UvA-DARE (Digital Academic Repository) Supramolecular control of selectivity in transition-metal catalysis through substrate preorganization

Supramolecular chemistry exploits multiple weak intermolecular interactions to assemble nano-sized molecular architectures, providing new possibilities for (transition metal) catalyst development. In this Perspective we focus on the application of such weak (directional) interactions between a substrate molecule and a (bifunctional) catalyst for structural preorganization prior to the catalytic reaction. As we discuss, such e ﬀ ects together with the con ﬁ nement properties of the nano-space of the ‘ active sites ’ play a crucial role for the exceptional selectivities and activities of natural enzymes. We will elaborate on the application of such supramolecular strategy to the more traditional transition-metal catalysis

Supramolecular control of selectivity in transitionmetal catalysis through substrate preorganization Paweł Dydio and Joost N. H. Reek * Supramolecular chemistry exploits multiple weak intermolecular interactions to assemble nano-sized molecular architectures, providing new possibilities for (transition metal) catalyst development. In this Perspective we focus on the application of such weak (directional) interactions between a substrate molecule and a (bifunctional) catalyst for structural preorganization prior to the catalytic reaction. As we discuss, such effects together with the confinement properties of the nano-space of the 'active sites' play a crucial role for the exceptional selectivities and activities of natural enzymes. We will elaborate on the application of such supramolecular strategy to the more traditional transition-metal catalysis, and we will compare it with the traditional substrate preorganization methods. Subsequently, literature examples of such bifunctional catalyst systems will be described in which the function of weak interactions was carefully designed a priori, as well as, the serendipitously found catalysts in which the presence of supramolecular effects was recognized post factum. The discussed examples demonstrate the power of the strategy for the control of selectivity in various types of metal catalyzed reactions, and the observation of the serendipitous findings can help to generate new leads for more efficient catalyst design.
Paweł Dydio (1985) studied Natural Sciences and Mathematics at the University of Warsaw, Poland (2004-2009 in Chemistry (summa cum laude). While at UW, he did undergraduate research in supramolecular chemistry with Professor Janusz Jurczak. Subsequently, he joined the group of Professor Joost Reek at the University of Amsterdam, the Netherlands, and working on the discovery and development of supramolecular strategies in homogeneous transition-metal catalysis he completed his PhD (cum laude) in 2013. He is currently a postdoctoral researcher in the group of Professor John Hartwig at the University of California Berkeley, USA.
Joost Reek (1967) completed his PhD in Nijmegen in 1996 in the area of supramolecular chemistry with Prof. Nolte. Aer a postdoctoral fellowship in Sydney, Australia, with Prof. M. Crossley, he joined the group of Prof. van Leeuwen at the University of Amsterdam as an assistant professor in 1998, with his research activities focusing on transition-metal catalysis. In 2005 he was elected to become a member of The Young Academy of Science (KNAW). In 2006 he became full professor (chair in supramolecular catalysis). He has received various prestigious grants, including VICI (2006) and an ERC advanced grant (2013). In 2009 he also started a spin-off company, InCatT, to explore the commercial potential of new supramolecular strategies for combinatorial approaches to nd catalysts for (asymmetric) chemical transformations. In 2013 he became an elected member of Royal Holland Society of Sciences and Humanities (KHMW), and scientic director of the van 't Hoff institute for molecular sciences. His current research activities include transition-metal catalysis, supramolecular catalysis and catalysis for green energy applications.

Introduction
Rapidly rising consumption of goods due to the ever increasing human population and the rise of living standards put enormous demands on the natural resources of the planet, which, if explored at the current rate, are expected to run out in the near future. This state of affairs calls for devoting more attention towards renewable resources and for using our current reserves of natural resources more efficiently. This requires the development of new technologies that are more environmentally friendly, and if possible also more economically efficient and as such attractive for industrial application. Catalysis is a key tool to control chemical transformations, especially in industry, and is therefore of crucial importance for sustainable development. 1 The central role of (transition metal) catalysts is to accelerate reactions that are otherwise very slow or even impossible, primarily by creating new reactivity pathways via a combination of simple elementary steps at the metal center. 2 Therefore, it can give access to new transformations and open up otherwise inaccessible, yet effective, shortcuts relevant to current synthetic schemes. In addition, on the long term new catalytic routes will be crucial for the conversion of renewable building blocks, replacing fossil-fuel-based materials.
Homogeneous transition-metal catalysis offers powerful and straightforward methods for carrying out selective and effective chemical transformations, 3 and as such this research eld attracts considerable academic attention that has resulted in tremendous progress in the last few decades. For transitionmetal catalysts the activity, selectivity (and the stability) is highly dependent on the ligands coordinated to the catalytic metal center. 4 Notable insights in various reaction mechanisms including the role of the ligands, has been obtained. Computational methods become increasingly sophisticated, and yet the ab initio design of a selective catalyst for most reactions with substrates of interest is still beyond our abilities. Therefore, the search for catalysts typically still involves the knowledge-supported trial-and-error screening of candidate systems. Oen, the screening of catalysts based on privileged ligands 5those that have been proven to provide good selectivities (and activities) for a broad range of reactions and substratesis a good starting point. If required, further structure optimization of the catalyst follows to nally obtain the catalyst with desired properties. This approach has provided many successes, but is not a general strategy that always results in success. There are reactions for which selectivity issues are difficult to solve with this approach; reactions, for example, in which the pathway to the desired product is higher in energy than the alternative pathways, or reactions with many competing pathways. Therefore, complementary approaches that allow for a more rational catalyst design for these challenging reactions would be of high value. Supramolecular chemistry describes chemical systems formed by a self-assembly of a number of molecular building blocks or components via reversible, relatively weak interactions. 6 Enormous progress in this eld has been achieved over the past few decades resulting in good understanding and insight into these weak, yet effective interactions that allow for formation of large self-assembled molecular architectures. 7 Therefore, supramolecular chemistry can provide powerful new tools for catalyst development and has attracted considerable attention in the last few years, giving rise to a group of strategies collectively called 'supramolecular catalysis'. 8 Much attention has been devoted to supramolecular capsules 9 the interior of which mimics the nite microenvironments of natural enzymes. 10 Such synthetic cavities can be used as 'molecular asks' inside of which the reactions experience a series of 'connement effects' that are otherwise not present in the bulk. 11 This approach has resulted in a number of elegant examples of capsule-driven reactions that display enhanced selectivity and/or activity. 11,12 For example, the Diels-Alder reactions within the octahedral coordination capsules, developed by 12d,13 lead to products that are not formed otherwise in these reactions in the bulk, demonstrating the capsule-directed selectivity effect. 14,15 Raymond and co-workers showed that the rate of the acid-catalyzed hydrolysis reaction of orthoesters is increased dramatically by the special microenvironment of the cage, and occurs even in a basic reaction medium, 16 demonstrating the potential of the strategy for the rate enhancement. Supramolecular interactions are also widely used to construct "supramolecular bidentate ligands" from easily accessible monodentate components that are programmed to self-assemble in solution. 17 The strategy gives easy access to wide libraries of ligands, particularly suitable for catalyst development based on high throughput screening and combinatorial methods. These methodologies are already wellmatured, complement traditional approaches, and in principle are ready to tackle practical problems for homogeneous catalysis. 18 Besides such approaches for which the supramolecular tools are used just to construct the catalysts from simpler subcomponents by self-assembly, recent breakthroughs demonstrate that application of supramolecular interactions between a catalyst and a substrate can be extremely powerful. Considering that the selectivity of catalytic reactions is determined by energy differences in the competing transition states that are as small as 3 kcal mol À1 , one can envision that the relatively weak supramolecular interactions can be used to change the selectivity of any catalytic transformation. In such approach the aim is no longer to affect the catalyzed reaction by just modifying secondary coordination sphere of the catalyst, but supramolecular chemistry tools are applied to control directly the chemical transformation taking place at the metal center via precise positioning of the substrate molecule. In this Perspective we will highlight some of the recent examples to demonstrate the power of this strategy.

Confinement effects and active substrate preorganization in natural enzymes
reaction takes place is typically buried within a specic proteomic microenvironment. Therefore, a substrate that is brought into a conned space of this 'cavity' experiences a series of 'connement effects' that are otherwise not present in the bulk. 10 For instance, the encapsulated substrate molecule can accept only specic conformations imposed by the size and shape of the cavity that also limit its motion, and restricting the number of possible reactions. This usually also results in reduced activation entropy of a reaction. The proximity and orientation of the reactive groups is usually restricted, which in turn determines the reaction selectivity. In some cases, the substrate or the reaction intermediate is forced to adopt a highenergy conformation required for increased reactivity, 19 which can effectively lower the free-energy reaction barrier, accelerating the reaction. Besides, the transition state of the effective reaction pathway can be stabilized more efficiently than the substrate in its ground state by the surrounding cavity, decreasing the overall energy barrier of the reaction. Mimicry of such effects was intensively explored with synthetic supramolecular capsules. This brought a number of elegant capsular catalysts that display unusual capsule-driven selectivity and/or enhanced activity in the catalyzed reactions. 11,12 In natural enzymes, however, aside from the connement effects intrinsic to the nano-space of the active sites, other factors oen play an important role in controlling the selectivity and the activity of the catalyzed reaction. Frequently the positioning of the substrate is determined not only by the size and shape of the enzymatic cavity, but also by directional interactions between functional groups of the substrate and of the active site, such as hydrogen bonding, ionic interactions or even reversible covalent bonding. 10 This ensures precise substrate orientation and dramatically limits its motion, both of which are essential for obtaining high selectivity. Importantly, this operational mode is less susceptible to changes in substrate size (as long as a substrate molecule bears the essential functional groups), and allows for selective transformations of a broad substrate scope. In contrast, substrate orientation based exclusively on shape and size should in principle be more substrate dependent, as is seen, for instance, for the Diels-Alder reactions within the cage-type catalysts. 14,15 Furthermore, such interactions with the functional groups of the substrate can substantially modulate their reactivity allowing for even better control over the selectivity and the activity of the reaction. Importantly, these directional interactions are well-understood and hence can be precisely manipulated, allowing for the rational design of the 'active sites' of such synthetic mimics of enzymes.

