DOI: 10.1002/chem.201402187
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& Polyoxometalates
Multivalent Recognition of Concanavalin A by {Mo132} Glyconanocapsules—Toward Biomimetic Hybrid Multilayers Mihail Barboiu,*[a] Zineb Mouline,[a] Mihaela Silion,[b] Erol Licsandru,[a] Bogdan C. Simionescu,[b] Eugene Mahon,[a] and Mariana Pinteala[b] Dedicated to Professor Achim Mller on the occasion of his 76th birthday
Abstract: Herein, we consider Mller’s spherical, porous, anionic, molybdenum oxide based capsule, (NH4)42[{(MoVI)MoVI5O21(H2O)6}12{MoV2O4(CH3COO)}30]·10 CH3COONH4· 300 H2O (NH4)42·1 a·crystal ingredients 1, {Mo132}, as an effective sugar-decorated nanoplatform for multivalent lectin recognition. The ion-exchange of NH4 + ions of 1 with cationic-sugars, d-mannose-ammonium chloride (2) or d-glucoseammonium chloride (3) results in the formation of glyconanocapsules (NH4)42n2n·1 a and (NH4)42m3m·1 a. The Mannose (NH4)42n2n·1 a capsules bind selectively Concanavalin A (Con A) in aqueous solution, giving an association avidity constant of K multi = 4.6 104 m1 and an enhancement factor a multi mono of b = Ka /Kass = 21.9, reminiscent of the formation of “gly-
coside clusters” on the external surface of glyconanocapsule. The glyconanocapsules (NH4)42n2n·1 a and (NH4)42m3m·1 a self-assemble in “hybrid multilayers” by successive layer-bylayer deposition of (NH4)42n2n·1 a or (NH4)42m3m·1 a and Con A. These architectures, reminiscent of versatile mimics of artificial tissues, can be easily prepared and quantified by using quartz crystal microgravimetry (QCM). The “biomimetic hybrid multilayers” described here are stable under a continual water flow and they may serve as artificial networks for a greater depth of understanding of various biological mechanisms, which can directly benefit the fields of chemical separations, sensors or storage-delivery devices.
Introduction
cles[16–20] have been used to generate multivalent carbohydrate nanoplatforms. Conclusions from these investigations typically reveal that multivalent binding is strongly dependent on the dynamic distribution of glycoside clusters self-assembled on the nanoplatform surface and vice versa. Within this context, the development of novel nanoscaled glycosystems[21–24] could provide new important insights into dynamic constitutional behaviour of glycocluster formation upon multivalent binding to lectins, observed in most of the biological scenarios. Herein we consider Mller’s spherical, anionic, molybdenum oxide based capsule, {Mo132}: (NH4)42[{(MoVI)MoVI5O21(H2O)6}12{MoV2O4(CH3COO)}30]·10 CH3COONH4·300 H2O (NH4)42·1 a·crystal ingredients 1,[25] which we hypothesised to be a potential nanoplatform of interest for sugar-cluster formation for lectin multivalent recognition. This capsule exhibits an advantage as a biomimetic nanocontainer in that it mimics the cell in terms of ionic/molecular exchanges with external media. Our goals are to quantify the glyconanocapsule–lectin interactions and to survey the formation of interconnected networks of glyconanocapsules through biomimetic protein–carbohydrate interactions. They may offer realistic interpretations close to in vivo tissue scenarios, which can also directly benefit the fields of chemical separations, sensors or storage-delivery devices.
Biological membranes present dense areas of carbohydrates (glycocalyx) that play a fundamental role in cell–cell recognition processes through the multivalent binding of lectins.[1–3] The enhancement in the activity beyond what would be expected due to the increase in local sugar density is known as the “cluster glycoside effect”.[4] Based on this understanding, many artificial multivalent carbohydrate-clustering systems have been reported.[5–20] Early evidence was observed by the group of Kiessling, using mannose-based polymers displaying inhibition of Concanavalin A (Con A) of erythrocytes.[5] Since then, molecular, dendrimeric or polymeric systems of increasing complexity have been extensively developed.[6] Up-scaled nanosystems like fullerenes,[7] metallosupramolecular spheres,[8] nanoparticles,[9–13] monolayers,[14] carbon nanotubes[15] and vesi[a] Dr. M. Barboiu, Dr. Z. Mouline, E. Licsandru, Dr. E. Mahon Adaptive Supramolecular Nanosystems Group Institut Europen des Membranes, ENSCM-UMII-CNRS UMR-5635 Place Eugne Bataillon, CC 047, F-34095, Montpellier (France) Fax: : (+ 33) 467149119 E-mail: [email protected] [b] Dr. M. Silion, Prof. B. C. Simionescu, Dr. M. Pinteala Centre of Advanced Research in Nanobioconjugates and Biopolymers “Petru Poni” Institute of Macromolecular Chemistry 41A Aleea Gr. Ghica Voda, Iasi (Romania) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402187. Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper Results and Discussion Synthesis of multivalent glyconanocapsules and their stability The ion-exchange of NH4 + cations of 1 with cationic ammonium sugars on their anionic external surface lead to the formation of multivalent glyconanocapsules, (NH4)42n2n·1 a and (NH4)42m3m·1 a (n and m were not experimentally determined)[26] (Scheme 1). They were prepared in situ, in milliQ
Scheme 1. Synthesis of mannoside (NH4)42n2n·1 a and glucoside (NH4)42m 3m·1 a glyconanocapsules.
