 Hz, 1H); 5.29–
13 5.43 (m, 4H); C NMR (75 MHz, CDCl3): δ 20.6, 28.3, 36.4, 37.0, 39.1, 41.67, 45.65, 59.7, 61.2, 66.9, 67.0, 67.2, 68.7, 69.1, 70.6, 101.1, 169.5, 169.6, 169.7, 169.9, 170.1, + 170.2; HRMS-MALDI (m/z): [M] Calculated for C22H34O13S: 538.1723; Found: 538.1721.
2-(2-(2-Thioethoxy)ethoxy)ethoxy-β-D-galactopyranoside (14). General procedure C with 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl- α-D galactopyranoside 13 (0.2 g, 0.33 mmol) and sodium methoxide (20 mg, 10%) afforded 0.11 g (87%) of 2-(2-(2- thioethoxy)ethoxy)ethoxy-β-D-galactopyranoside 14. r.t 1 [α]D = +0.22 (c = 1.0, MeOH); H NMR (300 MHz, CD3OD): δ 2.67 (t, J = 6.6 Hz, 2H); 3.56–3.67 (m, 12H); 3.81–3.88 (m, 4H); 4.26 (d, J = 7.2 Hz, 1H); 13C NMR (75 MHz,
75 2. Synthesis and Functionalization of Quantum Dots
CD3OD); δ 21.9, 60.2, 65.1, 65.8, 68.5, 68.6, 68.8, 69.3, 69.7, 71.4, 72.3, 72.9, 102.5; + HRMS-MALDI (m/z): [M] Calculated for C12H24O8S 328.1192; Found: 328.1192.
Phenyl seleno-2-azido-3,4,6-tri-O-acetyl-α-D-galactopyranoside (16).
Tri-O-acetyl-D galactal 15 (1.5 g, 5.51 mmol) was dissolved in 30 mL CH2Cl2 and diphenyl diselenide was added (1.72 g, 5.51 mmol). The solution was cooled to -30°C and bis(acetoxy)iodobenzene was added (1.77 g, 5.51 mmol). After addition of trimethylsilyl azide (1.45 ml, 11.02 mmol) the reaction was stirred for 12 h while warming to rt. The solvent was removed and the residue was purified over silica gel (10% to 30% EtOAc in cyclohexanes) to give galactosamine 17 as α-anomer (2.51 g, 5.35 mmol, 97%). The 33 analytical data were in agreement with those reported in the literature.
2-(2-(2-chloroethoxy)ethoxy)ethoxy-3,4,6-tri-O-acetyl-α-D-galactosamine (17).
D-galactosamine selenoglycoside 16 (522 mg, 1.1 mmol) was dissolved in 3 mL CH2Cl2 and 3 mL Et2O and cooled to 0 °C. (2-Chloroethoxy)bis ethanol (561 mg, 3.3 mmol) was added, followed by addition of N-iodosuccinimide (1.24 g, 5.5 mmol) and triflic acid (18 PL, 0.2 mmol). The mixture was stirred overnight while warming to r.t. The reaction was quenched with NEt3 and washed with sat. aq. sodium thiosulfate solution. The organic layer was separated and dried over Mg2SO4. The solvent was evaporated and the residue was purified over silica gel (30% to 60% EtOAc in cyclohexanes) to give galactosamine
17 as a separable α/β-mixture (1:1) (376 mg, 0.78 mmol, 71%). Rf 0.3 (cyclohexanes/ethyl rt acetate = 1:1); [α]D = 42.1 (c = 1, CHCl3); IR (thin film on NaCl): ν = 2874, 2112, 1747,
-1 1 1434, 1371, 1228, 1127, 1046 10 cm ; H NMR (300 MHz, CDCl3): δ 5.43-5.29 (m, 4H), 5.06 (d, J = 3.5 Hz, 1H), 4.74 (dd, J = 10.9, 3.4 Hz, 1H), 4.48 (d, J = 8.0 Hz, 1H), 4.30– 3.98 (m, 6H), 3.84–3.58 (m, 25H), 2.12 (s, 3H), 2.11 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 13 2.01 (s, 3H), 2.00 (s, 3H); C NMR (75 MHz, CDCl3): δ 170.2, 170.1, 169.9, 169.7, 169.6, 169.5, 102.4, 98.1, 71.3, 70.9, 70.8, 70.7, 70.6, 70.5, 70.4, 70.1, 69.3, 68.1, 67.7, 67.5, 66.5, 66.3, 61.5, 61.2, 60.7, 57.3, 42.8, 42.7, 21.0, 20.9, 20.8, 20.7, 20.6 20.5; + MALDI-HRMS (m/z): Calculated for C18H28ClN3O10Na : 504.1355, Found: 504.1361 [M+Na]+.
76 2. Synthesis and Functionalization of Quantum Dots
2-(2-(2-Thioacetylethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra-O-acetyl-α-D galactopyranoside (18a). General procedure A with 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra- O- acetyl-α-D-galactopyranoside 17 (0.1 g, 0.21 mmol) and potassium thioacetate (60 mg, 0.63 mmol), followed by purification over silica gel using hexane/ethyl acetate (20:80) afforded 85 mg (78%) of 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra-
r.t 1 Oacetyl- α-D-galactopyranoside 18a. [α]D = +12.3 (c = 1.0, CHCl3); H NMR (300 MHz,
CDCl3): δ 2.02 (s, 9H); 2.12 (s, 3H); 2.32 (s, 3H); 3.07 (t, J = 6.6 Hz, 2H); 3.58–3.69 (m, 9H); 3.99–4.16 (m, 1H); 4,46 (d, J = 7.5 Hz, 1H); 4.74 (dd, J = 3.3, 7.5 Hz, 1H); 5.07 (d, J 13 = 3.6 Hz, 1H); 5.28–5.41 ( m, 1H); C-NMR (75 MHz, CDCl3); δ 20.5, 28.7, 30.5, 42.6, 7.3, 60.6, 61.4, 66.3, 67.6, 68.1, 69.3, 70.0, 70.2, 70.4, 70.8, 71.2, 98.1, 169.7, 169.9, + 170.2; HRMS-MALDI (m/z): [M] Calculated for C20H31O11N3S: 521.1679; Found: 521.1678.
2-(2-(2-Thioethoxy)ethoxy)ethoxy-2-azido-α-D-galactopyranoside (18). General procedure B with 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra-O- acetyl-α-D-galactopyranoside 18a (80 mg, 0.17 mmol) and sodium methoxide (10 mg, 0.17 mmol) afforded (45 mg, 80%) of 2-(2-(2-thioethoxy)ethoxy)ethoxy-2-azido-α-D- r.t 1 galactopyranoside 18. [α]D = +0.96 (c = 1.0, MeOH); H NMR (300 MHz, CD3OD): δ 2.67 (t, J = 6.6 Hz, 2H); 3.58–3.71 (m,12H); 3.81-3.85 (m, 4H); 4.34 (d, J = 7.2 Hz, 1H); 13 4.96 (d, J = 3.6 Hz, 1H); C NMR (75 MHz, CD3OD); δ 21.9, 59.7, 66.9, 67.5, 67.6, 68.,
+ 6.7, 69.7, 71.3, 72.1, 73.9, 102.3; HRMS-MALDI (m/z): [M] Calculated for C12H23O7N3S: 353.1257; Found: 353.1255.
77 2. Synthesis and Functionalization of Quantum Dots
2-(2-(2-Thioethoxy)ethoxy)ethoxy-2-amino-α-D-galactopyranoside (19). Trimethylphosphine (0.1 mL of a 10 M solution) was added to 2-(2-(2- thioethoxy)ethoxy)ethoxy-2-azido- α-D-galactopyranoside 18 (40 mg, 0.11 mmol) in anhydrous THF. The solution was stirred at room temperature for 3 h before addition of water (1 mL) and stirring overnight. THF was removed in vacuo, and the residue was dissolved in 10 mL water. The water layer was washed with chloroform (4 x5 mL). Water was lyophilized to afford 30 mg (81%) of 2-(2-(2- thioethoxy)ethoxy)ethoxy-2-amino-α- r.t 1 D-galactopyranoside 19. [α]D = +1.25 (c = 1.0, MeOH); H NMR (300 MHz, CD3OD): δ 2.66 (t, J = 6.6 Hz, 2H); 3.45–3.75 (m, 14H); 3.99–4.04 (m, 1H); 4.26 (d, J = 7.2 Hz, 1H); 13 C NMR (75 MHz, CD3OD); δ 21.9, 59.7, 66.9, 67.5, 67.6, 68.4, 69.7, 69.7, 71.3, 72.1,
+ 73.9, 102.3; HRMS-MALDI (m/z): [M] Calculated for C12H25O7NS: 327.1352; Found: 327.1351.
2.7.6 Batch Functionalization of CdSe/ZnS QDs
QD635-DHLA-PEG2000-NH2 (22).
To 0.1 mL of QDs in chloroform (O.D > 50 at 400 nm) 50 µL of DHLA-PEG2000-NH2 6 in 50 µL of ethanol were added. The mixture was stirred at 60 °C for 12 h. In between, another 50 µL of ethanol were added. PEGylated-QDs were separated by 12 precipitation using ethanol/chloroform/hexane (1:0.5:5). Centrifugation at 3000 rpm for 3 min afforded a clear pellet-like suspension. This pellet was separated and dissolved in distilled water (1 mL), centrifuged at 3000 rpm for 3 min to afford a clear solution. This solution was lyophilized and pure PEGylated-QD was obtained from NAP-1 column chromatography using 1.0 µM borate-buffer pH 7.5 as the eluent. Concentration of the QDs was estimated using a previously published procedure.32
78 2. Synthesis and Functionalization of Quantum Dots
QD635-DHLA-PEG2000-maleimide (23). QD635-DHLA-PEG2000-NH2 22 (0.05 µM in 1 mL of 1.0 µM borate-buffer pH 8.5) was added to 3-maleimido propanoic acid N-hydroxysuccinimide ester (13 mg, 49.0 µM) and stirred at room temperature for 2 h. The aqueous layer was washed with chloroform (3 x 1 mL) to remove unreacted maleimido derivative. The aqueous layer was taken as such for the next step without further characterization.
QD635-DHLA-PEG2000-mannose (24a).
QD635-DHLA-PEG2000-maleimide 23 (0.02 µM in 1 mL of 1.0 µM borate-buffer pH 7.3) was added to 2-(2-(2 thioethoxy)ethoxy)ethoxy-α-D-mannopyranoside 10 (10 mg, 30.5 µM) and stirred at room temperature for 3 h. The crude product was purified by NAP-1 column using distilled water as the eluent. The final concentration of the sample was estimated by using a previously published procedure.32
QD635-DHLA-PEG2000-galactosamine (24b).
QD635-DHLA-PEG2000-maleimide (0.02 µM in 1 mL of 1.0 µM borate-buffer pH 7.3) was added to 2-(2-(2 thioethoxy)ethoxy)ethoxy-2-amino-β-Dgalactopyranoside 19 ( 10 mg, 30.5 µM) and stirred at room temperature for 3 h. The crude product was purified by NAP-1 column using distilled water as the eluent. The final concentration of the sample was estimated by using a previously published procedure.32
QD635-DHLA-PEG2000-galactose (24c).
QD635-DHLA-PEG2000-NH2 (0.02 µM in 1 mL of 1.0 µM borate-buffer pH 7.3) was added to 2-(2-(2 thioethoxy)ethoxy)ethoxy-2-amino-β-Dgalactopyranoside 14 (10 mg, 30.5 µM) and stirred at room temperature for 3 h. The crude product was purified by NAP-1 column using distilled water as the eluent. The final concentration of the sample was estimated by using a previously published procedure.32
79 2. Synthesis and Functionalization of Quantum Dots
QD635-DHLA-PEG2000-OH (24d). To 0.1 mL of QDs in chloroform (O.D > 50 at 400 nm) 50 µL of DHLA-PEG2000-OH 3 in 50 µL of ethanol were added. The mixture was stirred at 60 °C for 3 h. The crude product was purified by NAP-1 column using distilled water as the eluent. The final concentration of the sample was estimated by using a previously published procedure.32
2.7.7 Continuous Flow Synthesis of CdSe and CdTe QDs
Cadmium precursor was prepared by heating 100 mg (0.75 mmol) cadmium oxide with 600 mg (3.1 mmol) lauric acid at 150 °C until a clear solution was obtained. This solution was cooled to room temperature and 1.5 mL each of oleic acid and oleylamine were added to the flask. A solution containing 80 mg (1.0 mmol) selenium in 2 mL (6.76 mmol) of trioctylphosphine or 120 mg (0.97 mmol) tellurium in 2 mL (6.76 mmol) of trioctylphosphine was prepared. A solution of cadmium precursor (0.097 mmol) in 0.5 mL of squalene and 0.097 mmol of Se or Te precursor in 0.5 mL of squalene were pushed into the microreactor using two syringe pumps. CdSe or CdTe QDs were prepared at reaction times of 3, 10, 20 and 30 min at flow rates of 333 µL/min, 100 µL/min, 50 µL/min, 33.33 µL/min. The QDs were purified by precipitation with anhydrous
CHCl3/MeOH/hexane/isopropanol to afford 13 mg (72%, after 3 min), 15 mg (71%, after 10 min), 18 mg (67%, after 20 min) and 17 mg (66%, after 30 min) product.
2.7.8 Continuous Flow Synthesis of CdTe or CdSe/ZnS QDs
A solution of 20 mg of the CdTe or CdSe QDs in 1 mL of toluene and 2 mL of trioctylphosphine was used as nanocrystal precursor. Trioctylphosphine (2 mL), 50 µL
80 2. Synthesis and Functionalization of Quantum Dots
(0.28 mmol) of hexamethyldisilathiane and 400 µL of 10% diethylzinc in toluene were mixed. The two solutions were injected into the microreactor at 90-110 °C at a flow rate of 33.33 µL/min (residence time of 30 min) The resulting QDs were purified by precipitation with chloroform/methanol mixture to afford 22 mg (68%)32 product. Surface exchange of oleic acid–capped CdTe/ZnS QDs with pyridine was performed by dissolving 20 mg of the QDs in pyridine and passing the solution through the microreactor at 60 °C at a flow rate of 33.33 µL/min (30 min residence time). Precipitation with hexane followed by centrifugation afforded 12 mg (74%)32 of pyridine capped QDs.
2.7.9 Continuous Flow Functionalization of QDs
Preparation of mannose capped QDs (25) or galactose capped QDs (26). A solution of 10 mg of pyridine-capped CdTe-ZnS QDs in 1 mL ethylene dichloride and a freshly prepared solution of 35 mg 2-(2-(2-thioethoxy) ethoxy) ethoxy-α-D- mannopyranoside 10 (0.11 mmol) or 2-(2-(2-thioethoxy)ethoxy)ethoxy-β-D- galactopyranoside 14 (35 mg, 0.11 mmol) in 1 mL ethylenedichloride/ethanol (1:1) was prepared. The solutions (0.5 mL each) were simultaneously injected into the microreactor that was preheated at 50 °C at a flow rate of 33 µL/min (30 min residence time). The solvent was evaporated and carbohydrate coupled QDs were precipitated with hexane/chloroform/methanol (9:1:1). The final concentration of the sample was estimated using a published procedure.32 Preparation of dihydrolipoic acid capped QDs (27). A solution of 10 mg pyridine capped CdTe-ZnS QDs in 1 mL of ethylene dichloride and 20 mg (0.10 mmol) of dihydrolipoic acid in 1 mL of ethylene dichloride/ethanol (1:1) was prepared. The solutions (0.5 mL each) were simultaneously injected into the microreactor at 50 °C at a flow rate of 33.33 µL/min (residence time 30 min). The solvent was evaporated and dihydrolipoic acid coupled QDs were precipitated by addition of tetramethylammonium hydroxide. The final sample concentration was estimated using a published procedure.32
81 2. Synthesis and Functionalization of Quantum Dots
2.7.10 Three Steps Continuous Flow Synthesis of Mannose Capped CdSe/ZnS QDs
Preparation of mannose capped CdSe/ZnS QDs. Cadmium precursor was prepared by heating 100 mg (0.75 mmol) cadmium oxide with 600 mg (3.1 mmol) lauric acid at 150 °C until a clear solution was obtained. This solution was cooled to room temperature and 1.5 mL each of oleic acid and oleylamine were added to the flask. A solution containing 80 mg (1.0 mmol) selenium in 2 mL (6.76 mmol) of trioctylphosphine in 2 mL (6.76 mmol) of trioctylphosphine was prepared. A solution of cadmium precursor (0.097 mmol) in 0.5 mL of squalene and 0.097 mmol of Se precursor in 0.5 mL of squalene were pushed into the microreactor using two syringe pumps (15 min of residence time, flow rates of 66.66 µL/min). CdSe QDs were then flushed directly into another microreactor at 90-110°C. A solution of trioctylphosphine (2 mL), 50 µL (0.28 mmol) of hexamethyldisilathiane and 400 µL of 10% diethylzinc in squelene was prepared and injected separately. Finally CdSe-ZnS QDs in 1 mL ethylene dichloride and a freshly prepared solution of 35 mg 2-(2-(2-thioethoxy) ethoxy) ethoxy-α-D-mannopyranoside 10 (0.11 mmol) were flushed into a third microreactor at 60 °C o afforded 12 mg 52% final compound.
82 2. Synthesis and Functionalization of Quantum Dots
2.8 References
1 (a) Xie, R.; Kolb, U.; Li, J.; Basch, T.; Mews, A. J. Am. Chem. Soc. 2005, 127, 7480– 7488. (b) Susumu, K.; Mei, B. C.; Mattoussi, H. Nat. Prot. 2009, 4, 424–436. (c) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217– 224; (c) George, N.; Pick, H.; Vogel, H.; Johnsson, N.; Johnsson, K. J. Am. Chem. Soc. 2004, 126, 8896–8897. (d) Somers, R. C.; Bawendi, M. G.; Nocera, D. G. Chem. Soc. Rev. 2007, 36, 579–591. (e) Carion, O.; Mahler, B.; Pons, T.; Dubertret, B. Nat. Prot. 2007, 2,
2383–2390. (f) Zhong, H.; Scholes, G. D. J. Am. Chem. Soc. 2009, 131, 9170–9171. 2 (a) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc., 2000, 122, 12142–12150. (b) Peng, Z. A.; Peng, X. J. Am. Chem. Soc., 2001, 123, 183–184. (c) Liu, W.; Choi, H. S.; Zimmer, J. P.; Tanaka, E.; Frangioni, J. V.; Bawendi, M. J. Am. Chem. Soc., 2007, 129, 14530–1453. (d) Somers, R. C.; Bawendi, M. G.; Nocera, D. G. Chem. Soc. Rev., 2007, 36, 579–591. (e) Liu, W.; Howarth, M.; Greytak, A.; Zheng, B. Y.; Nocera, D. G.; Ting, A. Y.; Bawendi, M. G. J. Am. Chem. Soc., 2008, 130, 1274–1284. (f) Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino P.; Seeberger, P. H. J. Am. Chem. Soc., 2009, 131, 2110–2112. 3 Cohen, H.; Sarkar, S. K.; Hodes G. J. Phys. Chem. B 2006, 110, 25508–25513. 4 Ioannou, D.; Griffin, D. K. Nano Reviews 2010, 1, 5117–5131. 5 Murray, C. B.; Kanagan, C. R.; Bawendi, M. G. Annu. Rev. Mater. Sci. 2000. 30, 545–
610. 6 Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc., 1993, 115, 8706–8715. 7 Yang, Y. A.; Wu, H.; Williams, K. W.; Cao, Y. C. Angew. Chem. Int. Ed. 2005, 44,
6712–6715. 8 (a) Ekimov, A. I.; Onushchenko, A. A. JETP Lett. 1981, 34, 345–349. (b) Murray, C. B.;
Norris, D. J.; Bawendi, M. G., J. Am. Chem. Soc. 1993, 115, 8706–8715. 9 (a) Qu, L.; Peng, Z. A.; Peng, X. Nano Lett., 2001, 1, 333–337. (b) Talapin, D. V.; Rogach, A. L.; Kornowski, A.; Haase, A.; Weller, H. Nano Lett. 2001, 1, 207–211. (c)
Rempel, J. Y.; Bawendi, M. G.; Jensen, K. F. J. Am. Chem. Soc. 2009, 131, 4479–4489.
83 2. Synthesis and Functionalization of Quantum Dots
10 Reiss, P.; Protiere M.; Li, L. Small, 2009, 5, 154–168. 11 Altınoglu, E. I.; Adair, J. H. WIREs Nanomed Nanobiotechnol, 2010, 2, 461–477. 12 Piryatinski, A.; Ivanov, S. A.; Tretiak, S.; Klimov, V. I. Nano Lett. 2007, 7, 108–115. 13 Kim, S.; Fisher, B.; Eisler, H. J.; Bawendi, M. J. Am. Chem. Soc. 2003, 125, 11466–
11467 14 (a) Mews A., Eychmüller A., Giersig M., Schooss D., Weller H., J. Phys. Chem. 1994, 98, 934–941; (b) Battaglia D., Li J. J., Wang Y. J., Peng X. G., Angew. Chem. Int. Ed. 2003, 42, 5035–5039; (c) Zhong X. H., Xie R. G., Zhang Y., Bansche T.; Knoll W. Chem.
Mater. 2005, 17, 4038–4042. 15 Biju, V., Itoh, T. Ishikawa, M. Chem Soc. Rev, 2010, 39, 3031–3056. 16 (a) Gao, X. H.; Cui, Y. Y.; Levenson, R. M.; Chung, L. W. K., Nie, S. M. Nat. Biotechnol. 2004, 22, 969–976; (b) Hezinger, A.F.E.; Teßmar J.; Goepferich, A. Eur. J.