Traditional vs. bio-inspired supramolecular substrate preorganization
In traditional catalysis, substrate preorganization is realized via coordination of a 'directing group' to a metal center (Scheme 1). 20 The directing group can be either a functional group present in the substrate or it is temporarily introduced before the catalytic reaction. Although effective, this methodology is limited to substrates with directing groups spatially close to the reactive functionality, imposing limitations. Moreover, the reaction occurring at the metal center must be compatible with the directing group, and also requires one vacant coordination site at the metal center, which cannot be realized for all reactions.
Supramolecular chemistry provides ideal tools for substrate preorganization. In transition-metal catalysis, substrate preorganization can be realized by the supramolecular interactions between the 'directing group' of the substrate and a suitable bifunctional ligand from the catalyst (Scheme 1). In principle, a catalyst that is furnished with a specic recognition site will bind the substrate molecule and preorganize its reactive moiety with respect to the metal center. The location of the directing group and the reactive functionality are theoretically independent. This makes it possible for the strategy to be applied to substrates with directing groups in various positions of the molecule, allowing for remote control, and exceeding the possibilities of the traditional substrate preorganization approach. Thanks to a well-established understanding of weak interactions between molecules, the rational design of selective catalysts that operate predictively can be envisioned. The methodology is especially attractive for reactions for which the selectivity issues are notoriously difficult to solve with the traditional trial-and-error approach, e.g. reactions for which the pathway to the desired isomer is higher in energy than the alternative one or for which many reaction pathways compete simultaneously.
3.1 Non-covalent substrate preorganization 3.1.1 Predesigned bifunctional catalyst systems. Rich chemistry of molecular receptors operating with non-covalent interactions provides a full toolbox of potential recognition sites, that can be used directly for the development of catalyst, realizing supramolecular substrate preorganization strategy. In this vein, a catalyst that mimics the hydroxylating cytochrome P-450 enzyme was developed. 21 The Mn III -porphyrin 1 was functionalized with a cyclodextrin (CD) moiety on each of its meso-phenyl rings, and it was shown that the CDs bind to the hydrophobic groups of steroid derivative 2 (Scheme 2). The substrate binding precisely positions the steroid moiety on the manganese porphyrin such that its C6 a position is exposed to the catalytic center, resulting in highly regioselective hydroxylation of this CH function (regioselectivity >90%). Importantly, the porphyrin performs 187 turnovers and is not blocked by product inhibition. A substrate with different anchoring groups, which thus binds in a different geometry, reacts selectively at a different position (C9a). In further studies, it was shown that metalligand interactions can be also used for precise substrate preorganization. 22 In another study, another Mn-catalyzed, selective C-H oxidation at sp 3 -carbon atoms was targeted. In this case nonporphyrin system 3 was devised, which consists of a di-m-oxo dimanganese corea very active catalyst for oxidation chemistrybased on ligand 4 equipped with Kemp's triacid, a recognition site for carboxylic acids (Fig. 1). 23 Molecular modeling predicts that the catalyst binds a molecule of ibuprofen (5) in such a way that the benzylic proton of the more remote position is precisely oriented towards the catalytic manganese center (Fig. 1). As predicted by the model, the catalyst oxidizes ibuprofen at this position with very high regioselectivity (98.5%, Scheme 3). In contrast, the reaction with a catalyst devoid of the COOH recognition site leads to a mixture of products. Impressively, catalyst 3 can also oxidize (4-methylcyclohexyl)acetic acid 6 selectively (>99%) at the C(4)-H position, as predicted by the modeling. Interestingly, only the trans isomer of 6 reacts readily, since the cis isomer does not expose an oxidisable C-H group to the catalytic center when its COOH is bound to the recognition site. In a control experiment with the catalyst devoid of the COOH recognition site both isomers react similarly, and the reaction leads to a mixture of several oxidation products. This system shows that, along with providing an unprecedented level of reaction selectivity, bifunctional catalysts can allow for efficient substrate selection, including challenging diastereoselection.
In a subsequent study, the same catalyst and an analoguea manganese porphyrin catalyst furnished with the same Kemp's triacid recognition sitewere used as catalysts for selective epoxidation of alkenes containing a carboxylic group. 24 For two out of three substrates studied, molecular recognition directs oxidation to the olen moiety and prevents the unselective oxidation of C-H bonds observed in control experiments. However, poor diastereoselectivity is observed for the epoxide products, which on the basis of molecular modeling is attributed to the ill-dened preference between alternative orientations of the reactive double bond in the catalyst-substrate complex.
In a related study, a Ru(II)-porphyrin 7 equipped with a chiral tricyclic g-lactam that can form a complementary twopoint hydrogen bonding with the CONH amide functional group was synthesized (Fig. 2). 25 Molecular modeling showed Scheme 2 Mn-catalyst 1 with attached cyclodextrins, and selective hydroxylation of steroid 2 catalyzed by 1. that this two-point interaction orients 3-vinylquinolone (8) at the catalyst (Fig. 2) such that one prochiral site of the reactive double bond is exposed to the catalytic center preferentially due to steric requirements. As predicted by the model, substrate preorganization leads to high enantioselectivities (ee's up to 98%) in epoxidation for a series of 3-vinylquinolones. The epoxidation of substrate 9 with two reactive sites, 3,9-divinylquinolone, takes places with high regioselectivity on the vinyl group in position 3 (91% vs. 9% of the alternative product, Scheme 4). This selectivity is in line with the proposed mechanistic model, which predicts that only the vinyl group in position 3 can react at the catalytic center when the substrate is bound to the recognition site. The reaction with the same substrate, but with a catalyst devoid of the recognition site, leads to the two alternative products in a 62/38 ratio, revealing the crucial role of substrate orientation for the selectivity. The importance of the double hydrogen bonding was further conrmed by control reactions with either the N-methylated substrate or catalyst, which leads in both experiments to drastic deterioration of the selectivity. Studies on the substrate scope show that other substrates, such vinylpyridones, primary alkenoic amides and carbamates also experience the effect of the preorganization. However, a clear trend can be noted whereby the enantioselectivity gradually drops with increasing exibility of the substrate. The distance between the catalytic metal and the chiral groups is over 7.0Å, showing that the remote selectivity control via supramolecular interactions is viable. In a subsequent study, the same recognition site was used to preorganize sulde quinolone-type derivatives on a Mn(III)salen catalyst, resulting in an enantioselective sulfoxidation reaction (ee's up to 71%). 26 Again, control experiments conrmed the crucial role of the two-point hydrogen bonding in the prochiral face differentiation.
Recently it was also reported that a Rh(II) complex bearing the same two g-lactam binding sites catalyzes the regio-and enantioselective (ee's up to 74%) C-H amination reactions of 3-benzylquinolones. 27 It was conrmed that both the regio-and enantioselectivity of the reaction is controlled by substrate positioning imposed by the hydrogen bonding.
The chiral tricyclic g-lactam binding site was also used to preorganize prochiral substrates at photosensitizers (in metalfree reactions). This enabled unprecedented induction of enantioselectivity in the photoinduced conjugate additions of a-amino alkyl radicals to enones, 28 as well as in intra-and intermolecular [2 + 2] photocycloaddition reactions. 29,30 In another study, regioselectivity issues in hydroformylation reactions were addressed with phosphine ligand 10 bearing a guanidine-based recognition unit that is able to bind unsaturated carboxylic acids via two-point hydrogen bonding (Scheme 5). 31 The substrate binding results in increased activity and high regioselectivity for hydroformylation of 3-alkenoic acids 11-13. Remarkably, the system displays unprecedented high regioselectivity for hydroformylation of an internal alkene 12, introducing the aldehyde group with high selectivity at the carbon atom of the double bond more distant from the carboxylic moiety. In addition, the catalyst can discriminate between two similar reactive double bonds of substrate 13 by substrate preorganization (Scheme 5). The crucial role of binding to the recognition unit of the ligand was demonstrated by a series of control experiments with substrates or catalyst derivatives with Scheme 4 Epoxidation products of substrate 9 with 7 as catalyst.
Scheme 5 Binding of substrate 11 to ligand 10 in the hydride migration step of hydroformylation (above), and hydroformylation products of substrates 11-15 with Rh-10 catalyst (below). modied ligands that cannot form the supramolecular interaction. The system is highly susceptible to the relative position of the directing carboxylic group and the double bound, as the hydroformylation of an analogue of 11 that is one carbon longer (4-pentenoic acid 14) leads to a typical mixture of isomeric products. 2-Alkenoic acid 15 undergoes 2-regioselective decarboxylative hydroformylation, resulting in formation of the linear aldehyde, thus formally substituting the carboxylic group with an aldehyde (Scheme 5). 32 Furthermore, a related guanidiniumbased system was used for aldehyde hydrogenation and for tandem hydroformylation-hydrogenation of terminal olens, again with a crucial role for the hydrogen bonding functional groups. 33 Highly regioselective hydroformylation of unsaturated acids was also achieved with a series of bidentate phosphorus ligands -DIMPhos ligandsthat are equipped with an integral binding site for anions such as carboxylates (the DIM pocket). 34 It was shown that under hydroformylation conditions the ligands bind to a rhodium center in a bidentate fashion forming the typical rhodium-hydride complexes that are active in hydroformylation. Importantly, the metal complexes can strongly bind the anionic species in the DIM binding site of the ligand, without affecting the metal coordination sphere. The catalytic studies demonstrate that substrate preorganization results in unprecedented selectivities in hydroformylation of a broad range of terminal and internal alkenes functionalized with an anionic carboxylate or phosphate group (Scheme 6). Remarkably, the selectivity controlling anionic group can be even 10 bonds away from the reactive double bond, demonstrating the power of the supramolecular substrate orientation approach. Control experiments, in which formation of the hydrogen bonding is hindered, conrm the crucial role of the substrate binding to the DIM pocket for the selectivity. DFT studies reveal that the substrate binding in the DIM pocket restricts the rotational freedom of the reactive double bound (Fig. 3). As a consequence, the pathway to one product is strongly hindered, whereas that for the desired product is lowered in energy. Detailed kinetic studies, together with the in situ spectroscopy and the isotope-labelling studies support this mechanistic model and reveal that the supramolecular system follows enzymatic-type Michaelis-Menten kinetics, with competitive product inhibition. The system was further combined with an isomerization catalyst in the one-pot tandem isomerization-hydroformylation of terminal olens leading to valuable a-methyl-branched aldehyde products with unprecedented selectivity. 35 In a subsequent study, the DIMPhos catalyst system was used to control the regioselectivity in the hydroformylation of vinyl aryl derivatives. 36 Common hydroformylation catalysts generally produce the a-aldehyde products, due to the electronic properties of this class of substrate, and catalysts that overrule this natural selectivity to form preferably the linear aldehyde are scarce. 37 Remarkably, supramolecular substrate preorganization in the DIM pocket of the catalyst fully reverts the regioselectivity, and the b-aldehyde isomer is formed exclusively for substrate 30 (Scheme 7a). In sharp contrast, if the hydrogen bonding interaction is disrupted, for example in case of O-methylated substrate 31, the a-aldehyde is formed as the notably major product (95%), along with only a minor quantity of the b-isomer (Scheme 7b). Of note, substrate 30 reacts several times faster than ester analogue 31, demonstrating the effect of substrate preorganization on the rate of the reaction. The DIMPhos catalyst proved to be selective for a wide scope of vinyl arene substrates, including the most challenging substrates with an internal double bond. Importantly, this unprecedented selectivity opens new synthetic routes to valuable common intermediates for important synthetic targets, showing the added value of the supramolecular chemistry to the practical synthesis of ne-chemicals.

