Host-Guest of Dendrimers in the Gas Phase Zhenhui Qi, Christoph A. Schalley

To cite this version:

Zhenhui Qi, Christoph A. Schalley. Host-Guest Chemistry of Dendrimers in the Gas Phase. , Taylor & Francis: STM, Behavioural Science and Public Health Titles, 2010, pp.1. ￿10.1080/10610278.2010.486438￿. ￿hal-00599222￿

HAL Id: hal-00599222 https://hal.archives-ouvertes.fr/hal-00599222 Submitted on 9 Jun 2011

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Host-Guest Chemistry of Dendrimers in the Gas Phase

Journal: Supramolecular Chemistry

Manuscript ID: GSCH-2010-0032.R1

Manuscript Type: Special Issue Paper

Date Submitted by the 13-Apr-2010 Author:

Complete List of Authors: Qi, Zhenhui; Freie Universität Berlin, Institut für Chemie und Biochemie Schalley, Christoph; Freie Universität Berlin

host-guest chemistry, dendrimers, gas-phase chemistry, mass Keywords: spectrometry, supramolecular chemistry

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1 2 3 4 Host-guest chemistry of dendrimers in the gas phase 5 6 7 8 9 10 Zhenhui Qi and Christoph A. Schalley * 11 12 13 14 15 16 Institut für ForChemie und Peer Biochemie, FreieReview Universität Berlin, Only Takustraße 3, 14195 17 18 Berlin, Germany 19 20 21 Dedicated to the memory of Dmitry Rudkevich, who died much too early. Those who 22 have worked with him certainly know his very supportive optimism when showing 23 complicated NMR spectra to him: "Don't worry, I can see, it's in there!" 24 25 26 27 Corresponding author. E-mail: [email protected] 28 29 30 31 32 Since the early days of dendrimer chemistry, mass spectrometry has been an important 33 34 analytical method for determining the purity and the detection of defects in dendrimers. 35 36 Meanwhile, growing evidence demonstrates the great potential of mass spectrometry for 37 38 the investigation of non-covalent dendritic host-guest complexes. Mass spectrometry 39 40 provides an efficient means to isolate them in the high vacuum inside a mass 41 42 spectrometer under environment-free conditions. Gas-phase chemistry is particularly 43 44 beneficial for exploring the intrinsic properties which cannot easily be studied in solution. 45 46 This mini-review highlights the versatility of gas phase chemistry for (1) screening the 47 48 specificity and stability of multivalent dendritic host-guest complexes depending on the 49 50 nature of the guests, (2) revealing a dendritic effect during dendrimer-tweezer complex 51 52 fragmentation, and (3) monitoring an intra complex movement of small guests along the 53 54 dendrimer periphery. 55 56 57 58 59 Keywords: host-guest chemistry; supramolecular chemistry; dendrimers; mass spectrometry; 60 gas-phase chemistry; molecular mobility

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1 2 3 Introduction 4 5 6 The field of host-guest chemistry of dendritic has grown into a special area 7 8 9 of supramolecular chemistry during the last decades ( 1-2). The highly branched 10 11 architectures of dendrimers endow them with unique recognition properties, which are 12 13 appreciated for constructing diverse supramolecular host-guest systems. Insight into 14 15 16 these systemsFor opens upPeer new opportunities Review not only to understand Only the fundamental 17 18 principles in biological process ( 3-4), but also to develop functional materials ( 5-7). 19 20 21 Some ten to fifteen years ago, there was still a debate, whether dendrimers 22 23 would form specific host-guest complexes at all. Meanwhile it is well known that they 24 25 26 do: POPAM dendrimers have not only shown to bind metal , but even exhibit an 27 28 interesting kinetic behaviour in that metal ions quickly complex to the periphery and 29 30 then need significantly longer to enter the dendrimer core (8). Dendrimers have also 31 32 33 been equipped with specific binding sites in their peripheries, e.g. crown ethers ( 9), 34 35 urea units ( 10 ), ferrocenyl ( 11 ) and carbohydrate groups (12 ). For drug delivery, core- 36 37 38 shell particles are under intense investigation ( 13-15 ). 39 40 However, with the growing complexity of these systems, the dendritic host- 41 42 guest complexes are often large in size and molecular mass, and often display a highly 43 44 45 symmetrical arrangement of building blocks. Therefore it is often rather difficult to 46 47 characterize their exact structures and to determine the stoichiometries of the hosts 48 49 and guests involved. For a convincing analysis and characterization, usually a number 50 51 52 of different instrumental methods need to be applied ( 16-17 ). Moreover, many 53 54 researchers became aware of the often quite significant influence of the environment 55 56 18 57 on the formation of dendritic host-guest complexes ( ). They can be strongly 58 59 affected by the presence of the solvent or counter-ions. Stability constants and 60 thermodynamic parameters determined in solution are always influenced by solvent