aqueous and phosphate buffer (PBS, pH 7) solutions, from of d-mannose ammonium chloride (2) or d-glucose ammonium chloride (3) and the capsule 1 (molar ratio, sugar/capsule 40:1).[27] It is important to note that the initial pH 4.3 of the solutions of 1 become pH 6.3 after dilution with milliQ aqueous solutions of the cationic sugars. The final stock aqueous and PBS solutions (c = 0.5 mm) of 1 and of the glyconanocapsules, (NH4)42n2n·1 a and (NH4)42m3m·1 a present similar UV spectra (Figure 1 S in the Supporting Information). The UV spectrum of the PBS solution of 1 and both glyconanocapsule solutions present more intense absorbtion bands, probably due to the specific interactions of {Mo132} core with phosphate anions and cationic sugars, respectively, as previously observed for mixtures of {Mo132} and agarose samples.[28] The observation of similar spectra in the UV/Vis region suggest that the redox states of the {Mo132} core cluster is not affected by the value of pH nor by the cation sugar exchange in solutions at the mm concentration domain. The sugar multivalent clustering is favoured through {Mo132} macroanion/ammonium sugar charge interactions, stabilised by lateral sugar–sugar hydrogen-bonding interactions.
Figure 1. a) Time-dependent change in the fluorescence intensity emission at 500 nm and b) the decrease in the fluorescence intensity ratio (I/I0) of Con A (4.86 mm) on successive additions of aqueous solution of (NH4)42n2n·1 a (prepared in situ from 5 mL of each aqueous solutions 1.6 103 m of 2 and 4.0 105 m of 1).
centration of (NH4)42n2n·1 a is 60 mm and the pH 7.0). In this context it is important to note that the (NH4)42n2n·1 a/ConA mixture show no apparent agglutination (aggregation)[18] at mm concentrations as proved by fluorescence and dynamic light scattering (DLS) studies. The aggregation started to be observed at the mm concentration level, when despite the slower formation of small amounts of precipitate, the DLS spectra show new peak formation. Figure 1 shows that the intensity of the Con A fluorescence values decrease in the presence of the Mannoside hybrid (NH4)42n2n·1 a, due to the formation of (NH4)42n2n·1 a/ConA conjugates. The results of fitting the Stern–Volmer model[29] to the (NH4)42n2n·1 a conjugation on Con A in aqueous solution gives an association avidity constant of K multi = 4.6 104 m1 a (Figure 2 S in Supporting Information). It known that, the affinity constant is usually used for the monosaccharides as a binding equilibrium constant, K mono ass . In the case of (NH4)42n2n·1 clusters presented here, we consider a “collective affinity” or the avidity constant, K multi .[30, 31] Considering that the association a
Multivalent binding of glyconanocapsules to Con A Evidence for spontaneous Con A recognition by glyconanocapsules was demonstrated by the quenching of fluorescence of Con A (4.8 mm) at 500 nm, upon repetitive additions of aliquots of stock milliQ solution of hybrid (NH4)42n2n·1 a (the final con&
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Full Paper ing the enhancement factors and the avidity constants, the binding of (NH4)42n2n·1 a to Con A falls somewhere in between the previously calculated values for the interaction of monoand trisaccharide compounds with Con A, thus showing a disaccharide binding effect. The thermodynamic treatments consider the main driving force of the multivalent sugar–lectin recognition to be a reduced entropy cost on the binding of a second sugar molecule, as the binding enthalpy for the first sugar is almost constant.[32] Decreased conformational entropy cost would remain consistent in the present case, in which cationic mannoses 2 cluster with restricted entropy on the surface of the charged nanoanion 1 a. Multilayer hybrid architectures Our efforts have been concentrated in preparing composite multilayer architectures[13] by successive layer-by-layer deposition of (NH4)42n2n·1 a and Con A or (NH4)42m3m·1 a and Con A based on sugar–lectin multivalent interactions. These architectures can be easily prepared and quantified by using quartz crystal microgravimetry (QCM), which detects mass adsorption at sensor surfaces on the basis of the reciprocal piezoelectric effect.