Scheme 6
Binding of substrates to Rh-DIMPhos catalyst in the hydride migration step of hydroformylation (above), and hydroformylation products of substrates 16-29 with Rh-DIMPhos catalyst (below). Inspired by the cooperativity displayed by metalloenzymes such as carboxypeptidase, capable of peptide bond hydrolysis, highly active and selective bifunctional catalysts 32 for anti-Markovnikov hydration of terminal alkynes were developed (Scheme 8). 38 The full reaction mechanism has not yet been revealed, however, studies so far have shown the crucial role of pendant basic functional groups that take part in proton transfer and hydrogen bonding in the reaction intermediate. 39 3.1.2 Serendipitous ndings of bifunctional catalysts.
The previous examples demonstrate the general potential of catalysts that are carefully designed to control the selectivity of reactions via craed catalyst-substrate interactions. However, in many other cases the importance of non-covalent interactions on the catalyst selectivity was realized post factum. These serendipitous ndings suggest that non-covalent interactions play a crucial role in the selectivity control much more oen than it was previously assumed. This observation can help to generate new leads for catalyst design. For example, during the search for enantioselective catalysts for the hydrogenation of 3-hydroxy-2-methylpropionate to the Roche esteran important synthon for the synthesis of bioactive chemicalsit was found that the self-assembled bidentate ligand consisting of LEUPhos phosphoramidite and urea-phosphine furnishes a highly selective catalyst (99% ee). 40 Experimental and theoretical studies revealed that an additional single hydrogen bond between the substrate's OH function and the ester group of LEUPhos ligand is crucial for high selectivity (Fig. 4). Subsequently, related observations were made for other systems for the Rh-catalyzed enantioselective hydrogenation of prochiral alkenes. 41 Similarly, an additional hydrogen bond between the substrate and the catalyst's componentcofactorappears to mediate the enantio-induction in the hydrogenation reaction catalyzed by the catalyst-cofactor supramolecular assembly ( Fig. 5 and 6). 42 In principle, as shown by control experiments and DFT studies, a network of ve hydrogen bondsone between the substrate and the cofactor, and four between the cofactor and the ligandis required to achieve high enantioselectivity in this reaction. An effect of hydrogen bonding on reaction selectivity was also observed in the Co-catalyzed cyclopropanation reaction. 43 Recently, it was found that weak arene C-H/O hydrogen bonding between the substrate and the ligand plays an essential role in determining the enantioselectivity in the Pd-catalyzed arylation and vinylation of lactones (Scheme 9). 44 Based on this observation, a series of new ligands with better hydrogen bonding moieties was designed, which allowed to further improve the selectivity of that reaction. Furthermore, the hydrogen bonding between the substrate and the ligands was proposed to activate allylic alcohols for the Pd-catalyzed direct allylation of indoles, 45 and water molecules for the Ru-catalyzed hydration of nitriles. 46 In the search for a selective (and active) catalyst for challenging allylic alkylation of indoles with unsymmetrical 1,3disubstituted allyl acetates, P,S-bidentate ligands with an additional sulnyl group were explored. 47 It was found that the additional S]O moiety forms a hydrogen bond with the indole-NH, and presumably helps to orient the nucleophile during the reaction (Scheme 10), resulting in a high level of regio-and Scheme 7 Influence of substrate preorganization: (a) hydroformylation of substrate 30 that binds to the DIMPhos ligand, and (b) hydroformylation of substrate 31 that cannot bind to the DIMPhos ligand (both with Rh-DIMPhos as catalyst).
Scheme 8 Anti-Markovnikov hydration of terminal alkynes with bifunctional catalyst 32.  enantioselectivity (ee's 84-95%). This interaction seems to be crucial not only for the selectivity but also for the activity, as the reaction with N-substituted indole, which cannot form this interaction, does not take place. This successful example of a cooperative catalyst should encourage application of bifunctional ligands in catalyst discovery.

Covalent yet reversible substrate preorganization
The examples presented so far have primarily shown supramolecular substrate preorganization accomplished mainly through hydrogen bonding. However, in principle, any reversible bonding that does not interfere with either the metal center or the catalytic reaction can be used for that purpose. Reversible covalent bonding is an attractive tool to control selectivity of a reaction by modifying substrates with covalently attached ligand-type directing groups (e.g. diphenylphosphinobenzoate group). 20a Reversible bonding allows for the use of only catalytic amounts of these kinds of directing groups, improving the applicable value of the approach (from an atom-efficient and a commercial point of view).
To illustrate the potential of reversible covalent bonding, catalytic scaffolding ligands have been introduced and extensively studied in the Rh-catalyzed hydroformylation of unsaturated alcohols and sulfonamides (Scheme 11a and b). 48 In principle, under optimized conditions the scaffolding ligands extensively exchange with hydroxyl functionalities of substrates. Under catalytic conditions, the formed ligand-substrate composite reacts preferentially over the free substrate, since the ligand donor moiety coordinates to the metal bringing the reactive double bond near the catalytic center. Aer the transformation, the product formed is exchanged for a new substrate, which subsequently reacts. This efficient substrate preorganization allows for highly regio-, diastereo-and enantioselective reactions of various terminal and internal alkenes bearing either hydroxy or sulfonamide groups. 48 The strategy also facilitates hydroformylation of challenging tertiary carbon atoms that are difficult to convert otherwise, highlighting the power of the approach. Of note, the concept was previously developed to facilitate ortho-selective C-H activation and functionalization of phenol and aniline derivatives 49 and hydrogenation of acrylic acid derivatives (Scheme 11c and d). 50 The later study shows that scaffolding ligands allow for unprecedented (yet moderate) initial selectivity for a reaction at the C(2)]C(3) double bond in hydrogenation of 2,4-hexadienoic acid, while a control reaction without the scaffolding ligand shows the initial preference for the reaction at the C(4)]C(5) double bond (Scheme 11e).

Conclusions and outlook
In recent years the eld of supramolecular catalysis has grown signicantly, providing new tools for selective transformations. Whereas initial focus was mainly on the mimicry of enzymes, generally also exploring reactions that are important in nature such as hydrolysis, currently the attention has shied to the exploration of supramolecular tools in transition-metal catalysis. The concept of substrate organization, which is indeed also important for the operational mode of enzymes, has been broadly explored for transitionmetal catalysis, and is the main topic of this Perspective. Supramolecular substrate preorganization was demonstrated to be key in a number of highly selective catalytic transformations. Importantly, for several examples, such supramolecular bifunctional catalysts provide unprecedented reaction selectivities, with reaction rates that are sufficiently high for practical applications. Hence, supramolecular substrate preorganization with bifunctionalized catalysts clearly outlines a strategy that may quickly lead to applications in industrial synthesis. As in principle, substrate orientation at the catalyst is predictable, the design of selective catalysts for specic substrates of interest is at hand. However, further mechanistic studies are required to fully understand the principles that rule the reactions carried out with such supramolecular catalysts. This will further help researchers to take full advantage of the concept and will facilitate the step to practical applications. Such studies will substantially strengthen the potential of the approach, and we foresee a bright future for supramolecular transitionmetal catalysis in which substrate orientation directs the selectivity in these transformations.


extfieldsofstudy: Chemistry Medicine

text:
Regioselective synthesis, characterization, and antimicrobial activities of some new monosaccharide derivatives.