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1 2 3 effects. The intrinsic interaction between host and guest is thus inevitably challenging 4 5 6 to unravel. 7 8 For a long time, mass spectrometry was only considered as an important 9 10 11 analytical method in dendrimer chemistry. In most laboratory research, mass 12 13 spectrometry merely served as an excellent routine analytical tool to determine the 14 15 purity and to analyze the defects of dendrimers in higher generations ( 19-21 ). 16 For Peer Review Only 17 18 However, increasing evidence indicates the utility of mass spectrometry to go far 19 20 beyond the mere analytical characterization. The gas phase inside the mass 21 22 spectrometrer has proved to be particularly beneficial for the analysis of weakly 23 24 25 bound host-guest complexes ( 22-26 ). The high vacuum inside a mass spectrometer 26 27 provides an efficient means to isolate the complexes and to suppress the highly 28 29 dynamic guest exchange equilibria in solution. Once the desired complex ions are 30 31 32 generated, mass selection becomes possible and a large variety of tandem MS 33 34 (MS/MS) experiments is available which enable the dendrimer chemist to examine 35 36 37 the ions under environment-free conditions. Valuable information of the intrinsic 38 39 features of the complexes as well as the reactivity of non-covalent species can be 40 41 obtained. A comparison of these data from the gas phase, i.e. the intrinsic properties, 42 43 44 with results from condensed phase, i.e. the properties as influenced by the 45 46 environment, can consequently contribute significantly to the understanding of non- 47 48 covalent bonds, and in return to guide the design of supramolecular host-guest system. 49 50 51 The objective of this mini-review is to highlight an emerging area by 52 53 54 discussing studies which use tandem mass spectrometry to investigate dendrimer- 55 56 guest complexes. It advertises the great potential of gas-phase studies for dendrimer 57 58 host-guest chemistry of dendrimers. We will focus on three different aspects: The first 59 60 part will discuss a gas-phase approach to screen the relative binding strengths of

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1 2 3 multiple small guests that can bind to the dendrimer periphery in a dynamic 4 5 6 library. These gas-phase studies elegantly allow to compare different guests each of 7 8 which is bound in a multivalent fashion to more than one binding site in the 9 10 11 dendrimer. In the second example, dendritic viologen-tweezer complexes represent 12 13 another type of dendrimer host-guest structure in which the complexation occurred at 14 15 the core. In contrast to the solution behaviour, a quite pronounced dendritic effect is 16 For Peer Review Only 17 18 observed which has a major effect on the stability and on the fragment mechanisms of 19 20 the viologen dications in the dendrimer core. The differences in the gas-phase 21 22 behaviour of dendrimers of different generation can be traced back to backfolding of 23 24 25 the dendrimer branches. Finally, we will discuss a "spacewalk" at molecular level. 26 27 Crown ethers are able to walk along the periphery of POPAM dendrimers without 28 29 intermediate dissociation. A quite simple hydrogen/deuterium exchange (H/D 30 31 32 exchange) in the gas phase is capable of monitoring this highly dynamic 33 34 intramolecular movement in the gas phase. 35 36 37 38 39 40 Multivalency in the gas phase: multiple guest binding to urea-terminated POPAM 41 42 43 dendrimers 44 45 46 Recently, dendrimers are gaining popularity as frameworks for the study of 47 48 multivalent interactions in biological systems (4, 27 ). A particularly interesting 49 50 example is the third generation POPAM dendrimer D (Figure 1), which carries 16 51 52 53 peripheral adamantylurea groups (28). As shown in Figure 2, dendrimer D can serve 54 55 as the host scaffold for guests that can be bound through multivalent interaction. After 56 57 58 an initial transfer from the guest acid group to the dendrimer's tertiary amines, 59 60 strong electrostatic interactions exist between the acid anion of the guest molecule and

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1 2 3 the tertiary ammonium ions incorporated in host D. In addition, multiple hydrogen 4 5 6 bonds between the urea units of guest and host support binding. Therefore, host D 7 8 enables a variety of guest molecules based on urea-acid structures to attach on its 9 10 11 surface, even including some oligopeptides such as T ( 29). The most intriguing 12 13 feature of such a multivalent system is the non-covalent decoration of dendrimer 14 15 surface, giving rise to a dynamic binding equilibrium in solution, in which the guests 16 For Peer Review Only 17 18 can compete with each other and the guest with the optimal binding motif can easily 19 20 be detected. Moreover, due to the multibranched structures of dendrimers, a dynamic 21 22 combinatorial library of different complexes with various stoichiometries can 23 24 25 consequently form when at least two different guests compete for binding to the 26 27 dendrimer. Consequently, the multivalency of dendrimers surface is capable of 28 29 attaching multiple drug molecules, targeting groups and solubilizing moieties 30 31 32 coordinately ( 30 ). 33 34 35 Although the concept to dynamically modify the periphery of dendrimers is 36 37 very attractive, the characterization of such a system seems to be a quite challenging 38 39 task. Because up to eight of guests can bind to the same dendrimer in a dynamic 40 41 42 manner, the determination of binding stoichiometries for complexes, in particular in a 43 44 dynamic library with more than one type of guest is rather difficult. (28 ) 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Figure 1. Top left: Third-generation POPAM dendrimer D with 16 peripheral 51 52 admantylurea groups. Top right: The postulated binding mode of a dendrimer host 53 with (a) a urea-substituted phosphonate; (b) an N-terminal Boc-protected tripeptide. 54 Bottom: A variety of guests including urea-containing guests U1 , U2 , C1 , P1 , and S1 . 55 The three guests C2 , P2 , and S2 lack the urea group and thus serve as reference 56 compounds for examining the binding mode. The Boc-protected tripeptides T (Boc- 57 GGG, Boc-GGA, Boc-AAA, Boc-FGG, Boc-FFF, and Boc-GVV) were also 58 59 investigated in the gas phase (G = glycine, A = alanine, F = phenylalanine, V= valine, 60 Boc = tert-butoxycarbonyl).

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 2. (a) ESI-FTICR mass spectrum of a sample of dendrimer D with six 53 equivalents of guest U2 . Two species ions of host-guest complexes with 4+ and 3+ 54 charge states are observed respectively. (b) The deconvoluted mass spectrum shows 55 complexes [ D•(U2 )n] with n = 0 - 8. The mass differences of ∆m = 244 correspond to 56 3+ 57 individual guests U2 . (c) CID mass spectrum of mass-selected [ D•(U2 )4] . At 58 increasing collision energies, the guest molecules are successively removed as neutral 59 molecules, finally only bare dendrimer ions are left. Reproduced from ref. 31 with 60 kind permission from Wiley-VCH and the authors.

