Clustering of Giant Unilamellar Vesicles (GUVs) Promoted by

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Clustering of Giant Unilamellar Vesicles (GUVs) Promoted by Covalent and Non-Covalent Bonding of Functional Groups at Membrane-Embedded Peptides Nicolai Stuhr-Hansen, Charikleia Despoina Vagianou, and Ola Blixt Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00394 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 20, 2019

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Bioconjugate Chemistry

Clustering of Giant Unilamellar Vesicles (GUVs) Promoted by Covalent and Non-Covalent Bonding of Functional Groups at Membrane-Embedded Peptides

Nicolai Stuhr-Hansen,*† Charikleia-Despoina Vagianou,† and Ola Blixt* Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. E-mail: [email protected] SH Peptide 2

Peptide 2 /Streptavidin

O2

CrossBinding

Air oxidn.

Rh2+ and/or Streptavidin O

OPh H2NHN Peptide 2

Ligation GUV-clustering Peptide 2

Complexation

Peptide 2

+ = Peptide, = WGA-Lectin,

= Cholesteryl, = Biotin,

= GUV, SH = Thiol,

= BisPy-complex,

= GlcNAc,

= Triazole

ABSTRACT: Access to clusters of cell-sized globular objects such as giant unilamellar vesicles (GUVs) is of increasing interest due to their potential applications in prototissue and cell-cell adhesion studies. Aggregations of GUVs by four different approaches were observed via covalent as well as non-covalent bond participations of functional groups at membrane embedded cholesterylpeptides using optical microscopy. Passive air oxidation of GUV-surface thiols into trans-GUV disulfide bonds promoted multivesicle aggregation. Aggregations of GUVs into multi-clusters were also achieved by introduction of bispyridyl-ligand substituted peptides into GUV-membranes succeeded by rhodium diacetate mediated vesicle clustering, and furthermore, by co-installing a biotin moiety streptavidin addition attenuated the clustering effect visualized by formation of compact superaggregated GUV-multiclusters. Contacting between two different GUV-populations, i.e. GUV-hetero-connection, was achieved by trans-GUV phenyl ester-hydrazine ligations producing GUV-hetero-clusters. Indirectly, GUV-clustering was achieved by strain-promoted azide-alkyne cycloaddition (SPAAC) reacting bicyclononyne (BCN)-GUVs with azido-GlcNAc succeeded by biotinylated wheat germ agglutinin (WGA)- (lectin/streptavidin incubation arousing cross-binding of GUVs.

Keywords: Giant unilamellar vesicles (GUVs), vesicle aggregation, BCN-peptides, proto-tissue formation, membrane protein reconstitution.

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Introduction Giant unilamellar vesicles (GUVs)1, 2 can be produced by electroformation3, 4 as spheres consisting of one lipid bilayer membrane on micron scale. Despite GUVs are highly polydisperse only allowing for experiments with rather small numbers of GUVs they have been extensively utilized as models for natural cells.5-7 GUVs have been shown to have contact with their surroundings by transmembrane diffusion of small molecules8 or installation of protein complexes9 in the membrane such as ion channels10 introducing transmembrane transport. Interconnections between individual GUVs have been stimulated by physical techniques such as electrofusion11, 12 merging two smaller GUVs into one large GUV. Molecular recognition of bilayer vesicles13 has essentially been exploited as a key factor in making the vesicles achieve more natural cell-like behavior by insertions of diverse host-guest systems enabling them to interact specifically with their surroundings. Supramolecularly based fusion14 was achieved by DNA-base pairing between strands on different GUVs to constructively intercontact GUVs stimulating membrane fusion.15, 16 Kros et al.17 introduced a GUV-fusion18 concept based on supramolecular interactions by positioning small peptide hybrids, possessing all functional aspects of the biological membrane fusion regulator SNARE19 (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein-recognition domain at GUV surfaces, fixating GUVs by peptidepeptide interactions promoting liposome fusion allowing for fusogenicity20, 21 studies. Recently, this SNARE-peptide based fixation principle was extended to controlled fusion cascade processes involving different GUV-populations.22 Vesicle-clustering without observation of successive membrane fusion was first introduced in 199923 by metal-ligand recognition based assemblies upon treatment of chelator covered vesicles with iron(II) salt. Thorough metal-ligand complexation studies24 demonstrated that balancing of metal ions vs. chelator/vesicle systems determined if fusion or clustering of vesicles were dominant.25 Vesicle aggregations26 may contain valuable information regarding the mechanisms involved in cell-cell adhesion, GUV-cell communication,27 GUV-carbon nanotube interactions,28 GUVsurface nanostructure interactions,29 and ideally, be utilized in fabrication of prototissue.30 Assembling globular objects with dimensions similar to GUVs, synthetic protocells made from proteinosomes31, has most recently been utilized in production of advanced prototissues,32 which further stimulated our interest in methods for obtaining GUV-aggregates. Several chemically based methods for clustering of GUVs have been described via reactions of different molecular entities positioned at the vesicle surfaces, however, all based on a lipid anchor33 linked to a reactive group responsible for the chemical contacting with surroundings. Nevertheless, rather incomparable techniques have been utilized for monitoring the aggregation processes in each individual report covering recordings of electron micrographs23 to UV-spectroscopy34 measurements. By improving procedures for insertions of various chemically functionalizable entities into GUVmembranes succeeded by appropriate chemical stimulations the intention was to establish access to facile clustering procedures, and most importantly, to enable direct comparisons between diverse sets of GUV-clustering events utilizing microscopy monitoring. We envisioned that chemical reactivities of functionalized cholesterylpeptides equipped with different recognition moieties inserted in GUVmembranes would arouse clustering events upon chemical stimulations dependent of the nature of the specific chemically reactive head-groups groups, and that all individual clustering events could be monitored with the same microscopy technique. Publications by our group on synthesis of highly functionalized cholesterol substituted peptides35 via a cholesterylated Fmoc-amino acid36 studied in vesicles seemed well-suited starting points for synthesis of a series of diversely functionalized cholesterylpeptides subsequently inserted into GUVs studied undergoing aggregations upon appropriate chemical stimulations. The present paper describes four sets of GUV-clustering studies, all leading to formations of GUV-colonies,37 facilitated by covalent, i.e. disulfide formation and phenylesterhydrazine ligation, and non-covalent, i.e. metal-complexation and biotin-streptavin binding, chemical ACS Paragon Plus Environment