Pharm. Biopharm. 2008, 68, 138–152. 17 (a) Sun, X.–L.; Cui, W.; Haller, C.; Chaikof, E. L. ChemBioChem, 2004, 5, 1593–1596.
(b) Babu, P.; Sinha, S.; Surolia, A. Bioconj. Chem. 2007, 18, 146–151. 18 Rogach, A. L.; Nagesha, D.; Ostrander, J. W.; Giersig, M.; Kotov, N. A. Chem. Mater.
2000, 12, 2676–2685. 19 (a) Li, J. J.; Wang, Y. A.; Guo, W.; Keay, J. C.; Mishima, T. D.; Johnson, M. B.; Peng, X. J. Am. Chem. Soc. 2003, 125, 12567–12575. (b) Sanderson, K. Nature 2009, 459, 760–
761. 20 (a) Ehrfeld, W.; Hessel, V.; Löwe, H. Microreactors: New Technology for Modern Chemistry, Wiley-VCH, Weinheim, 2000. (b) Hessel, V.; Hardt, S.; Löwe, H. Chemical Micro Process Engineering, Wiley-VCH, Weinheim, 2004. (c) Geyer, K;. Codée, J. D. C.; Seeberger, P. H. Chem. Eur. J. 2006, 12, 8434–8442. (d) Mason, B. P.; Price, K. E.; Steinbacher, J. L.; Bogdan, A. R.; McQuade, D. T. Chem. Rev. 2007, 107, 2300–2318. (e) Wiles, C.; Watts, P. Eur. J. Org. Chem. 2008, 10, 1655–1671. (f) Brivio, M.; Verboom, W.; Reinhoudt, D. N. Lab Chip, 2006, 6, 329–344. (g) Jähnisch, K.; Hessel, V.; Löwe, H.; Baerns, M. Angew. Chem. Int. Ed. 2004, 43, 406–446. 21 (a) Marre, S., Park, J., Rempel, J., Guan, J., Bawendi, M. G. Jensen, K. F., Adv. Mater. 2008, 20, 4830–4834. (b) Yen B. K. H., Günther A., Schmidt M. A., Jensen K. F., Bawendi M. G., Angew. Chem. Int. Ed., 2005, 44, 5447–5451. (c) Shestopalov I., Tice J.
84 2. Synthesis and Functionalization of Quantum Dots
D., Ismagilov R. F., Lab Chip 2004, 4, 316–321. (d) Dideko Y. T., Suslick K. S. J. Am. Chem. Soc. 2005, 127, 12196–12197. 22 (a) Edel J. B., Fortt R., deMello J. C., deMello A. J., Chem. Commun. 2002, 1136–1137. (b) Sounart T. L., Safier P. A., Voigt J. A., Hoyt J., Tallant D. R., Matzke C. M., Michalske T. A., Lab Chip 2007, 7, 908–915. (c) Yen, B. K. H.; Stott, N. E. S.; Jensen, K.
F.; Bawendi, M. G. Adv. Mater. 2003, 15, 1858–1862. 23 Kikkeri, R.; Laurino, P.; Odedra, A.; Seeberger, P. H. Angew. Chem. Int. Ed. 2010, 49,
2054–2057. 24 Laurino, P.; Kikkeri, R.; Seeberger, P. H. Nat. Protoc., 2011, in press. 25 Syrris FRX, http://www.syrris.com. 26 (a) Rong, H.; Xiaogang, Y.; Hongye, T.; Feng, G.; Daxiang ,C.; Hongchen, G. Front. Chem. China 2006, 4, 378–383. (b) Zhong, H.; Scholes, G. D. J. Am. Chem. Soc. 2009, 31, 9170–9171. (c) Peng, X.; Manna, L.; Yang, W.; Wickham, J.; Scher, E.; Kadavanich, A.; Alivisatos, A. P. Nature, 2000, 404, 59–61. (d) Yu, W. W.; Wang, Y. A.; Peng, X. Chem.
Mater. 2003, 15, 4300–4308. 27 Yu, W.W. Chang, E. Drezeck, R. Colvin V.L. Biochem. Biophys. Res. Commun. 2006,
384, 781–786. 28 (a) Michalet, X.; Pinaud, F.F.; Bentolila, L.A.; Tsay, J.M.; Li, J.J.; Sundaresan, G.; Wu, A.M.; Gambhir, S.S.; Weiss, S. Science 2005, 307, 538–544. (b) Hild, W.A.; Breunig, M.;
Goepferich, A. Eur. J. Pharm. Biopharm. 2007, 68, 112–128. 29 (a) Lunney, J.; Ashwell, G. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 341–343. (b) Baenziger, J. U.; Fiete, D. Cell 1980, 22, 611–620. (c) Braun, J. R.; Willnow, T. E.; Ishibashi, S.; Ashwell, G.; Herz, J. J. Biol. Chem. 1996, 271, 21160–21166. 30 Higuchi, Y.; Oka, M.; Kawakami, S.; Hashida, M. J. Controlled Release 2008, 125, 131–136. 31 Xie, R.; Battaglia, D.; Peng, X. J. Am. Chem. Soc. 2007, 129, 15432–15433. 32 Leutherdale, C. A.; Woo, W.-K.; Mikulec, F. V.; Bawendi, M. G. J. Phys. Chem. 2002, 106, 7619–7622. 33 Mironov, Y. V.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. J. Am. Chem.
Soc. 2004, 45, 9107–9110.
85 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3. Continuous Flow Microreactors and Phosphine Oxide Initiators: an Unique Combination
This work was performed in collaboration with Dr. K. Tauer (discussions on the experiments and evaluation of data), the group of Prof. H. Grützmacher (synthesis of photoinitiator) and Dr. H. Hernandez (simulation studies).
Part of this chapter has been submitted for publication titled “Snowballing Radical Generation Leads to Ultrahigh Molecular Weight Polymers” Laurino, P.; Hernandez, H. F.; Bräuer, J.; Krüger, K.; Grützmacher, H.; Tauer, K.; Seeberger, P. H. 2011.
86 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.1 Introduction
Polymers are large molecules made of repeating units of smaller molecules of defined nature called monomers. The monomers are connected by covalent chemical bonds to form a polymer chain. Polymers can occur naturally or can be produced for specific purposes by chemical synthesis. The synthetic process of monomer compounds reacting together to generate a polymer chain is called polymerization. Polymerization is a well established technique for the fabrication of different polymers and material composites for various applications ranging from paper producing to well defined nanoparticles for drug delivery or specific biomedical applications.1 Of the many forms of polymerization, the most common type is the free radical mediated polymerization where the polymer is formed by the successive addition of free radical monomers. Three significant reactions for the generation of polymer by the free radical mediated polymerization are: initiation, propagation and termination (Figure 3.1).
Figure 3.1 Three key reactions in free radical polymerization (R = initiator; M = monomer)
In the first step of the polymerization process the free radical is generated by a molecule called initiator that upon thermal decomposition, photolysis, redox reaction or sonication can generate radical species that promote radical reactions. During the initiation, an active center is generated from the initiator and the radical is transferred to the monomer molecule. After the radical is transferred to a monomer molecule the polymer chain starts
87 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination to grow and the propagation occurs until no monomer is available or a termination reaction occurs. Termination can occur either via combination whereby two chains couple to form one single long chain or by disproportionation where a hydrogen is abstracted from a growing polymer chain giving an unsaturated terminal chain and a saturated terminal chain. The polymerization rate can be affected by a decrease of the initiator efficiency due to various side reactions. Common side reactions involving the initiator are: 1) Primary decomposition, when two radicals recombine before initiating a chain. 2) Other reaction pathways involving the recombination of the radicals with impurities. The most frequently used methods for radical polymerization are: 1) Bulk polymerization: reaction mixture comprised of initiator and monomer. 2) Solution polymerization where the reaction mixture is comprised of an initiator, monomer and solvent. 3) Emulsion polymerization: where the reaction mixture is comprised of an aqueous phase, water-insoluble monomer, initiator and emulsifying agent (surfactant). Emulsion polymerization in particular is a well investigated and characterized technique which in most cases is the method of choice for the synthesis of composite polymer particles with special morphologies to explore their potential as new materials for novel technologies.2 The most common type of emulsion polymerization is an oil-in-water emulsion where droplets of the hydrophobic monomer are emulsified with surfactants in an aqueous phase. It has to be noted that in emulsion polymerization, the polymerization events do not occur in the monomer droplets but occur in the polymer particles also called latex particles that are generated spontaneously in the early stages of the polymerization reaction. A key factor in radical-mediated emulsion polymerization is the average number of radicals per polymer particle that is defined by . This value depends on particle size, concentration of the monomer, rate of initiator decomposition and radical flux into and out of particles.3 Classical emulsion polymerization of polystyrene produces particles of less than 100 nm diameter, that contain, on average, one half radical per particle ( = 0.5). Such reactions are conformed to so-called zero–one kinetics, where each particle contains either one or no growing radical. In emulsion polymerizations, the latex particles obtained vary in diameter from less than 100 nm to above 1000 nm and contain many polymer chains. The latex particles are stabilized in many cases by the presence of the charged surfactant that generates a repulsion effect on the particle surface avoiding coagulation events. Polymerization parameters like temperature, reaction times, monomer, solvent,
88 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination surfactant and mixing have been investigated,4 and have an effect on the physical properties of the polymer latex including molecular weight, particle size and other properties.5 This chapter mainly focuses on the investigation of an emulsion polymerization mechanism mediated by phosphine oxide photoinitiators in a continuous flow microreactor.
3.1.1 Photo-initiated Polymerization in a Continuous Flow Microreactor
Continuous flow microreactors are widely used as alternative reactor systems6 for the formation of polymer particles or emulsions to achieve better control procedures. 7 Emulsion polymerization in continuous flow microreactors can generate particles with predetermined sizes and well defined shape in the nanometer range.8 The continuous flow devices can be efficiently employed to screen conditions, to use precious reagents, and to change reaction times based only on varying the flow rate.9 To emphasize the contribution of continuous flow on photo-polymerization a set of examples where the technical aspects of the continuous flow microreactor influenced the particle formation and facilitated the screening and optimization of polymerization conditions were reviewed. Doyle et al. reported on the formation of plug- or disk-shaped particles by the photo-polymerization of polyethylene glycol (PEG) gel using in situ UV- irradiation.10 The PEG-polymer solution is injected perpendicular to the water phase solution (Figure 3.2).
89 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.2 Schematic representation of the reaction zones where the particles are generated and irradiated. Irradiation can occur in (a) a first stage to generate plugs or in (b) a second stage where the droplet is able to relax to generate disks (100 W HBO mercury lamp, and UV filter to provide the desired wavelength).11
Key determinants for the different particle shapes are the flow rate, the capillary number which is related to viscosity and density of the fluids involved (eq. 6), and the channel dimension.
(6) Ca = µV/γ
Ca, capillary number; µ, viscosity of the liquid; V characteristic velocity; γ surface or interfacial tension between the two phases.
Injecting the PEG polymer solution into an aqueous continuous phase followed by the immediate irradiation of the formed droplets resulted in plug-shape particles. When the formed droplets were allowed to relax in a bigger channel before irradiation, disk shaped particles were formed. Constraining of droplets into the microreactor channel was exploited to define size and shape of the final particles. Lee et al. have described the preparation of microparticles in flow.11 In situ UV irradiation of 4-hydrobutyl acrylate (4-HBA) droplets in a sheath flow of poly(vinyl acrylate) (PVA) resulted in the generation of small particles of 10 µm diameter without the use of a surfactant, which is a key component for heterophase polymerization reaction
90 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
(Figure 3.3). Surfactant free particles are ideal candidates for various biomedical applications.
Figure 3.3 Reaction zones where the particles are generated and irradiated. The flow is constituted initially by the core flow (4-HBA) and then the sheath flow (PVA) is injected in diagonal. The irradiation occurs where the particles have defined spherical shape (UV light EXPO OmniCure S1000, Maritimes, Cadana, 324 nm, 300 mW/cm2).12
Lee et al. have described the formation of hydrogels via UV–initiated polymerization of a monomer (80 wt % 4-hydroxybuthyl acrylate, 11 wt % acrylic acid, 1 wt % ethyleneglycol dimethyl acrylate, and 3 wt % 2,2-dimethoxy-2-phenyl- acetonphenone) / enzyme solution in an immiscible non–polymerizable sheath fluid of mineral oil (Figure 3.4).12
91 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.4 Reaction zones for particles generation and irradiation (365 nm, 1.2 mW/cm2, Novacure, Photonic Solutions Inc.).16
The formed droplets were flashed by light (1 s irradiation time) in an unprotected part of the channel producing particles below 100 µm in diameter and higher flow rates of the flow sheath resulted in the formation of smaller particles.
3.1.2 Phosphine Oxide Photoinitiators
Acylphosphine oxides are well known and widely used photoinitiators for polymerization processes. 13 The photolysis of monoacylphosphine oxides (MAPOs, Scheme 3.1) is similar to that of arylalkylketone upon UV irradiation at 380 nm.
Scheme 3.1 Examples of monoacylphosphine oxides (MAPOs) as photoinitiators. MAPOs undergo α-cleavage upon irradiation with high efficiency to produce aroylphosphinoyl radical pair (quantum yield = 0.6, Scheme 3.2). 14, 15
92 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Scheme 3.2 Photolysis of (2, 4, 6-trimethylbenzoyl)diphenylphosphine oxide.
The arylphosphinoyl radical 7 exhibits 10–100 fold higher reactivity toward unsaturated compounds than the commonly used benzoyl radicals. This exceptional reactivity makes MAPOs excellent candidates for radical polymerization process. The absorption of MAPOs in the UV-visible region facilitates their use in the presence of pigments and results in many more applications.16 Nevertheless, subsequent investigations have lead to the development of a second generation of acylphosphine oxide initiators called bisacylphosphine oxide derivatives, BAPOs which are superior photoinitiators because of the additional chromophore present in them. (Scheme 3.3).17
Scheme 3.3 Examples of bisacylphosphine oxide (BAPOs) photoinitiators
The presence of a second chromophore shifts the absorption to a higher wavelength (ca. 420 nm) than MAPOs which absorb at 380 nm. This wavelength shift facilitates a photo- cleavage of the first chromophore at 420 nm and subsequent cleavage of the second chromophore at 380 nm.18 The enhanced absorption of BAPOs at higher wavelengths enables better performance using visible light. This property is important for biological applications as it allows for tissue curing without the extensive damage associated with the use of UV light.
93 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Despite their useful properties, the incompatibility of BAPOs with aqueous formulations presents a major drawback. In order to overcome this drawback, Grützmacher et al prepared two new water soluble bisacylphosphine oxide derivatives: (2-(bis(2, 4, 6- trimethylbenzoyl)phosphoryl)acetic acid (BAPO-AA), and bis(2, 4, 6- trimethylbenzoyl)phosphineoxide tris-oxyethylene (BAPO-PEG) (Scheme 3.4).
Scheme 3.4 Structures of (2-(bis(2, 4, 6-trimethylbenzoyl)phosphoryl)acetic acid 14 (BAPO-AA) and bis(2, 4, 6-trimethylbenzoyl)phosphineoxide tris-oxyethylene 15 (BAPO-PEG).
The derivatives described above are the first examples of water-soluble initiators having the photophysical properties of BAPOs.28 The availability of these initiators further expands the study of radical polymerization to water–soluble photoinitiators. To overcome difficulties associated with scale-up and reproducibility of emulsion polymerizations initiated by water soluble BAPOs, we decided to investigate the polymerization process in continuous flow microreactors.
94 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.2 Continuous Flow Microreactor Design
A detailed description of the continuous flow microreactor and its influence on the polymerization reaction are necessary to fully understand the emulsion polymerization investigation. The flow reactor set-up is comprised of two gas-tight syringes, a 2.5 mL syringe containing the monomer and a 10 mL syringe containing the aqueous phase where the photoinitiator and sodium dodecyl sulfate (SDS) were dissolved (Figure 3.5).
Figure 3.5 Continuous flow microreactor for photo-initiated emulsion polymerization. Syringe pump injection system (1, Harvard PHD 2000), gas tight syringe (2, Hamilton), countercurrent micromixer (3, Standard Slit Interdigital Micro Mixer), FEP tubing (4, fluorinated ethylene polymer, Tub FEP Nat 1/16 in x .030 in), quartz immersion well (5) thermostat (10), Pyrex filter (6), Hanovia medium pressure Hg lamp (7, 450 W), a power supply (9), and a collection vessel (11).
A syringe pump was used for flushing the reagents into the reactor through the gas-tight syringes. The emulsion was generated in a countercurrent micromixer19 (Standard Slit Interdigital Micro Mixer) resulting in a mean droplet diameter of about 50–100 µm. The mixing system was made of 15 lamellae, each with a height of 200 µm, width of 45 µm, and outflow width of 30 µm. The system combines the use of fluorinated ethylene propylene (FEP) tubing coiled around the cooling quartz jacket system containing a 450 W Hanovia medium pressure Hg Lamp with an arc length (27.9 cm) and a pyrex filter (1 mm
95 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination of wall thickness). The part of the reactor involving the lamp was placed in a box to protect the users from dangerous irradiation. The total reaction was divided into reaction zones with different volumes (Figure 3.6).
Figure 3.6 Detailed view of the reaction volumes in the tubing.
The first part of the reaction was placed out of the protection box and included two zones: an emulsification zone (E), followed by a relaxing and spontaneous emulsification zone (SE) with a volume of 0.075 mL. Next to the E and SE zones was the irradiated zone, with a volume of 2.432 mL including three parts: RZ1, MRZ and RZ2. RZ1 and RZ2 were reaction zones, irradiated but not coiled around the cooling system and MRZ was the reaction zone coiled around the cooling system. The last part of the reaction tubing outside of the protective box was the outflow zone of a volume of 0.193 mL. The initial stage of the investigations required some optimization of the continuous flow microreactor. The use of a T-connection was explored but results achieved with the micromixer were found to be better in terms of particle size and overall reproducibility. Optimization of the light source and tubing system was also performed. Medium pressure Hg lamps with different arc lengths (4.0 cm, 27.9 cm and 2.0 cm) and power (125 W, 450 W and 100W) were tested, as well as different kinds of Teflon tubing, PolyTetraFluoroEthylene (PTFE) and Fluorinated Ethylene Propylene (FEP).20 The system that afforded the best results for photon transmittance was FEP tubing and for light emission a 450 W medium pressure Hg lamp.
96 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.2.1 Compartmentalization of the Emulsion Polymerization Process in a Continuous Flow Microreactor
The possibility to transfer the emulsion polymerization reaction from a batch reactor to a continuous flow microreactor resulted in the change of certain parameters and conditions according to the characteristics of the microreactor system. The emulsion polymerization reaction in a continuous flow microreactor with a single micro-mixer and a finite capillary length is different from a traditional batch reactor, and may drastically influence the polymerization reaction. The differences between batch reactor and continuous flow microreactor were thought to be dependent on the reaction volume that is a defined volume at a precise reaction time. In a batch reactor, theoretically at any reaction time the reaction volume is identical while in the continuous flow microreactor, the same reaction time is characterized by different every reaction volumes that are unique in their properties and parameters since the system consists of different parts (injection system, mixing system, tubing, etc) (Figure 3.7).
97 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.7 Illustration of polymerization reaction volume in the continuous flow microreactor.
In a continuous flow microreactor the composition of the reaction mixture were changing along the length of the tubing. In addition, every part of the tubing with the corresponding reaction volume represented a different stage of the polymerization reaction. Assuming that every reaction volume moved independently along the tubing, each of these reaction volumes were unique with their conversion rate and kinetic situation and behaved as a small batch reactor at different reaction times along the length of the tubing. 21 The reduced diameter of the tubing influenced the reaction volume as well as the flow rate and the pressure generated by the injection system and the mixing system partially controlled the diffusion of the reactants between the two phases and the volume of the droplets at different reaction times. All these technical characteristics of the continuous flow microreactor influenced the emulsion polymerization process and the features of the resulting polymer product (particle size, particle size distribution, molecular weight of the polymer chains, solid content, etc.). The influence of continuous flow microreactors on the emulsion polymerization process had to be understood to evaluate the data and further it has also to be considered that this device afforded experimental conditions not achievable in batch reactor such as extremely short reaction times. In conclusion, a full understanding
98 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination and use of the continuous flow technique was essential to investigate the emulsion polymerization mechanism initiated by phosphine oxide photoinitiators.
99 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.3 Emulsification
Emulsion polymerization in general involves two immiscible phases. In our investigations an oil-in-water emulsion was used where the continuous phase is an aqueous phase and the discontinuous phase is constituted by the monomer and stabilized by a surfactant. The emulsification process plays a key role for emulsion polymerization since the hydrophobic monomers have to be transported in a multi-step process from a monomer droplet to a polymer particle as main reaction loci. The first step in this process is the passage of the phase boundary between the monomer droplets and water. In the second step the diffusion through the aqueous phase to a polymer particle occurs. The last step involves passing of the monomer the phase boundary between water and the particles. It is clear from these steps that larger the monomer droplet–water interface, faster the passage into the water phase. The passage of monomer as smallest possible droplets is essential for achieving high polymerization rates. In the continuous flow microreactor emulsification is influenced by mechanical emulsification, spontaneous emulsification and flow rate.