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1 2 3 Meijer and coworkers have studied the multivalent interaction between host D 4 5 6 and a series of guest molecules by mass spectrometry ( 31-32 ). The transfer of 7 8 dendrimer D and its host-guest complexes into gas phase is easily achieved by 9 10 11 electrospray ionization mass spectrometry (ESI-MS). A small amount of acetic acid is 12 13 added to the sample solution to achieve a net protonation of the dendrimer complexes. 14 15 Figure 2a shows the ESI spectrum of, for example, a mixture of dendrimer D with six 16 For Peer Review Only 17 18 equivalents of guest U2 in chloroform. The complexes appear in charge states 3+ and 19 3+/4+ 20 4+. For each charge state, all possible assemblies [D•( U2 )n] with n = 0 - 8 are 21 22 detected. Deconvolution of the different charge states provides a simpler and cleaner 23 24 25 picture (Figure 2b). Apart from the naked dendrimer D, eight peaks at higher mass 26 27 correspond to the dendrimer with one to eight guest molecules bound to it. Each mass 28 29 difference of ∆m = 244 between two adjacent signals corresponds to one U2 guest 30 31 32 molecule. The ESI-MS experiments unambiguously identify the individual 33 34 complexes. Similar spectra are observed for the other guests listed in Figure 1. 35 36 37 Once the desired complex ions are transferred successfully into the gas phase, 38 39 the stability of the ions of interest can be probed with tandem MS techniques, for 40 41 42 example by fragmenting them in collision-induced dissociation (CID) experiments. 43 44 After mass selection of the desired ions, the ions are accelerated, and subsequently 45 46 47 collided with a neutral gas in the analyzer cell of a Fourier-transform -cyclotron- 48 49 resonance (FTICR) mass spectrometer. During the collision, part of the kinetic energy 50 51 of the ions is converted into rovibrational excitation, which eventually gives rise to 52 53 54 the fragmentation with the weakest bond to represent the most probably cleavage site. 55 3+ 56 Figure 2c shows the detailed fragmentation picture of the [ D•(U2 )4] complex. At 57 58 increasing collision energies, the guests attached to the dendrimer can be removed as 59 60 neutral molecules one after the other until finally free D ions are left.

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1 2 3 In the experiments described so far, all guests in the complexes under study 4 5 6 were identical. No drastic change of the dissociation energies is thus expected, unless 7 8 the guests interact with each other. However, when mixed complexes are treated the 9 10 11 same way, a ranking of binding strengths in the gas phase can be obtained. Detailed 12 13 insight into the influence of the guest structure on the binding strength may provide 14 15 information about the binding mode. All guests in Figure 1 form complexes except for 16 For Peer Review Only 17 18 C2 ( 32 ), which lacks both the urea units and a strongly acidic end group. The fact that 19 20 the urea groups are not mandatory for complex formation indicates that the attractive 21 22 electrostatic interactions between the guests' phosphonate or sulphonate groups and 23 24 25 the tertiary ammonium in the dendrimer's binding pockets are strong enough to 26 27 generate host-guest complexes with D. For the weaker carboxylic acid guests, the urea 28 29 group is nevertheless required. This might indicate that no ion pair is formed between 30 31 32 host and guest. Instead, hydrogen bonding is likely responsible for guest binding. 33 34 Competition experiments are possible, when heterocomplexes are mass-selected and 35 36 37 fragmented in the gas phase. The guest which is more readily expelled from the 38 39 complex is the more weakly bound guest. Such CID experiments can be performed 40 41 with all possible guest pairs. The result is that guests are bound significantly more 42 43 44 strongly when they contain the urea groups. Consequently, hydrogen bonding 45 46 contributes its share to the binding energy. This finding is in excellent agreement with 47 48 the trend in solution-phase association constants gained for model systems in 49 50 51 chloroform and thus we conclude that binding mode to be the same in solution and in 52 53 the gas phase. 54 55 56 Finally, the binding strengths of a set of oligopetides with BOC protected 57 58 amino termini to the dendrimer were also examined. Already, slight structure 59 60 differences in the tripeptides lead to significant changes the binding strengths. For

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1 2 3 instance, dendrimer D shows a clear preference for BOC-Gly-Gly-Gly over BOC- 4 5 6 Ala-Ala-Ala. In the corresponding CID spectra, the initial loss of the latter tripeptide 7 8 is much more pronounced than that of the triglycine. 9 10 11 The studies discussed here demonstrate the utility of tandem mass 12 13 spectrometry for a fast screening of guest molecule binding to large molecules. Since 14 15 16 dendrimersFor are multibranched Peer structures, Review the effects of multivalent Only binding can thus 17 18 be examined in the gas phase. Moreover, the examples also nicely show how an 19 20 21 analysis of the gas-phase chemistry of dendrimer-guest complexes elegantly 22 23 contributes to a more profound understanding of both the binding motif (a structural 24 25 issue) and the binding strengths (a thermochemical issue). 26 27 28 29 30 31 32 A remarkable dendritic effect: complexes of a molecular tweezer and dendritic 33 34 viologen dications 35 36 37 Viologens are doubly charged bipyridinium ions which possess a quite electron- 38 39 deficient dicationic core. They are known to be good guests for molecular tweezer Tw 40 41 (33 ) with its extended aromatic systems (Figure 3). In dichloromethane, viologen- 42 43 44 tweezer complexes are quite stable, which is also true when the viologen is decorated 45 46 with Fréchet’s benzylether dendrons on both sides. The tweezer is located at the 47 48 49 central core and thus additional interactions of the aromatic rings within the dendrons 50 51 with the outer convex side of the tweezer may occur particularly in larger generations. 52 53 54 Upon electrospray ionization of the G0 viologen salt in the absence of the 55 56 tweezer, no bare G0 2+ dications could be detected. Only singly and doubly charged 57 58 59 clusters are observed in which the positive charges are counterbalanced by a 60 sufficiently high number of counterions. Density functional calculations for a simple