A regioselective acylation series of methyl α-D-glucopyranoside (1), methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-mannopyranoside (1A), and methyl 4,6-O-benzylidene-2-O-(3,5-dinitrobenzoyl)-α-D-mannopyranoside (1B) has been carried out by the direct acylation method and afforded the 2,6-di-O-glucopyranoside and 2 or 3-O-mannopyranoside derivatives in an excellent yield. In order to obtain newer products, the 2,6-di-O-glucopyranoside derivative was further transformed to a series of 3,4-di-O-acyl derivatives containing a wide variety of functionalities in a single molecular framework. The structures of the newly synthesized compounds were elucidated on the basis of IR, (1)H-NMR, (13)C-NMR, (13)C-DEPT spectral data, and elemental analysis. These synthesized derivatives were screened for in vitro antimicrobial activities against ten human pathogenic and five phytopathogenic microorganisms. A number of test compounds showed remarkable antimicrobial activity comparable to, and in some cases even higher than, the standard antibiotics employed. It was observed that methyl 3,4-di-O-(3-chlorobenzoyl)-2,6-di-O-hexanoyl-α-D-glucopyranoside (8) exhibited a varied range of MIC from 12.5 μg/disc to 25 μg/disc by the disk diffusion method and 1000 μg/mL to 1250 μg/mL by the broth macrodilution method.

Introduction
Carbohydrate chemistry is now found in the field of organic synthesis, protein and nucleic acid chemistry, enzymology, antibiotics, immunology, and biotechnology. Carbohydrates also play an important role in our industrial development and many industries are based on the utilization of carbohydrates. With the development of modern and sophisticated techniques, the isolation of various natural products from plants and other sources become easier. Of the carbohydrates isolated from natural sources, acyl-and alkyl-glycoses or glycoside derivatives are important and some of these have effective biological activity [1][2][3]. Selective acylation is considered by carbohydrate chemists as one of the most useful and versatile methods for the preparation of the hydroxyl groups [4,5]. Various selective acylation methods have so been developed and employed successfully in carbohydrate chemistry [6,7]. Of these, the direct method has been considered by the carbohydrate chemists as one of the most effective for selective acylation of carbohydrates. Selective acylation is also important because of its usefulness for the synthesis of biologically active carbohydrates [8][9][10] and nucleosides [11,12]. From the literature survey, it was revealed that a large number of biologically active compounds possess aromatic and heteroaromatic nuclei [13][14][15]. The benzene and substituted benzene nuclei play an important role as the common denominator for various biological activities [16,17]. Nitrogen (N)-and sulphur (S)-containing substitution products also showed marked antimicrobial activities [18,19]. As a continuation of a research project on the biological evaluation of carbohydrate derivatives and guided by some encouraging results obtained in this field [20][21][22], we deliberately synthesized some acylated derivatives of D-glucopyranoside (Scheme 1) and D-mannopyranoside (Scheme 2) containing a variety of substituents in a single molecular framework. We also evaluated the antibacterial and antifungal activities of the synthesized compounds using various bacterial and fungal strains and the results are reported here for the first time.
We then used a number of partially substituted monosaccharide derivatives, synthesized earlier in this laboratory, for acylation with a number of acylating agents in order to attain newer test chemicals for antimicrobial evaluation studies. Thus, pentanoylation of methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-mannopyranoside (1A) with pentanoyl chloride in pyridine gave compound 10 in 90% yield which is very similar to [23]. The IR spectrum of this compound indicated an absorption band at 1718 cm −1 corresponding to carbonyl stretching. In its 1 H-NMR spectrum, three two-proton multiplets at δ 2.41, 1.59, 1.32, and a three-proton multiplet at δ 0.86 corresponded to the presence of one pentanoyl group in the molecule. The introduction of the pentanoyl group at position 2 was demonstrated by a downfield shift of H-2 to δ 5.48. The 13 C-NMR spectrum of compound 10 also showed the presence of one pentanoyl group by displaying the following characteristic peaks: δ 172.52 {CH 3 (CH 2 ) 3 CO-}, δ 33.85, 26.98,22.11 {CH 3 (CH 2 ) 3 CO-}, and δ 13.62 {CH 3 (CH 2 ) 3 CO-}. The benzylidene derivative (1A) was then derivatized by using hexanoyl chloride in pyridine followed by the usual work-up and purification procedures. The hexanoyl derivative (11) was isolated in 92% yield as syrup.  (10)(11)(12)(13)(14).
The next substituted monosaccharide we used was methyl 4,6-O-benzylidene-2-O-(3,5dinitrobenzoyl)-α-D-mannopyranoside (1B) and employed pentanoyl chloride, hexanoyl chloride, and 4-methoxybenzoyl chloride as the acylating agents. Thus, the benzylidene derivative (1B) upon treatment with pentanoyl chloride in pyridine, followed by the usual work-up and purification, afforded the pentanoyl derivative (12)  Hexanoylation of the benzylidene derivative (1B) with hexanoyl chloride in pyridine provided compound 13 in 82% yield as needles, m.p. 137-138°C. The IR spectrum of this compound showed the following characteristic peak: 1712 cm −1 for -CO stretching. The introduction of one hexanoyl group was established by observing the following characteristic peaks in its 1 H-NMR spectrum: δ 2.26 (2H, m), δ 1.49 (2H, t, J= 7.1 Hz), δ 1.11 (4H, m), and δ 0.67 (3H, t, J= 6. 7 Hz). Also, we observed the downfield shift of H-3 to δ 5.79 (as t, J = 9. 4 Hz) showing the introduction of the hexanoyl group at position 3. Its 13 C-NMR spectrum also showed the presence of one hexanoyl group by displaying the following characteristic peaks: δ 172.80 {CH 3 (CH 2 ) 4 CO-}, δ 34. 19, 31.02, 24.66, 22.14 {CH 3 (CH 2 ) 4 CO-}, and δ 13.68 {CH 3 (CH 2 ) 4 CO-}. The benzylidene derivative (1B) was then allowed to react with 4-methoxybenzoyl chloride in pyridine and after the usual work-up and chromatographic purification, we obtained compound 14 in 85% yield as syrup. The IR spectrum of compound 14 showed an absorption band at 1716 cm −1 (-CO stretching), thereby suggesting the presence of a carbonyl group in the molecule. In its 1 H-NMR spectrum, the characteristic two-proton doublets at δ 8.08 and δ 6.97 (J = 8.8 Hz in each case) and a three-proton singlet at δ 3.88 were due to one 4-methoxybenzoyl group in the molecule. Also, the deshielding of H-3 to δ 6.04 (as t, J = 9.5 Hz) indicated the formation of the 3-substitution product. The 13 C-NMR spectrum also displayed the introduction of one 4-methoxybenzoyl group. Thus, the regioselective acylation of D-glucopyranoside (Scheme-1) and D-mannopyranoside (Scheme-2) by applying the direct acylation method was unique in that the reaction provided a single monosubstitution product in reasonably high yields.

Anibacterial Activity
The antibacterial evaluation results of the test compounds and the standard antibiotic, ampicillin, against Gram-positive and Gram-negative bacteria are listed in Table 1, Table  2

Fig. 1.
Zone of inhibition against B. cereus (A) and S. typhi (B) by the compounds 6-9. From the minimum inhibition concentration (MIC) experimental results, it was observed that chemical 8 exhibited a varied range of values from 12.5 µg/disc to 25 µg/disc and 1000 µg/mL to 1250 µg/mL by the disk diffusion (Table 3) and broth macrodilution methods, (Table 4) respectively. The lowest MIC (12.5 µg/disc) was recorded against B. cereus, B. megaterium, S. aureus, S. Typhi, and INABAET (Vibrio) by the disk diffusion method and the lowest MIC (1000 µg/mL) was recorded against B. cereus, B. megaterium, S. Aureus, and INABAET (Vibrio) by the broth macrodilution method. The MIC is indicative of the usefulness of these compounds as antimicrobial drugs, but some other experiments must be carried out before these can be used as effective drugs. As chemical 8 exhibited remarkable inhibitory activity against ten pathogenic bacteria, the efficacy of the chemical cannot be ignored. This chemical along with others, which showed promising inhibitory activity against particular bacterial strains, should be subjected to further experiments to evaluate their efficacy and this will be the subject of our future research works.

Tab. 3.
MIC test of compound 8 against ten human pathogenic bacteria by disk diffusion method.

Antifungal Activity
The results obtained from the present investigation of the antifungal studies as mentioned in Table 5 and Figure 2 clearly demonstrated that compound 8 showed the highest inhibition against all of these fungal strains. Here the percent inhibition of compound 8 is higher than the standard antibiotic, Nystatin in all the cases. The rest of the compounds was moderate or less sensitive towards the five tested fungal phytopathogens. The antifungal activities of our test compounds are in accordance with the results we observed before [24][25]. From the MIC test results reported in

Statistical Analysis
The standard deviation value is expressed in terms of ±SD. On the basis of the calculated value by using the ANOVA method, it has been observed that the differences below the 0.0001 level (p ≤ 0.0001) were considered as statistically significant.