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bond formations of functional groups at the GUV-membrane exteriors affecting gross cross-linking fixations into multi-vesicle aggregates. Results and discussion In order to install functional entities at GUV surfaces molecular designs were considered linking reactive groups to cholesterol in analogy to early work on vesicle functionalization with reactive cholesterylated small molecules.34 We have previously developed solid-phase peptide synthesis (SPPS) based procedures for preparations of diverse glycan functionalized fluorophore labelled cholesterylpeptides for insertions into GUV membranes experiencing specific recognitions with lectins,35, 38 and envisioned that far simpler minimalistic constructs equipped with molecular recognition units spaced to a cholesterol moiety with a short amino acid linker could be similarly prepared and subsequently inserted into GUV membranes. Short peptides consisting of a cholesterylated amino acid part PEG-spaced to a recognition function situated at the N-terminal were prepared by SPPS implementing our previously reported inherently cholesterylated Fmoc-amino acid, FmocAla(CholesterylTriazyl)-OH,36 and Fmoc-OEG2-OH (Fmoc-O2Oc-OH) as ordinary Fmoc-amino acids in SPPS sequences. For mounting a reactive head-group an amino acid containing a thiol or an azido moiety the sequences were terminated by coupling Fmoc-Tritylcysteine (Fmoc-Cys(Trt)-OH) or Fmocazidolysine (Fmoc-Lys(N3)-OH), affording the cholesterylated Cys-peptide 1 and azidopeptide 2, respectively (Scheme 1). BCN-peptides were synthesized by installation of BCN-carbamates at lysine side-chains, however, this transformation necessitated conductance as a post-SPPS step39 due to sensitivity of the BCN-moiety during acidic cleavage from resin.35 Lysine peptides were generated on resin omitting removal of the final N-terminal Fmoc-group at the end of the SPPS sequences. The peptides were cleaved off resin using a TFA-based cleavage cocktail, reacted with (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl Nsuccinimidyl carbonate (BCN-O-CO-OSu), and as a final solution step removal of the Fmoc-group by treatment with an excess of piperidine afforded the corresponding pure mono-BCN-peptide 3 and bisBCN-peptide 4 (Scheme 1). The procedure for synthesis of the bis-BCN peptide 4 is considered highly versatile for general synthesis of oligo-BCN-peptides by installation of BCN-units at lysine side-chains. Furthermore, the N-terminal amine remained protected throughout all conversions and released as a final step potentially opening for further post-SPPS functionalization.



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Scheme 1. Synthesis of head-functionalized cholesterylpeptides. Reagents and conditions: (i) SPPS omitting terminal Fmoc removal; (ii) TFA-TES-H2O (18:1:1); (iii) BCN-O(CO)-OSu, DIPEA, DMF, rt, 1 h; (iv) Piperidine, DMF, rt, 30 min. GUV-clustering by cysteine oxidation: GUVs were produced by co-electroformation (CoEF)36 from lipid solutions containing either of the three peptides 1-3 generating vesicles with standard dimensions (~10-20μm). However, microscopy showed that Cys-peptide covered GUVs (Cys-GUVs) behaved different from other vesicles, since clustering events were observed fixating numerous vesicles in multivesicle aggregates with close membrane-membrane contacts (Scheme 2a). The observed GUVaggregation phenomenon was most likely caused by spontaneous oxidation of Cys-thiols by passive diffusion of air forming trans-GUV cystine bonds fixating liposomes into clusters. It was assumed that variation of the cysteine peptide concentration in the lipid membrane would have an effect on the clustering tendency. By lowering the concentration of Cys-peptide in the CoEF mixture to 1/10 clustering was still observable even to the same extend. By further reducing the presence of membrane Cys-peptide to 1/100 of the original concentration, clustering was still predominant, but to a somewhat lower degree. At first upon dilution to 1/1000 of the original concentration significant decrease in clustering with fewer GUVs in each aggregate could be detected (see Supporting Information). Rather analogues, thioester covered GUVs was reported40 undergoing reversible aggregation upon reactions with dithiols, but it is not clear form these studies if thiol oxidations was partially responsible for causing clustering events. In order to confirm that trans-GUV interconnection of Cys-GUVs was resulting from cystine cross-linking by oxidations of Cys-thiols dithiothreitol (DTT) was added to the lipid mixture before CoEF. This reagent blocked disulfide bond formation resulting in production of GUVs displaying no clustering behavior at all (Scheme 2b), and solely non-aggregated vesicles could be observed after 14 days (see Supporting Information). Despite disulfide bonds were likely to be the actual inter-GUV fixating structural motifs responsible for inter-GUV fixations de-aggregations attempts by addition of DTT did not split up GUV-clusters but rather caused extensive GUV-bursting. The observed clustering of Cys-GUVs is the first example of covalent chemistry mediated vesicle aggregation. The membrane assemblies occurred spontaneously by passive diffusion of air into the serum oxidizing thiols with a sufficient rate to arouse fixation of vesicles. It is of high importance that clustering can be performed without external addition of chemicals, e.g. salts, since even small ionic and osmolarity changes may cause extensive GUV-decomposition and for instance GUVs have been reported decomposing under copper catalyzed azide-alkyne cycloaddition conditions.41


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Scheme 2. Cys-functionalized cholesterylpeptide. a) Cluster formations of GUVs decorated with cholesterylated Cys-peptide (Cys-GUVs) upon air oxidation; b) Behavior of Cys-GUVs coelectroformed in the presence of DTT. GUV-clustering via on-vesicle click chemistry: Azido-modifications of membrane lipids42 have been introduced for achieving liposome fusion. Rather similarly, installation of BCN-units in lipid membranes43 facilitated liposome functionalization with copper-free strain-promoted azide-alkyne cycloaddition44 (SPAAC) chemistry. Recently, SPAAC chemistry was reported triggering membrane fusion,45 but nevertheless, cycloaddition based chemistries seem yet unreported for facilitating GUVclustering. On the other hand, other single-walled objects with dimensions similar to GUVs such as polymersomes have been described46 experiencing shape transformations when undergoing SPAAC reactions between surface embedded azido functionalities and bis-BCN molecules. At cell-level SPAAC has recently been utilized47 for connecting BCN-functionalized nanoparticles at the exterior of azidecovered cell-membranes. GUV-clustering experiments were then conducted under copper-free SPAAC conditions by mixing azido-GUVs (prepared from peptide 2) with BCN-GUVs (prepared from peptide 3) without observation of dimerization/clustering tendency within several hours, even though these normally appearing GUVs displayed full stability. In analogy to above, homo-dimerization was attempted by adding the bis-BCN-peptide 4 to azido-GUVs and the diazidopeptide H-Lys(N3)-PheLys(N3)-Gly-OH 5 to BCN-GUVs, respectively. However, neither these dimerization mediators caused aggregations of GUVs (see Supporting Information). It was anticipated that a too low membrane surface representation of azido and BCN functionalities combined with a relatively low reaction rate of the SPAAC process obstructed sufficiently constructive inter-GUV fixations to arouse clustering. Instead, performing cycloadditions between BCN-GUVs and azido-glycans were utilized as alternative means for demonstrating SPAAC reactions at GUV surfaces, and furthermore, for indirect GUV-clustering. Thus, BCN-GUVs were treated with azido-GlcNAc (GlcNAc-N3) succeeded by incubation with its corresponding biotinylated glycan specific lectin, biotinylated wheat germ agglutinin (WGA) lectin, for qualitatively measuring the progress of the SPAAC glycosylation process after incubation with streptavidin (SA). When reacting the BCN-GUVs with a GlcNAc-azide solution ACS Paragon Plus Environment