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1 2 3 model compound indeed predict the C-N bond in the viologen dication to dissociate 4 5 6 due to the strong charge repulsion once the dication is formed in free form. Therefore, 7 8 the G0 2+ dication might be a short-lived metastable ion, which decomposes before it 9 10 11 can be detected on the timescale of the FTICR mass spectrometric experiments. The 12 13 two product ions, i.e. a singly substituted, singly charged bipyridinium ion and the 14 15 corresponding benzyl cation, are indeed observed in the ESI mass spectrum. Besides 16 For Peer Review Only 17 2+ 18 cluster formation and fragmentation, G0 has two other modes of avoiding being a 19 20 naked dication: Deprotonation which occurs presumably at the benzylic position and 21 22 one-electron reduction at the ESI spray capillary to yield the cation-radical. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

51 52 53 Figure 3. The detailed structures of the molecular tweezer Tw (inset) and dendron- 54 substituted viologens G0 2+ , G1 2+ , and G2 2+ . 55 56 57 58 2+ 59 In marked contrast, the generation of G1 dications is possible, but only 60 under as mild as possible ionization conditions. Finally, the generation of bare G2 2+ is

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1 2 3 even possible under somewhat harsher ionization conditions. Evidently, this marked 4 5 6 change indicates a trend toward higher dication stability with increasing dendron size. 7 8 9 When one equivalent of the tweezer Tw is added to the sample solutions, the 10 11 mass spectra change dramatically ( 34 ). Even for the least stable G0 2+ , quite intense 12 13 signals correspond to the intact doubly charged 1:1 complexes are observed. The 14 15 16 complexes Foralso form asPeer singly charged Review ions carrying one counterion.Only However, the 17 18 mere existence of the complex dication shows the tweezer to stabilize the dications 19 20 21 significantly through charge-transfer interaction. Simultaneously, this reduces the 22 23 strong charge-repulsion effects and thus renders the complex detectable as a free 24 25 dication. Similarly, dicationic tweezer-dendrimer complexes can be obtained for the 26 27 28 larger generations. 29 30 For an analysis of the fragmentation behavior of the viologen-tweezer 31 32 33 complexes, the desired ions were mass-selected and subjected to a CID experiment. 34 35 When monoisotopic tweezer-G0 2+ dications were isolated and examined, one would 36 37 38 probably expected to see an initial loss of the tweezer followed by an immediate 39 40 consecutive fragmentation of the remaining metastable dication (bottom pathway in 41 42 Figure 4). Indeed, the two expected fingerprint fragments from G0 2+ are observed 43 44 45 (Figure 5). However, an additional fragment at m/z 1059 corresponds to a singly 46 47 charged tweezer-bipyrindium ion and clearly indicates the loss of a di-t-butylbenyzl 48 49 cation from the complete complex to be operative in the fragmentation of [ Tw •G0 ]2+ . 50 51 52 Clearly, it is also possible that the supramolecular interaction is stronger than the 53 54 covalent N-benzyl bond. This finding raises the question, which of the two pathways 55 56 57 shown in Figure 4 contributes to what extent. A double resonance experiment 58 59 provides the answer: In this experiment, the ions with m/z 1059 are constantly 60 removed from the FTICR analyzer cell during the whole duration of the MS/MS

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1 2 3 experiment and thus all consecutive fragments formed through this ion as an 4 5 6 intermediate should vanish as well. The comparison of Figures 5a and 5b clearly 7 8 shows that almost all of the bipyridinium cations at m/z 359 vanish in the double 9 10 11 resonance experiment and thus must be due to a consecutive fragmentation through 12 13 the intermediate complex at m/z 1059. Instead, loss of tweezer followed by Coulomb 14 15 explosion of the G0 2+ dication contributes at best a few percent to the bipyridinium 16 For Peer Review Only 17 18 fragment at m/z 359. This experiment clearly identifies the upper fragmentation 19 20 channel in Figure 4 to the major one in the dissociation of the [ Tw •G0 ]2+ complex. 21 22 The covalent benzylic C-N bond is thus weaker in this complex than the 23 24 25 supramolecular interaction between host and guest. 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Figure 4. Two possible pathways for the fragmentation of the tweezer-viologen 53 54 complexes. The reactivity switches from the upper to the lower pathway depending on 55 the dendron sizes. 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 2+ 56 Figure 5. (a) Collision-induced decomposition (CID) of mass-selected tweezer-G0 57 complex. (b) Double-resonance experiment with the same ions, in which the fragment 58 at m/z 1059 was ejected from the reaction cell during the whole experiment. (c) CID 59 experiment with the tweezer-G1 2+ complex. (d) CID experiment with the tweezer- 60 G2 2+ complex revealing a complete change in reactivity. Tweezer loss is the major fragmentation pathway giving rise to dicationic G2 2+ at m/z 1005.