Materials and Methods
The 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded for solutions in deuteriochloroform (CDCl 3 ) using tetramethylsilane (TMS) as internal standard with a Bruker DPX-400 spectrometer at the Bangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories, Dhaka, Bangladesh. Evaporations were carried out under reduced pressure using a VV-1 type vacuum rotary evaporator (Germany) with a bath temperature below 40°C. Melting points were determined on an electro-thermal melting point apparatus (England) and are uncorrected. All reagents used were commercially available (Aldrich) and were used as received, unless otherwise specified. Column chromatography was performed with silica gel G 60 .

Synthesis of methyl 2,6-di-O-hexanoyl-α-D-glucopyranoside (2)
A solution of methyl α-D-glucopyranoside (1) (5 g, 25.75 mmol) in dry pyridine (60 mL) was cooled to −5°C whereupon hexanoyl chloride (3.9 mL, 28.97 mmol) was added to it. The mixture was stirred at the same temperature for 4 hours and then stirred overnight at room temperature. The progress of the reaction was monitored by TLC, which indicated the formation of two products, the slower-moving component being the major one. A few pieces of ice were added to the flask and then the product mixture was extracted with chloroform (3×10 mL). The combined chloroform layer was washed successively with dilute hydrochloric acid (10%), saturated aqueous sodium hydrogen carbonate (NaHCO 3 ) solution, and distilled water. The chloroform layer was dried (MgSO 4 ), filtered, and the filtrate was concentrated under reduced pressure to leave a syrup. The syrup was passed through a silica gel column and eluted with methanol-chloroform (1:20

General Procedure of the Synthesis of Compounds (3-9)
A stirred and cooled (0°C) solution of the 3,4-diol (2) 1:4) showed the complete conversion of reactant into a single product. Excess reagent was destroyed by the addition of a few pieces of ice and the reaction mixture was extracted with chloroform (3×10 mL). The combined organic extract was washed successively with dilute hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution, and water. The organic layer was dried (MgSO 4 ), filtered, and the filtrate was evaporated off. The resulting syrupy residue was passed through silica gel column chromatography and eluted with ethyl acetate-n-hexane to afford compounds 3,4-di-Oacetyl derivative (3), 4, 5, 6, 7, 8, and 9, respectively.

General Procedure for the Synthesis of Compounds 10 and 11
A cooled (0°C) and stirred solution of methyl 3-O-benzoyl-4,6-O-benzylidene-α-Dmannopyranoside (1A) [26] (100 mg, 0.26 mmol) in anhydrous pyridine (3 mL) was allowed to react with pentanoyl chloride (0.07 mL, 0.56 mmol) and stirring was continued for 8 hours. TLC examination indicated the formation of a faster-moving product. A few pieces of ice were added to the flask with constant shaking and the mixture was extracted three times with chloroform. The combined chloroform extract was washed successively with dilute hydrochloric acid, saturated aqueous sodium hydrogen carbonate solution, and distilled water. The organic layer was dried (Na 2 SO 4 ), filtered, and concentrated. Purification of the resulting syrupy residue was effected by silica gel column chromatography (ethyl acetate-hexane as eluant) to furnish the title compound (10). A similar reaction and purification procedure was applied to prepare compound 11.

General Procedure for the Synthesis of Compounds (12-14)
An ice-cooled solution of methyl 4,6-O-benzylidene-2-O-(3,5-dinitrobenzoyl)-α-D-mannopyranoside (1B) [27] (100 mg, 0.21 mmol) in dry pyridine (6 mL) was treated with pentanoyl chloride (0.052 mL, 0.42 mmol) and the solution was stirred at this temperature for 4 hours and then at room temperature for 12 hours. TLC examination indicated the full conversion of the starting material into a single product. Excess reagent was destroyed by the addition of a few pieces of ice and the reaction mixture was processed as usual. The resulting syrup was purified by column chromatography (with ethyl acetate-hexane as eluant) to afford the pentanoyl derivative (12). Recrystalization from ethyl acetate-hexane gave compound 12. A similar reaction and purification was used to isolate compounds 13, 14, and 15.

Antibacterial Activity Assay
The in vitro antibacterial activities of the synthesized compounds were detected by the disk diffusion method [28] with little modification [29]. Sterilized paper discs of 4 mm in diameter and Petri dishes of 150 mm in diameter were used throughout the experiment. The autoclaved Mueller-Hinton agar medium, cooled to 45°C, was poured into sterilized Petri dishes to a depth of 3 to 4 mm and after solidification of the agar medium; the plates were transferred to an incubator at 37°C for 15 to 20 minutes to dry off the moisture that developed on the agar surface. The plates were inoculated with the standard bacterial suspensions (as McFarland 0.5 standard) followed by the spread plate method and allowed to dry for 3 to 5 minutes. Dried and sterilized filter paper discs were treated separately with 50 µg dry weight/disc from 2% solution (in CHCl 3 ) of each test chemical using a micropipette, dried in air under aseptic condition, and were placed at equidistance in a circle on the seeded plate. A control plate was also maintained in each case without any test chemical. These plates were kept for 4-6 hours at low temperature (4-6°C) and the test chemicals diffused from the discs to the surrounding media by this time. The plates were then incubated at 35±2°C for 24 hours to allow maximum growth of the organisms. The antibacterial activity of the test agent was determined by measuring the mean diameter of the zones of inhibition in millimeters. Each experiment was repeated thrice. The standard antibiotic, ampicillin (BEXIMCO Pharm Bangladesh Ltd), was used as a positive control and compared with tested compounds under identical conditions. The MICs of the tested compounds were determined by the disk diffusion [28] method and broth macrodilution [30] method.

Antifungal Activity Assay
The in vitro antifungal activity of the acylated chemicals was done by the Poisons Food technique [31] with some modifications [29]. Two percent solution of the test chemical (in CHCl 3 ) was mixed with sterilized melted Saburaud agar medium to obtain the desired concentration (2%) and this was poured into sterilized Petri dishes. At the center of each plate, 5 days-old fungal mycelial block (4 mm in diameter) was inoculated and incubated at 27°C. A control set was also maintained in each experiment. The linear mycelial growth of fungus was measured after 3-5 days of incubation. The percentage inhibition of radial mycelial growth of the test fungus was calculated as follows: Where, I = Percentage of inhibition, C = Diameter of the fungal colony in the control (CHCl 3 ), T = Diameter of the fungal colony in the treatment. All the results were compared with the standard antifungal antibiotic Nystatin (100 µg/mL medium, BEXIMCO Pharm Bangladesh Ltd.).

Conclusion
In this paper we have explored the synthesis, characterization, and antibacterial screening studies of some acylated monosaccharide derivatives obtained from the direct acylation method. This method demonstrates a very simple and efficient method for the synthesis. Methyl 2,6-di-O-hexanoyl-3,4-di-O-methanesulphonyl-α-D-glucopyranoside (4) and methyl 3,4-di-O-(4-chlorobenzoyl)-2,6-di-O-hexanoyl-α-D-glucopyranoside (7) were found to be encouraging in terms of high selectivity and excellent yields as 95% and 93%, respectively. Thus, a good number of test compounds reported herein exhibited promising antibacterial activity. Methyl 3,4-di-O-(3-chlorobenzoyl)-2,6-di-O-hexanoyl-α-D-glucopyranoside (8) exhibited the highest antibacterial and antifungal activities against all of the tested microorganisms. So, this compound may be targeted for future studies for its usage as a broad-spectrum antibiotic. This piece of work, in our opinion, has created an opportunity for further work with these test compounds, ultimately leading to the development of new pesticides/medicines for human disease control with fewer environmental hazards.


text:
Crystal structure of dichlorido(2,2′:6′,2′′-terpyridine-κ3 N,N′,N′′)zinc: a redetermination

The crystal structure of the title compound, [ZnCl2(C15H11N3)], was redetermined based on modern CCD data. In comparison with the previous determination from photographic film data [Corbridge & Cox (1956 ▶). J. Chem. Soc. 159, 594–603; Einstein & Penfold (1966 ▶). Acta Cryst. 20, 924–926], all non-H atoms were refined with anisotropic displacement parameters, leading to a much higher precision in terms of bond lengths and angles [e.g. Zn—Cl = 2.2684 (8) and 2.2883 (11) compared to 2.25 (1) and 2.27 (1) Å]. In the title molecule, the ZnII atom is five-coordinated in a distorted square-pyramidal mode by two Cl atoms and by the three N atoms from the 2,2′:6′,2′′-terpyridine ligand. The latter is not planar and shows dihedral angles between the least-squares planes of the central pyridine ring and the terminal rings of 3.18 (8) and 6.36 (9)°. The molecules in the crystal structure pack with π–π interactions [centroid–centroid distance = 3.655 (2) Å] between pyridine rings of neighbouring terpyridine moieties. These, together with intermolecular C—H⋯Cl interactions, stablize the three-dimensional structure.