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containing 1:100 of the concentration of BCN-peptide utilized in the standard GUV co-production protocol for 16 hours, intense GUV-clustering was observed upon incubation with biotinylated WGAlectin/SA (Scheme 3a). This was interpreted as completion of SPAAC clicking the major fraction of GlcNAc onto the lipid membranes followed by lectin binding, and bySA incubation cross-linking of the biotin handles caused a clustering effect. By performing an analogue SPAAC experiment employing an only ten times lower concentration of the GlcNAc-azide compared to the total concentration of BCNpeptide used for the GUV production correspondingly incubating with WGA-lectin after 16 hours gave a somewhat similar result (see Supporting Information), however, with a less pronounced clustering effect. In this situation a fraction of the glycan remained non-clicked in solution partially inhibiting surface lectin binding of membrane glycans, and consequently, a far less cross-linking capacity resulted in diminished GUV-clustering. Analogously, one-hour SPAAC reactions were also performed with various GlcNAc-azide:BCN ratios succeeded by incubations with biotinylatedWGA-lectin/SA. However, excess of free non-membrane bound GlcNAc-entities in solution, due to limited progress of the SPAAC, inhibited biotin-lectin/SA mediated GUV-clustering (Scheme 3b).

Scheme 3. SPAAC at BCN-functionalized GUV-surfaces. a) BCN-GUVs reacting with GlcNAc-N3 under SPAAC conditions for 16 hours monitored by WGA-lectin mediated clustering (GUV-crossbinding); b) Identical SPAAC for 1 hour succeeded by WGA-lectin incubation (pseudo-negative control). GUV-clustering by Rh2+-complexation assisted biotin-streptavidin binding: Since metal-ligand complexation has been applied for vesicle clustering23 and biotin/SA binding has been utilized for gluing nanoparticles onto vesicles48 we envisioned that combinations of those effects would amplify aggregations. Rhodium/bipyridine complexes are utilized in e.g. hydrogenation catalysts,49 and highly importantly, they possess very high stabilities.50 Thus, formation of similar rhodium complexes with participating ligands situated at different GUV-membranes would presumably serve as means for dragging GUVs together into clusters. We observed intensive superaggregated GUV-multiclusters (Scheme 4c) events by sequential addition of SA and rhodium diacetate or vice versa to chelator/biotinGUVs formed by CoEF of the bispyridyl/biotin cholesterylpeptide. A schematic overview of rhodium diacetate mediated GUV-clustering experiments is depicted in Scheme 4.


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Scheme 4. Schematic representation of rhodium(II) acetate mediated GUV-clustering experiments at biotin/BisPy-GUVs; a) Co-electroformation (CoEF) succeeded by Rh2OAC4addition afforded GUV-multiclusters; GUV-clustering resulting from spiking GUVs with different concentrations of Rh2OAc4: 1 nM, 1 M, and 1 mM. b) Biotin-GUVs forming vesiclemulticlusters upon spiking with SA: No added SA (negative control), SA-incubation for 24 hours, and 72 hours. c) Rh2+-based multiclusters incubated with SA afforded superaggregated GUV-multiclusters, and similarly, SA-based multiclusters treated with Rh2OAC4 afforded superaggregated GUV-multiclusters. In order to generate a bipyridyl-like analogue equipped with a cholesteryl anchor for adhesion to lipid membranes a minimalistic peptide construct was considered an appropriate target module. Utilization of our procedure36 for introduction of cholesteryl moieties into peptides by incorporating a pre-synthesized cholesterylated Fmoc-amino acid as a standard building block in solid-phase peptide synthesis (SPPS) and sequentially coupling three Fmoc-OEG2-amino acid (Fmoc-O2Oc-OH) units and then a bis-pyridyl Fmoc-amino acid51 facilitated generation of a desired construct. Termination of the SPPS-sequence by final HBTU-coupling of biotin at the N-terminal prior to cleavage off resin afforded the biotinylated bispyridyl substituted cholesterylpeptide 6. Upon CoEF conditions36 a mixture of 6 and DOPC produced GUVs with standard dimensions. Metal-complexation studies were then performed by spiking with different concentrations of rhodium diacetate and the clustering behaviour of the GUVs were then studied by microscopy. Initially, addition of dilute Rh2OAC4 (1 nM) solutions did not have a pronounced effect on the behaviour of the GUVs and only sporadic small GUV-clusters were observed. It was assumed ACS Paragon Plus Environment