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1 2 3 The same reactivity pattern was also observed for the tweezer complex with 4 5 2+ 2+ 6 G1 . However, the fragmentation behavior reversed for the complex with the G2 7 8 viologen guest. Here, the loss of tweezer represents the first step and generates the 9 10 11 bare G2 dication at m/z 1005, which can undergo further fragmentation by benzyl 12 13 cation loss. The benzyl loss intermediate, however is not observed at all. 14 15 Consequently, the size of the dendrons plays an important role for the change in 16 For Peer Review Only 17 18 fragmentation behavior. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Figure 6. Lowest-energy conformations of the viologen derivatives substituted by 42 2+ 2+ 43 two 3,5-di-t-butylbenzyl groups ( G0 ), by methyl and the G1 dendron ( G1’ ), and 2+ 44 by methyl and the G2 dendron ( G2’ ) calculated by Monte-Carlo conformer search 45 using the MMFF force field implemented in SPARTAN 04. For the two larger 46 structures, one dendron was replaced by methyl in order to reduce the necessary 47 computer time. 48 49 50 51 52 At this point, we have encountered two interesting dendritic effects which 53 54 need to be explained: (1) A clear trend in dication stabilities emerges: The larger the 55 56 57 dendrons the better stabilized the dication is. (2) A reversal in the sequence of 58 59 fragmentation steps occurs between [ Tw •G1 ]2+ and [ Tw •G2]2+ . The origin of these 60 dendritic effects can be traced back to the backfolding of branches in the larger

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1 2 3 dendrons which leads to self-solvation in the gas phase (Figure 6). For G0 2+ , the 4 5 6 relatively small benzyl substituents are unable to backfold into the vicinity of the 7 8 dicationic viologen core. Consequently no self-solvation occurs and the cation 9 10 2+ 11 remains unstabilized causing the instability of the benzyl-N bond. In G1 , the naphtyl 12 13 methyl end groups can fold to approach the central viologen in a geometrically 14 15 favorable manner. However, only a minor stabilization of the dication is achieved, 16 For Peer Review Only 17 2+ 18 since the naphthalene moieties are not very electron-rich. In contrast, G2 is not only 19 20 more flexible, but also provides the electron-rich, dihydroxybenzyl branching units 21 22 which provide efficient internal solvation through the formation of intramolecular 23 24 25 charge-transfer-complexes. The backfolding of dendrimer branches not only explains 26 27 the dication stability order, but also the reversed fragmentation sequence of 28 29 [Tw •G2 ]2+ . The tweezer competes with the backfolded branches of the larger 30 31 32 dendrons for the binding to the viologen. Consequently, the supramolecular 33 34 interaction between tweezer and viologen decreases with increasing the dendron size. 35 36 37 At the same time, the reduced charge repulsion stabilizes the covalent benzyl-N bond 38 2+ 39 with increasing dendron size. For the complex of G2 , the covalent bond is finally 40 41 stronger than the non-covalent interaction between host and guest. 42 43 44 The gas-phase experiments with the tweezer-dendrimer host–guest complexes 45 46 47 add valuable insight into the reactivity of such species which cannot be gained from 48 49 solution studies. In solution, not only solvent molecules change the properties of the 50 51 complexes. Even more important, the counterions significantly stabilize the dications 52 53 54 and reduce charge repulsion. Also, reversible binding which constantly leads to an 55 56 exchange of the guest hampers the analysis of such a reaction. Such an exchange is 57 58 not possible in the gas phase, because the complexes are isolated from each other in 59 60 the high vacuum of a mass spectrometer.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Figure 7. Schematic representation of crown ether movement along a G4 POPAM 31 dendrimer. 32 33 34 35 36 A molecular spacewalk: 18-Crown-6 walks along the periphery of POPAM 37 38 39 dendrimers 40 41 42 Molecular mobility has attracted considerable attention in supramolecular chemistry 43 44 and biochemistry. Recently, a simple but challenging question (Figure 7) has been 45 46 answered: Can 18-crown-6 travel on the surface of a POPAM dendrimer? Can they 47 48 49 walk from binding site to binding site without intermediate dissociation (35-36 )? 50 51 Answering this question is difficult, if not impossible in solution, because the guest is 52 53 always involved in dissociation/reassociation equilibria and intercomplex exchange. 54 55 56 The critical issue of detecting such an intracomplex movement is to isolate the 57 58 complexes from each other and from their free components. From the two preceding 59 60 parts, one might assume the high vacuum of a mass spectrometry to be a good choice,

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1 2 3 since the gas phase is ideally suitable for studying the isolated complex. However, a 4 5 6 simple mass analysis is obviously unable to provide direct evidence, as the 7 8 intramolecular movement will not cause any mass change. Therefore, a gas-phase 9 10 11 reaction is required as a probe, which must meet the following criteria: (1) It has to 12 13 proceed energetically below the complex dissociation energy (for 18-crown-6 14 15 ammonium complexes ca. 180 - 200 kJ/mol), (2) must cause a mass change, and (3) 16 For Peer Review Only 17 18 needs to be directly linked to the guest movement. These requirements are met by a 19 20 gas-phase H/D exchange reaction (37 ). 18-crown-6 ( 18C6 ) is well known to bind to 21 22 primary ammonium ions in solution ( 38 ) as well as in the gas phase ( 39). 23 24 25 Furthermore, it efficiently protects the ammonium group to which it is attached 26 27 against H/D exchange in the gas phase (40-41 ). Consequently, one would expect that 28 29 a complete exchange of all labile hydrogen atoms in the dendrimer periphery is 30 31 32 possible, if the crown ether moves around. Instead, 3n hydrogen atoms should be 33 34 protected against exchange in a dendrimer-crown complex containing n crown ethers, 35 36 37 when the crown ether is positionally fixed. 38 39 Dendritic crown ether/ammonium complexes can easily be generated by ESI 40 41 42 when a mixture of host and guest in methanol with 1% formic or acetic acid is 43 44 sprayed. A broad charge state distribution (up to z = +8 for G5 ) with various 45 46 47 stoichiometries of crown molecule bound to dendrimer (up to n = 5 for G5 ) is 48 49 observed. The H/D exchange experiments were carried out with complexes of G1 to 50 51 G5 POPAM dendrimers. Figure 8a shows the H/D exchange results from G1 . When 52 53 54 there is no crown bound to singly protonated G1 , all labile hydrogen atoms are 55 56 rapidly exchanged. The exchange is almost complete after ca. 50 ms. A doubly 57 58 charged 1:1 complex which parallels the singly protonated POPAM since it also bears 59 60 one free ammonium site, undergoes a similarly fast exchange. Already after 50 ms,