3.3.1 Mechanical Emulsification
Mechanical emulsification is characterized by the constraining of the two phases in the reduced diameter channels of a mixer at well defined pressures and flow rates (Figure 3.8).
100 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.8 Droplet formation in a mixing system.22
In our continuous flow microreactor the mechanical emulsification occured in the interdigital micro mixer SSIMM within seconds. To investigate the effects of micro mixer on the emulsion polymerization process, a light microscopy image was taken one minute after the passage of a styrene solution and aqueous solution containing sodium dodecyl sulfate (SDS) and BAPO-AA into the mixer at a flow rate of 4 mL/min (Figure 3.9a).
Figure 3.9 (a) Styrene emulsion generated in the SSIMM interdigital micromixer. (b) TEM image of polystyrene particles stained with PTA-Ru and generated by the standard procedure (300 mg SDS, 10 mL water, 10 mg BAPO-AA, 2.5 mL styrene).
The results were remarkable since the average droplet size observed was smaller than the predicted (median droplet size of about 50–70 µm)19 and there was no correlation between the droplet size (average of 10 µm) and the final particle size (40 nm). The rate of polymerization of styrene using BAPO-AA as photoinitiator and varying SDS concentration was investigated. The rate of polymerization dropped when the
101 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination polymerization experiments were carried out with SDS concentration below 100 mg per 10 mL of water and at concentrations of 50 mg per 10 mL no polymerization was observed. A very efficient emulsification using 300 mg of SDS in 10 mL water for 2.5 mL styrene resulted high polymerization rates and complete conversion (18% of solid content in 36.5 s). A large interfacial area between the droplets and the aqueous phase guaranteed fast monomer transport to the growing polymer particles. In addition, high surfactant concentration stabilized the emulsion along the entire tubular reactor and guaranteed fast monomer transport along the entire reactor even far away from the micromixer. The observations were in accordance with studies on the effect of the surfactant concentration in the continuous flow microreactor that have been previously reported.9e
3.3.2 Spontaneous Emulsification
The spontaneous emulsification that could occur in the SE zone of the tubing in the continuous flow microreactor has to be considered (Figure 3.6). Spontaneous emulsification was facilitated by surfactant high concentration.23 A simple experiment illustrates the phenomena where the styrene or methyl methacrylate monomer was quiescently placed on the top of an aqueous solution containing surfactant (300 mg/10 mL of SDS) and UV spectra of the aqueous solution were recorded at different time points (Figure 3.10).
102 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.10 UV-vis spectra illustrating the uptake of A – styrene and B- methyl methacrylate via spontaneous emulsification into an aqueous surfactant solution (curve a - 36 sec, b - 72 sec, c - 10 min, and d - 3 h).
The concentration of the monomer increases very fast reflecting the growing monomer concentration in the aqueous phase without the application of mechanical shear. This process is called spontaneous emulsification since not only single monomer molecules, but also monomer aggregates and nano-droplets are transferred. 23 Increasing the concentration of SDS to 500 mg/10 mL led to higher conversion and smaller particles leading to the assumption that, the empty but still existing micelles contributed to stabilize the tiny formed particles.
3.3.3 Influence of the Flow Rate on the Emulsion Polymerization
The flow rate influenced the emulsification and consequentially the rate of polymerization. Decreased flow rates result in increased residence times in the continuous flow microreactor that generally induced the formation of more reactive radicals since the irradiation exposure of the photoinitiator is longer and therefore a higher initiation rate, polymerization rate, and number of active growing particles might be expected. A solution containing SDS, water and BAPO-AA and a styrene solution were flushed through the continuous flow microreactor at a flow rate of 1 mL/min and then at a flow rate of 4 mL/min. Decreasing the flow rate resulted in the formation of two distinct optically recognizable phases, signifying instability of the emulsion and consequentially resulting in a lower polymerization rate. In conclusion, the photo-induced polymerization in the continuous flow microreactor required a high flow rate and sequentially short residence time. Phosphine oxide photoinitiators decompose and initiate radical polymerizations fast enough for achieving an effective polymerization in the continuous flow microreactor.24
103 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.4 Extraordinarily High Molecular Weight Polymer in Small Particles
In continuous flow microreactors the polymerization of styrene occurred in an unusually short residence time (36.5 sec) resulting in small particles (< 50 nm) with a narrow particle size distribution (Figure 3.11) and polymer chains with extremely high molecular weight. Initially the emulsion polymerization of styrene initiated by BAPO-AA 14 in a continuous flow microreactor (Scheme 3.5) in presence of different concentration of sodium dodecyl sulfate (SDS, 200–500 mg/10 mL) was investigated.
Figure 3.11 SEM image of polystyrene particle generated in a microfluidic device.
Scheme 3.5 Structures of (2-(bis(2, 4, 6-trimethylbenzoyl)phosphoryl)acetic acid 14 (BAPO-AA).
104 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
At different surfactant concentrations an emulsion polymerization thermally-initiated with potassium peroxodisulfate (KPS) or photo-initiated with poly(ethylene glycol)-azo-initiator (16, PEGA200) in batch was performed. The size of the latex particles detected by dynamic light scattering (DLS), generated by the batch and continuous flow microreactor methods were compared and found to be similar (Figure 3.12).
Scheme 3.6 PEGA200 structure 16.
Figure 3.12 Correlation between average particle size (D) of polystyrene latex and the concentration of emulsifier (SDS) after thermal batch polymerization initiated by KPS (white square) and PEGA200 (grey square), and continuous flow polymerization photo-initiated by BAPO-AA (red square).
After emulsion polymerization of styrene photo-initiated with BAPO-AA in aqueous phase containing SDS in the continuous flow microreactor the latex was directly collected in a solution of tetrahydrofuran (THF) which is known to be able to break the colloidal dispersion and quench the polymerization reaction. The molecular weight distribution of the polystyrene collected was up to 30·106 g/mol with peak maximum at 3·106 g/mol (Figure 3.13).
105 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.13 Molecular weight distribution of polystyrene chains produced via photo-initiated emulsion polymerization in the continuous flow microreactor. GPC analysis of the polymer molecular weight used set of column B having a pore size of 106 Å allowing resolution of molecular weights of up to 107 g/mol (see experimental part, latex characterization).
The high solid content (18%) and ultrahigh molecular weight polymers (up to 30·106 g/mol) obtained within this short reaction time was unprecedented and triggered an in- depth investigation of the mechanism of BAPO-AA initiated process. The average molecular weight of polystyrene from the photo-initiated polymerizations was up to 30 times higher than that of the polystyrene product from the batch reaction photo-initiated by PEGA200 (Figure 3.14).
106 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.14 Correlation between cumulative number average molecular weight (Mn) of polystyrene latex and concentration of emulsifier (SDS) after thermal batch polymerization initiated by KPS (white circle) and PEGA200 (grey circle) and continuous flow photo- polymerization initiated by BAPO-AA (red circle). GPC analysis of the polymer molecular weight used set of column A having a pore size of 103 and 105 Å (see experimental part, latex characterization).
Emulsifier concentrations between 200–500 mg SDS/10 mL water, well above the critical micelle concentration (CMC = 23 mg SDS/10 mL water), were need for all photo-initiated continuous flow reactions. According to the classical kinetics, the degree of polymerization
(DPkin) of emulsion polymerization (eq 7) with a rate constant Kp of 107.3 L/mol·s at 9d 30 °C at a styrene concentration inside the particles (CM) of 3.5 M (estimated based on equilibrium swelling of the particles),25 was calculated.
(7)
(8)
Kp is the propagation rate constant; Ktr,m the rate constant of chain transfer to monomer; CM the monomer concentration at the reaction site; treac the reaction time.
The calculation of the DPkin indicated that the polystyrene chains should not grow larger than 1.5·106 g/mol, an order of magnitude smaller than the observed value of 3·107 g/mol
107 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination for emulsion polymerization of styrene photo-initiated with BAPO-AA in a continuous flow microreactor (Figure 3.15).
Figure 3.15 Comparison of the molecular weight distribution of polystyrene in the final latexes obtained in the continuous flow microreactor (curve a, solid red) and in a batch reactor (curve b, solid blue); the vertical lines indicate the kinetically accessible molecular weights (solid lines a1, b1) and the maximum molecular weights attainable in the polymerization time (dashed lines a2, b2) by multiplying DPkin and DPtime with the molecular weight of styrene. Polymerization conditions curve a: 2.5 mL of styrene, 10 mL of water, 300 mg of SDS, 10 mg of BAPO-AA, 36.5 seconds irradiation (polymerization) time, polymerization conditions curve b: 20 g of styrene, 80 g of water, 625 mg of SDS per 10 mL of water, 640 mg of KPS, 80 °C, batch reactor, 25 minutes polymerization time.
Upon comparison of the molecular weight distribution of both processes, it was also observed that the DP values for this process were inverted compared to a classical thermal emulsion polymerization. The kinetically accessible DPkin value for the thermal polymerization was lower than DPtime while for the BAPO-AA initiated emulsion polymerization in a continuous flow microreactor the situation was reversed (Figure 3.15, line b1 and line b2 vs a1 and a2). To understand the anomalous molecular weight of the polymer chain, an explanation on the polymer growth mechanism is required. It was clear that the classical
108 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination mechanism could not explain the results obtained during the emulsion polymerization in a continuous flow microreactor.
109 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.5 Ultrafast Polymerization of Styrene
The standard analysis of the molecular weight of the latex obtained by the emulsion polymerization BAPO-initiated in continuous flow microreactor, disclosed a high molecular weight of the polymer chain, which was surprisingly not due to an extended chain growth after irradiation time (post effect). The data collected proved that the emulsion polymerization of styrene using BAPO-AA as initiator occurred in 36.5 s to give full conversion. The rate of emulsion polymerization of styrene using BAPO-AA as phospine oxide photoinitiator has been investigated in detail in the microfluidic device and compared to a well known emulsion polymerization initiated by an azo-initiator (azodi[poly(ethylene glycol)] isobutyrate, PEGA200, 16). To investigate the rate of BAPO initiated polymerization and the eventual extended chain growth after irradiation time (post-effect), the polymer latex that was formed was checked by solid content and size exclusion chromatography (SEC) at different time intervals. Solid content of the samples were assessed few hours after sample collection, after three, five and 20 days. These results showed the absence of any post-polymerization reaction (Figure 3.16 a). On the contrary, the solid content for a standard emulsion polymerization photo-initiated by PEGA200 increased dramatically in the first three days (Figure 3.16 a). The molecular weight distribution increased as well from the initial value detected few hours after collecting the sample (black curve) during three (red curve), five (green curve) and 20 days (blue curve) for a standard polymerization initiated by PEGA200 (Figure 3.16 b). The emulsion polymerization was carried out and the molecular weight distribution checked few hours after polymerization (black curve), after three, five and 20 days (respectively red, green and blue curve, Figure 3.16 c). Overlapping curves indicates that the chain size of the polymer does not increase with time.
11 0 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.16 Time-dependent changes of properties of polystyrene latex particles following photo- initiated emulsion polymerization in a continuous flow reactor using BAPO-AA or PEGA200 as initiators; a) Solids content (spheres, %) and average molecular weight (squares, Mw), BAPO-AA (black) and PEGA200 (red); b) MWD shift for latex prepared using PEGA200; c) MWD shift for the latex prepared using BAPO-AA; d) MWD comparison of both latexes after 20 days (BAPO- AA – black, PEG200 – red). Light source: medium pressure Hg lamp 100 W (HBO 100).
Finally, molecular weight distribution of BAPO-AA and PEGA200 can be compared demonstrating that BAPO-AA polymerization can reach the same molecular weight distribution in 36.5 s as PEGA200 in 20 days (black curve vs red curve, Figure 3.16 d). In conclusion, the polymerization rate was extremely fast (36.5 s) and did not show any post- effect. Another direct confirmation that the polymerization terminated in 36.5 s was the scanning electron microscopy analysis (Figure 3.17a).
111 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.17 (a) Cryo-SEM image of polystyrene particles frozen immediately after removal from the reactor. (b) Standard SEM image of polystyrene particles 24 hours after polymerization.
The comparison between the cryo-SEM image of a polystyrene latex sample frozen immediately after collection and the high resolution image of the dried latex sample prepared 24 hours after the polymerization showed that the particle size in both cases was in a similar range. This result was a further confirmation that the entire polymerization process occurred in the continuous flow device. We investigated the decomposition mechanism of the initiators to explain the rationale behind this behavior. The absence of post-polymerization using BAPO-AA may be due to its decomposition pathway as acyl phosphine oxide photo-initiators decompose after excitation and intersystem crossing to the triplet state.24a, 26 The azo-group of PEGA200 16 decomposed, and generated free radicals, upon thermal treatment or irradiation, and surfactant micelles further enhanced the decomposition of azo-initiators.27 In conclusion, post-effect was observed only using PEGA200 as initiator but not using BAPO-AA.
Scheme 3.7 Decomposition of PEGA200 16.
112 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.6 Investigation of Emulsion Polymerization using BAPO-AA
The behavior of 2-(bis(2, 4, 6-trimethylbenzoyl)phosphoryl)acetic acid (BAPO- AA) under different polymerization conditions has been investigated. 28 A quite comprehensive study on BAPO-AA was necessary to understand its role as photoinitiator in emulsion polymerization reactions in continuous flow microreactors. The rate of decomposition of BAPO-AA, the influence of its concentration on the polymerization, its behavior at different irradiation times and finally the effect of different monomers on BAPO-AA have been studied in detail.
3.5.1 Decomposition Rate of BAPO-AA
It was important to understand the decomposition of BAPO-AA to establish the rate of its decomposition and the amount of radicals generated during the polymerization process (36.5 sec). The high reactivity of BAPO-AA was evaluated by taking UV-spectra after irradiating it in aqueous solution. The UV spectra were monitored in the range where BAPO´s chromophore groups absorb (350–450 nm).17 After 36 s irradiation in a quartz cuvette the absorption curve dropped considerably compared to the reference solution (curve b vs curve a, Figure 3.18). After 72 s the absorption curve overlapped curve b due to the absence of further decomposition, and longer irradiation (144 s) led to a slight decrease that could still be detected (curve c, Figure 3.18).
113 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.18 UV spectra of BAPO-AA (10 mg/10 mL of water) after different irradiation time; a – irradiation time zero, b – irradiation time 36 s and 72 s, c – irradiation time 144 s.
The absence of monomer in the solution allowed us to assume that most likely all radicals involved in the decomposition reaction of BAPO-AA recombined to different byproducts other than BAPO-AA or reacted with water. 29 Based on these data it can be concluded that the decomposition of BAPO is very fast during the polymerization.
114 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.5.2 Emulsion Polymerization at Different BAPO-AA Concentrations
The concentration of the initiator directly influences the number of radicals generated upon irradiation and has to be considered a fundamental aspect to elucidate the polymerization events. It is well known for free radical polymerization that the average molecular weight of the polymer chain depends on the square root of the initiator concentration.30 The unusual effects observed by varying the concentration of BAPO-AA, on the molecular weight of the final polymer are reported here. The molecular weight distribution of polystyrene did not change even though the BAPO-AA concentration was increased three times compared to the initial concentration (Figure 3.19).
Figure 3.19 Molecular weight distribution of polystyrene prepared in a continuous flow microreactor varying concentration of BAPO-AA, 5 mg (curve a), 10 mg (curve b), 15 mg (curve c), 50 mg (curve d) in 10 mL water solution containing 300 mg SDS.
Only when the photoinitiator concentration was ten times higher did the molecular weight shift slightly towards the lower value. The collected data showed that a special kinetic situation using BAPO photoinitiators in continuous flow emulsion polymerization can cause an unusual correlation between the photoinitiator concentration and the average molecular weight.
115 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.5.3 Emulsion Polymerization using BAPO-AA at Different Residence Times
Further investigations of the irradiation times were performed in an effort to understand the effect of this parameter on the average molecular weight distribution of the final polystyrene chain. Experiments were carried out blocking different portions of the irradiation zone with aluminum foil, but keeping all other parameters and condition the same. The original size exclusion chromatography (SEC) profile clearly showed an increase in the polymerization conversion by prolonging the irradiation time, expressed via the increase in the refractive index signal (Figure 3.20A).
Figure 3.20 Original SEC elugrams (A) and normalized molecular weight distributions (B) of polystyrene formed in a continuous flow microreactor with varying length irradiation zones. Molecular weight distributions of the final polymer chain after 14.6 s (curve a), 21.9 s (curve b), 29.2 s (curve c) and 36.5 s (curve d) of irradiation time. While this data exposes the significant influence of the irradiation time on the final polymerization conversions, there was not obviously correlation between the irradiation time and the molecular weight distributions of the polymer chains.
116 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.5.4 Screening of Different Monomers for Emulsion Polymerization
The effect of the monomers on the emulsion polymerization process photo-initiated by BAPO-AA in a continuous flow microreactor was also investigated. To understand the emulsion polymerization process, the behavior of three different monomers (styrene, STY 17; butyl methacrylate, BMA 18; methyl methacrylate, MMA 19; Scheme 3.8) in the continuous flow microreactor had to be analyzed.
Scheme 3.8 Styrene (STY) 17; butyl methacrylate (BMA) 18; methyl methacrylate (MMA) 19.
Initially a series of experiments were perfomed to optimize the emulsion polymerization conditions for STY, BMA and MMA. The optimum residence time for STY BMA and MMA was 36.5 s, 72 s and 144 s and this was determined by the screening different residence time in the continuous flow microreactor (Table 3.1).
117 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Table 3.1 Flow rates and residence times for different monomers in continuous flow microreactor during emulsion polymerization.
Solubility in Flow Rate Reaction -1 -1 Monomer Photoinitiator Kp (L mol s ) Water (mL/min) Time (s) (mol/L) STY BAPO-AA 4.0 36.5 88 0.003 BMA BAPO-AA 2.0 72 368 0.002 MMA BAPO-AA 1.0 144 318 0.160
Styrene (STY); Butyl methacrylate (BMA); Methyl-methacrylate (MMA); (2-(bis(2, 4, 6- 31 trimethylbenzoyl)phosphoryl)acetic acid (BAPO-AA), propagation rate constant at 25 °C (Kp), Solubility in Water detected at 25 °C.
In order to explain the different residence time exhibited by STY, BMA and MMA, the solubility in water and the propagation rate constant of the monomers could be considered. Apparently, lower the water-solubility of the monomer, shorter is residence time for the polymerization even though the water-solubility between styrene and BMA is only slightly different. Interestingly, the optimum average residence time shows no correlation with the value of the propagation rate constants of the monomers. All these data suggest that the difference in the irradiation time for the emulsion polymerization of different monomers could be due to the termination modes of the same monomers. The maximum peak in the high molecular weight part of the molecular weight distribution is inversely proportional to the residence time. It is at the highest molecular weight for STY (Figure 3.21, curve c), shifted towards lower values for BMA (Figure 3.21, curve b), and even further shifted for MMA (Figure 3.21, curve a).
118 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.21 Molecular weight distribution of polymer latexes prepared in a continuous flow microreactor with various monomers: styrene (curve c), butyl methacrylate (curve b) and methyl methacrylate (curve a).
In order to explain the molecular weight distribution results, the termination mode and polarity of the monomer have to be taken into account. The different termination behavior of styrene and methyl acrylate affected the reaction conditions and the polymer features. Styrene terminates exclusively by combination which occurs when the polymer growth is stopped by recombination of two growing radical to form a single unreactive polymer chain. Methyl methacrylate on the other hand terminates mainly by disproportionation event which halts the chain growth by the extraction of a proton leading to two unreactive polymers as a consequence of hydrogen transfers (Scheme 3.9).
Scheme 3.9 Termination mode by (a) combination and (b) disproportionation.
To explain the highest value of molecular weight for STY the lowest polarity of styrene should be considered which leads to the highest concentration in the latex particles and then the termination mode which goes exclusively via combination.32 For methacrylate the termination occurs via disproportionation or combination (generation of two chains or one
119 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination chain). Due to the different termination mode starting from the same reaction conditions, shorter chains are more likely for methacrylate than for STY.
120 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.7 Studies on Different Phosphine Oxide Initiators
In order to check the influence of the structure and reactivity of the photoinitiators on the emulsion polymerization, a study with different phosphine oxide photoinitiators varying their concentrations was carried out. In this regard three phosphine oxides initiators have been considered: 2, 4, 6-trimethylbenzoyl-diphenylphosphineoxide (MAPO 1), 2-(bis(2, 4, 6-trimethylbenzoyl)phosphoryl)acetic acid (BAPO-AA 14) and bis(2, 4, 6-trimethylbenzoyl)phosphineoxide tris-oxyethylene (BAPO-PEG 15) (Scheme 3.10). 13a, 13b The first photoinitiator is a mono(acyl) phopshine oxide and is soluble only in the monomer phase while the latter two are bis(acyl) phosphine oxide initiators that are water–soluble initiators.
Scheme 3.10 Photoinitiator structures: 2,4,6-trimethylbenzoyl-diphenylphosphineoxide (Lucirin TPO, MAPO) 1; 2-(bis(2, 4, 6-trimethylbenzoyl)phosphoryl)acetic acid (BAPO-AA) 14; bis(2, 4, 6-trimethylbenzoyl)phosphineoxide tris-oxyethylene (BAPO-PEG) 15.
The molecular weight distribution of the polystyrene chain obtained by emulsion polymerization using BAPO-AA and BAPO-PEG were comparable (Figure 3.22).
121 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.22 Molecular weight distribution of polymer latexes prepared in the continuous flow microreactor with different photoinitiators: BAPO-AA (curve a), BAPO-PEG (curve b), MAPO (curve c).