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that the Rh-concentration was to low and too few trans-GUV surface complexes were formed to establish sufficient trans-vesicle fixation to affect GUV-multiclustering. However, upon spiking with a thousand-fold higher rhodium salt concentration (1 M) a dramatic change in GUVbehaviour was observed manifested by intense clustering into multi-vesicle aggregates. At this Rh-concentration there was a higher number of surface chelators complexing rhodium salt. Thus, more rhodium mono-chelates were available seeking a non-chelated bis-pyridyl ligand and far more trans-GUV complexes were formed. Consequently, constructive relative fixations of GUVs were established multiplying this aggregation effect into multi-membered GUVclusters. By further elevating the Rh-concentration by a factor thousand (1 mM) GUV-clustering tendency was far less abundant and multi-aggregated GUVs could not be observed (Scheme 4a). An explanation for the non-abundant GUV-multiclustering was most likely rhodium oversaturation of the chelator units disabling formation of double ligated trans-GUV metalcomplexes in sufficient amounts to affect constructive GUV-fixation. Examination of GUVs produced incorporating 6 by the standard CoEF procedure without additives showed, besides gross formation of normally behaving individual non-contacted GUVs, sporadical presence of GUV-miniclusters most likely formed by complexations of residual trace-metals in the MilliQ-water. In order to completely prevent formations of background complexes, and possibly to de-complex GUV-clusters, GUV CoEFs were conducted both in the presence of ethylenediaminetetraacetic acid (EDTA) as well as 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). However, instead of producing single GUVs, free of mini-clusters, these chelator co-additions lead to no GUV-formations at all presumably caused by introduction of too large changes in osmolarity (see Supporting Information section). Since a biotin handle was attached to the peptide, vesicles made by CoEF including 6 could also be considered as biotin-GUVs. Thus, upon incubation of these GUVs-clusters with SA intensive aggregation into GUV-multiclusters was observed after 24 hours. This phenomenon could be interpreted as a result of multivalent cross-binding capacity of SA due to its four biotin binding sites.52 Remarkably, when incubating for 100 hours superaggregated GUV-multiclusters were still observable, and furthermore, the clusters appeared even more compact (Scheme 4b). This could be due to the fact that the multiclusters have had longer time to rearrange into more energetically favorable packings with less free space. Aiming for even larger aggregates of GUVs the GUV-multiclusters generated by Rh2OAc4addition to the chelator-GUVs were treated with SA. Already after incubation for a one-hour period intense compacting clustering into superaggregated GUV-multiclusters was observed. By continuous incubation for 24 hours the compacting effect was even more dramatic and at this point extremely superaggregated GUV-multiclusters had formed. In a reverse approach the GUV-multiclusters made by treatment of biotin-GUVs with SA were spiked with a 1 M Rh2OAc4 solution. After incubation for 1 hour superaggregated GUV-multiclusters had clearly formed and further compacting was resulting by incubation for 24 hours. Both paths show amplifications of GUV-assembly effects by assisted clusterings into superaggregated GUVmulticlusters (Scheme 4c). GUV-hetero-clustering: Despite effective procedures for GUV-clusterings had been developed these were solely involving the same GUV type and may therefore be considered as homo-clusterings. Since SPAAC chemistry seemed inappropriate for achieving hetero-clustering it seemed necessary to search for alternative ligation techniques. It was assumed that native chemical ligation53 (NCL) procedures reacting a cysteine-terminated peptide with a peptide thioester, like SPAAC reactions, would proceed too slow to effect constructive contacting between two GUVs overall leading to clustering. Furthermore, air oxidation of cysteine thiols aroused spontaneous clustering making NCL based methods less ACS Paragon Plus Environment

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valuable in terms of specific hetero-clustering. Hydrazines are much more nucleophilic than amines, even upon assistance by local thiols as in cysteines due to nitrogen-nitrogen lone-pair repulsion, undergoing efficient reactions with thioesters. Aryl esters, in analogy to reactions of thioesters, have been reported undergoing NCL upon reaction with cysteines54 producing less obnoxious phenols instead of smelly thiols as NCL byproducts. Liposome interconnections have been described by reacting a membrane bound hydroxamate on one vesicle with a membrane bound p-nitrophenyl ester on another vesicle.34 It was envisioned that rather similarly a hydrazine moiety at one GUV would react with a phenyl ester functionality at another GUV at a sufficiently high rate to arouse clustering (Scheme 5a). Thus, we initiated research towards production of phenyl ester GUVs as well as hydrazine GUVs by attaching a phenyl ester carboxylic acid and hydrazinoglycine,55 respectively, at the N-terminals of cholesteryl peptides.

Scheme 5. Phenyl ester-hydrazine ligation interconnecting GUVs. Phenyl ester- and hydrazineterminated cholesterylpeptides by SPPS; hetero-clustering of corresponding phenylester-GUVs and hydrazine-GUVs. Reagents and conditions: i) SPPS; ii) PhOCO(CH2)2CO2H or (Boc)2NNH(Boc)CH2CO2H, HBTU, DIPEA, DMF, rt, 30 min; iii) TFA-TES-H2O (18:1:1). a) PhenylesterGUVs without additives (red) and hydrazine-GUVs without additives (green); b) Multivesicle clustering of hydrazine-GUVs and phenylester-GUVs observed after 1h post mixing 1:1; c) Interaction of hydrazine-GUVs (green) and phenylester-GUVs (red) obtained after 1h post mixing 1:9.

Preparation of phenyl ester-GUVs necessitated access to a phenyl ester substituted cholesterylpeptide. Incorporation of Fmoc-Ala(CholTriaz)-OH in a SPPS sequence facilitated generation of a cholesterylpeptide 7 on resin. By standard HBTU coupling 4-oxo-4-phenoxybutanoic acid56 followed by cleavage off resin utilizing TFA/triethylsilane(TES)/H2O afforded the phenyl ester terminated cholesterylpeptide 8. In order to prepare a hydrazinoglycine terminated peptide a fully tris-Bocprotected hydrazinoacetic acid was employed i.e. HBTU-coupling the tris-Boc protected hydrazinoglycine, [N,N,N′-tris(tert-butyloxycarbonyl)hydrazino]acetic acid,57 onto the N-terminal of 6. As a final step cleavage off resin using TFA/TES/H2O afforded the hydrazinoacetyl-terminated cholesterylpeptide 9 (Scheme 5b). A complete list of peptides utilized in these studies, their preparation and characterization data are presented in the supporting information section. ACS Paragon Plus Environment


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Phenylester-GUVs and hydrazino-GUVs were prepared from incorporating the phenylester cholesterylpeptide 8 and the hydrazine cholesterylpeptide 9, respectively, utilizing the standard electroformation protocol. Both types of GUVs displayed normal behavior when examined by microscopy (Scheme 5c). GUV-hetero-clustering experiments were then conducted by mixing equal amounts of phenylester-GUVs and hydrazino-GUVs. By studying the GUV-behavior it became apparent that GUV-multivesicle clusters had formed after incubation for 1 hour (Scheme 5d). Heteroclustered GUVs were still present after 24 hours to a similar extent. From these 1:1 phenyl ester-GUVs and hydrazine-GUVs mixing experiments it seemed that more hydrazine-GUVs than phenyl esterGUVs were present in the GUV-clusters. This could be explained as competing background hydrolysis of the phenylester in the aqueous buffer system diminishing the overall amount of reactive ester at the GUV-surface, and thus, a lowered reactivity of phenyl ester-GUVs in ligations with hydrazine GUVs. Clustering experiments were then performed reacting phenyl ester-GUVs with hydrazine-GUVs in a 9:1 ratio. The excess of phenyl ester-GUVs seemed to have a negative effect on vesicle aggregation and only small clusters were spotted in these cases (Scheme 5e).