[35] The increase in absorbed mass of matter is correlated with a frequency shift (DF)—a shift of 1 Hz corresponds to a mass change of about 700 pg.[36] For this study, Con A was immobilised on QCM quartz crystals by combination of a non-specific hydrophobic interaction and by recognition of Mannan polysaccharide as previously reported by us[11, 13] or by Pei et al.[14b] We note that only mannoside ((NH4)42n2n·1 a) and glucoside ((NH4)42n3n·1 a) glyconanohybrids can be bound selectively to the protein layer. The Con A layer is resistant to the (NH4)42n1 a sugar-free capsules, which are anti-adhesive, showing no signs of adsorption (Figure 2 a). This proves that nonspecific binding of the (NH4)42n1 a sugar-free capsules can be avoided. The binding events are presumably a result of the tetrameric structure of Con A, which owing to shape constraints should only occupy two of its four binding sites upon surface mannan initial immobilisation, leaving two sites free presented to the aqueous media for further interactions with the sugar-decorated inorganic capsules (NH4)42n2n·1 a and (NH4)42 m3m·1 a. The Con A saturated surface once exposed to an aqueous solution of (NH4)42n2n·1 a can bind a stable layer of sugar-decorated inorganic capsules. Subsequent addition of Con A induces further formation of a new Con A layer. Alternating successive layers of a (NH4)42n2n·1 a and Con A or (NH4)42m3m·1 a and Con A may thus be repetitively built up by successive additions of each of the components. The plot in Figure 3 shows the initial six Con A injections (1 mm) on an adsorbed mannan film to give a saturated layer. This was followed by an injection of the mannoside (NH4)42n2n·1 a capsules at a concentration of 2 = 4 mm. A further Con A injection followed, which showed increased mass absorption of the Con A proving that thermodynamic equilibrium is not reached within a single-layer deposition. Interestingly, further injections of the mannoside (NH4)42n2n·1 a and
Figure 2. a) Frequency profile showing progressive Con A immobilisation on a mannan film on a ODT hydrophobic surface of a QCM gold electrode followed by the non-absorption of (NH4)421 a and the specific absorption of (NH4)42n2n·1 a (prepared at pH 4.3) on Con A layers. b) Experimental QCM monitoring of layer by layer formation (DF in Hz) associated with successive Con A/(NH4)42n2n·1 a and Con A/(NH4)42m3m·1 superposed deposition of layers by mono-injection mode.
constant for mannose is Kass = 2.1 103 m1,[32, 33] this results in /Kmono an enhancement factor,[30] b = Kmulti a ass = 21.9 for the mannose capsules (NH4)42n2n·1. A structural basis for this increased affinity can be described based on an increased number of favourable sugar–protein contacts. The previous determination of Con A structures with the disaccharides, Man-a-(1!6)-Man-a-OMe and Man-a-(1! 3)-Man-a-OMe, showed the O-1-linked mannose to occupy the monosaccharide binding site in an analogous fashion to aMeOMan.[33] The crystal structure of a trisaccharide, Man-a-(1! 6)-[Man-a-(1!3)]-Man[34] indicated that the 1!6 terminal mannose occupied the monosaccharide site. The central reducing sugar in the core and the 1!3 terminal mannose occupied an adjacent extended region of the binding groove, also making direct polar and apolar contacts with the protein. A resultant affinity of K multi = 4.9 105 m1 has been reported a for Man-a-(1!6)-[Man-a-(1!3)]-Man and an enhancement [33] factor related to mannose of b = Kmulti /Kmono Compara ass = 233. Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper packing within layers, being determinant for the increasing amounts of ConA/mannoside capsules at the thermodynamic equilibrium. These systems, which behave as hydrogel-like structures, are stable for days under the flow of an aqueous solution and in the absence of perturbing factors. Unfortunately, they collapse and are not stable in air or vacuum, which was the issue which caused problems in attempts at further characterisation and no-relevant results can be reported for the present work through electron microscopy (see some unrepresentative SEM images on cracked dried hydrogels on the QCM gold surface in Figure 5S in the Supporting Information), X-ray diffraction, ellipsometry and so forth.