As expected, the molecular weight distribution of the polymer generated using MAPO shifted towards a lower value due to the hydrophobicity of the photoinitiator that results in a higher concentration of radicals inside the particles. Despite these results, a significant fraction of chains produced by MAPO had an extremely high molecular weight (> 1.65·106 g/mol).
122 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.8 Molecular Weight Distribution and Average Sizes of Polymer Particles
Emulsion polymerization in a continuous flow microreactor initiated by phosphine oxide influence in an unusual way the relationship between molecular weight distribution of the polymer chain and the average particle size. The average particle size of polystyrene particles obtained by emulsion polymerizations in continuous flow microreactors using different concentrations of SDS (between 200–500 mg), 10 mL of water and 2.5 mL of the STY were compared to the average particle size diameter of polystyrene particles produced via thermal polymerization in a batch reactor reported previously (Figure 3.23 A).33
Figure 3.23 Experimental data comparing properties of latex particles prepared via thermal polymerization in the stirred tank reactor and photopolymerization in the microfluidic device. Log – log plot of the average particle size versus the surfactant concentration (A), black and grey circles are data for polymerization in the batch stirred tank and microfluidic reactor, respectively. The number average molecular weight versus the average particle size (B), black symbols are data for polymerization in the batch stirred tank and grey symbols for polymerization from microreactor; respectively, circles, squares, and triangles representing styrene, BMA, and MMA polymerizations, respectively; the lines are linear regressions. The relationship between the average particle size and the surfactant concentration did not depend on either the polymerization method or the initiation conditions (black spheres
123 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination represent data from thermal reactions in batch reactors and grey spheres emulsion polymerization photoinitiated in continuous flow microreactor; Figure 3.23 A). In contrast, considering all three monomers (STY, BMA, MMA) the relationship between the average molecular weight and the average particle size depended strongly on the polymerization process (Figure 3.23 B). The emulsion polymerization was run in a continuous flow microreactor using a solution of SDS, BAPO-AA in water and a solution of STY, BMA or MMA with a residence time of 36.5 s, 72 s and 144 s respectively and the obtained particles were analyzed. The data collected were compared with the data extrapolated by analysis of polystyrene particles produced using thermal polymerization in batch reactor and reported previously.33 The average molecular weight decreased with increasing average particle size for the thermal polymerization in stirred batch reactors in accordance with common emulsion polymerization mechanisms.34 For polymer particles with a size below 70 nm in diameter equals 0.5 and entry of a second radical instantly causes termination. As the entry rate increases with growing particle size, the average molecular weight decreases. With increasing average particle size the compartmentalization effect, which was responsible for the high molecular weight of emulsion polymers, was more and more weakened and hence, the average molecular weight decreased (black circles, line b, Figure 3.23 B). The photo-initiated emulsion polymerization in continuous flow microreactor showed the opposite effect (line b, Figure 3.23 B). For all three monomers the average molecular weight increased with increasing particle size (Figure 3.25 B, black triangles correspond to MMA, black squares to BMA, black circles to styrene). The relation between molecular weight distribution and average particle size was completely reversed in emulsion polymerizations initiated by phosphine oxide in a continuous flow microreactor compared to normal emulsion polymerizations in batch reactor.33 In conclusion, the chain growth follows different rules and the molecular weight is not determined by radical entry but by the monomer concentration inside the particles which increases with increasing particle size.35
124 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.9 Snowballing Radical Generation Mechanism
Established theories of the mechanism of emulsion polymerization do not explain how phosphine oxide-photoinitiated polymerization can generate ultrahigh molecular weight polymers in a continuous flow reactor in short residence time. Even though it is usually possible to achieve high molecular weight product due to a post-polymerization effect, (it is well known that the isolated polymeric radical can survive inside the latex particles for many hours after the radical generation is terminated),36 no post-effects were observed in BAPO-AA-initiated continuous flow emulsion polymerizations. The explanation of this result required a reconsideration of the mechanism of the polymer growth. Studies have shown that more than one polymer chain can grow per radical during the duration of a classical emulsion polymerization. It was clear that the classical mechanism does not explain the results obtained during the emulsion polymerization in a continuous flow reactor. A different decomposition of the bis(acyl)phosphine oxide photoinitiator, resulting in a new mechanism could provide a possible explanation for these observations.
3.9.1 Average Number of Radicals per Polymer Particle ( )
To justify the generation of an ultra fast polymerization yielding an ultra high molecular weight polymer chains, different parameters have to be considered compared to the traditional batch polymerization. Two fundamental values determined the average number of polymer chains per particle:
125 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
1) The number of chains that can grow to an average chain length during the propagation time (np,kin). This depends on the time a growing polymer chain resides in the reaction zone
(tres), compared to the time (tadd) it takes for a radical to add DP monomer units at a monomer concentration CM with propagation rate constant kp (eq. 9).
(9)
2) The geometric constraint (np,geo) the number of chains of density ρp with a given molecular weight (DP times the molecular weight of the monomer Mmon) that fill the volume of an average particle of diameter D (eq. 10).
(10)
The ratio of np,geo/np,kin constitutes an alternative definition of the average number of radicals per particle and allows for the calculation of from experimental data (eq. 11), where c1 is a geometric constant.
(11)
To understand if the results observed could be explained by a bigger number of radicals per particle, equation 11 was used to calculate the theoretical values for styrene and butyl methacrylate (BMA) polymerization initiated with BAPO-AA 1, BAPO-PEG 2, and the commercially available mono-acyl phosphine oxide 2, 4, 6-trimethylbenzoyl- diphenylphosphine oxide (Lucirin TPO or MAPO). Higher values were calculated for all photo-initiated polymerization reactions as compared to the zero-one scenario and confirmed the presence of many growing radicals per particles (Figure 3.24).
126 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.24 Average number of radicals per particle ( ) calculated using equation (5) for photoinitiated emulsion polymerizations in a continuous flow microreactor with different monomers, photoinitiators and residence times correlated with the average particle size.
The large number of radicals per particle can be best explained by the incorporation of the phosphine oxide units into the polymer backbone, which upon photolysis, results in “snowballing” radical generation (SRG) during the emulsion polymerization process.
3.9.2 Decomposition of BAPO-AA
For the purpose of the newly proposed mechanism, the following criteria were considered: 1) Radical formation is faster than the aggregation of particles during the nucleation process.37 2) Phosphorus-radicals are more reactive than the carbon radicals.24, 26, 38 3) Generation of diradical species during radical polymerization is also possible.39 Initially, decomposition of phosphine oxide initiator generates two primary radicals (20 and 22) before these radicals propagate the chain length to form polymer via monomer addition (21 and 23) (Scheme 3.11).
127 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Scheme 3.11 Photochemical decomposition of BAPO-AA and subsequent polymerization of styrene leading to polymer chains enabling snowballing radical generation
The polymer chain containing the phosphine oxide unit can then undergo decomposition in four possible ways generating eight different kinds of radicals, a phosphorus centered radical (22), a diradical chain presenting a phosphorus centered radical and a carbon
128 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination centered radical (24), three carbon centered radicals (20, 21 and 23) and three diradical chain presenting two carbon centered radicals (25, 26 and 27), each of which can undergo further propagation of the chain or further decomposition. For example, upon irradiation radical 26 can generate intermediates 24 and 25 upon irradiation (Figure 3.11). Finally radicals can combine: in case of monoradicals, this leads to termination (28) while for the diradicals the polymer chains can still propagate (26 and 29).40
Scheme 3.12 Possible pathway of combination of radical polymer chain resulting in (a) termination and (b) diradical polymer chains.
In the later diradical combination, if the phosphine oxide is already present in a diradical molecule, two different diradicals are obtained as a result (29).41, 39b The diradical chains are formed during decomposition when the phosphine oxide group is linked to at least one radical-containing chain (23 and 26).13b The rate of radical combination is dependent on the mobility of both the radicals involved in the reaction. 13C-NMR analysis of purified polystyrene polymers revealed a sharp signal at δ 13C ~31 ppm (Figure 3.25), an indication of tail-to-tail styrene additions (27). Polystyrene samples prepared by conventional means show no signal in this region, but only the expected aliphatic CH (40–41 ppm) and CH2 (42–48 ppm) resonances indicating the presence of atactic polystyrene in both samples.42, 34
129 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.25 13C-NMR spectra of the polystyrene generated by emulsion polymerization in continuous flow microreactor using BAPO-AA after purification (blue curve) and of thermally polymerized polystyrene (black curve).
To confirm the presence of 23 and 26 in the long polymer chain and the fact that this mechanism is universal for all the phosphine oxide initiators, 31P-NMR spectra of the reprecipitated polymer generated by di(acyl)phosphane oxide (Irgacure® 819, scheme 3.13, 8) were measured.
Scheme 3.13 Chemical structure of bis(2, 4, 6-trimethylbenzoyl)-phenylphosphine oxide 8 (Irgacure® 819).
The signals at 25-27 ppm show the presence of a phosphorus unit at the end of the chain (23, Figure 3.26 a). In this case the polymer was prepared at a 100 times higher concentration of BAPOs, only one α-cleavage occurs and the other chromophoric center is intact while the use of a lower concentration of BAPOs results in the α-cleavage also of the second chromophore. The signals at 39–41 ppm demonstrate the presence of the phosphine oxide residue in the middle of the polymer chain (26) (Figure 3.26 b).
130 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.26 (a) 31P-NMR spectra of polystyrene polymer containing compound 23; (b) 31P-NMR spectra of polystyrene polymer 26.
In addition, emulsion polymerization experiments using 2,4,6-tribenzyl- phosphineoxide (TBP, scheme 3.14, 30) as photoinitiator were carried out leading to cross- linked products but with lower efficiency under longer irradiation times.
131 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Scheme 3.14 Chemical structure of 2, 4, 6-tribenzyl-phosphinoxide (TBP) 30.
The observed behavior can be explained by the lower decomposition rate of the phosphorus-benzyl bond as compared to the phosphorus-acyl bond. The isolated cross- linked product could only have been because of the cleavage of all the three P-C bonds, and the growth polymer of the polymer from these sites. In addition, the molecular weight of the linear polymer increased with increasing residence time reaching up to 107 g/mol for residence times above 10 minutes (Figure 3.27).
Figure 3.27 Molecular weight distribution of the linear chain (soluble fraction) polymer in polystyrene particles prepared using 2, 4, 6-tribenzyl-phosphineoxide under varying residence times in the irradiation zone of the microfluidic device.
In conclusion, to explain the observed results, the new mechanism proposed demonstrates the concept of snowballing radical generation (SRG) by a repeated photo-induced cleavage of the phosphine oxide initiators producing unprecedented large number of radicals.
132 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.10 Snowballing Radical Generation Simulation
In order to understand the influence of snowballing radical generation on the emulsion polymerization kinetics and to provide a mechanistic rationale for the experimental results, a simulation based on a stochastic model was formulated.43 The simulation assumes an idealized situation where a radical enters a 40 nm spherical droplet of pure monomer and propagates for two seconds without additional radicals entering or exiting. The modeling frame considered the following kinetic events (represented as equations):
1. Primary BAPO decomposition:
2. Propagation of the polymer chain:
3. Decomposition of phosphine oxide-containing compounds:
4. Radical combination of monoradicals:
5. Radical combination of diradicals:
B= benzoyl group, PO= phosphine oxide group, h is Planck’s constant and v is the frequency of the irradiating light. The rate constant of BAPO photodecomposition is kd. Radicals are indicated using
133 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination the symbol (●). R● is an arbitrary molecule or macromolecule containing a radical, M is a monomer ● molecule and kp is the rate constant of radical propagation. R represents all possible radicals and ● ● ● ● ● ● ● diradicals present in the system, including: R’-B , R’-PO , R’-M , B-R’-B , B-R’-PO , etc.; kd2 is the kinetic rate constant of photodecomposition of all other phosphine oxide-containing molecules; kc is the rate constant of radical combination.
The simulation model was based on the the events occurring during the decomposition of phosphine oxide initiators. In the simulation model, the polymer chains were represented by a vector of varying length. Each vector contains information about length, structure and composition of the polymer chain. Vector manipulation rules have been defined on the basis of the events described above. In Figure 3.28, the results of the simulation are represented and compared to those for a typical radical polymerization.
Figure 3.28 Simulation of the snowballing radical polymerization mechanism. Final chain length distribution when a single radical with DP = 1 (green bar) starts to polymerize in a 40 nm sized monomer droplet. Distribution based on a phosphine oxide radical snowballing mechanism (red bars), growth of the starting radical based on normal radical polymerization kinetics (grey bar).
The average chain length based on a phosphine oxide radical snowballing mechanism is represented in red bars, the maximum chain length achieved by a polymer radical based on normal radical polymerization kinetics was almost one order of magnitude lower (grey bar). The green bar represents the starting point for the growth of a single radical entering a 40 nm size droplet. The simulation predicts the formation of 35 growing radicals from a
134 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination single phosphine oxide radical, and justifies the unusually high polymerization rate. On the other hand, the generation and further recombination of diradicals also explains the formation of polymers with extremely high molecular weights. The average chain length ( ) increases at similar rates for SRG (red line) and for traditional polymerization (black line) because it was assumed that the rate of propagation for both scenarios was the same (Figure 3.29).
Figure 3.29 Time dependent growth of the average chain length ( ) for snowballing (red line) and ordinary radical (black line) polymerization. DPmax simulated for snowballing kinetics (spheres).
The small differences in the average chain length observed were due to the stochastic model used during the simulation. Maximum chain lengths (DPmax) were simulated for polymers produced by snowballing kinetics (spheres), showing once more that the broad distribution of molecular weight of the resulting polymer chains is a results of the SRG mechanism.
3.10.1 Simulation of the Emulsion Polymerization at Different Initiator Concentration
135 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
The simulation of the emulsion polymerization was expanded to underpin also other experimental results collected during the investigation using BAPO-AA photoinitiator (see Section 3.14.8 for simulation assumption for the data). The influence of SRG on the chain length distribution in the final polymer using two different concentration of photoinitiator to start the polymerization was considered during the simulation (Figure 3.30 A).
Figure 3.30 Simulated chain length distributions for radical polymerization inside styrene droplets assuming SRG (graph A) and ordinary kinetics (graph B) for two different initial initiator concentrations (I0); grey bars I0 = 10 mM and black bars I0 = 1mM. Reaction rate constants: kd = -1 -1 -1 -1 -1 -1 100 s , kp = 240 lM s , kc = 300 lM s , kd2 = 100 s .
For both simulations there was an expected shift to higher values when the concentration decreased. This effect was more evident for the ordinary kinetics than for the snowballing radical generation (SRG). The minor influence of the change in the concentration of BAPO-AA was consistent with the experimental data that was collected (Figure 3.19, Section 3.5.2). The simulation showed that SRG leads to broader chain distribution length of the polymer than the traditional radical kinetics in accordance with the results collected during polymerization investigation. Same simulation study allowed results on the development of the chain length. These data were experimentally difficult to access due to the short residence time in the irradiation zone (Figure 3.31, symbols).
136 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.31 Simulated conversion dependence of average (graph A) and maximum (graph B) chain length in the chain length distribution for radical polymerization inside styrene droplets assuming
SRG (symbols) and ordinary kinetics (lines) for two different initial initiator concentrations (I0); grey circles and solid lines I0 = 10 mM; grey triangles and dashed lines I0 = 1mM.
The average degree of polymerization (DPave, graph A) and the maximum chain length achievable (DPmax, graph B) were plotted against the conversion at two concentrations of photoinitiator. All the data were compared to the classical kinetics for emulsion polymerization (Figure 3.31, lines). At low conversion the DPave and DPmax were almost overlapping independently from the photoinitiator concentrations, while at higher conversion the photoinitiator concentration played a clear role. From the simulation data it is clear that DPmax was strongly dependent on the photoinitiator concentration only in the classical polymerization (Figure 3.31 B). Moreover, DPmax increased smoothly with the conversion in the classical polymerization but as a scatter in the phosphine oxide mediated polymerization confirming the stochastic nature of the chain scission in SRG. These simulations interestingly revealed again the fundamental difference between SRG and common radical polymerization kinetics. The classical kinetic demonstrated higher DPave values but lower DPmax. These data supported and explained the experimental data where SRG lead to a much broader chain length distribution.
137 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.11 Seed Polymerization of Polymer Particles
According to the SRG mechanism, the use of phosphine oxide photoinitiators allows for the incorporation of phosphine oxide units in the polymer backbone, which enable, after extensive dialysis of the polymer latex and addition of a monomer, the restarting of the polymerization by irradiation without additional photoinitiator. Upon irradiation, the incorporated phosphine oxide units go through further cleavage allowing for a seed polymerization. Seed polymerization experiments are reported as support of the validity of the SRG mechanism. The purified polystyrene latex, generated in the continuous flow microreactor, was swollen with butyl methacrylate (BMA) and re-irradiated without the addition of additional photoinitiator. The black curve in figure 3.32 illustrates the molecular weight distribution of cleaned polystyrene seed latex prepared with BAPO-AA in a continuous flow reactor and the red line represents the molecular weight distribution of the latex after seed polymerization.
Figure 3.32 Molecular weight distributions of the starting polystyrene polymer generated using BAPO (black curve) and MAPO (black dashed curve) and after seed polymerization using BMA (red curve for the seed latex generated by BAPO and red dashed curve for the seed latex generated by MAPO).
138 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
The shift in the molecular weight distribution clearly shows the re-starting of the polymerization process due to the presence of phosphine oxide groups. To confirm the behavior of phosphine oxide initiators the same set of experiments were repeated using MAPO as photoinitiator (dashed curve, Figure 3.32). The black dashed curve shows the molecular weight distribution of purified polystyrene latex prepared with MAPO and the red dashed curve, which is shifted to lower values, the molecular weight distribution of the latex after seed polymerization using BMA. FT-IR spectroscopic analyses of the “seed” polymer, and of the polymers obtained after restarting the polymerization with BMA, showed that an IR absorption peak that corresponds to the carbonyl stretching frequency appears only after seeded polymerization (Figure 3.33).
Figure 3.33 FT-IR spectra of the seed polymer (black spectrum) and the copolymers after seeded polymerizations (red spectra).
This data clearly confirmed that an incorporation of butyl methacrylate units occurred during the seed polymerization. No further polymerization was detected when polystyrene latex generated by a non-phosphine oxide type photoinitiator was swollen and irradiated. Direct evidence through IR or 31P-NMR spectroscopy studies of the presence of the phosphine oxide units incorporated in the polymer chain prepared in continuous flow microreactor using BAPO-AA as photoinitiator, were difficult to obtain since the concentration of phosphorus unit in the extremely long polymer chain is too low to be detected.
139 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.12 Applications of SRG Mechanism
Emulsion polymerizations studies in continuous flow microreactor using phosphine oxide photoinitiator have lead to the discovery of SRG. This knowledge on the SRG mechanism was further used to show how the phosphine oxide units incorporated in the polymeric chains could be employed to reinitiate the polymerization as a new method for the synthesis of materials. The features of the emulsion polymerization process in combination with SRG mechanism allowed the incorporation only of few phosphine oxide units in the polymer chain, to obtain higher number of these active units in the polymer chain a different process is required. As the SRG mechanism should be applicable to every polymerization system we used a precursor polymer (PS-PO) prepared via bulk polymerization of styrene with the commercially available hydrophobic initiator bis(2, 4, 6-trimethylbenzoyl)-phenylphosphine oxide (8, Irgacure® 819). The concentration of photoinitiator was increased 100 times compared to the standard concentration because of the necessity to have a higher concentration of phosphine oxide repeating units in the polymer backbone. An analysis of the average molecular weight of the precursor polymer revealed shorter chain length than the chain lengths of the polymer prepared with standard concentration of BAPO (Figure 3.34).
Figure 3.34 Molecular weight distribution of PS-PO precursor polymers prepared via bulk polymerization, standard concentration of photoinitiator (curve 1) and 100 times higher (curve 2). These latter PS-PO precursor polymers, possessing a greater amount of photo-cleavable bonds per unit mass, as characterized by 31P-NMR signals at 25–27 ppm (Figure 3.25 a),
140 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination were used to initiate different polymerization reactions photolytically. Some possible applications using PS-PO precursor follow in this chapter. The PS-PO polymer was used for the preparation of a thermoplastic–elastomeric glass glue. 10 weight-% solution of PS-PO in butyl acrylate (BA) was prepared and spread between two thin glass plates. After irradiation both the glass plates were firmly stuck together by the completely transparent PS-PO-poly(butylacrylate) PBA block copolymer (Figure 3.35 a).
Figure 3.35 (a) thermoplastic–elastomeric glass glue composed of polystyrene-b-poly(butyl acrylate)(PBA) block copolymer generated by photo-initiated polymerization, and (b) glass slides glued together by a mixture of PS and PBA homopolymers produced by photo-polymerization of a PS solution using Irgacure® 819. (c) Light microscopy image of a portion of (a) and (d) Light microscopy image of a portion of (b).
If a mixture of non–reactive polystyrene generated by traditional polymerization, and BA was polymerized using Irgacure® 819, a strongly turbid layer was formed, due to the macroscopic demixing of the two homopolymers (Figure 3.35 b). The phosphine oxide units in the PS-PO precursor allowed for the re-initiation of the polymerization and the subsequent formation of the block copolymer. The presence of the copolymer was demostrated by the absence of macroscopic phase separation (Figure 3.35 c). The phase separation was clearly visible at the same magnification for the mixture of two homopolymers (Figure 3.35 d).