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1 2 3 the whole isotope pattern has moved behind the threshold at which is would be 4 5 6 expected to come to a halt when three were protected against exchange. 7 8 Instead, a special case is encountered, when both ammonium sites are occupied by 9 10 11 crown ethers: The exchange is almost completely suppressed. The last experiment 12 13 shows that the exchange proceeds through a so-called ( 40-41 ) relay mechanism 14 15 (Figure 8b). One ammonium site must remain unoccupied in order to permit a fast 16 For Peer Review Only 17 18 H/D-exchange process. 19 20 G5 21 The same scenario is found up to POPAM dendrimer complexes. The 22 23 results for G2 are shown in Figure 8c, those obtained with G3 and G4 in Figure 9. 24 25 The following G2 experiment further confirmed the exchange behavior observed in 26 27 28 G1 (Figure 8c). These results clearly demonstrate the crown ethers to move quite 29 30 rapidly along the dendrimer periphery. 31 32 33 Mechanistically, the crown ether spacewalk must proceed stepwise: A new 34 35 hydrogen bridge is formed with a neighboring amine followed by the release of one to 36 37 38 the old binding site. Then the next new hydrogen bond is formed and so on. A 39 40 dissociation-reassociation mechanism can safely be excluded, because the crown ether 41 42 would not return to the dendrimer once it has dissociated in the high vacuum inside 43 44 45 the mass spectrometer. Similarly, a back-side attack mechanism with an intermediate 46 47 in which both binding sites are fully bound is unlikely. This would require much 48 49 longer alkyl chains between the two binding sites. So far, the mechanism is quite 50 51 52 clear. One question, however, remains: Does the crown ether move as a neutral crown 53 54 from one ammonium site to another ammonium site or does it move together with a 55 56 57 proton from an ammonium to a neutral amino group? These two scenarios are shown 58 59 in Figures 10a,b and can be resolved by H/D exchange experiments on 1,12- 60 diaminododecane as a model compound mimicking the dendrimer (Figure 10c).

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Figure 8. (a) Left: Left: H/D exchange experiment with singly protonated G1 (0 and 55 50 ms reaction time). Center: H/D exchange of all ten NH protons of the doubly 56 protonated 1:1 18-crown-6/ G1 complex (0 and 50 ms). Right: An extremely slow H/D 57 exchange is observed for the doubly charged 2:1 18-crown-6/ G1 complex (0 and 1000 58 ms). (b) A "relay" mechanism for the gas-phase H/D exchange at protonated POPAM 59 2+ 60 dendrimers explains why the exchange is so slow in [18C6 2@G1 +2H] . (c) The same scenario is observed for higher generation dendrimers.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Figure 9. H/D exchange experiments conducted with 18-crown-6 complexes of (a) 50 G3 and (b) G4 POPAM dendrimers. Already after 50 ms, the exchange has proceeded 51 beyond the positions at which it would be expected to stop, if the crown ethers would 52 53 protect the ammonium protons against exchange (vertical solid arrows). Minor signals 54 in the spectra are due to the typical defects in the dendrimer structure which 55 unavoidably accumulate in the higher generations. 56 57 58 59 60 Now, the doubly protonated 1:1 complex bears two ammonium groups, one of which is bound to the crown ether. This corresponds to the situation in Figure 10a.

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1 2 3 Exchange of only three hydrogen atoms from the free ammonium ion would indicate 4 5 6 the crown to be positionally fixed and rule out the ammonium-ammonium scenario. 7 8 An exchange of all six protons would clearly show that a crown movement between 9 10 11 two ammonium ions is indeed possible. The singly protonated 1:1 complex, instead, 12 13 has one ammonium group carrying the crown ether and a free ammonium group and 14 15 thus corresponds to the situation in Figure 10b. Exchange of only two protons would 16 For Peer Review Only 17 18 again indicate the crown to be fixed at the ammonium ion and would rule out the 19 20 ammonium-to-amine scenario, while the exchange of all five protons would provide 21 22 evidence for the transfer of a protonated crown ether. The result is clearcut: The 23 24 25 doubly charged complex only exchanges three hydrogen atoms, while all five can be 26 27 exchanged in the singly protonated complex. This clearly indicates a protonated 28 29 crown ether to move from the ammonium to the amine terminus of the DAD chain. 30 31 32 Likely, charge repulsion prohibits the ammonium-to-ammonium scenario. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 10. Two possible mechanisms for crown’ spacewalk: (a) ammonium-to- 58 59 ammonium scenario; (b) ammonium-to-amine scenario. (c) The model compound 60 1,12-diaminododecane (DAD).