Conclusions Based on their complex structure, the (NH4)42·1 a porous spherical capsules are highly functional systems. 1) They present 20 {Mo9O9} crown-ether type pores that allow different types of metabolites (ions, water, large molecules, etc.) to enter/leave their cavities.[37] 2) Their self-assembly through non-specific hydrophobic or ionic interactions have been previously demonstrated: by using charged or surfactant-encapsulated {Mo132} capsules, we can generate different architectures, like cubic phases, honeycomb multilayers, core and shell or onion-like particles, and lamellar or columnar aggregates within lipophilic silica matriFigure 3. a) In situ QCM monitoring of layer-by-layer ConA/(NH4)42n2n·1 a multilayer arces, resulting in the formation of functional adaptive chitectures by specific multivalent recognition (see Figure 3 Sb in Supporting Information constitutional systems.[38, 39] Another impressive examfor the ConA/(NH4)42m3m·1 a multilayer architecture experiment). b) Schematic represenple within this context is provided by the self-recogtation of “inorganic cell tissues” interacting via specific sugar-lectin interactions. nition among the robust {Mo72Fe30} and {Mo72Cr30}, which form two different individual blackberry-type Con A produce successive layers of almost constant-weight of spherical structures in aqueous solution instead of mixed ones.[40] mannoside capsules (NH4)42n2n·1 a (red layers in Figure 3), while the layers of ConA constantly absorb increasing amounts Allied to in vitro cell-cell recognition mimic, the glyconanoof lectin (pink layers in Figure 3). They are probably redistributcapsules described here, specifically interact with lectins and ed within the next layers, resulting in the formation of inferior self-assemble in multilayer hybrid architectures (Figure 3 b) layers of higher density, reinforced via lateral hydrophobic inonly if their external multivalent carbohydrate presentation teractions between ConA biomolecules. and lectin recognition sites are compatible. This work is reportFurther results indicate that the glyconanocapsules are ing the formation of the multivalent glyconanocapsules that indeed specifically bind to the saccharide structure. The NMR selectively can recognise lectins with enhanced affinity. This is analysis show that d-mannose ammonium chloride (2), both in an important feature to emphasise complementary effects on aqueous solution and bonded on (NH4)42n2n·1 a, exists in constitutional distribution of glycoside clusters on the external surface of the {Mo132} upon multivalent binding at the Con A 100 % a-configuration, whereas for d-glucose ammonium chloride (3), which is initially 82 % in a-configuration in aqueous surface and vice-versa. solution, is bound to capsule 1 with 70 % in an a-configuration These systems are completely adapted to follow evolutional (as can be observed in 1H NMR spectra of sugar and capsule approaches toward biological selections of functions.[41, 42] As recorded in D2O). The preference of Con A for the a-configuraMller says: “With these materials we are bringing inorganic chemistry to life.”[43] More specifically in terms of the further intion of glycosides[33] can go some way towards rationalising the observed result: a higher successive increase in absorption vestigation, the systems described here can lead to a deeper of (NH4)42n2n·1 a/ConA mannoside layers, against a lower and understanding of their collective interaction with variation in guest ions/molecules distribution and their transport across constant absorption of (NH4)42n3n·1 a/ConA glucoside layers the hybrid multilayers, close to the natural selection of func(Figure 2 b and Figure 3S in Supporting Information). The sugar tions. affinity is of maximal importance for ConA/capsule multivalent &
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All of the materials were obtained from Aldrich/Fluka and used without purification. Compound 1[25] was kindly provided by Prof. Achim Mller’s group.
Quartz crystal microgravimetry (QCM) measurements These measurements were performed using the A100 instrument from Attana Biosensors. The experiments were run in the continuous-flow QCM system at a flow rate of 20 mL min1. The experiments were performed at 25 8C in deionised water or the PBS buffer at pH 7.0. Hydrophobic self-assembled monolayers were prepared directly on gold-coated quartz crystals acquired from Attana. The crystals were firstly immersed in a freshly prepared piranha solution (H2SO4/H2O2 7:3) for 1 min and then washed thoroughly with pure water. They were then dried under a nitrogen stream and immersed in a 1 mm solution of octadecanethiol (ODT) in EtOH/n-hexane (9:1) for at least 16 h followed by extensive rinsing with ethanol followed by n-hexane and then more ethanol and lastly water before being blown dry with a nitrogen stream and being placed in the QCM chip setup. Specific adsorption through mannoside recognition was performed for Con A immobilisation and was accomplished with self-assembled monolayers (SAMs) or adsorbed films of high specificity. The ODT monolayer surface was saturated with the mannan giving a stable film through repeated injections at a low concentration (50 mg mL1). Following this mannan–ODT film deposition, buffered Con A solutions at 1 mm concentration were passed over the films and resulted in the formation of dense lectin layers, which stabilised with time (20–30 min). This was followed by an injection of the mannoside capsules (NH4)42n2n·1 a (5 mL of 1.6 103 m of 2 and 4.0 105 m of 1) and so on.
Acknowledgements This work was financially supported by the Romanian National Authority for Scientific Research, CNCS–UEFISCDI grant, project number PN-II-ID-PCCE-2011-2-0028 contract 4/30.05.2012. Z.M. thanks the regional funds from Region Languedoc Roussillon (France). Keywords: “inorganic cells tissues” · glyconano hybrids · molybdates · polyoxometalates · sugar–protein interactions [1] [2] [3] [4]
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Sweet structures! Specific multivalent carbohydrate–protein interactions between sugar-decorated polyoxometalate capsules and Concavalin A can be used to generate biomimetic hybrid multilayers (see figure).