Related literature
The title compound is dimorphic, with one polymorph (form I) crystallizing in space group No. 15, and the second polymorph (type II) crystallizing in space group No. 14 (Corbridge & Cox, 1956). The crystal structure of the title compound was originally determined by Corbridge & Cox (1956) from photographic data (final R value = 0.24) and was later re-refined by Einstein & Penfold (1966) based on the original intensity data but using more advanced least-squares procedures (R = 0.14). In both reports, the setting in P2 1 /a of space group No. 14 was used. For background to terpyridine-based materials, see: Fermi et al. (2014); Song et al. (2014). For the biocompatibility of zinc compounds, see: Gao et al. (2009 Table 1 Hydrogen-bond geometry (Å , ).  (Sheldrick, 2008) and DIAMOND (Brandenburg, 2006); software used to prepare material for publication: SHELXTL (Sheldrick, 2008).

S1. Experimental
A solution of 2,2′:6′,2′′-terpyridine (0.23 g, 1 mmol) in acetonitrile (20 ml) was mixed with a solution of zinc chloride (0.14 g, 1 mmol) in methanol (5 ml) and refluxed for 4 h. The reaction mixture was the cooled to room temperature and filtered into a large test tube. Colorless crystals were obtained at room temperature after two weeks. Yield: 75%.

S2. Refinement
All hydrogen atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms, with C -H = 0.93 Å and U iso (H) = 1.2U eq (C).

Figure 1
The molecular structure of (I), with the atom-numbering scheme. Displacement ellipsoids are drawn at the 50% probability level. The arrangement of the molecules in the crystal structure of (I), showing π-π interactions (dashed red lines) and C-H···Cl hydrogen bonds (dashed green and turquoise lines).

Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.


s2fieldsofstudy: Chemistry Biology

text:
Investigation of Cytotoxicity of Phosphoryl Choline Modified Single-Walled Carbon Nanotubes under a Live Cell Station

Single-walled carbon nanotubes (SWCNTs) and various modified SWCNTs have drawn a lot of attention due to their potential applications in biomedical field. Before further moving on to real clinical applications, hydrophobicity and toxicity of SWCNTs should be investigated thoroughly. In this paper, 2-methacryloyloxy ethyl phosphorylcholine (MPC) was adopted to modify SWCNTs and phosphoryl choline was grafted onto SWCNTs as small molecule moieties and polymeric chains, which made SWCNTs dispersed stably both in water and in cell culture medium for a long time. Cytotoxicity of pristine and modified SWCNTs were assayed upon successful preparation of the designed modified SWCNT. Furthermore, the internalization of SWCNTs by three cells was investigated using a live cell station under normal culture temperature (37°C) and low temperature (4°C). The results showed that the internalization of modified SWCNTs was related to both the active transport and the passive transport. Although the modification with phosphoryl choline remarkably reduced the cytotoxicity of SWCNTs, the results were probably due to other reasons such as the decrease in the ratio of cells which internalized modified SWCNTs since the cells without SWCNTs occupation still exhibited normal states.

Introduction
Since the discovery of carbon nanotubes (CNTs) by Iijima in 1991 [1], both multiwalled CNTs and single-walled CNTs (SWCNTs) have drawn a lot of attention in different areas, among which biomedical engineering is one of the most prominent CNTs [2][3][4][5][6][7]. However, CNTs are not soluble or even dispersible in water due to their inclination of aggregation which resulted from their hydrophobicity. This phenomenon is especially troublesome to SWNTs by severely impeding their potential improvements for biomedical devices. Moreover, the concerns on the toxicity of nanomaterials including CNTs [8][9][10][11][12] are another problem that needs to be solved before real clinical application of CNTs as biosensor, drug delivery, and biomedicines. In order to deal with these problems, various physical and chemical modifications [13] on CNTs have been carried out for the purpose of improving their solubility in water, which will in turn partially resolve toxicity issue. For example, hydroxyl group [14], carboxyl group [15], polyethylene glycol [16][17][18][19][20][21][22], amino group [23], poly(allylamine) [24], DNA or RNA [25,26], and many others [13,27] have been used to functionalize CNTs. Meanwhile, the studies on the toxicities, bioeffects, and environment health risks about CNTs have also made great progresses in the past few years; the studies themselves have moved from in vitro model and statistical assay to in vivo animal studies and mechanisms investigations [11,[28][29][30][31].
Many researchers have been focusing on the cytotoxicity of CNTs and quite a few papers have been published, but controversies have been seen in their studies. For example, some studies showed that CNTs decrease cell viability [9,[32][33][34] and upregulate genes associated with inflammation and apoptosis [34][35][36][37], while others have shown minimal or no decrease in cell viability [38,39]. As a result, it is exceptionally difficult to evaluate CNTs toxicity based on current existing data. To make the case even more complicated, the designs of toxicity studies, such as the physicochemical characterizations [40], cytotoxicity assays [41], and the definition of 2 BioMed Research International toxicity itself [42], have aroused controversies. Unfortunately, despite the complexity and expense of evaluating toxicity of various CNTs modifications, it is essential to establish a general relationship between cytotoxicity and physicochemical properties of nanotubes as a method to lower the risk of commercializing CNTs biomedical devices. The establishment of cytotoxicity-physicochemical property relationship requires simultaneous material characterization and standardized toxicity evaluations. Deliberately documented and well-defined studies will also induce helpful mechanistic understanding of nanomaterial toxicity [43].
In our previous studies [44][45][46], phosphoryl choline moieties were grafted onto MWCNTs and then cytotoxicity of the modified CNTs was evaluated in cell and mice. It was found that the functionalization on MWCNTs with phosphoryl choline significantly reduced the toxicity of CNTs. Our results indicated that phosphoryl choline moiety was a valuable modifier for CNTs regarding their biomedical applications. In this paper, the similar modification was applied on SWCNTs; not only the basic cytotoxicity evaluation, but also the cell internalization mechanism was discussed.

Instruments and Methods
. The X-ray photoelectron spectra (XPS) of SWCNT-PC were recorded on a VG Scientific ESCALab MK-II spectrometer (West Sussex, England) equipped with a monochromatic Mg-K X-ray source. Raman spectra were recorded on a JY HR800 laser Raman spectroscopy (Jobin Yvon Ltd., France). Thermogravimetric analysis (TGA) was done with a PE Pyris 1 thermogravimetor (PerkinElmer Life and Analytical Sciences, Inc., MA, USA) in a N 2 atmosphere at heating a rate of 20 ∘ C/min from room temperature to 600 ∘ C. The content of metal and other impurity elements were recorded on ARL-9800 Xray fluorescence spectrometer (ARL Ltd. Switzerland). The micromorphology images were taken on a JEM-2100 (JEOL Ltd., Tokyo, Japan) transmission electron microscope (TEM). The cells used in cytotoxicity evaluations were cultivated in a CO 2 incubator (Heracell model 150i; Thermo Scientific, MA, USA) and the evaluations were carried out with a RT-6000 microplate reader (Rayto Ltd., Shenzhen, China) at a wavelength of 450 nm. The photographs were taken on an Axio Scope A1 microscope (Zeiss Ltd. Germany) with a water immersion/dipping lens (Zeiss W Plan-Apochromat 40x/1.0 DIC VIS-IR objective).

Preparation of Samples.
Two kinds of phosphoryl choline moieties modified SWCNTs were prepared according to the synthesis routes shown in Scheme 1. SWCNT-PC was synthesized following the steps including carboxylation, chloroacetylation, and amidation of SWCNTs; the brief reaction condition was presented in Scheme 1(a) and the details were described in reference [45]. For the step of Michael addition of MPC, ethanediamine grafted SWCNTs (100 mg) were reacted with MPC (1 g) in anhydrous ethanol (100 mL) for 48 hours at room temperature and then precipitated, centrifuged, and washed with acetone for three times to eliminate the unreacted reagents. In the final products, the phosphoryl choline moiety was grafted onto SWCNTs as a small molecule structure. As for SWCNT-PCn, the phosphoryl choline moiety was grafted onto SWCNTs as a polymeric chain on the grafting point of SWCNTs following the atom transfer radical polymerization (ATRP) of MPC as described by Narain et al. [48] and Zhu et al. [49] and briefly presented in Scheme 1(b).
In order to investigate the cell uptake of modified SWC-NTs, FITC was connected to the modified SWCNTs utilizing the residual second amine groups. In the label process, SWCNT-PCn was dispersed in deionized water at concentration of 20 mg/mL and the dispersion was modulated to pH 9; freshly prepared FITC/DMSO (1 mg/mL) was added and kept stirring for 24 hours in dark at 4 ∘ C. The reactants were dialyzed with deionized water for a week and then vacuum dried and marked as SWCNT-PCn-FITC. and 1% penicillin/streptomycin in 25 cm 2 cell culture flasks and incubated at 37 ∘ C with 5% CO 2 . In the culture of HUVEC, the cells were suspended in VEGF LS-1020 culture medium. When the cells adhered to and overspread on the plate, the culture medium was removed and 0.25% trypsin-EDTA solution was added. After 2-3 min the digestion was terminated by adding DMEM culture solution containing 10% FBS; the solution was bubbled to form a single-cell suspension and then diluted and counted. The viable cells were planted at a density of 5 × 10 3 /mL and cultivated for 24 hours before the cells in exponential phase of growth and were exposed to pristine SWCNTs, SWCNT-PC, and SWCNT-PCn.