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1 2 3 Conclusions 4 5 6 Three applications of tandem mass spectrometry in dendrimer host-guest chemistry 7 8 9 show the versatility and the potential of this method: Stoichiometries can be 10 11 determined. In competition experiments, the binding strengths of different guest can 12 13 be compared and yield information on the binding mode. Comparison with the 14 15 16 solution bindingFor parameters Peer may allow Review to draw conclusions Only on the similarities and the 17 18 solvation-mediated differences. In particular, however, it is interesting to investigate 19 20 21 the reactivity in the gas phase, because all dynamic host-guest exchange processes are 22 23 suppressed in the gas phase. Consequently, only direct dissociation reactions or 24 25 intramolecular processes can occur providing insight into a completely new reactivity 26 27 28 which cannot easily be examined in solution. In our examples, the energetic issues are 29 30 closely related to structural aspects as well as reactivity. Through a detailed analysis 31 32 of fragmentation reactions and their relative energies, indications for backfolding 33 34 35 could be obtained. With the help of bimolecular reactions, i.e. the H/D exchange with 36 37 methanol-OD, insight into the intramolecular dynamics and molecular mobility in 38 39 dendrimer guest complexes could be obtained. 40 41 42 43 44 45 References 46 47 48 (1) Baars, M.W.P.L.; Meijer, E.W. Top. Curr. Chem. 2000 , 210 , 131. 49 (2) Zimmerman, S.C.; Lawless, L.J. Top. Curr. Chem. 2001 , 217 , 95. 50 (3) Hecht, S.; Fréchet, J.M. Angew. Chem. 2001 , 113 , 76; Angew. Chem. Int. Ed. 2001 , 40 , 74. 51 (4) Martos, V.; Castreño, P.; Valero, J.; de Mendoza, J. Curr. Opin. Chem. Biol. 2008 , 12 , 698. 52 (5) Stiriba, S.E.; Frey, H.; Haag, R. Angew. Chem. 2002 , 114 , 1385; Angew. Chem. Int. Ed. 2002 , 41 , 53 1329. 54 (6) Longmire, M.; Choyke, P.L.; Kobayashi, H., Curr. Top. Med. Chem. 2008 , 8, 1180. 55 (7) Majoros, I.J.; Williams, C.R.; Baker Jr., J.R. Curr. Top. Med. Chem. 2008 , 8, 1165. 56 (8) Vögtle, F.; Gestermann, S.; Kauffmann, C.; Ceroni, P.; Vicinelli, V.; Balzani, V. J. Am. Chem. Soc. 57 2000 , 122 , 10398. 58 (9) Jones, J.W.; Bryant, W.S.; Bosman, A.W.; Janssen, R.A.J.; Meijer, E.W.; Gibson, H.W. J. Org. 59 Chem. 2003 , 68 , 2385. 60 (10) Paleos, C.M.; Tsiourvas, D.; Sideratou, Z.; Tziveleka, L. Biomacromolecules 2004 , 5, 524. (11) Nijhuis, C.A.; Huskens, J.; Reinhoudt, D.N. J. Am. Chem. Soc. 2004 , 126 , 12266.

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1 2 3 (12) Ashton, P.R.; Boyd, S.E.; Brown, C.L.; Nepogodiev, S.A.; Meijer, E.W.; Peerlings, H.W.I.; 4 Stoddart, J.F. Chem. Eur. J. 1997 , 3, 974. 5 (13) Haag, R. Angew. Chem. 2004 , 116 , 280; Angew. Chem. Int. Ed. 2004 , 43 , 278. 6 (14) Svenson, S.; Tomalia, D.A. Adv. Drug Deliver. Rev. 2005 , 57 , 2106. 7 (15) Calderón, M.; Quadir, M.A.; Sharma, S.K.; Haag, R. Adv. Mater. 2010 , 22 , 190. 8 (16) Shcharbin, D.; Pedziwiatr, E.; Bryszewska, M. J. Control. Release 2009 , 135 , 186. 9 (17) Biricova, V.; Laznickova, A. Bioorg. Chem. 2009 , 37 , 185. 10 (18) Giansante, C.; Mazzanti, A.; Baroncini, M.; Ceroni, P.; Venturi, M.; Klärner, F.G.; Vögtle, F. J. 11 Org. Chem. 2009 , 74 , 7335. 12 (19) Schalley, C.A.; Baytekin, B.; Baytekin, H.T.; Engeser, M.; Felder, T.; Rang, A. J. Phys. Org. 13 Chem. 2006 , 19 , 479. 14 (20) Blais, J.C.; Turrin, C.O.; Caminade, A.M.; Majoral, J.P. Anal. Chem. 2000 , 72 , 5097. 15 (21) Hummelen, J.C.; Dongen, J.L.J.; Meijer. E.W. Chem. Eur. J. 1997 , 3, 1489. 16 (22) Schalley,For C.A.; Springer, Peer A. Mass Spectrometry Review and Gas-Phase ChemistryOnly of Non-Covalent 17 Complexes. Wiley: Hoboken/NJ, 2009 . 18 (23) Baytekin, B.; Baytekin, H.T.; Schalley, C.A. Org. Biomol. Chem. 2006 , 4, 2825. 19 (24) Schalley, C.A. Mass. Spectrom. Rev. 2001 , 20 , 253. 20 (25) Weimann, D.P.; Schalley, C.A. Supramol. Chem. 2008 , 20 , 117. 21 (26) Schalley, C.A. Int. J. Mass Spectrom. 2000 , 194 , 11. 22 (27) Mulder, A.; Huskens, J.; Reinhoudt, D.N. Org. Biomol. Chem. 2004 , 2, 3409. 23 (28) Baars, M.W.P.L.; Karlsson, A.J.; Sorokin, V.; Waal, B.F.W.; Meijer, E.W. Angew. Chem. 2000 , 24 112 , 4432; Angew. Chem. Int. Ed. 2000 , 39 , 4262. 25 (29) Boas, U.; Sontjens, S.H.; Jensen, K.J.; Christensen, J.B.; Meijer, E.W. ChemBioChem 2002 , 3, 26 433. 27 (30) Galeazzi, S.; Hermans, T.M.; Paolino, M.; Anzini, M.; Mennuni, L.; Giordani, A.; Caselli, G.; 28 Makovec, F.; Meijer, E.W.; Vomero, S.; Cappelli, A., Biomacromolecules 2010 , 11 , 182. 29 (31) Broeren, M.A.; van Dongen, J.L.; Pittelkow, M.; Christensen, J.B.; van Genderen, M.H.; Meijer, 30 E.W. Angew. Chem. 2004 , 116 , 3579; Angew. Chem. Int. Ed. 2004 , 43 , 3557. 31 (32) Pittelkow, M.; Nielsen, C.B.; Broeren, M.A.; van Dongen, J.L.; van Genderen, M.H.; Meijer, 32 E.W.; Christensen, J.B. Chem. Eur. J. 2005 , 11 , 5126. 33 (33) Klärner, F.G.; Kahlert, B. Acc. Chem. Res. 2003 , 36 , 919. 34 (34) Schalley, C.A.; Verhaelen, C.; Klärner, F.G.; Hahn, U.; Vögtle, F. Angew. Chem. 2005 , 117 , 481; 35 Angew. Chem. Int. Ed. 2005 , 44 , 477. 36 (35) Winkler, H.D.; Weimann, D.P.; Springer, A.; Schalley, C.A. Angew. Chem. 2009 , 121 , 7382; 37 Angew. Chem. Int. Ed. 2009 , 48 , 7246. 38 (36) Weimann, D.P.; Winkler, H.D.; Falenski, J.A.; Koksch, B.; Schalley, C.A. Nature Chem. 2009 , 1, 39 573. 40 (37) Lifshitz, C. Int. J. Mass Spectrom. 2004 , 234 , 63. 41 (38) Cram, D.J.; Cram, J.M. Acc. Chem. Res. 1978 , 11 , 8. 42 (39) Maleknia, S.; Brodbelt, J. J. Am. Chem. Soc. 1993 , 115 , 2837. 43 (40) Lee, S.W.; Lee, H.N.; Kim, H.S.; Beauchamp, J.L. J. Am. Chem. Soc. 1998 , 120 , 5800. 44 (41) Wyttenbach, T.; Bowers, M.T. J. Am. Soc. Mass Spectrom. 1999 , 10 , 9. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 2010-03-15 4