M. Barboiu,* Z. Mouline, M. Silion, E. Licsandru, B. C. Simionescu, E. Mahon, M. Pinteala && – && Multivalent Recognition of Concanavalin A by {Mo132} Glyconanocapsules—Toward Biomimetic Hybrid Multilayers
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Multivalent Recognition of Concanavalin A by {Mo132} Glyconanocapsules—Toward Biomimetic Hybrid Multilayers Mihail Barboiu,*[a] Zineb Mouline,[a] Mihaela Silion,[b] Erol Licsandru,[a] Bogdan C. Simionescu,[b] Eugene Mahon,[a] and Mariana Pinteala[b] Dedicated to Professor Achim Mller on the occasion of his 76th birthday
Abstract: Herein, we consider Mller’s spherical, porous, anionic, molybdenum oxide based capsule, (NH4)42[{(MoVI)MoVI5O21(H2O)6}12{MoV2O4(CH3COO)}30]·10 CH3COONH4· 300 H2O (NH4)42·1 a·crystal ingredients 1, {Mo132}, as an effective sugar-decorated nanoplatform for multivalent lectin recognition. The ion-exchange of NH4 + ions of 1 with cationic-sugars, d-mannose-ammonium chloride (2) or d-glucoseammonium chloride (3) results in the formation of glyconanocapsules (NH4)42n2n·1 a and (NH4)42m3m·1 a. The Mannose (NH4)42n2n·1 a capsules bind selectively Concanavalin A (Con A) in aqueous solution, giving an association avidity constant of K multi = 4.6 104 m1 and an enhancement factor a multi mono of b = Ka /Kass = 21.9, reminiscent of the formation of “gly-
coside clusters” on the external surface of glyconanocapsule. The glyconanocapsules (NH4)42n2n·1 a and (NH4)42m3m·1 a self-assemble in “hybrid multilayers” by successive layer-bylayer deposition of (NH4)42n2n·1 a or (NH4)42m3m·1 a and Con A. These architectures, reminiscent of versatile mimics of artificial tissues, can be easily prepared and quantified by using quartz crystal microgravimetry (QCM). The “biomimetic hybrid multilayers” described here are stable under a continual water flow and they may serve as artificial networks for a greater depth of understanding of various biological mechanisms, which can directly benefit the fields of chemical separations, sensors or storage-delivery devices.
Introduction
cles[16–20] have been used to generate multivalent carbohydrate nanoplatforms. Conclusions from these investigations typically reveal that multivalent binding is strongly dependent on the dynamic distribution of glycoside clusters self-assembled on the nanoplatform surface and vice versa. Within this context, the development of novel nanoscaled glycosystems[21–24] could provide new important insights into dynamic constitutional behaviour of glycocluster formation upon multivalent binding to lectins, observed in most of the biological scenarios. Herein we consider Mller’s spherical, anionic, molybdenum oxide based capsule, {Mo132}: (NH4)42[{(MoVI)MoVI5O21(H2O)6}12{MoV2O4(CH3COO)}30]·10 CH3COONH4·300 H2O (NH4)42·1 a·crystal ingredients 1,[25] which we hypothesised to be a potential nanoplatform of interest for sugar-cluster formation for lectin multivalent recognition. This capsule exhibits an advantage as a biomimetic nanocontainer in that it mimics the cell in terms of ionic/molecular exchanges with external media. Our goals are to quantify the glyconanocapsule–lectin interactions and to survey the formation of interconnected networks of glyconanocapsules through biomimetic protein–carbohydrate interactions. They may offer realistic interpretations close to in vivo tissue scenarios, which can also directly benefit the fields of chemical separations, sensors or storage-delivery devices.
Biological membranes present dense areas of carbohydrates (glycocalyx) that play a fundamental role in cell–cell recognition processes through the multivalent binding of lectins.[1–3] The enhancement in the activity beyond what would be expected due to the increase in local sugar density is known as the “cluster glycoside effect”.[4] Based on this understanding, many artificial multivalent carbohydrate-clustering systems have been reported.[5–20] Early evidence was observed by the group of Kiessling, using mannose-based polymers displaying inhibition of Concanavalin A (Con A) of erythrocytes.[5] Since then, molecular, dendrimeric or polymeric systems of increasing complexity have been extensively developed.[6] Up-scaled nanosystems like fullerenes,[7] metallosupramolecular spheres,[8] nanoparticles,[9–13] monolayers,[14] carbon nanotubes[15] and vesi[a] Dr. M. Barboiu, Dr. Z. Mouline, E. Licsandru, Dr. E. Mahon Adaptive Supramolecular Nanosystems Group Institut Europen des Membranes, ENSCM-UMII-CNRS UMR-5635 Place Eugne Bataillon, CC 047, F-34095, Montpellier (France) Fax: : (+ 33) 467149119 E-mail: [email protected] [b] Dr. M. Silion, Prof. B. C. Simionescu, Dr. M. Pinteala Centre of Advanced Research in Nanobioconjugates and Biopolymers “Petru Poni” Institute of Macromolecular Chemistry 41A Aleea Gr. Ghica Voda, Iasi (Romania) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201402187. Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper Results and Discussion Synthesis of multivalent glyconanocapsules and their stability The ion-exchange of NH4 + cations of 1 with cationic ammonium sugars on their anionic external surface lead to the formation of multivalent glyconanocapsules, (NH4)42n2n·1 a and (NH4)42m3m·1 a (n and m were not experimentally determined)[26] (Scheme 1). They were prepared in situ, in milliQ
Scheme 1. Synthesis of mannoside (NH4)42n2n·1 a and glucoside (NH4)42m 3m·1 a glyconanocapsules.