Covalent chemistry based phenylester-hydrazine ligation proved useful for arousing various GUVhetero-clustering events and therefore the question arose if non-covalent interactions between different GUVs also would lead to hetero-clustering. Despite avidin has been used as glue connecting magnetic nanoparticles onto vesicles48 this multi-biotin binding protein has not been utilized for clustering GUVs. We then envisioned an alternative GUV-hetero-clustering strategy based upon non-covalent interactions was envisioned by uniting SA-GUVs and biotin-GUVs. Initially, SA labeling by a BCN-NHS ester. After installing this click-handle reacting the corresponding BCN-SA with azido-GUVs under SPAAC conditions. At this stage the major fraction of SA would supposedly be click attached to the GUVsurfaces. Incubating biotin-GUVs with SA-GUVs resulting in non-covalent SA-biotin overlapping hetero-connections (Scheme 6). In order to attach SA onto surfaces of GUVs BCN-units were covalently attached upon reaction with BCN-O(CO)OSu 10 in accordance with standard labeling protocols for attachment of fluorophore-NHSesters. After installing this click-handle the corresponding BCN-SA 11 was reacted overnight with azido-GUVs under SPAAC conditions. At this stage the major fraction of SA was supposedly click attached onto the GUV-surfaces. Clustering events were successively studied by incubation with various ratios of biotin-GUVs. By mixing SA-GUVs and biotin-GUVs in a 1:1-ratio no clustering was observed, but solely disrupted GUVs appeared. By adjustment of the SA-GUV:biotin-GUV ratio to 1:4 solely bursting of GUVs was again observed. However, by exchanging the SA-GUV:biotin-GUV ratio to 4:1 GUV-hetero-connections were observed manifested by formations of primarily hetero-dimers and hetero-trimers (Scheme 6). As proof of non-covalently based hetero-clustering microscopy clearly showed that contacting between individual GUVs were occurring solely between SA-GUVs and biotinGUVs.


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Scheme 6. Hetero GUV-connections by non-covalently binding SA/biotin head-groups. Schematic GUV-clustering event initiated by BCN-labeling of SA succeeded by SPAAC incubation onto azidoGUVs and then cross-reaction with biotin-GUVs; SA-GUVs:biotin-GUVs ratio 4:1: Predominantly GUV-hetero-dimer/trimers formed. It was thereby demonstrated that as alternatives to aggregations of one type of GUVs it was possible to expand the repertoire to encompass two different GUV-types. By ligations forming hydrazide bonds covalent connections between GUVs were formed and establishment of trans-GUV contacting based upon SA-biotin binding lead to non-covalent GUV-interconnections. These two chemically based GUVconnection types contain means for studying cell-cell contacting events on model basis without interference other cell-components. Furthermore, indirect SPAAC insertions of proteins on the surface of GUVs via cholesterol anchoring establish a new way of studying artificial cell surface proteins presumably perturbating the membrane structure to a lower degree than conventional membrane protein reconstitution.58-60 The procedure relies on the ability to BCN-label the protein without losing the activity of the protein and may be expandable to all proteins especially relevant for those involved in the construction of glycocalyx components. It is therefore possible to insert proteins onto the membranes lowering the overall GUV stabilities. If a protocol for labeling, i.e. with a fluorophore, of a protein with an NHS-ester has been established maintaining the overall activity of the protein, it can most certainly be extrapolated into GUV-surface covering of an active form of the corresponding protein. In conclusion, we successfully observed clustering of GUVs by chemically stimulating recognition units at membrane surface via four different pathways, two covalent routes forming trans-vesicle disulphide and hydrazide linkages, and two non-covalent routes based on metal-complex formations involving surface chelators and SA multi-binding of biotin-covered GUVs. Microscopy proved a valuable tool for independent evaluation of all four procedures since all multi-vesicle clustering events were clearly visible by microscopy. Furthermore, this technique gave sufficient qualitative measures of the aggregation degrees even for assessing attenuated degrees of clustering during metalcomplex/biotin-SA interplays. Trans-vesicle oxidations of surface cysteine units, despite requiring access to air, proceeded spontaneously like phenyl ester-hydrazine ligations producing multi-vesicle homo- and hetero-clusters, respectively, of same relative dimensions without added chemical stimuli making both methods directly applicable in aggregation studies due to the ease in preparation of the ACS Paragon Plus Environment


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active molecular entities inserted into the membranes. Two multi-binding phenomena gave the same outcome in terms of multi-vesicle clustering, metal-complexation of bis-Pyridyl-GUVs to rhodium salt and binding of several biotin-GUVs to one streptavidin unit. Both pathways were performed on identical GUVs covered with dually functionalized peptide entities proving independence between the two chemical recognition units, as further demonstrated by attenuation of vesicle aggregations by Ru2+/SA-interplays leading to clusters of clusters. The four developed clustering techniques are considered relatively orthogonal and may freely by combined as versatile ingredients in novel designs of prototissue assembly components. Materials and methods Materials: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) 18:1 Cis PC was purchased from Avanti Lipids and cholesterol from Sigma-Aldrich. The membrane marker Lissamine™ Rhodamine B 1,2Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium Salt (RhoB-DHPE) and N-(7Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt (NBD-PE) were purchased from Thermo Fisher Scientific. Lipids, cholesterol and cholesterylated peptides used for electroformation were dissolved in 9:1 chloroform/methanol. Streptavidin from Streptomyces avidinii was purchased from Sigma Aldrich. Solid-phase peptide synthesis: The peptides were manually synthesized on Fmoc-preprotected Rink TentaGel S-Ram™ resin (Rapp Polymere) (loading 0.23 mmol/gram) initially de-Fmoc’ed using 20% piperidine in DMF (1×5 and 1×15 min; 0.5 mL in each; DMF wash step in between) or on H-Glu(t-Bu)2-Chloro-trityl resin (Sigma-Aldrich) (loading 1.0 mmol/gram). Introductions of PEGylated, propargylated, azidofunctionalized, pyridyl-based chelator substituted, and cholesterylated amino acids were performed by utilization of Fmoc-OEG2-OH (O2Oc-OH) (Iris), Fmoc-propargylglycine-OH (Fmoc-Pra-OH) (Iris), Fmoc-azidolysine-OH (Fmoc-Lys(N3)-OH) (Iris), Fmoc-(BisPyridyl)lysine-OH51 (Fmoc-Lys(bisPy)-OH), and Fmoc-Ala(CholesterylTriazyl)-OH36 (Fmoc-Ala(CholTriaz)-OH), respectively, as standard protected Fmoc-amino acids in SPPS. Coupling of the consecutive amino acid was carried out with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and DIPEA (resin/amino acid/HBTU/DIPEA 1:4:4:8) in dry DMF (0.5 mL) for one hour. Dichloromethane (0.5 mL) was added to the coupling mixture when coupling FmocAla(CholesterylTriazyl)-OH. After draining and flow wash with DMF Fmoc deprotection was performed with 20% piperidine in DMF (1×5 and 1×15 min; 0.4 mL in each; DMF wash step in between). Standard Fmoc-protected amino acids with appropriate side-chain protection were used throughout. After final coupling the resins were washed with dichloromethane (5 x) and a cleavage cocktail consisting of a mixture of TFA/triethylsilane(TES)/H2O (18:1:1, 1 mL) was added. After vigorous shaking for 2 minutes moderate shaking was maintained for 1 h. Filtration, wash of the resin with additional dichloromethane (1 mL) and evaporation in a gentle stream of nitrogen gave the crude peptide. Ether-washes of crude cholesterylated peptides were completely omitted since the wet ether partly dissolved the peptide removing a major portion of it during standard double trituration. The peptides were dissolved in acetonitrile-water (1:1, 2 mL) and the solutions were filtered before HPLCpurifications succeeded by lyophilizations. Giant Unillamelar Vesicles (GUV) were grown61 by electroformation: a) GUVs containing cholesterylated Cys-peptide (8,4 mM, 0,84 mM, 0,084 mM or 0,0084 mM) preparation with or without DTT, cholesterylated BCN-peptide (1,3 mM), cholesterylated Azide-peptide (0,8 mM), biotinylated chelator-substituted cholesterylpeptide (1 mM), phenylester-terminated cholesterylpeptide (0,7mM), hydrazinoacetyl-terminated cholesterylpeptide (0,9 mM). Mix of DOPC 2,5 mM/ cholesterol 5,2 mM/ RhoB-DHPE 0,0125 mM/ cholesterylated modified-peptide were prepared in a ratio 90:5:0,5:4,5. Dithiothreitol (DTT) 10 mM was prepared in sucrose 290 mM for spiking in GUVs containing the ACS Paragon Plus Environment