141 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
The PS-PO prepared by this method was employed in the synthesis of a different block copolymer PS-b-poly(vinyl acetate), that is difficult to prepare by other polymerization methods. PS-b-poly(vinyl acetate) (PVA) copolymer can be prepared usually only by multi-step procedures based on controlled radical polymerization techniques using copper, cobalt or sulfur compounds.44 Irradiation of a solution of PS-PO precursor in vinyl acetate produced an elastic PS-PO-PVAc block copolymer that retains the shape of the reactor used for the preparation. The resulting PS-PO-PVAc block copolymer was dissolved in glacial acetic acid (Figure 3.36 a) and a light microscope image shows the block copolymer dispersed in the solvent (Figure 3.36 c).
Figure 3.36 (a) PS-PO—PVAc block copolymer disperses in glacial acetic acid; (b) PS-PO— PVAc block copolymer immediately after the polymerization; (c) light microscopy images of the PS-PO—PVAc block copolymer dispersed in glacial acetic acid.
The comparison of the dispersion of the obtained PS-PO-PVAc block copolymer in glacial acetic acid with a mixture of PS and PVA in the same acid provided further evidence for the formation of the co-polymer. While the mixture of the two homopolymers (PS and PVA) showed flocculated chunks in the solvent (Figure 3.37 a), the PS-PO-PVAc block copolymer formed a homogenous dispersion.
142 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.37 (a) Solution of polystyrene (PS) and polyvinylacetate (PVA) homopolymers in glacial acetic acid, and (b) of PS-PO-PVA block copolymer prepared using PS-PO as initiator.
The PS-PO precursor was also used as an initiator for the emulsion polymerization of styrene. In a standard procedure BAPO-AA was replaced with 500 mg of PS-PO. The resulting polymer was characterized by large particle size distribution, average particle size of 140 nm. The molecular weight distribution was slightly shifted towards higher molecular weights (Figure 3.38 and 3.39).
Figure 3.38 Transmission electron microscopy images of polystyrene latex particles obtained by photoinitiated polymerization with PS-PO as initiator.
143 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Figure 3.39 Molecular weight distribution of PS-PO precursor (red dashed curve) and polystyrene obtained after photoinitiated polymerization with PS-PO as initiator (black curve).
As visual proof-of-concept, a 50 weight-% PS-PO solution in a styrene-butyl acrylate mixture (3:2 g/g) containing a yellow dye (Hostasol Yellow 36, Clariant) was used as ink to photo-type through a PTFE stencil “SRG” after irradiation with a visible light for 30 min (Figure 3.40).
Figure 3.40 (a) Snapshots of the preparation of the photo-typing of “SRG” on a microscopy glass slide through a PTFE stencil (Osram, L18 W, light color 840, lumilux, cold white) and (b) of the photo-typed letters.
144 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.13 Conclusions
The use of continuous flow devices to study the phosphine oxide initiated emulsion polymerization process led to the discovery of the mechanism of these photoinitiators: snowballing radical generation (SRG). Photolysis of BAPOs rapidly generates an avalanche of radicals that produce polymer chains with ultra-high molecular weight up to 30·106 g/mol. The phosphine oxide photoinitiators decompose and integrate as units into the polymer chains allowing for repeated radical generation upon irradiation. This property was exploited to prepare materials with different morphologies that were difficult to produce previously. A stochastic model was used to simulate snowballing kinetics and to quantitatively rationalize the polymerization process. The discovery of this new mechanism was only possible by a unique combination of heterophase polymerization, phosphine oxide photoinitiators and the use of a continuous flow microreactor, will have a significant impact on the application of bis-acylphosphine oxide initiators and underscore the importance of using continuous flow reactor in reaction mechanism studies.
145 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.14 Experimental Part
3.14.1 Chemicals
The water used throughout was purified using a Seral purification system (PURELAB Plus) or an Integra UV plus (SG Reinstwassersysteme) system with a conductivity of 0.06 µS cm-1. The monomers styrene (Aldrich), methyl methacrylate (MMA, Aldrich), and butyl methacrylate (BMA, Aldrich) were purified by distillation under reduced pressure and stored in a refrigerator. Before use the monomers were checked regarding oligomers by instilling a drop into an excess of methanol. Only oligomer-free monomers were applied in the polymerizations. Sodium dodecyl sulfate, ultrapure, (SDS, Roth GmbH) and potassium peroxodisulfate (KPS, Aldrich) were employed as received. 2-(bis(2,4,6-trimethylbenzoyl)phosphoryl)acetic acid (BAPO-AA), bis(2,4,6-trimethylbenzoyl)phosphineoxide tris-oxyethylene (BAPO-PEG) were synthesized as described previously28c and 2,4,6-trimethylbenzoyl-diphenylphosphineoxide (MAPO) was a gift from BASF.
3.14.2 General Procedure for Emulsion Polymerizations
Emulsion polymerization in microfluidic device. A syringe was filled with monomers (2.5 mL as total volume) and a second syringe with a solution previously prepared of sodium dodecyl sulphate (SDS), photoinitiator (10 mg) and ultrapure water (10 mL). All operations involving the photoinitiator before and after the irradiation time were performed in the dark. The solutions were flushed into the reactor and the resulting reaction mixture was collected in a flask under stirring.
146 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
Emulsion polymerization in a stirred tank reactor. The polymerizations were carried out according to standard procedures either in glass reactor or a CPA200 reaction calorimeter (ChemiSens, Sweden).45
3.14.3 Latex Characterization
Solid content was determined using a HR73 Halogen Moisture Analyzer (Mettler Toledo). Average particle size (intensity-weighted diameter) was determined with a Nicomp particle sizer (PSS Santa Barbara, USA, model 370) at a fixed scattering angle of 90°. Molecular weight distributions (MWD) were determined by gel permeation chromatography (GPC) and were used to calculate weight and number average molecular weight of the polymers (Mw, Mn). GPC was carried out by injecting 100 µL polymer solutions (solvent tetrahydrofuran (THF)) through a Teflon-filter with a mesh size of 450 nm, into a Thermo Separation Products set-up equipped with ultra violet (UV) (TSP UV1000), and refractive index (RI) (Shodex RI-71) detectors in THF at 30 °C with a flow rate of 1 mL/min. Two column sets were employed. Column set A consists of two 300 x 8 mm columns filled with a MZ-SDplus spherical polystyrene gel (average particle size 5 µm) with a pore size of 103 and 105 Å, respectively. For column set B an additional column with a pore size of 106 Å was used allowing resolution of molecular weights of up to 107 g/mol. Average molecular weights, and number of average molecular weight polymers (Mw and, Mn) were calculated based on polystyrene standards (between 500 and 2·106 g mol-1 from PSS, Mainz, Germany). FT-IR spectra were recorded with a Varian 1000 FT-IR on an ATR Golden Gate (Pike) cell. Broadband decoupled 13C-NMR spectra were recorded using a Bruker Spectrospin (DPX400) spectrometer. The data acquisition time was 10 hours.
147 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
SEM images were performed with Omega 912, Zeiss AG apparatus. Light microscopy images taken by Keyence VHX100 microscope under oblique illumination in transmission mode.
3.14.4 Determination of Molecular Weight by Static Light Scattering
Static light scattering measurements were carried out in tetrahydrofuran using an ALV machine at 25 °C (ALV-7004 Multiple tau digital correlator equipped with CGS-3 Compact Goniometer system, 22 mW He-Ne laser, wavelength of 632.8 nm, and pair of avalanche photodiodes operated in a pseudo-cross-correlation mode). The program ALV- Stat was used for data evaluation. The Zimm plot resulted in apparent average molecular weights of 3.428·106 and 3.464·106 g/mol for polystyrene samples prepared in the microfluidic device under standard conditions with SDS concentrations of 300 and 500 mg per 10 mL water.
3.14.5 Monomer Concentration Inside Latex Particles
The CM value is an optimistic guess for fast monomer consumption. Under these conditions equilibrium thermodynamics are not suited to estimate the exact CM value as it does not remain constant during the period of time for average chain growth. CM = 3.5 M is likely too high, but constitutes the best approximation available today. Despite this uncertainty, using the standard IUPAC numerical value for the propagation frequency and a temperature of 30 °C, we were able to calculate an upper limit of the attainable molecular weight (see primary article for details).
148 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.14.6 Seed Polymerization
Seed polymers were produced by styrene photo-initiated polymerization in continuous flow device (10 g water, 2.25 g styrene, 0.01 g BAPO-AA or 0.01 g of MAPO, 25 °C, irradiation time 36.5 sec). For the second stage polymerization the BAOP-AA seed latex was swollen with BMA after dialysis and irradiated (shaking, 2 h, rt). After purification by repeated precipitation from a tetrahydrofuran solution in methanol the MAPO seed polymer was then dissolved in BMA. This solution was subjected to polymerization in the microfluidic device under standard conditions with an irradiation time of 144 sec.
3.14.7 Synthesis of the Precursor Polymer
The polymerizations were carried out according to standard procedures in a glass reactor using 18 g of styrene and 80 g or 8 g of Irgacure® 819. The sample was irradiated for 72 h with a light source (Osram, L18 W, light color 840, lumilux, cold white). The polymer was purified by repeated precipitation from a tetrahydrofuran solution in methanol.
3.14.8 Assumption for Simulation Data for Different Initial Initiator Concentrations
For the particular simulation of heterophase polymerization initiated by BAPO photodecomposition, the reaction volume was defined as the volume of a monomer droplet, containing initially only one single primary radical, either including or not, a phosphine oxide group. The only difference between the kinetic model based on SRG and
149 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination the traditional model based on common radical polymerization kinetics is the consideration of the decomposition of phosphine oxide groups inside the polymeric chain (reactions 14 a-c). In the traditional model, the kinetic rate coefficient for these reactions, kd2, is set to zero.
Table 3.4 Kinetic scheme and reactions rate constants of radical polymerization based on SRG used for the model simulations; the assignment of the particular chemical structures to the symbols used in the kinetic equations is possible with Scheme 2, Y is the phosphine oxide acetic acid unit, Z the trimethylbenzoyl unit, P and P’ are different polymer chains, and the open circle close to a letter denotes a single radical function
150 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
3.15 References
1 (a) Elaissari, A; Fessi, H. Braz. J. Phys. 2009, 39, 146–149. (b) Seo, M.; Nie, Z.; Xu, S.; Mok, M.; Lewis, P. C.; Graham, R.; Kumacheva, E. Langmuir 2005, 21, 11614–11622. 2 (a) Kitayama, Y.; Yorizane, M.; Kagawa, Y.; Minami, H.; Zetterlund, P. B.; Okubo, M. Polymer 2009, 50, 3182–3187. (b) Konishi, N.; Fujibayashi, T.; Tanaka, T; Minami, H.; Okubo, M. Polym. J. 2010, 42, 66–71. (c) Tanaka, T.; Komatsu, Y.; Fujibayashi, T.; Minami, H.; Okubo, M. Langmuir 2010, 26, 3848–3853. (d) Tanaka, T.; Okayama, M.; Kitayama, Y.; Kagawa, Y.; Okubo, M. Langmuir 2010, 26, 7843–7847. (e) Tanaka, T.; Okayama, M.; Minami, H.; Okubo, M. Langmuir 2010, 26, 11732–11736. (f) Lu, Y.; Wittemann, A.; Ballauff, M. Macromol. Rapid Commun. 2009, 30, 806–815.
3 Ugelstad, J.; Mork, P. C.; Aasen, J. O. J. Polym. Sci., A: Pol. Chem. 1967, 5, 2281–2288. 4 (a) Smith, W. V.; Ewart, R. H. J. Chem. Phys. 1948, 16, 592–599. (b) De Geest, B. G.; Urbanski, J. P.; Thorsen, T.; Demeester, J.; De Smedt, Langmuir 2005, 21, 10275–10279. (c) Xu, S.; Nie, Z;. Seo, M.; Lewis, P. C.; Kumacheva, E.; Stone, H. A.; Garstecki, P.; Weibel, D. B.; Gitlin, I.; Whitesides, G. M. Angew. Chem. Int. Ed. 2005, 44, 724–728. (d) Ugelstad, J.; Mork, P. C.; Aasen, J. O. J. Polym. Sci., A: Pol. Chem. 1967, 5, 2281–2288. 5 (a) Hubbell, J. A.; Thomas, S. N.; Swartz, M. A. Nature 2009, 462, 449–460. (b) Sharp,
F. A.; Lavelle, E. C. PNAS 2009, 106, 870–875.
6 (a) Esen, C.; Scweiger, G. J. Colloid Interface Sci 1995, 179, 276–280. (b) Whitesides, G. M. Nature, 2006, 442, 368–373. (c) Ouzineb, K.; Graillat, C.; McKenna, T. J. Applied Pol. Sci. 2004, 91, 2195–2207. (d) Paquet, D. A. Jr.; Ray, W. H. AIChE, 1994, 40, 73–87. (e) Ganan-Calvo, A. M.; Gordillo, J. M. Phys. Rev. Lett. 2001, 87, 274501–274505. (f) Garstecki, P.; Gitlin, I.; DiLuzio, W.; Whitesides, G. M.; Kumacheva, E.; Stone, Howard A. Appl. Phys. Lett. 2004, 85, 2649–2651. (g) Thorsen, T.; Roberts, R. W.; Arnold, F. H.; Quake, S. R. Phys. Rev. Lett. 2001, 86, 4163–4166. (h) Link, D. R.; Anna, S. L.; Weitz, D. A.; Stone, H. A. Phys. Rev. Lett. 2004, 92, 054503. (i) Anna, S. L.; Bontoux, N.; Stone, H. A. Appl. Phys. Lett. 2003, 82, 364–366. (l) Whitesides, G. M. Nature 2006, 442, 368–373. (m) Feldon, M.; McCann, R. F.; Laundrie, R. W. India Rubber World 1953, 128, 51–53.
151 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
(n) Ouzineb, K.; Graillat, C.; McKenna, T. J. Applied Pol. Sci. 2004, 91, 2195–2207. (o)
Paquet, D. A. Jr.; Ray, W. H. AIChE 1994, 40, 73–87. 7 (a) Sounart, T. L.; Safier, P. A.; Voigt, J. A.; Hoyt, J.; Tallant, D. R.; Matzke, C. M.; Michalske, A. Lab Chip 2007, 7, 908–915. (b) Ganan-Calvo, A. M.; Gordillo, J. M. Phys. Rev. Lett. 2001, 87, 274501–274505. (c) Thorsen, T.; Roberts, R. W.; Arnold, F. H.; Quake, S. R. Phys. Rev. Lett. 2001, 86, 4163–4166. (c) Link, D. R.; Anna, S. L.; Weitz, D. A.; Stone, H. A. Phys. Rev. Lett. 2004, 92, 054503–1–054503–3. (d) Anna, S. L.; Bontoux, N.; Stone, H. A. Appl. Phys. Lett. 2003, 82, 364–366. 8 (a) Okubo, Y.; Maki, T.; Nakanishi, F.; Hayashi, T.; Mae, K. Chem. Eng. Sci. 2010, 65, 386–391. (b) Tan, Y. C.; Fisher, J. S.; Lee, A. I.; Cristini, V.; Lee, A. P. Lab Chip 2004, 4: 292–298. (c) Xu, S.; Nie, Z.; Seo, M.; Lewis, P.; Kumacheva, E.; Stone, H. A.; Garstecki, P.; Weibel, D. B.; Gitlin, I.; Whitesides, G. M. Angew. Chem. Int. Ed. 2005, 44, 3799– 3799. (d) Sheperd, R. F.; Conrad, J. C.; Rhodes, S. K.; Link, D. R.; Marquez, M.; Weitz, D. A.; Lewis, J. A. Langmuir 2006, 22, 8618–8622. (e) Tan, Y.C.; Fisher, J. S.; Lee, A. I.; Cristini, V.; Lee, A. P. Lab Chip 2004, 4, 292–298. 9 (a) Mozharov, S.; Nordon, A.; Littlejohn, D.; Wiles, C.; Watts, P.; Dallin, P.; Girkin, J. M. J. Am. Chem. Soc. 2011, 133, 3601–3608. (b) Miyazaki, M.; Maeda, H. Trends Biotechnol. 2006, 24, 463–470. (c) Urban, P. L.; Goodall, D. M.; Bruce, N. C. Biotechnol. Adv. 2006, 24, 42–57. (d) Whitesides, G. M. Nature 2006, 442, 368–373. (e) Feldon, M.; McCann, R. F.; Laundrie, R. W. India Rubber World 1953, 128, 51–53. (f) Ouzineb, K.; Graillat, C.; McKenna, T. J. Applied Pol. Sci. 2004, 91, 2195–2207. (d) Paquet, D. A. Jr.;
Ray, W. H. AIChE 1994, 40, 73–87. 10 Dendukuri, D.; Tsoi, K.; Hatton, T. A.; Doyle, P. S. Langmuir 2005, 21, 2113–2116. 11 Shin, S.; Hong, J. S.; Lee, K-H; Lee, S-H. Langmuir 2009, 25, 12361–12366. 12 Jeong, W. J.; Kim, J. Y.; Choo, J.; Lee, E. K.; Han, C. S.; Beebe D. J.; Seong, G. H.;
Lee, S. H. Langmuir 2005, 21, 3738–3741. 13 (a) Griesser, M.; Neshchadin, D.; Dietliker, K.; Moszner, N.; Liska, R.; Gescheidt, G. Angew. Chem. Int. Ed. 2009, 48, 9359–9361. (b) Jockusch, S.; Turro, N. J. J. Am. Chem. Soc. 1998, 120, 11773–11777. 14 Sluggett, G. W.; Turro, C.; George, M. W.; Koptyug, I. V.; Turro, N. J. J. Am. Chem. Soc. 1995, 117, 5184–5187.
152 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
15 Urban, D.; Takamura, K. Polymer Dispersions and Their Industrial Applications, 2002,
Wiley-VCH Verlag GmbH, Weinheim. 16 Allen, N. S. J. Photochem. Photobiol. A: Chemistry 1996, 100, 101–107. 17 (a) Rutsh, W.; Dietliker, K.; Leppard, D.; Köhler, M.; Misev, L.; Kolczak, U.; Rist, G. Prog. Org. Coatings 1996, 27, 227–239. (b) Kolczak, U.; Rist, G.; Dietliker, K.; Wirrz, J. J. Am. Chem. Soc. 1996, 118, 6477–6489. (c) Jockusch, S.; Koptyug, I. V.; McGarry, P. F.;
Sliggett, G. W.; Turro, N. J.; Watkins, D. M. J. Am. Chem. Soc. 1997, 119, 11495–11501. 18 Günersel, E. D.; Hepuzer, Y.; Yusuf, Y. Angew. Makromol. Chem. 1999, 264, 88–91. 19 Löb, P.; Pennemann, H.; Hessel, V.; Men, Y. Chem. Eng. Science 2006, 61, 2959–2967. 20 Funayama, H.; Sugarawa, T. Bull. Chem. Soc. Jpn. 1987, 70, 2245–2249. 21 Hernandez, H. F.; Tauer, K. Macromol. Symp. 2008, 271, 64–74. 22 www.imtek.de 23 (a) Nishimi, T.; Miller, C. A. Langmuir 2000, 16, 9233–9241. (b) Tauer, K.; Kozempel, S.; Rother, G. J. Coll. Interf. Sci. 2007, 312, 432–438. 24 (a) Ikemura, K; Ichizawa, K.; Yoshida, M.; Ito, S.; Endo, T. Dent. Mat. J. 2008, 27, 765–774. (b) Shergill, R.; Haberler, M.; Vink, C. B.; Patten, H. V.; Woodward, J. R. Phys. Chem. 2009, 11, 7248–7256. (c) Sumiyoshi, T.; Schnabel, W.; Henne, A.; Lechtken, P. Polymer 1985, 26, 141–146. 25 Ouzineb, K.; Graillat, C.; McKenna, T. J. App. Pol. Sci. 2004, 91, 2195–2207. 26 Keskin, S.; Jockusch, S.; Turro, N. J.; Arsu, N. Macromol. 2008, 41, 4631–4634. 27 Tauer, K.; Öz, N. Macromol. 2004, 37, 5880–5888. 28 Grutzmacher, H.; Geier, J.; Stein, D.; Ott, T.; Schonberg, H.; Sommerlade, R. H.; Boulmaaz, S.; Wolf, J. P.; Murer, P.; Ulrich, T. Chimia 2008, 62, 18–22. 29 (a) Tauer, K. Pol. Adv. Tech., 1995, 6, 435–440. (b) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref. Data, 1988, 17, 513–886. 30 Tauer, K.; Yildiz, U. Macromol. 2003, 36, 8638–8647. 31 Brandrup, J.; Immergut, E. H.; Grulke, E. A Polymer Handbook 1999, 4th Ed, Wiley, New York. 32 (a) Moad, G.; Solomon, D. H. The Chemistry of Free Radical Polymerization 1995 Pergamon-press, Oxford. (b) Buback, M.; Egorov, M.; Gilbert, R. G.; Kaminsky, V.; Olaj, O. F.; Russell, G. T.; Vana, P.; Zifferer, G. Macromol. Chem. Phys. 2002, 203, 2570–2582. 33 Tauer, K.; Muller, H.; Colloid Polymer Sci. 2003, 281, 52–65.