aqueous and phosphate buffer (PBS, pH 7) solutions, from of d-mannose ammonium chloride (2) or d-glucose ammonium chloride (3) and the capsule 1 (molar ratio, sugar/capsule 40:1).[27] It is important to note that the initial pH 4.3 of the solutions of 1 become pH 6.3 after dilution with milliQ aqueous solutions of the cationic sugars. The final stock aqueous and PBS solutions (c = 0.5 mm) of 1 and of the glyconanocapsules, (NH4)42n2n·1 a and (NH4)42m3m·1 a present similar UV spectra (Figure 1 S in the Supporting Information). The UV spectrum of the PBS solution of 1 and both glyconanocapsule solutions present more intense absorbtion bands, probably due to the specific interactions of {Mo132} core with phosphate anions and cationic sugars, respectively, as previously observed for mixtures of {Mo132} and agarose samples.[28] The observation of similar spectra in the UV/Vis region suggest that the redox states of the {Mo132} core cluster is not affected by the value of pH nor by the cation sugar exchange in solutions at the mm concentration domain. The sugar multivalent clustering is favoured through {Mo132} macroanion/ammonium sugar charge interactions, stabilised by lateral sugar–sugar hydrogen-bonding interactions.
Figure 1. a) Time-dependent change in the fluorescence intensity emission at 500 nm and b) the decrease in the fluorescence intensity ratio (I/I0) of Con A (4.86 mm) on successive additions of aqueous solution of (NH4)42n2n·1 a (prepared in situ from 5 mL of each aqueous solutions 1.6 103 m of 2 and 4.0 105 m of 1).
centration of (NH4)42n2n·1 a is 60 mm and the pH 7.0). In this context it is important to note that the (NH4)42n2n·1 a/ConA mixture show no apparent agglutination (aggregation)[18] at mm concentrations as proved by fluorescence and dynamic light scattering (DLS) studies. The aggregation started to be observed at the mm concentration level, when despite the slower formation of small amounts of precipitate, the DLS spectra show new peak formation. Figure 1 shows that the intensity of the Con A fluorescence values decrease in the presence of the Mannoside hybrid (NH4)42n2n·1 a, due to the formation of (NH4)42n2n·1 a/ConA conjugates. The results of fitting the Stern–Volmer model[29] to the (NH4)42n2n·1 a conjugation on Con A in aqueous solution gives an association avidity constant of K multi = 4.6 104 m1 a (Figure 2 S in Supporting Information). It known that, the affinity constant is usually used for the monosaccharides as a binding equilibrium constant, K mono ass . In the case of (NH4)42n2n·1 clusters presented here, we consider a “collective affinity” or the avidity constant, K multi .[30, 31] Considering that the association a
Multivalent binding of glyconanocapsules to Con A Evidence for spontaneous Con A recognition by glyconanocapsules was demonstrated by the quenching of fluorescence of Con A (4.8 mm) at 500 nm, upon repetitive additions of aliquots of stock milliQ solution of hybrid (NH4)42n2n·1 a (the final con&
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Full Paper ing the enhancement factors and the avidity constants, the binding of (NH4)42n2n·1 a to Con A falls somewhere in between the previously calculated values for the interaction of monoand trisaccharide compounds with Con A, thus showing a disaccharide binding effect. The thermodynamic treatments consider the main driving force of the multivalent sugar–lectin recognition to be a reduced entropy cost on the binding of a second sugar molecule, as the binding enthalpy for the first sugar is almost constant.[32] Decreased conformational entropy cost would remain consistent in the present case, in which cationic mannoses 2 cluster with restricted entropy on the surface of the charged nanoanion 1 a. Multilayer hybrid architectures Our efforts have been concentrated in preparing composite multilayer architectures[13] by successive layer-by-layer deposition of (NH4)42n2n·1 a and Con A or (NH4)42m3m·1 a and Con A based on sugar–lectin multivalent interactions. These architectures can be easily prepared and quantified by using quartz crystal microgravimetry (QCM), which detects mass adsorption at sensor surfaces on the basis of the reciprocal piezoelectric effect.