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highest concentration of cholesterylated Cys-peptide (8,4 mM) and in chloroform:metahanol (9:1) for co-electroformation with cholesterylated Cys-peptide. Electroformation was performed in the Vesicle Prep Pro®, with ITO-coated glass slides and 8 mm rubber O-rings obtained from Nanion Technologies. Microscopy of GUVs: GUVs were imaged in a Nikon ECLIPSE 80i Microscope (Objective CFI Plan Achro 40X/N.A 0.65). Excitation wavelengths of 480 nm-FITC filter cube (for NBD-PE GUV membrane marker), 560 nm-Texas Red filter cube (for Rhodamine B-DHPE GUV membrane marker) were selected. All samples of GUVs were tested either on microscope slides immediately after electroformation or preincubated in Eppendorf tubes and tested afterwards. GUVs produced by DOPC/Cholesterol/ cholesterylated Cys-peptide with or without DTT co-electroformation were tested again 14 days post-electroformation. Modification of streptavidin with BCN-(CO)-OSu: Streptavidin (0.0182 mM in PBS, pH 7,34; 80 μl) was added to carbonate buffer (1 M, pH8,7; 10 μL). BCN-(CO)-OSu prepared in three different concentrations (0,1 M, 0,01 M and 0,001 M in DMF; 15 μL) and added in three different vials of streptavidin-carbonate buffer. The mixtures were left shaking for 3 hours at room temperature. Conjugated streptavidin used without further purification.

Supporting Information Experimental procedures are presented in the Supporting Information section. Furthermore, this section contains movies of spontaneous Cys-GUV-clustering events and details in SPAAC-based clustering processes. Author Information Corresponding Author * Dr. Nicolai Stuhr-Hansen, Department of Chemistry, Chemical Biology, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. E-mail: [email protected]. †These authors contributed equally. *Co-corresponding authors.

Acknowledgments The authors acknowledge support by the Danish Research Council (Innovationsfonden) and the framework of the EU ERASynBio project SynGlycTis to OB and the support from the Stiftelsen for Strategisk Forskning (SSF) to OB. Ms. Anette Andersen is thanked for carrying out high-resolution mass experiments.

References 1. Römer, W.; Berland, L.; Chambon, V.; Gaus, K.; Windschiegl, B.; Tenza, D.; Aly, M. R.; Fraisier, V.; Florent, J.-C.; Perrais, D., Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 2007, 450, 670-675. 2. Römer, W.; Pontani, L.-L.; Sorre, B.; Rentero, C.; Berland, L.; Chambon, V.; Lamaze, C.; Bassereau, P.; Sykes, C.; Gaus, K.; Johannes, L., Actin dynamics drive

membrane reorganization and scission in clathrinindependent endocytosis. Cell 2010, 140, 540-553. 3. Angelova, M. I.; Dimitrov, D. S., Liposome electroformation. Faraday Discuss. Chem. Soc. 1986, 81, 303-311. 4. Politano, T. J.; Froude, V. E.; Jing, B.; Zhu, Y., ACelectric field dependent electroformation of giant lipid vesicles. Colloids Surf. B. 2010, 79, 75-82. 5. Aroush, D. R.-B.; Yehudai-Resheff, S.; Keren, K.,