5 zhenhuiqi 6 7 8 发件人： Rights DE 9 发送时间： 2010-03-15 13:17:46 10 11 收件人： [email protected] 12 抄送： 13 14 主题： AW: Form: Permission request 15 16 Dear Customer,For Peer Review Only 17 Thank you for your email. 18 - 19 We hereby grant permission for the requested use expected that due credit is given to th 20 e original source. 21 - For material published before 2007 additionally: Please note that the (co- 22 )author's permission is also required. 23 If material appears within our work with credit to another source, authorisation from that 24 source must be obtained. 25 Credit must include the following components: 26 - 27 Books: Author(s)/ Editor(s) Name(s): Title of the Book. Page(s). Publication year. Copyr 28 ight Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission. 29 - 30 Journals: Author(s) Name(s): Title of the Article. Name of the Journal. Publication year. 31 32 Volume. Page(s). Copyright Wiley- 33 VCH Verlag GmbH & Co. KGaA. Reproduced with permission. 34 With kind regards 35 Bettina Loycke 36 ********************************************* 37 Bettina Loycke 38 Senior Rights Manager 39 Wiley-VCH Verlag GmbH & Co. KGaA 40 Boschstr. 12 41 69469 Weinheim 42 Germany 43 Phone: +49 (0) 62 01- 606 - 280 44 Fax: +49 (0) 62 01 - 606 - 332 45 Email: [email protected] 46 ______47 Wiley-VCH Verlag GmbH & Co. KGaA 48 Location of the Company: Weinheim 49 Chairman of the Supervisory Board: Stephen Michael Smith 50 Trade Register: Mannheim, HRB 432833 51 General Partner: John Wiley & Sons GmbH, Location: Weinheim 52 Trade Register Mannheim, HRB 432296 53 Managing Directors : Christopher J. Dicks, Bijan Ghawami, William Pesce 54 -----Ursprüngliche Nachricht----- 55 Von: [email protected] [mailto:[email protected]] 56 57 Gesendet: Montag, 15. März 2010 13:15 58 An: Rights DE 59 Betreff: Form: Permission request 60 Form: Permission request Path: Service Permission Request Language: en Adress

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1 2 3 First Name: Zhenhui 4 Surname: Qi 5 Street: Takustr.3 6 Zip code: 14159 7 City: Berlin 8 Country: Germany 9 Fax: ++49 (0)30 838 55 817 10 11 E-mail: [email protected] 12 Media Type: journal 13 Author or Editor: 14 ISBN: 15 Journal Title: Angewandte Chemie International Edition 16 Journal Month:For JulyPeer Review Only 17 Journal Year: 2004 18 Journal Volume: 43 19 Media Type: journal 20 Page No: Scheme 1 and Figure 1 21 Website or Intranet: yes 22 University/Institute: 23 Instructor: Christoph A. Schalley 24 Course: no 25 Wiley Author: no 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Dear Chris, 4

5 6 It is my great pleasure to give permission and many thanks for taking our work into account. 7 8 With best regards, 9 10 Bert 11 12 13 14 From: Prof. Dr. Christoph Schalley [mailto:[email protected]] 15 Sent: maandag 15 maart 2010 14:59 16 To: Meijer, E.W.For Peer Review Only 17 Cc: [email protected] 18 Subject: Copyright permission 19 20 Dear Bert, 21 22 Supramolecular Chemistry is organizing a memorial issue for Dmitry Rudkevich. We have put together 23 a mini-review on dendrimer host-guest chemistry in the gas phase (manuscript is attached) 24 highlighting your nice work “multivalency in the gas phase” in Angewandte. May we use the Figure 25 again (Figure 2 in the manuscript)? Some time ago, you already permitted me to use it in a book, but I 26 wanted to be sure that it is ok, if we use it again. 27 28 Best wishes, 29 Chris 30 31 32 33 ------34 Prof. Dr. Christoph A. Schalley 35 Institut für Chemie und Biochemie der Freien Universität Berlin 36 Takustr. 3 37 14195 Berlin 38 Germany 39 40 phone: +49-(0)30 838-52639 41 fax: +49-(0)30 838-55817 42 email: [email protected] 43 http://www.chemie.fu-berlin.de/~schalley 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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