[35] The increase in absorbed mass of matter is correlated with a frequency shift (DF)—a shift of 1 Hz corresponds to a mass change of about 700 pg.[36] For this study, Con A was immobilised on QCM quartz crystals by combination of a non-specific hydrophobic interaction and by recognition of Mannan polysaccharide as previously reported by us[11, 13] or by Pei et al.[14b] We note that only mannoside ((NH4)42n2n·1 a) and glucoside ((NH4)42n3n·1 a) glyconanohybrids can be bound selectively to the protein layer. The Con A layer is resistant to the (NH4)42n1 a sugar-free capsules, which are anti-adhesive, showing no signs of adsorption (Figure 2 a). This proves that nonspecific binding of the (NH4)42n1 a sugar-free capsules can be avoided. The binding events are presumably a result of the tetrameric structure of Con A, which owing to shape constraints should only occupy two of its four binding sites upon surface mannan initial immobilisation, leaving two sites free presented to the aqueous media for further interactions with the sugar-decorated inorganic capsules (NH4)42n2n·1 a and (NH4)42 m3m·1 a. The Con A saturated surface once exposed to an aqueous solution of (NH4)42n2n·1 a can bind a stable layer of sugar-decorated inorganic capsules. Subsequent addition of Con A induces further formation of a new Con A layer. Alternating successive layers of a (NH4)42n2n·1 a and Con A or (NH4)42m3m·1 a and Con A may thus be repetitively built up by successive additions of each of the components. The plot in Figure 3 shows the initial six Con A injections (1 mm) on an adsorbed mannan film to give a saturated layer. This was followed by an injection of the mannoside (NH4)42n2n·1 a capsules at a concentration of 2 = 4 mm. A further Con A injection followed, which showed increased mass absorption of the Con A proving that thermodynamic equilibrium is not reached within a single-layer deposition. Interestingly, further injections of the mannoside (NH4)42n2n·1 a and
Figure 2. a) Frequency profile showing progressive Con A immobilisation on a mannan film on a ODT hydrophobic surface of a QCM gold electrode followed by the non-absorption of (NH4)421 a and the specific absorption of (NH4)42n2n·1 a (prepared at pH 4.3) on Con A layers. b) Experimental QCM monitoring of layer by layer formation (DF in Hz) associated with successive Con A/(NH4)42n2n·1 a and Con A/(NH4)42m3m·1 superposed deposition of layers by mono-injection mode.
constant for mannose is Kass = 2.1 103 m1,[32, 33] this results in /Kmono an enhancement factor,[30] b = Kmulti a ass = 21.9 for the mannose capsules (NH4)42n2n·1. A structural basis for this increased affinity can be described based on an increased number of favourable sugar–protein contacts. The previous determination of Con A structures with the disaccharides, Man-a-(1!6)-Man-a-OMe and Man-a-(1! 3)-Man-a-OMe, showed the O-1-linked mannose to occupy the monosaccharide binding site in an analogous fashion to aMeOMan.[33] The crystal structure of a trisaccharide, Man-a-(1! 6)-[Man-a-(1!3)]-Man[34] indicated that the 1!6 terminal mannose occupied the monosaccharide site. The central reducing sugar in the core and the 1!3 terminal mannose occupied an adjacent extended region of the binding groove, also making direct polar and apolar contacts with the protein. A resultant affinity of K multi = 4.9 105 m1 has been reported a for Man-a-(1!6)-[Man-a-(1!3)]-Man and an enhancement [33] factor related to mannose of b = Kmulti /Kmono Compara ass = 233. Chem. Eur. J. 2014, 20, 1 – 7
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Full Paper packing within layers, being determinant for the increasing amounts of ConA/mannoside capsules at the thermodynamic equilibrium. These systems, which behave as hydrogel-like structures, are stable for days under the flow of an aqueous solution and in the absence of perturbing factors. Unfortunately, they collapse and are not stable in air or vacuum, which was the issue which caused problems in attempts at further characterisation and no-relevant results can be reported for the present work through electron microscopy (see some unrepresentative SEM images on cracked dried hydrogels on the QCM gold surface in Figure 5S in the Supporting Information), X-ray diffraction, ellipsometry and so forth.