Electrofusion of giant unilamellar vesicles to cells. Methods Cell Biol. 2015, 125, 409-422. 6. Karzbrun, E.; Tayar, A. M.; Noireaux, V.; Bar-Ziv, R. H., Programmable on-chip DNA compartments as artificial cells. Science 2014, 345, 829-832. 7. Blain, J. C.; Szostak, J. W., Progress toward synthetic cells. Annu. Rev. Biochem. 2014, 83, 615-640. 8. Martini, L.; Mansy, S. S., Cell-like systems with riboswitch controlled gene expression. Chem. Commun. 2011, 47, 10734-10736. 9. Gokel, G. W.; Negin, S., Synthetic ion channels: from pores to biological applications. Acc. Chem. Res. 2013, 46, 2824-2833. 10. Yanagisawa, M.; Iwamoto, M.; Kato, A.; Yoshikawa, K.; Oiki, S., Oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel KcsA. J. Am. Chem. Soc. 2011, 133, 11774-11779. 11. Yanai, G.; Hayashi, T.; Zhi, Q.; Yang, K.-C.; Shirouzu, Y.; Shimabukuro, T.; Hiura, A.; Inoue, K.; Sumi, S., Electrofusion of mesenchymal stem cells and islet cells for diabetes therapy: a rat model. PloS one 2013, 8, e64499. 12. Zhang, P.; Yi, S.; Li, X.; Liu, R.; Jiang, H.; Huang, Z.; Liu, Y.; Wu, J.; Huang, Y., Preparation of triple-negative breast cancer vaccine through electrofusion with day-3 dendritic cells. PloS one 2014, 9, e102197. 13. Voskuhl, J.; Ravoo, B. J., Molecular recognition of bilayer vesicles. Chem. Soc. Rev. 2009, 38, 495-505. 14. Ma, M.; Bong, D., Controlled fusion of synthetic lipid membrane vesicles. Acc. Chem. Res. 2013, 46, 29882997. 15. Stengel, G.; Zahn, R.; Höök, F., DNA-induced programmable fusion of phospholipid vesicles. J. Am. Chem. Soc. 2007, 129, 9584-9585. 16. Chan, Y.-H. M.; van Lengerich, B.; Boxer, S. G., Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 2009, pnas. 0812356106. 17. Robson Marsden, H.; Elbers, N. A.; Bomans, P. H.; Sommerdijk, N. A.; Kros, A., A reduced SNARE model for membrane fusion. Angew. Chem. Int. Ed. 2009, 48, 23302333. 18. Lira, R. B.; Robinson, T.; Dimova, R.; Riske, K. A., Highly efficient protein-free membrane fusion: a giant vesicle study. Biophys. J. 2019, 116, 79-91. 19. Witkowska, A.; Jablonski, L.; Jahn, R., A convenient protocol for generating giant unilamellar vesicles containing SNARE proteins using electroformation. Sci. Rep. 2018, 8, 9422. 20. Versluis, F.; Voskuhl, J.; van Kolck, B.; Zope, H.; Bremmer, M.; Albregtse, T.; Kros, A., In situ modification of plain liposomes with lipidated coiled coil forming

Page 14 of 17

peptides induces membrane fusion. J. Am. Chem. Soc. 2013, 135, 8057-8062. 21. Crone, N. S.; Minnee, D.; Kros, A.; Boyle, A. L., Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning. Int. J. Mol. Sci. 2018, 19, 211. 22. Vogel, S.; Löffler, P. M.; Ries, O.; Rabe, A.; Okholm, A. H.; Thomsen, R. P.; Kjems, J., A DNA‐Programmed Liposome Fusion Cascade. Angew. Chem. Int. Ed. 2017, 56, 13228-13231. 23. Constable, E. C.; Mundwiler, S.; Meier, W.; Nardin, C., Reversible metal-directed assembly of clusters of vesicles. Chem. Commun. 1999, 1483-1484. 24. Lim, C. W.; Crespo-Biel, O.; Stuart, M. C.; Reinhoudt, D. N.; Huskens, J.; Ravoo, B. J., Intravesicular and intervesicular interaction by orthogonal multivalent host–guest and metal–ligand complexation. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 6986-6991. 25. Richard, A.; Marchi-Artzner, V.; Lalloz, M.-N.; Brienne, M.-J.; Artzner, F.; Gulik-Krzywicki, T.; GuedeauBoudeville, M.-A.; Lehn, J.-M., Fusogenic supramolecular vesicle systems induced by metal ion binding to amphiphilic ligands. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 1527915284. 26. Webb, S. J.; Trembleau, L.; Mart, R. J.; Wang, X., Membrane composition determines the fate of aggregated vesicles. Org. Biomol. Chem. 2005, 3, 3615-3617. 27. Lentini, R.; Martín, N. Y.; Forlin, M.; Belmonte, L.; Fontana, J.; Cornella, M.; Martini, L.; Tamburini, S.; Bentley, W. E.; Jousson, O., Two-way chemical communication between artificial and natural cells. ACS Cent. Sci. 2017, 3, 117-123. 28. Pérez-Luna, V.; Moreno-Aguilar, C.; Arauz-Lara, J. L.; Aranda-Espinoza, S.; Quintana, M., Interactions of Functionalized Multi-Wall Carbon Nanotubes with Giant Phospholipid Vesicles as Model Cellular Membrane System. Sci. Rep. 2018, 8, 17998. 29. Cheeseman, S.; Truong, V. K.; Walter, V.; Thalmann, F.; Marques, C. M.; Hanssen, E.; Vongsvivut, J.; Tobin, M.; Baulin, V. A.; Juodkazis, S., Interaction of Giant Unilamellar Vesicles with the Surface Nanostructures on Dragonfly Wings. Langmuir 2019. 30. Mantri, S.; Sapra, K. T., Evolving protocells to prototissues: rational design of a missing link. Biochem. Soc. Trans. 2013, 41, 1159-1165. 31. Liu, X.; Zhou, P.; Huang, Y.; Li, M.; Huang, X.; Mann, S., Hierarchical proteinosomes for programmed release of multiple components. Angew. Chem. Int. Ed. 2016, 55, 7095-7100. 32. Gobbo, P.; Patil, A. J.; Li, M.; Harniman, R.; Briscoe, W. H.; Mann, S., Programmed assembly of synthetic protocells into thermoresponsive prototissues. Nat. Mater. 2018, 17, 1145-1153.

14


Page 15 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 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 56 57 58 59 60