Assessment of Cytotoxicity.
Pristine SWCNTs, SWCNT-PC, and SWCNT-PCn were dispersed in water by ultrasonication at a concentration of 10 mg/mL. To overcome the poor dispersibility of SWCNTs in water, the surfactant SDS was added at a concentration of 0.1 mg/mL, while no surfactant was added to the SWCNT-PC and SWCNT-PCn dispersion. Then five dilutions, 5, 10, 20, 40 and 80 g/mL, of the pristine SWCNTs, SWCNT-PC, and SWCNT-PCn were vortex-mixed, respectively, and each sample was added to each well of multiwell plates (200 L/well in 96 multi-well plates) and cultured for 24 h at 37 ∘ C. The supernatants were removed, and 0.01 mol/L PBS was used to clean the SWCNT residue. Then 20 L of 5 mg/mL CCK-8 in culture medium was added to each well and incubated for another 1 h at 37 ∘ C. The absorbance of the solution at 450 nm was recorded with a Rayto RT-6000 microplate reader. Every experiment was performed in sextuplicate.

Fluorescent Microscopy Investigation.
The viable cells were planted at density of 5 × 10 3 /mL in 6 cm cell culture dishes (5 mL/dish) and cultivated for 5 h; then 10 L SWCNT-PCn-FITC dispersions (10 g/mL) were added to each dish and continued being cultured at 37 ∘ C or 4 ∘ C. At 4th hour and 20th hour, the cells were investigated under microscopy with a live cell station which could control the temperature and CO 2 concentration, and the photographs were taken with a water immersion/dipping objective lens.

Sample Preparation and Characterizations.
The samples used in this study were firstly characterized for their size, purity, Zeta potential, and metal impurities. The results were summarized in Table 1.

XPS Analysis of SWCNTs and Modified SWCNTs.
The XPS of pristine SWCNTs, SWCNT-PC, and SWCNT-PCn was recorded to confirm the linkage between different elements. Figure 1 shows the survey scans and the region scans of SWCNT, SWCNT-PC, and SWCNT-PCn which confirms the existence of carbon, oxygen, nitrogen, and phosphorus elements in modified SWCNTs. But for pristine SWCNTs, only carbon and very seldom oxygen due to the structure defect could be observed. It indicated the successful introduction of phosphoryl choline moieties in SWCNT-PC and SWCNT-PCn samples. For both phosphoryl choline moieties modified samples, the O1s XPS spectrum showed a peak at the binding energy of 532.0 eV that belonged to the single bonds of C-O and/or P-O bonds and the humps at the binding energy of 530 eV which was the contribution of double bonds of C=O and/or P=O bonds [50]. Moreover, the higher ratio of double bonds to single bonds in SWCNT-PCn demonstrated that there are more phosphoryl choline structures, which in turn confirmed the existence of polymerized PC chain structure. Similar to O1s spectrum, the binding energy hump at 402 eV on the N1s XPS spectrum corresponded to N-H bonds in SWCNT-PC and SWCNT-PCn, while the binding energy peak at 399 eV corresponded to C-N bonds. A distinct peak for phosphorus 2p at 132.5 eV confirms the presence of the phosphorus containing bonds in both samples. The higher peak in SWCNT-PCn also proved the repeated structure of PC moieties.

Raman Spectrum Inspection.
The existence of covalent functionalization can be inspected by Raman spectra [17]. As shown in Figure 2(a), the Raman spectrum of the SWCNTs exhibits a tangential mode (G band) at 1590 cm −1 and a relative weak disorder mode at 1300 cm −1 (D band) that was probably caused by defects formed during either the synthesis or the purification of the nanotubes. After modification, the relative intensity of the G band at 1590 cm −1 decreased, corresponding to the covalent attachment of the functional groups. If D/G ratio was defined as the ratio of the integrated peak area of D bands divided by the integrated peak area of G bands, it could be found that the larger D/G ratio for SWCNT-PCn and SWCNT-PC (from 3.38 of SWCNT-PCn to 1.07 of SWCNT-PC and to 0.78 of SWCNT) implies that the reaction with phosphoryl choline moieties leads to a higher level of functionalization.

Thermogravimetric Analysis.
The TGA curves of raw SWCNTs and modified SWCNTs were recorded and shown in Figure 2(b). In the total heating process, the weight of pristine SWCNTs lost about 9% owning to the amorphous carbon residue. For the curves of modified SWCNTs, the hydrophilicity of samples increased after the grafting process. The weight losses of SWCNT-PC and SWCNT-PCn before 150 ∘ C were about 7.8% and 7.6%, respectively, which was mainly due to the saturated water absorption. At higher temperatures, the weight loss should be due to the thermal decomposition of the side groups. Up to 600 ∘ C, the value was 28% and 38%. It may be concluded that weight loss increases with increased amount and size of the side groups grafted onto SWCNTs.

Dispersibility and Micromorphology of SWCNTs, SWCNT-PC, and SWCNT-PCn.
After modification with phosphoryl choline moieties, the insoluble pristine SWCNTs could easily disperse homogenously in water and DMEM, a cell culture medium. As shown in Figure 3(a), the pristine SWCNTs at concentrations of 5 mg/mL aggregated on standing 1 h after being dispersed by ultrasonication. However, dispersions of SWCNT-PC and SWCNT-PCn at the same concentrations were stable 7 days after being dispersed. In fact, the samples were still stable even two months later with just a sign of precipitation. Additionally, we also investigated the dispersion under centrifugation. It    was found that SWCNT aggregated soon after ultrasonication and precipitated completely when centrifuged at 2500 ×g for 5 minutes. However, dispersion of SWCNT-PC and SWCNT-PCn at concentration of 5 mg/mL was stable after centrifugation and when left at least 10 days. The stable dispersion of SWCNT-PC and SWCNT-PCn can be attributed to the hydrophilic properties of the phosphoryl choline moieties. The side groups extend into the solution because of the strong solvation of water on the PC moiety making the modified SWCNTs dispersion stable.
Actually, modification of SWCNTs with grafting watersoluble substance is one of the most effective methods among many ways to help SWCNTs dispersing in water. The reason for our choice of phosphoryl choline moieties as the modifier to accomplish the mission is phosphoryl choline's well-known biocompatible and zwitterionic properties. Zhao et al. [51] reported the synthesis of water-soluble SWC-NTs with zwitterionic poly(m-aminobenzene sulfonic acid) graft copolymer and then found that its water solubility is about 5 mg/mL [18]. Similarly zwitterionic group, phosphoryl choline moieties, provides SWCNTs with similar solubility. Moreover, the biocompatibility of PC moiety is expected to improve the biocompatibility of SWCNT itself; thus by using three different kinds of cell lines, the cytotoxicity of SWCNTs and modified SWCNTs were investigated. Figure 3(b) shows the TEM images of SWCNTs and modified SWCNTs. The dispersibility and morphology of modified SWCNTs are similar but the pristine SWCNTs are slightly entangled. Although many recent studies show that SWCNTs toxicity is highly associated with morphological signatures, we do not think morphology in this experiment which had a major impact on toxicological effects.

Cytotoxicity of SWCNTs and Modified SWCNTs: CCK-8 Assay.
Three different cell lines, HUVEC, RAW264.7, and L929 cells, were chosen as our model cell lines to evaluate the cytotoxicity of SWCNTs as well as modified SWCNTs. In the in vivo toxicity evaluation of a hazardous materials, intravenous injection method is one of the most commonly used methods. In this method, endothelial cells were the first to contact the hazardous materials except for the blood components such as proteins, platelets, and red cells; therefore HUVEC was chosen by us to investigate the cytotoxicity for the purpose of evaluating their response to SWCNTs and modified SWCNTs. RAW264.7 and L929 are widely used in studies of cytotoxicity, endocytosis, metabolism, proliferation, and RNA expressions; we chose them to provide comparable results with the previous studies.
The cytotoxicity induced by pristine SWCNTs and the modified SWCNTs were determined by the CCK-8 assay, which is based on mitochondrial dehydrogenases activity. For a living cell, the succinate dehydrogenase in mitochondria may reduce exogenous CCK-8 and form water soluble orange formazan, while a dead cell, by definition, does not have such activity. The quantity of formazan formed is directly proportional to the quantity of living cells, which can be determined by directly measuring the absorbance at 450 nm. Figure 4 shows the results of the CCK-8 assay, the amounts of living cells exposed to SWCNTs, and modified Against RAW264.7 cell lines * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * P < 0.005, n = 5 * P < 0.005, n = 5 * P < 0.005, n = 6 SWCNTs for 24 h which were normalized according to the blank control group and the results were statistically analyzed with Student's t-test method. Due to the poor dispersion properties of SWCTS, surfactant, sodium dodecyl sulfate (SDS) at the concentration of 0.1 mg/mL was used to disperse the pristine SWCNTs, and no obvious cytotoxicity was found in the SDS group in all three cell lines.
Upon addition of pristine SWCNTs, the cell livability of three cell lines decreased remarkably and kept decreasing as the dosages of pristine SWCNTs increased. This observation indicated that pristine SWCNTs do shows dose-dependent cytotoxicity on the three cell lines tested. However, when SWCNTs were modified with grafting phosphoryl choline moieties, either PC or PCn cytotoxicity reduced: the cell livability of three cell lines increased compared to pristine SWCNTs at the same dosage. Moreover, the PCn moieties with chain structure attached to SWCNT showed much stronger effects on reducing the cytotoxicity than that of PC moieties with small molecule structure. Two possible explanations for these observations were made. The first possibility is that as more repeating phosphoryl choline units were introduced, the proportion of the hydrophilic groups in the modified moieties increased, which in turn led to higher hydrophilization of the SWCNTs' surface and weakened the interactions between carbon nanotube and cells. The other explanation is that, for the SWCNT-PCn sample, the hydrophobic polyacrylate carbon chains of PCn moieties tend to wrap the surface of SWCNT while the hydrophilic phosphoryl choline groups tend to detach the surface and be exposed to the environment, forming a cell mimetic membrane structure and reducing the transplant reaction of cell line cells to PCn moieties connected SWCNTs and ultimately improving cell compatibility of SWCNT-PCn. Moreover, the polyelectrolyte properties of PCn chains also changed the covalent-bond surface of SWCNTs, converting the covalent-bond surface to an electrolyte surface, wherein the compatibility of nanotubes with the cell membrane would be improved. Apparently, this conversion would certainly afford improvements in the cell compatibility and reduction in cytotoxicity.
Although the modification on SWCNTs with PC and PCn moieties reduced the cytotoxicity of SWCNTs, it should be noted that the modified SWCNTs still presented cytotoxicity at some level when higher exposure dosages were applied. For HUVEC and RAW264.7 cell lines, the phenomenon was investigated in repeated experiments. The results indicated that the modifications on SWCNTs were not able to completely remove the cytotoxicity of SWCNTs. We speculated that the cytotoxicity of modified SWCNTs at a high dosage was associated with their extremely high aspect ratio. The SWCNTs and modified SWCNTs can probably be embedded into cell membrane and influence its integrity and further disturb cell proliferation. This assumption has been proved in the following experiments to investigate the interaction of cells with carbon nanotubes using fluorescence microscope on a live cell station.
Using the same SWCNTs and modified SWCNTs with same dosage as we used in our experiments described above and under the same incubation conditions, the obtained cytotoxicity showed significant difference for different cell lines. In general, SWCNTs and modified SWCNTs showed the severest cytotoxicity against HUVEC, followed by RAW264.7, while L929 performed the best tolerance for the nanotubes. The difference is certainly related to the different characters of cell lines. HUVEC is a primary cell line coming from human beings, which presented a weak tolerance against hazardous materials such as carbon nanotubes. RAW264.7 and L929 are both murine tumor-cells which have been subcultured for several times, thereby exhibiting higher tolerance to hazardous materials. Moreover, as a macrophage, RAW264.7 tends to uptake while L929 tends to exclude foreign matters; therefore the former one is more susceptible to the effects of hazardous materials. Additionally, the differences of the cell membrane structures, protein expressions, and so forth would inevitably affect the cell proliferation, thereby impacting the cytotoxicity results. More works should be carried out to study the effects of hazardous materials on gene mutation, growth factors, cell receptors, transcription factors, cellular signaling pathways, and so on. The observation that different cell lines show different cytotoxicity results under the same conditions suggests that different conclusions, and sometimes even the opposite opinions, might be drawn based on which cell lines were chosen for nanotoxicological studies. Also, our results indicate that more careful studies on the bioeffects on these nanomaterials are necessary.