Conclusions Based on their complex structure, the (NH4)42·1 a porous spherical capsules are highly functional systems. 1) They present 20 {Mo9O9} crown-ether type pores that allow different types of metabolites (ions, water, large molecules, etc.) to enter/leave their cavities.[37] 2) Their self-assembly through non-specific hydrophobic or ionic interactions have been previously demonstrated: by using charged or surfactant-encapsulated {Mo132} capsules, we can generate different architectures, like cubic phases, honeycomb multilayers, core and shell or onion-like particles, and lamellar or columnar aggregates within lipophilic silica matriFigure 3. a) In situ QCM monitoring of layer-by-layer ConA/(NH4)42n2n·1 a multilayer arces, resulting in the formation of functional adaptive chitectures by specific multivalent recognition (see Figure 3 Sb in Supporting Information constitutional systems.[38, 39] Another impressive examfor the ConA/(NH4)42m3m·1 a multilayer architecture experiment). b) Schematic represenple within this context is provided by the self-recogtation of “inorganic cell tissues” interacting via specific sugar-lectin interactions. nition among the robust {Mo72Fe30} and {Mo72Cr30}, which form two different individual blackberry-type Con A produce successive layers of almost constant-weight of spherical structures in aqueous solution instead of mixed ones.[40] mannoside capsules (NH4)42n2n·1 a (red layers in Figure 3), while the layers of ConA constantly absorb increasing amounts Allied to in vitro cell-cell recognition mimic, the glyconanoof lectin (pink layers in Figure 3). They are probably redistributcapsules described here, specifically interact with lectins and ed within the next layers, resulting in the formation of inferior self-assemble in multilayer hybrid architectures (Figure 3 b) layers of higher density, reinforced via lateral hydrophobic inonly if their external multivalent carbohydrate presentation teractions between ConA biomolecules. and lectin recognition sites are compatible. This work is reportFurther results indicate that the glyconanocapsules are ing the formation of the multivalent glyconanocapsules that indeed specifically bind to the saccharide structure. The NMR selectively can recognise lectins with enhanced affinity. This is analysis show that d-mannose ammonium chloride (2), both in an important feature to emphasise complementary effects on aqueous solution and bonded on (NH4)42n2n·1 a, exists in constitutional distribution of glycoside clusters on the external surface of the {Mo132} upon multivalent binding at the Con A 100 % a-configuration, whereas for d-glucose ammonium chloride (3), which is initially 82 % in a-configuration in aqueous surface and vice-versa. solution, is bound to capsule 1 with 70 % in an a-configuration These systems are completely adapted to follow evolutional (as can be observed in 1H NMR spectra of sugar and capsule approaches toward biological selections of functions.[41, 42] As recorded in D2O). The preference of Con A for the a-configuraMller says: “With these materials we are bringing inorganic chemistry to life.”[43] More specifically in terms of the further intion of glycosides[33] can go some way towards rationalising the observed result: a higher successive increase in absorption vestigation, the systems described here can lead to a deeper of (NH4)42n2n·1 a/ConA mannoside layers, against a lower and understanding of their collective interaction with variation in guest ions/molecules distribution and their transport across constant absorption of (NH4)42n3n·1 a/ConA glucoside layers the hybrid multilayers, close to the natural selection of func(Figure 2 b and Figure 3S in Supporting Information). The sugar tions. affinity is of maximal importance for ConA/capsule multivalent &
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All of the materials were obtained from Aldrich/Fluka and used without purification. Compound 1[25] was kindly provided by Prof. Achim Mller’s group.
Quartz crystal microgravimetry (QCM) measurements These measurements were performed using the A100 instrument from Attana Biosensors. The experiments were run in the continuous-flow QCM system at a flow rate of 20 mL min1. The experiments were performed at 25 8C in deionised water or the PBS buffer at pH 7.0. Hydrophobic self-assembled monolayers were prepared directly on gold-coated quartz crystals acquired from Attana. The crystals were firstly immersed in a freshly prepared piranha solution (H2SO4/H2O2 7:3) for 1 min and then washed thoroughly with pure water. They were then dried under a nitrogen stream and immersed in a 1 mm solution of octadecanethiol (ODT) in EtOH/n-hexane (9:1) for at least 16 h followed by extensive rinsing with ethanol followed by n-hexane and then more ethanol and lastly water before being blown dry with a nitrogen stream and being placed in the QCM chip setup. Specific adsorption through mannoside recognition was performed for Con A immobilisation and was accomplished with self-assembled monolayers (SAMs) or adsorbed films of high specificity. The ODT monolayer surface was saturated with the mannan giving a stable film through repeated injections at a low concentration (50 mg mL1). Following this mannan–ODT film deposition, buffered Con A solutions at 1 mm concentration were passed over the films and resulted in the formation of dense lectin layers, which stabilised with time (20–30 min). This was followed by an injection of the mannoside capsules (NH4)42n2n·1 a (5 mL of 1.6 103 m of 2 and 4.0 105 m of 1) and so on.
Acknowledgements This work was financially supported by the Romanian National Authority for Scientific Research, CNCS–UEFISCDI grant, project number PN-II-ID-PCCE-2011-2-0028 contract 4/30.05.2012. Z.M. thanks the regional funds from Region Languedoc Roussillon (France). Keywords: “inorganic cells tissues” · glyconano hybrids · molybdates · polyoxometalates · sugar–protein interactions [1] [2] [3] [4]
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Chem. Eur. J. 2014, 20, 1 – 7
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Received: February 14, 2014 Published online on && &&, 0000
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Full Paper
FULL PAPER & Polyoxometalates
Sweet structures! Specific multivalent carbohydrate–protein interactions between sugar-decorated polyoxometalate capsules and Concavalin A can be used to generate biomimetic hybrid multilayers (see figure).
M. Barboiu,* Z. Mouline, M. Silion, E. Licsandru, B. C. Simionescu, E. Mahon, M. Pinteala && – && Multivalent Recognition of Concanavalin A by {Mo132} Glyconanocapsules—Toward Biomimetic Hybrid Multilayers
Chem. Eur. J. 2014, 20, 1 – 7
www.chemeurj.org
These are not the final page numbers! ÞÞ
7
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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