33. Khmelinskaia, A.; Franquelim, H. G.; Petrov, E. P.; Schwille, P., Effect of anchor positioning on binding and diffusion of elongated 3D DNA nanostructures on lipid membranes. J. Phys. D Appl. Phys. 2016, 49, 194001. 34. Menger, F. M.; Azov, V. A., Cytomimetic modeling in which one phospholipid liposome chemically attacks another. J. Am. Chem. Soc. 2000, 122, 6492-6493. 35. Stuhr-Hansen, N.; Vagianou, C.-D.; Blixt, O., Synthesis of BODIPY‐Labeled Cholesterylated Glycopeptides by Tandem Click Chemistry for Glycocalyxification of Giant Unilamellar Vesicles (GUVs). Chem. Eur. J. 2017, 23, 9472-9476. 36. Stuhr‐Hansen, N.; Madl, J.; Villringer, S.; Aili, U.; Römer, W.; Blixt, O., Synthesis of Cholesterol‐Substituted Glycopeptides for Tailor‐Made Glycocalyxification of Artificial Membrane Systems. ChemBioChem 2016, 17, 1403-1406. 37. Carrara, P.; Stano, P.; Luisi, P. L., Giant vesicles “colonies”: a model for primitive cell communities. ChemBioChem 2012, 13, 1497-1502. 38. Vagianou, C.-D.; Stuhr-Hansen, N.; Moll, K.; Bovin, N.; Wahlgren, M.; Blixt, O., ABO Blood Group Antigen-Decorated Giant Unilamellar Vesicles Exhibit Distinct Interactions with Plasmodium falciparum-Infected Red Blood Cells. ACS Chem. Biol. 2018, 13, 2421-2426. 39. Witte, C.; Martos, V.; Rose, H. M.; Reinke, S.; Klippel, S.; Schröder, L.; Hackenberger, C. P., Live‐cell MRI with Xenon Hyper‐CEST Biosensors Targeted to Metabolically Labeled Cell‐Surface Glycans. Angew. Chem. Int. Ed. 2015, 54, 2806-2810. 40. Berg, S. A.; Ravoo, B. J., Dynamic reactions of liposomes. Soft Matter 2014, 10, 69-74. 41. Noonan, P. S.; Mohan, P.; Goodwin, A. P.; Schwartz, D. K., DNA Hybridization‐Mediated Liposome Fusion at the Aqueous Liquid Crystal Interface. Adv. Func. Mater. 2014, 24, 3206-3212. 42. Lindner, S.; Gruhle, K.; Schmidt, R.; Garamus, V. M.; Ramsbeck, D.; Hause, G.; Meister, A.; Sinz, A.; Drescher, S., Azide-Modified Membrane Lipids: Synthesis, Properties, and Reactivity. Langmuir 2017, 33, 4960-4973. 43. Blenke, E. O.; Klaasse, G.; Merten, H.; Plückthun, A.; Mastrobattista, E.; Martin, N. I., Liposome functionalization with copper-free “click chemistry”. J. Control. Release 2015, 202, 14-20. 44. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R., A strain-promoted [3+ 2] azide− alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 2004, 126, 15046-15047. 45. Whitehead, S. A.; McNitt, C. D.; Mattern-Schain, S. I.; Carr, A. J.; Alam, S.; Popik, V. V.; Best, M. D., Artificial Membrane Fusion Triggered by Strain-Promoted

Alkyne–Azide Cycloaddition. Bioconjugate Chem. 2017, 28, 923-932. 46. van Oers, M. C.; Rutjes, F. P.; van Hest, J. C., Tubular polymersomes: a cross-linker-induced shape transformation. J. Am. Chem. Soc. 2013, 135, 16308-16311. 47. Lee, S.; Yoon, H. I.; Na, J. H.; Jeon, S.; Lim, S.; Koo, H.; Han, S.-S.; Kang, S.-W.; Park, S.-J.; Moon, S.-H., In vivo stem cell tracking with imageable nanoparticles that bind bioorthogonal chemical receptors on the stem cell surface. Biomaterials 2017, 139, 12-29. 48. de Cogan, F.; Booth, A.; Gough, J. E.; Webb, S. J., Conversion of Magnetic Impulses into Cellular Responses by Self‐Assembled Nanoparticle–Vesicle Hydrogels. Angew. Chem. Int. Ed. 2011, 50, 12290-12293. 49. Forato, F.; Belhboub, A.; Monot, J.; Petit, M.; Benoit, R.; Sarou‐Kanian, V.; Fayon, F.; Jacquemin, D.; Queffelec, C.; Bujoli, B., Phosphonate‐Mediated Immobilization of Rhodium/Bipyridine Hydrogenation Catalysts. Chem. Eur. J. 2018, 24, 2457-2465. 50. Oppelt, K. T.; Gasiorowski, J.; Egbe, D. A. M.; Kollender, J. P.; Himmelsbach, M.; Hassel, A. W.; Sariciftci, N. S.; Knör, G., Rhodium-Coordinated Poly(aryleneethynylene)-alt-Poly(arylene-vinylene) Copolymer Acting as Photocatalyst for Visible-Light-Powered NAD+/NADH Reduction. J. Am. Chem. Soc. 2014, 136, 12721-12729. 51. Armstrong, A. F.; Oakley, N.; Parker, S.; Causey, P. W.; Lemon, J.; Capretta, A.; Zimmerman, C.; Joyal, J.; Appoh, F.; Zubieta, J., A robust strategy for the preparation of libraries of metallopeptides. A new paradigm for the discovery of targeted molecular imaging and therapy agents. Chem. Commun. 2008, 44, 5532-5534. 52. Samanta, D.; Sarkar, A., Immobilization of biomacromolecules on self-assembled monolayers: methods and sensor applications. Chem. Soc. Rev. 2011, 40, 25672592. 53. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S., Synthesis of proteins by native chemical ligation. Science 1994, 266, 776-779. 54. Zheng, J. S.; Cui, H. K.; Fang, G. M.; Xi, W. X.; Liu, L., Chemical protein synthesis by kinetically controlled ligation of peptide O‐esters. ChemBioChem 2010, 11, 511515. 55. Kang, C. W.; Sarnowski, M. P.; Elbatrawi, Y. M.; Del Valle, J. R., Access to Enantiopure α-Hydrazino Acids for N-Amino Peptide Synthesis. J. Org. Chem. 2017, 82, 1833-1841. 56. Kita, Y.; Maeda, H.; Takahashi, F.; Fukui, S., A convenient synthesis of dicarboxylic monoesters using isopropenyl esters: synthesis of oxaunomycin derivatives. J. Chem. Soc., Chem. Commun. 1993, 410-412. 57. Bonnet, D.; Grandjean, C.; Rousselot-Pailley, P.; Joly, P.; Bourel-Bonnet, L.; Santraine, V.; Gras-Masse, H.;

15



Page 16 of 17

Melnyk, O., Solid-phase functionalization of peptides by an α-hydrazinoacetyl group. J. Org. Chem. 2003, 68, 70337040. 58. Jørgensen, I. L.; Kemmer, G. C.; Pomorski, T. G., Membrane protein reconstitution into giant unilamellar vesicles: a review on current techniques. Eur. Biophys. J. 2017, 46, 103-119. 59. Almendro-Vedia, V. G.; Natale, P.; Mell, M.; Bonneau, S.; Monroy, F.; Joubert, F.; López-Montero, I., Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 11291-11296. 60. Park, S.; Majd, S., Reconstitution and functional studies of hamster P-glycoprotein in giant liposomes. PloS one 2018, 13, e0199279. 61. Talbot, E. L.; Kotar, J.; Di Michele, L.; Cicuta, P., Directed tubule growth from giant unilamellar vesicles in a thermal gradient. Soft Matter 2019.

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90x66mm (300 x 300 DPI)


Clustering of Giant Unilamellar Vesicles (GUVs) Promoted by

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