153 3. Continuous Flow Microreactor and Phosphine Oxide Initiator: an Unique Combination
34 Gilbert, R. G. Emulsion Polymerization a Mechanistic Approach. 1995, Academic Press, London. 35 Tauer, K.; Kaspar, H.; Antonietti, M. Coll. Pol. Sci. 2000, 278: 814–820 36 Parker, H. Y.; Westmoreland, D. G.; Chang, H. R. Macromol. 1996, 29, 5119–5127. 37 Kühn, I.; Tauer, K. Macromol. 1995, 28, 8122–8128. 38 Sumiyoshi, T.; Schnabel, W.; Henne, A.; Lechtken, P. Polymer 1985, 26, 141–146. 39 (a) Wyzgoski, F.; Meng, H.; Rinaldi, P. L.; Harwood, H. J. Polymer Preprints (Am. Chem. Soc., Division of Polymer Chemistry) 1999, 40, 1028–1029. (b) Li, T.; Willis, T. J.; Padias, A. B.; Hall, H. K., Macromol. 1991, 24, 2485–2487. 40 (a) Russell, K. E.; Tobolsky, A. V. J. Am. Chem. Soc. 1954, 76, 395–399. (b) Zimm, B. H.; Bragg, J. K. J. Polym. Sci. 1952, 9, 476–478. 41 (a) Breitenbach, J. W.; Kastell, A. Monatshefte Chemie 1953, 84, 645–647. (b) Hall, H. K.; Padias, A. B.; Pandya, A.; Tanaka, H. A Macromol. 1987, 20, 247–254. (c) Percec, V.; Kim, H. J.; Barboiu, B., Macromol. 1997, 30, 6702–6705. 42 (a) Jasse, B.; Laupretre, F.; Monnerie, L. Macromol. Chem. Phys. 1977, 178, 1987– 1992. 43 Gillespie, D. T. J. Computational Phys. 1976, 22, 403–434. 44 (a) Destarac, M.; Pees, B.; Boutevin, B. Macromol. Chem. Phys. 2000, 201, 1189–1199. (b) Jeon, H. J.; Youk, J. H. Macromol. 2010, 43, 2184–2189. 45 Tauer, K.; Muller, H. Coll. Pol. Sci. 2003, 281, 52–65.
154 4. Conclusions and Outlook
4. Conclusions and Outlook
155 4. Conclusions and Outlook
This thesis has reported on the beneficial properties of continuous flow microreactors for chemical synthesis and mechanistic studies of reactions which are summarized below.
1. The synthesis of CdSe or CdTe quantum dots (QDs), covered by a shell of ZnS and further functionalized with carbohydrates and hydrophilic acid in a commercially available microreactor (1 mL glass microreactor) was described. The generation of the QD core was the fundamental step for the synthesis since the shape and size of the seed influenced the photophysical properties of the CdSe or CdTe QDs. The first step afforded CdSe or CdTe QD core, followed by screening of different conditions in a continuous flow microreactor to achieve different particles size distributions. Under these conditions, QDs of different absorption, emission spectra, and quantum yields were obtained. After covering the core with a shell of ZnS, the QDs were successfully functionalized with hydrolipoic acid or thio-linked carbohydrates. The carbohydrate functionalized QDs were used for in vitro and in vivo biological studies.
Figure 4.1 Schematic representation of carbohydrate capped QDs project.
The significance of this method can be summarized as follows: 1) A synthesis in single- phase with high control of nucleation and growth of the particles, at lower temperature than the previous synthesis reported in the literature was achieved. 2) Clogging of the continuous flow microreactor has been overcome by screening the best solvent, concentration of precursors, temperature and residence time. 3) The three step synthesis in continuous flow microreactor was successfully achieved requiring one purification step for the final QDs. 4) The functionalized QDs were ready to use for biological studies. Enormous improvements in the synthesis of QDs and their accessibility have further enhanced their applications in electronics and life sciences. Despite overcoming the
156 4. Conclusions and Outlook
problem of scaling up of QDs, maintaining consistent morphology and properties of the particles, the metals used in the synthesis of QDs are highly toxic and difficult to handle. Efforts are now focused on the use of less toxic metals but with similar photo-physical properties. Several studies have been reported in general, for the synthesis of particles using continuous flow microreactor. It is remarkable to notice how the highly controlled synthesis in continuous flow has a direct impact on the morphology of particle and on the final material. This area of research is now emerging and is already taking great advantage of the microreactor technology.
2. A continuous flow microreactor was designed for the synthesis of polymer particles via phosphine oxide photo-initiated emulsion polymerization reactions. The polymer particles were generated under short residence time and resulted in a very high molecular weight of the polymer chains. Microreactor technology facilitated the screening of different conditions and to propose a mechanism for phosphine oxide initiators in the polymerization reaction. Upon irradiation, the rapid generation of radicals and the repeated breaking of the polymer backbone where the phosphine oxide units were incorporated produced a snowballing generation of radicals (SRG). The SRG process was then exploited to generate block copolymer or to restart the polymerization via the use of the polymer precursor without additional initiators (Figure 4.2).
Figure 4.2 Schematic representation of emulsion polymerization in continuous flow microreactor project
157 4. Conclusions and Outlook
Two further developments can be seen on the basis of the knowledge gained on the reaction mechanism of phosphine oxide initiators: 1) Design new materials with novel morphologies. 2) The microreactor technology could be further improved to design a system which permits the use of compact light source with specific emission such as LEDs (light-emitting diode). To the best of our knowledge few examples of photochemical reactions are reported and the system can be further improved to achieve better results in this area using different sources of light or designing systems which can maximize the irradiation surface of microreactors. Alternatively kinetic studies of reactions using continuous flow microreactors are still rare examples in the literature and probably these fundamental investigations will find more space in this field in the future. Generally microreactor is the perfect tool to explore safe chemistry using limit conditions as high temperature and pressure. The design of a modular system easy to assemble and to use according to the required reaction with integrated analytical systems to characterize products and intermediates of reaction would further expand the use of microreactors. In conclusion, the two investigations reported in this thesis highlight the importance of the microreactor in addressing chemical phenomena and could encourage the development of other chemical reactions which previously could not be achieved.
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Appendix
159
160 5. Glyco-dendronized Polylysine for Bacteria Detection
5. Glyco-dendronized Polylysine for Bacteria Detection
This appendix summarizes the work performed in collaboration with Dr. R. Kikkeri (project design and evaluation of the data) and Dr. N. Azzouz (bacteria detection experiments). The E. Coli bacteria strain was provided by Professor P. E. Orndorff (College of Veterinary Medicine, Raleigh, NC, United States).
Parts of this chapter have been published in: • “Detection of Bacteria Using Glyco-dendronized Polylysine prepared by Continuous Flow Photo-Functionalization” Laurino, P.; Kikkeri, R.; Azzouz, N.; Seeberger, P. H. Nano Lett. 2011, 11, 73–78.
161 5. Glyco-dendronized Polylysine for Bacteria Detection
5.1 Introduction
Dendrimers are molecules with repeating branching units, symmetrically developed around a core with nanometer dimensions.1 The core is characterized by its functionality which is the number of chemical groups through which it can be connected to the external parts of the molecule. The origin of the branched structure starts from the core and when the structure of the dendrimer is developed in only one direction the dendrimer is called a dendron (Figure 5.1).2
Figure 5.1 Dendritic Polymer structure.
Dendrons can be used as lateral functional groups of a polymer main-chain and the resulting structure consists of a high density of functional groups and these are called dendronized polymer.3 The major drawback in the synthesis of dendrimers or dendrons is the tedious synthetic steps involved. An alternative to dendrimer and dendrons are the hyperbranched polymers which consist of imperfectly branched or irregular structures.4 Hyperbranched polymers present often an easier synthetic route compared to dendrimer structures but lack the regularity typical of the dendrimers which is the base for their physical, photophysical and supramolecular properties. A well defined dendrimer structures is characterized by a precise size and shape. All these features enable dendrimers, dendrons and dendronied polymer to be candidates for applications in sensors,
162 5. Glyco-dendronized Polylysine for Bacteria Detection catalysis, molecular electronics and photonics, and in recent times are considered one of the most important research topics nowdays in Material and Biomedical sciences.5 Dendronized polymers in particular are a new class of compounds.6 In these systems the polymer is used as a polyfunctional and polydisperse backbone functionalized by dendrons. The dendronized polymers are sterically hindered and lead to the formation of a rigid structure with different polymers compared to those of the polymer or dendrons precursors.7 An important feature of the dendronized polymer is the secondary interactions between the dendrons which can be attractive and lead to the generation of a self-assembly phenomena.8 Dendronized polymers can be prepared via three different strategies (Figure 5.2): 1. The graft-to approach, where a preformed polymer is functionalized by dendrons (convergent route). 2. The macromonomer approach, where the monomer are functionalized by a preformed dendrons and then polymerized. 3. The graft-from approach, where dendrons of the first generation are functionalized to the polymer backbone and then successively elongated (divergent route).
Figure 5.2 Schematic representation of the three synthetic strategies for dendronized polymer.
The graft-to approach has been choosen for this study because of the straightforward strategy for the formation of dendronized polymer. The dendronized polymer are multivalent and flexible tools that if functionalized with biologically active compounds (carbohydrates, peptides, nucleotides, etc.) can be used as biosensors.9 Consequently, we explored the photochemical [2 + 2] cycloaddition reaction,10 as an
163 5. Glyco-dendronized Polylysine for Bacteria Detection alternative graft-to approach for the synthesis of a dendronized polymer for biological applications (Figure 5.3).
Figure 5.3 Schematic representation of [2 + 2] cycloaddition between glycol-dendron and a polymer chain.
When functionalized with carbohydrate the dendronized polymer served as a powerful platform for the detection of bacteria by providing a multivalent system which was needed to enhance the generally weak protein-carbohydrate interactions. The glyco- dendronized polymer could further bend and adapt to the shape and morphology of the bacterial surface and optimize the binding to several bacterial carbohydrate receptors hence displaying a multivalent interaction (Figure 5.4).11
Figure 5.4 Schematic representation of the glyco-dendronized polymer binding to the bacterium surface.
Contamination of food with Escherichia coli, a pathogenic bacterium, results in frequent outbreaks of infections with grave consequences. Rapid, reliable and straightforward detection of pathogenic bacteria is a key step in avoiding the spread of contamination.12 Developing a detection system that meets these criteria has proven challenging and the glycodendronized polylysine has been demostrated to be a powerful candidate for the detection of bacteria.
164 5. Glyco-dendronized Polylysine for Bacteria Detection
5.2 Synthesis of Glyco-dendrons
Firstly two sets of dendrons with three and nine carbohydrates respectively, were synthesized bearing either mannose or galactose (Scheme 5.2).13
Scheme 5.2 Reagents and conditions: (a) Acrylonitrile/NaOH (40%), Conc HCl/EtOH, 51%; 5- Hexynoic acid/DIC/HOBT/DCM, 72%; (b) NaOH/MeOH; pentafluorophenol /DIC/ HOBT/DCM, 56%; (c) 2-(t-butoxycarbonylamino)ethoxy-2,3,4,6-tetra-O-acetyl-α-D-mannoside or 2-(t- butoxycarbonylamino)ethoxy-2,3,4,6-tetra-O-acetyl-α-D-galactoside/TFA/DCM (1:3) (53–61%) was added to 3/TEA/DCM, 67–74%; (d) tripod-mannose/tripod-galactose /DCM/TEA, 67–71%; (e) MeOH/ NaOMe, quant.; (f) MeOH/ NaOMe, quant.
Starting from commercial 2-amino-2-(hydroxymethyl)propane-1,3-diol 1, treatment with acrylonitrile under basic conditions, followed by hydrolysis and esterification, prior to coupling with 5-hexynoic acid provided structure 2. Ester hydrolysis under basic conditions, followed by coupling with pentafluorophenol yielded the active ester 3. Reaction of 3 with peracetylated amino linked mannose or galactose yielded first generation dendrons 4 or 5, while reaction with the peracetylated tri-pod mannose or galactose gave second generation dendrons 8 or 9. Removal of the acetate groups under basic conditions yielded 6 or 7 and 10 or 11.
165 5. Glyco-dendronized Polylysine for Bacteria Detection
5.3 Photo-functionalization of Polylysine
The [2+2] cycloaddition has been selected for the functionalization of a polymer chain. [2+2] Cycloaddition reactions are attractive since they can be carried out in water using inexpensive starting materials, and circumvent the use of heavy metals or other reagents that contaminate the polymer product, while avoiding the formation of side products. To improve the efficiency and to render easy scalability of the reaction a continuous flow device has been exploited for the functionalization of the polymer. As the polymer backbone, polylysine has been chosen due to its biocompatibility and biodegradability14 and two generations of glycodendrimer have been synthesized for the functionalization. The first and second generation dendrons were subsequently used for the functionalization of the PLL polymer backbone 12 (Scheme 5.2). The dendronized polymers 13-16 were created by photochemical functionalization using the [2 + 2] cycloaddition reaction in a continuous flow microreactor. Reactions were carried out at room temperature and in water with 40 min of irradiation time using a medium pressure Hg lamp (450 W). The first generation dendrons afforded the glyco-dendronized polylysine with higher yield (ca 68%) then the second generation (ca 50%). The major disadvantage of the functionalization was the steric hindrance of the second generation dendron on the polymer chain which did not allow the attachment of a second dendron because of proximity effect resulting in lower functionalization. Moreover, the self assembly of the second generation dendron can shield its own active group which serves for the attachment to the polymer chain and consequentially further decrease the conversion of the functionalization compared to the first generation dendron.
166 5. Glyco-dendronized Polylysine for Bacteria Detection
Scheme 5.2 Reagents and conditions: (a) 3-maleimidopropanoic acid N-hydroxysuccinimide ester, pH 8.0, 24 h; (b) or (c) Medium pressure Hg Lamp, (Hanovia ®), 450W, alkyn dendrimer mannose or galactose (6 or 7, 10 or 11), 40 min, rt.
The samples were immobilized on a gelatin coated mica surface and the size and shape of the dendronized polymers characterized by atomic force microscopy (AFM) analysis.15 The nonapod-mannose-functionalized polymer 15 adopted a globular structure, with a diameter of approximately 4 Å (Figure 5.5). The globular shape likely results from polymer and dendrimer integration.
Figure 5.5 AFM images of mannose nonapod PLL 15 on gelatin coated mica surfaces.
167 5. Glyco-dendronized Polylysine for Bacteria Detection
5.4 Bacteria Detection with Glyco- dendronized Polylysine
To demonstrate the clustering capability of the dendronized polymers, bacterial detection assays were performed.16 The dendronized polymers were incubated with either the mannose-binding, invasive strain of E. coli, ORN178 (positive control), or with the mutant E. coli strain, ORN208 that does not bind mannose (negative control). Binding, and the resultant clustering was detected by confocal microscopy using DAPI (4΄,6- diamidino-2-phenylindole), a fluorescent dye that stains DNA. Different numbers of E. coli bacteria were suspended in buffer and incubated with polymer 15 and single mannose containing polymer (17, see section 5.6.6). After centrifugation and washing, the cells were re-suspended and stained with DAPI (4΄, 6-diamidino-2-phenylindole). Confocal images showed no aggregation in case of single mannose polymer 17 (see section 5.6.6) whereas Figure 5.6 shows that, incubation of the nona-mannose functionalized polymer 15 with strain ORN178 resulted in large clusters of bacteria (Figure 5.6), while strain ORN208 did not aggregate with the polymer (Figure 5.6 c and d).
168 5. Glyco-dendronized Polylysine for Bacteria Detection
Figure 5.6 Confocal laser scanning microscope images for the incubation of bacteria E. coli strain ORN178 and dendronized polymer 15 (a); and ConA-FITC (b). Strain ORN208 (negative control) in the presence of 15 (c) and ConA-FITC (d).
We confirmed specific multivalent interactions between specific E. coli strains and polymer 15 by adding ConA-FITC (Concavanalin A-fluorescein isothiocyanate conjugate). ConA is a lectin well known for its strong interaction with α-mannose.17 Solutions containing different numbers of E. coli bacteria (105–107) were incubated with polymer 15. The cells were then incubated with ConA-FITC for approximately 10 min, centrifuged and washed. ConA-FITC staining successfully localized polymer 15 within a cluster of E. coli strain ORN178 (Figure 5.6b). Neither clustering nor significant ConA- FITC staining were observed in samples of E. coli strain ORN208 incubated with compound 15 (Figure 5.6d). Therefore, we conclude that the mannose-binding E. coli was aggregated by a matrix of the nona-mannose dendronized polymer 15, which was localized by fluorescently labeled ConA. E. coli bacteria that do not bind to mannose were not bound by the flexible polymer resulting in no visible aggregation.
169 5. Glyco-dendronized Polylysine for Bacteria Detection
5.5 Conclusions
To afford a glyco-dendronized polymer, a [2+2] cycloaddition reaction was carried out in a continuous flow microreactor. The [2+2] cycloaddition occurred under mild conditions upon irradiation in aqueous solution. The continuous flow microreactor allowed improved yields enhancing the photo-efficiency of the reaction compared to batch reactor. The reaction can be scaled up by maintaining the same conditions and extending the reaction time. Unfortunately the functionalization with the second generation glyco- dendrons occurred at lower yield than the first generation glyco-dendrons probably due to the steric hindrance of the dendron. The glyco-dendronized polymers were later used for bacteria detection. The multivalence of the system allowed for stronger carbohydrate-protein interactions and the flexibility of the polymer enhanced the sensitivity of detection. The glyco-dendronized polymer can be a powerful tool for efficient detection of bacteria in an early stage of contamination and furthermore serve as a platform to study pathogen-carbohydrate interactions.
170 5. Glyco-dendronized Polylysine for Bacteria Detection
5.6 Experimental Part
5.6.1 Continuous Flow Reactor Set Up
Figure 5.7 Schematic representation of the continuous flow microreactor
The flow reactor setup consists of a syringe pump injection system (1, Harvard PHD 2000), gas tight syringe (2, Hamilton), multiple loops of narrow FEP tubing (3, fluorinated ethylene polymer, Tub FEP Nat 1/16 in x .030 in)18 wrapped tightly around a quartz immersion well (4) cooled by a thermostat (9), a Pyrex filter (5), a Hanovia medium pressure Hg lamp (6, 450 W), a power supply (8), and a collection flask (10).
171 5. Glyco-dendronized Polylysine for Bacteria Detection
5.6.2 General Information
All chemicals were reagent grade and used as supplied except where noted.
Dichloromethane (CH2Cl2) was purified by a Cycle-Tainer Solvent Delivery System.
Triethylamine was distilled over CaH2 prior to use. Analytical thin layer chromatography
(TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm). Compounds were visualized by UV irradiation or dipping the plate in CAN solution followed by heating. Flash column chromatography was carried out using force flow of the indicated solvent on Fluka Kieselgel 60 (230-400 mesh). The water was taken from a Seral purification system (PURELAB Plus) with a conductivity of 0.06 µS/cm. 1H and 13C NMR spectra were recorded on a Varian VXR-300 (400 MHz) spectrometer. High-resolution mass spectra (HR-MALDI MS) and ESI-MS were performed by the Mass Spectrometry-service at the MPI Berlin. ESI-MS were run on an Agilent 1100 Series LC/MSD instrument. IR spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer. Optical rotation measurements were conducted using a Perkin-Elmer 241 polarimeter. Atomic Force Microscopic (AFM) was performed with multimode instrument with Nanoscope IIIa controller (Vecco Instruments) operating in the tapping mode. The samples were placed on a Mica disk and tips (Nano world TIP) with a spring constant of 42 N/m, and a resonance frequency of 285 kHz was used for air measurements and spring constant of 0.02 or 0.06 N/m, and resonance frequency of 10-20 kHz were used for liquid measurements. Confocal laser scanning microscope images were taken with Carl Zeiss confocal laser scanning microscope, LSM 700.
172 5. Glyco-dendronized Polylysine for Bacteria Detection
5.6.3 Synthesis of Dendrimers
General Procedure A: Synthesis of Sugar-nonapods. The Boc-protected sugar-amine or sugar-tripod (4.0 eq) was dissolved in 10 mL dichloromethane/trifluoroacetic acid (3:1) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure and the resulting oil was dissolved in anhydrous dichloromethane (20 mL). To this mixture was added tert-butoxycarbonyl-3- {N-{tris[3-[pentafluoro-phenyl-carboxyl-ethoxy) methyl]}methyl amine}-3-β-alanine or N-{tris[3-[pentafluoro phenyl carboxyl-ethoxy)methyl]}methyl amide}-5-hexynylamide (1.0 eq), adjusted to pH 8 with triethylamine (TEA) and the mixture was stirred at room temperature for 12 h. The solvent was evaporated in vacuo and purified by flash silica column chromatography. General Procedure B: Synthesis of sugar dendrons. Tripod or nonapod (1.0 eq) and sodium methoxide (10 eq) were dissolved in methanol (10 mL) and stirred at room temperature for 2 h. The solvent was then evaporated in vacuo, the residue was redissolved in water and dialyzed against water using 500 molecular weight cut-off resin. After two days of dialysis, the sample was lyophilized.