Fluorescent Microscopy Investigation at 37 ∘ C.
In order to further investigate the mechanism that causes cytotoxicity of the modified SWCNTs, fluorescent microscopy investigation was used. A fluorophores, FITC, was connected to SWCNT-PCn. Subsequently, a Carl Zeiss Axio Scope A1 fluorescent microscopy equipped with a water immersion/dipping objective lens was utilized to observe the interaction of the various cells and the fluorescently-labeled SWCNT-PCn (SWCNT-PCn-FITC) in a live cell station. Figure 5 shows the photographs taken 24 hours after incubating the cells with SWCNT-PCn-FITC at 37 ∘ C with a dosage of 20 g/mL.
As shown in Figure 5, it was obvious that SWCNT-PCn-FITC was internalized by cells in RAW264.7, L929, and HUVEC. As macrophage which is highly specialized in removing of dying cells, dead cells and cellular debris, RAW264.7 was also well known for its phagocytosis of ingesting foreign materials [52]. Carbon nanotubes, both pristine CNTs and modified ones, were subjected to uptake by RAW264.7 [28]. Our data of phosphoryl choline modified SWCNTs was in accordance with that phenomenon. However, in HUVEC and L929 cells without phagocytosis, the fluorescence was still strong enough to be regarded as that of SWCNT-PCn-FITC was taken up by the cells. Based on this observation, we assume that the internalization of SWCNT-PCn-FITC was not only related to the phagocytosis but also involved in passive transport. Besides our results of L929 and HUVEC, the passive transport was also documented by other researchers in various cells [33,53].
Besides the images shown in Figure 5, more data acquired during our observation of the living cells under microscope in live cell station showed that not all cells took up SWCNT-PCn-FITC. Those cells which internalized modified SWCNTs were abnormal cells showing irregular morphology, apoptotic and necrotic, and cell death, while the cells which were not occupied by modified SWCNT appeared to be normal. This difference suggested that the phosphoryl choline modified SWCNT still showed a certain degree of cytotoxicity just as concluded in the CCK-8 assay. In other words, the modification on SWCNTs by phosphoryl choline moieties can significantly reduce the cytotoxicity of SWCNTs but cannot eliminate it completely.
More remarkably, in CCK-8 assay with a dosage of 20 g/mL, SWCNT-PCn showed severest cytotoxicity against RAW264.7, which is followed by HUVEC, while cell viability of L929 was not significantly different from the control group. The same trend has also been seen in investigation under live cell station: under microscope, RAW264.7 group showed the largest number of cells which took up SWCNT-PCn-FITC. All these findings suggest that the use of only statistical CCK-8 cell viability assay is not enough for evaluating cytotoxicity of SWCNTs-like nanomaterials. Statistical CCK-8 cell viability assay probably only reflected the cell that internalized hazardous materials rather than the overall destructiveness on cells. This thought leads to another explanation of the mechanism of how cytotoxicity was attenuated by phosphoryl choline modifications on SWCNTs: the phosphoryl choline moieties grafted on SWCNTs rendered SWCNTs hydrophility and reduced the ratio of cells which internalized SWCNTs, resulting in a significant decrease in cytotoxicity. Based on that, more deliberate examinations of interactions of nanomaterials with cells, especially with subcellular organelles, should be carried out; since only in this way comprehensive and reliable results of cytotoxicity of these materials would be obtained.

Fluorescent Microscopy Investigation at 4 ∘ C.
When SWCNT-PCn-FITC was incubated with cells under normal incubation temperature, the internalization by cells could be seen clearly from the above sections. It is well known [54] that the internalization of nanoparticles can be subdivided into two categories: namely, receptor-mediated endocytosis and macropinocytosis. The former is fast and dependent on energy (active transport), while the latter shows no temperature dependence (passive transport). Thus, by incubating cells along with materials at different temperature and investigating the endocytosis of materials in cells, we can understand the subdivided pathway and the cytotoxicity caused by modified SWCNT nanoparticles.
The cells were incubated with SWCNT-PCn-FITC under lower temperature at 4 ∘ C. Unlike active transport, passive transport does not require an input of energy. The rate of passive transport depends on the permeability of the cell membrane, which, in turn, depends on the organization and characteristics of the membrane lipids and proteins [55]. If cells were incubated under low temperature, the active transport would be inhibited while passive transport would remain the same. Figure 6 shows the fluorescence microscopy images of HUVEC, RAW264.7, and L929 incubating with SWCNT-PCn-FITC for 4 h at 4 ∘ C with a 20 g/mL dose. The fluorescence was still strong enough for the confirmation that SWCNT-PCn-FITC has been taken by all three kinds of cells. This fact illustrated that passive transport contributes to the internalization of modified SWCNT by cells. Furthermore, as shown in the image of RAW264.7, the fluorescence in the cells decreased significantly, implying that the phagocytosis of the cell was inhibited by lowtemperature incubation, which in turn led to decrease in cell vitality followed by decreased chemical energy input and ultimately suppression of the active transport process. However, the strong fluorescence on cell membranes as well as the vesicular structures attached on cells still suggested the existence of SWCNT-PCn-FITC, which probably mainly resulted from passive transport. Continuing the observation at 4 ∘ C, we found that the cells which contained modified SWCNTs were also in their abnormal states just as what we found under higher temperature, revealing the impacts of nanomaterials on cells and the cytotoxicity nanomaterial would cause.
By comparing the images taken at 37 ∘ C and 4 ∘ C, we proposed that the internalization of modified SWCNTs in cells was caused by both active and passive transport and also depended on cell line itself. At lower temperature, the passive transport dominated the internalization process and could still cause the damage on cells.

Conclusions
In conclusion, two kinds of modified SWCNTs were prepared by grafting small molecule phosphoryl choline moiety and polymeric phosphoryl choline chains, respectively. The modified products showed great extent of hydrophilization. Moreover, the modified SWCNTs were found to be able to disperse stably in water and cell culture medium even a long time after incubation, which provided us with an opportunity to evaluate the cytotoxicity of SWCNTs with CCK-8 assay. The results indicated that the modification with biocompatible phosphoryl choline moieties greatly reduced but cannot completely eliminate the cytotoxicity of SWCNTs. Furthermore, the investigation under fluorescence microscope in a live cell station suggested that the internalization of modified SWCNTs was caused by both active transport, phagocytosis, and passive transport. The cell-internalized modified SWCNTs were found to be in their abnormal states, which were probably caused by the impacts of SWCNTs on cell membrane. But the percentage of cells which internalized modified SWCNTs decreased significantly due to the hydrophilization of SWCNTs, which is the observation that explained the improvements in cytotoxicity results in CCK-8 assay. Based on this discovery, we proposed that more deliberately designed experiments to investigate the interactions of nanomaterials with cells, especially to subcellular organelles, need to be carried out, since, only in this way, comprehensive and reliable results of cytotoxicity of these nanomaterials can be obtained.