N-{Tris[3-[ethylcarboxyl-ethoxy)methyl]}methylamide}-5-hexynylamide (2). To a solution of N-{tris[(3-[ethylcarboxyl-ethoxy)methyl]}methylamine (2 g, 4.7 mmol) and 5-hexynoic acid (0.53 g, 4.7 mmol) in dichloromethane (10 mL) at 0 °C, were added diisopropylcarbodiimide (0.87 mL, 5.6 mmol) and 1-hydroxybenzotriazole (0.07 g, 0.56 mmol) The reaction mixture was stirred at room temperature for 12 h and concentrated in vacuo. The crude residue was purified by flash silica column chromatography to yield N-
{tris[3-[ethylcarboxyl-ethoxy)methyl]}methylamide}-5-hexynylamide (1.75 g, 72%). Rf 1 = 0.5 (CH2Cl2/MeOH, 98:4); H NMR (400 MHz, CDCl3): δ 5.86 (br.s, 1H), 4.17 (q, J = 8.0 Hz, 6H), 3.94 (br. s, 11H), 2.88 (t, J = 4.0 Hz, 6H), 2.24-2.16 (m, 4H), 1.88 (t, J = 4.0 13 Hz, 1H), 1.78-1.74 (m, 2H), C NMR (100 MHz, CDCl3): δ 172.4, 171.6, 69.5, 66.5,
60.3, 45.2, 37.2, 35.4, 24.3, 14.3, FTIR(CHCl3): 3375, 2988, 2873, 1744, 1726, 1644, -1 1525 cm . HRMS (MALDI-ToF) (m/z) calcd. for C24H41NO10Na 538.2628, found: 538.2627.
173 5. Glyco-dendronized Polylysine for Bacteria Detection
N-{Tris[3-[pentafluoro phenyl carboxyl-ethoxy)methyl]}methyl amide}-5- hexynylamide (3). N-{Tris[3-[ethylcarboxyl-ethoxy)methyl]} methylamine}-5-azido pentamide (1.5 g, 2.9 mmol) was dissolved in ethanol (10 mL) and sodium hydroxide solution (aqueous, 1 N, 2 mL) was added and the mixture was stirred at room temperature for 1 h, concentrated in vacuo, adjusted to pH 5 with hydrochloric acid (aqueous 1 N) and extracted with ethyl acetate. The organic layer was dried with sodium sulfate and concentrated to dryness under reduced pressure. The residue was dissolved in dichloromethane (10 mL) and 2,3,4,5,6-pentafluorophenol (2.1 g, 11.6 mmol) was added. After cooling to 0 °C, diisopropyl carbadiazine (2.2 mL, 13.9 mmol) was added and the reaction mixture was stirred for 12 h at room temperature. The reaction mixture was concentrated in vacuo and purified by silica column flash chromatography to afford tert-butoxycarbonyl-3-{N- {tris[3-[pentafluorophenylcarboxyl-ethoxy)methyl]}methylamide}-5-azido pentamide 1 (1.56 g, 56%). Rf = 0.6 (CH2Cl2/EtOAc, 88:12); H NMR (400 MHz, CDCl3): δ 3.83 (br. s, 12H), 2.84 (t, J = 6.0 Hz, 6H), 2.22-2.17 (m, 4H), 1.71-1.62 (m, 3H), 13C NMR (100
MHz, CDCl3): δ 172.2, 171.1, 69.3, 66.5, 60.3, 45.2, 37.1, 35.4, 24.3, FTIR(CHCl3) : 3688, 3385, 1749, 1658, 1522, 1359 cm-1. HRMS (MALDI-ToF) (m/z) calcd. for
C37H28F15NO10Na 952.1215, found: 952.1218.
Tris[3-[2-ethoxy-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside- ethoxy]methyl]methylamide }-5-hexynylamide (4). General procedure A using 2-(tert-butoxycarbonylamino)ethoxy-2,3,4,6-tetra-O-acetyl-α-
D-mannopyranoside (0.97 g, 1.96 mmol), tris[3-[pentafluoro phenyl carboxyl- ethoxy)methyl]}methyl amide}-5-hexynylamide 2 (0.5 g, 0.53 mmol) and purified by flash chromatography to yield tris[3-[2-ethoxy-2,3,4,6-tetra-O-acetyl-α-D- mannopyranoside-ethoxy]methyl]methylamide }-5-hexynylamide (0.39 g, 47%). Rf = r.t 1 0.45 (CH2Cl2/MeOH, 93:7); [α]D = +23.4 (c = 1.0, CHCl3); H NMR (400 MHz,
CDCl3): δ 6.79 (br.s, 2H), 6.33 (br.s, 1H), 5.27-5.20 (m, 9H), 4.80 (d, J = 9.0 Hz, 3H), 4.25 (dd, J = 9.0, 5.1 Hz, 3H), 4.10 (dd, J = 2.1, 9.9 Hz, 3H), 3.90 (br.s, 3H), 3.76 (dd, J = 4.5, 5.4 Hz, 3H), 3.68 (dd, J = 5.4, 6.0 Hz, 6H), 3.64 (m, 6H), 3.54-3.52 (m, 6H), 3.37 (br.s, 8H), 2.72 (t, J = 5.4 Hz, 6H), 2.39 (t, J = 9.0 Hz, 4H), 2.28 (t, J = 5.4 Hz, 3H), 2.12 (t, J = 5.4 Hz, 2H), 2.07 (s, 9H), 2.02 (s, 9H), 1.96 (s, 9H), 1.71-1.62 (m, 2H), 13C
NMR (100 MHz, CDCl3): δ 171.4, 170.5, 170.0, 169.5, 155.8, 97.6, 69.3, 69.2, 68.6,
174 5. Glyco-dendronized Polylysine for Bacteria Detection
67.3, 65.1, 62.2, 60.4, 53.8, 39.0, 35.9, 34.5, 23.5, 20.3; FTIR(CHCl3): 3376, 2918, 1751, -1 1663, 1515, 1457, 1250 cm ; HRMS (MALDI-ToF) (m/z) calcd. for C67H98N9O37Na 1573.5808; found: 1573.5810.
Tris-[3-4-ethoxy-α-D-mannosepyranosyl-ethoxy}methyl]-5-hexynylamide (6).
General procedure B with tris[3-[2-ethoxy-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside- ethoxy]methyl]methylamide }-5-hexynyl amide 3 (100 mg, 97.5 µmol) sodium methoxide (10 mg, 0.18 mmol) yielded 45 mg, (65%) of cis- ruthenium(II)bis(bipyridine){1,1’-(2,2’-bipyridine-4,4’-diyl)bis-3-β-beta-propane-{tris- r.t [3-4-ethoxy-β-D-galactopyranosyl-ethoxy}methyl] methylamide. [α]D = +1.7 (c = 1.0, 1 H2O); H NMR (400 MHz, D2O/MeOD): δ 4.72 (s, 3H), 3.96-3.94 (m, 4H), 3.90 (d, J = 2.7 Hz, 12H), 3.75-3.65 (m, 34H), 3.61 (br.s, 24H), 3.49-3.46 (m, 12H), 3.73-3.21 (m, 44H), 2.37 (t, J = 6.0 Hz, 6H), 2.35 (t, J = 6.0 Hz, 3H), 2.12 (t, J = 6.0 Hz, 2H), 1.71- 13 1.62 (m, 2H), C NMR (100 MHz, CD3OD): δ 173.2, 172.6, 101.3, 74.8, 71.8, 71.5, 68.5, 67.6, 66.6, 62.4, 61.4, 40.0, 37.3, 34.9,27.3, 23.1, 21.2, 18.5. MALDI-ToF (m/z): + [M-1] Calcd for C43H174N4O2 1046.4642; Found: 1046.4645.
Tris[3-[2-ethoxy-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside- ethoxy]methyl]methylamide}-5-hexynylamide (5). General procedure A using 2-(tert-butoxycarbonylamino)ethoxy-2,3,4,6-tetra-O-acetyl-β-
D-galactopyranoside (0.42 g, 1.06 mmol), tert-butoxycarbonyl-3-{N-{tris[3- [pentafluoro-phenyl-carboxyl-ethoxy)methyl]}methyl amine}-5-hexynylamide 2 (0.25 g, 0.26 mmol) and purified by flash chromatography to yield tert-butoxycarbonyl-3-{tris[3-
[2-ethoxy-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside-ethoxy]methyl]methylamide}-3- r.t β-alanine (0.27 g, 57%). Rf = 0.45 (CH2Cl2/MeOH, 93:7); [α]D = +17.4 (c = 1.0, 1 CHCl3); H NMR (400 MHz, CDCl3): δ 6.47 (br.s, 2H), 5.37 (d, J = 4.0 Hz, 4H), 5.14 (t, J = 8.0 Hz, 3H), 4.99 (dd, J = 4.0 Hz, 3H), 4.49 (d, J = 8.0 Hz, 3H), 4.12-4.10 (m, 6H), 3.92 (t, J = 6.0 Hz, 3H), 3.84-3.83 (m, 3H), 3.70 (t, J = 6.0 Hz, 8H), 3.65 (s, 9H), 3.42 (t, J = 6.0 Hz, 6H), 3.33 (q, J = 6.0 Hz, 2H), 2.39 (t, J = 9.0 Hz, 4H), 2.28 (t, J = 5.4 Hz, 3H), 2.12 (t, J = 6.0 Hz, 2H), 2.07 (s, 9H), 2.02 (s, 9H), 1.96 (s, 9H), 1.71-1.62 (m, 2H); 13 C NMR (100 MHz, CDCl3): δ 171.1, 170.0, 169.8, 169.7, 155.7, 101.1, 70.6, 69.1, 68.7,
67.2, 67.0, 66.9, 61.2, 59.7, 45.65, 41.67, 39.1, 37.0, 36.4, 28.3, 20.6; FTIR(CHCl3):
175 5. Glyco-dendronized Polylysine for Bacteria Detection
3376, 2918, 1751, 1663, 1515, 1457, 1250 cm-1; HRMS (MALDI-ToF) (m/z) calcd. for
C67H98N9O37Na 1573.5808; found: 1573.5810.
Tris[3-4-ethoxy- β-D-galactopyranoside -ethoxy}methyl]-5-hexynylamide (7). General procedure D with cis-ruthenium(II)bis(bipyridine){1,1’-(2,2’-bipyridine-4,4’- diyl)bis-3-β-propane-{tris-[3-4-ethoxy-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside- ethoxy}methyl]methylamide 5 (100 mg, 97.7 µmol) sodium methoxide (10 mg, 2.2
µmol) yielded 51 mg, (76%) of tris[3-4-ethoxy- β-D-galactopyranoside -ethoxy}methyl]- r.t 1 5-hexynylamide . [α]D = +1.8 (c = 1.0, H2O); H NMR (400 MHz, D2O/MeOD): δ 4.36 (d, J = 8.0 Hz, 3H), 3.96-3.94 (m, 4H), 3.90 (d, J = 4.0 Hz, 12H), 3.75-3.65 (m, 17H), 3.61 (br.s, 12H), 3.49-3.46 (m, 6H), 3.43-3.41 (m, 2H), 2.37 (t, J = 6.0 Hz, 6H), 2.35 (t, J = 6.0 Hz, 3H), 2.12 (t, J = 6.0 Hz, 2H), 1.71-1.62 (m, 2H), 13C NMR (100 MHz,
CD3OD): δ 174.9, 173.2, 104.3, 77.8, 75.3, 73.5, 71.8, 71.8, 68.5, 67.6, 66.6, 62.4, 61.4, + 40.0, 37.3, 30.1, 26.4. MALDI-ToF (m/z): [M-1] Calcd for C43H174N4O2 1046.4642; Found: 1046.4645.
3-{Tris[3-carboxylethoxy]methyl]3’-{tris-[2-ethoxy-2,3,4,6-tetra-O-acetyl-α-D-manno pyranoside -ethoxy]methyl] methylamide}-5-hexynylamide (8). General procedure A using tert-butoxycarbonyl-3-{tris[3-[2-ethoxy-2,3,4,6-tetra-O-acetyl- α-D-mannopyranoside-ethoxy]methylamide}-3-β-alanine (0.34 g, 0.35 mmol), {N-{tris[3- [pentafluoro phenyl carboxyl-ethoxy)methyl]}methyl amide}-5-hexynlamide (50 mg, 0.053 mmol) and purified by flash chromatography to yield 3-{tris[3- carboxylethoxy]methyl]3’-{tris-[2-ethoxy-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside- r.t ethoxy] methyl] methylamide}-5-azido pentamide (0.22g, 57 %). [α]D = +19.4 (c =1.0, 1 CHCl3); H NMR (400 MHz, CDCl3): δ 6.84 (br.s, 3H), 6.13 (br.s, 1H), 4.87-5.8 (m, 27H), 4.25 (s, 9H), 3.81 (dd, J = 6.0, 4.0 Hz, 9H), 3.77 (d, J = 6.0 Hz, 9H), 3.55 (br.s, 9H), 3.31-
3.01 (m, 57H), 3.06 (m, J = 4.0 Hz, 34H), 2.47 (br, 32H), 2.32 (br.s, 2H), 2.21 (br.s, 2H), 2.13 (s, 27H), 2.06 (s, 27H), 2.04 (s, 27H), 1.96 (s, 27H), 1.38-1.32 (m, 2H); 13C-NMR
(100 MHz, CDCl3): δ 173.92, 172.2, 171.4, 102.7, 70.7, 69.6, 67.3, 63.4, 61.8, 54.5, 45.2, -1 39.7, 35.6, 20.6, 9.6. FTIR(CHCl3): 3332, 2734, 1745,1365,cm ;HRMS-MALDI (m/z): + [M+ Na] Calcd for C211H314N16O118 4989.9061; Found : 4989.9071.
176 5. Glyco-dendronized Polylysine for Bacteria Detection
Tris-[3-carboxyl-ethoxy]methyl]3’-{tris[2’-ethoxy-β-D-mannosepyranosyl- ethoxy]methyl]-5-hexynylamide (10). General procedure D with 3-{tris[3-carboxylethoxy]methyl]3’-{tris-[2-ethoxy-2,3,4,6- tetra-O-acetyl-α-D-manno pyranoside -ethoxy]methyl] methylamide}-5-hexynylamide 7 (200 mg, 40.0 µmol) and sodium methoxide (22 mg, 0.4 mmol) gave 108 mg (78%) of tris-[3-carboxyl-ethoxy]methyl]3’-{tris[2’-ethoxy-β-D-mannosepyranosyl-ethoxy]methyl]- r.t 1 5-hexynylamide. [α]D = +22.3 (c =1.0, H2O); H NMR (400MHz, MeOD): δ 4.79 (s, 9H), 3.78-3.6 (m, 98H), 3.54 (m, 24H), 3.41 (m, 24H), 2.46 (t, J = 4.0 Hz, 32H), 2.25 (t, J = 6.0 13 Hz, 3H), 2.16 (t, J = 6.0 Hz, 2H), 1.71-1.62 (m, 2H); C NMR (125MHz, CD3OD): δ 173.4, 104.3, 73.8, 71.5, 70.8, 68.8, 67.5, 65.6, 62.6, 60.4, 37.4, 35.1, 25.3, 16.4. MALDI- + HRMS (m/z): [M+1] Calcd for C139H243N16O82 3448.5258; Found: 3448.5251.
3-{Tris[3-carboxylethoxy]methyl]3’-{tris-[2-ethoxy-2,3,4,6-tetra-O-acetyl-β-D- galactopyranoside-ethoxy]methyl] methylamide}-5-hexynylamide (9). General procedure A using tert-butoxycarbonyl-3-{tris[3-[2-ethoxy-2,3,4,6-tetra-O- acetyl- β-D-galactopyranoside-ethoxy]methylamide}-3-β-alanine (0.15 g, 0.09 mmol), {N-{tris[3-[pentafluoro phenyl carboxyl-ethoxy)methyl]}methyl amide}-5-hexynlamide (21mg, 0.021 mmol) and purified by flash chromatography to yield 3-{tris[3- carboxylethoxy]methyl]3’-{tris-[2-ethoxy-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside- r.t ethoxy] methyl] methylamide}-5-azido pentamide (95 g, 54%). [α]D = +2.4 (c =1.0, 1 CHCl3); H NMR (400 MHz, CDCl3): δ 6.64 (br.s, 3H), 5.37 (d, J = 3.3 Hz, 9H), 5.12 (t,
J = 8.0 Hz, 9H), 4.49 (d, J = 4.0 Hz, 9H), 4.31-4.15 (m, 9H), 4.14-4.08 (m, 18H), 3.95- 3.92 (m, 9H), 3.84-3.78 (m, 9H), 3.66-3.56 (m, 64H), 3.41-3.36 (m, 34H), 2.41 (t, J = 5.4 Hz, 32H), 2.32 (br.s, 2H), 2.21 (br.s, 2H), 2.13 (s, 27H), 2.06 (s, 27H), 2.04 (s, 27H), 1.96 13 (s, 27H), 1.38-1.32 (m, 2H), C-NMR (100 MHz, CDCl3): δ 173.92, 172.2, 171.4, 102.7, - 70.7, 69.6, 67.3, 63.4, 61.8, 40.1, 35.6, 20.6, FTIR(CHCl3): 3332, 2734, 1745,1365,cm 1 + ;HRMS-MALDI (m/z): [M+ Na] Calcd for C211H314N16O118 4989.9061; Found : 4989.9071.
177 5. Glyco-dendronized Polylysine for Bacteria Detection
Tris-[3-carboxyl-ethoxy]methyl]3’-{tris[2’-ethoxy-β-D-galactopyranoside- ethoxy]methyl]-5-hexynylamide (11).
General procedure D with 3-{tris[3-carboxylethoxy]methyl]3’-{tris-[2-ethoxy-2,3,4,6- tetra-O-acetyl-β-D-galactopyranoside-ethoxy]methyl] methylamide}-5-hexynylamide 7 (90 mg, 18.0 µmol) and sodium methoxide (10 mg, 0.22 mmol) gave 41 mg (76%) of tris-[3-carboxyl-ethoxy]methyl]3’-{tris[2’-ethoxy-β-D-galactopyranoside- r.t 1 ethoxy]methyl]-5-hexynylamide. [α]D = + 1.3 (c =1.0, H2O); H NMR (400MHz, MeOD): δ 4.77 (s, 9H), 4.39 (d, J = 7.8 Hz, 9H), 4.15 (q, J =4.0 Hz, 8H), 3.91-3.85 (m, 18H), 3.76-3.60 (m, 93H), 3.47-3.45 (m, 24H), 3.41-3.35 (m, 26H), 2.47-2.42 (m, 32H); 2.25 (t, J = 6.0 Hz, 3H), 2.16 (t, J = 6.0 Hz, 2H), 1.71-1.62 (m, 2H), 13C NMR (125
MHz, CD3OD): δ 173.4, 104.3, 73.8, 71.5, 70.8, 68.8, 67.5, 65.6, 62.6, 60.4, 37.4, 35.1, + 25.3, 16.4. MALDI-HRMS (m/z): [M+1] Calcd for C139H243N16O82 3448.5258; Found: 3448.5251.
5.6.4 Functionalization of Polylysine
PLL-maleimido (12). Poly-L-Lysine hydrobromide (10 mg, 0.5 pmol) was added to 3- maleimidopropionic acid- N-hydrosuccinimide ester (133 mg, 0.5 mmol) in 6 mL of nano pure water. The solution was basified with 1 N KOH solution and shaken at room temperature. The aqueous layer was washed with dichloromethane (3 x 7 mL) to remove unreacted maleimido derivative gave 19 mg of pure yellow oil (98%). [α]D 25= -1.2 (c=1.5, MeOH/H2O 1:1); 1H NMR (300 MHz, MeOD) δ 2.2-2.4 (m, 10 H); 3.5 (t, J = 7 Hz, 2H); 5.8 (d, J = 13 Hz, 1H); 6.3 (d, J = 13 Hz, 1H); 13C NMR (300 MHz, MeOD) δ26.2. 37.8, 38.5, 125.5, 138.4, 167.5, 174.6, 178.6, 179.9.
General Procedure C: Synthesis of PLL-dendrone and nonapod. 3- Maleimidopropionic acid-N-poly-L-Lysine (0.2 µmol) and tripod or nonapod (0.4 mmol or 0.2 mmol)) were dissolved in water (3 mL) and flushed in a continuous flow reactor for 40
178 5. Glyco-dendronized Polylysine for Bacteria Detection min under irradiation (medium pressure Hg Lamp, Hanovia, 450W). The crude product was further purified with sephadex G-10 and finally the sample was lyophilized.
PLL-tripodmannose (13). General procedure C with tris-[3-4-ethoxy-α-D-mannosepyranosyl-ethoxy}methyl]-5- hexynylamide 6 (30 mg, 0.4 mmol) and maleimido-PLL (2 mg, 0.2 µmol) to yield 20 mg 1 of PLL-tripodmannose (75%). H NMR (300 MHz, D2O) δ 0.97 (d, J = 6.6 Hz, 1H); 1.69 – 1.59 (m, 3H); 1.78 (s, 1H); 1.87 (s, 1H); 2.11 (m, 3H); 2.21 (m, 3H); 2.39 (m, 6H); 3.24- 3.29 (m, 2H); 3.31 – 3.28 (m, 2H); 3.33-3.36 (m, 2H); 3.37-3.39 (m, 1H); 3.48 – 3.45 (m, 2H); 3.51 – 3.47 (m, 6H); 3.52 (s, 2H); 3.52 (s, 2H); 3.64 – 3.59 (m, 10H); 3.68 – 3.64 (m, 7H); 3.73 (d, J = 1.8 Hz, 2H); 3.76 (d, J = 2.0 Hz, 1H); 3.81 (m, 3H); 4.74 (m, 3H); 13C
NMR (300 MHz, D2O) δ 175.90, 174.19, 99.63, 72.79, 70.48, 69.97, 68.42, 67.43, 66.65, 65.76, 60.85, 60.13, 38.95, 36.08, 35.19, 24.06, 21.80, 16.97.
PLL- tripodgalactose (14).
General procedure C with Tris[3-4-ethoxy- β-D-galactopyranoside -ethoxy}methyl]-5- hexynylamide 7 (30 mg, 0.4 mmol) and PLL-maleimido (2 mg, 0.2 µmol) to yield 14 mg 1 of PLL-tripodgalactose (60%). H NMR (300 MHz, D2O) δ 0.93 (d, J = 6.4 Hz, 1H); 1.01 (t, J = 7.1 Hz, 1H); 1.11 (t, J = 7.2 Hz, 1H); 1.66 – 1.55 (m, 3H); 1.74 (s, 4H); 2.08 (t, J = 7.0 Hz, 3H); 2.18 (t, J = 7.4 Hz, 3H); 2.31-2.20 (m, 2H); 2.51 – 2.47 (m, 1H); 3.33 – 3.24 (m, 6H); 3.37-3.39 (m, 2H); 3.49 – 3.46 (m, 1H); 3.55 – 3.49 (m, 9H); 3.65 – 3.55 (m, 16H); 3.76 (d, J = 3.4 Hz, 3H); 3.85 – 3.78 (m, 4H); 3.91 (m, 1H); 4.24 (m, 3H); 4.84 (s, 13 1H); C NMR (300 MHz, D2O) δ 175.89, 174.22, 107.23, 102.94, 82.84, 80.92, 76.58, 75.10, 72.64, 70.70, 68.54, 68.32, 67.41, 66.17, 62.67, 60.91, 60.13, 39.33, 39.18, 36.08, 35.18, 24.05, 16.96.
PLL- nonapodmannose (15). General procedure C with tris-[3-carboxyl-ethoxy]methyl]3’-{tris[2’-ethoxy-β-D- mannosepyranosyl ethoxy]methyl]-5-hexynylamide 10 (30 mg, 0.2 mmol) and PLL- maleimido (2 mg, 0.2 µmol) to yield 14 mg of PLL-nonapodmannose (50%). 1H NMR
(300 MHz, D2O) δ 2.12 – 2.00 (m, 1H); 2.20 (s, 1H); 2.53 (t, J = 6.6 Hz, 1H); 2.59-2.64 (m, H); 2.69 (s, 1H); 2.86 – 2.72 (m, 6H); 2.95-3.00 (m, H); 3.74 – 3.65 (m, 3H); 3.83 – 3.73 (m, 3H); 3.90-3.97 (m, H); 3.99-4.07 (m, H); 4.26 – 4.13 (m, 5H); 5.20 – 5.13 (m,
179 5. Glyco-dendronized Polylysine for Bacteria Detection
13 2H); C NMR (300 MHz, D2O) δ 174.06, 173.79, 173.39, 129.11, 125.26, 99.59, 72.77, 70.45, 69.95, 68.39, 67.44, 66.62, 65.72, 60.82, 60.17, 60.06, 38.90, 36.02, 35.79, 35.60, 17.01.
PLL-nonapodgalactose (16).
General procedure C with tris-[3-carboxyl-ethoxy]methyl]3’-{tris[2’-ethoxy-β-D- galactopyranoside-ethoxy]methyl]-5-hexynylamide 11 (30 mg, 0.4 mmol) and PLL- maleimido (2 mg, 0.2 µmol) to yield 18 mg of PLL-nonapodgalactose (50%). 1H NMR
(300 MHz, D2O) δ 1.70 (dt, J = 14.6, 7.3 Hz, 1H); 1.84 (s, 1H); 1.91 (s, 1H); 2.17 (t, J = 7.0 Hz, 1H); 2.29 – 2.23 (m, 1H); 2.49 – 2.32 (m, 8H); 2.64 – 2.57 (m, 1H); 2.87 – 2.80 (m, 1H); 3.28 (t, J = 5.6 Hz, 1H); 3.33 - 3.44 (m, H); 3.78 – 3.53 (m, 25H); 3.86 (d, J = 3.3 Hz, 2H); 3.96 – 3.87 (m, 3H); 3.96 – 3.87 (m, 3H); 4.34 (d, J = 7.8 Hz, 1H); 4.94 (s, H); 13 C NMR (300 MHz, D2O) δ 174.10, 173.80, 173.39, 107.21, 104.98, 102.92, 82.82, 80.92, 76.57, 75.08, 72.62, 70.69, 68.52, 68.29, 67.43, 66.14, 62.64, 60.90, 60.19, 41.43, 39.31, 39.16, 36.04, 35.78, 35.57, 17.01.
PLL-mono-mannose (17). 3- Maleimidopropionic acid-N-poly-L-Lysine (10 mg, 0.2 µmol) and allyl-galactose (88 mg, 0.4 mmol) were dissolved in H2O and flushed in a continuous flow reactor for 20 min under irradiation (medium pressure Hg Lamp, Hanovia, 450W). The crude product was further purified with sephadex G-10 to yield 10 mg of PLL-mannose (90%). 1H
NMR (300 MHz, D2O) δ 1.14 (t, J = 7.1 Hz, 1H); 1.24 (t, J = 7.5 Hz, 2H); 1.97-1.78 (m, 10H); 2.40-2.36 (m, 1H); 2.65-2.61 (m, 8H); 2.68-2.71 (m, 8H); 2.86 (t, J = 6 Hz, 5H); 3.19-3.13 (m, 1H); 3.33-3.37 (m, 2H); 3.52-3.63 (m, 23H); 3.65-3.78 (m, 29H); 3.79-3.95 (m, 28H); 4.63 (s, 1H); 4.83 (s, 1H); 5.29 (dd, J = 25.0, 13.9 Hz, 2H); 5.84-6.02 (m, 1H); 13C NMR (300 MHz, MeOD) δ 44.80, 45.50, 50.23, 56.83, 71.69, 76.06, 77.09, 83.62, 87.01, 116.44, 116.48.
180 5. Glyco-dendronized Polylysine for Bacteria Detection
5.6.5 Cell Growth and Incubation with Polymer
The strains used in this study were a kind gift donated by Prof. Orndorff (College of Veterinary Medicine, Raleigh, NC United States), ORN178 for the mannose binding strain and ORN208 for the mutant strain that does not bind mannose. Cells were grown in LB media.19 The inoculated culture was incubated overnight at 37°C by shaking on an incubator shaker at 250 rpm. Intermittently, aliquots of bacteria were removed from the batch culture to monitor growth until they reached OD600 of 1.0. Aliquot of 2 ml cultures were then centrifuged for 10 min at 1,600 x g. The resulting pellets were then washed twice with PBS buffer and resuspended in 100 µL PBS. A 10 µg of the polymer was added in each incubation mixture or omitted from the control incubations. Each suspension was then incubated for 30 min at room temperature with gentle shaking and centrifuged to pellet the cells. To remove excess of polymer pellets were softly washed twice with PBS and centrifuged. This procedure was repeated twice for each culture.
5.6.6 Confocal Fluorescence Microscopy
The washed polymer-treated and non-treated bacteria were fixed with 100 µL with 4% (w/v) paraformaldehyde for 15 min. Bacteria were then centrifuged and the pellet resuspended in 100 µL of PBS containing the organic dye 4’,6-diamidino-2-phenylindole dihydrochloride, (DAPI) at dilution of 1:1000 and incubated for 15 min. DAPI forms fluorescent complexes with double-stranded DNA. Alternatively to image the polymers, following DAPI treatment bacteria were centrifuged and the pellet incubated for additional 15 min in the presence of 100 µL PBS containing fluorescein-labelled Concanavalin A (ConA-FITC) at a dilution of 1:1000. Bacteria were then centrifuged, washed twice as described above prior to analysis. Bacteria were imaged using a Carl Zeiss confocal laser scanning microscope (LSM 700) using a 63 x oil immersion or a 40 x objectives.
181 5. Glyco-dendronized Polylysine for Bacteria Detection
The single mannose-PLL was incubated with bacteria ORN178 strain and treated as described above for the dendronized polymer. Bacteria were then imaged with confocal fluorescence microscope, Figure 5.8 shows no aggregation. Diluted solution of bacteria ORN178 expressing mannose binding protein were treated as described for the previous experiments to determine the detection limit of aggregation of bacteria (Figure 5.9). Results show that aggregations are detectable to a limit of 105 of bacteria.
Figure 5.8 Confocal fluorescence microscopy image after incubation of single mannose-PLL (17) with E. coli ORN178 strain. Individual bacteria detected no aggregation.
Figure 5.9 Detection limit aggregation of bacteria ORN178 with nanopodmannose (15). The number of bacteria incubated with PLL-containing solution is indicated above each imagine.
182 5. Glyco-dendronized Polylysine for Bacteria Detection
5.7 References
1 Astruc, D.; Boisselier, E.; Ornelas, C. Chem. Rev., 2010, 110, 1857–1859. 2 Marcos, M.; Martín-Rapún, R.; Omenat, A.; Serrano, J. L. Chem. Soc. Rev., 2007, 36,
1889–1901. 3 Frauenrath, H. Prog. Polym. Sci., 2005, 30, 325–384. 4 Gao, C.; Yan, D. Prog. Polym. Sci., 2004, 29, 183–275. 5 (a) Fréchet, J. M. J.; Tomalia, D. A. Dendrimers and other Dendritic Polymers, John Wiley and Sons, Chichester, 2001. (b) Gingras, M.; Raimundo, J. M.; Chabre, Y. M.
Angew. Chem. Int. Ed., 2007, 46, 1010–1017. 6 Hawker, C. J.; Fréchet, J. M. J. Polymer, 1992, 33, 1507–1511. 7 Yin, R.; Zhu, Y.; Tomalia, D. A.; Ibuki, H. J. Am. Chem. Soc., 1998, 120, 2678–2679. 8 Schlüter, A. D.; Rabe, J. P. Angew. Chem. Int. Ed., 2000, 39, 864–883. 9 (a) Hein, C. D.; Liu, X. M.; Wang, D. Pharmaceutical Research, 2008, 25, 2216–2230. (b) Fournier, D.; Hoogenboom, R.; Schubert, U. S. Chem. Soc. Rev. 2007, 36, 1369–1380. (c) Soares, E. V.; Hebbelinck, K.; Soares, H. M. Can. J. Microbiol. 2003, 49, 336–343. (d) Chen, Z.; Meng, H.; Xing, G.; Chen, C.; Zhao, Y.; Jia, G.; Wang, T.; Yuan, H.; Ye, C.; Zhao, F.; Chai, Z.; Zhu, C., Fang, X.; Ma, B.; Wan, L. Toxicol. Lett., 2006, 163, 109–120. (e) Campos, L. M.; Kilops, K. L.; Sakai, R.; Paulusse, J. M. J.; Damiron, D.; Drockenmuller, E.; Messmore, B. W.; Hawker, C. J. Macromol. 2008, 41, 7063–7070. (f) Connal, L. A.; Kinnane, C. R.; Zelikin, A. N.; Caruso, F. Chem. Mater. 2009, 21, 576– 578. 10 (a) Booker-Milburn, K.; Anson, C. E.; Clissold, C.; Costin, N. J.; Dainty, R. F.; Murray, M.; Patel, D.; Sharpe, A. Eur. J. Org. Chem. 2001, 1473–1482. (b) Hook, B. D. A.; Dohle, W; Hirst P., L.; Pickworth, M.; Berry M. B.; Booker-Milburn, K.M; J. Org. Chem. 2005, 70, 7558–7564. 11 Ting, S. R. S.; Chen, G.; Stenzel, M. H. Polym. Chem. 2010, 1, 1392–1412. 12 (a) Sadik, O. A.; Aluoch, A. O.; Zhou, A. Biosens. Bioelectron. 2009, 24, 2749–2765. (b) Moldovan, C.; Mihailescu, C.; Stan D.; Ruta, L.; Iosub, R.; Gavrila, R.; Purica, M.; Vasilica, S. Applied Surface Science 2009, 255, 8953–8959. (c) Zhao, X.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.; Bagwe, R., P.; Jin, S.; Tan, W. PNAS 2004, 101, 15027–15032. (d) Disney, M. D.; Zheng, J.;Swager, T. M., Seeberger, P. H. J. Am. Chem. Soc. 2004,
183 5. Glyco-dendronized Polylysine for Bacteria Detection
126, 13343–13346. (e) So, H. M.; Park, D. W.; Jeon, E. K.; Kim, Y. H.; Kim, B. S.; Lee, C. K.; Choi, S. Y., Kim, S. C.; Lee, J. O. Small 2008, 197–201. 13 (a) Kikkeri, R.; Liu, X.; Adibekian, A.; Tsai, Y.-H.; Seeberger, P. H. Chem. Commun. 2010, 2197–2199. (b) Kikkeri, R.; Garcia-Rubio, I.; Seeberger, P. H. Chem. Commun. 2009, 235–237. 14 Orive, G.; Tam, S. K.; Pedraz, J. L.; Halle, J. P. Biomaterials 2006, 27, 3691–3700. 15 Doktycz, M. J.; Sullivan, C. J.; Hoyt, P. R.; Pelletier, D. A.; Wu, S.; Allison D. P. Ultramicroscopy 2003, 97, 209–216. 16 Altschuh, D.; Oncul, S.; Demchenko, P. A. J. Mol. Recognit. 2006, 19, 459–477. 17 (a) Kikkeri, R.; Kamena, F.; Gupta, T.; Hossain, L. H.; Boonyarattanakalin, S.; Gorodyska, G.; Beurer, E.; Coullerez, G.; Textor, M.; Seeberger, P. H. Langmuir, 2010, 26, 1520–1523. (b) Evangelista, K.; Andrich F.; Rezende, F. F.; Niland, S.; Cordeiro, M. N.; Horlacher, T.; Castelli, R.; Schmidt-Hederich, A.; Seeberger, P. H.; Sanchez, E. F.; Richardson, M.; Figueiredo, S.; Eble, J. A. J. Biol. Chem., 2009, 284, 34747–34759. 18 Upchurch Scientific, USA 19 Harris, S. L.; Spears, P. A., Havell, E. A.; Hamrick, T. S.; Horton, J. R.; Orndorff, P. E. J. Bacteriol. 2001, 183, 4099–4102.
184 List of Abbreviations
List of Abbreviations
Ac Acetyl ESI Electron spray ionization
aq Aqueous Et Ethyl
arom Aromatic FEP Fluorinated Ethylene
BMA Butylmethacrylate Propylene
br Broad FT Fourier Transformation
brs Broad singlet Gal galactose
c Concentration Glc Glucose
δ Chemical shift GlcN Glucosamine
d Doublet GPC Gel Permeation
dd Doublet of doublets Chromatography
ddd Double douplet of h Hour(s)
douplets HDA Hexadecylamine
dddd Double douplet douplet HRMS High Resolution Mass
of douplets Spectroscopy
ddt Double douplet of triplets Hz Hertz
DHLA Dihydrolipoic acid IR Infrared Spectroscopy
DMF N,N-Dimethylformamide J Coupling constant
DMSO Dimethylsulfoxide m Multiplet
DP Degree of polymerization M Molar
dt Doublet of triplets MALDI Matrix Assisted Laser
equiv Molar equivalents Desorption/Ionisation
185 List of Abbreviations
Me Methyl TEM Transmission Electronic min Minute(s) Microscope
MMA methylmethacrylate THF Tetrahydrofuran
Mn Molecular weight tlc Thin layer chromatography
MS Mass spectroscopy TMS2S Hexamethyldisilathiane
Mw Molecular weight TOF Time of flight m/z Mass to charge ratio TOP Trioctyl phosphine
NMR Nuclear Magnetic TOPO Trioctyl phosphine oxide
Resonance tt Triplet of triplets
PEG Polyethylene Glycol UV Ultraviolet
Ph Phenyl VA Vinylacrylate ppm Parts per million Vis Visible
PTFE Poly(tetrafluoroethylene) ZnEt2 Diethylzinc q Quartet
QDs Quantum Dots quint. Quintett rt Room temperature s Singlet s Second(s)
SEM Scanning Electron
Microscope
STY Stryrene t Triplet
T Temperature (°C)
186 Curriculum Vitae
Name: Paola Laurino Contact address: Arnimallee 22 Date of Birth: 01.04.1981 14195 Berlin, Germany Nationality: Italian Phone: +49-30-83859562 Email: [email protected]
Languages
Italian: Native language English: Fluent speaking, reading and writing German: Basic knowledge Spanish: Basic knowledge
Education and Research Experience
Since 10/2007 PhD studies, Department of Organic Chemistry ETH Zurich, Switzerland and Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Germany Research advisor: Professor P. H. Seeberger
06/2006-09/2007 MPhil, Department of Medicinal Chemistry University of Leiden, The Netherlands Research advisor: Professor A. IJzerman Project: Photoaffinity probes for adenosine A1 receptor.
9/2000-03/2006 Degree (Laurea) in Pharmaceutical Chemistry and Technology Department of Medicinal Chemistry, Pharmacy Faculty, University of Milan, Italy Research advisor: Professor E. Valoti
Thesis: Design, Synthesis and Characterization of antagonists for α1 adrenergic receptor.
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Publications
1. Laurino, P.; Tauer, K.; Hernandez, H.; Grützmacher, H.; Seeberger, P. H. “Ultrafast Photo-initiated Polymerization in Continuous Flow Reactor” Under preparation.
2. Laurino, P.; Hernandez, H.; Bräuer, J.; Grützmacher, H.; Tauer, K.; Seeberger, P. H. “Snowballing Radical Generation Leads to Ultrahigh Molecular Weight Polymers” 2011, Submitted.
3. Azzouz, N.; Kamena, F.; Laurino, P.; Kikkeri, R.; Mercier, C.; Cesbron-Delauw, M-F.; Dubremetz, J-F.; De Cola, L.; Seeberger, P. H. “Toxoplasma gondii secretory proteins bind to sulfated heparin structures” 2011, Submitted.
4. Laurino, P.; Kikkeri, R.; Azzouz, N.; Seeberger, P. H. “Detection of Bacteria Using Glyco-Dendronized Polylysine Prepared by Continuous Flow Photofunctionalization” Nano Lett. 2011, 11, 73–78.
5. Laurino, P.; Kikkeri, R.; Seeberger, P. H. “Continuous Flow Reactor Based Synthesis of Carbohydrate Capped Quantum Dots” Nat. Prot. 2011, In press.
6. Christina, D.; Laurino, P.; Azzouz, N.; Seeberger, P. H. “Accelerated Continuous Flow RAFT polymerization” Macromol. 2010, 43, 10311–10314.
7. Bernardes, G. J. L.; Kikkeri, R.; Maglinao, M.; Laurino, P.; Collot, M.; Hong, S. Y.; Lepenies, B.; Seeberger, P. H. “Design, synthesis and biological evaluation of carbohydrate functionalized cyclodextrins and liposomes for hepatocyte-specific targeting” Org. Biomol. Chem. 2010, 21, 4987–4996.
8. Kikkeri, R.; Laurino, P., Odedra, A.; Seeberger, P. H. “Synthesis of carbohydrate- functionalized quantum dots in microreactors”. Angew. Chem. Int. Ed. Engl. 2010, 49, 2054–2057.
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9. Laurino, P.; Odedra, A.; Mak, X. Y.; Gustafsson, T.; Geyer, K.; Seeberger, P. H. “Microfluidic devices for organic processes”. Chemical Reactions and Processes under Flow condition (RCS publishing 2010).
10. Mak, X. Y.; Laurino, P.; Seeberger, P. H. “Asimmetric reactions in continuous flow”. Beilstein J. Org. Chem. 2009, 5, 1–11.
11. Mak, X. Y.; Laurino, P.; Seeberger, P. H. “Synthesis of pharmaceutical and bio- active compounds in microreactors” Chemistry Today 2009, 27, 15–18.
12. Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger P. H. “In vitro imaging and in vivo liver targeting with carbohydrate capped quantum dots”. J. Am. Chem. Soc. 2009, 131, 2110–2112.
Patents
1. Seeberger, P. H.; Grützmacher, H.; Tauer, K.; Laurino, P.; Bräuer, J. “Ultra fast process for the preparation of polymer nanoparticles” Submitted.
2. Seeberger, P. H.; Grützmacher, H.; Tauer, K.; Laurino, P.; Bräuer, J. “Process for the modification of polymer nanoparticles” Submitted.
3. Seeberger, P. H.; Laurino, P.; Kikkeri, R. “Continuous Flow Photoconjugation” In preparation.
Poster Presentations
1. “Synthesis of Monodisperse Latex Particles”, IPCG conference. Il Ciocco, Italy. July 2009.
2. “Photochemical Reactions in Microreactor” at Industry Day “Micro and Nano Science – From Ideas to Innovations” ETH Zurich, Switzerland. 8th May, 2008.
189 3. “Photochemical Reactions in Mircoreactor” at 4th Workshop “Microtechnology for Chemistry and Biology Laboratories” Technische Universität Ilmenau, Germany. February 2008
Courses Attended
1. “Enzymes”, Professor D. Hilvert. Autumn Semester 2008, ETH Zurich, Switzerland.
2. “Advanced Methods and Strategies in Asymmetric Synthesis”, Professor E. Carreira. Autumn Semester 2008, ETH Zurich, Switzerland.
3. “Biomaterial Surfaces: Properties and Characterization”, Professor M. Textor. Autumn Semester 2008, ETH Zurich, Switzerland.
4. 2008 Annual course SPERU “Colloidal Chemistry for Materials Science”, Competence Centre for Materials Science and Technology, Luzern, Switzerland. 30-31 October 2008.
5. Short Course “Photochemistry of Materials” von Stefan Berndhard, Princeton, USA. Universität Zürich, Switzerland. November 2008
Teaching experiences
1. Spring Semester 2007/2008 Teaching assistant, Course of Organic Chemistry II. ETH Zurich, Switzerland. 2. Autumn Semester 2008/2009 Lab assistant, Praktikum Organic Chemistry I. ETH Zurich, Switzerland.
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